A lead compound of 2,5-disubstituted thiophene on-DNA and its synthesis method

Abstract

The application provides an On-DNA 2,5-disubstituted thiophene leading compound and a synthesis method thereof, and belongs to the field of gene coded compound library construction. The method has small DNA damage, good universality, simple operation, mild conditions and can obtain the On-DNA 2,5-disubstituted thiophene compound with high conversion rate. Under the reaction conditions in the application, the product On-DNA 2,5-disubstituted thiophene compound not only has high conversion rate, but also has good DNA integrity. The application enriches the chemical reaction type of the synthesis of the coded compound library on the DNA, constructs a new 2,5-disubstituted thiophene skeleton for the gene coded compound library, and has very good application prospect in the development of leading drugs.

Description

Technical Field

[0001] This invention belongs to the field of gene-encoded compound library construction, specifically involving an on-DNA 2,5-disubstituted thiophene lead compound and its synthesis method. Background Technology

[0002] After years of application, development, and improvement, high-throughput screening has established a comprehensive screening process and is an important pathway for major international drug development companies to obtain lead compounds for target proteins. However, traditional high-throughput screening of single molecule samples has drawbacks such as limited compound library size, long processing time, and high cost, and is increasingly unable to meet the needs of new drug development.

[0003] In 1992, Brenner and Lerner creatively proposed a method for screening bioactive compounds using gene-encoded library technology (DELT). The principle of DELT is to label each small molecule compound in the reaction process with gene fragments of different specific sequences. Using a combinatorial chemistry strategy, through splitting and pooling, millions to tens of billions of compounds linked to specific gene sequences can be synthesized in large quantities with limited cost and time. The resulting mixture of compounds is then incubated with a protein target. Physical separation is achieved by washing away compounds that do not bind to the protein target, thus identifying compounds with high affinity. The gene-encoded compound library required for incubating the target protein requires only extremely small doses (micrograms) and can be performed in a very short time (e.g., within one day). Furthermore, DELT technology can easily perform multiple biological screening experiments under different conditions. Currently, screening of gene-encoded compound libraries (DELs) has become a common method for discovering new protein ligands (Goodnow, RA; Dumerin, CE; Keefe, ADDNA-encoded chemistry: Enabling the deeper sampling of chemical space. Nat. Rev. Drug Discov. 2016; Neri, D.; Lerner, RADNA-Encoded Chemical Libraries: A Selection System Based on Endowing Organic Compounds with Amplifiable Information. Annu. Rev. Biochem. 2018, 87, 479-502). Compared with traditional chemistry, DELs offer significant advantages in cost, output, and the construction of larger chemical spaces through high-throughput screening, greatly increasing the number and diversity of compounds. In practical applications, DEL bioscreening has successfully identified affinities for many protein targets (Zimmermann, G.; Neri, D. DNA-encoded chemical libraries: Foundations and applications in lead discovery. Drug Discov. Today 2016, 21, 1828-1834), including small chemical molecules currently in clinical trials (Belyanskaya, SL; Ding, Y.; Callahan, JF; Lazaar, AL; Israel, DIChemBioChem. 2017, 18, 837-842).

[0004] One of the most important areas of research in gene-encoded compound library technology is the development of on-DNA chemical reactions (On-DNA chemical reactions). Several On-DNA chemical reactions have already been reported. For example, Shanghai WuXi AppTec New Drug Development Co., Ltd. (patent application CN201910609569.0) disclosed a method for obtaining On-DNA aromatic compounds via Suzuki coupling reaction: using On-DNA aryl halides as substrates, reacting with potassium organotrifluoroborate reagent in the presence of a Pd catalyst, ligands, and a base to prepare On-DNA aromatic compounds. This method increases the diversity of DNA-encoded compound libraries of On-DNA aryl halides, has high reaction yields, broad substrate versatility, mild conditions, and is easy to operate, making it suitable for the synthesis of DNA-encoded compound libraries in multi-well plates. Chengdu Pioneer Pharmaceuticals Co., Ltd. (patent application CN201910590679.7) disclosed a method for synthesizing On-DNA arylbenzyl-substituted compounds. This method uses On-DNA aldehyde compounds as raw materials, reacting them with indole under alkaline conditions to generate On-DNA indole alcohol compounds. Then, the On-DNA indole alcohol compounds are reduced to indole alkylated compounds by diethyl 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate under acidic conditions. The more diverse and abundant the chemical reactions realized in gene-encoded compound libraries, the more choices are available for the design and synthesis of these libraries, resulting in greater diversity. However, the currently reported types of On-DNA chemical reactions are limited and cannot yet meet the widespread demand for lead compound discovery.

