alexa Synthesis and Evaluation of Anti-HIV-1 Activities of Novel 7-Hydroxy-1,3- dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-4-carboxylate Derivatives | Open Access Journals
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Synthesis and Evaluation of Anti-HIV-1 Activities of Novel 7-Hydroxy-1,3- dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-4-carboxylate Derivatives

Guan-Nan LIU1,2, Rong-Hua LUO3, Xing-Jie ZHANG3, Yu ZHOU2, Jian LI2, Yong-Tang ZHENG3 and Hong LIU2*

1College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang, China

2State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

3Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China

*Corresponding Author:
Hong LIU
State Key Laboratory of Drug Research
Shanghai Institute of Material Medica
Chinese Academy of Sciences
555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park
Shanghai 201203, China
Tel: +86-21-50807042
Fax: +86-21-50807042
E-mail: [email protected]

Received date: July 03, 2014; Accepted date: July 27, 2014; Published date: July 29, 2014

Citation: LIU GN, LUO RH, ZHANG XJ, ZHOU Y, LI J, et al. (2014) Synthesis and Evaluation of Anti-HIV-1 Activities of Novel 7-Hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c]pyridine-4-carboxylate Derivatives. Med chem 4:573-580. doi:10.4172/2161-0444.1000196

Copyright: © 2014 LIU GN, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Aim: To design and synthesize a series of novel 7-hydroxy-1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-4- carboxylate derivatives and evaluate their anti-HIV-1 activities. Methods: Holding the same triad of metal-chelating heteroatoms for the catalytic site of IN and introducing a new hydroxyl group into the adjacent position of the amide to form another three-heteroatoms group for metal chelation, a series of novel 7-hydroxy-1,3-dioxo-2,3-dihydro-1Hpyrrolo[ 3,4-c]pyridine-4-carboxylate derivatives were designed and synthesized through multi-step chemical reactions. All the synthesized compounds were evaluated for their inhibitory activities against HIV-1 replication. Results: Thirtyfive new compounds (5–13) have been designed, synthesized and bioassayed. Their structural features were determined by 1H-NMR spectra, and low- and high-resolution mass spectra. Most of the synthesized compounds showed moderate to potent activities against HIV-1. Among the analogs, compounds 7d, 7f, 7i-7j, 8d and 9c exhibited potent anti-HIV-1 activities (EC50<10 μM). In particular, 7d exhibited significant anti-HIV-1 activities with EC50 values of 1.65 μM. Conclusion: This study provides a new template for further development of potent anti-HIV-1 drugs, and the preliminary SAR among the newly synthesized analogs provided useful indications for guiding further rational design of potent anti-HIV-1 agents.

Keywords

AIDS; Anti-HIV-1; Design; Integrase; SAR; Synthesis

Introduction

Acquired immunodeficiency syndrome (AIDS) and human immunodeficiency virus (HIV) infection are global health hazards with huge social, economic, and ethical consequences [1,2]. HIV-1 integrase (IN) is a virally encoded enzyme essential for replication, which catalyzes the integration of viral DNA into the host chromatin. As a result of this unique retroviral step, and the absence of any known human homolog, IN has become an attractive target for drug discovery in the treatment of HIV-1 infection [3-6]. After years of sustained effort, Raltegravir (RAL, 1) (October 2007)[7,8] and Elvitegravir (EVG, 2) (August 2012) [9,10] were the first generation of IN inhibitors (INIs) to be approved by the United States of America’s Food and Drug Administration (FDA), thus opening up a new class of antiretroviral agents (Figure 1). Unfortunately, treatment with RAL and EVG could lead to the development of resistance, and there is extensive shared cross-resistance [11-13]. The need to overcome these problems has driven the development of a second generation of INIs such as Dolutegravir (DTG, 3, Figure 1) approved on August 2013, which displays superior characteristics to RAL and EVG, but partially shares the resistance pathways [14-16]. Therefore, an intensified search for structurally novel INIs to combat resistance is urgently needed.

medicinal-chemistry-integrase-inhibitors

Figure 1: HIV-1 integrase inhibitors.

INIs share key structural features that trace their conceptual ancestry to diketoacid (DKA) progenitors containing a triad of heteroatoms, whose function is to chelate the two catalyticallyessential divalent metal ions at the IN active site [17]. Additionally, halo-substituted benzyl rings are present, appended through amine or amide groups, which interact with the penultimate cytosine base near the 3’-end of the viral DNA to prevent the insertion of the viral DNA into the host genome [18]. Based on the hits discovered using virtual screening and bioassays, we identified a new anti-HIV-1 compound, 1-pyrrolidineacetamide, through a new IN-binding site [19], which has a modified scaffold of 5,6-dihydroxypyrimidines and shows potent anti-HIV-1 activity, with therapeutic index (TI) values of >1000 [20]. Recently, the introduction of a hydroxyl group into the adjacent position of the carboxamide substituents resulted in analogs 4 (Figure 1), which is effective in suppressing the spread of HIV-1 infection in cells [21]. As a continuation of our research in this field, we have designed some novel small molecules 5-13 (Figure 1), by holding the same triad of metal-chelating heteroatoms for the catalytic site of IN (red color), and introducing a new hydroxyl group into the adjacent position of the amide substituted by halobenzyl substituents (blue color), to form another three-heteroatoms group for metal chelation (pink color). Herein, we report the synthesis and bioassay of these compounds, in which further explorations with hydrophobic groups at position N-2 and an ester or amide at the 4-position were performed.

Materials and Methods

Chemistry

Compound design: The inhibitory activity of DKA analogs against HIV depends on the triad of heteroatoms groups and halo-substituted benzyl rings of the DKA core. 35 new analogues (5–13) were designed by keeping the key groups of DKA and introducing a new hydroxyl group into the adjacent position of the amide (Table 1). We changed the length and bulk of the linker X, used various aromatic, heterocyclic, and aliphatic groups to substitute R1, and changed R2 from ester groups to amide groups.

Synthetic procedures: As outlined in Scheme 1, target compounds 5-13 were synthesized successfully from commercially available reagents. In a similar reported synthetic method, the treatment of glycine ethylester with diethyl oxalate and Et3N in ethanol followed by cyclization of the resulting ethyl 2-((2-ethoxy-2-oxoethyl)amino)- 2-oxoacetate M1 with phosphorous pentoxide (P2O5) in anhydrous acetonitrile without further purification, gave the intermediate ethyl 5-ethoxy-l,3-oxazole-2-carboxylate M2 [22]. Another intermediate M3 was obtained from maleic anhydride by treatment of the corresponding amines or hydrazines in acetic anhydride in the presence of sodium acetate [23]. Subsequently, reacting M3 with M2 in water under conditions of microwave irradiation produced target compounds 5-7 and 9-13 via a Diels-Alder reaction. Compound 8 was prepared from 7d, 7f and 7i by reacting them with the corresponding amine in anhydrous acetonitrile. 1H-nuclear magnetic resonance (1H-NMR) and mass spectroscopy (MS) characterized all the target compounds.

medicinal-chemistry-Synthetic-Procedures

Scheme 1: Synthetic Procedures. (a) Et3N/EtOH; (b) P2O5, CH3CN, N2; (c) NaOAc, (Ac)2O, Et2O, reflux; (d) H2O, microwave, 100oC, 30min; (e) CH3CN.

The reagents (chemicals) were purchased from commercial sources (Alfa, Acros, Sigma-aldrich and Shanghai Chemical Reagent Company), and used without further purification. Analytical thin layer chromatography (TLC) was HSGF 254 (0.15-0.2 mm thickness, Yantai Huiyou Company, China). The microwave reactor, chempower 18920570, was manufactured by Shanghai Chubo Instrument Limited Company. Column chromatography was performed with CombiFlash® Companion system (Teledyne Isco, Inc.). The products were characterized by their NMR and MS spectra. 1H NMR spectra were obtained on Varian Mercury-300 or Varian Mercury-400 spectrometers. Low- and high-resolution mass spectra (LRMS and HRMS) were measured on Finnigan MAT 95 and LCQ-DE-CA mass spectrometer.

