A method for preparing a fluorinated modified wang resin for immobilization of 25-hydroxyvitamin d3 feedstock cd loop fragments

Abstract

The application discloses a preparation method of a fluorine-modified Wang resin for fixing a 25-hydroxyvitamin D3 raw material CD ring fragment, and belongs to the technical field of solid-phase synthesis carriers. In view of the defect that the ester bond of a traditional Wang resin is prone to breaking under a strong alkaline (especially a deuterated methyl metal reagent) environment, leading to the falling of a substrate, fluorine atoms are introduced onto a benzene ring of the Wang resin through an electrophilic fluorination modification process, and the strong electron-withdrawing effect of the fluorine atoms is used to significantly enhance the alkali resistance of the ester bond. The resin can be combined with a CD ring carboxylic acid fragment of 25-hydroxyvitamin D3 with high efficiency, and the stable ester bond can effectively resist the damage of subsequent strong alkali addition reactions. Compared with a traditional process, the desorption yield is increased from 73.2% to 89.7% after the modified resin is applied, the overall production cost is reduced by 20%-40%, and the modified resin has remarkable industrial practicability.

Description

Technical Field

[0001] This invention relates to the fields of polymer chemical modification and solid-phase synthesis support technology, specifically to a method for preparing fluorinated modified Wang resin for immobilizing the CD ring fragment of 25-hydroxyvitamin D3 raw material. More specifically, it relates to a modification method that enhances the ester bond stability between the Wang resin and the raw material fragment by introducing fluorine atoms onto the benzene ring. This invention belongs to the field of solid-phase organic synthesis support material modification technology, and is particularly suitable for solid-phase synthesis applications requiring special tolerance to strongly alkaline reaction conditions. Background Technology

[0002] Since its inception by Merrifield in 1963, solid-phase synthesis has become an indispensable core tool in organic synthesis and medicinal chemistry due to its ability to separate and purify reaction substrates through simple filtration and washing. Among various solid-phase supports, Wang resin (hydroxymethyl polystyrene) has long been used as a classic linking strategy due to its simple structure and convenient functionalization. Its principle is to achieve covalent loading by utilizing the dehydration condensation of hydroxymethyl groups on the resin with carboxyl groups on the starting material fragments to form ester bonds.

[0003] However, in practical pharmaceutical intermediate applications, such as the solid-phase synthesis of the key CD ring fragment of deuterated 25-hydroxyvitamin D3, the structural defects of traditional Wang resins are drastically amplified. While the carbonyl carbon of the ester bond remains stable under normal conditions, it readily undergoes alkaline hydrolysis or acyl-oxygen cleavage when exposed to strongly basic nucleophiles (such as deuterated methyl metal reagents) required in subsequent synthetic steps. This chemical structural insensitivity directly leads to the premature detachment of a large number of loaded fragments, resulting in a solid-phase yield of only 73.2% under traditional processes. This inefficient conversion not only limits the synthetic purity of the target product but also leads to significant material waste during industrial scale-up, indirectly increasing overall production costs.

[0004] To address the aforementioned bottlenecks, those skilled in the art have attempted to circumvent alkaline hydrolysis by introducing sterically hindered connecting arms or replacing ester bonds with amide or ether bonds. However, these conventional modifications often result in negative impacts such as reduced resin loading capacity and stringent cutting conditions, and also limit the synthesis reaction rate, making it difficult to balance operational convenience and industrial economics. Therefore, how to enhance the resistance of ester bonds to strong bases at the molecular structure level while retaining the original advantages of Wang resin (such as mild cutting conditions and good compatibility) has become a key technical challenge that urgently needs to be overcome.

[0005] This invention, based on the theory of organic electronic effects, proposes a novel modification approach utilizing the strong electron-withdrawing inductive effect and small atomic radius of fluorine atoms. By introducing fluorine atoms at the ortho-position of the hydroxymethyl group on the benzene ring of Wang resin through an electrophilic fluorination process, the strong electron-withdrawing properties of fluorine effectively reduce the electron cloud density of the benzene ring and surrounding oxygen atoms. This structural modification fundamentally weakens the electrophilicity of the carbonyl carbon in the ester bond, giving it a stronger electron shielding effect against attacks from deuterated methyl metal reagents.

