Synthesis and biological evaluation of (3,4,5-trimethoxyphenyl)indol-3-ylmethane derivatives as potential antivascular agents

Bioorganic & Medicinal Chemistry 14 (2006) 4410–4426

Synthesis and biological evaluation of

(3,4,5-trimethoxyphenyl)indol-3-ylmethane derivatives

as potential antivascular agents

a

b

c

a

b

´

Gregory Dupeyre, Guy G. Chabot, Sylviane Thoret, Xavier Cachet, Johanne Seguin,

c

a

b

´

Daniel Guenard, Franc¸ois Tillequin, Daniel Scherman,

Michel Koch^aand Sylvie Michel^a,*

^aUniversite´ Paris 5, Faculte´ des Sciences Pharmaceutiques et Biologiques, UMR 8638 CNRS,

Laboratoire de Pharmacognosie, 4 avenue de l’Observatoire, 75006 Paris, France

^bUniversite´ Paris 5, Faculte´ des Sciences Pharmaceutiques et Biologiques, U 640 INSERM-UMR 8151 CNRS,

Laboratoire de Pharmacologie Chimique et Ge´ne´tique, 4 avenue de l’Observatoire, 75006 Paris, France

^cCentre National de la Recherche Scientiﬁque, Institut de Chimie des Substances Naturelles,

avenue de la Terrasse, 91198 Gif-sur-Yvette, France

Received 26 October 2005; revised 16 February 2006; accepted 21 February 2006

Available online 10 March 2006

Abstract—Combretastatin A-4 (CSA-4), a stilbene derivative, is a potent vascular disrupting agent (VDA) with the structural

requirement of a cis-conﬁguration to maintain a molecular geometry and a correct orientation of both phenyl groups. A series

of indolic analogues of CSA-4 was synthesized by means of an eﬃcient strategy. Six compounds (20b, 25b–27b, 32b, and 35b) were

identiﬁed as potent inhibitors of tubulin polymerization and also displayed cytotoxic activities on B16 melanoma cells at a nano-

molar level. Both activities were well correlated with the ability to induce morphological changes of EAÆhy 926 endothelial cells.

In conclusion, the cis-stilbene skeleton of CSA-4 could conveniently be replaced by the 3-aroylindolic moiety, thus avoiding any

isomerization leading to inactive trans compounds.

1. Introduction

Microtubules of the cytoskeleton also play a major role

in maintaining cell shape. The elongated endothelial cells

of the neovasculature are particularly sensitive to drugs

that cause depolymerization of the microtubules like

combretastatin A-4 (CSA-4) 2 and combretastatin A-1

(CA-1) 3 (Fig. 1), which are both natural compounds iso-

lated from the bark of the South African bush willow tree

Combretum caﬀrum Kuntze (Combretaceae).⁹

The mitotic spindle, whose formation and activity are

required for chromosome segregation and cell division,

is constituted by microtubules generated by polymeriza-

tion of tubulin a,b-dimers.¹Many drugs which interfere

with the dynamic behavior of the microtubules are

potential anticancer drugs.²Three main sites are

involved in the ligand binding on tubulin: the colchicine

1 site^3,4which is involved in binding of various natural

and synthetic compounds suggesting a high plasticity

of the target protein at this place,⁵the vinca alkaloid

site,⁶and the paclitaxel site.^7,8

Tumor metastasis, growth, and survival are highly

dependent on the development of new blood vessels.¹⁰

Therefore, several approaches have been explored to

selectively prevent the development of new blood vessels

in tumors, or to attack selectively the existing tumor vas-

culature with tubulin binding agents, of which CSA-4

emerged as the prototype. In contrast to drugs that

target tumor angiogenesis, CSA-4 targets the newly

formed tumor vasculature.^11–15

Keywords: Combretastatin A-4; Tubulin; 3-Aroylindole; EAÆhy 926

endothelial cells.

*

After administration of relatively non-toxic doses, CSA-

4 causes a rapid tubulin depolymerization of endothelial

Corresponding author. Tel.: +33 1 53 73 98 12; fax: +33 1 40 46 96

58; e-mail: sylvie.michel@univ-paris5.fr

doi:10.1016/j.bmc.2006.02.037

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

4411

derivative 9,³⁰the A-289099 (10)³¹and compound 11³²

developed by Abbott, the heterocombretastatin 12,³³

and the arylthioindole 13³⁴prepared, respectively, by

Medarde and De Martino (Fig. 2).

Other indolic agents, less obviously comparable to the

CSA-4 pattern, display powerful inhibitory activities of

tubulin polymerization: the indolyl-3-glyoxamide D-

24851 14,³⁵the 2-aroylindoles 15a–c³⁶discovered by

Baxter oncology, and the 3-formyl-2-phenylindole 16³⁷

designed by von Angerer.

The aim of the present study was to synthesize 3-aroy-

lindole analogues of CSA-4 in order to avoid the

problematic isomerization of the cis-conﬁguration.

The two-carbon linker of our parent compound was

maintained between the 3⁰,4⁰,5⁰-trimethoxyphenyl ring

and the phenyl part of the 3-indolyl moiety (Fig. 3).

This latter is thus considered as a mimic of CSA-4 B-

ring. To determine the optimal distance and angle

between the two rings, we chose to prepare indolylme-

thane derivatives with various substituted bridges

(X = O; S; H, OH; H, OCH₃; H, OCOCH₃; and H,

H). The inﬂuence of the substitution of the indolic

nitrogen was also studied. In order to mimic the

p-methoxy group, which is considered essential for

CSA-4 activity, derivatives bearing a 6-methoxy group

on the indole nucleus were prepared.

Figure 1. Structures of colchicines and combretastatin derivatives.

cells which induces morphological changes including

rounding up and membrane blebbing. This rapid col-

lapse in tumor blood ﬂow in vivo is most likely due to

morphological and functional changes associated with

endothelial cytoskeleton, similar to those that occur in

endothelial cells in vitro after short drug exposure to

CSA-4.¹⁶This cascade of events leads to extensive shut

down of tumor vasculature and consequently to tumor

cell necrosis.^17,18

The biological activities of the synthesized compounds

were assessed by the tubulin polymerization inhibition

assay, by their cytotoxicity against the B16 melanoma

cells, and also by their eﬀect on the morphology of

EAÆhy 926 endothelial cells,³⁸which are immortalized

human umbilical vein endothelial cells (HUVEC). We

report herein both the synthesis and biological activity

of 23 compounds. Six of them (20b, 25b–27b, 32b, and

35b) were identiﬁed as promising compounds on the

basis of their potent activities.

Combretastatins have received a great deal of atten-

tion¹⁹due to their relatively simple structures,²⁰high

potency as cytotoxic agents,²⁰and antivascular activi-

ty.^11,21Major problems are their poor water solubility

and the structural instability of the cis-conﬁguration

which is necessary for tubulin activity. To allow easier

administration in vivo, water-soluble derivatives have

been prepared, for example, the combretastatin A-4

sodium phosphate prodrug (CSA-4P) 4 and the combre-

tastatin A-1 sodium diphosphate prodrug (CA-1P or

Oxi4503) 5, which are both developed by Oxigene as

vascular-disrupting agents (VDAs).¹²

2. Chemistry

As a result of the SAR studies in the CSA-4 series, it was

deduced that a 3,4,5-trimethoxyphenyl and a 4-meth-

oxy-3-X-substituted-phenyl system (with X = OH and

NH₂), linked by a two-atom bridge and a cis-conﬁgura-

tion allowing a dihedral angle of about 66ꢁ between the

two rings,^22,23are the common structural characteristics

most important for the activity of these compounds.

These structural requirements have been achieved in

derivatives and analogues of diverse types, either con-

serving the cis oleﬁn, bearing an oleﬁn stabilized in the

cis conﬁguration by added substituents²⁴or bearing an

oleﬁn involved in a more or less unsaturated cycle.^19,25,26

Formal replacements of A- or B-ring by heteroaromatic

groups have been synthesized. Only a few results con-

cerning compounds bearing an indolic part have been

previously published. The indole nucleus has been used

to obtain cis-restricted analogues of CSA-4 and some

2,3-diarylindoles have been synthesized by Medarde

(6),²⁷Flynn and Pinney (7 and 8).^28,29The B-ring can

also be replaced by an indolyl part like the 5-indolyl

The key step of our synthesis was a Friedel–Crafts acyl-

ation of the corresponding N-benzenesulfonyl-1H-in-

dole (i.e., 19a,b) with the 3,4,5-trimethoxybenzoyl

chloride (Scheme 1). This reaction was quickly per-

formed in methylene chloride in a low excess of alumi-

num chloride at room temperature to aﬀord 20a,b with

a very good yield (80% and 86% respectively) due to

the phenylsulfonyl group which enhances the selectivity

of the substitution on the 3-position of the indole

nucleus.

The non-commercial 6-methoxy-N-benzenesulfonyl-1H-

indole 19b was prepared by means of a modiﬁed Bischler

strategy,^39,40optimized in order to obtain large amount

of 19b with an improved overall yield (Scheme 2). Ben-

zenesulfonylation of m-anisidine gave 17b in a quantita-

tive yield. Sulfonamide 17b was alkylated by the

bromoacetaldehyde diacetal in the presence of NaH to

aﬀord 18b. Finally, a boron triﬂuoride etherate promot-

ed cyclization aﬀords 19b in a quantitative yield.

4412

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

Figure 2. Structures of cis-restricted analogues of CSA-4.

dride in methanol at room temperature. Methylenes

24a,b were obtained from the corresponding carbinol

by reduction with triethylsilane in triﬂuoroacetic acid,

conditions previously published for the reduction of 3-

aroylindoles.⁴¹Two other modiﬁcations were obtained

from the carbinol 21a due to the extreme lability of its

benzylic hydroxyl group: the substitution by a methoxy

group (22a) occurred in methanol in the presence of

p-toluene sulfonic acid and the substitution by an acet-

oxyl group (23a) occurred in a mixture of acetic acid

and acetic anhydride in the presence of 4-DMAP (these

conditions also probably lead to a direct acetylation

of carbinol). Nevertheless, these two latter types of

substitution were not attempted on the 6-substituted ser-

ies due to the relative lack of stability of these products.

2.2. Deprotection of the 1-position of the indole nucleus

Figure 3. Modiﬁcations of the 3-indolyl methane skeleton.

The nitrogen substituent was also modiﬁed (Scheme 3).

Carbonyls 20a,b were easily deprotected in reﬂuxing

aqueous methanol in the presence of K₂CO₃to give

25a,b. Deprotection of the carbinol 21a in these condi-

tions led to the isolation of 1H-indole as major product.

The instable carbinol function probably underwent a

2.1. Modiﬁcation of the bridge

Carbinols 21a,b were obtained in a quantitative yield by

reduction of the carbonyl group with sodium borohy-

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

4413

Scheme 1. Synthesis of 3-aroyl indoles 20a and 20b.

Scheme 2. Synthesis of 6-methoxyindole 19b. Reagents and conditions: (a) PhSO₂Cl, pyridine, CH₂Cl₂, rt, 98%; (b) BrCH₂CH(OEt)₂, NaH, anhyd

DMF, reﬂux, 82%; (c) BF₃ÆEt₂O, CH₂Cl₂, rt, 97%.

Scheme 3. Modiﬁcations of the bridge and deprotection of the indole nucleus. Reagents and conditions: (a) NaBH₄, MeOH/THF, rt, 92–95%; (b)

PTSA, MeOH, rt, 85%; (c) Ac₂O, AcOH, 4-DMAP, rt, 95%; (d) Et₃SiH, TFA, CH₂Cl₂, 75–90%; (e) K₂CO₃, MeOH/H₂O, reﬂux, 94–99%; (f) KOH,

MeOH/H₂O, reﬂux, 88% or MeONa (0.1 M), MeOH, reﬂux, 81%; (g) Lawesson’s reagent, THF, rt, 91%.

retro-addition reaction in this basic medium. Conse-

quently, deprotections of neither the carbinol 21b in

the 6-methoxy series nor derivatives 22a and 23a were

not investigated anymore. Deprotection of the methyl-

enes 24a,b was only achieved with a stronger base

(KOH) to aﬀord 26a,b. This contrasts with the easier

deprotection of carbonyls 20a,b, where the carbonyl

group contributes to the formation of a more stable

indolyl anion by charge delocalization. The thioketone

27b was synthesized from the deprotected carbonyl 25b

using Lawesson’s reagent in THF at room temperature

in a quantitative yield.

(28a), –CH₂Ph (29a), and –CH₂Ph–p-OCH₃(30a); (b)

electron-withdrawing groups: –COCH₃(31a), –COPh

(32a,b), –COPh–p-OCH₃(33a), –SO₂Ph–p-OCH₃(34a),

and –COOPh (35a,b). All compounds were synthesized

according to the same general method (Scheme 4).

Carbonyls 25a,b were deprotonated by KOH in EtOH.

