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CN114057746B - Preparation of imidazo[2,1-F][1,2,4]triazine-4-amine derivatives as TLR7 agonists - Google Patents

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

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CN114057746B
CN114057746B CN202110885870.1A CN202110885870A CN114057746B CN 114057746 B CN114057746 B CN 114057746B CN 202110885870 A CN202110885870 A CN 202110885870A CN 114057746 B CN114057746 B CN 114057746B
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CN114057746A (en
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张国良
苗建壮
王策
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Baiji Shenzhou Beijing Biotechnology Co ltd
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Abstract

本文公开了一种制备可用作TLR7激动剂的咪唑并[2,1‑f][1,2,4]三嗪‑4‑胺衍生物或其立体异构体或其药学上可接受的盐的方法。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

Preparation of imidazo [2,1-F ] [1,2,4] triazin-4-amine derivatives as TLR7 agonists
Technical Field
Disclosed herein is a method of preparing imidazo [2,1-f ] [1,2,4] triazin-4-amine derivatives, or stereoisomers thereof, or pharmaceutically acceptable salts thereof, that are useful as TLR7 agonists.
Background
Toll-like receptors (TLRs) belong to a family of Pattern Recognition Receptors (PRRs) that play a key role in early innate immune responses by sensing a wide variety of pathogen molecular patterns (PAMP) that are highly conserved, as well as endogenous risk-related molecular patterns (DAMP) (Barton, G.M. and R.Medzhitov (2002), "oll-like receptors and their ligands.," Curr Top Microbiol Immunol 270:81-92.).
Ten different TLRs have been identified in humans. Wherein TLR7, TLR8 and TLR9 belong to the same TLR subfamily based on their genomic structure, sequence similarity and endosomal localization. They have a limited expression pattern, 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.).
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 maturation of Dendritic Cells (DCs) and secretion of pro-inflammatory cytokines (van Duin, d. et al (2006), "TRIGGERING TLR SIGNALING IN vaccination.," Trends Immunol 27 (1): 49-55.). Stimulated DCs further activate and proliferate CTL and NK cells through cytokine and antigen presentation. Thus, the properties of TLR agonists constitute an effective strategy for enhancing anti-cancer immunity (Adams, s. (2009), "Toll-like receptor agonists in cancer therapy.," Immunotherapy (6): 949-964.).
Imiquimod (TLR 7 agonist) was successfully used as a single anti-tumor agent with immunostimulating ability for the treatment of 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.).
WO 2016023511 discloses pyrrolopyrimidine compounds as TLR7 agonists for use in the treatment of antiviral drugs. Currently, preclinical and clinical development of TLR agonists for cancer therapy has invested a great deal of effort.
Disclosure of Invention
The present inventors developed methods for preparing imidazo [2,1-f ] [1,2,4] triazin-4-amines, especially compounds A1, A2, A3 and A4, with TLR7 agonist activity.
In a first aspect, disclosed herein is a method of preparing a TLR7 agonist having the structure of formula (I) by the following synthetic scheme,
Scheme I:
R 1 is-NHR 1a wherein R 1a is branched-C 4-8 alkyl;
R 2、R3 and R 4 are hydrogen;
ring a is phenyl or pyridinyl;
z is a direct bond;
R 6 is piperazinyl or piperazinyl further optionally substituted with one or two substituents R 6c, R 6c is-COR 6d or-C 1-8 alkyl, said-C 1-8 alkyl optionally being substituted with one or two or three substituents R 6g;
R 6d is-C 1-6 alkyl optionally substituted with one or two or three substituents R 6g;
R 6g is independently at each occurrence-OR 6h OR-NR 6hR6i,
R 6h and R 6i are independently hydrogen, -C 1-6 alkyl, or-C 1-6 alkoxy, each of said-C 1-6 alkyl or-C 1-6 alkoxy being independently and optionally substituted with one or two or three substituents selected from halogen, -C 1-4 alkyl, optionally amino-substituted-C 1-4 alkoxy, hydroxy, -NH 2、-C1-6 alkylamino, or di-C 1-6 alkylamino.
In some embodiments, R 1a is a branched-C 4-8 alkyl group selected from butan-2-yl, penta-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 in the alpha position relative to the nitrogen atom.
In some embodiments, R 5 and Z-R 6 are ortho to ring A.
