WO2021036937A1 - Pde3抑制剂与细胞因子联合治疗肿瘤的应用 - Google Patents

Pde3抑制剂与细胞因子联合治疗肿瘤的应用 Download PDF

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WO2021036937A1
WO2021036937A1 PCT/CN2020/110552 CN2020110552W WO2021036937A1 WO 2021036937 A1 WO2021036937 A1 WO 2021036937A1 CN 2020110552 W CN2020110552 W CN 2020110552W WO 2021036937 A1 WO2021036937 A1 WO 2021036937A1
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substituted
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hydrogen
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俞强
安然
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中国科学院上海药物研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of medicine. Specifically, the present invention relates to the application of a combination of PDE3 inhibitors and cytokines to treat tumors.
  • cytotoxic drugs such as DNA alkylating agents (cyclophosphamide, cisplatin, carboplatin), tubulin inhibitors (such as paclitaxel), and metabolic antagonists of DNA synthesis precursors (methotrexate) ), topoisomerase II inhibitors (such as etoposide), etc.
  • the purpose of the present invention is to provide a pharmaceutical composition of PDE3 inhibitor combined with cytokine with obvious synergistic anti-tumor effect.
  • composition comprising:
  • the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-50), preferably, 1:(0.1-20), more preferably, 1:( 0.15-10), best, 1: (0.2-5).
  • the PDE3 inhibitor is selected from the following group: the compound of formula I or a pharmaceutically acceptable salt thereof, the compound of formula II or a pharmaceutically acceptable salt thereof, or combination;
  • R1, R2, R3, R4, R5, R6, R7 and R8 are each independently selected from the following group: hydrogen atom, halogen atom, amino group, hydroxyl group, cyano group, aldehyde group, nitro group, substituted or unsubstituted C1-C4 Carboxy, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, substituted or unsubstituted Substituted C6-C10 aryl group, substituted or unsubstituted C5-C10 heteroaryl group (such as substituted or unsubstituted 5-membered or 6-membered heterocyclic group, 8-membered to 10-membered heteroaryl group), substituted or unsubstituted C1 -C10 alk
  • R9 is selected from the following group: hydrogen atom, oxygen atom, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C1-C10 heteroaryl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C6-C10 aryl-oxy, substituted or unsubstituted acyl (preferably -CO-C1-C10 alkane Group), substituted or unsubstituted C1-C10 sulfonyl;
  • any of the "substituted” means that one to four (preferably 1, 2, 3, or 4) hydrogen atoms on the group are substituted by a substituent selected from the following group: C1-C10 alkyl , C3-C10 cycloalkyl, C1-C10 alkoxy, halogen, hydroxyl, carboxyl (-COOH), C1-C10 aldehyde, C2-C10 acyl, C2-C10 ester, amino, phenyl, cyano, -OH, nitro, amino;
  • R11, R12, R13, R14, R15, R17, and R18 are each independently selected from the following group: a hydrogen atom, a halogen atom, an amino group, a hydroxyl group, a cyano group, an aldehyde group, a nitro group, a substituted or unsubstituted C1-C4 carboxyl group, Substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C5-C10 heteroaryl (such as substituted or unsubstituted 5- or 6-membered heterocyclic ring, 8- to 10-membered heteroaromatic bicyclic ring system), substitute
  • R16 is selected from the following group: hydrogen atom, oxygen atom, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C5-C10 heteroaryl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C6-C10 aryl-oxy, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1 -C10 Sulfonyl;
  • any of the "substituted” means that one to four (preferably 1, 2, 3, or 4) hydrogen atoms on the group are substituted by a substituent selected from the following group: C1-C10 alkyl , C3-C10 cycloalkyl, C1-C10 alkoxy, halogen, hydroxyl, carboxyl (-COOH), C1-C10 aldehyde, C2-C10 acyl, C2-C10 ester, amino, phenyl, cyano, OH, nitro and amino.
  • R1 is hydrogen
  • R2 is hydrogen
  • R3 is hydrogen
  • R4 is hydrogen
  • R5 is halogen
  • R6 is halogen
  • R7 is hydrogen
  • R8 is hydrogen
  • R9 is hydrogen
  • R11 is hydrogen
  • the halogen is Cl.
  • R11 is hydrogen
  • R12 is hydrogen or C1-C6 alkoxy.
  • R12 is hydrogen or methoxy.
  • R13 is a halogenated C1-C6 alkoxy group.
  • R13 is a dihalogenated C1-C6 alkoxy group.
  • R13 is halomethoxy
  • R13 is a dihalomethoxy group.
  • R13 is dichloromethoxy
  • R14 is hydrogen or C1-C6 alkoxy.
  • R14 is hydrogen or methoxy
  • R15 is hydrogen
  • R16 is hydrogen
  • R17 is hydrogen
  • R18 is hydrogen
  • the phenyl group includes an unsubstituted phenyl group or a substituted phenyl group having 1 to 3 substituents.
  • the PDE3 inhibitor is selected from the group consisting of Anagrelide or a pharmaceutically acceptable salt thereof, Zardaverine or a pharmaceutically acceptable salt thereof, or Its combination.
  • the cytokine is selected from the following group: antiviral cytokine, tumor necrosis factor, interferon, anti-DNA replication cytokine, or a combination thereof.
  • the cytokine is selected from the group consisting of interferon, tumor necrosis factor, or a combination thereof.
  • the interferon is selected from the group consisting of IFN- ⁇ , IFN- ⁇ , or a combination thereof.
  • the tumor necrosis factor is selected from the group consisting of tumor necrosis factor related apoptosis-inducing ligand (TRAIL), TNF- ⁇ , or a combination thereof.
  • TRAIL tumor necrosis factor related apoptosis-inducing ligand
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • the second aspect of the present invention provides a medicine kit comprising:
  • kit further includes instructions.
  • the instructions for use indicate that the first preparation and the second preparation are combined to prevent and/or treat cancer.
  • the first preparation and the second preparation are administered simultaneously, separately or sequentially in the prevention and/or treatment of tumors.
  • first preparation and the second preparation in the kit are independent of each other, or may be combined.
  • first preparation and the second preparation in the kit are independent of each other.
  • the first preparation is a solid preparation (such as a solid preparation for the gastrointestinal tract) or an injection preparation (such as an injection, an infusion or a lyophilized powder injection).
  • the second preparation is an injection preparation (such as an injection, an infusion solution or a lyophilized powder injection).
  • the first formulation and the second formulation are independent.
  • the kit of the present invention includes: a solid preparation (such as a solid preparation for the gastrointestinal tract) and a preparation for injection (such as an injection, an infusion or a lyophilized powder injection), wherein the solid preparation (such as a gastrointestinal
  • the solid preparation for road use contains a PDE3 inhibitor, and the preparation for injection (such as injection, infusion or lyophilized powder injection) contains cytokines.
  • an active ingredient combination is provided, and the active ingredient combination includes the following components:
  • the fourth aspect of the present invention provides the use of the pharmaceutical composition according to the first aspect of the present invention, the kit according to the second aspect of the present invention, and/or the use of the active ingredient combination according to the third aspect of the present invention, It is used to prepare drugs for preventing and or treating tumors.
  • the tumor is selected from the group consisting of cervical cancer, liver cancer, kidney cancer, colon cancer, glioma, melanoma, or a combination thereof.
  • the fifth aspect of the present invention provides a method for preventing and/or treating tumors, including the step of administering the composition according to claim 1 to a desired subject.
  • the subjects are humans and non-human mammals.
  • non-human mammals include (but are not limited to): pets (such as dogs, cats), domestic animals (such as cows, sheep, horses, pigs), various zoo animals (pandas, elephants, tigers) )Wait.
  • Figure 1A MTT detection set ANA (10nM) and ZARD (20nM) and interferon IFN- ⁇ (3000IU/mL), IFN- ⁇ (10ng/mL), apoptosis-related factors in SMMC7721, Bel7404, A498, and FHCC98 cells, respectively Combination of TRAIL (100ng/mL). After the drug is incubated for 72 hours, add 30 ⁇ L MTT to each well and incubate for 4-6 hours. The microplate reader detects the OD value at a detection wavelength of 492nM (blank group: detection medium OD value (not plated)).
  • Figure 1B shows the MTT detection of ANA (10nM) and ZARD (20nM) and interferon IFN- ⁇ (3000IU/mL), IFN- ⁇ (10ng/mL), apoptosis-related factor TRAIL in A2058, Hela, QGY7701, SW1116 cells (100ng/mL) combination.
  • ANA 10nM
  • ZARD 20nM
  • interferon IFN- ⁇ 3000IU/mL
  • IFN- ⁇ 10ng/mL
  • apoptosis-related factor TRAIL apoptosis-related factor TRAIL
  • Figure 2A shows the combined use of RTCA to detect IFN- ⁇ (3000IU/mL) and ANA (100nM) in Bel7404, A498, and SW1116.
  • the tumor cells are plated at 5000/well, and when the cells grow to the logarithmic growth phase, the test compounds of the specified concentration are added respectively.
  • RTCA detects the growth of the cells in real time, and the cell growth curve is obtained after the experiment is over.
  • Figure 2B shows the combined use of RTCA to detect IFN- ⁇ (3000IU/mL) and ZARD (100nM) in Bel7404, A498, and SW1116.
  • Tumor cells are plated at 5000/well, and when the cells grow to the logarithmic growth phase, the test compounds of the specified concentration are added.
  • RTCA detects the growth of the cells in real time, and the cell growth curve is obtained after the experiment is over.
  • Figure 2C shows the combined use of RTCA to detect IFN- ⁇ (10ng/mL) and ANA (100nM) in Bel7404, A498, and SW1116.
