WO2014101134A1

WO2014101134A1 - Folic acid derivative, and preparing method and application thereof

Info

Publication number: WO2014101134A1
Application number: PCT/CN2012/087891
Authority: WO
Inventors: 闫文广
Original assignee: Yan Wenguang
Priority date: 2012-12-28
Filing date: 2012-12-28
Publication date: 2014-07-03

Abstract

Provided are a folic acid derivative, a preparing method thereof, and a composition and a kit containing the folic acid derivative. The folic acid derivative is (R)-2-(2-(R)-3,4-dihydroxy-5-carbonyl-2,5-dihydrofuran)-2-hydroxyethyl-4-(6-(2-amino-4-carbonyl-3,4-dihydropteridine)-methylamino)benzoate ester. The folic acid derivative can be used for preparing a composition and a kit for diagnosing and treating tumors.

Description

Folic acid derivative, preparation method and application thereof

The present invention relates to a folic acid derivative, (R)-2-(2-(R)-3,4-dihydroxy-5-carbonyl-2,5-dihydrofuran)-2-hydroxyethyl-4- (6-(2-Amino-4-retho-3,4-dihydroacridine)methylamino)benzoate. The present invention also relates to a method of preparing the folic acid derivative, a composition and kit comprising the folic acid derivative, use of the folic acid derivative for treating and diagnosing cancer, and the folic acid derivative in preparation for diagnosis and Use in a composition for treating cancer. Background technique

Studies have shown that folate receptors on the surface of tumor cells have higher expression than normal cells, which is associated with rapid self-replication, division and proliferation of tumor cells. Folic acid has a high affinity with folate receptors, and when the γ-carboxyl group of folic acid is coupled with other small molecules, it still maintains high affinity with folate receptors, so folic acid can be used as an effective carrier for some drugs ^[2] By forming a folate complex or a folic acid derivative, the use of folate receptor mediated to localize the tumor becomes a positive path for tumor diagnosis and treatment.

Folic acid complexes or folic acid derivatives which have been experimentally studied include folic acid-mitomycin, folic acid-camptothecin, folic acid-deacetyl vinblastine monohydrazide, folic acid-epothilone and the like. These folate complexes or folic acid derivatives have excellent antitumor activity, and have high affinity with folate receptors, and can selectively act on tumor cells, and the toxicity is lower than that of the original drugs, and some are in clinical research. It shows a broad application prospect.

Design and synthesize folate complexes or folic acid derivatives with high affinity for folate receptors, explore the most suitable spacers for a certain tumor cell, find more active breakable bonds, and develop more practical value. Folic acid complexes or folic acid derivatives are of great significance and are also urgently needed in clinical practice. Summary of the invention

The inventors of the present invention have prepared a novel folic acid derivative, namely (R)-2-(2-(R)-3,4-dihydroxy-5-carbonyl-2,5-dihydrofuran)-2 -Hydroxyethyl-4-(6-(2-amino-4-reco-3,4-dihydroacridine)methanyl)benzoate having the structure of formula I below. ^^ According to the antitumor properties of furan compounds, the folic acid derivative can be used as a carrier for the diagnosis and treatment of tumors. In addition, The inventors discovered through a large number of experiments that under certain experimental conditions, by folic acid and ascorbic acid

(i.e., vitamin C) is decarboxylated by a series of process steps, and a folic acid derivative having the structure of the following formula I can be efficiently produced in a high yield, whereby the present invention has been completed.

Formula I

In another aspect, the invention provides a composition comprising a folic acid derivative of formula I of the invention. When the composition is applied to the diagnosis and treatment of tumor cells, the folic acid derivative of the present invention can exhibit high affinity with a folate receptor target site, and has an advantage of rapid cleavage in a cell. These properties have good application prospects, for example, the leaf S-derivative of the present invention and the composition comprising the folic acid derivative of the present invention can be used for tumor cell-specific diagnosis and treatment.

