Method for Preparing High-Purity Small-Molecule Peptidomimetic Compounds by Chromatography

Abstract

The present invention discloses a method for preparing high-purity small molecule peptide-like compounds by chromatography. Specifically, a purification method for small molecule peptide-like compounds is disclosed, which comprises the following steps: using ion exchange chromatography or reverse phase chromatography to elute the crude product of the small molecule peptide-like compound. The purification method provided by the present invention is applicable to the high-efficiency commercial production of high-purity small molecule peptide-like compounds with a chromatographic purity of 98.5% and low impurities.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical purification, and particularly relates to a purification method for small molecule peptide-like compounds. Background Art

[0002] Peptides play an important role in the growth, development and metabolism of organisms. They are composed of amino acids with a certain sequence through peptide bonds. A peptide composed of two amino acids is called a dipeptide. The compound of Formula I is a non-natural synthetic lysine derivative, with a dipeptide structure composed of two amino acids, and is also a common non-degradable ADC linker, which can be used for the synthesis of antibody-drug conjugates (ADCs).

[0003]

[0004] There is a literature (Tetrahedron Letters, Vol. 38, No. 30, pp. 5257-5260, 1997) that discloses a preparation method for the compound of Formula I. The compound of Formula I is synthesized according to the following synthetic route and purified by silica gel column chromatography.

[0005]

[0006] Through investigation, it is found that the purity range of the commercially available products of the compound of Formula I is between 95% and 98%. In view of the drug safety of the products, a more mature purification process is needed to obtain high-purity products. However, from the current literature, due to various defects, there is no production method for preparing high-purity peptide-like compounds suitable for commercial production. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the defect of low purity of small molecule peptide-like compounds in the prior art, and provide a method for high-purity (chromatographic purity reaching 98.5%) low-impurity small molecule peptide-like compounds with high efficiency and suitable for commercial production.

[0008] The present invention solves the above technical problem through the following technical solutions:

[0009] The present invention provides a purification method for small molecule peptide-like compounds, which comprises the following steps: using ion exchange chromatography or reverse phase chromatography to elute the crude product of the small molecule peptide-like compound.

[0010] The ion exchange chromatography conditions are as follows: the stationary phase of the ion exchange chromatography is a high molecular polymer; in the elution system of the ion exchange chromatography, eluent A is an acidic buffer solution, and eluent B is one or more of acetonitrile, methanol, ethanol, isopropanol and tetrahydrofuran.

[0011] The reverse-phase chromatography conditions are as follows: the stationary phase of the reverse-phase chromatography is reverse-phase silica gel or polymer microspheres; in the elution system of the reverse-phase chromatography, the eluent A is an acidic buffer solution, and the eluent B is one or more of acetonitrile, methanol, ethanol, isopropanol, and tetrahydrofuran.

[0012] Among them, the structure of the small molecule peptide compound is shown in Formula I.

[0013]

[0014] In the ion exchange chromatography, the stationary phase can be a cation exchange resin, preferably a weakly acidic cation exchange resin, such as a macroporous weakly acidic acrylic cation exchange resin; more preferably D113, D151 or D152; further preferably D151.

[0015] In the ion exchange chromatography, the particle size of the stationary phase packing can be conventional in the art. Preferably, the average particle size of the stationary phase packing is preferably 0.2 - 2 mm, more preferably 0.3 - 1.25 mm or 0.4 - 0.7 mm.

[0016] In the ion exchange chromatography, the eluent A can be a conventional acidic buffer solution in the art. Preferably, the eluent A is a buffer salt solution; among them, the cations are Na + , K + , Li + , H + , NH4 + and Et3NH + one or more of them, and the anions are PO4 3- (phosphate ion), HPO4 2- (monohydrogen phosphate ion), H2PO4 - (dihydrogen phosphate ion), CH3COO - (acetate ion), HCOO - (formate ion), Cl - , HCO3 - (bicarbonate ion), ClO - (hypochlorite ion) and SO3 2- (sulfite ion) one or more of them; the cations are preferably Na + and / or NH4 + ; the anions are preferably CH3COO - and / or H2PO4 - , such as: sodium dihydrogen phosphate, ammonium acetate or sodium acetate.

