Pyranoxanthones: Synthesis, growth inhibitory activity on human tumor cell lines and determination of their lipophilicity in two membrane models

European Journal of Medicinal Chemistry 69 (2013) 798e816

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Original article

Pyranoxanthones: Synthesis, growth inhibitory activity on human

tumor cell lines and determination of their lipophilicity in two

membrane models

,

c

,

a,

Carlos M.G. Azevedo ^{a b}, Carlos M.M. Afonso ^a, José X. Soares ^c, Salette Reis ^d,

*

Diana Sousa ^{a e}, Raquel T. Lima ^{a e}, M. Helena Vasconcelos ^{e f}, Madalena Pedro ^a

,

, g

João Barbosa ^g, Luís Gales ^{h i}, Madalena M.M. Pinto ^a

,

, c

^aCentro de Química Medicinal da Universidade do Porto (CEQUIMED-UP), Rua de Jorge Viterbo Ferreira n.^ꢀ228, 4050-313 Porto, Portugal

^bDepartment of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark

^cLaboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto,

Rua de Jorge Viterbo Ferreira n.^ꢀ228, 4050-313 Porto, Portugal

^dREQUIMTE, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n.^ꢀ228, 4050-313 Porto, Portugal

^eCancer Drug Resistance Group, Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Rua Dr. Roberto Frias s/n,

4200-465 Porto, Portugal

^fLaboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n.^ꢀ228,

4050-313 Porto, Portugal

^gGrupo de Biologia Molecular e Celular (GBMC), Centro de Investigação em Ciências da Saúde (CICS), Instituto Superior de Ciências da Saúde do Norte,

CESPU, Rua Central de Gandra 1317, 4585-116 Gandra, Portugal

^hGrupo de Biofísica Molecular, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal

ⁱInstituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Rua de Jorge Viterbo Ferreira n^ꢀ228, 4050-313 Porto, Portugal

a r t i c l e i n f o

a b s t r a c t

Article history:

The benzopyran and dihydrobenzopyran moieties can be considered as “privileged motifs” in drug

discovery being good platforms for the search of new bioactive compounds. These moieties are

commonly found fused to the xanthonic scaffold belonging to the biologically important family of the

generally designated prenylated xanthones. Several pyranoxanthones have shown promising antitumor

activity and since most of them are from natural origin, the biosynthetic pathway only allows a particular

pattern of substitution which limits their structural diversity and renders any broad structureeactivity

study hard to be established. Accordingly, with the aim of rationalizing the importance of the fused ring

orientation and oxygenation pattern in pyranoxanthones, this study describes the synthesis of 14 new

pyranoxanthones and evaluation of their cell growth inhibitory activity in four human tumor cell lines as

well as their lipophilicity in two membrane models. This systematic approach allowed establishing

structureeactivity and structureelipophilicity relationships for the obtained compounds in combination

with 6 previously described compounds. From this work an angular pyranoxanthone scaffold emerged as

particularly promising, presenting a potent cell growth inhibitory activity and suitable drug-like

lipophilicity.

Received 15 April 2013

Received in revised form

4 September 2013

Accepted 5 September 2013

Available online 18 September 2013

Keywords:

Xanthones

Xanthen-9-ones

Antitumor

Benzopyran

Chromans

Chromene

1. Introduction

motifs frequently found in secondary metabolites isolated from

nature [1,2]. In fact, several well-known bioactive molecules have

2,2-Dimethylbenzopyran (or 2,2-dimethylchromene) and 2,2-

dimethyl-3,4-dihydrobenzopyran (or 2,2-dimethylchroman) are

these motifs in their structure [1e4] and some representative ex-

amples are (Fig. 1): -lapachone (ARQ-501) (1), a compound iso-

b

lated from the heartwood of the Lapacho tree which exhibits a

potent antitumor activity [5,6]; nabilone (2), a synthetic cannabi-

noid used as an anti-emetic agent [7]; precocenes (3a and 3b)

which are used as insecticides [8]; calanolide A (4), an HIV-1

reverse transcriptase inhibitor isolated from Calophyllum

* Corresponding author. Laboratório de Química Orgânica

e Farmacêutica,

Departamento de Ciências Químicas, Rua de Jorge Viterbo Ferreira n.^ꢀ228, 4050-

313 Porto, Portugal. Tel.: þ351 220 428 000; fax: þ351 226 093 390.

E-mail address: cafonso@ff.up.pt (C.M.M. Afonso).

http://dx.doi.org/10.1016/j.ejmech.2013.09.012

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

799

Accordingly, in this work a systematic study of the following

structural aspects was envisaged: i) presence or absence of either

hydroxyl or methoxyl group on the xanthone core, ii) presence or

absence of a double bond in the ring fused to the xanthone core;

and iii) relative orientation of the fused ring along the xanthone

core (Fig. 3). In order to fulﬁll these requirements, a chemical li-

brary of 21 pyranoxanthones was obtained. The compounds were

synthesized either by chemical modiﬁcation of simple oxygenated

xanthones or by total synthesis through benzophenone and diaryl

ether routes [27,28]. The cell growth inhibitory activity of the

synthesized pyranoxanthones was evaluated in vitro in four human

tumor cell lines: MCF-7 (breast adenocarcinoma), NCI-H460 (non-

small cell lung cancer), A375-C5 (melanoma) and HL-60 (acute

myeloid leukemia).

In addition, a preliminary assessment of the drug-likeness of the

pyranoxanthones synthesized was also made by the evaluation of

their lipophilicity. This physicochemical property has a great

impact in the pharmacokinetic and pharmacodynamic behavior

being determined in an early stage of the drug discovery pipeline

[29e32]. In fact, lipophilicity has been correlated with several

pharmacokinetic parameters [33] and compounds with high lip-

ophilicity have an increased risk of attrition during clinical trials

[32]. Moreover, it is also one of the descriptors of the Lipinski “rule

of ﬁve” which is commonly used to roughly evaluate the oral

bioavailability [34]. In this work, the compounds lipophilicity was

determined using two membrane models, namely liposomes and

micelles which are able to mimic the anisotropic media found in

biomembranes and encode important interactions that take place

between the solute and the biomembranes [30,35e41]. This anal-

ysis provided an insight into the effects that oxygenation pattern

and fused ring orientation had on the lipophilicity, as well as to

select the most promising scaffolds, therefore serving as guidance

for further chemical modiﬁcations.

Fig. 1. Representative examples of bioactive compounds bearing 2,2-dimethyl-

benzopyran or 2,2-dimethyl-3,4-dihydrobenzopyran motifs.

lanigerum [9,10]; acronycine (5), a pyranoacridone alkaloid isolated

from Acronychia baueri with potent antitumor activity [11]; and

arugosin E (6), a compound isolated from the mycelial extract of

Aspergillus silvaticus with potent antibacterial activity [12].

These “privileged motifs” [1,2] are frequently found fused to the

xanthonic core both in secondary metabolites isolated from higher

plants and xanthones obtained by synthesis, with many of them

exhibiting promising biological activities [13e15], particularly

antitumor [16,17]. In fact, most of the described antitumor pyr-

anoxanthones are from natural origin [13,14,16] and representative

compounds [18e26] are depicted in Fig. 2 (only compound 14 is

from synthetic origin). Although these pyranoxanthones bear in

common the fused pyran or dihydropyran ring, the place of the ring

along the xanthone scaffold may vary as well as the position and

type of functional groups present. In addition, there has been a big

discrepancy in the methodologies and cell lines used when

studying the tumor cell growth inhibitory potential of pyranox-

anthones, which makes structureeactivity relationships hard to

establish. Consequently, there is a lack of a systematic study

focused on the orientation of the fused pyran and dihydropyran

ring as well as the pattern of substitution, which would be able to

provide a deeper comprehension of the structural features of pyr-

anoxanthones that are related to their biological activity.

2. Results and discussion

2.1. Chemistry

Pyranoxanthones are commonly obtained by two general ap-

proaches: i) synthesis of simple oxygenated xanthones and then

formation of the pyran ring; and ii) synthesis of benzopyran moiety

followed by the assembling of the xanthone core [14,42]. Most of

Fig. 2. Representative examples of pyranoxanthones with tumor cell growth inhibitory potential.

800

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

The pyranoxanthones 26e29 were synthesized using 1,3-

dihydroxyxanthone (25) as building block (Scheme 1 e II). The

pyranoxanthones 26 and 27 were synthesized using a previously

described procedure [50] and pyranoxanthones 28 and 29

were obtained in one step by the condensation of 1,3-

dihydroxyxanthone (25) with prenal [51].

The pyranoxanthones 32, 35 and 38 were successfully obtained

by the same approach followed for the synthesis of compounds 19

and 23 but using respectively 4-hydroxyxanthone (30), 2-

hydroxyxanthone (33) and 1,2-dihydroxyxanthone (36) as build-

ing blocks (Scheme 1 e III, IV and V). In the synthesis of the pyr-

anoxanthone 35, only one isomer was obtained from the cyclization

of the propargyl ether catalyzed by platinum and in the O-dime-

thylpropargylation of 1,2-dihydroxyxanthone (36), two by-

products were obtained besides compound 37 which were

formed by the intramolecular nucleophilic attack of the hydroxyl to

the alkyne of two propargyl aryl ethers intermediates (40 and 41).

The pyranoxanthone 39 was synthesized through the hydrogena-

tion of compound 38 by a transfer hydrogenation methodology

[52].

2.1.2. Synthesis of pyranoxanthones by total synthesis

The pyranoxanthone 51 was obtained via a diaryl ether inter-

mediate using benzopyran 45 and methyl 2-iodobenzoate (47) as

building blocks (Scheme 2). The benzopyran 45 was obtained in

three

steps

starting

from

the

reaction

of

2,4-

dihydroxybenzaldehyde (42) with prenal catalyzed by calcium

hydroxide [53,54] followed by an O-methylation with dime-

thylsulfate and oxidation by a BaeyereVilliger type-oxidation [55].

The other building block was synthesized from the esteriﬁcation of

2-iodobenzoic acid (46). The two compounds were coupled by an

Ullmann-ether synthesis [56] to give the diaryl ether 48 which was

then hydrolyzed with LiOH and transformed into the respective

N,N-diethylamide using TBTU as coupling reagent. Lastly, diaryl

ether 50 was cyclized to the pyranoxanthone 51 using a directed

remote metalation [57]. The pyranoxanthone 51 was reduced to

give pyranoxanthone 52 and O-demethylated with BBr₃to give

pyranoxanthone 53.

Fig. 3. Targeted compounds by variation of the pattern of oxygenation and the

orientation of the fused ring.

the synthetic pyranoxanthones referred in the literature used the

ﬁrst approach, being the later only applied for the total synthesis of

highly substituted pyranoxanthones, usually natural products [14].

In order to synthesize the envisaged compounds, both strategies

were applied and the synthesis of 21 pyranoxanthones is described

in the next two sections.

2.1.1. Synthesis of pyranoxanthones by chemical modiﬁcation of

simple oxygenated xanthones

The pyranoxanthones 62 and 63 were obtained by total syn-

thesis through the cyclization of the same benzophenone inter-

mediate (61), using as building blocks the benzopyran 57 and a

MOM-protected methyl salicylate (59) (Scheme 3). The synthesis

of benzopyran 57 started by the oxidation of 2,4-

dimethoxybenzaldehyde (54) to give 2,4-dimethoxyphenol (55)

which was then O-dimethylpropargylated through a DBU/CuCl₂/

MeCN/3-chloro-3-methyl-1-butyne methodology [58] and cyclized

thermally to give compound 57. After the synthesis of the two

building blocks, the next step was the synthesis of the benzophe-

none 60 which was accomplished through the 1,2-nucleophilic

addition of a lithiated intermediate of benzopyran 57 formed by

an ortho lithiation to the ester of the protected methyl salicylate

(59). The MOM group of benzophenone 60 was removed using

NbCl₅[59] and the respective benzophenone 61 cyclized to give

pyranoxanthones 62 and 63 by an intramolecular nucleophilic ar-

omatic substitution with cesium carbonate in DMF [60]. However,

pyranoxanthone 63 was obtained in a higher yield than 62 (81%

versus 8%) due to the inductive effect of the oxygen in position 1

which makes carbon 8 more prone to suffer a nucleophilic attack.

The pyranoxanthones 62 and 63 were reduced to give pyranox-

anthones 64 and 65 respectively. The pyranoxanthones 63 and 65

were also O-demethylated to give respectively 66 and 67.

In Scheme 1 e I is represented the synthesis of pyranoxanthones

19, 20 and 23 using 3,4-dimethoxyxanthone (15) as building block.

In the case of the pyranoxanthones 19 and 20, the ﬁrst step was the

mono O-demethylation of 3,4-dimethoxyxanthone (15) using an

odorless thiol reagent [43] to give 3-hydroxy-4-methoxyxanthone

(17). However, this reaction was slightly regioselective since 4-

hydroxy-3-methoxyxanthone (16) was also obtained but in lower

yield (ratio 17/16 of 2.7). Compound 17 was O-dimethylpropargy-

lated using a KI/CuI/K₂CO₃/3-chloro-3-methyl-1-butyne method-

ology [44,45] to give compound 18 which was then cyclized either

by thermal rearrangement, gold (Gagosz’s catalyst) [46,47] or

platinum (PtCl₄) [48,49] to give pyranoxanthone 19. Pyranox-

anthone 20 was obtained from compound 17 by reacting with

prenyl bromide under microwave heating and montmorillonite K-

10 catalysis, a methodology described in the literature for the

synthesis of compound 14 [25,50]. The approach followed for the

synthesis of 19 was applied for the synthesis of pyranoxanthone 23

using 3,4-dihydroxyxanthone (21) as building block which was

obtained from the O-demethylation of compound 15 with AlCl₃.

The O-dimethylpropargylation of compound 21 led to the desired

aryl dimethylpropargyl ether 22 and also to compound 24 which

was formed by the intramolecular nucleophilic attack of the hy-

droxyl of compound 22 to the alkyne (30% versus 47% respectively).

The cyclization of compound 22 led to pyranoxanthone 23 and also

to compound 24 (33% versus 53% respectively).

2.1.3. Structure elucidation

The structure elucidation of the compounds synthesized was

established on the basis of IR, HRMS and NMR techniques. The ¹H

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

801

Scheme 1. Synthesis of pyranoxanthones 19, 20, 23, 32, 35, 38 and 39.

and ¹³C NMR data of 14 pyranoxanthones are reported in Tables 1

and 2. The ¹³C NMR assignments of pyranoxanthones 19, 20, 51,

52, 53, 62, 63, 64, 65, 66 and 67 were determined by 2D hetero-

nuclear single quantum correlation (HSQC) and heteronuclear

multiple bond correlation (HMBC) experiments. The ¹³C NMR

chemical shifts assignments of compound 23 were established by

comparison with the assignments of compounds 14 [25], 19 and 20.

The ¹³C NMR chemical shifts assignments of compounds 38 and 39

were established by comparison with the ¹³C NMR assignments of

compounds 62 and 64. The compound 32 has been previously

isolated from Calophyllum caledonicum and has been given the

name caledinoxanthone B [61]. However, it has never been

Scheme 2. Synthesis of pyranoxanthones 51, 52 and 53.

802

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

Scheme 3. Synthesis of pyranoxanthones 62e67.

obtained by synthesis and the spectroscopic data of compound 32

were in accordance with the reported for caledinoxanthone B [61].

The intramolecular cyclization of benzophenone 61 led to xan-

thones 62 and 63. The structure of the two isomers was assigned by

a Nuclear Overhauser effect spectroscopy (NOESY) experiment

where a Nuclear Overhauser effect (NOE) was observed between

proton 5 and the methoxyl group (Fig. 4). The assignment of the

structure of compound 63 was also conﬁrmed by X-ray crystal-

lography (Fig. 4). The crystal structure of compound 63 reveals the

usual structural features [62] with the xanthone skeleton remain-

potent (GI₅₀of 3 mM). Most of the synthesized compounds were

shown to be more potent towards the HL-60 tumor cell line than

towards the other three cell lines tested.

The data obtained from the evaluation of the cell growth

inhibitory activity of the synthesized compounds in the four tumor

cell lines allowed to make structureeactivity relationships

regarding the orientation and type of fused ring (pyran or dihy-

dropyran) and presence or absence of hydroxyl or methoxyl on the

xanthone scaffold (Fig. 5). Considering the linear pyranoxanthones

A (Fig. 5), the presence of a hydroxyl in position 12 was associated

with the moderate activity observed in the four tumor cell lines

tested while the methoxyl group associated with growth inhibitory

effect only in the HL-60 cells. If the hydroxyl was instead in position

5, none or poor growth inhibitory activity was observed in the MCF-

7 and NCI-H460 cell lines. Regarding linear pyranoxanthones B, the

presence of a methoxyl group in position 5 was associated with a

moderate activity of these compounds in the four tumor cell lines

while the presence of a hydroxyl in this position was associated

with a loss of activity. Moreover, when comparing compounds 51

and 52, the presence of a double bond on ring D was associated

with a higher potency in the HL-60 cell line. Considering the pyr-

anoxanthone B with a hydroxyl or methoxyl in position 12, the

presence of a double bond in the fused ring D was associated with a

higher growth inhibitory effect in the four tumor cell lines and the

methoxyl with a higher potency observed in the HL-60 tumor cell

line. To sum up, in the case of the linear pyranoxanthones, the

orientation of the fused ring and the type and position of the hy-

droxyl or methoxyl may have an important role in the growth

inhibitory activity observed in the four human tumor cell lines

tested.

ing essentially planar and the

conformation.

g-pyrone in a half chair

2.2. Biological activity

The effect of 14 synthesized pyranoxanthones (19, 32, 35, 38, 39,

51, 52, 53, 62, 63, 64, 65, 66 and 67) was evaluated on the growth of

the following four human tumor cell lines: MCF-7 (breast adeno-

carcinoma), NCI-H460 (non-small cell lung cancer), A375-C5

(melanoma) and HL-60 (acute myeloid leukemia). The growth

inhibitory activity was determined using the Sulforhodamine B

(SRB) assay adopted from the National Cancer Institute (NCI, USA).

This assay uses the protein-binding dye SRB to indirectly assess cell

growth [63e65]. For each cell line, a doseeresponse curve was

established allowing the determination of the concentration that

caused a cell growth inhibition of 50% (GI₅₀) [66] and the results

obtained are shown on Table 3. In addition, Table 3 also shows the

GI₅₀concentration of compounds 14, 20, 26e29 which were pub-

lished elsewhere [67e69].

Regarding the results obtained in the adherent MCF-7, NCI-H460

and A375-C5 cell lines compound 32 was the most potent for MCF-

Considering the angular pyranoxanthone C, no growth inhibi-

tory activity was observed for MCF-7 and NCI-H460 cell lines.

