On-dna gamma-hydroxy amines and methods for their preparation

Abstract

The application provides an On-DNA-gamma-hydroxylamine compound and a preparation method thereof, and belongs to the field of gene coding compound library construction. The On-DNA-gamma-hydroxylamine compound is a compound shown in formula I. The method for synthesizing the On-DNA-gamma-hydroxylamine compound has small DNA damage, good universality, simple operation and mild conditions. The On-DNA-gamma-hydroxylamine compound has high yield, and the DNA integrity in the product is good. The application enriches the chemical reaction type for synthesizing the coding compound library on DNA, establishes a method for constructing a gene coding compound library with a new gamma-hydroxylamine skeleton, and has a very good application prospect in lead drug development.

Description

Technical Field

[0001] This invention belongs to the field of gene-encoded compound library construction, specifically relating to an On-DNA-γ-hydroxyamine compound and its preparation method. Background Technology

[0002] After many years of application, development, and improvement, high-throughput screening has established a highly automated and sophisticated screening process, along with improved quality and increased quantity of chemical libraries. It is a crucial pathway for leading international drug development companies to obtain lead compounds for target proteins. However, traditional high-throughput screening based on single molecule suffers from drawbacks such as limited compound library size, long screening cycles, and high costs, increasingly failing 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 methods, 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 binding 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 screening experiments under different conditions. DNA-encoded chemical libraries (DELs) have become a common method for discovering new protein ligands (1. Goodnow, RA; Dumerin, CE; Keefe, AD. DNA-encoded chemistry: Enabling the deeper sampling of chemical space. Nat. Rev. Drug Discov. 2016; 2. Neri, D.; Lerner, RA. DNA-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, yield, and the construction of larger chemical spaces through high-throughput screening, greatly increasing the number and diversity of compound libraries.

[0004] To further expand the chemical space of compound libraries, one of the most important tasks in gene-encoded compound library technology is the development of chemical reactions applicable to DNA (referred to as On-DNA chemical reactions). Several On-DNA chemical reactions have already been reported. For example, Shanghai WuXi AppTec New Drug Development Co., Ltd. (Chinese 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. (Chinese patent application No. CN201910590679.7) disclosed a method for synthesizing On-DNA arylbenzyl-substituted compounds. This method uses On-DNA aldehyde compounds as raw materials, reacts them with indole under alkaline conditions to generate On-DNA indole alcohol compounds, and then reduces the On-DNA indole alcohol compounds under acidic conditions with diethyl 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate to indole alkylated compounds. The more types of chemical reactions that can be realized on gene-encoded compound libraries, and the more diverse the conditions, the more choices are available for the design and synthesis of gene-encoded compound libraries, resulting in a more diverse library. However, the types of On-DNA chemical reactions currently reported are still limited and cannot yet meet the widespread needs for lead compound discovery.

[0005] Gamma-hydroxyamine compounds are compounds with a γ-hydroxyamine structure. Studies have found that γ-hydroxyamine compounds possess a variety of biological activities. For example, compound 1 developed by Bristol-Myers Squibb has therapeutic effects on arthritis (US2004063698), and compound 2 developed by the University of Bologna in Italy has therapeutic effects on Alzheimer's disease. Compound 3 developed by the Central Drug Research Institute (CDRI) in India has antithrombotic effects (Bioorg Med Chem 2004, 12(9):2059).

[0006]

[0007] Currently, the main method for synthesizing γ-hydroxyamine small molecule compounds is to react aldehydes and alkenes with DABCO as a base at room temperature for 30 minutes to obtain a Bayliss-Hillman adduct (DOI:10.1055 / s-2001-10815), followed by reaction with an amine in methanol solvent overnight to obtain the γ-hydroxyamine small molecule compound (DOI:10.1016 / j.bmc.2004.02.023). There are also reports of synthesizing γ-hydroxyamine small molecule compounds by reacting alkenyl alcohols and amines with a heavy metal catalyst, potassium phosphate as a base, isopropanol as a solvent, and under nitrogen protection (DOI:10.1002 / anie.202202972).

[0008] However, there are currently no reports of constructing γ-hydroxyamine gene-encoded compounds on DNA. This is because DNA must remain stable under specific conditions, including a certain proportion of aqueous phase, pH, temperature, metal ion concentration, and inorganic salt concentration. Furthermore, reactions used in the construction of DNA-encoded compound libraries require high yields for application in gene library synthesis. Therefore, developing a method for synthesizing On-DNA-γ-hydroxyamine compounds with minimal DNA damage and high yield is both challenging and significant. Summary of the Invention

[0009] The purpose of this invention is to provide an On-DNA-γ-hydroxyamine compound and its preparation method.

[0010] This invention provides On-DNA-γ-hydroxyamine compounds of Formula I:

[0011]

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

[0013] X is a connection unit;

[0014] L represents either not being replaced or being replaced by one or more R. L The following groups are substituted: 5-6 aryl, 5-6 heteroaryl, 3-8 saturated cycloalkyl, 3-8 saturated heterocyclic, C 1～6 Alkylene, C 2～6 imidene group, C 2～6 Ethyne group;

[0015] R L Each C is independently selected from halogenated, halogenated, or unhalogenated C 1～6 Alkyl, halogenated or unhalogenated C 1～6 Alkoxy, mercapto, carboxyl, ester, amide, cyano, or hydroxyl groups;

[0016] R aSelected from COOR a1 C 1～6 Alkyl, C 1～6 Alkyl group, cyano group;

