N, N-disubstituted hydroxylamine compound as well as synthesis method and application thereof

Abstract

The invention belongs to the field of chemical synthesis, particularly relates to an N, N-disubstituted hydroxylamine compound and a synthesis method and application thereof, and provides an efficient method for reducing 1, 3-dipolar nitrone into the N, N-disubstituted hydroxylamine compound, on the basis of nitrone synthesis, nitrone is reduced by taking trichlorosilane as a reducing agent, and the N, N-disubstituted hydroxylamine compound is obtained. The hydroxylamine compound is suitable for a plurality of different substituted benzene rings or heterocyclic compounds. The synthesis method has the characteristics of high efficiency, greenness, mild conditions, wide substrate application range and the like, and provides a new thought for synthesis of the hydroxylamine compound.

Description

Technical Field

[0001] The present invention belongs to the field of chemical synthesis, and specifically relates to a class of N,N-disubstituted hydroxylamine compounds, their synthesis methods and applications. Background Art

[0002] N,N-disubstituted hydroxylamine compounds are key intermediates for constructing complex molecules and are used in a variety of asymmetric synthesis reactions. After structural modification, such hydroxylamine compounds can act as antioxidants to protect drug molecules from oxidative decomposition and are used to treat neurodegenerative diseases; they can also inhibit the release of inflammatory mediators and are used as local anti-inflammatory drugs to treat diseases such as rheumatoid arthritis, such as zileuton and atreleuton, for relieving asthma; in addition, they can also be used as antibacterial agents, such as adepidyn.

[0003] Nitrones are important intermediates for synthesizing bioactive alkaloids and drugs. They can undergo various reactions, such as 1,3-dipolar cycloaddition reactions. Nitrones can also be reduced and transformed into corresponding amines, so they are also widely used in the synthesis of hydroxylamines. In recent years, silane-based reducing agents have received attention due to their high selectivity and mild conditions. However, reducing nitrones to corresponding amines using silane reagents is still challenging, for example, side reactions are difficult to control, post-treatment is complex and affects purity, the product is prone to decomposition, and the stereoselectivity of asymmetric reduction is insufficient. Therefore, there is still room for optimization. The present invention has developed an efficient method for synthesizing N,N-disubstituted hydroxylamine compounds by reducing nitrones using trichlorosilane, which solves most of the problems. Summary of the Invention

[0004] The object of the present invention is to provide a class of N,N-disubstituted hydroxylamine compounds, whose structural formula is shown in formula (I):

[0005]

[0006] The compound in formula (I) is a class of N,N-disubstituted hydroxylamine compounds, in which the nitrogen atom in the skeleton is replaced by a hydroxyl group; R1 is a substituted or unsubstituted phenyl, naphthyl or heterocycle; R2 is methyl or benzyl, and the compound is a racemate.

[0007] Furthermore, R1 is selected from one of p-bromophenyl, m-fluorophenyl, m-methylphenyl, o-chlorophenyl, o-methylphenyl, 2,4-dimethylphenyl, 4-(1,2-methylenedioxyphenyl), 1-naphthyl.

[0008] The present invention also provides a method for synthesizing the N,N-disubstituted hydroxylamine compound shown in the above formula (I):

[0009]

[0010] Using a nitrone intermediate Using [raw material] as the raw material, organic solvent A and reducing agent trichlorosilane are added, and the nitrone is reduced at room temperature under anhydrous and anaerobic conditions. After the reduction reaction is completed, the N,N-disubstituted hydroxylamine compound is obtained through extraction, separation and purification.

[0011] Furthermore, the organic solvent A is selected from one of 1,2-dichloroethane, dichloromethane, tetrahydrofuran, 1,2-dioxane, acetonitrile, and preferably 1,2-dichloroethane.