[0006] More importantly, this innovative approach directly translates the improvement in physicochemical properties into increased industrial efficiency. The Wang resin modified by this electrophilic fluorination process exhibits excellent strong alkali resistance when condensed with the CD ring fragment of deuterated 25-hydroxyvitamin D3. Experimental data fully demonstrate that this modification technology significantly increases the yield of the solid-phase reaction from 73.2% in the traditional process to 89.7% (a 16.5% increase), while effectively reducing material loss in subsequent purification stages and lowering overall production costs by 20%-40%. This invention not only provides a novel technical route to overcome the instability of ester bonds under strong alkaline conditions, but also, due to its extremely high overall yield and significant cost advantages, provides practical support for the industrial application of solid-phase synthesis technology in the development of sensitive drug intermediates and deuterated drugs. Summary of the Invention

[0007] This invention provides a method for preparing fluorinated modified Wang resin for immobilizing the CD ring fragment of 25-hydroxyvitamin D3 raw material. It primarily addresses the technical shortcomings of existing Wang resins based on hydroxymethyl polystyrene in solid-phase synthesis, namely, the poor stability of the ester bonds formed by the dehydration condensation of the resin and the raw material fragment. In particular, the ester bonds cannot withstand the strongly alkaline environment of the deuterated methyl metal reagent in subsequent synthesis steps, easily leading to side reactions or ester bond breakage, resulting in a low solid-phase yield (only 73.2% in the original process). This invention introduces fluorine atoms at the ortho position of the hydroxymethyl group on the benzene ring of the Wang resin. Utilizing the strong electron-withdrawing effect of fluorine atoms, the electron cloud density of oxygen atoms is effectively reduced, thereby enhancing the stability of the ester bonds to strong bases after the modified resin and the raw material fragment are combined.

[0008] To achieve the aforementioned technical objectives of this invention, on one hand, this invention provides a method for preparing fluorinated modified Wang resin for immobilizing the CD ring fragment of 25-hydroxyvitamin D3 raw material. The key lies in selectively substituting fluorine onto the aromatic ring of the Wang resin using specific electrophilic fluorinating reagents and reaction conditions. The synthetic route and modification strategy are shown below, and the specific implementation includes the following key steps: first, the Wang resin is pretreated and activated; then, it reacts with a selected electrophilic fluorinating reagent under suitable temperature and catalyst conditions; finally, after quenching, washing, and drying, the fluorinated modified Wang resin product is obtained. This method aims to fundamentally improve the stability and reliability of resin supports in solid-phase synthesis, especially in the assembly of peptides and complex organic molecular sequences.

[0009]

[0010]

[0011] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0012] Fluorine atoms are introduced onto the benzene ring of Wang resin via an electrophilic fluorination reaction. The strong electron-withdrawing inductive effect of fluorine atoms reduces the electron cloud density of the benzene ring adjacent to the ester bond, thereby reducing the possibility of nucleophilic attack on the carbonyl carbon of the ester bond. This results in a significantly enhanced alkali resistance to the ester bond formed between the modified Wang resin and the carboxyl group of the raw material fragment, effectively resisting the attack of strong bases such as deuterated methyl metal reagents during subsequent synthesis.

[0013] The modified Wang resin obtained has excellent industrial applicability, not only because the raw materials for the synthesis process are readily available and the post-processing is simple, but also because of its excellent performance in the synthesis of specific pharmaceutical intermediates. For example... Figure 2 As shown, the prepared modified Wang resin (compound VI), under the action of 1-methylimidazole (MeIm) and MSNT, can efficiently bind with the key raw material fragment of the CD ring of deuterated 25-hydroxyvitamin D3 (compound VII), forming a stable ester bond structure, which effectively resists the strong alkaline attack of the deuterated methyl metal reagent in subsequent reactions. In practical applications, using the modified Wang resin technology system of this invention, the yield of the solid-phase removal reaction is significantly increased from 73.2% in the original process to 89.7% (yield increase of 16.5%). This significant breakthrough in performance directly solves the purification difficulties and material losses caused by the easy breakage of ester bonds in traditional processes, reducing the production cost of the final product by 20%-40%, demonstrating significant economic benefits and broad industrial application prospects. Attached Figure Description

[0014] Figure 1This is a synthesis route diagram for Wang resin.

[0015] Figure 2 This is a route diagram for the synthesis of fluorinated modified Wang resin and the CD ring fragment from the raw material.

[0016] Figure 3 This is the 1H NMR spectrum of compound III.

[0017] Figure 4 This is the 1H NMR spectrum of compound V.

[0018] Figure 5 This is the 1H NMR spectrum of compound VI. Detailed Implementation

[0019] The technical solution of the present invention will now be described with reference to the embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0020] All weight-to-volume ratios in this invention are expressed in g:mL.

[0021] The synthetic route of the fluorinated modified Wang resin of this invention is shown below.

