Preparation of imidazo[2,1-F][1,2,4]triazine-4-amine derivatives as TLR7 agonists

Abstract

Disclosed herein is a method for preparing an imidazo[2,1-f][1,2,4]triazine-4-amine derivative or a stereoisomer thereof or a pharmaceutically acceptable salt thereof which can be used as a TLR7 agonist.

Description

Technical Field

[0001] Disclosed herein is a method for preparing an imidazo[2,1-f][1,2,4]triazine-4-amine derivative or a stereoisomer thereof or a pharmaceutically acceptable salt thereof which can be used as a TLR7 agonist. Background Art

[0002] Toll-like receptors (TLRs) belong to the pattern recognition receptor (PRR) family, which play a key role in the early innate immune response by sensing highly conserved multiple pathogen molecular patterns (PAMPs) as well as endogenous danger-associated molecular patterns (DAMPs) (Barton, GM and R. Medzhitov (2002). "Toll-like receptors and their ligands." Curr Top Microbiol Immunol 270: 81-92.).

[0003] Ten different TLRs have been identified in humans. Among them, TLR7, TLR8 and TLR9 belong to the same TLR subfamily based on their genomic structure, sequence similarity and endosomal localization. They have limited expression patterns and are limited to certain types of immune cells. TLR7 is expressed in B cells and plasmacytoid dendritic cells (pDC); TLR8 is expressed in monocytes and myeloid dendritic cells (mDC) (Iwasaki, A. and R. Medzhitov (2004). "Toll-like receptor control of the adaptive immune responses." Nat Immunol 5 (10): 987-995.).

[0004] In addition to the natural ligand single-stranded RNA, imidazoquinolones (or 'imiquimod-like' ligands) and guanosine analogs have also been shown to activate TLR7 and / or 8 with different specificities. Activation of TLR7 and / or TLR8 triggers the maturation of dendritic cells (DCs) and the secretion of proinflammatory cytokines (van Duin, D. et al. (2006). "Triggering TLR signaling invaccination." Trends Immunol 27 (1): 49-55.). Stimulated DCs further activate and proliferate CTL and NK cells through cytokine and antigen presentation. Therefore, the properties of TLR agonists constitute an effective strategy to enhance anti-cancer immunity (Adams, S. (2009). "Toll-like receptor agonists in cancer therapy." Immunotherapy 1 (6): 949-964.).

[0005] Imiquimod (TLR7 agonist) has been successfully used as a single antitumor agent with immunostimulatory ability to treat many primary skin tumors and skin metastases (Stary, G. et al. (2007). "Tumoricidal activity of TLR7 / 8-activated inflammatory dendritic cells." J Exp Med 204(6):1441-1451., Aranda, F. et al. (2014). "Trial Watch: Toll-like receptor agonists in oncological indications." Oncoimmunology 3:e29179.).

[0006] WO 2016023511 discloses pyrrolopyrimidine compounds as TLR7 agonists for use in treating antiviral drugs. Currently, a lot of effort has been invested in the preclinical and clinical development of TLR agonists for cancer therapy. Summary of the invention

[0007] The present inventors have developed a method for preparing imidazo[2,1-f][1,2,4]triazine-4-amines having TLR7 agonist activity, in particular compounds A1, A2, A3 and A4.

[0008] In a first aspect, disclosed herein is a method for preparing a TLR7 agonist having a structure of formula (I) by the following synthetic scheme,

[0009] Option I:

[0010]

[0011] R 1 Yes - NHR 1a , where R 1a It is a branched-C 4-8 alkyl;

[0012] R 2 , R 3 and R 4 It is hydrogen;

[0013] Ring A is phenyl or pyridyl;

[0014] Z is a direct key;

[0015] R 6 is piperazinyl or further optionally substituted by one or two substituents R 6c Substituted piperazinyl, R 6c Yes-COR6d or -C 1-8 Alkyl, the -C 1-8 The alkyl group is optionally substituted by one, two or three substituents R 6g replace;

[0016] R 6d is optionally substituted by one or two or three substituents R 6g Replaced-C 1-6 alkyl;

