Phosphaphenalen-gold(I) complex as a chemotherapeutic agent for glioblastoma

Abstract

Glioblastoma is one of the most deadly brain tumors. Difficulties in treating glioblastoma are related to, among other things, the brain's sensitivity to drugs, limited brain penetration of chemotherapy agents, and tumor cell resistance to conventional treatments. Provided are specific phosphaphenalene-gold(I) complexes, pharmaceutical compositions containing such complexes, and kits for use as pharmaceuticals, particularly in the treatment of brain cancers such as glioblastoma.

Description

[Technical Field]

[0001] The present invention relates to a phosphaphenalene-gold(I) complex used as a pharmaceutical, particularly in the treatment of brain cancers such as glioblastoma, pharmaceutical compositions and kits containing such a complex, and the use of such a complex to inhibit the activity of thioredoxin reductase (TrxR) in vitro / ex vivo. [Background technology]

[0002] Glioblastoma (GBM) is the most common and malignant human brain tumor, with a survival time of only about 15 months. Several main reasons for this include rapid tumor cell proliferation, tumor heterogeneity, genetic instability, and highly invasive growth. Due to its highly invasive growth in particular, systemic therapy targeting disseminated tumor cells that cannot be surgically removed is necessary. Therefore, current treatment consists of surgery followed by radiation and temozolomide-based combination chemotherapy. However, the 1-year, 2-year, and 5-year survival rates are still very low, at 40%, 17.4%, and 5.6%, respectively, and at least some populations of tumor cells show considerable resistance to this type of treatment.

[0003] It has been hypothesized that this may be due to an immature, highly oncogenic population of cells possessing stem cell-like characteristics such as reduced self-renewal and sensitivity to chemotherapy, and that this may be the cause of tumor recurrence.

[0004] These findings highlight the urgent need to develop more effective therapeutic agents that can prevent tumor regrowth by eradicating the entire tumor cell population.

[0005] A further challenge for novel therapeutic agents to overcome the resistance of brain tumor cells to current chemotherapy is the need for them to cross the blood-brain barrier (BBB) ​​and exert tumor-selective activity. In this respect, gold(I) complexes are promising compounds that possess the ability to cross the BBB (Non-Patent Literature 1).

[0006] One of the most widely accepted hypotheses regarding the mechanism of action of gold complexes involves the specific inhibition of thioredoxin reductase (TrxR), an enzyme involved in cellular redox homeostasis that has been shown to be overexpressed in tumor cells (Non-Patent Literature 2).

[0007] Gold(I) complexes hold great potential for selective inhibition of TrxR, as these complexes have the ability to specifically interact with the SH / Se center of the thioredoxin enzyme, inhibit its activity, and ultimately induce cell apoptosis (Non-Patent Literature 3).

[0008] Nevertheless, while the use of gold complexes in medicine and chrysotherapy dates back to ancient Egypt, the clinical application of the latter gold complexes is quite low today. Some authors attribute this fact to the "poor stability" and "solubility" of the compounds tested (Non-Patent Document 4; and Non-Patent Document 2 cited above).

[0009] Currently, the gold complexes most investigated for cancer treatment are auranofin™ and its derivatives. Their general structure consists of a linear molecule having a trisubstituted phosphine ligand (fragment A) bonded to an Au atom, to which an additional anionic ligand (fragment B) is attached. [ka]

[0010] Mechanistic investigations revealed that both ligands modulate the antitumor activity of the gold complex (Non-Patent Literature 2 cited above). First, fragments A and B must provide sufficient solubility in aqueous media to ensure the physiological activity of the complex. Second, fragment B must be unstable enough to allow the initial coupling of gold with a specific carrier enzyme. Next, the electronic properties of fragment A play a crucial role in actively inhibiting TrxR to reach the target species (i.e., R3P-Au +It must provide robust stability against [unspecified factors]. A weak P-Au bond leads to hydrolysis, irreversible oxidation of phosphorus, and formation of an inactive gold colloid. Furthermore, the lipophilicity and steric hindrance of fragment A are fundamental to the penetration of the gold portion into cells (Non-Patent Literature 3 cited above). Thus, enhancing the physiological activity of the molecular system is a challenging task because it is the result of various synergistic properties.

[0011] The most commonly used part of fragment A is the homotrisubstituted phosphine. The phosphorus-containing part, particularly the heterocycle of fragment A, has been little tested for cancer treatment. Only the 2,5-diarylphosphole complex, based on the five-membered heterocycle, has been used to date.

[0012] Non-patent document 5 reports the chlorogold complex "[1-phenyl-2,5-di(2-pyridyl)phosphole]AuCl" as a novel gold phosphole inhibitor (GoPI) capable of inhibiting human glutathione reductase, and further reports that GoPIs exhibit IC50 against NCH82 and NCH89, respectively. 50 The study demonstrated that values ​​of 12.5±0.8 μM and 10.8±0.8 μM inhibited the proliferation of glioblastoma cell lines.

[0013] The effects of phosphole-containing gold and platinum complexes on human glutathione reductase (hGR) and human thioredoxin reductase (hTrxR), as well as their growth inhibitory effects on tumor cells, were described in Non-Patent Literature 6. The antiproliferative effects of five different phosphole-containing complexes on NCH82 and NCH89 (IC) 50 The reported concentrations were 7.2 μM to 81.8 μM and 10.8 μM to 87.4 μM, respectively.

[0014] Furthermore, Non-Patent Document 7 evaluated the cytotoxic activity of different compounds, particularly phosphole-containing gold and platinum complexes, against the human breast cancer MCF-7 cell line.

[0015] Non-patent document 1 (cited above) describes the antitumor properties of the gold(I) complex 1-phenyl-bis(2-pyridyl)phosphorus gold chloride thio-β-d-glucosetetraacetic acid (GoPI-sugar), which exhibits antiproliferative effects against human (NCH82, NCH89) and rat (C6) glioma cell lines, and the effects of GoPI-sugar on thioredoxin reductase (IC). 50 4.3nM) and human glutathione reductase (IC) 50 It was reported that it inhibits (88.5 nM).

[0016] The cytotoxic activity of different phosphole gold complexes is also disclosed in Patent Document 1.

[0017] However, prior art has reported that some of these phosphole gold complexes are unstable in aqueous solutions (Non-Patent Document 7 cited above).

[0018] Therefore, the object of the present invention is to provide a compound that, apart from the prior art, has improved stability, can cross the blood-brain barrier, can inhibit the proliferation of tumor cells, and can sensitize tumor cells to induce apoptosis, and can be used as a pharmaceutical, particularly in the treatment of brain cancers such as glioblastoma. A further object is to provide pharmaceutical compositions and kits containing such compounds. [Prior art documents] [Patent Documents]

[0019] [Patent Document 1] U.S. Patent No. 7923434 [Non-patent literature]

[0020] [Non-Patent Document 1] Jortzik E. et al. "Antiglioma activity of GoPI-sugar, a novel gold(I)-phosphole inhibitor: Chemical synthesis, mechanistic studies, and effectiveness in vivo", Biochimica et Biophysica Acta (BBA).

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[0021] These objectives have been addressed by the compounds for use described in claim 1, the pharmaceutical compositions described in claim 10, and the kits described in claim 14. The subject of the present invention is further the use of these compounds to inhibit the activity of thioredoxin reductase (TrxR) described in claim 15 in vitro / ex vivo.

[0022] According to the present invention, formula (A) used as a pharmaceutical: [ka] (In the formula, ArI represents a monocyclic aromatic moiety selected from the group consisting of phenyl, pyridine, pyrrole, N-protected pyrrole, furan, thiophene, and a 7-membered aromatic monocycle, or a bicyclic aromatic moiety selected from the group consisting of naphthalene, indole, and benzothiophene. ArI is a five- or six-membered aromatic heterocycle containing a halogen atom (preferably selected from Cl, Br, I, and F), N, S, or O, and C 1~6 Aliphatic groups and C 3~6 Alicyclic group (C 1~6An aliphatic group and / or C 3~6 The alicyclic group may be further substituted by one or more substituents selected from the group consisting of (which may further contain one or more heteroatoms selected from N, S, and O). ArII and ArIII each independently represent a benzene group, a pyridine group, a pyrrole group, an N-protected pyrrole group, or a thiophene group. R 1 represents an aromatic group, a hydroxy group, a C1-C6 alkyl group or a C1-C6 alkoxy group, preferably a phenyl group. X is selected from the group consisting of a sugar, albumin, a halogen atom, CH3, NO3, CN, and SR 3 and is selected from the group consisting of R 3 is 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl, β-D-glucopyranosyl, 2,3,4,6-tetramesyl-β-D-glucopyranosyl, 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl, 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl, hepta-O-acetyl-β-maltosyl, 1,2-O-isopropylidene-5-α-D-xylofuranosyl, C1-C8 alkyl, CH(CO2H)CH2CO2H, 2-morpholinoethyl, 2'-ethyl-1-β-D-glucopyranosyl, 2'-ethyl-1-thio-β-D-glucopyranosyl, glutathione hydrochloride, CN, C(NH2)2·HCl, C(NH2)NHNH2, phenyl, 1-aminophenyl, 2-pyridyl, 6-methyl-2-pyridyl, 4-pyridyl, thiazolin-2-yl, 4,5-dihydrothiazol-2-yl, 1H-benzimidazol-2-yl, benzoxazol-2-yl, benzothiazol-2-yl, (CH2CH2OH)2NO3 - , pyrimidin-2-yl, 4-methylpyrimidin-2-yl, 4,6-dimethylpyrimidin-2-yl, 1,2-dihydropyridin-2-yl, 1,2-dihydropyrimidin-2-yl, 9H-purin-6-yl, 2-amino-9H-purin-6-yl, and (NH2)2C= and compounds selected from the group consisting of) are provided.

