WO2023031246A1 - Composés thiophènes substitués utilisés en tant qu'inhibiteurs de d-dopachrome tautomérase - Google Patents

Composés thiophènes substitués utilisés en tant qu'inhibiteurs de d-dopachrome tautomérase Download PDF

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WO2023031246A1
WO2023031246A1 PCT/EP2022/074153 EP2022074153W WO2023031246A1 WO 2023031246 A1 WO2023031246 A1 WO 2023031246A1 EP 2022074153 W EP2022074153 W EP 2022074153W WO 2023031246 A1 WO2023031246 A1 WO 2023031246A1
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pharmaceutically acceptable
compound
naphthalen
acceptable salt
branched
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Zhangping XIAO
Frans Jacobus DEKKER
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Rijksuniversiteit Groningen
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/38—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems

Definitions

  • the present invention relates to substituted thiophene compounds and methods useful for inhibiting D-dopachrome tautomerase (DDT or MIF2).
  • the invention also provides pharmaceutically acceptable compositions comprising compounds of the present invention and methods of using said compositions in the treatment of various disorders, notably cancer, such as NSCLC, and inflammation.
  • Cancer is one of the major public health challenges, which contributes to an estimated annual death toll of ten million worldwide in recent years.
  • targeted cancer treatment has achieved enormous progress over the last decades, its effectiveness is limited by the heterogeneity and acquired therapy resistance of cancers. Therefore, it is important to explore novel anti-cancer drug targets and to develop new therapeutic agents to target them. This could expand the possibilities to employ targeted therapeutic approaches and also increases the possibilities to develop combination therapy regimens.
  • MIF macrophage migration inhibitory factor
  • MIF macrophage migration inhibitory factor
  • MIF_HUMAN macrophage migration inhibitory factor
  • D-dopachrome tautomerase (DDT or MIF2, UniProtKB nr. P30046 (DOPD_HUMAN)) is a structural and functional homolog, but not a backup of MIF (see e.g. Sugimoto et al., Biochemistry 1999, 38, 3268-3279, and lllescas et al., Cytokine 2020, 133).
  • MIF2 and MIF share a high similarity in several aspects. Firstly, the 3D structure shows that the overall folding and a subunit topology of MIF2 and MIF are almost identical, with two p-a-p motifs related by pseudo-2-fold symmetry and similar trimeric p-sheet packing.
  • both MIF2 and MIF harbour enzyme activity to catalyze the keto-enol tautomerization of 4- hydroxylphenylpyruvate (4-HPP) in an active site centered around 1 -proline.
  • both MIF and MIF2 are ligands of CD74 and JAB, that could consequently endow these two proteins with a similar effect on cell growth and tumorigenesis.
  • MIF and MIF2 share just 34% percent amino acid sequence identity. These sequence differences provide differences in interaction sites. For instance, the difference of amino acids inside the tautomerase active sites cause differences in their activity towards keto-enol tautomerisation of 4-HPP.
  • MIF2 does not bind to MIF receptors CXCR2/4 because it lacks pseudo(E)LR motifs which mediate the interactions.
  • MIF2 plays a role that is similar or of even more importance than MIF in cell proliferation. Nevertheless, in contrast to MIF, the development of MIF2-directed therapeutics is lagging behind. The development of small-molecule inhibitors of MIF2 is almost non-explored. So far, 4-(3-carboxyphenyl)-2,5-pyridinedicarboxylic acid (4-CPPC) is the only reversible inhibitor reported to bind to MIF2. As reported in 2019, Bucala et al. (J. Biol. Chem. 2019, 294, 18522-18531) discovered selective MIF2 inhibitor 4-CPPC through virtual screening with an IC50 value of 27 pM on MIF2 tautomerase activity. More importantly, 4-CPPC can also inhibit MIF2-CD74 binding and MIF2 mediated activation of the MAPK pathway as determined by ERK phosphorylation.
  • 4-CPPC can also inhibit MIF2-CD74 binding and MIF2 mediated activation of the MAPK pathway as determined by ERK phosphorylation.
  • 4-iodo-6-phenylpyrimidine 4-IPP
  • 4-IPP covalently binds to Pro1 of MIF2 to interfere with its tautomerase enzyme activity and its biological function.
