Nitric oxide donor lanosterol derivative as well as preparation method and application thereof

Abstract

The invention discloses a nitric oxide donor type lanosterol derivative as shown in a formula I and a preparation method and application thereof, and the nitric oxide donor type lanosterol derivative has dual functions of reducing intraocular pressure and protecting optic nerves and can be used for preparing a medicine for treating glaucoma diseases. Wherein R is selected from one of C2-C5 straight chain alkylene groups.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a nitric oxide (NO) donor type lanosterol derivative and a preparation method and application thereof. BACKGROUND

[0002] Glaucoma is a group of diseases characterized by characteristic optic atrophy and visual field defects, and pathological intraocular pressure increase is the main risk factor. The treatment methods of glaucoma include intraocular pressure reduction treatment and optic nerve protection treatment. The commonly used intraocular pressure reducing drugs at present include prostaglandin analogs (such as latanoprost eye drops), β-adrenergic receptor blockers (such as timolol eye drops), carbonic anhydrase inhibitors (such as brinzolamide eye drops) and parasympathomimetic drugs (such as pilocarpine eye drops). The above drugs mainly focus on reducing intraocular pressure, but whether they can directly protect the optic nerve is not clear. Therefore, developing drugs that can effectively reduce intraocular pressure and protect the optic nerve has become an urgent need in clinical treatment. SUMMARY

[0003] The purpose of the present application is to provide a nitric oxide (NO) donor type lanosterol derivative and a preparation method and application thereof.

[0004] The present application is realized by the following technical solutions:

[0005] The nitric oxide (NO) donor type lanosterol derivative shown in formula I:

[0006]

[0007] , wherein R is selected from one of C2-C5 straight-chain alkylene, .

[0008] When R is selected from C2-C5 straight-chain alkylene, The equation is as follows:

[0009] The synthesis steps are as follows: compounds 1a-5a are reacted with silver nitrate to obtain corresponding intermediates 1b-5b. The intermediates 1b-5b are reacted under alkaline conditions of LiOH to remove the methyl group to obtain intermediates 1c-5c. The intermediates 1c-5c are reacted with lanosterol under alkaline catalytic conditions to obtain compounds L1-L5 through esterification.

[0010] When R is selected from The synthesis equation is as follows:

[0011]

[0012] comprising the following steps:

[0013] 1) Lanosterol (compound 1) reacts with succinic anhydride to obtain intermediate 2;

[0014] 2) Compounds 7a-10a react with silver nitrate to obtain corresponding intermediates 7b-10b;

[0015] 3) Intermediate 9b reacts under alkaline conditions of LiOH to remove acetyl group to obtain intermediate 9c;

[0016] 4) Isosorbide mononitrate (ISMN), intermediate 7b, intermediate 8b, intermediate 9c, intermediate 10b respectively react with intermediate 2 under alkaline catalytic conditions to obtain lanosterol derivatives L6-L10 through esterification.

[0017] The nitric oxide donor type lanosterol derivative of the present application has the dual functions of reducing intraocular pressure and protecting optic nerve, and is a potential new type of therapeutic drug for glaucoma, and therefore the present application also protects the use of the nitric oxide donor type lanosterol derivative in the preparation of a drug for treating glaucoma.

[0018] The drug contains a pharmaceutically acceptable adjuvant, and is prepared into various dosage forms, including liquid and solid dosage forms. The dosage forms include tablets, capsules, oral liquids, oral preparations, granules, instant preparations, pills, powders, ointments, Dan preparations, suspensions, powders, injections, suppositories, sprays, drops or patches.

[0019] Preferably, the concentration of the nitric oxide donor type lanosterol derivative in the drug is 10 mM.

[0020] The present application has the following beneficial effects: the present application designs and synthesizes a series of new NO donor type lanosterol derivatives, which have the dual functions of reducing intraocular pressure and protecting optic nerve, and can be used as a drug for treating glaucoma. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is compound L7 1 H NMR chart (CDCl3 303KAV-300);

[0022] Figure 2 is compound L7 13 C NMR chart (CDCl3 303KAV-300);

[0023] Figure 3 Statistical chart of compounds L1-L10 significantly improving the NO level of HTM cells;

[0024] Figure 4 Statistical chart of compounds L1-L10 significantly improving the cGMP level of HTM cells;

[0025] Figure 5 Figure 7 is a graph showing the effect of compound L7 on intraocular pressure (IOP) in mice. Figure 7A is a graph showing the change in IOP in each group of mice at 1, 2, 4, and 8 hours after eye drop administration of compound L7. Figure 7B is a graph showing the change in IOP in each group of mice at 2 hours after eye drop administration of compound L7. Figure 7C is a graph showing the change in IOP in each group of mice at 4 hours after eye drop administration of compound L7. Compound L7 significantly reduced IOP after eye drop administration, with the most significant effect at 4 hours after a single eye drop and no significant effect at 8 hours after a single eye drop. In Figure 7, A represents the fold line graph of the change in intraocular pressure (IOP) in each group of mice at 1, 2, 4, and 8 hours after eye drop administration of compound L7; B represents the change in IOP in each group of mice at 2 hours after eye drop administration of compound L7; and C represents the change in IOP in each group of mice at 4 hours after eye drop administration of compound L7.

