CN115667553A - Small molecule inhibitors of oncogenic CHD1L with preclinical activity against colorectal cancer - Google Patents

Small molecule inhibitors of oncogenic CHD1L with preclinical activity against colorectal cancer Download PDF

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CN115667553A
CN115667553A CN202180037594.1A CN202180037594A CN115667553A CN 115667553 A CN115667553 A CN 115667553A CN 202180037594 A CN202180037594 A CN 202180037594A CN 115667553 A CN115667553 A CN 115667553A
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D·V·拉巴贝拉
J·M·阿博特
周琼
A·D·亚伯拉罕
H·埃斯奎尔
B·J·普里加罗
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Abstract

CHD 1L-driven cancers, including TCF transcription-driven cancers and EMT-driven cancers, are treated with CHD1L inhibitors. Small molecule inhibitors of CHDL1 that inhibit CHD1L atpase and inhibit CHD 1L-dependent TCF transcription have been identified. CHD1L inhibitors prevent binding of the TCF complex to Wnt responsive elements and promoter sites. More specifically, CHD1L inhibitors induce the reversal of EMT. CHD1L inhibitors are useful for treating various cancers, particularly CRC and m-CRC. CHD1L driven cancers include CRC, breast cancer, glioma, liver cancer, lung cancer or Gastrointestinal (GI) cancer. Provided are CHD1L inhibitors of formulae I and XX as defined herein and salts thereof and pharmaceutical compositions containing CHD1L inhibitors. Synergistic combinations of CHD1L inhibitors with other antineoplastic agents are also described.

Description

Small molecule inhibitors of oncogenic CHD1L with preclinical activity against colorectal cancer
Cross Reference to Related Applications
This application claims the benefit of U.S. provisional application 62/994,259 filed on 24/3/2020 and U.S. provisional application 63/139,394 filed on 20/1/2021, both of which are incorporated herein by reference in their entirety.
Statement regarding U.S. government support
This invention was made with government support under grant number W81XWH1810142 awarded by the department of defense (DoD). The united states government has certain rights in this invention.
Background
Genome integrity is maintained by conformational changes in chromatin structure that regulate DNA accessibility for gene expression and replication. Chromatin structure is maintained by post-translational modification of histones and rearrangement of nucleosomes [ Lorch et al, 2010; kumar et al, 2016; swygert et al, 2014]. ATP-dependent chromatin remodelling agents are enzymes that alter chromatin by altering histone composition, as well as by expelling or translocating nucleosomes along the DNA. Their activity plays a key role in cellular function by regulating gene expression and DNA accessibility for replication, transcription and DNA repair [ Erdel et al, 2011; brownlee et al, 2015]. Dysregulation of chromatin remodeling has been associated with human disease, particularly cancer [ Zhang et al, 2016; valencia & Kadoc, 2019].
In the last decade, chromatin remodelling agents known as CHD1L (chromatin domain helicase/atpase DNA binding protein 1-like), also known as ALC1 (amplified in liver cancer 1), have become a cancer gene associated with significant human cancer pathology (Ma et al, 2008 cheng et al, 2013. CHD1L is also involved in multidrug resistance, up-regulation of drug-resistant efflux pumps (such as ABCB 1) [ Li et al, 2019] to PARP 1-mediated DNA repair [ Pines et al, 2012 tsuda et al, 2017] and anti-apoptotic activity [ Li et al, 2013 chen et al, 2009] in addition, amplification or overexpression of CHD1L is associated with poor prognosis in patients, including low overall survival rate (OS) and metastatic disease [ He et al, hyeon et al, 2013 su et al, man 2015 et al, mu et al, 2015 et al, with cancer as well as a mechanism of cancer progression in humans, cancer, ihc, 2015 et al, which has been implicated as a low-risk factor in cancers, no. cancer, no. h1, no. h1, no. hek 1, no. 1.
CRC is the third most common cancer diagnosed each year, with CRC patient mortality second worldwide [ Jemal et al, 2011]. Early detection of CRC combined with surgery and 5-fluorouracil (5 FU) -based combination chemotherapy had little improvement in overall survival rate [ Jemal et al, 2011; fakih,2015]. Current chemotherapy and targeted therapy are largely ineffective against metastatic CRC (mCRC), as evidenced by an overall survival rate as low as 11% in 5 years [ Jemal et al, 2011; fakih,2015]. There is an unmet need in the art to identify and characterize targets for pathologies involving CRC tumor progression and metastasis.
Most patients with CRC have mutations in the Wnt signaling pathway that result in aberrant T-cytokine/lymphokine transcription or TCF complex [ Kinzler & vollstein, 1996; cancer Genome Atlas,2012]. Such mutations can lead to translocation of constitutive β -catenin and transactivation of TCF transcription [ Clevers,2006; korinek et al, 1997]. The TCF complex is regulated by TCF4 (also known as TCFL 2), which is activated by interaction with a series of co-activators such as β -catenin, PARP1 and CREB Binding Protein (CBP) [ Shitashige et al, 2008]. Recently, TCF4 was demonstrated to be a specific driver for early metastasis of adenomas (i.e., polyps) and late mCRC [ Hyeo et al, 2013; su et al, 2014].
TCF transcription is reported to function as a primary regulator of epithelial-mesenchymal transition (EMT) [ S _ nch-Till _ al et al, 2011; zhou et al, 2016; abraham et al, 2019]. This process can transform relatively benign epithelial tumor cells into mesenchymal cells with increased Cancer Stem Cell (CSC) sternness (stemness) and other malignant properties that drive mCRC [ Chaffer et al, 2016]. Recently, it has been reported that certain CRC-driven gene changes are common in primary and metastatic tumor pairs [ Hu et al, 2019]. More specifically, it was reported that aberrant TCF4 is the specific driver of mCRC [ Hu et al, 2019], and that CRC can metastasize in early adenomas (i.e., polyps [ see also Magri & Bardelli,2019 ]), which may be caused by TCF-driven EMT [ Chaffer et al, 2016; chaffer & Weinberg,2011]. These reports indicate that TCF transcription is the driving force for all phases of CRC progression and metastasis.
EMT is a major driving force for many human diseases, in particular solid tumor progression, resistance to drugs and radiotherapy, immune response and escape of immunotherapy, and promotion of metastasis [ Chaffer et al 2016; chaffer & Weinberg,2011; scheel & Weinberg,2012].
Due to the importance of Wnt signaling pathways and TCF transcription in cancer and other diseases [ Clevers,2006], small molecule drugs that inhibit Wnt signaling pathways and TCF transcription have been detected [ Lee et al, 2011; polakis,2012]. Contemplated therapeutic strategies include receptor targets (e.g., frizzled); preventing Wnt ligand secretion (e.g., porcupine); inhibition of β -catenin disrupting complexes (e.g., tankyrases); and protein-protein inhibition (PPI) using beta-catenin and a co-activator (such as CBP). Although clinical trials may be in progress, no drugs have yet been clinically approved for the Wnt/TCF pathway [ Lu et al, 2016]. In contrast, the present invention describes a novel therapeutic strategy, in particular for identifying small molecule drugs for the treatment of Wnt/TCF driven CRC, where CHD1L is identified as a DNA binding factor required for TCF transcription that modulates the malignant phenotype in CRC.
For example, U.S. patent 9616047 reports small molecule inhibitors of β -catenin or disruptors of the β -catenin/TCF-4 complex, which are said to attenuate the onset of colon cancer. The inhibitors of beta-catenin reported therein include esculetin, and compounds designated HI-B1-HI-B20, HI-B22-HI-B-24, HI-B26, HI-B32, and HI-B34, the structures of which are provided in this patent. The patent further describes in some of its general chemical formulas compounds that are said to be useful as inhibitors of β -catenin and in the treatment of colon carcinogenesis. This patent is incorporated herein by reference in its entirety, where the structures, general formulas and variable definitions of specific compounds described therein are useful in the present invention. The compounds identified herein differ structurally from those described in that patent.
CN109761909 issued in 2019 at 17.5.7 (as described in the english abstract in the european patent office database), certain N- (4- (pyrimidin-4-amino) phenyl) sulfonamide compounds or certain salts of formula (la) inhibit the expression of client (client) proteins of Hsp90-Cdc37 (heat shock protein Hsp90 and its co-partner Cdc 37) interactions, and are reported for the treatment or prevention of various diseases mediated by Hsp90 signaling channels. The formula given in the published application is:
Figure BDA0003960268060000041
the variables are defined according to the European patent office database English machine translation as follows:
R 1 is mesitylene, 4-methylbenzenePhenyl, 4-trifluoromethyl-phenyl, naphthyl, 2,3,4, 5-tetramethyl-phenyl, 4-methoxy-phenyl, 4-tert-butylphenyl, 2, 4-dimethoxyphenyl, 2, 5-dimethoxyphenyl or 4-phenoxyphenyl;
R 2 is hydrogen, methyl acetate, acetoxy, aminoacetyl, 4-formic acid benzyl, 4-isopropylbenzyl, 4-chlorobenzyl or 4-methoxybenzyl;
R 3 is chlorine, -OR a or-NR b R c Wherein R is a Is C 1-3 Alkyl radical, C 5-6 Cycloalkyl, C 1-2 Alkoxy, mono-or di-C 1-2 Alkylamino, or C 5-6 A nitrogen-or oxygen-containing heterocyclic group; and R b And R c Are respectively C 1-5 An alkyl group. More specifically, the R 3 Is chloro, 2-hydroxytetrahydropyrrolyl, ethanolamine group, 2, 3-dihydroxy-1-methyl-propylamino, 2, 3-dihydroxypropylamino, piperazine group, N-methylpiperazino, azepinyl (azepyl), piperidine group, 2-methylpropylamine group, propoxy group, methylamino group, ethylamino group, cyclopropylamine group, 1-ethyl-propylamino group, tetrahydropyran-4-ylmethoxy group or 2-methoxyethoxy group. The reference also relates to compounds of formula I-5:
Figure BDA0003960268060000042
the published application is incorporated herein by reference in its entirety, wherein the structures of specific compounds, general formula of the compounds and definitions of variables described therein are useful in the present invention. The structures disclosed in this published application may be excluded from any of the formulae of the present application.
The present invention examines the clinical pathology of CHD1L in CRC, and the results herein indicate that CHD1L is a drug target involved in TCF transcription. The mechanism of CHD 1L-mediated TCF transcription is also presented herein. Small molecule inhibitors of CHD1L are identified herein that are capable of preventing TCF transcription, reversing EMT, and other malignant properties in a variety of cell models, including tumor organoids and patient-derived tumor organoids (PDTO). Certain CHD1L inhibitors identified herein exhibit drug-like pharmacological properties, including in vivo Pharmacokinetic (PK) and Pharmacodynamic (PD) profiles, important for the progression towards the treatment of CRC and other cancers.
Disclosure of Invention
The present invention relates to the treatment of CHD1L driven cancers, more specifically TCF transcription driven cancers, and still more specifically EMT driven cancers. CHD1L was found to be an important component of the TCF transcriptional complex. Small molecule inhibitors of CHDL1 that inhibit CHD1L atpase and inhibit CHD 1L-dependent TCF transcription have been identified. CHD1L inhibitors are thought to prevent binding of the TCF complex to Wnt responsive elements and promoter sites. More specifically, CHD1L inhibitors induce the reversal of EMT. CHD1L inhibitors are useful for treating various cancers, particularly CRC and m-CRC. With particular regard to CRC, inhibitors of CHD1L are shown in embodiments to inhibit the sternness and invasion potential of Cancer Stem Cells (CSCs). In embodiments, the CHD1L inhibitor induces cytotoxicity in CRC PDTO. In particular embodiments, the CHD 1L-driven cancer is CRC, breast cancer, glioma, liver cancer, lung cancer, or Gastrointestinal (GI) cancer. In particular embodiments, the TCF transcription driven cancer is CRC, including mCRC. In particular embodiments, the EMT-driven cancer is CRC, including mCRC.
The present invention provides a method of treating CHD1L driven cancers, more specifically TCF transcription driven cancers, and still more specifically EMT driven cancers, including GI cancers, particularly CRC and mCRC, comprising administering to a patient in need thereof an amount of a CHD1L inhibitor effective to inhibit CHD1L, effective to inhibit aberrant TCF transcription, or effective to induce reversal of EMT. In embodiments, the CHD1L inhibitor is a compound of any one of formulas I-XX or XXX-XVII. More specifically, the invention provides a method of inhibiting aberrant TCF transcription, particularly in CRC, by administering an effective amount of a CHD1L inhibitor. Still more particularly, the present invention provides a method of inducing EMT reversal, particularly in CRC or mCRC, by administering an effective amount of a CHD1L inhibitor. The present invention provides a method of inhibiting, particularly in CRC, the sternness and/or invasiveness potential of Cancer Stem Cells (CSCs) by administering an effective amount of a CHD1L inhibitor. The present invention provides a method of treating CHD1L driven cancer, TCF transcription driven cancer or EMT driven cancer, particularly CRC, by administering an effective amount of a CHD1L inhibitor.
In embodiments, the CHD1L inhibitor selectively inhibits CHD1L. In embodiments, the CHD1L inhibitor herein is not a PARP inhibitor. In embodiments, the CHD1L inhibitor herein is not a topoisomerase inhibitor. Specifically, the CHD1L inhibitors herein are not inhibitors of DNA topoisomerase. Specifically, the CHD1L inhibitors herein are not inhibitors of topoisomerase II α. In embodiments, the CHD1L inhibitor herein is not an inhibitor of β -catenin, in particular an inhibitor such as described in us patent 9616047. In embodiments, the CHD1L inhibitor herein is not an inhibitor of the expression of a client protein of the Hsp90-Cdc37 interaction, in particular an inhibitor as described in CN 109761909.
The invention also provides a method of preventing tumor growth, invasion and/or metastasis in a CHD 1L-driven, TCF transcription or EMT-driven cancer by administering to a patient in need thereof an amount of a CHD1L inhibitor of the invention effective to inhibit CHD1L inhibition, effective to inhibit aberrant TCF transcription, or effective to reverse EMT. In a specific embodiment, the tumor is a solid tumor. In embodiments, the tumor is a tumor associated with a GI cancer. In embodiments, the neoplasm is a neoplasm associated with CRC. In embodiments, the tumor is a tumor associated with mCRC.
In a specific embodiment, the present invention provides a method of treating CRC (including mCRC) comprising administering to a patient in need thereof an amount of a CHD1L inhibitor effective to inhibit CHD1L. In particular embodiments, the invention provides a method of treating CRC (including mCRC) comprising administering to a patient in need thereof an amount of a CHD1L inhibitor effective to inhibit aberrant TCF transcription. In particular embodiments, the present invention provides a method of treating CRC (including mCRC) comprising administering to a patient in need thereof an amount of a CHD1L inhibitor effective to induce reversal of EMT.
In a specific embodiment, the invention provides a method of inducing apoptosis in a cancer cell comprising contacting the cancer cell with an effective amount of a CHD1L inhibitor. In one embodiment, the CHD1L inhibitor is provided in an amount effective to inhibit aberrant TCF transcription. In one embodiment, the CHD1L inhibitor is provided in an amount effective to induce EMT reversal. In one embodiment, the cancer cell is a CRC cancer cell. In one embodiment, the cancer cell is a mCRC cancer cell. In one embodiment, the method is applied in vivo. In one embodiment, the method is applied in vivo in a patient. In one embodiment, the method is applied in vitro.
In embodiments herein that include methods of administering a CHD1L inhibitor, the CHD1L inhibitor is administered by any known method and dosing regimen to achieve the desired benefit. In one embodiment, the administration is oral administration. In one embodiment, administration is by intravenous injection.
The invention also provides a method of treating a drug resistant cancer comprising administering to a patient in need thereof a CHD1L inhibitor in an amount effective to inhibit CHD1L, effective to inhibit aberrant TCF transcription, or effective to induce reversal of EMT, in combination with a known treatment to which the cancer has become resistant. In particular embodiments, the treatment to which the cancer has become resistant is conventional chemotherapy and other targeted therapies. In a specific embodiment, the invention provides a method of increasing the efficacy of a DNA-damaging drug in cancer comprising treating the cancer with a DNA-damaging drug in combination with an inhibitor of CHD1L, wherein the CHD1L is used in an amount effective to decrease resistance to the DNA-damaging drug. In one embodiment, the DNA-damaging agent is a topoisomerase inhibitor. In particular, DNA damaging drugs are DNA topoisomerase inhibitors. In particular, DNA damaging drugs are topoisomerase II α inhibitors. In particular, the DNA-damaging agent is etoposide (etoposide) or teniposide (teniposide). In particular, the DNA-damaging drug is SN38 or a prodrug thereof. In one embodiment, the DNA-damaging agent is a thymidylate synthase inhibitor. In one embodiment, the thymidylate synthase inhibitor is a folate analogue. In one embodiment, the thymidylate synthase inhibitor is a nucleotide analog. In particular embodiments, the thymidylate synthase inhibitor is raltitrexed (raltitrexed), pemetrexed (pemetrexed), noratrixed (nolatrexed), or ZD9331. In a specific embodiment, the DNA damaging agent is 5-fluorouracil or capecitabine.
In one embodiment, the cancer is a CDH 1L-driven cancer. In one embodiment, the cancer is a TCF transcription driven cancer. In one embodiment, the cancer is an EMT-driven cancer. In one embodiment, the treatment is for CRC. In one embodiment, the treatment is for mCRC. In embodiments, the DNA-damaging agent and the CHD1 i inhibitor are administered by a dosing regimen adapted to any known method of achieving the benefits of the combination therapy. In embodiments, the CHD1L inhibitor is administered orally, and the DNA-damaging agent is administered by any known method of administration and dosing regimen. In embodiments, the CHD1L inhibitor is administered prior to administration of the DNA damaging agent. In embodiments, the CHD1L inhibitor is administered before and optionally after administration of the DNA-damaging agent. In embodiments, the CHD1L inhibitor is administered orally before and optionally after administration of the DNA damaging agent by intravenous injection.
The present invention provides methods of treating a CHD 1L-driven cancer, a TCF transcription-driven cancer, or an EMT-driven cancer comprising administering to a patient in need thereof an amount of a CHD1L inhibitor effective for CHD1L inhibition, or effective for aberrant TCF transcription inhibition, or effective for inducing EMT reversal, in combination with an alternative method of treating cancer. In one embodiment, the cancer is a GI cancer, or more specifically a CRC cancer, and still more specifically a mCRC. In one embodiment, an alternative method of treatment is administration of 5-fluorouracil, a combination of 5-fluorouracil and folinic acid (also known as aldehydic acid), a topoisomerase inhibitor, or one or more of a cytotoxic or antineoplastic agent. In embodiments, the CHD1L inhibitor is administered in combination with 5-fluorouracil, or in combination with 5-fluorouracil and folinic acid. In embodiments, the CHD1L inhibitor is administered in combination with a topoisomerase inhibitor, in particular irinotecan (prodrug of SN38, also known as camptothecin) or any other known prodrug of SN 38. In embodiments, the combination treatment with the CHD1L inhibitor and the topoisomerase inhibitor exhibits at least additive activity against cancer. In embodiments, the combination treatment of the CHD1L inhibitor with the topoisomerase inhibitor exhibits synergistic (greater than additive) activity against cancer.
In embodiments, the CHD1L inhibitor is administered in combination with a cytotoxic or antineoplastic agent, particularly a platinum-based antineoplastic agent, more particularly cisplatin, carboplatin, or oxaliplatin. In embodiments, the combination treatment with the CHD1L inhibitor and the platinum-based antineoplastic agent exhibits at least additive activity against cancer. In embodiments, the combination treatment of the CHD1L inhibitor and the platinum-based antineoplastic agent exhibits synergistic (greater than additive) activity against cancer. In embodiments, the platinum-based antineoplastic agent and CHD1L inhibitor are administered by a dosing regimen suitable for any known method of achieving the benefits of the combination therapy. In embodiments, the CHD1L inhibitor is administered orally, and the platinum-based neoplastic agent is administered by any known method of administration and dosing regimen. In embodiments, the CHD1L inhibitor is administered prior to administration of the platinum-based neoplastic agent. In embodiments, the CHD1L inhibitor is administered before and optionally after administration of the platinum-based antineoplastic agent. In embodiments, the CHD1L inhibitor is administered orally prior to and optionally after administration of the platinum-based neoplastic agent by intravenous injection.
In embodiments, the CHD1L inhibitor is administered in combination with a chemotherapeutic regimen for treating GI cancer, particularly CRC and mCRC. In embodiments, the CHD1L inhibitor is administered in combination with a chemotherapeutic regimen known as FOLFOX. In embodiments, the CHD1L inhibitor is administered in combination with a chemotherapeutic regimen known as FOLFIRI. In embodiments, the chemotherapeutic regimen and the CHD1L inhibitor are administered by a dosing regimen appropriate to any known method of achieving the benefits of the combination therapy. In embodiments, the CHD1L inhibitor is administered orally, and the chemotherapeutic regimen is administered by any known method of administration and dosing regimen. In embodiments, the CHD1L inhibitor is administered prior to administration of the chemotherapeutic regimen. In embodiments, the CHD1L inhibitor is administered prior to and optionally after administration of the chemotherapeutic regimen. In embodiments, the CHD1L inhibitor is administered orally before and optionally after the PARP inhibitor is administered by intravenous injection.
The present invention provides a method of treating cancer sensitive to poly (ADP) -ribose) polymerase I (PARPI), wherein a CHD1L inhibitor is used in combination with a PARP inhibitor. In embodiments, a CHD1L inhibitor in an amount effective for CHD1L inhibition, for aberrant TCF transcription inhibition, or for inducing EMT reversal is used in combination with a PARP inhibitor in an amount effective for treating cancer to at least enhance the effectiveness of the cancer treatment. In embodiments, combination treatment with a CHD1L inhibitor and a PARP inhibitor exhibits at least additive activity against cancer. In embodiments, the combination treatment of a CHD1L inhibitor with a PARP inhibitor exhibits synergistic activity (greater than additive activity) against cancer. In embodiments, the cancer is a cancer that is susceptible to treatment with a PARP inhibitor. In embodiments, the cancer is a cancer that is or has become resistant to PARP inhibitor treatment. In embodiments, the cancer is a cancer that is sensitive to or has developed resistance to PARP inhibitor treatment and is a CHD 1L-driven, TCF-driven, or EMT-driven cancer. In embodiments, the cancer is a homologous recombination-deficient cancer (see, e.g., zhou et al BioRxiv 2020). In an embodiment, the cancer treated is a cancer sensitive to PARP inhibitors, more specifically a cancer sensitive to breast or ovarian cancer. In a specific embodiment, the cancer is BRCA-deficient breast or ovarian cancer. In embodiments, the cancer treated is GI cancer, CRC, or mCRC. In embodiments, the combination treatment of a CHD1L inhibitor with a PARP inhibitor reverses the resistance of a cancer to PARP inhibitor treatment. In embodiments, the PARP inhibitor is olaparib, veliparib, or talazoparib. In embodiments, the PARP inhibitor is lucapanib (rucapanib) or nilapanib (nirapnib). The present invention also provides a method of treating cancer comprising administering a PARP inhibitor in an amount effective to treat cancer in combination with a CHD1L inhibitor in an amount effective to inhibit CHD1L. In embodiments, the PARP inhibitor and the CHD1 i inhibitor are administered by a dosing regimen suitable for any known method of achieving the benefits of combination therapy. In embodiments, the CHD1L inhibitor is administered orally and the PARP inhibitor is administered by intravenous injection. In embodiments, both the CHD1L inhibitor and PARP inhibitor are administered by intravenous injection. In embodiments, the CHD1L inhibitor is administered prior to administration of the PARP inhibitor. In embodiments, the CHD1L inhibitor is administered before and optionally after the PARP inhibitor. In embodiments, the CHD1L inhibitor is administered after the PARP inhibitor. In embodiments, the CHD1L inhibitor is administered orally before and optionally after the PARP inhibitor is administered by intravenous injection.
The invention also provides a method of identifying a CHD1L inhibitor that inhibits CHD 1L-dependent TCF transcription, comprising determining whether a selected compound inhibits CHD1L atpase, as described in embodiments herein. In a specific embodiment, the inhibition of cat-CHD1L ATPase is determined. In embodiments, compounds exhibiting a percent inhibition of 30% or more are selected as inhibitory CHD1L atpase. In embodiments, compounds exhibiting a percent inhibition of 80% or greater are selected as inhibitory CHD1L atpase. In particular embodiments, CHD1L inhibitors exhibit IC in a dose response assay against CHD1LATP enzymes, particularly cat-CHD1L ATPase 50 Less than 10. Mu.M. In particular embodiments, the CHD1L inhibitor exhibits IC in a dose response assay against CHD1L ATPase, particularly cat-CHD1L ATPase 50 Less than 5. Mu.M. In particular embodiments, the CHD1L inhibitor exhibits IC in a dose response assay against CHD1L ATPase, particularly cat-CHD1L ATPase 50 Less than 3. Mu.M. In particular embodiments, CHD1L inhibitors exhibit IC 50 Less than 5. Mu.M. In particular embodiments, CHD1L inhibitors exhibit IC 50 Less than 3. Mu.M. In particular embodiments, CHD1L inhibitors exhibit IC 50 Less than 1. Mu.M.
In particular embodiments, inhibition of TCF transcription by CHD1L inhibitors is assessed in a 2D cancer cell model, particularly using one or more CRC cell lines, as described in the examples herein. In a specific embodiment, inhibition of TCF transcription uses a TOPflash reporter gene construct and more specifically as described hereinAs determined by the topflash luciferase reporter construct described herein. In particular embodiments, the CHD1L inhibitor is dose-dependent on inhibition of TCF transcription in the cell model. In particular embodiments, inhibition of TCF transcription by the CHD1L inhibitor in the cell model is dose-dependent in the range of 1 to 50 μ Μ. In particular embodiments, the CHD1L inhibitor exhibits TCF-transcription% normalized to 75% or less of cell viability at 20 μ M. In particular embodiments, the CHD1L inhibitor exhibits TCF-transcription% normalized to 50% or less of cell viability at 40 μ M. In particular embodiments, the CHD1L inhibitor exhibits dose-dependent inhibition of TCF transcription, IC as measured in cancer cell lines using the TOPflash reporter 50 Less than 10. Mu.M. In particular embodiments, the CHD1L inhibitor exhibits dose-dependent inhibition of TCF transcription with an IC50 of less than 5 μ M as determined in cancer cell lines using the TOPflash reporter. In particular embodiments, CHD1L inhibitors exhibit dose-dependent inhibition of TCF transcription, IC as measured in cancer cell lines using TOPflash reporter genes 50 Less than 3. Mu.M. In an embodiment, the cancer cell line is a CRC cancer cell, a breast cancer cell, a glioma cell, a liver cancer cell, a lung cancer cell, or a GI cancer cell. In embodiments, the cancer cell line is a CRC cancer cell line. In a specific embodiment, the CRC cancer cell line is SW620.
In particular embodiments, CHD1L inhibitors are evaluated for their ability to reverse or inhibit EMT. In particular embodiments, the ability of a CHD1L inhibitor to reverse EMT is assessed in a tumor organoid. In embodiments, the reversal or inhibition of EMT is assessed in a tumor organoid that expresses vimentin, wherein a dose-dependent decrease in vimentin expression indicates reversal or inhibition of EMT. In embodiments, the reversal of EMT is assessed in a tumor organoid expressing E-cadherin, wherein a dose-dependent increase in E-cadherin expression indicates reversal or inhibition of EMT. In embodiments, the reversal of EMT is assessed in a tumor organoid expressing E-cadherin, vimentin, or both, wherein a dose-dependent decrease in vimentin and a dose-dependent increase in E-cadherin expression indicates reversal or inhibition of EMT. In specific embodiments, dose-dependent reversal or inhibition of EMT is measured at compound concentrations of 0.1 μ M to 100 μ M. In specific embodiments, dose-dependent reversal of EMT is measured at compound concentrations of 0.3 μ M to 50 μ M.
In particular embodiments, the ability of CHD1L inhibitors to inhibit clonogenic colony formation, a recognized assay for measuring cancer stem cell sternness, is evaluated. In embodiments, the cells are pretreated with a selected concentration of CHD1L inhibitor prior to plating. In embodiments, the cells are cultured at low density such that only CSCs form colonies in 10 days of culture. In embodiments, the cells are pretreated for 12-36 hours. In embodiments, the cells are pretreated for 24 hours. In embodiments, cells are pretreated with CHD1L inhibitor at a concentration ranging from 0.5 μ M to 50 μ M, with appropriate controls. In embodiments, the CHD1L inhibitor exhibits 40% or more inhibition of clonogenic colony counts for a CHD1L concentration of 40 μ M compared to no compound control. In embodiments, the CHD1L inhibitor exhibits an inhibition of colony counts of 40% or more clonogenic for a CHD1L concentration of 20 μ M compared to no compound control. In embodiments, the CHD1L inhibitor exhibits an inhibition of colony counts of 40% or more clonogenic as compared to no compound control for a CHD1L concentration of 2 μ M. In embodiments, inhibition of clonogenic colony formation persists over a 10 day culture.
In particular embodiments, CHD1L inhibitors are further evaluated for loss of invasive potential using any known method, particularly using the methods described in the embodiments herein.
In particular embodiments, the CHD1L inhibitor is further assessed for anti-tumor activity as measured by induction of cytotoxicity in tumor organoids. In embodiments, cells are treated with selected concentrations of CHD1L inhibitor (1 μ M to 100 μ M) for selected times (e.g., 24 to 96 hours, preferably 72 hours). In embodiments, cytotoxicity is measured using any of a variety of cytotoxic agents known in the art, e.g., entering damaged cells and exhibiting a measurable amount upon entrySmall molecules which are chemically modified (e.g. fluorescent, e.g. CellTox) TM Green reagent (Promega, madison, wis.), or IncuCyteCytox reagent (Sartorius, france). In embodiments, cytotoxicity is measured by measuring LDH (lactate dehydrogenase) released from dead cells.
In embodiments, CHD1L inhibitors useful in the methods of treatment herein are those of formulas I-XX and XXX-XLII, or pharmaceutically acceptable salts or solvates thereof. In an embodiment, the present invention provides novel compounds of any of formulae I-XX, XXXV-XLII herein, or salts or solvates thereof. In embodiments, the CHD1L inhibitors are those of formula I. In embodiments, the CHD1L inhibitors are those of formula XX. In embodiments, the CHD1L inhibitor is of formula I-IX, xi-XiX, XX, or XXXV-XLII.
In a specific embodiment, the methods of the invention administer a CHD1L inhibitor selected from one or more of compounds 1-73 or a pharmaceutically acceptable salt or solvate thereof. In the methods herein, two or more CHD1L inhibitors can be used in combination. In a specific embodiment, the CHD1L inhibitor used in the present invention is selected from one or more of compounds 6-39 or a pharmaceutically acceptable salt thereof. In a specific embodiment, the CHD1L inhibitor used in the methods of the present invention is selected from one or more of compounds 40-51 or a pharmaceutically acceptable salt or solvate thereof. In a particular embodiment, the CHD1L inhibitor used in the methods of the present invention is selected from one or more of compounds 52-68 or a pharmaceutically acceptable salt or solvate thereof. In a specific embodiment, the CHD1L inhibitor used in the methods of the present invention is selected from one or more of compounds 70-73 or a pharmaceutically acceptable salt or solvate thereof. In a particular embodiment, the CHD1L inhibitor used in the methods of the invention is compound 6, 8, 52, 54, 56, 61, 62, 65 or 66 or a pharmaceutically acceptable salt or solvate thereof. In a particular embodiment, the CHD1L inhibitor used in the methods of the invention is compound 6, 8, or a pharmaceutically acceptable salt or solvate thereof. In a particular embodiment, the CHD1L inhibitor used in the methods of the present invention is compound 52, 54, or a pharmaceutically acceptable salt or solvate thereof. In a specific embodiment, the CHD1L inhibitor used in the methods of the present invention is compound 22, 23, 26 or 27, or a pharmaceutically acceptable salt thereof.
