Melatonin derivatives, process for their preparation and use thereof

Abstract

The application provides a melatonin derivative, a preparation method and application thereof, and belongs to the technical field of organic synthesis. The melatonin derivative has the structural formula shown in the following formula (1): (1), wherein R is, or. The melatonin derivative is designed by taking melatonin as a prototype drug, and the melatonin derivative is used in eye drop preparations for treating ocular neovascular diseases.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, specifically relating to a melatonin derivative, its preparation method, and its application. Background Technology

[0002] With the development of society, economy, and medical standards, the aging population is intensifying, leading to an increase in the prevalence of systemic vascular diseases such as diabetes and hypertension. Simultaneously, the survival rate of premature infants is improving, resulting in a year-on-year increase in both the incidence and prevalence of fundus neovascular diseases, with the age of onset becoming increasingly younger. Based on the location of the lesion, fundus neovascular diseases are mainly divided into retinal neovascularization (RNV) diseases and choroidal neovascularization (CNV) diseases. RNV is mainly seen in proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), and retinopathy of prematurity (ROP). Among these, diabetic retinopathy is a leading cause of blindness in the working-age population. [1] The global prevalence of diabetic retinopathy, a vision-threatening condition, has reached 10.2% (28 million people). [2] Retinal vascular disease (RVV) has a high incidence and blindness rate, severely impacting patients' quality of life and imposing a heavy socioeconomic burden. A common pathological feature of RVV is abnormal neovascularization originating from the retinal capillary bed, which can break through the internal limiting membrane and proliferate into the vitreous body. [3] New blood vessels are prone to repeated leakage, rupture, bleeding, and organization, leading to complications such as vitreous hemorrhage and tractional retinal detachment, resulting in severe visual impairment or even blindness. CNV (Chronic Angiovascular Disease) is mainly seen in age-related macular degeneration (AMD), pathological myopia, and various other diseases such as vascular streaks. AMD accounts for 8.7% of all blinding eye diseases and is a leading cause of blindness in people over 50 years of age in Western countries. [4] With socioeconomic development and population aging, the number of AMD patients worldwide will reach 288 million by 2040. [5] This places an increasingly heavy burden on individuals, families, and society. In its later stages, AMD may involve choroidal neovascularization (CNV), a condition known as wet AMD (wAMD). [6,7] CNVs are formed when newly formed blood vessels from the choroid cross Bruch's membrane and grow beneath the retinal pigment epithelium (RPE) layer, or between the RPE layer and the retinal neurosensory layer. [8] Furthermore, it is highly susceptible to exudation, rupture, bleeding, and scar formation, severely damaging the structure and function of the retina and ultimately leading to irreversible vision loss. [9] .

[0003] Extensive basic research and clinical practice have confirmed that anti-VEGF therapy is one of the most effective methods for treating RNV and CNV. [10,11] However, because most cases require multiple intraocular injections, the incidence of endophthalmitis increases.

[12] Persistent elevation of intraocular pressure

[13] retinal detachment

[14] The risks of complications, frequent follow-up examinations make it difficult for patients to adhere to the treatment plan, and the treatment costs are high; some patients are not sensitive to anti-VEGF treatment or develop drug resistance during treatment. [15,16] Therefore, it is imperative that we conduct in-depth research on the pathogenesis of RNV and CNV, and search for safer and more effective new drugs or administration methods.

[0004] Melatonin (chemical name: N-acetyl-5-methoxytryptamine) is a hormone primarily secreted by the pineal gland.

[17] Its main function is to maintain the body's normal circadian rhythm. However, a growing body of research has found that melatonin can also be found in the skin, gastrointestinal tract, bone marrow, and respiratory epithelium.

[18] and the lens

[19] and retina

[20] Melatonin is synthesized and released in tissues or structures; melatonin receptors (MTs) are also widely distributed in the body. [21,22] Meanwhile, melatonin has also been shown to alleviate oxidative stress damage. [23 Anti-inflammatory

[24] Anti-apoptosis

[25] Inhibits angiogenesis, maintains vascular endothelial barrier function and normal vascular permeability.

[26] It has multiple functions. In ophthalmology, studies have reported the use of exogenous melatonin in glaucoma. [27,28] Retinitis pigmentosa

[29] In animal models of optic neuritis

[30] It has a neuroprotective effect and plays an antioxidant and prostaglandin-reducing role in experimental uveitis.

[31] In previous studies, our research group, through the construction of an OIR mouse model, found that intraperitoneal injection of melatonin can effectively inhibit RNV formation by downregulating the HIF1α-VEGF signaling pathway, while also playing a multiple role in protecting retinal glial cells, microglia, and retinal ganglion cells (RGCs). [32,33] Using a krypton-ion laser-induced CNV model in mice, this study found that melatonin can inhibit the RhoA / ROCK signaling pathway, promote M1 macrophage polarization, inhibit M2 macrophage polarization, thereby suppressing vascular endothelial cell proliferation and reducing vascular leakage, ultimately slowing down the development of CNV.

