A method for synthesizing a compound DDAO-Sulfate

Abstract

This invention provides a method for synthesizing the compound DDAO-Sulfate, belonging to the field of chemical synthesis technology. The method includes the following steps: mixing DDAO and sodium ethoxide with anhydrous dimethylformamide to obtain a mixed solution; adding pyridine sulfur trioxide to the mixed solution and heating and stirring the reaction under light-protected conditions; subsequently concentrating under vacuum to obtain a residue; and purifying the residue by column chromatography using an eluent to obtain the final product. This synthetic method effectively improves the yield while selecting and optimizing the reactants, avoiding the use of easily deteriorated reagents and interference caused by the introduction of irrelevant substances, thus providing an efficient pathway for the preparation of fluorescent detection probes for sulfatase.

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis technology, and in particular to a method for synthesizing the compound DDAO-Sulfate. Background Technology

[0002] Sulfatases, primarily located in lysosomes, endoplasmic reticulum, and Golgi apparatus, are mainly responsible for catalyzing the hydrolysis of sulfate ester bonds in various sulfate biomolecules to remove the sulfate moiety. They also participate in a variety of biological events, including cell signaling, hormone regulation, and bacterial pathogenesis. Abnormal levels of various sulfatase subtypes are closely related to the development and progression of various diseases, such as polysulfatase deficiency syndromes and cancer. For example, overexpression of heparanol sulfate-2 (Sulf-2) promotes the development of lung cancer in humans, and it is also upregulated in tumorigenesis and hepatocellular carcinoma. Furthermore, upregulation of steroid sulfatase (STS) levels leads to increased hormone production, inducing the pathophysiological conditions of hormone-dependent cancers, particularly breast cancer. STS inhibitors such as STX64 and EMATE have been used in the clinical treatment of breast cancer. Therefore, accurate quantification of sulfatase activity under biocompatible conditions is of great significance for the diagnosis and treatment of related diseases.

[0003] Fluorescent probes, with their advantages of high sensitivity, high selectivity, visualization, and ease of operation, have been widely used in the detection of biological samples. Notably, fluorescent probes can be used for non-invasive in vivo detection, thus attracting widespread attention in research fields such as medicine, biology, clinical diagnostics, and drug development. However, the excitation and emission wavelengths of many fluorescent probes are less than 600 nm, leading to problems such as poor tissue penetration, significant interference from autofluorescence in the body, and tissue damage, thus limiting their in vivo imaging applications. Encouragingly, near-infrared fluorescent (NIR) probes with emission and excitation wavelengths in the 600-900 nm range can effectively overcome these problems. For example, 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone (DDAO) possesses characteristics such as tunable excitation wavelength (600-650 nm), near-infrared emission (660 nm), good water solubility, and a low acid dissociation constant (pKa≈5). The NIR fluorescent probe DDAO-Sulfate, based on the DDAO core and synthesized using sulfatase, has been successfully used for rapid identification of mycobacterial species and strains. However, the current synthesis method of DDAO-Sulfate is relatively complex and has a low yield.

[0004] Therefore, this invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for synthesizing the compound DDAO-Sulfate. This method effectively improves the yield while selecting and optimizing the reactants, avoiding the use of easily deteriorated reagents and interference caused by the introduction of irrelevant substances, thus providing an efficient pathway for the preparation of sulfatase fluorescent detection probes.

[0006] The present invention also provides a method for synthesizing the compound DDAO-Sulfate and its application in the synthesis of DDAO-Sulfate.

[0007] To achieve the objectives of this invention, a method for synthesizing DDAO-Sulfate is provided, comprising the following steps:

[0008] S1. Mix DDAO and sodium ethoxide with anhydrous dimethylformamide to obtain a mixed solution;

[0009] S2. Add pyridine sulfur trioxide to the mixed solution and heat and stir to react under light-protected conditions;

[0010] S3. Subsequently, vacuum concentration was performed to obtain residue, which was then purified by column chromatography using an eluent to obtain the final product.

[0011] Furthermore, the molar ratio of pyridine sulfur trioxide: DDAO: sodium ethoxide is (0.2-0.6): (0.1-0.2): (0.4-1).

[0012] Furthermore, the molar ratio of pyridine sulfur trioxide:DDAO:sodium ethoxide is 0.3:0.1:0.5.