After evaporation of the solvent, the corresponding salts

were taken up with acetone or DMF and alkyl or acyl

halides were added.

3. Biological activity

2.3. Modiﬁcation of the 1-position of the indole nucleus

(N-substitution)

Twenty-three CSA-4 analogues were synthesized and

evaluated for their biological activities, including the

inhibition of tubulin polymerization (ITP), the cytotox-

icity against murine B16 melanoma cells, and their

eﬀects on the morphology of EAÆhy 926 endothelial cells

Diverse groups were introduced on the 1-position of

the indole ring: (a) electron-donating groups: –CH₃

4414

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

Scheme 4. Modiﬁcations of the N-position of the indole nucleus. Reagents and conditions: (a) KOH, EtOH, rt; (b) R₁X or R₁COX, acetone or

DMF, rt, 65–96% in one-pot.

(modiﬁed HUVEC), which are considered as a good

predictor of potential in vivo antivascular eﬀects.^42,43

(rounding up) of EAÆhy 926 endothelial cells at a rather

high concentration of 25 lg/mL for a 2 h exposure

time.

3.1. Modiﬁcations of the methane bridge of the 1H-indol-

3-yl-(3,4,5-trimethoxyphenyl) methane

The inﬂuence of a modiﬁcation of the bridge was ﬁrst

evaluated. Among the compounds tested, only the carbi-

nol 21a had a close ITP value of 9 compared to 20a, but

this modiﬁcation was accompanied by a 2-fold decrease

in cytotoxic activity. Eﬀects on the morphology of EAÆhy

926 endothelial cells were similar to 20a, with rounding

up activity noted at a high concentration of 25 lg/mL.

Compound 20a (R₁= SO₂Ph and R = O) was our starting

scaﬀold and therefore all the modiﬁcations were aimed at

improving the activity of that scaﬀold (Table 1).

Compound 20a presented a medium inhibition of tubulin

polymerization activity with an ITP value of 11 (results

expressed in terms of the relative IC₅₀/IC_50colchicineare

summarized in Table 1). Analogue 20a was moderately

cytotoxic in the micromolar range (8.4 lM) against

B16 melanoma cells and could alter the morphology

Other derivatives, the methoxy 22a, the acetoxy 23a, and

the methylene 24a, were rather ineﬀective on tubulin

inhibition (Table 1). However, compounds 23a and

24a presented cytotoxic activity similar to 20a. Interest-

ingly, the acetoxy derivative 23a exhibited a slightly

better activity on EAÆhy 926 cell rounding up

(at 2.5 lg/mL) than the starting compound 20a.

Table 1. Biological consequences of modiﬁcations of the methane

bridge for (1H-indol-3-yl)-3,4,5-trimethoxyphenyl-methane com-

pounds 20a–24b

Compound ITP

(IC₅₀/IC_50colchicine

Cytotoxicity Morphological

c

a,b

These results show the diﬃculty in trying to establish

structure–activity relationships for these types of modi-

ﬁcations since good ITP and cytotoxicity values are

not always correlated. No apparent correlation was also

noted with regard to morphological eﬀects on EAÆhy 926

endothelial cells. Therefore, the global activity of our

synthon was not markedly enhanced by replacing

the carbonyl group. It can be hypothesized that this

carbonyl group interacts at its binding site or that it

allows a better positioning between the two rings. It

was therefore decided to maintain the carbonyl group

on the bridge and to modify the nature of the indolic

nitrogen substituent.

)

IC₅₀(lM) activity on

EAÆhy 926^d

20a

21a

22a

23a

24a

11

9

8.4 0.9

16.2 0.8

20.9 6.7

7.5 1.2

6.1 0.7

1+

—

2+

—

Inactive

^aInhibition of tubulin polymerization (ITP).

^bCompounds were designated as ‘inactive’ when the measured ratio

was greater than 57.

^cIC₅₀SEM of 3–7 independent triplicate experiments (B16 melano-

ma cell line).

^dMorphological activity (rounding up) on modiﬁed HUVEC (EAÆhy

926) is expressed as the lowest concentration at which cell rounding

up was observed after a 2 h incubation period with the test com-

pound. The compounds were tested at concentrations ranging from

0.00025 to 25 lg/mL. The number of + sign indicates rounding up

more than 10% of cells at the following concentrations: 1+ at 25; 2+

at 2.5; 3+ at 0.25; 4+ at 0.025; and 5+ at 0.0025 lg/mL. A minus sign

(ꢀ) indicates no rounding up.

3.2. Modiﬁcations of the N-position of the 1H-indol-3-yl-

(3,4,5-trimethoxyphenyl) methane

Various nitrogen substitutions of the N-position were

studied (Scheme 4). As previously observed for the

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

4415

modiﬁcations of the methane bridge, correlations be-

tween the three biological tests were not clear, except

for compounds 29a and 30a which are devoid of signif-

icant activities.

of the indole nucleus was prepared in an attempt to

mimic the p-OCH₃of the CSA-4. Indeed, this substitu-

ent is well known to play a major role on the activities.²⁰

To facilitate comparisons, the results of the two series

are described in Table 3. The biological activity of col-

chicine and CSA-4 was included as reference

compounds.

In terms of ITP, the benzenesulfonyl 20a and the phenyl

carbamate 35a gave the best results (11 and 5, respec-

tively) (Table 2). In terms of cytotoxicity the deprotected

indole 25a (5.0 lM) and the indoles bearing a carbonyl

group (32a: COPh, 33a: p-OMe–COPh, and 35a:

COOPh) yielded the best results (3.5, 5.3, and 5.2 lM,

respectively). Only the phenyl carbamate 35a exhibits a

rather good correlation between both cytotoxic and

antitubulin activities (IC₅₀of 5.2 lM and ITP of 5). Sub-

stitution of the benzene sulfonyl by a methoxy group in

para position decreases ITP and cytotoxic activities

(compare 20a and 34a), indicating that the arylsulfonyl

group does not mimic the B-ring of CSA-4.

For most compounds, the presence of a 6-methoxy

group markedly increased the ITP activity compared

to the corresponding compound with no substituent

(Table 3). Several of the 6-methoxylated compounds

presented lower ITP values compared to the reference

colchicine, for example, compounds 25b, 26b, 27b, and

35b, which were 2.5 (ITP = 0.4) to 5 (ITP = 0.2) times

better than colchicine at inhibiting tubulin polymeriza-

tion. These values are very close to that measured for

CSA-4 (ITP = 0.7).

Derivatives 25a, 31a, and 32a were found to be the more

eﬃcient on cell shape rounding up of EAÆhy 926 endo-

thelial cells since they were still active at the concentra-

tion of 2.5 lg/mL (or 2+).

In parallel to their potent tubulin polymerization inhibi-

tion activity, the 6-methoxylated compounds 25b–27b

and 35b also presented nanomolar IC₅₀values against

the B16 melanoma cell line of 13, 45, 24, and 23 nM,

respectively (Table 3). These IC₅₀values were close to

that of colchicine (31 nM) or of reference compound

CSA-4 (3 nM). Compounds 20b and 32b present despite

their moderate ITP value a potent cytotoxicity with

respective values of 211 nM and 35 nM. The intermedi-

ate 19b, also evaluated under the same conditions, was

devoid of activity (ITP value of 32 lM, IC₅₀(B16) of

67 lM).

3.3. 6-Methoxy-1H-indol-3-yl-(3,4,5-trimethoxyphenyl)

methane derivatives

The above SAR showed that the best biologically active

molecules bear a carbonyl group on the bridge, and a

carbonyl, a sulfonyl, or no substituent on the nitrogen.

On the basis of these previous results, a new series of

compounds bearing a methoxy group on the 6 position

In addition, compounds which were most cytotoxic

were also the ones that presented the best activity on

the morphology of EAÆhy 926 endothelial cells, with

rounding up activity still observed at concentrations

as low as 25 ng/mL. For the best compounds, the cal-

culated concentrations for the rounding up of 50% of

EAÆhy 926 cells were as follows: CSA-4 = 1 ng/mL

(3.0 nM); compound 25b = 2 ng/mL (6.2 nM); com-

Table 2. Biological consequences of modiﬁcations of the N-position

for (1H-indol-3-yl)-3,4,5-trimethoxyphenyl-methane compounds (20a,

25a, and 28a–35a)

Compound ITP

(IC₅₀/IC_50colchicine

Cytotoxicity Morphological

c

a,b

)

IC₅₀(lM)

activity on

EAÆhy 926^d

pound 27b = 19 ng/mL

35b = 2 ng/mL (4.6 nM).

(52.6 nM);

compound

20a

25a

28a

29a

30a

31a

32a

33a

34a

35a

11

57

8.4 0.9

5.0 1.0

7.0 1.6

22.7 1.9

36.0 5.2

7.3 3.2

3.5 0.7

5.3 1.1

23.9 4.7

5.2 2.2

1+

2+

1+

—

Figure 4 depicts representative changes in the mor-

phology of EAÆhy 926 cells after exposure to CSA-4

and to compounds 27b and 35b. Compared to control

DMSO-treated cells (panel 1), it can be observed that

within 2 h, the cells presented a characteristic rounding

up morphology for CSA-4 (panel 2) used as a positive

control. Similarly, the synthesized compounds 27b and

35b (panels 3 and 4, respectively) presented a rounding

up aspect resembling that of CSA-4 (panel 2). The

dual staining used for tubulin and actin allowed us

to visualize the change in the distribution of these

cytoskeleton components: in control cells, tubulin

was relatively uniformly distributed within the entire

cell area, whereas actin was diﬀusely distributed in

the cell periphery; in cells treated with CSA-4, or com-

pounds 27b and 35b, the rounded cell area was mark-

edly reduced compared to controls, and the tubulin

was localized close to the nuclear margin, whereas ac-

tin was localized on the cell periphery with frequent

blebbing observed.

40

Inactive

41

35

31

24

5

—

2+

1+

^aInhibition of tubulin polymerization (ITP).

^bCompounds were designated as ‘inactive’ when the measured ratio

was greater than 57.

^cIC₅₀SEM of 3–7 independent triplicate experiments (B16 melano-

ma cell line).

^dMorphological activity (rounding up) on modiﬁed HUVEC (EAÆhy

926) is expressed as the lowest concentration at which cell rounding

up was observed after a 2 h incubation period with the test com-

pound. The compounds were tested at concentrations ranging from

0.00025 to 25 lg/mL. The number of + sign indicates rounding up

more than 10% of cells at the following concentrations: 1+ at 25; 2+

at 2.5; 3+ at 0.25; 4+ at 0.025; and 5+ at 0.0025 lg/mL. A minus sign

(ꢀ) indicates no rounding up.

Table 3. Biological consequences of modiﬁcations on the N- and the C-6 position of (1H-indol-3-yl)-3,4,5-trimethoxyphenyl-methane

Substitution R₁= H (a)

Cytotoxicity IC₅₀(lM)^c

R₁= OCH₃(b)

Compound

ITP^a,b

EAÆhy 926^d

Compound

ITP^a

Cytotoxicity

IC₅₀(lM)^c

EAÆhy 926^d

R₁= H

R = O

R = S

R = H, H

25a

26a

57

5.0 1.0

6.1 1.0

2+

25b

27b

26b

0.4

0.2

0.013 0.004

0.024 0.007

0.045 0.006

5+

4+

Inactive

R₁= SO₂Ph

R = O

R = H, OH

R = H, H

20a

21a

24a

11

9

Inactive

8.4 0.9

16.2 0.8

6.1 0.7

1+

—

20b

21b

24b

12

13

4

0.211 0.040

>51.70

7.0 0.4

4+

1+

2+

R₁= COPh

R = O

32a

35a

31

5

3.5 0.7

5.2 2.2

2+

1+

32b

35b

11

0.035 0.022

0.023 0.010

3+

5+

R₁= COOPh

0.2

Reference compounds

ITP^a

Cytotoxicity

IC₅₀(lM)

Morphological activity

on EAÆhy 926^d

c

Colchicine

CSA-4

1.0^e

0.7^f

0.031 0.003

0.003 0.001

3+

5+

^aInhibition of tubulin polymerization (ITP).

^bCompounds were designated as ‘inactive’ when the measured ratio was greater than 57.

^cIC₅₀SEM of 3–7 independent triplicate experiments (B16 melanoma cell line).

^dMorphological activity (rounding up) on modiﬁed HUVEC (EAÆhy 926) is expressed as the lowest concentration at which cell rounding up was observed after a 2 h incubation period with the test

compound. The compounds were tested at concentrations ranging from 0.00025 to 25 lg/mL. The number of + sign indicates rounding up of more than 10% of cells at the following concentrations: 1+ at

25; 2+ at 2.5; 3+ at 0.25; 4+ at 0.025; and 5+ at 0.0025 lg/mL. A minus sign (ꢀ) indicates no rounding up.

