The present disclosure belongs to the field of pharmaceutical biotechnology, and specifically, relates to a group of IMB-C5 series compounds having anti-coronavirus activity and application thereof.
Acute infectious diseases pose a major threat to public health, among which acute respiratory infection is a main cause of morbidity and mortality caused by infectious disease worldwide. Human coronavirus is one of the common pathogens that cause acute respiratory infection, and can cause zoonosis and may lead to a global pandemic. Since the 21st century, the world has experienced three epidemic outbreaks caused by highly pathogenic coronaviruses, including severe acute respiratory syndrome (SARS) in 2002, Middle East respiratory syndrome (MERS) in 2012 and COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) in late 2019. COVID-19 has become the fifth global pandemic recorded since the influenza pandemic in 1918 and the first coronavirus pandemic in human history, causing unprecedented damage to global health and economic development[1]. The “two-pronged approach” of vaccination and drug treatment will become the main means of fighting the virus at the next stage. Small molecule drugs have become the focus of antiviral drug research and development because of their advantages such as clear targets, easy large-scale production and distribution, and convenient oral administration.
Coronavirus is a class of enveloped positive-sense single-stranded RNA virus. Currently, there are 7 known coronaviruses that can infect human, including HCoV-229E and HCoV-NL63 of the genus α-coronavirus, and HCoV-OC43, HCoV-HKU1, SARS-COV, MERS-COV and SARS-COV-2 of the genus β-coronavirus, among which SARS-COV, MERS-COV and SARS-COV-2 are highly pathogenic. The coronavirus genome is about 26-32 Kb and encodes 4 structural proteins, including spike protein (S protein), envelope protein (E protein), membrane protein (M protein) and nucleocapsid protein (N protein), and non-structural proteins, such as RNA-dependent RNA polymerase (RdRp), 3-chymotrypsin like protease (3C-like protease, 3CLpro, also known as main protease (Mpro)), papain-like protease (PLpro) and the like. Two small molecule oral drugs, Paxlovid and Molnupiravir (MNP), targeting 3CLpro and RdRp, respectively, have recently been approved for marketing, and can decrease hospitalization rate and mortality of mild/non-severe patients at a high risk of hospitalization to some extent. These drugs that directly target the viral targets have high specificity, but they also face the challenges of rapid virus mutation and development of drug resistance.
Virus needs to invade host cells for survival and proliferation. Therefore, the strategy of developing an antiviral drug against a host target has increasingly received attention from researchers, which makes it possible to develop novel antiviral drugs which have broad-spectrum antiviral effects and are less likely to develop drug resistance. Virus replicates itself by hijacking and utilizing particular host proteins, and its energy supply and substrate metabolism, etc. also depend on the host. Lipid is a fundamental cellular component that plays an important biological role in composing cell structure, serving as a signaling molecule and energy storage, etc. and is crucial in the viral life cycle[2]. SARS-COV-2 is a virus enveloped by a lipid bilayer. Lipid is involved in the processes of the viral life cycle, including fusion of the viral membrane with the host cell, viral replication, viral endocytosis and exocytosis, etc. Coronavirus replication can be interfered by targeting lipid metabolism in the host cells, which is a new idea for developing novel anti-coronavirus drugs. It is found in the clinical data study that the levels of certain lipids in the plasma of COVID-19 patients are related to the severity of infection[3]. Some experts have proposed that lipid-lowering therapy such as statins can be one of the therapeutic strategies for complications in severe COVID-19 patients[4].
IMB-C5 is a novel small molecule inhibitor for proprotein convertase subtilisin/kexin type 9 (PCSK9), which was obtained by utilizing a PCSK9 transcriptional inhibitor screening model in our laboratory. IMB-C5 has been validated to have a good effect on lowering blood lipids and inhibiting atherosclerotic plaque formation[5]. Through a series of anti-coronavirus studies, it was found that IMB-C5 has a good inhibitory effect on the replication of common coronaviruses (HCoV-229E and HCoV-OC43), and also has an inhibitory effect on SARS-COV-2, suggesting that its antiviral mechanism may be related to the regulation of lipid metabolism. Based on the structure of IMB-C5 compound, we have performed a series of modifications in the chemical structure and obtained some active compounds with a better antiviral activity and a higher therapeutic index, which are expected to be developed as novel anti-coronavirus drugs. Such compounds, as host-targeting drugs, can also be combined with inhibitors that target virus such as 3CLpro and RdRp to form an antiviral “cocktail” to further improve the treatment for COVID-19.
Firstly, the present disclosure relates to a compound of Formula (1),
More preferably, the compound is IMB-ZH-2, IMB-ZH-11, IMB-ZH-12, IMB-ZHC-2, IMB-ZHC-15, IMB-2-26, IMB-2-31, IMB-2-32, IMB-3-19, IMB-4-6, IMB-4-12, IMB-4-13, IMB-ZHB-4x, IMB-2-3, IMB-2-8, IMB-68, IMB-82, IMB-83, IMB-84, IMB-85, IMB-92, IMB-93, IMB-3-6, IMB-3-13, IMB-3-15, IMB-3-16, IMB-3-30, IMB-3-45, IMB-3-46, IMB-3-47, IMB-3-57, IMB-3-58, IMB-3-71, IMB-3-72 or IMB-3-81.
The present disclosure further relates to use of the compound of Formula (1) in the preparation of a medicament for inhibiting coronavirus
Preferably, the coronavirus is a human coronavirus; more preferably, the coronavirus is a human α-coronavirus or a human β-coronavirus; most preferably, the coronavirus is HCoV-229E, HCoV-OC43 or SARS-COV-2.
The present disclosure further relates to use of the compound of Formula (1) in the preparation of a combined medicament for inhibiting coronavirus, wherein the combined medicament further comprises an agent that targets a further viral target; preferably, the further viral target is 3CLpro and/or RdRp and the like
Preferably, the coronavirus is a human coronavirus; more preferably, the coronavirus is a human α-coronavirus or a human β-coronavirus; most preferably, the coronavirus is HCoV-229E, HCoV-OC43 or SARS-COV-2.
The present disclosure further relates to a pharmaceutical composition and/or formulation for treating coronavirus, comprising a therapeutically effective amount of the compound of Formula (1), and an optional pharmaceutical excipient/diluent.
The present disclosure further relates to a method for treating a disease caused by coronavirus infection, comprising administering to a patient a therapeutically effective amount of the compound of Formula (1) or the pharmaceutical composition and/or formulation comprising the compound of Formula (1).
The present disclosure further relates to a method for synthesizing the compound of Formula (1), specifically comprising the following steps:
or the synthetic route as shown in Reaction Scheme 2:
or the synthetic route as shown in Reaction Scheme 3:
Con represents Control, which indicates the no-treatment virus control; RBV represents Ribavirin.
Mock represents the blank control of untreated cells; Con represents Control, which indicates the no-treatment virus control; RBV represents Ribavirin; RDV represents Remdesivir.
Con represents Control, which indicates the no-treatment virus control; RBV represents Ribavirin.
Mock represents the blank control of untreated cells; Con represents Control, which indicates the no-treatment virus control; RBV represents Ribavirin.
Mock represents the blank control of untreated cells; Con represents Control, which indicates the no-treatment virus control; MNP represents Molnupiravir. In the figure, Compound IMB-2-5 is marked as 2-5, and the rest are marked in the same way.
Con represents Control, which indicates the no-treatment virus control; RBV represents Ribavirin.
Mock represents the blank control of untreated cells; Con represents Control, which indicates the no-treatment virus control; RBV represents Ribavirin.
Mock represents the blank control of untreated cells; Con represents Control, which indicates the no-treatment virus control; RBV represents Ribavirin.
Mock represents the blank control of untreated cells; Con represents Control, which indicates the no-treatment virus control; MNP represents Molnupiravir.
