Synthesis and biological evaluation of benzo[4,5]imidazo[1,2-c]pyrimidine and benzo[4,5]imidazo[1,2-a]pyrazine derivatives as anaplastic lymphoma kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.01.007

Chromosomal translocations involving anaplastic lymphoma kinase (ALK) are the driving mutations for a range of cancers and ALK is thus considered an attractive therapeutic target. We synthesized a series of functionalized benzo[4,5]imidazo[1,2-c]pyrimidines and benzo[4,5]imidazo[1,2-a]pyrazines by an aza-Graebe-Ullman reaction, followed by palladium-catalyzed cross-coupling reactions. A sequential regioselective cross-coupling route is reported for the synthesis of unsymmetrically disubstituted benzo[4,5]imidazo[1,2-a]pyrazines. The inhibition of ALK was evaluated and compound 19 in particular showed good activity against both the wild type and crizotinib-resistant L1196M mutant in vitro and in ALK-transfected BaF3 cells.

Full text of DOI:10.1016/j.bmc.2014.01.007

Bioorganic & Medicinal Chemistry 22 (2014) 1303–1312
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of benzo[4,5]imidazo[1,2-c]
pyrimidine and benzo[4,5]imidazo[1,2-a]pyrazine derivatives
as anaplastic lymphoma kinase inhibitors
Sébastien Tardy a, Alexandre Orsato a, Luca Mologni b, William H. Bisson c,1, Carla Donadoni b,
Carlo Gambacorti-Passerini b, Leonardo Scapozza c, David Gueyrard a, Peter G. Goekjian a,
⇑
a Université de Lyon, Laboratoire Chimie Organique 2-Glycochimie, ICBMS, UMR-5246, CNRS-Université Claude Bernard Lyon 1, Bât. 308 CPE Lyon, 43 Bd du 11 Novembre 1918,
F-69622 Villeurbanne, France
b University of Milano-Bicocca, Department of Health Sciences, Via Cadore 48, 20052 Monza, Italy
c University of Geneva, School of Pharmaceutical Sciences, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Chromosomal translocations involving anaplastic lymphoma kinase (ALK) are the driving mutations for a
range of cancers and ALK is thus considered an attractive therapeutic target. We synthesized a series of
functionalized benzo[4,5]imidazo[1,2-c]pyrimidines and benzo[4,5]imidazo[1,2-a]pyrazines by an
aza-Graebe–Ullman reaction, followed by palladium-catalyzed cross-coupling reactions. A sequential
regioselective cross-coupling route is reported for the synthesis of unsymmetrically disubstituted
benzo[4,5]imidazo[1,2-a]pyrazines. The inhibition of ALK was evaluated and compound 19 in particular
showed good activity against both the wild type and crizotinib-resistant L1196M mutant in vitro and in
ALK-transfected BaF3 cells.
Received 24 August 2013
Revised 18 December 2013
Accepted 3 January 2014
Available online 10 January 2014
Keywords:
Anaplastic lymphoma kinase
Kinase inhibitors
Ó 2014 Elsevier Ltd. All rights reserved.
Aza-Graebe–Ullman
Regioselective cross coupling
Palladium catalyzed coupling
1. Introduction
attractive target for cancer therapies.7 Indeed, the first clinical
inhibitor of ALK, crizotinib, yielded impressive responses in
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase
which belongs to the insulin receptor family, initially identified as
the catalytic domain of the NPM/ALK fusion kinase, expressed in
most CD30+ anaplastic large-cell lymphoma (ALCL).1 Since then,
a variety of oncogenic fusion proteins resulting from chromosomal
translocations of the intracellular domain of ALK have been identi-
fied in a wide range of tumors, including non-small cell lung cancer
(NSCLC), inflammatory myofibroplastic tumor, diffuse large B-cell
lymphoma, breast cancer, colon cancer, and others.2,3 While the
amino terminal partner of the fusion varies from case to case, it al-
ways provides a strong homodimerization interface, thus causing
aberrant ligand-independent activation of ALK kinase activity. In
addition, kinase-activating point mutations of full-length ALK have
been reported and biologically validated in hereditary and sporadic
neuroblastomas as well as in anaplastic thyroid carcinomas.4,5 The
oncogenic role of ALK has been defined in numerous preclinical
models.6 Therefore, the selective inhibition of ALK emerged as an
patients with advanced NSCLC and ALCL, although acquired resis-
tance invariably develops, often as a result of point mutations
within the ALK catalytic domain.8,9 A large effort is ongoing to
develop more potent second-generation inhibitors that are able
to overcome such drug resistance.3
Chugai and Roche’s tetracyclic clinical candidate AF802 as well
as our own results with substituted a-carbolines have shown that
fused nitrogen-containing heterocyclic systems are privileged
skeletons for targeting the ALK tyrosine kinase domain (Fig. 1).10
We therefore turned our attention to the development of selective
NH
Cl
Me
N
N
N
N
F
N
O
N
Cl
O
HN
H2N
N
N
Crizotinib
AF802
N
⇑
Corresponding author. Tel.: +33 4 72448183; fax: +33 4 72448109.
