Synthesis and biological activities of aminopyrimidyl-indoles structurally related to meridianins

Article abstract of DOI:10.1016/j.bmc.2009.05.017

The synthesis of new meridianin derivatives substituted at the C-5 position of the 2-aminopyrimidine ring by various aryl groups and substituted or not by a methyl group on the indole nitrogen is described. These compounds were tested for their kinase inh

Full text of DOI:10.1016/j.bmc.2009.05.017

Bioorganic & Medicinal Chemistry 17 (2009) 4420–4424
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activities of aminopyrimidyl-indoles structurally
related to meridianins
Rufine Akue-Gedu a,b, Eric Debiton c, Yoan Ferandin d, Laurent Meijer d, Michelle Prudhomme a,b
,
a,b,
Fabrice Anizon a,b, , Pascale Moreau
*
*
a Clermont Université, Université Blaise Pascal, SEESIB, 24 avenue des Landais, F-63177 Aubière, France
b CNRS, UMR 6504, F-63177 Aubière, France
c Clermont Université, Université d’Auvergne, Centre Jean Perrin, EA 4231, F-63005 Clermont-Ferrand, France
d CNRS, Protein Phosphorylation & Human Disease Group, Station Biologique, BP 74, 29682 Roscoff, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of new meridianin derivatives substituted at the C-5 position of the 2-aminopyrimidine
ring by various aryl groups and substituted or not by a methyl group on the indole nitrogen is described.
These compounds were tested for their kinase inhibitory potencies toward five kinases (CDK5/p25, CK1d/
Received 30 March 2009
Revised 5 May 2009
Accepted 7 May 2009
Available online 12 May 2009
e
, GSK-3
blast primary culture and two human solid cancer cell lines (MCF-7 and PA 1).
Ó 2009 Elsevier Ltd. All rights reserved.
a/b, Dyrk1A and Erk2) as well as their in vitro antiproliferative activities toward a human fibro-
Keywords:
Meridianins
Suzuki cross-coupling
Kinase inhibitors
Dyrk1A inhibitors
1. Introduction
indole nitrogen were critical for either kinase inhibitory potencies
or in vitro antiproliferative activities. The meridianin derivatives A–
H and K–Q were tested toward a panel of 9 protein kinases (KDR,
IGF-1R, c-Met, RET, c-Src, c-Abl, PKA, CDK2/cyclin A and HER-1) and
their in vitro antiproliferative activities were examined toward a hu-
man fibroblast primary culture and two human solid cancer cell lines
(MCF-7 and PA 1). Despite weak kinase inhibitory potencies, com-
pounds G, H, L and M exhibited high in vitro antiproliferative activi-
ties toward PA 1 cells. It was found that these compounds do not
interfere with PA 1 cell-cycle and may be considered as direct cytol-
ysis or apoptosis inducers. Considering these first biological results, it
was of interest to pursue the study with new aryl substituents on the
pyrimidine ring in order to identify the substitution pattern provid-
ing the highest biological potencies.
Meridianins are marine alkaloids isolated and characterized
from the south Atlantic tunicate Aplidium meridianum.1 Meridianins
A–G1–3 (Fig. 1), indole derivatives substituted in the C-3 position by
a 2-aminopyrimidine ring, were successfully evaluated for their
ability to inhibit various protein kinases3 and to display antitumour
activity.4 The closely related meriolins, a family of 7-azaindole syn-
thetic meridianin analogues, which exhibit potent kinase inhibitory
activities and antiproliferative properties were also described.5,6
Since protein kinases are attractive targets for the discovery of
biologically active compounds of potential therapeutic interest, our
aim is to identify new ATP-competitive kinase inhibitors. Thus, we
were recently interested in meridianin derivatives substituted at
the C-5 position of the 2-aminopyrimidine ring. In recent papers,
we described the synthesis and biological activities of meridianin G
derivatives, substituted or not on the indole nitrogen with a methyl
group, and bearing various aryl moieties at the C-5 position of the
pyrimidine ring (compounds A–H and J–Q, Fig. 1).7,8 It was shown
that the substitution at this C-5 position and the methylation of the
In this paper, we described the synthesis of new meridianin
analogues diversely substituted at the C-5 position of the pyrimi-
dine ring and methylated or not on the indole nitrogen. Moreover
these compounds were tested for their kinase inhibitory potencies
toward five kinases (CDK5/p25, CK1d/e, GSK3a/b, Dyrk1A and
Erk2) as well as their in vitro antiproliferative activities toward a
human fibroblast primary culture and two human solid cancer cell
lines (MCF-7 and PA 1).
