Synthesis and antiproliferative activities of diversely substituted glycosyl-isoindigo derivatives

European Journal of Medicinal Chemistry 41 (2006) 88–100

http://france.elsevier.com/direct/ejmech

Short communication

Synthesis and antiproliferative activities

of diversely substituted glycosyl-isoindigo derivatives

Mathieu Sassatelli ^a, Fadoua Bouchikhi ^a, Samir Messaoudi ^a, Fabrice Anizon ^a, Eric Debiton ^b,

Chantal Barthomeuf ^b, Michelle Prudhomme ^a, Pascale Moreau ^a,*

a

Laboratoire SEESIB, Université Blaise-Pascal, UMR 6504 du CNRS, 24, avenue des Landais, 63177 Aubière, France

b

Laboratoire de Pharmacognosie–Biotechnologies, UMR Inserm 484, Université d’Auvergne,

Centre Jean-Perrin, rue Montalembert, B.P. 184, 63005 Clermont-Ferrand cedex, France

Received 25 July 2005; received in revised form 30 September 2005; accepted 6 October 2005

Available online 05 December 2005

Abstract

In the course of structure–activity relationship studies, diversely substituted 1-(β-^D-glucopyranosyl)-isoindigo derivatives were prepared from

commercially available indolines. Their antiproliferative activities were evaluated toward a panel of human solid cancer cell lines (PC 3, DLD-1,

MCF-7, M4Beu, A549, PA 1), a murine cell line (L929) and a human fibroblast primary culture to get an insight into the substitution pattern

required for the best biological potencies.

Keywords: Indigoids; Isoindigos; Antiproliferative activities

1. Introduction

FGFR and PDGFR [5,6] (e.g. SU6668, SU11248). Moreover,

some imidazo[2,1-b]thiazolylmethylene-2-indolinones such as

compound A or indolylmethylene-2-indolinones such as com-

pound B have been described as CDK1/cyclin B inhibitors [7].

Recently, SU9516, possessing an indolin-2-one framework was

described as an ATP competitive inhibitor of CDKs (Scheme

2) [8].

In the course of studies on the preparation of potential bio-

logically active compounds, we were interested in the synthesis

of indigoid derivatives. These heterocycles (indigo, indirubin,

isoindigo) containing a bis-indole framework are derived from

various natural sources (Scheme 1).

The bis-indole indirubin is the active ingredient of Danggui

Longhui Wan, a mixture of plants used in traditional Chinese

medicine to treat chronic diseases. Indirubins are potent inhibi-

tors of several kinases such as GSK-3β and cyclin dependent

kinases (CDKs) [1]. Indirubin is known to interact with the

ATP-binding site of CDK-2, CDK-5 and GSK-3 [2]. Moreover,

structure–activity relationship studies have shown that 5-nitro

and 5-bromoindirubin are more potent kinase inhibitors (GSK-

3β, CDK-1, CDK-5) than their parent indirubin [1]. Indirubin

and indigo are also potent aryl hydrocarbon receptor ligands

[3,4].

We have described, in a previous paper, a method for the

preparation of glycosyl-isoindigo derivatives substituted in the

5 position of one of the aromatic rings by either electron donor

or acceptor substituents [9]. In this paper, the synthesis of iso-

indigo derivatives (indirubin isomers possessing two indolin-2-

one moieties) bearing a sugar residue attached to one of the

aromatic nitrogens and diversely substituted on both aromatic

rings in the 5 or 5′ positions is described (Scheme 3).

It was reported in the literature [10,11] that the biological

activities of the isoindigo derivative called Natura (1-(β-^D-tri-

O-acetylxylopyranosyl)-isoindigo) (Scheme 3) were better than

those of the non-protected analogue, probably because of an

enhanced cellular penetration. Accordingly, we have prepared

glycosyl-isoindigo derivatives protected on the sugar residue

by either benzyl or acetyl groups (Scheme 3). To get an insight

into the effect of the substitution pattern on the biological ac-

Indolin-2-one derivatives are usually known as ATP compe-

titive inhibitors of receptor tyrosine kinase such as VEGFR,

^*Corresponding author. Tel.: +33 4 73 40 79 63; fax: +33 4 73 40 77 17.

E-mail address: Pascale.MOREAU@univ-bpclermont.fr (P . M oreau).

doi:10.1016/j.ejmech.2005.10.004

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 88–100

89

Scheme 1.

Scheme 2.

Scheme 3.

tivities of these compounds, their antiproliferative potencies

2. Chemistry

were examined in vitro by two fluorometric assays: resazurin

reduction test (RRT) and Hoechst 33342 dye DNA assay in a

panel of human solid cancer cell lines (PC 3: prostatic adeno-

carcinoma, DLD-1: colon carcinoma, MCF-7: breast adenocar-

cinoma, M4Beu: melanoma teratocarcinoma, A549: lung carci-

noma, PA 1: ovarian carcinoma), a murine cell line (L929) and

a human fibroblast primary culture.

Recently, we described the synthesis and spectral data of

compounds 23–25 [9]. The key intermediates in this approach

are the protected glycosyl-isatines 15–17 which were prepared

in four steps from the corresponding indolines. The preparation

of compound 15 has been previously detailed [12]. The deriva-

tive substituted in the 5 position by a phenyl group was pre-

90

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 88–100

pared using the same synthetic pathway. Indoline 1 was pre-

pared in 81% yield from the corresponding N-acetyl analogue

by basic hydrolysis and phenyl analogue 2 was obtained from

1 in 45% yield by a Suzuki coupling reaction using phenyl

boronic acid, palladium tetrakis(triphenylphosphine) and

sodium carbonate as described by Sun et al. [13] for bromo-

benzene derivatives. Glycosyl-indolines 3–6 were prepared by

glycosylation of the corresponding indolines [14]. After aroma-

tization with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ),

the hydroxy groups of the intermediates 7–10 were first pro-

tected before oxidation with chromium oxide to give the gly-

cosyl-isatines 15–18. As mentioned in our previous paper [9],

compared with reactions carried out with electron acceptor sub-

stituents, the first two steps were easier in the presence of elec-

tron donor substituents. Indeed, the glycosylation step was

achieved in 24 h for compounds 3, 5, 6 and required 6 days

for nitro analogue 4. The aromatization step was carried out at

room temperature during 12–18 h for compounds 7, 9, 10 and

at 50 °C during 48 h for nitro compound 8. Isatine derivatives

15–18 were prepared by oxidation of the corresponding indoles

using chromium oxide [12]. To obtain the corresponding gly-

cosyl-isoindigo derivatives, compounds 15–18 were treated in

an acidic medium [15] in the presence of oxindole. Deprotec-

tion of the hydroxy groups of the glycosyl moiety was per-

formed by reaction of derivatives 19–22 with boron tribromide

[16] to give compounds 23–26 (Scheme 4).

Glycosyl-isoindigo derivative 28, substituted in the 5′ posi-

tion, was obtained by acid-catalyzed coupling of isatine 15 and

substituted oxindole 27 which was prepared in 88% yield by

acylation of the oxindole in the presence of succinic anhydride

and aluminum chloride according to the procedure described

by Kakushima et al. [17] for pyrrole derivatives. Unfortunately,

the deprotection step performed in the same conditions as de-

scribed above did not allow the obtention of the corresponding

deprotected glycosyl-isoindigo (Scheme 5).

The synthesis of acetyl analogues 38–40 was performed by

a similar synthetic pathway (Scheme 6). The key intermediates,

acetylated glycosyl-isatines 35–37, were prepared in four steps

Scheme 4.

Scheme 5.

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 88–100

91

Scheme 6.

from the corresponding indolines. The glycosyl-indolines 3–5

were acetylated in the presence of pyridine and acetic anhy-

dride before aromatization with DDQ and oxidation with chro-

mium oxide which were performed in the same conditions as

described before for the benzylated series. Acetylated glycosyl-

isatines 35–37 were then coupled with the oxindole moiety in

an acidic medium [11] to give the acetylated glycosyl-isoindi-

gos (Scheme 6). In this case, the coupling was achieved in the

presence of p-toluenesulfonic acid (PTSA) instead of a mixture

of acetic acid and HCl to avoid deacetylation.

was a clear doublet with the usual 9 Hz coupling constant. The

measurement of the coupling constant for the H_1′signal clearly

indicate a β configuration. In contrast, the H_1′signal of the

deprotected analogue 24 was a broad signal. On the basis that

the presence or not of benzyl groups should not affect the β

configuration of the N-glycosidic bond, the hypothesis of an

isomerization was excluded and we assumed that the config-

uration of the N-glycosydic bond was β for all these com-

pounds.

The β configuration of the N-glycosyl derivatives 3–18 and

29–37 was determined by the measurement of the coupling

constant between the H_1′and H_2′of the sugar residue. In all

cases, the resulting signal of the H_1′was a doublet with a cou-

pling constant around 9 Hz.

3. Results and discussion

3.1. In vitro antiproliferative activities

Surprisingly, for several glycosyl-isoindigo derivatives 19,

21–26, 28, 38, 39, the signal of the H_1′of the sugar moiety

was not a clear doublet. Therefore these signals were assigned

as broad signals. NMR experiments were then run at higher

temperatures (70 °C) or in other solvents (CDCl₃or DMSO)

to investigate if solvatation effects or the presence of different

conformers could explain the shape of these signals. No

changes were observed on the H_1′signal in these conditions.

The unusual appearance of these signals could be due to iso-

merization to the α configuration but this was never observed

before in our previous works concerning glycosylated indole or

azaindole derivatives. Moreover, the H_1′signal of derivative

20, benzylated glycosyl-isoindigo substituted by a nitro group,

In vitro antiproliferative activities of compounds 19–26, 28

and 38–40 were evaluated toward a panel of human solid can-

cer cell lines (PC 3, DLD-1, MCF-7, M4Beu, A549, PA 1),

murine cell line (L929) and human fibroblast primary culture.

The IC₅₀, defined as the drug concentration required to inhibit

cell proliferation by 50%, was calculated from the curve of

concentration-dependent survival percentage, defined as fluor-

escence in experimental wells compared with fluorescence in

control wells, after subtraction of the blank values. The anti-

proliferative effect of the tested drug was assessed by both the

RRT [18] and the determination of DNA cellular content after

cell lysis by the Hoechst dye 33342 assay carried out according

to the method described by Rago et al. [19] with minor mod-

92

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 88–100

Table 1

Antiproliferative activities of compounds 34, 44 and 46 (IC₅₀in μM)

Compounds

28

Fibroblast

4.7

L929

6.5

A-549

12.4

DLD-1

6.0

M4Beu

7.0

MCF-7

8.0

PA 1

2.5

PC3

4.7

(5.1)

Inactive

(6.1)

8.7

(12.4)

Inactive

(5.3)

17.3

(7.1)

Inactive

(5.1)

25.1

(2.7)

6.2

(5.0)

25

38

40

(8.3)

8.1

(11.7)

17.0

(17.9)

24.0

(5.5)

6.1

(21.8)

3.7

Inactive

(7.3)

(13.5)

(12.0)

(5.5)

(3.1)

Inactive means an IC₅₀value > 30 μM (Rezazurin test results are given first and Hoechst test results are given in brackets).

ifications. Under the conditions used, only three of the tested

compounds have shown significant cytotoxic activities

(Table 1).

gel Geduran SI 60 (Merck) 0.040–0.063 mm column chroma-

tography. For purity tests, TLC were performed on fluorescent

silica gel plates (60 F254 from Merck).

The most potent compound was compound 28 which was

cytotoxic toward all the cell lines tested. This cytotoxity is

probably due to the presence of the carboxylic chain on the

upper oxindole moiety because compound 19, lacking this sub-

stitution, was completely inactive against the cell lines tested.

