ARCH PHARM

Arch. Pharm. Chem. Life Sci. 2015, 348, 786–795

Archiv der Pharmazie

Full Paper Design, Synthesis, and Molecular Docking of Novel Pyrrolooxazepinediol Derivatives with Anti-Influenza Neuraminidase Activity Ahmed O. H. El-Nezhawy1,2, Ahmad F. Eweas1,3, Ibrahim A. Maghrabi4, Ahmed S. Edalo5, and Sayed F. Abdelwahab6,7 1 2

3 4 5 6 7

Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, Taif, Saudi Arabia Department of Chemistry of Natural and Microbial Product, National Research Center, Dokki, Cairo, Egypt Department of Medicinal Chemistry, National Research Center, Dokki, Cairo, Egypt Department of Clinical Pharmacy, College of Pharmacy, Taif University, Taif, Saudi Arabia Clinical Pharmacology, College of Pharmacy, Taif University, Taif, Saudi Arabia Department of Microbiology, College of Pharmacy, Taif University, Taif, Saudi Arabia Department of Microbiology and Immunology, Faculty of Medicine, Minia University, Minia, Egypt

A series of novel pyrrolo[2,1-b][1,3]oxazepine-8,9-diol derivatives 12–15 were synthesized starting from L-tartaric acid, which was transformed into anhydride which then reacted with allylamine in xylene to afford the imide 2. The target molecules 12–15 were achieved via ring-closing metathesis with the Grubbs catalyst, followed by reduction of the carbonyl group and deprotection of hydroxyl groups. Finally, catalytic hydrogenation of the double bond afforded the title compounds 12–15. Molecular docking study of the title compounds 12–15 was carried out against neuraminidase as the target enzyme, in an attempt to understand the mechanism of action of the tested compounds as potential neuraminidase inhibitors. Molecular docking of the target compounds showed that all tested compounds bind to the active site of neuraminidase, with moderate to high binding energy. Compounds 12–15 were examined for their antiviral activity against H5N1 virus (A/chicken/Egypt/1/ 2008). Oseltamivir phosphate was used as a control for antiviral activity. The results show that compound 12 (EC50 ¼ 0.016 mg/mL) exhibited potent anti-influenza (H5N1) activity, which approximately equals that of oseltamivir (EC50 ¼ 0.012 mg/mL). Also, it had a therapeutic index similar to that of oseltamivir phosphate (20). The data also revealed that compounds 13, 14, and 15 had slightly lower antiviral activity and lower cytotoxicity than oseltamivir phosphate, with LD50 of 0.188, 0.162, and 0.176 mg/mL, respectively. However, 13, 14, and 15 had lower therapeutic indices than 12. In conclusion, we were able to synthesize cheap and potent anti-H5N1 compounds. Keywords: H5N1 / Influenza / Neuraminidase inhibitors / Oseltamivir phosphate / Pyrrolo[2,1-b][1,3]oxazepine-8,9-diol Received: June 18, 2015; Revised: August 6, 2015; Accepted: August 18, 2015 DOI 10.1002/ardp.201500209

:

Additional supporting information may be found in the online version of this article at the publisher’s web-site.

Correspondence: Dr. Ahmed O. H. El-Nezhawy, Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, Taif, Saudi Arabia and Department of Chemistry of Natural and Microbial Product, National Research Center, Dokki, Cairo 12622, Egypt. E-mail: [email protected], [email protected] Fax: þ 202 3337 0931

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Introduction Seasonal and pandemic influenza are major threats to global public health. During the past century, the 1918 Spanish flu, the 1957 Asian flu, and the 1968 Hong Kong flu pandemics

www.archpharm.com

786

ARCH PHARM

Arch. Pharm. Chem. Life Sci. 2015, 348, 786–795 Pyrrolooxazepinediol Derivatives with Anti-Influenza Activity

caused millions of fatalities [1]. Recent years have seen the emergence of the 1997 H5N1 virus in Hong Kong, or “bird flu”, which is known for its high fatality rate (even with low transmission to humans) [2], and the 2009 H1N1 virus in Mexico, or “swine flu”, which contributed to at least 16000 deaths [3, 4]. These two viruses have raised heightened concern, especially because of the fact that they can carry drug-resistant mutations [5, 6]. Influenza viruses are negativesense single-stranded RNA viruses, belonging to the Orthomyxoviridae family. Based on the antigenic difference in their nucleoproteins and matrix proteins, the Orthomyxoviridae viruses are classified into five genera including influenza viruses A, B, and C [7, 8]. Influenza A is the major pathogen responsible for epidemic influenza, which attracted the most attention. Vaccination is not a realistic plan for a rapidly spreading influenza pandemic, because of the substantial lead-time for vaccine production and protection. On the other hand, antiviral drugs provide alternative option to control influenza infections. To date, the FDA approved five antiviral drugs for treatment of influenza A infections, including three neuraminidase (NA) inhibitors including oseltamivir (Tami1 1 1 flu ), zanamivir (Relenza ), and peramivir (Rapivab ) (Fig. 1). The other two are M2 channel blockers, amantadine 1 1 (Symmetrel ) and rimantadine (Flumadine ). Rapid emergence of drug-resistant viral mutations has limited the use of the NA inhibitors [9–11] and rendered the M2 blockers ineffective [12–14]. There is an urgent need for development of novel anti-influenza drugs. The life cycle of influenza viruses has been well-studied and nearly all the viral proteins are becoming potential therapeutic targets [15, 16]. NA is a glycoprotein tetramer with relatively independent monomers, expressed at the surface of the influenza virus. It is responsible for releasing progeny viral particles by cleaving the terminal sialic acid from hemagglutinin (HA) receptors on cell membranes and facilitates the mobility of

Archiv der Pharmazie

viruses in the infected cells. A-315675 is an anti-influenza agent developed by Abbott scientists and has been reported to show high inhibitory activity against NA [17]. The unique structure of A-315675, a highly functionalized pyrrolidine core with a cispropenyl group as well as four contiguous stereogenic centers including a vicinal diamino moiety and a tertiary ether function, has been reported as crucial for its biological activity, and has offered synthetic challenges. It was previously reported that a series of tri-substituted pyrrolidine NA inhibitors have potent activities [18]. According to the studies on NA active site and structure–activity relationship (SAR) of published NA inhibitors, inhibition of the NA is mainly determined by the relative positions of the substituents (carboxylate, glycerol, acetamido, and hydroxyl domains) of the central ring. Currently, several pyrrolidine compounds have been found to possess potent NA inhibitory activities [19]. For example, A-315675 [20] is highly active in cell culture against a variety of strains of influenza A and B viruses (Fig. 1). This urged us to develop new NA inhibitors based on pyrrolidine derivatives which contain different substituents (carboxylate, guanidino-, acetamino-, and alkyl-) to interact with the four binding pockets of the NA active site [21]. There is a continuing need for the development of new anti-influenza therapies. As part of our continuing work in the area of drug discovery [22–25], we report in this article, a novel class of pyrrolo[2,1-b][1,3]oxazepine derivatives as potent NA inhibitors from cheap L-tartrate via a-amido-alkylation via a simple synthetic route with high yields.

