CN116173243A - Synthesis and application of alkynyl substituent of di (2-ethylhexyl) phthalate - Google Patents

Abstract

The present application discloses the synthesis and use of alkynyl substitutes for di (2-ethylhexyl) phthalate. Alkynyl substitutes having the following molecular structural formula are used in tracer analysis of di (2-ethylhexyl) phthalate and its metabolites,

the technical proposal can better realize the p-phthalic acidTracer analysis of di (2-ethylhexyl) acid esters and their metabolites.

Description

Synthesis and application of alkynyl substituent of di (2-ethylhexyl) phthalate

Technical Field

The application relates to the technical field of analysis of di (2-ethylhexyl) phthalate, in particular to synthesis and application of alkynyl substitutes of di (2-ethylhexyl) phthalate.

Background

Bis (2-ethylhexyl) phthalate (DEHP) is one of the most widely used plasticizers in the world and is added to plastic articles only in amounts lower than the high polymers. A large number of experimental researches show that DEHP easily enters human beings and animals through respiration, contact and other modes, so that reproductive toxicity, developmental toxicity, immune toxicity, embryotoxicity, hepatotoxicity, neurotoxicity, carcinogenicity and the like are generated, and the DEHP is classified as an environmental pollutant which is preferentially controlled by a plurality of countries. However, the metabolic status and toxicological mechanism of DEHP in organisms are not yet fully understood, and thus intensive studies on the metabolic status of DEHP in vivo are of great importance.

The tracer technique is an important means to study the metabolism of compounds in vivo, while the design of tracers is critical to influence the tracer performance in vivo. Copper-catalyzed azido-alkynyl cycloaddition (CuAAC) is a classical click chemistry. The reaction achieves the connection between molecules by a modular reaction of azide groups and terminal alkynes. The terminal alkynyl group is small in volume and almost does not exist in organisms, so that the terminal alkynyl group is an ideal label of an in-vivo metabolism tracer. Thus, the introduction of terminal alkynyl groups on the structure of target molecules is critical for the synthesis of click chemistry based tracers.

Disclosure of Invention

In view of this, the present application provides synthesis and use of alkynyl alternatives to di (2-ethylhexyl) phthalate, potentially enabling tracer analysis of di (2-ethylhexyl) phthalate and its metabolites.

In a first aspect, the present application provides the use of an alkynyl substituent having the following molecular structural formula,

in a second aspect, the present application provides a method for tracing di (2-ethylhexyl) phthalate and metabolites thereof, comprising the steps of:

carrying out alkynyl modification on di (2-ethylhexyl) phthalate to be traced and metabolites thereof to obtain an alkynyl substituent, wherein the alkynyl substituent has a molecular structural formula corresponding to the application of claim 1;

the alkynyl substituent was substituted for the di (2-ethylhexyl) phthalate and its metabolites for tracer analysis.

Suitably, but not limitatively, the tracer analysis comprises in particular the following steps:

A. marking tissues or organs with alkynyl substitutes by adopting an azido-based marking probe to form a target marker;

B. imaging or specifically displaying the target marker;

C. the distribution and the metabolic condition of alkynyl substitutes in tissues or organs are obtained by analyzing the imaging, namely, the metabolic condition of di (2-ethylhexyl) phthalate and metabolites thereof in the tissues or organs is obtained.

Suitably, but not limitatively, the labelled probe is a fluorescent probe or a mass spectrometric probe.

Suitably, but not limitatively, the means of imaging the target marker is fluorescence imaging or liquid chromatography-mass spectrometry.

In a third aspect, the present application provides a method of synthesizing an alkynyl substituent, as follows,

i, when the structure of the alkynyl substituent is shown as the formula,

the synthesis method comprises the following steps:

step 1. 3-hydroxyheptyne (30 mmol,3.4 g) was dissolved in dichloromethane (30 mL), and carbon tetrabromide (30 mmol,10 g) and triphenylphosphine (45 mmol,11 g) were added and reacted at room temperature for 1 hour. After the reaction is completed, ethyl acetate is diluted, filtered, and the filtrate is rotationally evaporated to obtain the compound with the structural formula shown as A.

