Application of tetrahydronaphthyridine derivative to preparation of product for improving chromatosis

Abstract

The invention provides an application of a compound as shown in a formula I, pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof in preparation of products for improving hyperpigmentation. The compound has a good application prospect in treatment of chloasma. # imgabs0 #

Description

Application of tetrahydronaphthyridine derivatives in preparing products for improving pigmentation

[0001] This application claims priority to a prior application, patent application number 202210597120.9, filed with the State Intellectual Property Office of China on May 30, 2022, entitled "Application of Tetrahydronaphthyridine Derivatives for the Preparation of Products for Improving Pigmentation." The entirety of the prior application is incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of chemistry, in particular to the use of tetrahydronaphthyridine derivatives in preparing products for improving pigmentation, and more particularly to the use of tetrahydronaphthyridine derivatives in preparing products for improving chloasma. Background Art

[0003] Skin pigmentation is a difficult dermatological condition. Examples include chloasma, age spots, freckles, melanosis, facial moles, and birthmarks. These conditions not only affect appearance but also severely impact people's well-being. With the continuous improvement of living standards, whitening, removing spots, and improving skin pigmentation have become common pursuits and are also a treatment requirement for many dermatological conditions.

[0004] Melasma is a very common, chronic, acquired skin disease characterized by increased melanin production on the face. Its primary characteristic is increased pigmentation. Melasma is most common in middle-aged women. Its primary symptom is the symmetrical distribution of dark brown or yellowish-brown patches of varying shades and unclear boundaries across the cheeks, forehead, and jawline. These patches are also known as liver spots or butterfly spots. Melasma is typically asymptomatic and requires no treatment, but it can severely impact aesthetics, causing both physical and psychological damage to patients. With rising living standards and a growing emphasis on appearance, the treatment of skin conditions like melasma is also receiving increasing attention.

[0005] While the pathogenesis of melasma remains unclear, numerous studies have shown that genetic susceptibility, ultraviolet (UV) exposure, endocrine dysfunction, skin barrier dysfunction, hormonal changes, psychological factors, and nutritional status are all closely linked to its development. In actual treatment, due to the varying size, severity, and severity of melasma among patients, clinical classification and categorization of chloasma lesions are typically employed to develop comprehensive treatment plans. Currently, treatment options for melasma include topical and systemic medications.

[0006] Topical medications generally aim to protect or restore the skin's barrier function through functional skincare products. Secondly, topical medications primarily targeted at epidermal melasma are used to inhibit and remove melanin from the lesions. A representative agent is hydroquinone, which inhibits DNA and RNA synthesis in melanocytes and competitively binds to tyrosinase, inhibiting melanosome formation or accelerating its degradation. Higher concentrations increase the depigmentation effect, but also increase skin irritation, potentially causing a burning sensation and occasionally local allergic reactions. Hydroquinone is commonly used at a concentration of 2% to 5%. In 2002, the FDA approved triple combination cream (TCC), consisting of 4% hydroquinone, 0.01% fluocinolone acetonide, and 0.05% tretinoin, for the treatment of melasma. It is currently the first-line treatment for melasma. However, due to its numerous adverse reactions, TCC should not be used for more than six months.

[0007] The most representative systemic medication is tranexamic acid (TXA). Tranexamic acid, a plasmin inhibitor, has recently demonstrated promising results in the treatment of melasma. Oral, topical, and microneedle injections have been shown to improve melasma. Tranexamic acid inhibits plasmin, reduces α-melanocyte-stimulating hormone (α-MSH), and reduces melanin production. It also competitively inhibits tyrosinase activity in melanosomes, resulting in lighter spots. Tranexamic acid also inhibits angiogenesis and reduces erythema. Some studies suggest that the improvement in melasma with tranexamic acid is related to its ability to inhibit endothelin-1 (ET-1) expression. However, tranexamic acid also has side effects in the treatment of melasma. There are reports of oral TXA causing headaches and severe abdominal distension, leading patients to abandon treatment due to intolerance. Topical TXA, on the other hand, can cause adverse reactions such as erythema and a burning sensation.

[0008] Although a variety of treatment options are currently available for melasma, their effectiveness is suboptimal, and the long and frequent use cycles make it difficult to meet the growing clinical needs. Developing safer and more effective products for pigmentation remains a key focus.

[0009] Summary of the Invention

[0010] One of the objects of the present invention is to provide a compound represented by formula I, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof for use in preparing a product for improving pigmentation.

[0011] wherein R1 is selected from carboxyl, phosphate, and sulfonic acid groups;

[0012] R2 is selected from hydrogen, substituted or unsubstituted amino, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, C1-C4 haloalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered alicyclic group, substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 6-10 membered aromatic heterocyclic group.

