CN109734708B - Pyrimidine indole Nur77 receptor modulator and preparation method and application thereof - Google Patents
Pyrimidine indole Nur77 receptor modulator and preparation method and application thereof Download PDFInfo
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Abstract
嘧啶吲哚类Nur77受体调控剂及其制备方法和应用，涉及嘧啶吲哚类衍生物。提供具有新结构的一类嘧啶吲哚类衍生物，及在药学上可接受的盐、水合物及药学上可接受的前药。提供结构式I所代表的嘧啶吲哚类衍生物或其可药用盐和前药的制备方法。提供一类具有新结构的嘧啶吲哚类衍生物在制备Nur77受体诱导剂的用途。提供一类新颖可调节Nur77信号通路活性的衍生物，它们可以通过调控Nur77相关信号通路活性，阻断肿瘤细胞增殖，诱导细胞凋亡，从而可以用于人和动物的多种疾病，如恶性肿瘤的治疗和预防。A pyrimidine indole class Nur77 receptor modulator and its preparation method and application relate to pyrimidine indole class derivatives. Provided are a class of pyrimidine indole derivatives with novel structures, as well as pharmaceutically acceptable salts, hydrates and pharmaceutically acceptable prodrugs. Provided are the preparation methods of pyrimidine indole derivatives represented by structural formula I or pharmaceutically acceptable salts and prodrugs thereof. Provided is the use of a class of pyrimidine indole derivatives with new structures in the preparation of Nur77 receptor inducers. Provide a new class of derivatives that can modulate the activity of Nur77 signaling pathway. They can block tumor cell proliferation and induce apoptosis by regulating the activity of Nur77-related signaling pathway, so that they can be used in various diseases of humans and animals, such as malignant tumors. treatment and prevention.
Description
Technical Field
The invention relates to pyrimidine indole derivatives, in particular to a pyrimidine indole Nur77 receptor regulator and a preparation method and application thereof.
Background
The orphan nuclear receptor Nur77, also known as NGFIB (nerve growth factor IB) or orphan nuclear receptor TR3, is a key regulator in the development of cancer, metabolism and inflammatory diseases. As a class of immediate early response genes, Nur77 plays an important role in a number of cellular processes, including cell survival, apoptosis, inflammation, and autophagy processes, which are caused by different stimuli such as cytokinins, hormones, stress, metabolic, and apoptotic signals. Nur77 is an ideal target for preventing and treating cancer and tumor. The research shows that Nur77 is a cell survival factor and can mediate a plurality of survival signal pathways, including MAPK, NF-kB, protein kinase A and protein kinase C pathways; nur77 is also an apoptosis factor, can mediate calcium ion carrier, endoplasmic reticulum osmotic pressure, mitochondrial osmotic pressure, P53, BCL-2 and the like to play a role in promoting apoptosis. The death effect of Nur77 was first discovered in 1994, that T cell receptor signaling was able to induce Nur77 expression, and Nur77 expression was increased during T cell receptor mediated apoptosis. In view of the important role of Nur77 in a variety of cellular processes as well as in a variety of disease processes, it has become an important target for the development of new therapies for such diseases. Therefore, there is a need in the art to find effective ligands that specifically bind to Nur77 to develop new therapies for the treatment of cancer.
Disclosure of Invention
The invention provides a pyrimidine indole derivative with a novel structure, and a pharmaceutically acceptable salt, a hydrate and a pharmaceutically acceptable prodrug, wherein the structural formula I is as follows:
wherein R1 represents a substituted or unsubstituted aromatic group, preferably
R2 represents a straight-chain or cyclic alkyl, adamantyl,morpholin-1-yl, substituted or unsubstituted heterocyclic compounds, substituted or unsubstituted 3-indole, preferably n-hexyl, adamantyl, morpholin-1-yl,
n is 2 or 3.
The pyrimidine indole derivatives of the invention are:
the pyrimidine indole derivatives are preferably as follows:
n- (3- (dimethylamino) propyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH117)
N- (2- (diisopropylamino) ethyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH133)
N- (2- (5-methoxy-1H-indol-3-yl) ethyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH129)
N- (3-Morpholinopropyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH102)
5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -N- (2- (pyrrolidin-1-yl) ethyl) -1H-indole-2-carboxamide (QH137)
N- (2- (1H-indol-3-yl) ethyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH138)
N-phenethyl-5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH141)
N- (2- (5-methyl-1H-indol-3-yl) ethyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH173)
N-butyl-5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH193)
N- (2- (2-methyl-1H-indol-3-yl) ethyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH130)
N- (4-hydroxyphenyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH158)
N- (4-methoxyphenyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH164)
N- (2- (5-hydroxy-1H-indol-3-yl) ethyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH170)
N- (3- (di-N-butylamino) propyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH167)
5- ((4- (2, 5-Dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -N- (2- (pyrrolidin-1-yl) ethyl) -1H-indole-2-carboxamide (QH188)
N- (3- (dimethylamino) propyl) -5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH146)
N- (3- (dimethylamino) ethyl) -5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH185)
5- ((4- (2, 5-Dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -N- (2-morpholinoethyl) -1H-indole-2-carboxamide (QH142)
N- (2- (1H-indol-3-yl) ethyl) -5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH147)
N- (5- (diethylamino) pentan-2-yl) -5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH125)
N- (3- (di-N-butylamino) propyl) -5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH178)
N- (2- (diisopropylamino) ethyl) -5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH133)
N- (3- (diethylamino) propyl) -5- ((4- (2-methoxyphenyl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH157)
N- (3- (dimethylamino) propyl) -5- ((4- (2-methoxyphenyl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH152)
N- (3- (dimethylamino) ethyl) -5- ((4- (2-methoxyphenyl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH149)
N- (3- (diethylamino) propyl) -5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH123)
N- (2-Morpholinoethyl) -5- ((4- (thien-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH118)
N- (2- (pyrrolidin-1-yl) ethyl) -5- ((4- (thiophen-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH101)
N- (3- (4-methylpiperazin 1-yl) propyl) -5- ((4- (thiophen-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH139)
N- (3- (dimethylamino) propyl) -5- ((4- (thiophen-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH 142).
The second object of the present invention is to provide a process for preparing pyrimidine indole derivatives represented by structural formula I or pharmaceutically acceptable salts and prodrugs thereof, which comprises the steps of:
1) taking p-nitrophenylhydrazine (1) as a raw material and absolute ethyl alcohol as a solvent;
2) dropwise adding ethyl pyruvate under the stirring state, heating to reflux after the dropwise adding is finished, and obtaining a yellow solid product ethyl pyruvate p-nitrophenylhydrazone (2);
3) heating the yellow solid product ethyl pyruvate p-nitrophenylhydrazone (2) obtained in the step 2) under the catalysis of polyphosphoric acid to obtain an intermediate 5-nitroindole-2-carboxylic acid ethyl ester (3);
4) reducing the nitro group into amino group to obtain 5-aminoindole-2-carboxylic acid ethyl ester (4);
5) converting the amino group of the 5-aminoindole-2-carboxylic acid ethyl ester (4) obtained in the step 4) into guanidine, and reacting with 3- (dimethylamino) -1- (substituted or unsubstituted aryl) -2-propen-1-one to obtain an intermediate ester (7);
6) hydrolyzing the intermediate ester (7) obtained in the step 5) into acid (8), and then condensing the acid (8) with different amines to obtain the target compound pyrimidine indole derivative.
The specific synthetic route is as follows:
wherein R1 represents a substituted or unsubstituted aromatic group, preferably
R2 represents a linear or cyclic alkyl, adamantyl, fatty amine, morpholin-1-yl, substituted or unsubstituted heterocyclic compound, substituted or unsubstituted 3-indole, preferably n-hexyl, adamantyl, morpholin-1-yl,
n represents 2 or 3.
The third purpose of the invention is to provide the application of the pyrimidine indole derivatives with the novel structure in preparing the Nur77 receptor inducer.
The fourth purpose of the invention is to provide a novel derivative capable of regulating the activity of Nur77 signal channel, which can block the proliferation of tumor cells and induce apoptosis by regulating the activity of Nur77 related signal channel, thereby being used for treating and preventing various diseases of human and animals, such as malignant tumor.
The invention relates to a novel pyrimidine indole derivative, a preparation method and medical application thereof, in particular to a pyrimidine indole compound serving as a Nur77 regulator, a preparation method and application thereof serving as a ligand of an orphan nuclear receptor Nur 77. The invention also relates to application of the pyrimidine indole derivatives and a pharmaceutical composition containing the derivatives in preventing or treating diseases related to the orphan nuclear receptor Nur77, in particular to application of the pyrimidine indole derivatives and the pharmaceutical composition containing the derivatives in preparing medicines for preventing or treating diseases such as tumors and inflammations as Nur77 regulating agents.
Drawings
FIG. 1 is a graph of single point binding of a QH series of compounds to Nur77-LBD protein using Biacore.
FIG. 2 is a graph showing the binding of a portion of the QH series of compounds to the concentration gradient of Nur77-LBD protein detected by Biacore.
Figure 3 is a graph of the effect of QH series compounds on the transcriptional activation of Nur77 using the dual luciferase reporter system.
FIG. 4 shows the transcriptional activation of QH117 on nuclear receptors Nur77, RAR α, RXR α, PPAR γ, etc. In figure 4, (a) QH117 is transcriptionally activated for Nur 77; (b) represents that QH117 has no transcriptional activation on RAR alpha, and uses RAR alpha ligand ATRA as positive control; (c) represents that QH117 has no transcriptional activation on RXR alpha, and takes RXR alpha ligand 9-cis as a positive control; (d) QH117 showed no transcriptional activation of PPAR γ and was used as a positive control with the PPAR γ ligand rosiglitazone.
FIG. 5 is a graph showing the effect of a portion of the QH series of compounds on the induction of PARP cleavage in Hela cells.
FIG. 6 shows the proliferation inhibitory effect of QH117 on various tumor cells. In FIG. 6, curve a is HT29, b is SW620, c is HCT116, d is MDA-MB-231, e is MCF-7, f is T47D, and g is HeLa.