4.1.1. 5-Bromoindoline 1

A 40% aqueous NaOH solution (40 ml) was added to a

solution of N-acetyl-5-bromoindoline (2 g, 8 mmol) in metha-

nol (40 ml). The reaction mixture was refluxed for 18 h before

hydrolysis. After extraction with dichloromethane, the organic

phases were dried over MgSO₄. The residue obtained after

concentration under vacuum, was purified by flash chromato-

graphy (eluent cyclohexane/EtOAc 70:30) to give 2 (1.28 g,

6.5 mmol) as a brown oil in 81% yield. C₈H₈BrN, IR (KBr)

Compounds 38 and 40 had similar inhibition profiles except

on PC3 cells on which compound 40 was slightly more active

than its non brominated analogue 38, they were both active on

PA 1, L929, DLD-1 and MCF-7 cells. In contrast to compound

28, they show selectivity between normal and tumor cell lines,

they are both inactive against healthy human fibroblast. As pre-

viously described for Natura^®[10,11], in our series, the acety-

lated derivatives 38 and 40, were more cytotoxic than their

benzylated analogues 19, 21 or parent compounds 23, 25. In

contrast, the nitro derivative 39 was inactive toward the cell

lines tested indicating that the presence of a nitro group in po-

sition 5 is detrimental to the cytotoxicity.

ν_NH3380 cm⁻¹, ν_C=C1600 cm⁻¹. H (400 MHz, DMSO-d₆):

1

2.91 (t, J = 8.5 Hz, 2H, CH₂), 3.42 (t, J = 8.5 Hz, 2H, CH₂),

5.65 (s, 1H, NH), 6.42 (d, J = 8.0 Hz, 1H), 7.02 (dd, J₁

= 8.0 Hz, J₂= 1.5 Hz, 1H), 7.14 (d, J = 1.0 Hz, 1H). ¹³C

(100 MHz, DMSO-d₆): 29.0, 46.6 (CH₂), 109.6, 126.8, 129.3

(CH), 107.1, 131.7, 151.9 (C).

In conclusion, we have synthesized diversely substituted 1-

(β-^D-glucopyranosyl)-isoindigos. The method described here

allows the substitution of both aromatic rings. The oxindole

moiety bearing the sugar residue was substituted by either elec-

tron donor or acceptor substituents in the 5 position. The re-

sults obtained in this structure–activity relationship studies

have shown that the pharmaceutical profile of this series could

be optimized by substitutions of the upper oxindole moiety and

the presence of acetyl groups on the sugar residue.

4.1.2. 5-Phenylindoline 2

Phenylboronic acid (332 mg, 2.72 mmol), 2 M aqueous so-

dium carbonate (2.5 ml) and palladium tetrakis (84.9 mg,

0.08 mmol) were added to a solution of 5-bromoindoline 1

(490 mg, 2.47 mmol) in a mixture of toluene (6 ml) and etha-

nol (3 ml). The reaction mixture was refluxed for 22 h. The

solid residue obtained after concentration under vacuum and

EtOAc addition was removed by filtration. The filtrate was

washed with water, dried over MgSO₄, and concentrated under

vacuum to give a residue, which was purified by flash chroma-

tography (eluent cyclohexane/EtOAc from 90:10 to 60:40) to

give the corresponding compound 2 (216 mg, 1.1 mmol) as a

brown oil in 45% yield. C₁₄H₁₃N, IR (KBr) ν_NH3417 cm⁻¹,

ν_C=C1599 cm⁻¹. ¹H (400 MHz, DMSO-d₆): 3.00 (t,

J = 8.5 Hz, 2H, CH₂), 3.49 (td, J₁= 8.5 Hz, J₂= 2.0 Hz, 2H,

CH₂), 5.63 (br s, 1H, NH), 6.59 (d, J = 8.0 Hz, 1H), 7.23–7.27

(m, 2H), 7.38–7.42 (m, 3H), 7.55–7.58 (m, 2H). ¹³C

(100 MHz, DMSO-d₆): 29.1, 46.5 (CH₂), 108.3, 122.6, 125.5

(2C), 125.6 (2C), 128.6 (2C) (CH), 128.9, 129.6, 141.1, 152.2

(C).

4. Experimental

4.1. Chemistry

IR spectra were recorded on a Perkin–Elmer 881 spectro-

meter (ν in cm⁻¹). NMR spectra were performed on a Bruker

AVANCE 400 (¹H: 400 MHz, ¹³C: 100 MHz) (chemical shifts

δ in ppm, the following abbreviations are used: singlet (s),

doublet (d), triplet (t), doubled triplet (dt), doubled doublet

(dd), doubled doubled doublet (ddd), multiplet (m), broad sig-

nal (br s). When necessary to identify all carbon atoms, com-

plementary NMR experiments (HSQC, HMBC) were per-

formed on a Bruker Avance 500. Mass spectra (ES) were

determined on a high resolution Waters Micro Q-toff apparatus.

Chromatographic purifications were performed by flash silica

4.1.3. Typical procedure for the glycosylation reaction

D-Glucose (3.05 mmol) was added to a solution of indoline

(6.1 mmol) in a mixture of ethanol (46.7 ml) and water (3.05

ml). The mixture was heated to 90 °C during 24 h for com-

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 88–100

93

pounds 3, 5, 6 and 6 days for compound 4. Water (0.6 ml) was

added after 7 and 14 h reflux. After concentration under va-

cuum, the resulting mixture was purified by flash chromatogra-

phy (eluent EtOAc/MeOH from 98:2 to 90:10) to give the cor-

responding glycosyl-indoline.

85.1 (CH_sugar), 107.6, 122.7, 125.6, 125.7 (2C), 125.9, 128.7

(2C) (CH), 129.9, 130.5, 140.9, 150.4 (C).

4.1.4. Aromatization with DDQ

1-(β-^D-Glucopyranosyl)-indoline 3: Obtained in 95% yield

as a white solid (m.p. = 113 °C), IR (KBr) υ_CO1270 cm⁻¹,

υ_C=C1610 cm⁻¹, υ_OH3100–3700 cm⁻¹. MS (EI) M⁺Calcd.

for C₁₄H₁₉NO₅, 281; Found: 281. ¹H NMR (400 MHz,

DMSO-d₆): 2.91 (m, 2H), 3.12 (m, 1H), 3.21 (m, 1H), 3.30

(m, 2H), 3.38–3.54 (m, 2H), 3.62 (m, 2H), 4.35 (t,

J = 5.0 Hz, 1H, OH), 4.64 (d, J = 8.0 Hz, 1H, H_1’), 4.91 (d,

J = 5.0 Hz, 1H, OH), 5.02 (br s, 2H, OH), 6.59 (t,

J = 8.0 Hz, 2H), 6.95 (d, J = 7.0 Hz, 1H), 7.00 (d,

J = 8.0 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆): 28.3

(CH₂), 45.7 (CH₂), 61.5 (CH_{2 sugar}), 70.7, 71.4, 78.4, 78.8,

85.7 (CH_sugar), 108.1, 118.3, 125.0, 127.6 (CH), 130.2, 151.3

(C).

4.1.4.1. For compounds 7–9. DDQ (1.1 or 0.4 mmol for com-

pound 9) was added to a solution of glycosyl-indoline

(0.36 mmol) in 1,4-dioxane (16 ml). Before hydrolysis with

saturated aqueous NaHCO₃, the mixture was stirred at room

temperature for 12 h for compound 7 or 18 h for compounds

9 and 10. The reaction mixture was heated at 50 °C for 48 h for

compound 8. The solid residue (DDQH₂and NaHCO₃) ob-

tained after concentration under vacuum and EtOAc addition

was removed by filtration. The filtrate was dried over

MgSO₄, and concentrated under vacuum to give a residue,

which was purified by flash chromatography (eluent EtOAc/

MeOH from 100:0 to 90:10) to give the corresponding glyco-

syl-indole.

1-(β-^D-Glucopyranosyl)-5-nitroindoline 4: C₁₄H₁₈N₂O₇ob-

tained in 80% yield as an orange powder (m.p. = 70–75 °C), IR

1

(KBr): ν_OH3680–3040 cm⁻¹; ν_C=C1610 cm⁻¹. H (400 MHz,

4.1.4.2. For compound 10. DDQ (0.4 mmol) was added to a

solution of glycosyl-indoline (0.36 mmol) in 1,4-dioxane (16

ml). The mixture was stirred at room temperature for 18 h be-

fore hydrolysis with saturated aqueous NaHCO₃. After extrac-

tion with EtOAc, the organic phases were dried over MgSO₄,

and concentrated under vacuum to give a residue, which was

purified by flash chromatography (eluent EtOAc/MeOH 98:2)

to give the corresponding glycosyl-indole.

DMSO-d₆): 3.00–3.15 (m, 3H), 3.28–3.36 (m, 3H), 3.36–3.48

(m, 1H), 3.60–3.76 (m, 2H), 3.80–3.91 (m, 1H), 4.49 (t,

J = 6.0 Hz, 1H, OH), 4.83 (d, J = 8.5 Hz, 1H, H_1’), 5.04 (d,

J = 5.5 Hz, 1H, OH), 5.15 (d, J = 4.5 Hz, 1H, OH), 5.28 (d,

J = 5.0 Hz, 1H, OH), 6.74 (d, J = 9.0 Hz, 1H), 7.91 (d,

J = 2.5 Hz, 1H), 8.02 (dd, J₁= 9.0 Hz, J₂= 2.5 Hz, 1H). ¹³C

(100 MHz, DMSO-d₆): 26.3, 46.0 (CH₂), 60.9 (CH_{2 sugar}),

69.9, 70.4, 77.3, 78.6, 84.2 (CH_sugar), 105.6, 120.5, 125.8

(CH), 130.6, 137.8, 157.0 (C).

1-(β-^D-Glucopyranosyl)-indole 7: C₁₄H₁₇NO₅obtained in

74% yield as a salmon colored solid (m.p. = 90–95 °C), IR

(KBr) υ_C=C1610 cm⁻¹, υ_OH3080–3700 cm⁻¹; ¹H NMR

(400 MHz, DMSO-d₆): 3.33 (m, 1H), 3.45 (m, 1H), 3.51 (m,

2H), 3.75 (m, 1H), 3.81 (m, 1H), 4.59 (br s, 1H, OH), 5.15 (d,

J = 5.0 Hz, 1H, OH), 5.25 (d, J = 5.5 Hz, 2H, OH), 5.46 (d,

J = 9.5 Hz, 1H, H_1′), 6.51 (d, J = 3.5 Hz, 1H), 7.09 (t,

J = 7.0 Hz, 1H), 7.18 (dt, J₁= 7.9 Hz, J₂= 1.5 Hz, 1H), 7.56

(d, J = 3.5 Hz, 1H), 7.59 (dd, J₁= 8.0 Hz, J₂= 1.0 Hz, 1H),

7.61 (d, J = 8.0 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆):

61.0 (CH_{2 sugar}), 69.9, 71.7, 77.6, 79.4, 84.8 (CH_sugar), 101.6,

110.9, 119.7, 120.3, 121.2, 126.3 (CH), 128.5, 136.2 (C).

1-(β-^D-Glucopyranosyl)-5-bromoindoline 5: C₁₄H₁₈BrNO₅

obtained in 80% yield as a brown gum, IR (NaCl): ν_OH

3402 cm⁻¹; ν_C=C1606 cm⁻¹. ¹H (400 MHz, DMSO-d₆):

2.89–3.04 (m, 2H), 3.09–3.16 (m, 1H), 3.21–3.27 (m, 1H),

3.28–3.36 (m, 2H), 3.41–3.56 (m, 2H), 3.63 (ddd, J₁

= 12.0 Hz, J₂= 5.5 Hz, J₃= 2.0 Hz, 1H), 3.66–3.72 (m, 1H),

4.43 (t, J = 6.5 Hz, 1H, OH), 4.68 (d, J = 8.5 Hz, 1H, H_1′),

4.99 (d, J = 5.0 Hz, 1H, OH), 5.09 (d, J = 4.5 Hz, 1H, OH),

5.11 (d, J = 4.5 Hz, 1H, OH), 6.59 (d, J = 8.5 Hz, 1H), 7.17

(dd, J₁= 8.5 Hz, J₂= 2.0 Hz, 1H), 7.22 (d, J = 2.0 Hz, 1H). ¹³

C

1-(β-^D-Glucopyranosyl)-5-nitroindole 8: C₁₄H₁₆N₂O₇ob-

tained in 65% yield as a yellow gum, IR (NaCl): ν_OH

3426 cm⁻¹; ν_C=C1615 cm⁻¹. ¹H (400 MHz, DMSO-d₆):

3.30–3.37 (m, 1H), 3.43–3.58 (m, 3H), 3.71–3.78 (m, 2H),

4.61 (t, J = 6.0 Hz, 1H, OH), 5.19 (d, J = 5.5 Hz, 1H, OH),

5.28 (d, J = 5.0 Hz, 1H, OH), 5.34 (d, J = 6.0 Hz, 1H, OH),

5.59 (d, J = 9.0 Hz, 1H, H_1′), 6.83 (d, J = 3.5 Hz, 1H), 7.78 (d,

J = 3.5 Hz, 1H), 7.84 (d, J = 9.0 Hz, 1H), 8.09 (dd, J₁

= 9.0 Hz, J₂= 2.0 Hz, 1H), 8.61 (d, J = 2.0 Hz, 1H). ¹³C

(100 MHz, DMSO-d₆): 60.9 (CH_{2 sugar}), 69.7, 72.0, 77.2,

79.6, 85.0 (CH_sugar), 104.2, 111.5, 116.6, 117.3, 130.1 (CH),

127.8, 139.2, 141.1 (C).

