Tellurium-isoindoline compounds for the preparation of pharmaceutical compositions for the treatment of lung cancer and head and neck cancer and their uses

Abstract

The purpose of this invention is to provide an isoindolin-1-one compound containing tellurium (Te) for use in the preparation of pharmaceutical compositions for treating lung cancer and head and neck cancer. This invention refers to the known synthetic method of ebselen (also known as PZ 51, DR3305, or SP-1005), replacing the selenium (Se) element used in the process with tellurium (Te), resulting in a drug with the same structure but completely different activity, named ebtellur. The invention also provides the use of ebtellur as an anticancer agent against human non-small cell lung cancer (A549) and head and neck cancer cells.

Description

Technical Field

[0001] This invention relates to the therapeutic efficacy of a tellurium-isoindoline compound—ebtellur—for cancer, particularly for lung cancer and head and neck cancer. Prior Technology

[0002] Tellurium is located below selenium in the periodic table, belonging to the metalloids. Compounds in this group, except for those containing oxygen, are generally called chalcogenides. Tellurium is considered a "niche" element in this group because it is not a necessary trace metal for humans. In contrast, humans cannot exist without compounds containing oxygen atoms; sulfur atoms are present in glutathione; and selenium atoms are present in related human proteins such as GPx, TrxR, and thyroxine deiodinase. Tellurium is considered a toxic, non-essential trace element, therefore chemists do not consider tellurium atoms as a pharmaceutical option. The toxicity of tellurium depends on its oxidation state; for example, tellurate (Te IV) is 100-1000 times more toxic than selenate. Because tellurium is rarely added to pharmaceuticals, reagents made from tellurium have the potential to be used as agents against multidrug-resistant pathogens.

[0003] Ebselen (2-phenyl-1,2-benzisoselenazol-3-(2H)-one), a selenium-containing selenium-isoindoline compound, was synthesized in 1924 but was not used in drug screening until around the 1980s. It reached clinical trial level II but did not reach clinical trial level III. It exhibited antibacterial activity against Staphylococcus aureus and could address the problem of multidrug resistance. Its research focused on oxidative-stress-related diseases, with Ebselen acting as an analogue to glutathione peroxidase (GPx), which protects cells from damage caused by reactive oxygen species (ROS), primarily peroxides. However, clinical evaluations revealed some cytotoxicity in Ebselen, preventing it from passing clinical trials.

[0004] Therefore, finding an epoepithecus substitute with low toxicity to normal cells is the problem that this invention aims to solve. Summary of the Invention

[0005] In view of this, the inventor of this case deeply understands the shortcomings and defects of the previous case, and is eager to improve and innovate. After years of research, the inventor has successfully developed this invention, which improves lung cancer and head and neck cancer by using a compound that replaces selenium in ibuprofen with tellurium, providing a novel strategy for cancer treatment.

[0006] To achieve the above objectives, the present invention provides the use of ebtellur or a pharmaceutically acceptable salt thereof for preparing a pharmaceutical composition for treating lung cancer or head and neck cancer.

[0007] In one embodiment of the present invention, the head and neck cancer is tongue cancer, oral squamous cell carcinoma, or human hypopharyngeal squamous cell carcinoma.

[0008] In one embodiment of the present invention, the head and neck cancer is human hypopharyngeal squamous cell carcinoma.

[0009] In one embodiment of the present invention, the treatment of lung cancer or head and neck cancer is achieved by inhibiting cancer cell proliferation, promoting cancer cell apoptosis, reducing the expression level of Bcl-2 protein in cancer cells, increasing the expression level of proteins selected from the group composed of Bax, cleaved-caspase 3, and cleaved-PARP1, or increasing the survival rate of the individual.

[0010] In one embodiment of the invention, the pharmaceutical composition for treating lung cancer or head and neck cancer further comprises a pharmaceutically acceptable carrier selected from one or more agents in the group consisting of solvents, buffers, emulsifiers, suspending agents, disintegrants, disintegrants, dispersants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delay agents, and liposomes.

[0011] In one embodiment of the present invention, the pharmaceutical composition for treating lung cancer or head and neck cancer is an oral dosage form or a non-enteric dosage form.

