Chemo-Diversification of Plant Extracts Using a Generic Bromination

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Chemo-diversification of plant extracts using a generic bromination reaction and monitoring by metabolite profiling Davide Righi, Laurence Marcourt, Alexey Koval, Verena Ducret, leonie PELLISSIER, Alice Mainetti, Vladimir Katanaev, Karl Perron, Jean-Luc Wolfender, and Emerson Ferreira Queiroz ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00132 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019

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ACS Combinatorial Science

Chemo-diversification of plant extracts using a generic bromination reaction and monitoring by metabolite profiling

Davide Righi,§ Laurence Marcourt,§ Alexey Koval,£ Verena Ducret,¥ Léonie Pellissier,§ Alice Mainetti,§ Vladimir L. Katanaev,£¶ Karl Perron,¥ Jean-Luc Wolfender§ and Emerson Ferreira Queiroz*§

§

School of Pharmaceutical Sciences, EPGL, University of Geneva, CMU, 1, Rue Michel

Servet, 1211 Geneva 4, Switzerland. ¥ Microbiology

unity, University of Geneva, CMU, 130 quai Ernest-Ansermet, 1211 Geneva 4,

Switzerland. £Department

of Cell Physiology and Metabolism, Translational Research Centre in

Oncohaematology, Faculty of Medicine, University of Geneva, CMU, 1, Rue Michel Servet, 1211 Geneva 4, Switzerland. ¶

School of Biomedicine, Far Eastern Federal University, 8 Sukhanova St. 690090, Vladivostok,

Russia.

1 ACS Paragon Plus Environment

ACS Combinatorial Science 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract

A generic procedure for direct bromination of polyphenol in crude plant extracts was developed in order to generate multiple ‘unnatural’ halogenated natural products for further bioassay evaluation. To better control the halogenation procedure, the bromination was optimized with a flavonoid standards and the reactions were monitored by HPLC-PDA coupled to the evaporative light scattering detection (ELSD). ELSD detection was successfully used for a relative yield estimation of the compounds obtained. From the halogenation of hesperitin (11), five brominated compounds were obtained. After optimization, the reaction was successfully applied to the methanolic extract of Citrus sinensis peels, a typical waste biomass and also to the methanolic extract of the medicinal plant Curcuma longa. In both cases, the methanolic extracts were profiled by NMR for a rapid estimation of the polyphenol versus primary metabolite content. An enriched secondary metabolites extract was obtained using vacuum liquid chromatography (VLC) and submitted to bromination. Metabolite profiling performed by UHPLC-TOF-HRMS revealed the presence of various halogenated products. To isolate these compounds, the reactions were scaled-up and six halogenated analogues were isolated and fully characterized by NMR and HRESIMS analyses. The antibacterial properties of these compounds were evaluated using in vitro bioassays against multi resistant’s strains of Staphylococcus aureus and Pseudomonas aeruginosa. Some of the halogenated derivatives obtained presented moderate antibacterial properties.


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Introduction It is estimated that twenty percent of all low molecular weight active principles (API) in pharmaceutical drugs are halogenated.1, 2 The introduction of a carbon halogen bond can have a number of effects such as the increase in thermal and oxidative stability, for less sensitivity towards oxidation by liver P450 detoxification enzymes and the increase in biological membrane permeability.1, 2 Historically, natural products (NPs) have been an important source of lead molecules in drug discovery.3 The huge structural diversity of NPs supports the belief that collections of NPs are not only more varied than those composed of synthetic compounds, but also that NPs better represent the ‘chemical space’ of drug-like molecules; consequently a significant number of new chemical entities registered as drugs are, or have been, inspired by NPs.4 In some specific therapeutic areas, such as anti-infective (antibacterial, antifungal, antiparasitic and antiviral) drugs, NPs or derived structures represent more than 50% of all current drugs.3 Novel type of NP scaffolds are particularly needed in drug discovery for finding efficient antibiotics against the increasing number multi-resistant pathogenic bacterial strains. This is particularly important against the Gram-positive Staphylococcus aureus that causes superficial and invasive infections potentially fatal, such as sepsis and pneumonia. 5-7 Classical antibiotic treatments become ineffective because of the antibiotic-resistant strains, such as the methicillin-resistant S. aureus (mecC-MRSA).6 In this context, halogenated NPs from marine origin frequently display interesting biological activities and several are strong antibacterial agents.8 For example some brominated NPs isolated from the marine red algae genus Laurencia presented strong antibacterial activity against vancomycin-resistant Staphylococcus aureus (VRSA).9



Based on such considerations and since NPs from terrestrial sources, mainly from plants, are very rarely halogenated, strategies for the generation of brominated ‘unnatural’ natural products directly from crude extracts have been developed in the present study. Different chemical reactions have been successfully used for the halogenation of pure NPs.10-12 The potential of plant extract’s chemical halogenation using an uncontrolled bromination reaction to obtain an acetylcholine esterase inhibitor has also been highlighted.13 In our study, a generic method of bromination of crude plant extracts is presented. The method has the advantage to cover a broad polarity of compounds for the generation of NP derivatives with different levels of bromine substitution. The way to adequately prepare the plant extracts for bromination was also investigated. For this, reactions were monitored overtime by HPLC-PDA-ELSD. Original and transformed extracts were also profiled by LC/HRMS for a rapid and accurate estimation of the bromination level of all constituents. Finally, the brominated compounds obtained were evaluated for their antibacterial activities against multi-resistant pathogenic bacterial strains of S. aureus.

Results and discussion

A survey of possible existing halogenation protocols10-13 led us to test in a first instance the eco-friendly methodology, based on aqueous middle reactions, proposed by Bernini and coworkers, i.e. halogenation of flavonoids, compounds which are commonly occurring in crude plant extracts.14 This method was first tested on a representative plant flavonoid hesperitin (7) before using it on complex crude extracts. The reaction was performed using a 30% aqueous solution of hydrogen peroxide (3.0 eq.), NaBr (1.0 eq.) in acetic acid (2.5 mL) during 4 hours at room


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temperature. HPLC-PDA and UHPLC-TOF-HRMS profiling of the brominated reaction revealed that hesperetin (7) was partially converted after 4 hours in two mono bromine derivatives (Figure S1, Supporting information). This assay confirmed the results obtained previously for the bromination of flavonoid derivatives.14 After this preliminary trial, the bromination procedure was applied to a typical plant polyphenol extract. For this, the pericarp of Citrus sinensis L. (Osbeck) was selected as a starting material. This material, rich in flavonoids, is indeed easily obtainable at large scale and considered as a waste material from the agroindustry sector.15 An enriched flavonoid extract was obtained by methanolic extraction of Citrus sinensis pericarp after hexane defatting. Prior to bromination this extract was analyzed by UHPLC-TOF-HRMS in negative ion mode for a preliminary metabolites profiling (Figure S2, Supporting information). Extraction of the molecular formulae from the HRMS data obtained enabled the dereplication of most of the polyphenols previously reported in C. sinensis.16 The extract was found to be mainly composed of the glycosylated flavonoids naringin (1) (Rt 6.33 min, m/z 625.1779 [M+HCO2]-, calcd for C28H33O16, 625.1749, Δ ppm = 1.6), hesperidin (2) (Rt 6.93 min, m/z 655.1862 [M+HCO2]-, calcd for C29H35O17, 655.1874, Δ ppm = -1.8) and poncirin (3) (Rt 8.93 min, m/z 639.1923 [M+HCO2]-, calcd for C29H35O16, 639.1925, Δ ppm = -0.3) and a series of polymethoxylated flavonoids aglycones: tetramethoxyflavone (Rt 13.31 min, m/z 343.1187 [M-H]-), pentamethoxyflavones (Rt 11.19 min, m/z 373.1342 [M-H]-; Rt 12.12 min, m/z 373.1384 [MH]-; Rt 14.41 min, m/z 373.1329 [M-H]-), hexamethoxyflavones (Rt 12.87 min, m/z 403.1416 [M-H]-; Rt 13.24 min, m/z 403.1396 [M-H]-) and heptamethoxyflavones (Rt 13.96 min, m/z 433.1523 [M-H]-). Citrusin III (4) (Rt 9.82 min, m/z 772.3966 [M-H]-)17 and limonin (5)18 (Rt 12.34 min, m/z 515.1906 [M-H]-) could also be annotated (Figure S3, Supporting information). This extract (2g) was submitted to the bromination reaction conditions developed by Bernini et al. with sodium bromide in an aqueous solution of hydrogen peroxide and acetic acid 5 ACS Paragon Plus Environment


