A pseudo-peptide compound and its preparation method and application

Abstract

The present invention provides a pseudo-peptide compound and a preparation method and application thereof, belonging to the field of drug development and synthesis technology. The present invention specifically provides a compound having structural formula I or a stereoisomer, crystal form or salt thereof, #imgabs0#Formula I wherein: R1 is a hydrogen atom, Fmoc-, Boc-, Cbz-, Trt- or Bsmoc-. The compound of formula I of the present invention is a pseudo-peptide, which can be used to synthesize a product containing the amino acid sequence GGS, and has good solubility and stability.

Description

Technical Field

[0001] The invention relates to a pseudo-peptide compound and a preparation method and application thereof. Background Art

[0002] Peptide bonds (-CONH-) are the characteristics of peptide molecules, and peptide bonds are easily recognized and degraded by proteases in the body. Pseudopeptides replace one or more atoms in the peptide bond with other atoms, which essentially changes the chemical structure of the amide bond. It is different from the homologous structure of proteins or polypeptides, so it can avoid the recognition and hydrolysis of proteases in the body, thereby improving the stability and activity of peptide molecules.

[0003] Pseudo-peptides have great application value and advantages in drug development and preparation, mainly including the following aspects: 1. By introducing non-natural amino acid residues, changing the connection mode of peptide bonds or modifying the side chains of amino acids, a variety of structures can be created, providing a broad space for drug development; 2. Pseudo-peptides can improve the binding affinity between drug molecules and targets, help enhance the activity of drugs, reduce the dosage of drugs, and reduce the side effects of drugs; 3. Pseudo-peptides can improve the selectivity of drugs and reduce the impact on other non-target tissues and cells, thereby reducing the toxic side effects of drugs; 4. Precisely design 1. The chemical structure of the pseudo-peptide can give it specific physical and chemical properties, improve the solubility, permeability and stability of the drug, and thus improve the pharmacokinetic properties of the drug; 2. Pseudo-peptides can increase resistance to enzymes, reduce enzymatic hydrolysis, prolong the drug's action time in the body, and reduce the frequency of administration; 3. The chemical structure of the pseudo-peptide is relatively stable and is not prone to chemical reactions such as oxidation and hydrolysis, thereby improving drug stability; 4. The special structure of the pseudo-peptide can be used to design drug preparations with controlled release functions. For example, the pseudo-peptide can be combined with the drug molecule, and the release rate of the drug can be controlled by the degradation or hydrolysis of the pseudo-peptide to achieve a long-lasting effect of the drug. Summary of the invention

[0004] One object of the present invention is to provide a novel pseudo-peptide compound or its stereoisomer, crystal form or salt.

[0005] Another object of the present invention is to provide a method for preparing the pseudo-peptide compound or its stereoisomer, crystal form or salt.

[0006] Another object of the present invention is to provide the use of the pseudo-peptide compound or its stereoisomer, crystal form or salt.

[0007] In one aspect, the present invention provides a compound having structural formula I or a stereoisomer, crystalline form or salt thereof,

[0008] Formula I

[0009] in:

[0010] R1 is a hydrogen atom (H), Fmoc-, Boc-, Cbz-, Trt- or Bsmoc-.

[0011] The compound of formula I of the present invention or its stereoisomer, crystal form or salt can be used to synthesize the linker of antibody-drug conjugate. Specifically, the compound of formula I of the present invention, its stereoisomer, crystal form or salt can be used to synthesize products containing the amino acid sequence GGS, for example, it can be used to synthesize the linker H[Aib]EGT FTSDY SSYLE EQAAKEFIAW LVKGG G(GGGGS)3, which can improve the synthesis efficiency and product quality. H[Aib]EGT FTSDY SSYLE EQAAKEFIAW LVKGG G(GGGGS)3 is the linker of the drug AMG133. AMG133 is a potential "first-in-class" bispecific antibody polypeptide conjugate (GIPR antibody conjugated to GLP-1) developed by Amgen. The GLP-1 analog is chemically coupled to the GIPR antibody heavy chain through the linker, which can activate GLP-1R and inhibit GIPR (in contrast to the dual agonism of GLP-1R / GIPR by tepoxetine). AMG is currently in Phase II clinical trials, with dosing once every 4 weeks, a lower dosing frequency and excellent efficacy.

[0012] According to some specific embodiments of the present invention, the compound of formula I of the present invention is .

[0013] According to some specific embodiments of the present invention, the compound of formula I of the present invention is .

[0014] According to a specific embodiment of the present invention, the stereoisomers of the compound of formula I of the present invention include but are not limited to:

[0015] Fmoc-Gly-Gly-D-Ser[Psi(Me,Me)Pro]-OH, Gly-Gly-D-Ser[Psi(Me,Me)Pro]-OH, Boc- Gly-Gly-D-Ser[Psi(Me,Me)Pro]-OH, Cbz- Gly-Gly-D-Ser[Psi(Me,Me)Pro]-OH, Trt- Gly-Gly-D-Ser[Psi(Me,Me)Pro]-OH or Bsmoc- Gly-Gly-D-Ser[Psi(Me,Me)Pro]-OH.

[0016] According to a specific embodiment of the present invention, the crystal form of the compound of formula I of the present invention includes, but is not limited to, one or more of: disordered molecular arrangement and polymorphism, molecular conformation and polymorphism, hydrate, and solvate.

[0017] According to some specific embodiments of the present invention, the crystalline form of the compound of formula I of the present invention has an X-ray diffraction pattern of 2θ WL=1.54.

[0018] According to some specific embodiments of the present invention, the present invention provides an oily substance of the compound of formula I, whose XRD spectrum has characteristic peaks as shown in Table 1.

[0019] Table 1

[0020]

[0021] According to some specific embodiments of the present invention, the present invention provides an oily substance of a compound of formula I, whose XRD is as follows Figure 5 shown.

[0022] According to some specific embodiments of the present invention, the present invention provides a hydrate of the compound of formula I, whose XRD spectrum has characteristic peaks as shown in Table 2.

