U.S. patent application number 14/087662 was filed with the patent office on 2014-07-10 for cdc42 inhibitor and uses thereof.
The applicant listed for this patent is Yanhua Chen, Amy Friesland, Qun Lu, Huchen Zhou. Invention is credited to Yanhua Chen, Amy Friesland, Qun Lu, Huchen Zhou.
Application Number | 20140194451 14/087662 |
Document ID | / |
Family ID | 47195390 |
Filed Date | 2014-07-10 |
United States Patent
Application |
20140194451 |
Kind Code |
A1 |
Lu; Qun ; et al. |
July 10, 2014 |
Cdc42 Inhibitor and Uses Thereof
Abstract
Compounds which inhibit the small G protein Rho GTPase cell
division cycle protein Cdc42 are provided. Morphological analyses
of filopodia, western blots of Ccd42 phosphorylation, and effects
on cellular wound healing and on growth cone formation all
demonstrate that the described compounds are able to inhibit all
tested Cdc42-mediated processes. The compounds effectively inhibit,
the effects of Cdc42 and effectively inhibit Cdc42-related cellular
functions involving actin, such as Golgi organization and cell
movement. Furthermore, the described Cdc42 inhibitor compounds may
be provided as a medicament for the treatment of various
conditions.
Inventors: |
Lu; Qun; (Taicang City,
CN) ; Zhou; Huchen; (Taicang City, CN) ; Chen;
Yanhua; (Taicang City, CN) ; Friesland; Amy;
(Taicang City, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lu; Qun
Zhou; Huchen
Chen; Yanhua
Friesland; Amy |
Taicang City
Taicang City
Taicang City
Taicang City |
|
CN
CN
CN
CN |
|
|
Family ID: |
47195390 |
Appl. No.: |
14/087662 |
Filed: |
November 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/CN2012/000708 |
May 21, 2012 |
|
|
|
14087662 |
|
|
|
|
Current U.S.
Class: |
514/275 ;
514/584; 544/297; 564/23 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 9/00 20180101; A61P 35/00 20180101; A61P 35/04 20180101; C07D
239/42 20130101; C07D 239/69 20130101; A61P 43/00 20180101; C07C
335/12 20130101; A61P 11/00 20180101 |
Class at
Publication: |
514/275 ;
544/297; 564/23; 514/584 |
International
Class: |
C07D 239/42 20060101
C07D239/42; C07C 335/12 20060101 C07C335/12 |
Claims
1. A compound of structure A: ##STR00009## structure B:
##STR00010## or a combination thereof, wherein R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8 are each
independently selected from the group consisting of fluoro, chloro,
bromo, nitro, cyano, amino, methyl, hydroxylmethyl,
trifluoromethyl, methoxy, trifluoromethyoxy, and ethyl; and A, D,
E, G, J, M, W, X, Y, Z are each selected from the group consisting
of nitrogen, carbon, and substituted carbon, and any of A, D, E, G,
J, M, W, X, Y, Z may be missing, such that the ring structure is
less than a six-membered ring.
2. A compound of structure C: ##STR00011## where R.sub.1 and
R.sub.2 are independently selected from the group consisting of
alkyl, cyclic alkyl, aryl, and substituted aryl.
3. The compound of claim 2 wherein R.sub.1 is selected from the
group consisting of ##STR00012## and R2 is selected from the group
consisting of ##STR00013##
4. The compound of claim 2 wherein the compound is according to
formula I: ##STR00014##
5. The compound of claim 1 wherein the compound is prepared as a
medicament for at least one of treatment of malignant tumors,
prevention and treatment of malignant tumor development and
invasion, treatment of diseases of the cardiovascular system
treatment of respiratory diseases, and treatment of diseases of
nervous system.
6. The compound of claim 2 wherein the compound is prepared as a
medicament for at least one of treatment of malignant tumors,
prevention and treatment of malignant tumor development and
invasion, treatment of diseases of the cardiovascular system,
treatment of respiratory diseases, and treatment of diseases of
nervous system.
7. The compound of claim 1 wherein the compound inhibits a function
of Cdc42.
8. The compound of claim 7 wherein the function of Cdc42 is
selected from the group consisting of cell movement, cell adhesion,
apoptosis, intracellular transport, cytoskeletal reorganization,
cellular endocytosis, regulation of cell cycle, cell transcription,
and combinations thereof.
9. The compound of claim 2 wherein the compound inhibits a function
of Cdc42.
10. The compound of claim 9 wherein the function of Cdc42 is
selected from the group consisting of cell movement, cell adhesion,
apoptosis, intracellular transport, cytoskeletal reorganization,
cellular endocytosis, regulation of cell cycle, cell transcription,
and combinations thereof.
11. A pharmaceutical composition comprising a compound of claim 1
and at least one excipient.
12. A pharmaceutical composition comprising a compound of claim 2
and at least one excipient.
13. A compound of formula I: ##STR00015##
14. The compound of claim 13 wherein the compound is prepared as a
medicament for at least one of treatment of malignant tumors,
prevention and treatment of malignant tumor development and
invasion, treatment of diseases of the cardiovascular system,
treatment of respiratory diseases, and treatment of diseases of
nervous system.
15. The compound of claim 13 wherein the compound inhibits a
function of Cdc42.
16. The compound of claim 15 wherein the function of Cdc42 is
selected from the group consisting of cell movement, cell adhesion,
apoptosis, intracellular transport, cytoskeletal reorganization,
cellular endocytosis, regulation of cell cycle, cell transcription,
and combinations thereof.
Description
[0001] This application is a continuation-in-part of co-pending
international Application Serial No. PCT/CN2012/000708 filed May
21, 2012, which claims priority to Chinese Application No.