Considering the angular pyranoxanthones D and E without sub-

stituents, the former was associated with a higher growth inhibi-

tory effect in the four tumor cell lines tested, which decreased with

the introduction of a methoxyl or hydroxyl in position 6 (although

less critical to the effect in the HL-60 cells). Moreover, the double

7

cells (GI₅₀

(GI₅₀¼ 6.2 ꢁ 0.6

in the NCI-H460 cells, presenting GI₅₀of 32.9 ꢁ 7.1

31.7 ꢁ 2.6 M respectively. When analyzing the results obtained in

the leukemia cell line (HL-60), compounds 20, 32, 51, 62, 63 and 66

presented GI₅₀below 12 M, with compound 32 being the most

¼

13.3

M). Compounds 32 and 38 were the most potent

M and

ꢁ

1.3

mM) and A375-C5 cells

m

Table 1

¹H NMR chemical shifts of compounds 19, 20, 23, 38, 39, 51, 52, 53, 62, 63, 64, 65, 66, and 67. (The compounds numbering is referred in Schemes 1e3).

19^a,b

20^a,b

23^a,c

38^a,e

39^a,e

51^a,b

52^a,b

53^b,d

62^a,b

63^a,b

64^a,b

65^a,b

66^a,b

67^a,b

H-3

H-4

5.75

1.90

5.73

5.94

1.94

5.86

1.86 (t, J ¼ 6.8) 5.77

5.95

5.89

1.89

1.91

5.86

1.92

(d, J ¼ 10.0) (t, J ¼ 6.8)

(d, J ¼ 10.0)

(d, J ¼ 9.9)

(t, J ¼ 6.8)

(d, J ¼ 10.1)

(d, J ¼ 10.0) (d, J ¼ 9.8) (d, J ¼ 9.8) (t, J ¼ 6.8) (t, J ¼ 6.9) (d, J ¼ 9.8)

(t, J ¼ 6.8)

6.46 2.64

6.47

6.40

2.90

6.71

2.93 (t, J ¼ 6.8) 6.80 6.41 6.38 2.91 2.88 6.31

2.90

(d, J ¼ 10.0) (td, J ¼ 6.8, 0.9)

(d, J ¼ 10.0)

7.59 (s)

e

(d, J ¼ 9.9)

6.65 (s)

e

(td, J ¼ 6.8; 0.5) (d, J ¼ 10.1)

(d, J ¼ 10.0) (d, J ¼ 9.8) (d, J ¼ 9.8) (t, J ¼ 6.8) (t, J ¼ 6.9) (d, J ¼ 9.8)

(t, J ¼ 6.8)

6.56 (s)

H-5

H-6

H-7

7.74 (s)

e

7.86 (t, J ¼ 0.9)

e

6.70 (broad s)

e

Not observed 6.91 (s)

6.47 (s)

e

7.00 (s)

e

6.50 (s)

e

6.42 (s)

12.14 [OH, s] 12.00 [OH, s]

e

8.32 (dd,

J ¼ 8.0, 1.7)

7.43 (dd,

7.44 (broad d, 7.46 (d,

7.53 (dd,

J ¼ 8.4, 1.0)

7.71 (ddd,

J ¼ 8.4,

7.1, 1.7)

7.36 (ddd,

J ¼ 8.0,

7.1, 1.0)

8.32 (dd,

J ¼ 8.0, 1.7)

7.51 (dd,

7.38 (dd,

e

7.37 (dd,

J ¼ 8.5, 0.8)

e

J ¼ 8.0, 1.7) J ¼ 7.9, 1.6)

J ¼ 8.6, 0.9) J ¼ 8.4)

J ¼ 8.6)

7.64 (ddd,

J ¼ 8.6,

7.1, 1.7)

7.29 (ddd,

J ¼ 8.0,

7.1)

8.24 (dd,

J ¼ 8.0,

1.7)

J ¼ 8.4, 0.9) J ¼ 8.5, 1.0)

H-8

7.37 (ddd,

J ¼ 8.0,

7.36 (ddd, J ¼ 7.9, 7.37 (ddd,

7.72 (ddd,

J ¼ 8.6,

7.71 (ddd,

J ¼ 8.4,

7.63 (ddd,

J ¼ 8.4,

7.65 (ddd, 8.29 (dd,

7.64 (ddd, 8.30 (dd,

8.19 (dd,

8.30 (dd,

7.0, 1.1)

J ¼ 8.0,

J ¼ 8.5,

J ¼ 8.0, 1.7) J ¼ 8.5,

J ¼ 8.0, 1.7) J ¼ 8.0, 1.7) J ¼ 8.0, 1.7)

7.0, 0.9)

7.71 (ddd,

J ¼ 8.5,

7.1, 1.1)

7.2, 1.7)

7.36 (ddd,

J ¼ 8.0,

7.1, 1.7)

7.35 (ddd,

J ¼ 8.0,

7.1, 1.7)

7.27 (ddd,

J ¼ 8.0,

7.1, 1.6)

7.0, 1.7)

H-9

7.70 (ddd, J ¼ 8.4, 7.70 (ddd,

7.32 (ddd, 7.32 (ddd, 7.31 (ddd, 7.33 (ddd, 7.31 (ddd,

7.40 (ddd,

J ¼ 8.0, 7.1,

1.1)

7.76 (ddd,

J ¼ 8.4, 7.1,

1.7)

7.0, 1.6)

J ¼ 8.5,

J ¼ 8.0,

7.0, 1.7)

7.57 (dd,

7.1, 1.7)

7.1, 0.9)

8.28 (dd,

7.0, 1.0)

8.24 (dd,

7.1, 0.9)

8.17 (dd,

7.1, 1.0)

8.28 (dd,

7.0, 1.0)

7.0, 0.8)

6.9, 1.1)

7.0, 1.0)

H-10

H-11

7.57 (dd,

7.56 (dd,

J ¼ 8.5, 1.1)

7.66 (ddd, 8.29 (dd,

7.65 (ddd, 7.67 (ddd,

J ¼ 8.5, 0.9) J ¼ 8.4, 1.1)

J ¼ 8.0, 1.7) J ¼ 8.0, 1.7)

J ¼ 8.0, 1.7) J ¼ 8.0, 1.6) J ¼ 8.5,

J ¼ 8.0, 1.6) J ¼ 8.4,

J ¼ 8.5,

7.0, 1.7)

6.9, 1.7)

7.55 (ddd, 7.52 (ddd,

7.0, 1.7)

e

7.53 (ddd,

J ¼ 8.5, 1.0)

e

7.64 (ddd,

J ¼ 8.4, 1.1) J ¼ 8.5, 1.0) J ¼ 8.4, 1.1)

H-12

e

5.67 [broad, OH] 12.66 [OH, s] 12.77 [OH, s]

1.55 (s)

7.39 (s)

1.40 (s)

7.49 (s)

1.36 (s)

7.03 (s)

1.36 (s)

e

H-1⁰a

1.55 (s)

1.46 (s)

1.54 (s)

1.49 (s)

1.52 (s)

1.46 (s)

1.42 (s)

1.46 (s)

and H-1⁰b

H-1⁰⁰

4.03 (s)

4.00 (s)

e

3.99 (s)

4.07 (s)

e

4.02 (s)

3.97 (s)

3.99 (s)

3.97 (s)

e

Abbreviations: singlet (s), doublet (d), triplet (t), quartet (q), quintet (qt), multiplet (m), doublet of doublets (dd), double doublet of doublets (ddd), doublet of triplets (dt) and triplet of doublets (td).

a

Values in ppm (d_H) measured at 300.13 MHz or 500.13 in CDCl₃, J values (Hz) are shown in parentheses.

Assignments were conﬁrmed by HSQC and HMBC experiments.

Assignments were established by comparison with the assignments of compounds 14, 19 and 20.

Values in ppm (d_H) measured at 300.13 MHz in DMSO-d₆, J values (Hz) are shown in parentheses.

b

c

d

e

Assignments were established by comparison with the assignments of compounds 62 and 64.

804

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

Table 2

¹³C NMR chemical shifts of compounds 19, 20, 23, 38, 39, 51, 52, 53, 62, 63, 64, 65, 66, and 67. (The compounds numbering is referred in Schemes 1e3).

19^a,b

20^a,b

23^a,c

38^a,e

39^a,e

51^a,b

52^a,b

53^b,d

62^a,b

63^a,b

64^a,b

65^a,b

66^a,b

67^a,b

C-2

C-3

C-4

C-4a

C-5

C-5a

C-6

C-6a

C-7

C-7a

C-8

C-9

C-10

C-10a

C-11

C-11a

C-12

C-12a

C-12b

C-1⁰a and C-1⁰b

C1⁰⁰

78.1

131.2

121.6

119.0

118.2

116.1

176.3

121.6

126.6

e

76.5

32.3

22.1

119.3

121.2

115.2

176.6

121.5

126.5

e

78.7

131.0

121.5

117.8

114.4

116.1

176.4

121.6

126.7

e

76.4

136.9

121.8

129.1

103.2

149.9

e

74.7

32.3

23.7

131.3

105.4

149.6

e

76.2

135.6

116.4

121.6

143.6

144.7

e

74.3

31.7

18.2

123.6

146.0

143.4

e

75.4

132.9

116.3

115.3

141.1

140.4

e

76.5

136.5

121.5

128.6

109.7

151.3

e

76.7

135.2

121.9

126.1

102.6

e

74.7

32.2

23.4

130.6

112.3

149.7

e

74.9

32.5

23.5

127.2

105.3

e

76.6

136.2

122.2

129.0

106.6

e

74.8

32.5

23.6

130.9

109.0

e

153.7

112.7

176.4

122.9

126.7

123.8

133.9

e

152.3

112.2

176.7

122.9

126.6

123.1

133.1

e

154.7

108.9

181.9

120.6

125.9

124.0

135.3

e

152.8

108.3

182.1

120.6

125.8

124.0

135.1

e

156.3

117.7

e

156.3

117.7

e

155.7

118.0

e

155.8

118.0

e

155.2

117.7

e

155.3

117.2

e

155.3

117.2

e

124.0

134.3

118.1

156.0

e

123.6

134.2

118.0

156.2

e

124.0

134.4

117.9

155.9

e

135.2

123.7

125.8

120.1

182.0

109.1

148.7

135.0

e

135.2

123.5

125.9

119.9

182.5

107.7

148.4

137.4

e

134.4

123.8

126.7

121.3

176.4

122.3

106.7

148.9

e

134.4

123.6

126.7

121.2

176.8

121.7

107.2

150.6

e

134.0

123.2

125.8

120.6

175.8

120.8

100.7

148.6

e

134.0

123.5

126.7

128.3

176.1

116.0

147.1

141.9

e

134.0

123.3

126.7

122.3

176.4

116.0

147.6

130.6

e

117.7

155.0

e

117.8

155.0

e

118.3

156.2

e

118.5

156.5

e

135.7

134.8

151.5

e

149.1

135.7

153.2

e

144.5

132.2

145.5

e

146.8

134.6

27.3

147.5

136.7

26.4

144.4

132.3

27.4

146.5

134.6

26.6

e

28.4

61.5

27.0

61.2

28.5

e

27.7

e

26.7

e

27.7

62.3

26.7

61.1

27.2

e

27.7

61.4

26.8

61.1

56.5

e

a

Values in ppm (d_C) measured at 75.45 MHz or 125.77 in CDCl₃.

Assignments were conﬁrmed by HSQC and HMBC experiments.

Assignments were established by comparison with the assignments of compounds 14, 19 and 20.

Values in ppm (d_C) measured at 75.45 MHz in DMSO-d6.

b

c

d

e

Assignments were established by comparison with the assignments of compounds 62 and 64.

bond in the fused ring D of analogs D was associated with a higher

growth inhibitory effect observed in the four tumor cell lines

studied. Pyranoxanthones with this particular fused ring orienta-

tion are not commonly found in nature [13] and, as far as we know,

no growth inhibitory activity has been previously reported for

pyranoxanthones with this geometry.

the aqueous to a non-polar media. This technique evaluate changes

in absorption parameters such as molar absorptivity (ε) or

maximum wavelength (l_max) and was calculated with the aid of

derivative spectroscopy to increase the noise-to-signal ratio and

eliminate the light scattering [71e75].

The K_pcan be calculated using the following equation

[72,74,75]:

2.3. Determination of the lipophilicity

Table 3

GI₅₀concentrations (

C5 and HL-60 cell lines.

The lipophilicity is commonly evaluated by the partition coef-

ﬁcient (K_p) of a solute in a biphasic octanolewater system and

designated as Log P [30,70]. In spite of being widely used in drug

discovery, it fails to mimic the anisotropic media found in mem-

branes and encode all the interactions that take place between a

solute and biomembranes [30,35]. As a result, membrane models

such as liposomes and micelles have been developed and showed

to be able to mimic to a better extent the biomembranes [36e41].

As a result, the lipophilicity of the synthesized compounds was

evaluated as the partition coefﬁcient (K_p) of the solute between

buffer (HEPES, pH ¼ 7.4) and liposomes or micelles.

mM) of the synthesized compounds in MCF-7, NCI-H460, A375-

Compound

GI₅₀(mM)

MCF-7

NCI-H460

A375-C5

HL-60

19

32

35

38

39

51

52

53

62

63

64

65

66

67

>150^a

32.9 ꢁ 7.1

44.5 ꢁ 1.4

31.7 ꢁ 2.6

>150^a

N.R.

>150^a

N.R.

13.3 ꢁ 1.3

50.9 ꢁ 3.5

39.6 ꢁ 0.6

>150^a

6.2 ꢁ 0.6

37.9 ꢁ 6.5

29.6 ꢁ 4.7

>150^a

3.2 ꢁ 0.7

36.7 ꢁ 3.3

38.9 ꢁ 9.8

26.1 ꢁ 9.5

11.8 ꢁ 3.8

31.8 ꢁ 6.1

>70^a

9.6 ꢁ 3.2

9.6 ꢁ 1.7

37.6 ꢁ 17.1

30.9 ꢁ 10.8

6.4 ꢁ 0.3

>50^a

N.R.

45.1 ꢁ 3.3

107.9 ꢁ 13.9

N.R.

47.3 ꢁ 6.0

>150^a

42.9 ꢁ 16.1

N.R.

42.5 ꢁ 5.5

>150^a

The method used for the determination of the K_pconsiders the

variations in the UVeVis spectra of a solute when it permeates from

47.4 ꢁ 4.1

69.5 ꢁ 5.9

>150^a

N.R.

>150^a

N.R.

>100^a

N.R.

>100^a

>50^a

14^b

20^b

26^b

27^b

28^b

29^b

39.7 ꢁ 3.2

40.3 ꢁ 3.3

28.9 ꢁ 8.1

23.4 ꢁ 1.1

8.8 ꢁ 5.9

N.D.

>150

88.6 ꢁ 12.9

>160

>150

>160

>150

N.D.

>150

N.D.

The values presented refer to mean ꢁ SE of at least three independent experiments.

N.R. e not-reproducible. N.D. e not determined. The maximum DMSO concentration

used was 0.25% for all compounds tested and was found to not interfere with cell

growth (data not shown). Doxorubicin was used as a positive control (MCF-7:

65.4 ꢁ 8.5 nM; NCI-H460: 64.1 ꢁ 6.8 nM, A375-C5: 144.8 ꢁ 9.8 nM and HL60:

28.0 ꢁ 0.6 nM).

Fig. 4. NOE correlation between proton in position 5 and the protons in the methoxyl

group bond to carbon 6 of compound 63. ORTEP view of the crystal structure of

compound 63 showing the atom-labeling scheme. Displacement ellipsoids are drawn

at the 50% probability level. Hydrogen atoms are represented by circles of arbitrary

size.

a

These values correspond to two experiments.

b

Previously published results in Refs. [67e69].

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

805

compounds 26e28, 66 and 67 with both low solubility and molar

absorptivity, the light scattering for wavelengths below 300 nm in

liposomes did not allow to calculate the Log K_pin this model.

The partition coefﬁcients determined in one or two membrane

models of the pyranoxanthones are summarized in Table 4. It can

be observed that all the compounds showed a Log K_pin liposomes

and micelles superior to 3 but below the critical 5 (upper limit

referred in the Lipinski “rule of ﬁve” [34]). Moreover, it can be

observed that the presence of a hydroxyl ortho to the carbonyl leads

to a dramatic increase in the lipophilicity (compare 14 with 26, 27,

38, 65; and 53 with 28, 29, 39, 66). This can be explained by the

intramolecular hydrogen bonding that is formed between the

carbonyl and the hydroxyl group. Considering the relation between

the hydroxyl or methoxyl group with the oxygen of the fused pyran

ring, the compounds with an ortho relationship seem in most of the

cases to have a lower partition coefﬁcient than the compounds with

a meta relationship (compare 19, 22 with 51, 52; and 39 with 26).

Comparing the linear with the angular arrangement of the pyr-

anoxanthones, it can be observed that in general, the latter have a

less partition coefﬁcient than the former (compare 26 with 27, 38

with 66; and 62, 64 with 63 and 65). Regarding the presence or

absence of the double bond in the fused ring, there is not a clear

tendency among the different set of compounds.

Fig. 5. Synthesized pyranoxanthones with different orientation of the fused ring D.

ðD_Iꢂ D_wÞK_p½LꢃV

f

D ¼ D_w

þ

(1)

1 þ K_p½LꢃV

f

where, [L] is the lipid concentration (mol L^ꢂ1), D_lthe derivative of

lipid absorbance, D_wthe derivative of water, V

f the lipid molar

volume (L mol^ꢂ1), K_pthe partition coefﬁcient (dimensionless) and:

In order to compare the liposome and micelle models for this

class of compounds, a linear regression analysis was made and the

following Equation (3) was established:

vⁿAbs

D ¼

(2)

v

lⁿ

Log K_{p liposomes}¼ ꢂ0:760ðꢁ0:604Þ

Accordingly, the partition coefﬁcient could be calculated by

ﬁtting the derivative spectrometric data (D vs [L]) to the Equation

(1) by a nonlinear regression method, being D_land K_pthe adjust-

able parameters [72,74,75].

þ 1:182ðꢁ0:164Þ Log K_{p micelles}

(3)

n ¼ 14; r²¼ 0:814; s ¼ 0:178; F ¼ 52

The K_pin liposomes/buffer was determined for 19, 32, 35, 38, 39,

51e56 and 62e65 and in micelles/buffer for all the compounds

with the exception of compound 23 which was obtained in low

yields (Table 4). The K_pwas determined using an adapted proce-

dure developed by Magalhães et al. using a 96-well plate [38] in

which DMSO was added to each plate to a maximum of 1% [76]. In

the case of the compounds having very low solubility and/or molar

absorptivity (ε), the Log K_pwas determined in a conventional

double-beam UVeVis spectrophotometer. Moreover, for

wherein 95% conﬁdence limits are in parentheses, n is the number

of compounds, r²the squared correlation coefﬁcient, s the standard

deviation, and F Fisher’s test. Considering the slope (1.182) and y-

intercept (ꢂ0.760), we can observe that there is not clear tendency

for this class of compounds to increase the afﬁnity for either of the

membrane models with an increase of hydrophobicity and that the

sum of interactions taking place between these compounds and

micelles are in general slightly higher than with liposomes. More-

over, a better correlation was obtained by removing two outliers,

namely compounds 64 and 65, which bare in common the presence

of a methoxyl ortho to the carbonyl and a fused 2,2-dimethyl-3,4-

dihydropyran ring.

Table 4

Partition coefﬁcients in liposomes-buffer and micelle-buffer for a chemical library of

pyranoxanthones.

