A preparation method of folic acid-coupled boron-containing compound

Abstract

The present invention belongs to the field of pharmaceutical synthesis and specifically provides a method for preparing a folic acid-coupled boron-containing compound. The method comprises reacting a compound of Formula I with a compound of Formula II in the presence of a carbodiimide condensing agent and a hydroxybenzotriazole compound to obtain a folic acid-coupled boron-containing compound of Formula III. The method further comprises using a sulfonic acid as a catalyst. Compared with the prior art, the technical solution of the present invention can obtain a high-purity, high-yield compound of Formula III. The reaction is clean and thorough, with no residual raw materials, and the yield can reach 91%, the purity can reach over 98%, and the formation of impurity A is effectively avoided. In addition, the technical solution of the present invention has mild reaction conditions, is easy to operate, and is more simple and efficient, making it more suitable for expanded industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical synthesis. Specifically, the present invention provides a preparation method of folic acid-coupled boron-containing compounds, which comprises using a carbodiimide condensing agent and a hydroxybenzotriazole compound as reaction reagents, and further comprises using sulfonic acid as a catalyst. Background Art

[0002] In recent years, boron neutron capture therapy (BNCT) has become an attractive treatment option for cancer, particularly malignant tumors, as it can selectively kill tumor cells using boron-containing agents while sparing normal cells. Specifically, BNCT is a novel radiotherapy method based on neutron capture and boron fission reactions. This treatment consists of two separate steps: delivery of a boron-containing agent and neutron irradiation. First, the boron-containing agent is injected externally and accumulates in tumor cells. Then, during neutron irradiation, the boron captures neutrons and undergoes nuclear fission, generating high-energy alpha ions and high-energy lithium-7. These neutrons then release gamma rays that kill tumor cells within a range of 5-9 μm. Compared to traditional chemotherapy and radiotherapy, BNCT offers several advantages: 1) the gamma ray range is narrow (5-9 μm), specifically targeting boron-containing cells without damaging surrounding tissues; 2) there is no radioresistance to hypoxic cells; and 3) it avoids the multidrug resistance often associated with chemotherapy and targeted drugs.

[0003] Although the concept of BNCT is well known, technical limitations associated with this type of treatment have slowed its development, with the primary barrier being the lack of ideal boron-containing reagents. It is well known in the art that folic acid (FA) can be internalized by cells through various mechanisms, including reduced folate carriers, proton-coupled folate transporters, and folate receptor-mediated endocytosis. Furthermore, folate receptor targeting has been successfully used in vitro to deliver many types of chemotherapeutic drugs and boron-containing nanoparticles to malignant cells. Therefore, in recent years, the use of folic acid to couple boron-containing compounds to improve delivery to cancer cells has attracted considerable attention.

[0004] However, there are few reports on the synthesis process of folic acid-coupled boron-containing compounds. <Folic Acid-Conjugated 4-Amino-Phenylboronate,a Boron-Containing CompoundDesigned for Boron Neutron Capture Therapy,is an Unexpected Agonist for HumanNeutrophils and Platelets> >, Chem Biol Drug Des 2014; 83: 532–540. discloses a method for preparing folic acid-coupled boron-containing compounds using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) / N-hydroxysuccinimide (NHS) as reaction reagents, but the yield of the reaction is only 46%. Andre Rosowsky et al. < <Synthesis and Biological Activity of Methotrexate Analogueswith Two Acid Groups and a Hydrophobic Aromatic Ring in the Side Chain> >, J. Med. Chem. 1991, 34, 574-579. discloses another method for preparing a folic acid-coupled boron-containing compound, using diphenylphosphoryl azide (DPPA) / triethylamine as reaction reagents, but the yield of the reaction is only 53%. CN101268085A also discloses a method for preparing a folic acid-coupled boron-containing compound, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / 4-dimethylaminopyridine (DMAP) as reaction reagents, but the yield of the reaction is also only 7%. In addition to the low yield, the above-disclosed prior art inevitably generates an isomeric impurity A similar to the following formula:

[0005]

[0006] The formation of this impurity is a major factor contributing to the low yields of existing preparation methods, and this impurity is difficult to remove. This makes it difficult for the folic acid-conjugated boron-containing compounds synthesized using existing methods to reach pharmaceutical grade and unsuitable for scaled-up industrial production. Therefore, it is necessary to find more rational reaction reagents and conditions that can effectively avoid the formation of this isomeric impurity and further improve the yield and purity of the product, which is of great significance for the large-scale industrial production of folic acid-conjugated boron-containing compounds. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a folic acid-coupled boron-containing compound, which can obtain a folic acid-coupled boron-containing compound with high yield and high purity and can effectively avoid the formation of isomer impurity A.

