U.S. patent application number 14/878330 was filed with the patent office on 2016-03-03 for thiadiazolidinone derivatives.
This patent application is currently assigned to University of Rochester. The applicant listed for this patent is Monica Guzman, Craig Jordan. Invention is credited to Monica Guzman, Craig Jordan.
Application Number | 20160058741 14/878330 |
Document ID | / |
Family ID | 38957376 |
Filed Date | 2016-03-03 |
United States Patent
Application |
20160058741 |
Kind Code |
A1 |
Jordan; Craig ; et
al. |
March 3, 2016 |
Thiadiazolidinone Derivatives
Abstract
The present invention relates to compounds of the formulae
herein, their acceptable salts, solvates, hydrates and polymorphs
thereof. The compounds of this invention are useful in treatment of
disease, particularly leukemia. The invention also provides
compositions comprising a compound of this invention and the use of
such compositions in methods of treating disease, disorders, or
symptoms thereof in a subject.
Inventors: |
Jordan; Craig; (Rochester,
NY) ; Guzman; Monica; (Rochester, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Jordan; Craig
Guzman; Monica |
Rochester
Rochester |
NY
NY |
US
US |
|
|
Assignee: |
University of Rochester
Rochester
NY
|
Family ID: |
38957376 |
Appl. No.: |
14/878330 |
Filed: |
October 8, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12374002 |
Nov 9, 2009 |
9180118 |
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PCT/US07/16391 |
Jul 18, 2007 |
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14878330 |
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60831893 |
Jul 18, 2006 |
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Current U.S.
Class: |
514/342 ;
514/361; 546/268.7; 548/130 |
Current CPC
Class: |
A61K 31/41 20130101;
A61K 31/4164 20130101; A61K 31/433 20130101; A61P 35/02 20180101;
A61K 31/4439 20130101; A61P 43/00 20180101 |
International
Class: |
A61K 31/433 20060101
A61K031/433; A61K 31/4439 20060101 A61K031/4439 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] Research supporting this application was carried out in part
by funding provided by the United States of America as represented
by the Secretary, Department of Health and Human Services under
grant NIH R01CA90446. The government may have certain rights in the
invention.
Claims
1. A method for treating leukemia in a subject comprising
administration to the subject of a compound of Formula (I):
##STR00015## or a salt thereof; or a prodrug or a salt of a prodrug
thereof; or a hydrate, solvate or polymorph thereof; wherein: A is
--C(R.sup.1).sub.2--, --O-- or --NR.sup.1--; E is --NR.sup.1-- or
--CR.sup.1R.sup.2-- and the substituent R.sup.2 is absent if - - -
is a second bond between E and G; G is --S--, --NR.sup.1-- or
--CR.sup.1R.sup.2-- and the substituent R.sup.2 is absent if - - -
is a second bond between E and G; - - - may be a second bond
between E and G where the nature of E and G permits and E with G
optionally then forms a fused aryl group; R.sup.1 and R.sup.2 are
each independently selected from hydrogen, alkyl, cycloalkyl,
haloalkyl, aryl, --(Z).sub.n-aryl, heteroaryl, --OR.sup.3,
--C(O)R.sup.3, --C(O)OR.sup.3, --(Z).sub.n--C(O)OR.sup.3 and
--S(O).sub.t-- or as indicated R.sup.2 can be such that E with G
then form a fused aryl group; Z is independently selected from
--C(R.sup.3)(R.sup.4)--, --C(O)--, --O--, --C(.dbd.NR.sup.3)--,
--S(O).sub.t-- and N(R.sup.3)--; n is zero, one or two; t is zero,
one or two; R.sup.3 and R.sup.4 are each independently selected
from hydrogen, alkyl, aryl and heterocyclic; and X and Y are each
independently selected from .dbd.O, .dbd.S, .dbd.N(R.sup.3) and
.dbd.C(R.sup.1)(R.sup.2).
2. A method for treating leukemia in a subject comprising
administration to the subject of a compound of Formula (I) in claim
1 capable of causing cell death of leukemia tumor cells.
3. A method for treating leukemia in a subject comprising
administration to the subject of a compound of Formula (I) in claim
1 capable of causing cell death of the rare leukemia stem cell
subpopulation.
4. A method for treating leukemia in a subject comprising causing
the cell death of leukemia stem cells in a subject by
administration to the subject of a compound of Formula (I) in claim
1.
5. A method for treating leukemia in a subject comprising
administration to the subject of a compound of Formula (I) in claim
1 capable of permeabilization of cell membranes and inducing
oxidative stress.
6. A method for treating leukemia in a subject comprising
administration to the subject of a compound of Formula (I) in claim
1 capable of causing cell death of both leukemia cells and leukemia
stem cells.
7. A method for treating a blood disease in a subject comprising
administration to the subject of a compound of Formula (I) in claim
1.
8. A method for treating leukemia in a subject comprising
administration to the subject of a compound of Formula (I) in claim
1 and an additional therapeutic agent.
9. The method of claim 1, wherein the leukemia is acute myelogenus
leukemia (AML), blast crisis leukemia (CML, both lymphoid and
meloid forms of the disorder), acute lymphocytic leukemia (ALL), or
chronic lymphocytic leukemia (CLL).
10. A method of treating a disorder in a subject, wherein the
disorder is cancer cell growth; lymphoma, multiple myeloma,
leukemia cell growth; proliferative diseases; blood cancers;
hematologic malignancies, or disorders such as acute myelogenus
leukemia (AML), blast crisis leukemia (CML, both lymphoid and
meloid forms of the disorder), acute lymphocytic leukemia (ALL), or
chronic lymphocytic leukemia (CLL), comprising administration of a
compound of Formula (I) in claim 1.
11. The method of claim 1 wherein A is --NR.sup.1-- and E is
--NR.sup.1--.
12. The method of claim 1 wherein G is --S--, A is --NR.sup.1-- and
E is --NR.sup.1--.
13. The method of claim 12 wherein X and Y are O.
14. The method of claim 13 wherein the compound is
4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8).
15. Use of a compound of Formula (I) in claim 1 for the manufacture
of a medicament for use in treatment or prevention of leukemia.
16. A composition for use in treatment or prevention of leukemia in
a subject comprising a compound of Formula (I) in claim 1 and a
pharmaceutically acceptable carrier.
17. An article of manufacture comprising separate dosage forms of a
composition comprising a compound of Formula I in claim 1, or a
pharmaceutically acceptable salt thereof; or a prodrug, or a
pharmaceutically acceptable salt of a prodrug thereof; or a
hydrate, solvate, or polymorph thereof; and an acceptable carrier;
and a second therapeutic agent, wherein both dosage forms are in a
single container.
18. A method for treating leukemia in a subject comprising
administration to the subject of a compound of Formula (II):
##STR00016## or a salt thereof; or a prodrug, or a salt of a
prodrug thereof; or a hydrate, solvate, or polymorph thereof;
wherein: each R.sup.1 and R.sup.2 are each independently selected
from hydrogen, alkyl, cycloalkyl, haloalkyl, aryl,
--(Z).sub.n-aryl, heteroaryl, --OR.sup.3, --C(O)R.sup.3,
--C(O)OR.sup.3, --(Z).sub.n--C(O)OR.sup.3 and --S(O).sub.t-- or as
indicated R.sup.2 can be such that E with G then form a fused aryl
group; Z is independently selected from --C(R.sup.3)(R.sup.4)--,
--C(O)--, --O--, --C(.dbd.NR.sup.3)--, --S(O).sub.t-- and
N(R.sup.3)--; n is zero, one or two; t is zero, one or two; R.sup.3
and R.sup.4 are each independently selected from hydrogen, alkyl,
aryl and heterocyclic; and X and Y are each independently selected
from .dbd.O, .dbd.S, .dbd.N(R.sup.3) and
.dbd.C(R.sup.1)(R.sup.2).
19. The method of claim 18, wherein each R.sup.1 is independently
alkyl or arylalkyl.
20. The method of claim 18, wherein one R.sup.1 is independently
alkyl and the other R.sup.1 is independently arylalkyl.
21. A method of treating a kinase-mediated disease or disorder in a
subject comprising administration to the subject of a compound of
Formula (I) in claim 1.
22. The method of claim 21, wherein the kinase is AKT1 (PKB alpha),
CHEK1 (CHK1), DYRK3, FLT3, GSK3B, KDR (VEGFR2), MAP4K4 (HGK),
MAPK14 (p38 alpha), MAPKAPK2, MET (cMet), PHKG2, PIM1, PRKCA (PKC
alpha), PRKCB1 (PKC beta1), PRKCB2 (PKC beta2), PRKCD (PKC delta),
PRKCE (PKC epsilon), PRKG (PKC gamma), PRKCH (PKC eta), PRKCI (PKC
iota), PRKCN (PKD3), PRKCQ (PKC theta), PRKCZ (PKC zeta), PRKCD1
(PKC mu), ROCK1, RPS6KA3 (RSK2), STK6 (Aurora A), or SYK.
Description
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to compounds of the formulae
herein, their acceptable salts, solvates, hydrates and polymorphs
thereof. The compounds of this invention are useful in treatment of
disease, particularly leukemia. The invention also provides
compositions comprising a compound of this invention and the use of
such compositions in methods of treating disease, disorders, or
symptoms thereof in a subject.
BACKGROUND OF THE INVENTION
[0003] Certain 2,4-disubstituted thiadiazolidinone (TDZD) compounds
have been reported to be useful as enzyme inhibitors of glycogen
synthase kinase 313, or GSK-3. See, e.g., US 2003/0195238A1, US
2005/0222220A1. Glycogen synthase kinase-3 (GSK-3) is a
serine/threonine protein kinase comprised of .alpha.- and
.beta.-isoforms that are each encoded by distinct genes (Coghlan et
al., Chemistry & Biology, 7, 793-803 (2000); Kim and Kimmel,
Curr. Opinion Genetics Dev., 10, 508-514 (2000)). The
threonine/serine kinase glycogen synthase kinase-3 (GSK-3) fulfills
a pivotal role in various receptor-linked signalling pathways
(Doble, B W, Woodgett, J R J. Cell Sci. 2003, 116:1175-1186).
Dysregulation within these pathways is considered a crucial event
in the development of several prevalent human disorders, such as
type II diabetes (Kaidanovich O, Eldar-Finkelman H, Expert Opin.
Ther. Targets, 2002, 6:555-561), Alzheimer's disease (Grimes C A,
Jope R S, Prog. Neurobiol. 2001, 65:391-426), CNS disorders such as
manic depressive disorder and neurodegenerative diseases, and
chronic inflammatory disorders (Hoeflich K P, Luo J, Rubie E A,
Tsao M S, Jin O, Woodgett J, Nature 2000, 406:86-90).
[0004] Additionally certain TDZD compounds are reported to be
non-competitive GSK-3.beta. inhibitors, which demonstrate promise
as AD pharmacotherapy agents. Martinez, A. et al. J. Med. Chem.,
45, 1292-1299 (2002).
[0005] It is now discovered that TDZD compounds are useful in
treating disorders different and distinguishable from those
previously reported.
SUMMARY OF THE INVENTION
[0006] The present invention relates to new treatment methods
relating to a compound of Formula I:
##STR00001##
or a salt thereof; or a prodrug, or a salt of a prodrug thereof; or
a hydrate, solvate, or polymorph thereof; wherein:
[0007] A is --C(R.sup.1).sub.2--, --O-- or --NR.sup.1--; E is
--NR.sup.1-- or --CR.sup.1R.sup.2-- and the substituent R.sup.2 is
absent if - - - is a second bond between E and G; G is --S--,
--NR.sup.1-- or --CR.sup.1R.sup.2-- and the substituent R.sup.2 is
absent if - - - is a second bond between E and G; - - - may be a
second bond between E and G where the nature of E and G permits and
E with G optionally then forms a fused aryl group; R.sup.1 and
R.sup.2 are each independently selected from hydrogen, alkyl,
cycloalkyl, haloalkyl, aryl, --(Z).sub.n-aryl, heteroaryl,
--OR.sup.3, --C(O)R.sup.3, --C(O)OR.sup.3,
--(Z).sub.n--C(O)OR.sup.3 and --S(O).sub.t-- or as indicated
R.sup.2 can be such that E with G then form a fused aryl group; Z
is independently selected from --C(R.sup.3)(R.sup.4)--, --C(O)--,
--O--, --C(.dbd.NR.sup.3)--, --S(O).sub.t-- and N(R.sup.3)--; n is
zero, one or two; t is zero, one or two; R.sup.3 and R.sup.4 are
each independently selected from hydrogen, alkyl, aryl and
heterocyclic; and X and Y are each independently selected from
.dbd.O, .dbd.S, .dbd.N(R.sup.3) and .dbd.C(R.sup.1)(R.sup.2).
[0008] The treatment methods include administration of a compound
of any of the formulae herein, or a composition including a
compound of any of the formulae herein, to a subject.
[0009] The invention also relates to compounds of any of the
formulae herein, and compositions thereof, for use in treatment of
a subject having a disease or disorder, and a compound of any
formulae herein, for manufacture of a composition including a
compound of any of the formulae herein useful for treatment of a
subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 illustrates results of human cell cultures treated
with 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8).
