Structure elucidation of anti-methicillin resistant Staphylococcus aureus (MRSA) flavonoids from balsam poplar buds

Accepted Manuscript Structure elucidation of anti-methicillin resistant Staphylococcus aureus (MRSA) flavonoids from balsam poplar buds François Simard, Charles Gauthier, Jean Legault, Serge Lavoie, Vakhtang Mshvildadze, André Pichette PII: DOI: Reference:

S0968-0896(16)30501-6 http://dx.doi.org/10.1016/j.bmc.2016.07.009 BMC 13122

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

9 May 2016 4 July 2016 5 July 2016

Please cite this article as: Simard, F., Gauthier, C., Legault, J., Lavoie, S., Mshvildadze, V., Pichette, A., Structure elucidation of anti-methicillin resistant Staphylococcus aureus (MRSA) flavonoids from balsam poplar buds, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.07.009

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Structure elucidation of anti-methicillin resistant Staphylococcus aureus (MRSA) flavonoids from balsam poplar buds

François Simarda, Charles Gauthiera,b, Jean Legaulta, Serge Lavoiea, Vakhtang Mshvildadzea and André Pichettea,*

a

Laboratoire LASEVE, Département des Sciences Fondamentales, Université du Québec à

Chicoutimi, 555, boul. de l'Université, Chicoutimi (Québec), Canada G7H 2B1

b

INRS-Institut Armand-Frappier, 531 boul. des Prairies, Laval (Québec), Canada H7V 1B7

*Corresponding author. Tel.: +1 418 545 5011; fax: +1 418 545 5012. E-mail address: [email protected] (A. Pichette).

1

GRAPHICAL ABSTRACT:

2

ABSTRACT:

There is nowadays an urgent need for developing novel generations of antibiotic agents due to the increased resistance of pathogenic bacteria. As a rich reservoir of structurally diverse compounds, plant species hold promise in this regard. Within this framework, we isolated a unique series of antibacterial flavonoids, named balsacones N–U, featuring multiple cinnamyl chains on the flavan skeleton. The structures of these compounds, isolated as racemates, were determined using extensive 1D and 2D NMR analysis in tandem with HRMS. Balsacones N–U along with previously isolated balsacones A–M were evaluated for their antibacterial activity against clinical isolates of methicillin resistant Staphylococcus aureus (MRSA). Several of the tested balsacones were potent anti-MRSA agents showing MIC values in the low micromolar range. Structureactivity relationships study highlighted some important parameters involved in the antibacterial activity of balsacones such as the presence of cinnamyl and cinnamoyl chains at the C-3 and C-8 positions of the flavan skeleton, respectively. These results suggest that balsacones could represent a potential novel class of naturally occurring anti-MRSA agents.

Keywords:

Flavonoids; dihydrochalcones; antibacterial activity; anti-MRSA; balsacones; structure-activity relationships

3

1. Introduction

The ever-increasing antibiotic resistance of bacteria is becoming a major health problem worldwide [1]. Among Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA) is a frequently reported microorganism in nosocomial diseases causing life-threatening and often fatal infections in patients [2]. In last years, some MRSA strains have even become resistant to last-resort antibiotics such as vancomycin, teicoplanin, linezolid and daptomycin. There is thus an urgent need to develop novel generations of antibiotics to fight against MRSA infections [3]. Owing to their huge chemical diversity, plant-derived natural products have emerged as a promising hope for the discovery of novel anti-MRSA agents [4, 5].

Based on the ethnomedical literature [6], we have undertaken the phytochemical investigation of balsam poplar (Populus balsamifera). Buds of trees from P. balsamifera are known as a traditional medicine largely used by North America Aboriginals for the treatment of various pathologies [7]. Earlier investigations on the chemical composition of P. balsamifera buds resulted in the identification of metabolites of various structures such as alkanes [8], fatty acids [8, 9], terpenes [8, 10], phenols [8, 11, 12], flavonoids [8, 11, 13], chalcones [8, 11-13], carbohydrates [14], and prostaglandins [15]. Our group has recently reported the presence of hydroxycinnamoylated dihydrochalcones and dihydrocinnamoylated flavans from P. balsamifera buds [16-18]. These polyphenolic compounds of the flavonoid family, named balsacones A–M, have been shown to exhibit significant in vitro antibacterial activity against S. aureus making them potentially promising new antibiotics.

4

Herein, we report the isolation and structural elucidation of novel structurally related flavonoids from an ethanolic extract of P. balsamifera buds. Balsacones N (1) and O (2) are two unprecedented hydroxycinnamate ester-substituted dihydrochalcones while balsacones P–U (3–8) are dihydrochalcones featuring two, three or four hydroxycinnamyl units. Similarly to the previously isolated balsacones, all isolates were obtained as racemic mixtures [16-18]. Balsacones N–U (1–8) together with balsacones A–M (9–22) (Figure 1) were evaluated in vitro for their antibacterial activity against S. aureus as well as against ten clinical isolate MRSA, and the structure-activity relationships (SAR) were established. As a preliminary investigation of the potential toxicity of balsacones, cytotoxicity was evaluated against human skin fibroblasts (WS1). We have also provided plausible pathways for the biosynthesis of these novel compounds.

2. Results and discussion

2.1. Isolation and structural elucidation of balsacones N–U

In two different experiments, frozen buds of P. balsamifera (1st and 2nd lot) were ground and extracted with 95% EtOH under reflux. The ethanolic extract was dried, suspended in MeOH and extracted with hexane. The dried MeOH fraction was then suspended in Et 2O and treated with water. In both experiments, the Et2O extract was fractionated by successive silica gel and reversed phase C18 silica gel column chromatographies (CC). Purification of the obtained fractions by semi-preparative reversed-phase C18 HPLC afforded compounds 1, 2, 5 and 6 (Figure 1) in the first experiment (1st lot) and compounds 3, 4, 7 and 8 in the second experiment (2nd lot). 5

Figure 1. Structures of balsacones N–U (1–8) and previously isolated balsacones A–M (9–22).

Balsacone N (1) was obtained as a greyish powder. Its molecular formula was established as C42H38O8 (24 degrees of unsaturation) based on the [M+Na] + quasimolecular ion at 693.2444 (calcd for C42H38O8Na , 693.2459) in the HRMS spectrum. The IR spectrum of 1 showed bands at 3332 (phenol), 1694 (carbonyl) and 1612 (carbonyl) cm−1. The

13

C NMR data (Table 1)

combined with information from DEPT135 suggested the presence of one ketone, one carboxylic carbon, five oxygenated aromatic quaternary carbons, six aromatic quaternary carbons, 15 sp2 methines, two sp3 methines and four methylenes. The HSQC spectrum indicated that some of these signals represented equivalent carbons. Detailed analysis of 1D 1H, 2D-COSY and HSQC experiments allowed to establish the presence of seven coupling systems (Figure 2): two monosubstituted aromatic rings (A and D) at H 7.24 (2H, m, H-2/H-6), 7.24 (2H, m, H-3/H-5), 7.14 (1H, m, H-4), 7.64 (2H, m, H-2''''/H-6''''), 7.44 (2H, m, H-3''''/H-5''''), and 7.44 (1H, m, 4''''), 6

two 1,4-disubstituted aromatic rings (B and C) at H 7.50 (2H, d, 8.5 Hz, H-2''/H-6''), 6.75 (2H, d, 8.5 Hz, H-3''/H-5''), 7.22 (2H, d, 8.3 Hz, H-2'''/H-6'''), and 6.75 (2H, d, 8.3 Hz, H-3'''/H-5'''), a CH2CH2 coupling system at H 2.90 (2H, dd, 8.7, 7.0 Hz, H-7) and 3.32 (2H, dd, 8.7, 6.9 Hz, H8), a trans-alkene at H 7.67 (1H, d, 16.1 Hz, H-7'''') and 6.50 (1H, d, 16.1 Hz, H-8''''), and a larger coupling system that includes six different signals: a trans-alkene at H 6.27 (1H, d, 15.8 Hz, H-7''') and 6.17 (1H, ddd, 15.8, 8.2, 5.8 Hz, H-8'''), two methines at H 4.61 (1H, d, 11.7 Hz, H-7'') and 3.43 (1H, m, H-8''), and two diastereotopic methylenes at H 4.15 (1H, dd, 11.0, 4.9 Hz, H-9''a) and 4.29 (1H, dd, 11.0, 3.3 Hz). HMBC correlations from H-7 to C 205.5 (C-9), and from H-2/H-6 to C 31.4 (C-7) indicated that the CH2CH2 coupling system was attached to the monosubstituted aromatic ring A and to a ketone thus resulting in a dihydrocinnamoyl unit (Figure 2). The trans alkene was found to be linked to the monosubstituted aromatic ring D and to an ester based on HMBC correlations from H-7'''' and H-3'''' to C 135.6 (C-1''''), and from H8'''' to C 167.0 (C-9'''') thus resulting in a cinnamate unit. The latter was confirmed by the formation of a fragment at 523 m/z [M−C9H7O2]+ in the APCI-MS spectrum (Figure 2). HMBC correlations from H-7''' and H-3''' to C 130.4 (C-1''') indicated that the other trans-alkene was attached to the disubstituted aromatic ring C. Furthermore, HMBC correlations from H-7'' and H3'' to C 135.7 (C-1''), and from H-9'' to C-9'''' indicated that the six signals coupling system was also bonded to the disubstituted aromatic ring B and to the cinnamate unit (D). Finally, the dihydrocinnamoyl unit (A) and the six signals coupling system were both found to be attached to the same penta-substituted aromatic ring according to HMBC correlations from H 6.07 (1H, s, H-5') to C-9 and C 110.2 (C-3'), and from H-7'' to C-3'. Assignations of this penta-substituted aromatic ring were determined according to the following HMBC correlations: from H-5' to C 160.6 (C-6'), 162.9 (C-4'), 105.0 (C-1'), and C-3', and from H-7'' to C-4' and C 165.7 (C-2'). 7

Based on the above evidences, balsacone N (1) was characterized as (E)-5-(4-hydroxyphenyl)-2[(4-hydroxyphenyl)(2,4,6-trihydroxy-3-(3-phenylpropanoyl)phenyl)methyl]pent-4-en-1-yl cinnamate.