[0012] Furthermore, the molar ratio of the reducing agent to the nitrone intermediate is 1.5:1;

[0013] Furthermore, the nitrone intermediate is prepared by the following method:

[0014] Using the compound as the raw material, N-hydroxyamine hydrochloride, sodium acetate, magnesium sulfate and organic solvent B are added, and the reaction is carried out overnight at room temperature under an inert gas atmosphere to synthesize the nitrone intermediate;

[0015] Furthermore, the has a molar ratio of 1:1.5:3:3 with N-hydroxyamine hydrochloride, sodium acetate, and magnesium sulfate.

[0016] Furthermore, the organic solvent B is one of absolute ethanol, absolute methanol, tetrahydrofuran, acetonitrile, and preferably absolute ethanol.

[0017] Furthermore, the N-hydroxyamine hydrochloride is N-methylhydroxyamine hydrochloride or N-benzylhydroxyamine hydrochloride.

[0018] The present invention also provides the application of the above N,N-disubstituted hydroxylamine compound in the preparation of a drug for inhibiting the growth of human lung cancer cell A549, human myelomonocytic leukemia cell MV4-11 or human acute myeloid leukemia cell MOLM13.

[0019] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0020] Based on the synthesis of nitrone, the present invention reduces the nitrone through trichlorosilane as a reducing agent, has high selectivity, finally generates a hydroxylamine compound, and is applicable to various differently substituted benzene rings or heterocyclic compounds, providing an efficient method for reducing 1,3-dipolar nitrone into N,N-disubstituted hydroxylamine compounds.

[0021] This synthetic method has the characteristics of high efficiency, greenness, mild reaction conditions, and a wide range of substrate applicability. N,N-disubstituted hydroxylamine compounds are generated by reducing nitrones with trichlorosilane. Most of the reported reactions involved in this method rely on noble metal catalysts or strong reducing agents, which are costly and environmentally unfriendly. The reduction with trichlorosilane used in this application has not been reported. Although chiral hydroxylamine has not been obtained, it provides a new idea for the synthesis of hydroxylamine compounds. The N,N-disubstituted hydroxylamine compounds provided by the present invention have important bioactivities against anti-tumor cell proliferation. The cytotoxicity tests of in vitro tumor cells on human lung cancer cell A549, human myelomonocytic leukemia cell MV4-11, and human acute myeloid leukemia cell MOLM13 show that the N,N-disubstituted hydroxylamine compounds shown in formula (I) have significant inhibitory effects on tumor cell growth and may be developed into new anti-tumor drugs. Description of the Drawings

[0022] Figure 1 1H NMR spectrum of compound 4a in Example 1;

[0023] Figure 2 13C NMR spectrum of compound 4a in Example 1;

[0024] Figure 3 1H NMR spectrum of compound 4b in Example 1;

[0025] Figure 4 13C NMR spectrum of compound 4b in Example 1;

[0026] Figure 5 19F NMR spectrum of compound 4b in Example 1;

[0027] Figure 6 1H NMR spectrum of compound 4c in Example 1;

[0028] Figure 7 13C NMR spectrum of compound 4c in Example 1;

[0029] Figure 8 1H NMR spectrum of compound 4d in Example 1;

[0030] Figure 9 13C NMR spectrum of compound 4d in Example 1;

[0031] Figure 10 1H NMR spectrum of compound 4e in Example 1;

[0032] Figure 11 13C NMR spectrum of compound 4e in Example 1;

[0033] Figure 121H NMR spectrum of compound 4f in Example 1;

[0034] Figure 13 13C NMR spectrum of compound 4f in Example 1;

[0035] Figure 14 1H NMR spectrum of compound 4g in Example 1;

[0036] Figure 15 13C NMR spectrum of compound 4g in Example 1;

[0037] Figure 16 1H NMR spectrum of compound 4h in Example 1;

[0038] Figure 17 13C NMR spectrum of compound 4h in Example 1. Detailed implementation manners

[0039] The following specific examples are only used to illustrate in detail the specific implementation manners of the present invention, and do not limit the scope of protection claimed in the claims of the present invention.

