CN112125951A

CN112125951A - Impurities in ubenimex bulk drug and preparation method thereof

Info

Publication number: CN112125951A
Application number: CN202011138447.7A
Authority: CN
Inventors: 吴子强; 刘东华; 于玉根; 雷光华; 陈齐阳; 袁文
Original assignee: SHENZHEN MAIN LUCK PHARMACEUTICALS Inc
Current assignee: SHENZHEN MAIN LUCK PHARMACEUTICALS Inc
Priority date: 2020-10-22
Filing date: 2020-10-22
Publication date: 2020-12-25

Abstract

The impurity prepared by the invention has the purity of 99 percent and can be used as a reference substance for the quality control process of ubenimex bulk drugs and preparation products thereof.

Description

Impurities in ubenimex bulk drug and preparation method thereof

Technical Field

The invention relates to the field of a medicine synthesis method, and particularly relates to an impurity generated in a ubenimex production process and a preparation method of the impurity.

Background

Ubenimex (Bestatin) is a low molecular dipeptide compound separated from the culture solution of Streptomyces olivaceus, which has the dual functions of inhibiting the action of aminopeptidase B, N and leucine aminopeptidase on the surface of tumor cells, inducing the apoptosis of tumor cells, promoting the immune function of host cells and enhancing the anti-cancer effect, and is marketed in Japan in 1987. Ubenimex is often used in combination with other chemotherapeutic drugs for the treatment of acute myelogenous leukemia, chronic myelogenous leukemia, lung squamous carcinoma, malignant melanoma, gastric tumor, etc. clinically; it can be used in combination with radiotherapy for treating tumor diseases such as nasopharyngeal carcinoma, and immunologic hypofunction caused by various factors, and is used for treating myelodysplastic syndrome and pure red blood cell aplasia. Various clinical results feed back that the medicine has obvious curative effect on enhancing the immunity of human bodies, and the ubenimex can obviously improve the survival rate of cancer patients and improve the life quality of the cancer patients. The ubenimex is chemically N- [ (2S,3R) -4-phenyl-3-amino-2-hydroxybutyryl ] -L-leucine and has the following structural formula:

the earliest method of synthesizing ubenimex was a biochemical bacterial fermentation process, disclosed by Umezawa H et al in j.anibiott.1976, 29, 97. With the increasing global demand for ubenimex, the production of ubenimex by fermentation cannot meet the demand of the drug market, but the chemical synthesis method is more beneficial to industrial production, and more chemical synthesis methods are reported. However, since ubenimex has a complex structure, peptide bonds in the structure are sensitive to acid and base, and are unstable at high temperature, some reported synthetic routes require the use of expensive actual and dangerous chemicals or have a low purity, so that only one route is available for industrial production, which is published by Journal of Antibiotics (1983, 36 (6): 695-one 699) and the reaction route is as follows:

ubenimex contains three chiral centers, so that the ubenimex raw material medicine obtained by synthesis may contain 7 optical isomer impurities, and in addition, a series of derivative impurities such as compounds shown in formulas VI and VII can be generated in the synthesis process.

In addition, the prior art CN109721505A also discloses an impurity compound in ubenimex synthesis, the structural formula is:

the ubenimex may generate so many impurities in the synthetic process, which causes difficulty in quality control of ubenimex raw material medicines and difficulty in producing high-quality ubenimex raw materials. In addition to the disclosed impurities, the impurities which have little influence on the quality of the bulk drug product exist in the process of preparing the ubenimex bulk drug. The quantity is very small, and the collection is difficult to carry out the structure confirmation, so that the difficulty is brought to the quality control of the ubenimex bulk drug. However, in order to obtain high-quality ubenimex bulk drug and further prepare safe and effective ubenimex preparations, it is necessary to confirm the structure of the impurity and synthesize an impurity compound with higher purity for the quality control process of ubenimex bulk drug.

Disclosure of Invention

The invention provides impurities in ubenimex bulk drug, which is characterized in that the structural formula of the impurities is shown as formula I:

in the production process of ubenimex, the generated target impurities are found to affect the quality of ubenimex, but because the generated amount is too small, a sufficient amount of samples meeting the requirement of carrying out structure confirmation cannot be collected.