In some embodiments, R 6c is
I) -COR 6d, wherein R 6d is-C 1-3 alkyl optionally substituted by one or two substituents R 6g, wherein R 6g is-NR 6hR6i or-C 1-3 alkyl, wherein R 6h and R 6i are each independently hydrogen or-C 1-3 alkyl optionally substituted by hydroxy, and
Ii) a-C 1-4 alkyl group optionally substituted with one OR two substituents R 6g, wherein R 6g is-OR 6h and-NR 6hR6i, wherein R 6h and R 6i are each independently hydrogen OR-C 1-3 alkyl, and the-C 1-3 alkyl group is optionally substituted with: -C 1-3 alkoxy OR amino substituted-C 1-3 alkoxy.
In some embodiments, Z-R 6 is 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-aminoethoxy) ethoxy) ethyl) piperazin-1-yl, 4- (2-aminoethyl) piperazin-1-yl, or 4- ((methylamino) methyl) piperidin-1-yl.
In some embodiments, ring a is pyridinyl and Z-R 6 are para to the phenyl or pyridinyl ring, each of said rings being further optionally substituted with one R 5, wherein Z, R 5 and R 6 are as defined in formula (I).
In some embodiments, intermediate 10 is prepared by the following scheme,
Scheme II.
In some embodiments, the TLR7 agonist is selected from compounds A1, A2, A3, and A4.
Detailed Description
Definition of the definition
The following terms have the indicated meanings throughout the specification:
as used herein, including the appended claims, the singular forms such as "a," "an," and "the" include plural references corresponding thereto, unless the context clearly dictates otherwise.
The term "or" is used to mean and be used interchangeably with the term "and/or" unless the context clearly dictates otherwise.
The term "alkyl" refers herein to a hydrocarbon group selected from straight and branched chain saturated hydrocarbon groups comprising from 1 to 18, such as from 1 to 12, further such as from 1 to 10, still 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 groups) 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-dimethylethyl or tert-butyl ("t-Bu"), 1-pentyl, 2-pentyl, 3-pentyl, 2-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, 3-dimethyl-2-butyl, and 3, 3-dimethyl-2-butyl.
The term "alkoxy" or "alkyloxy" refers to an alkyl group, as previously defined, attached to the parent molecular moiety through an oxygen atom.
The term "amino" refers to-NH 2. The term "alkylamino" refers to-NH (alkyl). The term "dialkylamino" refers to-N (alkyl) 2. The term "halogen" refers herein to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).
The term "alkyloxy" or "alkoxy" herein means an alkyl group, as defined above, appended to the parent molecular moiety through an oxygen atom. Examples of alkoxy groups such as C1-6 alkyl oxy or C1-4 alkyl oxy include, but are not limited to, methoxy, ethoxy, isopropoxy, propoxy, n-butoxy, t-butoxy, pentoxy, hexoxy, and the like.
Preparation of TLR7 agonists
In certain aspects, the drug moiety is a TLR7 agonist as disclosed herein, including salts thereof, and can be prepared using known organic synthetic techniques and can be synthesized according to any of a number of possible synthetic routes, examples of which are provided below.
The reactions for preparing the compounds disclosed herein can be carried out in suitable solvents that can be readily selected by those skilled in the art of organic synthesis. Suitable solvents may be substantially unreactive with the starting materials, intermediates, or products at the temperature at which the reaction is carried out (e.g., temperatures which may range from room temperature to the boiling temperature of the solvent). The given reaction may be carried out in one solvent or a mixture of solvents.
The selection of the appropriate protecting group can be readily determined by one skilled in the art.
The reaction may be monitored according to any suitable method known in the art, such as NMR, UV, HPLC, LC-MS and TLC. The compounds may be purified by a variety of methods, including HPLC and normal phase silica chromatography.
Chiral analytical HPLC was used for retention time analysis of different chiral examples, the conditions being divided into the following methods depending on the column, mobile phase, solvent ratios used.
TLR7 agonists disclosed herein can be prepared by the following synthetic schemes.
Scheme I:
Wherein R 1、R5, ring A, Z, R 6 and p are as defined for formula (I).
In scheme I, commercially available ethyl 1H-imidazole-2-carboxylate is reacted with 2-O- (4-nitrobenzoyl) hydroxylamine to form intermediate 2, intermediate 2 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 introduction of one Br atom using a brominating reagent, and then the diketone is chlorinated to form an intermediate. One chlorine atom is substituted with a protected amine and the other chlorine atom is reacted with R 1 -H to give intermediate 8, which is then reacted with a different aldehyde under basic conditions to form intermediate 10. The protected groups and hydroxyl groups on the amine are removed and, under basic conditions and deprotection, coupled with various acids, a compound of formula (I) is obtained.