[0026] Figure 6 Figure 8 is a graph showing the effect of compound L7 on the amplitude and peak time of N1 wave in mice. Figure 8A is a representative dark adaptation VEP curve of each group of mice. Figure 8B is a quantitative statistical result of the amplitude of N1 wave in each group of mice. Figure 8C is a quantitative statistical result of the peak time of N1 wave in each group of mice. Compound L7 significantly increased the amplitude of N1 wave and improved the peak time of N1 wave, with better effects than lanosterol. In Figure 8, A represents the representative dark adaptation VEP curve of each group of mice; B represents the quantitative statistical result of the amplitude of N1 wave in each group of mice; and C represents the quantitative statistical result of the peak time of N1 wave in each group of mice.

[0027] Figure 7 Figure 9 is a graph showing the effect of compound L7 on the survival rate of RGCs in mice. Figure 9A is a representative retinal mount and magnified view of the corresponding visual field of each group of mice. Figure 9B is a quantitative statistical result of the RBPMS-positive cell density of the retinal mount of each group of mice. Compound L7 significantly increased the survival rate of RGCs, with better effects than lanosterol. In Figure 9, A represents the representative retinal mount and magnified view of the corresponding visual field of each group of mice; and B represents the quantitative statistical result of the RBPMS-positive cell density of the retinal mount of each group of mice. DETAILED DESCRIPTION

[0028] The following is a further description of the present application, rather than a limitation thereof.

[0029] Example 1: Synthesis of compounds L1-L10

[0030] The synthesis route is as follows:

[0031]

[0032] The synthesis of compound L1 includes the following steps:

[0033] 1) Dissolve 4-bromobutyric acid methyl ester (compound la, 1.8 g, 1.0 equiv) in acetonitrile (0.1 mmol / mL) followed by silver nitrate (4250 mg, 2.5 equiv) and heat to reflux overnight in the dark. Monitor reaction progress by TLC, molybdenum phosphorus acid visualization (nitrate ester compounds appear yellow), remove acetonitrile by rotary evaporation, add excess saturated brine and EA, stir and let stand, transfer supernatant to separatory funnel, bottle solids washed three times with equal volumes of water and EA, transfer to separatory funnel, extract, combine organic phases, wash three times with saturated brine, dry over anhydrous sodium sulfate, concentrate and purify by column chromatography to yield intermediate lb. Dissolve intermediate lb (500 mg, 1.0 equiv) in 10 mL of methanol, stir at 0 °C for 15 min, add 2 M aqueous LiOH-H2O solution (257 mg, 2.0 equiv) dropwise, react overnight at 5 °C. Monitor by TLC (PE / EA = 10:1), molybdenum phosphorus acid visualization, after the starting material is completely reacted, remove methanol by rotary evaporation, add 15 mL of water, extract with EA (3 x 15 mL), wash with saturated brine (3 x 20 mL), dry over anhydrous sodium sulfate, concentrate to yield intermediate lc.

[0034] 2) Dissolve intermediate lc (149 mg, 1.0 equiv) with EDCI-HC1 (477.5 mg, 2.5 equiv), DMAP (13 mg, 0.1 equiv), and starting material lanosterol (compound 1, 42 mg, 1 equiv) in 10 mL of DMF and react overnight at room temperature. Monitor reaction progress by TLC (EA / MeOH = 40:1), purify by column chromatography at the end to yield compound LI. 1 HNMR (300 MHz, Chloroform-d) δ 5.10 (s, 1H), 4.52 (t, J = 6.4 Hz, 3H), 2.45 (t, J = 7.2 Hz, 2H), 2.07 (dt, J = 12.8, 6.3 Hz, 6H), 1.70 (d, J = 9.4 Hz, 7H), 1.43 (d, J = 104.5 Hz, 14H), 1.17 (s, 1H), 1.13 (s, 1H), 1.01 (s, 3H), 0.87 (d, J = 5.4 Hz, 14H), 0.69 (s, 3H). 13C NMR (75 MHz, Chloroform-d) δ 171.92, 134.57, 134.18, 125.25, 81.47, 72.10, 50.51, 50.40, 49.81, 44.48, 39.53, 37.84, 36.90, 36.48, 36.25, 35.24, 30.98, 30.83, 30.58, 28.20, 27.98, 26.37, 25.70, 24.93, 24.25, 24.19, 22.82, 22.54, 22.43, 21.02, 19.16, 18.63, 18.12, 17.61, 16.58, 15.75.

