Imidazole tetrazine compounds

Abstract

A novel synthetic approach provides previously unexplored functionality at the C8 position of imidazole tetrazines. A predictive model for the hydrolytic stability of imidazole tetrazines based on the Hammett constant of the C8 substituent was derived by synthesizing and evaluating a panel of compounds with a range of aqueous stabilities (0.5 to 40 hours). Promising compounds were identified that had activity against a panel of GBM cell lines, suitable hydrolytic and metabolic stabilities, and significantly improved brain-to-serum ratios relative to TMZ, resulting in a lower hematologic toxicity profile and higher activity than TMZ in a GBM mouse model.

Description

[0001] Related Applications

[0002] This application claims priority under USC §119(e) to the following prior applications: U.S. Provisional Patent Application Nos. 62 / 716,390 filed on August 9, 2018, 62 / 778,750 filed on December 12, 2018, and 62 / 873,669 filed on July 12, 2019. The above prior applications are hereby incorporated by reference.

[0003] Government support

[0004] This invention was made with government support through grant R21-CA195149 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Background Art

[0005] Glioblastoma multiforme (GBM) is the most prevalent, aggressive, and lethal primary malignant brain tumor, with only 10% of patients surviving five years. 1 The current standard of care for GBM is major surgical resection followed by radiation therapy combined with temozolomide (TMZ), a small molecule DNA alkylating agent. The antitumor effect of TMZ is ultimately through the O 6 -methylation and subsequent mismatch repair-dependent cell death 2–6 The beneficial properties of TMZ include good pharmacokinetic properties (including 100% oral bioavailability 7 ), nonenzymatic prodrug activation, and some accumulation in the brain (cerebrospinal fluid:blood ratio of 17:83 in human cancer patients 8,9 TMZ provides significant therapeutic benefits for some GBM patients. 6 -methylguanine DNA methyltransferase (MGMT, an enzyme that removes O 6 In patients with mitochondrial guanine depletion (MTD), TMZ was able to prolong median survival by about two years. 10 Even in the era of personalized anticancer therapy, TMZ remains the first-line therapy for oligodendroglioma, diffuse astrocytoma, and multiforme luteoastrocytoma in addition to GBM. 11 However, given the ineffectiveness of TMZ against tumors expressing MGMT and the inevitable recurrence of GBM after multimodality combination therapy, better treatment strategies are still greatly needed clinically.

[0006] TMZ is a prodrug activated in aqueous solution, which ultimately releases the active alkylating component, methyldiazo (Scheme 1(a)). The half-life of TMZ in aqueous solution is ~2 hours both in vivo and in vitro, and the drug has been shown to have a higher hydrolysis rate in the more alkaline environment of gliomas, providing some selectivity for cancerous versus noncancerous tumors. 12-15 . Although a 2-hour half-life allows TMZ to reach the central nervous system (CNS) and release methyldiazo, little information is available on the relationship between half-life and anticancer activity. Specifically, it is unclear whether 2 hours is optimal for maximal efficacy, and perhaps a shorter (or longer) half-life would be more effective. Given the advantages of TMZ, the relationship between its structure, hydrolytic stability, and anticancer activity should be understood.

[0007] Although TMZ has been approved by the FDA for two decades, there is a desire for a more effective drug against glioblastoma with less systemic toxicity. Compounds that can reach the entire diffuse tumor at sufficiently effective concentrations for treatment are sought. Therefore, there is a need for improved compounds that have the desirable properties of TMZ, but have better brain penetration, lower toxicity, and provide improved patient survival. Summary of the invention

[0008] This article describes the development of a model for accurately predicting the hydrolytic stability and half-life of imidazole tetrazines. The model was used to discover novel imidazole tetrazines that have superior BBB permeability and superior anticancer activity in a mouse model of GBM compared to TMZ.

[0009] Accordingly, the present invention provides compounds of formula I:

[0010] or its salts;

[0011] in:

[0012] X is O or S;

[0013] R 1 Halogen, –CN, –NO 2 ,–(C 1 -C 6 )alkyl, -C(=O)R a , phenyl, or a 5- or 6-membered heterocyclic group, wherein R a H, halogen, –(C 1 -C 6 )alkyl, –(C 3 -C 6 )cycloalkyl, OR b , SR b , or –NR b R c ;in:

[0014] R b is H, –(C 1 -C 6 )alkyl, or –(C 3 -C 6 )cycloalkyl;

[0015] R c is H, –(C 1 -C 6 )alkyl, or –(C 3 -C 6 )cycloalkyl; or

[0016] When R a Yes – NR b R c When R b and R c together optionally form a heterocyclic ring;

[0017] R 2 For –(C 1 -C 6 )alkyl,–(C 3 -C 6 ) cycloalkyl, propargyl, phenyl, or a 5- or 6-membered heterocyclyl; and

[0018] R 3 is H,–(C 1 -C 6 )alkyl, or–(C 3 -C 6 )cycloalkyl;

[0019] Where – (C 1 -C 6 )alkyl, –(C 3 -C 6 )cycloalkyl, propargyl, phenyl, and 5- or 6-membered heterocyclyl are each optionally substituted with one or more substituents, and -(C 1 -C 6 ) alkyl groups are each unbranched or optionally branched.

[0020] Also provided herein is a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the above-described compound, wherein the cancer is thereby treated.

[0021] The present invention provides novel compounds of formula I-III (A / B / C), intermediates for synthesizing compounds of formula I-III, and methods for preparing compounds of formula I-III. The present invention also provides compounds of formula I-III used as intermediates for synthesizing other useful compounds. The present invention provides the use of compounds of formula I-III in the preparation of drugs for treating bacterial infections in mammals such as humans.

[0022] The present invention provides the use of the compositions described herein for drug therapy. The drug therapy can treat cancer, such as breast cancer, lung cancer, pancreatic cancer, prostate cancer, colon cancer, or brain cancers such as glioblastoma. The present invention also provides the use of the compositions described herein in the preparation of a medicament for treating cancer of mammals such as humans. The medicament may include a pharmaceutically acceptable diluent, excipient or carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following drawings are part of the specification and are used to further describe some embodiments or different aspects of the present invention. In some examples, embodiments of the present invention can be better understood by referring to the drawings and in conjunction with the detailed description provided herein. These descriptions and drawings can highlight some specific embodiments or some aspects of the present invention. However, those skilled in the art will appreciate that the embodiments or aspects of these parts can also be used in conjunction with other embodiments or aspects of the present invention.

[0024] Figure 1 : Hydrolytic stability of C8 substituted imidazole tetrazines. (a) The remaining percentage of the compound relative to its C8 substituent after 2 hours is plotted against the Hammett constant. Compounds with hydrolytic stability similar to TMZ are enclosed in the oval (see Table 1c).

[0025] Figure 2: (a) Relative brain: serum ratios of TMZ, Me-TMZ and DiMe-TMZ (25 mg / kg) were measured 5 minutes after IV injection into mice. Values ​​are fold changes relative to the brain: serum ratio of TMZ. In a second experiment, brain (b) and serum (c) concentrations of TMZ and C8 analogs (25 mg / kg) were quantified 5 minutes after IV injection into mice. (d) Brain: serum ratios were calculated based on (b) and (c) assuming a mouse blood volume of 58.5 mL / kg (see Scheme 2b). Errors are SEM, number of mice per group = 3. Statistical significance was determined using a two-sample Student's t-test (two-tailed test, assuming equal variance).

[0026] Figure 3 : In vivo hematological toxicity assessment of imidazole tetrazine. Mice were intravenously injected with 125 mg / kg imidazole tetrazine. After 7 days, whole blood was collected and complete blood cell counts were obtained for each mouse. (a) Total leukocyte count. Control vs. Ox-TMZ: P = 0.7, Control vs. K-TMZ: P = 0.9. (b) Lymphocyte concentration. Control vs. Ox-TMZ: P = 0.5, Control vs. K-TMZ: P = 0.9. Error is SEM, number of mice per group = 4. Statistical significance was determined by using Student's t-test (two-tailed test, equal variances set) for two samples. The concentrations of other relevant blood components such as Figure 8 shown.

[0027] Figure 4 : Imidazolidine tetrazines were added to T98G cells with or without 3-hour pretreatment with O6BG (100 μM). IC50 values ​​after 7 days of incubation and fold changes between (+ / –)O6BG treatments are reported. For all compounds, P values ​​between IC50 values ​​(+ / –)O6BG < 0.02. Errors are SEM, n ≥ 3. Statistical significance was determined by using a two-sample Student's t-test (two-tailed test, assuming equal variances).

[0028] Figure 5: Evaluation of imidazotetrazine in the GBM intracranial mouse model. GBM Br23c oncospheres were implanted intracranially into female athymic nude mice. Treatment was initiated 5 days after implantation. (a) Mice were administered 15 mg / kg TMZ or an equimolar dose of Me-TMZ (16.1 mg / kg) or DiMe-TMZ (17.2 mg / kg) orally once a day, 5 times a week, for 7 weeks. Control vs. TMZ: P = 0.0014, TMZ vs. DiMe-TMZ: P = 0.061, TMZ vs. Me-TMZ: P = 0.016. (b) Mice were administered 15 mg / kg TMZ or an equimolar dose of DiMe-TMZ (17.2 mg / kg) or K-TMZ (14.9 mg / kg) orally once a day, for a total of 5 doses. Control vs. TMZ: P = 0.0007, DiMe-TMZ vs. TMZ: P = 0.7, K-TMZ vs. TMZ: P = 0.055. Compounds were prepared in 10% PEG in PBS immediately before each treatment. The number of mice in each treatment group was ≥ 5. Survival curves were compared using the log-rank test.

[0029] Figure 6 : Western blot of MGMT status of all cell lines used.

[0030] Figure 7 : The hydrolytic stability of TMZ and K-TMZ was evaluated in saline at pH 7.0, 7.4, and 8.0 by calculating the percentage of the parent compound remaining after 2 h at 37 °C.

[0031] Figure 8 : In vivo hematological toxicity assessment of imidazole tetrazine. Mice were treated with a single IV dose of 125 mg / kg imidazole tetrazine, and complete blood cell counts were obtained for each mouse 7 days later. (a) Neutrophil concentration (b) RBC concentration (c) Platelet concentration (see Table 2b).

[0032] Fig. 9 GBM Br23c cells were implanted intracranially into female athymic nude mice. Treatment was initiated 5 days after implantation. Compound 14 (12.8 mg / kg, equimolar to 15 mg / kg TMZ) was administered orally to mice once daily for 5 doses. Compounds were formulated in 10% PEG in PBS. n≥5. This experiment was performed with Figure 5 The experiment shown in b was performed simultaneously. Figure 5 b and Fig. 9 same.

[0033] Fig.10: Developmental pathways of MGMT-independent imidazole tetrazines. Brain: serum ratio graph: Mice were administered 25 mg / kg compound IV. After 15 minutes, mice were killed and blood and brain were collected. The concentration of each drug was quantified by LC-MS / MS. N≥3 mice per group, error is SEM. *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0034] Even in the era of personalized medicine and immunotherapy, temozolomide (TMZ), a small molecule DNA alkylating agent, remains the standard of care for glioblastoma (GBM). TMZ has an unusual mode of action and is spontaneously converted to its active moiety by hydrolysis in vivo. Despite 20 years of FDA approval, TMZ has little benefit in patients whose tumors express the resistance enzyme MGMT and cause systemic toxicity via myelosuppression. TMZ was first synthesized in 1984, but the unavailability of certain key derivatives due to TMZ's chemosensitivity has also prevented extensive exploration of the link between imidazole tetrazines' structure and biological activity. Therefore, understanding the relationship between hydrolytic stability of imidazole tetrazines and anticancer activity is needed to identify the optimal timing of prodrug activation and to develop suitable compounds with enhanced efficacy by increasing blood-brain barrier permeability.

[0035] This work required the development of new synthetic methods to access previously unexplored functionalities (e.g., lipids, ketones, halogens, and aryls) at the C8 position of imidazole tetrazines. A predictive model for the hydrolytic stability of imidazole tetrazines based on the Hammett constant of the C8 substituent was derived by synthesizing and evaluating a panel of compounds with a range of aqueous stabilities (0.5 to 40 h). Potential compounds were identified with activity against a panel of GBM cell lines, appropriate hydrolytic and metabolic stabilities, and significantly improved brain-to-serum ratios relative to TMZ, resulting in a lower hematotoxicity profile and superior activity to TMZ in a GBM mouse model. This work points the way forward for the development of novel and potent anticancer imidazole tetrazines.

[0036] Although it has been suggested in the past that the amide at C8 of TMZ is essential for activity 2,16 , conflicting reports since then have suggested that other features may be tolerated at this location 17-19Indeed, there are analyses leading to the belief that substitution at C8 could be used to modulate the hydrolytic stability of imidazole tetrazines, and in doing so could allow the construction of compounds with a range of half-lives. In addition to altering the stability of the prodrug, changes at the C8 position could result in compounds that retain the favorable pharmacokinetic properties of TMZ but have increased CNS penetration. Imidazole tetrazines with enhanced blood-brain barrier (BBB) ​​permeability would display less systemic toxicity and allow for higher doses and more effective dosing regimens, as the dose-limiting toxicity of TMZ (myelosuppression) is not associated with the CNS. 7,20,21 .

[0037] definition

[0038] The following definitions are intended to provide a clear and consistent understanding of the specification and claims. The terms used herein have the following meanings. Other terms and phrases used in this specification have their ordinary meanings as understood by those skilled in the art. Such ordinary meanings can be obtained by reference to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 14 th Edition, by RJ Lewis, John Wiley & Sons, New York, NY, 2001.

[0039] References to "one embodiment", "an embodiment", etc. in this specification indicate that the described embodiment may include specific aspects, features, structures, parts or characteristics, but not every embodiment must include these specific aspects, features, structures, parts or characteristics. In addition, these phrases may, but not necessarily, apply to the same embodiment mentioned in other parts of this specification. In addition, when specific aspects, features, structures, parts or characteristics are described in conjunction with a certain embodiment, those skilled in the art should be able to understand that these specific aspects, features, structures, parts or characteristics are applied to or associated with other embodiments, whether or not explicitly stated.

[0040] The singular articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, "a compound" may include a plurality of such compounds, such that compound X includes a plurality of compound Xs. It is further noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein and / or as a limitation on claim elements or as a "negative" limitation.

[0041] The term "and / or" refers to any one, any combination, or all of the related items. The phrases "one or more" and "at least one" are readily understood by those skilled in the art, especially in context. For example, these phrases may refer to one, two, three, four, five, six, ten, one hundred, or any upper limit that is approximately 10, 100, or 1000 times higher than the lower limit already recited. For example, one or more substituents on a benzene ring refers to one to five, or one to four in the case where, for example, the benzene ring is disubstituted.

[0042] Those skilled in the art will appreciate that any number, including those indicating ingredient amounts, properties such as molecular weight, reaction conditions, etc., may be approximate and may be modified by the term "about" in each case. These values ​​may vary depending on the properties desired by the skilled artisan through the methods described herein. It is also understood that these values ​​are inherently variable due to standard deviations in testing. When the antecedent "about" is used to indicate an approximate value, it is understood that a specific value not modified by "about" is also expressed in another aspect.

[0043] The term "about" means that a particular value may have a variation of ±5%, ±10%, ±20%, or ±25%. For example, a "about 50" percentage may vary to 45 to 55 percentages in some embodiments. For integer ranges, the term "about" may include one or two integers smaller or larger than the listed integers at each end of the indicated range. Unless otherwise specified herein, the term "about" is used to include values ​​close to the listed range that are equivalent to the function of a component, combination, or embodiment. The term "about" can also be used to adjust the endpoints of the aforementioned ranges in this paragraph.