[0017] R a1 Selected from C 1～6 alkyl;

[0018] R b Selected from hydrogen, unsubstituted or with one or more R... b1 The following genes are replaced: C1~ 12 Alkyl, C1-6 alkyloxy, 3-8 saturated cycloalkyl, 3-8 saturated heterocyclic, 5-6 aryl, 5-6 heteroaryl, benzyl;

[0019] R b1 Each is independently selected from halogen, ester group, cyano group, NR 4 R 5 Substituted or unsubstituted 5-6 aryl groups, substituted or unsubstituted 5-6 heteroaryl groups, halogenated or unhalogenated C1-6 alkoxy groups, halogenated or unhalogenated C1- 12 Alkyl groups; the substituents of the aryl and heteroaryl groups are selected from C1 to C2. 12 Alkyl groups, C1-6 alkyl oxygens, halogens, and cyano groups;

[0020] R c Selected from those that have not been replaced or have been replaced by one or more R c1 The following genes are replaced: C1~ 12 Alkyl, C1-6 alkyloxy, 3-8 saturated cycloalkyl, 3-8 saturated heterocyclic, 5-6 aryl, 5-6 heteroaryl, benzyl;

[0021] R c1 Each is independently selected from halogen, ester group, cyano group, NR 4 R 5 Substituted or unsubstituted 5-6 aryl groups, substituted or unsubstituted 5-6 heteroaryl groups, halogenated or unhalogenated C1-6 alkoxy groups, halogenated or unhalogenated C1- 12 Alkyl groups; the substituents of the aryl and heteroaryl groups are selected from C1 to C2. 12 Alkyl groups, C1-6 alkyl oxygens, halogens, and cyano groups;

[0022] R 4 Selected from hydrogen, C1~ 12 alkyl;

[0023] R 5 Selected from hydrogen, C1~ 12 alkyl;

[0024] Or R b R cConnected into unreplaced or by one or more R d1 Substituted 3- to 10-membered saturated heterocyclic groups and 5- to 10-membered heteroaryl groups;

[0025] R d1 Each is independently selected from substituted or unsubstituted C1~ 12 Alkyl, C1-6 alkoxy, halogen, 3-8 membered saturated cycloalkyl; the substituents of the alkyl group are selected from halogen, COOR d2 ;

[0026] R d2 Selected from C 1～6 alkyl.

[0027] Furthermore, the structure of the On-DNA-γ-hydroxyamine compound is shown in Formula I-1:

[0028]

[0029] DNA is a single-stranded or double-stranded nucleotide chain;

[0030] X is the connecting unit; X is selected from N(H)C(O)(CH2CH2O) m (CH2) n N(H)C(O); m is an integer selected from 1 to 4; n is an integer selected from 1 to 2;

[0031] R 0 Selected from hydrogen, halogens, C 1～3 Alkyl, C 1～3 Alkoxy;

[0032] R 1 Selected from the following genes: C1~, either unsubstituted or substituted with one or two substituents. 12 Alkyl, C1-6 alkyloxy, 3-8 saturated cycloalkyl, 3-8 saturated heterocyclic, 5-6 aryl, 5-6 heteroaryl; the substituents are selected from halogens, NR 4 R 5 Ester group, cyano group, substituted or unsubstituted 5-6 aryl group, substituted or unsubstituted 5-6 heteroaryl group, halogenated or unhalogenated C1-6 alkoxy group, halogenated or unhalogenated C1- 12 Alkyl; R 4 Selected from hydrogen, C1-6 alkyl, R 5 The substituents of the aryl and heteroaryl groups are selected from C1 to C6 alkyl groups. 12 Alkyl, C1-6 alkyl oxygen, halogen, cyano.

[0033] Furthermore, the structure of the On-DNA-γ-hydroxyamine compound is shown in Formula I-2:

[0034]

[0035] DNA is a single-stranded or double-stranded nucleotide chain;

[0036] X is the connecting unit; X is selected from N(H)C(O)(CH2CH2O) m (CH2) n N(H)C(O); m is an integer selected from 1 to 4; n is an integer selected from 1 to 2;

[0037] R 0 Selected from hydrogen, halogens, C 1～3 Alkyl, C 1～3 Alkoxy;

[0038] R 2 R 3 Each of the following genes, independently selected from those that are unsubstituted or substituted by one or two substituents: C1~ 12 Alkyl, benzyl; the substituents are selected from halogens, NR 4 R 5 Cyano, substituted or unsubstituted 5-6 aryl, halogenated or unhalogenated C1-6 alkoxy, halogenated or unhalogenated C1- 12 Alkyl; R 4 Selected from hydrogen, C1-6 alkyl, R 5 The aryl group is selected from hydrogen and C1-6 alkyl groups; the substituents of the aryl group are selected from C1-6 alkyl groups. 12 Alkyl groups, C1-6 alkyl oxygens, halogens, and cyano groups;

[0039] Or R 2 R 3 Connected into unreplaced or by one or more R d1 Substituted 3- to 10-membered saturated heterocyclic groups and 5- to 10-membered heteroaryl groups;

[0040] R d1 Each is independently selected from substituted or unsubstituted C1~ 12 Alkyl, C1-6 alkoxy, halogen, 3-8 membered saturated cycloalkyl; the substituents of the alkyl group are selected from halogen, COOR d2 ;

[0041] R d2 Selected from C 1～6 alkyl.