[0023] Table 2

[0024]

[0025] According to some specific embodiments of the present invention, the present invention provides a hydrate of a compound of formula I, whose XRD is as follows: Figure 6 shown.

[0026] According to some specific embodiments of the present invention, the present invention provides a solvate of the compound of formula I, whose XRD spectrum has characteristic peaks as shown in Table 3.

[0027] Table 3

[0028]

[0029] According to some specific embodiments of the present invention, the present invention provides a solvate of a compound of formula I, whose XRD is as follows: Figure 7 shown.

[0030] According to some specific embodiments of the present invention, the present invention provides the molecular conformation and polymorphic form of the compound of formula I, whose XRD spectrum has characteristic peaks as shown in Table 4.

[0031] Table 4

[0032]

[0033] According to some specific embodiments of the present invention, the present invention provides the molecular conformation and polymorphic form of the compound of formula I, whose XRD is as follows: Figure 8 shown.

[0034] According to a specific embodiment of the present invention, the salt of the compound of formula I of the present invention includes, but is not limited to, one or more of sodium salt, potassium salt, hydrochloride, and lithium salt.

[0035] In another aspect, the present invention also provides a method for preparing a compound of formula I or a stereoisomer, a crystalline form or a salt thereof, the method comprising:

[0036] R2-glycine-glycine and L-serine benzyl ester hydrochloride or L-serine methyl ester hydrochloride or L-serine tert-butyl ester hydrochloride are used as raw materials, a condensation reaction is carried out in the presence of a reaction solvent, a base and a condensation agent, and a ring-closing reaction and a hydrogenation reaction are performed to prepare the target compound (compound of formula I or its stereoisomer, crystal form or salt).

[0037] Wherein R2 represents a hydrogen atom, Fmoc-, Boc-, Cbz-, Trt-, Bsmoc- and the like and their derivative protecting groups.

[0038] According to a specific embodiment of the present invention, the method for preparing the compound of formula I or its stereoisomer, crystal form or salt of the present invention further comprises at least one of the following steps to prepare the crystal form or salt of the compound:

[0039] The hydrogenation product is purified and concentrated to no fraction to obtain an oily substance;

[0040] The hydrogenated product is purified, redissolved, crystallized, and slurried to obtain disordered molecular arrangement and polymorphs, molecular conformations and polymorphs, hydrates, or solvates;

[0041] The hydrogenation product is purified, redissolved, and then reacted with sodium methoxide, potassium methoxide, lithium hydroxide or hydrogen chloride to obtain the salt of the compound.

[0042] According to a specific embodiment of the present invention, in the method for preparing the compound of formula I or its stereoisomer, crystal form or salt thereof, during the condensation reaction, the reaction solvent is DMF.

[0043] According to a specific embodiment of the present invention, in the method for preparing the compound of formula I or its stereoisomer, crystal form or salt, during the condensation reaction, the base is DIEA or DIPEA, preferably DIEA. Preferably, the amount of the base is 2-4 eq, more preferably 2.5 eq.

[0044] According to a specific embodiment of the present invention, in the method for preparing the compound of formula I or its stereoisomer, crystal form or salt thereof, during the condensation reaction, the condensing agent is selected from one or more of HOBT, EDCI, HATU, TBTU, HBTU and TSTU. Preferably, the condensing agent is a mixture of HOBT and EDCI. More preferably, the amount of HOBT is 1-2eq, more preferably 1.1eq, and the amount of EDCI is 1.1-2.5eq, more preferably 1.1eq.

[0045] According to a specific embodiment of the present invention, in the method for preparing the compound of formula I or its stereoisomer, crystal form or salt thereof, during the condensation reaction, the amount of L-serine benzyl hydrochloride or L-serine methyl hydrochloride or L-serine tert-butyl hydrochloride is 1-2 eq, preferably 1.1 eq.

[0046] According to a specific embodiment of the present invention, in the method for preparing the compound of formula I or its stereoisomer, crystal form or salt thereof, during the condensation reaction, the temperature is maintained at -10 to 20°C for 15 to 30 minutes, and then L-serine benzyl hydrochloride or L-serine methyl hydrochloride or L-serine tert-butyl hydrochloride is added, and the temperature is naturally raised to room temperature for reaction.

[0047] According to a specific embodiment of the present invention, in the preparation method of the compound of formula I or its stereoisomer, crystal form or salt of the present invention, during the condensation reaction, TLC dot plate display or HPLC monitoring can be used to complete the reaction of the raw materials. Further, the reaction product can be added with water / EA and stirred, allowed to stand, and the organic phase is collected; the organic phase is washed with acid water, then washed with sodium bicarbonate solution, dried with anhydrous sodium sulfate, filtered, and concentrated; the concentrated solid is then filtered with petroleum ether / n-heptane: ethyl acetate / methyl tert-butyl ether / dichloromethane = 3-5:1, followed by pulping and filtration (petroleum ether / n-heptane: ethyl acetate / methyl tert-butyl ether / dichloromethane = 3-5:1). The filtered solid is placed in an oven at 40-60°C and dried to obtain an off-white solid condensation product.

[0048] According to a specific embodiment of the present invention, in the method for preparing the compound of formula I or its stereoisomer, crystal form or salt, the ring-closing reaction is carried out in the presence of a catalyst, and the catalyst is selected from 4-methylbenzenesulfonic acid pyridine, left-rotatory camphorsulfonic acid, 3-chloro-2-yl camphorsulfonic acid, Grubbs- Preferably, the catalyst is 4-methylbenzenesulfonic acid pyridine. The amount of the catalyst is 0.01-0.1eq, preferably 0.05eq.

[0049] According to a specific embodiment of the present invention, in the method for preparing the compound of formula I or its stereoisomer, crystal form or salt, during the ring-closing reaction, the auxiliary material used is selected from one or more of 2,2-dimethoxypropane and dimethoxypropylene. The auxiliary material is preferably 2,2-dimethoxypropane. The amount of the auxiliary material is 2-8 eq, preferably 3.5 eq.

