A process for synthesizing a cleavable linker Ala-Ala-Ala

Abstract

The present invention discloses a process for synthesizing an alanine-type cleavable linker of an ADC drug. Specifically, the present invention provides a process for synthesizing a compound of formula I, which specifically comprises the following steps: condensing compound 6 with compound SM3 to obtain a compound of formula II; and deprotecting the compound of formula II to obtain a compound of formula I.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a process for synthesizing a cleavable linker Ala-Ala-Ala. Background Art

[0002] (7S, 10S, 13S)-13-amino-7,10-dimethyl-6,9,12-trioxo-3-oxa-5,8,11-triazatetradecanoic acid (codenamed Ala-Ala-Ala, as shown in chemical formula Ⅰ) is a cleavable linker commonly used in multiple ADC drugs under development. It is coupled to the payload through an aminomethoxy-methylenecarbonyl (-NH-CH2-O-CH2-CO-) spacer and releases the payload under the action of tissue protease cleavage. It has very broad application prospects.

[0003]

[0004] WO2020219287 reports a milligram-scale synthesis method of Ala-Ala-Ala, wherein the starting material ((9H-fluorene-9-yl)methoxy)carbonyl)-L-alanyl-L-propylamino-L-propionylglycine is synthesized by solid phase synthesis using a standard procedure, and then reacted with lead tetraacetate to obtain (5S, 8S, 11S)-1-(9H-fluorene-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxo-2-oxa-4,7,10,1 3-tetraazatetradecane-14-yl acetate, the intermediate undergoes substitution reaction with benzyl glycolate to obtain the key intermediate (5S, 8S, 11S)-1-(9H-fluoren-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17 acid benzyl ester, the intermediate first uses morpholine to remove the fluorenylmethoxycarbonyl group, and then uses palladium carbon and hydrogen to remove the benzyl group to obtain the final product Ala-Ala-Ala. The synthesis of the starting material of this route requires solid phase synthesis, and the process production is relatively difficult; the purification of the first three steps all uses medium-pressure C18 reverse phase purification, the cost of scale-up production experiments is high, and the yield of each step of the reaction is not high; the patent application can only obtain unpurified milligram-level products, and there is no final product purification process, which is not conducive to scale-up production.

[0005] Summary of the invention

[0006] The present invention provides a process for synthesizing a compound of formula I, which is not only suitable for large-scale production but also has high yield and purity of the obtained final product (Ala-Ala-Ala).

[0007] A method for large-scale preparation of a compound of formula I, comprising:

[0008] Step (7): removing the protecting group of the compound of formula II to obtain the compound of formula I;

[0009] .

[0010] In some embodiments, the deprotection reaction of step (7) is carried out in a metal catalyst-hydrogen and solvent, preferably the metal catalyst is selected from palladium carbon, palladium hydroxide carbon, platinum carbon or nickel; the hydrogen pressure is selected from 0.001~2MPa; the solvent is selected from acetonitrile, a mixed solvent of acetonitrile and water, tetrahydrofuran, methyltetrahydrofuran, a mixed solvent of tetrahydrofuran and water, ether, dichloromethane, ethyl acetate, methanol, a mixed solvent of methanol and water, ethanol, a mixed solvent of ethanol and water, a mixed solvent of tetrahydrofuran-methanol-water or a mixed solvent of tetrahydrofuran-ethanol-water, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide or N-methylpyrrolidone; the reaction temperature is 0~100°C, preferably 20~80°C, more preferably 20~50°C, for example, room temperature reaction.

[0011] In some embodiments, the deprotection reaction is carried out in an acid and a solvent, preferably the acid is selected from trifluoroacetic acid, hydrochloric acid, sulfuric acid or p-toluenesulfonic acid; the solvent is selected from dichloromethane, tetrahydrofuran, methanol, ethanol, toluene, dioxane, ethyl acetate or not applicable statistics; the reaction temperature is 0~100°C, preferably 20~80°C, more preferably 20~50°C, such as room temperature reaction.

[0012] In some embodiments, the deprotection reaction is carried out in a base and a solvent, preferably the base is selected from dimethylamine, diethylamine, diisopropylamine, sodium hydroxide, potassium hydroxide, lithium hydroxide, lithium hydroxide hydrate, sodium bicarbonate, potassium bicarbonate, 1,8-diazabicyclo[5.4.0]undec-7-ene or 4-dimethylaminopyridine; the solvent is selected from acetonitrile, a mixed solvent of acetonitrile and water, tetrahydrofuran, methyltetrahydrofuran, a mixed solvent of tetrahydrofuran and water, dichloromethane, ethyl acetate, methanol, a mixed solvent of methanol and water, ethanol, a mixed solvent of ethanol and water, a mixed solvent of tetrahydrofuran-methanol-water or a mixed solvent of tetrahydrofuran-ethanol-water; the reaction temperature is 0-120°C, preferably 20-80°C, more preferably 20-50°C, for example, room temperature reaction.

