Synthesis of celacarfurine and key intermediates thereof

Abstract

The application provides a synthesis method of celacarfurine and key intermediates thereof, comprising the following steps: taking 4-amino butyric acid methyl ester hydrochloride as a starting material, sequentially performing ortho-nitrobenzenesulfonyl protection, alkylation, amide condensation, hydrolysis and deprotection, intramolecular cyclization, deprotection of ortho-nitrobenzenesulfonyl, and optional acylation to obtain a target product. The synthesis route is designed by an innovative strategy, and overcomes technical bottlenecks such as difficulty in obtaining raw materials and reagents, long steps, harsh conditions and low yield which exist in the existing macrocycle synthesis method, and the target product is prepared under mild conditions with high yield, simplicity and safety. The method is simple in operation, mild in conditions, and does not need special equipment, has significant industrial application potential, and provides a stable and reliable material basis for subsequent pharmacological research and new drug development of celacarfurine.

Description

Technical Field

[0001] This invention relates to the fields of organic synthesis and medicinal chemistry, and in particular to methods for synthesizing celacarfurine and its key intermediates. Background Technology

[0002] Macrocyclic spermidine alkaloids belong to the macrocyclic polyamine alkaloids and are commonly found in plants, bacteria, and marine organisms. They are a class of natural products with various pharmacological activities such as antiparasitic, antiplasmid DNA breakage, and anti-inflammatory effects. Their structural feature is that the macrocyclic skeleton contains two or more amino units, and they have broad application prospects in the field of drug development.

[0003] Tripterygium wilfordii ( Tripterygium wilfordii Hook.f., a representative medicinal plant of the Celastraceae family, has shown clear efficacy in treating autoimmune diseases such as rheumatoid arthritis, chronic nephritis, and lupus erythematosus. The natural product celacarfurine is a unique thirteen-membered macrocyclic alkaloid, first discovered and reported by the inventors' research group from Tripterygium wilfordii (reference: Jianqun Liu, Qiushan Wu, Jicheng Shu, Rui Zhang, Lifang Liu. A Novel Spermidine Macrocyclic Alkaloid from the Roots of Tripterygium wilfordii[J]. Chemistry of Natural Compounds, 2020, 56(3):496-499), with the molecular formula C. 21 H 25 N3O4. Currently reported thirteen-membered ring spermidine alkaloids generally contain only two amide bonds on the thirteen-membered ring. Unlike known thirteen-membered macrocyclic spermidine alkaloids, celacarfurine is characterized by having three amide bonds on the thirteen-membered ring, as shown in Formula I below.

[0004]

[0005] The key intermediate in the synthesis of celacarfurine is 2-phenyl-1,5,9-triazacyclotetrazane-4,13-dione, with the molecular formula C10. 16 H 23 N3O2 has the chemical structure shown in Formula II below.

[0006]

[0007] Currently, chemical synthesis methods for thirteen-membered macrocyclic spermidine alkaloids containing two amide bonds have been reported. However, these methods generally suffer from difficulties in sourcing raw materials and reagents (which are difficult to purchase commercially), lengthy procedures, low overall yields, and dependence on special reagents or harsh reaction conditions (such as ultra-low temperature environments), which severely restrict their large-scale application. Furthermore, no total synthesis method has been reported for the more structurally complex celacarfurine.

[0008] Studies have shown that celacarfurine possesses anti-inflammatory and other biological activities; however, its content in Tripterygium wilfordii is low, resulting in a very limited source. This "source bottleneck" significantly hinders in-depth pharmacological research and new drug development targeting this natural active compound. Therefore, developing an efficient and feasible total chemical synthesis method for celacarfurine is of crucial strategic significance for solving the problems of difficult and insufficient celacarfurine sources, overcoming the limitations of natural extraction, and promoting the scientific development of the synthesis of such complex macrocyclic natural products. Summary of the Invention

[0009] Because the probability of ring formation due to collisions at the ends of long-chain molecules is low, the rate of intermolecular reactions is generally much higher than the rate of intramolecular cyclization, making macrocyclic formation difficult. The efficient synthesis of macrocyclic molecules is recognized as highly challenging. The purpose of this invention is to provide a total chemical synthesis method for the macrocyclic natural product celacarfurine and its key intermediates. Through innovative synthetic route design, its unique thirteen-membered macrocyclic skeleton can be constructed efficiently and in high yield under mild conditions. The method is simple, the reaction conditions are mild, no special equipment or expensive reagents are required, and all raw materials are inexpensive and readily available, providing a stable and reliable source of materials for the subsequent development and application of celacarfurine.

[0010] This invention provides a synthetic method for celacarfurine and its key intermediates. Starting with methyl 4-aminobutyrate hydrochloride, the method involves the following steps: introducing an Ns protecting group via nucleophilic substitution; introducing an amino group and elongating the carbon chain via nucleophilic substitution; introducing a chiral amino acid and a Boc protecting group via amide condensation; removing the Boc substituent after ester hydrolysis; forming a thirteen-membered ring via intramolecular amide condensation cyclization; removing the Ns substituent; and introducing substituents via nucleophilic substitution. The resulting compound is shown in general formula (I). The specific synthetic route is detailed in the appendix. Figure 1 The structural formula of general formula (I) is: In the formula, R is hydrogen or acyl. When R is 3-furanoyl, the compound of general formula (I) is celacarfurine; when R is hydrogen, the compound of general formula (I) is the key intermediate 2-phenyl-1,5,9-triazacyclotridecane-4,13-dione; when R is other substituents, the compound of general formula (I) is a derivative of 2-phenyl-1,5,9-triazacyclotridecane-4,13-dione.

[0011] The method for synthesizing celacarfurine and its key intermediates includes the following steps:

[0012] Step 1: Methyl 4-aminobutyrate hydrochloride undergoes a nucleophilic substitution reaction with o-nitrobenzenesulfonyl chloride under alkaline conditions to generate compound 1, wherein the o-nitrobenzenesulfonyl group is abbreviated as Ns;

[0013] Step 2: Compound 1 undergoes a nucleophilic substitution reaction with N-(3-bromopropyl)carbamate tert-butyl ester under alkaline conditions upon heating to generate compound 2;

[0014] Step 3: Compound 2 undergoes an amide condensation reaction with (R)-3-(tert-butoxycarbonylamino)-3-phenylpropionic acid in the presence of a condensing agent to generate compound 3;

[0015] Step 4: Compound 3 undergoes ester hydrolysis under strongly alkaline conditions to generate a carboxylate, and then the Boc protecting group (tert-butyloxycarbonyl) is removed under strongly acidic conditions to generate compound 4;

[0016] Step 5: Compound 4 undergoes an intramolecular cyclization reaction in the presence of a condensing agent to generate compound 5;

[0017] Step 6: Compound 5 is reacted with thiols or thiophenols under alkaline conditions to remove the Ns substituent protecting group, and then treated with acid to form a salt, yielding the key intermediate compound 6.

[0018] Step 7: Compound 6 undergoes a nucleophilic substitution reaction with acyl chloride under alkaline conditions to generate compound 7;

[0019] Among them, the structural formula of compound 1 is Compound 2 has the following structural formula: Compound 3 has the following structural formula: Compound 4 has the following structural formula: Compound 5 has the following structural formula: Compound 6 has the following structural formula: Compound 7 has the following structural formula: The Ns substituent structure is as follows: The structural formula for celacarfurine is: .

[0020] Further, in step 1, methyl 4-aminobutyrate hydrochloride reacts with o-nitrobenzenesulfonyl chloride under set conditions to generate compound 1, wherein the o-nitrobenzenesulfonyl group is abbreviated as Ns. The reaction process is as follows:

[0021]

[0022] The set conditions are as follows:

[0023] The solvent is dichloromethane;

[0024] The base is one or a combination of at least two of triethylamine, N,N-diisopropylethylamine (DIPEA), pyridine, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU); preferably, the base is triethylamine;

[0025] The molar ratio of methyl 4-aminobutyrate hydrochloride to alkali is 1:2-3, preferably 1:2.2;

[0026] The molar ratio of methyl 4-aminobutyrate hydrochloride to o-nitrobenzenesulfonyl chloride is 1:1.

[0027] Furthermore, in step 2, compound 1 reacts with N-(3-bromopropyl)carbamate tert-butyl ester under specified conditions to generate compound 2, and the reaction process is as follows:

[0028]

[0029] The set conditions are as follows:

[0030] The solvent is one or a combination of at least two of acetonitrile, N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO); preferably, the solvent is acetonitrile.