as a solvent.14 To ensure that bromination occurs in such a complex extract, the number of equivalents of the reagents used in this reaction was 10 times higher than the one reported in the original protocol (citrus reaction n°1, experimental section).14 10 equivalents of NaBr and 30 equivalents of H2O2 in a solution of 25 ml of acetic acid and 40 mL of water were used. The equivalents were estimated assuming that the weight of the extract corresponded only to the major compound hesperidin (2). After 24 hours, the reaction was stopped (see experimental section). UHPLC-TOF-HRMS profiling of the brominated and the initial crude extract revealed that the major flavonoids naringin (1), hesperidin (2) and poncirin (3) were fully converted after reaction (Figure 1). An analysis of the isotopic pattern of the LC peaks that appeared after the reaction indicated a characteristic profile of brominated compounds (Figure 1). The molecular weight of these compounds was however lower than the one expected for brominated derivatives of the polyphenols found in the original extract. In order to identify these compounds the reaction mixture was purified by flash chromatography. Using this approach a series of five compounds (6-10) was obtained and fully characterized by spectroscopic techniques such as NMR and HRMS (Figure 1). Among them, four compounds (6-9) were found to contain bromine atoms. As expected, the isolated compounds were small molecular weight bromophenols probably generated by the degradation of the flavonoids present in the original extract.


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A)

100

Citrus sinensis methanolic extract

%

Hesperidin (2)

Citrus sinensis methanolic extract (2g)

Naringin (1) Poncirin (3)

0

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

AcOH (25 ml) NaBr (10eq), H2O2 (30eq) Water (40ml), 25°C, 24hs

20.00

After reaction *Brominated derivatives

%

B)

2.00

100

*

0

2.00

4.00

6.00

8.00

10.00

*

* *

**

12.00

14.00

16.00

O Br

Br

T ime 20.00

18.00

OCH3

Br

D)

Br

OH

(6)

C)

334.8366

100

Br

% ! 306.8795

0

O

3 Br 326.7654

300

305

310.8466

310

! 316.9524

315

320

Br OCH3

2 Br

336.8355

332.8389

332.7611

OCH3 Br

(8)

! 337.8380 325.0103

325

!;341.0984

330

335

340

352.8603

345

350

355

360.9417 367.1336

360

365

370

m/z

0

! ! 304.9166 308.1924 300

305

310

! 313.2419 324.8104 315

320

325

! 350.7914

333.7628 330

335

340

345

350

! 354.1763 355

! 362.9044 360

365

Br

(7)

6

328.7642 330.7618

100

%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O H 3C

OCH3 OCH3

OH O

Br

(9)

Br Br

O OH

(10)

! 372.7638 m/z 370

Figure 1. A) UHPLC-TOF-HRMS analysis of the enriched methanolic extract of C. sinensis before and B) after bromination. C) HR-MS (negative mode) spectra of some of the brominated derivatives. D) Structure of the isolated compounds.

These preliminary results indicated that the conditions used were too strong to ensure a controlled bromination reaction of the original polyphenols and therefore caused degradation, possibly due to a too large excess of reagents. To optimize the conditions for bromination, the reaction was carried out with a representative flavonoid aglycone hesperitin (11) derived from the major compound hesperidin (2) present in the citrus extract.15 In order to avoid possible problems of solubilization of the extract in water, acetonitrile was used as an inert organic solvent.14 In this first trial 50 mg of the standard was used for halogenation in different conditions (reaction time/number of equivalents) (Table 1). All reactions were monitored by HPLC-PDA coupled to the evaporative light scattering detection (ELSD) for a relative yield estimation of the compounds obtained 7 ACS Paragon Plus Environment


Page 8 of 43

based on the total ELSD response (see % of the different halogenated derivatives Table 1).19 Two reaction conditions were selected for further studies: reaction n°3, which afforded the highest amounts of compounds 12 and 13 and reaction n°4 which yielded compounds 14, 15 and 16 (Figure 2). Table 1. Experimental conditions for the bromination of hesperitin (11). Aliquots of each reaction (150 µL) was obtained at time 0, 1, 3, 5 and 24 hours and analyzed by HPLC-ELSD. Reaction (n°)

Time (h)

Co-solvent

NaBr

H2O2

AcOH

Temp

Product (% of the different halogenated derivatives and starting material based in the total ELSD response)a

1

0

MeCN:water (6.5 mL:6.5 mL)

1 eq.

21 eq.

5 mL

40°

11 (%) 100

1

1


1 eq.

21 eq.

5 mL

40°

82.9

10.1

7

0.0

0.0

0.0

1

3


1 eq.

21 eq.

5 mL

40°

45.9

31.9

22.2

0.0

0.0

0.0

1

24


1 eq.

21 eq.

5 mL

40°

4.3

51.8

35.6

8.3

0.0

0.0

2

0


2 eq.

21 eq.

5 mL

40°

100

0.0

0.0

0.0

0.0

0.0

2

1


2 eq.

21 eq.

5 mL

40°

100

0.0

0.0

0.0

0.0

0.0

2

3


2 eq.

21 eq.

5 mL

40°

95.4

2.5

2.0

0.0

0.0

0.0

2

24


2 eq.

21 eq.

5 mL

40°

18.9

46.7

32.1

2.3

0.0

0.0

3

0


10 eq.

21 eq.

5 mL

40°

100

0.0

0.0

0.0

0.0

0.0

3

1


10 eq.

21 eq.

5 mL

40°

96.7

2.0

1.3

0.0

0.0

0.0

3

3


10 eq.

21 eq.

5 mL

40°

90.1

5.8

4.1

0.0

0.0

0.0

3

24


10 eq.

21 eq.

5 mL

40°

1.9

50.3

35.8

12.0

0.0

0.0

4

0


100 eq.

21 eq.

5 mL

40°

100

0.0

0.0

0.0

0.0

0.0

4

1


100 eq.

21 eq.

5 mL

40°

87.5

4.9

7.6

0.0

0.0

0.0

4

3


100 eq.

21 eq.

5 mL

40°

47.7

32.9

19.4

0.0

0.0

0.0

4

24


100 eq.

21 eq.

5 mL

40°

0.0

0.0

0.0

45.7

33.8

20.5

5

0


1 eq.

21 eq.

1 mL

40°

100

0.0

0.0

0.0

0.0

0.0

5

1


1 eq.

21 eq.

1 mL

40°

100

0.0

0.0

0.0

0.0

0.0

5

3


1 eq.

21 eq.

1 mL

40°

100

0.0

0.0

0.0

0.0

0.0

5

24


1 eq.

21 eq.

1mL

40°

90.6

5.6

3.8

0.0

0.0

0.0

6

0


1 eq.

21 eq.

10 mL

40°

100

0.0

0.0

0.0

0.0

0.0

6

1


1 eq.

21 eq.

10 mL

40°

30.1

406

14.3

15.0

0.0

0.0

6

3


1 eq.

21 eq.

10 mL

40°

4.2

51.6

38.7

5.6

0.0

0.0

24


1 eq.

21 eq.

10 mL

40°

0.0

52.6

34.8

12.6

0.0

0.0

6 a calculated

12 (%) 0.0

13 (%) 0.0

14 (%) 0.0

15 (%) 0.0

16 (%) 0.0

based in the integrated area of the peaks obtained by HPLC-ELSD analysis.

