Groebke multicomponent reaction and subsequent nucleophilic aromatic substitution for a convenient synthesis of 3,8-diaminoimidazo[1,2-a]pyrazines as potential kinase inhibitors

Article abstract of DOI:10.1039/c1ob05336a

In a program aimed at discovering novel protein kinase inhibitors, a convenient synthesis of 3,8-diaminoimidazo[1,2-a]pyrazines has been developed exploiting the isocyanide-based multicomponent Blackburn reaction, followed by a nucleophilic aromatic subst

Full text of DOI:10.1039/c1ob05336a

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 4144
www.rsc.org/obc
PAPER
Groebke multicomponent reaction and subsequent nucleophilic aromatic
substitution for a convenient synthesis of 3,8-diaminoimidazo[1,2-a]pyrazines
as potential kinase inhibitors†
Margherita Guasconi,‡a Xiaoyun Lu,‡b,c Alberto Massarotti,a Antonio Caldarelli,a Elisa Ciraolo,b
Gian Cesare Tron,a Emilio Hirsch,b Giovanni Sorbaa and Tracey Pirali*a
Received 25th January 2011, Accepted 10th March 2011
DOI: 10.1039/c1ob05336a
In a program aimed at discovering novel protein kinase inhibitors, a convenient synthesis of
3,8-diaminoimidazo[1,2-a]pyrazines has been developed exploiting the isocyanide-based
multicomponent Blackburn reaction, followed by a nucleophilic aromatic substitution with ammonia
or primary and secondary amines. The potential of the reported scaffold is strengthened by the
inhibition of STAT5-dependent transcription displayed by four of the synthesized compounds.
us that the imidazo[1,2-a]pyrazine portion has been highly used
Introduction
as a general support, able to interact with the hinge region of the
Protein kinases represent one of the widest protein families in
enzyme with one or two hydrogen bonds, while the presence of
humans and play a pivotal role in cell growth, differentiation,
substituents at different positions was fundamental to issue the
apoptosis, and metabolism.1 Inhibition of kinases has become an
attractive therapeutic strategy for several diseases and a vast array
of molecules that target the hinge region of the ATP-binding
pocket in the catalytic domain have been identified.2 In this
context, the synthesis of chemicals with a nitrogen heterocyclic
core, resembling the adenine portion of ATP, is still the most
followed synthetic approach in the search for kinase inhibitors
that compete with ATP in the catalytic domain.3 Consequently,
the design of novel molecular scaffolds able to mimic the purine
nucleus is a primary target for medicinal chemists involved in the
identification of novel kinase inhibitors. The possibility to create
such a library using a single reaction sequence should furthermore
facilitate the search for inhibitors, reducing the time and the costs
required.
desired isoform-selectivity.6
In connection with our ongoing work on the application of
MCRs to the synthesis of biologically active compounds,7 in this
communication we describe the development of a facile route to
3,8-diaminoimidazo[1,2-a]pyrazines via MCR with the intention
to use this core structure as a general support in the discovery
of novel kinase inhibitors. Surprisingly, a survey on Sci-Finder
Scholar revealed that this scaffold and the use of a multicomponent
reaction strategy to prepare such analogues were missed by both
academic and industrial researchers involved in kinase projects,
despite the strong resemblance between the adenine ring and the
3,8-diaminoimidazo[1,2-a]pyrazines.8
As the purine skeleton can have a different binding mode
depending on the presence of substituents on the amino group,9
we compared our structure with adenine and olomoucine, a well-
known CDK2 kinase inhibitor (Fig. 1).10
In our view, multicomponent reactions (MCRs)4 can address
this problem. Indeed, over the last decades, MCRs have attained
an increasing importance in heterocyclic chemistry, allowing for
the assembly of molecular arrays with high molecular diversity.5 A
survey on the most exploited structures in the kinase field showed
aDiSCAFF, Universita` degli Studi del Piemonte Orientale, via
Bovio 6, 28100, Novara, Italy. E-mail: tracey.pirali@pharm.unipmn.it;
Fax: (+39)0321375821; Tel: (+39)0321375853
bMolecular Biotechnology Center, Universita` degli Studi di Torino, 10126,
Torino, Italy
cDepartment of Biological Science and Bioengineering, Key Laboratory of
Biomedical Information Engineering of Ministry of Education, School of
Life Science and Technology, Xi’an Jiaotong University, Xi’an, 710049,
Shaanxi, P. R. China
† Electronic supplementary information (ESI) available: proton and car-
bon NMR spectra. See DOI: 10.1039/c1ob05336a
Fig. 1 Schematic representation of ATP (a) and olomoucine (b) binding
the catalytic domain of protein kinases. The kinase hinge region (red), the
gatekeeper (purple) and the binding pockets are indicated.
‡ These authors equally contributed to this paper
4144 | Org. Biomol. Chem., 2011, 9, 4144–4149
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Preliminary computational studies performed with VegaZZ11
revealed similarity between the 3,8-diaminoimidazol[1,2-
a]pyrazine scaffold and both ATP and olomoucine: these good
superimposition (RMSD < 1 A) could maintain the key hydrogen
bonds with the hinge region (Fig. 2).
conditions was necessary. Our initial attempts to run the
Blackburn reaction with the representative starting materials 2-
amino-3-chloropyrazine, 3-methoxybenzaldehyde and cyclohexyl
isocyanide proved to be somewhat discouraging. In the presence
of a small set of catalysts such as ammonium chloride14 or silica
sulfuric acid15 in methanol as solvent, a complex mixture was
detected, and the target compound was present in less than 20%.
Pre-formation of the imine intermediate was attempted using
toluene as solvent,16 but only a small conversion was observed.