[0013] In certain specific embodiments, R2 is selected from hydrogen, unsubstituted or optionally substituted with one, two or more R 2a Substituted groups include amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C4 haloalkyl, C3-C6 cycloalkyl, 4-8 membered alicyclic group, 6-10 membered aryl, and 6-10 membered aromatic heterocyclic group.

[0014] In certain specific embodiments, the R 2a Selected from halogen, OH, NH2, NO2, CN, oxo (=O), C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 4-8 membered alicyclic group, 6-10 membered aryl, 6-10 membered aromatic heterocyclic group.

[0015] In certain specific embodiments, the R2 is selected from hydrogen, unsubstituted or optionally substituted with one, two or more R 2a Substituted phenyl, phenyl-C1-C6 alkyl-.

[0016] In certain specific embodiments, said R2 is selected from hydrogen,

[0017] In certain specific embodiments, the pigmentation may be a pigmentation-related disease, which refers to a disease caused by the deposition of melanin on the surface of the skin, including: chloasma, age spots, freckles, melanosis, facial moles, birthmarks, etc.

[0018] In certain specific embodiments, the pigmentation-related disease is melasma.

[0019] In certain specific embodiments, the compound of formula I is selected from the following compounds, pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof:

[0020] In certain specific embodiments, the pharmaceutically acceptable salt is the hydrochloride salt of the compound of formula I.

[0021] In certain specific embodiments, the product for improving pigmentation can be a medicine or a cosmetic.

[0022] In certain specific embodiments, the pigmentation-improving product refers to a pharmaceutical preparation for clinical use prepared using at least one of the compounds of Formula I, or pharmaceutically acceptable salts, hydrates, isomers, or prodrugs thereof, as an active ingredient, and one or more pharmaceutically acceptable excipients. The pharmaceutical preparation of the present invention can be prepared as an oral preparation, a topical preparation, or an injection for use. Beneficial effects

[0023] This study induced a rat melasma model using 280-320nm UVB irradiation combined with progesterone injection. The compound was administered via local injection and its efficacy was evaluated using epidermal markers, histopathological examination, and biochemical indices. The results showed that the compound of Formula I described in this invention effectively reduced the number of melanin granules in the surface cells of the skin in the modeling area of ​​rat melasma models, improved skin tissue morphology and inflammatory cell infiltration, and effectively reduced the levels of biochemical markers associated with melasma, demonstrating its promising application in the treatment of melasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 shows the effects of tranexamic acid and the test compound groups on a rat model of melasma induced by UV irradiation and progesterone injection. (A) shows the overall appearance of the rat skin in the experimental area; (B) shows the HE staining of the skin tissue in each group; (C) shows the distribution of melanin granules in the basal and basal layers of the epidermis in each group; (D) shows the expression of tyrosinase in the skin tissue in each group; (E) shows the grading of the chloasma model's apparent indicators; (F) shows the grading of melanin granule content in the chloasma rat model after skin tissue was harvested on day 58; (G) shows the average integrated optical density distribution of epidermal tyrosinase in each group after skin tissue was harvested on day 58; (H) shows the ET-1 content in the supernatant of skin tissue homogenates from each group after skin tissue was harvested on day 58; and (I) shows the weight changes of the rats in each group during the experimental period (Day 1 to Day 58). All scoring and counting results were obtained using a double-blind method. Compared with the control group, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001.

[0025] Definitions and Explanations of Terms

[0026] Unless otherwise indicated, the definitions of groups and terms in this specification and claims, including definitions used as examples, exemplary definitions, preferred definitions, definitions in tables, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with one another. The group definitions and compound structures resulting from such combinations and couplings should be understood to be within the scope of this specification and / or claims.

[0027] Unless otherwise indicated, the numerical ranges recited in this specification and claims are equivalent to reciting at least each specific integer value therein. For example, the numerical range "1-40" is equivalent to reciting each integer value in the numerical range "1-10," namely, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and the numerical range "11-40," namely, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40. In addition, when certain numerical ranges are defined as "numbers," it should be understood that both endpoints of the range, each integer within the range, and each decimal within the range are recited. For example, “a number from 0 to 10” should be understood as not only recording each integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, but also recording at least the sum of each of these integers and 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 respectively.

[0028] It should be understood that herein, when describing 1, 2 or more, "more" should refer to an integer greater than 2, such as an integer greater than or equal to 3, such as 3, 4, 5, 6, 7, 8, 9 or 10.

[0029] The term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0030] "C1-C6 alkyl" refers to a straight-chain or branched alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms. Examples of the alkyl group include methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or isomers thereof.

[0031] The term "alkoxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, butoxy.

