WO2020098443A1 - Bacterial chlorophyll derivative and preparation method therefor - Google Patents

Abstract

The present invention provides a bacterial chlorophyll derivative, a preparation method therefor and application thereof in medicine. Particularly, the present invention relates to a bacterial chlorophyll derivative represented by formula (I), a preparation method therefor and a pharmaceutically acceptable salt thereof, and application thereof in photodynamic therapy of tumors. The definition of each substituent in formula (I) is the same as that in the description. (I)

Description

Bacterial chlorophyll derivatives and preparation method thereof Technical field

The invention relates to the field of pharmaceuticals, in particular to a chlorophyll derivative of bacteria, a preparation method and application thereof.

Background technique

Photodynamic therapy (PDT) refers to a treatment method that uses visible light to sensitize a photosensitizer located inside a human diseased tissue to kill the diseased tissue. In clinical practice, intravenous injection of a photosensitizer that has an affinity for tumor cells and other biologically active tissues. The photosensitizer will selectively enrich in the lesion area, and then irradiate the lesion area with light of a certain wavelength. The photosensitizer absorbs light energy It is then converted into a highly reactive active substance, which, with the participation of oxygen, chemically reacts with the biological molecules of the diseased tissue to kill it. This is a new type of clinical treatment technology under research and development, which is mainly used in clinical treatment of various tumors.

Among all photosensitizers, porphyrin photosensitizers have always been a hot research object. Because chlorophyll (a) derivatives and bacterial chlorophyll derivatives have strong absorption characteristics in the 650-850nm spectral region and are easily degraded after treatment, they are considered as excellent photosensitizers for photodynamic therapy of tumors.

In photosensitizers of chlorophyll (a) derivatives and bacterial chlorophyll derivatives,

It is the first generation of photosensitizer. The photoperiod-free treatment period needs 4-6 weeks, the absorption wavelength is 630nm, and the penetration depth in the tissue is less than 4mm. HPPH, currently in Phase II clinical stage, is a second-generation photosensitizer with a maximum absorption wavelength of 665 nm and a penetration depth of about 0.5 cm in tissues. The photoperiod-free period after photodynamic therapy is one week. Long, in life, patients will inevitably be exposed to light, so HPHP treatment of the skin will cause erythema and other damage. WO2004 / 002476 reports a bacterial chlorophyll derivative with an absorption wavelength of 700-850 nm. Since it is a fat-soluble compound, it has a long metabolism time, high content in skin, and high skin phototoxicity. Similarly, the compounds reported in the literature (Bioconjug Chem. 2009 February; 20 (2): 274–282) have not been metabolized in the main tissues or organs after 96 hours of administration, indicating that the compounds are metabolized in vivo Slowness indicates that the compound has a longer light-shielding period.

It can be seen that the existing photosensitizers of chlorophyll (a) or bacterial chlorophyll derivatives have the limitations of slow metabolism and long photoperiod avoidance period after photodynamic therapy, which limits the wide application of photodynamic therapy in tumor therapy.

Summary of the invention

In order to solve the problems of slow metabolism of the existing photosensitizer and long light-proof period, the present invention provides a bacterial chlorophyll derivative with fast metabolism and short light-proof period, which can be used as a photosensitizer for photodynamic therapy research, and patients can achieve the same day Treatment, go home on the same day, thereby reducing the financial burden of patients and their families.

The object of the present invention is to provide a compound of formula (I) or its stereoisomer, or a pharmaceutically acceptable salt thereof:

among them,

R ¹ is hydrogen or (CH ₂ ) _m COOH;

R ^{2 is} selected from hydrogen, hydroxyl, halogen, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy;

R ³ and R ⁴ are each independently selected from C ₁ -C ₆ alkyl;

m is 1, 2, 3 or 4; and

n is 1, 2, 3 or 4.

In a preferred embodiment of the present invention, a compound of formula (I) or its stereoisomer, or a pharmaceutically acceptable salt thereof, is a compound of formula (II) or its stereoisomer, or its Medicinal salt:

Wherein, R ² , R ³ , R ⁴ , m and n are defined as in formula (I).

In a preferred embodiment of the present invention, a compound of formula (I) or its stereoisomer, or a pharmaceutically acceptable salt thereof, is a compound of formula (III) or its stereoisomer, or its Medicinal salt:

Wherein, R ² , R ³ , R ⁴ and n are as defined in formula (I).

In a preferred embodiment of the present invention, a compound of formula (I), formula (II), or formula (III) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein R ³ is methyl, ethyl Group, propyl, butyl or hexyl.

In a preferred embodiment of the present invention, a compound of formula (I), formula (II) or formula (III) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein R ⁴ is methyl, ethyl Group, propyl, butyl or hexyl.

In a preferred embodiment of the present invention, a compound of formula (I), formula (II) or formula (III) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein R ^{2 is} selected from hydrogen, halogen , C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy.

In a preferred embodiment of the present invention, a compound of formula (I), formula (II), or formula (III) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein R ^{2 is} selected from hydrogen, fluorine , Methyl or methoxy.

Representative compounds of the present invention include, but are not limited to:

Or its stereoisomers, or their pharmaceutically acceptable salts.

The present invention provides a method for preparing a compound of formula (II) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The method includes:

The compound of formula (IIA) undergoes a hydrolysis reaction under basic conditions, and then acid is added for acid hydrolysis to obtain the compound of formula (II);

Wherein: R ² , R ³ , R ⁴ , m and n are as defined in formula (I).

Further, the present invention provides a method for preparing a compound of formula (II) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, the method comprising: the base used in the alkaline condition is selected from alkali metal carbonates Or ammonium carbonate, preferably alkali metal carbonate, more preferably sodium carbonate, potassium carbonate or lithium carbonate;

The acid is selected from inorganic acid or organic acid, wherein the inorganic acid is selected from hydrochloric acid with a mass fraction of 1-30% or sulfuric acid with a mass fraction of 1-50%, preferably hydrochloric acid with a mass fraction of 1-10% or a mass Sulfuric acid with a fraction of 1-10%, the organic acid is selected from C ₁ -C ₄ organic acids, preferably formic acid, acetic acid or oxalic acid.

The present invention provides a method for preparing a compound of formula (III) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The method includes:

The compound of formula (IIIA) is hydrolyzed under basic conditions to obtain the compound of formula (III),

Wherein: R ² , R ³ , R ⁴ and n are as defined in formula (I).

Further, the present invention provides a method for preparing a compound of formula (III) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, the method comprising: the base used in the alkaline condition is selected from alkali metal carbonates , Ammonium carbonate or organic amine, preferably alkali metal carbonate or C ₁ -C ₆ primary amine, more preferably sodium carbonate, potassium carbonate, lithium carbonate, n-propylamine or butylamine.

The present invention provides a compound represented by formula (IIA) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

Wherein: R ² , R ³ , R ⁴ , m and n are as defined in formula (I).

The present invention provides a compound represented by formula (IIIA) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

Wherein: R ² , R ³ , R ⁴ and n are as defined in formula (I).

The present invention provides a pharmaceutical composition containing an effective dose of a compound of formula (I) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and excipient Or a combination of them.

The present invention provides a compound of formula (I) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for use in the preparation of a medicament for photodynamic therapy of cancer, wherein the cancer is preferably One or more of lung cancer, prostate cancer, esophageal cancer, melanoma, bile duct cancer, head and neck cancer, bladder cancer, stomach cancer, nasopharyngeal cancer, colon cancer.

The maximum absorption wavelength of the compound of the present invention is around 780nm, and the penetration depth of the tissue is 0.7-1cm, which can effectively kill tumor cells with deep tissues; due to the introduction of hydrophilic groups, carboxyl groups or The phenolic hydroxyl groups have good water solubility, so the metabolic time is short and the dark period is short; at the same time, the compounds of the present invention have good chemical stability.

Detailed description of the invention

Unless stated to the contrary, some terms used in the specification and claims of the present invention are defined as follows:

"Alkyl" when taken as a group or part of a group refers to a C ₁ -C ₆ linear or branched saturated aliphatic hydrocarbon group. For example, C ₁ -C ₆ refers to containing 1 to 6 carbon atoms, for example, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms . Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-di Methylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1 -Ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethyl Butyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl Wait.

