Imidazotetrazine compounds, methods of making, and uses thereof

Abstract

The application provides imidazotetrazine compounds or pharmaceutically acceptable salts thereof, and a preparation method and application thereof, and belongs to the technical field of medicines. The imidazotetrazine compounds or pharmaceutically acceptable salts thereof are obtained by connecting valproic acid, chlorambucil and borneol to an imidazotetrazine group through an ester bond or an amide bond by using a splicing principle. The imidazotetrazine compounds or pharmaceutically acceptable salts thereof have better antitumor activity, and a new generation of powerful antitumor drugs can be developed.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an imidazotetrazine compound or a pharmaceutically acceptable salt thereof, its preparation method, and its application. Background Technology

[0002] Gliomas are the most common primary brain tumors, arising from the cancerous transformation of glial cells in the brain and spinal cord. The World Health Organization classifies gliomas into four grades: Grades I and II are low-grade gliomas, mostly benign, while Grades III and IV are high-grade gliomas. Grade IV glioblastoma multiforme (GBM) is the most malignant and aggressive, with a median survival of approximately 12.1 months and a five-year survival rate of less than 5%. Currently, the main clinical treatment for GBM is maximally surgical resection of the tumor, followed by radiotherapy and chemotherapy. Postoperative chemotherapy has become one of the important means to prolong the survival time of patients with malignant gliomas.

[0003] Temozolomide (TMZ) is an orally administered imidazotetraazine compound or its pharmaceutically acceptable salt, and is currently a first-line drug for the clinical treatment of GBM. Due to the presence of the blood-brain barrier (BBB), novel molecular targeted therapies and immunotherapies are not very effective, while TMZ can cross the BBB and has become the gold standard for GBM treatment. At physiological pH, the methyldiazo ion released by TMZ can methylate guanine at the N7 and O6 positions and adenine at the N3 position, leading to base pair mismatches, DNA strand breaks, and ultimately apoptosis of tumor cells. However, due to the presence of methylguanine DNA methyltransferase (MGMT), drug resistance to TMZ can occur in clinical use.

[0004] In addition to temozolomide, ACT001, a compound derived from parthenolide through chemical structural modification, targets the NF-κB and STAT3 signaling pathways, exhibits immunomodulatory effects, can cross the blood-brain barrier, and directly act on brain tumor lesions; it is currently undergoing clinical trials. At present, new molecularly targeted drug therapies, immunotherapies, and gene therapies are also being explored in the treatment of glioblastoma, but their efficacy still needs to be verified through clinical trials.

[0005] Therefore, in order to overcome the shortcomings of TMZ in efficacy and further improve its efficacy, a large number of structural modification studies have been carried out using TMZ as a lead compound, and corresponding progress has been made. We modified the bicyclic structure of imidazotetrazine, the substituents at the 3-position nitrogen atom and the 8-position carbon atom in different ways, synthesized a variety of temozolomide derivatives, and studied their anti-glioma cell activity and mechanism of action. Summary of the Invention

[0006] On the one hand, the purpose of this invention is to provide an imidazotetrazine compound or a pharmaceutically acceptable salt thereof.

[0007] In a second aspect, the present invention provides pharmaceutical compositions comprising imidazotetrazine compounds or pharmaceutically acceptable salts thereof, and their use in the preparation of medicaments for the treatment of glioblastoma.

[0008] Thirdly, the present invention provides a method for preparing the imidazotetrazine compounds.

[0009] Fourthly, the present invention provides an intermediate of the imidazotetrazine compound or a pharmaceutically acceptable salt thereof.

[0010] Therefore, the technical solution of the present invention is implemented as follows:

[0011] This invention provides an imidazotetraazine compound or a pharmaceutically acceptable salt thereof, having a general structural formula of at least one of the following formulas I to IV:

[0012]

[0013] As a further improvement of the present invention, n = 1-10, preferably n = 1-6.

[0014] This invention provides an imidazotetraazine compound or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt includes organic or inorganic salts, including but not limited to: sodium salts, potassium salts, cesium salts, calcium salts, magnesium salts, triethylamine salts, pyridine salts, methylpyridine salts, ethanolamine salts, triethanolamine salts, dicyclohexylamine salts, N,N-dibenzylethylenediamine salts, hydrochloride salts, hydrobromide salts, sulfate salts, nitrate salts, phosphate salts, formate salts, acetate salts, trifluoroacetate salts, pantothenate, succinate, citrate, tartrate, fumarate, maleate, gluconate, glucuronide, glycosides, benzoate, lactate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, arginine salts, aspartate salts, glutamate salts, pantothenate, ascorbate, and combinations thereof.

[0015] The present invention also provides pharmaceutical compositions comprising an imidazotetrazine compound or a pharmaceutically acceptable salt thereof and thereof with a pharmaceutically acceptable carrier or excipient.

