U.S. patent application number 14/377205 was filed with the patent office on 2015-01-01 for 3-(2-amino-ethyl)-alkylidene)-thiazolidine-2,4-dione and 1-(2-amino-ethyl)-alkylidene-1,3-dihydro-indol-2-one derivatives as selective sphingosine kinase 2 inhibitors.
This patent application is currently assigned to Virginia Commonwealth University. The applicant listed for this patent is VIRGINIA COMMONWEALTH UNIVERSITY. Invention is credited to Sarah Spiegel, Shijun Zhang.
Application Number | 20150005353 14/377205 |
Document ID | / |
Family ID | 48947991 |
Filed Date | 2015-01-01 |
United States Patent
Application |
20150005353 |
Kind Code |
A1 |
Zhang; Shijun ; et
al. |
January 1, 2015 |
3-(2-AMINO-ETHYL)-ALKYLIDENE)-THIAZOLIDINE-2,4-DIONE AND
1-(2-AMINO-ETHYL)-ALKYLIDENE-1,3-DIHYDRO-INDOL-2-ONE DERIVATIVES AS
SELECTIVE SPHINGOSINE KINASE 2 INHIBITORS
Abstract
3-(2-amino-ethyl)-5-[3-(4-substituted-phenyl)-alkylidene)-thiazolidine-2,-
4-dione and 1-(2-amino-ethyl)-3-alkylidene-1,3-dihydro-indol-2-one
and derivatives thereof are provided for use as selective SphK2
inhibitors and for use in the treatment of human diseases, such as
cancer.
Inventors: |
Zhang; Shijun; (Richmond,
VA) ; Spiegel; Sarah; (Richmond, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VIRGINIA COMMONWEALTH UNIVERSITY |
Richmond |
VA |
US |
|
|
Assignee: |
Virginia Commonwealth
University
Richmond
VA
|
Family ID: |
48947991 |
Appl. No.: |
14/377205 |
Filed: |
February 7, 2013 |
PCT Filed: |
February 7, 2013 |
PCT NO: |
PCT/US2013/025093 |
371 Date: |
August 7, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61597344 |
Feb 10, 2012 |
|
|
|
Current U.S.
Class: |
514/369 ;
435/375; 548/183 |
Current CPC
Class: |
A61P 35/00 20180101;
C07D 209/34 20130101; C07D 277/34 20130101 |
Class at
Publication: |
514/369 ;
548/183; 435/375 |
International
Class: |
C07D 277/34 20060101
C07D277/34 |
Claims
1. A compound of Formula I: ##STR00023## wherein, R.sub.1 is
selected from the group consisting of: C.sub.3-C.sub.14 alkyl and
C.sub.3-C.sub.14 alkoxyl; R.sub.2, R.sub.3, R.sub.4 and R.sub.5 may
be the same or different and are independently selected from: H,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8
alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; V is S,
O, NH, or CH.sub.2; X is C.sub.1-C.sub.4 alkyl; Y is
C.sub.1-C.sub.4 alkyl; Z is S or O or NR.sup.6 in which R.sup.6 is
selected from the group consisting of: H, C.sub.1-C.sub.8 alkyl, or
isopropyl, tert-butyl, a saturated or unsaturated monocyclic ring
with ring size ranging from 3-7 carbons per ring, and phenyl which
may be substituted with one or more substituents selected from the
group consisting of: C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8
alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen, hydroxyl, amino,
nitro, and cyano; and W is NR.sub.7R.sub.8 where R.sub.7 and
R.sub.8 may be the same or different and are independently selected
from H; C.sub.1-C.sub.4 alkyl; a saturated heterocycle comprising N
bonded directly to Y; and an unsubstituted or substituted guanidine
moiety.
2. The compound of claim 1, wherein the number of carbon atoms in
said saturated or unsaturated monocyclic ring with ring size from
3-7 is selected from the group consisting of 3, 4, 5, 6, and 7.
3. The compound of claim 1, wherein said saturated heterocycle is
selected from the group consisting of morpholine, piperidine,
piperazine, and pyrrolidine.
4. The compound of claim 1, wherein said compound is selected from
3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-di-
one and
3-(2-aminoethyl)-5-[3-(4-octyl-phenyl)-propylidene]-thiazolidine-2-
,4-dione.
5. A compound of Formula II: ##STR00024## wherein, R.sub.10 is
selected from the group consisting of: C.sub.3-C.sub.14 alkyl,
C.sub.3-C.sub.14 alkoxyl; R.sub.11, R.sub.12, R.sub.13 and R.sub.14
may be the same or different and are independently selected from:
H, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8
alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano;
R.sub.15, R.sub.16, R.sub.17 and R.sub.18 may be the same or
different and are independently selected from the group consisting
of: C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8
alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; X is
C.sub.1-C.sub.4 alkyl; Y is C.sub.1-C.sub.4 alkyl; and W is
NR.sup.19R.sup.20 where R.sup.19 and R.sup.20 may be the same or
different and are selected from the group consisting of: H,
C.sub.1-C.sub.4 alkyl; a saturated heterocycle comprising N bonded
directly to Y, and an unsubstituted or substituted guanidine
moiety.
6. The compound of claim 5, wherein the number of carbon atoms in
said saturated or unsaturated monocyclic ring with ring size from
3-7 is selected from the group consisting of 3, 4, 5, 6, and 7.
7. The compound of claim 5, wherein said saturated heterocycle is
selected from the group consisting of morpholine, piperidine,
piperazine, and pyrrolidine.
8. A method of treating diseases or conditions associated with
positive SphK2 activity in a patient in need thereof, comprising
the step of administering to said patient a sufficient quantity of
at least one compound of Formula I or Formula II: ##STR00025##
wherein, in Formula I: R.sub.1 is selected from the group
consisting of: C.sub.3-C.sub.14 alkyl and C.sub.3-C.sub.14 alkoxyl;
R.sub.2, R.sub.3, R.sub.4 and R.sub.5 may be the same or different
and are independently selected from: H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; V is S, O, NH, or CH.sub.2; X is
C.sub.1-C.sub.4 alkyl; Y is C.sub.1-C.sub.4 alkyl; Z is S or O or
NR.sup.6 in which R.sup.6 is selected from the group consisting of:
H, C.sub.1-C.sub.8 alkyl, or isopropyl, tert-butyl, a saturated or
unsaturated monocyclic ring with ring size ranging from 3-7 carbons
per ring, and phenyl which may be substituted with one or more
substituents selected from the group consisting of: C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl,
halogen, hydroxyl, amino, nitro, and cyano; and W is
NR.sub.7R.sub.8 where R.sub.7 and R.sub.8 may be the same or
different and are independently selected from H; C.sub.1-C.sub.4
alkyl; a saturated heterocycle comprising N bonded directly to Y;
and an unsubstituted or substituted guanidine moiety; and wherein
in Formula II: R.sub.10 is selected from the group consisting of:
C.sub.3-C.sub.14 alkyl, C.sub.3-C.sub.14 alkoxyl; R.sub.11,
R.sub.12, R.sub.13 and R.sub.14 may be the same or different and
are independently selected from: H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; R.sub.15, R.sub.16, R.sub.17 and
R.sub.18 may be the same or different and are independently
selected from the group consisting of: C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; X is C.sub.1-C.sub.4 alkyl; Y is
C.sub.1-C.sub.4 alkyl; and W is NR.sup.19R.sup.20 where R.sup.19
and R.sup.20 may be the same or different and are selected from the
group consisting of: H, C.sub.1-C.sub.4 alkyl; a saturated
heterocycle comprising N bonded directly to Y, and an unsubstituted
or substituted guanidine moiety.
9. The method of claim 8, wherein the number of carbon atoms in
said saturated or unsaturated monocyclic ring with ring size from
3-7 is selected from the group consisting of 3, 4, 5, 6, and 7.
10. The method of claim 8, wherein said saturated heterocycle is
selected from the group consisting of morpholine, piperidine,
piperazine, and pyrrolidine.
11. The compound of claim 8, wherein said compound is selected from
3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-di-
one and
3-(2-aminoethyl)-5-[3-(4-octyl-phenyl)-propylidene]-thiazolidine-2-
,4-dione.
12. The method of claim 8, wherein said disease or condition
associated with positive SphK2 activity is selected from the group
consisting of: cancer, arthrosclerosis, arthritis, diabetes,
obesity, osteoporosis, inflammatory diseases and Alzheimer's
disease.
13. A method of inhibiting SphK2, comprising the step of exposing
said SphK2 to at least one compound of Formula I or Formula II:
##STR00026## wherein, in Formula I: R.sub.1 is selected from the
group consisting of: C.sub.3-C.sub.14 alkyl and C.sub.3-C.sub.14
alkoxyl; R.sub.2, R.sub.3, R.sub.4 and R.sub.5 may be the same or
different and are independently selected from: H, C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl,
halogen, hydroxyl, amino, nitro, and cyano; V is S, O, NH, or
CH.sub.2; X is C.sub.1-C.sub.4 alkyl; Y is C.sub.1-C.sub.4 alkyl; Z
is S or O or NR.sup.6 in which R.sup.6 is selected from the group
consisting of: H, C.sub.1-C.sub.8 alkyl, or isopropyl, tert-butyl,
a saturated or unsaturated monocyclic ring with ring size ranging
from 3-7 carbons per ring, and phenyl which may be substituted with
one or more substituents selected from the group consisting of:
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8
alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; and W is
NR.sub.7R.sub.8 where R.sub.7 and R.sub.8 may be the same or
different and are independently selected from H; C.sub.1-C.sub.4
alkyl; a saturated heterocycle comprising N bonded directly to Y;
and an unsubstituted or substituted guanidine moiety; and wherein
in Formula II: R.sub.10 is selected from the group consisting of:
C.sub.3-C.sub.14 alkyl, C.sub.3-C.sub.14 alkoxyl; R.sub.11,
R.sub.12, R.sub.13 and R.sub.14 may be the same or different and
are independently selected from: H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; R.sub.15, R.sub.16, R.sub.17 and
R.sub.18 may be the same or different and are independently
selected from the group consisting of: C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; X is C.sub.1-C.sub.4 alkyl; Y is
C.sub.1-C.sub.4 alkyl; and W is NR.sup.19R.sup.20 where R.sup.19
and R.sup.20 may be the same or different and are selected from the
group consisting of: H, C.sub.1-C.sub.4 alkyl; a saturated
heterocycle comprising N bonded directly to Y, and an unsubstituted
or substituted guanidine moiety.
14. The method of claim 13, wherein the number of carbon atoms in
said saturated or unsaturated monocyclic ring with ring size from
3-7 is selected from the group consisting of 3, 4, 5, 6, and 7.
15. The method of claim 13, wherein said saturated heterocycle is
selected from the group consisting of morpholine, piperidine,
piperazine, and pyrrolidine.
16. The compound of claim 13, wherein said compound is selected
from
3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-di-
one and
3-(2-aminoethyl)-5-[3-(4-octyl-phenyl)-propylidene]-thiazolidine-2-
,4-dione.
17. The method of claim 13, wherein said SphK2 is present in a
cell.
18. The method of claim 17, wherein said cell is selected but not
limited to a cancer cell, cardiocyte cell, epithelial cell,
pancreatic cell, and neuronal cell, and said method includes a step
of exposing said cell to said at least one compound of Formula I or
Formula II.
19. A method of inhibiting growth or killing or damaging cancer
cells, comprising the step of exposing said cancer cells to a
compound of Formula I or Formula II: ##STR00027## wherein, in
Formula I: R.sub.1 is selected from the group consisting of:
C.sub.3-C.sub.14 alkyl and C.sub.3-C.sub.14 alkoxyl; R.sub.2,
R.sub.3, R.sub.4 and R.sub.5 may be the same or different and are
independently selected from: H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; V is S, O, NH, or CH.sub.2; X is
C.sub.1-C.sub.4 alkyl; Y is C.sub.1-C.sub.4 alkyl; Z is S or O or
NR.sup.6 in which R.sup.6 is selected from the group consisting of:
H, C.sub.1-C.sub.8 alkyl, or isopropyl, tert-butyl, a saturated or
unsaturated monocyclic ring with ring size ranging from 3-7 carbons
per ring, and phenyl which may be substituted with one or more
substituents selected from the group consisting of: C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl,
halogen, hydroxyl, amino, nitro, and cyano; and W is
NR.sub.7R.sub.8 where R.sub.7 and R.sub.8 may be the same or
different and are independently selected from H; C.sub.1-C.sub.4
alkyl; a saturated heterocycle comprising N bonded directly to Y;
and an unsubstituted or substituted guanidine moiety; and wherein
in Formula II: R.sub.10 is selected from the group consisting of:
C.sub.3-C.sub.14 alkyl, C.sub.3-C.sub.14 alkoxyl; R.sub.11,
R.sub.12, R.sub.13 and R.sub.14 may be the same or different and
are independently selected from: H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; R.sub.15, R.sub.16, R.sub.17 and
R.sub.18 may be the same or different and are independently
selected from the group consisting of: C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; X is C.sub.1-C.sub.4 alkyl; Y is
C.sub.1-C.sub.4 alkyl; and W is NR.sup.19R.sup.20 where R.sup.19
and R.sup.20 may be the same or different and are selected from the
group consisting of: H, C.sub.1-C.sub.4 alkyl; a saturated
heterocycle comprising N bonded directly to Y, and an unsubstituted
or substituted guanidine moiety.