The compounds generated by reactions n°3 and n°4 were isolated by transfer of analytical to semi-preparative HPLC-UV conditions.20,

21

Reaction n°3 (10 eq.) yielded 8-

bromo-hesperitin (12), 6-bromo-hesperitin (13) and 6,8-di-bromo-hesperitin (14) confirming that flavanones were brominated regio-selectively at C-6 and C-8 (Figure 2).14 On the other hand bromination of hesperetin with 100 equivalents of NaBr at 24 hours afforded two tri-


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brominated derivatives 6,8,2’-tri-bromo-hesperitin (15) and 6,8,6’-tri-bromo-hesperitin (16) (Figure 2). mAU

A)

120

0h

11

Standard

140

100 80 60

5

mAU 100

10

15

Reaction n°3

90

20

12

25

30

35 min

13

24h

80 70

14

60 50

5

Reaction n°4

mAU 85 80 75 70 65 60 55 50 45

10

15

20

14

30

10

15

20

OH HO

OH O

HO

O

(14)

(13) OH O

OH Br

Br HO

Br

O

Br

OH O

OCH3

OH O

OCH3 HO

(12)

(11)

Br

35 min

OH

O

OH HO

30

OCH3

Br

O

25

OH

OCH3

35 min

24h

16

5

B)

25

15

OH OCH3

O

Br OH O

HO

(15)

OCH3

Br O Br

Br OH O

(16)

Figure 2. A) HPLC- ELSD analysis of the bromination reaction n°3 (conditions: 10 eq. of NaBr, 21 eq. of H2O2, 5 mL of acetic acid) and n°4 (conditions: 100 eq. of NaBr, 21 eq. of H2O2, 5 mL of acetic acid) of hesperitin (11) at time 0 h and 24 h. B) Brominated compounds isolated (12-16). These conditions have proved well adapted to the bromination of typical flavonoid standards. Since preliminary results obtained on citrus extract indicated that an excess reagent was used and led to the degradation of the compounds, the composition of the extract was more carefully estimated by NMR and UHPLC-HRMS profiling. Using this approach it was possible estimate the proportion of polyphenols versus primary metabolites and thus better evaluate the amount of reagents to be used. The 1H NMR analysis confirmed the presence of characteristic signals of flavonoids (e.g. H-6/H-8 protons between δH 6.4 and 6.0), but also revealed the 9 ACS Paragon Plus Environment


presence of an important amount of saccharides which clearly had to be taken into account for the estimation of the bromine equivalent calculation during the first reaction trial on the crude extract (Figure S3, Supporting information). To eliminate the sugars, the methanolic extract was submitted to filtration on reverse phase vacuum liquid chromatography (VLC) (see experimental section) and yielded an enriched extract mainly composed of flavonoids, as stated by HPLC-ELSD analysis (Figure S4, Supporting information). From 2 g of methanolic extract, 0.66 g of enriched extract was obtained. This roughly indicated that the equivalent calculation in the preliminary experiments were underestimated by a factor 3 and might explain the degradations observed. The bromination reaction were thus performed on 50 mg of this newly obtained enriched methanolic extract with 10 equivalents of NaBr and 21 equivalents of H2O2 (reaction conditions n° 3 of hesperetin named citrus reaction n°2, see experimental section). The reaction was monitored at 1, 3, 6, 18 and 24 hours by HPLC-PDA-ELSD and after 24 hours, the reaction was stopped and dried. UHPLC-TOF-MS profiling of the brominated and the initial crude extract revealed that the major flavonoids naringin (1), hesperidin (2) and poncirin (3) (Figure 3a) were fully converted after reaction (Figure 3b). Almost all peaks detected displayed typical isotopic pattern for brominated compounds as revealed by their negative ion mode HRMS spectra (Figure 3c). Several di-brominated isomers of hesperidin, two tri-brominated hesperitin derivatives (15 and 16), and one di-brominated derivative of poncirin (17) were identified (Figure 3b and 3c). In the positive ion mode UHPLC-TOF-MS profiling, compounds that do not react, such as the poly-methoxylated flavonoids, were highlighted (Figure S5, Supporting information).


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A) 2

5

1

3 4

Col 34

%

765.0094

2 Br

769.0035

15 di-brominated hesperidin

7 7 0 .0 0 5 6

C)

! ! ! 7 5 4 .9 6 0 97 6 4 .1 2 0 5 7 4 7 .1 1 7 27 4 9 .1 2 3 4

745

750

755

536.8039 538.8036

770

775

780

785

3 Br 534.8024

0 500

765

512.0868 521.3151 510 520

530

541.8043 !;542.7984 540

550

! 557.6176 560

16

790

m/z 800

795

17

Col 34

! ! 570.9797575.3052 588.3012 596.7737 m/z 570 580 590 600

16

536.8021

100

3 Br 534.8054

0 500

! 505.5769 510

! 520.1213 520

541.8077

! 534.4578 530

Col 34

17

442.8954

100

2 Br

!

440.8974

444.8952

540.8008

540.8039

! 534.4658

di-brominated

! ! ! 7 8 9 .2 1 7 87 9 3 .7 8 3 97 9 8 .3 9 0 6

7 7 2 .0 1 4 0 7 8 0 .8 7 2 7

15

Col 34 100

760

! 7 7 1 .0 1 6 7

%

0 740

tetra-brominated hesperetin

5

767.0049

100

%

B)

%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


! !;542.9996551.8054

540

550

! ! ! 570.9589 582.8009 564.9842

560

570

580

! 445.8978 ! 594.7672 590

m/z 600

409.1380 0 400

410

! 440.4521

423.2288 420

430

440

!;446.3944 ! 459.0172 450

460

! ! 487.0741491.2634495.2256 473.0534 m/z 480 490 500

470

Figure 3. A) UHPLC-TOF-HRMS analysis of the enriched methanolic extract of C. sinensis before and B) after bromination. C) HR-MS (negative mode) spectra of compounds 15, 16 and 17. In order to isolate these brominated compounds for full identification and further biological testing, the reaction was up-scaled, and 1g of the enriched extract was brominated and purified by reverse phase semi prep. HPLC chromatography. The profiling of the up-scaled reactions were very similar to the one obtained at the 50 mg scale. Using this approach, a series of tribrominated derivatives (15-17) was obtained together with the known methoxylated flavonoids (18-22)16 and one limonene derivative (5)22 already described from Citrus sinensis (Figure 4).



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2

A)

0hs

19

UV 280 nm 18 5

24hs

19

UV 280 nm

5 20 18

21 15 22

OH Br

Br HO

OH

OCH3 HO

B) Br

OH

OCH3

Br

O

OH O

O Br

Br

(15)

OH OH

OH OH

O

O

O

OH

OH

OCH3 OCH3 O

H3CO

OH O

OH O

R1 H3CO

O

Br

(16)

OH O

R2 OCH3O

(2)

OCH3

Br HO

OCH3 O

O

17 16

(17)

(18) R1 (19) R1 (20) R1 (21) R1

= H; R2 = OCH3 = OCH3; R2 = H = H; R2 = H = OCH3; R2 = OCH3

O O O

O H 3C

CH3

OCH3 O

H3CO O O

O

OCH3

H3CO OCH3O

(5)

(22)

Figure 4. A) HPLC-PDA analysis of the enriched methanolic extract of C. sinensis before (0 h) and after bromination (24 h). B) Compounds isolated after bromination.

Despite the fact that some of glycosylated brominated derivatives of hesperidin (2) were detected by UHPLC-HRMS analysis (Figure 3b), it was not possible to isolate them. This could be explained either because they are formed in small amounts or because the acidic reaction conditions induced hydrolysis of glycosylated hesperidin (2) since the corresponding aglycone was brominated (compounds 15 and 16).


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In order to check if the method could be applied to crude extracts containing other type of common NP scaffolds, the methanolic extract of Curcuma longa L. (Zingiberaceae) was selected for bromination. This extract is rich in curcuminoids and in sesquiterpene derivatives.23,

24

Prior to bromination the extract was analyzed by UHPLC-TOF-HRMS in

positive ion mode for metabolite profiling (Figure 5A). The dereplication procedure was similar to that applied to the Citrus extract. As expected, the C. longa extract was found to be mainly composed of curcuminoid derivatives such as curcumin (23) (Rt 14.75 min, m/z 369.1334 [M+H]+, calc. for C21H21O6, Δ 1.1 ppm), demethoxy curcumin (24) (Rt= 14.346 min, m/z 339.1215 [M+H]+, calc. 339.1232 for C20H18O5, Δ 2.1 ppm) and bis demethoxy curcumin (25)( Rt = 13.96 min, m/z 309.1095 [M+H]+, calc. 309.1095 for C19H17O4, Δ 10.4 ppm). The UHPLCTOF-HRMS also revealed the presence of less abundant sesquiterpene derivatives: dehydroturmerone (26) (Rt 19.151 min, m/z 217.1582 [M+H]+, calc. 217.1592 for C15H21O, delta 4.6 ppm), α-turmerone or β-turmerone (27) (Rt 20.79 min, m/z 219.1736 [M+H]+ calc. 219.1736 for C15H23O, delta 5.9 ppm) and α-turmerone or β-turmerone (28) (Rt 20.89 min, m/z 219.1719 [M+H]+ calc. 219.1749 for C15H23O, Δ 13.2 ppm). The bromination was performed with 1g of the C. longa methanolic extract. The reaction was performed in a solution of 130 mL of water and 130 mL of MeCN and stirred at 40°C. Hydrogen peroxide (21 eq.) was added to the solution and NaBr (10 eq.) in acetic acid (100 mL). As for the Citrus extract, the reaction was monitored at different timings. In this case, the reaction was faster and the different peaks corresponding to the curcuminoids disappeared 6hs after the initiation of the reaction. As shown in figure 5 B, new peaks appeared in the reaction mixture and their HR-MS spectra revealed typical pattern of mono-, di- and tri-brominated compounds (Figure 5C). Since the curcuma extract was not containing any glycoside derivatives and mainly lipophilic compounds, in this case the reaction mixture was extracted by liquid-liquid partition (H2O: Ethyl acetate). The ethyl acetate fraction containing the 13 ACS Paragon Plus Environment


brominated compounds was purified by semi-preparative HPLC. According to the polarity of the compounds generated, the separation was carried out by a normal phase chromatography. Using this approach, three brominated derivatives (29-31) were obtained together with the unmodified compounds dehydroturmerone (26)24 and sclareol (32)25.