[0017] R 6g In each occurrence, it is -OR independently 6h or -NR 6h R 6i ,

[0018] R 6h and R 6i are independently hydrogen, -C 1-6 Alkyl, or -C 1-6 Alkoxy, the -C 1-6 Alkyl or -C 1-6 Each of the alkoxy groups is independently and optionally substituted with one or two or three substituents selected from the group consisting of halogen, -C 1-4 alkyl, optionally substituted with amino-C 1-4 Alkoxy, hydroxy, -NH2, -C 1-6 Alkylamino, or di-C 1-6 Alkylamino.

[0019] In some embodiments, R 1a is a branched chain selected from the following 4-8 Alkyl: butan-2-yl, pentan-2-yl, pentan-3-yl, heptan-2-yl, heptan-3-yl, heptan-4-yl, octan-2-yl, octan-3-yl, octan-4-yl or octan-5-yl, wherein the branched substituent is alpha to the nitrogen atom.

[0020] In some embodiments, R 5 and ZR 6 In the ortho position on ring A.

[0021] In some embodiments, R 6c yes

[0022] i)-COR 6d , where R 6d is optionally substituted by one or two substituents R 6g Replaced-C 1-3 Alkyl, where R 6g Yes - NR 6h R 6i or -C 1-3 Alkyl, where R6h and R 6i are each independently hydrogen or -C 1-3 Alkyl; and

[0023] ii) which is optionally substituted by one or two substituents R 6g Replaced-C 1-4 Alkyl, where R 6g Yes-OR 6h and-NR 6h R 6i , where R 6h and R 6i are independently hydrogen or -C 1-3 alkyl, and the -C 1-3 The alkyl group is optionally substituted by: -C 1-3 Alkoxy or amino substituted -C 1-3 Alkoxy.

[0024] In some embodiments, ZR 6 Selected from:

[0025] 4-(2-((2-hydroxyethyl)amino)acetyl)piperazin-1-yl, 4-(2-(methylamino)acetyl)piperazin-1-yl), 4-(2-(ethylamino)acetyl)piperazin-1-yl), 4-(3-aminopropyl)piperazin-1-yl, 4-(2(2-(2-aminoethoxy)ethoxy)ethyl)piperazin-1-yl, 4-(2-aminoethyl)piperazin-1-yl, or 4-((methylamino)methyl)piperidin-1-yl.

[0026] In some embodiments, Ring A is pyridinyl, and Z and ZR 6 At the para position of the phenyl or pyridyl ring, each of the rings is further optionally represented by an R 5 Substitution, where Z, R 5 and R 6 As defined in formula (I).

[0027] In some embodiments, intermediate 10 is prepared by the following scheme,

[0028] Scheme II.

[0029]

[0030] In some embodiments, the TLR7 agonist is selected from Compounds A1, A2, A3, and A4. DETAILED DESCRIPTION

[0031] definition

[0032] The following terms have the indicated meanings throughout this specification:

[0033] As used herein, including the appended claims, singular forms such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0034] Unless the context clearly dictates otherwise, the term "or" is used to mean, and is interchangeable with, the term "and / or."

[0035] The term "alkyl" herein refers to a hydrocarbon group selected from straight and branched saturated hydrocarbon groups, which contains from 1 to 18, such as from 1 to 12, further such as from 1 to 10, further such as from 1 to 8, or from 1 to 6, or from 1 to 4 carbon atoms. Examples of alkyl groups containing from 1 to 6 carbon atoms (i.e., C1-6 alkyl) include, but are not limited to, methyl, ethyl, 1-propyl or n-propyl ("n-Pr"), 2- Propyl or isopropyl ("i-Pr"), 1-butyl or n-butyl ("n-Bu"), 2-methyl-1-propyl or isobutyl ("i-Bu"), 1-methylpropyl or sec-butyl ("s-Bu"), 1,1-dimethylethyl or tert-butyl ("t-Bu"), 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl and 3,3-dimethyl-2-butyl.

