API Synthesis Methods

Abstract

The invention discloses a synthesis method of an API, firstly condensing Fmoc-PEG4-propionic acid with diethanolamine to generate an intermediate 1; then reacting p-toluenesulfonyl chloride with the intermediate 1 to generate an intermediate 2; and finally reacting HO-PEG3-CH2CH2COOtBu with the intermediate 2 under alkaline conditions to obtain the API. The synthesis method of the invention has easy-to-obtain raw materials, mild reaction conditions, can reduce the generation of by-products, has high product purity, and high reaction yield.

Description

Technical Field

[0001] The present invention relates to the field of organic synthesis, in particular to a method for synthesizing Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu). Background Art

[0002] In recent years, the development of various forms of conjugate drugs has been in full swing, especially the antibody-drug conjugate (ADC)-led conjugate drugs have achieved great success in cancer treatment. There are many different forms of conjugate drugs in the drugs under development, including not only the common peptide-drug conjugate (PDC), but also small molecule-drug conjugate (SMDC) and other forms. The mechanism of action of these conjugate drugs is mainly to form a single compound by connecting two or more different drug molecules or functional groups in a specific way to achieve a synergistic effect or a drug form that achieves multiple target interventions.

[0003] The application areas of conjugated drugs are very wide, including cancer treatment, immunomodulation, anti-infection, neurological diseases, autoimmune diseases, etc. With the in-depth understanding of disease mechanisms and treatment needs, conjugated drugs will continue to become an important area of ​​drug research and development and treatment innovation, contributing to improving patients' quality of life and treatment effects.

[0004] Linkers play a role in connecting and stabilizing drug molecules in conjugated drugs, affecting the stability, pharmacokinetics, pharmacodynamics and safety of drugs. With the development iterations of conjugated drugs and the accumulation of successful and failed clinical experiences, it has been determined that linkers are a key element in the entire conjugated drug design.

[0005] The invention patent application with patent publication number CN115304582A mentions a drug multifunctional Linker, the synthesis route of which is as follows (see paragraph 0208 of its specification):

[0006]

[0007] The structural formula of the multifunctional Linker of this drug is as follows:

[0008] Fmoc-NH-PEG4-CH2CH2CONbis(PEG4-CH2CH2COtBu);

[0009] Its chemical structure is as follows:

[0010]

[0011] The Chinese name is: 16-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16-pentaoxa-4-azanonadecan-19-yl)-4,7,10,13,19,22,25,28-octaoxa-16-azatriacontanoate; the English name is: Di-tert-butyl 16-(1-(9H-fluroen-9-yl)-3-oxo-2,7,10,13,16-pentaoxa-4-azanonadecan-19-oyl)-4,7,

[0012] 10,13,19,22,25,28-octaoxa-16-azahentriacontanedioate;

[0013] The carboxyl groups protected by tert-butyl and the amino groups protected by Fmoc at both ends can be connected to different drug molecules or functional groups after deprotection, which can provide possibilities for different needs in drug research and development.

[0014] Fmoc-NH-PEG4-CH2CH2CONbis(PEG4-CH2CH2COtBu) is an important component in the development of conjugated drugs. Its purity will have a great impact on the quality and yield of subsequent products. At present, the existing synthesis methods of this substance have many by-products, low reaction yield, low safety of reaction conditions, high synthesis cost and other problems. Therefore, it is necessary to develop a high-purity and high-yield synthesis method. Summary of the invention

[0015] The object of the present invention is to provide a method for synthesizing Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu) which can reduce the generation of by-products and improve the purity and yield of the product in view of the above problems.

[0016] To achieve the above purpose, the technical solution provided by the present invention is:

[0017] A method for synthesizing Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu) comprises the following steps:

[0018] Step (1): Fmoc-PEG4-propionic acid and diethanolamine are subjected to a condensation reaction to generate an intermediate 1, wherein the intermediate 1 is 9-fluorenylmethyl (18-hydroxy-16-(2-hydroxyethyl)-15-oxo-3,6,9,12-tetraoxa-16-octadecyl) carbamate;

[0019] Step (2): reacting p-toluenesulfonyl chloride with the intermediate 1 obtained in step (1) to generate intermediate 2, wherein the intermediate 2 is 1-(9H-fluoro-9-yl)-3,19-dioxo-20-(2-(toluenesulfonyloxy)ethyl)-2,7,10,13,16-pentaoxa-4,20-diazacosyl-22-yl 4-methylbenzenesulfonate;

[0020] Step (3): react HO-PEG3-CH2CH2COOtBu with the intermediate 2 produced in step (2) under alkaline conditions to obtain Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu).

