CN112979647B - Azaindole derivative containing azaamino acid, preparation and application - Google Patents

Azaindole derivative containing azaamino acid, preparation and application Download PDF

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CN112979647B
CN112979647B CN202110271726.9A CN202110271726A CN112979647B CN 112979647 B CN112979647 B CN 112979647B CN 202110271726 A CN202110271726 A CN 202110271726A CN 112979647 B CN112979647 B CN 112979647B
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pyrrolo
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pyrimidin
pyridin
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陈文腾
应智敏
王思函
邵加安
杜雨棽
俞永平
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Zhejiang University ZJU
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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Abstract

The invention provides an azaindole derivative containing nitrogen and hetero amino acid, and preparation and application thereof. The compound has better inhibition effect on influenza virus in vitroEffect, IC in anti-influenza Virus polymerase and anti-influenza Virus replication50And EC50The nano-molar level is achieved, the compound of the invention has low toxicity, and most of the compound has CC (cancer cell center) of MDCK (Madin-Darby canine kidney) cells and A549 cells50Above 30 μ M, the selectivity of the compound is good. The compound provided by the invention aims at the PB2 subunit of influenza virus RNA polymerase, can be used for preparing anti-influenza drugs, can solve the drug resistance problem of the existing clinical anti-influenza drugs, and has a relatively high application value. The structural general formula (I) of the derivative is as follows:

Description

Azaindole derivative containing azaamino acid, preparation and application
Technical Field
The invention belongs to the field of pharmacy, and particularly relates to an azaindole derivative containing nitrogen and hetero amino acids, a preparation method, an intermediate and application thereof.
Background
The influenza viruses can be divided into four types of A, B, C and D, and the main types of the influenza viruses which can cause diseases for human beings are influenza A viruses and influenza B viruses. Approximately 300,000 to 500,000 deaths annually occur due to seasonal influenza infection. Vaccination against influenza has major limitations because there is no effective way to accurately predict influenza strains before each influenza season or pandemic. Therefore, small molecule anti-influenza drugs are very important in the prevention and treatment of influenza. Currently marketed antiviral drugs mainly comprise oseltamivir, zanamivir and peramivir which take a neuraminidase inhibitor as a target point; adamantane and amantadine targeting the M2 ion channel; and baroxavir taking the influenza polymerase PA subunit as a target point. Although these drugs can alleviate the impact of influenza to humans to some extent, the emergence of more and more resistant influenza virus strains has made the development of new influenza inhibitors urgent.
The influenza polymerase complex, which consists of PA, PB1 and PB2 subunits, is important in the replication and transcription of influenza viruses. In the process of transcription in eukaryotic cells, the capping of nascent mRNA is an essential process, but since influenza virus does not have a mechanism for synthesizing a cap structure, the virus steals its cap from host mRNA for the synthesis of self mRNA by means of a cap-scrambling mechanism. During viral transcription, the PB2 subunit in influenza polymerase binds to the 5 'cap of the pre-mRNA of the host cell first, and then the 5' cap of the pre-mRNA is cleaved off by the PA endonuclease for primers required for viral replication. The 3 'end of the capped RNA primer and the 3' end of the vRNA template are both located in the active cavity defined by PB1, and transcription begins with the viral genome as a template. Compared with the way that NAIs block the release of the virus, the research strategy taking influenza virus polymerase as a target block the initiation of the replication and transcription of the virus, which is equivalent to block the spread of the virus from the source, and provides a more effective antiviral action mechanism.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an azaindole derivative containing nitrogen hetero-amino acid, wherein the structural formula I is as follows:
Figure BDA0002974418840000011
wherein:
R1selected from H, F or Cl, CF3；
R2、R3Each independently hydrogen, methyl, tert-butyl, phenyl, cyclopropyl, or optionally R2And R3Together with the carbon atom to which they are attached form a 4-6 membered carbocyclic or heterocyclic ring;
R4selected from hydrogen, or optionally R2、R3And R4Together with the carbon atoms to which they are attached form a 5-10 membered carbon monocyclic, fused, spiro or bridged ring.
The azaindole derivative containing the side chain of the azahetero amino acid is any one of the following compounds:
n- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentyl glycine (Aza-01),
N-cyclopentyl-N- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-03),
N-cyclohexyl-N- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-04),
n- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N- (tetrahydro-2H-pyran-4-yl) glycine (Aza-07),
N-cyclobutyl-N- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-09),
N-cyclopentyl-N- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-14),
N-cyclohexyl-N- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-15), N-benzyl-N- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-16),
n- (1-cyclopropylethyl) -N- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-19),
(S) -2- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid (Aza-06),
1- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) piperidine-2-carboxylic acid (Aza-08),
(S) -2- (((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid (Aza-10),
1- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) piperidine-2-carboxylic acid (Aza-11),
(2S,3aS,7aS) -1- (((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) octahydro-1H-indole-2-carboxylic acid (Aza-12),
((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -L-proline (Aza-13),
(S) -5- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -5-azaspiro [2.4] heptane 6-carboxylic acid (Aza-17),
(2R,4R) -4-fluoro-1- (5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) pyrrolidine-2-carboxylic acid (Aza-18).
Another object of the present invention is to provide a preparation method of the compound I, which is realized by the following steps:
when R is4And when the compound is H, the synthesis of the compound I takes Boc-hydrazine 1 as a starting material, the Boc-hydrazine 1 reacts with substituted ketone at the temperature of 50 ℃ in toluene to generate imine compound 2, and the compound 2 is reduced under the conditions of sodium cyanoborohydride, acetic acid and tetrahydrofuran to generate substituted Boc-hydrazine compound 3. Reacting the compound 3 with ethyl bromoacetate under the reflux condition of potassium carbonate and toluene to generate a substituted compound 4, removing Boc from the compound 4 under the condition of trifluoroacetic acid to obtain a compound 5, and carrying out substitution reaction on the compound 5 and 2, 4-dichloro-5-fluoropyrimidine to obtain a compound 6. The compound 6 and azaindole borate (synthesis is according to the document J.Med.chem.2014,57,6668-6678) and Suzuki reaction are carried out to generate a compound 7, and the compound 7 is subjected to removal of two protecting groups under the action of lithium hydroxide to obtain a compound I (R, I and II are shown in the specification4Is H), the reaction formula:
Figure BDA0002974418840000031
reaction reagents and conditions: a) toluene, 50 ℃ for 1 hour, 90-95%; b) sodium cyanoborohydride, acetic acid, tetrahydrofuran, room temperature, overnight, 30-80%; c) potassium carbonate and toluene are refluxed for 24 hours, and the reaction time is 100 percent; d) trifluoroacetic acid, dichloromethane, room temperature, 2 hours, 100%; e) n, N-diisopropylethylamine and tetrahydrofuran, and the temperature is 20-86% at room temperature; f)1,1' -bis (diphenylphosphino) ferrocene palladium dichloride, cesium carbonate, 1, 4-dioxane-water, 110 ℃ for 4 hours, and 40-60%; g) lithium hydroxide, tetrahydrofuran-water, 50 ℃ for 4 hours, 50-60%. Wherein the substituents are as defined in the general formula.
