Endoplasmic reticulum targeted ferroptosis micromolecule inducer as well as preparation method and application thereof

Abstract

The invention discloses an endoplasmic reticulum targeted ferroptosis micromolecule inducer as well as a preparation method and application thereof. The structural formula of the inducer is shown in the specification. A COX2 inhibitor (indometacin) and an endoplasmic reticulum target head (2-(4-methyl phenyl sulfonamide) acetic acid) are connected through a disulfide bond, and targeted delivery to an endoplasmic reticulum is achieved. The compound can synergistically induce endoplasmic reticulum stress, so that the content of calcium ions in tumor cells is increased, and the levels of lipid peroxide and active oxygen are increased, thereby causing a severe ferroptosis phenomenon and improving the treatment effect on cancers.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of chemical medicine synthesis, and particularly relates to an endoplasmic reticulum targeted ferroptosis small molecule inducer and a preparation method and application thereof. BACKGROUND

[0002] Cancer is a major global public health problem, with high incidence and mortality rates that continue to rise, posing a serious threat to human health. Traditional cancer treatment methods include surgery, radiotherapy and chemotherapy, etc. However, many malignant tumors are prone to drug resistance, which limits the treatment effect. Therefore, exploring new cell death mechanisms has become a hot spot in the field of cancer research. Ferroptosis is a form of iron-dependent programmed cell death, and its core feature is the excessive accumulation of lipid peroxides (LPO) on the cell membrane, leading to cell membrane damage and cell death. Studies have shown that increasing LPO levels can effectively induce ferroptosis, thereby achieving the killing effect on cancer cells.

[0003] The endoplasmic reticulum (ER) is an important organelle in cells, responsible for lipid secretion, protein folding and calcium ion regulation. Existing literature shows that the LPO phenomenon occurs initially in the endoplasmic reticulum, so the endoplasmic reticulum is considered a key site for lipid peroxidation in ferroptosis. Cancer cells often exhibit high-intensity endoplasmic reticulum stress (ERS) to cope with the accumulation of unfolded or misfolded proteins. However, sustained strong ERS can cause the endoplasmic reticulum to release a large amount of calcium ions, which are transmitted to mitochondria through mitochondria-associated membranes, thereby promoting the generation of reactive oxygen species (ROS) and LPO accumulation, and driving the occurrence of ferroptosis. Existing literature reports that ERS and ferroptosis interact, for example, certain ferroptosis inducers (such as erastin) can induce ERS, thereby enhancing ROS / LPO levels and inhibiting cancer cell proliferation in tumor models. In addition, GPX4, as a key antioxidant enzyme, inhibits ferroptosis by reducing phospholipid peroxides, and its inhibitors (such as RSL3) have been used to induce cancer cell ferroptosis and show anti-tumor potential in in vitro and in vivo experiments. COX2 inhibitors, such as indomethacin, have also been explored in cancer treatment, which can enhance oxidative stress by regulating inflammatory pathways and promote cell death in certain tumor models, but their mechanisms of action are mostly limited to anti-inflammatory or adjuvant therapy.

[0004] In the prior art, various ferroptosis inducers (FINs) have been developed, such as inducing LPO accumulation by inhibiting the system Xc- (Cystine / glutamate antiporter xCT) or GPX4 pathway, and applied in the treatment of liver cancer, ovarian cancer and other tumors in preclinical studies. In addition, nanomaterials or small molecule compounds have been used to target ER to induce ERS and promote programmed cell death in cancer therapy, such as by regulating ER function to enhance ferroptosis sensitivity. However, the existing ferroptosis induction strategies still have limitations: on the one hand, many FINs lack specific targeting of ER, which cannot fully utilize the interaction between ERS and ferroptosis; on the other hand, although COX2 inhibitors and GPX4 inhibitors play a role in oxidative stress and LPO regulation, respectively, there is no scheme to achieve synergistic induction of ERS, ROS / LPO accumulation and ferroptosis by targeted delivery to ER. In addition, the existing treatment methods still need to be improved in reversing tumor drug resistance and improving specificity, and new small molecule ferroptosis inducers targeting ER need to be developed to improve the efficiency of cancer treatment. SUMMARY

[0005] The present application aims to overcome the defects of the prior art and provide an endoplasmic reticulum targeted ferroptosis small molecule inducer.