[0013] Furthermore, the heating temperature in S2 is 35℃-60℃.

[0014] Furthermore, the heating temperature in S2 is 40°C.

[0015] Furthermore, the reaction time in S2 is 5-8 hours.

[0016] Furthermore, the eluent is any one of a mixed solution of dichloromethane-methanol-acetic acid or a mixed solution of petroleum ether-ethyl acetate-trifluoroacetic acid.

[0017] Furthermore, in the dichloromethane-methanol-acetic acid mixed solution, the volume ratio of dichloromethane:methanol:ethanol is 10:1:0.1;

[0018] In the mixed solution of petroleum ether, ethyl acetate, and trifluoroacetic acid, the volume ratio of petroleum ether to ethyl acetate to trifluoroacetic acid is 5:1:0.05.

[0019] The present invention also provides the application of the method for synthesizing the compound DDAO-Sulfate in the synthesis of the compound DDAO-Sulfate.

[0020] The embodiments of the present invention have the following technical effects:

[0021] This invention solves the technical problem of complex synthesis steps in the prior art by optimizing reagent ratios, temperature and purification conditions, simplifies the synthesis route, and effectively improves yield and reaction efficiency, providing an efficient route for the preparation of sulfatase fluorescent detection probes. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 : NMR results for the synthesized DDAO-Sulfate. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] In a first aspect, the present invention provides a method for synthesizing the compound DDAO-Sulfate, which has a simple synthesis process, mild reaction conditions, low energy consumption, low synthesis cost, and is easy to operate; at the same time, it can also effectively improve the synthesis yield.

[0026] In some embodiments, the following steps are included:

[0027] S1. Mix DDAO and sodium ethoxide with anhydrous dimethylformamide to obtain a mixed solution;

[0028] S2. Add pyridine sulfur trioxide to the mixed solution and heat and stir to react under light-protected conditions;

[0029] S3. Subsequently, vacuum concentration was performed to obtain residue, which was then purified by column chromatography using an eluent to obtain the final product.

[0030] The phenolic hydroxyl group (-OH) in the DDAO molecule in S1 has weak acidity (pKa≈5). Under the action of the strong base sodium ethoxide, it undergoes deprotonation to generate the more nucleophilic phenoxy anion (DDAO-O). - Simultaneously, ethanol is formed as a byproduct. The nucleophilicity of the phenoxy anion is much higher than that of the neutral phenolic hydroxyl group, laying the foundation for the subsequent sulfonation reaction. At the same time, anhydrous dimethylformamide can provide an aprotic polar environment, preventing the protic solvent from protonating the phenoxy anion; it also prevents the sulfonating reagent (the sulfonating reagent in this invention is pyridine sulfur trioxide) from hydrolyzing and becoming ineffective.

[0031] Step S2 is the key step in the reaction, DDAO-O - The oxygen anion acts as a nucleophile, attacking the positively charged sulfur atom in pyridine sulfur trioxide. The sulfur atom forms a new SO bond with the oxygen atom, while the sulfur-oxygen double bond (S=O) of SO3 undergoes electron recombination to generate a sulfate monoester anion (-OSO3). - After the reaction, the sulfate ester group exists in the form of DDAO−OSO3, reacting with the pyridine cation (Py). + They form ion pairs.

[0032] Step S3 removes volatile components, resulting in a higher yield of DDAO-Sulfate. Simultaneously, this invention requires strict control of the anhydrous environment to minimize side reactions; the reaction process must be completely protected from light to prevent acridinone from undergoing ring-opening or oxidation under light irradiation.

[0033] The novel reaction pathway selected in this invention enables pyridine sulfur trioxide to specifically attack phenoxy anions, preventing other groups (such as alkyl hydroxyl groups) from being sulfonated, thereby effectively increasing the reaction rate. At the same time, the reaction conditions are milder, which can effectively reduce substrate decomposition and side reactions, thereby effectively improving the yield.

[0034] In some embodiments, the molar ratio of pyridine sulfur trioxide:DDAO:sodium ethoxide is (0.2-0.6):(0.1-0.2):(0.4-1).

[0035] In this invention, sodium ethoxide is used in excess to ensure complete deprotonation of DDAO and prevent unreacted DDAO from competing for side reactions; pyridine sulfur trioxide is used in excess to effectively promote the sulfonation reaction to completion.