When optionally R2、R3And R4When the compounds and carbon atoms connected with the compounds form 5-10 membered carbon monocyclic ring, fused ring, spiro ring or bridged ring, the compound I is synthesized by taking a compound 8 as a starting material, reacting the starting material with sodium nitrite and concentrated sulfuric acid to generate a nitroso compound 9, esterifying the compound 9 with methanol through the dehydration of the concentrated sulfuric acid to generate a compound 10, reducing the compound 10 through zinc powder to generate a compound 11, and dissolving the compound 11The compound 11 and 2, 4-dichloro-5-fluoropyrimidine are subjected to substitution reaction to obtain a compound 12. Azaindole borate (synthesis according to J.Med.chem.2014,57, 6668-one 6678) and compound 12 undergo a Suzuki reaction to generate compound 13, and compound 13 is subjected to removal of two protecting groups under the action of lithium hydroxide to obtain compound I (optional R is selected as R6678)2、R3And R4Together with the carbon atom to which they are attached form a 5-10 membered carbon monocyclic, fused, spiro or bridged ring), according to the formula:
Figure BDA0002974418840000032
reaction reagents and conditions: a) sodium nitrite, 3M sulfuric acid, water, ice bath-room temperature, 3 hours, 80-90%; b) concentrated sulfuric acid and methanol at room temperature, and the concentration is 80-95% at night; c) ammonium chloride, zinc powder, tetrahydrofuran-water, ice bath-room temperature, 10-20 minutes, 50-60%; d) n, N-diisopropylethylamine and tetrahydrofuran, and the reaction time is 50-55% at room temperature for 4 hours; e) 1,1' -bis-diphenylphosphino ferrocene palladium dichloride, cesium carbonate, 1, 4-dioxane-water, 110 ℃ for 4 hours, 40-60%; g) lithium hydroxide, tetrahydrofuran-water, 50 ℃ for 4 hours, 50-60%. Wherein the substituents are as defined in the general formula.
The starting materials or intermediates of the present invention can be purchased directly or prepared according to literature methods mentioned in the examples.
The invention further aims to provide application of the azaindole derivatives containing the azaamino acid side chain in preparing anti-influenza medicaments. The compounds of the invention are directed to the PB2 subunit of influenza virus RNA polymerase.
The invention has the beneficial effects that: firstly, the series of compounds have better inhibition effect on influenza virus in vitro and IC in anti-influenza virus polymerase and anti-influenza virus replication50And EC50The nanomolar level is reached. Secondly, the series of compounds have low toxicity, and most of the compounds have CC (continuous peptide) of MDCK (Madin-Darby Canada) cells and A549 cells50Above 30 μ M, the selectivity of the compound is good. Again, the compounds of this series are directed against the PB2 subunit of influenza virus RNA polymerase, which is currently availableThe drug is not on the market. The antiviral drug developed aiming at the target point can possibly solve the drug resistance problem of the existing clinical anti-influenza drug, and has a great application value.
Drawings
FIG. 1. ITC test results for Compound Aza-12.
Detailed Description
The present invention is further illustrated by the following examples, but is not intended to be limited thereby, and experimental procedures not specifically identified in the following examples, according to conventional procedures and conditions, or according to commercial specifications, are selected.
Example 1N- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine (Aza-01);
the method comprises the following steps:
raw material 1: 3-bromo-1-tosyl-1H-pyrrolo [2,3-b ] pyridine
Figure BDA0002974418840000041
3-bromo-1H-pyrrolo [2,3-b ] pyridine (10mmol) is dissolved in DMF, NaH (15mmol) is added in portions in an ice bath, and after reaction for 30min, p-toluenesulfonyl chloride is added. TLC monitoring, when the raw material disappeared, adding water into the system to stop the reaction, separating out a large amount of solid, and stirring for 30min at room temperature to completely separate out the solid. The filtrate gave a meat-pink solid with a yield of 93%.
1H NMR(500MHz,CDCl3)δ8.33(dd,J＝5.0,1.5Hz,1H),7.93(d,J＝8.5Hz,2H),7.67-7.64(m,2H),7.15(s,1H),7.13-7.10(m,2H),2.23(s,3H)。
Raw material 2: preparation of tert-butyl 2- (2, 2-dimethylpropylidene) hydrazine-1-carboxylate:
Figure BDA0002974418840000042
tert-Butoxycarbonylhydrazine (10mmol) was dissolved in 10mL of toluene, pivalaldehyde (10mmol) was added thereto at room temperature, and the reaction was allowed to warm to 50 ℃ for 1 hour. Solid is separated out in the reaction process. The reaction was monitored by TLC plate, directly filtered after the reaction was complete and washed with a small amount of dichloromethane to give a white solid with a yield of 95%.
1H NMR(500MHz,CDCl3)δ7.65(s,1H),7.05(s,1H),1.46(s,9H),1.09(s,9H)。
Raw materials 3: preparation of 2-neopentyl hydrazine-1-carboxylic acid tert-butyl ester
Figure BDA0002974418840000051
(E) Tert-butyl (2, 2-dimethylpropylidene) hydrazine-1-carboxylate (1mmol) was suspended in tetrahydrofuran (5mL), and acetic acid (0.2mL) was added at room temperature followed by addition of sodium cyanoborocyanide (2.5mmol) in portions. The reaction was carried out overnight at room temperature. After the reaction was complete, ethyl acetate (15mL) and saturated sodium bicarbonate (15mL) were added to the reaction mixture. After collecting the organic layer, the aqueous layer was neutralized with solid sodium bicarbonate and extracted twice with ethyl acetate. The organic fractions were combined, washed with saturated sodium bicarbonate and brine, dried over sodium sulfate and concentrated in vacuo. The residue was then dissolved in methanol (3mL) and 1N NaOH (3mL) was added. After stirring at room temperature for 2h, the methanol was evaporated and the aqueous layer was extracted 3 times with ethyl acetate. The combined organic portions were washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel chromatography to give a pale yellow liquid with a yield of 52%.