TDZD-8 induces death of primary human leukemic cells but not normal
cells.
[0011] FIG. 2 illustrates results of human cells treated with
TDZD-8. TDZD-8 inhibits growth of leukemic cells but not normal
progenitor cells.
[0012] FIG. 3 illustrates results of human cell engraftment in
NOD/SCID mice treated with TDZD-8. TDZD-8 inhibits the engraftment
and growth of leukemia stem cells, but not normal hematopoietic
stem cells.
[0013] FIG. 4 illustrates results of treating human AML cells with
TDZD-8. Exposure for as little as 30 minutes is sufficient to
induce irreversible death of primary human CD34+CD38-AML cells.
[0014] FIG. 5 illustrates results of treating human AML cells with
TDZD-8. Exposure for as little as 30 minutes is sufficient to
induce irreversible death in AML progenitor cells.
[0015] FIG. 6 illustrates results of treating human AML cells with
TDZD-8. TDZD-8 causes rapid loss of membrane integrity.
[0016] FIG. 7 illustrates results of treating human AML cells with
TDZD-8. N-acetylcysteine but not Z-VAD can block TDZD-8 toxicity in
primary AML cells.
[0017] FIG. 8 illustrates results of treating human AML cells with
TDZD-8. TDZD-8 induces oxidative stress in human leukemia cells by
rapid depletion of free thiol groups.
[0018] FIG. 9 TDZD-8 specifically induces cell death of primary
leukemia specimens. Primary AML (n=37), CLL (n=12), ALL (n=6),
bcCML (n=6) and normal specimens (n=13) obtained from BM (n=3), CB
(n=7) or MPB (n=3) specimens were cultured for 18-24 hours in the
presence of 20 .mu.M TDZD-8. Cell viability was assessed by Annexin
V/7-AAD staining. Percent viability is represented relative to
untreated control. Leukemia specimens were significantly
(p<0.001) more sensitive to TDZD-8 than normal specimens. Error
bars represent the SEM. All assays were performed in
triplicate.
[0019] FIG. 10 TDZD-8 ablates leukemia progenitor and stem cells.
(A) Primary AML (n=10), ALL (n=3), bcCML (n=3), and normal
specimens (n=7) obtained from BM, CB or MPB specimens were cultured
for 18-24 hours in the presence or absence of 20 .mu.M TDZD-8. Cell
viability was assessed by flow cytometry in CD34+CD38- populations
for AML and bcCML (CML) and CD34+CD10- cells for ALL using Annexin
V/7-AAD stain. Percent viability is represented relative to
untreated control. Specificity to leukemia specimens was
significant (**p<0.01). Error bars represent the SEM. (B)
primary cells from AML (n=11), bcCML (CML; n=3) and normal
specimens (n=12) were treated for 18 hours in suspension culture,
followed by plating in methylcellulose. Error bars represent the
SEM. Percent of colony-forming units (CFU). are normalized to
untreated controls. All assays were performed in triplicate.
Specificity to leukemia specimens was significant (**p<0.01,
*p<0.05). (C) Percent engraftment for NOD/SCID mice that
received a transplant with AML (upper panels) or normal CB (lower
panels) cells after 18 hours of culture with or without 20 .mu.M
TDZD-8. Each circle or triangle represents a single animal analyzed
at 6 weeks after transplantation. Each plot represents an
independent AML or CB specimen. Mean engraftment is indicated by
the horizontal bars. **p<0.01, *** p<0.001.
[0020] FIG. 11 TDZD-8 treatment induces oxidative stress. (A) Flow
cytometric overlays for mBBr fluorescence on primary AML, CLL, ALL
or normal mononuclear cells treated cells (20 .mu.M TDZD; black
bold lines) over untreated controls (black line/gray filling). (B)
Percent viability of primary AML cells pre-treated with NAC (grey
bars) for 1 h prior to treatment with 20 .mu.M TDZD-8. Viability
was determined 24 hours after the addition of each drug. Error bars
represent the SEM.
[0021] FIG. 12 TDZD-8 induces cell death with extremely rapid cell
death kinetics demonstrating loss of membrane integrity. (A)
Percent viability assessed at the indicated time points for
CD34+CD38- populations of primary AML specimens (n=8) treated with
TDZD-8 (left panel, black bars) or PTL (right panel, white bars).
Percent viability is represented relative to untreated control. (B)
Percent viability assessed at the indicated time points for
unfractionated primary AML specimens (n=17) treated with TDZD-8.
Percent viability is shown relative to untreated controls. Error
bars represent SEM. (C) Cells were treated with 20 .mu.M TDZD-8 for
the indicated periods of time, then washed and placed in culture
until analysis at 24 hours. Percent viability represented relative
to untreated control. (D) Percent CFU relative to untreated
control. Cells were washed and placed in methylcellulose culture
medium at the indicated time points after the addition TDZD-8. (E)
Loss of membrane integrity assessed by YoPro-1 uptake after 15 min
of TDZD-8 treatment. Multispectral imaging flow cytometry
demonstrates the internalization of YoPro-1. Cells were stained
with CD45 to delineate the plasma membrane and with the cell
permeable DNA dye Draq5 to identify the nucleus. (F) Flow
cytometric histograms for YoPro-1 and PI overlaying TDZD-8 treated
(20 .mu.M for 15 min) normal mononuclear cells or primary AML cells
over untreated controls. (G) Percent viability of primary AML cells
pre-treated with Z-VAD (white bars) for 1 h prior the treatment
with TDZD-8 20 .mu.M. Viability was determined 24 hours after the
addition of TDZD-8. Error bars represent the SEM.
[0022] FIG. 13 TDZD-8 inhibits PKC and FLT3 in primary AML
specimens. (A) Immunoblots for CD34+ AML and normal BM specimens to
determine PKC phosphorylation. Actin is shown as loading control.
(B) Primary CD34+ AML and ALL specimens treated with TDZD-8 for
1-hour were processed to obtain membrane fractions. Immunoblots
were performed to determine PKC.alpha. and PKC.beta. levels in the
membrane. HSP70 is shown as a control. (C) Titration curve and IC50
value for FLT3 kinase assay. (D) Overlays of flow cytometric
analysis for phospho-FLT3 in primary AML specimens. Light
gray-solid line histograms represent untreated cells. Dotted line
histograms represent TDZD-8 treated cells. Dark gray--no line
histogram represent controls. Cells were processed for analysis 30
minutes after the addition of drug. CD34+ (31% inhibition; left
panel) and CD34+CD38- (29.5% inhibition; right panel)
populations.
[0023] FIG. 14 illustrates % viability of normal specimens,
indicating that TDZD-8 does not induce cell death of primary
mononuclear cells from normal specimens.
[0024] FIG. 15 illustrates % viability of: (A) CLL specimens; (B)
ALL (total and CD34+CD10-) specimens indicating that TDZD-8 induces
cell death in primary lymphoid malignancies with rapid
kinetics.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides an isolated compound of
Formula I:
##STR00002##
or a salt thereof; or a prodrug, or a salt of a prodrug thereof; or
a hydrate, solvate, or polymorph thereof; wherein:
[0026] A is --C(R.sup.1).sub.2--, --O-- or --NR.sup.1--; E is
--NR.sup.1-- or --CR.sup.1R.sup.2-- and the substituent R.sup.2 is
absent if - - - is a second bond between E and G; G is --S--,
--NR.sup.1-- or --CR.sup.1R.sup.2-- and the substituent R.sup.2 is
absent if - - - is a second bond between E and G; - - - may be a
second bond between E and G where the nature of E and G permits and
E with G optionally then forms a fused aryl group; R.sup.1 and
R.sup.2 are each independently selected from hydrogen, alkyl,
cycloalkyl, haloalkyl, aryl, --(Z).sub.n-aryl, heteroaryl,
--OR.sup.3, --C(O)R.sup.3, --C(O)OR.sup.3,
--(Z).sub.n--C(O)OR.sup.3 and --S(O).sub.t-- or as indicated
R.sup.2 can be such that E with G then form a fused aryl group; Z
is independently selected from --C(R.sup.3)(R.sup.4)--, --C(O)--,
--O--, --C(.dbd.NR.sup.3)--, --S(O).sub.t-- and N(R.sup.3)--; n is
zero, one or two; t is zero, one or two; R.sup.3 and R.sup.4 are
each independently selected from hydrogen, alkyl, aryl and
heterocyclic; and X and Y are each independently selected from
.dbd.O, .dbd.S, .dbd.N(R.sup.3) and .dbd.C(R.sup.1)(R.sup.2).
[0027] In one aspect of the formulae herein, X and Y are
.dbd.O.
[0028] In another aspect of the formulae herein, A is
--NR.sup.1--.
[0029] In another aspect of the formulae herein, G is --S--.
[0030] In another aspect of the formulae herein, E is
--NR.sup.1--.
[0031] In another aspect of the formulae herein, A is --NR.sup.1--
and E is --NR.sup.1--.
[0032] In another aspect of the formulae herein, G is --S--, A is
--NR.sup.1-- and E is --NR.sup.1--.
[0033] In another aspect of the formulae herein, each R.sup.1 is
independently alkyl.
[0034] In another aspect of the formulae herein, A is --NR.sup.1--
and E is --NR.sup.1--, where one R.sup.1 is independently alkyl and
the other R.sup.1 is independently alkyl substituted with aryl.
[0035] Another aspect is a compound of Formula (II):
##STR00003##
or a salt thereof; or a prodrug, or a salt of a prodrug thereof; or
a hydrate, solvate, or polymorph thereof; wherein: [0036] each
R.sup.1 and R.sup.2 are each independently selected from hydrogen,
alkyl, cycloalkyl, haloalkyl, aryl, --(Z).sub.n-aryl, heteroaryl,
--OR.sup.3, --C(O)R.sup.3, --C(O)OR.sup.3,
--(Z).sub.n--C(O)OR.sup.3 and --S(O).sub.t-- or as indicated
R.sup.2 can be such that E with G then form a fused aryl group; Z
is independently selected from --C(R.sup.3)(R.sup.4)--, --C(O)--,
--O--, --C(.dbd.NR.sup.3)--, --S(O).sub.t-- and N(R.sup.3)--; n is
zero, one or two; t is zero, one or two; R.sup.3 and R.sup.4 are
each independently selected from hydrogen, alkyl, aryl and
heterocyclic; and X and Y are each independently selected from
.dbd.O, .dbd.S, .dbd.N(R.sup.3) and .dbd.C(R.sup.1)(R.sup.2).
[0037] In another aspect of the formulae herein, each R.sup.1 is
independently selected from alkyl and substituted alkyl.
[0038] In another aspect of the formulae herein, each R.sup.1 is
independently selected from alkyl and aryl-substituted alkyl.
[0039] In another aspect of the formulae herein, each R.sup.1 is
independently selected from alkyl and phenyl-substituted alkyl.
[0040] In another aspect of the formulae herein one R.sup.1 is
independently alkyl and the other R.sup.1 is independently
arylalkyl.
[0041] Other aspects are the specifically listed compounds in Table
I.
TABLE-US-00001 TABLE I ##STR00004## Com- pound No. R.sup.a R.sup.b
X Y 1 CH.sub.2Ph Me O O 2 Et Me O O 3 Et nPr O O 4 Et cyclohexyl O
O 5 Ph Me O O 6 CH.sub.2CO.sub.2Et Me O O 7 4-OMePh Me O O 8
CH.sub.2Ph Et O O 9 Et iPr O O 10 CH.sub.2Ph Et O S 11 CH.sub.2Ph
CH.sub.2Ph O S 12 Ph Ph O S 13 Et Et O S 14 Cyclohexyl Me O O 15
4-MePh Me O O 16 4-BrPh Me O O 17 4-FPh Me O O 18 4-ClPh Me O O 19
Et Me ##STR00005## O 20 Et Et ##STR00006## O 21 Et H ##STR00007## O
22 Me Me ##STR00008## O 23 Et Me ##STR00009## O 24 Et Me
##STR00010## O 25 ET Me ##STR00011## O 26 Et Me ##STR00012## S 27
Et Et O O 28 Et Et O S 29 Bn Bn O O 30 CH.sub.2CO.sub.2Et Et O O 31
CH.sub.2Ph COPh O O 32 Ph Et O NH 33 CH.sub.2Ph CH.sub.2CO.sub.2Et
O O 34 4-CF.sub.3Ph Me O O 35 n-Bu Et O O 36 CH.sub.2Ph Et O N--OH
37 3-BrPh Me O O 38 2-BrPh Me O O 39 Ph Et O NCONHEt 40 Ph
CO.sub.2Et S NCO.sub.2Et 41 CH.sub.2 CH.sub.2Ph Et O O 42
CH.sub.2Ph H O O 43 Ph Et O O 44 CH.sub.2CO.sub.2Et
CH.sub.2CO.sub.2Et O O 45 CH.sub.2CO.sub.2Et Me O O 46
CH.sub.2CO.sub.2Et iPr O O 47 CH.sub.2CO.sub.2Et Bz O O 48 Naphthyl
Me O O 49 4-NO.sub.2Ph Et O O 50 Ph Et O N--OH 51 CH.sub.2Ph iPr O
O 52 Ph Ph O O 53 4-MeOPh Et O O 54 4-MePh Et O O 55 4-BrPh Et O O
56 CH.sub.2Ph CH.sub.2CH.sub.2Ph O O 57 CH.sub.2Ph CH(Ph).sub.2 O O
58 CH.sub.2Ph naphthalen- O O 1-yl 59 CH.sub.2Ph 4-methoxy- O O
benzyl 60 CH.sub.2Ph 2-t-butyl-6- O O methyl- phenyl 61 CH.sub.2Ph
4-methyl O O benzyl 62 CH.sub.2Ph 2-benzyl- O O phenyl 63
CH.sub.2Ph 2-benzo[1,3] O O dioxol-5-yl 64 CH.sub.2Ph 4-phenoxy- O
O phenyl 65 Me CH.sub.2Ph O O
[0042] The term "compound" as used herein, is intended to mean
stable chemical compounds.
