key: cord-306633-69ljgkqy
authors: Završnik, Davorka; Muratović, Samija; Makuc, Damjan; Plavec, Janez; Cetina, Mario; Nagl, Ante; De Clercq, Erik; Balzarini, Jan; Mintas, Mladen
title: Benzylidene-bis-(4-Hydroxycoumarin) and Benzopyrano-Coumarin Derivatives: Synthesis, (1)H/(13)C-NMR Conformational and X-ray Crystal Structure Studies and In Vitro Antiviral Activity Evaluations
date: 2011-07-19
journal: Molecules
DOI: 10.3390/molecules16076023
sha: 
doc_id: 306633
cord_uid: 69ljgkqy

We report on the synthesis of 4-hydroxycoumarin dimers 1–15 bearing an aryl substituent on the central linker and fused benzopyranocoumarin derivatives 16–20 and on their in vitro broad anti-DNA and RNA virus activity evaluations. The chemical identities and structure of compounds 1–20 were deduced from their homo- and heteronuclear NMR measurements whereas the conformational properties of 5, 14 and 20 were assessed by the use of 1D difference NOE enhancements. Unequivocal proof of the stereostructure of compounds 7, 9, 16 and 18 was obtained by single crystal X-ray diffraction method. The X-ray crystal structure analysis revealed that two 4-hydroxycoumarin moieties in the 4-trifluoromethylphenyl- and 2-nitrophenyl derivatives (compounds 7 and 9, respectively) are intramolecularly hydrogen-bonded between hydroxyl and carbonyl oxygen atoms. Consequently, the compounds 7 and 9 adopt conformations in which two 4-hydroxy-coumarin moieties are anti-disposed. Antiviral activity evaluation results indicated that the 4-bromobenzylidene derivative of bis-(4-hydroxycoumarin) (compound 3) possesses inhibitory activity against HSV-1 (KOS), HSV-2 (G), vaccinia virus and HSV-1 TK(-) KOS (ACV(r)) at a concentration of 9–12 μM and at a minimum cytotoxic concentration (MCC) greater than 20 μM. Compounds 4–6, 8, and 20 were active against feline herpes virus (50% effective concentration, EC(50) = 5–8.1 μM), that is at a 4-7-fold lower concentration than the MCC.

Numerous experimental studies indicate that natural and synthetic coumarins (2H-1-benzopyran-2ones) and their derivatives are endowed with excellent chemical reactivity and different bioactivity. Thus, the natural coumarins play an important role in plant biochemistry and physiology. They act as antioxydants, enzyme inhibitors and precursors of toxic substances. They are also involved in the actions of plant growth hormones and growth regulators, the control over the respiration and photosynthesis, as well as in the defense against various infections [1] . Although most of the existing natural coumarins have been isolated from higher plants, some of them have been discovered in microorganisms, e.g., aminocoumarin antibiotics: Novobiocin, coumermycin A 1 and chlorobiocin (produced by the actinomycete Streptomyces niveus) [2] .

Synthetic coumarin derivatives have been obtained by chemical modification of the coumarin ring. As a substitution can conceptually occur at any of the six available sites of the basic molecule, these compounds are widely variable in structure and activity. The biological activities of coumarin derivatives, in particular their therapeutic application as anticoagulant and antibacterial agents [3] , has stimulated further interest for the synthesis of this class of compounds. A variety of synthesized coumarin derivatives have been experimentally shown to exert pharmacological activities including inhibition of platelet aggregation, cytochrome P450, and steroid 5-α-reductase.