Intermediate 10 was also prepared by scheme II below.
Scheme II.
In scheme II, intermediate 4 is chlorinated to form intermediate 6'. One chlorine atom is substituted with a protected amine to obtain intermediate 7 'and reacted with R 1 H to obtain the key intermediate 8', which is then reacted with a different aldehyde under basic conditions to form intermediate 10.
The following examples are intended to be purely exemplary and should not be considered limiting in any way. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Reagents and materials are commercially available unless otherwise specified. All solvents and chemicals used were of analytical grade or chemical purity. The solvents were redistilled before use. The anhydrous solvents are prepared according to standard or reference methods. Silica gel for column chromatography (100-200 mesh) and silica gel for Thin Layer Chromatography (TLC) (GF 254) are commercially available in china Tsingdao HAIYANG CHEMICAL co., ltd. Or YANTAI CHEMICAL co., ltd., all eluted with petroleum ether (60-90 ℃) in ethyl acetate (v/v) and visualized by iodine or phosphomolybdic acid in ethanol unless otherwise specified. All extraction solvents were dried over anhydrous Na 2SO4 unless specified otherwise. 1 H NMR spectra were recorded on a Bruck-400 NMR spectrometer, with TMS (tetramethylsilane) as internal standard. LC/MS data were recorded by using an Agilent1100 high performance liquid chromatography-ion trap mass spectrometer (LC-MSD trap) equipped with Diode Array Detectors (DAD) and ion traps (ESI sources) detecting at 214nm and 254 nm. All compound names except for the reagent were generated by ChemDraw.
Examples
The compounds in examples 1 and 2 below are intended to be purely exemplary and should not be considered limiting in any way. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Reagents and materials are commercially available unless otherwise specified. All solvents and chemicals used were of analytical grade or chemical purity. The solvents were redistilled before use. The anhydrous solvents are prepared according to standard or reference methods. Silica gel for column chromatography (100-200 mesh) and silica gel for Thin Layer Chromatography (TLC) (GF 254) are commercially available in china Tsingdao HAIYANG CHEMICAL co., ltd. Or YANTAI CHEMICAL co., ltd., all eluted with petroleum ether (60-90 ℃) in ethyl acetate (v/v) and visualized by iodine or phosphomolybdic acid in ethanol unless otherwise specified. All extraction solvents were dried over anhydrous Na 2SO4 unless specified otherwise. 1 H NMR spectra were recorded on a Bruck-400 NMR spectrometer, with TMS (tetramethylsilane) as internal standard. LC/MS data were recorded by using an Agilent1100 high performance liquid chromatography-ion trap mass spectrometer (LC-MSD trap) equipped with Diode Array Detectors (DAD) and ion traps (ESI sources) detecting at 214nm and 254 nm. All compound names except for the reagent are given byAnd (5) generating.
EXAMPLE 1 Synthesis of TLR7 agonists
Compound A1 (S) -1- (4- (5- ((4-amino-2- (pent-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
Step A (S) -1- (4- (5- ((4-amino-2- (pent-2-ylamino) imidazo [2,1-f ] [1,2,4] triazin-7-yl) methyl) -3-methylpyridin-2-yl) piperazin-1-yl) -2-chloroethan-1-one
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 ℃ 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. Purifying the residue by combi flash to obtain the target compound (320mg, 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),3.18-3.11(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)+.
Step B (S) -1- (4- (5- ((4-amino-2- (pent-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).
To a mixture of (S) -1- (4- (5- ((4-amino-2- (pent-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. Times.2), washed with brine, dried over Na 2SO4, filtered and concentrated. Purification of the residue by preparative HPLC gives the title compound (10mg,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).ppm MS:M/e 511 (M+1)+.
Compound A2 (S) -7- ((6- (4- (2- (2- (2-aminoethoxy) ethoxy) ethyl) piperazin-1-yl) -5-methylpyridin-3-yl) methyl) -N2- (pent-2-yl) imidazo [2,1-f ] [1,2,4] triazine-2, 4-diamine
Step A-4-methylbenzenesulfonic acid 2, 2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl ester.