[0005] 2,5-Disubstituted thiophenes are an important class of sulfur-containing aromatic heterocyclic compounds. On the one hand, they are important intermediates in organic synthesis; on the other hand, they have wide applications in medicine, agriculture, and materials chemistry. Examples include Sanofi's FC-3001 drug for treating inflammation and osteoarthritis; Bausch Health's CBS-211A, a Phase I clinical trial drug with efficacy in treating ophthalmological conditions and psoriasis; and Theralase Technologies' TLD-1433, a Phase II clinical trial drug with efficacy in treating bladder cancer, cervical cancer, gastric cancer, and acute respiratory distress syndrome.

[0006]

[0007] Various methods for the efficient synthesis of 2,5-disubstituted thiophene small molecule compounds have been reported in traditional chemistry. For example, Chinese patent application CN106008452A discloses a method for preparing 2,5-disubstituted thiophene, comprising the following steps: a sulfidation cyclization reaction of sodium sulfide and 1,4-disubstituted-1,3-butadiyne under organic solvent conditions to obtain 2,5-disubstituted thiophene. Specifically, Example 1 discloses the following specific operation: 1.2 mmol Na₂S·9H₂O, 1.0 mmol 1,4-diphenyl-1,3-butadiyne, and 2.0 mL DMF are added to a reaction flask equipped with a magnetic stirrer; the mixture is stirred and heated to 80°C in air for 4 hours; and the reaction is terminated by adding saturated sodium chloride.

[0008] However, the above method for synthesizing 2,5-disubstituted thiophene small molecule compounds is not applicable to the construction of 2,5-disubstituted thiophene gene-encoded compounds on DNA. This is because DNA must maintain stability under specific aqueous phase, pH, temperature, metal ion concentration, and inorganic salt concentration conditions, and reactions used for constructing DNA-encoded compound libraries require high conversion rates. Currently, there are no reported methods for constructing 2,5-disubstituted thiophene gene-encoded compounds on DNA. Therefore, developing a method for synthesizing On-DNA 2,5-disubstituted thiophene compounds with minimal DNA damage and high conversion rates is of great significance. Summary of the Invention

[0009] The purpose of this invention is to provide an On-DNA 2,5-disubstituted thiophene lead compound, a method for synthesizing the same, and the use of the method in constructing a gene-encoded compound library.

[0010] This invention provides an On-DNA 2,5-disubstituted thiophene lead compound of Formula I:

[0011]

[0012] DNA is a modified or unmodified single-stranded or double-stranded nucleotide chain;

[0013] L represents the connection unit;

[0014] R 1 Selected from None, M1, A1

[0015] M1 is selected from those that have not been replaced or have been replaced by one or more R. 3 Replacement C 1-6 Alkylene, unsubstituted or with one or more R 3 Replacement C 2-6 alkenyl, unsubstituted or with one or more R 3 Replacement C2-6 Ethyne group, -Z1-O-Z2-; R 3 Each was independently selected from C 1-6 Alkyl group, NHBoc, Z1 is selected from none, C 1-6 Alkylene, Z2 is selected from none, C 1-6 Alkylene, and Z1 and Z2 are not both absent;

[0016] A1 is selected from those that have not been replaced or have been replaced by one or more R. 4 Substituted groups include: 5-6 aryl, 5-6 heteroaryl, 3-8 saturated cycloalkyl, and 3-8 saturated heterocyclic groups; R 4 Selected from amino, nitro, cyano, hydroxyl, mercapto, halogen, and carboxyl groups;

