Synthesis and biological evaluation of 7-azaindole derivatives, synthetic cytokinin analogues

Article abstract of DOI:10.1016/j.bmcl.2007.01.033

Cytokinins, N6-substituted adenine derivatives, are plant hormones playing important roles in various processes in plant development. Furthermore, cytokinins and their derivatives are able to control mammalian cell apoptosis and differentiation. The aim of our study was the synthesis of 7-azaindole derivatives as cytokinin analogues with the Hartwig-Buchwald coupling reaction in order to evaluate their biological properties on human myeloblastic leukaemia cells (HL-60 cell line). All these compounds presented a cytotoxic activity on HL-60 cells especially the 4-phenylaminopyrrolo[2,3-b]pyridine and the 4-phenethylaminopyrrolo[2,3-b]pyridine.

Full text of DOI:10.1016/j.bmcl.2007.01.033

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1934–1937
Synthesis and biological evaluation of 7-azaindole derivatives,
synthetic cytokinin analogues
a
a
b,c
Jerome Guillard, Maylis Decrop, Nathalie Gallay, Claire Espanel,d Elodie Boissier,d
´
¨
Olivier Heraultb,c,d and Marie-Claude Viaud-Massuarda,*
aSPOT-EA3857, UFR des Sciences Pharmaceutiques, 31 Avenue Monge, 37200 Tours, France
bUniversite´ de Tours, e´quipe EA 3855, Tours, France
c
`
´
Inserm, e´quipe ESPRI, Microenvironnement de l’he´matopo¨ıese et cellules souches, 10 Boulevard Tonnelle, 37000 Tours, France
dCHRU de Tours, Service d’He´matologie, 10 Boulevard Tonnelle´, 37000 Tours, France
Received 25 October 2006; revised 9 January 2007; accepted 10 January 2007
Available online 19 January 2007
Abstract—Cytokinins, N6-substituted adenine derivatives, are plant hormones playing important roles in various processes in plant
development. Furthermore, cytokinins and their derivatives are able to control mammalian cell apoptosis and differentiation. The
aim of our study was the synthesis of 7-azaindole derivatives as cytokinin analogues with the Hartwig–Buchwald coupling reaction
in order to evaluate their biological properties on human myeloblastic leukaemia cells (HL-60 cell line). All these compounds pre-
sented a cytotoxic activity on HL-60 cells especially the 4-phenylaminopyrrolo[2,3-b]pyridine and the 4-phenethylaminopyrrolo
[2,3-b]pyridine.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Cytokinins are one of the known classes of plant growth
regulators, also called phytohormones. They play a
O
major role in many different processes in plant develop-
ment1 including cell growth and division control, and
the plant’s cell differentiation with auxins.2 Moreover,
they regulate the storage of various metabolites as alka-
loids. All naturally occurring cytokinins are N6-substi-
tuted adenine derivates which contain an aromatic ring
or an isoprenic chain in N6 position (Fig. 1).
NH
N
NH
N
NH
6
6
6
N
N
N
N
N
N
N
H
N
H
N
H
N
trans-Zeatin
Benzylaminopurine
Kinetin
Figure 1. Structures of three natural cytokinins.
Due to their important role in plant development, natu-
ral cytokinins have been tested for their activity on ani-
mal cells. Cytokinins and cytokinin nucleosides control
differentiation and apoptosis of human myeloid leukae-
mia cells.3 These results were likewise obtained on vari-
ous human cancer cell lines with certain cytokinin
analogues.4
atherosclerosis.5 Their antiproliferative and pro-apopto-
tic effects are related to their capacity to inhibit the cy-
clin-dependent kinase proteins (CDK) especially
CDK1 and CDK2.6
As we are interested in heterocyclic compounds with
potential pharmacological value, we decided to synthe-
sise new cytokinin analogues where the purine skeleton
was replaced by 7-azaindole. Our objective was to deter-
mine whether the nitrogen atom of the purine was
required for cytokinin-like activity. Here, we describe
the biological activity of eight 4-substituted
pyrrolo[2,3-b] pyridine compounds prepared via
Hartwig–Buchwald coupling.7–9
Cytokinins and their derivatives exert their biological
effects on several levels. Certain 6-alkynyl- and 6-alke-
nylpurines have a profound inhibiting effect on 15-lipo-
oxygenase which is implicated in the development of
Keywords: Cytokinin; 7-Azaindole; N-Arylamination; HL-60 cell line.
*
Corresponding author. Tel.: +33 247367227; fax: +33 247367229;
e-mail: mcviaud@univ-tours.fr
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.033