[0040] In the following specific implementation manners, NaOAc refers to Sodium acetate;

[0041] MgSO4 refers to Magnesium sulfate;

[0042] EtOH refers to Ethanol;

[0043] DCE refers to 1,2-Dichloroethane;

[0044] HSiCl3 refers to Trichlorosilane.

[0045] Synthesis of raw materials:

[0046] Preparation of compound 2:

[0047]

[0048] The preparation of the nitrone intermediate, i.e., compound 2, is carried out according to the above reaction formula. The preparation of p-bromophenylmethyl nitrone in Example 1 is described in detail as follows:

[0049] Add compound 1 p-bromoacetophenone (398 mg, 2.0 mmol), N-methylhydroxylamine hydrochloride (479 mg, 3.0 mmol), sodium acetate (492 mg, 6.0 mmol), magnesium sulfate (722 mg, 6.0 mmol) and absolute ethanol (20 mL) into a 100 mL round-bottom flask. After purging with nitrogen, stir the reaction overnight (12 h) at room temperature under a nitrogen atmosphere. After the reaction is completed, evaporate and spin-dry the solvent, add water to remove various salts generated during the reaction, then extract with DCM, collect the organic phase, dry it over anhydrous sodium sulfate, filter by suction, evaporate and spin-dry DCM, and recrystallize to obtain 173 mg of the target product 2 p-bromophenylmethyl nitrone with a yield of 80%.

[0050] The preparation process of the nitrone intermediates used in the preparation of the following compounds 4b - 4i is the same as above, except that the R1 group in compound 1 is different, specifically as follows:

[0051] Table 1

[0052]

[0053]

[0054] The yields in the table are the yields when preparing the corresponding nitrone intermediates of the compound type.

[0055] Example 1: Preparation of N,N-disubstituted hydroxylamine compounds

[0056] Preparation of compound 4a:

[0057]

[0058] Add nitrone intermediate p-bromophenylmethyl nitrone (91.2 mg, 0.4 mmol) and 1,2-dichloroethane (2 mL) into a 10 mL round-bottom flask dried with a heat gun. After dissolving trichlorosilane (80.7 mg, 0.6 mmol) in a small amount of dichloroethane (1 mL), slowly drip it into the above reaction flask with a syringe, and then stir at room temperature for 4 h. The above reaction needs to strictly control the anhydrous and anaerobic conditions. After the reaction is completed, remove the residual trichlorosilane in the reaction solution with sodium bicarbonate, then extract with DCM, collect the organic phase, dry it over anhydrous sodium sulfate, filter by suction, spin-dry, and separate through a silica gel column (200 - 300 mesh silica gel, petroleum ether:ethyl acetate = 10:1, volume ratio) to obtain 77.5 mg of the target product 4a with a yield of 84%. 1 H NMR(500MHz,CDCl3)δ7.5–7.4(m,2H),7.2(d,J=8.2Hz,2H),3.6(q,J=6.6Hz,1H),2.5(s,3H),1.4(d,J=6.9Hz,3H). 1313C NMR (126 MHz, CDCl3) δ 141.5, 131.7, 129.7, 121.4, 68.7, 46.0, 19.9. HRMS (ESI) m / z: [M+H] + calculated for C9H 13 BrNO, 230.0175; found, 230.0172.

[0059] The preparation methods of compounds 4b - 4i are the same as that of compound 4a, and the raw material feeding ratios are also the same as those in the preparation of compound 4a.

[0060] Compound 4b: Colorless oil (56.3 mg, 83% yield). 1 1H NMR (500 MHz, CDCl3) δ 7.3 (td, J = 8.0, 5.9 Hz, 1H), 7.1 (dd, J = 18.0, 9.5 Hz, 2H), 6.9 (td, J = 8.5, 2.7 Hz, 1H), 3.6 (q, J = 6.5 Hz, 1H), 2.5 (s, 3H), 1.4 (d, J = 6.7 Hz, 3H). 13 13C NMR (126 MHz, CDCl3) δ 163.0 (d, J = 245.4 Hz), 145.1, 130.0 (d, J = 8.3 Hz), 123.7, 114.8 (d, J = 22.1 Hz), 114.4 (d, J = 21.1 Hz), 68.9, 46.0, 19.9. 19 19F NMR (471 MHz, CDCl3) δ -113.1. HRMS (ESI) m / z: [M+H] + calculated for C9H 13 FNO, 170.0976; found, 170.0975.

