TWI534148B - Pyrazolopyrimidine prodrugs and methods of use - Google Patents

Description

Pyrazolopyrimidine prodrug and method of use thereof

The present invention relates to a pyrazolopyrimidine prodrug which acts as a precursor to pyrazolopyrimidine which modulates protein kinase CK2 and is therefore useful in the treatment of conditions which are directly or indirectly related to CK2 activity.

Casein kinase II, also known as protein kinase CK2 (referred to herein as "CK2"), is a broad and highly retained protein of serine/threonine kinase with many physiological targets. And participate in a range of complex cellular functions, including the maintenance of cell viability. CK2 is believed to play a role in cell growth and proliferation, and CK2 inhibitors are useful in the treatment of certain types of cancer, such as those described in PCT/US2007/077464, PCT/US2008/074820, and PCT/US2009/035609.

CK2 is unique in the diversity of biological programs it affects, and it is otherwise different from most kinases: CK2 has constitutively active properties, it can utilize ATP or GTP, in many tumors CK2 has a high performance in tissues with rapid proliferation. CK2 also has special structural features that distinguish it from most kinases, giving the opportunity to develop highly specific inhibitors. Many kinase inhibitors have been found to affect multiple kinases simultaneously, thus creating the potential for off-target effects or variability between individuals. Therefore, CK2 is a target molecule of particular interest in drug development.

U.S. Patent No. 8,575,177, filed on Nov. 15, 2010, which is hereby incorporated by reference in its entirety, the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of The pyrazolopyrimidine has the structure of formula A: It can be used to treat proliferative diseases such as cancer, as well as other kinase related conditions including inflammation, pain and certain immune diseases.

The invention relates to a novel pyrazolopyrimidine prodrug which has certain biological activity for inhibiting CK2, and the prodrug is characterized in that a pyrazolopyrimidine is improved in solubility, stability and bioavailability. And pharmacokinetic properties.

In one aspect, the invention provides a prodrug having the structure of Formula I:

Wherein R ¹ is selected from and One of the groups formed; and W is Among them, the prodrug is characterized in that a pyrazolopyrimidine having a biological activity of inhibiting CK2 is improved in solubility, stability, bioavailability and pharmacokinetic properties.

In the present invention, the prodrug can serve as a precursor of a CK2 inhibitor; therefore, the prodrug is effective for regulating the activity of the protein kinase CK2.

In some embodiments of the invention, the prodrug is effective for treating a disease or condition directly or indirectly related to CK2 activity, including proliferative diseases such as cancer, and other kinase-related conditions, including inflammatory conditions, infectious diseases , pain, immune disease or neurodegenerative disease.

In some embodiments of the invention, the prodrug is selected from The structure of the group formed.

In an embodiment of the invention, the prodrug has the following structure:

In another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a prodrug of the invention.

In yet another aspect, the invention provides a method of modulating CK2 activity comprising administering to a subject in need thereof a therapeutically effective amount of a prodrug of the invention and one or more pharmaceutically acceptable excipients.

In still another aspect, the invention provides a combination, composition or kit for treating cancer comprising a combination of a prodrug of the invention and a second anticancer agent.

These and other aspects of the invention are discussed in more detail below.

The foregoing summary, as well as the following detailed description of the invention, may In order to illustrate the invention, the specific embodiments shown in the drawings are presently preferred. However, it should be understood that the invention is not limited to this particular embodiment.

In the drawings: Figure 1 provides the results of treating xenograft mice with Compound 1 and both of erlotinib, Compound 1 as a single agent, and vehicle alone.

Figure 2 provides the results of treating xenograft mice with Compound 7 and soothing, Compound 7 as a single agent, and vehicle alone.

Figure 3 shows the plasma drug concentration after oral administration of Compound 1 and Compound 7.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning meaning

The singular forms "a" and "the" Not explicitly stated in this article. Thus, for example, reference to "the same" is intended to include a plurality of such references and the equivalents of those skilled in the art.

As used herein, the term "prodrug" refers to a pharmaceutically active compound precursor, wherein the precursor may or may not have pharmaceutically active activity, but may be metabolized or otherwise converted to pharmaceuticals when administered. An active compound or a desired drug. Prodrugs are often used to improve the absorption, distribution, metabolism, and excretion of the intended drug, and can also be used to improve how the intended drug selectively excludes interaction with unintended cells or procedures to reduce undesirable or non-exposure of the intended drug. The expected impact is particularly important in cancer chemotherapy.