They have also been shown to exert efficient anti-proliferative, antifungal, anti-psoriasis, antiinflammatory, as well as antiviral activities [4] [5] [6] [7] [8] . The interest in coumarins has recently increased significantly because it was found that they inhibit HIV (human immunodeficiency virus), by affecting integrase and reverse transcriptase, which play a critical role in the replicative cycle of HIV [9] [10] [11] . The present study is focused on the antiviral activity evaluation of the benzylidene-bis- 

The benzylidene-bis-(4-hydroxycoumarin) derivatives 1-15 and fused benzopyranocoumarin derivatives 16-20 were prepared by a sequence of reactions displayed in the Scheme 1. In the first step of the synthesis the aldol condensation of 4-hydroxycoumarin (4-HC) with an appropriately substituted aldehyde linker followed by dehydration of aldol product (AL) gave a chromone (CR). Subsequent in situ reaction of the chromon with 4-hydroxycoumarin already present in excess in the reaction mixture gave dimeric coumarin derivatives 1-15 bearing an aryl substituent in the central methylene linker. In contrast, the chromone derivatives containing an ortho-substituted phenyl moiety (R 1 = Cl, F, OH, OCH 3 ) gave under spontaneous cyclization the fused benzopyranocoumarin derivatives 16-20. The compounds 1 [11, 14, 16] , 2 [11, 13, 14] , 4 [14] , 5 [14] , 6 [13] , 8 [14] , 9 [11, 14] , 10 [14] , 11 [14] , 12 [11, 14] and 16 [17, 18] that have been described previously were also synthesized for their antiviral activity evaluations. 

+ R 2 R 1 R 4 R 3 H O (for R 1 -R 4 see Figure 1a) i O O O OH -H 2 O [R 1 -R 4 , c.f. Figure 1a] [R 5 -R 8 , c.f. Figure 1b] O OH O O OH O R 3 R 4 R 2 R 1 1-15 (c.f. Figure 1a) O OH O O O R 6 R 7 R 5 16-20 (c.f. Figure1b) O R 8 CR R 1 R 2 R 3 R 4 AL O O O R 1 R 2 R 3 R 4

The chemical identities and structures of 1-20 were confirmed by homo-and heteronuclear NMR measurements. 1 H-, 13 C-and 19 F-NMR chemical shifts are reported in the Experimental section. Proton-decoupled 13 C-NMR spectra showed C-F coupling constants that enabled straightforward identification of fluorinated carbon atoms and their neighbors. In the 1 H spectrum of 1, 2, 4 and 10, which were dissolved in non-polar solvents, well resolved hydroxyl protons were observed in the range from δ 11.3 to 11.5 ppm (see Experimental section for details). The strongly deshielded signals suggest that OH protons are most probably involved in hydrogen bond formation. In addition, two set of 1 H-NMR signals were observed for H5 and H5" protons which indicates a slight difference in magnetic environment for otherwise symmetrical moieties. Furthermore, hydroxyl protons, which were observed between δ 11.8 and 12.3 ppm for benzopyranocoumarin derivatives 16, 18 and 20, are most likely involved in hydrogen bonds.

Hydrogen bonds described above partially determined conformational preferences of the studied compounds. Conformational properties of 5, 14 and 20 were assessed with the use of 1D NOE difference experiments. Key NOE enhancements are shown in Figure 2 . The saturation of H* in 5 resulted in weak NOE at H6' (1.5%) and 2'-OCH 3 (0.8%, Figure 2a ). Likewise, the saturation of H* in 14 gave moderate NOE at H6' (3.7%) and weak NOE at 2'-OCH 3 (0.8%). These observations suggested nonrestricted rotation along C*-C1' bonds in 5 and 14. No NOEs indicative of relative orientations of individual heterocyclic moieties were observed for 20. Interestingly, benzopyranocoumarin derivatives 16, 18 and 20 showed two sets of signals in the 1 H spectrum for coumarin protons. One set of 1 H signals (H5-H8 protons) exhibited broader line-widths with respect to multiplets attributed to H5"-H8" protons. This phenomenon was studied in more detail for 20 by variable temperature experiments in the range from 298 to 358 K. As an example, broad multiplet at δ 8.00 ppm corresponding to H5 becomes sharper at higher temperatures ( Figure 3 ). This suggests that rotation along C*-C3 bond is restricted at lower temperatures, most likely due to formation of hydrogen bond between the C4-OH and C2" carbonyl group (for enumeration of atoms c.f. Figure 2 ).