E-mail address: goekjian@univ-lyon1.fr (P.G. Goekjian).
Present address: Department of Environmental and Molecular Toxicology,
N
N
H
R314
O
1
Environmental Health Sciences Center, Oregon State University, Corvallis, OR USA
Figure 1. Alk inhibitors.
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2014.01.007

1304
S. Tardy et al. / Bioorg. Med. Chem. 22 (2014) 1303–1312
Cl
Polyphosphoric acid (PPA)-mediated cyclization of intermedi-
ates 3 and 4 produced the desired compounds 1 and 2, respec-
tively, in satisfactory yields (Scheme 3). It should be noted that
the N-cyclized products 1 and 2 were obtained exclusively, rather
than the C-cyclized diazacarbazoles (9H-indolo[2,3-e]pyrimidine
and 9H-indolo[2,3-b]pyrazine). This selectivity is opposite that
which we observed with 1-pyrid-2-ylbenzotriazoles, which gave
exclusively the C-cyclized 9H-indolo[2,3-b]pyridines.10a Moreau
et al. observed a mixture of the N-cyclized 1H-Pyrrolo[30,20:
5,6]pyrido[1,2-a]benzimidazole and C-cyclized 1,9-dihydropyrrol-
o[30,20:5,6]pyrido[2,3-b]indole in the cyclization of 7-azaindol-
N
Cl
N
N
N
HN
HN
N
N
N
N
3
N
N
N
Cl
N
+
N
2
Cl
1
4,6-dichloro-pyrimidine
Cl
N
N
N
N
N
N
N
N
Cl
N
+
N
Cl
N
Cl
4
2,6-dichloro-pyrazine
Scheme 1. Synthetic route to target heterocyclic cores.
6-ylbenzotriazoles at high temperatures.12a
A
number of
C-cyclization and N-cyclization reactions have been reported in
the literature and the selectivity appears to depend on steric and
electronic factors as well as experimental conditions, although
the basis for the selectivity remains unclear.12
We then investigated the functionalization of these two hetero-
cyclic structures using palladium-catalyzed coupling reactions.
inhibitors of ALK based on two original heterocyclic cores: the
benzo[4,5]imidazo[1,2-c]pyrimidine and the benzo[4,5]imi-
1
dazo[1,2-a]pyrazine 2. These two tricyclic systems were synthe-
sized by a route analogous to that reported by Bergman and
Vallberg,11 with an added halogen substituent in order to incorpo-
rate diversity at a late stage through transition metal catalyzed
chemistry (Scheme 1). Condensation of either 4,6-dichloropyrimi-
dine or 2,6-dichloropyrazine with benzotriazole affords com-
pounds 3 and 4, which undergo a Graebe–Ullman-like cyclization
to produce 1 and 2, respectively.
Compound 1, for example, bearing a chlorine atom at the
a
position to a nitrogen, was expected to be highly reactive under
Pd-catalyzed coupling conditions. Surprisingly, however, no
Suzuki–Miyaura reaction13 was observed using either Pd(Ph3)4 or
Pd2(dba)3/X-Phos in the coupling reaction with boronic acid part-
ners. The starting material was recovered intact.
2. Results and discussion
In contrast, the Buchwald–Hartwig cross coupling reaction14
worked well using Pd2(dba)3/X-Phos in combination with aromatic
amines. Therefore, the reaction was performed with aniline and
two different substituted anilines to provide derivatives 5, 6 and
7 in good yields (Table 1). The difference in outcome between
the two reactions suggests that the transmetallation or reductive
elimination steps were at the origin of the lack of reactivity in
the Suzuki–Miyaura reaction, rather than the oxidative insertion.
In order to develop a sequential cross-coupling strategy, we
decided to investigate the regiochemistry of the bromination of
compound 1. Reaction of 3-chlorobenzo[4,5]imidazo[1,2-c]pyrimi-
dine (1) with N-bromosuccinimide (NBS) gave the dihalogenated
8-bromo derivative 8 in 68% yield (Scheme 4). The structure of
compound 8 was confirmed by NOE experiments.
