CN112500426A

CN112500426A - Pyrimidine boric acid derivative or pharmaceutically acceptable salt thereof, and preparation method and application thereof

Info

Publication number: CN112500426A
Application number: CN202011415791.6A
Authority: CN
Inventors: 王雪源; 杜晓
Original assignee: Suzhou Aihe Pharmaceutical Technology Co ltd
Current assignee: Suzhou Aihe Pharmaceutical Technology Co ltd
Priority date: 2020-12-03
Filing date: 2020-12-03
Publication date: 2021-03-16

Abstract

The invention discloses a VCP inhibitor derivative or a pharmaceutically acceptable salt thereof, and a preparation method and application thereof, wherein the VCP inhibitor derivative has a structure shown in the specification.

Description

Pyrimidine boric acid derivative or pharmaceutically acceptable salt thereof, and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicinal chemistry, in particular to a pyrimidine boric acid derivative or a pharmaceutically acceptable salt thereof, and a preparation method and application thereof.

Background

The AAA (atpase associated with multiple activities) atpase p97, which has a valosin containing protein, is conserved in all eukaryotes and essential for life in budding yeast and mice. People with a reduced function p97 allele suffer from syndromes that include inclusion body myopathy and frontal lobe degeneration. Loss of function studies in model organisms have shown that p97 plays a key role in a wide range of cellular processes including: golgi membrane reassembly, membrane transport, degradation of misfolded membranes and secreted proteins through the ubiquitin-proteasome system (UPS), regulation of myofibrillar assembly, and cell division. The broad range of cellular functions of this protein is believed to result from its ability to unfold the protein or dissociate protein complexes. The mechanochemical activity of p97 is related to the substrate protein by binding at least 14 UBX domain engager arrays of p97 and the non-UBX domain engagers Ufdl and Np 14. The p97 sequence is shown to have three domains (an N-domain, a D1 ATPase domain, and a D2 ATPase domain). X-ray crystallography of p97 shows that it forms a homohexamer of 97 kilodalton subunits, with 6 subunits assembled to form two stacked rings. These two loops are formed by the ATPase domains. The upper loop is formed by a hexamer of the D1 domain, while the lower loop is formed by a hexamer of the D2 domain. The N-domain extends outward from the D1 domain ring. Although it is clear that the D2 domain hydrolyses ATP in vitro, the levels of D1 specific atpase activity reported by different investigators vary. Nevertheless, genetic studies in yeast indicate that ATP hydrolysis by both the D1 and D2 domains is essential for the function of p 97. Binding of ATP to the D1 domain is also required for assembly of p 97. Although ATP hydrolysis by the D2 domain is not required for assembly of the p97 hexamer, it is believed that ATP hydrolysis by the D2 domain is a substrate conversion, causing their unfolding or dissociation from the binding partner.

The most detailed important cellular function of p97 was studied as its role in the turnover of misfolded secreted proteins via UPS (ubiquitin proteasome system). In a process called ERAD (for endoplasmic reticulum-associated degradation), proteins that fail to fold in the ER are translocated back into the cytoplasm in a p 97-dependent manner, and subsequently they are degraded by UPS. In this process, p97 is thought to mediate the extraction of substrate from the ER membrane. Complex p97 is also required for the turnover of cytoplasmic substrates of UPS, although its role in the turnover of cytoplasmic proteins is poorly understood.

The protein p97 containing valosin represents a suitable target for cancer therapeutics. Complex p97 and its function are essential for sustained cell viability, and therefore drugs that inhibit it should be antiproliferative. In other words, inhibition of p97 will result in undesirable protein aggregation within the target cell. The ensuing cellular response is often apoptosis or at least an improvement in cell growth and mitosis. Furthermore, it is known that overproduction of p97 in multiple cancers indicates that its activity may be limiting for the development of at least some cancers. P97 is known to be essential for ERAD, and recent studies have shown that cancer cells may be particularly dependent on ERAD. In addition, p97 has been implicated in the turnover of IKB and subsequent activation of NF-kB. NF-kB activity is important for the survival of some tumor cells, especially in multiple myeloma. Bortezomib has been shown to be active in multiple myeloma due to its ability to block protein turnover via the ERAD pathway and its ability to block IKB turnover, thereby inhibiting NF-kB activity. Given that p97 is involved in ERAD and IKB turnover, but additionally has a more limited effect in UPS than proteasome itself, drugs targeting p97 may retain most of the potency of bortezomib but have less toxicity.