[0044] Those skilled in the art will appreciate that any range provided herein also includes all possible sub-ranges and combinations of these sub-ranges, as well as individual values, especially integer values, constituting the range, for any of a variety of purposes, especially for providing textual descriptions. Therefore, it should be understood that each unit between two specific units is also disclosed. For example, if 10 to 15 are disclosed, 11, 12, 13 and 14 are disclosed separately, and as part of the range. The quoted range (such as weight percentage or carbon group) includes each specific numerical value, integer, decimal or characteristic within the range. It should be easily understood that any range listed can fully describe and enable the same range to be decomposed into equal halves, thirds, fourths, fifths or tenths. As a non-limiting example, each range described herein can be easily decomposed into the lower third, the middle third and the upper third, etc. Those skilled in the art will also appreciate that "up to", "at least", "higher than", "lower than", "more than", "or above" or similar terms include the numbers listed, and such terms also refer to the ranges that can be subsequently divided into the sub-ranges mentioned above. Likewise, any ratio listed herein also includes sub-ratios that fall within the broader ratio. Therefore, specific values ​​for radicals, substituents, and ranges are only for illustration; they do not exclude other specified values ​​for radicals and substituents or other specified ranges of values. It will also be understood that the endpoints of each range are meaningful relative to the other endpoint and independently of the other endpoint.

[0045] It should also be readily understood by those skilled in the art that when units are combined in the usual manner, such as in Markush groups, the present invention includes not only the whole of the listed unit combination, but also each unit of the group alone and any possible subgroup of the basic group. In addition, for all purposes, the present invention includes not only the basic group, but also the group in which one or more units are removed from the basic group. It can be seen that the present invention may include any one or more units that explicitly exclude the cited group. Therefore, the relevant constraints may be attached to any disclosed category or embodiment, wherein any one or more units, types or embodiments may be excluded from the category or embodiment, for example, for the occasion of explicit negative limitation.

[0046] The term "contacting" means touching, coming into contact, or being adjacent or in close proximity, including, for example, at the cellular or molecular level, such as in a solution or reaction mixture, in vitro or in vivo, to produce a physiological reaction, chemical reaction or physical change.

[0047] The term "effective amount" refers to an amount that is effective in treating a disease, disorder, and / or condition, or producing the described effect. For example, an effective amount can be a dose that is effective in slowing the progression or extent of the condition or symptom being treated. The therapeutically effective amount is within the capabilities of those skilled in the art to determine. The term "effective amount" refers to an amount of a compound described herein, or an amount of a combination of peptides described herein, for example, that is used to effectively treat or prevent a disease or disorder, or to treat a symptom of a disease or disorder, for a subject. Therefore, an "effective amount" generally refers to an amount that provides the desired effect.

[0048] Alternatively, the term "effective amount" or "therapeutically effective amount" herein refers to an agent or ingredient or combination of ingredients sufficient to relieve to some extent one or more symptoms of the disease or condition being treated. The result can be a reduction and / or alleviation of the signs, symptoms or causes of the disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic use is an amount required to provide a clinically significant reduction in the symptoms of the disease by containing the compounds described herein. In any case, techniques such as dose escalation studies can be used to determine an appropriate "effective" amount. The dose can be administered once or multiple times. However, the effective dose can be precisely determined based on individual factors for each patient, including but not limited to the patient's age, size, type or extent of disease, stage of disease, route of administration of the composition, type or extent of complementary therapies used, current disease process, and type of treatment required (e.g., active treatment versus conventional treatment).

[0049] The terms "treating," "treat" and "treatment" include (i) preventing the occurrence of a disease, pathology or medical condition (e.g., prophylaxis); (ii) arresting the occurrence of a disease, pathology or medical condition or controlling its development; (iii) alleviating a disease, pathology or medical condition; and / or (iv) alleviating a disease, pathology or medical condition. Thus, the term "treating" may include reducing, arresting or reversing the progression or extent of the condition or symptom being treated. Thus, the term "treating" may include medical treatment, convalescence and / or prophylactic medication, as appropriate.

[0050] As used herein, "subject" or "patient" refers to an individual who has symptoms of or is at risk of a disease or other malignancy. Patients can be human or non-human, and can include, for example, animal strains or species used as "model systems" for research purposes, such as the mouse model described herein. Likewise, patients can include adults or adolescents (e.g., children). In addition, patients can refer to any living organism that can benefit from the administration of the compositions described herein, preferably mammals (e.g., humans or non-humans). Examples of mammals include, but are not limited to, any member of the class of mammals: humans, non-human primates (e.g., chimpanzees), and other apes and monkeys; livestock, such as cattle, horses, sheep, goats, pigs; livestock, such as rabbits, dogs and cats; experimental animals include rodents, such as rats, mice and guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc. In one embodiment provided herein, the mammal is a human.

[0051] As used herein, the terms "providing", "administering", and "introducing" are used interchangeably herein and refer to placing the composition described herein in a subject's body by a certain manner or route so that the composition is at least partially placed in a desired location. The composition can be administered by any suitable route of delivery to a desired location in a subject. The composition described herein can be administered together with other compositions to prolong the stability and activity of the composition or in combination with other therapeutic agents.

[0052] The terms "inhibit, inhibiting and inhibition" refer to slowing, stopping or reversing the growth or progression of a disease, infection, condition or cell population. The inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95% or 99%, for example, compared to the degree of growth or progression that would occur in the absence of treatment or manipulation.

[0053] As used herein, the term "substantially" is a broad term and is used in its usual sense, including but not limited to a large extent but not necessarily all of what is specified. For example, the term can refer to a value that is not 100% of the overall value. The overall value can be less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%.

[0054] The term "substituted" or "substituent" means that one or more (e.g., 1, 2, 3, 4, or 5, in some embodiments 1, 2, or 3, and in other embodiments 1 or 2) hydrogen atoms on the group represented by "substituted" are replaced by a "substituent". The substituent may be selected from the described groups, or may be a suitable group known to those skilled in the art, provided that the normal valence of the substituted atom is not exceeded, and that the substitution produces a stable compound. Available substituents include, for example, alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, aroyl, heteroaryl, heterocyclic, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, amido, nitro, trifluoromethyl, trifluoromethoxy, carboxyl, carboxylalkyl, keto, thioketone, alkylthio, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, heteroarylsulfinyl, heteroarylsulfonyl, heterocyclic sulfinyl, heterocyclic sulfonyl, phosphate, sulfate, hydroxylamine, hydroxyl (alkyl) amine and cyano. In addition, non-limiting examples of substituents that can be bonded to the substituted carbon (or other) atom include F, Cl, Br, I, OR', OC(O)N(R') 2 ,CN,CF 3 ,OCF 3 ,R',O,S,C(O),S(O),methylenedioxy,ethylenedioxy,N(R') 2 ,SR',SOR',SO 2 R',SO 2 N(R') 2 ,SO 3 R',C(O)R',C(O)C(O)R',C(O)CH 2 C(O)R',C(S)R',C(O)OR',OC(O)R',C(O)N(R') 2 ,OC(O)N(R') 2 ,C(S)N(R') 2 ,(CH 2 ) 0-2 NHC(O)R',N(R')N(R')C(O)R',N(R')N(R')C(O)OR',N(R')N(R')CON(R') 2 ,N(R')SO 2 R',N(R')SO 2 N(R') 2 ,N(R')C(O)OR',N(R')C(O)R',N(R')C(S)R',N(R')C(O)N(R') 2,N(R')C(S)N(R') 2 ,N(COR')COR',N(OR')R',C(=NH)N(R') 2 , C(O)N(OR')R', or C(=NOR')R', where R' can be hydrogen or a carbonyl moiety, and where the carbonyl moiety itself can be further substituted. When the substituent is monovalent, such as F or Cl, it is bonded to the atom substituted by a single bond. When the substituent is more than monovalent, such as a divalent O, it can be bonded to the atom it replaces through more than one bond, that is, the divalent substituent is bonded by a double bond. For example, C substituted by O forms a carbonyl group, C=O, where C and O are double bonded. Alternatively, a divalent substituent such as O, S, C(O), S(O) or S(O) 2 It can be connected to two different carbon atoms through two single bonds. For example, a divalent substituent O can be bonded to two adjacent carbon atoms to provide an epoxy group, or O can form a bridging ether group between adjacent or non-adjacent carbon atoms, for example, bridging with the 1,4 carbon atoms of cyclohexyl to form a [2.2.1]-oxabicyclic ring system. In addition, any substituent can be connected to two different carbon atoms through two single bonds, such as (CH 2 ) n or (CR' 2 ) n A linker (where n is 1, 2, 3 or greater, and each R' is independently selected) is bonded to a carbon or other atom.

[0055] The term "halo" or "halogenated" refers to fluoro, chloro, bromo, or iodo. Similarly, the term "halogen" refers to fluoro, chloro, bromo, and iodine.

[0056] The term "alkyl" refers to a straight or branched chain alkyl group, for example, having 1-20 or a range of carbon atoms therebetween (e.g., 2-8 or 3-8), and often 1-12, 1-10, 1-8, 1-6 or 1-4 carbon atoms. As used herein, the term "alkyl" also encompasses "cycloalkyl" as defined below. Specific examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl (isopropyl), 1-butyl, 2-methyl-1-propyl (isobutyl), 2-butyl (sec-butyl), 2-methyl-2-propyl (tert-butyl), 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-2-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3 dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, etc. The alkyl group may be unsubstituted or may be optionally substituted, for example, substituted by the following substituents. The alkyl group may also be optionally partially or completely unsaturated. Thus, the listing of alkyl groups may include alkenyl and alkynyl groups. The alkyl group may also be a monovalent hydrocarbon group as described or listed above, or may be a divalent hydrocarbon group (ie, a hydrocarbylene group).

[0057] The term "cycloalkyl" refers to a cyclic alkyl group, for example, a cyclic alkyl group having a monocyclic ring or multiple condensed rings containing 3 to 10 carbon atoms. Cycloalkyl includes, for example, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc., or polycyclic structures such as adamantyl, pinenyl, etc. The cycloalkyl group can be monovalent or divalent, and can be selectively substituted by, for example, one or more alkyl groups. The cycloalkyl group can selectively include one or more unsaturated sites, for example, the cycloalkyl group can include one or more carbon-carbon double bonds, such as 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopentene-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, etc.

[0058] The term "heterocycloalkyl" refers to a saturated or partially saturated monocyclic, bicyclic or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably 1-3 heteroatoms in at least one ring. Each ring is preferably 3 to 10 members, more preferably 4 to 7 members. Examples of suitable heterocycloalkyl substituents include pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, morpholino, 1,3-diazapentane, 1,4-diazapentane, 1,4-oxaheptane and 1,4-oxaheptane. The group may be a terminal group or a bridging group.

[0059] The term "aryl" refers to an aromatic hydrocarbon group obtained by removing at least one hydrogen atom from a single carbon atom of a parent aromatic ring system. The group attachment site may be on a saturated or unsaturated carbon atom of the aromatic ring parent. The aryl group may have 6 to 30 carbon atoms, such as 6 to 10 carbon atoms, in its cyclic skeleton. In other embodiments, the aryl group may have 6 to 60 carbon atoms, 6 to 120 carbon atoms or 6 to 240 carbon atoms. The aryl group may have a monocyclic ring (such as a phenyl) or multiple condensed (fused) rings, wherein at least one ring is an aromatic ring (such as a naphthyl, a dihydrophenanthryl, a fluorenyl or anthracenyl). Typical aryl groups include, but are not limited to, groups derived from benzene, naphthalene, anthracene, biphenyl, etc. The aryl group may be unsubstituted or may be selectively substituted.

[0060] The term "heteroaryl" refers to a monocyclic, bicyclic or tricyclic ring system containing one, two or three aromatic rings and at least one nitrogen, oxygen or sulfur atom. The heteroaryl group may be unsubstituted or substituted, for example with one or more, in particular one to three, substituents, as defined by the term "substituted". Typical heteroaryl groups contain 2-20 carbon atoms in their cyclic backbone in addition to one or more heteroatoms. Illustrative examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinolyl, acridinyl, benzo[b]thienyl, benzothiazolyl, β -carbolyl, carbazolyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furanyl, imidazolyl, imino, indazolyl, indolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, phenanthrolinyl, phenanthrolinyl, phenothiazinyl, phenothiazinyl, phenothiazinyl, phenoxazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thienyl, thiazolyl, thienyl, triazolyl, tetrazolyl and xanthenyl. In one embodiment, the term "heteroaryl" refers to a monocyclic aromatic ring containing five or six ring atoms, the ring atoms including carbon and 1, 2, 3 or 4 heteroatoms independently selected from non-peroxide oxygen, sulfur and N (Z), wherein Z is absent or is H, O, alkyl, aryl or (C 1 -C 6 In some embodiments, the heteroaryl group represents an ortho-fused bicyclic heterocyclic group derived from about 8 to 10 ring atoms, especially a benzo derivative or fused through a propene, trimethylol or tetramethylol diradical.

[0061] Embodiments of the invention

[0062] The present invention provides compounds of formula I:

[0063] or its salts;

[0064] in:

[0065] X is O or S;

[0066] R 1 Halogen, –CN, –NO 2 ,–(C 1 -C 6 )alkyl, -C(=O)R a , phenyl, or a 5- or 6-membered heterocyclic group, wherein R a H, halogen, –(C 1 -C 6 )alkyl, –(C 3 -C 6 )cycloalkyl, OR b ,SR b or –NR b R c ;in

[0067] R b is H,–(C 1 -C 6 )alkyl, or –(C 3 -C 6 )cycloalkyl;

[0068] R c is H,–(C 1 -C 6 )alkyl, or –(C 3 -C 6 ) cycloalkyl; or

[0069] When R a For –NR b R c When R b and R c together optionally form a heterocyclic ring;

[0070] R 2 For –(C 1 -C 6 )alkyl,–(C 3 -C 6 ) cycloalkyl, propargyl, phenyl, or a 5- or 6-membered heterocyclic ring; and

[0071] R 3 is H, –(C 1 -C 6 )alkyl, or –(C 3 -C 6 )cycloalkyl;

[0072] Where – (C 1 -C 6 )alkyl, –(C 3 -C 6 )cycloalkyl, propargyl, phenyl, and 5- or 6-membered heterocyclic ring are each optionally substituted with one or more substituents, and -(C 1 -C 6 ) alkyl groups are each unbranched or optionally branched.

[0073] In some embodiments, phenyl and -(C 1 -C 6 ) alkyl is each independently substituted by, for example, but not limited to, halogen (e.g., one or more chlorine or fluorine), alkoxy or aminoalkyl. In some other embodiments, the substituent does not include phenyl, or the molecular weight of the substituent is less than about 100, about 90, about 80, about 70, about 60 or about 50. In still other embodiments, R b and R c Cannot be H.

[0074] In other embodiments, R 1 is –C(=O)-(C 1 -C 6 )alkyl, –C(=O)-NH(C 1 -C 6 )alkyl, or –C(=O)-N[(C 1 -C 6 )alkyl] 2 In other embodiments, X is O, R 3 is H, and R 1 is –C(=O)-(C 1 -C 6 )alkyl, –C(=O)-NH(C 1 -C 6 )alkyl or –C(=O)-N[(C 1 -C 6 )alkyl] 2 In other embodiments, X is O and R 1 is –C(=O)-(C 1 -C 6 In another embodiment, X is O, R 2 For –(C 1 -C 6 ) alkyl, and R 3 is H. In some other embodiments, R 2 For –(C 1 -C 6 ) alkyl, and R 3is H. In other different embodiments, R 2 is propargyl or substituted phenyl. In some embodiments, X is O and R 3 For H.

[0075] In various embodiments, R 1 For the portion described by formula IB:

[0076] in

[0077] W is O, S, or NR d ; where R d is H, –(C 1 -C 6 )alkyl or –(C 3 -C 6 )cycloalkyl;

[0078] V is N or CR x , where R x is H, –(C 1 -C 6 )alkyl or –(C 3 -C 6 )cycloalkyl;

[0079] Y is N or CR y , where R y is H, –(C 1 -C 6 )alkyl or –(C 3 -C 6 )cycloalkyl; and

[0080] Z is N or CH.