[0042] Furthermore, the On-DNA-γ-hydroxyamine compound is selected from one of the following compounds:

[0043]

[0044]

[0045] The present invention also provides a method for synthesizing the aforementioned On-DNA-γ-hydroxyamine compounds, the method comprising the following steps:

[0046] In a solvent, the compound shown in Formula II, the compound shown in Formula III, and a base are mixed and reacted. Then, the compound shown in Formula IV and a base are added, and the reaction is continued to obtain the On-DNA-γ-hydroxyamine compound shown in Formula I.

[0047]

[0048] Among them, X, L, R a R b and R c As shown above.

[0049] Furthermore, the compound represented by Formula II is one of the following compounds:

[0050]

[0051] The compound represented by Formula IV is one of the following compounds:

[0052]

[0053] Furthermore, the equivalent ratio of the compound shown in Formula II, the compound shown in Formula III, the base, and the compound shown in Formula IV is 1:200-500:400-1000:400-1000; the equivalent of the base is the sum of the two base equivalents; the equivalent of the base added each time is 50% of the total base equivalent;

[0054] The base is one or a mixture of two or more of the following: triethylenediamine, cesium carbonate, potassium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate, cesium hydroxide, potassium hydroxide, sodium hydroxide, lithium hydroxide, potassium phosphate, sodium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, cesium fluoride, potassium tert-butoxide, sodium tert-butoxide, triethylamine, n-butylamine, isobutylamine, 4-dimethylaminopyridine, pyridine, N,N-diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N,N',N'-tetramethylethylenediamine, 1,1,3,3-tetramethylguanidine, N,N-dicyclohexylmethylamine, dicyclohexylamine, tetrahydropyrrole, and boric acid buffer solution.

[0055] Furthermore, the equivalent ratio of the compound shown in Formula II, the compound shown in Formula III, the base, and the compound shown in Formula IV is 1:200:400:400; the equivalent of the base is the sum of the two base equivalents; the equivalent of the base added each time is 50% of the total base equivalent;

[0056] The base is triethylenediamine.

[0057] Further, the solvent for the reaction is 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, diethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1,4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol... Dimethyl alcohol ether, ethylene glycol 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, any one or a mixture of two or more thereof;

[0058] And / or, the reaction time after mixing the compound shown in Formula II, the compound shown in Formula III, and the base is 1 to 20 hours, and the reaction temperature is 0 to 90°C;

[0059] And / or, the reaction time after adding the compound of formula IV and the base is 1 to 10 hours, and the reaction temperature is 0 to 90°C.

[0060] Furthermore, the solvent for the reaction is a mixture of water and acetonitrile;

[0061] And / or, the reaction time after mixing the compound shown in Formula II, the compound shown in Formula III, and the base is 15 to 20 hours, and the reaction temperature is 4 to 70°C;

[0062] And / or, the reaction time after adding the compound of formula IV and the base is 1 to 5 hours, and the reaction temperature is 4 to 70°C.

[0063] Furthermore, in the mixture of water and acetonitrile, the volume percentage of acetonitrile is 83.3%.

[0064] And / or, the reaction time after mixing the compound shown in Formula II, the compound shown in Formula III, and the base is 16 hours, and the reaction temperature is 25-60°C;

[0065] And / or, the reaction time after adding the compound of formula IV and the base is 5 hours, and the reaction temperature is 25-60°C.

[0066] This invention also provides the application of the aforementioned On-DNA-γ-hydroxyamine compounds in gene-encoded compound libraries.

[0067] The compounds and derivatives provided in this invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0068] 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.

[0069] "Substitution" refers to the replacement of hydrogen atoms in a molecule by other different atoms or molecules.

[0070] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a ~ b Alkyl indicates any alkyl group containing "a" to "b" carbon atoms. Therefore, for example, "C 1～6 "alkyl" refers to an alkyl group containing 1-6 carbon atoms; "C" 1～6 "Alkoxy group" refers to an alkoxy group containing 1-6 carbon atoms.

[0071] "Alkyl" refers to a saturated hydrocarbon chain with a specified number of carbon atoms. For example, C1-6 alkyl refers to alkyl groups with 1 to 6 carbon atoms, i.e., 1, 2, 3, 4, 5, and 6 carbon atoms. Alkyl groups can be straight-chain or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl, etc.

[0072] "Alkenyl" refers to an aliphatic hydrocarbon group having at least one carbon-carbon double bond. Alkenyl groups can be straight-chain or branched.

[0073] "Alkyne" refers to an aliphatic hydrocarbon group having at least one carbon-carbon triple bond. The alkyne group can be straight-chain or branched.

[0074] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0075] The structure of the "ester group" is The structure of the amide group is Where R is C 1～6 alkyl.

[0076] "Saturated cycloalkyl" refers to saturated or unsaturated all-carbon monocyclic or polycyclic (including fused, spiro, or bridged rings) that do not possess a conjugated π-electron system, such as, but not limited to: 3- to 8-membered saturated cycloalkyl refers to saturated cycloalkyl groups that form a closed structure with 3 to 8 carbon atoms.

[0077] "Saturated heterocyclic group" refers to a cycloalkyl group in which at least one carbon atom on the ring is replaced by a heteroatom, which is O, N, or S, including but not limited to:

[0078] 3-8 membered saturated heterocyclic groups refer to saturated heterocyclic groups that form a closed structure with 3-8 atoms.