The present invention analyzes the molecular weight of the impurity obtained by LC-MS test, and presumes that the impurity may be impurity I generated by the presence of (2S,3R) -4-phenyl-3-amino-2-hydroxybutyric acid having unprotected amino group in the starting material (2S,3R) -4-phenyl-3-benzyloxycarbonylamino-2-hydroxybutyric acid and the following side reaction:

however, because the reaction has more active sites, the reaction products of the target impurities directly prepared by the method are too many, and the purity and the yield of the products cannot meet the requirements. Therefore, the inventors have selected three different amino protecting groups including Cbz (benzyloxycarbonyl), Boc (t-butyloxycarbonyl), Fmoc (fluorenylmethyloxycarbonyl) based on the presumed structure of the compound of formula i and prepared it according to the following three synthetic routes.

Route one:

and a second route:

and a third route:

however, experiments show that only the first route (adopting the carbobenzoxy protecting group) can prepare the target compound, and the second route and the third route can not prepare the impurity I product with qualified purity.

Therefore, the preparation method of the impurities in the ubenimex bulk drug shown in the formula I is characterized by being prepared according to the following synthetic route:

the method specifically comprises the following steps:

step 1): reacting the compound shown in the formula II with benzyl chloroformate under the action of an alkali reagent to obtain a compound shown in the formula III, namely N- [ (2S,3R) -3-benzyloxyamido-2-hydroxy-4-phenylbutyryl ] -L-leucine;

step 2): carrying out condensation reaction on the compound of the formula III under the action of a condensing agent to obtain a condensation compound of a formula IV;

step 3): removing a protecting group from the condensation compound under the condition of hydrogen/palladium carbon to obtain a crude product of the compound shown in the formula I;

step 4): dissolving the crude product of the compound shown in the formula I in a dilute hydrochloric acid solution, adjusting the pH value to 5-6 by using ammonia water, stirring for crystallization, filtering and drying.

Further optimized, step 1) is that the compound of formula II, namely N- [ (2S,3R) -3-amino-2-hydroxy-4-phenylbutyryl]The molar ratio of the L-leucine (ubenimex) to the amino protective agent benzyl chloroformate is 1: 1-3, preferably 1:2, and the reaction temperature is 0-10 ℃, preferably 0-4 ℃. The alkali reagent is NaHCO₃、Na₂CO₃Or K₂CO₃。

The condensing agent for the condensation reaction of step 2) may be generally selected from HOBt/DIC (1-hydroxybenzotriazole/N, N-diisopropylcarbodiimide), HOBt/DCC (1, 3-dicyclohexylcarbodiimide)/triethylamine, PyBOP (benzotriazol-1-yl-oxytrialkylphosphonium hexafluorophosphate)/HOBt/DIPEA (N, N-diisopropylethylamine), TBTU (O-benzotriazol-N, N, N ', N' -tetramethyluronium tetrafluoroborate)/HOBt/DIPEA, HBTU (2- (1H-benzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate)/HOBt/DIPEA, HOSU/DCC/Na₂CO₃(N-hydroxysuccinimide/N, N-dicyclohexylcarbodiimide/sodium carbonate), but only HOSU/DCC/Na was found by experiment₂CO₃Can produce a product in a higher yield, so step 2) the condensing agent selects HOSU/DCC/Na₂CO₃. The reaction temperature in the step is 10-30 ℃.

Step 3), the deprotection group is hydrogenated by adopting palladium-carbon, wherein the content of the palladium-carbon is 5-10%, and 5% is preferred; the dosage of the palladium-carbon is 5 to 25 percent of the weight of the condensate shown in the formula IV, and the optimal dosage is 20 percent; the hydrogen pressure is 0.5 to 2.0MPa, preferably 1.0 to 1.5 MPa.

The crystallization principle of the step 4) is that the structure of the compound of the formula I contains amino and carboxyl simultaneously, the compound has the chemical properties similar to amino acid and has isoelectric points, and the compound of the formula I is purified by an isoelectric point precipitation method by utilizing the characteristic of minimum solubility at the isoelectric points. The compound of formula I is dissolved by dilute hydrochloric acid, then the pH value is adjusted to be near the isoelectric point by ammonia water, the product can be separated out, and impurities are removed by filtration. The concentration of the dilute hydrochloric acid is 1-3 mol/L, and the preferable concentration is 1 mol/L. The compound shown in the formula I is most precipitated when the pH value is 5-6 by slowly dripping ammonia water to adjust the pH value, so that the pH value is adjusted to 5-6 when the compound shown in the formula I is purified and crystallized, but the pH value has little influence on the purity of the product. When the crystallization temperature is 0-10 ℃, the purity of the product is more than 99%, and the highest yield can be achieved at 0-5 ℃. The influence of pH and crystallization temperature on product yield and purity is examined in the following table.

