A class of temozolomide dextran derivatives and their use in the preparation of antitumor drugs

Abstract

The application discloses a temozolomide dexchlorpheniramine derivative and application thereof in preparation of an antitumor drug, and is based on new cognition of a TMZ drug resistance mechanism, designs and synthesizes two temozolomide derivatives. The target compounds retain the alkylating activity of TMZ, and metabolites do not produce 5-amino-imidazole-4 amide (AICA), but produce dexchlorpheniramine derivatives of AICA. Thus, the TMZ chemotherapy sensitivity is enhanced, and the TMZ drug resistance of glioma is delayed or reversed. The application first reconstructs the TMZ molecule from the metabolic product level, breaks through the action mode of the inherent drug resistance, and provides a brand-new drug design idea for overcoming the TMZ drug resistance. A new generation of TMZ derivative drugs can be developed, a single drug or a combined treatment scheme with higher efficacy and lower drug resistance is realized, and more breakthrough treatment options are provided for GBM patients.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, specifically relating to a class of temozolomide dextran derivatives and their use in the preparation of drugs for treating malignant brain tumors such as glioma. Background Technology

[0002] Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults, characterized by high invasiveness, high recurrence rate, and poor prognosis. Current standard treatment includes surgical resection, radiotherapy combined with temozolomide (TMZ) chemotherapy. TMZ, an alkylating prodrug capable of crossing the blood-brain barrier, has been a first-line chemotherapy drug since its approval for GBM treatment in 2005. However, the vast majority of patients experience recurrence after treatment, and effective treatment options are lacking after recurrence, resulting in an extremely low five-year survival rate.

[0003] TMZ is a small-molecule prodrug of the imidazotetrazine class. After oral administration, it readily crosses the blood-brain barrier and enters brain tissue. In vivo, TMZ is converted to methyltriazole imidazocarboxamide (MITC), which is further broken down into the metabolites 5-amino-imidazolium-4-amide (AICA) and methylhydrazine. Methylhydrazine transfers its methyl group to the nitrogen atoms at the 3rd and 7th positions of adenine and the oxygen atom at the 6th position of guanine in DNA, forming mispairings during DNA replication, causing DNA strand breaks, and inducing tumor cell death. However, GBM cells can develop resistance to TMZ through multiple mechanisms, primarily including enhanced DNA repair capabilities in tumor tissue and changes in the tumor microenvironment. Among these, O6-methylguanine-DNA methyltransferase (MGMT) can transfer methyl groups from DNA to itself, directly repairing TMZ-induced damage. Mismatch repair (MMR), homologous recombination repair (HR), and non-homologous end joining (NHEJ) DNA damage repair systems promote cell survival by repairing DNA breaks caused by TMZ. In addition, activation of pathways such as PI3K / AKT, Wnt / β-catenin, and Bcl-2, as well as abnormalities in glucose and lipid metabolism in tumor cells, also enhance the survival of tumor cells under drug stress.

[0004] In recent years, a study by Yin et al. (Nature Communications, 2023) revealed a novel metabolically dependent pathway for TMZ resistance: 5-aminoimidazolium-4-carboxamide (AICA), a byproduct of TMZ metabolism in vivo, can be converted into AICA ribonucleotides (AICAR) by hypoxanthine phosphoribosyltransferase 1 (HPRT1). AICAR, acting as an AMP analog, activates AMPK, which in turn phosphorylates RRM1, enhances ribonucleotide reductase (RNR) activity, promotes dNTP synthesis, and accelerates DNA damage repair, thereby mediating TMZ resistance. This study further found that high HPRT1 expression is significantly associated with poor prognosis in GBM patients, and that the HPRT1 inhibitor 6-mercaptopurine (6-MP) can block this pathway and enhance TMZ efficacy.

[0005] Although various strategies have been proposed to combat TMZ resistance, such as MGMT inhibitors, DNA repair inhibitors, signaling pathway targeted drugs, immunotherapy, and tumor-treating fields, no single approach has yet significantly improved survival outcomes in patients with relapsed GBM. Existing strategies primarily target downstream repair pathways or bypass signaling, failing to fundamentally address the resistance induced by TMZ metabolism itself. Furthermore, existing inhibitors, such as 6-MP, have non-specific mechanisms of action and may cause systemic toxicity, limiting their clinical application.