[0017] R 2 Selected from hydrogen, carboxyl, -Z3-OH, unsubstituted or surrounded by one or more R groups. 5 The following groups are substituted: 5-6 aryl, 5-6 heteroaryl, 3-8 saturated cycloalkyl, 3-8 saturated heterocyclic group;

[0018] R 5 Each was independently selected from C 1-6 Alkoxy, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -NR 6 R 7 Halogen, nitro, cyano, hydroxyl, mercapto, carboxyl, -Boc;

[0019] Z3 is selected from none, C 1-6 Alkylene;

[0020] R 6 R 7 Each is independently selected from hydrogen and C. 1-6 alkyl.

[0021] Furthermore, L is selected from NHCO, CONH, NH, or CO.

[0022] Furthermore, R 1 Selected from None, M1, A1

[0023] M1 is selected from those that have not been replaced or have been replaced by one or more R. 3 Replacement C 1-3 Alkylene, -Z1-O-Z2-, R 3 Each was independently selected from C 1-3 Alkyl group, NHBoc, Z1 is selected from none, C 1-3 Alkylene, Z2 is selected from none, C 1-3 Alkylene, and Z1 and Z2 are not both absent;

[0024] A1 is selected from 5-6 aryl, 5-6 heteroaryl, 5-6 saturated cycloalkyl, and 5-6 saturated heterocyclic groups;

[0025] Preferably, the 5-6 aryl group is phenyl, the 5-6 heteroaryl group is thiophene, and the 5-6 saturated heterocyclic group is selected from...

[0026] Furthermore, R 2 Selected from hydrogen, carboxyl, -Z3-OH, unsubstituted or surrounded by one or more R groups. 5 The following groups are substituted: 5-6 aryl, 5-6 heteroaryl, 5-6 saturated cycloalkyl, and 5-6 saturated heterocyclic groups;

[0027] R 5 Each was independently selected from C 1-3 Alkoxy, C 1-3 Alkyl, -NR 6 R 7 Halogens, Boc;

[0028] Z3 is selected from none, C 1-3 Alkylene;

[0029] R 6 R 7 Each is independently selected from hydrogen and C. 1-3 alkyl;

[0030] Preferably, the 5-6 aryl group is phenyl, and the 5-6 heteroaryl group is thiophene. The 5-6 member saturated heterocyclic group is

[0031] Furthermore, the On-DNA 2,5-disubstituted thiophene lead compound is selected from one of the following compounds:

[0032]

[0033] The present invention also provides a method for synthesizing the above-mentioned On-DNA 2,5-disubstituted thiophene lead compound, the method comprising the following steps:

[0034]

[0035] Using the compound shown in Formula Ia and the sulfide as raw materials, a reaction was carried out in a solvent to obtain the On-DNA 2,5-disubstituted thiophene lead compound shown in Formula I; DNA, L, R 1 R 2 As stated above.

[0036] Furthermore, the sulfide is sodium sulfide;

[0037] And / or, the equivalent ratio of the compound and the sulfide shown in Formula Ia is 1:(1-2000);

[0038] And / or, the reaction is carried out at a temperature of 0-90°C for a time of 0.5-24 hours;

[0039] And / or, the solvent is selected from water, methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2,2-difluoroethanol, 2,2,2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, ethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1,4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, ethylene glycol A solution containing one or more of the following: diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 4-methoxypyridine, toluene, xylene, inorganic salt buffer, and organic base buffer.

[0040] Furthermore, the equivalence ratio of the compound and the sulfide shown in Formula Ia is 1:(200-400), preferably 1:400;

[0041] And / or, the reaction temperature is 50-90°C, preferably 80°C, and the time is 0.5-6 hours, preferably 1 hour;

[0042] And / or, the solvent is a mixed solution of water and N,N-dimethylformamide; wherein the volume ratio of water to N,N-dimethylformamide in the mixed solution is preferably 3:(4-8).