[0035] Compounds L2-L5 were prepared according to the preparation of compound LI except that compound la was replaced by compounds 2a-5a.

[0036] NMR data for compound L2: 1 H NMR (300 MHz, Chloroform-d) δ 5.10 (s, 1H), 4.50 (dt, J = 18.1, 5.1 Hz, 3H), 2.38 (d, J = 6.5 Hz, 2H), 2.03 (s, 6H), 1.84 - 1.52 (m, 19H), 1.26 (s, 2H), 1.16 (d, J = 12.3 Hz, 3H), 1.01 (s, 4H), 0.88 (s, 14H), 0.69 (s, 3H). 13 C NMR (75 MHz, Chloroform-d) δ

[0037] 172.52, 134.59, 134.25, 125.26, 81.11, 72.71, 50.55, 49.83, 44.52, 37.86, 36.93, 36.47, 36.24, 35.28, 33.97, 31.02, 30.83, 28.18, 27.97, 26.34, 25.65, 24.24, 22.77, 22.51, 21.28, 21.03, 19.16, 18.63, 18.14, 17.59, 16.57, 15.76.

[0038] NMR data for compound L3: 1H NMR (300 MHz, Chloroform-d) δ 5.10 (s, 1H), 4.49 (dq, J = 17.5, 6.5, 5.8 Hz, 3H), 2.33 (s, 2H), 1.97 (d, J = 42.9 Hz, 6H), 1.83 - 1.37 (m, 21H), 1.27 (s, 2H), 1.16 (d, J = 12.0 Hz, 3H), 0.99 (d, J = 11.8 Hz, 4H), 0.88 (s, 14H), 0.69 (s, 3H). 13 C NMR (75 MHz, Chloroform-d) δ 172.93, 134.62, 134.32, 125.27, 80.93, 72.93, 50.59, 50.46, 49.85, 44.55, 39.53, 37.87, 36.95, 36.47, 36.36, 36.23, 35.30, 34.38, 31.04, 30.83, 28.16, 27.95, 26.56, 26.40, 25.61, 25.22, 24.93, 24.53, 24.23, 24.08, 22.74, 22.48, 21.03, 19.14, 18.62, 18.15, 16.55, 15.76.

[0039] Compound L4 NMR Spectroscopy Data: 1 H NMR (300 MHz, Chloroform-d) δ 8.07 (d, J = 5.5 Hz, 2H), 7.59 (d, J = 7.7 Hz, 1H), 7.49 (t, J = 7.9 Hz, 1H), 5.48 (s, 2H), 5.10 (t, J = 7.4 Hz, 1H), 4.77 (dd, J = 10.5, 4.2 Hz, 1H), 2.06 (s, 4H), 1.84 - 1.66 (m, 7H), 1.62 - 1.11 (m, 19H), 1.06 (s, 5H), 0.95 (s, 3H), 0.89 (s, 6H), 0.70 (s, 3H). 13C NMR (75 MHz, Chloroform-d) δ 165.48, 134.68, 134.29, 132.91, 132.77, 131.91, 130.46, 129.99, 128.97, 125.27, 82.06, 74.02, 50.68, 49.87, 44.57, 38.26, 37.00, 36.48, 36.25, 35.34, 31.05, 30.85, 28.18, 28.10, 26.42, 25.63, 24.95, 24.27, 24.09, 22.76, 22.50, 21.07, 19.19, 18.65, 18.18, 17.57, 16.81, 15.78.

[0040] Compound L5 NMR Spectroscopy Data: 1 H NMR (300 MHz, Chloroform-d) δ 8.08 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 5.48 (s, 2H), 5.1 1 (s, 1 H), 4.76 (dd, J = 1 1.2, 4.3 Hz, 1 H), 2.06 (s, 4H), 1.84 - 1.68 (m, 7H), 1.62 - 1.12 (m, 19H), 1.05 (d, J = 4.3 Hz, 5H), 0.95 (s, 3H), 0.90 (s, 6H), 0.71 (s, 3H). 13 C NMR (75 MHz, Chloroform-d) δ 165.48, 134.68, 134.29, 132.91, 132.77, 131.91, 130.46, 129.99, 128.97, 125.27, 82.06, 74.02, 50.68, 49.87, 44.57, 38.26, 37.00, 36.48, 36.25, 35.34, 31.05, 30.85, 28.18, 28.10, 26.42, 25.63, 24.95, 24.27, 24.09, 22.76, 22.50, 21.07, 19.19, 18.65, 18.18, 17.57, 16.81, 15.78.