* Corresponding authors. Tel.: +33 4 73 40 53 64; fax: +33 4 73 40 77 17 (F.A.),
tel.: +33 4 73 40 79 63; fax: +33 4 73 40 77 17 (P.M.).
2. Chemistry
E-mail addresses: Fabrice.ANIZON@univ-bpclermont.fr (F. Anizon), Pascale.MOR
EAU@univ-bpclermont.fr (P. Moreau).
Compounds 1–9 were synthesized from brominated derivatives
A or J, methylated or not on the indole nitrogen. A and J were
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.017

R. Akue-Gedu et al. / Bioorg. Med. Chem. 17 (2009) 4420–4424
4421
H2N
meridianin A, R1=OH, R2=R3=R4=H
N
meridianin B, R1=OH, R2=R4=H, R3=Br
N
R1
meridianin C, R1=R3=R4=H, R2=Br
meridianin D, R1=R2=R4=H, R3=Br
meridianin E, R1=OH, R2=R3=H, R4=Br
meridianin F, R1=H, R2=R3=Br, R4=H
meridianin G, R1=R2=R3=R4=H
R2
R3
N
H
R4
I R = Me, R' = H
A R = H, R' = Br
B R = H, R' = phenyl
J R = Me, R' = Br
K R = Me, R' = phenyl
H2N
N
C R = H, R' = 4-acetylphenyl
D R = H, R' = 4-biphenyl
E R = H, R' = 4-fluorophenyl
F R = H, R' = 2,4-difluorophenyl
G R = H, R' = 4-trifluoromethylphenyl
L R = Me, R' = 4-acetylphenyl
M R = Me, R' = 4-biphenyl
N R= Me, R' = 4-fluorophenyl
O R = Me, R' = 2,4-difluorophenyl
P R = Me, R' = 4-trifluoromethylphenyl
Q R = Me, R' = 4-trifluoromethoxyphenyl
N
R'
N
R
H R = H, R' = 4-trifluoromethoxyphenyl
Figure 1. Meridianins A–G and derivatives A–Q described by our group.
prepared according to previously described procedure (Scheme 1).7
Suzuki cross-coupling was performed using various commercially
pared in 77% yield from methylated meridianin G (compound I),
using iodine in DMSO.9
available
arylboronic
acids,
tetrakis(triphenylphosphine)
H2O/EtOH/toluene
Unfortunately, we never manage to substitute this position by
an amine derivative from compound 10 or its brominated
analogue J.
In summary, the method described here allowed the introduc-
tion, in the pyrimidine C-5 position, of heteroaryl moieties or phe-
nyl groups bearing both electron donor or electron acceptor
substituents in para or meta positions.
palladium(0) and sodium carbonate in
a
medium. Compounds 1–9 were obtained in 31ꢀ60% isolated
yields. Due to purification difficulties, the non-methylated ana-
logues of compounds 7–9 could not be isolated as pure
compounds.
Results were similar using compounds A or J (compare 1 and 2,
3 and 4, 5 and 6), and yields dropped using aryl boronic acid con-
taining a basic nitrogen atom.
3. Results and discussion
Moreover, in order to introduce various amines (aryl or alkyl-
amines) by Buchwald type coupling, in the C-5 position of the
pyrimidine ring, compound 10, the iodo analogue of J, was pre-
In vitro antiproliferative activities of compounds 1–10 were
evaluated toward a human fibroblasts primary culture and two
OCH3
1 R = H, 58%
Ar =
2 R = CH3, 60%
NH2
3 R = H, 41%
H2N
Ar =
Ar =
H2N
H2N
4 R = CH3, 35%
ArB(OH)2
Pd(PPh3)4, Na2CO3
H2O, EtOH, toluene
N
N
N
N
N
N
O
NBS, THF
Ref. 7
5 R = H, 49%
6 R = CH3, 54%
Br
Ar
Yields: 31-60%
NH2
O
N
R
N
R
N
R
7 R = CH3, 60%
8 R = CH3, 31%
Ar =
Ar =
Meridianin G R = H
I R = CH3
OH
A R = H
J R = CH3
1-9
N
N
9 R = CH3, 33%
Ar =
N
H2N
N
H2N
N
N
N
I2, DMSO
77%
I
N
CH3
N
CH3
I
10
Scheme 1. Synthesis of compounds 1–10.

