Covalent targeting of RhoA for cancer therapy

Abstract

Described herein are RhoA inhibitors that can covalently bind to RhoA. The disclosed RhoA inhibitors exhibit improved inhibitory efficacy (i.e., an IC50 value as low as about 400 nM). In addition, the disclosed RhoA inhibitors can specifically bind to RhoA rather than other GTPs, such as other Ras GTPs (e.g., K-Ras and N-Ras), and in particular other Rho GTPs (e.g., Cdc42 and Rac1). The combination of high RhoA inhibitory efficacy and high RhoA specificity of the disclosed RhoA inhibitors results in these compounds exhibiting high cytotoxicity and anti-metastatic effects in cancer cells in vitro and in vivo, with negligible toxicity in normal cells. Also described are pharmaceutical compositions containing the RhoA inhibitors and methods of treating cancer using the RhoA inhibitors or pharmaceutical compositions thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 579,246, filed on August 28, 2023, the disclosure of which is incorporated herein by reference. Technical Field

[0003] The present invention relates generally to the field of synthetic RhoA inhibitors and methods of use thereof. Background Art

[0004] Upregulation of RhoA has been reported in many malignancies, including melanoma, gastric cancer, breast cancer, and colorectal cancer (CRC), most of which are known to have poor prognosis and aggressive invasion. RhoA plays a role in controlling cell architecture by interacting with downstream effectors such as mDia1 and ROCK. RhoA has been reported to contribute to amoeboid and mesenchymal movement in 3D migration and invasion, indicating its importance in cancers using the Rho / ROCK pathway to infiltrate nearby tissues and escape anoikis. All of these promote cancer invasiveness. In addition, RhoA has been reported to be overactive in colorectal cancer and regulate cell proliferation, invasion, and cell cycle, especially the G1 / S transition. Therefore, RhoA inhibition should be a promising strategy for cancer therapy.

[0005] Despite its therapeutic significance, RhoA is considered undruggable due to its smooth surface and globular structure, which makes it difficult for small molecules to bind. Alternatively, attempts have been made to bypass RhoA but target its downstream proteins, such as the ROCK inhibitor Y27632 and fasudil, the latter of which is approved in Japan for the treatment of postoperative cerebral vasospasm. However, ROCK inhibitors cannot completely eliminate oncogenic signals from RhoA, such as RhoA-mDia1, which mediates chromosomal instability and drives tumor development. Therefore, interest in RhoA druggable for cancer therapy remains.

[0006] In 2014, Shang et al. screened a small molecule named Rhosin through in vitro docking. It has two aromatic ring parts that are believed to be located in a shallow groove on the surface of RhoA. Rhosin was selected because it can weaken the binding of RhoA to its guanine nucleotide exchange factor (GEF) LARG by targeting the GEF recognition site around the key residue Trp58, thereby interfering with the GEF-mediated GDP to GTP exchange and inactivating RhoA. Although some studies have reported the anticancer effects of Rhosin in vivo, Rhosin has not yet entered clinical trials from animal studies, partly due to its low anticancer efficacy (working concentration ≥30μM) and suboptimal pharmacokinetic profile.

[0007] Compared to non-covalent drug leads such as Rhosin, covalent ligands may provide an alternative solution without relying on high-affinity scaffolds for shallow binding pockets. Recently, Sun et al. reported a covalent ligand called DC-Rhoin that targets C107 of RhoA. They proposed that there is a shallow groove near C107 that can act as an allosteric site to interfere with the RhoA-LARG interaction. In addition, C107 is unique among Rho family proteins but not other Ras GTPases, thus increasing drug specificity. Thus, at working concentrations, DC-Rhoin targets Rho GTPases such as RhoA, Cdc42, and Rac1, but not Ras GTPases such as K-Ras and N-Ras. However, Rac1 and Cdc42 play important roles in controlling fundamental cellular processes such as the formation of lamellipodia and filopodia. Therefore, alterations in Rac1 and Cdc42 signaling by DC-Rhoin could lead to toxicity in cancer therapy.

[0008] There remains a need to develop compounds that bind RhoA with high potency and specificity compared to other GTPases such as Rac1 and Cdc42.

[0009] Therefore, an object of the present invention is to provide compounds which bind to RhoA with high specificity.

[0010] It is a further object of the present invention to provide compounds that bind to and inhibit RhoA with high efficacy.

[0011] A further object of the present invention is to provide compounds useful in cancer therapy with low toxicity.

[0012] It is a further object of the present invention to provide methods of using the compounds for cancer therapy. Summary of the invention

[0013] RhoA inhibitors (also referred to herein as "RhoA covalent inhibitors" or "compounds") are described herein. The disclosed RhoA inhibitors can covalently bind to RhoA, thereby achieving improved inhibitory efficacy (i.e., IC 50 Values ​​as low as about 400nM). In addition, the disclosed RhoA inhibitors can specifically bind to RhoA rather than other GTPases, such as other Ras GTPases (e.g., K-Ras and N-Ras) and in particular other Rho GTPases (e.g., Cdc42 and Rac1). The combination of high RhoA inhibitory efficacy and high RhoA specificity of the disclosed RhoA inhibitors allows these compounds to exhibit high cytotoxicity in cancer cells in vitro and in vivo, while the toxicity in normal cells is negligible. For example, as shown in the data in the Examples below, the exemplary compound CL16 exhibits high cytotoxicity in CRC cells, which is correlated with RhoA expression / activity in cancer cells, while its toxicity is much less for normal colon fibroblasts with lower RhoA levels. In addition, administration of CL16 in tumor-bearing mice inhibited tumor growth in vivo, while no observable toxicity was found in mice treated with the exemplary compound CL16, as shown by body weight measurements and immunohistochemistry of tissue sections from different organs. The superior efficacy and specificity of the disclosed RhoA inhibitors also make them superior to the reported RhoA inhibitor Rhosin. For example, as demonstrated by the data in the Examples below, the exemplary compound CL16 inhibited the growth of CRC spheroids with better efficacy than the reported RhoA inhibitor Rhosin. In addition, since the binding site of DC-Rhoin (i.e., Cys107) is conserved in Rac1 and Cdc42, DC-Rhoin alters Rac1 and Cdc42 signals, which leads to cytotoxicity.

[0014] Without being bound by any theory, it is believed that the excellent efficacy and specificity of the disclosed RhoA inhibitors are caused by the covalent binding of these compounds to Cys16, which is located near the nucleotide binding pocket of RhoA. After binding, the disclosed RhoA inhibitors are located next to the nucleotide binding pocket of RhoA, which leads to a significant reduction in the self-exchange of GDP to GTP and the LARG-mediated exchange, resulting in its high inhibitory efficacy. In addition, compared with other GTPases (such as Rac1 and Cdc42), the binding site of the disclosed RhoA inhibitors (i.e., Cys16) is unique to RhoA, resulting in its high binding specificity.

[0015] In some forms, the disclosed RhoA inhibitors can have the structure of Formula I:

[0016]

[0017] Where: (i) X 1 and X 2 Can be independently C(R 6 ) 2 , O, S or NR 6 , and R 6 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, alkoxy, or carbonyl; (ii) A 1 Can be C 5 -C 6 Aromatic ring or C 4 -C 5 heteroaromatic ring; (iii) n1 may be an integer from 0 to 4, 0 to 3, or 0 to 2, for example, 0, 1 or 2; (iv) n2 may be an integer from 0 to 3, 0 to 2, or 0 or 1; (v) R 1 , R 3 and R 5 may independently be hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; (vi) R 2 is a cysteine ​​reactive group (e.g., haloacetamide, acrylamide, maleimide, vinyl sulfone, epoxide, etc.) that allows chemical reaction with the cysteine ​​on RhoA; (vii) R 4 can be hydrogen, alkoxy, hydroxy, halogen, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, carbonyl, amino, or haloalkyl; and (viii) when substituents are present, the substituents can independently be unsubstituted alkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted polyaryl, unsubstituted heteropolyaryl, unsubstituted alkylaryl, unsubstituted ring, unsubstituted heterocycle, halide, amino, amide, thiol, hydroxyl, cyano, nitro, carbonyl, or alkoxy.

[0018] In some forms, the disclosed RhoA inhibitors can have the structure of Formula II:

[0019]

[0020] Where: A', n1, n2 and R 1 -R 5 May be as defined above for Formula I.

[0021] In some forms, the disclosed RhoA inhibitors can have the structure of Formula III:

[0022]

[0023] Where: n1 and R 1 -R 4 may be as defined above for formula I.

[0024] In some forms, for any formula described herein, n1 can be 0 or 1.

[0025] In some forms, for any formula described herein, R 1 It may be hydrogen.

[0026] In some forms, for any formula described herein, R 3 It may be hydrogen or a substituted or unsubstituted aryl, wherein when a substituent is present, the substituent is independently unsubstituted alkyl, unsubstituted aryl, or unsubstituted alkylaryl.

[0027] In some forms, for any formula described herein, R 2 Can be wherein n3 can be an integer from 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, such as 0, 1 or 2; R 7 and R 8 may independently be hydrogen, unsubstituted alkyl, or halogen; and R 9 It may be hydrogen or halogen.

[0028] In some forms, for any formula described herein, R 4 It may be hydrogen.

[0029] In some forms, for any formula described herein, R 4 Can be wherein n4 can be an integer from 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, such as 0, 1 or 2; R 10 and R 11 may independently be hydrogen or halogen; and R 12 can be hydrogen, halogen, amino, unsubstituted alkynyl, aldehyde, or carboxylic acid. 10 and R 11 may be hydrogen, and R 12 may be an unsubstituted alkynyl group. In these forms, R 4 Can serve as probes that allow further functionalization and / or binding to targets (e.g., detection moieties (e.g., fluorophores)) for biological studies (e.g., RhoA inhibitor binding and function in vitro and / or in vivo). An exemplary compound is a CL-alkyne as shown below.

[0030] In some forms, the RhoA inhibitor is CL16 or CL16-alkyne.

[0031]

[0032] Disclosed are pharmaceutical compositions containing one or more RhoA inhibitors and optionally one or more pharmaceutically acceptable excipients. The pharmaceutical compositions can be formulated for administration by a variety of routes, such as oral administration, intranasal administration, intramuscular administration, intravenous administration, intraperitoneal administration, or subcutaneous administration, or a combination thereof. Optionally, the pharmaceutical composition further contains one or more additional active agents, such as one or more additional anticancer agents.

[0033] Also disclosed are methods for treating cancer using RhoA inhibitors or pharmaceutical compositions containing RhoA inhibitors. Generally, the method for treating cancer includes the step of administering a RhoA inhibitor or a pharmaceutical composition thereof to a subject in need thereof. In some forms, the cancer is a solid cancer. Generally, after the administering step, an effective amount of a RhoA inhibitor is administered to the subject so as to kill cancer cells and / or reduce or prevent the growth or proliferation of cancer cells, thereby improving one or more symptoms associated with cancer in the subject, such as reducing tumor volume and / or tumor weight. For example, after the administering step or all administering steps (if the administration occurs more than once), an effective amount of a RhoA inhibitor is administered to the subject so that the tumor volume and / or tumor weight in the subject is at least 10%, at least 20%, at least 30%, at least 40% or at least 50% less than a control administered with only a pharmaceutically acceptable excipient. Optionally, the anti-cancer effect is achieved without observable toxicity to the subject, as indicated by physical vital measurements (e.g., body weight), standard hematological markers and / or blood biochemical parameters compared to the control. In some forms, the effective amount of the RhoA inhibitor administered to the subject is about 0.1 μg to about 1000 μg, about 0.1 μg to about 500 μg, about 0.1 μg to about 200 μg, about 0.1 μg to about 100 μg, about 0.5 μg to about 50 μg, about 1 μg to about 1000 μg, about 1 μg to about 500 μg, about 1 μg to about 100 μg, about 1 μg to about 50 μg, about 1 μg to about 25 μg, about 1 μg to about 10 μg, about 0.1 μg to about 50 μg, about 5 μg to about 50 μg, or about 0.1 μg to about 20 μg per gram of the subject. Optionally, the method further comprises administering an additional active agent before, during, and / or after administration of the pharmaceutical composition containing the RhoA inhibitor, optionally wherein the active agent is an anticancer agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1A Schematic illustration of the gel-based ABPP experimental procedure. Figure 1B The chemical structure of compound CL16 is shown. Figure 1C and Figure 1D Gel results from dose-dependency experiments are shown ( Figure 1C), which shows that CL16 strongly binds to RhoA in vitro, and the relative fluorescence intensity-concentration plot shows the IC of competitive binding 50 0.44μM( Figure 1D ).

[0035] Figure 2A Shown are the results of LC-MS / MS experiments of HCT116 cells treated with CL16, revealing that CL16 covalently binds to Cys16 of RhoA. Figure 2B Amino acid sequence alignment of human RHOA, RAC1 and CDC42 is shown. Cys16 of RhoA is unique compared to RAC1 and CDC42. Figure 2C CL16 is covalently docked onto RhoA (PDB: 1CC0). Through covalent targeting of Cys16, CL16 is located next to the nucleotide binding pocket of RhoA. Ligand interaction map showing CL16 with amino acid residues and Mg 2+ Interactions of cofactor ions. Figure 2D Results of co-immunoprecipitation experiments are shown, indicating that RhoA and ARHGEF1 / LARG interaction is disrupted after incubation with CL16.

[0036] Figure 3A Shown are Western blot results demonstrating inhibition of RhoA activation in HT29 cells after incubation with CL16 in a serum starvation-restimulation experiment. Figure 3B Western blot results are shown.In serum starvation-restimulation experiments, RhoA downstream signaling in HT29 cells was found to be inhibited by CL16, as indicated by decreased phosphorylation levels of MLC. Figure 3C is a graph showing that CL16 inhibits RhoA activity in live cells with serum restimulation, as demonstrated by FRET-RhoA biosensor. Quantitative data are shown as mean ± SD of n = 25 cells from 3 different biological replicates / group. ***P < 0.001 and ****P < 0.0001.

[0037] Figure 4A is a bar graph showing reduced colony formation and cell migration of HT29 cells with RhoA knockdown by siRNA using a wound healing assay. Figure 4B is a graph showing the cell viability of CRC and normal colon fibroblast CCD-18Co cells treated with CL16 for 48 h using MTT assay; n=3 different biological replicates / group. Figure 4C The IC values ​​of CL16 for different cell lines are shown. 50 Table of values. Figure 4Dis a bar graph showing wound healing assay of HT29 cells incubated with solvent vehicle, CL16 or Rhosin; n=3 different biological replicates / group. Figures 4E-4F The results showed that the HT29 cells ( Figure 4E ) and SW620( Figure 4F ) Graph of inhibition of spheroid growth; n=3 different biological replicates / group. Quantitative data are presented as mean±SD. *P<0.05, **P<0.01 and ***P<0.001.

[0038] Figure 5A-5B Is to show CL16 in HT29 ( Figure 5A ) and SW620( Figure 5B ) Graph of anticancer effects in spheroids. HT29 spheroids or SW620 spheroids were treated with CL16 at the indicated concentrations for 10 days. Spheroids were then imaged by microscopy using a 10x objective and size determined by ImageJ.

[0039] Fig. 6A The chemical structure of the CL16 molecular probe CL16-alkyne is shown. Figure 6B The results of the validation of CL16 covalently bound to RhoA are shown. HCT116 cells transiently expressing FLAG-RhoA were pretreated with solvent vehicle or CL16 for 2 h and then incubated with CL16-alkyne for another 2 h in complete medium. The cells were then lysed and immunoprecipitated by anti-FLAG beads. The enriched proteins were reacted with rhodamine azide by copper-catalyzed azide-alkyne cycloaddition (CuAAC), boiled with loading buffer and separated by SDS-PAGE. The protein bands were then visualized by their in-gel fluorescence or blotted for RhoA. Figure 6C Results for protein targeting of CL16 are shown. HCT116 cells were pretreated with solvent vehicle or CL16 for 2 h and then incubated with CL16-alkyne for an additional 2 h in complete medium. Cells were then lysed, reacted with rhodamine azide by CuAAC, boiled with loading buffer, separated by SDS-PAGE and imaged. The arrow points to the major protein target of CL16 at ~22 kDa, which matches the molecular weight of RhoA.

[0040] Fig. 7A is a graph showing that CL16 inhibited tumor growth in mouse HCT116 xenografts by intraperitoneal injection at 10 mg / kg (n=6 per group). Figure 7B is a graph showing that CL16 inhibits tumor growth in mouse HT29 xenografts by intraperitoneal injection at 10 mg / kg (n=7). Figure 7Cis a graph showing the body weight of mice bearing HT29 xenografts throughout treatment with CL16. Fig.7D is a graph showing that CL16 inhibited tumor growth in mouse SW620 xenografts by intraperitoneal injection at 10 mg / kg (n=6 per group). Fig. 7E is a bar graph showing tumor weights of tumors harvested from treated mice bearing SW620 xenografts. Figure 7F is a graph showing the body weight of mice bearing SW620 xenografts throughout treatment with CL16. Statistical analysis was performed using a two-tailed Student's t-test. *P<0.05.

[0041] Fig. 8A is a graph showing RhoA expression levels analyzed in tumor (red) and normal tissue (grey) in patients from the colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) datasets in The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx), respectively. Figure 8B is a graph showing the correlation between RhoA expression level and patient survival in TCGA and GTEx datasets. Figure 8C and 8D It is shown that for AARGHEF 25( Figure 8C ) and AKAP 13( Fig.8D ) Graph of analysis of the expression of GEFs and GAPs and their correlation with patient survival in TCGA and GTEx datasets. Fig. 8E and 8F It is shown that for ARGAP26 ( Fig. 8E ) and ARHGAP35( Fig.8F ) Graph of analysis of the expression of GEFs and GAPs and their correlation with patient survival in TCGA and GTEx datasets. Figure 8G and 8H It is shown that for AARGHEF 25( Figure 8G ) and AKAP13( Figure 8H ) Graph of GEF expression analysis in patients with different tumor stages in TCGA and GTEx datasets. Figure 8I and 8J is a graph showing gene set enrichment analysis (GESA) of RhoA-related pathways in COAD and READ patients with early stage (0 and I) and late stage (IV) tumors in the TCGA dataset. NES: Normalized Enrichment Score. Figure 8K and 8L is a graph showing GESA of RhoA-related pathways in COAD and READ patients in the TCGA data set with high epithelial-mesenchymal transition (EMT) scores (mesenchymal-like) and low EMT scores (epithelial-like).

[0042] Fig. 9 An amino acid sequence alignment of human RHOA, Rac1, and Cdc42 is shown. Cys16 on RhoA is highlighted in yellow, while other conserved cysteines are highlighted in blue.

[0043] Fig. 10A and Fig. 10B The results are shown in a buffer solution (20 mM Tris, 50 mM NaCl, 10 mM MgCl 2 Graph of a GTP binding assay of RhoA (0.75 μg), CL16 and GTP-MANT (5.6 μM) in PBS (pH 7.5).

[0044] Fig.11A The results show that HCT116 cells were in G o / G 1 , S and G 2 Graph of flow cytometric analysis of the percentage of cells in the M phase. Fig. 11B is a graph showing the results of apoptosis assay of HCT116 cells treated with CL16 using Caspase-3 / 7 Green detection reagent.

[0045] Fig. 12A is a volcano plot of an MS experiment on HCT116 cells probed by CL16-alkyne. Fig. 12B is a graph showing comparison of proteins in HCT116 cells modified by CL16 and proteins in HCT116 cells probed by CL16-alkyne.

[0046] Fig.13A is a graph showing cell viability examined by MTT assay. SL4 cells were treated with solvent vehicle or CL16 in complete medium for 48h. Fig. 13B is a graph showing the weight of tumors in mice treated with vehicle (n=7) or CL16 (n=8) at the end of the CL16 treatment study. Figures 13C-13H CD3 ( Fig. 13C )、CD4( Fig.13D )、CD8( Fig.13E )、Ki67( Fig.13F ), cleaved caspase-3 ( Figure 13G ) and VEGFR2( Fig.13H ) of the immunohistochemical staining scores, Fig.13Iis a graph showing the changes in body weight of mice with orthotopic SL4 CRC model treated with solvent vehicle or CL16 by ip injection.