[0017] In the ion exchange chromatography, the pH value of the eluent A is preferably 4.0 - 6.5. Preferably, the pH value of the eluent A is adjusted by acetic acid.

[0018] In the ion exchange chromatography, the eluent B is preferably methanol or acetonitrile.

[0019] In the ion exchange chromatography, in the elution system, the eluent is sodium dihydrogen phosphate - methanol, sodium acetate - acetonitrile or ammonium acetate - methanol, and the pH value of the eluent A is adjusted to 4.0 - 6.5 with acetic acid.

[0020] In the ion exchange chromatography, the volume ratio of the eluent A to the eluent B can be 5:95 - 95:5, preferably 40:60 - 80:20; more preferably 50:50 - 70:30, for example: 50:50, 60:40 or 70:30.

[0021] In the ion exchange chromatography, the crude small molecule peptide compound can be pretreated before elution to meet the injection standard. The pretreatment can be a conventional pretreatment in the art. Preferably, the pretreatment includes the following steps: dissolving the crude small molecule peptide compound in a mixed solvent of the eluent A and the eluent B, and adjusting the pH to complete dissolution with acid.

[0022] In the pretreatment, the acid can be acetic acid.

[0023] In the ion exchange chromatography, the elution method can be isocratic elution or gradient elution; preferably isocratic elution.

[0024] In the ion exchange chromatography, the elution temperature can be a conventional elution temperature in the art, preferably 20 - 35°C, for example 25°C.

[0025] In the ion exchange chromatography, the treatment method of the eluate after elution can be one or more of vacuum concentration, extraction and drying concentration, and freeze drying; preferably, vacuum concentration is carried out first and then freeze drying.

[0026] In the reverse phase chromatography, the stationary phase can be octadecylsilyl bonded reverse phase silica gel or styrene - divinylbenzene crosslinked polymer microspheres, such as Silicabio - C18 or UniPS40 - 300. Preferably, the stationary phase is octadecylsilyl bonded reverse phase silica gel.

[0027] In the reverse phase chromatography, the particle size of the stationary phase packing can be conventional in the art. Preferably, the average particle size of the stationary phase packing is 5 - 200 μm, preferably 20 - 50 μm, for example 20 μm or 50 μm.

[0028] In the reverse phase chromatography, the eluent A can be a conventional acidic buffer solution in the art. Preferably, the eluent A is a buffer salt solution; wherein, the cation is Na + 、K+ , Li + , H + , NH4 + and Et3NH + and one or more of the following, the anion being PO4 3- (phosphate ion), HPO4 2- (monohydrogen phosphate ion), H2PO4 - (dihydrogen phosphate ion), CH3COO - (acetate ion), HCOO - (formate ion), Cl - , HCO3 - (bicarbonate ion), ClO - (hypochlorite ion) and SO3 2- (sulfite ion) and one or more of the following; the cation is preferably Na + and / or NH4 + ; the anion is preferably CH3COO - and / or H2PO4 - , for example: sodium dihydrogen phosphate, ammonium acetate and sodium acetate.

[0029] In the reverse-phase chromatography, the pH value of the eluent A is preferably 4.0 - 6.5, and preferably, the pH value of the eluent A is adjusted by acetic acid.

[0030] In the reverse-phase chromatography, the eluent B is preferably methanol, acetonitrile or isopropanol.

[0031] In the reverse-phase chromatography, the elution system has eluents of sodium dihydrogen phosphate - methanol, sodium dihydrogen phosphate - isopropanol, sodium acetate - acetonitrile, ammonium acetate - methanol, ammonium acetate - acetonitrile or sodium acetate - methanol, and the pH value of the eluent A is adjusted to 4.0 - 6.5 by acetic acid.

[0032] In the reverse-phase chromatography, when the stationary phase is styrene - divinylbenzene cross-linked polymer microspheres, the eluent is sodium acetate - acetonitrile or ammonium acetate - methanol.

[0033] In the reverse-phase chromatography, the volume ratio of the eluent A to the eluent B can be 5:95 - 95:5, preferably 40:60 - 80:20; more preferably 50:50 - 70:30, for example: 50:50, 60:40 or 70:30.