Finally, TMSCl was used as a promoter, following the protocol
described by Krasavin et al.,17 and gratifyingly after 30 min at
reflux the reaction provided the desired product 1a in 68% of yield.
The imine pre-generation suppressed by-products formation and
avoided the undesired formation of the regioisomeric fused
imidazo-product due to the alternative reaction of the ring
nitrogen with the aldehyde, as was first reported by Bradley et al.18
Different isocyanides and aldehydes were reacted with the
2-amino-3-chloropyrazine in the MCR to give 3-amino-8-
chloroimidazo[1,2-a]pyrazines 1a–j in good yields (Fig. 3). Cy-
clohexyl, pentyl, and tert-butyl isocyanides participated in the
transformation. The reaction worked well with benzaldehyde,
[1,1¢-biphenyl]-4-carboxyaldehyde, electron-poor and electron-
rich aromatic aldehydes. Unfortunately, aliphatic aldehydes (hep-
tanal, isobutyraldehyde) failed to react under these conditions.
˚
Fig. 2 Superimposition between: (a) ATP (green) and 2c (yellow), (b)
olomoucine (cyan) and 2c, (c) olomoucine and 2g (pink). Structures of
ATP and olomoucine were obtained from Protein Data Bank, respectively
from crystal structure 1E8X and 1W0X.
To achieve the goal, our strategy involved the MCR discovered
in 1998 by the independent groups of Blackburn, Bienayme´,
and Groebke, where amidines (2-aminopyridine or pyrimidine
or pyrazine), aldehydes, and isocyanides react in the presence of
an acidic catalyst to give 3-amino-imidazoheterocycles.12 The 2-
aminoazine behaves as a bifunctional reagent, forming a Schiff
base with the aldehyde and providing a nucleophilic nitrogen to
intercept the nitrilium intermediate.
Results and discussion
We started from 2-amino-3-chloropyrazine, prepared from 2,3-
dichloropyrazine by nucleophilic aromatic substitution with aque-
ous ammonium hydroxide.13 By reacting it with different aldehydes
and isocyanides, we were able to synthesize the 3-amino-8-
chloroimidazo[1,2-a]pyrazines. Subsequent nucleophilic aromatic
substitution with aqueous ammonia or amines led to the desired
3,8-diaminoimidazo[1,2-a]pyrazines (Scheme 1).
Fig. 3 Synthesized intermediates.
The 3-amino-8-chloroimidazo[1,2-a]pyrazines were then re-
acted with aqueous ammonium hydroxide in a nucleophilic
aromatic substitution. The following optimized experimental
conditions were followed: the starting material was dissolved
in the minimum amount of dioxane, aqueous ammonium hy-
droxide (3 mL 100 mmol-1) was then added, and the reaction
was heated at 100 ◦C overnight in a Carius tube. Six 3,8-
diaminoimidazo[1,2-a]pyrazines 2a–f were synthesized in good to
excellent yields (Fig. 4). In order to demonstrate the generality
of our strategy, the reactions with primary and secondary amines
were also explored. The 3-amino-8-chloroimidazo[1,2-a]pyrazine
(1.0 equiv.) was dissolved in ethanol, the amine (benzylamine,
cyclohexylamine, cyclopropylamine, and morpholine) (3.0 equiv.)
was added, and the reaction was heated at reflux for 48 h. Five
3,8-diaminoimidazo[1,2-a]pyrazines 2g–k were prepared in good
yields (Fig. 4).
Scheme 1 Reagents and conditions: (i) 1. RCHO (1.2 equiv.), MeCN,
reflux, 2 h; 2. TMSCl (1.2 equiv.), MeCN/DCM, 30 min 3. R1NC
(1.2 equiv.), reflux, overnight; (ii) aq. NH4OH/dioxane, 100 ◦C, Carius
tube, overnight; (iii) R2R3NH (3 equiv.), EtOH, 48 h.
Most electron poor amidines, such as 2-amino-3-
chloropyrazine, are known to react slowly in the Blackburn
reaction and, indeed, a careful optimization of the experimental
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 4144–4149 | 4145
©

View Article Online
Fig. 5 HEK293 FLT3-ITD transfected with the reporter plasmid and
exposed for 24 h with increasing concentration of drugs. The reported
STAT5 activation is relative to the DMSO treated sample as 100%.
Fig. 4 Synthesized 3,8-diaminoimidazo[1,2-a]pyrazines.
The synthetic strategy used and the rationale of this preliminary
work were not aimed at identifying inhibitors of specific kinases.
Nonetheless, we felt it necessary to investigate the putative activity
of these compounds on two separate models. First, we investigated
all the synthesized molecules for inhibitory activity on PI3Ks.
None of the molecules displayed any significant inhibitory activity
on these kinases up to a concentration of 10 mM (not shown).
Second, as our aim was to demonstrate possible inhibitory activity
on kinases without investigating specificity, we opted for inves-
tigating a signalling cascade dependent on multiple kinases. In
brief, FLT3 is a receptor tyrosine kinase that plays a crucial role in
the maturation of hematopoietic progenitors.19 After stimulation
by its specific ligand FLT3, it dimerizes, autophosphorylates and
activates downstream signalling through AKT and ERK.20 In al-
most 25% of acute myeloid leukemias, the FLT3 receptor presents
a mutation of the juxtamembrane domain, named Internal
Tandem Duplication, which leads to a conformational change and
constitutive autophosphorylation.21 The activation of this cascade
leads directly or through other kinases to the phosphoryation and
activation of STAT5-dependent transcription.22 Such activation
can be detected via a specific reporter gene that expresses luciferase
(which yields light that can be read by a luminometer) when STAT5
is phosphorylated. Therefore, if upon addition of our compounds
a decrease in luminescence is recorded, it can be inferred that
one or more kinases are inhibited, although it does not allow to
pinpoint which specific kinase is inhibited. All compounds were
initially screened at a concentration of 100 nM. Of the eleven
compounds tested, four were able to reduce activity by at least
60% (2e, 2g, 2h, 2i). We therefore continued the characterization
of these latter compounds by performing full dose-response curves
(Fig. 5).