[0016] Each component in the pharmaceutical combination of the present invention may optionally be used in combination with one or more pharmaceutically acceptable carriers, wherein the components may be independent, or may contain, in part or in whole, a pharmaceutically acceptable carrier and / or excipients. The pharmaceutical combination of the present invention may be formulated separately, or may contain, in part or in whole, a formulation. The components in the pharmaceutical combination of the present invention may be administered individually, or may contain, in part or in whole, a formulation.

[0017] In this invention, depending on the desired route of administration, a pharmaceutically acceptable composition will comprise about 1 to about 99% by weight of the compound of the present invention or a pharmaceutically acceptable salt thereof, and 99 to 1% by weight of a suitable carrier or pharmaceutical excipient. Preferably, the composition comprises about 5 to 75% by weight of the conjugate of the present invention, with the remainder being a suitable carrier or pharmaceutical excipient. More preferably, the composition comprises about 10 to 50% by weight of the conjugate of the present invention, with the remainder being a suitable carrier or pharmaceutical excipient.

[0018] In embodiments of the invention, the combinations of the invention can be administered in pure compound form or in the form of a suitable pharmaceutical composition, using any acceptable route of administration or reagents for similar purposes. Therefore, the route of administration can be selected via oral, intranasal, enteral, topical, transdermal, or rectal routes, in the form of a solid, semi-solid, or liquid preparation, such as tablets, suppositories, pills, soft and hard gelatin capsules, powders, solutions, suspensions, and injections, preferably in a unit-dose form suitable for precise dosage.

[0019] The components in the pharmaceutical combination of the present invention may be individually, or some or all of them together, suitable dosage forms, including but not limited to tablets, lozenges, pills, capsules (e.g., hard capsules, soft capsules, enteric-coated capsules, microcapsules), elixirs, granules, syrups, injections (intramuscular, intravenous, intraperitoneal), granules, emulsions, suspensions, solutions, dispersants, and sustained-release formulations for oral or non-oral administration.

[0020] Pharmaceutical compositions that can be administered in liquid form can be formed by dissolving or dispersing the complex of the present invention (about 0.5% to about 20%) and selectively present pharmaceutical excipients in a carrier by means of dissolution, dispersion, etc. Examples of carriers are water, saline, aqueous glucose, glycerol, ethanol, etc., thereby forming a solution or suspension.

[0021] This invention further protects the use of an imidazotetrazine compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of glioblastoma.

[0022] Furthermore, the glioblastoma described in this invention includes, but is not limited to, primary glioblastoma and secondary glioblastoma.

[0023] Furthermore, the glioblastomas described in this invention include, but are not limited to, giant cell glioblastoma, epithelioid glioblastoma, and glioblastoma containing primitive neuronal components.

[0024] Furthermore, the glioblastomas described in this invention include, but are not limited to, locally advanced and / or advanced glioblastomas; the glioblastomas are those that have failed prior treatment; the glioblastomas are those that have failed prior surgical resection of the tumor; the glioblastomas are those that have failed radiotherapy and / or chemotherapy; the glioblastomas are those that have progressed or relapsed after prior treatment with at least one chemotherapy drug; and the glioblastomas are those that cannot tolerate chemotherapy drugs.

[0025] Specifically, the glioblastoma can be an IDH wild-type glioblastoma, an IDH mutant glioblastoma, or a glioblastoma with IDH1 and / or IDH2 mutations, with specific sites including but not limited to IDH1R132 and IDH2R172.

[0026] This invention further protects a method for preparing the above-mentioned imidazotetrazine compound, wherein the imidazotetrazine compound has the following structural formula: The synthesis route is as follows:

[0027]

[0028] The structural formula of the imidazotetraazine compound is as follows: The synthesis route is as follows:

[0029] The structural formula of the imidazotetraazine compound is as follows: The synthesis route is as follows:

[0030] The structural formula of the imidazotetraazine compound is as follows: The synthesis route is as follows:

[0031]

[0032] As a further improvement of the present invention, the structural formula of the imidazotetraazine compound is as follows: The preparation method is as follows: valproic acid, α-ω-diol, 4-dimethylaminopyridine, and dichloromethane are added to a flask, and carbodiimide is added under ice bath conditions. The reaction is carried out at room temperature for 24 hours, and the intermediate TMQX1 is purified by silica gel column chromatography. In a round-bottom flask, intermediate TMQX1, temozolomide, 4-dimethylaminopyridine, chloroform, and solvent are added sequentially, followed by the addition of carbodiimide under ice bath conditions. The reaction is carried out at room temperature for 24 hours, and the crude product is purified by silica gel column chromatography to obtain the final product.

[0033] As a further improvement of the present invention, the structural formula of the imidazotetraazine compound is as follows: The preparation method is as follows: Temozolomide, 1,2-dichloroethane, and oxalyl chloride are added sequentially and mixed evenly. The mixture is reacted at 85-95℃ for 2-4 hours. The solvent is evaporated, and the mixture is redissolved in 1,2-dichloroethane. Then, intermediate TMQX1 is added, and the mixture is reacted overnight at room temperature. The mixture is concentrated under reduced pressure and purified by silica gel column chromatography to obtain the product.

