Synthesis, kinase inhibitory potencies and in vitro antiproliferative activity of isoindigo and 7′-azaisoindigo derivatives substituted by Sonogashira cross-coupling

Article abstract of DOI:10.1016/j.ejmech.2009.01.027

In the course of structure-activity relationship studies we were interested in the synthesis of isoindigo and 7′-azaisoindigo derivatives substituted at the N-1 position by a 1-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl), at the 5′-position by various cha

Full text of DOI:10.1016/j.ejmech.2009.01.027

European Journal of Medicinal Chemistry 44 (2009) 2705–2710
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, kinase inhibitory potencies and in vitro antiproliferative activity
of isoindigo and 70-azaisoindigo derivatives substituted by Sonogashira
cross-coupling
*
Fadoua Bouchikhi, Fabrice Anizon, Pascale Moreau
´
`
Universite Blaise Pascal, SEESIB-UMR CNRS 6504, 24 Avenue des Landais, 63177 Aubiere Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
In the course of structure–activity relationship studies we were interested in the synthesis of isoindigo
and 70-azaisoindigo derivatives substituted at the N-1 position by a 1-(2,3,4,6-tetra-O-acetyl-
b-D-glu-
Received 9 October 2008
Accepted 23 January 2009
Available online 5 February 2009
copyranosyl), at the 50-position by various chains introduced by Sonogashira cross-coupling and
substituted or not at the 5-position by a bromine atom. To get an insight into the substitution pattern
required for the best biological potencies, their kinase inhibitory potencies and their in vitro anti-
proliferative activities were evaluated. The derivatives were tested toward four protein kinases (CDK5/
p25, GSK3, CK1, Dyrk1A) and their in vitro antiproliferative activity was tested against two human
myeloid leukaemia cell lines (K562 and HL60).
Keywords:
Indigoids
Isoindigos
Kinase inhibitors
Antitumour agents
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
cross-coupling. Moreover, to our knowledge, the functionalization
of the isoindigo or 7-azaisoindigo framework by palladium-cata-
In the course of studies on the preparation of potential biologi-
cally active compounds, we were interested in the synthesis of
indigoid derivatives. We have reported, in previous papers, the
synthesis and biological activities of isoindigo derivatives (indirubin
isomers possessing two indolin-2-one moieties) bearing a sugar
residue attached to one of the aromatic nitrogens and diversely
substituted on the aromatic rings at the 5-, 6- and/or 50-positions
[1–4]. 70-Azaisoindigo analogues were also prepared to evaluate the
influence of a 7-azaindolin-2-one moiety instead of an indolin-2-
one part on the biological activities of these compounds [4].
lyzed cross-coupling reactions has never been described.
The biological profile of these compounds was examined by in
vitro antiproliferative potencies’ testing toward two myeloid
leukaemia cell lines (K562, HL60) using the classic colorimetric
MTS test. Moreover, to identify the cellular target(s) implicated in
the in vitro antiproliferative activity of these derivatives, their
inhibitory potencies toward four different kinases (CDK5/p25,
GSK3, CK1, DyrK1A) were examined.
2. Chemistry
The results obtained in these previous structure–activity rela-
tionship studies have shown that the pharmaceutical profile of this
series could be optimized by substitutions of the upper oxindole
moiety, bromination in the 5-position and the presence of acetyl
groups on the sugar residue. Moreover, the 70-azaisoindigo deriv-
atives were more cytotoxic than their non-aza analogues, particu-
larly toward two myeloid leukaemia cell lines (K562, HL60).
In the isoindigo series, the most active compound was the one
bearing a 4-oxobutanoic acid side chain at the 50-position [3].
Therefore, to get an insight into the substitution pattern required
for the best biological potencies, we decided to introduce various
functionalized alkynyl side chains at the C-50 position of the upper
indolin-2-one or 7-azaindolin-2-one moiety by Sonogashira
In our previous work, we have described the synthesis of 50-
iodoglycosyl-isoindigo 3 by coupling in acidic conditions of the
glycosylated isatin 1 with 5-iodoindolin-2-one [4]. The brominated
analogue 4 was prepared in 68% yield, from 5-bromo glycosylated
isatine 2, using the same procedure (Scheme 1).
The alkynyl side chains were introduced in the 50-position of the
isoindigo or 70-azaisoindigo framework by Sonogashira coupling
using Pd(PPh3)4 in acetonitrile in the presence of CuI and Et3N. In
these conditions, compounds 5 and 6, brominated or not and
bearing a Cbz-protected aminopropyl side chain at the 50-position
were obtained in respectively 24% and 33% yields. Compounds 7 and
8 were obtained from 3 or 4 in 14 and 41% yield respectively, using
the same catalytic conditions in the presence of but-3-yn-1-ol.
To optimize the chemical yields of these reactions, the use of
Pd(PPh3)2Cl2, CuI, Et3N in acetonitrile as described previously for
* Corresponding author. Fax: þ33 4 73 40 77 17.
E-mail address: Pascale.MOREAU@univ-bpclermont.fr (P. Moreau).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.027

2706
F. Bouchikhi et al. / European Journal of Medicinal Chemistry 44 (2009) 2705–2710
H
N
H
N
O
R
O
O
H
N
O
I
O
R
R
N
I
CH2R'
CH R'
2
O
O
PTSA, toluene,
molecular sieves 4Å
O
3 mol% Pd(PPh3)4,
5 mol% CuI, Et3N,
CH3CN
N
N
OAc
AcO
O
O
OAc
OAc
AcO
AcO
OAc
OAc
OAc
OAc
OAc
OAc
1 R = H
2 R = Br
3 R = H, Yld = 61%
4 R = Br, Yld = 68%
5 R = H, R' = NHCO2Bn, Yld = 24%
6 R = Br, R' = NHCO2Bn, Yld = 33%
7 R = H, R' = CH2OH, Yld = 14%
8 R = Br, R' = CH2OH, Yld = 41%
Scheme 1.