[0013] In some embodiments, the deprotection reaction of step (7) is carried out under a hydrogen atmosphere in a metal catalyst-hydrogen and solvent, wherein the metal catalyst is palladium carbon; the reaction hydrogen pressure is 0.001-2 MPa, preferably 0.01-0.5 MPa; the solvent is a mixed solvent of tetrahydrofuran and water, preferably a mixed solvent of tetrahydrofuran and water in a volume ratio of 1-5:1, more preferably a mixed solvent of tetrahydrofuran and water in a volume ratio of 1:1; the reaction temperature is 10-50°C, preferably 20-40°C, for example 20°C, 25°C, 30°C, 35°C, 40°C.

[0014] In some embodiments, the deprotection reaction is carried out in palladium carbon and 0.04 MPa hydrogen atmosphere. In some embodiments, the yield of step (7) is 50% or more, further 90% or more. In some embodiments, the purity of the reaction product of step (7) is 50% or more, further 99% or more.

[0015] The method of the present invention further comprises step (6):

[0016] Step (6): subjecting compound 6 to a condensation reaction with compound SM3 in a condensing agent and a solvent to obtain a compound of formula II;

[0017]

[0018] Wherein R is selected from benzyloxycarbonyl, benzyl, tert-butyloxycarbonyl, fluorenylmethoxycarbonyl, allyloxycarbonyl or trityl, preferably benzyloxycarbonyl;

[0019] In some embodiments, the condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-carbonyldiimidazole, 1-propyl phosphoric anhydride, 1-butyl phosphoric anhydride, (7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate, tripyrrolidinylphosphonium bromide hexafluorophosphate, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluorophosphate. Fluoroborate, 2-(2-pyridone-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, (7-azabenzotriazole-1-oxy)tripyrrophosphonium hexafluorophosphate; the reaction solvent is selected from acetonitrile, a mixed solvent of acetonitrile and water, tetrahydrofuran, a mixed solvent of tetrahydrofuran and water, dichloromethane, N,N-dimethylformamide, a mixed solvent of N,N-dimethylformamide and water, N,N-dimethylacetamide, a mixed solvent of N,N-dimethylacetamide and water, dimethyl sulfoxide, N-methylpyrrolidone or dioxane; the reaction temperature is 0-100°C, preferably 20-80°C, more preferably 20-50°C, for example, room temperature reaction;

[0020] In some embodiments, the condensation agent in the above step (6) is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the solvent is a mixed solvent of acetonitrile and water, preferably a mixed solvent of acetonitrile and water in a volume ratio of 1-10:1, more preferably a mixed solvent of acetonitrile and water in a volume ratio of 6:1; and the reaction temperature is room temperature.

[0021] The method of the present invention further comprises step (5):

[0022] Step (5): Compound 5 is deprotected in a base and a solvent to obtain compound 6;

[0023]

[0024] The base is selected from dimethylamine, diethylamine, diisopropylamine, 4-dimethylaminopyridine, triethylenediamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylenediamine, triethylamine, diisopropylethylamine, pyridine, morpholine, N-methylmorpholine, potassium carbonate, sodium carbonate, potassium phosphate, sodium bicarbonate or potassium bicarbonate; the solvent is selected from acetonitrile, tetrahydrofuran, dichloromethane, ether, dioxane, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, methyltetrahydrofuran, methanol or ethanol; the reaction temperature is 0-100°C; preferably 20-80°C, more preferably 20-50°C, for example, room temperature reaction.

[0025] In some embodiments, in step (5), the base is selected from 1,8-diazabicyclo[5.4.0]undec-7-ene, the solvent is selected from acetonitrile, and the reaction temperature is preferably room temperature.

[0026] The method of the present invention further comprises step (4):

[0027] Step (4): Compound 4 and compound SM2 undergo a substitution reaction in a base and a solvent to obtain compound 5;

[0028]

[0029] In some embodiments, the base is selected from sodium hydroxide, potassium hydroxide, sodium tert-butoxide, potassium tert-butoxide, sodium carbonate, potassium carbonate, potassium phosphate, cesium carbonate or sodium hydride; the reaction solvent is selected from ethylene glycol dimethyl ether, ethylene glycol monomethyl ether, diethylene glycol dimethyl ether, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, methyltetrahydrofuran, dichloromethane, ether, dioxane, ethyl acetate, methanol or ethanol; the reaction temperature is -40~50°C, preferably 0~20°C, more preferably 0~10°C, for example, 5°C reaction.