[0023] 49>The compounds according to the present invention are based on a condensed six-membered phosphorus heterocycle, which is a derivative of phosphaphenalene. To date, six-membered phosphorus derivatives have not been investigated for cancer treatment. Furthermore, the compounds according to the present invention have structural and electronic properties that differ significantly from phosphine and phosphate, which are known to date as potential chemotherapeutic agents.

[0024] The preparation of chlorogold(I) complexes based on phosphaphenalene systems was described in Romero-Nieto C. et al., "Paving the Way to Novel Phosphorus-Based Architectures: A Noncatalyzed Protocol to Access Six-Membered Heterocycles", Angew. Chem. Int. Ed. 2015, 54(52), 15872-15875.

[0025] It has now been discovered that the compound according to the present invention not only exhibits unexpectedly high stability in a dimethyl sulfoxide / H2O solution, but also shows cytotoxic effects.

[0026] According to the present invention, in formula (A) above, it is preferable that both ArII and ArIII represent a naphthalene group, an indole group, an N-protected indole group, a quinoline group, an N-protected quinoline group, or a benzothiophene group.

[0027] According to a preferred embodiment of the present invention, both ArII and ArIII represent a naphthalene group.

[0028] ArI is a benzene group, naphthalene group, thiophene group, furan group, pyrrole group, benzothiophene group, or pyridine group, and ArI is a halogen atom (preferably selected from Cl, Br, I, and F), a 5-membered or 6-membered aromatic heterocycle containing N, S, or O, C 1~6 Aliphatic groups and C 3~6 Alicyclic group (C 1~6 Aliphatic groups and / or C 3~6It is even more preferable that the alicyclic group is substituted with one or more substituents selected from the group consisting of one or more heteroatoms selected from N, S, and O.

[0029] According to a preferred embodiment, ArI is not substituted. According to another preferred embodiment, ArI is substituted with one or two, more preferably one, substituents selected from the group described above.

[0030] In the above formula (A), ArI is preferably selected from the group consisting of phenyl, pyridine, pyrrole, N-protected pyrrole, furan, and thiophene. According to a preferred embodiment, ArI is a thiophene group, and more preferably an unsubstituted thiophene group.

[0031] According to a preferred embodiment, ArI is a pyrrole group, preferably having a methyl group or a phenylsulfonyl group as a substituent on the N atom, and particularly preferably an N-substituted pyrrole group having a methyl group as a substituent on the N atom.

[0032] According to a preferred embodiment, X in formula (A) above is selected from the group consisting of Cl, xanthate, thiocyanide, and 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate. It is more preferable that X is xanthate or 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate, and even more preferable that X is 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate.

[0033] According to a particular embodiment of the present invention, ArI is an N-substituted pyrrole group having a methyl group as a substituent on the N atom, and ArII and ArIII both represent a naphthalene group, R 1 X is a phenyl group, and X is 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate.

[0034] According to one embodiment of the present invention, the compound of the present invention is compound 1. According to another embodiment of the present invention, the compound of the present invention is compound 2. According to yet another embodiment of the present invention, the compound of the present invention is compound 3. According to yet another embodiment of the present invention, the compound of the present invention is compound 4.

[0035] According to another embodiment of the present invention, the compound of the present invention is compound 5. According to another embodiment of the present invention, the compound of the present invention is compound 6. According to another embodiment of the present invention, the compound of the present invention is compound 7. According to another embodiment of the present invention, the compound of the present invention is compound 8. It should be understood that compounds 5 through 8, regardless of their specific uses provided herein, constitute a compound of the present invention in themselves.

[0036] The protecting groups for ArI, ArII, and ArIII, namely N-protected pyrrole groups, N-protected indole groups, and / or N-protected quinoline groups, are preferably selected from Si(CH3)3, SO2Ph, and sugars. However, other suitable protecting groups commonly known in the art can also be used.

[0037] With respect to the position of the heteroatom (or possibly multiple heteroatom) in the aforementioned cyclic group containing the heteroatom, the compounds according to the present invention include all possible structural isomers.

[0038] According to one embodiment of the present invention, the present invention relates to formula (B): [ka] The present invention provides a compound of the form (wherein R is methyl or SO2Ph, X is chloride or 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate, preferably R is methyl and X is 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate). According to one embodiment of the present invention, the above compound is provided for use as a pharmaceutical.

[0039] According to a preferred embodiment, the compound according to the present invention is used in the treatment of cancer.

[0040] In a more preferred embodiment, the compound according to the present invention is used for the treatment of brain cancer, and preferably for the treatment of glioblastoma.

[0041] The subject of the present invention is further a pharmaceutical composition comprising a compound according to the present invention and at least one pharmaceutically acceptable excipient. The pharmaceutical composition is preferably characterized by being administered intravenously.

[0042] Furthermore, in the pharmaceutical composition according to the present invention, the compound is preferably soluble in an aqueous solution containing DMSO, preferably soluble in a mixture of water and DMSO, and more preferably soluble in water containing 5% to 20% by volume of DMSO. Preferably, the pharmaceutical composition described herein is used as a pharmaceutical.

[0043] According to one preferred embodiment, the pharmaceutical composition is used for the treatment of cancer, more preferably for the treatment of brain cancer, even more preferably for the treatment of glioblastoma, brain metastases, meningioma, IDH mutation glioma, or head and neck cancer, and particularly preferably for the treatment of glioblastoma.

[0044] The present invention further relates to a kit comprising at least the compound and container according to the present invention as described above.

[0045] The subject of the present invention is further the use of the compounds according to the present invention, as described above, to inhibit the activity of thioredoxin reductase (TrxR), and the compounds are used in vitro / ex vivo.

[0046] One aspect of the present invention further relates to a treatment method, wherein a compound, a pharmaceutical composition, or a kit described herein is used as part of the treatment.

[0047] The present invention will become more apparent from the following detailed description of the invention when interpreted in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0048] [Figure 1] This figure shows the dose-response curve at 48 hours for NCH82 cells obtained from one of three biological replicas of compound 4. In this replica, compound 4 inhibited the growth of NCH82 tumor cells with an IC50 of 1.55 μM. [Figure 2] This figure shows the effect of compound 4 on the migration of tumor cells NCH82, NCH89, NCH125, and NCH210 using a wound healing assay. [Figure 3] This figure shows the increase in the number of apoptotic / necrotic cells with increasing concentration of compound 4. Figure 3A shows flow cytometry analysis of untreated NCH89 cells and cells exposed to 1 μM, 2 μM, and 10 μM of compound 4 for 24 hours. Figure 3B is a stacked bar graph showing the relative proportions of apoptotic and necrotic cells in conventional glioblastoma cell lines NCH82 and NCH89. Figure 3C is a stacked bar graph showing the relative proportions of apoptotic and necrotic cells in glioma stem cell lines NCH421k, NCH644, and NCH660h. [Figure 4] This figure shows a stacked bar graph summarizing the flow cytometry analysis of (A) untreated cells with NCH93 and cells exposed to 1 μM, 2 μM, and 5 μM of compound 6 for 48 hours, as well as (B) brain metastasis cell lines, (C) meningioma cell lines, (D) IDH mutant glioma cell lines, and (E) head and neck cancer cell lines, showing the proportions of apoptotic and necrotic cells. [Modes for carrying out the invention]

[0049] During the evaluation of the compounds of the present invention, the inventors found that the gold-phosphaphenalene derivative was remarkably soluble and highly stable in dimethyl sulfoxide / H2O solution over several weeks.

[0050] Even the chloro derivative (compound 1 shown below) exhibited such high stability, in contrast to its analogue 2,5-diarylphosphole gold complex (non-patent document 7 cited above), which has been reported to be unstable in aqueous solution.

[0051] In the first stage, fragment B is Cl, and the following equation (I): [ka] The electronic properties of the compound of the present invention were investigated by comparing the above phosphaphenalene chloro derivative (compound 1), represented by [1-phenyl-2,5-di(2-pyridyl)phosphole]AuCl, triphenylphosphine-AuCl(Ph3PAuCl), and triethylphosphine-AuCl(Et3PAuCl) with a series of different chloro derivatives.