5-bromoindole derivatives under thermic conditions [5] or for
5-iodoindolin-2-one derivatives under microwaves irradiation [6]
was also tested. In these latter catalytic conditions, none of the
expected coupling products was observed.
As we had already described the preparation of the 7-aza-
5-bromoindolin-2-one 9 [4], in the aza series, the Sonogashira
cross-coupling reactions were first attempted with brominated
intermediates. Compound 11 was prepared in 51% yield by coupling
glycosylated isatin 2 and 7-aza-5-bromoindolin-2-one 9 in acidic
conditions as already described for the synthesis of the mono-
brominated analogue 10 [4] (Scheme 2).
From 70-aza-50-bromoisoindigo derivatives 10 and 11, no
coupling product was obtained when the Sonogashira reaction
was carried out in the same catalytic conditions than for
compounds 3 and 4. So we tried to introduce the alkynyl side
chain before the formation of the 70-azaisoindigo framework by
Then, we decided to activate the 5-position of the 7-aza-5-
bromoindolin-2-one 9 by preparing the iodinated analogue either
in the presence of KI and CuI in dimethylformamide as described
for 3,5-dibromopyridine [7] or with BuLi and iodine in THF as
described by Lavilla for 5-bromonicotinic acid [8]. In both condi-
tions, no halogen exchange was observed.
The halogen exchange was only observed when the reaction was
performed in the presence of CuI, NaI and a 1,2-diamine ligand (N,N0-
dimethyl-1,2-cyclohexanediamine) in dioxane [9,10] (Scheme 3). In
these conditions, compound 12 was obtained in 62% yield. The
halogen exchange performed in the same conditions with the azai-
soindigo derivative 10 led only to the recovery of starting material.
Compound 12 was reacted in acidic conditions with an isatine
derivative to give the corresponding glycosylazaisoindigos 13 and
14 in 61 and 74% yield respectively.
Finally, the coupling reaction performed between the iodo deriv-
atives 13,14 and the Cbz-protected propargylamine with Pd(PPh3)4 in
acetonitrile in the presence of CuI, Et3N led to the attempted products
15 and 16 in respectively 39 and 45% yields (Scheme 3).
treating the 7-aza-5-bromoindolin-2-one
9
in Sonogashira
conditions. Unfortunately, no coupling product was obtained
(Scheme 2).
H
H
O
N
N
N
N
R
O
O
H
O
N
N
Br
O
N
R
R
Br
CH2R'
CH2R'
9
O
O
O
OAc
AcO
PTSA, toluene,
molecular sieves 4Å
N
N
3 mol% Pd(PPh3)4,
5 mol% CuI, Et3N,
CH3CN
O
O
OAc
OAc
AcO
AcO
OAc
OAc
1 R = H
2 R = Br
OAc
OAc
OAc
OAc
10 R = H, Yld = 55%
11 R = Br, Yld = 51%
R' = NHCO2Bn or CH2OH
H
N
H
CH2R'
N
N
N
O
O
Br
3 mol% Pd(PPh3)4,
5 mol% CuI, Et3N,
CH3CN
9
CH2R'
R' = NHCO2Bn or CH2OH
Scheme 2.

F. Bouchikhi et al. / European Journal of Medicinal Chemistry 44 (2009) 2705–2710
2707
H
N
H
N
O
R
N
N
O
O
N
O
O
OAc
I
AcO
R
H
N
R
N
CH2R'
OAc
OAc
CH2R'
O
O
O
N
N
PTSA, toluene
molecular sieves 4Å
AcO
X
3 mol% Pd(PPh3)4,
5 mol% CuI, Et3N,
CH3CN
O
O
OAc
OAc
NHMe
AcO
9 X = Br
NHMe
CuI, NaI,
Dioxane
OAc
OAc
13 R = H, Yld = 61%
14 R = Br, Yld = 74%
OAc
OAc
12 X = I
Yld=62%
15 R = H, R' = NHCO2Bn, Yld = 39%
16 R = Br, R' = NHCO2Bn, Yld = 45%
17 R = H, R' = CH2OH, Yld = 25%
Scheme 3.
Compound 17 was obtained from 13 and but-3-yn-1-ol in 25%
yield by using the same reaction conditions. When this reaction
was applied to the brominated analogue 14, the formation of the
corresponding coupling product was observed but we never
succeed in an efficient purification of this compound.
In vitro kinase inhibitory potencies of compounds 3–7,10,11,13–
15 were evaluated toward CDK5/p25, GSK3, CK1 and DyrK1A as
already described in the literature [11] by Laurent Meijer’s group
(Station Biologique CNRS, Roscoff, France). Regarding the residual
kinase activity (more than 65%) when the compounds were tested at
No further chemical transformations of the reported
compounds 5–8 and 15–17 were carried out regarding the modest
overall yields of these synthetic approaches.
10
m
M, none of them was particularly active toward these kinases.
In conclusion, we have synthesized various 1-(2,3,4,6-tetra-
O-acetyl-
-glucopyranosyl)isoindigos and 70-azaisoindigos, substi-
b-D
tuted by Sonogashira reaction. The results obtained in this
structure–activity relationship study have shown that in both
series, the introduction of a functionalized side chain in the
50-position did not improve the cytotoxic activity of these series.
3. Results and discussion
In vitro antiproliferative activities of compounds 3–8, 10, 11,
13–17 were evaluted in triplicate toward human myeloid
leukaemia cell lines (K562, HL60) using the classic colorimetric
MTS test. Percentage of cell proliferation inhibition was defined
as absorbance in experimental wells compared with absor-
bance in control wells, after subtraction of the blank values.
