Combination therapy of anticancer drugs with hypoxia-responsive prodrugs and radiotherapy, and novel hypoxia-responsive prodrugs

Abstract

In the present invention, an anticancer drug / prodrug is used in a combination therapy with radiotherapy. In the chemical formula representing the same, the moiety in which R1 and R2 are bonded to an N atom results from the removal of an amino group or an imino group from an anticancer drug (selected from the group consisting of, e.g., gemcitabine, niraparib, crizotinib, dabrafenib, vemurafenib, entinostat, cobimetinib, palbociclib, and ribociclib) having the amino group or the imino group. Adverse effects from the effect of treating malignant tumors using radiotherapy are thereby reduced, or the effect of the treatment is enhanced.

Description

TECHNICAL FIELD

[0001] The present invention relates to a formulation containing a hypoxia-responsive prodrug as an active ingredient, for improving or enhancing the effect of radiotherapy in a combination therapy of an anticancer drug and radiotherapy. More specifically, the present invention relates to the above formulation, wherein the hypoxia-responsive prodrug is a compound having a structure covalently bonded by forming an amide or imide with 2-nitro-1-imidazolepropionic acid via an amino group (-NH2) or an imino group (-NH-) in the molecule of the anticancer drug, and to a novel hypoxia-responsive prodrug among the above compounds. BACKGROUND ART

[0002] Today, for some types of cancer, radiation therapy and drug therapy (anticancer drug treatment) are recommended as standard treatments. In such combination therapies, both side effects associated with radiation therapy and side effects associated with anticancer drugs can occur. For this reason, improvements to the radiation irradiation method have been considered, and furthermore, because cells in a hypoxic state contained in malignant tumors have little to no oxygen sensitization effect, the damaging effect of radiation on tumor cells is small, and generally, tumors containing many hypoxic cells are resistant to radiation therapy. For this reason, hypoxic cell radiosensitizers and hypoxic-active prodrugs have been proposed (IN Mistry et. al., Int J Radiation Oncol Bio Phys, Vol.98, No.5. pp. 1183-1196, 2017 (hereinafter referred to as Non-Patent Literature 1)). As such a prodrug, for example, the nitroimidazole-based TH-302 (evofosafamide) has been proposed and its use in combination therapy with radiation therapy has been investigated, but to the best of our knowledge, it has not reached clinical use. Furthermore, similar nitroimidazole-based compounds such as pimonidazole, etanidazole, and nimorazole have been proposed as sensitizers, but none of these compounds have achieved a statistically significant improvement in survival rates compared to conventional radiotherapy alone. Combining these compounds with radiotherapy has either not yielded significant benefits or has not led to clinical use due to their strong toxicity. It should be noted that TH-302 is described as potentially treating cancer when administered alone or in combination with other anticancer drugs and / or radiotherapy (however, no specific treatment data regarding combination therapy has been disclosed, and subsequent clinical trial results, as described in Non-Patent Document 1, have been disappointing). [Disclosure of the Invention] [Problems that the invention aims to solve]

[0003] However, the need remains to obtain compounds that produce more effective combined effects in combination therapy with radiation therapy and drug therapy.

[0004] Previously, some of the present inventors proposed a compound that belongs to the nitroimidazole class as a hypoxia-responsive prodrug for anticancer drugs, in which 2-nitro-1-imidazolepropionic acid is covalently bonded to the anticancer drug via an amino group (-NH2) or imino group (-NH-) within the anticancer drug molecule (Japanese Patent Publication No. 5676020). Such compounds (hereinafter sometimes abbreviated as PropHAPs) are thought to be able to contribute to cancer drug therapy because they exhibit cytotoxicity comparable to the parent anticancer drug under hypoxic conditions in malignant tumor cells, but show significantly reduced cytotoxicity compared to the parent anticancer drug in cells at normal oxygen concentrations.

