CN111848629B - mTOR/HDAC dual inhibitor and application thereof - Google Patents

Abstract

The invention discloses an mTOR/HDAC dual inhibitor and application thereof, wherein the dual inhibitor has dual inhibition effects of mTOR and HDAC, has better curative effect than the mTOR inhibitor, and can be used for preparing anti-tumor and anti-idiopathic pulmonary fibrosis drugs.

Description

mTOR/HDAC dual inhibitor and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to an mTOR/HDAC dual inhibitor and application thereof, and also relates to an mTOR/HDAC dual inhibitor composition and application thereof.

Background

Over-activation of the PI3K/Akt/mTOR (PAM) signaling pathway due to mutation of PIK3CA and abnormality of the cancer suppressor PTEN is one of the most common carcinogens. mTOR is a key signaling molecule of this pathway, present in the protein complexes mTORC1 and mTORC2, and aberrant activation of mTOR is seen in many malignancies. The application value of mTOR as a malignant tumor treatment target is fully proved by the marketing of first generation mTOR inhibitors Temsirolimus and Everolimus. However, the two drugs can only inhibit mTORC1, so the antitumor spectrum is narrow; in addition, the S6K/IRS1/PI3K negative feedback pathway can be activated, resulting in the attenuation of the antitumor effect itself. The second generation mTOR inhibitor is an ATP competitive inhibitor, can overcome the defects of the first generation of medicaments, and has a plurality of compounds entering clinical research. The research shows that tumor cells can mediate the drug resistance of the tumor cells to first-generation and second-generation drugs respectively through the mutation of an mTOR protein FRB region and an ATP binding pocket. At the same time, inhibitors of the PAM pathway, including mTOR inhibitors, may activate the associated signaling pathways, which in turn lead to the development of drug resistance. Therefore, mTOR inhibitors approved for marketing or clinical use have therapeutic deficiencies.

HDACs can induce the development and progression of tumors by epigenetic or catalytic deacetylation of non-histone substrates, and are also effective therapeutic targets for malignancies. A large number of researches prove that the combination of the HDAC inhibitor and the mTOR inhibitor can obtain a synergistic effect and weaken the cross-activation drug resistance caused by the HDAC inhibitor and the mTOR inhibitor, which provides a theoretical basis for the development of the mTOR/HDAC dual inhibitor. In view of the problems of interaction, accumulated toxic and side effects, complex pharmacokinetics, poor patient compliance and the like existing among medicaments of the medicament combination therapy, the mTOR/HDAC dual inhibitor is expected to obtain better curative effect than the mTOR inhibitor and weaken the drug resistance of the mTOR/HDAC dual inhibitor, and can avoid the problems of interaction, accumulated toxic and side effects, complex pharmacokinetics, poor patient compliance and the like of the medicament combination therapy of the mTOR inhibitor and the HDAC inhibitor, so that the research and development value is remarkable. However, to date, no dual mTOR/HDAC inhibitors are on the market or under clinical study.

HDAC has multiple subtypes, wherein, HDAC6 subtype has special structure and function, and the overexpression of the HDAC6 subtype can cause the abnormality of Ras, EGFR and other signal paths, promote oncogenic transformation and the growth, proliferation and angiogenesis of tumor cells, and enhance the invasiveness and the metastasis of the tumor cells. Recent studies have shown that selective inhibition of HDAC6 down-regulates the expression of Programmed death-ligand (PD-L1) and thus exerts immunotherapeutic effects. Currently, no compound has dual mTOR/HDAC inhibition effects and also exhibits selectivity for inhibiting HDAC6 subtype: the compound not only can obtain the synergistic effect of inhibiting mTOR and HDAC6, but also can reduce the toxic and side effect caused by inhibiting all or multiple HDAC subtypes, so that the compound is expected to provide a new therapy for treating malignant tumors.

In addition to being used against tumors, inhibition of mTOR or HDAC may also exert an anti-Idiopathic Pulmonary Fibrosis (IPF) effect. IPF is a chronic, progressive pulmonary interstitial disease, well developed in middle-aged and elderly populations, with an average survival time of only 2-3 years after diagnosis, with a mortality rate higher than that of most tumors. With the aging of the population in China, the number of IPF patients increases year by year. The existing anti-IPF drugs can only delay the disease process, but can not stop or reverse the fibrosis process, so the development of anti-IPF drugs with new action mechanisms is urgently needed. Because the pathogenesis process of IPF is complex and multiple channels are involved, the mTOR/HDAC dual inhibitor is expected to produce a better treatment effect, has a brand-new IPF resistant action mechanism, and can perform multi-target and multi-process intervention on epithelial cell injury, fibroblast proliferation, profibrotic matrix protein expression and epithelial mesenchymal transition in the IPF development process.

Disclosure of Invention

Aiming at the defects of the prior mTOR inhibitor in the aspect of anti-tumor curative effect and the problems of combined medication, the invention provides an mTOR/HDAC dual inhibitor, the molecular structure of the mTOR/HDAC dual inhibitor has structural units required for inhibiting mTOR and HDAC, and multiple activity tests prove that the compound has dual mTOR and HDAC inhibitory activity and obvious anti-tumor activity. Due to the single-molecule multi-target effect, the compound disclosed by the invention can avoid the problems of drug interaction, accumulated toxic and side effects, complex pharmacokinetics, poor patient compliance and the like of a combined therapy of an mTOR inhibitor and an HDAC inhibitor.

Meanwhile, aiming at the technical defect that no dual mTOR/HDAC inhibitor is adopted for IPF treatment at present, the invention provides the application of the dual mTOR/HDAC inhibitor in treating IPF, and multiple activity tests prove that the compound has obvious IPF resisting activity.

In order to achieve the purpose, the invention adopts the following technical scheme:

a dual mTOR/HDAC inhibitor is a compound shown in the following general formulas (I) to (III) and a pharmaceutically acceptable salt or a deuteride thereof:

in the above formula, R is at least 1R₁Substituted C6-14 aryl, C5-14 heteroaryl, X is

Wherein n is 1-10;

in the general formula (II), ring A is at least 1R₂Substituted C6-14 aryl, C5-14 heteroaryl;

in the general formula (III), ring B is at least 1R₃Substituted C6-14 aryl, C5-14 heteroaryl;

it is to be noted that, in the above substitution, when the corresponding position is substituted by 2 or more R₁、R₂Or R₃When substituted, substituted R₁、R₂Or R₃May be the same or different.