[0036] In some embodiments, the molar ratio of pyridine sulfur trioxide:DDAO:sodium ethoxide is 0.3:0.1:0.5.

[0037] This formulation maximizes the forward reaction, effectively avoids side reactions, and balances reaction efficiency with cost. This design addresses the issues of low yield and numerous steps inherent in traditional methods at the molecular level, representing a core innovation in the efficient synthesis of DDAO-Sulfate.

[0038] In some embodiments, the heating temperature in S2 is 35°C-60°C.

[0039] In some embodiments, the heating temperature in S2 is 40°C.

[0040] In this invention, reaction conditions of 35℃-60℃ are selected, with the optimal reaction temperature being 40℃. At this temperature, both reaction efficiency and the occurrence of side reactions can be effectively reduced. When the reaction temperature is too low, the reaction will be too slow; while at higher temperatures, DMF is prone to decomposition, or pyridine sulfur trioxide is prone to self-polymerization, thus leading to the occurrence of side reactions.

[0041] In some embodiments, the reaction time in S2 is 5-8 hours.

[0042] In some embodiments, the eluent is either a mixed solution of dichloromethane-methanol-acetic acid or a mixed solution of petroleum ether-ethyl acetate-trifluoroacetic acid.

[0043] As a polar molecule containing sulfonate groups, DDAO-Sulfate requires highly polar solvents for elution during purification due to the strong interaction between the sulfonate groups and the silica gel column. At the same time, the anions are easily ionized, causing chromatographic peak tailing, which requires additives for suppression.

[0044] When the eluent is a mixed solution of dichloromethane, methanol, and acetic acid, dichloromethane itself is a low-polarity main solvent, which can support the elution system and maintain basic elution power; methanol, as a strong polar solvent, can break the hydrogen bonds between silica gel and sulfonate and provide sufficient polarity to overcome retention; a small proportion of added acetic acid can synergistically regulate polarity and inhibit sulfonate ionization, and the addition of trace amounts can effectively improve tailing phenomenon.

[0045] When the eluent is a mixed solution of petroleum ether, ethyl acetate, and trifluoroacetic acid, petroleum ether, as a nonpolar solvent, adjusts the elution baseline and, as the main component of the eluent, controls the elution rate; ethyl acetate provides elution power, and trifluoroacetic acid protonates sulfonate ions to eliminate charge tailing. Furthermore, its low dosage effectively prevents column bed damage.

[0046] In some embodiments, the volume ratio of dichloromethane:methanol:acetic acid in the dichloromethane-methanol-acetic acid mixed solution is 10:1:0.1.

[0047] In the mixed solution of petroleum ether, ethyl acetate, and trifluoroacetic acid, the volume ratio of petroleum ether to ethyl acetate to trifluoroacetic acid is 5:1:0.05.

[0048] When the volume ratio of dichloromethane:methanol:acetic acid is 10:1:0.1, selective elution can be achieved, tailing can be reduced, and co-elution of impurities caused by excessive methanol ratio can be avoided.

[0049] When the volume ratio of petroleum ether: ethyl acetate: trifluoroacetic acid is 5:1:0.05, ionic interactions can be eliminated, thus resolving the tailing problem.

[0050] The dichloromethane-methanol-acetic acid system has a higher elution rate and yield than the petroleum ether-ethyl acetate-trifluoroacetic acid system.

[0051] On the other hand, the present invention also provides the application of the method for synthesizing the compound DDAO-Sulfate in the synthesis of the compound DDAO-Sulfate.

[0052] The following is a detailed explanation using specific embodiments:

[0053] Example 1: Synthesis method of the present invention

[0054]

[0055] DDAO (30.8 mg, 0.1 mmol), sodium ethoxide (0.034 g, 0.5 mmol), and anhydrous dimethylformamide (DMF, 5 mL) were mixed to obtain a mixture.

[0056] Pyridine sulfur trioxide (0.048 g, 0.3 mmol) was mixed with the above mixture and reacted at 40°C in the dark with stirring for 8 h.

[0057] After concentration in vacuum, the residue was obtained. The residue was purified by column chromatography using DCM-MeOH-CH3COOH (10 / 1 / 0.1, v / v / v) as eluent. The resulting probe was a deep orange solid with a yield of 60%.