[0081] In various other embodiments, R 1 One of the following:

[0082]

[0083] The 5-membered heterocyclic ring R 1 is optionally substituted (on one or the other carbon atom CH, thereby modifying the carbon to be a C-substituent, wherein the substituent is as defined herein).

[0084] In more embodiments, R 1 For i, ii or iii:

[0085]

[0086] wherein (i), (ii) and (iii) are optionally substituted at the 4 or 5 position.

[0087] In another embodiment, R 1 is a para-substituted phenyl, wherein each substituent has a molecular weight of less than about 300, about 200, or about 100 Daltons. In other embodiments, the para-substituent is a halogen, -CN, -CF 3 ,–CF 2 CF 3 , or –(C 1 -C 6 ) alkyl. In some other embodiments, R 1 is a halogen, –(C 1 -C 6 )alkyl or –(C 3 -C 6 )cycloalkyl.

[0088] In other embodiments, the substituted phenyl is substituted with halogen, alkyl, alkoxy, phenoxy, amine, alkylamine, dialkylamine, or a combination thereof.

[0089] In other embodiments, the compound is:

[0090]

[0091] In another embodiment, the compound of Formula I is a compound of Formula IC:

[0092]

[0093] Among them G 1 is halogen, alkyl, alkoxy, phenoxy or dialkylamine. 1 OCH 3 ,OCH 2 CH 3 ,OPh,or N(CH 3 ) 2 .

[0094] In other various embodiments, the compound of formula I is a compound of formula II:

[0095]

[0096] In another embodiment, R 2 For –(C 1 -C 6 ) alkyl, and R 3 is H. In other additional embodiments, the compound is K-TMZ:

[0097]

[0098] In a further embodiment, R aCH 3 ,CH 2 CH 3 ,NHCH 3 ,NHCH 2 CH 3 ,N(CH 3 ) 2 ,N(CH 2 CH 3 ) 2 ,N(CH 2 CH 2 CH 3 ) 2 ,N(CH 2 CH 2 CH 2 CH 3 ) 2 ,N(CH 2 CH 2 ) 2 ,N[(CH 2 CH 2 ) 2 O],OCH 3 ,OCH 2 CH 3 ,SCH 3 , or SCH 2 CH 3 In other embodiments, the compound is:

[0099]

[0100] In other embodiments, the compound of Formula I is a compound of Formula IIIA or IIIB:

[0101]

[0102] Where R z H, halogen, –(C 1 -C 6 )alkyl or –(C 3 -C 6 ) cycloalkyl. In other embodiments, R z CH 3 or CH 2 CH 3 In other embodiments, the compound is:

[0103]

[0104] The present invention also provides a method for treating cancer, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 19 to a subject in need thereof, wherein the cancer is treated. In other embodiments, the cancer is glioblastoma (GBM).

[0105] In some embodiments, the composition comprises the above disclosed compound and a second active agent. In other embodiments, the second active agent is a procaspase-3 activator, such as PAC-1:

[0106]

[0107] In other embodiments, the compound described herein and the second active agent are administered to the subject simultaneously or sequentially to treat cancer. In some embodiments, the compound and the second active agent are administered to the subject simultaneously. In other embodiments, the compound and the second active agent are administered to the subject sequentially. In some other embodiments, the compound is administered to the subject before the second active agent. In other embodiments, the compound is administered to the subject after the second active agent.

[0108] In some embodiments, the concentration of the compound described herein is from about 1 nM to about 10 μM. In other embodiments, the concentration of the second active agent is from about 1 nM to about 1 μM.

[0109] The scope, limit and deviation of variables such as volume, mass, percentage, ratio, etc. are described herein. It will be understood by those of ordinary skill in the art that a range such as "number 1" to "number 2" represents a continuous range of numbers, which includes integers and decimals. For example, 1 to 10 represents 1, 2, 3, 4, 5, ..., 9, 10, which also represents 1.0, 1.1, 1.2.1.3, ..., 9.8, 9.9, 10.0, and also represents 1.01, 1.02, 1.03, and so on. If the disclosed variable is a number less than "number 10", this means a continuous range of integers and fractions less than number 10, as described above. Similarly, if the disclosed variable is a number greater than "number 10", it represents a continuous range of integers and fractions greater than number 10. These ranges can be modified by the term "about", and its meaning has been described above.

[0110] Results and discussion

[0111] Construction of C8-substituted imidazotetrazines: The incorporation of an amide at the C8 position of TMZ is largely an artifact of the original synthesis of imidazotriazenes and imidazotetrazines. Both dacarbazine and TMZ are derived from the precursor 4-diazoimidazole-5-carboxamide (1, Scheme 1b). This diazo compound has a remarkable stability, reportedly lasting >2.5 years at room temperature. 22 , making it useful for exploratory chemical research, whereas other diazoimidazoles (e.g. 4-diazoimidazole (2)) would simply decompose 23 Thus, the first syntheses of dacarbazine in 1962 and TMZ in 1984 involved quenching of 1 with dimethylamine, respectively. 24 Or cyclize 1 with methyl isocyanate 25 , and the primary amide moiety is retained. Over time, it has been proposed that this amide is essential for anticancer activity. This claim is supported by theoretical studies that suggest that a hydrogen bond donor at the C8 position is necessary for activity 2,16 This interpretation is obscured by conflicting structure-activity relationships (SARs) established with derivatives of related compounds (mitozolomide) in non-CNS cancer models. 26 The establishment of general synthetic routes that can be used to construct novel derivatives at the C8 position poses a great challenge. These synthetic challenges have hampered the development of new imidazotetrazines, and outdated SARs are still used in the absence of new compounds and biological data.

[0112] Scheme 1: (a) Mechanism of activation of temozolomide (TMZ) in aqueous solution. (b) The favorable stability of compound 1 relative to related variations (eg, 2) is due to the introduction of an amide at C8 of TMZ.

[0113] (a)

[0114]

[0115] (b)

[0116]

[0117] Challenges of replacing C8 amides include: aqueous sensitivity; alkaline sensitivity; diazoimidazole degradation; and CH 3 Poor substitution of NCO.

[0118] Key challenges in preparing new imidazole tetrazines include sensitivity to protic solvents or basic reagents, instability of intermediate diazonium species, and lack of effective reagents to set the N3-methyl group. The sensitivity of the prodrug to conditions involving bases or water (pH>6) makes tetrazinones unstable to many practical cross-coupling or reduction conditions. As mentioned above, another challenge is the instability of the intermediate diazonium species. Specific 4-diazoimidazole-5-carboxamides (Scheme 1b, 1) are easily precipitated from solution as pure, stable compounds; however, other 4-diazoimidazoles (e.g., 2) remain in aqueous solution, are extremely prone to decomposition, and are sensitive to heat, shock, and light. 23 Finally, the installation of the N3-methyl group in the initial route to TMZ was achieved via methyl isocyanate cyclization. 25 However, methyl isocyanate is a toxic gas and is no longer commercially available. Therefore, an alternative route to methyl isocyanate must be used. 27 or less efficient substituents, such as N-succinimidyl N-methylcarbamate or N-methylcarbamoyl chloride, which would reduce the yield of the cyclization.

[0119] In order to obtain some derivatives of C8 amide, the existing approach mainly targeting mitozolamide was modified, thus starting the exploration of these types of compounds. 28 . The process begins with the hydrolysis of the amide of TMZ to the carboxylic acid 3 (Scheme 2a), which can then be converted to the acid chloride. From the intermediate, substitution with various nucleophiles can provide products with higher yields. This route is used to synthesize amide, ester, and thioester derivatives 4-10 (Scheme 2a). In addition, the cyano group (11) is directly installed from TMZ using existing reactions (Scheme 2a) 29 However, the creation of structurally diverse C8 analogs requires new synthetic routes, especially for those with aliphatic, ketone, halogen, and aryl groups; in the approximately 35-year history of TMZ, such substituents have not been described at this position. Therefore, an aliphatic group was introduced at C8 by diazotization of 5-amino-4-methylimidazole 12 to the diazo species 13 and subsequent cyclization with methyl isocyanate-substituted N-methylcarbamoyl chloride to give the C8-methyl derivative 14 (Scheme 2b).

[0120] Although various amides, esters, and thioamides have been set up at C8, a ketone has been completely absent, which is perhaps not surprising since initial attempts using Grignard or alkyllithium reagents resulted in complete degradation of the tetrazinone ring. Therefore, the methyl ketone derivative 17 was synthesized from its disubstituted precursor 16 by stepwise cyclization, which was prepared via 6-methylpurine N-oxide (15). 30) were obtained by hydrolytic degradation of (Scheme 2c). After decarboxylative halogenation of intermediate 3 using Dess-Martin periodinane and the corresponding tetraethylammonium salt, the bromine and chlorine substituents were directly combined at C8 in moderate yields (Scheme 2a, compounds 18 and 19). The strategy has not been previously applied to imidazoles and, in addition to representing novel derivatives per se, also confers potential diversity. However, the use of 18 as a cross-coupling ligand was unsuccessful due to the required alkaline, aqueous conditions. In contrast, Suzuki coupling generated 5-nitro-4-phenylimidazole (21) from the precursor 5-nitro-4-bromoimidazole (20), which could then be reduced to the corresponding amine 22 and cyclized to the phenyl-substituted imidazole tetrazine as described above (Scheme 2d). This approach provided 23 as well as minor para-substituted aryl derivatives 24-26. Finally, heterocyclic compounds 27 and 28 were synthesized after C8 amide or thioamide cyclization, respectively (Scheme 2e). A similar approach was used to generate 5-nitro-4-phenylimidazole (21) at C8. 19 The larger 4-substituted oxazoles and thiazoles, but not the smaller methyl groups, were introduced.

[0121] Anticancer activity of C8-substituted imidazole tetrazines: With a panel of imidazole tetrazines, each compound was evaluated against a panel of human GBM cell lines (Table 1a, Table 1b). The cell lines selected included those expressing and lacking MGMT ( Figure 6 ), and consistent with literature reports, those cell lines with negligible MGMT expression were sensitive to TMZ (IC 50~50μM or less), while those cell lines with significant MGMT expression were resistant (IC50>300μM). Amide substituted derivatives 4-8 as well as ester (9) and thioester (10) derivatives were comparable to TMZ in MGMT-deficient U87 and D54 cell lines. Notably, activity against the disubstituted amides (5-8) and esters (9) of the imidazole tetrazines was retained, confirming that a hydrogen bond donor is not required at C8. These derivatives were more active than TMZ in GBM cells expressing U118MG and T98G MGMT. The keto analog 17 was also effective against MGMT-deficient cell lines, indicating that an amide is not required at C8. Compounds that lacked a carbonyl group entirely (e.g., 14, 19, 23, and 27) were shown to be equally (or more) potent than TMZ in the absence of MGMT and significantly more potent in cell lines expressing MGMT. Methyl (14) and phenyl (23) substitutions were the most active in all cell lines. The cyano derivative 11 and the carboxylic acid derivative 3 were inactive in all cell lines tested (more than 7-fold less potent than TMZ), even in the absence of MGMT. In addition to these typical adherent GBM cell lines, most analogs were more active than TMZ in the patient-derived U3054MG GBM cell line cultured in serum-free stem cell conditions. 31 .

[0122] Scheme 2: Synthesis of novel C8 substituted imidazole tetrazines. DMP = Dess-Martin periodinane, SNMC = N-succinimidyl N-methylcarbamate.

[0123]

[0124]

[0125] Table 1a: A panel of C8-substituted imidazole tetrazines and their associated ICs in multiple GBM cell lines 50 Values ​​(μM); cell lines were incubated with compounds for 7 days and then viability was assessed using the Alamar Blue assay. Errors are SEM, n≥3, Pr1=pyrrolidine. A table with additional compounds (Table 1b) and Western blots of MGMT status for all cell lines used are disclosed herein ( Figure 6 ).

[0126]

[0127] Hydrolytic stability of C8-substituted imidazole tetrazines: The major factor determining the anticancer activity of imidazole tetrazines is the hydrolytic activation of the prodrug. As shown in Scheme 1(a), the half-life of TMZ in humans is about 2 hours. 7This timeline allows the intact prodrug to reach the brain and release the active methylated component prior to elimination. With the exception of TMZ, the relationship between imidazole tetrazines stability and anticancer activity is unknown; that is, although hydrolytic activation is required for cancer cell death, the optimal timing of said events in vitro and in vivo is unclear. To this end, the hydrolytic stability of various new compounds was evaluated in buffered saline, which simulates in vivo conditions (TMZ has a half-life of 119 minutes in PBS, pH 7.4, Figure 1 ). An HPLC assay was developed to quantify the proportion of intact prodrug remaining in solution after 2 hours at pH 7.4, 37°C. The experimental results show that the effect of the electron substituent at C8 is directly transferred through the bicyclic ring to the hydrolysis site C4. The extent of this effect is surprising and depends on the substituent at C8 ( Figure 1 ), and after 2 hours, the remaining stability ranged from 0% to 97%. Since the group at C8 appears to have such a significant effect on the aqueous stability of the prodrug, its Hammett constant (σ p ) is plotted against the remaining percentage after 2 hours. Figure 1 As shown, there is a clear relationship between these two parameters, which indicates that σ p It can be used to accurately predict the stability of C8-substituted imidazole tetrazines. p =0.36) similar electronic performance (0.23<σ p <0.50) of the substituents are amide derivatives 4 and 5, keto derivative 17 and chloro derivative 19. The half-life of TMZ in PBS at pH 7.4 was measured for each within one hour (Table 1c). The two extreme cases are the cyano analogue 11 (σ p =0.66), with a half-life of 0.5 h, and the methyl derivative 14 (σ p =–0.17), which maintained its prodrug form the longest with a half-life of 40 hours. The same assay was used to determine the effect of hydrolysis on C8-substituted derivatives (e.g., K-TMZ 17, Figure 7 ) still maintains pH dependence.

[0128] Table 1b: A panel of C8-substituted imidazole tetrazines and their associated 7-day IC in multiple GBM cell lines 50 Values ​​(μM); The following four compounds were tested and this table is supplementary to Table 1a. Cell viability was assessed using the Alamar Blue assay. Errors are SEM, n≥3.

[0129]

[0130] Table 1c: Half-life of some C8 derivatives in PBS (pH 7.4, 37°C).

[0131]

[0132] Relationship between hydrolytic stability and anticancer activity: The methyl and phenyl derivatives 14 and 23 were consistently the most potent compounds in various cell lines tested (Table 1a). Interestingly, they also possess electron-donating substituents and thus have the greatest water stability ( Figure 1 ), suggesting that longer-lived prodrugs are advantageous in cell culture. The opposite effect was observed for compound 11, which was the least stable in solution. Even in U87 cells lacking MGMT, its activity was tenfold lower than that of TMZ, suggesting that there is a critical aqueous stability threshold below which hydrolysis occurs too rapidly to methylate target DNA. Compounds with hydrolytic stability similar to that of TMZ (e.g., 4, 5, 17, 19, and 27) retained activity in culture. Notably, the keto derivative 17 was equivalent to TMZ, even with a shorter aqueous half-life, suggesting that σ p Compounds with a m / z of 0.50 can still retain significant anticancer activity.

[0133] Stability in liver microsomes: Fortunately, TMZ has several desirable pharmacokinetic properties, including avoidance of primary metabolism. 7 . To assess whether modification or substitution of the amide at C8 would result in significant metabolism, the stability of selected compounds was assessed after 2 h in the presence of mouse liver microsomes. Prodrug hydrolysis was accounted for by including a control experiment without liver microsomes. The slightly acidic pH of the working solution resulted in enhanced stability of TMZ compared to incubation in PBS alone. As expected, TMZ was insensitive to metabolic perturbations as its instability was entirely driven by hydrolysis (Table 2a). Addition of a methyl group to the amide (compounds 4 and 5) resulted in some sensitivity to microsomal effects, which was amplified for larger amide substitutions (compound 7), indicating improved aqueous stability but significantly reduced stability in liver microsomes. Ketone 17 and chloro 19 are generally stable to oxidative metabolism, suggesting that for these compounds, hydrolysis could drive in vivo pharmacokinetics, similar to TMZ.