(100 MHz, DMSO-d₆): 27.3, 45.3 (CH₂), 60.8 (CH_{2 sugar}),

69.9, 70.6, 77.5, 78.1, 84.8 (CH_sugar), 109.0, 126.9, 129.3

(CH), 108.1, 132.5, 150.1 (C).

1-(β-^D-Glucopyranosyl)-5-phenylindoline 6: C₂₀H₂₃NO₅

obtained in 85% yield as a brown gum, IR (NaCl): ν_OH

3400 cm⁻¹; ν_C=C1620 cm⁻¹. ¹H (400 MHz, DMSO-d₆):

2.96–3.09 (m, 2H), 3.13–3.21 (m, 1H), 3.26–3.31 (m, 1H),

3.32–3.42 (m, 2H), 3.44–3.51 (m, 1H), 3.58 (dd, J₁= 19.0 Hz,

J₂= 10.5 Hz, 1H), 3.66 (ddd, J₁= 12.0 Hz, J₂= 5.0 Hz, J₃

= 2.0 Hz, 1H), 3.70–3.77 (m, 1H), 4.42 (t, J = 6.5 Hz, 1H,

OH), 4.73 (d, J = 9.0 Hz, 1H, H_1’), 4.97 (d, J = 5.0 Hz, 1H,

OH), 5.08 (d, J = 4.5 Hz, 1H, OH), 5.09 (d, J = 5.0 Hz, 1H,

OH), 6.69 (d, J = 8.0 Hz, 1H), 7.27 (t, J = 7.0 Hz, 1H), 7.34

(dd, J₁= 8.5 Hz, J₂= 1.5 Hz, 1H), 7.37–7.44 (m, 3H), 7.59

(dd, J₁= 8.5 Hz, J₂= 1.0 Hz, 2H). ¹³C (100 MHz, DMSO-

d₆): 27.6, 45.3 (CH₂), 60.9 (CH_{2 sugar}), 70.0, 70.7, 77.7, 78.2,

1-(β-^D-Glucopyranosyl)-5-bromoindole 9: C₁₄H₁₆BrNO₅

obtained in 88% yield as a brown gum, IR (NaCl): ν_OH

3395 cm⁻¹; ν_C=C1606 cm⁻¹. ¹H (400 MHz, DMSO-d₆):

3.27–3.34 (m, 1H), 3.41–3.55 (m, 3H), 3.69–3.78 (m, 2H),

94

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 88–100

4.58 (t, J = 5.5 Hz, 1H, OH), 5.15 (d, J = 5.5 Hz, 1H, OH),

5.24 (d, J = 5.0 Hz, 1H, OH), 5.26 (d, J = 6.0 Hz, 1H, OH),

5.45 (d, J = 9.0 Hz, 1H, H_1′), 6.50 (d, J = 3.5 Hz, 1H), 7.29

(dd, J₁= 9.0 Hz, J₂= 2.0 Hz, 1H), 7.56 (d, J = 3.5 Hz, 1H),

7.59 (d, J = 9.0 Hz, 1H), 7.78 (d, J = 2.0 Hz, 1H). ¹³C

(100 MHz, DMSO-d₆): 60.9 (CH_{2 sugar}), 69.8, 71.8, 77.4,

79.4, 84.8 (CH_sugar), 101.2, 113.0, 122.5, 123.6, 127.8 (CH),

112.1, 130.3, 134.9 (C).

7.12–7.18 (m, 1H), 7.26 (dd, J₁= 7.5 Hz, J₂= 1.5 Hz, 2H),

7.30–7.41 (m, 13H), 7.92 (d, J = 9.0 Hz, 1H), 7.98 (d,

J = 3.0 Hz, 1H), 8.09 (dd, J₁= 9.0 Hz, J₂= 2.0 Hz, 1H), 8.67

(d, J = 2.0 Hz, 1H). ¹³C (100 MHz, DMSO-d₆): 68.5, 72.3,

73.6, 74.1, 74.6 (CH_{2 sugar + benzyl}), 76.5, 77.5, 80.3, 83.8, 84.6

(CH_sugar), 105.1, 111.5, 116.9, 117.6, 127.4–128.2, 130.0

(CH), 127.8, 137.2, 138.0, 138.1, 138.4, 138.9, 141.4 (C).

1-(2,3,4,6-tetra-O-benzyl-β-^D-glucopyranosyl)-5-bromoin-

dole 13: C₄₂H₄₀BrNO₅obtained in 82% yield as a beige gum,

1-(β-^D-Glucopyranosyl)-5-phenylindole 10: C₂₀H₂₁NO₅ob-

tained in 90% yield as a beige gum, IR (NaCl): ν_OH

3395 cm⁻¹; ν_C=C1600 cm⁻¹. ¹H (400 MHz, DMSO-d₆):

3.27–3.35 (m, 1H), 3.43–3.56 (m, 3H), 3.71–3.84 (m, 2H),

4.59 (t, J = 5.5 Hz, 1H, OH), 5.15 (d, J = 5.5 Hz, 1H, OH),

5.24 (d, J = 5.0 Hz, 1H, OH), 5.26 (d, J = 6.0 Hz, 1H, OH),

5.48 (d, J = 9.0 Hz, 1H, H_1’), 6.57 (d, J = 3.0 Hz, 1H), 7.34 (t,

J = 7.5 Hz, 1H), 7.46–7.51 (m, 3H), 7.54 (d, J = 3.5 Hz, 1H),

7.68 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 7.0 Hz, 2H), 7.85 (d,

J = 1.5 Hz, 1H). ¹³C (100 MHz, DMSO-d₆): 61.0 (CH_{2 sugar}),

69.9, 71.8, 77.5, 79.4, 84.9 (CH_sugar), 102.0, 111.4, 118.3,

120.5, 126.3, 126.7 (2C), 127.1, 128.8 (2C) (CH), 129.1,

132.2, 135.8, 141.6 (C).

1

IR (NaCl): ν_C=C1604 cm⁻¹. H (400 MHz, DMSO-d₆): 3.65

(d, J = 10.5 Hz, 1H), 3.71–3.75 (m, 2H), 3.78 (t, J = 9.5 Hz,

1H), 3.88–3.97 (m, 2H), 4.12 (t, J = 9.0 Hz, 1H), 4.29 (d,

J = 10.5 Hz, 1H), 4.50 (d, J = 12.0 Hz, 1H), 4.55 (d,

J = 12.5 Hz, 1H), 4.64 (d, J = 11.0 Hz, 1H), 4.83 (d,

J = 11.0 Hz, 1H), 4.87 (s, 2H), 5.82 (d, J = 9.0 Hz, 1H, H_1′),

6.63 (d, J = 3.5 Hz, 1H), 6.73 (d, J = 7.0 Hz, 2H), 7.09–7.15

(m, 2H), 7.16–7.22 (m, 1H), 7.23–7.28 (m, 2H), 7.29–7.41 (m,

14H), 7.70 (d, J = 8.5 Hz, 1H), 7.77 (d, J = 3.5 Hz, 1H), 7.85

(d, J = 2.0 Hz, 1H). ¹³C (100 MHz, DMSO-d₆): 68.6, 72.3,

73.4, 74.1, 74.5 (CH_{2 sugar + benzyl}); 76.3, 77.5, 80.2, 83.9, 84.6

(CH_sugar), 102.2, 113.1, 122.9, 124.1, 127.5–128.3 (CH),

112.5, 130.3, 134.6, 137.4, 138.0, 138.1, 138.4 (C).

1-(2,3,4,6-tetra-O-benzyl-β-^D-glucopyranosyl)-5-phenylin-

dole 14: C₄₈H₄₅NO₅obtained in 68% yield as a beige gum, IR

4.1.5. Typical procedure for the benzylation step

NaH (60% dispersion in mineral oil, 8.2 mmol) was added

at 0 °C to a solution of glycosyl-indole (0.86 mmol) in THF

(12 ml) before addition of nBu₄NI (0.07 mmol) and benzyl

bromide (6.7 mmol). The mixture was then stirred at 0 °C for

20 min before refluxing for 17 hours. After addition of florisil

(60/100 mesh) to the reaction mixture, the solvent was re-

moved under vacuum and the residue purified by flash chro-

matography (eluent: cyclohexane/EtOAc from 98:2 to 80:20)

to give the protected glycosyl-indole.

1

(NaCl): ν_C=C1450, 1490, 1500 cm⁻¹. H (400 MHz, DMSO-

d₆): 3.71 (d, J = 10.5 Hz, 1H), 3.73–3.77 (m, 2H), 3.79 (t,

J = 9.5 Hz, 1H), 3.91–3.98 (m, 2H), 4.16 (t, J = 9.0 Hz, 1H),

4.30 (d, J = 10.5 Hz, 1H), 4.51 (d, J = 12.0 Hz, 1H), 4.57 (d, J

= 12.5 Hz, 1H), 4.65 (d, J = 11.0 Hz, 1H), 4.84 (d, J = 11.0 Hz,

1H), 4.86 (d, J = 11.5 Hz, 1H), 4.90 (d, J = 11.5 Hz, 1H), 5.84

(d, J = 9.0 Hz, 1H, H_1′), 6.70 (d, J = 3.5 Hz, 1H), 6.74-6.78 (m,

2H), 7.08–7.13 (m, 2H), 7.14–7.19 (m, 1H), 7.24–7.27 (m,

2H), 7.31–7.40 (m, 14H), 7.48–7.54 (m, 3H), 7.72–7.77 (m,

3H), 7.80 (d, J = 8.5 Hz, 1H), 7.93 (d, J = 2.0 Hz, 1H). ¹³C

(100 MHz, DMSO-d₆): 68.7, 72.3, 73.4, 74.1, 74.6

1-(2,3,4,6-tetra-O-benzyl-β-^D-glucopyranosyl)-indole 11:

C₄₂H₄₁NO₅obtained in 78% yield as a yellow solid (m.p.

1

= 90 °C), IR (KBr) υ_C=C1610 cm⁻¹. H NMR (400 MHz,

(CH₂

_benzyl), 76.3, 77.6, 80.2, 84.0, 84.6 (CH_sugar),

+

sugar

DMSO-d₆): 3.65 (d, J = 9.5 Hz, 1H), 3.75 (m, 2H), 3.81 (d,

J = 9.5 Hz, 1H), 3.95 (m, 2H), 4.15 (t, J = 9.5 Hz, 1H), 4.25

(d, J = 11.0 Hz, 1H), 4.50 (d, J = 12.5 Hz, 1H), 4.55 (d,

J = 12.0 Hz, 1H), 4.65 (d, J = 11.0 Hz, 1H), 4.85 (d,

J = 10.5 Hz, 1H), 4.90 (br s, 2H), 5.82 (d, J = 9.0 Hz, 1H,

H_1’), 6.65 (d, J = 3.0 Hz, 1H), 6.74 (d, J = 7.0 Hz, 2H),

7.09–7.39 (m, 20H), 7.65 (d, J = 8.0 Hz, 1H), 7.70 (d,

J = 3.5 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H). ¹³C NMR

(100 MHz, DMSO-d₆): 68.7, 72.3, 73.3, 74.1, 74.6

(C_{sugar + benzyl}), 76.3, 77.6, 80.2, 83.9, 84.6 (CH_sugar), 102.5,

111.1, 120.0, 120.6, 121.6, 126.5, 127.5–128.2 (CH), 128.5,

135.9, 137.5, 138.1 (2C), 138.5 (C).