[0032] The term "C3-C6 cycloalkyl" is understood to mean a saturated monovalent monocyclic hydrocarbon ring having 3, 4, 5 or 6 carbon atoms. 3-10 The cycloalkyl group may be a monocyclic hydrocarbon group, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0033] Unless otherwise defined, the term "4-8 membered alicyclic radical" refers to a saturated ring or ring system, for example, a 4-, 5-, 6- or 7-membered monocycle, a 7- or 8-bicycle (e.g., fused, bridged, spirocyclic), and containing at least one, for example 1, 2, 3, 4, 5 or more heteroatoms selected from O, S and N, wherein N and S may also be optionally oxidized to various oxidation states to form nitrogen oxides, -S(O)- or -S(O)2- states. The alicyclic radical may be connected to the rest of the molecule via any one of the carbon atoms or a nitrogen atom, if present. The alicyclic radical may include fused or bridged rings and spirocyclic rings. In particular, the alicyclic group may include, but is not limited to: a 4-membered ring, such as azetidinyl, oxetanyl; a 5-membered ring, such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6-membered ring, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or a 7-membered ring, such as diazepanyl.

[0034] The term "6-10 membered aryl" is understood to mean preferably a monovalent aromatic or partially aromatic monocyclic or bicyclic ring having 6, 7, 8, 9 or 10 carbon atoms, in particular a ring having 6 carbon atoms ("C6 aryl"), such as phenyl; or biphenyl, or a ring having 9 carbon atoms ("C9 aryl"), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C 10 When the 6-10 membered aryl group is substituted, it may be monosubstituted or polysubstituted. Furthermore, there is no limitation on the substitution site, for example, it may be ortho-, para- or meta-substituted.

[0035] The term "6-10 membered aromatic heterocyclyl" is understood to include monovalent monocyclic or bicyclic (e.g., fused, bridged, spiro) aromatic ring systems having 6-10 ring atoms and containing 1-5 heteroatoms independently selected from N, O, and S, preferably containing 1-3 heteroatoms independently selected from N, O, and S. "Heteroaryl" also refers to a group in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, wherein the radical or point of attachment is on the heteroaromatic ring, for example, selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, and pyranyl. DETAILED DESCRIPTION

[0036] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0037] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0038] 1. Purpose of the experiment

[0039] The therapeutic effect of the compound of the present invention on the rat melasma model was tested.

[0040] 2. Experimental Materials and Instruments

[0041] Experimental reagents

[0042] Experimental instruments

[0043] 3. Experimental Methods

[0044] 3.1 Model Establishment: Prior to the experiment, on Day 0, SD rats (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., SPF-grade, female, 7-8 weeks old, weighing 200 ± 20 g) were randomly assigned to groups and numbered. The dorsal hair of each group was shaved with an electric shaver, and short hair was removed with a depilatory cream to fully expose the dorsal skin. The depilatory area was slightly larger than the actual modeling area (5 cm × 5 cm). Each rat was depilated every 2-3 days to ensure adequate dorsal skin exposure. The following day (Day 1), each rat was weighed, and body weight was recorded after the electronic scale reading stabilized. Progesterone injections were administered intramuscularly (20 mg / mL, 25 mg / kg) once daily into the groin (alternating between the thighs). Following intramuscular injection, the dorsal skin of the rats was irradiated with ultraviolet light at a wavelength of 280-320 nm, approximately 30 cm from the light source, for 60 minutes each time for a total of 4 weeks.

[0045] 3.2 Administration: After the rat model was established, 5 mg / mL of tranexamic acid (positive control group) and 5 and 1.5 mg / mL of the test compound were injected into the skin lesions using a 30G insulin needle at multiple points (the solvent was normal saline). The administration volume was 0.05 mL / cm 2 , 5 days / time, for 30 days, the control group (only hair removal, no modeling treatment) and the model group were given solvent.

