Compounds, pharmaceutical compositions, kits for capped RNA transcripts, and in vitro methods

Abstract

To provide compounds, pharmaceutical compositions, kits for capped RNA transcription, and in vitro transcription methods that can increase mRNA translation efficiency and are key players in the development of one-pot in vitro transcription processes.SOLUTION: A cap analog compound contacts a DNA template with a composition along with an RNA polymerase and a nucleoside triphosphate, thereby transcribing the DNA template in vitro into RNA.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to compounds, pharmaceutical compositions, kits, and in vitro methods for use in capping RNA transcripts. [Background technology]

[0002] The synthesis of messenger ribonucleic acid (mRNA) by in vitro transcription has become an important tool for introducing foreign genes and expressing genetic information, and is widely applied in the treatment and prevention of diseases. The industrial-scale production of messenger ribonucleic acid (mRNA) by one-pot in vitro transcription (IVT) reaction has the advantages of a simple process and low cost. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Patent No. 8,304,529 B2 Summary of the Invention [Problem to be solved by the invention]

[0004] Cap analogs can increase the translation efficiency of messenger ribonucleic acid (mRNA) and are important key raw materials for the development of one-pot in vitro transcription processes. [Means for solving the problem]

[0005] According to an embodiment of the present disclosure, the present disclosure provides compounds, such as cap analogs, which may have a structure shown in Formula (I), Formula (II), or Formula (III).

[0006] [ka] [ka] [ka]

[0007] In the formula, A 1 and A 2 are each independently [ka] and R 1 and R 2 may be hydrogen, a methyl group, or a phenyl group, and Q 1 may be a single bond or -CH2-, and Y 1 and Y 3 are each independently -O-, [ka] and Z may be [ka] may be; Y 2 and Y 4 are each independently -O-, [ka] may be Q 2 , Q 5 , Q 6 and Q 7 are each independently -CH2-, or [ka] Q may be 3 and Q 4may each independently be -O-, -CH2-, or -CCl2-; R 3 is a C1-C6 alkyl group, or [ka] R may be 4 may be hydrogen or a methyl group, R 5 , R 6 , and R 7 may each independently be hydrogen, a methyl group, or a phenyl group; Y 5 teeth [ka] R may be 8 may be a C1-C6 alkyl group, a C4-C8 cycloalkyl group, a phenyl group, a benzyl group, or a C3-C5 heterocyclic group; R 9 and R 10 are each independently hydrogen, a C1-C6 alkyl group, or a benzyl group.

[0008] According to an embodiment of the present disclosure, the present disclosure also provides a pharmaceutical composition, which may include a compound having a structure of Formula (I), Formula (II), or Formula (III) according to the present disclosure, and an RNA (ribonucleic acid) molecule.

[0009] According to an embodiment of the present disclosure, the present disclosure provides a kit for use in capped RNA transcription. The kit for capped RNA transcription includes a compound and an RNA polymerase. According to an embodiment of the present disclosure, the compound has a structure shown in Formula (I), Formula (II), or Formula (III).

[0010] According to embodiments of the present disclosure, the present disclosure also provides a method of in vitro transcription, comprising the steps of: providing a composition comprising an RNA polymerase, a nucleoside triphosphate, and a compound according to the present disclosure; and contacting a DNA template with the composition to in vitro transcribe the DNA template into RNA. [Effects of the Invention]

[0011] According to an embodiment of the present disclosure, the compounds of the present disclosure have a specific structure and can function as cap analogs, which can synchronize and complete the capping reaction of messenger ribonucleic acid (mRNA) during transcription. Therefore, the compounds of the present disclosure can be used in one-pot in vitro transcription (IVT) processes to increase yields, reduce process costs, and improve product stability and protein expression.

[0012] A detailed description will be given in the following embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0013] The compounds, pharmaceutical compositions, kits for use in capped RNA transcripts, and in vitro methods for forming electrode materials and methods for making the same are described in the following detailed description. In the following detailed description, for purposes of explanation, numerous specific details and embodiments are set forth to provide a thorough understanding of the present disclosure. The specific elements and configurations described in the following detailed description are set forth to clearly explain the present disclosure. However, it will be apparent that the exemplary embodiments shown herein are used for illustrative purposes only, and that the inventive concept may be embodied in various forms without being limited to these exemplary embodiments.

[0014] According to embodiments of the present disclosure, compounds according to the present disclosure may have a structure as shown in Formula (I), Formula (II), or Formula (III).

[0015] [ka] [ka] [ka]

[0016] In the formula, A 1 and A 2 are each independently [ka] and R 1 and R 2 may be hydrogen, a methyl group, or a phenyl group, and Q 1 may be a single bond or -CH2-, and Y 1 and Y 3 are each independently -O-, [ka] may be Z is [ka] and Y 2 and Y 4 are each independently -O-, [ka] Q may be 2 , Q 5 , Q 6 and Q 7 are each independently -CH2-, or [ka] may be Q 3 and Q 4 may each independently be —O—, —CH—, or —CCl—; R 3 is a C1-C6 alkyl group, or [ka] may be R 4 may be hydrogen or a methyl group, R 5 , R 6 , and R 7 may each independently be hydrogen, a methyl group, or a phenyl group; Y 5 teeth [ka] R may be 8 may be a C1-C6 alkyl group, a C4-C8 cycloalkyl group, a phenyl group, a benzyl group, or a C3-C5 heterocyclic group; R 9 and R 10 are each independently hydrogen, a C1-C6 alkyl group, or a benzyl group.

