A method for preparing on-dna beta-hydroxy thioether structured compounds

Abstract

This application belongs to the field of DEL library construction, specifically involving a method for synthesizing On-DNA β-hydroxy sulfide compounds via photocatalytic thiol-ene radical reaction. The reaction conditions are as follows: irradiation under a 365–535 nm light source, using ketones as catalysts, with a final concentration of the substrate thiol ranging from 25.0 mmol / L to 150.0 mmol / L. The reaction solvents are mixed solvents such as organic solvents DMSO, DMF, acetonitrile, ethanol, methanol, 1,4-dioxane, tetrahydrofuran, etc., and water. The conversion rate of the reaction product On-DNA β-hydroxy sulfide is over 90%, which not only fills the gap in the current methods for preparing On-DNA β-hydroxy sulfide compounds, but also enriches the On-DNA β-hydroxy sulfide compound library and the methods for preparing On-DNA sulfinimide compounds.

Description

Technical Field

[0001] This application belongs to the field of lead compound library construction in pharmaceutical research, specifically involving a method for synthesizing On-DNA β-hydroxy thioether structured compounds via photocatalytic thiol-ene radical reaction. Background Technology

[0002] The DNA-encoded compound library (DEL) technology, which has emerged in recent years, combines combinatorial chemistry and molecular biology techniques. It adds a DNA tag to each compound at the molecular level, enabling the synthesis of libraries containing hundreds of millions of compounds in a very short time. In the field of new drug development, high-throughput screening of biological targets is one of the main methods for rapidly obtaining lead compounds. DNA-encoded compound library technology significantly improves the efficiency and likelihood of lead compound discovery.

[0003] Currently, there are many known active compounds with β-hydroxythioether structures, such as the well-known clindamycin. The mechanism of action of clindamycin against *Escherichia coli* is as follows: the oxygen (O) of the hydroxyl group on the six-membered aliphatic ring forms a hydrogen bond with base 2058 of the RNA on the 50S ribosomal subunit of *E. coli*; while the sulfur (S) of the thioether on the six-membered aliphatic ring forms a hydrophobic interaction with base 2505. This blocks peptide bond formation or tRNA entry, inhibiting bacterial protein synthesis. The mechanism of action of clindamycin against *Propionibacterium acnes* is as follows: the oxygen (O) of the hydroxyl group on the six-membered aliphatic ring of clindamycin forms a hydrogen bond with base 2793 of the RNA on the 50S ribosomal subunit of *Propionibacterium acnes*; a water bridge is formed with base 2240; while the sulfur (S) of the thioether on the six-membered aliphatic ring forms a hydrophobic interaction with base 2687. This blocks peptide bond formation or tRNA entry, inhibiting its protein synthesis.

[0004] Another β-hydroxy sulfide with a relatively well-defined mechanism of action is leukotriene E4 (LTE4). Although present in trace amounts in the body, leukotriene E4 (LTE4) possesses high physiological activity and acts as a chemical mediator in certain allergic reactions, inflammation, and cardiovascular diseases. The mechanism of action of LTE4 can be simply summarized as follows: the oxygen (O) at the hydroxyl group of LTE4 forms a hydrogen bond with the α-ketoglutarate receptor 1 (OXGR1) residue Cys149; while the sulfur (S) at the thioether group of LTE4 forms a hydrophobic interaction with the OXGR1 residue Ala153. LTE4 is an agonist of OXGR1. Upon binding to OXGR1, it triggers the G protein-coupled receptor (GPCR) signaling cascade, leading to calcium ion influx, release of inflammatory factors, and airway hyperresponsiveness via the Gq protein pathway.

[0005] Other active compounds containing β-hydroxy sulfides include diltiazem. Naltiazem etc.

[0006] However, after extensive searching, no relevant literature reports were found. Among existing small molecule synthesis techniques, Anupam Das and KRJustin Thomas's paper, "Selective Regulation of Thiol-Alkene Coupling Reactions via EDA-TOCO Complex Formation Driven by Visible Light" (ACS Omega 2023, 8, 18275-18289), discloses a "two-step one-pot method for generating β-hydroxy thioether structures from thiols and alkenes." However, traditional high-throughput screening based on single molecule methods is time-consuming, requires significant equipment investment, and has a limited library of compounds (millions), clearly incomparable to DEL technology. Furthermore, this method is not only relatively cumbersome to operate, but also has a narrow substrate applicability; the narrow substrate applicability means that both thiols and alkenes must be aromatic compounds. Therefore, a broadly applicable method for preparing β-hydroxy thioether structures needs to be developed to meet the requirements of DEL technology for the construction and screening of lead compounds with β-hydroxy thioether structures. Summary of the Invention

[0007] To address the aforementioned technical problems, this application provides an On-DNA chemical reaction with broad substrate applicability compared to the prior art, namely, using light and ketone catalysts to co-catalyze the thiol-alkene radical reaction to synthesize On-DNA β-hydroxy thioether structural compounds.

[0008] The specific method for preparing the On-DNA β-hydroxy sulfide structured compound includes the step of reacting an On-DNA olefin with a thiol to generate the On-DNA β-hydroxy sulfide structured compound, wherein the reaction equation is as follows:

[0009] Where R 1 It is selected from any one of substituted or unsubstituted aryl and heterocyclic aryl groups; wherein the substituent is any one or any combination of halogen, C1 alkyl, and cyano, and the halogen is fluorine, chlorine, or bromine; the aryl group is a six-membered aryl group; the heterocyclic aryl group is a 5- to 6-membered heterocyclic aryl group, and the heteroatom in the heterocyclic aryl group is O, N, or S, and the number of heteroatoms is 1;