[0041] The synthesis of compound L7 comprises the following steps:

[0042] 1) Lanosterol (1707 mg, 4 mmol, 1 equiv), succinic anhydride (440 mg, 4.4 mmol, 1.1 equiv) and DMAP (489 mg, 4 mmol, 1 equiv) were dissolved in 20 mL of DMF and reacted at room temperature for 24 h, monitoring the reaction progress by TLC. After the reaction was completed, most of the DMF was removed by rotary evaporation, 50 mL of water was added to the reaction bottle, and a large amount of white solid was observed to precipitate. After filtration, the filter cake was washed with methanol (3 x 10 mL) and then vacuum dried to obtain intermediate 2;

[0043] 2) Compound 7a (1240 mg, 1.0 equiv) was dissolved in acetonitrile (0.1 mmol / mL), followed by the addition of silver nitrate (4175 mg, 2.5 equiv), and heated to reflux overnight in the dark. The reaction progress was monitored using TLC, molybdenum phosphoric acid coloration (nitrate compounds appear yellow), acetonitrile was removed by rotary evaporation, an excess of saturated brine and EA were added, stirred and allowed to stand, the supernatant was transferred to a separatory funnel, the in-bottle solid was washed with an equal volume of water and EA three times, transferred to the separatory funnel, extracted, the organic phase was washed with saturated brine three times, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain intermediate 7b;

[0044] 3) Intermediate 7b (107 mg, 1.0 equiv) was dissolved in 10 mL of DMF with EDCI-HCl (477.5 mg, 2.5 equiv), DMAP (13 mg, 0.1 equiv) and intermediate 2 (526 mg, 1 equiv) at room temperature overnight. The reaction progress was monitored by TLC (EA / MeOH = 40:1), and finally purified by column chromatography to obtain compound L7. 1 HNMR (300 MHz, Chloroform-d) δ 5.10 (s, 1H), 4.70 - 4.64 (m, 2H), 4.53 (dd, J = 11.2, 4.8 Hz, 1H), 4.43 - 4.35 (m, 2H), 2.66 (s, 4H), 2.03 (s, 4H), 1.70 (q, J = 13.0, 11.4 Hz, 7H), 1.58 (d, J = 15.0 Hz, 5H), 1.51 - 1.20 (m, 9H), 1.17 (s, 1H), 1.13 (s, 1H), 1.00 (s, 3H), 0.88 (s, 14H), 0.69 (s, 3H). 13CNMR (75 MHz, Chloroform-d) δ 171.91, 171.60, 134.60, 134.27, 130.80, 125.27, 81.52, 70.31, 60.38, 50.58, 49.84, 44.54, 37.88, 36.93, 35.27, 31.02, 30.84, 29.38, 29.02, 28.17, 27.89, 26.39, 25.64, 24.94, 24.23, 24.15, 22.76, 21.03, 19.15, 18.63, 18.13, 17.57, 16.51, 15.76. See the nuclear magnetic resonance spectrum of Figure 1 and Figure 2 .

[0045] The synthesis of compound L6 was prepared according to the preparation of compound L7, except that intermediate 7b was replaced by isosorbide mononitrate ISMN in step 3) to obtain compound L6.

[0046] The synthesis of compound L8 was prepared according to the preparation of compound L7, except that intermediate 7b was replaced by intermediate 8b in step 3) to obtain compound L8.

[0047] The synthesis of compound L9 was prepared according to the preparation of compound L7, except that intermediate 7b was replaced by intermediate 9c in step 3) to obtain compound L9. Wherein, the preparation of intermediate 9c is as follows, obtained by reacting intermediate 9b under alkaline conditions of LiOH to remove acetyl group, the specific steps are as follows, intermediate 9b (542 mg, 1.0 equiv) is dissolved in 10 mL of methanol, stirred at 0°C for 15 min, 2M LiOH·H2O aqueous solution (257 mg, 2.0 equiv) is added dropwise, and the reaction is carried out at 5°C overnight. TLC monitoring (PE / EA = 10:1) is used, phosphomolybdic acid is used for color development, after the raw material is completely reacted, the methanol is removed by rotary evaporation, 15 mL of water is added, extracted with EA (3 x 15 mL), washed with saturated brine (3 x 20 mL), dried over anhydrous sodium sulfate, and concentrated to obtain intermediate 9c. The synthesis of intermediate 9b is prepared according to the synthesis of intermediate 7b, except that compound 7a is replaced by compound 9a.