R₁、R₂、R₃Each independently selected from hydrogen, halogen, hydroxy, cyano, carbamoyl, trifluoromethyl, trifluoromethoxy, C1-6 alkyl, C1-6 alkoxy, C2-6 unsaturated aliphatic hydrocarbon, N (R)₄)₂、NR₄OR₄、NR₄N(R₄)₂、SO₂N(R₄)₂、NR₄SO₂R₄、NR₄CON(R₄)₂、NR₄COOR₄、NR₄COR₄、CON(R₄)₂Wherein R is₄Independently selected from hydrogen, C1-6 alkyl or C2-6 unsaturated aliphatic hydrocarbon, it is specifically noted that when 2R are present₄When substituted on the same atom, they may be the same or different.

Further, the dual mTOR/HDAC inhibitor is selected from the following compounds and pharmaceutically acceptable salts or deuterated compounds thereof, and it should be specifically noted that the following compounds are only used for example, so as to make the technical solution of the present invention clear, and the dual mTOR/HDAC inhibitor in the present invention includes not only the following compounds:

furthermore, some of the compounds of the present invention have dual mTOR/HDAC inhibitory effects and also exhibit HDAC6 inhibitory selectivity, and such compounds are represented by the following general formulas (II), (III) and pharmaceutically acceptable salts or deuterated compounds thereof, wherein R is defined in claim 1;

in the general formula (II), X is

Wherein n is₁1-6, ring a is as defined in claim 1;

in the general formula (III), X and ring B are defined as in claim 1. This is because it has aromatic ring or aromatic heterocyclic hydroxamic acid structure, and the structure can take better interaction with the catalytic channel of HDAC6 and zinc ion prosthetic group, and multiple activity tests show that the compound can also show selectivity of inhibiting HDAC6 while having dual mTOR/HDAC inhibition effect.

Furthermore, the invention discloses application of the mTOR/HDAC dual inhibitor in preparing an anti-tumor medicament or an anti-idiopathic pulmonary fibrosis medicament, wherein the tumor comprises a solid tumor and a blood tumor.

Furthermore, the invention discloses an mTOR/HDAC dual inhibitor composition, which comprises the mTOR/HDAC dual inhibitor and at least one medicinal carrier or excipient.

Further, the invention discloses an application of the dual mTOR/HDAC inhibitor composition in preparation of an anti-tumor drug or an anti-idiopathic pulmonary fibrosis drug.

Further, the dual mTOR/HDAC inhibitor composition also comprises at least one other therapeutic agent, and the dosage form of the dual mTOR/HDAC inhibitor composition is any clinically or pharmaceutically acceptable dosage form.

Further, the invention discloses an application of the dual mTOR/HDAC inhibitor composition in preparation of an anti-tumor drug or an anti-idiopathic pulmonary fibrosis drug.

The dosage of the compound of the invention is 1mg-1000 mg/day, and the dosage can be deviated from the range according to the severity of the disease or the dosage form.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.

Wherein "halogen" refers to fluorine, chlorine, bromine, iodine;

"C6-14 aryl" refers to an all-carbon monocyclic or fused polycyclic group of 6 to 14 carbon atoms with a fully conjugated pi-electron system, specific examples including, but not limited to, benzene, naphthalene, anthracene rings;

"C5-14 heteroaryl" refers to a non-all carbon monocyclic or fused polycyclic group of 5-14 ring atoms with a fully conjugated pi-electron system, specific examples including but not limited to pyridine, imidazole, thiophene, furan, thiazole, purine, indole, azaindole;

"C1-6 alkyl" refers to an alkyl group of 1 to 6 carbon atoms;

"C1-6 alkoxy" refers to an O-alkyl group containing 1 to 6 carbon atoms, specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy, and the like;

"C2-6 unsaturated aliphatic hydrocarbon group" means a straight or branched alkenyl, alkynyl or alkenynyl group of 2 to 6 carbon atoms containing a double or triple bond. Specific examples of unsaturated aliphatic hydrocarbon groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, ethynyl, and the like.

The compound or the pharmaceutically acceptable salt or the deutero-compound thereof has the same effect, wherein the pharmaceutically acceptable salt refers to the salt with the general formula (I), (II) or (III), and comprises alkali metal salt, alkaline earth metal salt, other metal salt, inorganic base salt, organic base salt, inorganic acid salt, organic acid salt, lower alkane sulfonate, aryl sulfonate and amino acid salt.

The "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable carrier, and includes diluents, excipients (e.g., water, etc.), fillers (e.g., starch, etc.), binders (e.g., cellulose derivatives, gelatin, etc.), humectants (e.g., glycerin, etc.), disintegrants (e.g., agar-agar, calcium carbonate, etc.), absorption enhancers (e.g., quaternary ammonium compounds, etc.), surfactants (e.g., cetyl alcohol, etc.), adsorbent carriers (e.g., kaolin, bentonite, etc.), lubricants (e.g., talc, etc.), and flavoring agents, sweeteners, etc., as necessary.

The "other therapeutic agent" refers to a therapeutic agent compatible with the dual mTOR/HDAC inhibitor, including but not limited to mitotic inhibitors (e.g., vinblastine, vindesine), tubulolysis inhibitors (e.g., taxol), bioalkylating agents (e.g., cyclophosphamide), antimetabolites (e.g., 5-fluorouracil, tegafur, methotrexate), antitumor antibiotics (e.g., doxorubicin, mitomycin), enzymes (e.g., asparaginase), topoisomerase inhibitors (e.g., etoposide and camptothecin), biological response modifiers (e.g., interferon), proteasome inhibitors (e.g., bortezomib).

The "pharmaceutically acceptable dosage form" is suitable for administration by any suitable route, such as the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, transdermal or inhalation), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. These formulations may be prepared by any method known in the art of pharmacy. For example, by admixing the active ingredient with a carrier or excipient.