1H NMR(500MHz,CDCl3)δ6.46(s,1H),4.48(s,1H),3.04-3.01(m,1H),2.95-2.90(m,1H),1.50(s,9H),1.04(s,9H)。
Raw materials 4: preparation of tert-butyl 2- (2-ethoxy-2-oxyethyl) -2-neopentylhydrazine-1-carboxylate
Figure BDA0002974418840000052
To a solution of tert-butyl 2-neopentylhydrazine-1-carboxylate (1mmol) in toluene (10mL) were added potassium carbonate (2mmol) and ethyl bromoacetate (1.5 mmol). The reaction was heated to reflux for 24 hours. The reaction mixture was cooled to ambient temperature, the solid removed by suction filtration and the filtrate partitioned between 1N hydrochloric acid and diethyl ether. The aqueous layer was extracted twice with ether and the organic layer was discarded. The pH of the aqueous layer was adjusted to 9 with 2.5N sodium hydroxide and extracted 3 times with ether. The combined organic extracts were washed with brine, dried over anhydrous sodium sulfate and the filtrate was spin-dried without further purification and directly charged to the next step.
Raw material 5: preparation of ethyl N- ((2-chloro-5-fluoropyrimidin-4-yl) amino) -N-neopentyl glycinate
Figure BDA0002974418840000053
Tert-butyl 2- (2-ethoxy-2-oxoethyl) -2-neopentylhydrazine-1-carboxylate (1mmol) was dissolved in dichloromethane (3mL) and trifluoroacetic acid (3mL) was added under ice bath and stirred at room temperature for 2 h. And after the reaction is completed, the system is dried by spinning, and then is dissolved in 5mL of tetrahydrofuran for later use. 2, 4-dichloro-5-fluoropyrimidine (1mmol) was dissolved in THF (10mL) at room temperature with stirring, and the above-mentioned stock solution was added to the stirred solution, followed by diisopropylethylamine (2.0equiv.) and reacted at room temperature for 2 hours. The reaction mixture was concentrated in vacuo to remove most of the THF, and the residue was dissolved in dichloromethane and washed with saturated NaHCO3And (4) washing the solution. The organics were dried and concentrated and the crude product was purified by silica gel chromatography (PET/EA ═ 15:1) to afford a brown solid in 60% yield.
1H NMR(500MHz,CDCl3)δ7.90(d,J＝3.0Hz,1H),7.33(s,1H),4.20(q,J＝7.0Hz,2H),3.75(s,2H),2.80(s,2H),1.28(t,J＝7.0Hz,3H),0.91(s,9H)。
Raw material 6: preparation of ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine
Figure BDA0002974418840000061
3-bromo-1-tosyl-1H-pyrrolo [2,3-b ]]Pyridine (1mmol) with pinacol ester of diboronic acid (1.5mmol) and potassium acetate(3mmol)，PdCl2(dppf) (0.11mmol) was placed in a three-necked flask, and nitrogen was substituted three times, after which 10mL of anhydrous dioxane was added, and the temperature was raised to 90 ℃ to react for about 3 hours. TLC monitoring reaction until the raw material disappears, naturally cooling, directly adding cesium carbonate (1.5equiv.), PdCl into the system without post-treatment2(dppf) (0.1equiv.) and ethyl N- ((2-chloro-5-fluoropyrimidin-4-yl) amino) -N-neopentylglycine (1.1equiv.), followed by addition of 0.5mL of water and stirring at 110 ℃ for 4 h. After the reaction was complete, the system was filtered through celite to remove most of the metal, most of the solvent was adjusted by rotation, extracted three times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, evaporated by rotation, and the crude product was purified by silica gel chromatography (PET/EA ═ 5:1) to give a brown solid with a yield of 54% in two steps.
1H NMR(500MHz,CDCl3)δ9.01(d,J＝7.5Hz,1H),8.53(s,1H),8.45(dd,J＝5.0,2.0Hz,1H),8.13–8.09(m,3H),7.27(s,1H),7.26–7.22(m,2H),7.17(d,J＝1.5Hz,1H),4.22(q,J＝7.5Hz,2H),3.83(s,2H),2.87(s,2H),2.36(s,3H),1.29(d,J＝7.5Hz,3H),0.95(s,9H)。
Aza-01: preparation of N- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine (Aza-01);
Figure BDA0002974418840000062
to a solution of ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine (1mmol) in THF (5mL) was added aqueous 1N LiOH (2.5 mL). The mixture was heated to 50 ℃ and reacted for 3.5 hours. The reaction mixture was cooled to room temperature and diluted with water. THF was removed under reduced pressure and the residue was washed twice with n-hexane. Ether was added and the layers were separated (ether layer discarded). The pH was adjusted to 5.5 with 1N HCl, the resulting solid was filtered and washed with water and dried to give a beige solid in 54% yield.
m.p.:>250℃；1H NMR(500MHz,DMSO-d6)δ12.12(s,1H),9.29(s,1H),9.00(d,J＝7.0Hz,1H),8.25(dd,J＝4.5,1.5Hz,1H),8.15(d,J＝4.0Hz,1H),8.07(s,1H),7.15–7.10(m,1H),3.32(s,2H),2.80(s,2H),0.92(s,9H).13C NMR(125MHz,DMSO-d6)δ174.1,158.6(d,J＝6.25Hz),151.4(d,J＝7.50Hz),149.1,143.2,142.7(d,J＝252.50Hz),130.5,128.1(d,J＝15.00Hz),125.6,118.3,116.5,113.9,70.2,62.6,32.4,27.7.HRMS(ESI):m/z calcd for(C18H21FN6O2+H)+:373.1783；found:373.1780.
The following compounds were prepared in the same manner as in example 1, using different starting materials.