4422
R. Akue-Gedu et al. / Bioorg. Med. Chem. 17 (2009) 4420–4424
Table 1
Inhibitory potencies toward five protein kinases (IC50 in
lM) and antiproliferative activity of compounds G, H, L, M and 1ꢀ10 (IC50 in lM)
Compounds
Kinase inhibition—IC50 in
l
M
Antiproliferative activity toward PA 1 cells (IC50 in lM)
CDK5/p25
CK1d/
e
GSK3
a/b
Dyrk1A
Erk2
G
H
L
M
1
2
3
4
5
6
7
8
9
11.0
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
5.3
8.3
1.9
3.0
1.4
>10
3.0
3.8
6.0
5.0
2.0
0.63
>10
6.1
1.2
4.2
2.3
2.9
5.0
>10
0.60
3.1
0.64
1.9
0.58
2.0
0.58
3.2
0.09
0.08
0.05
0.08
28
>10
>10
>10
0.49
2.2
3.3
3.1
3.4
4.1
>10
>10
3.10
>10
6.9
>10
1.90
7.00
>10
>10
1.30
4.0
34
>50
>50
38
>50
>50
>50
>50
31
>10
12.0
4.2
0.9
3.2
2.4
0.4
10
2.6
1.1
human solid cancer cell lines (MCF-7 and PA 1). The antiprolifera-
tive effect (IC50) of the tested drug was assessed by the resazurin
reduction test.10 Under the conditions used, none of the newly syn-
thesised compounds showed any significant cytotoxic activity
tion, due to the modification of the aryl moiety, with the cellular
target(s) involved in their antiproliferative activity.
Compounds G, 1, 3, 5–7 and 10 have shown sub-micromolar
inhibition potencies toward some of the kinases tested. The inter-
esting inhibition of Dyrk1A kinase, which is known to be involved
in Alzheimer’s disease11 and Down syndrome,12 leads us to expand
our effort in the synthesis of diversely substituted meridianin
derivatives as new potential inhibitors of this kinase.
(IC50 < 50
ties (IC50 around 30
l
M) on fibroblasts or MCF-7 cell lines. Only mild activi-
M) were observed on PA 1 cells with com-
l
pounds 1, 2, 5 and 10 (Table 1). These results indicate that the
substituents introduced at the C-50 position are detrimental to
the cytotoxic effect of these series on PA 1 cells.
The kinase inhibitory potencies of compounds 1–10 and that of
previously synthesised derivatives G, H, L and M which have
shown interesting antiproliferative activities toward PA1 cells
were evaluated as IC50 values toward five protein kinases (CDK5/
5. Experimental
5.1. In vitro Kinase inhibition assays
p25, CK1d/e, GSK-3a/b, Dyrk1A and Erk2).
5.1.1. Buffers
Only two compounds were mildly active toward CDK5/p25 with
IC50 values in the micromolar range. These derivatives are both
methylated on the indole nitrogen and substituted either by a pyr-
Buffer A: 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM Tris–
HCl pH 7.5, 50 lg heparin/ml.
Buffer C: 60 mM b-glycerophosphate, 15 mM p-nitrophenyl-
phosphate, 25 mM Mops (pH 7.2), 5 mM EGTA, 15 mM MgCl2,
1 mM DTT, 1 mM sodium vanadate, 1 mM phenylphosphate.
idyl group (compound 9, IC50 = 5.3
pound 10, IC50 = 2.6 M) in the C-5 position of the
aminopyrimidine moiety.
Considering the other kinases, the best inhibitory potencies
were sub-micromolar inhibition of CK1d/ (compound 1,
IC50 = 0.49 M), GSK-3 /b (compound 6, IC50 = 0.63 M), Dyrk1A
(compounds 1, IC50 = 0.6 M; 3, IC50 = 0.64 M; 5, IC50 = 0.58 M;
7, IC50 = 0.9 M; 10, IC50 = 0.4 M) and Erk2 (compound G,
IC50 = 0.58 M). Regarding these results, this series of compounds
lM) or by an iodine atom (com-
l
5.1.2. Kinase preparations and assays
Kinase activities were assayed in Buffer A or C, at 30 °C, at a final
ATP concentration of 15 lM. Blank values were subtracted and
activities expressed in % of the maximal activity, i.e. in the absence
of inhibitors. Controls were performed with appropriate dilutions
of DMSO.
e
l
a
l
l
l
l
l
l
l
could be particularly interesting in the development of new inhib-
itors of Dyrk1A kinase. Comparison of the activity of non-methyl-
ated compounds (1, 3, 5) toward Dyrk1A with that of their N–
CH3 analogues (2, 4, 6) indicate that the non-methylated analogues
are more active than their methylated counterparts. Nevertheless,
two methylated derivatives bearing either a p-carboxyphenyl moi-
ety (7) or an iodine atom (10) at the C-5 position of the pyrimidine
ring exhibited Dyrk1A sub-micromolar inhibition as well.