[0017] According to an embodiment of the present disclosure, a single bond according to the present disclosure refers to a state in which there is no single atom at the relevant position. For example, in the structures of formulas (I) to (III), Q 1 When is a single bond, Q 2 There is no single atom at the position represented by

[0018] According to embodiments of the present disclosure, the alkyl group according to the present disclosure may be a linear or branched alkyl group. According to embodiments of the present disclosure, the C1-C6 alkyl group may be a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, or an isomer thereof. For example, the C1-C6 alkyl group according to the present disclosure may be a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, a sec-butyl group, an isobutyl group, or a tert-butyl group.

[0019] According to an embodiment of the present disclosure, the C4-C8 cycloalkyl group according to the present disclosure may be a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, or a cyclooctyl group.

[0020] According to an embodiment of the present disclosure, the C3-C5 heterocyclic group of the present disclosure may be a pyrrolyl group, an imidazolyl group, a pyrazolyl group, a pyridinyl group, a pyrimidinyl group, a tetrahydrofuranyl group, or a piperidinyl group.

[0021] According to an embodiment of the present disclosure, the compound according to the present disclosure has a structure shown in formula (I): 2 When is -CH2-, Y 1 teeth [ka] Also, according to some embodiments of the present disclosure, Q 2 but [ka] When Y 1 teeth [ka] It could be.

[0022] According to an embodiment of the present disclosure, the compound according to the present disclosure has a structure shown in formula (II): 5 When is -CH2-, Y 2 -O-, [ka] Also, according to some embodiments of the present disclosure, Q 5 but [ka] When Y 2 teeth [ka] It may be.

[0023] According to an embodiment of the present disclosure, the compound according to the present disclosure has a structure shown in formula (II): 2 When is -O-, Z is [ka] and Y 4 teeth [ka] is.

[0024] According to an embodiment of the present disclosure, a compound according to the present disclosure has a structure represented by formula (II), wherein Z is [ka] According to an embodiment of the present disclosure, Q 7 When is -CH2-, Y 4 -O-, [ka] According to some embodiments of the present disclosure, Q 7 but [ka] When Y 4 teeth [ka] It may be.

[0025] According to an embodiment of the present disclosure, the compound according to the present disclosure has a structure shown in formula (III): 6 When is -CH2-, Y 3 teeth [ka] Also, according to some embodiments of the present disclosure, Q 6 but [ka] When Y 3 teeth [ka] It may be.

[0026] According to embodiments of the present disclosure, the compound may be any of the following:

[0027] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0028] In the formula, R 1 may be hydrogen, a methyl group, or a phenyl group, and Q 3 and Q 4 may each independently be -O-, -CH2-, or -CCl2-; R 4 may be hydrogen or a methyl group, and R 8 may be a C1-C6 alkyl group, a C4-C8 cycloalkyl group, a phenyl group, a benzyl group, or a C3-C5 heterocyclic group.

[0029] According to embodiments of the present disclosure, the compound may be any of the following:

[0030] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0031] In the formula, R 1 may be hydrogen, a methyl group, or a phenyl group, and Q 3 and Q 4may each independently be -O-, -CH2-, or -CCl2-; Q 5 are each independently -CH2-, or [ka] and Y 2 -O-, [ka] R may be 4 may be hydrogen or a methyl group, and R 8 may be a C1-C6 alkyl group, a C4-C8 cycloalkyl group, a phenyl group, a benzyl group, or a C3-C5 heterocyclic group.

[0032] According to embodiments of the present disclosure, the compound may be any of the following:

[0033] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0034] In the formula, Q 3 and Q 4 are each independently -O-, -CH2-, or -CCl2-, and R 4 is hydrogen or a methyl group, and R 6 and R 7 is hydrogen, a methyl group, or a phenyl group, and R 8 is a C1-C6 alkyl group, a C4-C8 cycloalkyl group, a phenyl group, a benzyl group, or a C3-C5 heterocyclic group.

[0035] According to embodiments of the present disclosure, the compound may be any of the following:

[0036] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0037] In the formula, R 1 is hydrogen, a methyl group, or a phenyl group, and R 2 is hydrogen or a methyl group, and Q 3 and Q 4 are each independently -O-, -CH2-, or -CCl2-, and Q 5 are each independently -CH2-, or [ka] and R 4 is hydrogen or a methyl group, R 8 is a C1-C6 alkyl group, a C4-C8 cycloalkyl group, a phenyl group, a benzyl group, or a C3-C5 heterocyclic group; R 9 and R 10 are each independently hydrogen, a C1-C6 alkyl group, or a benzyl group.

[0038] According to an embodiment of the present disclosure, there is no particular limitation on the method for producing the compound having the structure represented by formula (I) according to the present disclosure, and the compound can be produced using the following reaction scheme.

[0039] [ka]

[0040] According to an embodiment of the present disclosure, there is no particular limitation on the method for producing the compound having the structure represented by formula (II) according to the present disclosure, and the compound can be produced using the following reaction scheme.

[0041] [ka]

[0042] According to an embodiment of the present disclosure, there is no particular limitation on the method for producing the compound having the structure represented by formula (III) according to the present disclosure, and the compound can be produced using the following reaction scheme.

[0043] [ka]

[0044] According to an embodiment of the present disclosure, the present disclosure provides a pharmaceutical composition comprising a compound and an RNA (ribonucleic acid) molecule. According to an embodiment of the present disclosure, the compound has a structure represented by Formula (I), Formula (II), or Formula (III) and can be covalently bound to the RNA molecule (i.e., can react with the RNA molecule to form a covalent bond). According to an embodiment of the present disclosure, the RNA molecule is an mRNA molecule.

[0045] According to an embodiment of the present disclosure, the present disclosure provides a kit for use in capping RNA transcription, the kit comprising a compound and an RNA polymerase, wherein the compound has a structure shown in Formula (I), Formula (II), or Formula (III).