[0008] To achieve the above-mentioned objectives, the present inventors have investigated the synthesis process for coupling folic acid with a boron-containing compound of Formula III. Using a carbodiimide condensing agent and a hydroxybenzotriazole compound as reagents, and a compound of Formula I and a compound of Formula II as substrates, the reaction yield can be significantly improved. The present inventors surprisingly discovered that the addition of sulfonic acid as a catalyst to the reaction resulted in a product yield of 91% and a purity exceeding 98%.

[0009] To achieve the purpose of the present invention, the following embodiments are provided:

[0010] A method for preparing a folic acid-coupled boron-containing compound of formula III, characterized by comprising reacting a compound of formula I with a compound of formula II in the presence of a carbodiimide condensing agent and a hydroxybenzotriazole compound to produce the folic acid-coupled boron-containing compound of formula III, as shown in the following reaction formula:

[0011]

[0012] in,

[0013] When a is a single bond, a' is nothing or when a' is a single bond, a is nothing; when b is a single bond, b' is nothing or when b' is a single bond, b is nothing;

[0014] R1 is selected from -H, -CH3, -CHO or -CH=NH; R2 is selected from -H or -CHO; or the N at position 5 and the N at position 10 are connected by a methine or methylene group, that is, R1 and R2 are selected from -CH=, =CH- or -CH2-;

[0015] Z is selected from hydrogen or a protecting group, and the protecting group is preferably one of methyl, ethyl, tert-butyl or benzyl;

[0016] L is a connecting chain, preferably an alkyl group having 1 to 6 carbon atoms, a cycloalkyl group having a three-membered ring to a six-membered ring, or an aromatic group, which may be substituted or unsubstituted by a substituent. It is further preferably an aromatic group, which may be substituted or unsubstituted by a substituent; [ 10 B] is selected from carborane, dodecaborane or has the structure shown in the following formula 1:

[0017]

[0018] wherein R3 and R4 are independently hydrogen, C1-C3 alkyl or R3 and R4 are cyclized with the connected B and O atoms to form a 5-6 membered ring.

[0019] Preferably, the preparation method is characterized by comprising reacting a compound of formula I-1 with a compound of formula II-1 under the action of a carbodiimide condensing agent and a hydroxybenzotriazole compound to generate a folic acid-coupled boron-containing compound of formula III-1, as shown in the following reaction formula:

[0020]

[0021] wherein R2, R3, R4 and Z are as defined above.

[0022] Preferably, the carbodiimide condensing agent is selected from DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and a combination of two or more thereof, preferably EDCI.

[0023] Preferably, the hydroxybenzotriazole compound is selected from HOBt (1-hydroxybenzotriazole) or HOAt (1-hydroxy-7-azobenzotriazole).

[0024] Preferably, the preparation method further comprises using sulfonic acid as a catalyst.

[0025] Preferably, the sulfonic acid is selected from morpholineethanesulfonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and a combination of two or more thereof, preferably p-toluenesulfonic acid.

[0026] Preferably, the molar ratio of the carbodiimide condensing agent to the compound of formula I is (1.0-1.5):1, more preferably 1.1:1.

[0027] Preferably, the molar ratio of the hydroxybenzotriazole compound to the compound of formula I is (1.0-1.5):1, more preferably 1.1:1.

[0028] Preferably, the molar ratio of the sulfonic acid to the compound of formula I is (0.05-0.2):1, more preferably 0.1:1.

