A connector and its preparation method

Abstract

This application discloses a linker and its preparation method. The linker includes pharmacophore P, pharmacophore Q, and deoxynucleotides and / or deoxynucleotide derivatives connecting pharmacophore P and pharmacophore Q. Pharmacophore P is obtained by removing the protecting group from group B, and pharmacophore Q is obtained by removing the protecting group from group X. Group B is obtained by chemically modifying the benzylquinolone carboxylic acid pharmacophore, and group X is obtained by chemically modifying the zenomeprazole pharmacophore. The linker is prepared by linking group B and group X with deoxynucleotides and / or deoxynucleotide derivatives to form the linker, including solid-phase synthesis technology. The method of this application can automatically synthesize molecular probes or bivalent drugs containing two pharmacophores, thereby replacing the traditional stepwise linking method, enabling efficient construction of screening libraries, and allowing control of the spatial distance and spatial orientation of the pharmacophores by adjusting the number and base arrangement of deoxynucleotides.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to a linker and its preparation method. Background Technology

[0002] Constructing dual-recognition-site molecular probes or bivalent drugs composed of two pharmacophores has become a common strategy for selectively regulating protein function or constructing chimeric compounds that target protein degradation. Current pharmacophore modification strategies are limited to linker reactions of simple polymer structures, requiring multiple synthetic and purification steps for changing the linker between the two pharmacophores. This process is time-consuming, cumbersome, and difficult to automate, reducing the efficiency of candidate compound library construction. Furthermore, the spatial distance and orientation of the two pharmacophores after linking are difficult to precisely control, reducing the reliability of structural design and thus limiting the development of dual-recognition-site molecular probes and bivalent drugs. Summary of the Invention

[0003] This application provides a linker and its preparation method, which solves the problems of cumbersome steps in connecting two pharmacophores and the difficulty in accurately controlling the spatial distance and spatial orientation of the two pharmacophores after connection.

[0004] This application provides a linker comprising pharmacophore P, pharmacophore Q, and a deoxynucleotide and / or a derivative thereof connecting pharmacophore P and pharmacophore Q. The linker has the structure shown in Formula 12.

[0005] P-nt1-nt2...nt a-1 -nt a -Q

[0006] Formula 12

[0007] Wherein, nt1, nt2, ... nt a-1 nt a Each of the following is independently selected from adenine deoxynucleotide, guanine deoxynucleotide, cytosine deoxynucleotide, thymine deoxynucleotide, and derivatives of the above deoxynucleotides;

[0008] a is an integer between 1 and 100.

[0009] Optionally, in some embodiments of this application, the pharmacophore P is obtained by removing the protecting group from group B, and the pharmacophore P has the structure shown in Formula 13, while group B has the structure shown in Formula 4.

[0010]

[0011] Optionally, in some embodiments of this application, the pharmacophore Q is obtained by removing the protecting group from group X, and the pharmacophore Q has the structure shown in Formula 14, while group X has the structure shown in Formula 11.

[0012]

[0013] Optionally, in some embodiments of this application, the deoxynucleotide derivative includes an alkyne group, methoxy group, fluorine substituent, epoxy substituent, fluorescent group, radionuclide, biotin, digitalis or other modification of natural deoxynucleotides (adenine deoxynucleotide, guanine deoxynucleotide, cytosine deoxynucleotide, thymine deoxynucleotide), or deoxynucleotides containing artificial bases.

[0014] Optionally, in some embodiments of this application, group B is obtained by chemically modifying the benzyl quinolonecarboxylic acid (BQCA) pharmacophore, and group X is obtained by chemically modifying the xanomeline pharmacophore. The benzyl quinolonecarboxylic acid pharmacophore has the structure shown in Formula 1, and the xanomeline pharmacophore has the structure shown in Formula 8. A protecting group is introduced into the benzyl quinolonecarboxylic acid pharmacophore to obtain group B, and a protecting group is introduced into the xanomeline pharmacophore to obtain group X. Groups B and X can be directly and automatically linked with deoxynucleotides using a DNA synthesizer.

[0015]

[0016] Specifically, adenine deoxynucleotide is represented as A, cytosine deoxynucleotide as C, thymine deoxynucleotide as T, guanine deoxynucleotide as G, and the linker shown in Formula 12 is represented as "B-DNA-X". The linker can be BAX, BAAX, BACX, BAGX, BATX, BCCX, BCCGX, etc.

[0017] Optionally, in some embodiments of this application, nt1 is linked to P via a phosphodiester bond, nt a It is linked to Q via a phosphodiester bond.

[0018] Preferably, the linker contains pharmacophore P, two deoxynucleotides and pharmacophore Q; more preferably, the linker contains pharmacophore P, two adenine deoxynucleotides and pharmacophore Q.

[0019] Optionally, in some embodiments of this application, the linker containing pharmacophore P, two deoxynucleotides, and pharmacophore Q has a 2-3 times higher affinity for the M1 muscarinic acetylcholine receptor compared to the affinity of benzylquinolone carboxylic acid and zenomeprazole free drugs for the M1 muscarinic acetylcholine receptor; the linker containing pharmacophore P, two deoxynucleotides, and pharmacophore Q has a 19-20 times higher selectivity for the M1 muscarinic acetylcholine receptor compared to the selectivity of benzylquinolone carboxylic acid and zenomeprazole free drugs for the M1 muscarinic acetylcholine receptor.

[0020] Optionally, in some embodiments of this application, the linker containing pharmacophore P, two adenine deoxynucleotides, and pharmacophore Q exhibits 6-7 times higher agonistic activity against the M1 muscarinic acetylcholine receptor compared to the free drugs benzylquinolone carboxylic acid and phenomenin against the M1 muscarinic acetylcholine receptor; the linker containing pharmacophore P, two adenine deoxynucleotides, and pharmacophore Q exhibits 33-34 times higher selectivity against the M1 muscarinic acetylcholine receptor compared to the free drugs benzylquinolone carboxylic acid and phenomenin against the M1 muscarinic acetylcholine receptor.