  • the tumor cells are plated at 5000/well, and when the cells grow to the logarithmic growth phase, the test compounds of the specified concentration are added respectively.
  • RTCA detects the growth of the cells in real time, and the cell growth curve is obtained after the experiment is over.
  • Figure 2D shows the combined use of RTCA to detect IFN- ⁇ (10ng/mL) and ZARD (100nM) in Bel7404, A498, and SW1116.
  • the tumor cells are plated at 5000/well, and when the cells grow to the logarithmic growth phase, the test compounds of the specified concentration are added respectively.
  • RTCA detects the growth of the cells in real time, and the cell growth curve is obtained after the experiment is over.
  • Figure 2E shows the combined use of RTCA to detect TRAIL (100ng/mL) and ANA (100nM) in Bel7404, A498, and SW1116.
  • the tumor cells are plated at 5000/well, and when the cells grow to the logarithmic growth phase, the test compounds of the specified concentration are added respectively.
  • RTCA detects the growth of the cells in real time, and the cell growth curve is obtained after the experiment is over.
  • Figure 2F shows the combined use of RTCA to detect TRAIL (100ng/mL) and ZARD (100nM) in Bel7404, A498, and SW1116.
  • the tumor cells are plated at 5000/well, and when the cells grow to the logarithmic growth phase, the test compounds of the specified concentration are added respectively.
  • RTCA detects the growth of the cells in real time, and the cell growth curve is obtained after the experiment is over.
  • Figure 3A shows the combined use of TNF- ⁇ (10ng/mL) and ANA (100nM) in Bel7404 and SW1116 in Example 3.
  • the tumor cells are plated at 5000/well, and when the cells grow to the logarithmic growth phase, the test compounds of the specified concentration are added respectively.
  • RTCA detects the growth of the cells in real time, and the cell growth curve is obtained after the experiment is over.
  • Figure 3B shows the combined use of TNF- ⁇ (10ng/mL) and ZARD (100nM) in Example 3 in Bel7404 and SW1116.
  • the tumor cells are plated at 5000/well, and when the cells grow to the logarithmic growth phase, the test compounds of the specified concentration are added respectively.
  • RTCA detects the growth of the cells in real time, and the cell growth curve is obtained after the experiment is over.
  • PDE3 inhibitors such as ANA, or ZARD
  • cytokines such as TRAIL, IFN- ⁇ or IFN- ⁇
  • the synergistic therapeutic effect of PDE3 inhibitor and cytokine is significantly better than the single use of PDE3 inhibitor or cytokine.
  • the present invention has been completed.
  • the terms “including”, “including”, and “containing” are used interchangeably, and include not only closed definitions, but also semi-closed and open definitions. In other words, the term includes “consisting of” and “consisting essentially of”.
  • the term "pharmaceutically acceptable carrier” refers to components that are suitable for humans and/or animals without excessive side effects (such as toxicity, irritation, and allergic reactions), that is, those with a reasonable benefit/risk ratio substance.
  • the term "therapeutically effective amount” refers to an amount that produces function or activity on humans and/or animals and can be accepted by humans and/or animals. Those of ordinary skill in the art should understand that the “therapeutically effective amount” may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvants of the drug used, the severity of the disease, and the combination with other drugs. It's different.
  • the "prevention" and “treatment” in the present invention include delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal.
  • the composition or pharmaceutical composition of the present invention prevents, reduces, inhibits, and/or inhibits ischemia-reperfusion injury compared to the level observed in the absence of the composition or pharmaceutical composition of the present invention. Or reversal, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%.
  • the term "pharmaceutically acceptable salt” refers to a salt formed by a compound of the present invention and an acid or a base suitable for use as a medicine.
  • Pharmaceutically acceptable salts include inorganic salts and organic salts.
  • a preferred class of salts are the salts of the compounds of this invention with acids.
  • Acids suitable for salt formation include but are not limited to: toluenesulfonic acid, hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid and other inorganic acids, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, Fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid and other organic acids; and acidic amino acids such as aspartic acid and glutamic acid.
  • a preferred type of salt is the salt formed by the compound of the present invention and a base.
  • Suitable bases for salt formation include, but are not limited to, inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and sodium phosphate, ammonia, trihydrate, etc.
  • Organic bases such as ethylamine and diethylamine.
  • substituted or “substituted” means that the hydrogen atom on the group is replaced by a non-hydrogen atom group, but it needs to meet its valence requirements and be replaced to form a chemically stable compound, that is, it will not spontaneously undergo such as cyclization. , Elimination and other conversion compounds.
  • each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.
  • R1 the same meaning as “R 1” and “R 1” can be replaced with each other, the same as other similarly defined meanings.
  • C 1 -C 4 carboxyl group refers to a group of C 1 -C 3 alkyl-COOH structure, where the alkyl group can be linear or branched, such as CH 3 COOH, C 2 H 5 COOH, C 3 H 8 COOH, (CH 3 ) 2 CHCOOH, or similar groups.
  • alkyl refers to a linear (ie, unbranched) or branched saturated hydrocarbon group containing only carbon atoms, or a combination of linear and branched groups .
  • the alkyl group has a limited number of carbon atoms (such as a C 1 -C 10 alkyl group), it means that the alkyl group contains 1-10 carbon atoms.
  • a C 1 -C 6 alkyl group means that it contains 1 to 6 carbon atoms.
  • Representative examples of the alkyl group include but are not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or similar groups.
  • alkenyl refers to a straight or branched chain carbon chain group with at least one carbon-carbon double bond.
  • the alkenyl group has a limited number of carbon atoms (such as C 2 -C 10 ), it means that the alkenyl group contains 2-10 carbon atoms.
  • C 2 -C 6 alkenyl refers to an alkenyl group containing 2-6 carbon atoms, including vinyl, propenyl, 1,2-butenyl, 2,3-butenyl, butadienyl, or the like Group.
  • alkynyl refers to an aliphatic hydrocarbon group having at least one carbon-carbon triple bond.
  • the alkynyl group can be linear or branched, or a combination thereof.
  • the alkynyl group has a limited number of carbon atoms (such as C 2 -C 10 alkynyl), it means that the alkynyl group contains 2-10 carbon atoms.
  • C 2 -C 6 alkynyl refers to a straight-chain or branched alkynyl group having 2-6 carbon atoms, including ethynyl, propynyl, isopropynyl, butynyl, and isobutynyl. , Sec-butynyl, tert-butynyl, or similar groups.
  • cycloalkyl refers to a saturated or partially saturated unitary ring, bicyclic or polycyclic (fused, bridged, or spiro) ring system group .
  • a certain cycloalkyl group has a limited number of carbon atoms (such as C 3 -C 10 ), it means that the cycloalkyl group has 3-10 ring carbon atoms.
  • C 3 -C 10 cycloalkyl refers to a saturated or partially saturated monocyclic or bicyclic alkyl group with 3-10 ring carbon atoms, including cyclopropyl, cyclobutyl, Cyclopentyl, cycloheptyl, or similar groups.
  • aryl refers to an all-carbon monocyclic or fused polycyclic (that is, rings that share adjacent pairs of carbon atoms) with a conjugated ⁇ -electron system
  • the aryl group has a limit on the number of carbon atoms in front, such as C 6 -C 10 aryl, it means that the aryl group has 6-10 ring carbon atoms, such as phenyl and naphthyl.
  • the aryl ring can be fused to other cyclic groups (including saturated or unsaturated rings), but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of connection to the parent must be in a conjugated ⁇ -electron system On the carbon atom on the ring.
  • aryl groups including but not limited to:
  • heteroaryl refers to an aromatic heterocyclic ring system having one to more (preferably 1, 2, 3, or 4) heteroatoms, which may be Monocyclic (monocyclic) or polycyclic (bicyclic, tricyclic or polycyclic) fused together or covalently connected, the heteroatoms referred to herein include oxygen, sulfur and nitrogen.
  • the C5-C10 heteroaryl group refers to a heteroaryl group having 5-10 ring atoms.
  • examples of 5-membered heteroaryl include (but are not limited to): pyrrole, furan, thiophene, imidazole, oxazole, thiazole, and examples of 6-membered heteroaryl include (but are not limited to) pyridine, pyrazine, pyridazine, pyrimidine .
  • alkoxy refers to an RO- group, where R is an alkyl group, and alkyl is as defined herein above, when the alkoxy group has a limit on the number of carbon atoms before it
  • a C 1 -C 10 alkoxy group means that the alkyl group in the alkoxy group has 1-10 carbon atoms.
  • Representative examples of alkoxy include (but are not limited to): methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, or similar groups.
  • acyl refers to an R-CO- group, where R is an alkyl group, and the alkyl group is as defined herein, for example, "C 2 -C 10 acyl” refers to a C 1 -C 9 alkyl-CO- structure Groups, representative examples of acyl groups include (but are not limited to): CH 3 -CO-, C 2 H 5 -CO-, C 3 H 8 -CO-, or similar groups.
  • ester group refers to an R-CO-O- group or a -CO-OR group, where R is an alkyl group, and alkyl group is as defined herein above
  • C 2 -C 10 ester group refers to a group of C 1 -C 9 alkyl-CO-O- structure or a group of -CO-OC 1 -C 7 alkyl structure, representative of ester groups Examples include (but are not limited to): CH 3 COO-, C 2 H 5 COO-, C 3 H 8 COO-, (CH 3 ) 2 CHCOO-, -COOCH 3 , -COOC 2 H 5 , -COOC 3 H 8 , Or similar groups.
  • C1-C10 sulfonyl refers to -SO2-C1-C10 alkyl, and alkyl is as defined above.