[0036] The term "alkoxy" or "alkyloxy" refers to an alkyl group as previously defined, attached to the parent molecular moiety through an oxygen atom.

[0037] The term "amino" refers to -NH2. The term "alkylamino" refers to -NH(alkyl). The term "dialkylamino" refers to -N(alkyl). The term "halogen" as used herein refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0038] The term "alkyloxy" or "alkoxy" refers herein to an alkyl group as defined above attached to the parent molecular moiety through an oxygen atom. Examples of alkoxy groups such as C1-6 alkyloxy or C1-4 alkyloxy groups include, but are not limited to, methoxy, ethoxy, isopropoxy, propoxy, n-butoxy, tert-butoxy, pentyloxy, and hexyloxy groups, etc.

[0039] Preparation of TLR7 agonists

[0040] In certain aspects, the drug moiety is a TLR7 agonist as disclosed herein, including salts thereof, and can be prepared using known organic synthesis techniques and can be synthesized according to any of a variety of possible synthetic routes, examples of which are provided below.

[0041] The reaction for preparing the compounds disclosed herein can be carried out in a suitable solvent that can be easily selected by a technician in the field of organic synthesis. Suitable solvents can be substantially free from reacting with starting materials, intermediates or products at a temperature (e.g., a temperature in the range of room temperature to the boiling point of the solvent) for the reaction. A given reaction can be carried out in a solvent or a solvent mixture.

[0042] The selection of appropriate protecting groups can be readily determined by one skilled in the art.

[0043] The reaction can be monitored according to any suitable method known in the art, such as NMR, UV, HPLC, LC-MS and TLC. The compounds can be purified by a variety of methods, including HPLC and normal phase silica chromatography.

[0044] Chiral analytical HPLC was used for retention time analysis of different chiral examples. The conditions were divided into the following methods according to the column, mobile phase, and solvent ratio used.

[0045] The TLR7 agonists disclosed herein can be prepared by the following synthetic scheme.

[0046] Option I:

[0047]

[0048] Where R 1 , R 5 , Ring A, Z, R 6 and p are as defined for formula (I).

[0049] In Scheme I, commercially available 1H-imidazole-2-carboxylic acid ethyl ester is reacted with 2-O-(4-nitrobenzoyl)hydroxylamine to form intermediate 2, which is reacted with ethyl chloroformate and then ring-closed in the presence of ammonium hydroxide to give imidazo[2,1-f][1,2,4]triazine-2,4(1H,3H)-dione. Intermediate 5 is obtained after one Br atom is introduced using a brominating agent, and the diketone is then chlorinated to form intermediate 5. One chlorine atom is replaced with a protected amine, and the other chlorine atom is reacted with R 1 -H reaction to give intermediate 8, which is then reacted with different aldehydes under basic conditions to form intermediate 10. Removal of the protected groups on the amine and hydroxyl groups, coupling with different acids under basic conditions and deprotection affords a compound of formula (I).

[0050] Intermediate 10 was also prepared by Scheme II below.

[0051] Scheme II.

[0052]

[0053] In Scheme II, intermediate 4 is chlorinated to form intermediate 6'. One chlorine atom is replaced with a protected amine to obtain intermediate 7', and it is reacted with R 1 H reaction to give the key intermediate 8', which was subsequently reacted with different aldehydes under basic conditions to form intermediate 10.

[0054] The following examples are intended to be purely exemplary and should not be construed as limiting in any way. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, reagents and materials are commercially available. All solvents and chemicals used are of analytical grade or chemical purity. Solvents were redistilled before use. Anhydrous solvents were prepared according to standard methods or reference methods. Silica gel (100-200 mesh) for column chromatography and silica gel (GF254) for thin layer chromatography (TLC) are commercially available from Tsingdao Haiyang Chemical Co., Ltd. or Yantai Chemical Co., Ltd., China; all were eluted with petroleum ether (60°C-90°C) / ethyl acetate (v / v) and visualized by solutions of iodine or phosphomolybdic acid in ethanol, unless otherwise specified. Unless otherwise specified, all extraction solvents were dried over anhydrous Na2SO4. Recorded on a Bruck-400 nuclear magnetic resonance spectrometer 1 H NMR spectra with TMS (tetramethylsilane) as internal standard. LC / MS data were recorded using an Agilent 1100 high performance liquid chromatography-ion trap mass spectrometer (LC-MSD trap) equipped with a diode array detector (DAD) and ion trap (ESI source) detecting at 214 nm and 254 nm. All compound names except reagents were generated by ChemDraw.