8

Table 1. 1H and 13C NMR spectroscopic data of compounds 1 and 2 (acetone-d6,  in ppm, J in Hz). no.

1

C 1 2/6 3/5 4 7 8 9 4-OCH3 1' 2' 3' 4' 5' 6' 2'-OH 4'-OH 6'-OH 1'' 2''/6'' 3''/5'' 4'' 7'' 8'' 9''

142.9 129.2 129.1 126.5 31.4 46.5 205.5 105.0 165.7 110.2 162.9 95.4 160.6 135.7 130.8 115.5 156.2 42.1 39.1 65.9

4''-OH 1''' 2'''/6''' 3'''/5''' 4''' 7''' 8''' 9'''

130.4 128.0 116.1 157.4 132.1 126.2 34.6

4'''-OH 1'''' 2''''/6'''' 3''''/5'''' 4'''' 7''''

135.6 129.0 129.7 131.0 144.7

2

H 7.24 (m) 7.24 (m) 7.14 (m) 2.90 (m) 3.32 (m) 6.07 (s) 14.57 (s) 9.43 (s) 9.52 (s) 7.50 (d, 8.5) 6.75 (d, 8.5) 4.61 (d, 11.7) 3.43 (m) 4.29 (dd, 11.0, 3.3) 4.15 (dd, 11.0, 4.9) 8.07 (br s) 7.22 (d, 8.3) 6.75 (d, 8.3) 6.27 (d, 15.8) 6.17 (ddd, 15.8, 8.2, 5.8) 2.40 (ddd, 14.0, 5.8, 3.0) 2.23 (dt, 14.0, 8.2) 8.32 (br s) 7.64 (m) 7.44 (m) 7.44 (m) 7.67 (d, 16.1)

C 134.7 130.1 114.5 158.8 30.6 46.8 205.7 55.4 105.0 165.7 110.3 162.8 95.4 160.5

135.7 130.8 115.5 156.3 42.1 39.1 65.9 130.4 128.0 116.1 157.4 132.1 126.2 34.6

135.6 129.0 129.7 131.0 144.7

H 7.13 (d, 8.7) 6.80 (d, 8.7) 2.84 (m) 3.28 (m) 3.73 (s) 6.04 (s) 14.60 (s) 9.42 (s) 9.50 (s) 7.50 (d, 8.6) 6.75 (d, 8.6) 4.60 (d, 11.7) 3.43 (m) 4.29 (dd, 11.0, 3.3) 4.15 (dd, 11.1, 5.0) 8.08 (br s) 7.23 (d, 8.5) 6.76 (d, 8.5) 6.27 (d, 15.9) 6.17 (ddd, 15.7, 7.9, 6.0) 2.40 (m) 2.23 (dt, 14.0, 8.5) 8.32 (br s) 7.65 (m) 7.44 (m) 7.44 (m) 7.66 (d, 16.0) 9

8'''' 9''''

119.4 167.0

6.50 (d, 16.1) -

119.4 167.0

6.50 (d, 16.0) -

Figure 2. HMBC, COSY and MS key data for identification of compound 1

Balsacone O (2) was isolated as a greyish powder. Its molecular formula was established as C43H40O9 (24 degrees of unsaturation) based on the [M+Na] + quasi-molecular ion at 723.2553 (calcd for C43H40O9Na, 723.2565) in the HRMS spectrum. The 1H and

13

C NMR spectra of 2

were almost identical to those of 1 but with the presence of additional signals corresponding to a methoxy group at H 3.73 (3H, s, 4-OCH3) and C 55.4 (4-OCH3). This group was determined to be positioned at C-4 based on the HMBC correlations from the methoxy protons to C 158.9 (C4), and from H 7.13 (2H, d, J = 8.6 Hz, H-2) to C-4. Based on the above evidences, balsacone O (2) was characterized as (E)-5-(4-hydroxyphenyl)-2-[(4-hydroxyphenyl)(2,4,6-trihydroxy-3-(3(4-methoxyphenyl)propanoyl)phenyl)methyl]pent-4-en-1-yl cinnamate.

The stereochemistry of balsacones N (1) and O (2) could not be determined in this work. First, single bonds of the molecule did not rotate as freely as expected as demonstrated by some definite coupling constants (ex., 3JH-8'',H-9''' and 3JH8''',H9'''). Notwithstanding this rigidity, no 10

through-space correlations were observed between the different spin systems preventing any deduction regarding the relative stereochemistry. Furthermore, ECD spectra of both compounds did not show any Cotton effects and measurement of the optical rotations of 1 and 2 gave [α]D values of 0. The racemic nature of both compounds was confirmed by chiral analytical HPLC analysis.

Balsacone P (3) was isolated as a reddish powder and its molecular formula was established as C33H30O6 based on the [M+Na]+ quasimolecular ion peak at m/z 545.1943 (calcd for C33H30O6Na, 545.1935) in the HRMS spectrum. The IR spectrum showed bands at 3348 and 1598 cm−1, due to phenol and carbonyl functions, respectively. The 13C NMR data (Table 3) along with information from DEPT135 spectrum indicated the presence of one carbonyl, five oxygenated aromatic quaternary carbons, five aromatic quaternary carbons, ten sp2 methines, two sp3 methines and four sp3 methylenes. The 1D 1H and HSQC spectra indicated that some of these signals represented equivalent carbons. Detailed analysis of 1D 1H, 2D-COSY and HSQC experiments showed signals for two 1,4-disubstituted aromatic rings at δH 7.29 (2H, m, H-2'/6'), 6.90 (2H, d, 8.5 Hz, H-3'/5'), 7.23 (2H, d, 8.6 Hz, H-2'''/6'''), and 6.77 (2H, d, 8.6 Hz, H-3'''/5'''), and one monosubstituted aromatic ring at δH 7.28 (2H, m, H-2''/6''), 7.27 (2H, m, H-3''/5'') and 7.17 (1H, m, H-4''). The other coupling systems observed on the COSY spectrum were reminiscent of balsacone J (18) previously isolated from P. balsamifera buds [18]. Indeed, 1H and 13C spectra of 3 showed signals accounting for a flavan skeleton substituted by a p-hydroxycinnamyl moiety at position 3 of ring C and for a dihydrocinnamoyl moiety. The discrepancies between the spectral data of 3 and balsacone J (18) were attributed to a different position for the dihydrocinnamoyl moiety on ring A. HMBC correlations from δH 4.79 (1H, d, 8.8 Hz, H-2), δH 2.33 (1H, dd, 16.3, 10.3 Hz, H-4ax), δH 2.84 (1H, dd, 16.3, 4.7 Hz, H-4eq), and δH 5.94 (1H, s, H-8) to δC 162.3 (C-9), 11

and a W-coupling between H-8 and δC 205.5 (C-9'') observed in the HMBC spectrum indicated that the dihydrocinnamoyl moiety was attached at position 6 of ring A. Based on the previous observations, balsacone P (3) was characterized as 2,3-trans-6-dihydrocinnamoyl-3-(4hydroxycinnamyl)-5,7,4'-trihydroxyflavan.