20. The method of claim 19, wherein the number of carbon atoms in
said saturated or unsaturated monocyclic ring with ring size from
3-7 is selected from the group consisting of 3, 4, 5, 6, and 7.
21. The method of claim 19, wherein said saturated heterocycle is
selected from the group consisting of morpholine, piperidine,
piperazine, and pyrrolidine.
22. The compound of claim 19, wherein said compound is selected
from
3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-di-
one and
3-(2-aminoethyl)-5-[3-(4-octyl-phenyl)-propylidene]-thiazolidine-2-
,4-dione.
23. The method of claim 19, wherein said cancer cells are of a type
selected from the group consisting of: leukemia, lymphoma, sarcoma,
neuroblastoma, lung cancer, skin cancer, head squamous cell
carcinoma, neck squamous cell carcinoma, prostate cancer, colon
cancer, breast cancer, ovarian cancer, cervical cancer, brain
cancer, bladder cancer, and pancreatic cancer.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention generally relates to
5-alkylidenethiazolidine-2,4-dione and
3-alkylidene-1,3-dihydro-indol-2-one analogs and their use as
selective sphingosine kinase 2 (SphK2) inhibitors in clinical
implications. In particular, the invention provides
5-alkylidenethiazolidine-2,4-dione and
3-alkylidene-1,3-dihydro-indol-2-one analogs and derivatives
thereof as SphK2 inhibitors and for use in the treatment of
cancer.
[0003] 2. Background of the Invention
[0004] Sphingosine-1-phosphate (S1P), a lipid metabolite, has been
recognized and demonstrated as an important signaling mediator for
vital cellular and physiological processes, such as cell motility,
invasion, proliferation, angiogenesis and apoptosis. S1P is
produced from sphingosine by two kinases, namely, sphingosine
kinase 1 (SphK1) and sphingosine kinase 2 (SphK2). Upon production,
S1P is secreted and interacts with a family of G-protein coupled
receptors (S1P.sub.1-5) on the cell surface to impart a plethora of
roles in the regulation of diverse physiological functions such as
inflammation, immunity and angiogenesis. Recently, intracellular
targets, such as histone deacetylase (HDAC) and TRAF2, have been
identified for S1P produced by SphK1 and SphK2, respectively, thus
suggesting additional intracellular roles of this sphingolipid
metabolite.
[0005] S1P and its biosynthetic precursors ceramide and sphingosine
are the best characterized bioactive metabolites of sphingolipids.
Ceramide and sphingosine have been associated with growth arrest
and apoptosis. In contrast, S1P has been demonstrated to play
important pro-survival roles. Therefore, the levels of these lipid
metabolites need to be tightly controlled and a so called
sphingolipid rheostat has been proposed to be crucial in
determining cell fate. The regulation of the levels of these
metabolites is complex and a number of enzymes have been
demonstrated to play important roles, among which the SphKs have
emerged as a central player in this complex system. SphKs are the
key enzymes that catalyze the production of S1P. To date, two
isoenzymes, SphK1 and SphK2 have been identified in human tissues.
Although SphK1 and SphK2 share a high degree of homology, they have
significant differences in size, tissue distribution, and
subcellular localization, thus suggesting their distinct roles in
regulation of different physiological functions. For example, SphK1
is mainly localized in the cytosol while SphK2 is present in
several intracellular compartments, mainly in the nucleus,
endoplasmic reticulum, and mitochondria. Evidence has accumulated
that SphK1 promotes cell growth and survival while the function of
SphK2 is complex and controversial. Consistent with this notion,
numerous studies have shown that SphK1 is frequently up-regulated
and overexpressed in tumor tissues compared to normal tissues and
SphK1 has been associated with many aspects of cancer progression
such as proliferation, migration, invasion and angiogenesis.
SphK1/S1P has also been implicated in the pathology of asthma,
inflammatory diseases and sepsis. Compared to SphK1, much less is
known about SphK2 and the results are contrasting. Initially, SphK2
had been demonstrated to be pro-apoptotic. For example,
overexpression of SphK2 suppresses growth and promotes apoptosis.
However, it has also been demonstrated that downregulation of SphK2
inhibits the proliferation and migration of tumor cells such as
glioblastoma and breast cancer cells. These controversial results
strongly suggest developing powerful and selective pharmacological
tools for SphK2 to better understand the roles of SphK2 in
different pathological conditions. Even though a number of pan-SphK
and selective SphK1 inhibitors have been developed and reported,
the development of SphK2-selective inhibitors remains limited and
SphK2 inhibitors remain scarce, with only a few SphK2 inhibitors
having been reported in the literature (e.g. ABC294620, SG-12,
R-FTY720-OMe and trans-12). Therefore, there is an urgent need to
develop SphK2-selective inhibitors.
SUMMARY OF THE INVENTION
[0006] Development of isoform selective SphK inhibitors has
attracted extensive attention as they may serve as valuable
pharmacological tools to help decipher the physiological and
pathological roles of SphKs and as effective therapeutic agents for
human diseases, such as cancer. Even though several pan SphK
inhibitors and SphK1 selective inhibitors have been developed and
tested in preclinical experiments, selective SphK2 inhibitors
remain scarce. During efforts to design and develop novel
5-alkylidene-thiazolidine-2,4-dione derivatives as dual-pathway
inhibitors of the Raf/MEK/ERK and PI3K/Akt signaling pathways, a
family of 4-substituted-phenylpropylidene-thiazolidine-2,4-dione
analogs were discovered to be highly selective SphK2 inhibitors. In
addition, a series of 3-alkylidene-1,3-dihydro-indol-2-one analogs
has been designed to replace the thiazolidine-2,4-dione heterocycle
and they also function as selective SphK2 inhibitors. Thus, these
compounds, depicted in generic Formula I and generic Formula II,
are selective SphK2 inhibitors and represent novel therapeutic
agents, such as anticancer agents. In the Formulas which are
depicted herein, the letters N, V, W, X, Y and Z represent atoms in
the structures of the compounds which may vary as described herein,
and do not represent elements such a vanadium, tungsten, etc.
##STR00001##
[0007] It is an object of this invention to provide a compound of
Formula I:
##STR00002##
In Formula I, R.sub.1 is selected from the group consisting of:
C.sub.3-C.sub.14 alkyl and C.sub.3-C.sub.14 alkoxyl; R.sub.2,
R.sub.3, R.sub.4 and R.sub.5 may be the same or different and are
independently selected from: H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; V is S, O, NH, or CH.sub.2; X is
C.sub.1-C.sub.4 alkyl; Y is C.sub.1-C.sub.4 alkyl; Z is S or O or
NR.sup.6 in which R.sup.6 is selected from the group consisting of:
H, C.sub.1-C.sub.8 alkyl, or isopropyl, or tert-butyl, or a
saturated or unsaturated monocyclic ring with ring size ranging
from 3-7 carbons per ring, or unsubstituted or substituted phenyl
ring which may be substituted with one or more substituents
selected from the group consisting of: C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; W is NR.sup.16R.sup.17 where
R.sup.16 and R.sup.17 may be the same or different and are H;
C.sub.1-C.sub.4 alkyl; a saturated heterocycle comprising N bonded
directly to Y; or an unsubstituted or substituted guanidine
moiety.
[0008] It is also an object of this invention to provide a compound
of Formula II:
##STR00003##
[0009] wherein, R.sub.10 is selected from the group consisting of:
C.sub.3-C.sub.14 alkyl, C.sub.3-C.sub.14 alkoxyl; R.sub.11,
R.sub.12, R.sub.13 and R.sub.14 may be the same or different and
are independently selected from: H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; R.sub.15, R.sub.16, R.sub.17 and
R.sub.18 may be the same or different and are independently
selected from the group consisting of: H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; X is C.sub.1-C.sub.4 alkyl; Y is
C.sub.1-C.sub.4 alkyl; and W is NR.sup.16R.sup.17 where R.sup.16
and R.sup.17 may be the same or different and are H;
C.sub.1-C.sub.4 alkyl; a saturated heterocycle comprising N bonded
directly to Y; or an unsubstituted or substituted guanidine
moiety.
[0010] The invention also provides methods of treating cancer in a
patient in need thereof. The method comprises the step of
administering to the patient a sufficient quantity of a compound of
at least one compound of Formula I:
##STR00004##
[0011] where, R.sub.1 is selected from the group consisting of:
C.sub.3-C.sub.14 alkyl and C.sub.3-C.sub.14 alkoxyl; R.sub.2,
R.sub.3, R.sub.4 and R.sub.5 may be the same or different and are
independently selected from: H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; V is S, O, NH, or CH.sub.2; X is
C.sub.1-C.sub.4 alkyl; Y is C.sub.1-C.sub.4 alkyl; Z is S or O or
NR.sup.6 in which R.sup.6 is selected from the group consisting of:
H, C.sub.1-C.sub.8 alkyl, or isopropyl, or tert-butyl, or a
saturated or unsaturated monocyclic ring with ring size ranging
from 3-7 carbons per ring, or unsubstituted or substituted phenyl
ring which may be substituted with one or more substituents
selected from the group consisting of: C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; and W is NR.sup.16R.sup.17 where
R.sup.16 and R.sup.17 may be the same or different and are H;
C.sub.1-C.sub.4 alkyl; a saturated heterocycle comprising N bonded
directly to Y; or an unsubstituted or substituted guanidine
moiety.
[0012] In one embodiment of the method, the number of carbon atoms
in the alkoxyl substituent of R.sub.1 is 4, 7, or 8. In other
embodiments, W is NH.sub.2. The compound may be, for example,
3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-di-
one (Formula III, also referred to herein as "K145" or "compound
(30)") or
3-(2-aminoethyl)-5-[3-(4-octoxy-phenyl)-propylidene]-thiazolidine-2,4-dio-
ne (Formula IV, also referred to herein as "KLlll16" or "KLlll016"
or "compound (31)").
##STR00005##
[0013] The invention also provides methods of treating cancer in a
patient in need thereof. The method comprises the step of
administering to the patient a quantity of at least one compound of
Formula II sufficient to cure or ameliorate cancer symptoms:
##STR00006##
[0014] where, R.sub.10 is selected from the group consisting of:
C.sub.3-C.sub.14 alkyl, C.sub.3-C.sub.14 alkoxyl; R.sub.11,
R.sub.12, R.sub.13 and R.sub.14 may be the same or different and
are independently selected from: H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; R.sub.15, R.sub.16, R.sub.17 and
R.sub.18 may be the same or different and are independently
selected from the group consisting of: H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; X is C.sub.1-C.sub.4 alkyl; Y is
C.sub.1-C.sub.4 alkyl; and W is NR.sup.16R.sup.17 where R.sup.16
and R.sup.17 may be the same or different and are H;
C.sub.1-C.sub.4 alkyl; a saturated heterocycle comprising N bonded
directly to Y; or an unsubstituted or substituted guanidine
moiety.
[0015] The invention further provides a method of inhibiting the
growth or killing or damaging of human cancer cells. The method
comprises the step of exposing the cell to at least one compound of
Formula I and/or Formula II:
##STR00007##
[0016] In Formula I, R.sub.1 is selected from the group consisting
of: C.sub.3-C.sub.14 alkyl and C.sub.3-C.sub.14 alkoxyl; R.sub.2,
R.sub.3, R.sub.4 and R.sub.5 may be the same or different and are
independently selected from: H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; V is S, O, NH, or CH.sub.2; X is
C.sub.1-C.sub.4 alkyl; Y is C.sub.1-C.sub.4 alkyl; Z is S or O or
NR.sup.6 in which R.sup.6 is selected from the group consisting of:
H, C.sub.1-C.sub.8 alkyl, or isopropyl, or tert-butyl, or a
saturated or unsaturated monocyclic ring with ring size ranging
from 3-7 carbons per ring, or unsubstituted or substituted phenyl
ring which may be substituted with one or more substituents
selected from the group consisting of: C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; and W is NR.sup.16R.sup.17 where
R.sup.16 and R.sup.17 may be the same or different and are H;
C.sub.1-C.sub.4 alkyl; a saturated heterocycle comprising N bonded
directly to Y; or an unsubstituted or substituted guanidine
moiety.