Log K_{p liposomes}

Log K_{p micelles}

Log K_{p liposomes}¼ ꢂ0:308ðꢁ0:396Þ þ 1:075ðꢁ0:106Þ

Log K_{p micelles}n ¼ 12; r²¼ 0:902; s ¼ 0:118; F ¼ 102 (4)

19

26

27

28

29

32

35

38

39

51

52

53

62

63

64

65

66

67

14^a

20^a

3.88 ꢁ 0.09

e

3.83 ꢁ 0.05

4.32 ꢁ 0.04

4.27 ꢁ 0.06

4.06 ꢁ 0.01

4.02 ꢁ 0.03

3.50 ꢁ 0.12

4.10 ꢁ 0.04

4.15 ꢁ 0.08

3.88 ꢁ 0.08

4.07 ꢁ 0.10

3.88 ꢁ 0.02

3.29 ꢁ 0.06

3.76 ꢁ 0.03

3.33 ꢁ 0.02

3.58 ꢁ 0.08

3.46 ꢁ 0.01

4.08 ꢁ 0.04

4.28 ꢁ 0.03

3.28 ꢁ 0.02

3.59 ꢁ 0.06

e

3. Conclusions

3.42 ꢁ 0.02

4.14 ꢁ 0.08

4.17 ꢁ 0.06

3.96 ꢁ 0.03

3.94 ꢁ 0.09

3.92 ꢁ 0.04

3.06 ꢁ 0.16

3.54 ꢁ 0.01

3.32 ꢁ 0.12

3.09 ꢁ 0.18

3.08 ꢁ 0.06

e

In the present study, 21 pyranoxanthones were synthesized

either by molecular modiﬁcations of simple oxygenated xanthones

or by total synthesis. Regarding molecular modiﬁcations of simple

oxygenated xanthones, the pyran rings were formed either by the

cyclization with platinum of the dimethylpropargyl aryl ethers, or

by the condensation with prenal or by the reaction with prenyl

bromide catalyzed by Montmorillonite K-10 and microwave heat-

ing. The total synthesis of pyranoxanthones was accomplished via

diaryl ether and benzophenone using the appropriate benzopyrans

and carboxylic acid derivatives as building blocks.

e

3.35 ꢁ 0.02

3.60 ꢁ 0.08

The cell growth inhibitory activity of 14 of the synthesized

pyranoxanthones was evaluated in four human tumor cell lines,

namely MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell

Mean of three independent measurements.

a

These results have been published elsewhere [69].

806

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

lung cancer), A375-C5 (melanoma) and HL-60 (acute myeloid leu-

kemia). Among the cell lines tested, higher cell growth inhibitory

activity was generally observed in the HL-60 tumor cell line which

may indicate that a cellular target may be present or be more

relevant in this cell line than in the others. It was observed that the

orientation of the fused ring and the oxygenation pattern had an

effect in the cell growth inhibitory activity observed in the different

cell lines.

chromatography) or using Merck silica gel 60 (0.2e0.5 mm) (when

nothing is referred) and preparative thin layer chromatography

(TLC) using Merck silica gel 60 (GF₂₅₄) plates or GraceResolv^Òsilica

gel cartridges (5 g/25 mL).

4.2. Synthesis of pyranoxanthones 14, 19, 20 and 23

4.2.1. Synthesis of compound 15

The lipophilicity of the pyranoxanthones was evaluated for 20

compounds in two membrane models, namely liposomes and mi-

celles, and all the compounds showed a Log K_phigher than 3 and

below 5. Moreover, the micelle model showed a good correlation

with liposome model and as a result might be used as a surrogate of

liposomes for the determination of the partition coefﬁcient of

pyranoxanthones.

The present study allowed to describe, for the ﬁrst time, three

angular pyranoxanthones with a fused ring orientation which have

never been related to tumor cell growth inhibitory activity being

particularly active in the leukemia (HL-60) tumor cell line. Besides

the promising biological activity, the compounds with this partic-

ular fused ring orientation showed to have lower lipophilicity than

similar structural isomers. These facts make them interesting

scaffolds for further studies.

3,4-Dimethoxyxanthone (15) was synthesized according to the

published procedure [78] and the spectroscopic data were in

accordance with the literature [79].

4.2.2. Synthesis of compounds 16 and 17

2-(Diethylamino)ethanethiol HCl (397 mg/2.34 mmol) and 8 mL

of DMF anhydrous was placed in a two-necked round-bottom ﬂask

and under nitrogen atmosphere. The ﬂask was cooled in an ice bath

and when the internal temperature was below 5 ^ꢀC, solid NaO^tBu

(450 mg/4.68 mmol) was added in one portion. After 5 min the ice

bath was removed and it was allowed to warm to room tempera-

ture. After 15 min, a solution of 3,4-dimethoxyxanthone (15)

(500 mg/1.95 mmol) in 4 mL of DMF anhydrous was added. The

mixture was heated at reﬂux for 2 h under nitrogen atmosphere.

The mixture was allowed to cool to room temperature and then

poured over crushed ice. A solution of HCl 1 M was added to the

mixture until pH 1. The mixture was extracted with 3 ꢄ 50 mL of

ethyl acetate. The organic phase was washed with 3 ꢄ 50 mL of

water, dried over sodium sulfate anhydrous, ﬁltered and the

organic solvent evaporated. The crude product was puriﬁed by

silica gel ﬂash chromatography (petroleum ether (60e80^ꢀ)/ethyl

acetate 7:3). 3-Hydroxy-4-methoxyxanthone (17) was crystallized

from chloroform/petroleum ether (60e80^ꢀ) as white solid

(221.4 mg/46%) and 4-hydroxy-3-methoxyxanthone (16) from

methanol as a white solid (83 mg/17%).

4. Experimental

4.1. General methods

Microwave reactions were performed using glassware setup for

atmospheric-pressure reactions and also 12 mL, 50 mL, 100 mL or

270 mL closed glass reactors (internal reaction temperature mea-

surement with a ﬁber-optic probe sensor) and were carried out

using an Ethos MicroSYNTH 1600 Microwave Labstation from

Milestone. Reactions were monitored by TLC and/or GCeMS.

Melting points were obtained in a Köﬂer microscope and are un-

corrected. IR spectra were measured on an ATI Mattson Genesis

series FTIR (software: WinFirst v. 2.10) spectrophotometer in KBr

microplates (cm^ꢂ1). ¹H and ¹³C NMR spectra were taken in CDCl₃or

DMSO-d₆at room temperature, on Bruker Avance 300, 400 and 500

The spectroscopic data of 4-hydroxy-3-methoxyxanthone (16)

and 3-hydroxy-4-methoxyxanthone (17) was in accordance with

the literature [79].

4.2.3. Synthesis of compound 18

3-Hydroxy-4-methoxyxanthone (17) (120 mg/0.5 mmol), KI

(168.5 mg/1 mmol), CuI (11.8 mg/0.062 mmol), K₂CO₃(135.8 mg/

1 mmol) and 5 mL of anhydrous DMF were added to a round-

instruments. Chemical shifts are expressed in

d (ppm) values rela-

tive to tetramethylsilane (TMS) as an internal reference. ¹H NMR

spectra were measured at 300.13 MHz or 500.13 MHz and assign-

ment abbreviations are the following: singlet (s), doublet (d), triplet

(t), quartet (q), quintet (qt), multiplet (m), doublet of doublets (dd),

double doublet of doublets (ddd), doublet of triplets (dt) and triplet

of doublets (td). ¹³C NMR spectra were measured at 75.47 MHz,

100.63 MHz or 125.77 MHz. ¹³C NMR assignments were made by 2D

HSQC and HMBC experiments (long-range C, H coupling constants

were optimized to 7 and 1 Hz) or by comparison with the assign-

ments of similar molecules. The EI-MS were recorded on a Ther-

moQuest Finnigan GC 2000 series/GCQ plus. HRMS spectra were

recorded as ESI (electrospray ionization) mode either on an APEXQe

FT-ICRMS (Bruker Daltonics), equipped with a 7T actively shielded

magnet or VG Autoespec MicroTOF FOCUS (Bruker Daltonics)

spectrometer at C.A.C.T.I. e University of Vigo, Spain; or Bruker

micrOTOF-Q II (ESI) at the University of Southern Denmark. The X-

bottom ﬂask. 3-Chloro-3-methyl-1-butyne (430

mL/13.2 mmol)

was then added and the mixture was heated under nitrogen at-

mosphere at 75 ^ꢀC for 8 h. The solution was allowed to cool to room

temperature and poured over crushed ice and 5 mL of HCl 1 M. The

aqueous phase was extracted with 3 ꢄ 50 mL of ethyl acetate. The

organic layer was washed with 3 ꢄ 50 mL of distilled water, dried

over sodium sulfate anhydrous, ﬁltered and the organic solvent

evaporated. The crude product was then puriﬁed by silica gel ﬂash

chromatography (chloroform). Compound 18 was isolated as a

white solid (126 mg/83%).

4-Methoxy-3-((2-methylbut-3-yn-2-yl)oxy)xanthone (18). Mp:

121e123 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3432, 3249, 2984, 2934, 2836,

1655, 1602, 1498, 1464, 1448, 1424, 1332, 1278, 1140, 1068, 1034,

743. ¹H NMR (300.13 MHz, CDCl₃)

d

(ppm): 8.33 (dd, J ¼ 8.0, 1.7, He

C(8)), 8.04 (d, J ¼ 9.1, HeC(1)), 7.73 (ddd, J ¼ 8.5, 7.0, 1.7, HeC(6)),

7.62 (d, J ¼ 9.1, HeC(2)), 7.58 (d, J ¼ 7.8, HeC(5)), 7.39 (ddd, J ¼ 8.0,

7.1, 1.0, HeC(7)), 4.03 (s, CH₃OeC(4)), 2.67 (s, HeC(3⁰)), 1.78 (s, 6H,

Ray was determined in a Gemine PX ultra equipped with a CuK_a

ꢀ

radiation (

l

¼ 1.54184 A) and the structure was solved using

SHELXS-97 and reﬁned with SHELXL-97. All the reagents were

purchased from Sigma Aldrich or Acros and all the solvents were PA

used without further puriﬁcation. The anhydrous solvents were

either purchased from SigmaeAldrich or dried according to the

published procedures [77]. Puriﬁcations of compounds were per-

formed by column chromatography either by using Merck silica gel

60 (0.040e0.063 mm) (when it is referred to as ﬂash

HeC(1⁰⁰a/1⁰⁰b)). ¹³C NMR (75.47 MHz, CDCl₃)

d (ppm): 176.7 (C9),

156.2 (C10a), 154.2 (C3), 150.8 (C4a), 139.9 (C4), 134.5 (C6), 126.6

(C8), 124.0 (C7), 121.6 (C8a), 121.0 (C1), 118.1 (C5), 118.0 (C9a), 116.8

(C2), 85.2 (C2⁰), 74.9 (C3⁰), 74.2 (C1⁰), 61.5 (C1⁰⁰⁰), 29.7 (C1⁰⁰a/1⁰⁰b).

EIMS m/z (%): 309 (4, [M þ 1]^þ.), 308 (15, [M]^þ.), 294 (30), 293 (100),

278 (65), 194 (32), 165 (40), 89 (14), 76 (18), 63 (19). HRMS (ESI) m/z

calcd for C₁₉H₁₆NaO₄[M þ Na]^þ: 331.0941; found: 317.0925.

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

807

4.2.4. Synthesis of compound 19

150 g of crushed ice and 1 mL of concentrated HCl. The aqueous

phase was extracted with 3 ꢄ 100 mL of ethyl acetate. The organic

phase was washed 3 ꢄ 150 mL of distilled water, dried over sodium

sulfate anhydrous, ﬁltered and the organic solvent evaporated. The

crude product was puriﬁed by silica gel chromatography (chloro-

form/acetone 9:1). 3,4-Dihydroxyxanthone (21) was crystallized

from methanol as a pale yellow solid (534 mg/78%).

4.2.4.1. Thermal cyclization. A solution of 81 mg of compound 18 in

10 mL of anhydrous DMF was heated at 130 ^ꢀC under nitrogen at-

mosphere for 7 h. The solution was allowed to cool to room tem-

perature and the mixture was poured into 50 g of crushed ice and

1 mL of concentrated HCl. The aqueous phase was extracted with

3 ꢄ 50 mL of ethyl acetate. The organic phase was washed

3 ꢄ 50 mL of distilled water, dried over Na₂SO₄, ﬁltered and the

organic solvent evaporated. The crude product was puriﬁed by

silica gel ﬂash chromatography (petroleum ether (60e80^ꢀ)/ethyl

ether 7:3). The product was then further puriﬁed by preparative

TLC to give compound 19 as a white solid (6.3 mg/8%).

The spectroscopic data of 3,4-dihydroxyxanthone (21) was in

accordance with the literature [79].

4.2.7. Synthesis of compound 14

12-Hydroxy-2,2-dimethyl-3,4-dihydropyrano[3,2-b]xanthen-

6(2H)-one (14) was synthesized according to the published pro-

cedure (yield obtained of 21%) and the spectroscopic data was in

accordance with the literature [25].

4.2.4.2. Hydroarylation of alkynes by gold catalyst. Compound 18

(166.1 mg, 0.539 mmol) and (Ph₃P)AuNTf₂(4.2 mg, 2.695 mmol)

(Gagoz’s catalyst [47]) were placed in a two-necked round-bottom

ﬂask. The two solids were placed under nitrogen atmosphere and

5 mL of anhydrous toluene was added. The mixture was stirred for

7 h at 85 ^ꢀC. The reaction mixture was allowed to cool to room

temperature, ﬁltered, and washed with acetone. The mixture was

then concentrated under reduced pressure and the crude product

puriﬁed by silica gel ﬂash chromatography (n-hexane/ethyl acetate

9:1 to 5:5). Compound 19 was obtained as a white crystalline solid

(50.1 mg, 30%). Compound 17 was also isolated (83 mg/50%).

4.2.8. Synthesis of compound 22

3,4-Dihydroxyxanthone (21) (100 mg/0.44 mmol), potassium

carbonate (91 mg/0.66 mmol), potassium iodide (109 mg/

0.66 mmol) and copper iodide (13 mg/0.066 mmol) were placed in

a round-bottom ﬂask. The ﬂask was placed under nitrogen atmo-

sphere and 20 mL of anhydrous acetone was added. The mixture

was stirred for 15 min and 3-chloro-3-methyl-1-butyne (45 mg/

0.438 mmol) was added dropwise. The mixture was heated at 40 ^ꢀC

for 2 h and then allowed to cool to room temperature. The crude

product was ﬁltered and the organic solvent evaporated. The

extract was then puriﬁed by silica gel ﬂash chromatography (n-

hexane/ethyl acetate 9:1). Compound 22 was isolated as a white

solid (39 mg/30%) and compound 24 was isolated as a white solid

(60 mg/47%).

4.2.4.3. Hydroarylation of alkynes by PtCl₄. Compound 18 (110 mg,

0.35 mmol), PtCl₄(17.7 mg, 52.5 mmol, 15 mol %) and 15 mL dioxane

were placed in a round-bottom ﬂask. The mixture was stirred at r.t.

for 11 h and then ﬁltered. The solid was washed with diethyl ether

and dichloromethane. The organic phases were reunited and

concentrated under reduced pressure. The crude product was pu-

riﬁed by silica gel ﬂash chromatography (n-hexane/diethyl ether

8:2). Compound 19 was obtained as a white crystalline solid

(62.3 mg, 57%).

4-Hydroxy-3-((2-methylbut-3-yn-2-yl)oxy)xanthone (22). Mp:

168e169 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3254, 3152, 3114, 3071, 2992,

2934, 2361, 1639, 1604, 1577, 1455, 1344, 1277, 1247, 1217, 1135,

1065, 1004, 760, 707, 677. ¹H NMR (300.13 MHz, CDCl₃)

d (ppm):

12-Methoxy-2,2-dimethylpyrano[3,2-b]xanthen-6(2H)-one (19).

8.34 (dd, J ¼ 8.0, 1.7), 7.86 (d, J ¼ 8.9), 7.73 (ddd, J ¼ 8.5, 7.0, 1.7), 7.60

Mp: 129e130 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3254, 2973, 2932,1656,1607,

(dd, J ¼ 8.5,1.1), 7.55 (d, J ¼ 8.9), 7.39 (ddd, J ¼ 8.0, 7.0,1.1), 5.89 (OH),

1482, 1438, 1326, 1274,1128, 1074, 748. ¹H NMR data, see Table 1. ¹³

C

2.71 (s, 1H), 1.79 (s, 6H). ¹³C NMR (100.63 MHz, CDCl₃)

d (ppm):

NMR data, see Table 2. HRMS (ESI) m/z calcd for C₁₉H₁₇O₄[M þ H]^þ:

176.7, 156.2, 147.0, 145.3, 136.6, 134.6, 130.9, 126.7, 124.0, 121.3, 118.1,

116.8, 115.7, 84.7, 75.2, 74.9, 29.7. EIMS m/z (%): 295 (2, [M þ 1]^þ.),

294 (8, [M]^þ.), 279 (56), 205 (16), 165 (16), 146 (21), 121 (36), 115

(39), 102 (23), 92 (26), 91 (26), 77 (100), 76 (30), 75 (40), 74 (30), 65

(39), 63 (33), 51 (84). HRMS (ESI) m/z calcd for C₁₈H₁₄NaO₄

[M þ Na]^þ: 317.0784; found: 317.0780.

309.11214; found: 309.11204.

4.2.5. Synthesis of compound 20

To a Teﬂon vessel for microwave heating with 3-hydroxy-4-

methoxyxanthone (17) (110 mg/0.46 mmol), 2 g of clay montmo-

rillonite K-10 and approximately 10 mL of chloroform (just enough

to wet the clay), was added prenyl bromide (0.92 mmol). The

mixture was heated for 1 h and 15 min at 110 ^ꢀC under microwave

heating. The mixture was allowed to cool to room temperature and

the product was ﬁltered and washed with chloroform, acetone and

methanol. The organic solvents were reunited and evaporated. The

crude product was then puriﬁed by silica gel ﬂash chromatography

(petroleum ether (60e80^ꢀ)/ethyl acetate 95:5 to 5:5). Compound

20 was recrystallized from chloroform and petroleum ether (60e

80^ꢀ) as a white solid (17 mg/12%).

