Pyrimidine-hydroxamic acid compound and application thereof

Abstract

The invention discloses a pyrimidine-hydroxamic acid compound as well as a preparation method and application thereof. The compound has a general formula as shown in a formula I. The compound can be used as a small-molecule inhibitor of histone deacetylase (HDAC), has strong anti-malaria activity, has a remarkable killing effect on various wild and drug-resistant plasmodium, and has the potential of being developed into an anti-malaria drug with a novel action mechanism.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical chemistry, and in particular, the present application provides a class of small molecule compounds with 2,7-diaza-spiro-[3.5]-nonane pyrimidine-hydroxamic acid structure and the preparation method and use thereof. BACKGROUND

[0002] Malaria is a highly infectious and deadly disease caused mainly by the apicomplexan parasite Plasmodium falciparum. There are six species of Plasmodium that parasitize humans, namely P. falciparum, P. vivax, P. malariae, two species of P. ovale, and P. knowlesi. P. falciparum is the most harmful parasite in clinical practice because it is the only one that can cause serious complications and lead to death. Malaria is one of the most prominent problems in public health today and is the most widespread and harmful parasitic disease in the world. According to the World Health Organization (WHO) “2024 World Malaria Report” data released on December 11, 2024, there were approximately 263 million cases of malaria worldwide in 2023, an increase of about 11 million cases from 2022. Malaria not only puts a huge pressure on the public health system, but also has a significant negative impact on the economy of affected countries. Medical resources needed to treat malaria will squeeze other health projects, further burdening the public health system. In addition, malaria has a serious negative impact on the labor market, especially in Africa, where malaria is highly prevalent, a large number of labor forces cannot work normally due to illness or patient care, thereby affecting economic growth.

[0003] Despite the tremendous efforts made by humans to eradicate malaria over the past century, the prevention and control of malaria still face many difficult problems. Among them, the complex biological characteristics, multi-stage life cycle, and strong immune evasion ability of Plasmodium make it difficult to eradicate malaria at one stroke. More seriously, the resistance of Plasmodium to artemisinin-based combination therapies (ACTs) and other first-line drugs has shown a spreading trend. The latest monitoring data shows that the sensitivity of Plasmodium to artemisinin has decreased by 40% in the Cambodia-Thailand border area, and Plasmodium has a strong reproductive ability, producing about 10 12 substances per year, with a mutation rate 1000 times that of humans, and this evolution of drug resistance is shaking the foundation of the existing prevention and control system. This phenomenon not only weakens the effectiveness of existing treatment programs, but also casts a shadow over the global control and elimination goals of malaria.

[0004] Therefore, it is of great significance to find new targets of anti-malaria drugs, especially for key links in the life cycle of Plasmodium, and to develop anti-malaria drugs with new mechanisms of action different from existing chemotherapy drugs to solve the increasingly serious problem of drug resistance.

[0005] Histone deacetylase (HDAC) can catalyze the deacetylation of lysine and arginine residues in histone, thereby regulating the structure of cell chromatin, the process of transcription and gene expression. HDAC is a key epigenetic regulator involved in the progression and treatment of various human diseases. Currently, five malignant Plasmodium falciparum HDACs (PfHDAC) have been identified in malaria parasites. These enzymes modify the acetylation levels of several histone and non-histone proteins, and are essential for the basic biochemical processes of malaria parasites. In the field of prevention and treatment of parasitic diseases, HDAC inhibitors have shown potential for treating parasitic infections, such as Plasmodium, parasites, Trypanosoma and Toxoplasma.

[0006] Therefore, by developing new HDAC inhibitors, it is expected to produce excellent anti-malaria effects. SUMMARY

[0007] The present application provides a novel 2,7-diaza-spiro-[3.5]-nonane pyrimidine-hydroxamic acid compound with HDAC activity and anti-malaria efficacy.

[0008] In the first aspect of the present application, a pyrimidine-hydroxamic acid compound represented by Formula I and its optical isomer, non-racemic body, racemate, or a pharmaceutically acceptable salt thereof is provided:

[0009]

[0010] wherein R 1 is selected from the group consisting of H, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted -C0-C6 alkylene-C3-C18 carbocyclic ring, substituted or unsubstituted -C0-C6 alkylene-C6-C18 aryl, substituted or unsubstituted -C0-C6 alkylene-3-18 membered heterocyclic ring, substituted or unsubstituted -C0-C6 alkylene-5-18 membered heteroaryl;

[0011] wherein the substitution means that one or more hydrogen atoms on the group are replaced by a group selected from the group consisting of deuterium, halogen, hydroxyl, amino, cyano, oxo (C=O), C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C3-C7 carbocyclic ring, 3-7 membered heterocyclic ring, C6-C10 aryl, C5-C6 heteroaryl;

[0012] The carbon ring, heterocycle or heterocyclyl group can be a saturated or partially unsaturated structure, but not aromatic; the carbon ring, heterocycle, aromatic ring, heteroaromatic ring is optionally monocyclic, spirocyclic, annelated or bridged; the heterocyclyl or heteroaryl group each independently contains 1, 2, 3 or 4 heteroatoms selected from O, S and N.

[0013] In another preferred embodiment, the R 1 is

[0014] wherein L is a C0-C4alkylene group;

[0015] The connection site of the A ring to L is optionally a carbon atom or a heteroatom:

[0016] The A ring is selected from the group consisting of a substituted or unsubstituted C3-C16carbocyclic ring, a substituted or unsubstituted C6-C16aromatic ring, a substituted or unsubstituted 3-16 membered heterocyclic ring, a substituted or unsubstituted 5-16 membered heteroaromatic ring; and the A ring is optionally monocyclic, spirocyclic, annelated or bridged;

[0017] The hydrogen atoms on the A ring are optionally substituted by m R 2 ;

[0018] The R 2 is selected from the group consisting of deuterium, halogen, hydroxyl, amino, cyano, oxo (C=O), C1-C4alkyl, C1-C4alkoxy, C1-C4alkylamino, C3-C6cycloalkyl, phenyl;

[0019] m is selected from 0, 1, 2, 3, 4 or 5.

[0020] In another preferred embodiment, the A ring is a substituted or unsubstituted 3-16 membered fused cycloalkyl or 3-16 membered heterofused ring; wherein the heterofused ring has 1-3 heteroatoms selected from the group consisting of N, S and O; the carbon ring, heterocycle or heterocyclyl group can be a saturated or partially unsaturated structure, but not aromatic.

[0021] In another preferred embodiment, the A ring is

[0022] The connection site of the A ring to L is optionally a carbon atom or a heteroatom:

[0023] wherein A-1ring and A-2ring are each independently selected from the group consisting of a substituted or unsubstituted C3-C6carbocyclic ring, a substituted or unsubstituted phenyl ring, a substituted or unsubstituted 3-6 membered heterocyclic ring, a substituted or unsubstituted 5-6 membered heteroaromatic ring;

[0024] The R a and R bEach is independently selected from the following groups: deuterium, halogen, hydroxyl, amino, cyano, oxo (C=O), C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C3-C6 cycloalkyl, phenyl;

[0025] Or R a and R b It can be combined with the connected atoms to form a group consisting of one or more R atoms. c Substituted C3-C6 carbon rings, benzene rings, 3-6 membered heterocycles, and 5-6 membered heteroaromatic rings; wherein R c Each is independently selected from the following group: deuterium, halogen, hydroxyl, amino, cyano, oxo (C=O), C1-C4 alkyl;

[0026] p and q are each independently selected from 0, 1, 2, 3 or 4.

[0027] In another preferred embodiment, the A-1 ring in the compound is selected from the group consisting of: phenylcycloyl, pyridyl, pyridinyl, pyrimidinyl, pyrazinyl, pyrroleyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, imidazoleyl, or pyrazolyl.

[0028] In another preferred embodiment, the A-2 ring in the compound is selected from the group consisting of: phenylcycloyl, pyridinyl, pyridinyl, pyrazinyl, pyrrolithyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, cyclopentyl, cyclohexyl, tetrahydrofuranyl, pyrrolithyl, thiazolyl, dihydrofuranyl, pyrrolithyl, dihydrothiazolyl, pyrrolinyl, and oxazolinyl.

[0029] In another preferred embodiment, L in the compound is selected from the group consisting of none, methylene, and ethylene.

[0030] In another preferred embodiment, ring A is

[0031] It can be either a single bond or a double bond;

[0032] Z1, Z2, Z3, and Z4 are each independently selected from the following groups: chemical bond, CH2, CH2CH2, -CH=CH-, S, O, N, and NH;

[0033] The hydrogen atoms on ring A are optionally separated by one or more R atoms. 2 replace;

[0034] R 2 Selected from the following groups: deuterium, halogen, hydroxyl, amino, cyano, oxo (C=O), C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C3-C6 cycloalkyl, phenyl.

[0035] In another preferred embodiment, the A ring is a saturated or partially unsaturated C3-C8 carbon cyclic group;

[0036] A ring is optionally substituted by one or more R 2 ;

[0037] R 2 is selected from the group consisting of deuterium, halogen, hydroxyl, amino, cyano, oxo (C=0), C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C3-C6 cycloalkyl, phenyl.

[0038] In another preferred embodiment, the A ring is a C6-C18 aryl or a 5-18 membered heteroaryl, wherein the number of heteroatoms is 0-4, and the type of heteroatoms is selected from one, two or three of nitrogen, oxygen, sulfur.

[0039] In another preferred embodiment, the A ring is a C6-C18 aryl or a 5-18 membered heteroaryl comprising 1-4 fused rings, wherein each fused ring is independently a benzene ring or a 5-6 membered heteroaryl, and the number of heteroatoms in each fused ring is 0-2, and the type of heteroatoms is selected from one or two of nitrogen, oxygen, sulfur.

[0040] In another preferred embodiment, the A ring is a 7-15 membered carbocyclic or heterocyclic bridged ring; wherein the heterocyclic bridged ring has 1-3 heteroatoms selected from the group consisting of N, S and O;

[0041] A ring is optionally substituted by one or more R 2 ;

[0042] R 2 is selected from the group consisting of deuterium, halogen, hydroxyl, amino, cyano, oxo (C=0), C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C3-C6 cycloalkyl, phenyl.

[0043] In another preferred embodiment, the A ring is a group represented by formula II-1 or II-2:

[0044]

[0045] is optionally a single bond or a double bond;

[0046] wherein; X1, X2, X3and X4are each independently selected from the group consisting of CH2, CH2CH2, CH=CH-, O, S, N or NR A2 ; Y1, Y2, Y3and Y4are each independently CH or N;

[0047] wherein R A2 are each independently selected from the group consisting of hydrogen, C1-C4 alkyl;

[0048] A ring is optionally substituted by one or more R 2substituted;

[0049] R 2 selected from the group consisting of deuterium, halogen, cyano, C1-C4alkyl, C1-C4alkoxy, phenyl.

