Abstract
During the last decade, noroviruses have gained media attention as the cause of large scale outbreaks of gastroenteritis on cruise ships, dormitories, nursing homes, etc. Although noroviruses do not multiply in food or water, they can cause large outbreaks because approximately 10 – 100 virions are sufficient to cause illness in a healthy adult. Recently, it was shown that the activity of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) enzyme may be important in norovirus infection. In search of anti-noroviral agents based on the inhibition of ACAT1, we synthesized and evaluated the inhibitory activities of a class of pyranobenzopyrone molecules containing amino, pyridine, substituted quinolines, or 7,8-benzoquinoline nucleus. Three of the sixteen evaluated compounds possess ED50 values in the low M range. 2-Quinolylmethyl derivative 3A and 4-quinolylmethyl derivative 4A showed ED50 values of 3.4 and 2.4 M and TD50 values of >200 and 96.4 M, respectively. The identified active compounds are suitable for further modification for the development of anti-norovirus agents.
Keywords: acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1), caliciviruses, norovirus, anti-noroviruses, pyranobenzopyrones
Noroviruses, category B bioterrorism agents, are the leading cause of food- or water-borne gastroenteritis outbreaks. Studies have shown that noroviruses are responsible for approximate 90% of epidemic non-bacterial food-water-borne gastroenteritis outbreaks with an estimated 23 million cases annually in the US causing 50,000 hospitalizations and 300 deaths.1–3 Norovirus belongs to the Caliciviridae family, and may infect an individual with as few as 10 viral particles. Outbreaks often occur in closed environments such as dormitories, cruise ships, hospitals, and care facilities. Caliciviruses (Family Caliciviridae) are small, non-enveloped RNA viruses of 27–35 nm in diameter. They possess a single-stranded, plus-sense genomic RNA of 7–8 kb, which encodes a nonstructural polyprotein, a major structural capsid protein of 58–80 kD (VP1), and a small basic protein (VP2).4 Currently, there are no specific drugs for norovirus infection. Vaccine development for human noroviruses have faced challenges because noroviruses do not grow in cell culture, show high diversity, and immunity from heterologous strains do not seem to confer protection.5 Furthermore, repeat infections in adults indicate that long-term immunity may be absent. Consequently there is an urgent need for the development of effective anti-noroviral drugs. The recent development of replicon-harboring cells for norovirus6,7 has made possible the study of norovirus replication in cells. DNA microarray analysis of norovirus replicon-harboring cells (HG23) by an Affymetrix Gene Chip showed the up regulations of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) and other cholesterol modulating genes (> ±1.5 fold).9 Moreover, commercially available ACAT inhibitors were shown to inhibit norovirus replication.9 These findings suggest that cellular ACAT may be a potential therapeutic target for norovirus infection. Previously, we reported a class of pyranobenzopyrones that possesses ACAT inhibitory activity.10 Hence, anti-norovirus activities of pyranobenzopyrones were investigated. Herein, we report the synthesis and anti-norovirus activity of a small library of sixteen pyranobenzopyrones containing amino, pyridine, quinoline, and 7,8-benzoquinoline nucleus (Figure 1). Among the pyranobenzopyrones, several compounds possess anti-norovirus activity in low micromolar concentrations in vitro with therapeutic index values of ≥ 40.
Figure 1.
Synthesized and Bioevaluated Pyranobenzopyran Compounds 1 – 8.
The synthesis of pyranobenzopyrones 1 – 7 stems from a reductive amination reaction11 of amine 10 and various aldehydes, 11 – 17 as depicted in Scheme 1. Initially, amine 10 was prepared via a four-step sequence of reactions starting from pyranobenzopyrones 9 by hydroboration-hydroxylation reaction followed by mesylation, displacement with sodium azide, and reduction with H2/Pd.10 The synthesis was simplified by a one-pot hydroboration-amination reaction of 9 with BH3•THF followed by hydroxylamine-O-sulfonic acid12 in 50% yield (Scheme 1). A mixture of two diastereomers in a ratio of 1:1 resulted at the newly created carbon center C12 from the hydroboration reaction indicated by its 1H and 13C NMR spectra. The diastereomers are separable by HPLC but not silica gel column chromatograph. Alkylation of amine 10 with 1 equivalent each of aldehydes 11 – 17 separately in methanol followed by sodium cyanoborohydride afforded amines 1 – 7, respectively. Yields of compounds 1 – 7 range from 47 – 66%. Pyridinecarboxaldehydes, various substituted quinolinecarboxaldehydes, and 7,8-benzoquinoline-4-carboxaldehyde (17 or 4-azaphenanthrene-1-carboxaldehyde) were used in the reductive amination reaction, and functional groups such as primary alcohol, ester, and trioxane are stable under the reaction conditions.