Mock represents the blank control of untreated cells; Con represents Control, which indicates the no-treatment virus control; MNP represents Molnupiravir.
Con represents Control, which indicates the no-treatment virus control; Mock represents the blank control of untreated cells.
Mock represents the blank control of untreated cells; Con represents Control, which indicates the no-treatment virus control.
Mock represents the blank control of untreated cells; Con represents Control, which indicates the no-treatment virus control.
Human hepatocellular carcinoma cells C3A (ATCC, CRL-10741), preserved in our laboratory.
Human hepatocellular carcinoma cells Huh7, preserved in our laboratory.
Human hepatocellular carcinoma cells Huh7.5, preserved in our laboratory.
African green monkey kidney cells Vero E6 (ATCC, CRL-1586), preserved in our laboratory.
Human coronavirus HCoV-229E (ATCC, VR-740), preserved in our laboratory.
Human coronavirus HCoV-OC43 (ATCC, VR-1558), preserved in our laboratory.
Human coronavirus SARS-COV-2 Beta variant, preserved in Guangdong Provincial Center for Disease Control.
Ribavirin (RBV) injection, as a positive control, was purchased from Tianjin KingYork Group Hubei Tianyao Pharmaceutical Co. LTD (batch number: 31712252, specification: 100 mg/mL). Remdesivir (RDV), as a positive control, was purchased from MedChemExpress LLC (Cat. No. HY-104077). Molnupiravir (MNP), as a positive control, was purchased from Shanghai Taoshu Biotechnology Co., Ltd (Cat. No. T8309).
Antibody against coronavirus HCoV-OC43 nucleocapsid protein (NP) (mouse monoclonal antibody, MAB9013, Millipore).
Antibody against HCoV-229E NP (rabbit polyclonal antibody, 40640-T62, Sino Biological).
Antibody against human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (mouse monoclonal antibody, ZB2305, ZSGB-BIO).
Antibody against double strand RNA (dsRNA) (mouse monoclonal antibody J2, SCICONS).
Fluorescein Isothiocyanate (FITC)-labeled goat anti-mouse IgG (H+L) (HS211, TransGen).
CCK-8 Cell Counting Kit (CCK-8 Kit), purchased from Nanjing Vazyme Biotech Co., Ltd (Cat. No. A311).
Specific steps are described as follows:
Under the protection of nitrogen, 8-bromoxanthine (compound 8 in the reaction scheme, 1.0 equiv) was added into a round-bottom flask and dissolved in dry DMF (3.0 mL), followed by addition of anhydrous K2CO3 or NaH (1.2 equiv). The obtained solution was added with methoxybenzyl chloride (1.0 equiv), sealed and reacted at 0° C. for about 24 h until the reaction was complete and raw material spots disappear (monitored by TLC). The reaction solution was extracted with EA, washed with water, washed with saturated NaCl solution, dried and concentrated over anhydrous Na2SO4, and purified by column chromatography to obtain a product (compound 9 in the reaction scheme) (PE/EA=3/1−1/1).
Under the protection of nitrogen, the product obtained in step 1 (compound 9 in the reaction scheme, 1.0 equiv) was added into a round-bottom flask and dissolved in dry DMF (3.0 mL), followed by addition of DBU (1.2 equiv). The obtained solution was added with 2-(trimethylsilyl) ethoxymethyl chloride (1.0 equiv), sealed and reacted at 0° C. for about 24 h until the reaction was complete and raw material spots disappear (monitored by TLC). The reaction solution was extracted with EA, washed with water, washed with saturated NaCl solution, dried and concentrated over anhydrous Na2SO4 to obtain a crude product (compound 10 in the reaction scheme).
Under the protection of nitrogen, the crude product obtained in step 2 (compound 10 in the reaction scheme, 1.0 equiv) was added into a round-bottom flask and dissolved in dry DMF (3.0 mL), followed by addition of anhydrous K2CO3 or NaH (1.2 equiv). The obtained solution was added with methyl iodide (1.0 equiv), sealed and reacted at 60° C. for about 2 h until the reaction was complete and raw material spots disappear (monitored by TLC). The reaction solution was extracted with EA, washed with water, washed with saturated NaCl solution, dried and concentrated over anhydrous Na2SO4 to obtain a crude product (compound 11 in the reaction scheme).
Under the protection of nitrogen, the crude product obtained in step 3 (compound 11 in the reaction scheme, 1.0 equiv) was added into a round-bottom flask and dissolved in dry DCM (3.0 mL). The obtained solution was added with TFA (1.0 mL) and stirred at room temperature for about 1 h until the reaction was complete and raw material spots disappear (monitored by TLC). The reaction solution was adjusted to a neutral pH with 0.5 N NaOH, extracted with EA, washed with water, washed with saturated NaCl solution, dried and concentrated over anhydrous Na2SO4, and purified by column chromatography to obtain a product (compound 12 in the reaction scheme) (PE/EA=3/1−1/1).
Specifically, under the protection of nitrogen, 8-bromotheophylline (compound 1, 1.0 equiv) was added into a round-bottom flask and dissolved in dry DMF (3.0 mL), followed by addition of anhydrous K2CO3 or NaH (1.2 equiv). The obtained solution was added with halide (1.2 equiv), sealed and reacted at 60° C. for about 2 h until the reaction was complete and raw material spots disappear (monitored by TLC). The reaction solution was extracted with EA, washed with water, washed with saturated NaCl solution, dried and concentrated over anhydrous Na2SO4, and purified by column chromatography to obtain a product (compound 2) (PE/EA=3/1−1/1).
Specifically, under the protection of nitrogen, 8-bromo-3-methyl-3,7-dihydro-purine-2,6-dione (compound 4, 1.0 equiv) was added into a round-bottom flask and dissolved in dry DMF (3.0 mL), followed by addition of anhydrous K2CO3 or NaH (1.2 equiv). The obtained solution was added with methoxybenzyl bromide (1.0 equiv), sealed and reacted at 60° C. for about 2 h until the reaction was complete and raw material spots disappear (monitored by TLC). The reaction solution was extracted with EA, washed with water, washed with saturated NaCl solution, dried and concentrated over anhydrous Na2SO4, and purified by column chromatography to obtain a product (compound 5) (PE/EA=3/1−1/1).
Under the protection of nitrogen, compound 5 obtained in general synthesis method 2 or intermediate 12 (1.0 equiv) was added into a round-bottom flask and dissolved in dry DMF (3.0 mL), followed by addition of anhydrous K2CO3 or NaH (1.2 equiv). The obtained solution was added with different halide (3.0 equiv), sealed and reacted at 70° C. for about 7 h until the reaction was complete and raw material spots disappear (monitored by TLC). The reaction solution was extracted with EA, washed with water, washed with saturated NaCl solution, dried and concentrated over anhydrous Na2SO4, and purified by column chromatography to obtain a product (compound 6 or compound 13) (PE/EA=3/1−1/1).
Under the protection of nitrogen, compound 2, 6 or 13 (1.0 equiv) obtained in general synthesis method 1 or 3 was added into a round-bottom flask and dissolved in dry DMF (3.0 mL), followed by addition of anhydrous K2CO3 or NaH (1.2 equiv). The obtained solution was added with an amine compound (3.0 equiv), sealed and reacted at 130° C. for about 13 h until the reaction was complete and raw material spots disappear (monitored by TLC). The reaction solution was extracted with EA, washed with water, washed with saturated NaCl solution, dried and concentrated over anhydrous Na2SO4, and purified by column chromatography to obtain a product (compound 3, 7 or 14) (DCM/MeOH=30/1−20/1).