[0021] Accordingly, embodiments of this application also provide a method for preparing a linker, comprising: linking group B and group X with deoxynucleotides and / or deoxynucleotide derivatives to form a linker, the linker comprising pharmacophore P, pharmacophore Q, and deoxynucleotides and / or deoxynucleotide derivatives linking pharmacophore P and pharmacophore Q; pharmacophore P is obtained by removing a protecting group from group B, and pharmacophore Q is obtained by removing a protecting group from group X, the linker having the structure shown in Formula 12:

[0022] P-nt1-nt2...nt a-1 -nt a -Q

[0023] Formula 12

[0024] Wherein, nt1, nt2, ... nt a-1 nt a Each of the following is independently selected from adenine deoxynucleotide, guanine deoxynucleotide, cytosine deoxynucleotide, thymine deoxynucleotide, and derivatives of the above deoxynucleotides;

[0025] a is an integer between 1 and 100.

[0026] Optionally, in some embodiments of this application, the deoxynucleotide is selected from one or more of adenine deoxynucleotide, cytosine deoxynucleotide, thymine deoxynucleotide, or guanine deoxynucleotide.

[0027] Optionally, in some embodiments of this application, group B has the structure shown in Formula 4, and the method for synthesizing group B is as follows:

[0028]

[0029] Optionally, in some embodiments of this application, group X has the structure shown in Formula 11, and the method for synthesizing group X is as follows:

[0030]

[0031] Optionally, in some embodiments of this application, the methods for forming the linker and removing the protective base include solid-state synthesis techniques, but are not limited thereto.

[0032] Optionally, in some embodiments of this application, the solid-phase synthesis technique includes: after inputting sequence instructions into a DNA synthesizer, group B and group X are automatically linked to deoxynucleotides via phosphodiester bonds, and the linker is synthesized and output via a phosphonamide triester method.

[0033] This application has the following beneficial effects:

[0034] 1) This application enables the efficient construction of dual-recognition site molecular probes containing benzylquinolone carboxylic acid pharmacophores and zenomeprazole pharmacophores, as well as bivalent drug screening libraries. Compared with traditional modification strategies, the reaction conditions are mild, the yield is high, and it is suitable for general reactions in solid-phase synthesis, thereby improving the efficiency of constructing candidate compound libraries and showing broad application prospects in the biomedical field;

[0035] 2) This application can regulate the spatial distance and spatial orientation of the pharmacophore by regulating the number of deoxynucleotides and the base arrangement, respectively. The regulation method is efficient and simple. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 A schematic diagram illustrating the regulation of receptors by molecular probes with dual recognition sites;

[0038] Figure 2 The structural formula of a natural deoxynucleotide module;

[0039] Figure 3 The structural formulas are for module B and module X;

[0040] Figure 4A modular synthesis procedure for connectors;

[0041] Figure 5 A candidate library of connectors with different pharmacophore spacings and spatial orientations;

[0042] Figure 6 Competitive binding curves for B, BCG linkers, CG linkers, and M1Rs;

[0043] Figure 7 Competitive binding curves for the interaction of B, BCG linkers, CG linkers and M4Rs;

[0044] Figure 8 Concentration-response curves of intracellular calcium mobilization induced by B, BCG linkers, and CG linkers activating M1Rs;

[0045] Figure 9 Concentration-response curves of intracellular calcium mobilization induced by B, BCG linkers, and CG linkers activating M4Rs;

[0046] Figure 10 For X, CGX linker and competitive binding curves of CG linker and M1Rs;

[0047] Figure 11 For X, CGX linker and competitive binding curves of CG linker and M4Rs;

[0048] Figure 12 Concentration-response curves of intracellular calcium mobilization induced by the activation of M1Rs by X, CGX linkers and CG linkers;

[0049] Figure 13 Concentration-response curves of intracellular calcium mobilization induced by the activation of M4Rs by X, CGX linkers and CG linkers;

[0050] Figure 14 Competitive binding curves between B+X (a mixture of drugs B and X at equimolar concentrations) and B-DNA-X linkers of different lengths (B-1nt-X to B-7nt-X) and M1Rs;

[0051] Figure 15 Competitive binding curves between B+X (a mixture of drugs B and X at equimolar concentrations) and B-DNA-X linkers of different lengths (B-1nt-X to B-7nt-X) and M4Rs;

[0052] Figure 16 Concentration-response curves for intracellular calcium ion mobilization induced by B+X and B-DNA-X linkers of different lengths activating M1Rs.

[0053] Figure 17Concentration-response curves for intracellular calcium ion mobilization induced by B+X and B-DNA-X linkers of different lengths activating M4Rs.

[0054] Figure 18 pK for competitive binding of B+X and B-1nt-X to B-7nt-X with M1Rs i value([ 3 Comparison of the negative logarithm of the equilibrium dissociation constant of H]NMS;

[0055] Figure 19 pK for competitive binding of B+X and B-1nt-X to B-7nt-X with M4Rs i value([ 3 Comparison of the negative logarithm of the equilibrium dissociation constant of H]NMS;

[0056] Figure 20 pEC for intracellular calcium mobilization induced by B+X and B-DNA-X linkers of different lengths activating M1Rs. 50 Comparison of values ​​(negative logarithm of half effective concentration);

[0057] Figure 21 pEC for intracellular calcium mobilization induced by B+X and B-DNA-X linkers of different lengths activating M4Rs. 50 Comparison of values ​​(negative logarithm of half effective concentration);

[0058] Figure 22 A to 22D are concentration-response curves of intracellular calcium mobilization induced by B-2nt-X activating M1Rs with different base arrangements;