Figure 7 shows that PARP-promoting cleavage of tumor cells by QH117 is mediated by Nur 77.
Figure 8 shows that the proliferation inhibitory effect of QH117 on tumor cells is mediated by Nur 77.
Figure 9 shows that the pro-apoptotic effect of QH117 on tumor cells is mediated by Nur 77.
Detailed Description
In order to facilitate an understanding of the invention, the invention will now be further described with reference to specific embodiments.
Example 1: preparation of N- (3- (dimethylamino) propyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH117)
Intermediate I: preparation of 5-aminoindole-2-carboxylic acid ethyl ester
Adding p-nitrophenylhydrazine (10.00g,0.065mol) and absolute ethyl alcohol (85mL) into a 250mL dry double-neck bottle in sequence, dropwise adding ethyl pyruvate (8.14g,0.072mol) under a stirring state, heating to reflux after the dropwise adding is finished, carrying out reflux reaction for 2h, finishing TLC detection reaction, and stopping reaction. Cooling the reaction liquid to room temperature, performing suction filtration, collecting a filter cake, and drying to obtain 13.80g of a yellow solid product ethyl pyruvate p-nitrophenylhydrazone, wherein the yield is 89.6%, and the melting point (m.p.): 197.0-199.2 ℃.
In a dry 500mL reaction flask, ethyl pyruvate p-nitrophenylhydrazone (10.00g,0.042mol) and polyphosphoric acid (110.00g) are sequentially added, and then the temperature is raised to 100 ℃ under a stirring state, and the reaction is kept for 2 hours. The reaction was stopped by TLC detection. Cooling the reaction liquid to room temperature, adding a large amount of ice water, performing suction filtration, collecting a filter cake, and drying to obtain a green solid product, namely 5-nitroindole-2-carboxylic acid ethyl ester 8.90g, wherein the yield is 90.2%, and the m.p. is 222.2-224.3 ℃.
In a dry 250mL reaction flask, ethanol (80mL), water (20mL), acetic acid (10mL) and iron powder (8.62g,0.154mol) were added in that order; then, the temperature was raised to 75 ℃ while stirring, and Compound 4(9.00g,0.039mol) was added in portions, and after the addition, the reaction was allowed to proceed for 2 hours. The reaction was stopped by detecting the completion of the reaction by thin layer chromatography. Carrying out hot filtration on the reaction solution, cooling the filtrate, and then carrying out reduced pressure concentration to remove the solvent; adding 80mL of water into the concentrated viscous liquid, adding 80mL of ethyl acetate under stirring, and adjusting the pH value to 7-8 by using sodium bicarbonate; the liquid was filtered, separated, and the organic phase was dried over 15g of anhydrous sodium sulfate for 5 hours, filtered, and the solid obtained by concentrating the filtrate under reduced pressure was separated by silica gel column chromatography (eluent petroleum ether: ethyl acetate ═ 3: 1, v/v) to obtain 6.10g of ethyl 5-aminoindole-2-carboxylate as a red solid product, with a yield of 77.7%.
Intermediate II: preparation of 5-substituted- (4- (pyridin-3-yl) pyrimidin-2-ylamino) -1H-indole-2-carboxylic acid ethyl ester
100mL of ethanol, 5-aminoindole-2-carboxylic acid ethyl ester (3.39g,0.017mol) and cyanamide (1.59g,0.038mol) are sequentially added into a dry 250mL reaction bottle at room temperature, concentrated hydrochloric acid (2.1mL,0.025mol) is dropwise added under stirring, and after dropwise addition is finished, the temperature is raised to reflux reaction for 24 hours. The reaction was stopped by detecting the completion of the reaction by thin layer chromatography. Decompressing and concentrating the reaction liquid, removing the solvent, adding water, dropwise adding an aqueous solution of ammonium nitrate (2.64g,0.033mol) at 0 ℃, preserving heat for 1h after dropwise adding, filtering, washing the filter cake with diethyl ether for 2 times, drying the filter cake in a vacuum drying oven at 45 ℃ for 4-8 h, dissolving the filter cake in ethanol, adding 3- (dimethylamino) -1- (3-pyridyl) -2-propylene-1-ketone (2.92g,0.017mol), dropwise adding an ethanol solution of sodium hydroxide (0.80g,0.020mol), finishing dropwise adding, and heating to reflux reaction for 48 h. The reaction was stopped by TLC and cooled to give 4.12g of ethyl 5- (4- (pyridin-3-yl) pyrimidin-2-ylamino) -1H-indole-2-carboxylate as a yellow solid in 69.1% yield.
50mL of ethanol, 50mL of ethyl 5- (4- (pyridin-3-yl) pyrimidin-2-ylamino) -1H-indole-2-carboxylate (5.00g, 0.014mol) and 50mL of a 40% sodium hydroxide solution were sequentially added to a dry 250mL reaction flask at room temperature, and the mixture was heated to reflux with stirring for 1 hour. The reaction was stopped by detecting the completion of the reaction by thin layer chromatography. And pouring the reaction solution into ice water, and adjusting the pH value to 4-6 at the temperature of 0 ℃. The reaction solution was filtered by suction, and the filter cake was collected and dried to give 4.19g of an orange solid 5- (4- (pyridin-3-yl) pyrimidin-2-ylamino) -1H-indole-2-carboxylic acid in 91.0% yield.
Preparation of N- (3- (dimethylamino) propyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH117)
In a dry 50mL reaction flask, DMF 10mL, 5- (4- (pyridin-3-yl) pyrimidin-2-ylamino) -1H-indole-2-carboxylic acid (100.0mg,0.30mmol), EDCI (86.8mg,0.45mmol), and HOBT (41.5mg,0.30mmol) were added sequentially at room temperature. Stirring for 1h at room temperature. Then, N-dimethylpropane-1, 3-diamine (30.7mg, 0.30mmol) and triethylamine were added under stirring at room temperature over night to react. The reaction was completed by TLC detection and stopped. Pouring the reaction solution into 80mL of ice water, and separating out solids; and (4) carrying out suction filtration to obtain a filter cake (crude product). The obtained crude product was separated by silica gel column chromatography (eluent petroleum ether: ethyl acetate: 1, v/v) to obtain 89.0mg of solid product with a yield of 68.46%. Wherein the molar ratio of 5- (4- (pyridin-3-yl) pyrimidin-2-ylamino) -1H-indole-2-carboxylic acid to EDCI, HOBT, N, N-dimethylpropane-1, 3-diamine is 1: 2: 1.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.45(s,1H),9.57(s,1H),9.36(d,J＝1.83Hz,1H),8.73(dd,J＝1.65,4.77Hz,1H),8.57(d,J＝5.13Hz,1H),8.49(td,J＝1.88,7.98Hz,1H),8.46(t,J＝5.69Hz,1H),8.14(s,1H),7.58(ddd,J＝0.64,4.81,7.93Hz,1H),7.48(dd,J＝1.93,8.89Hz,1H),7.43(d,J＝5.13Hz,1H),7.38(d,J＝8.62Hz,1H),7.05(d,J＝1.47Hz,1H),3.31(q,J＝6.85Hz,2H),2.25～2.30(m,2H),2.15(s,6H),1.68(quin,J＝7.11Hz,2H)；13C NMR(151MHz,DMSO-d6):δ161.9,161.5,161.1,159.8,151.9,148.6,134.8,133.5,133.2,132.9,132.6,127.5,124.4,118.9,112.5,111.6,107.8,102.7,66.7,58.0,53.8(*2),36.6；ESI-MS(+):416.2[M+H]+。
example 2: preparation of N- (2- (diisopropylamino) ethyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH133)