A)

25

100

24

27 or 28

%

23

26

B)

0 -0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

26.00

28.00

30.00

24.00

26.00

28.00

Time 30.00

100

%

26

31

26 30

2.00

4.00

6.00

413.0247

100

C)

8.00

29

410.0493

16.00 380.1002

378.0950

100

18.00

30

414.0523

22.00 l

472.9182 474.9084

100

31 3Br

470.9214

376.2290

415.9963

375.6347

416.9863

0 m/z 406 407 408 409 410 411 412 413 414 415 416 417 418

20.00

1Br %

415.0575

409.0297

14.00

12.00

2Br

411.0227

406.9287

10.00

%

0 -0.00

%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 374

375

476.8971

377.2232 376

377

378

379.1011

379

381.0553 380

381

382

383

m/z 384

0 466

m/z 468

470

472

474

476

478

480

482

Figure 5. A) UHPLC-TOF-HRMS analysis of the enriched methanolic extract of C. longa before and B) after bromination. C) HR-MS spectra of compounds 29, 30 (pos. mode) 31 and (neg. mode). As also shown in the analytical profile of the crude reaction (Figure 5), no trace of the genuine curcuminoids were detected in the reaction. A search of brominated curcuminoid


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derivatives based on their estimated MW also did reveal their presence. This is probably due to the instability of these compounds in acidic conditions.26, 27 Based on their structure the three brominated compounds (29-31) most probably originated from the same sesquiterpene 4hydroxybisabola-2,10-diene-9-one previously described from the C. longa.24 O HO

O

OH

OH Br O

HO

O

Br

(26)

(23)

(29)

OCH3 HO

O

OH

O

HO

O

(24) OCH3

HO OCH3 OH

HO

(30) Br O

OH

Br Br

O

HO

O

(32)

(25)

Br

(31)

Figure 6. Compounds isolated after the bromination of the C. longa methanolic extract. These generic bromination conditions and the enrichment of the extract prior to the reactions enabled a good control and the upscale was readily achievable for isolation the main brominated derivatives. This provided a series of fully characterized brominated NPs that could be submitted to different bioassays. In the present study the antibacterial activity of the 13 isolated brominated derivatives has been tested against methicillin-resistant Staphylococcus aureus (MRSA, ATCC 33591) and Pseudomonas aeruginosa (ATCC 27853). The bromophenol derivatives 7 and 8 presented a very strong antibacterial activity against a S. aureus MRSA strain with a minimum inhibitory concentration (MIC) comparable to gentamicin and vancomycin antibiotics (Table 2). In order to check if the compounds



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presented a selective antibacterial activity they were additionally submitted to a general cytotoxicity assay. This was performed against breast cancer BT-20 cell lines (REF*) and revealed that both compound were cytotoxic in a comparable IC50 range to the antibacterial activity. Thus this degradation brominated compounds presented general toxicity. These compounds also moderate activity against Pseudomonas aeruginosa (ATCC 27853). The genuine flavonoids hesperidin (2) and hesperitin (11) were not active. Among the brominated flavonoids obtained, only the tri-brominated derivatives 15 and 16 showed a moderate activity at 128 and 64 µg/mL, respectively and no cytotoxicity. While such activities remain modest, this indicates that bromination can effectively enhance the antibacterial properties of genuine inactive NPs. Contrary to some of the flavonoids brominated compounds (29-31) obtained from the bromination of the methanolic extract of Curcuma longa remained inactive in these biological assays. Table 2. Minimum inhibitory concentration (MIC) of the halogenated derivatives against Staphylococcus aureus (ATCC33591) and Pseudomonas aeruginosa (ATCC 27853) and cytotoxic activity against breast cancer BT-20 cell line. Compound hesperidin (2) compound 6 compound 7 compound 8 compound 9 hesperitin (11) compound (12) compound (13) compound (14) compound 15 compound 16 compound (29) compound (30) compound (31) gentamicinRef

S. aureus ATCC 33591 (µg/mL) >256 32 1-2 4 128-256 >256 >256 >256 >256 128 64 >256 >256 >256 1.5*

P. aeruginosa ATCC 27853 (µg/mL) >256 >256 128 256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 2* 16

ACS Paragon Plus Environment

IC50 cytotoxicity against BT-20 cells 15.78±9.07 >40 1.71±0.31 2.85±0.71 11.14±4.4 ND >40 >40 >40 >40 >40 ND ND ND NA

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vancomycinRef

0.5-2*

NA

NA

ND = not determined. NA = not appropriate. Ref.= reference compound. *Minimum inhibitory concentration (MIC) according to the selected articles.28-30

Conclusion A procedure for a controlled bromination was developed and applied on two representative crude plant extract mainly composed of polyphenols and sesquiterpenes. The methodology includes the pre-treatment of the extract using vacuum liquid chromatography (VLC) for the elimination of the primary metabolites (mainly sugars) and the enrichment of the secondary metabolites. The approach also included the control of each reaction through a comprehensive metabolite profiling by HPLC-PDA-ELSD and UHPLC-TOF-HRMS. NMR was used to estimate the content of sugar, an aspect that has to be taken in consideration for an unbiased rough estimation of the equivalent of reagents when the reactions are carried out on polar plant extracts. The use of ELSD detection in particular was of great importance in order to estimate the relative percentage of the halogenated derivatives generated during the optimisation phase. The use of a mixture of MeCN and water (1:1) instead of pure water improved the solubility of the crude extract and thus the reaction yield. On the other hand, UHPLC-TOF-HRMS gives the possibility to localise the halogenated compounds directly in the crude reactions thanks to their characteristic isotopic pattern and directly estimate the extent of bromination. Among the 13 brominated compounds obtained, compounds 9, 13, 16, 17, 19, 29, 30 and 31 are described here for the first time. All of the halogenated derivatives obtained were evaluated for their antibacterial and cytotoxic activities. Some of the brominated flavonoids obtained, (15 and 16) presented a moderate antibacterial activity against a MRSA S. aureus (ATCC 33591). While such activities remain



modest, this indicates that bromination can effectively enhance the antibacterial properties of genuine inactive NPs. Two bromophenol derivatives 7 and 8 resulting from the degradation of the flavonoids in C. sinensis presented remarkable antibacterial activities against S. aureus comparable to the used antibiotics gentamicin and vancomycin. Such compounds exhibited however general toxicity against mammalian cells. It has to be noted that similar bromophenols derivatives occur naturally and are present in a large number of marine algae and marine sponges.31-34 These compounds possess many different biological activities including anticancer, antithrombotic, and anti-inflammatory activities.35 Some of these natural bromophenols were reported to exhibit antibacterial activity against Gram positive and Gram negative bacteria.36, 37 As a future perspective, the procedure developed here will be applied to other plant extracts containing other NP scaffolds. In addition other halogenation reactions with chlorine and iodine will be investigated since they can be performed with similar type of conditions.