Influence of pH on yield and purity

pH	4.0	4.5	5	5.2	5.5	6.0	7.0	8.0
									Yield of	3.1％	12.8％	68.4％	72.3％	85.9％	60.7％	46.9％	2.3％

Influence of crystallization temperature on yield and purity

Temperature of	10℃	5℃	0℃	-5℃	-10℃
						Yield of	61.5％	83.7％	85.9％	87.2％	88.1％
Purity of	99.05％	99.08％	99.06％	97.6％	96.5％

According to the invention, the ubenimex bulk drug and the preparation process thereof are analyzed, the structure of the target impurity which is found to affect the quality of the ubenimex bulk drug is predicted, a proper method is selected to prepare the compound shown in the formula I, and the compound shown in the formula I is confirmed to be the target impurity through liquid chromatography and mass spectrometry. The invention also provides a synthesis method of the compound shown in the formula I, the compound shown in the formula I with the purity of more than 99% is prepared by taking ubenimex as a raw material through a method of protecting group application, condensation and protecting group removal, and the compound can be used as a reference substance for quality control and related substance determination of ubenimex bulk drugs or preparations.

The invention is further described with reference to the following examples of specific embodiments and the accompanying drawings.

Drawings

FIG. 1 shows the liquid chromatogram of ubenimex bulk drug

FIG. 2 liquid chromatogram of a compound of formula I

FIG. 3 shows the mass spectrum of a compound of formula I

Detailed Description

Example 1 Synthesis of ubenimex drug substance

1000mL of tetrahydrofuran is added into a three-neck flask, stirring is started, 100g of Z-AHPA, 131.5g of L-leucine benzyl ester p-toluenesulfonate and 48.5g of 1-hydroxybenzotriazole (HOBt) are added in sequence, and then the temperature is reduced to 0 +/-2 ℃. Adding 33.6g of triethylamine and 74.5g of Dicyclohexylcarbodiimide (DCC), controlling the temperature to be 12 +/-2 ℃ after the triethylamine and the DCC are added, stirring and reacting for 20-24 hours, and filtering the reaction solution after the reaction is completed. Concentrating the filtrate under reduced pressure until no fraction is distilled off to obtain oil, adding ethyl acetate, and stirring until the oil is completely dissolved. The ethyl acetate solution was washed with 650mL of 0.5mol/L hydrochloric acid, 650mL of purified water, 650mL of a saturated sodium bicarbonate solution, and 650mL of a saturated sodium chloride solution, respectively. Adding anhydrous magnesium sulfate into the organic layer, drying, filtering, and concentrating the filtrate at 50 deg.C under reduced pressure until no fraction is evaporated; adding 270mL of ethyl acetate, heating to 70 +/-2 ℃, stirring to dissolve, cooling to 5 +/-2 ℃, stirring for 2 hours, and crystallizing. Filtration was carried out and the filter cake was washed 2 times with 100mL portions of ethyl acetate/petroleum ether (1: 2). After the filtration, the filter cake was taken out and dried to obtain 140g of a condensate product.

130mL of glacial acetic acid was added to a 2000mL hydrogenation reactor, and 140g of ubenimex condensate and 16g of 10% palladium on charcoal were added to replace the air in the reactor with nitrogen for 3 times and then with hydrogen for 3 times. Introducing hydrogen until the pressure in the reaction kettle is 1MPa, starting stirring, and supplementing hydrogen until hydrogen is hardly absorbed (the pressure in the reaction kettle is unchanged within half an hour). Emptying and discharging reaction liquid; washing the reaction kettle once by using 100mL of glacial acetic acid, filtering the reaction solution, washing a filter cake by using acetic acid, and concentrating the filtrate at 60 ℃ under reduced pressure; when no fraction is distilled off, 530mL of acetone is added and stirred at room temperature for 2 to 3 hours. Filtering, washing a filter cake with acetone, and drying to obtain 85g of crude ubenimex with the yield of 60.7%.