  • 4-IPP shows low potency on MIF2 inhibition with an IC50 value larger than 100 pM.
  • 4-IPP inhibits MIF with micromolar potency and binds covalently to the active site proline.
  • An over-activation of the MAP/ERK and/or the PI3K/Akt pathway by MIF family member proteins leads to the uncontrolled growth of many cancers (Med Res Rev. 2016, 36(3), 440- 640), including melanoma, acute myeloid leukemia (AML), non-small cell lung cancer (NSCLC), colorectal cancer, ovarian cancer, thyroid cancer, hairy cell leukemia, prostate cancer, glioblastoma, breast cancer, and oral cancer (I nt. J. Mol. Sci. 2020, 21 , 1102; BMC Cancer, 2014, 14(1), 30). Targeting the MAPK pathway in these cancers has provided enormous success in cancer treatment.
  • MIF2 is one of the key activators of the MAP/ERK pathway (PNAS 2011 , 108, E577) and was implicated in the pathogenesis of several types of cancers, such as neuroblastoma (Brain Sci. 2019, 9, 284), melanoma (FASEB J. 2021 , 35, e21671), renal tumor (JBC 2014, 289, 3713), pancreatic cancer (Int. J. Cancer2( ⁇ Q, 139, 2056), NSCLC (J. Immunol. 2008, 181 , 2330), glioblastoma (Oncol Lett. 2018, 16, 2881), and genitourinary cancer (Nat. Rev. Urol. 2019, 16, 318). Therefore, a promising strategy for cancer treatment is via tackling MIF2 activated MAPK pathway over-activation.
  • MIF2 inhibitor preferably a MIF2 inhibitor with an IC50 value of 100 pM or less, even more preferably less than 27 pM, most preferably less than 8 pM. It is a further objective of the present invention to provide a selective MIF2 inhibitor.
  • the present invention relates to compounds of structure (I) or pharmaceutically acceptable salts thereof, wherein
  • R 1 represents H or optionally substituted phenyl
  • R 2 represents H, straight or branched C1.8 alkyl, straight or branched C2-8 alkenyl, or optionally substituted phenyl;
  • R 3 represents straight or branched C1.8 alkyl, straight or branched C2-8 alkenyl, phenyl, benzyl, phenethyl, naphthyl, naphthalen-1-ylmethyl, naphthalen-2-ylmethyl, naphthalen-1-yl-2-ethyl, or naphthalen-2-yl-2-ethyl, all optionally substituted with one or more Cl, F, Br, CH3, C2H5, CC , CF 3 or CBr 3 groups;
  • R 5 represents H, and R 4 represents H or C1.8 alkanoyl; or
  • R 3 the amide group to which R 3 is attached, R 4 , R 5 , and the nitrogen atom to which R 4 and R 5 are attached together form a bivalent radical with structure wherein R 3 is as defined above;
  • R 3 , the amide group to which R 3 is attached, R 4 , R 5 , and the nitrogen atom to which R 4 and R 5 are attached together form a bivalent radical with structure wherein R 6 represents H, Cl, F, Br, or wherein R 6 represents straight or branched Ci-s alkyl, straight or branched C2-8 alkenyl, phenyl, benzyl, phenethyl, naphthyl, naphthalen-1-ylmethyl, naphthalen-2-ylmethyl, naphthalen-1-yl-2-ethyl, or naphthalen-2-yl-2-ethyl, all optionally substituted with one or more Cl, F, Br, C1.4 alkyl, C1.4 alkoxy, CC , CF3 or CBrs groups.
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof according to the invention, and a pharmaceutically acceptable adjuvant, carrier or vehicle.
  • compositions of this invention refers to a nontoxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated.
  • Pharmaceutically acceptable carriers, adjuvants or vehicles that are used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,
  • the invention further relates to a compound or pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the invention for use as a medicament.
  • the invention further relates to a compound or pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the invention for use in the treatment of a medical condition.
  • the invention further relates to a compound or pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the invention for use in therapy.
  • the invention further relates to a compound or pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the invention for use as medicament, wherein the use is as a MIF2 inhibitor.