[0052] All of these complexes contain a distinct phosphorus ligand reported as fragment A for the synthesis of auranofin-like structures. [1-phenyl-2,5-di(2-pyridyl)phosphole]AuCl was reported as a novel gold phosphole inhibitor (GoPI) by Non-Patent Document 5 cited above.

[0053] Reflecting the distinct electronic properties of the latter phosphorus ligand, 31 The P-NMR spectrum differed significantly within the AuCl complex series. [1-phenyl-2,5-di(2-pyridyl)phosphole]AuCl, Ph3PAuCl, and Et3PAuCl 31 The P signal is in the range of 32 ppm to 40 ppm, but compound 1 shows a singlet at 2.6 ppm (see Table 1). 31 Similar to P-NMR, the difference is quite limited, but the P-Au bond distance of compound 1 (2.225 Å) is smaller than that of other gold complexes in the range of 2.230 Å to 2.231 Å (Table 1). The latter indicates both the stronger electron-donating ability of phosphaphenalene and, therefore, a stronger P-Au bond to compound 1.

[0054] The steric requirements of phosphorus ligands are important characteristics because they play a crucial role not only in their stability but also in their penetration through cell membranes. Therefore, to provide insights into the steric hindrance of these compounds, we calculated the embedding volume percentage (V%) and mapped the electron density of phosphorus ligands penetrating the gold atom's coordination sphere.

[0055] The higher the V% value, the more the gold atom is shielded. Therefore, the V% value of the phosphaphenalene ligand in compound 1 is comparable to that of Ph3P (30.7%). 。 The lowest value (27.9%) was observed for triethylphosphine, while the highest value belonged to the 2,5-di(2-pyridyl)phosphole derivative (32.8%), which is likely due to the presence of the pyridyl substituent on the gold-coordinated sphere.

[0056] After confirming the contrasting properties of phosphaphenalene ligands versus phosphole and phosphine, the effect of fragment B was analyzed. For this purpose, three further compounds, compounds 2, 3, and 4, represented by the following formulas (II), (III), and (IV), were investigated. [ka]

[0057] Importantly, compounds 1 through 4 all readily dissolve in a DMSO / H2O mixture in a 1:9 ratio. Compound 1 was the least soluble, beginning to precipitate at concentrations higher than 0.1 M. Compound 4, on the other hand, was soluble in the most diverse solvents, namely methanol, ethanol, DCM, CHCl3, Et2O, and acetone, but insoluble in pentane and hexane.

[0058] 31 Regarding the characteristics of the P-NMR spectrum, changing fragment B shifted the signals of compound 1 from 2.56 ppm to 6.65 ppm, 7.1 ppm, and 8.17 ppm, respectively (see Table 1). Again, this is because, 31This is in stark contrast to phosphole and phosphine analogs, which exhibit P-NMR signals above 30 ppm. X-ray analysis was performed to further investigate the structural characteristics of compounds 1 to 4.

[0059] Therefore, we successfully crystallized compounds 2 and 3 and compared their properties with those of the parent compound 1 (Table 1). Unfortunately, attempts to crystallize compound 4 using a pool of techniques and solvents over several months were unsuccessful.

[0060] Among the series of compounds 1 to 3, the Au-P bond length increases slightly from 2.225 Å in compound 1 to 2.243 Å and 2.25 Å in compound 2 and compound 3, respectively. The Au-fragment B bond distance follows the same trend, from 2.293 Å in compound 1 to 2.326 Å and 2.332 Å in compound 2 and compound 3, respectively.

[0061] [Table 1]

[0062] 1 Experiments conducted to verify the stability of complexes 1-4 by monitoring 1H-NMR for 72 hours (exceeding the typical measurement time for bioassays) showed no signs of degradation.

[0063] After analyzing the characteristics of compounds 1 to 4, in vitro experiments were performed. First, the most effective fragment B of phosphaphenalene-gold(I) complexes 1 to 4 was systematically investigated for its inhibitory effect on glioma cell proliferation using a crystal violet proliferation assay. For this purpose, GBM cell lines NCH82 and NCH89 were incubated for 48 hours with increasing concentrations of compounds 1, 2, 3, and 4, respectively.

[0064] All derivatives were found to be active in inhibiting glioma cell proliferation (see Table 2). Compounds 1 to 3 showed mean IC50 concentrations in NCH82 and NCH89 ranging from 8.21±0.52 μM to 11.4±0.11 μM and 15.1±0.71 μM to 18.1±1.04 μM, respectively. 50 While the values ​​were shown, the strongest antiproliferative effect was observed for thiosaccharides containing compound 4, with average IC2 levels against GBM cell lines NCH82 and NCH89, respectively. 50 The values ​​were 1.44±0.16 μM and 2.9±0.41 μM.

[0065] Figure 1 illustrates one of three biological replicas of compound 4 applied to NCH82.

[0066] To further confirm the activity of the latter complex, additional primary glioblastoma cell lines NCH210 and NCH125 were treated with compound 4, yielding equivalent, and even better, average IC25 levels. 50 The values ​​obtained were 2.79 ± 0.07 μM and 0.78 ± 0.04 μM, respectively (Table 2).

[0067] [Table 2]

[0068] Based on the assumption that most drug-based therapies may fail due to treatment-resistant glioblastoma stem cell-like cells (GSCs), we further investigated the potential of targeting well-characterized GSC lines such as NCH421k, NCH644, and NCH660h. These cell lines were described in Campos B. et al., “Differentiation therapy exerts antitumor effects on stem-like glioma cells”, Clin Cancer Res. 2010 May 15;16(10), pages 2715-28.

[0069] For this purpose, considering its anti-proliferative properties, compound 4 was used on GSCs growing as floating neurospheres using the CellTiter-Glow® assay. As a result, treatment of GSCs yielded significant average IC50 levels of 6.95±1.95 μM, 6.60±1.98 μM, and 2.66±0.58 μM in NCH421k, NCH644, and NCH660h, respectively. 50 The value was shown. Slightly higher IC in this particular type of cell. 50 The values ​​may be caused by their deep self-renewal ability and reduced drug sensitivity, and yet the latter IC 50 The values ​​are within the same range as those observed in conventional GBM cells.

[0070] In addition to increased tumor cell proliferation, highly invasive cell growth causes malignant tumors of GBM. To obtain a more complete picture of the anticancer effects of the compounds according to the present invention, the effect of compound 4 on GBM cell infiltration was investigated using a conventional wound healing assay with NCH82, NCH89, NCH125, and NCH210 cells.

[0071] The application of compound 4 at different concentrations yields at least the corresponding IC 50 Only when the concentration was adopted did it significantly reduce wound closure compared to untreated control cells; the same was not observed with lower concentrations of compound 4.

[0072] Wound closure is achieved by reducing the concentration of c(IC). 50 ) / 2 and c(IC 50 ) / 10 showed no significant effect. Nevertheless, the short observation period of 24 hours, during which only limited tumor cell proliferation occurred, suggests that the mechanism of action of compound 4, in addition to its remarkable antiproliferative properties, is IC 50 The value still supports the possibility of additional anti-migratory components.

[0073] The results of the wound healing assay are shown in Figure 2. In the wound healing assay, a monolayer of GBM cells was scraped off with a pipette tip, and the concentration c(IC) was measured. 50 ) / 10, c(IC 50 ) / 2, c(IC50 ), and c(IC 50 The procedure was carried out by treating cells with compound 4 (2x) for 24 hours. Cells were imaged at 0 hours (t0) and 24 hours (t1) after the introduction of scrapings. Cell migration was assessed by measuring the cell-free areas at t0 and their decrease at t1.

[0074] The upper left corner of Figure 2 shows NCH82 p85 tumor cells untreated (control) and c(IC). 50 These are photographs taken after treating cells with compound 4 and incubation times of 0 and 24 hours. The bar graph in Figure 2 shows the data for individual cell lines as the mean ± standard deviation of three biological replicates.

[0075] Furthermore, we analyzed whether compound 4 could sensitize GBM cells (NCH82 and NCH89) and GSCs (NCH421k, NCH644, and NCH660h) and induce apoptosis. Figure 3 shows the increase in apoptotic / necrotic cells with increasing concentration of compound 4. Figure 3A shows flow cytometry analysis of NCH89 untreated cells, revealing a dose-dependent increase in apoptotic / necrotic cells after 24 hours of exposure to 1 μM, 2 μM, and 10 μM of compound 4.

[0076] Figure 3B is a stacked bar graph showing the relative proportions of apoptotic and necrotic cells in conventional glioblastoma cell lines NCH82 and NCH89, and Figure 3C is a stacked bar graph showing the relative proportions of apoptotic and necrotic cells in glioma stem cell lines NCH421k, NCH644, and NCH660h.

