Multidimensional optimization of promising antitumor xanthone derivatives

Bioorganic & Medicinal Chemistry 21 (2013) 2941–2959

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Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Multidimensional optimization of promising antitumor xanthone

derivatives

Carlos M. G. Azevedo ^a,b, Carlos M. M. Afonso ^a,b,, Diana Sousa ^a,c, Raquel T. Lima ^a,c

,

⇑

M. Helena Vasconcelos ^c,d, Madalena Pedro ^a,e, João Barbosa ^e, Arlene G. Corrêa ^f, Salette Reis ^g,

Madalena M. M. Pinto ^a,b

^aCentro de Química Medicinal da Universidade do Porto (CEQUIMED-UP), Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal

^bLaboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira no. 228, 4050-

313 Porto, Portugal

^cCancer Drug Resistance Group, Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal

^dLaboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal

^eGrupo 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

^fDepartamento de Química, Universidade Federal de São Carlos, Rodovia Washington Luís, km 235, CEP 13565-905, São Carlos, SP, Brazil

^gREQUIMTE, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira no. 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:

A promising antitumor xanthone derivative was optimized following a multidimensional approach that

involved the synthesis of 17 analogues, the study of their lipophilicity and solubility, and the evaluation

of their growth inhibitory activity on four human tumor cell lines. A new synthetic route for the hit xan-

thone derivative was also developed and applied for the synthesis of its analogues. Among the used cell

lines, the HL-60 showed to be in general more sensitive to the compounds tested, with the most potent

Received 10 December 2012

Revised 20 March 2013

Accepted 26 March 2013

Available online 6 April 2013

compound having a GI₅₀of 5.1 lM, lower than the hit compound. Lipophilicity was evaluated by the par-

Keywords:

tition coefﬁcient (K_p) of a solute between buffer and two membrane models, namely liposomes and

micelles. The compounds showed a logK_pbetween 3 and 5 and the two membrane models showed a

good correlation (r²= 0.916) between each other. Studies concerning relationship between solubility

and structure were developed for the hit compound and 5 of its analogues.

Xanthones

Xanthen-9-ones

Antitumor

Benzopyran

Chromans

Chromene

Lipophilicity

Solubility

1. Introduction

groups found in natural and synthetic xanthones are hydroxyl,

methoxyl, methyl, chloro, prenyl and carboxyl.^1–3For synthetic

Xanthones (or xanthen-9-ones) constitute a class of O-heterocy-

cles with a dibenzo- -pyrone scaffold commonly found as second-

xanthones, other substituents have also been introduced including

c

aminoalkyl, aminoalcohol, azido and furanyl groups.^2,3

ary metabolites in higher plants, fungi and lichens.¹They can also

be obtained by synthesis and several strategies to achieve this goal

have been described in the literature.²The members of the xan-

thone classes bear different types of substituents that are able to

interact with several biological targets exerting different pharma-

cological activities.³Indeed, the xanthone core is a rigid heteroaro-

matic tricyclic platform which may be considered as a ‘privileged

structure’ since it can provide potent and selective ligands through

modiﬁcation of functional groups, allowing them to interact with

different pharmacological targets.⁴The more frequent chemical

Prenylated xanthones are the most abundant family of naturally

occurring xanthones.^5,6This family comprises compounds that in-

clude one or more isoprenic moieties which can be found as an

open chain or cyclized to give a fused furan or pyran ring. More

importantly, this family of compounds has been described to pos-

sess a great variety of biological activities, in particular they have

been considered as promising antitumor agents.^5–8Representative

members of prenylated xanthones with potent antitumor activity

are a-mangostin (1) and c-mangostin (2) (Fig. 1) which have been

isolated from the pericarp of the mangosteen fruit.⁹These com-

pounds have previously been shown to exert a potent growth

inhibitory activity towards several tumor cell lines,¹⁰being capable

of inducing apoptosis in leukemia cell lines through a mechanism

⇑

Corresponding author. Tel.: +351 220 428 000; fax: +351 226 093 390.

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

http://dx.doi.org/10.1016/j.bmc.2013.03.079

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C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

Figure 1. Prenylated xanthones with promising antitumor activity.

which involves activation of caspases-3 and -9.^11,12Another well-

known family of prenylated xanthones with promising antitumor

activity are xanthones bearing a 4-oxo-tricyclo[4.3.1.0^3.7]dec-8-

en-2-one, from which gambogic acid (3) is the most representative

member of the so-called ‘caged xanthones’ (Fig. 1).¹³

evaluated for their in vitro growth inhibitory activity on four hu-

man tumor cell lines, namely MCF-7 (breast adenocarcinoma),

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

HL-60 (acute myeloid leukemia).

In addition to the study of cell growth inhibitory activity, a pre-

liminary estimation of the pharmacokinetic behavior of compound

4 analogues was made by evaluating their lipophilicity and solubil-

ity. Lipophilicity is one of the ﬁrst physicochemical properties to be

evaluated in the early phases of a drug discovery program²⁴and has

been correlated with several other physicochemical and pharmaco-

kinetic properties, for example, solubility,²⁵permeability,²⁶plasma

protein binding,²⁷metabolism,²⁸CNS penetration,²⁹volume of dis-

tribution²⁵and clearance.²⁸Consequently, it may extensively inﬂu-

ence the success of a drug discovery program. In fact, compounds

with high lipophilicity have shown an increased risk of attrition dur-

ing the clinical trials.³⁰Lipophilicity is commonly evaluated by the

partition coefﬁcient of a solute in a biphasic octanol–water system

which has some limitations since it fails to create the anisotropic

media that is found on biomembranes and encode some important

interactions that take place between the solute and the mem-

branes.³¹Therefore, other models have been developed such as lip-

osomes and micelles^32,33which have proved to be advantageous

when compared to octanol–water.³¹Consequently, it was decided

to evaluate the lipophilicity on these two models. Solubility is

emerging as one of the major issues in drug discovery and develop-

ment of new chemical entities.³⁴In fact, compounds with low solu-

bility have a higher risk of attrition in the drug discovery pipeline as

well as a higher cost during the drug development stage.³⁴There-

fore, the thermodynamic solubility in water at pH 7.4 (HEPES buffer)

of compound 4 and ﬁve of its analogues (36a–e) was evaluated.

In recent years, our research group has been synthesizing a

small library of prenylated xanthones^14–18and evaluating their

biological activity in different cell lines. This approach was shown

to be fruitful with many of the synthesized compounds presenting

encouraging antitumor activity. Among them, compound 4 (12-hy-

droxy-2,2-dimethyl-3,4-dihydropyran[3,2b]xanthene-6(2H)-one—

Fig. 1) was considered one of the most promising, exhibiting anti-

proliferative and pro-apoptotic activities in leukemia cell lines.¹⁷

Interestingly, it also showed an enhancement of the anti-estro-

genic effect of 4-hydroxytamoxifen in an estrogen-dependent

(MCF-7) tumor cell line.¹⁸Therefore, this molecule has the poten-

tial to be optimized and may be used as the starting point in the

search for more potent antitumor agents. To increase the likelihood

of success, attention must also be paid to their pharmacokinetic

behavior.¹⁹Consequently, it was decided to optimize this com-

pound following a multidimensional approach looking, in parallel,

at the activity and physicochemical properties, with the latter

being used as a tool to predict the pharmacokinetic behavior.²⁰

Compound 4 is composed of four fused rings that create a linear

tetracyclic system. This structure has an unsubstituted ring A

(Fig. 1) in which different groups can be introduced to increase

the interactions with a putative target, and consequently, its po-

tency. Furthermore, the introduction of substituents may also be

used to improve the pharmacokinetics. The described methodology

for the synthesis of compound 4 through a heterogeneous catalysis

methodology has only been employed with relative success for the

formation of a fused 2,2-dimethyl-3,4-dihydropyran ring in a few

simple substituted xanthones,^16–18ﬂavonoids,²¹benzophenones²²

and phenols.²³Moreover, low yields were generally obtained and

in some cases no regioselectivity was observed. Furthermore, the

scope and limitation of this reaction has not been thoroughly eval-

uated. In fact, for the synthesis of more complex molecules, this

methodology is expected to show serious limitations. Therefore,

in this paper the synthesis of compound 4 is reported by a new

and more effective route and its application for the synthesis of

17 structural analogues. The targeted modiﬁcations envisaged

the introduction of different functional groups on ring A and/or

ring D orientation (Fig. 2). The newly synthesized analogues were

2. Results and discussion

2.1. Chemistry

2.1.1. Synthesis of building blocks

Pyranoxanthones are generally obtained by two classic strate-

gies: Chemical modiﬁcation of simple oxygenated xanthones or to-

tal synthesis using a benzopyran derivative as building block.^2,5,8

The former is more commonly used and was already applied for

the synthesis of compound 4.¹⁸However, the reaction used has

some limitations and should not be applied for the synthesis of

compound 4 analogues bearing substituents on ring A. As a result,

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

2943

Figure 2. Targeted analogues of compound 4.

a total synthesis approach was undertaken and the syntheses of

the building blocks are represented on Scheme 1.

transfer hydrogenation protocol³⁷and brominated regioselectively

to give compound 14. Benzopyran 19 was synthesized in four steps

using 2,4-dihydroxybenzaldehyde (15) as starting point

(Scheme 1). The ﬁrst step consisted of the regioselective condensa-

tion of 2,4-dihydroxybenzaldehyde (15) with prenal catalyzed by

calcium hydroxide.³⁸This compound was then O-methylated with

dimethylsulfate, oxidized using a Baeyer–Villiger type oxidation

and again O-methylated with dimethylsulfate to yield compound

19. The O-protected MOM and benzyl salicylaldehydes or salicy-

lates were either bought already protected or protected under

the standard conditions (Scheme 1).

Benzopyran 9 was synthesized in four steps starting from

o-vanillin (5). The ﬁrst step was the oxidation of o-vanillin (5) to

3-methoxycatechol (6) using a Baeyer–Villiger-type oxidation. For-

mation of the benzopyran in one-step was accomplished by the

reaction of 3-methoxycatechol (6) with a very reactive 1,1-dimeth-

ylallyl cation generated from 2-methyl-3-buten-2-ol and catalyzed

by formic acid.³⁵The next step was the protection of the hydroxyl

with tert-butyldimethylsilyl (TBDMS) to yield compound 8 which

was then brominated regioselectively using a NBS/NH₄OAc/CH₃CN

protocol³⁶to produce the desired compound 9. Benzopyran 14 was

synthesized in four steps from 2,3-dimethoxyphenol (10)

(Scheme 1). The ﬁrst part was the synthesis of the 2,2-dimethyl-

benzopyran moiety (12) which was accomplished through the

cyclization of the appropriate aryl 1,1-dimethylpropargyl ether

(11). The 2,2-dimethylbenzopyran 31 was then reduced by a

2.1.2. Synthesis of compound 4 and analogues 32, 36a–e, 41c,

41e and 41f

Scheme 2 represents the total synthesis of compound 4. The

ﬁrst step involved the synthesis of the benzophenone 29 by con-

densation of the lithiated intermediate of compound 9 formed by

Scheme 1. Synthesis of the benzopyrans 9, 14 and 19, O-protected salicylaldehydes and methyl salicylate.

2944

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

halogen–lithium exchange and a MOM protected methyl salicy-

late (27). The MOM protective group was removed from benzo-

phenone 29 with niobium chloride³⁹which also removed the

TBDMS group for part of the starting material to give benzophe-

nones 30 and 31. Lastly, the two benzophenones were cyclized

to xanthone by an intramolecular nucleophilic aromatic substitu-

tion. The cyclization of benzophenone 31 produced compound 4

at an excellent (92%) yield while the cyclization of benzophone

30 gave rise to compound 4 and compound 32 with the formation

of the latter probably being due to an unexpected methyl transfer

triggered by the microwave heating and the cesium effect. In

summary, compound 4 was synthesized in 7 steps at a 15% yield.

The route was further applied for the synthesis of analogues bear-

ing substituents on ring A (Scheme 2). The ﬁrst step was the syn-

thesis of the diarylmethanol derivatives 33a–e which were then

oxidized to benzophenones with IBX in DMSO and O-deprotected

to give benzophenones 35a–e. In the case of benzophenone 34a,

besides the expected compound 35a, a hydrodechlorinated ben-

zophenone (30) was also obtained. The hydrodehalogenation cat-

alyzed by palladium on charcoal⁴⁰has been described previously

but to the best of our knowledge, no report exists in the literature

of hydrodechlorination using Pd/C-triethylsilane as a transfer

hydrogenation catalyst. The last step was the cyclization of benz-

ophenones 35a–e which led directly to the desired compounds

36a–e and in the case of benzophenone 35e also led to compound

37. Compound 4 analogues bearing a methoxyl in position 12 and

substituents on ring A were synthesized using the same approach

described previously but instead using benzopyran 14 as building

block (Scheme 2).

2.1.3. Synthesis of analogues 48a–c, 48e, 48g, 55a, 55f and 55g

The route for the synthesis of analogues of compound 4 with a

different D ring orientation is represented in Scheme 3 and used

benzopyran 19 and protected salicylaldehydes as building blocks.

The ortho-lithiation of benzopyran 19 took place at positions 7 or

8 (highlighted in Scheme 3) since both positions are ﬂanked by

an oxygen and as a result two isomeric diaryl methanols were ob-

tained. However, position 7 of the benzopyran 19 was favored for

lithiation and diarylmethanols 42a–c, 42e, 42g, 49a, 49f and 49g

were obtained at higher yields. This can be explained by lower ste-

ric hindrance at position 7 and the coordination effect of the two

methoxyls in positions 5 and 6⁴¹. The two diarylmethanol isomers

had a similar chromatographic behavior and were used as a mix-

ture in the following reactions. The diarylmethanols were oxidized

to benzophenones and O-deprotected. In the case of benzyl pro-

tected benzophenones, the double bond of the pyran ring was also

reduced and benzophenones 53a and 54a also underwent hyd-

rodehalogenation reaction. Lastly, the benzophenones with the

appropriate pattern of substitution were cyclized to xanthones

and separated easily from the remaining benzophenone.

Scheme 2. Synthesis of compound 4, analogues 32, 36a–e, 41c, 41e and 41f.

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

2945

Scheme 3. Synthesis of analogues 48a–c, 48e, 48g, 55a, 55f and 55g.

Table 1

2.2. Biological activity

Growth inhibitory activity of the synthesized analogues in MCF-7, NCI-H460, A375-C5

and HL-60 cell lines

The effect of compound 4 and of its 17 analogues was evaluated

in the growth of four human tumor cell lines. The cell lines used

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

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

kemia). The drug-screening procedure used was the Sulforhoda-

mine B (SRB) assay adopted from the National Cancer Institute

(NCI, USA) which uses the protein-binding dye SRB to indirectly as-

sess cell growth.^42,43For all the cell lines, a dose–response curve

was established for each compound studied and the concentration

which caused a cell growth inhibition of 50% (GI₅₀) was deter-

mined.⁴⁴The results obtained are summarized in Table 1.

Compound

GI₅₀

NCI-H460

lM

MCF-7

A375-C5

HL-60

4

32

39.7 3.2

>150

40.3 3.3

>150

28.9 8.1

>150

23.4 1.1^a

8.8 5.9

9.0 1.1

23.9 3.5

12.5 3.2

27.3 5.6

57.0 6.8

5.14 1.8

8.3 2.7

72.3 4.3

14.1 1.4

N.R.

36a

36b

36c

36d

36e

41c

41e

41f

48a

48b

48c

48e

48g

55a

55f

55g

>150

48.1 13.6

41.1 8.6

59.1 14.1

30.9 3.6

>150

>100

>150

>50

34.9 1.8

26.8 1.6

>75

13.7 0.47

17.6 4.9

68.0 4.5

40.7 8.5

29.7 4.7

>150

>100

>150

>50

27.1 1.7

21.9 2.7

>75

19.6 4.7

N.R.

82.5 15.0

31.8 5.4

>150

>100

>150

>50

36.9 4.7

34.3 1.1

>75

By analyzing Table 1, it can be observed that some of the com-

pounds studied were more active than compound 4 in the four tu-

mor cell lines tested. Regarding the results obtained in the MCF-7

and NCI-H460 cell lines, the compound which presented higher

>50

18.8 4.7

9.2 4.9

48.4 7.5

N.R.

activity (i.e., GI₅₀<20 lM) was the 55g. In the A375-C5 cell line,

the compounds that had a stronger growth inhibitory effect were

the 36c and the 55g. Regarding the HL-60 cell line, compounds

32, 36a, 36c, 41c, 41e, 48a, 48e, 48g and 55g presented a GI₅₀be-

>75

14.5 1.0

14.4 3.7

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

The maximum DMSO concentration used was 0.25% for all compounds tested and

did 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).

low 20

lM, with compounds 32, 36a, 41c, 41e and 48g presenting

a GI₅₀below 10

lM. In general, the synthesized compounds were

more active towards the HL-60 tumor cell line. Therefore, the

HL-60 cell line seems to be more sensitive to the effect of xanthon-

es with a fused 2,2-dimethylpyran and 2,2-dimethyl-3,4-dihydro-

pyran ring than the other cell lines tested.

N.R.—non-reproducible.

a

This compound has previously shown an IC₅₀of 6–7

lM in the HL-60 cell line

and determined with the Trypan blue exclusion assay.¹⁷

The evaluation of the growth inhibitory activity on the four tu-

mor cell lines allowed some assumptions to be made regarding

structure–activity relationship of the compounds studied. In the

case of the analogues of compound 4 with different substituents

on ring A, it was observed that the presence of an electronegative

substituent on position 8 (36a and 36b) was associated with an in-

crease in the effect in the HL-60 tumor cell line when compared to

the other three cell lines studied. On the other hand, the presence

of a methyl group in position 8 (36c) seems to have only a

detrimental effect on the activity on the MCF-7 tumor cell line in

comparison with the other tumor cell lines. The presence of a hy-

droxyl in position 9 (36e) or methoxyl in position 10 (36d) seemed

to be associated with a general decrease in activity. Comparing

compound 32 with compound 4, the presence of a methoxyl in-

stead of a hydroxyl in position 12 seemed to be associated with

an increase in activity for the HL-60 tumor cell line in comparison

with the other three cell lines tested. Moreover, for the compounds

bearing a methoxyl in position 12, the introduction of a methyl in

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C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

Table 2

position 8 (41c) or hydroxyl in position 9 (41e) seemed to be asso-

ciated with a slight increase in the effect in the HL-60 cells and in

the case of 41c with moderate activity on the other three tumor

cell lines also. Regarding the compounds bearing a different ring

D orientation, the presence of an electron-donating group in posi-

tion 8 of ring A (48e, 48g and 55g) seemed to be associated with a

moderate to good growth inhibitory activity in the four tumor cell

lines. Moreover, the presence of a 2,2-dimethyl-3,4-dihydropyran

seemed to be associated with a higher growth inhibitory activity

(comparing 48g with 55g). On the other hand, the presence of sub-

stituents on position 9 seemed to be detrimental although the

presence of chlorine was associated with moderate activity for

the HL-60 tumor cell line (48a and 55a).