[0010] R 2The alkyl group is selected from substituted or unsubstituted C1-C12 alkyl, cycloalkyl, six-membered heterocyclic alkyl, aryl, heterocyclic aryl, and fused cycloaryl groups; the substituent of the C1-C12 alkyl group is an amide group, halogen, phenyl, or carboxyphenyl, and the halogen is fluorine; the cycloalkyl group is a C5-C6 cycloalkyl or adamantane; the six-membered heterocyclic alkyl group is an N-heterocyclic ring with an N-Boc substituent; the substituent of the aryl group is an alkyl group, alkoxy group, halogen, cyano group, or phenyl group, and the halogen is fluorine, chlorine, or bromine; the heterocyclic aryl group is a 5-6 membered heterocycle or a benzo[5] five-membered heterocycle, and the heteroatom in the heterocyclic aryl group is N or S, and the number of heteroatoms is 1, 2, or 3;

[0011] The catalyst is a ketone catalyst; preferably, benzophenone, o-fluorobenzophenone, 2,4-dimethoxy-4-hydroxybenzophenone, 2,4-dichlorobenzophenone, 3-bromobenzophenone, bis(3-aminophenyl) methyl ketone, 3,4-diaminobenzophenone, 4-aminobenzophenone, 4-chlorobenzophenone, (4-fluorophenyl)(4-nitrophenyl) methyl ketone, 4,4'-... -Dihydroxybenzophenone, 4,4'-diaminobenzophenone, bis(3,4-diaminophenyl)benzophenone, 9-fluorenone, 2-amino-9-fluorenone, 2-bromo-9-fluorenone, 2,7-dibromo-9-fluorenone, 2,7-diaminoanthraquinone, dibenzocorne, doxorubicin, indolequinone, 2,2,2-trifluoroacetophenone, ethyl benzoylformate; most preferably 3,4-diaminobenzophenone;

[0012] The solvent comprises an organic solvent and water. The organic solvent includes one or more of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetonitrile (MeCN), ethanol, methanol, 1,4-dioxane, and tetrahydrofuran. Preferably, the volume ratio of water to DMSO is 1:4 to 4:1; more preferably, the volume ratio of water to DMSO is 1:2 to 2:1; and most preferably, the volume ratio of water to DMSO is 1:1.

[0013] The wavelength of the light source is 365 nanometers to 535 nanometers, preferably 365 to 455 nanometers, more preferably 365 to 425 nanometers, and most preferably 380 to 415 nanometers;

[0014] The temperature is no higher than 40°C.

[0015] In some embodiments of this application, the inventors experimented with 68 catalysts. For ease of representation, the ketone catalysts used in this application are numbered using the format C followed by a number. Their specific structures and corresponding numbers are shown in Table 1. The conversion rates of the catalytic reactions are listed below the corresponding structures and numbers. These conversion rates were obtained under identical conditions except for the catalyst.

[0016] Table 1 Ketone Catalysts

[0017] Among them, the preferred ketone catalysts are C9 (benzophenone), C11 (o-fluorobenzophenone), C16-C20 (C16: 2,4-dimethoxy-4-hydroxybenzophenone, C17: 2,4-dichlorobenzophenone, C18: 3-bromobenzophenone, C19: bis(3-aminophenyl) ketone, C20: 3,4-diaminobenzophenone), C23 (4-aminobenzophenone), C25 (4-chlorobenzophenone), C29 ((4-fluorophenyl)(4-nitrophenyl) ketone), C32 (4,4'-dihydroxybenzophenone), C34 (4,4'-diaminobenzophenone), C36-C37 (C36: bis(3,4-diaminophenyl) ketone, C37: The preferred ketone catalysts include C19 (9-fluorenone), C39 (2-amino-9-fluorenone), C41-C42 (C41: 2-bromo-9-fluorenone, C42: 2,7-dibromo-9-fluorenone), C44 (2,7-diaminoanthraquinone), C46 (dibenzocorne), C48 (doxorubicin), C51 (indolequinone), C54 (2,2,2-trifluoroacetophenone), and C56 (ethyl benzoylformate). These preferred ketone catalysts all exhibit conversion rates above 30%. More preferred catalysts with conversion rates above 40% include C9, C19-C20, C32, C34, C36-C37, C39, and C46. Among these, C20, i.e., 3,4-diaminobenzophenone, demonstrates the most outstanding performance. The preferred final concentration of the ketone catalyst is 2.5 mmol / L to 15.0 mmol / L.

[0018] In some embodiments of this application, researchers experimented with organic solvents such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetonitrile (MeCN), ethanol, methanol, 1,4-dioxane, and tetrahydrofuran. Experimental results showed that, under the same conditions except for the organic solvent, using a mixture of DMSO and water as the reaction solvent resulted in a significantly higher conversion rate of the On-DNA β-hydroxy sulfide compound compared to other solvents; the conversion rate reached a maximum of 72% in the DMSO and water solvent, while the conversion rates in other organic solvent and water mixtures were between 50% and 60%. Therefore, the ratio of organic solvent to water was optimized, with a preferred volume ratio of water to DMSO of 1:4 to 4:1; a more preferred volume ratio of water to DMSO of 1:2 to 2:1; and the most preferred volume ratio of water to DMSO of 1:1.

[0019] In some embodiments of this application, researchers experimented with multiple light source wavelengths, such as 365 nm, 380 nm, 405 nm, 415 nm, 425 nm, 455 nm, 475 nm, 500 nm, and 535 nm. Experiments showed that within the wavelength range of 365 nm to 535 nm, as the wavelength increases to 405 nm, the conversion efficiency decreases with increasing wavelength. The applicant believes that the preferred light source wavelength is 365–455 nm, more preferably 365–425 nm, and most preferably 380–415 nm.

[0020] In some embodiments of this application, researchers experimented with various final concentrations of the substrate thiol. Experiments showed that the final concentration of thiol in the range of 25.0 mmol / L to 150.0 mmol / L first increased and then decreased with the increase of the final concentration. Therefore, the final concentration of thiol is preferably 50.0 mmol / L to 150.0 mmol / L; more preferably 100.0 mmol / L to 150.0 mmol / L.

[0021] In some embodiments of this application, the reaction temperature has little effect on the conversion rate at a temperature that ensures the DNA strand does not break. For ease of operation, 0°C to 40°C (including the endpoint) is a convenient operating temperature depending on different regions, climates and other conditions.