[0005] Indeed, as described in Non-Patent Document 1, the nitroimidazole-based prodrugs described therein did not necessarily show satisfactory effects in combination therapy with radiotherapy that we conducted. Nevertheless, we investigated whether gemcitabine PropHAP had any merit in combination therapy with radiotherapy. Surprisingly, the results showed that gemcitabine PropHAP, in combination therapy with radiotherapy (in vivo), showed almost the same therapeutic effect as the parent compound in experimental animals at the radiation dose at which the parent compound caused side effects such as weight loss in the test animals, while not causing a significant decrease in weight. This was also true when the drug dose was increased. On the other hand, in experimental animals, at radiation doses and drug doses in which the parent compound did not substantially cause side effects such as weight loss in the test animals, the combination of radiotherapy and drug therapy showed a significantly superior anti-cancer effect compared to radiotherapy alone, parent compound alone, and combinations of parent compound and radiotherapy. Similar effects were also observed with palbociclib, niraparib, ribociclib, and doxorubicin, all PropHAPs whose chemical structures and mechanisms of action on tumors differ from those of gemcitabine.

[0006] The difference between the conventional nitroimidazole-based HAPs described above and PropHAPs is that, as described in the aforementioned patent publication, PropHAPs are understood to cleave residues or partial X derived from anticancer drugs under a hypoxic environment, as shown in the following reaction scheme. This is because their chemical structure is fundamentally different from the nitroimidazole-based prodrugs described in Non-Patent Document 1, and the cleavage mode of the nitroimidazole protecting group and the products resulting from the cleavage are also completely different. Therefore, in the subject matter of the present invention or disclosed herein, the use of Prop as the protecting group of the parent compound in PropHAPs is of extremely important significance. Reaction scheme:

[0007] TIFF0007923555000001.tif28163 [Means for solving the problem]

[0008] Therefore, the following can be cited as the principal aspects or features of the invention provided in this application or the subject matter disclosed herein:

[0009] Embodiment 1: A pharmaceutical preparation comprising a compound represented by Formula I as an active ingredient, for use in combination therapy of radiotherapy and drug therapy for malignant tumors, for improving or enhancing the therapeutic effect of said radiotherapy and other combination therapies.

[0010] Formula I: TIFF0007923555000002.tif44163

[0011] During the ceremony, The portion to which R1 and R2 are bonded to the N atom is the portion obtained by removing the amino group or imino group from an anticancer drug having an amino group or imino group. When an amino group is present, either R1 or R2 represents a hydrogen atom. The anticancer drug having the portion to which R1 and R2 are bonded to the N atom is an anthracycline selected from doxorubicin, idarubicin, epirubicin, daunorubicin, pirarubicin, amrubicin, acrasinomycin, anthramycin, and zolubicin, as well as bleomycin and actinocycline. Peptides selected from mycin, quinoline alkaloids selected from camptothecin, topotecan, and irinotecan, taxanes selected from docetaxel and paclitaxel, vinca alkaloids selected from vinorelbine, vincristine, vinblastine, and vindesine, deoxycytidines selected from gemcitabine and cytarabine, pyrimidines selected from 5-fluorouracil, capecitabine, and doxifluridine, fludarabine, and 6-mercaptopodium Phosphorus, purine ring derivatives selected from 6-thioguanine, macrolides selected from epotilone, niraparib, crizotinib, dabrafenib, vemurafenib, entinostat, panobinostat, cobimetinib, Pa The drug is selected from a group consisting of rubociclib and other anticancer drugs selected from ribociclib.

[0012] Embodiment 2: A pharmaceutical preparation according to Embodiment 1, wherein the compound represented by formula I is derived from each of the following anticancer drugs and is represented by each of the following structural formulas.

[0013] Derived from gemcitabine, structural formula:

[0014] TIFF0007923555000003.tif38163,

[0015] Derived from doxorubicin, structural formula:

[0016] TIFF0007923555000004.tif54163,

[0017] Derived from 5-fluorouracil, having the structural formula:

[0018] any one of TIFF0007923555000005.tif29163,

[0019] Derived from niraparib, having the structural formula:

[0020] TIFF0007923555000006.tif42163,

[0021] Derived from crizotinib, having the structural formula:

[0022] any one of TIFF0007923555000007.tif63163,

[0023] Derived from dabrafenib, having the structural formula:

[0024] TIFF0007923555000008.tif44163,

[0025] Derived from vemurafenib, having the structural formula:

[0026] TIFF0007923555000009.tif44163,

[0027] Derived from entinostat, having the structural formula:

[0028] TIFF0007923555000010.tif38163,

[0029] Derived from cobimetinib, having the structural formula:

[0030] TIFF0007923555000011.tif44163,

[0031] Pa Derived from ribociclib, having the structural formula:

[0032] TIFF0007923555000012.tif44163

[0033] , or Derived from ribociclib, structural formula:

