A method for constructing 2,4-imidazolidinedione compounds by DNA-encoded compound library

Abstract

The application relates to a synthesis method for constructing 2,4-imidazolidinedione compounds in a DNA coding compound library. The method takes 2-amino-N-acetamide compounds of On-DNA and p-nitrophenyl chloroformate as raw materials, reacts under the action of alkali, and obtains 2,4-imidazolidinedione compounds. The method has the advantages of mild reaction condition, simple operation, good substrate universality, good product yield and suitability for DNA coding compound synthesis in a multi-well plate.

Description

Technical Field

[0001] This invention belongs to the field of encoded compound library technology, specifically relating to a method for constructing On-DNA 2,4-imidazolidinedione compounds from a DNA-encoded compound library. Background Technology

[0002] Drug development is a lengthy process, and establishing a large compound library is essential for its advancement. However, the identification of compound structures still hinders the development of this method. To address this challenge, Sydney Brenner (winner of the 2002 Nobel Prize in Physiology or Medicine) and Richard Lerner (then director of the Scripps Research Institute) proposed the concept of DNA-encoded compound libraries at the Scripps Research Institute (Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5381). This technology utilizes the relative stability, measurability, and specificity of DNA to rapidly and accurately identify the active chemical structures of compounds linked to specific DNA fragments in new drug screening.

[0003] DNA-encoded compound libraries enable the rapid generation of giant compound libraries through combinatorial chemistry, and allow for high-throughput screening of lead compounds, making lead compound screening faster and more efficient than ever before. One of the challenges in constructing DNA-encoded compound libraries is the need for high-yield synthesis of chemically diverse small molecules on DNA. Since DNA requires specific conditions (solvent, pH, temperature, ion concentration) to maintain stability, and the On-DNA reactions used in the construction of DNA-encoded compound libraries also need to achieve high yields, the types of reagents, reaction types, and reaction conditions of chemical reactions performed on DNA (referred to as On-DNA reactions) directly affect the richness and selectivity of DNA-encoded compound libraries. Therefore, developing DNA-compatible chemical reactions has become a long-term exploration and research direction for DNA-encoded compound library technology, directly impacting the application and commercial value of DNA-encoded compound libraries. Currently, reactions that have been successfully applied to the construction of DNA-encoded compound libraries include: reductive amination, Suzuki reaction, nucleophilic substitution, acid-amine condensation, cycloaddition, and cross-coupling (Chem.Sci.2019 10,10481-10492; Annu.Rev.Biochem.2018,87,479).

[0004] 2,4-Imidazolidinones are intermediate structures in most analgesic drugs (J.Med.Chem.2011,54,7350-7362) and also an important intermediate structure for drugs treating other diseases (Bioorganic & Medicinal Chemistry27(2019) 2220-2227). Therefore, the synthesis of 2,4-imidazolidinediones is a crucial step in the development of most new drugs. This study aims to develop a novel on-DNA method for synthesizing 2,4-imidazolidinediones suitable for high-volume multi-well plate operations, thereby increasing the diversity of DNA-encoded compound libraries and further enhancing the application value of DNA-encoded compound library technology. Summary of the Invention

[0005] This invention provides a method for constructing 2,4-imidazolidinedione compounds from a DNA-encoded compound library. The method uses 2-amino-N-acetamide compounds from On-DNA and p-nitrophenyl chloroformate as raw materials, reacting them under alkaline conditions to obtain the 2,4-imidazolidinedione compounds. The structural formula of the 2-amino-N-acetamide compounds from the On-DNA is as follows: ;

[0006] Among them, R 1 Selected from groups with a molecular weight of less than 1000 that are directly linked to DNA; R 2 Selected from groups with a molecular weight of less than 1000 that are directly bonded to an amide nitrogen atom;

[0007] The DNA is 10-200 bp in length.

[0008] The DNA in the structural formula comprises single-stranded or double-stranded nucleotide chains obtained by polymerizing artificially modified and / or unmodified nucleotide monomers, which are bonded to R by one or more chemical bonds or groups. 1 Connected. When there is one chemical bond, it refers to the connection between DNA and R in the structural formula. 1 Directly connected; when there are multiple chemical bonds, it refers to the DNA and R in the structural formula. 1 They are connected by multiple chemical bonds, for example, DNA and R. 1 The amino groups of DNA are linked by a methylene group (-CH2-), i.e., they are connected by two chemical bonds; or DNA and R... 1 The amino group of DNA is linked by a carbonyl group (-CO-), which is also a connection between two chemical bonds; or DNA and R... 1 The amino group of DNA is linked via a methylene carbonyl group (-CH2CO-), or through three consecutive chemical bonds. Preferably, DNA is linked to R... 1 The amino groups of DNA are linked by a carbonyl group (-CO-).