CDK5/p25 (human, recombinant) was prepared as previously
described.13,14 Its kinase activity was assayed in buffer C, with
1 mg histone H1 /ml, in the presence of 15
(3,000 Ci/mmol; 10 mCi/ml) in a final volume of 30
30 min incubation at 30 °C, 25 l aliquots of supernatant were
lM [
c-
33P] ATP
l. After
l
l
spotted onto 2.5 ꢁ 3 cm pieces of Whatman P81 phosphocellulose
paper, and, 20 s later, the filters were washed five times (for at
least 5 min each time) in a solution of 10 ml phosphoric acid/liter
of water. The wet filters were counted in the presence of 1 ml
ACS (Amersham) scintillation fluid.
4. Conclusion
GSK-3ab (porcine brain, native) was assayed, as described for
Encouraging preliminary biological evaluation of diversely
substituted meridianins led us to synthesize new derivatives. Su-
zuki cross-coupling could be applied to compounds A or J, for cou-
pling with either aromatic boronic acids bearing both electron
donor and electron acceptor substituents or heteroaromatic boro-
nic acids.
Surprisingly, the cytotoxic activity of newly synthesised deriva-
tives 1–10 toward PA 1 cells is significantly smaller than that of the
previously described compounds G, H, L, M. This could be due to a
lower cellular penetration of compounds 1–10 or lower interac-
CDK5/p25 but in Buffer A and using a GSK-3 specific substrate
(GS-1: YRRAAVPPSPSLSRHSSPHQSpEDEEE) (pS stands for phos-
phorylated serine).15 GS-1 was synthesized by Millegen (Labege,
France).
CK1d/e (porcine brain, native) was assayed as described for
CDK5/p25 but using the CK1-specific peptide substrate
RRKHAAIGpSAYSITA,16 obtained from Millegen (Labege, France).
Erk2 (rat, recombinant) was assayed as described for CDK5/p25
but using the specific substrate Ets1 (amino acids 1–138) in
buffer A.

R. Akue-Gedu et al. / Bioorg. Med. Chem. 17 (2009) 4420–4424
4423
DYRK1A (rat, recombinant, expressed in Escherichia coli as a GST
5.3.2. 4-(1H-Indol-3-yl)-5-(3-methoxyphenyl)pyrimidin-2-
amine (1)
Purification of the residue by flash chromatography (cyclo-
fusion protein) was purified by affinity chromatography on gluta-
thione–agarose and assayed as described for CDK5/p25 using mye-
lin basic protein (1 mg/ml) as a substrate.
hexane/EtOAc 7:3) provided
powder.
1 (185 mg, 58%) as a yellow
5.2. In vitro antiproliferative assays
Mp 98ꢀ100 °C; HRMS (ESI+) calcd for C19H17N4O (M+H)+
317.1402, found 317.1385; IR (KBr): 3650ꢀ3150, 1605 cmꢀ1
;
1H
5.2.1. Cell cultures
NMR (400 MHz, DMSO-d6): 3.71 (3H, s, CH3), 6.52ꢀ6.58 (2H, br s,
NH2), 6.69 (1H, d, J = 3.0 Hz), 6.83ꢀ6.86 (2H, m), 6.91ꢀ6.95 (1H,
m), 7.06 (1H, ddd, J1 = 8.0 Hz, J2 = 7.0 Hz; J3 = 1.0 Hz), 7.12 (1H,
ddd, J1 = 8.0 Hz, J2 = 7.0 Hz, J3 = 1.0 Hz), 7.31 (1H, t, J = 8.0 Hz),
7.34 (1H, d, J = 8.0 Hz), 8.00 (1H, s), 8.46 (1H, d, J = 8.0 Hz),
11.23ꢀ11.33 (1H, br s, NH); 13C NMR (100 MHz, DMSO-d6): 55.0
(OCH3), 111.4, 112.6, 114.9, 120.0, 121.8, 121.9, 122.9, 128.9,
129.8, 157.8 (CH), 112.7, 120.9, 126.2, 135.8, 140.3, 159.5, 159.8,
162.5 (C).
