CN108299184B - Preparation method of deuterated ginkgoic acid - Google Patents

Preparation method of deuterated ginkgoic acid Download PDF

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CN108299184B
CN108299184B CN201810165424.1A CN201810165424A CN108299184B CN 108299184 B CN108299184 B CN 108299184B CN 201810165424 A CN201810165424 A CN 201810165424A CN 108299184 B CN108299184 B CN 108299184B
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deuterated
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rhodium
pentamethylcyclopentadienyl
iii
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CN108299184A (en
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陈波
文瑞芝
喻目千
蒋乐
冯丽丽
邓文杰
马铭
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Hunan Normal University
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Abstract

The invention discloses a preparation method of deuterated ginkgoic acid, which comprises the following steps: 1) dissolving dichloro (pentamethylcyclopentadienyl) rhodium (III) in a mixed solution of deuterated methanol and heavy water; 2) dissolving ginkgolic acid with heavy water, adding the prepared dichloro (pentamethylcyclopentadienyl) rhodium (III) solution, stirring, adjusting the pH value to be alkaline with deuterated sodium hydroxide, and heating by microwave; 3) adjusting the solution after the reaction to be acidic by an acidic regulator, extracting by diethyl ether for multiple times, combining diethyl ether layers, drying and concentrating to obtain the deuterated ginkgoic acid. The preparation method is simple, the operation is simple and convenient, and the cost is low; the prepared deuterated ginkgoic acid standard substance has high purity and stable chemical properties, is convenient to prepare, can be used as a deuterated internal standard substance used in the production, analysis and detection of ginkgoic acid, and fills the market gap of products.

Description

Preparation method of deuterated ginkgoic acid
Technical Field
The invention relates to the field of chemical analysis and detection, and particularly relates to a preparation method of deuterated ginkgoic acid.
Background
Ginkgo biloba (Ginkgo biloba.L) belongs to the deciduous tree of Ginkgo of Ginkgoaceae. Since ancient times, there are records of ginkgo leaves as a medicine and as a food therapy, as recorded in the meridian resource: ginkgo biloba enters lung meridian and can astringe lung to relieve asthma, benefit heart and alleviate pain. In recent decades, scholars at home and abroad have conducted a great deal of research on chemical, biological and pharmacological activities, clinical application and the like of active ingredients in ginkgo, and ginkgo extracts and preparations thereof have become internationally recognized botanical drugs for treating and preventing cardiovascular and cerebrovascular diseases at present and are expected to become effective drugs for treating senile dementia related diseases such as alzheimer disease and the like. Ginkgolic acid is a main component in ginkgo, and is a derivative of 6-alkyl or 6-alkenyl salicylic acid. The ginkgoic acid is considered as a main toxic and side effect component in the ginkgo biloba extract and the preparation thereof because of biological toxicity such as sensitization, embryotoxicity, immunotoxicity, cytotoxicity and the like, the limit quantity of the ginkgoic acid is not more than 5ppm, and the content of the ginkgoic acid is a main control index in the quality standard of the ginkgo biloba preparation.
The quality control method of the medicine analysis method comprises an external standard method and an internal standard method. The external standard method is to add a standard substance into a blank sample to monitor the analysis process and result, the external standard method requires strict control on the whole operation process, otherwise, analysis errors are caused; the internal standard method is a method for adding an internal standard substance into a sample to be detected for monitoring, and the influence of a plurality of objective factors is reduced. For the selection of the internal and external standard methods, it is generally considered that: for the conditions that the precision requirement is high and the result accuracy has a great influence, the internal standard method is selected first. The internal standard method can avoid the influence of factors such as complex matrix effect, pretreatment, mass spectrum detector and the like on the determination result of the analysis method in the drug analysis, particularly can effectively control the occurrence of false negative, can effectively correct the error in the method, and obviously improves the recovery rate of the target compound and the stability of the method. When the internal standard method is adopted for analysis, in order to obtain the most rigorous and accurate analysis result, the optimal internal standard substance is the deuterated standard substance of each analysis object.