  • the invention further relates to a compound or pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the invention for use in treating cancer or inflammatory disease.
  • Inflammatory diseases include a vast array of disorders and conditions that are characterized by inflammation. Examples include allergy, asthma, autoimmune diseases, coeliac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, preperfusion injury and transplant rejection.
  • Cancers that may particularly well be treated with the compounds of the invention include melanoma, AML, NSCLC, colorectal cancer, ovarian cancer, thyroid cancer, hairy cell leukemia, prostate cancer, glioblastoma, breast cancer, and oral cancer, neuroblastoma, renal tumors, pancreatic cancer and genitourinary cancer.
  • AML and/or NSCLC may particularly well be treated with the compounds of the invention.
  • the invention further relates to a compound or pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the invention for use in treating AML or NSCLC, most preferably for use in treating NSCLC.
  • the invention further relates to a compound or pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the invention for use in treating a medical disorder, preferably a disorder in which MIF2 inhibition relieves the pathological process, more preferably a disorder in which activation of the MAP/ERK and/or the PI3K/Akt pathway results in uncontrolled cell proliferation.
  • a medical disorder preferably a disorder in which MIF2 inhibition relieves the pathological process, more preferably a disorder in which activation of the MAP/ERK and/or the PI3K/Akt pathway results in uncontrolled cell proliferation.
  • a disorder may in certain cases be characterized by overexpression of MIF2.
  • Disorders in which MIF2 inhibition relieves the pathological process are generally disorders in which inhibition of cell proliferation relieves the pathological process. This is for example the case in cancers and inflammatory diseases.
  • the invention further relates to a method for treating cancer or inflammation in a patient in need thereof, comprising administering to said patient an effective amount of a compound or pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the invention.
  • the invention further relates to a method for treating NSCLC in a patient in need thereof, comprising administering to said patient an effective amount of a compound or pharmaceutically acceptable salt thereof or the pharmaceutical composition according to the invention.
  • the invention further relates to a method for treating AML in a patient in need thereof, comprising administering to said patient an effective amount of a compound or pharmaceutically acceptable salt thereof or the pharmaceutical composition according to the invention.
  • the invention further relates to a method for treating a disorder characterized by overexpression of MIF2 in a patient in need thereof, comprising administering to said patient an effective amount of the compound or pharmaceutically acceptable salt thereof or the pharmaceutical composition according to the invention.
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • R 1 represents wherein R 8 and R 9 each independently represent H, F, Cl, Br, straight or branched C1.4 alkyl, CC , CBrs, or CF3, or wherein R 8 and R 9 are attached to adjacent carbon atoms and together with these adjacent carbon atoms form a five- or six-membered homo- or heterocyclic ring, for example a dioxolane ring with structure .
  • at least one of R 8 and R 9 is F, Cl, Br, CCl3, CBr3, or CF3.
  • both of R 8 and R 9 are F, Cl, Br, CCl3, CBr3, or CF3.
  • both R 8 and R 9 are F, Cl or Br, preferably both R 8 and R 9 are Cl.
  • one of R 8 and R 9 is H, and the other is CCl 3 , CBr 3 , or CF 3 , preferably CF 3 .
  • R 1 represents .
  • R 2 represents H, straight or branched C1-4 alkyl or straight or branched C2-4 alkenyl. More preferably R 2 represents straight or branched C1-4 alkyl or straight or branched C2-4 alkenyl. Even more preferably R 2 represents methyl or ethyl. It is especially preferred that R 2 represents methyl.
  • R 3 represents benzyl, phenethyl, naphthyl, naphthalen-1-ylmethyl, naphthalen-2- ylmethyl, naphthalen-1-yl-2-ethyl, or naphthalen-2-yl-2-ethyl, all optionally substituted with one or more Cl, F, Br, CH 3 , C 2 H 5 , CCl 3 , CF 3 or CBr 3 groups.