[0077] Therefore, conventional GBM cells and GSCs were analyzed by flow cytometry after a 48-hour incubation period with different concentrations of compound 4. Annexin V and propidium iodide (PI) were used as indicators of apoptosis / necrosis. The obtained ICs are listed in Table 2. 50 Consistent with the values, NCH82 cells appeared to be the most sensitive GBM cell line.

[0078] Apoptosis was already induced in a high proportion of cells at a drug concentration of 2 μM (Figure 3B). In contrast, NCH89 cells were more resistant to programmed cell death, and no cell death was observed at a concentration of 2 μM of compound 4 (Figure 3B). In the GSCs analyzed (Figure 3C), induction of apoptosis was demonstrated, but to a low degree.

[0079] The compound defined in claim 1, namely the gold(I) complex based on a six-membered phosphorus heterocycle, has demonstrated remarkable potential for the development of novel chemotherapeutic agents not only for conventional glioblastoma cells but, importantly, for glioblastoma stem cell-like cells as well.

[0080] This is the result of a systematic evaluation of the cytotoxic effects of four different phosphaphenalene-gold(I) derivatives, with the best results obtained for the thioglycol derivative, namely compound 4. In particular, compound 4 showed significant inhibition of cell proliferation in both conventional GBM cells and GSCs.

[0081] Furthermore, compound 4 was shown to exhibit anti-migration effects against glioblastoma cells, sensitizing conventional GBM cells and GSC cells and inducing apoptosis. The compounds according to the present invention offer high stability, solubility in aqueous media, and synthetic versatility to meet possible further requirements.

[0082] Departing from the promising findings obtained with compounds 1 to 4 described above, the inventors have embarked on the development of more specific and improved phosphaphenalene-gold(I) complexes that exhibit superior spectroscopic properties, stability, and, most importantly, physiological activity of the compounds claimed herein.

[0083] Regarding this, see formula (V): [ka] Compound 5, which contains a pyrrole moiety condensed with a phosphaphenalene core coordinated to an AuCl fragment represented by , was tested.

[0084] Pyrrole-containing phosphaphenalene is stable, exhibits excellent photoelectronic properties relevant to materials science, and has a fluorescence quantum yield of up to 80%, making it suitable for use in photoelectrochemical cells, organic light-emitting diodes, and electrofluorochromic devices. Based on these properties, pyrrole-containing phosphaphenalene may offer additional advantages as a drug with important spectroscopic properties that are particularly valuable for in vivo mechanism investigations.

[0085] Compound 5 was further transformed into compound 6, an analog of compound 2 (see above), by replacing the chloride atom of compound 5 with the 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate in compound 6, as shown in formula (VI) below (also referred to herein as formula (B)): [ka]

[0086] To analyze the effect of additional structural modifications on physiological activity, the methyl substituent of pyrrole was replaced with a bulkier substituent, namely phenylsulfonyl. This yielded compound 7, supported by the chloride atom represented by formula (VII), and the corresponding derivative compound 8, having the 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate moiety represented by formula (VIII). [ka]

[0087] Structural modifications lead to changes in the electron distribution of molecules, and these 1 This is reflected in the 1H-NMR spectrum (data not shown). Even when compound 6 was produced by substituting the Cl atom of compound 5 with the sugar derivative 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate, only slight changes occurred.

[0088] However, N-substituents had a significant impact. Introducing a phenylsulfonyl group to the nitrogen of pyrrole-condensed phosphaphenalene resulted in dramatic descreening of specific protons in the pyrrole fragment and naphthalene.

[0089] Next, compared to the chloride derivatives of compounds 1 and 5, compound 7 exhibits a structural result derived from the electron-withdrawing effect of the phenylsulfonyl group. On the other hand, all sugar derivatives of compounds 4, 6, and 8 were clearly deshimulated at 8.2 ppm, 9.1 ppm, and 9.5 ppm. 31 The P-NMR spectrum is shown. These values ​​are significantly lower than those previously reported for bioactive phosphole and phosphine-based gold complexes (in the range of 32 ppm to 47 ppm).

[0090] To investigate the role of the condensed phosphaphenalene ring in the physiological activity of a drug, the ability of compound 5 to inhibit cell proliferation in glioblastoma cell lines NCH82, NCH89, and NCH149 was examined. For this purpose, cells were incubated with increasing concentrations of compound 5, and cell proliferation was evaluated by a crystal violet assay.

[0091] Compound 5 showed antiproliferative effects in all three cell lines. Average IC5 of cell lines NCH82, NCH89, and NCH149 50 The values ​​were 8.1 μM, 15.1 μM, and 8.87 μM, respectively (see Table 3 below). These values ​​correspond to the IC50 values ​​observed in compound 1, as shown in Table 2 above, i.e., 11.4 μM and 17.3 μM for NCH82 and NCH89, respectively. 50 It is slightly lower compared to

[0092] Therefore, modifying the phosphaphenalene core by substituting the thiophene moiety with a pyrrole ring results in improved antiproliferative activity in vitro. Consistent with the observations discussed above, replacing the chloride atom of the gold moiety with a sugar derivative significantly increases the physiological activity of the drug (Tables 2 and 3).

[0093] Compound 6 has an average IC that is an order of magnitude lower than Compound 5. 50 The values ​​were observed, reaching submicromolar concentrations of 0.73 μM, 4.00 μM, and 0.87 μM for cell lines NCH82, NCH89, and NCH149, respectively (Table 3). Here again, these values ​​are significantly lower than those for analog compound 4 (Table 3), which contains phosphaphenalene condensed on a thiophene ring instead of a pyrrole heterocycle.

[0094] Next, introducing a phenylsulfonyl fragment to the nitrogen of the phosphaphenalene core (compound 8) results in higher biological activity compared to compound 5. However, this value is slightly lower than that of the N-methyl derivative of compound 6.

[0095] [Table 3]

[0096] Average IC of compound 8 50 The values ​​were 1.37 μM, 4.49 μM, and 2.85 μM in glioblastoma cell lines NCH82, NCH89, and NCH149, respectively (Table 3). Overall, there appears to be flexibility in the condensed heterocycle of the phosphaphenalene core to maintain physiological activity, but specific types of condensed heterocycles in the phosphaphenalene core seem to affect the physiological activity of the drug. Specifically, pyrrole appears to result in further improvement of cytotoxic activity.

[0097] In contrast, further increasing the bulkiness of phosphaphenalene, which has an electron-accepting phenylsulfonyl group, largely maintains the drug's physiological activity but does not enhance it further.

[0098] Based on the remarkable results obtained with compound 6, its antiproliferative effects against a range of different cancer cell lines were investigated. For this purpose, in addition to the three glioblastoma cell lines mentioned above, compound 6 was used with 11 other cancer cell lines, including brain metastases (NCH517, NCH604a, and NCH466), meningiomas (NCH93 and BenMen-1), IDH-mutated gliomas (NCH511b, NCH1618, and NCH3763), and head and neck cancer cell lines (HNO210, HNO199, and HNO97) (Table 3).

[0099] Overall, compound 6 showed excellent antiproliferative activity in all cell lines, including average IC values ​​in several cell lines, even highly invasive brain metastatic cancer cells. 50 The value was approximately 1.5 μM. In the IDH-mutated glioma cell line NCH1681, IC 50 The result was impressive, with a value of only 0.88 μM.

[0100] Motivated by these results, we analyzed the ability of compound 6 to induce apoptosis in cancer cell lines derived from brain metastases, meningiomas, IDH-mutated gliomas, and head and neck cancers. For this purpose, cells were incubated with different concentrations of compound 6 for 48 hours and subsequently analyzed by flow cytometry (see representative example in Figure 4A). Annexin V and propidium iodide (PI) were used as indicators of apoptosis / necrosis.

[0101] As expected, compound 6 was able to induce apoptosis / necrosis in all cell lines analyzed (Figure 4). The obtained IC 50 Consistent with the values ​​(see Table 3 above), brain metastases and meningioma cell lines appeared to be more sensitive than head and neck cancer cell lines. A drug concentration of 2 μM induced apoptosis / necrosis in over 50% of NCH466, NCH517, NCH604a, and NCH93 cells (Figures 4B and 4C). In contrast, a drug concentration of 5 μM was required to induce apoptosis / necrosis in over 50% of head and neck cancer cell lines (Figure 4E).

[0102] In addition to these remarkable results, the compounds of the present invention exhibited extremely high stability against repeated illumination cycles, as demonstrated by experimental tests using compounds 6 and 8 under controlled thermodynamic conditions (data not shown).

[0103] Based on the high physiological activity and spectroscopic properties of compound 6, observed as an example of the present invention, the drug uptake kinetics in the NCH82 cell line were investigated. For this purpose, cells were treated with increasing concentrations of compound 6, and drug uptake was monitored using a fluorescence microscope (excitation / emission 350 nm / 455 nm; data not shown).