^eIC_50colchicinevaries from 1.8 to 6.7 lM for the diﬀerent experiments according to the tubulin concentration.

^fLit., IC₅₀(CSA-4) = 2.2 0.2 lM and IC₅₀(colchicine) = 3.2 0.4 lM.³⁴

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

4417

Figure 4. Representative photographs depicting the morphological aspects of EAÆhy 926 endothelial cells after treatment with analogues of

combretastatin A-4 (CSA-4). (1) Control cells exposed to 1% DMSO; (2) CSA-4 at 25 ng/mL; (3) compound 27b at 25 ng/mL; and (4) compound 35b

at 25 ng/mL. Cells were exposed to the compounds for 2 h and ﬁxed in ethanol. Tubulin (green) was visualized by employing a primary antibody to

tubulin and a secondary antibody coupled to ﬂuorescein. Actin (red) was visualized with phalloidin coupled to rhodamine. Photographs were taken

at a magniﬁcation of 360· under ﬂuorescent illumination.

4. Discussion

of a sp²center as linker between the two aryl parts of the

molecule. Interestingly, the phenyl carbamate 35b

exhibits potent eﬀects in both tests, whereas the benzam-

ide 32b displays a good cytotoxic activity and a moderate

inhibition of tubulin polymerization. However, possibili-

ty that these protecting groups were cleaved in the

conditions of experiments, leading to unsubstituted

indoles, could not be discarded. Indeed, the N-1 bond is

known to be very liable in the presence of a carbonyl

group at the 3-position of the indole nucleus.⁴⁶

Among the synthesized compounds, the most biological-

ly active were the ones bearing a sp²center between both

aromatic groups (except for 26b) and furthermore a

6-methoxy group, which markedly increased tubulin

polymerization inhibition, cytotoxic activities, and mor-

phological eﬀects on EAÆhy 926 cells. Compounds 20b

and 32b and particularly 25b, 27b, and 35b were found

to display the stronger activities. The SAR observed in

this study thus conﬁrms further the interest of substitut-

ing the indole nucleus with a 6-methoxy group to

increase its biological potency. In the course of the

biological evaluation of our molecules, Liou et al., in

the continuation of their previous work on phenstatin

analogues,⁴⁴have also recovered the potent activity of

compounds 25b and 26b. The compound 25b was submit-

ted to further in vivo development as an antimitotic

antitumoral agent under the reference number

BPR0L075.⁴⁵For both compounds 25b and 26b, good

cytotoxic and ITP activities were also recovered. Similar

potent activities were observed with the thioketone 27b

and the carbamate 35b, showing that the bridge and the

N-position accept some structural modiﬁcations. Results

obtained with 27b underline the importance for activities

Nevertheless, for the most active compounds, a good

correlation was observed between both their cytotoxic

and antitubulin activities.

The study of the morphological changes on EAÆhy 926

cells allowed us to identify six potent compounds pre-

senting a score greater than 3+ (colchicine value): 20b,

25b–27b, 32b, and 35b. Three of them, 25b, 27b, and

35b, were as eﬃcient as CSA-4 with a score of 5+. These

results are in good agreement with the aforementioned

data. It is the ﬁrst report on the morphological eﬀects

of 3-aroylindole derivatives on endothelial cells,

suggesting that these compounds may act in vivo as

antivascular antitumoral agents.

4418

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

5. Conclusion

(CDCl₃) d 3.68 (s, 3H, C₃–OCH₃), 6.61 (m, J_4–5=

8.1 Hz, 1H, H₄), 6.73 (d, J_6–5= 8.1 Hz, H₆), 6.77 (br s,

Of twenty-three CSA-4 analogues synthesized and eval-

uated for their biological activities, four new compounds

(20b, 27b, 32b, and 35b) emerged as leads that presented

activities as good as CSA-4, in terms of inhibition of

tubulin polymerization and cytotoxicity on B16 melano-

ma cells. In addition, these compounds were as active as

CSA-4 at altering the morphology of EAÆhy 926 endo-

thelial cells and therefore merit further testing in vivo

to ascertain their potential antivascular antitumor

activity.

1H, H₂), 7.08 (t, J_5–6= 8.1 Hz, 1H, H₅), 7.39 (m, 2H,

0

H₃and H₅), 7.48 (m, 1H, H₄), 7.87 (d, J_{2 –3}¼ 7:40

0

Hz, 2H, H₂and H₆), 8.01 (s, 1H, NH, D₂O exch.);

¹³C NMR (CDCl₃) d 55.4 (C₃–OCH₃), 106.9 (C₂),

0

111.0 (C₄), 113.4 (C₆), 127.4 (C₂and C₆), 129.2 (C₃

0

and C₅), 130.1 (C₅), 133.1 (C₄), 138.0 (C₁), 139.0

0

+

(C₁), 160.3 (C₃); MS (ESI) m/z 308 [M+2Na] , 286

0

[M+Na]⁺; Anal. (C₁₃H₁₃NO₃S) found: C, 59.05; H,

4.89; N, 5.21; calcd: C, 59.30; H, 4.98; N, 5.32.

6.2. N-(2,2-Diethoxyethyl)-N-(3-methoxyphenyl)benzene-

sulfonamide (18b)⁴⁰

6. Experimental

To a stirred solution of 17b (427 mg, 1.62 mmol) in

anhydrous DMF (20 mL), NaHCO₃(269 mg,

1.94 mmol) was added at room temperature. The sus-

pension was stirred for a few minutes, and then, a solu-

tion of bromoacetaldehyde diethyl acetal (584 lL,

3.88 mmol) was added dropwise, and the mixture was

kept at 110 ꢁC. After 20 h, 1.62 mmol (244 lL) of the

acetal was added and the mixture was kept at 110 ꢁC

for 20 h. Another 1.62 mol (244 lL) of acetal was added

and heating was continued for an additional 8 h. Then,

DMF was removed under reduced pressure and the res-

idue was taken up with CH₂Cl₂. The organic solution

was washed with water and brine, dried (MgSO₄), and

concentrated in vacuo to yield 565 mg of analytically

pure 18b (82%) as an amber oil. IR (NaCl ﬁlm) m⁰

(cm^ꢀ1) 1605, 1485, 1450, 1350, 1285, 1260, 1205, 1170,

1060, 955, 705, 690; ¹H NMR (CDCl₃) d 1.38 (t,

Melting points were determined on a LEICA VM micro-

scope equipped with a heating stage and are uncorrect-

ed. Infrared spectra were obtained with a Nicolet 510

FT-IR apparatus. UV spectra were obtained with a

BECKMAN DU 640 spectrophotometer apparatus.

NMR spectra were recorded at 300 or 400 MHz (¹H

NMR) and at 75 or 100 MHz (¹³C NMR) with an AC

300 and an Avance 400 BRUKER spectrometers; chem-

ical shifts are given in parts per million (ppm, d) relative

to solvent peaks as internal standards (d: CDCl₃:

7.27 ppm (¹H), 77 ppm. (¹³C); DMSO-d₆: 2.50 ppm

(¹H), 40.6 ppm (¹³C); acetone-d₆: 2.05 ppm (¹H), 29.8

and 206.0 ppm (¹³C)); ¹H and ¹³C signals were unambig-

uously attributed by 2D NMR experiments; coupling

constants are given in Hertz (Hz, J). Mass spectra

(MS) were measured with a Nermag R 10-10C mass

spectrometer (DIC/NH₃) or with a ZQ2000 Waters mass

spectrometer (ESI). Elemental analyses were performed

J = 7.0 Hz,

6H,

2· OCH₂CH₃),

3.50

(ddd,

²J = 14.1 Hz, J = 5.5 Hz, J = 5.5 Hz, 2H, CH₂CH),

3.64 (m, 4H, 2· OCH₂CH₃), 3.73 (s, 3H, C₃–OCH₃),

4.63 (t, J = 5.5 Hz, 1H, CH₂CH), 6.55–6.65 (m, 2H,

H₂and H₄), 6.83 (m, J_6–5= 8.0 Hz, 1H, H₆), 7.18 (t,

´

at the Laboratoire de microanalyses (Universite Paris

7—Denis Diderot, Paris, France). Flash chromatogra-

phy was performed using silica gel (SDS 60 ACC 35–

70 lM). The reactions were monitored by thin-layer

chromatography (TLC) using Merck Kieselgel 60 F₂₅₄

silica gel; zones were detected visually under ultraviolet

irradiation (254 and 366 nm) and read after spraying

with sulfuric vanillin followed by heating. All solvents

were dried according to standard procedures. All re-

agents were used as purchased without further treatment

unless otherwise stated.

0

J_5–6= 8.0 Hz, 1H, H₅), 7.46 (m, 2H, H₃and H₅), 7.57

0

(m, 1H, H₄), 7.63 (br d, J = 8.5 Hz, 2H, H₂and H₆);

¹³C NMR (CDCl₃) d 15.2 (OCH₂CH₃), 53.3 (CH₂CH),

55.3 (C₃–OCH₃), 62.2 (OCH₂CH₃), 100.9 (CH₂CH),

0

113.9 (C₂), 114.5 (C₄), 120.7 (C₆), 127.7 (C₂and C₆),

0

128.7 (C₃and C₅), 129.4 (C₅), 132.7 (C₄), 138.2 (C₁),

141.2 (C₁), 159.8 (C₃); MS (ESI) m/z 402 [M+Na]⁺.

6.3. N-Benzenesulfonyl-6-methoxy-1H-indole (19b)⁴⁰

6.1. N-Benzenesulfonamide-3-methoxyaniline (17b)

To a stirred mixture of 18b (188 mg, 0.49 mmol) in

CH₂Cl₂(7 mL), boron triﬂuoride etherate (93.5 lL,

0.74 mmol) was added dropwise at 0 ꢁC under argon.

The reaction monitored by TLC (cyclohexane/EtOAc,

(7:3)) was quenched after 15 min with a solution of sat-

urated aqueous NaHCO₃. The aqueous layer was

extracted with CH₂Cl₂and then washed with brine,

dried (MgSO₄), and concentrated in vacuo to give with-

out further puriﬁcation 138 mg of 19b (97%) as a crystal-

line transparent solid. Recrystallization from CH₂Cl₂

and MeOH gave large prismatic crystals. Mp 132–

135 ꢁC (lit.⁴⁷mp 140–142 ꢁC (CH₂Cl₂/n-hexane); lit.,⁴⁰

To a mixture of anhydrous pyridine (6 mL) and dry

CH₂Cl₂(25 mL), m-anisidine (1.82 mL, 16.0 mmol)

was added at room temperature. After a few minutes

of stirring, benzenesulfonyl chloride (2.5 mL,

19.5 mmol) was added dropwise. The reaction moni-

tored by TLC (toluene/EtOAc (5:5)) was stopped after

5 min. The solvents were evaporated to dryness and

the residue was taken up with CH₂Cl₂. The organic solu-

tion was washed with water, HCl 10%, water again, and

brine dried (MgSO₄), and concentrated in vacuo to yield

4.13 g of analytically pure 17b (98%). Recrystallization

from a 1:1 mixture of CH₂Cl₂and MeOH gave large

prismatic crystals. Mp 75–77 ꢁC; IR (NaCl ﬁlm) m⁰

(cm^ꢀ1) 3250, 3009, 2963, 2937, 1591, 1448, 1407, 1324,

mp 137–139 ꢁC (ether)); IR (NaCl ﬁlm) m⁰(cm^ꢀ1

)

3138, 3111, 2975, 2847, 1618, 1580, 1446, 1363, 1281,

1216, 1180, 1118, 997, 858, 810, 725; H NMR (CDCl₃)

1

1283, 1139, 1083, 1047, 939, 893, 754, 688; H NMR

d 3.89 (s, 3H, C₆–OCH₃), 6.59 (dd, J_3,2= 3.67 Hz,

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

4419

00 00

H₃), 7.54 (d, J_7,5= 2.3 Hz, 1H, H₇),7.62 (m, 1H, H₄),

J_3,7= 0.65 Hz, 1H, H₃), 6.87 (dd, J_5,4= 8.61 Hz,

J_5,7= 2.29 Hz, 1H, H₅), 7.39 (br d, J_4,5= 8.61 Hz, 1H,

00

7.92 (m, 2H, H₂and H₆), 7.95 (s, 1H, H₂), 8.12 (d,

0

H₄), 7.42–7.47 (m, 3H, H₃, H₅and H₂), 7.52–7.55 (m,

J_4,5= 8.8 Hz, 1H, H₄); ¹³C NMR (CDCl₃) d 55.8 (C₆–

2H, H₄and H₇), 7.85–7.89 (m, 2H, H₂and H₆); ¹³C

NMR (CDCl₃): d 55.9 (C₆–OCH₃), 98.0 (C₇), 109.3

(C₃), 112.7 (C₅), 121.9 (C₄), 124.5 (C_3a), 125.2 (C₂),

0

OCH₃), 56.3 (C_{3 –5}–OCH₃), 61.0 (C₄–OCH₃), 97.5

0

(C₇), 106.7 (C₂and C₆), 113.7 (C₅), 120.6 (C₃), 122.2

00

(C_3a), 123.4 (C₄), 126.9 (C₂and C₆), 129.6 (C₃and

00

0

126.8 (C₂and C₆), 129.3 (C₃and C₅), 133.9 (C₄),

00 0 00

C₅), 131.7 (C₂), 134.1 (C₁), 134.5 (C₄), 136.2 (C_7a),

0

00

0

137.7 (C_7a), 138.4 (C₁), 159.7 (C₆); MS (ESI) m/z 310

137.7 (C₁), 142.0 (C₄), 153.1 (C₃and C₅), 158.8 (C₆),

189.6 (CO); MS (ESI) m/z 504 [M+Na]⁺; Anal.