[0047] Fig.14A and 14B Figure 2 shows H&E and Ki67 immunohistochemical staining of tumor tissues from mice treated with vehicle or CL16 ( Fig.14A ) and cleaved caspase-3 staining ( Fig. 14B ) images. Fig. 14C and 14D Figure 2 shows H&E, Ki67 ( Fig. 14C ) and cleaved caspase-3( Fig.14D ) Graph of immunohistochemical staining scores of stained images.

[0048] Fig.15 is a schematic diagram illustrating the biology of RhoA and its regulation by guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs).

[0049] Fig.16 is a graph showing the analysis of the first screening results. Covalent ligands that exhibited less than 30% intensity ratio in silver staining and retained at least 70% intensity compared to the DMSO control were marked in red and regarded as lead compounds.

[0050] Fig.17 is a schematic diagram illustrating the mechanism of action of CL16 mediating anticancer and anti-metastatic effects through inhibition of RhoA.

[0051] Fig.18 is a schematic diagram illustrating the workflow for MS experiments using CL16-alkyne to identify protein targets of CL16 in HCT116 cells.

[0052] Fig.19A and 19B is shown in the expression of RhoA WT ( Fig.19A ) or C16A protein ( Fig.19B ) were incubated with vehicle, CL16 or Rhosin for 48 h in complete medium.

[0053] Fig. 20 is a schematic diagram illustrating the workflow for orthotopic mouse CRC model establishment and CL16 treatment studies. DETAILED DESCRIPTION OF THE INVENTION

[0055] It should be understood that, unless otherwise stated, the disclosed compounds, compositions and methods are not limited to specific synthetic methods, specific analytical techniques or specific reagents, and can therefore vary. It should also be understood that the terminology used herein is for the purpose of describing specific forms and embodiments only, and is not intended to be limiting.

[0056] As used herein, "substituted" refers to all permissible substituents of the compounds or functional groups described herein. In the broadest sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic group containing any number of carbon atoms (preferably 1-14 carbon atoms), and optionally contain one or more heteroatoms, such as oxygen, sulfur or nitrogen groups in a straight chain, branched or ring structure format. Representative substituents include substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted aralkyl, halogen, hydroxy, alkoxy, phenoxy, aryloxy, silyl, thiol, alkylthio, substituted alkylthio, phenylthio, arylthio, cyano, isocyano, nitro, substituted or unsubstituted carbonyl, carboxyl, amino, amido, oxo, sulfinyl, sulfonyl, sulfonic acid, phosphonium, phosphanyl, phosphoryl, phosphonyl, amino acid. Such substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted aralkyl, halogen, hydroxy, alkoxy, phenoxy, aryloxy, silyl, thiol, alkylthio, substituted alkylthio, phenylthio, arylthio, cyano, isocyano, nitro, substituted or unsubstituted carbonyl, carboxyl, amino, amide, oxo, sulfinyl, sulfonyl, sulfonic acid, phosphonium, phosphino, phosphoryl, phosphonyl and amino acid may be further substituted.

[0057] The heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatoms. It is understood that "substitution" or "substituted" includes the implicit proviso that such substitution is in accordance with the permissible valences of the substituted atom and the substituent, and that such substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation (e.g., rearrangement, cyclization, elimination, etc.).

[0058] As used herein, "alkyl" refers to a group of saturated aliphatic groups, including straight chain alkyl groups, branched chain alkyl groups, and cycloalkyl groups (alicyclic). In some forms, a straight chain or branched chain alkyl group has 30 or fewer carbon atoms in its backbone (e.g., a straight chain is C 1 -C 30 , the branch chain is C 3 -C 30 ), 20 or less, 15 or less, or 10 or less.Alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. Similarly, cycloalkyl is based on non-aromatic carbocyclic rings composed of at least three carbon atoms, for example, non-aromatic monocyclic or non-aromatic polycyclic rings containing 3-30 carbon atoms, 3-20 carbon atoms or 3-10 carbon atoms in its ring structure, and there are 5, 6 or 7 carbons in the ring structure.Cycloalkyl containing polycyclic ring systems can have two or more non-aromatic rings, wherein two or more carbons are common to two adjacent rings (i.e., "condensed cycloalkyl ring").Examples of cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.

[0059] "Substituted alkyl" refers to an alkyl moiety having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can be any of the above substituents, such as halogen (e.g., fluorine, chlorine, bromine or iodine), hydroxy, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (e.g., thioester, thioacetate or thioformate), aryl, alkoxy, aralkyl, phosphonium, phosphino, phosphonyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, oxo, sulfhydryl, thiol, alkylthio, silyl, sulfinyl, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aromatic or heteroaromatic moieties. -NRR', wherein R and R' are independently hydrogen, alkyl or aryl, and wherein the nitrogen atom is optionally quaternized; -SR, wherein R is phosphonyl, sulfinyl, silyl, hydrogen, alkyl or aryl; -CN; -NO 2 ; -COOH; carboxylate; -COR, -COOR or -CON(R) 2 , wherein R is hydrogen, alkyl or aryl; imino, silyl, ether, haloalkyl (e.g., -CF3, -CH 2 -CF 3 , -CCl 3 );-CN;-NCOCOCH 2 CH 2 ; -NCOCOCHCH; and -NCS; and combinations thereof.

[0060] It will be appreciated by those skilled in the art that the moiety substituted on the hydrocarbon chain itself may be substituted if appropriate. For example, the substituents of substituted alkyl groups may include halogen, hydroxyl, nitro, thiol, amino, aralkyl, azido, imino, amide, phosphonium, phosphino, phosphoryl (including phosphonates and phosphinates), oxo, sulfonyl (including sulfates, sulfonamido, sulfamoyl and sulfonates) and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates and esters), haloalkyl, -CN, etc. Cycloalkyl groups may be substituted in the same manner.

[0061] Unless the number of carbons is otherwise specified, "lower alkyl" as used herein refers to an alkyl group as defined above, but having 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

[0062] As used herein, "heteroalkyl" refers to an alkyl group or combination thereof containing a straight or branched chain or cyclic carbon containing at least one heteroatom on the carbon backbone. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, wherein nitrogen, phosphorus, and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. For example, the term "heterocycloalkyl" is a cycloalkyl group as defined above, wherein at least one carbon atom of the ring is replaced by a heteroatom (such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus).

[0063] As used herein, the term "alkenyl" is a hydrocarbon group having a structural formula of 2 to 24 carbon atoms and containing at least one carbon-carbon double bond. Alkenyl groups include straight chain alkenyl groups, branched alkenyl groups, and cycloalkenyl groups. Cycloalkenyl groups are based on non-aromatic carbocyclic rings composed of at least three carbon atoms and at least one carbon-carbon double bond, such as non-aromatic monocyclic or non-aromatic polycyclic rings containing 3-30 carbon atoms and at least one carbon-carbon double bond, 3-20 carbon atoms and at least one carbon-carbon double bond, or 3-10 carbon atoms and at least one carbon-carbon double bond in its ring structure, and having 5, 6 or 7 carbons and at least one carbon-carbon double bond in the ring structure. Cycloalkenyl groups containing polycyclic ring systems may have two or more non-aromatic rings, wherein two or more carbons are common to two adjacent rings (i.e., "fused cycloalkenyl rings") and contain at least one carbon-carbon double bond. Asymmetric structures such as (AB)C=C(C'D) are intended to include E and Z isomers. This can be inferred from the structural formula herein, where an asymmetric olefin is present, or it can be explicitly indicated by the bond symbol C. The term "alkenyl" as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," the latter referring to alkenyl moieties having one or more substituents replacing hydrogen on one or more carbons of the hydrocarbon backbone. The term "alkenyl" also includes "heteroalkenyl."

[0064] The term "substituted alkenyl" refers to an alkenyl moiety having one or more substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents may be any of the above substituents, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphonium, phosphino, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, oxo, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclic, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl and combinations thereof.

[0065] As used herein, "heteroalkenyl" refers to an alkenyl group or combination thereof containing a straight or branched chain or cyclic carbon containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, wherein nitrogen, phosphorus, and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. For example, the term "heterocycloalkenyl group" is a cycloalkenyl group in which at least one carbon atom of the ring is replaced by a heteroatom (such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus).

[0066] As used herein, the term "alkynyl group" is a hydrocarbon group having 2 to 24 carbon atoms, and the structural formula contains at least one carbon-carbon triple bond. Alkynyl groups include straight chain alkynyl groups, branched chain alkynyl groups, and cycloalkynyl groups. Cycloalkynyl groups are non-aromatic carbon-based rings consisting of at least three carbon atoms and at least one carbon-carbon triple bond, such as 3-30 carbon atoms and at least one carbon-carbon triple bond, 3-20 carbon atoms and at least one carbon-carbon triple bond, or 3-10 carbon atoms and at least one carbon-carbon triple bond in its ring structure, and non-aromatic monocyclic or non-aromatic polycyclic rings having 5, 6 or 7 carbons and at least one carbon-carbon triple bond in the ring structure. Cycloalkynyl groups containing polycyclic ring systems can have two or more non-aromatic rings, wherein two or more carbons are common to two adjacent rings (i.e., "fused alkynyl rings") and contain at least one carbon-carbon triple bond. Asymmetric structures such as (AB)C≡C(C"D) are intended to include both E and Z isomers. This may be inferred in the formulae herein where an asymmetric alkyne is present, or it may be explicitly indicated by the bond symbol C. The term "alkynyl" as used throughout the specification, examples and claims is intended to include both "unsubstituted alkynyls" and "substituted alkynyls", the latter referring to alkynyl moieties having one or more substituents replacing hydrogen on one or more carbons of the hydrocarbon backbone. The term "alkynyl" also includes "heteroalkynyls".

[0067] The term "substituted alkynyl" refers to an alkynyl moiety having one or more substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphonium, phosphino, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl, and combinations thereof.

[0068] As used herein, "heteroalkynyl" refers to an alkynyl group or combination thereof containing a straight or branched chain or cyclic carbon containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, wherein nitrogen, phosphorus, and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. For example, the term "heterocycloalkynyl group" is a cycloalkynyl group in which at least one carbon atom of the ring is replaced by a heteroatom (such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus).

[0069] As used herein, "aryl" refers to a C 4 -C 26 The term "aryl" refers to a ring system which is a fused ring system which contains one aromatic ring and optionally one or more non-aromatic rings. Examples of aryl groups are benzene, tetralin, indane, and the like.

[0070] The term "substituted aryl" refers to an aryl group in which one or more hydrogen atoms on one or more aromatic rings are replaced with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, carbonyl (e.g., ketone, aldehyde, carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, imino, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl (e.g., CF3, -CH 2 -CF 3 , -CCl 3 ), -CN, aryl, heteroaryl and combinations thereof.

[0071] "Heterocycle" and "heterocyclyl" are used interchangeably and refer to a cyclic group attached through a ring carbon or nitrogen atom of a monocyclic or polycyclic ring system containing 3-30 ring atoms, 3-20 ring atoms, 3-10 ring atoms, or 5-6 ring atoms, wherein the polycyclic ring system contains one or more non-aromatic rings and optionally one or more aromatic rings, wherein at least one non-aromatic ring contains carbon and 1 to 4 heteroatoms, each heteroatom selected from non-peroxidized oxygen, sulfur and N(Y), wherein Y is absent or is H, O, C 1 -C 10 Alkyl, phenyl or benzyl and optionally contain 1-3 double bonds and optionally substituted by one or more substituents.Heterocyclic radical is different from heteroaryl according to definition.Heterocycle can be heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl etc., such as piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, dihydrofuran and [2,3-b] tetrahydrofuran, morpholinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pyranyl, 2H-pyrrolyl, 4H-quinolizinyl, quinuclidinyl, tetrahydrofuranyl, 6H-1,2,5-thiadiazinyl.Heterocyclic radical can be optionally substituted by one or more substituents as defined above for alkyl and aryl.

[0072] The term "heteroaryl" refers to a C 3 -C 26The invention relates to a fused ring system of an aromatic ring or a ring containing an aromatic ring and optionally one or more non-aromatic rings, wherein one or more carbon atoms on the aromatic ring structure have been replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, oxygen, sulfur and nitrogen. Examples of heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, etc. Examples of heteroaryl rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, hexazinyl, decahydroquinolinyl, 2H,6H-1, 5,2-dithiazinyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, naphthyridinyl, octahydroisoquinolyl, 1,2, 3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl The term "heteroaryl" refers to 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl. One or more rings may be substituted as defined below for "substituted heteroaryl".

[0073] The term "substituted heteroaryl" refers to a heteroaryl radical in which one or more hydrogen atoms on one or more heteroaryl rings are replaced with one or more substituents including, but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, carbonyl (e.g., ketone, aldehyde, carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, imino, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl (e.g., CF3, -CH 2 -CF 3 , -CCl 3 ), -CN, aryl, heteroaryl and combinations thereof.

[0074] The term "polyaryl" refers to a fused ring system comprising two or more aromatic rings and optionally one or more non-aromatic rings. Examples of polyaryl groups are naphthalene, anthracene, phenanthrene, When the fused ring system contains two or more aromatic rings and optionally one or more non-aromatic rings, wherein one or more carbon atoms on one or more aromatic ring structures have been replaced by heteroatoms, the fused ring system may be referred to as "heteropolyaryl" or "polyheteroaryl". The terms "heteropolyaryl" and "polyheteroaryl" are used interchangeably herein.

[0075] The term "substituted polyaryl" refers to polyaryl groups in which one or more aryl groups are substituted with one or more substituents, including but not limited to halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (or quaternized amino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfoxide, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof. When referring to polyheteroaryl groups, the chemical moiety may be referred to as a "substituted polyheteroaryl".

[0076] The term "ring", "cyclic ring" or "cyclic group" refers to a substituted or unsubstituted monocyclic ring or a substituted or unsubstituted polycyclic ring (e.g., those formed by a monocyclic ring system or a fused ring system), such as a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkenyl, a substituted or unsubstituted cycloalkynyl, or a substituted or unsubstituted heterocyclyl, which, where geometric constraints permit, has 3 to 30 carbon atoms. Substituted cycloalkyl, cycloalkenyl, cycloalkynyl and heterocyclyl are substituted as defined above for alkyl, alkenyl, alkynyl, or heterocyclyl, respectively.

[0077] As used herein, the term "aralkyl" is an aryl or heteroaryl group having an alkyl, alkynyl or alkenyl group as defined above attached to an aromatic group (eg, aryl, heteroaryl, polyaryl or polyheteroaryl). An example of an aralkyl group is a benzyl group.

[0078] The terms "alkoxyl" or "alkoxy", "aroxy" or "aryloxy" generally describe a radical of the formula -OR v A compound represented by v Including but not limited to substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic radical, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted alkylheteroaryl, substituted or unsubstituted aralkyl, substituted or unsubstituted carbonyl, phosphonium, phosphino, phosphono, sulfinyl, silyl, thiol, amide and amino. Exemplary alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, etc. "lower alkoxy" groups are alkoxy groups containing 1 to 6 carbon atoms. "Ether" is two functional groups covalently linked by oxygen, as defined below. Thus, an alkyl substituent that makes an alkyl group an ether is or is similar to an alkoxy group, for example, represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O-arakyl, -O-aryl, -O-heteroaryl, -O-polyaryl, -O-polyheteroaryl, -O-heterocyclyl, etc.

[0079] The term "substituted alkoxy" refers to an alkoxy group having one or more substituents replacing one or more hydrogen atoms on one or more carbons of the alkoxy backbone. Such substituents can be any of the above substituents, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphonium, phosphino, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, oxo, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclic, alkylaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0080] As used herein, the term "ether" is a 2 OA 1 Indicates that A 2 and A 1 It may independently be a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, phosphonium, phosphino, phosphonyl, sulfinyl, silyl, thiol, substituted or unsubstituted carbonyl, alkoxy, amide or amino as described above.

[0081] As used herein, the term "polyether" is represented by the formula:

[0082]

[0083] Among them A 3 It may independently be a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, phosphonium, phosphino, substituted or unsubstituted carbonyl, alkoxy, amide or amino as described above; g may be a positive integer from 1 to 30.

[0084] The term "phenoxy" is art-recognized and refers to a radical of the formula -OR v A compound wherein R v It is C 6 H 5 (ie -OC 6 H 5 ). Those skilled in the art recognize that phenoxy is a type of aryloxy.

[0085] The term "substituted phenoxy" refers to a phenoxy group as defined above having one or more substituents replacing one or more hydrogen atoms on one or more carbons of a phenyl ring. Such substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphonium, phosphino, phosphino, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclic, alkylaryl, haloalkyl, -CN, aryl, heteroaryl and combinations thereof.

[0086] The terms "aroxy" and "aryloxy" used interchangeably herein are represented by -O-aryl or -O-heteroaryl, wherein aryl and heteroaryl are as defined herein.

[0087] The terms "substituted aroxy" and "substituted aryloxy" used interchangeably herein represent an -O-aryl or -O-heteroaryl group having one or more substituents replacing one or more hydrogen atoms on one or more ring atoms of the aryl and heteroaryl groups as defined herein. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphonium, phosphino, phosphino, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl, and combinations thereof.

[0088] As used herein, the term "amino" includes the following groups

[0089]

[0090] wherein E is absent or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, wherein independently of E, R x , R xi and Rxii Each independently represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted aralkyl group (e.g., a substituted or unsubstituted alkylaryl group, a substituted or unsubstituted arylalkyl group), a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted polyaryl group, a substituted or unsubstituted polyheteroaryl group, a substituted or unsubstituted heterocyclic group, a hydroxyl group, an alkoxy group, a phosphonium group, a phosphino group, a phosphono group, a sulfinyl group, a silyl group, a thiol group, an amide group, an amino group, or -(CH 2 ) m -R"'; R"' represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted polyaryl group, a substituted or unsubstituted polyheteroaryl group, an alkoxy group, a phosphonium group, a phosphine group, an amide group or an amino group; and m is 0 or an integer from 1 to 8. The term "quaternary amino group" also includes groups in which nitrogen, R x , R xi and R xii The N + A group forming a heterocyclyl or heteroaryl group having 3 to 14 atoms in the ring structure. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl).

[0091] The terms "amide" or "amido" are used interchangeably to refer to both "unsubstituted amide" and "substituted amide" and are represented by the following general formula:

[0092]

[0093] wherein E is absent or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, or substituted or unsubstituted heterocyclyl, wherein R and R' independently of E each independently represent hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclic, hydroxy, alkoxy, phosphonium, phosphino, phosphono, sulfinyl, silyl, thiol, amide, amino or -(CH 2 ) m -R"', or R and R' together with the N atom to which they are attached form a heterocyclic ring having 3 to 14 atoms in the ring structure; R"' represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted polyaryl group, a substituted or unsubstituted polyheteroaryl group, an alkoxy group, a phosphonium group, a phosphine group, an amide group or an amino group; and m is zero or an integer ranging from 1 to 8. In some forms, when E is oxygen, a carbamate is formed. Those skilled in the art understand that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl).

[0094] As used herein, "carbonyl" is art-recognized and includes moieties that can be represented by the following general formula:

[0095]

[0096] wherein X is a bond or represents oxygen or sulfur, and R represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aralkyl (e.g. substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, hydroxy, alkoxy, phosphonium, phosphino, amide, amino, or -(CH 2 ) m-R", or a pharmaceutically acceptable salt thereof; E" is absent, or E" is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl; R' represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, hydroxy, alkoxy, phosphonium, phosphino, amide, amino, or -(CH 2 ) m -R"; R" represents a hydroxyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted polyaryl group, a substituted or unsubstituted polyheteroaryl group, an alkoxy group, a phosphonium group, a phosphine group, an amide group or an amino group; and m is 0 or an integer ranging from 1 to 8. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphonium, phosphino, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, and combinations thereof. Those skilled in the art understand that the "E" groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl). When X is oxygen and R is as defined above, the moiety is also referred to as a carboxyl group. When X is oxygen and R is hydrogen, the formula represents a "carboxylic acid." When X is oxygen and R' is hydrogen, the formula represents a "format." When X is oxygen and R or R' is not hydrogen, the formula represents an "ester." In general, when the oxygen atom of the above formula is replaced by a sulfur atom, the formula represents a "thiocarbonyl" group. When X is sulfur and R or R' is not hydrogen, the formula represents a "thioester." When X is sulfur and R is hydrogen, the formula represents a "thioformic acid." When X is sulfur and R' is hydrogen, the formula represents a "thioformate." When X is a bond and R is not hydrogen, the above formula represents a "ketone." When X is a bond and R is hydrogen, the above formula represents an "aldehyde."