Such "solid or hematologic tumors" include, but are not limited to, breast cancer, sarcoma, lung cancer, prostate cancer, colon cancer, rectal cancer, kidney cancer, pancreatic cancer, neuroblastoma, glioma, head cancer, neck cancer, thyroid cancer, liver cancer, ovarian cancer, uterine cancer, endometrial cancer, stomach cancer, bladder cancer, gastrointestinal stromal tumors, nasopharyngeal cancer, leukemia, lymphoma, multiple myeloma.

Aiming at the condition that no mTOR/HDAC dual inhibitor is on the market or in a clinical research state at present, the mTOR/HDAC dual inhibitor can provide a new therapy for resisting tumors or idiopathic pulmonary fibrosis diseases. Through multiple experiments, the compounds in the invention have dual mTOR and HDAC inhibitory activities, wherein most of the compounds can inhibit mTOR, HDAC1 or/and HDAC6 with high intensity. Part of the compounds show significant anti-tumor cell proliferation activity and significant anti-lung fibroblast proliferation activity (fibroblasts are effector cells of IPF) while inhibiting mTOR, HDAC1 or/and HDAC6 at high intensity. And other compounds show excellent HDAC6 subtype selectivity while inhibiting mTOR and HDAC6 at high strength. Pharmacodynamic experiments show that the compound has a prospect of being developed into an anti-tumor drug or an anti-idiopathic pulmonary fibrosis drug.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description of specific embodiments. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The dual mTOR/HDAC inhibitor compounds of the present invention can be prepared in two routes, compound 3 (route one, compound 21 (route two):

route one:

in route one, the reactants and reaction conditions involved: a is 5-bromovaleric acid ethyl ester, K₂CO₃N, N-Dimethylformamide (DMF), 60 ℃;

b is 2-aminopyrimidine-5-boronic acid pinacol ester, Pd (PPh)₃)₄Saturated NaHCO₃Solution, ethylene glycol dimethyl ether (DME), H₂O, under the protection of nitrogen, and at 110 ℃;

c is 50% aqueous hydroxylamine, NaOH, Tetrahydrofuran (THF), MeOH, 0 deg.C-rt.

And a second route:

in route two, the reactants and reaction conditions involved: a is NaN₃，DMF，50℃；

b is propargyl bromide, K₂CO₃N, N-Dimethylformamide (DMF), 60 ℃;

c is 2-aminobenzoxazole-5-boronic acid pinacol ester, Pd (PPh)₃)₄Saturated NaHCO₃Solution, ethylene glycol dimethyl ether (DME), H₂O, under the protection of nitrogen, and at 110 ℃;

d is intermediate 27, CuSO₄Solution (1M), sodium ascorbate solution (1M), Dichloromethane (DCM), MeOH, rt;

e is aqueous hydroxylamine (50%), NaOH, Tetrahydrofuran (THF), MeOH, 0 deg.C-rt.

The preparation routes for the other compounds are similar to those described above, wherein compounds 1,2, 4-20 are prepared according to the first reference route; compound 22 was prepared according to scheme two.

Example 1

Synthesis of 5- (4-amino-3- (2-aminopyrimidine-5-) -1H-pyrazolo [3,4-d ] pyrimidin-1) -N-hydroxypentanamide (Compound 3).

(1) Synthesis of ethyl 5- (4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidine-1-) valerate (intermediate 24):

3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-4-amine (1.5g), ethyl 5-bromovalerate (1.3g), anhydrous potassium carbonate (1.1g) and anhydrous DMF (20mL) were sequentially added to a reaction flask, and the mixture was stirred at 60 ℃ for reaction for 12 hours. Adding appropriate amount of water into the system, standing, vacuum filtering to obtain crude product, and performing silica gel column chromatography to obtain white solid 1.7 g.

(2) Synthesis of ethyl 5- (4-amino-3- (2-aminopyrimidin-5-) -1H-pyrazolo [3,4-d ] pyrimidin-1-) valerate (Compound 25):

the intermediate 24(400mg), 2-aminopyrimidine-5-boronic acid pinacol ester (250mg) and Pd (PPh) were added in sequence to a reaction flask₃)₄(119mg) of saturated NaHCO₃Solution (2mL), DME (8mL), H₂O (2mL), the reaction system was sufficiently replaced with nitrogen, and the reaction was stirred at 110 ℃ for 6 hours under a nitrogen atmosphere. Removing organic solvent, adding appropriate amount of water, extracting product with DCM, washing organic phase with saturated saline solution, and extracting with anhydrous Na₂SO₄Drying, concentrating, and subjecting the crude product to silica gel column chromatography to obtain white solid 174 mg.

(3) Synthesis of 5- (4-amino-3- (2-aminopyrimidin-5-) -1H-pyrazolo [3,4-d ] pyrimidin-1) -N-hydroxypentanamide (Compound 3):

intermediate 25(60mg) was dissolved in THF/CH₃An OH mixture (2mL, V/V ═ 1:1) was added dropwise to a mixture of 50% aqueous hydroxylamine solution (1mL) and NaOH (54mg) with stirring in an ice bath, and the mixture was reacted at room temperature for 1 hour with stirring. After the reaction is finished, adjusting the pH of the system to be neutral by acetic acid, and removing THF and CH by spinning₃OH, adding a proper amount of water, performing suction filtration, washing a filter cake with a small amount of water, and drying to obtain a white-like solid of 46 mg; the nuclear magnetic resonance results are as follows:¹H NMR(400MHz,DMSO-d₆):δ10.34(s,1H),8.66(s,1H),8.46(s,2H),8.23(s,1H),7.05(brs,2H),6.92(s,2H),4.32(t,J＝6.8Hz,2H),1.99(t,J＝7.2Hz,2H),1.89-1.75(m,2H),1.57-1.40(m,2H)；ESI-MS:344.15[M+H]⁺。

example 2

Synthesis of 5- (4-amino-3- (6-aminopyridin-3-) -1H-pyrazolo [3,4-d ] pyrimidin-1) -N-hydroxypentanamide (Compound 1).

