阅读说明：本技术 一种放射性元素修饰的穿心莲内酯衍生物及其应用 (Radioactive element modified andrographolide derivative and application thereof ) 是由 崔庆新 于 2020-06-08 设计创作，主要内容包括：本发明公开了一种放射性元素修饰的穿心莲内酯及其应用,穿心莲内酯或穿心莲内酯衍生物的19位-OH被OTs取代后,与带有放射性元素的亲核试剂反应,制备得到所述的放射性元素修饰的穿心莲内酯衍生物,放射性核素为~(18)F、~(11)C、~(13)N、~(15)O、~(68)Ga、~(82)Rb或~(131)I,所述放射性元素修饰的穿心莲内酯衍生物可作为研究肺肠相互作用的一个工具分子,直观的揭示了“肺与大肠相表里”的现象。(The invention discloses andrographolide modified by radioactive elements and application thereof, wherein 19-OH of andrographolide or andrographolide derivatives is replaced by OTs and then reacts with nucleophilic reagent with radioactive elements to prepare the andrographolide derivatives modified by the radioactive elements, and the radionuclide is 18 F、 11 C、 13 N、 15 O、 68 Ga、 82 Rb or 131 The andrographolide derivative modified by the radioactive elements can be used as a tool molecule for researching lung-intestine interaction, and the phenomenon of 'lung and large intestine in exterior and interior' is intuitively disclosed.)

1. A radioactive element modified andrographolide, which is characterized in that hydroxyl connected to 19-position carbon of andrographolide or andrographolide derivative is replaced by a radioactive element X.

2. The radioelement-modified andrographolide of claim 1, wherein the andrographolide has the general structural formula:

the andrographolide derivative can be prepared from:

the andrographolide is obtained by dehydroxylation of a hydroxyl group connected with a carbon at the 14-position and/or double bond addition of carbon at the 12-position and the 13-position;

or the andrographolide is obtained by dehydroxylation of hydroxyl connected with carbon at position 14 and/or substitution of hydroxyl connected with carbon at position 3;

or the 8-carbon and 12-carbon of the andrographolide are oxygenated, and the 13-carbon and 17-carbon of the andrographolide are hydrogenated;

or 17-carbon hydrogenation and 9-carbon dehydrogenation of the andrographolide;

wherein X is¹⁸F、¹¹C、¹³N、¹⁵O、⁶⁸Ga、⁸²Rb or¹³¹I。

3. The radioelement-modified andrographolide of claim 1, wherein the radioelement-modified andrographolide has the general structural formula:

wherein X is¹⁸F、¹¹C、¹³N、¹⁵O、⁶⁸Ga、⁸²Rb or¹³¹I。

4. The radioelement-modified andrographolide of claim 1, wherein the radioelement-modified andrographolide is prepared by:

step 1, reacting andrographolide or andrographolide derivatives containing substituents with paratoluensulfonyl chloride to prepare 19-Ts-andrographolide;

and 2, reacting the nucleophilic reagent with the radioactive element with the 19-Ts-andrographolide to prepare the andrographolide modified by the radioactive element.

5. Use of the radioelement-modified andrographolide of any one of claims 1-4 as a tracer in noninvasive drug target localization.

6. The use of claim 5, wherein the distribution concentration of the radioelement-modified andrographolide in the tissues and organs of the body is obtained by dynamic detection using PET imaging within a predetermined period of time after the radioelement-modified andrographolide is injected into the body of the experimental animal.

7. Use of the radioelement-modified andrographolide of any one of claims 1-4 in the preparation or as a PET imaging agent.

8. Use of the radioelement-modified andrographolide according to any one of claims 1-4 as a tool molecule for studying lung and large intestine interaction relations.

9. The use according to claim 8, wherein after injecting the radioelement-modified andrographolide into a test animal, dynamic organ uptake detection and/or pathological detection is performed by using PET imaging within a predetermined period of time, and the distribution of the radioelement-modified andrographolide in the lung and large intestine and/or pathological changes of the lung and large intestine in vivo are obtained to study the interaction relationship between the lung and large intestine.

10. The use of claim 8, wherein the tool molecule is a PET imaging agent.

Technical Field

The invention relates to the technical field of medicines, in particular to andrographolide modified by radioactive elements and application thereof.