[0021] In order to optimize the above technical solutions, the specific limitations adopted also include:

[0022] In step (1), Fmoc-PEG4-propionic acid and diethanol undergo condensation reaction in the presence of a condensation agent and an acid binding agent.

[0023] In step (1), the reaction molar ratio of Fmoc-PEG4-propionic acid to diethanol is 1:1; the condensing agents are HOBt and EDCI, and the molar ratio of HOBt, EDCI and Fmoc-PEG4-propionic acid is 1.0-1.5:1.0-1.5:1; the acid binding agent is DIEA, and the molar ratio of DIEA to Fmoc-PEG4-propionic acid is 2.0-3.0:1; and dichloromethane is used as the reaction solvent.

[0024] In step (1), during the reaction, Fmoc-PEG4-propionic acid, condensation agent and acid binding agent are first added to the reaction solvent and stirred to dissolve, and the temperature is controlled at 0-10°C. After complete dissolution, stirring is continued for 30-60 minutes; the reaction temperature is room temperature, and the reaction time is 6-8 hours in total.

[0025] In step (2), intermediate 1 reacts with toluenesulfonyl chloride in the presence of an acid binding agent and a catalyst.

[0026] In step (2), the reaction molar ratio of intermediate 1 to toluenesulfonyl chloride is 1:1.0-2.0; triethylamine is used as the acid binding agent, and the molar ratio of triethylamine to intermediate 1 is 2.0-4.0:1; DMAP is used as the catalyst, and the molar ratio of DMAP to intermediate 1 is 0.1-0.2:1; and dichloromethane is used as the reaction solvent.

[0027] In step (2), during the reaction, the intermediate 1, the acid binding agent and the catalyst are first added to the reaction solvent and stirred to dissolve, and then the p-toluenesulfonyl chloride solution is added dropwise, and the temperature is controlled at -5-5°C during the addition process; the reaction temperature is 40-50°C, and the reaction time is 2-4h.

[0028] In step (3), the alkaline conditions for the reaction of intermediate 2 with HO-PEG3-CH2CH2COOtBu are inorganic bases, and the inorganic base is selected from potassium tert-butoxide, sodium hydroxide, potassium hydroxide, sodium hydride, and potassium carbonate.

[0029] In step (3), the reaction molar ratio of intermediate 2 to HO-PEG3-CH2CH2COOtBu is 1:2.

[0030] In some embodiments, the inorganic base is selected from potassium carbonate or sodium hydride, and the molar ratio of potassium carbonate or sodium hydride to intermediate 2 is 2-6:1.

[0031] In some preferred embodiments, the inorganic base is potassium carbonate, and the molar ratio of potassium carbonate to intermediate 2 is 3-4:1.

[0032] In step (3), the reaction temperature is 65-67° C. and the reaction time is 6-8 h; the reaction product is separated and purified by silica gel column.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention improves the problems existing in the existing synthesis method of Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu) and develops a new synthesis method; the present invention also studies and optimizes the reaction conditions in the developed new synthesis method to obtain significantly better method conditions.

[0035] Compared with the currently commonly used methods, the synthetic method of the present invention has readily available raw materials, mild reaction conditions, can reduce the generation of by-products, has high product purity, and has a high reaction yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : HPLC chromatogram of API purity determination under alkaline condition 1 in Example 1 of the present invention.

[0037] Figure 2 : HPLC chromatogram of API purity determination under alkaline condition 2 in Example 1 of the present invention.

[0038] Figure 3 : HPLC chromatogram of API purity determination under alkaline condition 3 in Example 1 of the present invention.

[0039] Figure 4 : HPLC chromatogram of API purity determination under alkaline condition 4 in Example 1 of the present invention.

[0040] Figure 5 : HPLC chromatogram of API purity determination under alkaline condition 5 in Example 1 of the present invention.

[0041] Figure 6 : Purity determination HPLC chromatogram of intermediate 1 of embodiment of the present invention 2.

[0042] Figure 7 : Purity determination HPLC chromatogram of intermediate 2 of embodiment of the present invention 2.

[0043] Figure 8 : HPLC chromatogram of purity determination of API in Example 2 of the present invention. DETAILED DESCRIPTION

[0044] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0045] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0046] In the description of the present invention, it is also necessary to explain:

[0047] The abbreviation API in this application refers to Fmoc-NH-PEG4-CH2CH2CONbis(PEG4-CH2CH2COtBu).

[0048] The present invention studies the drug multifunctional linker: Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu). The existing synthesis method of Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu) has the problems of more by-products, lower reaction yield, lower safety of reaction conditions, high synthesis cost, etc., as described in the following Comparative Examples 1 to 3.