"Alkoxy" refers to the group (alkyl-O-). Among them, alkyl refers to the relevant definitions herein. Examples of C ₁ -C ₆ alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, t-butoxy, n- Pentoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, 2-methylbutoxy Oxygen, 3-methylbutoxy, n-hexyloxy, 1-ethyl-2-methylpropoxy, 1,1,2-trimethylpropoxy, 1,1-dimethylbutoxy Group, 1,2-dimethylbutoxy, 2,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2-ethylbutoxy, 2-methylpentoxy , 3-methylpentoxy, 4-methylpentoxy, 2,3-dimethylbutoxy.

"Hydroxy" refers to the -OH group.

"Halogen" means fluorine, chlorine, bromine and iodine.

"Amino" refers to -NH ₂ .

"Cyano" means -CN.

"Nitro" refers to -NO ₂ .

"Benzyl" refers to -CH ₂ - phenyl.

"Carboxy" refers to -C (O) OH.

"Carboxylate group" refers to -C (O) O (alkyl), where alkyl is as defined above.

"Boc" refers to tert-butoxycarbonyl.

"DMSO" refers to dimethyl sulfoxide.

"Substituted" refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1-3 hydrogen atoms are independently substituted with a corresponding number of substituents for each other. It goes without saying that the substituents are only at their possible chemical positions, and those skilled in the art can determine (through experiment or theory) possible or impossible substitutions without undue effort. For example, an amino group or hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (eg, olefinic) bond.

"Pharmaceutically acceptable salts" refers to certain salts of the above compounds that can retain their original biological activity and are suitable for medical uses. The pharmaceutically acceptable salt of the compound represented by formula (I) may be a metal salt or a salt with a suitable acid.

"Stereoisomer" means including but not limited to diastereomers, enantiomers, atropisomers and mixtures thereof, such as racemic mixtures, forming part of the present invention. Diastereomers can be separated into individual diastereomers by chromatography, crystallization, distillation or sublimation based on their physical and chemical differences. Enantiomers can be converted into diastereoisomeric mixtures by separation, by reacting with appropriate optically active compounds (such as chiral auxiliary agents such as chiral alcohols or Mosher's acid chloride) , Separation of diastereomers, and the conversion of individual diastereomers to the corresponding pure enantiomers. The intermediates and compounds of the present invention may also exist in different tautomeric forms, and all such forms are included within the scope of the present invention. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane polarized light. When describing optically active compounds, the prefixes D, L or R, S are used to denote the absolute configuration of the molecular chiral center. The prefixes d, l, or (+), (-) are used to name the symbol for rotation of plane polarized light of the compound, (-) or l means the compound is left-handed, and the prefix (+) or d means the compound is right-handed. The atoms or groups of these stereoisomers are connected in the same order, but their stereostructures are different. The specific stereoisomer may be an enantiomer, and a mixture of isomers is generally called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or a racemate, which may result in no stereoselectivity or stereospecificity during the chemical reaction. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers, lacking optical activity.

"Alkali metal carbonate" means lithium carbonate, sodium carbonate, potassium carbonate, rubidium carbonate, cesium carbonate, and francium carbonate.

"Pharmaceutical composition" means a mixture comprising a compound of the present application or a physiologically acceptable salt or prodrug thereof with other chemical components, and other components such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert the biological activity.

BRIEF DESCRIPTION

Figure 1 shows the effect of different doses of compound (I-1) on the volume of NCI-H460 transplanted tumor;

Fig. 2 is a blood concentration-time curve of compound (I-1) (3.0 mg / kg) in different tissues;

Fig. 3 is the blood concentration-time curve of HPPH (0.6mg / kg) in different tissues.

detailed description

Hereinafter, the present invention will be described in detail with reference to specific embodiments. It should be understood that based on the content disclosed herein, those skilled in the art can make various modifications and improvements to the present invention without departing from the spirit and scope of the present invention, and they should all fall within the patent protection defined by the claims of the present application Within range. In addition, it should be understood that the embodiments provided herein are for illustrative purposes only and should not be construed as limiting the invention.

The following examples are used to further describe the present invention, but these examples are not intended to limit the scope of the present invention.

Examples

The examples give the preparation of representative compounds represented by formula (I) and related structural identification data. It should be noted that the following examples are for illustrating the present invention rather than limiting the present invention.

The NMR spectrum is measured with a Bruker instrument (400 MHz), and the chemical shift is expressed in ppm. Use tetramethylsilane internal standard (0.00 ppm). Representation method of ¹ H NMR: s = single peak, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of doublet, dt = doublet of triplet. If the coupling constant is provided, the unit is Hz.

Mass spectrometry is measured by LC / MS instrument, and the ionization method can be ESI or APCI.

Ultraviolet data was measured with SHIMADZU's UV-2600 ultraviolet spectrometer.

The thin-layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The specification of the thin-layer chromatography (TLC) silica gel plate is 0.15mm-0.2mm, and the specification of the thin-layer chromatography separation and purification product is 0.4mm. -0.5mm.

Column chromatography generally uses Yantai Yellow Sea silica gel 200-300 mesh silica gel as a carrier.

In the following examples, unless otherwise specified, all temperatures are in degrees Celsius. Unless otherwise specified, various starting materials and reagents are commercially available or synthesized according to known methods. Purification is used directly, unless otherwise specified, commercially available manufacturers include but are not limited to Aldrich Chemical Company, ABCR GmbH & Co. KG, Acros Organics and Sinopharm Chemical Reagent Co., Ltd. and other places to buy.

CDCl ₃ : deuterated chloroform.

DMSO: Deuterated dimethyl sulfoxide.

Nitrogen reaction means that the reaction bottle is connected to a nitrogen balloon with a volume of about 1L.

There is no special description in the examples, and the solution in the reaction refers to an aqueous solution.

To purify the compound, use silica gel column chromatography eluent system and thin layer chromatography, where the eluent system is selected from: petroleum ether and ethyl acetate system; dichloromethane and acetone system; dichloromethane and methanol, wash The ratio of the removal agent is the volume ratio, where the volume ratio of the solvent is different according to the polarity of the compound, and a small amount of acidic or basic reagents can also be added for adjustment, such as acetic acid or triethylamine.

The compound (IIB-1) in the present invention is synthesized according to the method of the literature (J. Med. Chem., 2016, 59, 9774-9787), compound (IIB-2), compound (IIB-3) and compound (IIB-4 ) Are synthesized according to the preparation method of compound (IIB-1), and the structural characterization data are as follows:

Example 1 Synthesis of Compound (I-1)

Step 1: Synthesis of methyl 2- (4-hydroxymethyl) phenoxyacetate (IV-1)

In a 500mL three-necked round bottom flask, add p-hydroxybenzaldehyde (10g, 82mmol), then add 120mL acetonitrile, stir to dissolve, then add methyl bromoacetate (10mL, 105mmol) and potassium carbonate (15g, 9.2mmol), The reaction was stirred at room temperature, and the reaction was monitored by TLC. After the reaction was completed, 150 mL of ethyl acetate was added to dilute the reaction solution, filtered, and the filtrate was concentrated under reduced pressure to obtain a yellow oil, which was directly subjected to the next reaction;

Transfer the obtained yellow oil to a 500mL three-necked round bottom flask, add 100mL of dichloromethane and 50mL of methanol, stir, and slowly add sodium borohydride (3.0g, 79mmol) under ice bath cooling. After the addition, Stir the reaction at room temperature and monitor the reaction by TLC. After the reaction is completed, quench the reaction with 10% dilute hydrochloric acid aqueous solution, then add 150mL of dichloromethane and 100mL of water, separate the organic phase, concentrate to dryness under reduced pressure, and use silica gel column chromatography Purification, eluent and volume ratio: petroleum ether: ethyl acetate = 4: 1, to obtain 8.5g methyl 2- (4-hydroxymethyl) phenoxyacetate (IV-1), compound (IV-1) It is a white solid; total yield: 52.86%.

¹ H NMR (400 MHz, CDCl ₃ ) δ: 2.18 (s, 1H), 3.77 (s, 3H), 4.56 (s, 2H), 4.6 (s, 2H), 6.85 (d, J = 8.36 Hz, 2H) , 7.25 (d, J = 8.28 Hz, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ: 52.30, 64.70, 65.31, 114.63, 128.63, 134.37, 157.25, 169.45.