[0034] As a further improvement of the present invention, the structural formula of the imidazotetraazine compound is as follows: The preparation method is as follows: chlorambucil, α-ω-diol, 4-dimethylaminopyridine, and dichloromethane are added to a flask. Carbodiimide is added under ice bath conditions, and the reaction is carried out at room temperature for 20-30 hours. The intermediate TMQX2 is purified by silica gel column chromatography. Temozolomide, 1,2-dichloroethane, and oxaloyl chloride are added sequentially to a round-bottom flask, and the reaction is carried out at 85-95℃ for 2-4 hours. The solvent is evaporated, and the product is redissolved in 1,2-dichloroethane. The intermediate TMQX2 is then added, and the reaction is carried out overnight at room temperature. The product is concentrated under reduced pressure and purified by silica gel column chromatography.

[0035] As a further improvement of the present invention, the structural formula of the imidazotetraazine compound is as follows: The preparation method is as follows: Borneol, phenoxyacetic acid, 4-dimethylaminopyridine, and dichloromethane are added sequentially. After dissolution, carbodiimide is added under ice bath and reacted at room temperature for 20-30 h. After the reaction is complete, the mixture is concentrated under reduced pressure. The crude product is purified by silica gel column chromatography. Anhydrous methanol is used as the solvent for Pd / C catalytic hydrogenation reduction reaction for 4-6 h. Pd / C is filtered off to obtain intermediate TMQX3. Temozolomide, 1,2-dichloroethane, and oxalyl chloride are added sequentially and reacted at 85-95℃ for 2-4 h. The solvent is evaporated, and the mixture is redissolved in 1,2-dichloroethane. Intermediate TMQX3 is added, and the mixture is reacted overnight at room temperature. The mixture is concentrated under reduced pressure and purified by silica gel column chromatography to obtain the product.

[0036] This invention further protects an intermediate TMQX1, having a structure as shown in Formula V:

[0037]

[0038] Where n = 1 - 10.

[0039] This invention further protects an intermediate TMQX2 having the structure shown in Formula VI:

[0040]

[0041] Where n = 1 - 10.

[0042] This invention further protects an intermediate TMQX3 having the structure shown in Formula VII:

[0043]

[0044]

[0045] Where n = 1 - 10.

[0046] The present invention has the following beneficial effects:

[0047] Valproic acid is commonly used clinically to treat epilepsy, a complication of glioma; chlorambucil is a commonly used nitrogen mustard antitumor drug; and borneol is often used to enhance BBB permeability. There are clinical records of their combined use with temozolomide. This invention connects valproic acid, chlorambucil, and borneol to an imidazotetrazine group via ester or amide bonds to obtain imidazotetrazine compounds. These imidazotetrazine compounds or their pharmaceutically acceptable salts exhibit better antitumor activity, especially against glioma, breast cancer, lung cancer, or human lymphoma. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 This is a comparison diagram of cytotoxicity against human breast cancer MDA-MB-231 cells in Example 5;

[0050] Figure 2 This is a comparison diagram of cytotoxicity against human lung cancer A549 cells in Example 5;

[0051] Figure 3 This is a comparison diagram of cytotoxicity against human lymphoma Raji cells in Example 5. Detailed Implementation

[0052] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] Example 1

[0054] The synthesis route is as follows:

[0055]

[0056] Where n = 2, QX102; n = 3, QX103; n = 4, QX104; n = 5, QX105; n = 6, QX106.

[0057] Valproic acid (1 mmol), α-ω-diol (5 mmol), 4-dimethylaminopyridine (1 mmol), and dichloromethane (5 mL) were added to a flask. Carbodiimide (1 mmol) was added under ice bath conditions, and the reaction was carried out at room temperature for 24 h. The intermediate TMQX1 was purified by silica gel column chromatography. In a round-bottom flask, TMQX1 (0.5 mmol), temozolomide (0.5 mmol), 4-dimethylaminopyridine (0.5 mmol), and chloroform (5 mL) were added sequentially. After dissolution, carbodiimide (2 mmol) was added under ice bath conditions, and the reaction was carried out at room temperature for 24 h. The crude product was purified by silica gel column chromatography to obtain the final product.

[0058] 2-((2-propylpentanoyl)oxy)ethyl 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetraazine-8-carboxylate (QX102)

[0059] Overall yield: 46.37%, HRMS: 388.1591 [M+Na] 1 H NMR (400MHz, CDCl3) δ8.48(s,1H),4.70(dd,J=5.7,3.9Hz,2H),4.49(dd,J=5.7,4.0Hz,2H),4.08(s,3 H),2.54–2.28(m,1H),1.67–1.55(m,2H),1.48–1.37(m,2H),1.35–1.24(m,4H),0.86(t,J=7.3Hz,5H). 13 C NMR (101MHz, CDCl3) δ176.25,160.05,138.46,135.86,128.80,128.52,63.39,61.48,45.05,36.61,34.43,20.51,13.91.