Accordingly, there is a need to develop compounds suitable for inhibiting p97 activity and methods of using such compounds to inhibit p97 activity. There is a need to develop such compounds for the treatment of oncological disorders.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a pyrimidine boric acid derivative or a pharmaceutically acceptable salt thereof, a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that: a pyrimidine boric acid derivative or a pharmaceutically acceptable salt thereof is characterized in that the structure of the pyrimidine boric acid derivative is shown as formula (I),

wherein: r is selected from the group consisting of a hydrogen atom, -CN, -R^a、-C(O)(R^a)₂、-S(O)_tR^a、-C(O)R^a、-S(O)_tOR^aand-PO₃(R^a)₂。

The invention is oneIn the preferred embodiment, each R^aIndependently hydrogen, alkyl, haloalkyl, alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, 8-10 membered bicyclic aromatic carbocyclic ring, 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and any combination thereof.

In a preferred embodiment of the invention, the substituted alkenyl or alkynyl group is represented by-R^c、-OR^c、-SR^c、-N(R^c)₂、-N(R^c)C(O)R^c、-C(O)N(R^c)₂、-N(R^c)C(O)N(R^c)₂、-N(R^c)C(O)OR^c、-OC(O)N(R^c)₂、-N(R^c)S(O)_tR^c、-C(O)R^c、-C(O)OR^c、-OC(O)R^c、-S(O)_tOR^c、-S(O)_tN(R^c)₂and-PO₃(R^c)₂And (c) substituted, each t is independently selected from an integer of 1-2.

In a preferred embodiment of the invention, each R is^cIndependently hydrogen, alkyl, haloalkyl, alkoxy, alkanyl, 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, 8-10 membered bicyclic aromatic carbocyclic ring, 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and any combination thereof.

In a preferred embodiment of the present invention, the frame

Comprises the following steps:

in a preferred embodiment of the present invention, m and n in the skeleton are each independently an integer of 0 to 4, and the sum of m and n is an integer of 2, 3 or 4.

In a preferred embodiment of the present invention, a in the skeleton is selected from H, C, N, O.

In a preferred embodiment of the invention, the pyrimidine boronic acid derivatives are selected from the following table:

in a preferred embodiment of the invention, the pharmaceutically acceptable salt is used for preparing a medicament for inhibiting the VCP protein.

In a preferred embodiment of the present invention, the pharmaceutically acceptable salt at least comprises a boronic acid compound capable of inhibiting the function of the VCP protein, and can block the proliferation of tumor cells and induce the apoptosis of tumor cells.

The invention solves the defects in the background technology, and has the following beneficial effects:

(1) the invention relates to a pyrimidine boric acid derivative or a pharmaceutically acceptable salt thereof, wherein the boric acid derivative is a boric acid compound with a novel structure and a VCP protein inhibiting function, and the boric acid derivative can be used as a VCP protein inhibitor and can block the proliferation of tumor cells and induce the apoptosis of the tumor cells, so that the pyrimidine boric acid derivative can be used for treating and preventing various diseases of human and animals such as malignant tumor, and has a remarkably better effect.

(2) The compounds of the present invention and their pharmaceutical compositions are capable of acting as "inhibitors" of p97, and are capable of blocking or reducing the activity of enzymes, such as inhibiting various activities of p 97.

(3) The concentration of the compound in the pharmaceutically acceptable salts of the present invention will vary depending on a variety of factors including the dose of the compound administered, the pharmacokinetic characteristics of the compound used, and the route of administration. Another aspect of the invention provides combination therapy wherein one or more additional therapeutic agents are administered with the proteasome inhibitor of the present invention. Such combination therapy may be achieved by the simultaneous, sequential or separate administration of the treatment components.