[0055] Example

[0056] The compounds in Examples 1 and 2 below are intended to be purely exemplary and should not be considered to be limiting in any way. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, reagents and materials are commercially available. All solvents and chemicals used are of analytical grade or chemical purity. Solvents were redistilled before use. Anhydrous solvents were prepared according to standard methods or reference methods. Silica gel (100-200 mesh) for column chromatography and silica gel (GF254) for thin layer chromatography (TLC) are commercially available from Tsingdao Haiyang Chemical Co., Ltd. or Yantai Chemical Co., Ltd., China; all were eluted with petroleum ether (60°C-90°C) / ethyl acetate (v / v) and visualized by solutions of iodine or phosphomolybdic acid in ethanol, unless otherwise specified. Unless otherwise specified, all extraction solvents were dried over anhydrous Na2SO4. Recorded on a Bruck-400 nuclear magnetic resonance spectrometer 1 H NMR spectra with TMS (tetramethylsilane) as internal standard. LC / MS data were recorded using an Agilent 1100 high performance liquid chromatography-ion trap mass spectrometer (LC-MSD trap) equipped with a diode array detector (DAD) detecting at 214 nm and 254 nm and an ion trap (ESI source). All compound names except reagents are generate.

[0057] Example 1: Synthesis of TLR7 agonists

[0058] Compound A1: (S)-1-(4-(5-((4-amino-2-(pentan-2-ylamino)imidazo[2,1-f][1,2,4]triazin-7-yl)methyl)-3-methylpyridin-2-yl)piperazin-1-yl)-2-((2-hydroxyethyl)amino)ethan-1-one

[0059]

[0060] Step A: (S)-1-(4-(5-((4-amino-2-(pentan-2-ylamino)imidazo[2,1-f][1,2,4]triazin-7-yl)methyl)-3-methylpyridin-2-yl)piperazin-1-yl)-2-chloroethan-1-one

[0061] To a mixture of (S)-7-((5-methyl-6-(piperazin-1-yl)pyridin-3-yl)methyl)-N2-(pentan-2-yl)imidazo[2,1-f][1,2,4]triazine-2,4-diamine (409 mg, 1 mmol) and TEA (202 mg, 2 mmol) in DCM (20 mL) at 0°C was added a solution of 2-chloroacetyl chloride (112 mg, 1 mmol) in DCM (1 mL) dropwise. The reaction was stirred at room temperature for 2 hours. The reaction solution was diluted with water, extracted with DCM (60 mL x 2), washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by combi flash to give the target compound (320 mg, 66%). (M+1)+. 1H NMR(400MHz,CD3OD)δ8.05(s,1H),7.51(s,1H),7.20 (s,1H),4.29(s,2H),4.09(s,2H),3.92-3.80(m,1H),3.78-3.65(m,4H),3.1 8-3.11(m,2H),3.10-3.03(m,2H),2.28(s,3H),1.70-1.30(m,4H),1.15(d,J= 6.8Hz, 3H), 0.91 (t, J = 6.8Hz, 3H) ppm. MS:M / e 486(M+1)+.

[0062] Step B: (S)-1-(4-(5-((4-amino-2-(pentan-2-ylamino)imidazo[2,1-f][1,2,4]triazin-7-yl)methyl)-3-methylpyridin-2-yl)piperazin-1-yl)-2-((2-hydroxyethyl)amino)ethan-1-one (Compound A1).