The Buchwald–Hartwig coupling reaction between compound 8
and aniline, using Pd2(dba)3/X-Phos, gave compound 9 in disap-
pointing 16% yield. We therefore investigated the Suzuki–Miyaura
coupling with Pd(Ph3)4 and 4-methoxyphenyl boronic acid. Once
again, the coupled product 10 was isolated in low yield (9%)
(Scheme 5). In these two reactions, the starting material was not
Optimization of the coupling of the dichlorodiazines with ben-
zotriazole was necessary, as both systems contain two equivalent
electrophilic sites (Scheme 2). Indeed, in the case of the more
reactive 4,6-dichloropyrimidine, heating a neat equimolar ratio of
the two reagents led predominantly to the double substitution
product. It was therefore necessary to work with 2 equiv of 4,
6-dichloropyrimidine in order to favor the formation of the
mono-substitution product 3. Compound 3 was thus obtained in
87% yield after 2 h by starting the reaction at 80 °C and then
increasing the temperature to 110 °C.
The reactivity of 2,6-dichloropyrazine, in contrast, was rather
poor. As the dichloropyrazine tended to sublimate at temperatures
below those required for the reaction, it was necessary to work in a
sealed tube at 155 °C for 24 h to provide the desired intermediate 4
in a respectable 60% yield (Scheme 2).
Cl
Cl
N
N
N
HN
N
i)
+
N
N
N
N
3
N
N
N
N
Cl
Table 1
Buchwald reactions on 3-chloro-benzo[4,5]imidazo[1,2-c]pyrimidine (1)
N
N
N
N
HN
N
N
ii)
Cl
NH2
+
N
i)
Cl
Cl
N
+
5-7
Cl
4
N
R
N
1
Scheme 2. Reagents and conditions: (i) 4,6-dichloropyrimidine (2 equiv), benzo-
triazole (1 equiv), neat, 80–110 °C, 2 h, 3 = 87%. (ii) 2,6-Dichloropyrazine (1 equiv),
benzotriazole (1.1 equiv), neat, sealed tube, 155 °C, 24 h, 4 = 60%.
Entry
1
Anilines
Products
Yield (%)
67
N
H2N
H2N
H2N
5
6
7
N
N
H
N
N
2
3
NO2
95
65
N
N
H
O2N
N
N
OMe
N
N
MeO
H
Reaction conditions: (i) Pd2(dba)3 (8 mol %), X-Phos (16 mol %), K2CO3 (3 equiv),
t-BuOH (C = 0.2 M), anilines (1.5 equiv), 100 °C, overnight.
Scheme 3. Synthesis of heterocyclic skeletons 1 and 2.

S. Tardy et al. / Bioorg. Med. Chem. 22 (2014) 1303–1312
1305
n.o.e.
N
Br
N
N
N
N
i)
N
N
Cl
Cl
N
N
N
N
1
N
8
N
Cl
Br
B
i)
O
O
Cl
Br
+
Scheme 4. Reagents and comditions: (i) N-bromo-succinimide (1.5 equiv), THF
(C = 0.1 M), rt, 15 h, 8 = 68%.
N
N
N
N
N
N
N
11
33%
N
recovered and the remaining material consisted of a complex
mixture of degradation products, which were not characterized.
Nevertheless, surprisingly, the reaction was completely regioselec-
tive in favor of the chlorine-substituted position, as neither the
8-substituted nor the disubstituted compound was detected.
Given the disappointing reactivity of compounds 1 and 8 in
cross coupling reactions, we turned our attention to the study of
the reactivity of core structure 2. We investigated the bromination
of compound 2 with bromine or N-bromo-succinimide to provide
11 (Scheme 6). Using bromine, the desired dihalogenated com-
pound was obtained in 53% yield, but compound 11 was not easily
separated from the remaining starting material. On the other hand,
bromination with NBS was slower and less efficient (only 29%
yield) but the purification was easier due to the absence of starting
material. The position of the bromine atom on the heterocyclic
structure was confirmed by 15N–1H NMR HMBC correlation.
When the dihalogenated benzo[4,5]imidazo[1,2-a]pyrazine 11
was submitted to Suzuki–Miyaura coupling with Pd(Ph3)4 at
70 °C using a slight excess of boronic acid or boronic acid pinacol
ester a lack of regioselectivity was observed. Indeed, using the
boronic acid, the conversion was satisfactory, but we obtained a
mixture of both mono- and disubstituted products. The corre-
sponding reaction using the boronic acid pinacol ester at 70 °C
(Scheme 7) provided an inseparable 7:3 mixture of the two
mono-substituted compounds in 33% yield in which the major
compound was substituted on the chlorinated position.
Scheme 7. Reagents and conditions: (i) Pd(Ph3)4 (10 mol %), K2CO3 (3 equiv), ester
pinacol borane (1.3 equiv), dioxane/H2O 5:1, 70 °C, overnight.
N
N
N
N
N
Cl
Br
N
i)
N
+
N
77%
N
11
B
N
O
O
N
N
12
Scheme 8. Reagents and conditions: (i) Pd(Ph3)4 (10 mol %), K2CO3 (4 equiv), ester
pinacol borane (3 equiv), dioxane/H2O 5:1, 100 °C, overnight.