[0050] In another preferred embodiment, the A ring is selected from the group consisting of substituted or unsubstituted:

[0051]

[0052] wherein each X is independently selected from the group consisting of O, S, N or NH;

[0053] substituted means that a hydrogen atom on a group is optionally replaced by one or more R 2 substituted;

[0054] R 2 selected from the group consisting of deuterium, halogen, hydroxyl, amino, cyano, oxo (C=0), C1-C4alkyl, C1-C4alkoxy, C1-C4alkylamino, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl.

[0055] In another preferred embodiment, R 2 selected from the group consisting of deuterium, halogen, cyano, C1-C4alkyl, C1-C4alkoxy, phenyl.

[0056] In another preferred embodiment, R 1 selected from the group consisting of substituted or unsubstituted:

[0057]

[0058] substituted means that a hydrogen atom on a group is optionally replaced by one or more R 2 substituted;

[0059] R 2 selected from the group consisting of deuterium, fluorine, chlorine, bromine, hydroxyl, amino, cyano, oxo (C=0), methyl, ethyl, methoxy, ethoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl.

[0060] In another preferred embodiment, R 2 selected from the group consisting of deuterium, halogen, cyano, C1-C4alkyl, C1-C4alkoxy, phenyl.

[0061] In another preferred embodiment, the compound is selected from the group consisting of:

[0062]

[0063]

[0064] In another preferred embodiment, the pharmaceutically acceptable salt is a hydrochloride salt.

[0065] In a second aspect of the present application, there is provided a pharmaceutical composition, characterized in that the composition comprises: a pyrimidine-hydroxamic acid compound as described in the first aspect of the present application, and an optical isomer, a diastereomer, a racemate thereof, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

[0066] In a third aspect of the present application, there is provided the use of a pyrimidine-hydroxamic acid compound as described in the first aspect of the present application, and an optical isomer, a diastereomer, a racemate thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in the second aspect of the present application, which use comprises:

[0067] (i) for the manufacture of a medicament or a preparation for use in the treatment of a disease associated with histone deacetylase (HDAC) activity; and / or

[0068] (ii) for the manufacture of a medicament or a preparation for use in the treatment of malaria; and / or

[0069] (iii) as an HDAC inhibitor.

[0070] In another preferred embodiment, the use is for the manufacture of a medicament or a preparation for use in the treatment of malaria.

[0071] In another preferred embodiment, the disease comprises a tumor.

[0072] In another preferred embodiment, the use of the pharmaceutical composition further comprises the use of a single agent or a combination of agents in the prevention or treatment of malaria.

[0073] In another preferred embodiment, the pharmaceutical composition can further comprise other pharmaceutical ingredients having anti-malaria activity, including but not limited to artemisinin, dihydroartemisinin, artesunate, artemether, lumefantrine, sulfadoxine, pyrimethamine, pyronaridine, atovaquone, quinine, chloroquine, primaquine, mefloquine, amodiaquine, piperaquine, and tafenoquine.

[0074] In another preferred embodiment, the pharmaceutical composition or medicament is in the form of an oral dosage form or an injectable dosage form.

[0075] In another preferred embodiment, the oral dosage form comprises a tablet, a capsule, a film, a granule, and the like, and further comprises a sustained-release or non-sustained-release dosage form.

[0076] In another preferred embodiment, the pharmaceutical composition comprises 0.001 to 99 wt%, preferably 0.1 to 90 wt%, and more preferably 1 to 80 wt% of the compound of Formula I or a pharmaceutically acceptable salt thereof, based on the total weight of the composition.

[0077] In another aspect of the present application, there is also provided a method for preparing the pyrimidine-hydroxamic acid and intermediate compounds having the general structure of Formula (I), which is specifically synthesized as follows

[0078]

[0079] 1) Dissolve 2-chloro-pyrimidine-5-carboxylic acid ethyl ester (compound 24) and 2,7-diaza-spiro-[3.5]-nonane-7-carboxylic acid tert-butyl ester (compound 25) in dichloromethane, cool to 0-5°C in an ice bath, add N,N-diisopropyl ethylamine, then remove the ice bath and wait until the reaction solution returns to room temperature, and stir for 5-6 hours. Add water to the reaction solution, shake and separate, remove the solvent under reduced pressure, and purify the remaining solid by silica gel column chromatography to obtain 7-(5-(ethoxycarbonyl)pyrimidin-2-yl)-2,7-diazaspiro[3.5]nonane-2-carboxylic acid tert-butyl ester (compound 26) as a light yellow solid.

[0080] 2) Dissolve the compound obtained in step 1 in dichloromethane, add hydrogen chloride-dioxane solution, and stir at room temperature for 5-6 hours. Remove the solvent under reduced pressure to obtain 2-(2,7-diazaspiro[3.5]nonan-7-yl)pyrimidine-5-carboxylic acid ethyl ester as an oil, which is directly used in the next step (compound 27).

[0081] 3) Dissolve the compound obtained in step 2 in dichloromethane, add N,N-diisopropyl ethylamine, cool to 0-5°C in an ice bath, slowly drop in chloroformic acid benzyl ester, then remove the ice bath and wait until the reaction solution returns to room temperature, and stir for 5-6 hours. After the reaction, add saturated aqueous sodium bicarbonate solution, shake and separate, remove the solvent under reduced pressure, and purify the remaining solid by silica gel column chromatography to obtain 7-(5-(ethoxycarbonyl)pyrimidin-2-yl)-2,7-diazaspiro[3.5]nonane-2-carboxylic acid benzyl ester (compound 28) as a light yellow solid.

[0082] 4) Dissolve the compound obtained in step 3 in a mixture of methanol and water, add potassium carbonate solid, and heat to react at 65-70°C for 5-6 hours. After the reaction, remove the solvent under reduced pressure, acidify the residue with hydrochloric acid to pH about 1, filter, wash the remaining solid with water to pH about 7, and dry to obtain 2-((benzyloxy)carbonyl)-2,7-diazaspiro[3.5]nonan-7-yl)pyrimidine-5-carboxylic acid (compound 29) as a white solid.

[0083] 5) The compound obtained in step 4 is dissolved in a mixture of N,N- dimethylformamide and dichloromethane, 1-hydroxybenzotriazole and l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added successively, and stirred at room temperature for 30 minutes. Then O-(tetrahydro-2H-pyran-2- yl)hydroxylamine and triethylamine are added successively, and stirred at room temperature for 48 hours. After the reaction, saturated aqueous sodium bicarbonate solution is added, and the mixture is shaken and separated. The solvent is removed by evaporation under reduced pressure, and the remaining solid is separated and purified by silica gel column chromatography to obtain 7-(((tetrahydro-2H-pyran-2-yl)oxy)carbamoyl)pyrimidin-2-yl)-2,7- diazaspiro[3.5]nonane-2-carboxylic acid benzyl ester (compound 30) as a white solid.

[0084] 6) The compound obtained in step 5 is dissolved in a mixture of methanol and dichloromethane, triethylsilane and palladium-carbon are added successively, and stirred at 35°C until the starting material disappears. After the reaction, Pd / C is removed by filtration, and the solvent is removed by evaporation under reduced pressure to obtain 2-(2,7-diazaspiro[3.5]nonan-7-yl)-N-((tetrahydro-2H-pyran-2- yl)oxy)pyrimidine-5-carboxamide as a white solid, which is directly used in the next step (compound 31).

[0085] 7) The compound obtained in step 6 is dissolved in methanol, and R1-substituted aldehyde and sodium cyanoborohydride are added, and stirred at room temperature for 24-60 hours. After the reaction, saturated aqueous sodium bicarbonate solution is added, and the mixture is shaken and separated. The solvent is removed by evaporation under reduced pressure, and the remaining solid is separated and purified by silica gel column chromatography. Then the obtained compound is dissolved in dichloromethane, and hydrogen chloride-dioxane solution is added, and stirred at room temperature for 30 minutes. The solid is filtered and washed with a large amount of dichloromethane to obtain I.

[0086] According to the teachings of the above preparation methods, all the compounds encompassed by formula I can be obtained by those of ordinary skill in the art without any inventive step.

[0087] It should be understood that all the technical features described above and in the following (e.g., in the examples) can be combined with each other within the scope of the present application to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0088] Figure 1 The results of the first animal experiment in vivo efficacy of compound I-3 are shown in the schematic diagram. (A) Parasite rate curve. (B) Survival curve.

[0089] Figure 2 The results of the second animal experiment in vivo efficacy of orally administered compound I-3 are shown in the schematic diagram. (A) Parasite rate curve. (B) Survival curve.

[0090] Figure 3Western blot analysis of the effect of compound I-3 on the acetylation level of histone H3. DETAILED DESCRIPTION

[0091] After long-term and in-depth research, a pyrimidine-hydroxamic acid compound and a preparation method and use thereof are developed. The compound has a 2,7-diaza-spiro-[3.5]-nonane pyrimidine-hydroxamic acid structure, as shown in formula I. The compound can be used as a small molecule inhibitor of histone deacetylase (HDAC), has strong anti-malaria activity, shows significant killing effect on various wild-type and drug-resistant Plasmodium, has high safety, has no killing effect on normal human cells, shows good pharmacokinetic characteristics in vivo, and is expected to be applied to clinical development as a new anti-malaria drug. Based on this, the present application is completed.

[0092] Main advantages of the present application

[0093] A pyrimidine-hydroxamic acid compound with a relatively simple structure is provided. The compound has a simple preparation process, low production cost, strong inhibitory activity in HDAC enzyme inhibition experiments and in vivo and in vitro insecticidal efficacy experiments related to the survival and reproduction of Plasmodium, low toxicity, no killing effect on normal human cells, and good pharmacokinetic characteristics in rats and humans. The novel 2,7-diaza-spiro-[3.5]-nonane pyrimidine-hydroxamic acid compound provided in the present application is expected to be developed into a new anti-malaria drug, or combined with other anti-malaria drugs to achieve the effect of combined anti-malaria therapy.

[0094] In addition, the present application also discloses a novel 2,7-diaza-spiro-[3.5]-nonane pyrimidine-hydroxamic acid compound.

[0095] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples without specific conditions are generally carried out according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0096] Example 1

[0097] Preparation of 7-(5-(ethoxycarbonyl)pyrimidin-2-yl)-2,7-diazaspiro[3.5]nonane-2-carboxylic acid tert-butyl ester (intermediate 26)

[0098]

[0099] Dissolve 24 g of tert-butyl 2,7-diazaspiro[3.5]nonane-7-carboxylate and 22.4 g of ethyl 2-chloro-pyrimidine-5-carboxylate in 500 mL of dichloromethane, cool to 0-5°C in an ice bath, add 25 mL of N,N-diisopropylethylamine, then remove the ice bath and allow the reaction to warm to room temperature. Stir for 6 hours. Add water to the reaction and shake to separate the layers. Remove the solvent under reduced pressure and purify the remaining solid by silica gel column chromatography to obtain a light yellow solid.