Drawings

The invention is further explained below with reference to the figures and examples;

FIG. 1 is a scheme for the synthesis of tert-butyl (2-methyl-1H-indol-4-yl) carbamate according to example one of the present invention;

FIG. 2 is a scheme showing the synthesis scheme of 2, 4-dichloro-6, 7-dihydro-5H-cyclopenta [ d ] pyrimidine, a first example of the present invention;

FIG. 3 is a schematic diagram of the synthesis of 2, 4-dichloro-5, 6,7, 8-tetrahydroquinazoline in accordance with the first embodiment of the present invention;

FIG. 4 is a synthetic scheme for tert-butyl (1- (4- ((3-hydroxybenzyl) amino) -6, 7-dihydro-5H-cyclopenta [ d ] pyrimidin-2-yl) -2-methyl-1H-indol-4-yl) carbamate according to example one of the present invention;

FIG. 5 is a scheme showing the synthesis of (3- ((((2- (4-amino-2-methyl-1H-indol-1-yl) -6, 7-dihydro-5H-cyclopenta [ d ] pyrimidin-4-yl) amino) methyl) phenyl) boronic acid according to a first example of the present invention;

FIG. 6 is a schematic synthesis of (3- ((((2- (2-methyl-2- (methylsulfonylamino) -1H-indol-1-yl) -6, 7-dihydro-5H-cyclopenta [ d ] pyrimidin-4-yl) amino) methyl) phenyl) boronic acid of example one of the present invention.

Detailed Description

The preparation of representative compounds of the invention is described in more detail below. The following examples are provided for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily recognize a variety of non-critical parameters that may be altered or modified to produce substantially the same result.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the syntheses of the compounds and methods of use thereof described herein. While certain exemplary embodiments are depicted and described herein, it will be appreciated that the compounds of the present invention may be prepared according to methods generally available to those of ordinary skill in the art. All of the above-cited references and publications are hereby incorporated by reference.

Unless otherwise specified, all solvents, chemicals and reagents were commercially available and used without purification.

Example one

Example one is the synthetic preparation of compounds of the invention, suitable pharmaceutically acceptable base addition salts of compounds of the invention include, for example, metal salts, including alkali metal, alkaline earth metal and transition metal salts, such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines, such as, for example, N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanates. Although pharmaceutically unacceptable salts are generally not suitable for use as pharmaceutical agents, such salts may be useful, for example, as intermediates in the synthesis of the compounds of formula (1), for example in their purification by recrystallization. All these salts can be prepared by conventional methods from the corresponding compounds according to formula (I) by reacting, for example, the appropriate acid or base with the compounds according to formula (I).

Synthesis of tert-butyl (2-methyl-1H-indol-4-yl) carbamate as shown in FIG. 1:

s1, adding (100mL) DMSO and acetone (7.5mL, 100mmol) to m-nitroaniline (10g, 74mmol), stirring at room temperature for 5 minutes, adding potassium tert-butoxide (19g, 169mmol) to the mixture, stirring for 24 hours, adding an appropriate amount of water until the pH is 4, adding (100mL x 3) DCM to the reaction, extracting the organic phases, combining the organic phases, washing with water, washing with saturated sodium chloride, drying over anhydrous sodium sulfate and concentrating in vacuum. By column chromatography on silica gel(PE: EA. RTM.8: 1) and the residue was purified to give the product 2-methyl-4-nitro-1H-indole (7g, 54% yield, 98% purity) as a brown solid, MS (ESI) M/z:177.4[ M + H ] M/z]⁺。

S2, adding (2g, 14.2mmol) Pd (OH) into a solution of 2-methyl-4-nitro-1H-indole (7g, 39.7mmol) in 200mL of ethanol at room temperature, replacing hydrogen by the reaction for three times, stirring for 3 hours, filtering, concentrating the filtrate in vacuum, and purifying the residue to obtain a brown solid product, 2-methyl-1H-indol-4-amine (5.5g, yield 94%, purity 97%), MS (ESI) M/z 147.6[ M + H ] M/z]⁺。