[0043] A salt of a compound of this invention is formed between an
acid and a basic group of the compound, such as an amino functional
group, or a base and an acidic group of the compound, such as a
carboxyl functional group. According to another preferred
embodiment, the compound is a pharmaceutically acceptable acid
addition salt.
[0044] As used herein and unless otherwise indicated, the term
"prodrug" means a derivative of a compound that can hydrolyze,
oxidize, or otherwise react under biological conditions (in vitro
or in vivo) to provide a compound of this invention. Prodrugs may
only become active upon such reaction under biological conditions,
or they may have activity in their unreacted forms. Examples of
prodrugs contemplated in this invention include, but are not
limited to, analogs or derivatives of compounds of any one of the
formulae disclosed herein that comprise biohydrolyzable moieties
such as biohydrolyzable amides, biohydrolyzable esters,
biohydrolyzable carbamates, biohydrolyzable carbonates,
biohydrolyzable ureides, and biohydrolyzable phosphate analogues.
Prodrugs can typically be prepared using well-known methods, such
as those described by Burger's Medicinal Chemistry and Drug
Discovery (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5th ed);
see also Goodman and Gilman's, The Pharmacological basis of
Therapeutics, 8th ed., McGraw-Hill, Int. Ed. 1992,
"Biotransformation of Drugs".
[0045] As used herein and unless otherwise indicated, the terms
"biohydrolyzable amide", "biohydrolyzable ester", "biohydrolyzable
carbamate", "biohydrolyzable carbonate", "biohydrolyzable ureide"
and "biohydrolyzable phosphate analogue" mean an amide, ester,
carbamate, carbonate, ureide, or phosphate analogue, respectively,
that either: 1) does not destroy the biological activity of the
compound and confers upon that compound advantageous properties in
vivo, such as uptake, duration of action, or onset of action; or 2)
is itself biologically inactive but is converted in vivo to a
biologically active compound. Examples of biohydrolyzable amides
include, but are not limited to, lower alkyl amides, .alpha.-amino
acid amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides.
Examples of biohydrolyzable esters include, but are not limited to,
lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl
esters, and choline esters. Examples of biohydrolyzable carbamates
include, but are not limited to, lower alkylamines, substituted
ethylenediamines, amino acids, hydroxyalkylamines, heterocyclic and
heteroaromatic amines, and polyether amines.
[0046] A prodrug salt is a compound formed between an acid and a
basic group of the prodrug, such as an amino functional group, or a
base and an acidic group of the prodrug, such as a carboxyl
functional group. In a preferred embodiment, the prodrug salt is a
pharmaceutically acceptable salt. According to another preferred
embodiment, the counterion to the saltable prodrug of the compound
of a formula herein is pharmaceutically acceptable.
Pharmaceutically acceptable counterions include, for instance,
those acids and bases noted herein as being suitable to form
pharmaceutically acceptable salts.
[0047] Particularly favored prodrugs and prodrug salts are those
that increase the bioavailability of the compounds of this
invention when such compounds are administered to a mammal (e.g.,
by allowing an orally administered compound to be more readily
absorbed into the blood) or which enhance delivery of the parent
compound to a biological compartment (e.g., the brain or central
nervous system) relative to the parent species. Preferred prodrugs
include derivatives where a group that enhances aqueous solubility
or active transport through the gut membrane is appended to the
structure of formulae described herein. See, e.g., Alexander, J. et
al. Journal of Medicinal Chemistry 1988, 31, 318-322; Bundgaard, H.
Design of Prodrugs; Elsevier: Amsterdam, 1985; pp 1-92; Bundgaard,
H.; Nielsen, N. M. Journal of Medicinal Chemistry 1987, 30,
451-454; Bundgaard, H. A Textbook of Drug Design and Development;
Harwood Academic Publ.: Switzerland, 1991; pp 113-191; Digenis, G.
A. et al. Handbook of Experimental Pharmacology 1975, 28, 86-112;
Friis, G. J.; Bundgaard, H. A Textbook of Drug Design and
Development; 2 ed.; Overseas Publ.: Amsterdam, 1996; pp 351-385;
Pitman, I. H. Medicinal Research Reviews 1981, 1, 189-214.
[0048] The term "pharmaceutically acceptable," as used herein,
refers to a component that is, within the scope of sound medical
judgment, suitable for use in contact with the tissues of humans
and other mammals without undue toxicity, irritation, allergic
response and the like, and are commensurate with a reasonable
benefit/risk ratio. A "pharmaceutically acceptable salt" means any
non-toxic salt that, upon administration to a recipient, is capable
of providing, either directly or indirectly, a compound or a
prodrug of a compound of this invention. A "pharmaceutically
acceptable counterion" is an ionic portion of a salt that is not
toxic when released from the salt upon administration to a
recipient.
[0049] Acids commonly employed to form pharmaceutically acceptable
salts include inorganic acids such as hydrogen bisulfide,
hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric
acid, as well as organic acids such as para-toluenesulfonic,
salicylic, tartaric, bitartaric, ascorbic, maleic, besylic,
fumaric, gluconic, glucaronic, formic, glutamic, methanesulfonic,
ethanesulfonic, benzenesulfonic, lactic, oxalic,
para-bromophenylsulfonic, carbonic, succinic, citric, benzoic and
acetic acid, and related inorganic and organic acids. Such
pharmaceutically acceptable salts thus include sulfate,
pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,
monohydrogenphosphate, dihydrogenphosphate, metaphosphate,
pyrophosphate, chloride, bromide, iodide, acetate, propionate,
decanoate, caprylate, acrylate, formate, isobutyrate, caprate,
heptanoate, propiolate, oxalate, malonate, succinate, suberate,
sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,
benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,
hydroxybenzoate, methoxybenzoate, phthalate, terephathalate,
sulfonate, xylenesulfonate, phenylacetate, phenylpropionate,
phenylbutyrate, citrate, lactate, .beta.-hydroxybutyrate,
glycolate, maleate, tartrate, methanesulfonate, propanesulfonate,
naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the
like salts. Preferred pharmaceutically acceptable acid addition
salts include those formed with mineral acids such as hydrochloric
acid and hydrobromlc acid, and especially those formed with organic
acids such as maleic acid.
[0050] Suitable bases for forming pharmaceutically acceptable salts
with acidic functional groups of prodrugs of this invention
include, but are not limited to, hydroxides of alkali metals such
as sodium, potassium, and lithium; hydroxides of alkaline earth
metal such as calcium and magnesium; hydroxides of other metals,
such as aluminum and zinc; ammonia, and organic amines, such as
unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines;
dicyclohexylamine; tributyl amine; pyridine; N-methyl,N-ethylamine;
diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower
alkyl amines), such as mono-, bis-, or tris-(2-hydroxyethyl)amine,
2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine,
N,N,-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such as
N,N-dimethyl-N-(2-hydroxyethyl)amine, or tri-(2-hydroxyethyl)amine;
N-methyl-D-glucamine; and amino acids such as arginine, lysine, and
the like.
[0051] As used herein, the term "hydrate" means a compound which
further includes a stoichiometric or non-stoichiometric amount of
water bound by non-covalent intermolecular forces.
[0052] As used herein, the term "solvate" means a compound which
further includes a stoichiometric or non-stoichiometric amount of
solvent such as water, acetone, ethanol, methanol, dichloromethane,
2-propanol, or the like, bound by non-covalent intermolecular
forces.
[0053] As used herein, the term "polymorph" means solid crystalline
forms of a compound or complex thereof which may be characterized
by physical means such as, for instance, X-ray powder diffraction
patterns or infrared spectroscopy. Different polymorphs of the same
compound can exhibit different physical, chemical and/or
spectroscopic properties. Different physical properties include,
but are not limited to stability (e.g., to heat, light or
moisture), compressibility and density (important in formulation
and product manufacturing), hygroscopicity, solubility, and
dissolution rates and solubility (which can affect
bioavailability). Differences in stability can result from changes
in chemical reactivity (e.g., differential oxidation, such that a
dosage form discolors more rapidly when comprised of one polymorph
than when comprised of another polymorph) or mechanical
characteristics (e.g., tablets crumble on storage as a kinetically
favored polymorph converts to thermodynamically more stable
polymorph) or both (e.g., tablets of one polymorph are more
susceptible to breakdown at high humidity). Different physical
properties of polymorphs can affect their processing. For example,
one polymorph might be more likely to form solvates or might be
more difficult to filter or wash free of impurities than another
due to, for example, the shape or size distribution of particles of
it.
[0054] The compounds of the present invention can contain one or
more asymmetric carbon atoms. As such, a compound of this invention
can exist as the individual stereoisomers (enantiomers or
diastereomers) as well a mixture of stereoisomers. Accordingly, a
compound of the present invention will include not only a
stereoisomeric mixture, but also individual respective
stereoisomers substantially free from one another stereoisomers.
The term "substantially free" as used herein means less than 25% of
other stereoisomers, preferably less than 10% of other
stereoisomers, more preferably less than 5% of other stereoisomers
and most preferably less than 2% of other stereoisomers, are
present. Methods of obtaining or synthesizing diastereomers are
well known in the art and may be applied as practicable to final
compounds or to starting material or intermediates. Other
embodiments are those wherein the compound is an isolated
compound.
[0055] The compounds herein may contain one or more asymmetric
centers and thus occur as racemates and racemic mixtures, single
enantiomers, individual diastereomers and diastereomeric mixtures.
All such isomeric forms of these compounds are expressly included
in the present invention. The compounds herein may also contain
linkages (e.g., carbon-carbon bonds) wherein bond rotation is
restricted about that particular linkage, e.g., restriction
resulting from the presence of a ring or double bond. Accordingly,
all cis/trans and E/Z isomers are expressly included in the present
invention. The compounds herein may also be represented in multiple
tautomeric forms, in such instances, the invention expressly
includes all tautomeric forms of the compounds described herein,
even though only a single tautomeric form may be represented. All
such isomeric forms of such compounds herein are expressly included
in the present invention. All crystal forms and polymorphs of the
compounds described herein are expressly included in the present
invention.
[0056] The compounds of the invention may be synthesized by
well-known techniques or are commercially available. The starting
materials and certain intermediates used in the synthesis of the
compounds of this invention are available from commercial sources
or may themselves be synthesized using reagents and techniques
known in the art, including those synthesis schemes delineated
herein. See, for instance, US 2003/0195238; US 2005/0222220, and
references cited therein.
[0057] In one aspect, compounds are synthesized according to Scheme
(I) or Scheme (H):
##STR00013##
##STR00014##
[0058] Definitions of variables in the structures in schemes herein
are commensurate with those of corresponding positions in the
formulae delineated herein.
[0059] The synthesized compounds can be separated from a reaction
mixture and further purified by a method such as column
chromatography, high pressure liquid chromatography, or
recrystallization.
[0060] As can be appreciated by the skilled artisan, further
methods of synthesizing the compounds of the formulae herein will
be evident to those of ordinary skill in the art. Additionally, the
various synthetic steps may be performed in an alternate sequence
or order to give the desired compounds. In addition, the solvents,
temperatures, reaction durations, etc. delineated herein are for
purposes of illustration only and one of ordinary skill in the art
will recognize that variation of the reaction conditions can
produce the desired bridged macrocyclic products of the present
invention. Synthetic chemistry transformations and protecting group
methodologies (protection and deprotection) useful in synthesizing
the compounds described herein are known in the art and include,
for example, those such as described in R. Larock, Comprehensive
Organic Transformations, VCH Publishers (1989); T. W. Greene and P.
G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John
Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's
Reagents for Organic Synthesis, John Wiley and Sons (1994); and L.
Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John
Wiley and Sons (1995).
[0061] The specific approaches and compounds shown above are not
intended to be limiting. Additional methods of synthesizing
compounds of the formulae herein and their synthetic precursors,
including those within routes not explicitly shown in Schemes
herein, are within the means of chemists of ordinary skill in the
art. In addition to the synthetic references cited herein, reaction
schemes and protocols may be determined by the skilled artisan by
use of commercially available structure-searchable database
software, for instance, SciFinder.RTM. (CAS division of the
American Chemical Society), STN.RTM. (CAS division of the American
Chemical Society), CrossFire Beilstein.RTM. (Elsevier MDL).