[0134] Blood-brain barrier (BBB) ​​permeability: It has been reported that >98% of small molecule drugs cannot penetrate the BBB 32 , making TMZ unusual, especially among anticancer drugs. In humans, TMZ is rapidly absorbed and reaches the brain within minutes, with an average concentration of 20% of that in plasma and cerebrospinal fluid. 8,9 Accumulating more drugs in the brain by increasing BBB permeability may be a feasible strategy to improve the efficacy of drugs against central nervous system tumors. To predict the BBB permeability of novel imidazotetrazines, the logBB value (cLogBB) was calculated based on the formula using cLogP and total polar surface area.33 When applied to stabilized drug-eluting scaffolds, these types of in silico metrics have been reliably used to predict relative changes in BBB permeability as well as other biological phenomena. 34-38 , although not always reflecting absolute concentrations. The cLogBB value for TMZ was –1.58 (Table 2a). Substitution of the primary amide resulted in a significant increase in cLogBB and a larger predicted brain:blood ratio relative to TMZ. Importantly, cLogBB does not account for molecular weight, and one should be wary of analogs with large hydrophobic functional groups (e.g., 7) even with large hydrophobicity predictions. CNS Multiparameter Optimization (MPO) Tool 39,40 is a more comprehensive metric that measures expected BBB penetration. The CNS MPO score is based on the optimal range of 6 physicochemical properties and ranges from 0 to 6.0. Although TMZ had an MPO of 4.9, the C8 analogs achieved higher scores, in some cases reaching the highest expected values ​​(Table 2a). The better cLogBB and CNS MPO values ​​predicted by the expert panel suggest that some derivatives may have higher drug concentrations in the brain than TMZ.

[0135] Therefore, the BBB permeability of the lead compounds (with promising anticancer activity, adequate hydrolysis and liver microsomal stability, and predicted BBB permeability, Scheme 2b) was assessed in vivo. In initial experiments, Me-TMZ (4) and DiMe-TMZ (5) were tested head-to-head with TMZ to explore whether alkylation of the C8 amide could increase the brain:blood ratio. Mice were administered 25 mg / kg of drug intravenously and sacrificed 5 min after injection. Serum and perfused brain samples were immediately acidified to prevent prodrug degradation before quantification of drug concentrations in each compartment by LC-MS / MS. After 5 min, the analogs Me-TMZ and DiMe-TMZ showed significantly elevated drug concentrations in the brain compared to TMZ, with a >3-fold increase in the brain:serum ratio for each compound ( Figure 2 a). The equivalent brain:serum ratios of Me-TMZ and DiMe-TMZ may be due to the rapid metabolism of the dimethylated amide to its monomethylated counterpart. The preliminary experiments described suggest that other derivatives with higher predicted BBB permeabilities may result in increased brain permeability in vivo. Therefore, the compounds Ox-TMZ (27) and K-TMZ (17) were evaluated head-to-head with DiMe-TMZ and TMZ. After 5 minutes, the cumulative concentrations of each derivative in the brain were higher than those of TMZ ( Figure 2 b). When paired with the corresponding serum concentrations ( Figure 2 c) The relative brain:serum ratio of TMZ was 0.23 ± 0.03 ng / g:ng / mL, which is comparable to the few other TMZ biodistribution experiments in the murine system. 41,42When blood volumes were equal to the unit, the absolute brain:serum ratio for TMZ was 8:92, while the brain:serum ratios for Ox-TMZ and K-TMZ were 55:45 and 69:31, respectively. ( Figure 2 d). The dramatic difference in drug partitioning suggests that amide substitution at C8 is a viable strategy to significantly increase local drug concentrations in the brain relative to blood, which could improve anti-brain tumor efficacy and reduce hematologic toxicity.

[0136] Scheme 2b: Structures of lead C8-substituted compounds. Blood-brain barrier permeability of imidazole tetrazines (see Figure 2 ).

[0137]

[0138] Table 2a: Metabolic stability, cLogBB and CNS MPO values ​​of relevant C8 analogs. Metabolic stability was assessed in mouse liver microsomes. Compounds were incubated in microsomes for 2 h and the remaining percentage was quantified relative to t0. The experiment to assess stability without microsomes was the same, but PBS was used instead of liver microsomes. Errors are SEM, n≥2. Internal standard = N3-propyl TMZ. CNS MPO = central nervous system multi-parameter optimization score.

[0139]

[0140] Hematologic toxicity assessment: With Ox-TMZ and K-TMZ, increased brain concentrations and significantly lower serum concentrations were observed after treatment compared with TMZ ( Figure 2 b, Figure 2 c), suggesting that these C8-modified imidazole tetrazines mitigate the dose-limiting hematological toxicity of TMZ observed in the clinic. To test this hypothesis, mice were treated intravenously with a single dose of 125 mg / kg TMZ, Ox-TMZ or K-TMZ, a dose that is non-lethal to mice. 43,44 Seven days after treatment, whole blood was collected and complete blood counts were obtained for each mouse. As expected, the 125 mg / kg dose of TMZ resulted in white blood cell (WBC) depletion ( Figure 3 a), indicating drug-induced bone marrow suppression. In TMZ-treated mice, lymphocytes ( Figure 3 b) and neutrophils ( Figure 8 In contrast, treatment with 125 mg / kg of Ox-TMZ or K-TMZ did not produce bone marrow suppression. The total number of leukocytes, lymphocytes, and neutrophils in mice treated with these compounds was the same as that of control mice. Notably, the novel imidazotetrazines did not induce other hematological symptoms, such as red blood cell (RBC) toxicity ( Figure 8b) or thrombocytopenia ( Figure 8 c), and did not result in weight loss after 7 days of treatment (Table 2b).

[0141] Table 2b: Group weights of mice before and at 7 days post-treatment blood draw. Errors are SEM, number of mice per group = 4 (see Figure 8 ).

[0142]

[0143] Novel imidazole tetrazines induce alkylation-mediated cancer cell death: TMZ cytotoxicity is mediated by O 6 Guanine methylation mediates the mismatch repair system, which promotes subsequent single-strand and double-strand breaks and apoptosis 2–6 To evaluate whether the new imidazole tetrazines kill via the same mechanism, we quantified O 6 -methylguanine adducts were treated with 100 or 1000 μM imidazotetrazine. After 8 h incubation with compounds, genomic DNA was isolated, quantified and hydrolyzed into its constituent deoxyribonucleosides, which were then quantified by LC-MS / MS analysis. Dose-dependent O 6 -The increase in the concentration of methylated deoxyguanosine (Table 2c) indicated that DNA methylation had occurred. 6 -Benzylguanine (O6BG) pretreatment yielded novel compounds that remained DNA alkylating agents. O6BG is a pseudosubstrate of MGMT, which terminates the cellular storage of the enzyme, leading to O 6 Preincubation of MGMT-expressing T98G cells with O6BG (100 μM) resulted in an eightfold increase in TMZ cytotoxicity ( Figure 4 ), which is consistent with the literature reports 45,46 Similarly, DiMe-TMZ, Ox-TMZ, and K-TMZ showed a significant increase in activity after O6BG administration, indicating that O 6 -Methylguanine damage is the cause of cell death.

[0144] The novel imidazotetrazines have excellent activity in GBM mouse models: amide derivatives (Me-TMZ and DiMe-TMZ), with increased BBB permeability observed relative to TMZ, suggesting that higher drug concentrations in the brain may result in greater efficacy in intracranial tumor models. GBM Hippocampal cell lines were selected for this study because they more accurately recapitulate the genetic and histopathological features of human GBM, are passaged in serum, and typically grow as dense clumps in vivo relative to traditional adherent cell lines. 47The Br23cGBM nematocyst cell line does not express MGMT and is sensitive to TMZ and novel C8-substituted imidazole tetrazines (Table 2d), and was therefore chosen as a model system. Mice implanted intracranially with these cells were administered 15 mg / kg of TMZ or equimolar equivalents of Me-TMZ or DiMe-TMZ by oral gavage once daily, 5 times per week. As expected, TMZ significantly improved median survival ( Figure 5 a). However, mice treated with Me-TMZ and DiMe-TMZ outperformed TMZ, with median survival increased by 24% and 46%, respectively, suggesting that improving the BBB permeability of imidazole tetrazine prodrugs is a viable strategy to improve efficacy. In the second experiment, K-TMZ was selected for evaluation due to its optimal brain:blood ratio ( Figure 2 d). K-TMZ treatment (by oral gavage) of mice implanted intracranially with Br23c cells prolonged the median survival of TMZ-treated mice by more than 50 days and was more effective than DiMe-TMZ ( Figure 5 b). Importantly, the methyl derivative 14, which has excellent efficacy in cell culture but a prolonged (40 h) half-life in aqueous solution, had no effect in this in vivo model ( Fig. 9 ), suggesting that a significantly prolonged half-life is detrimental in vivo, possibly due to compound clearance prior to hydrolytic activation.

[0145] Table 2c: O in U87 cells (10 μg DNA) after treatment with 100 or 1000 μM imidazole tetrazine for 8 h 6 Determination of -methylguanine concentration. IC 50 Value (μM)

[0146]

[0147] in conclusion

[0148] Despite being known since 1984, FDA approval in 1999, and sales reaching $1 billion in 2009, TMZ remains the only approved imidazole tetrazine anticancer drug. This is likely due to the lack of a general synthetic approach to this class of compounds, which has prevented conventional medicinal chemistry studies. Here we report new synthetic methods that enable the construction of novel C8-substituted imidazole tetrazines that were previously difficult to access. Evaluation of these compounds in systematic head-to-head assays leads to the unequivocal conclusion that a C8 amide is not required for anticancer activity, and indeed compounds lacking an H-bond donor or acceptor (or both) at C8 can still retain activity comparable to TMZ against cancer cells in culture. Freed from the necessity of a C8 amide, a group of imidazole tetrazines were synthesized in which this position was varied. Strikingly, the electronic properties of the substituents at C8 have a significant impact on the activation of the corresponding prodrugs, a phenomenon that could not have been previously determined. The relationship formed between the hydrolytic stability of imidazole tetrazines and the electronic properties at the C8 position enables the exploitation of the readily accessible σ p The stability of the prodrug can be modulated by adjusting the value of TMZ, leading to the rational design of TMZ derivatives with similar in vivo stability and helping to study the optimal timing of activation of imidazole tetrazine prodrugs.

[0149] From this work it appears that compounds with very short half-lives (e.g. 11, t 1 / 2 =0.57h) simply hydrolyzes too quickly, releasing diazomethane before accumulating in the DNA microenvironment, thereby reducing anticancer activity. Therefore, for cancer cell activity in culture, a half-life of 1 hour or longer is optimal. In contrast, compounds with very long half-lives (e.g., 14 or 23, t 1 / 2 >20h) may be more potent than TMZ in cell culture because the prodrug has enough time to distribute to the nucleus before being converted to the active species. However, these compounds with significantly improved hydrolytic stability are unlikely to be used in vivo because they will be cleared by other pathways (secretion of the prodrug intact, oxidative metabolism, etc.) before activation to the alkylating species. This hypothesis explains the lack of in vivo efficacy of compound 14.

[0150] A hallmark of GBM is its invasion of surrounding brain tissue at an early stage, making it incurable by surgical resection. Therefore, there is a clear need for improved compounds that can reach the entire diffuse tumor at concentrations sufficient to be effective. Importantly, the data herein demonstrate that the BBB permeability of imidazole tetrazines can be improved by modification at the C8 position. In particular, the brain:serum distribution of Ox-TMZ and K-TMZ was significantly enhanced, potentially offering a substantial improvement over TMZ in the treatment of CNS cancers. Both compounds retain the favorable features of TMZ (timely prodrug activation, stability to liver microsomes) while also accumulating higher drug concentrations in the brain and reducing concentrations in the blood. It is hypothesized that greater distribution of imidazole tetrazines to tumor sites and less to compartments that cause adverse effects will extend the therapeutic envelope by enhancing anticancer activity while reducing systemic toxicity. Myelotoxicity, which occurs in approximately 20% of TMZ-treated patients, is the major dose-limiting toxicity. 48 , and is exacerbated in elderly and female GBM patients 49,50 Ox-TMZ and K-TMZ were significantly less toxic to WBCs in vivo than TMZ, which may be a direct result of increased partitioning to the CNS. Imidazotetrazines with lower toxicity profiles such as these could allow for longer dosing with additional anticancer efficacy and / or make this class of drugs available to more patients.

[0151] Other imidazotetrazines of varying composition reported in the literature have failed to improve mid-term survival compared with TMZ in preclinical models, despite promising results in cell culture. 42,51,52 Only one derivative outperformed TMZ in an intracranial mouse model of GBM, resulting in a modest 10% increase in median survival 53 Clearly, the interplay between retaining the favorable properties of TMZ as a frontline treatment for GBM and modulating its structure is not trivial. The data presented here now demonstrate that imidazotetrazine can be substantially modified without losing these advantages and that such novel compounds are indeed able to significantly improve efficacy in vivo. TMZ remains the gold standard for the treatment of the most aggressive brain tumors and shows promise against brain metastases from other cancers. 54 , and its predictable activity (based on clinical biomarkers) has recently led to support for the widespread use of TMZ in the management of a variety of diseases. 55 Therefore, the novel imidazotetrazine compounds described herein have broad prospects in the treatment of GBM and other cancers.

[0152] Table 2d: 7-day IC of TMZ and lead C8-substituted imidazole tetrazines in the Br23c GBM nematode cell line 50 Values ​​(μM). Cell viability was assessed using the Alamar Blue assay. Errors are SEM, n≥3.

[0153]

[0154] General Synthesis Methods

[0155] The present invention also relates to methods for preparing the compounds and compositions. The compounds and compositions can be prepared by any suitable technique for organic synthesis, such as the techniques described herein. Many such techniques are well known in the art. Many such known techniques are described in the following literature: Compendium of Organic Synthetic Methods (John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, Jr., 1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6, Michael B. Smith; and standard organic reference books, such as March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 thEd.by MBSmith and J.March(John Wiley&Sons,New York,2001),Comprehensive Organic Synthesis;Selectivity,Strategy&Efficiency in Modern Organic Chemistry,in 9Volumes,Barry M.Trost,Ed.-in-Chief(Pergamon Press,New York,1993printing));Advanced Organic Chemistry,Part B:Reactions and Synthesis,Second Edition, Cary and Sundberg (1983); Protecting Groups in Organic Synthesis, Second Edition, Greene, TW, and Wutz, PGM, John Wiley & Sons, New York; and Comprehensive Organic Transformations, Larock, RC, Second Edition, John Wiley & Sons, New York (1999).

[0156] Many exemplary methods for preparing the compounds of the present invention are provided below. These methods are used to illustrate these preparation processes, but do not limit the scope of the related methods.

[0157] Typically, reaction conditions, such as temperature, reaction time, solvent, post-treatment procedures, etc., are common in the art for the desired specific reaction. The references cited herein and the materials cited therein all contain detailed descriptions of such conditions. Typically, the temperature is -100°C to 200°C, the solvent is aprotic or protic, depending on the desired conditions, and the reaction time is 1 minute to 10 days. The post-treatment typically includes quenching any unreacted reagents, then performing a phase (extraction) between the water / organic layer system and separating the layer containing the product.

[0158] Oxidation and reduction reactions are usually carried out at temperatures close to room temperature (about 20°C), although for reduction reactions of metal hydrides, the temperature is usually lowered to 0°C to -100°C. Heating can also be used when appropriate. Solvents are usually aprotic for reduction, while those for oxidation can be protic or aprotic. Reaction times are adjusted to obtain the desired conversion.