103.1, 111.6, 118.7, 121.0, 125.8–128.8 (CH), 129.1, 132.5,

135.5, 137.5, 138.1 (2C), 138.5, 141.4 (C).

4.1.6. Oxidation of the protected glycosyl-indole

to the corresponding isatine derivatives

Chromium oxide (5.89 mmol) was added slowly to a solu-

tion of indole derivative (0.45 mmol) in acetone (1 ml), acetic

acid (5 ml) and water (1.55 ml). The reaction mixture was stir-

red at room temperature for 2 hours before hydrolysis and ex-

traction with CH₂Cl₂. The organic phases were washed with

water and saturated aqueous NaCl until neutral pH. After dry-

ing over MgSO₄, the solvent was removed and the residue pur-

ified by flash chromatography (eluent cyclohexane/EtOAc

80:20) to give the corresponding isatines.

1-(2,3,4,6-tetra-O-benzyl-β-^D-glucopyranosyl)-5-nitroindole

12: C₄₂H₄₀N₂O₇obtained in 64% yield as a yellow gum, IR

(NaCl): ν_C=C1612 cm⁻¹. ¹H (400 MHz, DMSO-d₆): 3.71–3.77

(m, 3H), 3.81 (t, J = 9.5 Hz, 1H), 3.92–4.00 (m, 2H), 4.17 (t,

J = 9.0 Hz, 1H), 4.38 (d, J = 11.0 Hz, 1H), 4.50 (d,

J = 12.0 Hz, 1H), 4.56 (d, J = 12.0 Hz, 1H), 4.66 (d,

J = 11.0 Hz, 1H), 4.84 (d, J = 11.0 Hz, 1H), 4.89 (s, 2H),

5.96 (d, J = 9.0 Hz, 1H, H_1′), 6.70 (dd, J₁= 7.5 Hz, J₂

= 1.0 Hz, 2H), 6.94 (d, J = 3.0 Hz, 1H), 7.06–7.11 (m, 2H),

1-(2,3,4,6-tetra-O-benzyl-β-^D-glucopyranosyl)-indolin-2,3-

dione 15: Obtained in 62% yield as a yellow gum, IR (NaCl)

υ_C=C1610 cm⁻¹, υ_C=O1740 cm⁻¹, HRMS (ES) [M + Na]⁺

Calcd. for C₄₂H₃₉NNaO₇692.2618; Found: 692.2633; ¹H

NMR (400 MHz, DMSO-d₆): 3.71 (d, J = 3.0 Hz, 2H), 3.80

(m, 1H), 3.89 (dt, J₁= 9.5 Hz, J₂= 3.5 Hz, 1H), 3.96 (t,

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 88–100

95

J = 8.5 Hz, 1H), 4.11 (br s, 1H), 4.36 (d, J = 12.0 Hz, 1H),

4.50 (d, J = 3.0 Hz, 2H), 4.64 (d, J = 11.0 Hz, 1H), 4.69 (d,

J = 11.5 Hz, 1H), 4.81 (d, J = 11.0 Hz, 1H), 4.90 (s, 2H),

5.54 (d, J = 9.0 Hz, 1H, H_1′), 6.95 (d, J = 7.0 Hz, 2H), 7.04–

7.13 (m, 3H), 7.20 (t, J = 8.0 Hz, 1H), 7.25 (dd, J₁= 7.5 Hz, J₂

= 2.5 Hz, 1H), 7.29–7.40 (m, 15H), 7.55 (d, J = 7.5 Hz, 1H),

7.60 (t, J = 8.0 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆):

68.4, 72.2, 73.9, 74.2, 74.7 (CH_{2 sugar + benzyl}), 76.3, 76.5,

77.3, 80.0, 84.9 (CH_sugar), 123.7, 124.8, 127.4-128.3, 138.2

(CH), 117.5, 137.6, 138.0, 138.1 (2C), 138.4 (C), 157.4,

182.0 (C=O).

128.7, 129.2, 136.3 (CH), 118.2, 137.2, 137.5, 138.0, 138.2,

138.3, 139.2, 147.5 (C), 157.7, 182.7 (C=O).

4.1.7. Typical procedure for the acid-catalyzed coupling

reaction

A solution of glycosyl-isatine (0.19 mmol) and oxindole

(0.19 mmol) in a mixture of acetic acid (0.4 ml) and concen-

trated HCl (2.6 μl) was refluxed for 24 hours. After cooling,

EtOAc was added to the mixture, the organic phases were

washed twice with H₂O, dried over MgSO₄and concentrated

under vacuum to give a residue which was purified by flash

chromatography (eluent cyclohexane/EtOAc 80:20) to give

the corresponding protected glycosyl-isoindigo derivatives.

1-(2,3,4,6-tetra-O-benzyl-β-^D-glucopyranosyl)-5-nitroindo-

lin-2,3-dione 16: C₄₂H₃₈N₂O₉obtained in 66% yield as a yel-

1

low gum, IR (NaCl): ν_C=O1753 cm⁻¹; ν_C=C1615 cm⁻¹. H

1-(2,3,4,6-tetra-O-benzyl-β-^D-glucopyranosyl)-isoindigo 19:

Obtained in 40% yield as a red solid (m.p. = 205 °C), IR

(KBr): ν_NH3410 cm⁻¹, ν_C=O1700 cm ⁻¹, ν_C=C1600 cm⁻¹.

HRMS (ES) Calcd. for C₅₀H₄₅N₂O₇[M + H]⁺: 785.3227;

Found: 785.3224. ¹H (400 MHz, DMSO-d₆): 3.75 (d,

J = 2.5 Hz, 2H), 3.80–3.87 (m, 1H), 3.88–3.94 (m, 1H), 3.96

(t, J = 9.0 Hz, 1H), 4.10–4.28 (m, 2H), 4.54 (d, J = 12.0 Hz,

1H), 4.57 (d, J = 12.5 Hz, 1H), 4.57–4.63 (m, 1H), 4.65 (d,

J = 11.0 Hz, 1H), 4.84 (d, J = 11.0 Hz, 1H), 4.89 (s, 2H),

5.73 (br s, 1H, H_1′), 6.90–6.95 (m, 3H), 6.98 (t, J = 8.0 Hz,

2H), 7.01–7.10 (m, 2H), 7.13–7.18 (m, 1H), 7.27–7.47 (m,

18H), 8.97 (d, J = 8.0 Hz, 1H), 9.15 (d, J = 8.0 Hz, 1H), 11.0

(s, 1H, NH). ¹³C (100 MHz, DMSO-d₆): 68.5, 72.2, 73.9, 74.2,

74.6 (CH_{2 sugar + benzyl}), 76.5, 76.8, 77.5, 80.1, 84.8 (CH_sugar),

109.7, 121.2, 122.2, 127.4–128.2, 129.0, 129.3, 132.3, 133.2

(CH), 120.9, 121.6, 131.3, 134.6, 137.4, 138.0 (2C), 138.2,

138.5, 144.5 (C), 168.6 (2 C=O).

(400 MHz, DMSO-d₆): 3.68–3.78 (m, 2H); 3.88 (t;

J = 9.0 Hz, 1H), 3.93–3.99 (m, 1H), 4.00–4.14 (m, 2H), 4.41

(d, J = 12.0 Hz, 1H), 4.49 (d, J = 12.5 Hz, 1H), 4.53 (d,

J = 12.5 Hz, 1H), 4.66 (d, J = 11.0 Hz, 1H), 4.72 (d,

J = 12.0 Hz, 1H), 4.86 (d, J = 11.0 Hz, 1H), 4.94 (s, 2H),

5.63 (d, J = 9.0 Hz, 1H, H_1’), 6.94–7.07 (m, 5H), 7.27–7.45

(m, 15H), 7.53 (d, J = 9.0 Hz, 1H), 8.16 (d, J = 2.5 Hz, 1H),

8.32 (dd, J₁= 9.0 Hz, J₂= 1.5 Hz, 1H). ¹³C (100 MHz,

CDCl₃): 68.0, 73.3, 74.5, 75.2, 76.0 (CH_{2 sugar + benzyl}), 75.6,

76.8, 77.2, 80.8, 86.0 (CH_sugar), 113.9, 120.4, 127.4–128.6,

132.3 (CH), 117.2, 137.3, 137.7, 137.8 (2C), 143.7, 152.1

(C), 156.8, 180.4 (C=O).

1-(2,3,4,6-tetra-O-benzyl-β-^D-glucopyranosyl)-5-bromoin-

dolin-2,3-dione 17: C₄₂H₃₈BrNO₇obtained in 49% yield as

1

yellow gum, IR (NaCl): ν_C=O1747 cm⁻¹; ν_C=C1606 cm⁻¹. H

(400 MHz, DMSO-d₆): 3.66–3.74 (m, 2H), 3.81 (t, J = 9.0 Hz,

1H), 3.87–3.93 (m, 1H), 3.98 (t, J = 9.0 Hz, 1H), 4.02–4.10

(m, 1H), 4.39 (d, J = 12.0 Hz, 1H), 4.48 (d, J = 12.5 Hz,

1H), 4.52 (d, J = 13.0 Hz, 1H), 4.63 (d, J = 11.0 Hz, 1H),

4.69 (d, J = 12.5 Hz, 1H), 4.83 (d, J = 10.5 Hz, 1H), 4.91 (s,

2H), 5.53 (d, J = 9.5 Hz, 1H, H_1’), 6.97 (d, J = 7.0 Hz, 2H),

7.05 (t, J = 7.0 Hz, 2H), 7.09–7.14 (m, 1H), 7.23–7.43 (m,

16H), 7.65–7.70 (m, 2H). ¹³C (100 MHz, DMSO-d₆): 68.4,

72.2, 73.8, 74.2, 74.8 (CH_{2 sugar + benzyl}), 76.1, 76.2, 77.3,

79.9, 85.0 (CH_sugar), 127.0, 127.4–128.3, 139.5 (CH), 115.6,

119.2, 137.6, 138.0, 138.1, 138.3, 146.9 (C), 156.9, 180.7

(C=O).

1-(2,3,4,6-tetra-O-benzyl-β-^D-glucopyranosyl)-5-nitroisoin-

digo 20: Obtained in 25% yield as a red gum, IR (NaCl): ν_NH

3409 cm⁻¹, ν_C=O1702 cm⁻¹, ν_C=C1615 cm⁻¹. HRMS (ES)

Calcd. for C₅₀H₄₃N₃NaO₉(M + Na)⁺: 852.2897; Found:

852.2924. ¹H (400 MHz, DMSO-d₆): 3.72–3.76 (m, 2H),

3.84–3.96 (m, 2H), 4.00 (t, J = 9.5 Hz, 1H), 4.22–4.29 (m,

1H), 4.50 (d, J = 12.0 Hz, 1H), 4.55 (d, J = 12.0 Hz, 1H),

4.66 (d, J = 10.5 Hz, 1H), 4.62–4.69 (m, 1H), 4.85 (d,

J = 10.5 Hz, 1H), 4.92 (s, 2H), 5.76 (d, J = 9.0 Hz, 1H, H_1′),

6.84–6.90 (m, 4H), 6.94–6.98 (m, 2H), 7.04–7.10 (m, 1H),

7.26–7.45 (m, 15H), 7.47–7.53 (m, 2H), 8.15–8.23 (m, 1H),

9.00 (d, J = 8.0 Hz, 1H), 10.11 (d, J = 2.5 Hz, 1H), 11.10 (s,

1H, NH). The signal of one proton of the sugar moiety is

missing. ¹³C (100 MHz, DMSO-d₆): 68.4, 72.2, 73.8, 74.1,

1-(2,3,4,6-tetra-O-benzyl-β-^D-glucopyranosyl)-5-phenylin-

dolin-2,3-dione 18: C₄₈H₄₃NO₇obtained in 22% yield as a yel-

low gum, IR (NaCl): ν_C=O1741, 1694 cm⁻¹; ν_C=C1610 cm⁻¹.