In another aspect, the present invention provides a process for the preparation of a folic acid derivative of the formula I according to the invention, which process comprises dissolving folic acid in a solvent containing a hydrogen bond-destroying agent and pyridine, and oxidizing in the presence of a sulfur-nitrogen compound, Decarboxylation of ascorbic acid with ascorbic acid under high temperature conditions is carried out under the catalysis of sodium thiosulfate, and the solvent is removed by, for example, vacuum distillation or vacuum drying, and optionally purified.

The present invention also provides a composition and kit comprising the leaf-S-cross derivative of the formula I of the present invention, a method for diagnosing and treating tumor cells using the folic acid derivative, composition and kit, and the folic acid-derived Use of the compositions and compositions in the manufacture of a medicament or kit for the diagnosis and treatment of tumor cells.

In a preferred embodiment, the composition or kit comprising the folic acid derivative of Formula I of the present invention further comprises folic acid, a fluorene blue, a saccharide reducing agent for use in the diagnosis of epithelial tumors. Detailed description of the invention

Folic acid has a high affinity with folate receptors, and the folic acid derivative of the present invention maintains the high affinity of this folic acid with the folate receptor, while the active hydroxyl group on the furan ring in the structure allows the dihydrofuran ring to be in the cell. Internal separation is relatively easy and can be released quickly after entering the cell to function. The folic acid derivative of the formula I of the present invention, namely (R)-2-(2-(R)-3,4-dihydroxy-5-carboxy-2,5-dihydrofuran)-2-hydroxyethyl-4 - (6-(2-Amino-4-clustyl-3,4-dihydroacridine)methylamino)benzoate, a white crystal with two of the 3, 4 positions in the dihydrofuran structure The hydroxyl group is very active, prompting the folate derivative to bind with high affinity to the folate receptor on the surface of the cell membrane, and is rapidly separated from the folate receptor after being transported into the cell via endocytosis. After separation, the folic acid derivative is hydrolyzed intracellularly and participates in the biochemical reaction of the cells. .

The hydrogen bond breakers described in the present invention are those well known and commonly used in the art and may be selected from, but not limited to, urea, dimercaptocarboxamide (DMF), sodium hydroxide, dimethyl sulfoxide (DMSO), ten. Sodium dialkyl sulfate. In a preferred embodiment, the hydrogen bond breaker is dimethylformamide or dimethyl sulfoxide.

The sulfur-nitrogen compounds described in the present invention are those well known and commonly used in the art, and may be selected from, but not limited to, glutathione (GSSG), ammonium persulfate, thioindigo, and the like. In a preferred embodiment, the sulfur nitrogen compound is glutathione or ammonium persulfate.

The high temperature decarboxylation esterification conditions referred to in the production method of the present invention have a temperature in the range of 80 to 200 degrees Celsius, preferably 85 to 150 degrees Celsius, more preferably 90 to 100 degrees Celsius.

The removal of the solvent in the production method of the present invention can be carried out by distillation under reduced pressure. In a preferred embodiment, the vacuum distillation is carried out under conditions of a vacuum of > 0.9 and a boiling point of 80 to 90 °C. Removal of the solvent can also be carried out by vacuum drying in a manner well known to those skilled in the art.

After removal of the solvent, the compound of formula I of the present invention can be obtained. After obtaining the compound of the formula I of the present invention, it is preferably purified. In a preferred embodiment, the purification is carried out by washing. The lacquering may include washing with water, for example, 1-3 times, each washing is stirred for 5 to 30 minutes after adding water, and then allowed to stand for 72 hours, and the supernatant is removed by centrifugation.

In the composition of the present invention, in addition to the folic acid derivative of the formula I of the present invention, one or more of folic acid, methylene blue and a saccharide reducing agent may be contained. The composition of the present invention can be applied to the detection of tumors, such as the detection of epithelial tissue.