[0012] The present invention uses ibutellurium as an active ingredient to inhibit the proliferation of lung cancer and head and neck cancer cells and promote apoptosis of lung cancer and head and neck cancer cells, providing a novel and safe improvement strategy. Simple Explanation of the Diagram

[0013] Figure 1 shows the synthetic pathway of ibuselenium; Figure 2 compares the effects of ibutellurium and ibuselenium on inhibiting FaDu cell proliferation; Figure 3 compares the effects of ibutellurium and ibuselenium on inhibiting the proliferation of A549 cells; Figure 4 shows a comparison of the protein expression levels of Bcl-2 and cleaved-PARP in A549 cells after administration of ibutellurium and ibuselenium. Figure 5 shows a comparison of the protein expression levels of Bax and cleaved-caspase 3 in A549 cells after administration of ibutellurium and ibuselenium. Implementation

[0014] All technical and scientific terms used in this specification, unless otherwise defined, shall have the meaning commonly understood by one of ordinary skill in the art.

[0015] The singular terms “a,” “an,” and “the” used in this specification and the claims, unless otherwise stated, may refer to more than one object.

[0016] The term "medically acceptable salt" refers to a salt that retains the bioavailability and properties of a free base or free acid, and is not biologically or otherwise disadvantageous. This salt is formed from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and especially hydrochloric acid, as well as from organic acids, such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzyl acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetycysteine, etc. Furthermore, these salts can be prepared by adding inorganic or organic bases to free acids. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyimide resins, etc.

[0017] Unless otherwise specified, all materials used in this invention are commercially available and readily available.

[0018] [Example 1: Ibuselenium analogues in which tellurium is substituted for selenium]

[0019] There are multiple synthetic routes for ebselen, and related technologies have been comprehensively reviewed and reported, for example, Claudio Santi et al. (2021), Molecules. 26(14):4230. The synthetic routes of ebselen are summarized in Figure 1. The inventors attempted to replace the selenium used in the steps with tellurium to obtain ebtellurium. However, most preparation methods are not easy to adjust and have low yields. Among them, the routes with higher yields are route a and route e. After many attempts, the inventors found that route e is easier to adjust. Therefore, this invention uses the copper-catalyzed method (route e) to prepare the required Ebselen and Ebtellur.

[0020] The chemical formula of the ibutellite is shown in Chemical Formula 1. [Chemical Formula 1]

[0021] [Example 2: MTT cell proliferation assay (Methylthiazol tetrazolium cell proliferation assay)]

[0022] To compare the anticancer activities of ibutellurium and ibuselenium, lung cancer cells and normal cells were treated with ibutellurium or ibuselenium for 72 hours, and the effects of each compound on the activity of cancer cells and normal cells were compared by MTT assay to evaluate their direct anticancer effects.

[0023] IC50 is the half maximal inhibitory concentration, which is the concentration required to inhibit 50% of cell activity.

[0024] Selective antiproliferative activity can be determined using the selective index (SI), which is calculated as SI = IC50 of non-cancer cells / IC50 of cancer cells. An SI value > 3.0 indicates that, ideally, the drug can affect cancer cells but should not affect normal cells. In this invention, the non-cancer cells used to calculate the SI value are HEK293 normal human embryonic kidney cells.

[0025] The A549 human non-small cell lung cancer cell line and the HEK293 normal human embryonic kidney cell line were used. The results are shown in Table 1. Ibutellurium exhibited better selective antiproliferative effects against A549 lung cancer cells than ibuselenium, with IC50 values ​​of 6.02±1.62 and 22.09±1.27 μM, respectively (SI value > 3.0). Furthermore, the chemotherapeutic drugs doxorubicin (Dox) and cisplatin were used as positive controls; however, only cisplatin showed selective antiproliferative activity against A549 lung cancer cells, with an IC50 of 2.29±0.07 μM (SI value > 3.0). The IC50 of Dox's cytotoxic effect on A549 lung cancer cells was 3.04±0.59 μM (SI value = 2.80).