[0022] In summary, the reaction conditions of this application can yield a wide range of On-DNA β-hydroxy sulfide compounds in a single reaction. This not only significantly broadens the applicable range of raw materials compared to existing technologies, but also provides mild reaction conditions and a conversion rate exceeding 90%. Furthermore, the method for preparing On-DNA β-hydroxy sulfide compounds via the On-DNA olefin-thiol reaction of this application not only fills the current gap in methods for preparing On-DNA β-hydroxy sulfide compounds, but also enriches the On-DNA β-hydroxy sulfide compound library and methods for preparing On-DNA sulfinimide compounds. Attached Figure Description

[0023] Figure 1 3-Cyano-5-vinylbenzoic acid (S2g) 1 H NMR spectrum.

[0024] Figures 2 to 10 : On-DNA olefin compounds 1a~1q.

[0025] Figures 11 to 23 : On-DNA β-hydroxy thioether compounds 3a~3q, 3s~3z.

[0026] Figures 24 to 35 : On-DNA β-hydroxy thioether compounds 3aa~3ax.

[0027] Figure 36 : 4-(2-((2-chlorophenyl)sulfinyl)ethyl)benzoic acid (ca) 1 H NMR spectrum.

[0028] Figure 37 : 4-(2-((2-chlorophenyl)thio)-1-hydroxyethyl)benzoic acid (cb) 1 H NMR spectrum.

[0029] Figure 38 : 4-(2-((2-chlorophenyl)thio)acetyl)benzoic acid (cc) 1 H NMR spectrum.

[0030] Figure 39 The chromatograms of On-DNA-4-(2-((2-chlorophenyl)sulfinyl)ethyl)benzoic acid (3c-a), On-DNA-4-(2-((2-chlorophenyl)thio)-1-hydroxyethyl)benzoic acid (3c-b) and On-DNA-4-(2-((2-chlorophenyl)thio)acetyl)benzoic acid (3c-c) were analyzed by LC-MS after co-injection.

[0031] Figure 40 On-DNA β-hydroxy thioether compound 3c and 3c-c were co-injected for LC-MS analysis.

[0032] Figure 41 LCMS spectrum of 3c-a after the synthesis of sulfinimide.

[0033] Figure 42 LCMS spectrum of 3c-b after the synthesis of sulfinimide.

[0034] Figure 43 LCMS spectrum of 3c after the synthesis of sulfinimide. Detailed Implementation

[0035] Unless otherwise specified, all reagents or compounds used in this application are commercially available products, and all self-made products have preparation method instructions.

[0036] DMSO: Dimethyl sulfoxide;

[0037] DMF: N,N-dimethylformamide;

[0038] MeCN: Acetonitrile;

[0039] EtOH: Ethanol;

[0040] MeOH: Methanol;

[0041] 1,4-Dioxane: 1,4-dioxane;

[0042] THF: Tetrahydrofuran.

[0043] Sixty-eight ketone catalysts are denoted by C1 to C68, and their chemical structures are shown in Table 1. Their corresponding chemical names are as follows: C1: 2-Butanone, C2: Pyruvic acid, C3: Acetylacetone, C4: Acetone, C5: Cyclopentanone, C6: Cyclohexanone, C7: Acetophenone, C8: N-acetanilide, C9: Benzophenone, C10: 2-Methylbenzophenone, C11: o-fluorobenzophenone, C12: 2-Bromobenzophenone, C13: 2-Aminobenzophenone, C14: 2-Benzoylbenzoic acid, C15: 2-(3-amino-4-chlorobenzoyl)benzoic acid, C16: 2,4-Dimethoxy-4-hydroxybenzophenone, C17: 2,4-Dichlorobenzophenone. Ketones, C18: 3-bromobenzophenone, C19: bis(3-aminophenyl) benzophenone, C20: 3,4-diaminobenzophenone, C21: 3-benzoylbenzoic acid, C22: ketoibuprofen, C23: 4-aminobenzophenone, C24: mebendazole, C25: 4-chlorobenzophenone, C26: p-bromobenzophenone, C27: 4-benzoylbenzoic acid, C28: 4,4-difluorobenzophenone, C29: (4-fluorophenyl)(4-nitrophenyl) benzophenone, C30: fluorobenzamide, C31: 4,4-dibromobenzophenone, C32: 4,4'-dihydroxybenzophenone, C33: 4,4-dimethoxybenzophenone, C34: 4,4'-dihydroxybenzophenone - Diaminobenzophenone, C35: 4,4-bis(N,N-dimethylamino)benzophenone, C36: bis(3,4-diaminophenyl)benzophenone, C37: 9-fluorenone, C38: 1-amino-9-fluorenone, C39: 2-amino-9-fluorenone, C40: 9-fluorenone-2-carboxylic acid, C41: 2-bromo-9-fluorenone, C42: 2,7-dibromo-9-fluorenone, C43: anthrone, C44: 2,7-diaminoanthraquinone, C45: emodin, C46: dibenzocorne, C47: 6,13-pentabenzoquinone, C48: doxorubicin, C49: sodium anthraquinone-2-sulfonate, C50: 2-chlorothioxanone, C51: indolequinone, C52: Acenazoline, C53: 2-Butanone, C54: 2,2,2-Trifluoroacetophenone, C55: Benzoylcarboxylic acid, C56: Ethyl benzoylcarboxylate, C57: 4-Methylacetophenone, C58: 4-Fluoroacetophenone, C59: 4-Cyanoacetophenone, C60: 2-Methoxyacetophenone, C61: 3-Methoxyacetophenone, C62: 4-Methoxyacetophenone, C63: 2,3-Dimethoxyacetophenone, C64: 2,4-Dimethoxyacetophenone, C65: 3,4-Dimethoxyacetophenone, C66: 2,4,6-Trimethoxyacetophenone, C67: 2,3,4-Trimethoxyacetophenone, C68: 3',4',5'-Trimethoxyacetophenone.