Compound 1 was prepared according to example 1 by replacing 2-aminopyrimidine-5-boronic acid pinacol ester with 2-aminopyridine-5-boronic acid pinacol ester only in step (2) and then hydroxylamine-hydrolyzing in the same manner to obtain off-white solid.

The high resolution mass spectrometry results are: ESI-HRMS 343.1639[ M + H ]]⁺。

Example 3

Synthesis of 7- (4-amino-3- (6-aminopyridin-3-) -1H-pyrazolo [3,4-d ] pyrimidin-1) -N-hydroxyheptanamide (Compound 2).

Compound 2 was prepared according to example 1 by replacing ethyl 5-bromovalerate with ethyl 7-bromoheptanoate in step (1) and 2-aminopyrimidine-5-boronic acid pinacol ester with 2-aminopyridine-5-boronic acid pinacol ester in step (2) only and then hydroxylamine-hydrolyzing the same procedure to obtain an off-white solid.

¹H NMR(400MHz,DMSO-d₆):δ8.22(s,1H),8.18(d,2.0Hz,1H),7.64(dd,2.0Hz,8.8Hz,1H),6.80(brs,2H),6.58(d,8.8Hz,1H),6.24(s,2H),4.29(t,6.8Hz,2H),1.92(t,7.6Hz,2H),1.84-1.75(m,2H),1.51-1.40(m,2H),1.26-1.24(m,4H)；ESI-HRMS:371.1949[M+H]⁺。

Example 4

Synthesis of 7- (4-amino-3- (2-aminopyrimidine-5-) -1H-pyrazolo [3,4-d ] pyrimidine-1-) -N-hydroxyheptanamide (4)

Compound 4 was prepared by the same method as in example 1 except that ethyl 5-bromovalerate was replaced with ethyl 7-bromoheptanoate in the step (1) and then subjected to hydroxylamine hydrolysis.

An off-white solid;¹H NMR(400MHz,DMSO-d₆):δ10.33(s,1H),8.65(s,1H),8.45(s,2H),8.23(s,1H),7.04(brs,2H),6.92(s,2H),4.30(t,6.8Hz,2H),1.92(t,7.2Hz,2H),1.87-1.77(m,2H),1.52-1.40(m,2H),1.26-1.24(m,4H)；ESI-HRMS:372.1890[M+H]⁺。

example 5

Synthesis of 5- (4-amino-3- (quinoline-3-) -1H-pyrazolo [3,4-d ] pyrimidine-1-) -N-hydroxypentanamide (Compound 5).

Compound 5 was prepared according to example 1 by replacing the 2-aminopyrimidine-5-boronic acid pinacol ester with quinoline-3-boronic acid pinacol ester only in step (2) and then hydroxylamine-hydrolyzing in the same manner to obtain an off-white solid.

¹H NMR(400MHz,DMSO-d₆):δ10.36(brs,1H),9.18(d,2.0Hz,1H),8.60(d,1.6Hz,1H),8.30(s,1H),8.11(dd,2.8Hz,8.0Hz,1H),7.89-7.79(m,1H),7.74-7.66(m,1H),7.14(brs,2H),4.41(t,6.8Hz,2H),2.02(t,7.2Hz,2H),1.89-1.82(m,2H),1.58-1.49(m,2H)；ESI-HRMS:378.1671[M+H]⁺。

Example 6

Synthesis of 7- (4-amino-3- (quinoline-3-) -1H-pyrazolo [3,4-d ] pyrimidine-1-) -N-hydroxyheptanamide (Compound 6).

Compound 6 was prepared according to example 1 by replacing ethyl 5-bromovalerate with ethyl 7-bromoheptanoate in step (1) and replacing pinacol ester 2-aminopyrimidine-5-boronic acid with pinacol ester quinoline-3-boronic acid in step (2) only and then hydroxylamine-hydrolyzing the same to obtain a white-like solid.

¹H NMR(400MHz,DMSO-d₆):δ10.33(s,1H),9.17(d,2.0Hz,1H),8.65(s,1H),8.60(d,1.6Hz,1H),8.31(s,1H),8.16-8.08(m,2H),7.89-7.79(m,1H),7.73-7.64(m,1H),7.14(brs,2H),4.40(t,6.8Hz,2H),1.98-1.82(m,4H),1.54-1.41(m,2H),1.37-1.24(m,4H)；ESI-HRMS:406.2000[M+H]⁺。

Example 7

Synthesis of 5- (4-amino-3- (2-aminobenzo [ d ] oxazole-5-) -1H-pyrazolo [3,4-d ] pyrimidine-1-) -N-hydroxypentanamide (Compound 7).

Compound 7 was prepared according to example 1 by replacing 2-aminopyrimidine-5-boronic acid pinacol ester with 2-aminobenzoxazole-5-boronic acid pinacol ester only in step (2) and then hydroxylamine-hydrolyzing the same to obtain a white-like solid.

¹H NMR(400MHz,DMSO-d₆):δ10.34(s,1H),8.67(s,1H),8.25(s,1H),7.99-7.39(m,6H),7.25(d,8.4Hz,1H),4.34(t,6.8Hz,2H),2.00(t,6.8Hz,2H),1.91-1.76(m,2H),1.58-1.44(m,2H)；ESI-HRMS:383.1575[M+H]⁺。

Example 8

Synthesis of 7- (4-amino-3- (2-aminobenzo [ d ] oxazole-5-) -1H-pyrazolo [3,4-d ] pyrimidine-1-) -N-hydroxyheptanamide (Compound 8).

Compound 8 was prepared according to example 1 by replacing ethyl 5-bromovalerate with ethyl 7-bromoheptanoate only in step (1) and replacing pinacol ester 2-aminopyrimidine-5-boronic acid with pinacol ester 2-aminobenzoxazole-5-boronic acid in step (2), followed by hydroxylamine hydrolysis using the same method to prepare an off-white solid.

¹H NMR(400MHz,DMSO-d₆):δ10.33(s,1H),8.66(s,1H),8.25(s,1H),8.00-7.37(m,4H),7.25(d,7.6Hz,1H),4.49-4.23(m,2H),2.05-1.75(m,4H),1.57-1.39(m,2H),1.37-1.21(m,4H)；ESI-HRMS:411.1885[M+H]⁺。

Example 9

Synthesis of 7- (3- (2-acetamidobenzo [ d ] oxazole-5-) -4-amino-1H-pyrazolo [3,4-d ] pyrimidine-1-) -N-hydroxyheptanamide (Compound 9).