Partition coefﬁcients in liposomes-buffer and micelle-buffer for compound 4 and16 of

the analogues synthesized

Compound

LogK_pliposomes

LogK_pmicelles

4

32

3.35 0.02

3.60 0.08

—

3.25 0.08

3.86 0.08

3.53 0.04

3.32 0.10

4.02 0.03

3.43 0.04

—

3.28 0.02

3.59 0.06

4.39 0.12

3.35 0.04

3.92 0.01

3.58 0.01

3.76 0.07

4.01 0.06

3.49 0.06

3.88 0.03

4.34 0.04

4.21 0.04

4.29 0.02

3.90 0.06

4.41 0.03

3.83 0.10

4.70 0.04

36a

36b

36c

36d

36e

41c

41e

41f

48a

48b

48c

48e

55a

55f

55g

—

4.32 0.06

—

4.48 0.07

3.88 0.08

4.62 0.02

2.3. Physicochemical properties

2.3.1. Lipophilicity

The lipophilicity was determined as the partition coefﬁcient

(K_p) of the solute between buffer (pH 7.4) and micelles or lipo-

somes, and calculated without a phase separation. The method

used for the calculus considered the change in the UV–Vis spec-

trum produced by increasing concentrations of liposomes or mi-

celles at constant solute concentration. This technique considers

the change of an absorption parameter such as molar absorptivity

Mean of three independent measurements.

double-beam spectrophotometer we were unable to determine

the K_pfor 36a, 41f, 48a, 48b and 48e which had both a low solubil-

ity and molar absorptivity.

Among the xanthones tested, 55g was the most lipophilic and

compound 4 and 36b the less lipophilic. Considering the effect of

ring A substituents on the K_pof compound 4, it can be observed

that chlorine (36a) and methyl (36c) were associated with an in-

crease in K_p. In the case of the methoxyl, the introduction of this

group in position 8 (36b) did not interfere with the K_pwhile the

introduction in position 10 was associated with an increase of

the K_p(36d). For the hydroxyl group (36e) an unexpected result

was observed and no decrease in K_pwas determined. In fact, for

micelles, an increase in K_pwas in fact observed. Considering the

analogues of compound 4 with a methoxyl in position 12 (32), it

can be observed that this substituent was associated with an in-

crease in the K_p. In relation to the analogues with a different ring

D orientation, it can be observed that most of the compounds

had a K_pnear or higher than 4 with the chlorine (48a and 55a)

and the diethylamine (55g) derivatives representing the com-

pounds that showed a higher K_p.

(e) or maximum wavelength (k_max) of a solute when it permeates

from the aqueous to a non-polar media. However, it is only possi-

ble to calculate the partition coefﬁcient when there is a change of

k_maxof 5–10 nm and e

P10%.⁴⁵Moreover, in the case of liposomes,

it is difﬁcult to calculate the partition coefﬁcient due to the light

scattering at wavelengths below 300 nm which are not eliminated

even with the counterbalance of the sample and reference. To over-

come these limitations, it is necessary to use derivative spectro-

photometry (in order of wavelength) which not only eliminates

the light scattering but also leads to better resolution of overlap-

ping bands.⁴⁵Therefore, the K_pcan be calculated using the follow-

ing equation:⁴⁵

ðDl ꢀ D_wÞK_p½LꢁVø

D ¼ D_w

þ

1 þ K_p½LꢁVø

where, [L] is the lipid concentration (mol L^ꢀ1), Dl the derivative of

lipid absorbance, D_wthe derivative of water absorbance, V£ the li-

pid molar volume (L mol^ꢀ1), K_pthe partition coefﬁcient (dimension-

less) and:

For those compounds in which the partition coefﬁcient was

determined in the two models, the results obtained were com-

pared using a linear regression analysis and the following Eq. 1

was established:

@ⁿAbs

D ¼

@kⁿ

LogK_p_liposomes¼ ꢀ0:1614ðꢂ0:382Þ

Accordingly, the partition coefﬁcient could be calculated by ﬁt-

ting the derivative spectrometric data (D vs [L]) to the equation by

a nonlinear regression method, with Dl and K_pbeing the adjustable

parameters.⁴⁵

þ 1:03ðꢂ0:098ÞLogK_p_micelles

n

¼ 12; r²¼ 0:916; s ¼ 0:143; F ¼ 109

ð1Þ

The K_pin liposomes was determined for 4, 32, 36b–e, 41c, 41e,

48c, 55a, 55f, 55g and for all compounds in micelles with the

exception of compound 48g which was obtained at low yields (Ta-

ble 2). A procedure adapted from Magalhães et al.,⁴⁶which used a

96-well plate methodology was followed and 1% of DMSO was

added to each well to increase the solubility. The amount of DMSO

was limited to 1% since higher concentrations could interfere with

the partition between the two phases.⁴⁷In order to calculate the

K_p, several concentrations of lipid (with and without solute)

needed to be prepared and a UV–Vis spectrum traced. The 96-well

plate procedure made the determination of K_pquicker but could

not be applied for the compounds which had a very low solubility

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

ber of compounds, r²the squared correlation coefﬁcient, s the stan-

dard deviation, and F Fisher’s test. A good correlation between the

two models was observed (r²= 0.916) and the slope of the equation

is almost one (1.03), which implies that there is no clear tendency

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

membrane models with an increase in hydrophobicity. The results

obtained led to the observation that micelles can be used as a sur-

rogate for liposomes for these compounds with the advantage of

not having the light scattering limitation as well as a higher appli-

cability and easier preparation.

and/or molar absorptivity (e). In those cases, one at a time UV–Vis

2.3.2. Solubility

spectrum was traced for each in a conventional double-beam UV–

Vis spectrophotometer. Contrary to micelles, liposomes have a high

light scattering for wavelengths below 300 nm and even with a

The solubility is the maximum amount of a compound that can

remain in solution at a certain volume of solvent, temperature and

pressure, under equilibrium conditions. The equilibrium is a result

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

2947

of the net balancing of energy between the energy of the solvent

and the solute interacting with themselves against the energy of

solvent and solute interacting with each other.⁴⁸The solubility of

compound 4 and ﬁve ring A substituted analogues was determined

by a thermodynamic assay at pH 7.4 (using HEPES as buffer) and

the inﬂuence of chlorine, methoxyl, methyl and hydroxyl have on

the solubility of compound 4 was evaluated.

The values of solubility for all compounds tested were low as

can be depicted from Table 3. From the structure–solubility rela-

tionship for compounds 4 and 36a–e, only for compound 36e

was an increase in the solubility observed when compared to 4.

The introduction of chlorine (36a), methyl (36c), and methoxyl

(36b and 36c) had an adverse effect on the solubility. However,

the presence of a methoxyl in position 10 (compound 36d) has a

less detrimental effect on the solubility when compared to a meth-

oxyl in position 8 (compound 36b).

micelles, and all compounds showed a LogK_phigher than 3 and be-

low 5. The two models showed a good correlation between each

other and micelles could be used as a surrogate of liposomes for

the determination of the partition coefﬁcient for this type of com-

pounds. The solubility was also evaluated for compound 4 and ﬁve

analogues bearing substituents on ring A and all compounds

showed a low solubility. This was explained by the high planarity

and rigidity of these compounds as well as a moderate to high

lipophilicity.

The scaffold of compound 4 seemed to be associated with a high

potency for the HL-60 tumor cell line and a promising platform for

the discovery of new antitumor compounds. The high rigidity and

planarity could strongly inﬂuence the solubility. Nevertheless, this

study provided an insight of the positions which seemed to be crit-

ical for activity as well as the positions where molecular modiﬁca-

tions can be performed to decrease the lipophilicity and the

planarity without signiﬁcantly decreasing the activity.

The solubility in water depends mainly on two factors: the crys-

tallinity of the solute and its ability to interact with water and can

be related to the lipophilicity and melting point.⁴⁹Considering that

compound 4 and their analogues are all linear tetracyclic systems

with a high degree of planarity, this structural feature promotes

4. Experimental

4.1. General methods

high crystal packing and p–stacking interactions, corroborated by

Microwave reactions were performed using a 100 mL closed

Teﬂon reactor (internal reaction temperature measurement with

a ﬁber-optic probe sensor) and were carried out using an Ethos

MicroSYNTH 1600 Microwave Labstation from Milestone. Micro-

wave reactions were also carried out on a CEM Discover Bench-

Mate with 10-mL pressurized vials. Reactions were monitored by

TLC and/or GC–MS. Melting points were obtained in a Köﬂer micro-

scope and are uncorrected. IR spectra were measured on an ATI

Mattson Genesis series FTIR (software: WinFirst v. 2.10) spectro-

photometer in KBr microplates (cm^ꢀ1). ¹H and ¹³C NMR spectra

were taken in CDCl₃or DMSO-d₆at room temperature, on Bruker

Avance 300 instruments and Bruker ARX-400. Chemical shifts are

expressed in d (ppm) values relative to tetramethylsilane (TMS)

as an internal reference. ¹H NMR spectra were measured at

300.13 or 400.21 MHz and assignment abbreviations are the fol-

lowing: 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), triplet of doublets (td) and broad (b).

¹³C NMR spectra were measured at 75.47 or 100.63 MHz. The EI-

MS were recorded either in a Shimadzu GCMS-QP5000 or on a

ThermoQuest Finnigan GC 2000 series/GCQ plus. Elemental analy-

sis results were determined in an Analizador Elemental CarloErba

1108 at C.A.C.T.I., Vigo, Spain. 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.-University of Vigo, Spain. All reagents were purchased

from Sigma Aldrich or Acros and all solvents were PA used without

further puriﬁcation. The anhydrous solvents were either purchased

from Sigma–Aldrich or dried according to the published proce-

dures.⁵⁰Puriﬁcations of compounds were performed by column

chromatography either by using Merck silica gel 60 (0.040–

0.063 mm) (when it is referred to as ﬂash chromatography) or

using Merck silica gel 60 (0.2–0.5 mm) (when nothing is referred)

or by preparative thin layer chromatography (TLC) using Merck sil-

ica gel 60 (GF₂₅₄) plates.

the high melting point observed for these compounds (higher than

200 °C). Moreover, all compounds showed a moderate to high lipo-

philicity and as a result, these structural features have a crucial im-

pact on solubility and help to explain the low solubility found.

Moreover, when compounds 36e and 4 are compared, the intro-

duction of a polar and ionizable hydroxyl group did not lead to a

substantial increase in the solubility. This result can be explained

by the increase in crystal packing caused by the phenolic group

as veriﬁed by the high melting point (262–264 °C) determined

for this compound.

3. Conclusion

In the present work, a new route, through a benzophenone

intermediate was developed for the synthesis of compound 4

(12-hydroxy-2,2-dimethyl-3,4-dihydropyran[3,2b]xanthene-6(2H)-

one). This route was further applied for the synthesis of 17 ana-

logues bearing different substituents on ring A and different ring

D orientation. Subsequently, the growth inhibitory activity of com-

pound 4 analogues was evaluated in four human tumor cell lines,

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

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

kemia) and active compounds were found for all the cells lines

tested. However, a higher growth inhibitory activity was generally

observed for the HL-60 cells indicating that this compound may act

through a mechanism/target which is of particular relevance for

this cell line but not for the other cell lines tested. In addition,

the results obtained in the cell lines allowed us to establish struc-

ture–activity relationship considerations for the compounds

synthesized.

The lipophilicity of the 17 synthesized analogues of compound

4 was evaluated in two membrane models, namely liposomes and

Table 3

Solubility of compounds 4 and 36a–e

Compound

Solubility (lM)

4.2. Synthesis of benzopyran 9

4

0.40 0.1

<0.1

0.23 0.02

0.62 0.1

36a

36b

36c

36d

36e

4.2.1. 3-Methoxycatechol (6)

o-Vanillin (5) (25 g/164 mmol) was solubilized in 600 mL of

methanol and 22 mL of H₂O₂30% was added. Then, KHSO₄(3.3 g/

24.6 mmol) was added quickly and the solution was allowed to stir

at room temperature for 48 h. The reaction was quenched by the

Mean of three independent measurements.

2948

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

addition 200 mL of distilled water and the methanol evaporated.

The aqueous phase was extracted with 3 ꢃ 300 mL of diethyl ether.

The organic phase was washed with 2 ꢃ 250 mL of brine, dried

over sodium sulfate anhydrous, ﬁltered and the organic solvent

evaporated. The crude product was puriﬁed by silica gel ﬂash chro-

matography (dichloromethane) obtaining 3-methoxycatechol (6)

as an orange oil (20.96 g/91%)

ﬂask. The ﬂask was placed at 0 °C and then NBS (5.16 g/28.9 mmol)

was added in one portion. The mixture was allowed to stir for

15 min and warm to room temperature. The solution was parti-

tioned between water and ethyl acetate. The aqueous phase was

then extracted 2 ꢃ 250 mL of ethyl acetate. The organic phase

was washed 2 ꢃ 250 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 99:1). Compound 9 was obtained as orange oil that

solidiﬁed upon standing (10.68 g/96%).

3-Methoxycatechol (6). IR m

_max(cm^ꢀ1) (KBr): 3426, 2936, 1621,

1503, 1478, 1350, 1290, 1245, 1205, 1080, 765, 714. EIMS m/z (%):

141 (2, [M+1]⁺), 140 (24, [M]⁺), 125 (23), 110 (7), 107 (3), 97 (87),

79 (10), 77 (2), 68 (8), 65 (13), 63 (10), 53 (33), 52 (8), 51 (100).

((6-Bromo-7-methoxy-2,2-dimethyl-3,4-dihydro-8-yl)oxy)(tert-

butyl)dimethylsilane (9). Mp: 41–43 °C. IR m

_max(cm^ꢀ1) (KBr): 2971,

4.2.2. 7-Methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-8-ol (7)

3-Methoxycatechol (6) (1.5 g/10.7 mmol) and 40 mL of an 80%

formic acid solution were placed in a two necked round-bottom

ﬂask. The solution was heated to 80 °C and then 2-methyl-3-bu-

2925, 2889, 2851, 1462, 1441, 1338, 1249, 1197, 1119, 1092, 1040,

996, 870, 831, 777. ¹H NMR (300.13 MHz, CDCl₃): 6.87 (s), 3.77 (s,

OCH₃), 2.71 (t, J = 6.7, 2H), 1.75 (t, J = 6.7, 2H), 1.35 (s, 6H), 1.05 (s,

9H), 0.18 (s, 6H). ¹³C NMR (75.47 MHz, CDCl₃) d (ppm): 147.4,

146.0, 139.0, 124.2, 118.6, 106.6, 74.8, 60.2, 32.6, 26.8, 25.8, 22.1,

18.6. EIMS m/z (%): 402 (1, [M+2]⁺), 401 (0.5, [M+1]⁺), 400 (1,

[M]⁺), 289 (16), 288 (15), 287 (84), 174 (22), 272 (21), 265 (21),

264 (100), 249 (23),193 (36), 179 (23), 166 (23), 165 (83), 137

(34), 109 (19), 75 (56), 73 (45).

ten-2-ol was added dropwise (461 mg/5.35 mmol/560 lL) (over

10 min). The mixture was allowed to react for 1 h and 30 min and

then cooled to room temperature. The solution was poured over

100 mL of distilled water and neutralized with a saturated solution

of NaHCO₃until pH 7–8. The aqueous phase was extracted with

3 ꢃ 100 mL of diethyl ether. The organic phase was washed with

2 ꢃ 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) and

compound 7 was isolated as light orange oil (398 mg/36%).

7-Methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-8-ol (7). IR

m_max(cm^ꢀ1) (KBr): 3461, 2969, 2944, 2922, 2852, 2829, 1618,

1503, 1489, 1445, 1345, 1291, 1247, 1196, 1149, 1111, 1083,

962, 779, 642. ¹H NMR (300.13 MHz, CDCl₃): 6.56 (dt, J = 8.7, 0.9),

6.46 (d, J = 8.7), 3.87 (s, OCH₃), 2.74 (td, J = 6.7, 0.9, 2H), 1.81 (t,

J = 6.7, 2 H), 1.38 (6H, s). ¹³C NMR (75.47 MHz, CDCl₃) d (ppm):

145.3, 141.6, 134.1, 118.7, 114.5, 103.8, 75.1, 56.3, 32.9, 26.8,

21.7. EIMS m/z (%): 209 (17, [M+1]⁺), 208 (100 [M]⁺), 161 (15),

153 (84), 152 (17), 106 (16), 105 (27), 77 (26), 76 (23), 65 (17),

51 (18).

4.3. Synthesis of benzopyran 14

4.3.1. 2,4-Dimethoxy-1-((2-methylbut-3-yn-2-yl)oxy)benzene

(11)

To a solution of 2-methyl-3-butyn-2-ol (4.71 g, 56 mmol) in

100 mL of anhydrous CH₃CN under nitrogen, cooled in a salt

ice-bath (ꢀ5 °C) was added DBU (11.11 mL, 73 mmol) followed

by triﬂuoroacetic anhydride dropwise (7.79 mL, 56 mmol). The

reaction mixture was slowly warmed to 0 °C over 30 min. In an-

other ﬂask, 2,3-dimethoxyphenol (10) (7.5 g, 48.6 mmol) was dis-

solved in 100 mL of anhydrous CH₃CN and placed under nitrogen

at ꢀ5 °C. To this solution was added DBU (9.62 mL, 63.2 mmol)

followed by anhydrous CuCl₂(6.5 mg, 0.0486 mmol, 0.1 mol %).

To the later reaction mixture was added dropwise via canulla

the ice-cold solution of the freshly prepared 1,1-dimethyl-2-pro-

pynyl triﬂuoroacetate in CH₃CN. The resulting reaction mixture

was warmed to 0 °C and stirred for 7 h. The mixture was warmed

to room temperature and concentrated to 100 mL. Then it was

partitioned between 250 mL of distilled water and 250 mL of

diethyl ether. The aqueous phase was extracted with

2 ꢃ 250 mL of diethyl ether. The organic phase was washed with

2 ꢃ 150 mL of a 5% of solution of HCl, with 2 ꢃ 150 mL of a solu-

tion of NaOH 1 M and with 1 ꢃ 150 mL of brine. The organic

phase was dried over anhydrous sodium sulfate and the organic

solvent evaporated. The crude product was puriﬁed by silica gel

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

11 was obtained as colorless oil (6.22 g/58%).

4.2.3. tert-Butyl((7-methoxy-2,2-dimethyl-3,4-

dihydrobenzopyran-8-yl)oxy)dimethylsilane (8)

Compound

7

(500 mg/2.4 mmol), imidazole (408.5 mg/

6 mmol), and TBDMSCl (435 mg/2.9 mmol) were added to

a

round-bottom ﬂask of 100 mL. The mixture was placed under

nitrogen atmosphere and 5 mL of DMF anhydrous was added.