[0046] 3.3 Detection index: The first day of modeling was counted as Day 1, and the experimental period lasted for 58 days. After the end of the experiment (Day 58), 1 hour after the last administration, the skin of the modeling area of ​​each group of rats was photographed for apparent index scoring (scoring criteria are shown in Table 1). Then, the rats in each group were euthanized, the backs were depilated, 0.5 g of full-thickness skin tissue from the depilated back was taken and added with normal saline to make a 10% tissue homogenate in an ice bath. The homogenate was centrifuged at 3000 r / min at room temperature for 10 minutes to obtain the skin tissue homogenate supernatant. The expression of endothelin-1 in the rat skin tissue homogenate supernatant was detected by ELISA; the skin of the modeling area on the back of the rat was taken and immersed in 10% neutral formaldehyde for fixation at room temperature for 4 days. Four days later (Day 62), paraffin-embedded and sectioned skin paraffin sections were obtained from each group of rats. Skin histopathological changes in each group of rats were observed using HE staining. The following staining procedures were used: dried paraffin sections were dewaxed with conventional xylene, hydrated with gradient ethanol, and washed with distilled water. Nuclei were stained with hematoxylin for 2 minutes, differentiated with hydrochloric acid and alcohol for a few seconds, and washed with water to return to blue. Sections were stained with eosin solution for 1 minute, and residual stain was washed with water to remove. Sections were dehydrated with gradient ethanol, dried, transparentized with xylene, and mounted with neutral gum for microscopic examination. Skin tissue sections from each group were observed for epidermal hyperplasia, basal cell arrangement, inflammatory cell infiltration in the dermis, and changes in capillary count, according to evaluation criteria. Fontana–Masson staining was used to observe skin pigmentation in each rat group. The following experimental procedures were performed: After dewaxing and hydration, sections were immersed in Fontana silver nitrate solution and incubated in a dark incubator at 56°C for 30–40 minutes. Sections were then washed 5–6 times with distilled water for 1–2 minutes each time. Sections were then treated in Hypo solution for 1–5 minutes. Sections were then rinsed in tap water for 3–5 minutes. Sections were counterstained with neutral red stain for 5 minutes. Sections were then rinsed in tap water for 1 minute. Sections were then dehydrated with 95% ethanol and anhydrous ethanol, transparentized with xylene, and mounted with neutral gum for microscopic examination. Melanin granule distribution was scored (see Table 2 for scoring criteria).Immunohistochemistry was used to detect the expression of tyrosinase in the skin of each group. The experimental methods were as follows: 1) Baking: The prepared paraffin sections were placed in an electric constant temperature drying oven and baked at 60°C for 3 h; 2) The dried paraffin sections were dewaxed with conventional xylene, hydrated with gradient ethanol, and washed with distilled water; 3) Antigen retrieval; 4) Add 3% H2O2 and incubate at room temperature for 10 min to inactivate endogenous enzymes, then wash three times with PBS for 3 min each; 5) Block each section with normal goat immune serum, incubate at room temperature for 10 min, shake off excess liquid, do not wash, and add tyrosinase antibody diluted in a certain ratio (1:50) and refrigerate at 4°C overnight; 6) Remove from the refrigerator and return to room temperature, then wash three times with PBS for 3 min each; 7) Add polymer enhancer (reagent A) to each section, incubate at room temperature for 20 min, and wash three times with PBS for 3 min each; 8) Add enzyme-labeled anti-rabbit polymer (reagent B) to each section, incubate at room temperature for 10 min, and wash three times with PBS for 3 min each; 9) Develop with DAB, control the reaction time under a microscope, counterstain with hematoxylin, and wash thoroughly with distilled water to terminate the color development; 10) Dehydrate and dry the sections with graded alcohol, transparentize with xylene, and mount with neutral gum; 11) Photograph using a phase contrast microscope at 200× magnification. The area of ​​the tyrosinase-positive region (stained area) and the integrated optical density value were measured by ImageJ (1.8.0) software, and the average optical density value was calculated using the formula AOD=IOD / Area.

[0047] Table 1 Grading standards for apparent indicators of chloasma

[0048] Total integral = area integral + color integral

[0049] Table 2 Scoring criteria for local pathological observation using Fontana–Masson staining

[0050] 3.4 Experimental data were expressed as means ± SD. Statistical differences between groups were analyzed using one-way ANOVA and Tukey's test. The scoring data were analyzed using the Mann-Whitney U test. A P value less than 0.05 was considered to be significantly different.

[0051] 3.5 Preparation of test compounds

[0052] Preparation Example 1

[0053] Step 1: Preparation of tert-butyl 2-chloro-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate

[0054] 2-Chloro-5,6,7,8-tetrahydro-1,6-naphthyridine hydrochloride (0.9 g) was suspended in dichloromethane (15 mL). N,N-diisopropylethylamine (1.4 g) was added to dissociate the mixture. Di-tert-butyl dicarbonate (1.15 g) was then added and the mixture was allowed to react at room temperature for 1 h. TLC indicated complete consumption of the starting material. Purification by column chromatography afforded the title compound (1.12 g).

[0055] MS (ESI) m / z (M+H) + =269.0.

[0056] Step 2: Preparation of tert-butyl 2-cyano-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate

[0057] Weigh tert-Butyl 2-chloro-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (1.12 g) and dissolve it in N,N-dimethylformamide (20 mL). Add zinc cyanide (2.44 g) and tetrakis(triphenylphosphine)palladium (483 mg). Replace the atmosphere with argon three times and react at 120°C for 3 h. TLC shows complete consumption of the starting material. Dilute with ethyl acetate, filter through celite, extract twice with ethyl acetate, wash once with saturated brine, dry over anhydrous sodium sulfate, and purify with column chromatography to obtain the title compound (1.1 g).