Cl
Cl
Cl
i)
N
N
N
N
N
N
2
13
Scheme 9. Reagents and conditions: (i) NCS (1.6 equiv), DMF (C = 0.1 M), rt, 21 h,
13 = 54%.
cinimide to give the dichlorinated compound 13 in a moderate 54%
yield (Scheme 9).
Moreover, we obtained complete conversion of the starting
material to the disubstituted structure using an excess (3 equiv)
of the boronic acid pinacol ester and working at 100 °C overnight.
Compound 12 was obtained in a high 77% yield by this procedure
(Scheme 8).
In order to increase the regioselectivity and build an efficient
sequential route for functionalization of the heterocyclic core 11,
we replaced the bromine atom by chlorine. Thus, pyrazino-ben-
zimidazol 2 was chlorinated in the same position by N-chloro-suc-
As expected, when compound 13 was submitted to Suzuki–
Miyaura coupling reaction, the selectivity was strongly in favor
of position 4. The reaction occurred only on the activated chlori-
nated centre, leaving the chlorine on the phenyl moiety unreacted.
Using p-methoxyphenyl boronic acid and vinylphenyl boronic acid,
we synthesized compounds 14 and 15 in good yields (Table 2). As
expected, the best result was obtained using the more electron rich
4-methoxyphenyl boronic acid.
We then turned our attention to functionalizing the other chlo-
rinated position of 14 and 15. It was necessary to use more drastic
conditions using the Pd(OAc)2/S-Phos system, 2 equiv of the cou-
pling partner, at 100 °C in n-butanol for 16 h. Starting from com-
pound 14, three products 16, 17, and 18 were prepared in
moderate to good yields (Table 3). In order to use boronic acid
derivatives with a different range of reactivity, size and polarity,
we selected three different boronic reagents: 4-N-acetamido-phe-
nyl boronic acid, 4-(4-N-methyl-piperazin-1-yl)phenyl boronic
acid pinacol ester and E-styryl boronic acid.
Finally, in order to illustrate the scope of our strategy, we
synthesized the inverse-substituted analogue of compound 18
from 15, using 4-methoxyphenyl boronic acid under the same con-
ditions (Scheme 10). Thus, we obtained the derivative 19 in a fair
41% yield. These results demonstrate that the sequential function-
alization of the heterocyclic scaffold 2 was achieved, providing an
original strategy for the orthogonal substitution of compound 13.
The biological activities of compounds were assessed in a series
of biological assays against ALK. The compounds were tested in an
in vitro ELISA assay15 using a more physiologically relevant
NH2
Br
Br
Br
Br
N
N
N
N
i)
N
N
N
+
Cl
Cl
N
H
N
8
N
N
9
B(OH)2
O
ii)
N
+
O
N
8
10
Scheme 5. Reagents and conditions: (i) Pd2(dba)3 (8 mol %), X-Phos (16 mol %),
K2CO3 (3 equiv), t-BuOH (C = 0.2 M), aniline (1.5 equiv), 100 °C, overnight, 9 = 16%.
(ii) Pd(Ph3)4 (10 mol %), K2CO3 (3 equiv), dioxane/H2O 5:1, boronic acid (1.2 equiv),
70 °C, overnight, 10 = 9%.
Cl
Cl
Br
i) or ii)
N
N
N
N
N
2
N
11
concentration of ATP (300
alytic domain and the crizotinib-resistant gatekeeper mutant
lM), against both wild-type (wt) ALK cat-
Scheme 6. Reagents and conditions: (i) Br2 (2 equiv), DCM (C = 0.2 M), rt, 16 h,
11 = 53%. (ii) NBS (2.5 equiv), DMF (C = 0.1 M), rt, 24 h, 11 = 29%.

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S. Tardy et al. / Bioorg. Med. Chem. 22 (2014) 1303–1312
Table 2
Suzuki–Miyaura reactions on 4,7-dichloro-benzo[4,5]imidazo[1,2-a]pyrazine (13)
O
O
i)
+
Cl
Cl
Ar
Cl
Cl
N
N
i)
N
N
N
N
N
+ ArBH(OH)2
N
B(OH)2
N
N
19
N
N
15
13
14,15
Scheme 10. Reagents and conditions: (i) Pd(AcO)2 (10 mol %), S-Phos (20 mol %),
K3PO4 (3 equiv), n-BuOH (C = 0.05 M), boronic acid or pinacol ester (2 equiv), 100 °C,
14 h, (19) = 41%.
Entry
1
Boronic acid
Products
Yield (%)
86
OMe
OMe
parental Ba/F3 cells (control) and Ba/F3 cells transfected with either
native NMP/ALK or its L1196M mutant. Criztoinib (XalkoriÒ) was
used as a reference compound. It should be noted that the higher
14
15
Cl
B(OH)2
N
N
N
IC50 observed in our ELISA assay (1.6 lM) is by no means inconsis-
tent with literature reports for an ATP-competitive inhibitor, as
the [ATP] in our assay is considerably higher (see Supplementary
material).