Example 2N-cyclopentyl-N- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-03)
With reference to the procedure of example 1, except that ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine was replaced by ethyl N-cyclopentyl-N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycinate. White solid, yield 49%.
m.p.:>250℃；1H NMR(500MHz,DMSO-d6)δ12.13(s,1H),9.66(s,1H),8.9(d,J＝7.5Hz,1H),8.25(dd,J＝5.0,2.0Hz,1H),8.16(d,J＝4.0Hz,1H),8.10(s,1H),7.17-7.14(m,1H),3.70-3.65(m,1H),3.37(s,2H),1.67-1.65(m,6H),1.50-1.48(m,2H).13C NMR(125MHz,DMSO-d6)δ173.9,158.5(d,J＝6.25Hz),152.1(d,J＝7.50Hz),149.1,143.1,142.1(d,J＝261.25Hz),130.4,128.2(d,J＝3.75Hz),125.6,118.3,116.6,113.6,65.7,58.3,30.8,23.8.HRMS(ESI):m/z calcd for(C18H19FN6O2+H)+:371.1626；found:371.1623。
Example 3N-cyclohexyl-N- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-04)
With reference to the procedure of example 1, except that ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine was replaced by ethyl N-cyclohexyl-N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycinate. White solid, yield 51%.
m.p.:>205.5-206.9℃；1H NMR(500MHz,DMSO-d6)δ12.09(s,1H),8.69(dd,J＝8.0,1.5Hz,1H),8.27(dd,J＝4.5,1.5Hz,1H),8.17-8.16(m,2H),7.20-7.17(m,1H),4.79(s,2H),4.35–4.27(m,1H),1.86-1.82(m,2H),1.80-1.73(m,4H),1.68-1.66(m,1H),1.44–1.37(m,2H),1.17-1.17(m,1H).13C NMR(125MHz,DMSO-d6)δ157.5(d,J＝5.00Hz),152.4(d,J＝1.25Hz),149.1,143.3(d,J＝253.75Hz),143.1,129.6,128.7,125.5,118.0,116.6,113.5,57.4,49.4,32.1,28.9,25.3.HRMS(ESI):m/z calcd for(C19H21FN6O2+H)+:385.1783；found:385.1779。
Example 4N- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N- (tetrahydro-2H-pyran-4-yl) glycine (Aza-07)
Referring to the procedure of example 1 except substituting N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine ethyl ester for N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N- (tetrahydro-2H-pyran-4-ylglycine ethyl ester brown solid in 33% yield.
m.p.:221.2-223.9℃；1H NMR(500MHz,DMSO-d6)δ12.18(s,1H),9.55(s,1H),8.68-8.66(m,1H),8.35-8.27(m,2H),8.19(s,1H),7.23(s,1H),3.97(s,2H),3.53-3.35(m,4H),1.85-1.64(m,4H).13C NMR(125MHz,DMSO-d6)δ157.7(d,J＝6.25Hz),155.7,151.3,149.2,143.3,142.3(d,J＝225.00Hz),129.4,129.0,117.9,116.8,113.2,79.8,66.4,54.5,29.3.28.1.27.8.HRMS(ESI):m/z calcd for(C18H19FN6O2+H)+:387.1575；found:387.1569。
Example 5N-cyclobutyl-N- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-09)
With reference to the procedure of example 1, except that ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine was replaced with ethyl N-cyclobutyl-N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycinate. White solid, yield 47%.
m.p.:>211.3-212.1℃；1H NMR(500MHz,DMSO-d6)δ12.41(s,1H),9.31(s,1H),9.01-8.99(m,1H),8.45-8.37(m,1H),8.33-8.28(m,1H),7.49(d,J＝6.5Hz,1H),7.31-7.22(m,1H),7.11(d,J＝6.5Hz,1H),3.66(s,2H),2.31-2.24(m,1H),2.05-1.95(m,4H),1.65-1.57(m,2H).13C NMR(125MHz,DMSO-d6)δ171.1,155.7(d,J＝2.50Hz),153.3(d,J＝12.50Hz),148.9,143.8,141.1(d,J＝257.50Hz),130.7,129.6,128.1,125.5,118.2,117.4,59.4,54.7,26.2,13.8.HRMS(ESI):m/z calcd for(C17H17FN6O2+H)+:357.1470；found:357.1468.
Example 6N-cyclopentyl-N- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-14)
With reference to the procedure of example 1, except that ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycinate was replaced by N-cyclopentyl-N- ((5-fluoro-2- (5-fluoro-1-tolyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycinate. Brown solid, yield 61%.
m.p.:>250℃；1H NMR(500MHz,DMSO-d6)δ12.34(s,1H),9.00(s,1H),8.76-8.71(m,1H),8.25-8.24(m,1H),8.18(d,J＝4.5Hz,1H),8.16(d,J＝2.5Hz,1H),1.76–1.57(m,8H),1.53-1.43(m,3H).13C NMR(125MHz,DMSO)δ173.7,158.2(d,J＝6.25Hz),157.2,155.3,152.7,146.1,142.3(d,J＝253.75Hz),132.0(d,J＝28.75Hz),130.8,119.1(d,J＝7.50Hz),116.6(d,J＝22.50Hz),113.9(d,J＝3.75Hz),66.7,58.0,31.1,24.2.HRMS(ESI):m/z calcd for(C18H18F2N6O2+H)+:389.1532；found:389.1537。
Example 7N-cyclohexyl-N- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-15)
With reference to the procedure of example 1, except that ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine was replaced by N-cyclohexyl-N- ((5-fluoro-2- (5-fluoro-1-tolyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycinate. Brown solid in 54% yield.
m.p.:>250℃；1H NMR(500MHz,DMSO-d6)δ12.42(s,1H),9.59(s,1H),8.77(s,1H),8.25-8.22(m,1H),8.14(s,1H),7.51-7.45(m,1H),7.15-7.08(m,1H),3.09(s,2H),2.28(s,1H),1.94–1.86(m,2H),1.78-1.72(m,2H),1.54-1.48(m,1H),1.32-1.12(m,5H).13C NMR(125MHz,DMSO)δ174.4,156.7(d,J＝3.75Hz),154.8,152.1(d,J＝11.25Hz),145.9,145.7,142.3(d,J＝251.25Hz),137.8,131.5(d,J＝31.25Hz),130.3,118.7(d,J＝7.50Hz),115.9(d,J＝26.25Hz),113.8(d,J＝5.00Hz),61.9,56.3,30.4,29.1,25.8,24.1,20.9.HRMS(ESI):m/z calcd for(C19H20F2N6O2+H)+:403.1689；found:403.1691。
Example 8N-benzyl-N- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-16)
With reference to the procedure of example 1, except that ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycinate was replaced by N-benzyl-N- ((5-fluoro-2- (5-fluoro-1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycinate. White solid, yield 31%.