Background

Andrographolide is diterpene lactone component with antibacterial effect separated from herba Andrographitis, has effects of clearing heat and detoxicating, and relieving swelling and pain, and is suitable for acute bacillary dysentery, acute gastroenteritis, upper respiratory infection, etc. At present, when the in vivo metabolic process and the action target of andrographolide are researched, blood needs to be taken for analysis, time and labor are wasted, experimental materials are consumed, the workload for detecting the concentration of drugs in blood is heavy, and a simple and effective analysis method for andrographolide is lacked.

Disclosure of Invention

The invention aims to provide an andrographolide modified by radioactive elements, aiming at the problem that andrographolide is inconvenient to detect in vivo distribution in the prior art.

Another object of the present invention is to provide the use of the andrographolide modified by radioactive elements.

The technical scheme adopted for realizing the purpose of the invention is as follows:

a radioactive element modified andrographolide is prepared by substituting radioactive element X for hydroxyl group connected to 19-position carbon of andrographolide or andrographolide derivative.

The andrographolide derivative is a derivative derived from andrographolide and has similar or same curative effect as andrographolide, and can be an andrographolide derivative which is artificially synthesized or naturally separated and has antibacterial and anti-inflammatory effects in the prior art.

In the above technical scheme, the general structural formula of andrographolide is:

the andrographolide derivative can be prepared from:

the andrographolide is obtained by dehydroxylation of a hydroxyl group connected with a carbon at the 14-position and/or double bond addition of carbon at the 12-position and the 13-position;

or the andrographolide is obtained by dehydroxylation of hydroxyl connected with carbon at position 14 and/or substitution of hydroxyl connected with carbon at position 3;

or the 8-carbon and 12-carbon of the andrographolide are oxygenated, and the 13-carbon and 17-carbon of the andrographolide are hydrogenated;

or 17-carbon hydrogenation and 9-carbon dehydrogenation of the andrographolide;

wherein X is¹⁸F、¹¹C、¹³N、¹⁵O、⁶⁸Ga、⁸²Rb or¹³¹I。

In the above technical solution, the general structural formula of the andrographolide modified by the radioactive element is:

wherein the radioactive element is¹⁸F、¹¹C、¹³N、¹⁵O、⁶⁸Ga、⁸²Rb or¹³¹I。

In the technical scheme, the andrographolide modified by the radioactive element is prepared by the following method:

step 1, reacting andrographolide or andrographolide containing substituent groups with paratoluensulfonyl chloride to prepare 19-Ts-andrographolide;

and 2, reacting the nucleophilic reagent with the radioactive element with the 19-Ts-andrographolide to prepare the andrographolide modified by the radioactive element.

In another aspect of the invention, the application of the andrographolide modified by the radioactive element as a tracer in noninvasive drug target location is also included.

In the above technical solution, after the andrographolide modified by the radioactive element is injected into the body of the experimental animal, dynamic detection is performed by PET (positron emission tomography) imaging within a predetermined time period, so as to obtain the uptake concentration of the andrographolide modified by the radioactive element in each tissue and organ in the body.

In another aspect of the invention, the application of the andrographolide modified by the radioactive element in preparing or serving as a PET imaging agent is also included.

In another aspect of the invention, the use of said radioelement-modified andrographolide as a tool molecule for studying the interaction between the lung and the large intestine is provided.

In the technical scheme, after the andrographolide modified by the radioactive element is injected into an experimental animal body, the dynamic organ uptake detection and/or pathological detection is carried out by utilizing PET imaging in a preset time period to obtain the andrographolide¹⁸Distribution of F-andrographolide in lung and large intestine in vivo and pathological changes of lung and/or large intestine to study interaction relationship between lung and large intestine.

In another aspect of the invention, the use of the radioelement-modified andrographolide as a PET imaging agent in studying the interaction relationship between the lung and the large intestine.

In the technical scheme, after the andrographolide modified by the radioactive element is injected into an experimental animal body, dynamic organ uptake detection is carried out by utilizing PET imaging in a preset time period, and the distribution of the andrographolide modified by the radioactive element in the lung and the large intestine in the body is obtained, so that the interaction relationship between the lung and the large intestine is researched.