201110135073.8 filed May 23, 2011, each of which is expressly
incorporated by reference herein in its entirety.
TECHNICAL FIELD
[0002] The present invention relates to compounds which are
inhibitors of the small G protein Rho GTPase Cdc42 and uses of the
described compounds.
BACKGROUND
[0003] Cell division cycle protein Cdc42 is a sub-class of the
small G protein Rho GTPase family and is an important regulatory
protein of many cell biological functions. First identified in
Saccharomyces cerevisiae for its involvement in cell polarization,
Cdc42 was then recognized to play important roles in cytoskeletal
reorganization, cellular endocytosis, regulation of cell cycle and
cell transcription. Activation of Cdc42, like that of most GTPases,
is achieved through the exchange of guanosine-5'-diphosphate (GDP)
for guanosine-5'-triphsopahte (GTP) binding. Cycles of activation
and inactivation of Rho family of GTPases are regulated by three
important class of proteins, guanine nucleotide exchange factor
(GEF), which catalyzes the release of GDP for GTP binding; GTPase
activating protein (GAP) as negative regulatory factor to
accelerate the hydrolysis of GTP of Rho GTPases from the active to
inactive state; and Guanosine nucleotide dissociation inhibitors
(GDI) to prevent the separation of GDP from Rho GTPases, thereby
inhibiting Rho GTPase activity.
[0004] Recent studies reveal abnormal activity of Cdc42 widely
involved in the pathophysiology of human diseases including cancer
and neurodegenerative diseases. Interestingly, Cdc42 gene mutations
are not found in human tumors. Its alteration is mainly reflected
in its abnormal form and overexpression and is dependent on the
tissue microenvironment of disorders, closely related to tumor
transformation and progression. As a key regulator of neuronal
morphology, Cdc42 controls the fate of normal brain development.
Cdc42 knockout mice do not live to birth and show significant brain
malformations. Previous studies have shown that Cdc42 activates the
epithelial to mesenchymal cell transition (EMT), playing important
roles in intracellular transport and tumor cell invasion.
[0005] However, in three classical Rho GTPase family members,
studies of Cdc42 lag far behind the RhoA and Rac1. This is partly
due to fast activation/inactivation cycles of Cdc42, but also to
the lack of selective small molecule research tools to help
understand this process directly.
[0006] The Rho GTPase family proteins are involved in the signaling
pathways that regulate a variety of biochemical and cellular
functions, e. g. cell membrane and material transport, cell cycle
regulation and cytoskeletal organization which is related to the
control of cell morphology, cell motility and cell fate. In recent
years, studies have shown that deregulated Rho GTPase signaling is
involved in the pathogenesis of many diseases, and therefore it has
become an important target for drug development.
[0007] Applications of small molecule modulators also contributed
to the study of the functions of Rho GTPase family proteins. For
example, the new types of effective small molecule compounds
towards brain and cardiovascular system, fasudil and Y27632, target
RhoA downstream effector signaling molecules and are recognized
potent Rho/p160.sup.ROCK inhibitors. As a Rac1-selective inhibitor,
NSC23786 in the recent study of the small molecule compounds
targeting Rac1-GEF also greatly facilitate the understanding of
Rac1 protein function. However, there were almost no effective
Cdc42 selective inhibitors. Secramine analogues of natural products
galantamine recently showed to behave like a RhoGDI and inhibited
Cdc42-dependent Golgi-mediated protein transport through cell
membranes. Unlike the widely used Y27632 and NSC23766, the
secramine study has been very limited. Cdc42 alteration is closely
involved in tumorigenesis in many ways, including tumor
transformation and metastasis: in addition, the development and
maintenance of neurons is also heavily dependent on the normal
Cdc42 activity.
[0008] NSCG23766 was identified through a computer simulation of
the structure of the compounds screened, and it fits the Rac1
molecular surface structure and the known Rac1 essential binding
GEF (Gao, Y., J B Dickerson et al (2004) Rational design and
characterization of a Rac GTPase-specific small molecule inhibitor.
Proc Natl Acad Sci USA. 101 (20): 7618-7623). NSC23766 can inhibit
serum or growth factor-induced Rac1 activation and Rac1 mediated
lamellipodia formation. NSC23766 inhibits cell proliferation in
human prostate cancer cell lines and tumor growth, and reduces cell
invasion phenotype of the tumor cells which are dependent on the
activity of endogenous Rac1. In addition, new research shows that
NSC23766 treatment can improve Rac1-mediated spinal cord injury
(SCI)-induced neuropathic pain (Tan, A M, S. Stamboulian, et al
(2008). Neuropathic pain memory is maintained by Rac1-regulated
dendritic spine remodeling after spinal cord injury. J Neurosci. 28
(49): 13173-13183).
[0009] Thus, there is a need to develop inhibitors of Cdc42.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIGS. 1A-1C show characterization of described compounds
according to one embodiment, and referred to as ZCL compounds, in
targeting Cdc42-intersectin (ITSN) interaction: FIG. 1A shows
docked pose of ZCL278 in the Cdc42 binding pocket with protein
shown as gray surface (Arrow 1) and ligand is shown as sticks
(Arrow 2); FIG. 1B shows proposed interactions between ZCL278 and
Cdc42 residues, with ZCL278 shown as sticks (Arrow 3), Cdc42 is
shown as gray cartoon, residues of Cdc42 are shown as sticks (Arrow
4), and hydrogen bonds are represented as dashed lines (Arrow 5);
and FIG. 1C shows superposition of GMP-PCP (Protein DataBank ID
code 2QRZ) and the docked Cdc42-ZCL278 complex, with Cdc42
indicated by gray cartoon, ZCL278 by sticks, and GMP-PCP (GMP-PCP
is GTP analogs, beta, gamma-methylene diphosphate guanylate) by
sticks, as indicated.