S3, to a solution of 2-methyl-1H-indol-4-amine (5.5g, 37.6mmol) in DCM (150mL) was added (Boc)₂O, after stirring at room temperature for 24 hours, water was added (100mL), washed with saturated sodium chloride, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography on silica gel (PE: EA ═ 10:1) to give the product tert-butyl (2-methyl-1H-indol-4-yl) carbamate as a brown solid (7.8g yield 84%, purity 98%) ms (esi) M/z 247.3[ M + H ] M/z]⁺；¹HNMR(400MHz,DMSO-d₆)δ10.85(s,1H,NH),8.85(s,1H,NH),7.25(s,1H,Ph),6.96(d,J＝8.0Hz,1H,Ph),6.88(s,1H,Ph),6.37(s,1H,3Hofindole),2.35(s,3H,CH₃),1.49(s,9H,CH₃)。

As shown in fig. 2, synthesis of 2, 4-dichloro-6, 7-dihydro-5H-cyclopenta [ d ] pyrimidine:

s4, dissolving 2-oxocyclopentane-1-carboxylic acid ethyl ester (4g, 25.6mmol) in ethanol (20mL), adding Urea (2.3g, 38.4mmol) and HCl (0.4mL), heating the reaction to reflux and stirring for 4 hours, cooling the reaction to room temperature, concentrating the reaction in vacuum and evaporating the solvent, adding sodium hydroxide solution (20mL) into the obtained solid, heating the reaction to reflux and stirring for 30 minutes, filtering, collecting a filter cake, and drying to obtain the compound 6, 7-dihydro-5H-cyclopentyl [ d ] of the compound]Pyrimidine-2, 4-diol (2.6g, 69% yield, 98% purity) MS (ESI) M/z 153.1[ M + H ]]⁺。

S5, to 6, 7-dihydro-5H-cyclopenta [ d]To pyrimidine-2, 4-diol (2.6g, 17mmol) was added (4.12mL, 34mmol) DMA, dissolved to dryness and POCl was added₃(20mL), the reaction was warmed to 120 ℃ and stirred for 3 hours, cooled to room temperature and concentrated in vacuo to drynessThe resulting solid was poured into (100mL) water, (50mL x 3) DCM and extracted, the organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered and the solvent was evaporated in vacuo to give the compound 2, 4-dichloro-6, 7-dihydro-5H-cyclopenta [ d ] d]Pyrimidine (1.42g, yield 43%, purity 97%) MS (ESI) M/z 190.1[ M + H]⁺。

S6, to 2, 4-dichloro-6, 7-dihydro-5H-cyclopenta [ d]To pyrimidine (1g, 5.3mmol) and m-hydroxybenzylamine (0.8g, 6.8mmol) was added 10mL of IPA, followed by TEA (1g, 10.6mmol) and the mixture was placed in N₂The mixture was stirred at 80 ℃ for 12 hours. Then cooled to room temperature and concentrated. The residue was purified by chromatography (DCM/MeOH ═ 100:1) to give 3- (((2-chloro-6, 7-dihydro-5H-cyclopenta [ d)]Pyrimidin-4-acyl) amino) methyl) phenol as a white solid (0.57g, 40% yield, 97% purity).

As shown in fig. 3, synthesis of 2, 4-dichloro-5, 6,7, 8-tetrahydroquinazoline:

s7, adding 30mL of ethanol into ethyl 2-oxocyclohexane-1-carboxylate (2.5g, 14.7mmol) to dissolve until the mixture is clear, adding Urea (1.15g, 19.1mmol) and MeONa (5mL,5.4mol/Lin ethanol,29.4mmol), heating the reaction to 80 ℃, refluxing and stirring for 4 hours, cooling to room temperature, concentrating the evaporated solvent in vacuum, adding the obtained solid into sodium hydroxide solution (10mL), heating the reaction to reflux and stirring for 30 minutes, filtering, collecting a filter cake, and drying to obtain a

compound

5,6,7, 8-tetrahydroquinazoline-2, 4(1H, 3H) -diketone (2.03g, yield 83%, purity 97%) MS (ESI) M/z:167.6[ M + H ] -diketone]⁺。