The reaction procedure was the same as that of example 1 except for changing N, N-dimethylpropane-1, 3-diamine to N, N-diethylpropylethane-1, 2-diamine (43.3mg, 0.30mmol), and 47.2mg of a product was isolated as a pale yellow solid in a yield of 32.9%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.46(s,1H),9.57(s,1H),9.36(d,J＝1.83Hz,1H),8.73(dd,J＝1.56,4.68Hz,1H),8.57(d,J＝5.14Hz,1H),8.49(td,J＝1.88,7.98Hz,1H),8.35(t,J＝5.78Hz,1H),8.13(s,1H),7.59(dd,J＝4.77,7.89Hz,1H),7.49(dd,J＝1.93,8.90Hz,1H),7.43(d,J＝4.95Hz,1H),7.38(d,J＝8.80Hz,1H),7.03(d,J＝1.28Hz,1H),3.21～3.27(m,2H),2.96～3.02(m,2H),2.53～2.58(m,2H),0.97～1.03(m,12H)；13C NMR(151MHz,DMSO-d6):δ161.9,161.5,161.1,159.8,151.9,148.6,134.8,133.4,133.2,132.9,132.8,127.6,124.3,118.9,112.5,111.6,107.8,102.4,49.0(*2),44.8,41.1,21.2(*4)；ESI-MS(+):458.3[M+H]+。
example 3: preparation of N- (2- (5-methoxy-1H-indol-3-yl) ethyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH129)
The reaction procedure was as in the synthesis of example 1, substituting N, N-dimethylpropane-1, 3-diamine for 2- (5-methoxy-1H-indol-3-yl) ethane-1-amino (57.1mg, 0.30mmol), and finally isolating the product as a pale yellow solid 87.2mg, 55.5% yield.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.47(d,J＝1.47Hz,1H),10.67(d,J＝1.65Hz,1H),9.48(s,1H),8.57(t,J＝5.69Hz,1H),8.52(d,J＝5.14Hz,1H),8.16～8.20(m,2H),8.15(d,J＝1.28Hz,1H),7.54～7.58(m,3H),7.51(dd,J＝1.93,8.89Hz,1H),7.38(d,J＝8.80Hz,1H),7.33～7.35(m,1H),7.24(d,J＝8.80Hz,1H),7.17(d,J＝2.38Hz,1H),7.10(d,J＝2.38Hz,1H),7.07(d,J＝1.65Hz,1H),6.72(dd,J＝2.38,8.80Hz,1H),3.71～3.77(m,3H),3.55～3.61(m,2H),2.96(t,J＝7.43Hz,2H)；13C NMR(151MHz,DMSO-d6):δ163.9,161.6,161.1,159.5,153.5,137.4,133.7,133.1,132.8,131.8,131.2,129.3,128.1,127.6,127.3,123.8,118.8,112.5,112.5,112.2,111.6,111.4,107.6,102.6,100.6,60.2,55.7,25.8；ESI-MS(+):504.2[M+H]+。
example 4: preparation of N- (3-morpholinopropyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH102)
The reaction procedure was the same as that of example 1 except for changing N, N-dimethylpropane-1, 3-diamine to 3-morpholinopropane-1-amine (43.2mg, 0.30mmol), and finally isolating the product as a pale yellow solid in an amount of 65.1mg with a yield of 45.5%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.46(s,1H),9.57(s,1H),9.36(d,J＝1.83Hz,1H),8.73(dd,J＝1.47,4.77Hz,1H),8.57(d,J＝4.95Hz,1H),8.49(td,J＝1.86,8.02Hz,1H),8.44(t,J＝5.69Hz,1H),8.14(s,1H),7.59(dd,J＝4.77,7.89Hz,1H),7.48(dd,J＝1.93,8.90Hz,1H),7.43(d,J＝5.14Hz,1H),7.38(d,J＝8.80Hz,1H),7.06(d,J＝1.28Hz,1H),3.58(t,J＝4.40Hz,4H),3.30～3.34(m,2H),2.31～2.40(m,6H),1.71(quin,J＝7.06Hz,2H)；13C NMR(151MHz,DMSO-d6):δ161.9,161.5,161.1,159.8,151.9,148.6,134.8,133.4,133.2,132.9,132.7,127.5,124.4,118.9,112.5,111.6,107.8,102.5,66.7(*2),56.5,53.8(*2),37.7,26.7；ESI-MS(+):458.2[M+H]+。
example 5: preparation of 5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -N- (2- (pyrrolidin-1-yl) ethyl) -1H-indole-2-carboxamide (QH137)
The reaction procedure was the same as that for the synthesis of example 1, substituting N, N-dimethylpropane-1, 3-diamine for 2- (pyrrolidin-1-yl) propane-1-amine (34.2mg, 0.30mmol), and finally isolating the product as a pale yellow solid, 45.7mg, yield 34.5%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.48(s,1H),9.57(s,1H),9.33-9.38(m,1H),8.73(dd,J＝1.56,4.68Hz,1H),8.57(d,J＝5.14Hz,1H),8.49(td,J＝1.86,8.02Hz,1H),8.43(t,J＝5.69Hz,1H),8.14(s,1H),7.59(dd,J＝4.86,7.98Hz,1H),7.49(dd,J＝2.02,8.80Hz,1H),7.38(d,J＝8.62Hz,1H),7.08(d,J＝1.28Hz,1H),3.40～3.47(m,6H),2.61(t,J＝6.79Hz,2H),1.65～1.74(m,4H)；13C NMR(151MHz,DMSO-d6):δ161.9,161.5,161.1,159.8,151.9,148.6,134.8,133.4,133.2,132.9,132.6,127.5,124.4,118.9,112.5,111.6,107.8,102.8,55.5,54.2(*2),38.6,23.6(*2)；ESI-MS(+):428.2[M+H]+。
example 6: preparation of N- (2- (1H-indol-3-yl) ethyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH138)
The reaction procedure was as in the synthesis of example 1, substituting N, N-dimethylpropane-1, 3-diamine for 2- (1H-indol-3-yl) ethan-1-amine (48.0mg, 0.30mmol), and finally isolating the product as a light yellow solid, 88.7mg, 59.9% yield.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.48(s,1H),10.83(br.s.,1H),9.58(s,1H),9.36(d,J＝1.83Hz,1H),8.73(dd,J＝1.38,4.68Hz,1H),8.56～8.61(m,2H),8.50(td,J＝1.86,8.02Hz,1H),8.14(s,1H),7.62(d,J＝7.70Hz,1H),7.59(dd,J＝4.77,7.89Hz,1H),7.49(dd,J＝1.83,8.80Hz,1H),7.43(d,J＝5.14Hz,1H),7.39(d,J＝8.80Hz,1H),7.35(d,J＝8.07Hz,1H),7.21(d,J＝1.83Hz,1H),7.06～7.10(m,2H),6.98～7.02(m,1H),3.57-3.62(m,2H),2.99(t,J＝7.52Hz,2H)；13C NMR(151MHz,DMSO-d6):δ161.9,161.5,161.1,159.8,151.9,148.6,136.7,134.8,133.4,133.2,132.9,132.8,127.7,127.6,124.4,123.1,121.4,118.9,118.8,118.7,112.5,112.3,111.9,111.6,107.8,102.6,25.8,14.6；ESI-MS(+):474.2[M+H]+。
example 7: preparation of N-phenethyl-5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH141)
The reaction procedure was the same as that of example 1 except for changing N, N-dimethylpropane-1, 3-diamine to 2-phenylethane-1-amine (36.3mg, 0.30mmol), and finally isolating the product as a pale yellow solid in an amount of 98.2mg with a yield of 72.0%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.45(s,1H),9.52-9.62(m,1H),9.36(d,J＝1.65Hz,1H),8.73(dd,J＝1.65,4.77Hz,1H),8.57(d,J＝5.14Hz,1H),8.54(t,J＝5.59Hz,1H),8.49(td,J＝1.93,8.07Hz,1H),8.13(s,1H),7.59(ddd,J＝0.64,4.81,7.93Hz,1H),7.48(dd,J＝2.02,8.80Hz,1H),7.43(d,J＝5.14Hz,1H),7.37(d,J＝8.80Hz,1H),7.29～7.33(m,2H),7.26～7.29(m,2H),7.19～7.23(m,1H),7.06(d,J＝1.47Hz,1H),3.44～3.57(m,2H),2.84～2.94(m,2H)；13C NMR(151MHz,DMSO-d6):δ161.9,161.5,161.1,159.8,151.9,148.6,140.0,134.8,133.4,133.2,132.9,132.6,129.1(*2),128.8(*2),127.5,126.6,124.4,118.9,112.5,111.6,107.8,102.6,40.9,35.7；ESI-MS(+):438.2[M+H]+。
example 8: preparation of N- (2- (5-methyl-1H-indol-3-yl) ethyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH173)
The reaction procedure was as in the synthesis of example 1, substituting N, N-dimethylpropane-1, 3-diamine for 2- (5-methyl-1H-indol-3-yl) ethane-1-amino (52.3mg, 0.30mmol), and finally isolating the product as a pale yellow solid, 88.2mg, 58.0% yield.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.47(s,1H),10.67(s,1H),9.56(s,1H),9.36(d,J＝1.65Hz,1H),8.73(dd,J＝1.65,4.77Hz,1H),8.57(d,J＝5.13Hz,1H),8.55-8.56(m,1H),8.49(td,J＝1.95,8.02Hz,1H),8.14(s,1H),7.59(dd,J＝4.86,7.79Hz,1H),7.49(dd,J＝2.02,8.80Hz,1H),7.43(d,J＝4.95Hz,1H),7.37～7.40(m,2H),7.23(d,J＝8.07Hz,1H),7.15(d,J＝2.02Hz,1H),7.07(d,J＝1.47Hz,1H),6.90(dd,J＝1.10,8.25Hz,1H),3.53～3.60(m,2H),2.96(t,J＝7.52Hz,2H),2.35～2.39(m,3H)；13C NMR(151MHz,DMSO-d6):δ161.9,161.5,161.1,159.8,151.9,148.6,135.1,134.8,133.4,133.2,132.9,132.8,128.0,127.6,127.1,124.4,123.2,123.0,118.9,118.5,112.5,111.8,111.6,111.5,107.8,102.6,40.5,25.8,21.8；ESI-MS(+):487.2[M+H]+。
example 9: preparation of N-butyl-5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH193)
The reaction procedure was the same as that of example 1 except for changing N, N-dimethylpropane-1, 3-diamine to N-butane (21.9mg, 0.30mmol), and 98.2mg of a product was isolated as a pale yellow solid in a yield of 81.0%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.45(s,1H),9.57(s,1H),9.36(d,J＝1.83Hz,1H),8.73(dd,J＝1.56,4.68Hz,1H),8.57(d,J＝5.14Hz,1H),8.49(td,J＝1.86,8.02Hz,1H),8.40(s,1H),8.14(s,1H),7.59(dd,J＝4.77,7.89Hz,1H),7.49(d,J＝1.83Hz,1H),7.47～7.50(m,1H),7.43(d,J＝4.95Hz,1H),7.38(d,J＝8.80Hz,1H),7.08(d,J＝1.47Hz,1H),3.30(d,J＝6.24Hz,2H),1.54(t,J＝7.24Hz,2H),1.33～1.40(m,2H),0.93(t,J＝7.43Hz,3H)；13C NMR(151MHz,DMSO-d6):δ161.9,161.5,161.1,124.3,112.5,111.6,102.5,38.9,38.9,20.3,20.1；ESI-MS(+):386.2[M+H]+。