[0079] "Aryl" refers to an all-carbon monocyclic or polycyclic ring (including fused rings, spirocyclic or bridged rings) with a conjugated π-electron system, such as, but not limited to, phenyl, naphthyl, phenanthryl, anthracene, fluorenyl, and indene. The aromatic ring may be fused to other cyclic groups (including saturated and unsaturated rings), but cannot contain heteroatoms such as O, N, or S. Furthermore, the point of attachment to the parent group must be on a carbon atom of a ring with a conjugated π-electron system, such as, but not limited to, [other types of rings]. 5-6 aryl refers to an aryl group that has a closed structure consisting of 5-6 carbon atoms.

[0080] "Heteroaryl" refers to an aryl group in which at least one carbon atom on the ring of a conjugated π-electron system is replaced by a heteroatom, which is O, N, or S, including but not limited to:

[0081] 5-6 membered heteroaryl refers to a heteroaryl group that has a closed structure consisting of 5-6 atoms.

[0082] This invention discloses for the first time a method for synthesizing γ-hydroxyamine lead compounds on DNA. This method causes minimal damage to DNA, has good versatility, is simple to operate, operates under mild conditions, and can produce On-DNA-γ-hydroxyamine compounds in high yield. Furthermore, compared to existing methods for preparing small γ-hydroxyamine compounds that require two steps and heavy metal catalysts, this invention allows for the one-pot synthesis of On-DNA-γ-hydroxyamine compounds, avoiding cumbersome synthetic steps. Additionally, this invention does not require the use of heavy metal catalysts.

[0083] 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 yields. Under the reaction conditions of this invention, the resulting On-DNA-γ-hydroxyamine compounds not only have high yields but also exhibit good DNA integrity. The integrity of the On-DNA-γ-hydroxyamine 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, provides a novel γ-hydroxyamine backbone for constructing gene-encoded compound libraries, and has excellent application prospects in lead drug development.

[0084] 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.

[0085] 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

[0086] Figure 1 The structure of HP.

[0087] Figure 2 : Liquid chromatography-mass spectrometry detection chromatogram of S2.

[0088] Figure 3 : Liquid chromatography-mass spectrometry detection chromatogram of S3.

[0089] Figure 4 :S 4-1 The liquid chromatography-mass spectrometry detection chromatogram.

[0090] Figure 5 :S 4-2 The liquid chromatography-mass spectrometry detection chromatogram.

[0091] Figure 6 :S 4-3 The liquid chromatography-mass spectrometry detection chromatogram.

[0092] Figure 7 :S 4-4 The liquid chromatography-mass spectrometry detection chromatogram.

[0093] Figure 8 :S 4-5 The liquid chromatography-mass spectrometry detection chromatogram.

[0094] Figure 9 :S4-6 The liquid chromatography-mass spectrometry detection chromatogram.

[0095] Figure 10 :S 4-7 The liquid chromatography-mass spectrometry detection chromatogram.

[0096] Figure 11 :S 4-8 The liquid chromatography-mass spectrometry detection chromatogram.

[0097] Figure 12 : Liquid chromatography-mass spectrometry detection chromatogram of P1.

[0098] Figure 13 : Liquid chromatography-mass spectrometry detection chromatogram of P2.

[0099] Figure 14 : Liquid chromatography-mass spectrometry detection chromatogram of P3.

[0100] Figure 15 : Liquid chromatography-mass spectrometry detection chromatogram of P4.

[0101] Figure 16 : Liquid chromatography-mass spectrometry detection chromatogram of P5.

[0102] Figure 17 : Liquid chromatography-mass spectrometry detection chromatogram of P6.

[0103] Figure 18 : Liquid chromatography-mass spectrometry detection chromatogram of P7.

[0104] Figure 19 : Liquid chromatography-mass spectrometry detection chromatogram of P8.

[0105] Figure 20 : Liquid chromatography-mass spectrometry detection chromatogram of P9.

[0106] Figure 21 :P 10 The liquid chromatography-mass spectrometry detection chromatogram.

[0107] Figure 22 :P 11 The liquid chromatography-mass spectrometry detection chromatogram.

[0108] Figure 23 :P 12 The liquid chromatography-mass spectrometry detection chromatogram.

[0109] Figure 24 :P 13 The liquid chromatography-mass spectrometry detection chromatogram.

[0110] Figure 25 :P 14 The liquid chromatography-mass spectrometry detection chromatogram.

[0111] Figure 26 :P 15 The liquid chromatography-mass spectrometry detection chromatogram.

[0112] Figure 27 :P 16 The liquid chromatography-mass spectrometry detection chromatogram.

[0113] Figure 28 :P 17 The liquid chromatography-mass spectrometry detection chromatogram.

[0114] Figure 29 :P 18 The liquid chromatography-mass spectrometry detection chromatogram.

[0115] Figure 30 :P 19 The liquid chromatography-mass spectrometry detection chromatogram.

[0116] Figure 31 :P 20 The liquid chromatography-mass spectrometry detection chromatogram.

[0117] Figure 32 :P 21 The liquid chromatography-mass spectrometry detection chromatogram.

[0118] Figure 33 :P 22 The liquid chromatography-mass spectrometry detection chromatogram.

[0119] Figure 34 :P 23 The liquid chromatography-mass spectrometry detection chromatogram.

[0120] Figure 35 :P 24 The liquid chromatography-mass spectrometry detection chromatogram.

[0121] Figure 36 :P 25 The liquid chromatography-mass spectrometry detection chromatogram.

[0122] Figure 37 :P 26 The liquid chromatography-mass spectrometry detection chromatogram.

[0123] Figure 38 :P 27 The liquid chromatography-mass spectrometry detection chromatogram.