[0049] Comparative Example 1

[0050] Synthesis method of Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu):

[0051] Step 1: condensation of di(2-chloroethyl)amine hydrochloride with Fmoc-PEG4-propionic acid via HOBt;

[0052] Step 2: The condensed intermediate is reacted with HO-PEG3-CH2CH2COOtBu under alkaline conditions to generate Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu).

[0053] The reaction process is as follows:

[0054]

[0055] The reaction in step 2 of this method needs to be carried out under strong alkaline conditions (commonly used are sodium hydroxide or potassium hydroxide), but the amide bond in its structural formula is unstable in a strong alkaline environment and will degrade, resulting in a low yield of the product. If a weaker base is used, the reaction is difficult to proceed, and improvement is needed.

[0056] Comparative Example 2

[0057] Synthesis method of Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu):

[0058] Step 1: reacting TSO-PEG3- CH2CH2COOtBu with ammonia water under high temperature and high pressure conditions;

[0059] Step 2: Condensation with Fmoc-PEG4-propionic acid to obtain Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu).

[0060] The reaction process is as follows:

[0061]

[0062] The reaction conditions of step 1 of this method are usually carried out at 140° C. in a high-pressure reactor, which is a high-temperature and high-pressure reaction with low safety. At the same time, a by-product Ntri (PEG4-CH2CH2COtBu) is generated, which increases the difficulty of subsequent purification and has a low reaction yield.

[0063] The structural formula of Ntri(PEG4-CH2CH2COtBu) is as follows:

[0064]

[0065] Comparative Example 3

[0066] Synthesis method of Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu):

[0067] Step 1: Diethanolamine reacts with p-toluenesulfonyl chloride to generate azadiylbis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate);

[0068] Step 2: Azadiylbis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) is condensed with Fmoc-PEG4-propionic acid to generate 1-(9H-fluoro-9-yl)-3,19-dioxo-20-(2-(toluenesulfonyloxy)ethyl)-2,7,10,13,16-pentaoxa-4,20-diazacosyl-22-yl 4-methylbenzenesulfonate;

[0069] Step 3: 1-(9H-fluoro-9-yl)-3,19-dioxo-20-(2-(tosyloxy)ethyl)-2,7,10,13,16-pentaoxa-4,20-diazacosyl-22-yl 4-methylbenzenesulfonate reacts with HO-PEG3-CH2CH2COOtBu to generate Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu).

[0070] The reaction process is as follows:

[0071]

[0072] This synthetic route is to first protect the hydroxyl groups at both ends of diethanolamine with p-toluenesulfonic acid groups to avoid side reactions, and then condense the amino group with Fmoc-PEG4-propionic acid, and then with HO-PEG3-CH2CH2COOtBu to generate the target product. However, in the reaction process of diethanolamine and phenylmethylsulfonyl chloride in step 1, the amino group will react with phenylmethylsulfonyl chloride to form amide, resulting in low purity of the product. Because it is similar to the product structure, it increases the difficulty of purification, resulting in a low yield in this step, thereby affecting the overall reaction yield and increasing the cost.

[0073] The present invention designs a new synthesis route on the existing basis, and by optimizing the reaction conditions, Fmoc-NH-PEG4-CH2CH2CONbis (PEG4-CH2CH2COtBu) with a purity greater than 99.0% can be obtained, and the overall yield is above 90%, which is specifically introduced by Examples 1 and 2 below. Example 1

[0074] The scheme of the present invention uses Fmoc-PEG4-propionic acid and diethanolamine as starting materials, and reacts in three steps to finally obtain the target product. The reaction steps are as follows:

[0075] Step 1, Fmoc-PEG4-propionic acid is condensed with diethanolamine to generate 9-fluorenylmethyl (18-hydroxy-16-(2-hydroxyethyl)-15-oxo-3,6,9,12-tetraoxa-16-octadecyl) carbamate (intermediate 1);

[0076] Step 2, p-toluenesulfonyl chloride reacts with intermediate 1 to form sulfonate (intermediate 2);

[0077] Step 3: HO-PEG3-CH2CH2COOtBu reacts with intermediate 2 to obtain Fmoc-NH-PEG4-CH2CH2CONbis(PEG4-CH2CH2COtBu) (API).

[0078] This method first condenses the amino group of diethanolamine with Fmoc-PEG4-propionic acid to protect the amino group, avoid the next step of reaction with p-toluenesulfonyl chloride, reduce the generation of by-products, and thus improve the purity and yield of the product.