3,3-Dimethyl-2-methylene-2H-[1,4]dioxino[2,3-c]xanthen-7(3H)-

one (24). Mp: 173e175 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3008, 2972, 2920,

1651, 1605, 1501, 1449, 1332,1303, 1220, 1203, 1165, 1060, 1032, 899,

861, 748, 677. ¹H NMR (300.13 MHz, CDCl₃)

d (ppm): 8.32 (dd,

J ¼ 8.0, 1.7), 7.89 (d, J ¼ 8.9), 7.70 (ddd, J ¼ 8.5, 7.1, 1.7), 7.59 (dd,

J ¼ 8.5,1.0), 7.37 (ddd, J ¼ 8.0, 7.1,1.0), 6.89 (d, J ¼ 8.9), 4.97 (d, J ¼ 2.5,

1H), 4.65 (d, J ¼ 2.5, 1H), 1.58 (s, 6H). ¹³C NMR (100.63 MHz, CDCl₃)

d

(ppm): 176.2, 156.0, 155.1, 146.7, 145.3, 134.4, 130.0, 126.7, 124.1,

121.8, 119.6, 118.1, 116.8, 114.0, 91.3, 74.0, 25.1. EIMS m/z (%): 295 (1,

[M þ 1]^þ.), 294 (12, [M]^þ.), 251 (25), 228 (69), 200 (19), 172 (21), 171

(31), 115 (53), 114 (44), 88 (23), 77 (26), 76 (30), 75 (21), 67 (74), 65

(100), 63 (39), 62 (24). 53 (35), 51 (53). HRMS (ESI) m/z calcd for

12-Methoxy-2,2-dimethyl-3,4-dihydropyrano[3,2-b]xanthen-

6(2H)-one (20; 12%). Mp: 173e174 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 2967,

2936, 2829, 1655, 1609, 1443, 1326, 1114, 1077, 748. ¹H NMR data,

see Table 1. ¹³C NMR data, see Table 2. HRMS (ESI) m/z calcd for

C

₁₈H₁₅O₄[M þ H]^þ: 295.09649; found: 295.09787; m/z calcd for

₁₈H₁₄NaO₄[M þ Na]^þ: 317.07843, found: 317.07867.

C

₁₉H₁₉O₄[M þ H]^þ: 311.12779; found: 311,12772.

4.2.9. Synthesis of compound 23

4.2.6. Synthesis of compound 21

To a solution of compound 22 (30 mg/0.102 mmol) in 2.5 mL of

dioxane was added PtCl₄(0.015 mmol/5 mg). The mixture was

stirred for 6 h at room temperature. The mixture was ﬁltered and

washed with the ethyl ether. The organic solvents were reunited

and evaporated. The crude mixture was then puriﬁed by silica gel

ﬂash chromatography (n-hexane/ethyl acetate 9:1 to 7:3).

3,4-Dimethoxyxanthone (15) (769 mg/3 mmol) and 50 mL of

anhydrous toluene were added in a round-bottom ﬂask. The

mixture was placed at 0 ^ꢀC and aluminum chloride (1.2 g/9 mmol)

was added slowly. The mixture was heated for 2 h under reﬂux. The

mixture was allowed to cool to room temperature and poured over

808

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

Compound 23 was isolated as a light yellow solid (10 mg/33%).

Compound 24 (16 mg/53%) was also isolated.

ethyl acetate 9:1). Compound 31 was isolated as a white solid

(102 mg/52%).

12-Hydroxy-2,2-dimethylpyrano[3,2-b]xanthen-6(2H)-one (23).

Mp: 178e179 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3247, 2955, 2915, 1843, 1638,

1600, 1449, 1346, 1272, 1208, 1135, 753. ¹H NMR data, see Table 1.

¹³C NMR data, see Table 2. EIMS m/z (%): 294 (3, [M]^þ.), 279 (69),

205 (34), 165 (36), 152 (23), 139 (22), 121 (19), 115 (30), 102 (19), 91

(29), 89 (31), 88 (27), 87 (20), 77 (84), 75 (60), 63 (89), 53 (22), 51

(100). HRMS (ESI) m/z calcd for C₁₈H₁₅O₄[M þ H]^þ: 295.09649,

found: 295.09584.

4-((2-Methylbut-3-yn-2-yl)oxy)xanthone (31). Mp: 59e61 ^ꢀC. IR

n_max(cm^ꢂ1) (KBr): 3266, 2980, 2919, 2866, 2334, 1649, 1590, 1560,

1479, 1457, 1434, 1329, 1216, 1123, 904, 745, 626. ¹H NMR

(300.13 MHz, CDCl₃)

d

(ppm): 8.34 (dd, J ¼ 8.0, 1.70), 8.08 (dd,

J ¼ 8.0, 1.6), 7.82 (dd, J ¼ 8.0, 1.6), 7.73 (ddd, J ¼ 8.5, 7.1, 1.7), 7.55 (d,

J ¼ 8.5), 7.39 (ddd, J ¼ 8.0, 7.1, 1.1), 7.30 (t, J ¼ 8.0), 2.56 (s,1H), 1.78 (s,

6H). ¹³C NMR (100.63 MHz, CDCl₃)

d (ppm): 177.3, 155.8, 150.1,

144.3, 134.8, 127.8, 126.7, 124.0, 123.0, 121.6, 121.2, 118.2, 85.3, 75.2,

74.5, 29.6. EIMS m/z (%): 278 (5, [M]^þ.), 264 (20), 263 (100),179 (25),

178 (57), 115 (36), 77 (21), 76 (20), 63 (34), 51 (33). HRMS (ESI) m/z

calcd for C₁₈H₁₄NaO₃[M þ Na]^þ: 301.0835; found: 301.0834.

4.3. Synthesis of the pyranoxanthones 26, 27, 28 and 29

4.3.1. Synthesis of compound 25

4.4.3. Synthesis of compound 32

1,3-Dihydroxyxanthone (25) was synthesized according to the

published procedure [80] and the spectroscopic data was in

accordance with the literature [80,81].

To a solution of compound 31 (60 mg/0.216 mmol) in 2 mL of

dioxane was added PtCl₄(0.032 mmol/11 mg). The mixture stirred

overnight at room temperature. The solution was ﬁltered and the

solvent evaporated. The crude mixture was puriﬁed by silica gel

ﬂash chromatography (n-hexane/ethyl acetate 9:1). Compound 32

was isolated as yellow solid (45 mg/76%) and also 4-

hydroxyxanthone (30) as a white solid (7 mg/15%).

2,2-Dimethylpyrano[3,2-c]xanthen-7(2H)-one (32). Mp: 182e

184 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 2967, 2918, 2880, 1639, 1597, 1449,

1318, 1260, 1209, 1185, 1113, 1057, 905, 761, 737, 680. ¹H NMR

4.3.2. Synthesis of compounds 26 and 27

5-Hydroxy-2,2-dimethyl-3,4-dihydropyrano[3,2-b]xanthen-

6(2H)-one (26) and 6-hydroxy-3,3-dimethyl-2,3-dihydropyrano

[2,3-c]xanthen-7(1H)-one (27) were synthesized according to the

published procedure [50] and the spectroscopic data are in accor-

dance with the literature [67].

(300.13 MHz, CDCl₃)

d

(ppm): 8.32 (dd, J ¼ 8.0, 1.6), 7.82 (d, J ¼ 8.1),

4.3.3. Synthesis of compounds 28 and 29

7.71 (ddd, J ¼ 8.5, 8.0, 1.6), 7.61 (dd, J ¼ 8.5, 1.1), 7.37 (ddd, J ¼ 8.0, 6.9,

To a solution of compound 25 (200 mg/0.88 mmol) and Ca(OH)₂

(130.4 mg/1.76 mmol) in 50 mL of methanol was added prenal

(370 mg/4.4 mmol). The mixture was stirred for 66 h at room

temperature. Methanol was evaporated and the crude product was

partitioned between water and ethyl acetate. The aqueous phase

was extracted 2 ꢄ 50 mL of ethyl acetate. The organic phase was

washed 3 ꢄ 50 mL HCl 1 M, 3 ꢄ 50 mL of distilled water, dried over

sodium sulfate anhydrous, ﬁltered and the organic solvent evapo-

rated. The crude product was puriﬁed by silica gel chromatography

using Grace ResolvTM 5 g/25 mL cartridge (petroleum ether (60e

80^ꢀ)/ethyl acetate 9:1 to 5:5). Compound 28 (61.2 mg/24%) and 29

(10 mg/4%) were puriﬁed as yellow needles.

1.1), 7.01 (d, J ¼ 8.1), 6.44 (d, J ¼ 9.8), 5.82 (d, J ¼ 9.8), 1.57 (s, 6H). ¹³

C

NMR (125.77 MHz, CDCl₃)

d (ppm): 176.9, 156.1, 145.7, 141.3, 134.5,

133.5, 126.6, 126.0, 123.8, 122.4, 122.0, 121.7, 121.3, 118.4, 117.6, 77.5,

27.9. EIMS m/z (%): 278 (5, [M]^þ.), 264 (20), 263 (100), 178 (24), 89

(20), 77 (21), 76 (25), 75 (21), 63 (35), 51 (36). HRMS (ESI) m/z calcd

for C₁₈H₁₅O₃[M þ H]^þ: 279.10157; found: 279.10257; m/z calcd for

C

₁₈H₁₄NaO₄[M þ Na]^þ: 301.08352; found: 308.08303.

4.5. Synthesis of pyranoxanthone 35

4.5.1. Synthesis of compound 33

2-Hydroxyxanthone (33) was synthesized according to the

published procedure [82] and the spectroscopic data was in

accordance with the literature [79].

The spectroscopic data of 5-hydroxy-2,2-dimethylpyrano[3,2-b]

xanthen-6(2H)-one (28) and 6-hydroxy-3,3-dimethylpyrano[2,3-c]

xanthen-7(3H)-one (29)synthesized was in accordance with the

literature [68].

4.5.2. Synthesis of compound 34

2-Hydroxyxanthone (33) (155 mg/0.73 mmol), potassium car-

bonate (151 mg/1.1 mmol), potassium iodide (183 mg/1.1 mmol)

and copper iodide (21 mg/0.11 mmol) were added to a round-

bottom ﬂask. The ﬂask was placed under nitrogen atmosphere

and 15 mL of anhydrous DMF was added. The mixture was stirred

for 15 min and 3-chloro-3-methyl-1-butyne (150 mg/1.46 mmol)

was added dropwise. The mixture was heated to 75 ^ꢀC and stirred

for 24 h. The reaction was allowed to cool to room temperature and

poured over 100 g of crushed ice. The aqueous phase was extracted

with 3 ꢄ 100 mL of ethyl ether. The organic phase was washed with

2 ꢄ 100 mL of brine, dried over sodium sulfate anhydrous, ﬁltered

and the organic solvent evaporated. The crude product was puriﬁed

by silica gel ﬂash chromatography (n-hexane/ethyl acetate 9:1).

Compound 34 was isolated as a white solid (80 mg/39%).

4.4. Synthesis of pyranoxanthone 32

4.4.1. Synthesis of compound 30

4-Hydroxyxanthone (30) was synthesized according to the

published procedure [82] and the spectroscopic data was in

accordance with the literature [79].

4.4.2. Synthesis of compound 31

4-Hydroxyxanthone (30) (150 mg/0.707 mmol), potassium

carbonate (147 mg/1.06 mmol), potassium iodide (176 mg/

1.06 mmol) and copper iodide (20 mg/0.106 mmol) were placed in a

round-bottom ﬂask. The ﬂask was placed under nitrogen atmo-

sphere and 15 mL of anhydrous DMF was added. The mixture

stirred for 15 min and 3-chloro-3-methyl-1-butyne (145 mg/

1.415 mmol) was added dropwise. The mixture was heated to 75 ^ꢀC

and stirred for 14 h. The solution was allowed to cool to room

temperature and poured over 100 g of crushed ice. The aqueous

phase was extracted 3 ꢄ 100 mL of ethyl ether. The organic phase

was washed with 2 ꢄ 100 mL of brine, dried over sodium sulfate

anhydrous, ﬁltered and the organic solvent evaporated. The crude

product was puriﬁed by silica gel ﬂash chromatography (n-hexane/

2-((2-Methylbut-3-yn-2-yl)oxy)xanthone (34). Mp: 103e105 ^ꢀC.

IR n_max(cm^ꢂ1) (KBr): 3219, 2976, 2917, 2847, 2362, 1645, 1484, 1462,

1314, 1224, 1141, 899, 752. ¹H NMR (300.13 MHz, CDCl₃)

d (ppm):

8.34 (dd, J ¼ 8.0, 1.7), 8.15 (d, J ¼ 2.9), 7.72 (ddd, J ¼ 8.6, 7.1, 1.7), 7.57

(dd, J ¼ 8.9, 2.9), 7.48 (dd, J ¼ 8.6, 1.0), 7.44 (d, J ¼ 8.9), 7.37 (ddd,

J ¼ 8.0, 7.1, 1.0), 2.64 (s, 1H), 1.70 (s, 6H). ¹³C NMR (100.63 MHz,

CDCl₃)

d (ppm): 177.1, 156.2, 152.3, 151.6, 134.7, 130.0, 126.7, 123.8,

122.0, 121.3, 118.7, 117.9, 117.3, 85.4, 74.8, 63.4, 29.5. EIMS m/z (%):

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

809

279 (3, [M þ 1]^þ.), 278 (12, [M]^þ.), 264 (18), 263 (100), 235 (16), 220

(16), 205 (30), 180 (40), 152 (26), 131 (18), 115 (16), 103 (15), 89 (16).

HRMS (ESI) m/z calcd for C₁₈H₁₄NaO₃[M þ Na]^þ: 301.0835; found:

301.0826.

1-Hydroxy-2-((2-methylbut-3-yn-2-yl)oxy)xanthone (37). Mp:

103e104 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3465, 3241, 2978, 2917, 2847,

2324, 1647, 1606, 1580, 1460, 1283, 1232, 1133, 1053, 888, 760. ¹H

NMR (300.13 MHz, CDCl₃)

d

(ppm): 12.81 (OH), 8.29 (dd, J ¼ 8.0,1.7),

7.83 (d, J ¼ 9.0), 7.76 (ddd, J ¼ 8.3, 7.1, 1.7), 7.47 (d, J ¼ 8.3), 7.40 (ddd,

4.5.3. Synthesis of compound 35

J ¼ 8.0, 7.1, 0.9), 6.90 (d, J ¼ 9.0), 2.57 (s, 1H), 1.72 (s, 6H). ¹³C NMR

To a solution of compound 34 (60 mg/0.216 mmol) in 2 mL of

dioxane was added PtCl₄(0.032 mmol/11 mg). The mixture was

stirred overnight at room temperature. The solution was ﬁltered

and the solvent evaporated. The crude product was puriﬁed by

silica gel ﬂash chromatography (n-hexane/ethyl acetate 9:1).

Compound 35 was isolated as yellow solid (44 mg/75%) and 2-

hydroxyxanthone (33) as a pale yellow solid (7 mg/15%).

3,3-Dimethylpyrano[3,2-a]xanthen-12(3H)-one (35). Mp: 96e

98 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3063, 2968, 2915, 2853, 1642, 1608,

1576, 1466, 1449, 1300, 1234, 1115, 911, 824, 764, 749, 714. ¹H NMR

(100.63 MHz, CDCl₃) d (ppm): 182.7, 156.3, 155.1, 152.4, 137.6, 135.6,

132.6, 126.0, 124.0, 120.1, 117.8, 109.5, 105.7, 86.3, 75.3, 73.9, 29.3.

EIMS m/z (%): 295 (2, [M þ 1]^þ.), 294 (8, [M]^þ.), 279 (56), 121 (36),

115 (39), 77 (100), 76 (30), 75 (40), 74 (30), 65 (39), 63 (33), 51 (84).

HRMS (ESI) m/z calcd for C₁₈H₁₅O₄[M þ H]^þ: 295.0965, found:

295.0961.

3,3-Dimethyl-2-methylene-2H-[1,4]dioxino[2,3-a]xanthen-

12(3H)-one or 2,2-dimethyl-3-methylene-2H-[1,4]dioxino[2,3-a]

xanthen-12(3H)-one (40 or 41).

Compound 40. Mp: 90e93 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 2976, 2929,

(300.13 MHz, CDCl₃)

d

(ppm): 8.27 (dd, J ¼ 8.0,1.7), 8.09 (d, J ¼ 10.2),

2847, 1651, 1613, 1460, 1299, 1231, 1198, 1161, 1043, 859, 801, 761. ¹H

7.66 (ddd, J ¼ 8.4, 7.1, 1.74), 7.41 (dd, J ¼ 8.4, 1.1), 7.32 (ddd, J ¼ 8.0, 7.1,

NMR (300.13 MHz, CDCl₃)

d

(ppm): 8.28 (dd, J ¼ 8.0, 1.6), 7.66 (ddd,

1.1), 7.27 (d, J ¼ 9.2), 7.18 (d, J ¼ 9.2), 5.82 (d, J ¼ 10.2),1.46 (s, 6H). ¹³

C

J ¼ 8.5, 7.0,1.6), 7.40 (d, J ¼ 8.5,1.1), 7.33 (ddd, J ¼ 8.0, 7.0,1.1), 7.31 (d,

J ¼ 9.0), 7.03 (d, J ¼ 9.0), 4.72 (d, J ¼ 2.3, 1H), 4.52 (d, J ¼ 2.3, 1H), 1.64

NMR (125.77 MHz, CDCl₃)

d (ppm): 179.1, 155.3, 151.9, 149.1, 134.3,

132.5, 126.6, 124.1, 123.4, 122.2, 120.9, 120.0, 118.0, 117.4, 116.3, 75.4,

27.3. EIMS m/z (%): 278 (5, [M]^þ.), 264 (18), 263 (100), 180 (30), 178

(25), 152 (20), 88 (91), 77 (23), 76 (24), 75 (23), 74 (21), 65 (51), 62

(22), 51 (34). HRMS (ESI) m/z calcd for C₁₈H₁₅O₃[M þ H]^þ:

279.10157; found: 279.10227; m/z calcd for C₁₈H₁₄NaO₃[M þ Na]^þ:

301.08352; found: 308.09484.

(s, 6H). ¹³C NMR (100.63 MHz, CDCl₃)

d (ppm): 176.4, 155.7, 155.3,

152.2, 141.2, 137.5, 134.2, 126.6, 124.0, 123.7, 122.3, 122.0, 117.4,

109.8, 89.3, 73.7, 25.1. EIMS m/z (%): 295 (2, [M þ 1]^þ.), 294 (9,

[M]^þ.), 253 (27), 251 (45), 228 (70), 200 (27), 199 (47), 115 (35), 114

(24), 92 (12), 89 (10), 88 (16), 78 (19), 77 (100), 65 (38), 63 (40), 51

(62). HRMS (ESI) m/z calcd for C₁₈H₁₄NaO₄[M þ Na]^þ: 317.0784,

found: 317.0771.

4.6. Synthesis of pyranoxanthones 38 and 39

Compound 41. Mp: 116e119 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 2955, 2923,

2849, 1653, 1615, 1592, 1462, 1301, 1232, 1163, 1044, 862, 799, 761.

4.6.1. Synthesis of compound 36

¹H NMR (300.13 MHz, CDCl₃)

d

(ppm): 8.31 (dd, J ¼ 8.0, 1.7), 7.67

1,2-Dihydroxyxanthone (36) was synthesized according to the

published procedure [83] and the spectroscopic data was in

accordance with the literature [84].

(ddd, J ¼ 8.5, 7.1, 1.7), 7.42 (d, J ¼ 8.5), 7.35 (ddd, J ¼ 8.0, 7.1, 1.1), 7.24

(d, J ¼ 9.0), 7.03 (d, J ¼ 9.0), 5.01 (d, J ¼ 2.3, 1H), 4.61 (d, J ¼ 2.3, 1H),

1.64 (s, 6H). ¹³C NMR (100.63 MHz, CDCl₃)

d (ppm): 176.3, 155.5,

155.3, 151.7, 140.9, 137.0, 134.3, 126.7, 123.9, 123.7, 122.2, 117.4, 111.4,

110.6, 91.0, 72.3, 24.9. EIMS m/z (%): 295 (3, [M þ 1]^þ.), 294 (17,

[M]^þ.), 251 (50), 228 (90), 195 (54), 115 (34), 77 (100), 67 (31), 65

(44), 63 (40), 51 (69). HRMS (ESI) m/z calcd for C₁₈H₁₄NaO₄

[M þ Na]^þ: 317.0784, found: 317.0784.