General procedure for the synthesis of M1-M3

Ethyl 2-((2-ethoxy-2-oxoethyl)amino)-2-oxoacetate M1: To a solution of ethyl 2-aminoacetate (2 g, 19.4 mmol) and diethyl oxalate (5.29 mL, 38.8 mmol) in ethanol (25 mL), was added TEA (2.68 mL, 19.4 mmol). The reaction mixture was stirred at 50°C for 4 h. After removing of the solvent, the residue was dissolved in water (40 mL), extracted with CH2Cl2 (DCM) (20 mL*3), dried over Na2SO4, filtered and concentrated to give the title compound as white solid without further purification (2.72 g, 69%); LRMS (ESI) m/z 204 [M+H]+.

Ethyl 5-ethoxy-l,3-oxazole-2-carboxylate M2: To a solution of P2O5 (9.4 g, 67.0 mmol) in acetonitrile (50 mL) stirred under N2 at 0°C, was added M1 (2.72 g, 13.4 mmol) dropwise during 30 min. The reaction mixture was heated to 70°C for 4 h. Then the reaction was quenched with saturated brine, extracted with ethyl acetate (EtOAc) (20 mL*3), the combined organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by chromatography (4:1, Petro Ether(PE)-EtOAc) to afford the title compound as a white solid (1.72 g, 47.8%); 1H-NMR(300 MHz, CDCl3) δ 1.41 (t, 3H, J = 5.1 Hz), 1.47 (t, J = 5.4 Hz, 3H ), 4.25(m, 2H), 4.41(m,2H),6.34(s,1H);LCMS( ESI)m/z186[M+H]+.

General procedures for preparations of M3 (M3-1–35) are described as those for 1-benzyl-1H-pyrrole-2,5-dione M3-1.

In a 100 mL three-necked flask provided with a stirrer, a reflux condenser, and a dropping funnel were placed 0.98 g (10 mmol) of maleic anhydride and 25 mL of diethyl ether. The maleic anhydride dissolved upon stirring, at which point a solution of 1 equiv (10 mmol) of benzylamine in 5 mL of diethyl ether was run through the dropping funnel. The resulting thick suspension was stirred at room temperature for 1 h and was then cooled in an ice bath, filtered and dried. Subsequently, the residue was added to a flask containing a solution of anhydrous sodium acetate (0.33 g, 4 mmol) in acetic anhydride (5 mL) and stirred reflux for 30 min. The reaction mixture was then cooled to room temperature in a cold water bath and was then poured into 30 mL of an ice-water mixture. The precipitated product was recovered by filtration, washed three times with 10 mL portions of ice-cold water, and dried as white solid (1.50g, 80%); 1H NMR (300 MHz, CDCl3) δ 7.34-7.30 (m, 5H), 6.72 (s, 2H), 4.69 (s, 2H); MS (EI, m/z) 187 [M]+; HRMS (EI) m/z calcd C11H9NO2 [M]+ 187.0633, found 187.0638.

1-(2-fluorobenzyl)-1H-pyrrole-2,5-dione M3-2. Pale yellow solid (1.52 g, 74%); 1H NMR (300 MHz, CDCl3) δ 7.30-7.25 (m, 2H), 7.12- 7.04 (m, 2H), 6.74 (s, 2H), 4.77 (s, 2H); MS (EI, m/z) 205 [M]+; HRMS (EI) m/z calcd C11H8FNO2 [M]+ 205.0539, found 205.0542.

1-(4-fluorobenzyl)-1H-pyrrole-2,5-dione M3-3. Pale yellow solid (1.48 g, 72%); 1H NMR (300 MHz, CDCl3) δ 7.36-7.32 (m, 2H), 7.03- 6.97 (t, J = 8.7 Hz, 2H), 6.72 (s, 2H), 4.65 (s, 2H); MS (EI, m/z) 205 [M]+; HRMS (EI) m/z calcd C11H8FNO2 [M]+ 205.0539, found 205.0538.

1-(2-methoxybenzyl)-1H-pyrrole-2,5-dione M3-4. Pale yellow solid (1.65 g, 76%); 1H NMR (300 MHz, CDCl3) δ 7.25-7.22 (m, 1H), 7.12-7.09 (m, 1H), 6.92-6.85 (m, 2H), 6.73 (s, 2H), 4.74 (s, 2H), 3.84 (s, 3H); MS (EI, m/z) 217 [M]+; HRMS (EI) m/z calcd C12H11NO3 [M]+ 217.0739, found 217.0734.

1-(3-methoxybenzyl)-1H-pyrrole-2,5-dione M3-5. Pale yellow solid (1.52 g, 70%); 1H NMR (300 MHz, CDCl3) δ 7.26-7.21 (t, J = 8.1 Hz, 1H), 6.94-6.80 (m, 3H), 6.72 (s, 2H), 4.66 (s, 2H), 3.79 (s, 3H); MS (EI, m/z) 217 [M]+; HRMS (EI) m/z calcd C12H11NO3 [M]+ 217.0739, found 217.0740.

1-(4-methoxybenzyl)-1H-pyrrole-2,5-dione M3-6. Pale yellow solid (1.65 g, 76%); 1H NMR (300 MHz, CDCl3) δ 7.32-7.29 (d, J = 9.0 Hz, 2H), 6.86-6.83 (d, J = 8.7 Hz, 2H), 6.69 (s, 2H), 4.62 (s, 2H), 3.78 (s, 3H); MS (EI, m/z) 217 [M]+; HRMS (EI) m/z calcd C12H11NO3 [M]+ 217.0739, found 217.0733.

1-(3,4-dimethoxybenzyl)-1H-pyrrole-2,5-dione M3-7. Pale yellow solid (1.75 g, 71%); 1H NMR (300 MHz, CDCl3) δ 6.94-6.91 (d, J = 8.4 Hz, 2H), 6.81-6.78 (d, J = 8.1 Hz, 1H), 6.70 (s, 2H), 4.61 (s, 2H), 3.87 (s, 3H), 3.85 (s, 3H); MS (EI, m/z) 247 [M]+; HRMS (EI) m/z calcd C13H13NO4 [M]+ 247.0845, found 247.0844.

1-(naphthalen-1-ylmethyl)-1H-pyrrole-2,5-dione M3-8. Yellow solid (1.61 g, 68%); 1H NMR (300 MHz, CDCl3) δ 8.28-8.25 (d, J = 8.1 Hz, 1H), 7.89-7.79 (m, 2H), 7.61-7.41 (m, 4H), 6.71 (s, 2H), 5.16 (s, 2H); MS (EI, m/z) 237 [M]+; HRMS (EI) m/z calcd C15H11NO2 [M]+ 237.0790, found 237.0787.

1-(1-phenylethyl)-1H-pyrrole-2,5-dione M3-9. Yellow solid (1.25 g, 62%); 1H NMR (300 MHz, CDCl3) δ 7.44-7.41 (m, 2H), 7.36-7.27 (m, 3H), 6.64 (s, 2H), 5.40-5.33 (q, 1H), 1.85-1.82 (d, J = 7.2 Hz, 3H); MS (EI, m/z) 201 [M]+; HRMS (EI) m/z calcd C12H11NO2 [M]+ 201.0790, found 201.0792.