As used herein, the term "pharmaceutically acceptable" means applied to human or animal tissue without undue toxicity, irritation, allergic reaction and the like, with reasonable benefit/risk ratio and The intended use within the scope of reasonable medical judgment has utility.

As used herein, "an effective amount" or "a therapeutically effective amount" refers to an amount of a prodrug or composition of the invention sufficient to achieve the desired therapeutic response. The therapeutic response may be for the user (e.g., a clinician) to confirm any response that is therapeutically effective, to achieve a beneficial result when the compound is administered to the individual, or to produce the desired activity in vivo or in vitro. In the case of a proliferative disease, beneficial clinical outcomes include reducing the magnitude or severity of the symptoms associated with the disease or condition and/or increasing the life and/or quality of life of the patient compared to untreated conditions. For example, for cancer individuals, "beneficial clinical outcomes" include reducing tumor mass, reducing tumor growth rate, reducing metastasis, reducing the severity of cancer-related symptoms, and/or increasing individual lifespan compared to untreated conditions. . The exact amount of a compound administered to an individual will depend on the species or severity of the disease or condition and the characteristics of the subject, such as general health, age, sex, weight, and drug tolerance. It will also depend on the extent, severity and type of proliferative disease. Technology in the field Artists can determine the effective dose based on common technical knowledge.

The term "excipient" is used to mean a diluent, adjuvant, carrier or carrier with which the compound is administered. The excipients are compatible with the other ingredients of the formulation and are not deleterious to the individual to which the pharmaceutical composition is administered. Any excipient generally known or used in the art can be used in the present invention depending on the needs of the pharmaceutical preparation.

As used herein, the term "individual" refers to humans and other mammals, such as livestock (eg, cattle, pigs, sheep, dogs, and cats).

The terms "treatment" and "treatment" as used herein mean to ameliorate, alleviate, alleviate and eliminate the symptoms of a disease or condition. A candidate molecule or compound described herein can be a therapeutically effective amount in a formulation or medicament that results in a biological effect, such as apoptosis of a particular cell (eg, a cancer cell), reduces proliferation of a particular cell, or results in an improvement, The amount that relieves, alleviates, and eliminates the symptoms of a disease or condition. These terms may also refer to reducing or stopping the rate of cell proliferation (eg, slowing or preventing tumor growth) or reducing the number of proliferative cancer cells (eg, removing some or all of the tumor).

These terms may also mean reducing the microbial titer of one of the systems (ie, cells, tissues or individuals) infected by the microorganism, reducing the rate of microbial spread, reducing the number or symptoms of symptoms associated with microbial infection and/or The measurable amount of microorganisms is removed from the system. Examples of microorganisms include, but are not limited to, viruses, bacteria, and fungi.

The present invention provides a prodrug of Formula I: Wherein R ¹ is selected from and One of the groups formed; and W is

In the present invention, the prodrug is a derivative of a compound (Compound 1) having the following structure, and can be metabolized to Compound 1,

Compound 1 is a pyrazolopyrimidine having the biological activity of inhibiting CK2, as disclosed in U.S. Patent No. 8,575,177, the disclosure of which is incorporated herein in its entirety by reference.

Accordingly, the present invention provides a prodrug of Compound 1 to increase the bioavailability of pyrazolopyrimidine (e.g., Compound 1, which has significantly lower bioavailability when administered to an individual).

According to the present invention, the prodrug can be prepared by conventionally used organic synthesis techniques to improve solubility, stability, bioavailability, and pharmacokinetic properties.

Some specific embodiments of the invention include:

In an embodiment of the invention, the structure of the prodrug is

In another aspect, the invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I and one or more pharmaceutically acceptable excipients.

In another aspect, the invention provides a pharmaceutical composition comprising a prodrug of the invention in admixture with a pharmaceutically acceptable excipient.

In yet another aspect, the invention provides a method of using a prodrug of the invention to modulate the activity of protein kinase CK2. Accordingly, the method is effective for treating diseases or conditions directly or indirectly related to CK2 activity, including cell proliferation such as cancer, and inflammatory conditions, infectious diseases, pain, immune diseases, or neurodegenerative diseases.

The invention also provides the use of a prodrug of the invention as a medicament, and the use of a medicament for the treatment of a disorder as disclosed herein (such as a cancer, an inflammatory disorder, an infectious disease, a pain, an immune disease or a neurodegenerative disease). Pharmacy.