In particular embodiments, the methods of the invention employ CHD1L inhibitors of formula XX, and include all embodiments described herein for formula XX. The invention also provides novel compounds of formula XX, salts thereof, and pharmaceutical compositions containing such compounds and salts.
The invention also relates to the CHD1L inhibitors of the invention and pharmaceutically acceptable compositions comprising any such inhibitors. In embodiments, the present invention relates to any novel compound as described in the formulae herein or a pharmaceutically acceptable salt or solvate thereof. In particular, the invention relates to CHD1L inhibitors and pharmaceutically acceptable salts thereof as described in the formulae herein, with the exception that the CHD1L inhibitor is not compound 1-8 or a salt or solvate thereof. In particular, the invention relates to CHD1L inhibitors and pharmaceutically acceptable salts thereof as described in the formulae herein, with the exception that the CHD1L inhibitor is not compound 1-9 or a salt thereof. In embodiments, the present invention relates to any one of compounds 9-39, 40-68, 69-73, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically acceptable composition comprising such compounds, salts, or solvates. In embodiments, the present invention relates to any one of compounds 10-39 or 40-73, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically acceptable composition comprising such compounds, salts, or solvates. In embodiments, the invention relates to any one of compounds 52-73, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically acceptable composition comprising such a compound, salt or solvate. In embodiments, the CHD1L inhibitors of the invention have a Clog P of 5 or less. In embodiments, the CHD1L inhibitors of the invention have a Clog P of 3 to 4.
In particular embodiments, the present invention relates to the following compounds and methods of using these compounds herein to treat cancer (particularly CRC and mCRC): compounds 52-73; compound 52 or 53; compound 54, 55 or 67; or compound 57, 58 or 59; or a pharmaceutically acceptable salt or solvate thereof; any one of compound 8, compound 52, compound 53, compound 54, compound 55, compound 56, compound 57, compound 58, compound 59, compound 61, compound 62, compound 65, compound 66, or compound 67.
The invention also relates to the use of a CHD1L inhibitor for the manufacture of a medicament for the treatment of cancer, in particular for the treatment of cancer (CHD 1L-driven cancer, TCF-driven cancer or EMT-driven cancer, in particular GI cancer, and more in particular CRC or mCRC). The present invention also relates to CHD1L inhibitors herein for use in the treatment of cancer, CHD1L driven cancer, TCF driven cancer or EMT driven cancer, in particular GI cancer, and more particularly CRC or mCRC.
Other embodiments and aspects of the invention will be apparent to those of ordinary skill in the art upon review of the drawings, detailed description and examples herein.
Drawings
FIGS. 1A-B: validation of CHD1L inhibitors identified by HTS. (FIG. 1A) hit 1-7 cat-CHD1L ATPase C50 dose response. Calculation of average IC from three independent experiments 50 Values and shows a representative curve. (FIG. 1B) SW620, HCT-16 and DLD1CHD1L-OE cells with TOPflash reporter for measurement of inhibition of TCF transcription at 24 hours using 3 doses.
FIGS. 2A-2D: CHD1L inhibitors reverse EMT and malignant phenotypes in CRC. The dose response of CHD1L inhibitors of EMT was modulated by high content imaging measurements of (fig. 2A) down-regulation of VimPro-GFP reporter and (fig. 2B) up-regulation of ecappro-RFP reporter. Average EC 50 Values ± SEM were calculated from three independent experiments. (FIG. 2C) CSC dryness was measured by clonogenic colony formation after pretreatment with CHD1L inhibitors in DLD1CHD1L-OE and HCT-116 cells. (FIG. 2D) inhibition of the invasive potential of HCT-116 cells following CHD1L inhibitor treatment. Welch t-test statistical analysis was used to determine significance, where P = P ≦ 0.05, P ≦ 0.01, P ≦ 0.001, and P ≦ 0.0001.
FIGS. 3A-C: compound 6 induced apoptosis in CRC cell lines and PDTO. (drawing)3A) Induction of E-cadherin expression using Ecad-ProRFP reporter Gene assays and Cell-Tox TM Time course assessment of cytotoxicity of Green cytotoxicity assay (Promega, madison, wi). (FIG. 3B) analysis of annexin V-FITC staining for apoptosis 12 hours after SN-38 and 6 treatments. (FIG. 3C) use
Figure BDA0003960268060000151
Cell viability assay (Promega, madison, wis.) cytotoxicity of 6 in PDTO CRC 102. Average EC 50 Values ± s.d. were calculated from six independent experiments, and representative graphs show an inset representing PDTO. Welch t-test statistical analysis was used to determine significance, where @ = P ≦ 0.05,. Gtoreq.P ≦ 0.01,. Gtoreq.P ≦ 0.001, and ≦ 0.0001.
FIG. 4: accumulation of compound 6 in SW620 xenograft tumors. Compound 6 was administered by i.p. injection to athymic nude mice QD for 5 days to measure accumulation in SW620 xenograft tumors.
FIG. 5: proposed mechanism of action of CHD 1L-mediated transcription of TCF. CHD1L is activated by binding to TCF complex members PARP1 and TCF 4[ Abbott et al, 2020]. (1) Once activated, CHD1L is directed to hindered WRE located on chromatin. (2) Chromatin remodeling and DNA translocation occur to expose WRE sites. (3) Binding of TCF complex to exposed WRE is facilitated by CHD1L, promoting EMT genes and other genes associated with mCRC. CHD1L atpase inhibitors effectively prevent step 1, resulting in the reversal of EMT and other malignant properties of CRC.
FIGS. 6A-E: evaluation of compound 8. (FIG. 6A) Compound 8 showed effective low μ M dose-dependent inhibition of TCF transcription based on TOPFlash reported in SW260 cell cultures at 2D and over a 24 hour time course. Compound 8 was effective at 72 hours in reversing EMT in the dual reporter SW620 tumor organoids as evidenced by down-regulation of vimentin in a dose-dependent manner (fig. 6B) and up-regulation of E-cadherin promoter activity in a dose-dependent manner (fig. 6C). Compound 8 significantly inhibited clonogenic colony formation at 10 days (fig. 6D) and the invasive potential of HCT116 at 48 hours after 24 hours of pretreatment (fig. 6E). students t-test showed: = P ≦ 0.05
FIGS. 7A-B: viability of colorectal cancer tumor organoids after treatment with exemplary CHDIL inhibitors. These figures illustrate a representative plot of% activity as a function of log concentration for the compounds shown. (FIG. 7A) treatment with Compound 6.9; (FIG. 7B) treatment with Compound 6.11; the numbering of the alternative compounds used in scheme 1 is given in parentheses. Each figure is provided with an IC 50 In some cases, average IC 50 . Viability data for a number of exemplary compounds is provided in table 3.
FIGS. 8A-B: assessment of CHD1L mediated DNA repair and "targeting" effect of CHD1L inhibitor 6 alone and in combination with irinotecan (prodrug of SN 38). CHD1L is known to be critical for PARP-1 mediated DNA repair, resulting in resistance to chemotherapy of DNA damage [ Ahel et al, 2009; tsuda et al, 2017]. DLD1 CRC cells with low levels of CHD1L (DLD 1 empty vector, EV) expression compared to DLD1 cells engineered to overexpress CHD1L (DLD 1 overexpression, OE) were used. FIG. 8A is a Western blot comparing CHD1L expression in DLD1 (EV) and DLD1 (OE) and control expression of β -tubulin in these cells. Figure 8B shows a graph of γ -H2AX intensity (versus DMSO) in DLD1 empty vector cells and DLD1 overexpressing cells for compound alone, SN38 alone, and a combination of both. Compound 6 alone did not induce significant DNA damage nor did it act synergistically with SN38 in DLD1 cells with low CHD1L expression. The graph demonstrates that the synergistic effect of CHD1L inhibitors with SN38 exerts a "targeting" effect, inducing DNA damage in DLD1 cells overexpressing CHD1L.
FIGS. 9A-9C: synergy studies of exemplary CHD1L inhibitors and irinotecan (prodrug of SN 38). (FIG. 9A) synergy studies with compounds 6 and 6.3 in SW620 colorectal cancer (CRC) tumor organoids. (FIG. 9B) synergy studies with Compound 6.9 in SW620 colorectal cancer (CRC) tumor organoids. (FIG. 9C) synergy studies with compound 6.11 in SW620 colorectal cancer (CRC) tumor organoids. The combination of SN38 with 6 and 6.3 was 50-fold and 150-fold more potent in killing colon SW620 tumor organoids than SN38 alone. The efficacy of SN38 in combination with 6.9 and 6.11 was over 100 times that of SN38 alone. Each of compounds 6, 6.3, 6.9 and 6.11 showed synergistic effect with irinotecan (and SN 38) in killing SW620 tumor organoids.
FIG. 10: the in vivo synergy of the combination of compound 6 with irinotecan in mice was studied. Figure 10 includes a graph of tumor volume (fold) of SW620 tumor xenografts as a function of days (up to 28 days) treated with compound 6 (2) alone, irinotecan (3) alone, or combination thereof (4) compared to control (1). A data table showing statistical significance of the data is also provided. The combination of irinotecan and compound 6 significantly inhibited colon SW620 tumor xenografts to virtually no tumor volume within 28 days of treatment compared to the single agent treatment group.
FIG. 11: the in vivo synergistic effect of the CHD1L inhibitor compound 6 and irinotecan continues after treatment. Figure 11 includes a graph of tumor volume (fold) of SW620 tumor xenografts as a function of days (up to 41 days) treated with irinotecan alone (1), or the combination of compound 6 and irinotecan (2). A data table showing statistical significance of the data is also provided. The combination of irinotecan and compound 6 significantly inhibited colon SW620 tumor to almost no tumor volume after the last treatment (day 28) compared to irinotecan alone. Within 2 weeks of the last irinotecan treatment alone, tumor volume rose above the volume of the last treatment, indicating tumor recurrence. In contrast, the combination treatment maintained a lower tumor volume.
FIG. 12: compound 6 alone and in combination with irinotecan significantly increased survival of CRC tumor-bearing mice compared to vehicle and irinotecan alone. Figure 12 includes a graph of survival (%) versus time after the last treatment on day 28 with compound 6 (2) alone, irinotecan (3) alone or combination thereof (4) up to day 52, compared to control (1). A data table showing statistical significance of the data is also provided. The survival rate of the combination treatment was significantly higher compared to the single dose compound or control.
FIG. 13: compound 6.11 in combination with irinotecan synergy studies in mice. Figure 13 includes a graph of tumor volume (fold) of SW620 tumor xenografts as a function of days (up to 20 days) treated with compound 6.11 (2) alone, irinotecan (3) alone, or combination thereof (4) compared to control (1). A data table showing statistical significance of the data is also provided. The combination of irinotecan and compound 6.11 significantly inhibited colon SW620 tumor xenografts to virtually no tumor volume within 20 days of treatment compared to irinotecan alone.
FIG. 14: the in vivo synergy of the CHD1L inhibitor compound 6.11 and irinotecan continues after treatment. Figure 14 includes a graph of tumor volume (fold) of SW620 tumor xenografts as a function of days (up to 41 days) of treatment with irinotecan (1) alone, or a combination of compound 6 and irinotecan (2). Treatment (Tx release) was stopped on day 33. The combination of irinotecan and compound 6.11 significantly inhibited colorectal SW620 tumor after the last treatment (day 33) compared to irinotecan alone.
FIG. 15 is a schematic view of: the in vivo synergy of the CHD1L inhibitor 6.11 and irinotecan significantly increased survival benefit. The combination of compound 6.11 with irinotecan significantly improved survival in CRC tumor-bearing mice compared to vehicle and irinotecan alone. Figure 15 includes a graph of survival (%) versus time up to day 50 after the last treatment on day 33 with compound 6 (2) alone, erietide (3) alone, or combination thereof (4) as compared to control (1). A data table showing statistical significance of the data is also provided. The survival rate of the combination treatment was significantly higher compared to irinotecan alone or the control.
Detailed Description
The present invention relates generally to the characterization of a relatively novel oncogene, CHD1L, as a tumorigenic factor associated with poor prognosis and survival of CRC. The novel biological function of CHD1L as a DNA binding factor for the TCF transcription complex required to promote TCF-driven EMT and other malignant properties has been demonstrated. Abbott et al, 2020 and additional information in this article (available from journal web site (mct. Aacrjournals. Org)) provide a description of a portion of the experiments and data described herein, and are each incorporated by reference in its entirety.
CHD1L is amplified (Chr 1q 21) and overexpressed in many types of cancer [ Ma et al, 2008; cheng et al, 2013]. CHD1L overexpression is considered a poor prognostic and metastatic marker for many cancers [ Ma et al, 2008; cheng et al, 2008; hyeon et al, 2013; su et al 2014]. While the common literature demonstrates that CHD1L is convincing as a driver of oncogenes and malignancies, the stringency of previous studies and the hypothesis that CHD1L might be an oncogene that targets CRC molecules are tested herein. A computer analysis of transcriptome data obtained over a large cohort of 585 CRC patients obtained over 15 years was reported [ Marisa et al, 2013]. Notably, CHD1L expression is associated with poor survival, with patients with low CHD1L having significantly longer survival than patients with high CHD1L. Using the same cohort, marisa et al, 2013 identified six different subtypes for improving clinical stratification of CRC, CHD1L is ubiquitously expressed in all six subtypes, suggesting its potential as a therapeutic target for CRC. CHD1L is also associated with tumor lymph node metastasis, with an increase in expression from N0 (no regional spread) to N3 (distal regional spread). Analysis of transcriptome data from the UCCC patient cohort (n = 25) revealed that CHD1L expression was significantly associated with stage IV and mCRC. Literature reports and work herein indicate that CHD1L is an oncogene that promotes malignant CRC, and its high expression correlates with poor prognosis and low survival in CRC patients.
The novel biological function of CHD1L as a DNA binding factor for TCF transcription complexes required to promote TCF-driven EMT and other malignant properties has been demonstrated. Using HTS drug discovery, the first known CHD1L inhibitor has been identified and characterized, which shows good pharmacological effects in cell-based CRC models (including PDTO). CHD1L inhibitors are effective in preventing CHD 1L-mediated TCF transcription, leading to reversal of EMT and other malignant properties, including CSC sternness and invasive potential. Notably, CHD1L inhibitor 6 demonstrated the ability to induce cell death, consistent with the reversal of EMT and E-cadherin-mediated exogenous apoptosis via death receptor-induced cleavage. Furthermore, the synergistic effect of compound 6 with SN38 (i.e., irinotecan) showed potent DNA damage-inducing effects consistent with inhibition of PARP1/CHD 1L-mediated DNA repair compared to SN38 alone. CHD1L inhibitors have been identified that have physicochemical properties similar to drugs, good PK/PD profiles in vivo, and no acute hepatotoxicity.
Based on the data presented herein, the mechanism of action of CHD 1L-mediated TCF-driven EMT involved in CRC tumor progression and metastasis is shown (fig. 5). In this mechanism, the TCF complex specifically recruits CHD1L to dynamically regulate the expression of the transgene. Central to this mechanism is that CHD1L, when directed by the TCF complex via protein interactions with PARP1 and TCF4, binds to nucleosome-hindered WRE. Importantly, PARP1 is characterized as a major cellular activator of CHD1L bound by a macro-domain, which releases an auto-inhibitory action [ Lehmann et al, 2017; gottschalk et al, 2009]. In addition, PARP1 is an essential component of the interaction of the TCF complex with β -catenin and TCF 4[ Idogawa et al, 2005]. Thus, this mechanism suggests that CHD1L is recruited by the TCF complex and activated by PARP1 and TCF 4. Once activated, CHD1L exposes WRE through nucleosome translocation, promoting TCF complex binding to WRE and promoting transcription of malignant genes of EMT. CHD1L inhibitors have a unique mechanism of action by inhibiting CHD1L atpase activity, which prevents WRE exposure to TCF complexes, inhibiting transcription of TCF target genes associated with EMT (in particular mCRC).
The CHD1L small molecule inhibitors described herein have been identified in screens based on inhibition of CHD1L atpase activity. Certain identified inhibitors exhibit drug-like physicochemical properties and good PK/PD profiles in vivo, and are free of acute hepatotoxicity. Such inhibitors are effective in treating CRC and mCRC (metastatic CRC) as well as other CHD1L driven cancers.
Known methods for assessing druggability may be used to further assess the active compounds of the invention for a given therapeutic application. The term "druggability" relates to the pharmaceutical properties of administration, distribution, metabolism and excretion of a potential drug. The art evaluates drugginess in various ways. For example, the "Lipinski five-rate rule" can be applied to determine drug-like properties in molecules that are associated with absorption and permeability in vivo [ Lipinski et al, 2001; ghose, et al, 1999].
The present invention provides methods of combination therapy in which a CDH1L inhibitor is administered in combination with one or more anti-cancer agents other than a CDH1L inhibitor. In embodiments, the additional anti-cancer agent is a topoisomerase inhibitor, a platinum-based antineoplastic agent, a PARP inhibitor, or a combination of two or more such inhibitors and agents. In embodiments, the combination therapy combines administration of a CDH1L inhibitor with administration of a topoisomerase inhibitor. In embodiments, the combination therapy combines administration of a CDH1L inhibitor with a platinum-based antineoplastic agent. In embodiments, the combination therapy combines the administration of a CDH1L inhibitor with a PARP inhibitor. In embodiments, the combination therapy combines administration of a CDH1L inhibitor and a topoisomerase inhibitor with administration of a PARP inhibitor. In embodiments, the combination therapy combines administration of a CDH1L inhibitor with chemotherapy for the particular cancer being treated. In embodiments herein, the combination of a CDH1L inhibitor and another antineoplastic agent exhibits synergistic activity in combination.
In embodiments herein, therapy with CDH1L may be combined with radiation therapy appropriate for a given cancer.
Various PARP inhibitors are known in the art [ see, rouleau et al, 2010; yi et al, 2019; zhou et al, 2020; wahlberg et al, 2012; d' Andrea,2018]. Each of these references is incorporated by reference herein in its entirety for the purpose of describing PARP inhibitors, the mechanism of action of PARP inhibitors, cancers treated with PARP inhibitors, and resistance to PARP inhibitors. In particular embodiments herein, PARP resistant cancers are treated with a combination of a CDH1L inhibitor and a PARP inhibitor.
Various topoisomerase inhibitors are known in the art and have been used clinically (see, e.g., hevener, 2018). This reference is incorporated by reference herein in its entirety for the purpose of describing the type of topoisomerase inhibitor, the specific topoisomerase inhibitor, the mechanism of topoisomerase inhibition, the cancer treated with the topoisomerase inhibitor, and the combination therapy using the topoisomerase inhibitor. In embodiments, topoisomerase inhibitors useful in the methods herein include camptothecin (camptothecin) and prodrugs thereof, irinotecan (irinotecan), topotecan (topotecan), belotecan (belotecan), indotecan (indotecan), or indimititecan (indiitecan). In embodiments, topoisomerase inhibitors useful in the methods herein include etoposide (etoposide) or teniposide (teniposide). In embodiments, topoisomerase inhibitors useful in the methods herein include nanostaticon (namitecan), cilastacin (silatecan), vosarorin (vosaroxin), doxorubicin (aldoxorubicin), becker's (bicarin), or idarubicin (edotecarin).
Various platinum-based antineoplastic agents (also known as platinum-based antineoplastic agents) are known in the art and have been used clinically or in clinical trials [ see, e.g., white et al, 2010)]. This reference is incorporated by reference herein in its entirety for the description of the type of platinum-based antineoplastic agent, the particular platinum-based antineoplastic agent, the mechanism of action of such agent, the cancer treated with such agent, and combination therapies using platinum-based antineoplastic agents. In embodiments, the platinum-based antineoplastic agents for use in the methods herein include cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, or heptaplatin. In embodiments, the platinum-based antineoplastic agent comprises satraplatin or picoplatin. The platinum-based antineoplastic agent may be liposome-encapsulated (e.g., lypoplatin) TM ) Or incorporated in a nano-polymer (e.g. ProLindac) TM )。
Various thymidylate synthase inhibitors are known in the art and have been employed clinically, particularly in the treatment of CRC [ papamic, 2009; lehman,2002]. Thymidylate synthase inhibitors include folic acid analogs and nucleotide analogs. In particular embodiments, the thymidylate synthase inhibitor is raltitrexed (raltitrexed), pemetrexed (pemetrexed), noratrixed (nolatrexed), or ZD9331. In a more specific embodiment, the thymidylate synthase inhibitor is 5-fluorouracil or capecitabine.
The present invention provides CHD1L inhibitors of the formula:
compounds useful in the methods of the invention include compounds of formula I:
Figure BDA0003960268060000221
or a salt or solvate thereof, or a solvate thereof,
wherein:
ring B is a heteroaromatic ring or ring system having one, two or three 5-or 6-membered rings, any two or three of which may be fused, wherein the rings are carbocyclic, heterocyclic, aromatic or heteroaromatic and at least one ring is heteroaryl;
in the B ring, each X is independently selected from N or CH, and at least one X is N;
R P is a primary or secondary amine group [ -N (R) 2 )(R 3 )]Or is- (M) x A group of-P, wherein P is-N (R) 2 )(R 3 ) Or an aryl or heteroaryl group, wherein x is 0 or 1, meaning that M is absent or present, and M is an optionally substituted linking group- (CH) 2 ) n -or-N (R) (CH) 2 ) n -wherein each n is independently an integer from 1 to 6 inclusive;
y is selected from the group consisting of-O-, -S-, -N (R) 1 )-、-CON(R 1 )-、-N(R 1 )CO-、-SO 2 N(R 1 ) -or-N (R) 1 )SO 2 -a divalent atom or group of the group;
L 1 is an optional linking group of 1-4 carbons, which is optionally substituted, and is saturated or contains a double bond (which may be cis or trans), wherein x is 0 or 1, meaning that L is absent or present 1 ；
Ring a is a carbocyclic or heterocyclic ring or ring system having one, two or three rings, two or three of which may be fused, each ring having 3 to 10 carbon atoms and optionally 1 to 6 heteroatoms, and wherein each ring is optionally saturated, unsaturated or aromatic;
z is a divalent radical containing at least one nitrogen substituted with an R' group,
wherein in embodiments, Z is a divalent group selected from: -N (R ') -, -CON (R') -, -N(R′)CO-、–CSN(R′)-、–N(R′)CS-、-N(R′)CON(R′)-、–SO 2 N(R′)-、–N(R′)SO 2 -、-CH(CF 3 )N(R′)-、-N(R′)CH(CF 3 )-、-N(R′)CH 2 CON(R′)CH 2 -、-N(R′)COCH 2 N(R′)CH 2 -、
Figure BDA0003960268060000231
Or the divalent Z group comprises a 5-or 6-membered heterocyclic ring having at least one nitrogen ring member, for example,
Figure BDA0003960268060000232
L 2 is an optional linking group of 1-4 carbons, which is optionally substituted, and is saturated or contains a double bond (which may be cis or trans), wherein z is 0 or 1, meaning that L is absent or present 1 ；
R is selected from the group consisting of hydrogen, an aliphatic group, a carbocyclyl group, an aryl group, a heterocyclyl group, and a heteroaryl group, each of which is optionally substituted;
each R' is independently selected from the group consisting of hydrogen, an aliphatic group, a carbocyclyl group, an aryl group, a heterocyclyl group, and a heteroaryl group, each of which is optionally substituted;
R 1 selected from the group consisting of hydrogen, aliphatic groups, carbocyclyl groups, aryl groups, heterocyclyl groups, and heteroaryl groups, each of which is optionally substituted;
R 2 and R 3 Independently selected from the group consisting of hydrogen, aliphatic, carbocyclic, aryl, heterocyclic and heteroaryl groups, each of which is optionally substituted, or
R 2 And R 3 Together with the N to which they are attached form an optionally substituted 5-10 membered heterocyclic ring, the 5-10 membered heterocyclic ring being a saturated ring, a partially unsaturated ring, or an aromatic ring;
R A and R B Each represents hydrogen or 1-10 non-hydrogen substituents on the A and B rings or ring systems shown, wherein R is A And R B The substituents are independently selected from hydrogen, halogen, hydroxy, cyano, nitro, amino, mono-or di-substituted amino (-NR) C R D ) Alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy, acyl, haloalkyl, -COOR C 、-OCOR C 、-CONR C R D 、-OCONR C R D 、-NR C COR D 、-SR C 、-SOR C 、-SO 2 R C and-SO 2 NR C R D Wherein alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy, and acyl are optionally substituted;
each R C And R D Selected from hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with one or more halogen, alkyl, alkenyl, haloalkyl, alkoxy, aryl, heteroaryl, heterocyclyl, aryl-substituted alkyl, or heterocyclyl-substituted alkyl; and
R H is an optionally substituted aryl or heteroaryl group;
wherein the optional substitution comprises amino substituted with one or more halogen, nitro, cyano, amino, mono-or di-C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C1-C3 haloalkyl, C6-C12 aryl, C5-C12 heteroaryl, C3-C12 heterocyclyl, C1-C3 alkoxy, C1-C6 acyl, -COOR E 、-OCOR E 、-CONR E R F 、-OCONR E R D 、-NR E COR F 、-SR E 、-SOR E 、-SO 2 R E and-SO 2 NR E R F Wherein alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy, and acyl are optionally substituted, and
each R E And R F Selected from hydrogen, C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C1-C3 haloalkyl, C6-C12 aryl, C5-C12 heteroaryl, C3-C12 heterocyclyl, C1-C3 alkoxy, C1-C6 acyl, each of which is optionally substituted with one or more halogen, nitro, cyano, amino, mono-or di-C1-C3 alkyl-substituted amino, C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C1-C3 haloalkyl, C6-C12 aryl, C5-C12 heteroaryl, C3-C12 heterocyclyl, C1-C3 alkoxy, and C1-C6 acyl.
In embodiments of formula I:
r is selected from hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl or heteroaryl, wherein each is optionally substituted;
each R' is independently selected from hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, or heteroaryl, wherein each group is optionally substituted;
R 1 -R 3 independently selected from hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, or heteroaryl, wherein each group is optionally substituted;
R 1 -R 3 one or more of (a) is cycloalkyl-substituted alkyl, such as cyclopropylmethyl, cyclopentylmethyl or cyclohexylmethyl;
r is hydrogen or C1-C3 alkyl;
each R' is independently hydrogen or C1-C3 alkyl;
R 1 is hydrogen or C1-C3 alkyl;
R 2 and R 3 Independently selected from hydrogen or C1-C3 alkyl; or
R 2 And R 3 Together with the N to which they are attached form a saturated 5-7 membered heterocyclic ring.
In particular embodiments of formula I:
ring a is optionally substituted phenyl;
ring a is optionally substituted naphthyl;
ring B is an optionally substituted pyridyl group,
ring B is optionally substituted pyrimidinyl;
ring B is optionally substituted pyrazinyl;
ring B is optionally substituted triazinyl;
ring B is an optionally substituted quinazolinyl;
ring B is optionally substituted pteridinyl;
ring B is optionally substituted quinolinyl;
ring B is optionally substituted isoquinolinyl;
ring B is an optionally substituted naphthyridinyl group;
ring B is optionally substituted pyridopyrimidinyl;
ring B is optionally substituted pyrimidopyridyl;
ring B is optionally substituted pyranopyridyl;
ring B is optionally substituted pyranopyrimidinyl;
ring B is an optionally substituted purinyl group;
ring a is optionally substituted phenyl, ring B is optionally substituted pyrimidinyl; or alternatively
Ring a is optionally substituted phenyl and ring B is optionally substituted pteridinyl.
Preferred A and B ring substituents include one or more C1-C3 alkyl, C3-C7 cycloalkyl, C4-C10 cycloalkyl-substituted alkyl, C2-C4 alkenyl, C1-C3 alkoxy, C1-C3 acyl, halogen, hydroxy, C1-C3 haloalkyl, mono-or di-substituted phenyl, or mono-or di-substituted benzyl. More specific substituents for the A and B rings include methyl, ethyl, isopropyl, cyclopropyl, cyclopropylmethyl, methoxy, ethoxy, phenyl, benzyl, halophenyl, halobenzyl, cl, br, F, CF 3 -、HO-、CF 3 O-、CH 3 CO-and CHCO.
In a more specific embodiment, ring B has the structure shown as formula RBI in scheme 4, wherein X is 1 And X 2 Selected from CH and N, and X 1 And X 2 Is N, X 3 -X 6 Selected from CH, CH 2 O, S, N and NH, wherein the B ring is saturated, unsaturated or aromatic, depending on X 1 -X 6 And R is B Represents an optional substitution as defined in formula I. In embodiments, R B RepresentHydrogen and ring B is unsubstituted. In embodiments, R B Represents one or more halogens, C1-C3 alkyls, C1-C3 acylates, C1-C3 alkoxys. In embodiments, R B Represents one or more of F, cl or Br, methyl, ethyl, acetyl, or methoxy groups or combinations thereof. In embodiments, as shown in scheme 4, the B ring of formula I is selected from any one of RB2-RB 5.
In embodiments of formula I:
x is 1, and L 1 Is- (CH) 2 ) n -, where n is 1 or 2;
x is 0, and L1 is absent;
y is 1, and L 2 Is- (CH) 2 ) n -, wherein n is 1 or 2;
y is 1, and L 2 is-CH = CH-;
y is 1, and L 2 Is trans-CH = CH-;
x and y are both 0;
x is 1, and y is 0, and L 1 Is- (CH) 2 ) n -, wherein n is 1 or 2;
y is 1, and x is 0, and L 2 Is- (CH) 2 ) n -, where n is 1 or 2; or
x and y are both 1, and L 2 And L 1 Both are- (CH) 2 ) n -, where n is 1 or 2.
In embodiments of formula I:
y is-O-, -S-, -N (R) 1 )–、–CON(R 1 ) -or-N (R) 1 )CO–；
Y is-N (R) 1 )–、–CON(R 1 ) -, or-N (R) 1 )CO–；
R 1 Is hydrogen, C1-C3 alkyl or C1-C3 haloalkyl, especially C1-C3 fluoroalkyl;
R 1 is hydrogen, a methyl group or CF 3 -；
R 1 Is hydrogen;
y is-N (R) 1 )–、–CON(R 1 ) -or-N (R) 1 ) CO-, and R 1 Is hydrogen, methyl or CF 3 -；
Y is-NH-, -CONH-, or-NHCO-;
y is-N (R) 1 ) -, and R 1 Is hydrogen, C1-C3 alkyl or C1-C3 haloalkyl, especially C1-C3 fluoroalkyl; or
Y is-N (R) 1 ) -, and R 1 Is hydrogen, methyl or CF 3 -。
In embodiments of formula I, x and y are both 0, Y is-N (R) 1 ) -. In embodiments of formula I, x and Y are both 0, and Y is-NH-.