example 10: preparation of N- (2- (2-methyl-1H-indol-3-yl) ethyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH130)
The reaction procedure was as in the synthesis of example 1, substituting N, N-dimethylpropane-1, 3-diamine for 2- (2-methyl-1H-indol-3-yl) ethane-1-amino (52.0mg, 0.30mmol), and finally isolating the product as a pale yellow solid, 100.2mg, 66.0% yield.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.46(s,1H),10.72(s,1H),9.56(s,1H),9.36(d,J＝1.47Hz,1H),8.71-8.75(m,1H),8.57(d,J＝5.14Hz,1H),8.54～8.56(m,1H),8.49(d,J＝8.07Hz,1H),8.13(s,1H),7.58(dd,J＝4.77,7.89Hz,1H),7.52(d,J＝7.70Hz,1H),7.49(dd,J＝1.47,8.80Hz,1H),7.43(d,J＝5.13Hz,1H),7.38(d,J＝8.62Hz,1H),7.24(d,J＝7.70Hz,1H),7.04(d,J＝0.73Hz,1H),6.92～7.00(m,2H),3.45(q,J＝6.85Hz,2H),2.92(t,J＝7.34Hz,2H),2.33(s,3H)；13C NMR(151MHz,DMSO-d6):δ161.9,161.5,161.1,159.8,151.9,148.6,135.7,134.8,133.4,133.2,132.9,132.7,128.8,127.6,124.4,120.4,118.9,118.6,117.8,112.5,111.7,110.9,108.1,107.8,102.5,40.5,24.8,11.7；ESI-MS(+):487.2[M+H]+。
example 11: preparation of N- (4-hydroxyphenyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH158)
The reaction procedure was the same as that in example 1 except for changing N, N-dimethylpropane-1, 3-diamine to 4- (2-aminoethyl) phenol (41.0mg, 0.30mmol), and 60.6mg of a product was isolated as a pale yellow solid in a yield of 43.0%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.45(br.s.,1H),9.57(s,1H),9.36(br.s.,1H),9.19(br.s.,1H),8.73(d,J＝3.67Hz,1H),8.57(d,J＝5.14Hz,1H),8.49(d,J＝5.87Hz,2H),8.14(s,1H),7.59(dd,J＝4.77,7.70Hz,1H),7.49(dd,J＝1.28,8.62Hz,1H),7.43(d,J＝4.95Hz,1H),7.38(d,J＝8.80Hz,1H),7.03～7.09(m,3H),6.70(d,J＝8.25Hz,2H),3.46(q,J＝6.91Hz,2H),2.76(t,J＝7.43Hz,2H)；13C NMR(151MHz,DMSO-d6):δ161.9,161.5,161.1,159.8,156.1,151.9,148.6,134.8,133.4,133.2,132.9,132.7,130.0(*2),127.5,124.3,118.9,115.6(*2),112.5,111.6,107.8,102.6,41.3,35.0；ESI-MS(+):450.2[M+H]+。
example 12: preparation of N- (4-methoxyphenyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH164)
The reaction procedure was the same as that of example 1 except for changing N, N-dimethylpropane-1, 3-diamine to 2- (4-methoxyphenyl) ethane-1-ammonia (45.3mg, 0.30mmol), and 80.6mg of a product was isolated as a pale yellow solid in a yield of 55.0%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.45(br.s.,1H),9.57(br.s.,1H),9.36(br.s.,1H),8.73(d,J＝3.85Hz,1H),8.57(d,J＝4.58Hz,1H),8.50(br.s.,2H),8.14(br.s.,1H),7.57～7.61(m,1H),7.49(d,J＝8.62Hz,1H),7.43(d,J＝4.77Hz,1H),7.38(d,J＝8.62Hz,1H),7.18(d,J＝7.89Hz,2H),7.06(br.s.,1H),6.87(d,J＝8.07Hz,2H),3.72(s,3H),3.49(d,J＝6.05Hz,2H),2.81(t,J＝6.88Hz,2H)；13C NMR(151MHz,DMSO-d6):δ161.9,161.5,161.1,159.8,158.2,151.9,148.6,134.8,133.4,133.2,132.9,132.7,131.8,130.1(*2),127.5,124.3,118.9,114.2(*2),112.5,111.6,107.8,102.6,55.4,41.2,34.9；ESI-MS(+):464.2[M+H]+。
example 13: preparation of N- (2- (5-hydroxy-1H-indol-3-yl) ethyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH170)
The reaction procedure was as in the synthesis of example 1, substituting N, N-dimethylpropane-1, 3-diamine for 2- (5-hydroxy-1H-indol-3-yl) ethane-1-amino (57.6mg, 0.30mmol), and finally isolating the product as a pale yellow solid, 78.6mg, 50.0% yield.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.46(s,1H),10.50(d,J＝1.47Hz,1H),9.57(s,1H),9.36(d,J＝1.65Hz,1H),8.73(dd,J＝1.47,4.77Hz,1H),8.61(s,1H),8.57(d,J＝5.13Hz,1H),8.55(t,J＝5.69Hz,1H),8.50(td,J＝1.88,7.98Hz,1H),8.14(s,1H),7.59(dd,J＝4.86,7.79Hz,1H),7.49(dd,J＝1.83,8.80Hz,1H),7.43(d,J＝5.14Hz,1H),7.39(d,J＝8.80Hz,1H),7.14(d,J＝8.62Hz,1H),7.10(d,J＝2.02Hz,1H),7.08(d,J＝1.28Hz,1H),6.91(d,J＝2.02Hz,1H),6.61(dd,J＝2.20,8.62Hz,1H),3.53～3.58(m,2H),2.87～2.92(m,2H)；13C NMR(151MHz,DMSO-d6):δ161.9,161.5,161.1,159.8,151.9,150.7,148.6,134.8,133.4,133.2,132.9,132.8,131.3,128.4,127.6,124.4,123.6,118.9,112.5,112.1,111.8,111.6,111.3,107.8,102.8,102.6,40.1,25.9；ESI-MS(+):489.2[M+H]+。
example 14: preparation of N- (3- (di-N-butylamino) propyl) -5- ((4- (pyridin-3-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH167)
The reaction procedure was the same as that in example 1 except for changing N, N-dimethylpropane-1, 3-diamine to N, N-di-N-butylpropyl-1, 3-diamine (55.6mg, 0.30mmol), and 66.6mg of a product was isolated as a pale yellow solid in a yield of 42.8%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.44(s,1H),9.56(s,1H),9.36(d,J＝1.47Hz,1H),8.70-8.76(m,1H),8.57(d,J＝5.14Hz,1H),8.49(d,J＝7.89Hz,1H),8.40(t,J＝5.41Hz,1H),8.13(s,1H),7.58(dd,J＝4.86,7.79Hz,1H),7.48(dd,J＝1.47,8.80Hz,1H),7.43(d,J＝5.14Hz,1H),7.37(d,J＝8.80Hz,1H),7.05(s,1H),3.27～3.33(m,2H),2.43(t,J＝6.97Hz,2H),2.35(t,J＝7.15Hz,4H),1.66(quin,J＝6.92Hz,2H),1.37(quin,J＝7.24Hz,4H),1.27(qd,J＝7.28,14.65Hz,4H),0.86(t,J＝7.34Hz,6H)；13C NMR(151MHz,DMSO-d6):δ161.9,161.5,161.1,159.8,151.9,148.6,134.7,133.4,133.2,132.9,132.8,127.5,124.3,118.8,112.5,111.6,107.8,102.5,53.7(*2),51.8,37.9,29.4(*2),27.4,20.6(*2),14.4(*2)；ESI-MS(+):496.3[M+H]+。
example 15: preparation of 5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -N- (2- (pyrrolidin-1-yl) ethyl) -1H-indole-2-carboxamide (QH188)
Intermediate III: preparation of 5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid
100mL of ethanol, 5-aminoindole-2-carboxylic acid ethyl ester (3.39g,0.017mol) and cyanamide (1.59g,0.038mol) are sequentially added into a dry 250mL reaction bottle at room temperature, concentrated hydrochloric acid (2.1mL,0.025mol) is dropwise added under stirring, and after dropwise addition is finished, the temperature is raised to reflux reaction for 24 hours. The reaction was stopped by detecting the completion of the reaction by thin layer chromatography. Decompressing and concentrating the reaction liquid, removing the solvent, adding water, dropwise adding an aqueous solution of ammonium nitrate (2.64g,0.033mol) at 0 ℃, preserving heat for 1h after dropwise adding, filtering, washing the filter cake for 2 times by diethyl ether, drying the filter cake in a vacuum drying oven at 45 ℃ for 4-8 h, dissolving the filter cake in ethanol, adding an ethanol solution of sodium hydroxide (0.797g,0.020mol) after adding the filter cake in (E) -3- (dimethylamino) -1- (2, 5-dimethylthiazol-4-yl) butyl-2-propen-1-one (3.50g,0.017mol), and heating to reflux reaction for 48h after dropwise adding. And (3) detecting that the reaction is finished by thin layer chromatography, stopping the reaction, and cooling to obtain a yellow solid product, namely ethyl 5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylate, wherein the yield is 69.1 percent.
50mL of ethanol, 50mL of ethyl 5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylate (5.47g,0.014mol) and 50mL of 40.0% sodium hydroxide solution were sequentially added to a dry 250mL reaction flask at room temperature, and the mixture was heated to reflux with stirring for 1 hour. The reaction was stopped by detecting the completion of the reaction by thin layer chromatography. And pouring the reaction solution into ice water, and adjusting the pH value to 4-6 at the temperature of 0 ℃. The reaction solution was filtered by suction, and the filter cake was collected and dried to give 2.56g of an orange solid 5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid in 47.0% yield.