In a particular embodiment of the invention, the method or pharmaceutical composition is effective to treat a cell proliferative disorder. "Cellular proliferative disorder" is sometimes referred to as a tumor, such as a solid or circulatory tumor or a non-tumor cancer, including but not limited to, colorectal, breast, lung, liver, pancreas, lymph nodes, colon, prostate, brain, Cancer of the head and neck, skin, liver, kidneys, blood, and heart (eg, leukemia, lymphoma, and cancer).

As further described herein, the prodrugs and compositions of the present invention may be used alone or in combination with a second anticancer agent or other agent (e.g., a demulcent) that is typically administered to a cancer patient. In some embodiments, the therapeutic agents and molecules that inhibit CK2 can be administered simultaneously. Individuals sometimes use therapeutic agents and molecules that inhibit CK2. In some embodiments, the therapeutic agents and molecules that inhibit CK2 can be combined into a pharmaceutical composition; in other embodiments, the therapeutic agents and molecules can be administered separately in separate compositions. Accordingly, the present invention also provides a combination, composition or kit for treating cancer comprising a combination of a prodrug of the present invention and a second anticancer agent composition.

The second anticancer agent refers to any antitumor antibiotic, including reversible tyrosine kinase inhibitors such as erlotinib; anthracycline, such as daunorubicin (including liposome) Daunorubicin, doxorubicin (including liposome doxorubicin), epirubicin, idarubicin and valrubicin; Streptomyces Reagents such as bleomycin, actinomycin, mithramycin, mitomycin, waves Porfiromycin; and anthracenedione, such as mitoxantrone and pixantrone. Anthracycline has three mechanisms of action: intercalation between base pairs of DNA/RNA strands; inhibition of topoisomerase II enzymes; and production of iron-mediated oxygen radicals to destroy DNA and cell membranes. Anthracycline is generally characterized as a topoisomerase II inhibitor.