[0159] The condensation reaction is usually carried out at a temperature close to room temperature, although for non-equilibrium, kinetically controlled condensation reactions, reduced temperatures (0°C to -100°C) are also common. The solvent can be protic (common in equilibrium reactions) or aprotic (common in kinetically controlled reactions). Standard synthetic techniques common in the art, such as azeotropic removal of reaction by-products and use of anhydrous reaction conditions (e.g., an inert gas environment) are employed as appropriate.

[0160] Protecting group: The term "protecting group" refers to any group that, in combination with a hydroxyl or other heteroatom, prevents undesirable reactions from occurring on the group and can be removed by conventional chemical or enzymatic procedures to reconstruct the hydroxyl. The specific removable protecting group employed is not always critical, and preferred removable protecting groups for hydroxyl groups include conventional substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidene, benzoyl, methylmethoxy, silyl ethers (e.g., trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS) or tert-butyldimethylsilyl (TBS)), and any other group that can be chemically introduced to the hydroxyl functional group and subsequently selectively removed by chemical or enzymatic methods under mild conditions compatible with product properties.

[0161] Suitable hydroxy protecting groups are known to those skilled in the art and are described in detail in TW Greene, Protecting Groups In Organic Synthesis; Wiley: New York, 1981 ("Greene") and references cited therein, and in Kocienski, Philip J.; Protecting Groups (Georg Thieme Verlag Stuttgart, New York, 1994), both of which are incorporated herein by reference.

[0162] Protecting groups are available, commonly known and used, and can be selectively used to prevent side reactions with protected groups during synthesis (i.e., routes or methods for preparing compounds by the methods of the invention). In most cases, the decision of which groups to protect, when to protect, and the nature of the chemical protecting group "PG" depends on the chemical nature of the reaction to be protected (e.g., acidic, basic, oxidizing, reducing or other conditions) and the intended direction of the synthesis.

[0163] Pharmaceutical preparations

[0164] The compounds described herein can be used to prepare therapeutic drug combinations, for example, by combining the compounds with a pharmaceutically acceptable diluent, excipient or carrier. The compounds can be added to the carrier in the form of a salt or solvate. For example, in the case where the compound has sufficient alkalinity or acidity to form a stable non-toxic acid or base salt, it may be appropriate to administer the compound in the form of a salt. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form physiologically acceptable anions, such as toluenesulfonates, mesylates, acetates, citrates, malonates, tartrates, succinates, benzoates, ascorbates, alpha-ketoglutarate and -glycerophosphate. Suitable inorganic salts can also be formed, including hydrochlorides, halides, sulfates, nitrates, bicarbonates and carbonates.

[0165] Pharmaceutically acceptable salts can be obtained using standard methods well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (e.g., sodium, potassium or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared using similar methods.

[0166] The compounds of the formula described herein can be formulated into pharmaceutical compositions and administered to vertebrate or mammalian hosts, such as human patients, in a variety of forms. The forms can be particularly suitable for the selected route of administration, such as oral or parenteral administration by intravenous, intramuscular, topical or subcutaneous routes.

[0167] The compounds described herein can be systemically administered with a pharmaceutically acceptable carrier (e.g., an inert diluent or an absorbable edible carrier). For oral administration, the compound can be encapsulated in a hard or soft shell capsule, compressed into a tablet, or directly incorporated into the food of the patient's diet. The compound can also be combined with one or more excipients and used in the form of a digestible tablet, buccal tablet, lozenge, capsule, elixir, suspension, syrup, wafer, etc. Such compositions and preparations typically contain at least 0.1% of the active compound. The percentage of the composition and preparation can vary and can conveniently be about 0.5% to about 60%, about 1% to about 25%, or about 2% to about 10% of the weight of the set unit dosage form. The amount of active compound in such a therapeutically useful composition can be such that an effective dosage level is achieved.

[0168] Tablets, lozenges, pills, capsules, etc. may also contain one or more of the following substances: binders, such as gum tragacanth, gum arabic, corn starch or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; and lubricants, such as magnesium stearate. Sweeteners such as sucrose, fructose, lactose or aspartame may be added; or flavoring agents, such as mint, wintergreen oil or cherry flavoring. When the unit dosage form is a capsule, it may contain a liquid carrier, such as a vegetable oil or polyethylene glycol, in addition to the above-mentioned types of materials. Various other materials may be used as coatings or otherwise change the physical form of the solid unit dosage form. For example, tablets, pills or capsules may be coated with gelatin, wax, shellac or sugar, etc. Syrups or elixirs may contain the active compound, sucrose or fructose as sweeteners, methyl and propylparabens as preservatives, dyes and flavorings such as cherry or orange flavoring. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. Additionally, the active compound may be incorporated into sustained-release preparations and devices.

[0169] The active compound can be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salt can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycol, triacetin or a mixture thereof or in a pharmaceutically acceptable oil. Under ordinary storage and use conditions, the preparation may contain a preservative to prevent the growth of microorganisms.

[0170] The pharmaceutical dosage form suitable for injection or infusion can include sterile aqueous solution, dispersion or sterile powder, which contains active ingredients suitable for instant preparation of sterile injectable or infusion solutions or dispersions, optionally encapsulated in liposomes. The final dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or excipient can be a solvent or liquid dispersion medium, including for example water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oil, non-toxic glyceride and its suitable mixture. For example, suitable flow properties can be formed by forming liposomes, by maintaining the required particle size in the case of dispersions, or by using surfactants to maintain. The prevention of microbial action can be achieved by various antibacterial and / or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugar, buffer or sodium chloride. The extended absorption of injectable compositions can be by using agents that delay absorption, such as aluminum monostearate and / or gelatin.

[0171] The preparation of sterile injectable solutions can be carried out by combining the required amount of the active compound with various other ingredients listed above (if necessary) in an appropriate solvent, and optionally followed by filtered sterilization as required. In the case of sterile powders for preparing sterile injectable solutions, the preparation method may include vacuum drying and freeze drying techniques, which form a powder of the active ingredient plus any additional ingredients required in solution.

[0172] For topical administration, the compounds can be used in neat form, for example when they are liquids. However, it is often desirable to apply the active agent to the skin in the form of a composition or formulation, for example in combination with a dermatologically acceptable carrier, which may be a solid, liquid, gel, etc.

[0173] Available solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silicon dioxide, aluminum oxide, etc. Available liquid carriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, or water-alcohol / glycol mixtures, in which the compound can be dissolved or dispersed at an effective concentration, optionally with the aid of a nontoxic surfactant. Adjuvants such as spices and other antimicrobial agents may also be added to optimize performance for a given use. The resulting liquid composition can be applied from an absorbent pad, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump or aerosol sprayer.

[0174] Thickening agents, such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials, can also be employed with the liquid carrier to form spreadable pastes, gels, ointments, soaps and the like for application directly to the user's skin.

[0175] Examples of dermatological compositions for delivering active agents to the skin are known in the art; for example, see U.S. Pat. Nos. 4,992,478 (Geria), 4,820,508 (Wortzman), 4,608,392 (Jacquet et al.), and 4,559,157 (Smith et al.). Such dermatological compositions can be used in combination with the compounds described herein, wherein the ingredients of such compositions can be optionally replaced by the compounds described herein, or the compounds described herein can be added to the compositions.

[0176] Useful dosages of the compounds described herein can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known in the art; see, for example, U.S. Pat. No. 4,938,949 (Borch et al.). The amount of the compound or its active salt or derivative required for treatment will vary not only with the specific compound or salt selected, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be determined by the clinician or clinician.

[0177] However, in general, a suitable dosage of the active agent may be about 0.5 to about 100 mg / kg, for example about 10 to about 75 mg / kg body weight per day, for example about 3 to about 50 mg per day per kilogram body weight of the recipient, preferably 6 to 90 mg / kg / day, most preferably 15 to 60 mg / kg / day.

[0178] The compound may conveniently be administered in unit dosage form, for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently 50 to 500 mg of active ingredient per unit dosage form. In one embodiment, the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.

[0179] The compound may conveniently be administered in unit dosage form, for example, containing 5 to 1000 mg / m2 of the active ingredient. 2 , conveniently 10 to 750 mg / m 2 , most conveniently 50 to 500 mg / m 2 The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day. The sub-doses themselves may be further divided, for example as a plurality of discrete loosely spaced administrations.

[0180] The desired dose may conveniently be formulated as a single dose, or as divided doses for appropriate intervals, for example as two, three, four or more sub-doses per day. The sub-doses themselves may be further divided, for example into a plurality of individually approximately spaced administrations; for example by multiple inhalations from an insufflator or by instilling a plurality of drops of the drug solution into the eye.

[0181] The compounds described herein can be effective anti-tumor agents and have higher efficacy and / or lower toxicity than TMZ. Preferably, the compounds of the present invention are more effective and less toxic than TMZ, and / or avoid potential sites of catabolism of TMZ, i.e., have different metabolic levels than TMZ.

[0182] The present invention provides a method for treating cancer in a mammal, comprising administering an effective amount of a compound or composition described herein to a mammal suffering from cancer. The mammals include primates, humans, rodents, dogs, cats, cattle, sheep, horses, pigs, goats, cattle, etc. The cancer refers to any of various types of malignant tumors, such as colon cancer, breast cancer, melanoma and leukemia, and is generally characterized by uncontrolled cell proliferation, such as uncontrolled cell growth, insufficient differentiation, local tissue infiltration and metastasis.

[0183] The ability of the compounds of the invention to treat cancer can be determined using assays known in the art. For example, the design of treatment regimens, toxicity assessments, data analysis, quantification of tumor cell killing, and biological significance using transplantable tumor screening are known.

[0184] The following examples are used to illustrate the above invention content and should not be construed as narrowing the scope of the invention. Those skilled in the art should easily recognize that the embodiments can prompt the present invention to be implemented by many other methods. It should be understood that many changes and modifications can be made within the scope of the present invention.

[0185] Example

[0186] Example 1: Experimental information on biological data

[0187] Cell culture and reagents: All cell lines were cultured at 37°C, 5% CO 2 The cells were grown in a humidified environment in medium containing 1% penicillin / streptomycin. The cell culture conditions are as follows: Traditional cell lines U87 and T98G were grown in EMEM containing 10% FBS. Traditional cell lines D54 and U118MG were grown in DMEM containing 10% FBS. HGCC patient-derived cell line U3054MG 1 Cultivated in serum-free stem cell conditions (1:1 Neurobasal:DMEM / F12 medium, where DMEM / F12 medium is supplemented with B27, N2, hEGF and hFGF). GBM Hippocampal cell line Br23c 2 The cells were cultured using NeuroCult NS-A Proliferation Kit (Stem Cell Technologies) supplemented with 0.0002% heparin, hEGF, and hFGF. Temozolomide (TMZ) was purchased from AK Scientific. TMZ analogs were synthesized as described below. Compounds were dissolved in DMSO (final concentration 1%, Fisher Chemical) for cell culture studies.

[0188] Cell viability assay: Cells were harvested, seeded in 96-well plates and allowed to adhere. After three hours, compounds were added to each well in DMSO (final concentration 1%). Cells were incubated for 7 days and then viability was assessed by Alamar Blue Assay. Raptinal (20 μM) was used as a death control.

[0189] Mouse liver microsome stability assay: A mixture of PBS (pH 7.4), NADPH regeneration system solution A (Corning LifeSciences) and NADPH regeneration system solution B (Corning Life Sciences) was incubated in a shaking incubator at 37°C for 5 minutes. Next, the compound was added to DMSO (final concentration 50 μM, 0.5% DMSO), and then ice-cold mouse liver microsomes (Thermo Fisher, male CD-1 mice, pooled) were added (final protein concentration 1 mg / mL). An aliquot was immediately removed, quenched with an equal volume of 100 μM internal standard and 0.5% hydrochloric acid in ice-cold acetonitrile, and centrifuged at 13,000 rcf for 3 minutes. The supernatant was spun in ddH 2 The reaction was incubated at 37 °C for 2 h in a shaker. A second aliquot was removed, quenched and diluted as before, and analyzed by LC-MS. The area ratio of the analyte to the internal standard was calculated at 2 h vs. t 0 The samples were compared to determine the percentage of residual compound. Analysis was performed on an Agilent 6230 LC / MS TOF system equipped with a 1.8 μm, 2.1 x 50 mm Agilent ZORBAX Eclipse Plus C18 column. Internal standard = N3-propyl TMZ.

[0190] O 6 -Methyl deoxyguanosine quantification: U87 cells were counted at 1x10 6 c / w were seeded in 6-well plates and then treated with the indicated concentrations of compounds (final concentration 1% DMSO). After incubation for 8 hours, the cells were harvested and precipitated. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, ID: 69504). The DNA was then precipitated by the following steps: 1 / 10 v / v 3M sodium acetate (pH 5.2) and 2.5x v / v ethanol were added to each sample, which was then kept at -80°C for 1 h. The mixture was centrifuged at a maximum temperature of 4°C for 30 minutes, then decanted to obtain a DNA pellet and resuspended in ddH containing 10mM tris base (pH 7.5) and 1mM EDTA. 2O. The concentration of DNA in each sample was quantified by measuring absorbance on a NanoDrop 2000 UV-Vis Spectrophotometer (ThermoFisher). DNA (10 μg) from each sample was added to DNA hydrolysis buffer. 3 , and incubated at 37°C for 6 hours. The hydrolyzed samples were then submitted for LC-MS / MS quantification. The samples were analyzed using a 5500QTRAP LC / MS / MS system (AB Sciex) and a 1200 series HPLC system (Agilent).

[0191] In vivo blood-brain barrier permeability: All experimental procedures were reviewed and approved by the University of Illinois Institutional Animal Care and Use Committee. CD-1 IGS mice were injected with 1 mg DMSO ( Figure 2 a) or 10% DMSO ( Figure 2 bd) in PBS at a dose of 25 mg / kg. Five minutes after injection, mice were sacrificed and the right auricle was punctured with iris scissors to collect blood. An 18-gauge angiocatheter was inserted through the left ventricle and all residual circulating volume was removed by perfusing 0.9% saline solution using a simulated peristaltic pump. Blood samples were immediately centrifuged at 13,000 rcf for 5 minutes, and the supernatant was collected and washed with 8.5% H 3 PO 4 The brain was collected from the skull vault and acidified with 0.3% HO 3 PO 4 The aqueous solution was acidified and flash frozen. The homogenized brain samples were centrifuged twice at 13,000 rcf for 10 minutes, and the supernatant and tissue fragments were separated. The resulting supernatant and plasma were analyzed by LC-MS / MS to determine the compound concentration. In order to calculate the absolute brain: serum ratio (ng drug brain :ng drug serum ), assuming that the mouse blood volume of each mouse is 58.5 mL / kg.

[0192] In vivo efficacy model: Human GBM Br23c stem-like neurosphere cells were implanted intracranially into female athymic nude mice (150,000 cells / mouse). Starting on day 5 after tumor cell implantation, 15 mg / kg TMZ (or an equimolar dose of C8 analog) was administered by oral gavage once daily for 7 weeks in a saline solution containing 10% PEG 400. Figure 5 a) or once daily for a total of 5 treatments ( Figure 5b). TMZ and C8 analogs were freshly dissolved at each use. Mice were observed daily for any signs of deterioration, neurotoxicity, or motor impairment. They were examined for signs of pain and discomfort according to the Johns Hopkins Animal Care and Use Guidelines. If symptoms persisted and led to weakness, animals were euthanized according to the protocol.

[0193] Hematologic toxicity assessment: Male CD-1 IGS mice (n=4 mice / group) were intravenously injected with a single dose of 125 mg / kg of the compound. Immediately before injection, imidazotetrazine was prepared with SBEβCD in sterile water. Seven days after treatment, mice were humanely sacrificed and whole blood was collected to assess total leukocytes, lymphocytes, neutrophils, platelets, and erythrocytes.