[0124] Figure 39 :P 28 The liquid chromatography-mass spectrometry detection chromatogram.

[0125] Figure 40 :P 29 The liquid chromatography-mass spectrometry detection chromatogram.

[0126] Figure 41:P 30 The liquid chromatography-mass spectrometry detection chromatogram.

[0127] Figure 42 :P 31 The liquid chromatography-mass spectrometry detection chromatogram.

[0128] Figure 43 :P 32 The liquid chromatography-mass spectrometry detection chromatogram.

[0129] Figure 44 : Spectrum of an experiment verifying the integrity of oligonucleotides in oligonucleotide-γ-hydroxyamine structural compounds.

[0130] Figure 45 Oligonucleotide-aldehyde compound S 4-1 -S 4-8 Summary of molecular weight and yield.

[0131] Figure 46 Oligonucleotide-γ-hydroxyamine structural compound P1–P 32 A summary of molecular weight and conversion rate. Detailed Implementation

[0132] Unless otherwise specified, the raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0133] The amine reagent used in the embodiments of this invention is one of the following compounds, all of which are commercially available products:

[0134]

[0135] The oligonucleotide-aldehyde compound used in the embodiments of the present invention has the structure as shown in S. 4-1 -S 4-8 As shown:

[0136]

[0137] The following is a synthetic method for preparing oligonucleotide-aldehyde compounds.

[0138] (1) Synthesize oligonucleotide-NHFmoc raw material (S2) according to the following reaction formula:

[0139]

[0140] 100.0 nanomolar HP (also known as S1, with a structure as follows) Figure 1As shown, a commercially available product was dissolved in deionized water to prepare a 1.0 mmol / L HP solution (100.0 μL, 1.0 equivalent). 40.0 equivalents of a DMSO solution (20.0 μL, 200.0 mmol / L) of the starting head fragment compound (commercially available product), 250.0 equivalents of a sodium tetraborate (Na₂B₄O₇) buffer solution at pH 9.5 (100.0 μL, 250.0 mmol / L), and 40.0 equivalents of an aqueous solution of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) (20.0 μL, 200.0 mmol / L) were mixed and thoroughly vortexed. This mixture was then added to the HP 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 rpm for half an hour and discard the supernatant. Dry the remaining precipitate to obtain a DNA-NHFmoc solution (named S2). Detect the spectrum of S2 using liquid chromatography-mass spectrometry, as shown below. Figure 2 As shown, its molecular weight is 5406.

[0141] (2) Synthesize oligonucleotide-NH2 raw material (S3) according to the following reaction formula:

[0142]

[0143] 100.0 nanomolars of S2 were dissolved in deionized water to prepare a 1.0 mmol / L (100.0 μL, 1.0 equivalent) solution. 56.0 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 total 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 rpm for half an hour, and the supernatant was discarded. The remaining precipitate was dried to obtain an oligonucleotide-NH2 solution (named S3, abbreviated as DNA-NH2). The chromatogram of S3 was detected using liquid chromatography-mass spectrometry, as shown below. Figure 3 As shown, its molecular weight is 5184.

[0144] (3) Synthesis of oligonucleotide-aldehyde compound raw materials

[0145] (3.1) Oligomeric nucleic acid-aldehyde compound raw materials (S 4-1 Synthesis of )

[0146]

[0147] Dissolve 10.0 nanomolars of S3 in deionized water to prepare a 1.0 mmol / L (10.0 μL, 1.0 equivalent) S3 solution. Mix 40.0 equivalents of a DMSO solution of 3-fluoro-4-carboxymethylbenzoic acid (commercially available product) (2.0 μL, 200.0 mmol / L), 250.0 equivalents of a sodium tetraborate (Na2B4O7) buffer solution at pH 9.5 (10.0 μL, 250.0 mmol / L), and 40.0 equivalents of an aqueous solution of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) (2.0 μL, 200.0 mmol / L), and thoroughly mix the mixture using a vortex mixer. Add the mixture to the S3 solution, mix thoroughly, and react at room temperature for 2 hours. After the reaction was complete, 10% (by total volume) of 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, it was centrifuged at 4000 rpm for half an hour, and the supernatant was discarded. The remaining precipitate was dried to obtain S. 4-1 S was detected using liquid chromatography-mass spectrometry. 4-1 The spectrum, such as Figure 4 As shown, its molecular weight is 5334 and the yield is 76%.

[0148] (3.2) Oligomeric nucleic acid-aldehyde compound raw materials (S) 4-2 -S 4-8 Synthesis of )

[0149] Referring to the above S 4-1 The synthetic method differs only in that 3-fluoro-4-formyl-benzoic acid is replaced with the corresponding starting material to obtain oligonucleotide-aldehyde compound S. 4-2 -S 4-8 The detection was performed using liquid chromatography-mass spectrometry (LC-MS), and the results are shown below. Figures 5-11 Oligonucleotides-aldehyde compounds S 4-2 -S 4-8 A summary of structures, molecular weights, and yields can be found in [link to relevant documentation]. Figure 45 .

[0150] The following is a method for synthesizing oligonucleotide-γ-hydroxyamine compounds.