The second anticancer agent may also be a therapeutic antibody, such as a monoclonal antibody having anticancer activity, including, but not limited to, murine, chimeric or partially or fully humanized monoclonal antibodies. Therapeutic antibodies include, but are not limited to, antibodies directed against tumors or cancer antigens on the cell surface or inside the cell. Therapeutic antibodies also include, but are not limited to, antibodies directed against the target or pathway that are directly or indirectly related to CK2. Therapeutic antibodies can further include, but are not limited to, antibodies directed against the target or pathway of the target or pathway interactions directly associated with the compounds of the invention. In one variation, therapeutic antibodies include, but are not limited to, anticancer agents, such as Abagovomab, Adecatumumab, Afutuzumab, Aflatuzumab (Alacizumab pegol), Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Apolizumab, Batilizumab (Bavituximab), Belimumab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab, Berentuximab vedotin ), Cantuzumab mertansine, Catusmaxomab, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Edrecolomab, Elotuzumab, Epratuzumab, Ertumaxomab, Adalizumab (Etaracizumab), Farletuzumab, Figiturmumab, Fresolimumab, Galiximab, Gambitamumab (Glembatumumab) Vedotin), Ibritumomab tiuxetan, Intetumumab, Inotuzumab ozogamicin, Ipilimumab, Itozumab (Itolimumab) Iratumumab), Labetuzurnab, Lexatumumab, Lintuzumab, Lucatumumab, Lumiliximab, Mapam Mamumab (Mapatumumab), Matuzumab, Milatuzumab, Mitumomab, Nacolomab tafenatox, Nalumumab Estafenatox), Necitumumab, Nimotuzumab, Ofatumumab, Olazhudan (Olaratumab), Oportuzumab monatox, Oregovomab, Panitumumab, Pemtumomab, Pertuzumab, Pani Pantumomab, Pritumumab, Ramucirumab, Rilotumumab, Rituximab, Rotorumumab (Robatumumab) ), Sibrotuzumab, Tacatuzumab tetraxetan, Taplitumomab paptox, Tenatumomab, Ticilimumab, Tegafur Monoclonal antibody (Tigatuzumab), Tositumomab, Trastuzumab, Tremelimumab, Simo IL Anti-Tucotuzumab celmoleukin, Veltuzumab, Volociximab, Votoumumab, Zalutumumab, and Zanolimumab . In some embodiments, the therapeutic antibodies include alemtuzumab, bevacizumab, cetuximab, daclizumab, gemtuzumab (gemtuzumab), Ibritumomab tiuxetan, pantitumumab, rituximab, tositumomab, and trastuzumab; In other specific embodiments, the monoclonal antibodies include alemtuzumab, bevacizumab, cetuximab, ibimumab tiuxetan, rituximab (rituximab) and trastuzumab; or, antibodies include Daclizumab, gemtuzumab, and pantitumumab. In yet another embodiment, the therapeutic antibody for treating infection includes, but is not limited to, Afelimomab, Efungumab, Exbivirumab, non-dimensional bead Anti-Felvizumab, Foravirumab, Ibalizumab, Libivirumab, Motavizumab, Nebacumab, Par Pagibaximab, Palivizumab, Panobacumab, Rafivirumab, Raxibacumab, Regavirumab , Sevirumab, Tefibazumab, Tuvirumab, and Urtoxazumab. In another specific embodiment, the therapeutic antibody can be used to treat an inflammatory and/or autoimmune disease, including but not limited to, Adalimumab, Atlizumab, Atomilimumab (Atorolimumab) ), azeximumab (Aselizumab), Bapineuzumab, Basiliximab, Benralizumab, Bertilimumab, Besilesomab, Bay Burachimumab, Canakinumab, Cedelizumab, Certolizumab pegol, Clenoliximab, Daclizumab (Daclizumab) ), Desosumab, Eculizumab, Edobacomab, Efalizumab, Erlizumab, Fenzazumab ( Fezakinumab), Fontomizumab, Fresolimumab, Gantenerumab, Gavilimomab, Golimumab, Goli Gomiliximab, Infliximab, Inolimomab, Keliximab, Lebrikizumab, Lerdelimumab, Beauty Pericilumab (Mepolizumab), Metelimumab, Moromonab-CD3, Natalizumab, Orrelizumab, Odulimomab, Omalizumab, Otelixizumab, Pascolizumab, Plitizumab (Pascolizumab) Priliximab), Reslizumab, Rituximab, Rontalizumab, Rovelizumab, Ruplizumab, Sifalimumab ), Siplizumab, Solanezumab, Stamulumab, Talizumab, Tanezumab, Telibizumab ( Teplizumab), Tocilizumab, Toralizumab, Ustekinumab, Vedolizumab, Vepalimomab, Visilizumab, Zanolimumab, and Zolimomab aritox. In yet another embodiment, the therapeutic antibody includes, but is not limited to, adalimumab, basiliximab, certolizumab pegol, eculizumab (eculizumab) ), efalizumab, infliximab, molomonab-CD3, natalizumab, and omalizumab. Alternatively, the therapeutic antibody can include abciximab or ranibizumab. In general, the therapeutic antibody can be a non-binding type, or a binding antibody that binds to a radionuclide, an interleukin, a toxin, a drug activating enzyme, or a drug-filled liposome.

In terms of use in therapy, the prodrugs of the present invention can be formulated into pharmaceutical compositions for administration. Accordingly, the present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I and one or more pharmaceutically acceptable excipients.

According to the present invention, the pharmaceutical composition may be adapted to any suitable route of administration including, but not limited to, intravenous (IV), subcutaneous (SC), intramuscular (IM) or intraperitoneal (IP), oral, rectal, intranasal, topical , vaginal or parenteral route. In a particular embodiment of the invention, the pharmaceutical composition is formulated for oral administration. Such dosage forms can be prepared by any of the methods known in the art of pharmacy.

The invention will be more specifically described with reference to the following examples, which are illustrated by way of illustration and not limitation.

Instance

Example 1 - Preparation of Compound 1

1.1 Synthesis of 5-chloro-N-cyclopropylpyrazolo[1,5-a]pyrimidin-7-amine

Et ₃ N (125 ml) and cyclopropylamine (50 g) were added to isopropanol (750 ml) containing 5,7-dichloropyrazolo[1,5-a]pyrimidine (152.5 g) at room temperature. . The temperature is increased to 63.3 °C. The reaction was cooled and stirred at room temperature for 14 h. Water (1.5 l) was added and the mixture was stirred for 1 hour. The mixture was filtered and washed with water. The obtained solid was dried under vacuum to give 171.17 g of 5-chloro-N-cyclopropylpyrazolo[1,5-a]pyrimidine-7-amine.