[0100] 1 H NMR (400 MHz, DMSO) δ 8.77 (s, 2H), 4.26 (q, J = 7.1 Hz, 2H), 3.81 (d, J = 21.3 Hz, 4H), 3.61 (s, 4H), 1.71 (t, J = 5.6 Hz, 4H), 1.38 (s, 9H), 1.28 (t, J = 7.1 Hz, 3H).

[0101] Example 2

[0102] Preparation of ethyl 2-(2,7-diazaspiro[3.5]nonan-7-yl)pyrimidine-5-carboxylate (Intermediate 27)

[0103]

[0104] Dissolve 39 g of tert-butyl 7-(5-(ethoxycarbonyl)pyrimidin-2-yl)-2,7- diazaspiro[3.5]nonane-2-carboxylate (Intermediate 26) in 500 mL of dichloromethane, slowly add 100 mL of 4 M hydrogen chloride-dioxane solution, and stir at room temperature for 6 hours. Remove the solvent under reduced pressure to obtain an oil, which is used directly in the next step.

[0105] 1 H NMR (400 MHz, DMSO) δ 8.78 (s, 2H), 4.26 (q, J = 7.1 Hz, 2H), 3.84 - 3.80 (m, 4H), 3.75 (s, 4H), 1.82 - 1.78 (m, 4H), 1.29 (t, J = 7.1 Hz, 3H).

[0106] Example 3

[0107] Preparation of benzyl 7-(5-(ethoxycarbonyl)pyrimidin-2-yl)-2,7- diazaspiro[3.5]nonane-2-carboxylate (Intermediate 28)

[0108]

[0109] To a solution of 29 g of ethyl 2-(2,7-diazaspiro[3.5]nonan-7-yl)pyrimidine-5-carboxylate (Intermediate 27) in 500 mL of dichloromethane was added 41 mL of N,N-diisopropylethylamine, and the mixture was cooled to 0-5 °C in an ice bath. Then 17 mL of phenyl chloroformate was added dropwise slowly. After the addition was completed, the ice bath was removed, and the mixture was stirred for 6 h at room temperature. After the reaction was completed, the mixture was diluted with saturated aqueous sodium bicarbonate solution, and the mixture was shaken to separate the layers. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give a light yellow solid.

[0110] 1 H NMR (400 MHz, CDCl3) δ 8.81 (s, 2H), 7.35-7.24 (m, 5H), 4.33 (q, J = 7.1 Hz, 2H), 3.87-3.81 (m, 4H), 3.68 (s, 2H), 3.12 (s, 4H), 1.85-1.79 (m, 4H), 1.36 (t, J = 7.1 Hz, 3H).

[0111] Example 4

[0112] Preparation of 2-((benzyloxy)carbonyl)-2,7-diazaspiro[3.5]nonan-7-yl)pyrimidine-5-carboxylic acid (Compound 29)

[0113]

[0114] To a solution of 40 g of benzyl 7-(5-(ethoxycarbonyl)pyrimidin-2-yl)-2,7-diazaspiro[3.5]nonane-2-carboxylate (Intermediate 28) in a mixture of methanol-water (1:1) 500 mL was added 35 g of potassium carbonate solid, and the mixture was heated at 65-70 °C for 6 h. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was acidified with hydrochloric acid to pH about 1. The mixture was filtered, and the residue was washed with water to pH about 7. The solid was dried to give 2-((benzyloxy)carbonyl)-2,7-diazaspiro[3.5]nonan-7-yl)pyrimidine-5-carboxylic acid as a white solid.

[0115] 1 H NMR (400 MHz, DMSO) δ 11.64 (s, 1H), 8.75 (d, J = 2.3 Hz, 2H), 7.65-7.59 (m, 2H), 7.44 (d, J = 5.5 Hz, 3H), 4.40 (d, J = 5.8 Hz, 2H), 3.85 (t, J = 7.0 Hz, 6H), 3.77 (d, J = 6.0 Hz, 2H), 1.95 (t, J = 5.0 Hz, 2H), 1.84 (d, J = 8.5 Hz, 2H).

[0116] Example 5

[0117] Preparation of 7-(((tetrahydro2H-pyran-2-yl)oxy)carbamoyl)pyrimidin-2-yl)-2,7- diazaspiro[3.5]nonane-2-carboxylic acid benzyl ester (Compound 30)

[0118]

[0119] Dissolve 19.8 g of 2-((benzyloxy)carbonyl)-2,7-diazaspiro[3.5]nonan-7-yl)pyrimidine-5- carboxylic acid (Intermediate 29) in a mixed solution of N,N-dimethylformamide and dichloromethane (1:1) 250 mL, add 13.5 g of 1-hydroxybenzotriazole and 19.2 g of 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride successively, stir for 30 minutes at room temperature, add 17.8 g of O-(tetrahydro-2H-pyran-2-yl)hydroxylamine and 21 mL of triethylamine successively, and stir for 48 hours at room temperature. After completion of the reaction, add a saturated aqueous sodium bicarbonate solution, shake and separate, evaporate the solvent under reduced pressure, and purify the remaining solid by silica gel column chromatography to obtain a white solid.

[0120] 1 H NMR (400 MHz, DMSO) δ 8.67 (s, 2H), 7.41 - 7.30 (m, 5H), 5.04 (s, 2H), 4.95 (d, J = 3.3 Hz, 1H), 4.07 - 3.99 (m, 1H), 3.72 (d, J = 27.9 Hz, 8H), 3.53 (d, J = 11.2 Hz, 1H), 1.71 (q, J = 7.6 Hz, 7H), 1.59 - 1.50 (m, 3H).

[0121] Example 6

[0122] Preparation of 2-(2,7-diazaspiro[3.5]nonan-7-yl)-N-((tetrahydro2H-pyran-2-yl)oxy)pyrimidine- 5-carboxamide (Compound 31)

[0123]

[0124] Dissolve 5 g of 7-(((tetrahydro2H-pyran-2-yl)oxy)carbamoyl)pyrimidin-2-yl)-2,7- diazaspiro[3.5]nonane-2-carboxylic acid benzyl ester (Intermediate 30) in a mixed solution of methanol and dichloromethane (1:1) 50 mL, add 2.13 g of 10% palladium on carbon and 8 mL of triethylsilane successively, and stir until the starting material disappears at 35°C. After completion of the reaction, remove Pd / C by filtration, and evaporate the solvent under reduced pressure to obtain white solid 2-(2,7-diazaspiro[3.5]nonan-7-yl)-N-((tetrahydro2H-pyran-2-yl)oxy)pyrimidine-5- carboxamide, which is directly used in the next reaction.

[0125] 1 H NMR (600 MHz, DMSO) δ 8.66 (s, 2H), 4.95 (d, J = 3.2 Hz, 1H), 4.02 (ddd, J = 12.1, 9.0, 3.6 Hz, 1H), 3.78 - 3.73 (m, 4H), 3.54 - 3.50 (m, 1H), 3.17 (s, 1H), 1.76 - 1.66 (m, 7H), 1.58 - 1.50 (m, 3H).

[0126] Example 7

[0127] Preparation of final product

[0128]

[0129] Dissolve 2-(2,7-diazaspiro[3.5]nonan-7-yl)-N-((tetrahydro2H-pyran-2-yl)oxy)pyrimidine-5- carboxamide methanol, add R 1 substituted aldehyde, sodium cyanoborohydride, stir at room temperature for 24-60 hours. Upon completion, add saturated aqueous sodium bicarbonate solution, shake and separate, evaporate solvent under reduced pressure, and purify the remaining solid by silica gel column chromatography. Dissolve the obtained compound in dichloromethane, add hydrogen chloride-dioxane solution, and stir at room temperature for 30 minutes. Filter, and wash the solid with a large amount of dichloromethane to obtain I.

[0130] Example 8

[0131] Preparation of 2-(cyclopentylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N-hydroxypyrimidine-5- carboxamide hydrochloride (Compound I-1)

[0132]

[0133] Prepare I-1 from 31 and cyclopentylcarboxaldehyde according to the procedure of Example 7.

[0134] 1 H NMR (400 MHz, DMSO) δ 11.10 (s, 1H), 8.69 (s, 2H), 4.00 (dd, J = 10.6, 6.6 Hz, 2H), 3.86 - 3.79 (m, 4H), 3.73 (dd, J = 6.9, 4.5 Hz, 2H), 1.97 (t, J = 5.6 Hz, 2H), 1.77 (dt, J = 16.3, 5.9 Hz, 4H), 1.59 (tq, J = 9.1, 3.8 Hz, 2H), 1.50 (tt, J = 7.4, 3.4 Hz, 2H), 1.27 - 1.18 (m, 2H). HRMS (ESI) m / z calcd. for C 18 H 27N5O2, [M+H] + 346.2238, found 346.2244.

[0135] Example 9

[0136] Preparation of 2-(2-(cyclohexylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N- hydroxyprimidine-5-carboxamide hydrochloride (Compound I-2)

[0137]

[0138] I-2 was prepared from 31 and cyclohexylcarboxaldehyde following the procedure of Example 7.

[0139] 1 H NMR (400 MHz, DMSO) δ 11.08 (s, 1H), 8.69 (s, 2H), 4.03 (dd, J = 10.5, 6.5 Hz, 2H), 3.81 (td, J = 6.6, 2.8 Hz, 4H), 3.73 (dd, J = 7.4, 4.0 Hz, 2H), 3.05 (t, J = 6.5 Hz, 2H), 2.01 - 1.94 (m, 2H), 1.82 - 1.73 (m, 4H), 1.70 - 1.65 (m, 2H), 1.61 (d, J = 10.0 Hz, 2H), 1.25 - 1.19 (m, 1H), 1.14 (dd, J = 19.5, 10.9 Hz, 2H), 1.00 - 0.89 (m, 2H). HRMS (ESI) m / z calcd. for C 19 H 29 N5O2, [M+H] + 360.2394, found 360.2391.

[0140] Example 10

[0141] Preparation of 2-[2-(cycloheptylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl]-N- hydroxyprimidine-5-carboxamide hydrochloride (Compound I-3)

[0142]

[0143] I-3 was prepared from 31 and cycloheptylcarboxaldehyde following the procedure of Example 7.