Preparation of 5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -N- (2- (pyrrolidin-1-yl) ethyl) -1H-indole-2-carboxamide (QH188)
In a dry 50mL reaction flask, DMF 10mL, 5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid (110.0mg,0.30mmol), EDCI (86.8mg,0.45mmol), and HOBT (41.5mg,0.30mmol) were added sequentially at room temperature. Stirring for 1h at room temperature. Then, 2- (pyrrolidin-1-yl) propan-1-amine (34.2mg, 0.30mmol) and triethylamine were added thereto under stirring to react overnight at room temperature. The reaction was completed by TLC detection and stopped. Pouring the reaction solution into 80mL of ice water, and separating out solids; and (4) carrying out suction filtration to obtain a filter cake (crude product). The crude product was separated by silica gel column chromatography (eluent petroleum ether: ethyl acetate: 1, v/v) to give 87.0mg of solid product in 67.8% yield. Wherein the molar ratio of 5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid to EDCI, HOBT, 2- (pyrrolidin-1-yl) propan-1-amine is 1: 2: 1.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.47(s,1H),9.49(s,1H),8.47(d,J＝5.13Hz,1H),8.41(t,J＝5.69Hz,1H),8.09(s,1H),7.45(dd,J＝2.02,8.80Hz,1H),7.35(d,J＝8.80Hz,1H),7.06(d,J＝1.47Hz,1H),7.00(d,J＝5.32Hz,1H),3.41～3.44(m,2H),2.66(s,3H),2.64(s,3H),2.60(t,J＝6.88Hz,2H),2.50～2.52(m,4H),1.65-1.73(m,4H)；13C NMR(151MHz,DMSO-d6):δ166.6,161.5,160.6,159.4,158.3,152.3,133.3,133.2,132.6,131.4,127.5,118.8,112.4,111.5,108.2,102.7,55.6,54.2(*2),38.7,23.6(*2),19.5,18.4；ESI-MS(+):462.2[M+H]+。
example 16: preparation of N- (3- (dimethylamino) propyl) -5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH146)
The reaction procedure was as in the synthesis of example 15, substituting 2- (pyrrolidin-1-yl) propan-1-amine for N, N-dimethylpropane-1, 3-diamine (30.7mg, 0.30mmol), and finally isolating the product as a pale yellow solid, 98.2mg, yield 70.1%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.44(d,J＝0.92Hz,1H),9.48(s,1H),8.43-8.50(m,2H),8.09(s,1H),7.43(dd,J＝2.02,8.80Hz,1H),7.34(d,J＝8.80Hz,1H),7.03(d,J＝1.47Hz,1H),7.01(d,J＝5.13Hz,1H),3.29～3.33(m,2H),2.66(s,3H),2.61～2.65(m,3H),2.29(t,J＝7.15Hz,2H),2.15(s,6H),1.68(quin,J＝7.11Hz,2H)；13C NMR(151MHz,DMSO-d6):δ166.6,161.5,160.6,159.4,158.3,152.3,133.3,133.1,132.7,131.4,127.5,118.7,112.4,111.5,108.2,102.5,57.4,45.7(*2),37.8,27.7,19.5,18.4；ESI-MS(+):450.2[M+H]+。
example 17: preparation of N- (3- (dimethylamino) ethyl) -5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH185)
The reaction procedure was as in the synthesis of example 15, substituting 2- (pyrrolidin-1-yl) propan-1-amine for N, N-dimethylethane-1, 3-diamine (26.4mg, 0.30mmol), and finally isolating the product as a pale yellow solid, 48.2mg, yield 38.1%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.45(s,1H),9.48(s,1H),8.48(d,J＝5.14Hz,1H),8.35(t,J＝5.78Hz,1H),8.09(s,1H),7.44(dd,J＝2.02,8.80Hz,1H),7.35(d,J＝8.80Hz,1H),7.05(d,J＝1.47Hz,1H),7.01(d,J＝5.13Hz,1H),3.38～3.41(m,2H),2.66(s,3H),2.64(s,3H),2.43(t,J＝6.79Hz,2H),2.20(s,6H)；13C NMR(151MHz,DMSO-d6):δ166.6,161.5,160.6,159.4,158.3,152.3,133.3,133.2,132.6,131.4,127.5,118.8,112.4,111.5,108.2,102.7,58.8,45.8(*2),37.5,19.5,18.4；ESI-MS(+):435.2[M+H]+。
example 18: preparation of 5- ((4- (2, 5-dimethylthiazol-4-yl) pyrimidin-2-yl) amino) -N- (2-morpholinoethyl) -1H-indole-2-carboxamide (QH142)
The reaction procedure was as in the synthesis of example 15, substituting 2- (pyrrolidin-1-yl) propan-1-amine for N, N-dimethylethane-1, 3-diamine (26.4mg, 0.30mmol), and finally isolating the product as a pale yellow solid, 48.2mg, yield 38.1%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.45(d,J＝1.47Hz,1H),9.47(s,1H),8.46-8.49(m,1H),8.37(t,J＝5.78Hz,1H),8.09(d,J＝1.10Hz,1H),7.44(dd,J＝2.02,8.80Hz,1H),7.35(d,J＝8.62Hz,1H),7.05(d,J＝1.47Hz,1H),7.00(d,J＝5.32Hz,1H),3.58(t,J＝4.49Hz,4H),3.42(q,J＝6.66Hz,2H),2.66(s,3H),2.64(s,3H),2.48～2.49(m,2H),2.44(br.s.,4H)；13C NMR(151MHz,DMSO-d6):δ166.6,161.5,160.6,159.4,158.3,152.3,133.4,133.2,132.6,131.4,127.5,118.8,112.4,111.5,108.2,102.7,66.7(*2),58.0,53.8(*2),36.6,19.5,18.4；ESI-MS(+):477.2[M+H]+。
example 19: preparation of N- (2- (1H-indol-3-yl) ethyl) -5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH147)
An intermediate IV: preparation of 5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid
100mL of ethanol, 5-aminoindole-2-carboxylic acid ethyl ester (3.39g,0.017mol) and cyanamide (1.59g,0.038mol) are sequentially added into a dry 250mL reaction bottle at room temperature, concentrated hydrochloric acid (2.1mL,0.025mol) is dropwise added under stirring, and after dropwise addition is finished, the temperature is raised to reflux reaction for 24 hours. The reaction was stopped by detecting the completion of the reaction by thin layer chromatography. Decompressing and concentrating the reaction liquid, removing the solvent, adding water, dropwise adding an aqueous solution of ammonium nitrate (2.64g,0.033mol) at 0 ℃, preserving heat for 1h after dropwise adding, filtering, washing the filter cake with diethyl ether for 2 times, drying the filter cake in a vacuum drying oven at 45 ℃ for 4-8 h, dissolving the filter cake in ethanol, adding (E) -3- (dimethylamino) -1-phenyl-2-en-1-one (2.92g,0.017mol), dropwise adding an ethanol solution of sodium hydroxide (0.797g,0.020mol), finishing dropwise adding, and heating to reflux reaction for 48 h. And (3) detecting that the reaction is finished by thin layer chromatography, stopping the reaction, and cooling to obtain 4.12g of a yellow solid product, namely the ethyl 5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxylate, wherein the yield is 69.1%.
50mL of ethanol, 50mL of ethyl 5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxylate (5.0g,0.014mol) and 50mL of a 40% sodium hydroxide solution were sequentially added to a dry 250mL reaction flask at room temperature, and the mixture was stirred and heated to reflux for 1 hour. The reaction was stopped by detecting the completion of the reaction by thin layer chromatography. And pouring the reaction solution into ice water, and adjusting the pH value to 4-6 at the temperature of 0 ℃. And (3) carrying out suction filtration on the reaction solution, collecting a filter cake, and drying to obtain an orange solid 5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid 4.19g with the yield of 91.0%.
Preparation of N- (2- (1H-indol-3-yl) ethyl) -5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH147)
In a dry 50mL reaction flask, DMF 10mL, 5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid (99.0mg,0.30mmol), EDCI (86.8mg,0.45mmol), and HOBT (41.5mg,0.30mmol) were added sequentially at room temperature. Stirring for 1h at room temperature. Then, 2- (1H-indol-3-yl) ethan-1-amine (48.0mg, 0.30mmol) and triethylamine were added thereto under stirring to react overnight at room temperature. The reaction was completed by TLC detection and stopped. Pouring the reaction solution into 80mL of ice water, and separating out solids; and (4) carrying out suction filtration to obtain a filter cake (crude product). The crude product was separated by silica gel column chromatography (eluent petroleum ether: ethyl acetate: 1, v/v) to give 87.0mg of solid product in 67.8% yield. Wherein the molar ratio of 5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid, EDCI, HOBT, 2- (1H-indol-3-yl) ethane-1-amine is 1: 2: 1.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.47(s,1H),10.83(br.s.,1H),9.48(s,1H),8.57(t,J＝5.69Hz,1H),8.52(d,J＝5.14Hz,1H),8.18(dd,J＝2.93,6.79Hz,2H),8.15(s,1H),7.63(d,J＝7.89Hz,1H),7.54～7.58(m,3H),7.51(dd,J＝1.83,8.80Hz,1H),7.32～7.40(m,3H),7.21(d,J＝2.02Hz,1H),7.06～7.10(m,2H),7.00(t,J＝7.43Hz,1H),3.57～3.62(m,2H),3.00(t,J＝7.43Hz,2H)；13C NMR(151MHz,DMSO-d6):δ163.9,161.6,161.1,159.5,153.5,137.4,133.7,133.1,132.8,131.8,131.2,129.3(*2),128.1,127.6,127.3(*2),123.8,118.8,112.5,112.5,112.2,111.6,111.4,107.6,102.6,100.6,60.2,55.7,25.8；ESI-MS(+):473.2[M+H]+。
example 20: preparation of N- (5- (diethylamino) pentan-2-yl) -5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH125)
Reaction procedure the same synthesis as in example 19 was used to convert 2- (1H-indol-3-yl) ethan-1-amine to N, N-diethylpentane-1, 4-diamine (46.7mg, 0.30mmol), and the product was isolated as a light yellow solid, 88.2mg, 59.8% yield.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.43(br.s.,1H),9.41-9.51(m,1H),8.52(d,J＝4.95Hz,1H),8.37(t,J＝5.41Hz,1H),8.11～8.20(m,3H),7.53～7.60(m,3H),7.50(d,J＝8.07Hz,1H),7.31～7.40(m,2H),7.05(s,1H),3.30～3.34(m,2H),2.73(d,J＝11.00Hz,2H),2.12(s,3H),1.80(t,J＝11.00Hz,2H),1.68(d,J＝11.74Hz,2H),1.48(q,J＝6.79Hz,2H),1.24-1.33(m,1H),1.11-1.22(m,2H)；13C NMR(151MHz,DMSO-d6):δ163.9,161.4,161.1,159.5,137.4,133.6,133.1,132.7,131.2,129.3(*2),127.6(*2),127.3,118.8,112.5,111.4,107.6,102.5,55.9,46.7(*3),36.8,36.5,32.8,32.3(*2)；ESI-MS(+):471.3[M+H]+。
example 21: preparation of N- (3- (di-N-butylamino) propyl) -5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH178)