Step 2. Compound A (16 mmol,2.73 g) obtained was dissolved in tetrahydrofuran/water (30 mL, 1/1), and an aqueous formaldehyde solution (4 mL), elemental iodine (16.2 mmol,5 g), lithium iodide (16.2 mmol,2.7 g) and indium powder (4.8 mmol,4.6 g) were added in this order and reacted at room temperature for 3 hours. After the reaction was completed, ethyl acetate was added to dilute and filtered, and the filtrate was extracted three times with ethyl acetate. The combined organic layers were washed once with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was rotary evaporated to give the compound of formula B.

Step 3. Compound B (7.9 mmol,1 g) thus obtained was dissolved in methylene chloride, triethylamine (24 mmol,3.3 mL) and 4-dimethylaminopyridine (0.8 mmol,97 mg) were added thereto, and phthaloyl chloride (4 mmol,804 mg) was added dropwise under ice-bath conditions to react for 1 hour under ice-bath conditions. After the reaction, the mixture was poured into ice water, and extracted three times with methylene chloride. The combined organic layers were washed once with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was rotary evaporated to give the compound of formula C.

Step 4. Compound C (0.5 mmol,200 mg) obtained was dissolved in dimethyl sulfoxide/water (5 mL, 20/1), and 0.5M KOH (1.8 mL) was added under ice-bath conditions, and the reaction was carried out for 3 hours. After completion of the reaction, water was added for dilution, the pH was adjusted to 5 using 1M HCl, followed by extraction three times with ethyl acetate. The combined organic layers were washed with saturated brine, then dried over anhydrous sodium sulfate, and finally the filtrate was rotary evaporated to give the compound of formula D.

Ⅱ _、 When the structure of the alkynyl substituent is shown in the following formula,

the corresponding synthesis method comprises the following steps:

step 1. Reaction 1, 4-pentanediol (96 mmol,10 g) or 1, 3-pentanediol (96 mmol) or 5-hydroxypentanoic acid (96 mmol) or 3, 5-dihydroxypentanoic acid (96 mmol) or 4-hydroxybutyric acid (96 mmol) or 3-hydroxypropionic acid (96 mmol) was dissolved in dimethyl sulfoxide (100 mL) at room temperature, 2-iodoxybenzoic acid (0.24 mol,60 g) was added and reacted at room temperature under nitrogen protection for 12 hours. After completion of the reaction, the mixture was quenched by adding a saturated aqueous sodium sulfite solution (50 mL), then the pH was adjusted to 9 by adding a saturated aqueous sodium carbonate solution, and the mixture was extracted three times with methylene chloride. The combined organic layers were washed once with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrates were rotary evaporated to give the compounds of formula E, respectively.

Step 2. Compound E (12 mmol,1.2 g) obtained was dissolved in anhydrous tetrahydrofuran (30 mL), nitrogen protected, cooled to-5℃in an ice salt bath, and alkynylmagnesium bromide (0.5M, 12 mL) was added dropwise and reacted at-5℃for 0.5 hours. After the completion of the reaction, the reaction solution was poured into an ice saturated ammonium chloride aqueous solution, and extracted three times with ethyl acetate. The combined organic layers were washed once with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrates were rotary evaporated to give the compounds of formula F, respectively.

Step 3. Compound F (1.7 mmol,217 mg) obtained was dissolved in methylene chloride, and carbon tetrabromide (2.0 mmol,685 mg) and triphenylphosphine (3.4 mmol,902 mg) were added to react at room temperature for 2 hours. After the reaction is completed, ethyl acetate is added for dilution, filtration and rotary evaporation of the filtrate are carried out to obtain the compounds with the structural formulas shown as G.

