Small molecule active aldehyde scavenger-albumin conjugate drugs, preparation method and application thereof

Abstract

The application discloses a small-molecule active aldehyde scavenger-albumin coupled drug, a preparation method and application thereof, the coupled drug comprising a) the small-molecule active aldehyde scavenger compound or its derivative, and b) albumin, the albumin comprising at least one of human serum albumin, recombinant albumin, bovine serum albumin, ovalbumin and mouse serum albumin. The drug is further prepared into an ophthalmic preparation, the retention time of the drug in the cornea is improved, the efficiency of in-vitro scavenging active aldehyde is improved to 1.5 times of NS-2, the expression of inflammatory factor IL-6 of human corneal epithelial cells is significantly inhibited (greater than or equal to 50%), and the drug has no irritation to the eye. Through the long-acting sustained release of the albumin carrier and the synergistic effect of the targeted scavenging of NS, the technical defects of traditional dry eye drugs, such as short-acting and single mechanism, are overcome, and the drug has clear clinical transformation value.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a small molecule active aldehyde scavenger-albumin conjugate drug, its preparation method, and its application. Background Technology

[0002] Dry eye disease (DED) is a chronic ocular surface disease caused by insufficient tear secretion, excessive evaporation, or abnormal tear composition. Its core pathological mechanism involves a cascade of inflammatory responses (abnormally elevated levels of inflammatory factors such as IL-6 and TNF-α) and oxidative stress (accumulation of reactive aldehydes such as 4-hydroxynonenal) triggered by tear film homeostasis imbalance. DED severely impacts patients' quality of life and may lead to visual impairment. Current mainstream treatments have significant drawbacks. For example, cyclosporine A has a slow onset of action and is intolerant to some patients, causing a burning sensation after administration; long-term use of glucocorticoids can easily lead to increased intraocular pressure; and artificial tears cannot fundamentally treat the inflammatory pathways. Clinically, there is an urgent need for novel treatment strategies that combine rapid onset of action, long-term maintenance, and multi-mechanism intervention.

[0003] Reproxalap (NS-2), a novel active aldehyde small molecule inhibitor, exerts its anti-inflammatory effect through covalent binding with RASP. Preclinical studies have shown that it can rapidly inhibit the release of inflammatory mediators in tears. However, it has drawbacks such as short ocular retention time, limited corneal penetration, the need for frequent administration, and significant ocular irritation.

[0004] Human serum albumin (HSA), as a core component of tear film proteins, participates in ocular surface protection through multiple mechanisms: (1) In terms of physical barrier, its water-holding capacity (each molecule can bind ≥18 water molecules) and viscoelasticity (the viscosity of a 10 mg / mL solution reaches 3.2 mPa·s) can prolong the tear film breakup time by up to 8.5 seconds; (2) In terms of biological activity, 0.1 mM HSA inhibits corneal epithelial cell apoptosis by regulating the Bax / Bcl-2 protein ratio (decreasing to 0.31 times) (Annexin V positivity rate decreased by 65%), and increases mucin secretion by 2.4 times by activating MUC5AC gene expression. More importantly, as a drug carrier, HSA can prolong the plasma half-life of conjugates to 19-27 days (compared to free drugs <6 h), and its surface charge properties (pI = 4.7) can enhance corneal adhesion (increasing the amount of isolated porcine cornea retention by 3.8 times). This provides a new approach to addressing the shortcomings of Reproxalap (NS-2). Summary of the Invention

[0005] This invention addresses the technical bottlenecks in the treatment of eye diseases such as dry eye, including short-lived drug action, simple mechanisms of action, and strong irritation, by providing a bifunctional drug system based on albumin conjugation technology. The drug utilizes a warhead covalent chemical conjugation strategy to covalently couple the Reproxalap derivative NS with albumin, forming a novel molecular entity with synergistic effects of active aldehyde scavenging and antioxidant activity. The core construction scheme involves using maleimide as a linker to covalently couple NS to the cysteine ​​thiol group at position 34 of albumin, or using a lysine warhead as a linker to covalently couple NS to the surface lysine residue of albumin. The R1 group of the NS derivative is limited to halogen, C1-3 alkoxy, or cyano groups, etc.

[0006] The specific content of this invention is as follows:

[0007] In a first aspect, the present invention provides a small molecule active aldehyde scavenger-albumin conjugate, the conjugate comprising the following structure:

[0008] a) The small molecule active aldehyde scavenger compound or its derivative, wherein the structural formula of the small molecule active aldehyde scavenger compound is:

[0009]

[0010] R1 includes hydrogen and C. 1-3 Alkyl, C 2-3 alkenyl, C 2-3 alkynyl group, C 1-3 Alkoxy, C 3-5 cycloalkyl, C 3-5 Cycloalkyloxy, halogen, nitro, cyano, trifluoromethyl, 3-7 membered heterocyclic groups, unsubstituted or substituted C 6-10 At least one of the aryl groups, wherein the unsubstituted or substituted C 6-10 C substituted in aryl groups 6-10 The substituent group in the aromatic heterol group is named Ra. Optionally, each Ra may be the same or different, and each Ra is independently selected from halogens, C... 1-5 Alkyl, C 1-5 Alkoxy, C 6-10 At least one of the aryl acyl groups, preferably, R1 is a halogen, C 1-3 At least one of alkoxy, trifluoromethyl, nitro, and cyano groups;

[0011] b) Albumin, wherein the albumin comprises at least one of human serum albumin, recombinant albumin, bovine serum albumin, ovalbumin, and mouse serum albumin. Preferably, the recombinant albumin is recombinant human albumin.

[0012] Furthermore, in the connector, X is selected from at least one of carbon atom, nitrogen atom, and oxygen atom, and n is 0 to 3.

[0013] Furthermore, the small molecule active aldehyde scavenger compound or its derivative is coupled to albumin via chemical bonds or non-chemical bonds.

[0014] Furthermore, the small molecule active aldehyde scavenger compound or its derivative is covalently coupled to albumin via a warhead.

[0015] Furthermore, the small molecule active aldehyde scavenger is covalently coupled to the thiol group of cysteine ​​at position 34 of albumin via a cysteine ​​tip, or covalently coupled to the amino group of lysine in albumin via a lysine tip.

[0016] Furthermore, each albumin molecule is loaded with at least one small molecule active aldehyde scavenger compound or its derivative. Optionally, each albumin molecule is loaded with 1-4 small molecule active aldehyde scavenger compounds or their derivatives.

[0017] In a second aspect, the present invention provides a composition comprising the aforementioned conjugated drug and an additive acceptable in the pharmaceutical field.

[0018] Furthermore, the additives include at least one of the following: pH adjusters and buffers (such as boric acid, sodium hydroxide, phosphate buffer), osmotic pressure adjusters (such as sodium chloride, glycerol, mannitol), preservatives (such as benzalkonium chloride), antioxidants (such as sodium bisulfite, sodium ascorbate), thickeners (such as sodium carboxymethyl cellulose), surfactants (such as polysorbate 80), and stabilizers (α-cyclodextrin).

[0019] It should be noted that this invention does not specifically limit the additives in the pharmaceutical field. Any additives acceptable in the pharmaceutical field that can act as freeze-drying protectants, preservatives, or antioxidants are covered by this invention.

[0020] In a third aspect, the invention provides the use of the conjugated drug or the composition thereof in the preparation of a medicament for treating and / or preventing eye diseases.

[0021] Furthermore, the ocular diseases include, but are not limited to: dry eye syndrome, corneal injury, conjunctivitis, keratitis, scleritis, uveitis, post-cataract surgery inflammation, glaucoma, macular degeneration (including wet and dry age-related macular degeneration), diabetic retinopathy, retinal vein occlusion, intraocular neovascularization, ocular surface fibrosis, ocular scarring pemphigoid, refractive keratotomy (PRK) healing disorders, Fuch's endothelial dystrophy in the cornea, and ocular tumors.

[0022] Furthermore, the conjugated drug or the composition thereof treats eye diseases through synergistic effects in increasing the drug's residence time in the cornea, inhibiting the expression of the inflammatory factor IL-6 in human corneal epithelial cells, promoting tear secretion, promoting corneal damage repair, improving crystal morphology, and relieving eye irritation.

[0023] In a fourth aspect, the present invention provides a method for preparing the aforementioned conjugated drug, the method comprising the following steps:

[0024] S1: In a weakly alkaline buffer system, albumin and a small molecule active aldehyde scavenger compound are mixed in a molar ratio of 1:1 to 1:10. Preferably, albumin and a nitrite donor compound are mixed in a molar ratio of 1:1 to 1:8. More preferably, albumin and a nitrite donor compound are mixed in a molar ratio of 1:1 to 1:4.

[0025] S2: React at 4-25 ℃ in the dark for 8-12 h to generate the conjugated drug.

[0026] Further, the pH of the buffer system in step S1 is 7.0~8.0. Preferably, the buffer solution in the buffer system includes phosphate buffer (PBS) with 1-10 mM EDTA.

[0027] Furthermore, in step S1, the albumin concentration is 200-500 μM, and the concentration of the small molecule active aldehyde scavenger compound is 500-1000 μM.

[0028] Furthermore, step S2 also includes a co-solvent, the concentration of which is 1-5% (v / v). Preferably, the co-solvent includes at least one of DMSO, DMF, or PEG-400.

[0029] Furthermore, the method also includes the steps of purifying and drying the obtained conjugated drug.

[0030] In a specific embodiment of the present invention, the experimental step of purifying the conjugated drug is to use an ultrafiltration membrane with a molecular weight cutoff of 10-50 kDa to remove unbound small molecule compounds.

[0031] Furthermore, the method also includes a step of ultrafiltration centrifugation to concentrate the conjugated drug purified by ultrafiltration membrane. Preferably, the specific operation of ultrafiltration concentration is 4 °C, 10000-15000 rpm, 8-12 min.

[0032] Furthermore, the method also includes a step of washing the ultrafiltration concentrated conjugated drug solution, preferably by washing 3-5 times with PBS at pH 7.0-7.4, and collecting the final conjugated drug solution.

[0033] In a specific embodiment of the present invention, the drying step is freeze drying.

[0034] Furthermore, the freeze-drying step includes pre-freezing at -80 ℃ for 24 h, transferring to a freeze dryer pre-cooled to -50 ℃, and performing a gradient freeze-drying program: primary drying at -35 ℃ for 24 h (vacuum degree <10 Pa), followed by secondary drying at 25 ℃ for 24 h, finally obtaining a white, loose freeze-dried powder that is nitrogen-filled, capped, and sealed for storage.

[0035] Furthermore, the method also includes the step of preparing a small molecule active aldehyde scavenger compound or a derivative thereof.