S8, 5,6,7, 8-tetrahydroquinazoline-2, 4(1H, 3H) -dione (2.03g, 12.2mmol) was dissolved in (3g, 24.4mmol) DMA and POCl was added₃(13mL), the reaction temperature was raised to 120 ℃ and stirred under reflux for 3H, then cooled to room temperature, the solvent evaporated solid was concentrated in vacuo and poured into (50mL) water, extracted with DCM (50mL x 3), the organic phase washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered and the solvent evaporated in vacuo to give 2, 4-dichloro-5, 6,7, 8-tetrahydroquinazoline (1.1g, 45% yield, 98% purity) MS (ESI) M/z 204.7[ M + H ]]⁺。

Synthesis of tert-butyl (1- (4- ((3-hydroxybenzyl) amino) -6, 7-dihydro-5H-cyclopenta [ d ] pyrimidin-2-yl) -2-methyl-1H-indol-4-yl) carbamate as shown in figure 4:

s9 Synthesis of tert-butyl (2-methyl-1H-indol-4-yl) carbamate (1.3g,4.7mmol), 3- ((((2-chloro-6, 7-dihydro-5H-cyclopenta [ d ] d)]Pyrimidin-4-yl) amino) methyl) phenol (1.1g, 4.7mmol), Cs₂CO₃(2.3g，7.05mmol)、Pd₂(dba)₃(0.64g,0.7mmol) and X-Phos (0.33g, 0.7 mmol). Then 50mL of dioxane was added. The reaction was refluxed at 105 ℃ for 12 hours after replacing nitrogen by 3 times. The reaction was cooled to room temperature, filtered and the filtrate was collected and the solvent was evaporated under reduced pressure. The resulting solid was dissolved in DCM (50mL), washed with water (50mL), saturated sodium chloride solution (30mL), anhydrous Na₂SO₄Drying, filtering and evaporating the solvent under reduced pressure to obtain a crude product. Purification by column chromatography (PE/EtOAc ═ provides the product tert-butyl (1- (4- ((3-hydroxybenzyl) amino) -6, 7-dihydro-5H-cyclopenta [ d ] as a brown solid]Pyrimidin-2-yl) -2-methyl-1H-indol-4-yl carbamate (1.6g, 52% yield, 97% purity) MS (ESI) M/z 486.3[ M + H]⁺。

As shown in fig. 5, synthesis of (3- ((((2- (4-amino-2-methyl-1H-indol-1-yl) -6, 7-dihydro-5H-cyclopenta [ d ] pyrimidin-4-yl) amino) methyl) phenyl) boronic acid:

s10 Compound 13(2.3g, 4.74mmmol) was dissolved in THF (200mL) and PhNTf (5.08g, 14.22mmol) was added₂(2.8g, 28.44mmol) TEA, the resulting mixture was replaced with nitrogen 3 times and warmed to 50 ℃ for 12 hours, the reaction was directly evaporated to dryness under reduced pressure and the solvent was purified by column chromatography (PE/EtOAc ═ 5: 1) to give the product 3- ((((2- (4- ((tert-butoxycarbonyl) amino) -2-methyl-1H-indol-1-yl) -6, 7-dihydro-5H-cyclopenta [ d ] as a yellow solid]Pyrimidin-4-yl) amino) methyl) phenyl trifluoromethanesulfonate (1.68g, 58% yield, 97% purity) MS (ESI) M/z 618.4[ M + H]⁺。