Results and discussion Chemistry Cheap L-tartaric acid was transformed into anhydride followed by reaction with allylamine in xylene into imides 2. Protection of free alcoholic OH group using

Figure 1. Chemical structures of influenza neuraminidase inhibitors.

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.archpharm.com

anti-

787

ARCH PHARM

Arch. Pharm. Chem. Life Sci. 2015, 348, 786–795 A. O. H. El-Nezhawy et al.

t-butyldimethylsilyl chloride (TBSCl) in the presence of dimethylformamide and imidazole afforded 3 in a good yield (73%). Reduction of 3 with sodium borohydride in methanol afforded 5-hydroxypyrrolidinone derivative 4. Treatment of 5-hydroxy-pyrrolidin-2-one derivative 4 with trichloroacetonitrile in the presence of 1,8-diazabicylo [5.4.0]-undec-7-ene (DBU) as a base furnished trichloroacetimidate 5 in good yields (77%) (Scheme 1). Structures of all new compounds were confirmed by IR, MS, and NMR spectroscopy. Reaction of trichloroacetimidate 5 with allyl alcohol as O-nucleophile in the presence of the Lewis acid trimethylsilyl trifluoromethanesulfonate (TMSOTf) as a catalyst afforded a 1:1 ratio of 5-O-allyl derivatives 6 and 7 (Scheme 2). Separation of the diastereomers 6 and 7 was accomplished by silica gel chromatography and their structural assignments were based on the 1H NMR spectroscopic data (see Experimental section). Ring-closure metathesis of 6 and 7 with Grubbs’ catalyst in dichloromethane under argon atmosphere afforded 8 and 9 in a good yield. Reduction of the amide group to the corresponding amine was carried out by treatment of 8 and 9 with lithium aluminum hydride in tetrahydrofuran (THF) affording hexahydropyrrolo[2,1-b][1,3]oxazepine derivatives 10 and 11, respectively. Desilylation of 10 and 11 using tetrabutylammonium fluoride in THF at room temperature afforded 12 and 13 in an overall yield of 92 and 88%, respectively. The C –– C double bond in 12 and 13 was hydrogenated at 3.3 bar in 10% Pd on carbon in the presence of acid medium, furnishing compounds 14 and 15 in a good overall yield. The structural assignment was based on the 1H NMR, 13C NMR mass spectroscopic data, and elemental analysis (see Experimental section).

Biological evaluation Cytotoxicity and antiviral activity The target compounds 12–15 were examined for their cytotoxicity using Madian–Darby canine kidney (MDCK) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method with minor modifications. The antiviral activity of these compounds was examined using cytopathic effects (CPE) assay against H5N1 avian

Archiv der Pharmazie

influenza virus (A/chicken/Egypt/1/2008) taking oseltamivir phosphate as a control. The results of the antiviral assay are shown in Table 1. The antiviral activity showed that compound 12 was the most potent compound with EC50 value of 0.016 mg/mL, which is almost equivalent to that of oseltamivir phosphate (0.012 mg/mL). Compounds 13–15 showed slightly lower anti-NA activities than compound 12 and the activity decreased from compound 13 to compound 15. Consistent with these results, SAR data suggested that compounds 12 and 13 were more potent in antiviral activity due to the presence of a double bond as in the reference drug. Also, the stereocenters contribute to the activities of these compounds (Scheme 2). Compounds 13, 14, and 15 showed better cytotoxicity than the control oseltamivir phosphate with LD50 values of 0.188 mg/mL, 0.162 mg/mL, and 0.176 mg/mL, respectively. On the other hand, compound 12 showed a slightly higher cytotoxicity than oseltamivir phosphate and compounds 13–15 with an LD50 of 310 mg/mL. On the other hand, the therapeutic index of compound 12 was similar to that of oseltamivir phosphate. However, compounds 13–15 showed lower therapeutic indices than compound 12 and oseltamivir (Table 1).

Molecular docking study Molecular docking is a key tool in structural molecular biology and computer-assisted drug design. The goal of ligand– protein docking is to predict the predominant binding mode(s) of a ligand with a protein of known threedimensional structure. In order to better understand the activity of this series of compounds, we used 3D models to simulate the interactions between compounds 12, 13, 14, and 15 using oseltamivir phosphate as a control with NA (PDB entry code: 2HU0) in an open form based on the docking stimulation (Fig. 2). The scoring functions and hydrogen bonds formed with the surrounding amino acids of the receptor NA are used to predict tested compounds binding modes. Docking results revealed moderate binding affinity of all tested compounds against NA protein ranging between 46.72 and 50.16 kcal/mol; compared to that of the reference oseltamivir, which scores 78.50 kcal/mol (Table 2).

Scheme 1. Synthesis of trichloroacetimidates 5.

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.archpharm.com

788

ARCH PHARM

Arch. Pharm. Chem. Life Sci. 2015, 348, 786–795 Pyrrolooxazepinediol Derivatives with Anti-Influenza Activity

Archiv der Pharmazie

Scheme 2. Synthesis of the target compounds 12–15.

Compound 13 scores the best binding energy of 50.16 kcal/mol among all docked compounds. Also, docking results revealed the importance of the trans-dihydroxy groups in the pyrrolidine ring of all tested compounds to their antiviral activity, as they form hydrogen bonds with Glu 119, Tyr 406, Arg 156, and Trp 178 amino acids of the NA protein binding site (Fig. 2). The double bond of oxazepine ring in compounds 12 and 13 forms hydrophobic interactions with binding site amino acids. The docking view of

all tested compounds shows that they all lie nicely within the catalytic cavity of the NA protein. The moderate binding energy of the tested compounds compared to the reference oseltamivir can be attributed to the smaller molecular size of the tested compounds (Fig. 3) which lacks the required substituents to bind to the open 150 cavity of NA [26]. In addition, substitution on the oxazepine ring and the hydroxyl group on the pyrrolidine ring with some of the essential functional groups for binding to the NA catalytic

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.archpharm.com

789

ARCH PHARM

Arch. Pharm. Chem. Life Sci. 2015, 348, 786–795 A. O. H. El-Nezhawy et al.

Archiv der Pharmazie

Table 1. Cytotoxicity and antiviral activity of target compounds. Compound no. 12 13 14 15 Oseltamivir phosphate

EC50 (mg/mL) avian influenza virus (H5N1)

LD50 (mg/mL)

TI

0.016 0.021 0.026 0.029 0.012

0.310 0.188 0.162 0.176 0.240

19.36 8.95 6.23 6.07 20

LD50, 50% cytotoxicity; EC50, 50% effective antiviral concentration; TI, therapeutic index (calculated by dividing LD50 on EC50).

site, like acetyl, amino, and carboxylic groups may help in enhancing the antiviral activity of this class of compounds as potential NA inhibitors in the future.