At present, the deuterated standard products sold in domestic markets are mainly used for replacing foreign products and are high in price, but the deuterated ginkgolic acid products are blank and are in short supply in the market, and the research report of related preparation methods is not found.
Disclosure of Invention
In order to overcome the defects of the prior art and make up for the shortage of deuterated ginkgolic acid products on the market, the invention provides the deuterated ginkgolic acid preparation method which has the advantages of reasonable process design, strong operability, high product purity and high yield and can realize industrial production.
In order to achieve the above object, the present invention has the following technical means.
A preparation method of deuterated ginkgoic acid is characterized by comprising the following steps:
1) dissolving dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer with a mixed solution of deuteromethanol and heavy water to obtain dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution;
2) dissolving ginkgoic acid with heavy water, then adding dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution prepared in the step 1), stirring, adjusting the pH value to 10-12 with deuterated sodium hydroxide, and heating with microwave, wherein the structural formula of the ginkgoic acid is shown as follows:
Figure GDA0002600158030000021
3) adjusting the solution reacted in the step 2) to be acidic by an acidic regulator, extracting for 2-5 times by ether, combining ether layers, drying and concentrating to obtain deuterated ginkgoic acid;
preferably, the mass fraction of the dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution prepared in the step 1) is 1.6-3.3%, and the volume ratio of the deuterated methanol to the heavy water in the mixed solution is 1: 6-10;
preferably, the microwave heating temperature in the step 2) is 160-220 ℃, and the reaction time is 18-35 min.
The usual methods for preparing isotopically labelled compounds, which are synthesized using isotopically labelled reaction substrates, have advantages in terms of the desired availability of the product, but require more time and expensive reagents than natural isotopes. It would be an advantageous method to prepare deuterium containing compounds by subjecting the target molecule to H/D exchange, which is faster, more efficient and more cost effective. This method eliminates the need for subsequent purification steps, but must introduce deuterium atoms in a manner that is efficient and predictable. As a synthetic technique, H/D exchange is a reaction process that focuses primarily on pH-dependent catalysis and metal catalysis. In practice it has been found that: a certain mass fraction of soluble rhodium complex is used as a homogeneous catalyst and is particularly suitable for H/D exchange in a compound under an alkaline condition, but if the mass fraction of the rhodium complex is too high, the catalytic performance of the rhodium complex is too strong, and ginkgolic acid is promoted to be decomposed. In the above reaction environment, the alkalinity is favorable for the deuteration reaction of ginkgolic acid, and the deuteration rate of ginkgolic acid is increased along with the increase of the amount of deuterated sodium hydroxide at the beginning, when the amount of deuterated sodium hydroxide exceeds a certain value, the deuteration reaction occurs back, therefore, the addition amount of deuterated sodium hydroxide is strictly controlled. Microwave-assisted reactions are a common method in organic synthesis and give high yields of product in a short time. The microwave assistance has the effect of promoting the H/D exchange reaction, and the principle is that the energy released by the magnetron can directly influence the salt solution, so that the C-H bond is activated, and the H/D exchange efficiency is improved. In practice, the heating temperature and time of the microwave can influence the effect of the H/D exchange reaction, and the decomposition phenomenon is easy to generate due to the fact that the heating temperature and the heating time are not strictly controlled.
Preferably, in the step 2), the volume of the added heavy water is 1.6 mL-3.3 mL and the volume of the added dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution is 0.1 mL-0.7 mL in every 2.5mg of ginkgoic acid.
Preferably, the acidic regulator in step 3) is one of deuterated hydrochloric acid, deuterated phosphoric acid, deuterated formic acid, deuterated acetic acid and deuterated trifluoroacetic acid.
