Synthesis, protein kinase inhibitory potencies, and in vitro antiproliferative activities of meridianin derivatives

Article abstract of DOI:10.1021/jm200464w

The synthesis of new meridianin derivatives is described. The indolic ring system was substituted at the C-4 to C-7 positions either by a bromine atom or by nitro or amino groups. Additionally, an iodine atom or various aryl groups were introduced at the C-5 position of the 2-aminopyrimidine ring. These compounds as well as some of their synthetic intermediates were tested for their kinase inhibitory potencies and for their in vitro antiproliferative activities. We found that this series of compounds is particularly interesting in the development of new inhibitors of DYRK1A and CLK1 kinases. The most effective compounds toward these two kinase families are the 6- and 7-bromo derivatives 30, 33, and 34 that showed more than 45-fold selectivity toward DYRK1A/CLK1 kinases over the other kinases tested. Meridianin derivatives could thus be developed toward potent and selective inhibitors of key RNA splicing regulators and potential therapeutic agents.

Full text of DOI:10.1021/jm200464w

ARTICLE
pubs.acs.org/jmc
Synthesis, Protein Kinase Inhibitory Potencies, and in Vitro
Antiproliferative Activities of Meridianin Derivatives
Francis Giraud,‡,§ Georges Alves,||,^ Eric Debiton,# Lionel Nauton,‡,§ Vincent Thꢀery,‡,§ Emilie Durieu,3
Yoan Ferandin,3 Olivier Lozach,3 Laurent Meijer,*,3 Fabrice Anizon,‡,§ Elisabeth Pereira,‡,§ and
Pascale Moreau*,‡,§
‡Clermont Universitꢀe, Universitꢀe Blaise Pascal, Laboratoire SEESIB, BP 10448, F-63000 Clermont-Ferrand, France
§CNRS, UMR 6504, Laboratoire SEESIB, F-63177 Aubiꢁere, France,
Clermont Universitꢀe, Universitꢀe Blaise Pascal, GReD, BP 10448, F-63000 Clermont-Ferrand, France
^CNRS, UMR 6247, GReD, F-63001 Clermont-Ferrand, France
#Inserm, UMR 990, F-63005 Clermont-Ferrand, France
3CNRS, ‘Protein Phosphorylation & Human Disease’ Group, Station Biologique, BP 74, 29682 Roscoff, France
ABSTRACT: The synthesis of new meridianin derivatives is described. The
indolic ring system was substituted at the C-4 to C-7 positions either by a
bromine atom or by nitro or amino groups. Additionally, an iodine atom or
various aryl groups were introduced at the C-5 position of the 2-aminopyrimi-
dine ring. These compounds as well as some of their synthetic intermediates
were tested for their kinase inhibitory potencies and for their in vitro anti-
proliferative activities. We found that this series of compounds is particularly
interesting in the development of new inhibitors of DYRK1A and CLK1 kinases.
The most effective compounds toward these two kinase families are the 6- and
7-bromo derivatives 30, 33, and 34 that showed more than 45-fold selectivity
toward DYRK1A/CLK1 kinases over the other kinases tested. Meridianin
derivatives could thus be developed toward potent and selective inhibitors of
key RNA splicing regulators and potential therapeutic agents.
other hand, when compounds 8ꢀ14 (Figure 1) were tested toward
1. INTRODUCTION
the following kinases: CDK5/p25, casein kinase 1 (CK1)δ/ε,
Meridianins are marine alkaloids isolated from the south
Atlantic tunicate Aplidium meridianum.1 Meridianins AꢀG
glycogen synthase kinase 3 (GSK-3)R/β, dual-specificity
(1ꢀ7)1ꢀ4 (Figure 1) were characterized as indole derivatives
tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A),
and extracellular signal regulated kinase (Erk2), they showed
submicromolar inhibition potencies toward some of the tested
substituted in the C-3 position by a 2-aminopyrimidine ring and
were found to inhibit various protein kinases3 and to display
kinases.10 More particularly, some of them, including compounds
12ꢀ13, demonstrated interesting inhibition potencies toward
DYRK1A kinase which is known to be involved in Alzheimer’s
disease11 and Down syndrome.12 These promising results led us to
expand our effort in the synthesis of new diversely substituted
meridianin derivatives as potential kinase inhibitors. More particu-
larly, we focused on analogues of compound 10, which showed the
best antiproliferative activities toward PA1 cells and compound 8,
which was the only one to be active toward the five tested kinases
(CK1, GSK-3, DYRK1A, Erk2, CDK5/p25) and to exhibit an
interesting antiproliferative activity toward PA1 cells. More-
over, we were also interested in the preparation of analogues of
compounds 12 and 14 because they were active toward four of
the five tested kinases (CK1, GSK-3, DYRK1A, and Erk2) and
antitumor activity.5 The closely related meriolins, a family of
synthetic 7-azaindole meridianin analogues, were also described
for their potent kinase inhibitory activities and antiproliferative
properties.6,7
Our interest in the synthesis of new kinase inhibitors led us to
prepare meridianin analogues 8ꢀ14 (Figure 1) diversely sub-
stituted by aryl groups on the 2-aminopyrimidine ring.8ꢀ10
The meridianin derivatives 8, 9, 10, and 11 were previously
tested toward a panel of 9 protein kinases (kinase domain
receptor (KDR), insulin growth factor receptor (IGF-1R),
c-Met, RET, sarcoma kinase (c-Src), abelson leukemia oncogen
cellular homology (c-Abl), cAMP-dependent protein kinase
(PKA), cyclin-dependent protein kinase 2 (CDK2)/cyclin A
and HER-1 and their in vitro antiproliferative activities were
examined toward a human fibroblast primary culture and two
human solid cancer cell lines (MCF-7 and PA 1).9 Despite weak
kinase inhibitory potencies, compounds 8, 9, 10, and 11 exhibited
high in vitro antiproliferative activities toward PA 1 cells. On the
Received: January 26, 2011
Published: May 30, 2011
r
2011 American Chemical Society
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ARTICLE
Figure 1. Meridianins AꢀG (1ꢀ7) and derivatives 8ꢀ14 described by our group.
have shown modest antiproliferative activities with IC50 values in
the 28ꢀ38 μM range.
Scheme 1. Synthesis of Compounds 27ꢀ30 (Yields Are In-
dicated under Brackets)a
As shown in Figure 1, meridianins AꢀF (1ꢀ6) are substituted
at C-4 to C-7 positions of the indolic ring system either by a
hydroxyl and/or a bromine atom. It has been demonstrated that
the substitutions on these positions could have a major influence
on the biological potencies of meridianins.3 More particularly,
when tested toward CDKs, PKA and CK1, meridianin E,
substituted by a hydroxyl group at the C-4 and a bromine atom
at the C-7 positions was the most active, whereas the best
inhibitory potency against GSK-3 was found for meridianin B
bearing a hydroxyl group at the C-4 and a bromine atom at the
C-6 positions. For PKG, the best potency was obtained for
meridianin C substituted at the C-5 position by a bromine atom.
Thus, in this article, we describe the synthesis of brominated
analogues of compounds 8, 10, 12, and 14. The indolic nucleus of
these new compounds was substituted by a bromine atom at the
C-4, C-5, C-6, or C-7 positions. Moreover, to enlarge the
structureꢀactivity relationship study reported in this paper, the
C-4 to C-7 positions of the indolic ring were also substituted by
nitro or amino groups. These compounds as well as some of their
synthetic intermediates were tested for their kinase inhibitory
potencies toward five kinases (CDK5/p25, CK1δ/ε, GSK-3R/β,
DYRK1A, and cdc2-like kinase 1 (CLK1)) and for their in vitro
antiproliferative activities toward a human fibroblast primary culture,
a murine hippocampal cell line (HT22) and six human cancer cell
lines (PC3, DU145, PA1, SH-SY5Y, IMR-32, and MCF7).
a Reagents and conditions: (a) AcCl, SnCl4, toluene; (b) DMAP,
DIPEA, TsCl, CH2Cl2, or NaH, TsCl, DMF; (c) DMF-DMA, DMF,
or DMF/di-tert-butylacetal, DMF; (d) guanidine-HCl, K2CO3, 2-meth-
oxyethanol.
DMF/dimethylformamide-dimethylacetal (DMF-DMA) in 45%
yield was then treated with guanidine hydrochloride in 2-meth-
oxyethanol in the presence of potassium carbonate to give the
corresponding 2-aminopyrimidine derivative 29 in 47% yield.
The preparation of the corresponding 4-, 5-, and 7-bromo
derivatives was undertaken using a similar synthetic pathway
but without any purification of the enaminone intermediates 23,
24, and 26. Compounds 27 and 30 were then obtained from 19
and 22 in 48% and 27% yields, respectively, using the same
conditions as those described for compound 29. Compound 28
was obtained in 48% yield from 20 using DMF/di-tert-butylacetal
instead of DMF-DMA.
Bromo-meridianins 27ꢀ30 were then iodinated with iodine in
dimethylsulfoxide10 to give the corresponding compounds 31ꢀ
34 in 66% to 86% yield. Suzuki cross-coupling was then per-
formed using various commercially available arylboronic acids,
tetrakis(triphenylphosphine)palladium, and sodium carbonate
2. SYNTHESIS AND KINASE INHIBITORY ACTIVITY
The construction of the 2-aminopyrimidine ring was based on
the well-known Bredereck synthetic approach (Scheme 1).5,13ꢀ15
First, the C-3 position of commercially available 4-, 5-, 6-, and
7-bromoindoles was acetylated using acetyl chloride and tin(IV)
chloride in toluene to give derivatives 15ꢀ18 in 83% to 96%
yields. Then, protection of the indolic nitrogen was performed by
reaction with tosyl chloride in the presence of 4-(dimethylamino)-
pyridine (DMAP) and diisopropylethylamine (DIPEA) in
methylene chloride, leading to the formation of compounds 19ꢀ
21 in 98% to 99% yields. The same reaction conditions applied to
the 7-bromo analogue 18 gave compound 22 in only 27% yield.
Therefore, other reaction conditions were tested and, finally,
compound 22 was prepared in 72% yield using tosyl chloride
and sodium hydride in N,N-dimethylformamide (DMF). The
enaminone derivative 25 prepared by treatment of 21 with
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Scheme 2. Synthesis of Compounds 31ꢀ50 (Yields Are Indicated under Brackets)a
a Reagents and conditions: (a) I2, DMSO; (b) ArB(OH)2, Pd(PPh3)4, Na2CO3, H2O, EtOH, toluene.
in a H2O/EtOH/toluene medium.8 Thus, compounds 35ꢀ50
were obtained in 34ꢀ92% isolated yields (Scheme 2).
Table 1. Kinases Inhibitory Potencies (IC50 in μM) for
Compounds 27ꢀ50
The Suzuki cross-coupling products were obtained in either
modest or good chemical yields depending on both the position
of the bromine atom on the indolic moiety and the nature of the
boronic acid used. With all the tested boronic acids, the lowest
yields were observed with the 4-bromo derivatives. Moreover,
yields were usually better with the 3-methoxyphenylboronic acid.
This is probably due to the presence of an electron-donating
substituent on the phenyl moiety.
kinase inhibition, IC50 in μM
compd
CDK5/p25
CK1δ/ε
GSK-3R/β
CLK1
DYRK1A
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
10
5.3
>10
1.3
7.2
>10
>10
2.1
1.1
4
1.4
0.85
0.33
0.13
0.068
1.45
0.33
0.034
0.039
2.1
1.2
7.3
0.22
0.12
0.065
2.3
1.2
3.8
3.1
4.2
The kinase inhibitory potencies of compounds 31ꢀ50 as well
as those of their synthetic intermediates 27ꢀ30 were evaluated
as IC50 values toward five protein kinases (CDK5/p25, CK1δ/ε,
GSK-3R/β, DYRK1A, and CLK1) (Table 1).16
The best active compounds in this series were bromo-iodo
derivatives 33, 34, and noniodinated 7-bromo analogue 30.
These 6- and 7-bromo analogues were particularly active toward
DYRK1A and CLK1, with IC50 values in the nanomolar range
(DYRK1A IC50, 0.034ꢀ0.068 μM; CLK1, 0.032ꢀ0.065 μM). In
comparison, the corresponding 4- and 5-bromo analogues 27,
28, 31, and 32 were less active. Moreover, some of these bromo-
iodo derivatives 32 and 33 have shown submicromolar inhibition
of CDK5/p25 (compound 33) and CK1δ/ε (compounds 32
and 33).
Regarding the substitution of the 2-aminopyrimidine ring by
aryl groups, the 4-trifluoromethylphenyl series (compounds 43ꢀ
46) have shown the weakest inhibitory potencies without any sub-
micromolar IC50 values toward the tested kinases. 4-Acetylphenyl
(35ꢀ38), 4-acetamidophenyl (39ꢀ42), 4-trifluoromethylphe-
nyl (43ꢀ46), and 3-methoxyphenyl (47ꢀ50) derivatives have
only shown submicromolar IC50 values toward GSK-3R/β and
DYRK1A. In all cases, the general pattern was that 7-bromo
substituted compounds displayed both the highest inhibitory
activity and the strongest selectivity toward DYRK1A and CLK1.
According to these results, this series of compounds is
particularly interesting in the development of new inhibitors of
DYRK1A and CLK1 kinases, two families of kinases involved in
alternative mRNA splicing and neurodegenerative pathologies
such as Alzheimer’s disease. The most effective compounds are the
6- and 7-bromo derivatives 30, 33, and 34 that showed noteworthy
inhibitory potencies toward these kinases. Moreover, compounds
30, 33, and 34 demonstrated more than 45-fold selectivity
toward DYRK1A/CLK1 kinase versus the other kinases tested.