(C₂₅H₂₃NO₇S) found: C, 62.43; H, 4.71; N, 3.02; calcd:

C, 62.36; H, 4.81; N, 2.91.

[M+Na]⁺; Anal. (C₁₅H₁₃NO₃S) found: C, 63.05; H,

4.66; N, 4.98; calcd: C, 62.70; H, 4.56; N, 4.87.

6.4. (N-Benzenesulfonyl-1H-indol-3-yl)-(3,4,5-trimeth-

oxyphenyl) methanone (20a)

6.6. (N-Benzenesulfonyl-1H-indol-3-yl)-(3,4,5-trimeth-

oxyphenyl) methanol (21a)

To a magnetically stirred suspension of AlCl₃(517 mg,

3.88 mmol) in CH₂Cl₂at 25 ꢁC was added 3,4,5-trime-

thoxybenzoyl chloride (538 mg, 2.33 mmol), and the

mixture was stirred for 15 min to a red suspension. A

solution of 1-(phenylsulfonyl)indole 19a (500 mg,

1.94 mmol) in CH₂Cl₂was added dropwise; the mixture

was stirred at 25 ꢁC for 30 min and poured onto crushed

ice. The aqueous layer was extracted with CH₂Cl₂, and

the organic extract was washed with brine, saturated

aqueous NaHCO₃, and brine, dried (MgSO₄), and con-

centrated in vacuo. Flash chromatography using cyclo-

hexane/EtOAc (8:2) gave 707 mg of 20a (80%) as a

yellow powder. Recrystallization from CH₂Cl₂and

n-hexane gave transparent crystals. Mp 142–143 ꢁC;

UV (CH₂Cl₂) k_max(nm) 227.0 (loge = 4.48), 299.0

(loge = 4.23) nm; IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 2998, 2934,

To a magnetically stirred suspension of 20a (500 mg,

1.11 mmol) in a 20/1 mixture of MeOH and THF,

NaBH₄(84 mg, 2.22 mmol) was added portionwise at

room temperature. The reaction was monitored by

TLC (cyclohexane/EtOAc (5:5)) and stopped after

5 min. The solvents were evaporated in vacuo, and the

residue was taken up in CH₂Cl₂. The organic extract

was washed with water, then with brine, dried (MgSO₄),

and concentrated in vacuo to give 477 mg of analytically

pure 21a (95%). Recrystallization from THF and n-hex-

ane gave large prismatic crystals. Mp 140 ꢁC; UV

(CH₂Cl₂) k_max(nm) 227.0 (loge = 4.32); IR (NaCl ﬁlm)

m⁰(cm^ꢀ1) 3371, 2994, 2963, 2940, 2831, 1595, 1503,

1

1446, 1360, 1322, 1176, 1120; H NMR (DMSO-d₆) d

0

3.58 (s, 3H, C₄–OCH₃), 3.68 (s, 6H, C_{3 –5}–OCH₃),

0

2834, 1639, 1579, 1533, 1503, 1448, 1375, 1323, 1171,

5.84 (d, J_CH,OH= 4.0 Hz, 1H, CH), 5.97 (d,

0

1

0

1129; H NMR (CDCl₃) d 3.93 (s, 6H, C_{3 –5}–OCH₃),

J_OH,CH= 4.0 Hz, 1H, D₂O exch., OH), 6.73 (s, 2H, H₂

0

and H₆), 7.17 (dd, J_5,6= 7.7 Hz, J_5,4= 7.6 Hz,, 1H,

0

3.98 (s, 3H, C₄–OCH₃), 7.15 (s, 2H, H₂and H₆), 7.41

(t, J_5,4= 7.2 Hz, J_5,6= 7.2 Hz, 1H, H₅), 7.45 (dd,

H₅), 7.29 (dd, J_6,7= 8.3 Hz, J_6,5= 7.7 Hz, 1H, H₆),

00 00

7.53–7.56 (m, 3H, H₄, H₃and H₅), 7.66 (m, 1H,

00

J_6,5= 7.2 Hz, J_6,7= 8.2 Hz, 1H, H₆), 7.50 (m, 2H, H₅

00

and H₃), 7.62 (m, 1H, H₄), 7.95 (br d, J = 7.7 Hz,

00

H₄), 7.68 (s, 1H, H₂), 7.91 (br d, J_7,6= 8.3 Hz, 1H,

0

2H, H₂and H₆), 8.04 (d, J_7,6= 8.2 Hz, 1H, H₇), 8.07

H₇), 7.97 (br d, J = 7.7 Hz, 2H, H₂and H₆); ¹³C

00

(s, 1H, H₂), 8.26 (d, J_4,5= 7.2 Hz, 1H, H₄); ¹³C NMR

NMR (DMSO-d₆)

0

d

(C₄–OCH₃), 69.4 (CHOH), 104.7 (C₂and C₆), 114.3

0

56.9 (C_{3 –5}–OCH₃), 61.0

0

(CDCl₃) d 53.3 (C_{3 –5}–OCH₃), 61.0 (C₄–OCH₃), 106.7

0

(C₂and C₆), 113.2 (C₇), 120.5 (C₃), 122.9 (C₄), 124.8

0

(C₇), 122.3 (C₄), 124.1 (C₂), 124.3 (C₅), 125.8 (C₆),

00

(C₅), 126.0 (C₆), 127.0 (C₂and C₆), 128.5 (C_3a), 129.6

00

127.7 (C₂and C₆), 128.6 (C₃), 129.8 (C_3a), 130.8 (C₃

00

0

00

(C₃and C₅), 132.8 (C₂), 134.1 (C₁), 134.5 (C₄),

00 00 0

and C₅), 135.6 (C₄), 135.9 (C_7a), 137.5 (C₄), 138.1

00

135.1 (C_7a), 137.7 (C₁), 142.2 (C₄), 153.1 (C₃and

0

00

0

(C₁), 140.5 (C₁), 153.6 (C₃and C₅); MS (DIC/NH₃)

m/z 471 [M+NH₄]⁺; Anal. (C₂₄H₂₃NO₆S) found: C,

63.71; H, 5.09; N, 2.96; calcd: C, 63.56; H, 5.11; N, 3.09.

+

C₅), 189.5 (CO); MS (DIC/NH₃) m/z 452 [M+H] ,

0

312 [M+HꢀSO₂Ph]⁺; Anal. (C₂₄H₂₁NO₆S) found: C,

64.42; H, 4.59; N, 2.88; calcd: C, 63.8; H, 4.69; N, 3.10.

6.7. (N-Benzenesulfonyl-6-methoxy-1H-indol-3-yl)-

(3,4,5-trimethoxyphenyl) methanol (21b)

6.5. (N-Benzenesulfonyl-6-methoxy-1H-indol-3-yl)-

(3,4,5-trimethoxyphenyl) methanone (20b)

The same procedure as described above was performed

with 20b (400 mg, 0,83 mmol) and gave 370 mg of ana-

The same procedure as described above was performed

with 19b (340 mg, 1.18 mmol) and gave after ﬂash chro-

matography using cyclohexane/EtOAc (7:3) 490 mg of

analytically pure 20b (86%) as a yellowish crystal.

Recrystallization from CH₂Cl₂and MeOH gave large

yellowish crystals. Mp 162–165 ꢁC; IR (NaCl ﬁlm) m⁰

(cm^ꢀ1) 2998, 2939, 2830, 1581, 1492, 1378, 1220, 1127,

1003, 726; UV (CH₂Cl₂) k_max(nm) 228 (loge = 4.49),

246.0 (loge = 4.32), 268.0 (loge = 4.27), 295.0

(loge = 4.22); ¹H NMR (CDCl₃) d 3.92 (s, 3H, C₆–

lytically pure 21b (92%). IR (NaCl ﬁlm) m⁰(cm^ꢀ1

)

3371, 2994, 2963, 2940, 2831, 1595, 1503, 1446, 1360,

1322, 1176, 1120; ¹H NMR (DMSO-d₆) d 2.19 (d,

J_OH,CH= 4.1 Hz, 1H, D₂O exch., OH), 3.79 (s, 6H,

0

C_{3 –5}–OCH₃), 3.85 (s, 3H, C₄–OCH₃), 3.87 (s, 3H, C₆–

OCH₃), 5.91 (d, J_CH,OH= 4.1 Hz, 1H, CH), 6.62 (s,

0

2H, H₂and H₆), 6.82 (dd, J_5,4= 8.7 Hz, J_5,7= 2.3 Hz,

1H, H₅), 7.30 (d, J_4,5= 8.7 Hz, 1H, H₄), 7.36 (d,

00

J_2,CH= 0.9 Hz, 1H, H₂), 7.45 (m, 2H, H₅and H₃),

00

0

00

OCH₃), 3.93 (s, 6H, C_{3 –5}–OCH₃), 3.98 (s, 3H,

7.54–7.56 (m, 2H, H₇and H₄), 7.88 (m, 2H, H₂and

00

0

C₄–OCH₃), 7.02 (dd, J_5,4= 8.8 Hz, J_5,7= 2.3 Hz, 1H,

H₆); ¹³C NMR (CDCl₃) d 55.8 (C₆–OCH₃), 56.1

0

00

H₅), 7.13 (s, 2H, H₂and H₆), 7.50 (m, 2H, H₅and

0

0 0

(C_{3 –5}–OCH₃), 60.8 (C₄–OCH₃), 70.4 (CHOH), 98.2

4420

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

0

(C₇), 103.8 (C₂and C₆), 112.5 (C₅), 121.1 (C₄), 122.5

0

00

J_6,5= 7.8 Hz, 1H, H₆), 7.56–7.57 (m, 3H, H₄, H₅and

00

(C₂and C_3a), 125.6 (C₃), 126.7 (C₂and C₆), 129.2

00

00 00

H₃), 7.68 (m, 1H, H₄), 7.83 (s, 1H, H₂), 7.93 (br d,

00

(C₃and C₅), 133.8 (C₄), 136.8 (C_7a), 137.6 (C₁and

00

0

00

J_7,6= 8.3 Hz, 1H, H₇), 8.01 (br d, J = 8.2 Hz, 2H, H₂

C₄), 138.3 (C₁), 153.4 (C₃and C₅), 158.2 (C₆); MS

(ESI) m/z 506 [M+Na]⁺; Anal. (C₂₅H₂₅NO₇S) found:

C, 61.82; H, 5.11; N, 2.71; calcd: C, 62.10; H, 5.21; N,

2.90.

and H₆); ¹³C NMR (CDCl₃) d 21.9 (COCH₃), 57.0

0

00

0

00

0

(C_{3 –5}–OCH₃), 60.9 (C₄–OCH₃), 71.4 (CHOCOCH₃),

0

105.2 (C₂and C₆), 114.4 (C₇), 121.7 (C₄), 123.6 (C₃),

0

00

124.7 (C₅), 125.2 (C₂), 126.2 (C₆), 127.8 (C₂and C₆),

00

0

129.1 (C_3a), 130.9 (C₃and C₅), 135.4 (C₁), 135.6

00

(C_7a), 135.8 (C₄), 137.9 (C₁), 138.2 (C₄), 153.8 (C₃

00

0

6.8. N-Benzenesulfonyl-3-[methoxy-(3,4,5-trimethoxy-

phenyl) methyl]-1H-indole (22a)

0

and C₅), 170.6 (COCH₃); MS (DIC/NH₃) m/z 436

[M+HꢀCH₃COOH]⁺; Anal. (C₂₆H₂₅NO₇S) found: C,

66.59; H, 5.50; N, 3.92; calcd: C, 63.02; H, 5.09; N, 2.83.

