Dihydroisoindolo[2,1-a]quinazoline-5,11-dione derivatives as potent and selective inhibitors targeting hepatitis B virus
Abstract
The construction of dihydroisoindolo[2,1-a]quinazoline-5,11-dione derivatives (4a–4m), by the condensation isatoic anhydride, appropriate amines and 2-formylbenzoic acid by using silica sulfuric acid as catalyst was reported. These dihydroisoindolo[2,1-a]quinazoline-5,11-dione derivatives (DIQ) were identified as potent inhibitors of HBV capsid assembly. The newly synthe-sized dihydroisoindolo[2,1-a]quinazoline-5,11-dione derivatives 4a-4m were characterized by 1H NMR, 13C NMR, and Mass spectrum and evaluated for their anti-HBV activity. Majority of the synthesized compounds inhibited the expression of viral antigens at low concentration. But five compounds, 4a, 4b, 4c, 4f, and 4m were shown potent inhibition of HBV DNA replication at submicromolar range. Of these compounds, compound 4a was the most active when compared with lamivudine.
Introduction
Worldwide hepatitis B virus (HBV) becomes serious problem which is causing disease for more than 2 billion people. More than 360 million individuals were chronically infected from liver cirrhosis and hepatocellular carcinoma (Dény et al., 2010). The current therapies including vaccines, immunomodulators, interferon-a, polyethylene glycol interferon-a and nucleoside drugs for treating HBV are still unsatisfactory, due to high recurrence, drug resistance and inevitable side effects including influenza-like illness, myalgia, headache, reduction of neutrophilic granulocyte and blood platelet, etc (Sato et al., 2010; Locarnini et al., 2006; Wong et al., 1993; Fattovich et al., 1998). Therefore, it is important to explore novel classes of drugs with different antiviral targets and mechanisms for anti-HBV purposes.
On the other hand Hybrid molecules which can form by combining two heterocyclic cores of different nature often possess improved biological activities (Hyodo et al., 1995; Furumi et al., 1998). There are several literature precedence for the biologically active hybrids in the literature such as steroid-antibiotic (Oaksmith et al., 2009), steroid-nucleoside, (Kortylewicz et al., 2009) triterpenoidpeptide (Vasilevsky et al., 2011) and DNA-cleaving agent- amino acid (Breiner et al., 2007; Breiner et al., 2006; Kovalenko, 2005; Yang et al., 2011) etc.
Multicomponent reactions (MCRs), are the most powerful technique to access complex molecules in a single synthetic operation from easily available building blocks. (Foye, 1991; Bonsignore 1993) MCRs have led to interesting heterocyclic scaffolds, and are very useful in the construction of diverse chemical libraries of ‘drug-like’ molecules (Ellis, 1977; Arnesto et al., 1989). One of the hybrid molecule, 6,6a-dihydroisoindolo[2,1-a]quinazoline-5,11-diones which are synthesized from isatoic anhydride, appropriate amines and 2-formylbenzoic acid. These compounds are less focused heterocyclic compounds which are having biological importance (Siva Kumar et al., 2011).
Materials and Methods
Chemistry: Chemicals and reagents were purchased either from sigma or Merck, and all reagents were of analytical reagent grade. Thin-layer chromatography (TLC) was performed on Merck silica gel 60 F254 plates and visualized under UV light. 1H NMR spectra were recorded with Varian Mercury Plus 400 MHz instrument.13C NMR spectra were recorded with a Varian Gemini 100 MHz instrument. All the chemical shifts are reported in δ (ppm) using TMS as an internal standard. Multiplicity is indicated by one or more of the following abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad); the coupling constants (J) correspond to the order of the multiplicity assignment. Mass spectra were recorded with a PE Sciex model API 3000 instrument. All the reactions were carried out under nitrogen atmosphere.