[0104] Surprisingly, 10 μM (i.e., IC) 50 After treatment with compound 6 (at a concentration exceeding the specified value) for just one hour, cells were able to internalize the compound and begin to contract and detach. Cells treated with 1 μM compound 6 began absorbing the compound after one hour, but death was only observed after 24 hours. When used at a concentration of 0.1 μM, compound 6 began internalization after 24 hours, but dead cells were first observed 48 hours after the start of treatment.

[0105] In summary, the results presented herein demonstrate that the physiological activity of the phosphaphenalene gold complexes of the present invention can be influenced and improved by slight chemical modifications to their structural features. Their inherent properties allow for the tuning of electron distribution on the π-extended core, molecular bulk, and their photophysical properties.

[0106] In particular, pyrrole-condensed phosphaphenalene derivatives appear to offer even better performance than thiophene-based analogs, and it is noteworthy that all of these compounds remain stable for several weeks. Furthermore, sugar derivatives bonded to the gold atom provide even greater biological activity compared to those bonded to the chloride atom. Overall, the phosphophenalene-gold complexes described and claimed herein possess remarkable, unprecedented, and astonishing anti-proliferative capabilities.

[0107] Phosphophenalene gold complex inhibits the proliferation of 14 different tumor cell lines derived from glioblastoma, brain metastases, meningiomas, IDH-mutated gliomas, and head and neck cancers. Furthermore, the compound appears to sensitize cancer cell lines and induce apoptosis, demonstrating efficient uptake into cells.

[0108] In summary, the broad applicability and high physiological activity of the phosphaphenalene gold complexes of the present invention, coupled with their remarkable spectroscopic properties, suggest that these compounds can be appropriately used to provide promising therapeutic agents for improved treatment of lethal diseases.

[0109] The structural modifications included in the scope of this invention have substantially the same solvation shell radius as the experimentally tested claimed compounds. These modifications only slightly alter the lipophilicity of the complexes. However, since all the complexes according to this invention are relatively small in size, the changes in lipophilicity are not expected to result in substantial differences in terms of cytotoxic effects.

[0110] All compounds included in the scope of this invention exhibit similar electron densities at the phosphorus center in order to retain / coordinate the active gold atom responsible for the anticancer effect. Therefore, it is reasonable and appropriate that all currently claimed compounds exhibit similar technical effects based on similar structural properties. [Examples]

[0111] The following provides experimental details regarding the synthesis and characterization of the aforementioned compounds.

[0112] General information All reactions were carried out in dry glassware and under an inert atmosphere of purified argon or nitrogen using Schlenk technology. Solvents such as CH2Cl2 and THF were used directly from the MB SPS-800 solvent purification system. AcOEt and ethanolacetone were purchased from commercial suppliers and used as is. Ethyl potassium xanthogenic acid, AgNO3, KSCN, 1-thio-β-D-glucosetetraacetic acid, and magnesium sulfate were purchased from commercial suppliers and used as is.

[0113] NMR measurement: 1 H, 13 C, 1 and 31 P NMR spectra were recorded using a Bruker Avance DRX-300, Bruker Avance 500, or Bruker Avance 600. Chemical shifts are expressed in parts per million (ppm, δ), with respect to the external 85% H3PO4 ( 31 P), or solvent signal as an internal standard ( 1 H / 13 C): Refer to CD2Cl2 (5.33 ppm / 53.80 ppm). Signal descriptions include s=singlet, d=doublet, t=triplet, m=multiplet, and br=broad. All coupling constants are absolute values, and J values ​​are expressed in Hertz (Hz).

[0114] Mass spectrometry: MS and HRMS measurements were performed at the Organisch-Chemisches Institut at Heidelberg University. A Bruker ApexQe hybrid 9.4 T FT-ICR was used for DART spectra, and a JEOL AccuTOFGCx time-of-flight was used for EI spectra.

[0115] Steric hindrance of phosphorus ligands: The calculation of the buried volume percentage (%V) was performed using free web application software (http: / / www.molnac.unisa.it / OMtools / sambvca.php), considering a spherical radius of 3.50 Å. Hydrogen atoms were omitted, and the measured Bondi radius was 1.7. This software is presented in Poater, B. et al., "SambVca: A Web Application for the Calculation of the Buried Volume of N‐Heterocyclic Carbene Ligands", Eur. J. Inorg. Chem., 2009, pages 1759-1766.

[0116] X-ray crystallography: X-ray crystal structure analysis was performed using Mo-Kα emission with Bruker's Smart CCD or Bruker's Smart APEX instrument. Diffraction intensities were corrected for Lorentz and polarization effects. Empirical absorption correction was applied using SADABS, which is based on Laue symmetry in reciprocal space (absorption correction program SADABS 2008 / 1; GM Sheldrick, Bruker Analytical X-ray-Division, Madison, Wisconsin 2012).

[0117] Heavy atom diffraction was solved using a direct method and refined for F2 using a full matrix least-squares algorithm. Hydrogen atoms were isotropically refined or calculated. The structure is SHELXTL. [S2] The solutions were solved and refined using a software package. The crystal structure of compound 2 was obtained from DCM / pentane at room temperature, and the crystal structure of compound 3 was obtained from a DCM solution by slow evaporation at room temperature. Supplemental crystallographic data for these compounds are available free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk / data_request / cif-CCDC 1832813 (compound 2) and 1832814 (compound 3).

[0118] Cell culture conditions: Adherent growth cell lines derived from glioblastoma (NCH82, NCH89, and NCH149), brain metastases (NCH466, NCH517, and NCH604a), meningioma (NCH93 and Ben-Men-1 (DSMZ, Braunschweig, Germany)), and head and neck cancer (HNO97, HNO199, and HNO210), as well as stem cell-like cell lines derived from IDH-mutated glioma (NCH551b, NCH1618, and NCH3763), were characterized and cultured as previously described (B. Campos et al., Clin. Cancer Res. 2010, 16 (10), 2715-2728. https: / / doi.org / 10.1158 / 1078-0432.CCR-09-1800., P. Dao Trong et al., IJMS 2018, 19). (10), 2903. https: / / doi.org / 10.3390 / ijms19102903. Y. Jungwirth et al., Cancers 2019, 11 (4), 545. https: / / doi.org / 10.3390 / cancers11040545. S. Karcher et al., Int. J. Cancer 2006, 118 (9), 2182-2189. https: / / doi.org / 10.1002 / ijc.21648. and C. Rapp et al., Acta Neuropathol. 2017, 134 (2), 297-316. https: / / doi.org / 10.1007 / s00401-017-1702-1.).

[0119] Adherent growth cell lines (NCH82, NCH89, NCH210, and NCH125) and glioma stem cell-like cell lines (NCH421k, NCH644, and NCH660h) were established from glioblastoma samples obtained intraoperatively, characterized, and cultured as previously described (S. Karcher et al., Int. J. Cancer. 2006, 118, 2182-2189., C. Rapp et al., Acta Neuropathol. 2017, 134, 297-316., and B. Campos et al., Clin. Cancer Res. 2010, 16, 2715-2728.). The cell lines were validated, and written informed consent was obtained from patients in accordance with the research proposal approved by the Institutional Review Board of the Faculty of Medicine, Heidelberg University.

[0120] Growth assay - adherent cell lines: The effects of different compounds on cell growth were evaluated using the crystal violet method, as described above (JP Rigalli et al., Cancer Lett. 2016, 376, 165-172). Briefly, cells were seeded in 96-well plates (10,000 cells / well). After 24 hours, the cell culture medium was replaced with fresh compound-containing medium (10 different concentrations (0.01 μM to 200 μM)). The degree of cell proliferation inhibition after 48 hours of exposure was determined by crystal violet staining of viable cells. Therefore, cells were washed, stained with 0.5% crystal violet solution (2.5 g in 100 ml methanol, diluted with 400 ml of aqua bidest (re-distilled water)) for 15 minutes, rinsed, and dried overnight.

[0121] Next, crystal violet was solubilized in methanol, and its absorbance at 555 nm was measured. The proliferation index was calculated as the crystal violet absorption intensity as a percentage relative to baseline (no cells), as previously described (T. Peters et al., Naunyn Schmiedebergs Arch Pharmacol. 2006, 372, 291-299). Cell viability was plotted against the decimal logarithm of drug concentration in μM (c of compound x in μM) and fitted to a sigmoid dose-response curve using GraphPad Prism 7.02 (GraphPad Software, San Diego, USA).

[0122] Proliferation assay - Glioma stem cell-like (GSC) cell line: To evaluate the effects of compounds 4 and 6 on the cell growth of glioma stem cell-like cells (GSCs) and GSC strains derived from IDH-mutated gliomas, cellular ATP levels were measured using the luminescent CellTiter-Glo assay (Promega Corp, Madison, Wisconsin). GCS spheroid cultures were gently dissociated, and the cell suspension was seeded into 96-well tissue culture plates (8000 cells / well, 100 μl / well). After a 24-hour incubation period without the compounds, the cells were incubated for 48 hours with the addition of 10 different final concentrations of the compounds ranging from 0.01 μM to 200 μM.