The reaction procedure was as in the synthesis of example 19, substituting N, N-dimethylpropane for 2- (5-methoxy-1H-indol-3-yl) ethan-1-amino (55.8mg, 0.30mmol), and finally isolating the product as a light yellow solid, 90.9mg, 58.0% yield.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.43(s,1H),9.46(s,1H),8.50～8.53(m,1H),8.40(t,J＝5.59Hz,1H),8.17～8.19(m,1H),8.17(d,J＝2.38Hz,1H),8.14(d,J＝1.10Hz,1H),7.55(quin,J＝3.30Hz,3H),7.50(dd,J＝2.02,8.80Hz,1H),7.37(d,J＝8.80Hz,1H),7.34(d,J＝5.14Hz,1H),7.03(d,J＝1.28Hz,1H),3.28～3.33(m,2H),2.43(t,J＝7.06Hz,2H),2.33～2.38(m,4H),1.66(quin,J＝7.06Hz,2H),1.34～1.40(m,4H),1.27(sxt,J＝7.34Hz,4H),0.86(t,J＝7.24Hz,6H)；13C NMR(151MHz,DMSO-d6):δ163.9,161.5,161.1,159.4,137.4,133.6,133.1,132.7,131.2,129.3(*2),127.6,127.3(*2),118.8,112.5,111.4,107.6,102.4,53.7(*2),51.8,38.0,29.4(*2),27.4,20.6(*2),14.4(*2)；ESI-MS(+):498.3[M+H]+。
example 22: preparation of N- (2- (diisopropylamino) ethyl) -5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH133)
The reaction procedure was as in the synthesis of example 19, substituting N, N-dimethylpropane for N, N-diethylpropylethyl-1, 2-diamine (43.2mg, 0.30mmol), and finally isolating the product as a pale yellow solid, 78.9mg, 55.0% yield.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.44(s,1H),9.47(s,1H),8.47～8.55(m,1H),8.32(t,J＝5.87Hz,1H),8.17(dd,J＝2.93,6.60Hz,2H),8.14(d,J＝1.10Hz,1H),7.54～7.58(m,3H),7.50(dd,J＝1.83,8.80Hz,1H),7.36(d,J＝8.80Hz,1H),7.34(d,J＝5.32Hz,1H),7.01(d,J＝0.92Hz,1H),3.21～3.26(m,2H),2.99(td,J＝6.51,13.02Hz,2H),2.53～2.58(m,2H),1.00(d,J＝6.42Hz,12H)；13C NMR(151MHz,DMSO-d6):δ163.9,161.5,161.1,159.4,137.4,133.6,133.1,132.7,131.2,129.3(*2),127.6,127.3(*2),118.8,112.4,111.4,107.6,102.3,49.0(*2),44.8,41.1,21.2(*2)；ESI-MS(+)；456.3[M+H]+。
example 23: preparation of N- (3- (diethylamino) propyl) -5- ((4- (2-methoxyphenyl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH157)
An intermediate V: preparation of 5- ((4- (2-methoxyphenyl) pyrimidin-2-yl) -amino) -1H-indole-2-carboxylic acid
100mL of ethanol, 5-aminoindole-2-carboxylic acid ethyl ester (3.39g,0.017mol) and cyanamide (1.59g,0.038mol) are sequentially added into a dry 250mL reaction bottle at room temperature, concentrated hydrochloric acid (2.1mL,0.025mol) is dropwise added under stirring, and after dropwise addition is finished, the temperature is raised to reflux reaction for 24 hours. The reaction was stopped by detecting the completion of the reaction by thin layer chromatography. Decompressing and concentrating the reaction liquid, removing the solvent, adding water, dropwise adding an aqueous solution of ammonium nitrate (2.64g,0.033mol) at 0 ℃, preserving heat for 1h after dropwise adding, filtering, washing the filter cake with diethyl ether for 2 times, drying the filter cake in a vacuum drying oven at 45 ℃ for 4-8 h, dissolving the filter cake in ethanol, adding an ethanol solution of (E) -3- (dimethylamino) -1- (2-methoxyphenyl) but-2-en-1-one (3.32g,0.017mol), dropwise adding sodium hydroxide (0.797g,0.020mol), finishing dropwise adding, and heating to reflux reaction for 48 h. The reaction was stopped by TLC and cooled to give the yellow solid product ethyl 5- ((4- (2-methoxyphenyl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylate 4.12g, 78.1% yield ethyl 5- (4- (pyridin-3-yl) pyrimidin-2-ylamino) -1H-indole-2-carboxylate.
50mL of ethanol, 50mL of ethyl 5- ((4- (2-methoxyphenyl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylate (5.0g,0.014mol) and 50mL of a 40% sodium hydroxide solution were sequentially added to a dry 250mL reaction flask at room temperature, and the mixture was heated to reflux with stirring for 1 hour. The reaction was stopped by detecting the completion of the reaction by thin layer chromatography. And pouring the reaction solution into ice water, and adjusting the pH value to 4-6 at the temperature of 0 ℃. And (3) carrying out suction filtration on the reaction solution, collecting a filter cake, and drying to obtain an orange solid 5- ((4- (2-methoxyphenyl) pyrimidine-2-yl-) amino) -1H-indole-2-carboxylic acid 4.00g with the yield of 80.0%.
Preparation of N- (3- (diethylamino) propyl) -5- ((4- (2-methoxyphenyl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH157)
In a dry 50mL reaction flask, DMF 10mL, 5- ((4- (2-methoxyphenyl) pyrimidin-2-yl) -amino) -1H-indole-2-carboxylic acid (99.0mg,0.30mmol), EDCI (86.8mg,0.45mmol), and HOBT (41.5mg,0.30mmol) were added sequentially at room temperature. Stirring for 1h at room temperature. N, N-diethylpropane-1, 3-diamine (48.0mg, 0.30mmol) and triethylamine were added under stirring at room temperature over night as a catalyst. The reaction was completed by TLC detection and stopped. Pouring the reaction solution into 80mL of ice water, and separating out solids; and (4) carrying out suction filtration to obtain a filter cake (crude product). The crude product was separated by silica gel column chromatography (eluent petroleum ether: ethyl acetate: 1, v/v) to give 83.0mg of solid product in 67.8% yield. Wherein the molar ratio of 5- ((4-phenylpyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid, EDCI, HOBT, N, N-diethylpropane-1, 3-diamine is 1: 2: 1.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.41(br.s.,1H),9.41(s,1H),8.44(d,J＝5.14Hz,2H),8.15(s,1H),7.92(d,J＝6.97Hz,1H),7.42～7.52(m,2H),7.33(d,J＝8.80Hz,1H),7.26(d,J＝4.95Hz,1H),7.19(d,J＝8.25Hz,1H),7.11(t,J＝7.34Hz,1H),6.99(s,1H),3.85～3.92(m,3H),3.27～3.33(m,2H),2.46(td,J＝7.29,14.76Hz,6H),1.66(quin,J＝6.74Hz,2H),0.96(t,J＝7.06Hz,6H)；13C NMR(151MHz,DMSO-d6):δ163.2,161.5,160.9,158.2,158.1,133.8,133.0,132.6,131.9,130.7,127.6,126.8,121.0,118.6,112.6,112.4,111.1,102.3,56.1,50.6,46.8(*2),38.0,27.2,12.2(*2)；ESI-MS(+):473.3[M+H]+。
example 24: preparation of N- (3- (dimethylamino) propyl) -5- ((4- (2-methoxyphenyl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH152)
The reaction procedure was as in the synthesis of example 23, substituting N, N-diethylpropane-1, 3-diamine for N, N-dimethylpropane-1, 4-diamine (46.7mg, 0.30mmol), and finally isolating the product as a pale yellow solid, 83.2mg, 59.8% yield.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.42(br.s.,1H),9.41(s,1H),8.44(d,J＝5.13Hz,2H),8.15(br.s.,1H),7.92(d,J＝7.15Hz,1H),7.44～7.52(m,2H),7.34(d,J＝8.62Hz,1H),7.26(d,J＝4.95Hz,1H),7.19(d,J＝8.25Hz,1H),7.11(t,J＝7.34Hz,1H),7.00(s,1H),3.89(s,3H),3.27～3.33(m,2H),2.29(t,J＝6.97Hz,2H),2.16(s,6H),1.68(quin,J＝6.79Hz,2H)；13C NMR(151MHz,DMSO-d6):δ163.2,161.5,161.0,158.2,158.1,133.8,133.0,132.6,131.9,130.7,127.6,126.8,121.1,118.6,112.6,112.4,111.1,102.5,57.3,56.1,45.6(*2),37.7,27.7；ESI-MS(+):445.2[M+H]+。
example 25: preparation of N- (3- (dimethylamino) ethyl) -5- ((4- (2-methoxyphenyl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH149)
The reaction procedure was as in the synthesis of example 23, substituting N, N-diethylpropane-1, 3-diamine for N, N-dimethylethane-1, 4-diamine (26.7mg, 0.30mmol), and finally isolating the product as a pale yellow solid, 33.2mg, yield 26.8%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.40(d,J＝1.10Hz,1H),9.40(s,1H),8.44(d,J＝5.14Hz,1H),8.31(t,J＝5.69Hz,1H),8.14(d,J＝1.47Hz,1H),7.91(dd,J＝1.83,7.70Hz,1H),7.47～7.50(m,1H),7.46(dd,J＝2.02,8.80Hz,1H),7.34(d,J＝8.80Hz,1H),7.26(d,J＝5.14Hz,1H),7.19(d,J＝7.89Hz,1H),7.11(dt,J＝0.92,7.43Hz,1H),7.02(d,J＝1.47Hz,1H),3.89(s,3H),3.38-3.40(m,2H),2.42(t,J＝6.79Hz,2H),2.19(s,6H)；13C NMR(151MHz,DMSO-d6):δ163.2,161.5,160.9,158.2,158.1,133.8,133.0,132.5,131.9,130.7,127.5,126.8,121.1,118.7,112.6,112.4,111.1,102.7,58.8,56.1,45.8(*2),37.5；ESI-MS(+):430.2[M+H]+。
example 26: preparation of N- (3- (diethylamino) propyl) -5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH123)
Intermediate VI: preparation of 5- ((4- (5-methyl-2- (methylamino) thiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid
100mL of ethanol, 5-aminoindole-2-carboxylic acid ethyl ester (3.39g,0.017mol) and cyanamide (1.59g,0.038mol) are sequentially added into a dry 250mL reaction bottle at room temperature, concentrated hydrochloric acid (2.1mL,0.025mol) is dropwise added under stirring, and after dropwise addition is finished, the temperature is raised to reflux reaction for 24 hours. The reaction was stopped by detecting the completion of the reaction by thin layer chromatography. Decompressing and concentrating the reaction liquid, removing the solvent, adding water, dropwise adding an aqueous solution of ammonium nitrate (2.64g,0.033mol) at 0 ℃, preserving heat for 1h after dropwise adding, filtering, washing the filter cake for 2 times by diethyl ether, drying the filter cake in a vacuum drying oven at 45 ℃ for 4-8 h, dissolving the filter cake in ethanol, adding an ethanol solution of sodium hydroxide (0.797g,0.020mol), and heating to reflux for 48h after dropwise adding, wherein the ethanol solution is (E) -3- (dimethylamino) -1- (5-methyl-2- (methylamino) thiazol-4-yl) -but-2-en-1-one (3.73g,0.017 mol). And (3) detecting that the reaction is finished by thin layer chromatography, stopping the reaction, and cooling to obtain 5.12g of a yellow solid product, namely 5- ((4- (5-methyl-2- (methylamino) thiazole-4-yl) pyrimidine-2-yl) amino) -1H-indole-2-carboxylic acid ethyl ester, wherein the yield is 79.1%.