6-(2, 4-difluorophenyl)-6, 6a-dihydroisoindolo [2, 1-a] quinazoline-5, 11-dione (4a): Off-white solid; mp 201-203 oC; 1H NMR (400 MHz, CDCl3); δ 8.15 (dd, J = 7.04, 5.14 Hz, 2H), 7.96 (d, J = 7.66 Hz, 1H), 7.77-7.69 (m, 1H), 7.59 (m, 1H), 7.43-7.35 (m, 2H), 7.17-7.09 (m, 1H), 6.92-6.69 (m, 2H), 6.56 (s, 1H) 6.43 (m, 1H); 13C NMR (400 MHz, CDCl3); δ 166.1, 165.1, 139.1, 138.1, 135.2, 134.7, 134.4, 134.1, 133.1, 133.3, 133.2, 131.4, 129.9, 129.4, 126..3, 125.7, 121.3, 113.1, 113.0, 105.8, 105.0, 104.9, 71.8; Mass ESI calcd. C21H12F2N2O2; ([M+H] +), 362.09; found: ([M+H] +), 362.5.
6-(3-chloro-2-methylphenyl)-6, 6a-dihydroisoindolo [2, 1-a] quinazoline-5, 11-dione (4b): Pale yellow solid; mp 188-190 oC; 1H NMR (400 MHz, CDCl3); δ 8.23 (d, J = 7.9 Hz, 2H), 7.96 (d, J = 7.6 Hz, 1H), 7.74 (dd, J = 11.2, 4.4 Hz, 1H), 7.63-7.49 (m, 2H), 7.49-7.39 (m, 4H), 6.56 (s, 1H), 6.19 (d, J = 7.7 Hz, 1H), 1.67 (s, 3H); 13C NMR (400 MHz, CDCl3); δ166.2, 164.4, 139.3, 138.9, 138.3, 137.9, 136.9, 135.0, 131.1, 129.9, 128.6, 127.9, 126.4, 125.5, 125.4, 121.4, 121.1, 72.8, 14.9; Mass ESI calcd. C22H15ClN2O2; ([M+H] +), 374.08; found: ([M+H] +), 374.5.
2-(5, 11-dioxoisoindolo [2, 1-a] quinazolin-6(5H, 6aH, 11H)-yl) acetic acid (4c): White solid; mp 243-247 0C; 1H NMR (400 MHz, DMSO-d6); δ 8.08 (dd, J = 14.5, 7.9 Hz, 2H), 7.97 (d, J = 7.4 Hz, 1H), 7.88-7.73 (m, 4H), 7.42 (t, J = 7.5 Hz, 1H), 6.59 (s, 1H), 4.64 (d, J = 18.1 Hz, 1H), 4.34 (d, J = 18.1 Hz, 1H); 13C NMR (400 MHz, DMSO-d6); δ 171.0, 164.7, 163.6, 139.3, 137.2, 134.1, 133.6, 132.4, 131.1, 129.0, 125.8, 125.4, 120.3, 119.9, 70.6, 44.7; Mass ESI calcd. C17H12N2O4; ([M+H] +), 308.08; found: ([M-H]) -, 306.5.
6-methyl-6,6a-dihydroisoindolo[2,1-a]quinazoline-5,11-dione (4d): White solid, Mp = 188-189 °C 1 H NMR (400 MHz, CDCl3): 8.16 (dd, J=7.8, 1.0 Hz, 1H), 8.11 (dd, J=7.8, 1.0 Hz, 1H), 8.03 (dd, J=7.8, 1.0 Hz, 1H), 7.78-7.61 (m, 4H), 7.35-7.31 (m, 1H), 6.12 (s, 1H), 3.32 (s, 3H, CH3); 13C NMR (CDCl3, 50 MHz):164.8, 163.8, 137.9, 136.5, 133.3, 132.6, 132.4, 130.4, 128.9, 125.5, 125.0, 124.9, 120.0 (2C), 71.1, 29.9; Mass ESI calcd. C16H13N2O2; ([M+H] +), 265.0; found: ([M-H]) -, 265.6.