[0022]

[0023] Example 1

[0024] This embodiment provides a method for preparing fluorinated modified Wang resin, specifically including the following steps:

[0025] S1: Tetrahydrofuran (THF) used in the experiment must be refluxed with metallic sodium / benzophenone until a deep blue color is achieved, and then distilled before use (i.e., anhydrous and oxygen-free treatment). N,N-Dimethylformamide (DMF) must be distilled under reduced pressure with calcium hydride (CaH2) and stored with 4Å molecular sieves before use. Merrifield resin (100-200 mesh, chlorine content 1.0 mmol / g) must be thoroughly washed sequentially with dichloromethane, methanol, and acetone, and then vacuum dried to constant weight before use. Compound I (purity ≥98%, purchased from a reagent company) does not require further purification before use.

[0026] S2: In a 500 mL three-necked flask equipped with a magnetic stirrer, thermometer, and constant-pressure dropping funnel, add compound I (15.4 g, 100 mmol) and dry THF (150 mL). Place the reaction flask in an ice-water bath and, after the solution cools to 0–5 °C, add 60% sodium hydride (NaH, 4.8 g, 120 mmol) in portions. Carefully control the temperature during the addition process to avoid vigorous bubbling. After the hydrogen gas has been completely released and the reaction system has become a homogeneous suspension, slowly add chloromethyl methyl ether (MOM-Cl, 8.4 g, 105 mmol) dropwise over approximately 30 minutes. After the addition is complete, remove the ice bath and slowly heat the reaction solution to room temperature (approximately 25 °C) while stirring for 1 hour, then reflux at 80 °C for 1 hour. TLC (thin-layer chromatography, petroleum ether:ethyl acetate = 4:1) showed that the starting material spot had completely disappeared.

[0027] S3: Cool the reaction solution to room temperature and slowly quench the reaction in ice water containing 200 mL of saturated ammonium chloride solution. Extract with ethyl acetate (3 × 150 mL) and combine the organic phases. Wash the organic phase with saturated brine (150 mL) and dry overnight with anhydrous sodium sulfate. Filter and concentrate under reduced pressure using a rotary evaporator to remove the solvent, giving a pale yellow oily compound II (19.7 g). This product does not require further column chromatography purification and can be used directly in the next step. The calculated yield after purification in this step is approximately 93% (based on starting material I).

[0028] S4: In a 1 L single-necked flask, dissolve compound II (19.7 g) prepared in the previous step in 1,4-dioxane (150 mL), and add deionized water (100 mL). Slowly add a 10% sodium hypochlorite (NaClO) aqueous solution (150 mL, approximately 10% available chlorine) dropwise while stirring. After the addition is complete, heat the reaction system to 70°C and reflux at this temperature with vigorous stirring for 15 hours.

[0029] S5: In a dry 250 mL three-necked flask, add clean Merrifield resin (10 g, chlorine content 1.0 mmol / g, i.e., 10 mmol active sites), compound IV (2.24 g, approximately 12.0 mmol, 1.2 times excess relative to the resin active sites), and anhydrous cesium carbonate (Cs₂CO₃, 6.5 g, approximately 20.0 mmol). Add dry DMF (120 mL), heat to 80 °C under high-purity nitrogen protection, and stir the reaction for 12 hours.

[0030] S6: In a 500 mL three-necked flask, place the resin compound V (10 g) obtained in the previous step, add 100 mL of anhydrous THF under nitrogen protection, and allow it to swell at room temperature for 30 minutes. Subsequently, under ice-water bath cooling, carefully add lithium aluminum hydride (LAH, 1.5 g, approximately 40 mmol) in portions, controlling the temperature not to exceed 25°C during the addition process. After the addition is complete, remove the ice-water bath and stir the reaction at room temperature (25°C) for 2 hours.

[0031] Example 2

[0032] Comparative test of resin's resistance to strong alkalis

[0033] S1: Two resins were activated and grafted with the same small molecule fragments. The fragment linkers were connected via typical ester bonds (i.e., esterification with standard reagents via the -COOH groups on the resin). The loading of both resins was adjusted to 0.8 mmol / g, and 0.5 g of each was placed in a reaction tube.

[0034] Control group A: Commercially available Wang resin (unmodified), linked to the same small molecule carboxylic acid substrate.

[0035] Control group B: The fluorinated modified Wang resin (compound VI) of the present invention, linked to the same small molecule carboxylic acid substrate.