[0017] In Formula II, R.sub.10 is selected from the group
consisting of: C.sub.3-C.sub.14 alkyl, C.sub.3-C.sub.14 alkoxyl;
R.sub.11, R.sub.12, R.sub.13 and R.sub.14 may be the same or
different and are independently selected from: H, C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl,
halogen, hydroxyl, amino, nitro, and cyano; R.sub.15, R.sub.16,
R.sub.17 and R.sub.18 may be the same or different and are
independently selected from the group consisting of: H,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8
alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; X is
C.sub.1-C.sub.4 alkyl; Y is C.sub.1-C.sub.4 alkyl; and W is
NR.sup.16R.sup.17 where R.sup.16 and R.sup.17 may be the same or
different and are H; C.sub.1-C.sub.4 alkyl; a saturated heterocycle
comprising N bonded directly to Y; or an unsubstituted or
substituted guanidine moiety.
[0018] In one embodiment of the method, the number of carbon atoms
in the alkoxyl substituent of R.sub.1 is 4, 7, or 8. In other
embodiments, W is NH.sub.2. The compound may be, for example,
3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-di-
one (Formula III) or
3-(2-aminoethyl)-5-[3-(4-octoxy-phenyl)-propylidene]-thiazolidine-2,4-dio-
ne (Formula IV).
[0019] In yet another embodiment of the method, the cell that is
exposed to the compound is a cancer cell. In some embodiments, the
cancer cells are leukemia, lymphoma, sarcoma, neuroblastoma, lung
cancer, skin cancer, head squamous cell carcinoma, neck squamous
cell carcinoma, prostate cancer, colon cancer, breast cancer,
ovarian cancer, cervical cancer, brain cancer, bladder cancer,
and/or pancreatic cancer cells.
[0020] The invention also provides a method of inhibiting SphK2.
The method comprises the step of exposing the kinase enzyme to a
compound of Formula I or Formula II:
##STR00008##
[0021] In Formula I, R.sub.1 is selected from the group consisting
of: C.sub.3-C.sub.14 alkyl and C.sub.3-C.sub.14 alkoxyl; R.sub.2,
R.sub.3, R.sub.4 and R.sub.5 may be the same or different and are
independently selected from: H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; V is S, O, NH, or CH.sub.2; X is
C.sub.1-C.sub.4 alkyl; Y is C.sub.1-C.sub.4 alkyl; Z is S or O or
NR.sup.6 in which R.sup.6 is selected from the group consisting of:
H, C.sub.1-C.sub.8 alkyl, or isopropyl, or tert-butyl, or a
saturated or unsaturated monocyclic ring with ring size ranging
from 3-7 carbons per ring, or unsubstituted or substituted phenyl
ring which may be substituted with one or more substituents
selected from the group consisting of: C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl, halogen,
hydroxyl, amino, nitro, and cyano; and W is NR.sup.16R.sup.17 where
R.sup.16 and R.sup.17 may be the same or different and are H;
C.sub.1-C.sub.4 alkyl; a saturated heterocycle comprising N bonded
directly to Y; or an unsubstituted or substituted guanidine
moiety.
[0022] In Formula II, R.sub.10 is selected from the group
consisting of: C.sub.3-C.sub.14 alkyl, C.sub.3-C.sub.14 alkoxyl;
R.sub.11, R.sub.12, R.sub.13 and R.sub.14 may be the same or
different and are independently selected from: H, C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl,
halogen, hydroxyl, amino, nitro, and cyano; R.sub.15, R.sub.16,
R.sub.17 and R.sub.18 may be the same or different and are
independently selected from the group consisting of: H,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8
alkylcarbonyl, halogen, hydroxyl, amino, nitro, and cyano; X is
C.sub.1-C.sub.4 alkyl; Y is C.sub.1-C.sub.4 alkyl; and W is
NR.sup.16R.sup.17 where R.sup.16 and R.sup.17 may be the same or
different and are H; C.sub.1-C.sub.4 alkyl; a saturated heterocycle
comprising N bonded directly to Y; or an unsubstituted or
substituted guanidine moiety.
[0023] In one embodiment of the method, the number of carbon atoms
in the alkoxyl substituent of R.sub.1 is 4, 7, or 8. In other
embodiments, W is NH.sub.2. The compound may be, for example,
3-(2-aminoethyl)-5-[3-
(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (Formula
III) or
3-(2-aminoethyl)-5-[3-(4-octoxy-phenyl)-propylidene]-thiazolidine-2,4-dio-
ne (Formula IV).
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1A-B. K145 inhibits SphK2 but not SphK1. A) SphK1 and
SphK2 activities were measured with 5 .mu.M sphingosine in the
absence or presence of the indicated concentrations of K145 or 10
.mu.M DMS. Data are expressed as percentage SphK activity in the
absence of inhibitor; B) Effect of K145 (10 .mu.M) on activity of
the indicated enzymes was tested by SelectScreen Kinase Profiling
from Invitrogen. CaMKll.beta., Ca2+/calmodulin-dependent protein
kinase II; CDK2, cyclin-dependent kinase 2; EGFR, Epidermal Growth
Factor Receptor; Fyn, Fyn Kinase (p55); ERK2, extracellular
signal-regulated kinase 2; ERK1, extracellular signal-regulated
kinase 1; PKA, protein kinase A; PKC.alpha., protein kinase
C.alpha.; P13K, phosphatidylinositide 3-kinase. Data are expressed
as percentage of control activity averaged from 2 independent
experiments. Data are expressed as mean value .+-.SEM.
[0025] FIG. 2. Compound KLIII16 selectively inhibits SphK2 with a
IC.sub.50 of 1.9 .mu.M
[0026] FIG. 3. SphK2 was incubated with indicated compounds at 10
.mu.M, then the activity of SphK2 was determined. The data shown
here is a relative activity of SphK2 compared to control (no
inhibitor, column labeled Sph in the Figure). 96047 is a known SpK2
inhibitor. A5, B26, K25 and QLA13 are known compounds [Li et al.
(2009) Bioorg Med Chem Lett 19: 6042-6046; Liu et al. (2012) Eur J
Med Chem 47: 125-137] and were tested for purposes of comparison.
"A" and "B" refer to different batches of a compound.
[0027] FIG. 4. SphK1 was incubated with indicated compounds at 10
.mu.M, then the activity of SphK1 was determined. The data shown
here is a relative activity of SphK1 compared to control (no
inhibitor, column labeled Sph in the Figure). 96091 is a known Spk1
inhibitor. A5, B26, K25 and QLA13 are as above for FIG. 4. "A" and
"B" refer to different batches of a compound.
[0028] FIG. 5. SphK2 was incubated with indicated compounds at 10
.mu.M, then the activity of SphK2 was determined. The data shown
here is a relative activity of SphK2 compared to control (no
inhibitor).
[0029] FIG. 6. SphK1 was incubated with indicated compounds at 10
.mu.M, then the activity of SphK2 was determined. The data shown
here is a relative activity of SphK2 compared to control (no
inhibitor).
[0030] FIG. 7. Kinetic studies of Formula III (K145) in SphK2 and
Lineweaver-Burk plot. SphK2 activity was measured with increasing
concentrations of sphingosine and the indicated concentrations of
K145. Lineweaver-Burk analysis revealed a Vmax of 10820.+-.210
pmol/min per mg of protein, and a K., of 6.4.+-.0.7 .mu.M for
SphK2.
[0031] FIG. 8. Compound K145 dose-dependently inhibits the growth
of human leukemia U937 cells.
[0032] FIG. 9. Compound K145 inhibits the Raf/MEK/ERK and PI3K/Akt
signaling pathways.
[0033] FIG. 10. Compound S-II-71 and S-II-103 inhibit the
Raf/MEK/ERK and PI3K/Akt signaling pathways
[0034] FIG. 11. K145 induces apoptosis in U937 cells in dose- and
time-dependent manner.
[0035] FIG. 12. S-II-71 and S-II-103 induce apoptosis in U937 cells
in dose-dependent manner after 24 hrs treatment.
[0036] FIG. 13A-D. K145 suppresses the growth of U937 xenograft in
nude mice. BALB/c-nu mice (n=7) with palpable U937 xenograft were
treated daily with vehicle, tamibarotene (15 mg/kg), or K145 (15
mg/kg) for 17 days by i.p. injection. A) After treatment, animals
were sacrificed and tumors were removed and weighed and the TGI was
calculated; B) Tumor volumes were measured every other day during
the treatment course; C) Animal weights were measured every other
day during treatment course. D) Body weights of mice throughout
treatment. Data are expressed as mean value .+-.SD. *P<0.05
compared to control group.
[0037] FIG. 14A-E. K145 suppresses the growth of JC xenograft in
BALB/c mice. BALB/c mice (n=8) with palpable JC xenograft were
treated daily with vehicle or K145 (20 mg/kg and 35 mg/kg) for 15
days by i.p. injection. A) Tumor volumes were measured every other
day; B) After treatment, animals were sacrificed and tumors were
removed and weighed; C) The S1P and K145 levels in the tumor
samples from vehicle and treatment (20 mg/kg) groups (n=4) were
measured by ESI-MS/MS; D) Images of tumor samples from control and
treatment groups (n=7 for each group) after the experiments; E)
Tumor samples (20 mg/kg and control groups) were analyzed by
Western blot. Data are expressed as mean value .+-.SEM. *P<0.05
compared to control group.
[0038] FIG. 15A-D. K145 suppresses the growth of U937 tumors in
nude mice by oral administration. BALB/c-nu mice (n=7) with
palpable U937 xenograft were treated daily with vehicle,
tamibarotene (20 mg/kg), or K145 (50 mg/kg) for 15 days by oral
gavage. After treatment, animals were sacrificed and tumors were
removed, weighed and images were taken. A) Tumor weight and TGI
comparison; B) Images of tumor samples from control and treatment
groups (n=7 for each group) after the experiments; C) Tumor volumes
were measured every other day; D) Animal weights were measured
every other day. Data are expressed as mean value .+-.SEM.
*P<0.05 compared to control group.
[0039] FIG. 16A-E. K145 accumulates and suppresses the S1P level. A
and B) U937 cells were treated with K145 at the indicated
concentrations for 3 h and the levels of K145 and S1P were measured
by ESI-MS/MS. C) HEK293 cells were treated with K145 (10 .mu.M) for
2 h. Lipids were extracted and different chain length species of
ceramide were determined by LC-ESI-MS/MS. Numbers indicate chain
length followed by the number of double bonds in the fatty acid.
Data are averages of triplicate determinations and are expressed as
pmol lipid/10.sup.6 cells. D) U937 cells were treated with or
without K145 (10 .mu.M) for 3 h and levels of C1P species were
determined by ESI-MS/MS. E) U937 cells were treated with FTY720 (1
.mu.M) in the absence or presence of indicated K145 for 3 h, then
FTY720-P was measured by ESI-MS/MS. *P<0.05 compared to
control.
DETAILED DESCRIPTION
[0040] The invention provides compounds of the following Formula I
and Formula II:
##STR00009##
In Formula I:
[0041] R.sub.1 is selected from the group consisting of:
C.sub.3-C.sub.14 alkyl and C.sub.3-C.sub.14 alkoxyl;
[0042] R.sub.2, R.sub.3, R.sub.4 and R.sub.5 may be the same or
different and are independently selected from: H, C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl,
halogen, hydroxyl, amino, nitro, and cyano;
[0043] V is S, O, NH, or CH.sub.2;
[0044] X is C.sub.1-C.sub.4 alkyl;
[0045] Y is C.sub.1-C.sub.4 alkyl;
[0046] Z is S or O or NR.sup.6 in which R.sup.6 is selected from
the group consisting of: H, C.sub.1-C.sub.8 alkyl, or isopropyl, or
tert-butyl, a saturated or unsaturated monocyclic ring with ring
size ranging from 3-7 carbons per ring, an unsubstituted or
substituted phenyl ring which may be substituted with one or more
substituents selected from the group consisting of: C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl,
halogen, hydroxyl, amino, nitro, and cyano; and
[0047] W is NR.sub.7R.sub.8 where R.sub.7 and R.sub.8 may be the
same or different and are independently selected from H;
C.sub.1-C.sub.4 alkyl; a saturated heterocycle comprising N bonded
directly to Y; and an unsubstituted or substituted guanidine
moiety.