[0036] It should be noted that this invention does not specifically limit the method for preparing small molecule active aldehyde scavengers or their derivatives, and any method capable of preparing the small molecule active aldehyde scavenger compound or its derivative is protected by this invention.

[0037] The beneficial effects of the present invention include, but are not limited to:

[0038] The conjugated drug of the present invention exerts its efficacy through a triple mechanism: (1) albumin carrier prolongs ocular surface retention and enhances corneal permeability; (2) small molecule active aldehyde scavenger compound or its derivative targets and removes active aldehyde; (3) albumin itself inhibits oxidative stress and mucin secretion.

[0039] The conjugated drug described in this invention can significantly promote tear secretion, promote corneal damage repair, improve crystal morphology, inhibit the expression of pro-inflammatory factors, promote epithelial proliferation and accelerate corneal structural reconstruction, and has broad application prospects in the preparation of ophthalmic drugs.

[0040] The conjugated drug of this invention has significant efficacy for indications such as dry eye, allergic conjunctivitis, and post-corneal repair (in the mouse model Schirmer, tear secretion increased by 1.7 times and corneal fluorescein staining score decreased by 2 times), providing a solution that combines long-term efficacy and safety to overcome the limitations of existing therapies. Attached Figure Description

[0041] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0042] Figure 1 This is a schematic diagram illustrating the molecular principle of the recombinant human albumin-coupled active aldehyde scavenger in this invention.

[0043] Figure 2 This is a schematic diagram showing the stability results of the target compound in physiological saline for 72 h in an embodiment of the present invention.

[0044] Figure 3This is a schematic diagram showing the stability results of the target compound in sodium hyaluronate eye drops for 72 hours in an embodiment of the present invention.

[0045] Figure 4 These are schematic diagrams illustrating the ability of compounds 15, 16, 28a-b and the positive control drug NS-2 to scavenge active aldehydes in the embodiments of the present invention. Figure A shows the results of the ability of compounds 15, 16 and the positive control drug NS-2 to scavenge active aldehydes; Figure B shows the results of the ability of compounds 15, 28a-b and the positive control drug NS-2 to scavenge active aldehydes.

[0046] Figure 5 This is a schematic diagram showing the effect of each drug on tear secretion in mice with dry eye syndrome in the embodiments of the present invention.

[0047] Figure 6 Figure 1 is a schematic diagram showing the effect of each drug on the corneal damage repair level of dry eye mice in the embodiments of the present invention. Figures A and B are photographs of the corneal damage repair level of dry eye mice, and Figure C is a schematic diagram showing the effect of each drug on the corneal damage repair level of dry eye mice.

[0048] Figure 7 Figure A is a schematic diagram showing the effect of each drug on the recovery of tear crystallization in mice with dry eye syndrome in the embodiments of the present invention. Figure B is an electron microscope image of each drug on the recovery of tear crystallization in mice with dry eye syndrome, and Figure B is a statistical graph of the effect of each drug on the recovery of tear crystallization in mice with dry eye syndrome.

[0049] Figure 8 This is a schematic diagram showing the effects of various drugs on the levels of inflammatory factors in mice with dry eye syndrome in the embodiments of the present invention. In this diagram, Figure A shows the results of TNF-α and Figure B shows the results of IL-1β.

[0050] Figure 9 These are corneal HE staining images after each drug intervention in the embodiments of the present invention (n = 4, scale bar = 100 μm).

[0051] Figure 10 This is a schematic diagram of the irritation scores of the conjugate group, positive control group, and solvent group obtained by the Draize experiment in an embodiment of the present invention.

[0052] Figure 11 This is a schematic diagram of the in vitro antioxidant capacity test results of the target compound in the embodiments of the present invention. Detailed Implementation

[0053] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0054] Example 1: Synthesis of (8-aminooctyl)carbamate tert-butyl ester (YY-1)

[0055]

[0056] 1,8-Octadiamine (5 g, 34.68 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane and stirred until dissolved. Et3N (4.0 equiv) was then added, and the mixture was covered with a constant-pressure dropping funnel. Di-tert-butyl dicarbonate (0.6 equiv) was dissolved in anhydrous dichloromethane and added to the constant-pressure dropping funnel. Nitrogen gas was purged, and di-tert-butyl dicarbonate was added dropwise. The mixture was stirred overnight at room temperature. The reaction was monitored by TLC (DCM:MeOH = 6:1) with color development on alkaline KMnO4. After the starting material had reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 6:1) to give intermediate YY-1 (3.5 g). Yield: 41%, colorless oil.

[0057] Example 2: Synthesis of tert-butyl (8-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)octyl)carbamate (YY-2)

[0058]

[0059] Intermediate YY-1 (3.5 g, 14.33 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane, and Et3N (1.5 equiv) was added. After pre-cooling at 0 °C for 15 min, maleic anhydride (1.0 equiv) dissolved in anhydrous dichloromethane was added dropwise, purging with nitrogen, and the mixture was stirred at room temperature for 4 h. After the starting material reacted completely, the mixture was concentrated, dissolved in acetone, and Et3N (1.5 equiv) and acetic anhydride (1.5 equiv) were added. The mixture was then heated to 60 °C under nitrogen protection and stirred for 20 h. The reaction was monitored by TLC (DCM:MeOH = 8:1), and the reaction was developed with basic KMnO4. After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 8:1) to obtain intermediate YY-2 (1.6 g). The yield was 35%, and the product was a white solid powder.

[0060] Example 3: Synthesis of 1-(8-aminooctyl)-1H-pyrrole-2,5-dione (YY-3)

[0061]

[0062] Intermediate YY-2 (1.6 g, 4.97 mmol, 1.0 equiv) was dissolved in ethyl acetate, and a hydrogen chloride-ethyl acetate solution (2 M in ethyl acetate, 4 equiv) was added. Nitrogen gas was then introduced, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC (DCM: MeOH = 10:1), and a colorimetric reaction was performed with basic KMnO4. After the starting material had reacted completely, the reaction mixture was filtered and concentrated to give intermediate YY-3 (1.3 g). The yield was 98%, and the product was a white solid powder.

[0063] Example 4: Synthesis of 4-((8-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)octyl)amino)-4-oxobutyric acid (13)

[0064]

[0065] Intermediate YY-3 (1.3 g, 4.87 mmol, 1.0 equiv) was dissolved in anhydrous DMF, and succinic anhydride (1.5 equiv) and N,N-diisopropylethylamine (2.0 equiv) dissolved in anhydrous DMF were slowly added dropwise, purging with nitrogen, and stirred overnight at room temperature. The reaction was monitored by TLC (DCM:MeOH = 10:1) with color development on alkaline KMnO4. After the starting material had reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 10:1) to give linker compound 13 (1.31 g). Yield: 83%. White solid powder.

[0066] Example 5: Synthesis of tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (L-1)

[0067]

[0068] 1,8-Diamino-3,6-dioxane (5 g, 33.76 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane. After stirring and dissolving, Et3N (4.0 equiv) was added. The mixture was covered with a constant-pressure dropping funnel. Di-tert-butyl dicarbonate (0.6 equiv) was dissolved in anhydrous dichloromethane and added to the constant-pressure dropping funnel. Nitrogen gas was purged, and di-tert-butyl dicarbonate was added dropwise. The mixture was stirred overnight at room temperature. The reaction was monitored by TLC (DCM:MeOH = 6:1) and developed with alkaline KMnO4. After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 6:1) to give intermediate L-1 (3.6 g). Yield: 43%, white solid powder.

[0069] Example 6: Synthesis of tert-butyl (2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)ethoxy)ethyl)carbamate (L-2)

[0070]

[0071] Intermediate L-1 (3.6 g, 14.50 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane, and Et3N (1.5 equiv) was added. After pre-cooling at 0 °C for 15 min, maleic anhydride (1.0 equiv) dissolved in anhydrous dichloromethane was added dropwise, purging with nitrogen, and the mixture was stirred at room temperature for 4 h. After the starting material reacted completely, the mixture was concentrated, dissolved in acetone, and Et3N (1.5 equiv) and acetic anhydride (1.5 equiv) were added. The mixture was then heated to 60 °C under nitrogen protection and stirred for 20 h. The reaction was monitored by TLC (DCM:MeOH = 8:1), and the reaction was observed with basic KMnO4. After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 8:1) to give intermediate L-2 (1.5 g). The yield was 32%, and the product was a milky white oil.

[0072] Example 7: Synthesis of 1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-pyrrole-2,5-dione (L-3)

[0073]

[0074] Intermediate L-2 (1.5 g, 4.57 mmol, 1.0 equiv) was dissolved in ethyl acetate, and a hydrogen chloride-ethyl acetate solution (2 M in ethyl acetate, 4 equiv) was added. Nitrogen gas was then introduced, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC (DCM: MeOH = 10:1), and a colorimetric reaction was performed with basic KMnO4. After the starting material had reacted completely, the reaction mixture was filtered and concentrated to give intermediate L-3 (1.16 g). The yield was 96%, and the product was a yellow oil.

[0075] Example 8: Synthesis of 4-((2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)ethoxy)ethyl)amino)-4-oxobutyric acid (14)

[0076]

[0077] Intermediate L-3 (1.16 g, 4.39 mmol, 1.0 equiv) was dissolved in anhydrous DMF, and succinic anhydride (1.5 equiv) and N,N-diisopropylethylamine (2.0 equiv) dissolved in anhydrous DMF were slowly added dropwise, purging with nitrogen, and stirred overnight at room temperature. The reaction was monitored by TLC (DCM:MeOH = 10:1) with color development on alkaline KMnO4. After the starting material had reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 10:1) to give linker compound 14 (864 mg). Yield: 60%. Pale yellow oil.

[0078] Example 9: Synthesis of 1-bromo-3-hydroxy-3-methyl-2-butanone (2)

[0079]

[0080] Preparation of NaHSO4·SiO2 catalyst. SiO2 (5 g, 83.22 mmol, 1.0 equiv) was added to an aqueous solution of NaHSO4·H2O (11.49 g, 83.22 mmol, 1.0 equiv), and the reaction mixture was stirred for 15 min. The mixture was then slowly heated until a white solid formed. Finally, the solid was dried in an oven at 120 °C for 48 h to obtain the NaHSO4·SiO2 catalyst. Yield: 99%. White solid.

[0081] Starting material 1 (3-hydroxy-3-methyl-2-butanone) (2.00 g, 19.58 mmol, 1.0 equiv) was dissolved in anhydrous CCl4 (100 mL), and N-bromosuccinimide (NBS) (4.18 g, 23.50 mmol, 1.2 equiv) was added. The mixture was stirred and NaHSO4·SiO2 (1.76 g, 9.79 mmol, 0.5 equiv) was added. The mixture was then heated to 80 °C under reflux and nitrogen protection, and stirred for 2 h. The reaction was monitored by TLC (DCM:MeOH = 100:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 100:1) to give intermediate 2 (2.63 g). Yield: 75%. Pale yellow oil.