[0048] The synthesis of compound L10 was prepared according to the preparation of compound L7, except that intermediate 7b was replaced by intermediate 10b in step 3) to obtain compound L10.

[0049] Nuclear magnetic spectrum data of compound L6: 1H NMR (300 MHz, Chloroform-d) δ 5.35 (td, J = 5.5, 2.9 Hz, 1H), 5.25 (s, 1H), 5.10 (s, 1H), 4.97 (t, J = 5.2 Hz, 1H), 4.51 (dt, J = 8.6, 5.0 Hz, 2H), 4.03 (d, J = 9.2 Hz, 3H), 3.90 (dd, J = 11.2, 5.5 Hz, 1H), 2.64 (s, 4H), 2.03 (s, 4H), 1.80 - 1.09 (m, 26H), 1.00 (s, 3H), 0.87 (s, 11H), 0.69 (s, 3H). 13 C NMR (75 MHz, Chloroform-d) δ 171.64, 171.32, 134.61, 134.25, 125.26, 86.60, 81.54, 81.43, 81.25, 73.46, 69.19, 50.57, 49.83, 44.53, 37.89, 36.93, 36.47, 35.27, 31.02, 30.83, 29.66, 29.37, 29.19, 28.17, 27.91, 26.38, 25.64, 24.93, 24.17, 22.76, 22.50, 21.02, 19.14, 18.71, 18.13, 16.54, 15.76.

[0050] Compound L8 NMR spectra data: 1 H NMR (300 MHz, Chloroform-d) δ 5.10 (s, 1H), 4.55 (t, J = 6.3 Hz, 3H), 4.21 (t, J = 6.1 Hz, 2H), 2.64 (s, 4H), 2.06 (dd, J = 12.5, 6.4 Hz, 6H), 1.68 (s, 7H), 1.58 (d, J = 13.5 Hz, 5H), 1.49 - 1.24 (m, 9H), 1.17 (s, 1H), 1.13 (s, 1H), 1.00 (s, 3H), 0.88 (s, 14H), 0.69 (s, 3H). 13C NMR (75 MHz, Chloroform-d) δ 172.14, 171.84, 134.55, 134.21, 125.26, 81.43, 69.80, 60.55, 50.52, 50.39, 49.82, 44.49, 37.87, 36.90, 36.48, 36.36, 36.26, 35.23, 30.98, 30.83, 29.45, 29.14, 28.20, 28.00, 27.90, 26.41, 25.71, 24.25, 24.16, 22.83, 22.54, 21.02, 19.17, 18.12, 17.62, 16.54, 15.76.

[0051] Compound L9 NMR Spectroscopy Data: 1 H NMR (300 MHz, Chloroform-d) δ 5.17 (dd, J = 53.0, 7.4 Hz, 1H), 4.56 - 4.39 (m, 3H), 4.13 (t, J = 5.9 Hz, 2H), 2.62 (s, 4H), 2.01 (s, 4H), 1.72 (dd, J = 22.0, 7.7 Hz, 1 1H), 1.60 - 1.47 (m, 5H), 1.46 - 1.08 (m, 1 1H), 0.99 (s, 3H), 0.87 (s, 14H), 0.65 (d, J = 14.5 Hz, 3H). 13 C NMR (75 MHz, Chloroform-d) δ 172.17, 171.78, 134.60, 134.26, 81.40, 72.52, 63.62, 50.57, 44.53, 37.88, 36.93, 36.47, 36.24, 35.26, 31.03, 30.83, 29.51, 29.22, 28.17, 27.90, 26.40, 25.63, 24.98, 24.20, 23.65, 22.76, 22.50, 21.03, 19.14, 18.63, 18.13, 17.57, 16.52, 15.76.

[0052] Compound L10 NMR Spectroscopy Data: 1H NMR (300 MHz, Chloroform-d) δ 5.10 (s, 1H), 4.49 (dq, J = 18.9, 6.5, 5.7 Hz, 3H), 4.10 (t, J = 6.4 Hz, 2H), 2.63 (s, 4H), 2.03 (s, 4H), 1.80 - 1.63 (m, 11H), 1.62 - 1.25 (m, 16H), 1.17 (s, 1H), 1.12 (s, 1H), 1.00 (s, 3H), 0.88 (s, 14H), 0.69 (s, 3H). 13 C NMR (75 MHz, Chloroform-d) δ 172.32, 171.89, 134.54, 134.20, 125.25, 81.33, 72.96, 64.13, 50.52, 50.39, 49.81, 44.48, 37.86, 36.89, 36.47, 36.35, 35.22, 30.97, 30.82, 29.68, 29.53, 29.23, 28.14, 27.90, 26.44, 26.37, 25.70, 24.24, 24.15, 22.81, 22.25, 21.01, 19.16, 18.63, 18.11, 17.61, 16.55, 15.75.