##STR00010##
[0048] In Formula II:
[0049] R.sub.10 is selected from the group consisting of:
C.sub.3-C.sub.14 alkyl, C.sub.3-C.sub.14 alkoxyl;
[0050] R.sub.11, R.sub.12, R.sub.13 and R.sub.14 may be the same or
different and are independently selected from: H, C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkoxyl, C.sub.1-C.sub.8 alkylcarbonyl,
halogen, hydroxyl, amino, nitro, and cyano;
[0051] R.sub.15, R.sub.16, R.sub.17 and R.sub.18 may be the same or
different and are independently selected from the group consisting
of: H, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkoxyl,
C.sub.1-C.sub.8 alkylcarbonyl, halogen, hydroxyl, amino, nitro, and
cyano;
[0052] X is C.sub.1-C.sub.4 alkyl;
[0053] Y is C.sub.1-C.sub.4 alkyl;
[0054] and
[0055] W is NR.sup.16R.sup.17 where R.sup.16 and R.sup.17 may be
the same or different and are H; C.sub.1-C.sub.4 alkyl; a saturated
heterocycle comprising N bonded directly to Y; or an unsubstituted
or substituted guanidine moiety.
[0056] By "saturated heterocycle" we mean a saturated monocyclic
carbon ring containing at least one heteroatom atom N as part of
the ring. The monocyclic ring is fully saturated (i.e. it does not
contain any carbon-carbon double or triple bonds). In addition to N
bonded directly to Y, one or more additional positions in the
ring(s) may be substituted by other heteroatoms, examples of which
include but are not limited to: N, O, S, etc. Exemplary saturated
heterocycles that may be used in the practice of the invention
include but are not limited to morpholine, piperidine, piperazine,
pyrrolidine, etc.
[0057] By "saturated or unsaturated monocyclic ring" we mean a
fully saturated monocyclic carbon ring (i.e. it does not contain
any carbon-carbon double or triple bonds) without or with at least
one heteroatom, examples of which include but are not limited to:
one or more N, O, S, etc; as part of the ring. Unsaturated
monocyclic ring means a monocyclic carbon ring containing one or
more carbon-carbon or carbon-heteroatom double or triple bonds)
without or with at least one heteroatom, examples of which include
but are not limited to: one ore more N, O, S, etc; as part of the
ring.
[0058] C.sub.1-C.sub.4 alkyl includes include substituents with 1,
2, 3, or 4 carbon atoms and may be unbranched or branched isomeric
forms thereof, e.g. methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, sec-butyl, tert-butyl; C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxyl, and C.sub.1-C.sub.8 alkylcarbonyl include
substitutents with 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms in the
alkyl portion of the molecule (which may be unbranched or branched
isomeric forms thereof), e.g. they comprise methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,
isopentyl, neopentyl, hexyl, 2-methylpentane, 3-methylpentane,
2,3-dimethylbutane, 2,2-dimethylbutane and various 8-carbon octyl
component and branched isomers thereof, etc., as understood in the
art.
[0059] In one embodiment of the invention, the compound of Formula
I is the compound
3-(2-aminoethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-di-
one shown in Formula III. In another embodiment of the invention,
the compound of Formula I is
3-(2-aminoethyl)-5-[3-(4-octoxy-phenyl)-propylidene]-thiazolidine-2,4-dio-
ne shown in Formula IV. The compounds of Formulas III and IV
inhibit SphK2 selectively, i.e. they do not inhibit SphK1, or they
inhibit SphK2 to a greater extent, e.g. inhibition of SphK2 is at
least about 2 fold, and usually about 5, 10, 20, 30, 40, 50, 60,
70, 80, 90 or 100-fold or more greater than is inhibition of
SphK1.
##STR00011##
[0060] The synthesis and structural characterization of the
compounds represented by Formula III and Formula IV are described
in Examples 3, 10 and 15.
[0061] Other compounds that inhibited SphK2 include: S-11-73,
compound (61); KL11016, compound (31); and KL111023, compound (33);
(see FIGS. 3 and 4 for activity and Example 8 for structures of
(61), (31), (33); and KL11139, compound (34)]; KL1147, compound
(35); KL157, compound (32); S-11-103, compound (66); and S-11-104,
compound (70); (see FIGS. 5 and 6 for activity and Example 8 for
structures of (34), (35), (32), (66), (70)). These compounds
inhibited SphK2 selectively, compared to Sphk1, i.e. they did not
inhibit SphK1 or inhibited SphK1 to a lesser extent as described
above.
[0062] Compound K11167 [compound (42) herein] inhibits both Sphk1
and Sphk2 (see FIGS. 5 and 6 for activity and Example 8 for the
structure of compound (42)), and may be used for the non-selective
inhibition of one or both of the enzymes.
[0063] The invention also provides compositions for the treatment
of diseases or conditions associated with the over-activation or
over-expression of SphK2. In particular, the invention provides
compositions for the treatment of various cancers. The compositions
comprise at least one compound of Formula I and/or at least one
compound of Formula II and a pharmaceutically acceptable (i.e. a
physiologically compatible) carrier, e.g. saline, pH in the range
of about 6.5 to about 7.5, and usually about 7.2). Depending on the
route of administration, the compositions can take the form of
liquids suitable for injection or intravenous administration,
aerosols, cachets, capsules, creams, elixirs, emulsions, foams,
gels, granules, inhalants, liposomes, lotions, magmas,
microemulsion, microparticles, ointments, peroral solids, powders,
sprays, syrups, suppositories, suspensions, tablets and tinctures.
The amount of the compound of Formula 1 and/or Formula II present
in the composition can vary, but us usually in the range of from
about 1 to 99%.
[0064] The compositions may include one or more pharmaceutically
compatible additives or excipients. Commonly used pharmaceutical
additives and excipients which can be used as appropriate to
formulate the composition for its intended route of administration
include but are not limited to:
acidifying agents (examples include but are not limited to acetic
acid, citric acid, fumaric acid, hydrochloric acid, nitric acid);
alkalinizing agents (examples include but are not limited to
ammonia solution, ammonium carbonate, diethanolamine,
monoethanolamine, potassium hydroxide, sodium borate, sodium
carbonate, sodium hydroxide, triethanolamine, trolamine);
adsorbents (examples include but are not limited to powdered
cellulose and activated charcoal); aerosol propellants (examples
include but are not limited to carbon dioxide, CCl.sub.2F.sub.2,
F.sub.2ClC--CClF.sub.2 and CClF.sub.3); air displacement agents
(examples include but are not limited to nitrogen and argon);
antifungal preservatives (examples include but are not limited to
benzoic acid, butylparaben, ethylparaben, methylparaben,
propylparaben, sodium benzoate, propionic acids or its salts);
antimicrobial preservatives (examples include but are not limited
to benzalkonium chloride, benzethonium chloride, benzyl alcohol,
cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl
alcohol, phenylmercuric nitrate and thimerosal); antioxidants
(examples include but are not limited to ascorbic acid, ascorbyl
palmitate, butylated hydroxyanisole, butylated hydroxytoluene,
hypophosphorus acid, monothioglycerol, propyl gallate, sodium
ascorbate, sodium bisulfate, sodium formaldehyde sulfoxylate,
sodium metabisulfite, tocopherol, and vitamin E); binding materials
(examples include but are not limited to block polymers, natural
and synthetic rubber, polyacrylates, polyurethanes, silicones and
styrene-butadiene copolymers); buffering agents (examples include
but are not limited to potassium metaphosphate, potassium phosphate
monobasic, sodium acetate, sodium citrate anhydrous and sodium
citrate dihydrate); carrying agents (examples include but are not
limited to acacia syrup, aromatic syrup, aromatic elixir, cherry
syrup, cocoa syrup, orange syrup, syrup, corn oil, mineral oil,
peanut oil, sesame oil, bacteriostatic sodium chloride injection
and bacteriostatic water for injection); chelating agents (examples
include but are not limited to edetate disodium and edetic acid);
colorants (examples include but are not limited to FD&C Red No.
3, FD&C Red No. 20, FD&C Yellow No. 6, FD&C Blue No. 2,
D&C Green No. 5, D&C Orange No. 5, D&C Red No. 8,
caramel, ferric oxide red, natural colorants such as bixin,
norbixin, and carmine); clarifying agents (examples include but are
not limited to bentonite); emulsifying agents (examples include but
are not limited to acacia, cetomacrogol, cetyl alcohol, glyceryl
monostearate, lecithin, sorbitan monooleate, and polyethylene 50
stearate); encapsulating agents (examples include but are not
limited to gelatin and cellulose acetate phthalate); fillers
(examples include but are not limited to sugars, lactose, sucrose,
sorbitol, cellulose preparations, calcium phosphates, natural or
synthetic gums, solid starch, and starch pastes); flavorants
(examples include but are not limited to anise oil, cinnamon oil,
cocoa, menthol, orange oil, peppermint oil and vanillin);
humectants (examples include but are not limited to glycerin,
propylene glycol and sorbitol); levigating agents (examples include
but are not limited to mineral oil and glycerin); oils (examples
include but are not limited to arachis oil, mineral oil, olive oil,
peanut oil, sesame oil and vegetable oil); ointment bases (examples
include but are not limited to lanolin, hydrophilic ointment,
polyethylene glycol ointment, petrolatum, hydrophilic petrolatum,
white ointment, yellow ointment, and rose water ointment);
penetration enhancers (transdermal delivery) (examples include but
are not limited to monohydroxy or polyhydroxy alcohols, saturated
or unsaturated fatty alcohols, saturated or unsaturated fatty
esters, saturated or unsaturated dicarboxylic acids, essential
oils, phosphatidyl derivatives, cephalin, terpenes, amides, ethers,
ketones and ureas); plasticizers (examples include but are not
limited to diethyl phthalate and glycerin); solvents (examples
include but are not limited to alcohol, corn oil, cottonseed oil,
glycerin, isopropyl alcohol, mineral oil, oleic acid, peanut oil,
purified water, water for injection, sterile water for injection
and sterile water for irrigation); stiffening agents (examples
include but are not limited to cetyl alcohol, cetyl esters wax,
microcrystalline wax, paraffin, stearyl alcohol, white wax and
yellow wax); suppository bases (examples include but are not
limited to cocoa butter and polyethylene glycols (PEGS) (and
mixtures containing one or both of cocoa butter and PEGs));
surfactants (examples include but are not limited to benzalkonium
chloride, nonoxynol 10, oxtoxynol 9, polysorbate 80, sodium lauryl
sulfate and sorbitan monopalmitate); suspending agents (examples
include but are not limited to agar, bentonite, carbomers,
carboxymethylcellulose sodium, hydroxyethyl cellulose,
hydroxypropyl cellulose, hydroxypropyl methylcellulose, kaolin,
methylcellulose, tragacanth and veegum); sweetening agents
(examples include but are not limited to aspartame, dextrose,
fructose, glycerin, mannitol, propylene glycol, saccharin sodium,
sorbitol and sucrose); tablet anti-adherents (examples include but
are not limited to magnesium stearate and talc); tablet binders
(examples include but are not limited to acacia, alginic acid,
carboxymethylcellulose sodium, compressible sugar, ethylcellulose,
gelatin, liquid glucose, methylcellulose, povidone and
pregelatinized starch); tablet and capsule diluents (examples
include but are not limited to dibasic calcium phosphate, kaolin,
lactose, mannitol, microcrystalline cellulose, powdered cellulose,
precipitated calcium carbonate, sodium carbonate, sodium phosphate,
sorbitol and starch); tablet coating agents (examples include but
are not limited to liquid glucose, hydroxyethyl cellulose,
hydroxypropyl cellulose, hydroxypropyl methylcellulose,
methylcellulose, ethylcellulose, cellulose acetate phthalate and
shellac); tablet direct compression excipients (examples include
but are not limited to dibasic calcium phosphate); tablet
disintegrants (examples include but are not limited to alginic
acid, carboxymethylcellulose calcium, microcrystalline cellulose,
polacrillin potassium, sodium alginate, sodium starch glycollate
and starch); tablet glidants (examples include but are not limited
to colloidal silica, corn starch and talc); tablet lubricants
(examples include but are not limited to calcium stearate,
magnesium stearate, mineral oil, stearic acid and zinc stearate);
tablet/capsule opaquants (examples include but are not limited to
titanium dioxide); tablet polishing agents (examples include but
are not limited to carnuba wax and white wax); thickening agents
(examples include but are not limited to beewax, cetyl alcohol and
paraffin); tonicity agents (examples include but are not limited to
dextrose and sodium chloride); viscosity increasing agents
(examples include but are not limited to alginic acid, bentonite,
carbomers, carboxymethylcellulose sodium, methylcellulose,
povidone, sodium alginate and tragacanth); and wetting agents
(examples include but are not limited to heptadecaethylene
oxycetanol, lecithins, polyethylene sorbitol monooleate,
polyoxyethylene sorbitol monooleate, polyoxyethylene stearate).
[0065] Additional additives and excipients suitable for
pharmaceutical use such as those described in Remington's The
Science and Practice of Pharmacy, 21.sup.st Edition (2005), Goodman
& Gilman's The Pharmacological Basis of Therapeutics, 11.sup.th
Edition (2005) and Ansel's Pharmaceutical Dosage Forms and Drug
Delivery Systems (8.sup.th Edition), edited by Allen et al.,
Lippincott Williams & Wilkins, (2005) are also considered to be
within the scope of the invention. The complete contents of these
references are herein incorporated by reference in entirety.