[0034] In the reverse-phase chromatography, the elution temperature can be the conventional elution temperature in the art, preferably 20 - 35 °C, for example 25 °C.

[0035] In the reverse-phase chromatography, the elution method can be isocratic elution or gradient elution; preferably isocratic elution.

[0036] In the reversed-phase chromatography, the treatment method of the eluate after elution may be one or more of vacuum concentration, extraction and drying concentration, and freeze-drying operations; preferably, vacuum concentration is carried out first and then freeze-drying.

[0037] In the purification method, a pre-purification step may also be carried out before elution. The pre-purification may be a conventional pre-purification in the art. Preferably, the pre-purification includes the following steps: dissolving the crude product of the small molecule peptide compound to be treated in solvent A, then mixing with solvent B, standing, and concentrating the lower oily substance. Solvent A is a halogenated hydrocarbon solvent, and solvent B is an ether solvent.

[0038] In the pre-purification, solvent A is preferably dichloromethane.

[0039] In the pre-purification, solvent B is preferably methyl tert-butyl ether.

[0040] In the pre-purification, the mass ratio of solvent B to solvent A may be (1-25):1, such as 1:1, 3:1, 5:1, 7:1, 10:1 or 25:1, preferably (7-25):1, and more preferably (7-10):1.

[0041] In the pre-purification, the mixing time may be 1-16 h, preferably 2 h.

[0042] The crude product of the small molecule peptide compound to be treated can be prepared by the following method: in a solvent, compound of formula IB reacts with diethylamine, and then concentrate.

[0043]

[0044] In the reaction, preferably, the solvent is dichloromethane.

[0045] The molar ratio of the compound of formula IB to diethylamine is 1:(1-5), such as 1:3.

[0046] The temperature of the reaction is room temperature.

[0047] The progress of the reaction can be monitored by conventional testing methods in the art (such as TLC, HPLC, GC or NMR), and generally the end point of the reaction is when the compound of formula IB no longer reacts. The reaction time can be 2 hours.

[0048] In the present invention, "room temperature" refers to 10-30 °C.

[0049] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention. The positive and progressive effects of the invention are as follows:

[0050] 1) A highly pure peptidomimetic compound with a purity greater than 99.5% prepared by purification through ion exchange chromatography, with the impurity content greatly reduced.

[0051] 2) A peptidomimetic compound with a peptidomimetic compound content greater than 97.8% can be obtained through purification by reverse-phase chromatography, and the method is simple.

[0052] 3) For the treatment of fractions in this method, vacuum concentration and extraction drying concentration are adopted, avoiding the cumbersome operation of freeze-drying in reverse-phase separation. A high-purity sample powder can be obtained only through simple operations, the product state is good, and the post-treatment operation is simple and easy to perform.

[0053] 4) The two chromatographic packing stationary phases adopted in this method are simple to obtain, economical and applicable, and can be reused, improving the use efficiency and reducing the production cost.

[0054] 5) This purification method not only improves the product quality, but also this technology greatly reduces the use of environmentally unfriendly solvents to achieve the maximum degree of environmental protection. Specific embodiments

[0055] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions indicated in the following examples are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0056] Unless otherwise specified, the mass ratios of the various substances involved in the following examples are used.

[0057] The stationary phase parameters of the column chromatography packing used in the following examples are shown in Table 1.

[0058] Table 1. Packing stationary phase parameters

[0059]

[0060]

[0061] The parameters of the preparation and separation equipment used in the following examples are shown in Table 2.

[0062] Table 2. Preparation and separation equipment parameters

[0063]

[0064] In the following examples, the HPLC analysis conditions for analyzing the content of the compound of formula I are as follows:

[0065] Analysis column: Waters ACQUITY UPLCR BEH C18, specification 2.1×50mm, packing particle size 1.7μm.

[0066] Mobile phase: A—Sodium acetate / water = 20 mmol / L; B—Isopropanol. The elution gradient is shown in Table 3.