[0050] According to a specific embodiment of the present invention, in the method for preparing the compound of formula I or its stereoisomer, crystal form or salt of the present invention, during the ring-closing reaction, the solvent used is selected from one or more of ethyl acetate, toluene, tetrahydrofuran, methanol and dichloromethane. The solvent is preferably ethyl acetate. The amount of the solvent is generally 5-10 times the mass of the solute, preferably about 8 times.

[0051] According to a specific embodiment of the present invention, in the method for preparing the compound of formula I or its stereoisomer, crystal form or salt, the ring-closing reaction is a temperature-raising reaction in the range of 40-80°C, preferably 55°C.

[0052] According to a specific embodiment of the present invention, in the preparation method of the compound of formula I or its stereoisomer, crystal form or salt of the present invention, during the ring-closing reaction, TLC spot plate display or HPLC monitoring can be used to monitor the completion of the raw material reaction. The material after the reaction is completed is filtered while hot, and the filtrate is used for the next step of feeding, calculated according to the theoretical amount.

[0053] According to a specific embodiment of the present invention, in the method for preparing the compound of formula I or its stereoisomer, crystal form or salt thereof, during the hydrogenation reaction, palladium carbon is used as a catalyst, and the amount of the catalyst is 0.05eq to 0.3eq, preferably 0.1eq. Preferably, the palladium carbon is 5%-10% Pd / C, more preferably 5% Pd / C.

[0054] According to a specific embodiment of the present invention, in the method for preparing the compound of formula I or its stereoisomer, crystal form or salt thereof, during the hydrogenation reaction, an appropriate amount of Pd / C may be added after 2 hours of reaction.

[0055] According to a specific embodiment of the present invention, in the method for preparing the compound of formula I or its stereoisomer, crystal form or salt, during the hydrogenation reaction, TLC spot plate display or HPLC can be used to monitor the completion of the raw material reaction.

[0056] According to a specific embodiment of the present invention, in the method for preparing the compound of formula I or its stereoisomer, crystal form or salt thereof, after the hydrogenation reaction is completed, the reaction product is filtered by suction, the filtrate is added with water, sodium bicarbonate solution or DIEA is added to adjust the pH to 8-9, and the aqueous phase is collected by static separation; the aqueous phase is adjusted to pH 3-4, and methyl tert-butyl ether is added to extract the product until it is clean; and a preliminarily purified product is obtained. The preliminarily purified product can be further washed with saturated brine and concentrated to no fraction to obtain an oily substance. The oily substance can be further dissolved with a crystallization solvent (such as isopropanol, ethanol or methanol) as needed, and then water is added at 0-30°C for crystallization, filtered by suction, and then slurried to prepare a hydrate, a solvate or a polymorph.

[0057] According to a specific embodiment of the present invention, the oily substance of the compound of formula I of the present invention has a slightly poor solubility. 1g of the oily substance needs to be added with 4g of solvent, and ultrasonic heating can completely dissolve it. The solubility of the solvate and the polymorph is good, and 1g can be completely dissolved by adding 0.5g of solvent. The solubility of the hydrate is good, and 1g of the hydrate can be completely dissolved by adding 1.5g of solvent.

[0058] In some specific embodiments of the present invention, the present invention provides a method for preparing the sodium salt of the compound of formula I, which comprises: dissolving the target substance in methanol, adding sodium methoxide to saturation, precipitating solids, and filtering to obtain the sodium salt of the target substance.

[0059] In some specific embodiments of the present invention, the present invention provides a method for preparing the potassium salt of the compound of formula I, which comprises: dissolving the target substance in methanol, adding potassium methoxide to saturation, precipitating solids, and filtering to obtain the potassium salt of the target substance.

[0060] In some specific embodiments of the present invention, the present invention provides a method for preparing the lithium salt of the compound of formula I, which comprises: dissolving the target substance in methanol, adding lithium hydroxide solution, stirring evenly, evaporating the solvent, and precipitating a solid to obtain the lithium salt of the target substance.

[0061] In some specific embodiments of the present invention, the present invention provides a method for preparing the hydrochloride of the compound of formula I, which comprises: dissolving the target substance in ethyl acetate, passing hydrogen chloride gas to gradually precipitate solids, and filtering to obtain the hydrochloride of the target substance.

[0062] The preparation method of the present invention can obtain a product with a higher purity, the purity of which can be ≥99.0%, and the single impurity is less than 0.20%. 1- The chemical synthesis reaction conditions of Gly-Gly-Ser[Psi(Me,Me)Pro]-OH are mild, green and environmentally friendly, simple and fast to operate, and convenient for large-scale production. In addition, the preparation method of the present invention can reduce the need for silica gel elution and purification, thus saving costs.

[0063] According to a specific embodiment of the present invention, the compound of formula I or its stereoisomer, crystal form or salt has good solubility and stability.

[0064] On the other hand, the present invention also provides the use of the compound of formula I or its stereoisomer, crystal form or salt.

[0065] According to a specific embodiment of the present invention, the present invention provides the use of the compound of formula I or its stereoisomer, crystal form or salt in the synthesis of a product containing the amino acid sequence GGS.

[0066] According to a specific embodiment of the present invention, the product containing the amino acid sequence GGS can be any polypeptide and / or protein containing the GGS amino acid residue sequence, or can be a linker containing the amino acid sequence GGS.

[0067] The compound of formula I of the present invention or its stereoisomer, crystal form or salt can be used to synthesize the GGS amino acid residue sequence in any polypeptide / protein. For example, it can be used to synthesize the (GGGGS)n linker (linker) in the polypeptide / protein, wherein n is usually 1, 2, 3 or 4.

[0068] According to a specific embodiment of the present invention, the present invention provides the use of the compound of formula I or its stereoisomer, crystal form or salt in the synthesis of the drug AMG133 or its linker.

[0069] According to a specific embodiment of the present invention, in the present invention, the linker of AMG133 is H[Aib]EGT FTSDYSSYLE EQAAK EFIAW LVKGG G(GGGGS)3.