In embodiments of formula I:
z is-N (R ') -, -CON (R ') -, or-N (R ') CO-;
z is-CH (CF) 3 )N(R′)-；
Z is-SO 2 N(R′)-；
Z is-N (R ') CON (R') -;
z is-N (R') CH 2 CON(R′)CH 2 -；
Z is
Figure BDA0003960268060000281
Z is
Figure BDA0003960268060000282
Z is
Figure BDA0003960268060000283
R' is hydrogen, C1-C6 alkyl or C1-C3 haloalkyl, in particular C1-C3 fluoroalkyl;
r' is hydrogen or C1-C3 alkyl;
r' is hydrogen, methyl or CF3-;
r' is hydrogen or methyl;
r' is hydrogen;
z is-N (R ') -, -CON (R') -, or-N (R ') CO-, R' is hydrogen or methyl;
z is-CON (R ') -, or-N (R ') CO-, R ' is hydrogen or methyl;
z is-N (R ') CON (R ') -, both R ' are hydrogen;
z is
Figure BDA0003960268060000291
R' is hydrogen; or
Z is
Figure BDA0003960268060000292
R' is hydrogen.
In embodiments of formula I:
x is 0;
x is 1, L 2 Is- (CH) 2 ) n -, where n is 1 to 3;
y is 0, x is 1, L 2 Is- (CH) 2 ) n -, where n is 1 to 3;
x is 0, z is-N (R ') -, -CON (R ') -, or-N (R ') CO-;
x is 0, z is-N (R ') -, -CON (R') -, or-N (R ') CO-, R' is hydrogen or methyl;
x is 0, z is-CON (R') -;
x is 0, z is-CON (R ') -, R' is hydrogen or methyl;
x is 0, z is-CON (R ') -, R' is hydrogen;
x is 1, L 2 Is- (CH) 2 ) n -, where N is 1-3, z is-N (R) 1 ) -, -CON (R ') -, or-N (R') CO-;
x is 1,L 2 Is- (CH) 2 ) n -, wherein n is 1-3, z is-CON (R') -;
x is 1,L 2 is-CH 2 -z is-CON (R') -;
x is 1, L 2 is-CH 2 -CH 2 -z is-CON (R') -;
x is 0 or 1,L 2 If present, is-CH 2 -or-CH 2 -CH 2 -, z is-CON (R') -;
x is 0 or 1,L 2 If it exists, it is–CH 2 -or-CH 2 -CH 2 -, z is-CON (R ') -, R' is hydrogen;
y is 0, x is 0 or 1,L 2 If present, is-CH 2 -or-CH 2 -CH 2 -, z is-CON (R ') -, R' is hydrogen;
y is 0, Y is-N (R) 1 ) -, x is 0 or 1, L 2 If present, is-CH 2 -or-CH 2 -CH 2 -z is-CON (R') -; or
y is 0, Y is-N (R) 1 )-，R 1 Is hydrogen, x is 0 or 1, L 2 If present, is-CH 2 -or-CH 2 -CH 2 -, z is CON (R '), and R' is hydrogen.
In the embodiment of the formula I, the compound of formula I,
R P containing at least one nitrogen; or alternatively
When R is P Is- (M) x-P, and x =0, then P is-N (R) 2 )(R 3 ) Or P is a heterocyclyl or heteroaryl group having at least one ring N; or
When R is P Is- (M) x-P, x =1, and M = - (CH) 2 ) n When then P is-N (R) 2 )(R 3 ) Or P is a heterocyclyl or heteroaryl group having at least one ring N.
In embodiments of formula I, R P Comprises the following steps:
–N(R 2 )(R 3 )；
–(M)-N(R 2 )(R 3 ) Wherein M is an optionally substituted linking group- (CH) 2 ) n -or-N (R) (CH) 2 ) n -, wherein each n is independently an integer of 1 to 6 inclusive, R is hydrogen or an optionally substituted alkyl group having 1 to 3 carbon atoms;
–(M)-N(R 2 )(R 3 ) M is an optionally substituted linking group- (CH) 2 ) n -wherein each n is independently an integer from 1 to 6 inclusive, R is hydrogen or optionally substituted alkyl having from 1 to 3 carbon atoms;
–(M)-N(R 2 )(R 3 ) M is optionally substituted orLinking group- (CH) 2 ) n -wherein each n is independently an integer from 1 to 6 inclusive, R is hydrogen or optionally substituted alkyl having 1 to 3 carbon atoms, wherein the optional substitution is with one or more halogens or one or more C1-C3 alkyl groups;
–(M)-N(R 2 )(R 3 ) M is optionally substituted-N (R) (CH) 2 ) n -wherein each n is independently an integer from 1 to 6 inclusive, R is hydrogen or optionally substituted alkyl having from 1 to 3 carbon atoms;
–(M)-N(R 2 )(R 3 ) M is optionally substituted-N (R) (CH) 2 ) n -, wherein each n is independently an integer of 1 to 6 inclusive, R is hydrogen or optionally substituted alkyl having 1 to 3 carbon atoms, wherein the optional substitution is with one or more halogens or one or more C1-C3 alkyl groups;
–(M)-N(R 2 )(R 3 ) Wherein M is an optionally substituted linking group- (CH) 2 ) n N is 1, 2 or 3;
–(M)-N(R 2 )(R 3 ) Wherein M is an optionally substituted linking group-N (R) (CH) 2 ) n -n is 1, 2 or 3;
–(M)-N(R 2 )(R 3 ) Wherein M is- (CH) 2 ) n N is 1, 2 or 3;
–(M)-N(R 2 )(R 3 ) Wherein M is-N (R) (CH) 2 ) n N is 1, 2 or 3;
–(M) x -a P group, wherein P is an aryl or heteroaryl group, wherein x is 0 or 1 to indicate the absence or presence of M, M is an optionally substituted linking group- (CH) 2 ) n -or-N (R) (CH) 2 ) n -, wherein each n is independently an integer of 1 to 6 inclusive, R is H or an optionally substituted alkyl group having 1 to 3 carbon atoms;
a- (M) -P group, wherein P is an aryl or heteroaryl group and M is an optionally substituted linking group- (CH) 2 ) n -or-N (R) (CH) 2 ) n -, wherein each n is independently an integer of 1 to 6 inclusive, R is H or optionallySubstituted alkyl having 1 to 3 carbon atoms;
a- (M) -P group, wherein P is an aryl or heteroaryl group and M is an optionally substituted linking group-N (R) (CH) 2 ) n -wherein each n is independently an integer from 1 to 6 inclusive, R is H or optionally substituted alkyl having from 1 to 3 carbon atoms;
a- (M) -P group, wherein P is an aryl or heteroaryl group and M is an optionally substituted linking group- (CH) 2 ) n -, wherein each n is independently an integer of 1 to 3 inclusive, R is H or an optionally substituted alkyl group having 1 to 3 carbon atoms;
p is optionally substituted phenyl or naphthyl;
p is optionally substituted phenyl or naphthyl, and the optional substitution is with one or more halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkyl;
p is an optionally substituted heteroaryl group having a 5-or 6-membered ring or two fused 5-or 6-membered rings;
p is an optionally substituted heteroaryl group having a 5-or 6-membered ring or two fused 5-or 6-membered rings and having 1 to 3 nitrogen ring members;
R P r in (1) 2 Is hydrogen (i.e., -N (R) 2 )(R 3 ) Is a primary amine group);
R P r in (1) 2 And R 3 Are all radicals other than hydrogen (i.e. -N (R) 2 )(R 3 ) Is a secondary amine group);
R 2 is hydrogen, R 3 Is optionally substituted 3-8 membered cycloalkyl;
R 2 is hydrogen, R 3 Is C1-C3 alkyl substituted by 3-8 membered cycloalkyl;
R 2 is hydrogen, R 3 Is an optionally substituted aryl group having 6 to 12 carbon atoms;
R 2 is hydrogen, R 3 Is an optionally substituted heteroaryl group having 6 to 12 carbon atoms and 1 to 3 heteroatoms (N, O or S);
R 2 is hydrogen, R 3 Is optionally substituted having 6 to 12 carbon atoms and 1 to 3 ring nitrogensThe heteroaryl group of (a);
R 2 and R 3 Together with the N to which they are attached form an optionally substituted 5-10 membered heterocyclic ring which is a saturated, partially unsaturated, or aromatic ring;
R P is- (CH) 2 ) n -N(R 2 )(R 3 ) Wherein n is 1 or 2 2 And R 3 Together with the N to which they are attached form an optionally substituted 5-10 membered heterocyclic ring, which 5-10 membered heterocyclic ring is a saturated ring, a partially unsaturated ring, or an aromatic ring;
R P is-N (R) (CH) 2 ) n -N(R 2 )(R 3 ) Wherein n is 1 or 2, R is hydrogen or methyl, R 2 And R 3 Together with the N to which they are attached form an optionally substituted 5-10 membered heterocyclic ring, the 5-10 membered heterocyclic ring being a saturated ring, a partially unsaturated ring, or an aromatic ring;
R P is-M-N (R) 2 )(R 3 ) And R is 2 And R 3 Together with the N to which they are attached form an optionally substituted 5-10 membered heterocyclic ring, the 5-10 membered heterocyclic ring being a saturated ring, a partially unsaturated ring, or an aromatic ring;
R 2 and R 3 Together with the N to which they are attached form an optionally substituted 5-10 membered heterocyclic ring containing no double bonds;
R P is-N (R) 2 )(R 3 ) And R is 2 And R 3 Together with the N to which they are attached form an optionally substituted 5-10 membered heterocyclic ring containing no double bonds;
R 2 and R 3 Together with the N to which they are attached form an optionally substituted 5-10 membered heterocyclic ring containing one, two or three double bonds;
R P is-N (R) 2 )(R 3 ) And R is 2 And R 3 Together with the N to which they are attached form an optionally substituted 5-10 membered heterocyclic ring containing one, two or three double bonds;
R 2 and R 3 Together with the N to which they are attached form an optionally substituted 5-10 membered heteroaromatic ring; or alternatively
R P is-N (R) 2 )(R 3 ) And R is 2 And R 3 Together with the N to which they are attached form an optionally substituted 5-10 membered heteroaromatic ring.
In a particular embodiment of formula I, R P or-N (R) 2 )(R 3 ) The method comprises the following steps:
r of scheme 2 N 1-R N 31, any one of;
R N 1；
R N 3；
R N 2 or R N 4；
R N 5 or R N 6；
R N 7 or R N 8；
R N 9；
R N 10；
R N 11；
R N 12；
R N 13；
R N 14；
R N 15；
R N 16；
R N 17 or R N 18；
R N 19 or R N 20；
R N 21；
R N 22；
R N 23 or R N 24
R N 25；
R N 26-R N 29；
R N 30；
R N 31；
R N 1、R N 2、R N 3、R N 4、R N 11、R N 13 or R N 14; or
Unsubstituted R N 1-R N 31。
In embodiments of formula I, R H Comprises the following steps:
optionally substituted phenyl;
unsubstituted phenyl;
optionally substituted naphthyl;
an unsubstituted naphthyl group;
optionally substituted naphthalen-2-yl;
optionally substituted naphthalen-1-yl;
naphthalen-2-yl;
naphthalen-1-yl;
optionally substituted thienyl;
halo-substituted thienyl;
bromo-substituted thienyl;
optionally substituted thiophen-2-yl;
halo-substituted thiophen-2-yl;
bromo-substituted thiophen-2-yl;
4-halothiophen-2-yl;
4-bromothien-2-yl;
optionally substituted furyl;
optionally substituted furan-2-yl;
optionally substituted indolyl;
an unsubstituted indolyl group;
indol-3-yl;
indol-2-yl;
indol-1-yl;
optionally substituted pyridopyrrolyl;
optionally substituted pyridopyrrol-2-yl;
optionally substituted benzimidazolyl;
Figure BDA0003960268060000351
in a particular embodiment, R H Is optionally substituted with one or more halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C1-C3 fluoroalkyl, C4-C7 cycloalkylalkyl, OH, amino, C1-C6 acyl, -COOR E 、-OCOR E 、-CONR E R F 、-OCONR E R D 、-NR E COR F 、-SR E 、-SOR E 、-SO 2 R E and-SO 2 NR E R F Is substituted in which R E And R e As defined above, in particular hydrogen, C1-C3 alkyl, phenyl or benzyl. More specifically, R H Is optionally substituted with one or more halogens (especially Br or Cl), C1-C3 alkyl, C1-C3 alkoxy, C1-C3 fluoroalkyl (especially CF) 3 -) is substituted.
In embodiments, R H Having the formula:
Figure BDA0003960268060000352
wherein the content of the first and second substances,
X 11 is CH, CR T Or N; r is T Is optionally R as described above H Ring-substituted, R and R' are independently hydrogen, a C1-C6 alkyl group, a C4-C10 cycloalkylalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, each of which is optionally substituted. In a particular embodiment, R T Is hydrogen, or is substituted with one or more halogen, OH, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 alkyl substituted with C3-C6 cycloalkyl; r' is hydrogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 alkyl substituted with C3-C6 cycloalkyl; r is hydrogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 alkyl substituted with C3-C6 cycloalkyl.
In embodiments, R H Having the formula:
Figure BDA0003960268060000361
wherein, the first and the second end of the pipe are connected with each other,
X 11 is CH, CR T Or N; x 10 Is CH, CR T Or N; r T Is R as described above H Optionally substituted on the ring, R and R' are independently hydrogen, a C1-C6 alkyl group, a C4-C10 cycloalkylalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, each of which is optionally substitutedIs substituted. In a particular embodiment, R T Is hydrogen, or is substituted with one or more halogen, OH, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 alkyl substituted with C3-C6 cycloalkyl; r' is hydrogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 alkyl substituted with C3-C6 cycloalkyl; r is hydrogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 alkyl substituted with C3-C6 cycloalkyl.
In embodiments, R H The following formula selected from scheme 3:
r12-3, R12-4, R12-5, R12-7, R12-8, R12-10, R12-23, R12-25, R12-27, R12-29, or R12-31; or
R12-12, R12-13, R2-145, R12-15, R12-16, R12-17, R12-18, R12-19, R12-20, R12-21, or R12-22, wherein p is 0; or
R12-33, R12-34, R12-35, R12-36, R12-37, R12-38, R12-39, R12-40, R12-41, R12-42, wherein p is 0; or
R12-70 or R12-71, wherein p is 0.
In embodiments, R H A 5-membered heterocyclic group selected from the following general formulae:
Figure BDA0003960268060000371
wherein:
t, U, V and W are selected from O, S, C (R '), C (R') -/, C (R '), C-/, N (R'), or N-/;
wherein the group contains one or two double bonds, depending on the choice of T, U, V and W;
wherein R is H The group is bonded to- (L) in the compound of formula I through C-/, C (R' -/, or N-/bond 2 ) A y-Z-moiety; and
wherein R' represents an optional substitution on N or C.
More specifically, R H A 5 membered heterocyclic group selected from the following formulae:
Figure BDA0003960268060000372
wherein the content of the first and second substances,
t is C (R'), C-/, or N; or
U is O, S, C (R '), C (R ') -/, N (R '), or N-/;
v is CR', C-/, or N, and
w is CR', C-/, N, where R is H The group is bonded to- (L) in the compound of formula I through C-/, C (R' -/, or N-/bond 2 ) The moiety y-Z-is,
wherein R is H The group is bonded to- (L) in the compound of formula I through C-/, C (R' -/, or N-/bond 2 ) y-Z-moiety, and
wherein R' represents an optional substitution on N or C. The symbol "-/" denotes a divalent bond through which the heterocyclic group is bonded in the compounds herein, e.g., C-/denotes a divalent bond through which the heterocyclic group from a ring carbon is bonded in the compounds herein.
In embodiments, R H Is a fused ring heterocyclic group of the formula:
Figure BDA0003960268060000381
wherein, the first and the second end of the pipe are connected with each other,
u, V and W are selected from O, S, N, C (R '), C (R') -/, C (R '), C-/, N (R'), or N-/;
t ', U ', V ' and W are selected from C (R '), C-/, N (R '), or N-/;
wherein R is H The group is bonded to- (L) in the compound of formula I through C-/, C (R' -/, or N-/group 2 ) A y-Z-moiety;
wherein the group comprises a bond depending on the choice of U, V and W; and
wherein R' represents an optional substitution on N or C.
More specifically, R H Is a fused heterocyclic group of the formula:
Figure BDA0003960268060000382
wherein the content of the first and second substances,
u and V are selected from N, C (R'), or C-/,/;
w is selected from O, S, C (R '), C (R ') -/, N (R '), or N-/;
t ', U', V 'and W' are selected from C (R '), C-/, N (R'), or N-/;
wherein R is H The group is bonded to- (L) in the compounds of formula I through C-/, C (R' -/, or N-/in the ring shown 2 ) A y-Z-moiety; and
wherein R' represents an optional substitution on N or C.
Each R' is independently selected from hydrogen, halogen, nitro, cyano, amino, mono-or di-C1-C3 alkyl substituted amino, C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C1-C3 haloalkyl, C6-C12 aryl, C5-C12 heteroaryl, C3-C12 heterocyclyl. C1-C3 alkoxy, C1-C6 acyl, -COOR E 、-OCOR E 、-CONR E R F 、-OCONR E R D 、-NR E COR F 、-SR E 、-SOR E 、-SO 2 R E and-SO 2 NR E R F Wherein alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy, and acyl are optionally substituted;
wherein each R E And R F Selected from the group consisting of hydrogen, C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C1-C3 haloalkyl, C6-C12 aryl, C5-C12 heteroaryl, C3-C12 heterocyclyl, C1-C3 alkoxy, C1-C6 acyl, amino, each optionally substituted with one or more halogen, nitro, cyano, amino, mono-or di-C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C1-C3 haloalkyl, C6-C12 aryl, C5-C12 heteroaryl, C3-C12 heterocyclyl, C1-C3 alkoxy, and C1-C6 acyl.
In more specific embodiments, R H Selected from any one of the following:
Figure BDA0003960268060000401
wherein:
R T is R as described above H Optionally substituted on the ring, R and R' are independently hydrogen, a C1-C6 alkyl group, a C4-C10 cycloalkylalkyl group, an aryl group, a heterocyclyl group, or a heteroaryl group, each of which is optionally substituted. In a particular embodiment, R T Is hydrogen, or is substituted with one or more halogen, OH, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 alkyl substituted with C3-C6 cycloalkyl; r' is hydrogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 alkyl substituted with C3-C6 cycloalkyl; and R is hydrogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 alkyl substituted with C3-C6 cycloalkyl. In specific embodiments, R and R' are independently hydrogen, C1-C3 alkyl, or C4-C7 cycloalkylalkyl. In a particular embodiment, R T Represents hydrogen or is substituted by a halogen, in particular Br.
In embodiments, R H Is a 6-membered optionally substituted heterocyclyl or heteroaryl group having 1-3 nitrogens in the ring, 1 or 2 oxygens, sulfur or both in the ring, or 1 or 2 nitrogens and one oxygen or sulfur in the ring, wherein the optional substitution is as defined in formula I. The heterocyclic group may be an unsaturated group, a partially unsaturated group or a heteroaryl group.
In embodiments, R H Is an optionally substituted fused heterocyclic or heteroaryl group having two fused 6-membered rings having 1-5 nitrogens in the fused rings, 1-3 oxygens, sulfur or both in the fused rings, or 1-4 nitrogens and 1 or 2 oxygens, sulfur or both in the fused rings, wherein the optional substitution is as defined in formula I. In more particular embodiments, the fused ring has 1, 2,3, or 4 nitrogens in the fused ring. In more specific embodiments, the fused ring has 1 or 2 oxygens or sulfurs in the fused ring. In more specific embodiments, the fused rings have 1 or 2 nitrogens and one oxygen or sulfur in the fused ring. The fused ring heterocyclic group may be an unsaturated group, a partially unsaturated group, or a heteroaryl group.
In a particular embodiment, R H The group is selected from phenyl,
Figure BDA0003960268060000411
Oxazinyl, pyridyl, pyrimidinyl, thinly, pyranyl, thiazinyl, 4H-pyranyl, naphthyl, quinolyl, isoquinolyl, quinoxalyl, quinazolinyl, pteridinyl, purinyl and chromanyl, wherein R is H The group is attached to- (L) in the compounds of formula I at any available ring position 2 ) A y-Z-moiety. In a particular embodiment, R H The group being attached to- (L) in the compounds of formula I at a carbon in the ring 2 ) A y-Z-moiety.
In a particular embodiment of formula I, -Z- (L) 2 )y-R H Is free of-NH-SO 2 -R W A group other than, wherein R W Is mesitylene, 4-methylphenyl, 4-trifluoromethylphenyl, naphthyl, 2,3,4, 5-tetramethylphenyl, 4-methoxyphenyl, 4-tert-butylphenyl, 2, 4-dimethoxyphenyl, 2, 5-dimethoxyphenyl or 4-phenoxyphenyl. In a particular embodiment of formula I, -Z- (L) 2 ) y-is other than-NR X -SO 2 -a moiety other than, wherein R X Is H, hydrogen, methyl acetate, aminoacetyl, 4-carboxylic acid benzyl, 4-isopropylbenzyl, 4-chlorobenzyl or 4-methoxybenzyl. In embodiments of formula I, -Z-is not-NR X -SO 2 -, wherein R X Is H, hydrogen, methyl acetate, aminoacetyl, 4-formic acid benzyl, 4-isopropylbenzyl, 4-chlorobenzyl or 4-methoxybenzyl.
In embodiments of formula I, R H Is not phenyl or optionally substituted phenyl. In embodiments of formula I, R H Is a heterocyclic group substituted by a single halogen, especially Br.
In embodiments of formula I, R P or-N (R) 2 )(R 3 ) Is scheme 2 (R) N 1-R N 31 Optionally substituted amine groups as shown). Exemplary optional substitutions of groups are shown in scheme 2. The R substituents shown may be located at any available ring position. In the section of scheme 2, preferred alkyl groups are C1-C3 alkyl groups and acyl groups include formylPreferably acyl is C1-C6 acyl, more preferably acetyl, hydroxyalkyl is C1-C6 hydroxyalkyl, preferably-CH 2 -CH 2 OH, the preferred alkyl groups for the amine groups are C1-C3 alkyl, for-SO 2 Alkyl, preferred alkyl groups are C1-C3 alkyl, more preferably methyl.
In a particular embodiment of formula I, -N (R) 2 )(R 3 ) Is R N 1. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 3. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 2 or R N 4. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 5 or R N 6. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 7 or R N 8. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 9. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 10. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 11. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 12. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 13. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 14. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 15. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 16. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 17 or R N 18. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 19 or R N 20. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 21. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 22. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 23 or R N 24. In a specific embodiment, -N (R) 2 )(R 3 ) Is R N 25. In an embodiment, -N (R) 2 )(R 3 ) Is R N 1、R N 2、R N 3、R N 4、R N 11、R N 13 or R N 14. In an embodiment, -N (R) 2 )(R 3 ) Is R N 26-R N 29. In an embodiment, -N (R) 2 )(R 3 ) Is R N 30. In an embodiment, -N (R) 2 )(R 3 ) Is R N 31。
In embodiments of formula I, R H Are moieties shown in scheme 3 (R12-1 to R12-69). In embodiments, R H Is R12-35-R12-42. In embodiments, R H Is any one of R12-43-R12-69. In embodiments, R H Is any one of R12-43-R12-45. In embodiments, R H Is any one of R12-46-R12-48. In embodiments, R H Is any one of R12-49-R12-51. In embodiments, R H Is any one of R12-52-R12-54. In embodiments, R H Is any one of R12-55-R12-58. In embodiments, R H Is any one of R12-59-R12-62. In embodiments, R H Is any one of R12-63-R12-66. In embodiments, R H Is any one of R12-67-R12-69. In the section scheme 3, preferred alkyl groups are C-C6 alkyl groups or more preferred C1-C3 alkyl groups, preferred halogens are F, cl and Br, acyl groups include formyl, preferred acyl groups are-CO-C1-C6 alkyl, more preferably acetyl, phenyl is optionally substituted with one or more halogens, alkyl groups or acyl groups.
In particular embodiments, compounds useful in the methods herein include those of formula II:
Figure BDA0003960268060000431
or a salt or solvate thereof, or a solvate thereof,
wherein the variables are as defined in formula I and the dotted line represents a single or double bond. In embodiments, x is 1 and y is 1. In embodiments, both X are nitrogen. In embodiments, R P is-N (R) 2 )(R 3 ). In an embodiment, L 1 And L 2 Is- (CH) 2 ) n-, wherein n is independently 1, 2 or 3. In embodiments, R H Is a heterocyclic group or a heteroaryl group. In embodiments, Y is-N (R) 1 )-、-CON(R 1 ) -, or-N (R) 1 ) CO-. In embodiments, Z is-CON (R ') -or-N (R') CO-. In embodiments, R' is hydrogen, C1-C3 alkyl, or C1-C3 haloalkyl. In embodiments, R' is hydrogen, methyl, or trifluoromethyl. In embodiments, R A Is hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 acyl or C1-C3 haloalkyl. In embodiments, R' is hydrogen, methyl, methoxy, or trifluoromethyl. In embodiments, R 4 And R 5 Selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkyl. In embodiments, R 4 And R 5 Together form a 5-or 6-membered carbocyclic or heterocyclic ring which is saturated, partially unsaturated or heteroaromatic. In embodiments, R H Is any one of RH1-RH 12.
In particular embodiments, compounds useful in the methods herein include those of formula III:
Figure BDA0003960268060000441
or a salt or solvate thereof, or a solvate thereof,
wherein the variables are as defined in formula I and the dotted line represents a single or double bond.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are nitrogen. In embodiments, R P is-N (R) 2 )(R 3 ). In an embodiment, L 2 Is- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, R H Is a heterocyclic group or a heteroaryl group. In embodiments, Y is-N (R) 1 )-、-CON(R 1 ) -or-N (R) 1 ) CO-. In embodiments, Z is-CON (R ') -or-N (R') CO-. In embodiments, R' is hydrogen, C1-C3 alkyl, or C1-C3 haloalkyl. In embodiments, R' is hydrogen, methyl or triA fluoromethyl group. In embodiments, R A Is hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 acyl or C1-C3 haloalkyl. In embodiments, R' is hydrogen, methyl, methoxy, or trifluoromethyl. In embodiments, R 4 And R 5 Selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkyl. In embodiments, R 4 And R 5 Together form a 5-or 6-membered carbocyclic or heterocyclic ring which is saturated, partially unsaturated or heteroaromatic. In embodiments, R H Is any one of RH1-RH 12.
In particular embodiments, compounds useful in the methods herein include those of formula IV:
Figure BDA0003960268060000451
or a salt or solvate thereof;
wherein the variables are as defined in formula I and the dotted line represents a single or double bond.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are nitrogen. In embodiments, R P is-N (R) 2 )(R 3 ). In an embodiment, L 2 Is- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, R H Is a heterocyclic group or a heteroaryl group. In embodiments, R 1 Is hydrogen. In embodiments, R 1 Is hydrogen, methyl or trifluoromethyl. In embodiments, Z is-CON (R ') -or-N (R') CO-. In embodiments, R' is hydrogen, C1-C3 alkyl, or C1-C3 haloalkyl. In embodiments, R' is hydrogen, methyl, or trifluoromethyl. In embodiments, R A Is hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 acyl or C1-C3 haloalkyl. In embodiments, R' is hydrogen, methyl, methoxy, or trifluoromethyl. In embodiments, R 4 And R 5 Selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkyl. In embodiments, R 4 And R 5 Together form a 5-membered orA 6-membered carbocyclic or heterocyclic ring which is saturated, partially unsaturated or heteroaromatic. In embodiments, R H Is any one of RH1-RH 12.
In particular embodiments, compounds useful in the methods herein include those of formula V:
Figure BDA0003960268060000461
or a salt or solvate thereof, or a solvate thereof,
wherein the variables are as defined in formula I and the dotted line represents a single or double bond.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are nitrogen. In embodiments, R P is-N (R) 2 )(R 3 ). In an embodiment, L 2 Is- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, R H Is a heterocyclic group or a heteroaryl group. In embodiments, R 1 Is hydrogen. In embodiments, R 1 Is hydrogen, methyl or trifluoromethyl. In embodiments, rs is hydrogen, C1-C3 alkyl, optionally substituted C1-C3 alkyl, or aryl. In embodiments, R A Is hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 acyl or C1-C3 haloalkyl. In embodiments, R' is hydrogen, methyl, methoxy, or trifluoromethyl. In embodiments, R 4 And R 5 Selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkyl. In embodiments, R 4 And R 5 Together form a 5-or 6-membered carbocyclic or heterocyclic ring which is saturated, partially unsaturated or heteroaromatic. In embodiments, R H Is any one of RH1-RH 12.
In particular embodiments, compounds useful in the methods herein include those of formula VI:
Figure BDA0003960268060000471
or a salt or solvate thereof, or a solvate thereof,
wherein the variables are as defined in formula I and the dotted line represents a single or double bond.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are nitrogen. In embodiments, x is 1 and-N- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, y is 0. In an embodiment, L 2 Is- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, R H Is a heterocyclic group or a heteroaryl group. In embodiments, R 1 Is hydrogen. In embodiments, R 1 Is hydrogen, methyl or trifluoromethyl. In embodiments, rs is hydrogen, C1-C3 alkyl, optionally substituted C1-C3 alkyl, or aryl. In embodiments, R A Is hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 acyl or C1-C3 haloalkyl. In embodiments, R' is hydrogen, methyl, methoxy, or trifluoromethyl. In embodiments, R 4 And R 5 Selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkyl. In embodiments, R 4 And R 5 Together form a 5-or 6-membered carbocyclic or heterocyclic ring which is saturated, partially unsaturated or heteroaromatic. In embodiments, R H Is any one of RH1-RH 12.
In particular embodiments, compounds useful in the methods herein include those of formula VII:
Figure BDA0003960268060000481
or a salt or solvate thereof, or a solvate thereof,
wherein the variables are as defined in formula I and the dotted line represents a single or double bond.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are nitrogen. In embodiments, x is 1 and-N- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, y is 0. In an embodiment, L 2 Is- (CH) 2 ) n-, wherein n is 1, 2 or 3. In an embodiment，R H Is a heterocyclic group or a heteroaryl group. In embodiments, R 1 Is hydrogen. In embodiments, R 1 Is hydrogen, methyl or trifluoromethyl. In embodiments, rs is hydrogen, C1-C3 alkyl, optionally substituted C1-C3 alkyl, or aryl. In embodiments, R A Is hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 acyl or C1-C3 haloalkyl. In embodiments, R' is hydrogen, methyl, methoxy, or trifluoromethyl. In embodiments, R 4 And R 5 Selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkyl. In embodiments, R 4 And R 5 Together form a 5-or 6-membered carbocyclic or heterocyclic ring which is saturated, partially unsaturated or heteroaromatic. In embodiments, R H Is any one of RH1-RH 12.