[0006] Therefore, there is an urgent and important clinical need to develop a novel drug that can fundamentally block the drug resistance pathway mediated by TMZ metabolites, and is highly selective and low in toxicity. This invention is based on a deep understanding of the novel mechanisms of TMZ resistance. It aims to modify the TMZ molecule through structural design so that it can exert its alkylating and killing effects while avoiding the production of the resistance-promoting metabolite AICA. This, in turn, inhibits the activation of the HPRT1 / AMPK / RRM1 repair pathway at its source, enhancing the therapeutic effect of TMZ and prolonging patient survival.

[0007] References

[0008] Hypoxanthine phosphoribosyl transferase 1 metabolizes temozolomide toactivate AMPK for driving chemoresistance of glioblastomas. Nat Commun. 2023Sep 22;14(1):5913. Summary of the Invention

[0009] The purpose of this invention is to provide a novel temozolomide derivative and its application in the preparation of antitumor drugs. This temozolomide derivative does not produce AICA during metabolism, thereby enhancing the chemosensitivity of TMZ and delaying or reversing TMZ resistance in gliomas.

[0010] The objective of this invention can be achieved through the following technical solutions:

[0011] In a first aspect, the present invention seeks protection for a class of temozolomide dextran alcohol derivatives, the structural formula of which is shown in HL-1 or HL-2:

[0012] ,

[0013] .

[0014] Secondly, the present invention seeks protection for the use of the above-mentioned temozolomide dextranol derivative in the preparation of antitumor drugs.

[0015] Furthermore, the tumor is a glioma. Even further, the glioma is at least one of astrocytoma, oligodendroglioma, ependymoma, and other glial-derived tumors. Specifically, the astrocytoma is at least one of pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, and glioblastoma; the oligodendroglioma is at least one of oligodendroglioma and anaplastic oligodendroglioma; the ependymoma is at least one of subependymal tumor, myxopapillary ependymoma, ependymoma, and anaplastic ependymoma; and the other glial-derived tumors are at least one of choroid plexus tumors and astroblastoma.

[0016] The antitumor drugs used in the above applications use temozolomide dextran derivatives as the active ingredient or main active ingredient, and are prepared into pharmaceutically acceptable dosage forms with pharmaceutically acceptable carriers.

[0017] Thirdly, this invention claims protection for a pharmaceutical composition, wherein the aforementioned temozolomide dextran derivative is used as the active ingredient or main active ingredient, and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier. The dosage form is a tablet, capsule, powder, pellet, suspension, or nasal spray.

[0018] Recent studies have found that 5-aminoimidazole-4-carboxamide (AICA), a byproduct of TMZ metabolism in vivo, can be converted into AICA ribonucleotides (AICAR) by inosine phosphoribosyltransferase 1 (HPRT1). AICAR, acting as an AMP analog, activates AMPK, which in turn phosphorylates RRM1, enhances ribonucleotide reductase (RNR) activity, promotes dNTP synthesis, and accelerates DNA damage repair, thereby mediating TMZ resistance. Based on a deep understanding of this novel TMZ resistance mechanism, this invention modifies the TMZ molecular structure to ensure it exerts its alkylating and killing effects while avoiding the production of the resistance-promoting metabolite AICA. This inhibits the activation of the HPRT1 / AMPK / RRM1 repair pathway at its source, enhancing the therapeutic effect of TMZ.

[0019] Based on a new understanding of the TMZ resistance mechanism, this invention designs and synthesizes two temozolomide derivatives. While retaining the TMZ alkylation activity, the target compounds do not produce 5-amino-imidazol-4 amide (AICA) as their metabolites; instead, they produce dextromethorphanol derivatives of AICA.

[0020] In vitro cell experiments demonstrated that the temozolomide dextran derivative described in this invention exhibits a similar killing effect on temozolomide-sensitive U87 cells as temozolomide itself. However, its killing effect on temozolomide-resistant T98G cells is stronger than that of the temozolomide dextran derivative described in this invention. Further animal experiments showed that the target compound, alone or in combination with TMZ, can inhibit the growth of orthotopic xenografts, prolong the survival of tumor-bearing mice, and reduce the dosage of TMZ.

[0021] The beneficial effects of this invention are:

[0022] This invention is the first to reconstruct the TMZ molecule at the metabolite level, breaking through its inherent drug resistance mechanism and providing a novel drug design approach to overcome TMZ resistance. It holds promise for developing a new generation of TMZ-derived drugs, achieving more effective and less resistant monotherapy or combination therapy regimens, providing more groundbreaking treatment options for GBM patients. Attached Figure Description

[0023] Figure 1 Effects of compounds HL01, HL02 and TMZ on the viability of glioblastoma cell lines.