[0097] The term "phosphino" is represented by the formula

[0098]

[0099] wherein E is absent or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, wherein independently of E, R vi and R vii Each independently represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclic group, hydroxyl, alkoxyl, phosphonium, phosphino, phosphono, sulfinyl, silyl, thiol, amide, amino, or -(CH 2 ) m -R"', or R vi and R vii Together with the P atom to which they are attached, they form a heterocyclic ring having 3 to 14 atoms in the ring structure; R'' represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted polyaryl group, a substituted or unsubstituted polyheteroaryl group, an alkoxy group, a phosphonium group, a phosphine group, an amide group or an amino group; and m is 0 or an integer ranging from 1 to 8. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl) group, , alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl).

[0100] The term "phosphonium" is represented by the formula

[0101]

[0102] wherein E is absent or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, wherein independently of E, R vi , R vii and R viii Each independently represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclic group, hydroxyl, alkoxyl, phosphonium, phosphino, phosphono, sulfinyl, silyl, thiol, amide, amino, or -(CH 2 ) m -R"', or R vi , R vii and R viii The P +The atoms together form a heterocyclic ring having 3 to 14 atoms in the ring structure; R'' represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted polyaryl group, a substituted or unsubstituted polyheteroaryl group, an alkoxy group, a phosphonium group, a phosphino group, an amide group or an amino group; and m is 0 or an integer ranging from 1 to 8. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxy alkyl, alkyl, alkyl, alkylene ...

[0103] The term "phosphono" is represented by the formula

[0104]

[0105] wherein E is absent or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, oxygen, alkoxy, aryloxy, or substituted alkoxy or substituted aryloxy, wherein independently E, R vi and R vii are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, hydroxy, alkoxy, phosphonium, phosphino, phosphono, sulfinyl, silyl, thiol, amide, amino, or -(CH 2 )m -R"', or R vi and R vii Together with the P atom to which they are attached, they form a heterocyclic ring having 3 to 14 atoms in the ring structure; R'' represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted polyaryl group, a substituted or unsubstituted polyheteroaryl group, an alkoxy group, a phosphonium group, a phosphine group, an amide group or an amino group; and m is 0 or an integer ranging from 1 to 8. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl) group, , alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl).

[0106] The term "phosphoryl" defines a phosphonyl group wherein E is absent, oxygen, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy, as defined above and independently of E, R vi and R vii R is independently hydroxy, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy, as defined above. As will be appreciated by those of ordinary skill in the art, when E is oxygen, the phosphoryl group cannot be attached to another chemical species, such as to form an oxygen-oxygen bond or other unstable bond. vi and R viiWhen substituted, the substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl, and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl).

[0107] The term "sulfinyl" is represented by the formula

[0108]

[0109] wherein E is absent, or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, wherein R, independently of E, represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted arylalkyl ... substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, hydroxy, alkoxy, phosphonium, phosphino, phosphono, silyl, thiol, amide, amino, or -(CH 2 ) m-R"', or E and R together with the S atom to which they are attached form a heterocyclic ring having 3 to 14 atoms in the ring structure; R"' represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted polyaryl group, a substituted or unsubstituted polyheteroaryl group, an alkoxy group, a phosphonium group, a phosphine group, an amide group or an amino group; and m is 0 or an integer ranging from 1 to 8. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl).

[0110] The term "sulfonyl" is represented by the formula

[0111]

[0112] wherein E is absent or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, wherein R, independently of E, represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, hydroxy, alkoxy, phosphonium, phosphino, amide, amino, or -(CH 2)mR"', or E and R together with the S atom to which they are attached form a heterocyclic ring having 3 to 14 atoms in the ring structure; R"' represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted polyaryl group, a substituted or unsubstituted polyheteroaryl group, an alkoxy group, a phosphonium group, a phosphine group, an amide group or an amino group; and m is 0 or an integer ranging from 1 to 8. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl).

[0113] The term "sulfonic acid" refers to a sulfonyl group as defined above, wherein R is hydroxy and E is absent or E is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, or substituted or unsubstituted heteroaryl. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl).

[0114] The term "sulfate" refers to a sulfonyl group as defined above, wherein E is absent, oxygen, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy, all as defined above, and R is independently hydroxy, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy, all as defined above. As will be appreciated by those of ordinary skill in the art, when E is oxygen, sulfate cannot be connected to another chemical species, such as to form an oxygen-oxygen bond or other unstable bonds. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl).

[0115] The term "sulfonate" refers to a sulfonyl group as defined above, wherein E is oxygen, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy, all as defined above, and R is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted amino, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aralkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, -(CH 2 ) m-R"', R"' represents a hydroxyl group, a substituted or unsubstituted carbonyl group, an aryl group, a cycloalkyl ring, a cycloalkenyl ring, a heterocycle, an amide group, an amino group, or a polycycle; and m is zero or an integer ranging from 1 to 8. As will be appreciated by those of ordinary skill in the art, when E is oxygen, the sulfonate cannot be linked to another chemical species, for example to form an oxygen-oxygen bond or other unstable bonds. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl).

[0116] The term "sulfamoyl" refers to a sulfonamide or a sulfonamide represented by the formula

[0117]

[0118] wherein E is absent, or E is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted cycloalkyl, etc.), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclyl, wherein R and R' independently of E each independently represent hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted heterocyclic group, hydroxyl, alkoxy, phosphonium, phosphino, amide, amino or -(CH 2 ) m-R"', or R and R' together with the N atom to which they are attached form a heterocyclic ring having 3 to 14 atoms in the ring structure; R"' represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted polyaryl group, a substituted or unsubstituted polyheteroaryl group, an alkoxy group, a phosphonium group, a phosphine group, an amide group or an amino group; and m is 0 or an integer ranging from 1 to 8. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylene, cyclohexane-1,2-diyl).

[0119] The term "silyl group" as used herein is represented by the formula -SiRR'R", wherein R, R' and R" can independently be hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted carbonyl, phosphonium, phosphino, phosphono, sulfinyl, thiol, amide, amino, alkoxy or oxo as described above. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl, and combinations thereof.

[0120] The term "thiol" is used interchangeably and is represented by -SR, where R can be hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted carbonyl, phosphonium, phosphino, amido, amino, alkoxy, oxo, phosphono, sulfinyl, or silyl as described above. Such substituents can be any of the substituents described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silyl, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino (e.g., quaternized amino), amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaryl, polyheteroaryl, and combinations thereof.

[0121] The disclosed compounds and substituent groups can independently have two or more of the groups listed above. For example, if the compound or substituent group is a straight chain alkyl group, one hydrogen atom of the alkyl group can be substituted by a hydroxyl group, an alkoxy group, etc. Depending on the selected group, the first group can be incorporated into the second group, or the first group can be suspended (i.e., connected) to the second group. For example, for the phrase "an alkyl group comprising an ester group", the ester group can be introduced into the main chain of the alkyl group. Alternatively, the ester can be connected to the main chain of the alkyl group. The nature of the selected group will determine whether the first group is embedded in or attached to the second group.

[0122] The compounds and substituents may independently be substituted with substituents as described above in the definition of "substituted".

[0123] Numerical ranges disclose every possible number that such ranges can reasonably encompass, as well as any subranges and combinations of subranges encompassed therein. 3 -C 9 The carbon range also discloses C 3 , C 4 , C 5 , C 6 , C 7 , C 8 and C 9 , and any subranges between these numbers (e.g., C 4 -C6 ), and any possible range combination between these values. In yet another example, a given temperature range may be about 25°C to 30°C, wherein the range also discloses a temperature that can be independently selected from about 25, 26, 27, 28, 29, and 30°C, and any range between these numbers (e.g., 26 to 28°C), and any possible range combination between these values.

[0124] The use of the term "about" is intended to describe a value above or below the stated value, and the term "about" modifies the value within a range of about + / - 10%. When the term "about" is used before a numerical range (i.e., about 1-5) or before a series of numbers (i.e., about 1, 2, 3, 4, etc.), unless otherwise indicated, it is intended to modify both ends of the numerical range and / or each number listed in the entire series.

[0125] The disclosed compounds and substituent groups can independently have two or more of the groups listed above. For example, if the compound or substituent group is a straight chain alkyl group, one hydrogen atom of the alkyl group can be substituted by a hydroxyl group, an alkoxy group, etc. Depending on the selected group, the first group can be incorporated into the second group, or the first group can be suspended (i.e., connected) to the second group. For example, for the phrase "an alkyl group comprising an ester group", the ester group can be introduced into the main chain of the alkyl group. Alternatively, the ester can be connected to the main chain of the alkyl group. The nature of the selected group will determine whether the first group is embedded in or attached to the second group.

[0126] The compounds and substituents may independently be substituted with substituents as described above in the definition of "substituted".

[0127] II. Composition

[0128] Covalent inhibitors of RhoA (also referred to herein as "RhoA inhibitors" or "compounds") have been developed. The disclosed RhoA inhibitors are suitable for use in cancer therapy due to their high RhoA inhibitory efficacy and high RhoA specificity.

[0129] The disclosed RhoA inhibitors can covalently bind to RhoA, thereby achieving improved inhibitory efficacy (i.e., as low as about 400 nM). In addition, the disclosed RhoA inhibitors can specifically bind to RhoA, rather than other GTPases, such as other Ras GTPases (e.g., K-Ras and N-Ras) and in particular other Rho GTPases (e.g., Cdc42 and Rac1). This combination of high RhoA inhibitory efficacy and high RhoA specificity of the disclosed RhoA inhibitors enables these compounds to exhibit high cytotoxicity in cancer cells in vitro and in vivo, while having negligible toxicity in normal cells. Prior to the disclosed RhoA covalent inhibitors, there were no RhoA covalent inhibitors that had in vivo anti-tumor effects and good efficacy in a mouse colorectal cancer xenograft model.

[0130] For example, as shown in the data in the following examples, the exemplary compound CL16 exhibited high cytotoxicity in CRC cells, which correlated with RhoA expression / activity in cancer cells, while it was much less toxic to normal colon fibroblasts, which have lower levels of RhoA. In addition, administration of CL16 in tumor-bearing mice inhibited tumor growth in vivo, while no observable toxicity was found from mice treated with the exemplary compound CL16, as shown by body weight measurements and immunohistochemistry of tissue sections from different organs.

[0131] The superior efficacy and specificity of the disclosed RhoA inhibitors also make them superior to the reported RhoA inhibitors Rhosin and DC-Rhoin. For example, as shown by the data in the following examples, the exemplary compound CL16 inhibits the growth of CRC spheroids with better efficacy than the reported RhoA inhibitor Rhosin. In addition, since the binding site of DC-Rhoin (i.e., Cys107) is conserved in Rac1 and Cdc42, DC-Rhoin alters Rac1 and Cdc42 signals, which leads to cytotoxicity. In contrast, the disclosed compounds are relatively nontoxic in normal cells.

[0132] Without being bound by any theory, it is believed that the excellent efficacy and specificity of the disclosed RhoA inhibitors are caused by the covalent binding of these compounds to Cys16, which is located near the nucleotide binding pocket of RhoA. After binding, the disclosed RhoA inhibitors are located next to the nucleotide binding pocket of RhoA, which leads to a significant reduction in the self-exchange of GDP to GTP and the LARG-mediated exchange, resulting in its high inhibitory efficacy. In addition, compared with other GTPases (such as Rac1 and Cdc42), the binding site of the disclosed RhoA inhibitors (i.e., Cys16) is unique to RhoA, resulting in its high binding specificity.

[0133] In some forms, the disclosed RhoA inhibitors can contain probes, such as alkynyl groups, for biological studies of RhoA function and activity in cancer, as well as studies of the disclosed RhoA inhibitors in cancer therapy. For example, as shown in the following examples, a molecular probe of CL16 (CL16-alkynyl) was used to verify the specific binding of CL16 to RhoA.

[0134] Also disclosed are pharmaceutical preparations containing the RhoA inhibitor.

[0135] A. RhoA inhibitors and probes

[0136] The compounds disclosed herein are covalent RhoA inhibitors with high RhoA inhibitory efficacy and high RhoA specificity. These compounds are suitable for use in cancer therapy.

[0137] In some forms, the disclosed RhoA inhibitors can have the structure of Formula I:

[0138]

[0139] Where: (i) X 1 and X 2 Can be independently C(R 6 ) 2 , O, S or NR 6 , and R 6 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, alkoxy or carbonyl; (ii) A 1 Can be C 5 -C 6 Aromatic ring or C 4 -C 5 heteroaromatic ring; (iii) n1 may be an integer from 0 to 4, 0 to 3, or 0 to 2, for example, 0, 1 or 2; (iv) n2 may be an integer from 0 to 3, 0 to 2, or 0 or 1; (v) R 1 , R 3 and R 5 may independently be hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; (vi) R 2 is a cysteine ​​reactive group (e.g., haloacetamide, acrylamide, maleimide, vinyl sulfone, and epoxide) that allows chemical reaction with the cysteine ​​on RhoA; (vii) R 4can be hydrogen, alkoxy, hydroxy, halogen, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, carbonyl, amino, or haloalkyl; and (viii) when substituents are present, the substituents can independently be unsubstituted alkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted polyaryl, unsubstituted heteropolyaryl, unsubstituted alkylaryl, unsubstituted ring, unsubstituted heterocycle, halide, amino, amide, thiol, hydroxyl, cyano, nitro, carbonyl, or alkoxy.

[0140] In some forms, the disclosed RhoA inhibitors can have the structure of Formula II:

[0141]

[0142] Where: A', n1, n2 and R 1 -R 5 may be as defined above for formula I.

[0143] In some forms, the disclosed RhoA inhibitors can have the structure of Formula III:

[0144]

[0145] Where: n1 and R 1 -R 4 may be as defined above for formula I.

[0146] In some forms, for any formula described herein, n1 can be 0 or 1.

[0147] In some forms, for any formula described herein, R 1 It may be hydrogen.

[0148] In some forms, for any formula described herein, R 3 It may be hydrogen or a substituted or unsubstituted aryl, wherein when a substituent is present, the substituent is independently unsubstituted alkyl, unsubstituted aryl, or unsubstituted alkylaryl.

[0149] In some forms, for any formula described herein, R 2 Can be wherein n3 can be an integer from 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, such as 0, 1 or 2; R 7 and R 8 may independently be hydrogen, unsubstituted alkyl, or halogen; and R 9 It may be hydrogen or halogen.

[0150] In some forms, for any formula described herein, R 4 It may be hydrogen.

[0151] In some forms, for any formula described herein, R 4 Can be wherein n4 can be an integer from 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, such as 0, 1 or 2; R 10 and R 11 may independently be hydrogen or halogen; and R 12 can be hydrogen, halogen, amino, unsubstituted alkynyl, aldehyde or carboxylic acid. 10 and R 11 may be hydrogen, and R 12 may be an unsubstituted alkynyl group. In these forms, R 4 Can serve as probes that allow further functionalization and / or binding to a target (e.g., a detection moiety (e.g., a fluorophore)) for biological studies (e.g., RhoA inhibitor binding and function in vitro and / or in vivo). Exemplary compounds are CL-alkynes shown below.

[0152] For any formula described herein, the alkyl group (when present) can be a straight chain alkyl group, a branched chain alkyl group, or a cyclic alkyl group (monocyclic or polycyclic). The terms "cyclic alkyl group" and "cycloalkyl group" are used interchangeably herein. Exemplary alkyl groups include straight chain C 1 -C 30 Alkyl, branched chain C 4 -C 30 Alkyl, cyclic C 3 -C 30 Alkyl, straight chain C 1 -C 20 Alkyl, branched chain C 4 -C 20 Alkyl, cyclic C 3 -C 20 Alkyl, straight chain C 1 -C 10 Alkyl, branched chain C 4 -C 10 Alkyl, cyclic C 3 -C 10 Alkyl, straight chain C 1 -C 6 Alkyl, branched chain C 4 -C 6 Alkyl, cyclic C 3 -C 6 Alkyl, straight chain C 1 -C 4 Alkyl, cyclic C 3 -C 4 Alkyl groups, such as straight chain C 1 -C 10 , C1 -C 9 , C 1 -C 8 , C 1 -C 7 , C 1 -C 6 , C 1 -C 5 , C 1 -C 4 , C 1 -C 3 or C 1 -C 2 Alkyl group, branched chain C 3 -C 9 , C 3 -C 9 , C 3 -C 8 , C 3 -C 7 , C 3 -C 6 , C 3 -C 5 or C 3 -C 4 Alkyl groups or cyclic C 3 -C 9 , C 3 -C 9 , C 3 -C 8 , C 3 -C 7 , C 3 -C 6 , C 3 -C 5 or C 3 -C 4 The cyclic alkyl group can be a monocyclic or polycyclic alkyl group, such as C 4 -C 30 , C 4 -C 25 , C 4 -C 20 , C 4 -C 18 , C 4 -C 16 , C 4 -C 15 , C 4 -C 14 , C 4 -C 13 , C 4 -C 12 , C 4 -C 10 , C4 -C 9 , C 4 -C 8 , C 4 -C 7 , C 4 -C 6 or C 4 -C 5 A monocyclic or polycyclic alkyl group.

[0153] For any formula described herein, alkenyl (when present) can be straight chain alkenyl, branched chain alkenyl, or cyclic alkenyl (monocyclic or polycyclic). The terms "cyclic alkenyl" and "cycloalkenyl" are used interchangeably herein. Exemplary alkenyl groups include straight chain C 2 -C 30 Alkenyl, branched C 4 -C 30 Alkenyl, cyclic C 3 -C 30 Alkenyl, straight chain C 2 -C 20 Alkenyl, branched C 4 -C 20 Alkenyl, cyclic C 3 -C 20 Alkenyl, straight chain C 2 -C 10 Alkenyl, branched C 4 -C 10 Alkenyl, cyclic C 3 -C 10 Alkenyl, straight chain C 2 -C 6 Alkenyl, branched C 4 -C 6 Alkenyl, cyclic C 3 -C 6 Alkenyl, straight chain C 2 -C 4 Alkenyl, cyclic C 3 -C 4 Alkenyl, such as straight chain C 2 -C 10 , C 2 -C 9 , C 2 -C 8 , C 2 -C 7 , C 2 -C 6 , C 2 -C 5 , C 2 -C 4 , C 2 -C 3 Alkenyl group, branched C 3 -C9 , C 3 -C 9 , C 3 -C 8 , C 3 -C 7 , C 3 -C 6 , C 3 -C 5 , C 3 -C 4 Alkenyl groups, or cyclic C 3 -C 9 , C 3 -C 9 , C 3 -C 8 , C 3 -C 7 , C 3 -C 6 , C 3 -C 5 , C 3 -C 4 Cyclic alkenyl groups can be monocyclic or polycyclic alkenyl groups, such as C 4 -C 30 , C 4 -C 25 , C 4 -C 20 , C 4 -C 18 , C 4 -C 16 , C 4 -C 15 , C 4 -C 14 , C 4 -C 13 , C 4 -C 12 , C 4 -C 10 , C 4 -C 9 , C 4 -C 8 , C 4 -C 7 , C 4 -C 6 or C 4 -C 5 A monocyclic or polycyclic alkenyl group.