[0151] Example 1: Using oligonucleotide-aldehyde compound raw materials (S) 4-1 Synthesize oligonucleotide-γ-hydroxyamine structural compounds (P1-P) 17 P 24 -P 32 )

[0152]

[0153] At 1.0 nanomolar oligonucleotide-aldehyde compound (S 4-1 DABCO (triethylenediamine, commercially available, 1000.0 mmol / L acetonitrile solution, 200.0 equivalence, 2.0 μL) and ethyl acrylate (commercially available, 1000.0 mmol / L acetonitrile solution, 200.0 equivalence, 2.0 μL) and cyclohexylamine (commercially available, 1000.0 mmol / L acetonitrile solution, 200.0 equivalence, 2.0 μL) were added to an aqueous solution of 5.0 mmol / L acetonitrile, 1.0 equivalence, 2.0 μL. The mixture was thoroughly mixed by vortexing and reacted at 25°C for 16 hours. Subsequently, DABCO (1000.0 mmol / L acetonitrile solution, 200.0 equivalence, 2.0 μL) and cyclohexylamine (commercially available, 1000.0 mmol / L acetonitrile solution, 400.0 equivalence, 4.0 μL) were added to the reaction system, mixed, and reacted at 25°C for 5 hours. After the reaction was complete, 10% (total volume) of 5.0 mol / L sodium chloride solution and 3 times the volume of anhydrous ethanol were added to the reaction solution. After shaking well, the solution was placed in a -80°C freezer for 2 hours, followed by high-speed refrigeration centrifugation (4°C, 12000 rpm, 15 minutes). The supernatant was discarded, and the remaining precipitate was the product oligonucleotide-γ-hydroxyamine compound (P1). The product was detected by liquid chromatography-mass spectrometry (LC-MS). The results are shown in the figure. Figure 12 The molecular weight is 5534, and the conversion rate is 96%.

[0154] The conversion rate is calculated as: product purity / raw material purity. P1 has a raw material purity of 76% and a product purity of 73%, resulting in a conversion rate of 96% for P1. The calculation method for other products is the same. When calculating product purity, products that have undergone ester hydrolysis are also included in the product purity calculation.

[0155] The synthesis method described above is the same, except that the raw materials are replaced accordingly to synthesize the oligonucleotide-γ-hydroxyamine compound P2-P. 17 P 24 -P 32 The results were obtained by liquid chromatography-mass spectrometry (LC-MS). Figures 13-28 , Figures 35-43 .

[0156] Example 2: Using oligonucleotide-aldehyde compound raw materials (S) 4-1 Synthesize oligonucleotide-γ-hydroxyamine structural compounds (P) 18 -P 23 )

[0157]

[0158] At 1.0 nanomolar oligonucleotide-aldehyde compound (S 4-1DABCO (triethylenediamine, commercially available, 1000.0 mmol / L acetonitrile solution, 200.0 equivalence, 2.0 μL) and ethyl acrylate (commercially available, 1000.0 mmol / L acetonitrile solution, 200.0 equivalence, 2.0 μL) and ethyl acrylate (commercially available, 1000.0 mmol / L acetonitrile solution, 200.0 equivalence, 2.0 μL) were added to an aqueous solution of 5.0 mmol / L acetonitrile, 200.0 equivalence, 2.0 μL. The mixture was thoroughly mixed by vortexing and reacted at 25°C for 16 hours. Subsequently, DABCO (1000.0 mmol / L acetonitrile solution, 200.0 equivalence, 2.0 μL) and 3,5-difluorobenzylamine (commercially available, 1000.0 mmol / L acetonitrile solution, 400.0 equivalence, 4.0 μL) were added to the reaction system, mixed, and reacted at 60°C for 5 hours. After the reaction was complete, 10% (total volume) of 5.0 mol / L sodium chloride solution and 3 times the volume of anhydrous ethanol were added to the reaction solution. After shaking well, the solution was placed in a freezer at -80°C for 2 hours, and then centrifuged at high speed (4°C, 12000 rpm, 15 minutes). The supernatant was discarded, and the remaining precipitate was the product oligonucleotide-γ-hydroxyamine compound (P). 18 The results were obtained by liquid chromatography-mass spectrometry (LC-MS / MS) and are shown in the figure below. Figure 29 The molecular weight is 5577, and the conversion rate is 79%.

[0159] The synthesis method described above is the same, except that the raw materials are replaced accordingly to synthesize the oligonucleotide-γ-hydroxyamine compound P. 19 -P 23 The results were obtained by liquid chromatography-mass spectrometry (LC-MS) and are shown in the figure below. Figures 30-34 .

[0160] The oligonucleotide-γ-hydroxyamine compound P1-P synthesized in this invention 32 A summary of structures, molecular weights, and yields can be found in [link to relevant documentation]. Figure 46 .

[0161] The following experimental examples demonstrate the beneficial effects of the present invention.

[0162] Experimental Example 1: Verification Experiment of the Integrity of Oligonucleotides in the Oligonucleotide-γ-hydroxyamine Structural Compounds of the Present Invention

[0163] The integrity of the oligonucleotide was verified by linking the oligonucleotide compound P1 (a short-chain oligonucleotide with molecular weights of 4064 and 5884) with Tag A (a short-chain oligonucleotide with molecular weights of 4064 and 5884, respectively).

[0164]

[0165] 1.0 nanomolar P1 was dissolved in deionized water to prepare a 1.0 mmol / L solution (1.0 μL, 1.0 equivalent). 1.2 equivalents of Tag A (1 mmol / L aqueous solution, 1.2 μL), 1.0 μL of 10×T4 DNA ligation buffer, and 0.5 μL of T4 DNA ligase were added. The solution was then mixed thoroughly and reacted at room temperature for 1 hour. After the reaction, 10% of the total volume of 5.0 mol / L sodium chloride solution was added to the reaction solution, followed by 3 times the total volume of anhydrous ethanol. After shaking thoroughly, the reaction solution was frozen at -80°C for 2 hours. The solution was then centrifuged at 4000 rpm for half an hour, and the supernatant was discarded. The remaining precipitate was dissolved in deionized water, and the molecular weight of the product TagA-P1 was confirmed by liquid chromatography-mass spectrometry (LC-MS / MS) to be 15446. The mass spectrometry results are shown below. Figure 44 .

