Synthesis, antibacterial and cytotoxic activities of new biflorinbased hydrazones and oximes

Synthesis, antibacterial and cytotoxic activities of new biflorin-based

hydrazones and oximes

Luciana G. da S. Souza

a

, Macia C. S. Almeida

a

, Telma L. G. Lemos

a,

⇑

_{, Paulo R. V. Ribeiro}

b

_{, Edy S. de Brito}

b

_,

Vera L. M. Silva

c

_{, Artur M. S. Silva}

c,

_⇑

_{, Raimundo Braz-Filho}

d

_{, José G. M. Costa}

e

_{, Fábio F. G. Rodrigues}

e

_,

Francisco S. Barreto

f

, Manoel O. de Moraes

f

a_{Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Campus do Pici, 60451-970 Fortaleza, CE, Brazil} b_{Embrapa Agroindustria Tropical, R Dra Sara Mesquita, 2270, 60511-110 Fortaleza, CE, Brazil}

c_{Department of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal}

d_{Laboratório de Ciências Químicas, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28013-602 Campos dos Goytacazes, RJ, Brazil} e_{Laboratório de Pesquisa de Produtos Naturais, Universidade Regional do Cariri, 63105-000 Crato, CE, Brazil}

f_{Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, 60430-270 Fortaleza, CE, Brazil}

a r t i c l e

i n f o

Article history:

Received 6 October 2015 Revised 24 November 2015 Accepted 26 November 2015 Available online 27 November 2015

Keywords:

Biflorin Hydrazones Oximes Antibacterial Antitumoral

a b s t r a c t

Biflorin1is a biologically active quinone, isolated fromCapraria biflora. Five new biflorin-based nitrogen derivatives were synthesized, of which two were mixtures of (E)- and (Z)- isomers: (Z)-2a, (Z)-2b, (Z)-3a, (Z)- and (E)-3b, (Z)- and (E)-3c. The antibacterial activity was investigated using the microdilution method for determining the minimum inhibitory concentration (MIC) against six bacterial strains. Tests have shown that these derivatives have potential against all bacterial strains. The cytotoxic activity was also evaluated against three strains of cancer cells, but none of the derivatives showed activity.

Ó2015 Published by Elsevier Ltd.

Quinones represent a wide and varied family of secondary

metabolites of natural occurrence. The interest in these substances

has been intensified in recent years due to their pharmacological

importance and great structural variety. Many natural and

syn-thetic quinone derivatives possess potent and varied

pharmacolog-ical effects such as antitumor,

1–3

_{anti-inflammatory,}

4,5

_analgesic,

6

antifungal,

7,8

_{and trypanocidal}

9,10

_activities.

Biflorin

[6,9-dimethyl-3-(4-methylpent-3-en-1-yl)benzo[

de

]

chromene-7,8-dione]

1

is a prenylated

ortho

-naphthoquinone

isolated from the roots of

Capraria biflora

L. species. There two

naphthoquinone

isomers,

ortho

-naphthoquinones

and

para

-naphthoquinones. They are widespread in the plant kingdom

and, due to its redox properties they can interfere in different

biological oxidative processes.

11

_{Several naphthoquinones were}

found to exhibit interesting range of pharmacological properties

such as antimicrobial,

12,13

_antiviral,

14

_antifungal,

15

_{trypanocidal,}

16

antimalarial,

17,18

_{and anticancer}

19

_{activities. Some}

_ortho

-naphtho-quinones, are trypanosomatid growth inhibitors with high

cytotoxic activity.

20

_{Specifically, biflorin has shown antitumor}

3,21

and antibiotic activities.

22,23

_{Recent studies have shown that this}

compound also presents anticancer melanoma type activity

24

_as

well as anti-metastatic potential,

25

_{being able to inhibit tumor}

colonization.

Herein we present our results on the study of the reactivity of

biflorin

1

, towards different nucleophiles (hydrazines and

hydrox-ylamines) (Scheme 1). Novel nitrogenated derivatives of biflorin

1

were obtained, aiming to study its chemistry and the potential

pharmacological activities of the new derivatives

2

and

3

.

First efforts were focused on the reaction of biflorin

1

with

aryl-hydrazine derivatives (Scheme 2).