[0066] In one embodiment of the compositions of the invention, one
or more (i.e. at least one) additional anti-cancer agent can be
added to the composition. Representative anti-cancer agents
include, but are not limited to, Erbitux, methotrexate, taxol,
mercaptopurine, thioguanine, hydroxyurea, cytarabine,
cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin,
mitomycin, dacarbazine, procarbizine, etoposides, campathecins,
bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin,
plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine,
vinorelbine, paclitaxel, and docetaxel, .gamma.-radiation,
alkylating agents including nitrogen mustard such as
cyclophosphamide, ifosfamide, trofosfamide, chlorambucil,
nitrosoureas such as carmustine (BCNU), and lomustine (CCNU),
alkylsulphonates such as busulfan, and treosulfan, triazenes such
as dacarbazine, platinum containing compounds such as cisplatin and
carboplatin, plant alkaloids including vinca alkaloids,
vincristine, vinblastine, vindesine, and vinorelbine, taxoids
including paclitaxel, and docetaxol, DNA topoisomerase inhibitors
including epipodophyllins such as etoposide, teniposide, topotecan,
9-aminocamptothecin, campto irinotecan, and crisnatol, mitomycins
such as mitomycin C, anti-metabolites, including anti-folates such
as DHFR inhibitors, methotrexate and trimetrexate, IMP
dehydrogenase inhibitors including mycophenolic acid, tiazofurin,
ribavirin, EICAR, ribonucleotide reductase inhibitors such as
hydroxyurea, deferoxamine, pyrimidine analogs including uracil
analogs 5-fluorouracil, floxuridine, doxifluridine, and ratitrexed,
cytosine analogs such as cytarabine (ara C), cytosine arabinoside,
and fludarabine, purine analogs such as mercaptopurine,
thioguanine, hormonal therapies including receptor antagonists, the
anti-estrogens tamoxifen, raloxifene and megestrol, LHRH agonists
such as goscrclin, and leuprolide acetate, anti-androgens such as
flutamide, and bicalutamide, retinoids/deltoids, Vitamin D3 analogs
including EB 1089, CB 1093, and KH 1060, photodyamic therapies
including vertoporfin (BPD-MA), phthalocyanine, photosensitizer
Pc4, Demethoxy-hypocrellin A, (2BA-2-DMHA), cytokines including
Interferon, .alpha.-Interferon, .gamma.-interferon, tumor necrosis
factor, as well as other compounds having anti-tumor activity
including isoprenylation inhibitors such as lovastatin,
dopaminergic neurotoxins such as 1-methyl-4-phenylpyridinium ion,
cell cycle inhibitors such as staurosporine, alsterpaullone,
butyrolactone I, Cdk2 inhibitor, Cdk2/Cyclin Inhibitory Peptide I,
Cdk2/Cyclin Inhibitory Peptide II, Compound 52
[2-(2-hydroxyethylamino)-6-(3-chloroanilino)-9-isopropylpurine],
Indirubin-3'-monoxime, Kenpaullone, Olomoucine, Iso-olomoucine,
N.sup.9-isopropyl-olomoucine, Purvalanol A, Roscovitine, (5)-isomer
Roscovitine and WHI-P180
[4-(3'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, actinomycins
such as actinomycin D and dactinomycin, bleomycins such as
bleomycin A2, bleomycin B2, and peplomycin, anthracyclines such as
daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin,
pirarubicin, zorubicin, and mitoxantrone, MDR inhibitors including
verapamil, and Ca.sup.2+ ATPase inhibitors such as
thapsigargin.
[0067] In addition, the compounds or compositions of the invention
may be administered in conjunction with other health-related and/or
cancer treating substances or protocols, including but not limited
to: dietary modifications (e.g. vitamin or antioxidant therapy);
pain medication or procedures to lessen pain; radiation; various
forms of chemotherapy (e.g. administration of platinum drugs, etc.;
surgery; cryotherapy; and medications to lessen nausea, etc.).
[0068] The invention also provides methods of treating cancer in a
patient in need thereof. The methods comprise a step of
administering, to the patient, an effective amount of one or more
compounds of Formulas I and/or II, e.g. as a composition comprising
the compound(s). Methods of administration include but are not
limited to intradermal, intramuscular, intraperitoneal, intravenous
(IV), intratumoral, subcutaneous, intranasal, epidural, oral,
sublingual, intranasal, intracerebral, intravaginal, transdermal,
rectally, by inhalation, or topically, particularly to the ears,
nose, eyes, or skin. Frequently, administration will be IV,
although the mode of administration is left to the discretion of
the skilled practitioner (e.g. a physician). In most instances,
administration will result in the release of a compound of the
invention into the bloodstream. However, this need not always be
the case, e.g. with topical or intratumoral administration.
Further, modes of administration may be combined, e.g. intravenous
and intratumoral administration may both be carried out in a
patient.
[0069] The amount of the compound(s) of Formulas I and/or II that
is administered in one administration is generally in the range of
from about 0.1 to about 10 mg/kg of body weight of the patient, and
is usually in the range of from about 0.1 to about 10 mg/kg, with a
goal of achieving levels of from about 1 to about 5 .mu.M in the
blood stream. Those of skill in the art will recognize that
administration may be carried out according to any of several
protocols, and will generally be determined by a skilled
practitioner such as a physician. For example, administration may
be once per day, several times per day, or less frequent (e.g.
weekly, biweekly, etc.). The amount of the compound that is
administered and the frequency of administration may depend on
several factors, e.g. the characteristics of the patient (weight,
age, gender, overall state of health, etc.); the type and stage of
the cancer being treated; the response of the patient to the
treatment; etc.
[0070] By "an effective amount" we mean an amount that is
sufficient to ameliorate, lessen or eliminate symptoms of the
disease that is being treated. While in some cases, the patient may
be completely "cured" (disease symptoms disappear entirely), this
need not always be the case. Those of skill in the art will
recognize that substantial benefits may accrue if disease symptoms
are only partially mitigated, or if the progress of the disease is
slowed. For example, when treating cancer, substantial benefits re
quality of life and longevity are obtained by slowing or arresting
the growth of a tumor and/or preventing metastasis, shrinking
(decreasing) the size of a tumor, etc. even if the tumor itself is
not entirely destroyed by exposure to the compounds described
herein. In some cases, the cancer cells which are exposed to the
compounds of the invention are killed; in other embodiments, the
cancer cells are damaged, e.g. prevented from growing or rendered
incapable of cell division, etc.
[0071] Types of cancer that can be treated using the compounds and
methods described herein include but are not limited to: leukemia,
lymphoma, sarcoma, neuroblastoma, lung cancer, skin cancer,
squamous cell carcinoma of the head and neck, prostate cancer,
colon cancer, breast cancer, ovarian cancer, cervical cancer, brain
cancer, bladder cancer, pancreatic cancer. The cancer may be at any
stage of development, and pre-cancerous cells may also be
treated.
[0072] The patient or subject that is treated in this manner is
usually a mammal, although this is not always the case. Frequently,
the mammal is a human, although the methods may also be applied to
the treatment of other animals, e.g. in veterinary practice.
[0073] The invention also provides methods of inhibiting SphK2 and
downstream signaling pathways in a cell. In some embodiments,
inhibition of SphK2 is selective, e.g. the compound inhibits SphK2
but not other enzymes, for example, SphK1. In this embodiment,
compound K11167 is excluded since it inhibits both SphK2 and SphK1.
The methods involve exposing the cells to one or more compounds of
the invention, the one or more compounds being present in an amount
that is sufficient to inhibit the enzyme, SphK2, usually by at
least 50%, in some cases by 60%, 70%, 80%, 90%, 95% or more, or
even completely (i.e. 100% inhibition), compared to an untreated
control. Those of skill in the art are familiar with methods to
measure levels of inhibition of SphK2, e.g. by detecting the amount
of a metabolite of SphK2 substrate or compound that participates in
the downstream signaling pathway or that is made by or in the
pathway, e.g. by measuring an amount or degree of mRNA or protein
expression, or the amount of protein modification (e.g.
phosphorylation or de-phosphorylation), etc. In some cases, the
cells in which these pathways are inhibited are cancer cells.
[0074] The invention also provides methods of inhibiting SphK2. In
some embodiments, inhibition of SphK2 is selective, e.g. the
compound inhibits SphK2 but not other enzymes, for example, SphK1.
In this embodiment, compound Klll67 is excluded since it inhibits
both SphK2 and SphK1 (see FIGS. 5 and 6). The methods of the
invention involve bringing the enzyme into contact with one or more
compounds of the invention, e.g. by contacting, exposing or
otherwise providing access of the compound(s) to the enzyme(s). The
kinase may be an isolated purified or partially purified enzyme, or
may be within a cell (e.g. in a cell cultured in vitro), or within
and organism (in vivo). Generally, the activity of the kinase is
inhibited by at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, or 95% or even more, e.g. about 100%, compared
to a control enzyme which is not exposed to a compound of the
invention. Those of skill in the art are familiar with methodology
to measure the activity of enzymes, and of kinases in particular.
For example, the ability of a kinase to carry out its usual
enzymatic activity may be measured, e.g. by detecting a product of
that activity.
[0075] The invention also provides methods of inhibiting SphK1 and
SphK2 using the compound Klll67 (compound 42; see the Scheme in the
Examples entitled "Compounds 28-36, 39-40 and 42 of Schemes 2 and
3"). Compound 42 may be used for any of the purposes described
herein, e.g. for inhibiting SphK2, for treating diseases associated
with SphK2, for killing or damaging cancer cells, for treating
cancer, etc. However, its use is not selective for SphK2. Thus,
compound 42 may also be used to inhibit SphK1, to treat diseases or
conditions associated with SphK1, etc. In further embodiments,
compound 42 may be used to inhibit both SphK1 and SphK2, e.g. in
vitro or in cells.
[0076] The invention also provides methods of inhibiting growth or
killing or damaging cells exhibiting positive SphK2 activity. By
"positive" SphK2 activity we mean overactivation or overexpression
of the kinase. In some embodiments, the cells are cancer cells. The
methods involve exposing the cells to one or more compounds of the
invention, the one or more compounds being present in an amount
that is sufficient to cause the death of the cells, or to cause
damage to the cells, e.g. to slow the cells' metabolism, prevent
replication, prevent movement, induce apoptosis of the cells, etc.
The cells that are killed or damaged may be in vitro or in vivo,
i.e. this method may be carried out for clinical purposes (e.g. for
the treatment of disease) or in the laboratory (e.g. the compounds
of the invention may be used as laboratory reagents.) When a
population of cancer cells is exposed to the compounds of the
invention, generally about 25, 30, 35, 40, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, or more, e.g. even 100%, or the cells in the
population are damaged or killed, compared to a suitable control
population that is not exposed to the compounds.
[0077] Other embodiments of the invention include the treatment of
diseases or conditions associated with positive SphK2 activity.
These methods comprise the step of administering an effective
amount of the compound of Formula I and/or Formula II or a
composition thereof to a patient in need thereof to inhibit the
SphK2 activity. Examples of such disease or conditions include but
are not limited to cancer, arthrosclerosis, arthritis, diabetes,
obesity, osteoporosis, inflammatory diseases and Alzheimer's
disease. An "effective amount" or a "therapeutic amount" refers to
an amount that either cures (i.e. symptoms of disease disappear
completely or become undetectable), or ameliorates disease
symptoms, e.g. by lessening symptoms such as pain, slowing of the
growth of a tumor, lengthening the life of a patient, improving the
quality of life of a patient, etc.
[0078] The invention is further described by the following
non-limiting examples which further illustrate the invention, and
are not intended, nor should they be interpreted to, limit the
scope of the invention in any way.
EXAMPLES
[0079] The synthesis of the exemplary compounds is described in
Schemes 1-5.
Example 1
Preparation of Compound 26
[0080] To a stirred suspension of bromoethylamine hydrobromide 25
(20.5 g, 100 mmol), (Boc).sub.2O (21.8 g, 100 mmol) in
dichloromethane (200 mL) was added triethylamine (13.9 mL, 100
mmol) dropwise at 0.degree. C., after added the mixture was stirred
at room temperature (rt). overnight, water was added, the separated
CH.sub.2Cl.sub.2 layer was washed with brine, and dried by
Na.sub.2SO.sub.4, remove the solvent to give a colorless oil (20
g).
Example 2
Preparation of Compound 27
[0081] A mixture of 2,4-thiazolidinedione (7.9 g, 68 mmol),
compound 26 (17.9 g, 80 mmol), K.sub.2CO.sub.3 (11.1 g, 92 mmol),
TBAI (2.5 g, 6.8 mmol) in acetone (100 mL) was stirred at
40.degree. C. for 10 h, suction filter, the filtrate was
concentrated under vacuum, the residue was purified by flash column
chromatography (Hexane/EA=4/1 to 2/1) to give a white solid (12.3
g).