(S)-8-((1-hydroxypropan-2-yl)amino)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.22 (t, J=7.8, 1H), 6.88 (d, J=7.8, 1H), 6.82 (s, 2H), 6.77 (d, J=7.3, 1H), 5.28 (q, J=16.0, 2H), 4.74 (s, 1H), 3.98-3.92 (m, 1H), 3.71 (s, 3H), 3.47 (d, J=4.9, 1H), 3.35 (s, 3H), 3.15 (s, 3H), 1.14 (d, J=6.3, 3H), 13C NMR (126 MHz, DMSO-d6) δ=159.38, 153.67, 152.81, 151.10, 149.00, 138.85, 129.74, 119.23, 113.04, 112.68, 101.15, 64.49, 55.07, 50.64, 45.26, 29.45, 27.32, 17.64. HRMS (ESI) Calcd for C18H24N5O4 [M+H]+ 374.1828; Found 374.1822.
8-((3-hydroxypropyl)amino)-7(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.23 (t, J=8.1, 1H), 7.14 (s, 1H), 6.82 (s, 2H), 6.76 (d, J=7.3, 1H), 5.26 (s, 2H), 4.48 (s, 1H), 3.71 (s, 3H), 3.44 (d, J=5.1, 2H), 3.39 (d, J=5.8, 2H), 3.36 (s, 3H), 3.34 (s, 1H), 3.16 (s, 3H), 1.77-1.61 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ=159.41, 153.97, 152.87, 151.08, 148.93, 138.70, 129.78, 119.16, 113.15, 112.58, 101.29, 58.31, 55.09, 45.23, 40.51, 32.52, 29.44, 27.33. HRMS (ESI) Calcd for C18H24N5O4 [M+H]+ 374.1828; Found 374.1824.
7-(3-methoxybenzyl)-8-((2-methoxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.28 (d, J=5.0, 1H), 7.22 (t, J=7.8, 1H), 6.82 (d, J=7.4, 2H), 6.77 (d, J=7.5, 1H), 5.26 (s, 2H), 3.71 (s, 3H), 3.48 (dd, J=9.8, 4.5, 4H), 3.35 (s, 3H), 3.24 (s, 3H), 3.16 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ=159.29, 153.85, 152.76, 150.99, 148.87, 138.59, 129.67, 119.02, 113.17, 112.38, 101.19, 62.02, 55.52, 54.97, 45.10, 42.05, 29.33, 27.22. HRMS (ESD) Calcd for C18H24N5O4 [M+H]+ 374.1828; Found 374.2.
8-((4-fluoro-3-(trifluoromethyl)benzyl)amino)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.83 (t, J=5.5, 1H), 7.70 (d, J=6.7, 1H), 7.65 (s, 1H), 7.43 (t, J=9.6, 1H), 7.21 (t, J=7.8, 1H), 6.83 (d, J=8.2, 1H), 6.79 (s, 1H), 6.74 (d, J=7.4, 1H), 5.29 (s, 2H), 4.58 (d, J=5.4, 2H), 3.68 (s, 3H), 3.33 (d, J=7.8, 4H), 3.16 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ=159.33, 153.30, 152.93, 150.91, 148.45, 138.29, 136.69, 134.22, 129.61, 126.12, 123.52, 118.90, 117.06, 116.91, 116.21, 113.06, 112.42, 101.59, 54.89, 45.27, 44.61, 29.23, 27.19. HRMS (ESI) Calcd for C23H22N5O3F4 [M+H]+ 270.1658; Found 492.1645.
(R)-8-((1-hydroxy-3-methylbutan-2-yl)amino)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.21 (t, J=7.8, 1H), 6.85 (s, 1H), 6.80 (t, J=8.6, 2H), 6.70 (d, J=8.6, 1H), 5.41 (d, J=16.0, 1H), 5.28 (d, J=15.9, 1H), 4.59 (s, 1H), 3.74 (s, 1H), 3.70 (s, 3H), 3.51 (d, J=5.9, 2H), 3.33 (d, J=13.5, SH), 3.16 (s, 3H), 1.91 (dd, J=13.3, 6.7, 1H), 0.80 (dd, J=14.1, 6.7, 6H). 13C NMR (151 MHz, DMSO-d6) δ=159.28, 154.28, 152.67, 350.99, 148.78, 138.71, 129.52, 119.25, 112.59, 112.59, 101.02, 61.04, 60.05, 54.94, 45.08, 29.26, 28.49, 27.15, 19.43, 18.28. HRMS (ESI) Calcd for C20H28N5O4 [M+H]+ 402.2141; Found 402.2137.
methyl(7-(3-methoxybenzyl)-1,3-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)glycinate
1H NMR (500 MHz, CDCl3) δ=6.97-6.88 (m, 2H), 6.84 (d, J=8.1, 1H), 5.52 (d, J=5.2, 2H), 3.79 (s, 3H), 3.60-3.53 (m, 3H), 3.39 (t, J=8.1, 3H), 1.54 (s, 2H). 13C NMR (151 MHz, CDCl3) δ=160.07, 154.41, 151.41, 148.42, 147.47, 136.53, 130.13, 127.98, 320.08, 113.91, 113.70, 109.07, 55.40, 50.26, 49.36, 49.26, 30.01, 28.24. HRMS (ESI) Calcd for C18H22N5O5 [M+H]+ 388.1615; Found 388.1632.
(R)-8-((2-hydroxypropyl)amino)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (600 MHz, CDCl3) δ=6.82 (dd, J=11.3, 5.0, 2H), 6.78 (s, 1H), 5.32 (s, 2H), 4.71 (s, 1H), 3.98-3.88 (m, 1H), 3.77 (s, 3H), 3.60-3.48 (m, 4H), 3.37 (s, 3H), 3.30-3.20 (m, 1H), 1.14 (d, J=6.3, 3H). 13C NMR (151 MHz, CDCl3) δ=160.42, 154.31, 153.55, 151.77, 147.99, 136.78, 130.47, 119.33, 113.63, 113.18, 103.15, 67.63, 55.45, 50.64, 46.88, 29.99, 27.87, 20.83. HRMS (ESI) Calcd for C18H24N5O4 [M+H]+ 374.1823; Found 374.1832.
(S)-8-((2-hydroxypropyl)amino)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (600 MHz, CDCl3) δ=7.29-7.27 (m, 1H), 6.84 (dd, J=11.3, 5.3, 2H), 6.80 (d, J=1.7, 1H), 5.33 (s, 2H), 4.74 (t, J=5.4, 1H), 3.97-3.89 (m, 1H), 3.78 (s, 3H), 3.59-3.52 (m, 4H), 3.39 (s, 3H), 3.27 (ddd, J=13.8, 7.6, 4.9, 1H), 1.16 (d, J=6.3, 3H). 13C NMR (151 MHz, CDCl3) δ=160.40, 154.30, 153.66, 151.79, 148.19, 136.84, 130.44, 139.30, 113.57, 113.16, 103.16, 67.62, 55.43, 50.62, 46.84, 29.91, 27.85, 20.83. HRMS (ESI) Calcd for C18H24N5O4 [M+H]+ 374.1823; Found 374.1829.
((7-(3-methoxybenzyl)-1,3-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)amino)acetamide
1H NMR (500 MHz, CDCl3) δ=7.84 (s, 1H), 7.34 (d, J=7.8, 1H), 6.89 (dd, J=16.4, 8.9, 4H), 5.35 (s, 2H), 3.82 (d, J=4.9, 5H), 3.61-3.50 (m, 6H). 13C NMR (151 MHz, DMSO-d6) δ=171.26, 159.30, 154.29, 153.13, 150.95, 150.59, 138.54, 129.64, 119.41, 113.52, 112.45, 101.05, 55.01, 45.44, 44.98, 28.41, 28.32.
