WO2012159456A1 - 一种cdc42抑制剂及其应用 - Google Patents

一种cdc42抑制剂及其应用 Download PDF

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WO2012159456A1
WO2012159456A1 PCT/CN2012/000708 CN2012000708W WO2012159456A1 WO 2012159456 A1 WO2012159456 A1 WO 2012159456A1 CN 2012000708 W CN2012000708 W CN 2012000708W WO 2012159456 A1 WO2012159456 A1 WO 2012159456A1
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cdc42
zcl278
cell
cells
compound
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PCT/CN2012/000708
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French (fr)
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陆群
周虎臣
陈燕华
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昂科生物医学技术(苏州)有限公司
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Priority to EP12789644.7A priority Critical patent/EP2716292B1/en
Priority to JP2014511708A priority patent/JP6001650B2/ja
Priority to KR1020137034199A priority patent/KR20140028078A/ko
Publication of WO2012159456A1 publication Critical patent/WO2012159456A1/zh
Priority to US14/087,662 priority patent/US20140194451A1/en
Priority to US14/807,386 priority patent/US9725417B2/en
Priority to US15/671,388 priority patent/US20170334863A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/32—One oxygen, sulfur or nitrogen atom
    • C07D239/42—One nitrogen atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/04—Derivatives of thiourea
    • C07C335/06—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to acyclic carbon atoms
    • C07C335/10—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
    • C07C335/12—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to acyclic carbon atoms of an unsaturated carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/69—Benzenesulfonamido-pyrimidines

Definitions

  • the present invention relates to a compound, and in particular to a Cdc42 inhibitor and its use.
  • the cell division cycle protein Cdc42 is a subclass of the Rho GTPase family of small G proteins and is an important regulatory protein of many cellular biological functions. For the first time in Saccharomyces cerevisiae, it was involved in cell polarization and subsequently recognized its important role in cytoskeletal reorganization, cell endocytic transport pathways, cell cycle regulation and cell transcription. Like most GTPases, signal transduction is achieved by GTP binding through GDP exchange, activating Cdc42.
  • the cycle between this nucleotide-restricted conformation of the Rho GTPase family is regulated by three important proteins: guanine exchange factor (GEF), which catalyzes the release of GDP and the binding of GTP; GTPase activating protein (GAP), as The negative regulator accelerates the hydrolysis of the Rho GTPase, changing the Rho GTPase from an active state to an inactive state; the GDP dissociation inhibitor (GDI) prevents GDP from separating from the Rho GTPase and inhibits Rho GTPase activity.
  • GEF guanine exchange factor
  • GAP GTPase activating protein
  • GDI GDP dissociation inhibitor
  • Cdc42 is widely involved in the pathophysiology of human diseases including tumors and neurodegenerative diseases.
  • no Cdc42 mutant gene was found in human tumors. Its abnormal form is mainly characterized by dysregulation or overexpression in tissue and dependent microenvironment, which is closely related to tumor cell transformation and metastasis.
  • Cdc42 controls the fate of normal brain development, and Cdc42 knockout mice do not live to develop and exhibit significant brain malformations.
  • EMT epithelial mesenchymal transition
  • Rho GTPase family regulates a variety of biochemical functions of cells, such as cell membrane transport, cell cycle regulation, and cytoskeletal organization, which are related to cell morphology, cell movement, and cell fate. Recent studies have shown that the pathogenesis and progression of many diseases are related to the abnormality or loss of control of Rho GTPase family functional proteins, and thus become an important target for drug development.
  • Rho GTPase small molecule modulators of the Rho GTPase family has also facilitated the study of functional proteins.
  • the novel brain, cardiovascular active drug-fasudil and the small molecule compound Y27632 are recognized as potent inhibitors of RhoA pl60 RQeK, a downstream RhoA signaling molecule.
  • RhoA pl60 RQeK a downstream RhoA signaling molecule.
  • RhoA pl60 RQeK a downstream RhoA signaling molecule.
  • RhoA pl60 RQeK a downstream RhoA signaling molecule.
  • NSC23766 is a compound based on computer-simulated structural screening that is compatible with the surface structure of Racl molecules, and Racl is known to be critical for GEF binding (Gao, Y., JB 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 Racl activation and Racl flaky pseudo formation.
  • NSC23766 acts on human prostate cancer cell lines, inhibits cell proliferation, inhibits non-parking growth, and reduces the invasive phenotype of cells, which are dependent on the activity of endogenous Racl.
  • NSC23766 can ameliorate spinal cord injury (SCI)-induced neuropathic pain mediated by Racl (Tan, AM, S. Stamboulian, et al. (2008). Neuropathic pain memory is maintained by Racl - Regulated dendritic spine remodeling after spinal cord injury. J Neurosci. 28(49): 13173-13183 ).
  • SCI spinal cord injury
  • Racl Racl-Regulated dendritic spine remodeling after spinal cord injury. J Neurosci. 28(49): 13173-13183 ).
  • the ZCL278 provided by the invention can inhibit all the processes involved in Cdc42 and effectively inhibit the action of Cdc42. Because Cdc42 plays an important role in cell cycle, exercise, adhesion, apoptosis, and intracellular transport. It also plays an important role in the development and invasion of cancer, cardiovascular and respiratory diseases, nervous system diseases, and many other diseases. Therefore, the ZCL278 provided by the present invention can prepare a medicament for treating a malignant tumor, and specifically, a medicament for preventing the development and invasion of a malignant tumor can be prepared.
  • the above compounds can also be used for the preparation of a medicament for the treatment of diseases of the cardiovascular system.
  • the above compounds can also be used for the preparation of a medicament for the treatment of respiratory diseases.
  • the above compounds can also be used for the preparation of a medicament for the treatment of diseases of the nervous system.
  • the above compounds can exert the above effects by inhibiting the function of Cdc42.
  • Another object of the present invention is to provide a Cdc42 inhibitor comprising the compound of the above formula I.
  • the compound ZCL278 provided by the present invention is effective for inhibiting the activity of GTP-binding Cdc42.
  • compound ZCL278 affects two major effects of Cdc42 regulation of subcellular structure: elimination of cell microspin formation, disruption of GM130 docking The structure of the Golgi body.
  • NSC23766 Compared to Rac's selective inhibitor NSC23766, ZCL278 reduced the accumulation of nuclear ecto-active Cdc42.
  • ZCL278 inhibits Cdc42-mediated neuronal branching and its growth cone dynamics, and it is found that it can inhibit actin-based cell migration and migration of metastatic prostate cancer cell line PC-3 without destroying cell viability. Therefore, ZCL278 can effectively inhibit the regulation of Cdc42 on cell morphology and behavior, and can play an important role in cancer development and invasion, cardiovascular and respiratory diseases, and nervous system diseases.
  • Figure 1 A is a computer simulation of ZCL278 combined with Cck42 molecules (a color map generated in actual experiments): the gray surface (arrow 1 in the figure) shows the protein, and the green bar (arrow 2 in the figure) shows base.
  • Figure IB shows the binding of ZCL278 to Cdc42 amino acid residues: green rod is ZCL278 molecule (arrow 3 in the figure), gray (arrow 4 in the figure) indicates Cdc42 structure, orange line (arrow 5 in the figure) Hydrogen bonds are formed between the two molecules.
  • Figure 1C shows the structure of the Cdc42-ZCL278 complex with the GMP-PCP protein molecule (protein database: 2QRZ): green rod is ZCL278 molecule, gray indicates Cdc42 structure, cyan bar indicates GMP-PCP structure (GMP-PCP is GTP analog, beta, gamma-methylene diphosphate guanylate).
  • GMP-PCP protein database: 2QRZ
  • Figure 2A - Figure 2C shows the activity characteristics of ZCL278.
  • Figure 2 ⁇ shows that ZCL278 inhibits Cdc42-mediated cell microspin formation.
  • Figure 2B shows that ZCL278 inhibits the activation of endogenous Rac/Cdc42.