Conclusions
In conclusion, we have developed a straightforward synthetic
strategy for the synthesis of 3,8-diaminoimidazo[1,2-a]pyrazines,
starting from readily available starting materials. This synthesis
displays atom economy, efficiency and versatility, making its use
feasible for a combinatorial approach. Moreover, the reported
scaffold proved to be able to interfere with STAT5-dependent
transcription, demonstrating its inhibitory activity on kinases.
Experimental section
Commercially available reagents and solvents were used without
further purification and were purchased from Fluka–Aldrich or
Lancaster. Acetonitrile was dried by distillation from CaH2 and
stored on activated molecular sieves (4 A). Dichloromethane was
dried by distillation from P2O5 and stored on activated molecular
˚
˚
sieves (4 A). When needed, the reactions were performed in oven-
dried glassware under a positive pressure of dry nitrogen.
Melting points were determined in open glass capillary with
a Stuart scientific SMP3 apparatus and are uncorrected. All the
compounds were checked by IR (FT-IR THERMO-NICOLET
1
AVATAR), H and 13C APT (JEOL ECP 300 MHz), and mass
spectrometry (Thermo Finningan LCQ-deca XP-plus) equipped
with an ESI source and an ion trap detector. Chemical shifts are
reported in part per million (ppm). Column chromatography was
performed on silica gel (Merck Kieselgel 70-230 mesh ASTM)
using the indicated eluants. Thin layer chromatography (TLC) was
carried out on 5 ¥ 20 cm plates with a layer thickness of 0.25 mm
(Merck Silica gel 60 F254). When necessary they were developed
with KMnO4. Purity of tested compounds was established by
elemental analysis. Elemental analysis (C, H, N) of the target
compounds are within 0.4% of the calculated values.
The four compounds were able to inhibit fully STAT5-
dependent transcription at a concentration of 10 mM, suggesting
that at least a fundamental kinase in this process is inhibited. FLT3
inhibitors have been described previously23 and it is interesting
to note the structural similarity between purine derivatives (e.g.
AP23846)24 and the structures reported by us. It would therefore
not be surprising if our compounds were directly FLT3 inhibitors.
This is also supported by the complete abolishment of activity
observed at high concentrations.
General procedure for Groebke–Blackburn reaction (1a–j)
2-Amino-3-chloropyrazine (1.0 equiv.) is dissolved in acetonitrile
and aldehyde (1.2 equiv.) is added under nitrogen atmosphere. The
reaction is refluxed for 2 h. The solvent is removed under vacuum
4146 | Org. Biomol. Chem., 2011, 9, 4144–4149
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
and the resulting solid is taken up with toluene and evaporated
(¥ 3). A solution of TMSCl (1.2 equiv.) in dichloromethane is
added and the reaction is stirred at room temperature for 30 min
under nitrogen atmosphere. Then, a solution of the isocyanide
(1.2 equiv.) in acetonitrile is added and the reaction is stirred
at reflux overnight. The solvent is evaporated and the crude
material is purified by column chromatography to afford the
desired product.
brown solid; mp = 187–188 ◦C (found: C, 66.30; H, 45.95; N 17.00;
C18H19ClN4 requires C, 66.15; H, 5.86; N 17.14%); nmax(KBr)/cm-1
3303, 2931, 2850, 1536, 1455, 1355, 945, 775, 609; dH(300 MHz,
CDCl3) 7.99 (3 H, m), 7.66 (1 H, d, J 4.4), 7.47 (2 H, m), 7.37
(1 H, m), 2.97 (1 H, m), 1.81–1.56 (5 H, m), 1.25–1.12 (5 H,
m); dC(75 MHz, CDCl3) 142.7, 139.0, 132.9, 132.6 (2C), 128.2,
127.8, 127.0, 126.8, 115.1, 56.4, 33.8, 25.0, 24.3; MS (ESI) m/z
327 (M+H)+.
N-[8-chloro-2-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3-yl]-N-
cyclohexylamine, 1a. Eluent: PE/EtOAc 7 : 3, 68%, amorphous
yellow solid, (found: C, 63.99; H, 5.98; N 15.81; C19H21ClN4O
requires C, 63.95; H, 5.93; N 15.70%); nmax(KBr)/cm-1 3278, 2938,
2852, 1506, 1250, 1033, 836, 793; dH(300 MHz, CDCl3) 7.90 (3 H,
m), 7.57 (1 H, d, J 4.4), 6.93 (2 H, d, J 8.8), 3.82 (3 H, s), 3.20 (1
H, br s), 2.94 (1 H, m), 1.78–1.56 (5 H, m), 1.25–1.10 (5 H, m);
dC(75 MHz, CDCl3) 159.0, 142.1, 139.0, 132.6, 128.1, 126.9, 126.5,
124.9, 114.8, 113.3, 56.1, 54.6, 33.5, 24.0 (2C); m/z 357 (M+H)+.
N-(tert-butyl)-8-chloro-2-(4-nitrophenyl)imidazo[1,2-a]pyrazin-
3-amine, 1g. Column chromatography: PE/EtOAc 8 : 2; yield =
47%; yellow solid; mp = 167–168 ◦C (found: C, 55.71; H,
4.75; N 20.30; C16H16ClN2O2 requires C, 55.58; H, 4.66; N
20.25%); nmax(KBr)/cm-1 2924, 1598, 1518, 1346, 1201, 949, 866;
dH(300 MHz, CDCl3) 8.23 (4 H, m), 8.06, (1 H, d, J 4.4), 7.61 (1
H, d, J 4.4), 3.27 (1 H, br s), 1.04 (9 H, s); dC(75 MHz, CDCl3)
147.0, 143.4, 139.9, 139.8, 134.1, 128.6, 127.5, 127.3, 123.3, 116.0,
57.0, 30.1; MS (ESI) m/z 346 (M+H)+.