[34] The above findings indicate that melatonin can effectively inhibit the growth of new blood vessels in the fundus.

[0005] Besides regulating the HIF1α-VEGF and RhoA / ROCK signaling pathways, melatonin exerts multiple biological functions through different signaling pathways in various animal models of different diseases. Therefore, does melatonin regulate these multiple pathways through a common upstream signaling pathway? Literature studies suggest that melatonin mainly exerts these effects by binding to its melatonin receptor.

[34] Melatonin receptors are divided into membrane receptors and nuclear receptors. Melatonin membrane receptors are members of the G protein-coupled receptor family with a seven-transmembrane structure; three subtypes have been identified so far: MT1, MT2, and MT3.

[36] In addition to membrane receptors, researchers also discovered that melatonin has nuclear receptors RZR / ROR (retinoid Z receptor / retinoid acid receptor-related orphan receptor).

[37] After melatonin binds to its specific receptor, it enters the cell and activates the corresponding second signal transduction system to exert its biological functions.

[38] .

[0006] Melatonin receptors are widely expressed in retinal tissues, including vascular endothelial cells, photoreceptor cells, retinal ganglion cells, and macrophages. [39,40] It has been confirmed that melatonin, upon binding to its receptor, can reduce HIF-1α levels, improve hypoxia, and decrease VEGF expression, thereby inhibiting pathological angiogenesis. Antagonizing melatonin receptors weakens the effects of melatonin. [26,41] Furthermore, melatonin can inhibit the p38 / TXNIP / NF-κB pathway by binding to the receptor MT1, thereby suppressing the aggregation of inflammatory cells, reducing the level of inflammatory factors, upregulating the expression of tight junction proteins in retinal vascular endothelial cells, and ultimately improving vascular permeability in diabetic retinopathy and maintaining the integrity of the blood-retinal barrier.

[42] In our previous studies, we constructed an OIR model and found that the expression of melatonin receptors MT1 and MT2 in the retina of suckling mice was significantly decreased under ischemic and hypoxic conditions, while the expression of MT1 and MT2 increased after melatonin treatment. In a CNV model, we found that treatment with exogenous melatonin upregulated the expression of MT1 and MT2, thereby inhibiting RhoA / ROCK activation, regulating macrophage polarization, and inhibiting CNV formation. Based on the above studies, we propose the following hypothesis: melatonin plays a role in treating fundus vascular diseases by binding to melatonin receptors in vascular endothelial cells and macrophages, activating downstream signaling pathways, inhibiting inflammatory cell activation and pathological angiogenesis.

[0007] A growing body of research indicates that systemic application of melatonin can effectively inhibit the development and progression of various eye diseases. Meanwhile, topical eye drops are a crucial treatment method for eye diseases due to their convenience, high patient compliance, rapid onset of action, targeted application, and high safety. Therefore, we propose the possibility of using topical melatonin eye drops to treat neovascularization of the fundus. Current research on melatonin eye drops primarily focuses on the treatment of corneal diseases and glaucoma. Ahn, JH, et al. modified melatonin eye drops to reduce irritation to rabbit corneas, hoping to use them for the treatment of type 2 granular keratodystrophy.

[43] Studies by Martinez-Aguila, A. et al. have confirmed that eye drops containing melatonin and its analogue 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) can effectively reduce intraocular pressure (IOP) in a DBA / 2J glaucoma mouse model.

[44] Dal Monte, M. et al. found in a rat model of intraocular pressure induced by methylcellulose that both melatonin eye drops and its receptor agonist agomelatine eye drops effectively reduced IOP.

[45] However, whether topical application of melatonin eye drops can achieve effective drug concentrations in the vitreous cavity and retinal tissue, and exert the same therapeutic effect on fundus neovascularization diseases, remains to be seen. There are currently no reports on this.

[0008] The prerequisite for using melatonin eye drops to treat retinal neovascularization is that melatonin can penetrate anterior segment tissues and reach effective concentrations in the vitreous humor and retina. Dal Monte, M. et al., through testing drug concentrations in rat ocular tissues, found that after using melatonin eye drops, melatonin could penetrate to the fundus, but at extremely low concentrations, only 0.01 ng / ml in the vitreous humor and 0.004 ng / mg in the retina.

[45] In previous studies, our research group measured the drug concentration in the ocular tissue of New Zealand rabbits after administering melatonin eye drops. The results confirmed that melatonin could penetrate the vitreous and retinal tissues, but the concentrations were only 95.19 ng / ml and 355.29 ng / mg, respectively. This invention aims to enhance the permeability of melatonin to ocular tissues by altering its chemical structure. Building upon existing research on melatonin structural modification, we will synthesize different derivatives and ultimately screen for a novel melatonin derivative with better solubility and pharmacokinetic advantages, while also demonstrating comparable or superior efficacy to melatonin in treating neovascularization of the fundus.