4.6.2. Synthesis of compound 37

4.6.2.1. Method A. 1,2-Dihydroxyxanthone (36) (300 mg/1.31

mmol), potassium carbonate (181 mg/1.31 mmol), potassium io-

dide (328 mg/1.97 mmol) and copper iodide (38 mg/0.197 mmol)

were placed in a round-bottom ﬂask. The ﬂask was placed under N₂

atmosphere and 20 mL of anhydrous DMF was added. The mixture

was stirred for 15 min and 3-chloro-3-methyl-1-butyne

(202 mg/1.97 mmol) was added dropwise. The mixture was heat-

ed at 50 ^ꢀC for 1 h and then allowed to cool to room temperature.

The crude product poured over 100 g of crushed ice and extracted

3 ꢄ 100 mL of ethyl ether. The organic phase was washed with

2 ꢄ 100 mL of brine, dried over sodium sulfate anhydrous, ﬁltered

and the organic solvent evaporated. The crude product was puriﬁed

by silica gel ﬂash chromatography (n-hexane/ethyl acetate 95:5).

Compound 37 was isolated as an orange solid (38 mg/10%), com-

pound 40 (181 mg/47%) as an orange solid and also compound 41

(35 mg/10%) as an orange solid. 1,2-Dihydroxyxanthone (36) was

also recovered (90 mg/30%).

4.6.3. Synthesis of compound 38

To a solution of compound 37 (81 mg/0.275 mmol) in 2.5 mL of

dioxane was added PtCl₄(0.041 mmol/14 mg). The mixture was

stirred for 24 h at room temperature. The solution was ﬁltered and

the organic solvent evaporated. The crude product was puriﬁed by

silica gel ﬂash chromatography (n-hexane/ethyl acetate 95:5).

Compound 38 was isolated as an orange solid (60 mg/73%) and also

1,2-dihydroxyxanthone (36) (14 mg/22%).

12-Hydroxy-2,2-dimethylpyrano[2,3-b]xanthen-11(2H)-one (38).

Mp: 131e133 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3412, 2961, 2914, 2857, 1651,

1599,1475,1439,1303,1209,1135,1072, 902, 788, 610. ¹H NMR data,

see Table 1. ¹³C NMR data, see Table 2. EIMS m/z (%): 294 (24, [M]^þ.),

280 (17), 279 (84), 262 (10), 213 (28), 205 (48),197 (20),165 (42),151

(32), 121 (44), 115 (42), 77 (100), 63 (67), 51 (83). HRMS (ESI) m/z

calcd for C₁₈H₁₅O₄[M þ H]^þ: 295.09649, found: 295.09704; m/z

calcd for C₁₈H₁₄NaO₄[M þ Na]^þ: 317.07843; found: 317.07825.

4.6.2.2. Method B. 1,2-Dihydroxyxanthone (36) (250 mg/1.09

mmol), potassium carbonate (181 mg/1.31 mmol), potassium io-

dide (271 mg/1.635 mmol) and copper iodide (31 mg/0.163 mmol)

were placed in a round-bottom ﬂask. The ﬂask was placed under

nitrogen atmosphere and 20 mL of anhydrous acetone was added.

The mixture was stirred for 15 min and 3-chloro-3-methyl-1-

butyne (117 mg/1.314 mmol) was added dropwise. The mixture

was heated 40 ^ꢀC for 12 h and then allowed to cool to room tem-

perature. The crude product was ﬁltered and the organic solvent

evaporated. The crude product was then puriﬁed by silica gel ﬂash

chromatography (n-hexane/ethyl acetate 95:5). Compound 37 was

isolated as an orange solid (100 mg/31%).

4.6.4. Synthesis of compound 39

Compound 38 (25 mg/0.08 mmol) and 5 mL of anhydrous

methanol in nitrogen atmosphere were placed in a round-bottom

ﬂask. To the solution was added Pd/C 10% (15% weight/4 mg) in

one portion. Triethylsilane (0.136 mL, 0.8 mmol) was then added

dropwise. The mixture was stirred for 15 min at room temperature.

The product was ﬁltered over celite and washed 3 times with

methanol. The methanol was evaporated and the crude product

was puriﬁed by silica gel column chromatography (n-hexane/ethyl

810

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

acetate 95:5). Compound 39 was isolated as light orange solid

(23 mg/97%).

Then, KHSO₄(94.7 mg/0.7 mmol) was added in one portion and

the solution was allowed to stir at room temperature for 8 h. The

reaction was quenched by the addition 10 mL of distilled water

and the methanol evaporated. The aqueous phase was extracted

with 3 ꢄ 20 mL of dichloromethane. The organic phase was

washed with 2 ꢄ 50 mL of brine, dried over sodium sulfate

anhydrous, ﬁltered and the organic solvent evaporated. The crude

product was puriﬁed by silica gel ﬂash chromatography (n-hex-

ane/ethyl acetate 9:1). Compound 45 was isolated as orange oil

(898 mg/4.36 mmol/94%).

12-Hydroxy-2,2-dimethyl-3,4-dihydropyrano[2,3-b]xanthen-

11(2H)-one (39). Mp: 172e173 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3443, 2972,

2917, 2858,1650,1602,1478,1444,129,1182,1073, 921, 774. ¹H NMR

data, see Table 1. ¹³C NMR data, see Table 2. EIMS m/z (%): 297 (2,

[M þ 1]^þ.), 296 (100, [M]^þ.), 240 (43), 212 (48), 128 (77), 115 (27),

102 (41), 92 (46), 77 (85), 76 (31), 75 (32), 74 (26), 65 (32), 63 (100),

62 (39). HRMS (ESI) m/z calcd for C₁₈H₁₇O₄[M þ H]^þ: 297.11214;

found: 297.11353; m/z calcd for C₁₈H₁₆NaO₄[M þ Na]^þ: 319.09408;

found: 319.09250.

6-Hydroxy-5-methoxy-2,2-dimethyl-2H-benzopyran (45). IR n_max

(cm^ꢂ1) (KBr): 3422, 3045, 2973, 2934, 2836, 1728, 1634, 1584, 1470,

1437, 1373, 1295, 1260, 1215, 1152, 1114, 960, 811, 731. ¹H NMR

4.7. Synthesis of pyranoxanthones 51, 52 and 53

(300.13 MHz, CDCl₃)

d

(ppm): 6.72 (d, J ¼ 8.6), 6.55 (d, J ¼ 10), 6.50

4.7.1. Synthesis of compound 43

(d, J ¼ 8.6), 5.69 (d, J ¼ 9.9), 5.15 (OH), 3.82 (s, OCH₃), 1.41 (s, 6H). ¹³

C

2,4-Dihydroxybenzaldehyde (42) (3 g/20.17 mmol), calcium

hydroxide (1.543 g/20.8 mmol) and 150 mL of methanol were

placed in a round-bottom ﬂask. Then, prenal (9.135 g/108.6 mmol)

was added dropwise. The mixture was stirred at room temperature

for 48 h. The reaction was quenched with HCl 1 M until pH 1e2. The

methanol was evaporated and the aqueous phase was extracted

with 3 ꢄ 150 mL of ethyl acetate. The organic phase was washed

with 2 ꢄ 100 mL of brine, dried over sodium sulfate anhydrous,

ﬁltered and the organic solvent evaporated. The crude product was

puriﬁed by silica gel ﬂash chromatography (n-hexane/ethyl acetate

9:1). Compound 43 was crystallized from n-hexane/ethyl acetate

3:1 as a yellow solid (2.06 g/50%).

NMR (100.63 MHz, CDCl₃) d (ppm): 145.5, 142.5, 142.4, 131.6, 116.8,

114.90, 114.86, 112.4, 75.3, 62.2, 27.4. EIMS m/z (%): 208 (5,

[M þ 2]^þ.), 207 (15, [M þ 1]^þ.), 206 (60, [M]^þ.), 191 (100), 177 (50),

176 (30), 148 (18), 91 (10), 77 (5). HRMS (ESI) m/z calcd for C₁₂H₁₅O₃

[M þ H]^þ: 207.10139; found: 207.10157.

4.7.4. Synthesis of compound 47

To a solution of 2-iodobenzoic acid (46) (15 g/60 mmol) in

200 mL of methanol were added 8.8 mL of sulfuric acid dropwise.

The solution was reﬂuxed for 16 h. The mixture was allowed to cool

to room temperature and most of the methanol was evaporated by

rotary evaporator until approximately 20 mL of solution. The

product was then partitioned between water and dichloromethane.

The aqueous phase was extracted 2 ꢄ 150 mL of dichloromethane.

The organic layer was then washed with 3 ꢄ 100 mL of saturated

solution of bicarbonate, 3 ꢄ 100 mL of water, dried over sodium

sulfate anhydrous, ﬁltered and the organic solvent evaporated.

Compound 47 was isolated as light yellow oil (15.4 g/98%).

Methyl 2-iodobenzoate (47). IR n_max(cm^ꢂ1) (KBr): 3444, 3057,

2994, 2946, 2894, 2836, 1728, 1579, 1459, 1428, 1290, 1249, 1127,

1100, 1013, 738.

6-Formyl-5-hydroxy-2,2-dimethyl-2H-benzopyran (43). Mp: 68e

69 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3464, 2967, 2922, 2857, 1628, 1484,

1330, 1294, 1247, 1176, 1107, 1081, 748. ¹H NMR (300.13 MHz, CDCl₃)

d

(ppm): 11.65 (OH), 9.66 (s, CHO), 7.29 (d, J ¼ 8.6), 6.88 (d, J ¼ 10.0),

6.44 (d, J ¼ 8.6), 5.61 (d, J ¼ 10.0), 1.46 (s, 6H). ¹³C NMR (100.63 MHz,

CDCl₃)

d (ppm): 194.5, 160.6, 158.7, 134.7, 128.6, 115.2, 115.1, 109.4,

108.8, 78.2, 28.4. EIMS m/z (%): 205 (5, [M þ 1]^þ.), 204 (10 [M]^þ.),

190 (15), 189 (100), 187 (60), 159 (12), 131 (12), 103 (10), 77 (12), 51

(6). HRMS (ESI) m/z calcd for C₁₂H₁₃O₃[M þ H]^þ: 205.08570, found:

205.08592.

4.7.5. Synthesis of compound 48

4.7.2. Synthesis of compound 44

Methyl 2-iodobenzoate (47) (9.369 g/35.76 mmol), CuI (1.7 g/

8.94 mmol), cesium carbonate (19.4 g/59.6 mmol), N,N-dimethyl-

glycine (495.6 mg/3.576 mmol) were placed in a round-bottom

ﬂask and under nitrogen atmosphere. To this ﬂask was added

compound 45 (6.1 g/29.8 mmol) solubilized in 20 mL of anhydrous

dioxane. The reaction was heated for 18 h at 90 ^ꢀC and under ni-

trogen atmosphere. The reaction was allowed to cool to room

temperature and ﬁltered. The solid was washed several times with

acetone and then the organic phase was concentrated. The mixture

was partitioned between ethyl acetate and water. The aqueous

phase was then extracted with 2 ꢄ 50 mL of ethyl acetate. The

organic phase was washed 2 ꢄ 50 mL with brine, dried over sodium

sulfate anhydrous, ﬁltered and the organic solvent evaporated. The

crude product was puriﬁed by silica gel ﬂash chromatography (n-

hexane/ethyl acetate (9:1)). Compound 48 was recrystallized from

ethyl acetate/n-hexane 1:1 as a white solid (2.5655 g/26%).

Potassium carbonate (2.43 g/17.6 mmol) was placed in a round-

bottom ﬂask and under nitrogen atmosphere. Then, compound 43

(1.7976 g/8.8 mmol) solubilized in 20 mL of anhydrous acetone was

added by syringe. Dimethylsulfate (1.667/13.2 mmol) was added

dropwise and the mixture was stirred for 20 h at room temperature

and under nitrogen atmosphere. The reaction was quenched by the

addition of 20 mL of distilled water and the acetone was evapo-

rated. The aqueous phase was acidiﬁed with HCl 1 N until pH 2e3

and extracted with 3 ꢄ 100 mL of ethyl acetate. The organic phase

was washed with 2 ꢄ 50 mL of brine, dried over sodium sulfate

anhydrous, ﬁltered and the organic solvent evaporated. Compound

44 was obtained in quantitative yield as yellow oil.

6-Formyl-5-methoxy-2,2-dimethyl-2H-benzopyran (44). IR n_max

(cm^ꢂ1) (KBr): 2974, 2932, 2840, 2747, 1679, 1634, 1463, 1370, 1281,

1249, 1213, 1158, 1110, 1068, 984, 736. ¹H NMR (300.13 MHz, CDCl₃)

d

(ppm): 10.18 (s, CHO), 7.66 (d, J ¼ 8.6), 6.65 (d, J ¼ 8.6), 6.60 (dd,

Methyl

2-((5-methoxy-2,2-dimethyl-2H-benzopyran-6-yl)oxy)

J ¼ 9.9, 0.6), 5.70 (d, J ¼ 9.9), 3.90 (s, OCH₃), 1.47 (s, 6H). EIMS m/z

(%): 219 (5, [M þ 1]^þ.), 218 (10, [M]^þ.), 204 (18), 205 (100), 174 (10),

160 (60), 133 (16), 105 (5), 91 (8), 78 (10), 51 (6). ¹³C NMR

benzoate (48). Mp: 88e89 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3072, 2974,

2943, 2869, 2838, 1729, 1599, 1472, 1369, 1298, 1250, 1218, 1075,

959, 751. ¹H NMR (300.13 MHz, CDCl₃)

d

(ppm): 7.87 (dd, J ¼ 7.9, 1.8,

(100.63 MHz, CDCl₃)

d

(ppm): 188.3,160.0,159.9,130.5,129.8,122.5,

HeC(7⁰⁰)), 7.36 (ddd, J ¼ 8.4, 7.5, 1.8, HeC(5⁰⁰)), 7.05 (dd, J ¼ 7.5, 0.8,

HeC(6⁰⁰)), 6.78 (d, J ¼ 8.7, HeC(7)), 6.75 (d, J ¼ 8.4, HeC(4⁰⁰)), 6.63 (d,

J ¼ 9.9, HeC(4)), 6.54 (d, J ¼ 8.7, HeC(8)), 5.66 (d, J ¼ 9.9, HeC(3)),

3.90 (s, H₃COeC(1⁰⁰)), 3.85 (s, H₃COeC(5)), 1.44 (s, (CH₃)₂eC(2)). ¹³C

NMR (75.47 MHz, CDCl₃): 166.5 (C1⁰⁰), 157.8 (C3⁰⁰), 150.2 (C6), 147.2

(C5), 141.1 (C8a), 133.4 (C5⁰⁰), 131.6 (C7⁰⁰), 130.9 (C3), 121.7 (C6⁰⁰),

121.7 (C7),120.5 (C2⁰⁰),116.9 (C4),116.3 (C4⁰⁰),116.0 (C4a),112.0 (C8),

115.9, 114.4, 113.4, 64.4, 28.2. HRMS (ESI) m/z calcd for C₁₃H₁₅O₃

[M þ H]^þ: 219.10177; found: 219.10157.

4.7.3. Synthesis of compound 45

Compound 44 (1.0139 g/4.64 mmol) was solubilized in 20 mL

of methanol and H₂O₂(540 mL of a solution of H₂O₂30% in water).

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

811

75.9 (C2), 61.6 (C1⁰⁰⁰), 52.1 (C1⁰⁰⁰⁰), 27.7 (C1⁰a and C1⁰b); HRMS (ESI)

solvent evaporated. The crude product was puriﬁed by silica gel

ﬂash chromatography (n-hexane/ethyl acetate 9:1). Compound 51

was obtained as yellow solid (1.9 g/91%).

m/z calcd for C₁₉H₁₇O₄[M ꢂ OCH₃]^þ: 309.11214, found: 309.11203.

4.7.6. Synthesis of compound 49

Compound 48 (129.6 mg/0.38 mmol) and 10 mL of a solution of

THF/methanol 1:1 were placed in a round-bottom ﬂask. Then

5-Methoxy-2,2-dimethylpyrano[2,3-b]xanthen-11(2H)-one (51).

Mp: 124e125 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 2975, 2935, 2841,1652,1605,

1472, 1426, 1315, 1072, 961, 760. ¹H NMR data, see Table 1. ¹³C NMR

data, see Table 2. EIMS m/z (%): 308 (12, [M]^þ.), 294 (12), 293 (55),

279 (16), 278 (100), 235 (40), 221 (30), 179 (30), 165 (90), 152 (15),

77 (20). HRMS (ESI) m/z calcd for C₁₉H₁₇O₄[M þ H]^þ: 309.11286;

found: 309.11214.

250 mL of a solution of LiOH 3 N was added dropwise. The mixture

was stirred at room temperature for 46 h. To the ﬂask were added

50 mL of water and the aqueous phase was acidiﬁed with HCl 5%

until pH 2e3 and extracted with 3 ꢄ 50 mL of dichloromethane.

The organic phase was washed with 2 ꢄ 50 mL of brine, dried over

sodium sulfate anhydrous, ﬁltered and the organic solvent evapo-

rated. Compound 49 was obtained as a white solid (121.7 mg/98%).

2-((5-Methoxy-2,2-dimethyl-2H-benzopyran-6-yl)oxy)benzoic

acid (49). Mp: 143e144 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3065, 2974, 2934,

2869, 2809, 2640, 2592, 2809, 1684, 1565, 1464, 1250, 1233, 1060,

4.7.9. Synthesis of compound 52

Pd/C 10% (10e20% weight for mass) was added to a two-necked

round-bottom ﬂask and placed under N₂atmosphere. Then, MeOH

40 mL and compound 51 (270 mg/0.87 mmol) was added dropwise.

To this mixture was added triethylsilane (8.7 mmol/1 g) in a

pressure-equalizer dropping-funnel. The mixture was allowed to

react for 15 min and then the crude product was ﬁltered through a

pad of celite and washed with methanol. The organic solvent was

evaporated and the crude product was puriﬁed by silica gel ﬂash

chromatography (n-hexane/ethyl acetate 9:1). Compound 52 was

obtained as a white solid (267 mg, 98%).

953, 772, 730. ¹H NMR (300.13 MHz, CDCl₃)

d (ppm): 8.20 (dd,

J ¼ 7.9, 1.8), 7.45 (ddd, J ¼ 8.4, 7.1, 1.8), 7.19 (ddd, J ¼ 7.9, 7.1, 1.0), 6.79

(d, J ¼ 8.4), 6.92 (d, J ¼ 8.8), 6.63e6.57 (m, 2H), 5.71 (d, J ¼ 10.0), 3.74

(s, OCH₃), 1.46 (s, 6H). ¹³C NMR (75.47 MHz, CDCl₃): 166.7, 158.0,

151.7, 147.7, 138.9, 134.9, 133.5, 131.4, 123.0, 122.4, 117.8, 116.4, 115.5,

112.5, 76.3, 62.1, 27.7. HRMS (ESI) m/z calcd for C₁₉H₁₇O₄[M ꢂ OH]^þ:

309.11214, found: 309.11203.