The solution was allowed to react at 35 °C for 5 h. The solution

was allowed to cool to room temperature and poured over

100 mL of distilled water and extracted with 3 ꢃ 50 mL of diethyl

ether. The organic phase was washed with 2 ꢃ 50 mL of brine,

dried over anhydrous sodium sulfate, ﬁltered and the solvent evap-

orated. The crude product was puriﬁed by silica gel ﬂash chroma-

tography (n-hexane/ethyl acetate 95:5) and compound 8 was

isolated as colorless oil in quantitative yield.

tert-Butyl((7-methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-8-

yl)oxy)dimethylsilane (8). IR m_max(cm^ꢀ1) (KBr): 2965, 2922, 2890,

2847, 1493, 1454, 1289, 1242, 1148, 1097, 1051, 865, 826, 773. ¹H

NMR (300.13 MHz, CDCl₃): 6.60 (d, J = 8.4), 6.41 (d, J = 8.4), 3.76 (s,

OCH₃), 2.72 (t, J = 6.7, 2H), 1.76 (t, J = 6.7, 2H), 1.34 (s, 6H), 1.04 (s,

9H), 0.16 (s, 6H). ¹³C NMR (75.47 MHz, CDCl₃) d (ppm): 150.0,

146.2, 133.7, 120.5, 114.6, 103.9, 74.1, 55.9, 32.9, 29.9, 25.9, 22.1,

18.7. EIMS m/z (%): 322 (3, [M]⁺), 265 (10), 251 (2), 210 (17), 109

(100), 194 (16), 179 (2), 166 (7), 151 (3), 91 (2), 89 (3), 75 (6), 73

(3), 59 (2).

2,4-Dimethoxy-1-((2-methylbut-3-yn-2-yl)oxy)benzene (11).

IR m_max(cm^ꢀ1) (KBr): 3298, 3266, 2987, 2936, 2834, 2338, 2323,

1590, 1475, 1090, 740, 653. ¹H NMR (300.13 MHz, CDCl₃) d

(ppm): 7.13 (dd, J = 8.3, 1.4), 6.98 (t, J = 8.2), 6.67 (d, J = 8.3, 1.4),

3.86 (s, OCH₃), 3.85 (s, OCH₃), 2.55 (s, 1H), 1.68 (s, 6H). EIMS m/z

(%): 221 (8, [M+1]⁺), 220 (19, [M]⁺), 189 (15), 154 (100), 139

(50), 95 (21), 93 (32).

4.3.2. 7,8-Dimethoxy-2,2-dimethyl-2H-benzopyran (12)

Compound 11 (6.19 g/28.1 mmol) in anhydrous DMF (20 mL)

was placed in a closed vessel. The mixture was heated 145 °C for

4 h. After cooling to room temperature, the mixture was poured

into 150 g of crushed ice and extracted with 3 ꢃ 150 mL of diethyl

ether. The organic phase was washed with 2 ꢃ 150 mL of brine,

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

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

4.2.4. ((6-Bromo-7-methoxy-2,2-dimethyl-3,4-dihydro-8-

yl)oxy)(tert-butyl)dimethylsilane (9)

Compound 8 (8.9 g/27.6 mmol), NH₄OAc (212 mg/2.76 mmol)

and 200 mL of CH₃CN were placed in a two-necked round-bottom

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

2949

chromatography (n-hexane/ethyl acetate 95:5). Compound 12 was

obtained as colorless oil (4.11 g, 66%).

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 = 8.6), 6.88 (d, J = 10.0), 6.44 (d, J = 8.6), 5.61 (d, J = 10.0), 1.46 (s,

6H). EIMS m/z (%): 205 (5, [M+1]⁺), 204 (10 [M]⁺), 190 (15), 189

(100), 187 (60), 159 (12), 131 (12), 103 (10), 77 (12), 51 (6). HRMS

(ESI) m/z calcd for C₁₂H₁₃O₃[M+H]⁺: 205.08570, found: 205.08592.

7,8-Dimethoxy-2,2-dimethyl-2H-benzopyran (12). IR m_max

(cm^ꢀ1) (KBr): 2973, 2934, 2933, 1605, 1496, 1455, 1098, 797,

646. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 6.69 (d, J = 8.4), 6.42

(d, J = 8.4), 6.28 (d, J = 9.8), 5.52 (d, J = 9.8), 3.87 (s, OCH₃), 3.85 (s,

OCH₃), 1.48 (s, 6H). ¹³C NMR (75.47 MHz, CDCl₃): 153.5, 146.5,

137.5, 128.5, 122.1, 120.8, 116.3, 103.8, 76.4, 60.9, 56.0, 27.9. EIMS

m/z (%): 221 (6, [M+1]⁺), 220 (19, [M]⁺), 205 (100), 190 (21), 161

(38), 144 (19), 91 (11).

4.4.2. 6-Formyl-5-methoxy-2,2-dimethyl-2H-benzopyran (17)

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

bottom ﬂask and under nitrogen atmosphere. Then, compound 16

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

was added by syringe. Dimethyl sulfate (1.667/13.2 mmol) was

added dropwise and the mixture was allowed to stir 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 evaporated. The aqueous phase was acidiﬁed with

HCl 1 N until pH 2–3 and extracted with 3 ꢃ 100 mL of ethyl ace-

tate. The organic phase was washed with 2 ꢃ 50 mL of brine, dried

over sodium sulfate anhydrous, ﬁltered and the organic solvent

evaporated. Compound 17 was obtained in quantitative yield as

yellow oil.

6-Formyl-5-methoxy-2,2-dimethyl-2H-benzopyran (17). IR

m_max(cm^ꢀ1) (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 = 8.6),

6.65 (d, J = 8.6), 6.60 (dd, J = 9, 9, 0.6), 5.70 (d, J = 9.9), 3.90 (s,

OCH₃), 1.47 (s, 6H). EIMS m/z (%): 219 (5, [M+1]⁺), 218 (10, [M]⁺),

204 (18), 205 (100), 174 (10), 160 (60), 133 (16), 105 (5), 91 (8),

78 (10), 51 (6). HRMS (ESI) m/z calcd for C₁₃H₁₅O₃[M+H]⁺:

219.10177; found: 205.10157.

4.3.3. 7,8-Dimethoxy-2,2-dimethyl-3,4-dihydropyran (13)

Compound 12 (2.65 g/12 mmol) and 2 mL of anhydrous metha-

nol in nitrogen atmosphere were placed in a two-necked round-

bottom ﬂask. To the solution was added Pd/C 10% (15% weight/

395 mg) in one portion. Triethylsilane (19.2 mL, 120 mmol) was

then added dropwise. The mixture was allowed to stir for

15 min. The product was ﬁltered over celite and washed 3 times

with methanol. The methanol was evaporated and the crude prod-

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

ethyl acetate 9:1). Compound 13 was isolated as yellowish oil

(2.65 g/99%).

7,8-Dimethoxy-2,2-dimethyl-3,4-dihydropyran (13). IR m_max

(cm^ꢀ1) (KBr): 2975, 2932, 2833, 1609, 1496, 1457, 1293, 1153,

1100, 1058, 955, 788. ¹H NMR (400.21 MHz, CDCl₃) d (ppm): 6.67

(dt, J = 8.3, 0.9), 6.37 (d, J = 8.3), 3.75 (s, 2ꢃOCH₃), 2.66 (td, J = 6.7,

0.9, 2H), 1.71 (t, J = 6.7, 2H), 1.30 (s, 6H). EIMS m/z (%): 222 (6,

[M]⁺), 167 (100), 151 (17), 106 (12).

4.3.4. 6-Bromo-7,8-dimethoxy-2,2-dimethyl-3,4-

dihydrobenzopyran (14)

Compound 13 (2.6 g/11.7 mmol), NBS (2.19 g/12.28 mmol),

NH₄OAc (90 mg/1.17 mmol) and 100 mL of CH₃CN were placed in

a two-necked round-bottom ﬂask. The mixture was allowed to stir

for 20 min at room temperature and then the CH₃CN was evapo-

rated. The crude product was partitioned between water and ethyl

acetate. The aqueous phase was then extracted 2 ꢃ 250 mL of ethyl

acetate. The organic phase was washed 2 ꢃ 250 mL with brine,

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

vent evaporated. The crude product was puriﬁed by silica gel col-

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

14 was isolated as colorless oil (3.4 g/96%).

6-Bromo-7,8-dimethoxy-2,2-dimethyl-3,4-dihydrobenzopyran

(14). IR m_max(cm^ꢀ1) (KBr): 2975, 2933, 1461, 1419, 1339, 1205,

1097, 1050, 851. ¹H NMR (400.21 MHz, CDCl₃) d (ppm): 7.01 (t,

J = 1.0), 3.89 (s, OCH₃), 3.87 (s, OCH₃), 2.74 (td, J = 6.7, 1.0, 2H),

1.80 (t, J = 6.7, 2H), 1.39 (s, 6H). EIMS m/z (%): 302 (50, [M+2]⁺),

300 (50, [M]⁺), 247 (95), 245 (100), 231 (17), 229 (19).

4.4.3. 6-Hydroxy-5-methoxy-2,2-dimethyl-2H-benzopyran (18)

Compound 17 (1.0139 g/4.64 mmol) was solubilized in 20 mL of

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

Then, KHSO₄(94.7 mg/0.7 mmol) was added quickly and the solu-

tion 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 ꢃ 20 mL of dichloromethane. The organic phase was washed

with 2 ꢃ 50 mL of brine, dried over sodium sulfate anhydrous, ﬁl-

tered and the organic solvent evaporated. The crude product was

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

9:1). Compound 18 was isolated as orange oil (898 mg/4.36 mmol/

94%).

6-Hydroxy-5-methoxy-2,2-dimethyl-2H-benzopyran (18). IR

m_max(cm^ꢀ1) (KBr): 3422, 3045, 2973, 2934, 2836, 1728, 1634,

1584, 1470, 1437, 1373, 1295, 1260, 1215, 1152, 1114, 960, 811,

731. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 6.72 (d, J = 8.6), 6.55

(d, J = 10), 6.50 (d, J = 8.6), 5.69 (d, J = 9.9), 5.15 (OH), 3.82 (s,

OCH₃), 1.41 (s, 6H). EIMS m/z (%): 208 (5, [M+2]⁺), 207 (15,

[M+1]⁺), 206 (60, [M]⁺), 191 (100), 177 (50), 176 (30), 148 (18),

91 (10), 77 (5). HRMS (ESI) m/z calcd for C₁₂H₁₅O₃[M+H]⁺:

207.10139; found: 207.10157.

4.4. Synthesis of benxopyran 19

4.4.1. 6-Formyl-5-hydroxy-2,2-dimethyl-2H-benzopyran (16)

2,4-Dihydroxybenzaldehyde (15) (3 g/20.17 mmol), calcium

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

place in a two-necked round-bottom ﬂask. Then, prenal (9.135 g/

108.6 mmol) was added dropwise. The mixture was allowed to stir

at room temperature for 48 h. The reaction was quenched with HCl

1 M until pH 1–2. The methanol was evaporated and the aqueous

phase was extracted with 3 ꢃ 150 mL of ethyl acetate. The organic

phase was washed with 2 ꢃ 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 16 was crystallized from

n-hexane/ethyl acetate 3:1 as a yellow solid (2.06 g/50%).

4.4.4. 5,6-Dimethoxy-2,2-dimethyl-2H-benzopyran (19)

Potassium carbonate (10.6 g/76.8 mmol) was placed in a round-

bottom ﬂask and under nitrogen atmosphere. Then, compound 18

(8 g/38.4 mmol) was solubilized in 100 mL of anhydrous acetone

and added to the two necked round-bottom ﬂask. Dimethyl sulfate

(7.26 g/57.6 mmol) was added dropwise and the mixture was al-

lowed to stir for 20 h at reﬂux and under nitrogen atmosphere.

The reaction was quenched by the addition of 50 mL of distilled

water and the acetone evaporated. The aqueous phase was acidi-

ﬁed with HCl 1 N until pH 2–3 and extracted with 3 ꢃ 200 mL of

ethyl ether. The organic phase was washed with 2 ꢃ 100 mL of

brine, dried over sodium sulfate anhydrous, ﬁltered and the organ-

6-Formyl-5-hydroxy-2,2-dimethyl-2H-benzopyran (16). Mp:

68–69 °C. IR m_max(cm^ꢀ1) (KBr): 3464, 2967, 2922, 2857, 1628,

2950

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

ic solvent evaporated. The crude product was puriﬁed by silica gel

ﬂash chromatography (n-hexane/ethyl acetate 95:5) obtaining

compound 19 as light yellow oil (7.73 g/92%).

round bottom ﬂask and washed with anhydrous n-hexane. The

ﬂask was then placed under nitrogen atmosphere and on an ice-

bath. Methyl salicylate (25) (6.88 g/45.26 mmol) was solubilized

in 60 mL of anhydrous THF and added slowly to the ﬂask contain-

ing the sodium hydride. The mixture was allowed to stir for 15 min

and MOMCl (7.288 g/90.53 mmol) was added 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 ꢃ 150 mL of ethyl acetate. The organic

phase was washed with 2 ꢃ 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 27 was obtained as col-

ourless oil (8.1522 g/92%).

5,6-Dimethoxy-2,2-dimethyl-2H-benzopyran (19). IR m_max

(cm^ꢀ1) (KBr): 2974, 2932, 2831, 1576, 1474, 1215, 1063, 797. ¹H

NMR (300.13 MHz, CDCl₃) d (ppm): 6.69 (d, J = 8.8), 6.65 (d,

J = 9.9), 6.51 (d, J = 8.8), 5.66 (d, J = 9.9), 3.84 (s, OCH₃), 3.82 (s,

OCH₃), 1.41 (s, 6H). ¹³C NMR (75.47 MHz, CDCl₃): 147.0, 146.7,

131.4, 131.3, 117.1, 112.7, 112.6, 111.0, 75.4, 61.3, 55.5, 27.5. EIMS

m/z (%): 221 (22, [M+1]⁺), 220 (65, [M]⁺), 205 (100), 190 (34), 161

(34), 144 (9), 91 (14), 77 (5).

4.5. Protection of substituted salicylaldehydes with benzyl

group

Methyl 2-(methoxymethoxy)benzoate (27). IR: m_max(cm^ꢀ1

)

In a typical experiment: 5-chlorosalicylaldehyde (20) (1.0 g/

6.4 mmol) was placed in a two-necked round-bottom ﬂask and

then it was added Cs₂CO₃(3.13 g/9.6 mmol), benzyl bromide

(1.64 g/9.6 mmol) and 50 mL of acetone. The mixture was allowed

to stir at room temperature overnight. The product was then ﬁl-

tered and washed with acetone. The solvent was evaporated and

the crude product puriﬁed by column chromatography (silica gel,

n-hexane/ethyl acetate 95:5). Compound 24a was obtained as

white solid (1.42 g/90%).

(KBr): 2991, 2947, 2903, 2825, 1725, 1596, 1485, 1449, 1432,

1296, 1249, 1150, 1128, 1073, 984, 755. ¹H NMR (300.13 MHz,

CDCl₃) d (ppm): 7.78 (dd, J = 7.8, 1.8), 7.44 (ddd, J = 8.4, 7.5, 1.8),

7.20 (dd, J = 8.4, 0.9), 7.05 (ddd, J = 7.8, 7.5, 0.9), 5.26 (s, 2H), 3.89

(s, OCH₃), 3.52 (s, OCH₃). EIMS m/z (%): 197 (16, [M]⁺), 165 (100),

135 (26), 92 (45), 63 (34).

5-Chloro-2-(methoxymethoxy)benzaldehyde (28a) as a color-

less oil (88%). IR m_max(cm^ꢀ1) (KBr): 2922, 2852, 1667, 1628, 1472

1127, 866. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 10.43 (s, CHO),

7.80 (d, J = 2.8), 7.47 (dd, J = 9.0, 2.8), 7.20 (d, J = 9.0), 5.29 (s, 2H),

3.52 (s, OCH₃). EIMS m/z (%): 202 (10, [M+2]⁺), 200 (30, [M]⁺),

170 (16), 169 (30), 155 (35), 99 (16), 77 (30), 75 (82), 63 (100).

5-Methoxy-2-(methoxymethoxy)benzaldehyde (28b) as a col-

orless oil (78%). IR m_max(cm^ꢀ1) (KBr): 2927, 2879 1666, 1627,

1490. 813. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 10.47 (s, CHO),

7.33 (d, J = 3.2), 7.18 (dd, J = 9.1), 7.11 (dd, J = 9.0, 3.2), 5.24 (s,

2H), 3.81 (s, OCH₃), 3.52 (s, OCH₃). EIMS m/z (%): 197 (18,

[M+1]⁺), 196 (100, [M]⁺), 165 (52), 150 (98), 120 (25), 95 (36), 65

(38), 63 (58).

2-(Methoxymethoxy)-5-methylbenzaldehyde (28c) colorless oil

(88%). IR m_max(cm^ꢀ1) (KBr): 2925, 2853, 2790, 2665, 1627, 1450,

1386, 666. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 10.48 (s, CHO),

7.64 (d, J = 2.5), 7.34 (dd, J = 8.5, 2.5), 7.12 (d, J = 8.5), 5.27 (s, 2H),

3.52 (s, OCH₃), 2.32 (s, CH₃). EIMS m/z (%): 181 (10, [M+1]⁺), 180

(34, [M]⁺), 151 (20), 149 (45), 135 (100), 91 (44), 77 (55), 65

(25), 51 (56).

2,4-Bis(methoxymethoxy)benzaldehyde (28e) as white solid

(56%). Mp: 44–45 °C. IR m_max(cm^ꢀ1) (KBr): 3003, 2989, 2959,

2904, 1679, 1601, 1255, 1155, 999, 921. ¹H NMR (300.13 MHz,

CDCl₃) d (ppm): 10.34 (s, CHO), 7.80 (d, J = 8.7), 6.83 (d, J = 2.2),

6.74 (dd, J = 8.7, 2.2), 5.28 (s, 2H), 5.22 (s, 2H), 3.53 (s, OCH₃),

3.49 (s, OCH₃). EIMS m/z (%): 227 (10, [M+1]⁺), 226 (15, [M]⁺),

181 (28), 165 (35), 151 (22), 136 (40), 77 (23), 63 (100).

4-(Diethylamino)-2-(methoxymethoxy)benzaldehyde (28g) as

yellow oil (71%). IR m_max(cm^ꢀ1) (KBr): 2916, 2770, 1667, 1598,

1267, 1267, 990, 798. ¹H NMR (300.13 MHz, CDCl₃) d (ppm):

10.17 (s, CHO), 7.74 (d, J = 9.4), 6.39–6.37 (m, 2H), 5.27 (s, 2H),

3.52 (s, OCH₃), 3.45–3.38 (m, 4H), 1.21 (t, J = 7.1, 6H). EIMS m/z

(%): 238 (10, [M+1]⁺), 237 (25, [M]⁺), 222 (56), 192 (20), 178

(45), 176 (56), 162 (100), 148 (24), 134 (30), 122 (10), 77 (20),

65 (20).

2-(Benzyloxy)-5-chlorobenzaldehyde (24a) as white solid (90%).

Mp: 77–78 °C. IR m

_max(cm^ꢀ1) (KBr): 2867, 2757, 1678, 1590, 1451,

1383, 1266, 1174, 1123, 1019, 819, 733, 676. ¹H NMR

(300.13 MHz, CDCl₃) d (ppm): 10.44 (s, CHO), 7.75 (d, J = 2.8), 7.43–

7.3 (6H, m), 6.97 (d, J = 8.7), 5.13 (s, 2H). EIMS m/z (%): 248 (0.4,

[M+2]⁺), 246 (1.5, [M]⁺), 155 (27), 92 (11), 91 (100), 65 (34), 63 (16).