m.p.:>250℃；1H NMR(500MHz,DMSO-d6)δ12.37(s,1H),9.93(s,1H),8.78-8.69(m,1H),8.29-8.26(m,1H),8.20(s,1H),8.15(d,J＝4.0Hz,1H),7.55-7.50(m,2H),7.28-7.26(m,2H),7.23-7.18(m,1H),4.19(s,2H),3.30(s,2H).HRMS(ESI):m/z calcd for(C20H16F2N6O2+H)+:411.1376；found:411.1382。
Example 9N- (1-Cyclopropylethyl) -N- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycine (Aza-19)
With reference to the procedure of example 1, except that ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine was replaced by ethyl N- (1-cyclopropylethyl) -N- ((5-fluoro-2- (5-fluoro-1-p-sulfanyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) glycinate. White solid, yield 68%.
m.p.:214.8-215.6℃；1H NMR(500MHz,DMSO-d6)δ12.40(s,1H),12.31(s,1H),8.81-8.76(m,1H),8.72(s,1H),8.32(s,1H),8.28(d,J＝4.0Hz,1H),8.24-8.21(m,1H),3.92(s,2H),3.46-3.38(m,1H),1.96-1.95(m,1H),1.22(d,J＝6.5Hz,3H),0.94-0.86(m,1H),0.53-0.44(m,2H),0.17-0.14(m,1H).13C NMR(125MHz,DMSO)δ171.9,157.5(d,J＝5.00Hz),156.7,154.8,152.2,145.9,142.1(d,J＝253.75Hz),131.6(d,J＝28.75Hz),130.1,118.5(d,J＝7.50Hz),115.7(d,J＝22.50Hz),113.7(d,J＝2.50Hz),64.8,55.5,21.1,17.6,5.9,1.9.HRMS(ESI):m/z calcd for(C18H18F2N6O2+H)+:389.1532；found:389.1528。
Example 10(S) -2- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid (Aza-06)
Starting material 1: 3-bromo-1-tosyl-1H-pyrrolo [2,3-b ] pyridine:
Figure BDA0002974418840000101
3-bromo-1H-pyrrolo [2,3-b ] pyridine (10mmol) is dissolved in DMF, NaH (15mmol) is added in portions in an ice bath, and after reaction for 30min, p-toluenesulfonyl chloride is added. TLC monitoring, when the raw material disappeared, adding water into the system to stop the reaction, separating out a large amount of solid, and stirring for 30min at room temperature to completely separate out the solid. The meat was filtered off with suction to give a pink solid with a yield of 93%.
1H NMR(500MHz,CDCl3)δ8.33(dd,J＝5.0,1.5Hz,1H),7.93(d,J＝8.5Hz,2H),7.67–7.64(m,2H),7.15(s,1H),7.13–7.10(m,2H),2.23(s,3H)。
Preparation of starting material 2(S) -2-nitroso-1, 2,3, 4-tetrahydroisoquinoline-3-carboxylic acid:
Figure BDA0002974418840000102
(S) -1,2,3, 4-four hydrogen isoquinoline-3-carboxylic acid (10mmol) dissolved in water, added with sodium nitrite (10.0equiv.), ice bath under the dropwise addition of 20mL 3M sulfuric acid, yellow gas appears. When the reaction is complete, white solid is separated out, and the white solid obtained by suction filtration is the product, and the yield is 70%.
1H NMR(500MHz,DMSO-d6)δ13.23(s,1H),7.31–7.20(m,4H),5.99(dd,J＝5.5,3.0Hz,1H),4.94(d,J＝19.5Hz,1H),4.55(d,J＝19.5Hz,1H),3.41(dd,J＝8.0,5.5Hz,2H)。
Raw material 3: preparation of (S) -2-nitroso-1, 2,3, 4-tetrahydroisoquinoline-3-carboxylic acid methyl ester
Figure BDA0002974418840000103
2-nitroso-1, 2,3, 4-tetrahydroisoquinoline-3-carboxylic acid (1mmol) was dissolved in methanol, concentrated sulfuric acid was added thereto, and the mixture was stirred overnight. After the reaction is finished, the residue is dissolved in dichloromethane after the system is dried by spinning, is washed for multiple times by saturated sodium bicarbonate solution, organic layers are combined by extraction, and Na is added2SO4After drying, the organic layer was spin dried to give a colorless liquid with a yield of 87%.
1H NMR(500MHz,DMSO-d6)δ7.30–7.23(m,4H),6.15(t,J＝4.0Hz,1H),4.97(d,J＝19.0Hz,1H),4.56(d,J＝19.0Hz,1H),3.56(s,3H),3.43(d,J＝4.0Hz,2H)。
Raw materials 4: preparation of (S) -2- ((2-chloro-5-fluoropyrimidin-4-yl) amino) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid methyl ester
Figure BDA0002974418840000104
2-nitroso-1, 2,3, 4-tetrahydroisoquinoline-3-carboxylic acid methyl ester (1mmol) was dissolved in THF, and ammonium chloride (15mmol) and zinc powder (10mmol) in THF/H were added under ice bath2O system, room temperature reaction, TLC point plate monitoring. After the reaction is finished, removing residual zinc powder by suction filtration, and using dichloromethane for filtrateExtraction, saturated brine washing and Na2SO4Drying, spin-drying the organic phase at low temperature, and directly putting into the next step without further purification. The method is the same as N- ((2-chloro-5-fluoropyrimidin-4-yl) amino) -N-neopentyl glycine ethyl ester, except that 2- (2-ethoxy-2-oxyethyl) -2-neopentyl hydrazine-1-carboxylic acid tert-butyl ester is replaced by 2-amino-1, 2,3, 4-tetrahydroisoquinoline-3-carboxylic acid methyl ester, the reaction conditions and the molar weight of materials are unchanged, yellow solid is obtained, and the yield is 68%.