[0144] 1H NMR (400MHz, DMSO) δ10.85 (s, 1H), 8.68 (s, 2H), 4.02 (dd, J = 10.7, 6.6Hz, 2H), 3.82 (d t,J=11.1,5.3Hz,4H),3.73(dd,J=6.8,4.5Hz,2H),3.07(d,J=13.1Hz,2H),1.96(t,J= 5.7Hz,2H),1.87–1.78(m,3H),1.73(ddt,J=13.7,6.8,2.8Hz,2H),1.64–1.50(m,4H), 1.41(tdd,J=18.9,9.2,4.3Hz,4H),1.21(ddt,J=15.9,9.6,4.6Hz,2H).HRMS(ESI)m / z calcd.for C 20 H 31 N5O2,[M+H] + 374.2551, found 374.2557.

[0145] Example 11

[0146] Preparation of 2-(2-((4,4-difluorocyclohexyl)methyl)-2,7-diazaspiro[3.5]nonane-7-yl)-N-hydroxypyrimidine-5-carboxamide hydrochloride (compound I-4)

[0147]

[0148] I-4 was prepared by reacting 31 with 4,4-difluorocyclohexane-1-carboxaldehyde according to the steps in Example 7.

[0149] 1 H NMR (400MHz, DMSO) δ11.20(s,1H),10.67(d,J=4.9Hz,1H),8.96(s,1H),6.96(d,J=4.7Hz,1H),4.05(d,J=5.3Hz,2H),3.83(dd,J=10.8,6.3Hz,2H),3. 54(s,1H),3.45(d,J=5.5Hz,1H),3.14(t,J=6.7Hz,2H),2.03(t,J=9.7Hz, 4H),1.89–1.82(m,4H),1.79(s,2H),1.24(q,J=13.1Hz,3H).HRMS(ESI)m / z calcd.for C 19 H 27 F2N5O2,[M+H] + 396.2206, found 396.2212.

[0150] Example 12

[0151] N-hydroxy-2-(2-(tetrahydro-2H-pyran-4-ylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl)pyrimidine-5- carboxamide hydrochloride (Compound I-5)

[0152]

[0153] I-5 was prepared from 31 and tetrahydro-2H-pyran-4-carbaldehyde following the procedure of Example 7.

[0154] 1 H NMR (400 MHz, DMSO) δ 11.06 (s, 1H), 8.68 (s, 2H), 4.05 - 4.02 (m, 2H), 3.85 (d, J = 5.8 Hz, 2H), 3.83 - 3.79 (m, 4H), 3.73 (dd, J = 7.1, 4.2 Hz, 2H), 3.29 (d, J = 2.1 Hz, 1H), 3.23 (d, J = 2.2 Hz, 1H), 3.12 (t, J = 6.7 Hz, 2H), 1.96 (t, J = 5.6 Hz, 2H), 1.88 - 1.84 (m, 1H), 1.80 (t, J = 5.8 Hz, 2H), 1.68 (s, 1H), 1.64 (s, 1H), 1.25 (dd, J = 11.9, 4.2 Hz, 1H), 1.21 - 1.15 (m, 1H). HRMS (ESI) m / z calcd. for C 18 H 27 N5O3, [M+H] + 362.2187, found 362.2193.

[0155] Example 13

[0156] Preparation of 2-(2-(cyclohex-3-en-1-ylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N-hydroxypyrimidine-5- carboxamide hydrochloride (Compound I-6)

[0157]

[0158] I-6 was prepared from 31 and cyclohexenecarboxaldehyde following the procedure of Example 7.

[0159] 1H NMR (400 MHz, DMSO) δ 11.14 (s, 1H), 10.65 (s, 1H), 8.94 (s, 1H), 6.96 (d, J = 4.7 Hz, 1H), 5.71 - 5.58 (m, 2H), 4.10 - 3.96 (m, 4H), 3.90 - 3.81 (m, 2H), 3.59 - 3.51 (m, 2H), 3.50 - 3.42 (m, 2H), 3.15 - 3.09 (m, 2H), 2.13 (d, J = 17.2 Hz, 1H), 2.04 (s, 3H), 1.86 (t, J = 3.8 Hz, 2H), 1.82 - 1.72 (m, 2H), 1.30 - 1.16 (m, 1H). HRMS (ESI) m / z calcd for C 19 H 27 N5O2, [M+H] + 358.2238, found 358.2242.

[0160] Example 14

[0161] Preparation of (N-hydroxy-2-(2-((4-oxocyclohexyl)methyl)-2,7-diazaspiro[3.5]nonan-7- yl)pyrimidine-5-carboxamide hydrochloride (Compound I-7)

[0162]

[0163] I-7 was prepared from 31 and cyclohexanone formaldehyde following the procedure of Example 7.

[0164] 1 H NMR (400 MHz, DMSO) δ 11.16 (s, 1H), 8.68 (s, 2H), 4.08 (dd, J = 10.6, 6.6 Hz, 2H), 3.90 - 3.79 (m, 4H), 3.74 (s, 2H), 3.21 (t, J = 6.4 Hz, 2H), 2.37 (td, J = 13.5, 5.4 Hz, 2H), 2.22 (d, J = 14.7 Hz, 2H), 2.08 (d, J = 13.0 Hz, 2H), 1.99 (d, J = 7.3 Hz, 2H), 1.85 - 1.78 (m, 2H), 1.52 - 1.38 (m, 2H). HRMS (ESI) m / z calcd for C 19 H 27 N5O3, [M+H] + 374.2187, found 374.2184.

[0165] Example 15

[0166] 2-(2-(Bicyclo[2.2.1]hept-5-en-2-ylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N- hydroxyprimidine-5-carboxamide hydrochloride (Compound 1-8)

[0167]

[0168] I-8 was prepared from 31 and bicyclo[2.2.1]heptan-5-ene-2-carboxaldehyde following the procedure of Example 7.

[0169] 1 H NMR (400 MHz, DMSO) δ 8.68 (s, 2H), 6.21 (dd, J = 5.7, 3.0 Hz, 1H), 6.08 (dt, J = 16.0, 3.9 Hz, 1H), 4.00 (d, J = 16.0 Hz, 2H), 3.82 (q, J = 5.4 Hz, 4H), 3.72 (t, J = 5.6 Hz, 2H), 2.93 (s, 1H), 2.83 (d, J = 16.2 Hz, 1H), 1.96 (s, 2H), 1.88 (ddd, J = 12.4, 9.0, 3.7 Hz, 1H), 1.78 (t, J = 5.7 Hz, 2H), 1.41 - 1.19 (m, 4H), 0.58 (d, J = 12.0 Hz, 1H). HRMS (ESI) m / z calcd for C 20 H 27 N5O2, [M+H] + 370.2238, found 370.2228.

[0170] Example 16

[0171] 2-(2-(Bicyclo[2.2.1]hept-5-en-2-ylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N- hydroxyprimidine-5-carboxamide hydrochloride (Compound 1-8)

[0172]

[0173] I-9 was prepared from 31 and adamantane-1-carboxaldehyde following the procedure of Example 7.

[0174] 1H NMR (400 MHz, DMSO) δ 8.68 (s, 2H), 4.18 (dd, J = 10.7, 6.7 Hz, 2H), 3.85 (dd, J = 10.8, 5.8 Hz, 2H), 3.79 (dd, J = 7.2, 4.1 Hz, 2H), 3.76 - 3.70 (m, 2H), 3.01 (s, 2H), 2.02 - 1.97 (m, 2H), 1.95 (t, J = 3.4 Hz, 3H), 1.81 (t, J = 5.7 Hz, 2H), 1.68 (s, 1H), 1.65 (s, 2H), 1.61 (d, J = 2.9 Hz, 9H). HRMS (ESI) m / z calcd for C 23 H 33 N5O2, [M+H] + 412.2707, found 412.2716.

[0175] Example 17

[0176] Preparation of 2-(2-(benzyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N-hydroxy pyrimidine-5- carboxamide hydrochloride (Compound I-10)

[0177]

[0178] I-10 was prepared from 31 and benzaldehyde following the procedure of Example 7.

[0179] 1 H NMR (400 MHz, DMSO) δ 11.39 (s, 1H), 8.68 (s, 2H), 7.61 (dd, J = 6.5, 2.7 Hz, 2H), 7.44 (dd, J = 5.1, 1.9 Hz, 3H), 4.41 (d, J = 6.1 Hz, 2H), 3.91 - 3.84 (m, 4H), 3.82 - 3.78 (m, 2H), 3.76 - 3.72 (m, 2H), 1.93 (t, J = 5.7 Hz, 3H), 1.87 - 1.79 (m, 3H). HRMS (ESI) m / z calcd for C 19 H 23 N5O2, [M+H] + 354.1925, found 354.1924.

[0180] Example 18

[0181] Preparation of N-hydroxy-2-(2-(pyridin-4-ylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl) pyrimidine-5-carboxamide hydrochloride (Compound I-11)

[0182]

[0183] I-11 was prepared from 31 and 4-pyridinecarboxaldehyde following the procedure of Example 7.

[0184] 1 H NMR (400 MHz, DMSO) δ 8.99 (s, 1H), 8.89 (d, J = 5.6 Hz, 1H), 8.68 (d, J = 3.7 Hz, 2H), 8.25 (s, 1H), 8.02 (d, J = 5.5 Hz, 1H), 4.68 (d, J = 5.2 Hz, 2H), 3.98 (d, J = 6.6 Hz, 4H), 3.81 (s, 2H), 3.74 (s, 2H), 1.98 (s, 2H), 1.85 (s, 2H). HRMS (ESI) m / z calcd for C 18 H 22 N6O2, [M+H] + 355.1877, found 355.1874.

[0185] Example 19

[0186] Preparation of 2-(2-(furan-3-ylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N- hydroxy pyrimidine-5-carboxamide hydrochloride (Compound I-12)

[0187]

[0188] I-12 was prepared from 31 and furan-3-carboxaldehyde following the procedure of Example 7.

[0189] 1 H NMR (400 MHz, DMSO) δ 10.74 (s, 1H), 9.01 (s, 1H), 7.88 (d, J = 4.4 Hz, 1H), 7.73 (t, J = 1.7 Hz, 1H), 6.97 (d, J = 4.7 Hz, 1H), 6.74 (d, J = 1.8 Hz, 1H), 4.23 (t, J = 6.1 Hz, 2H), 3.84 (d, J = 3.4 Hz, 2H), 3.82 (s, 2H), 3.55 (t, J = 5.0 Hz, 2H), 3.51 - 3.46 (m, 2H), 2.00 (d, J = 6.0 Hz, 2H), 1.85 (s, 2H). HRMS (ESI) m / z calcd for C 17 H 21 N5O3, [M+H] + 344.1717, found 344.1717.