[0011] FIGS. 2A.-2C show properties of ZCL278: FIG. 2A shows the
inhibition of Cdc42-mediated cell microspike formation; FIG. 2B
shows ZCL278 inhibits the activation of endogenous Rac/Cdc42; and
FIG. 2C shows ZCL278 inhibits the stimulated Cdc42 activation.
[0012] FIGS. 3A-3C show immunofluorescence staining of active Cdc42
and the phosphorylated RhoA.
[0013] FIG. 4 shows ZCL278 disrupts the GM130-docked Golgi
organization.
[0014] FIGS. 5A-5C show ZCL278 suppresses cell migration without
affecting cell viability.
[0015] FIGS. 6A-6C show ZCL278 inhibits neuronal branching and
growth cone dynamics.
DETAILED DESCRIPTION
[0016] In one aspect, compounds which inhibit Cdc42 are provided.
In one embodiment, the compound has the following structure A:
##STR00001##
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, R.sub.8 are each independently selected from the group
consisting of fluoro, chloro, bromo, nitro, cyano, amino, methyl,
hydroxylmethyl, trifluoromethyl, methoxy, trifluoromethyoxy, and
ethyl.
[0017] In one embodiment, the compound has the following structure
B:
##STR00002##
where A, D, E, G, J, M, W, X, Y, Z are each selected from the group
consisting of nitrogen, carbon, and substituted carbon, and any of
A, D, E, G, J, M, W, X, Y, Z may be missing, such that the ring
structure is less than a six-membered ring.
[0018] In one embodiment, the compound is a combination of
structures A and B. For example, Y and Z are N and R.sub.1, R.sub.2
and R.sub.3, as described for structure A, are attached to X, W,
and M of structure B, respectively.
[0019] In one embodiment, the compound has the following structure
C:
##STR00003##
where R.sub.1 and R.sub.2 are independently selected from the group
consisting of alkyl, cyclic alkyl, aryl, and substituted aryl. In
one embodiment, R.sub.1 is selected from the group consisting
of
##STR00004##
In one embodiment, R2 is selected from the group consisting of
##STR00005##
[0020] In one embodiment, the compound has the structure of formula
I:
##STR00006##
The chemical name of Formula I is
4-(3-(2-(4-Bromo-2-chlorophenoxy)acetyl)
thioureido)-N-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide. For
the sake of simplicity, it will be referred to as ZCL278 in this
application.
[0021] In various embodiments, the compound may be a variant of the
compound of Formula I selected from the group consisting of Cl-2,
Cl-3, Cl-4, and Cl-5, as shown below:
##STR00007##
[0022] In another aspect, the present compounds, including ZCL278,
are used to inhibit Cdc42 and Cdc42 mediated cellular processes.
Because Cdc42 has important roles in cell cycle regulation, cell
movement, adhesion, apoptosis and intracellular transport, and in
the same time it plays important roles in the development of cancer
and invasion, cardiovascular system and respiratory diseases,
neurological diseases, and many other diseases, the presently
described compounds, including ZCL278, can be prepared for the
treatment of malignant tumors. In one embodiment, the described
compounds may be prepared for the prevention and treatment of
malignant tumor development and invasion. In one embodiment, the
described compounds can be prepared for the treatment of
cardiovascular diseases. In one embodiment, the described compounds
can be prepared for the treatment of pulmonary and respiratory
diseases. In one embodiment, the described compounds can be
prepared for the treatment of diseases of the nervous system. In
various embodiments, the described compounds function through
inhibition of Cdc42.
[0023] As used herein, the term "malignant tumor" is meant to
encompass any malignant proliferative cell disorder such as
carcinoma, sarcoma, lymphoma and blastoma. Thus, examples of
cancers that may be treated using the present method include, but
are not limited to, colorectal, prostate, testes, lung, stomach,
pancreas, uterine, cervix, bone, spleen, head and neck, brain such
as glioblastoma multiforme, breast, ovary, stem cell tumors,
non-Hodgkin's lymphoma, Kaposi's sarcoma and leukemia. In the
context of the invention, the terms "treat", "treating" or
"treatment" means alleviating, inhibiting the progression of, or
preventing the cancer, or one or more symptoms thereof.
[0024] In one embodiment, the present compound is provided as a
pharmaceutical composition. In various embodiments, the
pharmaceutical composition may also include one or more
pharmaceutically acceptable excipients such as, but not limited to,
carriers, diluents, adjuvants and vehicles. Excipients that may be
included in the present formulation include preserving or
antioxidant agents, fillers, disintegrating agents, wetting agents,
emulsifying agents, suspending agents, lubricants such as sodium
lauryl sulfate, stabilizers, solvents, dispersion media, tableting
agents, colouring and flavouring agents, coatings, antibacterial
and antifungal agents, isotonic agents and absorption delaying
agents. Supplementary active agents or ingredients may also be
included in the present pharmaceutical composition.
[0025] ZCL278 effectively suppressed GTP-binding activity of Cdc42.
In mouse Swiss 3T3 fibroblasts, ZCL278 affects Cdc42 regulation of
subcellular structures of two of the most important Cdc42
functions: the elimination of the formation of microspikes or
filopodia and disruption of GM130-docked Golgi structures. Compared
with Rac-selective inhibitor NSC23766, ZCL278 reduced the
peri-nuclear accumulation of active Cdc42. ZCL278 inhibits
Cdc42-mediated neuronal branching and growth cone dynamics, and
inhibits metastatic prostate cancer cells PC-3 cell actin-based
movement and migration without disrupting the cell survival. Thus,
because ZCL278 can effectively inhibit Cdc42 regulation of cell
morphology and behavior, the compound can play an important role in
modulating the development and invasion of cancer, cardiovascular
system and respiratory diseases, and nervous system diseases.