Stock cell cultures were maintained as monolayers in 75 cm2
culture flasks in Glutamax Eagle’s minimum essential medium
(MEM) with Earle’s salts supplemented with 10% fetal calf serum,
5 ml 100 mmol/l sodium pyruvate, 5 ml of 100ꢁ non-essential
amino acids and 2 mg gentamicin base. Cells were grown at
37 °C in a humidified incubator under an atmosphere containing
5% CO2.
5.2.2. Survival assays
Cells were plated at a density of 5 ꢁ 103 cells in 150
l
l culture
5.3.3. 4-(1-Methyl-1H-indol-3-yl)-5-(3-methoxyphenyl)
pyrimidin-2-amine (2)
medium in each well of 96-well microplates and were allowed to
adhere for 16 h before treatment with tested drug. A stock solution
20 mmol/l of each tested drug was prepared in DMSO and kept at
Purification of the residue by flash chromatography (cyclohex-
ane/EtOAc 7:3) provided 2 (198 mg, 60%) as a yellow powder.
Mp = 82ꢀ84 °C; HRMS (ESI+) calcd for C20H19N4O (M+H)+
ꢀ20 °C until use. Then 50
ll 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
the resazurin reduction test.
331.1559, found 331.1546; IR (KBr): 3650ꢀ3150, 1604 cmꢀ1
;
1H NMR (400 MHz, DMSO-d6): 3.61 (3H, s, NCH3), 3.72 (3H,
s, OCH3), 6.56ꢀ6.59 (2H, br s, NH2), 6.73 (1H, s), 6.83 (1H,
ddd, J1 = 7.5 Hz, J2 = 1.5 Hz, J3 = 1.0 Hz), 6.86 (1H, dd,
J1 = 2.5 Hz, J2 = 1.5 Hz), 6.93 (1H, ddd, J1 = 8.5 Hz, J2 = 2.5 Hz,
J3 = 1.0 Hz), 7.08 (1H, ddd, J1 = 8.0 Hz, J2 = 7.0 Hz, J3 = 1.0 Hz),
7.18 (1H, ddd, J1 = 8.0 Hz, J2 = 7.0 Hz, J3 = 1.0 Hz), 7.29 (1H, t,
J = 8.0 Hz), 7.40 (1H, d, J = 8.0 Hz), 8.03 (1H, s), 8.35 (1H, d,
J = 8.0 Hz); 13C NMR (100 MHz, DMSO-d6): 32.8 (CH3), 55.0
(OCH3), 109.8, 112.7, 114.8, 120.2, 121.7, 121.9, 122.8, 129.7,
132.6, 158.1 (CH), 112.0, 120.8, 126.5, 136.4, 140.0, 159.4,
159.5, 162.4 (C).
5.2.3. Resazurin reduction test
Plates were rinsed with 200
ll PBS at 37 °C and emptied by
overturning on absorbent toweling. Then 150
ll of a 25 g/ml
l
solution of resazurin in MEM without phenol red was added to
each well. Plates were incubated for 1 h at 37 °C in a humidified
atmosphere containing 5% CO2. Fluorescence was then measured
on an automated 96-well plate reader (Fluoroscan Ascent FL, Lab-
system) using an excitation wavelength of 530 nm and an emission
wavelength of 590 nm. Under 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 concentra-
tion-dependent survival percentage, defined as fluorescence in
experimental wells compared with fluorescence in control wells,
after subtraction of the blank values.
5.3.4. 4-(1H-Indol-3-yl)-5-(3-aminophenyl)pyrimidin-2-amine
(3)
Purification of the residue by flash chromatography (cyclohex-
ane/EtOAc 4:6) provided 3 (125 mg, 41%) as a yellow powder.
Mp >250 °C, decomposition; HRMS (ESI+) calcd for C18H16N5
(M+H)+ 302.1406, found 302.1397; IR (KBr): 3550–3100,
1614 cmꢀ1
;
1H NMR (400 MHz, DMSO-d6): 5.09ꢀ5.16 (2H, br s,
5.3. Chemistry
NH2), 6.42 (1H, ddd, J1 = 7.5 Hz, J2 = 1.5 Hz, J3 = 1.0 Hz), 6.45ꢀ6.51
(3H, br s, 1CH+NH2), 6.57 (1H, ddd, J1 = 8.0 Hz, J2 = 2.5 Hz,
J3 = 1.0 Hz), 6.75 (1H, d, J = 3.0 Hz), 7.05ꢀ7.16 (3H, m), 7.34 (1H,
d, J = 8.0 Hz), 7.91 (1H, s), 8.62 (1H, d, J = 8.0 Hz), 11.25ꢀ11.35
(1H, br s, NH); 13C NMR (100 MHz, DMSO-d6): 111.3, 112.9,
114.7, 116.8, 120.0, 121.8, 123.2, 129.2, 129.3, 157.4 (CH), 112.7,
121.7, 126.4, 135.8, 139.6, 149.1, 159.8, 162.3 (C).