[0123] Before measurement, the plates were equilibrated at room temperature for 30 minutes. Next, 100 μl of CellTiter-Glo reagent was added to each well, and the plates were placed in an orbital shaker for 2 minutes to mix the contents. The plates were then incubated at room temperature for 10 minutes, and finally, luminescence was measured. Cell viability was plotted against the decimal logarithm of drug concentration (c(compound 4 / compound 6) μM) in μM units, and fitted to sigmoid dose-response curves using GraphPad Prism 7.02 (GraphPad Software, San Diego, USA).

[0124] Drug effects on apoptosis (Annexin V apoptosis assay): To quantify the degree of apoptosis and necrosis, either annexin V staining combined with DAPI, or double labeling of cells with annexin V and propidium iodide (PI), was used. Double labeling allowed for the identification of apoptotic cells (annexin V staining). 陽性 / DAPI 陰性 or Annexin V 陽性 / PI 陰性 ) and necrotic cells (annexin V 陽性 / DAPI 陽性 or Annexin V 陽性 / PI 陽性 This makes it possible to distinguish the cells from other cells. 5 Cells were seeded in wells and left overnight. The culture medium was replaced with fresh medium containing the specified drug concentration or the compound solvent DMSO (untreated control), and incubated for 24 hours. As a positive control, cells were treated with 1 μM staurosporine (#9953, Cell Signaling Technology, Danbus, USA), an apoptosis-inducing reagent.

[0125] After collecting the supernatant containing apoptotic and necrotic cells, the cells were harvested, washed, and up to 1 × 10⁶ cells were collected. 6 Cells were incubated with FITC-conjugated annexin V antibody diluted in 1:1000 DAPI solution according to the manufacturer's instructions (#51-65874X, BD Bioscienes, Franklin Lakes, USA), or incubated with FITC-conjugated annexin V antibody and PI for 15 minutes according to the manufacturer's instructions (#51-65974X, BD Bioscienes, Franklin Lakes, USA). Cells were acquired by flow cytometry using a BD LSRII flow cytometer (BD Bioscienes, Franklin Lakes, USA) and analyzed using FlowJo Software v7.6.5 (TreeStar, Arland, USA).

[0126] Drug effects on glioma cell migration (scratch assay): To evaluate the effect of a drug on cell migration in vitro, cells were plated in a 6-well plate (5 × 10⁶). 5 Seeds were seeded and allowed to adhere to the cells in a well. The following day, the cell monolayer was scraped linearly, and a "scratch" was created using a p200 pipette tip. Debris was removed by washing the cells, and the culture medium was treated with three different cell line-specific concentrations (IC) for 24 hours. 50 ,I C 50 / 2, IC 50 The medium containing compound / 10) was replaced with 5 ml. Phase-contrast images were acquired at 0 and 12 hours after exposure to each drug concentration using a BX50 microscope equipped with an SC30 camera (both from Olympus Corporation, Tokyo, Japan). Cell-free regions were quantified at both time points and compared using imaging software cellSens (Olympus Corporation, Tokyo, Japan).

[0127] Drug uptake kinetics: To measure the uptake dynamics of compound 6 by NCH82 cells, NCH82 cells were seeded in 96-well plates (5000 cells / well), and after 24 hours, the cell culture medium was replaced with medium containing compound 6 or DMSO (0.1 μM, 1 μM, and 10 μM). Images were captured using a fluorescence microscope (Olympus Corporation, Shinjuku, Japan) at 1 hour, 24 hours, and 48 hours after the start of treatment. Images were captured using a laser with an excitation / emission spectrum of 350 nm / 455 nm and a 10x objective lens.

[0128] Synthesis procedure Compound 1 was prepared as previously reported by Romero-Nieto C. et al., Angew. Chem. Int. Ed. 2015 (cited above, shown as gold complex 16).

[0129] compound 2 [ka]

[0130] Compound 1 (1 equivalent, 0.076 mmol, 42 mg) was dissolved in 4 mL of DCM, and ethyl potassium xanthogene (1 equivalent, 0.076 mmol, 12 mg) was added at room temperature. The crude mixture was stirred for 1.5 hours. The mixture was then washed with water, dried over MgSO4, and volatile components were removed under reduced pressure. The crude mixture was washed three times with AcOEt and crystallized by slow evaporation from the DCM solution. Yield: 88 (42 mg, 0.066 mmol). 1 H-NMR (600 MHz, CD2Cl2): δ 8.26 (ddd, J = 18.8, 7.0, 1.2 Hz, 1H), 8.21 (dd, J = 7.4, 0.8 Hz, 1H), 8.17 (dd, J = 8.8, 2.9 Hz, 1H), 8.07 (dd, J = 2.9, 1.1 Hz, 1H), 8.04 (d, J = 8.2 Hz, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.63-7.61 (m, 1H), 7.42 (ddd, J = 13.7, 8.3, 1.1 Hz, 2H), 7.33 (td, J = 7.4, 2.0 Hz, 1H), 7.26 (td, J = 7.5, 2.5 Hz, 2H), 4.55-4.50 (m, 2H), 1.40 (d, J = 14.2 Hz, 3H). 13 C{ 1 H}{ 31P} NMR (151 MHz, CD2Cl2): δ 138.3 (s, 1C), 136.8 (s, 1C), 136.1 (s, 1C), 135.2 (s, 1C), 134.5 (s, 1C), 133.6 (s, 1C), 132.4 (s, 1C), 131.6 (s, 1C), 129.9 (s, 1C), 129.4 (s, 1C), 128.4 (s, 1C), 127.6 (s, 1C), 127.2 (s, 1C), 126.2 (s, 1C), 124.8 (s, 1C), 124.6 (s, 1C), 123.0 (s, 1C), 123.0 (s, 1C), 70.6 (s, 1C), 14.3 (s, 1C). 31 P-NMR (243 MHz, CD2Cl2): δ 6.55. HRMS(EI+) C 10 H4Br4O2 + The calculated value is 471.6939, and the measured value is 471.6940.

[0131] compound 3 [ka]

[0132] Compound 1 (1 equivalent, 0.273 mmol, 150 mg) was suspended in 4 mL of ethanol and mixed with AgNO3 (1 equivalent, 0.273 mmol, 46 mg) dissolved in 4 mL of water. The mixture was stirred for 1 hour, and 5 mL of DCM was added. After stirring for 30 minutes, 80 μL of 8 M KSCN in water was added. The mixture was stirred for 1 hour, the DCM phase was separated, and the aqueous phase was extracted three times with DCM. After drying over MgSO4, the crude product was concentrated under reduced pressure and filtered through Celite. The product was purified by column chromatography using a mixture of silica and DCM / pentane 6:4 eluents to pure DCM, and crystallized from the DCM / pentane mixture. Yield: 65% (102 mg, 0.179 mmol). 1H-NMR (600 MHz, CD2Cl2): δ 8.26 (d, J = 7.4 Hz, 1H), 8.18 (dd, J = 19.1, 7.0 Hz, 1H), 8.13 (dd, J = 6.6, 3.3 Hz, 2H), 8.09 (d, J = 8.2 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.71-7.65 (m, 2H), 7.42-7.37 (m, 3H), 7.31 (td, J = 7.8, 2.2 Hz, 2H). 13 C{ 1 H}{ 31 P} NMR (151 MHz, CD2Cl2): δ 138.2 (s, 1C), 136.9 (s, 1C), 136.3 (s, 1C), 134.6 (s, 1C), 134.1 (s, 1C), 134.0 (s, 1C), 132.6 (s, 1C), 132.1 (s, 1C), 130.1 (s, 1C), 129.6 (s, 1C), 128.3 (s, 1C), 127.4 (s, 1C), 126.2 (s, 1C), 125.0 (s, 1C), 123.3 (s, 1C), 123.3 (s, 1C), 121.9 (s, 1C). 31 P-NMR (243 MHz, CD2Cl2): δ 7.10. HRMS(EI+) C 10 H6Br2O2 + The calculated value is 315.87291 and the measured value is 315.8737.