1-((4-fluorophenyl) amino)-1H-pyrrole-2,5-dione M3-10. Yellow solid (1.07 g, 52%); 1H NMR (300 MHz, CDCl3) δ 6.98-6.93 (m, 2H), 6.82 (s, 2H), 6.77-6.73 (m, 2H); MS (EI, m/z) 206 [M]+; HRMS (EI) m/z calcd C10H7FN2O2 [M]+ 206.0492, found 206.0495.

1-((4-chlorophenyl)amino)-1H-pyrrole-2,5-dione M3-11. Yellow solid (1.22 g, 55%); 1H NMR (300 MHz, CDCl3) δ 7.22-7.19 (m, 2H), 6.83 (s, 2H), 6.40-6.31 (m, 2H); MS (EI, m/z) 222 [M]+; HRMS (EI) m/z calcd C10H7ClN2O2 [M]+ 222.0196, found 222.0201.

1-phenethyl-1H-pyrrole-2,5-dione M3-12. Pale yellow solid (1.69 g, 84%); 1H NMR (300 MHz, CDCl3) δ 7.29-7.19 (m, 5H), 3.79-3.75 (t, J = 7.5 Hz, 2H), 2.93-2.88 (t, J = 7.5 Hz, 2H); MS (EI, m/z) 201 [M]+; HRMS (EI) m/z calcd C12H11NO2 [M]+ 201.0790, found 201.0781.

1-(2-fluorophenethyl)-1H-pyrrole-2,5-dione M3-13. Pale yellow solid (1.73 g, 79%); 1H NMR (300 MHz, CDCl3) δ 7.23-7.12 (m, 2H), 7.07-6.99 (m, 2H), 6.66 (s, 2H), 3.83-3.77 (t, J = 7.2 Hz, 2H), 2.98- 2.93 (t, J = 6.9 Hz, 2H); MS (EI, m/z) 219 [M]+; HRMS (EI) m/z calcd C12H10FNO2 [M]+ 219.0696, found 219.0693.

1-(3-fluorophenethyl)-1H-pyrrole-2,5-dione M3-14. Pale yellow solid (1.80 g, 82%); 1H NMR (300 MHz, CDCl3) δ 7.29-7.22 (m, 1H), 6.99-6.89 (m, 3H), 6.68 (s, 2H), 3.79-3.74 (t, J = 7.5 Hz, 2H), 2.93- 2.88 (t, J = 7.5 Hz, 2H); MS (EI, m/z) 219 [M]+; HRMS (EI) m/z calcd C12H10FNO2 [M]+ 219.0696, found 219.0691.

1-(4-fluorophenethyl)-1H-pyrrole-2,5-dione M3-15. Pale yellow solid (1.80 g, 82%); 1H NMR (300 MHz, CDCl3) δ 7.18-7.13 (m, 2H), 7.00-6.94 (m, 2H), 6.66 (s, 2H), 3.77-3.72 (t, J = 7.2 Hz, 2H), 2.91- 2.88 (t, J = 7.2 Hz, 2H); MS (EI, m/z) 219 [M]+; HRMS (EI) m/z calcd C12H10FNO2 [M]+ 219.0696, found 219.0690.

1-(4-chlorophenethyl)-1H-pyrrole-2,5-dione M3-16. Pale yellow solid (1.69 g, 72%); 1H NMR (300 MHz, CDCl3) δ 7.27-7.24 (d, J = 8.4 Hz, 2H), 7.15-7.12 (d, J = 8.4 Hz, 2H), 6.67 (s, 2H), 3.77-3.72 (t, J = 7.5 Hz, 2H), 2.91-2.86 (t, J = 7.5 Hz, 2H); MS (EI, m/z) 235 [M]+; HRMS (EI) m/z calcd C12H10ClNO2 [M]+ 235.0400, found 235.0398.

1-(4-methylphenethyl)-1H-pyrrole-2,5-dione M3-17. Pale yellow solid (1.74 g, 81%); 1H NMR (300 MHz, CDCl3) δ 7.10 (s, 4H), 6.66 (s, 2H), 3.77-3.72 (t, J = 7.8 Hz, 2H), 2.89-2.84 (t, J = 7.8 Hz, 2H), 2.32 (s, 3H); MS (EI, m/z) 215 [M]+; HRMS (EI) m/z calcd C13H13NO2 [M]+ 215.0946, found 215.0942.

4-(2-(2,5-dioxo-2,5-dihydro-1H- p y r r o l - 1 - y l ) e t h y l ) benzenesulfonamide M3-18. Yellow solid (1.51 g, 54%); 1H NMR (300 MHz, CD3OD) δ 7.81-7.78 (d, J = 8.1 Hz, 2H), 7.37-7.34 (d, J = 8.1 Hz, 2H), 6.76 (s, 2H), 3.79-3.74 (t, J = 7.5 Hz, 2H), 3.00-2.95 (t, J = 7.8 Hz, 2H); MS (EI, m/z) 280 [M]+; HRMS (EI) m/z calcd C12H12N2O4S [M]+ 280.0518, found 280.0519.

1-(4-methoxyphenethyl)-1H-pyrrole-2,5-dione M3-19. Pale yellow solid (1.69 g, 72%); 1H NMR (300 MHz, CDCl3) δ 7.13-7.10 (d, J = 8.4 Hz, 2H), 6.84-6.81 (d, J = 9.0 Hz, 2H), 6.66 (s, 2H), 3.79 (s, 3H), 3.75- 3.70 (t, J = 7.5 Hz, 2H), 2.89-2.82 (t, J = 7.5 Hz, 2H); MS (EI, m/z) 231 [M]+; HRMS (EI) m/z calcd C13H13NO3 [M]+ 231.0895, found 231.0893.

1-(3,4-dimethoxyphenethyl)-1H-pyrrole-2,5-dione M3-20. Yellow solid (1.75 g, 67%); 1H NMR (300 MHz, CDCl3) δ 6.78-6.71 (m, 3H), 6.66 (s, 2H), 3.87 (s, 3H), 3.85 (s, 3H), 3.77-3.72 (t, J = 7.5 Hz, 2H), 2.88- 2.83 (t, J = 7.8 Hz, 2H); MS (EI, m/z) 261 [M]+; HRMS (EI) m/z calcd C14H15NO4 [M]+ 261.1001, found 261.0992.

1-(2-(1H-indol-3-yl)ethyl)-1H-pyrrole-2,5-dione M3-21. Pale yellow solid (1.68 g, 70%); 1H NMR (300 MHz, CDCl3) δ 7.69-7.66 (d, J = 7.5 Hz, 1H), 7.37-7.34 (d, J = 7.8 Hz, 2H), 7.21-7.14 (m, 2H), 7.06- 7.05 (br, 1H), 6.66 (s, 2H), 3.87-3.82 (t, J = 7.5 Hz, 2H), 3.10-3.05 (t, J = 7.5 Hz, 2H); MS (EI, m/z) 240 [M]+; HRMS (EI) m/z calcd C14H12N2O2 [M]+ 240.0899, found 240.0903.

1-(2-(4-bromophenyl)-2-oxoethyl)-1H-pyrrole-2,5-dione M3-22. Yellow solid (1.23 g, 42%); 1H NMR (300 MHz, CDCl3) δ 7.85-7.82 (d, J = 8.4 Hz, 2H), 7.68-7.65 (d, J = 8.4 Hz, 2H), 6.84 (s, 2H), 4.92 (s, 2H); MS (EI, m/z) 293 [M]+; HRMS (EI) m/z calcd C12H8BrNO3 [M]+ 292.9688, found 292.9691.