2-(Benzyloxy)-5-methoxybenzaldehyde (24b) as a white solid

in quantitative yield. Mp: 45–47 °C. IR m_max(cm^ꢀ1) (KBr): 2915,

2849, 1669, 1481, 1443, 1410, 1372, 1260, 1197, 1143, 1025,

1006, 719, 677, 540. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 10.50

(s, CHO), 7.42–7.33 (m, 5H), 7.31 (d, J = 3.1), 7.09 (dd, J = 9.1, 3.1),

6.98 (d, J = 9.1), 5.11 (s, 2H), 3.76 (s, OCH₃). EIMS m/z (%): 242

(0.8, [M]⁺), 214 (2), 213 (2), 151 (2), 150 (2), 95 (2), 92 (10), 91

(100), 89 (2), 77 (1), 65 (28), 53 (3), 52 (3), 51 (40).

2-(Benzyloxy)-5-methylsalicylaldehyde (24c). Mp: 55–56 °C. IR

m

_max(cm^ꢀ1) (KBr): 2919, 2864, 2760, 1677, 1606, 1490, 1453, 1381,

1280, 1244, 1154, 1110, 1025, 817, 733, 721, 686, 647. ¹H NMR

(300.13 MHz, CDCl₃) d (ppm): 10.52 (s, CHO), 7.67 (d, J = 2.7),

7.43–7.28 (m, 6H), 6.92 (d, J = 8.5), 5.12 (s, 2H), 2.28 (s, 3H). EIMS

m/z (%): 226 (2.5, [M]⁺), 198 (4), 197 (7), 135 (9), 92 (8), 91

(100), 65 (14), 51 (4).

2-(Benzyloxy)-3-methoxybenzaldehyde (24d). IR m_max(cm^ꢀ1

)

(KBr): 3086, 3060, 2959, 2938, 1896, 1874, 2837, 1690, 1582,

1479, 1454, 1439, 1367, 1309, 1268, 1248, 1217, 1184, 100,

1060, 963, 909, 780, 752, 696. ¹H NMR (300.13 MHz, CDCl₃) d

(ppm): 10.23 (s, CHO), 7.40–7.32 (6H, m), 7.20–710 (2H, m), 5.18

(s, 2H), 3.94 (s, OCH₃). EIMS m/z (%): 242 (3, [M]⁺), 214 (8), 213

(25), 181 (5), 151 (8), 122 (6), 92 (11), 91 (100), 77 (4), 65 (39),

63 (10), 51 (16).

2,4-Bis(benzyloxy)benzaldehyde (24e) as a light yellow solid in

quantitative yield. Mp: 75–77 °C. IR m

_max(cm^ꢀ1) (KBr): 2949, 2925,

2846, 1664, 1601, 1452, 1381, 1324, 1178, 1094, 1008, 827, 721,

691. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 10.38 (s, CHO), 7.84

(d, J = 8.6), 7.42–7.35 (10H, m), 6.63 (dd, J = 8.6, 2.2), 6.59 (d,

J = 2.2), 5.13 (s, 2H), 5.10 (s, 2H). EIMS m/z (%): 318 (0.5, [M]⁺),

298 (0.7), 199 (6), 181 (3), 92 (9), 91 (100), 65 (29), 63 (6), 51 (3).

4.7. Synthesis of compound 4

4.7.1. (8-((tert-Butyldimethylsilyl)oxy)-7-methoxy-2,2-

dimethyl-3,4-dihydrobenzopyran-6-yl)(2-(methoxymethoxy)

phenyl)methanone (29)

Compound 9 (1.5 g/3.74 mmol) was placed in an oven-dried

three-necked round-bottom ﬂask and solubilized in 50 mL of anhy-

drous THF. The mixture was then placed at ꢀ78 °C and nBuLi

(1.6 M, 5.6 mmol) was added dropwise. The mixture was allowed

4.6. Protection of methyl salicylate and substituted

salicylaldehydes with MOM group

In a typical experiment: sodium hydride (3.63 g/90.53 mmol of

60% sodium hydride in mineral oil) was placed in two-necked

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

2951

to react for 15 min at ꢀ78 °C. Then compound 27 (1.5 g/7.5 mmol)

4.7.3. 12-Methoxy-2,2-dimethyl-3,4-dihydropyrano[3,2-

b]xanthen-6(2H)-one (32) and 12-hydroxy-2,2-dimethyl-3,4-

dihydropyrano[3,2-b]xanthen-6(2H)-one (4)

solubilized in anhydrous THF at ꢀ78 °C was added dropwise and

allowed to react for 15 min at ꢀ78 °C and slowly warm to room

temperature. The reaction was quenched by the addition of a sat-

urated solution of NH₄Cl, extracted with 3 ꢃ 100 mL of ethyl ace-

tate. The organic phase was washed 2 ꢃ 100 mL of brine, dried

over sodium sulfate anhydrous, ﬁltered and the organic solvent

evaporated. The crude product was puriﬁed by silica gel ﬂash chro-

matography (n-hexane/ethyl acetate 8:2).Compound 29 was ob-

tained as a white solid (1.25 g/68%).

Cyclization of benzophenone 30 and 31.

In a typical experiment: to a solution of compound 30 (340 mg,

1.2 mmol) in DMF (5 mL) was added Cs₂CO₃(375 mg, 1.15 mmol).

The mixture was irradiated with microwaves for 15 min at 140 °C.

The mixture was allowed to cool to room temperature and poured

into crushed ice. The aqueous phase was then extracted with

3 ꢃ 100 mL of diethyl ether. The organic phase was washed with

2 ꢃ 30 mL of brine, dried over sodium sulfate anhydrous, ﬁltered

and the organic solvent evaporated. The crude product was puri-

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

7:3) and compound 4 was obtained as light yellow solid (83 mg,

36%) and compound 32 as a white solid (99 mg/32%).

(8-((tert-Butyldimethylsilyl)oxy)-7-methoxy-2,2-dimethyl-3,4-

dihydrobenzopyran-6-yl)(2-(methoxymethoxy)phenyl)methanone

(29). Mp: 83–84 °C. IR m_max(cm^ꢀ1) (KBr): 2963, 2925, 2852, 1662,

1595, 1465, 1349, 1252, 1232, 1201, 1150, 1116, 1099, 1074,

994, 834, 770. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 7.40 (dd,

J = 8.0, 1.7), 7.38 (ddd, J = 8.8, 7.5, 1.7), 7.14 (dd, J = 0.8, 8.8), 7.04

(ddd, J = 8.0, 7.5, 0.8), 7.00 (s), 5.05 (s, 2H), 3.51 (s, OCH₃), 3.33 (s,

OCH₃), 2.72 (t, J = 6.6, 2H), 1.78 (t, J = 6.6, 2H), 1.38 (s, 6H), 1.03

(s, 9H), 0.17 (s, 6H). ¹³C NMR (75.47 MHz, CDCl₃) d (ppm): 195.0,

155.1, 150.4, 150.4, 137.8, 131.4, 129.3, 125.5, 123.8, 121.6,

121.3, 116.6, 114.9, 95.0, 75.6, 60.8, 55.9, 32.7, 26.9, 25.8, 22.1,

18.6. EIMS m/z (%): 486 (1, [M]⁺), 383 (9), 309 (17), 298 (10), 297

(34), 277 (12), 264 (21), 263 (100), 253 (15), 245 (12), 223 (16),

165 (20), 121 (18), 75 (23), 73 (10).

Cyclization of benzophenone 31 (396 mg, 1.2 mmol) led to com-

pound 4 as a light yellow solid (330 mg/92%).

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

6(2H)-one (32) Mp: 173ꢀ174 °C. IR

m

_max(cm^ꢀ1) (KBr): 2967, 2936,

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

(300.13 MHz, CDCl₃) d (ppm): 8.32 (dd, J = 7.9, 1.6); 7.86 (t,

J = 0.9), 7.70 (ddd, J = 8.4, 7.0. 1.6), 7.57 (dd, J = 8.4, 1.1), 7.36 (ddd,

J = 7.9, 7.0, 1.1), 4.00 (s, OCH₃), 2.64 (td, J = 6.8, 0.9, 2H), 1.90 (t,

J = 6.8, 2H), 1.46 (s, 6H). ¹³C NMR (75.47 MHz, CDCl₃) d (ppm):

176.6, 156.2, 153.2, 149.1, 135.7, 134.2, 126.5, 123.6, 121.5,

121.2, 119.3, 118.0, 115.2, 76.4, 61.2, 32.3, 27.0, 22.1. HRMS (ESI)

m/z calcd for C₁₉H₁₉O₄[M+H]⁺: 311,12779; found: 311,12772.

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

6(2H)-one (4) The spectroscopic data was in accordance with the

4.7.2. (8-((tert-Butyldimethylsilyl)oxy)-7-methoxy-2,2-

dimethyl-3,4-dihydrobenzopyran-6-yl)(2-hydroxyphenyl)

methanone (30) and (8-hydroxy-7-methoxy-2,2-dimethyl-3,4-

dihydrobenzopyran-6-yl)(2-hydroxyphenyl)methanone (31)

To a solution of compound 29 (1.12 g, 2.3 mmol) in 30 mL of

CH₃CN at 0 °C was added in one portion NbCl₅(684 mg,

2.53 mmol). The mixture was stirred at 0 °C for 10 min and then al-

lowed to warm to room temperature and let stirring for more

45 min. The reaction was quenched by the addition of a saturated

solution of NaHCO₃. The mixture was extracted with 3 ꢃ 100 mL of

ethyl acetate. The organic phase was washed with 2 ꢃ 100 mL of

brine, dried over sodium sulfate anhydrous, ﬁltered and the organ-

ic solvent evaporated. The crude product was puriﬁed by silica gel

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

30 (390 mg/33%) was isolated as colorless oil and compound 31

(498 mg/67%) as a white solid

literature¹⁷

.

4.8. Synthesis of analogues 36a–e

4.8.1. Synthesis of diarylmethanols 33e–f

In a typical experiment: Compound 9 (1.5 g/3.74 mmol) was

placed in an oven-dried three-necked round-bottom ﬂask and sol-

ubilized in 50 mL of anhydrous THF. The mixture was then placed

at ꢀ78 °C and nBuLi (1.6 M, 5.6 mmol) was added dropwise. The

mixture was allowed to react for 15 min at ꢀ78 °C. Then the 2-ben-

zyloxy-5-chlorosalicylaldehyde (24a) (1.38 g/5.6 mmol) solubi-

lized in anhydrous THF was added dropwise and allowed to react

for 15 min at ꢀ78 °C and slowly warm to room temperature. The

reaction was quenched by a saturated solution of NH₄Cl, extracted

with 3 ꢃ 100 mL of ethyl acetate. The organic phase was washed

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

and the organic solvent evaporated. The crude product was then

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

8:2) and compound 33e was obtained as light yellow oil (1.62 g/

76%).

(8-((tert-Butyldimethylsilyl)oxy)-7-methoxy-2,2-dimethyl-3,4-

dihydrobenzopyran-6-yl)(2-hydroxyphenyl)methanone (30). IR

m

(cm^ꢀ1) (KBr): 3443, 2970, 2925, 2851, 1622, 1460, 1354, 1236,

1203, 1147, 1114, 1071, 830, 753. ¹H NMR (300.13 MHz, CDCl₃) d

(ppm): 12.27 (OH), 7.48–7.43 (m, 2H), 7.02 (dd, J = 8.8, 1.2), 6.81

(dd, J = 8.2, 1.1), 6.69 (s), 3.69 (s, OCH₃), 2.75 (t, J = 6.7, 2H), 1.80 (t,

J = 6.7, 2H), 1.40 (s, 6H), 1.04 (s, 9H), 0.20 (s, 6H). EIMS m/z (%): 443

(1, [M]⁺), 385 (1), 330 (5), 329 (21), 298 (11), 265 (6), 210 (7), 209

(45), 195 (7), 194 (7), 122 (9), 121 (100), 89 (4), 75 (17), 65 (6), 59 (4).

(8-Hydroxy-7-methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-

6-yl)(2-hydroxyphenyl)methanone (31). Mp: 173–174 °C. IR m_max

(cm^ꢀ1) (KBr): 3352, 2965, 2928, 2896, 2927, 1616, 1450, 1357,

1334, 1263, 1232, 1200, 1150, 1112, 1087, 983, 853, 759, 732,

655. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 12.22 (OH), 7.52–

7.45 (m, 2H), 7.02 (dd, J = 8.5, 1.8), 6.84 (ddd, J = 8.1, 7.0,1.1), 6.65

(t, J = 0.7), 5.62 (b, OH), 3.79 (s, OCH₃), 2.77 (td, J = 6.7, 0.7, 2H),

1.87 (t, J = 6.7, 2H), 1.43 (s, 6H). ¹³C NMR (75.47 MHz, CDCl₃) d

(ppm): 201.2, 162.8, 144.4, 143.0, 137.7, 136.3, 134.1, 123.9,

120.2, 119.9, 118.6, 117.9, 116.4, 76.3, 61.7, 32.6, 26.9, 21.7. EIMS

m/z (%): 329 (0.5, [M]⁺), 297 (14), 242 (19), 241 (99), 179 (78),

128 (21), 121 (54), 115 (24), 91 (26), 79 (27), 77 (51), 64 (100),

63 (40), 53 (31), 51 (54). HRMS (ESI) m/z calcd for C₁₉H₂₁O₅

[M+H]⁺: 329.13835; found: 329.13787; m/z calcd for C₁₉H₂₀NaO₅

[M+Na]⁺: 351.12029; found: 351.11901.

(2-(Benzyloxy)-5-chlorophenyl)(8-((tert-butyldimethylsilyl)oxy)-

7-methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-6-yl)methanol

(33e) as a yellow oil (1.62 g/76%). IR m

_max(cm^ꢀ1) (KBr): 3445, 2969,

2927, 2892, 2952, 1575, 1461, 1351, 1245, 1102, 1073, 1024, 1004,

944, 832, 780, 732, 694. ¹H NMR (300.13 MHz, CDCl₃) d (ppm):

7.43 (d, J = 8.7), 7.32–7.29 (m, 3H), 7.21–7.16 (m, 3H), 6.82 (d,

J = 8.7), 6.41 (s), 6.26 (s), 5.05 (d, J = 11.8, 1H), 4.99 (d, J = 11.8,

1H), 3.57 (s, OCH₃), 2.61 (t, J = 6.8, 2H), 1.73 (t, J = 6.8, 2H), 1.37

(s, 3H),1.33 (s, 3H), 1.04 (s, 9 H), 0.18 (s, 3H), 0.13 (s, 3H). EIMS

m/z (%): 198 (1), 196 (3), 167 (1), 141 (2), 92 (10), 91 (100), 89

(3), 77 (3), 75 (2), 65 (22), 63 (5), 51 (4).

(2-(Benzyloxy)-5-methoxyphenyl)(8-((tert-butyldimethylsilyl)

oxy)-7-methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-6-yl)meth-

anol (33b) as a colorless oil (1.9 g/98%). IR m

_max(cm^ꢀ1) (KBr): 3421,

2928, 2901, 2853, 1585, 1495, 1463, 1352, 1246, 1207, 1155, 1103,

1075, 1041, 834, 781, 734, 694. ¹H NMR (300.13 MHz, CDCl₃) d

2952

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

(ppm): 7.42–7.18 (m, 6H), 7.03 (d, J = 2.7), 6.85 (d, J = 8.7), 6.48 (s),

6.32 (s), 5.02 (d, J = 10.7, 1H), 4.93 (d, J = 11.3, 1H), 3.79 (s, OCH₃),

3.76 (s, OCH₃), 2.62 (t, J = 6.8, 2H), 1.73 (t, J = 6.8, 2H), 1.37 (s,

3H),1.35 (s, 3H), 1.05 (s, 9 H), 0.20 (s, 3H), 0.18 (s, 3H). EIMS m/z

(%): 299 (1.5), 283 (1.3), 214 (2), 193 (13), 192 (92), 191 (3), 177

(2), 163 (19), 138 (8), 137 (76), 136 (17), 121 (10), 110 (7), 107

(7), 92 (10), 91 (100), 71 (13), 65 (41), 53 (6), 51 (13).

(2-(Benzyloxy)-5-methylphenyl)(8-((tert-butyldimethylsilyl)oxy)-

7-methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-6-yl)methanol

(33c) as a colorless oil (1.91 g/93%). IR m_max(cm^ꢀ1) (KBr): 3439,

2969, 2926, 2894, 1606, 1495, 1462, 1351, 1244, 1209, 1102,

1074, 1024, 1004, 832, 779, 693. ¹H NMR (300.13 MHz, CDCl₃) d

(ppm): 7.34–7.25 (m, 4H), 7.23–7.20 (m, 2H), 7.02 (d, J = 2.1),

6.84 (d, J = 8.4), 6.52 (s), 6.34 (s), 5.07 (d, J = 11.7, 1H), 5.03 (d,

J = 11.7, 1H), 3.60 (s, OCH₃), 2.65 (t, J = 6.8, 2H), 2.31 (s, CH₃), 1.75

(t, J = 6.8, 2H), 1.39 (s, 3H),1.36 (s, 3H), 1.07 (s, 9 H), 0.22 (s, 3H),

0.20 (s, 3H). EIMS m/z (%): 199 (0.7), 176 (3), 175 (1), 147 (5),

135 (2), 121 (8), 119 (1), 115 (1), 92 (10), 91 (100), 79 (3), 65

(22), 51 (6).

(2-(Benzyloxy)-3-methoxyphenyl)(8-((tert-butyldimethylsilyl)

oxy)-7-methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-6-yl)meth-

anol (33d) as a yellow oil (1.9 g/90%). IR m_max(cm^ꢀ1) (KBr): 3443,

2967, 2928, 2894, 2851, 1578, 1469, 1351, 1261, 1205, 1102,

1077, 1025, 1003, 986, 832, 78, 745, 693. ¹H NMR (300.13 MHz,

CDCl₃) d (ppm): 7.39–7.17 (m, 6H), 7.06 (d, J = 8.0), 6.68 (d,

J = 8.4), 6.41 (s), 6.25 (b s), 5.01 (d, J = 10.7, 1H), 4.82 (d, J = 10.7,

1H), 3.89 (s, OCH₃), 3.49 (s, OCH₃), 2.91 (b d, J = 4.1, OH), 2.57 (t,

J = 6.8, 2H), 1.69 (t, J = 6.8, 2H), 1.33 (s, 3H),1.31 (s, 3H), 1.02 (s, 9

H), 0.16 (s, 3H), 0.14 (s, 3H). EIMS m/z (%): 283 (1.2), 214 (1.4),

193 (6), 192 (49), 163 (19), 150 (5), 145 (5), 137 (55), 131 (20),

124 (8), 121 (5), 103 (18), 92 (10), 91 (100), 79 (5), 78 (4), 77

(11), 65 (40), 51 (12).