[0009] Furthermore, the R 1 Selected from -C 1~6 Alkylene-.

[0010] Furthermore, the R 1 Selected from .

[0011] Furthermore, the R 2 Selected from -C 1~6 Alkyl, -C 0~4 Alkylene-OR a C 0~4 Alkylene-S(O)2NR a R b -C 0~4 Alkylene rings (3- to 10-membered carbon rings), -C 0~4 Alkylene (3- to 10-membered heterocycle), -C 0~4 Alkylene-(5-10 aryl), -C 0~4 Alkylene (5-10-membered heteroaryl); wherein the alkyl, alkylene, carbocyclic, heterocyclic, aryl, or heteroaryl group may be optionally surrounded by one, two, three, or four independent R groups. c replace;

[0012] R a R b Selected independently from hydrogen and -C 1-6 Alkyl, halogen-substituted -C 1~6 Alkyl groups, 3-10 membered carbon rings, 3-10 membered heterocycles;

[0013] R c Independently selected from halogen, cyano, nitro, -C 1-6 Alkyl, halogen-substituted -C 1~6 Alkyl, -C 1~6 Alkyl-OC 1~6 Alkyl, 3-10 membered carbon ring, 3-10 membered heterocyclic ring, 6-12 membered aryl, 5-12 membered heteroaryl.

[0014] Furthermore, the R 2 Selected from -C 1~6 Alkyl, -C 0~4 Alkylene-OR a -S(O) )2 NR a R b -C 0~1 Alkylene rings (3-6 membered carbon rings), -C 0~1 Alkylene-6-membered heterocycle, -C 0~1 Alkylene-7-membered heterocycle, -C 0~1 alkylene-8-membered heterocycle, -C0~1 Alkylene-9-membered heterocycle, -C 0~1 Alkylene-10-membered heterocycle, -C 0~1 Alkylene-(5-10 aryl), -C 0~3 Alkylene (5-10-membered heteroaryl);

[0015] R c Independently selected from halogen, cyano, nitro, -C 1-6 Alkyl, halogen-substituted -C 1~6 Alkyl, -C 1~6 Alkyl-OC 1~6 Alkyl, 3-6 membered carbon ring, 3-6 membered heterocyclic ring, 6 membered aryl, 6 membered heteroaryl.

[0016] More specifically, the R 2 Selected from , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

[0017] The present invention provides a method for constructing 2,4-imidazolidinedione compounds from a DNA-encoded compound library, comprising the following steps:

[0018]

[0019] Step 1: Dissolve the 2-amino-N-acetamide compound of On-DNA in 0.25 mM borate buffer;

[0020] Step 2: Dissolve the system obtained in Step 1 in an organic solvent, add alkali and p-nitrophenyl chloroformate, and react at 25~80℃ for 4~16h;

[0021] Step 3: The system obtained in Step 2 was subjected to alcohol precipitation, and then the product was centrifuged to obtain the 2,4-imidazolidinedione compound of On-DNA of formula (II).

[0022] Further, in step 1, the molar concentration of the 2-amino-N-acetamide compound of On-DNA is 0.5~2mM, and the molar equivalent is 1; in step 2, the molar equivalent of the base is 5~30; and in step 2, the molar equivalent of p-nitrophenyl chloroformate is 5~30.

[0023] Furthermore, the molar concentration of the 2-amino-N-acetamide compound of On-DNA in step 1 is 0.5 mM, 1 mM, 1.5 mM, or 2 mM; preferably, the molar concentration of the 2-amino-N-acetamide compound of On-DNA in step 1 is 1 mM; the molar equivalent of the base in step 2 is 5, 10, 15, 20, 25, or 30; preferably, the molar equivalent of the base in step 2 is 10; the molar equivalent of p-nitrophenyl chloroformate in step 2 is 5, 10, 15, 20, 25, or 30; preferably, the molar equivalent of p-nitrophenyl chloroformate in step 2 is 25.

[0024] Further, the reaction temperature of step 2 is: 25℃, 40℃, 50℃, 60℃, 65℃, 70℃, 75℃, 80℃; preferably, the reaction temperature of step 2 is 75℃.

[0025] Further, the reaction time of step 2 is 2h, 4h, 6h, 8h, or 16h; preferably, the reaction time of step 2 is 16h.