Step 4. Compound G (1.4 mmol,268 mg) obtained was dissolved in tetrahydrofuran/water (3 mL, 1/1), and an aqueous formaldehyde solution (37%, 0.6 mL), elemental iodine (1.5 mmol, 390 mg), lithium iodide (1.5 mmol,209 mg) and indium powder (4.2 mmol,268 mg) were added in this order, followed by reaction at room temperature for 6 hours. After the reaction was completed, filtration was performed, and ethyl acetate was added to the filtrate to extract three times. The combined organic layers were washed once with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrates were rotary evaporated to give the compounds of formula H, respectively.

Step 5. Compound H (0.37 mmol,52 mg) thus obtained was dissolved in methylene chloride (1 mL), and triethylamine (0.74 mmol,75 mg), 4-dimethylaminopyridine (0.04 mmol,9 mg) and phthalic anhydride (0.4 mmol,61 mg) were added in this order to react at room temperature for 3 hours. After the reaction was completed, the reaction was quenched with water and extracted three times with methylene chloride. The combined organic layers were washed twice with 1M HCl, once with saturated brine, dried over anhydrous sodium sulfate and filtered, and finally the filtrates were rotary evaporated to give the compounds of formula I,

drawings

Technical solutions and other advantageous effects of the present application will be made apparent from the following detailed description of specific embodiments of the present application with reference to the accompanying drawings.

FIGS. 1a-b provide NMR diagrams of Alkyne-DEHP for example 1 of the present application.

FIGS. 2a-b provide NMR diagrams of Alkyne-MEHP in example 2 of the present application.

FIGS. 3a-b provide NMR diagrams of Alkyne-5-Oxo-MEHP for example 3 of the present application.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below in connection with the embodiments of the present application. It will be apparent that the described embodiments are only some, but not all, of the embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments herein without making any inventive effort, are intended to be within the scope of the present application.

In the description of the present application, it should be understood that the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or an implicit indication of the number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more of the described features. In the description of the present application, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.

In the description of the present application, it should be noted that, unless explicitly specified and limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be either fixedly connected, detachably connected, or integrally connected, for example; can be mechanically connected, electrically connected or can be communicated with each other; can be directly connected or indirectly connected through an intermediate medium, and can be communicated with the inside of two elements or the interaction relationship of the two elements. The specific meaning of the terms in this application will be understood by those of ordinary skill in the art as the case may be.

The following disclosure provides many different embodiments or examples for implementing different structures of the present application. In order to simplify the disclosure of the present application, the components and arrangements of specific examples are described below. Of course, they are merely examples and are not intended to limit the present application. Furthermore, the present application may repeat reference numerals and/or letters in the various examples, which are for the purpose of brevity and clarity, and which do not in themselves indicate the relationship between the various embodiments and/or arrangements discussed. In addition, the present application provides examples of various specific processes and materials, but one of ordinary skill in the art may recognize the application of other processes and/or the use of other materials.

Example 1

Synthesis of di (2-acetylenehexyl) phthalate (Alkyne-DEHP)