1.2 Synthesis of 5-(5-chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidine-3-carbaldehyde

5-Chloro-2-fluorophenylamine (14.7 ml) and concentrated HCl (14.2 ml) were added to contain 5-chloro-N-cyclopropylpyrazolo[1,5-a]pyrimidin-7-amine (17.5 g) EtOH (175 ml). The mixture was heated to 90 ° C for 4 days and then cooled to room temperature. The obtained solid was separated by filtration, washed with ethanol and dried in vacuo to give 5-(5-chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1,5-a] Pyrimidine 23.45g. The solid was dissolved in DMF (125ml), followed by 5 ℃ water bath was added dropwise POCl ₃ (7.26ml). The mixture was stirred overnight at room temperature. Thereafter, POCl ₃ (3.63 ml) was further added at 0 ° C and the mixture was stirred overnight at room temperature. Ice was added to stop the excess POCl ₃ reaction, followed by neutralization of the mixture with 5N NaOH. The mixture was stirred for 1 hour, filtered, and the obtained solid was dried in vacuo to give 5-(5-chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1, 5-a]pyrimidine-3-carbaldehyde 20.9 g.

1.3(Z)-5-((5-(5-Chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidin-3-yl) Synthesis of methylene)imidazolidine-2,4-dione

Addition of carbendazim (1.74g) and piperidine (1.72ml) to 5-(5-chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazole [1,5 -a] Pyrimidine-3-carbaldehyde (5 g) in EtOH (150 ml). The mixture was refluxed overnight then cooled to 50 <0>C, filtered and washed with Et. The obtained yellow solid was dried under vacuum to give (Z)-5-((5-(5-chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1, 5-a]pyrimidin-3-yl)methylene)imidazolidine-2,4-dione 6.35 g. LCMS (ES): m / z 428 [M + 1] +.

Example 2 - Preparation of Compound 1 Pre-Pharmaceutical

2.1(Z)-5-((5-(5-Chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidin-3-yl) Synthesis of methylene)-3-(hydroxymethyl)imidazolidin-2,4-dione

Addition of formaldehyde (37% aq) (5.0 ml) to (Z)-5-((5-(5-chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[ 1,5-a]pyrimidin-3-yl)methylene)imidazolidine-2,4-dione (500 mg, 1.17 mmol) in acetonitrile (15 ml) and pyridine (1.5 ml). The reaction mixture was stirred at 65 ° C for 5 minutes and then cooled to room temperature. The resulting solid was isolated by filtration, washed with water and dried then evaporated tolulujjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj -(Cyclopropylamino)pyrazolo[1,5-a]pyrimidin-3-yl)methylene)-3-(hydroxymethyl)imidazolidin-2,4-dione.

2.2 (Z)-5-((4-((5-(5-Chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidine- Synthesis of 3-yl)methylene)-2,5-dioxoimidazolidine-1-yl)methoxy)-5-oxo-pentanoic acid

Glutaric anhydride (125 mg, 1.095 mmol) and DMAP (3 mg, 0.022) Addition of (Z)-5-((5-(5-chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidine-3 Methyl)-3-(hydroxymethyl)imidazolidine-2,4-dione (100 mg, 0.218 mmol) in pyridine (4.5 mL). The reaction mixture was stirred overnight at 75 ° C, after which the reaction was not completed. Further, glutaric anhydride (125 mg, 1.095 mmol) and DMAP (3 mg, 0.022 mmol) were added, and the reaction was stirred at 75 ° C for an additional 16 hours. After cooling to 0 ° C in an ice bath, 6 M HCl was added until a pH value of less than 3 was detected on a pH test paper. The solid obtained was washed with 0.1 M HCl and dried in vacuo to give 40% (32%) of (Z)-5-((4-(5-(5-chloro-2-fluorophenylamino) - 7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidin-3-yl)methylene)-2,5-dioxoimidazolidin-1-yl)methoxy) -5-oxo-valeric acid.

2.3 (Z)-(4-((5-(5-Chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidin-3-yl) Synthesis of methylene)-2,5-dioxoimidazolidine-1-yl)methyl 3-(4-methylpiperazin-1-yl)propionate

3-(4-Methylpiperazin-1-yl)propionic acid (75 mg, 0.436 mmol), dicyclohexylcarbodiimide (90 mg, 0.436 mmol) and DMAP (4.0 mg, 0.33 mmol) were added (Z) -5-((5-Chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidin-3-yl)methylene)-3 -(Hydroxymethyl)imidazolidine-2,4-dione (100 mg, 0.218 mmol) in DMF (3 mL). The reaction mixture was stirred overnight at room temperature then diluted with ethyl acetate and washed twice with water and brine. The organic layer was dried MgSO _4, filtered, and adsorbed on silica. The resulting crude material by column chromatography 0-10% MeOH / CH ₂ Cl ₂ gradient purification of the punch. Collect the purer liquid and remove the solvent. This material was crystallized from ethyl acetate and hexane to give (Z)-(4-(5-(5-chloro-2-fluorophenylamino)-7- ( Propylamino)pyrazolo[1,5-a]pyrimidin-3-yl)methylene)-2,5-dioxoimidazolidine-1-yl)methyl 3-(4-methylpiperidin Pyrazin-1-yl)propionate.