[0079] The key parameters of this synthesis method are the selection of the type and amount of base in the step 3 reaction. After research and optimization of the type and amount of base, the total yield can be increased to 90% and the product purity is above 99.0%. Compared with the existing synthesis method, the raw materials of the present invention are easy to obtain, the reaction conditions are mild, there are few by-products, and the product yield is high.

[0080] The present application optimizes the reaction conditions of the synthesis method to improve the total yield and product purity. It is found through research that in the entire synthesis route of the synthesis method, the reactions of step one and step two are relatively simple, and the yield and purity of these two steps are relatively high. The reaction of step three is an SN2 nucleophilic reaction between the sulfonic acid ester and the alcohol. This reaction needs to be carried out under alkaline conditions. Commonly used bases include potassium tert-butoxide, sodium hydroxide, potassium hydroxide, sodium hydride, potassium carbonate, triethylamine, etc. The present application selects potassium tert-butoxide, potassium hydroxide, sodium hydride, potassium carbonate, and triethylamine according to the strength of the alkalinity for testing. The intermediate 2 moles are used as the reference N0, and the theoretical amount of the base is 2.0N0 for testing. The reaction solvent is tetrahydrofuran, and the crude product post-treatment method is silica gel column purification. The results are shown in Table 1.

[0081] Table 1 Results of alkaline conditions in Example 1

[0082]

[0083] The purity charts of the products under alkaline conditions 1-5 are shown in Figure 1-Figure 5 From the test results, it can be seen that under strong alkaline conditions, the purity and yield of the product API are not high, which may be caused by the instability of the amide bond in its structure under strong alkaline conditions; when triethylamine is used, it may be that its alkalinity is relatively weak and the hydroxyl group of HO-PEG3-CH2CH2COOtBu cannot be completely deprotonated, resulting in a low overall reaction yield. Overall, potassium carbonate has the best purity and yield.

[0084] The reaction process is as follows:

[0085]

[0086] Example 2

[0087] The following is an example of the synthesis method using potassium carbonate as the alkaline condition:

[0088] (1) Synthesis of intermediate 1:

[0089]

[0090] Feeding amount:

[0091] Table 2 Synthesis feed amount of intermediate 1 in Example 2

[0092]

[0093] Process Description:

[0094] Weigh Fmoc-PEG4-propionic acid, HOBt, EDCI, and DIEA, add to dichloromethane, stir and dissolve, control the temperature at 0-10°C, continue stirring for 60 minutes after complete dissolution, add diethanolamine, heat to room temperature, react for 6-8 hours, and detect by TLC (ethyl acetate: toluene = 10:1).

[0095] Post-treatment: Take 1 ml of concentrated hydrochloric acid and add it to 100 ml of water to obtain a dilute hydrochloric acid solution. Add the prepared dilute hydrochloric acid to the reaction solution, stir for 5 minutes, let the reaction solution stand in a separatory funnel for 15 minutes, and retain the DCM layer. Prepare 100 ml of 5% NaHCO3 solution, add the 5% NaHCO3 solution to DCM, stir for 5 minutes, let the reaction solution stand in a separatory funnel for 15 minutes, and retain the DCM layer. Continue to wash the DCM with 100 ml of water, add anhydrous sodium sulfate to the washed DCM and dry it for 1 hour. After filtering, the DCM is concentrated under reduced pressure at 40°C to obtain intermediate 1 with a yield of 98.8% and a purity of 99.50%. The purity determination spectrum of intermediate 1 is shown as follows: Figure 6 shown.

[0096] (2) Synthesis of intermediate 2:

[0097]

[0098] Feeding amount:

[0099] Table 3 Synthesis feed amount of intermediate 2 in Example 2

[0100]

[0101] Process Description:

[0102] Weigh intermediate 1, DCM and triethylamine, add them into a three-necked flask, stir and dissolve, place the three-necked flask in a cold trap to control the temperature at -5-0°C, dissolve p-toluenesulfonyl chloride in DCM, add the DCM solution of p-toluenesulfonyl chloride dropwise at a temperature of -5-0°C, continue the reaction for 2-4 hours after the addition is complete, and detect by TLC (dichloromethane: methanol = 10:1).

[0103] Post-treatment: Prepare 100 ml of 5% NaHCO3 solution, add the 5% NaHCO3 solution to DCM, stir for 5 minutes, let the reaction solution stand in a separatory funnel for 15 minutes, and retain the DCM layer. Continue to wash the DCM with 100 ml of saturated brine, add anhydrous sodium sulfate to the washed DCM and dry it for 1 hour. After filtering, the DCM is concentrated under reduced pressure at 40°C to obtain intermediate 2 with a yield of 100.4% and a purity of 97.562%. The purity determination spectrum of intermediate 2 is shown below: Figure 7 shown.