5-Methoxy-2,2-dimethyl-3,4-dihydropyrano[2,3-b]xanthen-

11(2H)-one (52). Mp: 135e136 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 2983, 2936,

4.7.7. Synthesis of compound 50

2840,1658, 1473,1441,1312,1074, 758. ¹H NMR data, see Table 1. ¹³

C

TBTU (2.44 g/7.6 mmol) was placed in a round-bottom ﬂask and

then compound 49 (2.48 g/7.6 mmol) solubilized in 250 mL of THF

was added. Then triethylamine (1.538 g/15.2 mmol) was added

dropwise and the mixture stirred at room temperature for 15 min.

Diethylamine (1.112 g/15.2 mmol) was then added and stirred

overnight at room temperature. The mixture was concentrated and

partitioned between ethyl acetate (250 mL) and water (250 mL).

The aqueous phase was extracted with 2 ꢄ 250 mL of ethyl acetate.

The organic phase was washed 2 ꢄ 150 mL of a saturated solution of

NaHCO₃, 2 ꢄ 150 mL with brine, dried over sodium sulfate anhy-

drous, ﬁltered and the organic solvent evaporated. The crude

product was puriﬁed by silica gel ﬂash chromatography (n-hexane/

ethyl acetate 7:3). Compound 50 was obtained as colorless oil in

quantitative yield.

NMR data, see Table 2. EIMS m/z (%): 311 (12, [M þ 1]^þ.), 310 (30,

[M]^þ.), 255 (96), 254 (100), 225 (84), 211 (55),197 (48),169 (30), 141

(60), 128 (60), 115 (30), 77 (42), 51 (38). HRMS (ESI) m/z calcd for

C

₁₉H₁₉O₄[M þ H]^þ: 311.12779; found: 311.12767.

4.7.10. Synthesis of compound 53

Compound 51 (247.3 mg/0.8 mmol) was placed in a round-

bottom ﬂask and under nitrogen ﬂow. Then 30 mL of anhydrous

dichloromethane was added and the solution was placed in

at ꢂ78 ^ꢀC. It was allowed to stay for 15 min and BBr₃(2 mmol of a

solution 1 M in dichloromethane) was added dropwise. The

mixture was stirred for 1 h at ꢂ78 ^ꢀC and then allowed to warm

slowly to room temperature. After reaching room temperature, the

reaction was quenched by the addition of methanol. It was added

20 mL of water to the mixture and the organic solvent evaporated.

The aqueous phase was extracted with 2 ꢄ 50 mL of dichloro-

methane. The organic phase was washed with 2 ꢄ 50 mL of brine,

dried over sodium sulfate anhydrous, ﬁltered and the organic sol-

vent evaporated. The crude product was puriﬁed by silica gel ﬂash

chromatography (n-hexane/ethyl acetate 8:2). Compound 53 was

obtained as yellow solid (198.1 mg/84%).

2-((5-Methoxy-2,2-dimethyl-2H-benzopyran-6-yl)oxy)-N,N-

diethylbenzamide (50). IR n_max(cm^ꢂ1) (KBr): 2974, 2931, 2870, 1633,

1470, 1432, 1370, 1293, 1255, 1217, 1113, 1074, 960, 753. ¹H NMR

(300.13 MHz, CDCl₃)

d

(ppm): 7.28 (dd, J ¼ 7.4, 1.7), 7.22 (ddd, J ¼ 8.4,

7.6, 1.7), 7.03 (ddd, J ¼ 7.6, 7.4, 0.8), 6.79 (d, J ¼ 8.8), 6.67 (d, J ¼ 8.4),

6.63 (d, J ¼ 10.0), 6.53 (d, J ¼ 8.8), 5.66 (d, J ¼ 10.0), 3.80 (s, OCH₃),

3.50 (broad, 4H), 1.44 (s, 6H), 1.22 (t, 3H, J ¼ 7.1), 1.11 (t, 3H, J ¼ 7.1).

¹³C NMR (100.63 MHz, CDCl₃)

d

(ppm): 168.2, 154.1, 150.3, 147.6,

5-Hydroxy-2,2-dimethylpyrano[2,3-b]xanthen-11(2H)-one (53).

Mp: 245e246 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 2973, 2911, 2840, 1622, 1447,

754. ¹H NMR data, see Table 1. ¹³C NMR data, see Table 2. HRMS

140.1, 130.8, 129.9, 127.7, 127.3, 122.2, 122.0, 117.0, 116.0, 114.9, 111.9,

75.9, 61.7, 42.9, 39.0, 27.7, 14.2, 12.9. EIMS m/z (%): 382 (18, [M]^þ.),

366 (28), 295 (35), 263 (100), 235 (45), 189 (25), 176 (34), 160 (32),

72 (38). HRMS (ESI) m/z calcd for C₂₃H₂₈NO₄[M þ H]^þ: 382.20118;

found: 382.20128.

(ESI) m/z calcd for

295.09649.

C

₁₈H₁₅O₄[M

þ

H]^þ: 295.09653; found:

4.8. Synthesis of pyranoxanthones 62, 63, 64, 65, 66 and 67

4.7.8. Synthesis of compound 51

Compound 50 (2.555 g/6.7 mmol) was placed in a three-necked

round-bottom ﬂask and under nitrogen ﬂow. Then 150 mL of

anhydrous THF was added and the solution was placed in an ice

bath. After 10 min, LDA (16.75 mmol of a solution 2 M in THF) was

added dropwise and the reaction was stirred for 15 min at 0 ^ꢀC, and

allowed to warm slowly to room temperature. After 1 h the reaction

was quenched by the addition of a saturated solution of NH₄Cl. The

mixture was partitioned between ethyl acetate (150 mL) and water

(150 mL). The aqueous phase was extracted with 2 ꢄ 150 mL of

ethyl acetate. The organic phase was washed with 2 ꢄ 50 mL of

brine, dried over sodium sulfate anhydrous, ﬁltered and the organic

4.8.1. Synthesis of compound 55

To

a solution of 2,4-dimethoxybenzaldehyde (54) (5.0 g/

30 mmol) and 5 mL of H₂O₂30% in 50 mL of methanol, was added

dropwise 0.5 mL of sulfuric acid. The mixture was stirred for 20 h at

room temperature. Approximately 45 mL of methanol were evap-

orated by rotary evaporator and the solution was partitioned be-

tween dichloromethane and distilled water. The aqueous phase

was extracted 3 ꢄ 50 mL of dichloromethane. The organic phase

was washed with 3 ꢄ 50 mL of distilled water, dried over sodium

sulfate anhydrous, ﬁltered and the organic solvent evaporated. The

crude product was puriﬁed by column chromatography

812

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

(chloroform). 2,4-Dimethoxyphenol (55) was obtained as light

yellow oil (4.33 g/94%).

dropwise and the ﬂask placed under reﬂux for 16 h. The mixture

was cooled to room temperature and poured over 100 g of crushed

ice. The aqueous phase was extracted with 3 ꢄ 150 mL of ethyl

acetate. The organic phase was washed with 2 ꢄ 100 mL of brine,

dried over sodium sulfate anhydrous, ﬁltered and the organic sol-

vent evaporated. The crude product was puriﬁed by silica gel ﬂash

chromatography (n-hexane/ethyl acetate 95:5). Compound 59 was

obtained as colorless oil (8.1522 g/92%).

2,4-Dimethoxyphenol (55). IR n_max(cm^ꢂ1) (KBr): 3443, 2997,

2939, 2835, 1610, 1512, 1461, 1433, 1374, 1299, 1263, 1228, 1202,

1151, 1116, 1033, 918, 830, 792. ¹H NMR (300.13 MHz, CDCl₃)

d

(ppm): 6.75 (d, J ¼ 5.2), 6.42 (d, J ¼ 1.7), 6.31 (dd, J ¼ 5.2, 1.7), 3.79

(s, OCH₃), 3.69 (s, OCH₃). EIMS m/z (%): 156 (4, [M þ 2]^þ.), 155 (40,

[M þ 1]^þ.), 154 (100 [M]^þ.), 140 (4), 139 (58), 112 (4), 111 (90), 96

(22), 79 (10), 69 (10), 51 (20).

Methyl 2-(methoxymethoxy)benzoate (59). IR: n_max(cm^ꢂ1) (KBr):

2991, 2947, 2903, 2825, 1725, 1596, 1485, 1449, 1432, 1296, 1249,

4.8.2. Synthesis of compound 56

1150, 1128, 1073, 984, 755. ¹H NMR (300.13 MHz, CDCl₃)

d (ppm):

2,4-Dimethoxyphenol (55) (3 g/19 mmol) and CuCl₂anhydrous

(2.5 mg/0.019 mmol) were placed in a round-bottom ﬂask and

under N₂atmosphere. 25 mL of anhydrous CH₃CN were added and

the mixture was cooled to 0 ^ꢀC on an ice bath. Then, DBU (3.76 g/

24.7 mmol) was added and 10 min after 3-chloro-3-methyl-1-

butyne (2.53 g/24.7 mmol) was added dropwise to the mixture.

The mixture was stirred at 0 ^ꢀC for 10 h. The mixture was allowed to

warm to room temperature and then partitioned between 50 mL of

distilled water and 50 mL of diethyl ether. The aqueous solution was

extracted with 2 ꢄ 50 mL of diethyl ether. The organic phase was

washed with 2 ꢄ 50 mL of brine, dried over sodium sulfate anhy-

drous, ﬁltered and the organic solvent evaporated. The crude

product was puriﬁed by silica gel ﬂash chromatography (n-hexane/

ethyl acetate 95:5) obtaining compound 56 as yellowish oil

(3.5792 g/86%).

7.78 (dd, J ¼ 7.8, 1.8), 7.44 (ddd, J ¼ 8.4, 7.5, 1.8), 7.20 (dd, J ¼ 8.4, 0.9),

7.05 (ddd, J ¼ 7.8, 7.5, 0.9), 5.26 (s, 2H), 3.89 (s, OCH₃), 3.52 (s, OCH₃).

EIMS m/z (%): 197 (16, [M]^þ.), 165 (100), 135 (26), 92 (45), 63 (34).

4.8.5. Synthesis of compound 60

A solution of n-BuLi in hexanes (1.6 M, 6.25 mL, 11 mmol) was

placed in a dropping funnel with pressure equalizing tube. This

solution was then added dropwise over a 5 min period, to a solution

of compound 57 (2.350 g, 0.01 mol) in 70 mL of anhydrous THF at

0

^ꢀC under nitrogen and stirred for 30 min. Then, compound 59

(3.9 g, 0.02 mol) dissolved in 30 mL of anhydrous THF was added

dropwise, via cannula, at 0 ^ꢀC under nitrogen. The reaction mixture

was stirred for 5 h at 0 ^ꢀC. The reaction was quenched with a

saturated solution of NH₄Cl. The mixture was extracted with

3 ꢄ 100 mL of diethyl ether. The organic phase was washed with

2 ꢄ 50 mL of brine, dried over sodium sulfate anhydrous, ﬁltered

and the organic solvent evaporated. The crude product was puriﬁed

by silica gel ﬂash chromatography (n-hexane/ethyl acetate 95:5).

Compound 60 was obtained as orange oil (1.716 g, 45%).

2,4-Dimethoxy-1-(2-methylbut-3-yn-2-yloxy)benzene (56). IR:

n_max(cm^ꢂ1) (KBr): 3273, 2981, 2928, 2826, 2354, 2325, 1598, 1497,

1453, 143, 1200, 1148, 1127, 1030, 872. ¹H NMR (300.13 MHz, CDCl₃)

d

(ppm): 7.30 (d, J ¼ 8.7), 6.48 (d, J ¼ 2.8), 6.39 (dd, J ¼ 8.7, 2.8), 3.79

(s, OCH₃), 3.78 (s, OCH₃), 2.48 (s, 1H), 1.61 (s, 6H). EIMS m/z (%): 222

(10, [M þ 2]^þ.), 221 (20, [M þ 1]^þ.), 220 (42 [M]^þ.), 205 (25), 189

(10), 155 (15), 154 (100), 139 (40), 125 (30), 111 (30), 93 (15). HRMS

(ESI) m/z calcd for C₁₃H₁₇O₃[M þ H]^þ: 221.11722; found: 221.11705.

(6,8-Dimethoxy-2,2-dimethyl-2H-benzopyran-7-yl)(2-(methox-

ymethoxy)phenyl) methanone (60). IR n_max(cm^ꢂ1) (KBr): 2971, 2934,

2833, 1661, 1596, 1568, 1455, 1417, 1372, 1291, 1236, 1204, 1157, 1112,

1080, 980, 913, 756. ¹H NMR (300.13 MHz, CDCl₃)

d (ppm): 7.72 (dd,

J ¼ 7.7, 1.8; HeC(6⁰⁰)), 7.43 (ddd, J ¼ 8.5, 7.0, 1.8; HeC(4⁰⁰)), 7.12 (d,

J ¼ 8.5; HeC(3⁰⁰)), 7.04 (dd, J ¼ 7.7, 1.0; HeC(5⁰⁰)), 6.35 (s; HeC(5)),

6.31 (d, J ¼ 9.8; HeC(4)), 5.69 (d, J ¼ 9.8; HeC(3)), 5.05 (s, H2e

C(1⁰⁰⁰⁰⁰)), 3.71 (s, H₃COeC(6)), 3.66 (s, H₃COeC(8)), 3.35 (s, H₃e

C(2⁰⁰⁰⁰⁰)), 1.46 (s, (CH₃)₂eC(2)). ¹³C NMR (75.47 MHz, CDCl₃): 193.4

(C9), 156.6 (C2⁰⁰), 150.3 (C8), 145.7 (C6), 139.4 (C8a), 133.6 (C4⁰⁰),

132.1 (C3), 131.5 (C6⁰⁰), 129.6 (C1⁰⁰), 125.0 (C7), 123.9 (C4a), 122.3

(C4), 121.6 (C5⁰⁰), 115.8 (C3⁰⁰), 104.3 (C5), 94.7 (C1⁰⁰⁰⁰⁰), 76.1 (C2), 61.1

(C1⁰⁰⁰⁰), 56.4 (C1⁰⁰⁰), 56.0 (C2⁰⁰⁰⁰⁰), 27.5 (C2⁰a and C2⁰b). HRMS (ESI) m/

z calcd for C₂₂H₂₄NaO₆[M þ Na]^þ: 407.14651; found: 407.14638.

4.8.3. Synthesis of compound 57

Compound 56 (700 mg/2.3 mmol) was placed in a round-

bottom ﬂask and under nitrogen atmosphere. 15 mL of anhydrous

DMF were added and the solution was stirred for 4 h at 145 ^ꢀC. The

mixture was allowed to cool to room temperature and poured over

50 g of crushed ice. The aqueous phase was extracted with

3 ꢄ 50 mL of diethyl ether. The organic phase was washed with

2 ꢄ 50 mL of brine, dried over sodium sulfate anhydrous, ﬁltered

and the organic solvent evaporated. The crude product was puriﬁed

by silica gel ﬂash chromatography (n-hexane/ethyl acetate 85:15)

and compound 57 was obtained as yellow oil (649 mg/93%).

4.8.6. Synthesis of compound 61

6,8-Dimethoxy-2,2-dimethyl-2H-benzopyran (57). IR n_max(cm^ꢂ1

(KBr): 3033, 2967, 2930, 2834, 1631, 1602, 1578, 1478, 1382, 1251,

1199, 1151, 1085, 1047, 824. ¹H NMR (300.13 MHz, CDCl₃)

(ppm):

)

To a solution of compound 60 (152 mg, 0.396 mmol) in 10 mL of

CH₃CN at 0 ^ꢀC was added in one portion NbCl₅(107 mg,

0.396 mmol). The mixture was stirred at 0 ^ꢀC for 10 min and then

allowed to warm to room temperature and let stirring for more

50 min. After this time, the reaction mixture was quenched with

saturated solution of NaHCO₃. The mixture was extracted with

3 ꢄ 50 mL of ethyl acetate. The organic phase was washed with

2 ꢄ 50 mL of brine, dried over anhydrous sodium sulfate and

organic solvent evaporated. Compound 61 was obtained as orange

oil (131.9 mg/98%).

d

6.40 (d, J ¼ 2.8), 6.27 (d, J ¼ 9.8), 6.18 (d, J ¼ 2.8), 5.64 (d, J ¼ 9.8), 3.84

(s, OCH₃), 3.76 (s, OCH₃), 1.46 (s, 6H). ¹³C NMR (100.63 MHz, CDCl₃)

d

(ppm): 153.6, 150.0, 136.0, 131.7, 122.5, 121.9, 101.9, 100.4, 75.9,

56.2, 55.6, 27.5. EIMS m/z (%): 221 (10, [M þ 1]^þ.), 220 (24 [M]^þ.),

205 (100), 190 (20), 162 (20), 133 (22), 119 (26), 115 (32), 91 (80).

HRMS (ESI) m/z calcd for C₁₃H₁₇O₃[M þ H]^þ: 221.11722; found:

221.11707.

(6,8-Dimethoxy-2,2-dimethyl-2H-benzopyran-7-yl)(2-

4.8.4. Synthesis of compound 59

hydroxyphenyl)methanone (61). IR n_max(cm^ꢂ1) (KBr): 3415, 3343,

Sodium hydride (3.63 g/90.53 mmol of 60% sodium hydride in

mineral oil) was placed in a round-bottom ﬂask and washed with

anhydrous n-hexane. The ﬂask was then placed under nitrogen

atmosphere and on an ice-bath. Methyl salicylate (58) (6.88 g/

45.26 mmol) was solubilized in 60 mL of anhydrous THF and added

slowly to the ﬂask containing the sodium hydride. The mixture was

stirred for 15 min and MOMCl (7.288 g/90.53 mmol) was added

2973, 2935, 1623, 1568, 1461, 1119, 1071, 1029, 916, 808, 756. ¹H

NMR (300.13 MHz, CDCl₃)

d

(ppm): 12.09 (s, OH), 7.45 (ddd, J ¼ 8.5,

7.1, 1.7), 7.34 (dd, J ¼ 8.0, 1.7), 7.01 (dd, J ¼ 8.4, 1.0), 6.79 (ddd, J ¼ 8.2,

7.1, 1.0), 6.38 (s), 6.33 (d, J ¼ 9.8), 5.73 (d, J ¼ 9.8), 3.79 (s, OCH₃), 3.68

(s, OCH₃), 1.48 (s, 6H). ¹³C NMR (100.63 MHz, CDCl₃)

d (ppm): 200.6,

162.4, 150.0, 145.6, 139.2, 136.5, 133.3, 132.6, 124.0, 122.2, 121.6,

121.0, 119.0, 117.8, 104.2, 76.4, 61.4, 56.2. EIMS m/z (%): 342 (1,

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

813

[M þ 2]^þ), 325 (4), 309 (10), 294 (4), 279 (12), 253 (4), 207 (5), 206

(13), 205 (100), 190 (7), 173 (8),147 (11),121 (16), 91 (14), 77 (17), 65

(17). HRMS (ESI) m/z calcd for C₂₀H₂₁O₅[M þ H]^þ: 341.13835;

found: 341.13816; m/z calcd for C₂₀H₂₀NaO₅[M þ Na]^þ: 363.12029;

found: 363.12021.