[0060] 3-((2-propylpentanoyl)oxy)propyl 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetraazine-8-carboxylate (QX103)

[0061] Overall yield: 14.56%, HRMS: 402.1748 [M+Na] 1 H NMR(400MHz, CDCl3)δ8.48(s,1H),4.75–4.56(m,2H),4.51–4.38(m,2H),4.08(s,3H),2.63–2.28(m,1H), 1.62(td,J=15.2,8.6Hz,3H), 1.42(ddd,J=9.4,7.6,4.9Hz,2H), 1.36–1.23(m,4H), 0.87(t,J=7.3Hz,5H). 13 C NMR (101MHz, CDCl3) δ176.25,160.05,128.82,128.52,63.39,61.48,45.06,36.61,34.43,20.51,13.92.

[0062] 4-((2-propylpentanoyl)oxy)butyl-3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetraazine-8-carboxylate (QX104)

[0063] Overall yield: 36.87%, HRMS: 416.1904 [M+Na] 1 H NMR (400MHz, CDCl3) δ8.48(s,1H),4.52(t,J=6.4Hz,2H),4.15(t,J=6.3Hz,2H),4.07(s,3H),2.36(dd,J=9.7,4.4Hz,1H),1.93(dt ,J=10.6,6.4Hz,2H),1.85(dt,J=7.8,6.2Hz,2H),1.65–1.53(m,2H),1.47–1.38(m,2H),1.34–1.23(m,6H),0.90(t,J=7.3Hz,7H). 13 C NMR (101MHz, CDCl3) δ176.49,160.29,138.50,135.74,129.22,128.49,65.27,63.40,45.23,36.58,34.57,25.37,25.31,20.58,13.93.

[0064] 5-((2-propylpentanoyl)oxy)pentyl 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetraazine-8-carboxylate (QX105)

[0065] Overall yield: 18.50%, HRMS: 430.2057 [M+Na] 1 H NMR (400MHz, CDCl3) δ8.47(s,1H),4.49(t,J=6.7Hz,2H),4.09(t,J=6.6Hz,2H),4.07(s,2H),2. 41–2.31(m,1H),1.89(s,2H),1.73(s,2H),1.58(s,4H),1.42(s,2H),1.30(s,5H),0.89(s,6H). 13 C NMR (101MHz, CDCl3) δ176.54,160.35,138.51,135.71,129.29,128.47,65.59,63.70,45.23,36.57,34.59,28.26,28.20,22.38,20.56,13.92.

[0066] 6-((2-propylpentanoyl)oxy)hexyl 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetraazine-8-carboxylate (QX106)

[0067] Overall yield: 22.46%, HRMS: 444.2213 [M+Na] 1 H NMR(400MHz, CDCl3)δ8.47(s,1H),4.48(t,J=6.7Hz,2H),4.12–4.05(m,5H),2.41–2.31(m,1H),1.91–1.80(m,2H) ),1.67(dt,J=13.3,6.5Hz,2H),1.61–1.49(m,4H),1.48–1.37(m,4H),1.34–1.24(m,5H),0.90(t,J=7.3Hz,6H). 13 CNMR(101MHz,CDCl3)δ176.56,160.37,138.53,135.71,129.35,128.46,65 .74,63.84,45.26,36.56,34.61,28.49,28.47,25.53,25.48,20.56,13.93.

[0068] Example 2

[0069] The synthesis route is as follows:

[0070]

[0071] Where n = 2, QX202; n = 3, QX203; n = 4, QX204; n = 5, QX205; n = 6, QX206.

[0072] Temozolomide (0.5 mmol), 1,2-dichloroethane (5 mL), and oxalyl chloride (3 mmol) were added sequentially to a round-bottom flask. The mixture was reacted at 90 °C for 3 h. The solvent was evaporated and the product was redissolved in 1,2-dichloroethane (5 mL). Then, intermediate TMQX1 (0.5 mmol) was added and the mixture was reacted at room temperature for 12 h. The product was concentrated under reduced pressure and purified by silica gel column chromatography.

[0073] 2-(((((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)ethylvalerate 2-propyl ester (QX202)

[0074] Yield: 46.37%, HRMS: 409.1694 [M+H] 1 H NMR(400MHz, CDCl3)δ9.38(s,1H),8.44(s,1H),4.68–4.24(m,4H),4.09(s,3H),2.42(s,1H) ,1.68–1.53(m,2H),1.51–1.39(m,2H),1.30(dd,J=19.2,11.1Hz,6H),0.90(t,J=7.2Hz,6H). 13 C NMR (101MHz, CDCl3) δ176.24,156.20,149.83,138.30,135.05,129.56,127.85,63.96,61.31,45.05,36.81,34.48,20.53,13.92.