[0024] In Figure A, U87 cells were treated with different concentrations of compounds for 96 hours, and the inhibitory effect of the compounds on the cells was detected by the CCK8 assay. In Figure B, T98G cells were treated with different concentrations of compounds for 96 hours, and the inhibitory effect of the compounds on the cells was detected by the CCK8 assay. Data are mean ± SEM.

[0025] Figure 2Effects of compounds HL01, HL02 and TMZ on survival in mice with glioblastoma.

[0026] In this table, A represents the method of in situ glioma modeling and drug administration in mice. B represents the body weight of mice. C represents the survival curves of mice. C(a), C(b), and C(c) are the survival curves of mice in the HL-01 group, HL-02 group, and TMZ+HL-02 group, respectively (the solvent control group and the TMZ-only treatment group in C(a), C(b), and C(c) are the same set of data).

[0027] Figure 3 Compounds HL01, HL02, and TMZ slow down tumor growth.

[0028] In this diagram, A shows the bioluminescence in the brains of mice in each group, detected by a small animal in vivo imaging system, providing a visual representation of tumor growth. B shows a statistical graph of the fluorescence values ​​in the brains of mice in each group, quantifying the differences in tumor growth rates.

[0029] Figure 4 Effects of compounds HL01, HL02, and TMZ on glioblastoma volume in mice (Micro-CT in vivo imaging). Tumor shadows in the brains of mice in different treatment groups (solvent control, HL-01, HL-02, TMZ, TMZ+HL-02) are shown, allowing for a direct comparison of tumor size. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0032] Example 1: Design and Synthesis of Compounds

[0033] Example 1.1 Synthesis of (3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazodane-8-carboxyl)glycine dextranol ester (HL-1)

[0034] Synthesis route:

[0035]

[0036] Procedure: Boc-glycine (0.35 g, 2.01 mmol), dextranol (0.37 g, 2.41 mmol), and 4-dimethylaminopyridine (0.12 g, 1.00 mmol) were dissolved in dichloromethane (5 mL). The mixture was stirred at room temperature (25 ± 5 °C) for 10 minutes, followed by the addition of dicyclohexylcarbodiimide (0.62 g, 3.01 mmol), and stirring for another 5 hours. After the reaction was complete, the mixture was filtered, and the filtrate was evaporated to dryness. Water (40 mL) was added, and the mixture was extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated sodium chloride solution (50 mL × 3), and the organic phase was dried over anhydrous sodium sulfate. The solution was evaporated to dryness under reduced pressure to give a colorless oil, I-a (0.45 g, 1.43 mmol), in 72% yield. I-a (0.45 g, 1.43 mmol) was dissolved in ethyl acetate (3 mL), and 2 M hydrogen chloride-ethyl acetate solution (4 mL) was added at 0 °C. The mixture was stirred for 5 hours. A large amount of white solid precipitated out. The solid was evaporated to dryness under reduced pressure to obtain a white solid, which was Boc-glycine dextranol ester (0.34 g, 1.36 mmol). Temozolomide (0.30 g, 1.54 mmol), Boc-glycine dextranol ester (0.46 g, 1.83 mmol), and 4-dimethylaminopyridine (0.09 g, 0.77 mmol) were dissolved in dichloromethane (5 mL). After stirring at room temperature for 10 minutes, dicyclohexylcarbodiimide (0.48 g, 2.31 mmol) was added, and the mixture was stirred for another 5 hours. After the reaction was complete, the mixture was filtered, and the filtrate was evaporated to dryness. Water (40 mL) was added, and the mixture was extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated sodium chloride solution (50 mL × 3), and the organic phase was dried over anhydrous sodium sulfate. Column chromatography (petroleum ether:ethyl acetate = 5:1, v:v) yielded a white solid, HL-1 (0.41 g, 1.06 mmol). 1 HNMR (400 MHz, Chloroform-d) δ 8.38(s, 1H), 7.81 (t, J = 5.4 Hz, 1H), 4.95 (dt, J = 10.1, 2.7 Hz, 1H), 4.29 (d,J = 5.3 Hz, 2H), 4.00 (s, 3H), 2.34 (ddt, J = 13.9, 8.9, 4.0 Hz, 1H), 1.88 (ddd, J = 12.7, 9.0, 4.3 Hz, 2H), 0.90–0.78 (m, 10H).