  • C 3 -C 20 heterocycloalkyl refers to having 3-20 ring carbon atoms and 1-3 heteroatoms (preferably containing 1 nitrogen atom, that is, they are commonly adjacent to R 1 and R 2 The nitrogen atom) of monocyclic and polycyclic heterocycles (preferably monocyclic heterocycles), such as piperidine group, tetrahydropyrrolyl group, or similar groups.
  • aldehyde group refers to -CHO.
  • halogen refers to F, Cl, Br, and I.
  • halo refers to substitution by halogen.
  • the first active ingredient is a PDE3 inhibitor.
  • PDE3 belongs to the PDE (Phosphodiesterase) family, with two subtypes-PDE3A and PDE3B, located on chromosomes 12p12 and 11p15, respectively. PDE3 can hydrolyze both cAMP and cGMP, the second messenger of cells, and terminate the conduction of downstream signal pathways.
  • the PDE3 inhibitor is selected from the following group: the compound of formula I or a pharmaceutically acceptable salt thereof, the compound of formula II or a pharmaceutically acceptable salt thereof, or a combination thereof .
  • the PDE3 inhibitor is a compound of Formula I or a pharmaceutically acceptable salt thereof, and the compound of Formula I or a pharmaceutically acceptable salt thereof is as follows:
  • R1, R2, R3, R4, R5, R6, R7 and R8 are as defined above.
  • the PDE3 inhibitor is a compound of Formula II or a pharmaceutically acceptable salt thereof, and the compound of Formula II or a pharmaceutically acceptable salt thereof is as follows:
  • R11, R12, R13, R14, R15, R17 and R18 are as defined above.
  • the PDE3 inhibitor includes (but is not limited to): Anagrelide or a pharmaceutically acceptable salt thereof, Zardaverine or a pharmaceutically acceptable salt thereof , Or a combination thereof.
  • Anagrelide is referred to as ANA, and the structural formula is as follows:
  • Zardaverine (Zardaverine) is abbreviated as ZARD, and the structural formula is as follows:
  • the PDE3 inhibitor is Anagrelide or a pharmaceutically acceptable salt thereof.
  • the PDE3 inhibitor is Zardaverine or a pharmaceutically acceptable salt thereof.
  • the second active ingredient is a cytokine.
  • the cytokines include wild-type and mutant cytokines.
  • the cytokines include cytokines derived from human or non-human mammals.
  • the cytokine can be isolated, recombinantly produced, modified (such as PEG modification) or unmodified.
  • a preferred type of cytokine is a cytokine that has been modified to increase its half-life, such as a PEGylated cytokine, such as PEGylated TRAIL, PEGylated IFN.
  • representative cytokines include (but are not limited to): interferon, tumor necrosis factor, or a combination thereof.
  • the interferon includes (but is not limited to): IFN- ⁇ , IFN- ⁇ , or a combination thereof.
  • the tumor necrosis factor includes (but is not limited to): TRAIL, TNF- ⁇ , or a combination thereof.
  • TRAIL becomes a tumor necrosis factor-related apoptosis-inducing ligand (TNF-related apoptosis-inducing ligand).
  • TNF tumor necrosis factor
  • TRAIL is a member of the tumor necrosis factor (TNF) superfamily and can specifically bind to death receptors. Selectively induce apoptosis of cancer cells.
  • TNF tumor necrosis factor
  • TRAIL can be highly expressed in cancer cells and plays an important role in the occurrence, development and treatment of cancer. It is considered to be an anti-tumor cytokine with great development potential. .
  • IFN- ⁇ belongs to type I interferon, which can increase the expression of viral antigen polypeptides by up-regulating the expression of MHC I protein, enhance the activation of CD8+ T cells, and exert antiviral effects.
  • IFN- ⁇ can play an anti-tumor effect by changing the state of cell differentiation, interfering with the expression of oncogenes, changing the expression of cell surface antigens, increasing the phagocytic activity of macrophages, and enhancing the cytotoxicity of lymphocytes to target cells.
  • IFN- ⁇ is used clinically to treat hairy cell leukemia, malignant melanoma and AIDS-related Kaposi's sarcoma.
  • IFN- ⁇ belongs to type II interferon. IFN- ⁇ can stimulate the transcription and expression of immunoglobulin in B cells, up-regulate the antigen presentation pathway, activate the activity of macrophages, and enhance the antiviral and antiviral properties of type I interferon. Tumor effect. IFN- ⁇ is used clinically to treat chronic granulomatous disease and osteonecrosis.
  • TNF- ⁇ Tumor necrosis factor- ⁇
  • TNF- ⁇ Tumor necrosis factor- ⁇
  • TNF- ⁇ can induce apoptotic cell death, induce inflammation, and inhibit tumorigenesis and virus replication.
  • the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising:
  • the content of the first active ingredient ranges from 0.01% to 99.99%, based on the total weight of the active ingredients of the composition; preferably 0.1% to 99.9%; more preferably 1% to 99% ; Preferably 10% to 99%; more preferably 20% to 99%; more preferably 30-99%, more preferably 40-99%.
  • the content of the second active ingredient ranges from 0.01% to 99.99%, based on the total weight of the active ingredients of the composition; preferably 0.1% to 99.9%; more preferably 1% to 99%; It is preferably 1% to 90%; more preferably 1% to 80%; more preferably 1-70%, more preferably 1-60%.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition includes:
  • the pharmaceutical composition containing the first active ingredient and the second active ingredient of the present invention can be in various dosage forms suitable for oral administration, as well as various formulations for external administration or other parenteral administration formulations.
  • the external administration formulation of the present invention can also be added with auxiliary materials such as surfactants, transdermal absorption promoters, preservatives, solvents, antioxidants, moisturizers, pH adjusters, colorants, perfumes, etc.
  • Prepared into including but not limited to): liniment, tincture, oil, ointment, plaster, paste, iron, patch, patch, film, film, gel, cataplasm, Acupoint patch, spray, aerosol, implant, emulsion, etc.
  • Preferred dosage forms include: various dosage forms for oral administration, implants, and preparations for injection (such as injections, infusions or freeze-dried powder injections).
  • the carrier is not particularly limited, and is a commonly used material in the field, and its type, use method, and source are well known to those skilled in the art.
  • Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, etc.), gelatin, talc, and solid lubricants. (Such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), buffering agents, chelating agents, thickening agents, pH adjusters, penetration enhancers, coloring agents, flavoring agents, stabilizers, antioxidants, preservatives , Bacteriostatic agent, pyrogen-free water, etc.
  • cellulose and its derivatives such as methyl cellulose, ethyl cellulose, hydroxyprop
  • the present invention also provides a medicine box, which comprises:
  • kit further includes instructions.
  • the instructions for use indicate that the first preparation and the second preparation are combined to prevent and/or treat cancer.
  • the first preparation and the second preparation are administered simultaneously, separately or sequentially in the prevention and/or treatment of tumors.
  • first preparation and the second preparation in the kit are independent of each other, or may be combined.
  • first preparation and the second preparation in the kit are independent of each other.
  • the first preparation is a solid preparation (such as a solid preparation for the gastrointestinal tract) or an injection preparation (such as an injection, an infusion or a lyophilized powder injection).
  • the second preparation is an injection preparation (such as an injection, an infusion solution or a lyophilized powder injection).
  • the first formulation and the second formulation are independent.
  • the kit of the present invention includes: a solid preparation (such as a solid preparation for the gastrointestinal tract) and a preparation for injection (such as an injection, an infusion or a lyophilized powder injection), wherein the solid preparation (such as a gastrointestinal
  • the solid preparation for road use contains a PDE3 inhibitor, and the preparation for injection (such as injection, infusion or lyophilized powder injection) contains cytokines.
  • the present invention also provides an active ingredient combination, which includes the following components:
  • the first active ingredient and the second active ingredient may be independent of each other, or they may be combined together to form an active ingredient composition.
  • at least one active ingredient is independent.
  • the pharmaceutical composition, active ingredient combination, and medicine box of the present invention can all be prepared by using conventional methods and equipment.
  • the present invention provides a use of the pharmaceutical composition, kit or active ingredient combination described herein in the preparation of drugs for the prevention and or treatment of tumors.
  • the tumor includes (but is not limited to): cervical cancer, liver cancer, kidney cancer, colon cancer, glioma, melanoma, or a combination thereof.
  • the first active ingredient and the second active ingredient in the pharmaceutical composition, kit or active ingredient combination of the present invention can have a synergistic effect on the prevention and treatment of tumors, and enhance the effect of treating tumors.
  • kits or active ingredient combination of the present invention Before, at the same time or after using the pharmaceutical composition, kit or active ingredient combination of the present invention, other anti-tumor drugs can be used in combination.
  • the interaction of drugs is divided into additive, synergistic, and antagonistic according to the effects of the drugs when they are used together.
  • Synergistic action means that the effects of the combined drugs when used together are significantly stronger than when they are used alone.
  • Harmonic effect means that the effect of the combined drugs when used together is equivalent to that when used alone, and antagonism means that the effect of the combined drugs when used together is smaller than when used alone.
  • the administration mode of the present invention includes sequential administration of the first active ingredient and the second active ingredient, or simultaneous administration of the first active ingredient and the second active ingredient.
  • the pharmaceutical preparation should match the mode of administration.
  • a safe and effective amount of the drug is administered to the desired object (such as a human or non-human mammal), wherein the safe and effective date of the first active ingredient
  • the dosage used is usually at least about 0.1 mg, and in most cases does not exceed about 2500 mg.
  • the dosage is 1 mg-500 mg; the safe and effective amount of the second active ingredient is usually at least about 0.01 mg, and in most cases does not exceed 2500 mg.
  • the dosage range is 0.1 mg to 2500 mg, and the safe and effective amount of the third active ingredient is usually at least about 0.01 mg, and in most cases does not exceed 2500 mg.
  • the dosage range is 0.1 mg to 2500 mg.
  • the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.