  • R 1 represents , wherein R 8 and R 9 each independently represent H, F, Cl, Br, straight or branched C1-4 alkyl, CCl3, CBr3, or CF3, or wherein R 8 and R 9 are attached to adjacent carbon atoms and together with these adjacent carbon atoms form a five- or six-membered homo- or heterocyclic ring, for example a dioxolane ring with structure
  • R 2 represents straight or branched C1-4 alkyl or straight or branched C2-4 alkenyl, most preferably methyl
  • R 3 represents benzyl, phenethyl, naphthyl, naphthalen-1-ylmethyl, naphthalen-2-ylmethyl, naphthalen-1-yl-2-ethyl, or naphthalen-2-yl-2-ethyl, all optionally substituted with one or more Cl, F, Br, CH3, C2H5, CCl3, CF3 or CBr
  • R 3 represents naphthalen- r naphthalen-1-yl-2- It is especially preferred that R 3 represents .
  • R 4 and R 5 represent H.
  • R 3 , the amide group to which R 3 is attached, R 4 , R 5 , and the nitrogen atom to which R 4 and R 5 are attached together form a bivalent radical with structure wherein R 3 represents naphthalen-1-ylmethyl naphthalen-1-yl-2-ethyl
  • R 3 , the amide group to which R 3 is attached, R 4 , R 5 , and the nitrogen atom to which R 4 and R 5 are attached together form a bivalent radical with structure wherein R 6 represents Cl, F, Br, or phenyl optionally substituted with one or more Cl, F, Br, C 1-4 alkyl, C 1-4 alkoxy,, CCl 3 , CF 3 or CBr 3 groups, preferably wherein R 6 represents phenyl substituted with one or
  • the pharmaceutical compositions further comprises a MIF inhibitor. This enables dual targeting of MIF and MIF2 at the same time, which will provide a synergistic effect and thereby highly effective treatment of diseases in which inhibition of cell proliferation provides relief, such as cancer and inflammatory diseases.
  • Figure 1 A-D display the results of NSCLC cell proliferation assays with 5d.
  • Figure 2 A and B display the results of colony formation experiments with NSCLC cells treated with 5d.
  • Figure 3 A and B display the results of growth experiments with A549 cancer cells treated with 5d in a spheroid model.
  • Figure 4 A and B display the results of cell cycle arrest experiments with A549 cancer cells treated with 5d.
  • Figure 5 A and B display the results of MIF2-induced ERK phosphorylation experiments in A549 cancer cells treated with 5d.
  • FIG. 1 5d treatment inhibits cell proliferation of NSCLC cells.
  • DMSO vehicle
  • FIG. 5d treatment inhibits colony formation of NSCLC cells.
  • A A549 or other NSCLC cells were seeded in 12-well plate at a density of 1000 cells/well and incubated overnight. The cells were then treated with compounds or vehicle (DMSO) for 5 days. Afterwards, cells were fixed and stained with 0.5% crystal violet solution. The image of the representative well was scanned and shown.
  • FIG. 3 5d treatment inhibits growth of A549 cancer cells in a spheroid model.
  • A549 cells were seeded in an ultralow attachment 96-well round-bottomed plate (1000 cells/well) to generate tumor spheroids (a single spheroid per well). After initiation, the spheroids were treated with 5d at the indicated concentrations every three days. DMSO was used as vehicle control. The day of the first treatment was indicated as day 0.
  • A Representative images were obtained at the indicated intervals using an inverted microscope. Scale bar: 500 pm.
  • FIG. 5d induces cell cycle arrest in A549 cells.
  • A A549 cells were treated with 5d at the indicated concentrations for 48 h. The graphs show the representative cell cycle distribution of propidium-iodide stained cells assessed by flow cytometry.
  • B Relative number of cells in each stage of the cell cycle (G0/G1, S and G2/M phases) were analyzed by FlowJo. Data are shown as mean ⁇ SD of three replicates, t-test analysis was performed between G2/M phase of treated groups and control group. *p ⁇ 0.05 and **p ⁇ 0.01 vs vehicle group.
  • FIG. 5d inhibits the MIF2-induced ERK phosphorylation in A549 cells.
  • B Quantification of the pERK level using pERK:ERK ratio, normalized to control group. GAPDH was used as a loading control on western blots. Data are shown as mean ⁇ SD. **p ⁇ 0.01 and ***p ⁇ 0.001 vs vehicle group.