The preparation method of the deuterated ginkgolic acid is characterized by comprising the following steps of:
1) weighing dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer in a stoppered test tube, adding a mixed solution, wherein the mixed solution consists of deuterated methanol and heavy water in a volume ratio of 1: 6-8, and dissolving with the assistance of ultrasonic wave to obtain dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution with the mass fraction of 1.6% -2.8%;
2) respectively adding 2.5mg of ginkgolic acid, 1.6-2.8 mL of heavy water and 0.2-0.5 mL of dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution into a microwave reaction tank, adjusting the pH value to 10-12 by using deuterated sodium hydroxide, uniformly mixing and sealing;
3) placing the reaction tank in a microwave digestion instrument, and reacting at 190-210 ℃ for 26-31 min;
4) and after the reaction is finished, transferring the reaction liquid into a separating funnel, adjusting the pH value to 3-6 by using deuterated hydrochloric acid, adding 10-23 mL of ether for extraction, repeatedly extracting for 3 times, combining ether layers, drying by using anhydrous sodium sulfate, and carrying out rotary evaporation and concentration to obtain deuterated ginkgolic acid.
Drawings
FIG. 1 is a mass spectrum of ginkgolic acids;
FIG. 2 is a mass spectrum of deuterated ginkgoic acid;
FIG. 3 is an infrared spectrum of ginkgolic acid and deuterated ginkgolic acid;
FIG. 4 is a 1H-NMR chart of ginkgolic acids;
FIG. 5 is a 1H-NMR chart of deuterated ginkgolic acid.
Detailed Description
The present invention will be further described with reference to the following detailed description, which should be construed as illustrative only, and not limiting the scope of the invention, which is to be given the full breadth of the appended claims, and all changes that can be made by those skilled in the art and which are, therefore, intended to be embraced therein.
Example 1
Experimental reagents and instruments:
ginkgolic acids (13:0, purity: HPLC. gtoreq.98%) were purchased from Dow-Couma Biotech Ltd; dichloro (pentamethylcyclopentadienyl) rhodium (iii) dimer (99% purity), deuterium oxide (D2O), and deuterated hydrochloric acid (DCl) were all available from carbofuran corporation; methanol (CH3OH, chromatographically pure, Oceanpak, Sweden); 1mol/L NaOD/D2O is prepared by reacting sodium with heavy water; diethyl ether, n-hexane, anhydrous sodium sulfate, and hydrochloric acid were purchased from Beijing national medicine reagent group; spectrally pure potassium bromide was purchased from alatin reagent (shanghai) ltd; ultrapure water (>18.2M Ω) was prepared by Milli-Q ultrapure water instruments, USA;
bruker AVANCE-500 superconducting pulse Fourier transform nuclear magnetic resonance spectrometer (Bruker, Germany), LCQ Advantage Max mass spectrometer (Fennigan corp., CA, UK), ETHOS A microwave digestion instrument (Italy, MILESTONE), electrothermal blowing dry box type 101 (Youmiming medical instruments, Inc.), reaction tank (Red Plastic works, Binshai county), ultrasonic cleaner type KQ3200E (Shanghai Huqin instruments, Inc.), electronic balance type AL204 (Shanghai Meitler-Torilduo, Inc.).
The experimental method comprises the following steps:
and (3) paper spray mass spectrum detection: in the negative ion mode, the paper spray voltage is-3.5 kV, and the capillary voltage and the capillary temperature are respectively set to be 10V and 250 ℃. The sample amount is 15 mu L, methanol is taken as eluent, and the methanol solution of the deuterated ginkgolic acid sample is analyzed; the position of H substituted by D and the deuteration rate of ginkgolic acid (13:0) are characterized by analysis techniques such as mass spectrum, Fourier transform infrared spectrum, nuclear magnetic resonance and the like.
Selection of catalytic pH ambient conditions
250.8mg of dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer is weighed into a 25mL stoppered test tube, 1mL of deuterated methanol and 8.75mL of heavy water are added for ultrasonic dissolution, and a 2.5% dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution, called Rh complex solution for short, is obtained. Respectively adding 2.5mg of ginkgolic acid, 2.8mL of heavy water, 0-40 μ L of deuterated sodium hydroxide with the molar concentration of 1.0mol/L, 0-10 μ L of deuterated hydrochloric acid with the mass fraction of 35% and 0.2mL of dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution with the mass fraction of 2.5% into a plurality of microwave reaction tanks, uniformly mixing and sealing; placing the reaction tank in a microwave digestion instrument, and reacting for 30min at the temperature of 100 ℃; after the reaction is finished, transferring the reaction liquid into a separating funnel, adjusting the pH value to 6 by using 50% deuterated hydrochloric acid, adding 10mL of ether for extraction, repeatedly extracting for 3 times, combining ether layers, drying by using anhydrous sodium sulfate, and carrying out rotary evaporation and concentration to obtain deuterated ginkgolic acid, wherein the results are shown in table 1-1.