[0046] According to an embodiment of the present disclosure, the kit for use in producing capped RNA transcripts of the present disclosure further comprises an RNA molecule, and the RNA molecule is an mRNA molecule.

[0047] According to an embodiment of the present disclosure, the present disclosure provides a method of in vitro transcription, comprising the steps of: providing a composition comprising an RNA polymerase, a nucleoside triphosphate, and a compound according to the present disclosure, and contacting a DNA template with the composition to in vitro transcribe the DNA template into RNA.

[0048] The present invention will be described in detail below with reference to exemplary embodiments so that those skilled in the art can easily understand the present invention. The present invention is not limited to the exemplary embodiments described herein, but may be embodied in various forms.

[0049] A compound having a structure represented by formula (I) Table 1 lists compounds having the structure of formula (I) according to examples of the present disclosure.

[0050] [Table 1] JPEG2025118520000086.jpg182165JPEG2025118520000087.jpg187168JPEG2025118520000088.jpg182163JPEG20251185200 00089.jpg188169JPEG2025118520000090.jpg186166JPEG2025118520000091.jpg179161JPEG2025118520000092.jpg187167

[0051] A compound having a structure represented by formula (II) Table 2 lists compounds having the structure represented by formula (II) according to examples of the present disclosure.

[0052] [Table 2] JPEG2025118520000094.jpg209165JPEG2025118520000095.jpg214169

[0053] A compound having a structure represented by formula (III) Table 3 lists compounds having the structure represented by formula (III) according to examples of the present disclosure.

[0054] [Table 3] JPEG2025118520000097.jpg225165JPEG2025118520000098.jpg221166JPEG2025118520000099.jpg216167

[0055] To further explain the method for producing lipid compounds according to the present disclosure, the production flow of the compounds of Examples 1 to 4, 25, 26, and 33 to 35 will be described below with reference to examples.

[0056] Table 4 lists the structures and spectral data of compounds according to examples of the present disclosure. [Table 4] JPEG2025118520000101.jpg201166JPEG2025118520000102.jpg224162

[0057] Example 1 Compound (A) (2.30 mmol) was added to a stirred solution of 5 mL of water, and glacial acetic acid was slowly added dropwise to adjust the pH of the solution to 4.0. Dimethyl sulfate (23.0 mmol) was then slowly added dropwise. The resulting mixture was stirred at room temperature for 3 hours. As the methylation proceeded, the pH of the resulting mixture decreased to approximately 2.0. The pH of the mixture was then adjusted to 4.0 using aqueous sodium hydroxide (1 M). After stirring at room temperature for 5 hours, water (10 mL) was added, and the resulting mixture was washed with dichloromethane (30 mL), and the aqueous layer was collected. The resulting mixture was then purified using an ion exchange resin (DEAE) (eluent: aqueous triethylammonium bicarbonate (TEAB) solution (0.1 M)) to obtain compound (B) as a white solid.

[0058] The reaction formula for the above reaction is shown below.

[0059] [ka]

[0060] Compound (B) was analyzed by nuclear magnetic resonance spectroscopy, and the spectral data obtained are shown below. 1 H NMR(400MHz,D2O)δ5.98(d,J=7.2Hz,1H),4.55(t,J=4.0Hz,1H),4.49(t,J=5.4Hz,1H) ,4.27(m,1H),4.21(m,1H),4.02(s,3H),3.11(q,J=7.2Hz,19H),1.19(t,J=7.2Hz,29H)

[0061] Compound (B) (0.44 mmol), triphenylphosphine (PPh3) (2.18 mmol), imidazole (4.36 mmol), and 2-(pyridin-2-yldisulfanyl)pyridine, aldrithiol (2.18 mmol) were added to dimethylformamide (DMF) (2 mL). The mixture was stirred at room temperature under nitrogen for 6 hours. The resulting solution was then added dropwise to acetone (250 mL) at -20°C, producing a white solid. The precipitate was collected by centrifugation and washed five times with acetone (30 mL). The mixture was dried at room temperature under vacuum to obtain compound (C) (a white solid).

[0062] The reaction formula for the above reaction is shown below.

[0063] [ka]

[0064] Compound (C) was analyzed by nuclear magnetic resonance spectroscopy, and the spectral data obtained are shown below. 1 H NMR(400MHz,MeOD)δ8.02(s,1H),7.44(d,J=1.6Hz,1H),6.99(d,J=1.6Hz,1H),6 .02(d,J=2.8Hz,1H),5.51(s,1H),4.56(m,1H),4.46(m,1H),4.30-4.16(m,7H).

[0065] Compound (C) (0.06 mmol), compound (D) (0.05 mmol), and ZnCl2 (0.07 mmol) were added to dimethylformamide (DMF) (1 mL) at room temperature. After stirring under nitrogen for 48 hours, an aqueous solution of ethylenediaminetetraacetic acid (EDTA) (5 mL, 1 M concentration) was added. The resulting product was then purified using an ion exchange resin (DEAE) (eluent: an aqueous solution of triethylammonium bicarbonate (TEAB) (0.1 M concentration)) to obtain compound (1) (a white solid).

[0066] The reaction formula for the above reaction is shown below.

[0067] [ka]

[0068] Compound (1) was then analyzed using nuclear magnetic resonance spectroscopy, and the resulting spectral data are shown below. 1 H NMR (400 MHz, DO) δ 7.87 (s, 1H), 5.86 (s, 1H), 5.73 (s, 1H), 4.55 (s, 1H), 4.45 (d, J = 9.0 Hz, 1H), 4.37-4.43 (m, 2H), 4.05 (s, 3H), 4.04 (d, J = 8.5 Hz, 1H), 3.21 (J = 7.3 Hz, 18H), 1.28 (t, J = 7.5 Hz, 30H). Compound (1) was then analyzed by liquid chromatography-mass spectrometry (LC-MS). M / Z: [M] + =812(C 23 H 33 N 11 O 16 P3 + ) was measured.