8-Chloro-N-pentyl-2-phenylimidazo[1,2-a]pyrazin-3-amine, 1b.
Column chromatography: PE/EtOAc 5 : 5; yield = 40%; orange
solid; mp = 122–123 ◦C (dec) (found: C, 64.80; H, 5.99; N 17.71;
C17H19ClN4 requires C, 64.86; H, 6.08; N 17.80%); nmax(KBr)/cm-1
2921, 2856, 1464, 1350, 1198, 1076, 951, 702; dH(300 MHz, CDCl3)
7.86 (3 H, m), 7.57 (1 H, d, J 4.7), 7.39 (2 H, m), 7.30 (1 H, m),
3.35 (1 H, br s), 3.01 (2 H, t, J 7.1), 1.52 (2 H, quint, J 7.1), 1.28
(4 H, m), 0.84 (3 H, t, J 7.1); dC(75 MHz, CDCl3) 142.8, 138.1,
132.9, 132.5, 129.3, 128.3, 127.9, 127.0, 126.9, 114.9, 47.8, 30.0,
28.7, 22.1, 13.6; MS (ESI) m/z 315 (M+H)+.
N-[8-chloro-2-(4-nitrophenyl)imidazo[1,2-a]pyrazin-3-yl]-N-
cyclohexylamine, 1h. Column chromatography: PE/EtOAc 5 : 5;
yield = 52%; orange solid; mp = 157–158 ◦C (found: C, 58.23; H,
4.98; N 18.73; C16H16Cl2N4 requires C, 58.14; H, 4.88; N 18.84%);
n
max(KBr)/cm-1 3340, 3092, 2925, 2852, 1514, 1342, 1108, 953,
862, 719; dH(300 MHz, CDCl3) 8.30 (4 H, s), 7.95 (1 H, d, J 4.7),
7.67 (1 H, d, J 4.7), 2.99 (1 H, m), 1.84–1.57 (5 H, m), 1.27–1.15
(5 H, m); dC(75 MHz, CDCl3) 147.2, 143.9, 139.5, 138.0, 136.8,
133.8, 127.8, 127.7, 123.8, 115.4, 57.2, 34.3, 25.3, 24.7; MS (ESI)
m/z 372 (M+H)+.
N-(tert-butyl)-8-chloro-2-phenylimidazo[1,2-a]pyrazin-3-amine,
1c. Column chromatography: PE/EtOAc 8 : 2; yield = 77%;
brown solid; mp = 122–123 ◦C (found: C, 63.90; H, 6.05; N 18.60;
C16H17ClN4 requires C, 63.89; H, 5.70; N 18.63%); nmax(KBr)/cm-1
3287, 2967, 1604, 1459, 1348, 1197, 954, 780, 702; dH(300 MHz,
CDCl3) 7.88 (2 H, m), 7.39 (5 H, m), 1.05 (9 H, s); dC(75 MHz,
CDCl3) 143.0, 142.6, 133.7, 133.1, 128.9, 128.5 (2C), 128.3 (2C),
126.5, 56.6, 30.5; MS (ESI) m/z 301 (M+H)+.
N-[8-chloro-2-(4-chlorophenyl)imidazo[1,2-a]pyrazin-3-yl]-N-
cyclohexylamine, 1i. Column chromatography: PE/EtOAc 5 : 5;
yield = 70%; yellow solid; mp = 137–139 ◦C (found: C, 59.90; H,
5.10; N 15.48; C18H18Cl2N4 requires C, 59.84; H, 5.02; N 15.51%);
n
max(KBr)/cm-1 3295, 3065, 2925, 2852, 1466, 1349, 1088, 944,
793; dH(300 MHz, CDCl3) 7.89 (3 H, m), 7.56 (1 H, d, J 4.4), 7.35
(2 H, d, J 8.5), 3.23 (1 H, br s), 2.91 (1 H, m), 1.76–1.55 (5 H, m),
1.22–1.08 (5 H, m); dC(75 MHz, CDCl3) 140.7, 138.2, 134.1, 132.9,
131.4, 128.7, 128.6, 127.3, 128.1, 115.4, 56.8, 34.1, 25.4, 24.6; MS
(ESI) m/z 361 (M+H)+.
N-(2-[1,1¢-biphenyl]-4-yl-8-chloroimidazo[1,2-a]pyrazin-3-yl)-
N-(tert-butyl)amine, 1d. Column chromatography: PE/EtOAc
8 : 2; yield = 58%; orange solid; mp = 177–178 ◦C (found: C,
70.20; H, 5.70; N 14.90; C22H21ClN4 requires C, 70.11; H, 5.62;
N 14.87%); nmax(KBr)/cm-1 2969, 2933, 1545, 1466, 1352, 1217,
951, 776; dH(300 MHz, CDCl3) 7.97 (3 H, m), 7.58 (4 H, m), 7.50
(1 H, d, J 4.6), 7.38 (2 H, m), 7.30 (1 H, m), 3.25 (1 H, br s), 0.99 (9
H, s); dC(75 MHz, CDCl3) d 142.6, 142.0, 140.4 (2C), 139.9, 133.6,
132.2, 128.4, 128.3, 127.1, 126.7, 126.6 (2C), 116.0, 56.6, 30.0; MS
(ESI) m/z 377 (M+H)+.