[0194] Example 2: Synthesis method

[0195] Materials and Methods: Chemical reagents were purchased from commercial sources and used without further purification. Silica gel (230-400 mesh) was used for flash chromatography. Anhydrous solvents were dried by passing through a column filled with activated alumina under a positive pressure of nitrogen. Unless otherwise stated, all reactions were performed in oven-dried glassware under a nitrogen atmosphere with magnetic stirring. 1 H and 13 C NMR spectra were recorded on a Bruker 500 (500 MHz, 1 H; 125MHz, 13 C) or Varian Unity Inova 500 (500MHz, 1 The spectrum was referenced by residual chloroform (δ = 7.26 ppm, 1 H; 77.16ppm, 13 C) or dimethyl sulfoxide (δ=2.50ppm, 1 H; 39.52ppm, 13 C). Multiplicities are expressed as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad). Coupling constants J are expressed in Hertz (Hz). High-resolution mass spectrometry (HRMS) was performed on a Waters Q-Tof Ultima or Waters Synapt G2-Si instrument with electrospray ionization (ESI) or electron impact ionization (EI).

[0196] Preparation and characterization of C8 analogs: Compound 3, 4 9, 5 11, 6 15,7 16, 8 29, 9 31, 10 and 33 6 The experimental information has been reported previously.

[0197] General scheme for the preparation of amide, ester and thioester derivatives 4-10:

[0198]

[0199] General procedure for the preparation of 4-10: In an oven-dried 25 mL round-bottom flask, the acid chloride 29 (148.6 mg, 0.70 mmol, 1 eq.) was dissolved in anhydrous THF (2.8 mL, 0.25 M). Methylamine (33% w / w in ethanol, 0.09 mL, 0.73 mL, 1.05 eq.) was then added and the reaction was stirred at room temperature for 3 h. After completion, the reaction was stopped and the solvent was evaporated. The crude solid was purified by flash silica gel chromatography (100% ethyl acetate) to give 98.3 mg (68%) of pure 4 as a white solid.

[0200] The experimental data of compounds 3, 9 and 29 have been published. 4,5,9 .

[0201]

[0202] N,3-Dimethyl-4-oxo-3,4-dihydroimidazole[5,1-d][1,2,3,5]tetrazine-8-carboxamide (4,Me-TMZ):

[0203] 1 H NMR (500MHz, d-DMSO) δ8.84(s,1H),8.45(d,J=4.9Hz,1H),3.86(s,3H),2.81(d,J=4.8Hz,3H). 13 C(125MHz,d-DMSO)δ160.13,139.23,134.27,130.54,128.44,36.14,25.80.HRMS(ESI)calc.for C 7 H 8 N 6 O 2 Na,[M+Na] + :231.0606,found:231.0608.

[0204] N,N,3-Trimethyl-4-oxo-3,4-dihydroimidazole [5,1-d][1,2,3,5]tetrazine-8-carboxamide (5,DiMe-TMZ):

[0205] Yield 76%, white solid.

[0206] 1 H NMR (500MHz, d-DMSO) δ8.81(s,1H),3.85(s,3H),3.06(s,6H). 13 C NMR(125MHz,d-DMSO)δ161.76,139.22,133.57,132.05,128.59,38.12,36.05,34.84.HRMS(ESI)calc.for C 8 H 11 N 6 O 2 ,[M+H]:223.0938,found:223.0943.

[0207] N,N-Diethyl-3-methyl-4-oxo-3,4-dihydroimidazole [5,1-d][1,2,3,5] tetrazine-8-carboxamide (6):

[0208] Yield 91%, white solid.

[0209] 1 H NMR (500MHz, d-DMSO) δ8.81(s,1H),3.84(s,3H),3.49(q,J=7.1Hz,2H),3.38(q,J=7.0Hz,2H),1.18(t,J=7.1Hz,3H),1.11(t,J=7.0Hz,3H). 13 C NMR(125MHz,d-DMSO)δ161.35,139.24,133.54,132.72,128.45,42.53,36.01,14.43,12.80.HRMS(ESI)calc.forC 10 H 15 N 6 O 2 ,[M+H] + :251.1256,found:251.1250.

[0210] N,N-Dibutyl-3-methyl-4-oxo-3,4-dihydroimidazole[5,1-d][1,2,3,5]tetrazine-8-carboxamide (7):

[0211] Yield 85%, white solid.

[0212] 1H NMR(500MHz,d-DMSO)δ8.80(s,1H),3.84(s,3H),3.45(m,2H),3.34(m,2H),1.59(m,2H),1.49(m ,2H),1.35(h,J=7.4Hz,2H),1.11(h,J=7.4Hz,2H),0.94(t,J=7.4Hz,3H),0.76(t,J=7.4Hz,3H). 13 C NMR (125MHz, d-DMSO) δ161.74,139.23,133.35,132.80,128.42,47.66,44.41,35.99,30.54,29.20,19.65,19.17,13.79,13.55.HRMS(ESI)calc.for C 14 H 23 N 6 O 2 ,[M+H] + :307.1882,found:307.1881.

[0213] 3-Methyl-8-(pyrrolidine-1-carbonyl)imidazo[5,1-d][1,2,3,5]tetrazine-4(3H)-one (8):

[0214] Yield 55%, light yellow solid.

[0215] 1 H NMR (500MHz, d-DMSO) δ8.81(s,1H),3.85(s,3H),3.63(m,2H),3.53(m,2H),1.88(m,4H). 13 C NMR(125MHz,d-DMSO)δ159.73,139.21,134.07,132.54,128.33,48.05,46.09,36.05,25.80,23.63.HRMS(ESI)calc.for C 10 H 13 N 6 O 2 ,[M+H] + :249.1100,found:249.1105.

[0216] S-Ethyl 3-methyl-4-oxo-3,4-dihydroimidazole[5,1-d][1,2,3,5]tetrazine-8-thiosulfate (10):

[0217] Yield 92%, white solid.

[0218] 1 H NMR (500MHz, d-DMSO) δ8.86 (s, 1H), 3.89 (s, 3H), 3.02 (q, J = 7.4Hz, 2H), 1.28 (t, J = 7.4Hz, 3H). 13 C NMR(125MHz,d-DMSO)δ184.57,138.95,133.80,131.55,129.19,36.50,22.15,14.70.HRMS(ESI)calc.for C 8 H 10 N 5 O 2 S,[M+H] + :240.0555,found:240.0551.

[0219] 3,8-Dimethylimidazole[5,1-d][1,2,3,5]tetrazine-4(3H)-one (14):

[0220]

[0221] Procedure: 4-Methyl-1H-imidazole-5-amine dihydrochloride 12 (44.2 mg, 0.3 mmol, 1 eq.) was added to a 15 mL round bottom flask and dissolved in 1 M HCl (0.4 mL, 0.65 M), then sodium nitrite (26.2 mg, 0.4 mmol, 1.5 eq.) in water (0.4 mL, 0.65 M) was added at 0 °C in the dark. The solution was stirred for 30 minutes, then concentrated and azeotroped with toluene twice to give crude diazo 13. To the crude diazo suspended in ethyl acetate (1.3 mL, 0.2 M) was added anhydrous triethylamine (0.08 mL, 0.6 mmol, 2.2 eq.) and methylamine carboxylic acid chloride (79 mg, 0.8 mmol, 3.2 eq.) in the dark. The reaction was stirred overnight and then purified by flash silica gel chromatography (4:1 hexanes:ethyl acetate) to give 2.4 mg (6%) 14 as a light yellow solid. NOTE: To minimize decomposition of the crude diazo material, the concentration step (without heating) was performed in the dark as quickly as possible.

[0222] 1 H NMR (500 MHz, d-CHCl 3 )δ8.35(s,1H),3.94(s,3H),2.66(s,3H). 13 C NMR (125MHz, d-CHCl 3 )δ139.71,139.64,132.51,127.94,35.76,12.53.HRMS(EI)calc.forC 6H 7 N 5 O,[M] + :165.0651,found:165.0654.

[0223] 8-Acetyl-3-methylimidazo[5,1-d][1,2,3,5]tetrazine-4(3H)-one (17,K-TMZ):

[0224]

[0225] Procedure: An oven-dried 25 mL round-bottom flask was charged with 16 (186 mg, 0.89 mmol, 1 eq.) and N-succinimidyl N-methylcarbamate (321 mg, 1.86 mmol, 2.1 eq.) and suspended in anhydrous acetonitrile (1.5 mL, 0.6 M). Then, under nitrogen, dry triethylamine (0.34 mL, 2.4 mmol, 2.7 eq.) was slowly added and the solution was stirred at room temperature overnight. Upon completion, the mixture was concentrated and purified by silica gel flash chromatography (100% dichloromethane to 4:1 dichloromethane:methanol) to give 106 mg (66%) of intermediate 16a as a golden solid.

[0226] 1 H NMR (500MHz, d-DMSO) δ8.17 (br s, 1H), 7.97 (s, 1H), 7.56 (s, 2H), 3.36 (d, J = 4.5Hz, 3H), 2.73 (s, 3H).

[0227] In a 15 mL round bottom flask, LiCl (802 mg, 19 mmol, 36 eq.) was dissolved in distilled water (1.3 mL, 0.4 M) and AcOH (0.10 mL, 5.3 M) and stirred for 30 min until the exotherm disappeared. Intermediate 16a (96.3 mg, 0.53 mmol, 1 eq.) was added in one portion and stirred for 30 min. The suspension was cooled to 0 °C in an ice bath and then NaNO was added dropwise. 2 (57 mg, 0.8 mmol, 1.5 eq.) in a minimum amount of distilled water. The resulting mixture was stirred at 0°C for 30 minutes, then warmed to room temperature and stirred for an additional 5 hours. Upon completion, the reaction mixture was washed with CH 2 Cl 2 The organic layer was diluted and separated. The aqueous layer was extracted with dichloromethane (x6) and the combined organic layers were dried over sodium sulfate and concentrated to give a crude solid which was purified by flash silica gel chromatography (1:1 ethyl acetate:hexanes) to afford 36 mg (35%) of 17 as a white solid.

[0228] 1H NMR (500MHz, d-DMSO) δ8.86(s,1H),3.90(s,3H),2.68(s,3H). 13 C NMR(125MHz,d-DMSO)δ191.47,139.01,135.56,133.35,129.11,36.43,28.31.HRMS(ESI)calc.forC 7 H 8 N 5 O 2 ,[M+H] + :194.0678,found:194.0683.

[0229] Experimental data for intermediates 31, 15, and 16 have been published 7,8,10 .

[0230] 8-Bromo-3-methylimidazo[5,1-d][1,2,3,5]tetrazin-4(3H)-one (18):

[0231]

[0232] Process: To anhydrous CH 3 Tetraethylammonium bromide (240 mg, 1.12 mmol, 2.2 eq) was added to a stirred suspension of Dess-Martin periodinane (477 mg, 1.12 mmol, 2.2 eq) in CN (2.6 mL, 0.2 M). The reaction was stirred at room temperature for 5 minutes, and then 3 (100 mg, 0.51 mmol, 1 eq) was added. The resulting reaction mixture was heated at 50 ° C for 2 h. After completion, the solvent was concentrated under reduced pressure to give the crude product, which was purified by flash silica gel chromatography (9:1 hexane: ethyl acetate) to give 73 mg (58%) of 18 as a white solid.

[0233] 1 H NMR (500 MHz, CDCl 3 )δ8.37(s,1H),3.98(s,3H). 13 C NMR (125 MHz, CDCl 3 )δ138.74,133.32,128.56,117.16,36.43.HRMS(ESI)calc.for C 5 H 5 N 5 OBr,[M+H] + :229.9677,found:229.9684.

[0234] 8-Chloro-3-methylimidazole[5,1-d][1,2,3,5]tetrazine-4(3H)-one (19):

[0235]

[0236] Process: To anhydrous CH 3 To a stirred suspension of Dess-Martin periodinane (477 mg, 1,12 mmol, 2.2 eq.) in CN (2.6 mL, 0.2 M) was added tetramethylammonium chloride (123 mg, 1.12 mmol, 2.2 eq.). The reaction was stirred at room temperature for 5 minutes, and then 3 (100 mg, 0.51 mmol, 1 eq.) was added. The resulting reaction mixture was heated at 50 °C for 2 hours. After completion, the solvent was concentrated under reduced pressure to give the crude product, which was purified by flash silica gel chromatography (9:1 hexanes:ethyl acetate) to give 43 mg (45%) of 19 as a white solid.

[0237] 1 H NMR (500 MHz, CDCl 3 )δ8.33(s,1H),3.98(s,3H). 13 C NMR (125 MHz, CDCl 3 )δ138.74,130.84,129.81,127.25,36.37.HRMS(ESI)calc.for C 5 H 5 N 5 OCl,[M+H] + :186.0183,found:186.0186.

[0238] General scheme for the preparation of aryl derivatives 23–26:

[0239]

[0240] General procedure for preparing 23-26:

[0241] (a) Suzuki coupling: 4-bromo-5-nitro-1H-imidazole 20 (400 mg, 2.08 mmol, 1 eq.), phenylboronic acid (507 mg, 4.17 mmol, 2 eq.), XPhosPdG 1 (164 mg, 0.2 mmol, 0.1 eq.) and K 3 PO 4 A mixture of (1.32 g, 6.24 mmol, 3 eq.) was suspended in degassed 1:1 H 2O: dioxane (16 mL, 0.13 M). The resulting mixture was stirred at 110 °C for 16 h. The reaction was cooled to room temperature and H 2 The aqueous layer was extracted three times with ethyl acetate, and the combined organic layers were purified by Na 2 SO 4 Dried and concentrated.The residue was purified by flash silica gel chromatography (100% ethyl acetate) to give crude product 21 which was used in the next step without further purification.

[0242] (b) Nitro reduction: Crude 21 was dissolved in dry MeOH (10 mL, 0.2 M) containing 10% Pd / C, and then H 2 (1 atm). The reaction was stirred at room temperature for 16 hours, and then the catalyst was filtered through celite. The filtrate was concentrated under reduced pressure and purified by flash silica gel chromatography (95:5 DCM:MeOH) to give compound 22, which was used in the next step without further purification.

[0243] (c) Cyclization: To a suspension of intermediate 22 in 1 M HCl (2.9 mL, 0.7 M) was added dropwise the preformed 2 NaNO in O (2.9 mL, 0.9 M) 2 (186 mg, 2.7 mmol, 1.3 eq.) solution. The resulting mixture was stirred at 0 ° C in the dark for 30 minutes. After completion, the solvent was evaporated and the crude diazo compound was dissolved in ethyl acetate (9.6 mL, 0.2 M), and then triethylamine (544 μL, 4.6 mmol, 2 eq.) and methylcarbamic acid chloride (1010 mg, 10.8 mmol, 5.2 eq.) were added. The reaction mixture was stirred at room temperature for 16 hours in the dark. After the reaction was completed, the solvent was concentrated under reduced pressure and the residue was purified by flash silica gel chromatography (9:1 hexane: ethyl acetate) to obtain 28 mg (6%) of pure 23 as a white solid.

[0244] 3-Methyl-8-phenylimidazole[5,1-d][1,2,3,5]tetrazine-4(3H)-one (23):

[0245]

[0246] The product was obtained by the general procedure described. Yield 6% (4 steps), white solid.

[0247] 1 H NMR (500MHz, d-DMSO) δ8.84 (s, 1H), 8.31-8.29 (m, 2H), 7.57-7.53 (m, 2H), 7.44 (tt, J = 7.4, 1.3Hz, 1H), 3.85 (s, 3H). 13C NMR (125 MHz, CDCl 3 )δ140.02,137.02,132.30,131.88,129.86,129.48,129.43,127.07,36.29.HRMS(ESI)calc.for C 11 H 10 N 5 O,[M+H] + :228.0885,found:228.0878.

[0248] 8-(4-Fluorophenyl)-3-methylimidazo[5,1-d][1,2,3,5]tetrazine-4(3H)-one (24):

[0249]

[0250] The product was obtained by the general procedure described. Yield 3% (4 steps), yellow solid.