[0043] Furthermore, the preparation method of the compound shown in Formula Ia includes the following steps:

[0044]

[0045] Compound Xa reacts with compound d to give the compound shown in formula Ia.

[0046] The present invention also provides the use of the above-described synthesis method in constructing gene-encoded compound libraries.

[0047] In this invention, the structure of DNA can be...

[0048]

[0049] The HP structure is as follows:

[0050] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0051] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a-b Alkyl groups refer to any alkyl group containing one to two carbon atoms ("a" to "b"). For example, C 1-6 Alkyl groups are straight-chain or branched alkyl groups containing 1 to 6 carbon atoms.

[0052] "Aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group with a conjugated π-electron system, such as phenyl and naphthyl. The aryl ring can be fused to other cyclic groups (including saturated and unsaturated rings), but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of attachment to the parent group must be on a carbon atom of a ring with a conjugated π-electron system. The aryl group can be substituted or unsubstituted.

[0053] "Heteroaryl" refers to a heteroaryl group containing one or more heteroatoms. Heteratoms include oxygen, sulfur, and nitrogen. Examples include furanyl, thiophene, pyridinyl, pyrazolyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0054] "Connecting units" include chemical bonds and groups that can act as connectors.

[0055] Halogens include fluorine, chlorine, bromine, and iodine.

[0056] “Boc” refers to tert-butyloxycarbonyl.

[0057] This invention discloses for the first time a method for synthesizing 2,5-disubstituted thiophene lead compounds on DNA. This method causes little damage to DNA, has good versatility, is simple to operate, operates under mild conditions, and can produce On-DNA 2,5-disubstituted thiophene compounds with high conversion rates.

[0058] As is well known in the art, DNA must remain stable under certain conditions, and reactions used for constructing DNA-encoded compound libraries require high conversion rates. Under the reaction conditions of this invention, the product, On-DNA 2,5-disubstituted thiophene compounds, not only exhibit high conversion rates but also good DNA integrity. The integrity of the On-DNA 2,5-disubstituted thiophene compounds obtained in this invention can be confirmed not only by liquid chromatography-mass spectrometry but also by further verification through DNase-catalyzed coupling reactions. This invention enriches the types of chemical reactions for synthesizing DNA-encoded compound libraries, providing a novel 2,5-disubstituted thiophene backbone for constructing gene-encoded compound libraries, and has excellent application prospects in lead drug development.

[0059] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0060] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0061] Figure 1 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S1.

[0062] Figure 2 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S2.

[0063] Figure 3 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S3.

[0064] Figure 4 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S4.

[0065] Figure 5 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S5.

[0066] Figure 6 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S6.

[0067] Figure 7 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S7.

[0068] Figure 8Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S8.

[0069] Figure 9 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S9.

[0070] Figure 10 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S10.

[0071] Figure 11 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S11.

[0072] Figure 12 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S12.

[0073] Figure 13 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S13.

[0074] Figure 14 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S14.

[0075] Figure 15 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S15.

[0076] Figure 16 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S16.

[0077] Figure 17 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S17.

[0078] Figure 18 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S18.

[0079] Figure 19 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S19.

[0080] Figure 20 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S20.

[0081] Figure 21 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S21.

[0082] Figure 22 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S22.

[0083] Figure 23 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S23.

[0084] Figure 24 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S24.

[0085] Figure 25 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S25.

[0086] Figure 26 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S26.

[0087] Figure 27 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S27.

[0088] Figure 28 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S28.

[0089] Figure 29 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S29.

[0090] Figure 30 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S30.

[0091] Figure 31 Liquid chromatography-mass spectrometry (LC-MS) spectral data of oligonucleotide-1,3-diyne compound S31.

[0092] Figure 32 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P1.

[0093] Figure 33 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P2.

[0094] Figure 34 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P3.

[0095] Figure 35 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P4.

[0096] Figure 36Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P5.

[0097] Figure 37 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P6.