Compound 9 was prepared according to example 1 by replacing ethyl 5-bromovalerate with ethyl 7-bromoheptanoate only in step (1) and replacing pinacol ester 2-aminopyrimidine-5-boronic acid with pinacol ester 2-acetamidobenzoxazole-5-boronic acid in step (2), followed by hydroxylamine hydrolysis using the same method to give an off-white solid.

ESI-MS:453.23[M+H]⁺。

Example 10

Synthesis of 7- (4-amino-3- (5-hydroxy-1H-indol-2-) -1H-pyrazolo [3,4-d ] pyrimidine-1-) -N-hydroxyheptanamide (Compound 10).

Compound 10 was prepared according to example 1 by replacing ethyl 5-bromovalerate with ethyl 7-bromoheptanoate in step (1) and replacing pinacol ester 2-aminopyrimidine-5-boronic acid with pinacol ester 5-hydroxyindole-2-boronic acid in step (2) only and then hydrolyzing with hydroxylamine using the same method to obtain an off-white solid.

ESI-MS:410.17[M+H]⁺。

Example 11

Synthesis of 4- ((4-amino-3- (2-aminobenzo [ d ] oxazole-5-) -1H-pyrazolo [3,4-d ] pyrimidin-1-) methyl) -N-hydroxybenzamide (compound 11).

Compound 11 was prepared according to example 1 by replacing ethyl 5-bromovalerate with methyl 4-chloromethylbenzoate only in step (1) and 2-aminopyrimidine-5-boronic acid pinacol ester with 2-aminobenzoxazole-5-boronic acid pinacol ester in step (2), followed by hydroxylamine hydrolysis using the same method to prepare an off-white solid.

¹H NMR(400MHz,DMSO-d₆):δ11.16(s,1H),9.03(s,1H),8.29(s,1H),7.70(d,8.0Hz,2H),7.67-7.51(m,4H),7.47(d,8.4Hz,1H),7.42(s,1H),7.35(d,7.6Hz,2H),7.24(d,8.0Hz,1H),5.61(s,2H)；ESI-HRMS:417.1413[M+H]⁺。

Example 12

Synthesis of 6- ((4-amino-3- (2-aminobenzo [ d ] oxazole-5-) -1H-pyrazolo [3,4-d ] pyrimidin-1-) methyl) -N-hydroxynicotinamide (Compound 12).

Compound 12 was prepared according to example 1 by replacing ethyl 5-bromovalerate with methyl 6-bromomethylnicotinate in step (1) only and 2-aminopyrimidine-5-boronic acid pinacol ester with 2-aminobenzoxazole-5-boronic acid pinacol ester in step (2) and then hydroxylamine-hydrolyzing to give an off-white solid using the same method.

¹H NMR(400MHz,DMSO-d₆):δ11.25(brs,1H),9.24(brs,1H),8.83(s,1H),8.27(s,1H),8.05(d,7.2Hz,1H),7.54(s,2H),7.47(d,8.4Hz,1H),7.43(s,1H),7.26(d,7.6Hz,2H),7.19(d,8.0Hz,1H),5.71(s,2H)；ESI-HRMS:418.1375[M+H]⁺。

Example 13

Synthesis of 2- ((4-amino-3- (2-aminobenzo [ d ] oxazole-5-) -1H-pyrazolo [3,4-d ] pyrimidine-1-) methyl) -N-hydroxypyrimidine-5-carboxamide (Compound 13)

Compound 13 was prepared according to example 1 by replacing ethyl 5-bromovalerate with methyl 2- (bromomethyl) pyrimidine-5-carboxylate in step (1) and 2-aminopyrimidine-5-boronic acid pinacol ester with 2-aminobenzoxazole-5-boronic acid pinacol ester in step (2) only and then hydrolyzing with hydroxylamine using the same method to prepare a white-like solid.

ESI-HRMS:419.1334[M+H]⁺。

Example 14

Synthesis of 5- ((4-amino-3- (2-aminobenzo [ d ] oxazole-5-) -1H-pyrazolo [3,4-d ] pyrimidin-1-) methyl) -N-hydroxyfuran-2-carboxamide (Compound 14).

Compound 14 was prepared according to example 1 by replacing ethyl 5-bromovalerate with methyl 5- (chloromethyl) furan-2-carboxylate in step (1) and 2-aminopyrimidine-5-boronic acid pinacol ester with 2-aminobenzoxazole-5-boronic acid pinacol ester in step (2) only and then hydroxylamine-hydrolyzing to prepare an off-white solid using the same method.

¹H NMR(400MHz,DMSO-d₆):δ10.91(brs,1H),9.16(brs,1H),8.30(s,1H),7.75-7.51(m,4H),7.47(d,8.0Hz,1H),7.41(s,1H),7.24(d,8.0Hz,2H),7.04-6.95(m,1H),6.51(d,2.8Hz,1H),5.59(s,2H)；ESI-HRMS:407.1310[M+H]⁺。

Example 15

Synthesis of 5- ((4-amino-3- (2-aminobenzo [ d ] oxazole-5-) -1H-pyrazolo [3,4-d ] pyrimidine-1-) methyl) -N-hydroxythiophene-2-carboxamide (compound 15).

Compound 15 was prepared according to example 1 by replacing ethyl 5-bromovalerate with methyl 5- (bromomethyl) thiophene-2-carboxylate in step (1) and 2-aminopyrimidine-5-boronic acid pinacol ester with 2-aminobenzoxazole-5-boronic acid pinacol ester in step (2) only and then hydrolyzing with hydroxylamine using the same method to prepare a white-like solid.

¹H NMR(400MHz,DMSO-d₆):δ11.16(brs,1H),9.13(brs,1H),8.31(s,1H),7.75-7.35(m,7H),7.30-7.20(m,1H),7.14(d,3.2Hz,1H),5.74(s,2H)；ESI-HRMS:423.0970[M+H]⁺。

Example 16

Synthesis of 5- ((4-amino-3- (2-aminobenzo [ d ] oxazole-5-) -1H-pyrazolo [3,4-d ] pyrimidin-1-) methyl) -N-hydroxyisoxazole-3-carboxamide (compound 16).