[0154] For any formula described herein, the alkynyl group (when present) can be a straight chain alkynyl group, a branched chain alkynyl group, or a cyclic alkynyl group (monocyclic or polycyclic). The terms "cyclic alkynyl group" and "cycloalkynyl group" are used interchangeably herein. Exemplary alkynyl groups include straight chain C2 -C 30 Alkynyl, branched C 4 -C 30 Alkynyl, cyclic C 3 -C 30 Alkynyl, straight chain C 2 -C 20 Alkynyl, branched C 4 -C 20 Alkynyl, cyclic C 3 -C 20 Alkynyl, straight chain C 2 -C 10 Alkynyl, branched C 4 -C 10 Alkynyl, cyclic C 3 -C 10 Alkynyl, straight chain C 2 -C 6 Alkynyl, branched C 4 -C 6 Alkynyl, cyclic C 3 -C 6 Alkynyl, straight chain C 1 -C 4 Alkynyl, cyclic C 3 -C 4 Alkynyl, such as straight chain C 2 -C 10 , C 2 -C 9 , C 2 -C 8 , C 2 -C 7 , C 2 -C 6 , C 2 -C 5 , C 2 -C 4 , C 2 -C 3 Alkynyl group, branched C 3 -C 9 , C 3 -C 9 , C 3 -C 8 , C 3 -C 7 , C 3 -C 6 , C 3 -C 5 , C 3 -C 4 Alkynyl group, or cyclic C 3 -C 9 , C 3 -C 9 , C3 -C 8 , C 3 -C 7 , C 3 -C 6 , C 3 -C 5 , C 3 -C 4 The cyclic alkynyl group can be a monocyclic or polycyclic alkynyl group, such as C 4 -C 30 , C 4 -C 25 , C 4 -C 20 , C 4 -C 18 , C 4 -C 16 , C 4 -C 15 , C 4 -C 14 , C 4 -C 13 , C 4 -C 12 , C 4 -C 10 , C 4 -C 9 , C 4 -C 8 , C 4 -C 7 , C 4 -C 6 or C 4 -C 5 A monocyclic or polycyclic alkynyl group.

[0155] For any of the formulae described herein, the aryl group (when present) may be C 4 -C 30 Aryl, C 4 -C 20 Aryl, C 4 -C 12 Aryl, C 4 -C 11 Aryl, C 4 -C 9 Aryl, C 5 -C 30 Aryl, C 5 -C 20 Aryl, C 5 -C 12 Aryl, C 5 -C 11 Aryl, C 5 -C 9 Aryl, C6 -C 20 Aryl, C 6 -C 12 Aryl, C 6 -C 11 Aryl or C 6 -C 9 It should be understood that the aryl group can be a heteroaryl group, such as C 4 -C 30 Heteroaryl, C 4 -C 20 Heteroaryl, C 4 -C 12 Heteroaryl, C 4 -C 11 Heteroaryl, C 4 -C 9 Heteroaryl, C 5 -C 30 Heteroaryl, C 5 -C 20 Heteroaryl, C 5 -C 12 Heteroaryl, C 5 -C 11 Heteroaryl, C 5 -C 9 Heteroaryl, C 6 -C 30 Heteroaryl, C 6 -C 20 Heteroaryl, C 6 -C 12 Heteroaryl, C 6 -C 11 Heteroaryl or C 6 -C 9 For any of the formulas described herein, the polyaryl group may be C 8 -C 30 Polyaryl, C 8 -C 20 Polyaryl, C 8 -C 12 Polyaryl, C 8 -C 11 Polyaryl, C 10 -C 30 Polyaryl, C 10 -C 20 Polyaryl, C 10 -C 12 Polyaryl, C 10 -C 11 Polyaryl or C 12 -C 20 It should be understood that the aryl group can be a polyheteroaryl group, such as C 10 -C 30Polyheteroaryl, C 10 -C 20 Polyheteroaryl, C 10 -C 12 Polyheteroaryl, C 10 -C 11 Polyheteroaryl or C 12 -C 20 Polyheteroaryl.

[0156] Exemplary RhoA inhibitors are presented below.

[0157]

[0158] The compounds described herein may be neutral, or may be one or more pharmaceutically acceptable salts, crystalline forms, non-crystalline forms, hydrates, or solvates, or combinations thereof. Reference to a compound refers to the neutral molecule and / or those additional forms thereof, either collectively or individually, as derived from the context. Pharmaceutically acceptable salts of a compound include acid addition salts and base addition salts thereof.

[0159] Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include acetate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, edisylate, esylate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hyaluronate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthylate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, saccharate, stearate, succinate, tartrate, toluenesulfonate and trifluoroacetate.

[0160] Suitable base salts are formed from bases which form non-toxic salts. Examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts.

[0161] Hemisalts of acids and bases can also be formed, for example hemisulphate and hemicalcium salts.

[0162] The compounds described herein may be in the form of prodrugs. The term "prodrug" refers to a pharmacologically inactive substance that is converted in vivo (e.g., by enzymatic or non-enzymatic action, including pH-dependent bioactivation) into a pharmacologically active drug. In some forms of such prodrugs, the prodrug may contain a prodrug (promoeity) connected to an auxiliary group (auxophore) in the prodrug, which is intended to utilize a cell transporter (e.g., wherein the auxiliary group is a sugar or disaccharide, or an amino acid or dipeptide). Such prodrugs may exhibit enhanced active transport across cell membranes in vivo; or, such prodrugs may inhibit the efflux of drugs and prodrugs in vivo by interacting with cell efflux transporters. Exemplary groups that may convert disclosed compounds into prodrug forms include, but are not limited to, hydroxyl, carboxyl, amine, phosphate, phosphonate, amidine, guanine and / or carbohydrates. These may be attached to any suitable position of the disclosed compound to form a prodrug of the compound.

[0163] The in vitro and in vivo inhibitory activities and anticancer effects of the RhoA inhibitors disclosed herein can be evaluated using methods known in the art. For example, the in vitro inhibitory activities of the disclosed RhoA inhibitors can be obtained by measuring the cell viability of 2D and 3D cell culture models using MTT assays, wound healing assays, and / or Matrigel invasion assays. For example, the in vitro anticancer effects of the disclosed RhoA inhibitors can be obtained by measuring the tumor volume, tumor size, and / or tumor weight of a mouse xenograft model. Specific examples for evaluating the in vitro and in vivo inhibitory activities of RhoA inhibitors are described in the following examples.

[0164] B. Pharmaceutical Compositions Containing RhoA Inhibitors

[0165] Disclosed are pharmaceutical compositions containing one or more RhoA inhibitors described herein in a form suitable for in vitro or in vivo application (e.g., administration to a mammal). In some forms, the pharmaceutical compositions containing one or more RhoA inhibitors are used to treat cancer in a subject. The pharmaceutical compositions may include one or more pharmaceutically acceptable carriers and / or one or more pharmaceutically acceptable excipients. For example, the pharmaceutical composition may be in the form of a liquid, such as a solution or suspension, and contain a plurality of the disclosed RhoA inhibitors in an aqueous medium, and optionally one or more suitable excipients for liquid compositions. Optionally, the pharmaceutical composition is in solid form and contains a plurality of the disclosed RhoA inhibitors and one or more suitable excipients for solid compositions.

[0166] 1. Carriers and excipients

[0167] Suitable pharmaceutically acceptable carriers and excipients are generally recognized as safe (GRAS) and can be administered to an individual without causing undesirable biological side effects or unwanted interactions.

[0168] Representative carriers and excipients include solvents (including buffers), diluents, pH adjusters, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizers, and combinations thereof.

[0169] Excipients may be added to liquid or solid pharmaceutical compositions (for in vivo or in vitro use) to aid sterility, stability (e.g., shelf life), integration, and to adjust and / or maintain the pH or isotonicity of the RhoA inhibitor in the pharmaceutical composition, such as diluents, pH adjusters, preservatives, antioxidants, suspending agents, wetting agents, viscosity adjusters, tonicity agents and stabilizers, and combinations thereof.

[0170] The RhoA inhibitor for administration to a subject (e.g., a mammal) in need thereof can be dissolved or suspended in a suitable carrier to form a liquid pharmaceutical preparation, such as sterile saline, phosphate buffered saline (PBS), balanced salt solution (BSS), viscous gel or other pharmaceutically acceptable carriers for administration. The pharmaceutical preparation can also be a sterile solution, suspension or emulsion in a non-toxic, parenterally acceptable diluent or solvent.

[0171] 2. Form for administration

[0172] The disclosed RhoA inhibitors can be formulated as pharmaceutical compositions in liquid form or solid form as liquid preparations or solid preparations for oral administration, mucosal administration (e.g., intravaginal administration, pulmonary administration, and / or administration to other mucosal surfaces), or parenteral administration (e.g., intramuscular injection, intravenous injection, intraperitoneal administration, and subcutaneous administration) to a subject.

[0173] a. Oral preparations

[0174] Pharmaceutical compositions containing one or more disclosed RhoA inhibitors can be provided in a form suitable for oral administration to a subject (e.g., a mammal) (i.e., an oral composition). Oral administration can involve swallowing so that the RhoA inhibitor (optionally, an additional active agent included in the pharmaceutical composition) enters the gastrointestinal tract, or buccal or sublingual administration can be employed, by which the RhoA inhibitor enters the bloodstream directly from the oral cavity.

[0175] Compositions suitable for oral administration include solid compositions such as tablets, capsules containing microparticles, liquids, powders, lozenges (including liquid-filled lozenges), chewables, multi- and nanoparticulates, gels, solid solutions, liposomes, films, ovules, sprays, and liquid compositions.

[0176] Liquid compositions for oral administration include suspensions, solutions, syrups and elixirs. Such oral compositions can be used as fillers in soft or hard capsules and may contain one or more suitable carriers and / or excipients, such as water, ethanol, polyethylene glycol, propylene glycol, chitosan polymers and chitosan derivatives (e.g., N-trimethylene chloride chitosan, chitosan esters, chitosan modified with hydrophilic groups (e.g., amino groups, carboxyl groups, sulfate groups, etc.), methylcellulose, suitable oils, one or more emulsifiers, and / or suspending agents. Liquid compositions for oral administration can also be prepared by reconstitution of solids (e.g., from pouches).

[0177] Optionally, the RhoA inhibitor is contained in a rapidly dissolving and / or rapidly disintegrating dosage form.

[0178] For tablet or capsule dosage forms, in addition to the RhoA inhibitors described herein, the tablets typically contain a disintegrant, a binder, a diluent, a surfactant, a lubricant, a glidant, an antioxidant, a colorant, a flavoring agent, a preservative or a taste-masking agent, or a combination thereof.

[0179] Examples of suitable disintegrants for forming tablets or capsule dosage forms containing RhoA inhibitors include, but are not limited to, sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, cross-linked sodium carboxymethylcellulose, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropylcellulose, starch, pregelatinized starch, and sodium alginate. Typically, the concentration of the disintegrant can range from about 1 wt% to about 25 wt%, about 5 wt% to about 20 wt% of the tablet or capsule dosage form containing the RhoA inhibitor.

[0180] Binders are generally used to impart cohesiveness to the tablet composition containing the RhoA inhibitor. Suitable binders for forming tablet or capsule formulations containing the RhoA inhibitor include, but are not limited to, microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinyl pyrrolidone, pregelatinized starch, chitosan polymers and chitosan derivatives (e.g., N-trimethylene chloride chitosan, chitosan esters, chitosan modified with hydrophilic groups (e.g., amino groups, carboxyl groups, sulfate groups, etc.)), hydroxypropyl cellulose, and hydroxypropyl methylcellulose.

[0181] Suitable diluents for forming a tablet or capsule composition containing a RhoA inhibitor include, but are not limited to, lactose (such as, for example, the monohydrate, spray-dried monohydrate or anhydrous form), chitosan polymers and chitosan derivatives (e.g., N-trimethylene chloride chitosan, chitosan esters, chitosan modified with hydrophilic groups (e.g., amino groups, carboxyl groups, sulfate groups, etc.)), N-sulfonated derivatives of chitosan, quaternized derivatives of chitosan, carboxyalkylated chitosan, microcrystalline chitosan, mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.

[0182] Tablet or capsule compositions containing RhoA inhibitors may also contain surfactants, such as sodium lauryl sulfate and polysorbate 80, and glidants, such as silicon dioxide and talc, in the coating. When a surfactant is present, the surfactant concentration may range from about 0.2 wt% to 5 wt% of the tablet or capsule formulation.

[0183] Tablets or capsule compositions containing RhoA inhibitors generally also contain lubricants, such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and a mixture of magnesium stearate and sodium lauryl sulfate. The concentration of the lubricant may range from about 0.25 wt% to 10 wt%, about 0.5 wt% to 3 wt% of the tablet or capsule composition.

[0184] Other possible excipients included in the tablet or capsule formulation containing the RhoA inhibitor include glidants (e.g., talc or colloidal anhydrous silicon dioxide at about 0.1 wt % to about 3 wt % of the tablet or capsule formulation), antioxidants, colorants, flavoring agents, preservatives, and taste masking agents. When a glidant is present, the glidant concentration ranges from about 0.2 wt % to 1 wt % of the tablet or capsule composition.

[0185] Exemplary tablet compositions contain up to about 80 wt % of a RhoA inhibitor described herein, about 10 wt % to about 90 wt % of a binder, about 0 wt % to about 85 wt % of a diluent, about 2 wt % to about 10 wt % of a disintegrant, and about 0.25 wt % to about 10 wt % of a lubricant.

[0186] The tablet or capsule mixture (including the RhoA inhibitor and one or more suitable excipients) can be compressed directly or by roller to form tablets. The tablet or capsule mixture or a portion of the mixture can alternatively be wet, dry, or melt granulated, melt congealed or extruded before tableting. The final tablet or capsule composition can contain one or more layers and can be coated or uncoated; it can even be encapsulated in particles, such as polymer particles or liposome particles.

[0187] Solid dosage forms containing a RhoA inhibitor for oral administration can be formulated for immediate release and / or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release formulations.

[0188] b. Mucosal preparations

[0189] The disclosed RhoA inhibitors can be formulated for pulmonary or mucosal administration. Administration can include delivering the composition to the pulmonary, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa.

[0190] For example, the RhoA inhibitor can also be administered intranasally or by oral inhalation, typically from a dry powder inhaler in the form of a dry powder (alone, as a mixture, for example in a dry mixture with lactose, or as a mixed component particle, for example mixed with a phospholipid (e.g., phosphatidylcholine)) or as an aerosol spray from a pressurized container, pump, spray, nebulizer (preferably a nebulizer using electrohydrodynamics to produce a fine mist) or atomizer, with or without a suitable propellant, such as water, an ethanol-water mixture, 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal or oral inhalation use, the powder may contain a bioadhesive, such as chitosan or cyclodextrin. The term aerosol as used herein refers to any formulation of a fine particle mist, which may be in solution or suspension, whether or not it is made with a propellant. Aerosols can be made using standard techniques, such as ultrasonication or high pressure treatment.

[0191] The pressurized container, pump, spray, atomizer or nebulizer contains a solution or suspension of one or more RhoA inhibitors, including, for example, ethanol, aqueous ethanol or a suitable alternative for dispersing, solubilizing or prolonging the release of the active, one or more propellants as solvents and, optionally, a surfactant, such as sorbitan trioleate, oleic acid or oligolactic acid.

[0192] Capsules (e.g., made of gelatin or hydroxypropylmethylcellulose), blisters and cartridges for use in inhalers or insufflators can be formulated to contain a powder mix of a RhoA inhibitor as described herein, a suitable powder base (e.g., lactose or starch) and a performance modifier (e.g., l-leucine, mannitol or magnesium stearate). Lactose can be anhydrous or in the form of a monohydrate, the latter being preferred. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

[0193] Suitable solution formulations for use in atomizers that use electrohydrodynamics to produce a fine mist may contain 1 μg to 20 mg of one or more RhoA inhibitors per actuation, and the actuation volume may vary between 1 μl and 100 μl. A typical formulation may contain a variety of RhoA inhibitors disclosed herein, propylene glycol, sterile water, ethanol, and sodium chloride. Alternative solvents that may be used instead of propylene glycol include glycerol and polyethylene glycol.

[0194] Suitable flavoring agents, such as menthol and levomenthol, or sweetening agents, such as saccharin or saccharin sodium, may be added to those formulations intended for inhaled / intranasal administration.

[0195] Formulations for inhaled / intranasal administration can be formulated as immediate and / or modified release using, for example, PGLA. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release formulations.

[0196] In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve that delivers a metered amount. Units based on RhoA inhibitors are typically arranged to administer a metered dose or "puff". The total daily dose will be administered in a single dose or, more commonly, in divided doses throughout the day.

[0197] In some forms, the RhoA inhibitor can be formulated for pulmonary delivery, such as intranasal administration or oral inhalation. Carriers for pulmonary preparations can be divided into carriers for dry powder preparations and carriers for administration as solutions. Aerosols for delivering therapeutic agents to the respiratory tract are known in the art. For administration via the upper respiratory tract, the RhoA inhibitor can be formulated into an aqueous solution (e.g., water or isotonic saline, buffered or unbuffered), or as an aqueous suspension for intranasal administration as drops or as a spray. Such aqueous solutions or suspensions can be isotonic with respect to nasal secretions and have approximately the same pH, ranging, for example, from about pH 4.0 to about pH 7.4 or from pH 6.0 to pH 7.0. The buffer should be physiologically compatible and include, by way of example only, a phosphate buffer. Those skilled in the art can easily determine the appropriate salt content and pH of a harmless aqueous solution for nasal and / or upper respiratory tract administration.

[0198] In some forms, the aqueous solution is water, a physiologically acceptable aqueous solution containing salts and / or buffers (e.g., phosphate buffered saline (PBS)), or any other acceptable aqueous solution for administration to animals or humans. Such solutions are well known to those skilled in the art, and include, but are not limited to, distilled water, deionized water, pure or ultrapure water, saline, phosphate buffered saline (PBS). Other suitable aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl pyrrolidone and tragacanth, and wetting agents such as lecithin. Suitable preservatives for aqueous suspensions include ethyl p-hydroxybenzoate and n-propyl p-hydroxybenzoate.

[0199] In some forms, solvents of low toxicity organic (i.e., non-aqueous) Class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydrofuran, ether, and propanol, can be used in the formulation. The choice of solvent is based on its ability to easily aerosolize the formulation. The solvent should not react deleteriously with the RhoA inhibitor. An appropriate solvent should be used to form a suspension of the RhoA inhibitor. The solvent should have sufficient volatility to allow formation of an aerosol of the solution or suspension. Additional solvents or atomizing agents, such as freon, can be added as needed to increase the volatility of the solution or suspension.

[0200] In some forms, the pharmaceutical formulation may contain a small amount of surfactants or other excipients well known to those skilled in the art. In this context, "small amount" means that there are no excipients that may affect or mediate the penetration of the RhoA inhibitor in the tissue, and the excipients are present in an amount that does not adversely affect the penetration of the RhoA inhibitor in the tissue.

[0201] In some forms, pharmaceutical formulations containing one or more RhoA inhibitors can be administered directly to mucosal membranes (including the lining of the nose, lungs, and oral cavity), allowing the RhoA inhibitors to penetrate the mucosal layer and enter the underlying tissues.

[0202] Such formulations for direct application to mucosal membranes generally contain a dermatologically acceptable carrier that is suitable for application to the mucosa, has good aesthetic properties, is compatible with the active agent and any other ingredients, and does not raise any undesirable safety or toxicity issues.

[0203] The carrier can have a variety of forms. For example, emulsion carriers, including but not limited to oil-in-water, water-in-oil, water-in-oil-in-water, and silicone-in-water-in-oil emulsions, can be used herein. These emulsions can cover a wide range of viscosities, such as about 100 cps to about 200,000 cps. These emulsions can also be delivered in the form of sprays using mechanical pump containers or pressurized aerosol containers using traditional propellants. These carriers can also be delivered in the form of mousses or transdermal patches. Other suitable topical carriers include anhydrous liquid solvents, such as oils, alcohols, and silicones (e.g., mineral oil, ethanol isopropanol, polydimethylsiloxane, cyclomethicone, etc.); single-phase liquid solvents based on water (e.g., water-alcohol solvent systems, such as mixtures of ethanol and / or isopropanol and water); and thickened versions of these anhydrous and single-phase solvents based on water (e.g., where the viscosity of the solvent is increased to form a solid or semi-solid by adding appropriate gums, resins, waxes, polymers, salts, etc.). Examples of topical carrier systems that can be used in the formulations of the present invention are described in the following four references, all of which are incorporated herein by reference in their entirety: "Sun Products Formulary" Cosmetics & Toiletries, vol. 105, pp. 122 139 (December 1990); "Sun Products Formulary," Cosmetics & Toiletries, vol. 102, pp. 117 136 (March 1987); US Pat. No. 5,605,894 to Blank et al., and US Pat. No. 5,681,852 to Bissett.