[0061] Compound 4c: Colorless oil (44.6 mg, 67% yield). 1 1H NMR (500 MHz, CDCl3) δ 7.2 (t, J = 7.5 Hz, 1H), 7.2–7.0 (m, 3H), 3.6 (q, J = 6.6 Hz, 1H), 2.5 (s, 3H), 2.3 (s, 3H), 1.5 (d, J = 6.7 Hz, 3H). 13 13C NMR (126 MHz, CDCl3) δ 142.7, 138.2, 128.5, 128.3, 124.9, 69.7, 46.1, 29.8, 21.6, 20.3. HRMS (ESI) m / z: [M+H] + calculated for C10 H 16 NO, 166.1226; found, 166.1227.

[0062] Compound 4d: Colorless oil (65.2 mg, 88% yield). 1 H NMR (500 MHz, CDCl3) δ 7.5 (s, 1H), 7.4 (dd, J = 7.9, 1.7 Hz, 1H), 7.3 (td, J = 7.6, 1.5 Hz, 1H), 7.2 (td, J = 7.6, 1.9 Hz, 1H), 4.3 (q, J = 6.7 Hz, 1H), 2.5 (s, 3H), 1.4 (d, J = 6.7 Hz, 3H). 13 C NMR (126 MHz, CDCl3) δ 140.1, 133.8, 129.7, 128.9, 128.4, 127.2, 64.0, 46.1, 18.9. HRMS (ESI) m / z: [M+H] + calculated for C9H 13 ClNO, 186.0680; found, 186.0680.

[0063] Compound 4e: Colorless oil (43.4 mg, 66% yield). 1 H NMR (500 MHz, CDCl3) δ 7.4 (s, 1H), 7.2 (dt, J = 7.8, 4.3 Hz, 1H), 7.1 (dd, J = 4.0, 1.2 Hz, 2H), 3.9 (q, J = 6.6 Hz, 1H), 2.5 (s, 3H), 2.4 (s, 3H), 1.4 (s, 3H). 13 C NMR (126 MHz, CDCl3) δ 141.6, 135.6, 130.6, 126.9, 126.7, 126.5, 64.7, 46.1, 19.9. HRMS (ESI) m / z: [M+H] + calculated for C 10 H 16 NO, 166.1226; found, 166.1228.

[0064] Compound 4f: Colorless oil (51.8 mg, 72% yield). 1 H NMR (500 MHz, CDCl3) δ 7.0 (d, J = 7.9 Hz, 1H), 7.0 (s, 1H), 3.9 (q, J = 6.6 Hz, 1H), 2.5 (s, 3H), 2.3 (s, 3H), 2.3 (s, 3H), 1.4 (d, J = 4.6 Hz, 3H).13 C NMR (126 MHz, CDCl3) δ 138.5, 136.5, 135.4, 131.3, 127.2, 126.7, 64.4, 46.1, 21.1, 19.9, 19.8. HRMS (ESI) m / z: [M+H] + calculated for C 11 H 18 NO, 180.1383; found, 180.1383.

[0065] Compound 4g: Colorless oil (34.4 mg, 44% yield). 1 H NMR (500 MHz, CDCl3) δ 6.8 (s, 1H), 6.7 (d, J = 0.9 Hz, 2H), 5.9 (s, 2H), 3.6 (q, J = 6.7 Hz, 1H), 2.5 (s, 3H), 1.4 (d, J = 6.9 Hz, 3H). 13 C NMR (126 MHz, CDCl3) δ 147.8, 146.9, 136.5, 121.2, 108.2, 101.1, 69.1, 45.9, 29.8, 20.3. HRMS (ESI) m / z: [M+H] + calculated for C 10 H 14 NO3, 196.0968; found, 196.0969.