  • the 2-aminothiophenes were acylated by different acylchlorides to prepare the desired 2-amide substituted products 4a-c with yields of 44-58%.
  • the 2-aminothiophenes were also employed to synthesize thieno[2,3-d]pyrimidine-2,4(1/7,3/7)-diones 5a-e using 1 ,1'-carbonyldiimidazole (GDI) as coupling reagent with yields of 40-91%.
  • GDI 1 ,1'-carbonyldiimidazole
  • Same scaffold in 7a-i and 11a-b was synthesized using different method, in which isocyanates were reacted with 2-aminothiophenes to make ureas before cyclized by MeONa with overall yields of 20-62%.
  • the reaction mixture was stirred overnight at 40 °C, followed by the addition of 1 N HCl (25 mL) and then extracted with EtOAc (3 ⁇ 20 mL). The combined organic layers were washed with 1 N NaOH (25 mL), dried over MgSO4, filtrated and the solvent was removed under reduced pressure. The resulting mixture was dissolved in EtOH (5 mL), S8 (32mg, 1.0 mmol) and Et3N (0.2 mL) were added. The reaction mixture was refluxed overnight. Then, the mixture was diluted with EtOAc (25 mL) and washed with water (2 ⁇ 50 mL) and brine (2 ⁇ 50 mL).
  • N-allyl-2-amino- 5-(4-chlorophenyl)thiophene-3-carboxamide 0.3 g, 1 mmol was reacted with CDI (0.5g, 3.0 mmol) following a similar method for synthesis of 5a to provide 5b as 126 mg light-yellow solid as product. Yield 40%.
  • Ethyl 2-amino-4-methyl-5-phenylthiophene-3-carboxylate, 6a A mixture of phenolacetone (1f, 0.7 mL, 5 mmol), ethyl cyanoacetate (0.5 mL, 5mmol), ammonium acetate (0.1 g, 1 mmol), and acetic acid (0.2 mL, 4 mmol) in toluene (5 mL) was heated under reflux for 20 h, while water was removed using molecular sieve. After the mixture was cooled to room temperature, the mixture was concentrated in vacuo. The residue was diluted with saturated NaHCO3 (20 mL) and extracted with CHCl3 (3 ⁇ 25 mL).
  • the fractions containing MIF2 were brought to 1.7 M ammonium sulfate and loaded on a phenyl sepharose column (GE Healthcare) and eluted with a gradient to 0 M ammonium sulfate in a 20 mM sodium phosphate buffer, pH 8.0.
  • the proteins were purified by size exclusion chromatography on a Superdex75 column (GE Healthcare) in 20 mM sodium phosphate buffer, pH 8.0, with an elution volume characteristic for trimeric MIF2.
  • the collected protein was concentrated using a VivaSpin centrifugation column with a molecular weight cut off at 5000 Da (Sartorius Stedim Biotech GmbH).
  • Purified proteins were aliquoted, snap frozen in liquid nitrogen, and stored at -80 °C. Purity of obtained protein was tested by SDS-PAGE and coomassie staining.
  • MIF2-catalyzed tautomerization assay To facilitate the effective assessment of binding potency of MIF2 inhibitors, a convenient and reliable assay was needed.
  • the most widely used assay for MIF inhibitor evaluation is the 4-HPP based tautomerization assay, in which the potency on inhibition of MIF-catalyzed 4-HPP tautomerization is applied to reflect the binding affinity of tested compound to MIF (Ouertatani-Sakouhi et al., J. Biomol. Screen. 2010, 15, 347-358). This assay was also applied to assess MIF2 binder in previous studies (Tilstam et al., J. Biol. Chem. 2019, 294, 18522-18531).
  • MIF2 losses two hydrogen bonds formed between Asn97 of MIF and the 4-position hydroxyl group of 4-HPP, as the Arg98 locates at the corresponding placement of MIF2. Accordingly, it was hypothesized that MIF2 could exhibit different activity on catalysis the keto-enol tautomerization of 4-HPP analogues with different substituents at 4-position. Therefore, a more active substrate for MIF2 was sought in order to build up a convenient and sensitive enzymatic assay.