Step 2: Synthesis of compound (IIA-1)

In a 500mL three-necked round bottom flask, weigh compound (IIB-1) (1.4g, 2.15mmol), add 100mL of dichloromethane and 10mL of methanol, stir to dissolve, and slowly add sodium borohydride (0.815g) under ice bath cooling , 21.5mmol), after the addition, the reaction was stirred at room temperature, the reaction was monitored by TLC, after the reaction was completed, the reaction was quenched with 10% dilute acetic acid aqueous solution, followed by the addition of 150mL of methylene chloride and 100mL of water, the organic phase was separated, anhydrous sulfuric acid Sodium was dried, filtered, and the filtrate was concentrated under reduced pressure to obtain a black solid, which was directly subjected to the next reaction;

Transfer all the black solids into a 250mL single-necked round-bottom flask, add 150mL of dichloromethane, slowly stir, pass hydrogen bromide gas to saturation, continue stirring, TLC monitor the reaction, after the reaction is completed, concentrate to dryness under reduced pressure, directly Proceed to the next reaction;

The concentrate obtained above was transferred to a 500 mL three-necked round bottom flask, compound 2- (4-hydroxymethyl) phenoxyacetic acid methyl ester (IV-1) (1.9 g, 10 mmol) was added, and 100 mL of dichloromethane was added, After stirring and cooling in an ice bath, slowly add 50 mL of triethylamine in dichloromethane (containing triethylamine 2 mL, 14.35 mmol). After the addition, the reaction was performed at room temperature, and the reaction was monitored by TLC. After the reaction was completed, 100 mL of dichloromethane and 100mL of deionized water, the organic phase was separated, concentrated to dryness under reduced pressure, purified by silica gel column chromatography, eluent and volume ratio: dichloromethane: acetone = 100: 2, to obtain 1.5g compound (IIA-1 ), MS (ESI): [M + H ⁺ ]: 832.4250.

Step 3: Synthesis of compound (I-1)

The compound (IIA-1) was added to a 500 mL three-necked round bottom flask, 100 mL of acetonitrile and 40 mL of deionized water were added, stirred, and then potassium carbonate (3.8 g, 27.5 mmol) was added, and the reaction was stirred at 40 ° C under nitrogen protection. The reaction was monitored by TLC. After the reaction was completed, a 10% aqueous acetic acid solution was added to adjust the pH to 2-3. The reaction solution was transferred to a 500mL separatory funnel, 200mL ethyl acetate and 100mL deionized water were added, the organic phase was separated, concentrated to dryness under reduced pressure, purified by silica gel column chromatography, eluent and volume ratio is : Dichloromethane: methanol = 100: 10, 0.8 g of compound (I-1) was obtained, and the total yield was 45.1%.

The characterization data of compound (I-1) is as follows:

UV Wavelength (Absorbance): 781.5 (0.244), 537.0 (0.214), 416.0 (0.241), 365.5 (0.589), 344.0 (0.301), 291.5 (0.10), 225 (0.156), 201.0 (0.256), 194.5 (0.123) ;

MS (ESI): [M + H ⁺ ]: 818.4092, [M + Na ⁺ ]: 840.3926;

¹ H NMR (400M Hz, CDCl ₃ ) δ-0.28 (s, 1H), 0.06 (s, 1H), 0.90-0.97 (m, 6H), 1.36-1.46 (m, 2H), 1.58-1.66 (m, 6H), 1.70-1.77 (m, 3H), 1.83-1.93 (m, 4H), 2.17-2.32 (m, 3H), 2.47-2.55 (m, 1H), 3.22 (s, 3H), 3.38 (s, 3H), 3.42 (s, 3H), 3.84-3.86 (m, 1H), 4.02-4.13 (m, 3H), 4.24-4.30 (m, 1H), 4.43-4.54 (m, 2H), 4.64 (s, 2H), 5.06 (d, J = 7.96 Hz, 1H), 5.81-5.86 (m, 1H), 6.84-6.86 (m, 2H), 7.22 (d, J = 8.08 Hz, 2H), 8.58-8.61 (m , 2H), 8.78 (s, 1H), 8.86 (s, 1H), 13.01 (s, 1H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 10.77, 11.01, 11.08, 14.27, 20.48, 22.43, 22.81,23.68, 24.13, 24.36, 29.84, 30.89, 31.33, 32.07, 47.79, 48.76, 51.55, 53.55, 54.58, 64.95 , 70.24,70.49,71.40,95.73,99.28,101.48,113.13,114.62,128.19,129.79,131.03,131.24,132.54,134.06,138.03,138.41,141.58,157.69,161.25,162.84,167.02,170.62,171.37,171.60,172. , 173.43,175.45.

Example 2 Synthesis of Compound (I-2):

Step 1: Synthesis of methyl 5- (3-methyl-4-hydroxymethyl) phenoxyvalerate (IV-2)

In a 500mL three-necked round bottom flask, add 3-methyl-4-hydroxybenzaldehyde (5.0g, 36.7mmol), then add 80mL of acetonitrile, stir to dissolve, and then add 5-bromovaleric acid methyl ester (8.0g, 41.0mmol) and potassium carbonate (7.0g, 50.6mmol), the reaction was stirred at 60 ° C, the reaction was monitored by TLC, after the reaction was completed, 150mL of ethyl acetate was added to dilute the reaction solution, filtered, and the filtrate was concentrated under reduced pressure to obtain a yellow oil;

Transfer the resulting yellow oil to a 500mL three-necked round bottom flask, add 100mL of dichloromethane and 50mL of methanol, stir, and slowly add sodium borohydride (0.75g, 2.04mmol) under ice bath cooling. After the addition, Stir the reaction at room temperature and monitor the reaction by TLC. After the reaction is completed, quench the reaction with 10% dilute hydrochloric acid aqueous solution, then add 150mL of dichloromethane and 100mL of water, separate the organic phase, concentrate to dryness under reduced pressure, and use silica gel column chromatography Purification, eluent and volume ratio: petroleum ether: ethyl acetate = 4: 1, to obtain 7.0g 5- (3-methyl-4-hydroxymethyl) phenoxyvaleric acid methyl ester (IV-2), shallow Yellow liquid, total yield 76.9%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.73 (s, 1H), 1.86 (s, 4H), 2.23 (s, 3H), 2.43 (s, 2H), 3.69 (s, 3H), 3.99 (s, 2H), 4.59 (s, 2H), 6.78 (d, J = 8.0Hz, 1H), 7.13-7.16 (m, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 16.22, 21.73, 28.76, 33.69, 51.55, 65.10, 67.42, 110.77, 125.77, 127.01, 129.94, 132.70, 156.68, 173.99.

Step 2: Synthesis of compound (IIA-2)

In a 500 mL three-necked round bottom flask, weigh compound (IIB-1) (1.9 g, 2.91 mmol), add 100 mL of dichloromethane and 20 mL of methanol, stir to dissolve, and slowly add sodium borohydride (110 mg, 2.90mmol), after the completion of the reaction, the reaction was stirred at room temperature, the reaction was monitored by TLC, after the reaction was completed, the reaction was quenched with 10% dilute acetic acid aqueous solution, followed by the addition of 150mL of methylene chloride and 100mL of water, the organic phase was separated, anhydrous sodium sulfate Dry, filter, and concentrate the filtrate under reduced pressure to obtain a black solid, and proceed directly to the next reaction;

Transfer the black solid obtained above into a 250mL single-necked round-bottom flask, add 150mL of dichloromethane, slowly stir, pass hydrogen bromide gas to saturation, continue stirring, and monitor the reaction by TLC. After the reaction is completed, concentrate to dryness under reduced pressure , Proceed directly to the next reaction;

Transfer the concentrate obtained above into a 500mL three-necked round-bottom flask, weigh in compound (IV-2) (1.5g, 5.95mmol), add 100mL of dichloromethane, stir, under ice bath cooling, slowly add 50mL After the addition of ethylamine in dichloromethane (containing 5mL of triethylamine, 35.8mmol), the reaction was carried out at room temperature. The reaction was monitored by TLC. After the reaction was completed, 100mL of dichloromethane and 100mL of deionized water were added, the organic phase was separated, and the pressure was reduced. Concentrate to dryness and purify by silica gel column chromatography. The eluent and volume ratio are: dichloromethane: acetone = 100: 2 to obtain compound (IIA-2), MS (ESI): [M + H ⁺ ]: 888.55.