[0034] TIFF0007923555000013.tif44163

[0035] . Embodiment 3: A combination therapy of drug therapy and radiotherapy for malignant tumors, comprising the steps of administering an effective dose of a compound represented by formula I to a patient or individual requiring treatment for a malignant tumor, and irradiating with an effective dose of radiation. Formula I: TIFF0007923555000014.tif37161In formula, The portion to which R1 and R2 are bonded to the N atom is the portion obtained by removing the amino group or imino group from an anticancer drug having an amino group or imino group. When an amino group is present, either R1 or R2 represents a hydrogen atom. The anticancer drug having the portion to which R1 and R2 are bonded to the N atom is an anthracycline selected from doxorubicin, idarubicin, epirubicin, daunorubicin, pirarubicin, amrubicin, acrasinomycin, anthramycin, and zolubicin; a peptide selected from bleomycin and actinomycin; or a quinoline alkali selected from camptothecin, topotecan, and irinotecan. Taxanes selected from docetaxel and paclitaxel, vinca alkaloids selected from vinorelbine, vincristine, vinblastine, and vindesine, deoxycytidines selected from gemcitabine and cytarabine, pyrimidines selected from 5-fluorouracil, capecitabine, and doxyfluridine, purine ring derivatives selected from fludarabine, 6-mercaptopurine, and 6-thioguanine, and macrolides selected from epotilone, niraparib, crizotinib, dabrafenib, vemurafenib, entinostat, panobinostat, cobimetinib, Pa The drug is selected from a group consisting of rubociclib and other anticancer drugs selected from ribociclib. Aspect 4: A combination therapy according to Aspect 3, wherein the compound represented by formula I is derived from each of the following anticancer drugs and is represented by each of the following structural formulas. Derived from gemcitabine, structural formula: TIFF0007923555000015.tif31161, Derived from doxorubicin, structural formula: TIFF0007923555000016.tif48162, Derived from 5-fluorouracil, structural formula: One of the following: TIFF0007923555000017.tif25155 Derived from chiraparib, structural formula: TIFF0007923555000018.tif37157, Derived from crizotinib, structural formula: One of the following: TIFF0007923555000019.tif61158 Derived from dabrafenib, structural formula: TIFF0007923555000020.tif38160, Derived from vemurafenib, structural formula: TIFF0007923555000021.tif40158, Derived from entinostat, structural formula: TIFF0007923555000022.tif32160, Derived from cobimetinib, structural formula: TIFF0007923555000023.tif40159, Pa Derived from rubociclib, structural formula: TIFF0007923555000024.tif40160, or Derived from ribociclib, structural formula: TIFF0007923555000025.tif36157.

[0036] Embodiment 5: A compound represented by formula I-1 or a pharmaceutically acceptable salt thereof.

[0037] Formula I-1: TIFF0007923555000026.tif39163

[0038] During the ceremony, The portion to which R1 and R2 are bonded to the N atom is the portion obtained by removing the amino group or imino group from an anticancer drug having an amino group or imino group. When an amino group is present, either R1 or R2 represents a hydrogen atom. These are the portions of niraparib, crizotinib, taburafenib, vemurafenib, entinostat, cobimetinib, and others. Pa Compounds derived from anticancer drugs selected from the group consisting of rubocicrill and ribociclib, and each represented by the following structural formula: Derived from chiraparib, structural formula:

[0039] TIFF0007923555000027.tif39163,

[0040] Derived from crizotinib, structural formula:

[0041] One of the following: TIFF0007923555000028.tif63163

[0042] Derived from dabrafenib, structural formula:

[0043] TIFF0007923555000029.tif42163,

[0044] Derived from vemurafenib, structural formula:

[0045] TIFF0007923555000030.tif42163,

[0046] Derived from entinostat, structural formula:

[0047] TIFF0007923555000031.tif33163,

[0048] Derived from cobimetinib, structural formula:

[0049] TIFF0007923555000032.tif39163,

[0050] Pa Derived from rubociclib, structural formula:

[0051] TIFF0007923555000033.tif40163

[0052] , or Derived from ribociclib, structural formula:

[0053] TIFF0007923555000034.tif38163.