Example 3
Preparation of Compounds 28-36
[0082] A solution of compound 27 (12 mmol), aldehyde (12 mmol) and
piperidine (3.6 mmol) in MeOH (60 mL) was stirred at rt. overnight,
remove solvent under vacuum, the residue was purified by flash
column chromatography (Hexane/EA=8/1) to give a white solid, which
was subject to Boc deprotection conditions in ethyl acetate (30 mL)
by 4 M HCl in dioxane (15 mL), the solution to give 28-36 as white
solid.
Example 4
Preparation of 4-alkoxyphenylpropanal Compounds 14-19
[0083] To a stirred solution of compounds 2-7 (891 mg, 5 mmol) and
meldrum's acid (720 mg, 5 mmol) in EtOH (5 mL) was added piperidine
(two drops), the resulting solution was stirred at rt. overnight,
suction filter give a light yellow solid, (731 mg). To the solid
and acetic acid (3 mL) in DCM (20 mL) at 0.degree. C. was added
sodium borohydride (314 mg, 8.4 mmol) portion wise. The resulting
solution was stirred at rt. for 1 h. the solution was dissolved in
DCM and washed with brine and water. The organic layer was dried
over Na.sub.2SO.sub.4 and concentrated under vacuum, the residue
was purified by flash column chromatography (Hexane/acetone=5/2) to
give a light yellow solid (426 mg). To a solution of above
compounds (368 mg, 1.2 mmol) in tetrahydrofuran (7 mL) was added
triethylamine (0.334 mL, 2.4 mmol) followed by phenylsilane (0.444
mL, 3.6 mmol). The resulting solution was stirred for 2 hours at
room temperature. Water was added to the solution and stirred for
15 minutes. The reaction mixture was dissolved in ether and washed
with water, then with brine. The organic layer was dried over
Na.sub.2SO.sub.4 and concentrated, the residue was purified by
flash column chromatography (Hexane/acetone=10/1) to give a light
colorless oil.
Example 5
Preparation of 4-alkoxybenzaldehyde Compounds 2-7
[0084] A mixture of 4-hydroxybenzaldehyde (10 mmol), bromoalkane
(13 mmol), K.sub.2CO.sub.3 (13 mmol), DMF (20 mL) was refluxed for
18 h, cooled to rt., water was added, extracted by hexane,
concentrated under vacuum, the residue was purified by flash column
chromatography to give a light yellow oil.
Example 6
[0085] Preparation of Compounds 24, 46, 48 and 50 by Swern
Oxidation
[0086] DMSO (14 mmol) was added dropwise to a stirred solution of
oxalyl chloride (5 mmol) in DCM (20 mL) at -78.degree. C., after
added the mixture was stirred at -78.degree. C. for 20 min, then
alkanol (4 mmol) was added dropwise and stirred at -78.degree. C.
for 1 h, Et.sub.3N (1 mL) was added and the temperature was slowly
elevated to it, then water was added, the separated DCM layer was
washed with brine, concentrated under vacuum, the residue was
purified by flash column chromatography to give a colorless
oil.
Example 7
Preparation of Compound 39 by Reduction
[0087] A mixture of compound 31 (KLIII016) (100 mg) and 10% Pd/C
(50 mg) in methanol (15 mL) was hydrogenated at rt. overnight,
suction filter to remove the catalyst, the filtrate was
concentrated under vacuum, then ethyl acetate (1 mL) was added,
suction filter, and washed with ethyl acetate to give a white
solid.
Example 8
Preparation of Guanidine Analog 42
[0088] To a suspension of 31 (KLIII016) (0.2 mmol) in DCM (2 mL)
was added triethylamime (0.2 mmol) followed by
N,N'-Di-Boc-1H-pyrazole-1-carboxamidine (0.2 mmol), after added the
mixture was stirred at rt. overnight, water was added and extracted
by DCM, concentrated under vacuum, the residue was purified by
flash chromatography (Hexane/Acetone=10/1) give 41 as a colorless
oil (88 mg). Compound 41 (80 mg) was dissolved in 2 mL of DCM, 2 mL
of HCl (4M in dioxane) was added, the mixture was stirred at rt.
for 20 h, filtered to give 28 mg of compound 42 as white solid.
##STR00012##
##STR00013##
##STR00014##
Compounds 28-36 39-40 and 42 of Schemes 2 and 3
##STR00015## ##STR00016##
##STR00017##
##STR00018##
[0089] Compounds 59-70 of Scheme 5
##STR00019## ##STR00020## ##STR00021## ##STR00022##
[0090] Example 9
KII167 (42)
[0091] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.62 (m, 1H), 7.34
(brs, 3H), 7.15-7.13 (d, J=8.5 Hz, 2H), 7.01 (t, J=7.4 Hz, 1H),
6.86-6.83 (d, J=8.5 Hz, 2H), 3.91 (t, J=6.5 Hz, 2H), 3.67 (t, J=4.9
Hz, 2H), 3.39-3.35 (q, J=5.9 Hz, 2H), 2.76 (t, J=7.3 Hz, 2H),
2.53-2.45 (m, 2H), 1.72-1.65 (m, 2H), 1.41-1.39 (m, 2H), 1.37-1.28
(m, 6H), 0.87 (t, J=6.6 Hz, 3H); .sup.13C NMR (100 MHz,
DMSO-d.sub.6): 167.3, 164.3, 157.1, 156.9, 137.5, 132.0, 129.3,
125.1, 114.4, 67.3, 40.5, 38.2, 33.0, 32.1, 31.2, 28.7, 28.4, 25.5,
22.0, 13.9.
Example 10
KLIII16 (31)
[0092] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.13 (s, 3H), 7.15-7.13
(d, J=8.6 Hz, 2H), 7.01 (t, J=7.4 Hz, 1H), 6.85-6.83 (d, J=8.6 Hz,
2H), 3.91 (t, J=6.5 Hz, 2H), 3.83 (t, J=6.0 Hz, 2H), 3.00 (m, 2H),
2.76 (t, J=7.4 Hz, 2H), 2.53-2.45 (m, 2H), 1.70-1.66 (m, 2H),
1.41-1.37 (m, 2H), 1.35-1.26 (m, 6H), 0.87 (t, J=6.8 Hz, 3H);
.sup.13C NMR (100 MHz, DMSO-d.sub.6): 167.6, 164.5, 157.1, 137.4,
132.0, 129.2, 125.2, 114.4, 67.3, 36.5, 33.0, 32.1, 31.2, 28.7,
28.4, 25.5, 22.0, 13.9.
Example 11
KLII157 (32)
[0093] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.16 (s, 3H), 7.15-7.13
(d, J=8.6 Hz, 2H), 7.01 (t, J=7.4 Hz, 1H), 6.85-6.83 (d, J=8.6 Hz,
2H), 3.91 (t, J=6.5 Hz, 2H), 3.83 (t, J=6.0 Hz, 2H), 3.00 (m, 2H),
2.76 (t, J=7.4 Hz, 2H), 2.53-2.45 (m, 2H), 1.70-1.64 (m, 2H),
1.40-1.35 (m, 2H), 1.32-1.25 (m, 10H), 0.86 (t, J=6.8 Hz, 3H);
.sup.13C NMR (100 MHz, DMSO-d.sub.6): 167.6, 164.5, 157.1, 137.4,
132.0, 129.2, 125.2, 114.4, 67.3, 36.5, 33.0, 32.1, 31.2, 28.9,
28.7, 28.6, 28.5, 25.5, 22.0, 13.9.
Example 12
KLII123 (33)
[0094] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.11 (s, 3H), 7.15-7.13
(d, J=8.5 Hz, 2H), 7.01 (t, J=7.4 Hz, 1H), 6.85-6.83 (d, J=8.5 Hz,
2H), 3.91 (t, J=6.5 Hz, 2H), 3.83 (t, J=6.0 Hz, 2H), 3.00 (m, 2H),
2.76 (t, J=7.4 Hz, 2H), 2.53-2.48 (m, 2H), 1.69-1.66 (m, 2H),
1.40-1.37 (m, 2H), 1.27-1.25 (m, 14H), 0.85 (t, J=6.8 Hz, 3H);
.sup.13C NMR (100 MHz, DMSO-d.sub.6): 167.6, 164.5, 157.1, 137.4,
132.0, 129.2, 125.2, 114.4, 67.3, 36.6, 33.0, 32.1, 31.2, 28.9,
28.7, 28.68, 28.65, 25.5, 22.0, 13.9.
Example 13
KLII147 (35)
[0095] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.12 (s, 3H), 7.03 (t,
J=7.4 Hz, 1H), 3.85 (t, J=6.0 Hz, 2H), 3.02 (m, 2H), 2.24-2.19 (q,
J=7.4 Hz, 2H), 1.51-1.48 (m, 2H), 1.31-1.24 (m, 20H), 0.86 (t,
J=6.8 Hz, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 167.6, 164.6,
138.3, 124.9, 36.6, 31.3, 31.1, 29.0, 28.9, 28.8, 28.7, 28.6, 27.2,
22.0, 13.9.
Example 14
KLII139 (34)
[0096] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.10 (s, 3H), 7.15-7.13
(d, J=8.2 Hz, 2H), 7.12-7.10 (d, J=8.2 Hz, 2H), 7.02 (t, J=7.4 Hz,
1H), 3.83 (t, J=6.0 Hz, 2H), 3.00 (m, 2H), 2.79 (t, J=7.4 Hz, 2H),
2.55-2.49 (m, 4H), 1.55-1.51 (m, 2H), 1.40-1.37 (m, 2H), 1.26-1.23
(m, 10H), 0.85 (t, J=6.8 Hz, 3H); .sup.13C NMR (100 MHz,
DMSO-d.sub.6): 167.6, 164.5, 140.2, 137.4, 137.3, 128.3, 128.1,
125.2, 36.6, 34.7, 32.7, 32.6, 31.2, 30.9, 28.8, 28.6, 22.0,
13.9.
Example 15
K145 (30)
[0097] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.08 (brs, 3H),
7.15-7.13 (d, J=8.5 Hz, 2H), 7.01 (t, J=7.4 Hz, 1H), 6.86-6.84 (d,
J=8.5 Hz, 2H), 3.92 (t, J=6.4 Hz, 2H), 3.83 (t, J=6.0 Hz, 2H), 3.00
(m, 2H), 2.76 (t, J=7.3 Hz, 2H), 2.53-2.48 (m, 2H), 1.69-1.64 (m,
2H), 1.45-1.39 (m, 2H), 0.92 (t, J=7.3 Hz, 3H); .sup.13C NMR (100
MHz, DMSO-d.sub.6): 167.5, 164.4, 157.1, 137.3, 131.9, 129.2,
125.1, 114.3, 66.9, 36.5, 32.9, 32.1, 30.7, 18.6, 13.6.
Example 16
KL-SI-25 (36)
[0098] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.10 (s, 3H), 7.04 (t,
J=7.7 Hz, 1H), 3.85 (t, J=6.0 Hz, 2H), 3.04-3.01 (m, 2H), 2.25-2.19
(q, J=7.4 Hz, 2H), 1.52-1.47 (m, 2H), 1.27 (m, 10H), 0.86 (t, J=6.7
Hz, 3H), .sup.13C NMR (100 MHz, DMSO-d.sub.6): 167.6, 164.6, 138.3,
124.9, 36.6, 31.2, 31.1, 28.7, 28.6, 28.5, 27.2, 22.0, 13.9.
Example 17
B26
[0099] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.15 (s, 3H), 7.05 (t,
J=7.7 Hz, 1H), 3.04-3.00 (m, 2H), 2.20-2.14 (q, J=7.4 Hz, 2H), 1.94
(brs, 3H), 1.69-1.59 (m, 6H), 1.47-1.46 (d, J=2.3 Hz, 6H),
1.27-1.23 (m, 2H), .sup.13C NMR (100 MHz, DMSO-d.sub.6): 167.5,
164.5, 139.1, 124.3, 41.4, 36.5, 36.4, 31.8, 27.9, 25.0.
Example 18
KLII158 (40)
[0100] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.74 (s, 3H), 7.09-7.07
(d, J=8.5 Hz, 2H), 6.84-6.81 (d, J=8.5 Hz, 2H), 4.56-4.53 (m, 1H),
3.91 (t, J=6.5 Hz, 2H), 3.72 (t, J=6.2 Hz, 2H), 2.96-2.87 (m, 2H),
2.55 (t, J=7.4 Hz, 2H), 2.10-2.02 (m, 1H), 1.88-1.65 (m, 4H),
1.59-1.52 (m, 1H), 1.43-1.35 (m, 2H), 1.33-1.28 (m, 6H), 0.87 (t,
J=6.8 Hz, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6): 174.7, 171.5,
156.9, 133.0, 129.2, 114.3, 67.3, 49.6, 36.7, 33.6, 31.2, 31.1,
28.7, 28.5, 28.4, 25.5, 22.0, 13.9.