[0026] The above-mentioned, as well as other related features and
advantages, are further provided by the following examples.
Example 1
Virtual Screening for Cdc42 Inhibitors
[0027] Virtual screening for Cdc42 inhibitors was performed as
described in Friesland et al., PNAS 110; 4, 161-1266 (2013), which
is incorporated by reference herein in its entirety. Analysis of
the three-dimensional structure of Cdc42-ITSN (intersectin) complex
revealed a main binding region between Cdc42 and ITSN. Hydrogen
bonds were observed between Gin 1380 and Arg1384 of ITSN as well as
between Asn39 and Phe37 of Cdc42. Two clusters of hydrophobic
interactions were found between Leu1376, Met1379, and Thr1383 of
ITSN and Phe56, Tyr64, Leu67, and Leu70 of Cdc42. To screen for
Cdc42 inhibitors, the putative binding pocket on Cdc42 was created
within 7 .ANG. of the center of the above ITSN residues that
interact with Cdc42. The binding pocket consists of 16 Cdc42
residues, including Thr35, Val36, Asn39, Phe56, and Asp57 (FIGS.
1A-1C).
[0028] Glide program was applied to screen from SPECS database to
identify small molecules that can disrupt Cdc42-ITSN interaction.
The structure pose of Cdc42-ITSN complex was from the protein
databank (PDB: 1 Kl1). The ITSN residues that occupy the Cdc42
binding interface are Leu1376, Met1379, Gin1380, Thr1383, and
Arg1384. The binding pocket on Cdc42 was created with residues of
Cdc42 within 7.0 .ANG. of the center of the above five ITSN
residues. After the protein structure was prepared in Protein
Preparation Wizard, the docking grid was generated in the Receptor
Grid Generation module. The 197,000 compounds from SPECS were
screened using HTVS (high-throughput virtual screening) and SP
(standard precision) docking sequentially. From the top ranked
50,000- molecules, more stringent SP (standard precision) docking
resulted in the top ranked 100 molecules. The top ranked 100
molecules were subjected to manual inspection according to the
following criteria. ITSN-like binding posture and occupation for
the Leu1376, Gln1380, Arg1384, Met1379, and Thr1383 residue space
of ITSN should be observed; at least three hydrogen bonds should be
formed, a conserved hydrogen bond with Asn39 or Phe37 of Cdc42
should exist; and diversity of scaffolds should be considered. A
selection of 30 compounds was eventually tested on their ability to
disrupt Cdc42 activity and/or functions.
[0029] Computed binding mode of ZCL278 in Cdc42: as shown in FIG.
1A, one small molecule, termed ZCL278, bound to a well-formed Cdc42
pocket lined by residues Tbr35, Val36, Asp38, Asn38, Phe56, Tyr64,
Leu67, and Leu70. Extensive favorable interactions were found
between ZCL278 and Cdc42 residues. Five hydrogen bonds involving
residues Thr35, Asn39, and Asp57, as well as hydrophobic
interactions associated with residues Val36 and Phe58 were observed
(FIG. 1B). The bromophenyl ring was inserted into the adjacent
GTP/GDP binding pocket. The computed binding mode suggests that
ZCL278 should be able to disrupt the Cdc42-ITSN interaction as well
as GTP/GDP binding (FIG. 1C).
Example 2
Synthesis of Compound ZCL278
[0030] ZCL278 was synthesized by the following synthetic method.
This route of synthesis method is used for illustration, rather
than limiting the present invention. Those skilled in the art can
understand and expect other synthetic methods to synthesize ZCL278,
which also belong to the scope of protection of the present
invention.
[0031] Reaction Reagents and Conditions: (a) K.sub.2CO.sub.3, DMF
(N, N-dimethyl formamide), 70.degree. C.; (b) NaOH, dioxane
(dioxane. Embankments B)/H.sub.20; (c) SOCl.sub.2 (thionyl
chloride), DMF, reflux (reflux); (d) NaSCN (sodium thiocyanate),
acetone (acetone), 0 C-room temperature; (e) 4-amino-N-(
4,6-dimethylpyrimidin-2-yl) benzene-sulfonamide
(4-amino-N-(4,6-dimethyl-2-pyrimidinyl) benzenesulfonamide),
0.degree. C.-room temperature.
[0032] The reaction is as follows:
##STR00008##
Compound 1 (4-bromo-2-chlorophenol) and 2-ethyl bromoacetate in the
presence of K2CO.sub.3 occur under affinity substitution reaction
to give compound 2 (2 -(4-bromo-2-chlorophenoxy.)acetate). Compound
2 is hydrolyzed under alkaline conditions to give Compound 3
(2-(4-bromo-2-chlorophenoxy)acetic acid). Compound 3 in v,
v-dimethylformamide catalyst under reflux to give compound 4 can be
prepared in Thionyl. To form the corresponding isothiocyanate
intermediate compound 4 (2-(4-bromo-2-chlorophenoxy/acetyl
chloride) with sodium thiocyanate, further steps in the reaction
system was addition of 4-amino-N-(4,6-dimethyl-2-pyrimidinyl)
benzenesulfonamide to give compound 5 (i.e., ZCL278).