[0029] Preferably, the reaction solvent is selected from DMSO (dimethyl sulfoxide), diethylene glycol dimethyl ether, DMF (N,N-dimethylformamide), acetonitrile, acetone and a combination of two or more thereof, more preferably DMSO.

[0030] Preferably, the reaction temperature is controlled at 10-40°C, more preferably 20-25°C.

[0031] Preferably, the reaction time is controlled within 10-30 hours, more preferably within 19-25 hours.

[0032] The beneficial effects of the present invention are:

[0033] The present invention provides a method for preparing a folic acid-coupled boron-containing compound of Formula III. Compared with the prior art methods for preparing Formula III, the present invention can produce a high-purity, high-yield compound of Formula III. The reaction is clean and thorough, with no residual raw materials. The yield can reach 91%, the purity can reach over 98%, and the formation of impurity A is effectively avoided, with the amount of impurity A being controlled to below 2%. In addition, the present invention has mild reaction conditions, is easy to operate, and is more simple and efficient, making it more suitable for scaled-up industrial production. DETAILED DESCRIPTION

[0034] the term

[0035] In the present invention, "alkyl" refers to a saturated hydrocarbon group, which is a hydrocarbon group formed by removing one hydrogen atom from an alkane molecule, and is preferably an alkyl group having 1 to 6 carbon atoms, which may be substituted or unsubstituted, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, etc., but is not limited thereto.

[0036] In this specification, "aromatic group" refers to a group having aromatic properties, including monocyclic, bicyclic and polycyclic aromatic hydrocarbon groups, such as benzene, naphthalene, anthracene, and pyrene. The aromatic group may be substituted at one or more ring positions by one or more substituents, such as halogen, C1-C3 alkyl, hydroxyl, alkoxy, amino, nitro, thiol, imino, amide, carbonyl, carboxyl, aldehyde, etc., but is not limited to these.

[0037] In this specification, "carborane" refers to a carborane containing at least two carbon atoms and at least three boron atoms, or at least one carbon atom and at least five boron atoms, such as the structure shown in the following formula, but not limited thereto:

[0038]

[0039] In this specification, "dodecaborane" refers to BSH or B 12 H 12 2- , the structural formula is shown below:

[0040]

[0041] In order to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with specific embodiments. The embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0042] According to the preparation method of the present invention, the compounds shown in Table 1 below can be obtained:

[0043] Table 1

[0044]

[0045]

[0046] The specific preparation methods of the products in the above table are shown in the following examples, in which the compound of formula PL001 is used as a representative compound.

[0047] Example 1-9, Preparation of Folic Acid-Conjugated Boron-Containing Compound Formula PL001

[0048] Weigh 5.0g of the compound of Formula I into a 250ml reaction flask and dissolve it in 150ml of DMSO. Then, add 2.4g of EDCI, 1.7g of HOBt, and the catalyst. React at 25°C in the dark for 1-3 hours. Then, add 1.6g of the compound of Formula II and react at 20-25°C for 18-22 hours. After the reaction is complete, add excess methyl tert-ether to the reaction solution. The solid precipitates and is washed several times to obtain the compound of Formula III.

[0049] The catalysts used and their dosages are shown in Table 2 below:

[0050] Table 2

[0051] Example Catalyst and its dosage Output (yield) Product purity Impurity A content 1 - 5.0g(79％) 85.7％ 17.2％ 2 0.22g morpholineethanesulfonic acid 5.4g(86％) 86.1％ 10.5％ 3 0.11g methanesulfonic acid 5.3g(84％) 82.4％ 9.7％ 4 0.18g benzenesulfonic acid 5.5g(87％) 93.9％ 5.6％ 5 0.19g p-toluenesulfonic acid 5.8g(91％) 98.3％ 1.3％ 6 0.04g p-toluenesulfonic acid 4.7g(74％) 79.8％ 11.8％ 7 0.10g p-toluenesulfonic acid 5.4g(85％) 96.6％ 2.0％ 8 0.39g p-toluenesulfonic acid 5.6g(89％) 97.8％ 1.6％ 9 0.58g p-toluenesulfonic acid 4.1g(64％) 60.4％ 4.5％

[0052] Note: In Example 1, “-” means no catalyst was added.