[0054] Apparatus 6: A method for providing a hypoxia-responsive prodrug for improving or enhancing the therapeutic effect of a combination therapy including radiotherapy in a combination therapy of radiotherapy and drug therapy for malignant tumors, (1) The anticancer drug is transmitted via the amino group (NH2) or imino group (-NH-) in its molecule using formula II:

[0055] TIFF0007923555000035.tif35163

[0056] The steps include forming a prodrug for anticancer drugs by covalently bonding imidazole propionic acid, represented by [formula], (2) A step in which tumor experimental animals are treated with the anticancer drug prodrug obtained in step (1) and the corresponding parent compound in combination with radiotherapy, and the prodrug is selected that exhibits statistically significantly lower side effects or significantly higher therapeutic effects compared to radiotherapy alone. Methods that include... [Brief explanation of the drawing]

[0057] [Figure 1] This is a graph showing the results of in vitro test 1 (b) in Example 1. [Figure 2] This is a graph showing the results of in vitro test 2 in Example 1 (c). [Figure 3] This is a graph showing the results of the in vivo test in Example 1 (d). [Figure 4] This is a graph showing the results of the in vitro test in Example 2 (b). [Figure 5] This is a graph showing the results of the in vitro test in Example 3 (b). [Figure 6] This is a graph showing the results of the in vitro test in Example 4 (b). [Figure 7] This is a graph showing the results of the in vitro test in Example 5(b). [Figure 8] This is a graph showing the results of the in vitro test in Example 6 (b). [Figure 9] This is a graph showing the results of the in vitro test in Example 7(b). [Figure 10] Example 7(c) shows a graph of the results of the in vivo test. [Figure 11] This is a graph showing the results of the (b) in vitro test and (c) in vivo test in Example 8. [Figure 12] This is a graph showing the results of in vivo test 1 in Example 9. [Figure 13] This is a graph showing the results of in vivo study 2 in Example 9. [Figure 14] This is a graph showing the results of in vivo study 2 (part 2) of Example 9. [Figure 15] This is a graph showing the results of in vivo study 3 in Example 9. [Figure 16] This is a graph showing the results of the in vivo test in Example 10. Detailed description of the invention

[0058] Unless otherwise defined, the technical terms used herein or in connection with the description of the present invention have the meanings and content commonly used in the art.

[0059] The radiation used in the "radiotherapy method" of the present invention is not limited to the radiation used in conventional radiotherapy in the broad sense, but examples include X-rays, gamma (γ) rays, alpha (α) rays, beta (β) rays, proton beams, and heavy ion (carbon ion) beams, and the radiation irradiation method can be any method already used in the art.

[0060] In the combination therapy of the present invention, the patient or individual (which may be a mammal including a human) is administered a compound represented by formula I, and then irradiated with radiation for approximately 30 minutes to 48 hours, typically 1 hour to 24 hours. The irradiation may be external beam radiation, where radiation is applied from outside the patient or subject's body, or internal beam radiation, where radiation is applied to the cancer or its surrounding area from an implanted radiation source inside the body, or a combination of both. Furthermore, malignant tumors that can be treated or managed by such administration and irradiation may be, or may be, currently treated with radiation therapy and drug therapy or chemoradiotherapy, such as head and neck cancer, cervical cancer, esophageal cancer, lung cancer, pancreatic cancer, breast cancer, soft tissue sarcoma, and other tumors for which the desired effect is expected to be obtained by treatment with such therapy. On the other hand, the mode of administration of the compound represented by formula I may be oral or parenteral, and specifically, it may be appropriately determined by referring to the mode of administration that is clinically commonly used for the parent compound.

[0061] "To improve or enhance the effects of combination therapies, including radiotherapy" means that, in combination therapy of radiotherapy and anticancer drugs (in vivo), when the parent compound of the compound represented by formula I (an anticancer drug having a portion in which R1 and R2 are bonded to the N atom of the compound represented by formula I, or a compound that serves as a starting material for the said compound) is administered to experimental animals carrying malignant tumors and radiotherapy is performed, symptoms such as nausea, vomiting, neurological disorders, or weight loss that may result therefrom, which appear in the animals, are statistically significantly reduced when the parent compound is replaced with the compound of formula I, while the therapeutic effect is at least the same, or at least statistically significantly enhanced compared to radiotherapy alone. "Statistically significantly reduced" or "statistically significantly enhanced" means that when data obtained from the test results of five or more groups of subjects (including individual subjects) are statistically processed by a t-test, the reduction or enhancement is statistically significant.