8-(1H-imidazol-1-yl)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.86 (s, 1H), 7.24-7.18 (m, 2H), 6.82 (d, J=8.2, 1H), 6.58 (d, J=7.6, 1H), 6.54 (s, 1H), 5.53 (s, 2H), 3.74 (s, 3H), 3.59 (s, 3H), 3.43 (s, 3H). 13C NMR (151 MHz, CDCl3) δ=160.33 (s, 1H), 155.15 (s, 1H), 151.53 (s, 1H), 146.93 (s, 1H), 141.80 (s, 1H), 137.36 (s, 2H), 136.62 (s, 1H), 131.10 (s, 2H), 130.58 (s, 2H), 119.24 (s, 2H), 118.79 (s, 2H), 113.86 (s, 2H), 112.62 (s, 2H), 106.94 (s, 1H), 55.39 (s, 2H), 48.84 (s, 2H), 30.07 (s, 2H), 28.30 (s, 2H). HRMS (ESI) Calcd for C18H19N6O3 [M+H]+ 367.1519; Found 367.1527.
8-(4-(4-fluorophenyl)piperazin-1-yl)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.24 (d, J=7.9, 1H), 6.98 (t, J=8.6, 2H), 6.92-6.89 (m, 2H), 6.82-6.77 (m, 3H), 5.38 (s, 2H), 3.77 (s, 3H), 3.56 (s, 3H), 3.37 (s, 4H), 3.36 (d, J=4.7, 4H), 3.18 (d, J=4.4, 4H). 13C NMR (151 MHz, CDCl3) δ=160.11, 158.45, 156.86, 156.34, 154.86, 151.91, 147.81, 138.39, 130.07, 119.03, 115.88, 115.88, 135.73, 115.73, 112.97, 112.90, 105.27, 55.36, 50.53, 50.08, 48.68, 29.90, 27.99. HRMS (ESI) Calcd for C25H28N6O3F [M+H]+ 479.2207, Found 479.2203.
Methyl-1-(7-(3-methoxybenzyl)-1,3-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)-1H-indole-5-carboxylate
1H NMR (500 MHz, CDCl3) δ=8.05 (s, 1H), 7.94 (d, J=8.3, 1H), 7.71 (d, J=8.3, 1H), 7.26 (s, 1H), 7.10 (t, J=7.9, 1H), 6.77 (d, J=2.9, 1H), 6.72 (d, J=8.2, 1H), 6.50 (d, J=7.5, 1H), 6.36 (s, 1H), 5.45 (s, 2H), 3.92 (s, 3H), 3.62 (s, 3H), 3.59 (s, 3H), 3.47 (s, 3H). 13C NMR (151 MHz, CDCl3) δ=167.45, 159.97, 155.31, 151.65, 147.37, 143.29, 136.81, 136.12, 132.65, 130.16, 129.97, 126.02, 123.33, 121.34, 119.57, 114.05, 113.26, 112.89, 106.87, 106.70, 55.20, 52.29, 49.14, 30.14, 28.32. HRMS (ESI) Calcd for C25H24N5O5 [M+H]+ 474.1774; Found 474.1770.
7-(3-chloro-2-fluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.49 (s, 1H), 7.25 (s, 1H), 7.14 (s, 1H), 6.71 (s, 1H), 5.39 (s, 2H), 4.72 (s, 1H), 3.53 (s, 2H), 3.38 (d, J=1.6, 3H), 3.32 (s, 3H), 3.11 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ=153.42, 152.41, 151.05, 148.40 135.14, 130.74, 126.91, 126.46, 125.54, 123.96, 101.88, 57.90, 53.40, 44.47, 29.57, 27.33. HRMS (ESI) Calcd for C16H18N5O3FCl [M+H]+ 382.1082; Found 382.1073.
7-benzyl-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.18 (s, 2H), 5.26 (s, 2H), 4.70 (s, 1H), 3.51 (s, 2H), 3.36 (s, 2H), 3.31 (d, J=6.1, 3H), 3.11 (d, J=6.1, 3H). 13C NMR (126 MHz, DMSO-d6) δ=153.90, 151.00, 148.78, 137.19, 128.51, 128.51, 127.33, 127.02, 127.02, 101.28, 59.74, 45.29, 45.11, 29.33, 27.22. HRMS (ESI) Calcd for C16H20N5O3 [M+H]+ 330.1566; Found 330.1572
8-((2-hydroxyethyl)amino)-1,3-dimethyl-7-(2-methylbenzyl)-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.21 (d, J=7.3, 1H), 7.14 (t, J=7.4, 1H), 7.08 (dd, J=13.7, 6.3, 2H), 6.37 (d, J=7.2, 1H), 5.27 (s, 2H), 4.70 (t, J=5.3, 1H), 3.52 (dd, J=11.2, 5.4, 2H), 3.44-3.37 (m, 5H), 3.10 (s, 3H), 2.33 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ=153.87, 152.71, 150.97, 148.71, 137.52, 137.04, 128.38, 127.91, 127.53, 123.90, 101.27, 59.68, 45.21, 45.03, 29.29, 27.16, 21.02.
7-((2-hydroxyethyl)amino)-1,3-dimethyl-7-(3-nitrobenzyl)-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=8.22-8.09 (m, 2H), 7.63 (d, J=5.1, 2H), 7.37 (t, J=5.5, 1H), 5.43 (s, 2H), 4.73 (t, J=5.4, 1H), 3.53 (dd, J=11.5, 5.8, 2H), 3.43-3.39 (m, 2H), 3.37 (s, 3H), 3.16 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ=153.87, 152.83, 150.99, 148.98, 147.80, 139.38, 133.78, 130.25, 122.47, 122.05, 101.11, 59.63, 45.08, 44.77, 29.41, 27.27. HRMS (ESI) Calcd for C16H19N6O5 [M+H]+ 375.1411; Found 375.1416.
7-(3-fluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.37 (s, 1H), 7.25 (s, 1H), 7.06 (d, J=10.3, 3H), 5.30 (s, 2H), 4.73 (d, J=3.9, 1H), 3.53 (s, 2H), 3.38 (dd, J=16.4, 4.4, 5H), 3.16 (d, J=3.4, 3H). 13C NMR (126 MHz, DMSO-d6) δ=163.15, 161.21, 153.86, 152.79, 151.01, 148.88, 140.01, 130.61, 123.10, 114.11, 101.17, 59.67, 45.10, 44.85, 29.38, 27.25. Calcd for C16H19N5O3F [M+H]+ 348.1466; Found 348.1475.
7-(3-bromobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.40-7.34 (m, 1H), 7.25 (d, J=3.4, 1H), 7.06 (d, J=10.3, 3H), 5.30 (s, 2H), 4.73 (d, J=3.9, 1H), 3.57-3.51 (m, 2H), 3.42-3.38 (m, 2H), 3.36 (d, J=3.5, 2H), 3.32 (d, J=3.7, 1H), 3.16 (d, J=3.4, 3H). 13C NMR (126 MHz, DMSO-d6) δ=153.84, 152.78, 150.99, 148.88, 139.91, 130.82, 130.29, 129.90, 126.02, 121.72, 101.15, 59.66, 45.08, 44.75, 29.39, 27.26. HRMS (ESI) Calcd for C16H19N6O5 [M+H]+ 375.1411; Found 375.1422.
7-(2,3-difluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.34 (dd, J=17.4, 8.4, 1H), 7.24 (t, J=5.4, 1H), 7.12 (dd, J=12.8, 7.6, 1H), 6.58 (t, J=6.8, 1H), 5.41 (s, 2H), 4.71 (t, J=5.4, 1H), 3.53 (dd, J=11.5, 5.7, 2H), 3.43-3.34 (m, 5H), 3.12 (s. 3H). 13C NMR (126 MHz, DMSO-d6) δ=154.20, 152.66, 151.02, 148.89, 127.12, 127.12, 125.01, 122.34, 116.29, 116.29, 116.22, 101.22, 59.65, 45.12, 29.40, 27.17. HRMS (ESI) Calcd for C16H18N5O3F2 [M+H]+ 366.1372; Found 366.1374.