Scheme 1.
Synthesis of Compounds 1 – 7.
Amide 8 was synthesized from the coupling reaction of quinoline-4-carboxylic acid (18), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and amine 10 in 65% yield (Scheme 2). Aldehydes 11, 12, and 13A and carboxylic acid 18 along with various methylquinolines were obtained from commercial sources. Aldehydes 14A, 15, and 16 were prepared by benzylic oxidation with selenium dioxide13 or bromination followed by hydrolysis/oxidation14 of methylquinolines, and aldehydes 13B, 13C, 14B – 14F, and 17 were achieved from free-radical heteroaromatic trioxanylation15,16 with trioxane-t-BuOOH-ferrous sulfate (Schemes 2 – 4). Hence, oxidation of 4-methylquinoline (19) with SeO2 under refluxing toluene gave quinoline-4-carboxaldehyde (14A) in 73% yield along with a small amount of over oxidized carboxylic acid 18 (Scheme 2). Similarly, quinoline-6-carboxaldehyde (15) was obtained from 6-methylquinoline (20) in 54% yield after the treatment with SeO2 in refluxing xylene. To our surprise, oxidation of 8-methylquinoline (21) under similar reaction conditions provided only a trace amount of 8-quinoline-carboxaldehyde (16). Apparently, methyl group appended on ring A of quinoline is activated toward oxidation, but methyl group on ring B is not, and a sluggish oxidation resulted. To overcome the problem, benzylic bromination of 21 was carried out. Treatment of 21 with N-bromosuccinimide (NBS) and a catalytic amount of azobisisobutyronitrile (AIBN) followed by aqueous hydrolysis accompanying air oxidation gave aldehyde 16 (37% yield)14 along with the hydrolyzed product, 6-hydroxymethylquinoline (22) (53% yield). Oxidation of alcohol 22 with o-iodoxybenzoic acid (IBX) and DMSO17 furnished 16 in a 79% yield.
Scheme 2.
Syntheses of Compound 8 and quinolinecarboxaldehydes 14A, 15, and 16.
Scheme 4.
Syntheses of quinolinecarboxaldehydes 13B, 13C, 14E, 14F and 7,8-benzoquinolinecarboxaldehyde (17).
Substituted quinolinecarboxaldehydes 13B, 13C, 14B – 14F, and 7,8-benzoquinolinecarboxaldehyde (17) were obtained from heteroaromatic trioxanylation reactions (Schemes 3 and 4).15,16 Treatment of 2-methylquinoline (23) with trioxane, t-butyl hydroperoxide and trifluoroacetic acid (TFA) in the presence of a catalytic amount of ferrous sulfate afforded trioxanylquinoline 24, which underwent acidic hydrolysis to give aldehyde 14B. 2-Hydroxymethyl-4-quinolinecarboxaldehyde (14D) was obtained from the benzylic oxidation of 24 with selenium dioxide followed by the hydrolysis of the resulting trioxanyl aldehyde 25 with 2 N HCl to give dialdehyde 26. Subsequent reduction of 26 with sodium borohydride afforded 14D. In the reduction process, regioisomer, 4-hydroxymethyl-2-quinolinecarboxaldehyde (27) and 2,4-di(hydroxymethyl)quinoline (28) were also isolated. Acetylation of the hydroxyl function of 14D with acetic anhydride and zinc oxide18 produced acetate 14C (Scheme 3).
Scheme 3.
Syntheses of quinolinecarboxaldehydes 14B – 14D.
Similar trioxanylation of 4-methylquinoline (19) furnished 2-trioxanequinoline 29 in 43% yield along with a small amount of 4-methyl-2-quinolinecarboxaldehyde (13B) (3% yield), which derived from the hydrolysis of the trioxane moiety of 29 with TFA and water in the reaction mixture (Scheme 4). Alternatively, aldehyde 13B can be achieved in 50% yield from a two-step trioxanylation of 19 followed by acidic hydrolysis with 10% aqueous sulfuric acid of the resulting trioxane without purification. 2-Trioxanyl-4-carboxaldehyde (14E) was obtained from the benzylic oxidation of 29 with selenium dioxide. Similar to the selenylation reaction of 8-methylquinoline (21), trioxanylation of 6-methylquinoline (20) appeared to be sluggish. Hence, the reaction of 20 with trioxane, t-butyl hydroperoxide, TFA and ferrous sulfate followed by acidic hydrolysis furnished a mixture of regioisomers, 6-methyl-4-carboxaldehyde (14F) (17% yield) and 6-methyl-2-carboxaldehyde (13C) (16% yield), which were separated by silica gel column chromatography. The spectral data of compound 13C are similar to that reported,19 and the assignment of regiochemistry of 14F is based on its 1H NMR spectral data. The chemical shift of C2-H of 14F appears at 9.14 ppm as a doublet with coupling constant J value of 4.3 Hz, and that of C3-H at 7.77 ppm as a doublet with J value of 4.3 Hz, which are similar to that of 4-quinolinecarboxaldehyde (14A). Formylation of 4-azaphenanthrene (30) under similar reaction conditions followed by hydrolysis with 2 N HCl afforded 7,8-benzoquinoline-4-carboxaldehyde (17). Hence, methylquinolines can either be oxidized to the corresponding quinolinecarboxaldehydes or formylated to methylquinolinecarboxaldehydes.