[0031] The reaction temperature is 80-100℃; preferably, the reaction temperature is 80℃.

[0032] The reaction time is 24-120 hours; preferably, the reaction time is 96 hours.

[0033] The molar ratio of compound 1 to N-(3-bromopropyl)carbamate tert-butyl ester is 1:1;

[0034] The base is one or a combination of at least two of potassium carbonate, sodium carbonate, cesium carbonate, triethylamine, N,N-diisopropylethylamine (DIPEA), pyridine, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU); preferably, the base is potassium carbonate;

[0035] The molar ratio of compound 1 to the base is 1:3-5; preferably, the molar ratio of compound 1 to the base is 1:3.

[0036] Furthermore, in step 3, compound 2 reacts with (R)-3-(tert-butoxycarbonylamino)-3-phenylpropionic acid under specified conditions to generate compound 3, and the reaction process is as follows:

[0037]

[0038] The set conditions are as follows:

[0039] The molar ratio of compound 2 to (R)-3-(tert-butoxycarbonylamino)-3-phenylpropionic acid is 1:1;

[0040] The condensing agent is one or a combination of at least two of EDCI, HOBT, DMTMM, T3P, HATU, and TCFH; preferably, the condensing agent is one or a combination of HOBT and EDCI.

[0041] The molar ratio of compound 2 to any condensing agent is 1:1.1-1.5, preferably, the molar ratio of compound 2 to any condensing agent is 1:1.2;

[0042] The base is N,N-diisopropylethylamine (DIPEA) or triethylamine;

[0043] The molar ratio of compound 2 to the base is 1:1.1-2, preferably 1:2.

[0044] Furthermore, in step 4, compound 3 is converted into compound 4 under specified conditions, and the reaction process is as follows:

[0045]

[0046] The set conditions are as follows:

[0047] The solvent used in the ester hydrolysis process is a mixture of methanol and water;

[0048] The solvent for the deBoc substituenting process is one or a combination of at least two of dioxane hydrochloride, ethyl acetate hydrochloride, and trifluoroacetic acid; preferably, the solvent for the deBoc substituenting process is dioxane hydrochloride.

[0049] The alkali is one or a combination of at least two of lithium hydroxide, potassium hydroxide, and sodium hydroxide; preferably, the alkali is lithium hydroxide.

[0050] The molar ratio of compound 3 to the base is 1:1.1-3, preferably 1:1.5.

[0051] Furthermore, in step 5, compound 4 is converted into compound 5 under specified conditions, and the reaction process is as follows:

[0052]

[0053] The set conditions are as follows:

[0054] The condensing agent is one or a combination of at least two of EDCI, HOBT, DMTMM, T3P, HATU, and TCFH; preferably, the condensing agent is HATU.

[0055] The molar ratio of compound 4 to condensing agent is 1:1-5, preferably, the molar ratio of compound 4 to condensing agent is 1:4;

[0056] The solvent is acetonitrile;

[0057] The base is N,N-diisopropylethylamine (DIPEA) or triethylamine;

[0058] The molar ratio of compound 4 to the base is 1:5-10, preferably 1:10.

[0059] Further, in step 6, compound 5 reacts with a thiol-containing compound under set conditions, and is then acid-washed to generate compound 6, wherein the acid is hydrochloric acid, and the reaction process is as follows:

[0060]

[0061] The set conditions are as follows:

[0062] The thiol-containing compound is selected from thiols or thiophenols; preferably, the thiol-containing compound is 3-mercapto-1-propanol;

[0063] The molar ratio of compound 5 to the mercapto-containing compound is 1:5-10, preferably, the molar ratio of compound 5 to the mercapto-containing compound is 1:10;

[0064] The base is one or a combination of at least two of potassium carbonate, sodium carbonate, cesium carbonate, triethylamine, N,N-diisopropylethylamine, pyridine, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU); preferably, the base is 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0065] The molar ratio of compound 5 to the base is 1:5-10, preferably 1:10.

[0066] Furthermore, in step 7, compound 6 reacts with acyl chloride under set conditions to generate compound 7, and the reaction process is as follows:

[0067]

[0068] The set conditions are as follows:

[0069] The molar ratio of compound 6 to acyl chloride is 1:1-1.5, preferably 1:1.3;

[0070] The base is one or a combination of at least two of potassium carbonate, sodium bicarbonate, cesium carbonate, triethylamine, N,N-diisopropylethylamine, pyridine, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU); preferably, the base is sodium bicarbonate;

[0071] The solvent is one or a combination of water or dichloromethane.

[0072] Furthermore, the acyl chloride includes, but is not limited to, 3-furancarbamate chloride; wherein, when the acyl chloride is 3-furancarbamate chloride, compound 6 reacts with 3-furancarbamate chloride to generate celacarfurine; when the acyl chloride is other acyl chloride, a derivative of 2-phenyl-1,5,9-triazacyclotetrazane-4,13-dione is generated.

[0073] The technical solution of this invention has the following beneficial effects:

[0074] 1) For the first time, the total synthesis of celacarfurine, a 13-membered macrocyclic spermidine alkaloid with a unique "triamide on the ring" structure, was achieved, overcoming the technical gap in the artificial synthesis of this highly difficult natural product and laying the material foundation for subsequent research.

[0075] 2) The developed synthetic route is remarkably simple and efficient, and the target product can be obtained in just seven steps with a high yield (the total yield can reach 39.64%), which is far superior to the synthetic efficiency of most complex natural products.

[0076] 3) The method has mild reaction conditions, avoiding harsh requirements such as ultra-low temperature and special catalysts. It is safe and easy to operate, and environmentally friendly, which greatly reduces the difficulty of production control and safety risks.

[0077] 4) All raw materials and reagents are inexpensive and readily available commercial products, the supply chain is stable, the cost is controllable, and the synthesis route has outstanding potential for industrial production and broad market promotion prospects. Attached Figure Description

[0078] Figure 1 This is a synthetic route diagram for celacarfurine.

[0079] Figure 2 This is the high-resolution mass spectrum of compound 6.

[0080] Figure 3 This is the 1H NMR spectrum of compound 6.

[0081] Figure 4 This is the carbon NMR spectrum of compound 6.

[0082] Figure 5 This is the high-resolution mass spectrum of compound 7 (celacarfurine).

[0083] Figure 6 The image shows the 1H NMR spectrum of compound 7 (celacarfurine).

[0084] Figure 7 This is the carbon NMR spectrum of compound 7 (celacarfurine).

[0085] Figure 8 The HPLC chromatogram of natural celacarfurine (main chromatographic peak with retention time of 8.575 min).

[0086] Figure 9 The HPLC chromatogram of the synthesized celacarfurine (compound 7) (main chromatographic peak with a retention time of 8.416 min) is shown. Detailed Implementation

[0087] To enable those skilled in the art to better understand the technical solutions in this specification, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this specification, and not all embodiments. Based on the embodiments in this specification, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this specification.

[0088] This invention provides a synthetic method for celacarfurine and its key intermediates. Starting with methyl 4-aminobutyrate hydrochloride, the method involves the following steps: introducing an Ns protecting group via nucleophilic substitution; introducing an amino group and elongating the carbon chain via nucleophilic substitution; introducing a chiral amino acid and a Boc protecting group via amide condensation; removing the Boc substituent after ester hydrolysis; forming a thirteen-membered ring via intramolecular amide condensation cyclization; removing the Ns substituent; and introducing substituents via nucleophilic substitution. The resulting compound is prepared using general formula (I). The synthetic route for celacarfurine and its key intermediates is shown in the appendix. Figure 1 The structural formula of general formula (I) is: In the formula, R is hydrogen or acyl. When R is 3-furanoyl, the compound of general formula (I) is celacarfurine; when R is hydrogen, the compound of general formula (I) is the key intermediate 2-phenyl-1,5,9-triazacyclotridecane-4,13-dione; when R is other substituents, the compound of general formula (I) is a derivative of 2-phenyl-1,5,9-triazacyclotridecane-4,13-dione.