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Journal of pineal research2021: e12716. 34.Xu Y, Cui K, Li J, et al. Melatonin attenuates choroidalneovascularization by regulating macrophage / microglia polarization viainhibition of RhoA / ROCK signaling pathway. Journal of pineal research 2020;69(1): e12660. 35.Liu J, Clough SJ, Hutchinson AJ, Adamah-Biassi EB, Popovska-Gorevski M, Dubocovich ML. MT1 and MT2 Melatonin Receptors: A TherapeuticPerspective. Annual review of pharmacology and toxicology 2016; 56: 361-83. 36.Stauch B, Johansson LC, Cherezov V. Structural insights intomelatonin receptors. FEBS J 2020; 287(8): 1496-510. 37.Winczyk K, Pawlikowski M, Karasek M. Melatonin and RZR / RORreceptor ligand CGP 52608 induce apoptosis in the murine colonic cancer.Journal of pineal research 2001; 31(2): 179-82. 38.Pandi-Perumal SR, Trakht I, Srinivasan V, et al. Physiologicaleffects of melatonin: role of melatonin receptors and signal transductionpathways. Progress in neurobiology 2008; 85(3): 335-53. 39.Baba K, Pozdeyev N, Mazzoni F, et al. Melatonin modulates visualfunction and cell viability in the mouse retina via the MT1 melatoninreceptor. Proc Natl Acad Sci U S A 2009; 106(35): 15043-8. 40.Xia Y, Chen S, Zeng S, et al. Melatonin in macrophage biology:Current understanding and future perspectives. Journal of pineal research2019; 66(2): e12547. 41.Zonta YR, Martinez M, Camargo IC, et al. Melatonin ReducesAngiogenesis in Serous Papillary Ovarian Carcinoma of Ethanol-PreferringRats. Int J Mol Sci 2017; 18(4). 42.Tang L, Zhang C, Yang Q, et al. Melatonin maintains inner blood-retinal barrier via inhibition of p38 / TXNIP / NF-kappaB pathway in diabeticretinopathy. J Cell Physiol 2021. 43.Ahn JH, Kim HD, Abuzar SM, et al. Intracorneal melatonin deliveryusing 2-hydroxypropyl-β-cyclodextrin ophthalmic solution for granular cornealdystrophy type 2. International journal of pharmaceutics 2017; 529(1-2): 608-16. 44.Martinez-Aguila A, Fonseca B, Perez de Lara MJ, Pintor J. Effect of Melatonin and 5-Methoxycarbonylamino-N-Acetyltryptamine on the IntraocularPressure of Normal and Glaucomatous Mice. The Journal of pharmacology and experimental therapeutics 2016; 357(2): 293-9. 45. Dal Monte M, Cammalleri M, Amato R, et al. A Topical Formulation of Melatoninergic Compounds Exerts Strong Hypotensive and NeuroprotectiveEffects in a Rat Model of Hypertensive Glaucoma. Int J Mol Sci 2020; 21(23). Summary of the Invention Based on this, the present invention provides a melatonin derivative and its preparation method. The present invention designs a melatonin derivative based on melatonin as a prototype drug and uses the melatonin derivative in eye drop preparations for the treatment of ocular neovascular diseases.

[0010] On the one hand, the present invention provides a melatonin derivative, the structural formula of which is shown in formula (1) below: (1) Where R is , or .

[0012] On the other hand, the present invention provides a method for preparing the above-mentioned melatonin derivative: When R is or The preparation method includes the following steps: (1) After dissolving the reactants, add the amide condensing agent, stir, add 5-methoxytryptamine, and continue the reaction. After the reaction is completed by thin-layer chromatography, remove the solvent by rotary evaporation. Dissolve the crude product in DCM, wash, dry, and purify the intermediate by column chromatography. (2) After dissolving the intermediate in step (1), trifluoroacetic acid was added and the reaction continued. After the reaction was completed by thin-layer chromatography, the crude product was dissolved in sodium bicarbonate aqueous solution, extracted with DCM, the organic phases were combined, dried, and purified by column chromatography to obtain melatonin derivative.

[0013] The reactant mentioned in step (1) is Boc-sarcosine or Boc-glycine; The solvent used for dissolution in step (1) is tetrahydrofuran; the reaction temperature is room temperature; The amide condensing agent mentioned in step (1) is a mixture of TEA, EDCI·HCl and DMAP; The washing described in step (1) is performed by sequentially washing with 0.1M HCl, water and sodium chloride aqueous solution, and the drying is performed by drying with sodium sulfate. The molar ratio of the reactant to 5-methoxytryptamine in step (1) is 1-1.5:1; The molar ratio of TEA, EDCI·HCl and DMAP is 1-2:1-1.5:0.1-1; preferably 1.2:1.2:1.