None of the tested compounds have shown any cytotoxicity
toward the cell lines tested when the final concentration of the
tested compounds used was 10ꢀ6 M (Table 1). Only five
compounds (two isoindigo derivatives (3, 8) and three
70-azaisoindigo (10, 13, 15) analogues) have shown modest
antiproliferative activities when tested at 10ꢀ5 M. In the iso-
indigo series, the most active compound was compound 3,
bearing a sugar moiety and an iodine atom at the 50-position,
which inhibited the cell proliferation for both cell lines nearby
4. Experimental
4.1. Chemistry
IR spectra were recorded on a Perkin–Elmer Paragon 500 spec-
trometer (
AVANCE 400 (1H: 400 MHz, 13C: 100 MHz) or AVANCE 500 (1H:
500 MHz, 13C: 125 MHz), chemical shifts
in ppm, the following
n
in cmꢀ1). NMR spectra were performed on a Bruker
d
abbreviations are used: singlet (s), doublet (d), triplet (t), doubled
doublet (dd), doublet of triplets (dt), multiplet (m), broad signal (br s),
broad doublet (br d). When necessary to identify all carbon atoms,
complementary NMR experiments (HSQC, HMBC) were performed
on a Bruker AVANCE 500. Mass spectra (ES) were determined on
a high resolution Waters Micro Q-toff apparatus. Chromatographic
purifications were performed by flashsilica gel GeduranSI60(Merck)
0.040–0.063 mm column chromatography. For purity tests, TLC were
performed on fluorescent silica gel plates (60 F254 from Merck).
50%. Moreover, compound 8, bearing
a sugar moiety and
a hydroxybut-3-ynyl side chains in the 50-position, suppressed
the cell proliferation of K562 cells by 51%. In the 70-azai-
soindigo series, the most active compound was compound 10
bearing a sugar moiety and a bromine atom in the 50-position
which inhibited the cell proliferation in a 60–75% range for
both cell lines. Furthermore, compounds 13 and 15 suppressed
the cell proliferation of K562 cells in a 50–60% range.
4.1.1. 5-Iodo-1H-pyrrolo[2,3-b]pyridin-2(3H)-one 12
A mixture of 5-bromo-7-azaoxindole 9 (330 mg, 1.55 mmol),
copper iodide (15 mg, 0.079 mmol, 5 mol%), sodium iodide (465 mg,
3.10 mmol)
and
trans-N,N0-dimethyl-1,2-cyclohexanediamine
(22 mg, 0.155 mmol, 10 mol%) in anhydrous dioxane (15.8 mL)
under argon atmosphere was heated at 110 ꢁC in a sealed tube for
24 h in the darkness. After cooling, the mixture was diluted with
a 28–30% NH4OH solution (10 mL), waterwas added and the mixture
was extracted with dichloromethane. After drying over MgSO4, the
solvent was removed and the crude product was purified by flash
chromatography (cyclohexane/EtOAc 7:3 then 5:5) to give
compound 12 (250 mg, 0.96 mmol, 62%) as a white solid (mp 70 ꢁC).
Table 1
Antiproliferative activity of compounds 3–17 (percentage of inhibition of prolifer-
ation at 10ꢀ5/10ꢀ6 M). Taxotere was used as a positive control at a drug concen-
tration of 2.5 ꢂ10ꢀ10 M with 46% of HL60 cell growth inhibition and 32% of K562 cell
growth inhibition.
Compounds
3
4
5
6
7
8a
10
11 13
14 15
16 17
HL60
K562
45/0 0/0 1/0 0/0 8/0 28/0 73/3 0/0 1/0 0/0 11/0 0/0 0/0
49/16 3/0 6/0 2/1 33/2 51/7 64/19 4/3 47/0 3/0 59/4 5/0 10/5
IR (KBr) nC]O 1718 cmꢀ1; 1206 cmꢀ1
.
HRMS (ESþ) calcd for C7H6IN2O [M þ H]þ 260.9525, found
a
Compound 8 was tested as a mixture with oct-3,5-diyne-1,8-diol in a 94.3:5.7 w/
w ratio.
260.9522.

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F. Bouchikhi et al. / European Journal of Medicinal Chemistry 44 (2009) 2705–2710
1H NMR (400 MHz, DMSO-d6): 3.55 (2H, s), 7.85 (1H, m), 8.26
HRMS (ESþ) calcd for C29H27IN3O11 [M þ H]þ 720.0690, found
(1H, m), 11.10 (1H, s, NH).
720.0687.
13C NMR (100 MHz, DMSO-d6): 35.2 (CH2), 139.2, 151.3 (CHarom),
83.8, 123.3, 157.5 (Carom), 175.2 (C]O).
1H NMR (400 MHz, DMSO-d6): 1.78 (3H, s, CH3),1.96 (3H, s, CH3),
2.03 (3H, s, CH3), 2.05 (3H, s, CH3), 4.10–4.19 (2H, m), 4.32–4.38 (1H,
m), 5.29–5.39 (1H, m), 5.58 (1H, t, J ¼ 9.5 Hz), 5.55–5.71 (1H, br s),
6.11–6.20 (1H, m), 7.14 (1H, t, J ¼ 8.0 Hz), 7.50 (1H, t, J ¼ 7.5 Hz),
7.52–7.58 (1H, br s), 8.42 (1H, d, J ¼ 2.0 Hz), 9.15 (1H, d, J ¼ 8.0 Hz),
9.53 (1H, s), 11.73 (1H, s, NH).
4.1.2. General procedure for the preparation of compounds 4, 11, 13
and 14
To an anhydrous solution of PTSA (30 mol%) in toluene were
added indolin-2-one derivatives (1.1 eq; 1.8 eq for 9), acetylated
glycosylisatine (18 mM; 21 mM for 1), and 4 Å molecular sieves.