[0089] The synthesis method of celacarfurine and its key intermediates specifically includes the following steps:

[0090] Step 1: Dissolve methyl 4-aminobutyrate hydrochloride in an organic solvent. Under ice-water bath conditions, add an organic base, and slowly add an organic solution containing o-nitrobenzenesulfonyl chloride. After thin-layer chromatography until the starting material has completely disappeared, wash with hydrochloric acid solution until acidic, separate the organic phase, wash successively with saturated sodium bicarbonate solution, wash with water, dry with anhydrous sodium sulfate, and evaporate the organic phase to dryness to obtain compound 1. The structural formula of compound 1 is [insert structural formula here]. The organic base is selected from triethylamine, N,N-diisopropylethylamine, pyridine and DBU, with triethylamine being the preferred organic base; the organic solvent or organic solution is dichloromethane; the molar ratio of methyl 4-aminobutyrate hydrochloride, o-nitrobenzenesulfonyl chloride and the organic base is 1:1:(2-3), with a preferred molar ratio of 1:1:2.2.

[0091] Step 2: Compound 1, N-(3-bromopropyl)carbamate tert-butyl ester, and an inorganic base were added to a three-necked flask. An organic solvent was added to dissolve the compound, and the mixture was heated at a set temperature. Thin-layer chromatography was performed until the reactants were completely dissolved. After natural cooling, the mixture was filtered, and the filtrate was evaporated to dryness. Dichloromethane was added to dissolve the filtrate, and impurities were extracted with water. The organic phase was dried over anhydrous sodium sulfate, and the mixture was evaporated to dryness to obtain the crude compound. A small amount of dioxane was added to the crude compound to dissolve it. Under nitrogen protection, dioxane hydrochloride was added to remove the Boc substituent. Thin-layer chromatography was performed until the reactants were completely dissolved. The organic solvent was evaporated to dryness, and the compound was dissolved in water. The aqueous phase was extracted with dichloromethane to remove impurities. Triethylamine was added to the aqueous phase to adjust the pH to 9-10. The aqueous phase was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the organic phase was evaporated to dryness to obtain compound 2, with the structural formula [insert structural formula here]. The inorganic base is selected from potassium carbonate, sodium carbonate, and cesium carbonate, with potassium carbonate being the preferred inorganic base; the organic solvent is selected from high-boiling-point solvents such as acetonitrile, DMF, or DMSO, with acetonitrile being the preferred organic solvent; the heating temperature is set to 80~100℃, with 80℃ being the preferred setting; the heating time is 24h~120h, with 96h being the preferred heating time; the molar ratio of compound 1, N-(3-bromopropyl)carbamate tert-butyl ester, and the inorganic base is 1:1:(3-5); the preferred molar ratio is 1:1:3.

[0092] Step 3: Add compound 2, organic base, and condensing agent to a three-necked flask, dissolve in organic solvent until completely dissolved, then add (R)-3-(tert-butoxycarbonylamino)-3-phenylpropionic acid in portions. Detect by thin-layer chromatography until the starting material disappears completely. Wash the organic phase with water, saturated sodium bicarbonate solution, and dilute hydrochloric acid solution. Wash with saturated brine, dry with anhydrous sodium sulfate, and evaporate the organic phase to obtain compound 3. The structural formula of compound 3 is... The condensing agent is selected from commonly used condensing agents such as EDCI, HOBT, DMTMM, T3P, HATU, and TCFH, with a preferred combination of HOBT and EDCI; the organic base is DIPEA; the molar ratio of compound 2, (R)-3-(tert-butoxycarbonylamino)-3-phenylpropionic acid, condensing agent, and organic base is 1:1:(1.1-1.5):(1.1-2), with a preferred molar ratio of 1:1.2:2.

[0093] Step 4: Compound 3 was added to a three-necked flask and dissolved in a methanol-water mixture. An aqueous solution of a strong inorganic base was slowly added dropwise. Thin-layer chromatography was performed until the reactants were completely removed. The methanol was then evaporated to dryness. Impurities in the aqueous phase were extracted with ethyl acetate. The pH of the aqueous phase was adjusted to 2-3 with hydrochloric acid, and the solution was extracted with ethyl acetate. The solution was dried over anhydrous sodium sulfate to obtain the crude compound. The crude compound was dissolved in dioxane, and dioxane was removed from the Boc residue with hydrochloric acid. Thin-layer chromatography was performed until the reactants were completely removed. The organic solvent was evaporated to dryness. The compound was dissolved in water, and impurities in the aqueous phase were extracted with ethyl acetate. The aqueous phase was lyophilized to obtain compound 4. The structural formula of compound 4 is [insert structural formula here]. The inorganic strong base is selected from lithium hydroxide, potassium hydroxide, and sodium hydroxide, with lithium hydroxide being the preferred inorganic strong base; the solvent used for ester hydrolysis is a mixed solvent of methanol and water; the solvent for removing Boc substituents is selected from strong acid reagents such as dioxane hydrochloride, ethyl acetate hydrochloride, and trifluoroacetic acid, with dioxane hydrochloride being the preferred Boc substituent; the molar ratio of compound 3 to the inorganic strong base is 1:(1.1-3), with a preferred molar ratio of 1:1.5.

[0094] Step 5: A: Add compound 4, the organic base, and the condensing agent to a flask, and dissolve them with organic solvent; B: Add the organic base and the condensing agent to a three-necked flask, and dissolve them with a large amount of organic solvent. Cool the solution in B to 0°C under ice-water bath conditions. Slowly add the solution in A to the solution in B. Detect the reaction by thin-layer chromatography until the starting material has completely disappeared. After evaporating the organic solvent, purify the solution by silica gel column chromatography using dichloromethane and methanol as eluents to obtain compound 5. The structural formula of compound 5 is... The condensing agent is selected from commonly used condensing agents such as EDCI, HOBT, DMTMM, T3P, HATU and TCFH, with HATU being the preferred condensing agent; the organic solvent is acetonitrile; the organic base is DIPEA; the molar ratio of compound 4, condensing agent and organic base is 1:(1-5):(5-10), with a preferred molar ratio of 1:4:10.

[0095] Step 6: Add compound 5 and the base to a flask, add thiol or thiophenol (preferably 3-mercapto-1-propanol), and perform thin-layer chromatography until the starting material disappears completely. Reduce the solution to dryness, add water to dissolve, adjust the pH to acidic with hydrochloric acid solution, extract impurities from the aqueous phase with dichloromethane, reduce the aqueous phase to dryness, dissolve in a small amount of water, and purify by ODS column chromatography using water and acetonitrile as eluents to obtain compound 6. The structural formula of compound 6 is... The base is selected from potassium carbonate, sodium carbonate, cesium carbonate, triethylamine, N,N-diisopropylethylamine, pyridine, and DBU; the base is preferably DBU; the molar ratio of compound 5 to the base is 1:(5-10), preferably 1:10; the molar ratio of compound 5 to thiol or thiophenol is 1:(5-10), preferably 1:10.

[0096] Step 7: Add compound 6 to a three-necked flask, dissolve in water, adjust to alkalinity with alkali, dissolve the acyl chloride in anhydrous organic solvent, and slowly add it dropwise to the three-necked flask under nitrogen protection in an ice-water bath with vigorous stirring. After the reactants have completely disappeared according to thin-layer chromatography, dissolve in methanol, evaporate the organic solvent to dryness, extract the aqueous phase with dichloromethane, dry the organic phase with anhydrous sodium sulfate, and evaporate the organic phase to dryness to obtain compound 7. The structural formula of compound 7 is [insert structural formula here]. When the acyl chloride used is 3-furanoyl chloride, compound 7 is celacarfurine, which undergoes a nucleophilic substitution reaction with other acyl chlorides to generate a derivative of 2-phenyl-1,5,9-triazacyclotetrazane-4,13-dione. The base is selected from potassium carbonate, sodium bicarbonate, cesium carbonate, triethylamine, N,N-diisopropylethylamine, pyridine, and DBU; the base is preferably sodium bicarbonate; the molar ratio of compound 6 to the acyl chloride is 1:(1.1-5), preferably 1:1.3.

[0097] The present invention will be further described below with reference to specific embodiments. The abbreviations for reagents and testing methods in the embodiments are shown in Table 1, and the sources of key raw materials and reagents are shown in Table 2.

[0098] Table 1. Abbreviations for Reagents and Testing Methods

[0099]

[0100] Table 2 Sources of Key Raw Materials and Reagents

[0101]

[0102] Example 1

[0103] This embodiment specifically illustrates the total synthesis method of celacarfurine. The synthesis steps are as follows:

[0104] Step 1

[0105] ① Reagent preparation

[0106] 4-Aminobutyrate methyl hydrochloride, dichloromethane, triethylamine, o-nitrobenzenesulfonyl chloride, 1 mol / L hydrochloric acid solution, saturated sodium bicarbonate solution, anhydrous sodium sulfate.