[0062] "Pharmaceutical preparation" is a concept interchangeable with "pharmaceutical composition" and refers to a preparation that can be used in the field of pharmaceutical technology. In addition to the compound of formula I as the active ingredient, it may include, but is not limited to, diluents, carriers, excipients, etc., that can be used in the preparation of the preparation and are generally safe, non-toxic, and do not adversely affect the properties of the compound. Examples include sterile water, nonionic surfactants, ethanol, glycerol and mixtures thereof, and other compounds or mixtures that have been used as diluents, carriers, or excipients for individual active pharmaceutical ingredients.

[0063] The pharmaceutically acceptable salts of the compounds described in Embodiment 5 may be acid addition salts of mineral acids such as hydrochloric acid and sulfuric acid, or organic acids such as formic acid, acetic acid, citric acid, and methanesulfonic acid. On the other hand, if the compound has acidic groups such as carboxyl groups and hydroxyl groups, the salts may be addition salts of alkali metals such as sodium and potassium, or organic amines such as ammonium and methylamine.

[0064] The compound of formula I described in Embodiment 1 or the hypoxia-responsive prodrug described in Embodiment 6 can be produced by forming an amide bond or imide bond by reacting a corresponding or common anticancer drug with imidazolepropionic acid represented by formula II. Such a reaction may be carried out by reacting the anticancer drug and the compound of formula II in a suitable inactive solvent in the presence of a condensing agent known in the art (e.g., carbodiimides), or by reacting an active ester of the compound of formula II (halides, esters with N-hydroxysuccinimide, etc.) with the compound of formula I in a suitable solvent. Specific examples of the reaction include, if an amino group or a cyclic amino group and a hydroxyl group coexist in the molecule of the organic compound, either group may be protected by a method known in the art before carrying out one of the above reactions, if necessary. For specific examples of carrying out the reaction, please refer to the publication of Japanese Patent No. 5676020. For gemcitabine, doxilubicin, and 5-fluorouracil PropHAPs, the information provided in that patent publication can be used as is. Therefore, by citing that patent publication, its contents will be used exactly as described herein.

[0065] The effective radiation dose and the effective dose of the compound represented by formula I cannot be specified because the optimal amount varies depending on the type and severity of the malignant tumor being treated and the type of compound used. However, generally, the radiation dose, administration, and dosage can be referenced from cases where the corresponding parent compound has been specifically studied for the combination therapy in question. For example, these can be determined by medical professionals taking into account materials available from the U.S. Food and Drug Administration and data discussed in medical literature. [Examples]

[0066] The present invention will be described in more detail below with specific examples, but it is not intended to limit the present invention to these examples.

[0067] Example 1: Chive Parib HAP (a) Manufacturing example

[0068] TIFF0007923555000036.tif39163

[0069] Dissolve niraparib (compound 1,100 mg) in 10 mL of dichloromethane, and compound Compound 2 (105 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (71 mg), and dimethylaminopyridine (23 mg) were added and reacted at room temperature for 24 hours. After the reaction, the compound was purified by silica gel chromatography (chloroform:methanol = 97:3) to obtain 150 mg of compound 3 (niraparib HAP). ([M+H] + (Calculated value: 488.21, Observed value: 488.3) (b) In vitro test 1 Human breast cancer cells (MDA-MB-231) were seeded in 96-well plates at a density of 3000 cells / well, and cell viability was observed after incubation for 2 days at normal oxygen concentration and hypoxic concentration (0.2%) with the addition of niraparib or compound 3. The results are shown in Figure 1. Figure 1 confirms that cytotoxicity is restored under hypoxic conditions. (c) In vitro test 2 To confirm whether the targeted PARP activity was inhibited under hypoxic conditions, proteins were recovered from cells and poly-ADP-ribosylated (PAR-modified) proteins were detected by Western blotting. The results are shown in Figure 2. Figure 2 confirms that PARP activity is specifically inhibited under hypoxic conditions. (d) in vivo studies MDA-MB-231 cells were subcutaneously transplanted into Balb / c nude mice (6 or 7 mice per group), and niraparib or niraparib HAP was administered. The synergistic effect with radiotherapy was analyzed. The results are shown in Figure 3. Figure 3 shows that the drug-administered group exhibited enhanced tumor growth inhibition compared to radiotherapy alone. Furthermore, compared to the niraparib-administered group, the niraparib HAP-administered group did not experience weight loss and showed improved survival rates. <Drug dosage and administration, radiation dose> Medication: Oral administration of medication (Days 0, 1, 2, 7, 8, 9 (50 mg / kg), 10, 11, 14, 15, 16, 17, 18, 21, 22, 23, 24, 25 (100 mg / kg)) Irradiation: 0.5 Gy irradiation 1 hour after drug administration.