[0026] Table 1. Comparison of the inhibitory effects of ibutelluride and ibuselenium on human non-small cell lung cancer cells (A549) and normal cells (HEK293). Compound A549 HEK293 Lung cancer (EGFR wt) Normal human embryonic kidney IC 50 (µM) a SI IC 50 (µM) a Ebselen a 22.09±1.27 *** 4.53 >100 Ebtellur a 6.02±1.62 *** 13.28 79.97±0.77 ** Dox b 3.04±0.59 *** 2.80 8.50±0.59 *** Cisplatin b 2.29±0.07 *** 7.34 16.82±1.09 *** Results are presented as mean ± standard error, n=3. The concentration required to achieve 50% inhibition (IC50) is [a]. bDoxorubicin (DOX) and Cisplatin served as positive control groups. **p<0.01; ***p<0.0001. SI: Selective index.

[0027] To further compare the anticancer effects of ibutellurium and ibuselenium on head and neck cancer cells from different sources (tongue cancer, oral squamous cells, or oropharyngeal squamous cells), ibutellurium or ibuselenium was administered to cancer cells for 72 hours, and their direct anticancer effects were evaluated.

[0028] Head and neck cancer cells were selected using SAS tongue cancer cells, OECM-1 oral squamous cell carcinoma cells, and FaDu human hypopharyngeal squamous cell carcinoma cells. The results are shown in Table 2. Both ibutellurium and ibuselenium significantly reduced the cell viability of FaDu cells in a dose-dependent manner, with IC50 values ​​of 11.58±0.37 and 36.37±1.22 μM, respectively, and SI values ​​of 2.75 and 6.91, respectively. Ibutellurium also inhibited the cell viability of SAS and OECM-1 cells, with IC50 values ​​of 77.21±0.56 and 96.8±0.66 μM, respectively, while ibuselenium showed no significant inhibitory effect on the cell viability of SAS and OECM-1 cells.

[0029] The results of the MTT cell proliferation assay showed that ibutellurium has the effect of inhibiting the activity of cancer cells. When used to treat A549 lung cancer cells and FaDu cells, ibutellurium requires a lower dose than ibuselenium and has a higher SI value, while having less impact on normal cells.

[0030] Table 2. Comparison of the inhibitory effects and anticancer capabilities of ibutellurium and ibuselenium on human head and neck cancer cells. Compound SAS OECM-1 FaDu Tongue carcinoma Oral squamous cell carcinoma Human pharynx squamous cell carcinoma IC 50 (µM) a SI IC 50 (µM) a SI IC 50 (µM) a SI Ebselen a >100 *** >100 36.37±1.22 *** >2.75 Ebtellur a 77.21±1.56 *** 1.04 96.8±0.66 0.83 11.58±0.37 *** 6.91 Dox b 1.06±0.73 *** 8.02 2.56±0.62 *** 3.32 3.23±0.86 *** 2.63 Cisplatin b 7.05±1.43 *** 2.39 5.56±1.18 *** 3.03 6.38±0.78 *** 2.64 Results are presented as mean ± standard error, n=3. The concentration required to achieve 50% inhibition (IC50) is [a]. bDoxorubicin (DOX) and Cisplatin served as positive control groups. ***p<0.0001. SI: Selective index.

[0031] Example 3: Community Analysis Method [Clonogenic assay] [)]

[0032] The effects of ebutellurium and ebuselenium on FaDu cell proliferation were evaluated using community analysis, with Cisplatin as a positive control. FaDu cells were seeded in culture dishes and treated for 24 hours with ebuselenium (6.25, 12.5, 25, and 50 μM), ebutellurium (6.25, 12.5, and 25 μM), or Cisplatin (25 μM; control). After 14 days, cells were fixed in 1% formalin containing 1% crystal violet, and community formation was observed using an inverted microscope. The number of communities in the culture dishes was quantified and statistically analyzed, showing a significant difference compared to the control group. The results are shown in Figure 2 (*=p<0.05, **=p<0.01). At doses of 6.25, 12.5, 25, or 25 μM, the community-forming ability of FaDu cells treated with ebutellurium was significantly reduced, while ebuselenium had no effect on FaDu cell community formation.

[0033] The effects of ebutellurium and ebuselenium on the proliferation of A549 lung cancer cells were evaluated using community analysis, with Cisplatin as a positive control. A549 lung cancer cells were seeded in culture dishes and treated for 24 hours with ebuselenium (12.5, 25, and 50 μM), ebutellurium (6.25, 12.5, and 25 μM), or Cisplatin (25 μM; control). After 14 days, cells were fixed in 1% formalin containing 1% crystal violet, and community formation was observed using an inverted microscope. The number of communities in the culture dishes was quantified and statistically analyzed, showing a significant difference compared to the control group. The results are shown in Figure 3, *=p<0.05, **=p<0.01. Ebutellurium at a dose of 12.5 μM significantly inhibited the community formation of A549 lung cancer cells, indicating that ebutellurium has an anti-proliferative effect on FaDu cells.