In particular embodiments, compounds useful in the methods herein include those of formula VIII:
Figure BDA0003960268060000491
or a salt or solvate thereof, or a solvate thereof,
wherein the variables are as defined in formula I, the dotted line represents a single or double bond, R 6 -R 9 Independently selected from hydrogen and R as defined in formula I A A group. R M Represents an optional substitution on a condensed ring, R M Taking R in formula I A The value of (c).
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are nitrogen. In embodiments, x is 1 and-N- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, x is 0. In an embodiment, L 2 Is- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, R 1 Is hydrogen. In embodiments, R 1 Is hydrogen, methyl or trifluoromethyl. In embodiments, R 7 -R 9 Independently selected from hydrogen, C1-C3 alkyl, optionally substituted C1-C3 alkyl, or aryl. In embodiments, R 7 -R 9 Independently selected from hydrogenHalogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 acyl or C1-C3 haloalkyl. In embodiments, R 7 -R 9 Are all hydrogen. In embodiments, R 4 And R 5 Selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkyl. In embodiments, R 4 And R 5 Together form a 5-or 6-membered carbocyclic or heterocyclic ring which is saturated, partially unsaturated or heteroaromatic. In embodiments, R M Is one or more hydrogen, halogen, C1-C3 alkyl group, C4-C7 cycloalkylalkyl group, or C1-C3 haloalkyl group. In embodiments, R M Is one or more of hydrogen, halogen (especially Br), methyl or trifluoromethyl. In embodiments, R M Is hydrogen.
In particular embodiments, compounds useful in the methods herein include those of formula IX:
Figure BDA0003960268060000501
or a salt or solvate thereof, wherein the variables are as defined in formula I, the dotted line represents a single or double bond, R 6 -R 9 Independently selected from hydrogen and R as defined in formula I A Group, R M Represents an optional substitution as defined in formula I.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are nitrogen. In embodiments, x is 1 and M is-N- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, x is 1 and M is- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, x is 0. In an embodiment, L 2 Is- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, R 1 Is hydrogen. In embodiments, R 1 Is hydrogen, methyl or trifluoromethyl. In embodiments, R 7 -R 9 Independently selected from hydrogen, C1-C3 alkyl, optionally substituted C1-C3 alkyl, or aryl. In embodiments, R 7 -R 9 Independently selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 acyl or C1-C3 haloAn alkyl group. In embodiments, R 7 -R 9 Are all hydrogen. In embodiments, R 4 And R 5 Selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkyl. In embodiments, R 4 And R 5 Together form a 5-or 6-membered carbocyclic or heterocyclic ring which is saturated, partially unsaturated or heteroaromatic. In embodiments, R M Is hydrogen, halogen, a C1-C3 alkyl group or a C1-C3 haloalkyl group. In embodiments, R M Is hydrogen, halogen (especially Br), methyl or trifluoromethyl.
In other embodiments, the invention provides compounds of formula XI:
Figure BDA0003960268060000511
or a salt or solvate thereof, or a solvate thereof,
wherein:
each X is independently selected from N or CH, and at least one X is N;
ring a is a carbocyclic or heterocyclic ring having 3 to 10 carbon atoms and optionally 1 to 6 heteroatoms, and which is optionally saturated, unsaturated, or aromatic;
L 1 is an optionally substituted optional 1-3 carbon linking group, wherein x is 0 or 1, meaning that L is absent or present 1 ；
R 1 Selected from the group consisting of hydrogen, alkyl groups, alkenyl groups, cycloalkyl groups, cycloalkenyl groups, heterocyclyl groups, or aryl groups, each of which is optionally substituted;
R 2 and R 3 Independently selected from hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl or heterocyclyl, each of which is optionally substituted, or
R 2 And R 3 Together form an optionally substituted 5-8 membered heterocyclic ring which is a saturated ring, a partially unsaturated ring, or an aromatic ring;
R 4 and R 5 Independently selected from hydrogen, halogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, arylOr a heterocyclic radical, each radical being optionally substituted, or
R 4 And R 5 Together form an optionally substituted 5-or 6-membered ring, which optionally contains one or two double bonds, or is aromatic and optionally contains 1 to 3 heteroatoms;
wherein the dotted line is a single or double bond, depending on R 4 And R 5 Selecting; and
R A represents hydrogen or 1 to 10 substituents on the ring shown, wherein R A The substituents are independently selected from hydrogen, halogen, hydroxy, cyano, nitro, amino, mono-OR di-substituted amino, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, -OR 15 、-COR 15 、-COOR 15 、-OCOR 15 、-CO-NR 16 R 17 、-OCONR 16 R 17 、-NR 16 -CO-R 15 、-SR 15 、-SOR 15 、-SO 2 R 15 、-SO 2 -NR 16 R 17 、R10、–NH-CO-(L 2 ) y -R 12 or-NH-CO- (L) 2 ) y -R 12 Wherein L is 2 Is an optional linking group of 1 to 6 carbon atoms, wherein the linking group is optionally substituted, and wherein one or both carbons of the linking group is optionally substituted with O or S, wherein y is 0 or 1, meaning that L is absent or present 2 ；
R 10 Selected from alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl or aryl, each of which is optionally substituted with one or more halogen, alkyl, alkenyl, haloalkyl, alkoxy, aryl, heteroaryl, heterocyclyl, aryl-substituted alkyl, or heterocyclyl-substituted alkyl;
R 12 selected from cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl or aryl, each of which is optionally substituted, or R 12 Is C1-C3 alkyl substituted with cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl, each of which is optionally substituted, and wherein the optional substitution is one or more of halogen, alkyl, or heteroaryl alkenyl, haloalkyl, alkoxy, aryl,Heteroaryl or heterocyclyl;
each R 15 Independently selected from hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl or heterocyclyl, arylalkyl (alkyl substituted with aryl) and heterocyclylalkyl, cycloalkylalkyl, cycloalkenylalkyl, each of which is optionally substituted; and
each R 16 And R 17 Independently selected from hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl or heterocyclyl, arylalkyl (alkyl substituted with aryl) and heterocyclylalkyl, cycloalkylalkyl, cycloalkenylalkyl, each of which is optionally substituted;
wherein optional substitution comprises substitution with one or more of halogen, nitro, cyano, amino, mono-or di-C1-C3 alkyl substituted amino, C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C6-C12 aryl, and C6-C12 heterocyclyl.
In an embodiment, the compound has formula XII:
Figure BDA0003960268060000531
or a salt or solvate thereof, wherein the variables are as defined for formula XI.
In embodiments, the compound has formula XIII:
Figure BDA0003960268060000532
or a salt or solvate thereof, wherein the variables are as defined in formula XI, wherein:
each Y is independently selected from N or CH;
R B represents hydrogen or 1 to 10 substituents on the ring shown, wherein R A The substituents are independently selected from hydrogen, halogen, hydroxy, cyano, nitro, amino, mono-OR di-substituted amino, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, -OR 15 、-COR 15 、-COOR 15 、-OCOR 15 、-CO-NR 16 R 17 、-OCONR 16 R 17 、-NR 16 -CO-R 15 、-SR 15 、-SOR 15 、-SO 2 R 15 、-SO 2 -NR 16 R 17 Or (L) 2 ) y -R 10 Wherein L is 2 Is an optional linking group of 1 to 6 carbon atoms, which linking group is optionally substituted, and wherein y is 0 or 1, meaning that L is absent or present 2 。
In embodiments, the compound has formula XIV or XV:
Figure BDA0003960268060000541
or a salt or solvate thereof, or a solvate thereof,
wherein the variables are as defined in formula XI, XII or XIII.
In embodiments, the compound has formula XVI or XVII:
Figure BDA0003960268060000542
or a salt or solvate thereof
Wherein the variables are as defined in formula XI or XV, and
R 11 and R 12 Independently selected from hydrogen, halogen, alkyl groups, alkenyl groups, cycloalkyl groups, cycloalkenyl groups, or heterocyclyl groups, each of which is optionally substituted.
In embodiments, the compound has formula XVIII:
Figure BDA0003960268060000551
or a salt or solvate thereof, or a solvate thereof,
wherein:
R 1 selected from the group consisting of hydrogen, alkyl groups, alkenyl groups, cycloalkyl groups, cycloalkenyl groups, heterocyclic groupsA group of radicals or aryl radicals, each of which is optionally substituted (substitution is required to be defined);
R 2 and R 3 Together form an optionally substituted 5-or 6-membered heterocyclic ring which may contain one or two double bonds or be aromatic;
R 4 and R 5 Independently selected from hydrogen, halogen, alkyl groups, alkenyl groups, cycloalkyl groups, cycloalkenyl groups or heterocyclyl groups, each of which is optionally substituted, or
R 4 And R 5 Together form an optionally substituted 5-or 6-membered heterocyclic ring which may contain one or two double bonds or be aromatic;
the dotted line is a single or double bond, depending on R 4 And R 5 Selecting;
R 6 -R 9 independently selected from hydrogen, halogen, an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, or a heterocyclyl group, each of which is optionally substituted;
l is an optional linking group of 1 to 6 atoms, wherein x is 1 or 0, indicating the presence or absence of an L group; and
R 10 selected from an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, a heterocyclyl group, or an aryl group, each of which is optionally substituted.
For example, L is a 2-6 atom linking group; (e.g., -CH) 2 -O-、-CH 2 -CH 2 -O-、-O-CH 2 -、-O-CH 2 -CH 2 -、-CO-NH-、--NH-CO-、-CH 2 -CO-NH-、-CH 2 -CH 2 -CO-NH-)。
In embodiments, the compound has formula XIX:
Figure BDA0003960268060000561
or a salt (or solvate) thereof,
wherein:
R 1 -R 9 as defined above; the dotted line represents a single or double bond, depending on R 4 And R 5 Selecting;
y is 0 or an integer from 1 to 3 inclusive; and
R 10 selected from an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, a heterocyclyl group, or an aryl group, each of which is optionally substituted.
In embodiments, the CDH1L inhibitor is a compound of formula XX:
Figure BDA0003960268060000571
or a salt or solvate thereof, wherein R 1 -R 9 Represents hydrogen or an optional substituent, R 10 Is a moiety believed to be related to efficacy; and R is N Are moieties that are believed to be related to physicochemical properties such as solubility. In embodiments, R 5 Are substituents other than hydrogen, which are believed to be associated with metabolic stability. In a particular embodiment, R 5 Is halogen, in particular F or Cl, a C1-C3 alkyl radical, in particular a methyl radical. In embodiments, R 4 Are substituents other than hydrogen, in particular C1-C3 alkyl groups, more particularly methyl groups. In a particular embodiment, R 5 Is F, R 4 Is a methyl group. In embodiments, R 6 -R 9 Selected from hydrogen, C1-C3-alkyl, halogen, hydroxy, C1-C3-alkoxy, formyl, or C 1 -C 3 An acyl group. In embodiments, R 6 -R 9 One or both of which are moieties other than hydrogen. In embodiments, R 6 -R 9 Is halogen, in particular fluorine. In a particular embodiment, R 6 -R 9 All are hydrogen. In embodiments, R N Is an amino moiety-N (R) 2 )(R 3 ). In a particular embodiment, R N Is an optionally substituted heterocyclic group having a 5-7 membered ring, optionally containing a second heteroatom (N, S or O). In embodiments, R N Is optionally substitutedPyrrolidin-1-yl, piperidin-1-yl, aza-1-yl, piperazin-1-yl, or morpholinyl. R is N Substituted by a substituent selected from C1-C3 alkyl, formyl, C1-C3 acyl (especially acetyl), hydroxy, halogen (especially F or Cl), hydroxyC 1-C3 alkyl (especially-CH 2-OH). In embodiments, R N Is unsubstituted pyrrolidin-1-yl, piperidin-1-yl, aza-1-yl, piperazin-1-yl, or morpholinyl.
In embodiments, R 10 is-NRy-CO- (L) 2 )y-R 12 or-CO-NRy- (L) 2 )y-R 12 Wherein y is 0 or 1, denotes the absence or presence of L 2 Which is an optional linking group of 1 to 6 carbon atoms, which linking group is optionally substituted, and wherein one or two carbons of the linking group is optionally substituted with O, NH, NRy or S, wherein Ry is hydrogen or alkyl of 1 to 3 carbons, and R 12 Is an aryl group, a cycloalkyl group, a heterocyclic group, or a heteroaryl group, each of which is optionally substituted. In embodiments, y is 1.L is 2 Is- (CH) 2 ) p-, wherein p is 0-3. In embodiments, R 12 Is thiophen-2-yl, thiophen-3-yl, furan-2-yl, furan-3-yl, pyrrol-2-yl, pyrrol-3-yl,
Figure BDA0003960268060000581
An oxazol-4-yl group,
Figure BDA0003960268060000582
An oxazol-5-yl group,
Figure BDA0003960268060000583
Oxazol-2-yl, indol-3-yl, benzofuran-2-yl, benzofuran-3-yl, benzo [ b ]]Thiophen-2-yl, benzo [ b ]]Thien-3-yl, isobenzofuran-1-yl, isoindol-1-yl, or benzo [ c]Thiophen-1-yl. In embodiments, R 1 Is hydrogen or methyl. In embodiments, R 12 Is thiophen-2-yl, furan-2-yl, pyrrol-2-yl,
Figure BDA0003960268060000584
Azol-4-ylIndol-2-yl, benzofuran-2-yl or benzo [ b ]]Thiophen-2-yl. In embodiments, R 12 Is thiophen-2-yl or indol-2-yl. In embodiments, R 1 Is hydrogen or methyl.
In a more general embodiment of formula XX:
R 1 selected from the group consisting of hydrogen, an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, a heterocyclyl group, or an aryl group, each of which is optionally substituted;
R N is-NR 2 R 3 ，R 2 And R 3 Independently selected from hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl or heterocyclyl, each of which is optionally substituted, or R 2 And R 3 Together form an optionally substituted 5-8 membered heterocyclic ring which is a saturated, partially unsaturated or aromatic ring;
R 4 -R 9 independently selected from hydrogen, halogen, hydroxy, cyano, nitro, amino, mono-OR dialkyl substituted amino, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted aryl, optionally substituted heterocyclyl, -OR 15 、-COR 15 、-COOR 15 、-OCOR 15 、-CO-NR 15 R 16 、-OCONR 15 R 16 、-NR 15 -CO-R 16 、-SR 15 、-SOR 15 、-SO 2 R 15 and-SO 2 -NR 15 R 16 ；
R 10 is-NRy-CO- (L) 2 )y-R 12 ,-CO-NRy-(L 2 )y-R 12 Wherein L is 2 Is an optional linking group of 1 to 6 carbon atoms, wherein the linking group is optionally substituted, and wherein one or both of the carbons of the linking group is optionally replaced by O or S, wherein y is 0 or 1, indicating the absence or presence of L 2 ；
R 12 Selected from cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl or aryl, each of which is optionally substituted, or R 12 Is substituted by a cycloalkyl group,Cycloalkenyl, heterocyclyl, heteroaryl, or aryl substituted C1-C3 alkyl, each of which is optionally substituted, and wherein the optional substitution is one or more halogen, alkyl, alkenyl, haloalkyl, alkoxy, aryl, heteroaryl, or heterocyclyl;
each R 15 And R 16 Independently selected from hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl or heterocyclyl, arylalkyl and heterocyclylalkyl, cycloalkylalkyl, cycloalkenylalkyl, each of which is optionally substituted; and
wherein the optional substitution comprises amino substituted with one OR more halogen, nitro, cyano, amino, mono-OR di-C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C6-C12 aryl, C6-C12 heterocyclyl, -OR 17 、-COR 17 、-COOR 17 、-OCOR 17 、-CO-NR 17 R 18 、-OCONR 17 R 18 、-NR 17 -CO-R 18 、-SR 17 、-SOR 17 、-SO 2 R 17 and-SO 2 -NR 17 R 18 Wherein R is 17 And R 18 Independently of each other is hydrogen or C 1 -C 6 An alkyl group.
In embodiments of formula XX, R N Is selected from R N 1-R N 31 (scheme 2) an optionally substituted cyclic amine group of any one of (1). Exemplary optional substitutions of groups are shown in scheme 2. The R substituents shown may be located at any available ring position. In the section of scheme 2, preferred alkyl groups are C1-C3 alkyl groups, acyl groups include formyl, preferred acyl groups are C1-C6 acyl groups, more preferably acetyl, hydroxyalkyl groups are C1-C6 hydroxyalkyl groups, preferably-CH 2 -CH 2 -OH, the preferred alkyl groups for the amine group are C1-C3 alkyl, for-SO 2 Alkyl, preferred alkyl groups are C1-C3 alkyl, more preferably methyl.
In a particular embodiment of formula XX, R N Is R N 1. In a particular embodiment, R N Is R N 3. In a particular embodiment, R N Is R N 2 or R N 4. In a particular embodiment, R N Is R N 5 or R N 6. In a particular embodiment, R N Is R N 7 or R N 8. In a particular embodiment, R N Is R N 9. In a particular embodiment, R N Is R N 10. In a particular embodiment, R N Is R N 11. In a particular embodiment, R N Is R N 12. In a particular embodiment, R N Is R N 13. In a particular embodiment, R N Is R N 14. In a particular embodiment, R N Is R N 15. In a particular embodiment, R N Is R N 16. In a particular embodiment, R N Is R N 17 or R N 18. In a particular embodiment, R N Is R N 19 or R N 20. In a particular embodiment, R N Is R N 21. In a particular embodiment, R N Is R N 22. In a particular embodiment, R N Is R N 23 or R N 24. In a particular embodiment, R N Is R N 25. In embodiments, R N Is R N 1、R N 2、R N 3、R N 4、R N 11、R N 13. Or R N 14. In embodiments, R N Is R N 26-R N 29. In embodiments, R N Is R N 30. In embodiments, R N Is R N 31。
In embodiments of formula XX, R 12 Is optionally substituted thienyl, thienylmethyl, furyl, furylmethyl, indolyl or methylindolyl. In embodiments, R 12 Is a moiety shown in scheme 3 (R12-1 to R12-69). In the section scheme 3, preferred alkyl groups are C-C6 alkyl groups or more preferred C1-C3 alkyl groups, preferred halogens are F, cl and Br, acyl groups include formyl, preferred acyl groups are-CO-C1-C6 alkyl, more preferably acetyl, phenyl is optionally substituted with one or more halogens, alkyl groups or acyl groups. In embodiments, R 12 Is methyl, ethyl or propyl substituted with the moieties shown in R12-1 to R12-22 of scheme 3. In embodiments, R 12 Is R12-1.In embodiments, R 12 Is R12-2. In embodiments, R 12 Is R12-3. In embodiments, R 12 Is R12-4. In embodiments, R 12 Is R12-5. In embodiments, R 12 Is R12-6. In embodiments, R 12 Is R12-7. In embodiments, R 12 Is R12-8. In embodiments, R 12 Is R12-9. In embodiments, R 12 Is R12-10. In embodiments, R 12 Is R12-11. In embodiments, R 12 Is R12-12. In embodiments, R 12 Is R12-13. In embodiments, R 12 Is R12-14. In embodiments, R 12 Is R12-15. In embodiments, R 12 Is R12-16. In embodiments, R 12 Is R12-17. In embodiments, R 12 R12-18 in embodiments, R 12 Is R12-19. In embodiments, R 12 Is R12-20. In embodiments, R 12 Is R12-21. In embodiments, R 12 Is R12-22. In embodiments, R 12 Is R12-23-R12-26. In embodiments, R 12 Is R12-27-R12-30. In embodiments, R 12 Is R12-31-R12-34. In embodiments, R 12 Is R12-35-R12-42. In embodiments, R12 is any one of R12-43-R12-69. In embodiments, R12 is a methyl group, an ethyl group, or a propyl group substituted with the moieties shown in R12-43-R12-69 of scheme 3. In embodiments, R12 is any one of R12-43-R12-45. In embodiments, R 12 Is methyl, ethyl or propyl substituted with the moieties shown in R12-43-R12-45 of scheme 3. In embodiments, R12 is any one of R12-46-R12-48. In embodiments, R 12 Is methyl, ethyl or propyl substituted with the moieties shown in R12-46-R12-48 of scheme 3. In embodiments, R12 is any one of R12-49-R12-51. In embodiments, R 12 Is methyl, ethyl or propyl substituted with the moieties shown in R12-49-R12-51 of scheme 3. In embodiments, R12 is any one of R12-52-R12-54. In embodiments, R 12 Is substituted with moieties shown in R12-52-R12-54 of scheme 3Methyl, ethyl or propyl. In embodiments, R12 is any one of R12-55-R12-58. In embodiments, R 12 Is methyl, ethyl or propyl substituted with the moieties shown in R12-55-R12-58 of scheme 3. In embodiments, R12 is any one of R12-59-R12-62. In embodiments, R 12 Is methyl, ethyl or propyl substituted with the moieties shown in R12-59-R12-62 of scheme 3. In embodiments, R12 is any one of R12-63-R12-66. In embodiments, R 12 Is methyl, ethyl or propyl substituted with the moieties shown in R12-63-R12-66 of scheme 3. In embodiments, R12 is any one of R12-67-R12-69. In embodiments, R 12 Is methyl, ethyl or propyl substituted with the moieties shown in R12-67-R12-69 of scheme 3. In embodiments, R12 is a moiety as depicted in R12-70 or R12-71 of scheme 3.
In embodiments of formula XX herein, R N Is selected from R N 1-R N 25 (scheme 2) an optionally substituted cyclic amine group, R 12 Is thienyl, thienylmethyl, furyl, furylmethyl, indolyl or methylindolyl. In embodiments of formula XX, R N Is R N 1、R N 2、R N 3、R N 4、R N 11、R N 13、R N 14 or R N 25，R 12 Is thienyl, thienylmethyl, furyl, furylmethyl, indolyl or methylindolyl. In embodiments, R 10 is-NHCOR 12 . In embodiments, R 10 is-CONHR 12 . In embodiments of formula XX herein, R 10 is-CO-NH-R 12 ，R N Is R N 1-R N 25, R is any one of 12 Is any one of R12-1 to R12-22. In embodiments of formula XX herein, R 10 is-CO-NH-R 12 ，R N Is R N 1-R N 25, R is any one of 12 Is any one of R12-1 to R12-69.
In embodiments, the compound has formula XXX:
Figure BDA0003960268060000611
or a salt (or solvate) thereof,
wherein, the first and the second end of the pipe are connected with each other,
R 1 selected from hydrogen, alkyl groups, alkenyl groups, cycloalkyl groups, cycloalkenyl groups, heterocyclyl groups or aryl groups, each of which is optionally substituted (substitution is required to be defined);
R 2 and R 3 Together form an optionally substituted 5-or 6-membered heterocyclic ring which may contain one or two double bonds or be aromatic;
R 6 -R 9 independently selected from hydrogen, halogen, an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, or a heterocyclyl group, each of which is optionally substituted;
y is 0 or an integer from 1 to 3 inclusive;
R 10 selected from an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, a heterocyclyl group, or an aryl group, each of which is optionally substituted; and
R 11 and R 12 Independently selected from hydrogen, halogen, alkyl groups, alkenyl groups, cycloalkyl groups, cycloalkenyl groups, or heterocyclyl groups, each of which is optionally substituted. In embodiments, R 10 Is any one of RH1-RH 12.
In particular embodiments, compounds useful in the methods herein include compounds of formula XXXI:
Figure BDA0003960268060000621
or a salt or solvate thereof; wherein the variables are as defined in formula I, R 6 -R 9 Independently selected from hydrogen, and R as defined in formula I A Group, R M Represents optional substitution on a fused ring, R M Taking the value of RA in formula I, W 1 Is N or CH.
In embodiments, y is 1. In embodiments, y is 0. In embodiments, both X are nitrogen. In embodiments, x is 1, and M is-N- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, x is 1, and M is- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, x is 0. In an embodiment, L 2 Is- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, R 1 Is hydrogen. In embodiments, R 1 Is hydrogen, methyl or trifluoromethyl. In embodiments, R 7 -R 9 Independently selected from hydrogen, C1-C3 alkyl, optionally substituted C1-C3 alkyl, or aryl. In embodiments, R 7 -R 9 Independently selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 acyl or C1-C3 haloalkyl. In embodiments, R 7 -R 9 Are all hydrogen. In embodiments, R 4 And R 5 Selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkyl. In embodiments, R 4 And R 5 Together form a 5-or 6-membered carbocyclic or heterocyclic ring which is saturated, partially unsaturated or heteroaromatic. In embodiments, R M Is one or more hydrogen, halogen, C1-C3 alkyl groups or C1-C3 haloalkyl groups. In embodiments, R M Is one or more of hydrogen, halogen (especially Br), methyl or trifluoromethyl. In embodiments, R M Is hydrogen.
In embodiments, compounds useful in the methods of the invention include compounds of formula XXXII:
Figure BDA0003960268060000631
or a salt or solvate thereof, or a solvate thereof,
wherein the variables are as defined in formula I, R B Represents optionally substituted as defined in formula I, R 6 -R 9 Is hydrogen, or R in formula I A The value of (c).
In embodiments, y is 1. In embodiments, y is 0. In the embodiment(s) of the present invention,both X are nitrogen. In embodiments, x is 1, M is-N- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, x is 1, M is- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, x is 0. In an embodiment, L 2 Is- (CH) 2 ) n-, wherein n is 1, 2 or 3. In embodiments, R 1 Is hydrogen. In embodiments, R 1 Is hydrogen, methyl or trifluoromethyl. In embodiments, R 6 -R 9 Independently selected from hydrogen, C1-C3 alkyl, optionally substituted C1-C3 alkyl or aryl. In embodiments, R 6 -R 9 Independently selected from hydrogen, halogen C1-C3 alkyl, C1-C3 alkoxy, C1-C3 acyl or C1-C3 haloalkyl. In embodiments, R 7 -R 9 Are all hydrogen. In embodiments, R 4 And R 5 Selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkyl. In embodiments, R B Is one or more hydrogen, halogen, C1-C3 alkyl groups or C1-C3 haloalkyl groups. In embodiments, R B Is one or more of hydrogen, halogen (especially Br), methyl or trifluoromethyl. In embodiments, R B Is hydrogen. In embodiments, R H Is a heterocyclic group or a heteroaryl group. In embodiments, R H Is optionally substituted naphthyl, thienyl, indolyl or pyridopyrrolyl.
Compounds of formula XXXV-XLII may be used in the methods herein:
Figure BDA0003960268060000651
Figure BDA0003960268060000661
Figure BDA0003960268060000671
Figure BDA0003960268060000681
wherein the variables are as defined above in formulas I-XIX, X 5 Is a halogen, including F, cl, and Br, in particular embodiments Br. In a particular embodiment of formula XXXV-XLII, y is 0. In a particular embodiment of formula XXXV-XLII, y is 1,L 2 Is- (CH) 2 ) n-, n is 1, 2 or 3. In a particular embodiment of formula XXXV-XLII, ring A is a phenyl ring, wherein R is A Is hydrogen. In a particular embodiment, R P Is selected from R N -1 to R N -31, or a pharmaceutically acceptable salt thereof. In a specific embodiment, the B-ring of formula XLII is that of formula RBI as shown in scheme 4. In a more specific embodiment, ring B of formula XLII is ring B of RB2-RB5 of scheme 4.
The present invention provides salts, particularly pharmaceutically acceptable salts, of each of the compounds of any of formulas I-IX, XI-XIX, XXX-XXXII, XXXV-XLII, and formula XX below. The present invention provides solvates thereof and salts thereof, particularly pharmaceutically acceptable solvates of each of the compounds of any of the following formulas I-XIX, XXX-XXXII, XXXV-XLII and XX, and salts thereof. Preferred solvates are hydrates. The present invention provides a pharmaceutical composition comprising any compound of any of the formulae herein.
An aliphatic compound is an organic compound that contains carbon and hydrogen linked together in a straight, branched, or non-aromatic ring, and may contain single, double, or triple bonds. Aliphatic compounds are different from aromatic compounds. The term aliphatic group refers herein to a monovalent group that is not aromatic and contains carbon and hydrogen. Aliphatic groups include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl groups, as well as aliphatic groups substituted with other aliphatic groups, e.g., alkenyl groups substituted with alkyl groups, alkyl groups substituted with cycloalkyl groups.
The term alkyl or alkyl group refers to a monovalent group of a straight or branched chain saturated hydrocarbon. Alkyl groups include straight chain and branched alkyl groups. Unless otherwise specified, alkyl groups have from 1 to 8 carbon atoms (C1-C8 alkyl groups), preferably those containing from 1 to 6 carbon atoms (C1-C6 alkyl groups), more preferably those containing from 1 to 3 carbon atoms (C1-C3 alkyl groups). The alkyl group is optionally substituted with one or more non-hydrogen substituents as described herein. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, various branched pentyl, n-hexyl, various branched hexyl, all of which are optionally substituted, wherein substitution is defined elsewhere herein. Substituted alkyl groups include fully halogenated or semi-halogenated alkyl groups, such as alkyl groups in which one or more hydrogens are replaced with one or more fluorine, chlorine, bromine and/or iodine atoms. Substituted alkyl groups include perfluorinated or semi-fluorinated alkyl groups.
Cycloalkyl groups are alkyl groups having at least one 3 or higher carbon ring. Cycloalkyl groups include those having 3-12 membered carbocyclic rings. Cycloalkyl groups include those having 3 to 20 carbon atoms and those having 3 to 12 carbon atoms. More specifically, the cycloalkyl group may have at least one 3-to 10-membered carbocyclic ring. Cycloalkyl groups may have a single carbocyclic ring having from 3 to 10 carbons in the ring. Cycloalkyl groups are optionally substituted. The cycloalkyl group may be bicyclic having 6-12 carbons. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. Bicycloalkyl groups include fused bicyclic groups and bridged bicyclic groups. Exemplary bicycloalkyl groups include bicyclo [2.2.2] octyl, bicyclo [4.4.0] decyl (decalinyl), and bicyclo [2.2.2] heptyl (norbornyl).
A cycloalkylalkyl group is an alkyl group as described herein substituted with a cycloalkyl group as described herein. More specifically, the alkyl group is a methyl group or an ethyl group, and the cycloalkyl group is a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, or a cyclohexyl group. The cycloalkyl group is optionally substituted. In particular embodiments, optional substitution includes substitution with one or more of halogen, alkyl having 1 to 3 carbon atoms, alkoxy having 1 to 3 carbon atoms, hydroxyl, and nitro.
The term alkylene refers to a divalent group of a straight or branched chain saturated hydrocarbon. Unless otherwise specified, the alkylene group may have 1 to 12 carbon atoms. Alkylene groups include those having 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms. Linking groups (L1) herein include alkylene groups, particularly straight chain unsubstituted alkylene groups, - (CH 2) n-, wherein n is 1 to 12, n is 1 to 10, n is 1 to 9, n is 1 to 8, n is 1 to 7, n is 1 to 6, n is 1 to 5, n is 1 to 4, n is 1 to 3, n is 2 to 10, n is 2 to 9, n is 2 to 8, n is 2 to 7, n is 2 to 6, n is 2 to 5 or n is 2 to 4.
Alkoxy groups are alkyl groups (arylalkyl-O-) attached to oxygen as discussed extensively above. The alkoxy group is monovalent.