[0070] Example 2: Crizotinib HAP (a) Manufacturing example

[0071] TIFF0007923555000037.tif51163

[0072] Crizotinib (compound 4.50 mg) was dissolved in 2 mL of dichloromethane, and compound 2 (32 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (22 mg), and dimethylaminopyridine (7 mg) were added. The mixture was reacted at room temperature for 24 hours. After the reaction, the mixture was purified by silica gel chromatography (chloroform:methanol = 97:3) to obtain a mixture of compounds 5 and 6 (crizotinib HAP) of 20 mg. ([M+H] + (Calculated value: 618.2, Observed value: 618.3) (b) in vitro test EML-ALK mutant human non-small cell lung cancer (EML4-ALK fusion-A549) (ATCC model number CCL-185IG) was seeded in 96-well plates at a density of 3000 cells / well, and the resulting crizotinib or crizotinib HAP (a mixture of 5 and 6) was administered. Cell viability was observed after adding a substance and incubating for one day at normal oxygen concentration and low oxygen concentration (0.2%), followed by a change of culture medium and culturing under the same conditions for another two days. The results are shown in Figure 4. From Figure 4, it can be confirmed that cytotoxicity is restored in the low oxygen environment.

[0073] Example 3: Dabrafenib HAP (a) Manufacturing example

[0074] TIFF0007923555000038.tif38163

[0075] Dabrafenib (compound 7.50 mg) was dissolved in 2 mL of dichloromethane, compound 8 (35 mg) was added, and the mixture was reacted at room temperature for 24 hours. After the reaction, the mixture was purified by silica gel chromatography (chloroform:methanol = 98:2) to obtain 20 mg of compound 9 (dabrafenib HAP). ([MH] - (Calculated value: 685.1, Observed value: 685.3) (b) in vitro test Human malignant melanoma (A375) cells were seeded in 96-well plates at a density of 3000 cells / well. The cells were incubated for two days with either dabrafenib or compound 9 at normal oxygen and hypoxic (0.2%) concentrations, and cell viability was observed. The results are shown in Figure 5. Figure 5 confirms that cytotoxicity is restored under hypoxic conditions.

[0076] Example 4: Vemurafenib HAP (a) Manufacturing example

[0077] TIFF0007923555000039.tif45163

[0078] Vemurafenib (compound 10, 50 mg) was dissolved in 2 mL of dichloromethane, compound 8 (35 mg) was added, and the mixture was reacted at room temperature for 24 hours. After the reaction, the mixture was purified by silica gel chromatography (chloroform:methanol = 100:0 to 98:2) to obtain 20 mg of compound 11 (vemurafenib HAP). ([MH] - (Calculated value: 655.0, Observed value: 654.9) (b) in vitro test Human malignant melanoma (A375) cells were seeded in 96-well plates at a density of 3000 cells / well. Vemurafenib or compound 11 was added, and the cells were incubated for two days at normal oxygen and hypoxic (0.2%) concentrations. Cell viability was then observed. The results are shown in Figure 6. Figure 6 confirms that cytotoxicity is restored under hypoxic conditions.

[0079] Example 5: Entinostat HAP (a) Manufacturing example

[0080] TIFF0007923555000040.tif39163

[0081] Entinostat (compound 12, 100 mg) was dissolved in 2 mL of dichloromethane, and compound 2 (105 mg), 76 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and dimethylaminopyridine (24 mg) were added and the mixture was reacted at room temperature for 24 hours. After the reaction, the mixture was purified by silica gel chromatography (chloroform:methanol = 97:3) to obtain 50 mg of compound 13 (entinostat HAP). ([M+H] + (Calculated value: 544.2, Observed value: 544.5) (b) in vitro test Human breast cancer cells (MDA-MB-231) were seeded in 96-well plates at a density of 3000 cells / well. Cell viability was observed after incubation for 4 days under normal oxygen concentration and hypoxic concentration (0.2%) with the addition of the obtained entinostat or compound 13. The results are shown in Figure 7. Figure 7 confirms that cytotoxicity is restored under hypoxic conditions.