Step 3: Synthesis of compound (I-2)

Add the compound (IIA-2) obtained above to a 500mL single-necked round-bottom flask, add 100mL of acetonitrile and 40mL of deionized water, stir, and then add potassium carbonate (2.1g, 15.2mmol), stirring under nitrogen at 40 ° C After 4-6 hours of reaction, the reaction was monitored by TLC. After the reaction was completed, a 10% aqueous acetic acid solution was added to adjust the pH to 2-3. The reaction solution was transferred to a 500mL separatory funnel, 200mL ethyl acetate and 100mL deionized water were added, the organic phase was separated, concentrated to dryness under reduced pressure, purified by silica gel column chromatography, eluent and volume ratio is : Dichloromethane: methanol = 100: 10, 0.1 g of compound (I-2) was obtained, and the total yield was 3.9%.

The characterization data of compound (I-2) is as follows:

UV Wavelength (Absorbance): 782.2 (0.18), 537.0 (0.155), 416.0 (0.195), 365.6 (0.438), 202.0 (0.40);

^{MS (ESI): [M -} ]: 872.56, [M]: 873.56, [M + H +]: 874.56;

¹ H NMR (400M Hz, DMSO) δ-0.31 (s, 1H), 0.07 (s, 1H), 0.79 (t, J = 6.34 Hz, 3H), 0.94-0.98 (m, 6H), 1.15-1.19 ( m, 6H), 1.39-1.45 (m, 2H), 1.59-1.64 (m, 6H), 1.70-1.77 (m, 3H) 1.82-1.93 (m, 4H), 1.99-2.01 (m, 8H), 2.21 -2.28 (m, 3H), 3.20 (s, 3H), 3.42 (s, 3H), 3.77-3.86 (m, 1H), 3.84-3.86 (m, 1H), 4.02-4.05 (m, 3H), 4.13 -4.28 (m, 1H), 4.38-4.49 (m, 2H), 5.06 (s, 1H), 5.79 (s, 1H), 6.67-6.69 (m, 1H), 6.96-7.01 (m, 2H), 8.58 (s, 2H), 8.78 (d, J = 6.34 Hz, 1H);

¹³ C NMR (100MHz, DMSO) δ 10.77, 10.97, 11.83, 14.24, 14.51, 16.20, 20.46, 21.17, 21.67, 22.43, 22.72, 23.65, 24.15, 28.59, 29.91, 30.90, 31.33, 32.07, 33.72, 53.55, 60.18,67.55,70.79,99.28,101.45,111.19,113.12,125.86,127.22,128.14,128.23,130.08,130.76,131.02,132.55,134.03,138.07,138.64,138.73,141.62,156.53,161.24,162.87,167.05,170 171.32,171.57,172.68,173.42,175.46,176.08.

Example 3 Synthesis of Compound (I-3):

Step 1: Synthesis of methyl 2- (3-methyl- (4-hydroxymethyl)) phenoxyacetate (IV-3)

In a 500mL three-necked round bottom flask, add 3-methyl-4hydroxybenzaldehyde (5.02g, 25.5mmol), then add 80mL of acetonitrile, stir to dissolve, then add methyl bromoacetate (2.5mL, 2.55mmol) and Potassium carbonate (5.0g, 3.61mmol), stirring the reaction at room temperature, and monitoring the reaction by TLC. After the reaction was completed, 150mL of ethyl acetate was added to dilute the reaction solution, and the filtrate was concentrated to dryness under reduced pressure to obtain a yellow oil. ;

Transfer the obtained yellow liquid to a 500mL three-necked round bottom flask, add 100mL of methylene chloride and 50mL of methanol, stir, and slowly add sodium borohydride (0.7g, 1.9mmol) under ice bath cooling. Stir the reaction and monitor the reaction by TLC. After the reaction is completed, quench the reaction with 10% dilute hydrochloric acid aqueous solution, then add 150 mL of dichloromethane and 100 mL of water, separate the organic phase, concentrate to dryness under reduced pressure, and purify by silica gel column chromatography , The eluent and volume ratio are: petroleum ether: ethyl acetate = 4: 1, to obtain 5.8g of 2- (3-methyl- (4-hydroxymethyl)) phenoxyacetic acid methyl ester (IV-3), white Solid, total yield 75.3%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.77 (s, 1H), 2.31 (s, 3H), 3.81 (S, 3H), 4.59 (s, 2H), 4.67 (s, 2H), 6.69 (d, J = 8.24 Hz, 1H), 7.13 (d, J = 8.24 Hz, 1H), 7.19 (s, 1H);

¹³ CNMR (100MHz, CDCl ₃ ) δ 16.19, 52.2, 64.97, 65.63, 111.15, 125.67, 127.55, 130.27, 133.96, 155.66, 169.60.

Step 2: Synthesis of compound (IIA-3)

In a 500mL three-necked round bottom flask, weigh compound (IIB-2) (1.7g, 2.26mmol), add 100mL dichloromethane and 20mL methanol, stir to dissolve, and slowly add sodium borohydride (45mg, 1.13mmol), after the addition, the reaction was stirred at room temperature, and the reaction was monitored by TLC. After the reaction was completed, the reaction was quenched with 10% dilute acetic acid aqueous solution, followed by addition of 150mL of methylene chloride and 100mL of water, and the organic phase was separated, anhydrous sodium sulfate Dry, filter, and concentrate the filtrate under reduced pressure to obtain a black solid, and then proceed to the next reaction;

Transfer the black solid obtained above to a 250mL single-necked round-bottom flask, add 100mL of dichloromethane, slowly stir, pass hydrogen bromide gas to saturation, continue to stir, and monitor the reaction by TLC. After the reaction is completed, concentrate to dryness under reduced pressure , Proceed directly to the next reaction;

Transfer the concentrate obtained above into a 500mL three-necked round bottom flask, weigh in compound (IV-3) (1.5g, 7.14mmol), add 100mL of dichloromethane, stir, and slowly drop 50mL of trihydrate under ice bath cooling A solution of ethylamine in dichloromethane (containing 3mL of triethylamine, 21.5mmol). After the addition, the reaction was performed at room temperature. The reaction was monitored by TLC. After the reaction, 100mL of dichloromethane and 100mL of deionized water were added to separate the organic phase. Concentrate to dryness by pressure, purify by silica gel column chromatography, eluent and volume ratio: methylene chloride: acetone = 100: 2, to obtain compound (ⅡA-3), MS (ESI): [M + H ⁺ ] : 944.56.

Step 3: Synthesis of compound (I-3)

Add all the above-obtained compound (IIA-3) to a 500mL single-necked round bottom flask, add 100mL of acetonitrile and 40mL of deionized water, stir, and then add potassium carbonate (2.0g, 14.5mmol), under nitrogen protection, 40 ℃ The reaction was stirred, and the reaction was monitored by TLC. After the reaction was completed, a 10% aqueous acetic acid solution was added to adjust the pH to 2-3. Transfer the reaction solution to a 500 mL separatory funnel, add 200 mL of ethyl acetate and 100 mL of deionized water, separate the organic phase, concentrate to dryness under reduced pressure, and purify by silica gel column chromatography. The eluent and volume are: Dichloromethane: methanol = 100: 10, 0.33 g of compound (I-3) was obtained, and the total yield was 15.7%.

The characterization data of compound (I-3) is as follows:

UV Wavelength (Absorbance): 781.8 (0.123), 537.2 (0.108), 415.2 (0.135), 365.8 (0.309), 202.0 (0.212);

MS (ESI): [M + H ⁺ ]: 930.55;

¹ H NMR (400M Hz, DMSO) δ-0.30 (s, 1H), 0.10 (s, 1H), 0.61 (t, J = 6.34Hz, 3H), 0.74-0.84 (m, 4H), 0.88-1.02 ( m, 9H), 1.09-1.08 (m, 2H), 1.18-1.37 (m, 7H), 1.60-1.73 (m, 7H), 1.82-1.93 (m, 4H), 2.08-2.28 (m, 6H), 2.38-2.45 (m, 1H), 3.21 (s, 3H), 3.40 (s, 3H), 3.54-3.68 (m, 3H), 3.84-3.91 (m, 1H), 4.02-4.16 (m, 3H), 4.22-4.28 (m, 1H), 4.39-4.50 (m, 2H), 4.62-4.68 (m, 2H), 5.10 (d, J = 7.48Hz, 1H), 5.80-5.83 (t, J = 5.68Hz, 1H), 6.73 (d, J = 8.16 Hz, 1H), 6.96-7.12 (m, 2H), 8.60 (s, 2H), 8.88 (d, J = 19.8 Hz, 1H);

¹³ C NMR (100MHz, DMSO) δ 10.78, 11.04, 11.84, 14.02, 14.28, 16.35, 16.42, 22.18, 22.46, 22.72, 22.84, 23.66, 25.20, 26.82, 28.11, 28.64, 31.05, 31.49, 32.07, 53.49, 53.53,63.99,65.26,70.67,70.89,99.39,111.41,113.15,126.14,127.06,128.18,128.28,130.84,130.93,131.04,131.08,132.54,134.11,138.05,138.60,138.68,141.58,141.68,155.89,16 162.85,167.02,170.67,171.33,172.62,172.85,175.39,175.44,175.47,176.04.