These results prompted us to prepare new analogues bearing
an iodine atom at the C-5 position of the aminopyrimidine ring.
>10
1.8
1.3
0.77
0.7
0.18
0.032
0.042
1.5
0.68
2.1
1.6
7
>10
>10
>10
>10
>10
2.1
1.5
0.68
2.0
4.5
>10
0.4
1.6
2.2
1
>10
>10
>10
>10
6.8
1.6
8.45
>10
0.29
>10
4.15
3.1
1.8
0.38
>10
0.75
0.53
0.087
1.3
>10
7.5
8.1
>10
>10
>10
>10
>10
>10
2.2
0.19
1.85
>10
>10
1.25
0.81
5.65
8.2
4.7
>10
>10
>10
6
2
>10
>10
1.5
6.1
8.5
1.4
1.2
4.5
>10
1.3
7.6
2.5
>10
>10
3.2
2.3
4.2
0.87
0.85
Thus, we synthesized compounds 70ꢀ73 and 75ꢀ78 substi-
tuted at the C-4 to C-7 positions of the indolic moiety by nitro or
amino groups. Nonsubstituted compound 74 was also prepared
in order to evaluate the influence of indole substitution on the
biological potencies of the tested compounds. The 4- to 7-nitro
and amino derivatives (70, 73, 75, and 78) as well as the
nonsubstituted compound 74 were prepared using a similar
synthetic pathway as the one described above for the bromo
analogues (Scheme 3). First, the C-3 position of commercially
available 4-, and 7-nitroindoles was acylated using acetyl chloride
and ionic liquid generated from 1-ethyl-3-methylimidazolium
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Scheme 3. Synthesis of Compounds 51ꢀ78 (Yields Are Indicated under Brackets)a
a Reagents and conditions: (a) method A: EmimCl, AlCl3, AcCl; method B: Me2AlCl, AcCl, CH2Cl2; (b) TsCl, NaH, DMF; (c) Ac2O, AlCl3, CH2Cl2;
(d) DMF-DMA, DMF; (e) guanidine-HCl, K2CO3, 2-methoxyethanol; (f) I2, DMSO; (g) Fe, NH4Cl, iPrOH, H2O.
chloride (Emim Cl) and aluminum chloride17 or dimethylalu-
minium chloride18 to give acyl derivative 51 in 97% yield,
whereas compound 52 was not purified before the following
protection step. The tosylation of the indolic nitrogen of
compounds 51, 52, and commercially available 3-acetylindole
was carried out with tosyl chloride in the presence of NaH in
DMF to give compounds 55, 58, and 59 in 87%, 59% (2 steps),
and 89% yields, respectively.
Table 2. Kinases Inhibitory Potencies (IC50 in μM) for
Compounds 67ꢀ78
kinase inhibition, IC50 in μM
compd
CDK5/p25
CK1δ/ε
GSK-3R/β
CLK1
DYRK1A
67
68
69
70
71
72
73
74
75
76
77
78
>10
6.3
6.3
>10
>10
>10
>10
0.66
2.6
0.38
0.07
0.53
7.1
1.1
10
0.085
0.7
Regarding the synthesis of 5- and 6-nitro/amino derivatives,
the two first steps were inverted. Thus, the 5- and 6-nitroindoles
were previously tosylated in the presence of tosyl chloride and
sodium hydride before acylation with acetic anhydride and alumi-
num chloride19 to give compounds 56 and 57 in good yields.
Using a one-pot protocol, the corresponding enaminone
derivatives 60ꢀ64 prepared by treatment of the previous acy-
lated indoles with DMF-DMA were then treated with guanidine
hydrochloride in 2-methoxyethanol in the presence of potassium
carbonate to give the corresponding 2-aminopyrimidine deriva-
tive 65ꢀ69. Finally, treatment of 65ꢀ69 with iodine in di-
methylsulfoxide led to the formation of the corresponding
iodinated analogues 70ꢀ74 in 20% to 80% yields after one step
(72 and 73 from 67 and 68) or three steps (70, 71, and 74 from
55, 56, and 59). Finally, the reduction of the nitro group of com-
pounds 70ꢀ73 was performed in mild conditions using Fe powder
and NH4Cl.20 This method afforded the corresponding 4-, 5-, 6-,
and 7-amino analogues 75ꢀ78 in good yields (88 to 99%).
The kinase inhibitory potencies of compounds 67ꢀ78 were
evaluated as IC50 values toward five protein kinases (CDK5/p25,
CK1δ/ε, GSK-3R/β, DYRK1A, and CLK1) (Table 2). As in the
5
6.2
>10
1.6
>10
0.42
1
9.1
0.12
0.067
0.041
0.03
1
0.25
0.095
0.46
0.066
2.8
1.6
>10
1.4
>10
0.9
>10
3.7
>10
>10
6.2
>10
0.27
0.51
1.1
>10
>10
4.2
0.61
0.08
0.026
2.3
0.22
0.11
0.66
4.4
case of previously discussed brominated analogues, the most
sensitive kinases were DYRK1A and CLK1. The most active
compounds in this series were derivatives 68, 72, and 74 with
high inhibitory potencies toward both DYRK1A and CLK1
kinases (IC50, 0.03ꢀ0.095 μM). Moreover, compounds 73, 77,
and 78 that considerably inhibited CLK1 with IC50 in the range
of 0.026ꢀ0.08 μM only showed moderate inhibitory potencies
toward DYRK1A (0.11ꢀ0.46 μM), indicating that the inhibition
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Figure 2. Docking models of compounds 30 (yellow), 33 (cyan), and 34 (magenta) into the ATP binding site of DYRK1A (left) and CLK1 (right).
of these two kinases can be disconnected to some extent. These
results indicated that in this series (iodinated or not on the
aminopyrimidine ring), the introduction of a nitro/amino group
in the 4- or 5- position of the indolic nucleus is detrimental to
achieve a potent DYRK1A/CLK1 inhibition. On the contrary,
the introduction of a nitro group in the 6- or 7- position has
led to potent CLK1 and DYRK1A inhibitors. Moreover, the
6- and 7-amino derivatives have also shown good potencies
toward CLK1.
As in the brominated series, the 4- and 5-substitued derivatives
were less active than their 6- and 7-counterparts. The fact that
compounds 30, 33, 34, 68, 72, and 74 highly inhibited CLK1 and
DYRK1A prompted us to examine the relationship between
these two kinases that are both members of the CMCG branch of
the kinome. A sequence comparison between these two kinases
has pointed out 32.8% of homology.21,22 Regarding the ATP
binding site, a 70.4% of sequence identity was determined from
ATP docking experiments (see below). This was achieved by
sequence alignment of protein residues located at a distance e5 Å
around ATP atoms. Thus molecular modeling experiments were
performed to understand the molecular interactions involved
between this series of potent inhibitors and CLK1/DYRK1A
kinase active sites.
3. MOLECULAR MODELING EXPERIMENTS
The putative binding mode between the ATP binding site of
DYRK1A and the most active compounds of these series 30, 33,
and 34 was examined by molecular modeling experiments using a
model based on the 2wo6 DYRK1A structure available in the
Protein Data Bank (PDB).23 Hydrogen atoms were added for all
amino acid residues, the preferential position of hydrogen atoms
was determined by minimization, and the water molecules were
removed from the structure. The results obtained in the docking
experiments were sorted out, taking into account the preferred
ligand conformation. The selected structures obtained in these
docking assays were then minimized. As shown in Figure 2, the
meridianin derivatives 30, 33, and 34 adopt a similar binding
mode. The aminopyrimidine moiety is oriented toward the
bottom of the pocket making two hydrogen bonds with the
ATP binding site. Thus, the amino group is H-bonded with
Glu239 backbone carbonyl and the N-1 atom is H-bonded with
Leu241 backbone NH (Figure 2A). Moreover, the heteroaro-
matic scaffold of compounds 30, 33, and 34 is strongly associated
to the protein by hydrophobic interactions with Ile165, Val173,
Ala186, Leu241, Leu294, and Val306 side chains (Figure 2C).
The same approach was also used to propose a probable binding
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Figure 3. Docking models of ATP into the ATP binding site of DYRK1A (left) and CLK1 (right).
mode of meridianin derivatives 30, 33, and 34 with the CLK1
ATP binding site by using a X-ray cocrystal structure of CLK1
(PDB code 1z57).24 Again, the three meridianin derivatives
adopt the same binding mode. In this case, the amino group of
the aminopyrimidine moiety is H-bonded with Glu242 backbone
carbonyl and the N-1 atom is H-bonded with Leu244 backbone
NH (Figure 2B). For this protein kinase, the hydrophobic
interactions with compounds 30, 33, and 34 involve residue side
chains of Leu167, Val175, Ala189, Leu244, Leu295, and Val324
(Figure 2D).
In both cases, the interaction between the meridianin deriva-
tives 30, 33, and 34 and the ATP binding site of the kinase is
driven by the H-bonding of the aminopyrimidine moiety and the
enzyme as described above.
On the other hand, the orientation of the indolic ring system
differs from a complex model to another. The preferential
positioning of the indole moiety is directed by hydrophobic
interactions (see above) and steric effects due to the presence of a
bromine atom in the 6- or 7-position and/or the one of an iodine
atom at the 5-position of the aminopyrimidine ring.
4. IN VITRO ANTIPROLIFERATIVE ACTIVITY
As some of the kinases inhibited by compounds 27ꢀ50 and
67ꢀ78 are involved in the cellular proliferation, the antiproli-
ferative activities of these compounds were also evaluated
(Table 3). In vitro antiproliferative activities of compounds 27ꢀ
50 and 67ꢀ78 were tested toward a human fibroblast primary
culture, a murine hippocampal cell line (HT22), and six cancer
cell lines: PC3, DU145 (human prostate cancer), PA1 (human
ovarian teratocarcinoma), MCF7 (human breast cancer), and
SH-SY5Y, IMR-32 (human neuroblastoma). The antiprolifera-
tive effect of the tested drug was assessed by the resazurin
reduction test25 for fibroblast, PA1, PC3, and DU145 cell lines.
The MTS reduction assay was used for SH-SY5Y, IMR-32, and
HT22 cells. Under the conditions used, the most active com-
pounds were the 6- and 7-bromoderivatives 33, 37, and 38 with
submicromolar IC50 values for some of the cells line tested.
Moreover, compounds 34, 42, 70, 72, 73, 74, and 78 also showed
modest antiproliferative activities, with IC50 values in the micro-
molar range toward some of the cell lines tested.
To compare the ATP pocket binding mode of meridianin
derivatives 30, 33, and 34 with the one of ATP, docking experi-
ments were performed with ATP. As shown in Figure 3, the
adenine moiety of ATP interacted with DYRK1A and CLK1 in a
similar manner to that observed for meridianin derivatives. Two
hydrogen bonds were formed between the adenine moiety and
DYRK1A or CLK1. Specifically, the amino group was H-bonded
to the backbone carbonyl of Glu239 (for DYRK1A) or Glu242
(for CLK1) and the N1 atom was H-bonded with the backbone
NH of Leu241 (for DYRK1A) or Leu244 (for CLK1).
The morphological differences between both ATP binding
sites were examinated in more detail in order to explain the
higher activity toward CLK1 over DYRK1A observed for com-
pounds 73, 77, and 78. As shown in Figure 4, the aminopyr-
imidine moiety of meridianin derivatives 73, 77, and 78 adopt the
same binding mode with the ATP binding site of DYRK1A/
CLK1 than the one described above for compounds 30, 33, and
34. Unfortunately, taking into account the result of our molecular
modeling studies, it was not possible to explain the best inhibitory
potencies of compounds 73, 77, and 78 toward CLK1. This could
be due to diffences existing between the water molecule networks
in the ATP binding site of these two kinases. However, to validate
this hypothesis, the resolution of cocrystal structures of this series
of compounds in complex with CLK1/DYRK1A kinases will
be needed.
5. CONCLUSION
In conclusion, this work reports a structureꢀactivity relation-
ship study of new meridianin derivatives as kinase inhibitors and/
or antiproliferative agents. The synthetic approach reported in
this study allowed the substitution of the 4-, 5-, 6-, or 7-position
of the indolic moiety by a bromine atom or nitro or amino groups.
For this series of compounds, the most interesting inhibitory
potencies were observed toward DYRK1A and CLK1 kinases.
The results of this structureꢀactivity relationship study have
demonstrated that potent DYRK1A/CLK1 inhibitions were
observed for compounds substituted at the 6- or 7-position of
the indolic moiety by a bromine atom or a nitro or an amino
group. In addition, compounds 27ꢀ50 and 67ꢀ78 were evaluated
for their in vitro antiproliferative potencies toward six different
cancer cell lines, a fibroblast primary culture, and a murine hippo-
campal cell line. IC50 values in the micromolar/submicromolar
range were determined for compounds 33, 34, 37, 38, 42, 70,
72ꢀ74, and 78 toward the cell line tested. As there is no direct
correlation between the measured kinase inhibitory potencies
and the antiproliferative activities of these compounds, there must
be other biological target(s) involved in the antiproliferative effects
of this series. As is almost always seen with kinase inhibitors (and
probably also with any enzyme inhibitor), the efficacy on isolated
enzyme is higher than that on cells. This is likely the consequence
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Figure 4. Docking models of compounds 73 (orange), 77 (red), and 78 (blue) into the ATP binding site of DYRK1A (left) and CLK1 (right).
of the combination of several factors including interaction with
other targets within the cell (multitarget effects), nonspecific
binding to cellular proteins (ionic or hydrophobic interactions),
intracellular metabolism and possible inactivation, limitations in
the passage across biological membranes (plasma membrane,
nuclear envelope), and high intracellular ATP concentration. A
more precise evaluation of the effects of CLK/DYRK inhibition
by our inhibitors would require the generation of phospho-
specific antibodies that cross-react with sites that are specifically
and uniquely phosphorylated by CLK/DYRK. We are currently
attempting various approaches to design, generate, and validate
these tools. Co-crystallization assays between compounds 30, 33,
and 34 and DYRK1A and CLK1 will be undertaken to investigate
the mode of interaction of these compounds within the ATP
binding site of these kinases. This approach would provide
valuable information and facilitate further development of the
structure based design of new DYRK1A and/or CLK1 kinase
inhibitors in this series.