26,27

_{The reaction of biflorin}

₁

with phenylhydrazine hydrochloride resulted in the formation of

the corresponding hydrazone

2a

in moderate yield (42%).

27

When using 2,4-dinitrophenylhydrazine, the corresponding

hydrazone

2b

was obtained in good yield (63%), thus confirming

the more nucleophilic character of the terminal nitrogen of

2,4-dinitrophenylhydrazine.

27

_{Despite the possibility of the}

nucleophilic attack of hydrazine derivatives on any of the carbonyl

carbons C-7 or C-8 of

1

, only hydrazone compounds

2

were formed,

resulting from the nucleophilic addition to the more electrophilic

carbonyl group (C-7).

http://dx.doi.org/10.1016/j.bmcl.2015.11.095

0960-894X/Ó2015 Published by Elsevier Ltd.

⇑

Corresponding authors. Tel.: +55 85 3366 9369; fax: +55 85 3366 9782 (T.L.G.L.); tel.: +351 234 370 714; fax: +351 234 370 084 (A.M.S.S.).

E-mail addresses:[email protected](T.L.G. Lemos),[email protected](A.M.S. Silva).

Contents lists available at

ScienceDirect

Bioorganic & Medicinal Chemistry Letters

Then the reaction of biflorin

1

with substituted amine

hydrochlorides

28–30

_{was carried out and led to the formation of}

oxime derivatives

3

, in good yields (51–63%),

30

_{also resulting from}

the nucleophilic addition to the more electrophilic carbonyl group

(C-7) (Schemes 1 and 3). The structures of all compounds were

confirmed by NMR spectroscopic techniques (

1

_H,

13

_{C, COSY, HSQC}

and HMBC) as well as by high resolution mass spectrometry

(HRMS) analysis.

27,30

In this type of reactions it is possible to obtain a mixture of the

(

Z

)- and (

E

)-diastereomers. The configuration of the obtained

prod-ucts was confirmed by NMR. The

1

_{H NMR spectra of the}

hydra-zones

2a

and

2b

(Scheme 2) indicated that only the (

Z

)-isomer

was formed. The (

Z

)-configuration of these hydrazones was

estab-lished by observation of an intramolecular hydrogen bond forming

a six membered ring (Fig. 1), evidenced by the signals at

d

17.23

and 17.42 ppm (N

A

H

O) in the

1

H NMR spectra of hydrazones

2a

and

2b

, respectively.

27

_{The same applies to the oxime derivative}

3a

, where it is observed a signal at

d

19.91 ppm (O

A

H

O)

30

related to the hydrogen bond forming the six-membered ring, in

which only the (

Z

)-isomer is formed (Fig. 1).

In the synthesis of methyloxime

3b

and ethyloxime

3c

it was

observed the formation of a mixture of the two (

Z

)- and (

E

)-iso-mers (Scheme 3). The

1

_{H NMR spectrum of the mixture showed}

the presence of both (

Z

)- and (

E

)-isomers in the ratio 2:3 in both

cases, with methyloxime and ethyloxime. These mixtures were

evidenced by the presence of pairs of singlets at

d

_{4.17 (OC}

_H

₃

₎

and 4.18 ppm (OC

H

3

) assigned to the (

Z

)-

3b

and (

E

)-

3b

isomers,

and the pair of quartets at

d

4.42 (OC

H

2

CH

3

) and 4.46 ppm (OC

H

2

-CH

3

), assigned to the (

Z

)-

3c

and (

E

)-

3c

isomers, respectively.

30

These mixture of isomers can be easily separated by column

chro-matography, but they are gradually converted into the mixture

immediately after its separation into individual isomers.

31

All derivatives, including biflorin

1

,

32,33

_{were tested for}

antibac-terial activity against six bacantibac-terial strains, Gram-positive and

Gram-negative, by employing the microdilution method.

34,35

_The

corresponding minimum inhibitory concentration (MIC) values

are shown in

Table 1.

Previous studies reported the biflorin

1

antibacterial activity

against Gram-positive and Gram-negative bacteria.