[0070] The present invention also provides a method for synthesizing the drug AMG133 or its linker, the method comprising:

[0071] The linker H[Aib]EGT FTSDY SSYLE EQAAK EFIAW LVKGG G(GGGGS)3 is synthesized by using the compound of formula I of the present invention or its stereoisomer, crystal form or salt as one of the raw materials, or using the compound of formula I or its stereoisomer, crystal form or salt prepared by the method described in the present invention as one of the raw materials.

[0072] According to a specific embodiment of the present invention, the method for synthesizing the drug AMG133 or its linker can be liquid phase synthesis, solid phase synthesis, or a liquid-solid combination method.

[0073] Definition and Description

[0074] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0075] The term "pseudo-tripeptide" is not a traditional tripeptide formed by three amino acids connected by peptide bonds. It generally refers to a compound whose molecular structure simulates a tripeptide. For example, some pseudo-tripeptides appear in the ingredients of skin care products. Their structure has been modified to play a role similar to tripeptides in certain functions (such as stimulating collagen production), but their chemical composition is different from that of real tripeptides.

[0076] The term "Grubbs-II" refers to a ruthenium carbene complex catalyst discovered by Nobel Prize winner in Chemistry Robert H. Grubbs.

[0077] The term "extraction" refers to the process of transferring the target substance from one solvent to another by selecting an appropriate solvent, taking advantage of the different solubility of the components in the system. Extraction can be divided into two main types: liquid-liquid extraction and solid-liquid extraction.

[0078] The term "Trituration" is a purification method between washing and recrystallization, which uses the difference in solubility of the product and impurities in a single solvent (or mixed solvent) for purification.

[0079] The term "crystallization" refers to the process by which an organic compound precipitates from a solution into crystals under appropriate conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 This is the HPLC purity detection spectrum of Compound 1 of Experimental Example 1 of the present invention.

[0081] Figure 2 This is the HNMR spectrum of Compound 1 of Experimental Example 1 of the present invention.

[0082] Figure 3 This is the HPLC purity detection spectrum of Compound 1 in Experimental Example 20 of the present invention.

[0083] Figure 4 This is the HPLC purity detection spectrum of Compound 2 in Experimental Example 21 of the present invention.

[0084] Figure 5 The XRD pattern of the oily compound 1 of the present invention.

[0085] Figure 6It is the XRD pattern of the hydrate of compound 1 of the present invention.

[0086] Figure 7 It is the XRD pattern of the solvate of compound 1 of the present invention.

[0087] Figure 8 The XRD pattern of the molecular conformation and polymorphic form of compound 1 of the present invention. DETAILED DESCRIPTION

[0088] The embodiments of the present invention will be described in detail below in conjunction with the examples (experimental examples), but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0089] The structures of the compounds were determined by NMR, mass spectroscopy or infrared.

[0090] Experimental Example 1: Fmoc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH (Compound 1)

[0091] In this experimental example, Fmoc-L-glycine-L-glycine, L-serine benzyl ester hydrochloride, DIEA, and DMF were used as standard substrates to prepare (4S)-3-[1-(9H-fluoren-9-yl)-3,6,9-trioxylidene-4,7-diaza-2-oxanonan-9-yl]-2,2-dimethyl-1,3-oxazacyclopentane-4-carboxylic acid, i.e., Fmoc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH (Compound 1).

[0092] The synthetic route is as follows:

[0093]

[0094] The specific steps include:

[0095] 1) Add Fmoc-L-glycine-L-glycine (10 kg) into N,N-dimethylformamide (70 kg) and cool to below 10°C;

[0096] HOBT (4.2 kg), EDCI (5.6 kg), and DIEA (9.1 kg) were added in sequence, and the reaction was maintained at -10 to 20 °C for 30 mins. Then, L-serine benzyl ester hydrochloride (7.2 kg) was added, and the temperature was naturally raised to room temperature for reaction;

[0097] The reaction of the raw material Fmoc-L-glycine-L-glycine is completed by TLC spot plate display or HPLC monitoring (developing solvent is dichloromethane to methanol = 10:1), water (320kg): EA (96kg) = 5:1.5 is added to the reaction solution and stirred, and the mixture is allowed to stand, and the organic phase is collected; the organic phase is washed with 0.5N acid water twice, 40kg each time, and then washed with 5% sodium bicarbonate solution twice, 40kg each time, dried over anhydrous sodium sulfate, filtered, and concentrated at 30-50°C; the concentrated solid is then filtered with preferably petroleum ether: ethyl acetate = 4:1, 80kg / 20kg, and then pulped and filtered (preferably petroleum ether: ethyl acetate = 4:1, 80kg / 20kg). The filtered solid was placed in an oven and dried at 40-60°C to obtain 14.5 kg of off-white solid N-[1-(9H-fluoren-9-yl)-3,6,9-trioxy-4,7-diaza-2-oxanonan-9-yl]-DL-serine benzyl ester (intermediate product 1-1) with a yield of 96.8%.

[0098] 2) Add N-[1-(9H-fluorene-9-yl)-3,6,9-trioxy-4,7-diaza-2-oxanon-9-yl]-DL-serine benzyl ester obtained in the previous step to ethyl acetate (162 kg), add 4-methylbenzenesulfonate pyridine (0.35 kg), and then add 2,2-dimethoxypropane (6.8 kg), and heat the reaction in the range of 40-80°C, preferably 55°C;

[0099] The TLC spot plate showed that the reaction of the raw material N-[1-(9H-fluoren-9-yl)-3,6,9-trioxy-4,7-diaza-2-oxanon-9-yl]-DL-serine benzyl ester was completed, and it was filtered while hot. The filtrate (({2-[(2-{4-[(benzyloxy)carbonyl]-2,2-dimethyl-1,3-oxazacyclopent-3-yl}-2-oxyylideneethyl)amino]-2-oxyylideneethyl}amino)methane acid-9H-fluoren-9-ylmethyl ester) (intermediate product 2-1) was fed to the next step according to the theoretical amount.