N0,N0-tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT), 25 mM
Tris-HCl pH 7.5, 50 μg heparin/mL.
Buffer B. β-Glycerophosphate (60 mM), 15 mM p-nitrophenylpho-
sphate, 25 mM 3-(N-morpholino)propanesulfonic acid (Mops) (pH
7.2), 5 mM EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM sodium vanadate,
1 mM phenylphosphate.
Kinase Preparations and Assays. Kinase activities were assayed in
buffer A or B, at 30 ꢀC, at a final adenosine triphosphate (ATP) concentra-
tion of 15 μM. 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 dimethylsulfoxide (DMSO).
CDK5/p25. (Human, recombinant) was prepared as previously
described.26,27 Its kinase activity was assayed in buffer B, with 1 mg of
histone H1/mL, in the presence of 15 μM [γ-33P] ATP (3000 Ci/
mmol; 10 mCi/mL) in a final volume of 30 μL. After 30 min of incuba-
tion at 30 ꢀC, 25 μL aliquots of supernatant were spotted onto 2.5 cm ꢁ
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 of phosphoric acid/L of water. The wet filters were counted in
the presence of 1 mL ACS (Amersham) scintillation fluid.
GSK-3R/β. (Porcine brain, native) was assayed, as described for
CDK5/p25 but in buffer A and using a GSK-3 specific substrate (GS-
1: YRRAAVPPSPSLSRHSSPHQSpEDEEE) (pS stands for phosphory-
lated serine).28 GS-1 was synthesized by Millegen (Labege, France).
6. EXPERIMENTAL SECTION
6.1. In Vitro Kinase Inhibition Assays. Buffers. Buffer A.
MgCl2 (10 mM), 1 mM ethylene glycol-bis(2-aminoethylether)-N,N,
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Table 3. Antiproliferative Activity (IC50 in μM) of Compounds 27ꢀ50 and 67ꢀ78 (nd: Not Determined)
antiproliferative activity, IC50 in μM
compd
SHSY-5Y
IMR-32
MCF7
HT22
PA 1
nd
PC3
DU145
nd
fibro
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
67
68
69
70
71
72
73
74
75
76
77
78
>25
>25
>25
>25
>10
23
>25
>25
>25
25
>25
24
>25
9.3
nd
nd
nd
nd
nd
nd
nd
nd
>25
>25
>10
>25
5.2
>25
>25
>10
8
nd
nd
nd
nd
nd
nd
>10
13
>10
>10
nd
nd
>10
>10
nd
nd
3.2
3.8
5.1
<1
2.9 ( 0.3
15 ( 1
>10
<1
3.5 ( 0.3
10 ( 2
nd
8
7.3
17
12
2.0 ( 0.1
>10
4.2 ( 0.3
nd
>10
>25
0.4
>10
13
>10
4.2
>10
10
>10
>10
nd
nd
0.33
>10
>10
>10
>10
25
1
2.5
<0.6
<0.6
<0.6
<0.6
>10
>10
>10
>10
25
>10
>10
>10
>10
>25
>10
>10
>10
>10
>10
>25
>25
>10
>25
>25
>25
>10
>25
>25
>10
17
>10
>10
>10
>10
>25
>10
>10
>10
>10
>10
18
0.8 ( 0.2
>10
3.6 ( 0.4
>10
5.7 ( 0.8
nd
18.4 ( 0.8
nd
>10
>10
nd
nd
12 ( 2
3.0 ( 0.3
>10
14 ( 2
5 ( 3
>10
20 ( 1
13 ( 1
nd
24 ( 2
8 ( 1
nd
>10
>10
>10
>10
>10
>25
>25
>10
>25
>25
>25
>10
>25
12
>10
>10
>10
>10
>10
20
>10
>10
nd
nd
>10
>10
nd
nd
17 ( 3
>10
8 ( 2
>10
18 ( 1
nd
14 ( 2
nd
>10
>10
nd
nd
18
16
>10
>10
nd
nd
>10
>25
>25
>25
>10
10
>10
>25
>25
>25
>10
20
13 ( 3
nd
29 ( 3
nd
33 ( 4
nd
31 ( 3
nd
nd
nd
nd
nd
nd
nd
nd
nd
3.3 ( 0.7
8.4 ( 0.1
2.0 ( 0.5
3.2 ( 0.6
3.3 ( 0.1
18 ( 0
11 ( 1
13 ( 4
2.5 ( 0.2
7 ( 0
26 ( 3
11 ( 3
6 ( 2
10.6 ( 0.8
>50
5.4 ( 0.2
13.3 ( 0.4
6.3 ( 0.9
5.2 ( 0.4
5 ( 2
>50
5 ( 2
19 ( 6
13 ( 2
4.7 ( 0.6
8 ( 4
>50
10
18
>10
7.5
>10
10
>10
7.3
>10
>10
>10
9
>10
>10
>10
4
>10
>10
>10
20
>10
>10
>10
12
48 ( 6
47 ( 15
14 ( 3
40 ( 6
26 ( 6
6.1 ( 0.3
44 ( 3
36 ( 8
10.6 ( 0.6
CK1δ/ε. (Porcine brain, native) was assayed as described for CDK5/p25
but using the CK1-specific peptide substrate RRKHAAIGpSAYSITA,29
obtained from Millegen (Labege, France).
DYRK1A. (Rat, recombinant, expressed in E. coli as a glutathione
transferase (GST) fusion protein) was purified by affinity chromatog-
raphy on glutathione-agarose and assayed as described for CDK5/p25
using myelin basic protein (1 mg/mL) as a substrate.
CLK1. (Human, recombinant, expressed in E. coli as GST fusion
protein) was assayed in buffer A (þ 0.15 mg BSA/ml) with RS peptide
(GRSRSRSRSRSR) (1 μg/assay).
6.2. In Vitro Antiproliferative Assays. Cell Cultures. 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 of 100 mmol/L sodium
pyruvate, 5 mL of 100ꢁ nonessential amino acids and 2 mg of
gentamicin base. Cells were grown at 37 ꢀC in a humidified incubator
under an atmosphere containing 5% CO2.
Survival Assays. Cells were plated at a density of 5 ꢁ 103 cells in
150 μL culture 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 dimethylsulf-
oxide (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 the resazurin reduction test.
Resazurin Reduction Test. Plates were rinsed with 200 μL of PBS at
37 ꢀC and emptied by overturning on absorbent toweling. Then, 150 μL
of a 25 μg/mL 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, Labsystem)
using an excitation wavelength of 530 nm and an emission wavelength of
590 nm. Under the conditions used, fluorescence was proportional to
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the number of living cells in the well. The IC50, defined as the drug
concentration required to inhibit cell proliferation by 50%, was calcu-
lated from the curve of concentration-dependent survival percentage,
defined as fluorescence in experimental wells compared with fluores-
cence in control wells, after subtraction of the blank values.
Cell Lines and Culture Conditions. Cell Survival Quantification. SH-
SY5Y and HT22 cells were grown in DMEM medium from Invitrogen
(Cergy Pontoise, France), while IMR 32 was grown in RPMI (Invitrogen).
All media were supplemented with antibiotics (penicillinꢀstreptomycin)
from Lonza and 10% volume of fetal calf serum from Invitrogen. Cells
were cultured at 37 ꢀC with 5% CO2. Compound treatments were
performed on exponentially growing cultures at the indicated time and
concentrations. Control experiments were carried out also using appro-
priate dilutions of DMSO (maximum 1% DMSO). Cell viability was
determined by measuring the reduction of MTS as described in ref 30.
6.3. Chemistry. IR spectra were recorded on a Shimazu FTIR
obtained in 45% yield. Compounds 23, 24, and 26 were not purified
before the following cyclization step.
The spectroscopic data obtained for compound 25 were identical to
those described in literature.14
Compounds 27, 28, 29, and 30 were prepared according to a
procedure described by Fresneda et al.14 or minor modifications thereof
and were obtained in 48% (from 19), 48% (from 20), 47% and 27%
(from 22) yield, respectively.
The spectroscopic data obtained for compounds 27, 28, and 29 were
identical to those described in literature.1,14,19,33
Compounds 31ꢀ34 were prepared according to a procedure de-
scribed by Akue-Gedu et al.10 and Jovanovic et al.34 or minor modifica-
tions thereof.
Compounds 35ꢀ50 were prepared according to a procedure de-
scribed by Rossignol et al.8 or minor modifications thereof.
Compounds 51ꢀ52 were prepared according to a procedure de-
scribed by Yeung et al.17 and Okauchi et al.18 or minor modifications
thereof.
Compounds 53ꢀ55 and 58ꢀ59 were prepared according to proce-
dure described by Fresneda et al.14 or minor modifications thereof in
100%, 99%, 87%, 59% (2 steps), and 89% yield, respectively.
The spectroscopic data obtained for compound 59 were identical to
those described in literature.10
Compounds 56ꢀ57 were prepared according to procedure described
by Simon et al.19 or minor modifications thereof in 93% and 100% yield,
respectively.
84000S spectrometer; only structurally important peaks (ν) are pre-
h
sented in cmꢀ1. 1H (400 MHz) and 13C (100 MHz) NMR spectra were
obtained on a Bruker AC 400 spectrometer. Chemical shifts (δ) and
coupling constants (J) are given in ppm and Hz, respectively, using residual
solvent signals as reference. The following abbreviations are used: singlet
(s), doublet (d), triplet (t), quadruplet (q), doubled doublet (dd), broad
signal (br s), multiplet (m). HRMS spectra were recorded in electrospray
ionization mode (ESIþ) on a micro q-tof Micromass (3000 V) with an
internal lock mass (H3PO4) and an external lock mass (Leu-enkephalin).
Melting points were determined with a Reichert microscope appara-
tus and are uncorrected.
Compounds 60ꢀ64 were prepared according to a procedure de-
scribed by Fresneda et al.14 (DMF-DMA in DMF). Compound 63 was
obtained in 43% yield. Compounds 60, 61, 62, and 64 were not purified
before the following cyclization step.
Reactions were monitored by TLC using commercial plates of 0.25 mm
thickness silica gel on an aluminum support (60 F254 from Merck);
plates were visualized by UV fluorescence at 254 nm then revealed with a
vanillin solution. Retention factors (Rf) are given for such analyses. Chrom-
atographic purifications were performed on flash silica gel Geduran SI 60
(Merck) 0.040ꢀ0.063 mm column chromatography.
The spectroscopic data obtained for compound 64 were identical to
those described in literature.10
Compounds 65ꢀ69 were prepared according to a procedure de-
scribed by Fresneda et al.14 Compound 67 was obtained in 18% yield
(from 57). Compound 68 was obtained in 40% yield. Compounds 65,
66, and 69 were not purified before the following cyclization step.
The spectroscopic data obtained for compound 69 were identical to
those described in literature.10
Compounds 70ꢀ74 were prepared according to a procedure de-
scribed by Akue-Gedu et al.10 and Jovanovic et al.34 or minor modifica-
tions thereof. Compounds 70, 71, and 74 were obtained in 29%, 20%,
and 29% yield from 55, 56, and 59, respectively. Compounds 72 and 73
were obtained in 80% and 70% yield.
Purifications by preparative high performance liquid chromatography
(HPLC) were performed if necessary for tested compounds (compounds
34, 46, and 50) on a small amount using a Varian liquid chromatograph
(Varian Prep Star 218 equipped with a Varian Pro Star 335 photodiode
array detector) and a C18 Varian Dynamax Microsorb 60ꢀ8 column
(250 mm ꢁ 41.4 mm, 8 μm). Detection wavelength and flow rate was
indicated for each compound. Solvents were (A) water, 0.1% trifluoroacetic
acid (TFA); (B) acetonitrile and experiments were performed in an
isocratic mode.
The purity of tested compounds was established to be g95% by
HPLC analysis using a Dionex liquid chromatograph (TTC-100, 25 ꢀC;
P580; UVD340U) and a C18 Dionex Acclaim 120 column (4.6 mm ꢁ
250 mm, 5 μm, 120 Å). Detection wavelength was indicated for each
compound. Solvents were (A) water, 0.1% TFA; (B) acetonitrile; two
methods were used: method I, 95:5 A/B for 5 min then 95:5 A/B to 5:95
A/B in 20 min and then 5:95 A/B for 5 min; 0.5 mL/min; method II,
70:30 A/B in an isocratic mode.
Compounds 75ꢀ78 were prepared according to a procedure de-
scribed by Owa et al.20 or minor modifications thereof in 88% to
99% yield.
3-Acetyl-7-bromo-1H-indole (18). Compound 18 was obtained as a
white powder in 90% yield; mp 190ꢀ191 ꢀC; Rf 0.50 (cyclohexane/
EtOAc 50:50). 1H NMR (DMSO-d6): δ 2.48 (s, 3H, CH3), 7.12 (t, 1H,
J = 8.0 Hz), 7.43 (d, 1H, J = 8.0 Hz), 8.19 (d, 1H, J = 8.0 Hz), 8.35 (s,
1H), 12.15 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 27.3 (CH3), 104.5
(2C), 117.5 (C), 120.6 (CH), 123.0 (CH), 125.3 (CH), 126.9 (C),
135.1 (CH), 192.8 (CO). IR (ATR): 3200, 1631, 1432. HRMS: m/z
[M þ Na]þ calcd for C10H8 79BrNNaO, 259.9687; found, 259.9691.