[0059] Figure 23 pECs that induce intracellular calcium mobilization by B-2nt-X with different base arrangements activating M1Rs 50 Comparison of values ​​(negative logarithm of half effective concentration);

[0060] Figure 24 The structure of BAAX;

[0061] Figure 25 Concentration-response curves of intracellular calcium mobilization induced by BAAX activating M1Rs in the complete linker form of BAAX and fragmented combinations (BAA+X, B+AAX);

[0062] Figure 26 Binding curves of BAAX binding affinity to wild-type and mutant M1Rs obtained by micro-thermophoresis;

[0063] Figure 27 pKa represents the binding affinity of BAAX to wild-type and mutant M1Rs determined by micro-thermophoresis. dCompare values ​​(combined with the negative logarithm of the dissociation constant);

[0064] Figure 28 Dose-response curves for BAAX stimulating wild-type and mutant M1Rs;

[0065] Figure 29 pEC for BAAX to stimulate wild-type and mutant M1Rs 50 Comparison of values ​​(negative logarithm of half effective concentration). Detailed Implementation

[0066] The technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0067] This application provides a connector and a method for preparing the same. Detailed descriptions follow. It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments.

[0068] In this specification, the range of values ​​indicated by “~” represents the range containing the minimum and maximum values ​​recorded before and after “~”, respectively.

[0069] In this application, deoxynucleotides are small molecule compounds composed of purine or pyrimidine bases, deoxyribose, and phosphate, and are the material basis of DNA, the genetic material of organisms. The diversity of life is determined by the different sequences of the four bases in deoxynucleotides: adenine (A), thymine (T), cytosine (C), and guanine (G).

[0070] The M1 muscarinic acetylcholine receptor is a classic class A G protein-coupled receptor (GPCR). Endogenous ligands act on its transmembrane region. The amino acid sites in this transmembrane region are highly conserved among the various subtypes of muscarinic acetylcholine receptors (currently identified as M1-5), resulting in highly similar orthomeric sites across subtypes—the orthomeric sites—making it impossible to identify a single subtype receptor. For example, zenomeprazole, an M1 / M4-biased agonist, was discontinued in clinical trials due to adverse reactions caused by its action on other peripheral receptor subtypes. Near the orthomeric site, there are some non-conserved amino acid sites with structures that differ among subtypes—the allosteric sites. Therefore, acting on these allosteric sites can achieve regulation of a single receptor subtype. In recent years, the development of selective agonists targeting M1 receptors has been both a hot topic and a challenge in this field.

[0071] In the field of receptor subtype-selective drug development, allosteric modulators, as a class of ligands that recognize allosteric sites, exhibit strong selectivity for a single receptor subtype, thus becoming a hot area of ​​research and development. This class of drugs is vast, numbering as many as 89,554, and involves a wide range of drug targets, including GPCRs, ion channels, nuclear hormone receptors, kinases, ion transporters, transcription factors, cytokines, and molecular chaperones.

[0072] Among them, benzylquinoline carboxylic acid, an allosteric modulator of M1Rs, can exhibit M1Rs subtype selectivity, but due to its poor affinity, it cannot activate receptors when used alone. When combined with orthoform drugs, it can leverage both the selectivity of the allosteric drug and the intrinsic activity of the orthoform drug. Therefore, in recent years, constructing bivalent ligands composed of allosteric and orthoform pharmacophores has become a common strategy in drug development for selectively regulating single-subtype receptors. Current construction strategies have several limitations: First, the degree of polymerization of polymers is often difficult to control precisely, resulting in an inability to precisely regulate the spacing between pharmacophores. Second, most polymers consist of repeating units with a single structure, thus limiting the variability of pharmacophore spatial orientation and hindering the screening of bivalent ligands. Third, the traditional strategies for constructing candidate dual-recognition site molecules have several limitations: First, the degree of polymerization of polymers is often difficult to control precisely during synthesis, resulting in an inability to precisely regulate the spacing between pharmacophores. Second, most polymers consist of repeating units with a single structure, thus limiting the variability of pharmacophore spatial orientation and hindering the screening of bivalent ligands. Third, traditional strategies for constructing candidate bivalent ligand libraries require researchers to progressively synthesize polymer linkers with different lengths and chemical compositions, and then individually link each linker to one of the two pharmacophores. This process typically involves multiple synthesis and purification steps, which is laborious and time-consuming. All of these factors limit the construction and screening of bivalent ligands, hindering their development. Therefore, there is an urgent need to develop a precise, controllable, and efficient strategy for constructing candidate bivalent ligand libraries, enabling the two pharmacophores to be linked by pharmacophore linkers of precisely controllable length and variable spatial orientation, thereby allowing for fine-tuning to obtain the bivalent ligands with optimal activity.

[0073] Example 1

[0074] This embodiment provides a method for synthesizing group B (as shown in Formula 4) from the pharmacophore group of benzylquinolone carboxylic acid (as shown in Formula 1):

[0075] Synthetic route of group B:

[0076]

[0077] The reagents and conditions for synthesis were as follows: (i) 3-amino-1,2-propanediol, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, room temperature; (ii) 4,4-dimethoxytriphenylchloromethane, pyridine, room temperature; (iii) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonamide, N,N-diisopropylethylamine, dichloromethane, room temperature.

[0078] The synthesis method is as follows:

[0079] (1) Benzylquinolone carboxylic acid (compound 1), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine (0.5 mmol: 0.5 mmol: 1 mmol) were placed in a round-bottom flask. N,N-dimethylformamide was added to the flask, and the mixture was stirred at room temperature until completely dissolved. Then, 3-amino-1,2-propanediol (0.5 mmol) was added, and the reaction was carried out at room temperature. The reaction endpoint was confirmed by TLC analysis. After the reaction was completed, the product was dissolved in dichloromethane and extracted successively with saturated sodium bicarbonate solution, hydrochloric acid, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, separated by chromatography, and dried by rotary evaporation to obtain white crystals, 153.17 mg, yield 80.1%.