S11, reacting 3- (((((2- (4- ((tert-butoxycarbonyl) amino) -2-methyl-1H-indol-1-yl) -6, 7-dihydro-5H-cyclopenta [ d)]Pyrimidin-4-yl) amino) methyl) phenyl triflate (2.48g, 4.01mmol) was dissolved in 1.4 dioxane (80mL) and (1.5g, 6.01g) (BPin) was added₂，(0.59g，0.8mmol)Pd(dppf)₂Cl₂(0.78g, 8.02mmol) KOAc, and the reaction mixture was stirredThe reaction was cooled to 110 ℃ and stirred for 6H, the reaction was cooled to room temperature, the solvent was evaporated in vacuo and purified by column chromatography to give tert-butyl (2-methyl-1- (4- (((3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl) amino) -6, 7-dihydro-5H-cyclopenta [ d ] as a yellow solid]Pyrimidin-2-yl) -1H-indol-4-yl carbamate (1.72g, 74% yield, 98% purity) MS (ESI) M/z 469.7[ M + H]⁺。

S12, to tert-butyl (2-methyl-1- (4- (((3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzyl) amino) -6, 7-dihydro-5H-cyclopenta [ d ] o]Pyrimidin-2-yl) -1H-indol-4-yl) carbamate (1.72g, 2.96mmol) was added THF (10mL) and H (10mL)₂O is stirred and dissolved, and NaIO is added₄(1.26g，5.92mmol),NH₄OAc (0.45g, 5.92mmol), stirred at room temperature for 12H, then the reaction was poured into (50mL) water, added (40mL x 3) EtOAc, the organic phase was isolated by extraction, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated in vacuo to dryness to afford the product (3- (((2- (4- ((tert-butoxycarbonyl) amino) -2-methyl-1H-indol-1-yl) -6, 7-dihydro-5H-cyclopenta [ d ] d) as a yellow solid which was purified by column chromatography (DCM/MeOH ═ 100:1)]Pyrimidin-4-yl) amino) methyl) phenyl) boronic acid (0.9g, yield 60%, purity 99%) MS (ESI) M/z 514.2[ M + H ]]⁺。

S13 reaction of (3- (((2- (4- ((tert-butoxycarbonyl) amino) -2-methyl-1H-indol-1-yl) -6, 7-dihydro-5H-cyclopenta [ d)]Pyrimidin-4-yl) amino) methyl) phenyl) boronic acid (1.2g, 2.3mmol) was added DCM (20mL) and, when the reaction mass was clear, cooled to 0 ℃, TFA (5mL, 30.6mmol) was slowly added dropwise, then warmed to room temperature and stirred for 3 hours, the reaction mass was concentrated in vacuo to dryness and the solvent was purified by column chromatography to give the product (3- (((2- (4-amino-2-methyl-1H-indol-1-yl) -6, 7-dihydro-5H-cyclopenta [ d ] d) as a yellow solid]Pyrimidin-4-yl) amino) methyl) phenyl) boronic acid (0.4g, yield 44%, purity 98%) MS (ESI) M/z 414.2[ M + H]⁺。

As shown in fig. 6, synthesis of (3- ((((2- (2-methyl-2- (methylsulfonamido) -1H-indol-1-yl) -6, 7-dihydro-5H-cyclopenta [ d ] pyrimidin-4-yl) amino) methyl) phenyl) boronic acid:

s14, adding (3- ((((2- (4-amino-2-methyl-1H-indol-1-yl) -6, 7-dihydro-5H-cyclopentyl [ d) under ice-bath condition]Pyrimidines-4-yl) amino) methyl) phenyl) boronic acid (0.1g, 0.22mmol) is dissolved in DCM (5mL), MsCl (0.027g, 0.24mmol) and TEA (0.067g, 0.66mmol) are added and the mixture is stirred at rt for 3H, the reaction is evaporated to dryness under reduced pressure and purified by column chromatography (DCM/MeOH ═ 70:1) to give the product (3- (((2- (2-methyl-2- (methylsulfonamido) -1H-indol-1-yl) -6, 7-dihydro-5H-cyclopenta [ d ] as a yellow solid]Pyrimidin-4-yl) amino) methyl) phenyl) boronic acid (0.08g, 75% yield, 98% purity MS (ESI) M/z 514.1[ M + Na%]⁺。

The synthesis of all boronic acid end products of the present invention is similar to compound 19.

The specific compounds synthesized and their names are given in the table below.