[0033] Instruments and Reagents: Bruker Avance III 400 MHz NMR
spectrometer; SGWX-4 melting point apparatus; Agilent 1200-High
Performance Liquid Chromatography; ZORBAX Eclipse XDB-C18
Chromatography column (4.6 mm 150 mm, 5 .mu.M); All reagents were
of analytical grade or chemically pure.
[0034] Step 1: 2-(4-bromo-2-chlorophenoxy) acetate (i.e. Compound
2) Synthesis. Anhydrous K.sub.2CO.sub.3 (3.45 g, 25.0 mM) was added
to a solution of 4-bromo-2-chlorophenol (1) (5.2 g, 25.0 mM) and
ethyl 2-bromoacetate (4.3 g, 25.7 mM) in 50 mL DMF. After stirring
overnight at 70.degree. C., the mixture was poured into 150 mL
water and extracted with ethyl acetate (70 mL.times.4). The organic
layer was combined, washed with brine (100 ml.times.3), and dried
over anhydrous Na.sub.2SO.sub.4. The residue after rotary
evaporation was purified by column chromatography to give 2 (6.18
g, 84.1% yield) as a light oil. .sup.1H HMR (400 MHz, CDCl.sub.3):
7.53 (s, 1H), 7.30 (d, 1H, J=8.4 Hz), 6.72 (d, 1H, J=8.4 Hz), 4.68
(s, 2H), 4.26 (q, 2H, J=7.2 Hz) and 1.29 (t, 3H, J=7.2 Hz).
[0035] Step 2: 2-(4-bromo-2-chlorophenoxy) acetic acid (Compound 3)
Synthesis. 1 M NaOH (50 mL) was added to a solution of 2 (5.0 g,
17.0 mM) in 50 ml dioxane. After stirring at room temperature
overnight, the mixture was acidified with 1 M hydrochloric acid to
pH=3. After the reaction mixture was extracted with ethyl acetate
(50 mL.times.4), the organic layer was washed with brine (50 mL),
dried over anhydrous Na.sub.2SO.sub.4, and concentrated to give 3
(4.69 g, 94.3% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6):
7.66 (s, 1H), 7.44 (d, 1H, J=8.8 Hz), 6.97 (d, 1H, J=8.8 Hz), 4.72
(s, 2H).
[0036] Step 3: Preparation of 4-(3-(2-(4-bromo-2-chlorophenoxy)
acetyl) thioureido)-N-(4,6-dimethyl-2-pyrimidinyl:)
benzene--Synthesis of the sulfonamide (i.e., compound 5). Compound
3 (539 mg, 2.0 mM) in 25 mL SOCl.sub.2 and a drop of DMF were
heated to reflux. After 3 hours, SOCl.sub.2 was removed by
distillation and the residue was dried in vacuo for 5 minutes to
give crude acyl chloride 4. The solution of 4 in 10 mL dry acetone
was added drop-wise into sodium thiocyanate (326.8 mg, 4.0 mM) in
10 mL acetone at 0.degree. C. The mixture was stirred at 30.degree.
C. for 2 hours before 4-amino-N-(4,6-dimethyl-2-pyrimidinyl)
benzenesulfonamide (556 mg, 2.0 mM) was added at 0.degree. C. After
stirring at temperature overnight, the mixture was filtered and
washed with water and acetone to give compound 5 (276 mg, 23.6%
yield) as a yellow powder. .sup.1H NMR (400 MHz, DMSO-d6): 12.19
(s, 1H), 11.68 (s, 1H), 11.52 (br s, 1H), 7.99 (d, 2H, J=8.4 Hz),
7.86 ( d, 2H, J=8.4 Hz), 7.70 (d, 1H, J=1.6 Hz), 7.49 (d, 1H, J=8.8
Hz), 7.10 (d, 1H, J.sub.1=8.8 Hz, J.sub.2=1.6 Hz), 6.75 (s, 1H),
5.02 (s, 2H), 2.25 (s, 6H). HPLC: purity 95.9% (254 nm).
Example 3
Direct Binding of ZCL278 and Cdc42 Demonstrated by Fluorescence
Titration and Surface Plasmon Resonance.
[0037] The binding affinity of ZCL278 and Cdc42 was assessed by
using two independent biophysical methods. First, fluorescence
titration of purified Cdc42 by ZCL278 was carried out by monitoring
the change of fluorescence intensity of a tryptophan residue on
Cdc42 upon ZCL278 binding. As ZCL278 has a weak absorption peak at
310 nm, to avoid any experimental error that might result from
potential fluorescence quenching by ZCL278, the fluorescence
emission of Cdc42 was monitored at 350 nm, at which ZCL278 has a
negligible absorption. Thus, a Kd value of 6.4 .mu.M was obtained.
To further demonstrate the direct interaction between ZCL278 and
Cdc42, a surface plasmon resonance (SPR) experiment was performed
by covalently immobilizing purified Cdc42 onto CM5 chips and
varying ZCL278 concentration. The SPR response was observed to
increase along with elevated ZCL278 concentrations, and eventually
gave a Kd of 11.4 .mu.M. To support the Kd measured in our system,
the solubility of ZCL278 was determined to be 181 .mu.M and was
greater than the concentrations used in all examples. The
experimental pKa values of ZCL278 were determined to be
3.48.+-.0.04, 6.61.+-.0.02, and 7.45.+-.0.01. The pKa value of 3.48
is associated to pyrimidine nitrogen that should stay in a neutral
form at pH 7.4 The NH groups corresponding to pKa values of 6.61
and 7.45 should be partially deprotonated and give a population of
charged species in solution. These species in solution may have
modifying effects on membrane transport and binding to Cdc42.
Example 4
Activity Characteristic of the Embodiment of ZCL278
[0038] 1. ZCL278 inhibits Cdc42-mediated microspike formation.