Example 4 Synthesis of Compound (I-4):

Step 1: Synthesis of methyl 2- (4-hydroxybutyl) phenoxyacetate (IV-4)

In a 500mL three-necked round-bottom flask, add 4-hydroxyphenylbutyric acid (5.0g, 27.7mmol), then add 80mL of dichloromethane and 50mL of methanol, stir to dissolve, under ice water bath cooling, dropwise add dichlorosulfoxide ( After the addition of 6.5mL, 89.5mmol), the reaction was stirred at room temperature, and the reaction was monitored by TLC. After the reaction was completed, 200 mL of dichloromethane and 100 mL of water were added, and the organic phase was separated. The organic phase was washed twice with saturated aqueous sodium bicarbonate solution, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a yellow oil, which was directly subjected to the next reaction;

In a 500mL three-necked round-bottom flask, weigh lithium aluminum hydride (3.0g, 79.1mmol), add 150mL of tetrahydrofuran, stir, and cool in an ice bath, dropwise add the yellow oily tetrahydrofuran solution (containing 30mL) obtained in the above step Tetrahydrofuran), after the dropwise addition, the reaction was stirred at room temperature, and the reaction was monitored by TLC. After the reaction was completed, 10% aqueous hydrochloric acid was added. After stirring for 30 minutes without bubbles, 200 mL of dichloromethane was added to separate the organic phase and anhydrous magnesium sulfate. Dry, filter, and concentrate the filtrate to dryness under reduced pressure, which is directly used in the next reaction;

Transfer the above concentrate to a 500mL single-necked round bottom flask, add 150mL of acetonitrile, stir, then add methyl bromoacetate (6.0mL, 61.2mmol) and potassium carbonate (14.0g, 102mmol), and stir the reaction at room temperature, The reaction was monitored by TLC. After the reaction was completed, 150 mL of ethyl acetate was added and filtered. The filtrate was concentrated to dryness under reduced pressure and purified by silica gel column chromatography. The eluent and volume ratio were: petroleum ether: ethyl acetate = 4: 1. 5.0 g of methyl 2- (4-hydroxybutyl) phenoxyacetate (IV-4) was obtained as a pale yellow liquid with a total yield of 75.7%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.58-1.72 (m, 4H), 2.61 (t, J = 7.38 Hz, 2H), 3.67 (t, J = 6.36 Hz, 2H), 3.83 (s, 3H) , 4.63 (s, 2H), 6.86 (d, J = 8.64 Hz, 2H), 7.12 (d, J = 8.64 Hz, 2H);

¹³ CNMR (100MHz, CDCl ₃ ) δ 27.67, 32.21, 34.71, 52.22, 62.65, 65.48, 114.52, 129.40, 135.71, 155.94, 169.66.

Step 2: Synthesis of compound (IIA-4)

In a 500mL three-necked round bottom flask, weigh compound (IIB-3) (4.50g, 6.24mmol), add 100mL of dichloromethane and 20mL of methanol, stir to dissolve, and slowly add sodium borohydride (0.24g) under ice bath cooling , 6.3mmol), after the addition was completed, the reaction was stirred at room temperature for 1-2 hours, and the reaction was monitored by TLC. After the reaction was completed, the reaction was quenched with 10% dilute acetic acid aqueous solution, followed by addition of 150mL of dichloromethane and 100mL of water, and the organic phase was separated , Dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a black solid, which was directly subjected to the next reaction;

Transfer the black solid obtained above to a 250mL single-necked round-bottom flask, add 100mL of dichloromethane, slowly stir, pass hydrogen bromide gas to saturation, continue to stir, and monitor the reaction by TLC. After the reaction is completed, concentrate to dryness under reduced pressure , Proceed directly to the next reaction;

Transfer the concentrate obtained above into a 500mL three-necked round bottom flask, weigh in compound (IV-4) (1.0g, 4.20mmol), add 100mL of dichloromethane, stir, and slowly add 50mL After the addition of ethylamine in dichloromethane (containing 3mL of triethylamine, 21.5mmol), the reaction was carried out at room temperature. The reaction was monitored by TLC. After the reaction was completed, 100mL of dichloromethane and 100mL of deionized water were added, the organic phase was separated, and the pressure was reduced. Concentrate to dryness and purify by silica gel column chromatography. The eluent and volume ratio are: dichloromethane: acetone = 100: 2 to obtain compound (IIA-4), MS (ESI): [M + H ⁺ ]: 944.66.

Step 3: Synthesis of compound (I-4)

Add the above-obtained compound (IIA-4) to a 500mL three-necked round bottom flask, add 100mL of acetonitrile and 40mL of deionized water, stir, and then add potassium carbonate (1.9g, 13.7mmol), under nitrogen, stirring at 40 ℃ The reaction was monitored by TLC. After the reaction was completed, a 10% aqueous acetic acid solution was added to adjust the pH to 2-3. Transfer the reaction solution to a 500 mL separatory funnel, add 150 mL of ethyl acetate and 100 mL of deionized water, separate the organic phase, concentrate to dryness under reduced pressure, and purify by silica gel column chromatography. The eluent and volume are: Dichloromethane: methanol = 100: 10, 0.65 g of compound (I-4) was obtained, and the total yield was 11.2%.

The characterization data of compound (I-4) is as follows:

UV Wavelength (Absorbance): 781.8 (0.094), 536.6 (0.083), 416.0 (0.096), 365.6 (0.231), 200.4 (0.110);

^{MS (ESI): [M -} ]: 928.57, [M]: 929.63, [M + H +]: 930.64;

¹ H NMR (400M Hz, CDCl ₃ ) δ-0.26 (s, 1H), 0.04 (s, 1H), 0.86 (t, J = 7.16 Hz, 3H), 0.90-0.92 (m, 1H), 0.97 (t , J = 6.84 Hz, 3H), 1.07-1.17 (m, 3H), 1.20-1.34 (m, 5H), 1.42-1.54 (m, 6H), 1.58-1.84 (m, 12H), 1.92-2.12 (m , 6H), 2.30-2.46 (m, 3H), 2.46-2.56 (m, 1H), 2.58-2.68 (m, 1H), 3.24 (s, 3H), 3.31-3.40 (m, 1H), 3.52-3.73 (m, 5H), 3.94-3.45 (m, 3H), 4.16-4.28 (m, 2H), 4.36-4.48 (m, 2H), 4.58 (s, 1H), 5.28-5.30 (m, 1H), 5.67 -5.60 (m, 1H), 6.71-6.75 (m, 1H), 6.95-7.28 (m, 2H), 8.36 (s, 1H), 8.62 (s, 1H), 8.80 (d, J = 11.24 Hz, 1H );

¹³ C NMR (100 MHz, CDCl ₃ ) δ 10.77, 10.83, 11.90, 13.60, 14.11, 19.03, 22.65, 22.76, 23.57, 24.38, 27.19, 28.21, 28.97, 29.65, 30.25, 30.54, 31.17, 31.80, 32.46, 34.78 , 40.27,48.19,49.26,53.91,55.39,62.88,64.27,65.05,69.34,69.41,72.58,94.79,99.80,101.36,113.97,114.61,128.70,129.37,129.49,131.53,132.62,133.31,135.82,137.82,138.06,137.82 , 141.36,155.56,161.2,163.93,167.71,170.38,172.55,173.70,173.80,173.89.