[0058] MS (ESI) m / z (M+H) + =260.0.

[0059] Step 3: Preparation of 5,6,7,8-tetrahydro-1,6-naphthyridine-2-carboxylic acid hydrochloride

[0060] tert-Butyl 2-cyano-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (1.1 g) was dissolved in 6 M aqueous hydrochloric acid (25 mL) and reacted at 120°C overnight. LCMS showed complete consumption of the starting material. The reaction solution was concentrated to dryness and separated by pre-HPLC to obtain 5,6,7,8-tetrahydro-1,6-naphthyridine-2-carboxylic acid hydrochloride (test compound 1, 726 mg).

[0061] MS (ESI) m / z (M+H) + =179.0.

[0062] 1 H NMR (400MHz, Methanol-d4) δ8.21(d,J=8.1Hz,1H),8.12(d,J=8.0Hz,1H),4.60(s,2H),3.71(t,J=6.4Hz,2H),3.42(t,J=6.4Hz,2H).

[0063] Preparation Example 2

[0064] Step 1: Preparation of tert-butyl 2-chloro-7,8-dihydro-1,6-naphthyridine-6-(5H)-carboxylate

[0065] 2-Chloro-5,6,7,8-tetrahydro-1,6-naphthyridine hydrochloride (0.9 g) was suspended in dichloromethane (15 mL). N,N-diisopropylethylamine (1.4 g) was added, followed by di-tert-butyl dicarbonate (1.15 g). The mixture was allowed to react at room temperature for 1 hour. TLC indicated complete consumption of the starting material. The reaction mixture was diluted with water and extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude product was purified by column chromatography to yield the title compound (1.12 g).

[0066] MS (ESI) m / z (M+H) + =269.0.

[0067] Step 2: Preparation of tert-butyl 2-(diethoxyphosphoryl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate

[0068] Under an argon atmosphere, tert-butyl 2-chloro-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (100 mg) was dissolved in toluene (20 mL). Diethyl phosphite (102 mg), tris(dibenzylideneacetone)dipalladium (34 mg), 1,1'-bis(diphenylphosphino)ferrocene (41 mg), and triethylamine (75 mg) were added, and the system was reacted at 120°C overnight. TLC indicated complete consumption of the starting material. The mixture was diluted with ethyl acetate and filtered through celite. The filtrate was collected and concentrated. The crude product was purified by preparative TLC to obtain the title compound (70 mg).

[0069] MS (ESI) m / z (M+H) + =371.1.

[0070] Step 3: Preparation of (5,6,7,8-tetrahydro-1,6-naphthyridin-2-yl)phosphonic acid hydrochloride (test compound 2)

[0071] Dissolve tert-butyl 2-(diethoxyphosphoryl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (70 mg) in concentrated hydrochloric acid (5 mL) and react at 100°C overnight. LCMS showed complete consumption of the starting material. The reaction mixture was concentrated, and the crude product was purified by pre-HPLC to obtain the title compound (30 mg).

[0072] MS (ESI) m / z (M+H) +=215.0.

[0073] 1 H NMR (400MHz, D2O) δ8.35(dd,J=8.0,2.4Hz,1H),8.06(t,J=7.7Hz,1H),4.59(s,2H),3.67(t,J=6.0Hz,2H),3.49(t,J=6.4Hz,2H).

[0074] Preparation Example 3

[0075] (1) Preparation of 2-methyl-N-(4-phenylbutylidene)propane-2-sulfenamide

[0076] Dissolve 4-phenylbutanol (7.6 mL) in dichloromethane (200 mL), and add 2-iodoacylbenzoic acid (28.00 g), tert-butylsulfenamide (9.07 g), anhydrous magnesium sulfate (30.80 g), and 4-methylbenzenesulfonate pyridinium (0.627 g) in sequence. Heat to 40°C and react for 24 hours. Cool to room temperature, filter, and wash the filter cake with dichloromethane. Concentrate the filtrate to dryness and purify by column chromatography to obtain the title compound (5.1 g).

[0077] MS (ESI) m / z (M+H) + =252.1.

[0078] (2) Preparation of N-(1-(3-bromo-6-methoxypyridin-2-yl)-5-phenylpentan-2-yl)-2-methylpropane-2-sulfenamide

[0079] 3-Bromo-6-methoxy-2-methylpyridine (4.85 g) was weighed into a dry reaction flask and anhydrous tetrahydrofuran (100 mL) was added under a nitrogen atmosphere. The temperature was then cooled to -78°C. A solution of lithium diisopropylamide in tetrahydrofuran (12.00 mL, 2.0 M) was added dropwise, and the reaction was continued at -78°C for 40 minutes. 2-Methyl-N-(4-phenylbutylidene)propane-2-sulfenamide (5.02 g) in tetrahydrofuran (20 mL) was added dropwise, and the reaction was continued at -30°C for 30 minutes, then slowly warmed to room temperature. LCMS confirmed the reaction was complete, and the reaction was quenched by adding saturated ammonium chloride solution. Ethyl acetate and water were added, the layers were separated, extracted, and the organic phase was concentrated to dryness. Purification by column chromatography afforded the title compound (7.15 g).