As can be seen from Table 1-1, none of the Rh complexes have catalytic properties for the H/D exchange reaction of ginkgolic acids without the addition of acid or base. Wherein the serial numbers II and III show that the Rh complex has catalytic performance on ginkgolic acid H/D exchange by adding acid or alkali, and 40 mu L of 1mol/LNaOD/D is added2O, the mass spectrogram of ginkgolic acid shows that the main peak is [ M + 1]]Peak, i.e. D with one H atom substituted by D1The peak and the secondary peak are original substances d0Peak and D with two H atoms substituted by D2Peak, d0、d1And d2The relative percentage contents of the three components are respectively 23.50%, 54.64% and 21.86%; DCl of 10 mu L is added into the Rh complex system, the H/D exchange effect of the ginkgolic acid catalyzed by the Rh complex is not as good as that of the alkaline condition, and the mass spectrum shows that the main peak is the original substance peak D0Peak, only very small amount of d1Peak(s).
TABLE 1-1 selection of catalyst and reaction acidity and basicity
Figure GDA0002600158030000051
Note: the molecular weight is determined by paper jet mass spectrometry, and the ratio is determined by the relative percentage of mass spectrometry. d0、d1Refers to the number of ginkgolic acid H substituted by D.
Example 2
Selection of addition amount of deuterated sodium hydroxide
Respectively adding 2.5mg of ginkgolic acid, 2.8mL of heavy water, 20-80 μ L of deuterated sodium hydroxide with the molar concentration of 1.0mol/L and 0.2mL of dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution with the mass fraction of 2.5% into a plurality of microwave reaction tanks, uniformly mixing and sealing; placing the reaction tank in a microwave digestion instrument, and reacting for 30min at the temperature of 150 ℃; after the reaction is finished, transferring the reaction liquid into a separating funnel, adjusting the pH value to 6 by using 50% deuterated hydrochloric acid, adding 10mL of ether for extraction, repeatedly extracting for 3 times, combining ether layers, drying by using anhydrous sodium sulfate, and carrying out rotary evaporation and concentration to obtain deuterated ginkgolic acid, wherein the results are shown in tables 1-2.
As can be seen from tables 1-2, the deuteration ratio of ginkgolic acids increased with the increase of the addition amount of deuterated sodium hydroxide, and the content of reaction substrate d 0-ginkgolic acid was lower. When the addition amount of the deuterated sodium hydroxide is increased to 50 mu L, the H/D exchange reaction is rapidly accelerated and violent, the peaks of D0-ginkgolic acid and D1-ginkgolic acid disappear, the peak of D3-ginkgolic acid which exceeds 50 percent appears, the amount of the deuterated sodium hydroxide is continuously increased, the deuterated reaction degree is continuously increased, when 60 mu L of deuterated sodium hydroxide is added, namely the pH is 11, the D3-ginkgolic acid becomes a main peak which is 77.5 percent and contains D4-ginkgolic acid of 1/4, but when the amount of the deuterated sodium hydroxide is increased to 80 mu L, the H/D exchange reaction of the ginkgolic acid is not as good as the effect of adding 60 mu L of NaOD, and the deuterated sodium hydroxide has back reaction.
TABLE 1-2 NaOD/D2Effect of amount of O ginkgolic acid H/D reaction
Figure GDA0002600158030000061
Note: the molecular weight is determined by paper jet mass spectrometry, and the ratio is determined by the relative percentage of mass spectrometry. d0-d4The number of the ginkgolic acid H substituted by D is 0-4.