Compound 16 was prepared according to example 1 by replacing ethyl 5-bromovalerate with methyl 5- (bromomethyl) isoxazole-3-carboxylate in step (1) and 2-aminopyrimidine-5-boronic acid pinacol ester with 2-aminobenzoxazole-5-boronic acid pinacol ester in step (2) only and then hydroxylamine-cleaved using the same procedure to give a white-like solid.

ESI-HRMS:408.1181[M+H]⁺。

Example 17

Synthesis of 4- ((3- (2-acetamidobenzo [ d ] oxazole-5-) -4-amino-1H-pyrazolo [3,4-d ] pyrimidin-1-) methyl) -N-hydroxybenzamide (compound 17).

Compound 17 was prepared according to example 1 by replacing ethyl 5-bromovalerate with methyl 4-chloromethylbenzoate only in step (1) and replacing pinacol ester 2-aminopyrimidine-5-boronic acid with pinacol ester 2-acetamidobenzoxazole-5-boronic acid in step (2), followed by hydroxylamine hydrolysis using the same method to give an off-white solid.

ESI-HRMS:459.1531[M+H]⁺。

Example 18

Synthesis of 4- ((4-amino-3- (2- (methanesulfonamido) benzo [ d ] oxazole-5-) -1H-pyrazolo [3,4-d ] pyrimidin-1-) methyl) -N-hydroxybenzamide (compound 18).

Compound 18 was prepared by the same method as in example 1 except that ethyl 5-bromovalerate was replaced with methyl 4-chloromethylbenzoate in step (1) and pinacol ester 2-aminopyrimidine-5-boronic acid was replaced with 2- (methanesulfonamido) benzoxazole-5-boronic acid pinacol ester in step (2) and then subjected to hydroxylamine hydrolysis.

An off-white solid; ESI-HRMS 495.1211[ M + H ]]⁺。

Example 19

Synthesis of 4- ((4-amino-3- (5-hydroxy-1H-indol-2-) -1H-pyrazolo [3,4-d ] pyrimidin-1-) methyl) -N-hydroxybenzamide (Compound 19).

Compound 19 was prepared according to example 1 by replacing ethyl 5-bromovalerate with methyl 4-chloromethylbenzoate only in step (1) and replacing pinacol ester of 2-aminopyrimidine-5-boronic acid with pinacol ester of 5-hydroxyindole-2-boronic acid in step (2), followed by hydroxylamine hydrolysis using the same method to give an off-white solid.

ESI-HRMS:416.1475[M+H]⁺。

Example 20

Synthesis of 5- ((4-amino-3- (5-hydroxy-1H-indol-2-) -1H-pyrazolo [3,4-d ] pyrimidin-1-) methyl) -N-hydroxythiophene-2-carboxamide (Compound 20).

Compound 20 was prepared according to example 1 by replacing ethyl 5-bromovalerate with methyl 5- (bromomethyl) thiophene-2-carboxylate in step (1) and replacing pinacol ester 2-aminopyrimidine-5-boronic acid with pinacol ester 5-hydroxyindole-2-boronic acid in step (2) only and then hydrolyzing with hydroxylamine using the same method to prepare a white-like solid.

ESI-HRMS:422.1047[M+H]⁺。

Example 21

Synthesis of 4- ((4- ((4-amino-3- (2-aminobenzo [ d ] oxazole-5-) -1H-pyrazolo [3,4-d ] pyrimidine-1-) methyl) -1H-1,2, 3-triazole-1-) methyl) -N-hydroxybenzamide (compound 21).

(1) Synthesis of methyl 4- (azidomethyl) benzoate (intermediate 27):

methyl 4-chloromethylbenzoate (1.84g), sodium azide (0.85g) and anhydrous DMF (20mL) were added in this order to a reaction flask, and the reaction was stirred at 50 ℃. After the reaction, the system was extracted with Ethyl Acetate (EA), and washed with water and saturated brine in this order. The organic layer was passed over anhydrous Na₂SO₄Drying, concentrating under reduced pressure to obtain crude product, and performing silica gel column chromatography to obtain colorless oily substance 1.65 g.

(2) Synthesis of 3-iodo-1-propargyl-1H-pyrazolo [3,4-d ] pyrimidin-4-amine (intermediate 28):

synthesis of intermediate 28 referring to example 1, step (1), only ethyl 5-bromovalerate was replaced with propargyl bromide.

(3) Synthesis of methyl 4- ((4- ((4-amino-3-iodo-1H-pyrazolo [3,4-d ] pyrimidin-1-) methyl) -1H-1,2, 3-triazole-1-) methyl) benzoate (intermediate 29):

intermediate 27(0.40g), intermediate 28(0.48g), DCM (6mL) and MeOH (12mL) were added to the reaction flask in that order. After stirring uniformly, adding 1M CuSO into the obtained system in sequence₄The reaction mixture was stirred at room temperature for 5 hours in the presence of a solution (5mL) and a 1M sodium ascorbate solution (5mL), the reaction mixture was concentrated under reduced pressure, washed with water, extracted with a THF-EA mixed solvent (V/V ═ 1:4), and the organic layer was washed with saturated brine and dried over anhydrous Na₂SO₄Drying, concentrating under reduced pressure, and performing silica gel column chromatography to obtain white solid 0.38 g.

(4) Synthesis of methyl 4- ((4- ((4-amino-3- (2-aminobenzo [ d ] oxazole-5-) 1H-pyrazolo [3,4-d ] pyrimidine-1-) methyl) -1H-1,2, 3-triazole-1-) methyl) benzoate (intermediate 30):

synthesis of intermediate 30 referring to step (2) of example 1, only intermediate 24 was replaced with intermediate 29 and 2-aminopyrimidine-5-boronic acid pinacol ester was replaced with 2-aminobenzoxazole-5-boronic acid pinacol ester.

(5) Synthesis of Compound 21

Synthesis of Compound 21 Synthesis of intermediate 30 was subjected to hydroxylamine hydrolysis to give off-white solid in reference to step (3) of example 1.