B(OH)2
2
67
Cl
Neither of the two mono-chlorinated core compounds 1 and 2
themselves showed activity against ALK. Several of the imidazol-
o[1,2-c]pyrimidine compounds showed inhibition of the recombi-
nant ALK kinase domain in the 10 micromolar range in vitro,
such as the substituted compounds 6 and 10 (Table 4). The imidaz-
olo[1,2-a]pyrazine derivatives, for example, 16–19, gave more
promising results (Table 5). Significantly, while crizotinib was
approximately 10-fold less active against the L1196M gatekeeper
mutant compared to wt enzyme, the compounds from both series
were not significantly affected by this mutation. One derivative, 4-
(2-phenylvinyl)-7-methoxyphenylbenzo[4,5]imidazolo[1,2-a]pyr-
azine (19), showed submicromolar activity in vitro against wt ALK,
with comparable inhibition of the L1196M mutant, thus giving
superior results to crizotinib in our assay. Importantly, compound
19 inhibited Ba/F3-NPM/ALK cell proliferation with over 1-log
selectivity versus parental isogenic IL-3-dependent Ba/F3 cells (Ta-
N
N
N
(i) Pd(Ph3)4 (10 mol %), K2CO3 (3 equiv), boronic acid (1.3 equiv), THF/H2O 5:1,
70 °C, overnight.
Table 3
Suzuki coupling reactions of 7-chloro-4-(4-methoxy-phenyl)-benzo[4,5]imidazo[1,2-
a]pyrazine (14) under Buchwald conditions
O
O
i)
+ ArBH(OH)2
Cl
Ar
N
N
N
N
N
N
14
16-18
Table 4
In vitro inhibition of wild type and crizotinib-resistant ALK by benzo[4,5]imidazo[1,2-
c]pyrimidine derivatives
Entry
1
Boronic acid
Products
Yield (%)
80
O
OMe
H
O
N
Entry
Compound
IC50
(lM)
IC50 (lM)
HN
ALKwt
ALKL1196M
16
17
18
1
2
Crizotinib
1.6
10
N
N
N
N
B(OH)2
1
5
Cl
>100
52
n.t.
10
N
O
N
N
N
N
N
N
N
3
4
N
N
H
2
3
40
52
B
O
N
O
N
N
N
N
6
10
14
N
N
N
H
O2N
OMe
N
N
5
6
7
7
8
9
51
n.t.
n.t.
49
N
N
MeO
Cl
B(OH)2
H
N
Br
N
N
N
>50
69
N
(i) Pd(AcO)2 (10 mol %), S-Phos (20 mol %), K3PO4 (3 equiv), n-BuOH (C = 0.05 M),
boronic acid or pinacol ester (2 equiv), 100 °C, 14 h.
Br
N
N
N
H
N
Br
L1196M. While ATP concentrations close to the Km, near 10 lM, are
typically used in order to maximize the number of inhibitors de-
tected, we chose a more stringent assay, closer to the physiological
N
N
8
10
10
n.t.
O
N
concentration of 700
and specificity, selected compounds were tested against both
lM. In order to assess both cellular activity
n.t.-not tested.

S. Tardy et al. / Bioorg. Med. Chem. 22 (2014) 1303–1312
1307
Table 5
Table 7
In vitro inhibition of wild type and crizotinib-resistant ALK by benzo[4,5]imidazo[1,2-
ICM docking scores
a]pyrazine derivatives
Entry
Compound
IC50
(lM)
Docking score
Entry
Compound
IC50
(l
M)
IC50
(l
M)
wt-ALK
ALKwt
ALKL1196M
1
2
4
5
6
7
8
8
9
>50
>50
10
>50
3.5
ꢀ25.48
ꢀ29.54
ꢀ25.97
ꢀ26.23
ꢀ29.28
ꢀ31.30
ꢀ41.69
1
Crizotinib
1.6
10
Cl
10
11
18
19
TAE
N
2
3
2
>100
>50
n.t.
n.t.
N
N
0.65
0.056
N
N
Cl
Br
N
11
N
N
N
N
4
12
>100
29
N
N
N
N
OMe
H
N
O
5
6
16
17
18
14
n.t.
n.t.
N
N
O
Figure 2. Docking pose of 19 in the human wild type-ALK kinase catalytic domain.
The ligand is displayed as xstick and colored by atom type (carbon atoms in orange)
and residue M1199 is displayed as xstick and colored by atom type (carbon atoms
in green). The protein is displayed as Connolly Surface in green (ICM v. v3.7-3d). H-
bonds are represented by black dashed lines between the donor (D) and the
acceptor (A).