MATERIALS AND METHODS General Experimental Procedures. NMR spectroscopic data were recorded on a 500 MHz Varian (Palo Alto, CA, USA) INOVA NMR spectrometer and on a Bruker Avance III HD 600 MHz NMR spectrometer equipped with a QCI 5 mm Cryoprobe and a SampleJet automated sample changer (Bruker BioSpin, Rheinstetten, Germany). Chemical shifts are reported in parts per million (δ) using the residual CD3OD signal (δH 3.31; δC 49.0) or the DMSO-d6 signal (δH 2.50; δC 39.5) as internal standards for 1H and 13C NMR, respectively, and coupling constants (J) are reported in Hz. Complete assignments were obtained based on 2D-NMR experiments (COSY, NOESY, HSQC and HMBC). HRESIMS data were obtained on a Waters MicromassLCT Premier time-of-flight mass spectrometer with an electrospray (ESI) interface. Analytical HPLC was carried out on a HP 1260 system equipped with a photodiode array and an ELSD 18 ACS Paragon Plus Environment

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detectors (Sedere, Alfortville, France). Semi-preparative HPLC was performed on a Spot Prep Armen (Saint-Avé, France). Flash chromatography was performed on an Interchim system (Montluçon, France) equipped with a UV detector.

Chemicals. Hesperitin with 99% purity was purchased from Alpha Aesar (Karlsruhe, Germany). Hesperidin with 99% purity was purchased from Across organics (Geel, Belgium). Citrus sinensis L. (Osbeck) (Citrus aurantium L. ssp dulcis), Rubiaceae, dried peal was purchased from Dixa AG-herbs and spices (lot. 160375) (St. Gallen, Switzerland). NaBr with ≥99% (Fluka Chemika, Switzerland), H2O2 at 30%, 1.09 g/cm3 (Reactolab S.A., Servion, Switzerland), glacial acetic acid (Panreac Qumica S.A., Spain, Barcelone). CD3OD and DMSOd6 was purchased at Fluka Chemika, Switzerland.

HPLC-PDA-ELSD analysis of the crude reaction mixtures. HPLC-PDA-ELSD analyses were conducted on a HP 1260 system equipped with a photodiode array detector (Agilent Technologies, Santa Clara, CA, USA) connected to an ELSD Sedex 85 (Sedere, Oliver, France). The HPLC conditions were as follows: Interchim PF10 C18 column (250 × 4.6 mm i.d., 10 μm, Moluçon, France); solvent system MeOH (B) and H2O (A), both containing 0.1% formic acid. Flow rate 1 mL/min; injection volume 20 μL; sample concentration 10 mg/mL in the mobile phase. The UV absorbance was measured at 280 nm and the UV-PDA spectra were recorded between 190 and 600 nm (step 2 nm). The ELSD detection parameters were: pressure 3.5 bar, 40 °C, gain 8. This method was used for the profiling the crude plant extract and bromination reactions. The percentage of the compounds obtained were calculated by the integration of the ELSD peaks using the Agilent OpenLAB software (Agilent Technologies, Santa Clara, CA, USA).



UHPLC-TOF-ESI-HRMS analysis of the crude reaction mixtures. Aliquots (0.1 mL) of each reaction were collected each hour and analyzed by UHPLC-TOF-ESI-HRMS using the followed conditions: ESI conditions were as follows: capillary voltage 2400 V, cone voltage 40 V, MCP detector voltage 2500 V, source temperature 120 °C, desolvation temperature 300 °C, cone gas flow 20 L/h, and desolvation gas flow 700 L/h. Detection was performed in negative ion mode (PI) with a m/z range of 100–1300 Da and a scan time of 0.5 s in the W-mode. The MS was calibrated using sodium formate, and leucine encephalin (Sigma-Aldrich, Steinheim, Germany) was used as an internal reference at 2 μg/mL and infused through a Lock Spray™ probe at a flow rate of 10 μL/min with the help of a second LC pump. The separation was performed on an Acquity BEH C18 UPLC column (1.7 µm, 150 ×2.1 mm i.d.; Waters, Milford, MA, USA). For the plant extract profiling a linear gradient (solvent system: A) 0.1% formic acid–water, B) 0.1% formic acid–acetonitrile; gradient: 5-95% B in 30 min, then 95% B for 10 min re-equilibration step of 10 min at 5% B; flow rate 0.46 mL/min) was used. For a pure compound analysis, a linear gradient (solvent system: A) 0.1% formic acid–water, B) 0.1% formic acid–acetonitrile; gradient: 5-95% B in 4.8 min, flow rate 0.3 mL/min) was used. In all cases the temperature was set to 40 °C. The injected volume was kept constant (2 µL).

Extraction. Powdered peels (100 g) were extracted with MeOH by percolation at room temperature. The filtrate was concentrated to dryness under reduced pressure at 40 °C yielding the methanolic extract (15.4 g; 15.4% dry weight).

Enrichment of the C. sinensis extract by vacuum liquid chromatography (VLC). The Methanolic extract of C. sinensis peels (8.5 g) was fractionated by vacuum liquid chromatography (VLC) in a sintered glass filter funnel (250 mL) dry packed with 110 g Zeoprep 60 C18 as the stationary phase (40−63 μm, Zeochem, Uetikon am See, Switzerland) using a two 20 ACS Paragon Plus Environment

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isocratic steps elution with H2O 100% (900 mL) affording a 3.6 g of a fraction rich in sugars. A second elution step was performed with MeOH 100% (1200 mL) affording a 2.8 g of a fraction rich in secondary metabolites. This last fraction was characterized by UHPLC-TOFHRMS and used for the bromination reactions. The preparative VLC conditions were optimized using analytical solid phase extraction cartridge packed with the same stationary phase.

Bromination of the methanolic C. sinensis extract (citrus reaction N°1). The enriched VLC methanolic fraction of C. sinensis (2g) was solubilized using sonication in a solution of 40 mL of water and acetic acid (25 mL). Hydrogen peroxide (30 eq.) was added to the solution and NaBr (10 eq.). The mixture was stirred at room temperature at 25 °C for 24 h. The reaction was monitored by HPLC-PDA-ELSD. At the end, the crude reaction was transfer to a funnel and extracted by ethyl acetate (3 x 100 mL). The organic phases were pooled, washed with water (6 x 100 mL) and evaporated under reduced pressure yielding 2 g of the ethyl acetate fraction. This fraction was purified by normal phase flash chromatography using an Interchim normal phase column SIHP-80g (205 x 31 mm i.d., 10 µm, Montluçon, France). The flow rate was set at 20 mL/min. The UV detection was recorded at 280 nm. The injection was performed by dry load by mixing 2 g of the extract with 6 g of the Celite (Interchim, Montluçon, France). The mixture was conditioned in a dry-load cell (128 × 22 mm i.d., Interchim, Montluçon, France). The dry-load cell was connected subsequently between the pumps and the flash column. The solvent system used was (A) hexane and (B) ethyl acetate; gradient mode: 2% to 25% of B in 84 min followed by 25 to 100% B in 2 min, and the column was wash with 100% of B during 19 min, yielding compounds 6 (6 mg), 7 (1.3 mg), 8 (3.1 mg), 9 (1.3 mg) and 10 (3.6 mg).

Bromination of hesperitin (11). 50 mg of hesperitin was solubilized in a solution of 6.5 mL of water and 6.5 mL of MeCN. Hydrogen peroxide (21 eq.) was added to the solution and NaBr 21 ACS Paragon Plus Environment


(1.0-100 eq.) in acetic acid (1-10 mL). The mixture was stirred at room temperature or 40 °C for 1 to 24 h (see table 1). The reaction was monitored by HPLC-PDA-ELSD. At the end, the crude was treated neutralized with Na2CO3 until pH 6.7 and the solvent was evaporated under reduced pressure. The reaction was monitored by HPLC-PDA-ELSD. Final products were isolated and purified by reverse phase semi-preparative HPLC chromatography using an Interchim reverse phase column PFC18 HP (250 x 21.2 mm i.d., 10 µm, Montluçon, France). The flow rate was set at 20 mL/min and the UV detector was performed at 280 nm. The injection volume was 500 µL (22 mg, 2 injections). The solvent system used was (A) MeOH and (B) H2O, both containing 0.1% formic acid. Reaction n°3 (Table 1) was purified using isocratic elution: 55% of B in 110 min followed by 70 to 100% B in 10 min yielding 12 (12.7 mg), 13 (5.5 mg), 14 (2.6 mg). For the purification of reaction n°4 (Table 1) the injection volume was 500 µL (34 mg). The solvent system used was (A) MeOH and (B) H2O, both containing 0.1% formic acid. Purification was performed using a gradient elution: 5 to 100 % B in 60 min, followed 100% B during 10 min yielding 15 (9.2 mg), 16 (5.8 mg).