Example 3
Selection of reaction temperature
Respectively adding 2.5mg of ginkgolic acid, 2.8mL of heavy water and 60 mu L of deuterated sodium hydroxide with the molar concentration of 1.0mol/L, namely 0.2mL of dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution with the mass fraction of 2.5 percent when the pH value is 11 into a plurality of microwave reaction tanks, uniformly mixing and sealing; placing the reaction tank in a microwave digestion instrument, and reacting for 15min at the temperature of 120-200 ℃; after the reaction is finished, transferring the reaction liquid into a separating funnel, adjusting the pH value to 6 by using 50% deuterated hydrochloric acid, adding 10mL of ether for extraction, repeatedly extracting for 3 times, combining ether layers, drying by using anhydrous sodium sulfate, and carrying out rotary evaporation and concentration to obtain deuterated ginkgolic acid, wherein the results are shown in tables 1-3.
TABLE 1-3 Effect of temperature on Ginkgolic acid H/D exchange reaction
Figure GDA0002600158030000062
The molecular weight is determined by paper jet mass spectrometry, and the ratio is determined by the relative percentage of mass spectrometry. d1-d5Refers to the number of H of ginkgolic acid substituted by D.
As can be seen from tables 1-3, the high temperature favors the ginkgolic acid H/D exchange reaction, but it is considered that too high a temperature of ginkgolic acid causes more decomposition. The ginkgolic acid undergoes microwave reaction at 120 ℃ for 15min to generate H/D exchange, and D0Ginkgolic acid accounted for the main body, remaining 66.23%, minor peak d1Ginkgolic acids, with small amounts of d2-ginkgolic acids. Increase in temperature, marked increase in deuteration rate, d0Rapid decrease of peak, d1And d2The peak increases and when the temperature rises to 180 ℃, d appears3And (3) obtaining the product. Reaction substrate d when heated to 200 ℃0Ginkgolic acids are only 5%, the products are mainly d3And d4Ginkgolic acid, with a small amount of d present5Ginkgolic acid, estimated as 3 aromatic protons of the benzene ring and 2 protons of the methylene group at the benzyl position, undergoes deuteration and no significant decomposition or dimerization.
Example 4
Selection of reaction time
Respectively adding 2.5mg of ginkgolic acid, 2.8mL of heavy water and 60 mu L of deuterated sodium hydroxide with the molar concentration of 1.0mol/L, namely 0.2mL of dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution with the mass fraction of 2.5 percent when the pH value is 11 into a plurality of microwave reaction tanks, uniformly mixing and sealing; placing the reaction tank in a microwave digestion instrument, and reacting for 5-40 min at the temperature of 120 ℃; after the reaction is finished, transferring the reaction liquid into a separating funnel, adjusting the pH value to 6 by using 50% deuterated hydrochloric acid, adding 10mL of ether for extraction, repeatedly extracting for 3 times, combining ether layers, drying by using anhydrous sodium sulfate, and carrying out rotary evaporation and concentration to obtain deuterated ginkgolic acid, wherein the results are shown in tables 1-4.
TABLE 1-4 Effect of reaction time on Ginkgolic acid H/D exchange reaction
Figure GDA0002600158030000071
Note: the molecular weight is determined by paper jet mass spectrometry, and the ratio is determined by the relative percentage of mass spectrometry. d0-d4The number of H substituted by D in ginkgolic acid is 0-5.
As can be seen from tables 1-4, the microwave deuteration reaction is much faster than the conventional heating deuteration reaction, and the reaction time is greatly shortened. After 5min of reaction, a relatively large H/D exchange reaction of ginkgoic acid takes place, D1The product accounts for more than 30 percent, and the reaction substrate accounts for only about 60 percent. Properly prolonging the reaction time to facilitate the H/D exchange of ginkgolic acid, and detecting no substrate D after 30min reaction0Ginkgolic acids, the products being mainly d3Ginkgolic acids, and also about 22% of d4-ginkgolic acids. But the deuteration rate is not increased after the reaction time is prolonged, but the reaction is carried out again, and the product is formed by d0-ginkgolic acid and d1Ginkgolic acids predominate and the overall response values are almost an order of magnitude lower. This indicates that the high temperature will accelerate the H/D exchange rate of ginkgolic acids, but the reaction time will be long and the decomposition phenomenon will also occur.