The compounds of the present invention are useful in the treatment of cachexia and muscle wasting diseases. The compounds of the present invention are useful for treating hyperproliferative conditions, and the compounds of the present invention are useful in methods of preventing or reducing scarring. The compounds of the invention are useful for treating ischemic conditions or reperfusion injury, and the compounds of the invention are useful for inhibiting TNFa for the prevention and treatment of septic shock. The compounds of the invention are useful for antigen presentation in prepared places, and the compounds of the invention are useful in methods for altering the overall composition of antigenic peptides produced from antithrombogenic or other combinations of proteins having multicatalytic activity. The compounds of the invention are useful in methods of inhibiting IKB-alpha degradation. As described above, these methods involve administering to the patient an effective amount of a compound or pharmaceutical formulation disclosed above such that aberrant P97 metalloprotease domain activity is ameliorated, reduced, or inhibited.

The concentration of the compound in the pharmaceutically acceptable salts of the present invention will vary depending on a variety of factors including the dose of the compound administered, the pharmacokinetic characteristics of the compound used, and the route of administration. Another aspect of the invention provides combination therapy wherein one or more additional therapeutic agents are administered with the proteasome inhibitor of the present invention. Such combination therapy may be achieved by the simultaneous, sequential or separate administration of the treatment components.

Example two

Examples in vitro and in vivo bioassays were conducted to determine the anti-cancer properties of the fused pyrimidine compounds of the invention. Specifically comprises an in vitro P97 inhibition experiment and an in vitro cytotoxicity experiment.

In vitro P97 inhibition assay

The p97 assay is the primary screening assay for determining the inhibitory activity of fused pyrimidine compounds of the invention against the p97 complex. Inhibition of the activity of the p97 proteasome complex can lead to apoptosis and elimination of neoplastic cells (cancer cells).

Reagents for the p97 assay included: the assay buffer was 2. mu.L of 97 hexamease, 1% DMSO, 1. mu.LATP, ADP-GloTMMax. The well plate is a Corning3674, 384 well plate. The identification kit is ADP-G1o kit (Promega), a termination buffer solution and a detection reagent.

The assay protocol was carried out as follows:

s1, compound dissolved in DMSO and formulated as a concentration gradient with 10 μ M as the starting concentration, 3-fold serial dilutions, the tenth concentration point is the solvent control (no drug);

s2, 2. mu.L of p97 hexamease (60. mu.g/mL) and 1. mu.L of different concentrations of compound were added per well in 384-well plates, two parallel sets were set up for each gradient, and incubated for 10min at room temperature. 1% DMSO was used as a solvent control, CB-5083 as a positive control;

s3, adding 1 mu LATP (100 mu M), fully mixing the reaction system, and incubating for 60min at 30 ℃;

s4, adding 4 mu L of ADP-GloTMMax reagent into each hole, and incubating for 40min at 25 ℃;

s5, adding 8 mu L of ADP-GloTMMax detection reagent into each hole, and incubating for 60min at 25 ℃;

s6, absorbance at 570nm was measured and the IC50 value for each compound was calculated using GraphPadprism5.0 software.

In vitro cytotoxicity assay

1. Freezing and storing cells:

(1) after harvesting the cells, the cells were centrifuged at 1000 rpm for 5min and rinsed with PBS.

(2) 1640 medium containing 7% DMSO and 10% fetal bovine serum was resuspended.

(3) And (3) respectively loading into a freezing tube, placing into a cell freezing box, carrying out overnight treatment at-80 ℃, and then preserving by liquid nitrogen.

2. Cell recovery and culture:

(1) taking out the freezing tube from the liquid nitrogen tank, and placing the tube into warm water at 37 ℃ to shake the tube gently so as to thaw the cell sap quickly.

(2) Transferring into a sterile centrifuge tube, adding 1640 culture solution containing 10% fetal calf serum, and gently blowing and beating into suspension.

(3) Centrifuging at 1000 r/v for 5min, discarding the supernatant, adding 10% culture medium 1640 containing fetal calf serum, and gently blowing to obtain suspension.