[0062] Methods for optimizing reaction conditions, if necessary
minimizing competing by-products, are known in the art. Reaction
optimization and scale-up may advantageously utilize high-speed
parallel synthesis equipment and computer-controlled microreactors
(e.g. Design And Optimization in Organic Synthesis, 2.sup.nd
Edition, Carlson R, Ed, 2005; Elsevier Science Ltd.; Jahnisch, K et
al, Angew. Chem. Int. Ed. Engl. 2004 43: 406; and references
therein).
[0063] The synthetic methods described herein may also additionally
include steps, either before or after any of the steps described in
the synthetic schemes, to add or remove suitable protecting groups
in order to ultimately allow synthesis of the compound of the
formulae described herein. The methods delineated herein
contemplate converting compounds of one formula to compounds of
another formula. The process of converting refers to one or more
chemical transformations, which can be performed in situ, or with
isolation of intermediate compounds. The transformations can
include reacting the starting compounds or intermediates with
additional reagents using techniques and protocols known in the
art, including those in the references cited herein. Intermediates
can be used with or without purification (e.g., filtration,
distillation, sublimation, crystallization, trituration, solid
phase extraction, chromatography).
[0064] Combinations of substituents and variables envisioned by
this invention are only those that result in the formation of
stable compounds. The term "stable", as used herein, refers to
compounds which possess stability sufficient to allow manufacture
and which maintain the integrity of the compound for a sufficient
period of time to be useful for the purposes detailed herein (e.g.,
formulation into therapeutic products, intermediates for use in
production of therapeutic compounds, isolatable or storable
intermediate compounds, treating a disease or condition).
[0065] The term "alkyl" refers to a straight or branched
hydrocarbon chain radical consisting of carbon and hydrogen atoms,
containing no saturation, having one to eight carbon atoms, and
which is attached to the rest of the molecule by a single bond,
e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl,
n-pentyl, etc. Alkyl radicals may be optionally substituted by one
or more substituents independently selected from the group
consisting of a halo, hydroxy, alkoxy, carboxy, cyano, carbonyl,
acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.
[0066] "Alkoxy" refers to a radical of the formula --ORa where Ra
is an alkyl radical as defined above, e.g., methoxy, ethoxy,
propoxy, etc.
[0067] "Alkoxycarbonyl" refers to a radical of the formula
--C(O)ORa where Ra is an alkyl radical as defined above, e.g.,
methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, etc.
[0068] "Alkylthio" refers to a radical of the formula --SRa where
Ra is an alkyl radical as defined above, e.g., methylthio,
ethylthio, propylthio, etc.
[0069] "Amino" refers to a radical of the formula --NH.sub.2.
[0070] "Aryl" refers to a phenyl or naphthyl radical, preferably a
phenyl radical. The aryl radical may be optionally substituted by
one or more substituents selected from the group consisting of
hydroxy, mercapto, halo, alkyl, phenyl, alkoxy, haloalkyl, nitro,
cyano, dialkylamino, aminoalkyl, acyl and alkoxycarbonyl, as
defined herein.
[0071] "Aralkyl" refers to an aryl group linked to an alkyl group.
Preferred examples include benzyl and phenethyl.
[0072] "Acyl" refers to a radical of the formula --C(O)--Rc and
--C(O)--Rd where Rc is an alkyl radical as defined above and Rd is
an aryl radical as defined above, e.g., acetyl, propionyl, benzoyl,
and the like.
[0073] "Aroylalkyl" refers to an alkyl group substituted with
--C(O)--Rd, where Rd is as defined above. Preferred examples
include benzoylmethyl.
[0074] "Carboxy" refers to a radical of the formula --C(O)OH.
[0075] "Cyano" refers to a radical of the formula --CN.
[0076] "Cycloalkyl" refers to a stable 3- to 10-membered monocyclic
or bicyclic radical which is saturated or partially saturated, and
which consist solely of carbon and hydrogen atoms. Unless otherwise
stated specifically in the specification, the term "cycloalkyl" is
meant to include cycloalkyl radicals which are optionally
substituted by one or more substituents independently selected from
the group consisting of alkyl, halo, hydroxy, amino, cyano, nitro,
alkoxy, carboxy and alkoxycarbonyl.
[0077] "Fused aryl" refers to an aryl group, especially a phenyl or
heteroaryl group, fused to the five-membered ring.
[0078] "Halo" refers to bromo, chloro, iodo or fluoro.
[0079] "Haloalkyl" refers to an alkyl radical, as defined above,
that is substituted by one or more halo radicals, as defined above,
e.g., trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,
1-fluoromethyl-2-fluoroethyl, and the like.
[0080] "Heterocycle" refers to a heterocyclyl radical. The
heterocycle refers to a stable 3- to 15-membered ring which
consists of carbon atoms and from one to five heteroatoms selected
from the group consisting of nitrogen, oxygen, and sulfur,
preferably a 4- to 8-membered ring with one or more heteroatoms,
more preferably a 5- or 6-membered ring with one or more
heteroatoms. For the purposes of this invention, the heterocycle
may be a monocyclic, bicyclic or tricyclic ring system, which may
include fused ring systems; and the nitrogen, carbon or sulfur
atoms in the heterocyclyl radical may be optionally oxidised; the
nitrogen atom may be optionally quaternized; and the heterocyclyl
radical may be partially or fully saturated or aromatic. Examples
of such heterocycles include, but are not limited to, azepines,
benzimidazole, benzothiazole, furan, isothiazole, imidazole,
indole, piperidine, piperazine, purine, quinoline, thiadiazole,
tetrahydrofuran. The heterocycle may be optionally substituted by
R.sup.3 and R.sup.4 as defined above in the summary of the
invention.
[0081] "Heteroaryl" refers to an aromatic heterocycle.
[0082] "Mercapto" refers to a radical of the formula --SH.
[0083] "Nitro" refers to a radical of the formula --NO.sub.2.
[0084] "Stereoisomer" refers to both enantiomers and
diastereomers
[0085] "Boc" refers to tert-butoxycarbonyl
[0086] "alkylene" refers to a straight, branched, or partially or
wholly cyclic alkyl group which may contain one or more degrees of
unsaturation in the form of cis, trans, or mixed cis,trans-double
bonds, or triple bonds.
[0087] "Substituted" refers to any chemical structure or group
(e.g, alkyl, aryl, heteroaryl, etc.) referenced herein where one or
more atoms is replaced by one or more substituents independently
selected from the group consisting of a halo, hydroxy, alkoxy,
carboxy, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro,
mercapto and alkylthio.
[0088] The invention also provides compositions comprising an
effective amount of a compound of any one of the formulae herein or
a salt thereof; or a prodrug or a salt of a prodrug thereof; or a
solvate, hydrate, or polymorph thereof, if applicable; an
acceptable carrier. The carrier(s) must be "acceptable" in the
sense of being compatible with the other ingredients of the
formulation.
[0089] In a preferred embodiment, the invention provides a
composition comprising a compound of any of the formulae herein, or
a pharmaceutically acceptable salt, prodrug or pharmaceutically
acceptable prodrug salt thereof; or a solvate, hydrate or polymorph
of any of the foregoing and a pharmaceutically acceptable carrier,
wherein said composition is formulated for pharmaceutical use ("a
pharmaceutical composition"). A "pharmaceutically acceptable
carrier" is a carrier that is compatible with the other ingredients
of the composition and not deleterious to the recipient thereof in
amounts typically used in medicaments.
[0090] Pharmaceutically acceptable carriers, adjuvants and vehicles
that may be used in the pharmaceutical compositions of this
invention include, but are not limited to, ion exchangers, alumina,
aluminum stearate, lecithin, serum proteins, such as human serum
albumin, buffer substances such as phosphates, glycine, sorbic
acid, potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wool fat.
[0091] The pharmaceutical compositions of the invention include
those suitable for oral, rectal, nasal, topical (including buccal
and sublingual), vaginal or parenteral (including subcutaneous,
intramuscular, intravenous and intradermal) administration. In
certain embodiments, the compound of the formulae herein is
administered transdermally (e.g., using a transdermal patch or
iontophoretic techniques). Other formulations may conveniently be
presented in unit dosage form, e.g., tablets and sustained release
capsules, and in liposomes, and may be prepared by any methods well
known in the art of pharmacy. See, for example, Remington's
Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.
(17th ed. 1985).
[0092] Such preparative methods include the step of bringing into
association with the molecule to be administered ingredients such
as the carrier that constitutes one or more accessory ingredients.
In general, the compositions are prepared by uniformly and
intimately bringing into association the active ingredients with
liquid carriers, liposomes or finely divided solid carriers or
both, and then if necessary shaping the product.
[0093] In certain preferred embodiments, the compound is
administered orally. Compositions of the present invention suitable
for oral administration may be presented as discrete units such as
capsules, sachets or tablets each containing a predetermined amount
of the active ingredient; as a powder or granules; as a solution or
a suspension in an aqueous liquid or a non-aqueous liquid; or as an
oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or
packed in liposomes and as a bolus, etc. Soft gelatin capsules can
be useful for containing such suspensions, which may beneficially
increase the rate of compound absorption.
[0094] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active ingredient
in a free-flowing form such as a powder or granules, optionally
mixed with a binder, lubricant, inert diluent, preservative,
surface-active or dispersing agent. Molded tablets may be made by
molding in a suitable machine a mixture of the powdered compound
moistened with an inert liquid diluent. The tablets optionally may
be coated or scored and may be formulated so as to provide slow or
controlled release of the active ingredient therein. Methods of
formulating such slow or controlled release compositions of
pharmaceutically active ingredients, such as those herein and other
compounds known in the art, are known in the art and described in
several issued US patents, some of which include, but are not
limited to, U.S. Pat. Nos. 4,369,172; and 4,842,866; 5,807,574; and
references cited therein. Coatings can be used for delivery of
compounds to the intestine (see, e.g., U.S. Pat. Nos. 6,548,084,
6,638,534, 5,217,720, and 6,569,457, 6,461,631, 6,528,080,
6,800,663, and references cited therein), or they may be
non-eroding and designed to allow release of an active agent by
extrusion (see, e.g. U.S. Pat. No. 6,706,283).
[0095] In the case of tablets for oral use, carriers that are
commonly used include lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried cornstarch. When aqueous suspensions are administered
orally, the active ingredient is combined with emulsifying and
suspending agents. If desired, certain sweetening and/or flavoring
and/or coloring agents may be added. Surfactants such as sodium
lauryl sulfate may be useful to enhance dissolution and
absorption.
[0096] Compositions suitable for topical administration include
lozenges comprising the ingredients in a flavored basis, usually
sucrose and acacia or tragacanth; and pastilles comprising the
active ingredient in an inert basis such as gelatin and glycerin,
or sucrose and acacia.
[0097] Compositions suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example, sealed ampules and vials, and may be
stored in a freeze dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example water for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets.
[0098] Such injection solutions may be in the form, for example, of
a sterile injectable aqueous or oleaginous suspension. This
suspension may be formulated according to techniques known in the
art using suitable dispersing or wetting agents (such as, for
example, Tween 80) and suspending agents. 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-butanediol. Among the acceptable
vehicles and solvents that may be employed are mannitol, 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. Fatty
acids, such as oleic acid and its glyceride derivatives are useful
in the preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant such as Ph. Helv or a similar alcohol.
[0099] The pharmaceutical compositions of this invention may be
administered in the form of suppositories for rectal or vaginal
administration. These compositions can be prepared by mixing a
compound of Formula I with a suitable non-irritating excipient
which is solid at room temperature but liquid at the rectal
temperature and therefore will melt in the rectum to release the
active components. Such materials include, but are not limited to,
cocoa butter, beeswax and polyethylene glycols.
[0100] Topical administration of the pharmaceutical compositions of
this invention is especially useful when the desired treatment
involves areas or organs readily accessible by topical application.
For application topically to the skin, the pharmaceutical
composition will be formulated with a suitable ointment containing
the active components suspended or dissolved in a carrier. Carriers
for topical administration of the compounds of this invention
include, but are not limited to, mineral oil, liquid petroleum,
white petroleum, propylene glycol, polyoxyethylene polyoxypropylene
compound, emulsifying wax and water. Alternatively, the
pharmaceutical composition can be formulated with a suitable lotion
or cream containing the active compound suspended or dissolved in a
carrier. Suitable carriers include, but are not limited to, mineral
oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,
cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The
pharmaceutical compositions of this invention may also be topically
applied to the lower intestinal tract by rectal suppository
formulation or in a suitable enema formulation.
Topically-transdermal patches and iontophoretic administration are
also included in this invention.
[0101] The pharmaceutical compositions of this invention may be
administered by nasal aerosol or inhalation. Such compositions are
prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other solubilizing or dispersing agents known in the
art.
[0102] Application of the subject therapeutics may be local, so as
to be administered at the site of interest. Various techniques can
be used for providing the subject pharmaceutical compositions at
the site of interest, such as injection, use of catheters, trocars,
projectiles, pluronic gel, stents, sustained drug release polymers
or other device which provides for internal access.