[0053] Example 2: Determination of NO release level of cells treated with compounds L1-L10

[0054] A human trabecular meshwork (HTM) cell line was selected as a cell model to explore the NO release level of compounds L1-L10 in cells using DAF-FM DANO fluorescent probe.

[0055] 1. The HTM cell line was cultured, and then placed in serum-free and phenol red-free medium (Pricella, PM150316) for 12 h;

[0056] 2. The cell concentration was adjusted to 1 x 10 6 / mL using serum-free and phenol red-free medium, and the excess medium was removed by centrifugation;

[0057] 3. Lanosterol and each test compound were added to the cell suspension to a final concentration of 5 μM, and incubated in a 37°C biochemical incubator for 8 h;

[0058] 4. The cell precipitate was collected by centrifugation at 500 g for 5 min, and the precipitate was washed once with PBS;

[0059] 5. Dilute DAF-FM DA (Biotium, S0019S) fluorescent probe to 5 mM using probe diluent. Resuspend cells with diluted probe, incubate for 20 min at 37 °C in the dark, invert the cell suspension every 3-5 min to ensure the probe fully contacts with cells;

[0060] 6. Collect cell precipitate by centrifugation at 500g for 5 min, and wash the precipitate with PBS for three times;

[0061] 7. Resuspend cells using 300 pL PBS, and prepare single cell suspension by 40 pm cell screen;

[0062] 8. Detect the fluorescence intensity of cells under FITC channel using flow cytometry (BD LSRFortessa™ X-20);

[0063] 9. Take the mean fluorescence intensity as the analysis index to statistically analyze the results; the results are shown in Figure 3 As compared with the control group (DMSO), lanosterol failed to significantly increase the NO level of HTM cells, and compounds L1-L10 can significantly increase the NO level of HTM cells, among which compounds L7, L9 and L10 have the most significant effect.

[0064] III. Determination of cGMP level of cells treated by compounds L1-L10

[0065] cGMP is a key downstream signal molecule for NO to play physiological functions. By determining the cGMP level in HTM cell lines, the effect of the target compound on the cGMP level in HTM cell lines after incubation is explored. Human cyclic guanosine monophosphate (cGMP) quantitative detection kit (RUI XIN biotech, RXJ105816H) is used to determine the content of cGMP.

[0066] 1. Culture HTM cell lines, when the cell confluence reaches 80%, use serum-free medium to continue culture for 12 h;

[0067] 2. Treat cells with lanosterol and test compounds (final concentration of 5 mM) respectively for 24 h, collect cell supernatant, centrifuge to obtain supernatant (4 °C, 4000 rpm, 20 min);

[0068] 3. Let each reagent stand at room temperature for 30 min, take the concentrated washing solution, dilute it with distilled water at a ratio of 1:20, mix thoroughly and reserve;

[0069] 4. Take out the pre-coated plate from the sealed bag, set a blank control well without adding any liquid; set 2 wells for each calibrator, add 50 μL of corresponding calibrator to each well; add 50 μL of sample to be tested to the remaining detection wells;

[0070] 5. Add 50 μL of biotinylated antigen to all wells except the blank control well, mix well, and then paste the sealing film on the plate, and incubate at 37°C in the dark for 60 minutes;

[0071] 6. Discard the liquid in the wells, fill each well with washing solution, stand for 10 seconds, then shake dry, repeat this operation 3 times, then pat dry. Add 50 μL of enzyme-labeled avidin to each well (except the blank control well), mix well, and then paste the sealing film on the plate, and incubate at 37°C in the dark for 30 minutes;

[0072] 7. Discard the liquid in the wells, fill each well with washing solution, stand for 10 seconds, then shake dry, repeat this operation 3 times, then pat dry. Add 50 μL of color developing agent A and 50 μL of color developing agent B to each well, mix well, then incubate at 37°C in the dark for 15 minutes, and finally add 50 μL of stop solution to each well;

[0073] 8. Use the enzyme label instrument to take readings, set the wavelength to 450 nm, first use the blank control well to set the zero point, then measure the optical density value (OD value) of each well;

[0074] 9. After the detection is completed, use the standard sample concentration as the horizontal coordinate, and the corresponding OD value as the vertical coordinate, use the four-parameter Logistic curve fitting (4-pl) to establish the standard curve equation. Through the OD value of the sample, use the equation to calculate the concentration value of the sample; the results are shown in Figure 4 Lanosterol (Lanosterol) failed to significantly increase the cGMP level of HTM cells, and compounds L1-L10 could significantly increase the cGMP level of HTM cells, among which compound L7 had the most significant effect.