[0107] ②Preparation process of compound 1

[0108] Using a 50 mL three-necked flask as the reaction vessel, 5 g (32.55 mmol) of methyl 4-aminobutyrate hydrochloride was accurately weighed using an electronic balance and added to the reaction vessel. Anhydrous dichloromethane (30 mL) was added and stirred to dissolve the hydrochloride. The reaction system was placed in an ice-water bath (0–5 °C), and triethylamine (10 mL, 71.61 mmol) was slowly added under nitrogen protection. Subsequently, o-nitrobenzenesulfonyl chloride (7.21 g, 32.55 mmol) dissolved in anhydrous dichloromethane was slowly added dropwise over approximately 2 hours. After the addition was complete, the reaction was stirred for another hour under ice-water bath conditions. After the reaction was complete, the reaction solution was washed with 1 mol / L hydrochloric acid solution until the aqueous phase was acidic, and the organic phase was separated. The organic phase was washed successively with saturated sodium bicarbonate solution and deionized water, dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness to obtain compound 1. The prepared compound 1 was a pale yellow liquid with a mass of 9.77 g and a yield of 99.3%.

[0109] ③Analysis of structural characterization data of compound 1

[0110] High-resolution mass spectrometry (HR-ESI-MS): Measured m / z of the quasi-molecular ion peak [M+Na] in HR-ESI-MS + The value is 325.0464, m / z [M+Na]. + Theoretical value (C) 11 H 14 The N2O6SNa value is 325.0465, confirming the molecular formula of compound 1 as C. 11 H 14 N2O6S;

[0111] 1H NMR spectrum: 1 H-NMR (600 MHz, CD3OD) δ: 8.07-8.09 (1H, m), 7.85-7.87 (1H, m), 7.81-7.83 (2H, m), 3.36 (3H, s), 3.10 (2H, t, J =6.8 Hz), 2.38 (2H, t, J =7.3 Hz), 1.80 (2H, quint, J =7.1 Hz);

[0112] Carbon NMR spectrum: 13C-NMR (150MHz, CD3OD) δ: 175.20, 149.61, 134.95, 134.80, 133.53, 131.53, 125.87, 52.09, 43.46, 31.49, 26.05.

[0113] High-resolution mass spectrometry, proton NMR, and carbon NMR spectral data explanation: The measured quasi-molecular ion peak in the high-resolution mass spectrometry is in high agreement with the theoretical value, confirming its molecular formula as C. 11 H 14 N2O6S; the multiplet at δ7.81-8.09 in the 1H NMR spectrum is attributed to the aromatic proton on the o-nitrobenzenesulfonyl group; the singlet at δ3.36 is the methyl signal of the methyl ester; the series of signals at δ1.80-3.10 corresponds to the proton environment of the -CH2-CH2-CH2- chain in the molecule; the 1C NMR spectrum shows 11 independent carbon signals, consistent with the number of carbon atoms in the molecular formula, of which δ175.20 is the carbonyl carbon signal of the methyl ester, δ149.61-125.87 is the aromatic carbon signal, δ52.09 is the methyl carbon signal of the methyl ester, and the remaining aliphatic carbon signals are located in the δ43.46-26.05 range. These data collectively confirm that the structure of compound 1 is consistent with the target molecule.

[0114] Step 2

[0115] ① Reagent preparation

[0116] Compound 1, acetonitrile, N-(3-bromopropyl)carbamate tert-butyl ester, potassium carbonate, dichloromethane, anhydrous sodium sulfate, dioxane, dioxane hydrochloride, triethylamine.

[0117] ②Preparation process of compound 2

[0118] Compound 1 (7.27 g, 24.0 mmol) was accurately weighed into a 100 mL three-necked flask using an electronic balance, and 30 mL of acetonitrile was added for mixing. N-(3-bromopropyl)carbamate tert-butyl ester (5.73 g, 24.0 mmol) and potassium carbonate (9.97 g, 72.15 mmol) were weighed separately and added to the three-necked flask sequentially. The mixture was heated at 80 °C for 96 h. After the reaction was complete, the mixture was allowed to cool naturally and filtered to obtain the filtrate. The filtrate was subjected to a series of steps including rotary evaporation, dissolution in dichloromethane, water extraction for impurities, drying with anhydrous sodium sulfate, and rotary evaporation to obtain 10.44 g of the crude compound. 10.44 g of the crude compound was dissolved in a small amount of dioxane. Under nitrogen protection, 35 mL of dioxane hydrochloride (4 mol / L) was added to remove the Boc substituent. After the reaction was complete, thin-layer chromatography (TLC) confirmed that the starting material had been completely consumed. The reaction solution was then concentrated under reduced pressure by rotary evaporation to remove the organic solvent. The residue was dissolved in deionized water, and the aqueous phase was extracted three times with dichloromethane to remove impurities. The separated aqueous phase was alkalized to a pH of 9-10 with triethylamine, and then extracted three more times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure by rotary evaporation to obtain compound 2. Compound 2 was a pale yellow liquid, weighing 8.05 g, with a yield of 93.1%.

[0119] ③Analysis of structural characterization data of compound 2

[0120] High-resolution mass spectrometry (HR-ESI-MS): Measured quasi-molecular ion peak [M+H] in HR-ESI-MS + (m / z, 360.1220), theoretical value 360.1224, determining the molecular formula of compound 2 as C 14 H 21 N3O6S;

[0121] 1H NMR spectrum: 1 H-NMR (600 MHz, CD3OD) δ: 8.04 (1H, dd, 7.5, 1.7 Hz), 7.77-7.82 (2H, m), 7.76 (1H, dd, 7.5, 1.7 Hz), 3.36-3.40 (4H, m), 3.34 (3H, s), 2.65 (2H, t, J =7.0 Hz), 2.33(2H, t, J =7.2 Hz), 1.84 (2H, quint, J =7.2 Hz), 1.73 (2H, quint, J =7.1 Hz);

[0122] Carbon NMR spectrum:13 C-NMR (150MHz, CD3OD) δ: 173.62, 148.21, 133.88, 132.51, 131.71, 130.15, 124.03, 50.74, 46.54, 45.10, 38.04, 30.56, 29.96, 23.07.

[0123] High-resolution mass spectrometry, proton NMR, and carbon NMR spectra data show that the measured quasi-molecular ion peaks are in high agreement with the theoretical values, confirming that compound 2 has the molecular formula C. 14 H 21 N3O6S; the multiplet at δ7.76-8.04 in the 1H NMR spectrum is attributed to the aromatic proton on the o-nitrobenzenesulfonyl group, the singlet at δ3.34 is the methyl signal of the methyl ester, and the multiplet (4H) at δ3.36-3.40 and the series of signals at δ1.73-2.65 are consistent with the elongated carbon chain and newly added nitrogen substitution structure in the molecule; the 1C NMR spectrum shows 14 independent carbon signals, consistent with the number of carbon atoms in the molecular formula. Among them, δ173.62 is the carbonyl carbon signal of the methyl ester, δ148.21-124.03 is the aromatic carbon signal, δ50.74 is the methyl carbon signal of the methyl ester, and the remaining aliphatic carbon signals are located in the δ46.54-23.07 range. The above data collectively confirm that the structure of compound 2 is consistent with the target molecule.

[0124] Step 3

[0125] ① Reagent preparation

[0126] Compound 2, dichloromethane, DIPEA, HOBT, EDCI·HCl, (R)-3-(tert-butoxycarbonylamino)-3-phenylpropionic acid, saturated sodium bicarbonate aqueous solution, dilute hydrochloric acid aqueous solution, saturated saline solution, anhydrous sodium sulfate.

[0127] ②Preparation process of compound 3

[0128] In a 100 mL three-necked flask equipped with a magnetic stirrer, compound 2 (7.91 g, 22.0 mmol) and anhydrous dichloromethane (30 mL) were added under nitrogen protection in an ice-water bath (0–5 °C). While stirring continuously, N,N-diisopropylethylamine (DIPEA, 7.7 mL, 44.0 mmol), 1-hydroxybenzotriazole (HOBT, 3.57 g, 26.4 mmol), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI·HCl, 5.06 g, 26.4 mmol) were added sequentially via syringe. The resulting mixture was further stirred and activated in an ice-water bath for 1 hour until the system was homogeneous and clear. Subsequently, solid (R)-3-(tert-butoxycarbonylamino)-3-phenylpropionic acid (5.84 g, 22.0 mmol) was added to the reaction flask in three to four batches. After adding the ingredients, remove the ice-water bath and allow the reaction solution to rise naturally to room temperature. Stir the reaction solution at room temperature for about 12 hours.