900mL of 1mol/L hydrochloric acid solution is added into a container, heated to 55 ℃, added with 85g of crude ubenimex, and stirred until dissolved. Adding 3.35g of active carbon, stirring and decoloring for 15 minutes, and filtering. Adjusting the pH value of the filtrate to 5.5 by using ammonia water, cooling to 0-5 ℃, stirring for crystallization for 2 hours, filtering, washing a filter cake with 250mL of purified water for three times, transferring the filter cake to a three-neck flask, adding 900mL of acetone, and stirring at room temperature for 4 hours. Filtering, washing the filter cake with acetone for 2 times, each time 80mL, drying to obtain 72.2g of ubenimex, with purity of 99.82% by HPLC. The high performance liquid chromatogram is shown in figure 1.

The conditions of the high performance liquid chromatography used were as follows: octadecylsilane bonded silica gel was used as a column packing (Inertsil ODS-SP or Ecosil C18, 250 mm. times.4.6 mm, 5 μm); taking 0.05mol/L potassium dihydrogen phosphate solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution according to the following table; the column temperature was 30; the detection wavelength is 220 nm; the flow rate is 1.0 ml/min; the theoretical plate number is not less than 3000 calculated according to ubenimex peak. HPLC gradient elution flow rates are as follows:

EXAMPLE 2 preparation of the Compound of formula I

i, weighing 20g of ubenimex and 11.0g of sodium bicarbonate, adding the ubenimex and the sodium bicarbonate into a glass reaction bottle, adding 195 ml of water and 195 ml of tetrahydrofuran, stirring, cooling to 0 ℃, and dropwise adding 11g of benzyl chloroformate at the reaction temperature of 0-4. After the dropwise addition, raising the temperature to room temperature for continuous reaction overnight, adding 162 ml of water into the reaction solution, extracting twice with 162 ml of ethyl acetate, combining organic phases twice, washing with 162 ml of saturated sodium chloride solution, drying the organic phase with anhydrous magnesium sulfate, filtering, washing a filter cake with ethyl acetate, concentrating the filtrate under reduced pressure to dryness, adding 100ml of ethyl acetate, heating for dissolution, cooling to 0 ℃ for crystallization for 2 hours, filtering, and drying to obtain 22.3g of the compound shown in the formula III, wherein the yield is 77.8%.

ii weighing 10g of N- [ (2S,3R) -3-benzyloxyamido-2-hydroxy-4-phenylbutyryl ] -L-leucine, 3.4g of N-hydroxysuccinimide, 6.1g of N, N-dicyclohexylcarbodiimide and 3.1g of sodium carbonate of the compound shown in the formula III, adding 80ml of tetrahydrofuran into a glass reaction bottle, stirring at room temperature for reacting for 2 hours, then 4.4g of (2S,3R) -3-amino-2-hydroxy-4-phenylbutyric acid is added, the reaction is continued for 2 hours with stirring, stopping reaction, filtering, washing a filter cake with tetrahydrofuran, concentrating the filtrate under reduced pressure, adding 120 ml of ethyl acetate, washing with water and a saturated sodium bicarbonate solution respectively, drying an organic phase with anhydrous magnesium sulfate, filtering, and concentrating under reduced pressure; adding ethyl acetate to dissolve, cooling and crystallizing, filtering and drying to obtain 12.7g of the compound shown in the formula IV, wherein the yield is 90.7%.

And iii, weighing 10g of (2S,3R) -3- [ (S) -2- [ (2S,3R) -3-benzyloxyamido-2-hydroxy-4-phenyl-butyrylamino ] -4-methyl-pentanoylamino ] -2-hydroxy-4-phenyl-butyric acid of the compound shown in the formula IV, adding the weighed compound into a hydrogenation reaction kettle, adding 100ml of glacial acetic acid and 2g of 5% palladium-carbon, replacing air, adding hydrogen under the pressure of 1.2Mpa, stirring and reacting at room temperature for 3 hours, filtering after the medium control reaction is completed, concentrating the filtrate under reduced pressure to dryness, adding 50ml of acetone, pulping for 1 hour, filtering, washing a filter cake with acetone, and drying to obtain a crude compound shown in the formula I.