Bromination of the methanolic C. sinensis extract (citrus reaction N°2). The enriched VLC methanolic fraction of C. sinensis (1 g) was solubilized in a solution of 130 mL of water and 130 mL of MeCN. Hydrogen peroxide (21 eq.) was added to the solution and NaBr (10 eq.) in acetic acid (100 mL). The mixture was stirred at at 40°C for 24 h. The reaction was monitored by HPLC-PDA-ELSD. At the end, the crude was treated neutralized with Na2CO3 until pH 6.7 and the solvent was evaporated under reduced pressure. The resulting salt was removed by vacuum liquid chromatography using the same procedure developed to enrich the methanolic extract of Citrus described above. After this process, 0.77g was obtained. Final products were isolated and purified by reverse phase semi-preparative HPLC chromatography using an Interchim reverse phase column PFC18 HP (250 x 21.2 mm i.d., 10 µm, Montluçon, France).


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The flow rate was set at 20 mL/min and the UV detector was performed at 280 nm. The injection was performed by dry load by mixing 0.39 g of the extract with 1 g of the Zeoprep C18 stationary phase (two subsequent injections). The mixture was conditioned in a dry-load cell (250 × 10 mm i.d.). The dry-load cell was connected subsequently between the pumps and the HPLC column. The solvent system used was (A) MeOH and (B) H2O, both containing 0.1% formic acid. The elution followed a step gradient: 25% to 90% of A in 100 min followed by 90 to 100% A in 10 min. yielding the brominated derivatives 15 (2.2 mg), 16 (1.8 mg,) and 17 (2 mg).

Bromination of the methanolic extract of Curcuma longa. The methanolic extract of fraction of C. longa (1g) was solubilized in a solution of 130 mL of water and 130 mL of MeCN. Hydrogen peroxide (21 eq.) was added to the solution and NaBr (10 eq.) in acetic acid (100 mL). The mixture was stirred at 40°C for 6h. At the end, the crude extract was neutralized with Na2CO3 until pH 6.7. The crude reaction was transferred to a separating funnel and extracted with ethyl acetate (3 x 350 ml). The organic phases were pooled, and the solvent was evaporated under reduced pressure, yielding 0.91g. Final products were isolated and purified by normal phase semi prep. HPLC chromatography using an Interchim column PF-SIHP10 (250 x 21.2 mm i.d., 10 µm, Montluçon, France). The flow rate was set at 20 mL/min and the UV detection was set at 260 nm. The injection was performed by dry load by mixing 0.38g of the extract with 1g of the Zeoprep 60 stationary phase. The mixture was conditioned in a dry-load cell (250 × 10 mm i.d.). The dry-load cell was connected subsequently between the pumps and the HPLC column. The solvent system used was (A) hexane and (B) ethyl acetate, both containing. The elution followed a step gradient: 2% to 6% of B in 18 min followed by 6% to 50% of B in 60 min, followed by 50% to 100% B over 20 min. Yielding three brominated derivatives (29)(1.4 mg), (30)(1.5 mg), (31)(1.1 mg) and α-turmerone (27)(1.3 mg), and sclareol (1.2 mg)(32).



Page 24 of 43

Description of the isolated compounds 2,4,6-Tribromophenol (6). Amorphous solid; 1H NMR (DMSO-d6, 500 MHz) δ 7.77 (2H, s, H-3 and H-5). 13C NMR (DMSO-d6, 125 MHz) δ 111.4 (C-2 and C-6), 112.9 (C-4), 134.0 (C3 and C-5), 150.5 (C-1). HREIMS m/z 326.7681 [M-H]- (calcd for C6H2Br3O, 326.7656, ∆ ppm = 7.7).

2,2-dibromo-1-(4-methoxyphenyl)ethan-1-one (7). Amorphous solid; 1H NMR (DMSO-d6, 500 MHz) δ 3.88 (3H, s, OCH3), 7.11 (2H, d, J = 9.2 Hz, H-3, 5), 7.82 (1H, s, H-8), 8.07 (2H, d, J = 9.2 Hz, H-2, 6). 13C NMR (DMSO-d6, 125 MHz) δ 43.2 (C-8), 55.8 (OCH3), 114.5 (C-3 and C-5), 123.1 (C-1), 131.9 (C-2 and C-6), 164.2 (C-4), 185.4 (C-7). HREIMS m/z 306.8966 (calcd for C9H9Br2O2, 306.8969, ∆ ppm = -0.98).

2,2-dibromo-1-(3,4-dimethoxyphenyl)ethan-1-one (8). Amorphous solid; 1H NMR (DMSOd6, 500 MHz) δ 3.84 (3H, s, OCH33), 3.88 (3H, s, OCH310), 7.13 (1H, d, J = 8.5 Hz, H-5), 7.54 (1H, d, J = 2.1 Hz, H-2), 7.79 (1H, dd, J = 8.5, 2.1 Hz, H-6), 7.88 (1H, s, H-8).

13C

NMR

(DMSO-d6, 125 MHz) δ 43.1 (C-8), 55.7 (OCH33), 55.9 (OCH34), 111.2 (C-5), 111.5 (C-2), 123.0 (C-1), 124.4 (C-6), 148.9 (C-3), 154.3 (C-4), 185.5 (C-7). HREIMS m/z 336.9067 (calcd for C10H11Br2O3, 336.9075, ∆ ppm = -2.37).

8,8-dibromo-7-(3,5-dibromo-4-hydroxyphenyl)ethyl acetate (9). Amorphous solid; 1H NMR (DMSO-d6, 500 MHz) δ 2.18 (3H, s, COCH3), 6.08 (1H, d, J = 5.0 Hz, H-7), 6.54 (1H, d, J = 5.0 Hz, H-8), 7.66 (2H, s, H-2 and H-6).13C NMR (DMSO-d6, 125 MHz) δ 20.5 (COCH3), 47.6 (C-8), 76.0 (C-7), 111.6 (C-3 and C-5), 130.2 (C-1), 131.2 (C-2 and C-6), 151.3 (C-4), 169.0 (C-9). 24 ACS Paragon Plus Environment

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3,4-dimethoxybenzoic acid (10). Amorphous solid; 1H NMR (DMSO-d6, 500 MHz) δ 3.80 (3H, s, OCH33), 3.82 (3H, s, OCH34), 7.04 (1H, d, J = 8.5 Hz, H-5), 7.44 (1H, d, J = 2.0 Hz, H-2), 7.56 (1H, dd, J = 8.5, 2.0 Hz, H-6), 12.61 (1H, s, COOH).

13C

NMR (DMSO-d6, 125

MHz) δ 55.4 (OCH33), 55.6 (OCH34), 111.0 (C-5), 111.9 (C-2), 122.9 (C-1), 123.1 (C-6), 148.3 (C-3), 152.6 (C-4), 167.0 (COOH). HREIMS m/z 181.0497 [M-H]- (calcd for C9H9O4, 181.0501, ∆ ppm = -2.2).

8-bromo-hesperitin (12). Amorphous solid; 1H NMR (Methanol-d4, 600 MHz) δ 2.86 (1H, dd, J = 17.1, 3.3 Hz, H-3b), 3.10 (1H, dd, J = 17.1, 12.1 Hz, H-3a), 3.87 (3H, s, OCH3), 5.47 (1H, dd, J = 12.1, 3.3 Hz, H-2), 6.06 (1H, s, H-6), 6.95 (2H, m, H-5' and H-6'), 7.01 (1H, d, J = 1.3 Hz, H-2') 13C NMR (Methanol-d4, 151 MHz) δ 43.5 (C-3), 56.4 (OCH3), 80.6 (C-2), 89.9 (C8), 97.2 (C-6), 104.0 (C-10), 112.6 (C-5'), 114.5 (C-2'), 118.8 (C-6'), 132.7 (C-1'), 147.8 (C-3'), 149.4 (C-4'), 160.7 (C-9), 164.0 (C-7), 164.8 (C-5), 197.5 (C-4). HREIMS m/z 378.9816 [MH]- (calcd for C16H12O6Br, 378.9817, ∆ ppm = -0.42).

6-bromo-hesperitin (13). Amorphous solid; 1H NMR (Methanol-d4, 600 MHz) δ 2.76 (1H, dd, J = 17.2, 3.0 Hz, H-3b), 3.11 (1H, dd, J = 17.2, 12.7 Hz, H-3a), 3.87 (3H, s, OCH3), 5.35 (1H, dd, J = 12.7, 3.0 Hz, H-2), 6.07 (1H, s, H-8), 6.94 (3H, m, H-2', H-5' and H-6').