[0067] Table 3. HPLC elution gradient

[0068] Time(min) A％ B％ 0 90 10 2 50 50 8 10 90 10 10 90 10.5 90 10 15 90 10

[0069] (Column temperature: 30 °C, flow rate: 0.3 mL / min, detection wavelength: 254 nm)

[0070] Example 1 Pre-purification of crude product of Formula I compound with different solvent ratios

[0071] Preparation of crude product of Formula I compound: (Refer to the literature: Tetrahedron Letters, Vol. 38, No. 30, pp. 5257 - 5260, 1997):

[0072]

[0073] Dissolve the compound of Formula IB (1.0 equivalent) in dichloromethane (10 - 25 times the weight of the compound of Formula IB), then add diethylamine (3.0 equivalents). The resulting mixture is stirred at room temperature for 2.0 hours until the reaction is completed (monitored by TLC). The crude product of the compound of Formula I is obtained by concentration, which is an oily viscous substance with an HPLC purity of 63.95%.

[0074] Pre-purification of the crude product of Formula I compound:

[0075] The solvent ratios used for pre-purification in this example, the purity and recovery rate of the samples after pre-purification are shown in Table 4. Dissolve the concentrated oily crude product of the compound of Formula I in dichloromethane at 5 times the weight of the feeding amount of the compound of Formula IB at room temperature, then add methyl tert-butyl ether at 10 - 50 times the weight of the feeding amount of the compound of Formula IB, and stir for 2 hours at normal temperature. After standing, pour off the supernatant to obtain an off-white waxy solid, and vacuum concentrate to obtain the pre-purified crude product. The obtained crude product is further purified by chromatography.

[0076] Table 4. Influence of solvent ratios in the pre-purification step on the purity and recovery rate of the obtained crude product

[0077]

[0078] As can be seen from the results given in Table 4: When dichloromethane with a weight 5 times that of the feed amount of the compound of formula IB and methyl tert-butyl ether with a weight 25 - 50 times that of the feed amount of the compound of formula IB are used to pre-purify the compound of formula I according to the method of this example, a crude product of the compound of formula I with a purity greater than 65% can be obtained with a recovery rate greater than 80%; When dichloromethane with a weight 5 times that of the feed amount of the compound of formula IB and methyl tert-butyl ether with a weight 35 - 50 times that of the feed amount of the compound of formula IB are used to pre-purify the compound of formula I according to the method of this example, a crude product of the compound of formula I with a purity greater than 70% can be obtained with a recovery rate greater than 85%.

[0079] Example 2 Purification and Preparation of High-Purity Compound of Formula I by Normal-Phase and Reverse-Phase Chromatography

[0080] On the basis of pre-purifying the crude product of the compound of formula I according to the preferred pre-purification method of Example 1, this example further purifies the pre-purified sample by chromatography to obtain a high-purity compound of formula I. Specifically, the crude product of the compound of formula I is purified according to the following steps in this example:

[0081] Step 1. Pre-purify the crude product of the compound of formula I according to the pre-purification method of Example 1 with the solvent amounts numbered 1 - 5 in Table 4 to obtain a pre-purified crude product of the compound of formula I;

[0082] Step 2. Use normal-phase chromatography silica gel, reverse-phase chromatography silica gel, neutral alumina or polymer microspheres as the packing stationary phase, and adopt normal-phase or reverse-phase chromatography to prepare a high-purity compound of formula I.

[0083] Specific implementation method:

[0084] Normal-phase chromatography: Inject the crude product treated in Step 1 into a preparative normal-phase chromatography column. The specific chromatography column parameters are shown in Tables 1, 2 and 5. Elute at 25°C. The eluent is composed of components A and B, and their ratio is 5 / 95 - 95 / 5 by volume. Use the isocratic elution method to elute the compound of formula I and collect the eluate;

[0085] Reverse-phase chromatography: Inject the crude product treated in Step 1 into a preparative reverse-phase chromatography column. The specific chromatography column parameters are shown in Tables 1, 2 and 5. Elute at 25°C. The eluent is composed of components A and B, and their ratio is 5 / 95 - 95 / 5 by volume. Use the isocratic elution method to elute the compound of formula I and collect the eluate;

[0086] Step 3. Vacuum-concentrate the eluate with qualified purity obtained by normal-phase chromatography at 40°C to remove the organic solution contained in the eluate and obtain the finished product of the compound of formula I.