¹H (400 MHz, DMSO-d₆): 3.68–3.76 (m, 2H), 3.84 (t,

J = 9.5 Hz, 1H), 3.93 (dt, J₁= 9.5 Hz, J₂= 3.0 Hz, 1H), 4.00

(t, J = 9.0 Hz, 1H), 4.07–4.18 (br s, 1H), 4.42 (d, J = 11.5 Hz,

1H), 4.51 (d, J = 12.5 Hz, 1H), 4.54 (d, J = 12.5 Hz, 1H), 4.65

(d, J = 11.0 Hz, 1H), 4.71 (d, J = 12.0 Hz, 1H), 4.84 (d,

J = 11.0 Hz, 1H), 4.92 (s, 2H), 5.58 (d, J = 9.0 Hz, 1H, H_1′),

6.98 (d, J = 7.0 Hz, 2H), 7.04 (t, J = 7.5 Hz, 2H), 7.07–7.13

(m, 1H), 7.25–7.29 (m, 2H), 7.31–7.47 (m, 15H), 7.54 (t,

J = 7.5 Hz, 2H), 7.72 (d, J = 7.5 Hz, 2H), 7.77 (d,

J = 1.5 Hz, 1H), 7.83 (d, J = 7.5 Hz, 1H). ¹³C (100 MHz,

CDCl₃): 68.3, 73.5, 75.0, 75.4, 76.2 (CH_{2 sugar + benzyl}), 76.4,

77.4, 77.5, 81.0, 86.4 (CH_sugar), 114.1, 123.8, 126.6, 127.6–

74.6 (CH₂

_benzyl), 76.5, 76.6, 77.3, 80.1, 85.0

+

sugar

(CH_sugar), 120.9, 121.2, 128.6, 137.2, 137.3, 138.0, 138.2,

138.3, 142.3, 145.2, 145.9 (C), 110.0, 121.5, 124.3, 127.2–

128.3, 129.8, 134.3 (CH), 166.8, 168.6 (C=O).

1-(2,3,4,6-tetra-O-benzyl-β-^D-glucopyranosyl)-5-bromoi-

soindigo 21: Obtained in 43% yield as a red gum, IR (NaCl):

ν_NH3214 cm⁻¹; ν_C=O1696, 1661 cm⁻¹; ν_C=C1606 cm⁻¹.

HRMS (ES) Calcd. for C₅₀H₄₃BrN₂NaO₇(M + Na)⁺:

885.2151/887.2131;

Found:

885.2130/887.2128.

¹H

(400 MHz, DMSO-d₆): 3.70–3.76 (m, 2H), 3.79–3.86 (m,

1H), 3.87–3.93 (m, 1H), 3.96 (t, J = 8.5 Hz, 1H), 4.03–4.26

(m, 2H), 4.51 (d, J = 12.0 Hz, 1H), 4.55 (d, J = 12.0 Hz,

96

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 88–100

1H), 4.59–4.67 (m, 2H), 4.84 (d, J = 11.0 Hz, 1H), 4.90 (s,

2H), 5.70 (br s, 1H, H_1′), 6.88–6.99 (m, 5H), 7.00–7.07 (m,

2H), 7.25–7.56 (m, 18H), 8.98 (d, J = 8.5 Hz, 1H), 9.36 (d,

J = 2.0 Hz, 1H), 11.04 (s, 1H, NH). ¹³C (100 MHz, DMSO-

d₆): 68.4, 72.2, 73.9, 74.2, 74.6 (CH_{2 sugar + benzyl}), 76.5 (2C),

77.4, 80.0, 84.9 (CH_sugar), 109.9, 113.3, 121.4, 127.4–128.3,

129.6, 131.1, 133.8, 134.1 (CH), 114.1, 121.3; 122.7, 129.8,

136.1, 137.4, 138.0, 138.2, 138.4, 140.5, 144.8 (C), 166.4,

168.7 (C=O).

1-(β-^D-Glucopyranosyl)-5-nitroisoindigo 24: Obtained in

18% yield as a red solid (m.p. > 300 °C), IR (KBr): ν_{NH, OH}

3424 cm⁻¹; ν_C=O1720, 1703 cm⁻¹; ν_C=C1618 cm⁻¹. HRMS

(ES) Calcd. for C₂₂H₁₉N₃NaO₉[M + Na]⁺: 492.1019; Found:

492.1006. ¹H (400 MHz, DMSO-d₆): 3.30–3.45 (m, 3H),

3.47–3.58 (m, 1H), 3.80 (dd, 1H, J₁= 10.5 Hz, J₂= 5.5 Hz),

3.76–3.95 (m, 1H), 4.70 (t, J = 5.5 Hz, 1H, OH), 5.21 (d,

J = 5.0 Hz, 1H, OH), 5.23 (d, J = 4.5 Hz, 1H, OH), 5.45 (d,

J = 4.5 Hz, 1H, OH), 5.42 (br s, 1H, H_1′), 6.94 (d, J = 7.5 Hz,

1H), 7.09 (td, J₁= 8.0 Hz, J₂= 1.0 Hz, 1H), 7.47 (td, J₁

= 7.5 Hz, J₂= 1.0 Hz, 1H), 7.51 (d, J = 9.0 Hz, 1H), 8.38 (dd,

J₁= 9.0 Hz, J₂= 2.5 Hz, 1H), 9.05 (d, J = 8.0 Hz, 1H), 10.21

(d, J = 2.5 Hz, 1H), 11.10 (s, 1H, NH). ¹³C (100 MHz,

DMSO-d₆): 61.0 (CH₂), 68.7, 69.7, 77.0, 80.2, 82.2

(CH_sugar), 110.1, 111.7, 121.5, 124.2, 127.6, 130.0, 134.2

(CH), 121.1, 121.2, 129.2, 136.9, 142.1, 145.2, 147.0 (C),

167.1, 168.7 (C=O).

1-(2,3,4,6-tetra-O-benzyl-β-^D-glucopyranosyl)-5-phenyli-

soindigo 22: Obtained in 60% yield as a red gum, IR (NaCl):

ν_NH3410 cm⁻¹; ν_C=O1780 cm⁻¹; ν_C=C1610 cm⁻¹. HRMS

(ES) Calcd. for C₅₆H₄₈N₂NaO₇[M + Na]⁺: 883.3359; Found:

883.3333. ¹H (400 MHz, DMSO-d₆): 3.72–3.79 (m, 2H),

3.82–3.90 (m, 1H), 3.91–3.96 (m, 1H), 3.99 (t, J = 8.5 Hz,

1H), 4.12–4.30 (m, 2H), 4.54 (d, J = 12.0 Hz, 1H), 4.58 (d,

J = 12.0 Hz, 1H), 4.61–4.66 (m, 1H), 4.67 (d, J = 11.0 Hz,

1H), 4.85 (d, J = 10.5 Hz, 1H), 4.91 (s, 2H), 5.75 (br s, 1H,

H_1′), 6.90–6.99 (m, 5H), 7.01–7.07 (m, 2H), 7.27–7.48 (m,

18H), 7.55 (t, J = 8.0 Hz, 2H), 7.62–7.69 (m, 1H), 7.69 (d,

J = 8.0 Hz, 2H), 9.00 (d, J = 7.5 Hz, 1H), 9.51 (d,

J = 2.0 Hz, 1H), 10.95 (s, 1H, NH). ¹³C (100 MHz, DMSO-

d₆): 68.5, 72.3, 73.8, 74.2, 74.6 (CH_{2 sugar + benzyl}), 76.6, 76.7,

77.5, 80.1, 84.9 (CH_sugar), 109.8, 112.4, 121.2, 126.2, 127.0,

127.3–128.2, 129.0, 129.4, 130.4, 133.3 (CH), 121.5, 121.6,

131.3, 134.1, 135.0, 137.4, 138.0, 138.2, 138.4, 140.1, 140.7,

144.5 (C), 167.0, 168.7 (C=O).

1-(β-^D-Glucopyranosyl)-5-bromoisoindigo 25: Obtained in

59% yield as a red solid (m.p. > 300 °C), IR (KBr): ν_{NH, OH}

−1

3540, 3500–3200 cm⁻¹; ν_C=O1700, 1680 cm

; ν_C=C1620,

1600 cm⁻¹. HRMS (ES) Calcd. for C₂₂H₁₉BrN₂NaO₇(M

+ Na)⁺: 525.0273/527.0253; Found: 525.0261/527.0282. ¹H

(400 MHz, DMSO-d₆): 3.27–3.45 (m, 3H), 3.49–3.58 (m,

1H), 3.78 (dd, J₁= 11.0 Hz, J₂= 5.0 Hz, 1H), 3.79–3.96 (m,

1H), 4.66 (t, J = 5.5 Hz, 1H, OH), 5.17 (d, J = 5.0 Hz, 1H,

OH), 5.21 (d, J = 4.0 Hz, 1H, OH), 5.37 (br s, 1H, H_1′), 5.40

(d, J = 5.0 Hz, 1H, OH), 6.91 (d, J = 8.0 Hz, 1H), 7.03 (td, J₁

= 8.0 Hz, J₂= 1.0 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 7.43 (td,

J₁= 7.5 Hz, J₂= 1.0 Hz, 1H), 7.64 (dd, J₁= 8.5 Hz, J₂

= 2.0 Hz, 1H), 9.04 (d, J = 8.0 Hz, 1H), 9.43 (d, J = 2.0 Hz,

1H), 11.04 (s, 1H, NH). ¹³C (100 MHz, DMSO-d₆): 61.0

(CH₂), 68.4, 69.7, 77.2, 80.1, 82.0 (CH_sugar), 109.9, 113.3,

121.3, 129.8, 130.9, 133.6, 134.2 (CH), 113.7, 121.4, 122.9,

130.4, 135.6, 141.2, 144.7 (C), 166.6, 168.7 (C=O).

4.1.8. Typical procedure for the deprotection step

BBr₃(1 M solution in CH₂Cl₂, 1.5 mmol) was added to a

cooled (–80 °C) solution of protected glycosyl-isoindigo

(0.10 mmol) in CH₂Cl₂(9.5 ml). The mixture was stirred dur-

ing 2 hours (compounds 24–26) or 12 hours (compound 23)

before hydrolysis at –80 °C and warming up to room tempera-

ture. After decantation and extraction with EtOAc, the organic

phases were dried over MgSO₄and concentrated under va-

cuum to give a residue which was purified by flash chromato-

graphy (eluent EtOAc/MeOH from 95:5 to 85:15) to give the

corresponding non-protected glycosyl-isoindigo.

1-(β-^D-Glucopyranosyl)-5-phenylisoindigo 26: Obtained in

66% yield as a red powder (m.p. > 300 °C), IR (KBr):

ν_{NH, OH}3400 cm⁻¹; ν_C=O1706, 1683 cm⁻¹; ν_C=C1616 cm⁻¹.