Table 2. 1H NMR spectroscopic data of compounds 3-6 ( in ppm, J in Hz) 3a 4a 5b 6a No. 2 4.79 (d, 8.7) 4.76 d (9.6) 4.82 (d, 9.1) 4.86 (d, 9.2) 3 2.21 (m) 2.28 m 2.27 (m) 2.32 (m) c 4eq 2.84 (dd, 16.3, 4.7) 2.96 (dd, 16.2, 5.2) 2.75 (m) 2.91 (dd, 16.1, 4.7) 4ax 2.33 (dd, 16.3, 10.3) 2.46 (dd, 16.3, 10.9) 2.31 (m) 2.41 (dd, 16.1, 10.8) 5-OH 14.10 (s) 7-OH 9.80 (br s) 14.32 (s) 14.09 (s) 14.25 (s) 8 5.94 (s) c 2'/6' 7.29 (m) 7.37 (d, 8.0) 7.32 (d, 8.1) 7.40 (d, 8.2) 3'/5' 6.90 (d, 8.5) 6.94 (d, 8.0) 6.81 (d, 8.1) 6.94 (d, 8.2) c c 2''/6'' 7.28 (m) 6.69 (s) 7.12 (m) 6.72 (s) 3''/5'' 7.27c (m) 6.69 (s) 6.76 (m) 6.72 (s) c 4'' 7.17 (m) 7.11 (m) 7'' 2.99 (m) 2.75 (t, 8.1) 2.75 (m) 2.77 (m) 8'' 3.42 (m) 3.15 (m) 3.13 (m) 3.17 (m) 4-OCH3 3.72 (s) 3.74 (s) 2'''/6''' 7.23 (d, 8.6) 7.20 (d, 8.0) 7.09 (d, 8.3) 7.21 (d, 8.2) 3'''/5''' 6.77 (d, 8.6) 6.77 (d, 8.0) 6.73 (d, 8.3) 6.82 (d, 8.2) 7''' 6.29 (d, 15.4) 6.27 (d, 15.5) 4.94 (d, 6.3) 4.94 (d, 6.8) 8''' 6.02 (dt, 15.4, 7.2) 6.01 (dt, 15.5, 7.2) 3.97 (m) 4.09 (m) 9'''a 2.17 (m) 2.15 (m) 2.48c (m) 2.84 (dd, 16.3, 5.0) 9'''b 2.00 (m) 1.96 (m) 2.60 (dd, 16.3, 4.8) 2.60 (dd, 16.3, 7.6) 2''''/6'''' 7.16 (d, 8.2) 7.12 (d, 8.6) 7.16 (d, 8.2) 3''''/5'''' 6.74 (d, 8.2) 6.67 (d, 8.6) 6.75 (d, 8.2) 7'''' 6.36 (d, 15.8) 6.18 (d, 15.7) 6.25 (d, 15.5) 8'''' 6.17 (dt, 15.8, 6.2) 5.88 (dt, 15.1, 7.2) 5.98 (dt, 15.5, 7.2) 9''''a 3.52 (m) 1.95 (m) 2.19 (m) 9''''b 2.06 (m) 2.04 (m) a b c Recorded in acetone-d6. Recorded in DMSO-d6. Overlapped signals

12

Table 3. 13C NMR spectroscopic data of compounds 3-6 ( in ppm) No. 3a 4a 5b 6a 2 83.6 83.7 82.2 83.8 3 38.3 37.9 35.9 37.8 4 24.9 26.6 24.6 25.9 5 164.9 161.3 157.9 159.3 6 105.2 106.9 100.1 101.5 7 160.3 163.5 161.4 163.4 8 95.2 105.3 104.0 105.5 9 162.3 157.4 155.5 156.9 10 102.5 101.9 100.6 101.8 1' 131.4 131.0 129.0 131.2 2'/6' 129.5 129.8 128.6 129.8 3'/5' 116.1 116.4 115.2 116.4 4' 158.4 158.6 157.5 158.6 1'' 142.9 133.9 140.6 133.9 2''/6'' 129.3 129.8 127.9 129.8 3''/5'' 129.1 114.3 127.8 114.4 4'' 126.6 158.6 125.5 158.7 7'' 31.4 30.3 29.8 30.4 8'' 46.4 46.1 44.2 46.1 9'' 205.5 205.4 204.3 205.8 4-OCH3 55.3 55.3 1''' 130.1 130.1 129.0 130.7 2'''/6''' 128.1 128.1 127.5 129.0 3'''/5''' 116.1 116.1 114.9 116.0 4''' 157.6 157.6 157.0 158.2 7''' 132.6 132.5 81.4 83.2 8''' 124.6 124.4 65.2 67.6 9''' 36.3 36.2 25.7 27.3 1'''' 130.4 128.0 130.1 2''''/6'''' 127.9 127.0 128.1 3''''/5'''' 116.1 115.2 116.1 4'''' 157.3 156.6 157.6 7'''' 130.1 131.4 132.7 8'''' 126.1 123.1 124.5 9'''' 26.3 34.6 36.0 a Recorded in acetone-d6. bRecorded in DMSO-d6. cOverlapped signals

Balsacone Q (4) was isolated as a brownish powder and its molecular formula was established as C43H40O8 based on the [M+H]+ quasimolecular ion peak at m/z 685.2778 (calcd for C43H41O8,

13

685.2796) in the HRMS spectrum. The IR spectrum showed bands at 3335 and 1610 cm–1, due to phenol and carbonyl functions, respectively. The 13C NMR data (Table 3) along with information from DEPT135 spectrum indicated the presence of one carbonyl, seven oxygenated aromatic quaternary carbons, seven aromatic quaternary carbons, 12 sp2 methines, two sp3 methines and five methylenes. Spectral data of 4 showed many similarities with those of iryantherin-D (19) [19], which was recently identified in P. balsamifera buds [18]. Indeed, signals accounting for a flavan skeleton bearing a 4-methoxydihydrocinnamoyl moiety at position 8 and a phydroxycinnamyl moiety at position 3 were present in the 1H and 13C NMR spectra of 4. Only the signal corresponding to the hydrogen in position 6 of the flavan skeleton was missing, thus suggesting the presence of an additional substituent at this position. Furthermore, detailed analysis of 1D 1H, 2D-COSY and HSQC experiments (Figure 3) also showed the presence of a 1,4-disubstituted aromatic ring at δH 7.16 (2H, d, 8.2 Hz, H-2''''/H-6'''') and 6.74 (2H, d, 8.2 Hz, H-3''''/H-5''''), and a trans-allyl at δH 6.36 (1H, d, 15.8 Hz, H-7''''), 6.17 (1H, dt, 15.7, 6.2, H-8''''), and 3.52 (2H, m, H-9''''). HMBC correlations from H-7'''' to δC 127.9 (C-2''''/C-6''''), and from H2'''' to δC 157.3 (C-4'''') indicated that this 1,4-disubstituted aromatic ring was oxygenated and linked to the allyl thus resulting in another p-hydroxycinnamyl moiety. The attachment of this moiety at position 6 of the flavan skeleton was confirmed by the HMBC correlations from H-9'''' to δC 161.3 (C-5) and δC 163.5 (C-7). The exact substitution pattern on ring A of the flavan skeleton was also based on the following HMBC correlations: from δH 4.76 (1H, d, 9.6 Hz, H-2), 2.46 (1H, dd, 16.2, 10.9 Hz, H-4ax), and 2.96 (1H, dd, 16.2, 5.2 Hz, H-4eq) to δC 157.4 (C-9); from H-4ax and H-4eq to C-5 and C-9. Based on the above evidences, balsacone Q (4) was characterized

as

8-dihydrocinnamoyl-3-(4-hydroxycinnamyl)-6-(4-hydroxycinnamyl)-5,7,4'-

trihydroxyflavan.

14

Figure 3. HMBC, COSY, NOESY key data for identification of compounds 4, 5 and 7a.

Balsacone R (5) was obtained as a brownish powder and its molecular formula was established as C42H38O8 based on the [M+H]+ quasimolecular ion peak at m/z 671.2624 (calcd C42H39O8, 671.2639) in the HRMS spectrum. The IR spectrum showed bands at 3337 and 1612 cm−1, due to phenol and carbonyl functions, respectively. The 13C NMR data (Table 3) along with information from DEPT135 spectrum indicated the presence of one carbonyl, six oxygenated aromatic quaternary carbons, seven aromatic quaternary carbons, 11 sp2 methines, four sp3 methines and five methylenes. Detailed analysis of 1D 1H, 2D-COSY and HSQC experiments showed the presence of three 1,4-disubstituted aromatic rings at δH 7.32 (2H, d, 8.1 Hz, H-2'/H-6'), 6.81 (2H, d, 8.1 Hz, H-3'/H-5'), 7.12 (2H, d, 8.6, H-2''''/H-6''''), 6.67 (2H, d, 8.6 Hz, H-3''''/H-5''''), 7.09 (2H, d, 8.3, H-2'''/H-6''') and 6.73 (2H, d, 8.3 Hz, H-3'''/H-5'''), one monosubstituted aromatic ring at δH 15