2.4 (Z)-(4-((5-(5-Chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidin-3-yl) Synthesis of methylene)-2,5-dioxoimidazolidin-1-yl)methyl 2-aminoacetic acid

Glycine (153 mg, 0.873 mmol), dicyclohexylcarbodiimide (180 mg, 0.873 mmol) and DMAP (13 mg, 0.109 mmol) were added to contain (Z)-5-((5-(5-chloro-2) -fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidin-3-yl)methylene)-3-(hydroxymethyl)imidazolidin-2, 4-Dione (100 mg, 0.218 mmol) in DMF (3 mL). The reaction mixture was stirred overnight at room temperature then diluted with ethyl acetate and washed once with 1M EtOAc then three brine. The organic layer was dried MgSO _4, filtered, and adsorbed on silica. The resulting crude material by column chromatography 5-15% EtOAc / CH ₂ Cl ₂ gradient purification of the punch. Collect the purer liquid and remove the solvent. 4M HCl/dioxane (4 ml) was added to the residue and stirred at room temperature for 2 hr. The remaining HCl/dioxane was removed by evaporation under vacuum. Diethyl ether was added to the residue and the suspension was shaken with a supersonic wave. The resulting solid was filtered and washed with diethyl ether. Drying under vacuum to give (Z)-(4-((5-(5-chloro-2-fluorophenylamino))-7-(cyclopropylamino)pyrazole as a yellow solid (23%) And [1,5-a]pyrimidin-3-yl)methylene)-2,5-dioxoimidazolidine-1-yl)methyl 2-aminoacetic acid hydrogen chloride.

2.5(Z)-(4-((5-(5-Chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidin-3-yl) Synthesis of methylene)-2,5-dioxoimidazolidin-1-yl)methyl 2-isopropyl-2-aminoacetic acid

Preparation of (Z)-(4-((5-(5-chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1] by chemical synthesis method similar to the above examples , 5-a]pyrimidin-3-yl)methylene)-2,5-dioxoimidazolidin-1-yl)methyl 2-isopropyl-2-aminoacetic acid.

2.6 (Z)-(4-((5-(5-Chloro-2-fluorophenylamino)-7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidin-3-yl) Synthesis of methylene)-2,5-dioxoimidazolidin-1-yl)methyl 2-methyl-2-aminoacetic acid

D,L-Boc-Ala-OH (19g), EDCI (20.2g) and DMAP (1.3g) were all added together containing (Z)-5-((5-(5-chloro-2-fluorophenylamine) -7-(cyclopropylamino)pyrazolo[1,5-a]pyrimidin-3-yl)methylene)-3-(hydroxymethyl)imidazolidin-2,4-dione 24.13 g) of a DMF (600 ml) suspension. The reaction mixture was stirred for 30 minutes at room temperature. Additional D, L-Boc-Ala-OH (10 g) and EDCI (10 g) were added to the mixture to drive the reaction to completion. The mixture was stirred for 30 minutes at room temperature. Water (1.5 l) was added and stirred, and a yellow solid was filtered, washed with water (l.l), and then stirred in water (800 ml) for 30 minutes to remove residual DMF. The solid was isolated via filtration, washed with water (11) and dried over EtOAc. Diethyl ether (250 ml) containing 2M HCl was added dropwise to diethyl ether (600 ml) containing this yellow solid. The mixture was stirred at room temperature for 5 hours. The obtained solid was filtered, washed with diethyl ether (1.sub.1) and dried under vacuo overnight to afford (Z)-(4-((5-(5-chloro-2-fluorophenylamino))-7- (cyclopropylamino)pyrazolo[1,5-a]pyrimidin-3-yl)methylene)-2,5-dioxoimidazolidine-1-yl)methyl 2-methyl-2 - hydrochloride of amino acetic acid (30.52 g). LCMS (ES): m / z 529 [M + 1] +.