[0075] 3-(((((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)propyl 2-propane valerate (QX203)

[0076] Yield: 15.48%, HRMS: 423.1851 [M+H] 1H NMR (400MHz, CDCl3) δ9.33(s,1H),8.44(s,1H),4.36(t,J=6.0Hz,2H),4.23(t,J=5.9Hz,2H),4.09(s,3H),2.38(s,1H), 2.08(dd,J=12.3,6.3Hz,2H),1.68–1.56(m,2H),1.42(dd,J=13.1,7.2Hz,2H),1.35–1.22(m,5H),0.90(t,J=7.2Hz,6H). 13 C NMR (101MHz, CDCl3) δ176.42,156.24,150.25,138.31,135.04,129.62,127.84,62.98,60.26,45.18,36.80,34.54,27.99,20.58,13.92.

[0077] 4-(((((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)butyl-2-propane valerate (QX204)

[0078] Yield: 20.69%, HRMS: 437.2004 [M+H] 1 H NMR(400MHz, CDCl3)δ9.29(s,1H),8.42(s,1H),4.26(s,2H),4.08(s,2H),4.05(s,3H),2.33(s,1H),1 .76(s,4H),1.54(dt,J=15.1,7.5Hz,2H),1.44–1.33(m,2H),1.31–1.19(m,5H),0.86(t,J=7.1Hz,6H). 13 C NMR (101MHz, CDCl3) δ176.47,156.32,150.32,138.33,135.07,129.41,127.90,65.76,63.29,45.17,36.79,34.53,25.24,25.16,20.53,13.90.

[0079] 5-(((((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)pentyl-2-propane valerate (QX205)

[0080] Yield: 15.13%, HRMS: 451.2159 [M+H] 1H NMR(400MHz, CDCl3)δ9.30(s,1H),8.44(s,1H),4.27(t,J=6.1Hz,2H),4.09(d,J=9.0Hz,5H),2.36(s,1H),1.81–1.73(m,2H),1.72–1 .66(m,2H),1.58(dd,J=13.3,7.8Hz,2H),1.50(dd,J=15.2,8.4Hz,2H),1.45–1.36(m,2H),1.33–1.24(m,5H),0.89(t,J=7.1Hz,6H). 13 C NMR (101MHz, CDCl3) δ176.56,156.29,150.44,138.33,135.01,129.70,127 .82,66.17,63.64,45.23,36.78,34.59,28.23,28.14,22.26,20.56,13.93.

[0081] 6-(((((3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)propyl hexyl 2-pentanoate (QX206)

[0082] Yield: 35.01%, HRMS: 465.2318 [M+H] 1 H NMR (400MHz, CDCl3) δ9.28(s,1H),8.42(s,1H),4.23(t,J=6.3Hz,2H),4.04(d,J=11.0Hz,5H),2.38–2.26(m,1H),1.78–1.67(m,2H) ,1.62(dd,J=12.5,6.3Hz,2H),1.54(dt,J=15.1,7.5Hz,2H),1.38(dd,J=13.6,6.7Hz,6H),1.31–1.19(m,5H),0.87(t,J=7.1Hz,6H). 13 C NMR (101MHz, CDCl3) δ176.55,156.33,150.41,138.34,135.04,129.55,127.87 ,66.24,63.79,45.22,36.78,34.58,28.46,28.42,25.52,25.34,20.54,13.91.

[0083] Example 3

[0084] The synthesis route is as follows:

[0085]

[0086] Where n = 2, QX302; n = 3, QX303; n = 4, QX304; n = 5, QX305; n = 6, QX306.

[0087] Chlorobutyric acid mustard (1 mmol), α-ω-diol (5 mmol), 4-dimethylaminopyridine (1 mmol), and dichloromethane (5 mL) were added to a flask. Carbodiimide (2 mmol) was added under ice bath conditions, and the reaction was carried out at room temperature for 24 h. The intermediate TMQX2 was purified by silica gel column chromatography. Temozolomide (0.5 mmol), 1,2-dichloroethane (5 mL), and oxalyl chloride (3 mmol) were added sequentially to a round-bottom flask. The reaction was carried out at 90 °C for 3 h. The solvent was evaporated, and the product was redissolved in 1,2-dichloroethane (5 mL). TMQX2 (0.5 mmol) was then added, and the reaction was carried out overnight at room temperature. The product was concentrated under reduced pressure and purified by silica gel column chromatography.

[0088] 2-((((3-Methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)ethyl 4-(4-(bis(2-chloroethyl)amino)phenyl)butyrate (QX302)

[0089] Yield: 26.14%, HRMS: 568.1345 [M+H] 1 H NMR (400MHz, CDCl3) δ9.36 (s, 1H), 8.41 (s, 1H), 7.06 (d, J = 8.5Hz, 2H), 6.60 (d, J = 8.6Hz, 2H), 4.47 (dd, J = 5.7, 3.3Hz, 2H), 4.36 (dd, J = 5. 7,3.3Hz,2H),4.07(s,3H),3.69(t,J=6.4Hz,4H),3.61(t,J=6.3Hz,4H),2.56(t,J=7.5Hz,2H),2.37(t,J=7.4Hz,2H),1.98–1.86(m,2H). 13 C NMR (101MHz, CDCl3) δ173.26,156.20,149.96,144.29,138.27,135.05,130.42,129. 63,129.57,127.84,112.10,63.95,61.70,53.53,40.51,36.81,33.85,33.32,26.54.