[0026] Further, the base in step 2 is selected from one or more of the following: triethylamine, 4-dimethylaminopyridine, 1,8-diazabicycloundec-7-ene, N,N-diisopropylethylamine, cesium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, disodium hydrogen phosphate, and sodium phosphate; preferably, the base in step 2 is triethylamine.

[0027] Further, the organic solvent in step 2 is selected from one or more of 1,4-dioxane, acetone, acetonitrile, tetrahydrofuran, petroleum ether, dichloromethane, toluene, ethyl acetate, and diethyl ether; preferably, the organic solvent in step 2 is acetonitrile.

[0028] Furthermore, the method is used for batch operations of perforated plates.

[0029] Furthermore, the method is used for the synthesis of DNA-encoded compound libraries for multi-well plates.

[0030] This invention involves no catalyst, operates under mild conditions, is easy to operate, has good substrate versatility, and yields high products, providing a scientific basis for the rational construction and application of DNA-encoded compound libraries.

[0031] 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.

[0032] "Substitution" refers to the replacement of hydrogen atoms in a molecule by other different atoms or molecules.

[0033] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix (C a~b Alkyl groups indicate any alkyl group containing one to two carbon atoms ("a" to "b"). Therefore, for example, C 1～12 Alkyl groups are straight-chain or branched alkyl groups containing 1 to 12 carbon atoms.

[0034] "alkyl" refers to a straight-chain or branched hydrocarbon group in an alkane molecule, such as methyl-CH3, ethyl-CH2CH3, or methylene-CH2-; the alkyl group can also be part of other groups, such as C 1~6 Alkoxy, C 1~6 Alkylamino.

[0035] "Alkylene": refers to a saturated straight-chain or branched non-bridging divalent alkyl group containing 1-20 carbon atoms.

[0036] "Halogen": refers to fluorine, chlorine, bromine or iodine.

[0037] "Carbon ring": refers to a saturated or partially saturated cyclic group that has multiple carbon atoms and no heterocyclic atoms, and has a single ring or multiple rings (including fused, bridged and spirocyclic systems).

[0038] "Heterocyclic": is a saturated or partially saturated cyclic group carrying at least 3 to 8 saturated or unsaturated monocyclic or polycyclic hydrocarbon groups (including fused, bridged and spirocyclic systems) selected from O, S and N.

[0039] "Aryl": refers to an aromatic single or multiple cyclic group composed of carbon atoms without heteroatoms.

[0040] "Heteroary aryl" refers to a single or multiple cyclic group composed of 5 to 10 C, O, S, N atoms, etc., which has aromatic properties.

[0041] 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.

[0042] 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

[0043] Figure 1: Single-step yield distribution of the 22 2,4-imidazolidinedione compounds prepared in Example 10. Detailed Implementation Plan

[0044] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0045] The reagents used in this invention are abbreviated as follows: ACN: Acetonitrile. DMSO: Dimethyl sulfoxide. DMAP: 4-Dimethylaminopyridine. DBU: 1,8-Dazabicycloundec-7-ene. DIPEA: N,N-Diisopropylethylamine. TEA: Triethylamine.

[0046] In this invention, DNA-NH2 refers to a DNA structure with a -NH2 linker formed by single-stranded or double-stranded DNA and a linker group, such as the DNA-NH2 structure of "compound1" in WO2005058479. Other examples include the following DNA structures:

[0047]

[0048] Where A is adenine, T is thymine, C is cytosine, and G is guanine.

[0049] The present invention provides a method for constructing 2,4-imidazolidinedione compounds from a DNA-encoded compound library, comprising the following steps:

[0050] Step 1: Synthesis of DNA-encoded compounds.

[0051]

[0052] DNA-COOH (2 µmol), amine (600 µmol) were dissolved in 2 mL of ACN:H₂O (1:1), N,N-diisopropyldiimide (1.8 M) was dissolved in 2 mL of DMSO, and N-hydroxy-7-azabenzotriazole (1.8 M) was dissolved in 2 mL of DMSO. The mixture was then incubated at 25 °C for 16 h. After the reaction was complete, sodium chloride (5 M, 600 µL) and ethanol (20 mL) were added to precipitate the DNA encoding compound (I). The structure of the compound was determined by LC-MS.

[0053] Step 2: DNA-encoded compound formula (Ⅰ) undergoes intramolecular cyclization under catalyst-free conditions to generate DNA-encoded compound formula (Ⅱ).