[0036] S2: Add a high-concentration, strongly alkaline deuterated methyl reagent solution (simulating the actual reaction environment, such as a solution of deuterated methyl lithium in THF, 0.5 M, with an excess of 3 equivalents) to two reaction tubes. Stir the reaction under nitrogen protection at room temperature (25°C) for 4 hours. After the reaction, filter out the resin and wash thoroughly with large amounts of THF, methanol, and water to remove all free components. Then, cleave the remaining product on the resin with high-concentration trifluoroacetic acid (TFA), and quantitatively analyze the retention of the target product in the lysate using HPLC-MS (high-performance liquid chromatography-mass spectrometry) (calculated with the resin loading at 0 hours as 100%).

[0037] S3: Control group A (traditional Wang resin): After 4 hours of treatment with a strongly alkaline reagent, quantitative analysis based on HPLC-MS showed that the retention rate of the target product was only 25-35%. This indicates that the ester bonds in the traditional Wang resin undergo severe hydrolysis or alcoholysis under a strongly alkaline environment, and more than 2 / 3 of the product has been detached from the resin, leading to the risk of failure of the entire synthetic route.

[0038] Control group B (fluorinated modified Wang resin of this invention): Under exactly the same treatment conditions, the retention rate of the target product was as high as 92% or more. Even after exposure to an extremely alkaline environment for 4 hours, it could still maintain most of the covalent bonds.

[0039] Conclusion: The fluorinated modified Wang resin of the present invention, through electrophilic fluorination process to electronically modify the benzene ring, does indeed greatly enhance the tolerance of the ester bond linker to a strongly alkaline environment, and can effectively resist the attack of deuterated methyl metal reagents, thus fully achieving the purpose of the invention.

[0040] Example 3

[0041] Optimization of reaction parameters and exploration of conditions

[0042] S1: In this step, the amount and type of base significantly affect the reaction efficiency. Experimental data show that although NaOH can also be used as a base, for para-substituted phenolic substrates, NaH (and KH) provides a purer reaction and higher yield. When the amount of NaH is less than 1.2 equivalents, the reaction conversion drops below 80%; when the amount is greater than 3.0 equivalents, it exacerbates the formation of byproducts (such as dimerization). Therefore, the preferred amount of NaH in this invention is 1.5-2.0 equivalents.

[0043] S2: The effect of different volume ratios (10:0, 3:1, 1:1, 1:3, 0:10) of dioxane to water on the oxidation effect was investigated in a 10 mL small-scale experiment. The results showed that under pure dioxane (10:0) conditions, the reaction was very slow (requiring more than 24 hours). Under pure water (0:10) conditions, the substrate solubility was extremely poor, and the reaction was uneven. When the ratio was 1.5:1 to 2.5:1, the reaction reached complete conversion within 15 hours, with a yield as high as 99%. This indicates that this co-solubility ratio not only ensures sufficient substrate dissolution but also stabilizes the activity of NaClO.

[0044] S3: This invention focuses on the role of inorganic bases in the immobilization reaction. Although potassium carbonate (K₂CO₃) and sodium carbonate (Na₂CO₃) can also deprotonate phenolic hydroxyl groups, their solubility in DMF is limited. When using K₂CO₃, the reaction requires up to 48 hours to achieve a 90% grafting rate. However, when cesium carbonate (Cs₂CO₃) is used, its excellent solubility in DMF and the resulting Cs⁺ coordinating with the phenoxy anion significantly enhance nucleophilicity, allowing a grafting rate of over 85% to be achieved within 12 hours. Therefore, Cs₂CO₃ is the optimal base for this invention.

[0045] S4: In the LAH reduction step, the reaction progress cannot be monitored by conventional TLC due to the insolubility of the resin. The inventors developed a simple sampling and monitoring method: trace amounts of resin were sampled at different reaction time points (30 minutes, 1 hour, 2 hours, and 3 hours), cleaved using TFA, and then analyzed by MS. The results showed that after 2 hours, all methyl ester peaks disappeared from the MS spectrum, completely converting to the molecular weight of the hydroxyl peaks. Therefore, 2 hours is a suitable reaction time.

[0046] Industrial applicability

[0047] The fluorinated modified Wang resin prepared by this invention has significant industrial application value. This resin overcomes the technical defect of traditional Wang resin, where the ester bond formed between the hydroxymethyl group and the carboxyl group of the raw material fragment is easily hydrolyzed and detached under strongly alkaline conditions, by introducing fluorine atoms onto the benzene ring of the traditional Wang resin and utilizing the strong electron-withdrawing inductive effect of fluorine atoms.