To a solution of tert-butyl (2- (2- (2-hydroxyethoxy) ethoxy) ethyl) carbamate (1 g,4.02 mmol), DMAP (49 mg,0.402 mmol), etN 3 (487 mg,4.82 mmol) in DCM (20 mL) was added TsCl (917 mg, 4.83 mmol) in DCM (5 mL) dropwise under N 2. The reaction mixture was stirred at 25 ℃ 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％).1H 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)+.
Step B (S) - (2- (2- (2- (4- (5- ((4-amino-2- (pent-2-ylamino) imidazo [2,1-f ] [1,2,4] triazin-7-yl) methyl) -3-methylpyridin-2-yl) piperazin-1-yl) ethoxy) ethyl) carbamic acid tert-butyl ester
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-azatrideen-13-yl ester (118 mg,0.293 mmol) in DMF (3 mL) was added DMAP (3 mg,0.0245 mmol) under N 2. The reaction mixture was stirred at 80 ℃ for 12h. 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) +.
Step C (S) -7- ((6- (4- (2- (2- (2-aminoethoxy) ethoxy) ethyl) piperazin-1-yl) -5-methylpyridin-3-yl) methyl) -N2- (pent-2-yl) imidazo [2,1-f ] [1,2,4] triazine-2, 4-diamine (Compound A2)
To a solution of tert-butyl (S) - (2- (2- (4- (5- ((4-amino-2- (pent-2-ylamino) imidazo [2,1-f ] [1,2,4] triazin-7-yl) methyl) -3-methylpyridin-2-yl) piperazin-1-yl) 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 ℃ for 6h. The solvent was removed under vacuum. The residue was purified by preparative-TLC (DCM/MeOH (7M NH 3 in MeOH) =10/1) to provide the title compound (25mg,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)+.
Compound A3 (S) -7- ((6- (4- (3-aminopropyl) piperazin-1-yl) -5-methylpyridin-3-yl) methyl) -N2- (pent-2-yl) imidazo [2,1-f ] [1,2,4] triazine-2, 4-diamine
Step A (S) - (3- (4- (5- ((4-amino-2- (pent-2-ylamino) imidazo [2,1-f ] [1,2,4] triazin-7-yl) methyl) -3-methylpyridin-2-yl) piperazin-1-yl) propyl) carbamic acid tert-butyl ester
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 (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) 3 (627 mg,2.94 mmol). The reaction mixture was stirred at 25 ℃ for 12h. After completion, the reaction mixture was quenched with water (20 mL) and extracted with DCM (3×50 mL). The combined organic layers were dried over Na 2SO4 and concentrated in vacuo to give 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) +.
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)
To a solution of tert-butyl (S) - (3- (4- (5- ((4-amino-2- (pent-2-ylamino) imidazo [2,1-f ] [1,2,4] triazin-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 ℃ for 6h. The solvent was removed under vacuum. The residue was diluted with DCM (30 mL) and quenched with aqueous Na 2CO3 (15 mL) and extracted with DCM/MeOH (10/1, 3X 50 mL). The combined organic layers were dried over Na 2SO4 and concentrated in vacuo to give 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/CH 3 OH (7M NH 3 in MeOH) =8/1) to provide the title compound (52mg).1H 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/e 467.(M+H)+.
Compound A4 (S) -7- ((6- (4- (2-aminoethyl) piperazin-1-yl) -5-methylpyridin-3-yl) methyl) -N2- (pent-2-yl) imidazo [2,1-f ] [1,2,4] triazine-2, 4-diamine
Step A (S) - (2- (4- (5- ((4-amino-2- (pent-2-ylamino) imidazo [2,1-f ] [1,2,4] triazin-7-yl) methyl) -3-methylpyridin-2-yl) piperazin-1-yl) ethyl) carbamic acid tert-butyl ester
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 ℃ for 3h. After completion, the solvent was concentrated under vacuum to obtain a residue. The residue was purified by preparative TLC (DCM/meoh=15/1) to give the title compound (70 mg, 65%). MS: M/e 553. (M+H) +.
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)
To a solution of tert-butyl (S) - (2- (4- (5- ((4-amino-2- (pent-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 ℃ for 6h. The solvent was removed under vacuum. Purification of the residue by preparative HPLC provided the title compound (17mg,30％).1H 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)+.
Example 2 in vitro assay for determination of TLR7 stimulation using HEK-Blue Detection
This assay was designed to investigate stimulation of human TLR7 protein in HEK-BluehTLR tool cell lines by monitoring NF- κb activation.