[0082] Example 10: Synthesis of 1-(3-hydroxy-3-methyl-2-oxobutyl)pyridine-1-onium (3)

[0083]

[0084] Intermediate 2 (2.0 g, 11.11 mmol, 1.0 equiv) was dissolved in anhydrous ethanol and pre-cooled at 0 °C for 15 min. Pyridine (1.05 g, 13.33 mmol, 1.2 equiv) was then added, and stirring continued for 15 min. The mixture was then heated to 65 °C under nitrogen protection and stirred overnight. The reaction was monitored by TLC (DCM: MeOH = 10:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM: MeOH = 10:1) to give intermediate 3 (2.36 g). Yield: 82%. A pale yellow solid powder. 1 H NMR (300 MHz, DMSO- d 6) δ 9.00 – 8.89 (m, 2H), 8.69 (tt, J = 7.8, 1.4 Hz, 1H), 8.31 – 8.17 (m, 2H), 6.13 (s, 2H), 5.89 (s, 1H), 1.35 (s, 6H).

[0085] Example 11: Synthesis of 2-amino-5-iodobenzyl alcohol (5)

[0086]

[0087] The starting material 2-amino-5-iodobenzoic acid (compound 4, 15 g, 57.03 mmol, 1.0 equiv) was dissolved in anhydrous tetrahydrofuran. Lithium aluminum hydride (1M solution in THF, 1.2 equiv) was added to a constant-pressure dropping funnel, and nitrogen was purged. After pre-cooling at 0 °C for 15 min, lithium aluminum hydride was added dropwise. The mixture was stirred at room temperature for 6 h, and the reaction was monitored by TLC (PE:EA = 2:1). After the starting material reacted completely, 1 mL of water was added dropwise per gram of lithium aluminum hydride at 0 °C, followed by 1 mL of 15% sodium hydroxide solution, and finally 3 mL of water. The mixture was brought to room temperature and stirred for 30 min. Then, an appropriate amount of anhydrous sodium sulfate was added, and the mixture was stirred for another 30 min. The mixture was filtered, and the filter cake was washed 2 to 3 times with dichloromethane. The organic phases were combined and extracted with saturated brine (3 × 100 mL). The extract was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE:EA = 2). 1) Intermediate 5 (8.5 g) was obtained. Yield: 60%. Pale yellow solid powder.

[0088] Example 12: Synthesis of (2-amino-5-((trimethylsilyl)ethynyl)phenyl)methanol (6)

[0089]

[0090] Intermediate 5 (8.5 g, 34.14 mmol, 1.0 equiv), triphenylphosphine (0.02 equiv), bis-(triphenylphosphine)-dichloropalladium (0.01 equiv), and CuI (0.03 equiv) were dissolved in a DMF / Et3N solution that had been degassed by bubbling. Nitrogen gas was then introduced, followed by the addition of trimethylsilylacetylene (1.7 equiv). The reaction was stirred at room temperature for 22 h, and the reaction was monitored by TLC (PE:EA = 2:1). After the reactants had completely reacted, the reaction system was concentrated by vacuum distillation. The residue was dissolved in dichloromethane and washed with 1 M hydrochloric acid solution. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (PE:EA = 2:1) to obtain intermediate 6 (5.6 g). Yield: 75%. A pale yellow solid powder was obtained. 1 H NMR (400 MHz, DMSO- d 6) δ 7.17 (d, J = 2.1Hz, 1H), 7.05 (dd, J = 8.2, 2.1 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 5.37 (s, 2H), 5.06 (t, J = 5.5 Hz, 1H), 4.34 (d, J = 5.5 Hz, 2H), 0.19 (s, 9H).

[0091] Example 13: (2-amino-5-((trimethylsilyl)ethynyl)benzaldehyde (7)

[0092]

[0093] Intermediate 6 (5.6 g, 25.57 mmol, 1.0 equiv) was dissolved in ethyl acetate and pre-cooled at 0 °C for 15 min. Then, 2-iodobenzoic acid (3.0 equiv) was added, and stirring was continued for 15 min. The mixture was then heated to 80 °C under nitrogen protection and stirred for 4 h. The reaction was monitored by TLC (PE:EA = 3:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (PE:EA = 3:1) to give intermediate 7 (3.66 g). Yield: 66%. A pale yellow solid powder. 1 H NMR (300 MHz, CDCl3) δ 9.84 (d, J= 0.6 Hz, 1H), 7.67 (d, J = 2.0 Hz, 1H), 7.41 (dd, J = 8.6, 2.0 Hz, 1H), 6.60 (d, J = 8.6 Hz, 1H), 6.31 (s, 2H), 0.26 (s, 9H).

[0094] Example 14: Synthesis of 1-(2-(2-hydroxypropyl-2-yl)-6-((trimethylsilyl)ethynyl)quinoline-3-yl)pyridine-1-onium (8)

[0095]

[0096] Intermediate 7 (1.98 g, 9.11 mmol, 1.0 equiv) and intermediate 3 (2.36 g, 9.11 mmol, 1.0 equiv) were dissolved in anhydrous ethanol and pre-cooled at 0 °C for 15 min. Then, intermediate 3 (1.0 equiv) and pyridine (1.0 equiv) were added, and stirring was continued for another 15 min. The mixture was then heated to 85 °C under nitrogen protection and stirred for 36 h. The reaction was monitored by TLC (DCM:MeOH = 10:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 10:1) to give intermediate 8 (2.74 g). Yield: 69%. Yellow solid powder. 1 H NMR (400MHz, DMSO- d 6) δ 9.41 – 9.30 (m, 2H), 8.83 (m, 1H), 8.78 – 8.74 (m, 1H), 8.32– 8.27 (m, 2H), 8.25 (d, J = 1.9 Hz, 1H), 8.11 (dd, J = 8.8, 0.8 Hz, 1H), 7.95(dd, J = 8.8, 1.9 Hz, 1H), 5.27 (s, 1H), 1.58 (s, 6H), 0.29 (s, 9H).

[0097] Example 15: Synthesis of 2-(3-amino-6-((trimethylsilyl)ethynyl)quinoline-2-yl)prop-2-ol (9)

[0098]

[0099] Intermediate 8 (2.74 g, 6.24 mmol, 1.0 equiv) was dissolved in anhydrous ethanol, and morpholine (2.8 equiv) was added. The mixture was then heated to 85 °C under nitrogen protection and stirred overnight. The reaction was monitored by TLC (PE:EA = 2:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (PE:EA = 2:1) to give intermediate 9 (685 mg). Yield: 37%. Yellow solid powder. 1 H NMR (300 MHz, CDCl3) δ 7.85 (d, J = 8.6 Hz, 1H), 7.74 (d, J = 1.7 Hz, 1H), 7.48 (dd, J = 8.6, 1.8 Hz, 1H), 7.16 (s, 1H), 4.61 (s, 2H), 1.77 (s, 6H), 0.30 (s, 9H).

[0100] Example XVI: Synthesis of 2-(3-amino-6-ynylquinoline-2-yl)prop-2-ol (10)

[0101]

[0102] Intermediate 9 (685 mg, 2.30 mmol, 1.0 equiv) was dissolved in anhydrous methanol, and tetrabutylammonium fluoride (1 M solution in THF, 1.2 equiv) was added. The mixture was stirred at room temperature for 2 h under nitrogen protection. The reaction was monitored by TLC (PE:EA = 2:1). After the starting material reacted completely, saturated ammonium chloride was added to terminate the reaction. The reaction mixture was filtered, concentrated, dissolved in dichloromethane, extracted with saturated brine, dried over anhydrous sodium sulfate, concentrated in the organic phase, and purified by column chromatography (PE:EA = 2:1) to give intermediate 10 (442 mg). Yield: 85%. Yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.85(d, J = 8.6 Hz, 1H), 7.76 (d, J = 1.8 Hz, 1H), 7.49 (dd, J = 8.6, 1.8 Hz, 1H), 7.16(s, 1H), 4.66 (s, 2H), 3.94 (s, 1H), 3.16 (s, 1H), 1.77 (s, 6H).

[0103] Example 17: Synthesis of 2-(3-amino-6-[1-(2-(2-aminoethoxy)ethyl]-1H-1,2,3-triazol-4-yl)quinoline-2-yl)prop-2-ol (12)

[0104]

[0105] Intermediate 10 (442 mg, 1.96 mmol, 1.0 equiv) was dissolved in anhydrous tetrahydrofuran, and azido-PEG1-amine (compound 11, 1.1 equiv) and copper(I) 3-methylsalicylate (0.1 equiv) were added. The mixture was stirred at room temperature for 3 h under nitrogen protection. The reaction was monitored by TLC (DCM:MeOH = 5:1). After the starting material was completely reacted, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 5:1) to give intermediate 12 (557 mg). Yield: 80%. Yellow oil. 1 H NMR (400 MHz, DMSO-) d 6) δ 8.67 (s, 1H), 8.06 (d, J = 1.7 Hz, 1H), 7.85 – 7.67 (m, 2H),7.27 (s, 1H), 5.97 (s, 2H), 5.77 (s, 1H), 4.60 (t, J = 5.2 Hz, 2H), 3.86 (t, J =5.2 Hz, 2H), 3.40 (t, J = 5.7 Hz, 2H), 2.64 (t, J = 5.7 Hz, 2H), 1.63 (s, 6H).

[0106] Example 18: Synthesis of N1-(2-(2-(4-(3-amino-2-(2-hydroxypropen-2-yl)quinoline-6-yl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)-N4-(8-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)octyl)succinamide (15)

[0107]

[0108] Linker compound 13 (200 mg, 0.62 mmol, 1.2 equiv) was dissolved in anhydrous dichloromethane, and then... O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU, 2 equiv) and Et3N (2 equiv) were dissolved by stirring. After the solution became clear, intermediate 12 (183 mg, 0.51 mmol, 1.0 equiv) was added, and nitrogen was purged. The mixture was stirred at room temperature for 5 h. The reaction was monitored by TLC (DCM:MeOH = 20:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 20:1) to give target compound 15 (95 mg). Yield: 28%. Pale yellow solid powder. 1 H NMR (400 MHz, DMSO- d 6) δ 8.63 (s, 1H), 8.05 (d, J = 1.7 Hz, 1H), 7.91 (t, J = 5.7 Hz, 1H), 7.83 – 7.77 (m, 2H), 7.77 – 7.73 (m, 1H), 7.27 (s,1H), 7.00 (s, 2H), 5.96 (s, 2H), 5.75 (s, 1H), 4.59 (t, J = 5.1 Hz, 2H), 3.86(t, J = 5.1 Hz, 2H), 3.45 (t, J = 5.8 Hz, 2H), 3.38 (d, J = 7.0 Hz, 2H), 3.20 (q, J =5.4 Hz, 2H), 2.98 (q, J = 6.6 Hz, 2H), 2.28 (m, 4H), 2.00 (m, 2H), 1.62 (s, 6H), 1.51 – 1.27 (m, 10H).