7-(2,4-difluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.28 (t, J=10.0, 1H), 7.20 (d, J=5.3, 1H), 7.00 (t, J=8.3, 1H), 6.85 (dd, J=14.9, 8.3, 1H), 5.33 (s, 2H), 4.71 (t, J=5.4, 1H), 3.39 (d, J=11.4, 2H), 3.31 (s, 3H), 3 12 (s, 2H), 2.50 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ=160.53, 158.26, 154.18, 152.66, 151.03, 148.91, 128.70, 120.67, 111.53, 103.93, 101.21, 59.68, 45.11, 29.38, 27.17. HRMS (ESI) Calcd for C16H18N5O3F2 [M+H]+ 366.1372; Found 366.1378.
7-(4-(tert-butyl)benzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.33 (d, J=8.1, 2H), 7.21 (d, J=10.7, 1H), 7.15 (d, J=8.0, 2H), 5.25 (s, 2H), 4.73 (t, J=5.4, 1H), 3.55 (dd, J=11.6, 5.8, 2H), 3.42-3.38 (m, 2H), 3.35 (s, 2H), 3.32 (s, 1H), 3.15 (s, 3H), 1.24 (s, 9H). 13C NMR (101 MHz, DMSO-d6) δ=154.34, 153.20, 151.44, 150.16, 149.23, 134.66, 127.18, 125.72, 101.70, 60.16, 45.54, 45.38, 34.65, 31.54, 29.77, 27.66. HRMS (ESI) Calcd for C20H28N5O3 [M+H]+ 386.2187; Found 386.2193.
7-(4-butylbenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.15 (d, J=29.4, 5H), 5.25 (s, 2H), 4.72 (s, 1H), 3.54 (s, 2H), 3.37 (d, J=19.8, 5H), 3.31 (s, 2H), 3.15 (s, 3H), 1.50 (s, 2H), 3.28 (s, 2H), 0.87 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ=153.88, 152.77, 151.01, 148.79, 141.47, 134.39, 128.40, 128.40, 127.03, 127.03, 101.28, 59.72, 45,08. 34.47, 33.15, 29.34, 27.23, 21.78, 13.77. HRMS (ESI) Calcd for C20H28N5O3 [M+H]+ 386.2187; Found 386.2195.
7-(3,5-difluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.22 (d, J=8.5, 3H), 6.87 (s, 2H), 5.21 (s, 2H), 4.73 (d, J=3.8, 1H), 3.70 (d, J=3.5, 3H), 3.54 (s, 2H), 3.38 (d, J=14.4, 2H), 3.36 (s, 4H), 3.17 (d, J=3.3, 2H). 13C NMR (101 MHz, DMSO-d6) δ=158.62, 153.79, 152.80, 151.01, 148.82, 129.11, 128.74, 128.74, 113.88, 113.88, 101.19, 59.75, 55.07, 45.09, 44.74, 29.33, 27.25. HRMS (ESI) Calcd for C17H22N5O4 [M+H]+ 360.1666; Found 360.1575.
8-((2-hydroxyethyl)amino)-1,3-dimethyl-7-(3-(trifluoromethoxy)benzyl)-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.45 (t, J=7.9, 1H), 7.34-7.24 (m, 3H), 7.21 (d, J=7.5, 1H), 5.34 (s, 2H), 4.73 (t, J=5.3, 1H), 3.55-3.49 (m, 2H), 3.39 (d, J=5.7, 2H), 3.36 (s, 3H), 3.15 (s, 3H). 13C NMR (101 MHz, CDCl3) δ=159.09, 158.02, 156.21, 154.14, 153.65, 145.03, 135.83, 135.83, 131.21, 125.05, 124.90, 106.29, 64.85, 50.25, 50.01, 34.56, 32.43. HRMS (ESI) Calcd for C17H19N5O4F3 [M+H]+ 414.1384; Found 414.1380.
7-(2,6-difluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.43-7.30 (m, 1H), 7.12 (t, J=5.4, 1H), 7.04 (t, J=8.1, 2H), 5.35 (s, 2H), 4.74 (t, J=5.4, 1H), 3.55 (dd, J=11.6, 5.8, 2H), 3.46-3.39 (m, 2H), 3.35 (s, 1H), 3.31 (s, 3H), 3.08 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ=161.93 (d, J=8.1, 1H), 159.46 (d, J=8.0, 1H), 154.48, 152.53, 151.02, 148.76, 129.96, 112.77, 111.94, 111.51, 101.51, 59.85, 45.20, 36.54, 29.34, 27.18. HRMS (ESI) Calcd for C16H18N5O3F2 [M+H]+ 366.1372; Found 366.1365.
7-(4-fluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.29 (d, J=25.5, 2H), 7.17 (s, 2H), 5.30 (s, 2H), 4.75 (t, J=5.4, 1H), 3.55 (dd, J=10.3, 5.2, 2H), 3.41 (dd, J=9.4, 3.7, 2H), 3.38 (s, 3H), 3.18 (s, 3H). 13C NMR (101 MHz, CDCl3) δ=165.49, 159.02, 158.01, 156.21, 154.09, 138.52, 134.53, 120.51, 106.35, 64.88, 50.27, 49.81, 34.56, 32.45. HRMS (ESI) Calcd for C16H19N5O3F [M+H]+ 348.1466; Found 348.1459.
8-((2-hydroxyethyl)amino)-1,3-dimethyl-7-(4-(trifluoromethoxy) benzyl)-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.33 (s, 4H), 7.28 (t, J=4.6, 1H), 5.33 (s, 2H), 4.73 (t, J=5.1, 1H), 3.53 (dd, J=11.1, 5.1, 2H), 3.42-3.38 (m, 2H), 3.36 (s, 3H), 3.15 (s, 3H). 13C NMR (101 MHz, CDC3) δ=159.08, 158.01, 156.22, 154.30, 152.75, 152.75, 141.83, 134.15, 134.15, 126.40, 126.40, 106.37, 64.85, 50.28, 49.85, 34.57, 32.44. HRMS (ESI) Calcd for C17H19N5O4F3 [M+H]+ 414.1384; Found 414.1375.
7-(3-chlorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.38-7.31 (m, 3H), 7.26 (s, 1H), 7.15 (d, J=6.9, 1H), 5.30 (s, 2H), 4.73 (t, J=5.4, 1H), 3.53 (t, J=5.6, 2H), 3.53 (t, J=5.6, 2H), 3.43-3.37 (m, 2H), 3.31 (s, 4H), 3.16 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ=153.92, 152.85, 151.07, 148.93, 139.65, 133.15, 130.54 (, 127.44, 127.01, 125.71, 101.24, 59.72, 45.13, 44.87, 29.41, 27.29. HRMS (ESI) Calcd for C16H19N5O3Cl [M+H]+ 364.1171; Found 364.1163.
7-(2-bromobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.67 (d, J=7.8, 1H), 7.32-7.26 (m, 2H), 7.22 (t, J=7.6, 1H), 6.44 (d, J=7.3, 1H), 5.32 (s, 2H), 4.69 (t, J=5.4, 1H), 3.52 (d, J=5.7, 2H), 3.41 (s, 5H), 3.10 (s, 3H). 13C NMR (101 MHz DMSO-d6) δ=154.33, 152.66, 151.08, 148.92, 136.06, 132.60, 128.98, 128.12, 125.81, 121.23, 101.14, 59.68, 46.33, 45.11, 29.45, 27.16. HRMS (ESI) Calcd for C16H19N5O3Br [M+H]+ 408.0666; Found 408.0657.