[0204] Formulations for direct application on mucosa can be formulated for immediate release and / or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release formulations. Thus, RhoA inhibitors can be formulated as solids, semisolids, or thixotropic liquids for administration as implanted reservoirs providing modified release of active agents. Examples of such formulations include drug-coated stents.

[0205] c. Parenteral preparations

[0206] Optionally, the pharmaceutical composition containing one or more disclosed RhoA inhibitors is in a form suitable for direct administration into the bloodstream, muscle or internal organs. Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular and subcutaneous delivery. Suitable methods for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.

[0207] For example, pharmaceutical formulations containing one or more RhoA inhibitors are in a form suitable for intramuscular administration, intravenous administration, intraperitoneal administration, or subcutaneous administration, or a combination thereof.

[0208] Parenteral formulations containing the RhoA inhibitors described herein are typically aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (e.g., about pH 6.5 to about pH 8.0, about pH 6.5 to about pH 7.4, about pH 6.5 to about pH 7.0, about pH 7.0 to pH 8.0, or about pH 7.0 to about pH 7.4), however, for certain applications they may be more suitably formulated as sterile aqueous solutions or in dry form for use with a suitable vehicle (e.g., sterile, pyrogen-free water).

[0209] Liquid compositions containing a RhoA inhibitor for parenteral administration may be solutions, suspensions or emulsions.

[0210] The liquid pharmaceutically acceptable carrier forming the parenteral composition containing the RhoA inhibitor may include one or more physiologically compatible buffers, such as phosphate buffer. One skilled in the art can readily determine the appropriate salt content and pH of the aqueous carrier for administration (e.g., about pH 6.5 to about pH 8.0, about pH 6.5 to about pH 7.4, about pH 6.5 to about pH 7.0, about pH 7.0 to pH 8.0, or about pH 7.0 to about pH 7.4).

[0211] Liquid compositions containing RhoA inhibitors for parenteral administration may include one or more suspending agents, such as cellulose derivatives, sodium alginate, polyvinyl pyrrolidone, tragacanth gum or lecithin. Liquid compositions may also include one or more preservatives, such as ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate.

[0212] Optionally, the liquid composition containing the RhoA inhibitor contains one or more solvents that are low toxicity organic (i.e., non-aqueous) Class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydrofuran, ether, and propanol, and combinations thereof. Any such solvents included in the liquid formulation should not react deleteriously with the RhoA inhibitor or any additional active agent in the liquid composition. Solvents such as freons, alcohols, ethylene glycol, polyethylene glycol, or fatty acids may also be included in the liquid composition containing the RhoA inhibitor as desired to increase the volatility of the solution or suspension.

[0213] Liquid compositions containing RhoA inhibitors for parenteral administration may also contain small amounts of polymers, surfactants or other pharmaceutically acceptable excipients known to those skilled in the art. In this context, "small amounts" refers to amounts small enough to avoid adverse effects on the uptake of RhoA inhibitors by target cells (e.g., pituitary gonadotropins).

[0214] Preparation of parenteral compositions containing a RhoA inhibitor is typically carried out under sterile conditions, for example by lyophilization, which can be achieved using standard pharmaceutical techniques known to those skilled in the art.

[0215] Compositions for parenteral administration containing a RhoA inhibitor can be formulated to provide immediate and / or modified release of the active agent. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted- and programmed-release formulations.

[0216] d. Amount of RhoA inhibitor

[0217] The pharmaceutical composition generally contains an effective amount of a RhoA inhibitor and one or more pharmaceutically acceptable carriers and / or excipients. As used herein, the term "effective amount" refers to any amount of a RhoA inhibitor sufficient to achieve a desired therapeutic, preventive and / or diagnostic effect on a biological sample or in a subject to which it is administered. For example, the pharmaceutical composition contains an effective amount of a RhoA inhibitor to improve one or more symptoms associated with cancer in a subject.

[0218] Depending on the specific condition being treated and / or the route of administration, such an effective amount of a RhoA inhibitor can be from about 0.01 mg / kg to about 250 mg / kg body weight, from about 0.1 mg / kg to about 250 mg / kg body weight, from about 0.01 mg / kg to about 200 mg / kg body weight, from about 0.1 mg / kg to about 200 mg / kg body weight, from about 0.01 mg / kg to about 150 mg / kg body weight, from about 0.01 mg / kg to about 150 mg / kg body weight, from about 0.01 mg / kg to about 1 The invention relates to an agent that is preferably used in an amount of about 0.00 mg / kg body weight, about 0.1 mg / kg to about 100 mg / kg body weight, about 0.01 mg / kg to about 50 mg / kg body weight, about 0.1 mg / kg to about 50 mg / kg body weight, about 0.01 mg / kg to about 10 mg / kg body weight, about 0.1 mg / kg to about 10 mg / kg body weight, about 0.2 mg / kg to about 20 mg / kg body weight, or about 0.5 mg / kg to about 50 mg / kg body weight, which can be administered as a single daily dose, divided into more than one or more daily doses.

[0219] For example, an effective amount of a RhoA inhibitor is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 110 mg / kg, about 120 mg / kg, about 130 mg / kg, about 140 mg / kg, about 150 mg / kg, about 160 mg / kg, about 170 mg / kg, about 180 mg / kg, about 190 mg / kg, about 200 mg / kg, about 210 mg / kg, about 220 mg / kg, about 230 mg / kg, about 240 mg / kg, or about 250 mg / kg of the subject per day, which can be administered as a single daily dose, divided into more than one or more daily doses. The amount of RhoA inhibitor administered, the route of administration and further treatment regimen can be determined by the treating clinician or detection technician according to factors such as the age, sex and general condition of the subject, the nature and severity of the disease / symptom to be prevented, treated or diagnosed and / or the sample tested.

[0220] In some forms, the pharmaceutical composition is in unit dosage form and can be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or any other suitable single-dose or multi-dose holder or container (which can be appropriately labeled); optionally with one or more leaflets containing product information and / or instructions for use. Typically, such unit doses may contain 0.01 to 1000 mg, for example 0.01 to 500 mg, 0.01 to 100 mg, 0.01 to 50 mg, 0.01 to 10 mg, 0.01 to 1 mg, 0.1 to 500 mg, 0.1 to 100 mg, 0.1 to 50 mg, 0.1 to 10 mg, or 0.1 to 1 mg of a disclosed RhoA inhibitor, for example about 0.1, 0.2, 0.5, 1, 5, 10, 25, 50, 100, 200, 300 or 400 mg per unit dose.

[0221] e. Additional Active Agents

[0222] In some forms, a pharmaceutical composition containing a RhoA inhibitor also contains one or more additional active agents, such as one or more additional anti-cancer agents. For example, the one or more additional active agents are formulated into the same pharmaceutical composition containing one or more RhoA inhibitors described herein, as a single dosage unit or as multiple dosage units for coordinated, combined or concomitant administration, or formulated into separate pharmaceutical compositions for combination therapy.

[0223] Alternatively, one or more additional active agents can be formulated into a separate pharmaceutical composition, e.g., as a single dosage unit or as multiple dosage units, for co-administration with a pharmaceutical composition containing one or more RhoA inhibitors described herein. When formulated into a pharmaceutical composition separate from a pharmaceutical composition containing a disclosed RhoA inhibitor, one or more additional active agents can be formulated into a variety of formulations, e.g., injectable formulations, lyophilized formulations, liquid formulations, or oral formulations. The formulation can be selected based on the appropriate route of administration.

[0224] Examples of anticancer agents that can be used in a pharmaceutical composition containing a RhoA inhibitor or in a separate pharmaceutical composition include, but are not limited to, temozolomide, carmustine, bevacizumab, procarbazine, lomustine, vincristine, gefitinib, erlotinib, cisplatin, carboplatin, oxaliplatin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate, cytarabine, hydroxyurea, doxorubicin, bleomycin, doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mithramycin, long-acting Vinblastine, vindesine, vinorelbine, paclitaxel, taxol, docetaxel, etoposide, teniposide, amsacrine, topotecan, camptothecin, bortezomib, anagrelide, tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, fulvestrant, bicalutamide, flutamide, nilutamide, cyproterone acetate, goserelin, leuprolide, buserelin, megestrol acetate, anastrozole, letrozole, voroxazole, edoxifene Simestane, finasteride, marimastat, trastuzumab, cetuximab, dasatinib, imatinib, comprestatin, thalidomide, azacitidine, azathioprine, capecitabine, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, doxifluridine, epothilone, irinotecan, nitrogen mustard, mercaptopurine, mitoxantrone, pemetrexed, thioguanine, valrubicin and / or lenalidomide or a combination thereof, such as cyclophosphamide, methotrexate, 5-fluorouracil (CMF); doxorubicin, cyclophosphamide (AC) ; Nitrogen mustard, vinblastine, procarbazine, prednisolone (MOPP); adriamycin (sdriamycin), bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5-fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); and methotrexate, vincristine, doxorubicin, cisplatin (MVAC); and combinations thereof. Additional anticancer agents are described in U.S. Pat. No. 10,393,736, the disclosure of which is incorporated herein by reference in its entirety.

[0225] III. How to use

[0226] The disclosed RhoA inhibitors are suitable for cancer therapy. In particular, the RhoA inhibitors have high inhibitory efficacy and excellent binding specificity, which makes them superior to the reported RhoA inhibitors Rhosin and DC-Rhoin. For example, the RhoA inhibitors disclosed herein can strongly bind to RhoA in vitro, IC 50 The value is as low as about 400nM. Such a strong binding allows CL16 to inhibit RhoA in cancer cells with high efficacy. For example, the RhoA inhibitory effect is significantly higher than that reported for Rhosin. For example, as shown by the data in the examples, lower RhoA-GTP levels were observed in cells treated with 10μM CL16 than those treated with 30μM Rhosin. For example, compared with DC-Rhoin, which binds RhoA, Rac1, and Cdc42, the RhoA inhibitor disclosed herein can specifically bind to RhoA rather than other GTPases, such as other Ras GTPases (e.g., K-Ras and N-Ras) and in particular other Rho GTPases (e.g., Cdc42 and Rac1). This allows the disclosed RhoA inhibitor to exhibit high cytotoxicity in cancer cells both in vitro and in vivo, while the toxicity in normal cells is negligible. Prior to the disclosed RhoA covalent inhibitor, there was no RhoA covalent inhibitor that had an in vivo anti-tumor effect and good efficacy in a mouse colorectal cancer xenograft model.

[0227] In some forms, the disclosed RhoA inhibitors or pharmaceutical compositions containing RhoA inhibitors can be used to treat cancer. In some forms, the cancer is a solid cancer (also referred to herein as a "tumor"), such as melanoma, gastric cancer, breast cancer, and colorectal cancer. "Treatment" or "treating" refers to the medical management of a patient with the goal of curing, improving, stabilizing, or preventing a disease, pathological condition, or disorder, such as cancer. The term includes active treatment, i.e., treatment specifically directed to improving a disease, pathological condition, or disorder (e.g., cancer); and also includes etiological treatment, i.e., treatment directed to eliminating the cause of the relevant disease, pathological condition, or disorder (e.g., cancer). In addition, the term also includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition, or disorder (e.g., cancer); preventive treatment, i.e., treatment intended to minimize or partially or completely inhibit the development of the relevant disease, pathological condition, or disorder (e.g., cancer); and supportive treatment, i.e., treatment used to supplement another specific therapy directed to improving the relevant disease, pathological condition, or disorder (e.g., cancer).

[0228] In some forms, a method of treating cancer (eg, a solid cancer) using a pharmaceutical composition containing a disclosed RhoA inhibitor comprises: (i) administering the pharmaceutical composition to a subject in need thereof.

[0229] Administration in step (i) may be performed using any suitable technique, such as oral administration, intranasal administration, intramuscular administration, intravenous administration, intraperitoneal administration or subcutaneous administration, or a combination thereof.

[0230] Step (i) of the disclosed method (administration of the pharmaceutical composition) may occur one or more times. When the administration step is performed more than once, each administration may be performed regularly every 5 minutes, every 10 minutes, every 20 minutes, every 30 minutes, every hour, every 2 hours, every day, every two days, every three days, every week, every two weeks, every month, etc.; or irregularly at intervals of 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, etc. The specific number of treatments and the time intervals used for treatment can be determined by the treating clinician or the detection technology based on factors such as the age, sex and general condition of the subject, the nature and severity of the disease / symptom being prevented, treated or diagnosed, and / or the sample being detected.

[0231] Typically, following the administering step or all administering steps, an effective amount of a RhoA inhibitor in a pharmaceutical formulation is administered to the subject such that cancer cells are killed and / or the growth or proliferation of cancer cells is reduced or prevented, and thereby one or more symptoms associated with cancer are improved in the subject, such as a reduction in tumor volume and / or tumor weight.

[0232] For example, after the administering step or all administering steps (if the administering occurs more than once), the subject is administered an effective amount of the RhoA inhibitor such that the subject's tumor volume is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% less than a control administered with only a pharmaceutically acceptable excipient. For example, after the administering step or all administering steps (if the administering occurs more than once), the subject is administered an effective amount of the RhoA inhibitor such that the subject's tumor volume is unchanged compared to before the administration of the pharmaceutical composition, or the increase in the subject's tumor volume compared to before the administration of the pharmaceutical composition is less than the increase in the tumor volume of the control administered with only a pharmaceutically acceptable excipient.

[0233] For example, after the administering step or all administering steps (if the administering occurs more than once), an effective amount of a RhoA inhibitor is administered to the subject such that the tumor weight in the subject is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% less than a control administered with only the pharmaceutically acceptable excipient.

[0234] For example, after the administering step or all administering steps (if the administration occurs more than once), an effective amount of a RhoA inhibitor is administered to the subject such that the tumor volume and tumor weight of the subject is at least 10%, at least 20%, at least 30%, at least 40% or at least 50% smaller / less than a control administered with only the pharmaceutically acceptable excipient.

[0235] For example, an effective amount of a RhoA inhibitor is administered to a subject after the administration step or after all administration steps (if administration occurs more than once) such that the subject's tumor volume is unchanged compared to before administration of the pharmaceutical composition, or the increase in the subject's tumor volume compared to before administration of the pharmaceutical composition is less than the increase in tumor volume of a control administered with only a pharmaceutically acceptable excipient; and the subject's tumor weight is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% less than the control.

[0236] For example, after the administering step or all of the administering steps, the effective amount of RhoA administered to the subject is about 0.1 μg to about 1000 μg, about 0.1 μg to about 500 μg, about 0.1 μg to about 100 μg, about 0.5 μg to about 50 μg, about 1 μg to about 1000 μg, about 1 μg to about 500 μg, about 1 μg to about 100 μg, about 1 μg to about 50 μg, about 1 μg to about 25 μg, about 1 μg to about 10 μg, about 2 μg to about 20 μg, or about 5 μg to about 50 μg per gram of the subject.

[0237] The above cancer treatment effect can occur within 3 days, 5 days, 7 days, 10 days, 2 weeks, one month, three months or six months after the administration step or all administration steps. Optionally, the cancer treatment effect occurs without any observable toxicity to the subject, as shown by physical vital measurements (e.g., body weight), standard hematological markers and / or blood biochemical parameters compared to the control.

[0238] In some forms, the disclosed methods may further comprise administering to the subject one or more additional active agents, such as one or more of the additional anti-cancer agents described above, prior to administering the pharmaceutical composition containing the RhoA inhibitor in step (i), simultaneously with step (i), or after step (i), or a combination thereof.

[0239] The disclosed compositions and methods can be further understood through the following enumerated paragraphs.

[0240] 1. Compounds having the following structure:

[0241]

[0242] in:

[0243] (i)X 1 and X 2 Independently C(R 6 ) 2 , O, S or NR 6 , and R 6 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, alkoxy or carbonyl;

[0244] (ii)A 1 C 5 -C 6 Aromatic ring or C 4 -C 5 heteroaromatic ring;

[0245] (iii) n1 is an integer from 0 to 4;

[0246] (iv) n2 is an integer from 0 to 3;

[0247] (v)R 1 , R 3 and R 5 are independently hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl;

[0248] (vi)R 2 are cysteine-reactive groups (e.g., haloacetamide, acrylamide, maleimide, vinyl sulfone, and epoxide) that allow chemical reaction with the cysteine ​​on RhoA;

[0249] (vii) R 4 is hydrogen, alkoxy, hydroxy, halogen, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, carbonyl, amino, or haloalkyl; and

[0250] (viii) when substituents are present, they are independently unsubstituted alkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted polyaryl, unsubstituted heteropolyaryl, unsubstituted alkylaryl, unsubstituted cyclic, unsubstituted heterocyclic, halide, amino, amido, thiol, hydroxy, cyano, nitro, carbonyl, or alkoxy.

[0251] 2. The compound of paragraph 1, which has the following structure:

[0252]

[0253] 3. The compound of paragraph 1 or 2, having the following structure:

[0254]

[0255] 4. The compound of any one of paragraphs 1 to 3, wherein n 1 Is 0 or 1.

[0256] 5. The compound of any one of paragraphs 1 to 4, wherein R 1 It's hydrogen.

[0257] 6. The compound according to any one of paragraphs 1 to 5, wherein R 3 is hydrogen or substituted or unsubstituted aryl, wherein substituents, when present, are independently unsubstituted alkyl, unsubstituted aryl or unsubstituted alkylaryl.

[0258] 7. The compound according to any one of paragraphs 1 to 6, wherein R 2 for n3 is an integer from 0 to 6, R 7 and R 8 are independently hydrogen, unsubstituted alkyl or halogen, and R 9 is hydrogen or halogen.

[0259] 8. The compound of any one of paragraphs 1 to 7, wherein R 4 Hydrogen or n4 is an integer from 0 to 6, R 10 and R 11 are independently hydrogen or halogen, and R 12 is hydrogen, halogen, amino, unsubstituted alkynyl, aldehyde or carboxylic acid.

[0260] 9. The compound of paragraph 8, wherein R 10 and R 11 is hydrogen, and R 12 is an unsubstituted alkynyl group.

[0261] 10. The compound of any one of paragraphs 1 to 9, wherein the compound is not

[0262] 11. The compound of any one of paragraphs 1-10, wherein when X 1 O, X 2 For S, R 1 and R 3 -R 4 is hydrogen, and A 1 C 6 When the ring is aromatic, R 2 Not a haloacetamide.

[0263] 12. The compound of any one of paragraphs 1 to 9, which has the following structure:

[0264]

[0265] 13. The compound of any one of paragraphs 1 to 12, which has the following structure:

[0266]

[0267] 14. The compound of any of paragraphs 1-13, wherein the compound is covalently bound to RhoA.

[0268] 15. The compound of paragraph 14, wherein the compound is covalently bound to Cys16 of RhoA.

[0269] 16. The compound of paragraph 14 or 15, wherein the compound inhibits RhoA activity in living cells.

[0270] 17. A pharmaceutical composition comprising a compound according to any one of paragraphs 1 to 16, and optionally one or more pharmaceutically acceptable excipients.

[0271] 18. The pharmaceutical composition of paragraph 17, further comprising a second active agent, optionally wherein the second active agent is an anti-cancer agent.

[0272] 19. The pharmaceutical composition of paragraph 17 or 18, wherein the compound in the pharmaceutical composition is in an effective amount to improve one or more symptoms associated with cancer in a subject.

[0273] 20. The pharmaceutical composition of any of paragraphs 17-19, wherein the amount of the compound in the pharmaceutical composition ranges from about 0.1 μM to about 1 mM, about 0.1 μM to about 500 μM, about 0.1 μM to about 200 μM, about 0.1 μM to about 100 μM, about 0.1 μM to about 50 μM, or about 0.1 μM to about 20 μM.