1H NMR(500MHz,CDCl3)δ8.01(d,J＝6.0Hz,1H),7.27–7.18(m,4H),5.47(s,1H),5.07(d,J＝16.0Hz,1H),4.88(d,J＝16.0Hz,1H),3.63(s,3H),3.36(dd,J＝15.5,3.5Hz,1H),3.27(dd,J＝16.0,5.5Hz,1H)。
Raw material 5: preparation of methyl (S) -methyl 2- ((5-fluoro-2- (1-tolyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylate
Figure BDA0002974418840000111
3-bromo-1-tosyl-1H-pyrrolo [2,3-b ]]Pyridine (1mmol) with pinacol diboron (1.5mmol) and potassium acetate (3mmol), PdCl2(dppf) (0.11mmol) was placed in a three-necked flask, and nitrogen was substituted three times, after which 10mL of anhydrous dioxane was added, and the temperature was raised to 90 ℃ to react for about 3 hours. TLC monitoring reaction until the raw material disappears, naturally cooling, directly adding cesium carbonate (1.5equiv.), PdCl into the system without post-treatment2(dppf) (0.1equiv.) and (S) -methyl 2- ((2-chloro-5-fluoropyrimidin-4-yl) amino) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylate (1.1equiv.), then 0.5mL of water was added, and stirring was carried out at 110 ℃ for 4 hours. After the reaction was complete, the system was filtered through celite to remove most of the metal, most of the solvent was adjusted by rotation, extracted three times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, evaporated by rotation, and the crude product was purified by silica gel chromatography (PET/EA ═ 5:1) to give a brown solid with a yield of 57% in two steps.
1H NMR(500MHz,DMSO-d6)δ9.05(s,1H),8.88(dd,J＝8.0,1.0Hz,1H),8.37-8.34(m,3H),8.20(dd,J＝4.5,1.5Hz,1H),8.04(d,J＝8.5Hz,2H),7.96–7.92(m,1H),7.47(t,J＝3.0Hz,1H),7.41(d,J＝8.0Hz,2H),7.05-7.03(m,1H),6.43(dd,J＝3.5,2.0Hz,1H),4.40-4.37(m,1H),4.27–4.13(m,2H),3.55(s,3H),3.25(s,2H),2.32(s,3H)。
Aza-06: preparation of (S) -2- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid (Aza-06);
Figure BDA0002974418840000112
to a solution of (S) -methyl 2- ((5-fluoro-2- (1-tolyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid methyl ester (1mmol) in THF (5mL) was added 1N aqueous LiOH solution (2.5 mL). The mixture was heated to 50 ℃ and reacted for 3.5 hours. The reaction mixture was cooled to room temperature and diluted with water. THF was removed under reduced pressure and the residue was washed twice with n-hexane. Ether was added and the layers were separated (ether layer discarded). The pH was adjusted to 5.5 with 1N HCl, the resulting solid was filtered and washed with water and dried to give a brown solid in 67% yield.
m.p.:>250℃；1H NMR(500MHz,DMSO-d6)δ12.05(s,1H),10.04(s,1H),8.76(dd,J＝8.0,1.5Hz,1H),8.20(d,J＝4.0Hz,1H),8.18-8.17(m,1H),8.05(s,1H),7.48-7.46(m,1H),7.16-7.13(m,1H),7.12-7.08(m,2H),7.03-7.01(m,1H),6.87-6.85(m,1H),4.34(d,J＝14.0Hz,1H),4.17(d,J＝14.0Hz,1H),3.61(d,J＝5.0Hz,1H),3.45(d,J＝16.0Hz,1H),3.16(dd,J＝16.0,6.0Hz,1H).13C NMR(125MHz,DMSO-d6)δ174.2,158.6(d,J＝7.00Hz),150.5(d,J＝8.50Hz),149.0,143.0,142.6(d,J＝252.75Hz),134.4,133.7,130.3,128.5,128.0(d,J＝11.25Hz),126.2,125.8,125.5,125.0,118.3,116.4,113.8,61.9,52.4,32.1.HRMS(ESI):m/z calcd for(C21H17FN6O2+H)+:405.1470；found:405.1477。
Example 111- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) piperidine-2-carboxylic acid (Aza-08);
with reference to the procedure of example 10, except that ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine was replaced by methyl 1- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) piperidine-2-carboxylate. Yellow solid, yield 35%.
m.p.:>250℃；1H NMR(500MHz,DMSO-d6)δ12.17(s,1H),9.77(s,1H),8.96(d,J＝6.5Hz,1H),8.26-8.25(m,1H),8.16-8.14(m,2H),7.48(d,J＝7.5Hz,1H),7.18–7.11(m,1H),3.37-3.36(m,1H),3.19-3.10(m,1H),2.94-2.85(m,1H),2.30-2.28(m,1H),2.25-2.17(m,1H),1.71-1.66(m,2H),1.46-1.24(m,2H).13C NMR(125MHz,DMSO-d6)δ176.2,159.0(d,J＝3.75Hz),151.0(d,J＝5.0Hz),149.5,143.6,142.8(J＝250.00Hz),130.9,128.6(d,J＝16.25Hz),126.0,118.8,117.0,114.0,65.4,52.0,29.3,26.1,21.3.HRMS(ESI):m/z calcd for(C17H17FN6O2+H)+:357.1470；found:357.1473。
Example 12(S) -2- (((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid (Aza-10);
referring to the procedure of example 10, except for replacing N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine ethyl ester with (S) -2- ((5-fluoro-2- (5-fluoro-1-p-sulfanyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid methyl ester. Yellow solid, yield 65%.
m.p.:>228.7-230.5℃；1H NMR(500MHz,DMSO-d6)δ12.24(s,1H),10.14(s,1H),8.57-8.55(m,1H),8.20(d,J＝4.0Hz,1H),8.16-8.15(m,2H),7.16-7.05(m,4H),7.01(d,J＝7.5Hz,1H),4.35(d,J＝14.0Hz,1H),4.14(d,J＝14.0Hz,1H),3.62-3.61(m,1H),3.46-3.43(m,1H),3.20-3.15(m,1H).13C NMR(125MHz,DMSO)δ174.4,158.2(d,J＝6.25Hz),156.5,154.6,150.6(d,J＝5.00Hz),145.8,142.6(d,J＝252.50Hz),134.3,133.5,131.3(d,J＝28.75Hz),130.2,128.4,126.2,125.8,125.1,118.6(d,J＝7.50Hz),115.7(d,J＝21.25Hz),113.8(d,J＝2.50Hz),56.0,32.2,18.6.HRMS(ESI):m/z calcd for(C21H16F2N6O2+H)+:423.1376.；found:423.1369。
Example 131- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) piperidine-2-carboxylic acid (Aza-11);
with reference to the procedure of example 10, except that ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine was replaced with methyl 1- ((5-fluoro-2- (5-fluoro-1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) piperidine-2-carboxylate. Yellow solid, yield 45%.