[0082] Example 6: Cobimetinib HAP (a) Manufacturing example

[0083] TIFF0007923555000041.tif52163

[0084] Cobimetinib (compound 14, 50 mg) was dissolved in 2 mL of dichloromethane, and compound 2 (26 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (28 mg), and dimethylaminopyridine (9 mg) were added and the mixture was reacted at room temperature for 24 hours. After the reaction, the mixture was purified by silica gel chromatography (chloroform:methanol = 100:0 to 97:3) to obtain compound 15 (cobimetinib HAP) at a concentration of 30 mg. ([MH] - (Calculated value: 697.1, Observed value: 696.9) (b) in vitro test Human malignant melanoma (A375) cells were seeded in 96-well plates at a density of 3000 cells / well. Cell viability was observed after incubation for 2 days under normal oxygen concentration and hypoxic concentration (0.2%) with the addition of cobimetinib or compound 15. The results are shown in Figure 8. Figure 8 confirms that cytotoxicity is restored under hypoxic conditions.

[0085] Example 7: Palbociclib HAP (a) Manufacturing example

[0086] TIFF0007923555000042.tif42163

[0087] Dissolve palbociclib (compound 16,100 mg) in 2 mL of dichloromethane, and combine Compound 2 (75 mg), 51 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and dimethylaminopyridine (16 mg) were added and reacted at room temperature for 24 hours. After the reaction, the mixture was purified by silica gel chromatography (chloroform:methanol = 100:0 to 97:3) to obtain 120 mg of compound 17 (palbociclib HAP). ([M+H] + (Calculated value: 615.3, Observed value: 615.1) (b) in vitro test Human breast cancer cells (MCF-7) were seeded in 96-well plates at a density of 3000 cells / well. The cells were incubated for two days under normal oxygen and hypoxic (0.2%) conditions with either palbociclib or compound 17 added, and cell viability was observed. The results are shown in Figure 9. Figure 9 confirms that cytotoxicity is restored under hypoxic conditions. (c) in vivo study Human pancreatic cancer cells (MIA Paca-2) were subcutaneously transplanted into Balb / c nude mice (6 mice per group), and the synergistic effect of oral administration of palbociclib or palbociclib HAP (100 mg / kg, twice a week) with radiotherapy (2 Gy, twice a week, the day after drug administration) was analyzed. The results are shown in Figure 10. From Figure 10, it can be seen that the prodrug administration group showed enhanced tumor growth inhibition when combined with radiation therapy, compared to radiation therapy alone and the combination therapy of the active ingredient palbociclib with radiation.

[0088] Example 8: Ribociclib HAP (a) Manufacturing example

[0089] TIFF0007923555000043.tif42163

[0090] Ribociclib (compound 18, 100 mg) was dissolved in 2 mL of dichloromethane, and compound 2 (75 mg), 51 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and dimethylaminopyridine (16 mg) were added and the mixture was reacted at room temperature for 24 hours. After the reaction, the mixture was purified by silica gel chromatography (chloroform:methanol = 100:0 to 97:3) to obtain 100 mg of compound 19 (ribociclib HAP). ([M+H] + (Calculated value: 602.3, Observed value: 602.2) (b) in vitro test Human breast cancer cells (MCF-7) were seeded in 96-well plates at a density of 3000 cells / well. The cells were incubated for two days under normal oxygen and hypoxic (0.2%) conditions with either ribociclib or compound 19 added, and cell viability was observed. The results are shown in Figure 11. Figure 11 confirms that cytotoxicity is restored under hypoxic conditions. (c) in vivo study Balb / c nude mice (6 mice per group) were subcutaneously transplanted with 17β-estradiol sustained-release pellets (Innovative Research of America) and human breast cancer cells (MCF7). They were then orally administered ribociclib or ribociclib HAP (75 mg / kg, twice weekly), and the synergistic effect with radiotherapy (1 Gy, twice weekly, the day after drug administration) was analyzed. The results are shown in the lower panel of Figure 11. This figure shows that, compared to radiotherapy alone and radiotherapy combined with the active ingredient ribociclib, the prodrug administration group exhibited enhanced tumor growth inhibition when combined with radiotherapy.