[0087] The eluate with qualified purity obtained after reverse-phase chromatography elution is concentrated under vacuum at 40°C to remove the organic solution contained in the eluate, and then the remaining aqueous fraction is freeze-dried to obtain a white powder, thus obtaining the finished product of the compound of formula I.

[0088] Table 5. Chromatographic purification methods, purity and recovery rates of purified samples

[0089]

[0090] The chromatographic separation method, type of stationary phase, elution system used in this example, and the purity and recovery rate of the purified sample are shown in Table 5. It can be seen from the results in Table 5 that the chromatographic purity of the products prepared by the normal-phase chromatographic separation method is generally low (below 98%), which cannot meet the requirements for high-purity preparation, and the sample recovery rate is not high. In the reverse-phase chromatographic separation method, when octadecylsilyl-bonded reverse-phase silica gel is selected as the stationary phase, products with a purity above 98.5% can be obtained by using sodium acetate-acetonitrile, ammonium acetate-methanol, and sodium dihydrogen phosphate-isopropanol as the elution system. When styrene-divinylbenzene cross-linked polymer microspheres are selected as the stationary phase, products with a purity above 98.5% can be obtained by using sodium acetate-acetonitrile and ammonium acetate-methanol as the elution system.

[0091] Example 3 Purification of High-Purity Compound of Formula I by Ion Chromatography

[0092] Combining Example 1 and 2, after pre-purifying the crude product of the compound of formula I with the solvent dosages numbered 1-5 in Table 4 according to the pre-purification method of Example 1, and then further purifying it with the reverse-phase chromatographic separation methods numbered 2-9 in Table 5, a target product with a chromatographic purity of 98.78% can be obtained, which can basically meet the production target. However, the reverse-phase chromatographic separation method still has disadvantages such as poor sample recovery rate. The purification of the crude product of the compound of formula I in this example is carried out according to the following operating steps:

[0093] Step 1. Pre-purify the crude product of the compound of formula I with the solvent dosages numbered 1-5 in Table 4 according to the pre-purification method of Example 1 to obtain the pre-purified crude product of the compound of formula I;

[0094] Step 2. Dissolve the pre-purified crude product (5.0 g) of the compound of formula I obtained in Step 1 in a mixed solution (250 mL) of eluent A and B, adjust the pH to complete dissolution (pH range 4.0-6.5) with acetic acid, load the dissolved sample onto an equilibrated ion exchange chromatographic column for elution, the elution temperature is 25°C, the mobile phase ratio is 5 / 95-95 / 5 by volume, use the isocratic elution method to elute the compound of formula I, and collect the eluate;

[0095] Step 3. The eluents with qualified purity are combined and concentrated under vacuum at 40°C to remove the organic solvents contained in the eluents. Then, the remaining aqueous fraction is freeze-dried to obtain a white powder. The finished product of the compound of Formula I is obtained.

[0096] The separation conditions of the ion exchange chromatography used in Step 2 of this example and the purity and recovery rate results of the final product purified after Steps 1 to 3 are shown in Table 6.

[0097] Table 6. Ion exchange chromatography conditions and purity and recovery rate of the purified final product in Example 3

[0098]

[0099] It can be seen from the results in Table 6 that: using the ion exchange chromatography method of this example, the compound of Formula I with a purity greater than 99% can be obtained with a recovery rate greater than 60%, which basically meets the requirements for preparing the compound of Formula I with high purity; preferably, when the ion exchange chromatography separation stationary phase is weak acidic cation exchange resins D151 and D152, the compound of Formula I with a purity greater than 99% can be obtained with a recovery rate greater than 65%. Further preferably, when the ion exchange chromatography separation stationary phase is weak acidic cation exchange resin D151, the compound of Formula I with a purity greater than 99.5% can be obtained with a recovery rate greater than 65%. Using the weak acidic cation exchange resin in this method can directly purify and prepare the target product without the need for acidification reaction treatment during the exchange reaction.