The mixture was refluxed for 24 h (48 h for 11) in the darkness
(except for 11). After cooling, EtOAc was added, the organic phase
was washed with water. After drying over MgSO4, the solvent was
removed and the crude product was purified in different conditions
depending on the compounds:
13C NMR (100 MHz, DMSO-d6): 20.0, 20.3, 20.4, 20.5 (CH3), 61.9
(CH2), 67.2, 67.8, 72.5, 73.2, 78.2 (CH), 112.1, 122.7, 129.4, 133.7,
143.4, 155.0 (CHarom), 83.9, 118.4, 120.5, 131.2, 133.2, 141.7, 156.7
(Carom), 167.1, 167.7, 168.9, 169.4, 169.5, 170.0 (C]O).
4.1.6. 70-Aza-5-bromo-50-iodo-1-(2,3,4,6-tetra-O-acetyl-
glucopyranos-1-yl)isoindigo 14
b-D-
Compound 2 (150 mg, 0.270 mmol); compound 14 (159 mg,
0.199 mmol, 74%) as a red solid (mp > 260 ꢁC).
- Compounds 4, 13 and 14: flash chromatography (cyclohexane/
EtOAc 1:1) to give a residue which was precipitated in
a mixture of AcOH/toluene 1:1 with a few drops of water.
Filtration gave the attempted compound.
- Compound 11: flash chromatography (cyclohexane/EtOAc 6:4
then 5:5) to give the attempted compound.
IR (KBr) nNH 3460 cmꢀ1
1234 cmꢀ1
;
nC]O 1780–1710 cmꢀ1; 1601,1458,1365,
.
HRMS (ESþ) calcd for C29H2679BrIN3O11 [M þ H]þ 797.9795,
found 797.9814.
1H NMR (400 MHz, DMSO-d6): 1.79 (3H, s, CH3),1.96 (3H, s, CH3),
2.03 (3H, s, CH3), 2.05 (3H, s, CH3), 4.07–4.18 (2H, m), 4.31–4.37 (1H,
m), 5.30–5.41 (1H, m), 5.52–5.64 (2H, m), 6.12–6.20 (1H, m), 7.58
(1H, br d, J ¼ 8.0 Hz), 7.67 (1H, d, J ¼ 8.0 Hz), 8.45 (1H, d, J ¼ 2.0 Hz),
9.39 (1H, d, J ¼ 2.0 Hz), 9.54 (1H, s), 11.81 (1H, s, NH).
13C NMR (100 MHz, DMSO-d6): 20.0, 20.3, 20.4, 20.5 (CH3), 61.9
(CH2), 67.1, 67.8, 72.3, 73.2, 78.2 (CH), 114.1, 131.3, 135.4, 143.8, 155.6
(CHarom), 84.0, 114.7, 118.2, 122.3, 131.6, 132.8, 140.6, 157.0 (Carom),
166.6, 167.8, 169.0, 169.4, 169.5, 170.1 (C]O).
4.1.3. 5-Bromo-50-Iodo-1-(2,3,4,6-tetra-O-acetyl-
glucopyranos-1-yl)isoindigo 4
b-D-
Compound 2 (250 mg, 0.45 mmol); compound 4 (243 mg,
0.305 mmol, 68%) as a red solid (mp 270–273 ꢁC).
IR (KBr): nNH 3385 cmꢀ1 nC]O 1775–1695 cmꢀ1
; .
HRMS (ESþ) calcd for C30H2679BrIN2NaO11 [M þ Na]þ 818.9662,
found 818.9670.
1H NMR (400 MHz, DMSO-d6): 1.78 (3H, s, CH3),1.96 (3H, s, CH3),
2.03 (3H, s, CH3), 2.05 (3H, s, CH3), 4.09–4.18 (2H, m), 4.32–4.38 (1H,
m), 5.30–5.40 (1H, m), 5.52–5.64 (2H, m), 6.13–6.22 (1H, m), 6.72
(1H, d, J ¼ 8.0 Hz), 7.55 (1H, br d, J ¼ 7.5 Hz), 7.63 (1H, br d,
J ¼ 7.5 Hz), 7.71 (1H, dd, J1 ¼8.0 Hz, J2 ¼1.5 Hz), 9.34 (1H, s), 9.37
(1H, d, J ¼ 1.5 Hz), 11.15 (1H, s, NH).
4.1.7. General procedure for the preparation of compounds 5–8,
15–17
A mixture of iodinated isoindigo or 70azaisoindigo and alkyne in
acetonitrile in the presence of triethylamine (quantities indicated
below), CuI (5 mol%) and Pd(PPh3)4 (3 mol%) was refluxed (see
reaction time below). After cooling, water was added and the
mixture was extracted with EtOAc. After drying over MgSO4, the
solvent was removed and the crude product was purified as indi-
cated below to give the attempted compound.
13C NMR (125 MHz, DMSO-d6): 20.0, 20.3, 20.4, 20.5 (CH3), 61.9
(CH2), 67.2, 67.8, 72.3, 73.2, 78.2 (CH), 112.3, 114.1, 131.1, 134.9, 137.4,
141.5 (CHarom), 84.2, 114.5, 122.6, 123.5, 130.4, 134.8, 140.0, 144.3
(Carom), 166.6, 168.1, 168.9, 169.4, 169.5, 170.1 (C]O).
4.1.8. 50-(N-Benzyloxycarbonyl-3-aminoprop-1-ynyl)-1-(2,3,4,6-
4.1.4. 70-Aza-5,50-dibromo-1-(2,3,4,6-tetra-O-acetyl-
glucopyranos-1-yl)isoindigo 11
b
-D-
tetra-O-acetyl-
b
-D
-glucopyranos-1-yl)isoindigo 5
Compound
3
(200 mg, 0.278 mmol); Cbz-propargylamine
Compound 2 (50 mg, 0.090 mmol); compound 11 (34.6 mg,
(105 mg, 0.555 mmol); acetonitrile (0.64 mL); NEt3 (42 mL,
0.046 mmol, 51%) as a red solid (mp 280 ꢁC).