N
N
N
N
N
N
OMe
was first validated by docking NVP-TAE684 into the ALK kinase
catalytic domain (PDB 2XB7).16 The inhibitor docked with a score
of ꢀ41.69 (Table 1) reproducing the resolved crystallographic pose
with a low RMSD. Compounds 8, 9, 10, 11, 18 and 19 were then
docked into the same binding pocket. All derivatives established
one hydrogen bond with the NH of hinge Met 1199 providing the
scores listed in Table 7. The highest score of ꢀ31.3 was obtained
with 19 (Table 7, Fig. 2) in agreement with the experimental data
7
18
19
3.5
4.5
1.5
N
N
O
10
0.65
showing that this compound gives the best IC50 of 0.65 lM within
N
N
the series investigated. The vinylphenyl substituent is positioned
parallel to the gatekeeper L1196, which contributes to the favor-
able binding of this compound. Comparing the IC50 values of the
compounds in our assay to the corresponding docking scores
(Table 7) the derivatives screened can be clustered into four
N
n.t.-not tested.
groups: Group 1 (NVP-TAE684, score ꢀ41.69, IC50 <0.1
2 (19, score ꢀ31.3, IC50 <1 M), Group 3 (18, score ꢀ29.54, IC50
<10 M). The only
M), Group 4 (8, 10 and 11, score <ꢀ29, IC50 >10
outlier is compound 9 which should be part of Group 3 according
to the score but possesses an IC50 above 50 M. This can be ex-
plained by the overestimation of the contribution of the energy
interaction of the aromatic secondary amine group of 9 in the
docking pose obtained. More derivatives would be needed in order
lM), Group
ble 6), indicating selective cellular inhibition of NPM/ALK and
lower general cytotoxicity than Crizotinib. The activity against
the parent Ba/F3 cells is used as a test of selectivity, which may
be more representative than in vitro screens, and a selectivity
factor (SF) can be calculated by comparing the activity on the
NMP/ALK-dependent cells to the parent cells.
l
l
l
l
Modeling studies were performed to assess the binding mode of
the compounds to the ALK catalytic domain. The docking protocol
Table 6
Cell-based assays for ALK activity, as IC50 (lM)
Entry
Compound
ALKwt
ALKL1196M
BaF3 parent
BaF3 NPM/ALK
BaF3 NPM/ALKL1196M
SFa
1
2
3
4
5
6
Crizotinib
9
10
11
12
19
1.6
69
10
>50
>100
0.65
10
49
n.t.
n.t.
29
0.98
5.2
>25
2.4
0.87
>12.5
0.051
5.6
17
1.1
0.34
0.84
0.26
n.t.
9
2.2
0.20
0.78
6
1
>2
1
3
1.5
>15
a
SF: selectivity factor = BaF3 parent/avg (BaF3 NPM/ALK, BaF3 NPM/ALK L1196M).

1308
S. Tardy et al. / Bioorg. Med. Chem. 22 (2014) 1303–1312
to come up with a reliable SAR, yet the model can provide some
initial ideas for further elaboration of 19.
231.64 g molꢀ1 1H NMR (400 MHz, CDCl3) d 9.53 (s, 1H), 8.59
,
(s, 1H), 8.47 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.3 Hz, 1H), 7.65
(ft, J = 7.7 Hz, 1H), 7.50 (ft, J = 7.7 Hz, 1H); 13C NMR (101 MHz,
CDCl3) d 146.9 (C), 146.7 (2C), 141.6 (CH), 134.0 (CH), 131.3 (C),
129.9 (CH), 125.9 (CH), 120.4 (CH), 114.3 (CH); MS (ESI) m/z
232.0 [M+H]+, 254.0 [M+Na]+.
In conclusion, we have developed an original route to 3-substi-
tuted benzo[4,5]imidazo[1,2-c]pyrimidines and 4,7-disubstituted
benzo[4,5]imidazo[1,2-a]pyrazines. The parent monochlorinated
compounds were prepared by an aza-Graebe–Ullman reaction,
which after halogenation gave regioselectively dihalogenated
analogs. These heterocyclic cores were functionalized through
palladium-catalyzed coupling reactions to provide the mono- or
disubstituted products. We further developed a sequential regiose-
lective cross-coupling reaction using 4,7-dichlorobenzo[4,5]imi-
dazo[1,2-a]pyrazine, which is powerful method for the
preparation of large panels of unsymmetrically disubstituted com-
pounds. These compounds are inhibitors of ALK with, in the case of
compound 19, better in vitro activity in our assay than crizotinib
against both wt ALK and the crizotinib-resistant L1196M mutant.
This compound also shows excellent selectivity for NPM/ALK and
NPM/L1196M-ALK transfected Ba/F3 cells. Molecular docking
shows that the compounds are able to bind into the ATP binding
site of ALK forming a characteristic hydrogen bond with the hinge
region, typical of protein kinase inhibitors.17 These compound may
serve as useful scaffolds for the development of selective inhibitors
for ALK and other protein kinases.