[0098] Figure 38 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P7.

[0099] Figure 39 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P8.

[0100] Figure 40 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P9.

[0101] Figure 41 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P10.

[0102] Figure 42 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P11.

[0103] Figure 43 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P12.

[0104] Figure 44 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P13.

[0105] Figure 45 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P14.

[0106] Figure 46 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P15.

[0107] Figure 47 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P16.

[0108] Figure 48 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P17.

[0109] Figure 49 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P18.

[0110] Figure 50 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P19.

[0111] Figure 51 Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P20.

[0112] Figure 52 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P21.

[0113] Figure 53 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P22.

[0114] Figure 54 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P23.

[0115] Figure 55 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P24.

[0116] Figure 56 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P25.

[0117] Figure 57 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P26.

[0118] Figure 58 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P27.

[0119] Figure 59 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P28.

[0120] Figure 60 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P29.

[0121] Figure 61Liquid chromatography-mass spectrometry (LC-MS) chromatograms and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P30.

[0122] Figure 62 Liquid chromatography-mass spectrometry (LC-MS) chromatogram and conversion results of oligonucleotide-2,5-disubstituted thiophene compound P31.

[0123] Figure 63 : Liquid chromatography-mass spectrometry chromatogram of product P1-1 obtained by linking P1 with TagA. Detailed Implementation

[0124] Unless otherwise specified, the raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0125] The oligonucleotide-1,3-diyne used in this embodiment of the invention is one of S1-S31, and the structures of S1-S31 are as follows:

[0126]

[0127] The following are the synthesis methods for S1-S31:

[0128] 1. Synthetic raw material oligonucleotide-1,3-diyne compound (synthetic method refers to patent application CN 114957366 A)

[0129] (5.1) Synthesis of raw material oligonucleotide-1,3-diyne compound S1

[0130]

[0131] 20 nanomolar oligonucleotide-iodoalkynyl compound Y1 (synthetic method according to patent application CN 114106072A) was dissolved in deionized water to prepare a 0.7 mmol / L solution (20 μL, 20 nanomolar, 1 equivalent). 400 equivalents of alkyne B1 (400 mmol / L acetonitrile solution, 400 equivalents), 400 equivalents of tetrahydropyrrole (400 mmol / L acetonitrile solution, 400 equivalents), and 10 equivalents of cuprous iodide (20 mmol / L acetonitrile solution, 10 equivalents) were added to the above solution. The mixture was thoroughly mixed and reacted at 25°C for 48 hours. After the reaction was complete, 100 equivalents of a 100 mmol / L sodium diethyldithiocarbamate aqueous solution were added to the reaction mixture, and the reaction was carried out at room temperature for 10–30 minutes. Next, add 10% (5 mol / L) sodium chloride solution and 3 times (3 times) anhydrous ethanol to the above mixture. After shaking well, freeze the mixture at -80°C for 2 hours. Then, centrifuge at 4000 rpm for half an hour, discard the supernatant, and dry the remaining precipitate to obtain oligonucleotide-1,3-diyne compound S1. The liquid chromatography-mass spectrometry (LC-MS) chromatogram of S1 is shown below. Figure 1 As shown.

[0132] (5.2) Synthesis of oligonucleotide-1,3-diyne S2-S31

[0133] Following the synthetic method for oligonucleotide-1,3-diyne S1 described above, oligonucleotide-1,3-diyne S2-S31 were prepared (synthetic method referred to patent application CN 114957366 A). The liquid chromatography-mass spectra of 1,3-diyne compounds S2-S31 are shown below. Figure 2-31 As shown.