[0014] The stirring time mentioned in step (1) above is 20-40 min; preferably 30 min.

[0015] The reaction time described in step (1) above is 12-20 hours; preferably 16 hours.

[0016] The solvent used for dissolution in step (2) is DCM, the reaction temperature is room temperature, and the number of extractions is 2-3. The mass-to-volume ratio of the intermediate to trifluoroacetic acid in step (2) is 1-5:5.

[0017] The reaction time described in step (2) is 1-2 hours, preferably 1 hour.

[0018] When R is The preparation method includes the following steps: After dissolving the reactants, an amide condensing agent was added, followed by stirring and the addition of 5-methoxytryptamine. The reaction was continued, and the solvent was removed by rotary evaporation after the reaction was completed by thin-layer chromatography. The crude product was dissolved in DCM, washed, dried, and purified by column chromatography to obtain the melatonin derivative.

[0019] The reactant is N,N-dimethylglycine; The solvent used for dissolution is tetrahydrofuran; the reaction temperature is room temperature; The amide condensing agent is a mixture of TEA, EDCI·HCl and DMAP; The washing process involves sequentially washing with 0.1M HCl, water, and sodium chloride aqueous solution, and the drying process involves drying with sodium sulfate. The molar ratio of the reactant to 5-methoxytryptamine is 1-1.5:1; The molar ratio of TEA, EDCI·HCl and DMAP is 1-2:1-1.5:0.1-1; preferably 1.2:1.2:1.

[0020] The stirring time is 20-40 minutes; preferably 30 minutes.

[0021] The continued reaction time is 12-20 hours; preferably 16 hours.

[0022] In another aspect, the present invention provides the application of the above-mentioned melatonin derivative in the preparation of a drug for treating ocular neovascular diseases, wherein the drug is an eye drop preparation.

[0023] An eye drop formulation comprising the above-mentioned melatonin derivative.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention designs melatonin derivatives based on melatonin as a prototype drug and uses the melatonin derivatives in eye drop formulations for the treatment of ocular neovascular diseases. Attached Figure Description

[0025] Figure 1 Bar chart showing the inhibitory effect of melatonin and its isomers on the proliferation of human umbilical vein endothelial cells (HUVECs); Among them, A is the melatonin (MTN) group; B is the melatonin derivative M1 group; C is the melatonin derivative M2 group; and D is the melatonin derivative M3 group. Figure 2 Bar chart showing the toxic effects of melatonin and its isomers on human umbilical vein endothelial cells (HUVECs); Among them, A is the melatonin (MTN) group; B is the melatonin derivative M1 group; C is the melatonin derivative M2 group; and D is the melatonin derivative M3 group. Figure 3 Bar chart showing the toxic effects of melatonin and its isomers on human corneal epithelial cells (HCE2); Among them, A is the melatonin (MTN) group; B is the melatonin derivative M1 group; C is the melatonin derivative M2 group; and D is the melatonin derivative M3 group. Figure 4 A bar chart showing the effect of intravitreal injection of melatonin and its isomers on postnatal weight gain in mice. Figure 5Images showing the effects of intravitreal injection of melatonin and its isomers on neovascularization and non-perfusion areas of the retina in a mouse OIR model. Among them, A is the blank control group; B is the OIR modeling group; C is the OIR modeling + MTN intervention group; B is the OIR modeling + M1 intervention group; C is the OIR modeling + M2 intervention group; D is the OIR modeling + M3 intervention group. Figure 6 A graph showing the concentration-time variation of melatonin and its isomers in various tissues of the eye of a New Zealand rabbit. Among them, A is the melatonin (MTN) group; B is the melatonin derivative M1 group; C is the melatonin derivative M2 group; and D is the melatonin derivative M3 group. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0027] Example 1: Synthesis of compound M1: The reaction route is as follows:

[0028] (1) 2.39 g of 12.64 mmol Boc-sarcosine (SMB) was added to the reaction flask and dissolved in 20 mL of tetrahydrofuran. 1.07 g of 10.53 mmol TEA, 2.41 g of 12.64 mmol EDCI·HCl and 1.28 g of 10.53 mmol DMAP were added sequentially under stirring at room temperature. The mixture was stirred for 30 minutes, and then 2 g of 10.53 mmol 5-methoxytryptamine (SMA) was added. The mixture was stirred at room temperature for 16 hours. TLC was used to detect the disappearance of the starting material. Tetrahydrofuran was removed by rotary evaporation. DCM was dissolved and washed sequentially with 0.1 M HCl, water and sodium chloride aqueous solution. The product was dried over sodium sulfate and rotary evaporated. The crude product was purified by column chromatography to obtain 3.1 g of compound INT1, with a yield of 81.6%. (2) 3.1 g of compound and 8.59 mmol INT1 were added to the reaction flask, 30 mL of DCM was added to dissolve and stir, 10 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was detected by TLC until the starting material disappeared. The mixture was then evaporated by rotary evaporation, and sodium bicarbonate aqueous solution was added to dissociate the mixture. The mixture was extracted three times with DCM, the organic phases were combined, dried, and evaporated by rotary evaporation. The crude product was purified by column chromatography to obtain 1.8 g of compound M1, with a yield of 80.3%.