[0069] Example 2 Compound (C) (0.06 mmol), compound (E) (0.05 mmol), and ZnCl2 (0.07 mmol) were added to dimethylformamide (DMF) (1 mL) at room temperature. After stirring under nitrogen for 48 hours, an aqueous solution of ethylenediaminetetraacetic acid (EDTA) (5 mL, 1 M concentration) was added. The resulting product was then purified using an ion exchange resin (DEAE) (eluent: an aqueous solution of triethylammonium bicarbonate (TEAB) (0.1 M concentration)) to obtain compound (2) (a white solid).

[0070] The reaction formula for the above reaction is shown below.

[0071] [ka]

[0072] Compound (2) was then analyzed using nuclear magnetic resonance spectroscopy, and the resulting spectral data are shown below. 1 H NMR (400 MHz, DO) δ 7.87 (s, 1H), 5.86 (s, 1H), 5.73 (s, 1H), 4.55 (s, 1H), 4.45 (d, J = 9.0 Hz, 1H), 4.37-4.43 (m, 2H), 4.05 (s, 3H), 4.04 (d, J = 8.5 Hz, 1H), 3.21 (J = 7.3 Hz, 18H), 1.28 (t, J = 7.5 Hz, 30H). Compound (2) was then analyzed by liquid chromatography-mass spectrometry (LC-MS). M / Z: [M] - =815(C 22 H 30 N 10 O18P3 + ) was measured.

[0073] Example 3 Compound (F) (0.06 mmol), compound (G) (0.05 mmol), and ZnCl2 (0.07 mmol) were added to dimethylformamide (DMF) (1 mL) at room temperature. After stirring under nitrogen for 48 hours, an aqueous solution of ethylenediaminetetraacetic acid (EDTA) (5 mL, 1 M concentration) was added. The resulting product was then purified using an ion exchange resin (DEAE) (eluent: an aqueous solution of triethylammonium bicarbonate (TEAB) (0.1 M concentration)) to obtain compound (3) (a white solid).

[0074] The reaction formula for the above reaction is shown below.

[0075] [ka]

[0076] Compound (3) was then analyzed using nuclear magnetic resonance spectroscopy, and the resulting spectral data are shown below. 1 H NMR (500 MHz, DO) δ 7.97 (s, 1H), 5.71-5.65 (m, 1H), 5.64 (s, 1H), 4.79 (s, 2H), 4.59 (s, 1H), 4.41-4.38 (m, 3H), 4.30 (s, 1H), 4.24 (s, 2H), 3.38 (s, 3H), 3.11 (q, J = 7.2 Hz, 17H), 1.19 (t, J = 7.2 Hz, 27H). Compound (3) was then analyzed by liquid chromatography-mass spectrometry (LC-MS). M / Z: [M+H] + =829(C 23 H 32 N 10 O 18 P3 + ) was measured.

[0077] Example 4 Compound (M) (0.06 mmol) was dissolved in a mixture of water (0.03 mL) and dimethyl sulfoxide (0.28 mL) at room temperature, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) (0.10 mmol) and imidazole (0.19 mmol). The resulting mixture was stirred at room temperature for 4 hours, after which an aqueous magnesium chloride (MgCl2) solution (3.15 M, 0.03 mL) was added, followed by the addition of compound (O) (0.04 mmol). After stirring at room temperature for 16 hours, the resulting mixture was mixed with an aqueous ethylenediaminetetraacetic acid (EDTA) solution (5 mL, 1 M). The equivalent weight of EDTA was three times that of magnesium chloride. The resulting product was then purified using an ion exchange resin (DEAE) (the eluent was an aqueous solution of triethylammonium bicarbonate (TEAB) (concentration: 0.1 M)). After concentration and drying, the resulting product was redissolved in water, and acetone and sodium perchlorate were added. The formation of a precipitate was observed. The precipitate was centrifuged, washed with acetone, dissolved in water, and then freeze-dried to obtain compound (4).

[0078] The reaction formula for the above reaction is shown below.

[0079] [ka]

[0080] Compound (4) was then analyzed using nuclear magnetic resonance spectroscopy, and the resulting spectral data are shown below. 1H NMR (400 MHz, DO) δ 8.03 (s, 1H), 5.80-5.77 (2H), 4.89 (s, 1H), 4.68 (t, J = 6.0 Hz, 5.0 Hz, 1H), 4.48-4.45 (m, 2H), 4.40-4.35 (m, 2H), 4.32-4.26 (m, 2H), 4.09-4.07 (m, 4H), 3.62 (dd, J = 9.0 Hz, 21.0 Hz, 2H), 3.43 (s, 3H). Compound 42 was then analyzed by liquid chromatography-mass spectrometry (LC-MS). M / Z: [M+H] + =906.58(C 24 H 35 N 11 O 19 P3S + ) was measured.