  • Figure 2C shows that ZCL278 inhibits the activation of stimulatory Cdc42.
  • Figures 3A-3C show immunofluorescence staining of activated Cdc42/phosphorylated RhoA.
  • Figure 4 shows that ZCL278 disrupts the organization of intracellular GM130 protein docking Golgi.
  • Figure 5A-5C shows that ZCL278 blocks cell migration but does not affect cell viability.
  • FIGS 6A-6C show that ZCL278 inhibits neuronal branching and growth cone dynamics.
  • DETAILED DESCRIPTION OF THE INVENTION Example 1 Virtual Screening of Cdc42 Inhibitors
  • the Glide program was used to screen small molecule compounds in the SPECS database that disrupt Cdc42 and ITSN linkage.
  • the crystal structure of the Cdc42-ITSN complex is derived from the protein database, ID: 1KI1.
  • the amino acid residues in the ISTN occupying the Cdc42 binding site are Leu376, Met379, Glnl380, Thrl383, Argl384.
  • the distance from the five amino acid centers is within 7A.
  • the Cdc42 amino acid residue forms a binding pocket for Cdc42.
  • the initial structure of the model was processed using the Protein Preparation Wizard.
  • the Glide I Receptor Grid Generation module was used for molecular docking.
  • the ligand preparation module Ligprep was used to perform high-throughput virtual screening of 197,000 compounds in the Specs database and screened according to the following criteria.
  • the top 100 compounds will be selected: ITSN-like binding state, occupying the spatial structure of Leul376, Glnl380, Argl384, Metl379 and Thrl383 residues on ITSN; forming at least 3 hydrogen bonds; and conserving Asn39 in Cdc42 molecule or The Phe37 residue can form hydrogen bonds; the molecular skeleton is diverse.
  • the top 50000 molecules were subjected to standard precision calculations to exclude the first 100 molecules, and finally 30 compounds were selected to test their effects on the activity and/or function of Cdc42.
  • the ZCL278 chimeric Cdc42 protein has a pocket structure formed by the Thr35, Val36, Asp38, Asn39, Phe56, Tyr64, Leu67 and Leu70 residues.
  • a benign benign interaction is also found between the two molecules. It forms 5 hydrogen bonds with amino acid residues involved in Thr35, Asn39 and Asp57 residues, and forms a hydrophobic structure with Val36 and Phe56 residues.
  • the bromobenzene ring in the subunit is inserted into the binding pocket of GDP/GTP.
  • the combination of computer-simulated ZCL278 and Cdc42 can disrupt the Cdc42-ISTN GDP/GTP transition state.
  • ZCL278 can be synthesized by the following synthesis methods. The following synthesis methods are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art will understand and appreciate that other synthetic methods can be used to synthesize ZCL278, which also falls within the scope of the present invention. .
  • Reaction reagents and conditions (a) K 2 C0 3 , DMF (N, N-dimethylformamide), 70 ° C; (b) NaOH, dioxane (dioxane) / H 2 0; c) SOCl 2 (dichlorosulfoxide), DMF, reflux (d); (d) NaSCN (sodium thiocyanate), acetone (acetone), 0 °C-rt; (e) 4-amino-N-( 4,6-dimethylpyrimidin-2-yl)benzene-sulfonamide (4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide), 0°Cr.t.
  • Step 1 Synthesis of ethyl 2-(4-bromo-2-chlorophenoxy)acetate (ie compound 2)
  • Step 2 Synthesis of 2-(4-bromo-2-chlorophenoxy)acetic acid (ie compound 3)
  • Step 3 4-(3-(2-(4-Bromo-2-chlorophenoxy)acetyl)thioureido)-N-(4,6-dimethyl-2-pyrimidinyl)benzene Synthesis of sulfonamides (ie compound 5)
  • ZCL278 inhibits Cdc42-mediated micro-spin formation
  • the above agonist was added for 2 minutes, and then the cells were treated with 50 uM of ZCL278 for 1 hour, and ZCL278 significantly inhibited the formation of micro-spins compared to the agonist alone. This result indicates that the compound can be used as a candidate for a Cdc42 inhibitor for subsequent studies. ZCL278 showed the strongest inhibition in the test of 30 compounds.
  • FIG. 2A shows that ZCL278 inhibits Cdc42-mediated cell micro-spin formation.
  • the small molecule ligands screened by the computer were applied to serum-free cultured Swiss3T3 cells.
  • DMSO was used as a negative control, and 1 unit/mL of Cdc42 agonist (product of cytoskeleton) briefly stimulated cells (1 minute).
  • the agonist was applied for 1 minute and then the cells were treated with 50 uM ZCL278 for 1 hour.
  • the same conditions were treated with lOuM NSC23766 cells as a reference.
  • F-actin was identified by hybridization with rhodamine-Phalloidin fluorescent antibody. Very few micro-spinches are visible at the edge of the cell (arrow marks), and the asterisk indicates normal stress fiber distribution. Bar: 5um.
  • ZCL278 performed best in morphological tests related to Cdc42 activity, and then tested its activity at the biochemical level.
  • the human lysing prostate cancer cells PC3 were treated with Cdc42 agonist and ZCL278 (50uM) for 5, 10, and 15 minutes, and the cell lysate was used to extract protein samples.
  • Westemblot was used to detect the level of phosphorylated Rac/Cdc42 (upper layer), phosphorylated WASP protein (middle layer), and GAPDH protein as reference (lower layer).
  • Phosphorylation of the serine residue at position 71 is a negative regulatory mechanism of RAC/Cdc42 protein activity, so an increase in phosphorylated Rac/Cdc42 protein expression is indicative of a decrease in the activity Rac/Cdc42 (GTP-binding).
  • the expression of phosphorylated Rac/Cdc42 protein was continuously reduced by agonist, while 50 uM of ZCL278 increased the expression level of the protein in a time-dependent manner.
  • WASP protein is a downstream effector of Cdc42 activation. It induces the reorganization of cytoskeleton actin and the formation of micro-spindle and filopodia through the action of Arp2/3 complex. Its tyrosine phosphorylation is associated with rapid degradation after Cdc42 activation. As shown in Figure 2B, agonist action reduced phosphorylated WASP protein expression 15 minutes later, whereas ZCL278 did not inhibit expression of this protein over time. This data shows that ZCL278 inhibits the endogenous levels of phosphorylated Rac/Cdc42 protein in a time-dependent manner and maintains the level of tyrosine phosphorylated WASP protein.
  • Phosphorylation of the serine residue at position 71 can occur in both Rac and Cdc42 proteins.
  • the level of GTP-bound Cdc42 was quantified using the G-LISA kit. The cells were stimulated with 1 unit/mL of Cdc42 agonist for 1 minute, and then swiss 3T3 cells were treated with 50 uM of ZCL278 or 10 ⁇ M of Rac agonist NSC23766 for 1 hour, respectively, and cell protein samples were extracted, and GTP-binding type was quantitatively detected by G-LISA kit. The level of Cdc42.
  • Antibody incubation Primary antibody: Activation Cdc42 (murine antibody, Neweast Biosciences); Phosphorylated RhoA antibody (rabbit antibody, Santa Cruze Biotechnology); GM130 antibody (murine antibody, BD Biosciences), 1 : 100 dilution. After hybridization of the corresponding secondary antibody, rhodamine Phalloidin was incubated for 1 hour at room temperature to observe actin. Cell staining was observed by a Zeiss Axiovert electron microscope. Using image processing software MetaMorph randomly selected 5 cells to randomly select 5 points of pixels for the mean calculation to obtain the average pixel intensity value of each group.
  • Equal-concentration protein samples were subjected to Western blot analysis, and antibodies: phosphorylated Racl/Cdc42 (Milipore), phosphorylated WASP (Assay Biotech), were diluted in 1:1000 ratio, and hybridized with GAPDH antibody (Calbiochem) 1:2000.