(2-(Benzyloxy)-5-methoxyphenyl)(8-((tert-butyldimethylsilyl)

oxy)-7-methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-6-yl)meth-

anone (34b) as a white solid (1.14 g/60%). Mp: 69–70 °C. IR m_max

(cm^ꢀ1) (KBr): 2956, 2930, 2891, 2853, 1640, 1599, 1496, 1462,

1416, 1350, 1305, 1245, 1218, 1106, 1080, 1048, 996, 909, 835,

779, 733, 464. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 7.25–7.18

(m, 3H), 7.06–7.05 (m, 2H), 7.03 (d, J = 2.9), 7.01 (s), 6.93 (dd,

J = 8.9, 2.9), 6.87 (d, J = 8.9), 4.91 (s, 2H), 3.79 (s, OCH₃), 3.46 (s,

OCH₃), 2.72 (t, J = 6.7, 2H), 1.77 (t, J = 6.7, 2H), 1.36 (s, 6H), 0.99

(s, 9 H), 0.08 (s, 6H). ¹³C NMR (75.47 MHz, CDCl₃) d (ppm): 194.9,

153.5, 150.8, 150.4, 150.4, 137.7, 137.0, 132.7, 128.1, 127.4,

126.6, 125.8, 123.9, 117.2, 116.6, 114.3, 114.2, 75.5, 70.9, 60.9,

55.8, 32.7, 29.7, 27.0, 25.8, 22.1, 18.6. EIMS m/z (%): 448 (0.11),

431 (0.7), 399 (1), 355 (1), 339 (1), 327 (7), 300 (4), 299 (9), 277

(23), 263 (19), 255 (12), 241 (8), 225 (9), 219 (10), 209 (19), 207

(12), 151 (84), 121 (4), 105 (21), 92 (9), 91 (100), 75 (23), 73

(26), 65 (22), 59 (7), 51 (12).

(2-(Benzyloxy)-5-methylphenyl)(8-((tert-butyldimethylsilyl)

oxy)-7-methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-6-yl)meth-

anone (34c) as a white solid (1.14 g/64%). Mp: 110–111 °C. IR m_max

(cm^ꢀ1) (KBr): 2950, 2926, 2886, 2849, 1639, 1599, 1550, 1495,

1451, 1346, 1304, 1243, 1145, 1104, 1084, 1026, 992, 832, 799,

774, 731, 462. ¹H NMR (300,13 MHz, CDCl₃) d (ppm): 7.29 (d,

J = 2.2), 7.23–7.17 (m, 4H), 7.07–7.04 (m, 2H), 7.02 (s), 6.84 (d,

J = 8.4), 4.96 (s, 2H), 3.44 (s, OCH₃), 2.74 (t, J = 6.7, 2H), 2.31 (s,

CH₃), 1.79 (t, J = 6.7, 2H), 1.38 (s, 6H) 1.00 (s, 9 H), 0.09 (s, 6H).

¹³C NMR (75.47 MHz, CDCl₃) d (ppm): 195.5, 154.6, 150.2, 150.2,

137.7, 137.0, 132.1, 131.6, 130.1, 129.8, 128.1, 127.3, 126.5,

126.1, 123.7, 112.5, 75.4, 70.1, 60.8, 32.8, 27.9, 25.8, 22.1, 20.4,

18.6. EIMS m/z (%): 327 (7), 315 (2), 299 (20), 291 (4), 278 (6),

277 (26), 263 (25), 255 (13), 209 (9), 152 (10), 151 (100), 105

(22), 92 (9), 91 (92), 75 (14), 73 (15), 65 (18), 51 (6).

(2,4-Bis(benzyloxy)phenyl)(8-((tert-butyldimethylsilyl)oxy)-7-

methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-6-yl)methanol

(33e) as a light green oil (2.2 g).

(2-(Benzyloxy)-3-methoxyphenyl)(8-((tert-butyldimethylsilyl)

oxy)-7-methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-6-yl)meth-

anone (34d) as a colorless oil (1.37 g/71%). IR m_max(cm^ꢀ1) (KBr):

2972, 2929, 2892, 2854, 1658, 1594, 1578, 1562, 1469, 1349,

1312, 1259, 1210, 1116, 1064, 992, 907, 835, 780, 749, 695. ¹H

NMR (300.13 MHz, CDCl₃) d (ppm): 7.25–7.23 (m, 5H), 7.11 (dd,

J = 8.2, 7.3), 7.03 (dd, J = 8.2, 1.9), 7.00 (dd, J = 7.3,1.9), 6.92 (s),

4,93 (s, 2H), 3.89 (s, OCH₃), 3.54 (s, OCH₃), 2.67 (t, J = 6.6, 2H),

1.76 (t, J = 6.6, 2H), 1.37 (s, 6H), 1.01 (s, 9 H), 0.13 (s, 6H). ¹³C

NMR (75.47 MHz, CDCl₃) d (ppm): 195.0, 152.8, 150.6, 150.6,

145.9, 137.6, 128.1, 128.0, 127.5, 127.0, 125.2, 124.7, 123.7,

120.9, 116.5, 114.3, 75.6, 75.5, 60.8, 56.1, 32.7, 27.0, 25.8, 22.1,

18.6. EIMS m/z (%):355 (1), 339 (2), 329 (1), 327 (1), 312 (5), 311

(14), 299 (16), 277 (16), 263 (21), 239 (7), 225 (10), 203 (16),

136 (9), 135 (100), 105 (30), 92 (8), 91 (90), 77 (5), 75 (11), 65

(15), 51(3).

(2,4-Bis(benzyloxy)phenyl)(8-((tert-butyldimethylsilyl)oxy)-7-

methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-6-yl)methanone

(34e) as a colorless oil (1.4 g). IR m_max(cm^ꢀ1) (KBr): 2966, 2923,

2883, 2849, 1638, 1595, 1567, 1494, 1459, 1419, 1347, 1305,

1250, 1202, 1171, 1112, 1076, 1021, 860, 829. ¹H NMR

(300.13 MHz, CDCl₃) d (ppm): 7.53 (d, J = 8.5), 7.45–7.34 (m, 5H),

7.23–7.18 (m, 3H), 7.04–7.01 (m, 2H), 6.96 (s), 6.60 (dd, J = 8.5,

2.2), 6.56 (d, J = 8.5), 5.09 (s, 2H), 4.94 (s, 2H), 3.44 (s, OCH₃), 2.73

(t, J = 6.6, 2H), 1.77 (t, J = 6.6, 2H), 1.36 (s, 6H), 0.98 (s, 9 H), 0.06

(s, 6H). ¹³C NMR (75.47 MHz, CDCl₃) d (ppm): 194.2, 162.3, 158.9,

149.8, 149.7, 136.6, 136.4, 132.1, 128.7, 128.2, 127.7, 127.4,

127.0, 126.5, 124.7, 123.1, 116.6, 105.5, 100.4, 75.3, 70.2, 69.9,

61.0, 32.8, 29.7, 27.0, 25.8, 22.8, 18.6. EIMS m/z (%): 355 (2), 327

(11), 299 (20), 271 (5), 267 (7), 263 (19), 253 (9), 241 (13), 227

(10), 225 (20), 219 (12), 211 (16), 209 (11), 207 (29), 193 (11),

151 (69), 105 (20), 92 (9), 91 (100), 75 (16), 73 (31), 65 (15), 51

(14).

4.8.2. Synthesis of benzophenones 34a–e

In a typical experiment: Compound 33a (1.5 g/2.6 mmol) and

20 mL of DMSO were placed in a round-bottom ﬂask. Then it was

added IBX (1.1 g/3.9 mmol). After 24 h the reaction was quenched

by the addition of a 10% solution of Na₂S₂O₃, a saturated solution of

NaHCO₃and distilled water. The aqueous phase was then extracted

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

with a 2 ꢃ 100 mL solution of Na₂S₂O₃10%, 2 ꢃ 100 mL of a satu-

rated solution of NaHCO₃, dried over sodium sulfate anhydrous, ﬁl-

tered and the organic solvent evaporated. The crude product was

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

95:5) and compound 34a was recrystallized from ethyl acetate/n-

hexane as white solid (1.04 g/70%).

(2-(Benzyloxy)-5-chlorophenyl)(8-((tert-butyldimethylsilyl)

oxy)-7-methoxy-2,2-dimethyl-3,4-dihydrobenzopyran-6-yl)meth-

anone (34a) as a white solid (1.04 g/70%). Mp: 123–125 °C. IR m_max

(cm^ꢀ1) (KBr): 2948, 2925, 2880, 2877, 1637, 1590, 1544, 1453,

1342, 1301, 1239, 1206, 1137, 1101, 1077, 1016, 989, 963, 872,

825, 772, 728, 688, 458. ¹H NMR (300.13 MHz, CDCl₃) d (ppm):

7.42 (d, J = 2.6), 7.33 (dd, J = 8.8, 2.6), 7.24–7.20 (m, 3H), 7.07 (s),

7.07–7.04 (m, 2H), 6.88 (d, J = 8.8), 4.98 (s, 2H), 3.42 (s, OCH₃),

2.76 (t, J = 6.7, 2H), 1.80 (t, J = 6.7, 2H), 1.38 (s, 6H) 1.00 (s, 9 H),

0.08 (s, 6H). ¹³C NMR (75.47 MHz, CDCl₃) d (ppm): 193.5, 155.1,

150.9, 150.5, 137.7, 136.3, 133.4, 130.9, 129.0, 128.2, 127.6,

126.5, 125.6, 125.1, 123.8, 116.9, 113.7, 75.6, 70.3, 60.8, 32.7,

27.0, 25.7, 22.1, 18.6. EIMS m/z (%): 355 (1), 333 (2), 331 (4), 300

(4), 299 (19), 263 (7), 245 (3), 225 (4), 223 (4), 209 (6), 207 (4),

155 (12), 105 (33), 92 (9), 91 (100), 77 (6), 75 (14), 73 (19), 65

(17), 59 (4), 51 (7).

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

2953

4.8.3. Synthesis of benzophenones 34a–e and compound 30

In a typical experiment: Compound 34e (750 mg/1.32 mmol)

and 40 mL of anhydrous methanol/THF 3:1 were placed in a two-

necked round-bottom ﬂask and under nitrogen atmosphere. To

the solution was added Pd/C 10% (15% weight/112.5 mg) in one

portion. Triethylsilane (2.1 mL, 10.32 mmol) was then added drop-

wise. The mixture was allowed to stir for 15 min. The product was

ﬁltered over celite and washed 3 times with methanol. The meth-

anol was evaporated and the crude product was puriﬁed by silica

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

ture of compound 35a and 30 was isolated as yellowish oil

(564 mg). A small fraction of this mixture was puriﬁed by prepara-

tive thin layer chromatography and a small amount of compound

35a and compound 30 was isolated.

Mixture of compounds 35a and 30 as yellow oil (564 mg). (8-

((tert-Butyldimethylsilyl)oxy)-7-methoxy-2,2-dimethyl-3,4-dihy-

drobenzopyran-6-yl)(5-chloro-2-hydroxyphenyl)methanone (35a)

as a green solid. Mp: 107–109 °C. IR m_max(cm^ꢀ1) (KBr): 3434,

2943, 2879, 2840, 1614, 1582, 1452, 1346, 1238, 1183, 1102,

1062, 978, 890, 820, 769, 745, 454. ¹H NMR (300.13 MHz, CDCl₃)

d (ppm): 12.15 (OH), 7.43 (d, J = 2.8), 7.39 (dd, J = 8.9, 2.8), 6.97

(d, J = 8.9), 6.72 (s), 3.64 (s, OCH₃), 2.72 (t, J = 6.7, 2H), 1.80 (t,

J = 6.7, 2H), 1.41 (s, 6H), 1.04 (s, 9 H), 0.22 (s, 6H). EIMS m/z (%):

477 (0.6, [M]^+.], 365 (9), 363 (20), 332 (15), 266 (17), 265 (81),

210 (17), 209 (100), 195 (12), 194 (15), 167 (7), 166 (12), 157

(17), 155 (58), 99 (10), 75 (48), 73 (37), 59 (17).

(8-((tert-Butyldimethylsilyl)oxy)-7-methoxy-2,2-dimethyl-3,4-

dihydrobenzopyran-6-yl)(2-hydroxy-5-methoxyphenyl)metha-

none (35b) light yellow solid (816 mg/89%). Mp: 74–75 °C. IR m_max

(cm^ꢀ1) (KBr): 3437, 2970, 2939, 2893, 2849, 1628, 1591, 1462,

1416, 1355, 1328, 1294, 1195, 1150, 1110, 1068, 1036, 828, 780,

741, 657. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 11.90 (OH), 7.10

(dd, J = 9.1, 3.0), 6.96 (d, J = 9.1), 6.94 (d, J = 3.0), 6.71 (s), 3.67 (s,

OCH₃), 3.66 (s, OCH₃), 2.75 (t, J = 6.7, 2H), 1.80 (t, J = 6.7, 2H),

1.40 (s, 6H), 1.05 (s, 9 H), 0.20 (s, 6H). EIMS m/z (%): 473 (0.5,

[M]⁺], 359 (12), 328 (9), 250 (7), 209 (28), 152 (10), 151 (100), 75

(10).

(8-((tert-Butyldimethylsilyl)oxy)-7-methoxy-2,2-dimethyl-3,4-

dihydrobenzopyran-6-yl)(2-hydroxy-5-methylphenyl)methanone

(35c) as a white oil (900 mg/quantitative). IR m_max(cm^ꢀ1) (KBr):

3438, 2971, 2947, 2926, 2886, 2852, 1628, 1596, 1462, 1356,

1245, 1206, 1184, 1150, 1103, 1071, 910, 828, 778, 668. ¹H NMR

(300.13 MHz, CDCl₃) d (ppm): 12.07 (OH), 7.29–7.22 (2H, m),

6.92 (d, J = 8.4), 6.70 (s), 3.62 (s, OCH₃), 2.76 (t, J = 6.7, 2H), 2.20

(s, CH₃), 1.81 (t, J = 6.7, 2H), 1.40 (s, 6H), 1.04 (s, 9 H), 0.21 (s,

6H). EIMS m/z (%): 457 (1, [M]⁺], 345 (13), 312 (9), 209 (28), 136

(10), 135 (100), 77 (8), 75 (10).

4.8.4. Synthesis of xanthones 36a–e and compound 37

To a solution of a mixture 35a and 30 (530 mg) in DMF (10 mL)

was added Cs₂CO₃(543 mg, 1.67 mmol). The mixture was irradi-

ated with microwaves for 15 min at 140 °C. The mixture was al-

lowed to cool to room temperature and poured into crushed ice.

The aqueous phase was then extracted with 3 ꢃ 100 mL of diethyl

ether. The organic phase was washed with 2 ꢃ 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 8:1). Compound 36a was

recrystallized obtained as yellow solid (210 mg), and compound 4

as a light yellow solid (40 mg).

8-Chloro-12-hydroxy-2,2-dimethyl-3,4-dihydropyrano[3,2-b]

xanthen-6(2H)-one (36a) as a yellow solid (210 mg). Mp: 225–

227 °C. IR m_max(cm^ꢀ1) (KBr): 3322, 2973, 2937, 2854, 1636, 1614,

1471, 1353, 1259, 1239, 1214, 1185, 1157, 1124, 1073, 981, 887,

808, 645. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 8.28 (d, J = 2.6),

7.67 (t, J = 1.0), 7.62 (dd, J = 9.0, 2.6), 7.53 (d, J = 9.0), 5.75 (OH),

2.91 (td, J = 6.7, 1.0, 2H), 1.92 (t, J = 6.7, 2H), 1.45 (s, 6H). ¹³C

NMR (75.47 MHz, CDCl₃) d (ppm): 175.5, 154.5, 146.6, 143.1,

134.3, 132.5, 129.3, 126.0, 122.4, 119.7, 118.6, 117.1, 115.0, 77.5,

32.5, 27.0, 21.7. EIMS m/z (%): 331 (3, [M+1]⁺), 330 (5, [M]⁺), 275

(23), 190 (48), 184 (22), 162 (62), 155 (47), 139 (25), 128 (53),

127 (67), 126 (44), 99 (20), 77 (42), 75 (77), 74 (45), 62 (44), 53

(34), 51 (78). HRMS (ESI) m/z calcd for C₁₈H₁₆ClO₄[M+H]⁺:

331.07316; found: 331.07271; m/z calcd for

C₁₈H₁₆NaClO₄

[M+Na]⁺: 353.05511; found: 353.05410.

12-Hydroxy-8-methoxy-2,2-dimethyl-3,4-dihydropyrano[3,2-

b]xanthen-6(2H)-one (36b) as a white solid (478 mg/89%). Mp: de-

comp. IR m_max(cm^ꢀ1) (KBr): 3232, 2975, 2931, 2841, 1647, 1613,

1591, 1490, 1467, 1438, 1370, 1253, 1208, 1122, 1075, 1029,

787. ¹H NMR (300.13 MHz, CDCl₃/DMSO-d₆(10:1)) d (ppm): 8.12

(b, OH), 7.64 (d, J = 3.1), 7.58 (s), 7.52 (d, J = 9.1), 7.28 (dd, J = 9.1,

3.1), 3.91 (s, OCH₃), 2.94 (t, J = 6.7, 2H), 1.91 (t, J = 6.7, 2H), 1.45

(s, 6H). ¹³C NMR (75.47 MHz, CDCl₃/DMSO-d₆(10:1)): 175.5,

154.7, 150.0, 146.4, 143.3, 132.3, 123.0, 120.8, 118.5, 117.7;

115.1, 113.7, 105.1, 75.5, 55.9, 31.7, 26.1, 21.2. EIMS m/z (%): 327

(5, [M+1]⁺), 326 (10, [M]⁺), 272 (30), 271 (93), 270 (100), 267

(12), 255 (11), 254 (8), 242 (12), 228 (18), 215 (12), 214 (15),

171 (12), 115 (12), 77 (11), 63 (27), 62 (20), 53 (13), 51 (9). HRMS

(ESI) m/z calcd for

C

₁₉H₁₉O₅[M+H]⁺: 327.12270; found:

327.12371; m/z calcd for C₁₉H₁₈NaO₅[M+Na]⁺: 349.10464; found:

349.10323.

12-Hydroxy-2,2,8-trimethyl-3,4-dihydropyrano[3,2-b]xanthen-

6(2H)-one (36c) as a white solid (385 mg/65%). Mp: 227–228 °C. IR

m

_max(cm^ꢀ1) (KBr): 3239, 2981, 2934, 2856, 1652, 1616, 1490, 1465,

1421, 1365, 1252, 1220, 1197, 1153, 1131, 1096, 1073, 1021, 813,

783, 660. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 8.11–8.10 (m, 1H),

7.68 (t, J = 1.0), 7.49 (dd, J = 8.7, 2.0), 7.46 (dd, J = 8.7, 0.6), 5.85 (b,

OH), 2.91 (td, J = 6.7, 1.0, 2H), 2.45 (s, CH₃), 1.91 (t, J = 6.7, 2H),

1.43 (s, 6H). ¹³C NMR (75.47 MHz, CDCl₃): 176.7; 154.4, 146.1,

143.3, 135.5, 133.3, 132.3, 126.0, 121.1, 118.0, 117.7, 117.0,

115.4, 76.6, 32.6, 27.0, 21.2, 20.8. EIMS m/z (%): 311 (3, [M+1]⁺),

310 (8, [M]⁺), 255 (100), 254 (74), 199 (25), 198 (24), 170 (37),

142 (33), 141 (32), 128 (26), 115 (35), 77 (28), 63 (25), 51 (30).

HRMS (ESI) m/z calcd for C₁₉H₁₉O₄[M+H]⁺: 311.12779; found:

311.12851; m/z calcd for C₁₉H₁₈NaO₄[M+Na]⁺: 333.10973, found:

333.10805.