[0080] MS (ESI) m / z (M+H) + =453.1,455.1.

[0081] (3) Preparation of ethyl 2-(2-((tert-butylsulfinyl)amino)-5-phenylpentyl)-6-methoxynicotinate

[0082] Weigh N-(1-(3-bromo-6-methoxypyridin-2-yl)-5-phenylpentan-2-yl)-2-methylpropane-2-sulfenamide (5.42 g) and dissolve it in ethanol (150 mL). Add [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex (0.979 g) and N,N-diisopropylethylamine (4.12 mL). After addition, purge the system with carbon monoxide and reflux under a carbon monoxide atmosphere overnight. Remove the solvent under reduced pressure. Purify by column chromatography to obtain the title compound (2.01 g).

[0083] MS (ESI) m / z (M+H) + =447.2.

[0084] (4) Preparation of 2-methoxy-7-(3-phenylpropyl)-7,8-dihydro-1,6-naphthyridin-5(6H)-one

[0085] Weigh 2-(2-((tert-butylsulfinyl)amino)-5-phenylpentyl)-6-methoxynicotinate (2.00 g) and dissolve it in acetonitrile (50 mL). Add cesium carbonate (5.87 g). Heat to 80°C and stir overnight. Monitor the reaction for completion by LC-MS. Cool to room temperature, filter, and wash the filter cake with dichloromethane. Concentrate the filtrate to dryness and purify by column chromatography to obtain the title compound (1.1 g).

[0086] MS (ESI) m / z (M+H) + =297.1.

[0087] (5) Preparation of 2-methoxy-7-(3-phenylpropyl)-5,6,7,8-tetrahydro-1,6-naphthyridine

[0088] 2-Methoxy-7-(3-phenylpropyl)-7,8-dihydro-1,6-naphthyridin-5(6H)-one (1.10 g) was weighed and dissolved in tetrahydrofuran (70 mL). Lithium aluminum hydride (0.562 g) was added under ice-cooling and stirred at 70°C for 8 hours. The reaction was monitored for completion by LC-MS. Water (0.56 mL), sodium hydroxide solution (15%, 0.56 mL), and water (1.68 mL) were added dropwise under ice-cooling. The mixture was stirred at room temperature for 20 minutes, dried over anhydrous magnesium sulfate, filtered, and the filter cake was washed with dichloromethane and concentrated to dryness under reduced pressure. Purification by column chromatography afforded the title compound (0.41 g).

[0089] MS (ESI) m / z (M+H) + =283.2.

[0090] (6) Preparation of 7-(3-phenylpropyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ol

[0091] Weigh 2-methoxy-7-(3-phenylpropyl)-5,6,7,8-tetrahydro-1,6-naphthyridine (0.40 g), add hydrobromic acid in acetic acid (5 mL), heat to 80°C and stir for 5 hours, remove the solvent under reduced pressure, add ethyl acetate to slurry, filter and dry to obtain the title compound (0.295 g).

[0092] MS (ESI) m / z (M+H) + =269.1.

[0093] (7) Preparation of 2-chloro-7-(3-phenylpropyl)-5,6,7,8-tetrahydro-1,6-naphthyridine

[0094] Weigh 7-(3-phenylpropyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-2-ol (0.29 g), add phosphorus oxychloride (6 mL), heat to 100 ° C and stir for 10 hours, remove the solvent under reduced pressure, add ice water and dichloromethane, adjust the pH to 9-10 with Na2CO3 aqueous solution, separate the liquids and extract, and concentrate the organic phase to dryness to obtain the crude title compound (0.6 g).

[0095] MS (ESI) m / z (M+H) + =287.1.

[0096] (8) Preparation of tert-butyl 2-chloro-7-(3-phenylpropyl)-7,8-dihydro-1,6-naphthyridine-6-(5H)-carboxylate

[0097] Weigh 2-chloro-7-(4-methoxyphenethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine (0.6 g) and dissolve it in dichloromethane (5 ml) and water (5 ml). Add sodium carbonate solution to adjust the pH to 8-9. Add di-tert-butyl dicarbonate (0.50 mL). Stir at room temperature for 1 hour, concentrate to dryness under reduced pressure, and purify by column chromatography to obtain the title compound (0.22 g).

[0098] MS (ESI) m / z (M+H) + =387.1.