[0030] In some embodiments, in step (4), the base is selected from sodium hydroxide, the solvent is selected from ethylene glycol dimethyl ether, and the reaction temperature is preferably 0-5°C.

[0031] The method of the present invention further comprises step (3):

[0032] Step (3): Compound 3 reacts with lead tetraacetate in a solvent to obtain compound 4;

[0033]

[0034] In some embodiments, the reaction solvent is selected from acetic acid, a mixed solvent of acetic acid and tetrahydrofuran, tetrahydrofuran, toluene, benzene, ether, methyltetrahydrofuran, a mixed solvent of acetic acid and toluene, a mixed solvent of acetic acid and benzene, a mixed solvent of acetic acid and ether, or a mixed solvent of acetic acid and methyltetrahydrofuran; the reaction temperature is 0-100°C; preferably 20-80°C, more preferably 40-60°C, for example, 50°C reaction.

[0035] In some embodiments, the solvent in step (3) is selected from a mixed solvent of acetic acid and tetrahydrofuran, preferably a mixed solvent in which the volume ratio of tetrahydrofuran to acetic acid is 1-10:1, and more preferably a mixed solvent in which the volume ratio of tetrahydrofuran to acetic acid is 5:1; the reaction temperature is 40-60°C.

[0036] The method of the present invention further comprises step (2):

[0037] Step (2): Compound 2 and compound SM1 undergo amine ester exchange reaction under the action of a base to obtain compound 3;

[0038]

[0039] In some embodiments, the base is selected from sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, potassium phosphate, 4-dimethylaminopyridine, triethylenediamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine, diisopropylethylamine, pyridine, sodium hydroxide, potassium hydroxide, sodium tert-butoxide, potassium tert-butoxide or cesium carbonate; the reaction solvent is selected from acetonitrile, ether, methyltetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide or N-methylpyrrolidone, a mixed solvent of acetonitrile and water, tetrahydrofuran, a mixed solvent of tetrahydrofuran and water, dichloromethane, ethyl acetate, methanol, a mixed solvent of methanol and water, ethanol, a mixed solvent of ethanol and water, a mixed solvent of tetrahydrofuran-methanol-water or a mixed solvent of tetrahydrofuran-ethanol-water; the reaction temperature is 0~100°C; preferably 0~20°C, more preferably 0~10°C, for example, 5°C reaction.

[0040] In some embodiments, the base in step (2) is selected from sodium bicarbonate; the solvent is a mixed solvent of tetrahydrofuran and water, preferably a mixed solvent of tetrahydrofuran and water in a volume ratio of 1-5:1, more preferably a mixed solvent of tetrahydrofuran and water in a volume ratio of 1:1; the reaction temperature is 0-10°C.

[0041] The method of the present invention further comprises step (1):

[0042] Step (1): Compound 1 undergoes a condensation reaction with N-hydroxysuccinimide in the presence of a condensing agent to generate an active ester compound 2;

[0043]

[0044] In some embodiments, the condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-carbonyldiimidazole, 1-propyl phosphoric anhydride, 1-butyl phosphoric anhydride, (7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate, tripyrrolidinylphosphonium bromide hexafluorophosphate, 2-( 1H-benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, 2-(2-pyridone-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, (7-azabenzotriazole-1-oxy)tripyrrolphosphonium hexafluorophosphate; the solvent is selected from tetrahydrofuran, ether, methyltetrahydrofuran, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, toluene, dimethyl sulfoxide, ethyl acetate or dioxane; the reaction temperature is 0-100°C; preferably 20-600°C, preferably 30-50°C, for example 35°C.

[0045] In some embodiments, the condensing agent in step (1) is selected from N,N'-dicyclohexylcarbodiimide, the solvent is selected from tetrahydrofuran, and the reaction temperature is 30-50°C.

[0046] The present invention also provides an intermediate for synthesizing the compound of formula I, which has the following structure:

[0047]

[0048] Wherein, R is selected from benzyloxycarbonyl, tert-butyloxycarbonyl, fluorenylmethoxycarbonyl, allyloxycarbonyl or trityl, and is preferably benzyloxycarbonyl.