[0190] Example 20

[0191] Preparation of N-hydroxy-2-(2-(thiophen-3-ylmethyl)-2,7-diazaspiro[3.5]nonan-7- yl)pyrimidine-5-carboxamide hydrochloride (Compound 1-13)

[0192]

[0193] Compound 1-13 was prepared from 31 and 3-thiophenecarboxaldehyde following the procedure of Example 7.

[0194] 1 H NMR (400 MHz, DMSO) δ 11.12 (s, 1H), 8.67 (s, 2H), 7.79 (t, J = 3.4 Hz, 1H), 7.64 (dt, J = 5.4, 2.8 Hz, 1H), 7.33 (d, J = 10.4 Hz, 1H), 4.39 (d, J = 5.9 Hz, 2H), 3.86 (s, 4H), 3.80 (d, J = 5.9 Hz, 2H), 3.73 (t, J = 5.7 Hz, 2H), 1.90 (s, 2H), 1.79 (t, J = 5.7 Hz, 2H). HRMS (ESI) m / z calcd for C 17 H 21 N5O2S, [M+H] + 360.1489, found 360.1489.

[0195] Example 21

[0196] Preparation of N-hydroxy-2-(2-(1-methyl-1H-indol-3-ylmethyl)-2,7-diazaspiro[3.5]nonan-7- yl)pyrimidine-5-carboxamide hydrochloride (Compound 1-14)

[0197]

[0198] Compound 1-14 was prepared from 31 and 1-methylindole-3-carboxaldehyde following the procedure of Example 7.

[0199] 1 H NMR (400 MHz, DMSO) δ 11.02 (s, 1H), 8.67 (s, 2H), 7.87 (d, J = 7.9 Hz, 1H), 7.65 (s, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.13 (t, J = 7.4 Hz, 1H), 4.52 (d, J = 5.6 Hz, 2H), 3.91 - 3.69 (m, 11H), 1.90 - 1.68 (m, 4H). HRMS (ESI) m / z calcd for C22 H 26 N6O2,[M+H] + 407.2190, found 407.2180.

[0200] Example 22

[0201] Preparation of 2-(2-(benzofuran-3-ylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N- hydroxyprimidine-5-carboxamide hydrochloride (Compound I-15)

[0202]

[0203] I-15 was prepared from 31 and benzofuran-3-carboxaldehyde following the procedure of Example 7.

[0204] 1 H NMR (400 MHz, DMSO) δ 8.67 (s, 2H), 8.29 (s, 1H), 7.99 (d, J = 7.4 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.42 - 7.33 (m, 2H), 4.60 (s, 1H), 4.59 (s, 1H), 3.96 (t, J = 7.2 Hz, 4H), 3.81 (t, J = 5.4 Hz, 2H), 3.74 (t, J = 5.8 Hz, 2H), 1.92 (s, 2H), 1.85 - 1.80 (m, 2H). HRMS (ESI) m / z calcd for C 21 H 23 N5O3,[M+H] + 394.1874, found 394.1877.

[0205] Example 23

[0206] Preparation of 2-(2-(benzo[b]thiophen-3-ylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N- hydroxyprimidine-5-carboxamide hydrochloride (Compound I-16)

[0207]

[0208] I-16 was prepared from 31 and benzothiophene-3-carboxaldehyde following the procedure of Example 7.

[0209] 1H NMR (400 MHz, DMSO) δ 11.57 - 11.48 (m, 1H), 8.68 (s, 2H), 8.19 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.48 (dt, J = 19.9, 7.2 Hz, 2H), 4.74 (d, J = 5.9 Hz, 2H), 3.97 (d, J = 6.4 Hz, 4H), 3.84 - 3.79 (m, 2H), 3.74 (t, J = 5.4 Hz, 2H), 1.90 (dt, J = 38.2, 5.2 Hz, 4H). HRMS (ESI) m / z calcd for C 21 H 23 N5O 2S ,[M+H] + 410.1645, found 410.1642.

[0210] Example 24

[0211] Preparation of N-hydroxy-2-(2-(l-methyl-lH-benzo[d]imidazol-2-ylmethyl)-2,7- diazaspiro[3.5]nonan-7-yl)pyrimidine-5-carboxamide hydrochloride (Compound I-17)

[0212]

[0213] I-17 was prepared from 31 and l-methyl-2-formylbenzoimidazole following the procedure of Example 7.

[0214] 1 H NMR (400 MHz, DMSO) δ 8.69 (s, 2H), 7.78 (dd, J = 14.6, 8.0 Hz, 2H), 7.45 (dt, J = 19.7, 7.3 Hz, 2H), 5.03 (s, 2H), 4.19 (d, J = 32.6 Hz, 4H), 3.98 (s, 3H), 3.81 (s, 4H), 1.93 (s, 4H). HRMS (ESI) m / z calcd for C 21 H 25 N7O2,[M+H] + 408.2142, found 408.2138.

[0215] Example 25

[0216] Preparation of N-hydroxy-2-(2-(imidazo[l,2-a]pyridin-3-ylmethyl)-2,7- diazaspiro[3.5]nonan-7-yl)pyrimidine-5-carboxamide hydrochloride (Compound I-18)

[0217]

[0218] I-18 was prepared from 31 and imidazo[l,2-a]pyridine-3-carbaldehyde following the procedure of Example 7.

[0219] 1 H NMR (400 MHz, DMSO) δ 11.99 (s, 1H), 11.14 (s, 1H), 9.27 (d, J = 6.9 Hz, 1H), 8.68 (s, 2H), 8.55 (s, 1H), 8.04 (d, J = 4.1 Hz, 2H), 7.61 (dt, J = 7.5, 4.1 Hz, 1H), 5.08 - 5.01 (m, 2H), 4.12 (t, J = 8.0 Hz, 2H), 3.97 - 3.91 (m, 2H), 3.83 - 3.76 (m, 4H), 1.92 (dd, J = 14.1, 7.9 Hz, 4H). HRMS (ESI) m / z calcd for C 20 H 23 N7O2, [M+H] + 394.1986, found 394.1979.

[0220] Example 26

[0221] Preparation of 2-(2-(2,3-dihydro-lH-inden-5-ylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N- hydroxyprimidine-5-carboxamide hydrochloride (Compound I-19)

[0222]

[0223] I-19 was prepared from 31 and 2,3-dihydro-lH-inden-5-carbaldehyde following the procedure of Example 7.

[0224] 1 H NMR (400 MHz, DMSO) δ 11.99 (s, 1H), 11.14 (s, 1H), 9.27 (d, J = 6.9 Hz, 1H), 8.68 (s, 2H), 8.55 (s, 1H), 8.04 (d, J = 4.1 Hz, 2H), 7.61 (dt, J = 7.5, 4.1 Hz, 1H), 5.08 - 5.01 (m, 2H), 4.12 (t, J = 8.0 Hz, 2H), 3.97 - 3.91 (m, 2H), 3.83 - 3.76 (m, 4H), 1.92 (dd, J = 14.1, 7.9 Hz, 4H). HRMS (ESI) m / z calcd for C 22 H 27N5O2, [M+H] + 394.2238, found 394.2240.

[0225] Example 27

[0226] Preparation of 2-(2-(2,3-dihydrobenzofuran-5-ylmethyl)-2,7-diazaspiro[3.5]nonan-7- yl)-N-hydroxyprimidine-5-carboxamide hydrochloride (Compound I-20)

[0227]

[0228] I-20 was prepared from 31 and 2,3-dihydrobenzofuran-5-carbaldehyde following the procedure of Example 7.

[0229] 1 H NMR (400 MHz, DMSO) δ 11.29 (d, J = 39.9 Hz, 1H), 8.67 (s, 2H), 7.47 (s, 1H), 7.32 (d, J = 8.1 Hz, 1H), 6.80 (d, J = 8.1 Hz, 1H), 4.54 (d, J = 8.7 Hz, 2H), 4.27 (d, J = 6.0 Hz, 2H), 3.87 - 3.77 (m, 6H), 3.73 (t, J = 5.2 Hz, 2H), 3.17 (s, 2H), 1.91 (s, 2H), 1.84 - 1.77 (m, 2H). HRMS (ESI) m / z calcd. for C 21 H 25 N5O3, [M+H] + 396.2030, found 396.2030.

[0230] Example 28

[0231] Preparation of 2-(2-(2,3-dihydrobenzofuran-7-ylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N- hydroxyprimidine-5-carboxamide hydrochloride (Compound I-21)

[0232]

[0233] I-21 was prepared from 31 and 2,3-dihydrobenzofuran-7-carbaldehyde following the procedure of Example 7.

[0234] 1H NMR (400 MHz, DMSO) δ 11.20 (s, 1H), 8.67 (s, 2H), 7.33 - 7.27 (m, 2H), 6.89 (t, J = 7.5 Hz, 1H), 4.61 (t, J = 8.7 Hz, 2H), 4.32 (d, J = 5.8 Hz, 2H), 3.96 - 3.87 (m, 4H), 3.80 (dd, J = 7.4, 4.0 Hz, 2H), 3.73 (dd, J = 6.9, 4.4 Hz, 2H), 3.24 (t, J = 8.7 Hz, 2H), 1.90 (t, J = 5.7 Hz, 2H), 1.77 (t, J = 5.8 Hz, 2H). HRMS (ESI) m / z calcd for C 21 H 25 N5O3, [M+H] + 396.2030, found 369.2023.

[0235] Example 29

[0236] Preparation of 2-(2-(benzo[d][l,3]dioxol-5-ylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N- hydroxy pyrimidine-5-carboxamide (Compound I-22)

[0237]

[0238] I-22 was prepared from 31 and benzo[d][l,3]dioxol-5-carbaldehyde following the procedure of Example 7.

[0239] 1 H NMR (400 MHz, DMSO) δ 11.60 (s, 1H), 8.67 (d, J = 1.2 Hz, 2H), 7.27 - 7.22 (m, 1H), 7.08 (dt, J = 8.0, 1.9 Hz, 1H), 6.97 (dt, J = 8.0, 1.4 Hz, 1H), 6.06 (s, 2H), 4.28 (d, J = 6.1 Hz, 2H), 3.87 - 3.79 (m, 6H), 3.74 (t, J = 5.6 Hz, 2H), 1.92 (s, 2H), 1.81 (dd, J = 7.1, 4.4 Hz, 2H). HRMS (ESI) m / z calcd for C 20 H 23 N5O4, [M+H] + 398.1823, found 398.1814.

[0240] Example 30

[0241] Preparation of N-hydroxy-2-(2-(quinolin-3-ylmethyl)-2,7-diazaspiro[3.5]nonan-7- yl)pyrimidine-5-carboxamide hydrochloride (Compound 1-23)

[0242]

[0243] Compound 1-23 was prepared from 31 and quinoline-3-carbaldehyde following the procedure of Example 7.