[0103] Thus, according to another embodiment, a compound of the
formulae herein may be incorporated into a pharmaceutical
composition for coating an implantable medical device, such as
prostheses, artificial valves, vascular grafts, stents, or
catheters. Suitable coatings and the general preparation of coated
implantable devices are described in U.S. Pat. Nos. 6,099,562;
5,886,026; and 5,304,121. The coatings are typically biocompatible
polymeric materials such as a hydrogel polymer,
polymethyldisiloxane, polycaprolactone, polyethylene glycol,
polylactic acid, ethylene vinyl acetate, and mixtures thereof. The
coatings are optionally further covered by a suitable topcoat of
fluorosilicone, polysaccharides, polyethylene glycol, phospholipids
or combinations thereof to impart controlled release
characteristics in the composition. Coatings for invasive devices
are to be included within the definition of pharmaceutically
acceptable carrier, adjuvant or vehicle, as those terms are used
herein.
[0104] According to another embodiment, the invention provides a
method of coating an implantable medical device comprising the step
of contacting said device with the coating composition described
above. It will be obvious to those skilled in the art that the
coating of the device will occur prior to implantation into a
mammal.
[0105] According to another embodiment, the invention provides a
method of impregnating or filling an implantable drug release
device comprising the step of contacting said drug release device
with a compound of a compound of any of the formulae herein or a
pharmaceutical composition of this invention. Implantable drug
release devices include, but are not limited to, biodegradable
polymer capsules or bullets, non-degradable, diffusible polymer
capsules and biodegradable polymer wafers.
[0106] According to another embodiment, the invention provides an
implantable medical device coated with a compound of any of the
formulae herein or a pharmaceutical composition of this invention,
such that said compound is therapeutically active.
[0107] According to another embodiment, the invention provides an
implantable drug release device impregnated with or containing a
compound of any of the formulae herein or a pharmaceutical
composition of this invention, such that said compound is released
form said device and is therapeutically active.
[0108] Where an organ or tissue is accessible because of removal
from the patient, such organ or tissue may be bathed in a medium
containing a pharmaceutical composition of this invention, a
pharmaceutical composition of this invention may be painted onto
the organ, or a pharmaceutical composition of this invention may be
applied in any other convenient way.
[0109] The present invention further provides pharmaceutical
compositions comprising an effective amount of one or more compound
of any of the formulae herein, in combination with an effective
amount of one or more second therapeutic agents useful for treating
or preventing a disease or disorder herein.
[0110] Also within the scope of this invention are pharmaceutical
compositions comprising an effective amount of a compound of any of
the formulae herein, or a pharmaceutically acceptable salt thereof;
or a prodrug or a pharmaceutically acceptable salt of a prodrug
thereof; or a solvate, hydrate, or polymorph thereof; in
combination with an effective amount of a second therapeutic agent
useful for treating a disorder or symptom thereof, reducing side
effects due to a treatment regimen, and a pharmaceutically
acceptable carrier. Additional therapeutic agents useful in
combination with the compounds of this invention include, but are
not limited to: kinase inhibitors (e.g. Gleevec, CEP-701, PKC412,
etc.), heat shock protein inhibitors (17-AAG), farnesyltransferase
inhibitors (zarnestra), histone deacetylase inhibitors (SAHA,
depsipeptide, MS-275, etc), CDK inhibitors (flavopiridol),
proteasome inhibitors (bortezomib), demethylating agents
(decitabine, vidaza), Bcl-2 inhibitors (ABT-737), anthracyclines
(adriamycin, daunorubicin, doxorubicin, idarubicin), cytarabine,
etoposide, dcxamethasone, methotrexate, thioguanine,
6-mercaptopurine, ATRA, gemcitabine, cyclophosphamide, cisplatin,
vincristine, prednisone, mitoxantrone, bleomycin, 5-fluorouracil,
and rituxan; a pharmaceutically acceptable salt of any of the said
additional therapeutic agents; or combinations of two or more of
the foregoing.
[0111] In another embodiment, the invention provides separate
dosage forms of a compound of any of the formula herein and a
second therapeutic agent, wherein said compound and said second
therapeutic agent are associated with one another. The term
"associated with one another" as used herein means that the
separate dosage forms are packaged together in the same container
(e.g., in separate blister packs attached to one another, in
separate compartments of a compartmentalized container, in separate
vessels contained in the same box, etc.), or otherwise attached to
one another such that it is readily apparent that the separate
dosage forms are intended to be sold and administered together
(within less than 24 hours of one another, consecutively or
simultaneously).
[0112] In the pharmaceutical compositions of the invention, a
compound of any of the formulae herein is present in an effective
amount. As used herein, the term "effective amount" refers to an
amount which, when administered in a proper dosing regimen, is
sufficient to reduce or ameliorate the severity, duration or
progression, or enhance function compromised by a disorder, prevent
the advancement of a disorder, cause the regression of a disorder,
or enhance or improve the prophylactic or therapeutic effect(s) of
another therapy.
[0113] In one embodiment, the invention provides a method of
monitoring treatment progress. The method includes the step of
determining a level of diagnostic marker (Marker) (e.g., any target
or cell type delineated herein modulated by a compound herein) or
diagnostic measurement (e.g., screen, assay) in a subject suffering
from or susceptible to a disorder or symptoms thereof delineated
herein, in which the subject has been administered a therapeutic
amount of a compound herein sufficient to treat the disease or
symptoms thereof. The level of Marker determined in the method can
be compared to known levels of Marker in either healthy normal
controls or in other afflicted patients to establish the subject's
disease status. In preferred embodiments, a second level of Marker
in the subject is determined at a time point later than the
determination of the first level, and the two levels are compared
to monitor the course of disease or the efficacy of the therapy. In
certain preferred embodiments, a pre-treatment level of Marker in
the subject is determined prior to beginning treatment according to
this invention; this pre-treatment level of Marker can then be
compared to the level of Marker in the subject after the treatment
commences, to determine the efficacy of the treatment.
[0114] In certain method embodiments, a level of Marker or Marker
activity in a subject is determined at least once. Comparison of
Marker levels, e.g., to another measurement of Marker level
obtained previously or subsequently from the same patient, another
patient, or a normal subject, may be useful in determining whether
therapy according to the invention is having the desired effect,
and thereby permitting adjustment of dosage levels as appropriate.
Determination of Marker levels may be performed using any suitable
sampling/expression assay method known in the art or described
herein. Preferably, a tissue or fluid sample is first removed from
a subject. Examples of suitable samples include blood, mouth or
cheek cells, and hair samples containing roots. Other suitable
samples would be known to the person skilled in the art.
Determination of protein levels and/or mRNA levels (e.g., Marker
levels) in the sample can be performed using any suitable technique
known in the art, including, but not limited to, enzyme
immunoassay, ELISA, radiolabelling/assay techniques,
blotting/chemiluminescence methods, real-time PCR, and the
like.
[0115] The interrelationship of dosages for animals and humans
(based on milligrams per meter squared of body surface) is
described in Freireich et al., (1966) Cancer Chemother Rep 50: 219.
Body surface area may be approximately determined from height and
weight of the patient. See, e.g., Scientific Tables, Geigy
Pharmaceuticals, Ardley, N. Y., 1970, 537. An effective amount of a
compound of any of the formulae herein can range from about 0.001
mg/kg to about 500 mg/kg, more preferably 0.01 mg/kg to about 100
mg/kg, including a range with a high and low number within the
aforementioned ranges, inclusive. Effective doses will also vary,
as recognized by those skilled in the art, depending on the
diseases treated, the severity of the disease, the route of
administration, the sex, age and general health condition of the
patient, excipient usage, the possibility of co-usage with other
therapeutic treatments such as use of other agents and the judgment
of the treating physician.
[0116] For pharmaceutical compositions that comprise a second
therapeutic agent, an effective amount of that second therapeutic
agent is between about 20% and 100% of the dosage normally utilized
in a monotherapy regime using just that additional agent. The
normal monotherapeutic dosages of these second therapeutic agents
are well known in the art. See, e.g., Wells et al., eds.,
Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange,
Stamford, Corm. (2000); PDR Pharmacopoeia, Tarascon Pocket
Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma
Linda, Calif. (2000), each of which references are entirely
incorporated herein by reference.
[0117] It is expected that some of the second therapeutic agents
listed above will act synergistically with the compounds of this
invention. When this occurs, it will allow the effective dosage of
the second therapeutic agent and/or the compound any of the
formulae herein to be reduced from that required in a monotherapy.
This has the advantage of minimizing toxic side effects of either
the second therapeutic agent or a compound any of the formulae
herein, synergistic improvements in efficacy, improved ease of
administration or use and/or reduced overall expense of compound
preparation or formulation.
Methods of Treatment
[0118] In one embodiment, the present invention provides a method
of treating or preventing a disorder or symptom thereof in a
subject (e.g., human, animal) comprising the step of administering
to said subject an effective amount of a compound of any of the
formulae herein, preferably as part of a composition additionally
comprising a pharmaceutically acceptable carrier. Preferably this
method is employed to treat a subject suffering from or susceptible
to one or more diseases or disorders involving leukemia cells,
leukemia stem cells, or related hematologic disorders.
[0119] The method can also be employed to treat a subject suffering
from or susceptible to cancer cell growth, lymphoma, multiple
myeloma, leukemia cell growth, proliferative diseases, blood
cancers, cancers of the central nervous system, breast, prostate,
liver, lung, pancreas, kidney, colon, testes, ovary, thyroid, head
and neck, cervix, bone, skin, and stomach, and hematologic
malignancies, or disorders such as acute myelogenus leukemia (AML),
blast crisis leukemia (CML, both lymphoid and meloid forms of the
disorder), acute lymphocytic leukemia (ALL), and chronic
lymphocytic leukemia (CLL). Other embodiments include any of the
methods herein wherein the subject is identified as in need of the
indicated treatment.
[0120] The methods herein are useful to eradicate leukemia cells
(e.g., human leukemia cells) and in one aspect involve contacting
the leukemia cells with an agent (e.g., compounds and compositions
of any of the formulae herein) capable of causing two simultaneous
events: (i) permeabilization of cell membranes, and (ii) induction
of oxidative stress. The methods herein are also those that involve
one or more of: (i) rapid loss of membrane integrity, (ii)
depletion of free thiols, or (iii) inhibition of either or both the
PKC and FLT3 signaling pathways.
[0121] Another aspect of the invention is a compound of any of the
formulae herein for use in inhibiting (including causing cell
death) of leukemia cell (e.g., leukemia blast cells) populations
(e.g., tumors) in a subject. Preferably that use is in the
treatment or prevention in a subject of a disease, disorder or
symptom set forth herein.
[0122] Another aspect of the invention is a compound of any of the
formulae herein for use in inhibiting (including causing cell
death) of leukemia stem cell populations in a subject. Preferably
that use is in the treatment or prevention in a subject of a
disease, disorder or symptom set forth herein.
[0123] The preferred therapeutic methods of the invention (which
include prophylactic treatment) in general comprise administration
of a therapeutically effective amount of the compounds herein, such
as a compound of the formulae herein to a subject (e.g., animal,
human) in need thereof, including a mammal, particularly a human.
Such treatment will be suitably administered to subjects,
particularly humans, suffering from, having, susceptible to, or at
risk for a cancer or proliferative disease, disorder, or symptom
thereof. Determination of those subjects "at risk" can be made by
any objective or subjective determination by a diagnostic test or
opinion of a subject or health care provider (e.g., genetic test,
enzyme or protein marker, Marker (as defined herein), family
history, and the like).
[0124] Another aspect of the invention is the use of any of the
formulae herein in the manufacture of a medicament for treating
disease, causing cell death in leukemia cells, selectively causing
cell death in leukemia cells (e.g., while not adversely affecting
normal cells, with reduced or little toxicity to normal cells,
leukemia cells not usually destroyed by other therapies, leukemia
cells not usually destroyed by standard leukemia therapies) in a
subject. Preferably, the medicament is used for treatment or
prevention in a subject of a disease, disorder or symptom set forth
herein.
[0125] In another embodiment, the method of treatment further
comprises the step of administering to said patient one or more
additional therapeutic agents which, alone or in combination with a
compound of any of the formulae herein, are effective to treat a
disease, disorder or symptom thereof delineated herein; and for
reducing the side effects of a compound of any of the formulae
herein, enhancing or potentiating the activity of a compound of any
of the formulae herein, or for increasing the duration of
pharmacological action of a compound of any of the formulae
herein.
[0126] Additional agents include, for example, histone deacetylase
inhibitors (e.g., sodium butyrate, MS-275, SAHA, aphacidin,
depsipeptide, FK 228, trichostatin A), kinase inhibitors (e.g.
Gleevec, CEP-701, PKC412, etc.), heat shock protein inhibitors
(17-AAG), famesyltransferase inhibitors (zarnestra), CDK inhibitors
(flavopiridol), proteasome inhibitors (bortezomib), demethylating
agents (decitabine, vidaza), Bcl-2 inhibitors (ABT-737), etc.