[0063] To a mixture of (S)-1-(4-(5-((4-amino-2-(pentan-2-ylamino)imidazo[2,1-f][1,2,4]triazin-7-yl)methyl)-3-methylpyridin-2-yl)piperazin-1-yl)-2-chloroethan-1-one (48 mg, 0.1 mmol) and DIEA (25 mg, 0.2 mmol) in DMF (5 mL) was added 2-aminoethan-1-ol (12 mg, 0.2 mmol). The reaction was stirred at room temperature for 15 hours. The reaction was diluted with water, extracted with DCM / MeOH (4 / 1, 60 mL x 2), washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by preparative HPLC to give the target compound (10 mg, 16%). 1HNMR(400MHz,DMSO-d6)δ8.21(s,1H),8.08(s,1H),7.60-7.35(m,3H), 7.15(s,1H),5.99(d,J=8.8Hz,1H),3.98(s,2H),3.90-3.72(m,1H),3.65- 3.44(m,8H),3.05-2.86(m,5H),2.70-2.58(m,2H),2.20(s,3H),1.60-1.24(m,4H),1.09(d,J=6.0Hz,3H),0.90-0.80(m,3H).ppmMS:M / e 511 (M+1)+.

[0064] Compound A2: (S)-7-((6-(4-(2-(2-(2-aminoethoxy)ethoxy)ethyl)piperazin-1-yl)-5-methylpyridin-3-yl)methyl)-N2-(pentan-2-yl)imidazo[2,1-f][1,2,4]triazine-2,4-diamine

[0065]

[0066] Step A: 2,2-Dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl 4-methylbenzenesulfonate.

[0067] To a solution of tert-butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (1 g, 4.02mmol), DMAP (49mg, 0.402mmol), EtN (487mg, 4.82mmol) in DCM (20mL) was added dropwise TsCl (917mg, 4.83mmol) in DCM (5mL) under N2. The reaction mixture was stirred at 25°C for 12h. After completion, the solvent was concentrated under vacuum to obtain a residue. The residue was purified by column chromatography on silica gel eluting with ethyl acetate / petroleum ether (50%) to provide the title compound (1.48g, 91%). 1 H NMR(400MHz, DMSO-d6)δ7.79(d,J=8.0Hz,2H),7.48(d,J=7.6Hz,2H),6.77(s,1H),4.11 (s,2H),3.57(s,2H),3.43(s,4H),3.35(s,2H),3.04(d,J=5.6Hz,2H),2.42(s,3H),1.36(s,9H).MS:M / e 426(M+Na)+.

[0068] Step B: (S)-tert-butyl (2-(2-(2-(4-(5-((4-amino-2-(pentan-2-ylamino)imidazo[2,1-f][1,2,4]triazin-7-yl)methyl)-3-methylpyridin-2-yl)piperazin-1-yl)ethoxy)ethoxy)ethyl)carbamate

[0069] To a solution of (S)-7-((5-methyl-6-(piperazin-1-yl)pyridin-3-yl)methyl)-N2-(pentan-2-yl)imidazo[2,1-f][1,2,4]triazine-2,4-diamine (80 mg, 0.196 mmol), 4-methylbenzenesulfonic acid 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecane-13-yl ester (118 mg, 0.293 mmol) in DMF (3 mL) under N2 was added DMAP (3 mg, 0.0245 mmol). The reaction mixture was stirred at 80°C for 12 h. The mixture was purified by preparative TLC (DCM / MeOH=15 / 1) to provide the title compound (60 mg, 48%). MS: M / e 642 (M+H) + .

[0070] Step C: (S)-7-((6-(4-(2-(2-(2-aminoethoxy)ethoxy)ethyl)piperazin-1-yl)-5-methylpyridin-3-yl)methyl)-N2-(pentan-2-yl)imidazo[2,1-f][1,2,4]triazine-2,4-diamine (Compound A2)