[0053] Example 10-12, Preparation of Folic Acid-Conjugated Boron-Containing Compound Formula PL001

[0054] Weigh 5.0 g of the compound of Formula I into a 250 ml reaction flask and dissolve it in 150 ml of DMSO. Then, add a carbodiimide condensing agent, a hydroxybenzotriazole compound, and 0.19 g of p-toluenesulfonic acid. Incubate the mixture at 25°C in the dark for 1-3 hours. Add 1.6 g of the compound of Formula II and react at 20-25°C for 18-22 hours. After the reaction is complete, add excess methyl tert-ether to the reaction mixture. Solids precipitate and wash several times to obtain the compound of Formula III.

[0055] The selected carbodiimide condensing agents and their dosages, and hydroxybenzotriazole compounds and their dosages are shown in Table 3 below:

[0056] Table 3

[0057]

[0058] Comparative Example 1, Preparation of Folic Acid-Conjugated Boron-Containing Compound Formula PL001

[0059] 5.0 g of the compound of Formula I was weighed into a 250 ml reaction flask and dissolved in 150 ml of DMSO. 2.4 g of EDCI, 1.4 g of NHS, and 0.19 g of p-toluenesulfonic acid were then added sequentially. The mixture was allowed to react at 25°C in the dark for 1-3 hours. 1.6 g of the compound of Formula II was then added and allowed to react at 20-25°C for 18-22 hours. After the reaction was complete, excess methyl tert-ether was added to the reaction solution. Solids precipitated and were washed several times to obtain 3.3 g of the compound of Formula III, with a yield of 52%, a purity of 64.1%, and an impurity A content of 29.7%.

[0060] Comparative Example 2, Preparation of Folic Acid-Conjugated Boron-Containing Compound Formula PL001

[0061] 5.0 g of the compound of Formula I was weighed into a 250 ml reaction flask and dissolved in 150 ml of DMSO. 2.4 g of EDCI and 1.4 g of NHS were then added, and the mixture was allowed to react at 25°C in the dark for 1-3 hours. 1.6 g of the compound of Formula II was then added and allowed to react at 20-25°C for 18-22 hours. After the reaction was complete, excess methyl tert-ether was added to the reaction solution, and a solid precipitated. This solid was washed several times to obtain 3.7 g of the compound of Formula III, with a yield of 58%, a purity of 60.4%, and an impurity A content of 33.7%.

[0062] Example 13, Preparation of Folic Acid-Conjugated Boron-Containing Compound Formula PL007

[0063] Weigh 5.0g of the compound of Formula I into a 250ml reaction flask and dissolve it in 150ml of DMSO. Then, add 2.4g of EDCI, 1.7g of HOBt, and 0.19g of p-toluenesulfonic acid. Incubate the mixture at 25°C in the dark for 1-3 hours. Add 1.6g of the compound of Formula II and react at 20-25°C for 18-22 hours. After the reaction is complete, add excess methyl tert-ether to the reaction mixture. Solids precipitate and are washed several times to obtain 5.7g of the compound of Formula III, with a yield of 90% and a purity of 97.9%.

[0064] Example 14 Preparation of Folic Acid-Conjugated Boron-Containing Compound Formula PL012

[0065] Weigh 5.0g of the compound of Formula I into a 250ml reaction flask and dissolve it in 150ml of DMSO. Then, add 2.4g of EDCI, 1.7g of HOBt, and 0.19g of p-toluenesulfonic acid. Incubate the mixture at 25°C in the dark for 1-3h. Add 2.5g of the compound of Formula II and react at 20-25°C for 18-22h. After the reaction is complete, add excess methyl tert-ether to the reaction mixture. Solids precipitate and are washed several times to obtain 6.5g of the compound of Formula III, with a yield of 90% and a purity of 97.6%.