An alkenylene group is a divalent radical of a straight or branched chain alkylene group having one or more carbon-carbon double bonds. In particular embodiments, the same carbon atom will not be part of two double bonds. In an alkenylene group, one or more CH2-CH2 moieties of the alkylene group are substituted with a carbon-carbon double bond. In particular embodiments, alkenylene groups contain 2 to 12 carbon atoms or more preferably 3 to 12 carbon atoms. In particular embodiments, the alkenylene group contains one or two double bonds. In particular embodiments, the alkenylene group contains one or two trans double bonds. In particular embodiments, the alkenylene group contains one or two cis double bonds. Exemplary alkenylene groups include:
-(CH 2 )n-CH＝CH-(CH 2 ) n-, wherein n is 1-4, more preferably 2; and
-(CH 2 )n-CH＝CH-CH＝CH-(CH 2 ) n-, wherein n is 1 to 4, more preferably 1 or 2.
Alkoxyalkyl groups in which one or more are not adjacent internal-CH 2 Alkyl groups in which the radical is replaced by-O-, such radicals may also be referred to as ether radicals. The alkoxyalkyl groups are monovalent. These groups may be linear or branched, but linear groups are preferred. Alkoxyalkyl groups include those having 2 to 12 carbon atoms and 1, 2,3, or 4 oxygen atoms. More specifically, alkoxyalkyl groups include those having 3 or 4 carbons and 1 oxygen, or those having 4,5, or 6 carbons and 2 oxygens. Each oxygen in the group is bonded to a carbon in the group. The group passing through carbon in the groupTo synthesize a molecule.
The alkoxyalkylene group is a divalent alkoxyalkyl group. The group may be described as one or more of the internal-CH 2 Alkylene groups in which the group is replaced by oxygen. These groups may be linear or branched, but linear groups are preferred. Alkoxy alkylene groups include those having 2 to 12 carbon atoms and 1, 2,3, or 4 oxygen atoms. More specifically, alkoxyalkylene groups include those having 3 or 4 carbons and 1 oxygen, or those having 4,5, or 6 carbons and 2 oxygens. Each oxygen in the group is bonded to a carbon in the group. The group is bonded to the molecule through a bond to a carbon in the group. The linking group (L1) herein includes alkoxyalkylene groups, particularly linear unsubstituted alkoxyalkylene groups. Specific alkoxyalkylene groups include-CH 2 -O-CH 2 -、-CH 2- CH 2 -O-CH 2 -CH 2 -、-CH 2 -CH 2 -CH 2 -O-CH 2 -CH 2 -CH 2 -、-CH 2 -CH 2 -O-CH 2 -、-CH 2 -O-CH 2 -CH 2 -、-CH 2 -CH 2 -O-CH 2 -CH 2 -O-CH 2 -、-CH 2 -CH 2 -O-CH 2 -CH 2 -O-CH 2 -CH 2 -、-CH 2 -CH 2 -CH 2 -O-CH 2 -CH 2 -CH 2 -O-CH 2 -and-CH 2 -CH 2 -CH 2 -O-CH 2 -CH 2 -CH 2 -O-CH 2 -CH 2 -。
The term acyl group refers to the group-CO-R, where R is hydrogen, an alkyl group, or an aryl group, as described herein.
Aryl groups include monovalent groups having one or more 5-or 6-membered aromatic rings. The aryl group may comprise one, two or three 6-membered aromatic rings. The aryl group may comprise two or more fused aromatic rings. The aryl group may comprise two or three fused aromatic rings. The aryl group is optionally substituted with one or more non-hydrogen substituents. Substituted aryl groups include those substituted with alkyl or alkenyl groups, which in turn may be optionally substituted. Specific aryl groups include phenyl, biphenyl, and naphthyl, all of which are optionally substituted as described herein. Substituted aryl groups include fully halogenated or semi-halogenated aryl groups, for example aryl groups in which one or more hydrogens are replaced by one or more fluorine, chlorine, bromine and/or iodine atoms. Substituted aryl groups include perfluorinated or semi-fluorinated aryl groups, such as aryl groups in which one or more hydrogens are replaced with one or more fluorine atoms.
Alkyl groups include arylalkyl groups in which the alkyl group is substituted with an aryl group. Arylalkyl groups include benzyl groups, phenethyl groups, and the like. The arylalkyl group is optionally substituted, as described herein. Substituted arylalkyl groups include those in which the aryl group is substituted with 1 to 5 non-hydrogen substituents, particularly 1, 2, or 3 non-hydrogen substituents. Useful substituents include methyl, methoxy, hydroxy, halogen and nitro. Particularly useful substituents are one or more halogens. Specific substituents include F, cl and nitro.
Heterocyclic groups are monovalent groups having one or more saturated or unsaturated carbocyclic rings, and each ring of which contains one to three heteroatoms (e.g., N, O, or S). These groups optionally contain one, two or three double bonds. To satisfy valence requirements, the ring atoms may be bonded to one or more hydrogens, or substituted as described herein. One or more carbons in the heterocycle may be a-CO-group. Heterocyclic groups include those having from 3 to 12 carbon atoms and from 1 to 6 heteroatoms, where 1 or 2 carbon atoms are replaced by a-CO-group. Heterocyclic groups include those having 3 to 12 or 3 to 10 ring atoms, up to three of which may be heteroatoms other than carbon. Heterocyclic groups may contain one or more rings, each of which is saturated or unsaturated. Heterocyclic groups include bicyclic and tricyclic groups. Preferred heterocyclic groups have a 5-or 6-membered ring. The heterocyclic group is optionally substituted as described herein. In particular, the heterocyclic group may be substituted with one or more alkyl groups. Heterocyclic groups include those having 5-and 6-membered rings containing one or two nitrogens and one or two double bondsOf (c) is used. Heterocyclic groups include those having 5-and 6-membered rings containing oxygen or sulfur and one or two double bonds. Heterocyclic groups include those having a 5-or 6-membered ring and two different heteroatoms (e.g., N and O, O and S, or N and S). Specific heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl pyrrolyl, pyrrolinyl, furyl, thienyl, morpholinyl,
Figure BDA0003960268060000721
Azole group,
Figure BDA0003960268060000722
An oxazoline group,
Figure BDA0003960268060000723
Oxazolidinyl, indolyl, triazolyl and triazinyl.
A heterocycloalkyl group is an alkyl group substituted with one or more heterocyclic groups, wherein the alkyl group optionally bears additional substituents and the heterocyclic group is optionally substituted. Specific groups are methyl or ethyl groups substituted with heterocyclic groups.
Heteroaryl groups are monovalent groups having one or more aromatic rings in which at least one ring contains heteroatoms (atoms other than carbon rings). Heteroaryl groups include those having one or two heterocyclic rings with 1, 2 or 3 heteroatoms, and optionally having a 6-membered aromatic ring. Heteroaryl groups may contain 5-20, 5-12, or 5-10 ring atoms. Heteroaryl groups include those having one aromatic ring containing a heteroatom and one aromatic ring containing a carbon ring atom. Heteroaryl groups include those having one or more 5-or 6-membered heteroaromatic rings and one or more 6-membered carbocyclic aromatic rings. The heteroaromatic ring may include one or more N, O or S atoms in the ring. Heteroaryl rings can include those having one, two, or three N, those having one or two O, and those having one or two S, or a combination of one or two or three N, O, or S. Specific heteroaryl groups include furyl groups, pyridyl groups, pyrazinyl groups, pyrimidinyl groups, quinolinyl groups, purinyl groups, indolyl groups. In one embodiment, heteroaryl is an indolyl group, more specifically an indol-3-yl group.
Heteroatoms include O, N, S, P or B. More specifically, the heteroatom is N, O or S. In particular embodiments, one or more heteroatoms substitute for a carbon in an aromatic or carbocyclic ring. To satisfy valency, any heteroatom in such an aromatic or carbocyclic ring may be bonded to H or a substituent group, such as an alkyl group or other substituent.
A heteroarylalkyl group is an alkyl group substituted with one or more heteroaryl groups, wherein the alkyl group optionally bears additional substituents, and the aryl group is optionally substituted. Specific alkyl groups are methyl and ethyl groups.
The term amino group means-N (H) 2 Such as this. The term alkylamino refers to the class of-NHR ", wherein R" is an alkyl group, particularly an alkyl group having 1-3 carbon atoms. The term dialkylamino refers to-N (R ") 2 This class, wherein each R "is independently an alkyl group, particularly an alkyl group having 1 to 3 carbon atoms.
The groups herein are optionally substituted. Most typically, any of the alkyl groups, cycloalkyl groups, aryl groups, heteroaryl groups, and heterocyclic groups may be substituted with one or more halogen, hydroxyl groups, nitro groups, cyano groups, isocyano groups, oxo groups, thio groups, azido groups, cyanate groups, isocyanate groups, acyl groups, haloalkyl groups, alkyl groups, alkenyl groups, or alkynyl groups (particularly those having 1-4 carbons), phenyl groups or benzyl groups (including those substituted with halogen or alkyl), alkoxy groups, alkylthio groups, or mercapto groups (HS-). In particular embodiments, the optional substitution is with 1-12 non-hydrogen substituents. In particular embodiments, the optional substitution is with 1-6 non-hydrogen substituents. In particular embodiments, the optional substitution is with 1-3 non-hydrogen substituents. In particular embodiments, the optional substituents comprise 6 or fewer carbon atoms. In particular embodiments, the optional substitution is with one or more halogens, hydroxyl groups, cyano groups, oxo groups, thio groups, unsubstituted C1-C6 alkyl groups, or unsubstituted aryl groups. The terms oxo and thioxo refer to carbon atoms substituted with a = O or a = S to form a-CO- (carbonyl) or-CS- (thiocarbonyl) group, respectively.
Specific substituted alkyl groups include haloalkyl groups, particularly trihalomethyl groups, particularly trifluoromethyl groups. Specific substituted aryl groups include monohalo, dihalo, trihalo, tetrahalo and pentahalophenyl groups; monohalogenated, dihalogenated, trihalogenated, tetrahalogenated, pentahalogenated, hexahalogenated, and heptahalogenated naphthyl groups; 3-halo or 4-halophenyl groups, 3-alkyl or 4-alkyl substituted phenyl groups, 3-alkoxy or 4-alkoxy substituted phenyl groups, 3-RCO or 4-RCO substituted phenyl, 5-halo or 6-halonaphthyl groups. More specifically, substituted aryl groups include acetylphenyl groups, particularly 4-acetylphenyl groups; fluorophenyl groups, in particular 3-fluorophenyl groups and 4-fluorophenyl groups; chlorophenyl, especially 3-chlorophenyl and 4-chlorophenyl groups; a methylphenyl radical, in particular a 4-methylphenyl radical; and methoxyphenyl groups, in particular 4-methoxyphenyl groups.
The term "aromatic" as applied to cyclic groups refers to a ring structure that includes a double bond that may be conjugated around the entire ring structure through one or more heteroatoms, such as oxygen, sulfur, or nitrogen atoms. Aryl and heteroaryl groups are examples of aromatic groups. The conjugated system of the aromatic group contains a characteristic number of electrons, for example, 6 or 10 electrons occupying the electron orbitals constituting the conjugated system, which is typically an unhybridized p-orbital.
The term carbocycle refers to a monovalent group having a carbocycle or ring system, containing 3 to 12 carbon atoms, and may be monocyclic, bicyclic, or tricyclic. The ring does not contain any heteroatoms. The ring may be unsaturated, partially unsaturated or saturated.
The compounds and substituents of the formulae herein are optionally substituted. A substituent refers to a group of a single atom (e.g., a halogen atom) or two or more atoms covalently bonded to each other, which are typically covalently bonded to one or more atoms in the molecule in place of a hydrogen atom, to satisfy the valence requirements of one or more atoms in the molecule. Examples of the substituent include an alkyl group, a hydroxyl group, an alkoxy group, an acyloxy group, a mercapto group, and an aryl group. The substituents themselves may be substituted.
Substituted or substituted refers to the replacement of a hydrogen atom of a molecule or chemical group or moiety with one or more additional substituents such as, but not limited to, halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxyl, mercapto, carboxyl, aryloxy, aryl, aralkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidinyl, pyrrolidin-1-yl, piperazin-1-yl, nitro, sulfato, or other R-groups.
The carbocycle or heterocycle is optionally substituted as generally described for other groups, such as alkyl groups and aryl groups herein. If present, the substitution is typically on ring C, ring N, or both. In addition, in the case of the present invention, carbocycle and heterocycle may optionally contain in the ring-CO-,; -CO-O-, -CS-or-CS-O-moieties.
With respect to any substituted chemical group herein, i.e., containing one or more non-hydrogen substituents, it is to be understood that such groups do not contain any sterically impractical and/or synthetically non-feasible substitution or substitution patterns. In addition, the compounds of the present invention include all stereochemical isomers resulting from substitution of these compounds.
Protected derivatives of the disclosed compounds are also contemplated. Various suitable protecting Groups for use with the disclosed compounds are disclosed in Greene and Wuts, protective Groups in Organic Synthesis;3rd Ed; john Wiley&Sons, new York, 1999. In general, the protecting group is removed under conditions that do not affect the remainder of the molecule. These methods are well known in the art and include acid hydrolysis, hydrogenolysis, and the like. One preferred method involves removal of the ester, for example, cleavage of the phosphonate using lewis acidic conditions, for example in TMS-Br mediated ester cleavage, to produce a free phosphonate. A second preferred method involves removal of the protecting group, for example by use of a suitable solvent system such as an alcohol, acetic acid, or the like, or mixtures thereofPalladium on carbon removed the benzyl groups by hydrogenolysis. The tert-butoxy-based group, including the tert-butoxycarbonyl protecting group, may be in a suitable solvent system such as water, bis
Figure BDA0003960268060000751
Alkane and/or dichloromethane, and removed with inorganic or organic acids such as HCl or trifluoroacetic acid. Another exemplary protecting group suitable for protecting amino and hydroxyl functional amino groups is trityl. Other conventional protecting Groups are known, and suitable protecting Groups can be found by those skilled in the art in Greene and Wuts, protective Groups in Organic Synthesis;3rd Ed; john Wiley&Sons, new York, 1999. When the amine is deprotected, the resulting salt can be easily neutralized to yield the free amine. Similarly, when an acid moiety, such as a phosphonic acid moiety, is exposed, the compound can be isolated as an acid compound or salt thereof. Protected derivatives of the compounds herein may, for example, be used in the synthesis of structurally related compounds herein.
The present invention provides a novel therapeutic strategy targeting TCF-driven EMT, a method of promoting tumor cell heterogeneity, MDR, and metastasis. The inventors' structure-based drug design has resulted in new potent CHD1L inhibitors that, in embodiments, target TCF-driven EMT. When used in combination with cytotoxic chemotherapy and targeted antineoplastic drugs, as well as radiation therapy, CHD1L inhibitors reverse EMT would be an effective treatment. These EMT targeting agents can also sensitize primary tumors and metastatic foci to clinically relevant treatments and potentially inhibit tumor cell metastasis.
Accordingly, one aspect of the present invention is a CHD1L inhibitor that can be used to treat or prevent the metastasis of a variety of advanced solid tumors and blood cancers. Pharmaceutically acceptable salts, prodrugs, stereoisomers, and metabolites of all CHD1L inhibitor compounds of the present invention are also contemplated.
The present invention expressly includes pharmaceutically acceptable solvates of the compounds according to the formulae herein. In particular, useful solvates are hydrates. The compound of formula I or a salt thereof may be solvated (e.g. hydrated). Solvation may occur during the manufacturing process or may occur (e.g., due to hygroscopicity (hydration) of the initial anhydrous compound of the formula herein).
The compounds of the present invention may have a prodrug form. Prodrugs of the compounds of the invention may be used in the methods of the invention. Any compound that will be converted in vivo to provide a biologically, pharmaceutically or therapeutically active form of a compound of the invention is a prodrug. Various examples and forms of prodrugs are well known in the art. A prodrug is an active or inactive compound that is chemically modified to an active compound by physiological actions in vivo, such as hydrolysis, metabolism, etc., upon administration of the prodrug to a subject. The term "prodrug" as used herein refers to pharmacologically acceptable derivatives such as esters, amides and phosphates, such that the in vivo biotransformation product of the resulting derivative is the active drug as defined in the compounds described herein. The prodrugs preferably have excellent water solubility, increased bioavailability, and are readily metabolized in vivo into active TOP2A inhibitors. Prodrugs of the compounds described herein may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleavable to the parent compound by routine manipulation or in vivo. The suitability and techniques involved in making and using prodrugs are well known to those skilled in the art. Examples of Prodrugs can be found in Design of produgs, edited by h.bundgaard, (Elsevier, 1985), methods in Enzymology, volume 42, pages 309-396, editors, k.widder et al (Academic Press, 1985); a Textbook of Drug Design and Development, edited by Krosgaard-Larsen and H.Bundgaard, chapter 5, "Design and Application of Prodrugs," edited by H.Bundgaard, pages 113-191, 1991); bundgaard, advanced Drug Delivery Reviews, volume 8, pages 1-38 (1992); bundgaard et al, journal of Pharmaceutical Sciences, volume 77, page 285 (1988); and Nogrady (1985) Medicinal Chemistry A Biochemical Approach, oxford University Press, new York, pages 388-392).
Administration of a compound or composition and administering a compound or composition is understood to provide a compound or a salt thereof, a prodrug of a compound, or a pharmaceutical composition comprising a compound. The compound or composition may be administered to the patient by another person (e.g., intravenously), or may be self-administered by the subject (e.g., a tablet or capsule). The term "patient" refers to mammals (e.g., humans and veterinary animals such as dogs, cats, pigs, horses, sheep, and cattle). It is contemplated that the CHD1L inhibitors herein are administered in combination with other agents, such as alternative anti-cancer, anti-tumor or cancer cytotoxic drugs. Such combined administration includes administration of two or more active ingredients simultaneously or at a time separated by a few minutes, hours, or days, which is considered effective and consistent with administration of any known replacement therapy in which the CHD1L inhibitor is to be combined. Co-administration also includes administration by the same method and/or at the same site of the patient's body, or by different methods at different sites, which is also consistent with administration of known replacement therapies with which CHD1L inhibitors are to be combined.
The pharmaceutical compositions herein comprise the specified active ingredient in an amount effective to achieve the desired biological activity for a given form of administration to a given patient, and optionally comprise a pharmaceutically acceptable carrier. Pharmaceutical compositions can include an amount (e.g., unit dose) of one or more of the disclosed compounds and one or more non-toxic pharmaceutically acceptable additives, including carriers, diluents, and/or adjuvants, and optionally other biologically active ingredients. Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques, such as those disclosed in Remington's pharmaceutical Sciences, mack Publishing co., easton, pa. (19 th Edition).
Pharmaceutically acceptable carriers are those that are compatible with the other ingredients of the formulation and biologically acceptable. The carrier may be a solid or a liquid. It is presently contemplated that the preferred carrier is a liquid carrier. The carrier may include one or more substances that may also act as solubilizers, suspending agents, fillers, glidants, compression aids, binders, tablet-disintegrants or encapsulating materials. Liquid carriers may be used in preparing solutions, suspensions, emulsions, syrups and elixirs. The active ingredient may be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water (of suitable purity, e.g., pyrogen-free, sterile, etc.), an organic solvent, a mixture of the two, or a pharmaceutically acceptable oil or fat. The liquid carrier may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, coloring agents, viscosity regulators, stabilizers or osmo-regulators. Compositions for oral administration may be in liquid or solid form.
Suitable examples of liquid carriers for oral and parenteral administration include water of suitable purity, aqueous solutions (particularly containing additives such as cellulose derivatives, sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols such as glycols) and their derivatives, and oils. For parenteral administration, the carrier may also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are used in sterile liquid form compositions for parenteral administration. The liquid carrier for the pressurized composition may be a halogenated hydrocarbon or other pharmaceutically acceptable propellant. Liquid pharmaceutical compositions in the form of sterile solutions or suspensions can be administered, for example, by intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be injected intravenously. Compositions for oral administration may be in liquid or solid form. The carrier may also be in the form of creams and ointments, pastes and gels. Creams and ointments may be viscous liquid or semisolid emulsions of either the oil-in-water or water-in-oil type.
A "therapeutically effective amount" of a disclosed compound is a dose of the compound sufficient to achieve a desired therapeutic effect, such as an anti-tumor or anti-metastatic effect. In some embodiments, a therapeutically effective amount is an amount sufficient to achieve tissue concentrations at sites of action similar to those shown to modulate TCF transcription and/or epithelial-mesenchymal transition (EMT) in tissue culture, in vitro, or in vivo. For example, a therapeutically effective amount of a compound can be a dose that results in a subject receiving from about 0.1 μ g/kg body weight/day to about 1000mg/kg body weight/day, e.g., a dose from about 1 μ g/kg body weight/day to about 1000 μ g/kg body weight/day, e.g., a dose from about 5 μ g/kg body weight/day to about 500 μ g/kg body weight/day.
The term "modulate" refers to the ability of a disclosed compound to alter the amount, degree, or rate of a biological function, the development of a disease, or the amelioration of a disorder. For example, modulation may refer to the ability of a compound to cause an increase or decrease in angiogenesis, to inhibit TCF transcription and/or EMT, or to inhibit tumor metastasis or tumorigenesis.
Treatment refers to therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it begins to progress. As used herein, the term "ameliorating," in the context of a disease or pathological condition, refers to any observable beneficial effect of treatment. Such beneficial effects may be evidenced, for example, by a delayed onset of clinical symptoms of the disease in the susceptible subject, a reduction in the severity of some or all of the clinical symptoms of the disease, a slowing of the progression of the disease, an improvement in the overall health or well-being of the subject, or by certain other parameters known in the art to be possessed by a particular disease. The phrase "treating a disease" includes, for example, inhibiting the complete development of a disease or disorder in a subject at risk of the disease, or a subject having a disease such as cancer or a disease associated with an impaired immune system. Preventing a disease or condition refers to prophylactic administration of a composition to a subject who exhibits no signs of disease or only early signs of disease, with the aim of reducing the risk of developing a pathology or condition, or lessening the severity of a pathology or condition.
All references, such as patent documents, cited in this application are hereby incorporated by reference, including issued or granted patents or equivalents thereof; patent application publication documents; and non-patent literature documents or other starting materials; the entire contents of which are incorporated herein by reference as if individually incorporated by reference. All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. The references cited herein are incorporated by reference in their entirety to indicate the state of the art as of their filing date in certain instances and are intended to use this information to exclude (e.g., disclaim) specific embodiments in the prior art, if desired. For example, when a compound is claimed, it is to be understood that compounds known in the art, including certain compounds disclosed in the references disclosed herein (especially the cited patent documents), are not intended to be included in the claims.
Abbott et al, 2020, and additional information in this journal article are both incorporated herein by reference in their entirety for the purpose of describing biological and chemical methods that can be used to prepare and evaluate the activity and properties of the CHD1L inhibitors herein.
When a group of substituents is disclosed herein, it is to be understood that all individual members and all subgroups of this group, including any isomers and enantiomers of the members of this group, as well as the classes of compounds which can be formed using the substituents, are individually disclosed. When a compound is claimed, it is to be understood that it is not intended to include compounds known in the art, including the compounds disclosed in the references disclosed herein. When a markush group or other grouping is used herein, all individual members of the group and all possible combinations and subcombinations of the group are intended to be included individually in the invention.
When a compound is described herein such that a particular isomer, enantiomer or diastereomer of the compound is not specified, e.g., in a formula or chemical name, the description is intended to include each isomer and enantiomer (e.g., cis/trans isomer, R/S enantiomer) of the compound, alone or in any combination. Furthermore, unless otherwise indicated, all isotopic variations of the compounds disclosed herein are intended to be encompassed by the present invention. For example, it is understood that any one or more hydrogens in the disclosed molecule may be replaced with deuterium or tritium. Isotopic variants of a molecule are generally useful as standards in the analysis of such molecules and in chemical and biological studies involving such molecules or their use. Isotopic variants (including those carrying radioisotopes) are also useful in diagnostic assays and therapy. Methods for preparing such isotopic variations are known in the art
The molecules disclosed herein may contain one or more ionizable groups [ groups from which protons may be removed (e.g., -COOH) or groups to which protons may be added (e.g., amines), or groups that may be quaternized (e.g., amines) ]. All possible ionic forms of these molecules and their salts are intended to be included individually in the present invention. With respect to salts of the compounds herein, one of ordinary skill in the art can select from among a variety of available counterions those suitable for preparing a salt of the invention for a given application. In a particular application, the selection of a given anion or cation to make a salt may result in an increase or decrease in the solubility of the salt.
The CHD1L inhibitors of the invention are commercially available or can be prepared by the methods disclosed herein or by routine modification of these methods using starting materials and reagents that are commercially available or can be prepared by known methods without undue experimentation. It will be appreciated that depending on the compound to be synthesized, it may be necessary to protect potentially reactive groups in the starting materials from unwanted conjugation. Useful protecting groups for reactive groups are known in the art, for example, as described in Wutts & Greene, 2007.
The compounds herein may be in the form of a salt, such as an ammonium salt, having a selected anion or quaternized ammonium salt. The salt may be formed by adding an acid to the free base, as is known in the art. The salts may be formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, and the like.
In particular embodiments, the compounds of the invention may contain one or more negatively charged groups (free acids), which may be in the form of a salt. Exemplary salts of the free acid are formed with inorganic bases including, but not limited to, alkali metal salts (e.g., li) + 、Na + 、K + ) Alkaline earth metal salts (e.g. Ca) 2+ 、Mg 2+ ) Nontoxic heavy metal salts, and ammonium (NH) 4+ ) And substituted ammonium (N (R') 4 + Salts wherein R' is hydrogen, alkyl, or substituted alkyl, i.e., including methyl, ethyl, or hydroxyethyl, specifically, trimethylammonium, triethylammonium, and triethanolammonium salts), salts of the cationic forms of lysine, arginine, N-ethylpiperidine, piperidine, and the like. The compounds of the invention may also exist in zwitterionic form. The compounds herein may be in the form of pharmaceutically acceptable saltsIt refers to those salts that retain the biological effectiveness and properties of the free base or free acid, and which are biologically or otherwise undesirable.
The scope of the invention described and claimed includes racemic forms of the compounds as well as individual enantiomers and non-racemic mixtures thereof. The compounds of the present invention may contain one or more asymmetric carbon atoms and thus these compounds may exist in different stereoisomeric forms. The compounds may be, for example, in racemic or optically active form. The optically active forms can be obtained by resolution of the racemate or by asymmetric synthesis. In a preferred embodiment of the invention, the enantiomers of the invention exhibit a + (normal) specific rotation. Preferably, the (+) enantiomer is substantially free of the corresponding (-) enantiomer. Thus, an enantiomer substantially free of the corresponding enantiomer refers to a compound that is separated or isolated by separation techniques, or a compound prepared free of the corresponding enantiomer. By "substantially free" is meant that the compound consists of a significantly greater proportion of one enantiomer. In a preferred embodiment, the compound is made up of at least about 90% by weight of the preferred enantiomer. In other embodiments of the invention, the compound is made up of at least about 99% by weight of the preferred enantiomer. Preferred enantiomers may be separated from racemic mixtures by any method known to those skilled in the art, including High Performance Liquid Chromatography (HPLC) and the formation and crystallization of chiral salts, or prepared by methods described herein [ see, e.g., jacques et al, 1981; wilen et al, 1977; eliel,1962; wilen,1972].
The compounds of the present invention and their salts may exist in their tautomeric forms, wherein hydrogen atoms are replaced to other parts of the molecule, and thus the chemical bonds between the atoms of the molecule are rearranged. It is understood that all tautomeric forms which may be present are included herein.
Unless otherwise indicated, each formulation, compound, or combination of components described or illustrated herein can be used in the practice of the present invention. The specific names of the compounds are exemplary, as it is known that one of ordinary skill in the art may name the same compounds differently. When a compound is described herein such that a particular isomer or enantiomer of the compound is not specified, for example in the formula or chemical name, the description is intended to include each isomer and enantiomer of the compound, alone or in any combination.
One of ordinary skill in the art will appreciate that methods other than those specifically exemplified, alternative therapies, starting materials, and synthetic methods can be used in the practice of the present invention without resort to undue experimentation. All art-known functional equivalents of any such methods, apparatus elements, starting materials, and synthetic methods are intended to be included herein. Whenever a range such as a temperature range, a time range or a composition range, all intermediate ranges and subranges, and all individual values included in the range given are given in the specification, all values included in the range given are intended to be included in the present invention.
The singular terms indefinite articles ("a", "an") and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" means "including". Furthermore, "comprising a or B" is meant to include a or B, or both a and B, unless the context clearly dictates otherwise. It is also understood that all molecular weight or molecular mass values given for a compound are approximations and are provided for purposes of description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and embodiments are illustrative only and not intended to be limiting.
As used herein, "comprising" is synonymous with "including", "containing", or "characterized by", and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of 8230 \8230composition excludes any element, step or ingredient not specified in the claim element. As used herein, "consisting essentially of" \8230 ";" 8230 ";" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claims. Any recitation herein of the term "comprising," particularly in the context of a description of components of a composition or of elements of a device, is understood to include compositions and methods that consist essentially of and consist of the recited components or elements. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.
Without wishing to be bound by any particular theory, a concept or understanding of the underlying principles associated with the present invention may be discussed herein. It should be recognized that embodiments of the present invention are operable and useful regardless of the ultimate correctness of any mechanical interpretation or assumption.
The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
Examples
Example 1: clinicopathological characterization of CHD1L in CRC patients
In some cancers, CHD1L expression is associated with poor prognosis, but only limited information is known about the pathology of CHD1L in CRC. This example describes the pathogenic characteristics and pathological mechanisms of CHD1L in CRC patients. The clinical pathology of CHD1L expression (GEO: GSE 39582) was analyzed in 585 CRC patients by the Cartes' Identite des Tumeasurs (CIT) project [ Marisa et al, 2013]. These features are summarized in Abbott et al, 2020 supplementary information.
Additional data for this example is found in Abbott et al, 2020 and its supplementary information. Follow-up information is available for all patients in the CIT cohort over 15 years. High CHD1L expression in high CHD1L patients was associated with lower OS (P = 0.0167) and Median Survival (MS) of 8.8 years for the entire patient cohort. The low CHD1L cohort failed to reach median survival because 72% (115/159) of the patients were truncated (censored) and 26% (42/159) of the patients died. Patient data was evaluated using a TNM staging system. Since stage I and IV patients have a high likelihood of survival or death, respectively, survival of stage II and III CRC patients was evaluated. For stage II and III CRC, high CHD1L expression was associated with lower OS (P = 0.0191) and 11 years of MS, and the low CHD1L cohort also did not reach median survival. Survival of CHD1L expression was also analyzed for each phase of CRC. Survival of stage II patients showed significant differences (P = 00319), with 11 years of m.s., and no significant differences were observed for stage I, III or IV patients. Analysis of CHD1L expression showed significant differences in the stage of expressing cancer. Patients with stage I and II colorectal cancer were evaluated as compared to patients with stage III and IV and showed a significant increase in CHD1L expression in stage III and IV compared to the early cohort (P = 0.0051). Analysis of CHD1L expression for lymph node metastasis indicated that CHD1L was overexpressed in patients with increased regional lymph node metastasis (N1P =0.0128, N2P =0.05 compared to N0). Although the trends in CHD1L expression for the N3 cohort were the same, significance was not determined due to the limited number of patient samples available. There were no significant differences in CHD1L expression for tumor size, metastasis or location.