1-(2-phenylpropyl)-1H-pyrrole-2,5-dione M3-23. Yellow solid (1.08 g, 50%); 1H NMR (300 MHz, CDCl3) δ 7.31-7.08 (m, 5H), 6.61 (s, 2H), 3.74-3.58 (m, 2H), 3.27-3.22 (m, 1H), 1.28-1.26 (d, J = 6.9 Hz, 2H); MS (EI, m/z) 215 [M]+; HRMS (EI) m/z calcd C13H13NO2 [M]+ 215.0946, found 215.0941.

1-(2,2-diphenylethyl)-1H-pyrrole-2,5-dione M3-24. Pale yellow solid (1.80 g, 65%); 1H NMR (300 MHz, CDCl3) δ 7.29-7.20 (m, 10H), 6.55 (s, 2H), 4.64-4.58 (t, J = 8.7 Hz, 1H), 4.16-4.13 (d, J = 7.6 Hz, 2H); MS (EI, m/z) 277 [M]+; HRMS (EI) m/z calcd C18H15NO2 [M]+ 277.1103, found 277.1108.

methyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-phenylpropanoate M3-25. Pale yellow solid (1.58 g, 61%); 1H NMR (300 MHz, CDCl3) δ 7.27-7.21 (m, 5H), 6.60 (s, 2H), 4.99-4.94 (m, 1H), 3.79 (s, 3H), 3.50-3.43 (m, 2H); MS (EI, m/z) 259 [M]+; HRMS (EI) m/z calcd C14H13NO4 [M]+ 259.0845, found 259.0843.

1-(3-phenylpropyl)-1H-pyrrole-2,5-dione M3-26. White solid (1.59 g, 74%); 1H NMR (300 MHz, CDCl3) δ 7.28-7.26 (m, 2H), 7.20- 7.17 (m, 3H), 6.66 (s, 2H), 3.61-3.56 (t, J = 7.5 Hz, 2H), 2.66-2.61 (t, J = 7.5 Hz, 2H), 2.00-1.90 (m, 2H); MS (EI, m/z) 215 [M]+; HRMS (EI) m/z calcd C13H13NO2 [M]+ 215.0946, found 215.0945.

1-(1,2,3,4-tetrahydronaphthalen-1-yl)-1H-pyrrole-2,5-dione M3- 27. Pale yellow solid (1.41 g, 62%); 1H NMR (300 MHz, CDCl3) δ 7.16- 7.08 (m, 3H), 6.88-6.85 (d, J = 7.8 Hz, 1H), 6.71 (s, 2H), 5.38-5.33 (m, 1H), 3.04-2.93 (m, 1H), 2.84-2.76 (m, 1H), 2.35-2.23 (m, 1H), 2.11-2.00 (m, 2H), 1.84-1.79 (m, 1H); MS (EI, m/z) 227 [M]+; HRMS (EI) m/z calcd C14H13NO2 [M]+ 227.0946, found 227.0941.

1-propyl-1H-pyrrole-2,5-dione M3-28. Colorless oil (1.03 g, 74%); 1H NMR (300 MHz, CDCl3) δ 6.69 (s, 2H), 3.52-3.47 (t, J = 6.9 Hz, 2H), 1.66-1.58 (m, 2H), 0.93-0.88 (t, J = 7.8 Hz, 2H); MS (EI, m/z) 139 [M]+; HRMS (EI) m/z calcd C7H9NO2 [M]+ 139.0633, found 139.0634.

1-t-butyl-1H-pyrrole-2,5-dione M3-29. Colorless oil (1.04 g, 68%); 1H NMR (300 MHz, CDCl3) δ 6.52 (s, 2H), 1.58 (s, 9H); MS (EI, m/z) 153 [M]+; HRMS (EI) m/z calcd C8H11NO2 [M]+ 153.0790, found 153.0793.

General procedures for preparations of 5-7, 9-13 are described as those for ethyl 2-benzyl-7-hydroxy-1,3-dioxo-2,3-dihydro-1Hpyrrolo[ 3,4-c]pyridine-4-carboxylate 5a.

Compound M2 (100 mg, 0.54 mmol) and compound M3-1 (123 mg, 0.54 mmol) were dissolved in 4 mL water. The vial was sealed and the mixture was irradiated for 30 min at 100°C in the microwave reactor. The precipitated product was recovered by filtration, dried, and dissolved in DCM. The title compound was afford by chromatography (20:1, DCM-MeOH) as a yellow solid (130 mg, 74%); 1H NMR (300 MHz, CD3OD) δ 8.13 (s, 1H), 7.33-7.18 (m, 5H), 4.70 (s, 2H), 4.43-4.36 (dd, J = 6.9 and 14.1 Hz, 2H), 1.43-1.38 (t, J = 6.9 Hz, 3H); MS (ESI, m/z) 325 [M-H]+; HRMS (ESI) m/z calcd C17H13N2O5 [M-H]+ 325.0824, found 325.0842.

Ethyl 2-(2-fluorobenzyl)-7-hydroxy-1,3-dioxo-2,3-dihydro-1Hpyrrolo[ 3,4-c]pyridine -4-carboxylate 5b. Yellow solid (124 mg, 67%); 1H NMR (300 MHz, CDCl3) δ 8.69 (s, 1H), 7.43-7.37 (m, 1H), 7.34- 7.27 (m, 1H), 7.14-7.07 (m, 2H), 4.93 (s, 2H), 4.56-4.49 (q, 2H), 1.48- 1.43 (t, J = 7.2 Hz, 3H); MS (EI, m/z) 344 [M]+; HRMS (EI) m/z calcd C17H13FN2O5 [M]+ 344.0808, found 344.0792.

Ethyl 2-(4-fluorobenzyl)-7-hydroxy-1,3-dioxo-2,3-dihydro-1Hpyrrolo[ 3,4-c]pyridine -4-carboxylate 5c. Yellow solid (132 mg, 71%); 1H NMR (300 MHz, CD3OD) δ 8.12 (s, 1H), 7.37-7.33 (m, 2H), 7.02- 6.96 (t, J = 7.8 Hz, 2H), 4.72 (s, 2H), 4.42-4.39 (dd, J = 7.5 and 14.4 Hz, 2H), 1.42-1.37 (t, J = 7.2 Hz, 3H); MS (ESI, m/z) 343 [M-H]+; HRMS (ESI) m/z calcd C17H12FN2O5 [M-H]+ 343.0730, found 343.0733.

Ethyl 2-(2-methoxybenzyl)-7-hydroxy-1,3-dioxo-2,3-dihydro-1Hpyrrolo[ 3,4-c]pyri- dine-4-carboxylate 5d. Yellow solid (131 mg, 68%); 1H NMR (300 MHz, CDCl3) δ 8.68 (s, 1H), 7.31-7.25 (m, 2H), 6.94-6.86 (m, 2H), 4.90 (s, 2H), 4.56-4.48 (q, 2H), 3.84 (s, 3H), 1.48-1.43 (t, J = 6.9 Hz, 3H); MS (EI, m/z) 356 [M]+; HRMS (EI) m/z calcd C18H16N2O6 [M]+ 356.1008, found 356.1000.

Ethyl 2-(3-methoxybenzyl)-7-hydroxy-1,3-dioxo-2,3-dihydro-1Hpyrrolo[ 3,4-c]pyri- dine-4-carboxylate 5e. Yellow solid (144 mg, 75%); 1H NMR (300 MHz, CDCl3) δ 8.68 (s, 1H), 7.26-7.23 (m, 1H), 7.02-6.96 (m, 2H), 6.86-6.83 (m, 1H), 4.81 (s, 2H), 4.56-4.49 (q, 2H), 3.80 (s, 3H), 1.48-1.44 (t, J = 6.9 Hz, 3H); MS (EI, m/z) 356 [M]+; HRMS (EI) m/z calcd C18H16N2O6 [M]+ 356.1008, found 356.1006.