[0100] By adopting the purification method of this example, a higher purity product of the compound of Formula I can be obtained, and at the same time, it has a good sample recovery rate, which is suitable for commercial-scale production.

[0101] This method can not only improve the sample purity and yield, but also has the advantages of simple operation and good stability. Moreover, the used solid-phase filler is simple and easy to obtain and can be recycled repeatedly, saving production costs and reducing environmental pollution, which is suitable for industrial production.

[0102] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A purification method for small molecule peptide compounds, characterized in that, It includes the following steps: using ion exchange chromatography or reverse phase chromatography to elute the crude small molecule peptide compound; The ion exchange chromatography conditions are as follows: the stationary phase of the ion exchange chromatography is macroporous weakly acidic acrylic cation exchange resin D113, macroporous weakly acidic acrylic cation exchange resin D151 or macroporous weakly acidic acrylic cation exchange resin D152; in the elution system of the ion exchange chromatography, eluent A is sodium dihydrogen phosphate, ammonium acetate or sodium acetate, and eluent B is acetonitrile or methanol; In the ion exchange chromatography, in the elution system, the eluent is sodium dihydrogen phosphate - methanol, sodium acetate - acetonitrile or ammonium acetate - methanol, and the pH value of eluent A is adjusted to 4.0 - 6.5 by acetic acid; In the ion exchange chromatography, the volume ratio of eluent A to eluent B is 40:60 - 80:20; The reverse phase chromatography conditions are as follows: in the elution system of the reverse phase chromatography, eluent A is sodium dihydrogen phosphate, ammonium acetate or sodium acetate, and eluent B is acetonitrile, methanol or isopropanol; In the reverse phase chromatography, the volume ratio of eluent A to eluent B is 40:60 - 80:20; The stationary phase of the reverse chromatography is octadecylsilyl bonded reverse phase silica gel Silicabio - C18, the eluent is sodium dihydrogen phosphate - isopropanol, sodium acetate - methanol or ammonium acetate - acetonitrile, and the pH value of eluent A is adjusted to 4.0 - 6.5 by acetic acid; Or the stationary phase of the reverse chromatography is styrene - divinylbenzene crosslinked polymer microspheres UniPS40 - 300, the eluent is sodium acetate - acetonitrile or ammonium acetate - methanol, and the pH value of eluent A is adjusted to 4.0 - 6.5 by acetic acid; wherein, the structure of the small molecule peptide compound is as shown in formula I, 2. The purification method of the small molecule peptide compound according to claim 1, characterized in that, The purification method meets one or more of the following conditions: (1) In the ion exchange chromatography, the stationary phase is macroporous weakly acidic acrylic cation exchange resin D151; (2) In the ion exchange chromatography, the average particle size of the stationary phase filler is 0.2 - 2 mm; (3) The volume ratio of eluent A to eluent B is 50:50 - 70:30; (4) In the ion exchange chromatography, the elution method is isocratic elution or gradient elution; (5) In the ion exchange chromatography, the elution temperature is 20 - 35 °C; (6) In the ion exchange chromatography, the treatment method of the eluate after elution is one or several of vacuum concentration, extraction and drying concentration, and freeze drying.

3. The purification method of the small molecule peptide compound according to claim 1, characterized in that, The purification method meets one or more of the following conditions: (1) The volume ratio of eluent A to eluent B is 50:50, 60:40 or 70:30; (2) In the ion exchange chromatography, the average particle size of the stationary phase filler is 0.3 - 1.25 mm or 0.4 - 0.7 mm; (3) In the ion exchange chromatography, the elution method is isocratic elution; (4) In the ion exchange chromatography, the elution temperature is 25 °C; (5) In the ion exchange chromatography, the treatment method of the eluate after elution is to first perform vacuum concentration and then perform freeze-drying.

4. The purification method of the small molecule peptide compound according to claim 1, characterized in that, In the ion exchange chromatography, the crude small molecule peptide compound is pretreated before elution, and the pretreatment includes the following steps: dissolving the crude small molecule peptide compound in a mixed solvent of the eluent A and the eluent B, and adjusting the pH to complete dissolution by adding an acid.