In 7 ( Figure 4a ) and 9 (Figure 4b) , two 4-hydroxycoumarin moieties are linked through a methylene bridge on which one hydrogen atom has been replaced with a phenyl ring bearing p-trifluoromethyl and o-nitro groups, respectively. In general, the geometry of the molecules agrees with closely related structures [19] [20] [21] . This asymmetry, which is also found in similar structures, may be a consequence of steric crowding within the molecules. Because of the same reason all principal bond angles about C11 are widened over normal tetrahedral values, ranging from 112.5(2) to 115.4(2)° in 7 and 111.75 (12) to 115.63(12)° in 9. The coumarin rings are slightly distorted from planarity, with two planes inclined at 57.83(11)° and 59.55(5)° to each other in 7 and 9, respectively. The 4-hydroxycoumarin moieties are intramolecularly hydrogen bonded between hydroxyl and carbonyl oxygen atoms in both structures (see Figures 4a and 4b and Table S1 in ESI), thus forming two eight-membered rings. Thus, compounds 7 and 9 adopt a conformation in which two 4-hydroxycoumarin moieties are anti-disposed. Benzylidene bis (4-hydroxycoumarin) derivatives 7 and 9 form supramolecular self-assemblies by C−H···O hydrogen bonds (Figures S1 and S2, see ESI) in which infinite chains are extended by one F···F interaction between trifluoromethyl fluorine atoms in 7 [F2···F2 i = 2.934(5) Å; (i): −x, −y, −z] and one π···π interaction in 9. The distance between the ring centroids of coplanar C5'-C10' rings [α = 0 o ] in 9 is 3.6832(12) Å, the planes are separated by 3.5132(8) Å and centroids offset is ca. 1.11 Å.

Cyclized compound 16 ( Figure 5a ) crystallized with two independent molecules, two ethanol molecules and one water molecule in the asymmetric unit in monoclinic space group P 2 1 /c. The bond lengths in two independent molecules of 16, denoted as A and B, are within 2σ values, and agree very well with the corresponding ones in 18 ( Figure 5b ). (Table S1 , see ESI). Two independent molecules of 16, two ethanol molecules and water molecule are linked by six O−H···O and four C−H···O hydrogen bonds into three-dimensional network ( Figure S3 , see ESI). Two C−H···π interactions participate also in supramolecular aggregation. On the contrary, only one hydrogen bond of C−H···O type links the molecules of 18, thus forming discrete centrosymmetric dimers via 20-membered rings ( Figure S4 , see ESI). Of all evaluated compounds only the 3-bromobenzylidene derivative 3 of bis(4-hydroxycoumarin) showed potentially interesting inhibitory activity against HSV-1 (KOS), HSV-2 (G), vaccinia virus and HSV-1 TK − KOS (ACV r ) in the range of 9-12 μM at a minimum cytotoxic concentration (MCC) greater than 20 μM, whereas compound 19 showed only slight activity against HSV-1 (KOS), HSV-2 (G) and HSV-1 TK − KOS (EC 50 = 45-50 μM) in human embryonic lung HEL cell cultures and no cytotostatic activity at 100 µM (Table 1, Compounds 1-20 were also evaluated for their cytotoxicity and antiviral activities against Coxsackie virus B4 and respiratory syncytial virus (RSV) in HEL cell cultures; parainfluenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4 and Punta Toro virus in Vero cell cultures; and influenza virus subtypes A H1N1 and H3N2, and influenza virus B. No specific antiviral effects (i.e., antivirally effective concentration >5-fold lower than the minimal cytotoxic concentration) were noted for any of the evaluated compounds.