[0049] The process synthesis route of the invention is simple and easy to obtain, avoids the use of solid phase synthesis, and avoids medium-pressure C18 reverse phase purification. A crystallization purification method that is easier to produce is adopted, which reduces the process cost. Not only is the total yield high, but the obtained product purity is also very high, and it is suitable for large-scale production. DETAILED DESCRIPTION

[0050] The contents of the present invention are further illustrated by specific examples below, but it should not be understood that the scope of the present invention is limited to the following examples. All methods, steps, reagents, temperatures and other features disclosed in this specification can be combined or reasonably replaced in any way, except for obvious contradictions or mutually exclusive situations, which all belong to the scope of protection of the present invention.

[0051] The meanings of the abbreviations in the present invention are shown in the following table:

[0052] HOSu N-Hydroxysuccinimide ACN Acetonitrile DCC N,N'-Dicyclohexylcarbodiimide Pd / C Palladium on Carbon DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene <![CDATA[Cu(OAc)2]]> Copper acetate <![CDATA[Pd(OAc)4]]> Lead tetraacetate DMF N,N-Dimethylformamide HOBT 1-Hydroxybenzotriazole TFA Trifluoroacetic acid EDCI 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride DCM Dichloromethane THF Tetrahydrofuran MeOH Methanol <![CDATA[NaHCO3]]> Sodium bicarbonate h Hour AcOH Acetic acid min minute NaOH Sodium hydroxide HATU 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate DIPEA N,N-Diisopropylethylamine Room temperature 20~30℃ Example 1

[0053]

[0054] At room temperature, compound 1 (12 kg, 38.5 mol) and tetrahydrofuran (120 L) were added to a 200 L reactor, and then N-hydroxysuccinimide (4.9 kg, 42.4 mol) was added to the reaction solution. The reaction system was cooled to 5°C, and then N,N'-dicyclohexylcarbodiimide (9.5 kg, 46.3 mol) was added thereto. After the addition, the reaction system was heated to 35°C and the reaction was continued at this temperature for 2 h. The remaining compound 1 was controlled to be less than 0.5% by HPLC. The reaction system was cooled to room temperature, filtered, and the filter cake was washed with tetrahydrofuran (20 L). The filtrate was collected and concentrated to 48 L. Then n-heptane (155 L) was added, stirred at room temperature for 1 h, filtered, and the filter cake was washed with n-heptane (10 L). The filter cake was dried in an oven at 40°C for 24 h to obtain compound 2 as a white solid (15.4 kg, yield: 98%, purity 95%). LCMS: ESI-MS(m / z): [M+Na]+= 431.1.

[0055] 1H NMR (400 MHz, DMSO) δ 8.12 (d, J = 7.3 Hz, 1H), 7.89 (d, J = 7.5Hz, 2H), 7.71 (t, J = 6.7 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.4Hz, 2H), 4.52 (p, J = 7.3 Hz, 1H), 4.41 – 4.28 (m, 2H), 4.24 (t, J = 6.8 Hz,1H), 2.81 (s, 4H), 1.45 (d, J = 7.4 Hz, 3H).

[0056] Example 2

[0057]

[0058] Compound SM1 (2.83 kg, 37.7 mol), tetrahydrofuran (83 L) and water (83 L) were added to a 200 L reactor, followed by sodium bicarbonate (6.33 kg, 75.4 mol) to the reaction solution, the reaction system was cooled to 5 °C, compound 2 (15.4 kg, 37.7 mol) was added thereto, and the reaction was continued at 5 °C for 5 h. HPLC showed that the remaining amount of compound 2 was less than 0.5%, ethyl acetate (40 L) was added to the reaction solution, the pH was adjusted to about 4 with 1N HCl, the liquid was separated, the aqueous phase was extracted once with ethyl acetate (40 L), the organic phases were combined, and then washed once with saturated brine (40 L), the organic layer was dried with anhydrous sodium sulfate (2 kg), and concentrated to obtain compound 3 as a white solid (12.8 kg, yield: 92%, purity 95%). LCMS: ESI-MS (m / z): [M+H]+= 369.1.

[0059] 1H NMR (400 MHz, DMSO) δ 12.56 (s, 1H), 8.15 (t, J = 5.7 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.74 (t, J = 7.3 Hz, 2H), 7.55 (d, J = 7.8 Hz, 1H),7.41 (t, J = 7.3 Hz, 2H), 7.33 (t, J = 7.1 Hz, 2H), 4.31 – 4.18 (m, 3H), 4.10(p, J = 7.2 Hz, 1H), 3.77 (qd, J = 17.5, 5.8 Hz, 2H), 1.25 (d, J = 7.2 Hz, 3H).