Reactant 3-hydroxyheptyne 1a (30 mmol,3.4 g) was dissolved in dichloromethane (30 mL), carbon tetrabromide (30 mmol,10 g) and triphenylphosphine (45 mmol,11 g) were added and reacted at room temperature for 1 hour. After completion of the reaction, it was diluted with ethyl acetate, filtered, and the filtrate was dried by rotary evaporation to give a crude product, which was purified by silica gel column chromatography (petroleum ether as eluent) to give compound 2a (16 mmol,2.73 g). The obtained compound 2a was dissolved in tetrahydrofuran/water (30 mL, 1/1), and formaldehyde water was added thereto in this orderSolution (37%, 4 mL), elemental iodine (16.2 mmol,5 g), lithium iodide (16.2 mmol,2.7 g), indium powder (4.8 mmol,4.6 g), and reacted at room temperature for 3 hours. After completion of the reaction, filtration, extraction of the filtrate with ethyl acetate three times, washing the combined organic phases once with saturated brine, drying with anhydrous sodium sulfate and filtration, finally purification of the filtrate with rotary evaporation to give the crude product using silica gel column chromatography (petroleum ether/ethyl acetate=5/1) gives compound 3a (7.9 mmol,1 g). The obtained compound 3a was dissolved in methylene chloride (10 mL), and triethylamine (24 mmol,3.3 mL) and 4-dimethylaminopyridine (0.8 mmol,97 mg) were sequentially added, followed by dropwise addition of phthaloyl chloride 4a (4 mmol,804 mg) under ice-bath conditions, and the reaction was carried out under ice-bath conditions for 1 hour. After the reaction was completed, the reaction solution was poured into ice water, extracted three times with methylene chloride, the combined organic layers were washed once with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was rotary evaporated to give a crude product which was purified by silica gel column chromatography (petroleum ether/ethyl acetate=10/1) to give the product Alkyne-DEHP (1.24 g) as a yellow oil in 40.9% yield; ¹ H NMR(400MHz,DMSO-d6)δ7.84–7.63(m,4H),4.23(d,J＝6.2Hz,4H),2.99(d,J＝2.4Hz,2H),2.80-2.76(m,2H),1.59–1.26(m,12H),0.88(t,J＝7.2Hz,6H). ¹³ c NMR (101 MHz, DMSO-d 6) delta 167.0,132.3,131.8,129.3,84.5,73.8,67.4,31.0,30.8,29.0,22.3,14.3 (nuclear magnetic resonance spectroscopy is shown in FIGS. 1 a-b).

Example twoSynthesis of mono- (2-ethynylhexyl) phthalate (Alkyne-MEHP)

Alkyne-DEHP (0.5 mmol,200 mg) was dissolved in DMSO/H ₂ KOH (0.5M, 1.8 mL) was added to O (5 mL, 20/1) in ice bath and reacted for 3 hours in ice bath. After completion of the reaction, water (10 mL) was added for dilution, pH was adjusted to 5 using 1M HCl, followed by extraction with ethyl acetate three times, the combined organic layers were washed once with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was dried by spin-drying using a rotary evaporator to give a crude product, which was purified by thin layer silica gel chromatography (petroleum ether/ethyl acetate=1/1)The product Alkyne-MEHP (25 mg) was obtained as a yellow oil in 34.9% yield; ¹ H NMR(400MHz,DMSO-d6)δ13.25(s,1H),7.82–7.73(m,1H),7.68–7.60(m,3H),4.28-4.10(m,2H),2.98(d,J＝2.4Hz,1H),2.82-2.76(m,1H),1.59–1.23(m,6H),0.88(t,J＝7.2Hz,3H). ¹³ c NMR (101 MHz, DMSO-d 6) delta 168.39,167.83,132.64,132.60,131.85,131.66,129.39,128.64,84.56,73.72,67.21,31.02,30.79,29.00,22.30,14.32 (nuclear magnetic resonance spectroscopy is shown in FIGS. 2 a-b).

Example III

Synthesis of mono- (2-acetylene-5-carbohexyl) phthalate (Alkyne-5-Oxo-MEHP)