[0244] 1 H NMR (400 MHz, DMSO) δ 11.41 (s, 1H), 8.69 (s, 2H), 8.49 (d, J = 8.4 Hz, 1H), 8.06 (dd, J = 14.9, 8.2 Hz, 2H), 7.84 (t, J = 7.6 Hz, 1H), 7.66 (dd, J = 16.4, 8.1 Hz, 2H), 4.88 (d, J = 5.3 Hz, 2H), 4.10 (d, J = 18.0 Hz, 4H), 3.81 (s, 4H), 1.93 (s, 4H). HRMS (ESI) m / z calcd. for C 22 H 24 N6O2, [M+H] + 405.2034, found 405.2033.

[0245] Example 31

[0246] Preparation of N-hydroxy-2-(2-(naphthalen-2-ylmethyl)-2,7-diazaspiro[3.5]nonan-7- yl)pyrimidine-5-carboxamide hydrochloride (Compound 1-24)

[0247]

[0248] Compound 1-24 was prepared from 31 and 2-naphthaldehyde following the procedure of Example 7.

[0249] 1H NMR (400 MHz, DMSO) δ 12.12 (s, 1H), 8.70 (s, 2H), 8.18 (s, 1H), 8.00 - 7.95 (m, 2H), 7.93 (dd, J = 6.3, 3.3 Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.59 (dt, J = 6.2, 3.4 Hz, 2H), 4.58 (d, J = 6.1 Hz, 2H), 3.94 (dd, J = 10.4, 6.4 Hz, 2H), 3.85 (dd, J = 10.4, 6.4 Hz, 2H), 3.79 (t, J = 5.6 Hz, 2H), 3.76 - 3.71 (m, 2H), 1.97 (t, J = 5.5 Hz, 2H), 1.90 - 1.82 (m, 2H). HRMS (ESI) m / z calcd for C 23 H 25 N5O2, [M+H] + 404.2081, found 404.2075.

[0250] Example 32

[0251] Preparation of 2-(2-(l,l'-biphenyl-4-ylmethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N- hydroxyimidazole-5-carboxamide hydrochloride (Compound I-25)

[0252]

[0253] I-25 was prepared from 31 and l,l'-biphenyl-4-carboxaldehyde following the procedure of Example 7.

[0254] 1 H NMR (400 MHz, DMSO) δ 11.90 - 11.79 (m, 1H), 8.69 (s, 2H), 7.73 (s, 4H), 7.71 (d, J = 1.6 Hz, 1H), 7.69 (d, J = 1.8 Hz, 1H), 7.49 (t, J = 7.6 Hz, 2H), 7.42 - 7.37 (m, 1H), 4.45 (d, J = 6.1 Hz, 2H), 3.90 (tt, J = 10.6, 5.3 Hz, 4H), 3.84 - 3.79 (m, 2H), 3.75 (dd, J = 7.2, 4.1 Hz, 2H), 1.99 - 1.92 (m, 2H), 1.88 - 1.82 (m, 2H). HRMS (ESI) m / z calcd for C 25 H 27 N5O2, [M+H] + 430.2238, found 430.2228.

[0255] Example 33

[0256] Preparation of N-hydroxy-2-(2-(phenanthren-9-ylmethyl)-2,7-diazaspiro[3.5]nonan-7- yl)pyrimidine-5-carboxamide hydrochloride (Compound 1-26)

[0257]

[0258] I-26 was prepared from 31 and 9-phenylmethanal following the procedure of Example 7.

[0259] 1 H NMR (400 MHz, DMSO) δ 11.12 (s, 1H), 8.98 - 8.91 (m, 1H), 8.88 (d, J = 8.1 Hz, 1H), 8.68 (s, 2H), 8.36 - 8.30 (m, 1H), 8.17 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.80 (dt, J = 7.8, 4.7 Hz, 2H), 7.78 - 7.68 (m, 2H), 5.04 (d, J = 6.0 Hz, 2H), 4.11 (d, J = 6.2 Hz, 4H), 3.82 (t, J = 5.1 Hz, 2H), 3.77 (t, J = 4.8 Hz, 2H), 1.93 (dt, J = 16.9, 5.5 Hz, 4H). HRMS (ESI) m / z calcd for C 27 H 27 N5O2, [M+H] + 454.2238, found 454.2236.

[0260] Example 34

[0261] Preparation of N-hydroxy-2-(2-(2-(1-methyl-1H-indol-3-yl)ethyl)-2,7-diazaspiro[3.5]nonan-7- yl)pyrimidine-5-carboxamide hydrochloride (Compound 1-27)

[0262]

[0263] I-27 was prepared from 31 and 3-(1-methyl-1H-indol-3-yl)propanal following the procedure of Example 7.

[0264] 1H NMR (400MHz, DMSO) δ11.31(s,1H),8.68(s,2H),7.64(d,J=7.8Hz,1H),7.41(d,J=8.2 Hz,1H),7.22(s,1H),7.17(t,J=7.6Hz,1H),7.06(t,J=7.4Hz,1H),3.98(dd,J=10.3,6 .5Hz,2H),3.90–3.82(m,4H),3.75(s,3H),3.74–3.70(m,2H),3.43(q,J=7.0Hz,2H), 2.98(t,J=7.8Hz,2H),1.98–1.91(m,2H),1.84–1.77(m,2H).HRMS(ESI)m / zcalcd.for C 23 H 28 N6O2,[M+H] + 421.2347, found 421.2343.

[0265] Example 35

[0266] Preparation of N-hydroxy-2-(2-phenylethyl-2,7-diazaspiro[3.5]nonane-7-yl)pyrimidine-5-carboxamide hydrochloride (compound I-28)

[0267]

[0268] I-28 was prepared by reacting 31 with 3-phenylpropanal according to the steps in Example 7.

[0269] 1 H NMR (400MHz, DMSO) δ11.35(s,1H),8.69(s,2H),7.37–7.24(m,6H),3.95(dd,J=10.6,6.5Hz,2H),3.85(dt,J=11.8,6.2Hz,4H),3.73 (dd,J=6.9,4.5Hz,2H),3.47–3.39(m,2H),2.90–2.83(m,2H),1.94(t,J=5.6Hz,2H),1.80(t,J=5.6Hz,2H).HRMS(ESI)m / zcalcd.for C 20 H 25 N5O2,[M+H] + 368.2081, found 368.2075.

[0270] Example 36

[0271] 2-(2-(2-cyclohexylethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N-hydroxy pyrimidine-5- carboxamide hydrochloride (Compound 1-29)

[0272]

[0273] I-29 was prepared from 31 and 3-cyclohexylpropanal following the procedure of Example 7.

[0274] 1 H NMR (400 MHz, DMSO) δ 10.91 (s, 1H), 8.68 (d, J = 1.5 Hz, 2H), 3.94 (t, J = 8.4 Hz, 2H), 3.85 - 3.76 (m, 4H), 3.75 - 3.70 (m, 2H), 3.16 (q, J = 6.0 Hz, 2H), 1.91 (s, 2H), 1.79 (t, J = 5.8 Hz, 2H), 1.66 (d, J = 11.8 Hz, 6H), 1.38 (q, J = 7.3 Hz, 2H), 1.16 (p, J = 12.3 Hz, 5H), 0.90 (q, J = 11.3 Hz, 3H). HRMS (ESI) m / z calcd. for C 20 H 31 N5O2, [M+H] + 374.2551, found 374.2545.

[0275] Example 37

[0276] 2-(2-(2-(2,3-dihydrobenzofuran-5-yl)ethyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N- hydroxy pyrimidine-5-carboxamide hydrochloride (Compound 1-30)

[0277]

[0278] I-30 was prepared from 31 and 3-(2,3-dihydrobenzofuran-5-yl)propanal following the procedure of Example 7.

[0279] 1H NMR (400 MHz, DMSO) δ 11.50 (s, 1H), 8.69 (s, 2H), 7.16 (d, J = 1.8 Hz, 1H), 6.99 (dd, J = 8.1, 1.9 Hz, 1H), 6.71 (d, J = 8.1 Hz, 1H), 4.50 (t, J = 8.7 Hz, 2H), 3.92 (dd, J = 10.6, 6.5 Hz, 2H), 3.86 - 3.78 (m, 4H), 3.72 (dd, J = 6.9, 4.5 Hz, 2H), 3.36 (dq, J = 11.0, 5.7 Hz, 2H), 3.15 (t, J = 8.7 Hz, 2H), 2.78 (dd, J = 9.8, 6.3 Hz, 2H), 1.94 (t, J = 5.6 Hz, 2H), 1.79 (dd, J = 7.1, 4.4 Hz, 2H). HRMS (ESI) m / z calcd for C 22 H 27 N5O3, [M+H] + 410.2187, found 410.2186.

[0280] Example 38

[0281] Preparation of 2-(2-(3-(1H-indol-3-yl)propyl)-2,7-diazaspiro[3.5]nonan-7-yl)-N- hydroxy pyrimidine-5-carboxamide hydrochloride (Compound I-31)

[0282]

[0283] I-31 was prepared from 31 and 1H-indole-3-propanal following the procedure of Example 7.

[0284] 1 H NMR (400 MHz, DMSO) δ 10.88 - 10.83 (m, 1H), 8.67 (d, J = 1.5 Hz, 2H), 7.55 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.21 - 7.15 (m, 1H), 7.12 - 7.04 (m, 1H), 6.97 (q, J = 6.9 Hz, 1H), 4.01 - 3.95 (m, 4H), 3.82 (p, J = 6.0 Hz, 6H), 3.72 (t, J = 3.9 Hz, 2H), 3.22 (q, J = 7.0 Hz, 2H), 2.75 (q, J = 8.4 Hz, 2H), 1.88 (dq, J = 15.6, 7.2 Hz, 4H), 1.77 (d, J = 5.7 Hz, 2H). HRMS (ESI) m / z calcd for C 23 H 28N6O2, [M+H] + 421.2347, found 421.2347.

[0285] Example 39

[0286] Preparation of N-hydroxy-2-(2-(3-(1-methyl-1H-indol-3-yl)propyl)-2,7- diazaspiro[3.5]nonan-7-yl)pyrimidine-5-carboxamide hydrochloride (Compound I-32)

[0287]

[0288] I-32 was prepared from 31 and 4-(1-methyl-1H-indol-3-yl)propanal following the procedure of Example 7.