5. The purification method of the small molecule peptide compound according to claim 1, characterized in that, In the ion exchange chromatography, the crude small molecule peptide compound is pretreated before elution, and the pretreatment includes the following steps: dissolving the crude small molecule peptide compound in a mixed solvent of the eluent A and the eluent B, and adjusting the pH to complete dissolution by adding an acid; the acid is acetic acid.

6. The purification method of the small molecule peptide compound according to claim 1, characterized in that, The purification method satisfies one or more of the following conditions: (1) In the reverse phase chromatography, the average particle size of the stationary phase packing is 5 - 200 μm; (2) In the reverse phase chromatography, the volume ratio of the eluent A to the eluent B is 50:50 - 70:30; (3) In the reverse phase chromatography, the elution temperature is 20 - 35 °C; (4) In the reverse phase chromatography, the elution mode is isocratic elution or gradient elution; (5) In the reverse phase chromatography, the treatment method of the eluate after elution is one or several of vacuum concentration, extraction and drying concentration, and freeze-drying operations.

7. The purification method of the small molecule peptide compound according to claim 1, characterized in that, The purification method satisfies one or more of the following conditions: (1) In the reverse phase chromatography, the average particle size of the stationary phase packing is 20 - 50 μm; (2) In the reverse phase chromatography, the volume ratio of the eluent A to the eluent B is 50:50, 60:40 or 70:30; (3) In the reverse phase chromatography, the elution temperature is 25 °C; (4) In the reverse phase chromatography, the elution mode is isocratic elution; (5) In the reverse phase chromatography, the treatment method of the eluate after elution is to first perform vacuum concentration and then perform freeze-drying.

8. The purification method of the small molecule peptide compound according to claim 1, characterized in that, In the reverse phase chromatography, the average particle size of the stationary phase packing is 20 μm or 50 μm.

9. The purification method of the small molecule peptide compound according to claim 1, characterized in that, In the purification method, pre-purification is also performed before elution, and the pre-purification includes the following steps: dissolving the crude small molecule peptide compound to be treated with the solvent A, then mixing it with the solvent B, standing, and concentrating the lower oily substance; the solvent A is a halogenated hydrocarbon solvent, and the solvent B is an ether solvent.

10. The purification method of the small molecule peptide compound as described in claim 9, characterized in that, The purification method satisfies one or more of the following conditions: (1) In the pre-purification, the solvent A is dichloromethane; (2) In the pre-purification, the solvent B is methyl tert-butyl ether; (3) In the pre-purification, the mass ratio of the solvent B to the solvent A is (1 - 25):1; (4) In the pre-purification, the mixing time is 1 - 16 h.

11. The purification method of the small molecule peptide compound according to claim 9, characterized in that, The purification method satisfies one or more of the following conditions: (1) In the pre-purification, the mass ratio of the solvent B to the solvent A is 1:1, 3:1, 5:1, 7:1, 10:1 or 25:1; (2) In the pre-purification, the mixing time is 2 h.

12. The purification method of the small molecule peptide compound according to claim 9, characterized in that, In the pre-purification, the mass ratio of the solvent B to the solvent A is (7 - 25):

1.

13. The purification method of the small molecule peptide compound according to claim 9, characterized in that, In the pre-purification, the mass ratio of the solvent B to the solvent A is (7-10):

1.

14. The purification method of the small molecular peptide compound according to claim 1 or 9, characterized in that, The crude product of the small molecule peptidomimetic compound to be treated is prepared by the following method: in a solvent, the compound of formula IB reacts with diethylamine, and then it is concentrated.

15. The purification method of the small molecule peptide compound according to claim 14, wherein, The purification method satisfies one or more of the following conditions: (1) In the reaction, the solvent is dichloromethane; (2) The molar ratio of the compound of formula IB to the diethylamine is 1:(1-5); (3) The temperature of the reaction is room temperature; (4) The reaction time is 2 hours.

16. The purification method of the small molecule peptide compound according to claim 14, characterized in that, The molar ratio of the compound of formula IB to the diethylamine is 1:3.

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