0.301 mmol); 24 h. Purification by flash chromatography (cyclo-
hexane/EtOAc 6:4 then 4:6) to give a residue which was dissolved
in toluene and then precipitated by addition of cyclohexane.
Filtration gave 5 (52 mg, 0.067 mmol, 24%) as a red solid (mp
200 ꢁC).
IR (KBr): nNH 3390 cmꢀ1 nC]O 1790–1715 cmꢀ1; 1600, 1458,
;
1368, 1232 cmꢀ1
.
HRMS (ESþ) calcd for C29H2679Br2N3O11 [M þ H]þ 749.9934,
found 749.9951.
1H NMR (400 MHz, DMSO-d6): 1.79 (3H, s, CH3),1.96 (3H, s, CH3),
2.03 (3H, s, CH3), 2.04 (3H, s, CH3), 4.09–4.17 (2H, m), 4.31–4.38 (1H,
m), 5.29–5.41 (1H, m), 5.52–5.65 (2H, m), 6.11–6.19 (1H, m), 7.58
(1H, br d, J ¼ 7.5 Hz), 7.68 (1H, d, J ¼ 8.0 Hz), 8.37 (1H, d, J ¼ 2.0 Hz),
9.40 (2H, m), 11.86 (1H, s, NH).
IR (KBr) nNH 3386 cmꢀ1 nC]O 1790–1710 cmꢀ1 nC]C 1600 cmꢀ1
; ; .
HRMS (ESþ) calcd for C41H37N3NaO13 [M þ Na]þ 802.2224,
found 802.2202.
1H NMR (500 MHz, DMSO-d6): 1.77 (3H, s, CH3),1.96 (3H, s, CH3),
2.03 (3H, s, CH3), 2.05 (3H, s, CH3), 4.08 (2H, d, J ¼ 5.5 Hz), 4.10–4.18
(2H, m), 4.32–4.38 (1H, m), 5.07 (2H, s), 5.28–5.41 (1H, br s), 5.57
(1H, t, J ¼ 8.5 Hz), 5.56–5.67 (1H, br s), 6.10–6.23 (1H, br s), 6.85 (1H,
d, J ¼ 8.0 Hz), 7.11 (1H, t, J ¼ 8.0 Hz), 7.30–7.40 (5H, m), 7.42 (1H, d,
J ¼ 8.0 Hz), 7.46 (1H, t, J ¼ 7.5 Hz), 7.50–7.58 (1H, br s), 7.85 (1H, t,
J ¼ 5.5 Hz, NH), 9.04 (1H, s), 9.11 (1H, d, J ¼ 8.0 Hz), 11.17 (1H, s, NH).
13C NMR (100 MHz, DMSO-d6): 20.0, 20.3, 20.4, 20.5 (CH3), 30.6,
61.9, 65.6 (CH2), 67.3, 67.8, 72.5, 73.2, 78.3 (CH), 81.9, 85.3 (Calkyne),
110.1, 111.8, 122.5, 127.9 (3C), 128.4 (2C), 129.0, 132.2, 133.0, 136.1
(CHarom), 114.9, 120.7, 121.5, 131.8, 133.7, 136.9, 141.1, 144.4 (Carom),
156.0, 166.9, 168.5, 168.8, 169.4, 169.5, 170.0 (C]O).
13C NMR (100 MHz, DMSO-d6): 20.0, 20.3, 20.4, 20.5 (CH3), 61.9
(CH2), 67.1, 67.8, 72.3, 73.2, 78.2 (CH), 114.1, 131.4, 135.5, 138.5, 150.7
(CHarom), 112.0, 114.7, 117.7, 122.3, 131.9, 132.8, 140.5, 156.9 (Carom),
166.6, 168.0, 169.0, 169.4, 169.5, 170.1 (C]O).
4.1.5. 70-Aza-50-iodo-1-(2,3,4,6-tetra-O-acetyl-
1-yl)isoindigo 13
b-D-glucopyranos-
Compound 1 (100 mg, 0.209 mmol); compound 13 (91 mg,
0.127 mmol, 61%) as a red solid (mp 220–222 ꢁC).
IR (KBr) nNH 3450 cmꢀ1 nC]O 1770–1700 cmꢀ1; 1227 cmꢀ1
; .

F. Bouchikhi et al. / European Journal of Medicinal Chemistry 44 (2009) 2705–2710
2709
4.1.9. 50-(N-Benzyloxycarbonyl-3-aminoprop-1-ynyl)-5-bromo-1-
(2,3,4,6-tetra-O-acetyl- -glucopyranos-1-yl)isoindigo 6
Compound (50 mg, 0.063 mmol); Cbz-propargylamine
(16.5 mg, 0.087 mmol); acetonitrile (1 mL); NEt3 (20 L,
6.11–6.20 (1H, m), 6.83 (1H, d, J ¼ 8.0 Hz), 7.42 (1H, dd, J1 ¼8.0 Hz,
J2 ¼ 1.5 Hz), 7.55 (1H, br d, J ¼ 8.0 Hz), 7.63 (1H, br d, J ¼ 8.5 Hz), 9.02
(1H, s), 9.37 (1H, d, J ¼ 2.0 Hz), 11.17 (1H, s, NH).
b-D
4
m
13C NMR (100 MHz, DMSO-d6): 20.0, 20.3, 20.4, 20.5 (CH3), 23.2,
59.8, 61.9 (CH2), 67.2, 67.8, 72.4, 73.2, 78.2 (CH), 81.0, 86.6 (Calkyne),
110.2, 113.9, 131.0, 132.5, 134.7, 136.6 (CHarom), 114.5, 116.1, 121.3,
122.6, 130.0, 135.5, 140.2, 144.3 (Carom), 166.5, 168.6, 168.9, 169.4,
169.5, 170.1 (C]O).