[0104] (3) Synthesis of API (Fmoc-NH-PEG4-CH2CH2CONbis(PEG4-CH2CH2COtBu)):

[0105]

[0106] Feeding amount:

[0107] Table 4 Synthesis feed amount of API in Example 2

[0108]

[0109] Process Description:

[0110] Weigh intermediate 2, HO-PEG3-CH2CH2COOtBu and potassium carbonate into a three-necked flask, add THF, stir and dissolve, reflux for 6-8h, and detect by TLC (ethyl acetate:n-hexane=1:2).

[0111] Post-treatment: After filtering the reaction solution, it was concentrated to dryness under reduced pressure at 40°C, dissolved in ethyl acetate, washed with water 3 times, concentrated to dryness under reduced pressure at 40°C, loaded on a silica gel column, and eluted with ethyl acetate:n-hexane=1:4 (about 0.5 L), ethyl acetate:n-hexane=1:3 (about 0.5 L), ethyl acetate:n-hexane=1:2 (about 0.5 L), ethyl acetate:n-hexane=1:1 (about 1.0 L), ethyl acetate:n-hexane=2:1 (about 0.5 L), and the product eluate was collected and concentrated under reduced pressure at 40°C to obtain API with a yield of 92.6% and a purity of 99.422%. The API purity determination spectrum is as shown below: Figure 8 As shown; based on Fmoc-PEG4-propionic acid, the overall yield was 91.9%.

[0112] This method has high purity and yield, low production cost, simple process and easy operation, and is suitable for industrial production.

[0113] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for synthesizing an API, characterized in that: The following steps are involved: Step 1: reacting raw material 1 with diethanolamine to generate intermediate 1; Step 2: reacting p-toluenesulfonyl chloride with the intermediate 1 obtained in step 1 to generate intermediate 2; Step 3: reacting HO-PEG3-CH2CH2COOtBu with the intermediate 2 generated in step 2 under alkaline conditions to obtain API; in step 3, the reaction molar ratio of intermediate 2 to HO-PEG3-CH2CH2COOtBu is 1:2; the alkaline conditions for the reaction of intermediate 2 with HO-PEG3-CH2CH2COOtBu use an inorganic base, the inorganic base is selected from potassium carbonate or sodium hydride, and the molar ratio of the inorganic base to intermediate 2 is 2-6:1; The reaction process is as follows: 。 2. The method for synthesizing the API according to claim 1, characterized in that: In step 1, raw material 1 and diethanol undergo condensation reaction in the presence of a condensation agent and an acid binding agent.

3. The method for synthesizing the API according to claim 2, characterized in that: In step 1, the reaction molar ratio of raw material 1 to diethanol is 1:1; HOBt and EDCI are used as condensing agents, and the molar ratio of HOBt, EDCI and raw material 1 is 1.0-1.5:1.0-1.5:1; DIEA is used as an acid binding agent, and the molar ratio of DIEA to raw material 1 is 2.0-3.0:1; and dichloromethane is used as a reaction solvent.

4. The method for synthesizing the API according to claim 2, characterized in that: In step 1, during the reaction, raw material 1, condensing agent and acid binding agent are first added to the reaction solvent and stirred to dissolve, and the temperature is controlled at 0-10°C. After complete dissolution, stirring is continued for 30-60 minutes; the reaction time is 6-8 hours in total.

5. The method for synthesizing the API according to claim 1, characterized in that: In step 2, intermediate 1 reacts with toluenesulfonyl chloride in the presence of an acid binding agent and a catalyst.

6. The method for synthesizing the API according to claim 5, characterized in that: In step 2, the reaction molar ratio of intermediate 1 to toluenesulfonyl chloride is 1:1.0-2.0; triethylamine is used as an acid binding agent, and the molar ratio of triethylamine to intermediate 1 is 2.0-4.0:1; DMAP is used as a catalyst, and the molar ratio of DMAP to intermediate 1 is 0.1-0.2:1; and dichloromethane is used as a reaction solvent.

7. The method for synthesizing API according to claim 5, characterized in that: In step 2, during the reaction, the intermediate 1, the acid binding agent and the catalyst are first added to the reaction solvent and stirred to dissolve, and then the p-toluenesulfonyl chloride solution is added dropwise, and the temperature is controlled at -5-5°C during the addition process; the reaction temperature is 40-50°C, and the reaction time is 2-4h.

8. The method for synthesizing API according to claim 1, characterized in that: In step 3, the reaction temperature is 65-67° C., and the reaction time is 6-8 h; the reaction product is separated and purified by silica gel column.

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