Example 5 Synthesis of Compound (I-5):

Step 1: Synthesis of methyl 2- (3-methoxy-4-hydroxymethyl) phenoxyacetate (IV-5)

In a 500mL three-necked round bottom flask, add 3methoxy-4-hydroxybenzaldehyde (8.1g, 53.2mmol), then add 100mL of acetonitrile, stir to dissolve, and then add methyl bromoacetate (5.2mL, 53.08mmol) With potassium carbonate (10.0g, 72.3mmol), the reaction was stirred at room temperature, and the reaction was monitored by TLC. After the reaction was completed, 100mL of ethyl acetate was added to dilute the reaction solution, filtered, and the filtrate was concentrated under reduced pressure to obtain a yellow oil, which was directly subjected to the next reaction;

Transfer the resulting yellow oil to a 500mL three-necked round bottom flask, add 100mL of dichloromethane and 30mL of methanol, stir, and slowly add sodium borohydride (0.686g, 1.9mmol) under ice bath cooling. After the addition, The reaction was stirred at room temperature, and the progress of the reaction was monitored by TLC. After the reaction was completed, the reaction was quenched with 10% diluted hydrochloric acid aqueous solution, followed by addition of 150 mL of dichloromethane and 100 mL of water, the organic phase was separated, concentrated to dryness under reduced pressure, and silica gel column chromatography Method purification, eluent and volume ratio: petroleum ether: ethyl acetate = 3: 1, to obtain 10.9g methyl 2- (3-methoxy-4-hydroxymethyl) phenoxyacetate (IV-5) , Light yellow liquid, the total yield is 90.9%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 2.98 (s, 1H), 3.70 (s, 3H), 3.77 (s, 3H), 4.48 (s, 2H), 4.59 (s, 2H), 6.66-6.86 ( m, 2H), 7.28 (s, 1H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 52.13, 55.76, 64.58, 66.39, 110.99, 114.14, 118.99, 135.67, 146.38, 149.54, 169.57.

Step 2: Synthesis of compound (IIA-5)

In a 500mL three-necked round bottom flask, weigh compound (IIB-4) (1.2g, 1.76mmol), add 100mL of dichloromethane and 20mL of methanol, stir to dissolve, and slowly add sodium borohydride (0.1g) under ice bath cooling , 2.6mmol). After the addition, the reaction was stirred at room temperature for 1-2 hours. TLC monitored the progress of the reaction. After the reaction was completed, the reaction was quenched with 10% dilute acetic acid aqueous solution, followed by the addition of 150mL of dichloromethane and 100mL of water. Phase, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a black solid, which was directly subjected to the next reaction;

Transfer the black solid obtained above to a 250mL three-necked round-bottom flask, add 100mL of dichloromethane, slowly stir, pass hydrogen bromide gas to saturation, continue stirring, and monitor the reaction by TLC. After the reaction is completed, concentrate to dryness under reduced pressure , Proceed directly to the next reaction;

Transfer the concentrate obtained above into a 500mL three-necked round-bottom flask, weigh in compound (IV-5) (2.0g, 8.84mmol), add 100mL of dichloromethane, stir, under ice bath cooling, slowly add 30mL A solution of ethylamine in dichloromethane (containing 3mL of triethylamine, 21.5mmol). After the addition, the reaction was performed at room temperature. The reaction was monitored by TLC. After the reaction, 100mL of dichloromethane and 100mL of deionized water were added to separate the organic phase. Concentrate to dryness under pressure and purify by silica gel column chromatography. The eluent and volume ratio are: dichloromethane: acetone = 100: 2, to obtain compound (IIA-5), MS (ESI): [M + H ⁺ ] : 889.56;

Step 3: Synthesis of compound (I-5)

Add the above-obtained compound (IIA-5) to a 500mL three-necked round bottom flask, add 100mL of acetonitrile and 40mL of deionized water, stir, and then add potassium carbonate (3.7g, 26.7mmol), stirring under nitrogen at 40 ° C The reaction was monitored by TLC. After the reaction was completed, a 10% aqueous acetic acid solution was added to adjust the pH to 2-3. The reaction solution was transferred to a 500mL separatory funnel, 150mL ethyl acetate and 100mL deionized water were added, the organic phase was separated, concentrated to dryness under reduced pressure, purified by silica gel column chromatography, eluent and volume ratio is : Dichloromethane: methanol = 100: 10, 0.33 g of compound (I-5) was obtained, and the total yield was 21.3%.

The characterization data of compound (I-5) is as follows:

UV Wavelength (Absorbance): 782.2 (0.141), 537. (0.122), 415.6 (0.148), 365.8 (0.348), 202.8 (0.235);

MS (ESI): [M + H ⁺ ]: 876.55;

¹ H NMR (400M Hz, CDCl ₃ ) δ-0.28 (s, 1H), 0.04 (s, 1H), 0.98 (t, J = 6.66 Hz, 3H), 1.16 (t, J = 6.90 Hz, 3H), 1.42-1.51 (m, 4H), 1.60-1.64 (m, 3H), 1.71-1.84 (m, 6H), 1.96--2.13 (m, 7H), 2.32-2.43 (m, 3H), 2.65-2.71 ( m, 1H), 3.27 (s, 3H), 3.61 (s, 3H), 3.68 (s, 3H), 3.84 (s, 3H), 4.05 (br, 1H), 4.20-4.28 (m, 2H), 4.42 -4.58 (m, 3H), 4.66 (d, J = 7.92 Hz, 1H), 4.74 (s, 2H), 5.33 (d, J = 8.12 Hz, 1H), 5.80 (t, J = 6.04 Hz, 1H) , 6.88-6.92 (m, 3H), 6.98 (d, J = 13.44 Hz, 1H), 8.42 (s, 1H), 8.68 (s, 1H), 8.31 (d, J = 23.84 Hz, 1H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 10.77, 10.94, 14.13, 22.75, 23.56 ,, 24.19, 24.36, 27.19, 28.98, 30.24, 31.27, 31.81, 32.26, 40.30, 48.19, 49.32, 51.52, 53.84, 55.38, 55.93,67.38,70.99,71.52,95.01,98.98,99.16,99.78,101.49,112.19,114.15,115.76,120.79,129.91,131.75,132.77,133.44,137.10,137.45,140.79,146.84,146.87,149.84,161.17,163.93 167.74,170.33,171.80,172.53,173.65,174.11.

Example 6 Synthesis of Compound (I-6):

Step 1: Synthesis of 3-fluoro-4-acetoxybenzyl alcohol (IV-6)

In a 500mL three-necked round bottom flask, weigh in 3-fluoro-4hydroxybenzaldehyde (5g, 35.7mmol), add 250mL of dichloromethane and acetic anhydride (5mL, 52.9mmol), stir to dissolve, and cool in an ice bath, then Triethylamine (8mL, 70.6mmol) was added, the reaction was stirred at room temperature, and the reaction was monitored by TLC. After the reaction was completed, 20mL of ethanol was added to quench the reaction and concentrated under reduced pressure to obtain a yellow oil, which was directly subjected to the next reaction;

Transfer the yellow oil to a 500mL three-necked round-bottom flask, add 160mL of dichloromethane and 40mL of methanol, and slowly add sodium borohydride (0.76g, 20mmol) under ice-cooling. After the addition is complete, stir the reaction at room temperature, The reaction was monitored by TLC. After the reaction was completed, the reaction was quenched with 10% dilute hydrochloric acid aqueous solution, followed by addition of 150 mL of dichloromethane and 100 mL of deionized water, the organic phase was separated, concentrated to dryness under reduced pressure, and purified by silica gel column chromatography. The eluent and volume ratio are: petroleum ether: ethyl acetate = 4: 1, to obtain 4.5 g of 3-fluoro-4-acetoxybenzyl alcohol (IV-6), white solid, the total yield is 68.5%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 2.30 (s, 3H), 2.99 (br, 1H), 4.53 (s, 2H), 7.03-7.12 (m, 2H), 7.10 (d, J = 10.8 Hz, 1H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 20.40, 63.69, 114.82, 122.43, 123.49, 136.91, 140.84, 152.67, 155.15, 168.80.

Step 2: Synthesis of compound (IIIA-1)

In a 500mL three-necked round bottom flask, weigh compound (IIB-1) (0.65g, 0.99mmol), add 100mL of dichloromethane and 10mL of methanol, stir, and slowly add sodium borohydride (0.50g, 13.2mmol), after the addition, the reaction was stirred at room temperature, the reaction was monitored by TLC, after the reaction was completed, the reaction was quenched with 10% dilute acetic acid aqueous solution, followed by the addition of 150mL of methylene chloride and 100mL of water, the organic phase was separated, anhydrous sodium sulfate Dry, filter, and concentrate the filtrate under reduced pressure to obtain a black solid, and proceed directly to the next reaction;

Transfer the black body obtained above into a 250mL single-necked round-bottom flask, add 150mL of dichloromethane, slowly stir, pass hydrogen bromide gas to saturation, continue stirring, TLC monitor the reaction, after the reaction is completed, concentrate to dryness under reduced pressure , Proceed directly to the next reaction;

Transfer the concentrate obtained above into a 500mL three-necked round bottom flask, weigh in compound (IV-6) (0.40g, 2.17mmol), add 50mL of dichloromethane, stir, and ice-cooled, add 30mL of triethylbenzene dropwise A dichloromethane solution of amine (containing 2mL of triethylamine, 14.34mmol). After the addition, the reaction was performed at room temperature. The reaction was monitored by TLC. After the reaction was completed, it was concentrated to dryness under reduced pressure to obtain a concentrate containing compound (IIIA-1). Used directly in the next reaction.