In the technical scheme, the andrographolide modified by the radioactive element is andrographolide¹⁸F-andrographolide with the structural formula

Compared with the prior art, the invention has the beneficial effects that:

1. the andrographolide modified by the radioactive elements is a tool molecule, and has good application prospects in the aspects of research of the action mechanism of the traditional Chinese medicine and discovery of drug targets.

2. The invention carries out in-vivo drug tracing imaging on mice through MicroPET, is a novel and effective nondestructive tracing imaging analysis for andrographolide, and can visually obtain the uptake value of andrographolide modified by labeled radioactive elements in each tissue and organ.

3. By MicroPET, organ uptake detection and pathological detection of lung and large intestine tissues, the medical relationship between the lung and the large intestine can be simply, conveniently, quickly and effectively verified, and the proved pathological phenomenon of 'the exterior and interior of the lung and the large intestine' in the theory of traditional Chinese medicine is presented.

Drawings

In FIG. 1A is¹⁸Preparation of F-andrographolide roadmap, B is a radioactive thin layer detection map.

FIG. 2 is a MicroPET scan.

In FIG. 3, A is the maximum uptake value of radioactive substance in the tissue of the blank mouse group, and B is the maximum uptake value of radioactive substance in the tissue of the model mouse group.

In FIG. 4A is the proportion of bronchial damage, B is the pulmonary index, C is the proportion of intestinal damage, and D is the intestinal wall thickness.

FIG. 5 is a lung section of a blank group mouse and a model group mouse.

FIG. 6 is an intestinal section of a blank group mouse and a model group mouse.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1(¹⁸Preparation of F-andrographolide

¹⁸F labeling andrographolide to obtain positron labeled product¹⁸F-andrographolide, through marking condition optimization and quality control, reaches animal experiment standard. Each batch of [ 2 ]¹⁸F]-AG has a chemical purity of more than 98.5% and a radiochemical purity of 100%. Each batch of [ 2 ]¹⁸F]The radioactivity of the-AG is 2.33. + -. 0.28 millicurie (mCi), and the [ alpha ], [ beta ] -AG is injected into a mouse¹⁸F]The radioactivity of AG is 100 microCurie (. mu.Ci). All batches were free of insoluble impurities, sterile, pyrogen free. There was no significant change in the vital signs of the mice during the experiment.

As shown at a in figure 1 of the drawings,¹⁸the preparation method of the F-andrographolide comprises the following steps:

step 1, dissolve andrographolide (1g, 0.29mmol) in 20mL tetrahydrofuran, add p-toluenesulfonyl chloride (54mg, 0.29mmol), stir for 2h in an ice bath. After the reaction is finished, spin-drying the solvent, separating by silica gel column chromatography, and detecting by nuclear magnetic resonance to obtain a target product 19-Ts-andrographolide (19-Ts-AG), wherein the yield is 39%;

step 2, with 10mL (0.5M) of NaHCO₃Washing the QMA column by the solution, then washing by 20mL of injection water, and then drying; one branch C is taken₁₈The column, column B was washed with 10mL of methanol and then 20mL of water for injection, and blown dry. A10 mL penicillin bottle was taken, 1.5mL aminopolyether (Kryptofix2.2.2, k2.2.2) was added, and 2mg of 19-Ts-AG was weighed and dissolved in 1.2mL anhydrous acetonitrile. Produced by¹⁸F^-Under the action of positive pressure of helium gas,¹⁸F^-the aqueous solution was passed through QMA and captured by a QMA column. Rinsing QMA with k2.2.2 to remove impurities on QMA¹⁸F^-The nitrogen is introduced into the reaction tube for leaching, and the first water removal is carried out at the temperature of 110 ℃.2mL of dry acetonitrile was added and a second water removal was performed at 110 ℃. Adding the 19-Ts-andrographolide synthesized in the previous step, and reacting at 95 ℃ for 10 min. After the reaction, 8mL of ultrapure water was added, and the mixture was completely passed through a column C₁₈Column, eluting the product with 2mL acetonitrile into product vial to obtain¹⁸F-Andrographolide (b)¹⁸F-AG), the radioactivity was measured.

The eluted product was detected by TLC, which showed two peaks in TLC, with the former peak being free, as shown in B of FIG. 1¹⁸The peak of the F ion, the latter peak being the product peak. The two peaks are completely separated, and the product purity is high.