[0006] Another object of the present application is to provide a preparation method of the above-mentioned endoplasmic reticulum targeted ferroptosis small molecule inducer.

[0007] Still another object of the present application is to provide the use of the above-mentioned endoplasmic reticulum targeted ferroptosis small molecule inducer.

[0008] The technical solution of the present application is as follows:

[0009] An endoplasmic reticulum targeted ferroptosis small molecule inducer, whose structural formula is .

[0010] The preparation method of the above-mentioned endoplasmic reticulum targeted ferroptosis small molecule inducer, whose synthetic route is as follows: .

[0011] In a preferred embodiment of the present application, the following steps are included:

[0012] (1) Dissolve indomethacin (IND) and 2-hydroxyethyl disulfide (SS) in a first organic solvent, then add DCC and DMAP, and mix at room temperature, then sequentially extract, remove the first organic solvent by reduced pressure distillation, and separate and purify to obtain an intermediate compound (IND-SS);

[0013] (2) the intermediate compound obtained in step (1) and 2-(4-methylphenylsulfonamido) acetic acid (MSG) are dissolved in a second organic solvent, DCC and DMAP are added, and then mixed at room temperature, followed by removal of the solvent by reduced pressure distillation, extraction, removal of the second organic solvent by reduced pressure distillation, separation and purification, and vacuum drying, to obtain the compound.

[0014] Further preferably, the first solvent is dichloromethane (DCM), and the second organic solvent is tetrahydrofuran (THF).

[0015] Further preferably, the molar ratio of the indomethacin and 2-hydroxyethyl disulfide is 1:2, and the molar ratio of the intermediate compound and 2-(4-methylphenylsulfonamido) acetic acid is 1:2.

[0016] A self-assembled nanoparticle is prepared by dissolving the endoplasmic reticulum-targeting ferroptosis small molecule inducer in DMSO and then stirring and washing in deionized water.

[0017] The endoplasmic reticulum-targeting ferroptosis small molecule inducer and / or the self-assembled nanoparticle are used in the preparation of a drug for preventing and / or treating endoplasmic reticulum stress-related diseases, including cancer, inflammatory diseases, metabolic diseases, osteoporosis, and neurodegenerative diseases.

[0018] A drug for preventing and / or treating endoplasmic reticulum stress-related diseases, wherein the effective component comprises the endoplasmic reticulum-targeting ferroptosis small molecule inducer and / or the self-assembled nanoparticle.

[0019] The endoplasmic reticulum-targeting ferroptosis small molecule inducer and / or the self-assembled nanoparticle are used in the preparation of a ferroptosis induction composition.

[0020] A ferroptosis induction composition, wherein the effective component comprises the endoplasmic reticulum-targeting ferroptosis small molecule inducer and / or the self-assembled nanoparticle.

[0021] The beneficial effects of the present application are:

[0022] 1. The present application realizes targeted delivery to the endoplasmic reticulum by connecting the COX2 inhibitor (indomethacin) and the endoplasmic reticulum target (2-(4-methylphenylsulfonamido) acetic acid) through a disulfide bond. This compound can induce endoplasmic reticulum stress, increase the content of calcium ions in tumor cells, and increase the levels of lipid peroxide and reactive oxygen species, thereby triggering severe ferroptosis and improving the treatment effect on cancer. In vitro experiments show that after treating 4T1 cells with this compound, the fluorescence intensity of calcium ions is significantly enhanced, the degree of lipid peroxidation is deepened, and obvious cell killing effect is shown (cell viability is reduced compared with the existing control group).

[0023] 2、The self-assembled nanoparticles (particle size about 149.1 nm, spherical uniformity) of the application further improve the delivery efficiency and biocompatibility. In the in vivo tumor-bearing mouse model, the nanoparticles are injected into the tail vein at a dose of 20 mg / kg, once every two days, for 14 consecutive days, which can significantly inhibit the growth of tumor volume, and the tumor inhibition rate is higher than that of the single component control group, indicating its potential in anti-tumor therapy. The effect is better than the non-targeting strategy of existing ferroptosis inducers, providing a new way to overcome tumor drug resistance.