iv dissolving the product in 1mol/l hydrochloric acid, heating to 55 ℃ for dissolution, filtering to remove residues, adjusting the pH value to 5.5 by using ammonia water, separating out a large amount of white solid, cooling to 0 ℃ for crystallization for 2 hours, filtering, washing a filter cake by using precooled water, washing the filter cake by using acetone, and drying to obtain 6.7 g of a compound product of the formula I, wherein the yield is 85.9 percent, and the purity of the product is 99.07 percent by HPLC (according to the chromatographic conditions of the example 1). The HPLC chromatogram is shown in figure 2, and compared with the liquid phase chromatogram (figure 1) of ubenimex bulk drug obtained by the first preparation, the peak appearance time of the compound shown in formula I is the same as the peak appearance time of the impurity b in the bulk drug.

Mass spectrometric detection of compounds of formula i: (MS) (ESI +, m/z): m/z486.3 (base peak) is [ M + H ] + peak, MSm/z508.3 is [ M + Na ] + peak, M/z484.2 (base peak) is [ M-H ] -peak, the molecular weight of the compound of formula I is 485 (see figure 3), and is consistent with the predicted molecular weight of the impurity b structure.

¹H-NMR nuclear magnetic resonance assay assignment data (d-DMSO,500 MHz): 0.776(3H, -CH3), 0.793(3H, -CH3), 1.361(3H, -CH2), 2.543(1H, -CH2-Ph), 2.845(3H, -CH2-Ph), 3.515(1H, -CH-NH2), 3.555(1H, 1H, -CH-NH), 3.895(1H, -CH-CO), 4.164(2H, -CH-OH), 7.229(10H, -PH), 7.692(1H, -NH-CO), 8.263(1H, -NH-CO). The comparison is consistent with the predicted chemical structure of the target impurity.

COMPARATIVE EXAMPLE 1 preparation of the Compound of formula I (route two method)

i weighing 20g ubenimex and 7.2 g triethylamine, adding into a three-neck flask, adding 100ml methanol, then adding (Boc)₂17.1 g of O-anhydride, stirring and reacting for 2 hours at room temperature, after the central control reaction is completed, concentrating to be dry under reduced pressure, adding 100ml of water, extracting with 100ml of ethyl acetate, washing an organic phase once with 100ml of water, drying the organic phase with anhydrous sodium sulfate, concentrating to be dry under reduced pressure, adding 120 ml of ethyl acetate, heating and dissolving, crystallizing for 2 hours at 0 ℃, filtering, washing a filter cake with 20 ml of mixed solvent of ethyl acetate and petroleum ether (1:1), and drying to obtain 18.6 g of a product with the yield of 70.2%.

ii, weighing 10g of N- [ (2S,3R) -3-tert-butoxycarbonylamino-2-hydroxy-4-phenylbutyryl ] -L-leucine, 3.7g of N-hydroxysuccinimide, 6.6g of N, N-dicyclohexylcarbodiimide and 3.4g of sodium carbonate, adding the mixture into a glass reaction bottle, adding 80ml of tetrahydrofuran, stirring and reacting at room temperature for 2 hours, then adding 4.8g of (2S,3R) -3-amino-2-hydroxy-4-phenylbutyric acid, continuing stirring and reacting for 2 hours, stopping the reaction, filtering, washing a filter cake with tetrahydrofuran, concentrating the filtrate under reduced pressure, adding 120 ml of ethyl acetate, washing with water and a saturated sodium bicarbonate solution respectively, drying an organic phase with anhydrous magnesium sulfate, filtering and concentrating under reduced pressure; adding ethyl acetate to dissolve, cooling and crystallizing, filtering and drying to obtain 11.5g of a product with the yield of 80.4 percent.

iii weighing 10g of (2S,3R) -3- [ (S) -2- [ (2S,3R) -3-tert-butoxycarbonylamino-2-hydroxy-4-phenyl-butyrylamino ] -4-methyl-pentanoylamino ] -2-hydroxy-4-phenyl-butyric acid, adding 1mol/l HCl80 ml, stirring at room temperature for 1 hour, after the controlled reaction is completed, adding 80ml of ethyl acetate for extraction, discarding the organic phase, adjusting the pH value of the aqueous phase to 5.5 by ammonia water, separating out a large amount of white solid, crystallizing at 0 ℃ for 2 hours, filtering, washing the filter cake by 50ml of precooled water, then washing by 30ml of acetone, and drying to obtain 5.8 g of the product (formula I), wherein the yield is 70% and the purity is 90.2%.