To a solution of 21a (200 mg, 0.44 mmol) in a 20/1 mix-

ture of MeOH and THF, pyridinium p-toluenesulfonate

(223 mg, 0.89 mmol) was added and the reaction mix-

ture was magnetically stirred at room temperature dur-

ing 15 h. Solvents were evaporated under reduced

pressure. The residue was dissolved in CH₂Cl₂, washed

with brine, dried, and evaporated in vacuo. Flash chro-

matography using cyclohexane/EtOAc (6:4) gave

174 mg of 22a (85%) as a colorless oil. UV (CH₂Cl₂)

6.10. N-Benzenesulfonyl-3-(3,4,5-trimethoxybenzyl)-1H-

indole (24a)

To a magnetically stirred suspension of 21a (50 mg,

0.11 mmol) in CH₂Cl₂(5 mL) at 25 ꢁC was added triﬂu-

oroacetic acid (82 lL, 1.1 mmol) and then triethylsilane

(178 lL, 1.1 mmol). The reaction was followed by TLC

(cyclohexane/EtOAc (5:5)) and was stopped after

15 min. The solvent was evaporated in vacuo, and the

residue was dissolved in CH₂Cl₂. The organic extract

was washed with an aqueous saturated solution of NaH-

CO₃, brine, dried (MgSO₄), and concentrated in vacuo.

Recrystallization from THF and n-hexane gave analyti-

cally pure 24a (43 mg, 90%) as large prismatic crystals.

Mp 127–129 ꢁC; UV (CH₂Cl₂) k_max(nm) 228.0

(loge = 4.51); IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 3056, 2994,

k_max(nm) 227.0 (loge = 4.36); IR (NaCl ﬁlm) m⁰(cm^ꢀ1

)

2990, 2938, 2828, 1592, 1505, 1448, 1372, 1234, 1177,

1

1127, 1006, 986, 749, 728, 686; H NMR (DMSO-d₆) d

0

3.27 (s, 3H, –OCH₃), 3.59 (s, 3H, C₄–OCH₃), 3.69 (s,

0

6H, C_{3 –5}–OCH₃), 5.50 (s, 1H, CH), 6.71 (s, 2H, H₂

0

and H₆), 7.19 (dd, J_5,4= 7.9 Hz, J_5,6= 7.7 Hz, 1H,

H₅), 7.31 (dd, J_6,7= 8.3 Hz, J_6,5= 7.7 Hz, 1H, H₆),

00

7.51 (br d, J_4,5= 7.9 Hz, 1H, H₄), 7.55 (m, 2H, H₅

00

and H₃), 7.66 (m, 1H, H₄), 7.75 (s, 1H, H₂), 7.91 (br

00

d, J_7,6= 8.3 Hz, 1H, H₇), 7.98 (br d, J = 7.7 Hz, 2H,

00

2937, 2837, 1590, 1506, 1448, 1371, 1238, 1175, 1126,

0

1

H₂and H₆); ¹³C NMR (DMSO-d₆)

d

(C_{3 –5}–OCH₃), 57.3 (OCH₃), 61.0 (C₄–OCH₃), 78.9

56.9

725; H NMR (DMSO-d₆) d 3.57 (s, 3H, C₄–OCH₃),

00

0

3.66 (s, 6H, C_{3 –5}–OCH₃), 3.93 (s, 2H, CH₂), 6.58 (s,

0

(CH), 105.0 (C₂and C₆), 114.3 (C₇), 122.0 (C₄), 124.5

00

(C₅), 125.0 (C₂), 125.3 (C₃), 126.0 (C₆), 127.7 (C₂and

0

2H, H₂and H₆), 7.21 (dd, J_5,4= 7.5 Hz, J_5,6= 7.5 Hz,

1H, H₅), 7.31 (dd, J_6,7= 8.4 Hz, J_6,5= 7.5 Hz, 1H, H₆),

00

C₆), 129.5 (C_3a), 130.8 (C₃and C₅), 135.6 (C₄),

00

00 00

7.53–7.55 (m, 3H, H₄, H₅and H₃), 7.66 (m, 1H,

0

00

135.9 (C_7a), 137.1 (C₁), 137.9 (C₄), 138.0 (C₁), 153.8

00

H₄), 7.71 (s, 1H, H₂), 7.92 (br d, J_7,6= 8.4 Hz, 1H,

+

0

(C₃and C₅); MS (DIC/NH₃) m/z 485 [M+NH₄] ; Anal.

(C₂₅H₂₅NO₆S) found: C, 64.05; H, 5.92; N, 2.68; calcd:

C, 64.22; H, 5.39; N, 3.00.

H₇), 7.95 (br d, J = 7.5 Hz, 2H, H₂and H₆); ¹³C

0

NMR (CDCl₃) d 31.6 (CH₂), 56.8 (C_{3 –5}–OCH₃), 60.9

0

(C₄–OCH₃), 106.9 (C₂and C₆), 114.3 (C₇), 121.2

0

(C₄), 123.8 (C₃), 124.4 (C₅), 125.0 (C₂), 125.9 (C₆),

00 00 00 00

127.5 (C₂and C₆), 130.7 (C₃and C₅), 131.4 (C_3a),

00 0 0

135.5 (C₄), 135.7 (C_7a), 136.1 (C₁), 136.8 (C₄), 138.0

6.9. Acetic acid (N-benzenesulfonyl-1H-indol-3-yl)-(3,4,5-

trimethoxyphenyl) methyl ester (23a)

00

0

(C₁), 153.7 (C₃and C₅); MS (DIC/NH₃) m/z 455

[M+NH₄]⁺, 438 [M+H]⁺; Anal. (C₂₄H₂₃NO₅S) found:

C, 66.03; H, 5.27; N, 3.13; calcd: C, 65.89; H, 5.30; N,

3.20.

To a solution of 21a (200 mg, 0.44 mmol) in a mixture of

acetic anhydride (2.5 mL) and acetic acid (2.5 mL) was

added 4-DMAP (5 mg, 0.04 mmol) and the reaction

mixture was magnetically stirred at room temperature.

The reaction was monitored by TLC (cyclohexane/

EtOAc (5:5)) and was quenched after 48 h. Water

(2 mL) was added to hydrolyze the residual anhydride

and solvents were evaporated in vacuo after addition

of toluene (5 mL). The residue was dissolved in CH₂Cl₂,

washed with brine, dried, and evaporated in vacuo.

Compound 23a was obtained without further puriﬁca-

tion as a colorless oil (207 mg, 95%). Mp 176–177 ꢁC;

UV (CH₂Cl₂) k_max(nm) 223.0 (loge = 4.91), 229.0

(loge = 4.89), 258.0 (loge = 4.57), 301.0 (loge = 4.49);

IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 3002, 2959, 2939, 2835, 1723,

1581, 1542, 1449, 1383, 1324, 1219, 1197, 1127; ¹H

NMR (DMSO-d₆) d 2.16 (s, 3H, COCH₃), 3.60 (s, 3H,

6.11. N-Benzenesulfonyl-6-methoxy-3-(3,4,5-trimeth-

oxybenzyl)-1H-indole (24b)

The same procedure as described above was performed

with 21b (73 mg, 0.15 mmol) and gave after recrystalli-

zation from CH₂Cl₂and n-hexane 53.4 mg of 24b

(75%) as a greenish crystal. Mp 130–133 ꢁC; UV

(CH₂Cl₂) k_max(nm) 209.0 (loge = 4.62), 219.0

(loge = 4.67); IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 2998, 2959,

2938, 2831, 1590, 1506, 1448, 1364, 1238, 1174, 1126,

725; ¹H NMR (CDCl₃) d 3.75 (s, 6H, C_{3 –5}–OCH₃),

0

3.83 (s, 3H, C₄–OCH₃), 3.88 (s, 3H, C₆–OCH₃), 3.90

0

(s, 2H, CH₂), 6.39 (s, 2H, H₂and H₆), 6.83 (dd,

0

C₄–OCH₃), 3.70 (s, 6H, C_{3 –5}–OCH₃), 6.76 (s, 2H, H₂

J_5,4= 8.6 Hz, J_5,7= 2.3 Hz, 1H, H₅), 7.19 (s, 1H, H₂),

00

7.24 (d, J_4,5= 8.6 Hz, 1H, H₄), 7.43 (m, 2H, H₃and

0

and H₆), 6.96 (s, 1H, CH), 7.23 (dd, J_5,6= 7.8 Hz,

J_5,4= 7.6 Hz, 1H, H₅), 7.34 (dd, J_6,7= 8.3 Hz,

00

H₅), 7.54 (m, 1H, H₄), 7.55 (d, J_7,5= 2.3 Hz, 1H,

00

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

4421

H₇), 7.85 (m, 2H, H₂and H₆); ¹³C NMR (CDCl₃) d

342 [M+H]⁺; Anal. (C₁₉H₁₉NO₅) found: C, 65.81; H,

6.05; N, 3.69; calcd: C, 66.85; H, 5.61; N, 4.10.

00

0

31.7 (CH₂), 55.7 (C₆–OCH₃), 56.0 (C_{3 –5}–OCH₃), 60.8

0

(C₄–OCH₃), 98.1 (C₇), 105.4 (C₂and C₆), 112.3 (C₅),

0

00

120.3 (C₄), 122.5 (C₂and C₃), 124.5 (C_3a), 126.6 (C₂

6.14. 3-(3,4,5-Trimethoxybenzyl)-1H-indole (26a)

00

and C₆), 129.1 (C₃and C₅), 133.7 (C₄), 134.7 (C₁),

00

0

136.3 (C₄), 136.6 (C_7a), 138.2 (C₁), 153.1 (C₃and

00

0

To a magnetically stirred solution of 24a (60 mg,

0.20 mmol) in a 10/1 mixture of MeOH and H₂O,

KOH was added (112 mg, 2 mmol) and the reaction

mixture was reﬂuxed for 48 h. MeOH was evaporated

in vacuo, and the aqueous residue was thoroughly

extracted with CH₂Cl₂. The combined organic extracts

were washed with brine, dried (MgSO₄), and concentrat-

ed in vacuo. Flash chromatography using CH₂Cl₂gave

36 mg of 26a (88%) as a white powder. Recrystallization

from CH₂Cl₂and n-hexane gave a white solid. Mp 125–

128 ꢁC; UV (CH₂Cl₂) k_max(nm) 228.0 (loge = 4.45),

279.0 (loge = 3.77); IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 2990,

2936, 2835, 1590, 1505, 1420, 1122, 1126, 1091, 994,

+

C₅), 158.1 (C₆); MS (ESI) m/z 490 [M+Na] ; Anal.

0

(C₂₅H₂₅NO₆S) found: C, 62.45; H, 5.56; N, 2.95; calcd:

C, 64.22; H, 5.39; N, 3.00.

6.12. (1H-Indol-3-yl)-(3,4,5-trimethoxyphenyl) metha-

none (25a)

To a magnetically stirred solution of 20a (300 mg,

0.66 mmol) in a 4/1 mixture of MeOH and H₂O,

K₂CO₃was added (230 mg, 1.65 mmol) and the reaction

mixture was reﬂuxed under N₂. The reaction was moni-

tored by TLC (cyclohexane/EtOAc (5:5)) and stopped

after 1 h by evaporation of MeOH. The aqueous residue

was thoroughly extracted with CH₂Cl₂. The combined

extracts were washed with brine, dried (MgSO₄), and

concentrated in vacuo to give 208 mg of 25a (99%) as

a white powder. Recrystallization from CH₂Cl₂and

MeOH gave transparent crystals. Mp 174–176 ꢁC; UV

(CH₂Cl₂) k_max(nm) 228.0 (loge = 4.28), 250.0

(loge = 4.15), 272.0 (loge = 4.17), 306.0 (loge = 4.22);

IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 3189, 2927, 2828, 1574, 1503,

737; ¹H NMR (CDCl₃) d 3.82 (s, 6H, C_{3 –5}–OCH₃),

0

3.85 (s, 3H, C₄–OCH₃), 4.08 (s, 2H, CH₂), 6.58 (s,

0

2H, H₂and H₆), 7.12 (m, 1H, H₅), 7.20 (m, 1H, H₆),

6.95 (br s, 1H, H₂), 7.38 (br d, J_7,6= 8 Hz, 1H, H₇),

7.58 (br d, J_4,5= 7.5 Hz, 1H, H₄), 8.05 (br s, 1H, D₂O

exch., NH); ¹³C NMR (CDCl₃) d 32.1 (CH₂), 56.1

0

(C_{3 –5}–OCH₃), 61.0 (C₄–OCH₃), 105.8 (C₂and C₆),

111.3 (C₇), 115.6 (C₃), 119.1 (C₅), 119.4 (C₄), 122.1

0

(C₆), 122.5 (C₂), 127.5 (C_3a), 136.2 (C₄), 136.6 (C_7a),

1

0

1414, 1232, 1127, 744; H NMR (DMSO-d₆) d 3.75 (s,

137.1 (C₁), 153.2 (C₃and C₅); MS (DIC/NH₃) m/z

315 [M+NH₄]⁺, 298 [M+H]⁺; Anal. (C₁₈H₁₉NO₃)

found: C, 72.33; H, 6.98; N, 4.39; calcd: C, 72.60; H,

6.40; N, 4.70.