[0127] In yet another embodiment, the method of treatment comprises
the step of administering to said patient one or more therapeutic
agents which, alone or in combination with a compound of any of the
formulae herein, are effective to treat one or more of non-GSK
mediated diseases, disorders, or symptoms thereof.
[0128] In each of the above embodiments, the second therapeutic
agent or agents may be administered together with a compound a
compound of any of the formulae herein as part of a single dosage
form or as separate dosage forms. Alternatively, the second
therapeutic agent or agents may be administered prior to,
consecutively with, or following the administration of a compound a
compound of any of the formulae herein. In such combination therapy
treatment, both the compounds of this invention and the second
therapeutic agent(s) are administered by conventional methods. The
administration of the second therapeutic agent(s) may occur before,
concurrently with, and/or after the administration of the compound
of a compound of any of the formulae herein. When the
administration of the second therapeutic agent occurs concurrently
with a compound of a compound of any of the formulae herein, the
two (or more) agents may be administered in a single dosage form
(such as a composition of this invention comprising a compound a
compound of any of the formulae herein, a second therapeutic agent
or agents as described above, and a pharmaceutically acceptable
carrier), or in separate dosage forms. The administration of a
composition of this invention comprising both a compound of a
compound of any of the formulae herein and a second therapeutic
agent(s) to a subject does not preclude the separate administration
of said second therapeutic agent(s), any other therapeutic agent or
any compound of this invention to said subject at another time
during a course of treatment.
[0129] Effective amounts of second therapeutic agent or agents
useful in the methods of this invention are well known to those
skilled in the art and guidance for dosing may be found in patents
referenced herein, as well as in Wells et al., eds.,
Pharmacotherapy Handbook, 2.sup.nd Edition, Appleton and Lange,
Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket
Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma
Linda, Calif. (2000), and other medical texts. However, it is well
within the skilled artisan's purview to determine the optimal
effective-amount range of the additional agent(s).
[0130] Second therapeutic agents useful in the method of treatment
are the same as those described above as part of combination
compositions. The compounds herein are found to possess inhibitory
activity against members of the kinase family of enzymes, and thus
are useful in modulating kinase-mediated metabolic pathways and
disease/disorder processes. Another aspect is a method of treating
a kinase-mediated disease or disorder in a subject comprising
administration to the subject of a compound herein. In other
aspects, the kinase is, for example, AKT1 (PKB alpha), CHEK1
(CHK1), DYRK3, FLT3, GSK3B, KDR (VEGFR2), MAP4K4 (HGK), MAPK14 (p38
alpha), MAPKAPK2, MET (cMet), PHKG2, PIM1, PRKCA (PKC alpha),
PRKCB1 (PKC beta1), PRKCB2 (PKC beta2), PRKCD (PKC delta), PRKCE
(PKC epsilon), PRKG (PKC gamma), PRKCH (PKC eta), PRKCI (PKC iota),
PRKCN (PKD3), PRKCQ (PKC theta), PRKCZ (PKC zeta), PRKCD1 (PKC mu),
ROCK1, RPS6KA3 (RSK2), STK6 (Aurora A), or SYK.
[0131] Primary AML, blast crisis CML (bcCML), ALL, and CLL
specimens demonstrated rapid induction of cell death upon treatment
with TDZD-8. In addition, for myeloid leukemias, cytotoxicity was
observed for phenotypically described stem/progenitor cells, in
vitro colony-forming progenitors, and LSCs as defined by
xenotransplantation assays. In contrast, no significant toxicity
was observed for normal hematopoietic stem and progenitor cells.
Notably, cell death was frequently evident within 2 hours or less
of TDZD-8 exposure. Thus, other aspects of the compounds and
methods herein include those wherein leukemia cells undergo cell
death upon contact or exposure to the compounds/compositions
herein, while normal cells are not similarly impacted. Also,
methods wherein the leukemia cell death upon exposure to compounds
herein is rapid (e.g., <6 hours, <5 hours, <4 hours, <3
hours, <2 hours, <1 hour, <30 minutes, <15 minutes,
<5 minutes), and methods wherein the compound administered is
capable of causing rapid leukemia cell death upon contact or
exposure are contemplated.
[0132] According to another aspect, the invention provides a
compound of a compound of any of the formulae herein and one or
more of the above-described second therapeutic agents, either in a
single composition or as separate dosage forms for use in the
treatment or prevention in a subject of a disease, disorder or
symptom set forth above.
[0133] In yet another aspect, the invention provides the use of a
compound a compound of any of the formulae herein and one or more
of the above-described second therapeutic agents in the manufacture
of a medicament, either as a single composition or as separate
dosage forms, for treatment or prevention in a subject of a
disease, disorder or symptom set forth above.
[0134] The compounds of this invention may be readily assayed for
biological activity by known methods. For instance, in vitro
methods of determining cell cycle status (flow cytometry using
labeling with propidium iodide or similar dyes), cytotoxicity
(labeling with Annexin V, trypan blue, TUNEL, or similar reagents),
progenitor frequency (methylcellulose or soft agar colony-forming
unit--CFU--assays), cobblestone-area forming assays (CAFC), long
term culture-initiating cell (LTC-IC) assays, etc.
[0135] Animal models of some of the foregoing indications involving
aberrant proliferation of hematopoietic cells treatable by the
invention include for example: non-obese diabetic-severe combined
immune deficient (NOD/SCID) mice injected with primary human AML,
ALL, or CML cells; inbred Sprague-Dawley/Charles University Biology
(SD/Cub) rats (spontaneous T-cell lymphoma/leukemia model);
Emu-immediate-early response gene X-1 (IEX-1) mice (T-cell lymphoma
model); rabbits injected with cynomogulus-Epstein Barr virus
(T-cell lymphoma model); transgenic mice expressing p210bcr/abl
(founder mice, ALL model; progeny mice, CML model); transgenic mice
expressing TCL-1 (CLL model); NOD/SCID mice injected with OCI-Ly10
or related cell lines (Non-hodgkins lymphoma models);
NOD/SCID/gammac null (NOG) mice injected with U266 cells or primary
human myeloma cells (multiple myeloma model); and, C57B1/KaLwRij
mice injected with 5T33 cells (multiple myeloma model). Each of the
compounds of this invention may be tested in these or similar
animal models.
[0136] In order that the invention might be more fully understood,
the following examples are set forth. They are not intended to
limit the scope of the invention and further examples will be
evident to those of ordinary skill in the art.
Example 1
FIG. 1 Protocol
[0137] A. AML cells, bcCML cells, CLL, normal bone marrow (BM), and
umbilical cord blood (CB) were obtained from volunteer donors with
informed consent or from the National Disease Research Interchange
(NDRI). The cells were isolated and processed as described [1].
Briefly, samples were subjected to Ficoll-Paque (Pharmacia Biotech,
Piscataway, N.Y.) density gradient separation to isolate
mononuclear cells. The percent CD34 in the samples analyzed ranged
from 20% to 80%. Fresh or thawed cells were cultured in serum-free
medium (SFM) [2] for 1 h before the addition of drugs. All drug
treatments were performed in triplicate. TDZD-8 (Calbiochem) was
reconstituted in DMSO and subsequently diluted in phosphate buffer
saline (PBS). Total viable cell numbers were determined using a
flow cytometric apoptosis assay as described [3]. Briefly, after 18
h of treatment, specimens were labeled with anti-CD34-PE (Becton
Dickinson, San Jose, Calif.) for 20 minutes. Cells were then washed
in cold PBS and resuspended in 200 .mu.l of annexin-V buffer.
Annexin-V-fluorescein isothiocyanate (FITC) and 7-aminoactinomycin
(7-AAD; Molecular Probes, Eugene, Oreg.) were added and the samples
were incubated at room temperature for 15 minutes followed by
analysis using a Becton Dickinson LSRII flow cytometer. The total
number of events collected was 100,000. The percent viable cells
was defined as AnnexinV.sup.neg/7-AAD.sup.neg cells on total
(ungated) cells and on gates set for CD34.sup.+ populations. [0138]
Jordan, C. T., et al., The interleukin-3 receptor alpha chain is a
unique marker for human acute myelogenous leukemia stem cells.
Leukemia, 2000. 14(10): p. 1777-84. [0139] Lansdorp, P. M. and W.
Dragowska, Long-term erythropoiesis from constant numbers of CD34+
cells in serum-free cultures initiated with highly purified
progenitor cells from human bone marrow. J. Exp. Med., 1992. 175:
p. 1501-1509. [0140] Guzman, M. L., et al., Nuclear factor-kappaB
is constitutively activated in primitive human acute myelogenous
leukemia cells. Blood, 2001. 98(8): p. 2301-7.
[0141] B. Primary human AML, blast crisis CML, ALL and CLL cells,
mobilized peripheral blood (MPB) and normal bone marrow (BM) cells
were obtained from volunteer donors with informed consent.
Additional samples were obtained from the Quebec Leukemia Cell
Bank, which collects specimens from ten university and regional
hospitals. They obtained PB or BM cells with informed consent from
patients with different morphologic types of AML and ALL. Umbilical
cord blood (CB) was obtained from the National Disease Research
Interchange (NDRI), or with informed consent from volunteer donors
at Rochester General Hospital. Mononuclear cells were isolated from
the samples using Ficoll-Paque (Pharmacia Biotech, Piscataway,
N.Y.) density gradient separation. In some cases cells were
cryopreserved in freezing medium of Iscove's modified Dulbecco
medium (IMDM), 40% fetal bovine serum (FBS), and 10%
dimethylsulfoxide (DMSO) or in CryoStor.TM. CS-10 (VWR). Cells were
cultured in serum-free medium (SFM).sup.34 for 1 h before the
addition of drugs. TDZD-8 and Parthenolide were obtained from EMD
chemicals (San Diego, Calif.) from Biomol (Plymouth Meeting, Pa.)
respectively.
[0142] C. Flow Cytometry: Apoptosis assays were performed as
described..sup.19 Briefly, after 18-24 h of treatment, specimens
were labeling using anti-CD38-allophycocyanin (APC), CD34-PECy7,
CD123-phycoerythin (PE) or CD10-flurescein isothiocyanate (FITC)
(Becton Dickinson, San Jose, Calif.) for 15 minutes. Cells were
washed in cold PBS and resuspended in 200 .mu.l of annexin-V buffer
(0.01M HEPES/NaOH, 0.14M NaCl, 2.5 mM CaCl.sub.2) Annexin-V-FITC or
Annexin V-PE (Becton Dickinson) and 7-aminoactinomycin (7-AAD;
Molecular Probes, Eugene, Oreg.). Samples were then incubated at
room temperature for 15 minutes and analyzed on a BD LSRII flow
cytometer. Analyses for phenotypically described stem cell
subpopulations were performed by gating CD34+/CD38-/CD123+,
CD34+/CD10-, and CD34+/CD38- for AML, ALL and normal specimens
respectively. To assess human cell engraftment in the NOD/SCID
xenotransplant model, BM cells were blocked with the anti-Fc
receptor antibody 2.4G2 and 25% human serum and then labeled with
anti-human CD45-PE antibody (Becton Dickinson, San Jose, Calif.).
Free-thiol analysis was performed by labeling cells with
monobromobimane (mBBr) (Probes-Invitrogen). Phospho-FLT3 (Tyr591)
Alexa Fluor-488 conjugate (Cell Signaling, Danvers, Mass.) was used
for detection of active FLT3. For multispectral imaging flow
cytometry, cells were stained with YoPro-1 (Probes-Invitrogen),
7-AAD, Draq 5 and CD45-PE. Membrane integrity assays were also
performed by standard flow cytometry by staining with YoPro-1,
Hoescht-33342, Propidium Iodide (PI) (Probes-Invitrogen) and
annexin V-APC. Cells were analyzed using the Amnis Imagestream
imaging cytometer (Amnis Corporation; Seattle, Wash.).
Example 2
FIG. 2 Protocol
[0143] AML, normal cells or other specimens were cultured in SFM as
above for 18 h in the presence or absence of 20 micromolar TDZD-8.
Cells were then plated at 50,000 cells/ml in Methocult.TM. GF H4534
(1% methylcellulose in IMDM, 30% FBS, 1% BSA, 10.sup.-4M
2-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml rh stem cell factor,
10 ng/ml rh GM-CSF, 10 ng/ml rh IL-3--Stem Cell Technologies,
Vancouver, B.C.) supplemented with 3 units/ml of erythropoietin and
50 ng/ml G-CSF (R&D Systems, Minneapolis, Minn.). Colonies were
scored after 10-14 days of culture.
Example 3
FIG. 3 Protocol
[0144] NOD/SCID (NOD.CB17-prdkdc scid/J) mice (Jackson
Laboratories, Bar Harbor, Me.) were sub-lethally irradiated with
270 rad using a RadSource.TM. X-ray irradiator the day before
transplantation. Cells to be assayed (AML or normal umbilical cord
blood--CB) were injected via tail vein (5-10 million cells) in a
final volume of 0.2 ml of PBS with 0.5% FBS. After 6-8 weeks,
animals were sacrificed and BM was isolated. To analyze human cell
engraftment, BM cells were blocked with anti-Fc receptor antibody
2.4G2 and 25% human serum, labeled with anti-human CD45, CD33 or
CD19 antibodies (BD, San Jose, Calif.), and analyzed using a Becton
Dickinson LSRII flow cytometer.