Example 2 (examination on a Living animal level¹⁸In vivo process of F-andrographolide

Normal kunming mice were selected as 8 mice, divided into two groups, one group being a blank group (healthy mice, routinely bred) and designated as Con, and the other group being a model group (mice induced to produce inflammation) and designated as Mod. Wherein: the method for establishing the mouse acute pneumonia model in the model group comprises the following steps:

the induction of an inflammation model is carried out on a mouse by a nasopharynx instillation method, Lipopolysaccharide (LPS) physiological saline solution is prepared at a dose of 168 mu g/mL, the mouse is fixed on a foam plate, a tongue is pulled out by forceps, a nostril is pinched, 60 mu L of LPS physiological saline solution (about 10 mu g LPS/mouse) is sucked, the LPS physiological saline solution is instilled into an oral cavity through the pharyngeal wall, the operation is maintained for 30s, and the model building success is obtained when all liquid is inhaled into the nasal cavity and slight tracheal Ronchia occurs. The molding time is 72 h.

Mice were anesthetized by inhalation with isoflurane and 100 μ Ci of radiation was delivered via tail vein injection channel¹⁸F-andrographolide was injected into mice in the blank and model groups.

2.1(MicroPET scanning)

Taking blank group mice and model group mice respectively, fixing the blank group mice and the model group mice on a scanning bed for MicroPET scanning, scanning for two each time, scanning for one blank group and one model group for four times, uniformly recording scanning data from 5min for each scanning, and simultaneously carrying out dynamic data acquisition within a time range of 2h, wherein the result is shown in figure 2, and the concentration of model group andrographolide in lung and intestine is obviously higher than that of a control group. Comparing the images of the two groups, the radiation intensity of the lung and the intestinal tract of the mouse is obviously higher in the model group than in the control group along with the time, and the intake value of the mouse in the model group is continuously higher than that of other tissues and organs within 2 hours. Therefore, the main organs of andrographolide, which has an anti-inflammatory effect, are lung and intestine, which lays a foundation for the follow-up research on interaction between lung and intestine by andrographolide.

The blood concentration in the organ is shown by the measurement of the uptake value of the radioactive substance in each organ, so that the result of MicroPET scanning is more intuitive, the complicated processes of taking blood according to time points, killing animals and the like are avoided, and the blood concentration change in the organ can be reflected fast and faithfully.

2.2 (uptake detection of individual organs)

The maximum uptake value (% ID/g) of radioactive substance in each organ was recorded, and the change in tissue distribution after administration was compared.

Starting from 5min, respectively calculating the highest uptake value of radioactive substances of organs and tissues of a blank group mouse and a model group mouse within a time range of 2h, scanning the lung of the mouse at different time points, recording the radiation intensity, and drawing by using Prism GraphPad 5. The results are shown in FIG. 3, in which A in FIG. 3 is the experimental result of the blank group of mice and B is the experimental result of the model group of mice. Typical biodistribution curves of the radiotracer in mice from the first (5 min) to the last PET scan (120 min) are shown. As can be seen from the control group, AG is mainly distributed in heart, lung, stomach, liver, kidney, intestine of the mice, and gradually decreases with time, indicating that the drug undergoes normal primary metabolism in these organs and tissues. The concentration of the drug in the liver is increased and then decreased, and reaches a peak value within 15min, which indicates that the liver is the main metabolic organ of AG. The concentration of the drug in muscle and brain was always at a minimum level, indicating that AG was unable to penetrate the muscle or blood brain barrier.

The lung radiation intensity of the model group was significantly higher than that of the control group, which was 2.5 times higher than that of the control group, as shown in fig. 3C. The radiation intensity of the intestine was then compared in the same way, and the model group was again higher than the control group by 1.5 times as much as the control group as shown in FIG. 3D.

After modeling, the lung elevation is most obvious compared with the change of each organ value, and is 2.5 times of that of a control group. The intestinal tract is also increased significantly, 1.5 times that of the control group. While other organs did not change significantly. Indicating that the AG has specific distribution in the lung and intestine and indicating the correlation of the AG in the lung and intestine.

2.3 (Pathology test)

After PET scanning is finished, pathological detection of tissues of the lung and the large intestine is carried out on the blank group of mice and the model group of mice respectively, and a pathological phenomenon of 'the lung and the large intestine are out and inside' is shown. The results of the measurements are shown in tables 1 to 4 below.