  • the PVDF film was developed by chemiluminescence.
  • G-LISA kit analysis Swiss3T3 cells were cultured to a growth density of 40%, serum was removed for 48 hours, BCdc42 agonist was applied for 1 minute, and treated with 50 uM of ZCL278 and lOuM of NSC23766, respectively.
  • the cell extract protein was lysed according to the kit instructions, and the total protein concentration was determined to be 0.15 mg/ml for analysis.
  • Cells and buffers without any treatment were used as negative controls, and only agonist-treated cells and activated Cdc42 protein were used as positive controls.
  • the absorbance value at 490 nm of each sample was measured by enzyme labeling.
  • cytochemical immunofluorescence staining mouse monoclonal antibody GTP-binding Cdc42 antibody and Hoechest (Hurst) stain (identical nuclei).
  • the cells in the control group showed a distribution of activated Cdc42 in the periphery of the nucleus; the agonist effect increased its distribution and also distributed in the nucleus, which was consistent with the role of Cdc42 in Golgi protein transport; ZCL278 significantly destroyed this Tissue distribution and reduced immunoreactivity with anti-activated Cdc42 antibodies; NSC23766 has no effect on this.
  • Figure 3B shows the number of cells in each group of Golgi-like distributions: Quantification of the tissue distribution of the peripheral Golgi-endoplasmic reticulum network identified by activated Cdc42 antibody: 6 independent intervals of randomly selected cells. Golgi-endoplasmic reticulum Network (*: p ⁇ 0.05).
  • Figure 3C Quantification of the average pixel intensity value of the phosphorylated RhoA signal, data from 5 independent cells random 5 signal pixel intensity values are averaged. ⁇ indicates the mean difference, *: p ⁇ 0.03: The agonist, ZCL278, and NSC23766 did not show an effect on phosphorylated RhoA. This result indicates that ZCL278 selectively inhibits Cdc42 activity.
  • Swiss3T3 cells were cultured in serum, and Cdc42 agonist, ZCL278, NSC23766, and DMSO were added as negative controls, respectively.
  • Cells were immunofluorescently stained with Rhodamine-phalloidin (red), anti-GM130 antibody (green), Hoechest (blue:).
  • Rhodamine-phalloidin red
  • anti-GM130 antibody green
  • Hoechest blue
  • the arrow indicates the micro-spindle of the cell (red fluorescence in the actual test)
  • the asterisk indicates the Golgi structure of the GM130 antibody label (green fluorescence in the actual test).
  • the serum-suppressed Swiss3T3 cell control group showed characteristic stress fibers (shown by the arrow in the figure, red fluorescence in the actual test), corresponding to the distribution of GM130 antibody on one side of the nucleus (shown by the asterisk in the figure, actual test) Medium is green fluorescent).
  • the cells treated with Cdc42 agonist showed an increase in micro-spines, and GM130 increased in the outer nucleus of the nucleus.
  • ZCL278-treated cells showed significant reduction in micro-rats and a decrease in GM130 (ZCL278: red fluorescence in actual experiments) and dissipated to both sides of the nucleus (shown by an asterisk in the figure, green fluorescence in actual experiments).
  • Filamentous pseudopods are the movemental structures of cells, which are guided by cell migration and growth pathways, and are mainly regulated by Cdc42 activity.
  • the PC3 cell monolayer was over 6 well plates, and the serum was cultured. Three callus areas were made in each well of the lml tip, and the cells were removed by serum-free medium. Cells were treated with 1 unit/ml Cdc42 agonist, 50 uM, 5 uM ZCL278 and 10 uM NSC23766 for 24 hours, respectively.
  • the Cdc42 agonist was used as a positive control and no treatment was added as a negative control.
  • Drug effects Cell images were taken at 0 and 24 hours, respectively, and the distance of cell migration was analyzed using MetaMorph graphical software. Black lines indicate the boundaries of the callus area. Hypothesis testing p-value analysis (p>0 consent05) was performed for each experimental result.
  • PC3 cells were cultured at a cell density of 75000/mi for 48 hours, serum was removed and cultured with 50 uM of ZCL278 or iOuM NSC23766 for 24 hours, and cell viability was calculated by placental blue staining.
  • Figure 5A and Figure 5B (take the shortest distance between the borders of the callus to quantify the width of the area.
  • the histogram shows the percentage of the initial callus compared to the average, three independent experiments take the mean, ⁇ indicates the mean difference, **: p ⁇ 0.01, * :p ⁇ 0.05), compared with the negative control group (41% healing rate), the agonist significantly enhanced the healing ability of the cell wound (59%);
  • ZCL278 can inhibit the migration of cells at two concentrations, at high concentration The inhibitory effect is stronger (50uM-8%; 5uM ⁇ 30%); NSC23766 also shows a significant inhibition of cell migration (because Rac protein regulates cellular lamellipodia - also the formation of cellular motility). This result is consistent with biochemical test data, suggesting that ZCL278 is not only an inhibitor of Cdc42, but also effectively inhibits cell movement dependent on Cdc42.
  • Example 6 ZCL278 inhibits neuronal branching and growth cone dynamics
  • Cdc42 regulates the branching and growth of neurites.
  • Garvalor et al. confirmed the critical role of Cdc42 in neuronal morphogenesis by Cde42 gene knockout technology, which determines the fate of neurons.
  • the loss of Cdc42 in the collected neurons leads to a significant reduction in the number of neurites, and the function of filopodia Serious destruction. Therefore, the role of ZCL278 in inhibiting the branching of neurons will be examined.
  • mice born in one day old were taken from brain tissue and placed in HBSS buffer containing 0.25% trypsin for 15 minutes at 37°C. Carefully pipet the cells from the cells seeded with Poly-L-lysine. On the cover sheets, the cells were cultured for 16 hours in DMEM containing fetal bovine serum, and then cultured in the same manner as Neurobasal medium (Invitrogen). On the fifth day of culture, the cells were treated with DMSO or 50 uM of ZCL278 for 5 and 10 minutes respectively, and then the cells were fixed with 4% paraformaldehyde for 15 minutes. The fluorescent antibody Fluorescein-phalloidin was hybridized to label the F-actin structure, and the neurons were observed under a Zeiss microscope. form.
  • a microscopic time-lapse photography technique was used to record a 63-fold magnification of cell images in a 10-minute period with a Hamamatsu Orca digital camera, and a 300-millisecond exposure shot to minimize light on the cells.
  • Toxicity, ⁇ MetaMorph graphical software analyzes images and statistics and analysis.
  • FIG. 6A Primary cultured neurons in the neonatal center, as shown in Figure 6A, showed that the neurons grew a certain branch on the fifth day of culture. Neurons were treated with 50 uM of ZCL278 for 5 and 10 minutes, respectively, and DMSO was used as a negative control. Over time, ZCL278 inhibited the branching of neurons, and quantitative analysis showed a significant decrease in the number of neuronal branches after drug treatment (Fig. 6B: Quantification of number of neuronal branches after ZCL278 treatment: average of 3 independent experiments. ⁇ indicates mean difference, * : p ⁇ 0.01).
  • Cdc42 is recognized to regulate the formation of microspinous processes and filopodia in the growth cone-directed tip.
  • Time-lapse photographic images (Fig. 6C) show that control neuronal cells produce many microspinous processes or filopodia from the growth cone; however, ZCL278 causes rapid retraction of filopodia within four minutes. Bar: Lum. Therefore, this further demonstrates that ZCL278 is a small molecule inhibitor that effectively regulates Cdc42-mediated neuronal branching and growth cone dynamics.
  • the present invention employs a high throughput computational simulation method to screen for compounds in which the chimeric Cdc42 molecule binds to the critical structure of GEFs. Based on the structural characteristics of Cdc42 binding to its specific GEF molecule intersectin (ITSN), the three-dimensional structure of the compound just fills the pocket of the intersectin molecule is continuing to be studied. Thus, a compound ZCL278, a cell-permeable Cdc42-specific inhibitor, was successfully screened.