2-[1,1¢-Biphenyl]-4-yl-8-chloro-N-cyclohexylimidazo[1,2-a]pyra-
zin-3-amine, 1j. Column chromatography: PE/EtOAc 5 : 5;
yield = 68%; yellow solid; mp = 139–140 ◦C (found: C, 71.63; H,
5.79; N 14.01; C16H16Cl2N4 requires C, 71.54; H, 5.75; N 13.91%);
n
max(KBr)/cm-1 3307, 3277, 2925, 2852, 1484, 1357, 1201, 945,
769, 734; dH(300 MHz, CDCl3) 8.04 (2 H, d, J 8.2), 7.89 (1 H, d,
J 4.4), 7.64 (4 H, m), 7.54 (1 H, d, J 4.4), 7.43 (2 H, t, J 7.7), 7.33
(1 H, t, J 7.7), 3.32 (1 H, br d), 2.97 (1 H, m), 1.80–1.48 (5 H,
m), 1.27–1.13 (5 H, m); dC(75 MHz, CDCl3) 142.8, 140.6, 140.5,
140.1, 138.9, 133.2, 131.7, 128.6, 128.0, 127.4, 127.2, 126.9, 126.7,
115.3, 56.5, 34.0, 25.3, 24.4; MS (ESI) m/z 403 (M+H)+.
N-(tert-butyl)-8-chloro-2-(4-chlorophenyl)imidazo[1,2-a]pyra-
zin-3-amine, 1e. Column chromatography: PE/EtOAc 8 : 2;
yield = 54%; yellow solid; mp = 115–117 ◦C (found: C, 57.40; H,
4.90; N 16.85; C16H16Cl2N4 requires C, 57.33; H, 4.81; N 16.71%);
max(KBr)/cm-1 3317, 3284, 2962, 2924, 1603, 1462, 1342, 1205,
General procedure with ammonia (2a–f)
n
950, 832; dH(300 MHz, CDCl3) 8.00 (1 H, d, J 4.7), 7.84 (2 H,
d, J 9.6), 7.55 (1 H, d, J 4.7), 7.32 (2 H, d, J 9.6), 0.98 (9 H, s);
dC(75 MHz, CDCl3) 143.0, 141.5, 134.1, 133.8, 132.0, 129.5, 128.5,
127.1, 126.6, 116.1, 56.9, 30.2; MS (ESI) m/z 335 (M+H)+.
The 3-amino-8-chloroimidazo[1,2-a]pyrazine (0.200 mmol) is dis-
solved in dioxane in a Carius tube and 33% ammonium hydroxide
in water (5 mL) is added. The reaction is stirred at 100 ◦C for
three days. EtOAc is added and the organic phase is washed with
water (¥ 1), brine (¥ 1) and dried over sodium sulfate. The crude
material is purified by column chromatography.
8-Chloro-N-cyclohexyl-2-phenylimidazo[1,2-a]pyrazin-3-amine,
1f. Column chromatography: PE/EtOAc 7 : 3; yield = 56%;
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 4144–4149 | 4147
©

View Article Online
N-[8-amino-2-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3-yl]-N-
cyclohexylamine, 2a. Column chromatography: PE/EtOAc 4 : 6;
yield = 91%; brown solid; mp = 107–109 ◦C (found: C, 67.71; H,
6.93; N 20.72; C19H23N5O requires C, 67.63; H, 6.87; N 20.76%);
CDCl3) 149.8, 136.0, 133.8, 128.9, 128.6, 128.0 (2C), 127.5, 127.3,
108.1, 57.1, 34.3, 25.8, 25.0; MS (ESI) m/z 308 (M+H)+.
General procedure with amine (2g–k)
n
max(KBr)/cm-1 2926, 2852, 1611, 1507, 1250, 1174, 1031, 838;
The 3-amino-8-chloroimidazo[1,2-a]pyrazine (1.0 equiv.) is dis-
solved in ethanol and the amine (3.0 equiv.) is added. The reaction
is stirred at reflux for 24 h. The solvent is evaporated under vacuum
and the crude material is purified by column chromatography.
dH(300 MHz, CDCl3) 7.87 (2 H, d, J 8.8), 7.43 (1 H, d, J 4.9), 7.22
(1 H, d, J 4.9), 6.99 (2 H, d, J 8.8), 3.84 (3 H, s), 3.13 (1 H, d, J
5.8), 2.93 (1 H, m), 1.81–1.56 (5 H, m), 1.25–1.15 (5 H, m); MS
(ESI) m/z 337 (M+H)+.
N-benzyl-N-[3-(tert-butylamino)-2-(4-nitrophenyl)imidazo[1,2-
a]pyrazin-8-yl]amine, 2g. Column chromatography: PE/EtOAc
8 : 2; yield = 68%; orange oil; (found: C, 66.40; H, 5.95; N 20.17;
C23H24N6O2 requires C, 66.33; H, 5.81; N 20.18%); nmax(KBr)/cm-1
3399, 2973, 1599, 1547, 1341, 1203, 856, 739 cm-1; dH(300 MHz,
CDCl3) 8.24 (4 H, s), 7.43–7.27 (7 H, m), 6.41 (1 H, br t), 4.78 (2 H,
d, J 5.8), 2.95 (1 H, br s), 1.08 (9 H, s); dC(75 MHz, CDCl3) 148.9,
146.5, 141.3, 138.3, 136.1, 130.0, 128.6, 128.3, 128.0, 127.8, 127.4,
127.0, 123.5, 107.3, 56.8, 44.6, 30.4; MS (ESI) m/z 417 (M+H)+.