[0044] The LC-MS (electrospray ionization mass spectrometry) analysis conditions used in this application were as follows: The sample (approximately 100.0 picomoles, diluted with 40.0 μL of water) was injected into a reversed-phase column (XBridge Oligonucleotide BEH C18 column, 1.7 μm, 2.1 × 50 mm) and eluted with a gradient (solvent B increased from 10% to 90% within 1.2 min at a flow rate of 0.60 mL / min; solvent A: 0.75% hexafluoroisopropanol (HFIP) / 0.38% triethylammonium acetate (TEAA) / 10.0 μM ethylenediaminetetraacetic acid (EDTA) dissolved in deionized water; solvent B: 0.75% v / v HFIP / 0.38% v / v TEAA / 10.0 μM EDTA dissolved in 90 / 10 methanol / deionized water). The monitoring wavelength was UV 260.0 nm and the total ion current (TIC) was monitored.

[0045] The NMR detection conditions for this application are: DMSO-d6, 400MHz.

[0046] On-DNA olefin compounds are direct raw materials for preparing On-DNA β-hydroxy thioether structured compounds. The preparation method of On-DNA olefin compounds is as follows: (1) HP-Linker-NHFmoc is prepared from HP-NH2 (commercially available reagent), (2) HP-Linker-NH2 is prepared from HP-Linker-NHFmoc, and (3) in the third step, HP-Linker-NH2 is converted into On-DNA olefin compounds. This application uses the exact same method as CN118461019A to prepare HP-Linker-NH2, therefore the structural confirmation spectra of HP-NH2, HP-Linker-NHFmoc, and HP-Linker-NH2 are omitted.

[0047] 1. Synthesis of On-DNA-Linker-NH2 precursor (HP-Linker-NH2)

[0048] 1.1 The preparation of HP-Linker-NHFmoc from HP-NH2 (commercially available reagent) follows the following reaction equation:

[0049]

[0050] Specifically, 100.0 nanomolars of HP-NH2 (commercially available product) were dissolved in deionized water to prepare a 1.0 mmol / L solution (100.0 μL, 100.0 nanomolars, 1.0 equivalent). 40.0 equivalents of a DMSO solution of S1 (commercially available product) (concentration: 200.0 mmol / L), 250.0 equivalents of a sodium tetraborate (Na2B4O7) buffer solution at pH 9.5 (concentration: 250.0 mmol / L), and 40.0 equivalents of an aqueous solution of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) (concentration: 200.0 mmol / L) were mixed and thoroughly vortexed. This mixture was then added to the HP-NH2 solution, mixed thoroughly, and reacted at 4°C for 1 hour. After the reaction was complete, 10% by volume of a 5.0 mol / L sodium chloride solution was added to the reaction solution. Then, add three times the total volume of anhydrous ethanol, shake well, and freeze the reaction solution at -80°C for 2 hours. Afterward, centrifuge at 4000.0 rpm for half an hour and discard the supernatant. Dissolve the remaining precipitate in deionized water to obtain a solution of HP-Linker-NHFmoc. The molecular weight of HP-Linker-NHFmoc was determined to be 5406 using liquid chromatography-mass spectrometry.

[0051] 1.2 Preparation of HP-Linker-NH2 from HP-Linker-NHFmoc:

[0052]

[0053] 100.0 nanomolars of HP-Linker-NHFmoc were dissolved in deionized water to prepare a 1.0 mmol / L (100.0 μL, 100.0 nanomolars, 1.0 equivalent) solution. 36.0 μL of a 10% piperidine aqueous solution was added, and the mixture was thoroughly mixed and reacted at room temperature for 1 hour. After the reaction was complete, 10% (by volume) of a 5.0 mol / L sodium chloride solution was added to the reaction solution. Then, three times the total volume of anhydrous ethanol was added, and the mixture was shaken thoroughly. The reaction solution was then frozen at -80°C for 2 hours. Afterward, the solution was centrifuged at 4000.0 rpm for half an hour, and the supernatant was discarded. The remaining precipitate was dissolved in deionized water to obtain a solution of On-DNA-Linker-NH2 (HP-Linker-NH2). The molecular weight of HP-Linker-NH2 was determined to be 5184 using liquid chromatography-mass spectrometry.

[0054] 2. Preparation of On-DNA olefin compounds:

[0055] This application prepares On-DNA olefin compounds by reacting the carboxylated olefin compounds listed in Table 2 with HP-Linker-NH2.

[0056] Table 2 Carboxylated Alkenes

[0057] All compounds listed in Table 2, except for S2g, are commercially available products. S2g is a self-made compound, and the reaction equation for its preparation is as follows: The reaction conditions are as follows: a) potassium vinyltrifluoroborate, Pd(dppf)Cl2, K2CO3, 1,4-dioxane / water (2:1), 80℃; b) LiOH·H2O, acetonitrile / water (1:1). The specific operating steps are as follows:

[0058] Step (a): Vinylation reaction

[0059] Methyl 3-bromo-5-cyanobenzoate (590.0 mg, 2.47 mmol, 1.0 equivalent) and potassium vinyltrifluoroborate (1.5 equivalent) were dissolved in a mixed solvent of 1,4-dioxane and water, followed by the sequential addition of K₂CO₃ (3.0 equivalent) and Pd(dppf)Cl₂ (0.05 equivalent). The reaction was carried out under nitrogen protection and stirred at 80 °C for 4 hours. After the reaction was complete, the solvent was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to give methyl 3-vinyl-5-cyanobenzoate.

[0060] Step (b): Ester hydrolysis reaction

[0061] The above-mentioned methyl 3-vinyl-5-cyanobenzoate (1.0 equivalent) was dissolved in a 1:1 mixture of acetonitrile and water, and LiOH·H₂O (2.0 equivalent) was added at 25°C. The mixture was stirred for 2 hours. After the reaction was complete, the pH was adjusted to 2.0 with 1N hydrochloric acid, and the residue was purified by silica gel column chromatography to obtain a white solid 3-cyano-5-vinylbenzoic acid (S₂g, 325 mg, 76%). The NMR spectrum of S₂g is attached. Figure 1 The specific NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ13.56(s,1H),8.26(dt,J=4.5,1.7Hz,2H),8.16(t,J=1.6Hz,1H),6.86(d d,J=17.7,11.1Hz,1H),6.11(d,J=17.7Hz,1H),5.48(d,J=11.1Hz,1H).MS(ESI-MS)m / z:174[M+H] + .