[0109] Example 19: Synthesis of N1-(2-(2-(4-(3-amino-2-(2-hydroxypropen-2-yl)quinoline-6-yl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)-N4-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)ethoxy)ethyl)succinamide (16)

[0110]

[0111] Linker compound 14 (206 mg, 0.63 mmol, 1.2 equiv) was dissolved in anhydrous dichloromethane. O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU, 2 equiv) and Et3N (2 equiv) were added, and the mixture was stirred until dissolved. After the solution became clear, intermediate 12 (186 mg, 0.52 mmol, 1.0 equiv) was added, and the mixture was purged with nitrogen and stirred at room temperature for 5 h. The reaction was monitored by TLC (DCM:MeOH = 20:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 20:1) to give target compound 16 (87 mg). Yield: 25%. A pale yellow solid powder. 1 H NMR (600 MHz, DMSO- d 6) δ 8.65 (s, 1H), 8.05 (dd, J = 4.5, 1.8 Hz, 1H), 7.94 (t, J = 5.7 Hz, 1H), 7.89 – 7.78 (m, 2H), 7.77 (d, J = 8.6 Hz, 1H),7.36 – 7.17 (m, 2H), 7.12 – 6.58 (m, 1H), 5.96 (d, J = 5.1 Hz, 2H), 5.76 (d, J =3.5 Hz, 1H), 4.60 (t, J = 5.1 Hz, 2H), 3.87 – 3.85 (m, 2H), 3.58 – 3.49 (m,2H), 3.48 – 3.40 (m, 4H), 3.36 – 3.30 (m, 4H), 3.22 – 3.17 (m, 2H), 2.30 (s,2H), 2.10 – 1.89 (m, 4H), 1.76 (s, 2H), 1.63 (s, 6H).

[0112] Example 20: Synthesis of (2-amino-4-fluoro-5-iodophenyl)methanol (18)

[0113]

[0114] 2-Amino-4-fluoro-5-iodobenzoic acid (15 g, 57.03 mmol, 1.0 equiv) was dissolved in anhydrous tetrahydrofuran. Lithium aluminum hydride (1M solution in THF, 1.2 equiv) was added to a constant-pressure dropping funnel, and nitrogen was purged. After pre-cooling at 0 °C for 15 min, lithium aluminum hydride was added dropwise. The mixture was stirred at room temperature for 6 h, and the reaction was monitored by TLC (PE:EA = 2:1). After the reactants had reacted completely, 1 mL of water was added dropwise per gram of lithium aluminum hydride at 0 °C, followed by 1 mL of 15% sodium hydroxide solution, and finally 3 mL of water. The mixture was brought to room temperature and stirred for 30 min. Then, an appropriate amount of anhydrous sodium sulfate was added, and the mixture was stirred for another 30 min. The mixture was filtered, and the filter cake was washed 2 to 3 times with dichloromethane. The organic phases were combined and extracted with saturated brine (3 × 100 mL). The extract was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE:EA = 2:1). 2:1) Intermediate 18 (8 g) was obtained. Yield: 60%. Pale yellow solid powder.

[0115] Example 21: Synthesis of (2-amino-4-fluoro-5-((trimethylsilyl)ethynyl)phenyl)methanol (19)

[0116]

[0117] Intermediate 18 (7.8 g, 29.22 mmol, 1.0 equiv), triphenylphosphine (0.02 equiv), bis-(triphenylphosphine)-dichloropalladium (0.01 equiv), and CuI (0.03 equiv) were dissolved in a DMF / Et3N solution that had been degassed by bubbling. Nitrogen gas was then introduced, followed by the addition of trimethylsilylacetylene (1.7 equiv). The reaction was stirred at room temperature for 22 h, and the reaction was monitored by TLC (PE:EA = 2:1). After the reactants had completely reacted, the reaction system was concentrated by vacuum distillation. The residue was dissolved in dichloromethane and washed with 1M hydrochloric acid solution. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (PE:EA = 2:1) to obtain intermediate 19 (4.0 g). Yield: 58%. A pale yellow solid powder was obtained.

[0118] Example 22: Synthesis of 2-amino-4-methoxybenzaldehyde (21)

[0119]

[0120] The starting material 4-methoxy-2-nitrobenzaldehyde (compound 20, 15 g, 82.86 mmol, 1.0 equiv) was dissolved in anhydrous methanol. Palladium on carbon (1.5 g, 8.29 mmol, 0.1 equiv) and Et3N (16.8 g, 165.71 mmol, 2.0 equiv) were added. Nitrogen was first purged, then hydrogen was rapidly purged (using two balloons) at 0 °C. The reaction was allowed to proceed at room temperature for 18 h. The reaction was monitored by TLC (PE:EA = 2:1). After the starting material had completely reacted, the mixture was filtered through diatomaceous earth, concentrated, and purified by column chromatography (PE:EA = 2:1) to give intermediate 21 (10.6 g). Yield: 85%. A pale yellow solid powder.

[0121] Example 23: Synthesis of 2-amino-5-iodo-4-methoxybenzaldehyde (22)

[0122]

[0123] Intermediate 21 (10 g, 66.20 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane, and N-iodosuccinimide (14.9 g, 66.20 mmol, 1.0 equiv) was added. The mixture was stirred at room temperature for 1 h, and the reaction was monitored by TLC (PE:EA = 2:1). After the starting material reacted completely, the reaction system was concentrated by vacuum distillation. The residue was dissolved in ethyl acetate, washed three times with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE:EA = 2:1) to obtain intermediate 22 (16.1 g). Yield: 88%. A pale yellow solid powder.

[0124] Example 24: Synthesis of 2-amino-4-fluoro-5-((trimethylsilyl)ethynyl)benzaldehyde (23a)

[0125]

[0126] Intermediate 19 (4.0 g, 16.87 mmol, 1.0 equiv) was dissolved in ethyl acetate and pre-cooled at 0 °C for 15 min. Then, 2-iodobenzoic acid (3.0 equiv) was added, and stirring was continued for 15 min. The mixture was then heated to 80 °C under nitrogen protection and stirred for 4 h. The reaction was monitored by TLC (PE:EA = 3:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (PE:EA = 3:1) to give intermediate 23a (2.22 g). Yield: 56%. A pale yellow solid powder.

[0127] Example 25: Synthesis of 1-(7-fluoro-2-(2-hydroxypropyl-2-yl)-6-((trimethylsilyl)ethynyl)quinoline-3-yl)pyridin-1-onium (24a)

[0128]

[0129] Intermediate 23a (2.22 g, 9.44 mmol, 1.0 equiv) and intermediate 3 (2.45 g, 9.44 mmol, 1.0 equiv) were dissolved in anhydrous ethanol and pre-cooled at 0 °C for 15 min. Then, intermediate 3 (1.0 equiv) and pyridine (1.0 equiv) were added, and stirring was continued for another 15 min. The mixture was then heated to 85 °C under nitrogen protection and stirred for 36 h. The reaction was monitored by TLC (DCM:MeOH = 10:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 10:1) to give intermediate 24a (2.81 g). Yield: 65%. Yellow solid powder.

[0130] Example 26: Synthesis of 2-(3-amino-7-fluoro-6-((trimethylsilyl)ethynyl)quinoline-2-yl)prop-2-ol (25a)

[0131]

[0132] Intermediate 24a (2.81 g, 6.13 mmol, 1.0 equiv) was dissolved in anhydrous ethanol, and morpholine (2.8 equiv) was added. The mixture was then heated to 85 °C under nitrogen protection and stirred overnight. The reaction was monitored by TLC (PE:EA = 2:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (PE:EA = 2:1) to give intermediate 25a (872 mg). Yield: 45%. Yellow solid powder.

[0133] Example 27: Synthesis of 2-(3-amino-6-ethynyl-7-fluoroquinoline-2-yl)prop-2-ol (26a)

[0134]

[0135] Intermediate 25a (872 mg, 2.76 mmol, 1.0 equiv) was dissolved in anhydrous methanol, and tetrabutylammonium fluoride (1 M solution in THF, 1.2 equiv) was added. The mixture was stirred at room temperature for 2 h under nitrogen protection. The reaction was monitored by TLC (PE:EA = 2:1). After the starting material reacted completely, saturated ammonium chloride was added to terminate the reaction. The reaction mixture was filtered, concentrated, dissolved in dichloromethane, extracted with saturated brine, dried over anhydrous sodium sulfate, concentrated in the organic phase, and purified by column chromatography (PE:EA = 2:1) to give intermediate 26a (545 mg). Yield: 81%. Yellow oil.

[0136] Example 28: Synthesis of 2-(3-amino-6-[1-(2-(2-aminoethoxy)ethyl]-1H-1,2,3-triazol-4-yl)-7-fluoroquinoline-2-yl)prop-2-ol (27a)

[0137]

[0138] Intermediate 26a (545 mg, 2.23 mmol, 1.0 equiv) was dissolved in anhydrous tetrahydrofuran, and azido-PEG1-amine (1.1 equiv) and copper(I) 3-methylsalicylate (0.1 equiv) were added. The mixture was stirred at room temperature for 3 h under nitrogen protection. The reaction was monitored by TLC (DCM:MeOH = 5:1). After the starting material was completely reacted, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 5:1) to give intermediate 27a (618 mg). Yield: 74%. Yellow oil.