0

3H, C₄–OCH₃), 3.84 (s, 6H, C_{3 –5}–OCH₃), 7.08 (s, 2H,

0

H₂and H₆), 7.22-7.25 (m, 2H, H₅and H₆), 7.50 (br

d, J_7,6= 7.8 Hz, 1H, H₇), 8.09 (s, 1H, H₂), 8.23 (br d,

J_4,5= 7.2 Hz, 1H, H₄), 12.02 (br s, 1H, D₂O exch.,

NH); ¹³C NMR (CDCl₃) d 57.0 (C_{3 –5}–OCH₃), 61.1

0

6.15. 6-Methoxy-3-(3,4,5-trimethoxybenzyl)-1H-indole

(26b)⁴⁴

0

(C₄–OCH₃), 107.1 (C₂and C₆), 113.9 (C₇), 115.9

0

(C₃), 122.4 (C₄), 122.8 (C₅), 124.0 (C₆), 127.4 (C_3a),

0

136.5 (C₂), 136.8 (C₁), 137.7 (C_7a), 141.1 (C₄), 153.6

0

To a magnetically stirred solution of 24b (150 mg,

0.32 mmol) in MeOH, a 0.1 M methanolic solution of

MeONa (10 mL) was added and the reaction mixture

was reﬂuxed for 72 h. MeOH was evaporated in vacuo,

and the residue was taken up with CH₂Cl₂. The organic

extract was washed with brine, dried (MgSO₄), and con-

centrated in vacuo. Flash chromatography using cyclo-

hexane/EtOAc (7:3) gave 85 mg of 26a (81%) as a

greenish powder. Recrystallization from CH₂Cl₂and

n-hexane gave a white/greenish solid. Mp 95–97 ꢁC

(lit.⁴⁴mp 97–98 ꢁC); UV (CH₂Cl₂) k_max(nm) 228.0

(loge = 4.60), 272.0 (loge = 3.77), 293.0 (loge = 3.66);

IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 3372, 2994, 2938, 2901, 2834,

1629, 1591, 1504, 1457, 1237, 1125, 806; ¹H NMR

0

(C₃and C₅), 189.9 (CO); MS (DIC/NH₃) m/z 312

[M+H]⁺; Anal. (C₁₈H₁₇NO₄) found: C, 69.38; H, 5.35;

N, 4.32; calcd: C, 69.44; H, 5.50; N, 4.50.

6.13. (6-Methoxy-1H-indol-3-yl)-(3,4,5-trimethoxyphe-

nyl) methanone (25b)⁴⁴

The same procedure as described above was performed

with 20b (60 mg, 0.12 mmol). Flash chromatography

using EtOAc/cyclohexane (6:4) gave 40 mg of analytical-

ly pure 25b (94%) as a white powder. Recrystallization

from CH₂Cl₂and MeOH gave transparent crystals.

Mp 166–168 ꢁC (lit.⁴⁴mp 185–187 ꢁC (CH₂Cl₂/EtOAc));

IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 3418, 3005, 2951, 2928, 2835,

1633, 1578, 1502, 1414, 1323, 1236, 1156, 1126, 770;

¹H NMR (DMSO-d₆) d 3.88 (s, 3H, C₆–OCH₃), 3.90

0

(CDCl₃) d 3.80 (s, 6H, C_{3 –5}–OCH₃), 3.83 (s, 3H, C₆–

0

OCH₃), 3.85 (s, 3H, C₄–OCH₃), 4.03 (s, 2H, CH₂),

0

6.53 (s, 2H, H₂and H₆), 6.77 (dd, J_5,4= 8.6 Hz,

J_5,7= 2.2 Hz, 1H, H₅), 6.83 (br s, 1H, H₂), 7.41 (d,

J_4,5= 8.6 Hz, 1H, H₄), 7.89 (br s, 1H, D₂O exch.,

NH); ¹³C NMR (CDCl₃) d 32.1 (CH₂), 55.7 (C₆–

0

(s, 6H, C_{3 –5}–OCH₃), 3.94 (s, 3H, C₄–OCH₃), 6.93 (d,

J_7,5= 2.1 Hz, 1H, H₇), 6.99 (dd, J_5,4= 8.7 Hz,

0

J_5,7= 2.1 Hz, 1H, H₅), 7.12 (s, 2H, H₂and H₆), 7.63

(d, J_2,NH= 2.9 Hz, 1H, H₂), 8.26 (d, J_4,5= 8.7 Hz, 1H,

H₄), 8.61 (br s, 1H, D₂O exch., NH); ¹³C NMR

0

OCH₃), 56.0 (C_{3 –5}–OCH₃), 60.9 (C₄–OCH₃), 94.7

0

(C₇), 105.6 (C₂and C₆), 109.3 (C₅), 115.4 (C₃), 119.7

0

0 0

(C₄), 121.2 (C₂), 121.7 (C_3a), 136.0 (C₄), 137.1 (C₁),

(CDCl₃): d 55.5 (C₆–OCH₃), 56.2 (C_{3 –5}–OCH₃), 60.9

0

(C₄–OCH₃), 95.0 (C₇), 106.2 (C₂and C₆), 112.2 (C₅),

0

137.3 (C_7a), 153.1 (C₃and C₅), 156.5 (C₆); MS (ESI)

m/z 366 [M+K]⁺, 350 [M+Na]⁺; Anal. (C₁₉H₂₁NO₄)

found: C, 69.62; H, 6.09; N, 4.27; calcd: C, 69.71; H,

6.47; N, 4.28.

116.7 (C₃), 120.4 (C_3a), 122.8 (C₄), 133.0 (C₂), 135.9

0

(C₁), 137.4 (C_7a), 140.7 (C₄), 152.8 (C₃and C₅),

157.4 (C₆), 190.6 (CO); MS (ESI) m/z 364 [M+Na]⁺,

4422

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

0

0 0

(NCH₃), 57.1 (C_{3 –5}–OCH₃), 61.2 (C₄–OCH₃), 107.1

0 0

(C₂and C₆), 111.7 (C₇), 114.7 (C₃), 122.6 (C₄), 123.2

6.16. (6-Methoxy-1H-indol-3-yl)-(3,4,5-trimethoxyphe-

nyl) methanethione (27b)

0

(C₅), 124.2 (C₆), 127.8 (C_3a), 136.8 (C₁), 138.4 (C_7a),

0

To a magnetically stirred solution of 25a (65 mg,

0.19 mmol) in anhydrous THF at 25 ꢁC was added

Lawesson’s reagent (89 mg, 0.23 mmol). The mixture

monitored by TLC (cyclohexane/EtOAc (5:5)) was stir-

red for 1h. THF was evaporated in vacuo, and the resi-

due was dissolved in CH₂Cl₂. The organic extract was

washed with water, dried (MgSO₄), and concentrated

in vacuo. Flash chromatography using cyclohexane/

EtOAc (5:5) gave analytically pure 62 mg of 27b (91%)

as a reddish oil. UV (CH₂Cl₂) k_max(nm) 227.0

(loge = 4.40), 291 (loge = 3.96), 392 (loge = 4.04); IR

(NaCl ﬁlm) m⁰(cm^ꢀ1) 3311, 2934, 2835, 1625, 1579,

1487, 1448, 1411, 1324, 1232, 1161, 1125, 1028, 1001,

140.3 (C₂), 141.1 (C₄), 153.7 (C₃and C₅), 189.6 (CO);

MS (DIC/NH₃) m/z 326 [M+H]⁺; Anal. (C₁₉H₁₉NO₄)

found: C, 64.2; H, 5.97; N, 3.83; calcd: C, 70.14; H,

5.89; N, 4.31.

6.17.2. (N-Benzyl-1H-indol-3-yl)-(3,4,5-trimethoxyphe-

nyl) methanone (29a). The general procedure was per-

formed with 25a (50 mg, 0.16 mmol) and benzyl

bromide (24 lL, 0.19 mmol) and gave after ﬂash chro-

matography using cyclohexane and EtOAc (6:4),

62 mg of 29a (96%) as a colorless oil. UV (CH₂Cl₂) k_max

(nm) 226.0 (loge = 4.35), 260.0 (loge = 4.10), 274.0

(loge = 4.06), 314.0 (loge = 4.18); IR (NaCl ﬁlm) m⁰

(cm^ꢀ1) 3055, 3001, 2937, 2836, 1621, 1580, 1521, 1503,

822; ¹H NMR (CDCl₃) d 3.87 (s, 6H, C_{3 –5}–OCH₃),

0

1

0

3.89 (s, 3H, C₆–OCH₃), 3.92 (s, 3H, C₄–OCH₃), 6.93

1463, 1412, 1382, 1323, 1234, 1169, 1126, 813, 750; H

0

(d, J_7,5= 2.2 Hz, 1H, H₇), 6.96 (s, 2H, H₂and H₆),

7.01 (dd, J_5,4= 8.9 Hz, J_5,7= 2.2 Hz, 1H, H₅), 7.59 (d,

J_2,NH= 3.1 Hz, 1H, H₂), 8.58 (d, J_4,5= 8.9 Hz, 1H,

H₄), 8.64 (br s, 1H, D₂O exch., NH); ¹³C NMR

NMR (CDCl₃) d 3.85 (s, 6H, C_{3 –5}–OCH₃), 3.92 (s,

0

3H, C₄–OCH₃), 5.37 (s, 2H, CH₂), 7.08 (s, 2H, H₂

0

and H₆), 7.21 (m, 2H, H₂and H₆), 7.32–7.44 (m,

00

6H, H₃, H₅, H₄, H₅, H₆and H₇), 7.63 (s, 1H, H₂),

0

(CDCl₃): d 55.6 (C₆–OCH₃), 56.2 (C_{3 –5}–OCH₃), 60.9

8.41 (m, 1H, H₄); ¹³C NMR (CDCl₃) d 50.6 (CH₂),

0

(C₄–OCH₃), 95.4 (C₇), 106.4 (C₂and C₆), 112.7 (C₅),

0

0 0 0

56.2 (C_{3 –5}–OCH₃), 60.9 (C₄–OCH₃), 106.4 (C₂and

0

C₆), 110.0 (C₇), 115.9 (C₃), 122.7 (C₄and C₅), 123.8

120.9 (C₃), 123.7 (C₄), 129.3 (C_3a), 132.2 (C₂), 138.4

0

00

(C₆), 127.2 (C_3a, C₂and C₆), 128.3 (C₄), 129.0 (C₃

00

(C_7a), 139.7 (C₄), 145.1 (C₁), 152.3 (C₃and C₅),

157.8 (C₆), 222.6 (CS); MS (ESI) m/z 356 [MꢀH]^ꢀ;

Anal. (C₁₉H₁₉NO₄S) found: C, 64.42; H, 5.90; N, 3.76;

calcd: C, 63.85; H, 5.36; N, 3.92.

00

0

and C₅), 135.7 (C₁), 135.9 (C₁), 136.3 (C₂), 137.0

0

(C_7a), 141.0 (C₄), 152.9 (C₃and C₅), 192.2 (CO); MS

(ESI) m/z 424 [M+Na]⁺, 402 [M+H]⁺; Anal.

(C₂₅H₂₃NO₄) found: C, 70.6; H, 6.17; N, 3.12; calcd:

C, 74.79; H, 5.77; N, 3.49.

6.17. General procedure for the preparation of the N-

substituted methanones 28a–35a, 32b, and 35b

6.17.3. [N-(4-Methoxybenzyl)-1H-indol-3-yl]-(3,4,5-tri-

methoxyphenyl) methanone (30a). The general procedure

was performed with 25a (50 mg, 0.16 mmol) and

p-methoxybenzyl chloride (27 lL, 0.19 mmol) and gave

after ﬂash chromatography using cyclohexane/EtOAc

(6:4), 63 mg of 30a (91%) as a colorless oil. UV (CH₂Cl₂)

k_max(nm) 227.0 (loge = 4.53), 268.0 (loge = 4.17), 315.0

(loge = 4.21); IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 3045, 3000, 2937,

2836, 1613, 1580, 1515, 1463, 1412, 1382, 1324, 1249,

1175, 1127, 750; ¹H NMR (CDCl₃): d 3.79 (s, 3H,

A solution of 25a or 25b (50 mg) in 5 mL of EtOH was

magnetically stirred during 15 min at room temperature

in the presence of KOH (1.2 equiv). EtOH was evapo-

rated under reduced pressure. The residue was taken

up in 5 mL of dry acetone. Anhydrous sodium sulfate

(2 equiv) and a slight excess (1.2–1.5 equiv) of alkyl or

acyl halides were successively added. The reaction mix-

ture was monitored by TLC and stopped by ﬁltration

and solvent evaporation. The residue was taken up in

CH₂Cl₂and washed with brine, dried over MgSO₄,

ﬁltered, and evaporated to dryness. The crude residue

was then puriﬁed by ﬂash chromatography.