Example 5
The experimental method comprises the following steps:
infrared spectrum detection: mixing a proper amount of sample and potassium bromide uniformly, tabletting, and carrying out infrared spectrum qualitative analysis; nuclear magnetic resonance analysis: samples were subjected to 1H-NMR and 13C-NMR detection using DMSO-d6 as a solvent.
Characterization of deuterated ginkgolic acid
250.8mg of dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer is weighed into a 25mL stoppered test tube, 1mL of deuterated methanol and 8.75mL of heavy water are added for ultrasonic dissolution, and a 2.5% dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution, called Rh complex solution for short, is obtained. Respectively adding 2.5mg of ginkgolic acid, 2.8mL of heavy water and 60 mu L of deuterated sodium hydroxide with the molar concentration of 1.0mol/L, namely 0.2mL of dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution with the mass fraction of 2.5 percent when the pH value is 11 into a microwave reaction tank, uniformly mixing and sealing; placing the reaction tank in a microwave digestion instrument, and reacting for 30min at the temperature of 200 ℃; after the reaction is finished, transferring the reaction liquid into a separating funnel, adjusting the pH value to 6 by using 50% deuterated hydrochloric acid, adding 10mL diethyl ether for extraction, repeatedly extracting for 3 times, combining diethyl ether layers, drying by using anhydrous sodium sulfate, and performing rotary evaporation and concentration to obtain deuterated ginkgolic acid with the purity of 93.5% and the recovery rate of 88.6%. The deuterated ginkgolic acid prepared by the method is characterized by mass spectrum (see figures 1 and 2), infrared spectrum (see figure 3) and nuclear magnetic resonance spectrum (see figures 4 and 5), respectively.
As can be seen from fig. 1 and 2: the deuterated products after the ginkgolic acid H/D exchange reaction have different molecular masses due to different numbers of D atoms containing isotopes, so that the deuterated products can be analyzed and identified by mass spectrometry. FIGS. 1 and 2 are mass spectra of ginkgolic acid and deuterated ginkgolic acid. The molecular ion peak [ M-1] in the ginkgolic acid anion mode was 319.3. The main peaks in fig. 2 are 322.5 and 323.4, indicating that the deuterated product of ginkgoic acid is mainly d 3-ginkgoic acid with a content of 78.17%, and a small amount of d 4-ginkgoic acid. The reaction substrate d 0-ginkgolic acid and other deuterated products were rarely below the detection line. The d 3-ginkgoic acid and the original substance d 0-ginkgoic acid can be completely separated in a mass spectrogram and separated from a natural isotope peak of the d 0-ginkgoic acid, so that the method is completely suitable for quantitatively analyzing the content of ginkgoic acid by using a mass spectrum internal standard.
As can be seen from fig. 3: the ginkgolic acid and deuterated ginkgolic acid have the same functional group, so that the infrared spectrograms are basically consistent, the only difference is that the positions of the C-H bond and C-D bond stretching vibration peaks are different, and the vibration peak wavelength of the C-D bond (2200 cm)-1Nearby) is lower than the C-H bond (3000 cm) of the benzene ring-1Nearby). Because the ginkgolic acid (13:0) has a relatively large molecular weight and a relatively long branched C-H group, and the H/D exchange between the benzene ring proton and the branched alkyl proton is not complete, the infrared spectra of the deuterated ginkgolic acid and the substrate common ginkgolic acid in the figure 3 are very similar and cannot be greatly changed. Therefore, nuclear magnetic resonance spectroscopy is required to further accurately characterize the structure of the deuterated product.