In another aspect, the folic acid derivative of the formula I of the present invention and the composition comprising the folic acid derivative can also be applied due to the antiviral, antitumor and the like characteristics of the dihydrofuran ring in the folic acid derivative structure of the formula I of the present invention. Treatment of tumors, such as cervical cancer, ovarian cancer, etc.

In the detection method of the present invention, the detected tumor cells may be derived from a mammalian subject, including but not limited to humans.

In the method of treatment of the invention, the subject being treated can be a mammal, such as a human. The invention is further described by the following examples, which are not to be construed as limiting the scope of the invention. Example

EXAMPLES 1. Preparation of a compound of formula I

3 g of folic acid was added to a 100 ml solution containing 60% dimercaptoamide and lgpyridine as a solvent, and the mixture was stirred for 1 hour to be dissolved, and then lg glutathione was added thereto, stirred for 10 minutes to dissolve, and O.lg thiosulfuric acid was added thereto. The sodium was dissolved for 5 minutes, and after adding 3 g of ascorbic acid, it was dissolved at 90 ° C for 5 minutes, and evaporated under reduced pressure to have a vacuum of >0.9 and a boiling point of 83 °C. Thereafter, the mixture was stirred for 30 minutes with water, and after standing for 72 hours, the supernatant was discarded to carry out washing purification, and the washing process was repeated 3 times. The white crystals after the purification step, i.e., the folic acid derivative of the formula I of the present invention, were recovered in a 74% yield. Example 2. Preparation of a compound of formula I

A method similar to that of Example 1 was carried out except that 95 ml of dimethyl sulfoxide was used in place of dimercaptocarboxamide, and pyridine was adjusted to O.lg. The stirring time of folic acid dissolution was adjusted to 2 hours, and sodium thiosulfate was adjusted to 0.3 g, a yield of 71% was obtained for the folic acid derivative of the formula I of the present invention. Example 3. Preparation of a compound of formula I

A method similar to that of Example 1 was carried out except that 0.3 g of ammonium perchlorate was used in place of glutathione, and a yield of 75% was obtained for the folic acid derivative of the formula I of the present invention. Example 4. Preparation of a compound of formula I

A method similar to that of Example 1 was carried out, except that dimethylformamide and glutathione were replaced with 95 ml of dimethyl sulfoxide and 0.3 g of ammonium persulfate, respectively, and the stirring time of folic acid dissolution was adjusted to 2 hours, and then sulfur was added. The sodium sulphate was adjusted to 0.3 g, and a 76% yield was obtained for the folic acid derivative of the formula I of the present invention.

In the above Examples 1-4, the "percentage," is "percent by weight". Yield = synthetic target The theoretical reactant consumed by the product / the reactant actually participating in the reaction.

The invention is further illustrated by the examples and comparative examples in the application examples of the following compositions Compositions. Among them, cervical epithelial tissue was used as the examination object, and the results of cervical histopathological examination were used as reference standards. Sensitivity was used to indicate the detection rate of the detection agent containing the composition. The higher the sensitivity, the stronger the ability to find abnormal epithelial tissue; Specificity indicates the accuracy of the detection agent containing the composition, and the higher the specificity, indicating that the abnormal epithelial tissue detected has a high coincidence rate with the pathological examination result.

Specifically, in the present invention, sensitivity and specificity are defined and calculated as follows:

Sensitivity = number of true positives / (number of true positives + number of false negatives) 100%

Specificity = number of true negatives / (number of true negatives + number of false positives) X 100% of the components specified in Table 1 below, pharmaceutically acceptable grades of folic acid and folic acid derivatives (R) -2- ( 2- ( R ) -3,4-dihydroxy-5-rebel-2,5-dihydrofuran)-2-ylethyl-4-(6-(2-amino-4-indolyl-3,4-dihydroindole) The pyridinium methylamino)benzoate is dissolved at normal temperature and normal pressure, and the biodetector methylene blue is added and stirred, and the reducing agent glucose is added and stirred for 30 minutes, and then the analytically pure acetic acid is added and stirred to prepare a detecting agent composition.