[0062] The reaction equation for preparing On-DNA olefin compounds is as follows: The specific experimental conditions were as follows: Carboxyolefin S2 (200.0 mmol / L dissolved in DMSO, 10.0 μL, 40.0 equivalent) was mixed with DMT-MM (200.0 mmol / L aqueous solution, 10.0 μL, 40.0 equivalent), followed by the addition of DNA-NH2 (1.0 mmol / L aqueous solution, 50.0 μL, 50.0 nanomoles, 1.0 equivalent) and borate buffer (250.0 mmol / L, pH = 9.5, 50.0 μL). The reaction was quenched after 1 hour and precipitated with cold ethanol. The conversion rate was determined by LC-MS analysis. The structures of the prepared On-DNA olefin compounds 1a-1q are shown in Table 3 or the appendix. Figures 2 to 10 .

[0063] Table 3 On-DNA olefin compounds

[0064]

[0065] Screening of catalysts used in the preparation of On-DNA β-hydroxy thioether compounds in Examples 1-68

[0066] Taking the preparation of product 3av as an example, the catalyst used was screened, and the specific reaction equation is as follows: The reaction conditions were as follows: DNA starting material 1c (10.0 nmol, 1.0 equivalent, 1.0 mmol / L aqueous solution, 10.0 μL, final concentration 0.5 mmol / L), catalyst (20.0 mmol / L DMSO solution, 5.0 μL, final concentration 5.0 mmol / L), and 2av (200.0 mmol / L DMSO solution, 5.0 μL, final concentration 50.0 mmol / L) were added to a 0.2 mL octet PCR tube. After vortexing to mix, the reaction mixture was irradiated at 25 °C with a 405 nm LED lamp for 3 hours. After the reaction was completed, the samples were precipitated with ethanol, and the conversion rate was determined by LC-MS.

[0067] The catalysts used and the reaction results are shown in Table 1. Based on the results in Table 1, we can see that the preferred ketone catalysts are C9, C11, C16–C20, C23, C25, C29, C32, C34, C36–C37, C39, C41–C42, C44, C46, ​​C48, C51, C54, and C56, all of which have conversion rates above 30%. More preferred catalysts with conversion rates above 40% include C9, C19–C20, C32, C34, C36–C37, C39, and C46. The most outstanding ketone catalyst is C20, namely 3,4-diaminobenzophenone.

[0068] Examples 69-91: Optimization of conditions for preparing On-DNA β-hydroxy thioether compounds

[0069] Taking the synthesis of On-DNA β-hydroxy thioether compound (3av) from On-DNA olefinic compound raw material (1c) and 2av as an example, the catalyst used is C20, and the optimized reaction conditions are shown in Table 4.

[0070] Table 4. Reaction conditions and results of Examples 20, 69-91

[0071]

[0072]

[0073] a. Final concentration.

[0074] b Conversion rate was determined by LC-MS.

[0075] c In dark conditions.

[0076] d Reaction conditions: 1c (10.0 nanomoles, 1.0 equivalent, final concentration 0.5 mmol / L), C2O (final concentration 10.0 mmol / L) and 2av (final concentration 100.0 mmol / L) were dissolved in a water / DMSO (volume ratio 1:1) mixed solvent and irradiated at 25°C (405 nm) for 3 hours.

[0077] e Add TEMPO (tetramethylpiperidine oxide) to a final concentration of 100.0 mmol / L.

[0078] Compare the experimental conditions and results in Table 4:

[0079] (1) Examples 20, 69-73 explored different light sources. Example 69 was conducted in the dark. By comparing the results, we can see that a light source is necessary. In the wavelength range of 365 nm to 535 nm, when the wavelength increases to 405 nm, the conversion rate of 3av decreases with the increase of wavelength. Therefore, the applicant believes that the preferred wavelength of the light source is 365-455 nm, more preferably 365-425 nm, and most preferably 380-415 nm.

[0080] (2) Comparing Examples 20 and 74-79, we can see that when the organic solvent is DMSO, the 3av conversion rate is better than that of other organic solvents, while other reaction conditions remain unchanged.

[0081] (3) Comparing Examples 20 and 80-83, we can see that, under the condition that other reaction conditions remain unchanged, the conversion rate of 3av is significantly higher when DMSO and water are used as the reaction solvent; the conversion rate can reach up to 72% in the DMSO and water solvent, while the conversion rate in the mixed solvent of other organic solvents and water is between 50% and 60%. Therefore, the ratio of organic solvent to water is optimized. The preferred volume ratio of water to DMSO is 1:4 to 4:1, or the preferred reaction solvent is water to N,N-dimethylformamide (DMF), acetonitrile (MeCN), ethanol, methanol, 1,4-dioxane (1,4-dioxane) or tetrahydrofuran in a volume ratio of 1:1; more preferably, the volume ratio of water to DMSO is 1:2 to 2:1; and most preferably, the volume ratio of water to DMSO is 1:1.

[0082] (4) Comparing Examples 20 and 84-87, we can see that, under the condition that other reaction conditions remain unchanged, the final concentration of catalyst C20 in the range of 0.0 to 15.0 mmol / L first increases and then decreases with the increase of its final concentration. When it rises to 10.0 mmol / L, the conversion rate of 3av is the highest, and then the conversion rate of 3av decreases again with the increase of its final concentration. Based on the experimental results of Examples 20 and 84-87, the applicant believes that the final concentration of the catalyst is preferably 2.5 mmol / L to 15.0 mmol / L.