  • the present invention demonstrates that the activity profile of ZCL278, as the first small molecule Cdc42 inhibitor, selectively targets the attachment of Cdc42 to its GEF.
  • Cdc42 activation can promote the closure of callus, indicating that C (k42 promotes the transfer of tumor cells after activation.
  • ZCL278 significantly inhibits the migration of PC3 cells in a concentration-dependent manner.
  • ZCL278 is not a cytotoxic substance. , does not cause the cell migration effect caused by the death of tumor cells.
  • ZCL278 can reduce the number of branches of newborn central neurons and inhibit the dynamics of growth cones.
  • ZCL278 is the first small molecule inhibitor targeting Cdc42-ITSN linkage, which can be effectively used for the study of Cdc42 molecular function in tumors and neuropathy.

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Abstract

一种具有式I结构的化合物，该化合物能够用于制备Cdc42抑制剂。丝状伪足的形态学分析，Western blot Cdc42磷酸化及下游效应蛋白WASP的分析，以及细胞伤口愈合试验和生长锥的形成试验，均表明本发明提供的化合物能够抑制Cdc42参与的所有过程，有效抑制Cdc42的作用。有效抑制actin参与的细胞功能，如高尔基体组织和细胞运动。

Description

一种 Cdc42抑制剂及其应用 技术领域 本发明涉及一种化合物， 具体地涉及一种 Cdc42抑制剂及其应用。 背景技术 细胞分裂周期蛋白 Cdc42是小 G蛋白的 Rho GTP酶家族的一个亚类， 是许多细胞生物学功能的重要调控蛋白。 首次在 Saccharomyces cerevisiae 中发现， 参与细胞极化， 随后认识到其在细胞骨架重组、 细胞的胞吞运输 途径、 细胞周期调控和细胞转录中都发挥着重要作用。 与多数 GTP酶一 样， 通过 GDP交换为 GTP结合实现信号转导， 激活 Cdc42。 Rho GTP酶 家族的这种核苷酸限制型构象之间的循环由 3类重要蛋白调节： 鸟嘌呤 交换因子 （GEF ) , 催化 GDP的释放和 GTP的结合； GTP酶激活蛋白 ( GAP ) , 作为负向调节因子加速 Rho GTP酶的水解， 使 Rho GTP酶 由活性状态变为无活性状态； GDP解离抑制因子 (GDI) , 阻止 GDP从 Rho GTP酶上分离， 抑制 Rho GTP酶活性。
近年的研究揭示 Cdc42 的异常活性广泛参与了包括肿瘤和神经退行 性疾病等人类疾病的病理生理。有趣的是在人类肿瘤中没有发现 Cdc42的 突变基因，它的异常形式主要表现为在组织和依赖的微环境中失调或过表 达， 与肿瘤细胞的转化及转移密切相关。 作为关键的神经元形态形成的调 控器， Cdc42掌控着正常大脑发育的命运， Cdc42敲除的小鼠不能活至出 生并表现明显的大脑畸形。既往不同的研究显示 Cdc42的活化对上皮细胞 间充质细胞转变（EMT)和因此发生的胞内物质运输十分重要， 这对肿瘤 细胞的侵袭是非常必要的。
但是，在三大典型的 Rho超家族亚类中，对于 Cdc42的研究远滞后于 RhoA和 Racl。 这部分是由于 Cdc42的活化 /失活形式的转换非常快速， 也缺乏选择性的小分子研究工具来帮助直接了解这个过程。
Rho GTP酶家族参与的信号通路调控细胞多种生化功能， 诸如细胞膜 的物质运输、 细胞周期调控和细胞骨架的组织， 这关乎细胞形态、 细胞运 动以及细胞命运。 近年的研究显示很多疾病的病理发生和进程与 Rho GTP 酶家族功能蛋白的失常或失控有关， 因此成为药物开发的重要靶点。
同时， Rho GTP酶家族的小分子调节剂的应用也促进了对其中功能蛋 白的研究。 例如， 新型脑、 心血管活性药一法舒地尔和小分子化合物 Y27632是公认的 RhoA下游效应信号分子 Rho/pl60RQeK强效抑制剂。 作 为 Racl蛋白的选择性抑制剂， 近来对靶向 Racl-GEF连接的小分子化合 物 NSC23766的研究也大大促进了对 Racl蛋白功能的认识。 但是， 却几 乎没有有效的 Cdc42选择性抑制剂。 Secramine, 天然产物加兰他敏的类 似物， 近来认为它通过 RhoGDIl可抑制依赖于 Cdc42的高尔基体-细胞膜 间物质转运。不同于广泛应用的 Y27632 ( 1903篇相关文献）和 NSC23766 ( 115篇相关文献）， Secramine的获得十分有限， 研究甚少（仅有 9篇相 关文献）。 Cdc42 的失调在很多方面与肿瘤发生相关， 包括肿瘤的转化和 转移； 另外神经元的发展与维持也严重依赖于正常的 Cdc42活性。
NSC23766是一个基于计算机模拟的结构筛选的化合物，与 Racl分子 的表面结构契合， 而已知 Racl 对 GEF 的结合至关重要 （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可以抑制血清中或生长因子诱导的 Racl 活化和 Racl片状伪的形成。 NSC23766作用于人类前列腺癌细胞株， 能抑制细胞 增殖， 抑制非停泊性生长， 并且降低细胞的侵袭表型， 而肿瘤细胞的这些 表型均依赖于内源性 Racl的活性。此外， 新的研究表明， NSC23766可以 改善由 Racl所介导的脊髓损伤（SCI)诱发的神经性疼痛（Tan, A. M., S. Stamboulian, et al. (2008). Neuropathic pain memory is maintained by Racl -regulated dendritic spine remodeling after spinal cord injury. J Neurosci. 28(49): 13173-13183 )。 发明内容 本发明的目的在于提供一种化合物， 其具有下式 I的结构： 人 8"~ „ 、。 式 I， 其名称为：
4-(3-(2-(4-Bromo-2-chlorophenoxy)acetyl)thioureido)-N-(4,6-dimethylpy rimidin-2-yl)benzenesulfonamide， S卩 4-(3-(2-(4-溴 -2-氯 -苯氧基)-乙酰基) -硫 脲基:) -氮 -(4, 6-二甲基嘧啶 -2-基：)苯磺酰胺。 为了简便起见， 本申请文件中 将其命名为 ZCL278。