[0080] (2) The white crystals obtained in the previous step were mixed with 4,4-dimethoxytriphenylchloromethane (1.0 mmol: 1.0 mmol) in a round-bottom flask. Pyridine (10 mL) was added to the flask, and the mixture was stirred at room temperature. The reaction was monitored by TLC analysis to confirm the endpoint. After the reaction was complete, the product was rotary evaporated at room temperature. The solid product was then dissolved in dichloromethane and extracted successively with double-distilled water, saturated sodium bicarbonate solution, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and separated by chromatography. The product was a yellowish-white solid, 513.66 mg, with a yield of 75.0%.

[0081] (3) Dissolve 0.15 mmol of a yellowish-white solid powder in dichloromethane. Add 0.30 mmol of 2-cyanoethyl N,N-diisopropylphosphorimide and 100 μL of N,N-diisopropylethylamine sequentially to the flask. Stir in an ice bath for 5–10 min, then transfer to room temperature and react for 15 min. The reaction endpoint is confirmed by TLC analysis. After the reaction, extract with double-distilled water, saturated sodium bicarbonate solution, and saturated sodium chloride solution sequentially. Combine the organic phases, dry them over anhydrous sodium sulfate, and separate them by chromatography. Rotary evaporation and drying yields yellowish-white crystals, i.e., group B, 618.31 mg, yield 69.9%.

[0082] The following are the 1H NMR, 1C NMR, and 1P NMR spectra:

[0083] 1H NMR (500MHz, DMSO): δ8.64(d,J=6.7Hz,2H),8.26(d,J=7.5Hz,2H),7.64(t,J=9.5H z,4H),6.97(t,J=6.1Hz,3H),6.65(d,J=8.8Hz,4H),4.49(d,J=6.0Hz,4H),4.37(d dd,J=9.7,7.5,5.3Hz,2H),4.12-4.01(m,8H),2.61-2.54(m,1H),2.40(t,J=7.5Hz ,4H),2.11-2.02(m,2H),1.96(ddd,J=17.0,14.1,7.2Hz,2H),1.20-1.14(m,12H).

[0084] 13 C NMR (125MHz, DMSO): δ175.92(s),165.99(s),160.26(s),158.78(s),147.24(s),143.85(s),139.39(s),135.2 4(s),133.07(s),129.30(s),128.87(d,J=10.5Hz),128.47-128.13(m),127.96(s),124.30(s),123.91(s),11 8.58(d,J=4.7Hz),117.47(s),114.93(s),113.64(s),113.43(s),87.84(s),72.45(d,J=8.6Hz),61.50(d,J=4 .7Hz), 59.05 (d, J = 8.6Hz), 56.08 (s), 54.27 (s), 44.58 (d, J = 12.4Hz), 42.68 (d, J = 4.7Hz), 22.94 (d, J = 5.8Hz).

[0085] 31 P NMR (202MHz, DMSO): δ 149.55.

[0086] The synthesized group B can be automatically linked to deoxynucleotides directly using a DNA synthesizer.

[0087] Example 2

[0088] This embodiment provides a method for synthesizing group X (as shown in Formula 11) from the pharmacophore of nomereline (as shown in Formula 5):

[0089] Synthetic route of group X:

[0090]

[0091] The reagents and conditions for synthesis were as follows: (i) tert-butyl (3-hydroxypropyl)carbamate, sodium hydride, tetrahydrofuran, reflux for 18 h; (ii) sodium iodide (NaI), acetone, room temperature for 18 h; sodium borohydride, ethanol, reflux for 12 h; (iii) trifluoroacetic acid, dichloromethane, room temperature; (iv) 3-amino-1,2-propanediol, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, room temperature; (v) DMT-Cl, pyridine, room temperature; (vi) 2-cyanoethyl N,N,N',N'-tetraisopropylphosphonamide, N,N-diisopropylethylamine, dichloromethane, room temperature.

[0092] The synthesis method is as follows:

[0093] (1) Take 10 mL of dichloromethane in a round-bottom flask, cool to 0 °C, then add 8.50 mmol of 3-aminopropanol and 9.95 mmol of triethylamine, followed by 7.72 mmol of ditert-butyl dicarbonate. Stir well and continue the reaction at room temperature for 5 h. The next day, concentrate the product by rotary evaporation, then add 50 mL of anhydrous diethyl ether. Extract with citric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution in sequence. Dry the upper organic phase with anhydrous sodium sulfate and then dry by rotary evaporation again. The product is a colorless viscous liquid. Dissolve the colorless viscous liquid (1.32 mmol) and compound 5 (1.32 mmol) in tetrahydrofuran (15 mL), stir at 0 °C for about 10 min, then weigh out sodium hydride powder (105 mg, 2.63 mmol). Reflux for about 18 h, cool to room temperature, and concentrate by rotary evaporation. The product was dissolved in dichloromethane and extracted successively with sodium hydroxide solution and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and then concentrated by rotary evaporation. The product was then purified by silica gel chromatography with dichloromethane / ethyl acetate (9:1) as the mobile phase, yielding 6,221.92 mg of the compound (49.9%).

[0094] (2) Iodomethane (2.44 mmol) was added to a stirred acetone (5 mL) solution of compound 6 (1.22 mmol), and stirring was continued at room temperature for 18 h. The pyridinium salt precipitated from the solution, filtered, and washed with cold acetone. Sodium borohydride (3.4 mmol) was added to a stirred solution of the pyridinium salt in anhydrous ethanol (0.85 mmol) at 0 °C. The mixture was refluxed for 3 h, concentrated, dissolved in dichloromethane, and the aqueous layer was separated, followed by extraction with dichloromethane (2 × 50 mL). The combined organic matter was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to obtain a brown oil. Finally, purification was performed by silica gel column chromatography, eluting with ethyl acetate / methanol / triethylamine (9:1:0.2) to give the brown oil, compound 7, 67.60 mg, with a yield of 15.6%.