[0081] Example 25 Compound (H) (0.06 mmol) was dissolved in a mixture of water (0.03 mL) and dimethyl sulfoxide (0.28 mL) at room temperature, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) (0.10 mmol) and imidazole (0.19 mmol). The resulting mixture was stirred at room temperature for 4 hours, after which an aqueous magnesium chloride (MgCl2) solution (3.15 M, 0.03 mL) was added, followed by the addition of compound (I) (0.04 mmol). After stirring at room temperature for 16 hours, the resulting mixture was mixed with an aqueous ethylenediaminetetraacetic acid (EDTA) solution (5 mL, 1 M). The equivalent weight of EDTA was three times that of magnesium chloride. The resulting product was then purified using an ion exchange resin (DEAE) (the eluent was an aqueous solution of triethylammonium bicarbonate (TEAB) (concentration: 0.1 M)). After concentration and drying, the resulting product was redissolved in water, and acetone and sodium perchlorate were added. The formation of a precipitate was observed. The precipitate was centrifuged, washed with acetone, dissolved in water, and then lyophilized to obtain compound (25).

[0082] The reaction formula for the above reaction is shown below.

[0083] [ka]

[0084] Compound (25) was then analyzed by nuclear magnetic resonance spectroscopy, and the resulting spectral data are shown below. 1 H NMR (500 MHz, DO) δ 9.00 (s, 1H), 8.29 (s, 1H), 8.19 (s, 1H), 8.00 (s, 1H), 6.43 (s, 1H), 5.71 (s, 1H), 5.54 (s, 1H), 4.88 (s, 2H), 4.58 (s, 1H), 4.44 (br, 1H), 4.34-4.20 (m, 5H), 4.14-3.99 (m, 5H), 3.85 (s, 5H), 3.30 (s, 3H), 3.20 (s, 3H). Compound (28) was then analyzed by liquid chromatography-mass spectrometry (LC-MS). M / Z: [M+H] + =1171.7(C 34 H 47 N 16 O 24 P4 + ) was measured.

[0085] Example 26 Compound (H) (0.06 mmol) was dissolved in a mixture of water (0.03 mL) and dimethyl sulfoxide (0.28 mL) at room temperature, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) (0.10 mmol) and then imidazole (0.19 mmol). The resulting mixture was stirred at room temperature for 4 hours, followed by the addition of an aqueous magnesium chloride (MgCl2) solution (3.15 M, 0.03 mL) and then compound (J) (0.04 mmol). After stirring at room temperature for 16 hours, the resulting mixture was mixed with an aqueous ethylenediaminetetraacetic acid (EDTA) solution (5 mL, 1 M). The equivalent weight of EDTA was three times that of magnesium chloride. The resulting product was then purified using an ion exchange resin (DEAE) (the eluent was an aqueous solution of triethylammonium bicarbonate (TEAB) (concentration: 0.1 M)). After concentration and drying, the resulting product was redissolved in water, and acetone and sodium perchlorate were added. The formation of a precipitate was observed. The precipitate was centrifuged, washed with acetone, dissolved in water, and then lyophilized to obtain compound (26).

[0086] The reaction formula for the above reaction is shown below.

[0087] [ka]

[0088] Compound (26) was then analyzed by nuclear magnetic resonance spectroscopy, and the resulting spectral data are shown below. 1H NMR (500 MHz, DO) δ 9.13 (s, 1H), 8.33 (d, J = 12.5 Hz, 2H), 8.08 (s, 1H), 6.17 (s, 1H), 5.80 (s, 1H), 5.66 (s, 1H), 4.94 (s, 1H), 4.71-4.68 (m, 3H), 4.53-4.47 (m, 3H), 4.40-4.33 (m, 5H), 4.26 (s, 1H), 4.19-4.17 (m, 3H), 3.99 (s, 3H), 3.59-3.54 (m, 5H), 3.43 (s, 3H). Compound 26 was then analyzed by liquid chromatography-mass spectrometry (LC-MS). M / Z: [M+H] + =1235.8(C 34 H 47 N 16 O 25 P4S + ) was measured.

[0089] Example 33 Compound (K) (0.06 mmol) was added to a mixture of water (0.03 mL) and dimethyl sulfoxide (0.28 mL) at room temperature, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) (0.10 mmol) and then imidazole (0.19 mmol). The resulting mixture was stirred at room temperature for 4 hours, followed by the addition of an aqueous magnesium chloride (MgCl2) solution (3.15 M, 0.03 mL), followed by the addition of compound (L) (0.04 mmol). After stirring at room temperature for 16 hours, the resulting mixture was mixed with an aqueous ethylenediaminetetraacetic acid (EDTA) solution (5 mL, 1 M). The equivalent weight of EDTA was three times that of magnesium chloride. The resulting product was then purified using an ion exchange resin (DEAE) (the eluent was an aqueous solution of triethylammonium bicarbonate (TEAB) (concentration: 0.1 M)). After concentration and drying, the resulting product was redissolved in water, and acetone and sodium perchlorate were added. The formation of a precipitate was observed. The precipitate was centrifuged, washed with acetone, dissolved in water, and then lyophilized to obtain compound (33).

[0090] The reaction formula for the above reaction is shown below.

[0091] [ka]

[0092] Compound (33) was then analyzed by nuclear magnetic resonance spectroscopy, and the resulting spectral data are shown below. 1H NMR (500 MHz, DO) δ 8.20 (s, 1H), 7.91 (s, 1H), 7.77 (s, 1H), 5.80 (d, J = 4.0 Hz, 1H), 5.65 (d, J = 4.0 Hz, 1H), 5.54 (s, 1H), 4.75 (s, 1H), 4.66-4.58 (m, 3H), 4.35-4.31 (m, 3H), 4.26 (s, 3H), 4.22 (br, 3H), 4.16 (br, 1H), 4.09 (s, 3H), 3.84 (d, J = 14.0 Hz, 6H), 3.23 (s, 6H). Compound 33 was then analyzed by liquid chromatography-mass spectrometry (LC-MS). M / Z: [M+H] + =1172.7(C 34 H 46 N 15 O 24 P4 + ) was measured.