[0251] 1 H NMR (500 MHz, CDCl 3 )δ8.46(s,1H),8.44-8.40(m,2H),7.24-7.19(m,2H),4.01(s,3H). 13 CNMR (125MHz, CDCl 3 )δ164.57,162.58,139.20(d,J=86.4Hz,1C),131.00,129.43(d,J=8.3Hz,1C),128 .56,127.26(d,J=3.3Hz,1C),116.00(d,J=21.6Hz,1C),35.97.HRMS(ESI)calc.for C 11 H 9 FN 5 O,[M+H] + :246.0791,found:246.0788.

[0252] 3-Methyl-8-(4-(trifluoromethyl)phenyl)imidazo[5,1-d][1,2,3,5]tetrazine-4(3H)-one (25):

[0253]

[0254] The product was obtained by the general procedure described. Yield 5% (4 steps), yellow solid.

[0255] 1 H NMR (500 MHz, CDCl 3)δ8.52(d,J=8.2Hz,2H),8.48(s,1H),7.75(d,J=8.2Hz,2H),4.02(s,3H). 13 C NMR (125 MHz, CDCl 3 )δ139.34,137.89,134.30,131.91,131.03(q,J=32.3Hz,1C),128.77,127.61,125.80(q,J=3.8Hz,1C),122.96,36.15.HRMS(ESI)calc.forC 12 H 9 N 5 OF 3 ,[M+H] + :296.0759,found:296.0754.

[0256] 8-(4-Chlorophenyl)-3-methylimidazole[5,1-d][1,2,3,5]tetrazine-4(3H)-one (26):

[0257]

[0258] The product was obtained by the general procedure described. Yield 1.2% (4 steps), yellow solid.

[0259] 1 H NMR (500MHz, d-DMSO) δ8.86 (s, 1H), 8.30 (dt, J = 9.25, 2.5Hz, 2H), 7.62 (dt, J = 9.25, 2.5Hz, 2H), 3.86 (s, 3H). 13 C NMR(125MHz,d-DMSO)δ139.92,135.67,133.97,132.45,130.75,130.00,129.63,128.62,36.37.HRMS(ESI)calc.for C 11 H 9 N 5 OCl,[M+H] + :262.0496,found:262.0489.

[0260] 3-Methyl-4-oxo-N-(2-oxopropyl)-3,4-dihydroimidazole[5,1-d][1,2,3,5]tetrazine-8-carboxamide (32):

[0261]

[0262] Procedure: DMF (4.4 mL, 0.47 M) and pyridine (0.9 mL) were added to 29 (447 mg, 2.09 mmol, 1 eq.) and 2-aminoacetophenone hydrochloride (229 mg, 2.09 mmol, 1 eq.). The reaction mixture was stirred at room temperature for 16 hours. Water was added and the aqueous layer was extracted 5 times with ethyl acetate. The combined organic layers were purified by Na 2 SO 4 Dried and concentrated. The resulting residue was purified by flash silica gel chromatography (100% ethyl acetate) to afford 266 mg (51%) of 32 as an orange solid.

[0263] 1 H NMR (500MHz, d-DMSO) δ8.87 (s, 1H), 8.59 (t, J = 5.7Hz, 1H), 4.17 (d, J = 5.7Hz, 2H), 3.88 (s, 3H), 2.15 (s, 3H). 13 C NMR(125MHz,d-DMSO)δ204.55,160.15,139.65,135.10,130.18,129.09,49.57,36.67,27.52.LC-MS(ESI)calc.for C 9 H 11 N 6 O 3 [M+H] + :251.0893,found:251.09.

[0264] 3-Methyl-8-(5-methyloxazol-2-yl)imidazo[5,1-d][1,2,3,5]tetrazin-4(3H)-one (27,Ox-TMZ):

[0265] Procedure: Intermediate 32 (266 mg, 1.06 mmol, 1 eq.) was added to phosphorus oxychloride (6.5 mL, 0.16 M) and the stirred mixture was heated at 110 °C for 3 h. After completion, ice water was added and the aqueous layer was extracted with ethyl acetate × 4. The combined organic layers were purified by Na 2 SO 4 Dried and concentrated. The resulting residue was purified by flash silica gel chromatography (100% ethyl acetate) to afford 80 mg (32%) of the product 27 as a yellow solid.

[0266] 1 H NMR (500MHz, d-DMSO) δ8.89 (s, 1H), 7.13 (br d, J = 1.2Hz, 1H), 3.87 (s, 3H), 2.44 (d, J = 1.2Hz, 3H). 13C NMR(125MHz,d-DMSO)δ154.15,150.47,139.64,133.64,130.43,126.15,125.36,36.56,11.17.HRMS(ESI)calc.for C 9 H 9 N 6 O 2 ,[M+H] + :233.0782,found:233.0787.

[0267] The pathway to generate 4-substituted oxazol-2-yl groups at the C8 position of imidazole tetrazines is known. 11 However, the synthesis of compound 27 via intermediate 32 has never been reported.

[0268] 3-Methyl-4-oxo-3,4-dihydroimidazole [5,1-d] [1,2,3,5] tetrazine-8-thioamide (28):

[0269]

[0270] Procedure: To a solution of 33 (550 mg, 2.6 mmol, 1 eq.) in acetonitrile (40 mL, 0.07 M) was added α-bromoacetone (220 μL, 2.6 mmol, 1 eq.) and the solution was stirred at room temperature for 18 h. Upon completion, the reaction was stopped and the precipitate was filtered and purified by flash silica gel chromatography (4:6 hexanes:ethyl acetate) to give 167 mg (26%) of the desired product 28 as a yellow solid.

[0271] 1 H NMR (500MHz, d-DMSO) δ8.87 (s, 1H), 7.46 (d, J = 0.9Hz, 1H), 3.86 (s, 3H), 2.48 (d, J = 0.9Hz, 3H). 13 C NMR(125MHz,d-DMSO)δ158.63,154.58,139.75,131.84,131.80,130.24,116.56,36.52,17.45.HRMS(ESI)calc.for C 9 H 9 N 6 OS,[M+H] + :249.0559,found:249.0559.

[0272] The pathway to generate 4-substituted thiazol-2-yl groups at the C8 position of imidazole tetrazines is known. 11, but the synthesis of compound 28 has never been reported. The experimental data of intermediate 33 have been published 6 .

[0273] Example 3: Synthesis of Aromatic, Propargyl and Diazoalkane Compounds.

[0274] Aromatic and propargyl substituted imidazole tetrazines can be prepared as follows.

[0275]

[0276] Among them G 1 OCH 3 ,OCH 2 CH 3 ,OPh,N(CH 3 ) 2 , propargyl or a substituent as defined herein.

[0277] TMZ is a non-explosive, scalable alternative to diazomethane. TMZ and other imidazole tetrazines can be used as diazoalkane precursors in the synthesis shown below.

[0278]

[0279] Example 4: Pharmaceutical Dosage Form

[0280] The following formulations describe representative pharmaceutical dosage forms that can be used for the compound combinations described herein, specifically disclosed herein, or pharmaceutically acceptable salts or solvates thereof (hereinafter referred to as "Compound X'"):

[0281]

[0282]

[0283]

[0284] These preparations can be prepared by conventional methods known in the pharmaceutical field. It should be understood that the above-mentioned drug combination can be changed according to known pharmaceutical techniques to adapt to different amounts and different types of active ingredients "Compound X". Aerosol (vi) can be used in combination with a standard metered sprayer. In addition, specific ingredients and ratios are only used for illustration. Depending on the desired properties of the dosage form required, specific ingredients can be replaced with suitable equivalents (e.g., the above-mentioned ingredients), and the ratios can also be changed.

[0285] Citations

[0286] (1)Stupp,R.;Mason,W.;van den Bent,M.J.;Weller,M.;Fisher,B.M.;Taphoorn,M.J.B.;Belanger,K.;Brandes,A.A.;Marosi,C.;Bogdahn,U.;Curschmann,J.;Janzer,R.C.;Ludwin,S.K.;Gorlia,T.;Allgeier,A.;Lacombe,D.;Cairncross,J.G.;Eisenhauer,E.;Mirimanoff,R.O.(2005)Radiotherapy plus Concomitant and AdjuvantTemozolomide for Glioblastoma.N.Engl.J.Med.352,987–996。

[0287] (2)Denny,B.J.;Wheelhouse,R.T.;Stevens,M.F.;Tsang,L.L.;Slack,J.a.(1994)NMR and Molecular Modeling Investigation of the Mechanism of Activationof the Antitumor Drug Temozolomide and Its Interaction with DNA.Biochemistry33,9045–9051。

[0288] (3)Karran,P.;Macpherson,P.;Ceccotti,S.;Dogliotti,E.;Griffin,S.;Bignami,M.(1993)O6-Methylguanine Residues Elicit DNA Repair Synthesis byHuman Cell Extracts.J.Biol.Chem.268,15878–15886。

[0289] (4)Ceccotti,S.;Aquilina,G.;Macpherson,P.;Yamada,M.;Karran,P.;Bignami,M.(1996)Processing of O6-Methylguanine by Mismatch Correction in Human CellExtracts.Curr.Biol.6,1528–1531。

[0290] (5)Margison,G.P.;Santibanez Koref,M.F.;Povey,A.C.(2002)MechanismsofCarcinogenicity / Chemotherapy by O6-Methylguanine.Mutagenesis 17,483–487。

[0291] (6)Tisdale,M.J.(1987)Antitumour Imidazotetrazines-XV.Role of GuanineO6 Alkylation in the Mechanism of Cytotoxicity of Imidazotetrazinones.Biochem.Pharmacol.36,457–462。

[0292] (7)Newlands,E.S.;Blackledge,G.R.;Slack,J.A.;Rustin,G.J.;Smith,D.B.;Stuart,N.S.;Quarterman,C.P.;Hoffman,R.;Stevens,M.F.;Brampton,M.H.;Gibson,A.C.(1992)Phase I Trial of Temozolomide(CCRG 81045:M&B 39831:NSC 362856).Br.J.Cancer 65,287–291。

[0293] (8)Ostermann,S.;Csajka,C.;Buclin,T.;Leyvraz,S.;Lejeune,F.;Decosterd,L.a;Stupp,R.(2004)Plasma and Cerebrospinal Fluid Population Pharmacokineticsof Temozolomide in Malignant Glioma Patients.Clin.Cancer Res.10,3728–3736。

[0294] (9)Portnow,J.;Badie,B.;Chen,M.;Liu,A.;Blanchard,S.;Synold,T.W.(2009)TheNeuropharmacokinetics of Temozolomide in Patients with Resectable BrainTumors:Potential Implicationsfor the Current Approach toChemoradiation.Clin.Cancer Res.15,7092–7098。

[0295] (10)Stupp,R.;Hegi,M.E.;Mason,W.P.;van den Bent,M.J.;Taphoorn,M.J.;Janzer,R.C.;Ludwin,S.K.;Allgeier,A.;Fisher,B.;Belanger,K.;Hau,P.;Brandes,A.A.;Gijtenbeek,J.;Marosi,C.;Vecht,C.J.;Mokhtari,K.;Wesseling,P.;Villa,S.;Eisenhauer,E.;Gorlia,T.;Weller,M.;Lacombe,D.;Cairncross,J.G.;Mirimanoff,R.O.(2009)Effects of Radiotherapy with Concomitant and Adjuvant TemozolomideversusRadiotherapy Alone on Survival in Glioblastoma in a Randomised PhaseIII Study:5-Year Analysis of theEORTC-NCIC Trial.Lancet Oncol.10,459–466。

[0296] (11)Reifenberger,G.;Wirsching,H.-G.;Knobbe-Thomsen,C.B.;Weller,M.(2017)Advances in theMolecular Genetics of Gliomas—Implications forClassification and Therapy.Nat.Rev.Clin.Oncol.14,434–452。

[0297] (12)Vaupel,P.;Kallinowski,F.;Okunieff,P.(1989)Blood Flow,Oxygen andNutrient Supply,andMetabolic Microenvironment of Human Tumors:A Review.CancerRes.49,6449–6465。

[0298] (13)Rottenberg,D.A.,Ginos,J.Z.,Kearfott,K.J.,Junck,L.,and Bigner,D.D.(1984)In VivoMeasurement of Regional Brain Tissue PH Using Positron EmissionTomography.Ann.Neurol.15,S98–S102。

[0299] (14)Arnold,James B,Kraig,Richard P,Rottenberg,D.A.(1986)In VivoMeasurement of Regional Brainand Tumor pH Using[14 C]Dimethyloxazolidinedioneand Quantitative Autoradiography.II:Characterizationof the ExtracellularFluid Compartment Using PH-Sensitive Microelectrodes and[14 C]Sucrose.J.Cereb.Blood Flow Metab.6,435–440。

[0300] (15)McLean,L.A.;Roscoe,J.;Jorgensen,N.K.;Gorin,F.A.;Cala,P.M.(2000)Malignant GliomasDisplay Altered pH Regulation by NHE1 Compared withNontransformed Astrocytes.Am.J.Physiol.CellPhysiol.278,C676-88。

[0301] (16)Lowe,P.R.;Sansom,C.E.;Schwalbe,C.H.;Stevens,M.F.G.;Clark,A.S.(1992)AntitumorImidazotetrazines.25.Crystal Structure of 8-Carbamoyl-3-Methylimidazo[5,1-d]-1,2,3,5-Tetrazin-4(3H)-One(Temozolomide)and StructuralComparisons with the Related Drugs Mitozolomide and DTIC.J.Med.Chem.35,3377–3382。

[0302] (17)Suppasansatorn,P.;Wang,G.;Conway,B.R.;Wang,W.;Wang,Y.(2006)SkinDelivery Potencyand Antitumor Activities of Temozolomide EsterProdrugs.Cancer Lett.244,42–52。

[0303] (18)Liu,D.;Yang,J.G.;Cheng,J.;Zhao,L.X.(2010)Synthesis and AntitumorActivity of 3-Methyl-4-Oxo-3,4-Dihydroimidazo[5,1-d][1,2,3,5]Tetrazine-8-Carboxylates and-Carboxamides.Molecules 15,9427–9436。

[0304] (19)Hummersone,M.G.;Stevens,M.F.G.;Cousin,D.Preparation of 3-Substituted-3H-Imidazo[5,1-d][1,2,3,5]Tetrazin-4-One Compounds for TreatingProliferative Disorders.WO 2010149968,2010。

[0305] (20)O’Reilly,S.M.;Newlands,E.S.;Brampton,M.;Glaser,M.G.;Rice-Edwards,J.M.;Illingworth,R.D.;Richards,P.G.;Kennard,C.;Colquhoun,I.R.;Lewis,P.;Stevens,M.F.G.(1993)Temozolomide:ANew Oral Cytotoxic Chemotherapeutic Agentwith Promising Activity against Primary Brain Tumours.Eur.J.Cancer 29,940–942。

[0306] (21)Bleehen,N.M.;Newlands,E.S.;Lee,S.M.;Thatcher,N.;Selby,P.;Calvert,A.H.;Rustin,G.J.S.;Brampton,M.;Stevens,M.F.G.(1995)Cancer Research CampaignPhase II Trial of Temozolomide inMetastatic Melanoma.J.Clin.Oncol.13,910–913。

[0307] (22)Shealy,Y.F.;Struck,R.F.;Holum,L.B.;Montgomery,J.A.(1961)Synthesisof PotentialAnticancer Agents.XXIX.5-Diazoimidazole-4-Carboxamide and 5-Diazo-v-Triazole-4-Carboxamide.J.Org.Chem.26,2396–2401。

[0308] (23)Shechter,H.;Magee,W.L.;Rao,C.B.;Glinka,J.;Hui,H.;Amick,T.J.;Fiscus,D.;Kakodkar,S.;Nair,M.(1987)Dipolar Cycloaddition Reactions ofDiazoazoles with Electron-Rich and with StrainedUnsaturatedCompounds.J.Org.Chem.52,5538–5548。