22,23

_{In this}

work it was possible to prove this activity with very satisfactory

MIC values (in terms of clinical MIC

6

1.024

l

g/mL),

36

for all

strains of bacteria, using as positive control the antibiotics

Amika-cin (

Ami

), Gentamicin (

Gen

) and Neomycin (

Neo

). The most

satis-factory results were observed against strains of

Proteus vulgaris

(ATCC 13315), with a MIC value of 16

l

g/mL, showing to be more

efficient than antibiotics used as positive control, and for

Entero-coccus faecalis

(ATCC 4083), with values MIC 32

l

g/mL, similar to

that of Gentamicin (

Gen

).

The hydrazones were susceptible to all strains, yielding the best

result for the hydrazone

2a

, with the MIC value of 256

l

g/mL for

Staphylococcus aureus

(ATCC 25923), similar to MIC value of

Amika-cin (

Ami

). The MIC values for hydrazones were well above the

val-ues of biflorin

1

which was used as a standard.

The oximes showed more satisfactory results for all strains of

bacteria, among which methyloxime

3b

and ethyloxime

3c

showed

greater sensitivity against

Enterococcus faecalis

(ATCC 4083), with

MIC values of 32

l

g/mL and 16

l

g/mL, respectively, and

Staphylo-coccus aureus

(ATCC 25923) with MIC values 32

l

g/mL, both. The

MIC values of methyloxime

3b

and ethyloxime

3c

were similar

or even better than those of biflorin

1

, and antibiotics used as

pos-itive control, suggesting that the modifications made on the

biflorin structure led to an increase of the antibacterial potential

for these derivatives.

Cytotoxic activities in vitro of novel derivatives of biflorin

1

were evaluated using three human cancer cell lines, SF-295

(glioblastoma), OVCAR-8 (breast cancer) and HCT-116 (colon).

37,38

An intensity scale was used to assess the cytotoxic potential of the

samples tested; being considered samples without activity, with

little (cell growth inhibition 1–50%), moderate (cell growth

inhibi-tion 50–75%) and a high activity (growth inhibiinhibi-tion 75–100%). The

experiments were analyzed according to the mean ± standard

deviation of the percentage of inhibition of cell growth using the

GraphPad Prism program.

Doxorubicin (

Dox

) was used as a positive control, in addition to

biflorin

1

which has a high cytotoxic activity. The activity analysis

was performed by the MTT method used in the screening program

at the National Cancer Institute of the United States (NCI)

39

_and

allows to easily set the cytotoxicity of the substances.

40

_{The results}

obtained indicate that these derivatives showed no activity (Fig. 2),

suggesting that the carbonyl modification caused inactivation of

the substances against cancer cells.

O O O 3a 4 5 6 16 9b 9a 17 9 8 7 13 12 11 10

2 3 15

14 O N O N H R R O N O RO 1

a) R= H b) R= NO2

2 3 a) R= H

b) R= CH3

c) R= CH2CH3

1 6a Formation of hydrazones Formation of oximes

Scheme 1.Synthesis of biflorin derivatives: modification in carbonyl C-7.

O O O 1 3a 4 5 6 16 6a 9b 9a 17 9 8 7 13 12 11 10

2 3 15

14

1

and NHNH2.HCl

R O N O N H R R O N O N H

(Z)-2a, R=H (Z)-2b, R=NO2

Z-isomer E-isomer(Not formed) R

R

MeOH, r.t

R

(E)-2a, R=H (E)-2b, R=NO2

Scheme 2.Synthesis of biflorin-based hydrazone derivatives2a,b.

O O O 1 3a 4 5 6 16 6a 9b 9a 17 9 8 7 13 12 11 10

2 3 15

14 1 and O N O RO O N O OR

(Z)-3a, R=H (Z)-3b, R=CH3 (Z)-3c, R=CH2CH3

Z-isomer E-isomer (E)-3b. R=CH3 (E)-3c. R=CH2CH3 RONH2.HCl

MeOH, reflux

Scheme 3.Synthesis of biflorin-based oxime derivatives3a–c.