[0037] Example 1.2 Synthesis of 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazodane-8-carboxylic acid dextranol ester (HL-2)

[0038] Synthesis route:

[0039]

[0040] Procedure: Temozolomide (0.30 g, 1.54 mmol), dextran (0.24 g, 1.83 mmol), and 4-dimethylaminopyridine (0.09 g, 0.77 mmol) were dissolved in dichloromethane (5 mL). After stirring at room temperature for 10 minutes, dicyclohexylcarbodiimide (0.48 g, 2.31 mmol) was added, and the mixture was stirred for another 5 hours. After the reaction was complete, the mixture was filtered, and the filtrate was evaporated to dryness. Water (40 mL) was added, and the mixture was extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated sodium chloride solution (50 mL × 3), and the organic phase was dried over anhydrous sodium sulfate. The mixture was separated by column chromatography (petroleum ether:ethyl acetate = 5:1, v:v) to give a white solid HL-2 (0.33 g, yield 64.7%). 1 H NMR (400 MHz, DMSO-d6) δ 8.81 (d, J = 1.3 Hz, 1H), 4.56 (d,J = 1.7 Hz, 1H), 2.07 (dd, J = 16.8, 10.7 Hz, 1H), 1.76 – 1.59 (m, 4H), 1.52– 1.37 (m, 1H), 1.24 – 0.98 (m, 10H), 0.77 (s, 3H).

[0041] Example 2: Effect of the target compound on the viability of glioma cell lines

[0042] The cytotoxic effect of the target compound on tumor cells was detected using the CCK8 assay: TMZ-sensitive U87 and TMZ-resistant T98G cell lines were cultured in DMEM medium (containing 10% (v / v) fetal bovine serum). Cells were collected and seeded into 96-well plates at a density of 1500 cells / well and incubated overnight at 37°C. Different concentrations of the target compound and TMZ (800, 400, 200, 100, 50, and 25 μM) were added, and the cells were incubated at 37°C for another 96 hours. 10 μl of CCK8 solution was added to each well, and the cells were incubated for 30 minutes. The absorbance at 450 nm was measured using a microplate reader. The inhibition rate was calculated using the formula: Relative cell inhibition rate (%) = (Blank control group - Experimental group) / Blank control group × 100%. Each group was set up with 3 replicates, and the experiment was repeated three times.

[0043] Experimental results are as follows Figure 1 As shown, in TMZ-sensitive U87 cells, the cell-killing effects of the target compounds HL01 and HL02 are similar to those of TMZ. Figure 1 (A in the text). For the T98G cell line resistant to TMZ, the labeled compounds HL01 and HL02 showed stronger cytotoxic effects than TMZ. Figure 1 (B in the middle).

[0044] Example 3: Compounds prolong the survival of mice with glioblastoma.

[0045] Experimental Methods: Female BALB / c nude mice (4-6 weeks old) were acclimatized for one week in an SPF-grade animal laboratory. During this period, a constant temperature and humidity environment was maintained (22.0 ± 1.0℃, relative humidity 50% ± 10%), with a 12h / 12h diurnal light cycle. All animals had free access to sterilized feed and drinking water. A stable luciferase-expressing U87-luc cell line was constructed. When the cells reached 90% confluence in culture dishes, they were collected, and based on cell counts, an appropriate amount of PBS was added to adjust the cell concentration to 5 × 10⁻⁶ cells / mL. 4 2 μL of cells were placed on ice. Mice were anesthetized with 2% isoflurane. After the pain reflex disappeared, the mice were fixed on a small animal stereotaxic apparatus. A 1 mL syringe needle was used to pre-drill a hole slightly to the right of the midpoint between the anterior and posterior fontanelles. 2 μL of cells were drawn up with a microsyringe and the needle was inserted into the pre-drilled hole to a depth of 3.5 mm. The needle was stopped for 2 minutes. The cell suspension was then slowly and evenly injected into the mouse brain. After stopping the injection for 5 minutes, the microsyringe was slowly removed. The injection site was disinfected with povidone-iodine. The mice were placed in an incubator and awaited recovery, during which time their vital signs were observed. After recovery, the mice were returned to their cages and continued to be fed daily. The size of the tumor was observed using a small animal in vivo imaging system. When the fluorescence value reached 10... 6 Mice were divided into 5 groups: solvent control group (5% DMSO + 10% Solutol HS15 + 85% saline, intraperitoneally injected daily, where 5%, 10%, and 85% were volume ratios, i.e., v / v); compound HL01 group (51 μmol / kg, intraperitoneally injected); HL02 group (51 μmol / kg, intraperitoneally injected); TMZ group (51 μmol / kg, intraperitoneally injected); and TMZ+HL02 group (25 μmol / kg each, intraperitoneally injected). The administration frequency was daily for 5 consecutive days, followed by a 2-day break. Figure 2 (A) During the experiment, the mice's weight was recorded daily. The experiment was terminated when the weight loss was greater than 20%.