The ratio of bronchial damage was calculated, as shown in a in fig. 4, the ratio of bronchial damage is equal to the number of bronchi/total bronchi infiltrated by inflammatory cells × 100%), compared with the control group, the model group was significantly different from the control group (P <0.001), and the infiltration ratio of bronchitis was significantly increased, as shown in fig. 4B. This indicates that the bronchial and pulmonary functions of the model group mice are poor.

The lung index was calculated as shown in fig. 4B, which is the lung mass of the mouse/body mass of the mouse x 100%, the lung index of the model group (Mod) was significantly higher than that of the control group (Con), and the difference was statistically significant (P <0.001), indicating that the lung of the mouse is inflammatory and inflammatory exudation leads to lung weight gain.

The ratio of intestinal damage was calculated and, as shown in fig. 4C, the ratio of intestinal damage was equal to the length of intestinal mucosal damage/length of intestine × 100% in the visual field, and the morphology of the inner wall of the intestine was significantly changed in the model group (Mod) and the control group (Con). The damaged area of the inner wall of the control group is less than 20 percent, and the damaged area of the inner wall of the model group is more than 60 percent, which indicates that the intestinal tract of the mouse is influenced by inflammation and has damaged function.

The intestinal wall thickness was calculated as D in fig. 4, which is the average thickness of the intestinal wall in the field of view, and the intestinal wall thickness was significantly different between the model group (Mod) and the control group (Con) (P < 0.001). The thickness of the intestinal wall of the model group is obviously increased, which indicates that inflammation causes hyperplasia of intestinal wall glands and enteritis thickening.

The lung section and intestine section experiments are respectively carried out on a blank group mouse and a model group mouse, wherein the lung section is shown in figure 5, the control group lung tissue has clear and complete boundary, the alveolar form is uniform, the alveolar wall is not obviously exuded or hyperemic, the alveolar mesenchyme thickness is moderate, the secretion or inflammatory cells in the bronchial lumen are less, and the model group lung tissue is seriously damaged, unclear and incomplete. The number of alveoli is obviously reduced, the alveolar wall is thickened, and part of inflammatory cells obviously infiltrate the alveolar cavities. The bronchial mucosa epithelium is exfoliated, and a large amount of tissues, inflammatory cells and red blood cells are visible in the cavity.

Intestinal section experiments As shown in FIG. 6, the control group had intact intestinal mucosa and uniform folds. Has no obvious inflammatory exudation, clear running of blood vessels and no obvious ulcer. The intestinal wall is uniform, the intestinal cavity space is large, and no obvious inflammatory hyperplasia, gland hyperplasia or polyp formation is seen. The intestinal tissue of mucosal fold and intestine disappears, and congestion, edema and desquamation exist in the model group. There is an increase in mucosal inflammatory exudate, forming a polymorphous superficial ulcer. The intestinal wall thickens and the lumen narrows significantly. The gland is structurally disordered and has infiltration of various inflammatory cells.

In the above embodiments, use is made of¹¹C、¹³N、¹⁵O、⁶⁸Ga、⁸²Rb or¹³¹I substitution of radioactive elements¹⁸F, all showed similar experimental results to the above examples.

Example 3

The andrographolide is replaced by andrographolide derivant,

the andrographolide derivative can be prepared from:

the andrographolide is obtained by dehydroxylation of a hydroxyl group connected with a carbon at the 14-position and/or double bond addition of carbon at the 12-position and the 13-position;

or the andrographolide is obtained by dehydroxylation of hydroxyl connected with carbon at position 14 and/or substitution of hydroxyl connected with carbon at position 3;

or the 8-carbon and 12-carbon of the andrographolide are oxygenated, and the 13-carbon and 17-carbon of the andrographolide are hydrogenated;

or 17-carbon hydrogenation and 9-carbon dehydrogenation of the andrographolide;

and then reacting the andrographolide derivative with p-toluenesulfonyl chloride to prepare 19-Ts-andrographolide, and reacting a nucleophilic reagent with a radioactive element with the 19-Ts-andrographolide to prepare the radioactive element modified andrographolide.

Using the andrographolide modified with radioactive element obtained in this example, the method of example 2 was used to examine andrographolide at a living animal level¹⁸The in vivo process of F-andrographolide, (measurement of uptake in each organ) and pathological examination showed similar results to those in example 2.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.