[0071] To a solution of (S)-tert-butyl(2-(2-(2-(4-(5-((4-amino-2-(pentan-2-ylamino)imidazo[2,1-f][1,2,4]triazin-7-yl)methyl)-3-methylpyridin-2-yl)piperazin-1-yl)ethoxy)ethoxy)ethyl)carbamate (60 mg, 0.094 mmol) in DCM (6 mL) and MeOH (2 mL) was added HCl (0.6 mL). The reaction mixture was stirred at 25 °C for 6 h. The solvent was removed under vacuum. The residue was purified by preparative-TLC (DCM / MeOH (7M NH3 in MeOH)=10 / 1) to provide the title compound (25 mg, 49%). 1H NMR(400MHz,CD3OD)δ8.02(s, 1H),7.48(s,1H),7.19(s,1H),4.08(s,2H),3.85(s,1H),3.66(s,8H),3.11(s, 4H),3.02(s,2H),2.69(s,6H),2.24(s,3H),1.54(s,1H),1.40(s,3H),1.15(d,J =4.0Hz,3H),0.91(d,J=7.2Hz,3H)ppm. MS:M / e 541.(M+H)+.

[0072] Compound A3: (S)-7-((6-(4-(3-aminopropyl)piperazin-1-yl)-5-methylpyridin-3-yl)methyl)-N2-(pentan-2-yl)imidazo[2,1-f][1,2,4]triazine-2,4-diamine

[0073]

[0074] Step A: (S)-tert-butyl(3-(4-(5-((4-amino-2-(pentan-2-ylamino)imidazo[2,1-f][1,2,4]triazin-7-yl)methyl)-3-methylpyridin-2-yl)piperazin-1-yl)propyl)carbamate

[0075] To a solution of (S)-7-((5-methyl-6-(piperazine-1-yl)pyridin-3-yl)methyl)-N2-(pentan-2-yl)imidazo[2,1-f][1,2,4]triazine-2,4-diamine (600 mg, 1.47 mmol), tert-butyl (3-oxopropyl)carbamate (763 mg, 4.41 mmol) and AcOH (0.2 mL) in DCM (10 mL) and MeOH (3 mL) was added NaBH(OAc) (623 mg, 2.94 mmol). The reaction mixture was stirred at 25 °C for 12 h. After completion, the reaction mixture was quenched with water (20 mL) and extracted with DCM (3 x 50 mL). The combined organic layers were dried over NaSO and concentrated under vacuum to obtain a residue. The residue was purified by column chromatography on silica gel eluting with methanol in dichloromethane (14%) to provide the title compound (720 mg, 86%). MS: M / e 567. (M+H) + .

[0076] Step B: (S)-7-((6-(4-(3-aminopropyl)piperazin-1-yl)-5-methylpyridin-3-yl)methyl)-N2-(pentan-2-yl)imidazo[2,1-f][1,2,4]triazine-2,4-diamine (Compound A3)

[0077] To a solution of (S)-tert-butyl(3-(4-(5-((4-amino-2-(pentan-2-ylamino)imidazo[2,1-f][1,2,4]triazine-7-yl)methyl)-3-methylpyridin-2-yl)piperazin-1-yl)propyl)carbamate (720 mg, 1.27 mmol) in DCM (18 mL) and MeOH (6 mL) was added HCl (5 mL). The reaction mixture was stirred at 25 °C for 6 h. The solvent was removed under vacuum. The residue was diluted with DCM (30 mL) and quenched with Na2CO3 aqueous solution (15 mL) and extracted with DCM / MeOH (10 / 1, 3 x 50 mL). The combined organic layers were dried over Na2SO4 and concentrated under vacuum to obtain the crude product (500 mg). It was used in the next step without further purification. The crude product (80 mg) was purified by preparative TLC (DCM / CH3OH (7M NH3 in MeOH) = 8 / 1) to afford the title compound (52 mg). 1 H NMR(400MHz,CD3OD)δ8.02(s,1H),7.48(s, 1H),7.19(s,1H),4.07(s,2H),3.85(d,J=5.6Hz,1H),3.11(s,4H),2.81(t,J =6.8Hz,2H),2.64(s,4H),2.51(t,J=7.2Hz,2H),2.24(s,3H),1.83-1.68(m, 2H), 1.60-1.48 (m, 1H), 1.47-1.28 (m, 3H), 1.14 (d, J = 6.4Hz, 3H), 0.91 (d, J = 7.2Hz, 3H) ppm. MS:M / e467.(M+H)+.