本发明提供的 ZCL278, 能够抑制 Cdc42参与的所有过程， 有效抑制 Cdc42的作用。 因为 Cdc42在细胞周期、 运动、 黏附、 细胞凋亡和细胞内运输等中都 具有重要的作用。同时在癌症的发展和侵袭，心血管系统和呼吸系统疾病， 神经系统疾病， 以及其它许多疾病中均有重要的作用。所以本发明提供的 ZCL278可以制备用于治疗恶性肿瘤的药物， 具体说， 可以制备用于防治 恶性肿瘤的发展和侵袭的药物。
上述的化合物还可以制备用于治疗心血管系统疾病的药物。
上述的化合物还可以制备用于治疗呼吸系统疾病的药物。
上述的化合物还可以制备用于治疗神经系统疾病的药物。
其中， 上述的化合物可以通过抑制 Cdc42的功能发挥上述作用。
本发明的另一目的是提供一种 Cdc42抑制剂， 其包含上述式 I所示的 化合物。
本发明提供的化合物 ZCL278能够有效抑制 GTP结合 Cdc42 的活性。 在小鼠成纤维细胞 Swiss 3T3中， 化合物 ZCL278影响 Cdc42调控亚细胞 结构的两个最主要的效应表现： 消除细胞微棘的形成， 破坏 GM130对接 高尔基体的结构。 相比 Rac的选择性抑制剂 NSC23766, ZCL278可减少 细胞核外缘活性 Cdc42的积聚。 ZCL278抑制 Cdc42介导的神经元分支及 其生长锥动力学， 同时发现其在不破坏细胞活率的条件下即可抑制转移性 前列腺癌细胞 PC-3的 actin为基础的细胞运动及迁移。 所以， ZCL278能 够有效抑制 Cdc42对细胞形态及行为学的调控， 在癌症的发展和侵袭， 心 血管系统和呼吸系统疾病， 以及神经系统等疾病中均能发挥重要的作用。
为让本发明之上述和其它目的、 特征和优点能更明显易懂， 下文特举 较佳实施例， 并配合附图， 作详细说明如下。 附图说明 图 1A-图 1C: ZCL靶向 Cdc42- ITSN (交叉蛋白） 连接的结构鉴定。 其 中，
图 1 A为计算机模拟 ZCL278结合 Cck42分子（实际试验中生成的为彩色 图）： 灰色表面（如图中的箭头 1 )所示为蛋白质， 绿色棒（如图中的箭头 2 ) 所示为配基。 图 IB为 ZCL278与 Cdc42氨基酸残基的结合： 绿色棒为 ZCL278分子 （如图中的箭头 3 )， 灰色 (如图中的箭头 4)示意 Cdc42结构， 橙色线条 （如图中的箭头 5 ) 示意两分子间形成氢键。 图 1C表示将 Cdc42- ZCL278复合物与 GMP- PCP蛋白质分子（蛋白质数据库： 2QRZ) 的 结构进行叠合： 绿色棒为 ZCL278分子， 灰色示意 Cdc42结构， 青色棒示意 GMP- PCP结构 （GMP-PCP是 GTP类似物， β， γ-亚甲基二磷酸鸟苷酸）。
图 2Α-图 2C表示 ZCL278的活性特性。 其中： 图 2Α表示 ZCL278抑制 Cdc42介导的细胞微棘形成。 图 2B表示 ZCL278抑制内生型 Rac/Cdc42的活 化。 图 2C表示 ZCL278抑制刺激型 Cdc42的活化。
图 3A-图 3C表示活化 Cdc42/磷酸化 RhoA的免疫荧光染色。 图 4表示 ZCL278破坏细胞内 GM130蛋白对接高尔基体的组织。
图 5 A-图 5C表示 ZCL278阻碍细胞迁移， 但不影响细胞活率。
图 6A-图 6C表示 ZCL278抑制神经元分支和生长锥动力学。 具体实施方式 实施例 1: Cdc42抑制剂的虚拟筛选
分析 Cdc42-ITSN复合物的三维结构可发现介于两分子之间一个主要 的结合域， ITSN分子中 Glnl380残基和 Argl384残基之间以及 Cdc42上 Asn39和 Phe37之间的氢键， ITSN分子中 Leul376、 Metl379残基和 Thrl383 残基之间以及 Cdc42上 Phe56、 Tyr64、 Leu67和 Leu70之间的两个疏水簇。 为筛选 Cdc42抑制剂，推定 Cdc42分子上结合口袋为 ITSN蛋白表面与 Cdc42 结合的半径距离在 7A内的氨基酸残基形成的结构。 这其中含有 Cdc42蛋白 分子中 Thr35, Val36, Asn39, Phe56 和 Asp57等 16个氨基酸残基， 如图 1A- 图 1C所示。
用 Glide程序筛选 SPECS数据库中能破坏 Cdc42与 ITSN连接的小分子 化合物。 Cdc42-ITSN复合物的晶体结构来源于蛋白质数据库， ID号： 1KI1. 占据 Cdc42结合位点的 ISTN中氨基酸残基为 Leu376， Met379， Glnl380,Thrl383 , Argl384.距离这五个氨基酸中心在 7A内的 Cdc42氨基酸 残基形成 Cdc42的结合口袋。 运用蛋白准备模块 Protein Preparation Wizard 处理模型的初始结构， 使用 Glide I Receptor Grid Generation模块分子对 接， 使用配体准备模块 Ligprep将对 Specs数据库中 197000个化合物进行进 行高通量虚拟筛选， 按照以下标准筛选出来的排名前 100位的化合物将入 选： ITSN样的结合态， 可占据 ITSN上 Leul376, Glnl380, Argl384, Metl379 和 Thrl383残基的空间结构； 至少形成 3个氢键； 与 Cdc42分子中保守的 Asn39或 Phe37残基能形成氢键； 分子骨架的多样性。排名前 50000个分子 再进行标准精度计算排出前 100个分子， 最后选出 30个化合物测试它们对 Cdc42的活性和 /或功能的影响。
Cdc42分子上 ZCL278键合的电脑模拟模式： 如图 1A所示， ZCL278嵌 合 Cdc42 蛋白中 Thr35, Val36, Asp38,Asn39, Phe56, Tyr64, Leu67 和 Leu70 残基顺序形成的口袋结构。如图 IB所示， 两分子之间还发现外延的良性相 互作用。 与涉及 Thr35, Asn39 和 Asp57残基在内的氨基酸残基形成 5个氢 键， 同时与 Val36 和 Phe56残基形成有疏水作用的结构。 如图 1C所示， 分 子中的溴苯环插入到 GDP/GTP的结合口袋里。电脑模拟的 ZCL278与 Cdc42 的结合可破坏 Cdc42-ISTN的 GDP/GTP转换态结合。
实施例 2: 化合物 ZCL278的合成：
ZCL278可以通过下述合成方法合成， 以下合成方法仅用于示例， 而 非对本发明的限制，本领域的技术人员可以理解并想到可采用其它合成方 法来合成 ZCL278, 其也属于本发明的保护范围。
反应试剂和条件： （a) K2C03， DMF (N, N-二甲基甲酰胺）， 70 °C; (b) NaOH, dioxane (二氧杂环乙垸） /H20; (c) SOCl2 (二氯亚砜） ， DMF, reflux (回流） ； (d) NaSCN (硫氰酸钠） ， acetone (丙酮） ， 0 °C-rt; (e) 4-amino-N-(4,6-dimethylpyrimidin-2-yl)benzene- sulfonamide ( 4-氨基 -N- (4,6-二甲基 -2-嘧啶基) 苯磺酰胺） ， 0°C-r.t.