HRMS (ES) Calcd. for C₂₈H₂₄N₂NaO₇(M + Na)⁺: 523.1481;

1

Found: 523.1497. H (400 MHz, DMSO-d₆): 3.30–3.46 (m,

3H), 3.52–3.60 (m, 1H), 3.77–3.84 (m, 1H), 3.88–4.03 (br s,

1H), 4.66 (t, J = 5.5 Hz, 1H, OH), 5.15 (d, J = 5.0 Hz, 1H,

OH), 5.20 (d, J = 5.0 Hz, 1H, OH), 5.40 (br s, 1H, H_1′), 5.40

(d, J = 5.0 Hz, 1H, OH), 6.92 (d, J = 7.5 Hz, 1H), 7.03 (dt, J₁

= 8.0 Hz, J₂= 1.0 Hz, 1H), 7.35 (d, J = 8.5 Hz, 1H), 7.38–7.44

(m, 2H), 7.51–7.56 (m, 2H), 7.67–7.71 (m, 2H), 7.77 (dd, J₁

= 8.5 Hz, J₂= 2.0 Hz, 1H), 9.05 (d, J = 7.5 Hz, 1H), 9.55 (d,

J = 2.0 Hz, 1H), 10.94 (s, 1H, NH). ¹³C (100 MHz, DMSO-d₆)

: 61.1 (CH_{2 sugar}), 68.6, 69.9, 77.3, 80.1, 82.0 (CH_sugar), 109.8,

111.7, 121.2, 126.2 (2C), 127.0, 127.3, 129.0 (2C), 129.5,

130.5, 133.1 (CH), 121.6 (2C), 132.0, 133.9, 134.5, 140.3,

141.3, 144.4 (C), 167.1, 168.8 (C=O).

1-(β-^D-Glucopyranosyl)-isoindigo 23: Obtained in 60%

yield as a red solid (m.p. > 300 °C), IR (KBr): ν_NH,

OH

3377 cm⁻¹; ν_C=O1696; 1680 cm⁻¹; ν_C=C1604 cm⁻¹. HRMS

(FAB +) Calcd. for C₂₂H₂₁N₂O₇[M + H]⁺: 425.1349; Found:

425.1357. ¹H (400 MHz, DMSO-d₆): 3.29–3.46 (m, 3H),

3.52–3.59 (m, 1H), 3.78–3.84 (m, 1H), 3.86–4.02 (br s, 1H),

4.68 (t, J = 5.5 Hz, 1H, OH), 5.18 (d, J = 5.0 Hz, 1H, OH),

5.22 (d, J = 5.0 Hz, 1H, OH), 5.40 (d, J = 5.0 Hz, 1H, OH),

5.40 (br s, 1H, H_1′), 6.92 (d, J = 7.5 Hz, 1H), 7.04 (t,

J = 8.0 Hz, 1H), 7.13 (t, J = 7.5 Hz, 1H), 7.28 (d,

J = 8.0 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.48 (t, J = 7.5 Hz,

1H), 9.05 (d, J = 8.0 Hz, 1H), 9.19 (d, J = 7.5 Hz, 1H), 11.0

(s, 1H, NH). ¹³C (100 MHz, DMSO-d₆): 61.0 (CH₂), 68.4,

69.8, 77.3, 80.1, 81.9 (CH_sugar), 109.7, 111.5, 121.2, 121.7,

128.8, 129.4, 132.3, 132.9 (CH), 121.1, 121.6, 132.1, 134.0,

142.3, 144.3 (C), 167.0, 168.7 (C=O).

4-[5-(2-Oxo)-indolyl]-4-oxobutanoic acid 27: Succinic an-

hydride (225 mg, 2.25 mmol) was added to a solution of alu-

minum chloride (600 mg, 4.5 mmol) in CH₂Cl₂(3.1 ml). The

mixture was stirred at room temperature for 15 min before ad-

dition of a solution of oxindole (99.9 mg, 0.75 mmol) in

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 88–100

97

CH₂Cl₂(2 ml). The mixture was stirred at room temperature

for 24 hours before hydrolysis to give a solid which was iso-

lated by filtration and washed with water and CH₂Cl₂. The

corresponding oxindole 27 was obtained in 88% yield as a gray

solid (m.p. = 252–255 °C; Litt = 246–250 °C [20]), IR (KBr):

ν_{OH, NH}3560; 3300 cm⁻¹; ν_C=O1730, 1710, 1680 cm⁻¹; ν_C=C

2H), 4.97 (t, J = 9.0 Hz, 1H), 5.19 (t, J = 9.0 Hz, 1H), 5.46

(t, J = 9.0 Hz, 1H), 5.54 (d, J = 9.0 Hz, 1H, H_1′), 6.70 (t,

J = 7.0 Hz, 1H), 6.80 (d, J = 7.0 Hz, 1H), 7.05–7.12 (m, 2H).

¹³C (100 MHz, DMSO-d₆): 20.3, 20.4 (2C), 20.7 (CH_{3 acetate}),

27.5, 45.1 (CH_{2 indoline}), 61.9 (CH_{2 sugar}), 68.4, 68.6, 71.8,

73.1, 82.4 (CH_sugar), 108.1, 119.0, 124.6, 127.0 (CH), 129.9,

149.7 (C), 169.3, 169.4, 169.6, 170.0 (C=O_acetate).

+

1610 cm⁻¹. HRMS (ES) Calcd. for C₁₂H₁₁NNaO₄(M + Na) :

1

256.0586; Found: 256.0594. H (400 MHz, DMSO-d₆): 2.59

1-(2,3,4,6-tetra-O-acetyl-β-^D-glucopyranosyl)-5-nitroindo-

line 30: C₂₂H₂₆N₂O₁₁obtained in 78% yield as a yellow solid

(m.p. = 158 °C), IR (KBr): ν_C=O1750 cm⁻¹; ν_C=C1606 cm⁻¹.

¹H (400 MHz, DMSO-d₆): 1.94, 2.00, 2.04 (3 s, 12H,

CH_{3 acetate}), 2.95-3.06 (m, 1H), 3.08–3.18 (m, 1H), 3.63–3.73

(m, 2H), 4.03–4.20 (m, 3H), 5.03 (t, J = 9.5 Hz, 1H), 5.25 (t,

J = 9.5 Hz, 1H), 5.46 (t, J = 9.5 Hz, 1H), 5.67 (d, J = 9.5 Hz,

1H, H_1’), 6.89 (d, J = 9.0 Hz, 1H), 7.95 (d, J = 2.5 Hz, 1H),

8.09 (dd, J₁= 9.0 Hz, J₂= 2.5 Hz, 1H). ¹³C (100 MHz,

DMSO-d₆): 20.4 (2C), 20.5, 20.6 (CH_{3 acetate}), 26.5, 46.2

(CH_{2 indoline}), 61.9 (CH_{2 sugar}), 68.1, 68.4, 72.4, 72.8, 81.6

(CH_sugar), 106.4, 120.8, 125.6 (CH), 131.1, 139.1, 155.8, (C),

169.5–170.2 (C=O_acetate).

(t, J = 6.0 Hz, 2H, CH₂), 3.21 (t, J = 6.0 Hz, 2H, CH₂), 3.59

(s, 2H, CH₂), 6.95 (d, J = 8.0 Hz, 1H), 7.86 (s, 1H), 7.92 (d,

J = 8.0 Hz, 1H), 10.80 (s, 1H, NH), 12.15 (s, 1H, OH). ¹³C

(100 MHz, DMSO-d₆): 28.0, 32.6, 35.5 (CH₂), 108.7, 124.1,

129.0 (CH), 126.1, 130.1, 148.3 (C), 173.9, 176.8, 197.0

(C=O).

4-[5′-(1-(2,3,4,6-tetra-O-benzyl)-β-^D-glucopyranosyl)-isoin-

digoyl)-4-oxobutanoic acid 28: Obtained in 88% yield as a red

solid (m.p. = 105–106 °C) by reaction of glycosyl-isatine 15

with oxindole 27 using the same conditions as described

above. The only difference is that compound 28 precipitated

when the reaction mixture was hydrolyzed. This precipitate

was isolated by filtration and washed with water. IR (KBr):

ν_{NH, OH}3380, 3260 cm⁻¹; ν_C=O1740, 1710, 1680 cm⁻¹; ν_C=C

1610 cm⁻¹. HRMS (ES) Calcd. for C₅₄H₄₉N₂O₁₀(M + H)⁺:

1-(2,3,4,6-tetra-O-acetyl-β-^D-glucopyranosyl)-5-bromoindo-

line 31: C₂₂H₂₆BrNO₉obtained in 78% yield as a yellow solid

(m.p. = 142 °C), IR (KBr): ν_C=O1741 cm⁻¹; ν_C=C1597 cm⁻¹.

¹H (400 MHz, DMSO-d₆): 1.98, 1.99, 2.00, 2.04 (4s, 12H,

CH_{3 acetate}), 2.83–2.94 (m, 1H), 2.96–3.05 (m, 1H), 3.45–3.56

(m, 2H), 3.97–4.04 (m, 1H), 4.09–4.16 (m, 2H), 4.98 (t,

J = 9.5 Hz, 1H), 5.18 (t, J = 9.5 Hz, 1H), 5.44 (t, J = 9.5 Hz,

1H), 5.49 (d, J = 9.5 Hz, 1H, H_1′), 6.74 (d, J = 8.5 Hz, 1H),

7.23 (dd, J₁= 8.5 Hz, J₂= 2.0 Hz, 1H), 7.25 (d, J = 2.0 Hz,

1H). ¹³C (100 MHz, DMSO-d₆): 20.2, 20.3 (2C), 20.4

(CH_{3 acetate}), 27.2, 45.4 (CH_{2 indoline}), 61.8 (CH_{2 sugar}), 68.2,

68.4, 71.8, 72.9, 82.1 (CH_sugar), 109.7, 127.4, 129.4 (CH),

109.7, 132.8, 149.1, (C), 169.2, 169.3, 169.5, 169.9 (C=

O_acetate).

1

885.3387; Found: 885.3412. H (400 MHz, DMSO-d₆): 2.61

(t, J = 6.0 Hz, 2H, CH₂), 3.18–3.25 (m, 2H, CH₂), 3.72 (d,

J = 2.0 Hz, 2H, CH₂), 3.78–3.86 (m, 1H), 3.90–3.95 (m, 1H),

3.99 (t, J = 8.5 Hz, 1H), 4.08–4.24 (m, 2H), 4.47–4.55 (m,

3H), 4.62 (d, J = 11.0 Hz, 1H), 4.80 (d, J = 11.0 Hz, 1H),

4.86 (s, 2H), 5.79 (br s, 1H, H_1′), 6.86–6.95 (m, 4H), 6.96–

7.03 (m, 2H), 7.12 (dt, J₁= 8.0 Hz, J₂= 1.5 Hz, 1H), 7.19–

7.43 (m, 17H), 8.06 (dd, J₁= 8.5 Hz, J₂= 1.5 Hz, 1H), 9.11

(d, J = 8.0 Hz, 1H), 9.71 (s, 1H), 11.43 (s, 1H, NH). ¹³C

(100 MHz, DMSO-d₆): 27.9, 32.7 (CH₂), 68.5, 72.2, 73.9,

74.1, 74.6 (CH₂

_benzyl), 76.4, 76.9, 77.4, 80.0, 84.7

+

sugar

(CH_sugar), 109.6, 122.3, 123.7, 124.8, 127.4–128.2, 129.3,

129.8, 132.8, 133.2 (CH), 120.8, 121.3, 130.1, 132.6, 133.4,

137.6, 138.1 (2C), 138.2, 138.5, 148.1 (C), 167.0, 168.9,

173.9, 196.7 (C=O).

4.1.10. Aromatization of compounds 29–31

The aromatization of compounds 29–31 was performed

using the same procedure as described before for the prepara-

tion of compounds 7–10. The reaction mixture was stirred at

room temperature during 12 hours for compound 32 and

24 hours for compound 34. The aromatization was carried out

at 100 °C during 3 days for compound 33.

4.1.9. Typical procedure for the acetylation of the sugar

residue

A solution of indoline (3.55 mmol) in pyridine (10.7 ml)

was cooled to 0 °C before addition of acetic anhydride (7.9

ml). The reaction mixture was stirred at room temperature for

24 hours. After addition of water and extraction with EtOAc,

the organic phases were washed with water and saturated aqu-

eous NaHCO₃until neutral pH. After drying over MgSO₄, the

solvent was removed and the residue purified by flash chroma-

tography (eluent cyclohexane/EtOAc from 80:20 to 40:60) to

give the corresponding protected compounds.

1-(2,3,4,6-tetra-O-acetyl-β-^D-glucopyranosyl)-indole 32:

C₂₂H₂₅NO₉obtained in 98% yield as a white solid (m.p.