[0095] (3) After stirring product 7 (100 mg) in a mixture of trifluoroacetic acid and dichloromethane in equal proportions for 4 h, it was dissolved in 1 M potassium carbonate solution for extraction. The organic phase was collected and concentrated by rotary evaporation to obtain product 8, 21.53 mg, with a yield of 30.0%.

[0096] (4) Compound 8, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine (0.5 mmol: 0.5 mmol: 1 mmol) were placed in a round-bottom flask. N,N-dimethylformamide was added to the flask and stirred at room temperature until completely dissolved. Then, 3-amino-1,2-propanediol (0.5 mmol) was added and reacted at room temperature. The reaction endpoint was confirmed by TLC analysis. After the reaction was completed, the product was dissolved in dichloromethane and extracted successively with saturated sodium bicarbonate solution, hydrochloric acid, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, separated by chromatography, and dried by rotary evaporation to obtain a yellowish-white solid, compound 9, 122.51 mg, with a yield of 71.6%.

[0097] (5) Compound 9 and 4,4-dimethoxytriphenylchloromethane (1.0 mmol: 1.0 mmol) were placed in a round-bottom flask, and pyridine (10 mL) was added to the flask. The mixture was stirred at room temperature. The reaction was monitored by TLC analysis to confirm the reaction endpoint. After the reaction was completed, the product was rotary evaporated at room temperature. The solid product was then dissolved in dichloromethane and extracted successively with double-distilled water, saturated sodium bicarbonate solution, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and separated by chromatography. The product was then dried by rotary evaporation to obtain a pale yellow solid, compound 10, 313.76 mg, with a yield of 48.7%.

[0098] (6) Compound 10 (0.5 mmol) was dissolved in dichloromethane. 2-Cyanoethyl N,N-diisopropylphosphorimide (0.30 mmol) and N,N-diisopropylethylamine (100 μL) were added sequentially to the flask. After stirring in an ice bath, the mixture was transferred to room temperature and reacted for 15 min. The reaction endpoint was confirmed by TLC analysis. After the reaction was complete, the mixture was extracted sequentially with double-distilled water, saturated sodium bicarbonate solution, and saturated sodium chloride solution. The organic phases were combined, dried over anhydrous sodium sulfate, and separated by chromatography. Rotary evaporation and drying yielded yellowish-white crystals, i.e., group X, 215.48 mg, with a yield of 51.0%.

[0099] The following are the 1H NMR, 1C NMR, and 1P NMR spectra:

[0100] 1 H NMR (400MHz, DMSO): δ7.98(t,J=6.0Hz,1H),7.41(d,J=7.3Hz,3H),7.24(t,J=14.4,7.3Hz, 8H),7.01(d,J=4.0Hz,1H),6.86(dt,J=6.6,3.3Hz,4H),4.40(t,J=6.4Hz,2H),4.29-4.19( m,1H),3.73(d,J=2.7Hz,6H),3.29(s,3H),3.16(dd,J=9.3,3.3Hz,1H),2.73(t,J=5.9Hz,2 H), 2.32 (s, 4H), 1.99 (dd, J = 13.0, 6.5Hz, 2H), 1.15 (d, J = 6.6Hz, 6H), 1.08 (d, J = 6.7Hz, 6H).

[0101] 13 C NMR (125MHz, DMSO): δ169.67(s), 158.78(s), 158.22(s), 150.30(s), 143.85(s), 135. 24(s),129.30(s),128.29(d,J=12.0Hz),127.95(s),127.46(s),118.58(s),118.03(s ),113.43(s),87.84(s),71.66(s),65.45(s),64.05(s),59.05(s),56.66(s),56.08(s) ),53.79(s),45.00(s),44.58(s),37.33(s),30.61(s),25.98(s),22.93(d,J=0.9Hz).

[0102] δ 31 P NMR (202MHz, DMSO): 149.55.

[0103] The synthesized group X can be automatically linked to deoxynucleotides directly using a DNA synthesizer.

[0104] Example 3

[0105] This embodiment provides a method for constructing linkers based on DNA modularization:

[0106] After the sequence command is input into the DNA synthesizer, groups B and X can be automatically coupled to any natural deoxynucleotide module (A, G, C or T module) via phosphodiester bonds to synthesize and output a series of "B-DNA-X" linkers.

[0107] like Figure 1 The diagram illustrates the regulation of receptors by a molecular probe with dual recognition sites. BQCA is an ortho-articulate modulator; zenomeprazole is an ortho-articulate agonist. Figure 2 The image shows the structural formula of a natural deoxynucleotide module. Figure 3 The diagram shows the structural formulas of module B and module X. Figure 4 The diagram shows the modular synthesis procedure for connectors. For example... Figure 5 The diagram shows a library of linker candidates with varying pharmacophore spacing and spatial orientation. The pharmacophore spatial distance of the linkers can be adjusted by increasing or decreasing the number of intermediate deoxynucleotides. At the same pharmacophore spatial distance, the pharmacophore spatial orientation of the linkers can be modulated by changing the base arrangement.

[0108] Example 4

[0109] This embodiment provides a method for testing the affinity between a linker and a receptor:

[0110] Affinity was determined using radioligand competitive binding analysis with a 200 μL reaction system per well.