[0129] Thin-layer chromatography (TLC) confirmed that the starting material compound 2 had been completely consumed. Subsequently, the reaction mixture was transferred to a separatory funnel and subjected to the following washing operations: washing with deionized water to remove urea byproducts from EDCI hydrolysis; washing with saturated sodium bicarbonate aqueous solution to neutralize and remove HOBT byproducts and excess acid; washing with dilute hydrochloric acid aqueous solution to remove residual urea byproducts and excess organic base DIPEA; and washing with saturated brine to equilibrate the aqueous phase and reduce the water content in the organic phase. After washing, the resulting organic phase was collected and dried with anhydrous sodium sulfate to obtain compound 3. Compound 3 was a pale yellow liquid with a mass of 12.55 g, and the calculated yield was 94.0%.

[0130] ③Analysis of structural characterization data of compound 3

[0131] Specific rotation: ;

[0132] High-resolution mass spectrometry (HR-ESI-MS): Quasi-molecular ion peak [M+Na] in HR-ESI-MS + (m / z, 629.2271), theoretical value 629.2252, determining the molecular formula of compound 3 as C 28 H 38 N4O9S;

[0133] 1H NMR spectrum: 1 H-NMR (600 MHz, CD3OD) δ: 8.01 (1H, dd, J =7.5, 1.5 Hz), 7.79-7.83 (2H, m), 7.77 (1H, dd, J=7.5, 1.8 Hz), 7.21-7.33 (5H, m), 5.01 (1H, br.s), 3.66 (3H, s), 3.31-3.34 (2H, m), 3.22 (2H, t, J =7.5 Hz), 3.05-3.14(2H, m), 2.61(2H, d, J =7.6 Hz), 2.34(2H, t, J =7.2 Hz), 1.82 (2H, quint, J =7.2 Hz), 1.64 (2H, quint, J =7.1 Hz), 1.41 (9H, s);

[0134] Carbon NMR spectrum: 13 C-NMR (150MHz, CD3OD) δ: 174.99, 172.71, 157.33, 149.59, 143.60, 135.29, 133.80, 133.15, 131.46, 129.4 7, 128.30, 127.38, 125.43, 80.31, 52.18, 49.85, 48.27, 46.67, 44.10, 37.63, 31.37, 29.26, 28.74, 24.61.

[0135] Specific rotation, high-resolution mass spectrometry, proton NMR, and carbon NMR spectral data: The measured specific rotation value is positive, confirming that compound 3 is an optically active substance with a chiral center, maintaining the chiral configuration of the amino acid reactant; the measured quasi-molecular ion peak is in high agreement with the theoretical value, confirming its molecular formula as C3. 28 H 38N4O9S; In the 1H NMR spectrum, the multiplet (5H) at δ7.21–7.33 and the broad singlet at δ5.01 are attributed to the benzene ring and α-hydrogen signal of the chiral phenylalanine fragment; the singlet (9H) at δ1.41 is the characteristic signal of the tert-butoxycarbonyl (Boc); the multiplet at δ7.77–8.01 corresponds to the aromatic proton of the o-nitrobenzenesulfonyl group; the singlet at δ3.66 is the methyl signal of the methyl ester; and the remaining series of signals at δ1.64–3.34 are consistent with the proton environment of multiple methylene chains in the molecule; NMR The carbon resonance spectrum showed 28 independent carbon signals, consistent with the number of carbon atoms in the molecular formula. Among them, δ174.99 and 172.71 are two carbonyl carbon signals, δ157.33 is the carbonyl carbon of the Boc group, δ149.59-125.43 are aromatic carbon signals, δ80.31 is the tert-butyl quaternary carbon signal of the Boc group, δ52.18 is the methyl carbon signal of the methyl ester, δ28.74 is the methyl carbon signal of the Boc group, and the remaining aliphatic carbon signals are located in the δ49.85-24.61 range. These data collectively confirm that the structure of compound 3 is consistent with the target molecule.

[0136] Step 4

[0137] ① Reagent preparation

[0138] Compound 3, methanol, lithium hydroxide, ethyl acetate, hydrochloric acid, anhydrous sodium sulfate, dioxane, dioxane hydrochloride.

[0139] ②Preparation process of compound 4

[0140] In a 250 mL three-necked flask, compound 3 (12.55 g, 20.7 mmol) and a mixture of methanol and water (3:1, 100 mL, v / v) were added and stirred to dissolve. Then, a 1 mol / L lithium hydroxide aqueous solution (31 mL, 31.0 mmol) was slowly added dropwise with stirring. After the addition was complete, the reaction mixture was stirred at room temperature for approximately 12 hours. Thin-layer chromatography (TLC) confirmed that reactant 3 had been completely consumed. The reaction mixture was concentrated by rotary evaporation at 45 °C to remove methanol. The remaining aqueous phase was extracted three times with ethyl acetate to remove neutral or basic organic impurities. The aqueous phase was separated, and its pH was adjusted to 2-3 with 1 mol / L hydrochloric acid aqueous solution, followed by extraction with ethyl acetate. The combined ethyl acetate extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude hydrolysate with a mass of 12.26 g.

[0141] The crude product was dissolved in a suitable amount of dioxane, and a 4 mol / L dioxane hydrochloride solution (30 mL, 120 mmol) was added with stirring to carry out the Boc protecting group removal reaction at room temperature. After the reaction was completed as monitored by TLC, the reaction solution was evaporated to dryness to remove the organic solvent. The resulting residue was dissolved in water and extracted three times with ethyl acetate to remove lipid-soluble impurities. Finally, the aqueous phase was freeze-dried to give compound 4. Compound 4 was a white solid with a mass of 10.66 g, yield 97.4%.

[0142] ③Analysis of structural characterization data of compound 4

[0143] Specific rotation: ;

[0144] High-resolution mass spectrometry (HR-ESI-MS): Quasi-molecular ion peak [M+H] in HR-ESI-MS + (m / z, 493.1774), theoretical value 493.1751 (ESI ionization source shows molecular weight without hydrochloride), confirming compound 4's molecular formula as C 22 H 28 N4O7S.

[0145] 1H NMR spectrum: 1 H-NMR (600 MHz, CD3OD) δ: 7.89 (1H, d, J =7.3 Hz), 7.66-7.72(3H, m), 7.28-7.39(5H, m), 4.58(1H, t, J =6.8 Hz), 3.14-3.22(4H, m), 2.98-3.07(2H, m), 2.76-2.80(2H, m), 2.21(2H, t, J =7.0 Hz), 1.69 (2H, quint, J =7.0 Hz), 1.57 (2H, quint, J =6.8Hz);

[0146] Carbon NMR spectrum: 13 C-NMR (150MHz, CD3OD) δ: 174.99, 171.26, 149.49, 137.34, 135.36, 133.66, 133.27, 131.38 , 130.45, 130.30, 128.32, 125.42, 53.78, 48.26, 46.71, 40.09, 37.60, 31.33, 29.16, 24.58.

[0147] Specific rotation, high-resolution mass spectrometry, proton NMR, and carbon NMR spectra data indicate that the specific rotation value significantly increased to +35.8°, indicating that the stereochemical environment of the chiral center changed after the removal of the Boc protecting group, further confirming the chiral characteristics of compound 4. The specific rotation value remained positive, indicating that the chiral configuration was preserved. The measured quasi-molecular ion peak and the free form of the molecular formula C... 22 H 28 The theoretical value of N4O7S is highly consistent, and this signal corresponds to the free base form of compound 4, while the actual product exists in the form of hydrochloride, which is consistent with the salt formation step after Boc removal in the synthetic route. The disappearance of the characteristic tert-butyl singlet of the Boc group at δ1.41 in the 1H NMR spectrum confirms the successful removal of the protecting group; the multiplet (5H) at δ7.28-7.39 and the triplet at δ4.58 are attributed to the benzene ring and α-hydrogen signal of the chiral phenylalanine fragment; the multiplet at δ7.66-7.89 corresponds to the aromatic proton of the o-nitrobenzenesulfonyl group; the remaining series of complex signals at δ1.57-3.22 are consistent with the multiple methylene chains in the molecule and the proton environment attached to the amino group. The carbon NMR spectrum showed 20 independent carbon signals, consistent with the number of carbon atoms in the molecular formula (two carbon signals overlapped due to symmetry on monosubstituted benzene); the disappearance of the Boc group-related signals (δ157, 80, 28) further confirmed the structure; δ174.99 and 171.26 were two carbonyl carbon signals; δ149.49-125.42 were aromatic carbon signals; δ53.78 was a chiral α-carbon signal; the remaining aliphatic carbon signals were located in the δ48.26-24.58 range.