N-[4-(Methylphenyl)sulfonyl]-3-acetyl-7-bromo-1H-indole (22).
Purification of the crude residue by flash chromatography (cyclohexane/
EtOAc 85:15) provided 22 as a white powder in 72% yield; mp 159ꢀ
160 ꢀC; Rf 0.50 (cyclohexane/EtOAc 75:25). 1H NMR (DMSO-d6): δ
2.40 (s, 3H, CH3), 2.64 (s, 3H, COCH3), 7.27 (t, 1H, J = 8.0 Hz), 7.46
(d, 2H, J = 8.0 Hz), 7.58 (dd, 1H, J1 = 8.0 Hz, J2 = 0.8 Hz), 7.89 (d, 2H,
Compounds 15ꢀ18 were prepared according to the procedure
published by MacKay et al.31 or minor modifications thereof in 96, 83,
86, and 90% yield respectively.
The spectroscopic data for compounds 15ꢀ17 were identical to
those described in literature.14,31
Compounds 19ꢀ21 were prepared according to procedure described
by MacKay et al.31 or minor modifications thereof in 99, 98, and 98%
yield, respectively, and following the procedure reported by Fresneda
et al.14 for compound 22 which was obtained in 72% yield.
The spectroscopic data obtained for compounds 19ꢀ21 were
identical to those described in literature.14,31,32
J = 8.0 Hz), 8.35 (dd, 1H, J1 = 8.0 Hz, J2 = 0.8 Hz), 8.91 (s, 1H). 13
C
Compounds 23, 25, and 26 were prepared according to a procedure
described by Fresneda et al.14 (DMF-DMA in DMF) or by Rossignol
et al.8 (DMF/di-tert-butylacetal in DMF) for 24. Compound 25 was
NMR (DMSO-d6): δ 21.0 (CH3), 27.9 (COCH3), 118.7 (C), 131.6
(CH), 126.2 (CH), 127.3 (2CH), 130.1 (2CH), 131.1 (C), 131.4 (CH),
132.9 (C), 135.6 (C), 138.3 (CH), 145.6 (2C), 193.6 (CO). IR (ATR):
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1672, 1365, 1169. HRMS: m/z [Mþ Na]þ calcd for C17H14 79BrNNaO3S,
413.9775; found, 413.9785.
5-Iodo-4-(5-bromo-1H-indol-3-yl)pyrimidin-2-amine (32). Purifica-
tion of the crude residue by flash chromatography (cyclohexane/EtOAc
50:50) provided 32 as a yellow powder in 86% yield; mp 203ꢀ205 ꢀC; Rf
0.55 (cyclohexane/EtOAc 50:50). 1H NMR (DMSO-d6): δ 6.80 (br s,
2H, NH2), 7.29 (dd, 1H, J1 = 8.4 Hz, J2 = 2.0 Hz), 7.43 (d, 1H, J = 8.4
Hz), 8.38 (d, 1H, J = 2.0 Hz), 8.45 (s, 1H), 8.51 (s, 1H), 11.84 (br s, 1H,
NH). 13C NMR (DMSO-d6): δ 74.8 (C), 113.1 (C), 113.3 (C), 113.8
(CH), 124.3 (CH), 124.7 (CH), 127.8 (C), 130.7 (CH), 134.6 (C),
161.7 (C), 162.1 (C), 166.1 (CH). IR (ATR): 3365, 3319, 3174, 1645,
1550, 1537, 1518, 1458. HRMS: m/z [M þ H]þ calcd for C12H979BrI
N4, 414.9055; found, 414.9070. HPLC (method I): purity >99%, λ =
250 nm, tR = 22.9 min.
5-Iodo-4-(6-bromo-1H-indol-3-yl)pyrimidin-2-amine (33). Purifica-
tion of the crude residue by flash chromatography (cyclohexane/EtOAc
50:50) provided 33 as a yellow powder in 71% yield; mp 209ꢀ210 ꢀC; Rf
0.70 (cyclohexane/EtOAc 40:60). 1H NMR (DMSO-d6): δ 6.75 (br s,
2H, NH2), 7.22 (dd, 1H, J1 = 8.6 Hz, J2 = 1.6 Hz), 7.65 (d, 1H, J = 1.6
Hz), 8.25 (d, 1H, J = 8.8 Hz), 8.45 (d, 1H, J = 2.8 Hz), 8.50 (s, 1H), 11.76
(br s, 1H, NH). 13C NMR (DMSO-d6): δ 74.8 (C), 113.9 (C), 114.4
(CH), 114.7 (C), 123.0 (CH), 124.3 (CH), 125.2 (C), 130.3 (CH),
136.8 (C), 161.7 (C), 162.0 (C), 165.8 (CH). IR (ATR): 3396, 3306,
3157, 1552, 1539, 1516, 1450. HRMS: m/z [M þ H]þ calcd for
C12H979BrI N4, 414.9055; found, 414.9069. HPLC (method I): purity
>99%, λ = 250 nm, tR = 23.2 min.
5-Iodo-4-(7-bromo-1H-indol-3-yl)pyrimidin-2-amine (34). Purifica-
tion of the crude residue by flash chromatography (cyclohexane/EtOAc
60:40) provided 34 as a yellow powder in 66% yield. Purification of a
small amount was performed by preparative HPLC (system: 25:75 A/B,
flow 40 mL/min, λ = 254 nm, tR = 5.6 min); mp 236ꢀ237 ꢀC; Rf 0.50
(cyclohexane/EtOAc 60:40). 1H NMR (DMSO-d6): δ 6.78 (br s, 2H,
NH2), 7.07 (t, 1H, J = 8.0 Hz), 7.41 (d, 1H, J = 7.2 Hz), 8.28 (d, 1H, J =
8.0 Hz), 8.40 (d, 1H, J = 3.2 Hz), 8.52 (s, 1H), 11.89 (br s, 1H, NH). 13C
NMR (DMSO-d6): δ 75.1 (C), 104.3 (C), 114.9 (C), 121.6 (CH), 121.9
(CH), 124.7 (CH), 127.8 (C), 130.1 (CH), 134.2 (C), 161.7 (C), 162.0
(C), 165.9 (CH). IR (ATR): 3485, 3300, 3171, 1551, 1539, 1515, 1176.
HRMS: m/z [M þ H]þ calcd for C12H979BrI N4, 414.9055; found,
414.9075. HPLC (method I): purity >99%, λ = 250 nm, tR = 23.4 min.
4-(4-Bromo-1H-indol-3-yl)-5-(4-acetylphenyl)pyrimidin-2-amine
(35). Purification of the crude residue by flash chromatography (EtOAc
then EtOAc/MeOH 95:5) provided 35 as a yellow powder in 34% yield;
mp 245ꢀ246 ꢀC; Rf 0.25 (cyclohexane/EtOAc 20:80). 1H NMR
(DMSO-d6): δ 2.46 (s, 3H, CH3), 6.77 (br s, 2H, NH2), 6.98 (t, 1H,
J = 7.6 Hz), 7.14 (d, 1H, J = 7.6 Hz), 7.28 (d, 2H, J = 8.4 Hz), 7.31 (d, 1H,
J = 2.8 Hz), 7.39 (d, 1H, J = 8.0 Hz), 7.72 (d, 2H, J = 8.4 Hz), 8.29 (s,
1H), 11.51 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 26.5 (CH3), 111.3
(CH), 112.9 (C), 114.3 (C), 122.6 (CH), 123.4 (CH), 123.9 (C), 124.9
(C), 127.8 (2CH), 128.9 (2CH), 134.4 (C), 136.7 (3C), 141.8 (CH),
145.5 (CH), 148.4 (C), 197.3 (CO). IR (ATR): 1652, 1601, 1569, 1464,
1183. HRMS: m/z [M þ H]þ calcd for C20H1679BrN4O, 407.0507; found,
407.0507. HPLC (method I): purity >96%, λ = 230 nm, tR = 21.8 min.
4-(5-Bromo-1H-indol-3-yl)-5-(4-acetylphenyl)pyrimidin-2-amine
(36). Purification of the crude residue by flash chromatography (cyclo-
hexane/EtOAc 30:70) provided 36 as a yellow powder in 57% yield; mp
186ꢀ187 ꢀC; Rf 0.45 (cyclohexane/EtOAc 20:80). 1H NMR (DMSO-
d6): δ 2.59 (s, 3H, CH3), 6.77ꢀ6.79 (m, 3H), 7.24 (dd, 1H, J1 = 8.4 Hz,
J2 = 1.6 Hz), 7.33 (d, 1H, J = 8.4 Hz), 7.42 (d, 2H, J = 8.4 Hz), 7.95 (d,
2H, J = 8.4 Hz), 8.09 (s, 1H), 8.38 (d, 1H, J = 1.6 Hz), 11.47 (br s, 1H,
NH). 13C NMR (DMSO-d6): δ 26.6 (CH3), 112.2 (C), 112.9 (C),
113.5 (CH), 120.0 (C), 124.2 (CH), 124.5 (CH), 127.6 (C), 128.6
(2CH), 129.5 (2CH), 130.1 (CH), 134.5 (C), 135.2 (C), 143.6 (C),
158.3 (C), 159.2 (CH), 162.6 (C), 197.3 (CO). IR (ATR): 1699, 1600,
1538, 1524, 1458. HRMS: m/z [M þ H]þ calcd for C20H1679BrN4O,
407.0507; found, 407.0500. HPLC (method I): purity >98%, λ =
230 nm, tR = 22.7 min.
4-(4-Bromo-1H-indol-3-yl)pyrimidin-2-amine (27). Purification of
the crude residue by flash chromatography (CH2Cl2/MeOH from 98:1
to 98:2) provided 27 as a pale-yellow powder in 48% yield from
compound 19; mp 218ꢀ221 ꢀC; Rf 0.50 (CH2Cl2/MeOH 90:10). 1H
NMR (DMSO-d6): δ 6.43 (br s, 2H, NH2), 6.74 (d, 1H, J = 4.8 Hz), 7.07
(t, 1H, J = 8.0 Hz), 7.29 (d, 1H, J = 7.2 Hz), 7.49 (d, 1H, J = 8.0 Hz), 7.70
(s, 1H), 8.16 (d, 1H, J = 4.8 Hz), 11.84 (br s, 1H, NH). 13C NMR
(DMSO-d6): δ 111.6 (CH), 112.0 (CH), 113.0 (C), 116.1 (C), 122.7
(CH), 123.7 (C), 124.5 (CH), 128.6 (CH), 137.8 (C), 156.4 (CH),
161.9 (C), 163.0 (C). IR (ATR): 3408, 3306, 3161, 1647, 1577, 1527,
1465. HRMS: m/z [M þ H]þ calcd for C12H1079BrN4, 289.0089;
found, 289.0087. HPLC (method I): purity >99%, λ = 228 nm, tR
=
20.4 min.
4-(5-Bromo-1H-indol-3-yl)pyrimidin-2-amine (28). Purification of
the crude residue by flash chromatography (CH2Cl2/MeOH from 98:2
to 90:10) provided 28 as a pale-yellow powder in 48% yield from
compound 20; mp 100ꢀ102 ꢀC; Rf 0.50 (CH2Cl2/MeOH 90:10). 1H
NMR (DMSO-d6): δ 6.49 (br s, 2H, NH2), 7.00 (d, 1H, J = 5.2 Hz), 7.28
(dd, 1H, J1 = 8.4 Hz, J2 = 2.0 Hz), 7.40 (d, 1H, J = 8.8 Hz), 8.10 (d, 1H,
J = 5.2 Hz), 8.25 (d, 1H, J = 2.4 Hz), 8.75 (d, 1H, J = 1.6 Hz), 11.85 (br s,
1H, NH). 13C NMR (DMSO-d6): δ 105.3 (CH), 113.1 (C), 113.5 (C),
114.0 (CH), 124.7 (C), 124.9 (C), 126.9 (CH), 130.8 (C), 135.7 (CH),
155.4 (C), 162.3 (CH), 163.0 (CH). IR (ATR): 3383, 3371, 3120, 1575,
1452, 1419. HRMS: m/z [M þ H]þ calcd for C12H1079BrN4, 289.0089;
found, 289.0092. HPLC (method I): purity >99%, λ = 228 nm, tR
=
20.9 min.
4-(6-Bromo-1H-indol-3-yl)pyrimidin-2-amine (29). Purification of
the crude residue by flash chromatography (CH2Cl2/MeOH from 98:1
to 98:2) provided 29 as a pale-yellow powder in 47% yield from
compound 25; mp 219ꢀ222 ꢀC; Rf 0.50 (CH2Cl2/MeOH 90:10). 1H
NMR (DMSO-d6): δ 6.46 (br s, 2H, NH2), 7.01 (d, 1H, J = 5.2 Hz), 7.24
(dd, 1H, J1 = 8.6 Hz, J2 = 1.6 Hz), 7.63 (d, 1H, J = 1.6 Hz), 8.12 (d, 1H,
J = 5.2 Hz), 8.23 (d, 1H, J = 2.8 Hz), 8.56 (d, 1H, J = 8.8 Hz), 11.78 (br s,
1H, NH). 13C NMR (DMSO-d6): δ 105.5 (CH), 114.0 (C), 114.6
(CH), 114.9 (C), 123.2 (CH), 124.4 (CH), 124.6 (C), 129.5 (CH),
138.1 (C), 157.5 (CH), 162.4 (CH), 163.7 (C). IR (ATR): 3433, 3321,
3171, 1660, 1570, 1516, 1448. HRMS: m/z [M þ H]þ calcd for
C12H1079BrN4, 289.0089; found, 289.0091. HPLC (method I): purity
>96%, λ = 228 nm, tR = 21.6 min.