O N O O H 3a O N O N H NO₂ NO2 2b O N O N H H H 2a

δ17.23 δ17.42 δ19.91

Figure 1.Hydrogen bond forming the six-membered ring in (Z)-isomers2a,2band

Acknowledgments

The authors thank CNPq and CAPES (Brazil) for financial

support and study grants and also to the University of Aveiro

and the FCT/MEC (Portugal) for the financial support to the

QOPNA

research

Unit

(FCT

UID/QUI/00062/2013),

through

national funds and where applicable to those co-financed by

the FEDER, within the PT2020 Partnership Agreement, and

the Portuguese NMR Network. Finally, they thank Embrapa

Agroindustria Tropical, Ceará, for the high-resolution mass

spectrometry analysis.

References and notes

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Table 1

Values of the minimal inhibitory concentration (MIC) of biflorin1and derivatives2a,band3a–cagainst six bacterial strains

Bacterial strains MIC (

l

g/mL)

1 2a 2b 3a 3b(Z)/(E) 3c(Z)/(E) Ami Neo Gen

Enterococcus faecalis(ATCC 4083) 32 512 512 256 32 16 256 128 32

Proteus vulgaris(ATCC 13315) 16 P1024 512 512 64 512 512 512 64

Escherichia coli(27) 256 P1024 P1024 512 128 128 128 128 512

Escherichia coli(ATCC 10536) 128 P1024 512 P1024 P1024 512 512 512 128

Staphylococcus aureus(ATCC 25923) 64 256 512 256 32 32 256 32 32

Staphylococcus aureus(358) 128 P1024 512 P1024 64 64 256 32 8

HCT-116

2a 2b 3a 3b 3c 1

Dox

0 25 50 75 100

% growth inhibition

SF-295

2a 2b 3a 3b 3c 1

Dox

0 25 50 75 100

% growth inhibition

OVCAR-8

2a 2b 3a 3b 3c 1

Dox

0 25 50 75 100

% growth inhibition

Figure 2.Cell growth inhibition percentage (% CI) of the samples1,2a,band3a–cin three tumor cell lines. Values are means ± standard deviation.

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27. General procedure for the synthesis of hydrazones2a,b. Hydrazones2a,bwere synthesized following the methodology described by Campos and coworkers.26 The appropriate arylhydrazine hydrochloride (0.2 mmol) was added to a solution of biflorin1(0.1 mmol, 30.8 mg) in MeOH (2 mL). The mixture was stirred for 24 h at room temperature. After this period, the reaction mixture was concentrated under reduced pressure and the resulting residue was taken in dichloromethane and purified by TLC, using hexane and dichloromethane (4:6 v/v) as the eluent.

The reaction of biflorin 1 (0.1 mmol, 30.8 mg) with phenylhydrazine hydrochloride (0.2 mmol, 29.0 mg) yielded (16.7 mg, 42%) of an orange solid (Z)-6,9-dimethyl-3-(4-methylpent-3-en-1-yl)-7-(2-phenylhydrazono)benzo [de]chromen-8(7H)-one (2a): mp 115–117°C; IR (KBr): 3400, 2925, 1600, 1495, 1470, 1190 cm1_.1_{H NMR (500.13 MHz, CDCl}

3)d= 1.58 (s, 3H, H-14), 1.72 (s, 3H, H-15), 2.10 (s, 3H, H-17), 2.30 (q,J= 7.1 Hz, 2H, H-11), 2.46 (t,

J= 7.1 Hz, 2H, H-10), 2.83 (s, 3H, H-16), 5.19 (m, 1H, H-12), 6.94 (s, 1H, H-2), 7.12–1.16 (m, 2H, H-4 and H-40_{), 7.38–7.49 (m, 5H, H-5, H-2}0_{, H-3}0_{, H-5}0_and H-60_{), 17.23 (s, 1H, N-H) ppm.}13_{C NMR (125.77 MHz, CDCl3)}d= 7.4 (C-17), 17.9 14), 25.7 15), 26.7 11), 27.6 10), 27.9 16), 111.7 9), 116.3 (C-20_{and C-6}0_{), 116.5 (C-3), 117.7 (C-4}0_{), 119.3 (C-9b), 123.1 (C-12), 124.5 (C-4),} 128.3 (C-3a), 129.5 (C-50_{and C-3}0_{), 130.2 (C-6a), 131.5 (C-7), 133.0 (C-13),} 134.8 (C-6), 135.1 (C-5), 139.3 (C-2), 143.1 (C-10_{), 160.0 (C-9a), 179.2 (C-8)} ppm. ESI+_{-HRMS: calculated for C}

26H27N2O2 [M+H]+: 399.2073. Found: 399.2071 [M+H]+_.