ESI-HRMS:498.1759[M+H]⁺。

Example 22

Synthesis of 5- ((4- ((4-amino-3- (2-aminobenzo [ d ] oxazole-5-) -1H-pyrazolo [3,4-d ] pyrimidine-1-) methyl) -1H-1,2, 3-triazole-1-) methyl) -N-hydroxythiophene-2-carboxamide (compound 22).

Compound 22 was prepared with reference to example 21 by replacing methyl 4-chloromethylbenzoate with methyl 5- (bromomethyl) thiophene-2-carboxylate only in step (1), reacting intermediate 28 with the corresponding intermediate replacement intermediate 27 obtained in step (3), followed by Suzuki coupling and hydroxylamine hydrolysis to give an off-white solid.

ESI-HRMS:504.1326[M+H]⁺。

Test example 1: mTOR, HDAC inhibitory Activity

In the test example, a dual mTOR/PI3K inhibitor PI103 and a clinical ATP-competitive mTOR inhibitor BEZ235 are used as positive controls, and Lanth Ultra Assay is adopted to evaluate the mTOR inhibition activity of the compound. In addition, the compounds of the present invention were evaluated for HDAC1 and HDAC6 enzyme inhibitory Activity using a Fluorescent-based HDAC Activity Assay, with the above commercial broad-spectrum HDAC inhibitor SAHA as a positive control. Other compounds of the present invention have similar beneficial effects to those listed below, but this should not be understood as the only beneficial effects of the compounds of the present invention.

The test procedure for mTOR enzyme inhibitory activity was: preparing a DMSO solution of a compound to be detected, and preparing a kinase buffer solution according to the kit instruction; preparing a kinase solution and a substrate solution (containing ULight-4E-BP1 and ATP) by using a kinase buffer solution respectively; respectively adding the compound solution with gradient concentration, the kinase solution and the substrate solution into a 384-well plate to prepare a kinase reaction system; after reacting for a certain time at room temperature, terminating the reaction by adopting a Detection Solution Buffer (containing EDTA and Eu-anti-phosphorus-4E-BP 1), balancing, reading Lance signal values, and calculating the inhibition rate and IC (integrated circuit) under each concentration₅₀Obtained by fitting GraphPad Prism 5.

The HDAC1 enzyme inhibitory activity was tested by the following steps: preparing a sample to be testedPreparing a DMSO solution of the compound, and sequentially preparing a buffer solution, an enzyme solution and a corresponding Substrate/Trypsin mixed solution according to the kit instructions; adding the compound solution with gradient concentration, the enzyme solution and the Substrate/Trypsin mixed solution into a 384-hole plate respectively to prepare a catalytic reaction system (a compound-free control hole and an enzyme-free control hole are arranged); after incubation for a certain time at room temperature, continuously reading a fluorescence signal value by using a Synergy enzyme-labeling instrument, selecting a linear reaction section to obtain a slope (slope), and further calculating the inhibition rate and IC (integrated circuit) under each concentration₅₀Obtained by fitting GraphPad Prism 5 software.

Method for testing the inhibitory activity of compounds against HDAC6 referring to the method for testing the inhibitory activity of HDAC1, only the corresponding catalytic reaction system substrate needs to be replaced.

Inhibitory Activity of the Compounds of Table 1 on mTOR, HDAC1, HDAC6 enzymes

In table 1: "+ ++" represents 0-10 nM; "+ + + +" represents 10-100 nM; "+ +" represents 100 and 1000 nM; "+" represents 1000 + 10000 nM; "-" indicates not determined.

As can be seen from the enzyme inhibitory activity data in Table 1, most of the compounds of the present invention have significant dual mTOR/HDAC enzyme inhibitory activity. Among the compounds with remarkable dual mTOR/HDAC inhibitory activity, part of the compounds have mTOR inhibitory activity equivalent to or better than that of PI103 and BEZ-235, and have HDAC1 and HDAC6 inhibitory activity equivalent to or better than that of SAHA; the rest compounds with significant dual mTOR/HDAC inhibitory activity have mTOR inhibitory activity equivalent to or better than that of PI103 and BEZ-235, and HDAC6 inhibitory activity equivalent to or better than that of SAHA.

Test example 2: antitumor cell proliferation Activity

In the test example, BEZ235 and SAHA are used as positive controls, and the CCK-8 method is adopted to evaluate the antiproliferative activity of the compound with significant mTOR/HDAC dual-inhibitory enzyme activity on lung cancer cell strains A549 and colon cancer cell strains HCT 116. Other compounds of the present invention have similar beneficial effects to those listed below, but this should not be understood as the only beneficial effects of the compounds of the present invention.

The test procedure for the anti-tumor cell proliferation activity was: digesting and collecting tumor cells, inoculating the cells in a 96-well culture plate at a certain density, and placing the plate in an incubator (37 ℃, 5% CO)₂) Overnight. After the compound is applied for 72h, the culture medium is discarded, and the cells are washed gently with PBS 3 times. Subsequently, a certain volume of medium and CCK-8 were added to each well of the plate, and the incubation was continued for a certain period of time. Finally, a multifunctional microplate reader is adopted to measure the OD value of absorbance under the wavelength of 570nm, and the inhibition rate, GI is calculated₅₀Values were fitted by GraphPad Prism 5 software.

TABLE 2 antitumor cell proliferation Activity of Compounds with significant mTOR/HDAC Dual inhibitory Activity

In table 2: "+ ++" represents < 0.1. mu.M; "+ + + +" represents 0.1-1.0. mu.M; "+ +" represents 1.0-10. mu.M.

As can be seen from the antiproliferative activity data in Table 2, the compound having significant dual mTOR/HDAC (mammalian target of rapamycin/HDAC) inhibitory enzyme activity also shows significant antiproliferative activity on A549 and HCT116, and the antiproliferative activity of the antitumor cells is superior to or equivalent to BEZ235 and SAHA, so that the compound has good application prospect. Wherein, the anti-proliferation activity of most compounds on A549 and HCT116 is better than BEZ235 and SAHA.