Conclusion In this work, we designed and synthesized novel pyrrolo[2,1b][1,3]oxazepines from cheap commercially available L-tartrate via a-amido-alkylation through simple synthetic route with high yields. Molecular docking of the target compounds showed that all tested compounds bind to the active site of NA with moderate to high binding energy. The target compounds 12, 13, 14, and 15 were examined for their cytotoxicity using MTT assay and antiviral activity using the CPE assay against avian influenza virus (H5N1) taking oseltamivir phosphate as a control. The results revealed that compounds 13, 14, and 15 have lower cytotoxicity than the control oseltamivir phosphate with LD50 of 0.188 mg/mL,

0.162 mg/mL, and 0.176 mg/mL, respectively. The antiviral data showed that compound 12 was the most active with an EC50 value of 0.016 mg/mL, which is almost equivalent to that of oseltamivir phosphate. SAR suggested that compound 12 was more potent in antiviral activity due to the presence of a double bond as in the reference drug. In conclusion, we were able to synthesize cheap and potent anti-H5N1 compounds.

Experimental Chemistry All chemicals were purchased from common commercial suppliers and used without further purification. All reactions were carried out under argon with dry solvents. Also, all reactions were monitored by thin layer chromatography (TLC) carried out on Merck silica gel-coated plastic sheets (60 F254; E. Merck, layer thickness 0.2 mm) by using UV light as a visualizing agent. Detection was achieved by treatment either

Figure 2. Binding modes of oseltamivir and compounds 12–15 with N1 are shown in (A–E), respectively, showing intramolecular hydrogen bonds.

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.archpharm.com

790

ARCH PHARM

Arch. Pharm. Chem. Life Sci. 2015, 348, 786–795 Pyrrolooxazepinediol Derivatives with Anti-Influenza Activity

Archiv der Pharmazie

Table 2. The docking energy scores of products no. 12, 13, 14, 15, and oseltamivir phosphate with the amino acid residues of the target enzyme N1 forming hydrogen bonds. Docking score (kcal/mol)

No. of hydrogen bonds

12

46.72

3

13

50.16

4

14

48.29

5

15

48.09

2

Oseltamivir

78.50

4

Compound no.

with a solution of 20 g of ammonium molybdate and 0.4 g of cerium (IV) sulfate in 400 mL of 10% H2SO4 or with 15% H2SO4, and heating at 150°C. Melting points were determined on a Gallenkamp melting point apparatus and were uncorrected. Optical rotations were recorded with a PerkinElmer 241 MC polarimeter in a 1-dm cell at 22°C. IR spectra (KBr disks) were recorded on a Bruker Vector 22 instrument. 1 H NMR and 13C NMR spectra were recorded on a Jeol ECXspectrometer at 300 MHz, Bruker DRX 600 (600 MHz), and AC 250 (250 MHz) in CDCl3 or DMSO as a solvent and TMS as an internal standard. Mass spectra were recorded on Thermo Finnigan LCQ Advantage spectrometer in ESI mode, spray voltage 4.8 kV. Microanalyses were performed at the Microanalytical Center of Cairo University.

Amino acid residues forming hydrogen bonds in Å R156 hh12 -- m mL o3: 1.73 Y406 hh -- mL M o2: 2.74 E119 oe2 -- mL M h13: 2.38 R156 hh12 -- mL M o3: 1.99 R156 hh21 -- mL M o2: 1.22 R156 hh22 -- mL M o2: 2.61 E119 oe2 -- mL M h13: 2.14 W178 o -- mL M h12: 1.49 R156 hh12 -- mL M o2: 2.59 R156 hh21 -- mL M o2: 1.62 Y406 hh -- mL M o3: 2.66 E119 oe2 -- mL M h15: 2.19 W178 o -- mL M h14: 1.60 R156 hh12 -- mL M o2: 1.78 R156 hh21 -- mL M o2: 2.07 E119 oe2 -- mL M h14: 2.73 R292 hh12 -- mL M o1: 2.02 R292 hh21 -- mL M o1: 2.18 R371 hh12 -- mL M o1: 1.98 E276 oe2 -- mL M h23: 2.12

(3R,4R)-1-Allyl-3,4-dihydroxypyrrolidine-2,5-dione (2) A solution of L-tartaric acid (12.0 g, 80.0 mmol) and allylamine (3.04 mL, 70.0 mmol) in xylene (100 mL) was stirred at reflux for 20 h, cooled to 0°C and the solid was collected by filtration. The solid was purified by crystallization using ethanol followed by column chromatography using ethyl acetate/petroleum ether/methanol (9:1:1) which gave 2 (12.1 g, 88%) as white solid, mp 76°C. [a]D ¼ þ101.1 (c ¼ 1.8, MeOH). FT-IR n (cm1): 3400 (OH), 3100 (CH –– CH2), 1710 (C –– O). 1H NMR (DMSO, 300 MHz) d 4.00 (d, J ¼ 9.4 Hz, 2H, NCH2CH –– CH2), 4.40 (s, 2H, CHOH), 5.10 (m, 2H, NCH2CHCH2), 5.80 (m, 1H, NCH2CH –– CH2), 6.30 (br.s, 2H, OH). 13C NMR (DMSO, 75 MHz) d 45.8, 74.2, 120.5, 120.9, 168.1, 169.4 ppm. MS (EI) m/z (%): 171.15 (30, Mþ), 131 (100, M–40). Anal. calcd. for C7H9NO4: C, 49.12; H, 5.30; N, 8.18. Found: C, 48.92; H, 5.20; N, 8.10.

(3R,4R)-3,4-bis-(tert-Butyl-dimethyl-silyloxy)-1-allylpyrrolidine-2,5-dione (3)

Figure 3. Binding mode of compound 14 with the active site of N1, showing lack of substitution required to bind to the 150 cavity of N1.

To a solution of 2 (2.3 g, 13.5 mmol) and imidazole (4.8 g, 68.0 mmol) in DMF (15.0 mL), we added t-butyldimethylsilyl chloride (6.3 g, 40.0 mmol). After stirring for 10 h at 25°C, the reaction mixture was poured into water (250 mL) and extracted with CH2Cl2 (50 mL  4). The organic layer was dried over MgSO4 and concentrated in vacuo giving a residue that was subjected to flash column chromatography (ethyl acetate/petroleum ether at a ratio of 1:9) to give 3 (3.94 g, 73%) as colorless oil. [a]D ¼ þ132.5 (c ¼ 1.6, MeOH). FT-IR n (cm1): 3110 (CH –– CH2), 1715 (C –– O). 1H NMR (DMSO, 300 MHz) d 0.15 (s, 6H, 2CH3), 0.2 (s, 6H, 2CH3), 0.9 (s, 18H, 6CH3), 4.10 (d,

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.archpharm.com

791

ARCH PHARM

Arch. Pharm. Chem. Life Sci. 2015, 348, 786–795 A. O. H. El-Nezhawy et al.