4-(7-Bromo-1H-indol-3-yl)pyrimidin-2-amine (30). Purification of
the crude residue by flash chromatography (CH2Cl2/MeOH from 98:1
to 98:2) provided 30 as a yellow powder in 27% yield from compound
22; mp 218ꢀ219 ꢀC; Rf 0.10 (CH2Cl2/MeOH 98:2). 1H NMR
(DMSO-d6): δ 6.47 (br s, 2H, NH2), 7.05ꢀ7.09 (m, 2H), 7.40 (d,
1H, J = 8.0 Hz), 8.12 (d, 1H, J = 8.0 Hz), 8.25 (d, 1H, J = 8.0 Hz), 8.64 (d,
1H, J = 8.0 Hz), 11.87 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 104.3
(C), 105.4 (CH), 114.8 (C), 121.5 (CH), 121.9 (CH), 124.5 (CH),
127.0 (C), 129.1 (CH), 135.2 (C), 157.1 (CH), 162.0 (C), 163.4 (C).
IR (ATR): 1575, 1516, 1426, 1166. HRMS: m/z [M þ H]þ calcd for
C12H1079BrN4, 289.0089; found, 289.0087.
5-Iodo-4-(4-bromo-1H-indol-3-yl)pyrimidin-2-amine (31). Purifica-
tion of the crude residue by flash chromatography (cyclohexane/EtOAc
40:60) provided 31 as a yellow powder in 86% yield; mp 245ꢀ248 ꢀC; Rf
0.45 (cyclohexane/EtOAc 40:60). 1H NMR (DMSO-d6): δ 6.75 (br s,
2H, NH2), 7.05 (t, 1H, J =8.0 Hz), 7.21 (dd, 1H, J1 = 8.0 Hz, J2 = 0.8 Hz),
7.46 (dd, 1H, J1 = 8.0 Hz, J2 = 0.8 Hz), 7.53 (d, 1H, J = 2.8 Hz), 8.45 (s,
1H), 11.67 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 83.9 (C), 111.3
(CH), 113.0 (C), 117.1 (C), 122.6 (CH), 123.4 (CH), 124.3 (C), 126.6
(CH), 136.5 (C), 162.0 (C), 163.1 (CH), 165.4 (C). IR (ATR): 3399,
3308, 3173, 1645, 1558, 1548, 1512. HRMS: m/z [M þ H]þ calcd for
C12H979BrI N4, 414.9055; found, 414.9070. HPLC (method I): purity
>99%, λ = 250 nm, tR = 23.3 min.
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Journal of Medicinal Chemistry
ARTICLE
4-(6-Bromo-1H-indol-3-yl)-5-(4-acetylphenyl)pyrimidin-2-amine
(37). Purification of the crude residue by flash chromatography (cyclo-
hexane/EtOAc 50:50 then 40:60) provided 37 as a yellow powder in
58% yield; mp 249ꢀ250 ꢀC; Rf 0.30 (CH2Cl2/MeOH 95:5). 1H NMR
(DMSO-d6): δ 2.59 (s, 3H, CH3), 6.74 (br s, 2H, NH2), 6.76 (d, 1H, J =
2.8 Hz), 7.16 (dd, 1H, J1 = 8.8 Hz, J2 = 2.0 Hz), 7.43 (d, 2H, J = 8.4 Hz),
7.55 (d, 1H, J = 2.0 Hz), 7.96 (d, 2H, J = 8.4 Hz), 8.09 (s, 1H), 8.26 (d,
1H, J = 8.8 Hz), 11.38 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 26.6
(CH3), 113.6 (C), 113.9 (C), 116.8 (CH), 117.0 (C), 118.1 (CH),
120.8 (CH), 121.2 (C), 123.0 (C), 128.0 (C), 128.7 (CH), 129.6
(2CH), 131.3 (2CH), 132.5 (C), 132.6 (CH), 133.6 (C), 148.4 (C),
197.3 (CO). IR (ATR): 1677, 1598, 1532, 1457, 1136. HRMS: m/z
[M þ H]þ calcd for C20H1679BrN4O, 407.0507; found, 407.0502.
HPLC (method I): purity >95%, λ = 228 nm, tR = 22.8 min.
4-(7-Bromo-1H-indol-3-yl)-5-(4-acetylphenyl)pyrimidin-2-amine (38).
Purification of the crude residue by flash chromatography (cyclohexane/
EtOAc 50:50) provided 38 as a yellow powder in 75% yield; mp 220ꢀ
221 ꢀC; Rf 0.30 (cyclohexane/EtOAc 40:60). 1H NMR (DMSO-d6): δ
2.59 (s, 3H, CH3), 6.74 (d, 1H, J = 2.8 Hz), 6.77 (br s, 2H, NH2), 7.02 (t,
1H, J = 8.0 Hz), 7.36 (d, 1H, J = 8.0 Hz), 7.44 (d, 2H, J = 8.0 Hz), 7.96 (d,
2H, J = 8.0 Hz), 8.12 (s, 1H), 8.33 (d, 1H, J = 8.0 Hz), 11.51 (br s, 1H,
NH). 13C NMR (DMSO-d6): δ 26.7 (CH3), 104.2 (C), 113.8 (C), 117.4
(CH), 120.3 (C), 121.5 (CH), 121.9 (CH), 124.6 (CH), 127.7 (C), 128.7
(2CH), 129.6 (2CH), 130.6 (CH), 132.7 (C), 133.7 (C), 134.2 (C), 135.3
(C), 143.5 (C), 197.4 (CO). IR (ATR): 1682, 1578, 1536, 1455, 1141.
HRMS: m/z [M þ H]þ calcd for C20H1679BrN4O, 407.0507; found,
407.0508. HPLC (method I): purity >96%, λ = 228 nm, tR = 22.8 min.
4-[2-Amino-4-(4-bromo-1H-indol-3-yl)pyrimidin-5-yl]benzamide
(39). Purification of the crude residue by flash chromatography (EtOAc/
MeOH 95:5) provided 39 as a powder yellow in 41% yield; mp >
250 ꢀC; Rf 0.15 (EtOAc/MeOH 95:5). 1H NMR (DMSO-d6): δ 6.73
(br s, 2H, NH2), 6.98 (t, 1H, J = 8.0 Hz), 7.13 (d, 1H, J = 8.0 Hz), 7.19
(d, 2H, J = 8.0 Hz), 7.25 (br s, 1H, CONH2), 7.30 (d, 1H, J = 2.8 Hz),
7.38 (d, 1H, J = 8.0 Hz), 7.62 (d, 2H, J = 8.0 Hz), 7.81 (br s, 1H,
CONH2), 8.28 (s, 1H), 11.51 (br s, 1H, NH). 13C NMR (DMSO-d6): δ
111.2 (CH), 112.9 (C), 114.5 (C), 122.4 (CH), 123.2 (CH), 124.1 (C),
125.0 (C), 126.8 (CH), 126.9 (2CH), 128.5 (2CH), 131.7 (C), 136.6
(C), 140.7 (C), 157.5 (CH), 160.9 (C), 162.1 (C), 167.4 (CO). IR
(ATR): 3400ꢀ3100, 1670, 1579, 1549, 1513, 1476, 1194. HRMS: m/z
[M þ H]þ calcd for C19H1579BrN5O, 408.0460; found, 408.0467.
HPLC (method I): purity >99%, λ = 230 nm, tR = 19.6 min.
4-[2-Amino-4-(5-bromo-1H-indol-3-yl)pyrimidin-5-yl]benzamide
(40). Purification of the crude residue by flash chromatography
(EtOAc/MeOH 95:5) provided 40 as a powder in 51% yield; mp
174ꢀ175 ꢀC; Rf 0.55 (EtOAc/MeOH 90:10). 1H NMR (DMSO-d6): δ
6.70ꢀ6.72 (m, 3H), 7.24 (dd, 1H, J1 = 8.4 Hz, J2 = 2.0 Hz), 7.32ꢀ7.36
(m, 4H), 7.89 (d, 2H, J = 8.4 Hz), 7.99 (br s, 1H, CONH2), 8.07 (s, 1H),
8.46 (d, 1H, J = 2.0 Hz), 11.46 (br s, 1H, NH). 13C NMR (DMSO-d6): δ
112.2 (C), 112.9 (C), 113.5 (CH), 120.3 (C), 124.4 (CH), 124.5 (CH),
127.7 (C), 127.9 (2CH), 129.1 (2CH), 130.1 (CH), 132.6 (C), 134.5
(C), 141.6 (C), 158.2 (CH), 159.2 (C), 162.6 (C), 167.3 (CO). IR
(ATR): 3410ꢀ3175, 1680, 1608, 1585, 1533, 1456, 1149. HRMS: m/z
[M þ H]þ calcd for C19H1579BrN5O, 408.0460; found, 408.0475.
HPLC (method I): purity >99%, λ = 230 nm, tR = 20.8 min.
4-[2-Amino-4-(6-bromo-1H-indol-3-yl)pyrimidin-5-yl]benzamide
(41). Purification of the crude residue by flash chromatography
(CH2Cl2/MeOH 90:10) provided 41 as a yellow powder in 41% yield;
mp 176ꢀ177 ꢀC; Rf 0.35 (CH2Cl2/MeOH 90:10). 1H NMR (DMSO-
d6): δ 6.69ꢀ6.72 (m, 3H), 7.16 (dd, 1H, J1 = 8.4 Hz, J2 = 1.6 Hz), 7.35
(d, 2H, J = 8.0 Hz), 7.38 (br s, 1H, CONH2), 7.55 (d, 1H, J = 1.6 Hz),
7.89 (d, 2H, J = 8.0 Hz), 8.00 (br s, 1H, CONH2), 8.06 (s, 1H), 8.31 (d,
1H, J = 8.4 Hz), 11.39 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 112.8
(C), 114.1 (CH), 114.5 (C), 120.3 (C), 124.7 (CH), 124.5 (CH), 125.1
(C), 127.9 (2CH), 129.1 (2CH), 129.6 (CH), 132.6 (C), 136.7 (C),
141.6 (C), 158.1 (CH), 159.2 (C), 162.6 (C), 167.4 (CO). IR (ATR):
1665, 1610, 1582, 1536, 1462. HRMS: m/z [M þ H]þ calcd for
C19H1579BrN5O, 408.0460; found, 408.0477. HPLC (method I): purity
>99%, λ = 230 nm, tR = 20.9 min.
4-[2-Amino-4-(7-bromo-1H-indol-3-yl)pyrimidin-5-yl]benzamide
(42). Purification of the crude residue by flash chromatography
(CH2Cl2/MeOH 95:5 to 90:10) provided 42 as a yellow powder in
46% yield; mp 173ꢀ174 ꢀC; Rf 0.15 (CH2Cl2/MeOH 95:5). 1H NMR
(DMSO-d6): δ 6.68 (d, 1H, J = 3.2 Hz), 6.72 (br s, 2H, NH2), 7.02 (t,
1H, J = 7.6 Hz), 7.35ꢀ7.38 (m, 4H), 7.90 (d, 2H, J = 8.0 Hz), 8.01 (br s,
1H, CONH2), 8.09 (s, 1H), 8.39 (d, 1H, J = 8.0 Hz), 11.50 (br s, 1H,
NH). 13C NMR (DMSO-d6): δ 104.0 (C), 113.8 (C), 120.5 (C), 121.4
(CH), 122.0 (CH), 124.4 (CH), 127.8 (C), 127.9 (2CH), 129.2 (2CH),
129.6 (CH), 132.7 (C), 134.1 (C), 141.6 (C), 158.2 (CH), 159.2 (C),
162.6 (C), 167.4 (CO). IR (ATR): 3400ꢀ3110, 1657, 1608, 1582, 1531,
1459, 1132. HRMS: m/z [M þ H]þ calcd for C19H1579BrN5O,
408.0460; found, 408.0475. HPLC (method I): purity >98%, λ =
230 nm, tR = 20.7 min.
4-(4-Bromo-1H-indol-3-yl)-5-(4-trifluoromethylphenyl)pyrimidin-
2-amine (43). Purification of the crude residue by flash chromatography
(CH2Cl2/MeOH 95:5) provided 43 as a white powder in 43% yield; mp
> 250 ꢀC; Rf 0.30 (cyclohexane/EtOAc 20:80). 1H NMR (DMSO-d6):
δ 6.79 (br s, 2H, NH2), 6.99 (t, 1H, J = 8.0 Hz), 7.14 (dd, 1H, J1 = 8.0 Hz,
J2 = 0.8 Hz), 7.31 (d, 1H, J = 8.0 Hz), 7.34 (d, 2H, J = 8.0 Hz), 7.39 (dd,
1H, J1 = 8.0 Hz, J2 = 0.8 Hz), 7.51 (d, 2H, J = 8.0 Hz), 8.29 (s, 1H), 11.53
(br s, 1H, NH). 13C NMR (DMSO-d6): δ 111.2 (CH), 112.8 (C), 114.2
(C), 122.5 (CH), 123.3 (CH), 123.4 (C), 124.6 (2CH, q, JCF = 3,8 Hz),
125.1 (C, q, JCF = 270 Hz), 125.6 (C), 126.4 (C, q, JCF = 32 Hz), 126.9
(2CH), 129.4 (CH), 136.6 (C), 141.9 (C), 157.6 (CH), 161.0 (C),
162.2 (C). IR (ATR): 1575, 1515, 1472, 1319, 1110. HRMS: m/z [M þ
H]þ calcd for C19H1379BrF3N4, 433.0276; found, 433.0290. HPLC
(method I): purity >99%, λ = 230 nm, tR = 24.5 min.