0,143 mmol); 6 h. Purification by flash chromatography (cyclo-
hexane/EtOAc 6:4 then 4:6) to give 6 (17.9 mg, 0.021 mmol, 33%) as
a red solid (mp 130–132 ꢁC).
IR (KBr) nNH 3420 cmꢀ1 nC]O 1770–1700 cmꢀ1 nC]C 1610 cmꢀ1
; ; .
HRMS (ESþ) calcd for C41H3679BrN3NaO13 [M þ Na]þ 880.1329,
found 880.1333.
4.1.12. 70-Aza-50-(N-benzyloxycarbonyl-3-aminoprop-1-ynyl)-1-
1H NMR (400 MHz, DMSO-d6): 1.77 (3H, s, CH3),1.96 (3H, s, CH3),
2.03 (3H, s, CH3), 2.05 (3H, s, CH3), 4.08 (2H, d, J ¼ 5.5 Hz), 4.10–4.18
(2H, m), 4.32–4.37 (1H, m), 5.07 (2H, s), 5.30–5.40 (1H, m), 5.53–
5.64 (2H, m), 6.11–6.21 (1H, br s), 6.86 (1H, d, J ¼ 8.0 Hz), 7.29–7.40
(5H, m), 7.44 (1H, d, J ¼ 8.5 Hz), 7.52–7.58 (1H, m), 7.63 (1H, br d,
J ¼ 8.0 Hz), 7.83 (1H, t, J ¼ 5.5 Hz, NH), 9.05 (1H, s), 9.37 (1H, d,
J ¼ 2.0 Hz), 11.20 (1H, s, NH).
(2,3,4,6-tetra-O-acetyl-
Compound 13 (50.0 mg, 0.069 mmol); Cbz-propargylamine
(20.0 mg, 0.106 mmol); acetonitrile (1 mL); NEt3 (20 L,
b-D-glucopyranos-1-yl)isoindigo 15
m
0.143 mmol); 16 h. Purification by flash chromatography (cyclo-
hexane/EtOAc 6:4 then 5:5) to give 15 (21.2 mg, 0.027 mmol, 39%)
as a red solid (mp 248–250 ꢁC).
IR (KBr) nNH 3430 cmꢀ1 nC]O 1780–1690 cmꢀ1 nC]C 1610 cmꢀ1
; ; .
13C NMR (100 MHz, DMSO-d6): 20.0, 20.3, 20.4, 20.5 (CH3), 30.6,
61.9, 65.6 (CH2), 67.2, 67.8, 72.4, 73.2, 78.3 (CH), 81.7, 85.4 (Calkyne),
110.3, 113.9, 127.9 (3C), 128.4 (2C), 131.1, 132.6, 134.8, 136.6 (CHarom),
114.5, 115.1, 121.4, 122.6, 130.2, 135.3, 136.9, 140.1, 144.8 (Carom),
156.0, 166.5, 168.6, 168.9, 169.4, 169.5, 170.1 (C]O).
HRMS (ESþ) calcd for C40H37N4O13 [M þ H]þ 781.2357, found
781.2358.
1H NMR (400 MHz, DMSO-d6): 1.77 (3H, s, CH3),1.96 (3H, s, CH3),
2.02 (3H, s, CH3), 2.05 (3H, s, CH3), 4.11 (2H, d, J ¼ 5.5 Hz), 4.12–4.18
(2H, m), 4.31–4.38 (1H, m), 5.07 (2H, s), 5.28–5.41 (1H, br s), 5.57
(1H, t, J ¼ 9.0 Hz), 5.54–5.69 (1H, br s), 6.09–6.19 (1H, br s), 7.14 (1H,
t, J ¼ 8.0 Hz), 7.30–7.40 (5H, m), 7.49 (1H, t, J ¼ 7.5 Hz), 7.53–7.60
(1H, m), 7.88 (1H, t, J ¼ 5.5 Hz, NH), 8.28 (1H, d, J ¼ 1.5 Hz), 9.15 (1H,
d, J ¼ 8.0 Hz), 9.25 (1H, s), 11.83 (1H, s, NH).
4.1.10. 50-(4-Hydroxybut-1-ynyl)-1-(2,3,4,6-tetra-O-acetyl-
glucopyranos-1-yl)isoindigo 7
b-D-
Compound
3
(100 mg, 0.139 mmol); but-3-yn-1-ol (16
mL,
0.210 mmol); acetonitrile (2 mL); NEt3 (40
mL, 0.287 mmol); 14 h.
13C NMR (100 MHz, DMSO-d6): 20.0, 20.3, 20.4, 20.5 (CH3), 30.6,
61.9, 65.7 (CH2), 67.2, 67.8, 72.5, 73.2, 78.3 (CH), 78.9, 88.2 (Calkyne),
112.1, 122.7, 127.9 (3C), 128.4 (2C), 129.3, 133.6,138.7, 152.6 (CHarom),
112.7, 115.9, 120.5, 131.5, 133.1, 136.9, 141.6, 157.1 (Carom), 156.0,
167.0, 168.2, 168.9, 169.4, 169.5, 170.1 (C]O).
Purification by flash chromatography (cyclohexane/EtOAc 6:4 then
5:5) to give a residue which was dissolved in toluene and then
precipitated by addition of cyclohexane. Filtration gave 7 (12.5 mg,
0.0189 mmol, 14%) as a red solid (mp 304 ꢁC).
IR (KBr) nNH,OH 3530–3160 cmꢀ1 nC]O 1775–1685 cmꢀ1
; ; nC]C
1610 cmꢀ1
.