Example 3 - Inhibition of CK2 activity by Compound 1 and Compound 7 in vitro

Protein kinase CK2, or casein kinase II, is a widely-developed enzyme that functions to catalyze the biochemical and physiological responses essential for cell proliferation and growth. It has been reported that CK2 has a function of effectively inhibiting apoptosis. Specifically, CK2 was found to be associated with the development of certain cancers, including leukemia, lymphoma, and multiple myeloma, while enhancement or overexpression of CK2 activity was also observed in tumors of various organs and tissues. Since the structure of CK2 is composed of two regulatory subunits and two catalytic subunits, the activity of CK2 can be reduced by binding the inhibitor to the target position on the subunit. Therefore, the use of CK2 inhibitors for the treatment of certain types of cancer inhibits the growth and proliferation of tumor cells.

3.1 CK2 enzyme activity test of compound 1, compound 7 and other prodrugs

U.S. Patent No. 8,575,177, which discloses that Compound 1 has an activity of inhibiting CK2. Cell survival assays of different cancer cell lines were performed with test compounds, including Compound 1, Compound 7 (pre-drug 1 drug), and other prodrugs. Test compounds of a certain concentration range were cultured in cells for 96 hours, and their effects on proliferation and survival of the cells were evaluated and expressed in Table 1 as IC50. IC50 (or half maximal inhibitory concentration), which is defined as the concentration required to inhibit half of the enzyme in a given system, is used to assess the inhibitory effect of the compound. Compound 1 had an IC50 of 0.00079 μM, and Compound 1 had relatively good inhibitory activity relative to the prodrugs listed in Table 1.

Table 1 (Data is expressed in IC50 and the unit is μM; ND means not detected)

3.2 Pharmaceutical properties of Compound 1, Compound 7 and other prodrugs

Compound 1, Compound 7, and other prodrugs were administered orally to mice. Plasma samples from the administered mice were collected and analyzed for individual drug concentrations and expressed as AUC values. AUC represents the area under the plasma drug concentration-time curve and can be interpreted as the total amount of a drug over time in the body.

As shown in Table 2, the AUC (0-24 hrs) of Compound 1 was 2624 hr*ng/mL, and the AUC of Compound 7 was 6392 hr*ng/mL. After the compound 1 was modified to the compound 7 by the substituent, the bioavailability of the drug in the body was increased by 1.44 times, and the highest concentration value (Cmax) in the plasma was increased by 1.97 times (the Cmax of the compound 1 was 450 ng/mL and the compound 7) The Cmax is 1336 ng/mL).

(ND means not detected)

3.3 Anti-tumor activity of erlotinib combined with compound 1 or compound 7

A human tumor xenograft model was established in mice with human cancer cell lines. The tumor-producing mice were divided into three groups, each of which was orally administered once a day with Compound 1 and erlotinib, Compound 1 single agent, and vehicle alone. As shown in Figure 1, the development of the tumor (in terms of tumor volume) remained fairly consistent and did not grow further over time. This data demonstrates that the binding of Compound 1 to erlotinib has an activity of inhibiting tumor progression.

Tumor-producing mice were divided into three groups, each of which was orally administered once a day with Compound 7 and both erlotinib, Compound 7 single agent, and vehicle alone. As shown in Figure 2, mouse tumors treated with both Compound 7 and erlotinib maintained a fairly consistent volume over time, or even slightly decreased, while tumors of the other two groups grew relatively. In addition, each group of tumor growth inhibition (TGI) was calculated, which is a numerical indicator of tumor development. As shown in Table 3, the TGI of the mouse group administered with both Compound 7 and Erlotinib returned to 106%, which was higher than the group administered only with Compound 7, which had a TGI of 65%. These data demonstrate that erlotinib binding to compound 7 is effective in inhibiting tumor growth.

3.4 Pharmacokinetic Study of Compound 7

This study investigated four oral dosage forms, including: Compound 1 dissolved in 30% TPGS (vitamin E derivative) and 70% PEG400 (Formulation 1, called 30% TPGS); Compound 1 dissolved in 5% TPGS and 95% PEG400 (Formulation 2, referred to as 5% TPGS); Spray-Dried Dispersion (SDD) Compound 1 was dissolved in 0.5% HPMC and 0.2% Tween (Formulation 3, referred to as SDD); and Compound 7 (Compound 1) The prodrug was dissolved in 10% DMSO and 90% 0.5% HPMC (referred to as 10% DMSO). Compound 1 (5 mg/kg) was administered intravenously and used to calculate the bioavailability of the above four oral dosage forms. Each dog received five doses with a washout period of at least seven days between each dose. Plasma samples were collected after male and female dogs (four animals per sex) each received an oral dose of 10 mg/kg or an intravenous dose of 5 mg/kg of Compound 1. Samples were collected at time points such as 15 minutes, 30 minutes, 1, 2, and 3 hours before administration. Subsequently, additional samples were collected at time points of 4, 6, 8, 10, 12 and 24 hours. Bioanalytical tests were performed on the collected plasma samples and pharmacokinetic analysis was performed from these data.