m.p.:>187.3-189.1℃；1H NMR(500MHz,DMSO-d6)δ12.22(s,1H),8.90-8.88(m,1H),8.64-8.62(m,1H),8.29-8.24(m,1H),8.19-8.17(m,2H),3.24-3.16(m,1H),2.85-2.73(m,1H),1.98-1.92(m,1H),1.87-1.61(m,5H),1.44-1.31(m,1H).13C NMR(125MHz,DMSO)δ173.4,157.8(d,J＝6.25Hz),156.7,154.8,151.0(d,J＝11.25Hz),145.9,142.2(d,J＝253.75Hz),131.4(d,J＝28.75Hz),129.9,118.75(d,J＝7.50Hz),116.2(d,J＝25.00Hz),113.83(d,J＝2.50Hz),66.0,53.5,29.3,25.3,22.2.HRMS(ESI):m/z calcd for(C17H16F2N6O2+H)+:375.1376；found:375.1383。
Example 14(2S, 3aS,7aS) -1- (((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) octahydro-1H-indole-2-carboxylic acid (Aza-12);
with reference to the procedure of example 10, except that ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine is replaced by methyl (2S,3aS,7aS) -1- ((5-fluoro-2- (5-fluoro-1-p-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) octahydro-1H-indole-2-carboxylate. Yellow solid, yield 62%.
m.p.:>250℃；1H NMR(500MHz,DMSO-d6)δ12.94(s,1H),12.29(s,1H),9.03(s,1H),8.71-8.69(m,1H),8.28(s,2H),8.14-8.12(m,1H),3.77-3.73(m,1H),2.10-2.02(m,1H),1.74-1.69(m,2H),1.66-1.56(m,3H),1.51-1.32(m,3H),1.26-1.13(m,3H).13C NMR(125MHz,DMSO)δ175.2,157.4(d,J＝6.25Hz),156.7,154.8,152.5(d,J＝10.00Hz),145.8,141.9(d,J＝252.50Hz),131.7(d,J＝28.75Hz),129.9,118.3(d,J＝7.50Hz),115.9(d,J＝21.25Hz),113.4(d,J＝3.75Hz),66.0,54.9,35.3,33.7,28.2,25.1,24.7,19.9.HRMS(ESI):m/z calcd for(C20H20F2N6O2+H)+:415.1689；found:415.1689。
Example 15 ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -L-proline (Aza-13);
with reference to the procedure of example 10, except that ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine was replaced with methyl ((5-fluoro-2- (5-fluoro-1-tolyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -proline. Brown solid, yield 32%.
m.p.:>250℃；1H NMR(500MHz,DMSO-d6)δ10.32(s,1H),8.63(dd,J＝10.0,2.5Hz,1H),8.25-8.24(m,1H),8.19(s,1H),8.10(d,J＝3.5Hz,1H),7.33-7.28(m,1H),6.68(s,1H),3.52(t,J＝6.0Hz,1H),3.27-3.24(m,1H),2.99-2.95(m,1H),2.08-2.04(m,2H),1.84-1.79(m,2H).13C NMR(125MHz,DMSO)δ176.6,156.6(d,J＝6.25Hz),154.7,151.0,145.9,142.5(d,J＝252.5Hz),131.3(d,J＝28.75Hz),129.1(d,J＝263.75Hz),125.5,118.6(d,J＝7.50Hz),115.7(d,J＝22.50Hz),113.8(d,J＝2.50Hz),64.8,51.7,27.2,22.5.HRMS(ESI):m/z calcd for(C16H14F2N6O2+H)+:361.1219；found:361.1223。
Example 16(S) -5- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -5-azaspiro [2.4] heptane 6-carboxylic acid (Aza-17);
with reference to the procedure of example 10, except that ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine was replaced by methyl (S) -5- ((5-fluoro-2- (5-fluoro-1-p-sulfanyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -5-azaspiro [2.4] heptane-6-carboxylate. Brown solid in 73% yield.
m.p.:>250℃；1H NMR(500MHz,DMSO-d6)δ8.63-8.58(m,1H),8.28-8.26(m,1H),8.23(d,J＝3.5Hz,1H),8.17(s,1H),4.03-4.01(m,1H),3.29(d,J＝8.5Hz,1H),3.03(d,J＝8.5Hz,1H),2.42-2.33(m,1H),1.87-1.84(m,1H),0.62-0.50(m,4H).13C NMR(125MHz,DMSO)δ174.5,157.7(d,J＝5.00Hz),156.7,154.8,151.6(d,J＝8.75Hz),145.9,142.2(d,J＝252.50Hz),131.6(d,J＝28.75Hz),130.3,118.4(d,J＝7.50Hz),115.7(d,J＝22.50Hz),113.4(d,J＝5.00Hz),67.1,62.0,36.6,19.1,13.3.HRMS(ESI):m/z calcd for(C18H16F2N6O2+H)+:387.1376；found:387.1368。
Example 17(2R,4R) -4-fluoro-1- (5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) pyrrolidine-2-carboxylic acid (Aza-18);
with reference to the procedure of example 10, except that ethyl N- ((5-fluoro-2- (1-tosyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) amino) -N-neopentylglycine is replaced by methyl (2R,4R) -4-fluoro-1- (5-fluoro-2- (5-fluoro-1-p-toluenesulfonyl-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrimidin-4-yl) pyrrolidine-2-carboxylate. Yellow solid, yield 54%.
m.p.:>250℃；1H NMR(500MHz,DMSO-d6)δ12.50(s,1H),8.43-8.37(m,1H),8.25-8.21(m,1H),8.20-8.18(m,1H),8.07-8.02(m,1H),5.40-5.29(m,1H),4.64-4.53(m,1H),4.04-3.99(m,2H),3.16(s,2H).13C NMR(125MHz,DMSO)δ173.8,157.8(d,J＝7.50Hz),156.0(d,J＝240.00Hz),150.8(d,J＝20.00Hz),146.4,142.5(d,J＝321.25Hz),140.2(d,J＝23.75Hz),131.5(d,J＝28.75Hz),131.2(d,J＝6.25Hz),118.6(d,J＝7.50Hz),115.3(d,J＝17.50Hz),114.1(d,J＝7.5Hz),90.9(d,J＝178.75Hz),61.4(d,J＝2.50Hz),55.61,55.38(d,J＝28.75Hz),21.7(d,J＝30.00Hz).HRMS(ESI):m/z calcd for(C16H12F3N5O2+H)+:364.1016；found:364.1008。
EXAMPLE 18 toxicity of Compounds on MDCK cells and A549 cells
The toxic effect of the compounds on cells was tested using CCK 8. MDCK cells or A549 cells are inoculated in a 96-well plate, after the cells are attached to the wall, the compounds are added for treatment, the concentration gradient of the compounds is set to be 50, 30, 10, 3, 1, 0.3 and 0.1 mu M, the cytopathic effect caused by the compounds is observed under a mirror, and CCK8 is added for detection.