7.12 (2H, m, H-2''/H-6''), 6.76 (2H, m, H-3''/H-5'') and 7.11 (1H, m, H-4''), and an allyl at δH 6.18 (1H, d, 15.4 Hz, H-7''''), 5.88 (1H, dt, 15.4, 7.2, H-8''''), 2.06 (1H, m, H-9''''a), and 1.94 (1H, m, H-9''''b). As shown by HMBC correlations from H-2'''' to δC 131.4 (C-7''''), one of the 1,4disubstituted aromatic rings was linked to an allyl coupling system thus resulting in a phydroxycinnamyl unit. The monosubstituted aromatic ring was shown to be part of a dihydrocinnamoyl moiety based on HMBC correlations from δH 2.75 (2H, m, H-7'') to δC 204.3 (C-9''), 140.6 (C-1''), and 127.9 (C-2''). The two remaining 1,4-disubstituted aromatic rings were both linked to two coupling systems at δH 2.31 (1H, m, H-4ax), 2.75 (1H, m, H-4eq), 2.27 (1H, m, H-3), 4.82 (1H, d, 9.1 Hz, H-2), and at δH 2.48 (1H, m, H-9'''a), 2.60 (1H, dd, 16.3 and 4.8 Hz, H9'''b), 3.97 (1H, m, H-8''') and 4.94 (1H, d, 6.3 Hz, H-7'''). The first spin system was reminiscent of the signals accounting for the 3,4-dihydro-2H-pyran ring in 3 and 4 thus suggesting the presence of the same flavan skeleton. The second spin system was proposed to complete another 3,4-dihydro-2H-pyran ring with an oxygenated function at position 8''', as shown by the deshielded chemical shift of C-8'''. HMBC correlations from H-4ax, H-4eq, H-9'''a, H-9'''b and H7''' to δC 157.9 (C-5), and from H-9'''a and H-9'''b to δC 100.1 (C-6) indicated that this second 3,4dihydro-2H-pyran ring was fused with ring A of the flavan skeleton at positions 5 and 6 resulting in a 3,4-dihydro-2H-pyranoflavan skeleton. The signal of a chelated phenolic proton at δH 14.09 (1H, s, 7-OH) was observed on the 1H NMR spectrum and it was determined that this phenol was also situated on ring A based on HMBC correlations from 7-OH to δC 104.0 (C-8), 161.4 (C-7) and C-6. The carbonyl of the dihydrocinnamoyl unit was considered to be responsible for the chelation of this phenolic group through hydrogen bonding thus suggesting it was also attached to ring A on the final available position. A NOESY correlation between H-2'/6' and δH 3.12 (2H, m, H-8'') confirmed that the dihydrocinnamoyl unit was positioned at C-8. Finally, it was determined that the remaining p-hydroxycinnamyl unit was attached in position 3 as shown by the HMBC 16

correlation from H-9''''a to δC 35.9 (C-3) with thus resulting in the complete assignation for balsacone R (5) presented in Tables 2–3.

Balsacone S (6) was obtained as a brownish powder and its molecular formula was established as C43H40O9 based on the [M+Na]+ quasimolecular ion peak at m/z 723.2562 (calcd C43H40O9Na, 723.2565) in the HRMS spectrum. The IR spectrum showed bands at 3370 and 1611 cm−1, due to phenol and carbonyl functions, respectively. The 13C NMR data (Table 3) along with information from DEPT135 spectrum indicated the presence of one carbonyl, seven oxygenated aromatic quaternary carbons, seven aromatic quaternary carbons, ten sp2 methines, four sp3 methines and five methylenes. Spectral data of 6 were very similar to those of 5 except for the presence of an additional signal at δH 3.74 (3H, s, 4''-OCH3) corresponding to a methoxy group and the presence of a fourth 1,4-disubstituted aromatic ring instead of the monosubstituted ring present in 5. HMBC correlations from 4''-OCH3 and δH 6.72 (4H, s, H-2''/6'' and H-3''/5'') to δC 158.6 (C-4'') revealed that the additional methoxy group was positioned at C-4'' resulting in the structure presented in Figure 1 for balsacone S (6).

The presence of two enantiomers was also detected using chiral analytical HPLC for 4. Compound 3, 5 and 6 could not be separated on the chiral support used in this work but the absence of Cotton effect (CE) on its ECD spectrum suggested it was also racemic. Further purification of 4 was not undertaken because isolated racemic mixtures were not available in sufficient quantities. Relative configuration of 3-6 was established as trans for positions 2,3 based on the 3JH-2,H-3 coupling constants of 8.7, 9.6, 9.1 and 9.2 Hz, respectively. Such a high J value indicated that the additional hydroxycinnamyl moiety linked at position 3 of the flavan unit strongly reduced the conformational mobility [20, 21]. For compounds 5 and 6, relative 17

configuration at positions 7'''',8'''' was also established as trans based on the 3JH-7'''',H-8'''' coupling constant of 6.3 and 6.8 Hz, respectively.

Balsacone T1 (7a) was obtained as a brownish powder and its molecular formula was established as C51H46O8 based on the [M+H]+ quasimolecular ion peak at m/z 787.3245 (calcd C51H47O8, 787.3265) in the HRMS spectrum. The IR spectrum showed bands at 3216 and 1598 cm−1, due to phenol and carbonyl functions, respectively. The

13

C and 1H data (Tables 4–5) along with

information from DEPT135 and HSQC spectra indicated the presence of one carbonyl, seven oxygenated aromatic quaternary carbons, eight aromatic quaternary carbons, 15 sp2 methines, four sp3 methines and six methylenes. Detailed analysis of 1D 1H, 2D-COSY and HSQC experiments showed the presence of four 1,4-disubstituted aromatic rings and one monsubstituted aromatic ring. The monosubstituted aromatic ring was implicated in a dihydrocinnamoyl unit in the same manner as for 6. Two of the 1,4-disubstituted aromatic rings were both linked to very similar coupling systems at δH 2.40 (1H, m, H-4a), 2.80 (1H, m, H-4b), 2.26 (1H, m, H-3), 4.86 (1H, d, 9.4 Hz, H-2), 2.39 (1H, m, H-9'''a), 2.82 (1H, m, H-9'''b), 2.22 (1H, m, H-8''') and 4.88 (1H, d, 8.8 Hz, H-7'''). Signals accounting for these two coupling systems suggested the presence of the same 3,4-dihydropyranoflavan skeleton as in 6. The only difference was the absence of the hydroxy at position 8''' (δH 2.22). As shown by HMBC correlations from δH 7.22 (2H, d, 8.4 Hz, H-2, p-hydroxycinnamyl 2) to δC 132.6 (C-7, p-hydroxycinnamyl 2) and from δH 7.12 (2H, m, H2, p-hydroxycinnamyl 1) to δC 132.7 (C-7, p-hydroxycinnamyl 1), the two remaining 1,4disubstituted aromatic rings were linked to an allyl coupling system thus resulting in two phydroxycinnamyl units (Figure 3). These two remaining p-hydroxycinnamyl units were found to be attached at positions 3 and 8''' as shown by HMBC correlations from δH 2.03 (1H, m, H-9a, phydroxycinnamyl 1) and 5.94 (1H, m, H-8, p-hydroxycinnamyl 1) to δC 37.8 (C-3) and from δH 18

2.03 (1H, m, H-9a, p-hydroxycinnamyl 2), 2.19 (1H, m, H-9b, p-hydroxycinnamyl 2) and 6.03 (1H, d, 15.8 Hz, H-8, p-hydroxycinnamyl 2) to δC 83.6 (C-7'''). The signal of a chelated phenolic proton at δH 14.24 (1H, s, 7-OH) was also observed on the 1H NMR spectrum of 7a indicating the presence of a phenol on ring A. The position of the phenol was established as 7 based on HMBC correlations from 7-OH to δC 102.6 (C-6), 163.3 (C-7) and 105.3 (C8). A 2D NOESY correlation between δH 7.36 (2H, d, 8.4 Hz, H-2'/6') and 3.20 (2H, m, H-8'') allowed to confirm that the dihydrocinnamoyl unit was at position 8 thus resulting in the assignation presented in tables 4–5 for balsacone T1 (7a).