IR spectra were recorded on a Perkin–Elmer Paragon 500
spectrometer mmax in cmꢀ1). NMR spectra were performed on a
ꢀ
Bruker AVANCE 400 (1H: 400 MHz, 13C: 100 MHz), chemical
shifts d are noted in ppm and the following abbreviations are
used: singlet (s), doublet (d), triplet (t), doublet of doublet
(dd), doublet of doublet of doublet (ddd), doublet of triplet
(dt), multiplet (m), broad signal (br s). Mass spectra (ESI+) were
determined on a high resolution Waters Micro Q-Toff apparatus.
Chromatographic purifications were performed on flash silica gel
Geduran SI 60 (Merck) 0.040–0.063 mm column chromatography.
TLC were performed on fluorescent silica gel plates (60 F254
from Merck).
5.3.5. 4-(1-Methyl-1H-indol-3-yl)-5-(3-aminophenyl)
pyrimidin-2-amine (4)
Purification of the residue by flash chromatography (cyclohex-
ane/EtOAc 4:6) provided 4 (110 mg, 35%) as a yellow powder.
Mp = 183ꢀ185 °C; HRMS (ESI+) calcd for C19H18N5 (M+H)+
316.1562, found 316.1548; IR (KBr): 3600–3100, 1651,
1602 cmꢀ1
;
1H NMR (400 MHz, DMSO-d6): 3.60 (3H, s, CH3),
5.3.1. General procedure for the preparation of compounds 1–9
A solution of Na2CO3 (2.5 equiv) in H2O (2 ml/mmol) was
added to a suspension of bromide A (288 mg, 1.0 mmol) or J
(302 mg, 1.0 mmol), the required boronic acid (1.1 equiv),
Pd(PPh3)4 (57.8 mg, 5 mol %) in EtOH (3 ml/mmol) and toluene
(3 ml/mmol). The reaction mixture was refluxed overnight under
argon and the cooled resulting mixture was evaporated onto sil-
ica gel. Purification as indicated provided the desired
compounds.
5.08ꢀ5.16 (2H, br s, NH2), 6.41 (1H, d, J = 7.5 Hz), 6.48 (1H, t,
J = 1.5 Hz), 6.48ꢀ6.52 (2H, br s, NH2), 6.58 (1H, dd, J1 = 8.0 Hz,
J2 = 1.5 Hz), 6.73 (1H, s), 7.06 (1H, t, J = 7.5 Hz), 7.11 (1H, ddd,
J1 = 8.0 Hz, J2 = 7.0 Hz, J3 = 1.0 Hz), 7.19 (1H, ddd, J1 = 8.0 Hz,
J2 = 7.0 Hz, J3 = 1.0 Hz), 7.40 (1H, J = 8.0 Hz), 7.93 (1H, s), 8.53
(1H, d, J = 8.0 Hz); 13C NMR (100 MHz, DMSO-d6): 32.9 (CH3),
109.8, 112.8, 114.6, 116.8, 120.3, 121.9, 123.2, 129.3, 132.9,
157.6 (CH), 112.0, 121.7, 126.7, 136.4, 139.3, 149.0, 159.3,
162.3 (C).

4424
R. Akue-Gedu et al. / Bioorg. Med. Chem. 17 (2009) 4420–4424
5.3.6. 4-[2-Amino-4-(1H-indol-3-yl)pyrimidin-5-yl]benzamide
(5)
J3 = 1.0 Hz), 7.18 (1H, ddd, J1 = 8.0 Hz, J2 = 7.0 Hz, J3 = 1.0 Hz), 7.30
(2H, m), 7.42 (1H, d, J = 8.0 Hz), 8.02 (1H, d, J = 8.0 Hz), 8.14 (1H,
s), 8.49 (2H, d, J = 5.5 Hz); 13C NMR (100 MHz, DMSO-d6): 32.8
(CH3), 110.1, 120.3, 122.0, 122.1, 124.0 (2C), 132.6, 149.8 (2C),
158.6 (CH), 111.8, 118.5, 126.1, 136.6, 146.5, 159.5, 162.9 (C).