Cell activity (%) ═ (sample well-blank)/(cell control-blank) × 100%
Finally, fitting by software to obtain the half inhibitory concentration CC of the cell50。
Example 19 assay for anti-influenza Virus polymerase Activity
This example was conducted to determine the effect of the compounds of the present invention on the expression activity of influenza polymerase in HEK-293T cells into which plasmids of four segments of influenza polymerase components PB1, PB2, PA and NP were introduced, and the inhibition of the activity of the compounds against the viral polymerase was expressed by the inhibition rate of the activity of the influenza polymerase. The protocol was as follows: inoculating the cultured 293T cells on a 96-well culture plate; then treating the cells with the compound with the prepared concentration, adding the transfection system after 2 hours, placing the cells at 37 ℃ and 5% CO2After 24 hours, the cells were lysed and the fluorescence intensity was measured. The effect of the test compound on the activity of the influenza polymerase at a concentration of 10. mu.M was determined by the method of Dual Luciferase Reporter Assay, and the data are shown in Table 1. The inhibition rate of the compound on the activity of viral polymerase is more than or equal to 80 percent in A and more than or equal to 65 percent in B>80 percent, C represents the inhibition rate of 35 percent or less>65% and D represents the inhibition ratio<35％。
TABLE 1 influenza Virus polymerase inhibitory Activity
Numbering Inhibitory Rate (%) of influenza Virus polymerase activity
Aza-1 C
Aza-3 C
Aza-4 B
Aza-5 D
Aza-6 D
Aza-7 D
Aza-8 C
Aza-9 D
Aza-10 D
Aza-11 A
Aza-12 A
Aza-13 C
Aza-14 B
Aza-15 D
Aza-16 D
Aza-17 A
Aza-18 D
Aza-19 B
Aza-20 D
A portion of the compounds was also tested for inhibition of polymerase activity at 6 gradient concentrations (10. mu.M, 3. mu.M, 1. mu.M, 0.3. mu.M, 0.1. mu.M, 0.03. mu.M). The extent to which a compound inhibits half the polymerase activity is expressed as the half inhibitory concentration IC50To indicate.
Table 2 shows the inhibitory activity and cytotoxicity of the test compounds against influenza virus polymerase, wherein E represents IC50Less than 0.3. mu.M, F represents IC50Greater than 0.3. mu.M. G represents>30μM。
TABLE 2 influenza polymerase inhibitory Activity and cytotoxicity
Figure BDA0002974418840000151
Figure BDA0002974418840000161
Example 20 anti-influenza Virus replication assay
This example is used to determine the inhibitory effect of the compounds of the invention on viral replication in MDCK cells infected with influenza A virus A/WSN/33(H1N1), the inhibitory activity of the compounds on viral replication being determined at half the effective concentration EC50To indicate. MDCK cells and influenza A/WSN/33(H1N1) were obtained from Zhejiang university college of medicine. The protocol was as follows: inoculating MDCK on 96-well culture plates; the cells were then placed at 37 ℃ in 5% CO2After 24 hours, a series of concentration gradients of drug was added to the cultured cell culture medium and incubated for 2 hours, typically 6 concentrations (10. mu.M, 3. mu.M, 1. mu.M, 0.3. mu.M, 0.1. mu.M, 0.0.03. mu.M) were selected, then the wild-type strain (A/WSN/33) with an MOI of 0.1 was added, and then a sample of virus supernatant was collected 24 hours later to determine the TCID of the virus50The value is obtained. The effective drug concentration of the compound for inhibiting the replication of the median virus is EC50。
Table 3 shows the inhibitory activity of the test compounds against influenza A/WSN/33(H1N1), wherein H represents EC50Less than 1.0 μ M; i represents EC50Greater than 1.0. mu.M.
TABLE 3 inhibition of influenza virus replication activity
Numbering MDCK EC50(μM)
Aza-11 H
Aza-12 H
Aza-17 I
The results in tables 1-3 show that the compounds of the invention have inhibitory activity on influenza polymerase and influenza virus H1N1 replication, with the compounds Aza-11 and Aza-12 being most active at nanomolar levels.
Example 21 Isodifferential thermal titrator ITC determination of Compounds with PB2318-483This example was used to determine the interaction of compounds with PB2318-483Binding capacity of the protein. Test protocol: PB2 with different concentrations is prepared318-483Protein solutions and small molecule solutions. The final concentration of the protein solution is 30-60 mu M. Preparing small molecule solution according to the ratio of protein to small molecule concentration of 1: 10. And (3) centrifuging the protein solution and the small molecule solution at 13000rpm for 2min, slowly adding the protein solution into the ITC sample cell, adding the small molecule solution into a titration needle, and starting titration. The experimental results are as follows: FIG. 1 shows the ITC test results of Aza-12, which gave a signal corresponding to PB2318-483The Kd value of the protein was 202 nM.

Claims (2)

1. An azaindole derivative containing a nitrogen-containing heteroamino acid, which is selected from any one of the following compounds:
1- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2, 3-)b]Pyridin-3-yl) pyrimidin-4-yl) amino) piperidine-2-carboxylic acid,
(2S,3aS,7aS) -1- (((5-fluoro-2- (5-fluoro-1H-pyrrolo [2, 3-)b]Pyridin-3-yl) pyrimidin-4-yl) amino) octahydro-1H-indole-2-carboxylic acid,
(S) -5- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2, 3-)b]Pyridin-3-yl) pyrimidin-4-yl) amino) -5-azaspiro [2.4]Heptane 6-carboxylic acid.
2. Use of azaindole derivatives of aza amino acids as claimed in claim 1 for the preparation of a medicament for the treatment of influenza.
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