[0066] Example 15 Preparation of Folic Acid-Conjugated Boron-Containing Compound Formula PL058

[0067] 5.0 g of the compound of Formula I was weighed into a 250 ml reaction flask and dissolved in 150 ml of DMSO. 2.4 g of EDCI, 1.7 g of HOBt, and 0.19 g of p-toluenesulfonic acid were then added sequentially. The mixture was allowed to react at 25°C in the dark for 1-3 hours. 1.7 g of the compound of Formula II was then added and allowed to react at 20-25°C for 18-22 hours. After the reaction was complete, excess methyl tert-ether was added to the reaction solution. The solid precipitated and was washed several times to obtain 5.2 g of the compound of Formula III in an 81% yield and 90.4% purity.

[0068] Example 16 Preparation of Folic Acid-Conjugated Boron-Containing Compound Formula PL103

[0069] 5.0 g of the compound of Formula I was weighed into a 250 ml reaction flask and dissolved in 150 ml of DMSO. 2.4 g of EDCI, 1.7 g of HOBt, and 0.19 g of p-toluenesulfonic acid were then added sequentially. The mixture was allowed to react at 25°C in the dark for 1-3 hours. 2.2 g of the compound of Formula II was then added and allowed to react at 20-25°C for 18-22 hours. After completion of the reaction, excess methyl tert-ether was added to the reaction solution. Solids precipitated and were washed several times to obtain 5.1 g of the compound of Formula III, with a yield of 73% and a purity of 88.4%.

[0070] Example 17 Preparation of Folic Acid-Conjugated Boron-Containing Compound Formula PL204

[0071] Weigh 5.6g of the compound of Formula I into a 250ml reaction flask and dissolve it in 150ml of DMSO. Then, add 2.4g of EDCI, 1.7g of HOBt, and 0.19g of p-toluenesulfonic acid. Incubate the mixture at 25°C in the dark for 1-3 hours. Add 1.6g of the compound of Formula II and react at 20-25°C for 18-22 hours. After the reaction is complete, add excess methyl tert-ether to the reaction mixture. Solids precipitate and are washed several times to obtain 6.3g of the compound of Formula III, with a yield of 91% and a purity of 98.1%.

[0072] Example 18 Preparation of Folic Acid-Conjugated Boron-Containing Compound Formula PL226

[0073] 5.0 g of the compound of Formula I was weighed into a 250 ml reaction flask and dissolved in 150 ml of DMSO. 2.4 g of EDCI, 1.7 g of HOBt, and 0.19 g of p-toluenesulfonic acid were then added sequentially. The mixture was allowed to react at 25°C in the dark for 1-3 hours. 1.6 g of the compound of Formula II was then added and allowed to react at 20-25°C for 18-22 hours. After the reaction was complete, excess methyl tert-ether was added to the reaction solution. The solid precipitated and was washed several times to obtain 5.6 g of the compound of Formula III in an 88% yield and 97.8% purity.

[0074] Example 19 Preparation of Folic Acid-Conjugated Boron-Containing Compound Formula PL227

[0075] Weigh 5.0g of the compound of Formula I into a 250ml reaction flask and dissolve it in 150ml of DMSO. Then, add 2.4g of EDCI, 1.7g of HOBt, and 0.19g of p-toluenesulfonic acid. Incubate the mixture at 25°C in the dark for 1-3h. Add 1.6g of the compound of Formula II and react at 20-25°C for 18-22h. After the reaction is complete, add excess methyl tert-ether to the reaction mixture. Solids precipitate and are washed several times to obtain 5.6g of the compound of Formula III, with a yield of 89% and a purity of 97.4%.

[0076] Example 20 Preparation of Folic Acid-Conjugated Boron-Containing Compound Formula PL301

[0077] Weigh 5.1g of the compound of Formula I into a 250ml reaction flask and dissolve it in 150ml of DMSO. Then, add 2.4g of EDCI, 1.7g of HOBt, and 0.19g of p-toluenesulfonic acid. Incubate the mixture at 25°C in the dark for 1-3 hours. Add 1.6g of the compound of Formula II and react at 20-25°C for 18-22 hours. After the reaction is complete, add excess methyl tert-ether to the reaction mixture. Solids precipitate and are washed several times to obtain 5.4g of the compound of Formula III, with a yield of 85% and a purity of 98.4%.