反应式如下:
Figure imgf000008_0001
化合物 1 (4-溴 -2-氯酚）与 2-溴乙酸乙酯在 K2C03存在下发生亲核取 代反应可得到化合物 2 (2-(4-溴 -2-氯苯氧基：)乙酸乙酯）。化合物 2在碱性 条件下水解得到化合物 3 (2-(4-溴 -2-氯苯氧基)乙酸） 。 化合物 3在 Ν, Ν- 二甲基甲酰胺催化下在二氯亚砜中回流可制备得到化合物 4。 化合物 4 (2-(4-溴 -2-氯苯氧基)乙酰氯） 与硫氰酸钠反应后形成相应的硫氰酸酯中 间体， 进一歩在反应体系中加入 4-氨基 -Ν- (4， 6-二甲基 -2-嘧啶基） 苯磺 酰胺可得到化合物 5 (即本发明的 ZCL278) 。 仪器与试剂： Bruker Avance III 400 MHz核磁共振仪； SGWX-4熔点 仪； Agilent 1200型高效液相色谱； 201^ £(¾)86 08- 8色谱柱(4.6 mm l50 mm, 5μΜ); 所有试剂均为分析纯或化学纯。
歩骤 1 : 2-(4-溴 -2-氯苯氧基)乙酸乙酯 （即化合物 2) 的合成
在反应瓶中依次加入化合物 1 (4-溴 -2-氯酚）（5.2 g, 25.0 mmol), 50 mL N, N-二甲基甲酰胺 (DMF)， 2-溴乙酸乙酯（4.3, 25.7 mmol)以及 K2C03 (3.45 g, 25.0 mmol)。 在 70 搅拌过夜后将反应混合物倒入 150 mL水中， 用乙 酸乙酯萃取（70 mLx4)。 合并有机相， 用饱和食盐水洗涤（100 mL x3)后用 无水硫酸钠干燥， 过滤后减压除去溶剂， 柱层析纯化后得到浅色油状物 2 (6.18g)。 产率 84.1%。 NMR (400 MHz, CDC13): 7.53 (s, 1H), 7.30 (d, 1H, J= 8.4 Hz), 6.72 (d, , J= 8.4 Hz), 4.68 (s, 2H), 4.26 (q, 2R, J= 7.2 Hz) and 1.29 (t, 3Η, /= 7.2 Hz)。
歩骤 2: 2-(4-溴 -2-氯苯氧基)乙酸 （即化合物 3 ) 的合成
在反应瓶重加入化合物 2 (5.0 g, 17.0 mmol), 50 mL 二氧六环和 lM NaOH (50 mL), 室温下搅拌过夜后， 将反应混合物用 1M HC1酸化至 pH = 3 o将酸化后的反应液用乙酸乙酯 (50 mLx 萃取， 有机相合并后用饱 和食盐水洗涤（50 mL)， 无水硫酸钠干燥。 过滤除去干燥剂后， 减压浓縮 可得白色固体 3 (4.69 g)。 产率 94.3%。 ¾ NMR (400 MHz, DMSO-i 6): 7.66 (s, 1H), 7.44 (d, IR, J= 8.8 Hz), 6.97 (d, 1H, J= 8.8 Hz), 4.72 (s, 2H)。
歩骤 3: 4-(3-(2-(4-溴 -2-氯苯氧基)乙酰基)硫脲基) -N-(4,6-二甲基 -2-嘧 啶基：)苯磺酰胺 （即化合物 5 ) 的合成
在反应瓶中依次加入化合物 3， 25 mL二氯亚砜以及 1滴 DMF, 将反应 体系加热至回流。 回流 3小时后， 常压蒸馏除去二氯亚砜， 将剩余的液体 用油泵减压干燥 5分钟后即得化合物 4 (2-(4-溴 -2-氯苯氧基:)乙酰氯） 。 在 另一反应瓶中将硫氰酸钠（326.8 mg, 4.0 mmol)溶于 10 mL丙酮冰浴冷却至 0 °C， 将化合物 4用 10 mL丙酮稀释后逐滴加入到上述反应液中， 滴加完毕 后撤去冰浴， 用油浴加热反应体系至 30 °C反应。 在 30 °C反应 2小时后， 再 次将反应液冷却至 0 °C， 加入 4-氨基 -N-(4,6-二甲基 -2-嘧啶基 )-苯磺酰 胺（556 mg, 2.0 mmOi:>后升至室温搅拌过夜。反应完毕后，过滤反应液得到 的固体用水和丙酮洗涤后，可得黄色粉末 5 (276 mg 产率 23.6%。 ^ NMR (400 MHz, DMSO-i 6): 12.19 (s, IH), 1 1.68 (s, IH), 1 1.52 (br s, IH), 7.99 (d, 2R, J= 8.4 Hz), 7.86 ( d, 2R, J= 8.4 Hz), 7.70 (d, 1Η,/= 1.6 Hz), 7.49 (d, IR, J= 8.8 Hz), 7.10 (d, = 8.8 Hz, J2 = 1.6 Hz), 6.75 (s, IH), 5.02 (s,
2H), 2.25 (s, 6H). HPLC纯度 95.9% (254nm)。
实施例 3: ZCL278活性特性
1、 ZCL278抑制 Cdc42介导的微棘形成
以去血清培养的小鼠成纤维细胞 Swiss 3T3测试候选 30个化合物对 Cdc42介导的细胞微棘 /丝状伪足形成的作用。 成纤维细胞 actin组成的微棘 /丝状伪足是 Cdc42活性的特征。如图 2A所示， 对照组细胞边缘可见少数微 棘 （小箭头标识） 和特征性的 RhoA介导形成的应力纤维 （星号标识） 。 用 1单位 /mL的 Cdc42激动剂 （cytoskeleton公司产品） 短暂地刺激细胞后， 可见明显的微棘数量增多和应力纤维的减少。加入以上激动剂作用 2分钟， 然后以 50uM的 ZCL278处理细胞 1小时， 相比单独的激动剂作用 ZCL278明 显抑制微棘的形成。 这结果显示该化合物可作为 Cdc42抑制剂的候选进行 后续研究。 在 30个化合物的测试中 ZCL278显示最强的抑制作用。
具体如图 2A所示。 图 2A表示 ZCL278抑制 Cdc42介导的细胞微棘形成。 计算机虚拟筛选出的小分子配体分别作用于无血清培养的 Swiss3T3细胞。 DMSO作为阴性对照， 1单位 /mL的 Cdc42激动剂 （cytoskeleton公司产品） 短暂地刺激细胞 （1分钟） 。 激动剂作用 1分钟， 然后以 50uMZCL278处理 细胞 1小时。 同样条件用 lOuM的 NSC23766作用细胞作为参比。 细胞固定 后用 rhodamine-Phalloidin荧光抗体杂交标识 F-actin。 细胞边缘可见极少的 微棘 （箭头标识） ， 星号指示正常的应力纤维分布。 Bar: 5um。
2、 ZCL278抑制 Cdc42活性
Cdc42活性相关的形态学测试中 ZCL278表现最好，接下来则在生化水 平上测试它的活性。 首先， 分别以 Cdc42激动剂和 ZCL278 ( 50uM ) 作用 人转移性前列腺癌细胞 PC3 5、 10、 15分钟， 细胞裂解液提取蛋白样品进 行 Westemblot, 检测其中磷酸化 Rac/Cdc42 (上层） 、 磷酸化 WASP蛋白的 水平 （中层） ， GAPDH蛋白作参比 （下层） 。 71位丝氨酸残基的磷酸化 是 RAC/Cdc42蛋白活性的负调控机制， 所以磷酸化 Rac/Cdc42蛋白表达的 增加指示活性 Rac/Cdc42 (GTP结合型） 的减低。 如图 2B所示， 激动剂作 用下磷酸化 Rac/Cdc42蛋白的表达持续降低，而 50uM的 ZCL278则时间依赖 性地提高了该蛋白的表达水平。
WASP蛋白是 Cdc42活化的下游效应器，通过 Arp2/3复合物作用诱导细 胞骨架 actin重组和微棘、 丝状伪足的形成， 其酪氨酸磷酸化与 Cdc42活化 后快速降解相关。 如图 2B所示， 激动剂作用 15分钟后减少磷酸化 WASP蛋 白的表达， 而相应时间内 ZCL278并没抑制该蛋白的表达。 此数据显示 ZCL278能够抑制磷酸化 Rac/Cdc42蛋白的内生水平， 呈时间依赖性， 并可 维持酪氨酸磷酸化 WASP蛋白的水平。