[0039] 30 selected ZCL compounds were assessed for their ability to
inhibit Cdc42-mediated microspike/filopodia formation in
serum-starved Swiss 3T3 fibroblasts. Actin-based
microspikes/filopodia are characteristic of Cdc42 activity in
cultured fibroblastic cells. As shown in FIG. 2A DMSO-treated
(control) cells have few microspikes along its perimeter (Arrows)
as well as the characteristic presence of RhoA-mediated stress
fibers (Asterisk). When arrested fibroblasts were briefly
stimulated with 1 unit/mL of a commercial Cdc42 activator, a
dramatic increase in microspike number and decrease in stress
fibers occurred (FIG. 2A, Activator). Compound ZCL278 was applied
at 50 .mu.M for 1 hour and then stimulated with the Cdc42 activator
for 2 minute. The cell periphery of ZCL278-treated cells resembles
control cells with few microspikes (FIG. 2A, ZCL278). Following 1
hour of ZCL278 treatment and Cdc42 stimulation, there is obvious
inhibition of microspike formation (FIG. 2A, Activator+ ZCL278) as
compared to cells treated with only the activator (FIG. 2A,
Activator).
2 ZCL278 inhibits Cdc42 activity.
[0040] Since ZCL278 showed the direct binding to Cdc42 and
displayed most inhibitory effects in a morphological assay of Cdc42
function, the activity of the compound was examined at a
biochemical level. First, Cdc42 activation was investigated in
human metastatic prostate cancer PC-3 cells that were treated with
the Cdc42 activator or 50 .mu.M ZCL278 for 5, 10, and 15 minutes.
Serine 71 phosphorylation is known to negatively regulate Rac/Cdc42
activity, thus an increase in phospho-Rac/Cdc42 expression is
indicative of a decrease in active (GTP-bound) Rac/Cdc42. As
depicted in FIG. 2B, activation of Cdc42 shows an expected decrease
in phospho-Rac/Cdc42. However, the application of ZCL278 resulted
in a time-dependent increase in Rac/CDc42 phosphorylation.
[0041] Wiskott-Aldrich syndrome Protein (WASP) is a downstream
effector of Cdc42 activation. Tyrosine phosphorylation of WASP is
linked to rapid Cdc42 degradation following its activation. As
shown in FIG. 2B, the Cdc42 activator leads to a decreased
expression of phospho-WASP by 15 minutes while ZCL278 does not
suppress phospho-WASP activity. Thus, ZCL278 inhibits Rac/Cdc42
phosphorylation in a time-dependent manner and maintains tyrosine
phosphorylation of WASP.
[0042] Serine 71 phosphorylation can occur on both Rac and Cdc42.
To directly assess specific Cdc42 activation and inactivation, a
G-LISA, an ELISA-based assay that allows a quantitative
determination of the levels of GTP-bound (active) Cdc42 in cellular
lysates was utilized. Serum-starved Swiss 3T3 fibroblasts were
incubated for 1 hour with 50 .mu.M ZCL278 or 10 .mu.M NSC23766 (Rac
inhibitor), followed by 2 minutes of stimulation with 1 unit/mL
Cdc42 activator. This analysis revealed a significant increase
(70%) in GTP-bound Cdc42 in cells treated with the activator as
compared to control (untreated) cells (FIG. 2C). Cells treated with
ZCL278 showed a dramatic (nearly 80%) decrease in GTP-Cdc42 content
as compared with cells treated solely with the activator.
[0043] Finally, the ability of NSC23766 to cross-inhibit Cdc42
activation was analyzed. NSC23766 was developed in a similar manner
as ZCL278; however, it is specific to Rac and should therefore act
as an additional negative control in this assay. As expected,
NSC23766 does not reduce GTP-Cdc42 content (FIG. 2C). These data
establish that ZCL278 inhibits Cdc42 in two different cell
types.
Example 5
The Immunofluorescence Staining of Active Cdc42/Phosphorylated
RhoA
[0044] 1ZCL278, rather than NSC23766, disrupts perinuclear
distribution of active Cdc42.
[0045] Immunofluorescent staining: Swiss3T3 cells were grown on
coverslips to 30% confluence. In serum-deprived culture, cells were
treated with 10 uM NSC23766 or 50 uM ZCL278 for 1 hour followed by
1 unit/mL Cdc42 agonists (Cytoskeleton Products) treatment for 1
minute. Added agonist alone was used as a positive control while
DMSO was used as a negative control. Cells on the coverslips were
fixed in 4% paraformaldehyde for 15 minutes, and in 0.2% Triton
X-100 for permeabilization for 15 minutes and then blocked in 10%
BSA for 30 minutes.
[0046] Incubation with antibodies: anti-active Cdc42 (mouse
antibody, Neweast Biosciences); phosphorylated RhoA antibody
(rabbit antibody, Santa Cruz Biotechnology Company); GM130 antibody
(mouse antibody, BD Biosciences Company). The next step was to
apply corresponding secondary antibody. Rhodamine Phalloidin was
incubated for 1 hour at room temperature to observe filamentous
actin under the Zeiss Axiovert fluorescent light microscopy of
cells staining. Image processing software MetaMorph-5 was used to
randomly select cells in randomly selected five-point pixels to
obtain the average number of each group for pixel intensity
values.
[0047] The Western blot: PC3 cells cultured to approximately 70% of
the density, then removed serum and cultured for 16 hours, with
addition of drugs; 1 unit/mL Cdc42 agonist (cytoskeleton) 5 uM, 50
uM ZCL278 to treat cells for 5, 10, and 15 minutes respectively.