Assessment of CHD1L in CRC molecular subtypes
The association of CHD1L expression with six molecular subtypes of CRC [ Marisa et al, 2013] C1 (reduced immune system, n = 116), C2 (mismatch repair defect, n = 104), C3 (KRAS mutation, n = 75), C4 (CSC, n = 59), C5 (activated WNT pathway, n = 152) and C6 (chromosomal instability, n = 60) was studied. CHD1L expression was significantly different in six molecular subtypes (P < 0.001). CHD1L is highly expressed in C5, C4 and C3, and is less expressed in C2 and C6. The C2 subtype is associated with a reduction in the WNT signaling pathway and a defect in mismatch repair. The C4 and C6 subtypes are associated with poor relapse-free survival compared to other subtypes. The C4 subtype is associated with increased sternness of CSCs, while the C5 subtype is associated with activated WNT signaling and deregulated EMT pathways. C2 The lower CHD1L expression in the (mismatch repair-deficient) subtype is consistent with its known function in DNA damage response [ Ahel et al, 2009]. Furthermore, CHD1L expression is lower in patients with mismatch repair deficiency than in patients without mismatch repair deficiency (P < 0.001). The expression of CHD1L was also higher in KRAS mutant patients (P = 0.049). The expression of CHD1L in the C3, C4 and C5 molecular subtypes prompted further study of the function of CHD1L expression in EMT, CSC dryness and WNT/TCF pathways.
CHD1L expression is related to Wnt/TCF related gene
A similar trend was observed with a smaller cohort of CRC patients from the UCCC GI tumor tissue bank (n = 26), with a larger CIT cohort of CHD1L expression significantly correlated with late and metastatic CRC compared to early and primary CRC (Abbott et al, 2020, supplementary information). Expression was quantified as FPKM (fragments mapped per million fragments per kilobase exon). Metastatic tumor samples had significantly more CHD1L than primary tumors. In addition, CHD1L levels were higher during phase IV compared to phase II/III patient cohorts. When CHD1L expression was analyzed with genes involved in the KEGG WNT pathway, a significant positive correlation was observed with 65 of the 125 genes using Spearman correlation. Among these are putative genes involved in TCF-mediated transcription, such as topoisomerase II α (TOP 2A) (r =0.65, p =0.004, [ zhou et al, 2016, abraham et al, 2019]And TCF4 (r =0.61, p = 0.0012) (Abbott et al, supplementary fig. 2). Correlation value P of Gene<0.05. Log of transcript expression by FPKM (fragments mapped per million fragments per kilobase exon) 2 Normalization and quantization.
A significant positive correlation was observed between the known CSC markers CD44 (r =0.43, p = 0.038), LGR5 (r =0.55, p = 0.0075) and CHD1L. When comparing the CIT and UCCC queues, significant correlation was observed between TOP2A (r =404 0.1275, p = 0.0020) and TCF4 (r =0.1050, p = 0.011). Consistent with this result, TOP2A has been shown to be an essential component of the TCF-complex, promoting EMT in CRC [ Zhou et al, 2016; abraham et al, 2019]. Thus, CHD1L appears to be involved in TCF transcription and EMT in CRC patients.
Example 2: CHD1L mediates TCF-transcription in CRC
Based on the association of CHD1L with members of the TCF-complex, CHD1L may have a mechanistic role in TCF transcription. To evaluate this effect, SW620 and DLD1 cell lines with high and low endogenous CHD1L expression, respectively, were utilized. See Abbott et al, 2020 and its supplementary information for additional data for this example. Small hairpin RNA (shRNA) for knock-out of CHD1L (SW 620) in SW620 cells CHD1L-KD ). CHD1L is overexpressed in DLD1 cells (DLD 1) CHD1L-OE ). Transfection into SW620 CHD1L-KD Or DLD1 CHD1L-OE The TOPflash luciferase reporter gene of [ Morin et al, 1997; zhou et al, 2016]Determination of significant increase in TCF transcription resulting from over-expression of CHD1L (P)<0.0001 (Abbott et al, 2020). In contrast, SW620 CHD1L-KD Cells showed a significant decrease in TCF transcription (P = 0.0006). These results indicate that CHD1L is a potential factor directly involved in TCF transcription. Each of Morin et al, 1997 and Zhou et al, 2016 are herein incorporated by reference in their entirety for the description of the TOPflash reporter gene and the assays using it.
CHD1L interacts directly with the TCF transcriptional complex
Activation of TCF transcription is a dynamic process involving the shedding of co-repressor proteins, binding of co-activator proteins and remodeling of chromatin landscape [ Lorch et al, 2010, shitashige et al, 2008]. A Co-immunoprecipitation (Co-IP) study with TCF4 was performed to demonstrate that CHD1L binds directly to the TCF complex [ Abbott et al, 2020].
CHD1L has been well characterized as a binding partner for PARP1 in DNA damage response [ Pines,2012; ahel et al, 2009]. PARP1 is also a component of the TCF complex that binds to TCF4 and β -catenin [ Idogawa et al, 2005]. The results herein indicate that CHD1L binds to the TCF complex, probably through the interaction between TCF4 and PARP 1.
To further characterize CHD1L as a component of the TCF complex, chromatin immunoprecipitation (ChIP) of CHD1L against the WNT Response Element (WRE) of the TCF complex was performed in SW620 cells [ Abbott et al, 2020]. CHD1L is enriched in c-Myc, vimentin, slug, LEF1 and N-cadherin WRE, and further supports the direct action of CHD1L and TCF complex. Taken together, these data indicate that CHD1L is a key component of TCF transcription.
CHD 1L-mediated transcription of TCF promotes the sternness of EMT and CSC in CRC
Previously, TCF transcription was considered to be the major regulator of EMT in CRC [ Zhou et]. In addition, CHD1L is localized to the WRE of the EMT effector gene [ Abbott et al, 2020]. Therefore, the measurement SW620 CHD1L-KD And DLD1 CHD1L-OE Biomarker expression in cells to determine whether knock-out or overexpression of CHD1L modulates EMT. Knockout of CHD1L induces EMT reversal, decreases vimentin and slug, while increasing E-cadherin expression [ Abbott et al, 2020]. In contrast, EMT is at DLD1 CHD1L-OE Induced in cells as evidenced by decreased E-cadherin and increased vimentin and slug expression [ Abbott et al, 2020]. These results indicate that CHD1L is an EMT effector gene involved in promoting the mesenchymal phenotype in CRC. One feature of EMT is an increase in the dryness of CSCs.
Clonogenic colony formation assay [ Abbott et al, 2020]To characterize the effect of CHD1L expression on dryness [ Franken et al, 2006]. Measurement of DLD1 by colony formation CHD1L-OE CSC dryness in cells increased (P = 0.0001), whereas SW620 CHD1L-KD CSC dryness decreased in cells (P = 0.002).
Example 3: identification of small molecule inhibitors of CHD1L
As described in examples 1 and 2 herein, CHD1L is a driver of TCF-mediated EMT. Based on this, described herein are assays for identifying small molecule inhibitors of CHD1L. The goal of drug discovery is to target CHD1L DNA translocations or interactions with DNA, which are dependent on the activity of the catalytic domain atpase of CHD1L [ Ryan & Owen-Hughes,2011; flaus et al, 2011].
CHD1L belongs to the SNF2 (sucrose non-starter 2) atpase superfamily of chromatin remodelling players, comprising a bileaflet atpase domain [ Abbott et al, 2020 and its complement ]. CHD1L also has a macrodomain, which is unique relative to other chromatin remodelling players, that promotes an auto-inhibitory state through interactions between the macrodomain and the atpase domain [ Lehmann et al, 2017; gottschalk et al, 2009]. However, the binding of the macro domain to PARP1, a major activator of CHD1L, alleviates autoinhibition [ Lehmann et al, 2017; gottschalk et al, 2009].
Full-length CHD1L (fl-CHD 1L) and catalytic atpase domain (cat-CHD 1L) [ Abbott et al, 2020 and its complement ] were purified using the method of Lehmann et al, 2017. Protein constructs were used for recombinant expression and purification of CHD1L for in vitro HTS as described by Abbott et al, 2020. SDS page gels showed purified cat-CHD1L (68 kD) and fl-CHD1L (101 kD). The enzyme kinetics of cat-CHD1L were compared to fl-CHD 1L. cat-CHD1L provides a more robust atpase assay than fl-CHD1L, consistent with the report by Lehman et al, 2017. Thus, to identify direct inhibitors of CHD1L atpase, an exemplary High Throughput Screening (HTS) assay in the context of TCF transcription is described, which includes: cat-CHD1L, c-Myc DNA, ATP, and phosphate binding proteins that fluoresce when bound to inorganic phosphate (Pi).
This assay was validated and screened experimentally for clinically relevant kinase inhibitors [ Abbott et al, 2020 and its complement ]. No hits were found in the experimental screen, indicating that CHD1L is not a possible target for kinase inhibitors. After validation, a preliminary HTS was performed using 20,000 compounds in Life Chemicals Diversity Set, which was screened at 20 μ M in 1% DMSO and 10mM EDTA as a positive control [ Abbott et al, 2020 and its complement ]. This screen provided robust statistics with an average Z' -factor value of 0.57 ± 0.06 over 64 plates. The average compound activity was 92.3% ± 17.8. As a result, the hit limit was set to 39% of 3 standard deviations of the average value at the time of ATPase activity. This strict hit limit identified 64 hits, 53 of which were confirmed for recombinant CHD1L atpase activity.
Example 4: exemplary inhibitors
A subset of seven confirmed hits (compounds 1-7, see scheme 1) were purchased, representing a series of pharmacodynamic genes with greater than 50% inhibition of cat-CHD1L atpase. Dose response studies against cat-CHD1L ATPase were performed on compounds 1-7, which confirmed that these hits are potent CHD1L inhibitors with activities ranging from 900nM to 5 μ M (FIG. 1A). Additional structures of exemplary compounds 8-73 are provided in scheme 1, where SEM represents the protecting group trimethylsilylethoxymethyl. Note that in many cases in scheme 1, additional compound numbers are given in parentheses, which may be used in the tables and figures herein, or Abbott et al, 2020 and its supplemental information.
Testing of Compounds 1-7 at HCT116, SW620 and DLD1 Using the TOPflash reporter Gene System CHD1L-OE Ability to inhibit TCF transcription in cells (fig. 1B). Compounds 1-3 showed no significant activity in the cells. Compound 4 was shown to have the most moderate activity in cells, with no dose-dependent inhibition of TCF activity. However, compounds 5-7 showed superior dose-dependent activity against TCF transcription in all three CRC cell lines. Notably, in DLD1 CHD1L-OE In cells, a reduced inhibition of TCF transcription was observed at 2 μ M low doses of 5-7, which is evidence of cellular CHD1L target binding.
CHD1L inhibitors reverse EMT and malignant properties in CRC
After verifying hits 5-7 against CHD 1L-mediated TCF transcription, the ability of these compounds to reverse EMT and other malignant properties in CRC was evaluated. E-cadherin and vimentin are putative biomarkers of epithelial and mesenchymal phenotypes, respectively [ McDonald et al, 2015]. The absence of E-cadherin and the acquisition of vimentin are also clinical biomarkers of poor prognosis [ Yun et al, 2014; richardson et al, 2012; dhanasekaran et al, 2001; kashiwagi et al, 2010; toiyama et al, 2013](ii) a Thus, lentiviral promoter-driven reporter genes for E-cadherin (pCDH 1-ecaadpro-RFP) and vimentin (pCDH 1-VimPro-GFP) were developed, faithfully reporting the expression of E-cadherin and vimentin, respectively [ Zhou et al, 2016; abraham et al, 2019]. SW620 cells transduced with EcadpPro-RFP or VimPro-GFP were cultured as tumor organoids for 72 hours to reach a diameter of 600 μm. By usingCompounds 5-7 treatment of the tumor organoids for an additional 72 hours to determine an effective concentration of 50% (EC) to modulate promoter activity 50 ). Changes in promoter expression were quantified using a high content analysis algorithm based on 3D confocal images 507 (fig. 2A-2B) [ Zhou et. Abraham et al, 2019]。
Compounds 5-7 effectively down-regulate vimentin promoter activity, their EC 50 The values were 15.6. + -. 1.7. Mu.M (5), 4.7. + -. 510.5. Mu.M (6), and 12.8. + -. 1.3. Mu.M (7). In contrast, the E-cadherin promoter activity is up-regulated, EC 50 The values were 11.9. + -. 0.3. Mu.M (5), 11.4. + -. 0.3. Mu.M (6), and 28. + -. 0.003. Mu.M (7). Representative images showing that compound 6, measured by EMT reporter gene assay, reversed EMT in SW620 tumor organoids are shown in Abbott et al, 2020. These results indicate that small molecule inhibitors of CHD1L reverse TCF-driven EMT in CRC.
To confirm that CHD1L inhibitors reversed EMT, protein expression of two other putative biomarkers of EMT, slug (mesenchymal) and tight junction protein-1 (ZO-1, epithelial), were evaluated. Changes in slug and ZO-1 are considered to be the main criteria for EMT [ Zeisberg & Neilson,2009]. SW620 tumor organoids treated with CHD1L inhibitors down-regulated slug and up-regulated ZO-1, further indicating reversal of EMT. Western blot analysis showing changes in protein expression of additional EMT biomarkers slug and ZO1 is shown in Abbott et al, 2020.
EMT is characterized by an increase in CSC sternness and cell invasion. Thus, compounds 5-7 were tested at HCT-116 and DLD1 CHD1L-OE Ability to inhibit migration and invasion in cells. All three compounds showed significant inhibitory effect on the dryness of CSCs (fig. 2C). However, compounds 5 and 6 showed more potent dose-dependent inhibition. Note that DLD1 CHD1L-OE The cells formed twice as many colonies as HCT-116 cells, which had moderate CHD1L expression. The results of this study are consistent with the oncogenicity and tumorigenicity of CHD1L. Then, using embedding in
Figure BDA0003960268060000891
HCT-116 cells, treated with CHD1L inhibitor at the indicated concentrations, and monitored for invasion for 72 hours. Compounds 5-7 showed dose-dependent inhibition of invasion (fig. 2D), with compound 6 showing the most potent activity.
Scheme 1: exemplary Compounds of formula I or formula XX
Figure BDA0003960268060000901
Figure BDA0003960268060000911
Figure BDA0003960268060000921
Figure BDA0003960268060000931
Figure BDA0003960268060000941
Figure BDA0003960268060000951
Figure BDA0003960268060000961
Figure BDA0003960268060000971
Figure BDA0003960268060000981
Figure BDA0003960268060000991
Example 5: inhibition of the efficacy of CHD1L on DNA damaging agents
CHD1L is known to play a role in PARP 1-mediated DNA damage response repair, a mechanism that increases resistance to DNA damage chemotherapy [ Li et al, 2019; ahel et al, 2009; gottschalk et al, 2009]. For example, resistance of lung cancer to cisplatin is observed in cells that overexpress CHD1L. The therapeutic efficacy of cisplatin was restored after CHD1L knockout [ Li y.et al, 2019]. In addition, knockout of CHD1L alone did not increase DNA damage [ Ahel D et al, 2009]. To determine whether a CHD1L inhibitor can increase the efficacy of DNA damaging drugs on low CHD1L expressing DLD1 cells transduced with the empty vector (DLD 1CHD 1L-EV) and DLD1CHD1L-OE over-expressed in CRC cells, compound 6 was evaluated as a single dose alone and in combination with SN-38 (the active pharmacophore of the prodrug irinotecan), oxaliplatin and etoposide. To assess DNA damage, phosphorylation of H2AX (γ -H2 AX) was determined by immunofluorescence as shown in Abbott et al, 2020 and its complement, which is a biomarker for DNA damage chemotherapy [ Ahel d.et al, 2009]. Compound 6 alone showed no significant DNA damage when cells were treated at 10 μ M and γ -H2AX activity was measured, which was consistent with the previously reported CHD1L knockout study [ Ahel d.et al, 2009]. However, combined treatment of compound 6 with etoposide (10 μ M) and SN-38 (1 μ M) in synergy significantly increased DNA damage compared to etoposide alone and SN-38 alone in DLD1CHD1L-OE cells. In DLD1CHD1L-EV cells, the combination of etoposide and compound 6 alone showed significant synergy. Under the experimental conditions used, we observed no synergy with oxaliplatin. Nonetheless, SN-38 (i.e., irinotecan) combination therapy known as FOLFIRI is the standard treatment for treating CRC. Thus, the enhanced DNA damage that occurs when compound 6 is combined with SN-38 supports the hypothesis that CHD1L inhibitors can increase the efficacy of CRC standard therapy for DNA damage chemotherapy.
Example 6 chd1l inhibitors reversed EMT before inducing cell death.
CHD1L is reported to confer anti-apoptotic activity by inhibiting caspase (caspase) -dependent activation of apoptosis [ Li et al, 2013; sun et al, 2016]. Furthermore, it is known that reversing or inhibiting EMT can restore apoptotic activity in cancer cells [ Lu et al, 2014]. To determine whether CHD1L inhibitors reversed EMT prior to inducing cell death, E-cadherin expression was monitored by Ecadpro-RFP reporter gene activity and using CellTox TM The Green assay measures cytotoxicity. Cells were treated with CHD1L inhibitor for 72 hours, imaging every 2 hours. Compound 6 had a significant increase in E-cadherin expression before inducing cytotoxicity compared to DMSO (figure 3A).
To determine whether CHD1L inhibitors could induce apoptosis in CRC, western blots of SW620 tumor organoids were performed and it was observed that E-cadherin was cleaved after treatment with 5 and 6 [ Abbott et al, 2020]. Cleavage of E-cadherin is a hallmark of apoptosis [ Steinhuen et al, 2001].
Relative to the DMSO control, the more potent CHD1L inhibitor 6 showed an increase in cleaved PARP1, cleaved caspase 8 and cleaved caspase 3 [ Abbott et al, 2020]. These results indicate that compound 6 induces exogenous apoptosis, consistent with E-cadherin mediated apoptosis through death receptors [ Lu et al, 2014].
To further characterize the apoptotic activity of CHD1L inhibitors, SW620 cells were examined for annexin-V staining for 12 hours. Compound 6 induced significant apoptosis compared to DMSO alone and had similar activity to the positive control SN-38 (an active metabolite of irinotecan) (fig. 3B).
CHD1L inhibitors are effective against patient-derived tumor organoids (PDTO). The use of PDTO in the development of preclinical drugs has been established as a predictive in vitro cell model for clinical efficacy [ Drost J & Clevers H,2018]. After determining the ability of compound 6 to reverse EMT and induce apoptosis using a cell line-based model, the efficacy of compound 6 was evaluated in PDTO generated from CRC102, a patient sample obtained from the Gastrointestinal (GI) tissue bank of the University of Colorado Cancer Center (UCCC) (fig. 3C). Consistent with the results in the CRC cell line, compound 6 showed potent cytotoxicity in PDTO with an EC50 of 11.6 ± 2 μ M.
Example 7: exemplary inhibitor compound 6 is PK, PD and hepatotoxicity in vitro and in vivo.
To evaluate the drug-like potential and properties of compound 6 in silico, in vitro and in vivo PK studies were performed, evaluating CLogP, water solubility, stability in mouse liver microsomes and PK in CD-1 mice.
Table 1 provides a summary of the in vivo and in vitro pharmacokinetic parameters of compound 6. Consensus LogP (CLOGP) values were obtained using the SwissaDME network tool [ Daina et al, 2017]. Compound 6 was administered intraperitoneally to athymic nude mice QD for 5 days to measure accumulation in SW620 xenograft tumors (fig. 4) and to assess histopathology of hepatotoxicity. Abbott et al, 2020 shows a representative H for the liver of vehicle and Compound 6 treated animals&E staining of the micrograph sections (5 Xmagnification). The images show normal hepatic chordal and lobular structure with no evidence of hepatocyte degeneration, necrosis, hyperplasia or parenchymal inflammation. Compound 6 has an excellent balance of lipophilicity (CLogP = 3.2) and water solubility that are relatively stable to liver metabolizing enzymes, and an excellent PK profile (displacement) when administered to CD-1 mice. High plasma drug concentration C was achieved following intraperitoneal (i.p.) administration of Compound 6 Maximum of (. About.30,000ng/mL) and AUC (about.80,000ng/mL/h), half-life (T 1/2λ ) Relatively long, 3 hours.
In the initial study, compound 6 had a half-life of less than 20 minutes in liver microsomes. In a similar in vitro liver microsomal half-life experiment subsequently performed with different liver microsomal preparations (data not shown), compound 6 exhibited a longer half-life of 67 minutes, compound 6.3 exhibited an improved (over 6) in vitro half-life of 98 minutes, and compound 6.11 exhibited a further improved (over 6) in vitro half-life of 130 minutes. Initial half-life studies of compound 6 were performed with different liver microsome preparations, which were not comparable to the later in vitro microsome half-life experiments. Table 2 provides the results of a second series of in vitro and in vivo half-life measurements, which included data for several additional compounds shown.
The second acute in vivo experiment was administered to athymic nude mice by intraperitoneal injection of QD for five days with a maximum tolerated dose of 6 (50 mg/kg). The objectives of this experiment were (1) to determine whether compound 6 caused acute toxicity to the liver, (2) accumulation in VimPro-GFP SW620 xenograft tumors, and (3) to determine PD effects. Compound 6 accumulated in SW620 tumors at a concentration of 10,533 ± 5,579ng/mL (n = 4). As expected, when comparing the rate of compound 6 accumulation in tissue/plasma, it was observed that its accumulation in the liver was 2.7-fold higher than in the tumor (fig. 4). However, compound 6 did not produce significant hepatotoxicity at the doses and schedule administered (table 3). Overall, there were no significant histological differences between the livers of vehicle or compound 6 treated mice. The major histological changes observed in vehicle and compound 6 treated animals were minimal fibrosis and inflammation of the liver envelope. This indicates a very low level of subclinical peritonitis, consistent with peritonitis secondary to peritoneal drug administration.
The effect of PD on tumor tissue was measured by western blot analysis based on the accumulation of compound 6 in the tumor, indicating significant down-regulation of the mesenchymal markers vimentin, vimentin reporter (VimPro-GFP) and slug [ Abbott et al, 2020]. Although not statistically significant, up-regulation of the epithelial marker ZO-1 and induction of cleaved caspase 3 (a putative biomarker of apoptosis) were also observed. Taken together, these observations of the PD effect of compound 6 indicate reversal of EMT and apoptosis in vivo, consistent with the cell-based in vitro anti-tumor activity of compound 6. Compound 6 showed good PK-class drug profile and the ability to alter EMT and induce apoptosis in vivo without observing hepatotoxicity.
In contrast, compound 6.11 exhibited a significantly longer half-life (t.p.) following intraperitoneal (i.p.) administration compared to compound 6 1/2λ ) Much greater than 6 hours.
Table 1: PK parameters for Compound 6
Figure BDA0003960268060001031
Table 2: pharmacokinetics of CHD1L inhibitors
Figure BDA0003960268060001032
Table 3: raw scores were histologically assessed by QD treatment of livers of athymic nude mice with vehicle or compound 6 (50 mg/kg) for 5 days.
Figure BDA0003960268060001033
1 Evaluation: n = normal background lesions of mouse strain; a = abnormal. 2 Inflammation score (performed when tissue assessment is abnormal): 0= none, 1= lowest, 2= mild, 3= moderate, 4= severe
Example 8: biological evaluation of Compound 8
Compound 8 was evaluated in many of the biological assays described above. The results are shown in FIGS. 7A-E. Compound 8 exhibited more potent dose-dependent inhibition of CHD 1L-mediated TCF transcription compared to compound 6 (fig. 7A). Likewise, compound 8 reversed EMT, as evidenced by down-regulation of vimentin and up-regulation of E-cadherin promoter activity (fig. 7B and 7C, respectively). Compound 8 significantly inhibited clonogenic colony formation at 10 days (fig. 7D). Compound 8 significantly inhibited the invasive potential for HCT116 at 48 hours (fig. 7E).
Example 9: methods applied in the examples herein
Other materials and methods
An antibody. Monoclonal mouse anti-TCF 4 antibody was purchased from EMD Millipore (Billerica, MA, USA) (catalog # 05-511) at 1:1000 dilution for Western blot and 2. Mu.g antibody/300. Mu.g protein for IP. Monoclonal rabbit anti-CHD 1L antibody was purchased from Abcam (Cambridge, MA, USA) (catalog # ab 197019) at 1: 5000 dilution for Western blotting and 1.5. Mu.g antibody/300. Mu.g protein for IP. Monoclonal rabbit anti-vimentin (cat # 5741), anti-Slug (cat # 9585), anti-E-cadherin (cat # 3195), anti-ZO-1 (cat # 8193), anti-histone H3 (cat # 4620) were purchased from Cell Signaling (Danvers, MA, USA), and mouse anti-a-tubulin (cat # 3873) was purchased from Cell Signaling, 1. Monoclonal rabbit anti- β -catenin (catalog # 9582) was purchased from Cell Signaling, 1. Monoclonal rabbit anti-phospho- β -catenin was purchased from Cell Signaling (catalog # 5651). Monoclonal rabbit anti-TCF 4 (catalog # 2569) and anti-histone H3 (catalog # 4620) were purchased from Cell Signaling,2 μ g antibody/1 mg protein for ChIP. Anti-rabbit IgG HRP-linked secondary antibody (catalog # 7074) was purchased from Cell Signaling, diluted 1: 3000 for western blotting. Anti-goat and anti-mouse IgG HRP-linked secondary antibodies (catalog #805-035-180 and # 115-035-003) were from Jackson ImmunoResearch (West Grove, pa.) diluted 1:10,000 for Western blotting.
Clinical pathological characterization of CHD1L
Transcriptome expression data from CIT cohort (GEO: GSE 39582) for 585 CRC patients for computer validation (GSE 39582) [ Marisa et al, 2013]. Gene expression analysis by Affymetrix GeneChip TM Human genome U133 Plus 2.0 arrays (Thermo Fisher Scientific, waltham, mass.). Robust multi-array analysis (RMA) was used for data pre-processing and ComBat (empirical bayesian regression) was used for batch corrections. The signal intensity is normalized by log 2. The CHD1L cut-off for CRC risk stratification based on disease-specific survival was determined from the Receiver Operating Characteristic (ROC) curve. The cutoff value for CHD1L expression was set to 6.45. Differences in OS were assessed by Kaplan-Meier method and compared using log rank test. The Fisher exact test is used for comparison of categorical variables. The Mann-Whitney U test was used for two sets of continuous variables. In the case of two or more groups, the data were analyzed by Kruskal-Wallis test. For all bilateral P values, an unadjusted significance level of 0.05 was applied.
CHD1L cut-off and clinical pathology were assessed by multiple cox regression analysis. Only the variables with significance in univariate analysis were integrated into the cox regression model and significance was determined using the Wald Forward Algorithm. For more than 2 groupsAll variables are classified, and the gradual entry standard of covariates is P<0.05, removal criteria is P>0.1. Use of
Figure BDA0003960268060001051
SPSS Statistics(IBM,Armonk,NY)、Prism8(GraphPad Software,San Diego,CA)、
Figure BDA0003960268060001052
(SAS Institute, cary, NC) and RStudio TM Statistical analysis was performed on IDE (RStudio Inc, boston, MA).
UCCC patient sample RNA-seq analysis
RNA-seq data from tumor xenografts from CRC patients were obtained from UCCC (University of Colorado Cancer Center) GI tumor tissue banks and analyzed as described previously [ Scott et al, 2017]. Briefly, gene expression was normalized by Log2 and measured by FPKM (transcription per kilobase per million mapped read fragments). The Wnt signaling pathway defined by Kyoto Encyclopedia of Genes and Genomics (KEGG) was used as the gene set in this study. Samples with CHD1L expression <1FPKM were considered low expression and were removed from this study. Genes with significant Spearman correlation (P < 0.05) were displayed as heatmaps using matrix2png (gene Z normalized) [ see Abbott et al, 2020 and its complement ].
CHD1L overexpression and shRNA knock-out
Full-length CHD1L was synthesized in pGEX-6P-1 plasmid (GenScript, piscataway, NJ). The EcoRI and NotI flanked CHD1L sequences were digested and ligated to the lentiviral backbone to create pCDH1-CMV-CHD1L-EF1-puro plasmid for overexpression of CHD1L in human CRC cells.
Figure BDA0003960268060001053
shrnas (Sigma-Aldrich co.llc, st.louis, MO) (scrambled) and TRCN0000013469 and TRCN0000013470 (sh 69 and sh 70) specific for CHD1L were purchased from Sigma-Aldrich (st.louis, MO). Use of
Figure BDA0003960268060001061
The-293 reagent (Mirus, madison, wis.) and plasmids pHRdelta8.9 and pVSV-G produced virus in HEK293T cells. CRC cells were transduced with over-expressed or shRNA knock-out virus and selected with 2. Mu.g/ml puromycin for 7 days.
Western blot
The CRC cell lines and homogenized tumor tissue samples from mice were resuspended in RIPA lysis buffer (20 mM Tris-HCl (pH 7.5), 150mM NaCl, 1mM Na 2 EDTA, 1mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5mM sodium pyrophosphate, 1mM b-glycerophosphate, 1mM Na 3 VO 4 0.1mM PMSF). Use of Pierce TM The BCA protein assay kit (ThermoFisher, waltham, mass.) measures protein concentration. 40 micrograms of samples were run on 10% bis-Tris gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes. The membrane is kept at room temperature
Figure BDA0003960268060001062
20 (TBST contained 20mM Tris, 150mM NaCl and 0.1%
Figure BDA0003960268060001063
20 (Croda International PLC, snaith, UK) was blocked with 5% skim milk for 1 hour. Membranes were washed three times with TBST. The blot was incubated with the appropriate primary antibody in TBST containing 5% skim milk overnight at 4 ℃. Membranes were washed three times with TBST and then incubated with appropriate secondary antibodies for one hour. The membrane was washed three more times with TBST. The blots were blotted with SuperSignal TM West Pico PLUS chemiluminescent substrate (ThermoFisher, waltham, mass.) was exposed and imaged using the ChemiDoc imaging System (Bio-Rad, hercules, calif.) [ see Abbott et al, 2020 and its complementary information on Western blotting ]]。
TOPflash TCF-transcriptional reporter gene detection
The TOPflash assay (Millipore, billerica, MA) was used to assess TCF transcriptional activity in CRC cells. A total of 20,000 cells/well were seeded in a 96-well white plate and used
Figure BDA0003960268060001064
LT1 transfection reagent (Mirus, madison, wis.) for transfection. Cells were incubated with the transfection mixture for 24 hours. Next, the cells were washed with Phosphate Buffered Saline (PBS), and PBS: ONE-Glo was added at a ratio of 1:1 TM Luciferase reagent Promega (Madison, wis.) detected luminescence within 10 minutes. Use of
Figure BDA0003960268060001065
Luminescent Cell Viability Assay (Promega, madison, WI) was performed in duplicate to measure Cell Viability, which was used to normalize TOPflash luminescence to obtain fold-change in TCF activity. The experiment was repeated 2 times (n =3 per experiment).