Ethyl 2-(4-methoxybenzyl)-7-hydroxy-1,3-dioxo-2,3-dihydro-1Hpyrrolo[ 3,4-c]pyri- dine-4-carboxylate 5f. Yellow solid (135 mg, 70%); 1H NMR (300 MHz, CD3OD) δ 8.16 (s, 1H), 7.16-7.14 (d, J = 6.9 Hz, 2H), 6.74-6.71 (d, J = 7.5 Hz, 2H), 4.62 (s, 2H), 4.42-4.36 (dd, J = 7.5 and 14.4 Hz, 2H), 3.72 (s, 3H), 1.43-1.38 (t, J = 6.9 Hz, 3H); MS (ESI, m/z) 355 [M-H]+; HRMS (ESI) m/z calcd C18H15N2O6 [M-H]+ 355.0930, found 355.0938.

Ethyl 2-(3,4-dimethoxybenzyl)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c] pyridine-4-carboxylate 5g. Yellow solid (110 mg, 53%); 1H NMR (300 MHz, CDCl3) δ 8.67 (s, 1H), 7.03-6.99 (m, 2H), 6.82-6.79 (m, 1H), 4.77 (s, 2H), 4.56-4.49 (q, 2H), 3.88 (s, 3H), 3.86 (s, 3H), 1.48-1.44 (t, J = 6.9 Hz, 3H); MS (EI, m/z) 386 [M]+; HRMS (EI) m/z calcd C19H18N2O7 [M]+ 386.1114, found 386.1120.

Ethyl 2-(naphthalen-1-ylmethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c] pyridine-4-carboxylate 5h. Yellow solid (128 mg, 63%); 1H NMR (300 MHz, CDCl3) δ 8.66 (s, 1H), 8.34-8.32 (d, J = 8.4 Hz, 1H), 7.89-7.83 (m, 2H), 7.66-7.43 (m, 4H), 5.32 (s, 2H), 4.56-4.49 (q, 2H), 1.48- 1.43 (t, J = 7.2 Hz, 3H); MS (EI, m/z) 376 [M]+; HRMS (EI) m/z calcd C21H16N2O5 [M]+ 376.1059, found 376.1062.

Ethyl 2-(1-phenylethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro-1Hpyrrolo[ 3,4-c]pyridine- 4-carboxylate 5i. Yellow solid (92 mg, 50%); 1H NMR (300 MHz, CDCl3) δ 8.65 (s, 1H), 7.51-7.48 (m, 2H), 7.35-7.27 (m, 3H), 5.58-5.51 (q, 1H), 4.56-4.48 (q, 2H), 1.94-1.92 (d, J = 7.2 Hz, 3H), 1.48-1.44 (t, J = 6.9 Hz, 3H); MS (EI, m/z) 340 [M]+; HRMS (EI) m/z calcd C18H16N2O5 [M]+ 340.1059, found 340.1052.

Ethyl 2-((4-fluorophenyl)amino)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c] pyridine-4-carboxylate 6a. Yellow solid (129 mg, 69%); 1H NMR (300 MHz, CDCl3) δ 8.77 (s, 1H), 6.97-6.88 (m, 4H), 4.55-4.48 (q, 2H), 1.47-1.42 (t, J = 6.9 Hz, 3H); MS (EI, m/z) 345 [M]+; HRMS (EI) m/z calcd C16H12FN3O5 [M]+ 345.0761, found 345.0758.

Ethyl 2-((4-chlorophenyl)amino)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c] pyridine-4-carboxylate 6b. Yellow solid (125 mg, 64%); 1H NMR (300 MHz, CDCl3) δ 8.79 (s, 1H), 7.25-7.22 (d, J = 9.0 Hz, 2H), 6.81-6.78 (d, J = 9.0 Hz, 2H), 4.93 (s, 2H), 4.55-4.48 (q, 2H), 1.47-1.42 (t, J = 7.2 Hz, 3H); MS (EI, m/z) 361 [M]+; HRMS (EI) m/z calcd C16H12ClN3O5 [M]+ 361.0465, found 361.0460.

Ethyl 2-phenethyl-7-hydroxy-1,3-dioxo-2,3-dihydro-1H-pyrrolo [3,4-c]pyridine-4-carboxylate 7a. Yellow solid (138 mg, 75%); 1H NMR (300 MHz, CDCl3) δ 8.68 (s, 1H), 7.30-7.22 (m, 5H), 4.57-4.50 (q, 2H), 3.96-3.91 (t, J = 7.2 Hz, 2H), 3.02-2.97 (t, J = 7.2 Hz, 2H), 1.50-1.45 (t, J = 7.2 Hz, 3H); MS (EI, m/z) 340 [M]+; HRMS (EI) m/z calcd C18H16N2O5 [M]+ 340.1059, found 340.1069.

Ethyl 2-(2-fluorophenethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c]pyridine-4-carboxylate 7b. Yellow solid (151 mg, 78%); 1H NMR (300 MHz, CD3OD) δ 8.10 (s, 1H), 7.19-7.17 (m, 2H), 7.05-6.99 (m, 2H), 4.40-4.33 (dd, J = 7.2 and 13.8 Hz, 2H), 3.86-3.81 (t, J = 6.9 Hz, 2H), 3.00-2.95 (t, J = 6.9 Hz, 2H), 1.40-1.35 (t, J = 7.2 Hz, 3H); MS (ESI, m/z) 357 [M-H]+; HRMS (ESI) m/z calcd C18H14FN2O5 [M-H]+ 357.0887, found 357.0887.

Ethyl 2-(3-fluorophenethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c]pyridine-4-carboxylate 7c. Yellow solid (145 mg, 75%); 1H NMR (300 MHz, CD3OD) δ 8.14 (s, 1H), 7.25-7.20 (m, 1H), 7.01-6.89 (m, 3H), 4.41-4.34 (dd, J = 7.8 and 14.1 Hz, 2H), 3.84-3.79 (t, J = 7.5 Hz, 2H), 2.96-2.91 (t, J = 7.5 Hz, 2H), 1.41-1.37 (t, J = 6.9 Hz, 3H); MS (ESI, m/z) 357 [M-H]+; HRMS (ESI) m/z calcd C18H14FN2O5 [M-H]+ 357.0887, found 357.0897.

Ethyl 2-(4-fluorophenethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c]pyridine-4-carboxylate 7d. Yellow solid (130 mg, 67%); 1H NMR (300 MHz, CDCl3) δ 8.69 (s, 1H), 7.22-7.17 (m, 2H), 7.02-6.96 (m, 2H), 4.57-4.50 (q, 2H), 3.93-3.88 (t, J = 7.8 Hz, 2H), 3.00- 2.95 (t, J = 7.2 Hz, 2H), 1.49-1.44 (t, J = 7.2 Hz, 3H); MS (EI, m/z) 358 [M]+; HRMS (EI) m/z calcd C18H15FN2O5 [M]+ 358.0965, found 358.0957.

Ethyl 2-(4-chlorophenethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c]pyridine-4-carboxylate 7e. Yellow solid (150 mg, 74%); 1H NMR (300 MHz, CD3OD) δ 8.10 (s, 1H), 7.25-7.16 (m, 4H), 4.40-4.33 (dd, J = 7.2 and 14.1 Hz, 2H), 3.82-3.77 (t, J = 6.9 Hz, 2H), 2.93-2.88 (t, J = 7.5 Hz, 2H), 1.41-1.36 (t, J = 7.2 Hz, 3H); MS (ESI, m/z) 373 [M-H]+; HRMS (ESI) m/z calcd C18H14ClN2O5 [M-H]+ 373.0591, found 373.0584.