[0289] 1 H NMR (400 MHz, DMSO) δ 11.27 (d, J = 8.4 Hz, 1H), 8.68 (s, 2H), 7.56 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 7.18 - 7.10 (m, 2H), 7.06 - 6.98 (m, 1H), 4.01 - 3.92 (m, 2H), 3.86 - 3.79 (m, 4H), 3.74 (s, 5H), 3.21 (q, J = 7.1 Hz, 2H), 2.75 (t, J = 7.5 Hz, 2H), 1.91 (dt, J = 23.9, 6.6 Hz, 4H), 1.78 (t, J = 5.7 Hz, 2H). HRMS (ESI) m / z calcd for C 24 H 30 N6O2, [M+H] + 435.2503, found 435.2506.

[0290] Bioactivity Test Example 1

[0291] Determination of the in vitro growth half maximal effective inhibitory concentration (IC 50 ) of compounds on two strains of Plasmodium 3D7 and Dd2

[0292] (1) Experimental Methods

[0293] 1. Plasmodium culture: Plasmodium culture uses PRMI (containing NaHCO3, HEPES, Albumax I, Hypoxanthine, Genaotamicin) complete culture medium, cultured in a 37°C incubator (5% CO2, 5% O2).

[0294] 2. In vitro growth half maximal effective inhibitory concentration (IC 50Determination of the activity of the compounds: 100 μL of complete medium was added to a 96-well plate, an appropriate amount of 200 μM compound was added to the first well and diluted with complete medium to 200 μL to make the final concentration of the compound 1000 nM, and then gradient dilution was performed at a ratio of 1 / 2 (11 concentration gradients), dihydroartemisinin (DHA) was used as a positive drug, no compound was added as a negative group, and no plasmodium and compound was added as a blank group. Then 100 μL of plasmodium culture (hematocrit 4%, parasite rate 1%) was added to each well to make the final hematocrit 2% and the parasite rate 1%, and the final concentration gradient of the compound was 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, 3.90625 nM, 1.953125 nM, 0.9765625 nM, 0.48828125 nM. After the addition was completed, the 96-well plate was placed in a 37°C incubator (5% CO2, 5% O2) for incubation for 72 hours. After incubation, 100 μL of supernatant was removed from each well, 100 μL of lysis solution (10×SYBR Green I, 0.5% v / v Triton X-100, 0.5 mg / mL saponin, 0.75% EDTA / Tris-Cl buffer) was added, mixed well, and incubated at room temperature for 2 hours in the dark. The value of each well was read by a plate fluorescence reader (maximum excitation light / maximum acceptance light: 485 nm / 535 nm). The inhibition rate of each well was calculated according to the fluorescence value, and the inhibition rate % = (negative group-experimental group) / (negative group-blank group) × 100%, and the concentration-inhibition rate was plotted in Graphpad Prism to calculate the IC 50 value.

[0295] (2) Test results of the activity of the compounds

[0296] Table 1 IC 50 values of the compounds on 3D7 and Dd2

[0297]

[0298] 3D7 is a wild type strain of plasmodium and has no obvious resistance to drugs; Dd2 has resistance to chloroquine, quinine, sulfadoxine, pyrimethamine and amodiaquine.

[0299] Table 1 shows that the compounds have strong in vitro insecticidal activity, and the IC 50 values of some compounds on 3D7 and Dd2 are comparable to DHA, and some compounds can effectively inhibit the activity of Dd2, showing an inhibitory effect on drug-resistant plasmodium.

[0300] Biological activity test example 2

[0301] Determination of the half effective inhibitory concentration (IC 50 ) of the compounds on two human cell lines, HepG-2 and 293-T

[0302] (1) Determination of the inhibitory activity of the compounds on two human cell lines

[0303] Prepare HepG-2 and 293-T cells and culture them in 10 cm dishes at 37°C in a 5% CO2 cell incubator.

[0304] Day 1

[0305] Trypsinize the cells and count them, then transfer the cells to a 96-well plate at a volume of 100 μL / well and a cell count of 7000 cells. Incubate at 37°C in a 5% CO2 cell incubator for 24 hours.

[0306] Day 2

[0307] 1. Prepare a gradient concentration system of the compound, with a 2-fold dilution, and a system volume of 100 μL / well. (The highest concentration should be determined according to the toxicity of the drug. In general, the IC 50 concentration should be in the middle of the concentration gradient. In general, a high concentration can be selected for the first time for gradient dilution, and the results can be adjusted accordingly.)

[0308] 2. Remove the supernatant from the 96-well plate cell culture system on the first day, and add the newly prepared drug concentration system to the culture plate wells containing the cells (set up double duplicate wells). Incubate at 37°C in a 5% CO2 cell incubator for 72 hours.

[0309] Day 5

[0310] 1. After the cell culture is complete, remove the supernatant from the 96-well plate cell culture system, and add 100 μL of detection solution (culture medium containing 10% CCK-8) to each well. Incubate at 37°C in a 5% CO2 cell incubator for 1 hour, then remove and measure the absorbance at 450 nm using a microplate reader.

[0311] 2. Process the data and calculate the inhibition rate of the compound on cell growth at different concentrations. Enter the inhibition rate into GraphPad Prism, and calculate the IC 50 readings for each drug using the non-linear regression method. The inhibition rate calculation formula is as follows:

[0312] Inhibition rate % = [A(0) - A(drug)] / [A(0) - A(blank)] x 100%

[0313] A(drug): absorbance of the well containing cells, CCK-8 solution, and drug solution

[0314] A (blank): absorbance of wells with medium and CCK-8 solution without cells

[0315] A (0): absorbance of wells with cells, CCK-8 solution without drug solution

[0316] Calculation of selectivity index SI

[0317] SI = IC 50 (IC 50 (3D7)

[0318] (2) Compound test results

[0319] Table 2 IC of compounds on HepG-2 and 293-T and SI values 50

[0320]

[0321] Table 2 shows that the compounds have weak growth inhibition on two cells of HepG-2 and 293-T, and the selectivity index can reach 200000.

[0322] Biological activity test example 3

[0323] Determination of half effective inhibition concentration (IC 50 ) of part of compounds on five clinical strains 50 According to the method of biological activity test example 1, test the IC 50 values and RI of the compounds with better insecticidal activity and cytotoxicity on five clinical strains with different drug resistance, wherein RI = IC 50 (3D7) to represent the strength of drug resistance.

[0324] The results are as follows:

[0325] Table 3 Inhibition effect of compound I-3 on different resistant strains

[0326]

[0327] Note: The structure of Quisinostat is

[0328] GB4 is resistant to chloroquine; C2A is resistant to quinine; CP286 is resistant to sulfadoxine, pyrimethamine and mefloquine; as can be seen from table 3, preferred compound I-3 shows significant inhibition effect on three drug-resistant strains of GB4, C2A and CP286, and the IC 50 ​At about 10-30 nM, and the RI values are all less than 2. This shows that the preferred compound I-3 exhibits good potential to overcome drug resistance and is expected to provide a new solution for anti-malaria treatment.

[0329] Bioactivity test example 4

[0330] Compound I-3 mouse liver microsomal metabolic stability experiment

[0331] (1) Compound mouse liver microsomal metabolic stability test

[0332] The mouse liver microsomes (0.5 mg / mL) were selected for the experiment and purchased from Corning Company. The positive control was selected as ketanserin, and the test compound was first prepared into a 10 mM DMSO solution, diluted with acetonitrile to 0.5 mM; 30 μL of the above 0.5 mM solution was added to the buffer containing the liver microsomes, so that the concentration of the compound was 1.5 μM; 15 μL of 6 mM NADPH solution was added to the above 1.5 μM compound / liver microsome mixture, so that the final concentration of the compound was 1.5 μM and the final concentration of NADPH was 2 mM. The compound / liver microsome test solution was placed on the test plate, incubated in a 37°C water bath, and 135 μL of acetonitrile was added at each time point (0, 5, 15, 30, 45 minutes) to quench. After all the samples were quenched, the samples were shaken for 10 minutes (600 rpm / min) on a shaker, and then centrifuged at 4495g for 15 minutes. The supernatant was diluted with distilled water at a ratio of 1:1, and analyzed by LC-MS. The peak area response ratio (PARR) of the compound at 5, 15, 30, and 45 minutes was compared with the PARR at time 0 to determine the percentage of test compound remaining at each time point. The half-life was calculated using the single-phase exponential decay equation fitted by Excel software.

[0333] (2) Compound test results

[0334] Table 4 Metabolic stability of some compounds in mouse liver microsomes

[0335]

[0336] Compared with the control compound Quisinostat, the half-life of compound I-3 in rats and humans is longer and the clearance rate is lower, indicating that compound I-3 can stay in the body for a longer time, which is helpful for clinical application and prolongs the drug administration interval and improves compliance.

[0337] Bioactivity test example 5

[0338] Some compounds mouse in vivo efficacy experiment

[0339] (1) Mouse in vivo efficacy experiment method

[0340] The in vivo efficacy experiment was performed by using the P. berghei infected balb-c mice model. The mice were 6-8 weeks old female, and 5 mice were set in each dose group (dose grouping is shown in Table 1). Figure 1 The positive drug was piperaquine phosphate (PPQ, structure is shown in ), and the control compounds were JX21108 and Quisinostat, wherein the structure of JX21108 is shown in The administration method was intraperitoneal injection (100 mg / kg high dose was changed to oral administration), and the average body weight of each group was measured before administration, and the corresponding volume of drug solution was injected at 15 μL / g. The preparation method of the compound injection solution was as follows: first dissolved in 5% v / v dimethyl sulfoxide, and oscillated vigorously to partially or completely dissolve the solid part, then 95% v / v 20% wt β-hydroxypropyl cyclodextrin aqueous solution was added, and mixed to obtain the injection solution. The P. yoelii was thawed from -78°C, and then restored virulence by transferring to two mice, and blood was taken and diluted with PBS. Each mouse was inoculated with 10 5 malaria parasites, and the administration was started 24 hours after infection, and a total of 5 administrations were performed, with an interval of 24 hours each time.

[0341] The blood smears were taken from the tail vein of the mice regularly from 24 hours after infection, and the parasitemia was observed and calculated:

[0342] Parasitemia = number of red blood cells infected with malaria parasites / total number of red blood cells x 100%

[0343] The blood smears were observed 30 days after infection, and the corresponding parasitemia was calculated.

[0344] (2) Results of in vivo efficacy experiment of mice

[0345] As shown in Figure 1 , the parasitemia curve shows that the compound has good insecticidal activity at the doses of 60 mg / kg intraperitoneal injection and 100 mg / kg oral gavage, and the parasitemia of the surviving mice is 0 at 30 days, indicating that the malaria parasites in vivo have been cleared. The parasitemia curve and the survival curve show that I-3 has a good balance between efficacy and toxicity.