[0093] Example 34 Compound (M) (0.06 mmol) was added to a mixture of water (0.03 mL) and dimethyl sulfoxide (0.28 mL) at room temperature, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) (0.10 mmol) and then imidazole (0.19 mmol). The resulting mixture was stirred at room temperature for 4 hours, followed by the addition of an aqueous magnesium chloride (MgCl2) solution (3.15 M, 0.03 mL), followed by the addition of compound (L) (0.04 mmol). After stirring at room temperature for 16 hours, the resulting mixture was mixed with an aqueous ethylenediaminetetraacetic acid (EDTA) solution (5 mL, 1 M). The equivalent weight of EDTA was three times that of magnesium chloride. The resulting product was then purified using an ion exchange resin (DEAE) (the eluent was an aqueous solution of triethylammonium bicarbonate (TEAB) (concentration: 0.1 M)). After concentration and drying, the resulting product was redissolved in water, and acetone and sodium perchlorate were added. The formation of a precipitate was observed. The precipitate was centrifuged, washed with acetone, dissolved in water, and then lyophilized to obtain compound (34).

[0094] The reaction formula for the above reaction is shown below.

[0095] [ka]

[0096] Compound (34) was then analyzed by nuclear magnetic resonance spectroscopy, and the resulting spectral data are shown below. 1H NMR(400MHz,D2O)δ8.24(s,1H),7.95(s,1H),7.80(s,1H),5.84(d,J=6.0Hz,1H),5.67(d,J=5. 6Hz,1H),5.61(s,1H),4.81-4.72(m,1H),4.69(s,1H),4.35-4.31(m,3H),4.27-4.23(m,2H),4 3.17-4.11 (m, 4H), 4.08-4.03 (m, 2H), 3.89-3.85 (m, 4H), 3.42 (d, J = 9.6 Hz, 1H), 3.37 (d, J = 9.6 Hz, 1H), 3.26 (s, 3H), 3.24 (s, 3H), 3.04 (q, J = 7.2 Hz, 12H), 2.99 (s, 3H), 1.12 (t, J = 7.2 Hz, 19H). Compound 34 was then analyzed by liquid chromatography-mass spectrometry (LC-MS). M / Z: [M+H] + =1249.17(C 35 H 49 N 16 O 25 P4S + ) was measured.

[0097] Example 35 Compound (M) (0.06 mmol) was dissolved in a mixture of water (0.03 mL) and dimethyl sulfoxide (0.28 mL) at room temperature, followed by the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) (0.10 mmol) and imidazole (0.19 mmol). The resulting mixture was stirred at room temperature for 4 hours, followed by the addition of an aqueous magnesium chloride (MgCl2) solution (3.15 M, 0.03 mL), followed by the addition of compound (N) (0.04 mmol). After stirring at room temperature for 16 hours, the resulting mixture was mixed with an aqueous ethylenediaminetetraacetic acid (EDTA) solution (5 mL, 1 M). The equivalent weight of EDTA was three times that of magnesium chloride. The resulting product was then purified using an ion exchange resin (DEAE) (the eluent was an aqueous solution of triethylammonium bicarbonate (TEAB) (concentration: 0.1 M)). After concentration and drying, the resulting product was redissolved in water, and acetone and sodium perchlorate were added. The formation of a precipitate was observed. The precipitate was centrifuged, washed with acetone, dissolved in water, and then lyophilized to obtain compound (35).

[0098] The reaction formula for the above reaction is shown below.

[0099] [ka]

[0100] Compound (35) was then analyzed by nuclear magnetic resonance spectroscopy, and the resulting spectral data are shown below. 1H NMR(400MHz,D2O)δ8.28(s,1H),8.01(s,1H),7.86(s,1H),5.91(d,J=5.5Hz,1H),5.75(d,J=5. 5Hz,1H),5.63(s,1H),4.86-4.84(m,3H),4.75(s,2H),4.66(t,J=5.5Hz,5.5Hz,1H),4.45(s,1 1H), 4.41 (t, J = 3.5 Hz, 5.0 Hz, 1H), 4.37-4.33 (m, 2H), 4.27-4.25 (m, 4H), 4.21-4.13 (m, 2H), 3.93 (s, 4H), 3.50 (dd, J = 9.0 Hz, 19.5 Hz, 3H), 3.38 (s, 3H), 3.32 (s, 3H), 3.08 (s, 3H), 3.00 (s, 3H). Compound 35 was then analyzed by liquid chromatography-mass spectrometry (LC-MS). M / Z: [M+H] + =1263.8(C 36 H 51 N 16 O 25 P4S + ) was measured.

[0101] Assessment of mRNA yield Using in vitro transcription (IVT) technology, mRNA containing cap analogs of compounds (25), (26), (33), and (35) of the present disclosure and TriLink CleanCap cap analog (product number #N-7113) was generated. First, the DNA template plasmid required for IVT was generated, and the enzymatic reaction was carried out using T7 enzyme to produce mRNA. The yield of the resulting mRNA was evaluated. The results are shown in Table 5.

[0102] [Table 5]

[0103] Evaluation of capping efficiency (1): Using anti-cap antibodies Nuclease-free water was added to the lyophilized carrier RNA to obtain a stock RNA solution (concentration: 2 μg / μL). The stock RNA solution was then heated at 80°C for 2 minutes with various cap analogs (compound (3) and Jena-ARCA cap analog (product number #NU-855L, purchased from Jena Bioscience)) and cooled in ice water at 0°C for 5 minutes. The resulting product was then placed on a nitrocellulose membrane and irradiated with UV light to obtain crosslinked RNA samples.