6-ethyl-6,6a-dihydroisoindolo[2,1-a]quinazoline-5,11-dione (4e): White solid, Mp = 156-159 °C 1 H NMR (DMSO-d6, 400 MHz): 8.01 (d, J=7.4 Hz, 1H), 7.99-7.95 (m, 3H), 7.87-7.84 (m, 1H), 7.77 (d, J=7.4 Hz, 1H), 7.74-7.69 (m, 1H), 7.40-7.36 (m, 1H), 6.60 (s, 1H), 3.90-3.83 (m, 1H), 3.72-3.67 (m, 1H), 1.03 (t, J=7.4 Hz, 3H, CH3) 13C NMR (DMSO-d6, 100 MHz): 164.3, 162.6, 138.4. 136.4, 133.2, 133.1, 131.8, 130.6, 128.3, 126.0, 125.0, 124.3, 120.3, 119.9, 69.7, 37.2, 13.4 IR (KBr): 3403, 1711, 1668, 1603, 1490, 1469 cm-1 HRMS (ESI): calcd for C17H15N2O2 (M+H)+ 279.1134, found 279.1135
6-cyclopropyl-6,6a-dihydroisoindolo[2,1-a]quinazoline-5,11-dione (4f): White solid, Mp = 155-158 °C 1 H NMR (CDCl3, 400 MHz): 8.19-8.14 (m, 2H), 8.05 (d, J-6.9Hz, 1H), 7.92 (d, J-6.9Hz, 1H), 7.70-7.60 (m, 3H), 7.32-7.28 6-(4-chlorobenzyl)-6,6a-dihydroisoindolo[2,1-a]quinazoline-5,11-dione (4l): White solid, Mp = 178-181 °C 1 H NMR (DMSO-d6, 400 MHz): 8.06-8.04 (m, 2H), 7.86-7.84 (m, 1H), 7.78-7.73 (m, 2H), 7.65- 7.61 (m, 2H), 7.44-7.40 (m, 1H), 7.24 (d, J-8.8 Hz, 2H), 7.07 (d, J-8.8 Hz, 2H), 6.71 (s, 1H), 5.12-4.99 (m, 2H) 13C NMR (DMSO-d6, 100 MHz): 164.3, 163.4, 138.0, 136.7, 136.6, 133.6, 133.0, 131.7, 131.1, 130.6, 128.7, 128.3 (2C), 127.9 (2C), 126.1, 125.2, 124.1, 120.0 (2C), 70.2, 45.1 IR (KBr): 3429, 2932, 1726, 1664, 1490, 1470 cm-1.
6-(1-methyl-1H-pyrazol-3-yl)-6, 6a-dihydroisoindolo [2, 1-a] quinazoline-5, 11-dione (4m): White solid; mp 139-152.5 0C; 1H NMR (400 MHz, CDCl3); δ 8.19 (d, J = 8.0 Hz, 2H), 7.96 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 15.6 Hz, 1H), 7.55 (t, J = 14.8 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.39 (m, 2H), 6.65 (s, 1H), 6.59 (d, J = 7.6 Hz, 1H), 6.01 (s, 1H), 4.00 (s, 3H); 13C NMR (400 MHz, CDCl3); δ 165.3,164.5,145.6,138.8,137.0,133.9,132.2,132.1,130.1,129.4,125.19,125.14, 124.4, 120.3, 119.9,105.8, 71.8, 39.6; Mass ESI calcd. C19H14N4O2; ([M+H] +), 330.11; found: ([M+H] +), 330.6.
Biological evaluation
Cell culture: HepG2 2.2.15 cells, derived from HepG2 human hepatocellular carcinoma cells, were stably transfected with a head-to-tail HBV DNA dimer and were maintained in MEM with heat-inactivated 10% fetal bovine serum (FBS) and 1% antibiotics. In parallel experiments, human Huh7 hepatoma cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with heat-inactivated 10% FBS and 1% antibiotics. HepG2 2.2.15 and Huh7 cells were both grown at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
Cell viability assay: The cytotoxic effects of synthesized compounds were determined using a Cell Titer 96 cell proliferation assay kit (MTS) (Promega, Madison, WI, USA). In order to pinpoint the toxicity limits in HepG2 2.2.15 and Huh7 cells, they were plated into 96-well plates at a density of 4 x 104 cells/mL for 24 hours. Cells were then treated with serial dilutions of compounds ranging 2.5-160 mg/mL, and the mixture was incubated for 3 days. Cell toxicity was calculated according to the maker’s protocol. All the results were performed in triplicates, and results are presented as relative percentages over that of the control group.