[0046] Experimental results are as follows Figure 2As shown, during the administration period, the body weight of mice in the TMZ-only treatment group decreased significantly ( Figure 2 (B) Compared with the control group, the survival time of mice in the compound treatment group was significantly prolonged ( Figure 2 (C) Among them, the average survival time of mice in the solvent control group was 30.5 days, while that in the TMZ-only group was extended to 59.5 days. The average survival time of mice in the HL01 group was 70.5 days, longer than that of the temozolomide-only group. More importantly, the average survival time of mice in the HL02 and TMZ combination group was 63.5 days. With the TMZ dosage halved, the survival time of mice was similar to that of the TMZ-only group (59.5 days), indicating that HL02 can effectively reduce the dosage of temozolomide (Table 1).

[0047] Table 1. Mean lifespan of mice

[0048] Solvent control HL01 HL02 Temozolomide Temozolomide + HLO2 Mean survival time (days) 30.5 70.5 54 59.5 63.5

[0049] Example 4: Compounds slow down tumor growth in mice

[0050] Experimental Methods: The mouse modeling of orthotopic glioblastoma and the drug administration regimen were the same as in Example 3. Small Animal In vivo Imaging Observation Method: Mice were anesthetized with 2% (v / v) isoflurane after intraperitoneal injection of D-fluorescein potassium solution, and then placed in the instrument for visible light imaging. The brain was the quantitative site for visible light, and the fluorescence values ​​of each mouse's brain were measured and statistically analyzed. During the experiment, in vivo imaging was performed every 7 days to monitor tumor growth rate.

[0051] Experimental results are as follows Figure 3 As shown, during the experiment, the tumor volume in the solvent control group mice continued to increase until the mice died. The tumor growth rate in the drug-treated group mice was slower. The tumor growth rates in the TMZ and TMZ+HL02 combined drug-treated groups were comparable. This indicates that the compound can significantly delay the tumor growth rate in mice.

[0052] Example 5: Compound induces tumor volume reduction

[0053] Experimental methods: The mouse orthotopic glioblastoma model and drug administration regimen were the same as in Example 3. Twenty-one days after drug administration, tumor size was observed using a small animal in vivo Micro-CT imaging system.

[0054] Experimental results are as follows Figure 4 As shown, compared with the solvent control group, the tumors in mice treated with HL01 and HL02 compounds alone were significantly shrunk, indicating that the target compounds have a good anti-glioblastoma effect. Meanwhile, the tumors in the TMZ+HL02 combined treatment group shrank further, approaching the size of the TMZ-treated group, indicating that the HL02 compound can achieve a good anti-glioblastoma effect with reduced TMZ dosage.

Claims

1. A class of temozolomide dextran alcohol derivatives, the structural formula of which is shown in HL-1 or HL-2: 、 。 2. The use of the temozolomide dextranol derivative according to claim 1 in the preparation of antitumor drugs.

3. The application according to claim 2, characterized in that, The tumor in question is a glioma.

4. The application according to claim 3, characterized in that, The glioma is at least one of astrocytoma, oligodendroglioma, ependymoma, and other glial-derived tumors.

5. The application according to claim 4, characterized in that, The astrocytoma is at least one of pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, and glioblastoma; the oligodendroglioma is at least one of oligodendroglioma and anaplastic oligodendroglioma; the ependymoma is at least one of subependymal tumor, myxopapillary ependymoma, ependymoma, and anaplastic ependymoma; the other glial-derived tumors are at least one of choroid plexus tumors and astroblastoma.

6. The application according to claim 2, characterized in that, The drug uses temozolomide dextran derivative as the active ingredient or main active ingredient, and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition uses the temozolomide dextran derivative of claim 1 as the active ingredient or main active ingredient, and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The dosage form is tablet, capsule, powder, drop pill, suspension or nasal spray.

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