J. Guillard et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1934–1937
1935
Cl
i
ii
iii
N
H
N
H
N+
O-
N
N
H
N
H
N
N
55 %
75 %
1
3
4
2
4a : 57 %
4b : 60 %
4c : 38 %
4d : 39 %
iv
81 %
H
N
a
Cl
N
R
N
H
iii
b
c
H
N
R =
N
Me
N
Me
N
5
6
6a : 44 %
6b : 80 %
6c : 43 %
6d : 33 %
N
N
d
Scheme 1. Synthesis of 4-arylamino-substituted 7-azaindoles. Reagents and conditions: (i) m-CPBA, AcOEt, rt; (ii) MsCl, DMF, 70 ꢁC; (iii)
corresponding amine RH, Pd2(dba)3, t-BuONa, Xantphos, Dioxane, sealed tube; (iv) NaH, CH3I, DMF, rt.
All compounds were synthesised from 4-chloro-7-azain-
dole 3 (Scheme 1). The compound 3 was prepared in two
steps from 7-azaindole 1.10,11 The first step involved an
N-oxidation with m-CPBA in ethyl acetate to provide
2. This latter was then treated with methanesulfonyl
chloride at 50 ꢁC in N,N-dimethylformamide to give
Briefly, the cells were maintained in RPMI 1640 medium
supplemented with 10% foetal bovine serum, 1% penicil-
lin G/streptomycin, 1% L-glutamine and 0.2% b-mercap-
toethanol at 37 ꢁC in a humidified atmosphere of 5%
CO2 in air. Cell lines were incubated at 37 ꢁC with differ-
ent concentrations of 7-azaindole derivatives.
4-chloro-1H-pyrrolo[2,3-b]pyridine
3 in 75% yield.
Treatment of 3 with methyl iodide in the presence of
sodium hydride led to product 5. Then compounds
4a–d and 6a–d were obtained with moderate to good
yields (33–80%), by the condensation of N-substituted
4-chloropyrrolo[2,3-b]pyridine 3 or 5 with arylamines
in the presence of 2% Pd2(dba)3 and Xantphos as the li-
gand and t-BuONa as base in these reactions.12
After 72-h incubation, viable cell number was deter-
mined by Trypan blue exclusion, and the concentrations
lethal for 50% of the tumour cells (IC50) were estimated
from dose–response curves.
The IC50 values show interesting cytotoxic properties of
compound 4b, in contrast to derivatives 6a–d which dis-
played no activity in this assay. On the other hand, the
activities of 4a, 4c, 4d and kinetin were similar. Overall
the results show that the replacement of the purine ring
by 7-azaindole led to an increased inhibitory activity
compared to BAP.3,4
Compounds 4a–d and 6a–d were evaluated in vitro
for their antiproliferative activity using the human
myeloblastic leukaemia HL-60 cell line.13,14 The results
expressed as IC50 (concentration reducing cell prolifera-
tion by 50%) are reported in Table 1. Cell cycle
perturbations are shown in Figure 2.
After 72 h of culture, the cellular cycle was analyzed
with flow cytometry (Facscaliburꢂ, Becton–Dickinson,
Le Pont de Claix, France) after cell incubation with pro-
pidium iodide for 30 min at 37 ꢁC. The apoptosis was
evaluated by pre-G1 hypoploidy and the cellular prolif-
eration by S/G2/M fraction.
Table 1. Characteristics and pharmacological activity of synthesised
compounds
Compound
Formula
IC50 (lM)
Figure 2 presents a representative experiment studying
the effects of three different concentrations of analogues
on the cell cycle of HL-60 cells. The cells were cultured
in 25-cm2 tissue culture flasks at 37 ꢁC in 5% CO2
humidified air. HL-60 cells were cultured at a density
of 5 · 105 cells/mL in RPMI-1640 medium with
20 lmol/L glutamine (Life Technologies, Cergy-Pon-
toise, France) supplemented with 15% heat-inactivated
foetal calf serum (Life Technologies), 100 U/mL of pen-
icillin G and 100 lg/mL of streptomycin (Boehringer–
Kinetin
BAP
4a
C10H9N5O
C12H11N5
21.9
>100
23.5
12.6
C13H11N3 HCl
C14H12N3 HCl
C15H15N3 HCl
C17H18N4 HCl
C14H13N3 HCl
C15H15N3 HCl
C16H17N3 HCl
C18H20N4 HCl
4b
4c
23.7
23.6
4d
6a
>100
>100
>100
>100
6b
6c
6d