[0166] LCMS analysis showed that the oligonucleotide-γ-hydroxyamine compound P1 could be successfully coupled with Tag A. Other oligonucleotide-γ-hydroxyamine compounds of this invention could also be successfully coupled with Tag A. This indicates that the oligonucleotide-γ-hydroxyamine compounds obtained according to the synthesis method of this invention have good DNA chain integrity. Further LCMS mass spectrometry accurately displayed their molecular weight, further confirming that their nucleotides were not damaged. Experimental results show that the reaction method in this invention does not damage the basic structure and activity of DNA, and the DNA chain integrity is good.

[0167] Experiment Example 2: Screening Experiment for Synthesis of Oligonucleotide-γ-hydroxyamine Compounds

[0168]

[0169] P was prepared according to the method described in Example 1. 24 The preparation process only changed the type and amount of alkali, the amount of cyclohexylamine, the reaction temperature, and the reaction time in the second step (specific changes are shown in Table 1). The calculated product P... 24 The conversion rates are shown in Table 1.

[0170] Table 1. Preparation of oligonucleotide-γ-hydroxyamine structural compound P 24 Conditions and conversion rate

[0171]

[0172] As shown in Table 1, the highest conversion rate was achieved in the second step of the preparation process when the base was DABCO, the base equivalent was 200, the cyclohexylamine equivalent was 400, the reaction temperature was 25℃, and the reaction time was 5 hours.

[0173] In summary, this invention discloses for the first time a method for synthesizing γ-hydroxyamine lead compounds on DNA. This method causes minimal DNA damage, has good versatility, is simple to operate, and operates under mild conditions. The obtained On-DNA-γ-hydroxyamine compounds not only have high yields but also exhibit good DNA integrity. This invention enriches the types of chemical reactions for synthesizing DNA-encoded compound libraries, constructs novel γ-hydroxyamine backbones for gene-encoded compound libraries, and has excellent application prospects in lead drug development.