N -(8-amino-2-phenylimidazo[1,2-a]pyrazin-3-yl)-N -pentyl-
amine, 2b. Column chromatography: PE/EtOAc 5 : 5; yield =
88%; yellow solid; mp = 132–133 ◦C (dec) (found: C, 69.21; H,
7.23; N 23.59; C17H21N5 requires C, 69.12; H, 7.17; N 23.71%);
n
max(KBr)/cm-1 2956, 2924, 2853, 1516, 1028; dH(300 MHz,
CDCl3) 7.87 (2 H, m), 7.50–7.26 (5 H, m), 3.20 (1 H, br t), 3.04 (2
H, q, J 6.9), 1.57 (2 H, quint, J 6.9), 1.35–1.18 (4 H, m), 0.85 (3
H, t, J 7.1); dC(75 MHz, CDCl3) 149.9, 135.7, 134.1, 129.6, 129.2,
128.6, 128.0, 127.4, 127.1, 108.3, 48.9, 30.7, 30.0, 29.5, 14.3; MS
(ESI) m/z 296 (M+H)+.
N-cyclohexyl-N-[3-(cyclohexylamino)-2-(4-nitrophenyl)imida-
zo[1,2-a]pyrazin-8-yl]amine, 2h. Column chromatography:
PE/EtOAc 8 : 2; yield = 78%; orange solid; mp = 149–150 ◦C
(dec) (found: C, 66.40; H, 6.97; N 19.30; C24H30N6O2 requires
C, 66.34; H, 6.96; N 19.34%); nmax(KBr)/cm-1 2929, 2850, 1508,
1340, 1108, 854; dH(300 MHz, CDCl3) 8.28 (4 H, s), 7.34 (1 H, d,
J 4.9), 7.25 (1 H, d, J 4.9), 5.93 (1 H, br d), 3.02 (1 H, br d), 2.94
(1 H, m), 2.12 (1 H, m), 1.82–1.15 (20 H, m); dC(75 MHz, CDCl3)
148.6, 146.5, 140.9, 132.9, 129.9, 128.9, 128.8, 127.0, 124.0, 106.0,
57.4, 49.3, 34.4, 33.3, 25.7, 25.6, 25.1, 24.9; MS (ESI) m/z 435
(M+H)+.
N-(8-amino-2-phenylimidazo[1,2-a]pyrazin-3-yl)-N-(tert-butyl)-
amine, 2c. Column chromatography: PE/EtOAc 4 : 6; yield =
81%; yellow solid; mp = 133–134 ◦C (dec) (found: C, 68.35; H,
6.73; N 24.92; C16H19N5 requires C, 68.30; H, 6.81; N 24.89%);
n
max(KBr)/cm-1 3467, 3292, 2923, 1742, 1637, 1517, 1203, 755,
699; dH(300 MHz, CDCl3) 7.84 (2 H, m), 7.54 (1 H, d, J 4.7), 7.40
(2 H, m), 7.29 (2 H, m), 1.02 (9 H, s); dC(75 MHz, CDCl3) 149.1,
139.1, 134.2, 128.8, 128.0, 127.6, 127.0, 126.8, 125.9, 108.5, 56.1,
29.8; MS (ESI) m/z 282 (M+H)+.
N-[2-(4-chlorophenyl)-3-(cyclohexylamino)imidazo[1,2-a]pyra-
zin-8-yl]-N-cyclopropylamine, 2i. Column chromatography:
PE/EtOAc 8 : 2; yield = 50%; brown solid; mp = 79–80 ◦C (found:
C, 66.08; H, 6.40; N 18.40; C21H24ClN5 requires C, 66.04; H, 6.33;
N 18.34%); nmax(KBr)/cm-1 2929, 2852, 1541, 1490, 1091, 1013,
836; dH(300 MHz, CDCl3) 7.92 (2 H, d, J 8.2), 7.40–7.28 (4 H,
m), 2.95 (3 H, m), 1.84–1.55 (5 H, m), 1.22–1.05 (5 H, m), 0.90
(4 H, m); dC(75 MHz, CDCl3) 150.9, 135.9, 135.0, 134.6, 133.7,
130.1 (2C), 129.5, 129.0, 108.6, 58.4, 35.6, 27.0, 26.1, 25.0, 8.60;
MS (ESI) m/z 382 (M+H)+.
N-(8-amino-2-[1,1¢-biphenyl]-4-ylimidazo[1,2-a]pyrazin-3-yl)-
N-(tert-butyl)amine, 2d. Purification: filtration and water
washes; yield = 83%; pale yellow solid; mp = 150–152 ◦C (found:
C, 73.99; H, 6.38; N 19.61; C22H23N5 requires C, 73.92; H, 6.49;
N 19.59%); nmax(KBr)/cm-1 3475, 3286, 3096, 2971, 1635, 1519,
1199, 844, 759; dH(300 MHz, CDCl3) 7.96 (3 H, m), 7.67 (4 H,
m), 7.56 (1 H, d, J 4.6), 7.46 (2 H, m), 7.29 (1 H, m), 5.72 (1 H,
br s), 2.02 (2 H, br s), 1.07 (9 H, s); dC(75 MHz, CDCl3) 153.6,
144.6, 144.5, 143.0, 137.2 (2C), 132.8, 132.4, 131.5, 131.1, 130.9
(2C), 130.7, 112.8, 60.5, 34.1; MS (ESI) m/z 358 (M+H)+.