J ¼ 9.0 Hz, 2H, NCH2CH –– CH2), 4.50 (s, 2H, CHOSi), 5.20 (m, 2H, NCH2CHCH2), 5.80 (m, 1H, NCH2CH –– CH2). 13C NMR (DMSO, 75 MHz) d 4.1, 4.6, 19.8, 24.8, 26.2, 45.8, 74.8, 120.3, 120.9, 168.7, 169.8 ppm. MS (EI) m/z (%): 399.67 (10, Mþ), 139 (100, M–260). Anal. calcd. for C19H37NO4Si2: C, 57.10; H, 9.33; N, 3.50. Found: C, 57.14; H, 9.43; N, 3.55.

(3R,4R)-3,4-bis-(tert-Butyl-dimethyl-silyloxy)-1-allyl-5hydroxy-pyrrolidin-2-one (4) To a solution of 3 (3.4 g, 8.5 mmol) in methanol (100 mL), we added sodium borohydride (1.1 g, 28.1 mmol) at 7°C. The resulting solution was stirred for 12 min. Then, the reaction mixture was partitioned between 200 mL of CH2Cl2, 100 mL of saturated aqueous sodium bicarbonate, and water (100 mL). The layers were separated and the aqueous phase was extracted with three 100 mL portions of CH2Cl2. The combined organic phases were dried (MgSO4) and concentrated in vacuo followed by column chromatography using (petroleum ether/ethyl acetate, at a ratio of 1:1) to give 4 as a pale yellow oil (yield 2.94 g, 86%). [a]D ¼ þ32.1 (c ¼ 1.3, MeOH). FT-IR n (cm1): 3430 (OH), 3115 (CH –– CH2), 1712 (C –– O). 1H NMR (CDCl3, 300 MHz): d 0.06 (s, 3H, CH3), 0.07 (s, 3H, CH3), 0.11 (s, 3H, CH3), 0.13 (s, 3H, CH3), 0.84 (s, 9H, 3CH3), 0.86 (s, 9H, 3CH3), 3.40 (d, J ¼ 12.3 Hz, 1H, OH), 3.60 (dd, J ¼ 7.2, 15.0 Hz, 1H, NCH2CH –– CH2), 3.86 (m, 1H, NCH2CH –– CH2), 3.94 (d, J ¼ 4.3 Hz, 1H, 3-H), 4.15 (dd, J ¼ 5.4, 5.4 Hz, 4-H), 4.68 (dd, J ¼ 2.7, 2.7 Hz, 5-H), 5.18 (m, 2H, NCH2CHCH2), 5.68 (m, 1H, NCH2CH¼CH2) ppm. 13C NMR (CDCl3, 75 MHz): d 3.4, 3.6, 18.6, 18.7, 26.3, 26.4, 42.5, 78.2, 79.6, 87.8, 118.3, 132.7, 172.0 ppm. MS (EI) m/z (%): 301.67 (15, Mþ), 139 (100, M–262). Anal. calcd. for C19H39NO4Si2: C, 56.81; H, 9.79; N, 3.49. Found: C, 56.91; H, 9.71; N, 3.39.

(3R,4R)-3,4-bis-(tert-Butyl-dimethyl-silyloxy)-1-allyl-5oxopyrrolidin-2-yl-2,2,2-trichloroacetimidate (5) Stirred solution of 4 (4.0 g, 10 mmol) and trichloroacetonitrile (5 mL, 50 mmol) in dry CH2Cl2 (20 mL) was treated with DBU (72 mL) at 0°C and then left for 15 min. The reaction mixture was concentrated in vacuo followed by column chromatography using 5% triethylamine in toluene to give 5 as a pale yellow oil (yield 4.2 g, 77%). [a]D ¼ þ82.1 (c ¼ 1.6, MeOH). FT-IR n (cm1): 3420 (NH), 3110 (CH –– CH2), 1708 (C –– O). 1H NMR (CDCl3, 300 MHz): d 0.05 (s, 3H, CH3), 0.06 (s, 3H, CH3), 0.11 (s, 3H, CH3), 0.12 (s, 3H, CH3), 0.82 (s, 9H, 3CH3), 0.84 (s, 9H, 3CH3), 3.65 (dd, J ¼ 7.1, 7.2 Hz, 1H, NCH2CH –– CH2), 3.90 (d, J ¼ 1.2 Hz, 3-H), 4.05 (d, J ¼ 1.2 Hz, 4-H), 4.31 (m, 1H, NCH2CH –– CH2), 5.18 (m, 2H, NCH2CHCH2), 5.75 (m, 1H, NCH2CH –– CH2), 5.90 (d, J ¼ 1.3 Hz, 1H, 5-H), 8.50 (br.s, 1H, NH) ppm. 13C NMR (CDCl3, 75 MHz): d 3.3, 3.4, 18.7, 19.0, 26.5, 43.2, 76.9, 78.4, 93.8, 118.7, 132.9, 162.4, 174.0 ppm. MS (EI) m/z (%): 546.07 (6, Mþ), 283 (20, M–262), 139 (100, M–407). Anal. calcd. for C21H39Cl3N2O4Si2: C, 46.19; H, 7.20; Cl, 19.48; N, 5.13. Found: C, 46.06; H, 7.31; Cl, 19.38; N, 5.02.

Archiv der Pharmazie

reaction was quenched by addition of solid sodium bicarbonate, diluted with dichloromethane, filtered, and concentrated. The crude residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, at a ratio of 1:1) to give 6 and 7.

(3R,4S,5S)-3,4-Bis((tert-butyldimethylsilyl)oxy)-1-allyl-5(allyloxy)pyrrolidin-2-one (6) Colorless oil (yield 1.04 g, 47%) [a]D ¼ þ60.2 (c ¼ 0.6, MeOH). FT-IR n (cm1): 3115 (CH –– CH2), 1700 (C –– O). 1H NMR (CDCl3, 300 MHz): d 0.12 (s, 6H, 2CH3), 0.13 (s, 6H, 2CH3), 0.88 (s, 9H, 3CH3), 0.89 (s, 9H, 3CH3), 3.50 (dd, J ¼ 5.0, 13.1 Hz, 1H, OCH2CH –– CH2), 3.90 (m, 3H), 4.60 (d, J ¼ 7.2 Hz, 1H, 5-H), 5.15– 5.30 (m, 4H,), 5.33 (dd, J ¼ 5.0, 7.1 Hz, 1H, 4-H), 5.60 (d, J ¼ 7.0 Hz, 1H, 3-H), 5.69 (m, 2H, CHCH2CH –– CH2, NCH2CH –– CH2) ppm. 13C NMR (CDCl3, 75 MHz): 18.5, 18.9, 26.4, 26.5, 42.6, 69.2, 78.2, 79.3, 92.9, 117.9, 118.4, 132.6, 134.4, 171.7 ppm. MS (EI) m/z (%): 441 (20, Mþ), 179 (60, M–262), 139 (100, M–302). Anal. calcd. for C22H43NO4Si2: C, 59.82; H, 9.81; N, 3.17. Found: C, 59.64; H, 9.61; N, 3.27.