7-(4-(difluoromethoxy)benzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.29 (s, 3H), 7.12 (d, J=8.8, 2H), 5.28 (s, 2H), 3.54 (s, 2H), 3.39 (s, 2H), 3.35 (d, J=7.3, 3H), 3.16 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ=153.80, 152.75, 150.96, 150.15, 148.83, 134.11, 128.85, 128.85, 118.85, 118.85, 116.30, 101.11, 59.65, 45.06, 44.61, 29.28, 27.18. HRMS (ESI) Calcd for C17H20N5O4F2 [M+H]++396.1478; Found 396.1469.
8-bromo-7-(4-chlorobenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.39 (s, 2H), 7.25 (s, 3H), 5.29 (s, 2H), 4.74-4.71 (m, 1H), 3.53 (s, 2H), 3.35 (d, J=4.4, 5H), 3.15 (s, 3H). HRMS (ESI) Calcd for C16H19N5O3Cl [M+H]+ 364.1176; Found 364.1172.
((2-hydroxyethyl)amino)-1,3-dimethyl-7-((tetrahydro-2H-pyran-4-yl)methyl)-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, DMSO-d6) δ=7.00 (s, 1H), 5.75 (s, 1H), 4.73 (s, 1H), 3.90 (d, J=5.0, 2H) 3.81 (d, J=9.8, 2H), 3.54 (s, 2H), 3.39 (s, 2H), 3.34 (s, 4H), 3.26-3.08 (m, 5H), 1.99 (s, 1H), 1.32 (dd, J=38.0, 11.2, 4H). 13C NMR (126 MHz, DMSO-d6) δ=153.82, 152.57, 150.98, 148.47, 101.73, 66.58, 66.58, 59.77, 47.29, 45.06, 34.92, 29.38, 29.38, 29.28, 27.22. HRMS (ESI) Calcd for C15H24N5O4 [M+H]+ 338.1828; Found 338.1824.
8-((2-hydroxyethyl)amino)-1,3-dimethyl-7-(3-methylbenzyl)-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.24 (d, J=8.0, 1H), 7.12 (d, J=7.6, 1H), 7.04 (d, J=12.0, 2H), 5.33 (s, 2H), 4.50 (s, 1H), 3.76-3.69 (m, 2H), 3.53 (d, J=6.7, 5H), 3.40 (s, 3H), 2.33 (s, 3H). 13C NMR (151 MHz, CDCl3) δ=154.29, 153.73, 151.81, 148.28, 139.21, 135.22, 129.20, 129.17, 127.74, 124.09, 103.29, 62.69, 46.85, 46.00, 29.84, 27.85, 21.52. HRMS (ESI) Calcd for C17H22N5O3 [M+H]+ 344.1723; Found 344.1701.
ethyl3-((8-((2-hydroxyethyl)amino)-1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)methyl)benzoate
1H NMR (500 MHz, CDCl3) δ=8.01-7.91 (m, 2H), 7.51 (d, J=7.2, 1H), 7.45 (d, J=7.6, 1H), 5.40 (s, 2H), 4.66 (s, 1H), 4.38 (q, J=7.2. 2H), 3.78 (t, J=3.8, 2H), 3.59 (dd, J=9.9, 5.7, 2H), 3.52 (s, 3H), 3.38 (s, 3H), 3.39 (t, J=7.1, 3H). 13C NMR (151 MHz, CDCl3) δ=166.58, 154.25, 153.63, 151.80, 148.65, 136.08, 131.92, 131.19, 129.48, 129.38, 128.15, 102.95, 62.27, 61.54, 46.35, 45.80, 29.85, 27.83, 14.37. HRMS (ESI) Calcd for C19H24N5O5 [M+H]+ 402.1777; Found 402.1775.
8-((2-hydroxyethyl)amino)-1,3-dimethyl-7-(thiophen-3-ylmethyl)-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.41-7.36 (m, 1H), 7.27 (s, 1H), 7.08 (d, J=4.9, 1H), 5.40 (s, 2H), 4.65 (s, 1H), 3.84-3.76 (m, 2H), 3.61 (dd, J=9.5, 5.3, 2H), 3.55 (s, 3H), 3.43 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ=153.56, 152.77, 150.97, 148.75, 137.56, 127.41, 126.59, 123.21, 101.02, 59.70, 45.03, 40.85, 29.27, 27.19. HRMS (ESI) Calcd for C14H18N5O3S [M+H]+ 336.1130; Found 336.1124.
8-((2-hydroxyethyl)amino)-7-(3-methoxyphenethyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.27 (d, J=8.0, 1H), 6.85 (d, J=6.4, 1H), 6.75 (d, J=7.4, 1H), 6.66 (s, 1H), 4.27 (t, J=6.2, 2H), 3.80 (s, 3H), 3.75 (s, 1H), 3.61-3.56 (m, 2H), 3.53 (s, 3H), 3.45 (s, 3H), 3.27 (d, J=9.5, 5.2, 2H), 3.10 (t, J=6.1, 2H). 13C NMR (151 MHz, DMSO-d6) δ=159.22, 153.46, 152.66, 151.00, 348.61, 139.48, 129.24, 121.34, 114.50, 111.84, 101.20, 59.75, 54.89, 44.95, 43.59, 35.11, 29.24, 27.21. HRMS (ESI) Calcd for C18H24N5O4 [M+H]+ 374.1828; Found 374.1821.
8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.28 (d, J=7.9, 1H), 6.87-6.79 (m, 3H), 5.35 (s, 2H), 4.79 (s, 1H), 3.93 (t, J=11.6, 4H), 3.78 (s, 3H), 3.77-3.73 (m, 2H), 3.60 (s, 2H), 3.54 (s, 3H), 3.34 (t, J=11.6, 2H), 2.61 (s, 1H), 2.07 (ddd, J=11.4, 7.4, 4.0, 1H), 1.56 (d, J=12.4, 2H), 1.51-1.42 (m, 2H). 13C NMR (151 MHz, CDCl3) δ=160.40, 154.40, 153.78, 151.83, 148.43, 136.83, 130.44, 119.30, 113.58, 113.20, 103.19, 67.82, 67.82, 62.74, 55.40, 46.86, 46.26, 46.01, 34.43, 30.91, 30.91, 29.84. HRMS (ESI) Calcd for C22H30N5O5 [M+H]+ 444.2247; Found 444.2241.
1-(4-fluorobenzyl)-8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (600 MHz, CDCl3) δ=7.48-7.44 (m, 2H), 7.28 (d, J=7.9, 1H), 6.87-6.77 (m, 5H), 5.33 (s, 2H), 5.30 (s, 1H), 5.12 (s, 2H), 4.59 (s, 1H), 3.76 (s, 3H), 3.74-3.71 (m, 2H), 3.53 (dd, J=9.7, 5.0, 2H), 3.51-3.49 (m, 3H). 13C NMR (151 MHz, CDCl3) δ=160.41, 159.01, 154.16, 153.72, 151.69, 148.39, 136.80, 130.47, 130.47, 130.42, 130.25, 119.28, 113.80, 113.80, 113.77, 113.03, 103.33, 62.76, 55.38, 46.88, 46.02, 43.74, 29.84.
8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-1-(4-methoxybenzyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.16 (dt, J=20.1, 8.0, 2H), 6.98-6.92 (m, 2H), 6.80-6.68 (m, 4H), 5.46 (s, 1H), 5.25 (s, 2H), 5.09 (s, 2H), 3.71 (s, 3H), 3.68 (d, J=9.1, 5H), 3.49 (d, J=3.8, 2H), 3.44 (s, 3H).