Ethyl 2-(4-methyphenethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c]pyridine-4-carboxylate 7f. Yellow solid (155 mg, 81%); 1H NMR (300 MHz, CDCl3) δ 8.68 (s, 1H), 7.06-7.02 (m, 4H), 4.46-4.39 (q, 2H), 3.85-3.80 (t, J = 7.5 Hz, 2H), 2.92-2.87 (t, J = 7.8 Hz, 2H), 2.26 (s, 3H), 1.42-1.37 (t, J = 7.2 Hz, 3H); MS (EI, m/z) 354 [M]+; HRMS (EI) m/z calcd C19H18N2O5 [M]+ 354.1216, found 354.1220.

Ethyl 2-(4-sulfamoylphenethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c]pyridine-4-carboxylate 7g. Yellow solid (174 mg, 77%); 1H NMR (300 MHz, CD3OD) δ 8.07 (s, 1H), 7.80-7.77 (d, J = 8.1 Hz, 2H), 7.41-7.39 (d, J = 8.4 Hz, 2H), 4.40-4.33 (dd, J = 6.9 and 14.1 Hz, 2H), 3.88- 3.83 (t, J = 8.1 Hz, 2H), 3.06-3.01 (t, J = 6.9 Hz, 2H), 1.41-1.36 (t, J = 7.2 Hz, 3H); MS (ESI, m/z) 418 [M-H]+; HRMS (ESI) m/z calcd C18H16N3O7S [M-H]+ 418.0709, found 418.0730.

Ethyl 2-(4-methoxyphenethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c]pyridine-4-carboxylate 7h. Yellow solid (146 mg, 73%); 1H NMR (300 MHz, CD3OD) δ 8.16 (s, 1H), 7.10-7.07 (d, J = 8.1 Hz, 2H), 6.80-6.77 (d, J = 8.1 Hz, 2H), 4.42-4.35 (dd, J = 6.9 and 14.1 Hz, 2H), 3.79-3.72 (m, 5H), 2.86-2.81 (t, J = 7.5 Hz, 2H), 1.41-1.37 (t, J = 7.2 Hz, 3H); MS (ESI, m/z) 369 [M-H]+; HRMS (ESI) m/z calcd C19H17N2O6 [M-H]+ 369.1087, found 369.1100.

Ethyl 2-(3,4-dimethoxyphenethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4- c]pyridine-4-carboxylate 7i. Yellow solid (171 mg, 79%); 1H NMR (300 MHz, CDCl3) δ 8.67 (s, 1H), 6.77-6.75 (m, 3H), 4.57-4.49 (q, 2H), 3.94-3.89 (t, J = 7.5 Hz, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 2.97-2.92 (t, J = 7.8 Hz, 2H), 1.49-1.44 (t, J = 7.2 Hz, 3H); MS (EI, m/z) 400 [M]+; HRMS (EI) m/z calcd C20H20N2O7 [M]+ 400.1271, found 400.1281.

Ethyl 2-(2-(1H-indol-3-yl)ethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c]pyridine-4-carboxylate 7j. Yellow solid (149 mg, 73%); 1H NMR (300 MHz, CDCl3) δ 8.67 (s, 1H), 7.71-7.68 (d, J = 8.4 Hz, 1H), 7.27 (s, 1H), 7.18-7.13 (m, 3H), 4.58-4.51 (q, 2H), 4.04-3.99 (t, J = 7.5 Hz, 2H), 3.19-3.15 (t, J = 7.5 Hz, 2H), 1.50-1.45 (t, J = 7.2 Hz, 3H); MS (EI, m/z) 379 [M]+; HRMS (EI) m/z calcd C20H17N3O5 [M]+ 379.1168, found 379.1171.

Ethyl 2-(2-(1H-indol-3-yl)ethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c]pyridine-4-carboxylate 7j. Yellow solid (149 mg, 73%); 1H NMR (300 MHz, CDCl3) δ 8.67 (s, 1H), 7.71-7.68 (d, J = 8.4 Hz, 1H), 7.27 (s, 1H), 7.18-7.13 (m, 3H), 4.58-4.51 (q, 2H), 4.04-3.99 (t, J = 7.5 Hz, 2H), 3.19-3.15 (t, J = 7.5 Hz, 2H), 1.50-1.45 (t, J = 7.2 Hz, 3H); MS (EI, m/z) 379 [M]+; HRMS (EI) m/z calcd C20H17N3O5 [M]+ 379.1168, found 379.1171.

Ethyl 2-(2-phenylpropyl)-7-hydroxy-1,3-dioxo-2,3-dihydro-1Hpyrrolo[ 3,4-c]pyridine-4-carboxylate 9b. Yellow solid (140 mg, 73%); 1H NMR (300 MHz, CDCl3) δ 8.65 (s, 1H), 7.31-7.21 (m, 5H), 4.56-4.48 (q, 2H), 3.90-3.74 (m, 2H), 3.37-3.29 (m, 1H), 1.48-1.43 (t, J = 6.9 Hz, 3H), 1.33-1.31 (d, J = 8.1 Hz, 3H); MS (EI, m/z) 354 [M]+; HRMS (EI) m/z calcd C19H18N2O5 [M]+ 354.1216, found 354.1210.

Ethyl 2-(2,2-diphenylethyl)-7-hydroxy-1,3-dioxo-2,3-dihydro- 1H-pyrrolo[3,4-c]pyridine-4-carboxylate 9c. Yellow solid (175 mg, 78%); 1H NMR (300 MHz, CDCl3) δ 8.62 (s, 1H), 7.30-7.19 (m, 10H), 4.74-4.68 (t, J = 8.1 Hz, 1H), 4.55-4.48 (q, 2H), 4.32-4.29 (d, J = 8.4 Hz, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 2.97-2.92 (t, J = 7.8 Hz, 2H), 1.49- 1.44 (t, J = 7.2 Hz, 3H); MS (EI, m/z) 416 [M]+; HRMS (EI) m/z calcd C24H20N2O5 [M]+ 416.1372, found 416.1368.

Ethyl 2-(1-methoxy-1-oxo-3-phenylpropan-2-yl)-7-hydroxy- 1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-4-carboxylate 9d. Yellow solid (148 mg, 69%); 1H NMR (300 MHz, CDCl3) δ 8.65 (s, 1H), 7.26-7.14 (m, 5H), 5.17-5.12 (m, 1H), 4.53-4.46 (q, 2H), 3.81 (s, 3H), 3.63-3.47 (m, 2H), 1.47-1.42 (t, J = 7.2 Hz, 3H); MS (EI, m/z) 398 [M]+; HRMS (EI) m/z calcd C20H18N2O7 [M]+ 398.1114, found 398.1115.

Ethyl 2-(3-phenylpropyl)-7-hydroxy-1,3-dioxo-2,3-dihydro-1Hpyrrolo[ 3,4-c]pyridine-4-carboxylate 10. Yellow solid (145 mg, 76%); 1H NMR (300 MHz, CD3OD) δ 8.24 (s, 1H), 7.20-7.02 (m, 5H), 4.45- 4.38 (dd, J = 6.9 and 14.1 Hz, 2H), 3.66-3.61 (m, 2H), 2.64-2.57 (m, 2H), 1.98-1.90 (m, 2H), 1.43-1.39 (t, J = 7.2 Hz, 3H); MS (ESI, m/z) 353 [M+H]+; HRMS (ESI) m/z calcd C19H17N2O5 [M-H]+ 353.1137, found 353.1146.