The reaction of biflorin1(0.1 mmol, 30.8 mg) with 2,4-dinitrophenylhydrazine hydrochloride (67%) (0.1 mmol, 29.0 mg) yielded (30.8 mg, 63%) of a brown solid (Z )-7-(2-(2,4-dinitrophenyl)hydrazono)-6,9-dimethyl-3-(4-methylpent-3-en-1-yl)benzo[de]chromen-8(7H)-one (2b): mp 246–248°C; IR (KBr): 3425, 2920, 1600, 1430 cm1_.1_{H NMR (500.13 MHz, CDCl}

3)d= 1.60 (s, 3H, H-14), 1.81 (s, 3H, H-15), 2.11 (s, 3H, H-17), 2.33 (q,J= 7.2 Hz, 2H, H-11), 2.55 (t,

J= 7.2 Hz, 2H, H-10), 2.86 (s, 3H, H-16), 5.19 (t,J= 7.2 Hz, 1H, H-12), 7.09 (s, 1H, H-2), 7.39 (d,J= 8.1 Hz, 1H, H-4), 7.56 (d,J= 8.1 Hz, 1H, H-5), 8.26 (d,J= 9.4 Hz, 1H, H-60_{), 8.48 (dd,J= 2.4 and 9.4 Hz, 1H, H-5}0_{), 9.21 (d,J= 2.4 Hz, 1H, H-3}0_), 17.42 (s, 1H, N-H) ppm.13_{C NMR (125.77 MHz, CDCl}

3)d= 7.5 17), 17.9 (C-14), 25.7 (C-15), 26.9 (C-11), 27.5 (C-10 and C-16), 112.5 (C-9), 116.5 (C-3), 117.4 (C-60_{), 120.7 (C-9b), 121.8 (C-4), 122.7 (C-12), 123.2 (C-3}0_{), 128.9 (C-6a),} 129.2 (C-3a), 129.6 (C-50_{), 133.0 (C-2}0_{), 133.4 (C-13), 136.4 (C-5), 137.0 (C-6),} 138.5 (C-7), 140.0 (C-2), 140.5 (C-40_{), 144.2 (C-1}0_{), 161.4 (C-9a), 180.3 (C-8)} ppm. ESI+_{-HRMS: calculated for C}

26H25N4O6 [M+H]+: 489.1774. Found: 489.1770 [M+H]+_.

28. Oliveira, M. F. Contribuição ao Conhecimento Químico das Espécies Tabebuia serratifolia Nichols e Tabebuia rosea Bertol; Universidade Federal do Ceará: Fortaleza, Ceará, Brazil, 2000; p 1 (Ph.D. thesis).

29. Silva, A. R.; Herbst, M. H.; Ferreira, A. B. B.; Silva, A. M.; Visentin, L. C.Molecules 2011,16, 1192.

30. General procedure for the synthesis of oximes3a–c. The oximes were synthesized following the methodology proposed by Oliveira and coworkers.28,29_Biflorin1 (0.1–0.16 mmol) was added to a methanolic solution (1–2 mL) of the appro-priate amine hydrochloride (0.1–0.16 mmol) under magnetic stirring. The reaction mixture was left at reflux (70°C) for a period of 6 h. After this period the reaction mixture was evaporated to the dryness. The obtained residue was taken in dichloromethane and purified by TLC using hexane/ethyl acetate (6:4 v/v).