19

Table 4. 1H NMR spectroscopic data of compounds 7a, 7b, 8a and 8b in acetone-d6 (δH in ppm, J in Hz) No. 2 3 4a 4b 2'/6' 3'/5' 2''/6'' 3''/5'' 4'' 7'' 8'' 2'''/6''' 3'''/5''' 7''' 8''' 9'''

7a 7b 8a 8b 4.86 (d, 9.6) 4.82 (d, 9.1) 4.83 (d, 8.8) 4.81 (d, 9.0) 2.25 (m)a 2.29 (m)a 2.27 (m)a 2.29 (m)a a a a 2.80 (m) 2.84 (m) 2.80 (m) 2.84 (m)a 2.39 (m)a 2.36 (m)a 2.40 (m)a 2.36 (m)a 7.36 (d, 8.3) 7.37 (d, 8.1) 7.37 (d, 8.0) 7.37 (d, 8.1) 6.90 (d, 8.3) 6.91 (d, 8.1) 6.93 (d, 8.0) 6.93 (d, 8.1) 6.84 (d, 7.6) 6.82 (d, 7.4) 6.71 (s) 6.70 (s) a a 7.14 (m) 7.14 (m) 6.71 (s) 6.70 (s) 7.09 (m) 7.09 (m) a a a 2.83 (m) 2.82 (m) 2.75 (m) 2.75 (m) 3.21 (m) 3.19 (m) 3.15 (m) 3.15 (m 7.26 (d, 8.1) 7.25 (d, 8.0) 7.25 (d, 8.1) 7.26 (d, 8.1) 6.87 (d, 8.1) 6.87 (d, 8.0) 6.87 (d, 8.1) 6.87 (d, 8.1) 4.88 (d, 8.7) 4.87 (d, 8.2) 4.86 (d, 7.7) 4.86 (d, 8.2) 2.22 (m)a 2.17 (m)a 2.20 (m)a 2.17 (m)a a a a 2.82 (m) 2.84 (m) 2.82 (m) 2.84 (m)a 2.40 (m) 2.36 (m) 2.38 (m)a 2.36 (m)a p-hydroxycinnamyl 1 2 7.13 (d, 8.2) 7.12 (d, 8.1) 7.13 (d, 8.2) 7.13 (d, 8.3) 3 6.73 (d, 8.2) 6.75 (d, 8.1) 6.73 (d, 8.2) 6.75 (d, 8.3) 7 6.22 (d, 15.4) 6.21 (d, 15.4) 6.21 (d, 15.5) 6.21 (d, 15.5) 8 5.94 (dt, 15.4, 7.2) 5.93 (dt, 15.4, 7.2) 5.93 (dt, 15.5, 7.2) 5.94 (dt, 15.5, 7.1) 9a 2.19 (m)a 2.17 (m)a 2.19 (m)a 2.17 (m)a 9b 2.03 (m)a 2.00 (m)a 2.02 (m)a 2.01 (m)a p-hydroxycinnamyl 2 2 7.22 (d, 8.1) 7.22 (d, 8.1) 7.22 (d, 8.2) 7.22 (d, 8.3) 3 6.77 (d, 8.1) 6.77 (d, 8.1) 6.77 (d, 8.2) 6.77 (d, 8.3) 7 6.30 (d, 15.4) 6.30 (d, 15.5) 6.29 (d, 15.3) 6.29 (d, 15.4) 8 6.03 (dt, 15.4, 7.1) 6.03 (dt, 15.5, 7.0) 6.02 (dt, 15.3, 7.1) 6.03 (dt, 15.4, 7.1) 9a 2.19 (m)a 2.18 (m)a 2.19 (m)a 2.18 (m)a 9b 2.03 (m)a 2.01 (m)a 2.03 (m)a 2.02 (m)a 7-OH 14.24 (s) 14.25 (s) 14.30 (s) 14.30 (s) 4''3.73 (s) 3.73 (s) OCH3 a Overlapped signals

20

Table 5. 13C and 1H NMR spectroscopic data of compounds 7a, 7b, 8a and 8b in acetone-d6 ( in ppm) No. 7a 2 83.7 3 37.8 4 25.6 5 159.9 6 102.6 7 163.2 8 105.2 9 156.7 10 101.8 1' 131.2 2'/6' 129.7 3'/5' 116.3 4' 158.6 1'' 142.1 2''/6'' 128.9 3''/5'' 129.0 4'' 126.3 7'' 31.2 8'' 45.8 9'' 205.5 1''' 131.6 2'''/6''' 129.2 3'''/5''' 116.1 4''' 158.3 7''' 83.6 8''' 38.2 9''' 24.5 p-hydroxycinnamyl 1 1 130.1 2 128.1 3 116.1 4 157.6 7 132.6 8 124.5 9 36.1 p-hydroxycinnamyl 2 1 130.0 2 128.1 3 116.1 4 157.6 7 132.6

7b 83.7 37.9 25.8 160.0 102.7 163.2 105.2 156.6 101.8 131.1 129.7 116.3 158.6 142.1 128.9 129.0 126.3 31.2 45.8 205.5 131.5 129.2 116.1 158.4 83.7 38.3 24.7

8a 83.7 37.7 25.7 159.9 102.6 163.3 105.2 156.7 101.8 131.1 129.7 116.3 158.6 133.9 129.8 114.3 158.6 30.4 46.1 205.7 131.5 129.2 116.1 158.4 83.6 38.2 24.6

8b 83.7 37.8 25.9 159.9 102.7 163.2 105.2 156.6 101.8 131.2 129.8 116.3 158.6 133.9 129.8 114.3 158.6 30.3 46.0 205.7 131.6 129.2 116.1 158.3 83.7 38.3 24.7

130.1 128.1 116.1 157.6 132.7 124.5 35.9

130.0 128.1 116.1 157.6 132.6 124.5 36.1

130.1 128.1 116.1 157.6 132.7 124.5 36.0

130.1 128.1 116.1 157.6 132.6

130.0 128.1 116.1 157.6 132.6

130.1 128.1 116.1 157.6 132.6 21

8 9 4''-OCH3

124.7 36.4 -

124.7 36.3 -

124.7 36.4 55.3

124.7 36.3 55.3

Purification of 7a also yielded a second compound (7b, balsacone T2), for which spectral data (1H, 13C, IR, MS) were very similar to those of 7a (Tables 4–5). Indeed, detailed analysis of 2DCOSY, HSQC, HMBC and 2D-NOESY experiments of 7b led to the same planar structure than that of 7a. Both compounds were found to be racemic using analytical chiral HPLC. The relative configuration of both compounds were established as trans for positions 2,3 and 7''',8''' based on coupling constants ranging from 7.6 to 9.4 Hz for 3JH-2,H-3 and 3J H-7''',H-8'''. Since both compounds were obtained as racemic mixtures, further chiroptical analysis was impossible. However, it seems likely that the two enantiomers of racemic 7b were diastereomers of those of racemic 7a. From the 1H NMR data, the largest difference between 7a and 7b were obtained for 3JH-7''',H-8''' (8.8 Hz for 7a and 8.2 Hz for 7b) indicating slight differences of configuration between the two compounds.

Balsacone U1 (8a) was obtained as a brownish powder and its molecular formula was established as C52H48O9 based on the [M+H]+ quasimolecular ion peak at m/z 817.3370 (calcd C52H49O9, 817.3371) in the HRMS spectrum. The IR spectrum showed bands at 3265 and 1608 cm−1, due to phenol and carbonyl functions, respectively. The 13C NMR data (Table 5) along with information from DEPT135 spectrum indicated the presence of one carbonyl, eight oxygenated aromatic quaternary carbons, eight aromatic quaternary carbons, 14 sp2 methines, four sp3 methines, six methylenes and one methoxy. Spectral data of 8a were very similar to those of 7a except for the presence of an additional signal at δH 3.73 (3H, s, 4''-OCH3) corresponding to a methoxy group and the presence of a fifth 1,4-disubstituted aromatic ring instead of the monosubstituted ring 22

present in 7a. HMBC correlations from 4-OCH3 and δH 6.71 (4H, s, H-2''/6'' and H-3''/5'') to δC 158.7 (C-4'') revealed that the additional methoxy group was at C-4'' resulting in the structure presented in Figure 1 for balsacone U1 (8a).

Purification of 8a also yielded a second compound (8b, balsacone U2). Again, spectral data (1H, 13

C, IR, MS) of 8a and 8b were very similar (Tables 4–5) and the same planar structures were

found for both compounds. They were also found to be racemic and their relative configurations were the same as for 7a and 7b. Slight differences between the values of 3JH-7''',H-8''' (7.7Hz for 8a and 8.2 Hz for 8b) were also observed thus suggesting they could also be diastereomers.

2.2. Plausible biosynthetic pathways for balsacones N-U

We have proposed a possible biosynthetic pathway for balsacones N (1) and O (2) as shown in Figure 4. Coumaryl cinnamate has been proposed as a likely precursor since it was previously identified in bud exudates from Populus species [22]. Addition of hydroxycinnamoyl and dihydrochalcone moieties on the double bound at C-7''-C-8'' followed by reduction of the ketone at C-9''' would lead to 1 and 2, depending on the substitution pattern at position 4 on the dihydrochalcone moiety. Dihydrochalcones are also known components of P. balsamifera buds [23].