[0090] 3-(((3-Methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)propyl 4-(4-(bis(2-chloroethyl)amino)phenyl)butyrate (QX303)

[0091] Yield: 32.00% HRMS: 582.1506 [M+H] 1 HNMR (400MHz, CDCl3) δ9.33(s,1H),8.44(s,1H),7.08(d,J=8.4Hz,2H),6.65(d,J=8.5Hz,2H),4.38(t,J=6.2Hz,2H),4.24(t,J=6.2Hz,2H),4 .09(s,3H),3.72(t,J=6.5Hz,4H),3.64(t,J=6.4Hz,4H),2.57(t,J=7.5Hz,2H),2.35(t,J=7.5Hz,2H),2.17–2.04(m,2H),1.98–1.86(m,2H). 13 C NMR (101MHz, CDCl3) δ173.43,156.23,150.31,144.22,138.30,135.03,130.56,129. 62,127.84,112.23,62.99,60.55,53.60,40.47,36.81,33.90,33.47,27.89,26.60.

[0092] 4-(((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)butyl-4-(4-(bis(2-chloroethyl)amino)phenyl)butyrate (QX304)

[0093] Yield: 64.06%, HRMS: 596.1658 [M+H] 1 H NMR (400MHz, CDCl3) δ9.31 (s, 1H), 8.43 (s, 1H), 7.07 (d, J = 8.3Hz, 2H), 6.63 (d, J = 8.3Hz, 2H), 4.31 (t, J = 5.5Hz, 2H), 4.13 (t, J = 5.6Hz, 2H) ,4.08(s,3H),3.71(t,J=6.8Hz,4H),3.63(t,J=6.7Hz,4H),2.56(t,J=7.5Hz,2H),2.34(t,J=7.4Hz,2H),1.97–1.87(m,2H),1.81(s,5H). 13C NMR (101MHz, CDCl3) δ173.53,156.29,150.44,144.29,138.31,135.04,130.49,129.60, 127.85,112.12,65.88,63.62,53.53,40.53,36.80,33.93,33.56,26.68,25.28,25.15.

[0094] 5-((((3-Methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)pentyl 4-(4-(bis(2-chloroethyl)amino)phenyl)butyrate (QX305)

[0095] Yield: 24.24%, HRMS: 610.1814 [M+H] 1 H NMR (400MHz, CDCl3) δ9.30 (s, 1H), 8.43 (s, 1H), 7.07 (d, J = 8.5Hz, 2H), 6.63 (d, J = 8. 6Hz,2H),4.29(t,J=6.5Hz,2H),4.13–4.05(m,5H),3.71(t,J=6.4Hz,4H),3.63(dd,J =10.3,3.7Hz,4H),2.56(t,J=7.6Hz,2H),2.33(t,J=7.5Hz,2H),1.97–1.86(m,2H), 1.82–1.74(m,3H),1.70(dd,J=14.6,7.0Hz,2H),1.51(ddd,J=18.8,9.1,6.1Hz,2H). 13 C NMR (101MHz, CDCl3) δ173.57,156.29,150.48,144.28,138.31,135.01,130.54,129.69,129.59 ,127.84,112.11,66.17,63.97,53.54,40.52,36.80,33.93,33.60,28.21,28.18,26.70,22.31.

[0096] 6-((((3-Methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)hexyl-4-(4-(bis(2-chloroethyl)amino)phenyl)butyrate (QX306)

[0097] Yield: 65.35%, HRMS: 624.1973 [M+H] 1H NMR (400MHz, CDCl3) δ9.30 (s, 1H), 7.06 (d, J = 8.4Hz, 2H), 6.62 (d, J = 8.4Hz, 2H),4.26(t,J=6.6Hz,2H),4.10–4.04(m,5H),3.70(t,J=6.9Hz,4H),3.62(t ,J=6.8Hz,4H),2.55(t,J=7.5Hz,2H),2.32(t,J=7.4Hz,2H),1.89(p,J=7.7 Hz,3H),1.80–1.71(m,2H),1.70–1.61(m,2H),1.43(dd,J=11.2,7.7Hz,4H). 13 C NMR (101MHz, CDCl3) δ173.59,156.36,150.46,144.28,138.33,135.05,130.51,129.58,127.89 ,112.09,66.27,64.14,53.51,40.55,36.80,33.92,33.60,28.45,28.42,26.71,25.54,25.38.

[0098] Example 4

[0099]

[0100]

[0101] Where n = 1, QX401; n = 2, QX402; n = 3, QX403; n = 4, QX404; n = 5, QX405.