Reactant 1, 4-pentanediol 5a (96 mmol,10 g) was dissolved in dimethylsulfoxide (100 mL) at room temperature, 2-iodoxybenzoic acid (0.24 mol,60 g) was added, and the reaction was carried out at room temperature under nitrogen protection for 12 hours. After completion of the reaction, a saturated aqueous sodium sulfite solution (50 mL) was added to quench, followed by addition of a saturated aqueous sodium carbonate solution to adjust the pH to 9, extraction with methylene chloride was performed three times, the combined organic layers were washed once with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was dried by spin-drying using a rotary evaporator to give a crude product, which was purified by silica gel column chromatography (pentane/diethyl ether=5/1) to give compound 6a (3.5 g). Compound 6a (12 mmol,1.2 g) was dissolved in anhydrous THF (30 mL), nitrogen-protected, cooled to-5℃in an ice-salt bath, and alkynylmagnesium bromide 7a (0.5M in THF,12 mL) was added dropwise and reacted at-5℃for 0.5 h. After the completion of the reaction, the reaction solution was poured into an ice saturated ammonium chloride aqueous solution, extracted three times with ethyl acetate, the combined organic layers were washed once with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was dried by spin-drying using a rotary evaporator to give a crude product, which was purified by silica gel column chromatography (petroleum ether/ethyl acetate=2/1) to give compound 8a (1.7 mmol,217 mg). Compound 8a (1.7 mmol,217 mg) was dissolved in dichloromethane, carbon tetrabromide (2.0 mmol,685 mg) and triphenylphosphine (3.4 mmol,902 mg) were added and reacted at room temperature for 2 hours. After the reaction is completedEthyl acetate was added for dilution, filtration, and the filtrate was dried by spin-drying using a rotary evaporator to give a crude product, which was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 15/1) to give compound 9a (1.4 mmol,268 mg). Compound 9a was dissolved in tetrahydrofuran/water (3 mL, 1/1), and an aqueous formaldehyde solution (37%, 0.6 mL), elemental iodine (1.5 mmol, 390 mg), lithium iodide (1.5 mmol,209 mg), and indium powder (4.2 mmol,488 mg) were added in this order, followed by reaction at room temperature for 6 hours. After completion of the reaction, filtration, extraction of the filtrate with ethyl acetate three times, washing the combined organic layers with saturated brine once, drying over anhydrous sodium sulfate and filtration, spin-drying of the filtrate on a rotary evaporator, gave a crude product, which was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5/1) to give compound 10a (0.37 mmol,52 mg). Compound 10a was dissolved in methylene chloride (1 mL), and triethylamine (0.74 mmol,75 mg), 4-dimethylaminopyridine (0.04 mmol,9 mg) and phthalic anhydride 11a (0.4 mmol,61 mg) were added in this order to react at room temperature for 3 hours. After the reaction was completed, the reaction was quenched with water, extracted three times with dichloromethane, the combined organic layers were washed twice with 1M HCl, then once with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was spin-dried on a rotary evaporator to give a crude product which was purified by thin layer silica gel chromatography (dichloromethane/methanol=10/1) to give the product Alkyne-5-Oxo-MEHP (43 mg) as a yellow oil in 36.2% yield; ¹ H NMR(400MHz,Chloroform-d)δ7.87–7.80(m,1H),7.73–7.68(m,1H),7.58–7.52(m,2H),4.45(dd,J＝10.8,5.6Hz,1H),4.22(dd,J＝10.8,7.8Hz,1H),2.95–2.85(m,1H),2.75–2.66(m,2H),2.18(s,3H),2.16(d,J＝2.8Hz,1H),2.08–1.97(m,1H),1.74–1.64(m,1H). ¹³ c NMR (101 MHz, chloride-d) delta 209.25,167.82,132.25,131.80,131.31,131.03,129.34,128.80,82.67,71.68,66.87,40.50,30.45,30.17,25.08 (nuclear magnetic resonance spectroscopy is shown in FIGS. 3 a-b).

Application example

When the molecular structural formula of the alkynyl substituent is Alkyne-DEHP shown in the following formula,

the method is characterized in that the rat is subjected to gastric lavage by Alkyne-DEHP (dissolved in corn oil), all organs and tissues are collected, the organs and tissues of the rat are respectively marked by using an azido fluorescent probe reagent or an azido mass spectrometry probe reagent, the spatial distribution condition of the DEHP in the organs of the rat is explored by a fluorescent imaging technology or the metabolite condition of the DEHP in the body is researched by a liquid chromatography-mass spectrometry technology, and therefore the understanding of the metabolism mechanism and the toxicology mechanism of the DEHP in the body is promoted.

The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.