[0148] The above data collectively confirm that the structure of compound 4 (in its hydrochloride form) is consistent with the target molecule.

[0149] Step 5

[0150] ① Reagent preparation

[0151] Compound 4, DIPEA, acetonitrile, HATU, dichloromethane, methanol.

[0152] ②Preparation process of compound 5

[0153] In a 100 mL flask, compound 4 (1.74 g, 3.29 mmol) and N,N-diisopropylethylamine (DIPEA, 1.15 mL, 6.58 mmol) were accurately weighed and dissolved in acetonitrile (80 mL) to obtain solution A. In another 1000 mL three-necked flask, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HATU, 5.0 g, 13.2 mmol) and DIPEA (4.6 mL, 26.3 mmol) were dissolved in acetonitrile (300 mL) and stirred until completely dissolved. The reaction system was cooled in an ice-water bath and the internal temperature was maintained at 0 °C.

[0154] Solution A was slowly added dropwise to a three-necked flask using a constant-pressure dropping funnel at 0°C with continuous stirring, and the dropping rate was controlled to continue the process for approximately 10 hours. After the addition was complete, the reaction was continued at 0°C for another 5 hours. Thin-layer chromatography (TLC) confirmed that the starting material compound 4 had been completely consumed. After the reaction solution was evaporated to remove the organic solvent, the residue was purified by normal silica gel column chromatography. The target fraction was collected using a dichloromethane-methanol (80:1 v / v) mixture as the eluent, and after concentration, compound 5 was obtained. Compound 5 was a white solid (0.98 g), with a yield of 62.8%.

[0155] ③ Structural characterization data analysis of compound 5

[0156] Specific rotation: ;

[0157] High-resolution mass spectrometry (HR-ESI-MS): Quasi-molecular ion peak [M+Na] in HR-ESI-MS + (m / z, 497.1467), theoretical value 497.1465, confirming compound 5's molecular formula as C 22 H 26 N4O6S.

[0158] 1H NMR spectrum: 1 H-NMR (600MHz, DMSO-d6) δ: 8.39 (1H, d, J =8.6 Hz), 8.09 (1H, d, J =7.6 Hz), 7.97 (1H, d, J =7.6 Hz), 7.86-7.90 (3H, m), 7.32 (4H, s), 7.23 (1H, s), 5.29 (1H, br.s), 3.43 (2H, m), 3.22 (2H, m), 2.88 (2H, m), 2.49 (2H, d, J=11.6 Hz), 2.32(1H, m), 1.98(1H, m), 1.64-1.81(3H, m), 1.39(1H, s);

[0159] Carbon NMR spectrum: 13 C-NMR(150 MHz, DMSO-d6)δ: 170.42, 169.88, 147.43, 143.09, 134.61, 132.49, 132.48, 129.90, 128 .39, 126.84, 126.06, 124.16, 50.22, 44.81, 44.42, 43.23, 35.44, 32.72, 26.64, 24.69.

[0160] Specific rotation, high-resolution mass spectrometry, proton NMR, and carbon NMR data indicate that the specific rotation measured in DMF was +46.0°, confirming that compound 5 retains its original chiral characteristics. The measured quasi-molecular ion peak is in high agreement with the theoretical value, accurately confirming that compound 5 has the molecular formula C5. 22 H 26 N4O6S. The doublets at δ8.39, 8.09, and 7.97 and the multiplet at 7.86–7.90 in the 1H NMR spectrum are attributed to the aromatic protons of the o-nitrobenzenesulfonyl group; the singlets at δ7.32 and 7.23 and the broad singlet at δ5.29 are consistent with the proton environment of the chiral phenylalanine fragment and the macrocyclic structure; the complex multiplets, broad peaks, and singlets in the δ3.43–1.39 range correspond to the protons of multiple methylene and methine groups in the macrocyclic ring and side chain, reflecting the structural complexity. The 1C NMR spectrum shows 20 independent carbon signals, consistent with the number of carbon atoms in the molecular formula. Among them, δ170.42 and 169.88 are two carbonyl carbon signals; δ147.43–124.16 are aromatic carbon signals; δ50.22 is a chiral α-carbon signal; and the remaining aliphatic carbon signals are located in the δ44.81–24.69 range.

[0161] The above data collectively confirm that the structure of compound 5 is consistent with the target macrocyclic intermediate molecule protected by Ns.

[0162] Step 6

[0163] ① Reagent preparation

[0164] Compound 5, N,N-dimethylformamide, DBU, 3-mercapto-1-propanol, hydrochloric acid, dichloromethane, acetonitrile.

[0165] ②Preparation process of compound 6

[0166] In a 100 mL three-necked flask, compound 5 (1.04 g, 2.2 mmol) was accurately weighed and dissolved in 20 mL of N,N-dimethylformamide (DMF). Under stirring at room temperature, 3.3 mL of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 3.3 mL, 22.0 mmol) and 1.9 mL of 3-mercapto-1-propanol (22.0 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 2 hours. Thin-layer chromatography (TLC) confirmed complete consumption of starting compound 5. The reaction mixture was then evaporated to dryness to remove most of the DMF. The residue was dissolved in water and adjusted to acidity with a 1 mol / L aqueous hydrochloric acid solution. The aqueous phase was extracted (three times) with dichloromethane to remove lipid-soluble impurities. The aqueous phase was evaporated to dryness, the resulting solid was redissolved in a small amount of water, and purified by reversed-phase ODS column chromatography. Using water-acetonitrile (95:5 v / v) as the eluent, the fraction containing the target product was collected and concentrated to give compound 6. Compound 6 was a white solid (0.60 g), with a yield of 83.7%. Compound 6 is the hydrochloride form of the key intermediate 2-phenyl-1,5,9-triazacyclotetrazane-4,13-dione.

[0167] ③Analysis of structural characterization data of compound 6

[0168] Specific rotation: ;

[0169] High-resolution mass spectrometry (HR-ESI-MS): HR-ESI-MS (with appendix) Figure 2 The quasi-molecular ion peak [M+H] in ) + (m / z, 290.1878), theoretical value 290.1863 (ESI ionization source shows molecular weight without hydrochloride), confirming compound 6's molecular formula as C. 16 H 23 N3O2;

[0170] 1H NMR spectrum (with appendix) Figure 3 ): 1 H-NMR (600 MHz, D2O) δ: 7.17-7.25 (5H, m), 5.25 (1H, d, J =11.9 Hz), 3.37 (1H, d, J=14.2 Hz), 3.00-3.03 (1H, m), 2.92-2.95 (1H, m), 2.81-2.86 (1H, m), 2.74-2.78 (2H, m), 2.56-2.59 (1 H, m), 2.47-2.52 (1H, m), 2.37-2.41 (1H, m), 2.13-2.17 (1H, m), 1.84-1.90 (2H, m), 1.75 (2H, m);

[0171] Carbon NMR Spectroscopy (with appendix) Figure 4 ): 13 C-NMR (150 MHz, D2O) δ: 174.17, 173.60, 140.22, 129.03, 128.03, 126.01, 50.93, 44.04, 43.33, 42.80, 36.72, 32.97, 23.83, 21.02.

[0172] Specific rotation, high-resolution mass spectrometry, proton NMR, and carbon NMR spectral data indicate that the specific rotation measured in water was +17.1°, confirming the optical activity of compound 6 as a chiral macrocyclic molecule. The measured quasi-molecular ion peak and the free base form of the molecule (C6) in the high-resolution mass spectrometry data further confirm this. 16 H 23 The theoretical value for N3O2 is highly consistent, and this signal corresponds to the free base form of compound 6, while the actual product exists in the form of hydrochloride. The ¹H NMR and ¹³C NMR spectra (detailed attribution shown in Table 3) are highly consistent with the target macrocyclic structure. The multiplet (5H) at δ7.17–7.25 is attributed to the phenyl proton; the complex aliphatic region proton and carbon signals shown in the spectrum (¹³C NMR shows 14 distinct carbon signals, including two amide carbonyl carbons at δ174.17 and 173.60) are consistent with the expected thirteen-membered macrocyclic skeleton and the chemical environment of the substituents.