[0054]

[0055] The DNA-encoded compound (formula (I), 10 nmol) was dissolved in 10 mL of 0.25 mM BBS buffer, and a base (50 mM) and p-nitrophenyl chloroformate (50 mM) dissolved in the solvent were added. The mixture was reacted at 75 °C for 16 h. After the reaction was completed, ethanol and sodium chloride were added to precipitate the DNA, resulting in the DNA-encoded compound (II).

[0056] Example 1:

[0057] Step 1: Synthesis of DNA-encoded compound (I-a).

[0058]

[0059] DNA-COOH (2 µmol), p-toluidine (600 µmol) were dissolved in 2 mL ACN:H2O = 1:1, N,N-diisopropyldiimide (1.8 M) was dissolved in 2 mL DMSO, and N-hydroxy-7-azabenzotriazole (1.8 M) was dissolved in 2 mL DMSO. The mixture was placed together and reacted at 25 °C for 16 h. After the reaction was completed, sodium chloride (5 M, 600 µL) and ethanol (20 mL) were added to precipitate the DNA-encoded compound (I-a). The structure of the compound was determined by LCMS, with a single-step yield of 95%.

[0060] Step 2: DNA-encoded compound formula (Ⅰ-a) undergoes intramolecular cyclization under catalyst-free conditions to generate DNA-encoded compound formula (Ⅱ-a).

[0061]

[0062] The DNA-encoded compound (Ⅰ-a, 10 nmol) was dissolved in 10 mL of 0.25 mM BBS buffer, and triethylamine (50 mM) and p-nitrophenyl chloroformate (50 mM) dissolved in acetonitrile were added. The reaction was carried out at 75 °C for 16 h. After the reaction was completed, ethanol and sodium chloride were added to precipitate the DNA, and the DNA-encoded compound (Ⅱ-a) was obtained with a single-step yield of 93%.

[0063] Example 2

[0064] Using the same method as in Example 1, but with toluene instead of acetonitrile as the reaction solvent in step 2, the DNA-encoded compound (Ⅱ-a) was obtained with a single-step yield of 55%.

[0065] Example 3

[0066] Using the same method as in Example 1, except that the reaction temperature in step 2 was replaced with 60°C, the DNA-encoded compound (Ⅱ-a) was obtained with a single-step yield of 92%.

[0067] Example 4

[0068] Using the same method as in Example 1, except that the concentration of the reaction DNA in step 2 was replaced with 0.5 mM, the DNA-encoded compound (II-a) was obtained with a single-step yield of 64%.

[0069] Example 5

[0070] Using the same method as in Example 1, but with DBU instead of TEA as the base in step 2, the DNA-encoded compound (Ⅱ-a) was obtained with a single-step yield of 90%.

[0071] Example 6

[0072] Step 1:

[0073]

[0074] Using the same method as in Example 1, but replacing p-toluidine with 2-ethoxyethylamine as the starting material for the primary amine compound in step 1, the DNA-encoded compound I-b was obtained with a single-step yield of 84%.

[0075] Step 2:

[0076]

[0077] Using the same method as in Example 1, the DNA-encoded compound in step 2 was replaced with I-b instead of I-a, resulting in DNA-encoded compound II-b with a single-step yield of 96%.

[0078] Example 7

[0079] Step 1:

[0080]

[0081] Using the same method as in Example 1, but replacing p-toluidine with 1-(2-thiazolyl)-ethylamine in step 1, the DNA-encoded compound I-d was obtained with a single-step yield of 98%.

[0082] Step 2:

[0083] Using the same method as in Example 1, the DNA-encoded compound in step 2 was replaced with I-d instead of I-a, resulting in DNA-encoded compound II-d with a single-step yield of 40%.

[0084] Example 8

[0085] Step 1:

[0086]

[0087] Using the same method as in Example 1, but replacing p-toluidine with 2,5-dichlorobenzylamine in step 1, the primary amine compound was used to obtain DNA-encoded compound I-e, with a single-step yield of 92%.

[0088] Step 2:

[0089]

[0090] Using the same method as in Example 1, the DNA-encoded compound in step 2 was replaced with I-e instead of I-a, resulting in DNA-encoded compound II-e with a single-step yield of 75%.

[0091] Example 9

[0092] Step 1:

[0093]

[0094] Using the same method as in Example 1, but replacing p-toluidine with 2-naphthylamine as the primary amine compound in step 1, the DNA-encoded compound I-h was obtained with a single-step yield of 80%.

[0095] Step 2:

[0096]

[0097] Using the same method as in Example 1, the DNA-encoded compound in step 2 was replaced with I-h.