N-[2-[1,1¢-biphenyl]-4-yl-3-(cyclohexylamino)imidazo[1,2-a]py-
razin-8-yl]-N-cyclopropylamine, 2j. Column chromatography:
PE/EtOAc 5 : 5; yield = 42%; brown oil (found: C, 76.66; H,
6.98; N 16.65; C27H29N5 requires C, 76.56; H, 6.90; N 16.53%);
N-[8-amino-2-(4-chlorophenyl)imidazo[1,2-a]pyrazin-3-yl]-N-
(tert-butyl)amine, 2e. Column chromatography: PE/EtOAc 5 : 5;
yield = 50%; yellow solid; mp = 154–155 ◦C (dec) (found: C,
60.89; H, 5.81; N 22.20; C16H18ClN5 requires C, 60.85; H, 5.75;
N 22.18%); nmax(KBr)/cm-1 2961, 2884, 2806, 1635, 1522, 1482,
1439, 1202, 1012, 832; dH(300 MHz, CDCl3) 7.88 (2 H, d, J 8.5),
7.51 (1 H, d, J 4.7), 7.39 (2 H, d, J 8.5), 7.28 (1 H, d, J 4.7), 5.76 (2
H, br s), 2.98 (1 H, br s), 0.99 (9 H, s); dC(75 MHz, CDCl3) 149.8,
138.4, 133.5, 133.4, 129.5, 129.3, 128.6, 127.7, 108.8, 126.1, 56.5,
30.5; MS (ESI) m/z 316 (M+H)+.
n
max(KBr)/cm-1 2950, 2852, 1538, 1512, 1091, 1013, 836, 720;
dH(300 MHz, CDCl3) 8.03 (2 H, d, J 8.2), 7.70–7.35 (9 H, m),
2.95 (2 H, m), 1.86–1.56 (5 H, m), 1.22–1.05 (5 H, m), 0.96 (2 H,
m), 0.83 (2 H, m); dC(75 MHz, CDCl3) 149.4, 146.3, 140.9 (2C),
140.6 (2C), 132.4, 129.2, 127.9, 127.7, 127.6, 127.5, 127.3, 107.6,
57.4 (2C), 34.6, 25.9, 25.1, 7.67; MS (ESI) m/z 424 (M+H)+.
N-cyclohexyl-8-morpholino-2-(4-nitrophenyl)imidazo[1,2-a]py-
razin-3-amine, 2k. The product precipitates during the reaction
and is filtered under vacuum; yield = 77%; yellow amorphous solid
(found: C, 62.60; H, 6.23; N 19.91; C22H26N6O3 requires C, 62.54;
H, 6.20; N 19.89%); nmax(KBr)/cm-1 2936, 2856, 1598, 1510, 1338,
1115; dH(300 MHz, CDCl3) 8.27 (4 H, m), 7.38 (2 H, m), 4.32 (4
H, t, J 4.1), 3.89 (4 H, t, J 4.1), 2.98 (1 H, m), 1.80–1.58 (5 H, m),
1.27–1.13 (5 H, m); dC(75 MHz, CDCl3) 151.7, 148.6, 142.8, 134.5,
N-(8-amino-2-phenylimidazo[1,2-a]pyrazin-3-yl)-N-cyclohexyl-
amine, 2f. Column chromatography: PE/EtOAc 5 : 5; yield =
74%; brown solid; mp = 134–135 ◦C (found: C, 70.39; H,
6.90; N 22.89; C18H21N5 requires C, 70.33; H, 6.89; N 22.78%);
n
max(KBr)/cm-1 2360, 2341, 764, 749; dH(300 MHz, CDCl3) 7.94
(2 H, m), 7.45 (3 H, m), 7.31 (1 H, m), 7.26 (1 H, m), 3.16 (1 H, br d),
2.98 (1 H, m), 1.80–1.58 (5 H, m), 1.27–1.13 (5 H, m); dC(75 MHz,
4148 | Org. Biomol. Chem., 2011, 9, 4144–4149
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
132.8, 130.2, 130.0, 129.1, 126.0, 109.7, 69.2, 59.3, 48.9, 36.5, 27.7,
27.0; MS (ESI) m/z 423 (M+H)+.
(d) WO 2007028051 pan-kinase inhibitors; (e) WO 2005085252 JNK
inhibitors; (f) WO 2004072081 EphB4 inhibitors.
7 (a) T. Pirali, V. Faccio, R. Mossetti, A. A. Grolla, S. Di Micco, G.
Bifulco, A. A. Genazzani and G. C. Tron, Mol. Diversity, 2010, 14, 109–
121; (b) A. A. Grolla, V. Podesta`, M. G. Chini, S. Di Micco, A. Vallario,
A. A. Genazzani, P. L. Canonico, G. Bifulco, G. C. Tron, G. Sorba and
T. Pirali, J. Med. Chem., 2009, 52, 2776–2785; (c) T. Pirali, G. Callipari,
E. Ercolano, A. A. Genazzani, G. B. Giovenzana and G. C. Tron, Org.
Lett., 2008, 10, 4199–4202; (d) T. Pirali, G. C. Tron, G. Masson and J.
Zhu, Org. Lett., 2007, 9, 5275–5278; (e) M. Giustiniano, T. Pirali, A.
Massarotti, B. Biletta, E. Novellino, P. Campiglia, G. Sorba and G. C.
Tron, Synthesis, 2010, 4107–4118; (f) O. Mesenzani, A. Massarotti, M.
Giustiniano, T. Pirali, V. Bevilacqua, A. Caldarelli, P. L. Canonico, G.
Sorba, E. Novellino, A. A. Genazzani and G. C. Tron, Bioorg. Med.
Chem. Lett., 2011, 21, 764–768.
8 To the best of our knowledge only three substituted 3,8-
diaminoimidazo[1,2-a]pyrazines have been synthesized in a more
general patent: WO 2005/120513 A1.
9 J. Shen and D. Borcherding, Med. Chem. Res., 2003, 12, 435–443.
10 L. Havlicek, J. Hanus, J. Vesely, S. Leclerc, L. Meijer, G. Shaw and M.
Strnad, J. Med. Chem., 1997, 40, 408–412.