Ethyl 2-(1,2,3,4-tetrahydronaphthalen-1-yl)-7-hydroxy-1,3-dioxo- 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-4-carboxylate 11. Yellow solid (134 mg, 68%); 1H NMR (300 MHz, CDCl3) δ 8.68 (s, 1H), 7.19-7.07 (m, 3H), 6.95-6.92 (d, J = 4.5 Hz, 1H), 5.57-5.52 (m, 1H), 4.56-4.49 (q, 2H), 3.03-2.79 (m, 2H), 2.40-2.29 (m, 1H), 2.17-2.05 (m, 2H), 1.91-1.82 (m, 1H), 1.48-1.44 (t, J = 7.2 Hz, 3H); MS (EI, m/z) 366 [M]+; HRMS (EI) m/z calcd C20H18N2O5 [M]+ 366.1216, found 366.1218.

Ethyl 2-propyl-7-hydroxy-1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c] pyridine-4-carboxylate 12. Yellow solid (99 mg, 66%); 1H NMR (300 MHz, CD3OD) δ 8.10 (s, 1H), 4.42-4.35 (dd, J = 7.5 and 14.4 Hz, 2H), 3.57-3.52 (t, J = 7.5 Hz, 2H), 1.67-1.60 (m, 2H), 1.42-1.37 (t, J = 7.2 Hz, 3H), 0.93- 0.88 (t, J = 7.5 Hz, 3H); MS (ESI, m/z) 277 [M-H]+; HRMS (ESI) m/z calcd C13H13N2O5 [M-H]+ 277.0824, found 277.0815.

Ethyl 2-t-butyl-7-hydroxy-1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c] pyridine-4-carboxylate 13. Yellow solid (100 mg, 63%); 1H NMR (300 MHz, CD3OD) δ 8.05 (s, 1H), 4.41-4.34 (dd, J = 7.2 and 14.1 Hz, 2H), 1.64 (s, 9H), 1.41-1.36 (t, J = 7.2 Hz, 3H); MS (ESI, m/z) 291 [M-H]+; HRMS (ESI) m/z calcd C14H16N2O5 [M-H]+ 292.1059, found 292.1063.

General procedures for preparations of 8 are described as those for N-(4-fluorobenzyl)-7-hydroxy-2-(4-methylphenethyl)-1,3-dioxo-2,3- dihydro-1H-pyrrolo[3,4-c]pyridine-4-carboxamide 8a.

Compound 7f (50 mg, 0.14 mmol) and (4-fluorophenyl) methanamine (21 mg, 0.17 mmol) were dissolved in 10 mL EtOH. The reaction mixture was refluxed for 5 h. After removing of the solvent, the residue was dissolved in DCM (20 mL), washed with water and brine, dried over Na2SO4, filtered and concentrated. The title compound was afford by chromatography (20:1, DCM-MeOH) as a yellow solid (44 mg, 73%); 1H NMR (300 MHz, CD3OD) δ 8.52 (s, 1H), 7.14-7.00 (m, 8H), 4.57 (s, 2H), 3.71-3.68 (m, 2H), 3.05-3.08 (m, 2H), 2.28 (s, 3H); MS (EI, m/z) 433 [M]+; HRMS (EI) m/z calcd C24H20FN3O4 [M]+ 433.1438, found 433.1431.

N-(2-methoxybenzyl)-7-hydroxy-2-(4-methylphenethyl)-1,3- dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-4-carboxamide 8b. Yellow solid (31 mg, 50%); 1H NMR (300 MHz, CD3OD) δ 8.56 (s, 1H), 7.14-7.03 (m, 8H), 4.58 (s, 2H), 3.83 (s, 3H), 3.69-3.65 (t, J = 6.9 Hz, 2H), 3.94-2.89 (t, J = 7.2 Hz, 2H), 2.28 (s, 3H); MS (EI, m/z) 445 [M]+; HRMS (EI) m/z calcd C25H23N3O5 [M]+ 445.1638, found 445.1632.

N-(cyclopropylmethyl)-7-hydroxy-2-(4-methylphenethyl)-1,3- dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-4-carboxamide 8c. Yellow solid (32 mg, 61%); 1H NMR (300 MHz, CD3OD) δ 8.56 (s, 1H), 7.18-7.08 (m, 4H), 3.73-3.68 (t, J = 6.9 Hz, 2H), 3.56-3.53 (d, J = 6.9 Hz, 2H), 2.97-2.92 (t, J = 6.9 Hz, 2H), 2.25 (s, 3H), 1.31- 1.28 (m, 1H), 0.55-0.50 (m, 2H), 0.40-0.35 (m, 2H); MS (EI, m/z) 379 [M]+; HRMS (EI) m/z calcd C21H21N3O4 [M]+ 379.1532, found 379.1528.

N-(4-fluorobenzyl)-2-(4-fluorophenethyl)-7-hydroxy-1,3-dioxo- 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-4-carboxamide 8d. Yellow solid (41 mg, 67%); 1H NMR (300 MHz, CD3OD) δ 8.56 (s, 1H), 7.36- 7.33 (m, 2H), 7.22-7.17 (m, 2H), 7.07-6.95 (m, 4H), 4.84 (s, 2H), 3.69- 3.65 (t, J = 6.9 Hz, 2H), 2.86-2.81 (t, J = 7.5 Hz, 2H); MS (ESI, m/z) 436 [M-H]+; HRMS (ESI) m/z calcd C23H16F2N3O4 [M-H]+ 436.1109, found 436.1118.

N-(2-methoxybenzyl)-2-(4-fluorophenethyl)-7-hydroxy-1,3- dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-4-carboxamide 8e. Yellow solid (33 mg, 53%); 1H NMR (300 MHz, CD3OD) δ 7.91 (s, 1H), 7.24-7.15 (m, 3H), 7.02-6.87 (m, 4H), 6.73-6.68 (m, 1H), 4.57 (s, 2H), 3.91-3.82 (m, 5H), 2.94-2.92 (t, J = 7.2 Hz, 2H); MS (EI, m/z) 449 [M]+; HRMS (EI) m/z calcd C24H20FN3O5 [M]+ 449.1387, found 449.1395.

N-(2-methoxybenzyl)-2-(3,4-dimethoxyphenethyl)-7-hydroxy- 1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-4-carboxamide 8f. Yellow solid (33 mg, 48%); 1H NMR (300 MHz, d6-DMSO) δ 8.55 (s, 1H), 7.29-7.24 (m, 1H), 7.07-7.01 (m, 2H), 6.90-6.74 (m, 4H), 4.72 (s, 2H), 3.82 (s, 3H), 3.69 (s, 6H), 3.49-3.43 (m, 2H), 2.79-2.74 (t, J = 7.5 Hz, 2H); MS (EI, m/z) 491 [M]+; HRMS (EI) m/z calcd C26H25N3O7 [M]+ 491.1693, found 491.1694.

Anti-HIV-1 evaluation

Target compounds were evaluated for their inhibitory activities against HIV-1 replication in acutely infected C8166 cells in vitro according to the previously described method [24,25]; AZT was used as a positive control.

Cytotoxicty assay: The cytotoxicty assay was performed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) method. Briefly, 100μl 4×104 C8166 cells were seeded each well with 100 μl different concentrations compounds in 96-well plate. The plate was incubated 72 hours at 37℃, 5% CO2 incubator. 20 μl MTT (5 mg/ ml) was added each well and the plate was incubated at 37℃ for 4 h. 100 μl supernatant was discarded and 100 μL 20% SDS-50% DMF was added. The plates were read at 570/630 nm by Bio-Tek Elx 800 reader. The 50% cytotoxic concentration (CC50) was calculated.

Syncytia inhibition assay: The syncytia inhibition assay was applied by counting syncytia which were formed by HIV-1IIIB infected C8166 cells. Briefly, HIV-1IIIB supernatant and C8166 cells were seeded in 96-well plate with different concentration of compounds. The plate was incubated in a 37℃, 5% CO2 incubator for 72 hours. Syncytia were counting under an inverted microscope and 50% effective concentration (EC50) was calculated. Therapeutic index (TI) was calculated by the ratio of CC50/EC50.