[0173] Table 3 Compound 6 1 H-NMR (600MHz, D2O) and 13 C-NMR (150MHz, D2O) nuclear magnetic resonance spectral data assignment

[0174]

[0175] The above data confirm that the synthesized compound 6 is 2-phenyl-1,5,9-triazacyclotetrazane-4,13-dione, with the structural formula [structure not provided]. It was identified as a key intermediate in the target pathway.

[0176] Step 7

[0177] ① Reagent preparation

[0178] Compound 6, sodium bicarbonate, 3-furanoyl chloride, dichloromethane, methanol, dichloromethane, anhydrous sodium sulfate.

[0179] ②Preparation process of compound 7

[0180] Prepare a 50 mL three-necked flask as the reaction vessel. Accurately weigh compound 6 (82 mg, 0.25 mmol) and add it to the reaction vessel. Add 10 mL of water to dissolve it, and then add 5 mL of saturated sodium bicarbonate solution to adjust the reaction system to alkalinity. Dissolve 3-furanoyl chloride (32 μL, 0.32 mmol) in 10 mL of anhydrous dichloromethane. Under an ice-water bath (0–5 °C) and nitrogen protection, slowly add this solution dropwise to the three-necked flask over approximately 2 hours, maintaining vigorous stirring throughout the process. After the addition is complete, continue the reaction in the ice-water bath for 1 hour. Thin-layer chromatography (TLC) confirms that the starting material compound 6 has been completely consumed. Add an appropriate amount of methanol to the reaction solution to aid dissolution, and then evaporate to dryness at 45 °C to remove the organic solvent. Extract the remaining aqueous phase with dichloromethane (5 times), combine the organic phases, dry with anhydrous sodium sulfate, filter, and evaporate to dryness to obtain compound 7. Compound 7 was a white solid (86 mg) in 89.1% yield.

[0181] ③ Structural characterization data analysis of compound 7

[0182] Specific rotation: ;

[0183] High-resolution mass spectrometry (HR-ESI-MS): HR-ESI-MS (with appendix) Figure 5 The quasi-molecular ion peak [M+H] in ) + (m / z, 384.1910), theoretical value 384.1918, confirming the molecular formula of compound 7 as C. 21 H 25 N3O4.

[0184] 1H NMR spectrum (with appendix) Figure 6 ): 1 H-NMR (600MHz, CF3COOD) δ: 8.12 (1H, br.s), 7.67 (1H, d, J=7.4Hz), 7.35-7.42 (5H, m), 6.74 (1 H, s), 5.74 (1H, s), 3.48-4.04 (6H, m), 3.19 (2H, br.s), 2.63-2.74 (2H, m), 2.13-2.33 (4H, m);

[0185] Carbon NMR Spectroscopy (with appendix) Figure 7 ): 13C-NMR(150 MHz, CF3COOD) δ: 178.08, 177.33, 170.90, 148.85, 147.73, 139.76, 131.24, 130.96, 127.64, 127.60, 110.40, 54 .56, 54.48, 51.41, 50.72, 48.63, 47.91, 43.79, 39.73, 39.56, 34.96, 34.84, 31.39, 28.87, 27.33, 26.29, 24.61.

[0186] High-resolution mass spectrometry, proton NMR, and carbon NMR spectral data description: High-resolution mass spectrometry data accurately confirmed the molecular composition (C60-240 nm) of compound 7. 21 H 25 N3O4). The low-field signals (including characteristic signals of furanamide and phenyl fragments) in its proton and carbon NMR spectra are basically consistent with the spectral data of celacarfurine from natural sources, confirming the successful construction of the target macrocyclic core skeleton. In the high-field region (aliphatic carbon region) of the carbon NMR spectrum of compound 7, two sets of signals with similar chemical shifts were observed, corresponding to a total of 8 carbon atoms on the spermidine unit (detailed assignment is shown in Table 4). This phenomenon is attributed to the fact that the CN bond in macrocyclic amide compounds has a certain double bond property due to the resonance effect of the amide bond, resulting in a hindered rotation isomer. Due to the inherent conformational rigidity of the thirteen-membered macrocyclic skeleton of celacarfurine, the rotation of the amide bond on the ring is restricted under NMR measurement conditions, resulting in two stable conformational isomers with similar energies and slow interconversion in solution, leading to the "double" phenomenon of some carbon atom signals. This characteristic further confirms its expected macrocyclic structure, and this phenomenon has been reported in the study of such structures in this field (Reference 1: Wu Hao, Liu Zhen, Bai Dachang. 1H NMR spectra of amide compounds [J]. University Chemistry, 2024, 39(03):231-238. Reference 2: ELLERAAS J, EWANICKI J, JOHNSON TW, et al. Conformational studies and atropisomerismkinetics of the ALK clinical candidate lorlatinib (PF-06463922) and desmethylcongeners [J]. Angewandte Chemie International Edition, 2016, 55(11):3590-3595).

[0187] Compound 7 in Table 4 1 H-NMR and13 C-NMR nuclear magnetic resonance spectroscopy data assignment

[0188]

[0189] Compound 7 synthesized in this invention was compared with natural celacarfurine isolated from Tripterygium wilfordii using HR-ESI-MS and HPLC-DAD. The HPLC chromatogram of natural celacarfurine is shown in the appendix. Figure 8 The HPLC chromatogram of the synthesized celacarfurine is shown in the appendix. Figure 9 The results showed that the specific rotation, high-resolution mass spectrum, HPLC-DAD retention time and ultraviolet spectrum of the two were completely identical.

[0190] Conclusion: Based on the above spectral data, isomerism analysis, and direct comparison with natural products, the synthesized compound 7 is confirmed to be the target natural product celacarfurine.

[0191] Example 2

[0192] The difference between this embodiment and Example 1 lies in the following process parameters in step 2: the initial feed amount of compound 1, the molar ratio of compound 1 to N-(3-bromopropyl)carbamate tert-butyl ester, and the heating time; other steps and conditions are the same as in Example 1.

[0193] The difference between this embodiment and Example 1 lies only in the preparation process of compound 2 in step 2. The specific process parameters are adjusted as follows: the initial feed amount of compound 1 is 3.02 g, and the molar amount is 9.98 mmol; the molar ratio of compound 1 to N-(3-bromopropyl)carbamate tert-butyl ester is 1:0.7; the heating time is 72 h; the specific process is as follows:

[0194] Compound 1 (3.02 g, 9.98 mmol) was accurately weighed into a 100 mL three-necked flask using an electronic balance, and 30 mL of acetonitrile was added for mixing. N-(3-bromopropyl)carbamate tert-butyl ester (2.38 g, 7.07 mmol) and potassium carbonate (4.15 g, 30.0 mmol) were weighed separately and added to the three-necked flask sequentially. The mixture was heated at 80 °C for 72 h. After a series of reaction treatments, 3.05 g of compound 2 was obtained, with a yield of 90.7%.

[0195] Example 3

[0196] The difference between this embodiment and Embodiment 1 lies in the following process parameters in step 2: the initial feed amount of compound 1 and the molar ratio of compound 1 to potassium carbonate; other steps and conditions are the same as in Embodiment 1.

[0197] The difference between this embodiment and Example 1 lies only in the preparation process of compound 2 in step 2. The specific process parameters are adjusted as follows: the initial feed amount of compound 1 is 9.61 g, and the molar amount is 31.8 mmol; the molar ratio of compound 1 to N-(3-bromopropyl)carbamate tert-butyl ester remains 1:1; the molar ratio of compound 1 to potassium carbonate is 1:5; the specific process is as follows:

[0198] Compound 1 (9.61 g, 31.8 mmol) was accurately weighed into a 100 mL three-necked flask using an electronic balance, and 30 mL of acetonitrile was added for mixing. N-(3-bromopropyl)carbamate tert-butyl ester (7.57 g, 31.8 mmol) and potassium carbonate (22.0 g, 159.0 mmol) were weighed separately and added to the three-necked flask sequentially. The mixture was heated at 80 °C for 96 h. After a series of reactions and treatments, 9.87 g of compound 2 was obtained, with a yield of 86.4%.

[0199] Example 4

[0200] The difference between this embodiment and Example 1 lies in the following process parameters in step 2: the initial feed amount of compound 1, the type of alkali added, and the molar ratio of compound 1 to alkali; other steps and conditions are the same as in Example 1.