  • the first active ingredient, the second active ingredient and the third active ingredient are applied sequentially, there is no special requirement for the interval of administration.
  • the composition, the active ingredient combination, the pharmaceutical composition and the first active ingredient, the second active ingredient and the third active ingredient in the kit of the present invention are administered simultaneously or sequentially by the same or different routes, including but not Limited to: oral administration, injection administration, intratumoral administration, implantation administration, intracavity administration, anal administration, transdermal administration, internal and external application;
  • Preferred injection administration includes: intravenous injection, intramuscular injection, subcutaneous injection, and intracavity injection.
  • the present invention also provides a method for preventing and/or treating tumors by administering the pharmaceutical composition, kit or active ingredient combination of the present invention to a desired subject.
  • the subjects are humans and non-human mammals.
  • non-human mammals include (but are not limited to): pets (such as dogs, cats), domestic animals (such as cows, sheep, horses, pigs), various zoo animals (pandas, elephants, tigers) )Wait.
  • PDE3 inhibitors such as ANA, or ZARD
  • cytokines such as TRAIL, TNF- ⁇ , IFN- ⁇ or IFN- ⁇
  • the microplate reader detects the OD value at the detection wavelength of 492nM (PS blank group: detection medium OD value (not plated cells) control group: the same volume of DMSO as the drug is added to the medium (cell plated)) to calculate the cell survival rate
  • the cells are adhered to the wall overnight, and when the growth curve enters the logarithmic growth phase. Take out the E-plate from the detection platform and add 50ul of the test compound to each well to the specified concentration (the final concentration of each group is shown in Table 3), and then put it back into the detection platform to detect cell growth in real time after the treatment.
  • the experimental method is basically the same as that in Example 2.
  • TNF- ⁇ (10ng/mL), ANA (100nM) and TNF- ⁇ (10ng/mL)+ANA (100nM) were used to treat Bel7404 and SW1116 cell lines;
  • TNF- ⁇ (10ng/ mL), ZARD(100nM) and TNF- ⁇ (10ng/mL)+ZARD(100nM) were treated with Bel7404 and SW1116 cell lines.

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Abstract

一种PDE3抑制剂与细胞因子联合治疗肿瘤的应用。一种药物组合物，所述组合物包括：（a）治疗有效量的第一活性成分，所述第一活性成分为PDE3抑制剂；和（b）治疗有效量的第二活性成分，所述第二活性成分为细胞因子。PDE3抑制剂与细胞因子联合应用具有明显的协同抗癌作用。

Description

PDE3抑制剂与细胞因子联合治疗肿瘤的应用 技术领域
本发明涉及药物领域，具体地，本发明涉及PDE3抑制剂与细胞因子联合治疗肿瘤的应用。
背景技术
肿瘤成为人类健康的头号杀手，根据美国癌症学会《2018全球癌症统计数据》报告，2018年全球大约有1810万癌症新生病例和960万癌症死亡病例。肿瘤治疗一直以来都是人类亟需攻克的难题，传统的肿瘤治疗包括手术、化疗、放疗三种方式，这三种治疗方式取得了很大的成就也具有很大的局限性。传统化疗药物主要是细胞毒药物，例如DNA的烷化剂(环磷酰胺、顺铂、卡铂)、微管蛋白抑制剂(如紫杉醇)、DNA合成前体的代谢拮抗剂(甲氨蝶呤)、拓扑异构酶II抑制剂(如依托泊苷)等等，这些药物在靶向快速增殖分裂的肿瘤细胞的同时也抑制正常细胞生长，因而产生了很多严重副反应例如心脏毒性、呕吐、脱发、免疫抑制等等。因此，寻求特异性靶向肿瘤细胞的治疗方式成为肿瘤治疗的研发热点，靶向治疗成为新一代肿瘤治疗方式。靶向治疗区别传统化疗方式是在分子水平上针对明确癌变的位点设计相应的治疗药物，使肿瘤细胞发生特异性的死亡而并不影响周围正常组织。
因此，本领域需要开发一种提供肿瘤治疗效果的药物。
发明内容
本发明的目的是提供具有明显协同抗肿瘤效果的PDE3抑制剂与细胞因子联用的药物组合物。
本发明的第一方面，提供了一种药物组合物，所述组合物包括：
(a)治疗有效量的第一活性成分，所述第一活性成分为PDE3抑制剂；和
(b)治疗有效量的第二活性成分，所述第二活性成分为细胞因子。
在另一优选例中，所述的第一活性成分和第二活性成分的重量比为1：(0.1-50)，较佳地，1：(0.1-20)，更佳地，1：(0.15-10)，最佳地，1：(0.2-5)。
在另一优选例中，所述的PDE3抑制剂选自下组：式I所述的化合物或其药学上可接受的盐、式II所述的化合物或其药学上可接受的盐，或其组合；
Figure PCTCN2020110552-appb-000001
其中，
R1、R2、R3、R4、R5、R6、R7和R8各自独立地选自下组：氢原子、卤素原子、氨基、羟基、氰基、醛基、硝基、取代或未取代的C1-C4羧基、取代或未取代的C1-C10烷基、取代或未取代的C3-C10环烷基、取代或未取代的C2-C10烯基、取代或未取代的C2-C10炔基、取代或未取代的C6-C10芳基、取代或未取代的C5-C10杂芳基(如取代或未取代的5元或6元杂环、8元至10元杂芳基)、取代或未取代的C1-C10烷氧基、取代或未取代的C6-C10芳基-氧基、取代或未取代的C5-C10杂芳基-氧基、取代或未取代的C 2-C 10酰基(、取代或未取代的C 2-C 10酯基、取代或未取代的C1-C10磺酰基(-SO2-C1-C10烷基)；
或R1和R2、R3和R4共同构成选自下组的基团：取代或未取代的C3-C20环烷基(优选为C3-C10环烷基)、取代或未取代的C3-C20杂环烷基(优选为取代或未取代的5元或6元杂环、8元至12元杂芳二环环系)、羰基(＝O)；
R9选自下组：氢原子、氧原子、取代或未取代的C1-C10烷基、取代或未取代的C3-C10环烷基、取代或未取代的C6-C10芳基、取代或未取代的C1-C10杂芳基、取代或未取代的C1-C10烷氧基、取代或未取代的C6-C10芳基-氧基、取代或未取代的酰基(优选为-CO-C1-C10烷基)、取代或未取代的C1-C10磺酰基；
其中，所述的任一“取代”是指基团上的一个到四个(优选为1、2、3或4个)氢原子被选自下组的取代基所取代：C1-C10烷基、C3-C10环烷基、C1-C10烷氧基、卤素、羟基、羧基(-COOH)、C1-C10醛基、C2-C10酰基、C2-C10酯基、氨基、苯基、氰基、-OH、硝基、氨基；
Figure PCTCN2020110552-appb-000002
其中，