[0083] (5) Comparing Examples 86 and 88-90, we can see that, under the condition that other reaction conditions remain unchanged, the final concentration of thiol 2av increases and then decreases with the increase of its final concentration in the range of 25.0 to 150.0 mmol / L. When it reaches 100.0 mmol / L, the conversion rate of 3av is the highest, and then the conversion rate of 3av decreases again with the increase of its final concentration. Based on the experimental results of Examples 86 and 88-90, the applicant believes that the final concentration of thiol 2av is preferably 50.0 mmol / L to 150.0 mmol / L; more preferably 100.0 mmol / L to 150.0 mmol / L.

[0084] (6) Comparing Examples 89 and 91, we can see that the reaction no longer occurs after the addition of the free radical scavenger TEMPO, indicating that the reaction is a free radical reaction.

[0085] Examples 92-139: Preparation of On-DNA β-hydroxy sulfide compounds. On-DNA β-hydroxy sulfide compounds were prepared by reacting the On-DNA olefin compounds in Table 3 with the thiols in Table 5. The reaction equations are as follows:

[0086] Table 5 Thiols

[0087]

[0088] The procedure was as follows: Take 0.2 mL of an eight-tube PCR apparatus and add DNA starting material 1 (10.0 nanomoles, 1.0 equivalent, 1.0 mmol / L aqueous solution, 10.0 μL, final concentration 0.5 mmol / L), C20 (40.0 mmol / L DMSO solution, 5.0 μL, final concentration 10.0 mmol / L), and 2 (400.0 mmol / L DMSO solution, 5.0 μL, final concentration 100.0 mmol / L). After vortexing the reaction mixture, irradiate it at 25°C using a 405 nm LED lamp for 3 hours. After the reaction was complete, precipitate the mixture with ethanol and analyze it by LC-MS. The conversion rate was confirmed based on the LC-MS results. The structures of the On-DNA β-hydroxy thioether compounds obtained in Examples 89, 92–139 are shown in Table 6 or the appendix. Figures 11 to 35 As shown.

[0089] Table 6. On-DNA β-hydroxy thioether compounds prepared in this application

[0090]

[0091] Validation example: Structural confirmation of on-DNA β-hydroxythioether

[0092] Based on literature and product molecular weight information, the product structure synthesized in this application via photocatalytic thiol-alkene radical reaction may be diverse. Therefore, it is necessary to determine that the β-hydroxy sulfide structure is unique and definite. Taking product 3c as an example, 3c may have three structures, which we have named 3c-a, 3c-b, and 3c-c, as shown in Table 7:

[0093] Table 7. Structural formulas of 3c-a, 3c-b, and 3c-c

[0094] Therefore, it is necessary to verify that product 3c has a β-hydroxy thioether structure. However, since On-DNA β-hydroxy thioether compounds are macromolecules, it is not feasible to directly use NMR. Therefore, we indirectly proved that 3c has a β-hydroxy thioether structure through the following method.

[0095] The structural confirmation of On-DNA β-hydroxy sulfide involves two steps: the first step is to synthesize 3c-a, 3c-b, or 3c-c with a defined structure; the second step is to synthesize On-DNA sulfinimide compounds with a defined structure, and then use the defined On-DNA sulfinimide structure to conduct structural verification of the On-DNA β-hydroxy sulfide compounds prepared in this application.

[0096] Step 1: Prepare 3c-a, 3c-b, or 3c-c structures with defined structures.

[0097] Step 1.1: Prepare raw materials ca, cb, cc with defined 3c-a, 3c-b, or 3c-c structures.

[0098] (1) 4-(2-((2-chlorophenyl)sulfinyl)ethyl)benzoic acid (ca): The preparation reaction equation is as follows:

[0099] ①Methyl 4-(2-((2-chlorophenyl)sulfinyl)ethyl)benzoate (ca-1)

[0100] Under air atmosphere, Selectfluor (1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane difluorophosphate, 329.5 mg, 0.93 mmol, 1.5 equivalent) was added to a stirred methanol mixture (4.0 mL) of methyl 4-vinylbenzoate (103.0 mg, 0.62 mmol, 1.0 equivalent) at 60 °C and the mixture was stirred for 12 hours. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, the mixture was concentrated under reduced pressure to obtain a crude product, which was then purified by column chromatography to obtain the target compound methyl 4-(2-((2-chlorophenyl)sulfinyl)ethyl)benzoate (ca-1, 141.0 mg, 71%) as a white solid. MS (ESI-MS) m / z: 323.0 [M+H] + .

[0101] ②4-(2-((2-chlorophenyl)sulfinyl)ethyl)benzoic acid (ca)

[0102] Methyl 4-(2-((2-chlorophenyl)sulfinyl)ethyl)benzoate (ca-1, 141.0 mg, 0.44 mmol, 1.0 equivalent) was dissolved in a stirred solution of acetonitrile / water (1:1). Lithium hydroxide monohydrate (LiOH·H₂O, 36.8 mg, 0.88 mmol, 2.0 equivalent) was added at 40 °C, and the reaction was continued with stirring for 1 hour. After the reaction was complete, the mixture was acidified to pH 2 with 1N hydrochloric acid. The resulting residue was purified by silica gel column chromatography to obtain the target product, 4-(2-((2-chlorophenyl)sulfinyl)ethyl)benzoic acid (ca, 109.0 mg, 81%), as a white solid. 1H NMR(400MHz,DMSO-d6)δ12.94(s,1H),7.98–7.78(m,3H),7.75–7.53(m,3H),7.49–7.29(m ,2H),3.51–3.37(m,1H),3.22–3.04(m,2H),2.93–2.81(m,1H).MS(ESI-MS)m / z:307.0[MH] + .

[0103] (2) Preparation of 4-(2-((2-chlorophenyl)thio)-1-hydroxyethyl)benzoic acid (cb) and 4-(2-((2-chlorophenyl)thio)acetyl)benzoic acid (cc) with defined structures.