[0078] Example 21 Preparation of Folic Acid-Conjugated Boron-Containing Compound Formula PL321

[0079] 5.6 g of the compound of Formula I was weighed into a 250 ml reaction flask and dissolved in 150 ml of DMSO. 2.4 g of EDCI, 1.7 g of HOBt, and 0.19 g of p-toluenesulfonic acid were then added sequentially. The mixture was allowed to react at 25°C in the dark for 1-3 hours. 1.6 g of the compound of Formula II was then added and allowed to react at 20-25°C for 18-22 hours. After the reaction was complete, excess methyl tert-ether was added to the reaction solution. The solid precipitated and was washed several times to obtain 6.2 g of the compound of Formula III, with a yield of 89% and a purity of 97.3%.

Claims

1. A method for preparing a folic acid-coupled boron-containing compound of formula (III), characterized in that: The process comprises reacting a compound of formula (I) with a compound of formula (II) under the action of a carbodiimide condensing agent and a hydroxybenzotriazole compound to generate a folic acid-coupled boron-containing compound of formula (III). The reaction formula is shown below: in, When a is a single bond, a' is nothing or when a' is a single bond, a is nothing; when b is a single bond, b' is nothing or when b' is a single bond, b is nothing; R1 is selected from -H, -CH3 or -CH=NH; R2 is selected from -H or -CHO; Z is selected from hydrogen or a protecting group, wherein the protecting group is one of methyl, ethyl, tert-butyl or benzyl; L is a connecting chain, which is selected from an unsubstituted alkyl group having 1 to 6 carbon atoms, a six-membered cycloalkyl group or an unsubstituted aromatic group; [ 10 B] is selected from carborane, dodecaborane or has the structure shown in the following formula (1): wherein R3 and R4 are each independently hydrogen, C1-C3 alkyl, or R3 and R4 are cyclized with the connected B and O atoms to form a 5-6 membered ring; The carbodiimide condensing agent is selected from one of DCC, DIC or EDCI; The hydroxybenzotriazole compound is selected from HOBt or HOAt; The reaction further comprises using sulfonic acid as a catalyst, and the molar ratio of the sulfonic acid to the compound of formula (I) is (0.05-0.2):

1.

2. The preparation method according to claim 1, characterized in that The process comprises reacting a compound of formula (I-1) with a compound of formula (II-1) under the action of a carbodiimide condensing agent and a hydroxybenzotriazole compound to generate a folic acid-coupled boron-containing compound of formula (III-1). The reaction formula is shown below: Wherein, the definitions of R2, R3, R4 and Z are the same as those in claim 1.

3. The preparation method according to claim 1, wherein the carbodiimide condensing agent is EDCI. The preparation method according to claim 1 , wherein the hydroxybenzotriazole compound is selected from HOBt. 5 . The preparation method according to claim 1 , wherein the molar ratio of the carbodiimide condensing agent to the compound of formula (I) is (1.0-1.5):

1.

6. The preparation method according to claim 5, wherein the molar ratio of the carbodiimide condensing agent to the compound of formula (I) is 1.1:

1.

7. The preparation method according to claim 1, wherein the molar ratio of the hydroxybenzotriazole compound to the compound of formula (I) is (1.0-1.5):

1.

8. The preparation method according to claim 7, wherein the molar ratio of the hydroxybenzotriazole compound to the compound of formula (I) is 1.1:

1.

9. preparation method according to claim 1, described sulfonic acid is selected from one among morpholineethanesulfonic acid, methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid.

10. The preparation method according to claim 9, wherein the sulfonic acid is p-toluenesulfonic acid. The preparation method according to claim 1 , wherein the molar ratio of the sulfonic acid to the compound of formula (I) is 0.1:

1.

12. The preparation method according to any one of claims 1 to 11, characterized in that: The reaction solvent is selected from DMSO, diethylene glycol dimethyl ether, DMF, acetonitrile or acetone; the reaction temperature is controlled at 10-40° C.; and the reaction time is controlled at 10-30 hours.

13. The preparation method according to claim 12, characterized in that The reaction solvent is DMSO; the reaction temperature is 20-25°C; and the reaction time is 19-25h.

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