[0201] The difference between this embodiment and Example 1 lies only in the preparation process of compound 2 in step 2. The specific process parameters are adjusted as follows: the initial feed amount of compound 1 is 1.03 g, and the molar amount is 3.4 mmol; the molar ratio of compound 1 to N-(3-bromopropyl)carbamate tert-butyl ester remains 1:1; the alkali is changed to triethylamine, and the molar ratio of compound 1 to the alkali is 1:5; the specific process is as follows:

[0202] Compound 1 (1.03 g, 3.4 mmol) was accurately weighed into a 100 mL three-necked flask using an electronic balance, and 30 mL of acetonitrile was added for mixing. N-(3-bromopropyl)carbamate tert-butyl ester (0.80, 3.4 mmol) and triethylamine (2.4 mL, 17.0 mmol) were weighed separately and added sequentially to the three-necked flask. The mixture was heated at 80 °C for 96 h. After a series of reactions and treatments, HPLC monitoring revealed a low product yield and a large amount of unreacted starting material.

[0203] By comparing the reaction conditions and target product yields of Examples 1-4, it can be found that potassium carbonate is the preferred alkali in step 2. Changing the initial feed amount of compound 1, the molar ratio of compound 1 to alkali, the molar ratio of compound 1 to N-(3-bromopropyl)carbamate tert-butyl ester, and the heating time has little effect on the product yield in step 2. Furthermore, by comparing Examples 1-4, it can be found that the highest target product yield is achieved when the heating time is 96 h and the molar ratio of compound 1, N-(3-bromopropyl)carbamate tert-butyl ester, and potassium carbonate is 1:1:3.

[0204] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A process for the synthesis of celacarfurine and key intermediates thereof, characterized in that, The synthesis route is as follows: Starting with methyl 4-aminobutyrate hydrochloride, the compound was subjected to a series of reactions including o-nitrobenzenesulfonyl protection, alkylation, amide condensation, hydrolysis and deprotection, intramolecular cyclization, removal of the o-nitrobenzenesulfonyl protecting group, and optional acylation, to obtain the compound shown in general formula I; wherein, the structural formula of general formula I is [insert structural formula here]. In this formula, R is hydrogen or an acyl group. When R is 3-furanoyl, the compound of general formula I is celacarfurine; when R is hydrogen, the compound of general formula I is the key intermediate 2-phenyl-1,5,9-triazacyclotetane-4,13-dione. The specific preparation steps for compound I of general formula are as follows: Step 1: Methyl 4-aminobutyrate hydrochloride undergoes a nucleophilic substitution reaction with o-nitrobenzenesulfonyl chloride under alkaline conditions to generate compound 1, wherein the o-nitrobenzenesulfonyl group is abbreviated as Ns; Step 2: Compound 1 undergoes a nucleophilic substitution reaction with N-(3-bromopropyl)carbamate tert-butyl ester under alkaline conditions upon heating to generate compound 2; Step 3: Compound 2 undergoes an amide condensation reaction with (R)-3-(tert-butoxycarbonylamino)-3-phenylpropionic acid in the presence of a condensing agent to generate compound 3; Step 4: Compound 3 undergoes ester hydrolysis under strongly alkaline conditions to generate a carboxylate, and then the Boc protecting group is removed under strongly acidic conditions to generate compound 4. Step 5: Compound 4 undergoes an intramolecular cyclization reaction in the presence of a condensing agent to generate compound 5; Step 6: Compound 5 is reacted with thiols or thiophenols under alkaline conditions to remove the Ns substituent protecting group, and then treated with acid to form a salt, yielding the key intermediate compound 6. Step 7: Compound 6 undergoes a nucleophilic substitution reaction with acyl chloride under alkaline conditions to generate compound 7; Among them, the structural formula of compound 1 is Compound 2 has the following structural formula: Compound 3 has the following structural formula: Compound 4 has the following structural formula: Compound 5 has the following structural formula: Compound 6 has the following structural formula: Compound 7 has the following structural formula: The Ns substituent structure is as follows: The structural formula for celacarfurine is: .

2. The method according to claim 1, characterized in that, In step 1, methyl 4-aminobutyrate hydrochloride reacts with o-nitrobenzenesulfonyl chloride under set conditions to generate compound 1. The reaction process is as follows: The set conditions are as follows: The solvent is dichloromethane; The base is one or a combination of at least two of triethylamine, N,N-diisopropylethylamine, pyridine, and 1,8-diazabicyclo[5.4.0]undec-7-ene; The molar ratio of methyl 4-aminobutyrate hydrochloride to base is 1:2-3; The molar ratio of methyl 4-aminobutyrate hydrochloride to o-nitrobenzenesulfonyl chloride is 1:

1.

3. The method of claim 1, wherein, In step 2, compound 1 reacts with N-(3-bromopropyl)carbamate tert-butyl ester under specified conditions to generate compound 2. The reaction process is as follows: The set conditions are as follows: The solvent is one or a combination of at least two of acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide; The reaction temperature is 80-100℃; The reaction time is 24-120 hours; The molar ratio of compound 1 to N-(3-bromopropyl)carbamate tert-butyl ester is 1:1; The base is one or at least a combination of two of the following: potassium carbonate, sodium carbonate, cesium carbonate, triethylamine, N,N-diisopropylethylamine, pyridine, and 1,8-diazabicyclo[5.4.0]undec-7-ene; The molar ratio of compound 1 to the base is 1:3-5.

4. The method of claim 1, wherein, In step 3, compound 2 reacts with (R)-3-(tert-butoxycarbonylamino)-3-phenylpropionic acid under set conditions to generate compound 3. The reaction process is as follows: The set conditions are as follows: The molar ratio of compound 2 to (R)-3-(tert-butoxycarbonylamino)-3-phenylpropionic acid is 1:1; The condensing agent is one or a combination of at least two of the following: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-propylphosphonic anhydride, O-(7-azabenzotriazine-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate, and tetramethylchlorourea hexafluorophosphate. The molar ratio of compound 2 to any condensing agent is 1:1.1-1.5; The base is N,N-diisopropylethylamine or triethylamine; The molar ratio of compound 2 to the base is 1:1.1-2.

5. The method of claim 1, wherein, In step 4, compound 3 is converted into compound 4 under specified conditions, and the reaction process is as follows: The set conditions are as follows: The solvent used in the ester hydrolysis process is a mixture of methanol and water; The solvent for the de-Boc substituenting process is one or a combination of at least two of dioxane hydrochloride, ethyl acetate hydrochloride, and trifluoroacetic acid. The base is one or a combination of at least two of lithium hydroxide, potassium hydroxide, and sodium hydroxide; The molar ratio of compound 3 to the base is 1:1.1-3.

6. The method of claim 1, wherein, In step 5, compound 4 is converted into compound 5 under the specified conditions, and the reaction process is as follows: The set conditions are as follows: The condensing agent is one or a combination of at least two of the following: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-propylphosphonic anhydride, O-(7-azabenzotriazine-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate, and tetramethylchlorourea hexafluorophosphate. The molar ratio of compound 4 to the condensing agent is 1:1-5; The solvent is acetonitrile; The base is N,N-diisopropylethylamine or triethylamine; The molar ratio of compound 4 to the base is 1:5-10.

7. The method of claim 1, wherein, In step 6, compound 5 reacts with a thiol-containing compound under set conditions, and after acid washing, compound 6 is generated. The acid is hydrochloric acid, and the reaction process is as follows: The set conditions are as follows: The thiol-containing compound is selected from thiols or thiophenols; The molar ratio of compound 5 to the mercapto-containing compound is 1:5-10; The base is one or at least a combination of two of the following: potassium carbonate, sodium carbonate, cesium carbonate, triethylamine, N,N-diisopropylethylamine, pyridine, and 1,8-diazabicyclo[5.4.0]undec-7-ene; The molar ratio of compound 5 to the base is 1:5-10.

8. The method of claim 1, wherein, In step 7, compound 6 reacts with acyl chloride under set conditions to generate compound 7. The reaction process is as follows: The set conditions are as follows: The molar ratio of compound 6 to acyl chloride is 1:1-1.5; The base is one or at least a combination of two of the following: potassium carbonate, sodium bicarbonate, cesium carbonate, triethylamine, N,N-diisopropylethylamine, pyridine, and 1,8-diazabicyclo[5.4.0]undec-7-ene; The solvent is one or a combination of water or dichloromethane.

9. The method according to claim 1 or 8, characterized in that, The acyl chloride includes, but is not limited to, 3-furancarbamate chloride; wherein, when the acyl chloride is 3-furancarbamate chloride, compound 6 reacts with 3-furancarbamate chloride to generate celacarfurine; when the acyl chloride is other acyl chloride, a derivative of 2-phenyl-1,5,9-triazacyclotetane-4,13-dione is generated.

Images