[0274] 21. A method for treating cancer using the pharmaceutical composition of any one of paragraphs 17-20, comprising:

[0275] (i) administering the pharmaceutical composition to a subject in need thereof,

[0276] wherein step (i) occurs one or more times.

[0277] 22. The method of paragraph 21, wherein the pharmaceutical composition is administered orally, intramuscularly, intravenously, intraperitoneally or subcutaneously, or a combination thereof.

[0278] 23. The method of paragraph 21 or 22, wherein in step (i), the dose of the compound administered is from about 0.1 μg to about 1000 μg, from about 0.1 μg to about 500 μg, from about 0.1 μg to about 100 μg, from about 0.5 μg to about 50 μg, from about 1 μg to about 1000 μg, from about 1 μg to about 500 μg, from about 1 μg to about 100 μg, from about 1 μg to about 50 μg, from about 1 μg to about 25 μg, from about 1 μg to about 10 μg, from about 0.1 μg to about 50 μg, from about 5 μg to about 50 μg, or from about 0.1 μg to about 20 μg per gram of subject.

[0279] 24. The method of any of paragraphs 21-23, wherein the cancer is a solid cancer.

[0280] 25. The method of any of paragraphs 21-24, wherein the cancer is colorectal cancer.

[0281] 26. The method of paragraph 24 or 25, wherein after step (i) or the entire step (i) (if step (i) occurs more than once), the tumor volume and / or tumor weight is at least 10%, at least 20%, at least 30%, at least 40% or at least 50% smaller / less than a control administered with only a pharmaceutically acceptable excipient.

[0282] 27. The method of any of paragraphs 21-26, wherein after step (i) or the entire step (i) (if step (i) occurs more than once), there is no observable toxicity to the subject as demonstrated by physical vital measurements (e.g. body weight), standard hematological markers and / or blood biochemical parameters compared to a control.

[0283] 28. The method of any of paragraphs 21-27, further comprising administering an active agent before, during and / or after step (i), optionally wherein the active agent is an anti-cancer agent.

[0284] The invention will be further understood with reference to the following non-limiting examples. Example

[0285] Example 1. Synthetic RhoA covalent inhibitor targeting Cys16 for cancer therapy

[0286] The design, synthesis, and biological activity of exemplary RhoA covalent inhibitors targeting Cys16 for cancer therapy are provided. The chemical proteomics platform ABPP is used to discover RhoA covalent inhibitors. By covalently targeting RhoA Cys16, which has never been used as a binding site for drug compounds, the exemplary compound CL16 was shown to selectively bind to RhoA, but not to other Rho family proteins, such as Rac1 and Cdc42. LC-MS / MS experiments showed covalent binding of CL16 on Cys16, which is adjacent to the nucleotide binding pocket of RhoA. This covalent binding places CL16 next to the nucleotide binding pocket, as shown by computational docking, and results in the prohibition of GTP binding to RhoA. The covalent binding of CL16 on RhoA Cys16 also disrupts the RhoA-GEF interaction. The selective covalent binding of CL16 to RhoA results in specific inhibition of RhoA activity by prohibiting GDP to GTP exchange in vitro and in cancer cells, as demonstrated using FRET-RhoA biosensor imaging, immunofluorescence, and Western blot experiments. For example, as shown by the data below, the selective covalent binding of CL16 to RhoA results in inhibition of RhoA signaling in colorectal cancer (CRC) cells after CL16 treatment, resulting in high cytotoxicity and significantly slowed cell migration and invasion of CRCs with overexpressed RhoA. In particular, CL16 treatment was shown to be effective in shrinking CRC spheroids and slowing tumor growth in vivo, with no observable toxicity. These demonstrate the use of the disclosed RhoA covalent inhibitors as anticancer and anti-metastatic agents.

[0287] CL16 was found to be more potent in RhoA inhibition than the reported RhoA inhibitor Rhosin. In addition, the good efficacy of RhoA inhibition enabled CL16 to mediate anticancer and anti-metastatic properties in 2D cancer cell cultures and 3D spheroid models with overactive RhoA, while the toxicity was much lower in normal cells. CL16 also exhibited potent in vivo antitumor effects in mouse cancer xenograft models with no observable toxicity. CL16 is believed to be the first RhoA covalent inhibitor with in vivo antitumor effects in a mouse colorectal cancer xenograft model with good efficacy. A molecular probe of CL16, CL16-alkyne, was developed, which can act as a probe of RhoA to verify the specific binding of CL16 to RhoA. The results indicate that the RhoA inhibitors disclosed herein can be used for biological studies of RhoA function and activity in cancer, as well as as candidate drugs for targeting RhoA for cancer therapy.

[0288] Materials and methods

[0289] Activity-Based Protein Profiling (ABPP)

[0290] Recombinant human RhoA (0.1 μg; ab101594) was pretreated with cysteine-reactive compounds or DMSO for 1 hour at room temperature and then incubated with iodoacetamide-rhodamine (IA-Rh; 1 μM) for 1 hour at room temperature. The reaction was then quenched by boiling in loading buffer. The samples were subjected to SDS-PAGE to separate protein bands. In-gel fluorescence intensity was measured with Chemidoc using the rhodamine setting. The gel was subsequently stained with silver stain (Pierce) for visualization of protein content and covalent modifications. Band intensities were quantified using ImageJ.

[0291] Covalent docking

[0292] Maestro 13.4 was used for covalent docking. The structure of GDP-bound RhoA (PDB: 1CC0) was retrieved from the Protein Data Bank (PDB). The protein structure was prepared using the Protein Preparation Wizard tool by adding missing side chains and loops, removing water proteins, and adjusting hydrogen orientations at pH 7.0 using PROPKA. The protein was minimized using the OPLS4 force field. The CL16 ligand structure was prepared using the LigPrep module by performing Epik at pH 7.0 (±2.0) followed by OPLS4 force field minimization. Covalent docking was performed by the CovDoc algorithm. The receptor was selected for the A:16 (Cys16) residue and the box size was such that Ligand docking of length. Nucleophilic substitution was selected as the reaction type. No restrictions were imposed.

[0293] Co-immunoprecipitation

[0294] 10 μg of purified GST-RhoA was immobilized on glutathione resin (Pierce) and incubated with CL16 in GTP exchange buffer (20 mM HEPES, 150 mM NaCl, 5 mM MgCl 2 , pH 7.4) for 1 hour. Then, the mixture was centrifuged at 700g for 2min and the supernatant was discarded. 120μg of total cell lysate with overexpressed GEFs (Arhgef1 p-115, Arhgef12 LARG DH-PH domain, Arhgef13Akap Lbc) was added to the resin. The mixture was placed on an end-to-end rotator and incubated at 4°C for 4 hours. The resin was then washed, eluted and immunoblotted.

[0295] GDP-GTP exchange assay

[0296] Recombinant human RhoA protein (0.2 μg; ab101594) was incubated with GDP (50 nM) at room temperature in 100 μL of GTP exchange buffer (20 mM HEPES, 150 mM NaCl, 5 mM MgCl 2 , pH 7.4) and incubated for 1 h. CL16 or solvent control was then added to the solution mixture and incubated for another hour at room temperature. For self-exchange assays, BODIPY-GTP (100 nM) was added to the solution and the fluorescence from the plate was measured every 30 s. For LARG-mediated GDP to GTP exchange, BODIPY-GTP (100 nM) and LARG DH-PH protein (30 nM) were added to the solution mixture and the fluorescence from the plate was measured every 30 s. BODIPY-GTP was excited at 477 ± 14 nm and its fluorescence was measured at 525 ± 30 nm using a plate reader (Clariostar).

[0297] RhoA GTP-rhotekin pull down

[0298] RhoA Pull-Down Activation Assay Biochemical Kit (#BK036) was purchased from Cytoskeleton. Total cell lysate (0.5 mg / mL; 300 μL) was added to 7.5 μL Rhotekin-Rho binding domain beads in a 0.6 mL tube according to the manufacturer's protocol. The solution mixture was incubated on an end-to-end rotator at 4°C for 1 hour. The beads were washed once with 500 μL of wash buffer, then centrifuged at 5000 g for 3 min, and the supernatant was discarded. RhoA-GTP was then eluted in 2X laemmli buffer and resolved in 10% SDS-PAGE.

[0299] Cell culture

[0300] The cells were cultured in a 5% CO 2 HT29 cells were cultured in DMEM (Gibco, with 10% FBS and 1% penicillin and streptomycin). SW620, SW480, LOVO and HCT116 cells were cultured in RPMI (Gibco, with 10% FBS and 1% penicillin and streptomycin). CCD-18Co cells were cultured in MEM (Gibco, with 10% FBS and 1% penicillin and streptomycin). These cell lines were obtained from Professor SY Leung and Dr. Helen Yan, Department of Pathology, University of Hong Kong.

[0301] Gene set enrichment analysis (GSEA)

[0302] The analysis was performed using non-stranded TPM values ​​from open access colorectal adenocarcinoma tumor tissue RNA sequencing results obtained from TCGA and calculated using GSEA v4.3.3 Mac App. When comparing stages 0-I and IV, the results were divided according to the AJCC pathological stage and analyzed using relevant samples. To compare high and low EMT scores, the EMT scores of all samples were calculated using the single sample gene set enrichment score (ssGSEA) using the hacksig R package. The scores were divided into two groups relative to the median score.

[0303] Immunoblotting

[0304] Anti-RhoA (#2117), anti-GAPDH (#2118), anti-phosphorylated MLC (#3671), anti-MLC (#3672), anti-FLAG (#8146), anti-GFP (#2956) and anti-GST (#2624) were purchased from Cell Signaling Technology. Cells were washed with PBS and then harvested using cell lysis buffer (1% Triton X100, 10mM β-glycerophosphate, 10mM sodium pyrophosphate, 40mM HEPES, 4mM EDTA, pH 7.4) with 4% protease / phosphatase inhibitor cocktail (Pierce). Protein samples were quantified using BCA assay (Pierce), normalized, and then boiled at 95°C for 5 min with 4X Laemmli loading buffer. The samples were resolved in 10% polyacrylamide gels (Biorad), then transferred to PVDF membranes (Biorad) and blocked with blocking buffer (5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TBST)) for 1 h. The membranes were washed in TBST and then incubated with the corresponding primary antibodies (5% BSA / milk in Tris-buffered saline with 1% NaN) at 4°C. 3 The membrane was incubated overnight with 1:1000 dilution in TBST. After washing three times with TBST, the membrane was incubated with HRP-conjugated secondary antibody (#7074 or 7076) at room temperature for one hour. The membrane was then incubated with LumiGlo reagent (Cell Signaling Technology) and chemiluminescence was captured using Chemidoc (Biorad).

[0305] Rac1-GTP and Cdc42-GTP assays

[0306] The procedure is similar to the RhoA-GTP assay procedure, except that Rac1-GTP and Cdc42-GTP in CRC cells are first enriched by Rac1 Pull-Down Activation Assay Biochemical Kit (Cytoskeleton#BK035) instead of using the RhoA kit. The enriched Rac1-GTP and Cdc42-GTP are then immunoblotted using Rac1 / 2 / 3 antibodies (CST#2465) and Cdc42 antibodies (CST#2462), respectively.

[0307] MTT assay

[0308] In a 96-well plate, HCT116 cells were seeded at 2,500 cells / well; HT29, LOVO, and SW620 cells were seeded at 4,000 cells / well; CCD-18Co cells were seeded at 10,000 / well; and SW480 cells were seeded at 12,000 cells / well in triplicate. The cells were treated with a concentration gradient of CL16 for 48 hours. The cells were then incubated with 10% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (50 mg / mL) at 37°C for 4 hours. The cells were then lysed with 10% SDS in 0.01 M HCl. The absorbance of a single well was measured at 570 nm. IC was analyzed by nonlinear best fit regression using Prism Graphpad 9 50 value.

[0309] Wound healing and Matrigel invasion assays

[0310] For the wound healing assay, HT29 cells were seeded at 90% confluency and the next day the wound was scratched using a P-20 tip. Cell debris was washed with PBS and then aspirated. Then, complete DMEM medium with corresponding concentrations of CL16 or Rhosin was added to the cells. Wound closure was recorded every 24 hours under a light microscope with a 10X objective, and the wound area was measured by ImageJ.

[0311] For the Matrigel invasion assay, SW620 cells (100,000 cells per well) in serum-free medium with CL16 or DMSO were seeded into Boyden chambers. Full RPMI was loaded into the lower chamber as a chemoattractant. After 24 h, the bottom of the Boyden chamber was fixed by 4% PFA for 10 min, and then stained with crystal violet solution for another 10 min. Excess stain was washed off with tap water. Invaded cells were recorded with a 10X objective under an optical microscope, and the area of ​​cells with penetration was measured by ImageJ.

[0312] siRNA knockdown

[0313] A set of 4 siRNAs targeting different RHOA sequences was purchased from Horizon (Horizon L-003560-00-0010). HT-29 cells were transfected with 5 μM siRNA using Dharmafect reagent (Horizon) (3×10 cells / well). 6 The knockdown efficiency was quantified by immunoblotting.

[0314] Spheroid formation assay

[0315] 5,000 cells of HT29 and SW620 cells were seeded in serum-free DMEM / F12 medium with 20 ng / mL epithelial growth factor (EGF) in 96-well round-bottom low-affinity plates (Nunc). When spheroids formed an opaque core, the medium was supplemented with CL16 or Rhosin. The morphology was recorded under an optical microscope with a 10X objective.

[0316] Flow cytometry experiments on the cell cycle of CRC cells treated with CL16

[0317] HCT116 cells were seeded into 6-well plates (500,000 cells) and cultured at 37°C, 5% CO 2 Incubate overnight in complete medium. After 24h of treatment with DMSO control or specified concentration of CL16, cells were harvested by trypsinization, washed with PBS, and fixed in 70% cold ethanol on ice for 30 minutes. The cells were then centrifuged at 250g for 5min and resuspended in a staining buffer (PBS with 0.05% TritonX-100) containing propidium iodide (50μg / mL) and RNase (100μg / mL) at 37°C in the dark for 30min. The cells were then filtered through a 40μm filter and analyzed by flow cytometry. Representative FACS graphs were developed to illustrate the gating strategy for studying the cell cycle. Cell events were acquired using a NovoCyte Advanteon BVYG analyzer, and data were processed using NovoExpress software.

[0318] Apoptosis assay by confocal fluorescence microscopy

[0319] HCT116 cells were seeded onto poly-D-lysine-coated Ibidi 8-well chamber slides (20,000 cells) and cultured at 37°C, 5% CO 2 Cells were cultured overnight in serum-free medium with the indicated concentrations of DMSO control, Rhosin or CL16, and 5 μM CellEvent TMCells were treated with Caspase-3 / 7 Green for 24 h. Before imaging, the culture medium was replaced with Hanks balanced salt solution (HBSS). Confocal fluorescence microscopy was performed using a Zeiss laser scanning microscope 880 with a 20× water immersion objective and ZEN 2.3 (Black Edition) software (CarlZeiss). TM Caspase-3 / 7Green was excited by a 488nm diode laser and emission was collected on a META detector between 490 and 525nm. Image analysis was performed on ImageJ, where the number of cells expressing fluorescence within the field of view was counted against the total number of cells to report the percentage of apoptotic cells. The average percentage was calculated from 3 independent images from the same well.

[0320] Mouse xenograft model

[0321] Seven-week-old mice weighing approximately 20 g were treated with HCT116 (2 × 10 per mouse) in serum-free RMPI or Matrigel in PBS. 6 cells) or HT29 (3x10 cells per mouse 6 CL16 was dissolved in PET (60% PEG400, 30% ethanol, 10% Tween 80, v / v) and sonicated until the solution became clear. Then the PET vehicle or CL16 in PET was mixed with PBS at a ratio of 1:1 (v / v) to prepare the solution for injection. When the tumor reached 100 mm 3 After the tumor size reached 400 μg / cm2, CL16 (200 μL) was injected intraperitoneally every 2 days until the humane endpoint was reached. The tumor volume and body weight of nude mice were measured throughout the treatment. Tumor volume was calculated by the following equation: Volume (mm 3 )=l×w 2 / 2, where l and w are the length and width of the tumor. At the end of treatment, mice were sacrificed and tumor weights were measured. Tumor tissues were then processed into PEFF blocks.

[0322] Gel ABPP

[0323] HCT116 and SW620 cells were cultured at 5 × 10 5The density of cells was plated on a 6-well plate overnight. The cells were then treated with CL16 at a specified concentration for 2 hours, followed by CL16-alkyne (1 μM) for 2 hours. After treatment, the cells were washed with PBS and lysed by probe sonication. Cell debris was removed by centrifugation at 4 ° C for 10 min at 5,000 g. 50 μL of standardized protein lysate (2 mg / mL) was labeled with azide-fluorescence 545 for 1 hour at room temperature in the dark by copper-catalyzed azide-alkyne cycloaddition. Then, the reaction was quenched by boiling in the loading buffer, and the sample was subjected to SDS-PAGE to separate protein bands to measure intra-gel fluorescence. After imaging, the gel was washed with MilliQ and incubated with Coomassie blue dye with gentle shaking.

[0324] Stable HT29 cells expressing RhoA C16A

[0325] HT29 cells with RhoA C16A knock-in were prepared by BrainVTA using CRISPR-Cas9 technology. Briefly, HT29 cells were transfected with a RhoA construct with a mutation site in a CRISPR / Cas9 vector (U6-sa-sgRNA) using PEI. Cells were subsequently screened for 2 weeks with 2 μg / mL puromycin and 800 μg / mL neomycin. Monoclonal cells were then screened, propagated, and Sanger sequenced to verify the homozygous C16A amino acid substitution (GCT to TGT) in the RhoA gene.

[0326] Physical Measurement and Instrumentation

[0327] 1 H NMR and 13 C{ 1 H} spectra were collected on a Bruker AVANCE NEO600 MHz spectrometer at the Department of Chemistry or the Department of Microbiology at the University of Hong Kong. All chemical shifts are reported in standard δ notation relative to the residual solvent peak as an internal reference. Splitting patterns are denoted as follows: br, broad; s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of doublets; tt, triplet of triplets. UV spectra of the chemicals were acquired by an ACQUITY UPLC I-Class PLUS system (Waters), while high-resolution mass spectra were acquired by a TripleTOF Mass Spectrometer provided by the Biomedical Technology Support Center of the Hong Kong Science Park. TM6600 (Sciex). UV-Vis absorption and fluorescence of microplates were recorded on Perkin Elmer Victor 3 (Molecular Devices) or CLARIOstar Plus (BMG Labtech). In-gel fluorescence images were recorded on ChemiDoc MP gel imaging system (Bio-Rad). Confocal microscopy images were recorded on Zeiss laser scanning microscope 880 with 20× air objective lens at 3× magnification using ZEN 2.3 (Black Edition) software (Carl Zeiss). Flow cytometry experiments were performed on NovoCyte Advanteon BVYG analyzer (Agilent).

[0328] LC-MS / MS experiments on HCT116 cells treated with CL16

[0329] HCT116 cells were treated with DMSO or CL16 (25 or 50 μM) in complete medium for 4 hours, washed with PBS, harvested, and lysed in PBS by sonication. The protein amount of the samples was determined by BCA analysis and normalized. Cell lysates in PBS were reduced by TCEP (1 mM) at 65 ° C for 30 min, and then alkylated with iodoacetamide (18 mM) at 37 ° C for 30 min in the dark. The mixture was added with pre-cooled acetone (600 μL) and the protein was precipitated at -20 ° C overnight. The samples were centrifuged at 8000g for 10 min at 4 ° C. The supernatant was discarded and the dried precipitate was resuspended in 2M urea in PBS (100 μL). Sequencing grade trypsin (Promega # V5111) was added to the sample and digested overnight at 37 ° C with constant shaking. The solution was then acidified with formic acid (FA; final concentration 5%) and centrifuged at 13,200 rpm for 30 min at 4 ° C. The supernatant was collected and stored at -80 °C prior to MS analysis.