[0029] 1H NMR(400 MHz, DMSO-d6): δ 10.62 (s, 1H), 8.02 (s, 1H), 7.24 (d, J= 8.7 Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 6.74 (dd,J = 8.7, 2.4 Hz, 1H), 3.95 (s, 3H), 3.81 (s, 3H), 3.32 (m, 3H), 3.20 (m, 2H), 2.81 (t, J = 7.4 Hz, 2H); MS(ESI) m / z 262.2 (MH + ).

[0030] Example 2: Synthesis of compound M2: The reaction route is as follows:

[0031] 1.3 g of 12.64 mmol N,N-dimethylglycine (SMC) was added to a reaction flask and dissolved in 20 mL of tetrahydrofuran. While stirring at room temperature, 1.07 g of 10.53 mmol TEA, 2.41 g of 12.64 mmol EDCI·HCl, and 1.28 g of 10.53 mmol DMAP were added sequentially. The mixture was stirred for 30 minutes, followed by the addition of 2 g of 10.53 mmol 5-methoxytryptamine (SMA). The mixture was stirred at room temperature for 16 hours. TLC was performed until the starting material disappeared. Tetrahydrofuran was removed by rotary evaporation. The dissolved DMC was washed sequentially with 0.1 M HCl, water, and sodium chloride aqueous solution. The product was dried over sodium sulfate and rotary evaporated. The crude product was purified by column chromatography to give 2.1 g of compound M2, with a yield of 72.5%.

[0032] 1H NMR(400 MHz, DMSO-d6): δ 10.60 (s, 1H), 8.01 (s, 1H), 7.22 (d, J= 8.7 Hz, 1H), 7.09 (d, J = 2.4 Hz, 1H), 7.02 (d, J = 2.3 Hz, 1H), 6.72(dd, J= 8.7, 2.4 Hz, 1H), 3.91 (s, 3H), 3.30 (m, 4H), 2.90 (m, 6H), 2.82 (t, J = 7.3Hz, 2H); MS(ESI) m / z 276.3 (MH + ).

[0033] Example 3: Synthesis of compound M3: The reaction route is as follows:

[0034] (1) 2.21 g of 12.64 mmol Boc-glycine (SMD) was added to the reaction flask and dissolved in 20 mL of tetrahydrofuran. 1.07 g of 10.53 mmol TEA, 2.41 g of 12.64 mmol EDCI·HCl and 1.28 g of 10.53 mmol DMAP were added sequentially under stirring at room temperature. The mixture was stirred for 30 minutes, and then 2 g of 10.53 mmol 5-methoxytryptamine (SMA) was added. The mixture was stirred at room temperature for 16 hours. TLC was used to detect the disappearance of the starting material. Tetrahydrofuran was removed by rotary evaporation. DCM was dissolved and washed sequentially with 0.1 M HCl, water and sodium chloride aqueous solution. The product was dried over sodium sulfate and rotary evaporated. The crude product was purified by column chromatography to obtain 3.1 g of compound INT2, with a yield of 84.9%.

[0035] (2) 3.1 g of compound and 8.59 mmol INT2 were added to the reaction flask, 30 mL of DCM was added to dissolve and stir, 10 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was detected by TLC until the starting material disappeared. The mixture was then evaporated by rotary evaporation, and sodium bicarbonate aqueous solution was added to dissociate the mixture. The mixture was extracted three times with DCM, the organic phases were combined, dried, and evaporated by rotary evaporation. The crude product was purified by column chromatography to obtain 1.9 g of compound M3, with a yield of 86.1%.

[0036] 1H NMR(400 MHz, DMSO-d6): δ 10.66 (s, 1H), 8.02 (s, 1H), 7.21 (d, J= 8.7 Hz, 1H), 7.12 (d, J = 2.3 Hz, 1H), 7.03 (d, J = 2.4 Hz, 1H), 6.72 (dd,J = 8.7, 2.4 Hz, 1H), 3.80 (s, 3H), 3.36 (q, J = 7.1 Hz, 2H ), 3.10 (s, 2H), 2.83 (t, J = 7.4 Hz, 2H), 2.29 (brs, 2H ); MS(ESI) m / z 248.1 (MH + ).