[0346] In order to further explore the oral efficacy of the compound I-3, a second animal experiment was carried out, and the experimental operation was the same as described above, only the administration method was changed to oral administration, and the control compound was increased to JX21108, and the structure is shown in The experimental results are shown in Figure 2 , and the compound I-3 shows a clear dose-dependent effect under the condition of oral administration, and can effectively clear the malaria infection at the doses of 60 mg / kg and 100 mg / kg, showing excellent antimalarial activity and good safety, and having the potential to be further developed as a new antimalarial candidate drug.

[0347] Bioactivity Test Example 6

[0348] Verification of preferred compounds inhibiting the activity of malaria parasite deacetylase

[0349] (1) Western Blot experimental procedure

[0350] 1. Malaria parasite culture

[0351] The malaria parasite culture was carried out using RPMI (containing NaHCO3, HEPES, Albumax I, Hypoxanthine Genaotamicin) complete medium, and the culture was carried out in a 37°C incubator.

[0352] 2. Drug preparation

[0353] I-3 was selected for the experiment, and according to the IC 50 value measured in the early stage of the drug, the drug was dissolved in DMSO to prepare an initial concentration of 200*20*IC 50 .

[0354] 3. Drug treatment (carried out in a biological safety cabinet)

[0355] About 44h of P. falciparum was taken, and a mixture was prepared in a 50mL tube with complete medium and red blood cells (the content of red blood cells was 2%, and the parasite rate was 8%-10%). 6mL of the mixture was added to each well of a 6-well plate, and 30uL of the drug (the working concentration was 20*IC 50 ) was added to each well. After adding, the plate was mixed and placed in a three-gas incubator for incubation for 4h.

[0356] 4. Protein sample collection

[0357] Take out the 6-well plate, discard the 4 mL supernatant, and transfer the remaining 2 mL of the worm blood mixture to a 2 mL EP tube. Centrifuge (4000 r / 2 min, room temperature) to remove the supernatant. Resuspend the residue in each well with 1 mL of PBS and transfer it to the original EP tube to reduce loss. Centrifuge again to remove the supernatant (Note: After centrifugation at 4000 rpm, to prevent protein degradation, subsequent centrifugation should be performed at 4°C, and the EP tube should be continuously placed on ice during operation). Add 2 mL of lysis buffer to each EP tube, mix well with a vortex oscillator, and lyse on ice for 10 min. Centrifuge (12000 r / 1 min, 4°C) to discard the supernatant. Add 1 mL of PBS to each EP tube, mix well with a vortex oscillator, and centrifuge (12000 r / 1 min, 4°C) to discard the supernatant. Repeat this step twice. Resuspend and transfer 90 μL of 1×PBS to a 1.5 mL ultrasonic tube, then add 10 μL of 10% SDS. Mix well and ultrasonic for 5 min (30 s on / 30 s off). Centrifuge (12000 r / 10 min, 4°C) to take the supernatant. Add loading, shake, heat at 100°C for 10 min, and store the sample at -20°C.

[0358] 5. Western Blot

[0359] Load 10 uL of pre-prepared gel into each well. After running at 80V for 30 min, adjust the voltage to 120V and continue running until the loading liquid approaches the lower edge of the separation gel. Cut the corresponding PVDF membrane with the same coverage area. Use wet transfer method, fast transfer buffer, 400 mA constant current for 35 min. After the end, take out the PVDF membrane. Place the membrane in blocking solution (add 5% skim milk powder in TBST), and shake the incubator for 2 hours. Dilute the histone H3 antibody and H3K9 acetylation antibody with 5% skim milk powder at a ratio of 1:2000, and incubate the PVDF membrane on the shaker for 2 hours. Discard the incubation solution, and wash the membrane with TBST three times for 10 minutes each time. Dilute the secondary antibody at a ratio of 1:5000, and incubate the PVDF membrane on the shaker for 1 hour. Discard the incubation solution. Prepare the developing solution immediately and evenly spread it on the PVDF membrane. Adjust the exposure time according to the brightness of the band.

[0360] (2) Western Blot experimental results of the compound

[0361] As shown in Figure 3 , Quisinostat is a positive control compound. Comparing the Plasmodium histone H3 acetylation bands after treating compound I-3 and DMSO at a concentration of 1×IC 50 and 10×IC 50 for 4 hours, it can be seen that compound I-3 up-regulates the acetylation level, i.e. inhibits the activity of deacetylase, which indirectly proves that the compound is a pan-pfHDAC inhibitor.

[0362] The compounds provided by the present application overcome the shortcoming of the lead compounds being cytotoxic as anticancer drugs, and through testing, the IC50 values of the compounds for Plasmodium and human cells are obtained to obtain a selectivity index (SI), the selectivity index is improved, and it is relatively safer; the problem of poor oral bioavailability of the previous compounds is also avoided, and oral effectiveness is achieved.

[0363] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. In addition, it should be understood that, after reading the above teaching of the present application, those skilled in the art can make various modifications or changes to the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. Pyrimidine-isohydroxamic acid compounds of Formula I and their optical isomers, non-racemic, racemic, or pharmaceutically acceptable salts thereof: in, R 1 Selected from the following group: H, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted -C0-C6 alkylene-C3-C18 carbon ring, substituted or unsubstituted -C0-C6 alkylene-C6-C18 aryl, substituted or unsubstituted -C0-C6 alkylene-3-18 heterocyclic, substituted or unsubstituted -C0-C6 alkylene-5-18 heteroaryl; The substitution refers to the substitution of one or more hydrogen atoms on the group by a group selected from the group consisting of: deuterium, halogen, hydroxyl, amino, cyano, oxo (C=O), C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C3-C7 carbocyclic, 3-7 membered heterocyclic, C6-C10 aryl, C5-C6 heteroaryl. The carbon ring, heterocycle, or heterocyclic group may be a saturated or partially unsaturated structure, but does not possess aromaticity; the carbon ring, heterocycle, aromatic ring, or heteroaromatic ring may optionally be a monocyclic, spirocyclic, fused, or bridged ring; each of the heterocyclic group or heteroaromatic group independently contains 1, 2, 3, or 4 heteroatoms selected from O, S, and N.

2. The pyrimidine-isohydroxamic acid compound of formula I as described in claim 1, and its optical isomers, non-racemic, racemic, or pharmaceutically acceptable salts thereof, characterized in that, The R 1 for Where L is a C0-C4 alkylene group; The connection site between ring A and L can be optionally a carbon atom or a heteroatom: Ring A is selected from the following group: substituted or unsubstituted C3-C16 carbon rings, substituted or unsubstituted C6-C16 aromatic rings, substituted or unsubstituted 3-16 membered heterocycles, substituted or unsubstituted 5-16 membered heteroaromatic rings; and ring A is optionally monocyclic, spirocyclic, fused, or bridged. Hydrogen atoms on ring A are optionally separated by m R atoms. 2 replace; R 2 Selected from the following groups: deuterium, halogen, hydroxyl, amino, cyano, oxo (C=O), C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C3-C6 cycloalkyl, phenyl; m is selected from 0, 1, 2, 3, 4 or 5.

3. The pyrimidine-isohydroxamic acid compound of formula I as described in claim 2, and its optical isomers, non-racemic, racemic, or pharmaceutically acceptable salts thereof, characterized in that, The A ring is The connection site between ring A and L can be optionally a carbon atom or a heteroatom: The A-1 ring and A-2 ring are each independently selected from the following group: substituted or unsubstituted C3-C6 carbon rings, substituted or unsubstituted benzene rings, substituted or unsubstituted 3-6 membered heterocycles, and substituted or unsubstituted 5-6 membered heteroaromatic rings. R a and R b Each is independently selected from the following groups: deuterium, halogen, hydroxyl, amino, cyano, oxo (C=O), C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C3-C6 cycloalkyl, phenyl; Or R a and R b It can be combined with the connected atoms to form a group consisting of one or more R atoms. c Substituted C3-C6 carbon rings, benzene rings, 3-6 membered heterocycles, and 5-6 membered heteroaromatic rings; wherein R c Each is independently selected from the following group: deuterium, halogen, hydroxyl, amino, cyano, oxo (C=O), C1-C4 alkyl; p and q are each independently selected from 0, 1, 2, 3 or 4.

4. The pyrimidine-isohydroxamic acid compound of formula I as described in claim 2, and its optical isomers, non-racemic, racemic, or pharmaceutically acceptable salts thereof, characterized in that, The A ring is It can be either a single bond or a double bond; Z1, Z2, Z3, and Z4 are each independently selected from the following groups: chemical bond, CH2, CH2CH2, -CH=CH-, S, O, N, and NH; The hydrogen atoms on ring A are optionally separated by one or more R atoms. 2 replace; R 2 Selected from the following groups: deuterium, halogen, hydroxyl, amino, cyano, oxo (C=O), C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, C3-C6 cycloalkyl, phenyl.

5. The pyrimidine-isohydroxamic acid compound of formula I as described in claim 2, and its optical isomers, non-racemic, racemic, or pharmaceutically acceptable salts thereof, characterized in that, Ring A is selected from the following group, whether substituted or unsubstituted: Each of X is independently selected from the following groups: O, S, N, or NH; Substitution refers to the optional substitution of hydrogen atoms on a group with one or more R atoms. 2 replace; R 2 Selected from the following group: deuterium, halogen, hydroxyl, amino, cyano, oxo (C=O), C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl.

6. The pyrimidine-isohydroxamic acid compound of formula I as described in claim 1, and its optical isomers, non-racemic, racemic, or pharmaceutically acceptable salts thereof, characterized in that, R 1 Selected from the following group of groups, whether substituted or unsubstituted: Substitution refers to the optional substitution of hydrogen atoms on a group with one or more R atoms. 2 replace; R 2 Selected from the following groups: deuterium, fluorine, chlorine, bromine, hydroxyl, amino, cyano, oxo (C=O), methyl, ethyl, methoxy, ethoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzene.

7. The pyrimidine-isohydroxamic acid compound of formula I as described in claim 1, and its optical isomers, non-racemic, racemic, or pharmaceutically acceptable salts thereof, characterized in that, The compounds are selected from the group consisting of:

8. A pharmaceutical composition, characterized in that, The composition comprises: a pyrimidine-isohydroxamic acid compound as described in any one of claims 1-7, and its optical isomers, non-racemic, racemic, or pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable carrier.

9. Use of the pyrimidine-hydroxamic acid compounds as described in any one of claims 1-7, their optical isomers, non-racemic, racemic, or pharmaceutically acceptable salts thereof, or the pharmaceutical composition of claim 8, wherein the use includes: (i) Used in the preparation of drugs or formulations related to histone deacetylase (HDAC) activity; and / or (ii) Drugs or preparations used to inhibit malaria parasites or treat malaria; and / or (iii) As an HDAC inhibitor.

10. The use as described in claim 9, characterized in that, The diseases mentioned include tumors.

Images