Claims (7)

1. Use of an alkynyl substituent in tracer analysis of di (2-ethylhexyl) phthalate and its metabolites, characterized in that the alkynyl substituent has the following molecular structural formula,

2. an in vivo tracer method for di (2-ethylhexyl) phthalate and its metabolites, comprising the steps of:

carrying out alkynyl modification on di (2-ethylhexyl) phthalate to be traced and metabolites thereof to obtain an alkynyl substituent, wherein the alkynyl substituent has a molecular structural formula corresponding to the application of claim 1;

the alkynyl substituent was substituted for the di (2-ethylhexyl) phthalate and its metabolites for tracer analysis.

3. An in vivo tracer method according to claim 2, characterized in that the tracer analysis comprises in particular the following steps:

A. marking tissues or organs with alkynyl substitutes by adopting an azido-based marking probe to form a target marker;

B. imaging or specifically displaying the target marker;

C. the distribution and the metabolic condition of alkynyl substitutes in tissues or organs are obtained by analyzing the imaging, namely, the metabolic condition of di (2-ethylhexyl) phthalate and metabolites thereof in the tissues or organs is obtained.

4. An in vivo tracking method as claimed in claim 3, wherein the labelled probe is a fluorescent probe or a mass spectrometric probe.

5. An in vivo tracer method according to claim 3, wherein the imaging or specific display of the target marker is by fluorescence imaging or liquid chromatography-mass spectrometry.

6. A method for synthesizing an alkynyl substituent is characterized in that the alkynyl substituent has the structural formula shown in the specification,

the synthesis method comprises the following steps:

step 1, dissolving a reactant 3-hydroxyheptyne into dichloromethane, adding carbon tetrabromide and triphenylphosphine, and reacting at room temperature to obtain a compound with a structural formula shown as A;

step 2, dissolving the obtained compound A in tetrahydrofuran/water, sequentially adding formaldehyde aqueous solution, iodine simple substance, lithium iodide and indium powder, and reacting at room temperature to obtain a compound with a structural formula shown as B;

step 3, dissolving the obtained compound B in dichloromethane, adding triethylamine and 4-dimethylaminopyridine, dropwise adding phthaloyl chloride under the ice bath condition, and reacting under the ice bath condition to obtain a compound with a structural formula shown as C;

step 4, dissolving the obtained compound C in a mixed solution of dimethyl sulfoxide and water, and adding KOH under the ice bath condition, so as to obtain a compound with a structural formula shown as D;

7. a method for synthesizing an alkynyl substituent is characterized in that the alkynyl substituent has the structural formula shown in the specification,

the synthesis method comprises the following steps:

step 1, under the condition of room temperature, dissolving reactants 1, 4-pentanediol or 1, 3-pentanediol or 5-hydroxy valeric acid (96 or 3, 5-dihydroxyvaleric acid or 4-hydroxybutyric acid or 3-hydroxy propionic acid) in dimethyl sulfoxide (100 mL), adding 2-iodized benzoic acid, and reacting at room temperature under the condition of nitrogen protection to obtain a compound with a structural formula shown as E;

step 2, dissolving the obtained compound E in anhydrous tetrahydrofuran, cooling to-5 ℃ in an ice salt bath under the protection of nitrogen, dropwise adding alkynyl magnesium bromide, and reacting at-5 ℃ to obtain a compound with a structural formula shown as F;

step 3, dissolving the obtained compound F in dichloromethane, adding carbon tetrabromide and triphenylphosphine, and reacting at room temperature to obtain a compound with a structural formula shown as G;

step 4, dissolving the obtained compound G in tetrahydrofuran and water mixed solution, sequentially adding formaldehyde aqueous solution, iodine simple substance, lithium iodide and indium powder, and reacting at room temperature to obtain a compound with a structural formula shown as H;

step 5, dissolving the obtained compound H in dichloromethane, sequentially adding triethylamine, 4-dimethylaminopyridine and phthalic anhydride, reacting at room temperature to obtain a compound with a structural formula shown as I,

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