Claims

1. A method for synthesizing the On-DNA-γ-hydroxyamine compound shown in Formula I, characterized in that: The method includes the following steps: In a solvent, the compound shown in Formula II, the compound shown in Formula III, and a base are mixed and reacted. Then, the compound shown in Formula IV and a base are added, and the reaction continues to yield the On-DNA-γ-hydroxyamine compound shown in Formula I. The base in the mixture of the compound shown in Formula II, the compound shown in Formula III, and the base is triethylenediamine. The base in the addition of the compound shown in Formula IV and the base is selected from borate buffer solution with pH 9.5, N,N-diisopropylethylamine, or triethylenediamine. DNA is a single-stranded or double-stranded nucleotide chain; X is a connection unit; L represents either not being replaced or being replaced by one or more R. L The following groups are substituted: 5-6 aryl, 5-6 heteroaryl, 3-8 saturated cycloalkyl, 3-8 saturated heterocyclic, C 1~6 Alkylene, C 2~6 imidene group, C 2~6 Ethyne group; R L Each C is independently selected from halogenated, halogenated, or unhalogenated C 1~6 Alkyl, halogenated or unhalogenated C 1~6 Alkoxy, mercapto, carboxyl, ester, amide, cyano, or hydroxyl groups; R a Selected from COOR a1 C 1~6 Alkyl, C 1~6 Alkyl group, cyano group; R a1 Selected from C 1~6 alkyl; R b Selected from hydrogen, unsubstituted or with one or more R... b1 The following genes are replaced: C1~ 12 Alkyl, C1-6 alkyloxy, 3-8 saturated cycloalkyl, 3-8 saturated heterocyclic, 5-6 aryl, 5-6 heteroaryl, benzyl; R b1 Each is independently selected from halogen, ester group, cyano group, NR 4 R 5 Substituted or unsubstituted 5-6 aryl groups, substituted or unsubstituted 5-6 heteroaryl groups, halogenated or unhalogenated C1-6 alkoxy groups, halogenated or unhalogenated C1-6 alkoxy groups 12 Alkyl groups; the substituents of the aryl and heteroaryl groups are selected from C1~ 12 Alkyl, C1-6 alkyl oxygen, halogen, cyano; R c Selected from those that have not been replaced or have been replaced by one or more R c1 The following genes are replaced: C1~ 12 Alkyl, C1-6 alkyloxy, 3-8 saturated cycloalkyl, 3-8 saturated heterocyclic, 5-6 aryl, 5-6 heteroaryl, benzyl; R c1 Each is independently selected from halogen, ester group, cyano group, NR 4 R 5 Substituted or unsubstituted 5-6 aryl groups, substituted or unsubstituted 5-6 heteroaryl groups, halogenated or unhalogenated C1-6 alkoxy groups, halogenated or unhalogenated C1-6 alkoxy groups 12 Alkyl groups; the substituents of the aryl and heteroaryl groups are selected from C1~ 12 Alkyl, C1-6 alkyl oxygen, halogen, cyano; R 4 Selected from hydrogen, C1~ 12 alkyl; R 5 Selected from hydrogen, C1~ 12 alkyl; Or R b R c Connected into unreplaced or by one or more R d1 Substituted 3-10 saturated heterocyclic groups and 5-10 heteroaryl groups; R d1 Each is independently selected from substituted or unsubstituted C1~ 12 Alkyl, C1-6 alkoxy, halogen, 3-8 membered saturated cycloalkyl; the substituents of the alkyl group are selected from halogen, COOR d2 ; R d2 Selected from C 1~6 alkyl; The structure of the On-DNA-γ-hydroxyamine compound is shown in Formula I-1: Formula I-1 DNA is a single-stranded or double-stranded nucleotide chain; X is the connecting unit; X is selected from N(H)C(O)(CH2CH2O) m (CH2) n N(H)C(O); m is an integer selected from 1 to 4; n is an integer selected from 1 to 2. R 0 Selected from hydrogen, halogens, C 1~3 Alkyl, C 1~3 Alkoxy; R 1 Selected from the following genes that are either unsubstituted or substituted with one or two substituents: C1~ 12 Alkyl, C1-6 alkyloxy, 3-8 saturated cycloalkyl, 3-8 saturated heterocyclic, 5-6 aryl, 5-6 heteroaryl; the substituents are selected from halogens, NR 4 R 5 Ester group, cyano group, substituted or unsubstituted 5-6 aryl group, substituted or unsubstituted 5-6 heteroaryl group, halogenated or unhalogenated C1-6 alkoxy group, halogenated or unhalogenated C1-6 alkoxy group 12 Alkyl; R 4 Selected from hydrogen, C1~6 alkyl, R 5 The substituents of the aryl and heteroaryl groups are selected from C1 to C6 alkyl groups. 12 Alkyl, C1-6 alkyl oxygen, halogen, cyano; Alternatively, the structure of the On-DNA-γ-hydroxyamine compound is shown in Formula I-2: Formula I-2 DNA is a single-stranded or double-stranded nucleotide chain; X is the connecting unit; X is selected from N(H)C(O)(CH2CH2O) m (CH2) n N(H)C(O); m is an integer selected from 1 to 4; n is an integer selected from 1 to 2. R 0 Selected from hydrogen, halogens, C 1~3 Alkyl, C 1~3 Alkoxy; R 2 R 3 Each of the following genes, independently selected from those that are unsubstituted or substituted with one or two substituents: C1~ 12 Alkyl, benzyl; the substituents are selected from halogens, NR 4 R 5 Cyano, substituted or unsubstituted 5-6 aryl, halogenated or unhalogenated C1-6 alkoxy, halogenated or unhalogenated C1- 12 Alkyl; R 4 Selected from hydrogen, C1~6 alkyl, R 5 The aryl group is selected from hydrogen and C1-6 alkyl groups; the substituents of the aryl group are selected from C1-6 alkyl groups. 12 Alkyl, C1-6 alkyl oxygen, halogen, cyano; Or R 2 R 3 Connected into unreplaced or by one or more R d1 Substituted 3-10 saturated heterocyclic groups and 5-10 heteroaryl groups; R d1 Each is independently selected from substituted or unsubstituted C1~ 12 Alkyl, C1-6 alkoxy, halogen, 3-8 membered saturated cycloalkyl; the substituents of the alkyl group are selected from halogen, COOR d2 ; R d2 Selected from C 1~6 alkyl.

2. The method according to claim 1, characterized in that: The On-DNA-γ-hydroxyamine compound is selected from one of the following compounds: 。 3. The method according to claim 1, characterized in that: The compound represented by Formula II is one of the following compounds: ； The compound represented by Formula IV is one of the following compounds: 。 4. The method according to claim 1, characterized in that: The equivalent ratio of the compound shown in Formula II, the compound shown in Formula III, the base, and the compound shown in Formula IV is 1:200~500:400~1000:400~1000; the equivalent of the base is the sum of the two base equivalents; the equivalent of the base added each time is 50% of the total base equivalent.

5. The method according to claim 4, characterized in that: The equivalent ratio of the compound shown in Formula II, the compound shown in Formula III, the base, and the compound shown in Formula IV is 1:200:400:400; the equivalent of the base is the sum of the two base equivalents; the equivalent of the base added each time is 50% of the total base equivalent.

6. The method according to claim 1, characterized in that: The solvents used in the reaction are 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, diethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1,4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, and ethylene glycol dimethyl ether. Dimethyl ether, ethylene glycol 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, any one or a mixture of two or more thereof; And / or, the reaction time after mixing the compound shown in Formula II, the compound shown in Formula III, and the base is 1 to 20 hours, and the reaction temperature is 0 to 90°C; And / or, the reaction time after adding the compound of formula IV and the base is 1 to 10 hours, and the reaction temperature is 0 to 90°C.

7. The method according to claim 6, characterized in that: The solvent for the reaction is a mixture of water and acetonitrile; And / or, the reaction time after mixing the compound shown in Formula II, the compound shown in Formula III, and the base is 15 to 20 hours, and the reaction temperature is 4 to 70°C; And / or, the reaction time after adding the compound of formula IV and the base is 1 to 5 hours, and the reaction temperature is 4 to 70°C.

8. The method according to claim 7, characterized in that: In the mixture of water and acetonitrile, acetonitrile accounts for 83.3% by volume. And / or, the reaction time after mixing the compound shown in Formula II, the compound shown in Formula III, and the base is 16 hours, and the reaction temperature is 25 ~ 60°C; And / or, the reaction time after adding the compound of formula IV and the base is 5 hours, and the reaction temperature is 25~60℃.

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