[0111] (1) In the saturation binding experiment, first add 100 μL of KHB buffer to each well of a U-shaped 96-well plate. Then take [ 3 H]N-methylscopolamine ([ 3 [H]N-methylscopolamine (NMS) radioligand stock solution (14.3 μM) was prepared according to the set [ 3 The final concentration gradient range of the H]NMS radioligand (0–6.4 nM) was prepared by 1:1 dilution, and 20 μL of radioligand dilution was added to each well. The frozen membrane proteins were removed, thawed, and repeatedly mixed to a concentration of 0.25 μg / μL. Then, 80 μL of membrane protein dilution was added to each well of a 96-well plate, for a total volume of 200 μL.

[0112] (2) In the nonspecific binding experiment, first add 80 μL of KHB buffer to each well of a U-shaped 96-well plate, then add 20 μL of 10 μM atropine solution to each well (final concentration 10 μM), followed by 20 μL of […]. 3 H]NMS radioligand working solution (final concentration is the Ksaturation experiment) d (Value), and finally add 80 μL of membrane protein diluent to each well to make up a total volume of 200 μL (Note: When adding membrane protein diluent, the liquid in the well needs to be mixed repeatedly with a pipette; the pipette tip is radioactive waste). Then, after capping the U-bottom 96-well plate, transfer it to a constant temperature shaking water bath at 37°C for incubation for 30 min. During incubation, a paper filter membrane (printed filtermat A) can be prepared and immersed in 0.3% polyethyleneimine solution to reduce non-specific binding. After about 30 seconds, remove the paper membrane and place it directly above the suction port of the vacuum filter, so that A1 is located to the left front. After incubation, remove the 96-well plate, open the lid, and place it under the vacuum filter, oriented in the same direction as the paper membrane. Then, lift the 96-well plate until it contacts the aspiration port, secure the device, turn on the vacuum filter power switch, turn the knob to Trap, and create a vacuum. Press the aspiration button to draw the reaction mixture from the 96-well plate onto the paper membrane. After aspiration, remove the 96-well plate and add 200 μL of pre-cooled ultrapure water per well. Then aspirate the water onto the paper membrane to rinse away any residue in the wells and quickly rinse the membrane three times to remove free ligands. Repeat once. After aspiration, turn off the vacuum filter switch, remove the paper membrane, and lay it flat in an oven. Heat on medium heat for 4 minutes. After the paper film is dried, place it in a special packaging bag (PE sample bag for MicroBeta), ensuring the edges of the paper film are tightly sealed against the bag. Trim any excess material from the bag and seal the opening with a heat sealer, leaving one side open. Then, in a dark environment, add scintillation fluid (2-3 mL) using a pipette, ensuring the well containing the sample is completely submerged. Seal the well again with a heat sealer. Next, place the packaged paper film in a special clamp to secure it, minimizing the exposure of the black edges. For instrument testing, insert the holder into the slot and place the termination plate above the detection plate with the barcode facing the door. Close the door and wait for the liquid scintillation counter to initialize. Set the detection program and start the test. During routine testing, equilibration is achieved after 2 minutes of reading per well.

[0113] (3) In the competitive binding experiment, first, add 80 μL of KHB buffer to each well of a U-shaped 96-well plate. Then, replace 20 μL of 10 μM atropine solution per well with 20 μL of the ligand solution to be tested (10 times the final concentration). Next, add 20 μL of […] 3H]NMS radioligand working solution (final concentration equal to the Kd value of the saturation experiment) was added to each well, and finally, 80 μL of membrane protein diluent was added to each well to bring the total volume to 200 μL. We selected the complete agonist carbachol as a positive control, with the gradient settings identical to those for the test ligand. Incubation and post-treatment were performed as described in the saturation experiment.

[0114] Example 5

[0115] This embodiment provides a method for testing the receptor agonistic activity of the linker:

[0116] (1) Intracellular calcium mobilization detection procedure: A wash-free calcium flow assay kit was used. The stock solutions of each component were prepared in advance according to the kit instructions, including: 2mM Fluo-8 AM; 50X component B; 250mM probenecid; and Hank's balanced salt solution (HBSS). On the day of detection, the Fluo-8 Calcium assay loading solution was prepared, including 2mM Fluo-8 AM (20μL), 50X component B (200μL), 250mM probenecid (100μL), and HBSS (9.7mL). After mixing, the solution was temporarily stored at 4℃.

[0117] (2) In the ligand screening experiment, the culture medium in the 96-well plate was discarded, and 100 μL of the prepared loading solution was added to each well. The 96-well plate was then transferred to a CO2 incubator and incubated for 30 min, followed by incubation at room temperature for 15–30 min. This experiment used a Flexstation III multi-functional microplate reader, which automatically added gradient concentrations of ligand solutions to the 96-well plate according to the programmed settings for intracellular calcium flux detection. Carbachol was used as a positive control, with a final concentration of up to 10 μM. Detection conditions: excitation wavelength 485 nm, detection wavelength 525 nm, cut-off: 515 nm, continuous measurement for 150 s, with a set of data collected every 2.0 s.

[0118] Figure 6 and Figure 7 Competitive binding curves of B, BCG, and CG linkers to M1Rs and M4Rs are shown. Experiments were performed using CHO cells stably expressing human M1Rs or M4Rs. Data are expressed as mean ± standard error (n = 3). Table 1 shows the K-axis binding modulotropic enzymes (K-axis) of B, BCG, and CG linkers to M1Rs and M4Rs. i Value (the ligand concentration required to replace 50% of a fixed concentration of radioactive ligand). (From...) Figure 6 , Figure 7As shown in Table 1, group B and the BCG linker have an affinity for M1Rs and M4Rs, while the CG linker does not have an affinity for M1Rs and M4Rs. This indicates that group B retains the affinity of the original drug for M1 muscarinic acetylcholine receptors and M4 muscarinic acetylcholine receptors.

[0119] Table 1

[0120]

[0121] Data are expressed as mean ± standard error (n=3).