[0100] 3) Add 5% Pd / C 0.8 kg to the filtrate of the previous step ({2-[(2-{4-[(benzyloxy)carbonyl]-2,2-dimethyl-1,3-oxazacyclopent-3-yl}-2-oxyylideneethyl)amino]-2-oxyylideneethyl}amino)methane acid-9H-fluoren-9-ylmethyl ester, replace the gas with nitrogen 3 times, replace the gas with hydrogen 3 times, react for 2 hours, add 5% Pd / C (0.8 kg), replace the gas with hydrogen 3 times, and continue to react for 2 hours;

[0101] TLC spot plate showed that the reaction of the starting material ({2-[(2-{4-[(benzyloxy)carbonyl]-2,2-dimethyl-1,3-oxazacyclopent-3-yl}-2-oxyylideneethyl)amino]-2-oxyylideneethyl}amino)methanoic acid-9H-fluoren-9-ylmethyl ester was complete;

[0102] Filter by suction, add 100 kg of water to the filtrate, add sodium bicarbonate solution or DIEA to adjust the pH to 8-9, collect the aqueous phase by static separation; adjust the pH of the aqueous phase to 3-4 with 1N brine, add 100 kg of methyl tert-butyl ether to extract the product until it is clean; wash twice with saturated brine (125 kg); concentrate until there is no fraction, add 32.5 kg of preferably isopropanol to dissolve, add water (195 kg) to crystallize at 20°C, filter by suction; then slurry (n-heptane: dichloromethane = 3:1) (90 kg: 30 kg);

[0103] Finally, 13.0 kg of (4S)-3-[1-(9H-fluorene-9-yl)-3,6,9-trioxy-4,7-diaza-2-oxanon-9-yl]-2,2-dimethyl-1,3-oxazacyclopentane-4-carboxylic acid was obtained (HPLC purity test see Figure 1 , HNMR spectrum see Figure 2 ), the yield is 95.7%.

[0104] Experimental Examples 2 to 5

[0105] The condensing agent HOBT / EDCI in step 1) of Experimental Example 1 was replaced with HATU (Experimental Example 2), TBTU / HOBT (Experimental Example 3), HBTU / HOBT (Experimental Example 4), and TSTU (Experimental Example 5), respectively, and the other reaction conditions remained unchanged to synthesize Fmoc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH (Compound 1).

[0106] The product purity and yield of Experimental Examples 1 to 5 are shown in Table 5.

[0107] Table 5

[0108]

[0109] In the synthetic routes of Experimental Examples 1 to 5 of the present invention, the purity of the post-treatment of step 1) reaction will determine the purity of the final product, so the purity of the condensation is crucial.

[0110] Experimental Examples 6 to 8

[0111] The catalyst 4-methylbenzenesulfonate pyridine in step 2) of Experimental Example 1 was replaced by L-camphorsulfonic acid (Experimental Example 6), 3-camphorsulfonic acid (Experimental Example 7) or Grubbs- (Experimental Example 8), the other reaction conditions remained unchanged, and Fmoc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH (Compound 1) was synthesized.

[0112] The product purity of Experimental Examples 1 and 6 to 8 is shown in Table 6.

[0113] Table 6

[0114]

[0115] Experimental Example 9

[0116] The auxiliary material 2,2-dimethoxypropane in step 2) of Experimental Example 1 was replaced with dimethoxypropylene (Experimental Example 9), and the other reaction conditions remained unchanged to synthesize Fmoc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH (Compound 1).

[0117] The remaining raw materials of Experimental Examples 1 and 9 are shown in Table 7.

[0118] Table 7

[0119]

[0120] Experimental Examples 10 to 13

[0121] The solvent ethyl acetate in step 2) of Experimental Example 1 was replaced with toluene (Experimental Example 10), tetrahydrofuran (Experimental Example 11), methanol (Experimental Example 12), and dichloromethane (Experimental Example 13), respectively, and the other reaction conditions remained unchanged to synthesize Fmoc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH (Compound 1).

[0122] The product purity of Experimental Example 1 and Experimental Examples 10-13 is shown in Table 8.

[0123] Table 8

[0124]

[0125] Experimental Examples 14 to 16

[0126] The palladium carbon equivalent in step 3) of Experimental Example 1 was replaced with 0.05eq (Experimental Example 14), 0.2eq (Experimental Example 15), and 0.3eq (Experimental Example 16), respectively, and the reaction time was adjusted with ice. The other reaction conditions remained unchanged to synthesize Fmoc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH (Compound 1).

[0127] The reaction conditions of each experimental example are shown in Table 9.

[0128] Table 9

[0129]

[0130] As can be seen from the table above, the amount of 5% palladium carbon will affect the yield. In addition, the preferred feeding method of the present invention is to add once after 2 hours of reaction (the amount added is 0.1 eq). If only 0.1 eq (0.8 kg) of palladium carbon is added at one time, part of the raw material will be decomposed into impurities, which will also affect the yield.

[0131] Experimental Examples 17 to 19

[0132] The solvent volumes in step 3) of Experimental Example 1 were replaced with 1 V (Experimental Example 17), 2 V (Experimental Example 18), and 3 V (Experimental Example 19), respectively, and the reaction time was adjusted with ice. The other reaction conditions remained unchanged to synthesize Fmoc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH (Compound 1).

[0133] The reaction conditions of each experimental example are shown in Table 10.

[0134] Table 10

[0135]

[0136] Experimental Example 20: Fmoc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH (Compound 1)

[0137] Synthesis route:

[0138]

[0139] The specific steps include:

[0140] 1) Preparation of intermediate 1-2:

[0141]

[0142] Add Fmoc-L-glycine-L-glycine (10 kg) to N,N-dimethylformamide (70 kg) and cool to below 10°C;

[0143] HOBT (4.2 kg), EDCI (5.6 kg), and DIPEA (9.1 kg) were added in sequence, and the reaction was maintained at -10 to 20 °C for 30 mins. Finally, L-serine methyl ester hydrochloride (6.2 kg) was added, and the temperature was naturally raised to room temperature for reaction;

[0144] The TLC spot plate showed that the reaction of the raw material Fmoc-L-glycine-L-glycine was complete. Water (320 kg): EA (96 kg) was added to the reaction solution and stirred. The mixture was allowed to stand and the organic phase was collected. The organic phase was washed twice with 0.5N acid water and then washed twice with 5% sodium bicarbonate solution, dried, filtered, and concentrated at 30-50°C. The concentrated solid was filtered with petroleum ether: ethyl acetate = 80 kg / 20 kg, and then pulped and filtered with petroleum ether: ethyl acetate = 80 kg / 20 kg. The filtered solid was dried in an oven at 40-60°C to obtain 13.1 kg of an off-white solid, namely intermediate 1-2, with a yield of 93.2%.