[0024] 3、The preparation method of the application is simple, controllable, stable in yield (about 60% of the intermediate and about 30% of the target product), and the structural purity is verified by NMR and HRMS. The technical scheme is not only suitable for cancer, but also can be extended to the prevention and treatment of other endoplasmic reticulum stress related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of the intermediate compound IND-SS of embodiment 1 of the application.

[0026] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum of the target product IND-SS-MSG of embodiment 1 of the application.

[0027] Figure 3 It is the high resolution mass spectrum of the target product IND-SS-MSG of embodiment 1 of the application.

[0028] Figure 4 It is the TEM diagram of the nanoparticles of ISSM of embodiment 2 of the application.

[0029] Figure 5 It is the particle size diagram of ISSM of embodiment 2 of the application.

[0030] Figure 6 It is the intracellular calcium ion content diagram of embodiment 3 of the application.

[0031] Figure 7 It is the intracellular lipid peroxidation content diagram of embodiment 3 of the application.

[0032] Figure 8 It is the cytotoxicity diagram of embodiment 3 of the application.

[0033] Figure 9 It is the tumor volume change diagram of tumor-bearing mice in embodiment 4 of the application.

[0034] Figure 10 It is the tumor inhibition rate diagram of tumor-bearing mice in embodiment 4 of the application. DETAILED DESCRIPTION

[0035] The technical solutions of the present application are further described and explained with the specific embodiments in conjunction with the accompanying drawings.

[0036] Example 1

[0037] This example relates to a preparation method of endoplasmic reticulum targeting ferroptosis small molecule inducer compound IND-SS-MSG, and the synthetic route thereof is as follows:

[0038] Specifically includes the following steps:

[0039] (1) IND (714 mg, 2 mmol) and SS (489 μL, 4 mmol) were dissolved in 20 mL of DCM, and then DCC (495 mg, 2.4 mmol) and DMAP (73 mg, 0.6 mmol) were added, and the reaction was stirred at room temperature overnight (12-15 h). The reaction solution was extracted, distilled under reduced pressure, column chromatography and vacuum drying to obtain the intermediate compound IND-SS (590 mg, 60%). The nuclear magnetic resonance hydrogen spectrum of IND-SS is shown in Figure 1 , and the spectral data is as follows: 1 H NMR (600 MHz, DMSO): δ (ppm) 7.69-7.66 (m, 1H), 7.65-7.62 (m, 1H), 7.03 (d, J = 2.5 Hz, 1H), 6.93 (d, J = 9.0 Hz, 1H), 6.72 (dd, J = 9.0, 2.5 Hz, 1H), 4.88 (t, J = 5.5 Hz, 1H), 4.30 (t, J = 6.2 Hz, 2H), 3.79 (s, 2H), 3.76 (s, 3H), 3.59 (dd, J = 12.0, 6.3 Hz, 2H), 2.97 (t, J = 6.2 Hz, 2H), 2.77 (t, J = 6.4 Hz, 2H), 2.22 (s, 3H).

[0040] (2) The IND-SS (1170 mg, 2.37 mmol) and MSG (1086 mg, 4.14 mmol) obtained in step (1) were dissolved in 35 mL of THF, and then DCC (247 mg, 2.84 mmol) and DMAP (86 mg, 0.741 mmol) were added, and the reaction was stirred at room temperature overnight (12-15 h). The reaction solution was subjected to pressure reduction distillation, extraction, pressure reduction distillation, column chromatography, and vacuum drying in sequence to obtain the target product IND-SS-MSG (500 mg, 30%). The proton nuclear magnetic resonance spectrum thereof is shown in Figure 2 1 HNMR (600 MHz, CDCl3): δ (ppm) 7.65 (d, J = 8.2 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.1 Hz, 2H), 6.88 (d, J = 2.3 Hz, 1H), 6.81 (d, J = 9.0 Hz, 1H), 6.60 (dd, J = 9.0, 2.4 Hz, 1H), 5.16 (t, J = 5.5 Hz, 1H), 4.27 (t, J = 6.5 Hz, 2H), 4.15 (t, J = 6.4 Hz, 2H), 3.76 (s, 3H), 3.70 (d, J = 5.7 Hz, 2H), 3.62 (s, 2H), 2.81 (t, J = 6.5 Hz, 2H), 2.68 (t, J = 6.4 Hz, 2H), 2.34 (s, 3H), 2.31 (s, 3H).