Rac和 Cdc42蛋白都可以发生 71位丝氨酸残基的磷酸化。 为直接检测 Cdc42的活化与失活， 接下来用 G-LISA试剂盒定量检测 GTP结合型 Cdc42 的水平。 用 1单位 /mL的 Cdc42激动剂刺激细胞 1分钟， 然后分别用 50uM的 ZCL278或 lOuM的 Rac激动剂 NSC23766处理 swiss 3T3细胞 1小时， 提取细 胞蛋白样品， 用 G-LISA试剂盒定量检测 GTP结合型 Cdc42的水平。 未加任 何处理的细胞作为阴性对照， 仅激动剂处理的细胞作为阳性对照， 另外缓 冲液和活化 Cdc42蛋白也分别作为阴性对照和阳性对照。 如图 2C所示 （图 中数据为三次独立实验的平均结果， ±标示平均差， **: p<0.01,*:p<0.05 )， 相比阴性对照， 激动剂作用使活化的 Cdc42蛋白水平明显增高 (70%);对比 阳性对照， ZCL278则使活化的 Cdc42蛋白水平明显降低 (80%)。 NSC23766 如预期地对 Cdc42没有影响。 这些数据说明在两种类型的细胞上， ZCL278 既可抑制刺激型也可抑制内生型的 Cdc42活性。
实施例 4: 活化 Cdc42/磷酸化 RhoA的免疫荧光染色
1、 ZCL278, 而非 NSC23766, 破坏活化 Cdc42在细胞核外周的分布 免疫荧光染色： Swiss3T3细胞在盖片上生长密度大约 30%.加药时去血 清培养， 加入 1单位 /mL的 Cdc42激动剂 （ cytoskeleton公司产品） 作用 1分 钟， 然后以 50uM的 ZCL278或 lOuM的 NSC23766分别处理细胞 1小时。单独 加激动剂作为阳性对照， DMSO作为阴性对照。 4%多聚甲醛固定细胞盖片 15分钟， 0.2%Triton X-100透化 15分钟， 10%BSA 37度封闭 30分钟。
抗体孵育： 一抗： 活化 Cdc42 (鼠抗， Neweast Biosciences公司） ； 磷 酸化 RhoA抗体（兔抗， Santa Cruze Biotechnology公司）； GM130抗体（鼠 抗， BD Biosciences公司）， 1 : 100稀释使用。相应二抗杂交后， rhodamine Phalloidin室温孵育 1小时用以观察 actin。 Zeiss Axiovert电子显微镜观察细 胞染色。 用图像处理软件 MetaMorph随机选取 5个细胞随机选取 5个点的 pixel进行均数计算即得各组平均像素强度值。
Westernblot: PC3细胞培养至约 70%的密度， 撤除血清继续培养 16小 时，分别加药物： 1单位 /mL的 Cdc42激动剂（cytoskeleton公司产品）， 5uM、 50uM的 ZCL278处置细胞 5、 10、 15分钟。细胞裂解缓冲液（配方： 50mMTris 缓冲液 PH7.5 , 10mM氯化镁， 0.5M氯化钠， l%Triton X-100,蛋白酶抑制剂） 裂解细胞， 14000转 4度离心提取细胞蛋白样品。 等浓度蛋白样品进行 Westernblot分析，抗体：磷酸化 Racl/Cdc42 (Milipore公司）、磷酸化 WASP ( Assay Biotech公司） ， 均以 1： 1000比例稀释杂交， GAPDH抗体 ( Calbiochem公司） 1 : 2000稀释杂交。 PVDF膜用化学发光法显影。
G-LISA试剂盒分析： Swiss3T3细胞培养至 40%的生长密度，撤除血清 培养 48小时，力 BCdc42激动剂作用 1分钟，再分别以 50uM的 ZCL278和 lOuM 的 NSC23766处理。 按照试剂盒说明书裂解细胞提取蛋白、 定量总蛋白浓 度 0.15mg/ml进行分析。 未加任何处理的细胞和缓冲液作为阴性对照， 仅 激动剂处理的细胞和活化的 Cdc42蛋白作为阳性对照。 酶标法测定各样品 光波 490nm时的吸光度值。
细胞水平上检测 ZCL278对 Cdc42活化的选择性抑制： 去血清的 Swiss3T3细胞加 1单位 /ml Cdc42激动剂作用 2分钟， 再分别以 50uM的 ZCL278或 lOuM的 NSC23766处理， DMSO做阴性对照。 为检测 ZCL278的 作用是选择性抑制 Cdc42活性而非对 RhoA的作用， 细胞分别用活化 Cdc42 (图 3A、 图 3B ) 和磷酸化 RhoA (图 3C )抗体免疫杂交。 箭头： 细胞核外 缘高尔基体-内质网网络； Hoechest染色剂标识细胞核。 Bar: 15um。如图 3A所示， 细胞化学免疫荧光染色： 小鼠单抗 GTP结合型 Cdc42抗体和 Hoechest (赫斯特）染色剂（标识细胞核） 。 对照组细胞呈现活化的 Cdc42 在细胞核外周有组织的分布； 激动剂作用使其分布明显增加， 并在核内也 有分布， 这与 Cdc42参与高尔基体蛋白运输的作用相一致； ZCL278明显破 坏了这种组织性的分布， 并降低与抗活化 Cdc42抗体的免疫反应性； 而 NSC23766对此没有影响。 图 3B显示各组高尔基体样分布的细胞数： 活化 Cdc42抗体识别的细胞核外周高尔基体 -内质网网络有组织分布的量化： 随 机选取细胞的 6个各自独立的区间计数高尔基体 -内质网网络（*:p<0.05 ) 。 图 3C: 磷酸化 RhoA信号的平均像素强度值定量， 数据来自 5个独立细胞随 机 5个信号的像素强度值取平均值。 ±标示平均差， *:p<0.03 : 激动剂、 ZCL278、 NSC23766均未显示对磷酸化 RhoA的影响。 这结果表明 ZCL278 选择性抑制 Cdc42活性。
2、 ZCL278, 而非 NSC23766, 破坏细胞内 GM130蛋白对接高尔基体 的组织
为了解 ZCL278破坏细胞核外周的活化 Cdc42的分布是否反映其对高 尔基体的组织也有作用， 设计实验检测 GM130蛋白-一种细胞质外缘的蛋 白质， 紧密连接高尔基体膜， 以维持高尔基体的顺式结构。
Swiss3T3细胞去血清培养， 分别加入 Cdc42激动剂、 ZCL278、 NSC23766, DMSO作为阴性对照。 细胞用 Rhodamine-phalloidin (红色） 、 抗 GM130抗体（绿色） 、 Hoechest (蓝色：)免疫荧光染色。 如图 4所示， 其中 箭头指示细胞微棘（实际试验中为红色荧光） ， 星号指示 GM130抗体标记 的高尔基体结构 （实际试验中为绿色荧光） 。 去血清的 Swiss3T3细胞对照 组显示特征性应力纤维 （如图中的箭头所示， 实际试验中为红色荧光） ， 对应细胞核单侧边的 GM130抗体分布（如图中的星号所示， 实际试验中为 绿色荧光） 。 Cdc42激动剂处理的细胞可见微棘的增多， GM130在细胞核 外缘分布增多。 ZCL278处理的细胞表现明显的微棘减少和 GM130的减少 (ZCL278: 实际试验中为红色荧光） ， 并且向细胞核两侧驱散 （如图中 的星号所示， 实际试验中为绿色荧光） 。 NSC23766对 GM130的表达以及 分布没有明显影响。 Bar: 10um。 这结果不仅更加说明 ZCL278对 Cdc42的 选择性抑制作用， 还证明了 Cdc42在高尔基体的组织和物质运输中发挥重 要作用。
实施例 5: 细胞伤口愈合实验： ZCL278阻碍细胞伤口愈合， 但不影响细胞 活率
丝状伪足是细胞的运动结构， 是细胞迁移导向和生长路径导引， 主要 由 Cdc42活性调控。 PC3细胞单层长满 6孔板， 去血清培养， 用 lml枪头每 孔分别做三个愈伤区域， 无血清培养基洗去剥落下的细胞。 分别以 1单位 /mlCdc42激动剂.、 50uM、 5uM的 ZCL278和 10uM的 NSC23766处置细胞 24 小时。 Cdc42激动剂作为阳性对照， 未加任何处置的作为阴性对照。 药物 作用 0时和 24小时分别拍下细胞影像，用 MetaMorph图形软件分析细胞迁移 的距离。 黑色线条指示愈伤区域的界限。 每次实验结果均进行假设检验 p 值分析 （p>0„05 )。
细胞活率检测： 以 75000/mi细胞密度种下培养 PC3细胞 48小时， 撤除 血清与 50uM的 ZCL278或 iOuM的 NSC23766继续培养 24小时， 胎盘蓝染色 计算细胞活率。