  • the protocol for measuring MIF tautomerase enzyme activity and enzyme kinetics was described in Xiao et al., Eur. J. Med. Chem. 2020, 186, 111849-111862.
  • the methods of enzyme study on MIF2 was adapted from the protocol of the MIF study. Briefly, 180 pL of a 500 nM MIF2 solution in boric acid buffer (435 mM, pH 6.2) was mixed with 10 pL of a 20 mM EDTA solution in demiwater and 10 pL of a solution of the desired compound dissolved in DMSO or blank DMSO. This mixture was pre-incubated at room temperature for 10 min.
  • the window coefficient (Z-factor) of this assay was proved to be 0.75 in this setup, which indicates the quality of this assay is sufficient for medium- to high-throughput applications (0.5-1) (Zhang et al., J. Biomol. Screen. 1999, 4, 67-73).
  • the IC50 values of synthesized compounds on inhibition of MIF2 tautomerase activity are shown in Table 1 - 3.
  • 5c has the lowest IC50 value.
  • solubility of 5c is only 3.3 pg/mL (7.4 pM), which would be a limitation for the following cell assays.
  • Both 5d and 5e have improved solubility in aqueous solution with saturated concentrations of 16 pg/mL (36 pM) and 15 pg/mL (33 pM) for 5d and 5e, respectively.
  • 5d exhibits a comparable potency as 5c, while 5e is less active.
  • the toxicity of 5d was investigated using an MTS assay, which indicated that 5d did not inhibit cell viability from a concentrations of 10 pM or lower for a treatment of 24 hours.
  • Cell proliferation assay Cell proliferation was measured with the CyQUANT® Direct Cell Proliferation Assay Kit (Thermo Fisher, #C35011) by following the protocol. Cells were cultured in 96-well plates at a density of 1 ,000 cells/well and treated with different concentrations of 5d (0.25-10 pM) for 72 h. Cells were incubated with detection reagent (100 pL) for 60 min at 37 °C with 5% CO2. The fluorescence of each well was read at 485/535nm by plate reader (BioTek).
  • Clonogenic assay Cells were seeded in 12-well plates (1000 cells per well in 2 mL of RPMI medium (#61970-010, Gibco) containing 10% (v/v) fetal bovine serum (FBS) and 100 U/rnL penicillin/streptomycin (#10378016, Gibco)) and incubated overnight. The cells were treated with corresponding inhibitors for 5 days. Subsequently, the medium was carefully removed, and cells were fixed with 4% (v/v) paraformaldehyde for 20 min and stained with 0.5% (w/v) crystal violet for 20 min. After washing, the image of each well was photographed. To quantify the staining, 10% acetic acid was utilized to dissolve the colonies. The absorbance at a wavelength of 590 nm was measured to represent the relative cell number by comparing with the DMSO-treated group.
  • Tumor spheroid assay A 3D spheroid model was employed to investigate the effect of longer-term 5d treatment in a more complex model of tumor growth.
  • the 3D spheroid model was established using A549 cancer cells by a method adapted from Feng et al. (J. Med. Chem. 2015, 58, 6456-6480). Each spheroid was prepared from 1000 A549 cells. The A549 cells (1000 cells/well) were seeded onto a 96-well round-bottomed ultra low attachment plate (Corning). After 2 days of incubation without disturbance, the spheroid was treated with indicated compound every 3 days. Images were captured and the diameter of each tumor spheroid were measured on the indicated days post-treatment using an inverted microscope (Nikon Eclipse Ti) connected with a NIS-elements software. The data were analyzed and plotted with Graphpad Prism8.
  • these spheroids were treated with 1, 2, or 5 pM of 5d with 72 hours intervals over 12 days. Spheroid growth was monitored by measuring the diameter and this was compared to day 0 of the treatment. The tumor spheroids treated with 5d were significant smaller compared to the control group (see Figure 3). With continuous exposure to 1 , 2, or 5 pM of 5d for 12 days, the growth of the spheroid tumor volume was inhibited by 40%, 63%, and 79%, respectively. These results indicate that the MIF2 tautomerase inhibitor 5d effectively inhibits proliferation of A549 cancer cells in a spheroid tumor model.