[0330] The peptide solution was dried and desalted by passing through C18 Stage tips, and the peptides were loaded onto an Aurora C18 UHPLC column (75 μm id×25 cm length×1.6 μm particle size (Ion Opticks Australia)) coupled to a timsTOF Pro mass spectrometer (Bruker). Chromatographic separation was performed using buffer A (98:2 water:acetonitrile, 0.1% formic acid) and B (acetonitrile, 0.1% formic acid) with a gradient of 2% to 30% buffer B in 90 min at a flow rate of 300 nL / min, followed by an increase from 30% to 50% buffer B in 25 min, an isocratic gradient of 95% buffer B in 8.5 min, a decrease of buffer B to 2% in 0.5 min, and then an isocratic gradient of 2% buffer B for 5.5 min. MS data were collected over the m / z range of 100 to 1700 and the MS / MS range of 100 to 1700. During MS / MS data collection, each TIMS cycle was 1.1 s and included 1MS + averaging of 10 PASEF MS / MS scans.

[0331] Raw files were searched using MaxQuant (v2.0.3.0) with the Uniprot human database (UP000005640). The search was specified as trypsin digestion (up to 3 cuts allowed). Cysteine ​​carbamidomethylation (+57.02146) was selected as a fixed modification, while N-terminal acetylation (+42.01057), methionine oxidation (+15.99491), and CL16-cysteine ​​adduct (+235.06333) were set as variable modifications. The peptide false discovery rate (FDR) was set to 1%. Modified peptides with spectral counts less than 2, PEP greater than 0.01, and spectral counts of CL16 at 50 μM lower than 25 μM were filtered out.

[0332] LC-MS / MS for CL16 Target Identification Using CL16-Alkyne

[0333] HCT116 cells were treated with DMSO or CL16 (25 μM) in complete medium for 2 h. Cells were washed with PBS and then incubated with CL16-alkyne (50 μM) for 2 h. After treatment, cells were washed with PBS and lysed by probe sonication in PBS. Cell debris was removed by centrifugation at 5,000 g for 10 min at 4 °C. After protein determination using BCA and protein standardization, cell lysate (4 mg / mL, 2 mL) was mixed with master mix solution for CuAAC reaction. The master mix solution contained CuSO 4, TBTA, DTB-PEG-azide and TECP were added to the final concentrations of 1 mM, 100 μM, 100 μM and 1 mM, respectively. After the samples were vortexed and incubated with the master mix solution at room temperature for 1 hour, pre-cooled acetone (12 mL) was added to the samples and the proteins were allowed to precipitate overnight at -20°C. The samples were centrifuged at 5,000 g for 10 min at 4°C and the supernatant was discarded. The protein precipitate was then redispersed in 1.2% SDS in PBS (w / v) and heated at 80°C for 5 min. Any insoluble solids were discarded by centrifugation at 6,500 g for 5 min and the supernatant was transferred to a 4% PBS containing Pierce TM Streptavidin agarose beads (Thermo Scientific #20349) in PBS with a final concentration of 0.2% SDS (w / v). The sample and beads were incubated overnight at 4°C with rotation. The beads were then washed with PBS and water and redispersed in 6M urea in PBS. The sample was reduced by TCEP (1mM) at 65°C for 20min and then alkylated with iodoacetamide (18mM) at 37°C in the dark for 30min. The beads were then spun down by centrifugation at 1,400g for 2min, washed with PBS, and resuspended in 2M urea in PBS. The proteins on the beads were digested overnight at 37°C by sequencing grade trypsin (Promega). The solution mixture was then transferred to a BioSpin column (Bio-rad), and the trypsin-digested peptides were collected by centrifugation at 1,400g for 2min. The beads were washed twice with PBS (100 μL). The wash solution was collected by centrifugation at 1,400g for 2min and combined with the trypsin-digested peptide solution. The combined solution was dried and desalted by C18 Stage tips. Finally, the peptides were sent to the timsTOF Pro mass spectrometer for LC-MS / MS analysis and analyzed using the protocol described above for LC-MS / MS experiments.

[0334] The raw files were searched using MSFragger (v3.7) with the Uniprot human database (UP000005640). The search was specified as trypsin digestion (up to 3 cuts allowed). Cysteine ​​carbamidomethylation (+57.02146) was selected as a fixed modification, while N-terminal acetylation (+42.01057) and methionine oxidation (+15.99491) were set as variable modifications. The peptide false discovery rate (FDR) was set to 1%. For proteins with zero LFQ intensity in all 3 replicate runs in the control group and proteins with non-zero LFQ intensity in all 3 runs in the treatment group, they were assigned an LFQ ratio of 20. To eliminate false positives, proteins with an average intensity less than 100,000, a combined spectral count less than 10, or a protein probability less than 1 were filtered out.

[0335] Synthesis of exemplary RhoA inhibitors

[0336]

[0337] 1a was synthesized according to the reported literature (Wang, X.-L. et al. European Journal of Medicinal Chemistry 45, 4631-4639 (2010)).

[0338]

[0339] Hydrazine (2 eq.) was added to 1a (1 eq.) in ethanol and heated under reflux for 3-4 h. Any organic volatiles were evaporated under reduced pressure and the residue was extracted with dichloromethane. The dichloromethane layer was filtered through MgSO 4 The intermediate was then dissolved in anhydrous dichloromethane. Chloroacetyl chloride (1.2 equivalents) and triethylamine (2 equivalents) were slowly added dropwise on ice. The reaction was allowed to proceed overnight. Any volatile organic solvents were evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography using hexane / ethyl acetate (80:20, v / v) as eluent to give CL16. 1 H NMR (600 MHz, chloroform-d) δ7.66-7.54 (m, 2H), 7.44-7.39 (m, 1H), 7.34-7.27 (m, 2H), 7.16 (s, 1H), 6.45-6.29 (m, 2H), 5.73 (dd, J = 11.7, 4.8 Hz, 1H), 4.65-4.53 (m, 2H), 3.70-3.53 (m, 2H). ESI-MS (m / z): [M+H] + 329.3. 13 C NMR (151 MHz, chloroform-d) δ 164.14, 155.63, 150.46, 147.68, 147.18, 142.37, 127.68, 126.71, 123.74, 121.87, 111.80, 110.73, 109.72, 108.73, 53.82, 42.35, 37.72. HRMS (ESI+): m / zC 17 H 14 C1N 2 O 3 [M+H]+ calculated value is 329.0693; found value is 329.0698.

[0340]

[0341] 1b was synthesized according to the same procedure as 1a, except that 2-acetyl-7-hydroxybenzofuran was used instead of 2-acetylbenzofuran. The crude product was purified by silica gel column chromatography using hexane / ethyl acetate (60:40, v / v) as eluent to give 1b as a yellow solid. 1 H NMR (600MHz, chloroform-d) δ7.70(d,J=15.3Hz,1H),7.65(s,1H),7.57(d,J=1.8Hz,1H),7.43(d,J=15.3Hz,1H),7.28(dd,J=7.9,1.0Hz,2H ),7.21(t,J=7.8Hz,1H),7.03(ddd,J=7.0,5.9,1.1Hz,1H),6.79(d,J=3.4Hz,1H),6.55(dd,J=3.4,1.8Hz,1H).ESI-MS(m / z):[M+H] + 255.1.

[0342]

[0343] 2b (1.0 equiv.) was dissolved in acetonitrile. Propargyl bromide (1.5 equiv.) and K 2 CO 3 (2.5 equiv) was added to the solution mixture and heated under reflux overnight. The reaction mixture was quenched by adding water and dried under reduced pressure, and then the aqueous layer was extracted with dichloromethane. 4 The organic layer was dried, filtered and evaporated under reduced pressure.The crude product was purified by silica gel column chromatography using hexane / ethyl acetate (80:20, v / v) as eluent to afford compound 2b. 1 H NMR (600MHz, chloroform-d) δ7.70(d,J=15.4Hz,1H),7.62(s,1H),7.57(s,1H),7.52(d,J=15.4Hz,1H),7.35(d,J=7.4Hz,1H),7.22-7.27(m,1 H),7.11(d,J=7.9Hz,1H),6.78(d,J=3.4Hz,1H),6.53-6.54(m,1H),4.97(d,J=2.4Hz,2H),2.58(t,J=2.4Hz,1H).ESI-MS(m / z):[M+H] + 293.3.

[0344]

[0345] Hydrazine (2 eq.) was added to 2b (1 eq.) in ethanol and heated under reflux for 3-4 h. Any organic volatiles were evaporated under reduced pressure and the residue was extracted with dichloromethane. The dichloromethane layer was purified by MgSO 4 The intermediate was then dissolved in anhydrous dichloromethane. Chloroacetyl chloride (1.2 equivalents) and triethylamine (2 equivalents) were slowly added dropwise on ice. The reaction was allowed to proceed overnight. Any volatile organic solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography using hexane / ethyl acetate (80:20, v / v) as eluent to give CL16-alkyne. 1 HNMR (600 MHz, chloroform-d) δ7.30 (d, J = 1.8 Hz, 1H), 7.27 (d, J = 7.4 Hz, 1H), 7.21 (d, J = 7.9 Hz, 1H), 7.16 (d, J = 3.6 Hz, 1H), 7.04 (dd, J = 7.9, 1.1 Hz, 1H), 6.40 (d, J = 3.3 Hz, 1H), 6.32 (dd, J = 3.3,1.8Hz,1H),5.71(dd,J=11.6,4.9Hz,1H),4.93(d,J=2.4Hz,2H),4.59(q,J=14.3Hz, 2H), 3.66 (dd, J=17.6, 11.7Hz, 1H), 3.57 (dd, J=17.6, 4.9Hz, 1H), 2.57 (t, J=2.4Hz, 1H). 13 C NMR (151 MHz, chloroform-d) δ 171.21, 164.20, 164.18, 150.47, 148.04, 147.88, 147.26, 147.16, 145.18, 144.46, 143.23, 142.34, 132.89, 130.03, 129.72, 129.55, 124.36, 124.29, 118.35, 116.32, 114.98, 11 4.83,114.13,110.69,110.42,109.98,109.54,109.48,109.40,108.65,108.62,78.06,76.31,76. 22,69.94,60.41,56.76,53.83,42.35,42.30,37.85,37.82,29.69,21.05,14.19.HRMS(ESI+):m / z C 20 H 16 C1N 2 O 4 [M+H]+ calculated value is 383.0799; found value is 383.0796.

[0346] result

[0347] Expression of RhoA, GEF, and GAP is associated with CRC development and / or patient survival

[0348] RhoA is considered a molecular switch that controls cytoskeletal organization and cell motility and is therefore closely related to cancer invasion and metastasis. When bound to GTP, it exists in an active form, thereby enhancing the interaction with downstream effector proteins for signal transduction. On the other hand, when bound to GDP, it becomes an inactive form. The balance between the GDP-bound and GTP-bound states is mainly regulated by two protein families, namely guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs promote the exchange of GDP to GTP, thereby activating RhoA. GAPs catalyze the hydrolysis of GTP on RhoA to GDP, resulting in its inactivation ( Fig.15 ). Therefore, the cellular activity of RhoA depends not only on its expression level, but also on the critical level of the active GTP-bound form of RhoA.

[0349] Based on the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx; Fig. 8A ) dataset, found higher RhoA expression in tumor tissues of patients with colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) compared with normal tissues. Although there was no correlation between RhoA expression and patient survival ( Figure 8B ), but found that higher expression of two GEF proteins, ARHGEF25 and AKAP13, was associated with lower patient survival ( Figure 8C-8D On the other hand, lower GAP levels (ARHGAP26 and ARHGAP35) were beneficial for patient survival ( Figures 8E-8F ). It was observed that ARHGEF23 and AKAP13 levels increased with increasing tumor stage in COAD and READ patients with statistically significant ( Figure 8G-8H ). Notably, AKAP13 is a RhoA GTPase-specific GEF and has no guanine nucleotide exchange activity toward Cdc42, Rac, and Ras. In addition, the RhoA signaling pathway was more enriched in CRC patients with stage IV tumors than in patients with earlier stage tumors ( Figures 8I-8J ), indicating elevated RhoA activity in advanced stage tumors. and enrichment of the Rho pathway in CRC patients with higher metastatic potential, as indicated by higher epithelial-mesenchymal transition (EMT) scores ( Figure 8K-8L ), all of these results suggest that RhoA activity is associated with the development and invasiveness of CRC.

[0350] Gel-based ABPP assay to identify CL16 as a lead compound with strong and specific binding to RhoA in vitro.

[0351] In a covalent ligand screening experiment performed by ABPP, CL16 was identified for its strong and specific binding to RhoA. ABPP is considered a powerful platform for covalent ligand screening, especially for identifying lead compounds targeting new hot spots for therapeutics. Competitive gel-based activity protein profiling (ABPP) experiments were performed on purified RhoA protein to screen its interaction with a library of cysteine-reactive covalent ligands with acrylamide or chloroacetamide warheads. The first screen was performed on RhoA protein, and a second screen was performed on Rac1 and Cdc42 to identify lead compounds that specifically bind to RhoA Cys16. RhoA protein was pretreated with covalent ligand or DMSO control and then incubated with iodoacetamide-rhodamine for cysteine ​​labeling. The reaction mixture was then resolved by SDS-PAGE and the in-gel fluorescence was imaged ( Figure 1A The RhoA binding cues were identified by the disappearance of the fluorescence signal or the shift of the protein band in silver staining due to covalent modification. Since Cys16 is the only unique cysteine ​​on RhoA ( Fig. 9 ), and RhoA, Rac1, and Cdc42 have similar protein structures, so compounds that bind to RhoA but not to Rac1 and Cdc42 should target RhoA-Cys16. Dose-dependent experiments showed that CL16 has a strong binding affinity for RhoA, and the IC 50 Significantly reduced to 440nM ( Figure 1B-1D CL16 had no observable binding to other GTPases such as Rac1 and Cdc42. Therefore, CL16 was identified as a lead compound due to its strong and specific binding to RhoA.

[0352] For the analysis of the first screening results, covalent ligands that showed an intensity ratio lower than 30% compared to the DMSO control and maintained at least 70% intensity in silver staining were considered lead compounds.

[0353] In the first screening experiment of RhoA protein, six lead compounds were found ( Fig.16 ).

[0354] LC-MS / MS experiments showed that CL16 covalently bound to Cys16 of RhoA in HCT116 cells.

[0355] Liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS) experiments were performed on colorectal cancer HCT116 cells treated with CL16. It was found that the only covalent modification of RhoA by CL16 was Cys16 ( Figure 2A Cys16 is a unique residue on RhoA that is not present in Rac1 or Cdc42 ( Figure 2B ), thus explaining the high specificity of CL16 for RhoA rather than other Rho GTPases.

[0356] Covalent docking showed that CL16 is located next to the nucleotide binding pocket of RhoA.

[0357] Cys16 on RhoA is close to the nucleotide binding pocket, which is crucial for the regulation of RhoA activity. Therefore, the structural characteristics of CL16 binding to RhoA were investigated using covalent docking ( Figure 2C CL16 was found to be located next to the nucleotide binding pocket, adjacent to Phe30 and Lys118 as well as Mg 2+ This suggests that covalent targeting of CL16 to RhoA can interfere with nucleotide binding to RhoA.

[0358] Covalent targeting of RhoA Cys16 by CL16 disrupted GDP-GTP exchange and RhoA-GEF interaction in vitro.

[0359] Covalent docking of CL16 with RhoA protein (PDB: 1CC0) revealed π-π stacking, π-cation and hydrogen bonding interactions of CL16 with Thr19, Phe30 and Lys118 in the nucleotide binding pocket, as well as Mg 2+ Metal coordination and π-cation interactions of cofactor ions ( Figure 2C ). Although Rho GTPases usually bind tightly to GTP, covalent inhibitors have been reported to compete with GTP for binding to genetically engineered KRAS GTPase via irreversible covalent binding, suggesting that covalent binding of CL16 to RhoA Cys16 can interfere with GTP loading and inhibit RhoA to some extent. This was supported by fluorescence measurements of MANT-GTP binding to RhoA, where preincubation of RhoA with CL16 significantly slowed the increase in fluorescence intensity, indicating that CL16 binding prevented GTP loading onto RhoA ( Fig. 10A). In addition, when analyzing the interaction of CL16 with the co-crystal structure of the DH / PH domain of RhoA and LARG (PDB:1X86), it was found that CL16 interacted with both RhoA and LARG, and its docking model was that CL16 covalently bound to Cys16 of RhoA in the RhoA-LARG protein complex (PDB:1X86). This suggests that CL16 binding may interfere with the RhoA-GEF interaction and therefore interfere with GTP loading onto RhoA. In addition, according to this covalent docking model, fluorescence-based assays showed that the kinetics of Dbs-mediated GTP binding to RhoA decreased significantly with increasing CL16 concentration ( Fig. 10B Co-immunoprecipitation experiments (i.e., a mixture of purified human GST-RhoA (10 μg) and HEK293T cell lysate containing ARHGEF1 or LARG-GFP (360 μg) was incubated with CL16 or Rhosin, a reported RhoA inhibitor) supported that CL16 disrupts the interaction of RhoA with ARHGEF1, the LARG DH-PH domain, AKAP13, and ARHGAP4 ( Figure 2D ).

[0360] Inhibition of RhoA activation in colorectal cancer cells incubated with CL16.

[0361] GTP loading onto RhoA is critical for protein activation and mediating downstream signaling. Given the above-discussed inhibition of GDP to GTP exchange of RhoA by CL16 in vitro, the effect of CL16 on RhoA biology in colorectal cancer cells was studied. In a serum starvation-restimulation experiment, HCT116 cells were grown in serum-free medium containing CL16, Rhosin, or solvent controls for 24 h and then stimulated with 10% FBS for 10 min. The cells were then lysed and RhoA activity was determined by the level of RhoA-GTP, which is the active form of RhoA. In the solvent control, increased RhoA-GTP levels were observed in serum-stimulated cells, indicating that RhoA signaling was activated ( Figure 3A ). CL16 treatment resulted in much lower cellular RhoA-GTP levels after serum stimulation compared to solvent controls, indicating that CL16 inhibits RhoA activation. Surprisingly, CL16 was more effective than Rhosin in inhibiting RhoA activity, as shown by lower RhoA-GTP levels in cells treated with 10 μM CL16 than in those treated with 30 μM Rhosin ( Figure 3A). This indicates that CL16 is a more effective RhoA inhibitor than Rhosin in CRC. RhoA activity was specifically inhibited in HT29 cells treated with CL16, while no significant changes were observed in the activity of other Rho GTPases (such as Rac1 and Cdc42), while Rhosin inhibited Cdc42 in HT29 cells at its working concentration (30 μM) for RhoA inhibition. HT29 cells were incubated with solvent vehicle, CL16 or Rhosin in complete medium for 24 h. The cells were then washed, lysed and subjected to rhotekin pull-down to imprint RhoA-GTP, or PAK-PBD pull-down to imprint Rac1-GTP and Cdc42-GTP. Confocal fluorescence images of HCT116 and HT29 cells expressing the FRET RhoA biosensor were taken in serum starvation-restimulation experiments.

[0362] The effect of CL16 on RhoA downstream signaling in serum starved-restimulated CRC was investigated. HCT116 cells incubated with solvent control showed increased levels of phosphorylated MLC, a downstream effector of RhoA, after serum restimulation ( Figure 3B ). In contrast, CL16 treatment significantly abolished the increase in MLC phosphorylation.

[0363] To further validate the inhibitory effect of CL16 on RhoA, HCT116 and HT29 cells were transfected with the FRET-RhoA biosensor, where cells with increased RhoA activity would show higher FRET / mTFP1. Specifically, HCT116 cells transfected with the FRET-RhoA biosensor were treated with CL16 or solvent in serum-free medium for 24 h, washed with PBS, restimulated with 10% FBS for 10 min, washed, and then imaged in HBSS by confocal fluorescence microscopy. CL16 treatment resulted in the disappearance of the increase in FRET / mTFP1 from transfected cells, as found in the serum restimulation experiment ( Figure 3C). In addition, HCT116 cells were serum starved and treated with CL16 or Rhosin for 24h, followed by restimulation with 10% FBS for 10min. The cells were then washed and fixed with 4% paraformaldehyde, washed with PBS, and stained with Alexa Fluor 488-Phalloidin (1:20, v / v) in PBS for 15min at room temperature. The cells were washed with PBS, incubated with Hoechst (8.2μM) in PBS for 15min, washed, and then imaged in PBS by confocal fluorescence microscopy. The results showed that in the serum restimulation experiment, a reduction in F-actin fibers was observed in cells treated with CL16 starting from 5μM compared to the solvent control (imaging data not shown). Since RhoA regulates cytoskeletal dynamics and RhoA activation can induce the formation of contractile F-actin, the reduction in F-actin fibers by CL16 treatment supports its inhibitory effect on RhoA signaling.