Example 19
KLII159 (39)
[0101] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.91 (s, 3H), 7.10-7.07
(d, J=8.5 Hz, 2H), 6.84-6.82 (d, J=8.5 Hz, 2H), 4.56-4.53 (m, 1H),
3.92 (t, J=6.5 Hz, 2H), 3.73 (t, J=6.2 Hz, 2H), 2.99-2.88 (m, 2H),
2.55 (t, J=7.4 Hz, 2H), 2.11-2.02 (m, 1H), 1.88-1.63 (m, 4H),
1.61-1.52 (m, 1H), 1.47-1.38 (m, 2H), 0.92 (t, J=6.8 Hz, 3H);
.sup.13C NMR (100 MHz, DMSO-d.sub.6): 174.7, 171.5, 156.9, 133.0,
129.2, 114.3, 67.0, 49.6, 36.6, 33.6, 31.1, 30.8, 28.5, 18.7,
13.7.
Example 20
Compound 41
[0102] .sup.1H NMR (400 MHz, CDCl3): 11.4 (s, 1H), 8.44 (t, J=5.5
Hz, 1H), 7.08-7.04 (m, 3H), 6.84-6.82 (d, J=8.6 Hz, 2H), 3.94-3.89
(m, 4H), 3.69-3.65 (q, J=5.8 Hz, 2H), 2.78 (t, J=7.4 Hz, 2H),
2.52-2.47 (q, J=7.6 Hz, 2H) 1.79-1.75 (m, 2H), 1.48-1.34 (m, 20H),
1.32-1.30 (m, 6H), 0.88 (t, J=6.8 Hz, 3H).
Example 21
1-(2-aminoethyl)-3-(3-cyclohexylpropylidene)indolin-2-one
(S-II-36)
[0103] .sup.1H NMR (400 MHz, DMSO): .delta. 8.1279 (s, 3H),
7.65-7.63 (d, J=7.48, 1H), 7.35-7.31 (t, J=7.2 Hz, 1H), 7.22-7.20
(d, J=7.72 Hz, 1H), 7.11-7.07 (t, J=8.16 Hz, 1H), 6.90-6.86 (t,
J=7.76 Hz, 1H), 4.01-3.98 (t, J=6.48, 2H), 3.03-3.00 (m, 2H),
2.71-2.65 (q, J=7.64 Hz, 2H), 1.74-1.51 (m, 5H), 1.49-1.39 (q,
J=7.56 Hz, 2H), 1.26-1.11 (m, 4H), 0.97-0.88 (m, 2H)
[0104] .sup.13C NMR (100 MHz, DMSO): 167.1, 142.2, 142.2, 128.9,
128.4, 126.8, 123.4, 122.2, 121.8, 108.7, 36.8, 36.7, 35.5, 32.6,
30.7, 26.1, 25.7
Example 22
1-(2-aminoethyl)-3-(3-cyclopropylpropylidene)indolin-2-one
(S-II-40)
[0105] .sup.1H NMR (400 MHz, MeOD): .delta. 7.35-7.33 (d, J=6.88
Hz, 1H), 7.35-7.33 (t, J=6.84 Hz, 1H), 7.16-7.07 (m, 3H), 4.11-4.08
(t, J=6.00 Hz, 2H), 3.27-3.24 (t, J=5.72 Hz, 3H) 2.88-2.83 (q,
J=7.44 Hz, 2H), 1.60-1.55 (q, J=7.16 Hz, 2H), 0.51-0.46 (m, 2H),
0.16-0.12 (m, 2H)
[0106] .sup.13C NMR (100 MHz, DMSO): 170.5, 144.4, 143.1, 130.2,
128.5, 125.1, 124.0, 123.7, 109.4, 39.3, 38.7, 34.8, 30.6, 11.7,
5.2.
Example 23
3-(3-((3r,5r,7r)-adamantan-1-yl)propylidene)-1-(2-aminoethyl)indolin-2-one
(S-II-73)
[0107] .sup.1H NMR (400 MHz, DMSO): .delta. 8.11 (s, 1H), 7.62-7.61
(d, J=7.52 Hz, 1H), 7.35-7.31 (t, J=7.72 Hz, 1H), 7.22-7.20 (d,
J=7.84 Hz, 1H), 7.12-7.09 (t, J=7.52 Hz, 1H), 6.91-6.87 (t J=7.84
Hz, 1H), 4.00-3.97 (t, J=6.56, 2H), 3.04-3.00 (q, J=5.44, 2H),
2.65-2.59 (q, J=8.04 Hz, 2H), 1.99 (s, 3H), 1.71-1.60 (q, J=12.04
Hz, 6H), 1.54 (s, 6H), 1.36-1.32 (m, 2H).
[0108] .sup.13C NMR (100 MHz, DMSO): 167.0, 157.1, 142.1, 140.9,
132.4, 129.3, 129.0, 127.2, 123.5, 122.2, 121.6, 114.4, 114.3,
108.7, 67.0, 66.3, 37.0, 36.6, 32.8, 30.8, 30.4, 18.7, 13.7.
Example 24
1-(2-aminoethyl)-3-nonylideneindolin-2-one (S-II-103)
[0109] .sup.1H NMR (400 MHz, DMSO): .delta.7.96 (s, 1H), 7.67-7.65
(d, J=7.56, 1H), 7.34-7.17 (t, J=7.68, 1H), 7.17-7.16 (d, J=7.8,
3H), 7.11-7.07 (t, J=7.56, 1H), 6.91-6.87 (t, J=7.80, 1H),
3.99-3.96 (t, J=6.32, 2H), 2.71-2.65 (q, J=7.40, 2H), 1.61-1.57 (m,
2H), 1.39-1.26 (m, 11H), 0.87-0.83 (t, J=6.60, 3H).
[0110] .sup.13C NMR (100 MHz, DMSO): 142.1, 129.0, 127.0, 123.5,
122.2, 121.7, 36.8, 31.2, 28.8, 28.6, 28.0, 22.0, 13.9.
Example 25
1-(2-aminoethyl)-3-(3-(4-methoxyphenyl)propylidene)indolin-2-one
(S-II-71)
[0111] .sup.1H NMR (400 MHz, DMSO): .delta. 8.14 (s, 1H), 7.65-7.64
(d, J=7.48, 1H), 7.35-7.31 (t, J=7.72, 1H), 7.24-7.20 (t, J=8.48,
3H), 7.10-7.06 (t, J=7.60, 1H), 6.89-6.85 (m, 3H), 4.00-3.96 (t,
J=6.52, 2H), 3.01-2.95 (m, 4H), 2.88-2.85 (t, J=6.92, 2H),
2.51-2.49 (m, 3H) .sup.13C NMR (100 MHz, DMSO): 167.0, 157.7,
142.1, 141.9, 140.9, 140.6, 132.6, 129.3, 129.2, 129.1, 128.7,
127.2, 123.5, 122.2, 121.8, 121.6, 119.4, 113.8, 108.7, 108.4,
55.0, 37.0, 36.8, 36.8, 36.6, 33.4, 32.8, 30.4, 30.4, 29.3
Example 26
1-(2-aminoethyl)-3-(3-(4-ethoxyphenyl)propylidene)indolin-2-one
(S-II-102)
[0112] .sup.1H NMR (400 MHz, DMSO): .delta. 8.14 (s, 1H), 7.65-7.64
(d, J=7.48, 1H), 7.33-7.31 (t, J=7.72 Hz, 1H), 7.24-7.20 (m, 3H),
7.10-7.06 (t, J=7.60 Hz, 1H), 6.89-6.85 (m, 3H), 4.00-3.96 (t,
J=6.52, 2H), 4.00-3.96 (t, J=6.52 Hz, 3H), 3.01-2.95 (m, 4H),
2.88-2.85 (t, J=6.92 Hz, 2H), 2.51-2.49 (m, 3H)
[0113] .sup.3C NMR (100 MHz, DMSO): 167.1, 142.1, 132.4, 129.3,
129.0, 127.2, 123.6, 121.7, 114.2, 108.6, 62.9, 37.1, 36.8, 32.8,
30.4, 14.7
Example 27
1-(2-aminoethyl)-3-(3-(4-butoxyphenyl)propylidene)indolin-2-one
(S-II-78)
[0114] .sup.1H NMR (400 MHz, DMSO): .delta. 8.16 (s, 1H), 7.66-7.64
(d, J=7.48 Hz, 1H), 7.35-7.31 (t, J=7.72 Hz, 1H), 7.22-7.16 (m,
3H), 7.08-7.07 (t, J=6.92 Hz, 1H), 6.89-6.84 (m, 3H), 4.00-3.97 (t,
J=6.56 Hz, 2H), 3.94-3.91 (t, J=6.44 Hz, 3H), 3.03-2.95 (m, 4H),
2.87-2.84 (t, J=6.96, 2H), 1.70-1.63 (m, 2H), 1.45-1.39 (m, 2H),
0.94-0.90 (t, J=7.36 Hz, 3H)
[0115] .sup.13C NMR (100 MHz, DMSO): 167.1, 142.9, 142.1, 128.9,
126.5, 123.3, 122.2, 121.7, 108.7, 66.3, 108.7, 37.0, 36.7, 36.6,
36.5, 32.0, 28.0, 22.4
Example 28
1-(2-aminoethyl)-3-(3-(4-(heptyloxy)phenyl)propylidene)indolin-2-one
(S-II-103)
[0116] .sup.1H NMR (400 MHz, DMSO): .delta. 8.06 (s, 1H), 7.66-7.64
(d, J=7.48 Hz, 1H), 7.35-7.31 (t, J=7.72 Hz, 1H), 7.22-7.18 (m,
3H), 7.10-7.06 (t, J=7.56 Hz, 1H), 6.89-6.84 (m, 3H), 3.99-3.95 (t,
J=6.32, 2H), 3.93-3.90 (t, J=6.52 Hz, 3H), 3.02-2.95 (m, 4H),
2.87-2.84 (t, J=7.04 Hz, 2H), 1.72-1.65 (m, 2H), 1.43-1.17 (m, 8H),
0.88-0.85 (t, J=6.64 Hz, 3H)
[0117] .sup.3C NMR (100 MHz, DMSO): 167.0, 157.1, 142.1, 140.9,
132.4, 129.3, 129.0, 127.2, 123.6, 122.2, 121.7, 114.4, 108.6,
37.0, 36.7, 32.8, 31.2, 30.4, 28.7, 28.7, 28.4, 25.5, 22.0,
13.9.
Example 29
1-(2-aminoethyl)-3-(3-(4-fluorophenyl)propylidene)indolin-2-one
(S-II-39)
[0118] .sup.1H NMR (400 MHz, MeOD): .delta. 7.66-7.64 (d, J=7.56,
1H), 7.35-7.27 (m, 3H), 7.13-6.97 (m, 5H), 7.10-7.06 (t, J=6.96 Hz,
1H), 6.90-6.86 (t, J=7.24 Hz, 1H), 4.09-4.06 (t, J=5.88 Hz, 2H),
3.26-3.23 (t, J=6.12 Hz, 2H), 3.08-3.03 (m, 2H), 2.99-2.95 (m,
2H).
[0119] .sup.13C NMR (100 MHz, DMSO): 170.0, 164.2, 161.8, 143.1,
138.0, 131.2, 130.4, 129.0, 125.0, 124.1, 123.5, 116.3, 116.1,
109.5, 39.3, 38.7, 34.6, 32.1
Example 30
1-(2-aminoethyl)-3-(3-(4-chlorophenyl)propylidene)indolin-2-one
(S-II-60)
[0120] .sup.1H NMR (400 MHz, DMSO): .delta. 7.97 (s, 3H), 7.67-7.65
(d, J=7.52 Hz, 1H), 7.36-7.32 (m, 5H), 7.17-7.15 (d, J=7.80 Hz,
2H), 7.10-7.06 (t, J=6.96 Hz, 1H), 6.87-6.84 (t, J=7.04 Hz, 1H),
3.98-3.94 (t, J=6.28 Hz, 2H), 3.04-2.99 (m, 4H), 2.95-2.91 (m,
2H).