[0048] In practical solid-phase synthesis applications, the fluorinated modified Wang resin prepared by this invention can specifically react with carboxyl-containing raw material fragments. Specifically, the hydroxymethyl group of the modified Wang resin can bind to the CD ring fragment of deuterated 25-hydroxyvitamin D3. The specific reaction binding mechanism is shown in the following equation:

[0049]

[0050] As shown in the reaction pathway in the formula, the hydroxymethyl group on the modified Wang resin (corresponding to compound VI in the formula) and the carboxyl group in the CD ring fragment of deuterated 25-hydroxyvitamin D3 (corresponding to compound VII in the formula) form a stable ester bond under the action of a condensing agent, generating a combined resin fragment (corresponding to compound VIII in the formula).

[0051] In this strongly basic addition reaction, the introduction of fluorine atoms significantly increases the difficulty for the carbonyl carbon of the ester bond to be attacked by nucleophiles. This allows the modified Wang resin to effectively resist the destructive effects of the strongly basic deuterated methyl metal reagent in the subsequent addition reaction. Practical application data shows that the modified Wang resin prepared using this invention significantly increases the solid-phase yield from 73.2% to 89.7% compared to the original process. This technical effect not only significantly improves the synthetic purity and yield of the product but also directly brings significant economic benefits, reducing overall production costs by 20%-40%.

[0052] Furthermore, this modified fluorinated Wang resin is not only suitable for the solid-phase synthesis of specific drugs such as deuterated 25-hydroxyvitamin D3-D6, but can also be widely applied in many fields such as the synthesis of isotope-labeled drugs requiring extremely strong basic reaction conditions, the total synthesis of complex natural products, and the construction of combinatorial chemistry libraries. Simultaneously, thanks to the introduction of fluorine atoms on the benzene ring, this resin can also serve as a 19F NMR probe for real-time monitoring of solid-phase synthesis reactions and for the analysis of reaction kinetics and process optimization, possessing extremely high industrial application value and broad market prospects. In summary, the modified Wang resin provided by this invention has extremely strong industrial applicability.

Claims

1. A method for preparing a fluorinated modified Wang resin for immobilizing the CD ring fragment of 25-hydroxyvitamin D3 raw material, characterized in that, Includes the following steps: S1: The phenolic hydroxyl group in compound I is protected by MOM and reacted with MOM-Cl in an organic solvent under strong base conditions to generate a MOM ether protected intermediate; S2: The MOM ether protected intermediate obtained in step S1 is subjected to an oxidation reaction to oxidize the substituents on the substrate to carboxyl groups, thereby obtaining acidic compound III; S3: The acidic compound III obtained in step S2 is subjected to deprotection and esterification reaction in an acidic alcohol solution. The MOM ether bond is broken and the phenolic hydroxyl group is released again. At the same time, the carboxyl group reacts with the alcohol in situ to generate methyl ester, and a small molecule intermediate containing phenolic hydroxyl group and methyl ester is obtained. S4: The small molecule intermediate obtained in step S3 is reacted with chloromethylated polystyrene resin under alkaline conditions to undergo a Williamson ether synthesis reaction. Through the nucleophilic substitution reaction between the phenolic hydroxyl group and the benzyl chloride on the resin, the small molecule is covalently linked to the resin backbone to obtain a supported resin intermediate. S5: The supported resin intermediate obtained in step S4 is reduced by a reducing agent to selectively reduce the methyl ester group on it to a primary alcohol, thereby obtaining a modified Wang resin containing fluorine atoms on the benzene ring.

2. The method according to claim 1, characterized in that, The strong base in step S1 is sodium hydride, the organic solvent is tetrahydrofuran, the reaction temperature is reflux to 80°C, and the reaction time is 1 hour.

3. The method according to claim 1, characterized in that, The oxidation reaction in step S2 uses sodium hypochlorite as the oxidant, a mixture of water and dioxane as the reaction solvent, a reaction temperature of 70°C, and a reaction time of 15 hours.

4. The method according to claim 1, characterized in that, The modified Wang resin has a fluorine atom introduced at the C2 position of the hydroxymethyl side chain of the benzene ring. Based on its strong electron-withdrawing inductive effect, this fluorine atom can significantly enhance the bond energy of the ester bond formed after the resin and the raw material fragment undergo carboxyl condensation (or coupling), thereby improving the ester bond's resistance to strongly basic nucleophiles. 。 5. The application according to claim 4, characterized in that, When the modified Wang resin is used instead of the conventional Wang resin without fluorine atoms for solid-phase reaction, the solid-phase yield increases from 73.2% to 89.7%. Furthermore, due to the reduction of side reactions and the lower difficulty of product purification, the overall production cost based on this solid-phase synthesis is reduced by 20% to 40%.

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