[0134] Example 1: Synthesis of oligonucleotide-2,5-disubstituted thiophene compound P1

[0135]

[0136] One nanomolar of oligonucleotide-1,3-diyne compound S1 was dissolved in deionized water to prepare a 1 mmol / L solution (1 μL, 1 nanomolar, 1 equivalent). 400 equivalents of sodium sulfide aqueous solution (0.5 μL, 800 mmol / L, 400 equivalents) and 2 μL of N,N-dimethylformamide were added to the above solution. The mixture was thoroughly mixed and reacted at 80°C for 1 hour. After the reaction was complete, 10% (total volume) of 5 mol / L sodium chloride aqueous solution and 3 times (total volume) of anhydrous ethanol were added sequentially to the above reaction solution. After shaking thoroughly, the reaction mixture was placed in a -80°C freezer for 2 hours. Then, the mixture was centrifuged at 4000 rpm for half an hour, the supernatant was discarded, and the remaining precipitate was dried to obtain a solution of oligonucleotide-2,5-disubstituted thiophene P1 with a molecular weight of 5461. Detection by liquid chromatography-mass spectrometry (LC-MS / MS) revealed the molecular weight of the corresponding product, indicating that oligonucleotide-1,3-diyne compound S1 can react with sodium sulfide to yield oligonucleotide-2,5-disubstituted thiophene P1. The LC-MS / MS chromatogram of oligonucleotide-2,5-disubstituted thiophene compound P1 is shown below. Figure 32 As shown.

[0137] This invention utilizes liquid chromatography-mass spectrometry (LC-MS) to accurately detect the conversion rate of the target product. The conversion rate was determined to be 100% using LC-MS.

[0138] Example 2: Synthesis of oligonucleotide-2,5-disubstituted thiophene compounds P2-P31

[0139] The synthesis method of Example 1 is used as a reference, except that oligonucleotide-1,3-diyne S1 is replaced with one of oligonucleotide-1,3-diyne S2-S31 to synthesize oligonucleotide-2,5-disubstituted thiophene compounds P2-P31 respectively.

[0140] P2-P31 was detected using liquid chromatography-mass spectrometry (LC-MS). The LC-MS chromatograms and conversion results of the oligonucleotide-2,5-disubstituted thiophene compound P2-P31 are shown below. Figure 33-62 As shown.

[0141] The following experimental examples demonstrate the beneficial effects of the present invention.

[0142] Experimental Example 1: Screening Experiment for Synthesis of Oligonucleotide-2,5-Disubstituted Thiophene Compounds

[0143] The method for synthesizing oligonucleotide-2,5-disubstituted thiophene compound P1 in Example 1 was followed, with the only difference being the control of the following parameters according to Table 1: equivalent amount of sodium sulfide, type of base, type and amount of organic solvent in the reaction co-solvent, reaction time, and temperature. The conversion rate of P1 under different parameters was calculated. The results are shown in Table 1.

[0144] Table 1. Conversion rates of oligonucleotide-2,5-disubstituted thiophene compound P1 synthesized under different conditions

[0145]

[0146] It can be seen that under reaction conditions 1 and 7, the conversion rate of the obtained oligonucleotide-2,5-disubstituted thiophene compounds is as low as 29%-30%; under reaction conditions 8-10, the conversion rate of the obtained oligonucleotide-2,5-disubstituted thiophene compounds is as high as over 85%; and under reaction condition 10, the conversion rate of the obtained oligonucleotide-2,5-disubstituted thiophene compounds is the highest, reaching 100%. This indicates that the method using the specific parameters of the embodiments of the present invention yields the oligonucleotide-2,5-disubstituted thiophene compounds with the highest product conversion rate.

[0147] Experimental Example 2: Verification of the integrity of oligonucleotides in the method for synthesizing oligonucleotide-2,5-disubstituted thiophene compounds of the present invention.