The reaction of biflorin1(0.16 mmol, 50.0 mg) with hydroxylamine hydrochlo-ride (0.16 mmol, 11.1 mg) yielded (31.9 mg, 58%) of an orange residue (Z )-7-(hydroxyimino)-6,9-dimethyl-3-(4-methylpent-3-en-1-yl)benzo[de ]chromen-8(7H)-one (3a): IR (KBr): 3400, 2920, 1600, 1530, 1030 cm1_. 1_{H NMR} (500.13 MHz, CDCl3)d= 1.59 (s, 3H, 14), 1.72 (s, 3H, 15), 2.03 (s, 3H, H-17), 2.32 (q,J= 7.2 Hz, 2H, H-11), 2.54 (t,J= 7.2 Hz, 2H, 10), 2.70 (s, 3H, H-16), 5.18 (t,J= 7.2 Hz, 1H, H-12), 7.09 (s, 1H, H-2), 7.36 (d,J= 8.1 Hz, 1H, H-4), 7.53 (d,J= 8.1 Hz, 1H, H-5), 19.91 (s, 1H, N-OH) ppm.13_{C NMR (125.77 MHz,} CDCl3)d= 6.9 (C-17), 18.0 (C-14), 25.1 (C-15), 26.9 (C-11), 26.8 (C-16), 27.5 (C-10), 110.7 (C-9), 117.8 (C-3), 119.7 (C-9b), 121.3 (C-4), 122.8 (C-12), 128.0 (C-6a), 128.9 (C-3a), 133.5 (C-13), 136.9 (C-5), 138.5 (C-6), 147.4 (C-7), 139.8 (C-2), 163.2 (C-9a), 179.5 (C-8) ppm. ESI+_{-HRMS: calculated for C}

20H21NO3[M +H]+_{: 324.1601. Found: 324.1600 [M+H]}+_.

The reaction between biflorin1(0.1 mmol, 30.8 mg) andO -methylhydroxy-lamine hydrochloride (0.1 mmol, 8.35 mg) yielded (21.3 mg, 63%) of an orange residue, consisting of a mixture of (Z )-7-(methoxyimino)-6,9-dimethyl-3-(4-methylpent-3-en-1-yl)benzo[de]chromen-8(7H)-one (Z-3b) and (E )-7-(methoxyimino)-6,9-dimethyl-3-(4-methylpent-3-en-1-yl)benzo[de]chromen-8 (7H)-one (E-3b): IR (KBr): 3410, 2930, 1600, 1195, 1015 cm1_{. (Z-3b):}1_{H NMR} (500.13 MHz, CDCl3)d= 1.59 (s, 3H, 14), 1.71 (s, 3H, 15), 1.95 (s, 3H, H-17), 2.25 (m, 2H, H-11), 2.47 (m, 2H, H-10), 2.61 (s, 3H, H-16), 4.18 (s, 3H, OCH3), 5.18 (m, 1H, H-12), 6.93 (s, 1H, H-2), 7.24 (d,J= 8.6 Hz, 1H, H-4), 7.35 (d,

J= 8.6 Hz, 1H, H-5) ppm.13_{C NMR (125.77 MHz, CDCl}

3)d= 7.5 17), 17.9 (C-14), 24.0 (C-16), 25.7 (C-15), 27.0 (C-11), 27.1 (C-10), 64.8 (OCH3), 112.4 (C-9), 115.7 (C-3), 121.2 (C-4), 121.4 (C-9b), 122.9 (C-12), 128.0 (C-6a), 128.8 (C-3a), 133.1 (C-13), 134.9 (C-5), 138.2 (C-6), 139.7 (C-2), 147.3 (C-7), 160.1 (C-9a),

178.9 (C-8) ppm. (E-3b):1_{H NMR (500.13 MHz, CDCl}

3)d= 1.59 (s, 3H, H-14), 1.71 (s, 3H, H-15), 1.96 (s, 3H, H-17), 2.25 (m, 2H, H-11), 2.27 (s, 3H, H-16), 2.47 (m, 2H, H-10), 4.18 (s, 3H, OCH3), 5.18 (m, 1H, H-12), 6.47 (s, 1H, H-2), 7.33 (s, 1H, H-4), 7.33 (s, 1H, H-5) ppm.13_{C NMR (125.77 MHz, CDCl}

3)d= 7.6 (C-17), 17.9 (C-14), 23.0 (C-16), 25.7 (C-15), 27.0 (C-11), 27.1 (C-10), 63.7 (OCH3), 110.5 (C-9), 115.5 (C-3), 121.3 (C-9b), 122.8 (C-12), 123.2 (C-4), 126.3 (C-6a), 127.6 (C-3a), 133.2 (C-13), 133.5 (C-5), 140.1 (C-2), 140.7 (C-6), 151.5 (C-7), 160.5 (C-9a), 185.2 (C-8) ppm. ESI+_{-HRMS: calculated for C}

21H24NO3[M+H]+: 338.1756. Found: 338.1753 [M+H]+_.