(3R,4S,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-1-allyl-5(allyloxy)pyrrolidin-2-one (7) Colorless oil (yield 1.04 g, 47%) [a]D ¼ þ160 (c ¼ 1.6, MeOH). FT-IR n (cm1): 3113 (CH –– CH2), 1701 (C –– O). 1H NMR (CDCl3, 300 MHz): d 0.11 (s, 6H, 2 CH3), 0.12 (s, 6H, 2 CH3), 0.87 (s, 9H, 3CH3), 0.88 (s, 9H, 3CH3), 3.53 (dd, J ¼ 8.4, 14.0 Hz, 1H, OCH2CH –– CH2), 3.94 (m, 3H), 4.65 (d, J ¼ 5.0 Hz, 1H, 5-H), 5.10– 5.25 (m, 4H,), 5.40 (dd, J ¼ 5.0, 7.0 Hz, 1H, 4-H), 5.60 (d, J ¼ 7.0 Hz, 1H, 3-H) 5.73 (m, 2H, CHCH2CH –– CH2, NCH2CH –– CH2) ppm. 13C NMR (CDCl3, 75 MHz): 18.0, 18.7, 26.1, 26.1, 43.6, 69.7, 78.5, 79.6, 93.6, 118.9, 119.0, 131.9, 134.8, 171.2 ppm. MS (EI) m/z (%): 441 (15, Mþ), 179 (47, M–262), 139 (100, M–302). Anal. calcd. for C22H43NO4Si2: C, 59.82; H, 9.81; N, 3.17. Found: C, 59.67; H, 9.69; N, 3.03.

General procedure for the synthesis of 8 and 9 via ringclosing olefin metathesis using the Grubbs catalyst To a solution of 6 or 7 (1.1 g, 2.5 mmol) in dichloromethane (30 mL), under an argon atmosphere, we added (bis(tricyclohexylphosphane)benzylidene)-ruthenium(IV) dichloride (Grubbs’ catalyst) (0.005 g, 0.007 mmol) in dichloromethane (10 mL) under an argon atmosphere. The mixture was refluxed for 10 h. The solvent was removed under reduced pressure and the residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, 10:1) to give 8 or 9, respectively.

(8S,9R,9aS)-8,9-Bis(tert-butyldimethylsilyloxy)2,5,7,8,9,9a-hexahydropyrrolo[2,1-b][1,3]oxazepin-7-one (8)

A solution of trichloroacetimidate 5 (2.73 g, 5 mmol) and allyl alcohol (0.34 mL, 5 mmol) in dry CH2Cl2 (30 mL) was treated with TMSOTf (0.2 mL) and then stirred for 30–120 min. The

Colorless oil (yield 0.74 g, 72%) [a]D ¼ þ36.8 (c ¼ 0.8, MeOH). FT-IR n (cm1): 3112 (CH –– CH2), 1698 (C –– O). 1H NMR (CDCl3, 300 MHz): d 0.10 (s, 6H, 2CH3), 0.11 (s, 6H, 2CH3), 0.85 (s, 9H, 3CH3), 0.87 (s, 9H, 3CH3), 3.45 (br d, J ¼ 18.6 Hz, 1H, 5-H), 4.10 (m, 1H, 5-H), 4.30 (m, 2H, 2-H), 4.45 (d, J ¼ 7.0 Hz, 1H, 8-H), 4.60

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.archpharm.com

Synthesis of 6 and 7

792

ARCH PHARM

Arch. Pharm. Chem. Life Sci. 2015, 348, 786–795 Pyrrolooxazepinediol Derivatives with Anti-Influenza Activity

(dd, J ¼ 7.0, 6.2 Hz, 1H, 9-H), 4.75 (d, J ¼ 7.1 Hz, 1H, 9a-H), 5.60 (m, 2H, 3-H, 4-H) ppm. 13C NMR (CDCl3, 75 MHz): 18.8, 18.9, 26.4, 42.2, 68.9, 75.8, 76.0, 88.8, 127.2, 129.2, 171.9 ppm. MS (EI) m/z (%): 413 (10, Mþ), 185 (100, M–228), 157 (50, M–256). Anal. calcd. for C20H39NO4Si2: C, 58.06; H, 9.50; N, 3.39. Found: C, 57.96; H, 9.43; N, 3.30.

(8S,9R,9aR)-8,9-Bis(tert-butyldimethylsilyloxy)2,5,7,8,9,9a-hexahydropyrrolo[2,1-b][1,3]oxazepin-7-one (9) Colorless oil (yield 0.68 g, 67%) [a]D ¼ þ168.0 (c ¼ 1.1, MeOH). FT-IR n (cm1): 3116 (CH –– CH2), 1705 (C –– O). 1H NMR (CDCl3, 300 MHz): d 0.11 (s, 6H, 2CH3), 0.12 (s, 6H, 2CH3), 0.86 (s, 9H, 3CH3), 0.88 (s, 9H, 3CH3), 3.65 (br d, J ¼ 18.8 Hz, 1H, 5-H), 4.05 (m, 1H, 5-H), 4.20 (m, 2H, 2-H), 4.40 (d, J ¼ 5.0 Hz, 1H, 8-H), 4.40 (dd, J ¼ 5.0, 6.6 Hz, 1H, 9-H), 4.85 (d, J ¼ 5.0 Hz, 1H, 9a-H), 5.65 (m, 2H, 3-H, 4-H) ppm. 13C NMR (CDCl3, 75 MHz): 18.6, 18.9, 26.4, 41.2, 64.7, 78.2, 79.6, 93.5, 126.1, 129.6, 170.0 ppm. MS (EI) m/z (%): 413 (15, Mþ), 185 (100, M–228), 157 (30, M–256). Anal. calcd. for C20H39NO4Si2: C, 58.06; H, 9.50; N, 3.39. Found: C, 58.13; H, 9.57; N, 3.32.