Melting points (uncorrected) were determined with Büchi melting point B-545. Precoated Merck silica gel 60F-254 plates were used for thin-layer chromatography (TLC) and the spots were detected under UV light (254 nm). Solvent system used for TLC was chloroform:methanol = 9:1. Mass spectra were recorded with an Autospec: ESI/Q-TOF Premier instrument. 1 H-NMR spectra were recorded at 600 MHz, in deuterated DMSO-d6, on Bruker (UXNMR/XwinNMR) spectrometer, using tetramethylsilane (TMS) as internal reference. 1D and 2D NMR spectra were recorded at 25 °C on Varian Unity Inova 300 spectrometers. 1 H and 13 C chemical shifts were referred to residual signal of CDCl 3 , CD 2 Cl 2 and DMSO-d 6 (δ TMS 0.0 ppm). 19 F chemical shifts were referenced externally with respect to CCl 3 F (δ 0.0 ppm). 1 H, 13 C and 19 F-NMR resonances were assigned on the basis of signal intensities and multiplicities in 1D spectra as well as correlation signals in 2D 1 H-1 H COSY, 1 H-13 C HSQC and 1 H-13 C HMBC NMR spectra. Elemental analyses were performed in the Central Analytic Service, Rudjer Bošković Institute Zagreb, Croatia, using a Perkin Elmer 2400 Elemental Analyser. The infrared spectra were obtained from potassium bromide triturate containing 0.5% of the product on a Perkin-Elmer 1000 FT-IR spectrophotometer. All data were recorded at 25 °C unless specified otherwise.

3.2.1. The compounds 1-20 were prepared by the following general procedure 4-Hydroxycoumarin (2 mmol) was dissolved in hot ethanol (6 mL), the corresponding aldehyde (1 mmol) was added, and the reaction mixture was refluxed for 24 h. After cooling to room temperature, the solid was filtered off and crystallized to give the product benzylidene-bis-(4hydroxycoumarin) derivatives 1-15 and 3-[6-oxo-(1H)-benzopyrano[4,3-b]benzopyran-7-yl]-4hydroxycoumarin derivatives 16-20 (Figure 1 ). 

(2) [11, 13, 14] . Yield 74%; mp 267. (9) [11, 14] . (12) [11, 14] . (16) [17, 18] . program was used for data collection and processing. The intensities were corrected for absorption using the multi-scan absorption correction method [22] . The crystal structures were solved by direct methods [23] and all non-hydrogen atoms were refined anisotropically by full-matrix least-squares calculations [23] based on F 2 using the programs integrated in WinGX [24] program package. The hydrogen atoms attached to the O3 and O3' atoms in 7 and 9, O3A and O3B atoms in 16 and O3 atom in 18 were found in a difference Fourier map and are refined with O-H distance restraint of 0.82 Å. All other hydrogen atoms were treated using appropriate riding models, with SHELXL97 defaults [23] . Fluorine atoms of trifluoromethyl group in 7 are heavily disordered and restraints on anisotropic displacement parameters were therefore used in their refinement. The C29 and C31 atoms of ethanol molecules in 16 are disordered with the site occupancy factors refined to 0.62(3)/0.38(3) and 0.52(3)/0.48(3) ratio, respectively. Details of crystal data, data collection and refinement parameters are given in Table 3 . PLATON [25] program was used for structure analysis and molecular and crystal structure drawings preparation. CCDC 818091-818094 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. 

The antiviral assays, other than the anti-HIV assays, were based on inhibition of virus-induced cytopathic effect in human lung fibroblast [herpes simplex virus type 1 (HSV-1) [strain KOS], herpes simplex virus type 2 (HSV-2) [strain G], vaccinia virus (VV) and vesicular stomatitis virus (VSV)], African green monkey kidney (Vero, parainfluenza-3, reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus), human cervix carcinoma (vesicular stomatitis virus, Coxsackie virus B4 and respiratory syncytial virus (RSV)), feline Crandell-Rees kidney (CRFK) (feline herpes virus, feline corona virus (FIPV)) or Madin-Darby canine kidney (MDCK) (influenza A [H1N1; H3N2] and influenza B) cell cultures. Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID 50 of virus (1 CCID 50 being the virus dose to infect 50% of the cell cultures) in the presence of varying concentrations (100, 40, 8, 1.6 and 0.32 µM) of the test compounds. Viral cytopathicity was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. The anti-HIV activity and cytotoxicity of the compounds were evaluated against wild-type HIV-1 strain III B in MT-4 cell culture using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) method. MT-4 cells were suspended in culture medium at 1 × 10 5 cells/mL and infected with HIV at a multiplicity of infection of 0.02. Immediately after viral infection, 100 µL of the cell suspension was placed in each well of a flat-bottomed microtiter tray containing various concentrations of the test compounds. After four days of incubation at 37 °C, the number of viable cells was determined using the MTT method. Compounds were tested in parallel for cytotoxic effects in uninfected MT-4 cells.