= 140 °C), IR (KBr): υ_C=O1790–1720 cm⁻¹; ν_C=C1650,

1

1610 cm⁻¹. H (400 MHz, DMSO-d₆): 1.67, 2.00, 2.02, 2.08

(4s, 12H, CH_{3 acetate}), 4.10–4.20 (m, 2H), 4.32–4.38 (m, 1H),

5.27 (t, J = 10.0 Hz, 1H), 5.58 (t, J = 9.5 Hz, 1H), 5.65 (t,

J = 9.5 Hz, 1H), 6.26 (d, J = 9.0 Hz, 1H, H_1’), 6.55 (d,

J = 3.5 Hz, 1H), 7.12 (dt, J₁= 8.0 Hz, J₂= 1.0 Hz, 1H), 7.24

(dt, J₁= 8.0 Hz, J₂= 1.0 Hz, 1H), 7.50 (d, J = 3.5 Hz, 1H),

7.58 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.5 Hz, 1H). ¹³C

(100 MHz, DMSO-d₆): 19.8, 20.2, 20.4, 20.5 (CH_{3 acetate}), 62.1

(CH_{2 sugar}), 68.1, 69.9, 72.7, 73.0, 81.3 (CH_sugar), 103.2, 110.4,

120.2, 120.6, 121.8, 125.7 (CH), 128.4, 136.0 (C), 168.4,

169.4, 169.6, 170.1 (C=O_acetate).

1-(2,3,4,6-tetra-O-acetyl-β-^D-glucopyranosyl)-indoline 29:

C₂₂H₂₇NO₉obtained in 96% yield as a white solid (m.p.

= 100 °C), IR (KBr): υ_C=O1780–1730 cm⁻¹; ν_C=C

1

1610 cm⁻¹. H (400 MHz, DMSO-d₆): 1.95, 1.99, 2.00, 2.03

(4s, 12H, CH₃acetate), 2.80–2.90 (m, 1H), 2.93–3.04 (m, 1H),

3.42–3.55 (m, 2H), 3.95–4.04 (m, 1H), 4.14 (m, J = 9.0 Hz,

98

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 88–100

1-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-5-nitroindole

20.6 (2C), 20.8 (CH_{3 acetate}), 61.5 (CH_{2 sugar}), 67.6, 68.0,

72.5, 75.2, 80.2 (CH_sugar), 114.2, 121.3, 133.4 (CH), 117.7,

144.7, 152.1 (C), 156.9, 179.8 (C=O_isatine), 169.6, 169.7,

169.9, 170.3 (C=O_acetate).

33: C₂₂H₂₄N₂O₁₁obtained in 98% yield as a yellow solid (m.

1

p. = 163 °C), IR (KBr): ν_C=O1749 cm⁻¹; ν_C=C1616 cm⁻¹. H

(400 MHz, DMSO-d₆): 1.67, 2.01, 2.04, 2.09 (4s, 12H,

CH_{3 acetate}), 4.13–4.23 (m, 2H), 4.33–4.39 (m, 1H), 5.34 (t,

J = 9.5 Hz, 1H), 5.58 (t, J = 9.5 Hz, 1H), 5.66 (t, J = 9.5 Hz,

1H), 6.36 (d, J = 9.0 Hz, 1H, H_1′), 6.86 (d, J = 3.5 Hz, 1H),

7.81 (d, J = 3.5 Hz, 1H), 7.92 (d, J = 9.5 Hz, 1H), 8.16 (dd,

J₁= 9.0 Hz, J₂= 2.5 Hz, 1H), 8.61 (d, J = 2.5 Hz, 1H). ¹³C

(100 MHz, DMSO-d₆): 19.7, 20.2, 20.4, 20.5 (CH_{3 acetate}), 62.0

(CH_{2 sugar}), 67.9, 70.0, 72.4, 73.3, 81.8 (CH_sugar), 105.5, 111.1,

117.2, 117.6, 129.6 (CH), 127.9, 138.7, 141.6 (C), 168.4,

169.4, 169.6, 170.0 (C=O_acetate).

1-(2,3,4,6-tetra-O-acetyl-β-^D-glucopyranosyl)-5-bromoindo-

line-2,3-dione 37: C₂₂H₂₂BrNO₁₁obtained in 75% yield as a

1

yellow gum, IR (NaCl): ν_C=O1752 cm⁻¹; ν_C=C1607 cm⁻¹. H

(400 MHz, CDCl₃): 1.91, 2.01, 2.07, 2.09 (4 s, 12H,

CH_{3 acetate}), 3.94 (ddd, , J₁= 10.0 Hz, J₂= 4.5 Hz, J₃

= 2.0 Hz,1H), 4.17 (dd, J₁= 12.5 Hz, J₂= 2.5 Hz, 1H), 4.27

(dd, J₁= 12.5 Hz, J₂= 4.5 Hz, 1H), 5.23 (t, J = 9.5 Hz, 1H),

5.40 (t, J = 9.5 Hz, 1H), 5.48 (t, J = 9.0 Hz, 1H), 5.67 (d,

J = 9.5 Hz, 1H, H_1′), 7.20 (dd, J₁= 8.0 Hz, J₂= 1.0 Hz, 1H),

7.74 (dd, J₁= 8.0 Hz, J₂= 2.5 Hz, 1H), 7.75 (d, J = 2.0 Hz,

1H). ¹³C (100 MHz, CDCl₃): 20.3, 20.6 (2C), 20.7

(CH_{3 acetate}), 61.6 (CH_{2 sugar}), 67.7 (2C), 72.8, 74.9, 79.9

(CH_sugar), 115.4, 128.5, 140.9 (CH), 117.8, 119.1, 146.8 (C),

156.8, 180.5 (C=O_isatine), 169.6, 169.7, 169.8, 170.4 (C=

1-(2,3,4,6-tetra-O-acetyl-β-^D-glucopyranosyl)-5-bromoin-

dole 34: C₂₂H₂₄BrNO₉obtained in 90% yield as a white solid

(m.p. = 168 °C), IR (KBr): υ_C=O1740 cm⁻¹. (400 MHz,

DMSO-d₆): 1.68, 2.00, 2.03, 2.08 (4s, 12H, CH_{3 acetate});

4.10–4.21 (m, 2H), 4.30–4.35 (m, 1H), 5.29 (t, J = 10.0 Hz,

1H), 5.55 (t, J = 9.5 Hz, 1H), 5.63 (t, J = 9.0 Hz, 1H), 6.23

(d, J = 9.0 Hz, 1H, H_1′), 6.54 (d, J = 3.5 Hz, 1H), 7.37 (dd,

J₁= 9.0 Hz, J₂= 2.0 Hz, 1H), 7.57 (d, J = 3.5 Hz, 1H), 7.69

(d, J = 9.0 Hz, 1H), 7.79 (d, J = 2.0 Hz, 1H). ¹³C (100 MHz,

DMSO-d₆): 19.8, 20.2, 20.4, 20.5 (CH_{3 acetate}), 62.0

(CH_{2 sugar}), 67.9, 69.8, 72.5, 73.1, 81.6 (CH_sugar), 102.7,

112.6, 122.9, 124.4, 127.5 (CH), 112.7, 130.3, 134.6 (C),

168.4, 169.4, 169.6, 170.0 (C=O_acetate).

O_acetate).

4.1.12. General procedure for the preparation of the acetylated

glycosyl-isoindigos 38–40

To a solution of acetylated glycosyl-isatine (0.17 mmol) in

toluene (9 ml) were added successively, 4 Å molecular sieves,

oxindole (0.17 mmol) and PTSA (0.03 mmol). The mixture

was refluxed for 24 hours. After cooling, EtOAc was added,

the organic phase was washed with saturated aqueous

NaHCO₃. After drying over MgSO₄, the solvent was removed

and the residue purified by flash chromatography (eluent cy-

clohexane/EtOAc 50: 50) to give a red solid which was crystal-

lized in a mixture of acetic acid, toluene and water. The acety-

lated glycosyl-isoindigos were isolated by filtration. The

chemical yields for this coupling reaction were not optimized.

4.1.11. Oxidation of compounds 32–34

Oxidation of compounds 32–34 using chromium oxide was

performed according to the procedure described before.

1-(2,3,4,6-tetra-O-acetyl-β-^D-glucopyranosyl)-indoline-2,3-

dione 35: Obtained in 88% yield as a yellow gum, IR (NaCl):

υ_C=O1750 cm⁻¹; υ_C=C1612 cm⁻¹. MS (IE): Calcd. for

C₂₂H₂₄NO₁₁(M + H⁺): 478; Found: 478. HRMS (ES) [M +

Na]⁺Calcd. for C₂₂H₂₃NNaO₁₁, 500.1163; Found: 500.1170,

¹H (400 MHz, CDCl₃): 1.92, 2.03, 2.09, 2.11 (4s, 12H,

CH_{3 acetate}), 3.96 (ddd, , J₁= 10.0 Hz, J₂= 4.5 Hz, J₃

= 2.0 Hz, 1H), 4.20 (dd, J₁= 12.5 Hz, J₂= 2.0 Hz, 1H), 4.28

(dd, J₁= 12.5 Hz, J₂= 4.5 Hz, 1H), 5.26 (t, J = 10.0 Hz, 1H),

5.41 (t, J = 9.5 Hz, 1H), 5.57 (t, J = 9.5 Hz, 1H), 5.70 (d,

J = 9.5 Hz, 1H, H_1′), 7.22 (t, J = 8.0 Hz, 1H), 7.30 (d,

J = 8.0 Hz, 1H), 7.63–7.68 (m, 2H). ¹³C (100 MHz, CDCl₃):

20.3, 20.6, 20.7, 20.8 (CH₃acetate), 61.7 (CH_{2 sugar}), 67.7,

67.8, 73.0, 74.9, 79.9 (CH_sugar), 113.6, 124.7, 125.8, 138.7

(CH), 117.9, 148.0 (C), 157.6, 181.7 (C=O_isatine), 169.5,

169.6, 169.8, 170.5 (C=O_acetate).

1-(2,3,4,6-tetra-O-acetyl-β-^D-glucopyranosyl)-isoindigo 38:

Obtained in 15% yield as a red solid (m.p. = 277 °C), IR

(KBr): υ_NH3446 cm⁻¹; υ_C=O1742, 1699 cm⁻¹; υ_C=C

1618 cm⁻¹. HRMS (ES) Calcd. for C₃₀H₂₈N₂NaO₁₁(M +

Na)⁺: 615.1591; Found: 615.1616. ¹H (400 MHz, CDCl₃):

1.85, 2.02, 2.09, 2.10 (4s, 12H, CH_{3 acetate}), 3.92–3.99 (m,

1H), 4.19–4.31 (m, 2H), 5.30 (t, J = 10.0 Hz, 1H), 5.42 (t,

J = 9.5 Hz, 1H), 5.70-5.78 (m, 1H), 5.86 (br s, 1H, H_1’), 6.81

(d, J = 7.5 Hz, 1H), 7.05 (dt, J₁= 8.0 Hz, J₂= 1.0 Hz, 1H),

7.12 (t, J = 8.0 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 7.34 (dt, J₁

= 7.5 Hz, J₂= 1.0 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.57 (s,

1H, NH), 9.03 (d, J = 8.0 Hz, 1H), 9.14 (d, J = 8.0 Hz, 1H). ¹³

C

(100 MHz, CDCl₃): 20.4, 20.7 (2C), 20.8 (CH_{3 acetate}), 61.9

(CH_{2 sugar}), 67.7, 68.1, 73.7, 74.9, 79.7 (CH_sugar), 121.7, 122.5,

132.3, 134.2, 141.3, 142.7 (C), 109.5, 111.0, 122.5, 123.2,

129.8, 130.1, 132.7, 133.0 (CH), 167.7, 169.0, 169.2, 169.6,

170.1, 170.6 (C=O).