4.1.13. 70-Aza-50-(N-benzyloxycarbonyl-3-aminoprop-1-ynyl)-5-
HRMS (ESþ) calcd for C34H33N2O12 [M þ H]þ 661.2034, found
bromo-1-(2,3,4,6-tetra-O-acetyl-
Compound 14 (53.0 mg, 0.066 mmol); Cbz-propargylamine
(13.0 mg, 0.069 mmol); acetonitrile (1 mL); NEt3 (20 L,
b-D-glucopyranosyl)isoindigo 16
661.2043.
1H NMR (500 MHz, DMSO-d6): 1.77 (3H, s, CH3),1.96 (3H, s, CH3),
2.03 (3H, s, CH3), 2.05 (3H, s, CH3), 2.56 (2H, t, J ¼ 7.0 Hz), 3.59 (2H,
dt, J1 ¼7.0 Hz, J2 ¼ 5.5 Hz), 4.11–4.18 (2H, m), 4.33–4.38 (1H, m),
4.90 (1H, t, J ¼ 5.5 Hz), 5.29–5.38 (1H, br s), 5.57 (1H, t, J ¼ 9.0 Hz),
5.56–5.68 (1H, br s), 6.10–6.20 (1H, br s), 6.82 (1H, d, J ¼ 8.0 Hz),
7.10 (1H, t, J ¼ 8.0 Hz), 7.39 (1H, d, J ¼ 8.0 Hz), 7.46 (1H, t, J ¼ 7.5 Hz),
7.49–7.56 (1H, br s), 9.01 (1H, s), 9.11 (1H, d, J ¼ 8.0 Hz), 11.10 (1H, s,
NH).
m
0,143 mmol); 6 h. Purification by flash chromatography (cyclo-
hexane/EtOAc 6:4 then 4:6) to give 16 (25.5 mg, 0.030 mmol, 45%)
as a red solid (mp 210–212 ꢁC).
IR (KBr) nNH 3414 cmꢀ1 nC]O 1770–1700 cmꢀ1 nC]C 1618 cmꢀ1
; ; .
HRMS (ESþ) calcd for C40H3679BrN4O13 [M þ H]þ 859.1462,
found 859.1464.
1H NMR (500 MHz, DMSO-d6): 1.78 (3H, s, CH3),1.96 (3H, s, CH3),
2.03 (3H, s, CH3), 2.05 (3H, s, CH3), 4.11 (2H, d, J ¼ 5.5 Hz), 4.09–4.17
(2H, m), 4.31–4.37 (1H, m), 5.07 (2H, s), 5.30–5.42 (1H, br s), 5.52–
5.64 (2H, m), 6.10–6.20 (1H, br s), 7.30–7.40 (5H, m), 7.55–7.62 (1H,
br s), 7.67 (1H, br d, J ¼ 7.5 Hz), 7.88 (1H, t, J ¼ 5.5 Hz, NH), 8.30 (1H,
s), 9.25 (1H, s), 9.39 (1H, d, J ¼ 1.5 Hz), 11.90 (1H, s, NH).
13C NMR (100 MHz, DMSO-d6): 20.0, 20.2, 20.4, 20.5 (CH3), 30.6,
61.9, 65.7 (CH2), 67.1, 67.8, 72.4, 73.3, 78.3 (CH), 78.8, 88.3 (Calkyne),
114.1, 127.9 (3C), 128.4 (2C), 131.3, 135.4, 139.0, 153.1 (CHarom), 112.9,
114.7, 115.7, 122.3, 131.5, 133.1, 136.9, 140.4, 157.4 (Carom), 156.0,
166.6, 168.3, 169.0, 169.4, 169.5, 170.0 (C]O).
13C NMR (100 MHz, DMSO-d6): 20.0, 20.3, 20.4, 20.5 (CH3), 23.2,
59.9, 61.9 (CH2), 67.3, 67.8, 72.5, 73.1, 78.3 (CH), 81.1, 86.5 (Calkyne),
110.0, 111.8, 122.4, 129.0, 132.1, 132.9, 136.0 (CHarom), 116.0, 120.8,
121.5, 131.6, 133.9, 141.3, 143.9 (Carom), 166.9, 168.5, 168.8, 169.4,
169.5, 170.1 (C]O).
4.1.11. 5-Bromo-50-(4-hydroxybut-1-ynyl)-1-(2,3,4,6-tetra-O-
acetyl-
Compound
0.105 mmol); acetonitrile (1 mL); NEt3 (20
b
-
D
-glucopyranos-1-yl)isoindigo 8
(50 mg, 0.063 mmol); but-3-yn-1-ol (8
L, 0.143 mmol); 3 h.
4
mL,
m
Purification by flash chromatography (cyclohexane/EtOAc 6:4 then
5:5) to give 8 in a 94.3:5.7 w/w mixture with oct-3,5-diyne-1,8-diol
(20.4 mg, 0,026 mmol, 41% – the ratio 8/oct-3,5-diyne-1,8-diol was
estimated from the 1H NMR spectra).
4.1.14. 70-Aza-50-(4-hydroxybut-1-ynyl)-1-(2,3,4,6-tetra-O-acetyl-
b-
D-glucopyranos-1-yl)isoindigo 17
Compound 13 (90 mg, 0.125 mmol); but-3-yn-1-ol (14 mL,
0.184 mmol); acetonitrile (2 mL); NEt3 (38 mL, 0.273 mmol); 14 h.
IR (KBr) nNH 3630–3160 cmꢀ1
,
nC]O 1770–1700 cmꢀ1
,
nC]C
1610 cmꢀ1
.
Purification by flash chromatography (cyclohexane/EtOAc 6:4 then
4:6) to give 17 (20.6 mg, 0.031 mmol, 25%) as a red solid (mp
160 ꢁC).
HRMS (ESþ) calcd for C34H3279BrN2O12 [M þ H]þ 739.1139,
found 739.1147.