[0145] Using Fmoc-L-glycine-L-glycine, L-serine methyl ester hydrochloride, DMF, and condensing agent as standard substrates, the base in the reaction was studied without changing the temperature and reaction time. The results are shown in Table 11.

[0146] Table 11

[0147]

[0148] 2) Preparation of intermediate 2-2:

[0149]

[0150] The intermediate 1 obtained in the previous step was added to toluene / tetrahydrofuran / ethyl acetate (162 kg) (preferably ethyl acetate), 4-methylbenzenesulfonate pyridine (0.31 kg) was added, and then 2,2-dimethoxypropane (5.6 kg) was added, and the temperature was raised to react;

[0151] The TLC plate showed that the raw material reaction was complete, and the filtrate was filtered while hot and concentrated to dryness.

[0152] The catalyst in the reaction was studied using intermediate 1, ethyl acetate, and 2,2-dimethoxypropane at a constant temperature. The results are shown in Table 12.

[0153] Table 12

[0154]

[0155] Using the intermediate 1,4-methylbenzenesulfonate pyridine, the solvent in the reaction was studied under the condition of constant temperature. The results are shown in Table 13.

[0156] Table 13

[0157]

[0158] The solvent in the reaction was studied using the intermediate 1,4-methylbenzenesulfonate pyridine and 2,2-dimethoxypropane at a constant temperature. The results are shown in Table 14.

[0159] Table 14

[0160]

[0161] This step takes a long time to react, which will result in a more complex reaction. TLC monitoring is required at any time. When the raw material reaction is complete, it is processed immediately. The present invention uses TLC monitoring for purification, avoiding the need for column brushing for purification.

[0162] 3) Preparation of intermediate 3-2:

[0163]

[0164] Add tetrahydrofuran / ethyl acetate / methanol / DMF / dichloromethane (preferably tetrahydrofuran, 150 kg) to the concentrated intermediate 2-2 of the previous step to dissolve, add lithium hydroxide / sodium hydroxide / sodium carbonate / DIEA solution (preferably lithium hydroxide, 800 g) to react for 2-4 hours at a temperature of 5-35°C;

[0165] TLC plate showed that the reaction of the raw material intermediate 2-2 was complete.

[0166] The reaction solution was adjusted to pH 6-7 with 1N acid water, solids were precipitated, filtered, and the solids were slurried with acetonitrile / methyl tert-butyl ether / ethyl acetate (preferably acetonitrile, 80 kg), filtered, and dried to obtain 10 kg of intermediate 3-2.

[0167] Using intermediate 3-2, tetrahydrofuran, and the reaction temperature unchanged, the type of base added in the reaction was studied. The results are shown in Table 15.

[0168] Table 15

[0169]

[0170] From the above table, we can see that the type of base will affect the yield and the length of the reaction time.

[0171] With intermediate 3-2, reaction temperature and type of base unchanged, the addition of solvents in the reaction was studied and the results are shown in Table 16.

[0172] Table 16

[0173]

[0174] From the above table, it can be seen that the solvent will also affect the yield and reaction time, and some reaction solvents will even consume the base, resulting in an increase in the amount of base used.

[0175] Taking intermediate 3-2 as an example, the type of crystallization solvent added in the reaction was studied under the condition that the crystallization temperature remained unchanged. The results are shown in Table 17.

[0176] Table 17

[0177]

[0178] It can be seen from the above table that different crystallization solvents affect the crystal form of the product, which will also bring unnecessary difficulties to production, cause the loss of part of the product, and affect the purity of the product.

[0179] 4) Preparation of target object:

[0180]

[0181] Add tetrahydrofuran / ethyl acetate / methanol / DMF / dichloromethane (preferably tetrahydrofuran, 150 kg) to dissolve the intermediate 3-2, then add Fmoc-OSU (11.7 kg), add sodium bicarbonate / sodium hydroxide / sodium carbonate / DIEA solution (preferably DIEA, 800 g) and react for 6 hours at 15°C;

[0182] The TLC spot plate showed that the reaction of the raw material intermediate 3 was complete;

[0183] The reaction solution was adjusted to pH 6 with 1N acid water, solids were precipitated, filtered, and the solids were slurried with acetonitrile / methyl tert-butyl ether / ethyl acetate / isopropanol / methanol (preferably isopropanol), filtered, and dried to obtain 11.7 kg of finished product with a purity of 99% and a yield of 90%, i.e., the target final product pseudo-tripeptide (HPLC purity test spectrum see Figure 3 ).

[0184] Experimental Example 21: Cbz-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH (Compound 2)

[0185] Synthesis route:

[0186]

[0187] The specific steps include:

[0188] 1) Preparation of intermediate 2-1:

[0189]

[0190] Add Cbz-L-glycine-L-glycine (10 kg) to N,N-dimethylformamide (70 kg), cool down, and then add HOBT (3.8 kg), EDCI (4.5 kg), DIEA (7.3 kg), L-serine benzyl ester hydrochloride (8.7 kg)

[0191] 2) TLC spot plate showed that the reaction of the raw materials was complete. Water (320kg):EA (96kg) was added to the reaction solution, stirred, allowed to stand, and the organic phase was collected; the organic phase was washed with 0.5N acid water, then washed with 5% sodium bicarbonate solution, dried, filtered, and concentrated; petroleum ether:ethyl acetate = 80kg / 20kg was added to the concentrate, filtered, and then slurried, petroleum ether:ethyl acetate = 80kg / 20kg, filtered, and dried to obtain an off-white solid.