As can be seen from fig. 4: FIG. 4 shows ginkgolic acid GA (13)0) of1H-NMR spectrum, the broad peak at chemical shift (. delta.) of 10.196 is the-COOH proton peak of GA (13:0) as shown by the NMR spectrum of GA (13:0), and the chemical shifts of 3 benzene ring protons are as follows: h4(δ7.15，m，1H，J4,3＝8.5HZ，J4,5＝7.5HZ，)、H3(δ6.714d，1H，J＝8.5HZ)、H5(δ6.67，d，1H，J＝7.5HZ). One broad peak between delta 3 and delta 4 is a ginkgolic acid-OH proton and a moisture peak in a sample, a triplet of delta 2.592 is a No. 7 benzyl methylene proton peak connected with a benzene ring, a multiplet at delta 1.5 is a No. 8 methylene proton peak adjacent to No. 7 methylene, a large envelope peak at the position of delta 1.23 is 10 methylene proton peaks between No. 8 methylene and No. 9 methyl of a ginkgolic acid branched chain, and a triplet of delta 0.856 is a No. 9 methyl peak at the tail end of the branched chain.
As is clear from FIGS. 4 and 5, GA (13:0) methyl-CH3Protons are not H/D exchanged, so no other internal standard is added to calculate deuteration, and the integral value of other protons is used together with-CH3The integrated values of 3 protons are compared to calculate the deuteration ratio. It can thus be seen that: h4The H/D exchange reaction of (2) is not complete, and the deuteration rate is about 57 percent; h3And H5The H/D exchange of the (D) is basically complete, and the deuteration rate is 95 percent; h7The integral after the reaction was reduced by approximately 1, and thus it was found that 1 of 2 protons in the benzyl group was substituted with D, and the deuteration rate was approximately 94%. Thus, the H/D exchange of 3 protons at positions 3, 5, and 7 has been substantially complete, and more than half of the protons at position 4 have been replaced with D, consistent with the mass spectrometry results.

Claims (4)

1. A preparation method of deuterated ginkgoic acid is characterized by comprising the following steps:
1) dissolving dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer with a mixed solution of deuteromethanol and heavy water to obtain dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution;
2) dissolving ginkgoic acid with heavy water, then adding dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution prepared in the step 1), stirring, adjusting the pH value to 10-12 with deuterated sodium hydroxide, and heating with microwave, wherein the structural formula of the ginkgoic acid is shown as follows:
Figure FDA0002600158020000011
3) adjusting the solution reacted in the step 2) to be acidic by an acidic regulator, extracting for 2-5 times by ether, combining ether layers, drying and concentrating to obtain deuterated ginkgoic acid;
wherein the mass fraction of the dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution prepared in the step 1) is 1.6-3.3%, and the volume ratio of the deuterated methanol to the heavy water in the mixed solution is 1: 6-10;
wherein the microwave heating temperature in the step 2) is 160-220 ℃, and the reaction time is 18-35 min.
2. The method of claim 1, wherein in step 2), the volume of the added heavy water is 1.6 mL-3.3 mL and the volume of the solution of dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer is 0.1 mL-0.7 mL per 2.5mg of ginkgolic acid.
3. The method as claimed in claim 1, wherein the acidic modifier in step 3) is one of deuterated hydrochloric acid, deuterated phosphoric acid, deuterated formic acid, deuterated acetic acid and deuterated trifluoroacetic acid.
4. The method of any one of claims 1 to 3, comprising the steps of:
1) weighing dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer in a stoppered test tube, adding a mixed solution, wherein the mixed solution consists of deuterated methanol and heavy water in a volume ratio of 1: 6-8, and dissolving with the assistance of ultrasonic wave to obtain dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution with the mass fraction of 1.6% -2.8%;
2) respectively adding 2.5mg of ginkgolic acid, 1.6-2.8 mL of heavy water and 0.2-0.5 mL of dichloro (pentamethylcyclopentadienyl) rhodium (III) dimer solution into a microwave reaction tank, adjusting the pH value to 10-12 by using deuterated sodium hydroxide, uniformly mixing and sealing;
3) placing the reaction tank in a microwave digestion instrument, and reacting at 190-210 ℃ for 26-31 min;
4) and after the reaction is finished, transferring the reaction liquid into a separating funnel, adjusting the pH value to 3-6 by using deuterated hydrochloric acid, adding 10-23 mL of ether for extraction, repeatedly extracting for 3 times, combining ether layers, drying by using anhydrous sodium sulfate, and carrying out rotary evaporation and concentration to obtain deuterated ginkgolic acid.
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