[0139] Example 29: Synthesis of N1-(2-(2-(4-(3-amino-7-fluoro-2-(2-hydroxypropen-2-yl)quinoline-6-yl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)-N4-(8-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)octyl)succinamide (28a)

[0140]

[0141] Linker compound 13 (135 mg, 0.42 mmol, 1.2 equiv) was dissolved in anhydrous dichloromethane, and then... O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethylurea tetrafluoroborate (TBTU, 2 equiv) and Et3N (2 equiv) were dissolved by stirring. After the solution became clear, intermediate 27a (130 mg, 0.35 mmol, 1.0 equiv) was added, and nitrogen was purged. The mixture was stirred at room temperature for 5 h. The reaction was monitored by TLC (DCM:MeOH = 20:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 20:1) to give the target compound 28a (60 mg). Yield: 25%. Pale yellow solid powder. 1 H NMR (600 MHz, DMSO- d 6) δ 8.43 (d, J = 4.0 Hz, 1H), 8.31 (d, J = 7.9Hz, 1H), 7.83 (t, J = 5.5 Hz, 1H), 7.71 (t, J = 5.6 Hz, 1H), 7.57 (d, J = 12.4 Hz,1H), 7.36 (s, 1H), 6.98 (s, 2H), 6.43 – 6.05 (m, 1H), 6.00 – 5.53 (m, 2H),4.63 (t, J = 5.3 Hz, 2H), 3.87 (t, J = 5.3 Hz, 2H), 3.44 (t, J = 5.9 Hz, 2H), 3.36(t, J = 7.1 Hz, 4H), 3.17 (t, J = 5.8 Hz, 2H), 3.09 (q, J = 7.3 Hz, 4H), 2.99 –2.96 (m, 2H), 2.35 – 2.17 (m, 6H), 1.62 (s, 6H), 1.42 – 1.48 (m, 2H), 1.37 –1.31 (m, 2H).

[0142] Example 30: Synthesis of N1-(2-(2-(4-(3-amino-7-fluoro-2-(2-hydroxypropen-2-yl)quinoline-6-yl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)-N4-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)ethoxy)ethyl)succinamide (29a)

[0143]

[0144] Linker compound 14 (135 mg, 0.41 mmol, 1.2 equiv) was dissolved in anhydrous dichloromethane, and then... O -(benzotriazol-1-yl)-N,N,N′,N′-tetramethylurea tetrafluoroborate (TBTU, 2 equiv) and Et3N (2 equiv) were dissolved by stirring. After the solution became clear, intermediate 27a (128 mg, 0.34 mmol, 1.0 equiv) was added, and nitrogen was purged. The mixture was stirred at room temperature for 5 h. The reaction was monitored by TLC (DCM:MeOH = 20:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 20:1) to give the target compound 29a (70 mg). Yield: 30%. The product was a pale yellow solid powder. 1 H NMR (600 MHz, DMSO- d 6) δ 7.60 – 7.56 (m, 1H), 7.52 – 7.49 (m,1H), 7.42 (s, 1H), 7.35 (q, J = 5.5, 4.2 Hz, 1H), 7.26 – 7.21 (m, 2H), 7.01 (s,2H), 6.87 (s, 2H), 5.75 (s, 1H), 4.74 (d, J = 2.7 Hz, 4H), 4.66 – 4.62 (m,10H), 4.59 (d, J = 10.9 Hz, 2H), 3.09 (q, J = 7.3 Hz, 4H), 2.32 – 2.26 (m, 4H), 1.60 (s, 6H).

[0145] Example 31: Synthesis of 2-amino-4-methoxy-5-((trimethylsilyl)ethynyl)benzaldehyde (23b)

[0146]

[0147] Intermediate 22 (16 g, 57.77 mmol, 1.0 equiv), triphenylphosphine (0.02 equiv), bis-(triphenylphosphine)-dichloropalladium (0.01 equiv), and CuI (0.03 equiv) were dissolved in a DMF / Et3N solution that had been degassed by bubbling. Nitrogen gas was then introduced, followed by the addition of trimethylsilylacetylene (1.7 equiv). The reaction was stirred at room temperature for 22 h, and the reaction was monitored by TLC (PE:EA = 2:1). After the reactants had completely reacted, the reaction system was concentrated by vacuum distillation. The residue was dissolved in dichloromethane and washed with 1 M hydrochloric acid solution. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (PE:EA = 2:1) to obtain intermediate 23b (11.0 g). Yield: 77%. The product was a pale yellow solid powder.

[0148] Example 32: Synthesis of 1-(2-(2-hydroxypropyl-2-yl)-7-methoxy-6-((trimethylsilyl)ethynyl)quinoline-3-yl)pyridine-1-onium (24b)

[0149]

[0150] Intermediate 23b (11.0 g, 44.52 mmol, 1.0 equiv) and intermediate 3 (11.53 g, 44.52 mmol, 1.0 equiv) were dissolved in anhydrous ethanol and pre-cooled at 0 °C for 15 min. Then, intermediate 3 (1.0 equiv) and pyridine (1.0 equiv) were added, and stirring was continued for another 15 min. The mixture was then heated to 85 °C under nitrogen protection and stirred for 36 h. The reaction was monitored by TLC (DCM:MeOH = 10:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 10:1) to give intermediate 24b (15.49 g). Yield: 74%. Yellow solid powder.

[0151] Example 33: Synthesis of 2-(3-amino-7-methoxy-6-((trimethylsilyl)ethynyl)quinoline-2-yl)prop-2-ol (25b)

[0152]

[0153] Intermediate 24b (15.49 g, 32.95 mmol, 1.0 equiv) was dissolved in anhydrous ethanol, and morpholine (2.8 equiv) was added. The mixture was then heated to 85 °C under nitrogen protection and stirred overnight. The reaction was monitored by TLC (PE:EA = 2:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (PE:EA = 2:1) to give intermediate 25b (4.11 g). Yield: 38%. Yellow solid powder.

[0154] Example 34: Synthesis of 2-(3-amino-6-ethynyl-7-methoxyquinoline-2-yl)prop-2-ol (26b)

[0155]

[0156] Intermediate 25b (4.11 mg, 12.52 mmol, 1.0 equiv) was dissolved in anhydrous methanol, and tetrabutylammonium fluoride (1M solution in THF, 1.2 equiv) was added. The mixture was stirred at room temperature for 2 h under nitrogen protection. The reaction was monitored by TLC (PE:EA = 2:1). After the starting material reacted completely, saturated ammonium chloride was added to terminate the reaction. The reaction mixture was filtered, concentrated, dissolved in dichloromethane, extracted with saturated brine, dried over anhydrous sodium sulfate, concentrated in the organic phase, and purified by column chromatography (PE:EA = 2:1) to give intermediate 26b (2.5 g). Yield: 78%. It was a pale yellow solid powder.

[0157] Example 35: Synthesis of 2-(3-amino-6-[1-(2-(2-aminoethoxy)ethyl]-1H-1,2,3-triazol-4-yl)-7-methoxyquinoline-2-yl)prop-2-ol (27b)

[0158]

[0159] Intermediate 26b (2.5 g, 9.76 mmol, 1.0 equiv) was dissolved in anhydrous tetrahydrofuran, and azido-PEG1-amine (1.1 equiv) and copper(I) 3-methylsalicylate (0.1 equiv) were added. The mixture was stirred at room temperature for 3 h under nitrogen protection. The reaction was monitored by TLC (DCM:MeOH = 5:1). After the starting material was completely reacted, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 5:1) to give intermediate 27b (2.78 g). Yield: 72%. Pale yellow solid.

[0160] Example 36: Synthesis of N1-(2-(2-(4-(3-amino-2-(2-hydroxypropen-2-yl)-7-methoxyquinoline-6-yl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)-N4-(8-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)octyl)succinamide (28b)

[0161]

[0162] Linker compound 13 (136 mg, 0.42 mmol, 1.2 equiv) was dissolved in anhydrous dichloromethane, and then... O -(benzotriazol-1-yl)-N,N,N′,N′-tetramethylurea tetrafluoroborate (TBTU, 2 equiv) and Et3N (2 equiv) were dissolved by stirring. After the solution became clear, intermediate 27b (135 mg, 0.35 mmol, 1.0 equiv) was added, and nitrogen was purged. The mixture was stirred at room temperature for 5 h. The reaction was monitored by TLC (DCM:MeOH = 20:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 20:1) to give the target compound 28b (56 mg). Yield: 23%. Pale yellow solid powder. 1 H NMR (600 MHz, DMSO- d 6) δ 8.44 (s, 1H), 8.33 (s, 1H), 7.83 (t, J =5.6 Hz, 1H), 7.71 (t, J = 5.6 Hz, 1H), 7.33 (dd, J = 28.3, 22.6 Hz, 2H), 6.99 (s,2H), 5.75 (s, 2H), 5.69 – 5.34 (m, 1H), 4.61 (t, J = 5.3 Hz, 2H), 4.00 (s, 3H), 3.86 (t, J = 5.3 Hz, 2H), 3.44 (t, J = 6.0 Hz, 2H), 3.37 (t, J = 7.1 Hz, 2H), 3.19(q, J = 5.8 Hz, 2H), 2.98 (q, J= 6.7 Hz, 2H), 2.35 – 2.21 (m, 6H), 1.63 (s, 6H), 1.49 – 1.42 (m, 2H), 1.37 – 1.29 (m, 2H), 1.25 – 1.17 (m, 6H).

[0163] Example 37: N1-(2-(2-(4-(3-amino-7-fluoro-2-(2-hydroxypropen-2-yl)quinoline-6-yl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)N4-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy)ethoxy)ethyl)succiamide (29b)

[0164]

[0165] Linker compound 14 (136 mg, 0.41 mmol, 1.2 equiv) was dissolved in anhydrous dichloromethane. O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU, 2 equiv) and Et3N (2 equiv) were added, and the mixture was stirred until dissolved. After the solution became clear, intermediate 27b (133 mg, 0.35 mmol, 1.0 equiv) was added, and the mixture was purged with nitrogen and stirred at room temperature for 5 h. The reaction was monitored by TLC (DCM:MeOH = 20:1). After the starting material reacted completely, the reaction mixture was filtered, concentrated, and purified by column chromatography (DCM:MeOH = 20:1) to give target compound 29b (76 mg). Yield: 31%. A pale yellow solid powder. 1 H NMR (600 MHz, DMSO- d 6) δ 8.46 – 8.42 (m, 1H), 8.34 (s, 1H), 7.83(t, J = 5.7 Hz, 1H), 7.79 (t, J = 5.7 Hz, 1H), 7.14 – 7.10 (m, 1H), 7.03 –7.08(m,1H), 7.01 (s, 2H), 5.83 (d, J = 76.2 Hz, 2H), 5.75 (s, 1H), 4.61 (t, J = 5.3 Hz,2H), 4.00 (s, 3H), 3.86 (t, J = 5.4 Hz, 2H), 3.56 (t, J= 5.5 Hz, 2H), 3.51 (t, J =6.0 Hz, 2H), 3.49 – 3.41 (m, 8H), 3.33 (d, J = 6.0 Hz, 2H), 3.19 (d, J = 5.8 Hz, 2H), 3.15 – 3.12 (m, 2H), 1.64 (s, 6H), 1.24 (d, J = 5.7 Hz, 2H).

[0166] Example 38: Preparation of rHA-15, a conjugate of compounds 15 and 16 with recombinant human albumin.

[0167] a) Dissolve compound 15 or 16 in PBS buffer (pH 7.4, containing 1 mM EDTA) containing 5% DMSO (v / v) to prepare a 300 mM stock solution, which is then sterilized by passing through a 0.22 μm filter membrane for later use.