Example 4
FIG. 4 Protocol
[0145] The same procedure as described in Example 1 was used except
after the timepoints indicated on the horizontal axis, cells were
washed in PBS to remove excess TDZD-8, and replated in pre-warmed
SFM for a total of 24 hours.
Example 5
FIG. 5 Protocol
[0146] The same procedure as described in Example 2 was used
except, after the timepoints indicated on the horizontal axis,
cells were washed in PBS to remove excess TDZD-8, and plated at
50,000 cells/ml in Methocult.TM. GF H4534.
Example 6
FIG. 6 Protocol
[0147] AML cells were cultured as described in Example 1 for the
indicated times with 20 micromolar TDZD-8. Cells were then washed
with PBS and stained to assess membrane permeability using annexin
V-Allophycocyanin (APC) and the vital dyes: Yo-Pro-1 (0.1 uM),
Hoescht 33342 (2.5 ug/ml), and Propidium Iodide (0.5 ug/ml)
(Molecular Probes).
Example 7
FIG. 7 Protocol
[0148] Cultures were established and analyzed as described for
Example 1; however, cells were pre-incubated with either
n-acetylcysteine (Sigma) or z-vad (Calbiochem) for one hour prior
to addition of TDZD-8.
Example 8
FIG. 8 Protocol
[0149] AML or normal cells were cultured in SFM as above for the
indicated time (horizontal axis) in the presence or absence of 20
micromolar TDZD-8. Intracellular thiol levels were assessed by flow
cytometry after labeling with 50 .mu.M monobromobimane (molecular
probes).
[0150] Analysis of primary AML, blast crisis CML (bcCML), ALL, and
CLL specimens demonstrated rapid induction of cell death upon
treatment with TDZD-8. In addition, for myeloid leukemias,
cytotoxicity was observed for phenotypically primitive cells, in
vitro colony-forming progenitors, and LSCs as defined by
xenotransplantation assays. In contrast, no significant toxicity
was observed for normal hematopoietic stem and progenitor cells.
Notably, cell death was frequently evident within 2 hours or less
of TDZD-8 exposure. Cellular and molecular studies indicate that
the mechanism by which TDZD-8 induces cell death involves rapid
loss of membrane integrity, depletion of free thiols, and
inhibition of both the PKC and FLT3 signaling pathways. We conclude
that TDZD-8 employs a unique and previously unknown mechanism to
rapidly target leukemia cells, including malignant stem and
progenitor populations.
[0151] Kinase Assays:
[0152] Single point and titration assays are performed using
protocols (or essentially using protocols) know in the art for
kinase activity profiling against a battery of kinase targets,
including, for example, those specifically delineated herein. Such
assays include, for example, those available from commercial
sources, contract research laboratories, and the like.
Representative results for TDZD-8 against a battery of kinase
targets is delineated at Table 3.
[0153] Immunoblots.
[0154] Cells were prepared and analyzed as previously
described..sup.35 Membrane fractions were prepared using mem-PER
eukaryotic membrane protein extraction kit as per manufacturer's
instructions (Pierce; Rockford, Ill.). Blots were probed with
phospho-PKC (pan) (beta II; Ser660), phospho-PKCalpha/betaII
(Thr638/641), total PKC.alpha., PKC.beta. and FLT3 (Santa Cruz
Biotechnology, CA), Caspase-3 (Cell Signaling technologies;
Danvers, Mass.); caspase-8 and PARP (BD bioscience), cleaved PARP
(abcam) or anti-actin (AC-15; Sigma) antibodies.
[0155] Statistical Analysis.
[0156] Statistical analyses and graphs were performed using
GraphPad Prism software (GraphPad Software, San Diego, Calif.). For
statistical analysis the data was log transformed and analyzed by
one-way ANOVA followed by Tukey post-hoc test. For 2 group
comparisons, significance was determined by paired t-tests.
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[0211] Results
[0212] TDZD-8 Induces Leukemia Specific Cell Death.
[0213] Initial studies were performed to determine the effects of
TDZD-8 on different types of primary human leukemia (AML, blast
crisis CML, CLL and ALL), as well as normal hematopoietic cells.
FIG. 9 shows the percent viability relative to untreated controls
for primary human specimens treated with 20 .mu.M TDZD-8 for 24
hours. All forms of leukemia were strongly impaired by TDZD-8, with
mean viability of 15% for AML (n=37), 7.2% for CLL (n=12), 12.4%
for ALL (n=6) and 21.6% for bcCML (n=6). In contrast the cell
viability for normal specimens was 79.5% (n=13). Moreover, the lack
of toxicity towards normal specimens was not significantly
different for CB, BM and MPB when each tissue type was analyzed
separately (FIG. 14). Thus, the cytotoxicity of TDZD-8 was
significantly (p<0.001) more specific to leukemia specimens.
Given the broad efficacy towards leukemia cells, we further
determined the range of activity for different types of tumor cells
by submitting TDZD-8 for screening against the NCI-60 panel..sup.36
Interestingly, TDZD-8 activity was specific to cell lines derived
from hematologic malignancies, where the average concentration to
achieve 50% growth inhibition (GI50) was 8.3 .mu.M (Table 1). All
other tumor lines showed no growth inhibition up to concentrations
of 100 .mu.M. Together, these data indicate that while TDZD-8 is
highly cytotoxic to leukemia and related diseases, the compound
does not substantially harm normal hematopoietic cells or tumors
derived from other non-hematopoietic tissues.
[0214] TDZD-8 Anti-Leukemia Effects are Observed at the Progenitor
and Stem Cell Levels.
[0215] While many agents show efficacy towards bulk tumor
populations, eradication of more primitive stem and progenitor
cells can represent a significant challenge. Given the established
role of LSCs in several forms of leukemia,.sup.1,3,4,37 we examined
the effect of TDZD-8 on phenotypically described stem cells from
AML, bcCML and ALL specimens. Treatment with 20 .mu.M TDZD-8 for 24
hours resulted in a mean viability of 7.6% for CD34+CD38- from AML
specimens (n=10); 2.8% for CD34+CD38- from bcCML specimens (n=3);
and 22.3% for CD34+CD10- from ALL specimens (n=3) (FIG. 10A). In
contrast, the viability of CD34+CD38- cells from healthy specimens
(n=7) was 80.2% after TDZD-8 treatment (FIG. 10A, grey bar). Thus,
the specificity of the compound for phenotypically described LSCs
was highly significant (p<0.001). To determine whether TDZD-8
could also target functionally defined myeloid progenitor cells, we
performed methylcellulose colony assays. FIG. 10B shows that the
ability of normal specimens to form colonies was not substantially
affected by treatment with 20 .mu.M TDZD-8 (84.14% myeloid colonies
and 94.79% erythroid colonies; n=12). In contrast, a significant
decrease in colony formation was observed for both AML and blast
crisis CML, with only 7.3% CFU for AML (n=11, p<0.001) and 16.1%
CFU for bcCML (n=3, p<0.01) after TDZD-8 treatment. It should be
noted that for 6 out of the 11 AML samples assayed, no colonies
whatsoever were evident after treatment with TDZD-8. Further, we
analyzed stem cell activity for AML and normal specimens using the
NOD/SCID xenotransplant model..sup.38 These studies demonstrated
that AML cells treated with 20 .mu.M TDZD-8 for 18 hours
significantly decreased their ability to engraft into NOD/SCID mice
(FIG. 10C). Analysis of 3 independent specimens demonstrated
engraftment of leukemic cells decreased to 11% (p<0.001), 1%
(p=0.001) and 8.5% (p<0.01) respectively relative to untreated
controls. In contrast, little to no effect on the engraftment of
normal specimens (n=3) was observed after treatment with TDZD-8.
Together these data demonstrate that TDZD-8 is highly cytotoxic
towards leukemic but not normal hematopoietic progenitor and stem
cells.
[0216] TDZD-8 Activity Involves Oxidative Stress.
[0217] Previous studies have indicated that leukemia-specific
agents may function via mechanisms involving the induction of
oxidative stress..sup.39,14 Therefore, we performed studies to
examine whether TDZD-8 might also modulate the oxidative state of
target cells. Shown in FIG. 11A is labeling with the dye mBBr,
which detects free thiol groups. Reduced labeling intensity
signifies loss of free thiols, an indication of increased oxidative
stress. Upon treatment with TDZD-8, reduction in mBBr labeling is
evident in primary AML, ALL and CLL specimens as early as 30
minutes after exposure, suggesting rapid thiol depletion in the
cell. Moreover, only slight changes in mBBr staining were observed
in normal specimens. To further examine the role of oxidative
state, we pretreated target cells with the anti-oxidant
N-acetylcysteine (NAC), which completely blocked the cell death
response in primary AML cells induced by TDZD-8 (FIG. 11B). Taken
together, these data indicate that TDZD-8 induces oxidative stress
and that this activity is important for the anti-leukemia
properties shown above.
[0218] TDZD-8 Anti-Leukemia Activity is Observed with Very Rapid
Kinetics.
[0219] We noted that changes in oxidative state (FIG. 11A) occurred
with relatively rapid kinetics, suggesting that other cellular
changes may also occur quickly. To further test the rate at which
TDZD-8 may affect leukemic cells we performed additional studies
using primary AML specimens to determine viability at various times
post-exposure (0.5, 1, 2, 4, 6 and 24 hours). For comparison,
parallel studies were performed with parthenolide (PTL), a drug we
have previously shown can also specifically target primary human
LSC..sup.14 As shown in FIG. 12A (left panel), primitive AML
CD34+/CD38- cells treated with TDZD-8 displayed an extremely rapid
loss of viability, with a mean time of only 2 hours to achieve
.gtoreq.50% cell death. In contrast, PTL did not significantly
change cell viability until 6 hours of treatment where the mean
viability was still over 70% (FIG. 12A, right panel). The rapid
cell death induced by TDZD-8 treatment was also observed for bulk
leukemia blast populations, where an analysis of 17 primary
specimens also showed a mean time of 2 hours to achieve .gtoreq.50%
cell death (FIG. 12B). Interestingly, while all 17 specimens
responded relatively fast, three specimens showed a particularly
dramatic reduction in viability to below 30% within 30 minutes. We
also tested lymphoid specimens and observed rapid cell death
kinetics for primary CLL and ALL samples (both total blast
populations and phenotypically described stem cells) (FIG. 15).
Next we examined the minimum time of exposure required for the
commitment of AML populations to cell death. For these studies,
cells were treated with 20 .mu.M TDZD-8 for varying times and then
immediately washed and re-plated in fresh culture medium. Cell
viability was evaluated 24 hours after initial treatment.
Strikingly, as little as 30 minutes exposure to TDZD-8 was
sufficient to commit primary human AML cells to death (FIG. 12C).
The 30-minute exposure time was also sufficient to inhibit the
ability of AML progenitor cells to form colonies in methylcellulose
culture (FIG. 12D). Together, the data indicate that primary AML
bulk and progenitor cell populations are irreversibly committed to
cell death within 30 minutes of exposure to TDZD-8.
[0220] The short exposure time for commitment to cell death
suggests that TDZD-8 may be rapidly binding and/or internalized by
cells. Since the hydrophobic chemical structure of TDZD-8 predicts
that the drug is likely to intercalate in membranes, experiments
were performed to analyze plasma membrane integrity. For this
purpose, several nucleic acid dyes of varying sizes were employed
(YoPro-1, Hoescht-33342, 7-AAD and propidium iodide (PI)). The
uptake of smaller dyes, YoPro-1 and Hoescht-33342, can be altered
by relatively moderate changes in membrane permeability, whereas
larger dyes such as 7-AAD and PI are only internalized when
profound loss of membrane integrity occurs. FIG. 12E shows a
representative example of a primary AML specimen treated with
TDZD-8 for 15 minutes and analyzed by nucleic acid dye labeling and
multispectral imaging flow cytometry (Amnis Imagestream). The left
panels show dye uptake analysis in control or TDZD-8 treated cells
where R1 represents intact cells (impermeable to YoPro-1), R2 shows
cells with compromised membrane integrity (YoPro-1 permeable), and
R3 represents dead cells (permeable to YoPro-1 and 7-AAD). The
percent of YoPro-1 positive cells (R2) increased from 7% to 78%
upon treatment with TDZD for 15 min. In addition, cells were also
labeled with anti-CD45 to delineate the plasma membrane and the
cell permeable DNA dye Draq5 to identify the nucleus. FIG. 12E
(right panel) shows representative pictures of cells present in the
R1, R2 and R3 gates, where the nuclear localization of YoPro-1 is
evident in R2 but not R1. Additional studies in FIG. 12F
demonstrate that the rapid uptake of YoPro-1 observed for primary
AML cells is not evident in normal specimens (BM or CB). These data
indicate that TDZD-8 mediates a rapid alteration in membrane
permeability in primary AML cells but not in normal hematopoietic
cells.