Co-immunoprecipitation (Co-IP)
The nuclear cell lysate was produced 138 from untreated SW620 cells. For the import control, 100. Mu.L of 1mg/mL nuclear extract was saved and used as the import. Immunoprecipitation (IP) Using Dynabeads TM Protein A IP Kit (Thermoscientific, waltham, mass.). Briefly, 300 μ g of lysate was incubated with 2 μ g of anti-TCF 4 and anti-CHD 1L IP antibody, anti-rabbit IgG and anti-mouse IgG as non-specific binding control and spun at 4 ℃ for 2h. After pre-incubation, 50 μ L of beads were transferred to the pre-incubated antibody/lysate mixture and then incubated overnight at 4 ℃. The stream was collected and the beads were washed 3 times with PBST. The protein was eluted with 20 μ L of 50mM glycine (pH = 2.8) for 10 min at 70 ℃.
Chromatin immunoprecipitation (ChIP)
Using the detailed procedure described previously [ Zhou et al, 2016]Cells were cross-linked with 1.42% formaldehyde for 15 min and quenched with 125mM glycine for 5 min. Cells were lysed with Szak RIPA (radioimmunoprecipitation assay buffer) buffer and sonicated. The IP step was carried out at 4 ℃ as follows: 50 μ L protein A/G agarose beads were pre-washed with cold Szak RIPA buffer and incubated with 1mg lysate for 2 hours. 0.3mg/mL salmon sperm DNA was added and incubated for 2 hours. Lysates (100 μ L) were left as input controls. To the residue was added anti-CHD 1L (2. Mu.g) and incubated overnight. Washing beadSeparately, the supernatant was pipetted to 100. Mu.L, followed by addition of 200. Mu.L of 1.5X-Talianidis elution buffer (70 mM Tris-Cl pH 8.0,1mM EDTA pH 8.0,1.5%w/v SDS). To elute the immunocomplexes and reverse cross-links, 12 μ L of 5M NaCl was added and the mixture was incubated at 65 ℃ for 16 hours. The supernatant was mixed with 20. Mu.g proteinase K and incubated at 37 ℃ for 30 minutes. DNA was extracted with phenol/chloroform and precipitated with ethanol. Using known public primers, with PowerUp TM SYBR TM Green Master Mix (Applied Biosystems, austin, TX) amplified IP product [ Zhou et al, 2016]。
Clone formation assay
As described previously, CHD1L was knocked out in SW620 cells or assessed for colony formation after overexpression in DLD1 cells [ Zhou et al, 2016; abraham et al, 2019]. Cells were plated at 1000 cells/well in 6-well plates and media was changed 2 times per week over the course of a 10 day period. Colony formation assays were also performed as described previously [ Zhou et al, 2016; abraham et al, 2019].
To assess the ability of CHD1L inhibitors to inhibit CSC dryness, HCT-116 or CHD1L overexpressing DLD1 cell lines were pretreated in monolayer cultures for 24 hours with either vehicle control (0.5% dmso) or CHD1L inhibitor at the concentrations shown in fig. 2C. The pretreated live cells were plated at 1,000 cells/well in 6-well plates, or at 200 cells/well in 24-well plates. Use of
Figure BDA0003960268060001081
The S3 2018A (Sartorius, france) software analyzed colonies (with the following parameters modified from default values to (1) segmentation adjustment =0.6 for HCT116 cells, minimum area (μm 2) =3 × 104, maximum area (μm 2) =1.6 × 106, maximum eccentricity =0.9, (2) segmentation adjustment =1 for DLD1CHD1L OE cells, minimum area (μm 2) =1 × 104, maximum area is not restricted, maximum eccentricity =0.95. The experiment was repeated 2 times (n =2 per experiment).
Tumor organoid culture
Cell lines were cultured [ Zhou et al, 2016; abraham et al, 2019]For tumor organoids, phenol-free containing 5% FBS was usedRed RPMI-1640 and seeded at 5,000 cells/well in uncoated 96-well U-bottom ultra-low adsorption microplates (Perkin-Elmer, hopkinton, mass.), followed by centrifugation at 1,000rpm for 15 minutes to promote cell aggregation. Adding to a final concentration of 2%
Figure BDA0003960268060001082
matrix (Corning Incorporated, corning, new York) and incubating tumor organoids prior to treatment (5% CO) 2 37 ℃, humidity) for 72 hours and maintained under standard cell culture conditions during the treatment time.
VimPro-GFP and Ecadpro-RFP reporter gene 3D high content imaging analysis
As previously reported, pCDH imPro-GFP-EF1-puro viruses or pCDH-ecadapro-mCherry-EF 1-puro viruses were used to generate stable VimPro-GFP or ecadapro-RFP SW620 reporter cells [ Zhou et al, 2016; abraham et al, 2019]. As described herein, stable fluorescently labeled reporter cells are used to generate tumor organoids. Tumor organoids were treated with 10 μ M of CHD1L inhibitor for 72 hours more. After treatment, tumor organoids were stained with 16 μ M Hoechst33342 for 1 hour (nuclear staining). The image was taken with a 5x aerial objective. The Z-stacks were set 26.5 μm apart for a total of 15 optical slices. Using Opera Phenix TM And
Figure BDA0003960268060001083
the software was used for imaging and high content analysis (PerkinElmer, hopkinton, MA). Nuclei were identified in each layer and cells were found to have GFP or mCherry channels. The fluorescence intensity of each channel was calculated and a threshold was set based on the background intensity. The percentage of GFP or mCherry RFP positive cells was calculated and normalized to DMSO treated groups.
Cytotoxicity of tumor organoids
SW620 tumor organoids were cultured as described herein. CellTox TM The Green cytotoxicity assay solutions were prepared according to the manufacturer's protocol (Promega, madison, wis.). Briefly, cellTo was usedx TM Tumor organoids were treated with Green reagent (0.5X) and different doses of CHD1L inhibitor (range 0-100 μ M) for 72 hours. Using Opera Phenix TM 207 screening system (PerkinElmer Cellular Technologies, hamburg, germany) imaged organoids with an excitation wavelength of 488nm and an emission wavelength of 500-550nm. Cytotoxicity was calculated using the average intensity of the whole well, using lysis buffer (Promega, madison, WI) as 100% cytotoxicity control, 0.5% dmso as 0% cytotoxicity control. Intensity values were normalized to these controls using Prism8 (GraphPad, san Diego, CA).
Invasion test
HCT116 cells were plated at 60,000 cells/well
Figure BDA0003960268060001091
ImageLock 96 well plates (Sartorius, france) and allowed to attach overnight. Use of
Figure BDA0003960268060001092
WoundMaker formed wounds in all wells and was then washed 2 times with PBS. The plates were brought to 4 ℃ using Corning XT Cool Core to avoid the need for aggressive conditioning during the preparation of the conditions
Figure BDA0003960268060001093
matrix (Corning Life Sciences, corning, N.Y.) was polymerized. Using 50. Mu.L in RPMI-1640 medium
Figure BDA0003960268060001094
matrix coats the wells. Plates were centrifuged at 150rpm for 3 minutes at 4C using a swinging blade rotor to ensure uniform substrate coating, without air bubbles. The plate was then placed in a cell culture chamber (5% CO) 2 37 ℃ C., moist) for 10 minutes, and then adding CHD1L inhibitor dissolved in 50. Mu.L of RPMI-1640 medium containing 5% FBS. Finally, the plate is placed on
Figure BDA0003960268060001095
S3 Living cell imager (Sartorius, france) for 48 hours. Wounds were imaged hourly in wide mode using a phase contrast channel and a 10 x objective.
Cloning and purification of recombinant human CHD1L
The Cat-CHD1L (residues 16-61) and fl-CHD1L (residues 16-879) constructs were generously gifted by Helena Berglund of the Carolinsca Institute Medical Biochemistry and Biophysics system (Karolinska Institute, department of Medical Biochemistry and Biophysics). Rosetta of proteins in Terrific Broth (ThermoFischer, waltham, mass.) TM 2 (DE 3) pLysS cells (Novagen available from Sigma-Aldrich, st. Louis, mo.). Cultures were induced with 0.2mM IPTG (OD 600= 2.0) for 16 hours at 18 ℃. Cells were harvested and resuspended in buffer-A containing 20mM HEPES, pH7.5, 500mM NaCl, 50mM KCl, 20mM imidazole, 10mM MgCl 2 1mM TCEP (tris (2-carboxyethyl) phosphine), 10% glycerol and 500. Mu.M PMSF. Cells were lysed by sonication and cell debris was removed by centrifugation. The supernatant was loaded onto a Ni-NTA resin column (Qiagen, hilden, germany). The proteins bound to the column were washed 1 time with buffer-A, 1 time with buffer-A containing 10mM ATP and once again with buffer-A. The protein was eluted with a gradient of 20-500mM imidazole using buffer-B (buffer-A containing 500mM imidazole). After affinity purification, cat-CHD1L was dialyzed overnight against 50mM Tris, pH7.5, 200mM NaCl and 1mM DTT. Similarly, fl-CHD1L was dialyzed overnight in 20mM MES, pH 6.0, 300mM NaCl, 10% glycerol and 1mM DTT. The protein was then purified by ion exchange chromatography. cat-CHD1L binds to Q-sepharose column (GE Healthcare, chicago, IL), fl-CHD1L binds to S-sepharose column (GE Healthcare, chicago, IL), and for cat-CHD1L, the protein is eluted using a 0.2-1M NaCl gradient and for fl-CHD1L, a 0.3-1M NaCl gradient. The pure fractions were combined, concentrated and Superdex was used TM 200 columns (GE Healthcare, chicago, IL) were further purified by size exclusion chromatography using 20mM HEPES, pH7.5, 100mM NaCl, 1mM TCEP and 10% glycerol. Use of
Figure BDA0003960268060001101
Start FPLC (GE Healthcare, chicago, IL) for protein purification.
CHD1L ATPase assay
All reactions were performed using low volume non-binding surface 384-well plates (Corning inc., corning NY). cat-CHD1L or fl-CHD1L (100 nM) and 200nM c-Myc DNA or mononucleosomes (Active Motif, carlsbad, CA) were added to a buffer containing 50mM Tris pH7.5, 50mM NaCl, 1mM DTT, 5% glycerol, the reaction was initiated by the addition of 10 μ M ATP (New England Biolabs, ipswich, MA) to a total volume of 10 μ L, and incubated at 37 ℃ for 1 hour. ATPase activity was measured by the addition of 500nM of a phosphate sensor (Life Technologies, carlsbad, calif.) containing a labeled phosphate-binding protein, specifically labeled with the fluorophore MDCC
Figure BDA0003960268060001111
Excitation (430 nm) was measured immediately and irradiated (450 nm) on a plate reader (Perkinelmer, hopkinton, MA). Conversion of fluorescence to [ Pi using inorganic phosphate Standard Curve]Enzyme kinetics was determined using Prism8 (GraphPad Software, san Diego, calif.).
HTS drug discovery for inhibitors of CHD1L
The assay composition was identical to that described above using cat-CHD1L, except that the volume of the reaction mixture was varied to accommodate the addition of drug or DMSO. Use of
Figure BDA0003960268060001112
Liquid handler (Perkinelmer, hopkinton, MA), selected amounts of compound dissolved in 100% DMSO were mixed with 50mM Tris pH7.5, 50mM NaCl, 1mM DTT, 5% glycerol buffer to 200. Mu.M in 10% DMSO. Next, 1. Mu.L of each compound was added to the enzyme mixture to a final concentration of 20. Mu.M. The negative control used was 1% DMSO, and 1mM EDTA was used as a positive control. The reaction was initiated by the addition of 10. Mu.M ATP and incubated at 37 ℃ for 1 hour. ATPase activity was measured by fluorescence by adding 500nM phosphate sensor. Diversity set of 20,000 compounds from Life Chemicals (Woodbridge, CT) and Selleck CCat-CHD1L was screened from a kinase inhibitor library of chemicals (Houston, TX). Both libraries were pre-screened prior to purchase to remove Pan-screened (Pan-assay) interfering compounds (PAINS) that tend to react non-specifically with many biological targets, rather than selectively with the desired target [ Baell ]&Nissink,2018；Baell&Holloway,2010]。
Patient-derived tumor organoid (PDTO) culture and viability assays
CRC patient tumor tissue was obtained from the UCCC GI tissue bank and amplified according to established protocols [ Morin et al, 1997)]. Briefly, cells were seeded at 5,000 cells/well in 96-well plates and cultured by established methods [ Franken et al, 2006]PDTO was allowed to form over 72 hours. PDTO was treated with DMSO (0.5%) or different concentrations of compound 6 for an additional 72 hours to obtain a dose response. Use of
Figure BDA0003960268060001113
Reagents (Promega, madison, WI) measure PDTO cell viability. Media (80 μ L) was aspirated from the wells, 80 μ L of reagent was added and incubated for 4 hours, and cell viability was measured by fluorescence intensity using excitation (560 excitation and 590 radiation).
Assessment of apoptosis
SW620 cells were plated at 30,000 cells/well in 96-well plates. Cells were treated with DMSO (negative control), SN-38 (apoptosis positive control) and compound 6 at the indicated concentrations for 12 hours. The cells were then washed 2 times with cold PBS and stained with cold Annexin-V staining buffer (10 mM HEPES, pH 7.4, 140mM NaCl, 2.5mM CaCl) 2 ) Cells were washed 1 time and then incubated with Annexin-V FITC 1:100 for 30 minutes in the dark. Cells were then washed twice with Annexin-V staining buffer and used
Figure BDA0003960268060001121
FITC strength was measured with a plate reader (Perkinelmer, hopkinton, MA).
Assessment of DNA Damage by Gamma-H2 AX
Will DLD1 CHD1L-OE Cells were seeded into 96-well PerkinElmer cell vector plates and allowed to attach overnight. Cells were then treated with 10 μ M of the appropriate compound (0.5% DMSO) or with CHD1L inhibitor in combination with SN-38 (1 μ M), oxaliplatin (10 μ M) and etoposide (10 μ M). Cells were treated for 6 hours. The medium was aspirated and the cells were washed with cold PBS. The cells were then fixed with 3% paraformaldehyde for 15 minutes at room temperature and the fixed cells were washed three times with PBS. The cells were blocked for 1 hour at room temperature in PBS containing 5% BSA, 0.3% Triton X-100. Cells were then immunostained using phosphorylated- (S139) -g-H2AX rabbit mAb at 1: 800 dilution in PBS containing 1% BSA, 0.3% Triton X-100, overnight at 4 ℃. Aspirate primary antibody and wash cells with PBS. The cells were incubated at room temperature with goat anti-rabbit Alexa Fluor PlusTM 647 fluorescent secondary antibody at a concentration of 5. Mu.g/mL in PBS containing 1% BSA, 0.3% Triton X-100 for 2 hours. The cells were then washed with PBS, hoechst33342 stain diluted 1:1000 in PBS and added to the cells for 15 minutes at room temperature. Then using Opera Phenix TM Cells were imaged with a 20X water objective on a HCS imaging system (PerkinElmer). Synergy was determined using the drug interaction Coefficient (CDI) equation: CDI = (a + B)/(AB). Synergistic effects were defined in these experiments as CDI<0.8. The additive effect is 0.8-1.2, and the antagonistic effect is caused by CDI>1.2 definition.
Water solubility and CLOGP
The water solubility of compound 6 was measured using the recently reported detailed method [ Abraham et al, 2019]. The PBS UV absorption spectrum was compared to a fully saturated solution in 1-propanol and the solubility of Compound 6 in 10% DMSO in PBS (pH 7.4) was determined using linear regression analysis. The measurement of solubility in PBS was performed in repeated experiments. Consensus LogP (CLogP) values were obtained using the swissanedme network tool [ Daina et al, 2017].
Microsome stability study
Compound 6 was assayed for microsomal stability using female CD-1 mouse microsomes (M1500) purchased from Sekisui XenoTech (Kansas City, KS) according to the recently reported method [ Abraham et al, 2019]. Samples were centrifuged at 20,000g for 10 minutes and the supernatant transferred to an autosampler vial for LCMS analysis. Compound 6 (molecular weight = 393.5) was monitored for the following mass transitions (m/z, amu).
In vivo pharmacology
All Animal studies were conducted according to the Institutional Animal Care and Use Committee (IACUC) approved Animal protocol procedures at the University of Colorado anshutz Medical Campus (Aurora, CO) and Colorado State University (Colorado State University) (Fort Collins, CO).
Pharmacokinetics
9 week old female CD-1 mice purchased from Charles River (Wilmington, MA) were used for PK studies using the recently reported method [ Abraham et al, 2019]. Briefly, PK studies were designed to cover the range of 0.25-24 hours, 3 mice per time point, for a total of 21 mice per compound 6. Each mouse was intraperitoneally injected with 6 compounds at 50mg/kg prepared in 100% DMSO. Whole blood was collected at specific time points and the separated plasma was either cryopreserved at-80 ℃ or used for LC-MS/MS analysis.
Pharmacodynamics and hepatotoxicity
Suspending in 100 μ L
Figure BDA0003960268060001131
Figure BDA0003960268060001131
200 ten thousand VimPro-GFP SW620 cells in a 1:1 mixture of matrix (Corning Life Sciences, corning, N.Y.) and RPMI 1640 were injected into the flank of 9 week old female athymic Nude mice (Nude-Foxn 1nu (069)) (Envigo, huntingdon, cambridge shire, UK). Growth was monitored by caliper measurements 3 times per week. At the fourth week, mice were randomly divided into 2 groups, treated with 50mg/kg of compound 6 in 200 μ L of vehicle (10% dmso, 40 % peg 400, 50% pbs ph = 7.4), or treated with vehicle control. Qd application treatment for 5 days. On the fifth day of treatment, mice were sacrificed 2h after the last dose. Tumor and liver were collected for analysis of accumulation of compound 6 by LCMS measurement, western blot analysis to measure effects on EMT and apoptosisLoud, and hepatotoxicity.
Statistical analysis
Unpaired two-tailed Student's t-test was performed on the data using Welch's corrected statistical analysis, or on the data using Prism8 (GraphPad, laJolla, CA). All experiments were repeated 3 times (n = 3) or as described in the methods.
Example 10: other Experimental methods for assessing Compound Activity
Microsomal stability.CD-1 mouse microsomes were purchased commercially and the reactions were performed as described previously. Briefly, a master mix was prepared as follows: microsomes (0.5 mg/mL), 10 μ M CHD1Li in DMSO (0.1%), 5mM UDPGA, 25 μ g doxorubicin, and 1mM MgCl2 in 100mM phosphate buffer (pH = 7.4). The master mix was preincubated at 37 ℃ for 5 minutes, then 1mM NADPH was added to start the microsomal activity reaction and was maintained at 37 ℃ throughout the time. The reaction was stopped at 0,5, 15, 30, 45 and 60 minutes by addition of 200 μ L acetonitrile and analyzed by mass spectrometry. Appropriate microsomal controls were also performed under the same reaction conditions.
Combined studies of γ -H2AXDNA damage with irinotecan (SN 38).As previously reported [ Abbott et al, 2020; abraham et al, 2019]For DNA damage, CHD1L inhibitor 6 alone and a combination of inhibitor 6 and SN38 were evaluated. DNA damage studies were performed using DLD1 colorectal cancer cells with low endogenous expression of CHD1L, and DLD1 cells engineered to overexpress CHD1L, measuring immunofluorescence of γ -H2AX (a recognized biomarker of DNA damage) [ Ji et al, 2017; ivashkevich et al, 2012]. Cells were seeded into 96-well plates as monolayers and treated with 10 μ M (0.5% DMSO) of Compound 6, or SN-38 (1 μ M), or a combination of Compound 6 and SN38 for 6 hours. The medium was aspirated and the cells were washed with cold PBS. Cells were then fixed with 3% paraformaldehyde for 15 minutes at room temperature and washed three times with PBS. The cells were blocked for 1 hour at room temperature in PBS containing 5% BSA, 0.3% Triton X-100. The cells were then diluted 1: 800 in PBS containing 1% BSA, 0.3% Triton X-100 using phosphorylated- (S139) -gamma-H2 AX rabbit mAbImmunostaining was performed overnight at 4 ℃. Primary antibody was aspirated and cells were washed with PBS. The cells were incubated with goat anti-rabbit Alexa Fluor Plus at a concentration of 5. Mu.g/mL TM 647 fluorescent Secondary antibody was incubated in PBS containing 1% BSA, 0.3% Triton X-100% at room temperature for 2 hours. The cells were then washed with PBS; hoechst33342 stain was diluted to a concentration of 1. Cells were then imaged using a 20X water objective lens on a PerkinElmer Phenix HCS imaging system. We observed a synergistic effect of compound 6 and SN38 in inducing DLD1 cell damage overexpressing CHD1L using the drug interaction Coefficient (CDI) equation: CDI = (a + B)/(AB) determination. Synergistic effect is caused by CDI<0.8, additive effect of 0.8-1.2, antagonism by CDI>1.2 definition. Welch t-test statistical analysis was used to determine significance, where × = P ≦ 0.01.
Cell-based cytotoxic dose response and combination studies.The CHD1L inhibitor and SN38 (the active pharmacophore of irinotecan) were evaluated for antitumor activity against colorectal cancer cell lines, alone or in combination. As previously reported [ Abbott el al, 2020]The cell lines were cultured as monolayers or 3D tumor organoids using RPMI-1640 containing 5% fetal bovine serum. For cytotoxicity studies of 3DSW620 tumor organoids, 2000 cells in 100 μ Ι _ were seeded into each well of a 96-well U-bottom ultra-low adsorption microplate (Corning inc., corning, NY, USA). Plates were centrifuged at 1000rpm for 15 minutes to facilitate cell aggregation. The final 2% matrigel concentration was achieved by coating the centrifuged cells with 25 μ Ι 10% matrigel in each well. The plates were then incubated for 3 days before treatment. The 3D organoids were treated with 25 μ L of various concentrations of drug. 3 days after treatment, organoids and 40. Mu.L of medium were manually transferred to 96-well white solid plates. Equal amounts of Celltiter-glo 3D (Promega) were added and the plates were held on a plate shaker at 400rpm for 45 minutes before the luminescence was read with an Envision plate reader (Perkinelmer). Synergy scores for the combination studies were determined using the Combenefit assay [ De Veroli et al, 2016]。
In vivo studies.As described in previous reports [ Abbott el al.,2020]Pharmacokinetic evaluation of CHD1L inhibitor Compounds 6 and 6.11Estimated to determine the plasma half-life of 9-week-old female CD-1 mice. Compound 6 was further evaluated for anti-tumor activity against SW620 tumor xenografts in athymic nude mice, alone and in combination with irinotecan. Using the previously reported method [ Zhou et al, 2016]Generating a xenograft. Briefly, compound 6 was administered at 5mg/kg i.p. 2 times/day, 7 days/week, for a total of 5 weeks. Beginning one week after compound 6 treatment, irinotecan was administered at 60mg/kg intraperitoneally once a week for 3 weeks. Body weight and tumor volume were monitored 2 times per week. When a single tumor reaches 2000mm 3 Or total tumor volume reaches 3000mm, mice are sacrificed and tissue is collected [ Ji et al, 2017]. The antitumor activity of compound 6.11 against SW620 tumor xenografts was similarly evaluated, both alone and in combination with irinotecan. Recent reports [ Esquer et al, 2021 and supplementary information thereof]It was shown that the CRC M-phenotype is significantly more tumorigenic than the other CRC EMT-phenotypes, and that the M-phenotype also has significantly higher TCF transcription. The xenografts used in this study were generated using isolated double reporter mesenchymal cells (M-phenotype) as described by espquer et al, 2021. Compound 6.11 was 8 hours stable 2.7 fold in CD-1 mice compared to compound 6 (half-life =3 hours). Thus, the number of treatments was reduced from 2 to 1 treatments per day. In addition, irinotecan was administered at 50mg/kg by intraperitoneal injection.
FIGS. 7A and 7B illustrate dose response studies of cytotoxicity of representative single doses in SW620 colorectal cancer (CRC) tumor organoids and provide IC's of the exemplary compounds shown 50 . Tables 4A and 4B below provide a summary of cytotoxicity data for exemplary compounds. Table 4A provides cytotoxicity data for representative individual compounds in several different CRC tumor organoids.
FIG. 4A: cytotoxicity of tumor organoids
Figure BDA0003960268060001161
Figure BDA0003960268060001171
Table 4B provides the results of the indicated combination therapy of representative CHD1L inhibitors (CHD 1 Li) with SN38 or Olaparib. Treatment was performed in four different CRC tumor organoid types. The concentration of CHD1L inhibitor was varied as indicated. IC of combination therapy compared to SN38 and Olaparib alone 50 Generally decreases.
TABLE 4B tumor organoid cytotoxic combination therapy
Figure BDA0003960268060001172
Figure 8B shows a graph of γ -H2AX intensity (relative to DMSO) in DLD1 Empty Vector (EV) cells and DLD1 (OE) overexpressing cells for compound 6 alone, irinotecan alone (SN 38) and a combination of the two. Fig. 8A is a western blot showing the relative expression of CHD1L for DLD1 (EV) cells compared to DLD1 (OE) cells compared to control expression of β -tubulin in these cells. CHD1L is known to be critical for PARP-1 mediated DNA repair, and may lead to resistance to DNA damage chemotherapy [ Ahel el el al, 2009; tsuda el al, 2017]. The data in fig. 8B demonstrates the "targeting" effect of CHD1L inhibitors in synergy with SN38 to induce DNA damage.
Fig. 9A-9C illustrate the results of a synergy study with exemplary CHD1L inhibitors 6, 6.3, 6.9, and 6.11 in SW620 colorectal cancer (CRC) tumor organoids. SN38 in combination with 6 and 6.3 was 50 and 150 times more effective in killing colon SW620 tumor organoids than SN38 alone, respectively. The combination of SN38 with 6.9 and 6.11 is more than 100 times that of SN38 alone. Each of compounds 6, 6.3, 6.9 and 6.11 showed synergistic effects with irinotecan (and SN 38) in killing SW620 tumor organoids.
Synergy scores for exemplary CHD1L inhibitors were determined as described in the report of De Veroli et al, 2016 and provided in table 5. To account for the score value of synergy, since SynergyFinder has normalized the input data to a percent inhibition, they can be directly interpreted as the proportion of cellular responses that can be attributed to drug interactions (e.g., synergy score of 20 corresponds to a response of 20% over expected). According to our experience, synergy scores close to 0 have limited confidence in synergy or antagonism. When the synergistic effect is evaluated as follows:
less than-10: the interaction between the two drugs is likely to be antagonistic;
from-10 to 10: the interaction between the two drugs is likely to be additive;
greater than 10: the interaction between the two drugs is likely to be a synergistic effect.
Table 5: exemplary synergy scores for SN38 with representative Compounds
Figure BDA0003960268060001181
Figure 10 includes a graph of tumor volume (fold) of SW620 tumor xenografts as a function of days (up to 28 days) treated with compound 6 alone, irinotecan alone, or combinations thereof. The combination of irinotecan and compound 6 significantly inhibited colon SW620 tumor xenografts to nearly no tumor volume within 28 days of treatment compared to the single agent treatment group or control.
Figure 11 includes a graph of tumor volume (fold) of SW620 tumor xenografts as a function of days (up to 28 days) treated with irinotecan alone (1), or a combination of compound 6 and irinotecan (2). The combination of irinotecan and compound 6 significantly inhibited colon SW620 tumor to virtually no tumor volume after the last treatment compared to irinotecan alone. Within 2 weeks of the last irinotecan treatment alone, tumor volume rose above the volume of the last treatment, indicating tumor recurrence. In contrast, the combination maintained a lower tumor volume.
Figure 12 shows that compound 6 alone and in combination with irinotecan (4) significantly increased survival of CRC tumor-bearing mice compared to vehicle (1), compound 6 (2) alone and irinotecan (3) alone.
Figure 13 includes a graph of tumor volume (fold) of SW620 tumor xenografts as a function of days (up to 20 days) treated with compound 6.11 alone, irinotecan alone, or combinations thereof. The combination of irinotecan and compound 6.11 significantly inhibited colorectal cancer SW620 tumor xenografts compared to irinotecan alone or control.
Figure 14 includes a graph of tumor volume (fold) of SW620 tumor xenografts as a function of days (up to 33 days) of treatment with irinotecan alone, or compound 6.11 in combination with irinotecan. The combination of irinotecan and compound 6.11 significantly inhibited colorectal SW620 tumor after the last treatment (day 33) compared to irinotecan alone. At 8 days post-treatment (Tx release), irinotecan alone treated tumor volume increased approximately 3-fold, indicating tumor recurrence. In contrast, the tumor volume of the combination treatment of 6.11 and irinotecan continued to decrease (approximately 1.5-fold) after treatment. The difference in tumor volume between irinotecan treatment alone and the combination of 6.11 and irinotecan at day 8 post-treatment was 3.4-fold.
Figure 15 shows that the combination of compound 6.11 with irinotecan significantly increased survival of CRC tumor-bearing mice compared to irinotecan alone and the control.
Example 11: summary of the currently preferred structural activity relationships of inhibitors
Based on formula I of the CDH1L inhibitors of the present invention, the presently preferred structural activity relationships are as follows:
Figure BDA0003960268060001201
for ring B, the presently preferred ring is a 6-membered aromatic ring or a fused 6,6-membered aromatic ring, and both X are N. If a second fused ring is present, the fused ring may contain one or two additional N. Preferred R B (B ring substitution, if present) is not an electronegative group. Preferred R B Is hydrogen or C1-C3 alkyl. Preferred A rings are optionally substituted phenyl, more preferably unsubstituted phenyl (wherein R is A Is hydrogen). R P The group is believed to be involved in water solubility, where-N (R) 2 )(R 3 ) Radicals are generally preferred, more particularly optionally substituted N-containing heterocycles in which R is 2 And R 3 Together with the N to which they are attached form a 5-8 membered ring, which may contain one or more additional heteroatoms, and which may be saturated (no double bonds) or contain one or more double bonds. R H Is thought to be associated with activity, potency and metabolic stability. R H Preferred are aromatic groups, more particularly ring-substituted heteroaryl groups having a stable aromatic or heteroaromatic ring. R in NR is preferably Y, more preferably hydrogen. Preferably, x is 0. A preferred Z is-CO-NH-. Preferred L 2 is-CH 2 -or-CH 2 -CH 2 -。
In one embodiment, HTS screening of CHD1L identifies a phenylaminopyrimidine pharmacophore represented by formula XX:
Figure BDA0003960268060001202
and salts thereof, wherein R 1 -R 9 Represents hydrogen or an optional substituent, R 10 Is a moiety believed to be associated with efficacy; and R is N Is a moiety that is believed to be related to a physicochemical property such as solubility. In embodiments, R 5 Are substituents other than hydrogen, which are believed to be associated with metabolic stability. In a particular embodiment, R 5 Is halogen (in particular F or Cl), a C1-C3 alkyl radical (in particular a methyl radical). In embodiments, R 4 Are substituents other than hydrogen, in particular C1-C3 alkyl groups, more particularly methyl groups. In specific embodiments, R 5 Is F, R 4 Is methyl. In embodiments, R 6 -R 9 Selected from hydrogen, C1-C3-alkyl, halogen, hydroxy, C1-C3-alkoxy, formyl, or C1-C3-acyl. In embodiments, R 6 -R 9 One or both of which are moieties other than hydrogen. In embodiments, R 6 -R 9 Is halogen, in particular fluorine. In specific embodiments, R 6 -R 9 All are hydrogen. In embodiments, R N Is an amino moiety-N (R) 2 )(R 3 ). In a particular embodiment, R N Is an optionally substituted heterocyclic group having a 5-7 membered ring optionally containing a second heteroatom (N, S or O). In embodiments, R N Is optionally substituted pyrrolidin-1-yl, piperidin-1-yl, aza-1-yl, piperazin-1-yl, or morpholinyl. R N Substituted with one substituent selected from: C1-C3 alkyl, formyl, C1-C3 acyl (in particular acetyl), hydroxy, halogen (in particular F or Cl), hydroxyC 1-C3 alkyl (in particular-CH) 2 -CH 2 -OH). In embodiments, R N Is unsubstituted pyrrolidin-1-yl, piperidin-1-yl, aza-1-yl, piperazin-1-yl, or morpholinyl.