[0168] b) In a temperature-controlled stirred reactor, the rHA solution (30 mg / mL, pH 7.2 PBS buffer) and the compound stock solution were mixed at a molar ratio of 1.5-4:1. The mixture was added directly or in a gradient feeding strategy (added in 3 batches at 2 h intervals). The mixture was incubated at 4 °C or room temperature with constant shaking at 200 r / min for 8-12 h to generate covalent conjugates.

[0169] c) Purify the protein using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to remove unbound small molecule compounds. Concentrate the protein solution in an ultrafiltration centrifuge tube (4 °C, 12000 rpm, 10 min), wash four times with PBS at pH 7.2, and collect the final coupling solution.

[0170] d) The concentrate was dispensed into 2R borosilicate glass vials (2 mL / vial), pre-frozen at -80 °C for 24 h, and then transferred to a freeze dryer pre-cooled to -50 °C. A gradient freeze-drying program was performed: primary drying at -35 °C for 24 h (vacuum <10 Pa), followed by secondary drying at 25 °C for 24 h. The final product was a white, loose freeze-dried powder that was nitrogen-filled, crimped, and sealed for storage.

[0171] Experimental Example 1: Compound Stability Experiment

[0172] After successfully synthesizing target compounds 15, 16, 28a-b, and 29a-b, they were covalently conjugated with recombinant human albumin, and the stability characteristics of the conjugates and the positive control were systematically evaluated. To simulate the actual function of albumin conjugates, covalent conjugates of small molecules with N-acetyl-L-cysteine ​​(15-Cys, 16-Cys, 28a-Cys, 28b-Cys, 29a-Cys, 29b-Cys) were used as research subjects. First, the stability of each conjugate and the positive control NS-2 in a physiological saline system was tested, and a 5 mM DMSO stock solution of the target compounds was prepared. EP tubes were filled with 1800 μL of solvent (physiological saline, sodium hyaluronate eye drops, and rat plasma), followed by 200 μL of DMSO stock solution, yielding 2000 μL of a 500 μM target compound solution. The mixture was incubated in a shaker (37 ℃, 200 rpm). At each time point after incubation, 200 μL of the solution was pipetted into 200 μL of mass spectrometry grade methanol. After centrifugation, the supernatant was collected, filtered through a 0.22 μm organic filter, and analyzed by HPLC. The concentration was calculated, and stability curves of the compounds were plotted. All tested compounds showed good stability within 72 h, and their peak areas did not decrease significantly. Figure 2 ).

[0173] The research system was further extended to the artificial tear environment simulated by sodium hyaluronate eye drops (trade name: Waterkeying). Experimental data showed that the stability of the seven tested compounds in this medium was highly consistent with that of the physiological saline system, and all compounds remained stable within 72 hours. Figure 3 It is worth noting that the comparative analysis results show that neither molecular structure modification nor the implementation of the albumin coupling strategy had a statistically significant impact on the chemical stability of the compound.

[0174]

[0175] Experimental Example 2: In vitro scavenging experiment of a compound to remove reactive aldehydes

[0176] To verify the effect of structural modifications to compounds 15, 16, and their derivatives on their ability to scavenge reactive aldehydes, a parallel comparative experiment was conducted using the positive control drug NS-2 (Reproxalap drug: 2-(3-amino-6-chloroquinoline-2-yl)prop-2-ol). The experiment was based on the detection principle of malondialdehyde (MDA), an end product of lipid peroxidation, and quantitative analysis was performed using the MDA detection kit from Beyotime Biotechnology Co., Ltd. MDA, as an endogenous lipid peroxidation end product, reflects the degree of lipid oxidation under oxidative stress in the body. When cells are subjected to oxidative damage, the oxidative decomposition of polyunsaturated fatty acids generates various metabolites, including MDA. Existing patent data have confirmed that the MDA content in the tears of dry eye patients is significantly higher than that of healthy individuals, and due to the characteristics of reactive aldehyde compounds, this study selected MDA as an evaluation index for reactive aldehyde scavenging ability.

[0177] The structural formula of NS-2:

[0178]

[0179] The experiment monitored the residual MDA levels at different time points (initial, 30 min, 1 h, 2 h, 4 h, 12 h, 24 h, and 48 h), and found that the clearance efficiency of compounds 15, 16, and the positive control drug NS-2 did not show a significant difference at the 48 h endpoint. Figure 4 This result indicates that the structural modification of the target molecule did not significantly affect its scavenging ability for reactive aldehydes, providing crucial experimental evidence for subsequent drug evaluation. To further investigate the effect of substituent electronic effects on aldehyde scavenging ability, the results showed that the scavenging rate of compound 15 was 1.3 times higher than that of NS-2. Compound 15, with superior activity, and its ortho-substituted derivatives (fluorine-substituted 28a and methoxy-substituted 28b) were selected for comparative analysis. Experimental data showed that the electron-donating methoxy derivative 28b exhibited the best scavenging efficiency, with a scavenging rate 1.5 times higher than NS-2 and a scavenging efficiency 1.2 times higher than that of 15; while the electron-withdrawing fluorine-substituted derivative 28a showed the lowest scavenging activity. Therefore, experimental analysis indicates that electron-donating groups can accelerate the nucleophilic addition reaction with the carbonyl group by enhancing the nucleophilicity of the amino group, while electron-withdrawing groups inhibit the reaction process by reducing the electron density of the amino group.

[0180] Experimental Example 3: In vivo anti-mouse dry eye activity experiment of the compound

[0181] Based on the stability and scavenging of active aldehydes, the conjugate rHA-15 of compound 15 was first selected for in vivo efficacy verification. A benzalkonium chloride-induced dry eye mouse model was constructed to systematically evaluate the in vivo anti-dry eye efficacy of rHA-15. The experiment included gradient dosing groups: low / high dose rHA-15 groups (1%, 5%), rHA control group (1%, 5%), and a 0.25% NS-2 positive control group. A Normal group (treated with an equal volume of physiological saline) and a model group (treated with 0.2% benzalkonium chloride) were also established. On days 0, 3, and 7 of administration, tear secretion volume was measured, ocular surface integrity was assessed, and a tear fern test was performed. On day 7, corneal histopathological examination (HE staining) and inflammatory factor levels were simultaneously measured to evaluate the compound's anti-dry eye activity.

[0182] 1. Benzalkonium chloride-induced dry eye syndrome in mice experiment

[0183] 1.1 Laboratory Animals

[0184] C57BL / 6 35 birds (18~20g, female), purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.

[0185] 1.2 Administration method

[0186] eye drops

[0187] 1.3 Experimental Equipment

[0188] 1 mL syringe, 20 mL syringe, LYL-S handheld slit lamp microscope, sodium fluorescein ophthalmic test strip (Qianwanli), phenol red cotton thread for tear testing (Tianjin Jingming), electronic digital caliper, pathological microscope slides, 2-10 μL pipettes, cotton, padding cloth, 0.3 mm capillary tube.

[0189] 1.4 Experimental Reagents

[0190] Isoflurane, physiological saline, PBS buffer (1×, pH = 7.2~7.4), 4% paraformaldehyde fixative, and the target compound.

[0191] 1.5 Experimental Methods

[0192] C57BL / 6 mice were acclimatized for 3 days (temperature 25 ℃, humidity 40%). Their eyes were observed daily to ensure they were normal.

[0193] Mice were first anesthetized with isoflurane, and then bilateral modeling was performed in the mice with 0.2% benzalkonium chloride (5 μL / eye / time, twice a day, 2-10 μL pipette, 8:30; 19:30) for 7 consecutive days. After 7 days of modeling, the modeling operation was continued, and bilateral administration was started (5 μL / eye / time, three times a day, 2-10 μL pipette, 10:00; 14:00; 18:00) for 7 consecutive days.

[0194] 1.6 Evaluation Indicators

[0195] On days 0, 3, and 7 after drug administration, mice should undergo ocular surface and tear film examinations. After the seventh day of drug administration, mice should be sacrificed and their eyeballs and corneas removed for inflammatory factor and corneal hematoxylin and eosin (HE) staining.

[0196] 1.6.1 Ocular surface examination

[0197] Symptoms of dry eye in animals are assessed by examining changes in the morphology of the cornea and conjunctiva using a slit lamp.

[0198] The main observations of a slit lamp include: (1) whether the width of the tear duct has narrowed; (2) the degree of keratinization of the corneal epithelium, and whether there are abnormalities such as blisters, ulcers, or pannus; (3) whether there are debris on the surface of the cornea and the lower fornix; (4) whether there is congestion or papillary hyperplasia of the conjunctiva; (5) whether the conjunctival sac is loose and wrinkled, whether the eyelid margin is congested, or whether there is irregularity, thickening, or eversion; (6) whether there is clear or yellow serous fluid obstruction at the gland opening, or whether the gland duct is blurred.

[0199] Simultaneously, ocular surface examination should be combined with conjunctival fluorescein staining for further examination. The conjunctival fluorescein staining test involves instilling 1 μL of 1% sodium fluorescein into the conjunctival sac and assessing the staining using a slit-lamp microscope. Based on the degree and area of ​​staining, four levels are assigned: no staining (0 points); scattered sparse punctate staining or staining area less than or equal to 1 / 8 of the total area (1 point); relatively dense punctate staining or staining area between 1 / 8 and 1 / 4 of the total area (2 points); patchy staining or staining area between 1 / 4 and 1 / 2 of the total area (3 points); and staining area greater than or equal to 1 / 2 of the total area (4 points). Finally, the scores for each group are statistically analyzed to create a corneal damage severity map.

[0200] 1.6.2 Tear examination

[0201] (1) Tear secretion test (Schirmer test)

[0202] This experiment measures the total secretion of basal and reflex tears after conjunctival stimulation, serving as a direct indicator for assessing tear secretion function. The specific procedure is as follows: On days 0, 3, and 7 after drug administration, mice were anesthetized with isoflurane. Using ophthalmic microforceps, a phenol red cotton suture for tear detection was precisely placed into the conjunctival sac at the outer third of the lower eyelid. After holding for 30 seconds, the suture was removed. Because the suture reacts with the tears upon contact, a color change occurs. The tear secretion level can be quantified by measuring the length (in mm) of the reddened area of ​​the suture.

[0203] (2) Tear ferning test (TFT)

[0204] The tear fernification test effectively assesses changes in tear composition and mucin function by analyzing the morphological characteristics of tear crystals. The experimental procedure is as follows: Mice anesthetized with isoflurane are given 1 μL of physiological saline to the ocular surface. A 0.3 mm capillary glass tube is used to collect tear samples from the caruncle of the lower palpebral conjunctiva using a siphon effect (care should be taken to avoid contact with ocular surface tissue during the procedure). The sample is evenly spread on a pathological microscope slide and dried at 25 ℃ for 10–20 min. The fern-like crystal morphology of the tear is then observed and recorded using an upright fluorescence microscope.