[0133] Compound 4

change

[0134] NaH was added to 1-thio-β-D-glucose tetraacetate (1 equivalent, 0.182 mmol, 66 mg) in 5 mL of THF at room temperature. The mixture was stirred for 1 hour, filtered through celite via a cannula, and added to compound 1 (0.9 equivalent, 0.164 mmol, 90 mg) dissolved in 5 mL of THF at room temperature. The solution was stirred for 1.5 hours and the solvent was removed under vacuum. The crude material was dissolved in DCM, filtered through celite, and subjected to column chromatography using silica and AcOEt / acetone 8:2 as the eluent. Yield: 72% (104 mg, 0.118 mmol). 1 1H-NMR (600 MHz, CD2Cl2): δ 8.31-8.24 (m, 3H), 8.12 (ddd, J = 6.2, 2.9, 1.1 Hz, 1H), 8.06 (dd, J = 8.2, 1.2 Hz, 1H), 7.94 (s, 1H), 7.69-7.64 (m, 2H), 7.45-7.39 (m, 2H), 7.35-7.32 (m, 1H), 7.30-7.26 (m, 2H), 5.20-5.14 (m, 2H), 5.08 (dt, J = 13.1, 9.5 Hz, 2H), 4.14 (qd, J = 12.0, 3.7 Hz, 2H), 3.77 (ddd, J = 10.0, 5.0, 2.5 Hz, 1H), 2.07 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H), 1.91 (s, 3H). 13 C{ 1 H}{ 31P} NMR (151 MHz, CD2Cl2): δ 170.3 (s, 1C), 169.9 (s, 1C), 169.5 (s, 1C), 169.4 (s, 1C), 137.9 (s, 1C), 137.9 (s, 1C), 136.4 (s, 1C), 136.32(s, 1C), 135.6 (s, 1C), 135.5 (s, 1C), 135.1 (s, 1C), 135.1 (s, 1C), 134.2 (s, 1C), 134.1 (s, 1C), 133.0 (s, 1C), 132.0 (s, 1C), 132.0 (s, 1C), 131.1 (s, 1C), 131.1 (s, 1C), 129.5 (s, 1C), 129.0 (s, 1C), 128.9 (s, 1C), 128.1 (s, 1C), 127.2 (s, 1C), 126.7 (s, 1C), 124.3 (s, 1C), 122.8 (s, 1C), 122.8 (s, 1C), 122.5 (s, 1C), 122.4 (s, 1C), 83.0 (s, 1C), 77.6 (s, 1C), 75.7 (s, 1C), 73.9 (s, 1C), 68.7 (s, 1C), 62.6 (s, 1C), 20.9 (s, 1C), 20.4 (s, 1C), 20.4 (s, 1C), 20.4 (s, 1C). 31 P-NMR (122 MHz, CDCl3) HRMS(EI+) C 10 H6Br2O2 + The calculated value is 315.8729, and the measured value is 315.8728.

[0135] Synthesis of Compound 5 In a bake-out 10 mL round-bottom flask connected to a Schlenk adapter, 2-(8-bromonaphthalene-1-yl)-1-methyl-1H-pyrrole (1.0 equivalent, 0.18 mmol, 52 mg) was dissolved in 3.6 mL of dry Et2O and cooled to -80°C. Then, t BuLi (1.0 equivalent, 0.18 mmol, 0.11 mL, 1.7 M in pentane) was added dropwise.

[0136] Immediately thereafter, the lithiated 2-(8-bromonaphthalen-1-yl)-1-methyl-1H-pyrrole was reacted with PhPCl2 (1.05 eq, 0.19 μmol, 26 μL, 97%), and the reaction mixture was stirred at room temperature for 1 h. The solvent was removed under vacuum, and then 4.5 mL of DCM and chloro(dimethylsulfide)gold(I) (DMS-AuCl) (1.0 eq, 0.18 mmol, 53 mg) were added, and the reaction mixture was stirred for an additional 1.5 h.

[0137] The solvent was removed again under vacuum to afford a light brown solid. After purification by column chromatography using alumina and a mixture of DCM:pentane (7:3) as the eluent, 76 mg (0.14 mmol) of a yellow solid was isolated (yield: 78%). 1 H NMR (400 MHz, CDCl3): δ 8.17 (dd, J = 18.4, 6.7 Hz, 2H), 8.02 (d, J = 7.3 Hz, 1H), 7.96 (d, J = 8.1, 1H), 7.80 (d, J = 8.51 Hz, 1H), 7.61 - 7.58 (m, 2H), 7.49 (dd, J = 14.0, 7.0 Hz, 2H), 7.33 - 7.30 (m, 1H), 7.26 - 7.24 (m, 2H), 6.92 (t, J = 7.3 Hz, 1H), 6.53 (dd, J = 5.5, 2.8 Hz, 1H), 4.12 (s, 3H). 13 C{ 1H} and DEPT 135 NMR (101 MHz, CDCl3): δ 136.9 (d, CH), 135.3 (s, C), 134.7 (s, C), 134.3 (d, C), 133.2 (d, CH), 133.0 (d, CH), 132.3 (d, CH), 131.2 (d, CH), 129.8 (d, CH), 129.0 (d, CH), 128.9 (s, CH), 128.7 (d, CH), 126.1 (s, C), 126.0 (d, CH), 125.0 (d, C), 124.2 (s, C), 123.6 (s, C), 127.7 (d, CH), 112.4 (d, CH), 106.0 (d, C), 39.4 (s, CH3). 31 P{ 1 H} NMR (162 MHz, in CDCl3): δ 2.64. HRMS(FAB)m / z:[C 21 H 16 AuClNP] ·+ of [M ·+ Calculated value: 545.0374, measured value: 545.0404.

[0138] Synthesis of Compound 6 In a baked-out Schlenk tube, 1-thio-β-D-glucosetetraacetic acid (1.0 equivalent, 0.061 mmol, 22 mg) was dissolved in 2 mL of dry THF. Then, NaH (2.0 equivalent, 0.122 mmol, 3 mg) was added, and the reaction mixture was stirred for 1 hour. The resulting suspension was filtered through Celite under an inert atmosphere into a baked-out Schlenk tube containing compound 5 (0.9 equivalent, 0.055 mmol, 30 mg) dissolved in 1.6 mL of dry THF.

[0139] After stirring for 1.5 hours, the solvent was removed under vacuum. Purification by column chromatography using alumina and an siRNA:DCM(2:8) mixture as the eluent yielded 35 mg (0.04 mmol) of a yellow solid (yield: 73%). 1H NMR (600 MHz, CDCl3): δ 8.28 (dd, J = 18.2, 7.0 Hz, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.65 - 7.57 (m, 2H), 7.56 - 7.50 (m, 2H), 7.35 - 7.28 (m, 3H), 6.94 (dt, J = 4.1, 2.7 Hz, 1H), 6.65 (ddd, J = 8.0, 5.4, 2.8 Hz, 1H), 5.23 - 5.10 (m, 4H), 4.22 (dd, J = 12.2, 4.7 Hz, 1H), 4.14 (d, J = 2.1 Hz, 1H), 4.12 (d, J = 1.4 Hz, 3H), 3.77 (ddd, J = 9.2, 4.5, 2.2 Hz, 1H), 2.09 (s, 3H), 2.01 (d, J = 10.7 Hz, 6H), 1.88 (s, 3H)。 13 C{ 1 H} and DEPT 135 NMR (101 MHz, CDCl3): δ 171.2 (s, C), 170.7 (s, C), 170.0 (d, C), 137.0 (dd, CH), 134.6 dd, C), 133.6 (d, C), 133.3 (0, CH), 132.6 (dd, CH), 131.2 (s, CH), 129.9 (dd, CH), 129.4 (s, C), 129.2 (d, CH), 129.0 (s, CH), 127.3 (d, CH), 126.4 (d, CH), 126.3 (s, CH), 125.6 (d, C), 122.7 (s, CH), 112.8 (dd, CH), 83.4 (s, CH), 77.9 (s, CH), 76.0 (s, CH), 74.6 (s, CH), 69.2 (s, CH), 63.1 (s, CH2), 39.7 (s, CH3), 21.5 (s, CH3), 21.0 (d, CH3)。 31 P{ 1H} NMR (162 MHz, in DCl3): δ 9.11. HRMS(DART + ):[C 35 H 35 AuNO 9 PS] ·+ of [M ·+ Calculated value: 873.1436, measured value: 874.1494.

[0140] Synthesis of Compound 7 In a baked-out Schlenk tube, 2-(8-bromonaphthalene-1-yl)-1-(phenylsulfonyl)-1H-pyrrole (1.0 equivalent, 102 μmol, 42 mg) was dissolved in 5 mL of dry Et2O and cooled to -90°C. Then, t BuLi (1.0 equivalent, 102 μmol, 0.06 mL, 1.7 M in pentane) was added dropwise. Immediately thereafter, the lithiated intermediate was reacted with PhPCl2 (1.0 equivalent, 102 μmol, 14 μL, 97%), and the reaction mixture was stirred at room temperature for 1 hour.