[0078] Compound A4: (S)-7-((6-(4-(2-aminoethyl)piperazin-1-yl)-5-methylpyridin-3-yl)methyl)-N2-(pentan-2-yl)imidazo[2,1-f][1,2,4]triazine-2,4-diamine

[0079]

[0080] Step A: (S)-tert-butyl(2-(4-(5-((4-amino-2-(pentan-2-ylamino)imidazo[2,1-f][1,2,4]triazin-7-yl)methyl)-3-methylpyridin-2-yl)piperazin-1-yl)ethyl)carbamate

[0081] To a solution of (S)-7-((5-methyl-6-(piperazin-1-yl)pyridin-3-yl)methyl)-N2-(pentan-2-yl)imidazo[2,1-f][1,2,4]triazine-2,4-diamine (80 mg, 0.196 mmol), tert-butyl (2-oxoethyl)carbamate (32 mg, 0.201 mmol) and AcOH (20 uL) in DCM (3 mL) and MeOH (1 mL) was added NaBH(OAc)3 (85 mg, 0.401 mmol). The reaction mixture was stirred at 25 °C for 3 h. After completion, the solvent was concentrated under vacuum to obtain a residue. The residue was purified by preparative TLC (DCM / MeOH=15 / 1) to provide the title compound (70 mg, 65%). MS: M / e553. (M+H) + .

[0082] Step B: (S)-7-((6-(4-(2-aminoethyl)piperazin-1-yl)-5-methylpyridin-3-yl)methyl)-N2-(pentan-2-yl)imidazo[2,1-f][1,2,4]triazine-2,4-diamine (Compound A4)

[0083] To a solution of (S)-tert-butyl(2-(4-(5-((4-amino-2-(pentan-2-ylamino)imidazo[2,1-f][1,2,4]triazin-7-yl)methyl)-3-methylpyridin-2-yl)piperazin-1-yl)ethyl)carbamate (70 mg, 0.127 mmol) in DCM (9 mL) and MeOH (3 mL) was added HCl (1 mL). The reaction mixture was stirred at 25 °C for 6 h. The solvent was removed under vacuum. The residue was purified by preparative HPLC to provide the title compound (17 mg, 30%). 1 H NMR (400MHz, CD3OD) δ8.02(s, 1H),7.48(s,1H),7.19(s,1H),4.07(s,2H),3.86(s,1H),3.11(s,4H),2.84(s, 2H), 2.64 (s, 4H), 2.55 (d, J = 6.0Hz, 2H), 2.24 (s, 3H), 1.53 (s, 1H), 1.40 (s, 3H), 1.14 (d, J = 6.4Hz, 3H), 0.91 (d, J = 7.2Hz, 3H) ppm. MS:M / e 454.(M+H)+.

[0084] Example 2: In vitro assay: Determination of TLR7 stimulation using HEK-Blue Detection

[0085] This assay is designed to study the stimulation of human TLR7 protein in the HEK-BluehTLR7 tool cell line by monitoring the activation of NF-κB.

[0086] HEK-BluehTLR7 cells are obtained by co-transfecting the hTLR7 gene and the optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene into HEK293 cells. The SEAP reporter gene is placed under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Stimulation with TLR7 ligands activates NF-κB and AP-1, which induce the expression of SEAP. The level of SEAP can be easily determined with HEK-Blue Detection, which is a cell culture medium that allows real-time detection of SEAP. HEK-Blue Detection contains all the nutrients required for cell growth and a specific SEAP color substrate. The hydrolysis of the substrate by SEAP produces a purple / blue color that can be measured with a spectrophotometer.