Synthesis of 10 and 11 A solution of 8 or 9 (0.46 g, 1.13 mmol) dissolved in THF (15 mL) was added under argon to a suspension of LiAlH4 in THF (0.9 g, 2.3 mmol in 5 mL) and heated at reflux for 4 h. Excess hydride was destroyed at 0°C with 10% aq. NH4Cl (0.4 mL). The solid was filtered and washed with ethyl acetate (2  30 mL). The organic phase was dried (MgSO4) and after solvent evaporation, the crude material was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, 10:1) to give 10 or 11, respectively.

(8S,9R,9aS)-8,9-Bis(tert-butyldimethylsilyloxy)2,5,7,8,9,9a-hexahydropyrrolo[2,1-b][1,3]oxazepine (10) Colorless oil (yield 0.34 g, 76%) [a]D ¼ þ16.2 (c ¼ 0.4, MeOH). 1 H NMR (CDCl3, 300 MHz): d 0.10 (s, 6H, 2CH3), 0.12 (s, 6H, 2CH3), 0.83 (s, 9H, 3CH3), 0.86 (s, 9H, 3CH3), 2.62 (d, J ¼ 7.4, 1H, 7-H), 2.92 (d, J ¼ 11.2 Hz, 1H, 7-H), 3.45 (br d, J ¼ 18.6 Hz, 1H, 5H), 4.10 (m, 1H, 5-H), 4.30 (m, 2H, 2-H), 4.40 (m, 1H, 8-H), 4.54 (dd, J ¼ 7.1, 6.1 Hz, 1H, 9-H), 4.75 (d, J ¼ 7.0 Hz, 1H, 9a-H), 5.61 (m, 2H, 3-H, 4-H) ppm. 13C NMR (CDCl3, 75 MHz): 18.1, 18.3, 26.9, 40.2, 43.8, 68.3, 75.0, 76.7, 88.3, 127.9, 129.7 ppm. MS (EI) m/z (%): 399 (20, Mþ), 167 (100, M–232). Anal. calcd. for C20H41NO3Si2: C, 60.10; H, 10.34; N, 3.50. Found: C, 59.98; H, 10.40; N, 3.39.

(8S,9R,9aR)-8,9-Bis(tert-butyldimethylsilyloxy)2,5,7,8,9,9a-hexahydropyrrolo[2,1-b][1,3]oxazepine (11)

Archiv der Pharmazie

77.9, 79.3, 94.5, 126.8, 128.6 ppm. MS (EI) m/z (%): 399 (10, Mþ), 167 (100, M-232). Anal. calcd. for C20H41NO3Si2: C, 60.10; H, 10.34; N, 3.50. Found: C, 60.17; H, 10.29; N, 3.44.

Synthesis of 12 and 13 To a solution of 10 or 11 (1.8 mmol) in THF (30 mL), we added tetrabutylammonium fluoride (1.0 mol in THF, 5 mL) at room temperature and stirred for 2 h. The reaction mixture was quenched with water (20 mL) and then extracted with ethyl acetate (25 mL  4). The organic phase was dried (MgSO4) and after solvent evaporation, the crude material was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, at a ratio of 10:1) to give 12 or 13, respectively.

(8S,9R,9aS)-2,5,7,8,9,9a-Hexahydropyrrolo[2,1-b][1,3] oxazepine-8,9-diol (12) Colorless oil (yield 0.25 g, 92%) [a]D ¼ þ76.0 (c ¼ 1.4, MeOH). 1 H NMR (CDCl3, 300 MHz): d 2.60 (d, J ¼ 7.0, 1H, 7-H), 2.90 (d, J ¼ 11.0 Hz, 1H, 7-H), 3.44 (br d, J ¼ 16.6 Hz, 1H, 5-H), 4.13 (m, 1H, 5-H), 4.33 (m, 2H, 2-H), 4.44 (dd, J ¼ 4.1, 7.1 Hz, 1H, 8-H), 4.55 (dd, J ¼ 7.2, 6.3 Hz, 1H, 9-H), 4.85 (d, J ¼ 7.2 Hz, 1H, 9a-H), 5.66 (m, 2H, 3-H, 4-H) ppm. 13C NMR (CDCl3, 75 MHz): 42.6, 44.5, 66.9, 76.2, 78.3, 88.9, 128.2, 130.1 ppm. MS (EI) m/z (%): 171 (100, Mþ). Anal. calcd. for C8H13NO3: C, 56.13; H, 7.65; N, 8.18. Found: C, 56.18; H, 7.49; N, 8.23.

(8S,9R,9aR)-2,5,7,8,9,9a-Hexahydropyrrolo[2,1-b][1,3]oxazepine-8,9-diol (13) Colorless oil (yield 0.29 g, 88%) [a]D ¼ þ126.0 (c ¼ 2.5, MeOH). 1 H NMR (CDCl3, 300 MHz): d 2.70–2.88 (m, 2H, 7-H), 3.66 (br d, J ¼ 15.9 Hz, 1H, 5-H), 4.15 (m, 1H, 5-H), 4.24 (m, 2H, 2-H), 4.45 (t, 1H, 8-H), 4.49 (dd, J ¼ 6.0, 6.8 Hz, 1H, 9-H), 4.83 (d, J ¼ 6.1 Hz, 1H, 9a-H), 5.69 (m, 2H, 3-H, 4-H) ppm. 13C NMR (CDCl3, 75 MHz): 40.9, 43.4, 64.8, 75.9, 78.1, 89.1, 129.8, 131.0 ppm. MS (EI) m/z (%): 171 (100, Mþ). Anal. calcd. for C8H13NO3: C, 56.13; H, 7.65; N, 8.18. Found: C, 56.20; H, 7.79; N, 8.08.

Synthesis of 14 and 15 To a solution of 12 or 13 (0.6 mmol) in methanol (30 mL), we added concentrated HCl (10 drops) and the mixture was hydrogenated at 3.3 bar (0.08 g of 10% Pd on carbon). TLC monitoring indicated the reaction was complete after 10 h and then a solution of sodium hydroxide (2.0 mL, 3 M) was added followed by filtration through Celite. The crude material was purified by column chromatography on silica gel (dichloromethane/methanol/aq. NH3, at a ratio of 85:10:5) to give 14 or 15, respectively.