(4) Transferring into a culture bottle, and culturing in an incubator containing 5% CO 2at 37 deg.C and saturated humidity for 1-2 days.

3. Cell passage:

discarding the original culture medium, adding sterile PBS to wash once, adding 1 mL0.25% pancreatin to incubate for about 1 minute, observing under a mirror, carefully sucking the pancreatin after most cells begin to become round, adding fresh culture medium to stop digestion, blowing and beating the cells into uniform cell suspension, and transferring the cell suspension into a cell incubator to continue culture.

4. Cytotoxicity test

The specific measurement protocol is as follows

(1) A549 cells in logarithmic growth phase were seeded in 96-well plates at 190. mu.L per well volume, 2000 cells per well, and incubated at 37 ℃ under 5% CO2 for 24 h.

(2) mu.L of different concentrations of compound (DMSO concentration not exceeding 0.1%) were added to each well in 4 replicates per concentration with a maximum concentration of 10. mu.M and diluted in a 3-fold gradient, with the tenth concentration point being the solvent control (no drug). 1% DMSO was used as a solvent control and CB-5083 as a positive control.

(3) After incubation at 37 ℃ for 72h, 10. mu.L of CCK-8 detection reagent was added to each well and incubation was continued for 30 min.

(4) After shaking and mixing, absorbance was measured at 450nm and IC50 value was calculated using GraphPadPrism5.0 software.

5. Compound biological data:

in light of the foregoing description of the preferred embodiment of the present invention, it is to be understood that various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.

Claims

1. A pyrimidine boric acid derivative or a pharmaceutically acceptable salt thereof is characterized in that the structure of the pyrimidine boric acid derivative is shown as formula (I),

2. A pyrimidine boronic acid derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein: each R^aIndependently hydrogen, alkyl, haloalkyl, alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, 8-10 membered bicyclic aromatic carbocyclic ring, 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and any combination thereof.

3. A pyrimidine boronic acid derivative or a pharmaceutically acceptable salt thereof according to claim 2, wherein: said substituted alkenyl or alkynyl group is represented by-R^c、-OR^c、-SR^c、-N(R^c)₂、-N(R^c)C(O)R^c、-C(O)N(R^c)₂、-N(R^c)C(O)N(R^c)₂、-N(R^c)C(O)OR^c、-OC(O)N(R^c)₂、-N(R^c)S(O)_tR^c、-C(O)R^c、-C(O)OR^c、-OC(O)R^c、-S(O)_tOR^c、-S(O)_tN(R^c)₂and-PO₃(R^c)₂And (c) substituted, each t is independently selected from an integer of 1-2.

4. A pyrimidine boronic acid derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein: each of said R^cIndependently hydrogen, alkyl, haloalkyl, alkoxy, alkanyl, 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, 8-10 membered bicyclic aromatic carbocyclic ring, 4-8 membered saturated having 1-2 heteroatoms independently selected from nitrogen, oxygen or sulfurAnd or a partially unsaturated monocyclic heterocycle, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and any combination thereof.

5. A pyrimidine boronic acid derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein:

wherein the skeleton

Comprises the following steps:

6. a pyrimidine boronic acid derivative or a pharmaceutically acceptable salt thereof according to claim 5, wherein: m and n in the skeleton are each independently an integer of 0 to 4, and the sum of m and n is an integer of 2, 3 or 4.

7. A pyrimidine boronic acid derivative or a pharmaceutically acceptable salt thereof according to claim 5, wherein: the skeleton has A selected from H, C, N, O.

8. A pyrimidine boronic acid derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein: the pyrimidine boronic acid derivatives are selected from the following table:

9. a pyrimidine boronic acid derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein: the application of the pharmaceutically acceptable salt in preparing VCP protein inhibiting medicines.

10. A pyrimidine boronic acid derivative or a pharmaceutically acceptable salt thereof according to claim 9, wherein: the pharmaceutically acceptable salt at least contains a boric acid compound capable of inhibiting the function of VCP protein, and can block the proliferation of tumor cells and induce the apoptosis of the tumor cells.