Results

In total, 35 new compounds (5–13) were designed and synthesized whose chemical structures are shown in Table 1. These compounds were synthesized through the routes outlined in Scheme 1, and all the compounds were purified by silica gel thin-layer chromatography (>95%), and their structures were determined by 1H-NMR and lowand high-resolution mass spectra.

image
Entry Comp. X R1 R2 EC50b (μM) CC50c (μM) TId
1 5a CH2 Ph OEt 63.37 234.02 3.69
2 5b CH2 2-FPh OEt 252.56 >581.40 >2.30
3 5c CH2 4-FPh OEt 88.89 323.32 3.64
4 5d CH2 2-OMePh OEt 57.58 395.39 6.87
5 5e CH2 3-OMePh OEt 213.17 >561.80 >2.64
6 5f CH2 4-OMePh OEt 69.18 243.69 3.52
7 5g CH2 3,4-OMePh OEt 288.91 >518.13 >1.79
8 5h CH2 naphthalen-1-yl OEt 48.32 >531.91 >11.01
9 5i CH(CH3) Ph OEt 68.79 320.09 4.65
10 6a NH 4-FPh OEt 32.61 213.01 6.53
11 6b NH 4-ClPh OEt 14.59 135.46 9.28
12 7a CH2CH2 Ph OEt 55.91 376.18 6.73
13 7b CH2CH2 2-FPh OEt 44.83 148.80 3.32
14 7c CH2CH2 3-FPh OEt 52.26 176.42 3.38
15 7d CH2CH2 4-FPh OEt 1.65 13.16 7.98
16 7e CH2CH2 4-ClPh OEt 15.16 145.43 9.59
17 7f CH2CH2 4-MePh OEt 2.34 15.45 6.60
18 7g CH2CH2 4-SO2NH2 OEt 136.42 173.46 1.27
19 7h CH2CH2 4-OMePh OEt 163.92 232.97 1.42
20 7i CH2CH2 3,4-OMePh OEt 9.15 33.52 3.66
21 7j CH2CH2 indol-3-yl OEt 9.37 26.73 2.85
22 8a CH2CH2 4-MePh NH(4-FBn) 40.37 164.60 4.08
23 8b CH2CH2 4-MePh NH(2-OMeBn) 32.70 215.82 6.60
24 8c CH2CH2 4-MePh NHCH2CH(CH2CH2) 36.41 306.17 8.41
25 8d CH2CH2 4-FPh NH(4-FBn) 5.70 30.76 5.40
26 8e CH2CH2 4-FPh NH(2-OMeBn) 63.01 224.25 3.56
27 8f CH2CH2 3,4-OMePh NH(2-OMeBn) 149.25 217.70 1.46
28 9a CH2CO 4-BrPh OEt 54.27 >464.04 >8.55
29 9b CH2CH(CH3) Ph OEt 25.14 146.10 5.81
30 9c CH2CH(Ph) Ph OEt 3.10 25.22 8.14
31 9d CH(COOMe)CH2 Ph OEt 185.55 346.88 1.87
32 10 CH2CH2CH2 Ph OEt 59.15 206.41 3.49
33 11 1,2,3,4-tetrahydronaphthalen-1-yl OEt 36.64 132.92 3.63
34 12 propyl OEt 352.81 >719.42 >2.03
35 13 t-butyl OEt 119.38 >684.93 >5.74
36 AZTe - - - 0.0085 3779 444588

Table 1: Anti-HIV activity of compounds 5-13 in vitroa.

The assay results of the target compounds are presented in Table 1, expressed as EC50, CC50 and TI values. Among all the target compounds, 7d, 7f, 7i-7j, 8d and 9c exhibited potent anti-HIV-1 activities (EC50 < 10 μM). Compound 7d showed the most potent anti-HIV activity, with EC50 values of 1.65 μM.

Discussion

The structure–activity relationships (SARs) in these novel compounds were investigated by increasing the length of the linker X between N-2 and the side chain phenyl ring R1, introducing substituents on the terminal aryl ring R1, and changing R2 from an ester group to an amide group. We initiated our work by extending the chain length between N-2 and substituting group R1. As shown in Table 1, the length of the linker appears to be is important for the compounds’ biological activities against HIV-1, appending an ethylidene and hydrazine linker could offer a better anti-HIV-1 activity (6a vs. 5c, 7b vs. 5b, 7d vs. 5c, 7i vs. 5g), compared with that produced by introducing a methylene linker. The substitution style of the R1 groups is also has a key impact on anti-HIV-1 activities. Compared with the unsubstituted compound 7a, introduction of a sulfamoyl (7g) and methoxyl (7h) in the 4-position of phenyl group decreased the anti-HIV-1 activities, whereas fluorine (7d), chlorine (7e) and methyl (7f) groups enhanced the antiviral potency against HIV-1. In particular, the 4-F-substituted compound 7d displayed the most potent biological activity among the nine analogs (EC50 = 1.65 μM). However, introducing a fluorine atom into the 2- or 3-position of the phenyl group (7b and 7c) had a negative impact on HIV-1 inhibition. Although the 4-methoxyl substituent showed poor anti-HIV-1 potency (7h), compound 7i with a 3,4-dimethoxyl group showed an improved inhibitory profile, with an EC50 value of 9.15 μM. Interestingly, when we replaced the terminal benzene group with an indole ring (7j), good anti-HIV-1 activities were obtained (EC50 = 9.37 μM).

Encouraged by the improved potency of compounds 7d, 7f and 7i-7j, we continued our structural optimization effort by introducing some amino groups into the R2 position (8). Although most of the tested compounds exhibited poor inhibitory potency against HIV- 1, good biological activity was observed when 4-F-benzylamine was introduced into the molecule (8d, EC50 = 5.7 μM).

Subsequently, we have screened a diversity of linkers, and found that introducing bulky groups into the linker enhances the biological activities. In particular, a bulky phenyl substituent in the linker caused a large improvement in anti-HIV-1 activity (9c vs 7a), with an IC50 value of 3.10 μM. We further lengthened the linker and introduced less flexible groups to explore the biological activities toward HIV-1. However, these strategies resulted in a slight decrease in the biological activity against HIV-1 (10 vs. 7a and 11 vs. 7a, respectively). Finally, as exemplified by compounds 12 and 13, replacement of the benzene ring with adipic groups led to loss of potency.

Conclusion

In summary, we designed and synthesized a series of 7-hydroxy- 1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-4-carboxylate derivatives (5-13) that exhibited anti-HIV-1 activities in vitro. Among the analogs, compounds 7d, 7f, 7i-7j, 8d and 9c exhibited potent anti-HIV-1 activities (EC50<10 μM). In particular, 7d exhibited significant anti-HIV-1 activities with EC50 values of 1.65 μM. On the basis of this work, it would be possible to keep the ethylidene linker and 4-F-substituted phenyl group and systematically replace the other fragments with functional isosteres, potentially leading to more potent and less toxic compounds. The preliminary SAR among the newly synthesized analogs provided useful indications for guiding further rational design of potent anti-HIV-1 agents.

Conflict of Interest

The authors confirm that this article content has no conflict of interest.

Acknowledgements

We gratefully acknowledge financial support from the National Natural Science Foundation of China (Grants 21021063, 91229204, 81102483, and 81025017), National S&T Major Projects (2012ZX09103101-072, 2012ZX09301001-005, 2013ZX09507-001, and 2014ZX09507002-001), Zhejiang Provincial Natural Science Foundation (LQ14B020004), Sponsored by Program of Shanghai Subject Chief Scientist (Grant 12XD1407100).

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