Purification of the residue by flash chromatography (cyclohex-
ane/EtOAc 4:6) provided 5 (160 mg, 49%) as a yellow powder.
Mp >250 °C; HRMS (ESI+) calcd for C19H16N5O (M+H)+ 330.1355,
found 330.1344; IR (KBr): 3600–3100, 1665, 1610 cmꢀ1 1H NMR
;
(400 MHz, DMSO-d6): 6.62ꢀ6.68 (2H, br s, NH2), 6.67 (1H, d,
J = 3.0 Hz), 7.05 (1H, ddd, J1 = 8.0 Hz, J2 = 7.0 Hz, J3 = 1.0 Hz), 7.12
(1H, ddd, J1 = 8.0 Hz, J2 = 7.0 Hz, J3 = 1.5 Hz), 7.33ꢀ7.40 (4H, m),
7.89 (2H, d, J = 8.5 Hz), 7.98ꢀ8.02 (1H, br s, NH), 8.05 (1H, s),
8.37 (1H, d, J = 8.0 Hz), 11.26ꢀ11.32 (1H, br s, NH); 13C NMR
(100 MHz, DMSO-d6): 111.5, 120.1, 121.9, 122.6, 127.9 (2C),
128.9, 129.2 (2C), 158.0 (CH), 112.7, 120.4, 126.1, 132.6, 135.9,
142.0, 159.9, 162.6 (C), 167.5 (C@O).
5.3.11. 5-Iodo-4-(1-Methyl-1H-indol-3-yl)pyrimidin-2-amine
(10)
Iodine (1.13 g, 4.46 mmol) was added to the solution of com-
pound I (1.0 g, 4.46 mmol) in DMSO (50 ml). The mixture was stir-
red overnight at 25 °C. The reaction was quenched with a saturated
aqueous solution of sodium thiosulfate and was extracted with
EtOAc. The organic phase was washed with a brine solution and
then dried over MgSO4, filtrated and evaporated onto silica gel.
Purification by flash chromatography (cyclohexane/ethyl acetate
1:9) provided 10 (1.20 g, 3.43 mmol, 77%) as a yellow powder.
Mp = 185ꢀ188 °C; HRMS (ESI+) calcd for C13H12IN4 (M+H)+
5.3.7. 4-[2-Amino-4-(1-methyl-1H-indol-3-yl)pyrimidin-5-
yl]benzamide (6)
Purification of the residue by flash chromatography (cyclohex-
ane/EtOAc 4:6) provided 6 (184 mg, 54%) as a yellow powder.
Mp >250 °C; HRMS (ESI+) calcd for C20H18N5O (M+H)+ 344.1511,
351.0107, found 351.0091; IR (KBr): 3650ꢀ3200, 1633 cmꢀ1
;
1H
NMR (400 MHz, DMSO-d6): 3.89 (3H, s, CH3), 6.67ꢀ6.77 (2H, br s,
NH2), 7.16 (1H, ddd, J1 = 8.0 Hz, J2 = 7.0 Hz, J3 = 1.0 Hz), 7.25 (1H,
ddd, J1 = 8.0 Hz, J2 = 7.0 Hz, J3 = 1.0 Hz), 7.51 (1H, dt, J1 = 8.0 Hz,
J2 = 1.0 Hz), 8.32 (1H, ddd, J1 = 8.0 Hz, J2 = 1.0 Hz, J3 = 0.5 Hz), 8.43
(1H, s), 8.49 (1H, s); 13C NMR (100 MHz, DMSO-d6): 33.0 (CH3),
110.1, 120.5, 122.1, 122.7, 133.2, 165.8 (CH), 74.7, 112.8, 126.6,
136.5, 161.9, 162.1 (C).
found 344.1494; IR (KBr): 3600–3100, 1681, 1594 cmꢀ1 1H NMR
;
(400 MHz, DMSO-d6): 3.62 (3H, s, CH3), 6.62ꢀ6.70 (2H, br s,
NH2), 6.80 (1H, s), 7.06 (1H, t, J = 7.5 Hz), 7.18 (1H, t, J = 7.5 Hz),
7.36 (2H, d, J = 8.0 Hz), 7.35ꢀ7.41 (1H, br s, NH), 7.41 (1H, d,
J = 8.0 Hz), 7.87 (2H, d, J = 8.0 Hz), 7.97ꢀ8.02 (1H, br s, NH), 8.07
(1H, s), 8.20 (1H, d, J = 8.0 Hz); 13C NMR (100 MHz, DMSO-d6):
32.8 (CH3), 109.9, 120.2, 121.9, 122.5, 127.9 (2C), 129.0 (2C),
132.6, 158.4 (CH), 112.0, 120.3, 126.3, 132.5, 136.5, 141.7, 159.4,
162.6 (C), 167.5 (C@O).