[0141] The solvent was removed under vacuum, and 2 mL of DCM and chloro(dimethyl sulfide) gold(I) (DMS-AuCl) (1.0 equivalent, 102 μmol, 30 mg) were added. The reaction mixture was stirred for a further 1 hour. The solvent was removed again under vacuum to obtain a brown solid. After purification by column chromatography using silica and DCM as the eluent, 24 mg (36 μmol) of a yellow solid was isolated (yield: 51%). 1 H NMR (400 MHz, CDCl3): δ 8.70 (d, J = 7.4 Hz, 1H), 8.05 (dd, J = 18.7, 7.1 Hz, 1H), 7.96 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.80 (t, J = 3.6 Hz, 1H), 7.65 - 7.54 (m, 3H), 7.49 (dd, J = 7.6, 3.8 Hz, 3H), 7.39 - 7.28 (m, 6H), 6.67 (dd, J = 4.5, 3.6 Hz, 1H). 13 C{1 H} and DEPT 135 NMR (101 MHz, CDCl3): δ 137.7 (d, C), 137.0 (s, C), 136.5 (d, CH), 134.6 (s, CH), 133.6 (d, C), 133.3 (s, CH), 132.4 (s, CH), 132.3 (s, CH), 131.9 (s, CH), 130.8 (s, CH), 130.6 (s, CH), 129.2 (s, CH), 129.1 (s, CH), 128.6 (s, CH), 126.9 (s, CH), 126.1 (s, CH), 125.9 (s, CH), 122.2 (d, C), 115.6 (d, CH). 31 P{ 1 H} NMR (162 MHz, in CDCl3): δ 5.57. HRMS(ESI + )m / z:[C 26 H 18 AuClNO2PS] ·+ of [M ·+ Calculated value: 671.0150, measured value: 671.0205.

[0142] Synthesis of compound 8 1-thio-β-D-glucosetetraacetic acid (1.0 equivalent, 0.041 mmol, 15 mg) was dissolved in a baked-out Schlenk tube containing 1.4 mL of dry THF. Then, NaH (2.0 equivalent, 0.082 mmol, 2 mg) was added, and the reaction mixture was stirred for 1 hour. The resulting suspension was filtered through Celite under an inert atmosphere into a baked-out Schlenk tube containing compound 7 (0.9 equivalent, 0.037 mmol, 25 mg) dissolved in 1.1 mL of dry THF.

[0143] After stirring for 1.5 hours, the solvent was removed under vacuum. Purification by column chromatography using alumina and an siRNA:pentane (6:4) mixture as the eluent yielded 20 mg (0.02 mmol) of a yellow solid (yield: 54%). 11H NMR (600 MHz, CDCl3): δ 8.70 (dd, J = 7.4, 4.3 Hz, 1H), 8.16 (ddd, J = 18.2, 6.3, 3.5 Hz, 1H), 7.94 (d, J = 8.1 Hz, 1H), 7.82 (dt, J = 7.2, 3.5 Hz, 2H), 7.61 - 7.55 (m, 2H), 7.53 - 7.48 (m, 2H), 7.47 (d, J = 7.5 Hz, 1H), 7.42 - 7.28 (m, 6H), 6.82 - 6.77 (m, 1H), 5.21 - 5.09 (m, 4H), 4.23 (dd, J = 12.2, 4.7 Hz, 1H), 4.13 (d, J = 12.0 Hz, 1H), 3.78 (dd, J = 7.2, 2.2 Hz, 1H), 2.09 (s, 3H), 2.02 (d, J = 14.8 Hz, 7H), 1.88 (s, 3H). 13 13C{ 1 1H} and DEPT 135 NMR (101 MHz, CDCl3): δ 171.6 (d, C), 170.6 (s, C), 170.1 (s, C), 170.0 (s, C), 137.5 (d, C), 136.7 (t, CH), 134.8 (s, CH), 133.8 (t, C), 133.3 (s, CH), 132.7 (dd, CH), 131.9 (s, CH), 130.8 (dd, CH), 129.5 (dd, CH), 129.2 (s, CH), 128.5 (d, CH), 127.3 (d, CH), 126.4 (d, CH), 126.2 (s, CH), 12,2.6 (t, C), 116.0 (s, CH), 83.5 (s, CH), 76.1 (s, CH), 74.5 (s, CH), 69.2 (s, CH), 63.1 (s, CH2), 21.5 (s, CH3), 21.1 (d, CH3). 31 31P{ 1 1H} NMR (162 MHz, in CDCl3): δ 9.45. HRMS(ESI +)m / z:[C 40 H 37 AuNO 11 [PS2Na] ·+ of [M ·+ Calculated value: 1022.1109, measured value: 1022.1122.

Claims

1. Formula (A): 【Chemistry 1】 (In the formula, ArI represents a monocyclic aromatic moiety selected from the group consisting of phenyl, pyridine, pyrrole, furan, thiophene, and a 7-membered aromatic monocycle, or a bicyclic aromatic moiety selected from the group consisting of naphthalene, indole, and benzothiophene. ArI is a five- or six-membered aromatic heterocycle containing a halogen atom, N, S, or O, and C １～６ Aliphatic groups and C ３～６ Alicyclic group (the C １～６ Aliphatic groups and / or the C ３～６ The alicyclic group may be substituted with one or more substituents selected from the group consisting of one or more heteroatoms selected from N, S, and O. ArII and ArIII each independently represent a benzene group, a pyridine group, a pyrrole group, or a thiophene group. R １ represents an aromatic group, a hydroxy group, a C １ to C ６ alkyl group or a C １ to C ６ alkoxy group, and X is a sugar, albumin, halogen atom, CH ３ NO ３ , CN, SR ３ Selected from the group consisting of xanthate and 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate, R ３ 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl, β-D-glucopyranosyl, 2,3,4,6-tetramesyl-β-D-glucopyranosyl, 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl, 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl, hept-O-acetyl-β-maltosyl, 1,2-O-isopropylidene-5-α-D-xylofuranosyl, C １ ~C ８ Alkyl, CH(CO) ２ H) CH ２ CO ２ A pharmaceutical product comprising a compound selected from the group consisting of H,2-morpholinoethyl, 2'-ethyl-1-β-D-glucopyranosyl, 2'-ethyl-1-thio-β-D-glucopyranosyl, glutathioneyl hydrochloride, CN,phenyl, 1-aminophenyl, 2-pyridyl, 6-methyl-2-pyridyl, 4-pyridyl, thiazolin-2-yl, 4,5-dihydrothiazole-2-yl, 1H-benzimidazole-2-yl, benzoxazole-2-yl, benzothiazole-2-yl, pyrimidine-2-yl, 4-methylpyrimidine-2-yl, 4,6-dimethylpyrimidine-2-yl, 1,2-dihydropyridine-2-yl, 1,2-dihydropyrimidine-2-yl, 9H-purine-6-yl, and 2-amino-9H-purine-6-yl.

2. The pharmaceutical product according to claim 1, wherein ArII and ArIII both form a condensed bicyclic aromatic moiety selected from a naphthalene group, an indole group, a quinoline group, and a benzothiophene group.

3. The pharmaceutical product according to claim 1 or 2, wherein ArII and ArIII both form a naphthalene group.

4. ArI is a benzene group, naphthalene group, thiophene group, furan group, pyrrole group, benzothiophene group, or pyridine group, and ArI is a halogen atom, a 5-membered or 6-membered aromatic heterocycle containing N, S or O, C １～６ Aliphatic groups and C ３～６ Alicyclic group (the C １～６ Aliphatic groups and / or the C ３～６ The pharmaceutical product according to any one of claims 1 to 3, wherein the alicyclic group may be substituted with one or more substituents selected from the group consisting of one or more heteroatoms selected from N, S, and O.

5. The pharmaceutical product according to any one of claims 1 to 4, wherein ArI is a thiophene group.

6. The pharmaceutical product according to any one of claims 1 to 4, wherein ArI is a pyrrole group.

7. The pharmaceutical product according to any one of claims 1 to 6, wherein X is selected from the group consisting of Cl, xanthate, thiocyanide, and 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxan-2-thiolate.

8. The pharmaceutical product according to any one of claims 1 to 7, wherein X is xanthate or 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxan-2-thiolate.

9. ArI is an N-substituted pyrrole group having a methyl group as a substituent on the N atom, and R １ The pharmaceutical product according to claim 3, wherein is a phenyl group and X is 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate.

10. Formula (B): 【Chemistry 2】 (In the formula, R is methyl or SO ２ A compound of which is pH, where X is a chloride or 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate.

11. The compound according to claim 10, wherein R is methyl and X is 3,4,5-triacetyloxy-6-(acetyloxymethyl)oxane-2-thiolate.

12. A pharmaceutical product comprising the compound described in claim 10 or 11.

13. A pharmaceutical product according to any one of claims 1 to 9 or 12, for the treatment of cancer.

14. The pharmaceutical product according to claim 13, wherein the compound is for the treatment of glioblastoma.

15. A pharmaceutical product comprising a compound according to any one of claims 1 to 11 and at least one pharmaceutically acceptable excipient.

16. The pharmaceutical product according to claim 15, wherein the compound is dissolved in an aqueous solution containing DMSO.

17. A pharmaceutical product according to claim 15 or 16, for the treatment of cancer.

18. The pharmaceutical product according to claim 17, wherein the compound is used to be administered intravenously.

19. A kit comprising at least one compound and a container according to any one of claims 1 to 14.

20. A composition comprising a compound according to any one of claims 1 to 11 for in vitro / ex vivo inhibition of thioredoxin reductase (TrxR) activity.

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