[0102] Borneol (1 mmol), phenoxyacetic acid (1 mmol), 4-dimethylaminopyridine (1 mmol), and dichloromethane (5 mL) were added sequentially to a round-bottom flask. After dissolution, carbodiimide (2 mmol) was added under ice bath conditions, and the reaction was carried out at room temperature for 24 h. After the reaction was complete, the mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography. 5 mL of anhydrous methanol was added, and hydrogenation was carried out for 5 h using 0.1 mmol Pd / C catalysis. The Pd / C was filtered off to obtain intermediate TMQX3. Temozolomide (0.5 mmol), 1,2-dichloroethane (5 mL), and oxalyl chloride (3 mmol) were added sequentially to a round-bottom flask. The mixture was reacted at 90 °C for 3 h, the solvent was evaporated, and the product was redissolved in 1,2-dichloroethane (5 mL). Intermediate TMQX3 (0.5 mmol) was then added, and the reaction was carried out overnight at room temperature. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography to obtain the product.

[0103] (1R,2S,4R)-1,7,7-trimethylbicyclo[2.2.1]heptane-2-yl-2-(((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)acetate (QX401)

[0104] Yield: 28.36%, HRMS: 433.1693 [M+H] 1 H NMR (400MHz, CDCl3) δ9.53 (s, 1H), 8.44 (s, 1H), 5.00 (d, J = 8.9Hz, 1H), 4.77 (s, 2H), 4.07 (s, 3H), 2.37(s,1H),1.87(d,J=8.3Hz,1H),1.72(d,J=16.1Hz,2H),1.38–1.20(m,4H),0.94–0.80(m,9H). 13 C NMR (101MHz, CDCl3) δ167.54,156.19,149.58,138.31,135.14,129.32,127.97,81 .63,61.87,48.88,47.84,44.74,36.84,36.39,27.85,26.93,19.60,18.74,13.38.

[0105] (1R,2S,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl 3-(((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)propionate (QX402)

[0106] Yield: 10.23%, HRMS: 447.1987 [M+H] 1 H NMR (400MHz, CDCl3) δ9.34(s,1H),8.44(s,1H),4.97(d,J=9.7Hz,1H),4.57(t,J=6.3Hz,2H),4.09(s,3H),2.80(t,J=6.3Hz,2H ), 2.38(ddd,J=13.8,9.0,4.0Hz,1H),2.00–1.90(m,1H),1.82–1.65(m,3H),1.39–1.19(m,3H),0.87(dd,J=22.9,10.7Hz,9H). 13C NMR (101MHz, CDCl3) δ170.54,156.23,149.90,138.31,135.02,129.62,127.84,80.56 ,61.71,48.76,47.77,44.81,36.79,36.65,34.07,27.95,27.04,19.62,18.76,13.43.

[0107] (1R,2S,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl 4-(((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)butyrate (QX403)

[0108] Yield: 66.25%, HRMS: 461.2143 [M+H] 1 H NMR (400MHz, CDCl3) δ9.32 (s, 1H), 8.43 (s, 1H), 4.90 (d, J = 9.5Hz, 1H), 4.31 (t, J=6.3Hz,2H),4.07(s,3H),2.49(t,J=7.4Hz,2H),2.35(td,J=9.8,4.9Hz,1H), 2.10–2.03(m,2H),1.97–1.88(m,2H),1.75(ddd,J=11.1,7.3,3.6Hz,1H),1.67 (t,J=4.4Hz,1H),1.31(d,J=12.5Hz,1H),1.28–1.20(m,2H),0.92–0.81(m,9H). 13 C NMR (101MHz, CDCl3) δ172.91,156.34,150.30,138.32,135.08,129.50,127.89,80.06,65 .39,48.68,47.73,44.79,36.81,36.71,30.90,27.94,27.04,24.07,19.62,18.75,13.45.

[0109] (1R,2S,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl5-(((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)valerate (QX404)

[0110] Yield: 31.49%, HRMS: 475.2163 [M+H] 1H NMR (400MHz, CDCl3) δ9.31 (s, 1H), 8.44 (s, 1H), 4.92 (d, J = 9.7Hz, 1H), 4.32 (s, 2H), 4.09 (s, 3H), 2.41 (s ,3H),1.94(d,J=10.2Hz,1H),1.81(s,5H),1.67(d,J=20.2Hz,2H),1.40–1.17(m,3H),0.93–0.81(m,9H). 13 C NMR (101MHz, CDCl3) δ173.42,156.33,150.33,138.34,135.04,129.52,127.87,79.79,65 .80,48.67,47.71,44.80,36.80,36.75,33.94,27.96,27.04,21.35,19.62,18.75,13.46.