Comparative example 2 preparation of a Compound of formula I (route three methods)

i, weighing 20g of ubenimex and 5.8 g of sodium bicarbonate, adding 80ml of water into a three-neck flask, adding 23.5 g of Fmoc-Cl, stirring and reacting for 2 hours at room temperature, adding 100ml of ethyl acetate for extraction after the middle control reaction is completed, washing an organic phase once by using 100ml of water, drying the organic phase by using anhydrous sodium sulfate, concentrating under reduced pressure to dryness, adding 80ml of ethyl acetate, heating and dissolving, crystallizing for 2 hours at 0 ℃, filtering, washing a filter cake by using 20 ml of a mixed solvent of ethyl acetate and petroleum ether (1:1), and drying to obtain 20.5 g of a product, wherein the yield is 61.4%.

ii, weighing 10g of N- [ (2S,3R) -3-fluorenylmethoxyamido-2-hydroxy-4-phenylbutyryl ] -L-leucine, 2.9g of N-hydroxysuccinimide, 5.2g of N, N-dicyclohexylcarbodiimide and 2.7g of sodium carbonate, adding the materials into a glass reaction bottle, adding 80ml of tetrahydrofuran, stirring and reacting at room temperature for 2 hours, then adding 4.8g of (2S,3R) -3-amino-2-hydroxy-4-phenylbutyric acid, continuing stirring and reacting for 2 hours, stopping the reaction, filtering, washing a filter cake with tetrahydrofuran, concentrating the filtrate under reduced pressure, adding 120 ml of ethyl acetate, washing with water and a saturated sodium bicarbonate solution respectively, drying an organic phase with anhydrous magnesium sulfate, filtering and concentrating under reduced pressure; adding ethyl acetate to dissolve, cooling and crystallizing, filtering and drying to obtain 9.5g of a product. The yield thereof was found to be 70.4%.

iii weighing 10g of (2S,3R) -3- [ (S) -2- [ (2S,3R) -3-fluorenylmethoxyamido-2-hydroxy-4-phenyl-butyrylamino ] -4-methyl-pentanoylamino ] -2-hydroxy-4-phenyl-butyric acid, adding into a three-neck flask, then adding 50ml of dimethylformamide and 10 ml of piperidine, stirring and reacting for 1 hour at room temperature, after the control reaction is completed, adding 80ml of ethyl acetate for extraction, removing an organic phase, adjusting the pH value of a water phase to 5.5 by using ammonia water, separating out a large amount of white solid, crystallizing for 2 hours at the temperature of 0 ℃, filtering, washing a filter cake by using 50ml of precooled water, then washed with 30ml of acetone and dried to obtain 3.7g of the product (formula I), the yield is 52.9% and the purity is 91.0%.

Claims

1. The impurity in the ubenimex bulk drug is characterized in that the structural formula of the impurity is shown as formula I:

2. the preparation method of the impurities in the ubenimex bulk drug shown in the formula I is characterized by comprising the following steps of:

the method specifically comprises the following steps:

3. The method of claim 2, wherein the alkaline reagent of step 1) is NaHCO₃、Na₂CO₃Or K₂CO₃。

4. The preparation method of claim 2, wherein the molar ratio of the compound of formula II in the step 1) to benzyl chloroformate is 1: 1-3.

5. The method according to claim 2, wherein the condensing agent for the condensation reaction in step 2) is HOSU/DCC/Na₂CO₃。

6. The method according to claim 2, wherein the reaction temperature in the step 2) is 10 to 30 ℃.

7. The preparation method of claim 2, wherein the palladium-carbon in step 3) is 5-10% by weight of the condensate of formula IV, and the hydrogen pressure is 0.5-2.0 MPa.

8. The method according to claim 2, wherein the concentration of the dilute hydrochloric acid in the step 4) is 1 to 3 mol/L.

9. The method according to claim 2, wherein the pH in the step 4) is adjusted to 5.5.

10. The method according to claim 2, wherein the crystallization temperature in the step 4) is 0 to 10 ℃.