Test example 3: HDAC6 Selectivity

The assay for HDAC1, HDAC2, HDAC3, HDAC6, HDAC8, HDAC10, HDAC11 enzyme inhibitory activity may reflect the selectivity of the compound for inhibition of the HDAC6 subtype. The selectivity of the compounds of the present invention for HDAC6 is further illustrated by the inhibition activity data of the compounds with significant mTOR, HDAC6, and antiproliferative activities on HDACs 1,2,3, 6, 8, 10, 11. It is not to be understood that the present invention is such that only the following compounds are selective for HDAC 6.

Method for testing inhibitory activity of compounds against other HDAC isoforms the catalytic reaction system substrate was only replaced when the corresponding enzyme inhibitory activity was tested, with reference to the HDAC1 inhibitory activity test method.

HDAC6 Selectivity of the Compounds of Table 3

In table 3: "+ + + +" represents 100-; "+ +" represents 30-100; "+" represents 10-30.

As is clear from tables 1 and 3, compounds 11 and 16 corresponding to formula (II) and compound 21 corresponding to formula (III) exhibit excellent selectivity for HDAC6 while significantly inhibiting HDAC 6; the broad spectrum HDACs inhibitor SAHA inhibits the activity of each HDAC subtype relatively closely, lacking selectivity for HDAC6 subtype. The compounds 11, 16 and 21 of the present invention are useful for reducing the toxicity of inhibiting all or many HDAC isoforms while significantly inhibiting HDAC6 isoform.

Test example 4: anti-proliferative Activity of Lung fibroblasts

Fibroblast proliferation can promote fibrosis, so the fibroblast proliferation inhibition activity of the compound can reflect the anti-IPF effect of the compound. In this test example, BEZ235 and SAHA were used as positive controls, and the anti-proliferative activity of the compound having significant mTOR/HDAC dual inhibitory activity of the present invention on lung fibroblast MLg2908 was evaluated by MTT method. Other compounds of the present invention have similar beneficial effects to those listed below, but this should not be understood as the only beneficial effects of the compounds of the present invention.

The test procedure for the anti-tumor cell proliferation activity was: MLg2908 cells are packed at a certain densityInoculating to 96-well culture plate, and placing in incubator (37 deg.C, 5% CO)₂) Overnight. Treating cells with compound solutions of different concentrations, allowing the compound to act for 72h, adding MTT solution into each well, culturing for a certain time, discarding supernatant of each well, adding DMSO into each well, shaking for a certain time, dissolving crystals completely, and measuring OD with enzyme-labeling instrument₅₇₀Calculating the inhibition ratio, GI₅₀Values were fitted by GraphPad Prism 5 software.

TABLE 4 anti-lung fibroblast proliferation Activity of Compounds with significant mTOR/HDAC Dual inhibitory Activity

In table 4: "+ ++" represents < 0.01. mu.M; "+ + + +" represents 0.01-0.1. mu.M; "+ +" represents 0.1-1. mu.M.

As can be seen from the data in table 4, the compound having significant mTOR/HDAC dual inhibitory activity of the present invention also exhibits significant antiproliferative activity to MLg2908, and the activity is superior to or equivalent to BEZ235 and SAHA, which has good application prospects.

The results show that the compound has good application prospects in the aspects of tumor resistance and IPF resistance.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (8)

1. A class of dual mTOR/HDAC inhibitors characterized by: the dual mTOR/HDAC inhibitor is a compound shown in the following general formulas (I) to (III) and a pharmaceutically acceptable salt or a deuteron thereof:

in the above formula, R is at least 1R₁Substituted C6-14 aryl, C5-14 heteroaryl, X is

Wherein n is 1-10;

in the general formula (II), ring A is at least 1R₂Substituted C6-14 aryl, C5-14 heteroaryl;

in the general formula (III), ring B is at least 1R₃Substituted C6-14 aryl, C5-14 heteroaryl;

R₁、R₂、R₃each independently selected from hydrogen, halogen, hydroxy, cyano, carbamoyl, trifluoromethyl, trifluoromethoxy, C1-6 alkyl, C1-6 alkoxy, C2-6 unsaturated aliphatic hydrocarbon, N (R)₄)₂、NR₄OR₄、NR₄N(R₄)₂、SO₂N(R₄)₂、NR₄SO₂R₄、NR₄CON(R₄)₂、NR₄COOR₄、NR₄COR₄、CON(R₄)₂Wherein R is₄Independently selected from hydrogen, C1-6 alkyl or C2-6 unsaturated aliphatic hydrocarbon.

2. A dual mTOR/HDAC inhibitor class according to claim 1, characterized in that: the dual mTOR/HDAC inhibitor is selected from the following compounds and pharmaceutically acceptable salts or deuterates thereof:

3. the dual mTOR/HDAC inhibitor class according to claim 1, wherein: the dual mTOR/HDAC inhibitor is a compound shown as general formulas (II) and (III) and a pharmaceutically acceptable salt or a deuteron thereof, wherein R is defined as claim 1;

in the general formula (II), X is

Wherein n is₁1-6, ring a is as defined in claim 1;

in the general formula (III), X and ring B are defined as in claim 1.

4. Use of a dual mTOR/HDAC inhibitor according to any one of claims 1-3 for the preparation of an anti-tumor medicament or an anti-idiopathic pulmonary fibrosis medicament, wherein the tumor comprises a solid tumor, a hematologic tumor.

5. A dual mTOR/HDAC inhibitor composition, characterized by: a dual mTOR/HDAC inhibitor comprising according to any of claims 1-3, further comprising at least one pharmaceutically acceptable carrier or excipient.

6. The use of an mTOR/HDAC dual inhibitor composition as claimed in claim 5 for the preparation of an anti-tumor drug or an anti-idiopathic pulmonary fibrosis drug.

7. The dual mTOR/HDAC inhibitor composition according to claim 5, further comprising at least one additional therapeutic agent, wherein the dual mTOR/HDAC inhibitor composition is in any clinically or pharmaceutically acceptable dosage form.

8. Use of a dual mTOR/HDAC inhibitor composition according to claim 7 for the preparation of an anti-tumor drug or an anti-idiopathic pulmonary fibrosis drug.