  • Flow cytometry For cell cycle analysis, A549 cells were seeded in 6-well plates at a density of 1x10 5 per well. The next day, the cells were treated with 5d or vehicle for 48h. Subsequently, the cells were washed with PBS (3x) and then harvested after trypinzation. After centrifugation at 300g, the cells were incubated with a solution containing 20 pg/mL propidium iodide (PI) (Sigma, P4864) and 0.1% (v/v) Triton-X100 (Sigma, T8787) for 15 min at room temperature. Fluorescence was detected by a Cytoflex flow cytometer (Beckman Coulter, Woerden, the Netherlands) immediately. 30,000 cells were collected for each sample. Data were analyzed using FlowJo software (Tree start, Ashland, USA).
  • the effect of 5d on cell cycle progression was analyzed using flow cytometry.
  • A549 cells were treated with different concentration of 5d for a duration of 48 hours before analysis.
  • the results showed that 5d dose-dependently induced cell cycle arrest at the G0/G1 phase (see Figure 4).
  • the percentage of A549 cells in G0/G1 phases was 56% for the control group. This percentage increased to 58%, 63%, and 67% upon treatment with 5, 7.5, and 10 pM 5d, respectively. This result indicate 5d induces inhibition of cell cycle progression, which can explain the observed inhibition of cell proliferation.
  • A549 cells (3x10 5 cells per well) were seeded into each well of a 6-well plate with 2 mL RPMI-1640 medium containing 0.5% FBS (Costar Europe, Badhoevedorp, The Netherlands), and 1 % penicillin/streptomycin solution (Corning). After overnight culturing, the cells were stimulated with MIF2 (100ng/mL in FBS free medium) or a mixture of MIF2 and different concentration of 5d for 15 minutes. After that, cells were lysed by RIPA buffer containing 1 * PhosSTOP and protease inhibitor (PI) cocktail (Roche, Mannheim, Germany).
  • PI protease inhibitor
  • the BCA Protein Assay Kit (Pierce, Rockford IL, USA) was used to determine the protein concentration. 20 pg protein was separated by a pre-cast 10% NuPAGE Bis-Tris gel (Invitrogen, USA) and then transferred to a polyvinylidene difluoride (PVDF) membrane.
  • PVDF polyvinylidene difluoride
  • the membrane was treated with an HRP-conjugated secondary goat anti-rabbit antibody (#P0448, Dako, 1 :2000) or rabbit anti-mouse antibody (#P0260, Dako, 1 :2000) at room temperature for 1 hour.
  • the protein bands were visualized with enhanced chemiluminescence (ECL) solution (GE Healthcare).
  • ECL enhanced chemiluminescence
  • 5d inhibits ERK signaling.
  • the effect of treatment with 5d on MIF2-related signaling pathways was investigated by assessment of MIF2-induced ERK phosphorylation using western blot analysis.
  • A549 cells were stimulated with MIF2 or 5d preincubated MIF2 for 15 minutes, subsequently ERK phosphorylation was detected using western blot.
  • MIF2 stimulated the ERK phosphorylation level of A549 cells to about 4.5 folds of control. This stimulation was attenuated by 5d in a dose-dependent manner (see Figure 5). After pre-incubation with 10 pM 5d, MIF2 only activated the ERK phosphorylation level to two folds of control.
  • R 1 represents H or optionally substituted phenyl
  • R 2 represents H, straight or branched C1-8 alkyl, straight or branched C2-8 alkenyl, or optionally substituted phenyl
  • R 3 represents straight or branched C1-8 alkyl, straight or branched C2-8 alkenyl, phenyl, benzyl, phenethyl, naphthyl, naphthalen-1-ylmethyl, naphthalen-2-ylmethyl, naphthalen-1-yl-2-ethyl, or naphthalen-2-yl-2-ethyl, all optionally substituted with one or more Cl, F, Br, CH 3 , C 2 H 5 , CCl 3 , CF 3 or CBr 3 groups
  • R 5 represents H
  • R 4 represents H or C 1-8 alkanoyl
  • R 3 the amide group to which R 3 is attached, R 4 , R 5 , and the nitrogen atom to which R
  • R 1 represents , wherein R 8 and R 9 each independently represent H, F, Cl, Br, straight or branched C1-4 alkyl, CCl3, CBr3, or CF3, or wherein R 8 and R 9 are attached to adjacent carbon atoms and together with these adjacent carbon atoms form a five- or six-membered homo- or heterocyclic ring, for example a dioxolane ring with structure .