[0309] (24)Fulmer Shealy,Y.;Krauth,C.A.;Montgomery,J.A.(1962)Imidazoles.I.Coupling Reactions of 5-Diazoimidazole-4-Carboxamide.J.Org.Chem.27,2150–2154。

[0310] (25)Stevens,M.F.;Hickman,J.A.;Stone,R.;Gibson,N.W.;Baig,G.U.;Lunt,E.;Newton,C.G.(1984)Antitumor Imidazotetrazines.1.Synthesis and Chemistry of 8-Carbamoyl-3-(2-Chloroethyl)Imidazo[5,1-d]-1,2,3,5-Tetrazin-4(3 H)-One,a NovelBroad-Spectrum Antitumor Agent.J.Med.Chem.27,196–201。

[0311] (26)Lunt,E.;Newton,C.G.;Smith,C.;Stevens,G.P.;Stevens,M.F.G.;Straw,C.G.;Walsh,R.J.A.;Warren,P.J.;Fizames,C.;Lavelle,F.;Langdon,S.P.;Vickers,L.M.(1987)Antitumor Imidazotetrazines.14.Synthesis and Antitumor Activity of 6-and 8-Substituted Imidazo[5,l-d]-l,2,3,5-Tetrazinones and 8-SubstitutedPyrazolo[5,l-d]-l,2,3,5-Tetrazinones.J.Med.Chem.30,357–366。

[0312] (27)Moseley,C.K.;Carlin,S.M.;Neelamegam,R.;Hooker,J.M.(2012)AnEfficient and PracticalRadiosynthesis of[11C]Temozolomide.Org.Lett.14,5872–5875。

[0313] (28)Horspool,K.R.;Stevens,M.F.G.;Baig,G.U.;Newton,C.G.;Lunt,E.;Walsh,R.J.A.;Pedgrift,B.L.;Lavelle,F.;Fizames,C.(1990)Antitumor Imidazotetrazines.20.Preparation of the 8-Acid Derivative ofMitozolomide and Its Utility in thePreparation of Active Antitumor Agents.J.Med.Chem.33,1393–1399。

[0314] (29)Langnel,DAF;Arrowsmith,J.andStevens,MFG(2000)AntitumorImidazotetrazines.38.New 8-Substituted Derivatives of the Imidazo[5,1-d]-1,2,3,5-Tetrazines Temozolomide and Mitozolomide.FILE No. iii, 421–437.

[0315] (30)Stevens,MA;Giner-Sorolla,A.Smith,HW,Bosworth Brown,G.(1962)Purine N-Oxides.X.

[0316] (31)Xie,Y. T.Jiang,Y.Johansson,P.Marinescu,VDLindberg,N.Segerman,A.Wicher,G.Niklasson,M.Baskaran,S.Sreedharan,S.Everlien,I.Kastemar,M.Her mansson,A.Elfineh,L.Libard,S.Holland,ECHesselager,G.Alafuzoff,I.Westermark,B.Nelander,S.Forsberg-Nilsson,K.Uhrbom,L.(2015)TheHuman Glioblastoma Cell Culture Resource:Validated Cell ModelsRepresentingAll Molecular Subtypes.EBioMedicine 2,1351–1363.

[0317] (32)Pardridge,WM(2005)The Blood-Brain Barrier:A Bottleneck in BrainDrug Development.NeuroRX2,3–14.

[0318] (33)Norinder,U.;Haeberlein,M.(2002)Computational Approaches to thePrediction of the Blood-BrainDistribution.Adv.Drug Deliv.Rev.54,291–313。

[0319] (34)West,D.C.;Qin,Y.;Peterson,Q.P.;Thomas,D.L.;Palchaudhuri,R.;Morrison,K.C.;Lucas,P.W.;Palmer,A.E.;Fan,T.M.;Hergenrother,P.J.(2012)Differential Effects of Procaspase-3 ActivatingCompounds in the Induction ofCancer Cell Death.Mol.Pharm.9,1425–1434。

[0320] (35)Egan,W.J.;Merz,K.M.;Baldwin,J.J.(2000)Prediction of DrugAbsorption Using MultivariateStatistics.J.Med.Chem.43,3867–3877。

[0321] (36)Tian,S.;Wang,J.;Li,Y.;Li,D.;Xu,L.;Hou,T.(2015)The Application ofin Silico Drug-LikenessPredictions in Pharmaceutical Research.Adv.DrugDeliv.Rev.86,2–10.

[0322] (37)Daina,A.;Zoete,V.(2016)A BOILED-Egg To Predict GastrointestinalAbsorption and BrainPenetration of Small Molecules.ChemMedChem 11,1117–1121。

[0323] (38)Alifrangis,L.H.;Christensen,I.T.;Berglund,A.;Sandberg,M.;Hovgaard,L.;Frokjaer,S.(2000)Structure-Property Model-for MembranePartitioning of Oligopeptides.J.Med.Chem.43,103–113。

[0324] (39)Wager,T.T.;Hou,X.;Verhoest,P.R.;Villalobos,A.(2010)Moving beyondRules:TheDevelopment of a Central Nervous System Multiparameter Optimization(CNS MPO)Approach to EnableAlignment of Druglike Properties.ACSChem.Neurosci.1,435–449。

[0325] (40)Wager,T.T.;Hou,X.;Verhoest,P.R.;Villalobos,A.(2016)CentralNervous SystemMultiparameter Optimization Desirability:Application in DrugDiscovery.ACS Chem.Neurosci.7,767–775。(41)Reyderman,L.;Statkevich,P.;Thonoor,C.M.;Patrick,J.;Batra,V.K.;Wirth,M.(2004)Dispositionand Pharmacokinetics ofTemozolomide in Rat.Xenobiotica 34,487–500。

[0326] (42)Rai,R.;Banerjee,M.;Wong,D.H.;McCullagh,E.;Gupta,A.;Tripathi,S.;Riquelme,E.;Jangir,R.;Yadav,S.;Raja,M.;Melkani,P.;Dixit,V.;Patil,U.;Shrivastava,R.;Middya,S.;Olivares,F.;Guerrero,J.;Surya,A.;Pham,S.M.;Bernales,S.;Protter,A.A.;Hung,D.T.;Chakravarty,S.(2016)TemozolomideAnalogs withImproved Brain / Plasma Ratios–Exploring the Possibility of Enhancing theTherapeutic Indexof Temozolomide.Bioorganic Med.Chem.Lett.26,5103–5109。

[0327] (43)Kim,S.S.;Rait,A.;Kim,E.;DeMarco,J.;Pirollo,K.F.;Chang,E.H.(2015)Encapsulation ofTemozolomide in a Tumor-Targeting Nanocomplex Enhances Anti-Cancer Efficacy and Reduces Toxicity in aMouse Model of Glioblastoma.CancerLett.369,250–258。

[0328] (44)Stevens,M.F.;Hickman,J.A.;Langdon,S.P.;Chubb,D.;Vickers,L.;Stone,R.;Baig,G.;Goddard,C.;Gibson,N.W.;Slack,J.A.(1987)Antitumor Activity andPharmacokinetics in Mice of 8-Carbamoyl-3-Methyl-Imidazo[5,1-d]-1,2,3,5-Tetrazin-4(3H)-One(CCRG 81045;M&B 39831),a Novel Drug withPotential as anAlternative to Dacarbazine.Cancer Res.47,5846–5852。

[0329] (45)Bobola,M.S.;Tseng,S.H.;Blank,a;Berger,M.S.;Silber,J.R.(1996)Roleof O6-Methylguanine-DNA Methyltransferase in Resistance of Human Brain TumorCell Lines to the Clinically RelevantMethylating Agents Temozolomide andStreptozotocin.Clin.Cancer Res.2,735–741。

[0330] (46)Kanzawa,T.;Bedwell,J.;Kondo,Y.;Kondo,S.;Germano,I.M.(2003)Inhibition of DNA Repairfor Sensitizing Resistant Glioma Cells toTemozolomide.J.Neurosurg.99,1047–1052。

[0331] (47)Binder,Z.A.;Wilson,K.M.;Salmasi,V.;Orr,B.A.;Eberhart,C.G.;Siu,I.M.;Lim,M.;Weingart,J.D.;Quinones-Hinojosa,A.;Bettegowda,C.;Kassam,A.B.;Olivi,A.;Brem,H.;Riggins,G.J.;Gallia,G.L.(2016)Establishment and BiologicalCharacterization of a Panel of Glioblastoma Multiforme(GBM)andGBM VariantOncosphere Cell Lines.PLoS One 11,e0150271。

[0332] (48)Scaringi,C.;De Sanctis,V.;Minniti,G.;Enrici,R.M.(2013)Temozolomide-Related HematologicToxicity.Onkologie 36,444–449。

[0333] (49)Thomas,R.P.;Recht,L.;Nagpal,S.(2013)Advances in the Management ofGlioblastoma:The Roleof Temozolomide and MGMT Testing.Clin.Pharmacol.,5,1–9。

[0334] (50)Chamberlain,M.C.(2010)Temozolomide:Therapeutic Limitations in theTreatment of Adult High-Grade Gliomas.Expert Rev.Neurother.10,1537–1544。

[0335] (51)Li,R.;Tang,D.;Zhang,J.;Wu,J.;Wang,L.;Dong,J.(2014)TheTemozolomide Derivative 2T-P400 Inhibits Glioma Growth via AdministrationRoute of Intravenous Injection.J.Neurooncol.116,25–30。

[0336] (52)Cheng,Y.;Sk,U.H.;Zhang,Y.;Ren,X.;Zhang,L.;Huber-Keener,K.J.;Sun,Y.W.;Liao,J.;Amin,S.;Sharma,A.K.;Yang,J.M.(2012)Rational Incorporation ofSelenium into Temozolomide Elicits SuperiorAntitumor Activity Associated withBoth Apoptotic and Autophagic Cell Death.PLoS One 7,e35104。

[0337] (53)Cho,H.-Y.;Wang,W.;Jhaveri,N.;Lee,D.J.;Sharma,N.;Dubeau,L.;Schonthal,A.H.;Hofman,F.M.;Chen,T.C.(2014)NEO212,Temozolomide Conjugated toPerillyl Alcohol,Is a Novel Drug for EffectiveTreatment of a Broad Range ofTemozolomide-Resistant Gliomas.Mol.Cancer Ther.13,2004–2017。

[0338] (54)Owonikoko,TK;Arbiser,J.Zelnak,A.Shu,H.-KG.Shim,H.Robin,AM.Kalkanis,SN.Whits ett,TGSalhia,B.Tran,NLRyken,T.Moore,MKEgan,KMOlson,JJ(2014)CurrentApproaches to the Treatment of Metastatic Brain Tumours.Nat.Rev.Clin.Oncol.11,203–222.

[0339] (55)Thomas,A.Tanaka,M.Trepel,J.Reinhold,WC;Rajapakse,VN,Pommier,Y.(2017)Temozolomide in the Era of Precision Medicine.Cancer Res.,77,823–826.

[0340] References to Examples

[0341] (1)Xie,Y. T.Jiang,Y.Johansson,P.Marinescu,VDLindberg,N.Segerman,A.Wicher,G.Niklasson,M.Baskaran,S.et al.(2015)The Human Glioblastoma Cell Culture Resource:Representing Validated Cell Models AllMolecular Subtypes.EBioMedicine 2,1351–1363.

[0342] (2)Binder,Z.A.;Wilson,K.M.;Salmasi,V.;Orr,B.A.;Eberhart,C.G.;Siu,I.M.;Lim,M.;Weingart,J.D.;Quinones-Hinojosa,A.;Bettegowda,C.;et al.(2016)Establishment and Biological Characterization of aPanel of GlioblastomaMultiforme(GBM)and GBM Variant Oncosphere Cell Lines.PLoS One 11,e0150271。(3)Quinlivan,E.P.;Gregory,J.F.(2008)DNA Digestion to Deoxyribonucleoside:ASimplified One-Step Procedure.Anal.Biochem.373,383–385。

[0343] (4)Arrowsmith,J.;Jennings,S.A.;Clark,A.S.;Stevens,M.F.G.(2002)Antitumor Imidazotetrazines.41.1Conjugation of the Antitumor AgentsMitozolomide and Temozolomide to Peptides and Lexitropsins Bearing DNA Majorand Minor Groove-Binding Structural Motifs.J.Med.Chem.45,5458–5470。

[0344] (5)Wang,Y.;Conway,B.;Suppasansatorn,P.Synthesis of TemozolomideEsters as Potent Anticancer Pro-Drugs for Topical and TransdermalApplications in Treatments of Cancers.US 2006 / 0047117 A1,2006。

[0345] (6)Langnel,D.A.F.;Arrowsmith,J.;Stevens,M.F.G.(2000)AntitumorImidazotetrazines.38.New 8-Substituted Derivatives of the Imidazo[5,1-d]-1,2,3,5-Tetrazines Temozolomide and Mitozolomide.ARKIVOC No.iii,421–437。

[0346] (7)Jiao,Y.G.;Yu,H.T.(2001)Methyltrioxorhenium(MeReO3)CatalyzedSelective Oxidation of Purine and Related Compounds into Their N-Oxides.Synlett No.1,73–74。

[0347] (8)Stevens,M.A.;Giner-Sorolla,A.;Smith,H.W.;Bosworth Brown,G.(1962)Purine N-Oxides.X.The Effect of Some Substituents on Stability andReactivity1.J.Org.Chem.27,567–572。

[0348] (9)Liu,D.;Yang,J.G.;Cheng,J.;Zhao,L.X.(2010)Synthesis and AntitumorActivity of 3-Methyl-4-Oxo-3,4-Dihydroimidazo[5,1-d][1,2,3,5]Tetrazine-8-Carboxylates and-Carboxamides.Molecules 15,9427–9436。

[0349] (10) Marasco, CJ; Pera, PJ; Spiess, AJ; Bernacki, R.; Sufrin, JR (2005) Improved Synthesis of β-D-6-Methylpurine Riboside and Antitumor Effects of the β-D-andα-D-Anomers. Molecules 10, 1015–1020.

[0350] (11)Hummersone, Marc Geoffery; Stevens, Malcolm Francis Graham; Cousin, D.

[0351] The above embodiments and examples are described herein, but these descriptions are only for illustration and explanation and do not limit the scope of the present invention. Some changes and modifications may be made according to the ordinary skills in the art, but they do not depart from the broader scope of the present invention as defined by the following claims.

[0352] All publications, granted patents and patent application documents are incorporated herein by reference, just as each is individually incorporated herein by reference. No limitations inconsistent with the disclosure herein should be introduced by these publications. The description of the present invention quotes various specific and preferred embodiments and technical solutions. It should be understood that there are many variations and modifications that can be adopted, which still all belong to the spirit and scope of the present invention.

Claims

1. A compound of formula I: or its salts; in: X is O or S; R 1 is a halogen; R 2 is -(C1-C6)alkyl or -(C3-C6)cycloalkyl; and R 3 is H or -(C1-C6)alkyl; Wherein, -(C1-C6)alkyl and -(C3-C6)cycloalkyl are each optionally substituted by one or more substituents, and -(C1-C6)alkyl is each unbranched or optionally branched; wherein alkyl may optionally include alkenyl or alkynyl.

2. The compound according to claim 1, wherein R 1 For chlorine.

3. The compound according to claim 1, wherein X is O, R 2 is -(C1-C6)alkyl, and R 3 For H.

4. The compound according to claim 1, wherein R 2 is -(C1-C6)alkyl, and R 3 For H.

5. The compound according to claim 1, wherein R 2 It is propargyl.

6. The compound according to claim 1, wherein X is O, R 3 For H.

7. The compound according to claim 1, wherein the compound is CPZ:

8. Use of a compound in the preparation of a medicament, wherein the medicament comprises the compound according to any one of claims 1 to 7, and the medicament is used to treat cancer; wherein the cancer is glioblastoma (GBM).

Images