[0192] 2) Preparation of intermediate 2-2:

[0193]

[0194] Add intermediate 2-2 to ethyl acetate (162 kg), add 4-methylbenzenesulfonate pyridine (0.28 kg), and then add 2,2-dimethoxypropane (6.8 kg), and heat to react; TLC spot plate shows that the raw material reaction is complete, filter while hot, and use the filtrate for the next step of feeding according to the theoretical amount.

[0195] 3) Preparation of intermediate 2-3:

[0196]

[0197] The filtrate from the previous step was added with 5% Pd / C (0.6 kg), and the atmosphere was replaced with nitrogen and hydrogen; the TLC plate showed that the reaction of the raw materials was complete;

[0198] Filter with suction, add water to the filtrate, add sodium bicarbonate solution to adjust the pH to 8-9, collect the aqueous phase; adjust the pH of the aqueous phase to 3-4, add methyl tert-butyl ether to extract the product until it is clean; wash with saturated brine (125 kg); concentrate until there is no fraction, add methyl tert-butyl ether for crystallization, filter with suction, and dry to obtain 9.3 kg of solid.

[0199] 4) Preparation of target object:

[0200]

[0201] The intermediate 2-3 was added with tetrahydrofuran (130 kg) to dissolve, and Cbz-OSU (8.3 kg) and DIEA solution (600 g) were added to react; TLC plate showed that the reaction of the raw intermediate 3 was complete.

[0202] The reaction solution was adjusted to pH 6-7 with acid water, solids were precipitated, filtered, and the solids were slurried with isopropanol, filtered, and dried to obtain 12.3 kg of finished product with a purity of 97.6% and a yield of 93%, i.e., the target product (HPLC purity test spectrum see Figure 4 ).

[0203] Experimental Example 22: Bsmoc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH (Compound 3)

[0204] Synthesis route:

[0205]

[0206] Bsmoc-L-glycine-L-glycine is added to N,N-dimethylformamide, the temperature is lowered, and then HOBT, EDCI, DIEA, and L-serine benzyl hydrochloride are added. After the reaction is completed, water and EA are added to the reaction solution and stirred, and the organic phase is collected; the organic phase is washed with acid water and sodium bicarbonate solution, dried, filtered, and concentrated; petroleum ether:ethyl acetate is added for crystallization, filtered, and dried to obtain an off-white solid intermediate. The intermediate is added to ethyl acetate, 4-methylbenzenesulfonate pyridine is added, 2,2-dimethoxypropane is added, and the temperature is raised to react; after the reaction is completed, the product is filtered while hot, 5% Pd / C is added to the filtrate, and nitrogen and hydrogen are replaced. After the reaction is completed, the product is filtered, water is added to the filtrate, and sodium bicarbonate solution is added to adjust the pH to 8-9, and the aqueous phase is collected; the aqueous phase is adjusted to pH 3-4, and ethyl acetate is added to extract the product until it is clean; the product is washed with saturated brine, concentrated to no fraction, and isopropanol is added to crystallize, filtered, and dried to obtain the target product Bsmoc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH.

[0207] Experimental Example 23: Boc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH (Compound 4)

[0208] Synthesis route:

[0209]

[0210] Add Boc-L-glycine-L-glycine to N,N-dimethylformamide, cool down, then add HOBT, EDCI, DIEA, L-serine benzyl ester hydrochloride, react, add water and EA to the reaction solution, stir, collect the organic phase; wash the organic phase with acid water and sodium bicarbonate solution, dry, filter, and concentrate; add petroleum ether: ethyl acetate for crystallization, filter, and dry to obtain an off-white solid intermediate. Add the intermediate to ethyl acetate, add 4-methylbenzenesulfonic acid pyridine, add 2,2-dimethoxypropane, and heat to react; after the reaction is completed, filter while hot, add 5% Pd / C to the filtrate, replace with nitrogen and hydrogen, after the reaction is completed, filter, add water to the filtrate, add sodium bicarbonate solution to adjust the pH to 8-9, collect the aqueous phase; adjust the acidity of the aqueous phase, add ethyl acetate to extract the product; wash with saturated brine, concentrate until there is no fraction, add isopropanol for crystallization, filter, and dry to obtain the target Boc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH.

[0211] Experimental Examples 24 to 28

[0212] With different raw materials and similar operating steps to Experimental Example 1, the yield of the final product was studied. The results are shown in Table 18.

[0213] Table 18

[0214]

[0215] It can be seen from the above table that the purity and yield of the finished products obtained from different starting materials are slightly different. The best choice is Fmoc-L-glycine-L-glycine and L-serine benzyl ester hydrochloride as the starting materials.

[0216] Experimental Example 29, Preparation Example of Crystal Form

[0217] In Experimental Example 1, step 3) is concentrated until there is no fraction, to obtain an oily substance.

[0218] The oily substance was dissolved in water / methanol (1:4), stirred and precipitated to obtain a hydrate.

[0219] The oily substance was dissolved in n-heptane / methyl tert-butyl ether (4:1) to precipitate and obtain a solvate.

[0220] The oily substance was dissolved in isopropanol, cooled, and polymorphs were precipitated.

[0221] The XRD pattern of the oily substance in this experimental example is shown in Figure 5 .

[0222] The XRD pattern of the hydrate in this experimental example is shown in Figure 6 .

[0223] The XRD pattern of the solvate of this experimental example is shown in Figure 7 .

[0224] The XRD patterns of the molecular conformation and polymorphic forms of the structure of this experimental example are shown in Figure 8 .