(8-((tert-Butyldimethylsilyl)oxy)-7-methoxy-2,2-dimethyl-3,4-

dihydrobenzopyran-6-yl)(2-hydroxy-3-methoxyphenyl)metha-

none (35d) as a yellow solid (717 mg/85%). Mp: 138–139 °C. IR

m_max(cm^ꢀ1) (KBr): 3454, 2967, 2918, 2878, 2843, 1617, 1599,

1447, 1348, 1249, 1106, 1084, 1050, 982, 896, 825, 774, 746,

728. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 12.55 (OH), 7.07 (dd,

J = 8.1, 1.3), 7.05 (dd, J = 8.1, 1.3), 6.76 (t, J = 8.1), 6.69 (s), 3.93 (s,

OCH₃), 3.67 (s, OCH₃), 2.74 (t, J = 6.7, 2H), 1.80 (t, J = 6.7, 2H),

1.40 (s, 6H), 1.04 (s, 9 H), 0.19 (s, 6H). EIMS m/z (%): 473 (0.8,

[M]⁺], 359 (13), 250 (10), 209 (14), 152 (10), 151 (100), 75 (10),

51 (12).

(8-((tert-Butyldimethylsilyl)oxy)-7-methoxy-2,2-dimethyl-3,4-

dihydrobenzopyran-6-yl)(2,4-dihydroxyphenyl)methanone (35e)

as a yellow oil (850 mg/quantitative). IR m_max(cm^ꢀ1) (KBr): 3383,

2968, 2925, 2851, 1622, 1594, 1459, 1360, 1296, 1242, 1207,

1113, 1071, 985, 830, 779, 625. ¹H NMR (300.13 MHz, CDCl₃) d

(ppm): 12.75 (OH), 7.35 (d, J = 8.8), 6.66 (s), 6.40 (d, J = 2.5), 6.28

(dd, J = 8.8, 2.5), 5.71 (OH), 3.66 (s, OCH₃), 2.74 (t, J = 6.7, 2H),

1.79 (t, J = 6.7, 2H), 1.39 (s, 6H), 1.03 (s, 9 H), 0.20 (s, 6H).

12-Hydroxy-10-methoxy-2,2-dimethyl-3,4-dihydropyrano[3,2-

b]xanthen-6(2H)-one (36d) as a light yellow solid (387 mg/83%).

Mp: 207–208 °C. IR m_max(cm^ꢀ1) (KBr): 3308, 3271, 2976, 2931,

2843, 1650, 1597, 1473, 1445, 1372, 1265, 1223, 1197, 1118,

1069, 937, 755. ¹H NMR (300.13 MHz, CDCl3) d (ppm): 7.88 (dd,

J = 7.8, 1.8), 7.67 (t, J = 1.0), 7.27 (t, J = 7.8), 7.20 (dd, J = 7.8, 1.8),

6.03 (b, OH), 4.03 (s, OCH₃), 2.93 (td, J = 1.0, 6.7, 2H), 1.92 (t,

J = 6.7, 2H), 1.44 (s, 6H). ¹³C NMR (75.47 MHz, CDCl₃): 176.6,

2954

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

148.6, 146.5, 146.4, 132.8, 123.0, 122.4, 118.5, 117.6, 116.7, 115.1,

114.9, 76.9, 56.4, 32.6, 26.9, 21.6. EIMS m/z (%): 327 (11, [M+1]⁺),

326 (11, [M]⁺), 271 (50), 270 (33), 214 (24), 200 (28), 199 (28),

185 (33), 171 (22), 157 (25), 151 (26), 144 (31), 128 (37), 116

(24), 115 (100), 89 (21), 77 (40), 75 (22), 63 (59), 62 (24), 53

(2-(Benzyloxy)-4,5-dimethoxyphenyl)(7,8-dimethoxy-2,2-di-

methyl-3,4-dihydrobenzopyran-6-yl)methanol (38f) as a yellow

oil (652 mg/48%). IR m_max(cm^ꢀ1) (KBr): 3518, 3493, 2965, 2926,

2832, 1537, 1504, 1457, 1337, 1202, 1104, 1019, 876. ¹H NMR

(400.21 MHz, CDCl₃) d (ppm): 7.31–7.23 (m, 5H), 7.04 (s), 6.57

(s), 6.52 (s), 6.25 (d, J = 3.9, 1H), 5.00 (d, 1H, J = 11.5), 4.96 (d, 1H,

J = 11.5), 3.85 (s, OCH₃), 3.84 (s, OCH₃), 3.83 (s, OCH₃), 3.71 (s,

OCH₃), 3.05 (d, J = 3.9, OH), 2.59 (t, J = 6.8, 2H), 1.75 (t, J = 6.8,

2H), 1.37 (s, 3H),1.34 (s, 3H).

(24), 51 (64). HRMS (ESI) m/z calcd for

C

₁₉H₁₉O₅[M+H]⁺:

327.12270; found: 327.12325; m/z calcd for

C₁₉H₁₈NaO₅

[M+Na]⁺: 349.10464; found: 349.10362.

9,12-Dihydroxy-2,2-dimethyl-3,4-dihydropyrano[3,2-b]xanthen-

6(2H)-one (36e) as a white solid (407 mg/71%). Mp: 262–264 °C. IR

m

_max(cm^ꢀ1) (KBr): 3334, 3288, 3241, 2972, 2925, 2845, 1645, 1611,

4.9.2. Synthesis of benzophenones 39c, 39e and 39f

1588, 1457, 1266, 1324, 1256, 1217, 1192, 1162, 1118, 1017, 850,

699. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 10.80 (b, OH), 9.50 (b;

OH), 8.02 (d, J = 8.5), 7.42 (s), 6.89 (d, J = 2.0), 6.87 (dd, J = 8.5, 2.0),

2.87 (t, J = 6.6, 2H), 1.84 (t, J = 6.6, 2H), 1.38 (s, 6H). ¹³C NMR

(75.47 MHz, CDCl₃) d (ppm): 174.5, 163.4, 147.2, 143.9, 133.1,

127.9, 118.4, 115.3, 114.4, 113.8, 113.5, 102.1, 75.8, 31.9, 26.6,

21.6. HRMS (ESI) m/z calcd for C₁₉H₁₇O₅[M+H]⁺: 313.10705;

found: 313.10696.

In a typical experiment: Compound 38e (450 mg/1.0 mmol) and

10 mL of DMSO were place in a round-bottom ﬂask. Then it was

added IBX (420 mg/1.5 mmol). After 12 h the reaction was

quenched with the addition of a 10% solution of Na₂S₂O₃, a satu-

rated solution of NaHCO₃and distilled water. The aqueous phase

was then extracted with 3 ꢃ 50 mL of ethyl acetate. The organic

phase was washed with 2 ꢃ 50 mL of a 10% solution of Na₂S₂O₃,

2 ꢃ 50 mL of a saturated solution of NaHCO₃, 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 8:2) and compound 39e was isolated as

yellow oil (380 mg/85%).

(2-(Benzyloxy)-5-methylphenyl)(7,8-dimethoxy-2,2-dimethyl-

3,4-dihydrobenzopyran-6-yl)methanone (39c) as a yellow solid

(460 mg/66%). Mp: 114–115 °C. IR m_max(cm^ꢀ1) (KBr): 2975, 2924,

2865, 1649, 1604, 1498, 1459, 1340, 1247, 1149, 1108, 1026,

987, 804, 734. ¹H NMR (400.21 MHz, CDCl₃) d (ppm): 7.33 (d,

J = 2.3), 7.22–7.18 (m, 4H), 7.14 (s), 7.01–6.98 (m, 2H), 6.87 (d,

J = 8.3), 4.94 (s, 2H), 3.71 (s, OCH₃), 3.52 (s, OCH₃), 2.73 (t, J = 6.7,

2H), 2.83 (s, CH₃), 1.81 (t, J = 6.7, 2H), 1.39 (s, 6H).

(2,4-dihydroxyphenyl)(8-hydroxy-7-methoxy-2,2-dimethyl-

3,4-dihydrobenzopyran-6-yl)methanone (37) as a white solid (125

mg, 20%). Mp: 220–222 °C. IR m

_max(cm^ꢀ1) (KBr): 3378, 3283, 2971,

2930, 2895, 2849, 1630, 1583, 1492, 1371, 1313, 1295, 1251, 1201,

1154, 1090, 1054, 983, 948, 786, 623. ¹H NMR (300.13 MHz, CDCl₃)

d (ppm): 12.61 (OH), 10.90 (b, OH), 8.70 (b, OH), 7.23 (d, J = 8.6),

6.51 (s), 6.33 (dd, J = 2.3, 8.6), 6.30 (d, J = 2.3), 3.61 (s, OCH₃), 2.60

(t, J = 6.6, 2H), 1.77 (t, J = 6.6, 2H), 1.33 (s, 6H). HRMS (ESI) m/z calcd

for C₁₉H₂₁O₆[M+H]⁺: 345.13326; found: 345.13402; m/z calcd for

C

₁₉H₂₀NaO₆[M+Na]⁺: 367.11521; found: 36.11345.

4.9. Synthesis of analogues 41c, 41e and 41f

(2,4-Bis(methoxymethoxy)phenyl)(7,8-dimethoxy-2,2-dimethyl-

3,4-dihydrobenzopyran-6-yl)methanone (39e) as a yellow oil (380

4.9.1. Synthesis of diarylmethanols 38c, 38e and 38f

In a typical experiment: Compound 14 (800 mg/2.7 mmol) was

placed in a two-necked round-bottom ﬂask and 25 mL of anhy-

drous THF was added. The mixture was then placed at ꢀ78 °C

and nBuLi (1.6 M, 3.24 mmol) was added dropwise. The mixture

was allowed to react for 15 min at ꢀ78 °C. Then the benzaldehyde

24f (882 mg/3.24 mmol) solubilized in anhydrous THF was added

dropwise and allowed to react for 1 h and 30 min at ꢀ78 °C and

slowly warm to room temperature (about 6 h). The reaction was

quenched by addition of a saturated solution of NH₄Cl. The aque-

ous phase was extracted with 3 ꢃ 50 mL of ethyl acetate. The or-

ganic phase was washed 2 ꢃ 50 mL of brine, dried over sodium

sulfate anhydrous, ﬁltered and the organic solvent evaporated.

The crude product was then puriﬁed by silica gel ﬂash chromatog-

raphy (n-hexane/ethyl acetate 8:2). Compound 38f was obtained

(652 mg/48%) as yellow oil.

(2-(Benzyloxy)-5-methylphenyl)(7,8-dimethoxy-2,2-dimethyl-

3,4-dihydrobenzopyran-6-yl)methanol (38c) as a yellow oil (730

mg/58%). IR m_max(cm^ꢀ1) (KBr): 3474, 3442, 2975, 2931, 2860,

1494, 1463, 1343, 1241, 1106, 736. ¹H NMR (400.21 MHz, CDCl₃)

d (ppm): 7.32–7.21 (m, 6H), 7.02 (dd, J = 8.3, 2.1), 6.81 (d, J = 8.3),

6.59 (s), 6.25 (b s), 5.03 (d, J = 11.4, 1H), 4.99 (d, 1H, J = 11.4),

3.82 (s, OCH₃), 3.67 (s, OCH₃), 3.00 (b, OH), 2.60 (t, J = 6.8, 2H),

2.30 (s, CH₃), 1.75 (t, J = 6.7, 2H), 1.37 (s, 3H),1.34 (s, 3H).

mg/85%). IR m

_max(cm^ꢀ1) (KBr): 2973, 2935, 2830, 1652, 1601, 1464,

1343, 1308, 1156, 1116, 1075, 1000, 923, 848, 793. ¹H NMR

(400.21 MHz, CDCl₃) d (ppm): 7.44 (d, J = 8.5), 7.06 (s), 6.80 (d,

J = 2.3), 6.72 (dd, J = 8.5, 1.4), 5.20 (s, 2H), 5.03 (s, 2H), 3.82 (s,

OCH₃), 3.62 (s, OCH₃), 3.49 (s, OCH₃), 3.32 (s, OCH₃), 2.75 (t,

J = 6.9, 2H), 1.82 (t, J = 6.9, 2H), 1.40 (s, 6H). EIMS m/z (%): 447 (3,

[M+1]⁺), 446 (10, [M]⁺), 416 (25), 415 (100), 385 (38), 383 (19),

359 (12), 315 (31), 249 (12), 235 (18), 211 (26), 193 (14), 179

(12), 69 (10).

(2-(Benzyloxy)-4,5-dimethoxyphenyl)(7,8-dimethoxy-2,2-di-

methyl-3,4-dihydrobenzopyran-6-yl)methanone (39f) as a yellow

solid (580 mg/96%). Mp: 128–129 °C. IR m_max(cm^ꢀ1) (KBr): 2973,

2930, 2843, 1642, 1604, 1513, 1450, 1404, 1341, 1276, 1210,

1111, 1026 732. ¹H NMR (400.21 MHz, CDC_l3) d (ppm): 7.23–7.20

(m, 4H), 7.03 (s), 6.97–6.95 (m, 2H), 6.55 (s), 4.90 (s, 2H), 3.92 (s,

OCH₃), 3.90 (s, OCH₃), 3.69 (s, OCH₃), 3.60 (s, OCH₃), 2.72 (t,

J = 6.7, 2H), 1.79 (t, J = 6.7, 2H), 1.38 (s, 6H).

4.9.3. Synthesis of benzophenones 40c, 40e and 40f

Benzyl ether removal. In a typical experiment: Compound 39f

(344 mg/0.68 mmol) and 25 mL of anhydrous methanol/THF (1:1)

were place in a two-necked round-bottom ﬂask and under nitrogen

atmosphere. To the solution was added Pd/C 10% (15% weight/

52 mg) in one portion. Triethylsilane (1.1 mL, 6.8 mmol) was then

added dropwise. The mixture was allowed to stir for 15 min. The

product was ﬁltered over celite and washed 3 times with metha-

nol. The methanol was then evaporated and the crude product

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

acetate 8:2). Compound 40f was isolated as yellowish oil (180 mg/

67%).

(2,4-Bis(methoxymethoxy)phenyl)(7,8-dimethoxy-2,2-dimethyl-

3,4-dihydrobenzopyran-6-yl)methanol (38e) as a yellow oil (460

mg/38%). IR m_max(cm^ꢀ1) (KBr): 3503, 3447, 2971, 2934, 2829,

1606, 1464, 1153, 1106, 1072, 1010, 923. ¹H NMR (400.21 MHz,

CDCl₃) d (ppm): 7.23 (d, J = 8.5), 6.78 (d, J = 2.3), 6.70 (dd, J = 8.5,

2.3), 6.69 (b s), 6.25 (d, J = 3.7, 1H), 5.19 (d, J = 6.7, 1H), 5.15 (s,

2H), 5.12 (d, J = 6.7, 1H) 3.83 (s, OCH₃), 3.78 (s, OCH₃), 3.48 (s,

OCH₃), 3.35 (s, OCH₃), 3.00 (d, J = 3.7, OH), 2.68 (td, J = 6.9, 2.0,

2H), 1.74 (t, J = 6.9, 2H), 1.34 (s, 3H), 1.32 (s, 3H).

(7,8-Dimethoxy-2,2-dimethyl-3,4-dihydrobenzopyran-6-yl)(2-

hydroxy-5-methylphenyl)methanone (40c) as yellow oil (110 mg/

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

2955

77%). IR m_max(cm^ꢀ1) (KBr): 3328, 2976, 2934, 1629, 1601, 1474,

1349, 1110, 1072, 989, 828, 767, 670. ¹H NMR (400.21 MHz, CDCl₃)

d (ppm): 12.02 (OH), 7.28 (dd, J = 8.4, 2.3), 7.21 (d, J = 2.3), 6.93 (d,

J = 8.4), 6.80 (t, J = 0.8), 3.88 (s, OCH₃), 3.79 (s, OCH₃), 2.77 (td,

J = 6.7, 0.8, 2H), 2.23 (s, CH₃), 1.85 (t, J = 6.7, 2H), 1.43 (s, 6H). EIMS

m/z (%): 357 (12, [M+1]⁺), 356 (1, [M]⁺), 355 (2), 325 (16), 270 (20),

269 (100), 226 (8), 222 (10), 193 (47), 168 (11), 167 (63), 165 (10),

135 (20), 115 (10), 91 (11), 79 (11), 77 (31), 51 (11).

115 (10), 91 (4), 77 (4). HRMS (ESI) m/z calcd for C₂₀H₂₁O₄

[M+H]⁺: 325.14344; found: 325.14330.

9-Hydroxy-12-methoxy-2,2-dimethyl-3,4-dihydropyrano[3,2-

b]xanthen-6(2H)-one (41e) as a white solid (64 mg/42%). Mp: 286–

287 °C. IR m_max(cm^ꢀ1) (KBr): 3227, 2973, 2940, 2840, 1646, 1611,

1574, 1444, 1349, 1202, 1116. ¹H NMR (400.21 MHz, DMSO-d₆) d

(ppm): 7.99 (d, J = 8.5), 7.66 (s), 6.87 (dd, J = 8.5, 2.3), 6.85 (d,

J = 2.3), 3.87 (s, OCH₃), 2.89 (t, J = 6.7, 2H), 1.86 (t, J = 6.7, 2H),

1.38 (s; 6H). ¹³C NMR (100.63 MHz, DMSO-d₆) d (ppm): 174.1,

163.7, 157.5, 148.3, 147.8, 135.2, 127.8, 120.3, 119.3, 114.4,

113.8, 113.8, 102.2, 76.3, 60.7, 31.5, 26.7, 21.5. HRMS (ESI) m/z

calcd for C₁₉H₁₉O₅[M+H]⁺: 327.12270; found: 327.12258.

8,9,12-Trimethoxy-2,2-dimethyl-3,4-dihydropyrano[3,2-b]xan-

then-6(2H)-one (41f) as a yellowish solid (140 mg/83%). Mp: 228–

229 °C. IR m_max(cm^ꢀ1) (KBr): 2968, 2928, 2832, 1644, 1613, 1483,

1432, 1309, 1274, 1202, 1148, 1202, 1148, 1089, 1103, 784. ¹H

NMR (400.21 MHz, CDCl₃) d (ppm): 7.84 (t, J = 1.0), 7.66 (s), 7.03

(s), 4.02 (s, OCH₃), 4.00 (s, 2ꢃOCH₃), 2.94 (td, J = 6.8, 1.0, 2H), 1.96

(t, J = 6.8, 2H), 1.45 (s, 6H). ¹³C NMR (100.63 MHz, CDCl₃) d (ppm):

175.6, 154.9, 152.5, 152.4, 149.0, 146.4, 135.7, 121.0, 119.2, 115.0,

114.5, 105.5, 100.0, 76.3, 61.2, 56.5, 56.4, 32.5, 27.0, 22.2. EIMS m/z

(%): 371 (14, [M+1]⁺), 370 (57, [M]⁺), 355 (12), 316 (20), 315 (100),

314 (28), 313 (9), 300 (9), 299 (7), 285 (9), 170 (5). HRMS (ESI) m/z

calcd for C₂₁H₂₃O₆[M+H]⁺: 371.14891; found: 371.14872.