  • R 8 and R 9 is F, Cl, Br, CCl3, CBr3, or CF3.
  • R 3 represents benzyl, phenethyl, naphthyl, naphthalen-1- ylmethyl, naphthalen-2-ylmethyl, naphthalen-1-yl-2-ethyl, or naphthalen-2-yl-2-ethyl, all optionally substituted with one or more Cl, F, Br, CH 3 , C 2 H 5 , CCl 3 , CF 3 or CBr 3 groups.
  • composition comprising a compound according to any one of the preceding clauses or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant, carrier or vehicle.
  • composition according to clause 15 further comprising a macrophage migration inhibitory factor (MIF) inhibitor.
  • MIF macrophage migration inhibitory factor
  • Clause 17 Compound or pharmaceutically acceptable salt thereof according to any one of clauses 1 - 14, or a pharmaceutical composition according to clause 15 or 16 for use as a medicament.
  • Clause 18 Compound or pharmaceutically acceptable salt thereof according to any one of clauses 1 - 14 or a pharmaceutical composition according to clause 15 or 16 for use in treating cancer or inflammatory disease, preferably for use in treating cancer, more preferably for use in treating a cancer chosen from the group consisting of melanoma, non-small-cell lung carcinoma (NSCLC), colorectal cancer, ovarian cancer, thyroid cancer, hairy cell leukemia, prostate cancer, glioblastoma, breast cancer, oral cancer, neuroblastoma, renal tumors, pancreatic cancer and genitourinary cancer, more preferably for use in treating NSCLC.
  • NSCLC non-small-cell lung carcinoma
  • Clause 19 Compound or pharmaceutically acceptable salt thereof according to any one of clauses 1 - 14 or a pharmaceutical composition according to clause 15 or 16 for use as medicament, wherein the use is as a MIF2 inhibitor.
  • Clause 20 Compound or pharmaceutically acceptable salt thereof according to any one of clauses 1 - 14 or a pharmaceutical composition according to clause 15 or 16 for use in treating a disorder in which DDT tautomerase inhibition relieves the pathological process, preferably a disorder in which activation of the MAP/ERK and/or the PI3K/Akt pathway results in uncontrolled cell proliferation, more preferably a disorder which is characterized by overexpression of MIF2.
  • Clause 21 Method for treating cancer or inflammatory disease in a patient in need thereof, comprising administering to said patient an effective amount of the compound or pharmaceutically acceptable salt thereof of any one of clauses 1 - 14 or the pharmaceutical composition according to clause 15 or 16.
  • Clause 22 Method for treating NSCLC in a patient in need thereof, comprising administering to said patient an effective amount of the compound or pharmaceutically acceptable salt thereof of any one of clauses 1 - 14 or the pharmaceutical composition according to clause 15 or 16.
  • Clause 23 Method for treating a disorder characterized by overexpression of MIF2 in a patient in need thereof, comprising administering to said patient an effective amount of the compound or pharmaceutically acceptable salt thereof of any one of clauses 1 - 14 or the pharmaceutical composition according to clause 15 or 16.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des composés thiophènes substitués et des procédés utiles pour inhiber la D-dopachrome tautomérase. L'invention concerne également des compositions pharmaceutiquement acceptables comprenant des composés de la présente invention et des procédés d'utilisation desdites compositions dans le traitement de divers troubles, notamment le cancer, tels que NSCLC et l'inflammation.
PCT/EP2022/074153 2021-09-01 2022-08-31 Composés thiophènes substitués utilisés en tant qu'inhibiteurs de d-dopachrome tautomérase WO2023031246A1 (fr)

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NL2029098 2021-09-01
NL2029098A NL2029098B1 (en) 2021-09-01 2021-09-01 Substituted thiophene compounds as D-dopachrome tautomerase inhibitors

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