[0225] Experimental Example 30: Compound 1 is used to prepare AMG133

[0226] This experimental example provides a method for preparing the drug AMG133 using the solvate of the compound of formula I of Experimental Example 29 of the present invention, specifically the synthesis of the linker H[Aib]EGT FTSDY SSYLE EQAAK EFIAWLVKGG G(GGGGS)3 for the drug AMG133. The specific process includes:

[0227] 1) Preparation of Fmoc-Aib-CTC Resin

[0228] Take 2-Cl-CTC resin and add it to a solid phase reactor, add DMF to swell the resin; drain the solvent, add DMF solution of Fmoc-Aib-OH and DIEA to stir the reaction, drain the solvent, add DIEA / methanol / DCM solution, and seal; wash with DCM and methanol alternately, and obtain Fmoc-Aib-CTC Resin after vacuum drying;

[0229] 2) Preparation of the first fragment

[0230] Take Fmoc-Aib-CTC Resin, add it to the reaction column, add DMF to swell, drain the solvent, add piperidine / DMF solution to react; drain the solvent, add DMF to wash; indene test is positive; take Fmoc-Glu (OtBu) -OH, HOBt, DIC, dissolve in DMF, activate in ice bath; add activation solution to the reaction column to react, after the indene test result is negative, drain the solvent; add DMF to wash; repeat the above steps, and add Fmoc-Gly-OH, Fmoc-Thr (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Thr(OtBu)-OH, Boc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(OtBu)-OH, Boc-Ser(tBu)-OH, Boc-Se r(tBu)-OH, Fmoc-Tyr(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(trt)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Glu(OtBu)-OH are subjected to coupling reaction; after the coupling is completed, DCM and methanol are washed alternately; vacuum drying is performed to obtain the first fragment peptide resin of AMG133, and the peptide resin is added to TFE / DCM for reaction; the filtrate is collected and the filtrate is vacuum-dried to obtain the first fragment of AMG133;

[0231] 3) Preparation of Fmoc-Phe-PS-Wang Resin

[0232] Take 2-Cl-CTC resin and add it to a solid phase reactor, add DMF to swell the resin; drain the solvent, add DMF solution of p-hydroxybenzyl alcohol, stir evenly, add DIEA, react at 30°C for 2h, then add MeOH to the reaction solution to block the Cl that has not been completely reacted, and wash the resin with DMF after completion; add DMF solution of Fmoc-Phe-OH and DIEA to stir and react, drain the solvent, add DIEA / methanol / DCM solution to block; wash with DCM and methanol alternately, and obtain Fmoc-Phe-PS-Wang Resin after vacuum drying;

[0233] 4) Preparation of the second fragment

[0234] Take Fmoc-Phe-PS-Wang Resin, add it to the reaction column, add DMF to swell, drain the solvent, add piperidine / DMF solution to react; drain the solvent, add DMF to wash; indene test is positive; take Fmoc-Ile-OH, HOBt, DIC, dissolve them in DMF, and activate them in an ice bath; add the activation solution to the reaction column to react, and after the indene test result is negative, drain the solvent; add DMF to wash; repeat the above steps, and add Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc- Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Gly-Gly-OH, Fmoc-Gly-Gly-OH, and Fmoc-Gly-Gly-Ser[Psi(Me,Me)Pro]-OH are subjected to coupling reaction; after the coupling is completed, DCM and methanol are washed alternately; vacuum drying is performed to obtain the second fragment peptide resin of AMG133, and the peptide resin is added to 3% TFA / DCM for reaction; the filtrate is collected and the filtrate is vacuum-dried to obtain the second fragment of AMG133;

[0235] 5) Preparation of AMG133 fully protected peptide

[0236] The first fragment, the second fragment, and PyBoP were dissolved in a mixed solution of DMF / DCM, and the pH was adjusted to about 8 using DIEA, and the reaction was carried out for 3 hours. After completion, the mixture was rotary evaporated to an oily state, dissolved in ethyl acetate, and the organic phase was alternately washed with 5% NaHCO3 and 5% citric acid aqueous solutions, and then washed with pure water, and the fully protected peptide AMG133 was obtained after rotary evaporation and drying.

[0237] 6) AMG133 preparation

[0238] Prepare a lysis solution of TFA / TIS / m-cresol = 90 / 5 / 5, add peptide resin to react, filter after completion, take the filtrate, add the filtrate to isopropyl ether for precipitation, centrifuge and dry to obtain crude AMG133, and purify and prepare to obtain finished product AMG133.

[0239] The compound of formula I of the present invention has the characteristics of high purity and low isomers. Compared with the compound of formula I with low purity, the synthesis efficiency and quality of the linker (Linker) H[Aib]EGT FTSDY SSYLE EQAAK EFIAW LVKGG G(GGGGS)3 can be improved.

[0240] The above description is only a specific implementation of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a compound of formula I' or a crystal form, hydrate, solvate or salt thereof, characterized in that: The method includes: Formula I' In formula I', R1 is Fmoc-, Boc-, Cbz-, or Bsmoc-; Using R2-glycine-glycine and L-serine benzyl ester hydrochloride or L-serine methyl ester hydrochloride or L-serine tert-butyl ester hydrochloride as raw materials, a condensation reaction is carried out in the presence of a reaction solvent, a base and a condensation agent, and a ring-closing reaction and a hydrogenation reaction are performed to prepare the target compound; wherein R2 is selected from Fmoc-, Boc-, Cbz- or Bsmoc-; The method for preparing the crystalline form, hydrate, solvate or salt of the compound further comprises the following steps: The hydrogenation product is purified and concentrated to no fraction to obtain an oily substance; The oil was dissolved in 1:4 water / methanol and stirred to precipitate to obtain a hydrate; The oil was dissolved in 4:1 n-heptane / methyl tert-butyl ether to precipitate and obtain a solvate; The oily substance is dissolved by isopropanol, cooled, and a polymorph is precipitated. The polymorph has the molecular conformation and polymorphic form of the structure with characteristic peaks shown in Table 4; The hydrogenated product is purified and redissolved, and then reacted with sodium methoxide, potassium methoxide, lithium hydroxide or hydrogen chloride to obtain a salt of the compound; in: During the condensation reaction, the reaction solvent is DMF; the base is DIEA; the condensation agent is selected from HOBT / EDCI; The ring-closing reaction is carried out in the presence of a catalyst, wherein the catalyst is selected from 4-methylbenzenesulfonate pyridine; During the ring-closing reaction, the auxiliary material used is selected from one or more of 2,2-dimethoxypropane and dimethoxypropylene; the solvent used is selected from ethyl acetate; During the hydrogenation reaction, palladium on carbon is used as a catalyst.

2. The method according to claim 1, characterized in that During the hydrogenation reaction, the amount of catalyst used is 0.05eq to 0.3eq.

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