1936
J. Guillard et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1934–1937
Figure 2. Effects of compounds 4a–d and 6a–d on HL-60 cell apoptosis and cellular cycle.
Mannheim, Mannheim, Germany). The cells were incu-
bated with three different concentrations (10, 100 and
500 lM) of synthesised analogues. The cell cycle status
and apoptosis was studied using propidium iodide (PI)
for nuclear staining. PI is a fluorogenic compound that
binds stoichiometrically to nucleic acids so that fluores-
cence emission is proportional to the DNA content of a
cell. When apoptotic cells are stained with PI and ana-
lyzed with a flow cytometer, they display a broad hypo-
diploid (sub-G1) peak, which can be easily discriminated
from the narrow peak of cells with normal (diploid)
DNA content in the red fluorescence channels.15 Propi-
dium iodide staining was realized using Cycle TestTM kit
(BD-Biosciences, Le Pont de Claix, France) according to
the manufacturer’s instructions. All cytometric analyses
were performed with a 488-nm laser flow cytometer. In
comparison to untreated cells (grey histogram), the three
different concentrations used in these experiments pro-
vided an evaluation of the biological effects of the syn-
thesised analogues.
(BAP) was replaced with 7-azaindole. The biological
tests showed that the NH group is essential for potent
activity and the benzylamino substitution was the opti-
mal structure. At the present time these structures are
being submitted to further biological tests in order to
determine their precise action on the cell cycle.
Acknowledgment
We are grateful to IFR 135 ‘Imagerie Fonctionnelle’,
(University Franc¸ois Rabelais) 37000 Tours, France,
for their financial support.
References and notes
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Mol. Biol. 2001, 52, 89.
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Differ. 2002, 13, 19.
All of these compounds were anti-proliferative as shown
by the decrease of the S/G2/M fraction. N-Methyl-
substituted analogues showed a pro-apoptotic effect at
concentrations of 500 lM, whereas NH analogues had
this effect at 10 lM. The 1H-4-(phenylamino)-pyr-
rolo[2,3-b]pyridine 4a increased the pre-G1 fraction by
39.9% versus 19.9% without cytokinin.
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˚
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In summary, we have prepared eight new cytokinin ana-
logues where the purine ring of benzylaminopurine

J. Guillard et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1934–1937
1937
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12. Typical procedure for Pd-catalysis. A 100-mL oven-dried
resealable flask capped with a rubber septum was evacu-
ated and backfilled with Argon. The flask was charged
with 4-chloro-1H-pyrrolo[2,3-b]pyridine (3) (0.131 mmol),
sodium tert-butoxide (0.367 mmol), Pd2dba3 (0.00262
mmol), Xantphos (0.00524 mmol), and evacuated and
backfilled with Argon. 1,4-Dioxane (10 mL) and N-alkyl-
amine (0.393 mmol) were added and Argon was bubbled
through the mixture for 20 min. The septum was replaced
with a Teflon screwcap, the flask was sealed and the
mixture was heated at 100 ꢁC for 24 h. The mixture was
cooled to room temperature, diluted with ethyl acetate
(50 mL), filtered through Celite and concentrated in
vacuo. The resulting oil was dissolved in dichloromethane
(25 mL), washed twice with water, dried over Na2SO4,
filtered and concentrated in vacuo. The crude material was
purified by flash column chromatography (ethyl acetate/
light petroleum) on silica gel to give the desired
4-substituted pyrrolo[2,3-b]pyridine (4a–d) and (6a–d) as
solid. After, the HCl salts were prepared from the
corresponding compound in hydrogen chloride 2.0 M
solution in diethyl ether for 2 h at rt.
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