The reaction of biflorin1(0.1 mmol, 30.8 mg) with O-ethylhydroxylamine hydrochloride (0.1 mmol, 9.75 mg) yielded (17.9 mg, 51%) of an orange residue, consisting of a mixture of (Z )-7-(ethoxyimino)-6,9-dimethyl-3-(4-methylpent-3-en-1-yl)benzo[de]chromen-8(7H)-one (Z-3c) and (E )-7-(ethoxy-imino)-6,9-dimethyl-3-(4-methylpent-3-en-1-yl)benzo[de]chromen-8(7H )-one (E-3c): IR (KBr): 3395, 2915, 1600, 1185, 1050 cm1_{. (Z-3c):}1_{H NMR} (500.13 MHz, CDCl3)d= 1.43 (m, 3H, CH3), 1.59 (s, 3H, H-14), 1.71 (s, 3H, H-15), 1.95 (s, 3H, H-17), 2.25 (m, 2H, H-11), 2.47 (m, 2H, H-10), 2.61 (s, 3H, H-16), 4.42 (q,J= 7.1 Hz, 2H, OCH2), 5.16 (m, 1H, H-12), 6.93 (s, 1H, H-2), 7.24 (d,

J= 8.2 Hz, 1H, H-4), 7.35 (d,J= 8.2 Hz, 1H, H-5) ppm.13_{C NMR (125.77 MHz,} CDCl3)d= 7.5 (C-17), 14.8 (CH3), 17.9 14), 24.3 16), 25.7 15), 27.0 (C-11), 27.4 (C-10), 72.8 (OCH2), 112.5 (C-9), 115.6 (C-3), 121.4 (C-9b), 121.5 (C-4), 122.9 (C-12), 128.8 (C-6a), 128.0 (C-3a), 133.1 (C-13), 134.9 (C-5), 138.0 (C-6), 139.7 (C-2), 147.0 (C-7), 159.9 (C-9a), 178.7 (C-8) ppm.

(E-3c):1_{H NMR (500.13 MHz, CDCl}

3)d= 1.35 (t,J= 7.1 Hz, 3H, CH3), 1.59 (s, 3H, 14), 1.71 (s, 3H, 15), 1.96 (s, 3H, 17), 2.25 (m, 2H, 11), 2.27 (s, 3H, H-16), 2.46 (m, 2H, H-10), 4.46 (q,J= 7.1 Hz, 2H, OCH2), 5.16 (m, 1H, H-12), 6.96 (s, 1H, H-2), 7.33 (s, 1H, H-4), 7.33 (s, 1H, H-5) ppm.13_{C NMR (125.77 MHz,} CDCl3)d= 7.6 (C-17), 14.6 (CH3), 17.9 (C-14), 23.3 (C-16), 25.7 (C-15), 27.1 (C-11), 27.4 (C-10), 72.2 (OCH2), 110.5 (C-9), 110.6 (C-3), 121.3 (C-9b), 122.8 (C-12), 123.0 (C-4), 126.4 (C-6a), 127.6 (C-3a), 133.2 (C-13), 133.5 (C-5), 140.0 (C-2), 140.6 (C-6), 150.7 (C-7), 160.5 (C-9a), 185.4 (C-8) ppm. ESI+_-HRMS: calculated for C22H26NO3[M+H]+: 352.1919. Found: 352.1913 [M+H]+. 31. Nicolaides, D. N.; Gautam, D. R.; Litinas, K. E.; Hadjipavlou-Litina, D. J.;

Kontogiorgis, C. A. J.Heterocycl. Chem.2004,41, 605.

32. Souza, L. G. S.; Almeida, M. C. S.; Monte, F. J. Q.; Santiago, G. M. P.; Braz-Filho, R.; Lemos, T. L. G.Quim. Nova2012,35, 2258.