[0364] Anticancer and anti-metastatic effects of CL16 on CRC via inhibition of RhoA.

[0365] As discussed above, overexpression / overactivity of RhoA is associated with poor prognosis and aggressive behavior in CRC ( Fig. 8A Therefore, the anticancer and anti-metastatic properties of CL16 in CRC were explored. First, it was confirmed by using staining (data not shown) and wound healing assays that genetic knockdown of RhoA by siRNA slowed down the colonization and migration of CRC ( Figure 4A MTT assay was used to determine the cell viability of CRC and normal colon fibroblast CCD-18Co cells treated with CL16 for 48 h (n = 3 different biological replicates / group). It was also determined by Western blotting (data not shown) that the expression level of RhoA was much lower in normal colon fibroblast CCD-18Co cells compared with CRC. CL16 showed cytotoxic effects in CRC, and IC 50 Values ​​correlated with RhoA levels in CRC ( Figure 4B and 4C HT29 cells were transfected with siRNA (5 μM) using Dharmafect1 for 48 h, and protein expression was then investigated by immunoblotting. CL16 was much less toxic in normal CCD-18Co cells with lower RhoA levels, demonstrating that there is a good therapeutic window for the use of CL16 in the treatment of CRC. In addition, CL16 showed good efficacy in reducing HT29 cell migration and invasion, as shown in the wound healing assay ( Figure 4D ) and transwell invasion assay (HT29 cells were treated with CL16 or Rhosin for 24 h; data not shown).

[0366] To further investigate the anticancer effects of CL16, HT29 and SW620 cells were seeded as spheroids in low affinity plates with 20 ng / mL EGF and incubated with different concentrations of CL16 or Rhosin. CL16 treatment resulted in significant inhibition of the growth of HT29 and SW620 spheroids and was found to be more effective than Rhosin treatment, as reflected by the similar anticancer effects mediated by a lower working dose of CL16 ( Figure 4E and 4F ). Low cytotoxicity IC of CL16 in SW620 and HT29 spheroids 50 values ​​(4.10 and 5.57 μM, respectively; Figure 5A and 5B ) demonstrated good penetration of CL16 across the tumor mass to mediate anticancer effects through inhibition of RhoA and its use for the treatment of CRC in vivo.

[0367] The molecular mechanism of the anticancer effect of CL16 was investigated. Flow cytometric analysis of HCT116 cells treated with CL16 showed that G 2 The cell population increased significantly ( Fig.11A ). Western blotting experiments showed that p21 and p27 protein levels increased in HCT116, HT29, and SW620 cells after CL16 treatment, while no significant changes were observed for cyclin A2 and cyclin B1. Immunoblotting of cell cycle checkpoint proteins was performed in HCT116 cells subjected to CL16 treatment for 24 h. The effect of CL16 treatment on cell cycle checkpoint proteins was investigated, and cell cycle checkpoint proteins were immunoblotted in SW620 and HT29 cells after CL16 treatment for 24 h. CL16 treatment also mediated apoptosis, as found by confocal fluorescence imaging experiments using Caspase-3 / 7Green detection reagent ( Fig. 11B ). Caspase-3 / 7Green detection reagent was used by confocal fluorescence imaging to study apoptosis in HCT116 cells treated with Rhosin. Since p21 and p27 expression is regulated by Dia1, a downstream target of RhoA, the increased levels of p21 and p27 are attributed to the downregulation of RhoA-Dia1 signaling by CL16. This results in the inhibition of CDK1-cyclin B complex by p21 and p27, thereby preventing CRC cells from progressing to mitosis and inducing G2 arrest, leading to apoptotic cell death ( Fig.17 ).

[0368] Molecular probes and chemical proteomics experiments of CL16 indicated that RhoA Cys16 was the major target of CL16 in CRC.

[0369] To identify protein targets and any off-targets of CL16 in CRC, a molecular probe of CL16, CL16-alkyne ( Fig. 6A ). CL16-alkyne contains an alkyne handle that allows functionalization by copper-catalyzed azide-alkyne cycloaddition (CuAAC) to study CL16-protein binding. HCT116 cells transiently expressing FLAG-RhoA were pretreated with solvent vehicle or CL16 for 2 h and then incubated with CL16-alkyne in complete medium for 2 h. The cells were then lysed and immunoprecipitated by anti-FLAG beads. The enriched proteins were reacted with rhodamine azide by copper-catalyzed azide-alkyne cycloaddition (CuAAC), boiled with loading buffer and separated by SDS-PAGE. The protein bands were then visualized by their in-gel fluorescence or blotted against RhoA. The results confirmed that CL16-alkyne was able to bind to RhoA by incubating the probe with HCT116 cells expressing FLAG-RhoA, as shown by strong fluorescence from the FLAG-RhoA protein after immunoprecipitation and CuAAC ( Figure 6B ). Notably, preincubation of HCT116 cells expressing FLAG-RhoA with CL16 competed with alkyne probe labeling ( Figure 6B ), supporting the target binding of CL16 to RhoA. Next, CL16-alkyne was used to examine any off-target binding of CL16 in CRC. HCT116 cells were pretreated with solvent vehicle or CL16 for 2 h and then incubated with CL16-alkyne for an additional 2 h. The probe-labeled proteins were then mounted with desthiobiotin by CuAAC, enriched, trypsinized, and analyzed by LC-MS / MS ( Fig. 12A and 18 Peptides with a fold change (control / CL16) > 4 and p < 0.05 (i.e. at least 75% occupancy and statistical significance) were considered targets for binding to CL16. RhoA was found to be one of the protein targets of CL16 ( Fig. 12A ), and was the only protein identified in LC-MS / MS experiments on CL16-treated HCT116 cells and CL16-alkyne-probed HCT116 cells ( Fig. 12B ), indicating that RhoA is the primary target of CL16. Gel-based ABPP experiments also supported that CL16 binds to RhoA with minimal off-target binding, as other proteins showed weaker fluorescence or no significant changes in fluorescence intensity in cells pretreated with CL16 compared to the DMSO control ( Figure 6C). The probe-labeled proteins were then conjugated to rhodamine by CuAAC and visualized by their in-gel fluorescence after SDS-PAGE. A protein band with strong fluorescence was found at ∼22 kDa in the sample incubated only with CL16-alkyne, which is consistent with the molecular weight of RhoA ( Figure 6C ). Pretreatment with 10 μM CL16 resulted in the complete disappearance of the protein band, indicating that CL16 was tightly bound to the protein ( Figure 6C Other proteins showed much weaker fluorescence or no significant competitive binding with CL16 ( Figure 6C ), indicating that CL16 has very little off-target binding in HCT116 cells.

[0370] CL16 exhibits biological activity in CRC by covalently targeting RhoA Cys16.

[0371] The effects of CL16 in HT29 cells expressing RhoA WT protein and HT29 cells with RhoAC16A knock-in by CRISPR-Cas9 were studied separately to verify the molecular mechanism of CL16 inhibition of RhoA and the anticancer properties of CL16. CL16 treatment resulted in a significant decrease in the level of active RhoA-GTP in HT29 cells with RhoA WT, but not in RhoA C16A knock-in cells. HT29 cells expressing RhoA WT or C16A protein were incubated with solvent vehicle, CL16 or Rhosin in complete medium for 24 h. The cells were then washed, lysed, subjected to rhotekin pull-down and immunoblotted. After 48 h of treatment with CL16, a decrease in cell migration was observed in HT29 cells expressing RhoA WT, while no inhibitory effect was observed in CL16-treated HT29 cells with C16A knock-in. Wound healing assays were performed on HT29 cells expressing RhoA WT or C16A proteins incubated with vehicle, CL16 or Rhosin in complete medium for 48 h. Fig.19A and 19B These results suggest that CL16 inhibits RhoA activity and mediates anticancer properties in CRC primarily by covalently targeting RhoA Cys16.

[0372] CL16 exhibits antitumor effects against CRC in vivo.

[0373] After confirming the anticancer properties of CL16 on CRC cells and RhoA Cys16 as its major molecular target, the anticancer effect of CL16 in vivo was investigated. In nude mice with CRC xenografts, CL16 at a dose of 10 mg / kg significantly inhibited the proliferation of HCT116 xenografts ( Fig. 7A), HT29 xenograft ( Figure 7B ) and SW620 xenografts ( Fig.7D and 7E ) tumor growth. The body weight of treated mice was monitored throughout the experiment and no observable differences were observed between treated and untreated groups, indicating that CL16 has minimal toxicity ( Figure 7C and 7F In addition, H&E and Ki67 immunohistochemical staining of tumor tissues from vehicle-treated mice or CL16-treated mice showed that Ki67 was reduced in the treatment groups ( Figures 14A-14D ), indicating that proliferating cancer cells were reduced after CL16 treatment. Increased levels of cleaved caspase-3 in tumor tissues of CL16-treated mice indicated induction of apoptosis in CRC cells, highlighting that CL16 effectively kills cancer cells in vivo. H&E staining showed no observable lesions on major organs of mice treated with CL16, further supporting the low systemic toxicity associated with CL16 treatment.

[0374] The antitumor effects of CL16 were examined in an immunocompetent, orthotopic mouse CRC model established by intraintestinal inoculation of mouse CRC cells SL4 ( Fig. 20 ). Prior to the mouse experiments, it was confirmed that Cys16 is conserved on the mouse RhoA protein, and CL16 treatment significantly reduced the activity of SL4 ( Fig.13A ), similar to what was found in human CRC cells. In this immunocompetent mouse CRC model, a significant reduction in tumor weight was observed after CL16 treatment ( Fig. 13B ). H&E staining of tumor tissues from CL16-treated mice revealed nuclear atypia, such as nuclear condensation and fragmentation, suggesting induction of apoptosis. This was supported by immunohistochemistry for cleaved caspase-3, which was found at elevated levels in tumor tissues from treated mice ( Figures 13C-13H ). Tumor tissues from vehicle-treated mice or CL16-treated mice were immunohistochemically stained for CD3, CD4, CD8, and Ki67 with cleaved caspase-3 and VEGFR2. The reduction of Ki67 in tumor tissues is consistent with the good antitumor effect of CL16 ( Figures 13C-13H On the other hand, there was no significant change in the body weight of the treated mice ( Fig.13I ), and no organ damage was found in kidney tissues in treated mice, indicating that CL16 treatment had minimal toxicity. In addition, tumor metastasis to the liver was observed in control mice, while CL16 treatment inhibited tumor metastasis. Combined with the decrease in VEGFR2 levels in immunohistochemistry experiments ( Figures 13C-13H), which indicates the anti-metastatic properties of CL16 in vivo. Interestingly, increased CD3, CD4, and CD8 were found in immunohistochemistry of tumor tissues from CL16-treated mice ( Figures 13C-13H ), indicating that the infiltration of cytotoxic T cells into the tumor microenvironment increased after CL16 treatment. To our knowledge, this is the first reported study showing that small molecule compounds improve immunity by inhibiting RhoA in CRC cells.

[0375] CL16 achieves RhoA inhibition and anticancer properties by covalently targeting Cys16 of RhoA.

[0376] RhoA serves as a very valuable therapeutic target due to its role in regulating cancer migration and invasion. Clinical data also show that RhoA is upregulated in melanoma, breast cancer and colorectal cancer samples. K-Ras covalent inhibitors targeting small Rho GTPases have attracted people's interest. A RhoA covalent inhibitor targeting Cys107, DC-Rhoin, has been reported. In contrast, the RhoA covalent inhibitors disclosed herein (e.g., CL16) target another Cys on RhoA to mediate a different RhoA inhibitory mode of action compared to DC-Rhoin. By covalently binding to Cys16 (Cys16 is located near the nucleotide binding pocket), CL16 is located next to the nucleotide binding pocket in RhoA, as found in covalent docking. This leads to a significant reduction in self-exchange of GDP to GTP and LARG-mediated exchange, which is essential for RhoA activation, as demonstrated using a fluorescence-based exchange assay. This unique mechanism of action enables the disclosed RhoA covalent inhibitors (eg, CL16) to exhibit better efficacy than Rhosin in inhibiting cellular RhoA activity in CRC cells, as demonstrated using Western blotting, FRET-RhoA biosensor imaging, and immunofluorescence experiments.

[0377] Compared with other cysteine ​​residues, Cys16 is an ideal target site for RhoA with covalent inhibitors, which can achieve high specificity superior to other small GTPases. According to sequence homology analysis, this may be the reason for the uniqueness of Cys16 on RhoA, while Cys107 (which is a reported binding site for DC-Rhoin) is also conserved in Rac1 and Cdc42. Covalent targeting of Cys16 to achieve high specificity for RhoA is important for cancer therapy with minimal toxic side effects, especially Rac1 and Cdc42 control many important cellular processes, including cell proliferation, polarity and migration. RhoA inhibition superior to other Rho GTPases and good RhoA specificity make the disclosed RhoA covalent inhibitors (such as CL16) show high cytotoxicity in CRC cells, which is related to RhoA expression / activity in cancer cells, while it is much less toxic to normal colon fibroblasts with lower RhoA levels. Consistent with the excellent anticancer and anti-metastatic properties of CL16 in 2D cell culture, CL16 inhibited the growth of CRC spheroids with a potency superior to that of the reported RhoA inhibitor Rhosin. More importantly, the exemplary compound CL16 is the first RhoA covalent inhibitor to show antitumor effects against mouse CRC xenografts in vivo. No observable toxicity was found from the treated mice, as shown by body weight measurements and immunohistochemistry of tissue sections from different organs. These in vivo data highlight that covalent targeting of RhoA with good specificity is an effective strategy for treating CRC.

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Claims

1. Compounds having the following structure: in: (i) X1 and X2 are independently C(R6)2, O, S or NR6, and R6 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, alkoxy, or carbonyl; (ii) A1 is a C5-C6 aromatic ring or a C4-C5 heteroaromatic ring; (iii) n1 is an integer from 0 to 4; (iv) n2 is an integer from 0 to 3; (v) R1, R3 and R5 are independently hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; (vi) R2 is a cysteine-reactive group (e.g., haloacetamide, acrylamide, maleimide, vinyl sulfone, and epoxide) that allows chemical reaction with the cysteine ​​on RhoA; (vii) R4 is hydrogen, alkoxy, hydroxy, halogen, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, carbonyl, amino, or haloalkyl; and (viii) when substituents are present, they are independently unsubstituted alkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted polyaryl, unsubstituted heteropolyaryl, unsubstituted alkylaryl, unsubstituted cyclic, unsubstituted heterocyclic, halide, amino, amido, thiol, hydroxy, cyano, nitro, carbonyl, or alkoxy, The conditions are: (i) the compound is not and (ii) When X1 is O, X2 is S, R1 and R3-R4 are hydrogen and A1 is a C6 aromatic ring, then R2 is not a haloacetamide.

2. The compound of claim 1, having the following structure:

3. The compound of claim 1, having the following structure:

4. The compound of claim 1, wherein n1 is 0 or 1.

5. The compound of claim 1, wherein R1 is hydrogen.

6. The compound of claim 1, wherein R3 is hydrogen or substituted or unsubstituted aryl, wherein when substituents are present, the substituents are independently unsubstituted alkyl, unsubstituted aryl, or unsubstituted alkylaryl.

7. The compound of claim 1, wherein R2 is n3 is an integer from 0 to 6, R7 and R8 are independently hydrogen, unsubstituted alkyl, or halogen, and R9 is hydrogen or halogen.

8. The compound of claim 1, wherein R4 is hydrogen or n4 is an integer from 0 to 6, R 10 and R 11 are independently hydrogen or halogen, and R 12 is hydrogen, halogen, amino, unsubstituted alkynyl, aldehyde or carboxylic acid.

9. The compound of claim 8, wherein R 10 and R 11 is hydrogen, and R 12 is an unsubstituted alkynyl group.

10. The compound of claim 1, having the following structure:

11. The compound of claim 1, wherein the compound is covalently bound to RhoA.

12. The compound of claim 11, wherein the compound is covalently bound to Cys16 of RhoA.

13. The compound of claim 11, wherein the compound inhibits RhoA activity in living cells.

14. A pharmaceutical composition for treating cancer in a subject, comprising a compound having the structure: in: (i) X1 and X2 are independently C(R6)2, O, S or NR6, and R6 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, alkoxy, or carbonyl; (ii) A1 is a C5-C6 aromatic ring or a C4-C5 heteroaromatic ring; (iii) n1 is an integer from 0 to 4; (iv) n2 is an integer from 0 to 3; (v) R1, R3 and R5 are independently hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; (vi) R2 is a cysteine-reactive group (e.g., haloacetamide, acrylamide, maleimide, vinyl sulfone, and epoxide) that allows chemical reaction with the cysteine ​​on RhoA; (vii) R4 is hydrogen, alkoxy, hydroxy, halogen, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, carbonyl, amino, or haloalkyl; and (viii) when substituents are present, the substituents are independently unsubstituted alkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted polyaryl, unsubstituted heteropolyaryl, unsubstituted alkylaryl, unsubstituted cyclic, unsubstituted heterocyclic, halide, amino, amido, thiol, hydroxyl, cyano, nitro, carbonyl, or alkoxy, and optionally one or more pharmaceutically acceptable excipients, The compound is in an effective amount to improve one or more symptoms associated with the cancer in the subject.

15. The pharmaceutical composition of claim 14, further comprising a second active agent, optionally wherein the second active agent is an anti-cancer agent.

16. The pharmaceutical composition of claim 14, wherein the amount of the compound in the pharmaceutical composition ranges from about 0.1 μM to about 1 mM, about 0.1 μM to about 500 μM, about 0.1 μM to about 200 μM, about 0.1 μM to about 100 μM, about 0.1 μM to about 50 μM, or about 0.1 μM to about 20 μM.

17. A method for treating cancer using the pharmaceutical composition of claim 14, comprising: (i) administering the pharmaceutical composition to a subject in need thereof, wherein step (i) occurs one or more times.

18. The method of claim 17, wherein the pharmaceutical composition is administered orally, intramuscularly, intravenously, intraperitoneally, or subcutaneously, or a combination thereof.

19. The method of claim 17, wherein in step (i), the dose of the compound administered is from about 0.1 μg to about 1000 μg, from about 0.1 μg to about 500 μg, from about 0.1 μg to about 100 μg, from about 0.5 μg to about 50 μg, from about 1 μg to about 1000 μg, from about 1 μg to about 500 μg, from about 1 μg to about 100 μg, from about 1 μg to about 50 μg, from about 1 μg to about 25 μg, from about 1 μg to about 10 μg, from about 0.1 μg to about 50 μg, from about 5 μg to about 50 μg, or from about 0.1 μg to about 20 μg per gram of subject.

20. The method of claim 17, wherein the cancer is a solid cancer.

21. The method of claim 17, wherein the cancer is colorectal cancer.

22. The method of claim 20, wherein after step (i) or the entire step (i) (if step (i) occurs more than once), the tumor volume and / or tumor weight is at least 10%, at least 20%, at least 30%, at least 40% or at least 50% smaller / less than a control administered with a pharmaceutically acceptable excipient alone.

23. The method of claim 17, wherein after step (i) or the entire step (i) (if step (i) occurs more than once), there is no observable toxicity to the subject as demonstrated by physical vital measurements (e.g., body weight), standard hematological markers, and / or blood biochemical parameters compared to a control.

24. The method of claim 17, further comprising administering an active agent before, during, and / or after step (i), optionally wherein the active agent is an anti-cancer agent.

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