13C

NMR

(Methanol-d4, 151 MHz) δ 43.7 (C-3), 56.4 (OCH3), 80.4 (C-2), 91.3 (C-6), 96.6 (C-8), 103.3 (C-10), 112.6 (C-5'), 114.5 (C-2'), 119.0 (C-6'), 132.9 (C-1'), 147.8 (C-3'), 149.4 (C-4'), 161.7 (C-5), 163.3 (C-9), 165.7 (C-7), 197.3 (C-4). HREIMS m/z 378.9816 [M-H]- (calcd for C16H12O6Br, 378.9817, ∆ ppm = -0.34).



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6,8-di-bromo-hesperitin (14). Amorphous solid; 1H NMR (Methanol-d4, 600 MHz) δ 2.84 (1H, dd, J = 17.1, 3.2 Hz, H-3b), 3.09 (1H, dd, J = 17.1, 12.1 Hz, H-3a), 5.44 (1H, dd, J = 12.1, 3.2 Hz, H-2), 6.95 (2H, m, H-5' and H-6'), 7.01 (1H, s, H-2').

13C

NMR (Methanol-d4, 151

MHz) δ 43.1 (C-3), 56.4 (OCH3), 80.4 (C-2), 92.2 (C-6/8), 93.1 (C-6/8), 102.5 (C-10), 112.6 (C-5'), 114.5 (C-2'), 118.8 (C-6'), 132.9 (C-1'), 147.8 (C-3'), 149.3 (C-4'), 159.4 (C-9), 160.5 (C-5/7), 196.1 (C-4). HREIMS m/z 456.8921 [M-H]- (calcd for C16H11O6Br2, 456.8922, ∆ ppm = -0.4).

6,8,2’-tri-bromo-hesperitin (15). Amorphous solid; 1H NMR (DMSO-d6, 600 MHz) δ 2.88 (1H, dd, J = 17.1,2.9 Hz, H-3b), 3.42 (1H, overlapped, H-3a), 3.86 (3H, s, OCH3), 5.83 (1H, dd, J = 13.2, 2.9 Hz, H-2), 7.10 (1H, d, J = 8.6 Hz, H-5'), 7.14 (1H, d, J = 8.6 Hz, H-6'), 9.65 (1H, brs, OH-3´), 12.8 (1H, s, OH-5).

13C

NMR (DMSO-d6, 151 MHz) δ 40.3 (C-3), 56.2

(OCH3), 78.5 (C-2), 90.9 (C-6), 102.7 (C-10), 110.5 (C-2'), 110.6 (C-5'), 118.1 (C-6'), 129.0 (C-1'), 143.9 (C-3'), 148.6 (C-4'), 157.7 (C-7/9), 158.4 (C-5), 159.0 (C-7/9), 196.4 (C-4). HREIMS m/z 534.8049 [M-H]- (calcd for C16H10O6Br3, 534.8027, ∆ ppm = 4.1).

6,8,6’-tri-bromo-hesperitin (16). Amorphous solid; 1H NMR (DMSO-d6, 600 MHz) δ 2.82 (1H, dd, J = 17.1, 2.9 Hz, H-3b), 3.34 (1H, overlapped, H-3a), 3.82 (3H, s, OCH3), 5.75 (1H, dd, J = 13.2, 2.9 Hz, H-2), 7.13 (1H, s, H-2'), 7.19 (1H, s, H-5'), 12.8 (1H, s, OH-5). 13C NMR (DMSO-d6, 151 MHz) δ 40.5 (C-3), 56.1 (OCH3), 78.4 (C-2), 91.0 (C-6), 110.1 (C-6'), 114.7 (C-2'), 116.0 (C-5'), 128.6 (C-1'), 146.3 (C-3'), 148.9 (C-4'), 158.4 (C-5), 158.4 (C-5), 196.1 (C-4). HREIMS m/z 534.8013 [M-H]- (calcd for C16H10O6Br3, 534.8027, ∆ ppm = -2.6).


Page 27 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6,8-di-bromo-5,7-hydroxy-4’-methoxy-flavanone (17). Amorphous solid; 1H NMR (DMSOd6, 600 MHz) δ 2.63 (1H, overlapped, H-3b), 2.97 (1H, dd, J = 16.9, 11.3 Hz, H-3a), 3.76 (3H, s, OCH3), 5.39 (1H, dd, J = 11.3, 3.4 Hz, H-2), 6.96 (2H, d, J = 8.7 Hz, H-3' and H-5'), 7.43 (2H, d, J = 8.7 Hz, H-2'and H-6'), 13.5 (1H, s, OH-5). HREIMS m/z 440.8963 [M-H]- (calcd for C16H11O5Br2, 440.8973, ∆ ppm = -2.3).

3,11-dihydroxy-4,10-dibromo-9-oxo-1-bisabolen (29). 1H NMR (CDCl3, 600 MHz) δ 0.95 (3H, d, J=7.0 Hz, H-14A), 0.96 (3H, d, J=6.9 Hz, H-14B), 1.40, 1.40 and 1.41 (6H, s, H-12, H13), 1.48 (3H, s, H-15A), 1.49 (3H, s, H-15B), 2.04 (1H, m, H-5''), 2.17 (1H, m, H-5'), 2.29 (1H, m, H-7), 2.46 (1H, m, H-6), 2.54 (1H, dd, J=17.7, 7.6 Hz, H-8''A), 2.69 (2H, d, J=6.8 Hz, H-8B), 2.87 (1H, dd, J=17.7, 5.8 Hz, H-8'A), 4.23 (1H, s, H-10A), 4.24 (1H, s, H-10B), 4.32 (1H, dd, J=7.2, 3.2 Hz, H-4), 5.66 (1H, m, H-1), 5.73 (1H, m, H-2); 13C NMR (CDCl3, 151 MHz) δ 16.7 (C-14), 27.0 and 27.6 (C-12, C-13), 27.4 (C-15), 30.4 (C-5), 32.0 (C-7), 37.7 (C6), 45.8 and 46.0 (C-8), 58.8 (C-4), 60.7 and 61.3 (C-10), 70.2 (C-3), 71.2 (C-11), 131.4 (C-1), 131.7 (C-2), 204.9 and 205.1 (C-9). HREIMS m/z 428.0405 [M+NH4]+ (calcd for C15H28O3NBr2, 428.0436, ∆ = 1.2 ppm)

3-hydroxy-4-bromo-9-oxo-1,10-bisaboladien (30). 1H NMR (CDCl3, 600 MHz) δ 0.93 (3H, d, J=6.7 Hz, H-14), 1.47 (3H, s, H-15), 1.89 (3H, d, J=1.3 Hz, H-13), 2.05 (1H, m, H-5''), 2.14 (3H, d, J=1.3 Hz, H-12), 2.18 (1H, m, H-5'), 2.23 (1H, m, H-7), 2.27 (1H, d, J=15.1 Hz, H-8''), 2.42 (1H, m, H-6), 2.45 (1H, dd, J=15.1, 5.3 Hz, H-8'), 4.32 (1H, ddt, J=7.4, 3.3, 0.8 Hz, H-4), 5.67 (1H, ddd, J=10.2, 2.6, 0.7 Hz, H-1), 5.71 (1H, ddd, J=10.2, 2.2, 0.9 Hz, H-2), 6.06 (1H, hept, J=1.3 Hz, H-10); 13C NMR (CDCl3, 151 MHz) δ 17.2 (C-14), 20.8 (C-12), 27.2 (C-15), 27.7 (C-13), 30.8 (C-5), 32.9 (C-7), 38.2 (C-6), 48.7 (C-8), 59.2 (C-4), 70.2 (C-3), 124.0 (C27 ACS Paragon Plus Environment


10), 131.4 (C-2), 131.6 (C-1), 155.8 (C11), 200.3 (C-9). HREIMS m/z 378.1045 [M+ACN+Na]+ (calcd for C17H26O2NBrNa , 378.1056, ∆ = 2.9 ppm)

3-hydroxy-4,10,11-tribromo-9-oxo-1-bisabolen (31). 1H NMR (CDCl3, 600 MHz) δ 0.96 (3H, d, J=6.9 Hz, H-14), 1.48 (3H, s, H-15), 1.95 (3H, s, H-13), 2.03 (3H, s, H-12), 2.07 (1H, m, H-5''), 2.18 (1H, m, H-5'), 2.31 (1H, m, H-7), 2.49 (1H, tdt, J=6.8, 4.6, 2.4 Hz, H-6), 2.61 (1H, dd, J=17.7, 8.4 Hz, H-8''), 2.73 (1H, dd, J=17.7, 4.9 Hz, H-8'), 4.32 (1H, dd, J=7.2, 3.2 Hz, H-4), 4.68 (1H, s, H-10), 5.68 (1H, ddd, J=10.1, 2.6, 0.7 Hz, H-1), 5.73 (1H, ddd, J=10.1, 2.3, 1.0 Hz, H-2); 13C NMR (CDCl3, 151 MHz) δ 16.5 (C-14), 27.3 (C-15), 28.3 (C-12), 30.6 (C-5), 31.6 (C-7), 34.6 (C-13), 37.7 (C-6), 46.9 (C-8), 58.7 (C-10), 59.0 (C-4), 62.4 (C-11), 70.2 (C-3), 131.6 (C-1, C-2), 200.8 (C-9). HREIMS m/z 470.9170 [M-H]- (calcd for C15H22O2Br3, 470.9196, ∆ = 5.5 ppm).