A series of the benzylidene-bis-(4-hydroxycoumarin) derivatives 1-15 and fused benzopyranocoumarin derivatives 16-20 were synthesized and evaluated for their antiviral activities on a broad panel of DNA and RNA viruses. X-ray crystal structure analysis of 4-trifluoromethylphenyl-and 2-nitrophenyl derivatives 7 and 9 revealed intramolecular hydrogen bonding between hydroxyl and carbonyl oxygen atoms of two 4-hydroxycoumarin moieties resulting in the formation of two eight-membered rings. Accordingly, two 4-hydroxycoumarin moieties in compounds 7 and 9 are anti-disposed. The 4-bromobenzylidene derivative of bis(4-hydroxycoumarin) (3) exerted some inhibitory activity against HSV-1 (KOS), HSV-2 (G), vaccinia virus and HSV-1 TK -KOS (ACV r ) in the range of 9-12 μM at minimum cytotoxic concentration (MCC) greater than 20 μM, whereas the compounds 4-6, 8, and 20 exhibited rather pronounced anti-Feline Herpes Virus activity (EC 50 = 5-8.1 μM) but with MCC at only 4-7-fold higher than EC 50 values.

Tables S1 with hydrogen-bonded geometries, Figures S1-S4 presenting hydrogen bonds for compounds 7, 9, 16 and 18.

32 (H8, m, 1H), 7.34 (H3', d, J = 8.7, 1H), 7.36 (H6, m, 1H), 7.45 (H8", m, 1H), 7.49 (H6", m, 1H), 7.52 (H4', m, 1H), 7.61 (H7, m, 1H), 7.71 (H7", m, 1H), 8.03 (H5, b, 1H), 8.11 (H5

16-methoxybenzopyrano

DMSO-d 6 ): δ 3.93 (OCH 3 , s, 3H), 5.72 (H*, s, 1H), 6.75 (H6', m, 1H), 7.02 (H4', m, 1H), 7.08 (H5', m, 1H), 7.33 (H8, m, 1H), 7.36 (H6, m, 1H), 7.45 (H8", d, J = 8.2, 1H), 7.51 (H6", m, 1H), 7.60 (H7, m, 1H), 7.70 (H7", dd, J = 7.5, 1.7, 1H), 7.99 (H5", dd, J = 7.8, 1.5, 1H), 8.06 (H5, m, 1H), 12.26 ppm

Yield 69%; mp 315

OH); 1 H-NMR (DMSO-d 6 ): δ 5.79 (H*, b, 1H)

Elemental analysis. Calc. for C 25 H 13 O 8 N: C 65.93, H 2.88, N 3.07, Found: C 66.02, H 2.96

5.57 (H*, b, 1H), 6.36 (H5', d, J = 2.4, 1H), 6.59 (H3', d, J = 2.4, 1H), 7.27 (H8, d, J = 8.1, 1H), 7.34 (H6, m, 1H), 7.44 (H8", m, 1H), 7.48 (H6", m, 1H), 7.57 (H7, m, 1H), 7.69 (H7", dd, J = 7.5, 1.7, 1H), 8.00 (H5, m, 1H)

Elemental analysis. Calc. for C 27 H 18 O 8 : C 68

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Support of this study by the Ministry of Science, Education and Sport Republic of Croatia (project Nos. 125-0982464-2922 and 119-1193079-3069) is gratefully acknowledged.