1H NMR (400 MHz, DMSO-d6): 1.78 (3H, s, CH3),1.96 (3H, s, CH3),
2.03 (3H, s, CH3), 2.05 (3H, s, CH3), 2.56 (2H, t, J ¼ 7.0 Hz), 3.58 (2H,
dt, J1 ¼7.0 Hz, J2 ¼ 5.5 Hz), 4.09–4.18 (2H, m), 4.32–4.38 (1H, m),
4.91 (1H, t, J ¼ 5.5 Hz), 5.30–5.40 (1H, m), 5.51–5.65 (2H, m),
IR (KBr) nNH,OH 3580–3150 cmꢀ1 nC]O 1770–1695 cmꢀ1
; ; nC]C
1609 cmꢀ1
.
HRMS (ESþ) calcd for C33H32N3O12 [M þ H]þ 662.1986, found
662.1983.

2710
F. Bouchikhi et al. / European Journal of Medicinal Chemistry 44 (2009) 2705–2710
1H NMR (400 MHz, DMSO-d6): 1.78 (3H, s, CH3),1.96 (3H, s, CH3),
(Promega Corp) according to the manufacture’s instructions.
Absorbance was measured at 490 nm with a microplate reader.
2.03 (3H, s, CH3), 2.05 (3H, s, CH3), 2.60 (2H, t, J ¼ 6.5 Hz), 3.61 (2H,
dt, J1 ¼6.5 Hz, J2 ¼ 5.5 Hz), 4.10–4.19 (2H, m), 4.32–4.38 (1H, m),
4.93 (1H, t, J ¼ 5.5 Hz), 5.28–5.40 (1H, m), 5.57 (1H, t, J ¼ 9.5 Hz),
5.54–5.73 (1H, br s), 6.09–6.18 (1H, m), 7.14 (1H, t, J ¼ 8.0 Hz), 7.50
(1H, t, J ¼ 7.5 Hz), 7.52–7.59 (1H, m), 8.25 (1H, d, J ¼ 1.5 Hz), 9.15
(1H, d, J ¼ 8.0 Hz), 9.23 (1H, s), 11.76 (1H, s, NH).
Acknowledgements
The authors thank Laurent Meijer’s Group from the Station
Biologique-CNRS in Roscoff (France) for kinase testing. The authors
are also grateful to Prof. Michelle Prudhomme for helpful discus-
13C NMR (100 MHz, DMSO-d6): 20.0, 20.2, 20.4, 20.5 (CH3), 23.3,
59.7, 61.9 (CH2), 67.2, 67.8, 72.5, 73.2, 78.3 (CH), 78.1, 89.6 (Calkyne),
112.2, 122.7, 129.3, 133.5, 138.6, 152.5 (CHarom) 113.7, 115.8, 120.5,
131.6, 133.0, 141.4, 156.6 (Carom), 167.0, 168.2, 168.9, 169.3, 169.5,
170.0 (C]O).
´
sions and to Bertrand Legeret for mass spectral analysis.
References
[1] M. Sassatelli, E. Saab, F. Anizon, M. Prudhomme, P. Moreau, Tetrahedron Lett.
45 (2004) 4827.
4.2. Antiproliferative activities
[2] M. Sassatelli, F. Bouchikhi, S. Messaoudi, F. Anizon, E. Debiton, C. Barthomeuf,
M. Prudhomme, P. Moreau, Eur. J. Med. Chem. 41 (2006) 88.
[3] M. Sassatelli, F. Bouchikhi, B. Aboab, F. Anizon, D. Fabbro, M. Prudhomme,
P. Moreau, Anti-Cancer Drugs 18 (2007) 1069.
[4] F. Bouchikhi, F. Anizon, P. Moreau, Eur. J. Med. Chem. 43 (2008) 755.
[5] T. Balle, J. Perregaard, M.T. Ramirez, A.K. Larsen, K. Krojer Soby, T. Liljefors,
K. Andersen, J. Med. Chem. 46 (2006) 265.
[6] M. Koenig, J. Cui, C.C. Wei, S.H. Do, F.-J. Zhang, T. Vojkovsky, J. Ramphal,
G. Yang, M. Mattson, C. Nelson, P.C. Tang, PCT Int. Appl. WO2005005378 A2,
2005. Chem. Abstr. 142 (2005) 155814.
[7] Y. Kosaka, H.M. Yamamoto, A. Nakao, M. Tamura, R. Kato, J. Am. Chem. Soc. 129
(2007) 3054.
[8] R. Lavilla, F. Gullon, J. Bosch, Eur. J. Org. Chem. (1999) 373.
[9] A. Klapars, X. Huang, S.L. Buchwald, J. Am. Chem. Soc. 124 (2002) 7241.
[10] A. Klapars, S.L. Buchwald, J. Am. Chem. Soc. 124 (2002) 14844.
[11] A. Echalier, K. Bettayeb, Y. Ferandin, O. Lozach, M. Cle´ment, A. Valette, F. Liger,
B. Marquet, J.C. Morris, J.A. Endicott, B. Joseph, L. Meijer, J. Med. Chem. 51
(2008) 737.
4.2.1. Cell cultures and proliferation assays
Stock cell cultures were maintained as monolayers in 75-cm2
culture flasks in Glutamax RPMI medium with 10% fetal calf serum
containing penicillin and streptomycin. Cells were grown at 37 ꢁC
in a humidified incubator under an atmosphere containing 5% CO2.
Cells were plated at a density of 500–800 cells in 200 ml culture
medium in each well of 96-well microplates and were allowed to
adhere for 24 h before treatment with tested drugs in DMSO
solution (10ꢀ5 M final concentration). Cell viability was assayed
after 72 h continous drug exposure with a tetrazolium compound
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium salt; MTS; Promega Corp] by Cell-
Titer 96 Aqueous One Solution Cell Proliferation Assay kit