Step 3: Synthesis of compound (I-6)

Transfer the concentrate from the previous reaction to a 500mL three-necked round bottom flask, add 150mL of dichloromethane, stir, then add n-butylamine (3mL, 30.35mmol), continue stirring at room temperature, TLC monitor the reaction, after the reaction, It was concentrated to dryness under reduced pressure and purified by silica gel column chromatography. The eluent and volume ratio were: dichloromethane: acetone = 100: 4, 0.3 g of compound (I-6) was obtained, and the total yield was 48.3%.

The characterization data of compound (I-6) are as follows:

MS (ESI): M / Z [M + H ⁺ ]: 778.46

¹ H NMR (400MHz, CDCl ₃ ) δ-0.37 (s, 1H),-0.03 (s, 1H), 1.08-1.13 (m, 5H), 1.26-1.29 (m, 1H), 1.58-1.67 (m, 6H), 1.71-1.73 (m, 3H), 1.78-1.82 (m, 1H), 1.92-2.06 (m, 6H), 2.20 (s, 1H), 2.28-2.39 (m, 3H), 2.63-2.67 ( m, 1H), 3.24 (s, 2H), 3.58 (s, 3H), 3.66 (s, 3H), 4.04-4.091 (m, 1H), 4.14-4.23 (m, 2H), 4.43-4.48 (m, 2H), 4.51-4.63 (m, 1H), 5.15-5.22 (m, 1H), 5.29-5.32 (m, 1H), 5.71-5.78 (m, 1H), 6.94-7.09 (m, 2H), 8.38 ( s, 1H), 8.65 (s, 1H), 8.77 (s, 1H), 8.84 (s, 1H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 10.70, 10.91, 11.94, 14.06, 20.75, 22.74, 23.59, 24.18, 30.22, 31.10, 32.19, 40.06, 48.16, 49.29, 51.50, 53.81, 55.37, 70.18, 70.95, 71.51 , 94.93,99.77,101.45,114.12,115.43,117.15,124.63,129.8,131.06,132.78,133.40,137.01,137.30,140.79,143.29,149.83,152.20,161.18,163.93,167.73,170.25,170.34,172.46,173.59,174. .

Biological evaluation

Experimental animals and feeding instructions

Strain: SPF grade BALB / c nude mouse

Gender: male

Weeks of age: 28-41 days

Source: Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

License number: SCXK (Beijing) 2012-0001

Feeding conditions: Animals are housed in the IVC system of the experimental animal house, 5 animals / cage, light period 10h / 14, indoor temperature 20-26 ℃, relative humidity 40-70%, all cages, bedding and drinking water are required before use Sterilization, the feed is special sterilized feed, cages, feed and water are replaced once a week, all replacement operations are carried out on the ultra-clean workbench.

Test Example 1 Tumor inhibition rate of different doses of compound (I-1) on nude mice transplanted with NCI-H460 (human lung cancer)

NCI-H460 cells were cultured in RPMI-1640 complete culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in 5% CO ₂ , 37 ° C, saturated humidity constant temperature incubator. Cells in the logarithmic growth phase were planted in the axilla of the right forelimb of nude mice, and the number of cells inoculated was 2 × 10 ⁶ cells. Take the NCI-H460 tumor mass (generations 3-10) inoculated in the axillary of nude mice at a rapid proliferation stage, remove the necrotic tissue in the center of the tumor body, and cut the tumor mass into small tumor masses of 1mm × 1mm × 1mm under sterile conditions Use a trocar to inoculate subcutaneously in the right forelimb of nude mice. When the tumor grows to 60-120 mm ^{3 and} then start grouping according to body weight, a total of 5 groups, 8 animals in each group, are tested.

After grouping the animals, re-weigh the next morning, take a picture, and after measuring the tumor volume, use the insulin injection needle at 20mL / kg to inject the drugs of different doses in sequence through the tail vein. Except for HPPH, all other groups use semiconductor 780nm PDT (Photodynamic Therapeutic Apparatus), and irradiation treatment was performed 0.5h after administration, and irradiation was performed 24h after HPPH administration, using 665nm PDT. Animals were anesthetized with a 3.5% chloral hydrate intraperitoneal injection (0.1mL / 10g) about 5 minutes before irradiation. After anesthesia, they were fixed on a foam board with transparent tape and wrapped in tin foil to expose only the tumor site to ensure the light process The medium spot will not deviate from the tumor. The optical fiber probe is fixed with a fixed frame, the spot area is one square centimeter, the light intensity is set to 150mw / cm ² , the energy density is 150J / cm ² , the illumination time is 20min; 780nm PDT, the probe is about 7.0cm away from the tumor surface, the spot area is one square In centimeters, the light intensity is set to 100mw / cm ² and the light duration is 15min. After the animals were irradiated, they were kept in the IVC system, and the volume of the transplanted tumor was measured every 4 days from the 11th day after the light irradiation until the end of the experiment on the 31st day.

The effects of different doses of compound (I-1) on the NCI-H460 transplanted tumor volume (TV) are shown in Figure 1 and Table 1, respectively. The doses are 0.67mg / kg, 1.0mg / kg or 1.5mg / At kg, compound (I-1) can produce a good inhibitory effect on NCI-H460 transplanted tumors.

The inhibition rate of compound (I-1) at different doses on NCI-H460 transplanted tumors is shown in Table 2, where the calculation formula of inhibition rate (%):

Tumor inhibition rate (%) = [(average tumor weight of model control-average tumor weight of experimental group) / average tumor weight of experimental group] × 100%

Table 1 The effect of different doses of compound (I-1) on the volume of NCI-H460 transplanted tumor (unit: mm ³ )

Table 2 Tumor inhibition rate (%) of different doses of compound (I-1) on NCI-H460 transplanted tumor

Group	Average tumor weight / g	Tumor inhibition rate (%)
Model group	1.5901 ± 0.9172	N / A
HPPH (1.5mg / kg)	1.2366 ± 0.7939	22.23
Compound (I-1) (0.67mg / kg)	0.2679 ± 0.2146	83.15
Compound (I-1) (1.0mg / kg)	0.1422 ± 0.1159	91.06
Compound (I-1) (1.5mg / kg)	0.0954 ± 0.0958	94.01

As can be seen from the data in Table 1, after photodynamic therapy, the tumor volume of each treatment group of compound (I-1) gradually decreased, reaching a minimum value in about 11-15 days, and the HPPH group also reflected the inhibition of nude mouse transplantation. The role of the tumor, but only makes the tumor grow slower. At the same time, when the dose of compound (I-1) is 0.67mg / kg-1.5mg / kg, it shows a good dose effect. It can be seen from the data in Table 2 that when compound (I-1) is administered at a dose of 0.67 mg / kg-1.5 mg / kg, it has better inhibition of NCI-H460 transplanted tumor than HPPH at a dose of 1.5 mg / kg. The tumor growth effect, and the inhibition rate can reach more than 80%, compound (I-1) has a great advantage in inhibiting the tumor growth of NCI-H460 transplanted tumors.

The structural formula of HPPH is as follows:

Test Example 2 Pharmacokinetic experiment of compound (I-1) of the present application

NCI-H460 cells were cultured in RPMI-1640 complete culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in 5% CO ₂ , 37 ° C, saturated humidity constant temperature incubator. Cells in the logarithmic growth phase were planted in the axilla of the right forelimb of nude mice, and the number of cells inoculated was 2 × 10 ⁶ cells. Take the NCI-H460 tumor mass (generations 3-10) inoculated in the axillary of nude mice at a rapid proliferation stage, remove the necrotic tissue in the center of the tumor body, and cut the tumor mass into small tumor masses of 1mm × 1mm × 1mm under sterile conditions Use a trocar to inoculate subcutaneously in the right forelimb of nude mice. After the tumor grows to 80-150 mm ³ , combine the body weight to start grouping experiments, so that the average tumor volume of each group of mice tends to be consistent. The tumor-bearing nude mice have a tumor volume of 80-150 mm ³ , a male body weight of 21-25 g.