[0037] Efficacy Experiment: In vivo and in vitro experiments on the inhibition of ocular angiogenesis by melatonin and its derivatives 1. Inhibitory effect of melatonin and its derivatives on the proliferation of human umbilical vein endothelial cells (HUVECs) Cell proliferation function was determined using the CCK8 assay. (1) Add 100 μL of cells (about 1000 cells) to each well of a 96-well plate; 3 replicates per sample. Incubate the cells at 37°C, 5% CO2, and 21% O2 until the cells are nearly 70%-80% confluent, then switch to serum-free medium to synchronize the cells to the G0 phase.

[0038] (2) Add 10 μL of drug of different concentrations to each well for stimulation.

[0039] (3) Continue to culture the 96-well plate in a cell culture incubator at 37°C, 5% CO2, and 21% O2 for 12 hours.

[0040] (4) Add 10 μL of CCK-8 solution to each well. Incubate at 37℃, 5% CO2, 21% O2 for 2 h.

[0041] (5) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0042] The test results are attached. Figure 1 .

[0043] Depend on Figure 1 It can be seen that: when the melatonin concentration reaches 10... -2 At M, it has a significant inhibitory effect on the proliferation of HUVECs; the concentrations of the three melatonin isomers reach 10. -3 At time M, it has a significant inhibitory effect on the proliferation of HUVECs.

[0044] 2. Toxicity of melatonin and its isomers on human umbilical vein endothelial cells (HUVECs) Experimental methods: (1) Add 100 μL of cells (approximately 5000 cells) to each well of a 96-well plate; repeat 3 times for each sample. Incubate the cells at 37°C, 5% CO2, and 21% O2 until the cells are fully adhered.

[0045] (2) Add 10 μL of drug of different concentrations to each well for stimulation.

[0046] (3) Continue to culture the 96-well plate in a cell culture incubator at 37°C, 5% CO2, and 21% O2 for 12 hours.

[0047] (4) Add 10 μL of CCK-8 solution to each well. Incubate at 37℃, 5% CO2, 21% O2 for 2 h.

[0048] (5) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0049] The test results are attached. Figure 2 .

[0050] Depend on Figure 2 It can be seen that: MTN at concentrations ≤10 -2M showed no significant toxicity to HUVECs; the three melatonin isomers showed no significant toxicity at concentrations of 10. -2 At M, it has a toxic effect on HUVECs.

[0051] 3. Toxic effects of melatonin and its isomers on human corneal epithelial cells (HCE2). Experimental method: Same as above The test results are attached. Figure 3 .

[0052] Depend on Figure 3 It can be seen that: MTN and its isomers at concentrations <10 -2 At M, there was no significant toxic effect on HCE2.

[0053] 4. Inhibitory effects of melatonin and its isomers on angiogenesis in mouse oxygen-induced retinopathy (OIR). Experimental methods: 1) Establishment of the OIR model in C57BL / 6J mice: On postnatal day 7 (P7), C57BL / 6J suckling mice were placed in a hyperoxia chamber with an oxygen partial pressure of 75% (75% O2) along with their mothers and kept there for 5 days (the mothers were replaced every other day to avoid hyperoxia injury to the mothers). On P12, they were returned to a normal oxygen environment (21% O2) and continued to be raised until the observation time point. Control group: Age-matched newborn suckling mice were raised in a normal oxygen environment (21% O2) until the observation time point.

[0054] 2) Intravitreal injection of MTN, M1, M2, and M3: Newborn mice in each group were injected intravitreally once with MTN, M1, M2, and M3 at P12 (on the day after leaving the oxygen chamber), and were then fed until the observation time point. The control group was injected intravitreally once with an equal volume of physiological saline.

[0055] 3) Observe the changes in neovascularization and non-perfusion areas in the retina of mice in each group: Retinal tissues of mice in each group were collected at P17, retinal slides were prepared, vascular endothelium was immunofluorescently labeled, and the changes in the area of ​​non-perfusion areas and neovascularization areas in the retina of mice in each group were compared. The test results are attached. Figure 4-5 .

[0056] Depend on Figure 4 It can be seen that the body weight of mice P17 injected with melatonin and its isomers intravitreal was not significantly different from that of the control group.

[0057] Depend on Figure 5 It can be seen that, compared with the normal control group, after OIR modeling (Figure BF), the mouse retina showed obvious central non-perfusion area and peripheral neovascularization area. After intravitreal injection of M2 into the OIR mouse cavity, the area of ​​retinal neovascularization was reduced compared with other OIR model groups.

[0058] 5. Analysis of ocular pharmacokinetic biological samples of melatonin and its isomers from New Zealand rabbits after a single dose. Experimental methods: 1. Solution preparation (1) Homogenization extract (methanol: ethanol: water = 2:2:1, v:v:v): Measure 40 mL of methanol, 40 mL of ethanol and 20 mL of ultrapure water, mix them well and prepare the appropriate amount as needed.