The test agent composition is taken with a large cotton swab, applied to the cervical epithelial tissue, and the cotton swab is immediately taken out, and the color change of the cotton swab is immediately observed. The color of the cotton swab is yellowish brown, indicating that the epithelial tissue has no lesion; the color of the cotton swab is green or blue. Green, suggesting inflammatory lesions, condyloma acuminata (HPV virus infection) and CIN I lesions; cotton swabs are brownish green, purple-black, suggesting CINII+ lesions.

At the same time, the histopathological examination was performed on the epithelial tissue at multiple points, and then the sensitivity and specificity of the test agent were tested based on the histopathological test results. The test results are listed in Table 1. Example 6

The detecting agent composition was prepared similarly to that in Example 5. The composition of the specific components was referred to the contents of the respective components specified in Table 1 below, and the preparation method and the detection method were the same as those of Example 5, specifically, the composition was carried out. The glucose in Example 5 was replaced with a reducing agent hexose derivative. The test results are listed in Table 1. Comparative example 1

At normal temperature and pressure, add 5ml of acetic acid to 95ml of distilled water to make 5% acetic acid solution, draw with a large cotton swab, apply on the cervical epithelial tissue, wait a minute, at the scalp junction of the cervix Observe the presence or absence of acetic acid bleaching on the epithelial tissue, and observe its boundary, thickness, color, reaction time, etc. If there is a clear whitening area of acetic acid, it suggests a lesion above CIN I. Place The observed epithelial tissue was taken at the same time for multi-point biopsy to histopathological examination. The sensitivity and specificity of the 5% acetic acid solution were tested according to the histopathological test results. The test results are listed in Table 1. Comparative example 2

10 g of potassium citrate was dissolved in 100 ml of distilled water at normal temperature and normal pressure, 5 g of iodine was added, stirred and dissolved, and Lugo's iodine solution was prepared, which was taken with a large cotton swab and applied to the cervical epithelial tissue to observe the epithelium. Whether the tissue is iodine stained, the normal epithelium is reddish brown or black, and the abnormal epithelium is thick with mustard yellow or khaki, suggesting a lesion above CIN I. At the same time, all the observed epithelial tissues were subjected to histopathological examination by multi-point biopsy, and the sensitivity and specificity of Lug's iodine solution were tested by histopathological test results. The test results are listed in Table 1. Table 1

references

1. Tran T, Shatnawi A, Zheng X, et al. (2005) Enhancement of folate receptor alpha expression in tumor cells through the glucocorticoid receptor: a Promising means to improved tumor detection and targeting. Cancer Res 65: 4431-4441.

2. Leamon CP. Reddy JA. Folate-targeted chemotherapy [J]. Adv Drug Deliv Rev, 2004, 56: 1127-1141.

Claims

Claim

A folic acid derivative having the structure of formula I:

Formula I.

A method for producing a folic acid derivative according to claim 1, which comprises dissolving folic acid in a solvent containing a hydrogen bond-destroying agent and pyridine, and oxidizing with a sulfur-nitrogen compound under the catalysis of sodium thiosulfate. Ascorbic acid is decarboxylated and esterified under high temperature conditions to remove the solvent.

3. The method of claim 2 further comprising the step of purifying.

4. A method according to claim 2 or 3 wherein the solvent is removed by steaming or under vacuum.

5. A composition comprising the folic acid derivative of claim 1.

6. The composition of claim 5 further comprising one or more of folic acid, fluorenyl blue and a saccharide reducing agent.

A kit comprising the folic acid derivative of claim 1 or the composition of claim 6.

8. A method of detecting tumor cells comprising using the folic acid derivative of claim 1.

9. A method of treating a tumor comprising the use of the folic acid derivative of claim 1.

10. Use of a folic acid derivative according to claim 1 in the manufacture of a composition or kit for the diagnosis and treatment of a tumor.