(7,8-Dimethoxy-2,2-dimethyl-3,4-dihydrobenzopyran-6-yl)(2-

hydroxy-4,5-dimethoxyphenyl)methanone (40f) as yellow oil (180

mg/67%). IR m

_max(cm^ꢀ1) (KBr): 3473, 2974, 2936, 2857, 1619, 1505,

1461, 1350, 1268, 1203, 1153, 1110, 1071, 829. ¹H NMR

(400.21 MHz, CDCl₃) d (ppm): 12.70 (OH), 6.86 (s), 6.83 (s), 6.51

(s) 3.94 (s, OCH₃), 3.88 (s, OCH₃), 3.79 (s, OCH₃), 3.70 (s, OCH₃),

2.77 (t, J = 6.7, 2H), 1.84 (t, J = 6.7, 2H), 1.42 (s, 6H). EIMS m/z (%):

402 (12, [M]⁺), 372 (28), 371 (79), 355 (6), 315 (30), 281 (19),

222 (41), 209 (10), 208 (14), 207 (68), 193 (10), 191 (11), 181

(17), 168 (10), 167 (100), 166 (13), 165 (11), 147 (11), 133 (10),

96 (11), 73(22).

Methoxymethyl ether removal

To a solution of compounds 39e (227 mg, 0.51 mmol) in 5 mL of

CH₃CN at 0 °C was added in one portion NbCl₅(138 mg,

0.19 mmol). The mixture was stirred at 0 °C for 10 min and then al-

lowed to warm to room temperature and let stirring for more

45 min and the reaction mixture was quenched with a saturated

solution of NaHCO₃. The mixture was extracted with 3 ꢃ 25 mL

of ethyl acetate. The organic phase was washed with 2 ꢃ 50 mL

of brine, dried over sodium sulfate anhydrous, ﬁltered and the or-

ganic solvent evaporated. Compound 40e was obtained as yellow

oil (172 mg/94%) and used in the next reaction without further

puriﬁcation

(2,4-Dihydroxyphenyl)(7,8-dimethoxy-2,2-dimethyl-3,4-dihy-

drobenzopyran-6-yl)methanone (40e). IR m_max(cm^ꢀ1) (KBr): 3328,

2975, 2932, 2857, 1606, 1460, 1353, 1239, 1114, 989, 799. ¹H NMR

(400.21 MHz, CDCl₃) d (ppm): 12.68 (OH), 7.34 (d, J = 8.8), 6.78 (s),

6.41 (d, J = 2.5), 6.30 (dd, J = 8.8, 2.5), 3.88 (s, OCH₃), 3.79 (s, OCH₃),

2.75 (t, J = 6.7, 2H), 1.83 (t, J = 6.7, 2H), 1.41 (s, 6H). EIMS m/z (%):

345 (5, [M]⁺), 328 (21), 327 (100), 303 (7), 272 (10), 271 (52),

192 (26), 151 (4), 137 (42), 123 (6), 115 (5), 107 (5), 107 (5), 91

(6), 81 (13), 79 (6), 77 (7), 69 (11), 53 (10).

4.10. Synthesis of analogues 48a–c, 48e and 48g

4.10.1. Synthesis of diarylmethanols 42a–c, 42e, 42g, 43a–c, 43e

and 43g

In a typical Experiment: Compound 19 (193 mg/0.87 mmol) sol-

ubilized in 5 mL of anhydrous THF and TMEDA (1.05 mmol,

121 mg) were added to a two-necked round-bottom ﬂask under

nitrogen atmosphere. The mixture was then placed at 0 °C and

nBuLi (1.6 M, 1.05 mmol) was added dropwise. The mixture was al-

lowed to react for 30 min at 0 °C. Then the benzaldehyde 28a

(176 mg/0.87 mmol) was added and allowed to react for 1 h

30 min at 0 °C and 6 h at room temperature. The reaction was

quenched with a saturated solution of ammonium chloride, ex-

tracted with 3 ꢃ 20 mL of ethyl acetate. The organic phase was

washed 2 ꢃ 25 mL of brine, dried over sodium sulfate anhydrous,

ﬁltered and the organic solvent evaporated. The crude product

was then separated by silica gel ﬂash chromatography (n-hex-

ane/ethyl acetate 8:2). Compounds 42a and 43a were obtained as

yellow oil (137 mg/38%), although compound 42a was obtained

in higher yield. The mixture was used in the next reaction without

further puriﬁcation.

Combined yield for compounds 42a and 43a (yellow oil, 137

mg/38%). (5-Chloro-2-(methoxymethoxy)phenyl)(5,6-dimethoxy-

2,2-dimethyl-2H-benzopyran-7-yl)methanol (42a). EIMS m/z (%):

422 (15, [M+2]⁺), 420 (35, [M]⁺), 407 (40), 405 (100), 373 (10),

345 (10), 329 (20), 327 (40), 312 (20), 297 (10), 205 (20), 155

(20). (5-Chloro-2-(methoxymethoxy)phenyl)(5,6-dimethoxy-2,2-

dimethyl-2H-benzopyran-8-yl)methanol (43a). EIMS m/z (%):422

(20, [M+2]⁺), 420 (65, [M]⁺), 407 (30), 405 (75), 373 (20), 359

(35), 345 (50), 315 (25), 247 (20), 221 (65), 219 (55), 205 (100),

189 (30), 155 (62).

4.9.4. Synthesis of xanthones 41c, 41e and 41f

Synthesis of xanthones 41c, 41e and 41f. In a typical experi-

ment: To a solution of compound 40c (186 mg, 0.52 mmol) in

3 mL of DMF was added K₂CO₃(142 mg, 1.03 mmol). The mixture

was irradiated with microwave (200 W max) for 15 min at

140 °C. The mixture was allowed to cool to room temperature

and poured into crushed ice. The aqueous phase was then ex-

tracted with 3 ꢃ 50 mL of diethyl ether. The organic phase was

washed with 2 ꢃ 50 mL of brine, dried over sodium sulfate anhy-

drous, ﬁltered and the organic solvent evaporated. The crude prod-

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

ethyl acetate 8:2). Compound 41c was obtained as light yellow so-

lid (160 mg, 95%).

12-Methoxy-2,2,8-trimethyl-3,4-dihydropyrano[3,2-b]xanthen-

6(2H)-one (41c) as a yellowish solid (160 mg/95%). Mp: 101–

102 °C. IR m_max(cm^ꢀ1) (KBr): 2972, 2926, 2846, 1655, 1609, 1480,

1446, 1312, 1117, 1083, 781. ¹H NMR (400.21 MHz, CDCl₃) d

(ppm): 8.09 (d, J = 2.1), 7.85 (t, J = 1.0), 7.50 (dd, J = 8.3, 2.1), 7.46

(d, J = 8.3), 3.99 (s; OCH₃), 2.93 (td, J = 6.8, 1.0), 2.46 (s; CH₃), 1.89

(t, J = 6.8, 2H), 1.45 (s, 6H). ¹³C NMR (100.63 MHz, CDCl₃) d

(ppm): 176.7, 154.5, 153.1, 149.2, 135.7, 135.5, 133.4, 125.9,

121.3, 121.2, 119.2, 117.8, 115.2, 76.4, 61.2, 32.4, 27.0, 22.2, 20.8.

EIMS m/z (%): 325 (10, [M+1]⁺), 324 (47, [M]⁺), 307 (4), 293 (4),

270 (18), 269 (100), 268 (25), 267 (15), 239 (9), 197 (6), 141 (5),

Combined yield for compounds 42b and 43b (yellow oil, 185

mg/41%). (5,6-Dimethoxy-2,2-dimethyl-2H-benzopyran-7-yl)(5-

methoxy-2-(methoxymethoxy)phenyl)methanol (42b). EIMS m/z

(%): 416 (15, [M]⁺), 401 (20), 369 (15), 353 (15), 323 (100), 308

(20), 293 (15), 205 (15), 151 (20). (5,6-Dimethoxy-2,2-dimethyl-

2H-benzopyran-8-yl)(5-methoxy-2-(methoxymethoxy)phenyl)

methanol (43b). EIMS m/z (%): 417 (10, [M+1]⁺), 416 (40, [M]⁺), 401

(25), 354 (70), 311 (100), 247 (40), 247 (35), 219 (95), 195 (35), 175

(30), 151 (70), 137 (25).

2956

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

Combined yield for compounds 42c and 43c (yellow oil, 261

445 (10, [M+1]⁺), 444 (45, [M]⁺), 429 (100), 413 (40), 381 (50),

353 (20), 309 (25), 249 (15). (2,4-bis(methoxyme-

mg/45%). (5,6-Dimethoxy-2,2-dimethyl-2H-benzopyran-7-yl)(2-

(methoxymethoxy)-5-methylphenyl)methanol (42c). EIMS m/z

(%): 401 (10, [M+1]⁺), 400 (40, [M]⁺), 385 (90), 353 (30), 307

(100), 292 (25), 277 (20), 205 (30), 135 (55). (5,6-dimethoxy-2,2-

dimethyl-2H-benzopyran-8-yl)(2-(methoxymethoxy)-5-methyl-

phenyl)methanol (43c). EIMS m/z (%): 401 (10, [M+1]⁺), 400 (50,

[M]⁺), 385 (45), 355 (10), 325 (20), 395 (20), 247 (35), 219 (30),

205 (100), 179, 135 (65).

thoxy)phenyl)(5,6-dimethoxy-2,2-dimethyl-2H-benzopyran-8-

yl)methanone (45e). EIMS m/z (%): 445 (5, [M+1]⁺), 444 (30, [M]⁺),

429 (40), 400 (25), 385 (60), 369 (100), 341 (25), 205 (20).

Combined yield for compounds 44g and 45g (yellow oil, 135

mg/74%). Both isomers were separated by silica gel ﬂash chroma-

tography but were not characterized by GC–MS since they were

not sufﬁciently volatile.

Combined yield for compounds 42e and 43e (yellow oil, 1.03 g/

68%).

(2,4-Bis(methoxymethoxy)phenyl)(5,6-dimethoxy-2,2-di-

4.10.3. Synthesis of benzophenones 46a–c, 46e, 46g, 47a–c, 47e

and 47g

methyl-2H-benzopyran-7-yl)methanol (42e). EIMS m/z (%): 447

(10, [M+1]⁺), 446 (45, [M]⁺), 432 (25), 431 (100), 399 (15), 353

(35), 247 (50), 205 (25), 181 (20), 151 (20). (2,4-bis(methoxyme-

thoxy)phenyl)(5,6-dimethoxy-2,2-dimethyl-2H-benzopyran-8-yl)

methanol (43e). EIMS m/z (%): 446 (20, [M]⁺), 415 (25), 397 (40),

369 (20), 354 (30), 353 (100), 323 (30), 293 (35), 281 (20), 263

(80), 247 (55), 207 (30), 165 (25).

Combined yield for compounds 42g and 43g (yellow oil, 165

mg/33%). Both isomers were separated from the starting materials

by silica gel ﬂash chromatography but were not characterized by

GC–MS since they were not sufﬁciently volatile.

In a typical experiment: Compounds 44a and 45a (80.6 mg,

0.19 mmol) and 1.5 mL of CH₃CN were place in a round-bottom

ﬂask. The solution was placed at 0 °C and NbCl₅(52 mg,

0.19 mmol) was added in one portion. The mixture was stirred at

0 °C for 10 min and then allowed to warm to room temperature

and let stirring for more 45 min. After this time, the reaction was

quenched with saturated solution of NaHCO₃. The mixture was ex-

tracted with 3 ꢃ 15 mL of ethyl acetate. The organic phase was

washed with 2 ꢃ 100 mL of brine, dried over sodium sulfate anhy-

drous and the organic solvent evaporated. The mixture of the two

isomers (46a and 47a) was obtained as yellow oil in quantitative

yield and used in the next reaction without further puriﬁcation.

Compounds 46a and 47a as yellow oil in quantitative yield. (5-

Chloro-2-hydroxyphenyl)(5,6-dimethoxy-2,2-dimethyl-2H-benzo-

pyran-7-yl)methanone (46a). EIMS m/z (%): 376 (5, [M+2]⁺), 374

(30, [M]⁺), 361 (40), 359 (100), 343 (40), 219 (20), 164 (25), 155

(30). (5-Chloro-2-hydroxyphenyl)(5,6-dimethoxy-2,2-dimethyl-

2H-benzopyran-8-yl)methanone (47a). EIMS m/z (%): 376 (5,

[M+2]⁺), 374 (30, [M]⁺), 361 (40), 359 (100), 331 (20), 219 (20),

205 (20), 155 (30).

Compounds 46b and 47b as yellow oil in quantitative yield.

(5,6-Dimethoxy-2,2-dimethyl-2H-benzopyran-7-yl)(2-hydroxy-5-

methoxyphenyl)methanone (46b). EIMS m/z (%): 370 (20, [M]⁺),

355 (60), 339 (100), 309 (25), 205 (20), 162 (20). (5,6-dime-

thoxy-2,2-dimethyl-2H-benzopyran-8-yl)(2-hydroxy-5-methoxy-

phenyl)methanone (47b). EIMS m/z (%): 371 (10, [M+1]⁺), 370 (40,

[M]⁺), 355 (100), 327 (20), 295 (15), 205 (20), 151 (40).

Compounds 46c and 47c as yellow oil in quantitative yield. (5,6-

Dimethoxy-2,2-dimethyl-2H-benzopyran-7-yl)(2-hydroxy-5-meth-

ylphenyl)methanone (46c). EIMS m/z (%): 354 (25, [M]⁺), 339 (90),

323 (100), 293 (40), 154 (60), 135 (50). (5,6-dimethoxy-2,2-di-

methyl-2H-benzopyran-8-yl)(2-hydroxy-5-methylphenyl)metha-

none (47c). EIMS m/z (%): 354 (35, [M]⁺), 339 (100), 281 (30), 205

(20), 135 (40).

4.10.2. Synthesis of benzophenones 44a–c, 44e, 44g, 45a–c, 45e

and 45g

In a typical experiment: the two isomers, 42a and 43a (46.2 mg/

0.11 mmol) and 1 mL of DMSO were place in two-necked round-

bottom ﬂask. Then it was added IBX (37 mg/0.132 mmol). After

12 h the reaction was quenched with the addition of a solution

of Na₂S₂O₃10%, saturated NaHCO₃and water. The aqueous phase

was then extracted with 3 ꢃ 15 mL of ethyl acetate. The organic

phase was washed with 2 ꢃ 25 mL of a 10% solution of Na₂S₂O₃,

2 ꢃ 25 mL of a saturated solution of NaHCO₃, dried over sodium

sulfate anhydrous, ﬁltered and the organic solvent evaporated.

The crude product was puriﬁed by silica gel column chromatogra-

phy (n-hexane/ethyl acetate 9:1) and both benzophenones (44a

and 45a) were obtained as yellow oil (33.6 mg/73%).

Combined yield for compounds 44a and 45a (yellow oil, 137

mg/73%). (5-Chloro-2-(methoxymethoxy)phenyl)(5,6-dimethoxy-

2,2-dimethyl-2H-benzopyran-7-yl)methanone (44a). EIMS m/z

(%): 420 (5, [M+2]⁺), 418 (30, [M]⁺), 405 (30), 403 (100), 387 (30),

373 (5), 371 (30), 359 (40), 327 (40), 327 (40). (5-chloro-2-

(methoxymethoxy)phenyl)(5,6-dimethoxy-2,2-dimethyl-2H-benz-

opyran-8-yl)methanone (45a). EIMS m/z (%): 420 (10, [M+2]⁺), 418

(70, [M]⁺), 405 (35), 403 (100), 373 (25), 345 (35), 343 (95), 315

(25), 219 (50), 207 (40), 189 (30), 155 (70).

Combined yield for compounds 44b and 45b (yellow oil, 113

mg/86%). (5,6-Dimethoxy-2,2-dimethyl-2H-benzopyran-7-yl)(5-

methoxy-2-(methoxymethoxy)phenyl)methanone (44b). EIMS m/

z (%): 414 (15, [M]⁺), 399 (40), 383 (50), 367 (95), 351 (100), 323

(50), 249 (30). (5,6-Dimethoxy-2,2-dimethyl-2H-benzopyran-8-

Combined yield for compounds 46e and 47e (yellow oil, 76 mg/

85%).

(2,4-Dihydroxyphenyl)(5,6-dimethoxy-2,2-dimethyl-2H-

benzopyran-7-yl)methanone (46e). EIMS m/z (%): 357 (5, [M+1]⁺),

356 (30, [M]⁺), 341 (100), 325 (70), 295 (20), 205 (25), 176 (20),

155 (30), 137 (50). (2,4-dihydroxyphenyl)(5,6-dimethoxy-2,2-di-

methyl-2H-benzopyran-8-yl)methanone (47e). EIMS m/z (%): 357

(5, [M+1]⁺), 356 (40, [M]⁺), 341 (100), 313 (20), 281 (30), 207

(40), 137 (60).

yl)(5-methoxy-2-(methoxymethoxy)phenyl)methanone

(45b).

EIMS m/z (%): 415 (10, [M+1]⁺), 414 (55, [M]⁺), 399 (60), 367

(35), 339 (100), 311 (30), 295 (35), 219 (40), 189 (35), 175 (60),

151 (90).

Combined yield for compounds 46g and 47g (yellow oil, 60 mg/

56%). Both isomers were separated by silica gel ﬂash chromatogra-

phy but were not characterized by GC–MS since they were not suf-

ﬁciently volatile.

Combined yield for compounds 44c and 45c (yellow oil, 66 mg/

87%).

(5,6-Dimethoxy-2,2-dimethyl-2H-benzopyran-7-yl)(2-

(methoxymethoxy)-5-methylphenyl)methanone (44c). EIMS m/z

(%): 399 (5, [M+1]⁺), 398 (40, [M]⁺), 383 (100), 367 (60), 351 (50),

335 (55), 307 (40). (5,6-dimethoxy-2,2-dimethyl-2H-benzopyran-

4.10.4. Synthesis of xanthones 48a–c, 48e and 48g

8-yl)(2-(methoxymethoxy)-5-methylphenyl)methanone

(45c).

With conventional heating

EIMS m/z (%): 399 (10, [M+1]⁺), 398 (70, [M]⁺), 383 (80), 353

(20), 323 (100), 135 (65).

In a typical experiment: a solution of compound 46a and 47a

(68 mg, 0.18 mmol) in 1 mL of DMF were place in a two-necked

round-bottom ﬂask and then it was added Cs₂CO₃(88 mg,

0.27 mmol). The mixture was stirred at 50 °C for 21 h. The mixture

was allowed to cool to room temperature and poured into crushed

Combined yield for compounds 44e and 45e (yellow oil, 870

mg/88%). (2,4-bis(methoxymethoxy)phenyl)(5,6-dimethoxy-2,2-

dimethyl-2H-benzopyran-7-yl)methanone (44e). EIMS m/z (%):

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

2957

ice. The aqueous phase was then extracted with 3 ꢃ 50 mL of

diethyl ether. The organic phase was washed with 2 ꢃ 50 mL of

brine, dried over sodium sulfate anhydrous, ﬁltered and the organ-

ic solvent evaporated. The crude product was puriﬁed by silica gel

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

48a was obtained as yellow solid (35 mg, 65%) and compound

47a (7.1 mg) was also isolated.