8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-1-(pyridin-4-ylmethyl)-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.40 (d, J=7.5, 2H), 7.25-7.15 (m, 4H), 6.78 (dd, J=13.4, 8.1, 3H), 5.45 (s, 1H), 5.26 (s, 2H), 5.12 (s, 2H), 3.68 (d, J=9.4, 5H), 3.50 (s, 2H), 3.45 (s, 3H). 13C NMR (151 MHz, CDCl3) δ=160.40, 154.11, 153.76, 151.69, 148.47, 137.94, 136.80, 330.40, 128.76, 128.76, 128.46, 128.46, 127.42, 119.28, 113.78, 113.03, 103.27, 62.72, 55.39, 46.87, 46.00, 44.31, 29.88. HRMS (ESI) Calcd for C22H25N5O4 [M+H]+ 437.1937; Found 437.1931
8-bromo-1-ethyl-7-(3-methoxybenzyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=6.94 (d, J=12.0, 2H), 6.85 (d, J=8.2, 1H), 5.53 (s, 2H), 4.07 (q, J=7.0, 2H), 3.78 (s, 2H), 3.55 (s, 3H), 1.24 (t, J=7.0, 3H). 13C NMR (151 MHz, CDCl3) δ=160.07, 154.15, 151.03, 148.52, 136.64, 130.11, 127.88, 120.19, 113.81, 109.37, 55.38, 50.25, 36.87, 29.90, 13.36. HRMS (ESI) Calcd for C16H18N4O3Br [M+H]+ 393.0562; Found 393.0570.
1-(but-3-yn-1-yl)-8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.33-7.27 (m, 1H), 6.83 (dd, J=15.7, 7.6, 3H), 5.32 (s, 2H), 4.57 (s, 1H), 4.21 (t, J=6.2, 2H), 3.78 (d, J=1.9, 3H), 3.75 (s, 2H), 3.55 (s, 2H), 3.51 (d, J=1.9, 3H), 2.886 (s, J=2.886, 1H), 1.96 (d, J=2.2, 1H), 1.26 (s, 1H). 13C NMR (151 MHz, CDCl3) δ=160.38, 153.84, 153.76, 151.42, 148.51, 136.84, 130.41, 119.31, 113.49, 113.28, 103.16, 81.26, 69.78, 62.71, 55.42, 46.84, 46.00, 39.43, 29.80, 17.91. HRMS (ESI) Calcd for C20H24N5O4 [M+H]+ 398.1828; Found 398.1826.
ethyl-8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.28 (d, J=7.9, 1H). 6.83 (dd. J =: 15.4, 7.7, 3H), 5.33 (s, 2H), 4.55 (s, 1H), 4.07 (q, J=6.8, 2H), 3.78 (s, 3H), 3.74 (s, 2H), 3.53 (dd, J=9,6, 5.3, 2H), 3.50 (s, 3H), 3.00 (s, 1H), 1.24 (t, J=6.9, 3H). 13C NMR (151 MHz, CDCl3) δ=160.39, 154.05, 153.66, 151.42, 148.24, 136.89, 130.43, 119.28, 113.57, 113.13, 103.35, 62.80, 55.41, 46.83, 46.05, 36.35, 29.75, 13.56. HRMS (ESI) Calcd for C18H24N5O4 [M+H]+ 374.1828; Found 374.1807
8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-1-propyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.28 (d, J=7.8, 31), 6.83 (dd, J=14.7, 7.0, 3H), 5.33 (s, 2H), 4.58 (s, 1H), 4.00-3.92 (m, 2H), 3.78 (s, 3H), 3.74 (s, 2H), 3.54 (dd, J=9.6, 5.2, 2H), 3.50 (s, 3H), 3.04 (s, 1H), 1.73-1.64 (m, 2H), 0.95 (t, J=7.4, 3H). 13C NMR (151 MHz, CDCl3) δ=160.37, 154.24, 153.67, 151.57, 148.25, 136.91, 130.40, 119.27, 113.58, 113.09, 103.29, 62.78, 55.40, 46.80, 46.04, 42.78, 29.77, 21.55, 11.47. HRMS (ESI) Calcd for C19H26N5O4 [M +H]+ 388.1985; Found 388.1979.
tert-butyl(2-(8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)ethyl)carbamate
1H NMR (500 MHz, CDCl3) δ=7.36-7.30 (m, 1H), 6.87 (t, J=7.4, 2H), 6.83 (s, 1H), 5.34 (d, J=3.9, 2H), 4.21 (s, 2H), 3.82 (s, 3H), 3.80-3.74 (m, 2H), 3.58 (dd, J=9.6, 5.2, 2H), 3.54 (s, 3H), 3.46 (s, 2H), 1.64 (s, 1H), 1.42 (s, 9H). 13C NMR (151 MHz, CDCl3) δ=160.27, 156.17, 154.23, 153.79, 151.64, 148.51, 136.78, 130.32, 119.19, 113.49, 313.04, 103.04, 78.94, 62.48, 55.30, 46.71, 45.86, 38.23, 37.26, 29.75, 28.49, 28.49, 28.49.
tert-butyl(3-(8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)propyl)carbamate
1H NMR (500 MHz, CDCl3) δ=7.28 (d, J=6.8, 1H), 6.86-6.81 (m, 2H), 6.79 (s, 1H), 5.31 (s, 2H0, 4.68 (t, J=5.4, 1H), 4.07 (t, J=6.1, 2H), 3.78 (s, 3H), 3.76-3.71 (m, 2H), 3.54 (dd, J=9.7, 5.2, 2H), 3.50 (s, 3H), 3.09 (d, J=5.3, 2H), 1.87-1.79 (m, 2H), 1.43 (s, 9H). 13C NMR (151 MHz, CDCl3) δ=360.22, 156.15, 354.18, 153.80, 151.63, 148.53, 136.78, 130.27, 119.35, 113.43, 113.00, 102.99, 78.93, 62.40, 55.26, 46.65, 45.82, 38.20, 37.25, 29.71, 28.46, 28.46, 28.46, 28.36. HRMS (ESI) Calcd for C24H35N6O6 [M+H]+ 503.2618, Found 503.2603.
allyl-8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.24 (d, J=7.9, 1H), 6.80 (dd, J=15.9, 7.5, 3H) 5.91 (dq, J=10.7, 5.6, 1H), 5.29 (d, J=3.8, 2H), 5.22 (d, J=17.2, 1H), 5.14 (d, J=10.2, 1H), 4.70 (t, J=5.3, 1H), 4.59 (d, J=5.5, 2H), 3.75 (s, 3H), 3.72 (dd, J=13.6, 8.9, 2H), 3.52 (dd, J=9.8, 5.2, 3H). 13C NMR (151 MHz, CDCl3) δ=160.38, 353.84, 153.78, 151.41, 148.51, 136.83, 132.92, 130.41, 119.27, 117.13, 113.62, 113.10, 103.22, 62.72, 55.41, 46.84, 46.00, 43.18, 29.80. HRMS (ESI) Calcd for C19H24N5O4 [M+H]+ 386.1828; Found 386.1816.
8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-1-(prop-2-yn-1-yl)-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.25 (d, J=4.3, 1H), 6.81 (dd, J=15.1, 7.3, 3H), 5.30 (s, 2H), 5.29 (s, 1H), 4.76 (d, J=2.2, 2H), 4.66 (t, J=5.3, 1H), 3.77 (s, 3H), 3.75-3.68 (m, 2H), 3.54 (dd, J=9.5, 4.9, 2H), 3.48 (s, 3H), 2.15 (t, J=2.1, 1H). 13C NMR (151 MHz, CDCl3) δ=160.44, 153.87, 153.11, 151.04, 148.73, 136.67, 130.49, 119.34, 113.75, 113.17, 103.16, 79.32, 70.29, 62.67, 55.46, 46.99, 45.98, 30.34, 29.91. HRMS (ESI) Calcd for C19H22N5O4 [M+H]384.1672; Found 384.1662.
benzyl-8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-1-methyl-3,7-dihydro-1H-purine-2,6-dione
1H NMR (500 MHz, CDCl3) δ=7.51 (d, J=7.1, 2H), 7.31 (d, J=8.0, 3H), 6.87-6.82 (m, 2H), 6.81 (s, 1H), 5.32 (s, 2H), 5.21 (s, 2H), 3.78 (s, 3H), 3.76 (dd, J=7.1, 2.8, 2H), 3.55 (dd, J=9.5, 4.5, 2H), 3.39 (s, 3H). 13C NMR (151 MHz, CDCl3) δ=160.43, 154.38, 153.61, 153.59, 147.92, 136.83, 136.81, 130.51, 128.97, 128.97, 128.63, 128.63, 127.92, 119.40, 113.64, 113.25, 103.39, 62.70, 55.45, 46.95, 46.75, 46.05, 27.91. HRMS (ESI) Calcd for C23H26N5O4 [M+H]+ 436.1985; Found 436.1987.