[0041] The high-resolution mass spectrum thereof is shown in Figure 3 32 H 33 ClN2O8S3 + [M] + 705.1166, found 705.1164.

[0042] Example 2

[0043] This example relates to a preparation method of self-assembled nanoparticles of the endoplasmic reticulum-targeting ferroptosis small-molecule inducer prepared in Example 1, comprising the following steps:

[0044] ​​IND-SS-MSG prepared in Example 1 was used as the prodrug compound molecule, and self-assembled nanoparticles ISSM were prepared by nano-precipitation method. A certain amount of IND-SS-MSG was dissolved in a proper amount of DMSO, and after ultrasonic, it was slowly and uniformly added into deionized water under stirring for 12-15 h, and then centrifuged and washed, and the precipitate was self-assembled nanoparticles ISSM. Figure 4 For the TEM image of the self-assembled nanoparticles ISSM, it can be seen that the nanoparticles are spherical particles with uniform morphology. Figure 5 For the particle size diagram of the self-assembled nanoparticles ISSM, the size is about 149.1 nm.

[0045] Example 3

[0046] This example relates to the test of the self-assembled nanoparticles ISSM prepared in Example 2 on the intracellular calcium ion, lipid peroxide content and cell killing effect of 4T1 cells, as follows:

[0047] Logarithmic growth phase 4T1 cells were inoculated into 35 mm confocal culture dishes (2 × 10 6 cells per dish), complete culture medium was added, and the cells were cultured in a 37℃, 5% CO2 incubator for 24 h. The culture medium was replaced, and different drugs (60 μM) set according to the experiment were added to each group for continuous culture for 24 h. Subsequently, the cells were incubated with calcium ion fluorescent probe Fluo-4 (2 μM) for 30 min, washed with PBS for 3 times, and then incubated at 37℃ for 30 min. The sample was immediately observed using CLSM to obtain Figure 6 . As can be seen from Figure 6 , the red fluorescence of the tumor cells treated with ISSM was significantly enhanced, indicating that the intracellular calcium ion content was significantly increased, and it was proved that ISSM significantly induced the endoplasmic reticulum stress response of 4T1 tumor cells.

[0048] Logarithmic growth phase 4T1 cells were inoculated into 6-well plates (2 × 10 5 cells per well), complete culture medium was added, and the cells were cultured in a 37℃, 5% CO2 incubator for 24 h. The culture medium was replaced, and different drugs (60 μM) set according to the experiment were added to each group for continuous culture for 24 h. Subsequently, the cells were incubated with C11-BODIPY 581 / 591 fluorescent probe (10 μM) for 30 min, and then washed and observed using CLSM to obtain Figure 7 . As can be seen from Figure 7 , the red fluorescence of the tumor cells treated with ISSM was weakened, and the green fluorescence was enhanced, indicating that ISSM significantly induced the lipid peroxidation in 4T1 tumor cells.

[0049] Logarithmic growth phase 4T1 cells were inoculated into 96-well plates (1 × 108 Cells were added to complete culture medium and incubated at 37°C in a 5% CO2 incubator for 24 h. The culture medium was then changed, and different doses of the drug (5 μM, 10 μM, 20 μM, 40 μM, and 80 μM) were added to each group, with three replicates per group. After 24 h of incubation, the old drug-containing culture medium was aspirated, the cells were washed three times with PBS, and then CCK8 solution (100 μL / well) was added. The cells were incubated at 37°C for another 0.5 h, and finally, the absorbance was measured at 450 nm to calculate the relative cell viability. The results are as follows: Figure 8 As shown, ISSM has the most significant tumor cell killing effect.