276 (22), 265 (85), 349 (64), 221 (25), 191 (35), 173 (65), 165 (100),

152 (24), 121 (30), 115 (50), 95 (25), 89 (40), 77 (65), 63 (50), 51 (28).

HRMS (ESI) m/z calcd for C₁₉H₁₉O₄[M þ H]^þ: 311.12779; found:

311.12769.

4.8.10. Synthesis of compound 66

4.8.7. Synthesis of compound 62 and 63

N,N-2-(Diethylamino)ethanethiol HCl (98.9 mg/0.584 mmol)

and 5 mL of DMF anhydrous were placed on a round-bottom ﬂask

and under nitrogen atmosphere. The ﬂask was cooled in an ice bath

and when the internal temperature was below 5 ^ꢀC, solid NaOtBu

(117.2 mg/1.22 mmol) was added in one portion. After 5 min the ice

bath was removed and it was allowed to warm to room tempera-

ture. After 15 min, a solution of compound 63 (150 mg/0.487 mmol)

in 2 mL of DMF anhydrous was added. The mixture was heated at

reﬂux for 30 min under nitrogen atmosphere. The mixture was

allowed to cool to room temperature and then was placed in an ice

bath. A solution of HCl 1 M was added to the mixture until pH 1 and

20 mL of water was added to the mixture. The mixture was

extracted with 3 ꢄ 50 mL of diethyl ether. The organic phase was

washed with 3 ꢄ 50 mL of brine, dried over sodium sulfate anhy-

drous, ﬁltered and the organic solvent evaporated. The crude

product was puriﬁed by silica gel ﬂash chromatography (n-hexane/

ethyl acetate 9:1). Compound 66 was obtained as orange crystalline

solid (102.8 mg/72%).

To a solution of compound 61 (119 mg, 0.35 mmol) in 15 mL of

DMF was added Cs₂CO₃(171 mg, 0.525 mmol). The mixture was

stirred at 50 ^ꢀC for 25 h. The mixture was allowed to cool to room

temperature and poured into crushed ice. The aqueous phase was

then extracted with 3 ꢄ 50 mL of diethyl ether. The organic phase

was washed with 2 ꢄ 50 mL of brine, dried over anhydrous sodium

sulfate, ﬁltered and the organic solvent evaporated. The crude

product was puriﬁed by silica gel ﬂash chromatography (n-hexane/

ethyl acetate 8:2). Compound 63 was obtained as deep green solid

(87.6 mg, 81%) and compound 62 as deep green oil (8.6 mg, 8%).

12-Methoxy-2,2-dimethylpyrano[2,3-b]xanthen-11(2H)-one (62).

IR n_max(cm^ꢂ1) (KBr): 2958, 2923, 2853, 1653, 1614, 1463, 1429, 1111,

1083. ¹H NMR data, see Table 1. ¹³C NMR data, see Table 2. EIMS m/z

(%): 308 (10, [M]^þ), 293 (18), 276 (22), 265 (85), 349 (64), 221 (25),

191 (35), 173 (65), 165 (100), 152 (24), 121 (30), 115 (50), 95 (25), 89

(40), 77 (65), 63 (50), 51 (28). HRMS (ESI) m/z calcd for C₁₉H₁₇O₄

[M þ H]^þ: 309.11214, found: 309.11206.

6-Methoxy-2,2-dimethylpyrano[3,2-c]xanthen-7(2H)-one

(63).

6-Hydroxy-2,2-dimethylpyrano[3,2-c]xanthen-7(2H)-one

(66).

Mp: 164e165 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 2974, 2927, 1652, 1615, 1554,

1468, 1420, 1344, 1305, 1265, 1219, 1120, 1077. ¹H NMR data, see

Table 1. ¹³C NMR data, see Table 2. EIMS m/z (%): 308 (20, [M]^þ), 294

(20), 293 (45), 276 (52), 265 (45), 250(55), 249 (60), 221 (40), 205

(35), 178 (45),165 (100),152 (20),121 (55), 115 (35), 89 (20), 77 (45),

63 (40), 51 (45). HRMS (ESI) m/z calcd for C₁₉H₁₇O₄[M þ H]^þ:

309.11214, found: 309.11203.

Mp: 191e192 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3488, 2970, 2919,1648, 1606,

1464, 1356, 1272, 1214, 1110, 762. ¹H NMR data, see Table 1. ¹³C NMR

data, see Table 2. HRMS (ESI) m/z calcd for C₁₈H₁₅O₄[M þ H]^þ:

295.09649; found: 295.09614.

4.8.11. Synthesis of compound 67

N,N-2-(Diethylamino)ethanethiol HCl (35.6 mg/0.193 mmol)

and 3 mL of DMF anhydrous were placed in a round-bottom ﬂask

and under nitrogen atmosphere. The ﬂask was cooled in an ice bath

and when the internal temperature was below 5 ^ꢀC, solid NaO^tBu

(41.7 mg/0.43 mmol) was added in one portion. After 5 min the ice

bath was removed and it was allowed to warm to room tempera-

ture. After 15 min, a solution of compound 65 (54 mg/0.16 mmol) in

2 mL of DMF anhydrous was added. The mixture was heated at

reﬂux for 2 h under nitrogen atmosphere. The mixture was allowed

to cool to room temperature and then was placed in an ice bath. A

solution of HCl 1 M was added to the mixture until pH 1 and 10 mL

of water was added to the mixture. The mixture was extracted with

3 ꢄ 25 mL of diethyl ether. The organic phase was washed with

3 ꢄ 25 mL of brine, dried over sodium sulfate anhydrous, ﬁltered

and the organic solvent evaporated. The crude product was puriﬁed

by silica gel ﬂash chromatography (n-hexane/ethyl acetate 9:1).

Compound 67 was obtained as orange crystalline solid (21 mg/

45%).

4.8.8. Synthesis of compound 64

Pd/C 10% (10e20% weight for mass) was placed in a round-

bottom ﬂask and under nitrogen atmosphere. Then, compound

62 (28 mg/0.09 mmol) solubilized in 10 mL of methanol was added

dropwise. To this mixture was added triethylsilane (0.9 mmol/

104 mg) in a pressure-equalizing dropping-funnel. The mixture

was allowed to react for 10 min and then the crude product was

ﬁltered through celite and washed repeatedly with methanol. The

solvent was evaporated and the crude product was puriﬁed by

Grace cartridge ﬂash chromatography (n-hexane/ethyl acetate 8:2).

Compound 64 was obtained as a green solid in quantitative yield.

12-Methoxy-2,2-dimethyl-3,4-dihydropyrano[2,3-b]xanthen-

11(2H)-one (64). Mp: 132e133 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 2972, 2919,

2849, 1655,1616,1458,1421,1320,1122,1078, 746. ¹H NMR data, see

Table 1. ¹³C NMR data, see Table 2. HRMS (ESI) m/z calcd for

C

₁₉H₁₉O₄[M þ H]^þ: 311.12779; found: 311.12769.

6-Hydroxy-2,2-dimethyl-3,4-dihydropyrano[3,2-c]xanthen-

4.8.9. Synthesis of compound 65

7(2H)-one (67). Mp: 205e206 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 3500, 2924,

Pd/C 10% (10e20% weight for mass) was placed on a round-

bottom ﬂask and under nitrogen atmosphere. Then, 20 mL of

methanol and compound 63 (166 mg/0.54 mmol) was added

dropwise. To this mixture was added triethylsilane (5.4 mmol/

627 mg) in a pressure-equalizing dropping-funnel. The mixture

was allowed to react for 10 min and then the crude product was

ﬁltered by celite and washed repeatedly with methanol. The

organic solvent was evaporated and the crude product was puriﬁed

by Grace cartridge chromatography (n-hexane/ethyl acetate 8:2).

Compound 65 was obtained as a light yellow solid (138 mg, 81%).

6-Methoxy-2,2-dimethyl-3,4-dihydropyrano[3,2-c]xanthen-

2857, 1654, 1607, 1467, 1375, 1271, 751. ¹H NMR data, see Table 1. ¹³

C

NMR data, see Table 2. HRMS (ESI) m/z calcd for C₁₈H₁₇O₄[M þ H]^þ:

297.11214; found: 297.11204.

4.9. X-ray crystallography

Crystals of compound 63 suitable for X-ray diffraction were

obtained by slow evaporation of a solution in acetone. They were

orthorhombic, had a P2₁2₁2₁space group and a cell volume of

3

ꢀ

1508.90(11) A , and unit cell dimensions were a ¼ 5.6701(2) A,

ꢀ

b ¼ 15.5612(6) A and c ¼ 17.1013(9) A (uncertainties in parenthesis).

7(2H)-one (65). Mp: 204e205 ^ꢀC. IR n_max(cm^ꢂ1) (KBr): 2971, 2933,

2874, 2844, 1661, 1603, 1481, 1082, 749. ¹H NMR data, see Table 1.

¹³C NMR data, see Table 2. EIMS m/z (%): 308 (10, [M]^þ), 293 (18),

The calculated density was 1.357 g/ml. Diffraction data were

collected at 293 K with a Gemini PX Ultra equipped with CuK_ara-

ꢀ

diation (

l

¼ 1.54184 A). A total of 4847 reﬂections were measured,

814

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

of which 2484 were independent, and 2393 were observed

(I > 2 (I)). The structure was solved by direct methods using

bottomed 96-well UV-plates acquired from BD Biosciences or

fused quartz cuvettes of 1.4 mL from Hellma.

s

SHELXS-97 and reﬁned with SHELXL-97. Carbon and oxygen atoms

were reﬁned anisotropically. C1⁰⁰bound hydrogens were positioned

with idealized geometry and reﬁned riding on their parent C atom

4.11.2. Liposome preparation

Liposomes were prepared by evaporation to dryness with a ni-

trogen stream of an EPC solution prepared with chloroform/

methanol (9/1). The resulting dried lipid ﬁlm was dispersed with a

buffer (HEPES: 10 mmol L^ꢂ1, I ¼ 0.1 mol L^ꢂ1with NaCl, pH 7.4) and

the mixture was vortex mixed to give multilamellar liposomes

(MLVs). The MLVs were extruded 10 times through polycarbonate

ﬁlters with a pore diameter of 100 nm to form large unilamellar

vesicles (LUVs).

ꢀ

at a distance of 0.93 A, with U_iso(H) ¼ 1.2 U_eq(C). Other hydrogen

atoms were reﬁned freely with isotropic displacement parameters.

The reﬁnement converged to R (all data) ¼ 5.15% and wR₂(all

data) ¼ 13.50%. Tables containing the ﬁnal fractional coordinates,

temperature parameters, bond distances, and bond angles were

deposited with the Cambridge Crystallographic Data Centre (CCDC

reference number 933804).

4.10. Biological activity

4.11.3. Micelle preparation

Micelle solutions were prepared by dissolution of hex-

4.10.1. Cell culture

adecylphosphocholine (HDPC) in buffer (HEPES: 10 mmol L^ꢂ1

,

The adherent cell lines MCF-7 (breast adenocarcinoma, ECACC,

UK), NCI-H460 (non-small cell lung cancer, a kind gift from NCI,

Bethesda, USA), A375-C5 (melanoma, ECACC, UK) and the suspen-

sion cell line HL-60 (acute myeloid leukemia, DSMZ, Germany)

were routinely maintained in RPMI-1640 (with Glutamax, Lonza),

supplement with 5% heat inactivated fetal bovine serum (FBS, PAA)

at 37 ^ꢀC in a humidiﬁed incubator with 5% CO₂. Cell number and

viability were routinely determined with Trypan blue (Sigma). All

the experiments were performed with cells in exponential growth

and presenting more than 90% viability.

I ¼ 0.1 mol L^ꢂ1with NaCl, pH 7.4) and mixed by vortex.

4.11.4. General procedure for K_pdetermination

The procedure used was adapted from the literature [38] and

was the following: i) 3

mL of compound solubilized in DMSO

(concentration established before in 4.11.5) was added to each well;

ii) increasing volumes of liposome or micelles were added to each

well and the ﬁnal concentration for 300

mL were 0 (only buffer), 50,

100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000

m

M. In the

end, for the same concentration of xanthone, increasing concen-

trations of lipid were prepared; iii) HEPES buffer was added to

4.10.2. Cell growth inhibition assay

make up 300 mL of ﬁnal volume; iv) a blank was used for each

Cells were plated into 96-well tissue culture plates at appro-

priate densities (MCF-7 and NCI-H460 at 5 ꢄ 10³cells/well, A375-

C5 at 7.5 ꢄ 10³cells/well and HL-60 cells at 1 ꢄ 10⁴cells/well)

and incubated for 24 h. Cells were then treated with serial dilutions

concentration of lipid containing 1% of DMSO; v) the plate was

incubated at 37 ꢁ 0.1 ^ꢀC for 30 min and then the spectrum was

traced from 240 nm to 600 nm. For poorly soluble compounds, the

solutions were prepared with the same concentration but in

eppendorf (1.5 mL) instead. Each solution was mixed by vortex and

incubated for 30 min. The spectrum ranging from 240 nm to

600 nm was traced individually in a double-beam spectropho-

tometer for each sample at 37 ^ꢀC. The compound concentration

of the different compounds (ranging from 0 to 150 mM whenever

possible). Following 48 h of incubation, the effect of the compounds

in the growth of the different cell lines was analyzed with the

Sulforhodamine B (SRB) assay, as adopted by the National Cancer

Institute (NCI, USA) [63e65]. Brieﬂy, following incubation, cells

were ﬁxed in situ with ice cold trichloroacetic acid (at 10% for

adherent cells and at 16.7% for the cells growing in suspension).

Following the SRB staining, plates were washed with 1% acetic acid,

the bound dye was solubilized with 10 mM Tris Base and absor-

bance was measured at 510 nm in a microplate reader (Biotek In-

struments Inc, Synergy XS, Winooski, USA). The GI₅₀values for the

compounds synthesized (concentration causing a 50% inhibition of

cell growth) were calculated from the plotted results. Doxorubicin

was used as a positive control. The effect of the solvent (DMSO) on

the growth of these cell lines was evaluated in preliminary exper-

iments, by exposing untreated control cells to the maximum con-

centration of DMSO used in each assay (0.25%).

used in the determination of the K_pvaried from 2.5

mM for the most

poorly soluble compounds to 20 M for the most soluble.

m

Partition coefﬁcient (K_p) was calculated by adjusting experi-

mental data through a nonlinear regression of LevenbergeMar-

quardt in where the adjustable parameter was K_p. The program was

available free of charge in the Supplementary information from Ref.

[38].

All experimental data are presented as means ꢁ SE from at least

three independent experiments. The linear regression analysis was

calculated using the IBM SPSS Statistics 20.

Acknowledgments

All experimental data is presented as means of GI₅₀values ꢁ SE

from at least three independent experiments (except when indi-

cated otherwise).

The authors would like to thank FCT e Fundação para a Ciência e

Tecnologia under the project CEQUIMED e PESt.OE/SAU/UI4040/

2011 and FEDER funds through the COMPETE program under the

project FCOMP-01-0124-FEDER-011057. The authors would also

like to thank FCT for the grants of: Diana Sousa (PTDC/SAU-FCT/

100930/2008), C.M.G. Azevedo (SFRH/BD/41165/2007) and R.T.

Lima (SRH/BPD/68787/2010), and also to U. Porto/Santander Totta

for the ﬁnancial support. The authors would also like to thank Sara

Cravo and Gisela Adriano for the technical support. IPATIMUP is an

Associate Laboratory of the Portuguese Ministry of Science, Tech-

nology and Higher Education and is partially supported by FCT.

4.11. Lipophilicity

4.11.1. Materials

The egg yolk phosphatidylcholine (EPC), HEPES, DMSO and NaCl

were acquired from SigmaeAldrich, the hexadecylphosphocholine

(HDPC) from Cayman chemicals and the water used was double-

deionized with conductivity less than 0.1

m

S cm^ꢂ1. The 96-well

plate reader used was a Synergy HT from Bio-Tek Instruments

and the double beam spectrophotometer was a JASCO V660. The

extrusion device used was a Lipex^ÒExtruder manufactured from

Northern Lipids and the ﬁlters were acquired from Whatman. The

determination of the UV-spectrum was made either in a ﬂat-

Appendix A. Supplementary data

Supplementary data associated with this article can be found in

the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.09.

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

815

[26] A. Paiva, M. Sousa, A. Camões, M. Nascimento, M. Pinto, Prenylated xanthones:

antiproliferative effects and enhancement of the growth inhibitory action of

4-hydroxytamoxifen in estrogen receptor-positive breast cancer cell line,

Med. Chem. Res. 21 (2012) 552e558.

012. These data include MOL ﬁles and InChiKeys of the most

important compounds described in this article.

[27] K.-S. Masters, S. Bräse, Xanthones from fungi, lichens, and bacteria: the nat-

ural products and their synthesis, Chem. Rev. 112 (2012) 3717e3776.

[28] M.E. Sousa, M.M.M. Pinto, Synthesis of xanthones: an overview, Curr. Med.

Chem. 12 (2005) 2447e2479.

[29] P.D. Leeson, B. Springthorpe, The in ﬂ uence of drug-like concepts on decision-

making in medicinal chemistry, Nat. Rev. Drug Discov. 6 (2007) 881e890.

[30] X. Liu, B. Testa, A. Fahr, Lipophilicity and its relationship with passive drug

permeation, Pharm. Res. 28 (2011) 962e977.

References

[1] K.C. Nicolaou, G.Q. Cao, J.A. Pfefferkorn, Selenium-based solid-phase syn-

thesis of benzopyrans II: applications to combinatorial synthesis of

medicinally relevant small organic molecules, Angew. Chem. Int. Ed. 39

(2000) 739e743.

[2] K.C. Nicolaou, J.A. Pfefferkorn, A.J. Roecker, G.Q. Cao, S. Barluenga, H.J. Mitchell,

Natural product-like combinatorial libraries based on privileged structures. 1.

General principles and solid-phase synthesis of benzopyrans, J. Am. Chem.

Soc. 122 (2000) 9939e9953.

[31] N.A. Meanwell, Improving drug candidates by design: a focus on physico-

chemical properties as a means of improving compound disposition and

safety, Chem. Res. Toxicol. 24 (2011) 1420e1456.

[32] M.P. Gleeson, A. Hersey, D. Montanari, J. Overington, Probing the links be-

tween in vitro potency, ADMET and physicochemical parameters, Nat. Rev.

Drug Discov. 10 (2011) 197e208.

[33] M.P. Gleeson, Generation of a set of simple, interpretable ADMET rules of

thumb, J. Med. Chem. 51 (2008) 817e834.

[34] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and

computational approaches to estimate solubility and permeability in drug

discovery and development settings, Adv. Drug Deliv. Rev. 23 (1997) 3e25.