4-(5-Bromo-1H-indol-3-yl)-5-(4-trifluoromethylphenyl)pyrimidin-
2-amine (44). Purification of the crude residue by flash chromatography
(cyclohexane/EtOAc 50:50) provided 44 as a white powder in 63%
yield; mp 108ꢀ109 ꢀC; Rf 0.40 (cyclohexane/EtOAc 40:60). 1H NMR
(DMSO-d6): δ 6.78ꢀ6.79 (m, 3H), 7.24 (dd, 1H, J1 = 8.8 Hz, J2 = 2.0
Hz), 7.33 (d, 1H, J = 8.8 Hz), 7.50 (d, 2H, J = 8.0 Hz), 7.72 (d, 2H, J = 8.0
Hz), 8.12 (s, 1H), 8.32 (d, 1H, J = 2.0 Hz), 11.51 (br s, 1H, NH). 13C
NMR (DMSO-d6): δ 112.3 (C), 113.1 (C), 113.6 (CH), 119.8 (C),
124.0 (C, q, JCF = 270 Hz), 124.2 (CH), 124.6 (CH), 125.5 (CH), 125.7
(2CH, q, JCF = 4.0 Hz), 127.7 (C), 129.5 (C, q, JCF = 32 Hz), 130.2
(2CH), 134.5 (C), 143.2 (C), 158.5 (CH), 159.3 (C), 162.8 (C). IR
(ATR): 3428ꢀ3199, 1579, 1530, 1457, 1323, 1123. HRMS: m/z [M þ
H]þ calcd for C19H1379BrF3N4, 433.0276; found, 433.0269. HPLC
(method I): purity >95%, λ = 230 nm, tR = 24.7 min.
4-(6-Bromo-1H-indol-3-yl)-5-(4-trifluoromethylphenyl)pyrimidin-
2-amine (45). Purification of the crude residue by flash chromatography
(cyclohexane/EtOAc 50:50) provided 45 as a yellow powder in 86%
yield; mp 201ꢀ202 ꢀC; Rf 0.30 (cyclohexane/EtOAc 40:60). 1H NMR
(DMSO-d6): δ 6.73 (d, 1H, J = 2.8 Hz), 6.75 (br s, 2H, NH2), 7.16 (dd,
1H, J1 = 8.4 Hz, J2 = 2.0 Hz), 7.50 (d, 2H, J = 8.0 Hz), 7.55 (d, 1H, J = 1.2
Hz), 7.73 (d, 2H, J = 8.0 Hz), 8.10 (s, 1H), 8.25 (d, 1H, J = 8.8 Hz), 11.42
(br s, 1H, NH). 13C NMR (DMSO-d6): δ 112.6 (C), 114.2 (CH), 114.6
(C), 119.6 (C), 122.8 (CH), 124.1 (CH), 124.9 (C), 125.5 (2CH, q,
JCF = 3.8 Hz), 127.0 (C, q, JCF = 271 Hz), 127.4 (C, q, JCF = 31 Hz),
129.8 (CH), 130.1 (3CH), 131.8 (C), 136.7 (C), 159.5 (C), 162.8 (C).
IR (ATR): 3329ꢀ3224, 1579, 1532, 1458, 1324, 1115. HRMS: m/z
[M þ H]þ calcd for C19H1379BrF3N4, 433.0276; found, 433.0259.
HPLC (method I): purity >99%, λ = 230 nm, tR = 24.8 min.
4-(7-Bromo-1H-indol-3-yl)-5-(4-trifluoromethylphenyl)pyrimidin-
2-amine (46). Purification of the crude residue by flash chromatography
(CH2Cl2/MeOH 99:1 to 97:3) provided 46 as a yellow powder in 71%
yield. Purification of a small amount was performed by preparative
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ARTICLE
HPLC (system: 30:70 A/B, flow 38 mL/min, λ = 228 nm, tR = 5.8 min);
mp 104ꢀ105 ꢀC; Rf 0.45 (cyclohexane/EtOAc 40:60). 1H NMR
(DMSO-d6): δ 6.73 (d, 1H, J = 2.8 Hz), 6.78 (br s, 2H, NH2), 7.01
(t, 1H, J = 7.6 Hz), 7.36 (d, 1H, J = 7.2 Hz), 7.52 (d, 2H, J = 8.4 Hz), 7.74
(d, 2H, J = 8.4 Hz), 8.13 (s, 1H), 8.31 (d, 1H, J = 8.0 Hz), 11.54 (br s, 1H,
NH). 13C NMR (DMSO-d6): δ 113.7 (C), 119.8 (C), 120.6 (CH),
122.2 (CH), 124.2 (C, q, JCF = 271 Hz), 124.5 (CH), 125.5 (2CH, q,
JCF = 4.0 Hz), 127.6 (C), 127.8 (C, q, JCF =30 Hz), 128.5(C), 129.6(CH),
130.2 (CH), 134.2 (C), 138.7 (2CH), 142.9 (C), 164.5 (C), 179.5 (C).
IR (ATR): 1576, 1538, 1456, 1322, 1123. HRMS: m/z [M þ H]þ calcd
for C19H1379BrF3N4, 433.0276; found, 433.0295. HPLC (method I):
purity >99%, λ = 230 nm, tR = 25.21 min.
4-(4-Bromo-1H-indol-3-yl)-5-(3-methoxyphenyl)pyrimidin-2-amine
(47). Purification of the crude residue by flash chromatography (cyclo-
hexane/EtOAc 40:60) provided 47 as a yellow powder in 57% yield; mp
224ꢀ225 ꢀC; Rf 0.25 (cyclohexane/EtOAc 20:80). 1H NMR (DMSO-
d6): δ 3.32 (s, 3H, OCH3), 6.63ꢀ6.68 (m, 4H), 6.74 (dd, 1H, J1 =
7.6 Hz, J2 = 1.2 Hz), 6.99 (t, 1H, J = 8.0 Hz), 7.06 (t, 1H, J = 8.0 Hz), 7.14
(d, 1H, J = 8.0 Hz), 7.27 (d, 1H, J = 2.8 Hz), 7.39 (dd, 1H, J1 = 8.0 Hz,
J2 = 0.8 Hz), 8.25 (s, 1H), 11.45 (br s, 1H, NH). 13C NMR (DMSO-d6): δ
54.5 (OCH3), 111.2 (CH), 111.9 (CH), 112.9 (2C), 114.3 (CH), 121.1
(2CH), 122.4 (CH), 123.2 (CH), 124.8 (C), 125.2 (C), 128.8 (2CH),
136.6 (2C), 138.7 (C), 158.5 (2C). IR (ATR): 1599, 1576, 1467, 1196.
HRMS: m/z [M þ H]þ calcd for C19H1679BrN4O, 395.0507; found,
395.0510. HPLC (method I): purity >98%, λ = 230 nm, tR = 22.8 min.
4-(5-Bromo-1H-indol-3-yl)-5-(3-methoxyphenyl)pyrimidin-2-amine
(48). Purification of the crude residue by flash chromatography (cyclo-
hexane/EtOAc 50:50) provided 48 as a yellow powder in 69% yield; mp
166ꢀ167 ꢀC; Rf 0.40 (cyclohexane/EtOAc 40:60). 1H NMR (DMSO-
d6): δ 3.72 (s, 3H, OCH3), 6.65 (br s, 2H, NH2), 6.74 (d, 1H, J = 2.8
Hz), 6.82ꢀ6.85 (m, 2H), 6.93 (dd, 1H, J1 = 7.6 Hz, J2 = 2.0 Hz), 7.24
(dd, 1H, J1 = 8.4 Hz, J2 = 2.0 Hz), 7.29ꢀ7.34 (m, 2H), 8.03 (s, 1H), 8.52
(d, 1H, J = 2.0 Hz), 11.46 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 55.0
(OCH3), 112.3 (C), 112.7 (CH), 113.1 (C), 113.5 (CH), 114.9 (CH),
120.8 (C), 121.7 (CH), 124.5 (CH), 124.6 (CH), 127.8 (C), 129.8
(CH), 130.2 (CH), 134.5 (C), 139.9 (C), 157.6 (CH), 159.4 (C), 159.5
(C), 162.2 (C). IR (ATR): 3339ꢀ3210, 1587, 1528, 1471. HRMS: m/z
[M þ H]þ calcd for C19H1679BrN4O, 395.0507; found, 395.0500.
HPLC (method I): purity >99%, λ = 230 nm, tR = 23.5 min.
4-(6-Bromo-1H-indol-3-yl)-5-(3-methoxyphenyl)pyrimidin-2-amine
(49). Purification of the crude residue by flash chromatography (cyclo-
hexane/EtOAc 50:50) provided 49 as a yellow powder in 92% yield; mp
156ꢀ157 ꢀC; Rf 0.30 (cyclohexane/EtOAc 40:60). 1H NMR (DMSO-
d6): δ 3.72 (s, 3H, OCH3), 6.62 (br s, 2H, NH2), 6.71 (d, 1H, J = 2.8
Hz), 6.82ꢀ6.85 (m, 2H), 6.93 (dd, 1H, J1 = 8.0 Hz, J2 = 1.6 Hz), 7.17
(dd, 1H, J1 = 8.8 Hz, J2 = 1.6 Hz), 7.31 (t, 1H, J = 8.0 Hz), 7.54 (d, 1H, J =
1.6 Hz), 8.02 (s, 1H), 8.40 (d, 1H, J = 8.8 Hz), 11.38 (br s, 1H, NH). 13C
NMR (DMSO-d6): δ 55.0 (OCH3), 112.7 (CH), 112.8 (C), 114.0
(CH), 114.5 (C), 114.8 (CH), 120.8 (C), 121.7 (CH), 122.7 (CH),
124.5 (CH), 125.2 (C), 129.6 (CH), 129.8 (CH), 136.6 (C), 139.9 (C),
157.9 (CH), 159.2 (C), 159.5 (C), 162.3 (C). IR (ATR): 1577, 1538, 1457,
1133. HRMS: m/z [M þ H]þ calcd for C19H1679BrN4O, 395.0507; found,
395.0507. HPLC (method I): purity >96%, λ = 230 nm, tR = 23.6 min.
4-(7-Bromo-1H-indol-3-yl)-5-(3-methoxyphenyl)pyrimidin-2-amine
(50). Purification of the crude residue by flash chromatography (cyclo-
hexane/EtOAc 60:40 then 50:50) provided 50 as a yellow powder in
71% yield. Purification of a small amount was performed by preparative
HPLC (system: 30:70 A/B, flow 38 mL/min, λ = 228 nm, tR = 4.8 min);
mp 163ꢀ164 ꢀC; Rf 0.30 (cyclohexane/EtOAc 40:60). 1H NMR
(DMSO-d6): δ 3.72 (s, 3H, OCH3), 6.64 (br s, 2H, NH2), 6.73 (d,
1H, J = 2.8 Hz), 6.83ꢀ6,87 (m, 2H), 6.94 (dd, 1H, J1 = 8.0 Hz, J2 = 2.8
Hz), 7.02 (t, 1H, J = 7.6 Hz), 7.32 (t, 1H, J = 8.0 Hz), 7.36 (d, 1H, J = 7.6
Hz), 8.04 (s, 1H), 8.49 (d, 1H, J = 8.0 Hz), 11.48 (br s, 1H, NH). 13C
NMR (DMSO-d6): δ 55.0 (OCH3), 103.9 (C), 112.7 (CH), 113.8 (C),
114.8 (CH), 120.9 (C), 121.4 (CH), 121.7 (CH), 122.3 (CH), 124.4
(CH), 127.9 (C), 129.6 (CH), 129.8 (CH), 134.1 (C), 139.9 (C), 158.0
(CH), 159.1 (C), 159.5 (C), 162.4 (C). IR (ATR): 1595, 1578, 1464,
1163. HRMS: m/z [M þ H]þ calcd for C19H1679BrN4O, 395.0507;
found, 395.0501. HPLC (method I): purity >98%, λ = 230 nm, tR
=
23.7 min.
3-Acetyl-4-nitro-1H-indole (51). Purification of the crude residue by
flash chromatography (cyclohexane/EtOAc 20:80) provided 51 as a yellow
powder in 97% yield; mp 218ꢀ219 ꢀC; Rf 0.30 (cyclohexane/EtOAc
20:80). 1H NMR (DMSO-d6): δ 2.47 (s, 3H, CH3), 7.38 (t, 1H, J = 8.0
Hz), 7.59 (d, 1H, J = 8.0 Hz), 7.80 (d, 1H, J = 8.0 Hz), 8.56 (s, 1H), 12.57
(br s, 1H, NH). 13C NMR (DMSO-d6): δ 27.5 (COCH3), 115.2 (C),
116.3 (C), 116.6 (2CH), 122.5 (CH), 136.4 (CH), 138.7 (C), 144.1 (C),
190.9 (CO). IR (ATR): 3231, 1638, 1623, 1514, 1313, 1167. HRMS: m/z
[M þ Na]þ calcd for C10H8N2 NaO3, 227.0433; found, 227.0421.
3-Acetyl-7-nitro-1H-indole (52). Compound 52 was obtained as a
sufficiently pure brown powder and directly used in the next step; mp
228ꢀ229 ꢀC; Rf 0.40 (cyclohexane/EtOAc 50:50). 1H NMR (DMSO-
d6): δ 2.53 (s, 3H, CH3), 7.42 (t, 1H, J = 8.0 Hz), 8.19 (d, 1H, J = 8.0
Hz), 8.44 (s, 1H), 8.66 (d, 1H, J = 8.0 Hz), 12.56 (br s, 1H, NH). 13C
NMR (DMSO-d6): δ 27.5 (CH3), 117.3 (2C), 119.7 (CH), 121.6
(CH), 128.9 (C), 129.5 (CH), 136.6 (CH), 136.7 (C), 193.2 (CO). IR
(ATR): 3284, 1640, 1629, 1534, 1357, 1109. HRMS: m/z [M þ H]þ
calcd for C10H9N2O3, 205.0613; found, 205.0614.