[0221] With respect to the death mechanism, TDZD-8 treated cells
show a rapid increase in Annexin V labeling, a marker of apoptosis
(data not shown). Since loss of membrane integrity and Annexin V
binding are early events associated with apoptosis, we tested
downstream events involved in caspase-dependent apoptotic events
(e.g. pro-caspase and PARP cleavage). Interestingly, no cleavage of
pro-caspases 3, 8 or PARP was detected (data not shown). Moreover,
no abrogation of death was observed upon treatment with the
pan-caspase inhibitor Z-VAD (FIG. 12G, white bars). These data
suggest that death occurs via a caspase-independent pathway.
[0222] TDZD-8 Activity as a Kinase Inhibitor.
[0223] TDZD-8 has been reported to be a GSK-313 kinase inhibitor
(IC.sub.50=2 .mu.M) and to not significantly affect the activities
of Cdk-1/cyclin B, CK-II, PKA, and PKC (IC.sub.50>100
.mu.M)..sup.24 Therefore, to determine whether the anti-leukemia
activity observed with TDZD-8 involves GSK3.beta. inhibition, we
tested other commercially available GSK30 inhibitors. Seven of the
eight agents tested failed to induce AML cell death (Table 2). The
single other GSK3.beta. inhibitor
(2-chloro-1-(4,5-dibromo-thiophen-2-yl-ethanone) that could induce
AML cell death was also toxic to normal cells. In addition, we
tested different compounds that share the thiazolidinedione ring
structure and did not observe the AML-specificity or cell death
kinetics obtained with TDZD-8 (Table 2). Since the concentrations
that induce leukemia specific cell death are 10 times higher than
the IC50 reported for inhibition of GSK3.beta., it is likely that
the TDZD-8 anti-leukemia activity is due to an off-target effect.
To test this hypothesis, a commercial kinase profiling service was
employed to identify other potential targets of TDZD-8. A broad
range of 44 different transmembrane and intracellular kinases was
examined at a drug concentration of 20 .mu.M. Greater than 90%
inhibition was observed for 14 different classes of kinase. Of
those, the most evident enzymes with potential ties to hematologic
malignancy were PKC and FLT3. Notably, other related kinases
implicated in hematologic diseases (c-kit, PDGF-R, Jak2, Src, and
Tie2) showed little to no inhibition. To further examine PKC and
FLT3, preliminary studies were performed to examine the activity of
each in primary specimens and in response to drug treatment.
[0224] First, since PKC was previously reported to not be a target
of TDZD-8 (IC50>100 .mu.M), we analyzed one family member from
each of the three major PKC classes (conventional, novel, and
atypical). The in vitro IC50 for PKC isoforms was: PKC beta I=1.4
.mu.M, PKC delta=1.1 .mu.M, and PKC iota=5.5 .mu.M. Thus, at least
in vitro, TDZD-8 appears to be a broad inhibitor of the PKC family.
As a control for drug activity, GSK3.beta. was also tested, and
consistent with previous reports had an IC50 of 1.4 .mu.M. Next, to
examine the activity of PKC in primary cells, immunoblots of
purified CD34+ populations from AML and normal BM specimens were
performed. As shown in FIG. 13A, the levels of both total and
phosphorylated-PKC were much higher in primary CD34+ AML specimens
compared to normal controls, suggesting a role for the PKC family
in primitive leukemic cells. Since active PKCs are localized in the
plasma membrane.sup.40,41 and PKC.alpha. and PKC.beta. plasma
membrane localization has been reported in leukemia cell
lines,.sup.42 we examined their levels upon TDZD-8 treatment. FIG.
13B shows that plasma membrane localized PKC.alpha. and .beta.
decreases with TDZD-8 exposure, suggesting that TDZD-8 induces PKC
inactivation in primary CD34+ AML and ALL cells. We did not observe
a difference in the levels of HSP70 suggesting that reduction of
PKC in the membrane is not due to a general loss of membrane
proteins. The data in FIGS. 13A and B indicates the PKC family
members are active in primary AML cells and that TDZD-8 potentially
inhibits their function.
[0225] To examine the inhibitory effect of TDZD-8 on FLT3 activity,
titration assays for FLT3 kinase activity were performed to
estimate the in vitro IC50 of TDZD-8. FIG. 13C shows the titration
curve, which demonstrates an IC50 of 673 nM in vitro. Next, to
assess whether TDZD-8 inhibitory FLT3 activity could be detected in
vivo, FACS analyses were performed to measure the phospho-specific
(i.e. activated) FLT3 form. As shown in FIG. 13D, phosphorylated
FLT3 is readily detected in primary AML specimens (as previously
reported.sup.43-45), and treatment with TDZD-8 induced a .about.30%
reduction in phosphorylation for both CD34+ and CD34+,CD38-
populations. These data suggest TDZD-8 may also function as an
intracellular inhibitor of FLT3 activation.
[0226] In the present study we describe unique anti-leukemia
properties of the compound TDZD-8, which was originally developed
as a non-ATP competitive inhibitor of GSK3.beta.. The compound has
undergone preclinical analysis as a cyto-protective agent in
numerous models, including studies of type-2 diabetes, Alzheimer's
disease, spinal cord injury, and several forms of inflammation. Our
findings add an entirely new dimension to the activities of TDZD-8
by demonstrating that the compound selectively induces death of
several major forms of leukemia cells, including malignant myeloid
stem and progenitor populations, while sparing normal hematopoietic
tissue. Further, the rate of cell death is exceptionally fast, with
most toxicity evident within 1-6 hours. Based on these data, we
believe that TDZD-8 may function via a novel mechanism to induce
death of malignant hematopoietic cells.
[0227] Another striking feature of TDZD-8 is its apparent affinity
to target cells. Experiments demonstrated that exposure to the drug
of only 30 minutes was sufficient to mediate all cytotoxic
activity. Additional preliminary data indicates that treatments as
brief as 5 minutes may also be effective (data not shown). Given
the hydrophobic nature of TDZD-8, it appears that the molecule is
rapidly inserted into cellular membranes. This premise in turn
indicates that one component of the drug's mechanism might be a
direct effect on the plasma membrane. Subsequent studies confirmed
that TDZD-8 induces a rapid change in membrane integrity, such that
small nucleic acid dyes are readily internalized. These findings
support the concept the TDZD-8 directly modulates membrane
integrity.
[0228] Aside from the novel membrane-directed biology described
above, TDZD-8 also functions as a multi-kinase inhibitor. Among
those targets is, as described, GSK3.beta...sup.24 Notably,
analysis of several other known GSK3.beta. inhibitors failed to
induce leukemia-specific cell death (Table 2), suggesting that the
activity of TDZD-8 is independent of GSK3.beta. or at least
combined with other activities. Other targets of TDZD-8, both in
vitro and in vivo, are PKC family members and FLT3. PKC is known to
play a role in growth and differentiation of hematopoietic
cells..sup.42 Moreover, it has been reported that PKC.alpha.
over-expression confers chemoresistance to leukemia cells and is
associated with poor survival,.sup.49,50 and that PKC.beta. is
important for differentiation of HL-60 and U937 cells..sup.51,52 In
addition, PKC is involved in protection of K562 cells against
drug-induced apoptosis..sup.53 These findings support a role for
PKC in malignant hematopoiesis and suggest the PKC family may
represent an important target for therapy. Similarly, aberrant
activation of FLT3 is a well described feature of AML cell types
and inhibition of FLT3 clearly mediates an anti-leukemic effect in
several systems..sup.44,54 Thus, the activity of TDZD-8 towards
FLT3 and PKC are potentially important component of its overall
mechanism of action.
[0229] Finally, we note that previous studies have described TDZD-8
as a moderate NF-.kappa.B inhibitor,.sup.27-29,32 an activity we
have confirmed in leukemia cells (data not shown). Further, our
data indicate the compound is a strong oxidant (FIG. 11).
Therefore, TDZD-8 fulfills the two criteria described above that
have previously been reported for regimens that selectively target
LSC (i.e. inhibition of NF-.kappa.B and induction of oxidative
stress)..sup.47 However, as shown in FIG. 12, in addition to the
established mechanisms of LSC death induction TDZD-8 also confers a
rapid alteration in membrane permeability for malignant cells of
hematologic origin. Thus, it is indicated that loss of membrane
integrity substantially accelerates the rate of cell death and is
integral to the overall effects observed for TDZD-8.
[0230] All references cited herein, whether in print, electronic,
computer readable storage media or other form, are expressly
incorporated by reference in their entirety, including but not
limited to, abstracts, articles, journals, publications, texts,
treatises, technical data sheets, internet web sites, databases,
patents, patent applications, and patent publications.
[0231] The recitation of a listing of chemical groups in any
definition of a variable herein includes definitions of that
variable as any single group or combination of listed groups. The
recitation of an embodiment for a variable herein includes that
embodiment as any single embodiment or in combination with any
other embodiments or portions thereof. The recitation of an
embodiment herein includes that embodiment as any single embodiment
or in combination with any other embodiments or portions
thereof.
[0232] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
TABLE-US-00002 TABLE 1 NCI-60 screen Cell lines Log.sub.10 GI50
.mu.M Leukemia CCRF-CEM -4.94 11.5 HL-60 -6.63 0.2 K-562 -5.19 6.5
RPMI-8226 -4.63 23.4 SR -8 0.01 Non-small cell lung cancer 9 cell
lines -4 >100.0 Colon cancer 7 cell lines -4 >100.0 CNS
cancer 6 cell lines -4 >100.0 Melanoma 8 cell lines -4 >100.0
Ovarian cancer 5 cell lines -4 >100.0 Renal cancer 7 cell lines
>100.0 Prostate cancer 2 cell lines -4 >100.0 Breast cancer 7
cell lines -4 >100.0
TABLE-US-00003 TABLE 2 Inhibitor tested on primary AML and Normal
specimens Cytotoxic Rapid Activity Compound AML Normal kinetics
GSK-3.beta. inhibitor TDZD-8 (4-Benzyl-2-methyl-1,2,4- Yes No Yes
thiadiazolidine-3,5-dione_ GSK-3.beta. inhibitor BIO No No No
GSK-3.beta. inhibitor (5-Methyl-1H-pyrazol-3-yl)-(2- No No No
phenylquinazolin-4-yl)amine GSK-3.beta. inhibitor
2-Chloro-1-(4,5-dibromo-thiophen-2-yl)- Yes Yes No ethanone
GSK-3.beta. inhibitor TWS119 No No No GSK-3.beta. inhibitor
SB-216763 No No No GSK-3.beta. inhibitor AR-A014418 No No No
GSK-3.beta. inhibitor 1-Azakenpaullone No No No GSK-3.beta.
inhibitor 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione No No No
thiazolidin ring Anthrax Lethal Factor protease inhibitor No No No
(thiazolidinedione 2,4-thiazolidinedione No No No ring)
(thiazolidinedione DRF 2519 No No No ring) PPAR.alpha./.gamma.
agonist (thiazolidinedione Troglitazone Yes Yes No ring)
PPAR.alpha./.gamma. agonist (thiazolidinedione
-(2-Aminoethyl)-5-((4- No No No ring) Erk inhibitor
ethoxyphenyl)methylene)-2,4- thiazolidinedione, HCl
(thiazolidinedione 5-(2,2-Difluoro-benzo[1,3]dioxol-5- No No No
ring) PI3-K ylmethylene)-thiazolidine-2,4-dione inhibitor
TABLE-US-00004 TABLE 3 TDZD-8 as test compound % Inhibition Kinase
Tested mean ABL1 70 AKT1 (PKB alpha) 102 BTK 72 CDK1/cyclin B 15
CHEK1 (CHK1) 87 CSNK1G2 (CK1 gamma 2) 22 CSNK2A1 (CK2 alpha 1) 27
DYRK3 85 EGFR (ErbB1) 1 EPHA2 3 ERBB2 (HER2) 12 FGFR1 1 FLT3 99
GSK3B (GSK3 beta) 98 GSK3B (GSK3 beta) 99 IGF1R -2 INSR 9 IRAK4 8
JAK3 -1 KDR (VEGFR2) 100 KIT -6 LCK -9 MAP2K1 (MEK1) 80 MAP4K4
(HGK) 99 MAPK14 (p38 alpha) 97 MAPK3 (ERK1) 61 MAPKAPK2 99 MET
(cMet) 90 NTRK1 (TRKA) -9 PDGFRB (PDGFR beta) -4 PHKG2 80 PIM1 95
PRKACA (PKA) 18 PRKCA (PKC alpha) 102 PRKCB1 (PKC beta I) 98 PRKCB1
(PKC beta I) 98 PRKCB2 (PKC beta II) 99 PRKCD (PKC delta) 102 PRKCE
(PKC epsilon) 99 PRKCG (PKC gamma) 103 PRKCH (PKC eta) 104 PRKCI
(PKC iota) 100 PRKCN (PKD3) 94 PRKCQ (PKC theta) 101 PRKCZ (PKC
zeta) 100 PRKD1 (PKC mu) 97 RET 34 ROCK1 113 RPS6KA3 (RSK2) 101 SRC
27 STK6 (Aurora A) 99 SYK 99 TEK (Tie2) 9
* * * * *