HEK-BluehTLR cells were obtained by co-transfection of the hTLR7 gene and an 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-. Kappa.B and AP-1-binding sites. Stimulation with TLR7 ligands activates NF- κb and AP-1, which thereby induces expression of SEAP. The level of SEAP can be readily determined using HEK-Blue Detection, a cell culture medium that allows real-time Detection of SEAP. HEK-Blue Detection contains all nutrients and specific SEAP color substrates required for cell growth. Hydrolysis of the substrate by SEAP produces a purple/blue color that can be measured spectrophotometrically.
When grown to 50% -80% confluence, HEK-Blue hTLR7 cells were plated into 96-well plates (costar 3599) at a density of 40000 cells/well. Test compounds were then added in serial dilutions at 10 points in the final concentration range of 0.04 nM-10. Mu.M in 0.1% DMSO/HEK-Blue Detection. Plates were then incubated at 37 ℃ in 5% co 2 for 16h and vortexed for 30s before measurement. The optical density at 620-655nm was read on BMG PHERAstar FSX instrument. EC 50 for each compound was determined by calculating the percent maximum activation identified with resiquimod. EC 50 for each compound is reported in table 1.
TABLE 1
While the present disclosure has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications, and other variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications, and variations are intended to fall within the spirit and scope of the present disclosure.

Claims (8)

1. A method of preparing a TLR7 agonist having the structure of formula I, wherein the TLR7 agonist is prepared by the following synthetic scheme,
Scheme I:
R 1 is-NHR 1a wherein R 1a is branched-C 4-8 alkyl;
ring a is phenyl or pyridinyl;
(R 5)p is methyl;
z is a direct bond;
R 6 is piperazinyl or piperazinyl further optionally substituted with one or two substituents R 6c,
R 6c is-COR 6d or-C 1-8 alkyl, said-C 1-8 alkyl being optionally substituted by one or two or three substituents R 6g;
R 6d is-C 1-6 alkyl optionally substituted with one or two or three substituents R 6g;
R 6g is independently at each occurrence-OR 6h OR-NR 6hR6i,
R 6h and R 6i are independently hydrogen, -C 1-6 alkyl, or-C 1-6 alkoxy, each of said-C 1-6 alkyl or-C 1-6 alkoxy being independently and optionally substituted with one or two or three substituents selected from halogen, -C 1-4 alkyl, optionally amino-substituted-C 1-4 alkoxy, hydroxy, -NH 2、-C1-6 alkylamino, or di-C 1-6 alkylamino.
2. The method of claim 1, wherein R 1a is a branched-C 4-8 alkyl group selected from the group consisting of butan-2-yl, penta-2-yl, heptan-2-yl, and octan-2-yl, wherein the branched substituent is in the alpha position relative to the nitrogen atom.
3. The method of claim 1, wherein R 5 and Z-R 6 are ortho to ring a.
4. The method of claim 1, wherein R 6c is
Iii) -COR 6d, wherein R 6d is-C 1-3 alkyl optionally substituted by one or two substituents R 6g,
Wherein R 6g is-NR 6hR6i or-OC 1-3 alkyl, wherein R 6h and R 6i are each independently hydrogen or-C 1-3 alkyl optionally substituted with hydroxy, and
Iv) a-C 1-4 alkyl group optionally substituted with one OR two substituents R 6g, wherein R 6g is-OR 6h and-NR 6hR6i, wherein R 6h and R 6i are each independently hydrogen OR-C 1-3 alkyl, and the-C 1-3 alkyl group is optionally substituted with: -C 1-3 alkoxy OR amino substituted-C 1-3 alkoxy.
5. The method of claim 1, wherein Z-R 6 is 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-aminoethoxy) ethoxy) ethyl) piperazin-1-yl, 4- (2-aminoethyl) piperazin-1-yl, or 4- ((methylamino) methyl) piperidin-1-yl.
6. The method of claim 1, wherein ring a is pyridinyl and Z-R 6 are para to the pyridinyl ring, the ring optionally substituted with one R 5, wherein Z, R 5 and R 6 are as defined in formula (I).
7. The process of claim 1, wherein intermediate 10 is prepared by the following scheme, scheme II:
8. The method of claim 1, wherein the TLR7 agonist is selected from the group consisting of compounds
A1
A2
A3And
A4
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