[0066] Compound 4h: Colorless oil (60.7 mg, 75% yield). 1 H NMR (500 MHz, CDCl3) δ 8.3 (d, J = 8.4 Hz, 1H), 7.9 (dd, J = 7.9, 2.0 Hz, 1H), 7.8 (d, J = 8.2 Hz, 1H), 7.6 (d, J = 8.1 Hz, 1H), 7.6–7.4 (m, 3H), 4.5 (q, J = 6.7 Hz, 1H), 2.6 (s, 3H), 1.6 (d, J = 6.7 Hz, 3H). 13 C NMR (126 MHz, CDCl3) δ 139.2, 134.1, 131.4, 129.1, 127.8, 126.1, 125.7, 125.6, 124.8, 123.5, 64.9, 46.3, 19.9. HRMS (ESI) m / z: [M+H] + calculated for C 13 H 16 NO, 202.1226; found, 202.1226.

[0067] Example 2: Cytotoxicity Experiment

[0068] 1. Experimental Materials

[0069] The human lung cancer cell line A549, human myelomonocytic leukemia cell line MV4-11, and human acute myeloid leukemia cell line MOLM13 used in this experiment were purchased from the European Collection of Authenticated Cell Cultures; RPMI1640 medium was purchased from Hyclone, USA; fetal bovine serum (FBS) was purchased from Biontech, Germany; penicillin-streptomycin solution (100×) was purchased from Beyotime Biotechnology Co., Ltd.; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide were purchased from Sigma-Aldrich, USA.

[0070] 2. Experimental Methods

[0071] 2.1 Cell Culture

[0072] A549, MV4-11, and MOLM13 cells were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (hereinafter referred to as RPMI1640 medium) in an incubator at 37°C, 45-65% humidity, and 5% CO2. When the cells grew to the logarithmic growth phase, they were passaged, used for experiments, and cryopreserved.

[0073] 2.2 Detection of Cell Viability by MTT Method

[0074] Cell Seeding and Drug Administration: Cells in the logarithmic growth phase were collected and seeded into 96-well plates at a density of 3×10 3 cells / well, and cultured in a cell incubator at 37°C and 5% CO2 for 24 h. Then, the old medium in the wells was aspirated, and the test compound dissolved in DMSO was diluted to the corresponding concentrations (0.1, 1, 10, 100 μM) with RPMI 1640 medium and added to the 96-well plates, 100 μL per well. Three replicates were set for each concentration, and cisplatin was set as the positive control group (the drug administration method was the same as that of the test compound group), the normal cell group (only containing cells and RPMI 1640 medium), and the blank control group (only containing RPMI1640 medium without cells) were set on each plate at the same time. After drug administration, the 96-well plates were incubated in a cell incubator at 37°C and 5% CO2 for 72 h.

[0075] Detection of Cell Viability: After 72 h of incubation, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated for another 1.5 h. Subsequently, the medium in the well plates was removed, 150 μL of DMSO was added to each well, and the plates were placed on a horizontal shaker and shaken at medium speed for 5 min. The absorbance at a wavelength of 562 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0076] Data processing: Calculate the relative inhibition rate of the drug on cell growth according to the following formula, that is, the cell inhibition rate:

[0077] Cell inhibition rate = [1 - (X - C0) / (C - C0)] × 100%

[0078] Wherein, C, C0, and X respectively represent the average absorbance values of three wells in the normal cell group, blank control group, and drug treatment group. Finally, use Graphpad Prism 5.0 software to fit the cell inhibition rate curve and calculate the IC 50 value of the cell growth inhibition rate of the compound to be tested.

[0079] 2.3 Experimental results

[0080] The IC 50 of cisplatin in the positive control group against A549 tumor cells was 22.0 μmol / L.