[0121] .sup.13C NMR (100 MHz, DMSO): 140.4, 139.8, 130.7, 129.1,
128.3, 127.4, 123.6, 122.2, 121.6, 108.7, 37.1, 36.7, 33.0,
29.9
Example 31
1-(2-aminoethyl)-3-(3-(4-nitrophenyl)propylidene)indolin-2-one
(S-II-61)
[0122] .sup.1H NMR (400 MHz, MeOD): .delta. 8.75-8.74 (d, J=7.48
Hz, 3H), 7.67-7.65 (d, J=7.52 Hz, 1H), 7.35-7.2890 (m, 5H),
7.23-7.20 (m, 2H), 7.10-7.06 (t, J=6.96 Hz, 1H), 6.90-6.86 (t,
J=7.24 Hz, 1H), 4.00-3.96 (t, J=6.48 Hz, 2H), 3.05-2.99 (m, 4H),
2.95-2.91 (m, 2H).
[0123] .sup.13C NMR (100 MHz, DMSO): .delta. 168.7, 147.4, 145.1,
135.1, 131.2, 129.9, 123.8, 124.2, 123.2, 115.6, 109.5, 37.9, 38.7,
26.6, 32.1
Example 32
In Vitro Test
SphK1 and SphK2 Test
[0124] The results from FIGS. 1-6 demonstrate that Formula III
(K145) and some other compounds of Formula I and Formula II
significantly inhibit SphK2, but not SphK1, thus demonstrating they
are selective SphK2 inhibitors. As shown in FIG. 1, Formula III
(K145) dose-dependently inhibited SphK2 with a IC.sub.50 of 5 uM.
Significantly, the results of FIG. 2 demonstrates that the potency
of this type of SphK2 inhibitors from Formula I can be
significantly improved by structural modification as Formula IV
(KLIII16) with a longer alkyl chain has an IC.sub.50 of 1.9 uM
compared to that of Formula III (K145).
Kinetic Studies of Formula III (K145)
[0125] As shown in FIG. 7, the kinetic studies of Formula III
revealed that Formula III (K145) is a competitive inhibitor (with
the substrate sphingosine) of SphK2 with a Ki value of
4.3.+-.0.7M.
Cell Viability Assays in Human Leukemia U937 Cells
[0126] Cells were cultured at a density of 5.times.10.sup.4 (U937)
or 1.times.10.sup.4 (PC-3, DU145, M12, HT29) cells per well in flat
bottomed 96-well plates and treated with various concentrations of
test compound at 37.degree. C. (5% CO.sub.2). After 24 h, 20 .mu.L,
of CellTiter 96.RTM. Aqueous One Solution Reagent (Promega,
Madison, Wis.) was added to each well according to the
manufacturer's instructions. After 1 hour, the cell viability was
determined by measuring the absorbance at 490 nm using a
micro-plate reader.
[0127] The results are presented in FIG. 8. These results show that
Formula III (K145) inhibited the proliferation of tested cancer
cells with an IC.sub.50 at single digit micromolar
concentrations.
[0128] The results of Table 1 also show that Formula II compounds
inhibited the proliferation of human leukemia U937 cells.
TABLE-US-00001 TABLE 1 Inhibition of U937 cell proliferation by
indicated compounds.* Compound IC.sub.50 .+-. SEM Compound
IC.sub.50 .+-. SEM Compound IC.sub.50 .+-. SEM S-II-129 2.128 .+-.
0.006 S-II-103 3.5 .+-. 0.3 S-II-104 3.9 .+-. 0.3 S-II-36 2.7 .+-.
0.2 S-II-71 0.92 .+-. 0.04 S-II-39 2.69 .+-. 0.06 S-II-40 1.16 .+-.
0.05 S-II-102 0.95 .+-. 0.11 S-II-60 4.0 .+-. 0.7 S-II-73 4 .+-. 1
S-II-78 4.6 .+-. 0.8 S-II-61 3.6 .+-. 0.2 *U937 cells were treated
with indicated compounds at various concentrations for 24 hrs,
after which cell viability was measured using MTT assay and
IC.sub.50 was calculated.
Western Blot Analysis
[0129] Cells (5.times.10.sup.5 per ml) were treated with various
concentrations of test compound at 37.degree. C. (5% CO.sub.2) for
3 hrs, then stimulated with TPA at a final concentration of 200 nM
for 20 min. Samples from whole-cell pellets were prepared and 30
.mu.g protein for each condition was subjected to SDS-PAGE,
transferred onto a PVDF membrane, and blocked with 5% fat-free milk
for 30 min. The membrane is probed with primary antibodies
overnight at 4.degree. C. followed by incubation with horseradish
peroxidase-labeled anti-mouse IgG (1:5000, BD Bioscience). The
immunoreactive bands are detected by chemilluminescence methods
(Pierce) and visualized on Kodak Omat film. The following primary
antibodies were used: phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204),
p44/42 MAPK, phospho-p90RSK (Thr359/Ser363), RSK1/RSK2/RSK3 (Cell
Signaling). Blots were reprobed with antibodies against
.alpha.-tubulin to ensure equal loading and transfer of
proteins.
[0130] The results are presented in FIG. 9, which shows that
Formula III (K145) significantly inhibited the phosphorylation of
both ERK and Akt at 3 .mu.M concentrations. The results are
consistent with reported results that SphK execute their effects
through, at least through, the Raf/MEK/ERK and PI3K/Akt signaling
pathways. As shown in FIG. 10, compounds S-II-71 and S-II-103 also
inhibit the Raf/MEK/ERK and PI3K/Akt signaling pathways, which
indicates that inhibition of SphK2 by compounds from Formula II,
interferes with the Raf/MEK/ERK and PI3K/Akt signaling
pathways.
In Vitro Kinase Screening
Cell Apoptosis Assays.
[0131] Apoptosis was measured by flow cytometry using annexin
V/propidium iodide (PI) as staining reagent. Briefly, after
treatment with test compound of varying concentrations for varying
intervals (4, 8, 18, 36 hrs), cells were washed twice with cold PBS
and then resuspended in 1.times. binding buffer (10 mM HEPES
[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]/NaOH, pH 7.4,
140 mM NaOH, 2.5 mM CaCl.sub.2). The cells were then incubated with
annexin V--fluorescein isothiocyanate (FITC) (BD PharMingen, San
Diego, Calif.) and 5 .mu.g/mL propidium iodide (PI), and incubated
for 15 minutes at room temperature in the dark per the
manufacturer's instructions. The samples were analyzed by flow
cytometry using a Becton Dickinson FACScan (Becton Dickinson, San
Jose, Calif.) within 1 hr to determine the percentage of cells
displaying annexin V staining (early apoptosis) or both annexin V
and PI staining (late apoptosis).
[0132] The results are presented in FIG. 11, which shows that
Formula III (K145) significantly and dose-dependently induced
apoptosis in U937 cells. After 18 hrs, Formula III (K145)
significantly induced apoptosis in U937 cells at 5 .mu.M. At 10
.mu.M concentration, Formula III (K145) significantly induced
apoptosis at as early as 4 hrs treatment. As shown in FIG. 12,
S-II-71 and S-II-103 from Formula II also induced apoptotic effects
in U937 cells after 24 hrs treatment. Interestingly, S-II-71
induced both early and late apoptotic effect while S-II-103 mainly
induced late apoptotic effects.
Example 18
In Vivo Studies
Anti-Proliferation of U937 Xenografts in Nude Mice.
[0133] Female nude mice (BALB/c-nu, n=7) were inoculated with
3.times.10.sup.7 U937 cells subcutaneously in the right flank.
After 7 days of implant of U937 cells, mice were treated daily with
Vehicle (control), or Tamibarotene (15 mg/Kg, positive control), or
Formula III (K145) (15 mg/Kg) by i.p. administration. Tumor size
was measured every other day. After 18 days treatment, mice were
sacrificed and the tumor were removed and weighed.
[0134] The results are presented in FIG. 13A, K145 significantly
inhibited the growth of U937 tumors in nude mice with a TGI of
44.2%, slightly less potent than tamibarotene (TGI=50.4%) after 17
days treatment. This is also reflected by the tumor weights of
treatment groups (FIG. 13B). The tumor growth curve during the
treatment course (FIG. 13C) also attests to the anti-tumor effects
of K145 in this model. Lastly, as shown in FIG. 13D, the body
weights of K145-treated mice remained the same as that of
vehicle-treated mice, while tamibarotene treatment caused body
weight decreases in the mice. These results strongly suggest that
K145 exhibits comparable in vivo anti-tumor activity to
tamibarotene, while concomitantly exhibiting less toxicity in this
U937 xenograft cancer model.
Anti-Growth of JC Xenografts in Syngenic BALB/c Mice.
[0135] Female BALB/c mice (n=12) were injected with
1.times.10.sup.6 JC cells subcutaneously in the flank, and
treatment with the compound produced as described in Example 15
i.e. 3-(2-amino
ethyl)-5-[3-(4-butoxyphenyl)-propylidene]-thiazolidine-2,4-dione
(Formula III-K145, 20 mg/kg and 35 mg/Kg dosage daily (i.p.) was
started seven days after the tumor cell injection and stopped at
day 15 after tumor injection. Mice were sacrificed at 15 days
treatment.
[0136] As illustrated in FIG. 14A, treatment of BALB/c mice (n=8)
bearing the JC xenograft significantly inhibited tumor growth at
both doses with the higher dose being more potent. After 15 days
treatment, the mean volume of the JC tumors in the treated-mice at
both doses was >50% smaller than that in the vehicle-treated
mice. Tumor weights of K145-treated mice were also significantly
less than that in vehicle-treated mice in a dose-dependent manner
(FIG. 14B). Post-experiment visual evaluation of the tumor samples
also confirms the results (FIG. 14D). We analyzed the tumor samples
to detect K145, the change of S1P by ESI-MS/MS and the change of
signaling pathways by Western blot. As shown in FIG. 14C, K145 was
detected in JC tumors and the S1P level was suppressed compared to
vehicle, consistent with the results from U937 cells assays.
Notably, the p-ERK and p-Akt levels were decreased in the tumor
samples compared to the vehicle controls (FIG. 14E), which is
consistent with the results from U937 cells (FIG. 9). We did not
observe significant changes in body weights and the major organs,
such as heart, lung, liver and kidney, thus indicating a lack of
general toxicity of K145.
Anti-Proliferation of U937 Xenografts in Nude Mice by K145 Through
Oral Administration.
[0137] We examined the anticancer activity of K145 to inhibit the
tumor growth of U937 cells in nude mice (BALB/c-nu) by oral
administration to investigate whether it is orally available. In
this experiment, K145 was given at 50 mg/kg by oral gavage daily
for 15 days and tumor volume and animal weights were measured every
other day. Again, tamibarotene (20 mg/kg) was used as positive
control. As shown in FIG. 15A, tumor weights of K145-treated mice
were significantly less than that in vehicle-treated mice and K145
exhibited better antitumor activity than tamibarotene at tested
doses by oral administration (TGI for K145 and tamibarotene are
51.25% and 33.37%, respectively). Visual examination of the tumor
samples also confirmed the significant inhibition of U937 tumor
growth by K145 (FIG. 15B). Tumor growth curve also demonstrated the
superior anti-tumour activity of K145 in these experimental
settings (FIG. 15C). As shown in FIG. 15D, at the beginning of the
treatment, there was a slight decrease of body weights in
K145-treated group but the body weights of this group came back in
the remaining course of the study. Collectively, the results of in
vivo studies with K145 by oral administration demonstrated that
K145 is orally available to inhibit the growth of U937 tumors at 50
mg/kg dose and no apparent toxicity was observed, which is
consistent with the results from in vivo studies by i.p. injection
administration.
Example 19
In Vitro Target Validation of K145
[0138] Next we examined whether K145 affects cellular levels of
S1P. Human leukemia U937 cells have been demonstrated to be a good
model to test compounds that interfere with the SphK/S1P system and
it has previously been shown that S1P is protective against
apoptosis of U937 cells. Therefore, we further characterized K145
in U937 cells. As shown in FIG. 16A, K145 is readily taken up by
U937 cells in a concentration dependent manner. As shown in FIGS.
16B and 16C, treatment with K145 (10 .mu.M) caused a decrease of
total cellular S1P without significant effects on ceramide levels.
The inhibitory potency of K145 on cellular level of S1P is somewhat
less than its IC.sub.50 (4.3 .mu.M) determined at recombinant
SphK2. This might be due to the fact that many enzymes such as
SphK1, SphK2, S1P lyase, and S1P phosphatise, not just SphK2, are
involved in the regulation of cellular S1P. The level of
ceramide-1-phosphate (C1P) was not significantly affected upon
treatment with K145 (10 .mu.M, FIG. 16D), which indicates that K145
does not interfere with CERK and/or ceramide synthase, consistent
with the results from recombinant CERK studies. To further confirm
its SphK2 selectivity, we then tested the effects of K145 on the
phosphorylation of FTY720, a SphK2 specific substrate [35]. As
shown in FIG. 16E, K145 inhibited the phosphorylation of FTY720,
which further indicates the SphK2 specificity of K145.
* * * * *