[0075] Four, the effect of compound L7 on chronic high intraocular pressure mouse model

[0076] Based on the results of NO release level and cGMP level, compound L7 was selected as a representative to explore the effect of this series of compounds at the animal level.

[0077] (I) Establishment of chronic high intraocular pressure mouse model

[0078] 1. Select 6-8 week old C57BL / 6J male mice for the experiment, and anesthetize them with 1% sodium pentobarbital solution at a dose of 10 μL / g;

[0079] 2. Topical anesthesia with hydrochloric acid propamocaine on the right eye surface;

[0080] 3. Perform anterior chamber paracentesis using a 30G insulin needle, followed by injection of 10 μΐ of 6 μιη magnetic beads using a microsyringe (Hamilton, 7654-01, 25 μΐ);

[0081] 4. Place a ring magnet around the eye to direct the beads to form a ring and immobilize them evenly in the angle;

[0082] 5. Apply tobramycin ointment to the right eye of the mouse, and place the mouse on a homeothermic pad until it recovers.

[0083] (B) Compound L7 eye drop administration

[0084] 1. The above chronic high intraocular pressure mice were randomly divided into three groups, L7 group, lanosterol group and vehicle group. From the first day after modeling, 5 μΐ of 10 mM L7, 10 mM lanosterol and DMSO / PBS mixture (v:v = 1:9) were added to the right eye of the mice in the three groups, respectively, three times a day, for a total of 7 consecutive days;

[0085] 2. Before each administration, the mice were anesthetized with isoflurane gas, and 5 μΐ of drug was added to the right eye of the mice in each group, which was kept for 30 seconds and then put back into the cage. The first administration was completed between 9:00 am and 10:00 am, the second administration was completed between 13:00 pm and 14:00 pm, and the third administration was completed between 19:00 pm and 20:00 pm.

[0086] (C) Measurement of intraocular pressure of compound L7 single eye drop administration

[0087] The rebound tonometer (Icare, TV02) was used to measure the intraocular pressure of the chronic high intraocular pressure model mice, and the average value was obtained by measuring six times.

[0088] 1. The mice were anesthetized with isoflurane gas, and the right eye intraocular pressure of the mice in each group was measured as the baseline intraocular pressure;

[0089] 2. 5 μΐ of 10 mM L7, 10 mM lanosterol and DMSO / PBS mixture (v:v = 1:9) were added to the right eye of the mice in the three groups, respectively, which was kept for 30 seconds and then put back into the cage;

[0090] 3. The right eye intraocular pressure of the mice in each group was measured again at 1, 2, 4 and 8 hours after eye drop administration, respectively, and the results were recorded;

[0091] 4. Data analysis: ΔIOP = intraocular pressure at each time point after eye drop administration - baseline intraocular pressure;

[0092] 5. The results are as follows Figure 5As shown in the results, compared with the control group (Vehicle), lanosterol eye drops had no significant effect on reducing intraocular pressure, and compound L7 eye drops could significantly reduce intraocular pressure, and the effect was most significant 4 hours after a single eye drop, and there was no significant effect on reducing intraocular pressure 8 hours after a single eye drop.

[0093] (IV) Detection of visual function of mice after compound L7 eye drops

[0094] The visual evoked potential (VEP) of the above three groups of chronic high intraocular pressure model mice and 6-8 week old C57BL / 6J male mice (Normal group) without treatment was recorded by using a small animal visual electrophysiology instrument (Diagnosys, Celeris).

[0095] 1. All mice ready for VEP detection were dark adapted overnight in advance;

[0096] 2. Anesthesia: 1% sodium pentobarbital solution was injected intraperitoneally at a dose of 10 μL / g;

[0097] 3. Mydriasis and moisturizing: compound tropicamide eye drops were added to the eye surface of the mouse to dilate the pupils, and after confirming the mydriasis, the compound tropicamide eye drops on the eye surface were wiped off with a cotton swab, and hydroxypropyl methylcellulose was applied to keep the eye moist;

[0098] 4. Place the electrode: insert the ground electrode into the subcutaneous tissue of the mouse tail, and the two positive electrodes are respectively attached to the mouse cornea, and the two negative electrodes are respectively inserted into the subcutaneous tissue of the mouse neck and the subcutaneous tissue of the nose, and ensure that the resistance of all electrodes is lower than 10.0 kΩ, to confirm that the electrode connection is normal;

[0099] 5. Start detection and record;

[0100] 6. After the experiment, the mouse eye surface was smeared with tobramycin ointment, and the mouse was placed on a constant temperature heating pad to recover; the results are shown in the table below. Figure 6 As shown in the results, compared with the control group (Vehicle), lanosterol eye drops could significantly increase the amplitude of N1 wave, but had no significant effect on the peak time of N1 wave; L7 eye drops could significantly increase the amplitude of N1 wave and shorten the peak time of N1 wave, and the effect was better than that of lanosterol.