Cell lysis buffer (Formulation: 50 mM Tris buffer solution pH 7.5,
10 mM magnesium chloride, 0.5 M sodium chloride. 1% Triton X-100
and protease inhibitors) cell lysis and centrifuged at 14000 rpm
and 4.degree. C to obtain extracted protein samples. Western blot
analysis was conducted using antibody: phosphorylated Rac1/Cdc42
(Milipore Company), phosphorylated WASP (Assay Biotech Company),
all at 1:1000, and GAPDH antibody (Calbiochem Company) on PVDF
membrane for chemiluminescence development.
[0048] G-LISA kit: Swiss 3T3 cells were cultured to 40% of the
growth density, then removed serum and cultured for 48 hours,
treated with Cdc42 agonist for 1 minute, and treated with the 50 uM
ZCL278 and 10 uM NSC23766. Cell lysis proteins were extracted
according to kit instructions, and analysis of the quantification
of total protein concentration to be 0.15 mg/ml. Untreated cells or
cells treated with buffer were used as negative control while the
agonist treated cells and active Cdc42 protein were used as a
positive control. Enzyme linked immunosorbent assay measured the
absorbance value of each sample 490 nm light wave.
[0049] Determination of selective inhibition of Cdc42 activation by
ZCL278 at the cellular level. Serum starved Swiss3T3 cells were
treated with 1 unit/ml Cdc42 agonist for 2 minutes, then treated
with 50 uM ZCL278 or NSC23766 10 uM, while DMSO to be used as a
negative control. To determine the role of ZCL278 to selectively
inhibit Cdc42 activity rather than the role of RhoA, cells were
immunostained with anti-active Cdc42 (FIG. 3A, FIG. 3B) and
phosphorylated RhoA (FIG. 3C): Arrow: perinuclear Golgi-endoplasmic
reticulum network; Hoechst staining identifies the nucleus. Bar; 15
um. As shown in FIG. 3A, immunofluorescence staining: mouse
monoclonal antibody against active Cdc42, and Hoechst (identified
nucleus). While control group cells showed activation of Cdc42 in
the nucleus and perinuclear organization, agonists significantly
increased its distribution and distribution in the nucleus, which
is consistent with the role of Cdc42 involved in Golgi protein
transport. ZCL278 obviously reduced this distribution, and reduce
immunoreactivity to active Cdc42. NSC23786 did not have similar
effects. FIG. 3B shows the number of cells of each group with
Golgi-like distribution, randomly selected 6 cells were
independently counted for Golgi-endoplasmic reticulum network (*;
p<0.05). FIG. 3C: Pixel intensity of phospho-RhoA in cells after
treatments with Activator, ZCL278, or NSC23766 was quantified.
Results reflect the averaged intensity generated at five random
points in five independent cells (n=5/group).+-.S.E. (*.
p<0.03). Agonists, ZCL278, and NSC23766 did not alter the
phosphorylation of RhoA. This result suggests that ZCL278 selective
inhibits Cdc42 activity.
2. ZCL278, but not NSC23766, disrupts G130 docked Golgi
organization.
[0050] To determine whether the ZCL278-induced disruption of
peri-nuclear distribution of active Cdc42 reflected its effects on
Golgi organization, GM130, a peripheral cytoplasmic protein that is
tightly bound to Golgi membranes and helps to maintain cis-Golgi
structures, was examined.
[0051] Control, serum-starved Swiss 3T3 cells showed well-developed
stress fibers (FIG. 4, Red) and GM130 immunoreactivity polarizing
to one side of the nucleus (FIG. 4, Green-asterisk). Treatment with
the Cdc42 activator led to increased microspikes, as expected (FIG.
4, Red-arrows and insert, arrowheads), and intense peri-nuclear
GM130 immuroreactivity (FIG. 4, Green-asterisk). As depicted in
FIG. 4 (also see FIG. 2A); ZCL278-treated cells showed not only
fewer microspikes but also a clear reduction of GM130
immunoreactivity as well as its dissipation to both sides of the
nucleus (FIG. 4, Green-asterisk). Rac inhibitor NSC23766 did not
significantly alter GM130 expression or distribution (FIG.
4-Green-asterisk). These results not only further confirm ZCL278 as
a specific Cdc42 inhibitor, but also demonstrate the importance of
Cdc42 in Golgi organization and protein trafficking.
Example 8
[0052] ZCL278 impedes wound healing without disruption of cell
viability.
[0053] Filopodia are dynamic structures that aid cells in
pathfinding and migration, and are largely controlled by Cdc42
activity. Using a metastatic line of human prostate cancer cells
(PC-3), a wound healing assay was used in order to elucidate the
effects of ZCL278 on cellular migration. 1 unit/ml Cdc42 activator;
50 uM or 5 uM of ZCL278; and 10 uM of NSC23786 respectively, were
used to treat cells for 24 hours, with Cdc42 activator as positive
control, and no treatment as negative control. Cells were
photographed at 0 and 24 hours following drug treatments and
MetaMorph software was used to determine the distances cells had
migrated. Black line indicates the boundary of wounded area. Each
experiment was tested with p value (p<0.05).
[0054] Examination of cell survival: PC3 cells were plated at 75000
/ml for 48 hours, serum was removed and 50 uM ZCL278 or 10 uM
NSC23766 was added, and then continue to culture for 24 hours
before staining with trypan blue to determine cell survival
rate.