[0122] Figure 8 and Figure 9 Concentration-response curves are shown for the effect of B, BCG, and CG linkers on intracellular calcium mobilization induced by the activation of M1Rs and M4Rs. Experiments were performed using CHO cells stably expressing human M1Rs or M4Rs. Data are expressed as mean ± standard error (n = 3). Table 2 shows the half-maximal effective concentrations (MCCs) (compound concentrations producing a 50% maximal effect) of the B, BCG, and CG linkers on M1Rs and M4Rs. Figure 8 , Figure 9 As shown in Table 1, the B group and the BCG linker have agonistic activity against M1Rs, while the B group and the BCG linker do not have agonistic activity against M4Rs. The CG linker does not have agonistic activity against either M1Rs or M4Rs. This indicates that the B group retains the agonistic activity of the original drug against M1 muscarinic acetylcholine receptors.

[0123] Table 2

[0124]

[0125] Data are expressed as mean ± standard error (n=3).

[0126] Figure 10 and Figure 11 Table 3 shows the competitive binding curves of X, CGX linkers and CG linkers with M1Rs and M4Rs. Experiments were performed using CHO (Chinese hamster ovary) cells stably expressing human M1Rs or M4Rs. Data are expressed as mean ± standard error (n = 3). Table 3 shows the K values ​​of the competitive binding curves of X, CGX linkers and CG linkers with M1Rs and M4Rs. i Value. (By) Figure 10 , Figure 11 As shown in Table 3, the X group and the CGX linker have an affinity for M1Rs and M4Rs, while the CG linker does not have an affinity for M1Rs and M4Rs. This indicates that the X group retains the affinity of the original drug for M1 muscarinic acetylcholine receptors and M4 muscarinic acetylcholine receptors.

[0127] Table 3

[0128]

[0129] Data are expressed as mean ± standard error (n=3).

[0130] Figure 12 and Figure 13 Concentration-response curves were generated for intracellular calcium mobilization induced by the X, CGX, and CG linkers activating M1Rs and M4Rs. Experiments were performed using CHO cells stably expressing human M1Rs or M4Rs. Data are presented as mean ± standard error (n = 3). Table 4 shows the ECGs mobilization of M1Rs and M4Rs by the X, CGX, and CG linkers. 50 Value. (By) Figure 12 , Figure 13 As shown in Table 4, both the X group and the CGX linker exhibit agonistic activity against both M1Rs and M4Rs, while the CG linker does not exhibit agonistic activity against either M1Rs or M4Rs. This indicates that the X group retains the agonistic activity of the original drug against both M1 and M4 muscarinic acetylcholine receptors.

[0131] Table 4

[0132]

[0133] Data are expressed as mean ± standard error (n=3).

[0134] Figures 14-21 This study presents the affinity and agonistic activity of linkers of different deoxynucleotide lengths for M1Rs and M4Rs. Experiments were performed using CHO cells stably expressing human M1Rs or M4Rs. Data are presented as mean ± standard error (n = 3). pK i Value refers to [ 3 The negative logarithm of the equilibrium dissociation constant of H]NMS. Statistical differences between each group and the B-2nt-X linker were analyzed by one-way ANOVA and Dunnett's multiple comparison test. Corrected P-values ​​are shown in the figure. NS, no significant difference. Table 5 shows the K values ​​of B-1nt-X to B-7nt-X bound to M1Rs and M4Rs. i Values. Table 6 shows the ECG values ​​of different lengths of B-DNA-X linkers (B-1nt-X to B-7nt-X) activating M1Rs and M4Rs. 50 Value. (By) Figures 14-21 As shown in Tables 5 and 6, the linker containing two deoxynucleotides has a higher affinity and agonistic activity for M1Rs, even higher than that of B+X. This indicates that selective agonistic activity of M1Rs can be achieved by regulating the spatial distance between the B and X groups by controlling the number of deoxynucleotides.

[0135] Table 5

[0136]

[0137]

[0138] Data are expressed as mean ± standard error (n=3).

[0139] Table 6

[0140]

[0141] Data are expressed as mean ± standard error (n=3).

[0142] Figure 22 Concentration-response curves of intracellular calcium mobilization induced by B-2nt-X with different base arrangements activating M1Rs; Figure 23 pECs that induce intracellular calcium mobilization by B-2nt-X with different base arrangements activating M1Rs 50 Values. Experiments were conducted using CHO cells stably expressing human M1Rs. Data are expressed as mean ± standard error (n=3). Statistical differences between each group and BAAX were calculated using Dunnett's multiple comparisons via one-way ANOVA. Corrected p-values ​​are shown in the figure. NS, no significant difference was found. Figure 22 and Figure 23 It is evident that BAAX exhibits high agonistic activity towards M1Rs, demonstrating that efficient agonistic activity towards M1Rs can be achieved by regulating the spatial orientation of groups B and X through the base arrangement of natural deoxynucleotides.

[0143] Figure 24 The structure of BAAX, Figure 25 Concentration-response curves of intracellular calcium mobilization induced by BAAX activation of M1Rs in the complete linker form of BAAX and fragmented combinations (BAA+X, B+AAX). Experiments were performed using CHO cells stably expressing human M1Rs or M4Rs. Data are presented as mean ± standard error (n = 3). Table 7 shows the concentration-response curves of intracellular calcium mobilization induced by BAAX activation of M1Rs in EC2 cells using the complete linker form of BAAX and fragmented combinations. 50 Value. (By) Figure 25 As shown in Table 7, the complete BAAX connector exhibits the best excitatory activity for M1Rs, indicating that the BAAX connector is crucial for efficient M1R excitation.

[0144] Table 7

[0145]

[0146] Data are expressed as mean ± standard error (n=3).