[0111] (1R,2S,4R)-1,7,7-trimethylbicyclo[2.2.1]heptane-2-yl6-(((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)hexanoate (QX405)

[0112] Yield: 32.46%, HRMS: 489.2318 [M+H] 1 H NMR(400MHz, CDCl3)δ9.27(s,1H),8.42(s,1H),5.03–4.76(m,1H),4.24(t,J=6.6Hz,2H),4.06(s,3H),2.33(dd,J=9.6,5.3Hz,3 H),1.97–1.86(m,1H),1.80–1.62(m,6H),1.50–1.40(m,2H),1.31(dd,J=9.9,6.8Hz,1H),1.26–1.14(m,2H),0.92–0.77(m,9H). 13 C NMR (101MHz, CDCl3) δ173.69,156.33,150.37,138.34,135.04,129.53,127.87,79.63,66.12,48 .66,47.69,44.80,36.79,36.74,34.36,28.23,27.96,27.04,25.28,24.60,19.62,18.75,13.44.

[0113] Example 5 Antitumor Activity Test

[0114] (1) Human breast cancer MDA-MB-231 cells and human lung cancer A549 cells were seeded into culture dishes at a rate of 5000 cells / well, and human lymphoma Raji cells were seeded at a rate of 20000 cells / well. Cells were cultured to 90% confluence, digested with trypsin, collected, and counted. The required cell suspension volume was calculated at 4000 cells / well and 100 μl / well. The cell suspension was transferred to 15 ml centrifuge tubes and seeded into 96-well plates (with three blank control wells). The plates were incubated overnight at 37°C in a 5% CO2 incubator.

[0115] (2) After 24 hours, the drug (concentration of 100mM) was diluted to 600μM with culture medium. The drug was diluted to the corresponding final concentration at 100μl / well and added to the well. 1% DMSO was used as the drug control group.

[0116] (3) After 72 hours, the culture medium was aspirated, and 100 μl of complete culture medium and 10 μl of CCK-8 were added to each well. Two hours later, the OD450 value was measured using a microplate reader. Data analysis: The OD value of the cells was subtracted from the OD value of the blank control well to obtain the OD value of the cells after background subtraction, and then the cell viability was calculated. Cell viability = OD value of the drug-treated group / OD value of the control group (no drug-treated) * 100%.

[0117] See results Figures 1-3 The results show that QX302 and QX404 at a drug concentration of 10 μM have better inhibitory activity against tumor cells from three different sources: MDA-MB-231, A549, and Raji than TMZ at 300 μM.

[0118] Example 6: Determination of anti-glioma activity

[0119] (1) Day 1: U251 cells (5000 cells / well) were seeded into 96-well plates. Drug concentration gradient groups were set up, with three auxiliary wells in each group and three blank control wells in each 96-well plate.

[0120] (2) Day 2: Remove the culture medium and replace it with a drug concentration gradient culture medium.

[0121] (3) Day 5: After 72 hours, add 10 μl of CCK-8 to each well, and after another 2 hours, measure the OD using a microplate reader. 450 The read value.

[0122] (4) Data Analysis: First, the OD value of the cells was subtracted from the OD value of the blank control wells to obtain the OD value of the cells after background subtraction. Then, the cell viability was calculated. Cell viability = (OD value of the drug-treated group / OD value of the control group (no drug)) * 100%. GraphPad was then used to calculate the IC50 of the drug.50 The results are shown in Table 1.

[0123] Table 1 IC50 of the target compound 50

[0124]

[0125]

[0126] Note: / indicates that the compound is IC 50 The value is higher than TMZ.

[0127] As shown in Table 1, temozolomide (TMZ) has an IC50 effect on glioma U251 cells. 50 The concentration was 357.6 ± 52.1 μM. Most of the compounds in this invention showed an IC50 concentration of 357.6 ± 52.1 μM against glioma U251 cells. 50 <100μM, with activity superior to TMZ, for example, compound QX302 has an IC50 value of <100μM. 50 It is 8.38±0.48μM, which is 1 / 42.7 of TMZ.

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An imidazotetraazine compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound is shown below: 2-((((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)ethyl 4-(4-(bis(2-chloroethyl)amino)phenyl)butyrate, 3-(((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)propyl 4-(4-(bis(2-chloroethyl)amino)phenyl)butyrate, 4-(((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)butyl-4-(4-(bis(2-chloroethyl)amino)phenyl)butyrate, 5-((((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)pentyl 4-(4-(bis(2-chloroethyl)amino)phenyl)butyrate, 6-((((3-methyl-4-oxo-3,4-dihydroimidazolium[5,1-d][1,2,3,5]tetraazine-8-carbonyl)carbamoyl)oxy)hexyl-4-(4-(bis(2-chloroethyl)amino)phenyl)butyrate.

2. A pharmaceutical composition comprising an imidazotetrazine compound as described in claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

3. Use of an imidazotetrazine compound as described in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of glioblastoma.

4. The application according to claim 3, characterized in that, The glioblastoma is selected from primary glioblastoma or secondary glioblastoma.

5. The application according to claim 4, characterized in that, The glioblastoma is selected from giant cell glioblastoma, epithelioid glioblastoma, or glioblastoma containing primitive neuronal components.

Images