58 °C. IR m_max(cm^ꢀ1) (KBr): 2967, 2929, 2868, 1640, 1607, 1435,

114, 1073, 813. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 8.12 (d,

J = 9.1), 7.46 (s), 6.77 (d, J = 10.2), 6.71 (dd, J = 9.1, 2.4), 6.59 (d,

J = 2.4), 5.89 (d, J = 10.2), 4.05 (s, OCH₃), 3.49 (q, J = 7.1, 4H), 1.47

(s, 6H), 1.27 (t, J = 7.1, 6H). ¹³C NMR (75.47 MHz, CDCl₃): 174.8,

158.4, 152.4, 148.5, 144.4, 143.3, 134.7, 128.1, 122.8, 120.4,

116.6, 111.1, 109.5, 106.9, 96.1, 75.9, 62.3, 44.8, 27.6, 12.5. HRMS

9-Chloro-5-methoxy-2,2-dimethylpyrano[2,3-b]xanthen-11(2H)-

one (48a) yellowish solid (35 mg/65%). Mp: 163–164 °C. IR m_max

(cm^ꢀ1) (KBr): 2954, 2913, 2846, 1644, 1603, 1478, 1435, 1256,

1116, 1079, 1017, 806, 699. ¹H NMR (300.13 MHz, CDCl₃) d

(ppm): 8.31 (d, J = 2.6)), 7.69 (dd, J = 8.8, 2.6), 7.54 (d, J = 8.8),

7.48 (s), 6.82 (d, J = 10.8), 5.99 (d, J = 10.8), 4.09 (s, OCH₃), 1.51 (s,

6H). ¹³C NMR (75.47 MHz, CDCl₃): 175.3, 154.1, 149.3, 144.6,

143.6, 135.8, 134.5, 129.6, 126.0, 122.1, 121.9, 121.9, 119.7,

116.3, 106.7, 76.3, 62.3, 27.8. EIMS m/z (%): 344 (10, [M+2]⁺), 342

(70, [M]⁺), 329 (35), 327 (100), 314 (10), 312 (40), 156 (20). HRMS

(ESI) m/z calcd for C

₂₃H₂₆NO₄[M+H]⁺: 380.18563; found:

380.18546.

4.11. Synthesis of analogues 55a, 55f and 55g

4.11.1. Synthesis of diarylmethanols 49a, 49f, 49g, 50a, 50f and

50g

Same experimental procedure described in Section 4.10.1.

Combined yield for compounds 49a and 50a (yellow oil, 1.48 g/

40%) Both isomers were separated from the starting materials by

silica gel ﬂash chromatography but were not characterized by

GC–MS since they were not sufﬁciently volatile.

(ESI) m/z calcd for

C

₁₉H₁₆ClO₄[M+H]⁺: 343.07316; found:

343.07304. Compound 47a (7.1 mg).

5,9-Dimethoxy-2,2-dimethylpyrano[2,3-b]xanthen-11(2H)-one

Combined yield for compounds 49f and 50f (yellow oil, 951 mg/

50%) Both isomers were separated from the starting materials by

silica gel ﬂash chromatography but were not characterized by

GC–MS since they were not sufﬁciently volatile.

Combined yield for compounds 49g and 50g (yellow oil, 750

mg/16%). Both isomers were separated from the starting materials

by silica gel ﬂash chromatography but were not characterized by

GC–MS since they were not sufﬁciently volatile.

(48b) yellow solid (46 mg/51%). Mp: 174–175 °C. IR m_max(cm^ꢀ1

(KBr): 2978, 2943, 2845, 1642, 1617, 1488, 1444, 1202, 1127,

783. ¹H NMR (300.13 MHz, CDCl₃) d (ppm): 7.72 (d, J = 3.1), 7.53

(d, J = 9.2), 7.52 (s), 7.36 (dd, J = 9.2, 3.1), 6.83 (d, J = 10.0), 5.98 (d,

J = 10.0), 4.10 (s, OCH₃), 3.97 (s, OCH₃), 1.52 (s, 6H). ¹³C NMR

(75.47 MHz, CDCl₃): 176.3, 155.9, 150.7, 148.8, 144.8, 143.6,

135.6, 124.7, 121.7, 121.5, 121.4, 119.4, 116.4, 106.5, 105.6, 76.2,

62.2, 55.9, 27.7. EIMS m/z (%): 338 (30, [M]⁺), 323 (100), 308

)

(40), 161 (20). HRMS (ESI) m/z calcd for

C

₂₀H₁₉O₅[M+H]⁺:

4.11.2. Synthesis of benzophenones 51a, 51f, 51g, 52a, 52f and

52g

339.12270, found: 339.12259. Compound 47b (5 mg).

5-Methoxy-2,2,9-trimethylpyrano[2,3-b]xanthen-11(2H)-one

Same experimental procedure described in Section 4.10.2.

Combined yield for compounds 51a and 52a (yellow oil, 1.13 g/

91%). Both isomers were separated by silica gel ﬂash chromatogra-

phy but were not characterized by GC–MS since they were not suf-

ﬁciently volatile.

Combined yield for compounds 51f and 52f (yellow oil, 489 mg/

59%). Both isomers were separated silica gel ﬂash chromatography

but were not characterized by GC–MS since they were not sufﬁ-

ciently volatile.

Combined yield for compounds 51g and 52g (yellow oil, 135

mg/74%). Both isomers were separated by silica gel ﬂash chroma-

tography but were not characterized by GC–MS since they were

not sufﬁciently volatile.

(48c) yellowish solid (85 mg/72%). Mp: 181–182 °C. IR m )

_max(cm^ꢀ1

(KBr): 2973, 2924, 1642, 1616, 1484, 1437, 1305, 1119. ¹H NMR

(300.13 MHz, CDCl₃) d (ppm): 8.13 (d, J = 2.0), 7.56 (dd, J = 8.4,

2.0), 7.51 (s), 7.48 (d, J = 8.4), 6.83 (d, J = 10.1), 5.97 (d, J = 10.1),

3.98 (s, OCH₃), 2.38 (s, CH₃), 1.39 (s, 6H). ¹³C NMR (75.47 MHz,

CDCl₃): 176.5, 154.0, 148.8, 144.7, 143.6, 135.7, 135.5, 133.6,

125.9, 122.2, 121.4, 120.9, 117.7, 116.4, 106.7, 76.1, 62.2, 27.7,

20.8. EIMS m/z (%): 323 (5, [M+1]⁺), 322 (70, [M]⁺), 308 (20), 307

(100), 292 (40), 153 (20). HRMS (ESI) m/z calcd for C₂₀H₁₉O₄

[M+H]⁺: 323.12779, found: 323.12768. Compound 47c (4.8 mg).

With microwave heating.

In a typical experiment: To a solution of compounds 46e and 47e

(210 mg, 0.11 mmol) in 1.5 mL of DMF was added K₂CO₃(163 mg,

1.18 mmol). The mixture was irradiated with microwave (200 W

max) for 15 min at 140 °C. The mixture was allowed to cool to room

temperature and poured into crushed ice. The aqueous phase was

then extracted with 3 ꢃ 30 mL of diethyl ether. The organic phase

was washed with 2 ꢃ 30 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 8:2). Compound 48e was obtained as light yellow

solid (87 mg, 22%).

4.11.3. Synthesis of benzophenones 53a, 53f, 53g, 54a, 54f and

54g

In a typical experiment: Compounds 51f and 52f (575 mg/

1.14 mmol) and 25 mL of anhydrous methanol were placed in a

two-necked round-bottom ﬂask and under nitrogen atmosphere.

To the solution was added Pd/C 10% (15% weight/86 mg) in one por-

tion. Triethylsilane (2.7 mL, 17.1 mmol) was then added dropwise.

The mixture was allowed to stir for 15 min. The product was ﬁltered

over celite and washed 3 times with methanol. The methanol was

then evaporated and the crude product was puriﬁed by silica gel

column chromatography (n-hexane/ethyl acetate 9:1). Compounds

53f and 54f were isolated as yellowish oil (380 mg/83%).

Combined yield for compounds 53a and 54a (yellow oil, 400 mg

of impure mixture). From the GC–MS chromatogram we observed

not only the desired compounds but also hydrodechlorinated prod-

ucts which we were not able to isolate.

8-Hydroxy-5-methoxy-2,2-dimethylpyrano[2,3-b]xanthen-11(2H)-

one (48e) as a light yellow solid (87 mg/22%). Mp: 193–194 °C. IR

m

_max(cm^ꢀ1) (KBr): 3112, 2975, 2936, 2845, 1609, 1435, 1234, 1101,

1005, 846, 780. ¹H NMR (400.21 MHz, CDCl₃) d (ppm): 8.25 (d,

J = 8.7), 7.44 (s), 7.27 (d, J = 2.3), 7.13 (dd, J = 8.7, 2.3), 6.78 (d,

J = 9.9), 5.93 (d, J = 9.9), 4.06 (s; OCH₃), 1.47 (s, 6H). ¹³C NMR

(100.63 MHz, CDCl₃) d (ppm): 175.6, 161.4, 157.3, 149.1, 143.6,

135.5, 128.7, 122.4, 121.4, 116.9, 116.4, 113.9, 106.9, 103.5, 76.3,

Combined yield for compounds 53f and 54f (yellow oil, 380 mg/

83%).

(5,6-Dimethoxy-2,2-dimethyl-3,4-dihydrobenzopyran-7-

62.4, 27.8. HRMS (ESI) m/z calcd for

C

₁₉H₁₇O₅[M+H]⁺:

yl)(2-hydroxy-4,5-dimethoxyphenyl)methanone (53f). EIMS m/z

(%): 403 (1, [M+1]⁺), 402 (4, [M]⁺), 372 (20), 371 (100), 315 (7),

303 (4), 301 (5), 287 (5), 222 (3), 207 (5), 180 (9), 166 (5), 151

(4), 125 (4), 69 (5), 53 (3). (5,6-dimethoxy-2,2-dimethyl-3,4-dihy-

325.10705; found: 325.10695.

8-(Diethylamino)-5-methoxy-2,2-dimethylpyrano[2,3-b]xan-

then-11(2H)-one (48g) as a yellowish solid (20 mg/44%). MP: 56–

2958

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

drobenzopyran-8-yl)(2-hydroxy-4,5-dimethoxyphenyl)metha-

none (54f). EIMS m/z (%): 403 (2, [M+1]⁺), 402 (8, [M]⁺), 221 (6),

207 (8), 182 (10), 181 (100), 125 (6), 110 (3), 95 (3).

Combined yield for compounds 53g and 54g (yellow oil,

250 mg/69%). Compounds decomposed with the conditions used

in the GC–MS.

incubated for 24 h. Cells were then treated with serial dilutions of

the different compounds (ranging from 0 to 150 M whenever pos-

l

sible). Following 48 h of incubation, the effect of the compounds in

the growth of the different cell lines was analyzed using the Sulfo-

rhodamine B (SRB) assay, as adopted by the National Cancer Insti-

tute (NCI, USA).^42,43Brieﬂy, 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 SRB

staining, plates were washed with 1% acetic acid, the bound dye

was then solubilized with 10 mM Tris Base and absorbance was

measured at 510 nm in a microplate reader (Biotek Instruments

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 vehicle solvent (DMSO) on the

growth of these cell lines was evaluated in preliminary experi-

ments, by exposing untreated control cells to the maximum con-

centration of DMSO used in each assay (0.25%).

4.11.4. Synthesis of xanthones 55a, 55f and 55g

Same experimental procedure using microwave heating de-

scribed in Section 4.10.4.

9-Chloro-5-methoxy-2,2-dimethyl-3,4-dihydropyrano[2,3-b]

xanthen-11(2H)-one (55a) as a white solid (105 mg/37%). Mp:

167–168 °C. IR m_max(cm^ꢀ1) (KBr): 2976, 2933, 1841, 1658, 1615,

1466, 1432, 1120, 1075, 942, 821. ¹H NMR (300.13 MHz, CDCl₃) d

(ppm): 8.28 (d, J = 2.6), 7.65 (dd, J = 8.9, 2.6), 7.52 (d, J = 8.9), 7.48

(s), 4.09 (s, OCH₃), 2.95 (t, J = 6.8, 2H), 1.89 (t, J = 6.8, 2H), 1.40 (s,

6H). ¹³C NMR (75.47 MHz, CDCl₃): 175.6, 154.0, 150.9, 146.9,

143.2, 134.4, 129.2, 125.9, 124.1, 121.9, 121.2, 119.7, 107.2, 74.4,

61.1, 31.5, 26.6, 18.2. EIMS m/z (%): 346 (2, [M+2]⁺), 345 (9,

[M+1]⁺), 344 (16, [M]⁺), 290 (24), 289 (51), 288 (50), 259 (34),

231 (36), 217 (51), 168 (57), 161 (29), 139 (52), 126 (84), 77

(33), 75 (58), 74 (41), 63 (100), 51 (61). HRMS (ESI) m/z calcd for

4.12.3. Statistical analysis

All experimental data are presented as means SE from at least

three independent experiments.

C

₁₉H₁₈ClO₄[M+H]⁺: 345.08881, found: 345.08865.

5,8,9-Trimethoxy-2,2-dimethyl-3,4-dihydropyrano[2,3-b]xan-

4.13. Lipophilicity

then-11(2H)-one (55f) as a light yellow solid (130 mg/46%). Mp:

216–217 °C. IR m_max(cm^ꢀ1) (KBr): 2979, 2933, 2830, 1642, 1615,

1479, 1447, 1432, 1270, 1097, 1004, 779. ¹H NMR (400.21 MHz,

CDCl₃) d (ppm): 7.65 (s), 7.49 (s), 6.96 (s), 4.06 (s, OCH₃), 4.03 (s,

OCH₃), 3.99 (s, OCH₃), 2.92 (t, J = 6.8, 2H), 1.86 (t, J = 6.8, 2H),

1.37 (s, 6H). ¹³C NMR (100.63 MHz, CDCl₃) d (ppm): 175.7, 155.2,

152.1, 150.5, 146.6, 145.9, 143.4, 122.8, 121.5, 114.3, 107.1,

105.4, 99.7, 74.3, 61.1, 56.5, 56.3, 31.8, 26.7, 18.2. EIMS m/z (%):

371 (25, [M+1]⁺), 370 (100, [M]⁺), 355 (8), 316 (11), 316 (62),

314 (50), 299 (8), 286 (8), 285 (26), 271 (16), 257 (10), 243 (10),

207 (7), 170 (11). HRMS (ESI) m/z calcd for C₂₁H₂₃O₆[M+H]⁺:

371.14891; found: 371.14872.

8-(Diethylamino)-5-methoxy-2,2-dimethyl-3,4-dihydropyrano

[2,3-b]xanthen-11(2H)-one (55g) as a light yellow solid (100 mg/

48%). Mp: 126–128 °C. IR m_max(cm^ꢀ1) (KBr): 2067, 2921, 2876,

1644, 1609, 1438, 1343, 1098, 773. ¹H NMR (300.13 MHz, CDCl₃)

d (ppm): 8.12 (d, J = 9.1), 7.47 (s), 6.69 (dd, J = 9.1, 2.5), 6.54 (d,

J = 2.5), 4.05 (s, OCH₃), 3.48 (q, J = 7.1, 4H), 2.92 (t, J = 6.9, 2H),

1.85 (t, J = 6.9, 2H), 1.37 (s, 6H), 1.27 (t, J = 7.1, 6H). ¹³C NMR

(75.47 MHz, CDCl₃): 175.2, 158.3, 152.6, 150.0, 145.6, 143.3,

128.1, 122.1, 122.0, 110.8, 109.2, 107.4, 96.1, 74.0, 61.1, 44.7,

4.13.1. Materials

The egg yolk phosphatidylcholine (EPC), HEPES, DMSO and NaCl

were acquired from Sigma–Aldrich, the hexadecylphosphocoline

(HDPC) from Cayman chemicals and the water used was double-

deionised with conductivity less than 0.1 l

S cm^ꢀ1. 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-

bottomed 96-well UV-plates acquired from BD Biosciences or fused

quartz cuvettes of 1.4 mL from Hellma.

4.13.2. Liposome preparation

Liposomes were prepared by evaporation to dryness with a nitro-

gen 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^ꢀ1, I = 0.1 mol L^ꢀ1withNaCl, pH 7.4) and the mix-

ture 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).

31.8, 26.7, 18.1, 12.5. HRMS (ESI) m/z calcd for

C₂₃H₂₈NO₄

[M+H]⁺: 382.20128; found: 382.20109.

4.13.3. Micelle preparation

4.12. Biological activity

4.12.1. Cell culture

Micelle solutions were prepared by dissolution of hexadec-

ylphosphocholine (HDPC) in buffer (HEPES: 10 mmol L^ꢀ1

,

I = 0.1 mol L^ꢀ1with NaCl, pH 7.4) and mixed by vortex.

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.

4.13.4. General procedure for K_pdetermination

The procedure used was adapted from the literature.⁴⁶and was

the following: (i) 3 lL of compound solubilized in DMSO (concen-

tration 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

lL were 0 (only buffer),

50, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000

the end, for the same concentration of xanthone, increasing con-

centrations of lipid were prepared; (iii) HEPES buffer was added

l

M. In

to make up 300 lL of ﬁnal volume; (iv) a blank was used for each

4.12.2. Cell growth inhibition assay

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 to 600 nm. For poorly soluble compounds, the

solutions were prepared with the same concentration but in

Cells were plated into 96-well tissue culture plates at appropri-

ate densities (MCF-7 and NCI-H460 at 5 ꢃ 10³cells/well, A375-C5

at 7.5 ꢃ 10³cells/well and HL-60 cells at 1 ꢃ 10⁴cells/well) and

C. M. G. Azevedo et al. / Bioorg. Med. Chem. 21 (2013) 2941–2959

2959

eppendorf (1.5 mL) instead. Each solution was mixed by vortex and

incubated for 30 min. The spectrum ranging from 240 to 600 nm

was traced individually in a double-beam spectrophotometer for

each sample at 37 °C. The compound concentration used in the

These data include MOL ﬁles and InChiKeys of the most important

compounds described in this article.

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4.14.2. Standard calibration curve determination

The standard calibration curve for each compound was pre-

pared using a stock solution of 20 mM in DMSO. The ﬁnal solutions

for each standard were prepared containing 15

lL of DMSO and

1485 L of buffer. An UV/Spectrum ranging from 240 to 500 nm

l

was traced for each standard and a calibration curve was estab-

lished using the second derivative. The concentrations of the stan-

dards ranged from 0.1 to 20 lM.

4.14.3. Solubility procedure

To 2 mL of an HEPES beffer (10 mmol L^ꢀ1, I = 0.1 mol L^ꢀ1with

NaCl, pH 7.4) was added each compound until a saturated solution

was obtained. The suspension of each compound (in triplicate) was

agitated at 37 °C for 24 h. Each sample was ﬁltered through a

0.1

l

m ﬁlter and 1.485

lL of each ﬁltrate was added to another

eppendorf containing 15

l

L of DMSO. The UV/Vis spectrum was

traced in a double-beam spectrophotometer and the concentration

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Acknowledgments

The authors would like to thank FCT—Fundação para a Ciência e

Tecnologia under the project CEQUIMED—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 Santander Totta for the

ﬁnancial support. The authors would also like to thank Sara Cravo

and Gisela Adriano for the technical support. A.G. Corrêa thanks

FAPESP and CAPES (Brazil) for ﬁnancial support. IPATIMUP is an

Associate Laboratory of the Portuguese Ministry of Science, Tech-

nology and Higher Education and is partially supported by FCT.

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Supplementary data

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Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.bmc.2013.03.079.

Article Doi

Multidimensional optimization of promising antitumor xanthone derivatives

DOI: 10.1016/j.bmc.2013.03.079

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