50mL of ethanol, 50mL of ethyl 5- ((4- (5-methyl-2- (methylamino) thiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylate (5.68g,0.014mol) and 50mL of 40% sodium hydroxide solution were sequentially added to a dry 250mL reaction flask at room temperature, and the mixture was heated to reflux with stirring for 1 hour. The reaction was stopped by detecting the completion of the reaction by thin layer chromatography. And pouring the reaction solution into ice water, and adjusting the pH value to 4-6 at the temperature of 0 ℃. The reaction solution was filtered by suction, and the filter cake was dried to give 3.20g of an orange solid 5- ((4- (5-methyl-2- (methylamino) thiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid in 57.0% yield.
Preparation of N- (3- (diethylamino) propyl) -5- ((4- (4-methyl-2- (methylamino) thiazol-5-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH123)
In a dry 50mL reaction flask, DMF 10mL, 5- ((4- (5-methyl-2- (methylamino) thiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid (99.0mg,0.30mmol), EDCI (86.8mg,0.45mmol), and HOBT (41.5mg,0.30mmol) were added sequentially at room temperature. Stirring for 1h at room temperature. N, N-diethylpropane-1, 3-diamine (48.0mg, 0.30mmol) and triethylamine were added under stirring at room temperature over night as a catalyst. The reaction was completed by TLC detection and stopped. Pouring the reaction solution into 80mL of ice water, and separating out solids; and (4) carrying out suction filtration to obtain a filter cake (crude product). The crude product was separated by silica gel column chromatography (eluent petroleum ether: ethyl acetate: 1, v/v) to give 83.0mg of solid product in 67.8% yield. Wherein the molar ratio of 5- ((4- (5-methyl-2- (methylamino) thiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid to EDCI, HOBT, N, N-diethylpropane-1, 3-diamine is 1: 2: 1.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.40(br.s.,1H),9.23(br.s.,1H),8.44(br.s.,1H),8.30(d,J＝5.13Hz,1H),7.99～8.09(m,2H),7.46(d,J＝8.44Hz,1H),7.32(d,J＝8.62Hz,1H),7.00(br.s.,1H),6.83(d,J＝4.95Hz,1H),3.28～3.33(m,3H),2.87(d,J＝3.48Hz,3H),2.42～2.49(m,9H),1.61～1.73(m,2H),0.96(t,J＝6.88Hz,6H)；13C NMR(151MHz,DMSO-d6):δ169.6,161.5,160.3,159.0,158.1,152.4,133.7,133.0,132.6,127.5,118.6,118.5,112.3,111.2,106.6,102.4,50.6,46.8(*2),38.0,31.2,27.2,19.1,12.2(*2)；ESI-MS(+):493.2[M+H]+。
example 27: preparation of N- (2-morpholinoethyl) -5- ((4- (thien-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH118)
Intermediate VII: preparation of 5- ((4- (thien-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid
100mL of ethanol, 5-aminoindole-2-carboxylic acid ethyl ester (3.39g,0.017mol) and cyanamide (1.59g,0.038mol) are sequentially added into a dry 250mL reaction bottle at room temperature, concentrated hydrochloric acid (2.1mL,0.025mol) is dropwise added under stirring, and after dropwise addition is finished, the temperature is raised to reflux reaction for 24 hours. The reaction was stopped by detecting the completion of the reaction by thin layer chromatography. Concentrating the reaction solution under reduced pressure to remove the solvent, adding water, dropwise adding an aqueous solution of ammonium nitrate (2.64g,0.033mol) at 0 ℃, keeping the temperature for 1H after dropwise adding, filtering, washing the filter cake with diethyl ether for 2 times, drying the filter cake in a vacuum drying oven at 45 ℃ for 4-8H, dissolving the filter cake in ethanol, adding an ethanol solution of sodium hydroxide (0.797g,0.020mol), heating to reflux reaction for 48H after dropwise adding, stopping the reaction, and cooling to obtain a yellow solid product 5- ((4- (5-methyl-2- (methylamino) thiazol-4-yl) pyrimidin-2-yl) amino) -1H-indole-2-one Ethyl carboxylate 5.12g, yield 79.1%.
50mL of ethanol, 50mL of ethyl 5- ((4- (thien-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylate (5.06g,0.014mol) and 50mL of a 40% sodium hydroxide solution were sequentially added to a dry 250mL reaction flask at room temperature, and the mixture was heated to reflux with stirring for 1 hour. The reaction was stopped by detecting the completion of the reaction by thin layer chromatography. And pouring the reaction solution into ice water, and adjusting the pH value to 4-6 at the temperature of 0 ℃. And (3) carrying out suction filtration on the reaction solution, collecting a filter cake, and drying to obtain an orange solid 5- ((4- (thiophene-2-yl) pyrimidine-2-yl) amino) -1H-indole-2-carboxylic acid (3.01 g), wherein the yield is 57.0%.
Preparation of N- (2-morpholinoethyl) -5- ((4- (thien-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH118)
In a dry 50mL reaction flask, DMF 10mL, 5- ((4- (thiophen-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid (100mg,0.30mmol), EDCI (86.84mg,0.45mmol), and HOBT (41.54mg,0.30mmol) were added sequentially at room temperature. Stirring for 1h at room temperature. 2-Morpholinoethane-1-ammonia (39.0mg, 0.30mmol) was added with stirring and triethylamine was added as a catalyst for reaction overnight at room temperature. The reaction was completed by TLC detection and stopped. Pouring the reaction solution into 80mL of ice water, and separating out solids; and (4) carrying out suction filtration to obtain a filter cake (crude product). The crude product was separated by silica gel column chromatography (eluent petroleum ether: ethyl acetate: 1, v/v) to give 63.0mg of solid product in 45.3% yield. Wherein the molar ratio of 5- ((4- (thiophen-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxylic acid to EDCI, HOBT and 2-morpholinoethane-1-ammonia is 1: 2: 1.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.42(br.s.,1H),9.45(br.s.,1H),8.45(d,J＝4.95Hz,1H),8.37(t,J＝5.04Hz,1H),8.23(br.s.,1H),7.98(d,J＝3.12Hz,1H),7.80(d,J＝4.77Hz,1H),7.46(d,J＝8.62Hz,1H),7.35(d,J＝8.80Hz,1H),7.27(d,J＝5.14Hz,1H),7.24(t,J＝4.13Hz,1H),7.06(s,1H),3.59(br.s.,4H),3.42(q,J＝6.11Hz,2H),2.48(br.s.,2H),2.44(br.s.,4H)；13C NMR(151MHz,DMSO-d6):δ161.5,160.7,159.2,159.0,143.4,133.6,133.1,132.6,130.8,129.2,128.5,127.6,118.6,112.4,111.1,105.9,102.7,66.7,58.1(*2),53.8(*2),36.6；ESI-MS(+):448.2[M+H]+。
example 28: preparation of N- (2- (pyrrolidin-1-yl) ethyl) -5- ((4- (thiophen-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH101)
Reaction procedure the same synthesis as in example 27 was performed, substituting 2-morpholinoethane-1-ammonia for 2- (pyrrolidin-1-yl) ethane-1-ammonia (34.2mg, 0.30mmol), and finally isolating the product as a pale yellow solid, 55.6mg, yield 42.8%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.43(br.s.,1H),9.46(br.s.,1H),8.45(d,J＝4.95Hz,1H),8.40(br.s.,1H),8.24(br.s.,1H),7.98(d,J＝2.57Hz,1H),7.80(d,J＝4.59Hz,1H),7.46(d,J＝8.62Hz,1H),7.35(d,J＝8.62Hz,1H),7.27(d,J＝4.77Hz,1H),7.25(d,J＝4.04Hz,1H),7.08(s,1H),3.41(d,J＝6.05Hz,2H),2.60(t,J＝6.60Hz,2H),2.50(br.s.,4H),1.69(br.s.,4H)；13C NMR(151MHz,DMSO-d6):δ161.5,160.7,159.2,159.0,143.4,133.6,133.0,132.6,130.8,129.2,128.5,127.6,112.4,111.1,105.9,102.7,55.6,54.2(*2),38.7,23.6(*2)；ESI-MS(+):432.2[M+H]+。
example 29: preparation of N- (3- (4-methylpiperazin 1-yl) propyl) -5- ((4- (thien-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH139)
Reaction procedure the same synthesis as in example 27 was performed to exchange 2-morpholinoethane-1-ammonia for 3- (4-methylpiperazin-1-yl) propane-1-ammonia (47.1mg, 0.30mmol), and the product was isolated as a pale yellow solid, 62.1mg, yield 42.8%.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.43(s,1H),9.42～9.51(m,1H),8.43～8.47(m,2H),8.24(br.s.,1H),7.96～8.00(m,1H),7.80(dd,J＝0.73,4.95Hz,1H),7.46(dd,J＝2.02,8.80Hz,1H),7.34(d,J＝8.80Hz,1H),7.27(d,J＝5.14Hz,1H),7.25(dd,J＝3.85,4.95Hz,1H),7.06(d,J＝0.92Hz,1H),3.30～3.33(m,2H),2.20～2.48(m,10H),2.13～2.18(m,3H),1.70(quin,J＝6.97Hz,2H)；13C NMR(151MHz,DMSO-d6):δ161.5,160.6,159.2,159.0,143.4,133.6,133.0,132.7,130.8,129.2,128.5,127.6,118.5,112.4,111.1,105.9,102.6,56.2,55.3,53.2,46.2,37.9,26.9；ESI-MS(+):475.2[M+H]+。
example 30: preparation of N- (3- (dimethylamino) propyl) -5- ((4- (thien-2-yl) pyrimidin-2-yl) amino) -1H-indole-2-carboxamide (QH142)
The reaction procedure was as in the synthesis of example 27, substituting 2-morpholinoethane-1-ammonia for N, N-dimethylpropane-1, 3-diamine (30.6mg, 0.30mmol), and finally isolating the product as a pale yellow solid, 56.1mg, 43.2% yield.