3.1.3. 3-Chlorobenzo[4,5]imidazo[1,2-c]pyrimidine (1)
In
a 100 mL round-bottom flask, a mixture of the pyr-
imidinylbenzotriazole derivative 3 (297 mg, 1.28 mmol) and poly-
phosphoric acid (sufficient volume to cover the white solid) was
heated from room temperature to 150 °C until the production of
nitrogen ceased (2 h). The reaction mixture was then cooled to
room temperature and suspended by trituration with a saturated
solution of Na2CO3. The aqueous layer was slowly neutralized with
a
hydrochloride solution to pH 7 and extracted with DCM
(2 ꢁ 60 mL) and AcOEt (2 ꢁ 60 mL). The combined organic layers
were washed with brine, dried over MgSO4, filtered and solvents
were removed in vacuo. The crude product was triturated in MeOH
and filtered. The filtrate was purified by silica gel flash chromatog-
raphy (DCM/AcOEt: 100:0–90:10) to afford the desired compound,
which combined to the solid resulting from the trituration, gave
37% yield (97 mg) as
a
light orange solid.
C10H6ClN3,
203.63 g molꢀ1 1H NMR (400 MHz, DMSO) d 9.91 (d, J = 1.0 Hz,
,
1H), 8.42 (d, J = 8.2 Hz, 1H), 7.84–7.86 (m, 2H), 7.61 (t, J = 7.7 Hz,
1H), 7.50 (t, J = 7.7 Hz, 1H); 13C NMR (101 MHz, DMSO) d 147.2
(C), 146.0 (C), 144.7 (C), 142.2 (CH), 127.1 (CH), 126.7 (C), 122.5
(CH), 119.3 (CH), 112.9 (CH), 110.0 (CH); MS (ESI) m/z 204.2 [M+H]+.
3. Experimental
3.1. Synthesis
General experimental procedures are provided in the Supple-
mentary material.
3.1.4. 4-Chloro-benzo[4,5]imidazo[1,2-a]pyrazine (2)
In a 100 mL round-bottom flask, a mixture of the pyra-
zinylbenzotriazole derivative 4 (560 mg, 2.42 mmol) and poly-
phosphoric acid (sufficient volume to cover the white solid) was
heated from room temperature to 150 °C until the production of
nitrogen ceased (2 h). The reaction mixture was then cooled to
room temperature and suspended by trituration with a saturated
solution of Na2CO3. The aqueous layer was extracted with DCM
(50 mL, 4 ꢁ 25 mL). The combined organic layers were washed
with a brine/water 1:1 mixture (2 ꢁ 35 mL), dried over MgSO4, fil-
tered and the solvent was removed under reduced pressure. A yel-
lowish solid was obtained in 74% yield (364 mg) after purification
by silica gel flash chromatography (PE/AcOEt: 70:30–60:40). C10H6-
ClN3, 203.63 g molꢀ1, 1H NMR (400 MHz, CDCl3) d 9.19 (s, 1H), 8.58
(d, J = 8.7 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.94 (s, 1H), 7.67 (ft,
J = 7.8 Hz, 1H), 7.51 (ft, J = 7.9 Hz, 1H); 13C NMR (101 MHz, CDCl3)
d 144.5 (C), 143.5 (C), 143.1 (CH), 128.8 (C), 127.5 (CH), 126.6
(CH), 126.0 (C), 123.8 (CH), 121.5 (CH), 115.7 (CH); MS (ESI) m/z
204.2 [M+H]+.
3.1.1. 1-(6-Chloropyrimidin-4-yl)-1H-benzo[d][1,2,3]triazole (3)
4,6-Dichloropyrimidine (2.32 g, 15.6 mmol, 2 equiv) and benzo-
triazole (0.928 g, 7.78 mmol) were placed in a 25 mL round-bottom
flask attached to an open Vigreux condenser. The mixture was
placed directly in an oil bath at 80 °C and then warmed to
110 °C. After stirring 2 h at 110 °C, the reaction mixture was cooled
to room temperature and cautiously quenched with 25% aqueous
ammonia. Then, 15 mL of DCM were added to the aqueous layer
and stirred for 15 min. The organic layer was collected and the
aqueous layer was extracted with DCM (60, 30 and 5 ꢁ 20 mL).
The combined organic layers were dried over MgSO4, filtered and
the solvent was removed under reduced pressure. A white powder
was obtained in 87% yield (1.58 g) after purification by silica gel
flash chromatography (PE/DCM: 50:50–30:70).