[0050] Example 4

[0051] This embodiment relates to the in vivo pharmacodynamic evaluation of the self-assembled nanoparticles ISSM prepared in Example 2, as detailed below:

[0052] Healthy 4T1 cells were digested with trypsin, counted, centrifuged, and redispersed with cold PBS before being stored on ice. The PBS-dispersed tumor cell suspension was then inoculated into the subcutaneous tissue of the hind leg of BALB / c nude mice (6 × 10⁻⁶ cells / year). 6 To establish a 4T1 tumor-bearing mouse model, 200 μL of 100 cells were used. The tumor volume was increased to ~100 mm. 3 Twenty-five mice were randomly divided into five groups (n = 5): (1) PBS; (2) IND; (3) SS; (4) MSG; and (5) ISSM. The drugs in each group were administered via tail vein injection every two days at a dose of 20 mg / kg for 14 consecutive days (all tumor-bearing mice in the IND group died within 12 hours of the first administration). Tumor volume and mass were monitored, and the tumor volume was calculated using the formula: Tumor volume = (long axis × short axis × short axis) / 2. Tumor growth and size results are shown below. Figure 9 The results of tumor inhibition rate in mice are as follows: Figure 10 The results showed that the self-assembled nanoparticles ISSM exhibited the most significant tumor-suppressive effect within 14 days of drug administration.

[0053] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. An endoplasmic reticulum-targeted ferroptosis small molecule inducer, characterized by: The structural formula is 。 2. The preparation method of the endoplasmic reticulum targeting ferroptosis small molecule inducer of claim 1, characterized in that: The synthetic route is: 。 3. The production method according to claim 2, characterized by: The method comprises the following steps: (1) dissolving indometacin and 2-hydroxyethyl disulfide in a first organic solvent, then adding DCC and DMAP, mixing and reacting at room temperature, and then sequentially performing extraction, removing the first organic solvent by reduced pressure distillation, and separating and purifying to obtain an intermediate compound; (2) dissolving the intermediate compound obtained in step (1) and 2-(4-methylphenylsulfonamido) acetic acid in a second organic solvent, then adding DCC and DMAP, mixing and reacting at room temperature, and then sequentially performing removal of the solvent by reduced pressure distillation, extraction, removal of the second organic solvent by reduced pressure distillation, separation and purification, and vacuum drying.

4. The production method according to claim 3, characterized by: The first solvent is dichloromethane, and the second organic solvent is tetrahydrofuran.

5. The production method according to claim 3 or 4, characterized by: The molar ratio of indometacin to 2-hydroxyethyl disulfide is 1:2, and the molar ratio of the intermediate compound to 2-(4-methylphenylsulfonamido) acetic acid is 1:

2.

6. A self-assembling nanoparticle, characterized by: The endoplasmic reticulum-targeted ferroptosis small-molecule inducer in claim 1 is dissolved in DMSO, and then prepared by stirring and washing in deionized water.

7. Use of the endoplasmic reticulum-targeted ferroptosis small molecule inducer of claim 1 and / or the self-assembled nanoparticle of claim 6 in the manufacture of a medicament for preventing and / or treating an endoplasmic reticulum stress-related disease, characterized in that: The endoplasmic reticulum stress-related diseases include cancer, inflammatory diseases, metabolic diseases, osteoporosis, and neurodegenerative diseases.

8. A medicine for preventing and / or treating an endoplasmic reticulum stress-related disease, characterized by: The effective components include the endoplasmic reticulum-targeted ferroptosis small-molecule inducer in claim 1 and / or the self-assembled nanoparticles in claim 6.

9. Use of the endoplasmic reticulum-targeted ferroptosis small-molecule inducer in claim 1 and / or the self-assembled nanoparticles in claim 6 in the preparation of a ferroptosis induction composition.

10. An iron death inducing composition, characterized by: The effective components include the endoplasmic reticulum-targeted ferroptosis small-molecule inducer in claim 1 and / or the self-assembled nanoparticles in claim 6.

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