[0091] Example 9: Gemcitabine HAP In vivo study 1: Human pancreatic cancer cells (MIA Paca-2) were subcutaneously transplanted into Balb / c nude mice (6 or 7 mice per group), and gemcitabine or gemcitabine HAP (manufactured according to the method described in the examples of Japanese Patent Publication No. 5676020) were administered intraperitoneally (120 mg / kg, twice / week). The synergistic effect of combination therapy with radiotherapy (2 Gy, twice / week, the day after drug administration) was analyzed. The results are shown in Figure 12. Figure 12 shows that the drug administration group exhibited enhanced tumor growth inhibition compared to radiotherapy alone. Furthermore, compared to the gemcitabine administration group, the gemcitabine HAP administration group did not show weight loss and had an improved survival rate. In vivo study 2: Balb / c nude mice (6 mice per group) were subcutaneously transplanted with human pancreatic cancer cells (MIA Paca-2), and each group received gemcitabine or gemcitabine HAP intraperitoneally (10 mg / kg, twice / week) in combination with radiotherapy (1 Gy, twice / week, administered the day after drug administration). The synergistic effects were analyzed. The results are shown in Figure 13. From Figure 13, it can be seen that the tumor growth inhibitory effect was enhanced in the gemcitabine HAP administration group when combined with radiation, compared to radiation therapy alone and the gemcitabine HAP administration group. When the dose was reduced to 10 mg / kg, it was confirmed that it was more effective than gemcitabine when combined with radiation. In vivo study 2 (part 2): Human cancer cells (MIA Paca-2) were subcutaneously transplanted into Balb / c nude mice (7 mice per group), and each group was administered intraperitoneally with 2-nitroimidazole (10 mg / kg, twice / week), 2-nitroimidazole and gemcitabine (10 mg / kg each, twice / week), or gemcitabine HAP (10 mg / kg, twice / week). The synergistic effects of combination therapy with radiation therapy (1 Gy, twice / week, the day after drug administration) were analyzed. The results are shown in Figure 14. From Figure 14, it can be seen that the gemcitabine HAP administration group showed enhanced tumor growth inhibition when combined with radiation therapy, compared to radiation therapy alone and the radiosensitizer 2-nitroimidazole and the active ingredient gemcitabine. In vivo study 3: Human cholangiocarcinoma cells (HuCCT1) were introduced into Balb / c nude mice (5 mice per group). Each is subcutaneously transplanted, and gemcitabine or gemcitabine HAP is administered intraperitoneally (30 mg). The drug was administered (2 Gy per kg, twice per week) and the synergistic effect of combination therapy with radiotherapy (2 Gy, twice per week, the day after drug administration) was analyzed. The results are shown in Figure 15. From Figure 15, it can be seen that the drug administration group showed enhanced tumor growth inhibition compared to radiotherapy alone. In addition, compared to the gemcitabine administration group, no weight loss was observed in the gemcitabine HAP administration group. Example 10: Doxorubicin HAP Balb / c nude mice (6 or more mice per group) were subcutaneously transplanted with 17β-estradiol sustained-release pellets (Innovative Research of America) and human breast cancer cells (MCF7). Doxorubicin (4 mg / kg, Day 0, 2) or doxorubicin HAP (4 mg / kg, or 16 mg / kg, Day 0, 2, 7, 9) were administered intraperitoneally, and the synergistic effect with radiotherapy (0.5 Gy, Day 1, 3, 8, 10) was analyzed. The results are shown in the lower panel of Figure 16. From this figure, it can be seen that the tumor growth inhibitory effect was enhanced in the prodrug administration group when radiotherapy was used in combination with radiotherapy, compared to radiotherapy alone and when doxorubicin (the active ingredient) was used in combination with radiotherapy. In addition, compared to the doxorubicin-administered group, no weight loss was observed in the doxorubicin HAP-administered group.

Claims

[Claim 1] A pharmaceutical preparation for use in combination therapy of radiotherapy and drug therapy for malignant tumors, comprising a compound represented by the following structural formula as an active ingredient, for improving or enhancing the therapeutic effect of said combination therapy, The aforementioned compound is derived from gemcitabine, and its structural formula is: Compounds represented by, Derived from doxorubicin, structural formula: Compounds represented by, Derived from chiraparib, structural formula: Compounds represented by, Derived from palbociclib, structural formula: Compounds represented by, Derived from ribociclib, Structural formula: Selected from the group consisting of compounds represented by The aforementioned pharmaceutical preparation.

Images