Determination of HBV expression levels: After treating HepG2 2.2.15 cells or HBV-transfected Huh7 cells, levels of the HBsAg and HBeAg proteins were measured in culture media using an EIA kit (Johnson and Johnson, Skillman, NJ, USA) according to the manufacturer’s instructions.
SEC general procedure: Capsid assembly was initiated by mixing Cp149 with test compounds in 2 buffer incubation 24 h, respectively. Assembly reactions were examined on a Superose column (Biosep-SEC-S3000) mounted on HPLC system equipped with an auto injection module. The column was equilibrated with 100 mM HEPES pH 7.5, 300 mM NaCl at 0.6 mL/min.
Result and Discussion
In the initial studies we investigated the cylclization reaction of 2-formylbenzoic acid (3) with isatoic anhydride (1) and 2,4-difluoro aniline (2a) using SSA as catalyst in ethanol under reflux (Scheme-1) for 2 hours which yielded the required compound 4a in 89% (Table I, entry 1). Several amines were used for checking the compatibility of the method. All the amines including aliphatic, aromatic and also heterocyclic amines were well tolerated with the current methodology to yield the desired products in good to better yields. Thus, the structures of the synthesized target compounds are listed in Table I.
Table I:Synthesis of a novel series of substituted 5-(aminomethylene)thiazolidine-2,4-diones
| Entry | 2o Amine (3) | Product | Yield (%)b | |
|---|---|---|---|---|
| 1 |  |
 4a |
89 | |
| 2 |  |
 4b |
93 | |
| 3 |  |
 4c |
81 | |
| 4 |  |
 4d |
78 | |
| 5 |  |
 4e |
80 | |
| 6 |  |
 4f |
87 | |
| 7 |  |
 4g |
82 | |
| 8 |  |
 4h |
85 | |
| 9 |  |
 4i |
92 | |
| 10 |  |
 4j |
96 | |
| 11 |  |
 4k |
96 | |
| 12 |  |
 4l |
97 | |
| 13 |  |
 4m |
92 | |
Scheme 1: Synthesis of 5-(aminomethylene)thiazolidine-2,4-diones (a) Ac2O, HC(OEt)3, reflux (b) acetonitrile, 45°C, 30 min
Inhibition activities for HBV replication of the compounds 4a-4m were determined in the HepG2.2.15 cells, which constitutively produces HBV genomes, and secretes virus-like particles (Korba et al., 1992). Lamivudine (3TC) was used as positive control. To ascertain the cytotoxic effects of the tested compounds, the cell viability was determined after the cells exposed to the compounds for 48 hours (Table I).
Table II: Anti-HBV activity and cytotoxicity of target compounds in vitro
| Compound | TC50 (µM) | HBsAg | HBeAg | HBV DNA Replication | |||
|---|---|---|---|---|---|---|---|
| IC50 (µM) | SI | IC50 (µM) | SI | IC50 (µM) | SI | ||
| 4a | >1500 | 920 | 41 | 846 | 55.3 | 4.13 | 456 |
| 4b | 147 | 203 | 5.8 | 594 | 42.5 | 13.6 | 245 |
| 4c | 1339 | 458 | 4.6 | 215 | 39.7 | 12.6 | 265 |
| 4d | 225 | 564 | - | 678 | - | 69.5 | 3.2 |
| 4e | 171 | - | - | 177 | - | 36.6 | 4.7 |
| 4f | 420 | 289 | 3.4 | 143 | 3.4 | 22.3 | 164 |
| 4g | 126 | 880 | - | 12.3 | - | 60.6 | 14.2 |
| 4h | 225 | 565 | - | 23.4 | - | 69.5 | 9.3 |
| 4i | 1276 | >1598 | - | >974 | - | 58.3 | 7.6 |
| 4j | 326 | 162 | - | 49.2 | - | 221 | 12.6 |
| 4k | 122 | 92 | - | 77.5 | - | 49.3 | 18.7 |
| 4l | 92 | 57 | - | 56.8 | - | 42.6 | 16.5 |
| 4m | 157 | 768 | 41 | 296 | 123.4 | 8.4 | 386 |
| 3TC | >1634 | 1243 | >1.6 | 1389 | >1.8 | 0.95 | >1289 |
HBV replication than 3TC (13.9 µM). When the attachments on dihydroisoindolo[2,1-a]quinazoline-5,11-dione was replaced by less rigid alkyl groups to give compounds 4c, 4d, 4e, 4f, 4g, 4h (the IC50 values 12.6 µM, 69.5 µM, 36.6 µM, 22.3 µM, 60.6 µM and 69.5 µM respectively), their anti-HBV activities were reserved or even decreased. Replacing the alkyl attachment to benzylic groups 4j to 4l there is no improvement in anti-HBV activity. There is no effect on the anti HBV activity when the dihydroisoindolo[2,1-a]quinazoline-5,11-dione compounds was substituted with cyclohexane 4i. Compounds, 4a (IC50 = 4.13 µM), 4b (IC50 = 13.6 µM), 4c (IC50 = 12.6 µM), 4f (IC50 =22.3 µM) and 4m (IC50 = 8.4 µM) showed high inhibition of HBV replication. It illuminated that the anti-HBV activity largely depends on the size, length and character of dihydro-isoindolo[2,1-a]quinazoline-5,11-dione substituent.