1-(2,3,4,6-tetra-O-acetyl-β-^D-glucopyranosyl)-5-nitroindo-

line-2,3-dione 36: C₂₂H₂₂N₂O₁₃obtained in 57% yield as a

yellow solid (m.p. = 60 °C), IR (KBr): ν_C=O1754 cm⁻¹; ν_C=C

1

1616 cm⁻¹. H (400 MHz, CDCl₃): 1.93, 2.04, 2.10, 2.12 (4s,

12H, CH_{3 acetate}), 3.99 (ddd, J₁= 10.0 Hz, J₂= 4.5 Hz, J₃

= 2.0 Hz,1H), 4.20 (dd, J₁= 12.5 Hz, J₂= 2.0 Hz, 1H), 4.32

(dd, J₁= 12.5 Hz, J₂= 4.5 Hz, 1H), 5.28 (t, J = 10.0 Hz, 1H),

5.42-5.52 (m, 2H), 5.73 (d, J = 9.0 Hz, 1H, H_1’), 7.50 (d,

J = 9.0 Hz, 1H), 8.53 (d, J = 2.5 Hz, 1H), 8.58 (dd, J₁

= 9.0 Hz, J₂= 2.5 Hz, 1H). ¹³C (100 MHz, CDCl₃): 20.3,

1-(2,3,4,6-tetra-O-acetyl-β-^D-glucopyranosyl)-5-nitroisoin-

digo 39: Obtained in 48% yield as a red solid (m.p. >300 °C),

IR (KBr): ν_NH3463 cm⁻¹; ν_C=O1749 cm⁻¹; ν_C=C1614 cm⁻¹.

HRMS (ES) Calcd. for C₃₀H₂₇N₃NaO₁₃(M + Na)⁺: 660.1442;

Found: 660.1467. H (400 MHz, DMSO-d₆): 1.82, 2.01, 2.08,

2.09 (4s, 12H, CH_{3 acetate}), 4.14–4.24 (m, 2H), 4.39–4.49 (m,

1

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 88–100

99

1H), 5.39–5.49 (m, 1H), 5.55–5.72 (m, 2H), 6.22 (br s, 1H,

H_1′), 6.93 (d, J = 7.5 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.47

(t, J = 7.5 Hz, 1H), 7.83–7.90 (m, 1H), 8.33–8.40 (m, 1H),

8.98 (d, J = 8.0 Hz, 1H), 10.19 (d, J = 2.0 Hz, 1H), 11.12 (s,

1H, NH). ¹³C (100 MHz, DMSO-d₆): 19.9, 20.2, 20.4, 20.5

(CH_{3 acetate}), 61.8 (CH_{2 sugar}), 67.4, 67.7, 72.2, 73.3, 78.3

(CH_sugar), 121.1, 121.2, 128.0, 137.7, 142.6, 145.4, 145.6

(C), 110.2, 112.3, 121.6, 124.1, 127.5, 129.8, 134.5 (CH),

166.8, 168.6, 169.0, 169.3, 169.5, 170.1 (C=O).

in a humidified atmosphere containing 5% CO₂. Fluorescence

was then measured on an automated 96-well plate reader

(Fluoroscan Ascent FL, Labsystem) using an excitation wave-

length of 530 nm and an emission wavelength of 590 nm. Un-

der the conditions used, fluorescence was proportional to the

number of living cells in the well. The IC₅₀, defined as the

drug concentration required to inhibit cell proliferation by

50%, was calculated from the curve of concentration-depen-

dent survival percentage, defined as fluorescence in experimen-

tal wells compared with fluorescence in control wells, after

subtraction of the blank values.

1-(2,3,4,6-tetra-O-acetyl-β-^D-glucopyranosyl)-5-bromoisoin-

digo 40: Obtained in 13% yield as a red solid (m.p. = 285 °C),

IR (KBr): ν_NH3425 cm⁻¹; ν_C=O1747, 1699 cm⁻¹; υ_C=C

1618 cm⁻¹. HRMS (ES) Calcd. for C₃₀H₂₇BrN₂NaO₁₁(M +

Na)⁺: 693.0696/695.0675; Found: 693.0714/695.0713. ¹H

(400 MHz, CDCl₃): 1.86, 2.02, 2.09, 2.10 (4s, 12H,

CH_{3 acetate}), 3.93–3.99 (m, 1H), 4.21 (dd, J₁= 12.5 Hz, J₂

= 2.5 Hz, 1H), 4.27 (dd, J₁= 12.5 Hz, J₂= 4.0 Hz, 1H), 5.28

(t, J = 9.5 Hz, 1H), 5.42 (t, J = 9.5 Hz, 1H), 5.63–5.70 (m, 1H),

5.84 (br s, 1H, H_1′), 6.83 (d, J = 7.5 Hz, 1H), 7.03–7.11 (m,

2H), 7.36 (t, J = 7.5 Hz, 1H), 7.52 (dd, J₁= 8.5 Hz, J₂

= 2.0 Hz, 1H), 7.64 (s, 1H, NH), 9.03 (d, J = 8.5 Hz, 1H),

9.39 (d, J = 2.0 Hz, 1H). ¹³C (100 MHz, CDCl₃): 20.4, 20.7

(2C), 20.8 (CH_{3 acetate}), 61.8 (CH_{2 sugar}), 67.7, 68.0, 73.4, 74.9,

79.7 (CH_sugar), 116.2, 122.2, 123.3, 130.7, 135.6, 140.1, 143.2

(C), 109.8, 112.6, 122.7, 130.5, 132.4, 133.7, 135.0 (CH),

167.2, 169.0, 169.2, 169.6, 170.0, 170.6 (C=O).

After reading, cells were prepared for cellular DNA quanti-

tation with Hoechst dye 3342. They were rinsed with PBS,

resazurin solution was then eliminated and plates were stored

at –80 °C.

4.2.4. Hoechst dye 3342 test

On the day of assay, plates were thawed at room tempera-

ture for 10 min. Hundred microliters of a 0.01% (m/v) SDS

solution in distilled water was then distributed into each well,

the plates were incubated for 1 h at room temperature and fro-

zen again at –80 °C for 1 h. After thawing, 100 μl of Hoechst

dye 33342 solution at 30 μg ml^–1in a hypersaline buffer

(10 mM Tris–HCl, pH 7.4, 1 mM EDTA and 2 M NaCl) were

added to each well. The plates protected from light were incu-

bated in this solution at room temperature for 1 h on a plate

shaker. Fluorescence was then measured at 360/460 nm on a

microplate fluorescence reader.

4.2. Antiproliferative activities

4.2.1. Cell cultures

Under the conditions used, fluorescence was proportional to

the amount of biomass and the IC₅₀was calculated as above.

Stock cell cultures were maintained as monolayers in 75-

cm²culture flasks in Glutamax Eagle’s minimum essential

medium (MEM) with Earle’s salts supplemented with 10% fe-

tal calf serum, 5 ml 100 mM sodium pyruvate, 5 ml of 100×

non-essential amino acids and 2 mg gentamicin base. Cells

were grown at 37 °C in a humidified incubator under an atmo-

sphere containing 5% CO₂.

Acknowledgements

The authors are grateful to Bertrand Légeret for mass spec-

tra analysis.

References

4.2.2. Survival assays

Cells were plated at a density of 5 × 10³cells in 190 μl cul-

ture medium in each well of 96-well microplates and were al-

lowed to adhere for 16 h before treatment with tested drug. A

stock solution 20 mM of each tested drug was prepared in

DMSO and kept at –20 °C until use. Then 50 μl of each tested

solution were added to the cultures. A 48 h continuous drug

exposure protocol was used. The antiproliferative effect of the

tested drug was assessed by both the RRT and determination of

DNA cellular content after cell lysis.

[1] S. Leclerc, M. Garnier, R. Hoessel, D. Marko, J.A. Bibb, G.L. Snyder,

P. Greengard, J. Biernat, Y.-Z. Wu, E.-M. Mandelkow, G. Eisenbrand,

L. Meijer, J. Biol. Chem. 276 (2001) 251–260.

[2] R. Hoessel, S. Leclerc, J.A. Endicott, M.E.M. Nobel, A. Lawrie, P. Tun-

nah, M. Leost, E. Damiens, D. Marie, D. Marko, E. Niederberger, W.

Tang, G. Eisenbrand, L. Meijer, Nat. Cell Biol. 1 (1999) 60–67.

[3] J. Adachi, Y. Mori, H. Takigami, J. Fujino, H. Kitagawa, C.A. Miller III,

T. Kato, K. Saeki, T. Matsuda, J. Biol. Chem. 276 (2001) 31475–31478.

[4] M. Knockaert, M. Blondel, S. Bach, M. Leost, C. Elbi, G. Hager, S.R.

Naggy, D. Han, M. Denison, M. Ffrench, X.P. Ryan, P. Magiatis, P. Poly-

chronopoulos, P. Greengard, L. Skaltsounis, L. Meijer, Oncogene 23

(2004) 4400–4412.

4.2.3. RRT

[5] A.D. Laird, J.M. Cherrington, Expert Opin. Investig. Drugs 12 (2003)

Plates were rinsed with 200 μl PBS at 37 °C and emptied by

overturning on absorbent toweling. Then 150 μl of a

25 μg ml^–1solution of resazurin in MEM without phenol red

was added to each well. Plates were incubated for 1 h at 37 °C

51–64.

[6] L. Sun, C. Liang, S. Shirazian, Y. Zhou, T. Miller, J. Cui, J.Y. Fukuda,

J.-Y. Chu, A. Nematalla, X. Wang, H. Chen, A. Sistla, T.C. Luu, F. Tang,

J. Wei, C. Tang, J. Med. Chem. 46 (2003) 1116–1119.

100

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 88–100

[7] A. Andreani, A. Cavalli, M. Granaiola, A. Leoni, A. Locatelli, R. Morigi,

M. Rambaldi, M. Recanatini, M. Garnier, L. Meijer, Anticancer Drug

Des. 15 (2000) 447–452.

[13] L. Sun, N. Tran, C. Liang, F. Tang, A. Rice, R. Schreck, K. Waltz, L.K.

Shawver, G. Mc Mahon, C. Tang, J. Med. Chem. 42 (1999) 5120–5130.

[14] N. Suvorov, M.N. Preobrazhenskaya, Zh. Obs. Khim 31 (1961) 2839–

[8] D.J. Moshinsky, C.R. Bellamacina, D.C. Boisvert, P. Huang, T. Hui, J.

Jancarik, S.-H. Kim, A.G. Rice, Biochem. Biophys. Res. Commun. 310

(2003) 1026–1031.

2845.

[15] C. Papageorgiou, X. Borer, Helv. Chim. Acta 71 (1988) 1079–1083.

[16] D.E. Ward, Y. Gai, B.F. Kaller, J. Org. Chem. 60 (1995) 7830–7836.

[9] M. Sassatelli, E. Saab, F. Anizon, M. Prudhomme, P. Moreau, Tetrahe-

dron Lett. 45 (2004) 4827–4830.

[17] M. Kakushima, P. Hamel, R. Frenette, J. Rokach, J. Org. Chem. 48

(1983) 3214–3219.

[10] L. Wang, X. Liu, R. Chen, US Patent (2003) US 6566341, Chem. Abstr.

138: 379213.

[18] J. O’Brien, I. Wilson, T. Orton, F. Pognan, Eur. J. Biochem. 267 (2000)

5421–5426.

[11] L. Wang, X. Liu, R. Chen, WO Patent (2003) WO 03051900, Chem.

Abstr. 139: 47135.

[19] R. Rago, J. Mitchen, G. Wilding, Anal. Biochem. 191 (1990) 31–34.

[20] K. Nakagawa, T. Sato, T. Nishi, Y. Oshiro, K. Yamamoto, JP Patent

(1977) JP 52073866, Chem. Abstr. 87: 167899.

[12] S. Messaoudi, M. Sancelme, V. Polard-Housset, B. Aboab, P. Moreau,

M. Prudhomme, Eur. J. Med. Chem. 39 (2004) 453–458.

Article Doi

Synthesis and antiproliferative activities of diversely substituted glycosyl-isoindigo derivatives

DOI: 10.1016/j.ejmech.2005.10.004

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2005.10.004

Full text of DOI:10.1016/j.ejmech.2005.10.004

Products guided by the article

R&D Labs maybe for 727651-30-1

Relevant to this article

Hot Product