[0147] Table 8 shows the EC50 of B+X and BAAX stimulating M1Rs and M4Rs. 50 Values. As shown in Table 8, BAAX can selectively activate M1Rs better than the original free form (B+X).

[0148] Table 8

[0149]

[0150] Data are expressed as mean ± standard error (n=3).

[0151] Figure 26 Binding curves of BAAX binding affinity to wild-type and mutant M1Rs obtained by micro-thermophoresis; Figure 27 pKa represents the binding affinity of BAAX to wild-type and mutant M1Rs determined by micro-thermophoresis. d Value comparison. Data are expressed as mean ± standard error (n=3). pK values ​​between wild-type M1Rs and mutant M1Rs. d Statistical significance of the comparisons was determined using Dunnett's multiple comparisons analysis via one-way ANOVA, and the corrected p-values ​​are shown in the figure. NS, no significant difference. Figure 26 and Figure 27 It is known that mutation sites such as D105A, Y106A, and W157A ( Figure 26 The mutation at the boxed mutation site (the mutation site in the middle) causes pK d The values ​​decreased significantly, indicating that these amino acid sites and the binding of BAAX to M1Rs are closely related.

[0152] Figure 28 pEC for BAAX to stimulate wild-type and mutant M1Rs 50 The dose-response curve of the value; Figure 29 pEC for BAAX to stimulate wild-type and mutant M1Rs 50 pEC value 50 Value comparison. Data are expressed as mean ± standard error (n=3). pEC values ​​between wild-type M1Rs and mutant M1Rs. 50 Statistical significance of the comparisons was determined using Dunnett's multiple comparisons analysis via one-way ANOVA, and the corrected p-values ​​are shown in the figure. NS, no significant difference. Figure 28 and Figure 29 It is known that mutation sites such as D105A, Y106A, and W157A ( Figure 28 Mutations at the mutation sites marked with boxes in the middle cause pEC 50 The values ​​decreased significantly, indicating that these amino acid sites are closely related to the activation of M1Rs by BAAX.

[0153] This application enables the efficient construction of dual-recognition site molecular probes or bivalent drug screening libraries containing two pharmacophores by linking the benzylquinolone carboxylic acid pharmacophore and the zenomeline pharmacophore after the introduction of a protectant via deoxynucleotide linkage. The spatial distance and spatial orientation of the pharmacophores can be controlled by regulating the number of deoxynucleotides and the base arrangement.

[0154] The foregoing provides a detailed description of a connector and its preparation method according to the embodiments of this application. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A connector, characterized in that, The linker includes pharmacophore P, pharmacophore Q, and a deoxynucleotide linking pharmacophore P and pharmacophore Q, and the linker has the structure shown in Formula 12: Formula 12 Wherein, nt1, nt2, ... nt a-1 nt a Each of the following is independently selected from adenine deoxynucleotide, guanine deoxynucleotide, cytosine deoxynucleotide, and thymine deoxynucleotide; a is an integer selected from 1 to 7.

2. The connector according to claim 1, characterized in that, The pharmacophore P is obtained by removing the protecting group from group B. The pharmacophore P has the structure shown in Formula 13, and the group B has the structure shown in Formula 4. 。 3. The connector according to claim 1, characterized in that, The pharmacophore Q is obtained by removing the protecting group from group X. The pharmacophore Q has the structure shown in Formula 14, and group X has the structure shown in Formula 11. 。 4. The connector according to claim 1, characterized in that, The linker contains the pharmacophore P, two deoxynucleotides, and the pharmacophore Q.

5. The connector according to claim 1, characterized in that, The linker contains the pharmacophore P, two adenine deoxynucleotides, and the pharmacophore Q.

6. The connector according to claim 4, characterized in that, The linker containing pharmacophore P, two deoxynucleotides, and pharmacophore Q exhibits a 2-3 times higher affinity for the M1 muscarinic acetylcholine receptor compared to the free drugs benzylquinolone carboxylic acid and phenomenin. Furthermore, the linker containing pharmacophore P, two deoxynucleotides, and pharmacophore Q demonstrates a 19-20 times higher selectivity for the M1 muscarinic acetylcholine receptor compared to the free drugs benzylquinolone carboxylic acid and phenomenin.

7. The connector according to claim 5, characterized in that, The linker containing pharmacophore P, the two adenine deoxynucleotides, and pharmacophore Q exhibits 6-7 times greater agonistic activity against the M1 muscarinic acetylcholine receptor compared to the free drug benzylquinolone carboxylic acid and phenomenin against the M1 muscarinic acetylcholine receptor; the linker containing pharmacophore P, the two adenine deoxynucleotides, and pharmacophore Q shows 33-34 times greater selectivity against the M1 muscarinic acetylcholine receptor compared to the free drug benzylquinolone carboxylic acid and phenomenin against the M1 muscarinic acetylcholine receptor.

8. A method for preparing a connector, characterized in that, include: Group B and group X are linked with a deoxynucleotide to form a linker, the linker comprising pharmacophore P, pharmacophore Q, and a deoxynucleotide linking pharmacophore P and pharmacophore Q; pharmacophore P is obtained by deprotecting group B, and pharmacophore Q is obtained by deprotecting group X; the linker has the structure shown in Formula 12. Formula 12 Where nt1, nt2, ...nt a-1 nt a Each of the following is independently selected from adenine deoxynucleotide, guanine deoxynucleotide, cytosine deoxynucleotide, and thymine deoxynucleotide; a is an integer selected from 1 to 7.

9. The preparation method according to claim 8, characterized in that, The group B has the structure shown in Formula 4, and the method for synthesizing the group B is as follows: 。 10. The preparation method according to claim 8, characterized in that, The group X has the structure shown in Formula 11, and the method for synthesizing the group X is as follows: 。 11. The preparation method according to claim 8, characterized in that, The method for forming the linker or removing the protective group includes solid-phase synthesis techniques.

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