00

0

C₄–OCH₃), 3.85 (s, 6H, C_{3 –5}–OCH₃), 3.92 (s, 3H,

0

C₄–OCH₃), 5.29 (s, 2H, CH₂), 6.87 (m, 2H, H₃and

00

H₅), 7.07 (s, 2H, H₂and H₆), 7.16 (m, 2H, H₂and

0

00

H₆), 7.32–7.37 (m, 2H, H₅and H₆), 7.42 (m, 1H, H₇),

6.17.1. (N-Methyl-1H-indol-3-yl)-(3,4,5-trimethoxyphe-

nyl) methanone (28a). The general procedure was per-

formed with 25a (207 mg, 0.66 mmol) and

dimethylsulfate (63 lL, 0.79 mmol). The reaction was

monitored by TLC (cyclohexane/EtOAc (5:5)). The res-

idue was puriﬁed by ﬂash chromatography using cyclo-

hexane/EtOAc (6:4) to give 195 mg of analytically pure

28a (90%) as an oil. UV (CH₂Cl₂) k_max(nm) 228.0

(loge = 4.42), 260.0 (loge = 4.11), 314.0 (loge = 4.12);

IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 2932, 2831, 1633, 1581, 1464,

7.59 (m, 1H, H₂), 8.40 (m, 1H, H₄); ¹³C NMR (CDCl₃)

00

0

d 50.2 (CH₂), 55.3 (C₄–OCH₃), 56.2 (C_{3 –5}–OCH₃), 61.0

0

(C₄–OCH₃), 106.4 (C₂and C₆), 110.1 (C₇), 114.4 (C₃

00

and C₅), 115.7 (C₃), 122.7 (C₄and C₅), 123.7 (C₆),

00

127.5 (C₁and C_3a), 128.9 (C₂and C₆), 136.0 (C₁),

00

0

136.3 (C₂), 137.1 (C_7a), 140.9 (C₄), 152.9 (C₃and C₅),

0

+

159.6 (C₄), 189.8 (CO); MS (ESI) m/z 454 [M+Na] ;

00

Anal. (C₂₆H₂₅NO₅) found: C, 72.01; H, 6.52; N, 2.82;

calcd: C, 72.37; H, 5.84; N, 3.25.

1

1367, 1323, 1218, 1125; H NMR (DMSO-d₆) d 3.75

6.17.4. [N-Acetyl-1H-indol-3-yl]-(3,4,5-trimethoxyphe-

nyl) methanone (31a). The general procedure was per-

formed with 25a (50 mg, 0.16 mmol) and acetyl

chloride (14 lL, 0.20 mmol) and gave after ﬂash chro-

matography using cyclohexane/EtOAc (6:4), 48 mg of

31a (85%) as a crystalline solid. Recrystallization from

CH₂Cl₂and n-hexane gave large prismatic crystals.

0

(s, 3H, C₄–OCH₃), 3.86 (s, 6H, C_{3 –5}–OCH₃), 3.88 (s,

0

3H, NCH₃), 7.07 (s, 2H, H₂and H₆), 7.28 (dd,

J_5,4= 7.6 Hz, J_5,6= 7.3 Hz, 1H, H₅), 7.32 (dd,

J_6,7= 7.6 Hz, J_6,5= 7.3 Hz, 1H, H₆), 7.57 (br d,

J_7,6= 7.6 Hz, 1H, H₇), 8.13 (s, 1H, H₂), 8.26 (br d,

J_4,5= 7.6 Hz, 1H, H₄); ¹³C NMR (DMSO-d₆) d 34.2

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

4423

0

(C₂), 134.3 (C₁), 137.6 (C_7a), 141.9 (C₄), 153.1 (C₃

0

Mp 180–182 ꢁC; UV (CH₂Cl₂) k_max(nm) 229.0

(loge = 4.49), 256.0 (loge = 4.22), 306.0 (loge = 4.25);

IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 3002, 2967, 2939, 2835, 1722,

1634, 1581, 1449, 1383, 1324, 1219, 1197, 1126; ¹H

NMR (DMSO-d₆) d 2.74 (s, 3H, COCH₃), 3.77 (s, 3H,

0

and C₅), 159.0 (C₆), 168.9 (CO), 189.8 (CO); MS

(ESI) m/z 468 [M+Na]⁺, 446 [M+H]⁺; Anal.

(C₂₆H₂₃NO₆) found: C, 70.14; H, 5.53; N, 3.22; calcd:

C, 70.10; H, 5.20; N, 3.14.

0

C₄–OCH₃), 3.87 (s, 6H, C_{3 –5}–OCH₃), 7.23 (s, 2H, H₂

0

and H₆), 7.42 (dd, J_5,4= 7.3 Hz, J_5,6= 7.1 Hz, 1H,

H₅), 7.45 (dd, J_6,5= 7.1 Hz, J_6,7= 7.0 Hz, 1H, H₆),

8.20 (br d, J_4,5= 7.3 Hz, 1H, H₄), 8.40–8.41 (m, 2H,

H₇and H₂); ¹³C NMR (DMSO-d₆) d 25.0 (COCH₃),

6.17.7. [N-(4-Methoxybenzoyl)-1H-indol-3-yl]-(3,4,5-tri-

methoxyphenyl) methanone (33a). The general procedure

was performed with 25a (24 mg, 0.08 mmol) and p-

methoxybenzoyl chloride (16 mg, 1.00 mmol) and gave

after recrystallization from CH₂Cl₂and n-hexane

23 mg of 33a (67%) as a white solid. Mp 137–139 ꢁC;

UV (CH₂Cl₂) k_max(nm) 225.0 (loge = 4.49), 260.0

(loge = 4.37), 309.0 (loge = 4.27); IR (NaCl ﬁlm) m⁰

(cm^ꢀ1) 3000, 2963, 2929, 2829, 1691, 1635, 1602, 1575,

0

57.2 (C_{3 –5}–OCH₃), 61.2 (C₄–OCH₃), 107.7 (C₂and

0

C₆), 116.9 (C₇), 119.3 (C₃), 122.6 (C₄), 125.7 (C₅),

0

126.9 (C₆), 129.0 (C_3a), 135.0 (C₁), 136.5 (C₂and C_7a),

141.8 (C₄), 153.9 (C₃and C₅), 171.7 (COCH₃), 190.3

(CO); MS (DIC/NH₃) m/z 354 [M+H]⁺, 312

[M+HꢀCOCH₃]⁺; Anal. (C₂₀H₁₉NO₅) found: C,

67.99; H, 5.41; N, 4.00; calcd: C, 67.98; H, 5.42; N, 3.96.

0

1

1445, 1322, 1258, 1165, 1130; H NMR (DMSO-d₆) d

0

3.89 (s, 6H, C_{3 –5}–OCH₃), 3.92 (s, 3H, C₄–OCH₃),

0

00

0

3.93 (s, 3H, C₄–OCH₃), 7.02 (br d, J = 8.7 Hz, 2H,

00 00 0 0

H₃and H₅), 7.14 (s, 2H, H₂and H₆), 7.45–7.47 (m,

00

2H, H₅and H₆), 7.79 (br d, J = 8.7 Hz, 2H, H₂and

6.17.5. (N-Benzoyl-1H-indol-3-yl)-(3,4,5-trimethoxyphe-

nyl) methanone (32a). The general procedure was per-

formed with 25a (50 mg, 0.16 mmol) and benzoyl

chloride (23 lL, 0.19 mmol) and gave after ﬂash chro-

matography using cyclohexane/EtOAc (6:4), 56 mg of

32a (84%) as a white powder. Recrystallization from

CH₂Cl₂and n-hexane gave colorless needles. Mp 138–

140 ꢁC; UV (CH₂Cl₂) k_max(nm) 228.0 (loge = 4.55),

310.0 (loge = 4.23); IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 2998,

2940, 2837, 1698, 1637, 1581, 1541, 1504, 1450, 1377,

00

H₆), 7.87 (s, 1H, H₂), 8.25 (m, 1H, H₇), 8.29 (m, 1H,

13

00

H₄); C NMR (CDCl₃) d 55.6 (C₄–OCH₃), 56.3

0

(C_{3 –5}–OCH₃), 60.9 (C₄–OCH₃), 106.6 (C₂and C₆),

00

114.3 (C₃and C₅), 115.7 (C₇), 119.9 (C₃), 122.4 (C₄),

00

125.0 (C₅), 125.3 (C₁), 126.0 (C₆), 128.5 (C_3a), 131.9

00

(C₂and C₆), 134.4 (C₂and C₁), 136.8 (C_7a), 141.8

0

00

(C₄), 153.1 (C₃and C₅), 163.7 (C₄), 168.3 (NCO),

190.4 (CO); MS (ESI) m/z 468 [M+Na]⁺; Anal.

(C₂₆H₂₃NO₆) found: C, 70.93; H, 5.47; N, 3.18; calcd:

C, 70.10; H, 5.20; N, 3.14.

1327, 1128; ¹H NMR (CDCl₃)

d 3.88 (s, 6H,

0

C_{3 –5}–OCH₃), 3.93 (s, 3H, C₄–OCH₃), 7.13 (s, 2H, H₂

0

and H₆), 7.45–7.50 (m, 2H, H₅and H₆), 7.55 (m, 2H,

00

H₃and H₅), 7.67 (m, 1H, H₄), 7.79 (br d,

00

6.17.8. [N-(4-Methoxybenzenesulfonyl)-1H-indol-3-yl]-

(3,4,5-trimethoxyphenyl) methanone (34a). The general

procedure was performed with 25a (40 mg, 0.13 mmol)

and p-methoxybenzenesulfonyl chloride (32 mg,

0.16 mmol) and gave after ﬂash chromatography using

cyclohexane/EtOAc (6:4), 54 mg of 34a (87%) as a yellow

powder. Recrystallization from CH₂Cl₂and n-hexane

gave yellow crystals. Mp 161–162 ꢁC; UV (CH₂Cl₂) k_max

(nm) 228.0 (loge = 4.38), 249.0 (loge = 4.42), 299.0

(loge = 4.24); IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 3001, 2938,

2834, 1635, 1577, 1496, 1414, 1374, 1168, 1130; ¹H

00

J = 7.2 Hz, 2H, H₂and H₆), 7.82 (s, 1H, H₂), 8.29

(m, 1H, H₄), 8.35 (m, 1H, H₇); ¹³C NMR (CDCl₃) d

0

56.3 (C_{3 –5}–OMe), 61.0 (C₄–OMe), 106.6 (C₂and C₆),

0

116.0 (C₇), 120.3 (C₃), 122.5 (C₄), 125.3 (C₅), 126.2

00

(C₆), 128.6 (C_3a), 128.9 (C₃and C₅), 129.2 (C₂and

00

C₆), 132.9 (C₄), 133.5 (C₁), 134.1 (C₂), 134.3 (C₁),

00

0

136.6 (C_7a), 141.7 (C₄), 153.1 (C₃and C₅), 168.6

(NCO), 191.2 (CO); MS (ESI) m/z 438 [M+Na]⁺; Anal.

(C₂₅H₂₁NO₅) found: C, 72.51; H, 5.28; N, 2.75; calcd: C,

72.28; H, 5.10; N, 3.37.

00

NMR (DMSO-d₆) d 3.82 (s, 3H, C₄–OCH₃), 3.93 (s,

0

0 0

6H, C_{3 –5}–OCH₃), 3.98 (s, 3H, C₄–OCH₃), 6.92 (br d,

6.17.6. (N-Benzoyl-6-methoxy-1H-indol-3-yl)-(3,4,5-tri-

methoxyphenyl) methanone (32b). The general procedure

was performed with 25b (52 mg, 0.15 mmol) and benzo-

yl chloride (22 lL, 0.18 mmol) and gave after ﬂash chro-

matography using cyclohexane/EtOAc (7:3), 50 mg of

32b (74%) as a yellowish powder. Recrystallization from

CH₂Cl₂and n-hexane gave yellowish crystals. Mp 118–

121 ꢁC; UV (CH₂Cl₂) k_max(nm) 229.0 (loge = 4.54),

273.0 (loge = 4.42); IR (NaCl ﬁlm) m⁰(cm^ꢀ1) 2998,

2940, 2835, 1698, 1634, 1581, 1492, 1324, 1217, 1127;

00

0

J = 9.0 Hz, 2H, H₃and H₅), 7.14 (s, 2H, H₂and H₆),

7.39 (ddd, J_5,4= 7.5 Hz, J_5,6= 7.3 Hz, J_5,7= 1.0 Hz,

1H, H₅), 7.43 (ddd, J_6,7= 7.5 Hz, J_6,5= 7.3 Hz,

00

J_6,4= 1.0 Hz, 1H, H₆), 7.88 (br d, J = 9.0 Hz, 2H, H₂

00

and H₆), 8.01 (br d, J_7,6= 7.5 Hz, 1H, H₇), 8.06 (s,

1H, H₂), 8.26 (br d, J_4,5= 7.5 Hz, 1H, H₄); ¹³C NMR

00

0

(CDCl₃) d 55.8 (C₄–OCH₃), 56.3 (C_{3 –5}–OCH₃), 61.0

0

(C₄–OCH₃), 106.7 (C₂and C₆), 113.2 (C₇), 114.8 (C₃

00

and C₅), 120.2 (C₃), 122.8 (C₄), 124.7 (C₅), 125.9 (C₆),

00 00 00

128.5 (C_3a), 128.9 (C₁), 129.4 (C₂and C₆), 133.0 (C₂),

¹H NMR (CDCl₃): 3.87 (s, 6H, C_{3 –5}–OCH₃), 3.92 (s,

0

6H, C₆–OCH₃and C₄–OCH₃), 7.10 (dd, J_5,4= 8.8 Hz,

0 0 0 0

134.2 (C₁), 135.0 (C_7a), 142.1 (C₄), 153.2 (C₃and C₅),

+

164.4 (C₄), 189.6 (CO); MS (ESI) m/z 504 [M+Na] ,

0

J_5,7= 2.3 Hz, 1H, H₅), 7.11 (s, 2H, H₂and H₆), 7.55

0

00

482 [M+H]⁺; Anal. (C₂₅H₂₃NO₇S) found: C, 61.06; H,

4.90; N, 3.03; calcd: C, 62.36; H, 4.81; N, 2.91.