[0104] The reaction equations for preparing 4-(2-((2-chlorophenyl)thio)-1-hydroxyethyl)benzoic acid (cb) and 4-(2-((2-chlorophenyl)thio)acetyl)benzoic acid (cc) are as follows:

[0105] ①Methyl 4-(2-((2-chlorophenyl)thio)-1-hydroxyethyl)benzoate (cb-1)

[0106] Under an oxygen atmosphere, a mixed solution of methyl 4-vinylbenzoate (103.0 mg, 0.62 mmol, 1.0 equivalent) and 2-chlorothiophenol (2ay, 134.5 mg, 0.93 mmol, 1.5 equivalent) dissolved in DMSO (4 mL) was stirred at 40 °C. Tetrabutylammonium tribromide (TBATB, 89.3 mg, 0.19 mmol, 0.3 equivalent) and water (H₂O, 55.5 mg, 3.09 mmol, 5.0 equivalent) were added sequentially, and the reaction was stirred continuously for 12 hours. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was complete, the reaction mixture was concentrated under reduced pressure to obtain a crude product, which was further purified by column chromatography to obtain the target compound methyl 4-(2-((2-chlorophenyl)thio)-1-hydroxyethyl)benzoate (cb-1, 163.0 mg, 82%) as a white solid. MS (ESI-MS) m / z: 323.0 [M+H] + .

[0107] ②4-(2-((2-chlorophenyl)thio)-1-hydroxyethyl)benzoic acid (cb)

[0108] Methyl 4-(2-((2-chlorophenyl)thio)-1-hydroxyethyl)benzoate (cb-1, 50.0 mg, 0.16 mmol, 1.0 equivalent) was dissolved in an acetonitrile / water (1:1) mixture. Lithium hydroxide monohydrate (LiOH·H₂O, 13.0 mg, 0.31 mmol, 2.0 equivalent) was added with stirring at room temperature, and the reaction was allowed to proceed for 4 hours. After the reaction was complete, the reaction solution was acidified to pH 2 with 1N hydrochloric acid. The resulting residue was purified by silica gel column chromatography to give the target compound 4-(2-((2-chlorophenyl)thio)-1-hydroxyethyl)benzoic acid (cb, 42.6 mg, 89%) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ12.88(s,1H),7.99–7.83(m,2H),7.61–7.48(m,2H),7.47–7.36(m,2H),7.29(td,J=7.6,1.4Hz,1H ),7.16(td,J=7.6,1.5Hz,1H),5.91(d,J=4.7Hz,1H),4.88(d,J=5.5Hz,1H),3.31–3.25(m,2H).MS(ESI-MS)m / z:307.0[MH] + .

[0109] ③Methyl 4-(2-((2-chlorophenyl)thio)acetyl)benzoate (cc-1)

[0110] Methyl 4-(2-((2-chlorophenyl)thio)-1-hydroxyethyl)benzoate (cb-1, 100.0 mg, 0.31 mmol, 1.0 equivalent) was dissolved in dichloromethane (DCM, 3 mL), and Dess-Martin periodane (264.0 mg, 0.62 mmol, 2.0 equivalent) was added with stirring at room temperature. The reaction mixture was reacted for 1 hour. After the reaction was complete, the reaction mixture was concentrated under reduced pressure to obtain a crude product, which was further purified by column chromatography to obtain the target compound methyl 4-(2-((2-chlorophenyl)thio)acetyl)benzoate (cc-1, 85.5 mg, 86%) as a white solid. MS (ESI-MS) m / z: 321.0 [M+H] + .

[0111] ④4-(2-((2-chlorophenyl)thio)acetyl)benzoic acid (cc)

[0112] Methyl 4-(2-((2-chlorophenyl)thio)acetyl)benzoate (cc-1, 50.0 mg, 0.16 mmol, 1.0 equivalent) was dissolved in an acetonitrile / water (1:1) mixture. Lithium hydroxide monohydrate (LiOH·H₂O, 13.1 mg, 0.31 mmol, 2.0 equivalent) was added with stirring at room temperature, and the reaction was allowed to proceed for 4 hours. After the reaction was complete, the reaction solution was acidified to pH 2 with 1N hydrochloric acid. The resulting residue was purified by silica gel column chromatography to give the target compound 4-(2-((2-chlorophenyl)thio)acetyl)benzoic acid (cc, 39.7 mg, 83%) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ13.37(s,1H),8.16(d,J=8.0Hz,2H),8.08(d,J=8.1Hz,2H),7.43(dd ,J=23.1,7.9Hz,2H),7.25(dt,J=42.1,7.6Hz,2H),4.84(s,2H).MS(ESI-MS)m / z:305.0[MH] + .

[0113] Step 1.2: Compounds ca, cb and cc were coupled to DNA using the same procedure as in the synthesis of 1a-1q, to ​​obtain DNA-coupled products 3c-a, 3c-b and 3c-c with defined structures, respectively.

[0114] LC-MS analysis of co-injected samples 3c-a, 3c-b, and 3c-c revealed that the elution times of 3c-a and 3c-b overlapped, and 3c-c eluted before 3c-c, as shown in the attached figure. Figure 39 As shown. Therefore, the On-DNA β-hydroxy compound 3c and 3c-c obtained in Example 94 were co-injected for LC-MS analysis, and the results are as follows. Figure 40 As shown. Figure 39 and Figure 40 Comparison revealed that the peak position of On-DNA β-hydroxy compound 3c was the same as that of 3c-a (3c-b). Therefore, it could not be determined whether the On-DNA β-hydroxy compound 3c obtained in Example 94 was 3c-a or 3c-b. Thus, the second step of structural confirmation, "synthesis of On-DNA sulfinimide structure compound with a defined structure", was carried out. Since the structure that is not 3c-c has been excluded, it is only necessary to determine whether 3c is 3c-a or 3c-b.