[0055] As shown in FIG. 5A and quantified in FIG. 5B, Cdc42
activation resulted in a significant increase (58%) in wound
healing ability in comparison to controls (41%). Bar graph shows
the wound area compared to that before drug treatment. The
experiment was repeated three times and the means were obtained:
**; p<0.01, *:p<0.05. Wound closure was less pronounced at 50
.mu.M (8%) than 5 .mu.M (30%) concentrations. Cellular migration
was also significantly reduced with NSC23766 treatment. This result
is to be expected since Rac regulates the formation of
lamellipodia, which are well-described motile structures. These
data, which are in agreement with our biochemical analysis,
suggests that ZCL278 is not only a selective inhibitor of Cdc42
activation but also a potent suppressor of Cdc42-dependent cell
motility.
[0056] In order to ensure that decreases in cellular migration seen
with ZCL278 treatment was due to Cdc42 inhibition (or Rac
inhibition when treated with NSC23766) rather than cell death, cell
viability was tested using the trypan blue dye exclusion assay,
PC-3 cells were arrested in G0, and then 50 .mu.M ZCL278 or 10
.mu.M NSC23766 was applied for 24 hours. FIG. 5C demonstrates that
there was no difference in viability between treated and
non-treated (control) cells. Therefore, the differences seen in
migratory ability is due to ZCL278-mediated Cdc42 inhibition or
NSC23766-mediated Rac inhibition and not cell death.
Example 7
[0057] ZCL278 inhibits neuronal branching and growth cone
dynamics
[0058] Cdc42 plays a crucial role in the establishment of neuronal
morphogenesis. Cdc42's absence in neurons results in a
significantly reduced number of neurites and severely disrupted
filopodia function. Therefore, the ability of ZCL278 to inhibit
neuronal branching in primary neonatal cortical neurons was
tested.
[0059] Primary neonatal day-one mouse brain was incubated with
0.25% trypsin in HBSS for 15 minutes at 37.degree. C. gently
separated neurons were plated on poly-L-lysine coated coverslips,
incubated with DMEM containing 10% bovine serum for 16 hours, then
the media was changed to Neurobasal (Invitrogen). At 5 days in
vitro, the cells were treated with either DMSO or 50 uM ZCL278 for
5 or 10 minutes, then fixed in 4% paraformaldehyde for 15 minutes.
Fluorescein-phalloidin was applied to stain for F-actin structure
and the neuronal morphology was observed under the Zeiss light
microscope.
[0060] Time-lapsed video light microscopy was used to observe the
effects of ZCL278 on neuronal growth cone dynamics. Hamamatsu Orea
digital camera was used to record the cell images under 63.times.
magnification for 10 minute. 300 ms exposure for imaging was used
to minimize phototoxicity. The images were analyzed using MetaMorph
software and statistical analysis was performed. At 5 days cultured
in vitro, cortical neurons extended neurites with multiple branches
(FIG. 6A, Control). 50 .mu.M of ZCL278 was applied for 5 and 10
minutes, while DMSO-treated neurons were maintained as negative
controls. As demonstrated in FIG. 6A, neuronal branching was
suppressed in ZCL278-treated neurons over the time course in
comparison to the highly branched neurites of control cells.
Quantitative measurements found the branch number to be
significantly reduced in ZCL278 treated neurons (FIG. 6B:
Quantitation of neuronal branching number following ZCL278
treatment: The data represent the means after three independent
experiments.+-.standard errors. *: p<0.01).
[0061] Cdc42 is also widely known to control filopodia and
microspikes at the leading edge of migrating growth cones.
Time-lapse video light microscopy shows a control cortical neuron
with multiple microspikes or filopodia extended from the growth
cone (FIG. 6C). However, ZCL278 treatments resulted in rapid
retraction of filopodia within 4 minutes (FIG. 6C). Thus, these
studies further support ZCL278 as an effective small-molecule
inhibitor of Cdc42-mediated neuronal branching and growth cone
motility.
[0062] The present description provided computer simulation methods
which were applied for high-throughput in silico screening of
compounds that target chimeric Cdc42-GEF structures. Based on the
structure characteristics of Cdc42 and its specific GEF intersectin
(ITSN), the three-dimensional structure of the described compounds
can fit exactly into the pocket that intersectin interacts with
Cdc42. Additional research successfully screened and identified a
compound ZCL278 as a cell permeable Cdc42 specific inhibitor.
[0063] The present description confirmed the active properties of
ZCL278 as the first small molecule inhibitor of Cdc42, selectively
targeting Cdc42 and its GEF intersectin. Cell wound healing
experiments showed that activated Cdc42 promotes the wound closure
and tumor cell metastasis. ZCL278 significantly inhibits the
migration of PC3 cells in a concentration-dependent manner. ZCL278
inhibits cell migration but it is not cytotoxic and does not cause
cell death.
[0064] Also, the neonatal central neurons experiments have proved
that the Cdc42 plays an important role in the development of
neurons. Garalov et al. (J. Neurosci. 27(48): 13117-13129) showed
that Cdc42-deficient mice exhibited brain and neuronal development
which was severely disrupted. These mice showed a series of brain
malformations, including the reduction of the axon bundles, as well
as neurons filamentous pseudopodia dynamics and reduced growth
cone, and suppression of axonal extension. In fact, the movement of
axons and dendrites is mainly actin-based, the process also
regulated by Cdc42. ZCL278 can reduce the number of branches of the
newborn central neurons, and inhibition of the growth cone
dynamics. In summary, ZCL278, which targets Cdc42-ITSN, is the
first small molecule inhibitor that can be effectively used in the
studies of molecular functions of Cdc42 in cancer and neurological
disorders.
[0065] Although the present invention has been presented with the
disclosed embodiments, it is not intended to limit the present
invention. A person having ordinary skill in the art, without
departing from the spirit and scope of the present invention, will
recognize that modifications and improvements of these embodiments
are within the scope of the present description.
* * * * *