00

(m, 2H, H₃and H₅), 7.69 (s, 1H, H₂), 7.78 (m, 2H,

00

H₂and H₆), 7.95 (d, J_7,5= 2.3 Hz, 1H, H₇), 8.12 (m,

00

1H, H₄), 8.15 (d, J_4,5= 8.8 Hz, 1H, H₄); ¹³C NMR

0

(CDCl₃) 55.7 (C₆–OCH₃), 56.3 (C_{3 –5}–OCH₃), 61.0

0

6.17.9. [N-Phenyloxycarbonyl-1H-indol-3-yl]-(3,4,5-tri-

methoxyphenyl) methanone (35a). The general procedure

was performed with 25a (60 mg, 0.19 mmol) and phenyl-

chloroformate (30 lL, 0.23 mmol) and gave after ﬂash

0

(C₄–OCH₃), 99.9 (C₇), 106.6 (C₂and C₆), 114.6 (C₅),

0

00

120.3 (C₃), 122.1 (C_3a), 123.0 (C₄), 128.9 (C₃and C₅),

00

129.2 (C₂and C₆), 130.2 (C₄), 132.8 (C₁), 133.1

00

4424

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

chromatography using cyclohexane/EtOAc (8:2), 40 mg

of 35a (67%) as a white powder. Recrystallization from

CH₂Cl₂and n-hexane gave prismatic colorless crystals.

Mp 155–156 ꢁC; UV (CH₂Cl₂) k_max(nm) 228

(loge = 4.51), 249 (loge = 4.24), 299 (loge = 4.24); IR

(NaCl ﬁlm) m⁰(cm^ꢀ1) 3002, 2936, 2835, 1755, 1639,

compound. The IC₅₀of all compounds were compared

to the IC₅₀of colchicine, measured the same day under

the same conditions. The results are presented as inhibi-

tion of tubulin polymerization (or ITP), which is the ra-

tio of the IC₅₀value of a given synthesized compound to

the IC₅₀value of colchicine (ITP = IC₅₀test compound/

IC_50colchicine). Compounds with a ratio value greater

than 57C are designated as ‘inactive’ in Tables 1–3.

1

1581, 1452, 1225, 1127, 1004; H NMR (acetone-d₆) d

0

3.83 (s, 3H, C₄–OCH₃), 3.93 (s, 6H, C_{3 –5}–OCH₃),

0

00

7.29 (s, 2H, H₂and H₆), 7.39 (m, 1H, H₄), 7.44–7.55

00

(m, 6H, H₂, H₆, H₃, H₅, H₅and H₆), 8.28 (br d,

J_7,6= 7.8 Hz, 1H, H₇), 8.32 (br d, J_4,5= 7.8 Hz, 1H,

H₄), 8.47 (s, 1H, H₂); ¹³C NMR (acetone-d₆) 57.2

8. Evaluation of cytotoxicity in murine B16 melanoma

cells

0

(C_{3 –5}–OCH₃), 61.3 (C₄–OCH₃), 108.2 (C₂and C₆),

0

00

116.4 (C₇), 121.2 (C₃), 123.1 (C₂and C₆), 123.8 (C₄),

00

126.0 (C₅), 127.3 (C₆), 128.1 (C₄), 130.0 (C_3a), 131.1

00

Murine B16 melanoma cells were grown in DMEM con-

taining 2 mM ^L-glutamine, 10% fetal bovine serum,

100 U/mL penicillin, and 100 lg/mL streptomycin

(37 ꢁC, 5% CO₂). All compounds were initially dissolved

in DMSO at a stock concentration of 2.5 mg/mL and

were further diluted in cell culture medium. For compar-

ative purposes, combretastatin A-4 phosphate (CSA-4)

and colchicine were routinely included in the experi-

ments as internal standards. Exponentially growing

B16 cells were plated onto 96-well plates at 5000 cells

per well in 100 lL of culture medium. Twenty-four

hours after plating, 100 lL of medium containing the

compound of interest (ﬁnal concentrations ranging from

0.25 ng/mL to 25 lg/mL, in 10-fold dilutions) was added

to the wells (in triplicate) containing the cells and incu-

bated for 48 h at 37 ꢁC and 5% CO₂. After the 48 h

exposure period to the test compounds, cell viability

was assayed using the MTT test⁵⁰and absorbance was

read at 562 nm in a microplate reader (BioKinetics

Reader, EL340). Appropriate controls with DMEM

only and MTT were run to substract background absor-

bance. Results are presented as percent of controls con-

taining 1% DMSO, which was not cytotoxic at this

concentration. The concentration of compound that

inhibited cell viability by 50% (inhibitory concentration

for 50% of cells, or IC₅₀) was determined using the

GraphPad Prism software. Results are presented as

means SEM of 3–7 independent experiments each

run in triplicate.

00

0

(C₃and C₅), 135.1 (C₂and C₁), 135.9 (C_7a), 141.9

0

00

0

(C₄), 148.9 (NCOO), 150.5 (C₁), 154.9 (C₃and C₅),

190.7 (CO); MS (ESI) m/z 454 [M+Na]⁺; Anal.

(C₂₅H₂₁NO₆) found: C, 69.49; H, 4.75; N, 3.21; calcd:

C, 69.60; H, 4.91; N, 3.25.

6.17.10. [N-Phenyloxycarbonyl-6-methoxy-1H-indol-3-

yl]-(3,4,5-trimethoxyphenyl) methanone (35b). The gener-

al procedure was performed with 25b (57 mg,

0.17 mmol)

and

phenylchloroformate

(26 lL,

0.20 mmol) and gave after ﬂash chromatography using

cyclohexane/EtOAc (8:2), 50 mg of 35b (65%) as a white

powder. Recrystallization from CH₂Cl₂and n-hexane

gave colorless needle crystals. Mp 90–93 ꢁC; UV

(CH₂Cl₂) k_max(nm) 228.0 (loge = 4.55), 264.5

(loge = 4.36); 299.5 (loge = 4.17); IR (NaCl ﬁlm) m⁰

(cm^ꢀ1) 2998, 2940, 2837, 1760, 1637, 1581, 1493, 1391,

1

1326, 1214, 1127, 1003; H NMR (CDCl₃) 3.90 (s, 3H,

0

C₆–OCH₃), 3.93 (s, 6H, C_{3 –5}–OCH₃), 3.96 (s, 3H,

0

C₄–OCH₃), 7.08 (dd, J_5,4= 8.8 Hz, J_5,7= 2.3 Hz, 1H,

00

H₅), 7.30 (m, 2H, H₂and H₆), 7.37 (m, 1H, H₄),

00

7.50 (m, 2H, H₃and H₅), 7.85 (d, J_7,5= 2.3 Hz, 1H,

H₇), 8.16 (s, 1H, H₂), 8.17 (d, J_4,5= 8.8 Hz, 1H, H₄);

¹³C

NMR

(CDCl₃)

55.7

0

(C_{3 –5}–OCH₃), 61.0 (C₄–OCH₃), 99.4 (C₇), 106.7 (C₂

(C₆–OCH₃),

56.4

0

and C₆), 114.1 (C₅), 121.0 (C₃), 121.3 (C₂and C₆),

00

121.5 (C_3a), 123.3 (C₄), 126.9 (C₄), 129.9 (C₃and

00

C₅), 131.2 (C₂), 134.4 (C₁), 137.0 (C_7a), 142.1 (C₄),

0

00

0

149.1 (NCOO), 150.0 (C₁), 153.2 (C₃and C₅), 159.1

(C₆), 190.0 (CO); MS (ESI) m/z 484 [M+Na]⁺, 462

[M+H]⁺; Anal. (C₂₆H₂₃NO₇) found: C, 65.36; H, 5.25;

N, 2.71; calcd: C, 67.67; H, 5.02; N, 3.04.

9. Eﬀect on the morphology of transformed HUVEC

(EAÆhy 926 cells)

To assess the eﬀects of the compounds on the morphol-

ogy of endothelial cells, we used the EAÆhy 926 cell line

which is derived from the fusion of human umbilical vein

endothelial cells (HUVEC) with the permanent human

cell line A549.³⁸The EAÆhy 926 cell line is considered

as one of the best immortalized HUVEC lines because

these cells express most of the biochemical markers of

parental HUVEC.⁵¹EAÆhy 926 cells, originally obtained

from Dr. Cora-Jean S. Edgell (Pathology Department,

University of North Carolina, Chapel Hill, NC 27599-

7525, USA), were used with her permission and were

grown in DMEM containing 2 mM ^L-glutamine, 10% fe-

tal bovine serum, 100 U/mL penicillin and 100 lg/mL

streptomycin (37 ꢁC, 5% CO₂). Exponentially growing

EAÆhy 926 cells were plated onto 96-well plates at 5000

cells/100 lL/well. Twenty-four hours after plating, the

7. Tubulin binding assay

Calf brain tubulin was puriﬁed according to the method

of Shelanski et al.,⁴⁸by three cycles of assembly–disas-

sembly, and then dissolved at a ﬁnal concentration of

2–3 mg/mL in the assembly buﬀer (pH 6.6) containing

0.1 M MES, 0.5 mM MgCl₂, 2 mM EGTA, and 1 mM

GTP. Tubulin assembly was monitored and recorded

continuously by turbidimetry at 400 nm in a UV spec-

trophotometer, equipped with a thermostated cell at

37 ꢁC.⁴⁹The IC₅₀value, deﬁned as the concentration

of inhibitor which decreased by 50% the maximum

assembly rate of tubulin in control cell without test

compound, was determined for each newly synthesized

G. Dupeyre et al. / Bioorg. Med. Chem. 14 (2006) 4410–4426

4425

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medium was aspirated, and 100 lL of medium contain-

ing the test compound was added to the well containing

the cells (in triplicate) at ﬁnal concentrations ranging

from 0.25 ng/mL to 25 lg/mL, in 10-fold dilutions, and

incubated for 2 h at 37 ꢁC and 5% CO₂. After the 2 h

incubation period, digital photographs were taken of

representative center areas of each well at a magniﬁca-

tion of 360·. The presence of rounded cells was scored

for each concentration, and the results are presented as

the presence of rounded cells for more than 10% of cells

in a given ﬁeld, because the DMSO-treated cells present-

ed about 10% of rounded cells. The number of + sign

indicates rounding up at the following concentrations:

1+ at 25; 2+ at 2.5; 3+ at 0.25; 4+ at 0.025; and 5+ at

0.0025 lg/mL. A minus sign (ꢀ) indicates no rounding

up. Combretastatin A-4 (CSA-4) and colchicine were

routinely included in the experiments as internal stan-

dards. For the best compounds, the concentrations for

the rounding up of 50% of EAÆhy 926 cells were calculat-

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the logarithm of the concentrations of compounds by

using the GraphPad Prism software.

´

24. Borrel, C.; Thoret, S.; Cachet, X.; Guenard, D.; Tillequin,

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´

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Chem. 2005, 13, 2097–2107.

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Acknowledgments

28. Flynn, B. L.; Hamel, E.; Jung, M. K. J. Med. Chem. 2002,

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29. Hadimani, M. B.; Kessler, R. J.; Kautz, J. A.; Ghatak, A.;

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We thank Dr Julia Kaﬀy of the Institut Curie, Paris, for

providing us with synthetic CSA-4. G.D. was supported

`

by a studentship from the Ministere de l’Education et de

la Recherche.

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Article Doi

Synthesis and biological evaluation of (3,4,5-trimethoxyphenyl)indol-3-ylmethane derivatives as potential antivascular agents

DOI: 10.1016/j.bmc.2006.02.037

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Full text of DOI:10.1016/j.bmc.2006.02.037

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