As shown in Figure 3, samples were collected and analyzed for the concentration of Compound 1. After oral administration of Compound 7 (pre-compound 1), the concentration of plasma compound 1 at each experimental time point was relatively higher than that of the other three reagents.

As shown in Table 4, the bioavailability of Compound 7 obtained by the test was 19.0%. The other three oral dosage forms of Compound 1 were relatively poor: Formulations 1, 2 and 3 were only 0.8%, 0.4% and 3.0%, respectively. Based on this higher bioavailability, Compound 7 was confirmed to be a more potent pharmaceutical dosage form.

It is believed that those having ordinary skill in the art to which the invention pertains can use the invention to the full scope thereof. Therefore, the description and claims of the present invention are intended to be illustrative and not restrictive.

Claims (22)

a prodrug having a selected from A compound of the composition of the group, wherein the prodrug is characterized in that a pyrazolopyrimidine having inhibition of protein kinase CK2 activity improves its bioavailability. As claimed in the first paragraph of the patent application, which has Structure. As a prodrug of the first aspect of the patent application, it is a precursor of an inhibitor of protein kinase CK2. A prodrug as described in claim 1 of the patent application, when administered to a subject, is metabolized to an active compound which is effective to treat a disease or condition directly or indirectly related to protein kinase CK2 activity. The prodrug as described in claim 4 of the patent application, when administered to a body, is metabolized to an active compound which is effective for inhibiting cell proliferation. The prodrug as described in claim 4 of the patent application, when administered to a body, is metabolized into an active compound which is effective for treating an inflammatory condition, an infectious disease, a pain, an immune disease or a nerve. Degenerative diseases. If the prior drug mentioned in item 5 of the patent application is administered to a body, it is passed through the generation. It is an active compound which is effective in treating cancer. A pharmaceutical composition comprising a prodrug as described in claim 1 and at least one pharmaceutically acceptable excipient. The pharmaceutical composition according to claim 8, which comprises the prodrug as described in claim 2 and at least one pharmaceutically acceptable excipient. The pharmaceutical composition according to claim 8 when the pharmaceutical composition is administered to a body, wherein the drug is metabolized into an active compound as described in claim 1 of the patent application, which is effective for inhibiting Cell proliferation. The pharmaceutical composition according to claim 8, wherein the drug is metabolized into an active compound as described in claim 1 of the patent application, which is effective for treating an inflammatory condition, an infectious disease, pain, and immunity. Disease or neurodegenerative disease. The pharmaceutical composition according to claim 10, wherein the drug is metabolized into an active compound as described in claim 1 of the patent application, which is effective for treating cancer. A use of a prodrug as described in claim 1 of the patent application for the preparation of a medicament effective for inhibiting the individual protein kinase CK2. The use according to claim 13, wherein the medicament is effective for inhibiting cell proliferation in an individual. The use according to claim 13, wherein the medicament is effective for treating an inflammatory condition, an infectious disease, a pain, an immune disease or a neurodegenerative disease in an individual. The use according to claim 15, wherein the medicament is effective for treating cancer in an individual. For example, the use of the prodrug is as follows: A combination, composition or kit for treating cancer comprising a combination of a prodrug as described in claim 1 of the patent application and a second anticancer agent. The combination, composition or kit of claim 18, wherein the second anticancer agent is selected from the group consisting of a reversible tyrosine kinase inhibitor, anthracycline, and doxorubicin a group consisting of (doxorubicin), Streptomyces-related agents, and anthracenedione. The combination, composition or kit of claim 19, wherein the second anticancer agent is selected from the group consisting of erlotinib, daunorubicin, doxorubicin , epirubicin, idarubicin, valrubicin, bleomycin, actinomycin, mithramycin, mitos A group consisting of mitomycin, porfiromycin, mitoxantrone, and pixantrone. The combination, composition or kit of claim 20, wherein the second anticancer agent is erlotinib. The combination, composition or kit of claim 18, wherein the second anticancer agent is a therapeutic antibody.