(8S,9R,9aS)-Octahydropyrrolo[2,1-b][1,3]oxazepine-8,9diol (14)

Colorless oil (yield 0.29 g, 65%) [a]D ¼ þ116.0 (c ¼ 1.0, MeOH). 1 H NMR (CDCl3, 300 MHz): d 0.12 (s, 6H, 2CH3), 0.13 (s, 6H, 2CH3), 0.88 (s, 9H, 3CH3), 0.91 (s, 9H, 3CH3), 2.70–2.88 (m, 2H, 7H), 3.65 (br d, J ¼ 18.8 Hz, 1H, 5-H), 4.05 (m, 1H, 5-H), 4.20 (m, 2H, 2-H), 4.40 (t, 1H, 8-H), 4.42 (dd, J ¼ 5.1, 6.7 Hz, 1H, 9-H), 4.85 (d, J ¼ 5.1 Hz, 1H, 9a-H), 5.66 (m, 2H, 3-H, 4-H) ppm. 13 C NMR (CDCl3, 75 MHz): 18.8, 19.4, 26.9, 42.2, 44.6, 64.9,

Colorless oil (yield 0.20 g, 72%) [a]D ¼ þ46.0 (c ¼ 2.5, MeOH). 1 H NMR (CDCl3, 600 MHz): d 1.42–1.55 (m, 4H, 2 3-H, 2 4-H), 2.07 (ddd, J ¼ 2.8, 11.5, 16.3 Hz, 1H, 5-H), 2.53 (dd, J ¼ 7.5, 11.1 Hz, 1H, 7-H), 2.75 (br d, J ¼ 11.2 Hz,1H, 7-H), 2.90 (br d, J ¼ 11.3 Hz, 1H, 5-H), 2.91 (br d, J ¼ 11.1 Hz, 1H, 5-H), 3.61 (dd, J ¼ 3.8, 8.6 Hz, 1H, 9-H), 3.65–3.73 (m, 2H, 2-H), 4.10 (m, 2H,

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.archpharm.com

793

ARCH PHARM

Arch. Pharm. Chem. Life Sci. 2015, 348, 786–795 A. O. H. El-Nezhawy et al.

8-H, 8a-H) ppm. 13C NMR (CDCl3, 150.9 MHz): 25.6, 30.5, 53.5, 56.9, 66.3, 73.9, 78.2, 97.1 ppm. MS (EI) m/z (%): 173 (100, Mþ). Anal. calcd. for C8H15NO3: C, 55.47; H, 8.73; N, 8.09. Found: C, 55.38; H, 8.69; N, 8.20.

(8S,9R,9aR)-Octahydropyrrolo[2,1-b][1,3]oxazepine-8,9diol (15) Colorless oil (yield 0.20 g, 72%) [a]D ¼ þ26.0 (c ¼ 2.5, MeOH). 1 H NMR (CDCl3, 600 MHz): d 1.44–2.25 (m, 6H, 2 3-H, 2 4-H, 1 5H, 1 7-H), 2.07 (ddd, J ¼ 2.8, 11.5, 16.3 Hz, 1H, 5-H), 2.94 (br d, J ¼ 11.0 Hz, 1H, 5-H), 3.30 (dd, J ¼ 7.0, 10.6 Hz, 1H, 7-H), 3.81 (d, J ¼ 8.6 Hz, 1H, 9-H), 3.65–3.73 (m, 2H, 2-H), 4.19 (m, 2H, 8-H, 8a-H) ppm. 13C NMR (CDCl3, 150.9 MHz): 24.9, 29.6, 50.5, 58.6, 66.3, 72.8, 76.9, 92.8 ppm. MS (EI) m/z (%): 173 (100, Mþ). Anal. calcd. for C8H15NO3: C, 55.47; H, 8.73; N, 8.09. Found: C, 55.58; H, 8.58; N, 8.03.

Anti-viral screening Influenza A virus (H5N1) NA inhibition assay Virus and cells: The H5N1 avian influenza A virus used in this study (A/chicken/Egypt/1/2008) was used to prepare low pathogenic rH5N1 [27]. H5N1 was titered on MadinDarby canine kidney (MDCK) cells at a multiplicity of infection (MOI) ¼ 0.001. The MDCK cell line was kindly provided by Dr. Richard Webby from St. Jude Children’s Research Hospital, Memphis, TN, USA. MDCK cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin, and incubated at 37°C in a humidified CO2 incubator. In vitro anti-influenza virus assay: The antiviral activity of the target compounds was determined against low pathogenic reassortant avian influenza virus (rH5N1) [27] from the National Research Center (NRC) using the CPE assay as previously described [28, 29] with oseltamivir phosphate as a control. Stock solutions of the test compounds were prepared in DMSO at a concentration of 10 mg/mL. The results were expressed as the 50% effective concentration (EC50). The 50% effective antiviral concentration (EC50) was defined as the compound concentration required for protecting 50% of the virus-infected cells against viral cytopathic effects. The therapeutic index was calculated by dividing 50% cytotoxicity (LD50) over EC50.

Cytotoxicity assay (MTT assay) The compounds tested were prepared in 10% DMSO in ddH2O to a 10 mg/mL concentration. Stock solutions of the test compounds were used for further dilutions according to the assay applied. Samples were diluted with DMEM to the desired concentrations (5, 10, 20, 40, and 80 mg/100 mL). The cytotoxic activity of the new compounds were tested in MDCK cell line by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method [29, 30] with minor modifications. Briefly, the cells were seeded in 96-well plates (100 mL/well at a density of 3  105 cells/mL), and then incubated for 24 h at 37°C in the presence of 5% CO2. After

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Archiv der Pharmazie

24 h, cells were treated with various concentrations of the tested compounds in triplicates. After further 24 h, the supernatant was discarded and cell sheets were washed with sterile phosphate buffer saline (PBS) three times and MTT solution (20 mL of 5 mg/mL stock solution) was added to each well and incubated at 37°C for 4 h followed by medium aspiration. In each well, the formed formazan crystals were dissolved with 200 mL of acidified 2-propanol (0.04 M HCl in absolute 2-propanol). Absorbance of formazan solutions was detected at lmax 540 nm with 620 nm as a reference wavelength using a multi-well plate reader. The percentages of cytotoxicity compared to the untreated cells were determined with the following equation: % Cytotoxicity ¼

Absorbance of cells without treatment  Absorbance of cells with treatment  100 Absorbance of cells without treatment

The plot of percentage cytotoxicity versus sample concentration for each compound was used to calculate the concentration, which exhibited 50% cytotoxicity (LD50).

Molecular docking All docking studies were performed using “Internal Coordinate Mechanics” [Molsoft ICM 3.5-0a]. A set of novel pyrrolo[2,1-b][1,3]oxazepine-8,9-diol derivatives was compiled by us using ChemDraw. 3D structures were constructed using Chem 3D ultra 12.0 software [Molecular Modeling and Analysis; Cambridge Soft Corporation, USA (2010)]. The selected compounds were energetically minimized by using MOPAC (semi-empirical quantum mechanics), Job Type with 100 iterations, and a minimum RMS gradient of 0.01, and saved as MDL MolFile ( .mol). The “open” form crystal structure 2HU0 [15] of NA bound to oseltamivir is downloaded from PDB data bank [20]. All bound water ligands and cofactors were removed from the protein prior to the docking process. The authors gratefully acknowledge the financial support provided by Taif University, KSA, through Project No.2300/ 434/1. The authors have declared no conflicts of interest.