[0087] When grown to 50%-80% confluence, HEK-Blue hTLR7 cells were plated at a density of 40,000 cells / well in 96-well plates (costar 3599). Test compounds were then added in a 10-point serial dilution with a final concentration range of 0.04nM-10μM in 0.1% DMSO / HEK-Blue Detection. The plates were then incubated at 37°C in 5% CO2 for 16h and vortexed for 30s before measurement. The optical density at 620-655nm was read on a BMG PHERAstar FSX instrument. The EC for each compound was determined by calculating the maximum activation percentage identified with resiquimod. 50 The EC values ​​for each compound are reported in Table 1. 50 .

[0088] Table 1

[0089]

[0090] Although the present disclosure has been described in conjunction with the specific embodiments set forth above, many substitutions, modifications and other changes will be apparent to those skilled in the art. All such substitutions, modifications and changes are intended to fall within the spirit and scope of the present disclosure.

Claims

1. A method for preparing a TLR7 agonist having a structure of Formula I, wherein the TLR7 agonist is prepared by the following synthetic scheme, Option I: R 1 Yes - NHR 1a , where R 1a It is a branched-C 4-8 alkyl; Ring A is phenyl or pyridyl; (R 5 ) p It is methyl; Z is a direct key; R 6 is piperazinyl or further optionally substituted by one or two substituents R 6c Substituted piperazinyl, R 6c Yes-COR 6d or -C 1-8 Alkyl, the -C 1-8 The alkyl group is optionally substituted by one, two or three substituents R 6g replace; R 6d is optionally substituted by one or two or three substituents R 6g Replaced-C 1-6 alkyl; R 6g -OR independently at each occurrence 6h or -NR 6h R 6i , R 6h and R 6i are independently hydrogen, -C 1-6 Alkyl, or -C 1-6 Alkoxy, the -C 1-6 Alkyl or -C 1-6 Each of the alkoxy groups is independently and optionally substituted with one or two or three substituents selected from the group consisting of halogen, -C 1-4 alkyl, optionally substituted with amino-C 1-4 Alkoxy, hydroxy, -NH2, -C 1-6 Alkylamino, or di-C 1-6 Alkylamino.

2. The method according to claim 1, wherein R 1a is a branched chain selected from the following 4-8 Alkyl: butan-2-yl, pentan-2-yl, heptan-2-yl or octan-2-yl, wherein the branched substituent is in the alpha position relative to the nitrogen atom.

3. The method according to claim 1, wherein R 5 and ZR 6 In the ortho position on ring A.

4. The method according to claim 1, wherein R 6c yes iii) -COR 6d , where R 6d is optionally substituted by one or two substituents R 6g Replaced-C 1-3 alkyl, Where R 6g Yes - NR 6h R 6i or -OC 1-3 Alkyl, where R 6h and R 6i are each independently hydrogen or -C 1-3 Alkyl; and iv) which is optionally substituted by one or two substituents R 6g Replaced-C 1-4 Alkyl, where R 6g Yes-OR 6h and-NR 6h R 6i , where R 6h and R 6i are independently hydrogen or -C 1-3 alkyl, and the -C 1-3 The alkyl group is optionally substituted by: -C 1-3 Alkoxy or amino substituted -C 1-3 Alkoxy.

5. The method according to claim 1, wherein ZR 6 Selected from: 4-(2-((2-hydroxyethyl)amino)acetyl)piperazin-1-yl, 4-(2-(methylamino)acetyl)piperazin-1-yl), 4-(2-(ethylamino)acetyl)piperazin-1-yl), 4-(3-aminopropyl)piperazin-1-yl, 4-(2(2-(2-aminoethoxy)ethoxy)ethyl)piperazin-1-yl, 4-(2-aminoethyl)piperazin-1-yl, or 4-((methylamino)methyl)piperidin-1-yl.

6. The method according to claim 1, wherein ring A is pyridyl, and Z and ZR 6 In the para position of the pyridyl ring, the ring is optionally replaced by an R 5 Substitution, where Z, R 5 and R 6 As defined in formula (I).

7. The method according to claim 1, wherein intermediate 10 is prepared by the following scheme, Scheme II:

8. The method according to claim 1, wherein the TLR7 agonist is selected from the group consisting of compounds A1 A2 A3 and A4

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