[0148] The integrity of the oligonucleotide was verified by linking the oligonucleotide-2,5-disubstituted thiophene compound P1 with Tag A (a short-chain oligonucleotide with molecular weights of 4063 and 5884, respectively):

[0149]

[0150] Procedure: Dissolve 10 nanomoles of P1 in deionized water to prepare a 1 mmol / L solution (1 μL, 1 nanomole, 1 equivalent). Add 1.2 equivalents of TagA (1 mmol / L aqueous solution, 1.2 equivalents), 5 μL of 10×T4 DNA ligation buffer, and 2 μL of T4 DNA ligase. Mix the solutions thoroughly and react at room temperature for 1 hour. After the reaction, add 10% (total volume) of 5 mmol / L sodium chloride solution and 3 times (total volume) of anhydrous ethanol to the reaction solution, shake well, and freeze at -80°C for 2 hours. Then centrifuge at 4000 rpm for half an hour and discard the supernatant. Dissolve the remaining precipitate in deionized water and analyze and confirm the molecular weight of the product using liquid chromatography-mass spectrometry (LC-MS). The LC-MS results of the reactant P1 and product P1-1 are shown below. Figure 32 and Figure 63 .

[0151] The results showed that the oligonucleotide-2,5-disubstituted thiophene compound P1 could successfully undergo a DNase-catalyzed coupling reaction with Tag A, indicating that the method for synthesizing compound P1 in this invention did not damage the structure of the oligonucleotide moiety. At the same time, liquid chromatography-mass spectrometry analysis showed that the reaction product had high purity and the mass spectrometry detection could accurately obtain experimental values ​​that matched the calculated values, further verifying the good integrity of the DNA.

[0152] In summary, this invention discloses for the first time a method for synthesizing 2,5-disubstituted thiophene lead compounds on DNA. This method causes minimal DNA damage, has good versatility, is simple to operate, operates under mild conditions, and can produce On-DNA 2,5-disubstituted thiophene compounds with high conversion rates. Under the reaction conditions of this invention, the On-DNA 2,5-disubstituted thiophene compounds not only have high conversion rates but also exhibit good DNA integrity. This invention enriches the types of chemical reactions for synthesizing DNA-encoded compound libraries, constructs new 2,5-disubstituted thiophene backbones for gene-encoded compound libraries, and has excellent application prospects in lead drug development.

Claims

1. A method for synthesizing On-DNA 2,5-disubstituted thiophene lead compounds, characterized in that: The method includes the following steps: Using the compound shown in Formula Ia and the sulfide as raw materials, the On-DNA 2,5-disubstituted thiophene lead compound shown in Formula I was obtained by reacting in a solvent. DNA is a modified or unmodified single-stranded or double-stranded nucleotide chain; L is selected from NHCO, CONH, or CO; When L is CO, R 1 Selected from , , ; When L is selected from NHCO or CONH, R 1 Selected from M1, A1, , ; M1 is selected from those that have not been replaced or have been replaced by one or more R. 3 Replacement C 1-3 Alkylene, -Z1-O-Z2-, R 3 Each was independently selected from C 1-3 Alkyl group, NHBoc, Z1 is selected from none, C 1-3 Alkylene, Z2 is selected from none, C 1-3 Alkylene, and Z1 and Z2 are not simultaneously absent; A1 is selected from phenyl and thiophene groups; R 2 Selected from hydrogen, carboxyl, -Z3-OH, unsubstituted or surrounded by one or more R groups. 5 The following groups are substituted: phenyl, thiophene, , , , , , , , ; R 5 Each was independently selected from C 1-3 Alkoxy, C 1-3 Alkyl, -NR 6 R 7 Halogens, Boc; Z3 is selected from none, C 1-3 Alkylene; R 6 R 7 Each is independently selected from hydrogen and C. 1-3 alkyl; The equivalence ratio of the compound and the sulfide shown in Formula Ia is 1:(200-400); The reaction was carried out at a temperature of 80 °C for 1-6 hours. The solvent is a mixed solution of water and N,N-dimethylformamide.

2. The method according to claim 1, characterized in that: The On-DNA 2,5-disubstituted thiophene lead compound is selected from one of the following compounds: 。 3. The method according to claim 1, characterized in that: The sulfide is sodium sulfide.

4. The method according to claim 1, characterized in that: The equivalence ratio of the compound and the sulfide shown in Formula Ia is 1:400; And / or, the reaction time is 1 hour; And / or, in the mixed solution of water and N,N-dimethylformamide, the volume ratio of water to N,N-dimethylformamide is 3:(4-8).

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