33. Plant Material:The speciesCapraria bifloraL. (Scrophulariaceae) was collected in Itapiúna, Ceará, Brazil and was identified by Dr. Edson Nunes. A voucher specimen (No. 30848) was deposited in the Herbarium Prisco Bezerra of the Biology Department of the Federal University of Ceará.

Isolation of biflorin:The isolation of biflorin1was performed as described by Souza et al.32_{Air-dried powdered roots (700 g) were extracted with petroleum} ether and the solvent was evaporated under reduced pressure to yield 1.7 g of extract. The extract (1.0 g) was chromatographed on silica gel by elution using binary mixtures of hexane/EtOAc and EtOAc/MeOH, with increasing polarity. Fourteen fractions were collected with a volume of 200 mL. Fractions 8–10 were pooled according to thin-layer chromatographic (TLC) analysis, yielding 58 mg of biflorin1.

34. NCCLS (2006): Clinical and Laboratory Standards Institute, Performance standards for antimicrobial susceptibility testing, 16th Informational Supplement, CLSI document M100-S16, CLSI, Wayne, PA, 2006.

35. Antibacterial activity evaluation:The antibacterial activity of the samples was evaluated using the broth microdilution method based on the document M100-S16 (CLSI, 2006)34_{for bacteria. In the tests, six strains of Gram-positive,}

Enterococcus faecalis(ATCC 4083),Staphylococcus aureus(ATCC 25923), and multidrug-resistantStaphylococcus aureus(358) and Gram-negative, Escher-ichia coli(ATCC 10536),Proteus vulgaris(ATCC 13315) and multidrug-resistant

Escherichia coli (27) were used. The Oswaldo Cruz Foundation-FIOCRUZ provided the six standard bacteria strains used.

The bacterial strains were activated in the midst Brain Heart Infusion Broth (BHI) for 24 h at 35 ± 2°C. Next, a bacterial suspension was prepared in BHI at 3.8%, corresponding to the turbidity of 0.5 McFarland scale (1108CFU/ mL). Then the suspension was diluted to 1106CFU/mL in BHI broth at 10%, and volume of 100

l

L, and homogenized in microdilution 96 well plates, plus different concentrations of the samples resulting in a final inoculum of 5105CFU/mL. The samples were solubilized in distilled water and DMSO to obtain a stock solution of 1024

l

g/mL. The final concentrations of the samples were in the culture medium 512–8

l

g/mL. The tests were performed in triplicate. The plates were incubated at 35 ± 2°C for 24 h. The antibacterial activity was detected using a colorimetric method by adding 25

l

L of the resauzurin staining (0.01%) aqueous solution to each one of the wells at the end of the incubation period. The minimal inhibitory concentration (MIC) was defined as the lowest extract concentration able to inhibit the bacteria growth, as indicated by resauzurin staining (dead bacterial cells are not able to change the staining color by visual observation—blue to red). The antibiotics Amikacin, Gentamicin and Neomycin were utilized as a positive control.

36. Nascimento, P. G. G.; Lemos, T. L. G.; Bizerra, A. M. C.; Arriaga, A. M. C.; Ferreira, D. A.; Santiago, G. M. P.; Braz-Filho, R.; Costa, J. G. M.Molecules2014,19, 1317. 37. Mosmann, T.J. Immunol. Methods1983,65, 55.

Samples were diluted in DMSO and tested at a concentration of 5

l

g/mL. Cells were plated at a concentration of 0.1106cells/mL for strains SF-295 and OVCAR-8 and 0.7105_{cells/mL for the strain HCT-116 and incubated} for 72 h in an oven. Subsequently the samples were centrifuged and the supernatant was removed. Then 150

l

L of 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution were added and the plates were incubated for 3 h. The absorbance was measured after dissolution of the precipitate with 150

l

L of DMSO in plate

spectrophotometer at 595 nm. Cell viability was evaluated by reduction of the yellow dye (MTT) to a blue product as described by Mosmann.37 Biflorin1and Doxorubicin, each one were utilized as a positive control. 39. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;

Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R.J. Natl. Cancer Inst.1990,82, 1107.

40. Berridge, M. V.; Tan, A. S.; McCoy, K. D.; Wang, R.Biochemica1996,4, 14.