[0099] (9) Preparation of tert-butyl 2-(benzylthio)-7-(3-phenylpropyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate

[0100] tert-Butyl 2-chloro-7-(3-phenylpropyl)-7,8-dihydro-1,6-naphthyridine-6-(5H)-carboxylate (147 mg), benzyl mercaptan (71 mg), tris(dibenzylideneindeneacetone)dipalladium (35 mg), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (44 mg) and triethylamine (113 uL) were weighed into a reaction flask, 1,4-dioxane (5 mL) was added, the atmosphere was replaced with nitrogen, the reaction was carried out at 100°C overnight, and the mixture was concentrated to dryness under reduced pressure. The title compound (159 mg) was purified by column chromatography.

[0101] MS (ESI) m / z (M+H) + =475.2.

[0102] (10) Preparation of tert-butyl 2-(chlorosulfonyl)-7-(3-phenylpropyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate

[0103] Weigh tert-Butyl 2-(benzylthio)-7-(3-phenylpropyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (156 mg) and dissolve it in acetonitrile (5 mL). Add acetic acid (79 mg), water (48 mg), and 1,3-dichloro-5,5-dimethylhydantoin (130 mg) in sequence under ice-cooling. Stir under ice-cooling for 1 hour, then add ice water and dichloromethane. Separate the mixture for extraction, and concentrate the organic phase to dryness to obtain the crude title compound (148 mg).

[0104] MS (ESI) m / z (M+H) + =451.1.

[0105] (11) Preparation of sodium 6-(tert-butyloxycarbonyl)-7-(3-phenylpropyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-2-sulfonate

[0106] Weigh tert-Butyl 2-(chlorosulfonyl)-7-(3-phenylpropyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxylate (148 mg), dissolve it in tetrahydrofuran (5 mL) and water (5 mL), add sodium hydroxide (27 mg), stir at 80°C for 1 hour, add ethyl acetate and water, separate the layers and extract, discard the organic phase, and concentrate the aqueous phase to dryness to obtain the crude title compound (50 mg).

[0107] MS (ESI) m / z (M+H) + =433.1.

[0108] (12) Preparation of 7-(3-phenylpropyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-2-sulfonic acid hydrochloride

[0109] The crude product of sodium (tert-butyloxycarbonyl)-7-(3-phenylpropyl)-5,6,7,8-tetrahydro-1,6-naphthyridine-2-sulfonate (50 mg) was dissolved in 6 M hydrochloric acid solution (3 mL) and stirred at room temperature for 1 hour. The system was concentrated to dryness and separated by pre-HPLC to obtain the title compound (test compound 3, 2.25 mg).

[0110] MS (ESI) m / z (M+H) + =333.0.

[0111] 1 H NMR(400MHz,D2O)δ7.74(dd,J=18.5,8.1Hz,2H),7.24(ddd,J=21.9,14.9,7.3Hz,5H),4.42(s,2H), 3.72-3.62(m,1H),3.32-3.22(m,1H),3.01-2.92(m,1H),2.63(d,J=3.6Hz,2H),1.83-1.58(m,4H).

[0112] 4. Experimental Results

[0113] The pharmacological activity of the test compound of the present invention was verified by the aforementioned experimental method. Through statistical analysis of the epidermal, biochemical and pathological indicators, the results showed that the application of the test compound of the present invention can effectively reduce the pigmentation of rat skin and has a significant effect on improving the symptoms of chloasma in model rats.

[0114] According to the experimental methods under 3.1-3.4, the experimental data collected on the 58th day, which is the end of the experiment, were analyzed. The specific results are shown in Figure 1:

[0115] 4.1 The grading evaluation of the apparent indicators in Figures 1A and 1E showed that there was no significant change in the back skin of the rats in the control group; the rats in the model group had obvious dark brown lesions and the lesion area was larger; compared with the model group, the lesion areas of the rats in the 5 mg / mL tranexamic acid and test compound groups were observed to be significantly lighter in color, showing light brown, and the lesion area was significantly reduced (P < 0.05).

[0116] 4.2 As shown in Figure 1B, HE staining results show that the epidermal tissue structure of the control rats is intact, the stratum corneum cells are neatly arranged, there is no obvious fibrosis, and no inflammatory cell infiltration; compared with the control rats, the epidermal tissue of the model group rats showed obvious thickening of the granular layer, spinous layer and stratum corneum, disordered skin tissue arrangement and uneven distribution, and inflammatory cell infiltration was visible; compared with the model group, the thickness of the basal layer and spinous layer of the epidermal tissue in the 5 mg / mL tranexamic acid and test compound groups was significantly reduced, the skin tissue structure gradually became flatter, and the inflammatory cell infiltration was improved.