Among all the compounds 4a, 4b, and 4m which are showing better anti-HBV activities, compounds containing the electron-withdrawing groups such as fluorine or chlorine and heterocyclic system.
To gain better understand into the mechanisms of our compounds, 4a was investigated to examine the effect on preformed Hepatitis B virus (HBV) capsid and on HBV capsid assembly by size exclusion chromatography (SEC) (Stray et al., 2005). Recovered protein was assigned to the void (aberrant capsid induced by 4a, 8.6 min), capsid (9.6 min) and dimer (12.6 min) based on the HPLC chromatogram.
By SEC, we had observed the effect of compounds 4a on Cp149 (Figure 1). There was no change of the capsid morphology detected at low concentration of 4a (Cp149:4a = 4:1 or 2:1). At higher concentration (Cp149:4a = 1:2 or 1:4), the increasing continuous spectrum of void and the decreasing dimers were observed. The results suggested that 4a can break the equilibrium and change the product of HBV core protein self-assembly. By structural biology, we established a screening system for anti-HBV compounds that target on nucleocapsid. SEC would be a much better method to discover the strong antiviral compounds because of its objectivity, convenience and precision.
Figure 1:SEC showed the effect of 4a on capsid assembly
Conclusion
The present study describes the synthesis of dihydroisoindolo[2,1-a]quinazoline-5,11-dione derivatives (4a–4m). The newly synthesized analogues 4a-4m were characterized by 1H NMR, 13C NMR, and MS and evaluated for their anti-HBV activity, which provided 5 active derivatives inhibiting HBsAg secretion, 5 active derivatives suppressing HBeAg secretion and 5 active derivatives inhibiting HBV DNA replication. Interestingly, compound 4a could inhibit not only HBsAg and HBeAg secretions but also HBV DNA replication with SI values of 41, 55.3 and 456. In view of their observed anti HBV activity, these compounds are seemed to have potential medicinal value.
References
Arnesto D, Horspool WM, Martin N, Ramos A, Seaone C. Synthesis of cyclobutenes by the novel photochemical ring contraction of 4-substituted 2-amino-3,5-dicyano-6-phenyl-4H-pyrans. J Org Chem. 1989; 54: 3069-72.
Bonsignore L, Loy G, Secci D, Calignano A. Synthesis and pharmacological activity of 2-oxo-(2H) 1-benzopyran-3-carboxamide derivatives. Eur J Med Chem. 1993; 28: 517-20.
Breiner B, Schlatterer JC, Kovalenko SV, Greenbaum NL, Alabugin IV. DNA damage-site recognition by lysine conjugates. Proc Natl Acad Sci USA. 2007; 104: 13016-21.
Breiner B, Schlatterer JC, Kovalenko SV, Greenbaum NL, Alabugin IV. Protected 32P-Labels in Deoxyribonucleotides: Investigation of sequence selectivity of DNA photocleavage by enediyne–, fulvene–, and acetylene–lysine conjugates. Angew Chem Int Ed. 2006; 45: 3666-70.
Dény P, Zoulim F. Hepatitis B virus: From diagnosis to treatment. Pathol Biol. 2010; 58: 245-53.