[0098] I-a yielded DNA-encoded compound II-h with a single-step yield of 91%.

[0099] Example 10

[0100] Step 1: Synthesis of DNA-encoded compounds.

[0101]

[0102] DNA-COOH (2 µmol), 22 amines (600 µmol), N,N-diisopropyldiimide (1.8 M), and N-hydroxy-7-azabenzotriazole (1.8 M) were dissolved in 2 mL of ACN:H₂O (1:1), respectively. The mixtures were then incubated at 25 °C for 16 h. After the reaction, sodium chloride (5 M, 600 µL) and ethanol (20 mL) were added to precipitate 22 DNA-encoded compounds (Formula I). ​​The structures of these compounds were determined using LC-MS.

[0103] Step 2: DNA-encoded compound formula (Ⅰ) undergoes intramolecular cyclization under catalyst-free conditions to generate DNA-encoded compound formula (Ⅱ).

[0104]

[0105] Twenty-two DNA-encoded compounds (formula (I), 10 nmol) were dissolved in 10 mL of 0.25 mM BBS buffer, and triethylamine (50 mM) and p-nitrophenyl chloroformate (50 mM) dissolved in acetonitrile were added. The mixture was reacted at 75 °C for 16 h. After the reaction was completed, ethanol and sodium chloride were added to precipitate the DNA, yielding 22 precipitated DNA-encoded compounds of formula (II).

Claims

1. A method for constructing 2,4-imidazolidinedione compounds from a DNA-encoded compound library, characterized in that: This method uses 2-amino-N-acetamide compounds of On-DNA and p-nitrophenyl chloroformate as raw materials, reacting them under alkaline conditions to obtain 2,4-imidazolidinedione compounds; wherein, the structural formula of the 2-amino-N-acetamide compounds of On-DNA is as follows: ; R 1 Selected from -C 1~6 alkylene-; R 2 Selected from -C 1~6 Alkyl, -C 0~4 Alkylene-OR a -C 0~4 Alkylene-S(O)2NR a R b -C 0~4 Alkylene rings (3- to 10-membered carbon rings), -C 0~4 Alkylene (3-10 membered heterocycle); wherein the alkyl, alkylene, carbocyclic, and heterocycle may be optionally divided by one, two, three, or four independent R... c replace; R a R b Selected independently from hydrogen and -C 1-6 Alkyl, halogen-substituted -C 1~6 Alkyl groups, 3-10 membered carbon rings, 3-10 membered heterocycles; R c Independently selected from halogen, cyano, nitro, -C 1-6 Alkyl, halogen-substituted -C 1~6 Alkyl, -C 1~6 Alkyl-OC 1~6 Alkyl groups, 3-10 membered carbon rings, 3-10 membered heterocycles; The DNA in the structural formula comprises single-stranded or double-stranded nucleotide chains obtained by polymerizing artificially modified and / or unmodified nucleotide monomers, which are bonded to R by one or more chemical bonds or groups. 1 Connected; The method includes the following steps: Step 1: Dissolve the 2-amino-N-acetamide compound of On-DNA in 0.25 mM borate buffer; Step 2: Dissolve the system obtained in Step 1 in acetonitrile, add alkali and p-nitrophenyl chloroformate, and react at 60~75℃ for 4~16 h; Step 3: The system obtained in Step 2 was subjected to alcohol precipitation, and then the product was centrifuged to obtain the 2,4-imidazolidinedione compound of On-DNA of formula (II); In step 2, the base is triethylamine or 1,8-diazabicycloundec-7-ene.

2. The method according to claim 1, characterized in that: The R 1 Selected from ; The R 2 Selected from , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

3. The method according to claim 1, characterized in that: The molar concentration of the 2-amino-N-acetamide compound in On-DNA in step 1 is 0.5~2mM, and the molar equivalent is 1. The molar equivalent of the base in step 2 is 5~30, and the molar equivalent of p-nitrophenyl chloroformate in step 2 is 5~30.

4. The method according to claim 1, characterized in that: The reaction temperatures in step 2 are: 60℃, 65℃, 70℃, and 75℃.

5. The method according to claim 1, characterized in that: The reaction time for step 2 is 4 hours, 6 hours, 8 hours, or 16 hours.

6. The method according to any one of claims 1-5, characterized in that: The method is used for batch operations of multi-hole plates.

7. The method according to any one of claims 1-5, characterized in that: The method is used for the synthesis of DNA-encoded compound libraries for multi-well plates.

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