After inoculated with tumors, nude mice were divided into 3 groups of 10 groups. Conventional tail vein injection, cardiac puncture and blood collection, dipotassium ethylenediaminetetraacetate (EDTA-K2) as anticoagulant. Killed at 1, 24, 48, and 96 hours after administration of compound (I-1) (3.0 mg / kg) at 1, 2, 4, 8, 12 hours, and after HPPH (0.6 mg / kg) administration, respectively Dead nude mice obtained anticoagulated plasma; after tumor, skin and muscle tissue homogenate and methanol precipitation extraction, the sample was injected into a high-performance liquid system for content determination. The drug concentration-time curve of compound (I-1) in different tissues is shown in the figure As shown in 2, the drug concentration at the corresponding time point is shown in Table 3; the drug concentration-time curve of HPPH in different tissues is shown in Figure 3, and the drug concentration of each HPPH tissue is shown in Table 4.

Table 3 Time-concentration data of compound (I-1) pharmacokinetic parameters

BLOQ: It means that the drug content is extremely low, which is lower than the detection limit of high performance liquid phase.

Table 4 Time-concentration data of HPPH pharmacokinetic parameters

BLOQ: It means that the drug content is extremely low, which is lower than the detection limit of high performance liquid phase.

Table 5 Pharmacokinetic parameters of 3.0 mg / kg dose of compound (I-1) in plasma

K el	t 1/2	AUC 0-T	AUV 0-inf	AUMC 0-T	AUMC 0-inf	MRT IV	Cl	VD ss
0.453	1.5	24358	24424	30120	31055	1.3	2.05	0.156

As can be seen from the data in Table 3, at a dose concentration of 3.0 mg / kg, compound (I-1) has a better tissue distribution selectivity after about 1 hour of administration, which can reduce side effects and weaken the Damage to surrounding tissues; it is rarely detected in the skin and the drug is metabolized faster, which indicates a shorter period of avoiding light after clinical treatment.

It can be seen from the data in Table 4 that at 24 hours after injection, the content of HPPH in the tumor and skin is similar and the selectivity is poor. 48 hours after the injection, HPPH can still be detected in the skin tissue, which means that HPPH is metabolized slowly in the body, and a longer period of light avoidance is needed after photodynamic therapy in clinic.

Comparing the experimental data in Tables 3 and 4, the plasma drug concentration of compound (I-1) decreases from 7217 ng / ml at the first hour to 2600 ng / kg at the second hour, and the drug concentration decreases by about 2/3; The 1873ng / ml at 5 hours dropped to 633ng / ml at 24h, and the drug concentration also decreased by about 2/3 after 19h. Converted in the same ratio, the half-life (t _1/2 ) of HPPH is about 19 times that of compound (I-1). It can be seen that, compared with HPPH, Compound (I-1) significantly shortens the half-life of the drug, and can clinically significantly reduce the time for avoiding light after photodynamic therapy.

It can be seen from the data in Table 5 that the half-life of compound (I-1) in plasma is extremely short, 1.5 hours. At the same time, as can be seen from FIG. 2, after about 8 hours of administration, the drug concentration in each tissue Low, which indicates that compound (I-1) is rapidly metabolized in various tissues in the body.

According to the literature (Molecular Pharmaceutics, 2011, 8 (4): 1186-1197), after injection of HPPH into the tail vein of mice, photodynamic therapy needs to be performed 24 hours later, and it still takes 48h after treatment to reduce the amount of skin fluorescence to Weak (fluorescence is proportional to the HPPH content). From this, it can be judged that the compound (I-1) has a shorter half-life, and its light avoidance period (to avoid skin phototoxicity of drugs in the skin under sunlight) is relatively short, and it has a great effect in photodynamic therapy. The advantages.

in conclusion:

In Test Example 1, the compound (I-1) exemplified the dose-effect relationship of the tumor suppressive effect on nude mice xenografts between 0.67 mg / kg and 1.5 mg / kg, and proved its effectiveness; At lower doses, the tumor inhibition rate of the tumor strains in this test case was significantly higher than that of the HPPH group, which had an advantage.

In Test Example 2, after the compound (I-1) was administered at a dose of 3.0 mg / kg in the tail vein, its distribution in the skin and muscle tissue was extremely small, and it had good selectivity. It can be calculated from the data in Tables 3 and 4 that the half-life (t _1/2 ) of HPPH is about 19 times that of compound (I-1), and compound (I-1) is metabolized faster than HPPH In the clinic, it can greatly reduce the time for patients to avoid light and stay in hospital after treatment.

In summary, the compound of the present invention has advantages over HPPH: the compound of the present invention has better tumor suppressing effect and better tumor selectivity; the compound of the present invention has a shorter half-life, which is beneficial to shorten the treatment cycle in clinical application.

Claims (16)

1. A compound of formula (I) or its stereoisomer, or a pharmaceutically acceptable salt thereof:

   

   among them,

   R ¹ is hydrogen or (CH ₂ ) _m COOH;

   R ^{2 is} selected from hydrogen, hydroxyl, halogen, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy;

   R ³ and R ⁴ are each independently selected from C ₁ -C ₆ alkyl;

   m is 1, 2, 3 or 4; and

   n is 1, 2, 3 or 4.
2. The compound according to claim 1 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, which is a compound represented by formula (II) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

   

   Wherein R ² , R ³ , R ⁴ , m and n are as defined in claim 1.
3. The compound according to claim 1 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, which is a compound represented by formula (III) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

   

   Wherein, R ² , R ³ , R ⁴ and n are as defined in claim 1.
4. The compound according to any one of claims 1 to 3, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein R ³ is methyl, ethyl, propyl, butyl, or hexyl.
5. The compound according to any one of claims 1 to 3, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein R ⁴ is methyl, ethyl, propyl, butyl, or hexyl.
6. The compound according to any one of claims 1-3, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein R ^{2 is} selected from hydrogen, halogen, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy.
7. The compound according to claim 6, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein R ^{2 is} selected from hydrogen, fluorine, methyl or methoxy.
8. The compound according to claim 1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

   

   
9. A method for preparing a compound of formula (II) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof according to claim 2, the method comprising:

   

   The compound of formula (IIA) is hydrolyzed under basic conditions, and then acidified by adding acid to obtain the compound of formula (II);

   Wherein: R ² , R ³ , R ⁴ , m and n are as defined in claim 1.
10. The preparation method according to claim 9, wherein the base used in the alkaline condition is selected from alkali metal carbonate or ammonium carbonate, preferably alkali metal carbonate, more preferably sodium carbonate, potassium carbonate or lithium carbonate The acid is selected from inorganic acids or organic acids, wherein the inorganic acid is selected from hydrochloric acid with a mass fraction of 1-30% or sulfuric acid with a mass fraction of 1-50%, preferably hydrochloric acid with a mass fraction of 1-10% or content of 1-10% sulfuric acid, organic acids selected from C ₁ -C _4, preferably formic acid, acetic acid or oxalic acid.
11. A method for preparing a compound of formula (III) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof according to claim 3, the method comprising:

    

    The compound of formula (IIIA) is hydrolyzed under basic conditions to obtain the compound of formula (III),

    Wherein: R ² , R ³ , R ⁴ and n are as defined in claim 1.
12. The preparation method according to claim 11, characterized in that the base used in the alkaline conditions is selected from alkali metal carbonates, ammonium carbonates or organic amines, preferably alkali metal carbonates or C ₁ -C ₆ primary The amine is more preferably sodium carbonate, potassium carbonate, lithium carbonate, n-propylamine or butylamine.
13. A compound represented by formula (IIA), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

    

    Wherein: R ² , R ³ , R ⁴ , m and n are as defined in claim 1.
14. A compound represented by formula (IIIA) or its stereoisomer, or a pharmaceutically acceptable salt thereof:

    

    Wherein, R ² , R ³ , R ⁴ and n are as defined in claim 1.
15. A pharmaceutical composition containing an effective dose of a compound according to any one of claims 1-8 or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable Carrier, excipient or combination thereof.
16. The compound according to any one of claims 1-8 or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 15 in the preparation of a drug for photodynamic therapy of cancer In the application, wherein the cancer is preferably one or more of lung cancer, prostate cancer, esophageal cancer, melanoma, cholangiocarcinoma, head and neck cancer, bladder cancer, gastric cancer, nasopharyngeal cancer, colon cancer.

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