R11、R12、R13、R14、R15、R17和R18各自独立地选自下组：氢原子、卤素原子、氨基、羟基、氰基、醛基、硝基、取代或未取代的C1-C4羧基、取代或未取代的C1-C10烷基、取代或未取代的C3-C10环烷基、取代或未取代的C2-C10烯基、取代或未取代的C2-C10炔基、取代或未取代的C6-C10芳基、取代或未取代的C5-C10杂芳基(如取代或未取代的5元或6元杂环、8元至10元杂芳二环环系)、取代或未取代的C1-C10烷氧基、取代或未取代的C6-C10芳基-氧基、取代或未取代的C5-C10杂芳基-氧基、取代或未取代的C2-C10酰基、取代或未取代的C2-C10酯基、取代或未取代的C1-C10磺酰基(-SO2-C1-C10烷基)；
R16选自下组：氢原子、氧原子、取代或未取代的C1-C10烷基、取代或未取代的C3-C10环烷基、取代或未取代的C6-C10芳基、取代或未取代的C5-C10杂芳基、取代或未取代的C1-C10烷氧基、取代或未取代的C6-C10芳基-氧基、取代或未取代的C1-C10酰基、取代或未取代的C1-C10磺酰基；
Figure PCTCN2020110552-appb-000003
表示单键或不存在；
其中，所述的任一“取代”是指基团上的一个到四个(优选为1、2、3或4个)氢原子被选自下组的取代基所取代：C1-C10烷基、C3-C10环烷基、C1-C10烷氧基、卤素、羟基、羧基(-COOH)、C1-C10醛基、C2-C10酰基、C2-C10酯基、氨基、苯基、氰基、OH、硝基和氨基。
在另一优选例中，R1为氢。
在另一优选例中，R2为氢。
在另一优选例中，R3为氢。
在另一优选例中，R4为氢。
在另一优选例中，R5为卤素。
在另一优选例中，R6为卤素。
在另一优选例中，R7为氢。
在另一优选例中，R8为氢。
在另一优选例中，R9为氢。
在另一优选例中，R11为氢。
在另一优选例中，所述的卤素为Cl。
在另一优选例中，R11为氢。
在另一优选例中，R12为氢或C1-C6烷氧基。
在另一优选例中，R12为氢或甲氧基。
在另一优选例中，R13为卤代C1-C6烷氧基。
在另一优选例中，R13为二卤代C1-C6烷氧基。
在另一优选例中，R13为卤代甲氧基。
在另一优选例中，R13为二卤代甲氧基。
在另一优选例中，R13为二氯甲氧基。
在另一优选例中，R14为氢或C1-C6烷氧基。
在另一优选例中，R14为氢或甲氧基
在另一优选例中，R15为氢。
在另一优选例中，R16为氢。
在另一优选例中，R17为氢。
在另一优选例中，R18为氢。
在另一优选例中，所述的苯基包括未取代的苯基或具有1-3个取代基的取代苯基。
在另一优选例中，所述的PDE3抑制剂选自下组：阿那格雷(Anagrelide)或其药学上可接受的盐、扎达维林(Zardaverine)或其药学上可接受的盐，或其组合。
在另一优选例中，所述的细胞因子选自下组：抗病毒的细胞因子、肿瘤坏死因子、干扰素、抗DNA复制的细胞因子，或其组合。
在另一优选例中，所述的细胞因子选自下组：干扰素、肿瘤坏死因子，或其组合。
在另一优选例中，所述的干扰素选自下组：IFN-α、IFN-γ，或其组合。
在另一优选例中，所述的肿瘤坏死因子选自下组：肿瘤坏死因子相关诱导凋亡配体(TRAIL)、TNF-α，或其组合。
在另一优选例中，所述的药物组合物还包括药学上可接受的载体。
本发明第二方面提供了一种药盒，所述药盒包括：
(A)含有PDE3抑制剂的第一制剂；和
(B)含有细胞因子的第二制剂。
在另一优选例中，所述的药盒还包括说明书。
在另一优选例中，所述的使用说明书中注明将所述第一制剂和第二制剂进行联用，从而预防和/或治疗癌症。
在另一优选例中，所述的第一制剂和第二制剂在预防和/或治疗肿瘤中同时给药、分别给药或顺序给药。
在本发明所述的药盒中，应当理解的是，所述的药盒中第一制剂和第二制剂是相互独立的，也可以是是合并的。
在另一优选例中，所述的药盒中第一制剂和第二制剂是相互独立的。
在另一优选例中，所述的第一制剂为固体制剂(如胃肠道用固体制剂)或注射用制剂(如注射剂、输液剂或冻干粉针剂)。
在另一优选例中，所述的第二制剂为注射用制剂(如注射剂、输液剂或冻干粉针剂)。
例如，第一制剂和第二制剂为是独立的。典型地，本发明药盒包括：一固体制剂(如胃肠道用固体制剂)和一注射用制剂(如注射剂、输液剂或冻干粉针剂)，其中，所述的固体制剂(如胃肠道用固体制剂)含有PDE3抑制剂，所述的注射用制剂(如注射剂、输液剂或冻干粉针剂)含有细胞因子。
本发明第三方面，提供了一种活性成分组合，所述的活性成分组合包括以下组分：
(1)治疗有效量的第一活性成分，所述第一活性成分为PDE3抑制剂；和
(2)治疗有效量的第二活性成分，所述第二活性成分为细胞因子。
本发明第四方面，提供了如本发明第一方面所述的药物组合物、本发明第二方面所述的药盒、和/或如本发明第三方面所述的活性成分组合的用途，用于制备预防和或治疗肿瘤的药物。
在另一优选例中，所述的肿瘤选自下组：宫颈癌、肝癌、肾癌、结肠癌、脑胶质瘤、黑色素瘤，或其组合。
本发明第五方面，提供了一种预防和/或治疗肿瘤的方法，包括步骤：给所需的对象施用如权利要求1所述的组合物。
在另一优选例中，所述对象为人和非人哺乳动物。
代表性地，所述的非人哺乳动物包括(但并不限于)：宠物(如狗、猫)、家畜(如牛、羊、马、猪)、各种动物园动物(熊猫、大象、虎)等。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1A MTT检测在SMMC7721、Bel7404、A498、FHCC98细胞中分别设置ANA(10nM)与ZARD(20nM)与干扰素IFN-α(3000IU/mL)、IFN-γ(10ng/mL)、凋亡相关因子TRAIL(100ng/mL)的联用。药物孵育72h后，每孔加入30μL MTT孵育4-6h,酶标仪在检测波长492nM检测OD值(空白组：检测培养基OD值(并不铺细胞)对照组：培养基中加入与药物同体积的DMSO(铺细胞)计算细胞存活率＝((实验组OD-空白组OD))/((对照组OD-空白组OD))x100％)。
图1B为MTT检测在A2058、Hela、QGY7701、SW1116细胞中ANA(10nM)与ZARD(20nM)与干扰素IFN-α(3000IU/mL)、IFN-γ(10ng/mL)、凋亡相关因子TRAIL(100ng/mL)的联用。药物孵育72h后，每孔加入30μL MTT孵育4-6h,酶标仪在检测波长492nM检测OD值(空白组：检测培养基OD值(并不铺细胞)对照组：培养基中加入与药物同体积的DMSO(铺细胞)计算细胞存活率＝((实验组OD-空白组OD))/((对照组OD-空白组OD))x100％)
图2A为RTCA检测IFN-α(3000IU/mL)与ANA(100nM)在Bel7404、A498、SW1116中联用。肿瘤细胞5000/孔铺板，待细胞生长到对数生长期时分别加入指定浓度的待测化合物，RTCA实时检测细胞的生长，实验结束后得到细胞生长曲线。
图2B为RTCA检测IFN-α(3000IU/mL)与ZARD(100nM)在Bel7404、A498、SW1116中联用。肿瘤细胞5000/孔铺板，待细胞生长到对数生长期时分别加入 指定浓度的待测化合物，RTCA实时检测细胞的生长，实验结束后得到细胞生长曲线。
图2C为RTCA检测IFN-γ(10ng/mL)与ANA(100nM)在Bel7404、A498、SW1116中联用。肿瘤细胞5000/孔铺板，待细胞生长到对数生长期时分别加入指定浓度的待测化合物，RTCA实时检测细胞的生长，实验结束后得到细胞生长曲线。
图2D为RTCA检测IFN-γ(10ng/mL)与ZARD(100nM)在Bel7404、A498、SW1116中联用。肿瘤细胞5000/孔铺板，待细胞生长到对数生长期时分别加入指定浓度的待测化合物，RTCA实时检测细胞的生长，实验结束后得到细胞生长曲线。
图2E为RTCA检测TRAIL(100ng/mL)与ANA(100nM)在Bel7404、A498、SW1116中联用。肿瘤细胞5000/孔铺板，待细胞生长到对数生长期时分别加入指定浓度的待测化合物，RTCA实时检测细胞的生长，实验结束后得到细胞生长曲线。
图2F为RTCA检测TRAIL(100ng/mL)与ZARD(100nM)在Bel7404、A498、SW1116中联用。肿瘤细胞5000/孔铺板，待细胞生长到对数生长期时分别加入指定浓度的待测化合物，RTCA实时检测细胞的生长，实验结束后得到细胞生长曲线。
图3A为实施例3中TNF-α(10ng/mL)与ANA(100nM)在Bel7404、SW1116中联用。肿瘤细胞5000/孔铺板，待细胞生长到对数生长期时分别加入指定浓度的待测化合物，RTCA实时检测细胞的生长，实验结束后得到细胞生长曲线。
图3B为实施例3中TNF-α(10ng/mL)与ZARD(100nM)在Bel7404、SW1116中联用。肿瘤细胞5000/孔铺板，待细胞生长到对数生长期时分别加入指定浓度的待测化合物，RTCA实时检测细胞的生长，实验结束后得到细胞生长曲线。
具体实施方式
本发明人经过广泛而深入地研究，意外地发现了，PDE3抑制剂(如ANA、或ZARD)和细胞因子(如TRAIL、IFN-α或IFN-γ)联合应用能够明显提高对肿 瘤的治疗效果，PDE3抑制剂和细胞因子的协同治疗效果明显优于PDE3抑制剂或细胞因子的单独使用。在此基础上，完成了本发明。
术语
除非另有定义,否则本文中所用的所有技术和科学术语的含义与本发明所属领域普通技术人员普遍理解的含义相同。
如本文所用，术语“包含”、“包括”、“含有”可互换使用，不仅包括封闭式定义，还包括半封闭、和开放式的定义。换言之，所述术语包括了“由……构成”、“基本上由……构成”。
如本文所用，术语“药学上可接受的载体”的成分是指适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)的，即有合理的效益/风险比的物质。