[0059] (2) Preparation method of tissue homogenate: After weighing, cut the tissue into small pieces and place it in a spiral tube. Add 4 times (cornea, sclera) or 9 times (retina) the weight of the tissue homogenate extract (methanol: ethanol: water = 2:2:1, v:v:v), and add 1 spherical meson. Homogenize for 30 seconds using a Fastprep-24 5G sample processing system at a speed of 6 m / sec. Centrifuge at 14000 rpm / min for 10 min and control the temperature at 4℃. Take the supernatant as the corresponding tissue homogenate.

[0060] (3) Melatonin (MTN) reference stock solution: Weigh a certain amount of melatonin reference standard (greater than 2 mg), dissolve it in a small amount of ethanol, and then dilute it with 50% methanol to obtain a melatonin reference stock solution with a concentration of 1.00 mg / mL. Store at 2-8℃.

[0061] (4) M1 reference stock solution: Take a certain volume of M1 reference (greater than 20 μL), dissolve it in a small amount of methanol, and then dilute it with 50% methanol to obtain an M1 reference stock solution with a concentration of 1.00 mg / mL. Store at 2-8℃.

[0062] (5) M2 reference stock solution: Weigh a certain amount of M2 reference (greater than 2 mg), dissolve it in a small amount of methanol, and then dilute it with 50% methanol to obtain an M2 reference stock solution with a concentration of 1.00 mg / mL. Store at 2-8℃.

[0063] (6) M3 reference stock solution: Weigh a certain amount of M3 reference (greater than 2 mg), dissolve it in a small amount of methanol, and then dilute it with 50% methanol to obtain an M3 reference stock solution with a concentration of 1.00 mg / mL. Store at 2-8℃.

[0064] (7) Phenacetin reference stock solution: Weigh a certain amount of phenacetin (greater than 2 mg), add a certain amount of methanol to dissolve it, and dilute it with 50% methanol solution to obtain a phenacetin reference stock solution with a concentration of 1.00 mg / mL.

[0065] (8) Mobile phase (0.1% formic acid aqueous solution): Measure 500 mL, add 500 L of formic acid, and mix well. Prepare the appropriate amount as needed.

[0066] (9) Preparation of diluent (50% acetonitrile solution): Measure acetonitrile and ultrapure water, mix them in a 1:1 ratio and shake well.

[0067] (10) Internal standard working solution: Pipette 50 μL of 1.00 mg / mL phenacetin reference stock solution and add it to 950 L of 50% acetonitrile solution to prepare an internal standard solution with a concentration of 50 μg / mL. Then, pipette 10 L of the 50 μg / mL internal standard solution and add it to 990 L of 50% acetonitrile solution to prepare an internal standard working solution with a concentration of 0.500 μg / mL. Prepare the solution according to the required amount.

[0068] (11) Preparation of standard curve sample A (SJ-202016): Use blank matrix (rabbit plasma, aqueous humor, vitreous body, corneal homogenate or retinal homogenate) and MTN, M1, M2 and M3 reference stock solutions to prepare a series of standard curve samples with different concentrations according to the table below. The required amount can be prepared according to the ratio of reference volume to blank matrix volume.

[0069]

[0070] Note: Standard solutions in tubes S1-S9 are used for constructing standard curves. The linear range of MTN, M1, M2, and M3 is 1.00-400 ng / mL.

[0071] (12) Preparation of Quality Control (QC) Sample A (SJ-202016): Use blank matrix (rabbit plasma, aqueous humor, vitreous body, corneal homogenate, scleral homogenate or retinal homogenate) and MTN, M1, M2 and M3 reference stock solutions to prepare a series of quality control (QC) samples according to the table below. The required amount can be prepared according to the ratio of standard solution volume to blank matrix volume.

[0072]

[0073] (13) Preparation of standard curve sample B (SJ--202111): Use blank matrix (rabbit vitreous body, corneal homogenate, scleral homogenate or retinal homogenate) and MTN, M2 reference stock solution to prepare a series of standard curve samples with different concentrations according to the table below. The required amount can be prepared according to the ratio of reference volume to blank matrix volume.

[0074]

[0075] Note: Standard solutions in tubes S1-S9 are used for constructing standard curves. The linear range of MTN, M1, M2, and M3 is 1.00-400 ng / mL.

[0076] (14) Preparation of Quality Control (QC) Sample B (SJ-202111): Use blank matrix (rabbit vitreous body, corneal homogenate, scleral homogenate or retinal homogenate) and MTN, M2 reference stock solution to prepare a series of quality control (QC) samples according to the table below. The required amount can be prepared according to the ratio of standard liquid volume to blank matrix volume.