[0060] Example 3

[0061]

[0062] At room temperature, compound 3 (12.8 kg, 34.7 mol), tetrahydrofuran (190 L) and acetic acid (38 L) were added to a 300 L reactor. After the addition, the reaction system was heated to 45 ° C. After the system was dissolved, lead tetraacetate (24.6 kg, 55.6 mol) was slowly added. Then the reaction system was heated to 50 ° C for 30 min. HPLC showed that the remaining compound 3 was less than 0.5%. After the reaction system was cooled to room temperature, it was continued to stir for 1.5 h, filtered, and the filter cake was washed with ethyl acetate (60 L). The mother liquor obtained by filtration was washed three times with a 20% aqueous solution of trisodium citrate dihydrate (60 L). The volume of the organic phase was concentrated to about 128 L, water (38 L) was added to the organic phase, stirred for 30 min, and then water (128 L) was added dropwise. The temperature was lowered to 0 ° C. Stir for 5 h, filtered, and washed with a cold 30% tetrahydrofuran aqueous solution (25 L) Wash the filter cake, and place the filter cake in a 40°C oven to dry for 48 hours to obtain a crude pale yellow solid compound 4 (12.6 kg, yield: 95%, purity 88%). LCMS: ESI-MS (m / z): [M+Na]+ = 405.1.

[0063] 1H NMR (400 MHz, DMSO) δ 8.89 (t, J = 6.9 Hz, 1H), 7.89 (d, J = 7.5Hz, 2H), 7.73 (t, J = 6.6 Hz, 2H), 7.58 (d, J = 7.6 Hz, 1H), 7.42 (t, J = 7.2Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 5.15 – 5.03 (m, 2H), 4.31 – 4.16 (m, 3H), 4.06 (p, J = 7.2 Hz, 1H), 1.99 (s, 3H), 1.21 (d, J = 7.2 Hz, 3H).

[0064] Embodiment 4:

[0065]

[0066] At room temperature, compound 4 (12.6 kg, 32.9 mol) was added to a 300 L reactor to neutralize ethylene glycol dimethyl ether (190 L), and then compound SM2 (5.5 kg, 32.9 mol) was added to the reaction system. The reaction system was cooled to 5 ° C. The temperature was controlled, and sodium hydroxide aqueous solution (18 L, 36 mol, 2 mol / L) was slowly added dropwise to the reaction system. Stirring was performed for 1.5 h. HPLC detected that compound 4 was less than 0.5%. Acetic acid (2.1 kg, 36 mol) was added to the reaction system, and stirring was continued for 1 h. Water (130 L) and ethyl acetate (130 L) were added to the reaction system. The liquid was separated, and the organic phase was washed once with saturated brine (70 L). The organic layer was dried and concentrated, and then column chromatography was performed. The mobile phase was (petroleum ether: ethyl acetate = 10:1-2:1) to obtain compound 5 as a white solid (12.2 kg, yield: 75%, purity 97%). LCMS: ESI-MS(m / z): [M+Na]+= 511.2.

[0067] 1H NMR (400 MHz, DMSO) δ 8.76 (t, J = 6.4 Hz, 1H), 7.89 (d, J = 7.5Hz, 2H), 7.72 (t, J = 7.7 Hz, 2H), 7.60 (d, J = 7.2 Hz, 1H), 7.37 (td, J =14.8, 7.0 Hz, 9H), 5.11 (d, J = 12.8 Hz, 2H), 4.63 (d, J = 6.5 Hz, 2H), 4.23(dt, J = 11.9, 6.7 Hz, 3H), 4.14 (s, 2H), 4.07 – 3.89 (m, 1H), 1.22 (d, J =7.1 Hz, 3H).

[0068] Embodiment 5:

[0069]

[0070] At room temperature, compound 5 (6.5 kg, 13.3 mol) and acetonitrile (133 L) were added to a 200 L reactor, and then 1,8-diazabicyclo[5.4.0]undec-7-ene (1013 g, 6.6 mol) was added to the reaction system. The reaction was continued for 4 h. HPLC detected that compound 5 was less than 0.5%. Then 1-hydroxybenzotriazole (4.5 kg, 29.3 mol) was added to the reaction system in batches, stirred for 1 h, and the reaction system was cooled to 0 ° C., stirred for 10 h, filtered, and the filter cake was washed with 15 L of cold acetonitrile. It was dried in an oven at 40 ° C for 12 h to obtain a light yellow solid, which was the 1-hydroxybenzotriazole salt of compound 6 (4.8 kg, yield: 90%, purity 98%). LCMS: ESI-MS (m / z): [M+H]+= 267.1.