如本文所用，术语“治疗有效量”，是指对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。本领域的普通技术人员应该理解，所述的“治疗有效量”可随着药物组合物的形式、给药途径、所用药物的辅料、疾病的严重程度以及与其他药物联合用药等情况的不同而有所不同。
本发明所述的“预防”和“治疗”包括延缓和终止疾病的进展，或消除疾病，并不需要100％抑制、消灭和逆转。在一些实施方案中，与不存在本发明所述组合物或药物组合物时观察到的水平相比，本发明所述组合物或药物组合物将缺血再灌注损伤预防，减轻、抑制和/或逆转了例如至少约10％、至少约30％、至少约50％、或至少约80％。
在本发明中，术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：甲苯磺酸、盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。一类优选的盐是本发明化合物与碱形成的盐，适合形成盐的碱包括但并不限于：氢氧化钠、氢氧化钾、碳酸钠、碳酸氢钠、磷酸钠等无机碱，氨水、三乙胺、二乙胺等有机碱。
应当理解，本领域的普通技术人员可以选择本发明的式I或式II化合物上的 取代基和取代型式以产生化学上稳定的化合物，所述化合物可以通过本领域己知的技术以及下文所阐述的方法合成。如果被超过一个取代基团取代，应当理解，这多个基团可以是在同一个碳上或在不同碳上，只要产生稳定的结构即可。
如本文所用，术语“取代”或“取代的”是基团上的氢原子被非氢原子基团，但需要满足其化合价要求并且由取代生成化学稳定的化合物，即不会自发进行诸如环化、消除等转变的化合物。
除特别说明之处，本发明的所有化合物之中，各手性碳原子(手性中心)可以任选地为R构型或S构型，或R构型和S构型的混合物。
如本文所用，“R1”、“R 1”和“R 1”的含义相同，可相互替换，其它类似定义的含义相同。
术语“C 1-C 4羧基”是指C 1-C 3烷基-COOH结构的基团，其中烷基可以为直链或支链的，例如CH 3COOH、C 2H 5COOH、C 3H 8COOH、(CH 3) 2CHCOOH，或类似基团。
如本文所用，在单独或作为其他取代基一部分时，术语“烷基”指只含碳原子的直链(即，无支链)或支链饱和烃基，或直链和支链组合的基团。当烷基前具有碳原子数限定(如C 1-C 10烷基)指所述的烷基含有1-10个碳原子，例如，C 1-C 6烷基指含有1-6个碳原子的烷基，代表性实例包括但不限于甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、或类似基团。
如本文所用，在单独或作为其他取代基一部分时，术语“烯基”是指直链或支链，具有至少一个碳-碳双键的碳链基团。当烯基前具有碳原子数限定(如C 2-C 10)时，指所述的烯基含有2-10个碳原子。例如，C 2-C 6烯基指含有2-6个碳原子烯基，包括乙烯基、丙烯基、1,2-丁烯基、2,3-丁烯基、丁二烯基、或类似基团。
如本文所用，在单独或作为其他取代基一部分时，术语“炔基”是指具有至少一个碳-碳三键的脂肪族碳氢基团。所述的炔基可以是直链或支链的，或其组合。当炔基前具有碳原子数限定(如C 2-C 10炔基)时，指所述的炔基含有2-10个碳原子。例如，术语“C 2-C 6炔基”指具有2-6个碳原子的直链或支链炔基，包括乙炔基、丙炔基、异丙炔基、丁炔基、异丁炔基、仲丁炔基、叔丁炔基、或类似基团。
如本文所用，在单独或作为其他取代基一部分时，术语“环烷基”指具有饱和的或部分饱和的单元环，二环或多环(稠环、桥环或螺环)环系基团。当某个环烷基前具有碳原子数限定(如C 3-C 10)时，指所述的环烷基具有3-10个环碳原子。在一些优选实施例中，术语“C 3-C 10环烷基”指具有3-10个环碳原子的饱和或部分饱和的单环或二环烷基，包括环丙基、环丁基、环戊基、环庚基、或类似基团。
如本文所用，在单独或作为其他取代基一部分时，术语“芳基”指具有共轭的π电子体系的全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，当芳基前面具有碳原子数限定，如C 6-C 10芳基，则指所述的芳基具有6-10个环碳原子，例如苯基和萘基。所述芳基环可以稠合于其它环状基团(包括饱和或不饱和环)，但不能含有杂原子如氮、氧、或硫，同时连接母体的点必须在具有共轭的π电子体系的环上的碳原子上。如下是芳基代表性实例，包括但不限于：
Figure PCTCN2020110552-appb-000004
如本文所用，在单独或作为其他取代基一部分时，术语“杂芳基”指具有一个到多个(优选为1、2、3或4个)杂原子的芳族杂环系，其可以是单环(单环的)或者稠合在一起或共价地连接的多环(二环的、三环的或多环的)，这里所指的杂原子包括氧、硫和氮。当杂芳基前被限定时，C5-C10杂芳基指的是具有5-10个环原子的杂芳基。例如5元杂芳基的实例包括(但不限于)：吡咯、呋喃、噻吩、咪唑、恶唑、噻唑，6元杂芳基的实例包括(但不限于)吡啶、吡嗪、哒嗪、嘧啶。
如本文所用，在单独或作为其他取代基一部分时，术语“烷氧基”指R-O-基团，其中R为烷基，烷基为如上本文所定义，当烷氧基前具有碳原子数限定，如C 1-C 10烷氧基基指所述的烷氧基中的烷基具有1-10个碳原子。烷氧基的代表性示例包括(但不限于)：甲氧基、乙氧基、正丙氧基、异丙氧基、叔丁氧基，或类似基团。
术语“酰基”是指R-CO-基团，其中R为烷基，烷基为如上本文所定义，例如“C 2-C 10酰基”指具有C 1-C 9烷基-CO-结构的基团，酰基的代表性示例包括(但不限于)：CH 3-CO-、C 2H 5-CO-、C 3H 8-CO-，或类似基团。
如本文所用，在单独或作为其他取代基一部分时，术语“酯基”指具R-CO-O-基团或-CO-O-R基团，其中R为烷基，烷基为如上本文所定义，例如“C 2-C 10酯基”是指C 1-C 9烷基-CO-O-结构的基团或者-CO-O-C 1-C 7烷基结构的基团，酯基的代表性示例包括(但不限于)：CH 3COO-、C 2H 5COO-、C 3H 8COO-、(CH 3) 2CHCOO-、-COOCH 3、-COOC 2H 5、-COOC 3H 8，或类似基团。
如本文所用，在单独或作为其他取代基一部分时，术语“C1-C10磺酰基”指-SO2-C1-C10烷基，烷基如上所定义。
如本文所用，术语“C 3-C 20杂环烷基”指具有3-20个环碳原子和1-3个杂原子(优选含有1个氮原子，即与R 1和R 2共同相邻的氮原子)的单环和多环杂环(优选单环杂环)，例如哌啶基团、四氢吡咯基，或相似基团。
如本文所用，术语“醛基”指-CHO。
如本文所用，术语“卤素”指F、Cl、Br和I。
如本文所用，术语“卤代”指被卤素所取代。
第一活性成分
本发明中，第一活性成分为PDE3抑制剂。
PDE3属于PDE(Phosphodiesterase)家族，共有两个亚型-PDE3A和PDE3B，分别位于染色体12p12 and 11p15。PDE3能同时水解细胞第二信使cAMP和cGMP，终止下游信号通路的传导。
在一个优选例中，所述的PDE3抑制剂选自下组：式I所述的化合物或其药学上可接受的盐、式II所述的化合物或其药学上可接受的盐，或其组合。
优选地，所述的PDE3抑制剂为式I所述的化合物或其药学上可接受的盐，所述的式I所述的化合物或其药学上可接受的盐如下所示：
Figure PCTCN2020110552-appb-000005
R1、R2、R3、R4、R5、R6、R7和R8如上定义。
优选地，所述的PDE3抑制剂为式II所述的化合物或其药学上可接受的盐，所述的式II所述的化合物或其药学上可接受的盐如下所示：
Figure PCTCN2020110552-appb-000006
R11、R12、R13、R14、R15、R17和R18如上定义。
在另一优选例中，所述的PDE3抑制剂包括(但不限于)：阿那格雷(Anagrelide) 或其药学上可接受的盐、扎达维林(Zardaverine)或其药学上可接受的盐，或其组合。
在本发明中，阿那格雷(Anagrelide)简称ANA，结构式如下所示：
Figure PCTCN2020110552-appb-000007
在本发明中，扎达维林(Zardaverine)简称ZARD，结构式如下所示：
Figure PCTCN2020110552-appb-000008
优选地，所述的PDE3抑制剂为阿那格雷(Anagrelide)或其药学上可接受的盐。
优选地，所述的PDE3抑制剂为扎达维林(Zardaverine)或其药学上可接受的盐。
第二活性成分
本发明中，第二活性成分为细胞因子。
优选地，所述的细胞因子包括野生型和突变型的细胞因子。所述的细胞因子包括人或非人哺乳动物来源的细胞因子。所述的细胞因子可以是分离的，也可以是重组产生的，也可以是修饰(如PEG修饰)或未修饰的。
一类优选的细胞因子是经修饰的从而半衰期延长的细胞因子，如PEG化的细胞因子，例如PEG化的TRAIL、PEG化的IFN。
在本发明中，代表性的细胞因子包括(但不限于)：干扰素、肿瘤坏死因子，或其组合。
优选地，所述的干扰素包括(但不限于)：IFN-α、IFN-γ，或其组合。
优选地，所述的肿瘤坏死因子包括(但不限于)：TRAIL、TNF-α，或其组合。
在本发明中，TRAIL成为肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand)，TRAIL是肿瘤坏死因子(TNF)超家族中的一员,能与死亡受体特异性结合后选择性诱导癌细胞发生凋亡。TRAIL作为靶向癌细胞的选择性杀伤配体,可在癌细胞中高度表达,在癌症的发生、发展和治疗方面扮演着重要的角色,被认为是一种非常具有开发潜力的抗肿瘤细胞因子。
在本发明中，IFN-α属于I型干扰素，可以通过上调MHC I蛋白的表达，从而增加病毒抗原多肽的表达,增强了CD8+T细胞的活化，发挥抗病毒作用。IFN-α可以通过改变细胞分化状态、干扰癌基因表达、改变细胞表面抗原表达、增加巨噬细胞吞噬活性、增强淋巴细胞对靶细胞的细胞毒性等方式来发挥抗肿瘤作用。IFN-α在临床上用于治疗毛细胞白血病、恶性黑色素瘤和艾滋病相关的卡波西肉瘤。