23

Figure 4. Possible biosynthetic pathway for compounds 1 and 2

We proposed in our previous work that the dihydro-2H-pyran ring of balsacones originated from a 1,4-Michael addition of hydroxycinnamoylated dihydrochalcones [18]. The formation of the hydroxycinnamyl substituted dihydro-2H-pyran rings in 3–10 is proposed to proceed via the same

pathway

(Figure

5):

non-stereospecific

1,4-Michael

addition

followed

by

hydroxycinnamoylation and reduction of both carbonyls from the former hydroxycinnamoyl units. In the case of 3, a 1,4-Michael addition would occur between the phenol at C-4' and C-7'' and would be followed by hydroxycinnamoylation at C-8'' and reduction at both C-9'' and C-9'''

24

(Figure 5). The pathway leading to compounds 4–8 would also start with the formation of a hydroxycinnamyl substituted dihydro-2H-pyran ring but with the 1,4-Michael addition occurring between the phenol at C-2' and C-7''. Additional hydroxycinnamoylation at C-5' followed by reduction at C-9''' would explain the formation of 4. Another 1,4-Michael addition occurring between the phenols at C-4' and C-7''' of the supplementary dihydrocinnamoyl at C-5', followed by hydroxylation at C-8''' and reduction at C-9''' would lead to 5 and 6. Finally, the formation of 7a, 7b, 8a and 8b could be explained by the formation of a second hydroxycinnamyl substituted dihydro-2H-pyran ring with the supplementary dihydrocinnamoyl unit at C-5' (Figure 5).

25

Figure 5. Possible biosynthetic pathways for compounds 3–8

26

2.3. SAR of anti-MRSA balsacones

The anti-S. aureus (strain ATCC 25923) and anti-MRSA activities of isolated compounds (1–8) along with previously isolated balsacones 9–22 were evaluated in vitro in terms of their minimum inhibitory concentrations (MICs, µM), which was determined using the liquid microdilution method as previously reported [24]. The ten MRSA strains used in this study were clinical isolates obtained from the Chicoutimi hospital in Saguenay, Province of Québec, Canada (see supporting information for isolation and identification details). The antibacterial activities are summarized in Table 6 along with cytotoxicity results against human normal skin fibroblasts (WS1). Gentamycin was used as a control for the bacterial growth inhibition assay.

As depicted in Table 6, balsacone R (5) was the most active of the newly isolated compounds with MIC <10 µM against all MRSA strains. Balsacone Q (4) was also a good candidate showing MIC <10 µM for half of the MRSA strains. Regarding the previously isolated balsacones, dihydrochalcone 11 together with compounds (–)-12, (+)-12, (–)-18, (+)-18, (–)-20, (–)-21, (+)21 and (±)-22 were strongly potent to inhibit the growth of MRSA. Furthermore, almost all of the isolated balsacones (1–22) were not cytotoxic against WS1 cell line (IC50 >10 µM), with the exception of compounds 7a and (–)-21, which is a good point for their use as non-toxic antibiotics.

Examination of the biological data in terms of SARs led us to establish some crucial structural parameters involved in the anti-MRSA activity of balsacones that can be resumed as depicted in Figure 6. First, inversion of the absolute configuration of compounds 12, 14-19 and 21, did not

27

impact the antibacterial activity. Compound 13, for which the 2S configuration [(–)-13] was more active than its enantiomer, and compound 20 for which the 2S,3S configuration [(–)-20] was more active than its enantiomer were exceptions in this regard. Second, the presence of a cinnamoyl or dihydrocinnamoyl chain at C-8 was of crucial importance for the antibacterial activity. The aromatic ring on the dihydrocinnamoyl chain can be para-substituted with either a hydroxy or methoxy group without loss of activity. Third, the flavan skeleton can bear a supplemental p-hydroxycinnamyl group at the C-3 position, but its presence was not of primordial importance for the activity. However, balsacones featuring a hydroxy group at C-3, such as for compounds 15 and 16, were significantly less active than their unsubstituted or cinnamylated counterparts. Fourth, the presence of an additional 3,4-dihydropyran ring fused with the A-ring at positions C-5 and C-6 was not detrimental for the activity if no phydroxycinnamyl chain were found at the C-8 position, such as the inactive balsacones 7a, 7b, 8a and 8b. Fifth, the most active compounds were unsubstituted at the C-6 position of the A-ring, but the presence of a supplemental p-hydroxycinnamyl group was well tolerated.

Another important aspect highlighted by this SAR study deals with the negative impact of methylation on the anti-MRSA activity of balsacones. For instance, the presence of a methoxy group at the C-4 position of compound 6 significantly decreased its activity compared to its unsubstituted analogue (5). A similar behavior was observed for the ester-containing dihydrochalcones: compound 2 bearing a methoxy group at C-4 was less active than unsubstituted 1. Methylation of the hydroxy groups at both C-4 and C-4 in dihydrochalcones 9 and 10 also decreased the antibacterial activity compared to the structurally related compound 11. The presence of methoxy groups at C-4 or C-4 was also detrimental to the activity of

28

compounds 13 and 14 compared to the related compound 12, and compound 19 compared to unsubstituted 18. These results suggest that the hydrogen bond donor potential of some hydroxy groups found on the balsacones might be crucial for interacting effectively with their bacterial targets.

Table 6. Antibacterial activity of balsacones (1–22) against S. aureus (ATCC 25923) and clinical isolate MRSA together with cytotoxicity against human fibroblast cell line WS1.

Compd (±)-1 (±)-2 (±)-3 (±)-4 (±)-5 (±)-6 (±)-7a (±)-7b (±)-8a (±)-8b 9 10 11 (-)-12 (+)-12 (-)-13 (+)-13 (-)-14 (+)-14 (-)-15 (+)-15 (-)-16 (+)-16 (-)-17 (+)-17 (-)-18 (+)-18 (-)-19 (+)-19 (-)-20 (+)-20 (-)-21 (+)-21 (±)-22 Gentamycin

S. aureus

50 25 50 13 6 50 50 50 50 50 50 50 6 6 6  50 25 50 50 25 25 25 25 25 50 3 3 25 25 6 13 2 6 3 0.03

#1 25 50 50 6 6 25 50 50 50 50 50 50 6 6 13  50 13 50 50 50 25 50  50 50 50 3 3 25 25 3 25 2 2 3 0.05

#2 25 50 50 6 6 25 50 50 50 50 50 25 6 6 13  50 13 50 50 25 25 25 50 50 50 3 3 25 25 3 13 2 6 2 0.04

#3 13 50 50 3 6 25 50 50 50 50 50 13 6 6 6  50 13 50 50 50 25 25  50 50 50 3 3 13 6 3 13 2 3 3 0.1

Antibacterial activity (MIC, µM) MRSA #4 #5 #6 25 3 25 50 50 50 50 50 50 13 13 6 6 6 6 50 13 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 13 13 25 6 3 50 6 6 13 13 6 25  50  50  50 25 13 25 50 50 50 50 50 50 50 25 50 50 25 25 13 13 50 50 50  50 50 50 50 50 50 50 3 6 3 6 3 6 25 13 25 25 25 25 3 3 3 25 13 13 2 2 3 3 3 3 3 1 2 0.1 0.08 0.05

Cytotox. (IC50, µM) #7 25 50 50 6 6 25 50 50 50 50 50 25 6 6 6  50 13 50 50 25 25 25 25 25 50 3 3 25 13 3 6 0.8 2 2 0.07

#8 25 50 50 13 6 25 50 50 50 50 50 25 3 6 13  50 13 50 50 50 25  50 50  50 50 3 13 25 25 3 13 2 3 3 0.09

#9 25 50 50 13 6 25 50 50 50 50 50 25 3 13 13  50 13 50 50 50 25 50 50  50  50 6 3 13 25 3 13 2 3 2 0.07

#10 25 50 50 13 6 25 50 50 50 50 50 25 6 13 13  50 25 50 50 25 25 25  50 50  50 13 3 25 25 3 13 3 2 2 0.07

WS1 >10 >10 >10 >10 >10 >10 2.3 ± 0.5 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 8±1 >10 >10 nd

29

Figure 6. Crucial structural parameters for the anti-MRSA activity of balsacones

3. Conclusions

In summary, a unique series of naturally occurring flavonoids bearing multiple cinnamyl units were isolated as racemates from the buds of P. balsamifera. Thorough 1D and 2D NMR analysis led to the characterization of their structures. Some of these compounds were shown to possess strong antibacterial activity against MRSA with MIC values in the low micromolar range. The SAR study highlighted the importance of crucial structural parameters for enhancing the antiMRSA activity of balsacones, such as the presence of a cinnamoyl chain at C-8 and a phydroxycinnamyl unit at C-3. Additionally, the presence of methoxy groups at specific positions was shown to reduce the activity for almost all of the isolated balsacones with the exception of the potent compound 22. These preliminary results demonstrate the interest of further developing cinnamylated flavonoids such as balsacones as a potentially novel class of anti-MRSA agents.