[0077]

[0078] 2. Chromatographic conditions Column: Waters ACQUITY UPLC BEH C18 (2.1mm × 50mm, 1.7μm) Mobile phase: A is 0.1% formic acid aqueous solution; B is methanol. Flow rate: 0.4 mL / min The flow ratios are as follows:

[0079] Column temperature: 40℃ Injection volume: 5 μL 3. Mass spectrometry method The system employs an electrospray ionization (ESI) power supply, positive ion mode, and multiple reaction monitoring. Key parameters include: Capillary voltage: 2.00 kV; Sample cone: 30 V; Source temperature: 150 °C; Desolvation temperature: 500 °C; Desolvation gas flow: 1000 L / h; Cone gas flow: 10 L / h.

[0080] The mass spectrometry conditions for MTN, M1, M2, M3 and the internal standard phenacetin are as follows:

[0081] 4. Sample pretreatment Accurately transfer 50 μL of blank matrix or standard curve sample, quality control sample, or unknown sample, add 5 L of internal standard working solution (0.5 g / mL phenacetin), add 150 L of acetonitrile, vortex for 30 s, centrifuge at 14000 r / min for 15 min, control the temperature at 4℃, and transfer the supernatant to the sample vial.

[0082] The experimental results are shown in Tables 1-4 below. Figure 6 : Table 1. Average values ​​of major pharmacokinetic parameters of MTN in various tissues (Mean ± SD)

[0083] Note: "—" indicates no such data, and "±" indicates n=1.

[0084] Table 2. Mean values ​​of major pharmacokinetic parameters of M1 in each tissue (Mean ± SD)

[0085] Note: "—" indicates no such data, and "±" indicates n=1.

[0086] Table 3. Mean values ​​of major pharmacokinetic parameters of M2 in each tissue (Mean ± SD)

[0087] Note: "—" indicates no such data, and "±" indicates n=1.

[0088] Table 4. Average values ​​of major pharmacokinetic parameters (Mean ± SD) for M3 in each tissue.

[0089] Note: "—" indicates no such data, and "±" indicates n=1.

[0090] According to Table 1-4 above and Figure 6 It is understood that the present invention designs melatonin derivatives based on melatonin as a prototype drug, and uses melatonin derivatives in eye drop formulations to treat ocular neovascular diseases.

[0091] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A melatonin derivative, characterized in that: Its structural formula is shown in equation (1) below: （1） Where R is , or .

2. The method for preparing the melatonin derivative according to claim 1, characterized in that: When R is or The preparation method includes the following steps: (1) After dissolving the reactants, add the amide condensing agent, stir, add 5-methoxytryptamine, and continue the reaction. After the reaction is completed by thin-layer chromatography, remove the solvent by rotary evaporation. Dissolve the crude product in DCM, wash, dry, and purify the intermediate by column chromatography. (2) After dissolving the intermediate in step (1), trifluoroacetic acid was added and the reaction continued. After the reaction was completed by thin-layer chromatography, the crude product was dissolved in sodium bicarbonate aqueous solution, extracted with DCM, the organic phases were combined, dried, and purified by column chromatography to obtain melatonin derivative.

3. The preparation method according to claim 1, characterized in that: The reactant in step (1) is Boc-sarcosine or Boc-glycine; the molar ratio of the reactant to 5-methoxytryptamine is 1-1.5:

1.

4. The preparation method according to claim 1, characterized in that: The solvent used for dissolution in step (1) is tetrahydrofuran; the reaction temperature is room temperature.

5. The preparation method according to claim 1, characterized in that: The amide condensing agent mentioned in step (1) is a mixture of TEA, EDCI·HCl and DMAP; the molar ratio of TEA, EDCI·HCl and DMAP is 1-3:1-2:0.1-1.

6. The method for preparing the melatonin derivative according to claim 1, characterized in that: When R is The preparation method includes the following steps: After dissolving the reactants, an amide condensing agent was added, followed by stirring and the addition of 5-methoxytryptamine. The reaction was continued, and the solvent was removed by rotary evaporation after the reaction was completed by thin-layer chromatography. The crude product was dissolved in DCM, washed, dried, and purified by column chromatography to obtain the melatonin derivative.

7. The preparation method according to claim 6, characterized in that: The reactant is N,N-dimethylglycine.

8. The preparation method according to claim 6, characterized in that: The amide condensing agent is a mixture of TEA, EDCI·HCl and DMAP; the molar ratio of TEA, EDCI·HCl and DMAP is 1-2:1-1.5:0.1-1.

9. The use of the melatonin derivative of claim 1 or the melatonin derivative prepared by any one of claims 2-8 in the preparation of a drug for treating ocular neovascular diseases, wherein the drug is an eye drop preparation.

10. An eye drop formulation comprising the melatonin derivative of claim 1 or the melatonin derivative prepared by any one of claims 2-8.

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