[0104] A 2.5% nonfat dry milk solution was prepared and allowed to stand at room temperature for 30 minutes. The primary antibody (anti-7-methylguanosine (m7G)-Cap mAb) was then diluted with the 2.5% nonfat dry milk and incubated at room temperature for 1 hour. The resulting material was then washed twice (10 minutes each) with TBST buffer. The secondary antibody (anti-mouse IgG-HRP) was then diluted with the 2.5% nonfat dry milk and incubated at room temperature for 1 hour. The resulting material was then washed three times (10 minutes each) with TBST buffer. The material was then incubated for 1 to 3 minutes with a chemiluminescent reagent (Femto). Finally, image analysis was performed using a luminometer image analyzer system (Fujifilm LAS 4000). The image analysis results indicated that compound (3) of the present disclosure possesses capping ability similar to that of commercially available ARCA.

[0105] Evaluation of cap efficiency (2): Using IP-RP-UPLC (1) Nuclease-free water was added to the lyophilized carrier RNA to obtain a stock RNA solution (concentration: 2 μg / μL). Deionized water (0.1 mL) and the stock RNA solution (10 μL) were mixed with various cap analogs (compound (3) and Jena-ARCA cap analog (product number #NU-855L, purchased from Jena Bioscience)), respectively, to obtain samples.

[0106] Next, the sample (10 μL) was injected onto the column and subjected to ion-pair reversed-phase ultra-high performance liquid chromatography (IP-RP UPLC) (45 minutes) using the gradient of the solutions in Table 6. The absorbance at UV260 was monitored. The column used was an oligonucleotide column (Acquity Premier Oligonucleotide C18, manufactured and sold by Waters Corp.), with a water-based buffer solution (triethylamine acetate buffer, pH 7.0, 100 mM) as solution A and an acetonitrile (ACN)-based buffer solution (a solution containing solution A and acetonitrile; the volume ratio of solution A to acetonitrile was 3:1) as solution B.

[0107] [Table 6]

[0108] As can be seen from the analysis results, the capping efficiency of compound (3) according to the present disclosure was about 60.8%, which was equivalent to the capping efficiency of commercially available Jena-ARCA (about 63%).

[0109] Evaluation of CAP efficiency (3): Use of IP-RP-UPLC In vitro transcription (IVT) technology was used to generate mRNAs containing the presently disclosed compounds (25), cap analogs (33) to (35), and TriLink CleanCap cap analog (product number #N-7113). The DNA template used was a 40-nucleotide nucleic acid fragment, and the enzymatic reaction proceeded with T7 enzyme to generate 40-nt mRNAs.

[0110] Nuclease-free water was added to the lyophilized carrier RNA to obtain a stock RNA solution (concentration: 2 μg / μL). Deionized water (0.1 mL) and the stock RNA solution (10 μL) were mixed with the 40-nt mRNAs obtained from the cap analogs to obtain samples.

[0111] Next, 10 μL of the sample was injected onto the column and subjected to ion-pair reversed-phase ultra-high-performance liquid chromatography (IP-RP UPLC) (45 min) using the gradient of the solutions shown in Table 7. The absorbance at UV260 was monitored, and the capping efficiency (%) was evaluated based on the results. The results are shown in Table 8. The column used was an oligonucleotide column (Acquity Premier Oligonucleotide C18, manufactured and sold by Waters Corp.). Solution A was a water-based buffer solution (triethylamine acetate buffer, pH 7.0, concentration 100 mM), and Solution B was an acetonitrile (ACN)-based buffer solution (a solution containing Solution A and acetonitrile; the volume ratio of Solution A to acetonitrile was 3:1).

[0112] [Table 7]

[0113] [Table 8]

[0114] As can be seen from the analytical results, the capping efficiency of compounds (25), (33) to (35) according to the present disclosure was approximately 71.3% to 100%, which was equivalent to the capping efficiency of commercially available Trilink CleanCap BTfLuc.

[0115] Assessment of transfection efficiency Nuclease-free water was added to the lyophilized carrier RNA to obtain a stock RNA solution (concentration: 2 μg / μL). Deionized water (0.1 mL) and the stock RNA solution (10 μL) were mixed with various cap analogs (compounds 3, 25, 33, 34, 35, and Jena-ARCA cap analog (#NU-855L, purchased from Jena Bioscience)) to obtain samples.

[0116] HEK293 cells were seeded into a 96-well culture plate at 4 x 104 cells / well and cultured for 24 hours. Then, transfection reagent (Lipofectamine MessengerMax, purchased from Thermo Fisher Scientific) (0.3 μL per well) and sample (50 ng per well) were added to each well. The plates were cultured for 5 hours in an incubator at 37°C with 5% carbon dioxide. Next, 100 μL of signal excitation and fluorescence detection reagent (One-Glo Luciferase) was added to each well and incubated for 5 minutes at room temperature to measure luciferase activity. The product (100 μL) was then aspirated with a pipette, and the resulting fluorescence was recorded and measured using a microplate reader (GloMax Microplate Reader). The results showed that after transfection of HEK293 cells with luciferase mRNA capped with compounds (3), (25), (33) to (35) of the present disclosure, the performance of nucleic acid translation into protein was superior to that of commercially available Jena-ARCA.