30

4. Experimental

4.1. General procedures

Optical rotations were determined at the sodium D line (590 nm) on a Rudolph Research Analytical Autopol IV automatic polarimeter. Absorption UV spectra were performed using an Agilent 8453 diode-array spectrophotometer. ECD spectra were recorded on a Jasco J-815 CD spectrometer. IR spectra were conducted on a Perkin-Elmer SpectrumOne (neat, thin films, on NaCl plates). 1H NMR,

13

C NMR and 2D NMR (1H-1H, COSY, NOESY, HSQC, and HMBC)

spectra were performed using an Avance 400 Bruker spectrometer (400.13 MHz for 1H, 100.61 MHz for 13C spectra) equipped with a 5 mm QNP probe. Chemical shifts values were reported in ppm () relative to TMS. High resolution ESIMS spectra were obtained with an Agilent Technologie 6210 TOFMS system. Low resolution APCIMS (positive mode) were obtained with an Agilent G1946 VL Mass Selective Detector. Ultra pure silica gel (40–63 µm) for column chromatography and glass TLC plates (40–63 µm with F254 indicator) were supplied by Silicycle Inc. (Ville de Québec, Québec, Canada). Reversed phase flash chromatography was performed on a Biotage Flash+ system using a C18 40iM cartridge (17%, 135 g) from Silicycle Inc. (Ville de Québec, Québec, Canada). Semi-preparative HPLC was carried out on an Agilent 1100 Series equiped with a UV-vis detector at 254 nm with an Inerstil Prep ODS column (20 mm  250 mm, 10 µm). Chiral HPLC was conducted using a tris-(3,5-dimethylphenyl)carbamoyl amylose chiral coated column (RegisPackTM, 10  250 mm, 5 µm). Reagents and analytical grade solvents were purchased from VWR International (Ville Mont-Royal, Québec, Canada) and were used as received.

31

4.2. Plant material

Buds from different trees of P. balsamifera were collected in March 2005 near Chicoutimi, Québec, Canada. The plant was authenticated by Mr. Patrick Nadeau (Université du Québec à Chicoutimi) and a voucher specimen (No. 499678) was deposited at the Louis-Marie Herbarium of Université Laval, Ville de Québec, Canada. After collection, buds were kept frozen until extraction.

4.3. Extraction and isolation of compounds 1, 2, 5 and 6

Buds previously immersed in liquid nitrogen were coarsely ground in an electrical blender. The obtained residue (1.04 kg) was extracted with refluxing 95% EtOH (5 times, 5 h, 2.5 L). The combined extracts were concentrated using a rotary evaporator, suspended in MeOH and washed with hexanes. After concentration of the MeOH phase using a rotary evaporator, the obtained residue was suspended in Et2O and extracted with H2O. The Et2O phase was dried giving a crude extract (355.1 g), which was fractionated by silica gel CC (CHCl3/MeOH, 40:1, 20:1, 10:1, 0:1) yielding 7 fractions (A–G). Fraction F (59.5 g) was submitted to another silica gel CC (CHCl3/MeOH, 40:1, 30:1, 20:1, 10:1, 0:1) to afford 6 subfractions (F1–F6). Subfraction F4 (45.6 g) was fractionated by reversed phase C18 flash chromatography (MeOH/H2O, 65:35, 70:30, 72:28, 0:100) yielding subfractions F4a to F4e. Subfraction F4b (6.33 g) was submitted to silica gel CC (CHCl3/MeOH, 40:1, 30:1, 10:1, 0:1) yielding 7 subfractions (F4b.I to F4b.VII). Subfraction F4b.VI (960.7 mg) was applied to silica gel CC (CHCl3/EtOAc, 2:1, then MeOH) to obtain 6 other subfractions (F4b.VI.1 to F4b.VI.6). F4b.VI.3 (92.4 mg) was submitted to semi32

preparative HPLC (MeOH/H2O, 80:20) to obtain (±)-1 (tr = 20.6 min, 32.4 mg). Semi-preparative HPLC (MeOH/H2O, 80:20) of subfraction F4b.VI.4 (147.1) yielded (±)-2 (tr = 18.3 min 29.2 mg). Subfraction F4c (2.93 g) was submitted to another silica gel CC (CHCl3/EtOAc, 4:1→3:1, then MeOH) to afford 9 subfractions (F4c.І–F4c.IX). Subfraction F4c.III (379.5 mg) was purified first by silica gel CC (CHCl3/MeOH, 30:1 then MeOH) followed by preparative HPLC (MeOH + 0.1% HCOOH/H2O + 0.1% HCOOH, 71:29, flow rate 10 mL/min) to afford (±)-5 (tr = 82.6 min, 9.2 mg). Subfraction A4c.IX was purified by silica gel CC (CHCl3/MeOH, 30:1, 20:1, then MeOH), reversed phase C18 flash chromatography (MeOH/H2O, 80:20, 100:0) and by two preparative HPLC separations (MeOH/H2O, 73:27, flow rate 10 mL/min followed by CH3CN/H2O, 50:50, flow rate 10 mL/min) to afford (±)-6 (tr = 56.5 min, 7.3 mg).

4.4. Extraction and isolation of compounds 3, 4, 7 and 8

Frozen buds were coarsely ground using an electrical blender. The resulting crude resinous material (2.69 kg) was divided in two flasks and extracted 4 times (3 h each time) with a total of 6 L of refluxing 95% EtOH. Extracts were combined and concentrated using a rotary evaporator giving a resinous residue. Part of this residue (1.00 kg) was suspended in MeOH and washed with hexanes. The concentrated MeOH residue was suspended in Et 2O and extracted with water. The concentrated Et 2O phase was submitted to silica gel CC (CHCl3/MeOH, 1:0, 80:1, 40:1, 20:1, 10:1, 0:1) yielding 6 fractions (A–F). Fraction D (112.0 g) was submitted to another silica gel CC (CH2Cl2/EtOAc, 10:1, 8:1, 6:1, 4:1, 2:1, then MeOH) to afford 8 subfractions (D1–D8). Subfraction D1 was fractionated by silica gel CC (CHCl3/EtOAc, 7:1, 5:1, 2:1, then MeOH) to afford 5 fractions (D1a–D1e). Subfraction D1c (959.6 mg) was purified by preparative HPLC (MeOH/H2O, 80:20, flow rate 10 mL/min) to afford 3 (tr = 22.5 min, 129.0 mg). Subfraction D4 33

was submitted to reversed phase C18 flash chromatography (MeOH/H2O, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 95:5, 0:100) yielding 9 subfractions (D4a–D4i). Subfraction D4g (1.69 g) was submitted to preparative HPLC (MeOH/H2O, 80:20  100:0, 30 min, flow rate 10 mL/min) to afford impure 4 (159.1 mg), which was repurified by preparative HPLC (CH3CH/H2O, 60:40, flow rate 10 mL/min) yielding racemic 4 (tr = 35.5 min, 50.5 mg). Subfraction D5 was fractionated by reversed phase C18 flash chromatography (MeOH/H2O, 70:30, 75:25, 80:20, 90:10, 100:0) to afford 8 subfractions (D5a–D5h). Subfraction D5g was submitted to preparative HPLC purification (CH3CN/H2O, 60:40, 20 mL/min) yielding 7a (tr = 66.3 min, 15.8 mg) and 7b (tr = 70.2 min, 22.8 mg). Fraction D7 (3.34 g) was fractionated by reversed phase C18 flash chromatography (MeOH/H2O, 50:50, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, 100:0) yielding 13 subfractions (D7a–D7m). Subfraction D7l (211.2 mg) was purified by semipreparative HPLC (CH3CN/H2O, 60:40, flow rate 10 mL/min) giving 8a (tr = 61.6 min, 18.5 mg) and 8b (tr = 65.2 min, 21.3 mg).

4.5. Resolution of racemic mixtures using chiral HPLC Chiral HPLC using isocratic elution (hexane-IPA, 70:30) allowed to resolved the two enantiomers of racemic mixutre (±)-1 (tr = 7.4 and 13.0 min), (±)-2 (tr = 9.3 and 25.8 min) and (±)-4 (tr = 18.0 and 29.5). Racemic mixtures (±)-7a (hexane-IPA, 50:50, tr = 8.8 and 13.2 min), (±)-7b (hexane-IPA, 40:60, tr = 9.7 and 26.3 min), (±)-8a (hexane-IPA, 50:50, tr = 14.6 and 19.7 min) and (±)-8b (hexane-IPA, 60:40, tr = 13.1 and 20.2 min) were also resolved using chiral HPLC.

4.6. Characterization of balsacones N–U (1–8)

34

(±)-Balsacone N (±)-1: greyish powder; UV (MeOH) λmax (log Ɛ) 203 (4.84), 275 (4.68), 326 (3.98) nm; IR (neat, thin film) νmax 3338, 3027, 2952, 1697, 1611, 1512, 1439, 1366, 1244, 1175, 1109, 1076, 1033, 977, 830, 769, 684 cm−1; for 1H and 13C NMR spectroscopic data, see Table 1; APCIMS 671 [M+H]+ (28), 523 (100), 419 (17) 259 (17), 133 (56); HRESIMS m/z 693.2444 [M+Na]+ (calcd for C42H38O8Na, 693.2459).

(±)-Balsacone O (±)-2: greyish powder; UV (MeOH) λmax (log Ɛ) 217 (4.72), 274 (4.69), 330 (4.18) nm; IR (neat, thin film) νmax 3338, 3027, 2952, 1697, 1611, 1512, 1439, 1366, 1244, 1175, 1109, 1076, 1033, 977, 830, 769, 684 cm−1; for 1H and 13C NMR spectroscopic data, see Table 1; APCIMS 701 [M+H]+ (12), 553 (100), 391 (7), 289 (21), 133 (80); HRESIMS m/z 723.2553 [M+Na]+ (calcd for C43H40O9Na, 723.2565).