[0081] The IC 50 of compound 4b against A549 tumor cells was 2.108 μmol / L.

[0082] The IC 50 of compound 4e against MV4-11 cells was 4.203 μmol / L, and the IC 50 of cisplatin in the positive control group against MV4-11 cells was 5.2 μmol / L.

[0083] The IC 50 of compound 4h against MOLM-13 cells was 0.7212 μmol / L, the IC 50 of compound 4d against MOLM-13 cells was 1.961 μmol / L, the IC 50 of compound 4e against MOLM-13 cells was 2.635 μmol / L, and the IC 50 of cisplatin in the positive control group against MOLM-13 cells was 3.5 μmol / L.

[0084] Table 2: IC 50 values of each compound against tumor cells A549, MV4-11, and MOLM-13

[0085]

[0086]

[0087] 3. Experimental conclusions

[0088] A549, MV4-11 and MOLM-13 cells are effective tools and evaluation indicators for testing the cytotoxicity of test compounds against tumor cells. This experiment shows that the N,N-disubstituted hydroxylamine compounds represented by formula (I) have certain cytotoxicity against A549, MV4-11 and MOLM-13 cells, have the activity of inhibiting the proliferation of A549, MV4-11 and MOLM-13 cells, and may be developed into new anti-tumor drugs.

Claims

1. A class of N,N-disubstituted hydroxylamine compounds, the structural formula of which is shown in formula (I): In the skeleton of the N,N-disubstituted hydroxylamine compound shown in formula (I), the nitrogen atom is substituted by a hydroxyl group; R1 is a substituted or unsubstituted phenyl, naphthyl or heterocycle; R2 is methyl or benzyl, and the compound is a racemate.

2. The N,N-disubstituted hydroxylamine compound according to claim 1, wherein The R1 is one of p-bromophenyl, m-fluorophenyl, m-methylphenyl, o-chlorophenyl, o-methylphenyl, 2,4-dimethylphenyl, 4-(1,2-methylenedioxyphenyl), 1-naphthyl.

3. The synthesis method of the N,N-disubstituted hydroxylamine compound according to claim 1 or 2, the specific steps are as follows: Using a nitrone intermediate as a raw material, adding organic solvent A and reducing agent trichlorosilane, reducing the nitrone under room temperature and anhydrous and anaerobic conditions, and obtaining the N,N-disubstituted hydroxylamine compound after extraction, separation and purification at the end of the reduction reaction.

4. The synthesis method according to claim 3, characterized in that, The organic solvent A is selected from one of 1,2-dichloroethane, dichloromethane, tetrahydrofuran, 1,2-dioxane, acetonitrile.

5. The synthesis method according to claim 3, characterized in that, The molar ratio of the reducing agent to the nitrone intermediate is 1.5:

1.

6. The synthesis method according to claim 3, wherein The nitrone intermediate is prepared by the following method: Using the compound as the raw material, N-hydroxyamine hydrochloride, sodium acetate, magnesium sulfate and organic solvent B are added, and the reaction is carried out overnight at room temperature under an inert gas atmosphere to synthesize a nitrone intermediate.

7. The synthesis method according to claim 6, wherein The has a molar ratio of 1:1.5:3:3 with N-hydroxyamine hydrochloride, sodium acetate, and magnesium sulfate; and / or The organic solvent B is one of absolute ethanol, absolute methanol, tetrahydrofuran, acetonitrile.

8. The application of the N,N-disubstituted hydroxylamine compound according to claim 1 or 2 in the preparation of anti-tumor drugs.

9. The application of the N,N-disubstituted hydroxylamine compound synthesized by the synthesis method according to any one of claims 3-7 in the preparation of anti-tumor drugs.

10. The application according to claim 8 or 9, characterized in that, The anti-tumor drug is a drug that inhibits the growth of human lung cancer cell A549, human myelomonocytic leukemia cell MV4-11 or human acute myelogenous leukemia cell MOLM13.

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