如图 5A和图 5B (取愈伤区域边界间最短距离定量区域宽度。 柱状图 表示对比初始愈伤区域的百分比， 三次独立实验取平均值， ±标示平均差， **: p<0.01,*:p<0.05 )所示， 对比阴性对照组 （41%愈合率）， 激动剂显著 增强细胞伤口的愈合能力 （59%); ZCL278两个浓度条件下均能捭制细胞 的迁移， 高浓度时抑制效应更强（50uM- 8%; 5uM~30%); NSC23766也显 示了明显的抑制细胞迁移效应（因为 Rac蛋白调控细胞片状伪足--同样是细 胞运动的结构的形成）。 这一结果与生化检测数据结果一致， 提示 ZCL278 不仅是 Cdc42的抑制剂， 还可有效抑制依赖于 Cdc42的细胞运动。
为说明 ZCL278 ίΦ制细胞迁移是源于抑制 Cdc42活性的作用 (NSC23766抑制 Rac活性作用）， 而非因为导致细胞死亡， 以胎盘蓝染色 法测试细胞活率。 PC3细胞阻滞于 G。期， 分别用 50uM的 ZCL278和 iOuM的 NSC23766处置细胞 24小时， 如图 5Cjff示（PC3细胞以 75000/ml细胞密度种 下， 培养 48小时， 撤除血清与 50uM的 ZCL278或 lOuM的 NSC23766继续培 养 24小时， 胎盘蓝染色计算细胞活率）， 对比阴性对照组， 药物处理的细 胞活率没有差别。因此可以认为药物未影响细胞生长而是抑制 Cdc42或 Rac 蛋白活性导致的抑制细胞迁移作用。
实施例 6: ZCL278抑制神经元分支和生长锥动力学
Cdc42调控神经突的分支与生长。 Garvalor等人通过 Cde42基因敲除技 术证实了 Cdc42对于神经元形态生成的关键性作用， 决定着神经元命运， 采集的神经元中 Cdc42的缺失导致明显的神经突数量减少， 丝状伪足功能 的严重破坏。 所以将检测 ZCL278抑制神经元分支的作用。
原代培养一日龄出生小鼠取其大脑组织，放置于含 0.25%胰酶的 HBSS 缓冲液中 37度培养 15分钟， 小心吹打分离神经元铺种细胞于 Poly-L-lysine 包被的细胞盖片上， 用含胎牛血清的 DMEM培养液培养 16小时， 然后换 Neurobasal培养液（Invitrogen公司）继续培养。至培养第五天分别用 DMSO 或 50uM的 ZCL278作用细胞 5、 10分钟， 随后即以 4%多聚甲醛固定细胞 15 分钟， 荧光抗体 Fluorescein-phalloidin杂交标记 F-actin结构， Zeiss显微镜下 观察神经元形态。
为观察 ZCL278对神经元生长锥动力学的作用采用了显微镜下的縮时 摄影技术， 用 Hamamatsu Orca数字照相机记录十分钟内放大 63倍的细胞 影像， 300毫秒曝光拍摄以最大程度减低对细胞的光毒性， ^ MetaMorph 图形软件分析影像并统计 ·分析。
原代培养新生中枢的神经元， 如图 6A所示， 培养的第五天， 可见神经 元长出一定的分支， 用 50uM的 ZCL278分别处置神经元 5、 10分钟， DMSO 作为阴性对照。 随时间推移， ZCL278抑制神经元的分支， 定量分析显示 药物处置后神经元分支数量明显减少 （图 6B: 定量 ZCL278处置后神经元 分支数量： 3次独立实验取平均值。 ±标示平均差， *: p<0.01 )。
Cdc42被公认可调控生长锥导向端微棘突和丝状伪足的形成。 缩时摄 影图像 （图 6C ) 展示对照组神经元细胞从生长锥处外生出不少微棘突或 丝状伪足； 但是， ZCL278在四分钟内即引起丝状伪足快速的收回。 Bar: lum。 所以， 这进一歩说明 ZCL278是一个有效调控 Cdc42介导神经元分 支和生长锥动力学的小分子抑制剂。
本发明应用高通量计算模拟方法筛选嵌合 Cdc42分子结合 GEFs的关 键结构的化合物。 基于 Cdc42 结合其特异性 GEF分子 intersectin (ITSN) 的结构特征， 化合物的三维结构恰好可填充 intersectin 分子连接的口袋 的被继续进行相关研究。 由此成功筛选出一个化合物 ZCL278 , 具有细 胞透性的 Cdc42特异性抑制剂。
本发明证实 ZCL278的活性特性， 作为第一个小分子 Cdc42抑制剂， 选 择性地直接靶向作用于 Cdc42与其 GEF的连接。 利用细胞伤口愈合实验， 可见 Cdc42激活后促进愈伤区域的合拢，说明 C(k42活化后促进肿瘤细胞的 转移。 ZCL278则显著抑制 PC3细胞的迁移， 具浓度依赖性。 而且 ZCL278 不是细胞毒类物质， 并非引起肿瘤细胞的死亡而导致的抑制细胞迁移效 应。
本发明中应用新生中枢神经元的实验也证明了 Cdc42在神经元发展 中所起的重要作用。 Garalov等的实验显示 Cdc42缺陷型小鼠的大脑和神 经元发展被严重破坏， 这些小鼠表现一系列的大脑畸形， 包括轴突束的减 少等， 以及神经元丝状伪足动力学降低， 生长锥变大， 轴突生成抑制。 其 实轴突和树突的运动主要是以 actin为基础，这一进程是由 Cdc42调控的。
ZCL278能够减少新生中枢神经元的分支数， 并抑制生长锥的动力学。 综 上所述， ZCL278是第一个靶向 Cdc42-ITSN连接的小分子抑制剂， 能够 有效用于肿瘤和神经病变中 Cdc42分子功能研究。
虽然本发明已以较佳实施例披露如上， 然其并非用以限定本发明， 任 何所属技术领域的技术人员， 在不脱离本发明之精神和范围内， 当可作些 许之更动与改进， 因此本发明之保护范围当视权利要求所界定者为准。

Claims

1. 一种化合物， 其特征在于， 具有下式 I的结构:
、 (j p1 f H
式 I。
2. 权利要求 1 所述的化合物在制备用于治疗恶性肿瘤的药物中的应 用。
3. 根据权利要求 2所述的应用，其特征在于，所述药物用于防治恶性 肿瘤的发展和侵袭。
4. 权利要求 1 所述的化合物在制备用于治疗心血管系统疾病的药物 中的应用。
5. 权利要求 1 所述的化合物在制备用于治疗呼吸系统疾病的药物中 的应用。
6. 权利要求 1 所述的化合物在制备用于治疗神经系统疾病的药物中 的应用。
7. 根据权利要求 2至 7中任一项所述的应用，其特征在于，所述的化 合物抑制 Cdc42的功能。
8. —种 Cdc42抑制剂， 其特征在于， 包含权利要求 1所述的化合物。
PCT/CN2012/000708 2011-05-23 2012-05-21 一种cdc42抑制剂及其应用 WO2012159456A1 (zh)

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EP3999053A4 (en) * 2019-07-17 2023-07-19 Children's Hospital Medical Center METHOD OF TREATING NEOPLASTIC DISEASES USING A SPECIFIC CDC42 INHIBITOR
CA3151110A1 (en) * 2019-08-16 2021-02-25 Children's Hospital Medical Center Methods of treating a subject with a cdc42-specific inhibitor
CN115068480B (zh) * 2022-08-09 2023-10-20 郑州大学第一附属医院 细胞分裂周期蛋白42小分子抑制剂在制备治疗慢性肾脏病药物中的应用

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US20170334863A1 (en) 2017-11-23
EP2716292B1 (en) 2017-09-06
US20150329496A1 (en) 2015-11-19
CN102796050B (zh) 2014-07-23
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EP2716292A1 (en) 2014-04-09
US20140194451A1 (en) 2014-07-10

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