[0205] Crystallization morphology was classified into grades I to IV. Grade I crystallography showed uniform, fern-like branching with relatively narrow spatial intervals, scoring 1 point; Grade II crystallography showed small crystals with fewer branches and increased intervals, scoring 2 points; Grade III crystallography showed a significant reduction in branches, with intervals increasing to approximately the size of the crystals, scoring 3 points; Grade IV showed no fern-like crystallography, but a small number of amorphous crystals were visible, scoring 4 points. Dry eye model animals generally exhibited typical characteristics of Grade III and IV crystallization. By systematically collecting crystallization images from each group and statistically analyzing the scoring data, a tear fern-like degeneration atlas was finally constructed.

[0206] 1.6.3 Corneal HE staining

[0207] Hematoxylin-Eosin (HE) staining, through nuclear-cytoplasmic contrast, is used to assess corneal histopathological changes, including epithelial integrity, stromal structure, and inflammatory cell infiltration. After the experiment, mice were euthanized by cervical dislocation, and corneal tissue was fixed in 4% paraformaldehyde fixative for 72 h. The tissue was then dehydrated with graded ethanol (70%–100%), cleared with xylene, embedded in paraffin, and prepared into tissue sections approximately 5 μm thick. The sections were cleared and hydrated, stained with HE, mounted, and photographed under an upright fluorescence microscope to observe the histopathological changes in the corneal tissue.

[0208] The characteristics of a normal cornea are: (1) Epithelial layer: composed of 4-5 layers of tightly packed squamous epithelial cells; (2) Matrix layer: parallel collagen fiber bundles, without inflammatory cells; (3) Endothelial layer: a single layer of flat cells continuously covering the cornea. Pathological changes diagnostic criteria: (1) Epithelial damage: reduction or excessive proliferation of basal cells, nuclear pyknosis or cell shedding; (2) Matrix abnormalities: disordered collagen structure, activation of keratinocytes; (3) Inflammatory infiltration: aggregation of neutrophils / lymphocytes; (4) Neovascularization: formation of endothelial cell lumen structures.

[0209] 1.6.4 Detection of corneal and conjunctival inflammatory factors

[0210] The occurrence of dry eye syndrome is closely related to ocular surface inflammation. Abnormal expression of inflammatory factors (such as IL-1β and TNF-α) is an important biomarker for assessing disease progression and treatment efficacy. The detection of corneal and conjunctival inflammatory factors can be achieved through the following procedure:

[0211] 1. Sample Collection and Processing

[0212] After the experiment, the mice were euthanized by cervical dislocation, and the entire eyeball was separated. Then, the eyeball homogenate was lysed, centrifuged, and the protein extract was collected.

[0213] 2. Detection Method

[0214] The experiment employed a double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) to detect the concentration of inflammatory factors (including IL-1β and TNF-α) in the supernatant of centrifuged tissue homogenates using targeted specific antibodies. The general procedure was as follows: after standardizing the total protein concentration of the sample using the BCA method, the expression levels of the target factors were quantitatively analyzed according to a standard curve. The final data were statistically analyzed in pg / mg of total protein. The specific procedure is as follows:

[0215] Preparations before testing:

[0216] (1) Weigh the mouse eyeballs and add the corresponding tissue homogenate according to the weight of the eyeballs for homogenization.

[0217] (2) Take the kit out of the refrigerator 20 minutes in advance and allow it to equilibrate to room temperature.

[0218] (3) Dilute the 20x concentrated washing solution with double-distilled water to make a 1x working solution.

[0219] (4) Preparation of TNF-α standard: Before opening, centrifuge at 1000 rpm for 1 min. Add 0.5 mL of the standard and sample universal diluent, let stand for 15 min until dissolved and clear, then gently mix. Prepare a standard curve with the following concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, and 0 pg / mL. 0 pg / mL is the blank well.

[0220] (5) Preparation of IL-1β standard: Add 1.2 ml of the standard and sample universal diluent again, let stand for 15 min, and after it dissolves and becomes clear, mix gently. Prepare a standard curve with the following concentrations: 1000, 500, 250, 125, 62.5, 31.25, 15.6, and 0 pg / mL. 0 pg / mL is the blank well.

[0221] (6) Biotinylated antibody working solution: 20 min before use, dilute the 30x concentrated biotinylated antibody with biotinylated antibody diluent to a 1x working solution.

[0222] (7) Enzyme conjugate working solution: 20 min before use, dilute the 30x concentrated enzyme conjugate to 1x working solution with enzyme conjugate diluent.

[0223] Testing work:

[0224] (1) Remove the required strips from the sealed bag that has been equilibrated to room temperature.

[0225] (2) Add standard and general sample diluent to blank wells, and add standard or sample of different concentrations (100 µL / well) to other wells. Seal the reaction wells with sealing tape and incubate at 37 ℃ in the dark for 90 min.

[0226] (3) Prepare the biotinylated antibody working solution 20 minutes in advance.

[0227] (4) Wash the plate 5 times: Wash the plate manually, shake off the liquid in the wells, and pat dry with clean absorbent paper. Add 350 µL of washing solution to each well, let it stand for 30 seconds, shake off the liquid, and then pat dry with thick absorbent paper. Wash a total of 5 times.

[0228] (5) Add biotinylated antibody diluent to the blank wells and biotinylated antibody working solution (100 µL / well) to the remaining wells. Seal the reaction wells with new sealing tape and incubate at 37 °C in the dark for 60 min.

[0229] (6) Prepare the enzyme conjugate working solution 20 minutes in advance and place it in the dark at room temperature (22~25 ℃).

[0230] (7) Wash the plate 5 times.

[0231] (8) Add enzyme conjugate dilution to blank wells and enzyme conjugate working solution (100 µL / well) to the remaining wells. Seal the reaction wells with new sealing tape and incubate at 37 °C in the dark for 30 min.

[0232] (9) Wash the plate 5 times.

[0233] (10) Add 100 µL of chromogenic substrate (TMB) to each well and incubate at 37 °C in the dark for 15 min.

[0234] (11) Add 100 µL of reaction termination solution to each well, mix well, and measure OD immediately. 450 Value (within 3 minutes).

[0235] 3. Data Analysis

[0236] Concentration Quantitative Analysis: Based on the ELISA standard curve, the optical density value (OD) is detected. 450 The expression levels of inflammatory factors were converted to absolute concentration values ​​and normalized to per unit weight of tissue (pg / mg).

[0237] 1.7 Statistics

[0238] All data were statistically analyzed using GraphPad Prism 8 software. For comparisons among multiple groups, two-way ANOVA was used, combined with Tukey's test, to perform pairwise comparisons to determine significant differences between groups. This statistical method is suitable for handling complex data involving multiple independent variables and can provide more accurate results for difference analysis. For all statistical analyses, the significance level was set at p < 0.05 to ensure the reliability and scientific validity of the results. Where p < 0.05 indicates a significant difference (usually marked with *), p < 0.01 indicates a highly significant difference (usually marked with **), p < 0.001 indicates an extremely significant difference (usually marked with ***), and p < 0.0001 indicates an exceptionally significant difference (usually marked with ****).

[0239] 2 Results

[0240] 2.1 Tear secretion volume detection experiment

[0241] The phenol red cotton thread method was used to quantitatively detect tear secretion, objectively reflecting the level of tear production by measuring changes in the length of the wetted cotton thread. Experimental results are as follows: Figure 5 As shown, the tear secretion in the model group and each drug administration group after modeling showed statistically significant differences compared with the Normal group (p<0.01), confirming the successful establishment of the benzalkonium chloride-induced dry eye model.

[0242] On day 3 after drug intervention, tear secretion recovered in all treatment groups. Notably, the efficacy of the albumin conjugate rHA-15 showed a dose-dependent increase, and the 5% concentration group showed varying degrees of increase in tear production compared to the 0.25% NS-2 positive drug group and the control groups of different concentrations of pure recombinant human albumin, indicating a synergistic effect of the conjugation strategy on efficacy. By day 7 of drug administration, tear secretion in all treatment groups tended to stabilize at a plateau. Quantitative analysis showed that on day 7, tear secretion in the 5% rHA-15 group, 1% rHA-15 group, and 0.25% NS-2 group recovered to levels comparable to the normal group, with the 5% rHA-15 group showing a 1.7-fold increase in tear volume compared to the model group. These results suggest that high-dose rHA-15 not only has the advantage of a rapid onset of action (significant efficacy within 3 days) but also achieves therapeutic effects comparable to physiological states.

[0243] 2.2 Ocular surface examination

[0244] The degree of damage to the cornea and conjunctiva is assessed using a slit-lamp microscope system to reflect the pathological state of dry eye. Ocular surface examination is performed using white light to obtain the morphological characteristics of the unstained ocular surface. Fluorescein staining assessment involves instilling 1% fluorescein solution (1 μL) and recording the fluorescein-stained area of ​​the cornea using cobalt blue light excitation mode. Damage is scored based on the extent of corneal damage (0-4 points, with higher scores indicating more severe damage).

[0245] The results are as follows Figure 6 As shown, seven days after modeling, the corneal damage scores of the model group and all treatment groups were statistically significantly higher than those of the Normal group, confirming the successful establishment of the dry eye model. On day 3 of drug administration, the corneal repair efficiency of the 5% rHA-15 group was significantly higher than that of other intervention groups, with its damage score reduced by 61.8% compared to the model group (p<0.0001), which was superior to the 1% rHA-15 group and the 0.25% NS-2 positive control group. Notably, the pure rHA control groups (1% and 5%) only showed a low level of damage repair rate. With continued intervention until day 7, the 5% rHA-15 group achieved almost complete corneal epithelial repair, with a repair level 25% higher than that of the 1% rHA-15 group, and significantly better than the positive drug group (p<0.05), representing a two-fold improvement compared to the positive drug group. These results indicate that the albumin-conjugated strategy accelerates the corneal damage repair process in a dose-dependent manner, and its therapeutic effect is significantly better than that of traditional positive drugs and recombinant human albumin alone.

[0246] 2.3 Tear-induced fern experiment

[0247] The tear fern test was used to quantitatively evaluate the crystallization characteristics of the tear film. Tear samples were collected from the lacrimal caruncle of the lower eyelid conjunctival sac using a capillary tube, and after natural drying, the crystal morphology was observed and a crystallization index (0-4) was assigned using an upright fluorescence microscope (200×). The normal group showed a typical Grade I fern-like structure, characterized by dense, fern-like branches. The model group exhibited Grade III crystallization characteristics, with broken branches, increased lattice spacing, and randomly distributed amorphous crystals.