Antibacterial assay. Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 33591) and Pseudomonas aeruginosa (ATCC 27853) were used for the antibacterial assay. The minimum inhibitory concentration (MIC) of the different compounds were determined in triplicate using the broth dilution method in 96-well microtiter plates as previously described (Wiegand et al., 2008). Briefly, compounds were resuspended at 10.24 mg/mL in DMSO and serially diluted in Mueller−Hinton broth (MHB, Oxoid). The maximum initial concentration used for this assay was 256 µg/mL. After an incubation of 24 h at 37 °C, Iodonitrotetrazolium chloride (INT, Sigma-Aldrich) was added to each wells, as growth indicator, and incubated for several hours (Eloff, 1998). The highest dilution of a compound in which no growth appears corresponds to its MIC. Gentamicin and Vancomycin (source) were used as control of inhibition and compared to the reference values.


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Cytotoxic assays. BT-20 TNBC cells were detached by Trypsin/EDTA solution (Invitrogen) and re-suspended at 40’000 cells/ml and were added into each well of a transparent 384-well plate (VWR) using cell dispenser/washer (BioTek). The cells were maintained in DMEM containing 10% FBS and incubated at 37°C, 5% CO2 overnight. The next day medium of each well was replaced by 50μl fresh medium containing serial dilutions of the compounds starting from 41 μg/ml. After incubation for 3 days, the medium in each well was replaced by 30μl of 0.5 mg/ml Thiazolyl blue (Carl Roth) solution in 1xPBS. The plates were incubated for 3h at 37°C. Then the solution was removed, and 50μl DMSO was added into each well. Absorbance at 570 nm was measured in the plate reader (M Plex, Tecan).

Acknowledgements The School of Pharmaceutical Sciences of the University of Geneva (Prof. J-L. Wolfender) is thankful to the Swiss National Science Foundation for the support in the acquisition of the NMR 600 MHz (SNF R’Equip grant 316030_164095). The authors would like to thanks Mr. Frédéric Borlat for his

assistance during some reaction experiments.

ASSOCIATED CONTENT

Supporting Information UHPLC-PDA-TOF/MS and HPLC-PDA-ELSD analyses and NMR spectra of the brominated compounds

Author information Corresponding Author *Tel: +41 223793641. Fax: +41 223793399. E-mail: [email protected] (E.F.Q). 29 ACS Paragon Plus Environment


ORCID

Emerson Ferreira Queiroz: 0000-0001-9567-1664 Jean-Luc Wolfender: 0000-0002-0125-952X

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

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Graphical Table of Contents OH

Citrus sinensis

Before reaction

OCH3 HO

OH

O

OH OCH3

Br HO

Plant extract bromination

Curcuma longa

O

Br

O

Br

After reaction

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

OH O O Br Br HO

Analytical reaction monitoring

Br


Page 36 of 43

Citrus sinensis Plant extract bromination

After reaction

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41


Before reaction

Page 37 of 43

Curcuma longa

Analytical reaction monitoring



A)

100

Citrus sinensis methanolic extract

%

Hesperidin (2)

Citrus sinensis methanolic extract (2g)

Naringin (1) Poncirin (3)

AcOH (25 ml) NaBr (10eq), H2O2 (30eq) Water (40ml), 25°C, 24hs

0

2.00

B)

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

100

After reaction

*Brominated derivatives

%

*

0 2.00

4.00

6.00

*

* *

8.00

10.00

D)

**

12.00

14.00

16.00

Time 20.00

18.00

(6) 334.8366

100

2 Br

336.8355

332.8389

6

328.7642 330.7618

100

(7)

3 Br %

C)

%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Page 38 of 43

326.7654 332.7611

! 306.8795

0

300

305

310.8466

310

! 316.9524

315

320

(8)

! 337.8380 325.0103

325

!;341.0984

330

335

340

352.8603

345

350

355

360.9417

360

! 304.9166

367.1336

365

370

m/z

! 308.1924

313.2419

! 324.8104

! 350.7914

333.7628

0 300

305

310

315

320

325

330

335

340

345

350

! 354.1763 355

! 362.9044 360

365

! 372.7638 m/z 370


(9)

(10)

Page 39 of 43

ACS Combinatorial Science mAU

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

A)

140 120

0h

11

Standard

100 80 60

mAU 100 90

5

10

15

Reaction n°3

20

12

25

35 min

30

13

24h

80

70

14

11

60 50

5

mAU 85 80 75 70 65 60 55 50 45

Reaction n°4

10

15

20

14

25

15

24h

16

5

10

15

35 min

30

20

25

35 min

30

B) (11)

(12)

(13)

(14)

(15)

(16)



A) 2

5

1

3 4

B)

767.0049

100

tetra-brominated hesperetin

5

Col 34

%

2 Br 765.0094

! 747.1172

0 740

745

749.1234

750

755

di-brominated hesperidin

16

772.0140

765

770

775

! ! ! 789.2178 793.7839 798.3906

780.8727

780

785

790

m/z 800

795

17

Col 34

536.8039 538.8036

100

3 Br 534.8024

Col 34

3 Br 534.8054

512.0868 521.3151 510

520

! 534.4658 530

17

442.8954

100

!

2 Br

444.8952 440.8974

540.8008

540.8039

!;542.7984 540

550

! 445.8978

541.8077

541.8043 0 500

16

536.8021

100

%

15

Col 34

%

C)

di-brominated

! 771.0167

! 764.1205

760

15

769.0035

770.0056

! 754.9609

%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Page 40 of 43

! 557.6176 560

! ! 570.9797 575.3052 588.3012 596.7737 m/z 570 580 590 600

0 500

! 505.5769 510

! 520.1213 520

! 534.4578 530

! !;542.9996551.8054 540

550

! 564.9842

560

! ! 570.9589 582.8009

570

580


! 594.7672 590

m/z 600

409.1380 0 400

410

! 440.4521

423.2288 420

430

440

!;446.3944 ! 459.0172 450

460

! 473.0534 470

! 487.0741491.2634495.2256 m/z 490 500

480

Page 41 of 43

ACS Combinatorial Science 2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

A)

0hs

19

UV 280 nm 18

5

19

UV 280 nm

24hs

5 20 18

21 15 22

17 16

B) (16)

(15)

(18) R1 = H; R2 = OCH3 (19) R1 = OCH3; R2 = H (20) R1 = H; R2 = H (21) R1 = OCH3; R2 = OCH3

(2)

(5)

(17)

ACS Paragon Plus (22)Environment


A)

100

25

24

27 or 28

%

23

26 0 -0.00

B)

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

26.00

28.00

30.00

24.00

26.00

28.00

Time 30.00

100

26 %

31

26 30

2.00

4.00

6.00

8.00

413.0247

100

29

C)

12.00

18.00

380.1002

378.0950

100

20.00

30

414.0523

472.9182 474.9084

100

31 3Br

470.9214

376.2290

476.8971 375.6347

416.9863

0 m/z 406 407 408 409 410 411 412 413 414 415 416 417 418

22.00 l

1Br

415.9963 410.0493

16.00

%

415.0575

406.9287

14.00

2Br

411.0227

409.0297

10.00

%

0 -0.00

%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Page 42 of 43

0 374

375

377.2232 376

379.1011

381.0553

ACS Paragon Plus Environment 377

378

379

380

381

382

383

m/z 384

0 466

m/z 468

470

472

474

476

478

480

482

Page 43 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41


(23)

(26)

(29)

(30)

(24)

(25)

(32)


(31)

Chemo-Diversification of Plant Extracts Using a Generic Bromination

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