[0117] Assessment of cell immunogenicity HEK293(RIGI + )-IFNβ-fLuc cells were added to 1 × 10 4Cells were seeded into a 96-well culture plate at 1 cell per well and cultured for 24 hours. Then, transfection reagent (Lipofectamine MessengerMax, purchased from Thermo Fisher Scientific) (0.15 μL per well) and sample (200 ng per well) were added to each well. Culture was continued for 5 hours in an incubator at 37°C and 5% carbon dioxide. Next, 100 μL of signal excitation fluorescence detection reagent (One-Glo Luciferase) was added to each well, and the mixture was incubated at room temperature for 5 minutes to measure luciferase activity. The product (180 μL) was then aspirated with a pipette, and the fluorescence intensity of the resulting product was recorded and measured using a microplate reader (GloMax Microplate Reader). The results demonstrated that luciferase mRNA capped with compound (3) of the present disclosure transduced HEK293 (RIGI + )-IFNβ-fLuc transfection into cells elicited a lower immune response than commercially available Jena-ARCA-capped mRNA.

[0118] It will be apparent that various modifications and variations can be made to the methods and materials of this disclosure. It is intended that the specification and embodiments be considered as exemplary only, with the true scope of the disclosure being indicated by the following claims and their equivalents.

Claims

1. A compound having a structure represented by formula (I), formula (II) or formula (III). 【Chemical 1】 【Chemistry 2】 (In the formula, A １ and A ２ are each independently 【Chemistry 3】 and R １ and R ２ are each independently a hydrogen atom, a methyl group, or a phenyl group; １ is a single bond or -CH ２ - and Y １ and Y ３ are each independently -O-, 【Chemistry 4】 and Z is 【Chemistry 5】 and Y ２ and Y ４ are each independently -O-, 【Chemistry 6】 and Q ２ , Q ５ , Q ６ and Q ７ are each independently -CH ２ -or 【Chemistry 7】 and Q ３ and Q ４ are each independently —O—, —CH ２ -or-CCl ２ - and R ３ is a C1-C6 alkyl group or 【Chemistry 8】 and R ４ is hydrogen or a methyl group, and R ５ , R ６ and R ７ are each independently a hydrogen atom, a methyl group, or a phenyl group; Y ５ teeth 【Chemistry 9】 and R ８ is a C1-C6 alkyl group, a C4-C8 cycloalkyl group, a phenyl group, a benzyl group, or a C3-C5 heterocyclic group, and R ９ and R １０ are each independently hydrogen, a C1-C6 alkyl group, or a benzyl group.

2. The compound of claim 1, wherein the compound is any of the following: 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 (In the formula, R １ is hydrogen, a methyl group, or a phenyl group, and Q ３ and Q ４ are each independently —O—, —CH ２ -or-CCl ２ - and R ４ is hydrogen or a methyl group, and R ８ is a C1-C6 alkyl group, a C4-C8 cycloalkyl group, a phenyl group, a benzyl group, or a C3-C5 heterocyclic group.

3. The compound of claim 1, wherein the compound is any of the following: 【Chemistry 18】 【Chemistry 19】 【Chemistry 20】 【Chemical 21】 【Chemical 22】 【Chemical 23】 【Chemistry 24】 【Chemistry 25】 【Chemical 26】 【Chemical 27】 【Chemical formula 28】 (In the formula, R １ is hydrogen, a methyl group, or a phenyl group, and Q ３ and Q ４ are each independently —O—, —CH ２ -or-CCl ２ - and Q ５ are each independently -CH ２ -or 【Chemical 29】 and Y ２ Ha-O-, 【Chemistry 30】 and R ４ is hydrogen or a methyl group, and R ８ is a C1-C6 alkyl group, a C4-C8 cycloalkyl group, a phenyl group, a benzyl group, or a C3-C5 heterocyclic group.

4. The compound of claim 1, wherein the compound is any of the following: 【Chemical 31】 【Chemical 32】 【Chemical 33】 【Chemical 34】 【Chemical 35】 【Chemical 36】 【Chemical 37】 【Chemical Formula 38】 【Chemical Formula 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 (In the formula, Q ３ and Q ４ are each independently —O—, —CH ２ -or-CCl ２ - and R ４ is hydrogen or a methyl group, and R ６ and R ７ is hydrogen, a methyl group, or a phenyl group, and R ８ is a C1-C6 alkyl group, a C4-C8 cycloalkyl group, a phenyl group, a benzyl group, or a C3-C5 heterocyclic group.

5. The compound of claim 1, wherein the compound is any of the following: 【Chemistry 43】 【Chemical 44】 【Chemistry 45】 【Chemistry 46】 【Chemistry 47】 【Chemistry 48】 【Chemistry 49】 【Chemistry 50】 【Chemistry 51】 【Chemistry 52】 【Chemistry 53】 【Chemical 54】 (In the formula, R １ is hydrogen, a methyl group, or a phenyl group, and R ２ is hydrogen or a methyl group, and Q ３ and Q ４ are each independently —O—, —CH ２ -or-CCl ２ - and Q ５ are each independently -CH ２ -or 【Chemistry 55】 and R ４ is hydrogen or a methyl group, R ８ is a C1-C6 alkyl group, a C4-C8 cycloalkyl group, a phenyl group, a benzyl group, or a C3-C5 heterocyclic group, and R ９ and R １０ are each independently hydrogen, a C1-C6 alkyl group, or a benzyl group.

6. 1. A pharmaceutical composition comprising: A compound according to claim 1; an RNA molecule; 10. A pharmaceutical composition comprising the compound of claim 1, wherein the compound of claim 1 is capable of binding to the RNA molecule in a covalent manner.

7. 1. A kit for use in capped RNA transcripts, comprising: A compound according to claim 1; RNA polymerase and A kit for use in capped RNA transcripts comprising:

8. 1. A method of in vitro transcription comprising: providing a composition comprising an RNA polymerase, a nucleoside triphosphate, and a compound of claim 1; contacting a DNA template with the composition and in vitro transcribing the DNA template into RNA; A method of in vitro transcription comprising:

Images