[0071] 1H NMR (400 MHz, DMSO) δ 9.08 (t, J = 6.3 Hz, 1H), 7.77 – 7.68 (m,1H), 7.48 – 7.27 (m, 6H), 7.27 – 7.12 (m, 2H), 5.15 (s, 2H), 4.67 (d, J = 6.4Hz, 2H), 4.18 (s, 2H), 3.64 (q, J = 7.0 Hz, 1H), 1.27 (d, J = 7.0 Hz, 3H).

[0072] Example 6

[0073]

[0074] At room temperature, compound 6 (4.8 kg, 12 mol), acetonitrile (58 L) and water (10 L) were added to a 200 L reactor, followed by compound SM3 (3.47 kg, 11.8 mol) to the reaction system, the reaction system was cooled to 5 ° C, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.5 kg, 13.2 mol) was added to the reaction, the temperature was raised to room temperature and stirred for 2 h, after HPLC detection of the reaction was complete, ethanol (10 L) and water (72 L) were added to the reaction system, stirred for 2 h, filtered, and the filter cake was washed with water (50 L), and the filter cake was dried in an oven at 40 ° C for 48 h to obtain a white solid compound 7-1 (6 kg, yield: 93%, purity 97%). LCMS: ESI-MS (m / z): [M+Na]+= 565.2.

[0075] 1H NMR (400 MHz, DMSO) δ 8.68 (t, J = 6.6 Hz, 1H), 7.99 (t, J = 6.5Hz, 2H), 7.49 (s, 1H), 7.34 (dt, J = 10.4, 2.8 Hz, 10H), 5.14 (s, 2H), 5.06 – 4.96 (m, 2H), 4.69 – 4.54 (m, 2H), 4.33 – 4.15 (m, 2H), 4.13 (s, 2H), 4.04 (p, J = 7.0 Hz, 1H), 1.20 (dd, J = 8.8, 5.3 Hz, 9H).

[0076] Example 7

[0077]

[0078] At room temperature, compound 7-1 (6 kg, 11 mol), tetrahydrofuran (120 L) and water (120 L) were added to a 300 L reactor, stirred, 10% palladium carbon (600 g) was added to the reaction system, hydrogen was replaced 3 times, and the reaction was carried out for 12 h in a 0.04 MPa hydrogen atmosphere. HPLC showed that compound 7-1 was less than 0.5%, palladium carbon was filtered, the filter cake was washed with water (50 L), the filtrate was combined, the filtrate was rotary evaporated to 24 L, ethanol (96 L) was added to the remaining aqueous phase, stirred overnight, the precipitate was filtered, the filter cake was washed with ethanol (10 L), and the filter cake was dried in an oven at 40 ° C for 48 h to obtain white solid Ala-Ala-Ala (3.1 kg, yield: 90%, purity 99.4%). ESI-MS (m / z): [M+H]+=319.1.

[0079] 1H NMR (400 MHz, D2O) δ 4.76-4.72 (m, 2H), 4.34 (dq, J = 25.4, 7.2Hz, 2H), 4.12 (q, J = 7.1 Hz, 1H), 3.93 (s, 2H), 1.57 (d, J = 7.1 Hz, 3H),1.44 (dd, J = 7.2, 1.7 Hz, 6H).

[0080] Example 8

[0081]

[0082] Step 1: Dissolve compound 6 (4 g, 10 mmol) in acetonitrile (40 mL), start stirring, add compound SM4 (3.8 g, 10 mmol) to the system, place the reaction system in an ice bath, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.1 g, 11 mmol) to the reaction, remove the ice bath, and stir for another 2 h. HPLC shows that compound 6 is less than 0.5%. Pour into water and extract with ethyl acetate. After the organic phase is concentrated, directly purify by reverse phase to obtain compound C-7, 4.2 g, yield 67%, purity 95%, LCMS: ESI-MS (m / z): [M+Na]+= 653.2.

[0083] Step 2: Add methanol (45 mL) to compound 7-2 (4.2 g, 6.7 mmol), stir, add palladium carbon (0.4 g, 10%) to the reaction system, replace hydrogen, react at 25°C for 24 hours, HPLC shows that compound 7-2 is less than 0.5%, filter palladium carbon, wash the filter cake with a large amount of water, combine the aqueous phases, concentrate the aqueous phases and directly purify by reverse phase to obtain a white solid compound of formula I, 1.2 g, yield 56%, purity 95%, ESI-MS (m / z): [M+H]+=319.1.