References [1] J. Taubenberger, D. Morens, Rev. Sci. Tech. 2009, 28, 187. [2] A. S. Monto, D. A. Iacuzio, J. R. La Montagne, J. Infect. Dis. 1997, 176 (Supplement 1), S1–S3. [3] R. J. Garten, C. T. Davis, C. A. Russell, B. Shu, S. Lindstrom, A. Balish, W. M. Sessions, X. Xu, E. Skepner, V. Deyde, M. Okomo-Adhiambo, L. Gubareva, J. Barnes, C. B. Smith, S. L. Emery, M. J. Hillman, P. Rivailler, J. Smagala, M. de Graaf, D. F. Burke, R. A. Fouchier, C. Pappas, C. M. Alpuche-Aranda, H. Lopez-Gatell, H. Olivera, I. Lopez, C. A. Myers, D. Faix, P. J. Blair, C. Yu, K. M. Keene, P. D. Dotson, Jr., D. Boxrud,

www.archpharm.com

794

ARCH PHARM

Arch. Pharm. Chem. Life Sci. 2015, 348, 786–795 Pyrrolooxazepinediol Derivatives with Anti-Influenza Activity

[4] [5] [6]

[7] [8]

[9]

[10] [11]

[12] [13]

[14]

A. R. Sambol, S. H. Abid, K. St George, T. Bannerman, A. L. Moore, D. J. Stringer, P. Blevins, G. J. DemmlerHarrison, M. Ginsberg, P. Kriner, S. Waterman, S. Smole, H. F. Guevara, E. A. Belongia, P. A. Clark, S. T. Beatrice, R. Donis, J. Katz, L. Finelli, C. B. Bridges, M. Shaw, D. B. Jernigan, T. M. Uyeki, D. J. Smith, A. I. Klimov, N. J. Cox, Science 2009, 325, 197–201. G. Neumann, T. Noda, Y. Kawaoka, Nature 2009, 459, 931–939. L. V. Gubareva, L. Kaiser, M. N. Matrosovich, Y. Soo-Hoo, F. G. Hayden, J. Infect. Dis. 2001, 183, 523–531. N. J. Dharan, L.V. Gubareva, J. J. Meyer, M. OkomoAdhiambo, R. C. McClinton, S. A. Marshall, K. S. George, S. Epperson, L. Brammer, A. I. Klimov, JAMA 2009, 301, 1034–1041. P. Palese, J. F. Young, Science 1982, 215, 1468–1474. E. Ghedin, N. A. Sengamalay, M. Shumway, J. Zaborsky, T. Feldblyum, V. Subbu, D. J. Spiro, J. Sitz, H. Koo, P. Bolotov, D. Dernovoy, T. Tatusova, Y. Bao, K. St George, J. Taylor, D. J. Lipman, C. M. Fraser, J. K. Taubenberger, S. L. Salzberg, Nature 2005, 437, 1162–1166. W. Chen, P. A. Calvo, D. Malide, J. Gibbs, U. Schubert, I. Bacik, S. Basta, R. O’Neill, J. Schickli, P. Palese, P. Henklein, J. R. Bennink, J. W. Yewdell, Nat. Med. 2001, 7, 1306–1312. V. P. Mishin, F. G. Hayden, L. V. Gubareva, Antimicrob. Agents Chemother. 2005, 49, 4515–4520. A. Meijer, A. Lackenby, O. Hungnes, B. Lina, S. Van Der Werf, B. Schweiger, M. Opp, J. Paget, J. van de Kassteele, A. Hay, Emerg. Infect. Dis. 2009, 15, 552. R. A. Bright, D. K. Shay, B. Shu, N. J. Cox, A. I. Klimov, JAMA 2006, 295, 891–894. V. M. Deyde, X. Xu, R. A. Bright, M. Shaw, C. B. Smith, Y. Zhang, Y. Shu, L. V. Gubareva, N. J. Cox, A. I. Klimov, J. Infect. Dis. 2007, 196, 249–257. Y. Furuse, A. Suzuki, H. Oshitani, Antimicrob. Agents Chemother. 2009, 53, 4457–4463.

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Archiv der Pharmazie

[15] R. M. Krug, J. M. Aramini, Trends Pharmacol. Sci. 2009, 30, 269–277. [16] K. Das, J. M. Aramini, L.-C. Ma, R. M. Krug, E. Arnold, Nat. Struct. Mol. Biol. 2010, 17, 530–538. [17] T. Momose, N. Hama, C. Higashino, H. Sato, N. Chida, Tetrahedron Lett. 2008, 49, 1376–1379. [18] C. J. Maring, V. S. Stoll, C. Zhao, M. Sun, A. C. Krueger, K. D. Stewart, D. L. Madigan, W. M. Kati, Y. Xu, R. J. Carrick, J. Med. Chem. 2005, 48, 3980–3990. [19] D. Young, C. Fowler, K. Bush, Biol. Sci. 2001, 356, 1905–1913. [20] S. Hanessian, M. Bayrakdarian, X. Luo, J. Am. Chem. Soc. 2002, 124, 4716–4721. [21] G. T. Wang, Y. Chen, S. Wang, R. Gentles, T. Sowin, W. Kati, S. Muchmore, V. Giranda, K. Stewart, H. Sham, D. Kempf, W. G. Laver, J. Med. Chem. 2001, 44, 1192–1201. [22] A. O. H. El Nezhawy, S. T. Gaballah, M. A. A. Radwan, Tetrahedron Lett. 2009, 50, 6646–6650. [23] S. A. Galal, S. I. El-Naem, A. O. El-Nezhawy, M. A. Ali, H. I. El-Diwani, Arch. Pharm. (Weinheim) 2011, 344, 255–263. [24] A. O. El-Nezhawy, F. G. Adly, A. F. Eweas, A. G. Hanna, Y. M. El-Kholy, S. H. El-Sayed, T. B. El-Naggar, Arch. Pharm. (Weinheim) 2011, 344, 648–657. [25] A. O. el-Nezhawy, F. G. Adly, A. F. Eweas, A. G. Hanna, Y. M. el-Kholy, S. H. el-Syed, T. B. el-Naggar, Med. Chem. 2011, 7, 624–638. [26] N. Han, Y. Mu, PloS ONE 2013, 8, e73344. [27] M. M. Bahgat, M. A. Kutkat, M. H. Nasraa, A. Mostafa, R. Webby, I. M. Bahgat, M. A. Ali, J. Virol. Methods 2009, 159, 244–250. [28] A. Rashad, M. Ali, Nucleosides Nucleotides Nucleic Acids 2006, 25, 17–28. [29] R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Herdewijn, J. Desmyter, E. De Clercq, J. Virol. Methods 1988, 20, 309–321. [30] T. Mosmann, J. Immunol. Methods 1983, 65, 55–63.

www.archpharm.com

795