Ellis GP. In: The chemistry of heterocyclic compounds: Chromenes, chromanes and chromones. Weissberger A, Taylor EC (eds). New York, John Wiley, 1977, p 11.
Fattovich G, Brollo L, Alberti A, Pontisso P, Giustina G, Realdi G. Long-term follow-up of anti-HBe positive chronic active hepatitis B. Hepatology 1998; 8; 1651-54.
Foye WO. Principidi chemico farmaceutica; Piccin, Padora, Italy, 1991, p 416.
Furumi K, Fujioka T, Fujii H, Okabe H, Nakano Y, Matsunaga H, Katano M, Mori M, Mihashi K. Novel antiproliferative falcarindiol furanocoumarin ethers from the root of Angelica japonica. Bioorg Med Chem Lett. 1998; 8: 93-96.
Hyodo S, Fujita K, Kasuyaa O, Takahashia I, Uzawab J, Koshino H. Structures of phospholipase A2 inhibitors, ergo-philones A and B. Tetrahedron 1995; 51: 6717-24.
Kortylewicz ZP, Nearman J, Baranowska-Kortylewicz J. Radio-labeled 5-Iodo-3′-O-(17β-succinyl-5α-androstan-3-one)-2′-deoxyuridine and Its 5′-monophosphate for imaging and therapy of androgen receptor-positive cancers: Synthesis and biological evaluation. J Med Chem. 2009; 52; 5124-43.
Kovalenko SV, Alabugin IV. Lysine–enediyne conjugates as photochemically triggered DNA double-strand cleavage agents. Chem Commun. 2005; 1444-46.
Korba BE, Gerin JL. Use of a standardized cell culture assay to assess activities of nucleoside analogs against hepatitis B virus replication. Antiviral Res. 1992; 19: 55-70.
Locarnini S, Mason WS. Cellular and virological mechanisms of HBV drug resistance. J Hepatol 2006; 44; 422-31.
Oaksmith JM, Ganem B. Synthesis of a COMC–estradiol conjugate for targeted, tissue-selective cancer chemotherapy. Tetrahedron Lett. 2009; 50; 3497-98.
Sato K, Mori M. Current and novel therapies for hepatitis B virus infection. Mini Rev Med Chem. 2010; 10; 20-31.
Siva Kumar K, Mahesh Kumar P, Anil Kumar K, Sreenivasulu M, Ahamed AJ, Rambabu D, Rama Krishna G, Malla Reddy C, Ravikumar K, Shivakumar K, Krishna Priya K, Kishore VLP, Pal M. A new three-component reaction: green synthesis of novel isoindolo[2,1-a]quinazoline derivatives as potent inhibitors of TNF-α. Chem Commun. 2011; 47; 5010-12.
Stray SJ, Bourne CR, Punna S, Lewis WG, Finn MG, Zlotnick A. A heteroaryl dihydropyrimidine activates and can misdirect hepatitis B virus capsid assembly. Proc Natl Acad Sci USA. 2005; 102: 8138-43.
Vasilevsky SF, Govdi AI, Sorokina IV, Tolstikova TG, Baev DS, Tolstikov, GA, Mamatuyk, VI, Alabugin IV. Rapid access to new bioconjugates of betulonic acid via click chemistry. Bioorg Med Chem Lett. 2011; 21: 62-65.
Wong DKH, Cheung AM, Orourke K, Naylor CD, Detsky AS. Effect of alpha-interferon treatment in patients with heaptitis B e antigen-positive chronic hepatitis B: A meta-analysis. Ann Intern Med. 1993; 119: 312-23.
Yang WY, Breiner B, Kovalenko SV, Ben C.; Singh M, Le Grand SN, Sang QX, Strouse GF, Copland JA, Alabugin IV. C-Lysine conjugates: pH-controlled light-activated reagents for efficient double-stranded DNA cleavage with implications for cancer therapy. J Am Chem Soc. 2009; 131; 11458-70.
Yang WY, Marrone SA, Minors N, Zorio DAR, Alabugin IV. Fine-tuning alkyne cyclo additions: Insights into photochemistry responsible for the double-strand DNA cleavage via structural perturbations in diaryl alkyne conjugates. Beilstein J Org Chem. 2011; 7; 813-23.