N-[4-(Methylphenyl)sulfonyl]-5-nitro-1H-indole (53). Purification
of the crude residue by flash chromatography (CH2Cl2) provided 53
as a white powder in 100% yield; mp 150ꢀ151 ꢀC; Rf 0.40 (cyclohexane/
EtOAc 80:20). 1H NMR (DMSO-d6): δ 2.37 (s, 3H, CH3), 6.82 (d, 1H,
J = 3.6 Hz), 7.29 (d, 2H, J = 7.6 Hz), 7.75 (d, 1H, J = 3.6 Hz), 7.81 (d, 2H,
J = 8.4 Hz), 8.09 (d, 1H, J = 9.2 Hz), 8.19 (dd, 1H, J1 = 9.2 Hz, J2 = 2.0
Hz), 8.46 (d, 1H, J = 2.0 Hz). 13C NMR (DMSO-d6): δ 21.7 (CH3),
109.5 (CH), 113.6 (CH), 117.8 (CH), 119.8 (CH), 127.0 (CH), 129.3
(CH), 130.3 (CH), 130.6 (C), 134.7 (C), 137.7 (C), 144.3 (C), 146.0
(C). IR (ATR): 1518, 1441, 1373, 1348, 1172, 1121. HRMS: m/z [M þ
Na]þ calcd for C15H12N2NaO4S, 339.0415; found, 339.0417.
N-[4-(Methylphenyl)sulfonyl]-6-nitro-1H-indole (54). Purification
of the crude residue by flash chromatography (cyclohexane/EtOAc
70:30 then 50:50) provided 54 as a yellow powder in 99% yield; mp
196ꢀ197 ꢀC; Rf 0.45 (cyclohexane/EtOAc 70:30). 1H NMR (DMSO-
d6): δ 2.32 (s, 3H, CH3), 7.05 (d, 1H, J = 3.6 Hz), 7.41 (d, 2H, J = 8.0
Hz), 7.85 (d, 1H, J = 8.8 Hz), 7.92 (d, 2H, J = 8.0 Hz), 8.13 (dd, 1H, J1 =
8.8 Hz, J2 = 1.6 Hz), 8.22 (d, 1H, J = 3.6 Hz), 8.74 (s, 1H). 13C NMR
(DMSO-d6): δ 20.9 (CH3), 108.8 (CH), 109.5 (CH), 118.5 (CH),
122.4 (CH), 126.7 (2CH), 130.5 (2CH), 132.5 (CH), 132.8 (C), 133.7
(C), 135.4 (C), 144.4 (C), 146.1 (C). IR (ATR): 1527, 1508, 1430,
1364, 1341, 1169, 1099. HRMS: m/z [M þ Na]þ calcd for C15H12N2Na-
O4S, 339.0415; found, 339.0410.
3-Acetyl-N-[4-(methylphenyl)sulfonyl]-4-nitro-1H-indole (55). Puri-
fication of the crude residue by flash chromatography (cyclohexane/
EtOAc 70:30 then 50:50) provided 55 as a yellow powder in 87% yield;
mp 189ꢀ190 ꢀC; Rf 0.50 (cyclohexane/EtOAc 70:30). 1H NMR
(DMSO-d6): δ 2.34 (s, 3H, CH3), 2.61 (s, 3H, COCH3), 7.47 (d,
2H, J = 8.4 Hz), 7.61 (t, 1H, J = 8.0 Hz), 7.84 (d, 1H, J = 8.0 Hz), 8.09 (d,
2H, J = 8.4 Hz), 8.31 (d, 1H, J = 8.0 Hz), 9.00 (s, 1H). 13C NMR
(DMSO-d6): δ 21.1 (CH3), 28.4 (COCH3), 117.4 (CH), 117.7 (C),
119.7 (CH), 120.4 (C), 126.1 (CH), 127.1 (2CH), 130.6 (2CH), 132.9
(C), 135.5 (CH), 143.9 (C), 146.9 (2C), 192.3 (CO). IR (ATR): 1672,
1530, 1381, 1173, 1089. HRMS: m/z [M þ Na]þ calcd for
C17H14N2NaO5S, 381.0521; found 381.0516.
3-Acetyl-N-[4-(methylphenyl)sulfonyl]-5-nitro-1H-indole (56). Pur-
ification of the crude residue by flash chromatography (cyclohexane/
EtOAc 70:30 then 50:50 and finally pure CH2Cl2) provided 56 as a
white powder in 93% yield; mp 231ꢀ232 ꢀC; Rf 0.70 (cyclohexane/
EtOAc 60:40). 1H NMR (DMSO-d6): δ 2.36 (s, 3H, CH3), 2.63 (s, 3H,
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COCH3), 7.48 (d, 2H, J = 8.4 Hz), 8.10 (d, 2H, J = 8.4 Hz), 8.19 (d, 1H,
J = 9.6 Hz), 8.27 (dd, 1H, J1 = 9.6 Hz, J2 = 2.0 Hz), 8.98 (d, 1H, J = 2.0
Hz), 9.06 (s, 1H). 13C NMR (DMSO-d6): δ 21.0 (CH3), 27.7
(COCH3), 113.9 (CH), 117.9 (CH), 120.6 (C), 120.7 (CH), 126.9
(C), 127.3 (2CH), 130.6 (2CH), 132.9 (C), 136.8 (C), 136.9 (CH),
144.6 (C), 146.9 (C), 193.7 (CO). IR (ATR): 1680, 1520, 1386, 1343,
1165, 1079. HRMS: m/z [M þ Na]þ calcd for C17H14N2NaO5S,
381.0521; found, 381.0526.
129.1 (C), 129.3 (C), 130.9 (CH), 131.2 (CH), 132.8 (C), 157.6 (CH),
161.4 (C), 163.4 (C). IR (ATR): 3464ꢀ3218, 1568, 1526, 1483, 1365,
1122. HRMS: m/z [M þ H]þcalcd for C12H10N5O2, 256.0834; found,
256.0826. HPLC (method I): purity >96%, λ = 228 nm, tR = 20.4 min.
5-Iodo-4-(4-nitro-1H-indol-3-yl)pyrimidin-2-amine (70). Purifica-
tion of the crude residue by flash chromatography (CH2Cl2/MeOH
95:5) provided 70 as a black powder in 29% yield (3 steps); mp 196ꢀ
197 ꢀC; Rf 0.45 (CH2Cl2/MeOH 90:10). 1H NMR (DMSO-d6): δ 6.67
(br s, 2H, NH2), 7.36 (t, 1H, J = 8.0 Hz), 7.91ꢀ7.96 (m, 3H), 8.44 (s,
1H), 12.30 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 115.8 (C), 117.2
(C), 117.3 (CH), 118.8 (CH), 120.8 (CH), 131.7 (CH), 138.1 (2C),
141.6 (C), 161.8 (C), 163.4 (CH), 164.8 (C). IR (ATR): 3500ꢀ3350,
1647, 1557, 1546, 1509, 1335, 1109. HRMS: m/z [M þ H]þcalcd for
C12H9N5O2I, 381.9801; found, 381.9814. HPLC (method I): purity
>95%, λ = 230 nm, tR = 21.8 min.
5-Iodo-4-(5-nitro-1H-indol-3-yl)pyrimidin-2-amine (71). Purifica-
tion of the crude residue by flash chromatography (cyclohexane/EtOAc
50:50) provided 71 as a yellow powder in 20% yield (3 steps); mp
244ꢀ245 ꢀC; Rf 0.50 (CH2Cl2/MeOH 90:10). 1H NMR (DMSO-d6):
δ 6.86 (br s, 2H, NH2), 7.66 (d, 1H, J = 8.8 Hz), 8.07 (d, 1H, J = 8.8 Hz),
8.53 (s, 1H), 8.57 (s, 1H), 8.99 (s, 1H), 12.30 (br s, 1H, NH). 13C NMR
(DMSO-d6): δ 75.5 (C), 112.5 (CH), 115.9 (C), 117.3 (CH), 118.7
(CH), 125.3 (C), 132.5 (CH), 139.0 (C), 141.6 (C), 161.3 (C), 162.2
(C), 166.1 (CH). IR (ATR): 3509, 3324, 1639, 1532, 1506, 1452, 1331,
1121. HRMS: m/z [M þ H]þ calcd for C12H9N5O2I, 381.9801; found,
381.9805. HPLC (method I): purity >96%, λ = 230 nm, tR = 22.4 min.
5-Iodo-4-(6-nitro-1H-indol-3-yl)pyrimidin-2-amine (72). Purifica-
tion of the crude residue by flash chromatography (CH2Cl2/MeOH
95:5) provided 72 as a orange powder in 80% yield; mp 259ꢀ260 ꢀC; Rf
0.55 (CH2Cl2/MeOH 90:10). 1H NMR (DMSO-d6): δ 6.85 (br s, 2H,
NH2), 7.96 (dd, 1H, J = 8.4 Hz, J = 2.4 Hz), 8.39 (d, 1H, J = 8.4 Hz), 8.42
(d, 1H, J = 2.4 Hz), 8.55 (s, 1H), 8.74 (d, 1H, J = 2.4 Hz), 12.30 (br s, 1H,
NH). 13C NMR (DMSO-d6): δ 75.0 (C), 108.9 (CH), 114.7 (C), 114.9
(CH), 122.4 (CH), 130.8 (C), 134.4 (C), 134.9 (CH), 142.4 (C), 161.2
(C), 162.1 (C), 166.1 (CH). IR (ATR): 3350, 3180, 1652, 1553, 1501,
1460, 1343, 1073. HRMS: m/z [M þ H]þ calcd for C12H9N5O2I,
381.9801; found, 381.9820. HPLC (method I): purity >99%, λ =
230 nm, tR = 22.7 min.
5-Iodo-4-(7-nitro-1H-indol-3-yl)pyrimidin-2-amine (73). Purifica-
tion of the crude residue by flash chromatography (cyclohexane/EtOAc
50:50) provided 73 as a yellow powder in 70% yield; mp 246ꢀ247 ꢀC; Rf
0.50 (cyclohexane/EtOAc 50:50). 1H NMR (DMSO-d6): δ 6.86 (br s,
2H, NH2), 7.36 (t, 1H, J = 8.0 Hz), 8.19 (d, 1H, J = 8.0 Hz), 8.50 (d, 1H,
J = 2.4 Hz), 8.56 (s, 1H), 8.77 (d, 1H, J = 8.0 Hz), 12.36 (br s, 1H, NH).
13C NMR (DMSO-d6): δ 111.3 (C), 115.3 (C), 119.5 (CH), 120.1
(CH), 128.2 (C), 130.1 (C), 131.0 (CH), 131.7 (CH), 132.9 (C), 161.0
(C), 162.2 (C), 166.2 (CH). IR (ATR): 3512ꢀ3392, 1611, 1535, 1516,
1480, 1361, 1134. HRMS: m/z [M þ H]þ calcd for C12H9N5O2I,
381.9801; found, 381.9784. HPLC (method II): purity >98%, λ =
230 nm, tR = 40.7 min.
5-Iodo-4-(1H-indol-3-yl)pyrimidin-2-amine (74). Purification of the
crude residue by flash chromatography (cyclohexane/EtOAc 50:50)
provided 74 as a yellow powder in 29% yield (3 steps); mp 167ꢀ168 ꢀC;
Rf 0.70 (CH2Cl2/MeOH 90:10). 1H NMR (DMSO-d6): δ 6.71 (br s,
2H, NH2), 7.10 (t, 1H, J = 7.2 Hz), 7.18 (t, 1H, J = 7.2 Hz), 7.46 (d, 1H,
J = 7.8 Hz), 8.30 (d, 1H, J = 7.8 Hz), 8.42 (d, 1H, J = 2.8 Hz), 8.49 (s, 1H),
11.67 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 74.8 (C), 111.6 (CH),
113.7 (C), 120.1 (CH), 121.9 (CH), 122.4 (CH), 126.1 (C), 129.4
(CH), 135.9 (C), 162.0 (2C), 165.8 (CH). IR (ATR): 3490ꢀ3280,
1554, 1515, 1456, 1121. HRMS: m/z [M þ H]þcalcd for C12H10N4I,
336.9950; found, 336.9964. HPLC (method II): purity >99%, λ =
230 nm, tR = 16.5 min.
3-Acetyl-N-[4-(methylphenyl)sulfonyl]-6-nitro-1H-indole (57). Puri-
fication of the crude residue by flash chromatography (cyclohexane/
EtOAc 70:30 then 50:50) provided 57 as a white powder in 100% yield;
mp 200ꢀ201 ꢀC; Rf 0.10 (cyclohexane/EtOAc 80:20). 1H NMR
(DMSO-d6): δ 2.35 (s, 3H, CH3), 2.63 (s, 3H, COCH3), 7.48 (d,
2H, J = 8.0 Hz), 8.09 (d, 2H, J = 8.0 Hz), 8.25 (dd, 1H, J1 = 8.8 Hz, J2 =
0.8 Hz), 8.39 (d, 1H, J = 8.8 Hz), 8.72 (d, 1H, J = 0.8 Hz), 9.17 (s, 1H).
13C NMR (DMSO-d6): δ 21.9 (CH3), 27.9 (COCH3), 108.7 (CH),
119.9 (CH), 120.3 (C), 123.0 (CH), 127.3 (2CH), 130.7 (2CH), 131.8
(C), 132.9 (C), 133.1 (C), 138.5 (CH), 144.9 (C), 146.9 (C), 193.6
(CO). IR (ATR): 1675, 1517, 1385, 1338, 1168, 1088. HRMS: m/z
[M þ H]þ calcd for C17H15N2O5S, 359.0702; found, 359.0696.