[0101] (V) Detection of the number of retinal ganglion cells (RGCs) of mice after compound L7 eye drops

[0102] RGCs damage and death caused by glaucoma is a key factor of vision loss in patients, therefore, the antibody of RGCs specific biomarker RBPMS was used to evaluate the survival rate of RGCs after eye drop administration of compound L7.

[0103] 1. After 7 days of continuous eye drop administration, the mice in the above three groups and the normal group were sacrificed by cervical dislocation, the eyeballs were carefully taken out under a microscope using surgical scissors, and the corneas were peeled off;

[0104] 2. The eyeballs were fixed in pre-cooled 4% PFA for 40 min, and the retinas were peeled off, which were white and transparent under normal conditions;

[0105] 3. The removed retinas were washed in PBS solution for 3 times, each for 10 min, to completely remove the fixing solution;

[0106] 4. The PBS blocking solution containing 5% BSA and 0.5% Trixon X-100 was added to the EP tube and blocked at 4°C overnight;

[0107] 5. The blocking solution was removed, and the RBPMS (Abeam, #ab152101) primary antibody diluted with the blocking solution (1:200) was added, and incubated at 4°C for 48 h;

[0108] 6. After incubation, the primary antibody was removed, and the retinas were washed with PBS for 3 times, each for 10 min, to completely remove the excess primary antibody staining agent;

[0109] 7. After washing, the Alex 555 labeled rabbit secondary antibody (1:500) was used for incubation at room temperature for 2 h;

[0110] 8. The excess secondary antibody was removed, and the retinas were washed with PBS for 3 times, each for 10 min;

[0111] 9. The retinas were cut into clover shapes under a microscope, and were mounted and fixed using an anti-fluorescence quencher (Beyotime, P0126-5 mL);

[0112] 10. Image shooting was performed at 555 nm wavelength using a high-speed large-size panoramic confocal tissue imaging analysis system (Tissue Gnostics, TissueFAXS Q+);

[0113] 11. For each retina sample, 1 field of view was selected from the middle area of each leaf of the clover-shaped retina mount, and the RGCs were counted, and the number of RBPMS positive cells in each 1 mm 2 was counted; the results are shown in Figure 7Compared with the control group (Vehicle), lanosterol eye drops can improve the survival rate of RGCs to some extent, but there is no statistical difference; L7 eye drops can significantly improve the survival rate of RGCs, and its effect is better than that of lanosterol.

Claims

1. Nitric oxide donor-type lanosterol derivatives as shown in Formula I: in, R is selected from C2-C5 straight-chain alkylene groups. One of them.

2. The method for preparing the nitric oxide donor-type lanosterol derivative according to claim 1, characterized in that, When R is selected from C2-C5 straight-chain alkylene, The equation is as follows: The synthesis steps are as follows: Compounds 1a-5a react with silver nitrate to obtain the corresponding intermediates 1b-5b. Intermediates 1b-5b are reacted under alkaline conditions of LiOH to remove the methyl group and obtain intermediates 1c-5c. Intermediates 1c-5c are then reacted with lanosterol under alkaline catalysis to obtain compounds L1-L5.

3. The method for preparing the nitric oxide donor-type lanosterol derivative according to claim 1, characterized in that, When R is selected The synthesis equation is as follows: Includes the following steps: 1) Lanosterol reacts with succinic anhydride to give intermediate 2; 2) Compounds 7a-10a react with silver nitrate to give the corresponding intermediates 7b-10b; 3) Intermediate 9b reacts under alkaline conditions in LiOH to remove the acetyl group, yielding intermediate 9c; 4) Isosorbide mononitrate, intermediate 7b, intermediate 8b, intermediate 9c, and intermediate 10b were esterified with intermediate 2 under alkaline catalysis to obtain lanosterol derivatives L6-L10.

4. The use of the nitric oxide donor-type lanosterol derivative of claim 1 in the preparation of a medicament for treating glaucoma.

5. The application according to claim 4, characterized in that, The drug contains pharmaceutically usable excipients and is formulated into various dosage forms, including liquid and solid dosage forms.

6. The application according to claim 4, characterized in that, The dosage forms include tablets, capsules, oral liquids, lozenges, granules, powders, pills, powders, ointments, elixirs, suspensions, powders, injections, suppositories, sprays, drops, or patches.

7. The application according to claim 4, characterized in that, The concentration of the nitric oxide donor type lanosterol derivative in the drug is 10 mM.

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