Spectral data:1H NMR(600MHz,DMSO-d6):δ11.42(s,1H),9.46(s,1H),8.44～8.47(m,2H),8.23(br.s.,1H),7.98(dd,J＝1.01,3.76Hz,1H),7.80(dd,J＝1.01,5.04Hz,1H),7.46(dd,J＝2.02,8.99Hz,1H),7.34(d,J＝8.80Hz,1H),7.27(d,J＝5.14Hz,1H),7.25(dd,J＝3.67,4.95Hz,1H),7.05(d,J＝0.73Hz,1H),3.29～3.33(m,2H),2.25～2.30(m,2H),2.15(s,6H),1.68(quin,J＝7.11Hz,2H)；13C NMR(151MHz,DMSO-d6):161.5,160.6,159.2,159.0,143.4,133.6,133.0,132.7,130.8,129.2,128.5,127.6,118.5,112.4,111.1,105.9,102.5,57.4,45.7(*2),37.7,27.8；ESI-MS(+):420.2[M+H]+。
example 31: binding experiment of partial QH series compounds of the invention with Nur77-LBD protein in vitro
50.0. mu.g of His-Nur77-LBD protein was coupled to a Biacore chip, 72 compounds were selected from the synthesized pyrimidine indole derivatives (QH series compounds) to pass through the chip with His-Nur77-LBD protein at a concentration of 10.0. mu.M, and tripterine (10.0. mu.M) was used as a positive control compound, and response values were preliminarily screened using a Biacore T200 instrument (FIG. 1). The binding constant (Kd value) was fitted to the compounds having the better response (including 10 compounds such as QH102 and QH117) by sequentially passing the concentration gradients of 0.156. mu.M, 0.3125. mu.M, 0.625. mu.M, 1.25. mu.M, 2.5. mu.M, 5.0. mu.M, 10.0. mu.M and 20.0. mu.M through the chip (FIG. 2). These results show that the QH series compounds can be well combined with Nur77-LBD protein in vitro.
Example 32: experiment of partial QH series compound of the invention on Nur77 transcriptional activation in 293T cell
Selecting 48-well plate, digesting 293T cells with length of 10cm plate being 80-90%, taking appropriate amount of 293T cells in 10mL serum-containing culture solution, adding 200 muL of 293T cells into 48-well plate by using pipette, culturing, selecting cell density of 50-70%, adding 20 muL of transfection system into each well, gently shaking 48-well plate, mixing, placing in 5% CO-containing medium2At 37 ℃ in an incubator. After culturing for 6-10 h, changing the culture medium into a serum-containing culture medium preheated at 37 ℃, continuously culturing for about 16h, and adding the medicine. After a certain period of time of drug treatment, the sample can be collected, the 48-well plate is taken out of the cells, the culture medium is discarded, the cells are washed twice by 200 mu L of precooled PBS, and the PBS is completely absorbed. mu.L of 1 Xreporter lysine buffer was added to each well, and Luciferase activity was measured using the Luciferase assay kit. Figure 3 shows the effect of the QH series of compounds on the transcriptional activation of Nur77 as detected by the dual luciferase reporter system, and experiments show that the QH series of compounds activate the transcriptional activation function of Nur 77.
Example 33: specific experiment of QH117 on transcriptional activation of nuclear receptor Nur77
In the same manner as in example 32, the dual-luciferase reporter gene system was used to detect the binding activity of compound QH117 to Nur77, RXR α -LBD (ligand binding domain of retinoid X receptor α), RAR α -LBD (ligand binding domain of retinoid α receptor), and PPAR γ -LBD (ligand binding domain of peroxisome proliferator-activated receptor γ). The results show (FIG. 4), that compound QH117 can activate the transcriptional activation function of Nur77, but has no obvious effect on the transcriptional activation functions of RXR alpha-LBD, RAR alpha-LBD and PPAR gamma-LBD.
Example 34: compound QH117 anti-tumor effect
Pro-apoptotic effects of QH117 on tumor cells: the results of immunoblot analysis of PARP in HeLa cells treated with 1.0 μ M QH117 are shown in fig. 5. The results show that the QH series compounds can induce PARP cleavage and promote apoptosis.
Inhibition of proliferation of tumor cells by QH 117: various tumor cells (SW620, HCT116, HT29, MDA-MB231, MCF-7, T47D, HeLa) were treated with different concentrations of QH117 (0.3125. mu.M, 0.625. mu.M, 1.25. mu.M, 2.5. mu.M, 5.0. mu.M, 7.5. mu.M, 10.0. mu.M), and the effect of different concentrations of QH117 on the proliferation of each tumor cell is shown in FIG. 6. Experimental results show that QH117 can obviously inhibit the proliferation of tumor cells.
Example 35: the pro-apoptotic effect of QH117 on tumor cells was mediated by Nur77
HeL-con control cells and HeLa-Nur77KD cells are constructed by a CRISPR/Cas9 system, treated by QH117 with different concentrations, and the apoptosis-promoting effect indexes of the QH117 on the two cells are analyzed. FIG. 7 shows the results of immunoblot analysis of PARP of HeLa cells and Nur77-/-HeLa cells treated with different concentrations of QH117 (0.5. mu.M, 1.0. mu.M, 2.5. mu.M). FIG. 8 shows the proliferation ratio of different concentrations of QH117 (0.3125. mu.M, 0.625. mu.M, 1.25. mu.M, 2.5. mu.M, 5.0. mu.M, 7.5. mu.M, 10.0. mu.M) treated HeLa cells and Nur77-/-HeLa cells versus the concentration of QH 117. FIG. 9 shows the results of analysis of the positive ratios of Annexin V and PI for HeLa cells and Nur77-/-HeLa cells treated with different concentrations of QH117 (2.5. mu.M, 5.0. mu.M). Apoptosis is a pathway of programmed cell death and plays an important role in maintaining tissue organ homeostasis. Annexin V is selectively combined with Phosphatidylserine (PS), the PS is mainly distributed on the inner side of a cell membrane, the PS can outwards turn out of the surface of the cell membrane in the early apoptosis stage of the cell, and the early apoptosis phenomenon of the cell can be reflected when FITC green fluorescence is detected. Propidium Iodide (PI) stains necrotic or late apoptotic cells, primarily with nucleotides, and when the integrity of the cell membrane is destroyed it can bind to DNA and fluoresce red. As shown in fig. 9, as the concentration of HeLa cells treated by QH117 increased, the positive ratio of Annexin V and PI increased, indicating that QH 117-induced apoptosis had a significant concentration dependence; meanwhile, nonspecific cell death caused by QH117 is low, which indicates that the apoptosis induced by QH117 shows programmed death. The results in FIGS. 7-9 show that the pro-apoptotic effect of QH117 on tumor cells is Nur77 dependent.
Example 36: in vitro tumor cell inhibition activity test of QH series compounds
Cell lines: MDA-MB-231 (triple negative breast cancer cells), HeLa (human cervical cancer cells).
Cells were confluent (approximately 90%), counted, and plated at 6000 cells per well. Culturing for 4-6 h in a cell culture box, adding 100 mu L of medicine after the cells adhere to the wall, culturing for 24h with the concentrations of 50.0 mu M, 10.0 mu M, 2.0 mu M, 0.4 mu M and 0.08 mu M respectively, and adding 20 mu L of MTT (5mg/mL) into each hole. After 4h, the 96-well plate was aspirated and 100. mu.L of DMSO was added. Finally, the absorbance was measured at a wavelength of 570 nm. Processing data, computing IC50. The results of the in vitro tumor cell inhibitory activity test of some of the compounds tested are shown in table 1.
TABLE 1 inhibition of tumor cell proliferation by partial QH series of compounds
Experimental results show that the QH series compounds have certain antitumor activity.
Claims (4)
1. A pyrimidine indole derivative and pharmaceutically acceptable salt thereof are characterized in that the structural formula of the pyrimidine indole derivative is as follows:
2. a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt of claim 1 and a pharmaceutically acceptable carrier.
3. Use of a compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as claimed in claim 2 in the manufacture of a medicament for the treatment of human cervical cancer, breast cancer.
4. Use of a compound as described in claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as described in claim 2 for the preparation of a Nur77 receptor inducer; the application is used for treating human cervical cancer and breast cancer malignant tumor.
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