3-Acetyl-N-[4-(Methylphenyl)sulfonyl]-7-nitro-1H-indole (58). Puri-
fication of the crude residue by flash chromatography (CH2Cl2)
provided 58 as a yellow powder in 59% yield (2 steps); mp 213-
214 ꢀC; Rf 0.75 (CH2Cl2). 1H NMR (DMSO-d6): δ 2.43 (s, 3H, CH3),
2.64 (s, 3H, COCH3), 7.54 (d, 2H, J = 8.0 Hz), 7.59 (t, 1H, J = 8.0 Hz),
7.93 (d, 1H, J = 8.0 Hz), 8.02 (d, 2H, J = 8.0 Hz), 8.58 (d, 1H, J = 8.0 Hz),
9.13 (s, 1H). 13C NMR (DMSO-d6): δ 21.1 (CH3), 28.0 (COCH3),
120.9 (C), 121.5 (CH), 124.5 (C), 125.4 (CH), 127.1 (2CH), 127.2
(CH), 129.9 (2CH), 130.1 (C), 134.0 (C), 138.7 (CH), 146.1 (2C),
193.7 (CO). IR (ATR): 1676, 1535, 1388, 1364, 1169, 1090. HRMS:
m/z [M þ Na]þ calcd for C17H14N2O5NaS, 381.0521; found, 381.0533.
(2E)-3-(Dimethylamino)-1-{7-nitro-1-[4-(methylphenyl)sulfonyl]-
1H-indol-3-yl}-2-propen-1-one (63). Purification of the crude residue
by flash chromatography (cyclohexane/EtOAc 25:75 then pure EtOAc)
provided 63 as a yellow powder in 43% yield; mp 218ꢀ219 ꢀC; Rf 0.15
1
(cyclohexane/EtOAc 25:75). H NMR (DMSO-d6): δ 2.41 (s, 3H,
CH3), 2.97 (br s, 3H, NCH3), 3.15 (br s, 3H, NCH3), 5.96 (d, 1H, J =
12.4 Hz), 7.49ꢀ7.54 (m, 3H), 7.70 (d, 1H, J = 12.4 Hz), 7.86 (d, 1H, J =
8.0 Hz), 7.91 (d, 2H, J = 8.4 Hz), 8.71 (d, 1H, J = 8.0 Hz), 8.89 (s, 1H).
13C NMR (DMSO-d6): 21.1 (CH3), 92.6 (CH), 120.9 (CH), 123.3 (C),
124.6 (CH), 124.8 (C), 126.8 (2CH), 127.9 (CH), 129.9 (2CH), 131.9
(C), 133.9 (CH), 134.3 (C), 138.3 (C), 145.8 (C), 153.3 (CH), 181.3
(CO). IR (ATR): 1644, 1547, 1379, 1367, 1173, 1039. HRMS: m/z
[M þ Na]þ calcd for C20H19N3NaO5S, 436.0943; found, 436.0959.
4-(6-Nitro-1H-indol-3-yl)pyrimidin-2-amine (67). Purification of the
crude residue by flash chromatography (CH2Cl2/MeOH 97:3 then
95:5) provided 67 as a yellow powder in 18% yield (2 steps); mp >
250 ꢀC; Rf 0.50 (CH2Cl2/MeOH 90:10). 1H NMR (DMSO-d6): δ 6.46
(br s, 2H, NH2), 7.07 (d, 1H, J = 5.2 Hz), 7.97 (d, 1H, J = 8.8 Hz), 8.17
(d, 1H, J = 5.2 Hz), 8.38 (s, 1H), 8.60 (s, 1H), 8.80 (d, 1H, J = 8.8 Hz),
12.37 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 105.3 (CH), 108.5
(CH), 114.5 (C), 114.9 (CH), 122.6 (CH), 130.0 (C), 134.0 (CH),
135.5 (C), 142.3 (C), 157.5 (CH), 161.4 (C), 163.5 (C). IR (ATR):
3481ꢀ3347, 1582, 1557, 1498, 1456, 1336, 1061. HRMS: m/z [M þ
H]þ calcd for C12H10N5O2, 256.0834; found, 256.0822. HPLC
(method I): purity >96%, λ = 228 nm, tR = 20.2 min.
4-(7-Nitro-1H-indol-3-yl)pyrimidin-2-amine (68). Purification of the
crude residue by flash chromatography (CH2Cl2/MeOH 97:3 then 95:5
and finally 90:10) provided 68 as a orangeꢀyellow powder in 40% yield;
mp decomposition; Rf 0.50 (CH2Cl2/MeOH 90:10). 1H NMR
(DMSO-d6): δ 6.58 (br s, 2H, NH2), 7.15 (d, 1H, J = 5.2 Hz), 7.36
(t, 1H, J = 8.0 Hz), 8.17 (d, 1H, J = 5.2 Hz), 8.18 (d, 1H, J = 8.0 Hz), 8.35
(d, 1H, J = 2.0 Hz), 9.20 (d, 1H, J = 8.0 Hz), 12.36 (br s, 1H, NH). 13C
NMR (DMSO-d6): δ 105.6 (CH), 115.0 (C), 119.3 (CH), 120.0 (CH),
4-(4-Amino-1H-indol-3-yl)-5-iodo-pyrimidin-2-amine (75). Purifi-
cation of the crude residue by flash chromatography (EtOAc) provided
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75 as a yellow powder in 96% yield; mp > 250 ꢀC; Rf 0.30 (cyclohexane/
EtOAc 30:70). 1H NMR (DMSO-d6): δ 5.47ꢀ5.49 (br s, 2H, NH2 indole),
6.33 (d, 1H, J = 8.0 Hz), 6.69 (d, 1H, J = 8.0 Hz), 6.85 (br s, 2H,
NH2 pyrimidine), 6.89 (t, 1H, J = 8.0 Hz), 7.88 (s, 1H), 8.51 (s, 1H), 11.42
(br s, 1H, NH). 13C NMR (DMSO-d6): δ 76.7 (C), 100.6 (CH), 105.0
(CH), 113.6 (C), 114.6 (C), 123.3 (CH), 128.6 (CH), 137.6 (C), 142.7
(C), 161.8 (C), 163.0 (C), 165.7 (CH). IR (ATR): 3470ꢀ3280, 1546,
1525, 1456, 1101. HRMS: m/z [M þ H]þcalcd for C12H11N5I,
352.0059; found, 352.0054. HPLC (method I): purity >95%, λ =
230 nm, tR = 18.4 min.
4-(5-Amino-1H-indol-3-yl)-5-iodo-pyrimidin-2-amine (76). Purifi-
cation of the crude residue by flash chromatography (EtOAc) provided
76 as a yellow powder in 99% yield; mp 197ꢀ198 ꢀC; Rf 0.45 (EtOAc).
1H NMR (DMSO-d6): δ 4.51ꢀ4.72 (br s, 2H, NH2 indole), 6.56 (br s,
3H, NH2 pyrimidine þ 1H), 7.13 (d, 1H, J = 8.4 Hz), 7.55 (s, 1H), 8.24 (s,
1H), 8.45 (s, 1H), 11.25 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 74.8
(C), 106.0 (CH), 111.6 (CH), 112.3 (CH), 112.4 (C), 127.3 (C), 128.8
(CH), 129.5 (C), 142.3 (C), 161.9 (C), 162.6 (C), 165.5 (CH). IR
(ATR): 3500ꢀ3200, 1553, 1514, 1470, 1158. HRMS: m/z [M þ H]þ
calcd for C12H11N5I, 352.0059; found, 352.0047. HPLC (method II):
purity >99%, λ = 230 nm, tR = 5.9 min.
without constraints. These minimizations were carried out with the
Tripos force field, Gasteiger-H€uckel charges, dielectric constants set to
78 and conjugate gradient method.
’ AUTHOR INFORMATION
Corresponding Author
*For chemistry (P.M.): phone, þ33 (0) 4 73 40 79 63; fax, þ33
(0) 4 73 40 77 17; E-mail, Pascale.MOREAU@univ-bpclermont.
fr. For biology (L.M.): phone, þ33 (0) 2 98 29 23 39; E-mail,
meijer@sb-roscoff.fr.
’ ACKNOWLEDGMENT
We thank Aurꢀelie Job for HPLC analysis, Bertrand Lꢀegeret for
mass spectrometry analysis, and Billel Khelfaoui for technical
assistance. This research was also supported by grants from
“Association France-Alzheimer Finistꢁere” (L.M.), CRITT Santꢀe
Bretagne (L.M.), “Fondation Jꢀer^ome Lejeune” (L.M.). The
authors (G.A., E.D., F.A., E.P., P.M.) also thank “Rꢀegion Au-
vergne” (Synbio Project) for financial support. L.M.’s fundings
on this project included “CRITT Santꢀe Bretagne”, “Fondation
Jꢀer^ome Lejeune”, “Association France-Alzheimer (Finistꢁere)”,
and the FUI PharmaSea project.
4-(6-Amino-1H-indol-3-yl)-5-iodo-pyrimidin-2-amine (77). Purifi-
cation of the crude residue by flash chromatography (EtOAc) provided
77 as a yellow powder in 88% yield; mp 137ꢀ138 ꢀC; Rf 0.30 (CH2Cl2/
1
MeOH 90:10). H NMR (DMSO-d6): δ 4.84ꢀ4.89 (br s, 2H, NH2
indole), 6.49 (d, 1H, J = 8.0 Hz), 6.58ꢀ6.62 (m, 3H), 8.04 (d, 1H, J = 8.0
Hz), 8.21 (d, 1H, J = 2.0 Hz), 8.43 (s, 1H), 11.12 (br s, 1H, NH). 13C
NMR (DMSO-d6): δ 74.2 (C), 94.9 (CH), 110.8 (CH), 113.6 (C),
117.7 (C), 123.0 (CH), 126.9 (CH), 137.6 (C), 144.6 (C), 161.9 (C),
162.1 (C), 165.6 (CH). IR (ATR): 3424ꢀ3280, 1542, 1533, 1446, 1124.
HRMS: m/z [M þ H]þ calcd for C12H11N5I, 352.0059; found,
352.0062. HPLC (method I): purity >99%, λ = 230 nm, tR = 17.3 min.
4-(7-Amino-1H-indol-3-yl)-5-iodo-pyrimidin-2-amine (78). Purifi-
cation of the crude residue by flash chromatography (EtOAc) provided
78 as a yellow powder in 99% yield; mp 193ꢀ194 ꢀC; Rf 0.10
(cyclohexane/EtOAc 30:70). 1H NMR (DMSO-d6): δ 5.10ꢀ5.11 (br s,
2H, NH2 indole), 6.39 (d, 1H, J = 8.2 Hz), 6.65 (br s, 2H, NH2 pyrimidine),
6.82 (t, 1H, J = 8.2 Hz), 7.49 (d, 1H, J = 8.2 Hz), 8.29 (d, 1H, J = 2.0 Hz),
8.47 (s, 1H), 11.23 (br s, 1H, NH). 13C NMR (DMSO-d6): δ 105.7
(CH), 110.7 (CH), 114.2 (C), 121.2 (CH), 125.4 (C), 126.9 (C), 127.9
(CH), 133.7 (2C), 162.0 (C), 162.6 (C), 165.6 (CH). IR (ATR): 3520ꢀ
3180, 1564, 1511, 1438, 1167. HRMS: m/z[Mþ H]þ calcd for C12H11N5I,
352.0059; found, 352.0055. HPLC (method II): purity >99%, λ =
230 nm, tR = 5.8 min.
6.4. Molecular Modeling Experiments. For docking experi-
ments, all protein structures were prepared using chimera35 and Sybyl-
X1.136 from 2wo6 PBD file for DYRK1A23 and 1z5724 for CLK1. All the
structures of ligands used were first designed with Sybyl-X1.1 and then
optimized with Gaussian0337 (DFT/B3LYP/6-31 g).
Docking experiments were performed with the surflex module of
Sybyl-X1.1. The GeomX docking option was used with Threshold set to
0.20 and Bloat set to 3. Docking performed in these conditions
considered potential hydrogen bonding without taking into account
electrostatic charges.
’ ABBREVIATIONS USED
c-Abl, Abelson leukemia oncogen cellular homologue; ATP,
adenosine triphosphate; BSA, bovin serum albumin; CDK,
cyclin-dependent protein kinase; CK, casein kinase; CLK, cdc2-
like kinase; DIPEA, diisopropylethylamine; DMA, dimethylace-
tal; DMAP, 4-(dimethylamino)pyridine; DMF, dimethylforma-
mide; DMSO, dimethylsulfoxide; DTT, dithiothreitol; EGTA,
ethylene glycol-bis(2-aminoethylether)-N,N,N0,N0-tetraacetic
acid; DYRK, dual-specificity tyrosine-(Y)-phosphorylation regulated
kinase; Emim, 1-ethyl-3-methyl imidazolium; Erk, extracellular
signal regulated kinase; GSK-3, glycogen synthase kinase 3; GST,
glutathione transferase; His6, hexahistidine; IGF-1R, insulin
growth factor receptor-1; KDR, kinase domain receptor; MEM,
minimum essential medium; Mops, 3-(N-morpholino)propane-
sulfonic acid; PKA, cAMP-dependent protein kinase; PKG, protein
kinase G; Src, sarcoma kinase; TFA, trifluoroacetic acid
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