[0115] Step 2: Synthesis of the On-DNA sulfinimide compound with a defined structure: On-DNA β-hydroxy sulfide compound 3 (5.0 nmol, 1.0 equivalent, 2.0 mmol / L aqueous solution, 2.5 μL, final concentration 0.5 mmol / L), 5 (ammonium carbamate, 800.0 mmol / L aqueous solution, 2.5 μL, final concentration 200.0 mmol / L), and 6 (diacetoxyiodobenzene, 200.0 mmol / L DMSO solution, 5.0 μL, final concentration 100.0 mmol / L) were added sequentially to the reaction tube. The reaction solution was shaken at 25.0 °C for 2 hours. After ethanol precipitation, the precipitate of compound 4 (On-DNA sulfinimide compound) was directly dissolved in distilled water without further purification. The conversion rate was determined by LC-MS.

[0116] Through the synthesis of sulfinimide on DNA, compounds 3c-a, 3c-b, and 3c were converted into their corresponding products, labeled 4c-a, 4c-b, and 4c, respectively. The reaction equations are as follows: Where 3 represents 3c-a, 3c-b, and 3c, and the corresponding reaction products 4 are named 4c-a, 4c-b, and 4c, respectively. LC-MS analysis was performed on 4c-a, 4c-b, and 4c, and the analytical results are as follows: Figures 41-43 As shown. The peak time of 4c-a is related to... Figure 39 The peak positions of 3c-a (or 3c-b) overlap, and their molecular weights are the same (5475.3) as 3c-a, leading to the conclusion that 3c-a did not react. The molecular weight of 4c-b is 5507.2, meaning that if 3c-b were to convert to 4c-b according to the above reaction, the structure would be... The molecular weight should also be 5507. 4c and 4c-b have the same molecular weight and the same peak position; therefore, 4c and 4c-b are identical, i.e., 4c-b. This proves that product 3c from Example 94 has the same β-hydroxy thioether structure as 3c-b. The reaction types for generating On-DNA in this application are the same; therefore, the structures can all be confirmed using the above methods.

[0117] Application Examples: Application of On-DNA β-hydroxythioether structured compounds in this application

[0118] Based on the discussion of the verification examples, we can definitively conclude that the On-DNA β-hydroxy thioether compound prepared by the On-DNA olefin-thiol reaction of this application can be used to prepare On-DNA sulfinylimide compounds. Therefore, the method of preparing On-DNA β-hydroxy thioether compounds by the On-DNA olefin-thiol reaction of this application not only fills the current gap in the lack of methods for preparing On-DNA β-hydroxy thioether compounds, but also enriches the On-DNA β-hydroxy thioether compound library and the method for preparing On-DNA sulfinylimide compounds.

[0119] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing On-DNA β-hydroxythioether structured compounds, characterized in that, include: The steps for reacting On-DNA olefins with thiols to generate On-DNA β-hydroxythioether compounds are described by the following reaction equation: ; R 1 It is selected from any one of substituted or unsubstituted aryl and heterocyclic aryl groups; wherein the substituted substituent is one or more of halogen, C1 alkyl, and cyano; the aryl group is a six-membered aryl group; the heterocyclic aryl group is a 5- to 6-membered heterocyclic aryl group; the heteroatom in the heterocyclic aryl group is O, N, or S, and the number of heteroatoms is 1; the halogen is fluorine, chlorine, or bromine. R 2 The alkyl group is selected from substituted or unsubstituted C1-C12 alkyl, cycloalkyl, six-membered heterocyclic alkyl, aryl, heterocyclic aryl, and fused cycloaryl groups; the substituent of the C1-C12 alkyl group is an amide group, halogen, phenyl, or carboxyphenyl, and the halogen is fluorine; the cycloalkyl group is a C5-C6 cycloalkyl or adamantane; the six-membered heterocyclic alkyl group is an N-heterocyclic ring with an N-Boc substituent; the substituent of the aryl group is an alkyl group, alkoxy group, halogen, cyano group, or phenyl group, and the halogen is fluorine, chlorine, or bromine; the heterocyclic aryl group is a 5-6 membered heterocycle or a benzo[5] five-membered heterocycle, and the heteroatom in the heterocyclic aryl group is N or S, and the number of heteroatoms is 1, 2, or 3; The catalyst is a ketone catalyst, doxorubicin, or ethyl benzoylformate; the ketone catalyst is benzophenone, o-fluorobenzophenone, 2,4-dimethoxy-4-hydroxybenzophenone, 2,4-dichlorobenzophenone, 3-bromobenzophenone, bis(3-aminophenyl) ketone, 3,4-diaminobenzophenone, 4-aminobenzophenone, 4-chlorobenzophenone, (4-fluorophenyl)(4-nitrophenyl) ketone, 4,4'-dihydroxybenzophenone, 4,4'-diaminobenzophenone, bis(3,4-diaminophenyl) ketone, 9-fluorenone, 2-amino-9-fluorenone, 2-bromo-9-fluorenone, 2,7-dibromo-9-fluorenone, 2,7-diaminoanthraquinone, dibenzocorne, indolequinone, or 2,2,2-trifluoroacetophenone; With a final concentration of 0.5 mmol / L for the On-DNA olefin as 1.0 equivalent, the final concentration of the catalyst is 2.5 mmol / L to 15.0 mmol / L, and the final concentration of the substrate thiol is 25.0 mmol / L to 150.0 mmol / L. The reaction solvent is water to DMSO in a volume ratio of 1:4 to 4:1, or water to N,N-dimethylformamide, acetonitrile, ethanol, methanol, 1,4-dioxane or tetrahydrofuran in a volume ratio of 1:

1. The wavelength of the light source is 365 nanometers to 455 nanometers; Temperature range: 0°C to 40°C, including endpoints; The reaction time is 3 hours.

2. The method for preparing On-DNA β-hydroxythioether structured compounds according to claim 1, characterized in that, The solvent is water and DMSO in a volume ratio of 1:2 to 2:

1.

3. The method for preparing On-DNA β-hydroxythioether structured compounds according to claim 1, characterized in that, The wavelength of the light source is 365 nanometers to 425 nanometers.

4. The method for preparing On-DNA β-hydroxythioether structured compounds according to claim 1, characterized in that, The final concentration of the substrate thiol is 50.0 mmol / L to 150.0 mmol / L.

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