[35] G.P. van Balen, C.A.M. Martinet, G. Caron, G. Bouchard, M. Reist, P.-A. Carrupt,

R. Fruttero, A. Gasco, B. Testa, Liposome/water lipophilicity: methods, infor-

mation content, and pharmaceutical applications, Med. Res. Rev. 24 (2004)

299e324.

[3] S.B. Ferreira, F.d.C. da Silva, A.C. Pinto, D.T.G. Gonzaga, V.F. Ferreira, Syntheses

of chromenes and chromanes via o-quinone methide intermediates,

J. Heterocycl. Chem. 46 (2009) 1080e1097.

[4] M.E. Welsch, S.A. Snyder, B.R. Stockwell, Privileged scaffolds for library design

and drug discovery, Curr. Opin. Chem. Biol. 14 (2010) 347e361.

[5] S.M. Wuerzberger, J.J. Pink, S.M. Planchon, K.L. Byers, W.G. Bornmann,

D.A. Boothman, Induction of apoptosis in MCF-7:WS8 breast cancer cells by b -

lapachone, Cancer Res. 58 (1998) 1876e1885.

[6] C. Ríos-Luci, E.L. Bonifazi, L.G. León, J.C. Montero, G. Burton, A. Pandiella,

R.I. Misico, J.M. Padrón, -Lapachone analogs with enhanced antiproliferative

b

activity, Eur. J. Med. Chem. 53 (2012) 264e274.

[7] M.A. Ware, P. Daeninck, V. Maida, A review of nabilone in the treatment of

chemotherapy-induced nausea and vomiting, Ther. Clin. Risk Manag. 4 (2008)

99e107.

[8] G.E. Pratt, R.C. Jennings, A.F. Hamnett, G.T. Brooks, Lethal metabolism of

precocene-I to a reactive epoxide by locust corpora allata, Nature 284 (1980)

320e323.

[9] Y. Kashman, K.R. Gustafson, R.W. Fuller, J.H. Cardellina, J.B. McMahon,

M.J. Currens, R.W. Buckheit, S.H. Hughes, G.M. Cragg, M.R. Boyd, HIV inhibitory

natural products. Part 7. The calanolides, a novel HIV-inhibitory class of

coumarin derivatives from the tropical rainforest tree, Calophyllum lanigerum ,

J. Med. Chem. 35 (1992) 2735e2743.

[36] T. Hartmann, J. Schmitt, Lipophilicity

e beyond octanol/water: a short

comparison of modern technologies, Drug Discov. Today Technol. 1 (2004)

431e439.

[37] C. Giaginis, A. Tsantili-Kakoulidou, Alternative measures of lipophilicity: from

octanolewater partitioning to IAM retention, J. Pharm. Sci. 97 (2008) 2984e

3004.

[38] L.M. Magalhães, C. Nunes, M. Lucio, M.A. Segundo, S. Reis, J.L. Lima, High-

throughput microplate assay for the determination of drug partition co-

ef ﬁ cients, Nat. Protoc. 5 (2010) 1823e1830.

[39] B. de Castro, P. Gameiro, J.L.F.C. Lima, C. Matos, S. Reis, Interaction of drugs

with hexadecylphosphocholine micelles. Derivative spectroscopy, acidebase

and solubility studies, Mat. Sci. Eng. C 18 (2001) 71e78.

[10] I.P. Singh, H.S. Bodiwala, Recent advances in anti-HIV natural products, Nat.

Prod. Rep. 27 (2010) 1781e1800.

ꢁ

ꢂ

[40] O. Cudina, J. Brboric, I. Jankovic, K. Karljikovic-Rajic, S. Vladimirov, Study of

valsartan interaction with micelles as a model system for biomembranes,

Colloids Surf. B. 65 (2008) 80e84.

[11] G.H. Svoboda, G.A. Poore, P.J. Simpson, G.B. Boder, Alkaloids of Acronychia

Baueri Schott I: isolation of the alkaloids and a study of the antitumor and

other biological properties of acronycine, J. Pharm. Sci. 55 (1966) 758e768.

[12] N. Kawahara, K. Nozawa, S. Nakajima, K.-i. Kawai, Studies on fungal products.

Part 15. Isolation and structure determination of arugosin E from Aspergillus

silvaticus and cycloisoemericellin from Emericella striata , J. Chem. Soc. Perkin

Trans. 1 (1988) 907e911.

[13] M.M.M. Pinto, R.A.P. Castanheiro, Natural prenylated xanthones: chemistry

and biological activities, in: G. Brahmachari (Ed.), Natural Products: Chemis-

try, Biochemistry and Pharmacology, Narosa Publishing House PVT, LTD, New

Delhi, 2009, pp. 520e675.

[41] E. Örnskov, J. Gottfries, M. Erickson, S. Folestad, Experimental modelling of

drug membrane permeability by capillary electrophoresis using liposomes,

micelles and microemulsions, J. Pharm. Pharmacol. 57 (2005) 435e442.

[42] C.M.G. Azevedo, C.M.M. Afonso, M.M.M. Pinto, Routes to xanthones: an update

on the synthetic approaches, Curr. Org. Chem. 16 (2012) 2818e2867.

[43] J. Magano, M.H. Chen, J.D. Clark, T. Nussbaumer, 2-(Diethylamino)ethanethiol,

a new reagent for the odorless deprotection of aromatic methyl ethers, J. Org.

Chem. 71 (2006) 7103e7105.

[44] D. Bell, M.R. Davies, G.R. Geen, I.S. Mann, Copper(I) iodide: a catalyst for the

improved synthesis of aryl propargyl ethers, Synthesis (1995) 707e712.

[45] R. Pernin, F. Muyard, F. Bevalot, F. Tillequin, J. Vaquette, Ef ﬁ cient synthesis of

octandrenolone and related dipyranoacetophenones, J. Nat. Prod. 63 (2000)

245e247.

[46] C. Nevado, A.M. Echavarren, Intramolecular hydroarylation of alkynes cata-

lyzed by platinum or gold: mechanism and endo selectivity, Chem. Eur. J. 11

(2005) 3155e3164.

[14] M.M.M. Pinto, R.A.P. Castanheiro, Synthesis of prenylated xanthones: an

overview, Curr. Org. Chem. 13 (2009).

[15] Y.-W. Chin, A.D. Kinghorn, Structural characterization, biological effects,

and synthetic studies on xanthones from mangosteen (Garcinia man-

(2008) 355e364.

[16] N. Pouli, P. Marakos, Fused xanthone derivatives as antiproliferative agents,

Anti-cancer Agents Med. Chem. 9 (2009) 77e91.

[47] N. Mézailles, L. Ricard, F. Gagosz, Phosphine gold(I) bis-(tri-

ﬂ uoromethanesulfonyl)imidate complexes as new highly ef ﬁ cient and air-

stable catalysts for the cycloisomerization of enynes, Org. Lett. 7 (2005)

4133e4136 .

[17] H.T. Nguyen, M.C. Lallemand, S. Boutefnouchet, S. Michel, F. Tillequin, Anti-

tumor psoropermum xanthones and sarcomelicope acridones: privileged

structures implied in DNA alkylation, J. Nat. Prod. 72 (2009) 527e539.

[18] E.K. Seo, M.E. Wall, M.C. Wani, H. Navarro, R. Mukherjee, N.R. Farnsworth,

A.D. Kinghorn, Cytotoxic constituents from the roots of Tovomita brevistami-

nea , Phytochemistry 52 (1999) 669e674.

[19] F. Zelefack, D. Guilet, N. Fabre, C. Bayet, S. Chevalley, S. Ngouela, B.N. Lenta,

A. Valentin, E. Tsamo, M.G. Dijoux-Franca, Cytotoxic and antiplasmodial

xanthones from Pentadesma butyracea , J. Nat. Prod. 72 (2009) 954e957.

[20] Y.S. Zou, A.J. Hou, G.F. Zhu, Y.F. Chen, H.D. Sun, Q.S. Zhao, Cytotoxic iso-

prenylated xanthones from Cudrania tricuspidata , Bioorg. Med. Chem. 12

(2004) 1947e1953.

[48] S.J. Pastine, S.W. Youn, D. Sames, Pt^IV-catalyzed cyclization of areneealkyne

substrates via intramolecular electrophilic hydroarylation, Org. Lett. 5 (2003)

1055e1058.

[49] S.J. Pastine, S.W. Youn, D. Sames, Pt(IV)-catalyzed cyclization of areneealkyne

substrates via CeH bond functionalization, Tetrahedron 59 (2003) 8859e

8868.

[50] R.A.P. Castanheiro, M.M.M. Pinto, S.M.M. Cravo, D.C.G.A. Pinto, A.M.S. Silva,

A. Kijjoa, Improved methodologies for synthesis of prenylated xanthones by

microwave irradiation and combination of heterogeneous catalysis (K10 clay)

with microwave irradiation, Tetrahedron 65 (2009) 3848e3857.

[51] M. Mondal, V.G. Puranik, N.P. Argade, Facile synthesis of 1,3,7-

trihydroxyxanthone and its regioselective coupling reactions with prenal:

simple and ef ﬁ cient access to osajaxanthone and nigrolineaxanthone F, J. Org.

Chem. 71 (2006) 4992e4995.

[21] S.

N. Lartpornmatulee, A. Suksamrarn, Cytotoxic prenylated xanthones from the

young fruit of Garcinia mangostana , Chem. Pharm. Bull. 54 (2006) 301e305.

[22] H.K. Wabo, H. Kikuchi, Y. Katou, P. Tane, Y. Oshima, Xanthones and

a

benzophenone from the roots of Pentadesma butyracea and their anti-

proliferative activity, Phytochem. Lett. 3 (2010) 104e107.

[23] J.J. Chen, I.S. Chen, C.Y. Duh, Cytotoxic xanthones and biphenyls from the root

of Garcinia linii , Planta Med. 70 (2004) 1195e1200.

[52] P.K. Mandal, J.S. McMurray, PdeC-induced catalytic transfer hydrogenation

with triethylsilane, J. Org. Chem. 72 (2007) 6599e6601.

[53] H. Saimoto, K. Yoshida, T. Murakami, M. Morimoto, H. Sashiwa, Y. Shigemasa,

Effect of calcium reagents on aldol reactions of phenolic enolates with alde-

hydes in alcohol, J. Org. Chem. 61 (1996) 6768e6769.

[24] L.B.S. Kardono, H. Muhammad, G. Sherley, S. Kosela, L.J. Harrison, Bioactive

constituents of Garcinia porrecta and G. parvifolia grown in Indonesia, Pak. J.

Biol. Sci. 9 (2006) 483e486.

[25] A. Palmeira, A. Paiva, E. Sousa, H. Seca, G.M. Almeida, R.T. Lima,

M.X. Fernandes, M. Pinto, M.H. Vasconcelos, Insights into the in vitro anti-

tumor mechanism of action of a new pyranoxanthone, Chem. Biol. Drug Des.

76 (2010) 43e58.

[54] M. Mondal, V.G. Puranik, N.P. Argade, A facile phenol-driven intramolecular

diastereoselective thermal/base-catalyzed dipolar [2

actions: an easy access to complex bioactive natural and unnatural benzo-

þ

2] annulation re-

pyran congeners, J. Org. Chem. 72 (2007) 2068e2076.

816

C.M.G. Azevedo et al. / European Journal of Medicinal Chemistry 69 (2013) 798e816

[55] M. Matsumoto, K. Kobayashi, Y. Hotta, Acid-catalyzed oxidation of benzal-

dehydes to phenols by hydrogen peroxide, J. Org. Chem. 49 (1984) 4740e

4741.

[71] P.I. Lelkes, I.R. Miller, Perturbations of membrane structure by optical probes:

I. Location and structural sensitivity of merocyanine 540 bound to phospho-

lipid membranes, J. Membr. Biol. 52 (1980) 1e15.

[56] D. Ma, Q. Cai, H. Zhang, Mild method for ullmann coupling reaction of amines

and aryl halides, Org. Lett. 5 (2003) 2453e2455.

[57] O.B. Familoni, I. Ionica, J.F. Bower, V. Snieckus, Intramolecular anionic Friedele

Crafts equivalents. A general regiospeci ﬁ c route to substituted and naturally

occurring xanthen-9-ones, Synlett (1997) 1081e1083.

[58] J.D. Godfrey Jr., R.H. Mueller, T.C. Sedergran, N. Soundararajan, V.J. Colandrea,

Improved synthesis of aryl 1,1-dimethylpropargyl ethers, Tetrahedron Lett. 35

(1994) 6405e6408.

[59] J.S. Yadav, B. Ganganna, D.C. Bhunia, P. Srihari, NbCl₅mediated deprotection of

methoxy methyl ether, Tetrahedron Lett. 50 (2009) 4318e4320.

[60] L. Hintermann, R. Masuo, K. Suzuki, Solvent-controlled leaving-group selec-

tivity in aromatic nucleophilic substitution, Org. Lett. 10 (2008) 4859e4862.

[61] C. Morel, D. Séraphin, J.-M. Oger, M. Litaudon, T. Sévenet, P. Richomme,

J. Bruneton, New xanthones from Calophyllum caledonicum , J. Nat. Prod. 63

(2000) 1471e1474.

[72] H. Ferreira, M. Lúcio, B. de Castro, P. Gameiro, J. Lima, S. Reis, Partition and

location of nimesulide in EPC liposomes: a spectrophotometric and ﬂ uores-

cence study, Anal. Bioanal. Chem. 377 (2003) 293e298.

[73] B. de Castro, P. Gameiro, J. Lima, C. Matos, S. Reis, A fast and reliable spec-

troscopic method for the determination of membrane-water partition co-

ef ﬁ cients of organic compounds, Lipids 36 (2001) 89e96.

[74] K. Kitamura, N. Imayoshi, Second-derivative spectrophotometric determina-

tion of the binding constant between chlorpromazine and

aqueous solutions, Anal. Sci. (1992).

b -cyclodextrin in

[75] K. Kitamura, N. Imayoshi, T. Goto, H. Shiro, T. Mano, Y. Nakai, Second deriv-

ative spectrophotometric determination of partition coef ﬁ cients of chlor-

promazine and promazine between lecithin bilayer vesicles and water, Analyt

Chim. Acta 304 (1995) 101e106.

[76] E. Kerns, L. Di, Drug-like Properties: Concepts, Structure Design and Methods

from ADME to Toxicity Optimization, Academic Press, 2008.

[62] L. Gales, A. Damas, Xanthones e a structural perspective, Curr. Med. Chem. 12

(2005) 2499e2515.

[77] W.L.F. Armarego, D.D. Perrin, Puri ﬁ cation of Laboratory Chemicals, third ed.,

Pergamon, 1988.

[63] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paul, D. Vistica, C. Hose,

J. Langley, P. Cronise, A. Vaigro-Wolf, M. Gray-Goodrich, H. Campbell, J. Mayo,

M. Boyd, Feasibility of a high-ﬂ ux anticancer drug screen using a diverse panel

of cultured human tumor cell lines, J. Natl. Cancer Inst. 83 (1991) 757e776.

[64] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,

J.T. Warren, H. Bokesch, S. Kenny, M.R. Boyd, New colorimetric cytotoxicity

assay for anticancer-drug screening, J. Natl. Cancer Inst. 82 (1990) 1107e1112.

[65] V. Vichai, K. Kirtikara, Sulforhodamine B colorimetric assay for cytotoxicity

screening, Nat. Protoc. 1 (2006) 1112e1116.

[78] E. Sousa, A. Paiva, N. Nazareth, L. Gales, A.M. Damas, M.S.J. Nascimento,

M.M.M. Pinto, Bromoalkoxyxanthones as promising antitumor agents: syn-

thesis, crystal structure and effect on human tumor cell lines, Eur. J. Med.

Chem. 44 (2009) 3830e3835.

[79] E.G.R. Fernandes, A.M.S. Silva, J.A.S. Cavaleiro, F.M. Silva, M.F.M. Borges,

M.M.M. Pinto, ¹H and ¹³C NMR spectroscopy of mono-, di-, tri- and tetra-

substituted xanthones, Magn. Reson. Chem. 36 (1998) 305e309.

[80] R.K.M. Pillai, P. Naiksatam, F. Johnson, R. Rajagopalan, P.C. Watts,

R. Cricchio, S. Borras, Thermorubin II

e 1,3-dihydroxy-9 H -xanthone and

[66] L.J.J. Farrugia, ORTEP-3 for windows e a version of ORTEP-III with a graphical

user interface (GUI), J. Appl. Crystallogr. 30 (1997) 565.

1,3-dihydroxy-9 H -xanthenes -new methods of synthesis, J. Org. Chem. 51

(1986) 717e723.

[67] R.A.P. Castanheiro, M.M.M. Pinto, A.M.S. Silva, S.M.M. Cravo, L. Gales,

A.M. Damas, N. Nazareth, M.S.J. Nascimento, G. Eaton, Dihydroxyxanthones

prenylated derivatives: synthesis, structure elucidation, and growth inhibi-

tory activity on human tumor cell lines with improvement of selectivity for

MCF-7, Bioorg. Med. Chem. 15 (2007) 6080e6088.

[81] G.S.R. Subba Rao, S.A. Raghavan, Synthetic studies on morellin. Part 4:

synthesis of 2,2-dimethyl-12-[3-methylbut-2-enyl]-2 H ,6 H -pyrano[3,2-b ]

xanthen-6-one, J. Indian Inst. Sci. 81 (2001) 393e401.

[82] R.A. Finnegan, P.L. Bachman, Natural occurrence of 2-hydroxyxanthone,

J. Pharm. Sci. 54 (1965) 633e635.

[68] R.A.P. Castanheiro, A.M.S. Silva, N.A.N. Campos, M.S.J. Nascimento,

M.M.M. Pinto, Antitumor activity of some prenylated xanthones, Pharma-

ceuticals 2 (2009) 33e43.

[69] C.M.G. Azevedo, C.M.M. Afonso, D. Sousa, R.T. Lima, M.H. Vasconcelos,

M. Pedro, J. Barbosa, A.G. Corrêa, S. Reis, M.M.M. Pinto, Multidimensional

optimization of promising antitumor xanthone derivatives, Bioorg. Med.

Chem. (2013). http://dx.doi.org/10.1016/j.bmc.2013.03.079.

[83] L. Gales, M.E. Sousa, M.M.M. Pinto, A. Kijjoa, A.M. Damas, Naturally occurring

1,2,8-trimethoxyxanthone and biphenyl ether intermediates leading to 1,2-

dimethoxyxanthone, Acta Crystallogr. Sect. C: Cryst. Struct. Commun. 57

(2001) 1319e1323.

[84] E. Sousa, A.M.S. Silva, M.M.M. Pinto, M.M. Pedro, F.A.M. Cerqueira,

M.S.J. Nascimento, Isomeric kielcorins and dihydroxyxanthones: synthesis,

structure elucidation, and inhibitory activities of growth of human cancer cell

lines and on the proliferation of human lymphocytes in vitro , Helv. Chim. Acta

85 (2002) 2862e2876.

[70] A. Leo, C. Hansch, D. Elkins, Partition coef ﬁ cients and their uses, Chem. Rev. 71

(1971) 525e616.

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Pyranoxanthones: Synthesis, growth inhibitory activity on human tumor cell lines and determination of their lipophilicity in two membrane models

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