From our initial screening of the effects of pyranobenzopyrone compounds on the reduction of NV replicon-harboring cells (HG23 cells), 3-pyridyl analog 1 showed promising results with ED50 value (effective dosage at reducing NV genome levels by 50% at 24 h post-treatment) and TD50 value (cytotoxic dosage in killing 50% HG23 cells at 48 h post-treatment determined by cytotoxicity assay at 48 h of treatment)7,9,20 of 4 and >200 M, respectively. Hence, a small library of pyridylmethyl, quinolylmethyl, quinolylcarbonyl, and 4-azaphenanthrenylmethyl derivatives along with their synthetic precursor, amine 10, was evaluated for their anti-norovirus activities. Results of the inhibition of NV RNA replication are summarized in Table 1. To our surprise, amine 10, 4-pyridylmethyl 2, 8-quinolylmethyl 6, and 2-acetoxymethyl- and 2-hydroxymethyl-4-quinolylmethyls 4C and 4D have ED50 values >10 M. Due to high ED50 values, TD50 values of these compounds were not determined except compound 10. Other quinolylmethyl, substituted quinolylmethyl, and 4-azaphenanthrenylmethyl derivatives along with 4-quinolyl amide 8 possess ED50 values ranging from 2 – 8 M and TD50 values of 61 – >200 M. In particular, 2-quinolylmethyl 3A, 4-quinolylmethyl 4A, and 6-methyl-4-quinolylmethyl 4F possess the strongest anti-norovirus activities with respective ED50 values of 3.4, 2.4, and 3.4 M and TI (therapeutic index; derived from TD50/ED50) values of 58.8, 40.2, and 18.0, respectively. 6-Methyl-2-quinolylmethyl 3C on the other hand showed a lower ED50 value of 8.1 M. Hence, addition of substituent at C6 or ring B of 4-quinolylmethyl analog appears to retain its antiviral activity and substituent on ring A diminishes its activity.
Table 1.
Effects of pyranobenzopyrone compounds on the reduction of NV RNA replication (ED50) and toxicity (TD50) in HG23 cells (NV replicon-haboring cells) and their therapeutic indexes (TI).
| Compound | ED50 value in μM | TD50 value in μM | Therapeutic index (TI) |
|---|---|---|---|
| 1 | 4.1 | >200 | >50 |
| 2 | 9.6 | ND* | - |
| 3A | 3.4 | >200 | >58.8 |
| 4A | 2.4 | 96.4 | 40.2 |
| 5 | 8.1 | ND | - |
| 6 | >10 | >160 | >16 |
| 4B | 8.2 | ND | - |
| 4C | 9.7 | ND | - |
| 4D | 9.5 | ND | - |
| 4E | 8.4 | ND | - |
| 3B | 5.3 | >200 | >40 |
| 4F | 3.4 | 61.2 | 18.0 |
| 3C | 8.1 | 83.7 | 10.3 |
| 7 | 5.3 | >200 | >40 |
| 8 | 5.5 | 103.5 | 18.8 |
| 10 | >10 | >200 | - |
ND: not determined due to high ED50 values. Each value is the average of at least 2 independent tests.
In conclusion, various pyranobenzopyrone molecules containing pyridine, quinoline, or 4-azaphenanthrene nucleus were synthesized and evaluated. 4-Quinolylmethyl analogs 4A and 4F and 2-quinolylmethyl analog 3A possess the strongest anti-norovirus activities having ED50 values of 2.4 – 3.4 M and TI values of 18.0 – >58.8. The identified hits are suitable for further optimization via medicinal chemistry and molecular modeling.
Supplementary Material
Acknowledgments
This work has been supported by the National Institutes of Health, National Institute of Allergy and Infectious Diseases (U01 AI081891).
Footnotes
Synthetic procedure, analytical data, and protocols for antiviral evaluation are included. Supplementary data associate with this article can be found in the online version, at …
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References and notes
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