In embodiments, R 10 is-NRy-CO- (L) 2 )y-R 12 or-CO-NRy- (L) 2 )y-R 12 Wherein y is 0 or 1, denotes the absence or presence of L 2 Which is an optionally substituted linking group of 1 to 6 carbon atoms and wherein one or two carbons of the linking group are optionally replaced by O, NH, NRy or S, wherein Ry is hydrogen or alkyl of 1 to 3 carbons, and R 12 Is an aryl group, a cycloalkyl group, a heterocyclic group, or a heteroaryl group, each of which is optionally substituted. In embodiments, y is 1.L is a radical of an alcohol 2 Is- (CH) 2 ) p-, wherein p is 0-3. In embodiments, R 12 Is thiophen-2-yl, thiophen-3-yl, furan-2-yl, furan-3-yl, pyrrol-2-yl, pyrrol-3-yl,
Figure BDA0003960268060001211
An oxazol-4-yl group,
Figure BDA0003960268060001212
An oxazol-5-yl group,
Figure BDA0003960268060001213
Oxazol-2-yl, indol-3-yl, benzofuran-2-yl, benzofuran-3-yl, benzo [ b ]]Thiophen-2-yl, benzo [ b ]]Thien-3-yl, isobenzofuran-1-yl,Isoindol-1-yl, or benzo [ c]Thien-1-yl. In embodiments, R 1 Is hydrogen or methyl. In embodiments, R 12 Is thiophen-2-yl, furan-2-yl, pyrrol-2-yl,
Figure BDA0003960268060001214
Oxazol-4-yl, indol-2-yl, benzofuran-2-yl, or benzo [ b ]]Thiophen-2-yl. In embodiments, R 12 Is thiophen-2-yl or indol-2-yl. In embodiments, R 1 Is hydrogen or methyl.
Exemplary compounds of the invention are shown in scheme 1.
Exemplary R P and-N (R) 2 )(R 3 ) The groups are shown in scheme 2.
Exemplary R 12 And R H The groups are shown in scheme 3.
An exemplary B ring of formula I is shown in scheme 4.
Scheme 1: exemplary Compounds of formula I or formula XX
Figure BDA0003960268060001221
Scheme 1 (continue)
Figure BDA0003960268060001231
Scheme 1 (continue)
Figure BDA0003960268060001241
Scheme 1 (continue)
Figure BDA0003960268060001251
Scheme 1 (continue)
Figure BDA0003960268060001261
Scheme 1 (continue)
Figure BDA0003960268060001271
Scheme 1 (continue)
Figure BDA0003960268060001281
Scheme 1 (continue)
Figure BDA0003960268060001291
Scheme 1 (continue)
Figure BDA0003960268060001301
Scheme 1 (continue)
Figure BDA0003960268060001311
Scheme 2 (exemplary R) P and-N (R) 2 )(R 3 ) Radical)
Figure BDA0003960268060001321
Scheme 2 (continue)
Figure BDA0003960268060001331
Scheme 2 (continue)
Figure BDA0003960268060001341
Scheme 3: exemplary R 12 And R H Radical (I)
Figure BDA0003960268060001351
Scheme 3 (continue)
Figure BDA0003960268060001361
Scheme 3 (continue)
Figure BDA0003960268060001371
Scheme 3 (continue)
Figure BDA0003960268060001381
Scheme 3 (continue)
Figure BDA0003960268060001391
Scheme 3 (continue)
Figure BDA0003960268060001401
Scheme 4 (exemplary B Ring for formula I)
Figure BDA0003960268060001411
Wherein X 1 And X 2 Selected from CH and N and X 1 And X 2 At least one of which is N, X 3 -X 6 Is selected from CH 2 CH, O, S, N and NH, and the B ring is saturated, partially unsaturated or aromatic, depending on X 3 -X 6 Is selected from, and R B Represents an optional substitution in the ring carbon and/or nitrogen as defined in formula I.
Example 12: exemplary synthetic methods
The compounds of formula XX, as well as many other compounds of the invention, are prepared by, for example, the methods shown in scheme 5, where the variables are as defined above. The three-step synthesis begins with the use of p-phenylenediamine B in the presence of 2, 4-dichloro-pyrimidine A (e.g., 2, 4-dichloro-6-methylpyrimidine, wherein R is 4 Is methyl or 2, 4-dichloro-5-fluoropyrimidine, in which R 5 Is fluorine) to form intermediate C. Exemplary reaction conditions are shown in scheme 5, where the reaction is added with trimethylamine to ice-cold ethanol and stirred at room temperature for 15 hours [ Kumar et al, 2014; odingo et al, 2014]. The chlorinated intermediate C is then reacted with any amine (HNR) 2 R 3 ) D generates an intermediate E through amination reaction. Exemplary amination conditions are shown in scheme 5, where the reactant is at K 2 CO 3 In the presence of DMF at elevated temperature. Step three, using acid F to react R 10 The group is coupled to intermediate E. Various known synthetic methods can be employed to introduce the selected R 10 Groups such as cross-coupling, click chemistry, or displacement reactions (e.g., SN2, aromatic, electrophilic) [ Li et al, 2014a; li et al, 2014b; labarbera et al, 2007]. Scheme 5 shows the coupling of the amine group of E with a selected carboxylic acid F to form R 10 Which is-NH-CO-R in Compound G 12 . Exemplary R 12 Are aryl, aryl-substituted alkyl, heteroaryl and heteroaryl-substituted alkyl. Exemplary coupling conditions are shown in scheme 5, where coupling is carried out in the presence of propylphosphonic anhydride (T3P) and triethylamine at room temperature to form the desired compound G. The methods shown have been used, for example, to prepare compound 6 and compound 8 (see scheme 6).
Various substituted starting materials a, B, D and F are commercially available or can be prepared using known methods. In embodiments, compounds already designated by R may be used 10 (B') substituted aniline derivative instead of p-phenylenediamine derivative B to form the corresponding R 10 -substituted intermediate C'. To proceed withStep 2 of the reaction shown, by reacting intermediate C 'with D, will yield the desired corresponding compound G' (wherein R is 10 Substituted for R 12 -CO-NH-). As will be appreciated by those of ordinary skill in the art, it may be useful or necessary to protect certain groups in the starting materials or intermediates during the reactions shown to prevent undesirable side reactions. For example, ring N in reactant F may be protected with a suitable amine protecting group. The use of suitable protecting groups is generally conventional in the art. A variety of primary or secondary amines (D) are commercially available or can be prepared by well-known methods. Alternatively, the chlorinated intermediate C may be reacted with a suitable nucleophile to increase the selected-NR at the 4-chloro position 2 R 3 A group. For example, D may be a cyclic amine, such as pyrrolidine. As another possible option, suzuki coupling can be used to add amine-containing groups by forming C-C bonds [ Li et al, 2014a]. As another possible alternative, buchwald-Hartwig cross-coupling may be used to form carbon and amine bonds in such intermediates.
Figure BDA0003960268060001431
Scheme 5
Detailed Synthesis of Compounds 6 and 8 (scheme 6)
N- (4-aminophenyl) -2-chloro-6-methyl-pyrimidin-4-amine (102). To 0.5g (3.06 mmol) of 2, 4-dichloro-6-methylpyrimidine (100) dissolved in 10mL of ethanol was added 513.7. Mu.L (1.2 eq, 372.5mg, 3.68 mmol) of Triethylamine (TEA) and 330.5mg (3.06 mmol) of p-phenylenediamine (101) at 0 ℃. The reaction mixture was warmed to room temperature and stirred at that temperature overnight. The solvent was removed in vacuo and the resulting residue was chromatographed on silica gel using 40% hexane in ethyl acetate as eluent to give 500mg (70% yield) of the pure product (102). 1 H NMR:(400MHz,CDCl 3 ):δ7.19(broad s,1H),7.02(d,J＝8.8Hz,2H),6.70(d,J＝8.8Hz,2H),6.18(s,1H),3.77(broad s,2H),2.26(s,3H)。
N- (4-aminophenyl) -6-methyl-2- (pyrrolidin-1-yl) pyrimidin-4-amine (104).To 1.2g of N- (4-aminophenyl) -2-chloro-6-methyl-pyrimidin-4-amine (102) dissolved in 120mL of DMF at room temperature was added 777.3mg (5.62 mmol) of potassium carbonate and 3.63mg (4.12 mL, 51.1 mmol) of pyrrolidine (103). The reaction mixture was heated at 80 ℃ for 8 hours. The reaction was cooled to room temperature and diluted with water. The product was extracted with ethyl acetate (3X 100 mL). The organic layers were combined and washed with brine, then Na 2 SO 4 Drying, filtration and concentration gave the crude product as an oil which was chromatographed on silica gel using 10% methanol in DCM (containing a few drops of TEA) to give 1.31g (96% yield) of the pure product (104). 1 H NMR:(400MHz,CDCl3):δ7.11(d,J＝8.4Hz,2H),6.67(d,J＝8.4Hz,2H),6.25(broad s,1H),5.68(s,1H),3.63(broad s,2H),3.56(t,J＝6.8Hz,4H),2.19(s,3H),1.93(t,J＝6.8Hz,4H)。
N- (4- ((6-methyl-2- (pyrrolidin-1-yl) pyrimidin-4-yl) amino) phenyl) -2- (thiophen-2-yl) acetamide (6). To 700mg (2.60 mmol) of N- (4-aminophenyl) -6-methyl-2- (pyrrolidin-1-yl) pyrimidin-4-amine (104) (15 mL) at 0 ℃ were added 406mg (2.86 mmol) of 2-thiopheneacetic acid (105), 906.8. Mu.L (657.5 mg, 6.50 mmol) of TEA and 3.06mL (50 wt% in ethyl acetate solution) of T3P. The mixture was warmed to room temperature and stirred for 15 hours. The reaction was quenched by the stepwise addition of water and the product was extracted with DCM (3 × 150 mL) and then washed with brine. The organic layers were combined and washed with Na 2 SO 4 Drying, filtration and concentration under reduced pressure gave the crude product, which was purified using silica gel and 10% methanol in DCM to give 864.5mg (84% yield) of pure product (6). 1 HNMR (400MHz, DMSO-d 6): delta 10.07 (s, 1H), 9.06 (broad s, 1H), 7.64 (d, J =8.8Hz, 2H), 7.49 (d, J =9.2Hz, 2H), 7.39-7.37 (m, 1H), 6.98-6.96 (m, 2H), 3.84 (s, 2H), 3.47 (s, 4H), 2.13 (s, 3H), 1.89 (t, J =6.6Hz, 4H): HPLC:98% purity.
Boc-protected indole-3-carboxylic acid 106-Boc was used in a peptide coupling procedure with compound 104 in the presence of T3P and TEA to achieve synthesis of Boc-protected indole derivative 8-Boc, which was converted to indole derivative 8 in good yield by TFA deprotection of the Boc protecting group. Note that the boc protecting group is-COO-t-butyl K2Co3, KI, etOH.
N- (4- { [ 6-methyl-2- (1-pyrrolidinyl)) -4-pyrimidines]Amino } phenyl) -1- { [ (2-methyl-2-propyl) oxy]Carbonyl } -1H-indole-3-carboxamide (8-boc). To 80mg (0.297 mmol) of compound 104 in 8mL DCM at 0 deg.C were added 85.36mg (0.3267 mmol) boc-protected indole-3-carboxylic acid (106-boc), 103.6. Mu.L (75.13 mg, 0.742 mmol) TEA, 350. Mu.L (189 mg, 0.594 mmol) T3P. The mixture was warmed to room temperature and stirred for 20 hours. The reaction was quenched by the stepwise addition of water and the product was extracted with DCM (3 × 50 mL) and washed with brine. The organic layers were combined and washed with Na 2 SO 4 Drying, filtration and concentration under reduced pressure gave the crude product which was purified using silica gel and 10% methanol in DCM to give 76mg (50% yield) of pure product (8-boc). 1 H NMR:(400MHz,CDCl 3 ):δ8.28(broad s,1H),8.24-8.13(m,3H),7.57(q,J＝4.8,8.8Hz,4H),7.40 -7.31m,3H),5.92(s,1H),3.54(s,4H),2.23(s,3H),1.86(s,4H),1.66(s,9H)。
N- (4- { [ 6-methyl-2- (1-pyrrolidine) -4-pyrimidine]Amino } phenyl) -1H-indole-3-carboxamide (8). 70mg (0.136 mmol) of N- (4- { [ 6-methyl-2- (1-pyrrolidine) -4-pyrimidine]Amino } phenyl) -1H-indole-3-carboxamide (8-boc) was dissolved in DCM (5 mL) 25% TFA. The solution was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure and the crude product was purified using silica gel and 10% methanol in DCM to give 43.78mg (78% yield) of pure product. 1 H NMR:(400MHz,DMSO-d6):δ11.7(s,1H),9.65(s,1H),9.09(s,1H),8.28(broad s,1H),8.20(d,J＝7.6Hz,1H),7.67(s,4H),7.47(d,J＝8.0Hz,1H),7.20-7.12(m,2H),5.88(s,1H),3.50(s,4H),2.14(s,3H),1.91(s,4H)。
Figure BDA0003960268060001451
Scheme 6
Scheme 7 shows another synthetic approach optimized for the yield of compound 6. In this process, a t-butyl protected carbamate, e.g., compound 35, is reacted with a selected aromatic carboxylic acid, e.g., compound 36, to form a protected carbamate intermediate, e.g., compound 37. As is known in the art, the intermediate is deprotected, for example with trifluoroacetic acid (TFA), and the deprotected carbamate is reacted with a chlorinated heterocyclic group bearing a primary or secondary amine group (e.g., a pyrrolidinyl group), such as compound 38, to form the desired compound of formula XX, such as compound 6. The process can also be used to prepare a variety of compounds of formula XX by selecting the starting aromatic carboxylic acid and chlorinated heterocyclic compound bearing a primary or secondary amine group.
In scheme 7, the reagents used to synthesize compound 6 are shown, where in the first reaction DCC is N, N' -dicyclohexylcarbodiimide, DMAP is dimethylaminopyridine, and the solvent is DCM dichloromethane. In a second reaction, after TFA deprotection, potassium carbonate and potassium iodide in ethanol are used. Reagents and reaction conditions for preparing the desired compound of formula XX can be readily adjusted by one of ordinary skill in the art.
Figure BDA0003960268060001461
Scheme 7
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Claims (25)

1. A method of treating a TCF transcription driven cancer comprising administering to a patient in need thereof an amount of a CHD1L inhibitor effective for inhibition of CHD1L or for inhibition of aberrant TCF transcription.
2. The method of claim 1, wherein the CHD1L inhibitor is a compound, salt or solvate of formula I:
Figure FDA0003960268050000011
or a salt thereof, or a solvate thereof,
wherein the content of the first and second substances,
ring B is a heteroaromatic ring or ring system having one, two or three 5-or 6-membered rings, any two or three of which may be fused, wherein the rings are carbocyclic, heterocyclic, aromatic or heteroaromatic, and at least one of the rings is heteroaryl;
in the B ring, each X is independently selected from N or CH, and at least one X is N;
R P is a primary or secondary amine group [ -N (R) 2 )(R 3 )]Or- (M) x A group of-P, wherein P is-N (R) 2 )(R 3 ) Or an aryl or heteroaryl group, wherein x is 0 or 1, meaning that M is absent or present, and M is an optionally substituted linking group- (CH) 2 ) n -or-N (R) (CH) 2 ) n -wherein each n is independently an integer from 1 to 6 inclusive;
y is selected from the group consisting of-O-, -S-, -N (R) 1 )-、-CON(R 1 )-、-N(R 1 )CO-、-SO 2 N(R 1 ) -or-N (R) 1 )SO 2 -a divalent atom or group of the group;
L 1 is an optional C1 to C4 linking group, which is optionally substituted, and is saturated or contains a double bond (which may be cis or trans), wherein x is 0 or 1, meaning that L is absent or present 1 ；
Ring a is a carbocyclic or heterocyclic ring or ring system having one, two or three rings, two or three of which may be fused, each ring having 3 to 10 carbon atoms and optionally 1 to 6 heteroatoms, and wherein each ring is optionally saturated, unsaturated or aromatic;
z is a divalent radical containing at least one nitrogen substituted with an R' group,
wherein in embodiments, Z is a divalent group selected from: -N (R ') -, -CON (R ') -, -N (R ') CO-, -CSN (R ') -, and-N (R ') CS-, -N (R ') CON (R ') -, -SO 2 N(R′)-、–N(R′)SO 2 -、-CH(CF 3 )N(R′)-、-N(R′)CH(CF 3 )-、-N(R′)CH 2 CON(R′)CH 2 -、-N(R′)COCH 2 N(R′)CH 2 -、
Figure FDA0003960268050000021
Or the divalent Z group comprises a 5-or 6-membered heterocyclic ring having at least one nitrogen ring member, e.g.,
Figure FDA0003960268050000022
L 2 is an optional 1 to 4 carbon linking group, which is optionally substituted, and is saturated or contains a double bond (which may be cis or trans), wherein z is 0 or 1, meaning that L is absent or present 1 ；
R is selected from the group consisting of hydrogen, aliphatic groups, carbocyclyl groups, aryl groups, heterocyclyl groups, and heteroaryl groups, each of which is optionally substituted;
each R' is independently selected from the group consisting of hydrogen, an aliphatic group, a carbocyclyl group, an aryl group, a heterocyclyl group, and a heteroaryl group, each of which is optionally substituted;
R 1 selected from the group consisting of hydrogen, aliphatic groups, carbocyclyl groups, aryl groups, heterocyclyl groups, and heteroaryl groups, each of which is optionally substituted;
R 2 and R 3 Independently selected from the group consisting of hydrogen, aliphatic groups, carbocyclic groups, aryl groups, heterocyclic groups, and heteroaryl groups, each of which is optionally substituted, or
R 2 And R 3 Together with the N to which they are attached form an optionally substituted 5-10 membered heterocyclic ring, which 5-10 membered heterocyclic ring is a saturated ring, a partially unsaturated ring, or an aromatic ring;
R A and R B Each represents hydrogen or 1-10 non-hydrogen substituents on the A and B rings or ring systems shown, wherein R is A And R B The substituents are independently selected from hydrogen, halogen, hydroxy, cyano, nitro, amino, mono-or di-substituted amino (-NR) C R D ) Alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy, acyl, haloalkyl, -COOR C 、-OCOR C 、-CONR C R D 、-OCONR C R D 、-NR C COR D 、-SR C 、-SOR C 、-SO 2 R C and-SO 2 NR C R D Wherein alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy, and acyl are optionally substituted;
each R C And R D Selected from hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with one or more halogen, alkyl, alkenyl, haloalkyl, alkoxy, aryl, heteroaryl, heterocyclyl, aryl-substituted alkyl, or heterocyclyl-substituted alkyl; and
R H is an optionally substituted aryl or heteroaryl group;
wherein the optional substitution comprises amino substituted with one or more halogen, nitro, cyano, amino, mono-or di-C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C1-C3 haloalkyl, C6-C12 aryl, C5-C12 heteroaryl, C3-C12 heterocyclyl, C1-C3 alkoxy, C1-C6 acyl, -COOR E 、-OCOR E 、-CONR E R F 、-OCONR E R D 、-NR E COR F 、-SR E 、-SOR E 、-SO 2 R E and-SO 2 NR E R F Wherein alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy, and acyl are optionally substituted, and
each R E And R F Selected from the group consisting of hydrogen, C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C1-C3 haloalkyl, C6-C12 aryl, C5-C12 heteroaryl, C3-C12 heterocyclyl, C1-C3 alkoxy, C1-C6 acyl, each of which is optionally substituted with one or more halogen, nitro, cyano, amino, mono-or di-C1-C3 alkyl substituted amino, C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C1-C3 haloalkyl, C6-C12 aryl, C5-C12 heteroaryl, C3-C12 heterocyclyl, C1-C3 alkoxy, and C1-C6 acyl.
3. The method of claim 1, wherein the CHD1L inhibitor is not a PARP inhibitor, an inhibitor of topoisomerase, or an inhibitor of β -catenin.
4. The method of claim 1, wherein the CHD1L inhibitor is a compound, salt or solvate of formula XX:
Figure FDA0003960268050000041
and salts or solvates thereof, wherein:
R 10 is-NRy-CO- (L) 2 )y-R 12 or-CO-NRy- (L) 2 )y-R 12 Wherein y is 0 or 1, denotes the absence or presence of L 2 ，L 2 Is an optional linking group of 1 to 6 carbon atoms, which linking group is optionally substituted, and wherein one or two carbons of said linking group is optionally replaced by O, NH, NRy or S, wherein Ry is hydrogen or alkyl of 1 to 3 carbons, and R is 12 Is an aryl group, a cycloalkyl group, a heterocyclyl group, or a heteroaryl group, each of which is optionally substituted;
R N is an amino moiety-N (R) 2 )(R 3 ) Wherein R is 2 And R 3 Is hydrogen or an optionally substituted alkyl group, or R 2 And R 3 Together with the nitrogen to which they are attached form a 5-10 membered heterocyclic ring, which 5-10 membered heterocyclic ring is saturated or partially unsaturated; and
R 1 、R 6 -R 9 represents hydrogen or an optional substituent.
5. A method of preventing tumor growth, invasion and/or metastasis in a CHD 1L-driven, EMT-driven or TCF transcription-driven cancer by administering to a patient in need thereof a CHD1L inhibitor in an amount effective for inhibition of CHD1L or inhibition of aberrant TCF transcription.
6. The method of claim 5, wherein the CHD1L inhibitor is a compound, salt, or solvate of any of formula I or formula XX.
7. The method of claim 5, wherein the CHD1L inhibitor is not a PARP inhibitor, an inhibitor of topoisomerase, or an inhibitor of β -catenin.
8. A method for treating CRC, including mCRC, comprising administering to a patient in need thereof an amount of a CHD1L inhibitor effective to inhibit aberrant TCF transcription.
9. The method of claim 8, wherein the CHD1L inhibitor is a compound, salt or solvate of formula I or formula XX.
10. The method of claim 8, wherein the CHD1L inhibitor is not a PARP inhibitor, an inhibitor of topoisomerase, or an inhibitor of β -catenin.
11. A method of treating a drug resistant cancer comprising administering to a patient in need thereof a CHD1L inhibitor in an amount effective for inhibition of CHD1L or inhibition of aberrant TCF transcription, in combination with a known treatment to which the cancer has developed resistance.
12. The method of claim 11, wherein the CHD1L inhibitor is a compound, salt or solvate of formula I or formula XX.
13. The method of claim 11, wherein the CHD1L inhibitor is not a PARP inhibitor, an inhibitor of topoisomerase, or an inhibitor of β -catenin.
14. The method of claim 11, wherein the treatment to which the cancer has become resistant is conventional chemotherapy.
15. A combination method for treating cancer comprising administering a CHD1L inhibitor in combination with a PARP inhibitor, a topoisomerase inhibitor, a platinum-based antineoplastic agent, or a thymidylate synthase inhibitor.
16. A method of identifying a CHD1L inhibitor that inhibits CHD 1L-dependent TCF transcription comprising determining whether a selected compound inhibits CHD1L atpase.
17. A method of identifying a compound useful for treating cancer comprising determining whether the compound inhibits CHD1L atpase.
18. A pharmaceutical composition for the treatment of cancer comprising one or more compounds, salts or solvates of formula I or formula XX.
19. A compound, salt or solvate of formula I:
Figure FDA0003960268050000051
wherein:
ring B is a heteroaromatic ring or ring system having one, two or three 5-or 6-membered rings, any two or three of which may be fused, wherein the rings are carbocyclic, heterocyclic, aromatic or heteroaromatic, and at least one of the rings is heteroaryl;
in the B ring, each X is independently selected from N or CH, and at least one X is N;
R P is a primary or secondary amine group [ -N (R) 2 )(R 3 )]Or- (M) x A group of-P, wherein P is-N (R) 2 )(R 3 ) Or an aryl or heteroaryl group, wherein x is 0 or 1, represents the absence or presence of M, and M is an optionally substituted linking group- (CH) 2 ) n -or-N (R) (CH) 2 ) n -, wherein each n is independently an integer from 1 to 6 inclusive;
y is selected from the group consisting of-O-, -S-, -N (R) 1 )-、-CON(R 1 )-、-N(R 1 )CO-、-SO 2 N(R 1 ) -or-N (R) 1 )SO 2 -a divalent atom or group of the group consisting;
L 1 is an optional C1 to C4 linking group, which is optionally substituted, and is saturated or contains a double bond (which may be cis or trans), wherein x is 0 or 1, meaning that L is absent or present 1 ；
Ring a is a carbocyclic or heterocyclic ring or ring system having one, two or three rings, two or three of which may be fused, each ring having 3 to 10 carbon atoms and optionally 1 to 6 heteroatoms, and wherein each ring is optionally saturated, unsaturated, or aromatic;
z is a divalent radical containing at least one nitrogen substituted with an R' group,
wherein in embodiments, Z is a divalent group selected from: -N (R ') -, -CON (R ') -, -N (R ') CO-, -CSN (R ') -, -N (R ') CS-, -N (R ') CON (R ') -, -SO 2 N(R′)-、–N(R′)SO 2 -、-CH(CF 3 )N(R′)-、-N(R′)CH(CF 3 )-、-N(R′)CH 2 CON(R′)CH 2 -、-N(R′)COCH 2 N(R′)CH 2 -、
Figure FDA0003960268050000061
Or the divalent Z group comprises a 5-or 6-membered heterocyclic ring having at least one nitrogen ring member, e.g.,
Figure FDA0003960268050000071
L 2 is an optional 1 to 4 carbon linking group, which is optionally substituted, and is saturated or contains a double bond (which may be cis or trans), wherein z is 0 or 1, meaning that L is absent or present 1 ；
R is selected from the group consisting of hydrogen, aliphatic groups, carbocyclyl groups, aryl groups, heterocyclyl groups, and heteroaryl groups, each of which is optionally substituted;
each R' is independently selected from the group consisting of hydrogen, aliphatic groups, carbocyclyl groups, aryl groups, heterocyclyl groups, and heteroaryl groups, each of which is optionally substituted;
R 1 selected from the group consisting of hydrogen, aliphatic groups, carbocyclyl groups, aryl groups, heterocyclyl groups, and heteroaryl groups, each of which is optionally substituted;
R 2 and R 3 Independently selected from the group consisting of hydrogen, aliphatic, carbocyclic, aryl, heterocyclic and heteroaryl groups, each of which is optionally substituted, or
R 2 And R 3 Together with the N to which they are attached form an optionally substituted 5-10 membered heterocyclic ring, which 5-10 membered heterocyclic ring is a saturated, partially unsaturated or aromatic ring;
R A and R B Each represents hydrogen or 1-10 non-hydrogen substituents on the A and B rings or ring systems shown, wherein R A And R B The substituents are independently selected from hydrogen, halogen, hydroxy, cyano, nitro, amino, mono-or di-substituted amino (-NR) C R D ) Alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy, acyl, haloalkyl, -COOR C 、-OCOR C 、-CONR C R D 、-OCONR C R D 、-NR C COR D 、-SR C 、-SOR C 、-SO 2 R C and-SO 2 NR C R D Wherein alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy, and acyl are optionally substituted;
each R C And R D Selected from hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl or heteroaryl, each of which is an alkane optionally substituted with one or more halogen, alkyl, alkenyl, haloalkyl, alkoxy, aryl, heteroaryl, heterocyclyl, arylAlkyl substituted by a group or a heterocyclic group; and
R H is an optionally substituted aryl or heteroaryl group;
wherein the optional substitution comprises amino substituted with one or more halogen, nitro, cyano, amino, mono-or di-C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C1-C3 haloalkyl, C6-C12 aryl, C5-C12 heteroaryl, C3-C12 heterocyclyl, C1-C3 alkoxy, C1-C6 acyl, -COOR E 、-OCOR E 、-CONR E R F 、-OCONR E R D 、-NR E COR F 、-SR E 、-SOR E 、-SO 2 R E and-SO 2 NR E R F Wherein alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, alkoxy, and acyl are optionally substituted, and
each R E And R F Selected from the group consisting of hydrogen, C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C1-C3 haloalkyl, C6-C12 aryl, C5-C12 heteroaryl, C3-C12 heterocyclyl, C1-C3 alkoxy, C1-C6 acyl, each of which is optionally substituted with one or more halogen, nitro, cyano, amino, mono-or di-C1-C3 alkyl substituted amino, C1-C3 alkyl, C2-C4 alkenyl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C1-C3 haloalkyl, C6-C12 aryl, C5-C12 heteroaryl, C3-C12 heterocyclyl, C1-C3 alkoxy, and C1-C6 acyl;
with the exception that the compound is not one of compounds 1-9.
20. A compound of formula XX, or a salt or solvate thereof:
Figure FDA0003960268050000081
and salts or solvates thereof, wherein:
R 10 is-NRy-CO- (L) 2 )y-R 12 or-CO-NRy- (L) 2 )y-R 12 Wherein y is 0 or 1, representsPresence or presence of L 2 ，L 2 Is an optional linking group of 1 to 6 carbon atoms, which linking group is optionally substituted, and wherein one or two carbons of said linking group are optionally replaced by O, NH, NRy or S, wherein Ry is hydrogen or alkyl of 1 to 3 carbons, and R is 12 Is an aryl group, a cycloalkyl group, a heterocyclic group, or a heteroaryl group, each of which is optionally substituted;
R N is an amino moiety-N (R) 2 )(R 3 ) Wherein R is 2 And R 3 Is hydrogen or an optionally substituted alkyl group, or R 2 And R 3 Together with the nitrogen to which they are attached form a 5-10 membered heterocyclic ring, which 5-10 membered heterocyclic ring is saturated or partially unsaturated;
R 1 、R 6 -R 9 represents hydrogen or an optional substituent, with the exception that the compound is not a compound 1-9.
21. A compound selected from compounds 6.5, 6.11-6.29, having the structure given in scheme 1, or a salt or solvate thereof.
22. A compound selected from compounds 6.5, 6.11, 6.16, 6.18, 6.20 and 6.21 having the structures given in scheme 1, or a salt or solvate thereof.
23. A pharmaceutical composition comprising: a compound selected from compounds 6.5, 6.11-6.29, or a salt or solvate thereof, and a pharmaceutically acceptable carrier.
Use of a chd1l inhibitor for the manufacture of a medicament for the treatment of cancer, in particular for the treatment of CRC and mCRC.
25. A CHD1L inhibitor for use in the treatment of cancer, in particular for use in the treatment of CRC and mCRC.
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