11 A. Pedretti, l. Villa and G. Vistoli, VEGA: a versatile program to
convert, handle and visualize molecular structure on Windows-based
PCs, J. Mol. Graphics Modell., 2002, 21, 47–49.
12 (a) C. Blackburn, B. Guan, P. Fleming, K. Shiosaki and S. Tsai,
Tetrahedron Lett., 1998, 39, 3635–3638; (b) H. Bienayme´ and K. Bouzid,
Angew. Chem., Int. Ed., 1998, 37, 2234–2237; (c) K. Groebke, L. Weber
and F. Mehlin, Synlett, 1998, 6, 661–663; (d) for a recent review on the
synthesis of bicyclic imidazo[1,2-x]-heterocycle, see: C. Hulme, Mol.
Diversity, 2008, 12, 1–15.
Cell culture and detection of STAT5 phosphorylation and
activation
HEK293 stable expressing FLT3-ITD (kind gift of Prof. Thomas
Fisher) where cultured in RPMI1640 10% FCS, 1% Pen/Strep
and Glutamine. 10 000 cells where plated in a 96 well plate
in 50 mL of medium 6 h before transfection. For transfection,
0.2 mL of Lipofectamine 2000 (Invitrogen) was mixed in 20 mL
of Opti-MEM (Invitrogen) with 50 ng of LHRE-FF plasmid
(Lactogene Hormone Responsive Element-Firefly luciferase) and
50 ng of pGL4.73 renilla luciferase plasmid (Promega) that served,
respectively, as reporter of STAT5 activation and normalization
of transfection and viability. After 18 h, the cells were exposed
for 24 h to the compounds dissolved in DMSO, which served
as a negative control. Luciferase activity was then measured by
a commercial luciferase assay (Promega). All experiments were
conducted at least in triplicate.
Acknowledgements
We gratefully acknowledge Regione Piemonte, DRUIDI project.
13 C. R. Hopkins and N. Collar, Tetrahedron Lett., 2004, 45, 8631–
8633.
14 A. Shaabani, F. Rezazadeh and E. Soleimani, Monatsh. Chem., 2008,
Notes and references
139, 931–933.
1 G. Manning, D. B. Whyte, R. Martinez, T. Hunter and S. Sudarsanam,
Science, 2002, 298, 1912–1934.
15 A. Shaabani, E. Soleiman and A. Maleki, Monatsh. Chem., 2007, 138,
73–76.
2 (a) P. Cohen, Nat. Rev. Drug Discovery, 2002, 1, 309–315; (b) Kinase
inhibition drugs, ed. R. Li and J. A. Stafford, Wiley-VCH: Weinheim,
2009 and references therein.
16 V. Z. Parchynsky, O. Shulvalova, O. Ushakova, D. V. Kravchenko and
M. Krasavin, Tetrahedron Lett., 2006, 47, 947–951.
17 M. Krasavin, S. Tsirulnikov, M. Nikulnikov, V. Kysil and A.
Ivachtchenko, Tetrahedron Lett., 2008, 49, 5241–5243.
18 G. S. Mandair, M. Light, A. Russell, M. Hursthouse and M. Bradley,
Tetrahedron Lett., 2002, 43, 4267–4269.
19 S. D. Lyman and S. E. Jacobsen, Blood, 1998, 91, 1101–1134.
20 C. Hannum, J. Culpepper, D. Campbell, T. McClanahan, S. Zurawski,
S. J. F. Bazan, R. Kastelein, S. Hudak, J. Wagner and J. Mattson,
Nature, 1994, 368, 643–648.
3 (a) N. S. Gray, L. Wodicka, A. M. Thunnissen, T. C. Norman, S. Kwon,
F. H. Espinoza, D. O. Morgan, G. Barnes, S. LeClerc, L. Meijer, S. H.
Kim, D. J. Lockhart and P. G. Schultz, Science, 1998, 281, 533–538;
(b) S. P. Davies, H. Reddy, M. Caivano and P. Cohen, Biochem. J., 2000,
351, 95–105; (c) M. E. M. Noble, J. A. Endicott and L. N. Johnson,
Science, 2004, 303, 1800–1805; (d) H. Weinmann and R. Metternich,
ChemBioChem, 2005, 6, 455–459; (e) J. J.-L. Liao, J. Med. Chem., 2007,
50, 409–424.
4 Multicomponent Reactions, ed. J. Zhu and H. Bienayme´, Wiley-VCH:
Weinheim, 2005 and references therein.
5 Synthesis of heterocycles via multicomponent reactions I and II, ed.
R. V. A. Orru and E. Ruijeter, Springer GmbH, Berlin, 2010 and
references therein.
21 J. Griffith, J. Black, J. C. Faerman, L. Swenson, M. Wynn, F. Lu, J.
Lippke and K. Saxena, Mol. Cell, 2004, 13, 169–178.
22 K. Spiekermann, K. Bagrintseva, R. Schwab, K. Schmieja and W.
Hiddemann, Clin Cancer Res., 2003, 9, 2140–2150.
23 S. Schenone, C. Brullo and M. Botta, Curr. Med. Chem., 2008, 15,
3113–3132.
6 For examples of using the imidazo[1,2-a]pyrazine nucleus, see the
following patents: (a) WO 2009135885 MAPKAPK5 inhibitors; (b) WO
2009102468 SYK inhibitors; (c) WO 2009021990 PI3K/Akt inhibitors;
24 S. Schnittger, C. Schoch, M. Dugas, W. Kern, P. Staib, C. Wuchter,
H. Lo¨ffler, C. Sauerland, H. Serve, T. Bu¨chner, T. Haferlach and W.
Hiddemann, Blood, 2002, 100, 59–66.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 4144–4149 | 4149
©