[0084] Example 9

[0085]

[0086] Step 1: Add N,N-dimethylformamide (100 mL) to compound 5 (20 g, 20.5 mmol), start stirring, add morpholine (1.8 g, 20.5 mmol) to the mixture, continue stirring for 1 h, HPLC shows that compound 5 is less than 0.5%, and the reaction solution is concentrated with an oil pump to obtain a pale yellow oily crude product of compound 6, 10.5 g, purity 85%, LCMS: ESI-MS (m / z): [M+H]+= 267.1.

[0087] Step 2: Dissolve the crude compound 6 (10.5 g, 39.5 mmol) in N,N-dimethylformamide (100 mL), start stirring, add compound SM3 (11.6 g, 39.5 mmol) to the system, place the reaction system in an ice bath, add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (16.5 g, 43.4 mmol) and N,N-diisopropylethylamine (6.1 g, 47.4 mmol) to the reaction, remove the ice bath, and stir for another 2 h. HPLC shows that compound 6 is less than 0.5%. Pour it into water and extract it with ethyl acetate. After the organic phase is concentrated, it is directly purified by reverse phase to obtain compound 7-1, 5.3 g, with a yield of 25% and a purity of 95%. LCMS: ESI-MS (m / z): [M+Na]+= 565.2.

[0088] Step 3: Add methanol (53 mL) to compound 7-1 (5.3 g, 9.8 mmol), stir, add palladium carbon (0.5 g, 10%) to the reaction system, replace hydrogen, react at 25°C for 24 hours, HPLC shows that compound 7-1 is less than 0.5%, after the reaction is completed, filter the palladium carbon, wash the filter cake with a large amount of water, combine the aqueous phase, concentrate the aqueous phase and directly purify by reverse phase to obtain the final product, a white solid compound of formula I, 2.1 g, yield 67%, purity 95%, ESI-MS (m / z): [M+H]+=319.1.

[0089] Example 10

[0090]

[0091] Step 1: Dissolve compound 6 (3 g, 11 mmol) in N,N-dimethylformamide (30 mL), start stirring, add compound SM4 (4.3 g, 11 mmol) to the system, place the reaction system in an ice bath, add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.7 g, 12.4 mmol) and N,N-diisopropylethylamine (1.7 g, 13.2 mmol) to the reaction, remove the ice bath, and stir for another 2 h. HPLC showed that compound 6 was less than 0.5%. Pour it into water and extract it with ethyl acetate. After the organic phase is concentrated, it is directly purified by reverse phase to obtain white solid compound 7-2, 3.5 g, with a yield of 50% and a purity of 95%. LCMS: ESI-MS (m / z): [M+Na]+= 653.2.

[0092] Step 2: Add methanol (35 mL) to compound 7-2 (3.5 g, 5.6 mmol), stir, add palladium carbon (0.35 g, 10%) to the reaction system, replace hydrogen, react at 25°C for 24 hours, HPLC shows that compound 7-2 is less than 0.5%, filter palladium carbon, wash the filter cake with a large amount of water, combine the aqueous phases, concentrate the aqueous phases and directly purify by reverse phase to obtain a white solid compound of formula I, 1 g, yield 56%, purity 95%, ESI-MS (m / z): [M+H]+=319.1.

Claims

1. A method for large-scale preparation of a compound of formula I, the method comprising: Step (6): subjecting compound 6 to a condensation reaction with compound SM3 in a condensing agent and a solvent to obtain a compound of formula II; ,in, R is selected from benzyloxycarbonyl; The condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the solvent is selected from acetonitrile, a mixed solvent of acetonitrile and water; the reaction temperature is room temperature; Step (7): removing the protecting group of the compound of formula II to obtain the compound of formula I; , wherein the deprotection reaction of step (7) is carried out under a hydrogen atmosphere in a metal catalyst-hydrogen and a solvent, the metal catalyst is palladium carbon; the hydrogen pressure is 0.001~2MPa; the solvent is a mixed solvent of tetrahydrofuran and water; the reaction temperature is 10~50°C; The yield of step (7) is above 90%.

2. The method according to claim 1, wherein the deprotection reaction is carried out under palladium carbon and 0.04 MPa hydrogen atmosphere.