[0058] NMR confirmation of the target product:

[0059] 1 H NMR (DMSO, 400 MHz)

[0060] 7.83 (s, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.46 (d, J=4.0 Hz, 1H), 7.33 (dd, J=8.0, 2.8 Hz, 1H), 1.81 (s, 6H).

[0061] Comparative Example 1: Synthesis methods reported in the literature

[0062]

[0063] DDAO (61 mg, 198 µmol) was dissolved in anhydrous DMF (2.8 mL). The flask was placed on ice, and sodium hydride (22.5 mg, 563 µmol) with a dispersion of 60% was added to the stirred mixture under argon atmosphere. After 15 minutes, the flask was removed from the ice bath and heated to room temperature (15 minutes). Trimethylamine trioxide (SO3-TMA, 135 mg, 970 µmol) was added, and the reaction was heated at 55 °C for 4 hours. The reaction was then cooled and quenched with methanol.

[0064] After solvent evaporation, silica gel column chromatography was performed using a SiliaFlash P60 (SiliCycle) in a solution of ethyl acetate:acetone (gradient 8:1 to 2:1) and 0.05% triethylamine to give a red oil, which was identified as DDAO sulfate-sulfate and triethylamine counterion (79 mg, 161 µmol, yield 81.5%). DDAO-sulfate was further purified by Varian Pro Star reversed-phase high-performance liquid chromatography on a Dynamax 100 Å C18 semi-preparative column using a linear gradient of water (solvent A) and methanol (solvent B) (100% to 0% water). A singlet corresponding to DDAO-sulfate was obtained, and solvent evaporation yielded pure DDAO-sulfate as a deep orange solid (47 mg, 96 µmol, overall yield 48.5%).

[0065] NMR confirmation of the target product:

[0066] 1 H NMR (CD3OD, 400 MHz): δ 7.636 (s, 1 H), 7.56 (d, J = 5.6 Hz, 1 H),7.52 (s, 1 H), 7.27 (d, J = 5.2 Hz, 1 H), 1.84 (t, J = 4.8 Hz, 6 H).

[0067] From the reaction process and results of the comparative examples and embodiments, it can be seen that the present invention can simplify the reaction process and effectively improve the product yield.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A method for synthesizing the compound DDAO-Sulfate, characterized in that, Includes the following steps: S1. Mix DDAO and sodium ethoxide with anhydrous dimethylformamide to obtain a mixed solution; S2. Add pyridine sulfur trioxide to the mixed solution and heat and stir to react under light-protected conditions; S3. Subsequently, vacuum concentration was performed to obtain residue, which was then purified by column chromatography using an eluent to obtain the final product. The molar ratio of pyridine sulfur trioxide: DDAO: sodium ethoxide is (0.2-0.6): (0.1-0.2): (0.4-1). The heating temperature in S2 is 35℃-60℃.

2. The method for synthesizing the compound DDAO-Sulfate according to claim 1, characterized in that, The molar ratio of pyridine sulfur trioxide:DDAO:sodium ethoxide is 0.3:0.1:0.

5.

3. The method for synthesizing the compound DDAO-Sulfate according to claim 1, characterized in that, The heating temperature in S2 is 40°C.

4. The method for synthesizing the compound DDAO-Sulfate according to claim 1, characterized in that, The reaction time in S2 is 5-8 hours.

5. The method for synthesizing the compound DDAO-Sulfate according to claim 1, characterized in that, The eluent is either a mixed solution of dichloromethane-methanol-acetic acid or a mixed solution of petroleum ether-ethyl acetate-trifluoroacetic acid.

6. The method for synthesizing the compound DDAO-Sulfate according to claim 5, characterized in that, In the dichloromethane-methanol-acetic acid mixed solution, the volume ratio of dichloromethane:methanol:acetic acid is 10:1:0.1; In the mixed solution of petroleum ether, ethyl acetate, and trifluoroacetic acid, the volume ratio of petroleum ether to ethyl acetate to trifluoroacetic acid is 5:1:0.

05.

7. The application of a method for synthesizing the compound DDAO-Sulfate as described in any one of claims 1-6 in the synthesis of the compound DDAO-Sulfate.

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