[0248] Experimental results are as follows Figure 7 As shown, seven days after modeling, the tear crystallization scores of the model group and each treatment group were statistically significantly higher than those of the Normal group, confirming the successful establishment of the dry eye model. On day 3 of drug administration, the rHA-15 conjugate group (1% and 5%) showed a significant improvement in crystal morphology (p<0.0001), and its recovery efficiency was superior to that of the pure rHA group at the same concentration (1% and 5%). Notably, the crystal repair rate of the positive control drug NS-2 group was not statistically different from that of the model group (p>0.05), suggesting a delayed onset of action in small molecule treatment regimens. By day 7 after intervention, the crystal repair effect of the rHA-15 conjugate group (1% and 5%) continued to be superior to that of the pure recombinant human albumin group and the positive control drug group, and the tear volume of the 5% rHA-15 group was 1.53 times better than that of the positive control drug group. Experimental analysis shows that albumin conjugate technology significantly improves tear film stability by prolonging drug retention time, and its therapeutic advantage is closely related to the enhanced bioavailability mediated by the conjugate strategy.

[0249] 2.4 Inflammatory Factor Measurement Experiment

[0250] In this experiment, mice were sacrificed on day 7 after drug administration and mouse eyeball tissue was collected. Tissue lysate was prepared by low-temperature homogenization, and the concentrations of pro-inflammatory cytokines TNF-α and IL-1β were quantitatively detected by double antibody sandwich ELISA (kit purchased from Xinbosheng).

[0251] Experimental results are as follows Figure 8As shown, the expression levels of pro-inflammatory cytokines TNF-α and IL-1β in the model group were 2.5-fold and 2.0-fold higher than those in the normal group, respectively (p<0.0001), confirming that dry eye syndrome is accompanied by a significant inflammatory response. The 5% rHA-15 treatment group showed the strongest anti-inflammatory effect, with no statistically significant difference in TNF-α and IL-1β levels compared to the normal group (p>0.05), and the TNF-α level was reduced by 27.8% compared to the pure rHA group at the same concentration (p<0.05). Notably, the inflammatory factor inhibition rate of the positive control drug NS-2 group was weaker than that of the rHA-15 conjugate groups (1% and 5%) and the pure rHA group (1% and 5%). Specifically, the tear volume in the 5% rHA-15 group was 1.55-fold better than that in the positive control group and 1.3-fold better than that in the 1% rHA-15 group. Experimental analysis shows that the albumin conjugate strategy exerts its therapeutic advantages through a dual synergistic mechanism: on the one hand, the high half-life of albumin significantly enhances drug retention in ocular surface tissues; on the other hand, the antioxidant properties of the albumin carrier and the overall free radical scavenging capacity of the conjugate significantly inhibit the expression levels of pro-inflammatory factors (TNF-α, IL-1β, etc.). The efficacy of this albumin conjugate exhibits a clear concentration-dependent effect, indicating that the therapeutic effect is positively correlated with the administered dose.

[0252] 2.5 Corneal HE staining experiment

[0253] This study used hematoxylin and eosin (H&E) staining to systematically analyze the structural integrity of corneal tissue. The experimental results are as follows: Figure 9 As shown, after 7 days of drug intervention, the neatness of the corneal epithelial cell layer arrangement improved in all treatment groups. The 0.25% NS-2 positive control group still exhibited local epithelial discontinuity, while the 1% rHA-15 and 5% rHA-15 groups showed good restoration of epithelial cell polarity and a reduction in the area of ​​inflammatory cell infiltration in the stromal layer. Notably, the corneal epithelial layer thickness was increased in the 1% rHA group and the conjugate groups (1% and 5% rHA-15) compared to the model group, suggesting that albumin conjugates can accelerate corneal structural reconstruction by promoting epithelial proliferation. These results indicate that rHA-15 has superior tissue repair activity in the 1%–5% concentration range.

[0254] Test Example 4: Compound Eye Irritation Test

[0255] Ophthalmic medications often require ocular irritation testing to assess their safety. The Draize test is the primary method for evaluating ocular irritation. This experiment used the Draize ocular irritation score and the McDonald-Shadduck scoring system to evaluate the irritation of compounds. Rabbits have large, colorless eyes, making them easy to manipulate, and their corneal thickness (approximately 0.37–0.4 mm) is similar to that of humans (approximately 0.5–0.6 mm), resulting in high irritation to sensitizing substances. Reversibility is a crucial indicator of ocular irritation in the Draize test, referring to the time it takes for the eye to return to its normal state after external stimulation, typically at 24-hour intervals. Therefore, a rabbit model with multiple doses was established to evaluate the ocular irritation of the compound (25 μL / dose, three times daily for seven days). Slit-lamp photography was used to record data 30 minutes after each dose. The degree of corneal damage, iris congestion, conjunctival edema, and conjunctival secretions were scored according to the ocular irritation scoring criteria. Higher scores indicate greater irritation, and the severity of irritation was determined accordingly. The experiment recorded the condition of the rabbit's eyes, including the front, upper, and lower parts, nictitating membrane, corner of the eye, and corneal damage. Green arrows indicate eye irritation or discharge. The images were taken under cobalt blue light from a slit lamp to observe for corneal damage (if damage is present, it will produce yellow-green fluorescence).

[0256] The experiment used 0.25% NS-2 as a positive control to evaluate the ocular irritation of the compound conjugate 1% rHA-15. Results showed that 1% rHA-15 significantly improved ocular irritation compared to the positive control group starting from day four (p<0.05). On day seven, the positive control group showed significant subcorneal ocular damage, and persistent ocular discharge began on day five, while the conjugate group showed almost no ocular discharge, exhibiting fewer side effects. 1% rHA-15 showed a 2.27-fold improvement in ocular irritation compared to the positive control group. Experimental analysis indicates that the conjugate has better tolerability than the positive control group, and the introduction of albumin effectively alleviates ocular irritation; this process may be related to the biocompatibility of albumin. Figure 10 ).

[0257] Experimental Example 5: In vitro antioxidant experiment of compounds

[0258] This experiment used a total antioxidant capacity assay kit (Beyotime) to analyze the in vitro antioxidant activity of the positive control drug NS-2, small molecule compound 15, conjugate rHA-15, and rHA and HSA. Based on the physiological albumin concentration of humans (35–52 g / L), three doses (30, 40, and 50 mg / mL) were designed to compare the in vitro antioxidant levels of the compounds. The experimental results are as follows: Figure 11As shown, recombinant human albumin has a significantly higher free sulfhydryl group content than human serum albumin, and its antioxidant capacity is also significantly higher (p<0.05). The albumin-small molecule conjugation strategy significantly enhances the antioxidant capacity of the conjugate rHA-15 compared to the small molecule drugs NS-2 and NS-15.

[0259] Experimental Example 6: Optimization of the coupling process between compound 15 and recombinant human albumin

[0260] To optimize the coupling process of compound 15 with recombinant human albumin, this study focused on three key parameters: coupling ratio, reaction temperature, and time, designing 10 different coupling conditions (Table 1). The products were systematically characterized using high-resolution mass spectrometry (MS, Thermo Scientific Q Exactive) combined with ultra-high performance liquid chromatography (UPLC, Thermo Scientific Vanquish Flex). Experimental data showed that condition 8 exhibited the best coupling effect (small molecule 15: rHA ratio of 1.5:1, temperature of 4 ℃, and reaction time of 12 h). Mass spectrometry analysis showed that under this condition, the proportion of rHA single-molecule modified product reached 100%, completely avoiding multi-molecule modification and unreacted prototype residues (Table 1). In subsequent process validation, this condition showed good reproducibility, with the single-molecule modification yield consistently maintained above 95%.

[0261] During the coupling reaction, recombinant human albumin contains not only cysteine ​​sulfhydryl groups but also amino groups of amino acids such as lysine, which may lead to multiple modification sites during small molecule coupling. Previous peptide mass spectrometry analysis demonstrated that maleimide achieved a coupling efficiency of 97.2% with Cys 34 in a pH 7.2 buffer system, thus ensuring batch-to-batch consistency of albumin coupling sites. Given the sensitivity of protein carriers to reaction conditions, studies have found that excessively low pH may induce conformational changes in albumin, while pH > 8.5 promotes deprotonation of amino groups on the albumin surface, increasing the risk of side reactions. While increased temperature improves the reaction rate, prolonged reactions may compromise albumin stability. Therefore, neutral pH (7.2) and low temperature (4 °C) were chosen as the optimal reaction environment, maintaining both the conformation and activity of recombinant human albumin while enabling directional coupling via Cys 34. Meanwhile, through concentration gradient decreasing experiments, it was found that when the coupling ratio of small molecules to albumin was greater than 1.5, the original albumin could be basically completely reacted. Based on this, the experiment adjusted the coupling ratio by 0.5 times and combined it with time variable control to optimize the coupling ratio of small molecules to albumin, and finally determined the optimal coupling condition 8.

[0262] Table 1. Coupling conditions of compound 15 with rHA

[0263]

[0264] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A small molecule active aldehyde scavenger-albumin conjugate, characterized in that: The conjugated drug comprises the following structure: a) A small molecule active aldehyde scavenger compound, wherein the structural formula of the small molecule active aldehyde scavenger compound is: Where R1 is selected from at least one of -H, -F, and -OCH3, X is selected from at least one of carbon atom and oxygen atom, and n is 0~3; b) Albumin, wherein the albumin is selected from at least one of human serum albumin and recombinant albumin; The small molecule active aldehyde scavenger compound is chemically coupled to albumin.

2. A composition, characterized in that, The composition comprises the conjugated drug of claim 1 and pharmaceutically acceptable additives.

3. The use of the conjugated drug of claim 1 or the composition of claim 2 in the preparation of a medicament for treating ocular diseases, wherein, The eye condition described is dry eye syndrome.

4. A method for preparing the conjugated drug according to claim 1, characterized in that, The method includes the following steps: S1: In a weakly alkaline buffer system, the albumin described in claim 1 (b) and the small molecule active aldehyde scavenger compound described in claim 1 (a) are mixed at a molar ratio of 1:1 to 1:10; S2: React at 4-25 ℃ in the dark for 8-12 h to generate the conjugated drug.

5. The method according to claim 4, characterized in that, The method also includes the steps of purifying and drying the obtained conjugated drug.

6. The method according to claim 4, characterized in that, The pH of the buffer system in step S1 is 7.0~8.

0.

7. The method according to claim 4, characterized in that, Step S2 also includes a co-solvent, wherein the volume concentration of the co-solvent is 1%-5%.

Images