CPE evaluation criteria: 4+(cell death percentage of 75% to 100%), 3+(cell death percentage of 50% to 75%), 2+(cell death percentage of 25% to 50%), 1+(cell death percentage of 0 to 25%) and 0+(all cells survive), according to cell death percentage.
The results are as shown in Table 1 and Table 2. IMB-C5 series compounds exhibited a good activity of inhibiting HCoV-229E in Huh7 and Huh7.5 cells. Furthermore, most compounds showed an activity superior to that of the positive control Ribavirin (RBV) and comparable or superior to that of the commercially available RdRp inhibitor Molnupiravir (MNP). Among them, compound IMB-85 had the best activity, and the EC50 of IMB-85 for inhibiting HCoV-229E in Huh7 and Huh7.5 cells was 0.09 μM and 4.05 μM, respectively, superior to that of the positive control and other IMB-C5 homologous compounds.
Compounds IMB-ZH-2, IMB-ZH-11, IMB-ZH-12, IMB-ZHC-2, IMB-ZHC-15, IMB-2-26, IMB-2-31, IMB-2-32, IMB-3-19, IMB-4-6, IMB-4-12, IMB-4-13, IMB-ZHB-4x, IMB-2-3, IMB-2-8, IMB-68, IMB-82, IMB-83, IMB-84, IMB-85, IMB-92, IMB-93, IMB-3-6, IMB-3-13, IMB-3-15, IMB-3-16, IMB-3-30, IMB-3-45, IMB-3-46, IMB-3-47, IMB-3-57, IMB-3-58, IMB-3-71, IMB-3-72 and IMB-3-81 in the table are the compounds with new structures.
In the in vitro pharmacodynamic assay, the effect of IMB-C5 on mRNA level of coronavirus HCoV-229E N protein was first determined (
Huh7 and Huh7.5 cells were infected at a viral load of MOI=0.035, and simultaneously administered with three concentrations of IMB-C5 or 200 μM of the positive drug (RBV). After 24 h, RNA was extracted for RT-qPCR. As shown in
Subsequently, the effect of IMB-C5 on the double-stranded RNA (dsRNA) of HCoV-229E was determined by immunofluorescence assay (
Huh7 cells were infected at a viral load of MOI=0.035, and simultaneously administered with gradient concentrations of IMB-C5 or the positive drug. After cultured at 35° C. for 24 h, the treated cells were washed with PBS for 3 times, added with 4% paraformaldehyde and incubated at room temperature for 15 min for fixation. The cells were then washed and incubated with PBS buffer containing 0.5% Triton X-100 at room temperature for 20 min for permeabilization. The cells were then washed and added with TBST buffer containing 1% bovine serum albumin (BSA) for blocking at room temperature for 1 h. The antibody against dsRNA were added and incubated at 4° C. overnight. The cells were then washed with PBST buffer, added with the FITC fluorescence-labeled secondary antibody, and incubated at room temperature in dark for 1 h. Finally, the cells were added with the nuclei-staining solution (Hoechst 33342), incubated at room temperature for 10 min, and observed under a fluorescence microscope for dsRNA level. As shown in
C3A cells were used as viral hosts and the effect of IMB-C5 on mRNA level of coronavirus HCoV-OC43 N protein was determined (
Further, the effect of IMB-C5 on N protein level of coronavirus HCoV-OC43 was determined (
In the in vitro pharmacodynamic assay, the effect of IMB-C5 series compounds on N protein level of coronavirus HCoV-229E was determined. 8 compounds with SI>80 were selected from the CPE results, including IMB-2-5, IMB-2-8, IMB-2-14, IMB-68, IMB-92, IMB-93, IMB-3-19 and IMB-85, and IMB-C5 was used as homologue control. Huh7 cells were infected at a viral load of MOI=1, and simultaneously administered with 5 μM of IMB-C5 series compounds and 15 μM of the positive control Molnupiravir (MNP, a commercially available oral coronavirus RdRp inhibitor). After 24 h, the protein was extracted for Western blotting. As shown in
In order to determine the possible cytotoxicity of IMB-C5 and IMB-85, the CCK-8 assay was first used to detect the effect of the two compounds at different concentrations on viability of different cells 48 hours after administration. As shown in
The effect of IMB-C5 and IMB-85 on mRNA level and protein level of coronavirus HCoV-229E N protein was determined. Huh7 cells were infected at a viral load of MOI=17 and simultaneously administered with IMB-C5 (3 μM), IMB-85 (3 μM, 0.6 μM and 0.12 μM) and the positive drug (RBV, 200 μM). After 24 h, RNA and protein were extracted for RT-qPCR and Western blotting, respectively. As shown in
Under the same experimental conditions, the effect of IMB-C5 and IMB-85 on HCoV-229E double-stranded RNA (dsRNA) was determined by immunofluorescence assay (
Huh7 cells were used as viral hosts and the effect of IMB-C5 and IMB-85 on N protein level of coronavirus HCoV-229E was determined (
Huh7.5 cells were used as viral hosts and the effect of IMB-C5 and IMB-85 on N protein level of coronavirus HCoV-229E was determined (
The effect of IMB-C5 and IMB-85 on N protein level of coronavirus HCoV-OC43 was determined. C3A cells were infected at a viral load of MOI=0.037, and simultaneously administered with IMB-C5 (10 μM), IMB-85 (10 μM, 2 μ, 0.4 μM and 0.08 μM) and MNP (the positive control, 15 μM). After 24 h, the protein was extracted for Western blotting. As shown in
The anti-SARS-COV-2 activity of IMB-C5 was determined by CPE assay. Vero E6 cells were seeded into 96-well culture plates and cultured at 37° C. overnight. The culture medium was then discarded. The cells in the 96-well culture plates were infected with SARS-COV-2 Beta variant (MOI=0.05) and simultaneously administered with the compounds diluted in the DMEM medium without FBS. After 1 h of infection, the culture medium was discarded, and the cells in the 96-well culture plates were administered with the compounds diluted in DMEM medium containing 2% FBS at a concentration gradient and cultured. Cytopathic effect in each group was observed when CPE in the virus control group reached 4+. The results showed that IMB-C5 had an inhibitory activity against the SARS-COV-2 Beta variant.
C3A cells were infected with HCoV-OC43 (MOI=0.28) and then added with 50 μM IMB-C5 at different time points. The viral N protein level was determined by immunofluorescence assay (
Huh7 cells were infected with HCoV-229E (MOI=10) for 2 h, and added with 5 M IMB-85 at the time of infection and at different time points after infection. The viral N protein level (
Finally, it should be noted that the above examples are provided only to assist those skilled in the art in understanding the essence of the present disclosure, but not to limit the protection scope of the present disclosure.
Number | Date | Country | Kind |
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202110668715.4 | Jun 2021 | CN | national |
Filing Document | Filing Date | Country | Kind |
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PCT/CN2022/099223 | 6/16/2022 | WO |