Acknowledgments
The authors thank the European Union Prokinase Research Con-
sortium for financial support and Bertrand Légeret for mass spec-
trometry analysis. This research was also supported by grants
from ‘Association France-Alzheimer Finistère’ (LM), CRITT Santé
Bretagne (LM) and ‘Fondation Jérôme Lejeune’ (LM).
5.3.8. 4-[2-Amino-4-(1-methyl-1H-indol-3-yl)pyrimidin-5-
yl]benzoic acid (7)
Purification of the residue by flash chromatography (EtOAc/
MeOH 9:1) provided 7 (207 mg, 60%) as a yellow powder.
Mp >250 °C; HRMS (ESI+) calcd for C20H17N4O2 (M+H)+
References and notes
345.1352, found 345.1342; IR (KBr): 3250ꢀ2800, 1608 cmꢀ1
;
1H
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NMR (400 MHz, DMSO-d6): 3.63 (3H, s, CH3), 6.66ꢀ6.72 (2H, br s,
NH2), 6.83 (1H, s), 7.05 (1H, ddd, J1 = 8.0 Hz, J2 = 7.0 Hz,
J3 = 1.0 Hz), 7.17 (1H, ddd, J1 = 8.0 Hz, J2 = 7.0 Hz, J3 = 1.0 Hz),
7.38ꢀ7.42 (3H, m), 7.91 (2H, d, J = 8.5 Hz), 8.08 (1H, s), 8.16 (1H,
d, J = 8.0 Hz), 12.73ꢀ13.09 (1H, br s, COOH); 13C NMR (100 MHz,
DMSO-d6): 32.8 (CH3), 109.9, 120.2, 121.9, 122.4, 129.3 (2C),
129.7 (2C), 132.7, 158.4 (CH), 111.9, 120.1, 126.3, 129.1, 136.5,
143.3, 159.4, 162.6 (C), 167.2 (C@O).
5.3.9. 4-(1-Methyl-1H-indol-3-yl)-5,50-bipyrimidin-2-amine (8)
Purification of the residue by flash chromatography (cyclohex-
ane/EtOAc 2:8) provided 8 (95 mg, 31%) as a yellow powder.
Mp >250 °C; HRMS (ESI+) calcd for C17H15N6 (M+H)+ 303.1358,
found 303.1344; IR (KBr): 3400ꢀ3100, 1638 cmꢀ1
;
1H NMR
(400 MHz, DMSO-d6): 3.69 (3H, s, CH3), 6.80ꢀ6.87 (2H, br s,
NH2), 7.04 (1H, s), 7.05 (1H, t, J = 7.5 Hz), 7.18 (1H, t, J = 7.5 Hz),
7.43 (1H, d, J = 8.0 Hz), 7.99 (1H, d, J = 8.0 Hz), 8.21 (1H, s,), 8.70
(2H, s), 9.09 (1H, s); 13C NMR (100 MHz, DMSO-d6): 32.8 (CH3),
110.1, 120.3, 122.0, 122.1, 132.7, 156.5 (3C), 159.1 (CH), 111.7,
114.2, 125.9, 132.5, 136.6, 160.1, 163.2 (C).
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Hashimoto, R.; Tanaka, T.; Kudo, T.; Yamagata, H.; Tabara, Y.; Miki, T.;
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5.3.10. 4-(1-Methyl-1H-indol-3-yl)-5-(pyridin-4-yl)pyrimidin-
2-amine (9)
13. Leclerc, S.; Garnier, M.; Hoessel, R.; Marko, D.; Bibb, J. A.; Snyder, G. L.;
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Purification of the residue by flash chromatography (cyclohex-
ane/EtOAc 4:6) provided 9 (98 mg, 33%) as a yellow powder.
Mp >250 °C; HRMS (ESI+) calcd for C18H16N5 (M+H)+ 302.1406,
found 302.1393; IR (KBr): 3350ꢀ3100, 1649 cmꢀ1
;
1H NMR
(400 MHz, DMSO-d6): 3.67 (3H, s, CH3), 6.76ꢀ6.83 (2H, br s,
NH2), 7.01 (1H, s), 7.04 (1H, ddd, J1 = 8.0 Hz, J2 = 7.0 Hz,