在本发明中，IFN-γ属于II型干扰素，IFN-γ可以刺激B细胞中免疫球蛋白转录表达，上调抗原呈递途径，激活巨噬细胞的活性，增强I型干扰素的抗病毒和抗肿瘤作用。IFN-γ在临床用来治疗慢性肉芽肿病和骨坏死。
TNF-α(Tumor necrosis factor-α)的主要作用是调节免疫细胞。此外TNF-α可以诱导凋亡细胞死亡，诱导炎症，抑制肿瘤发生和病毒复制。
药物组合物、药盒、活性成分组合
本发明提供一种药物组合物，所述药物组合物包括：
(a)治疗有效量的第一活性成分，所述第一活性成分为PDE3抑制剂；和
(b)治疗有效量的第二活性成分，所述第二活性成分为细胞因子。
在另一优选例中，第一活性成分的含量范围为0.01％至99.99％，以组合物活性成分的总重量计；较佳地0.1％至99.9％所述；更佳地1％至99％；较佳地10％至99％；更佳地20％至99％；更佳地30-99％，更佳地40-99％。
在另一优选例中，第二活性成分的含量范围为0.01％至99.99％，以组合物活性成分的总重量计；较佳地0.1％至99.9％；更佳地1％至99％；更佳地1％至90％；更佳地1％至80％；更佳地1-70％，更佳地1-60％。
在一个优选例中，所述的药物组合物还包括药学上可接受的载体。
代表性地，所述药物组合物包括：
(a)治疗有效量的第一活性成分，所述第一活性成分为PDE3抑制剂；
(b)治疗有效量的第二活性成分，所述第二活性成分为细胞因子；和
(c)药学上可接受的载体。
本发明所述的含有第一活性成分和第二活性成分药物组合物，可以是适宜口服给药的各种剂型外，还可以是各种外用给药制剂或其它胃肠道外给药制剂。例如，本发明所述的外用给药制剂，还可以通过添加表面活性剂、透皮吸收促进剂、防腐剂、溶剂、抗氧剂、保湿剂、pH调节剂、着色剂、香料等辅料，进一步制备成(包括但不限于)：搽剂、酊剂、油剂、软膏剂、硬膏剂、糊剂、熨剂、贴膏、贴片、涂膜剂、膜剂、凝胶剂、巴布剂、穴位贴敷剂、喷雾剂、气雾剂、植入剂、乳剂等。优选的剂型包括：口服给药的各种剂型、植入剂、注射用制剂(如注射剂、输液剂或冻干粉针剂)。
应理解，在本发明中，所述的载体没有特别的限制，为本领域常用材料，其种类、使用方法、来源为本领域技术人员所熟知。
药学可接受的载体部分例子有纤维素及其衍生物(如甲基纤维素、乙基纤维素、羟丙甲基纤维素、羧甲基纤维素钠等)、明胶、滑石粉、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油、等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温)、润湿剂(如十二烷基硫酸钠)、缓冲剂、螯合剂、增稠剂、pH调节剂、透皮促进剂、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、抑菌剂、无热原水等。
本发明还提供一种药盒，所述药盒包括：
(A)含有PDE3抑制剂的第一制剂；和
(B)含有细胞因子的第二制剂。
在另一优选例中，所述的药盒还包括说明书。
在另一优选例中，所述的使用说明书中注明将所述第一制剂和第二制剂进行联用，从而预防和/或治疗癌症。
在另一优选例中，所述的第一制剂和第二制剂在预防和/或治疗肿瘤中同时给药、分别给药或顺序给药。
在本发明所述的药盒中，应当理解的是，所述的药盒中第一制剂和第二制剂是相互独立的，也可以是是合并的。
在另一优选例中，所述的药盒中第一制剂和第二制剂是相互独立的。
在另一优选例中，所述的第一制剂为固体制剂(如胃肠道用固体制剂)或注射用制剂(如注射剂、输液剂或冻干粉针剂)。
在另一优选例中，所述的第二制剂为注射用制剂(如注射剂、输液剂或冻干粉针剂)。
例如，第一制剂和第二制剂为是独立的。典型地，本发明药盒包括：一固体制剂(如胃肠道用固体制剂)和一注射用制剂(如注射剂、输液剂或冻干粉针剂)，其中，所述的固体制剂(如胃肠道用固体制剂)含有PDE3抑制剂，所述的注射用制剂(如注射剂、输液剂或冻干粉针剂)含有细胞因子。
本发明还提供一种活性成分组合，所述的活性成分组合包括以下组分：
(1)治疗有效量的第一活性成分，所述第一活性成分为PDE3抑制剂；和
(2)治疗有效量的第二活性成分，所述第二活性成分为细胞因子。
在所述的活性成分组合中，第一活性成分和第二活性成分可以是相互独立的，也可以使组合在一起，以活性成分组合物的形成存在。优选地，所述的活性成分组合中，至少有一种活性成分是独立的。
本发明的药物组合物、活性成分组合、药盒均可采用常规方法和设备进行制备。
用途和给药方式
本发明提供了一种本文所述药物组合物、药盒或活性成分组合在制备预防和或治疗肿瘤的药物方面中的用途。
优选地，所述的肿瘤包括(但不限于)：宫颈癌、肝癌、肾癌、结肠癌、脑胶质瘤、黑色素瘤，或其组合。
本发明所述的药物组合物、药盒或活性成分组合中的第一活性成分和第二活性成分可对肿瘤的预防和治疗产生协同作用，增强治疗肿瘤的作用。
在使用本发明的药物组合物、药盒或活性成分组合之前、同时或之后，可配合使用其它抗肿瘤药物联合使用。
在联合用药过程中，药物的相互作用根据药物共同使用时的效应分为加合作用、协同作用、拮抗作用，协同作用是指联合用药的药物共同使用时的效应要比单独使用显著增强，加和作用是指联合用药的药物共同使用时的效应要与单独使用相 当，拮抗作用是指联合用药的药物共同使用时的效应要比单独使用小。在本发明中，首次发现第一活性成分、第二活性成分和第三活性成分联合使用具有协同作用。
在预防和/或治疗肿瘤过程中，本发明的给药方式包括先后依次施用第一活性成分和第二活性成分，或同时施用第一活性成分和第二活性成分。
药物制剂应与给药方式相匹配，使用药物组合物或制剂时，是将安全有效量的药物施用于所需对象(如人或非人哺乳动物)，其中，第一活性成分的安全有效日使用剂量通常至少约0.1mg，而且在大多数情况下不超过约2500mg。较佳地，该剂量是1mg-500mg；第二活性成分的安全有效量通常至少约0.01mg，而且在大多数情况下不超过2500mg。较佳地，该剂量范围是0.1mg至2500mg，第三活性成分的安全有效量通常至少约0.01mg，而且在大多数情况下不超过2500mg。较佳地，该剂量范围是0.1mg至2500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是在熟练医师技能范围之内的。当先后依次施用第一活性成分、第二活性成分和第三活性成分时，施用的间隔时间无特别要求。本发明的组合物、活性成分组合、药物组合物以及药盒中的第一活性成分、第二活性成分和第三活性成分分别以相同或不同的途径同时或相继给药，其中包括但并不限于：口服给药、注射给药、瘤内给药、植入给药、腔内给药、肛门给药、透皮给药、内外敷；
优选的注射给药包括：静脉注射、肌肉注射、皮下注射、腔内注射。
本发明还提供一种预防和/或治疗肿瘤的方法，给所需的对象施用本发明所述的药物组合物、药盒或活性成分组合。
在另一优选例中，所述对象为人和非人哺乳动物。
代表性地，所述的非人哺乳动物包括(但并不限于)：宠物(如狗、猫)、家畜(如牛、羊、马、猪)、各种动物园动物(熊猫、大象、虎)等。
本发明的主要优点包括：
1、本发明中，PDE3抑制剂(如ANA、或ZARD)和细胞因子(如TRAIL、TNF-α、IFN-α或IFN-γ)联合应用明显协同增强对肿瘤的治疗效果，PDE3抑制剂和细胞因子的协同治疗效果明显优于PDE3抑制剂或细胞因子的单独使用。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明 本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1
细胞毒性实验
1.取对数生长期的肿瘤细胞消化计数，3000-5000/孔接种于96孔板，每孔50ul培养基，在恒温37℃、5％CO 2细胞培养箱贴壁过夜。
2.细胞贴壁稳定后，每孔加入50ul如表1所示药物浓度的化合物(其中ANA表示阿那格雷(Anagrelide)，ZARD表示扎达维林(Zardaverine))孵育72h。
3.药物孵育时间结束后，首先显微镜下观察细胞对药物的响应(细胞数量、细胞状态)，然后向每孔加入30uL MTT(5mg/ml in PBS)作用3-5小时。
4.待MTT孵育完成，到水槽边甩出96孔板液体(一次性甩出，尽可能甩出板内液体，但不能甩掉板内蓝紫色结晶)，将96孔板反向扣在一次性纸巾上，吸取96孔板残留液体。
5.在每孔加入150ul DMSO然后以600rpm速度水平震荡3min待结晶甲瓒(Formazan)完全溶解。
6.酶标仪在检测波长492nM检测OD值(PS空白组：检测培养基OD值(并不铺细胞)对照组：培养基中加入与药物同体积的DMSO(铺细胞))计算细胞存活率
Figure PCTCN2020110552-appb-000009
表1
IFN-α 3000IU/mL(30ng/mL)
IFN-γ 10ng/mL
TRAIL 100ng/mL
ANA 10nM(2.56ng/mL)
ANA+IFN-α 10nM+3000IU/mL
ANA+IFN-γ 10nM+10ng/mL
ANA+TRAIL 10nM+100ng/mL
ZARD 10nM(2.68ng/mL)
ZARD+IFN-α 10nM+3000IU/mL
ZARD+IFN-γ 10nM+10ng/mL
ZARD+TRAIL 10nM+100ng/mL
实验结果
MTT实验中，各个药物组对不同肿瘤细胞的治疗结果如图1A、图1B和表2所示，从图1A和图1B中可以看出，与单独的ANA、ZARD、TRAIL、IFN-α和IFN-γ给药相比，ANA、ZARD与干扰素IFN-α、IFN-γ、凋亡相关因子TRAIL的联用能够发挥协同作用，协同抗肿瘤效果明显优于PDE3抑制剂或细胞因子的单独使用，尤其是ANA+TRAIL的联合应用具有优异的协同抗肿瘤作用。