(±)-Balsacone P (3): reddish powder; UV λmaxMeOH (log ɛ): 204 (4.54), 273 (4.24), 290 (4.21); IR νmax 3348, 2963, 1598, 1513, 1438, 1261, 1090, 1030, 799, 701 cm−1 ; For 1H and

13

C NMR

spectroscopic data, see Tables 2 and 3, respectively; APCIMS: m/z 523 [M+H]+ (100), 271 (7.8) ; HRESIMS: m/z 545.1943 [M+Na]+ (calcd for C33H30O6Na, 545.1935).

(±)-Balsacone Q (4): brownish powder; UV λmaxMeOH (log ɛ): 203 (4.72), 223 (4.59), 272 (4.35); IR νmax 3335, 2963, 1610, 1512, 1446, 1260, 1093, 1030, 799 cm−1; For 1H and

13

C NMR

spectroscopic data, see Tables 2 and 3, respectively; APCIMS: m/z 685 [M+H]+ (64), 433 (19), 401 (14); HRESIMS: m/z 685.2778 [M+H]+ (calcd for C43H40O9, 685.2796).

(±)-Balsacone R (5): brownish powder; UV λmaxMeOH (log ɛ): 202 (4.51), 262 (4.02), 296 (3.96); IR νmax 3337, 2926, 1700, 1612, 1516, 1447, 1423, 1379, 1360, 1264, 1235, 1149, 1132, 1072, 35

1056, 1023, 834 cm−1; For 1H and 13C NMR spectroscopic data, see Tables 2 and 3, respectively; APCIMS: m/z 671 [M+H]+ (7), 535 (7), 419 (100), 283 (14); HRESIMS: m/z 671.2624 [M+H]+ (calcd for C42H39O8, 671.2639).

(±)-Balsacone S (6): brownish powder; UV λmaxMeOH (log ɛ): 205 (4.65), 224 (4.56), 272 (4.26), 296 (4.26); IR νmax 3370, 2924, 1698, 1611, 1513, 1446, 1426, 1377, 1242, 1152, 1130, 1061, 1031, 831 cm−1; For 1H and

13

C NMR spectroscopic data, see Tables 2 and 3, respectively;

APCIMS: m/z 701 [M+H]+ (5), 565 (9), 449 (100); HRESIMS: m/z 723.2562 [M+Na]+ (calcd C43H40O9Na, 723.2562).

(±)-Balsacone T1 (7a): brownish powder; UV λmaxMeOH (log ɛ): 208 (4.87), 264 (4.59), 296 (4.47); IR νmax 3216, 2962, 1598, 1510, 1414, 1260, 1089, 1030, 799 cm−1; For 1H and 13C NMR spectroscopic data, see Tables 4 and 5, respectively; APCIMS: m/z 787 [M+H]+ (47), 534 (23); HRESIMS: m/z 787.3245 [M+H]+ (calcd for C51H46O9, 787.3265).

(±)-Balsacone T2 (7b): brownish powder; UV λmaxMeOH (log ɛ): 208 (4.87), 264 (4.59), 296 (4.47); IR νmax 3239, 2960, 1608, 1512, 1444, 1362, 1260, 1091, 1029, 799 cm−1; For 1H and 13C NMR spectroscopic data, see Tables 4 and 5, respectively; APCIMS: m/z 787 [M+H]+ (54), 535 (40); HRESIMS: m/z 787.3229 [M+H]+ (calcd for C51H47O9, 787.3265).

(±)-Balsacone U1 (8a): brownish powder; UV λmaxMeOH (log ɛ): 202 (5.06), 264 (4.68), 295 (4.55); IR νmax 3265, 1608, 1511, 1444, 1417, 1260, 1091, 1026, 798 cm−1; For 1H and 13C NMR spectroscopic data, see Tables 4 and 5, respectively; APCIMS: m/z 817 [M+H]+ (17), 565 (12); HRESIMS: m/z 817.3370 [M+H]+ (calcd C51H49O9, 817.3371). 36

(±)-Balsacone U2 (8b): brownish powder; UV λmaxMeOH (log ɛ): 203 (4.81), 269 (4.39), 295 (4.31); IR νmax 3240, 2959, 1608, 1511, 1442, 1259, 1093, 1028, 800 cm−1; For 1H and 13C NMR spectroscopic data, see Tables 4 and 5, respectively; APCIMS: m/z 817 [M+H]+ (28), 565 (17), 391 (100); HRESIMS: m/z 817.3391 [M+H]+ (calcd C51H49O9, 817.3371).

4.7. Bacterial growth inhibition assay

Antibacterial activity against S. aureus (ATCC 25923) and clinical isolate MRSA (see SI for strain details) were evaluated using the microdilution method as previously reported [24]. Gentamycin was used as a positive control. The results are reported as the minimum inhibitory concentration (MIC) in µM.

4.8. Cytotoxicity assay

Cytotoxicity was evaluated against the human fibroblast cell line (WS1) using the resazurin reduction test with slight modifications as previously reported [25]. Cytotoxicity was expressed as the concentration (µM) inhibiting 50% of cell growth (IC50). Etoposide was used as a positive control and showed an IC50 value of 34 ± 4 µM.

Acknowledgments

The project was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and by the Fonds de Recherche Nature et Technologies (PhD fellowships to FS). We 37

aknowledge the Chaire de Recherche sur les Agents Anticancéreux d’Origine Naturelle for funding. The authors would like to thank C. Dusseault for her technical assistance in the evaluation of antibacterial and cytotoxic activities.

Supplementary data

Supplementary data (1D and 2D NMR, and HRMS spectra for new compounds 1–8) associated with this article can be found in the online version of the manuscript.

References and notes

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[13] V.A. Kurkin, G.G. Zapesochnaya, V.B. Braslavskii, Flavonoids of the buds of Populus balsamifera, Chem. Nat. Compd., 26 (1990) 224-225. [14] E.V. Isaeva, T.V. Ryazanova, Group composition of carbohydrates of poplar buds, Khimiya Rastitel'nogo Syr'ya, (2006) 33-36. [15] E.D. Levin, E.V. Isaeva, V.E. Cherepanova, Arachidonic acid and prostaglandins in buds of Populus balsamifera, Phytochemistry, 29 (1990) 2325-2326. [16] S. Lavoie, J. Legault, F. Simard, É. Chiasson, A. Pichette, New antibacterial dihydrochalcone derivatives from buds of Populus balsamifera, Tetrahedron Lett., 54 (2013) 1631-1633. [17] F. Simard, J. Legault, S. Lavoie, A. Pichette, Balsacones D-I, dihydrocinnamoyl flavans from Populus balsamifera buds, Phytochemistry, 100 (2014) 141-149. [18] F. Simard, C. Gauthier, É. Chiasson, S. Lavoie, V. Mshvildadze, J. Legault, A. Pichette, Antibacterial Balsacones J–M, Hydroxycinnamoylated Dihydrochalcones from Populus balsamifera Buds, J. Nat. Prod., 78 (2015) 1147-1153. [19] L.M. Conserva, M. Yoshida, O.R. Gottlieb, J.C. Martinez V, H.E. Gottlieb, Iryantherins, lignoflavonoids of novel structural types from the myristicaceae, Phytochemistry, 29 (1990) 3911-3918. [20] R.W. Hemingway, F.L. Tobiason, G.W. McGraw, J.P. Steynberg, Conformation and complexation of tannins: NMR spectra and molecular search modeling of flavan-3-ols, Magn. Reson. Chem., 34 (1996) 424-433. [21] A.L. Davis, Y. Cai, A.P. Davies, J.R. Lewis, 1H and 13C NMR Assignments of Some Green Tea Polyphenols, Magn. Reson. Chem., 34 (1996) 887-890. [22] W. Greenaway, S. English, F.R. Whatley, Relationships of Populus × acuminata and Populus × generosa with their parental species examined by gas chromatography – mass spectrometry of bud exudates, Canadian Journal of Botany, 70 (1992) 212-221. [23] W. Greenaway, J. May, T. Scaysbrook, F.R. Whatley, Compositions of bud and leaf exudates of some Populus species compared, Zeitschrift für Naturforschung, 47 (1992) 329-334. [24] E. Banfi, G. Scialino, C. Monti-Bragadin, J. Antimicrob. Chemother., 52 (2003) 796. [25] C. Gauthier, J. Legault, K. Girard-Lalancette, V. Mshvildadze, A. Pichette, Haemolytic activity, cytotoxicity and membrane cell permeabilization of semi-synthetic and natural lupane- and oleananetype saponins, Bioorganic & Medicinal Chemistry, 17 (2009) 2002-2008.

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