3. The method according to claim 1, wherein the purity of the reaction product in step (7) is above 99%.

4. The method according to claim 1, wherein the method further comprises step (5): Step (5): Compound 5 is deprotected in a base and a solvent to obtain compound 6; The base is selected from dimethylamine, diethylamine, diisopropylamine, 4-dimethylaminopyridine, triethylenediamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylenediamine, triethylamine, diisopropylethylamine, pyridine, morpholine, N-methylmorpholine, potassium carbonate, sodium carbonate, potassium phosphate, sodium bicarbonate or potassium bicarbonate; the solvent is selected from acetonitrile, tetrahydrofuran, dichloromethane, ether, dioxane, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, methyltetrahydrofuran, methanol or ethanol; and the reaction temperature is 0~100°C.

5. The method according to claim 4, wherein the base in step (5) is selected from 1,8-diazabicyclo[5.4.0]undec-7-ene, the solvent is selected from acetonitrile, and the reaction temperature is room temperature.

6. The method according to claim 4, wherein the method further comprises step (4): Step (4): Compound 4 and compound SM2 undergo a substitution reaction in a base and a solvent to obtain compound 5; in, The base is selected from sodium hydroxide, potassium hydroxide, sodium tert-butoxide, potassium tert-butoxide, sodium carbonate, potassium carbonate, potassium phosphate, cesium carbonate or sodium hydride; the solvent is selected from ethylene glycol dimethyl ether, ethylene glycol monomethyl ether, diethylene glycol dimethyl ether, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, methyltetrahydrofuran, dichloromethane, ether, dioxane, ethyl acetate, methanol or ethanol; the reaction temperature is -40~50°C.

7. The method according to claim 6, wherein the base in step (4) is selected from sodium hydroxide, the solvent is selected from ethylene glycol dimethyl ether, and the reaction temperature is 0-5°C.

8. The method according to claim 6, wherein the method further comprises step (3): Step (3): Compound 3 reacts with lead tetraacetate in a solvent to obtain compound 4; ,in, The solvent is selected from acetic acid, a mixed solvent of acetic acid and tetrahydrofuran, tetrahydrofuran, toluene, benzene, ether, methyltetrahydrofuran, a mixed solvent of acetic acid and toluene, a mixed solvent of acetic acid and benzene, a mixed solvent of acetic acid and ether, or a mixed solvent of acetic acid and methyltetrahydrofuran; and the reaction temperature is 0-100°C.

9. The method according to claim 7, wherein the solvent in step (3) is selected from a mixed solvent of acetic acid and tetrahydrofuran; and the reaction temperature is 40-60°C.

10. The method according to claim 8, wherein the method further comprises step (2): Step (2): Compound 2 and compound SM1 undergo amine ester exchange reaction in the presence of a base and a solvent to obtain compound 3; in, The base is selected from sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, potassium phosphate, 4-dimethylaminopyridine, triethylenediamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine, diisopropylethylamine, pyridine, sodium hydroxide, potassium hydroxide, sodium tert-butoxide, potassium tert-butoxide or cesium carbonate; the solvent is selected from acetonitrile, ether, methyltetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide or N-methylpyrrolidone, a mixed solvent of acetonitrile and water, tetrahydrofuran, a mixed solvent of tetrahydrofuran and water, dichloromethane, ethyl acetate, methanol, a mixed solvent of methanol and water, ethanol, a mixed solvent of ethanol and water, a mixed solvent of tetrahydrofuran-methanol-water or a mixed solvent of tetrahydrofuran-ethanol-water; the reaction temperature is 0-100°C.

11. The method according to claim 10, wherein the base in step (2) is selected from sodium bicarbonate; the solvent is a mixed solvent of tetrahydrofuran and water; and the reaction temperature is 0-10°C.

12. The method according to claim 10, wherein the method further comprises step (1): Step (1): Compound 1 undergoes a condensation reaction with N-hydroxysuccinimide in a solvent under the action of a condensing agent to generate an active ester compound 2; in, The condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-carbonyldiimidazole, 1-propyl phosphoric anhydride, 1-butyl phosphoric anhydride, (7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate, tripyrrolidinophosphonium bromide, Hexafluorophosphate, 2-(1H-benzotriazo L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, 2-(2-pyridone-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, (7-azabenzotriazole-1-oxy)tripyrrolphosphonium hexafluorophosphate; the solvent is selected from tetrahydrofuran, ether, methyltetrahydrofuran, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, toluene, dimethyl sulfoxide, ethyl acetate or dioxane; the reaction temperature is 0-100°C.

13. The method according to claim 12, wherein the condensing agent in step (1) is selected from N,N'-dicyclohexylcarbodiimide, the solvent is selected from tetrahydrofuran, and the reaction temperature is 30-50°C.

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