表2
Figure PCTCN2020110552-appb-000010
Figure PCTCN2020110552-appb-000011
Figure PCTCN2020110552-appb-000012
实施例2
RTCA实验
1.在E-plate板中每孔加入50ul待检测细胞的细胞培养基，检测背景阻抗值
2.在每孔中加入50ul含有3000-5000个待检测细胞培养基，置入检测台中(检测台整体放置在细胞培养箱中)，设置程序每5min扫描检测一次阻抗值。
3.细胞过夜贴壁培养，待生长曲线进入对数生长期时。从检测台取出E-plate板子每孔再加入50ul待测化合物到指定浓度(各组最终浓度如表3)，处理结束后重新放回检测台实时检测细胞生长。
表3
IFN-α 3000IU/mL(30ng/mL)
IFN-γ 10ng/mL
TRAIL 100ng/mL
ANA 100nM(25.5ng/mL)
ANA+IFN-α 100nM+3000IU/mL
ANA+IFN-γ 100nM+10ng/mL
ANA+TRAIL 100nM+100ng/mL
ZARD 100nM(26.8ng/mL)
ZARD+IFN-α 100nM+3000IU/mL
ZARD+IFN-γ 100nM+10ng/mL
ZARD+TRAIL 100nM+100ng/mL
实验结果
RTCA实验中，各个药物组对不同肿瘤细胞的治疗结果如图2A-2F所示，从图2A-2F中可以看出，与单独的ANA、ZARD、TRAIL、IFN-α和IFN-γ给药相比，ANA、ZARD与干扰素IFN-α、IFN-γ、凋亡相关因子TRAIL的联用能够发挥协同作用，协同抗肿瘤效果明显优于PDE3抑制剂或细胞因子的单独使用，尤其是ANA+TRAIL的联合应用具有优异的协同抗肿瘤作用。
实施例3
实验方法与实施例2基本相同，使用TNF-α(10ng/mL)、ANA(100nM)及TNF-α(10ng/mL)+ANA(100nM)处理Bel7404、SW1116细胞株；TNF-α(10ng/mL)、ZARD(100nM)及TNF-α(10ng/mL)+ZARD(100nM)处理Bel7404、SW1116细胞株。
实验结果如图3A-3B所示，结果表明ANA、ZARD与肿瘤坏死因子TNF-α，能够发挥协同作用，协同抗肿瘤效果明显优于PDE3抑制剂或肿瘤坏死因子TNF-α的单独使用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种药物组合物，其特征在于，所述组合物包括：
    (a)治疗有效量的第一活性成分，所述第一活性成分为PDE3抑制剂；和
    (b)治疗有效量的第二活性成分，所述第二活性成分为细胞因子。
  2. 如权利要求1所述的药物组合物，其特征在于，所述的第一活性成分和第二活性成分的重量比为为1：(0.1-50)，较佳地，1：(0.1-20)，更佳地，1：(0.15-10)，最佳地，1：(0.2-5)。
  3. 如权利要求1所述的药物组合物，其特征在于，所述的PDE3抑制剂选自下组：式I所述的化合物或其药学上可接受的盐、式II所述的化合物或其药学上可接受的盐，或其组合；
    Figure PCTCN2020110552-appb-100001
    其中，
    R1、R2、R3、R4、R5、R6、R7和R8各自独立地选自下组：氢原子、卤素原子、氨基、羟基、氰基、醛基、硝基、取代或未取代的C1-C4羧基、取代或未取代的C1-C10烷基、取代或未取代的C3-C10环烷基、取代或未取代的C2-C10烯基、取代或未取代的C2-C10炔基、取代或未取代的C6-C10芳基、取代或未取代的C5-C10杂芳基(如取代或未取代的5元或6元杂环、8元至10元杂芳基)、取代或未取代的C1-C10烷氧基、取代或未取代的C6-C10芳基-氧基、取代或未取代的C5-C10杂芳基-氧基、取代或未取代的C 2-C 10酰基(、取代或未取代的C 2-C 10酯基、取代或未取代的C1-C10磺酰基(-SO2-C1-C10烷基)；
    或R1和R2、R3和R4共同构成选自下组的基团：取代或未取代的C3-C20环烷基(优选为C3-C10环烷基)、取代或未取代的C3-C20杂环烷基(优选为取代或未取代的5元或6元杂环、8元至12元杂芳二环环系)、羰基(＝O)；
    R9选自下组：氢原子、氧原子、取代或未取代的C1-C10烷基、取代或未取代的C3-C10环烷基、取代或未取代的C6-C10芳基、取代或未取代的C1-C10杂芳基、取代或未取代的C1-C10烷氧基、取代或未取代的C6-C10芳基-氧基、取代或未取代的酰基(优选为-CO-C1-C10烷基)、取代或未取代的C1-C10磺酰基；
    其中，所述的任一“取代”是指基团上的一个到四个(优选为1、2、3或4个)氢原子被选自下组的取代基所取代：C1-C10烷基、C3-C10环烷基、C1-C10烷氧基、卤素、羟基、羧基(-COOH)、C1-C10醛基、C2-C10酰基、C2-C10酯基、氨基、苯基、氰基、-OH、硝基、氨基；
    Figure PCTCN2020110552-appb-100002
    其中，
    R11、R12、R13、R14、R15、R17和R18各自独立地选自下组：氢原子、卤素原子、氨基、羟基、氰基、醛基、硝基、取代或未取代的C1-C4羧基、取代或未取代的C1-C10烷基、取代或未取代的C3-C10环烷基、取代或未取代的C2-C10烯基、取代或未取代的C2-C10炔基、取代或未取代的C6-C10芳基、取代或未取代的C5-C10杂芳基(如取代或未取代的5元或6元杂环、8元至10元杂芳二环环系)、取代或未取代的C1-C10烷氧基、取代或未取代的C6-C10芳基-氧基、取代或未取代的C5-C10杂芳基-氧基、取代或未取代的C2-C10酰基、取代或未取代的C2-C10酯基、取代或未取代的C1-C10磺酰基(-SO2-C1-C10烷基)；
    R16选自下组：氢原子、氧原子、取代或未取代的C1-C10烷基、取代或未取代的C3-C10环烷基、取代或未取代的C6-C10芳基、取代或未取代的C5-C10杂芳基、取代或未取代的C1-C10烷氧基、取代或未取代的C6-C10芳基-氧基、取代或未取代的C1-C10酰基、取代或未取代的C1-C10磺酰基；
    Figure PCTCN2020110552-appb-100003
    表示单键或不存在；
    其中，所述的任一“取代”是指基团上的一个到四个(优选为1、2、3或4个) 氢原子被选自下组的取代基所取代：C1-C10烷基、C3-C10环烷基、C1-C10烷氧基、卤素、羟基、羧基(-COOH)、C1-C10醛基、C2-C10酰基、C2-C10酯基、氨基、苯基、氰基、OH、硝基和氨基。
  4. 如权利要求3所述的药物组合物，其特征在于，所述式I所述的化合物具有选自下组的一种或多种特征：
    R1为氢；
    R2为氢；
    R3为氢；
    R4为氢；
    R5为卤素；
    R6为卤素；
    R7为氢；
    R8为氢；和/或
    R9为氢。
  5. 如权利要求3所述的药物组合物，其特征在于，所述式II所述的化合物具有选自下组的一种或多种特征：
    R11为氢；
    R12为氢或C1-C6烷氧基；
    R13为卤代C1-C6烷氧基；
    R14为氢或C1-C6烷氧基；
    R15为氢；
    R16为氢；
    R17为氢；和/或
    R18为氢。
  6. 如权利要求1所述的药物组合物，其特征在于，所述的PDE3抑制剂选自下组：阿那格雷(Anagrelide)或其药学上可接受的盐、扎达维林(Zardaverine)或其药学上可接受的盐，或其组合。
  7. 如权利要求1所述的药物组合物，其特征在于，所述的细胞因子选自下组：干扰素、肿瘤坏死因子，或其组合。
  8. 如权利要求7所述的药物组合物，其特征在于，所述的干扰素选自下组：IFN-α、IFN-γ，或其组合；和/或
    所述的肿瘤坏死因子选自下组：肿瘤坏死因子相关诱导凋亡配体(TRAIL)、TNF-α，或其组合。
  9. 如权利要求1所述的药物组合物，其特征在于，所述的药物组合物还包括药学上可接受的载体。
  10. 一种药盒，其特征在于，所述药盒包括：
    (A)含有PDE3抑制剂的第一制剂；和
    (B)含有细胞因子的第二制剂。
  11. 一种活性成分组合，其特征在于，所述的活性成分组合包括以下组分：
    (1)治疗有效量的第一活性成分，所述第一活性成分为PDE3抑制剂；和
    (2)治疗有效量的第二活性成分，所述第二活性成分为细胞因子。
  12. 如权利要求10所述的药盒、和/或如权利要求11所述的活性成分组合，其特征在于，所述的PDE3抑制剂选自下组：阿那格雷(Anagrelide)或其药学上可接受的盐、扎达维林(Zardaverine)或其药学上可接受的盐，或其组合；和/或
    所述的细胞因子选自下组：干扰素、肿瘤坏死因子，或其组合。
  13. 如权利要求1所述的药物组合物、如权利要求10所述的药盒、和/或如权利要求11所述的活性成分组合的用途，其特征在于，用于制备预防和或治疗肿瘤的药物。
  14. 如权利要求13的用途，其特征在于，所述的肿瘤选自下组：宫颈癌、肝癌、肾癌、结肠癌、脑胶质瘤、黑色素瘤，或其组合。
  15. 一种预防和/或治疗肿瘤的方法，包括步骤：给所需的对象施用如权利要求1所述的药物组合物、如权利要求10所述的药盒、和/或如权利要求11所述的活性成分组合。
PCT/CN2020/110552 2019-08-23 2020-08-21 Pde3抑制剂与细胞因子联合治疗肿瘤的应用 WO2021036937A1 (zh)

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