[0034] Community analysis experiments showed that ibutellurium had a stronger inhibitory effect on the proliferation of lung cancer and human hypopharyngeal squamous cell carcinoma compared to ibuselenium.

[0035] Example 4: Western Ink Dot Method Western Blotting [)]

[0036] The effects of ebutellurium and ebuselenium on the expression levels of proteins related to apoptosis and cell death in A549 lung cancer cells were confirmed using the Western ink dot method. A549 lung cancer cells were treated with 12.5, 25, and 50 μM ebutellurium and 25 and 50 μM ebuselenium for 48 hours, with Cisplatin as a positive control. The results are shown in Figures 4 and 5. Figure 4 shows the Western ink dot method results for Bcl-2 and cleaved-PARP, and Figure 5 shows the Western ink dot method results for Bax and cleaved-caspase 3.

[0037] Western ink dot assay results showed that the 25 and 50 μM ibutelluride concentrations significantly reduced the expression of Bcl-2 protein, which inhibits programmed cell death, and significantly increased the expression of the apoptosis regulator Bax, the apoptosis protease cleaved-caspase 3, and the apoptosis response marker cleaved-PARP. Moreover, ibutelluride had a more significant effect than ibuselenium in all these cases.

[0038] The inventors replaced selenium in ebuselenium with tellurium, obtaining a tellurium-isoindoline compound called ebuselenium tellurium. Ebuselenium tellurium exhibits anticancer activity and significant selective antiproliferative activity against A549 lung cancer cells and FaDu hypopharyngeal carcinoma cells, with a higher selectivity index than ebuselenium. Compared to ebuselenium, ebuselenium tellurium shows stronger antiproliferative effects against A549 lung cancer cells, FaDu hypopharyngeal carcinoma cells, SAS tongue cancer cells, and OECM-1 oral cancer cells. Ebuselenium tellurium can regulate the expression levels of apoptosis-related proteins, inducing cancer cell apoptosis, and exhibits a stronger regulatory effect than ebuselenium. Ebuselenium tellurium shows greater potential than ebuselenium in the treatment of lung cancer and head and neck cancer.

[0039] Based on the preferred embodiments disclosed in this specification, those skilled in the art will clearly understand that the foregoing embodiments are merely illustrative; those skilled in the art can implement the invention through numerous modifications and substitutions without differing from the technical features of the invention. According to the embodiments in this specification, the invention can be modified in various ways without hindering its implementation. The claims provided in this specification define the scope of the invention, which covers the foregoing methods and structures and equivalent inventions.

[0040] The aforementioned multiple benefits fully meet the statutory requirements for novelty and inventiveness for patentability. Therefore, this application is filed in accordance with the law, and we respectfully request your approval of this invention patent application to encourage invention.

Claims

1. Use of an ebtellurium or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition for treating cancer; wherein the cancer is lung cancer or head and neck cancer.

2. The use as described in claim 1, wherein the lung cancer is non-small cell lung cancer.

3. The use as described in claim 1, wherein the head and neck cancer is tongue cancer, oral squamous cell carcinoma, or human hypopharyngeal squamous cell carcinoma.

4. The use as described in claim 1, wherein the head and neck cancer is human hypopharyngeal squamous cell carcinoma.

5. The use as described in claim 1, which is used to inhibit cancer cell proliferation, promote cancer cell apoptosis, reduce the expression level of Bcl-2 protein in cancer cells, increase the expression level of proteins selected from the group composed of Bax, cleaved-caspase 3, and cleaved-PARP1, or increase the survival rate of the individual.

6. The use as described in claim 1, wherein the pharmaceutical composition for treating lung cancer or head and neck cancer further comprises a pharmaceutically acceptable carrier selected from one or more agents comprising solvents, buffers, emulsifiers, suspending agents, disintegrants, disintegrants, dispersants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delay agents, and liposomes.

7. As claimed in claim 1, wherein the pharmaceutical composition for treating lung cancer or head and neck cancer is an oral or non-enteric dosage form.

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