[0117] 4.3 Figure 1C Fontana–Masson staining results show that melanin granules are positively expressed and appear black, with cell nuclei appearing red. There were almost no black-positive areas in the stained sections of the control group. In the epidermal sections of the model group, the black-positive areas were the largest, darkest, and distributed in a continuous, band-like pattern, indicating a significant increase in melanin granules. Numerous melanin granules appeared in the basal cells and stratum spinosum, with densely distributed melanin granules and melanin caps in the epidermis. Figure 1F shows a graded evaluation of melanin granule content in rat skin tissue. Compared with the model group, the black-positive areas in the sections of the 5 mg / mL tranexamic acid group were smaller and lighter in color, indicating a decrease in melanin granules in the skin tissue. The melanin granules were distributed in a band-like pattern, but were primarily concentrated in the basal layer and stratum spinosum. Compared with the model group, the black-positive areas in the sections of the 5 mg / mL test compound 1 and 3 groups were significantly smaller and lighter in color, indicating a significant decrease in the number of melanin granules in the skin tissue, with only discontinuous melanin granules visible in the basal layer.

[0118] 4.4 The immunohistochemical tyrosinase staining results in Figures 1D and 1G showed that tyrosinase-positive expression was yellow-brown and the cell nucleus was blue; there was almost no yellow-brown area in the immunohistochemical sections of the control group, indicating that the expression of tyrosinase in the rat skin tissue was low; the tyrosinase-positive area in the skin tissue sections of the rats in the model group was the largest, and the yellow-brown color was obvious, which showed that the tyrosinase-positive rate in the skin tissue of the rats in the model group was higher; compared with the model group, the tyrosinase-positive expression area in the skin tissue sections of the 5 mg / mL tranexamic acid, test compound 1 group and test compound 3 group was significantly reduced and the color was significantly lighter, which showed that the tyrosinase-positive rate in the skin tissue was significantly reduced (P<0.001); 1.5 and 5 mg / mL test compound 2 showed significant differences compared with the model group (P<0.01).

[0119] 4.5 Figure 1H The ET-1 content in skin tissue homogenate was detected using the ELISA method. The ET-1 content in the skin tissue of rats in the model group was significantly increased compared with the control group, while the ET-1 content in the 5 mg / mL tranexamic acid and test compound groups was significantly reduced compared with the model group, with significant differences (P<0.01).

[0120] 4.6 Figure 1I shows that there was no significant difference in body weight among the rats in each group throughout the experiment, indicating that the animals tolerated the test substance well.

[0121] The above is an exemplary description of the implementation methods of the technical solution of the present invention. It should be understood that the scope of protection of the present invention is not limited to the above implementation methods. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention shall be included in the scope of protection of the claims of this application.

Claims

1. Use of a compound of formula I, a pharmaceutically acceptable salt, hydrate, isomer, prodrug, or a mixture thereof for preparing a product for improving pigmentation, in, R1 is selected from carboxyl, phosphate, and sulfonic acid; R2 is selected from hydrogen, substituted or unsubstituted amino, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, C1-C4 haloalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered alicyclic group, substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 6-10 membered aromatic heterocyclic group.

2. The use according to claim 1, characterized in that R2 is selected from hydrogen, unsubstituted or optionally substituted by one, two or more R 2a Substituted groups: amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C4 haloalkyl, C3-C6 cycloalkyl, 4-8 membered alicyclic group, 6-10 membered aryl, 6-10 membered aromatic heterocyclic group; The R 2a Selected from halogen, OH, NH2, NO2, CN, oxo (=O), C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 4-8 membered alicyclic group, 6-10 membered aryl, 6-10 membered aromatic heterocyclic group.

3. The use according to claim 1 or 2, characterized in that The R2 is selected from hydrogen, unsubstituted or optionally substituted by one, two or more R 2a Substituted phenyl, phenyl-C1-C6 alkyl-.

4. The use according to any one of claims 1 to 3, characterized in that Said R2 is selected from hydrogen, 5. The use according to any one of claims 1 to 4, characterized in that The pigmentation may be a pigmentation-related disease, and the pigmentation-related disease refers to a disease caused by the deposition of melanin on the surface of the skin.

6. The use according to claim 5, characterized in that The pigmentation-related diseases include one or more of chloasma, age spots, freckles, melanosis, facial moles, and birthmarks.

7. The use according to claim 5, characterized in that The pigmentation-related disease is melasma.

8. The use according to any one of claims 1 to 7, characterized in that The compound of formula I is selected from the following compounds, pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof:

9. The use according to any one of claims 1 to 8, characterized in that The pharmaceutically acceptable salt is the hydrochloride of the compound of formula I.

10. The use according to any one of claims 1 to 9, characterized in that The pigmentation-improving product may be a medicine or a cosmetic.

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