C10H6ClN5,
231.64 g molꢀ1 1H NMR (300 MHz, CDCl3) d 8.98 (d, J = 0.9 Hz,
,
1H), 8.59 (d, J = 8.3 Hz, 1H), 8.28 (d, J = 1.0 Hz, 1H), 8.12 (d,
J = 8.2 Hz, 1H), 7.65 (ddd, J = 8.3, 7.1, 1.0 Hz, 1H), 7.49 (ddd,
J = 8.2, 7.1, 1.0 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 162.8 (C),
158.9 (CH), 158.3 (C), 147.0 (C), 131.2 (C), 130.1 (CH), 126.0 (CH),
120.5 (CH), 115.1 (CH), 110.2 (CH); MS (ESI) m/z 232.0 [M+H]+,
254.0 [M+Na]+.
3.1.5. N-Phenyl-benzo[4,5]imidazo[1,2-c]pyrimidin-3-ylamine
(5)
In a Schlenk tube with a stirring bar, 3-chloro-benzo[4,5]imi-
dazo[1,2-c]pyrimidine
3 equiv),
1 (40 mg, 0.196 mmol), K2CO3 (81 mg,
2-dicyclohexylphosphino-20,40,60-triisopropylbiphenyl
3.1.2. 1-(6-Chloropyrazin-2-yl)-1H-benzo[d][1,2,3]triazole (4)
(X-phos, 15 mg, 0.16 equiv), and Pd2(dba)3 (14 mg, 0.08 equiv) were
placed. The tube was evacuated and back-filled withargon (repeated
In
a sealed tube of 5 mL, 2,6-dichloropyrazine (359 mg,
2.41 mmol) and benzotriazole (316 mg, 2.65 mmol, 1.1 equiv)
were heated at 155 °C for 24 h. After cooling to room temperature,
the reaction mixture was cautiously quenched with 25% aqueous
ammonia and diluted with DCM. After 15 min stirring, the organic
layer was collected and the aqueous layer was extracted with DCM
(3 ꢁ 20 mL). The combined organic layers were washed with a
brine/water 1:1 mixture (20 mL), dried over MgSO4, filtered and
the solvent was removed in vacuo. A white powder was obtained
in 60% yield (335 mg) after purification by silica gel flash
three additional times). Then, 1 mL of t-BuOH and aniline (27
lL,
1.5 equiv) were introduced (degassed solvents were used). The reac-
tion mixture was allowed to stir at 100 °C for 15 h. After cooling to
room temperature and quenching with water, the aqueous layer
was extracted with DCM (2 ꢁ 15 mL) and AcOEt (2 ꢁ 15 mL). The
combined organic extracts were washed with brine, dried over
MgSO4, filtered and the solvent was removed in vacuo. The crude
product was purified by silica gel flash chromatography (PE/AcOEt
50:50–30:70) to provide the desired compound (34 mg) in 67% yield
as a pale yellow solid. C16H12N4, 260.29 g molꢀ1, 1H NMR (400 MHz,
chromatography
(PE/DCM:
50:50–30:70).
C10H6ClN5,

S. Tardy et al. / Bioorg. Med. Chem. 22 (2014) 1303–1312
1309
CDCl3) d 8.98 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.1 Hz, 1H),
7.47 (t, J = 7.7 Hz, 1H), 7.39 (t, J = 7.7 Hz, 2H), 7.35–7.27 (m, 3H),
7.15 (t, J = 7.2 Hz, 1H), 7.01 (s, NH), 6.85 (s, 1H); 13C NMR
(101 MHz, CDCl3) d 152.8 (C), 150.8 (C), 146.1 (C), 139.0 (CH),
138.9 (C), 129.7 (CH), 127.0 (C), 126.6 (CH), 124.6 (CH), 121.6 (CH),
120.8 (CH), 119.0 (CH), 109.8 (CH), 85.2 (CH); HRMS calcd for
succinimide (180 mg, 1.5 equiv) was added and the mixture was
stirred for 15 h at room temperature. Then, the reaction was
quenched with a water/saturated aqueous Na2S2O3 1:1 mixture.
The aqueous layer was extracted with AcOEt (60 mL, 2 ꢁ 20 mL).
The combined organic layers were washed with brine, dried over
MgSO4, filtered and the solvent was removed under reduced pres-
sure. The crude product was purified by silica gel flash chromatog-
raphy (DCM/AcOEt 100:0–97:3) to afford the brominated
compound (130 mg) in 68% yield as a pale yellow solid. C10H5-
C
16H13N4 [M+H]+ 261.1135 found 261.1137.
3.1.6. N-(3-Nitrophenyl)-benzo[4,5]imidazo[1,2-c]pyrimidin-
3-ylamine (6)
BrClN3, 282.52 g molꢀ1 1H NMR (300 MHz, CDCl3) d 9.13 (d,
,
In a Schlenk tube with a stirring bar, 3-chlorobenzo[4,5]imi-
J = 1.2 Hz, 1H), 8.16 (d, J = 1.7 Hz, 1H), 7.81 (d, J = 8.8 Hz, 1H),
7.72 (dd, J = 8.8, 1.8 Hz, 1H), 7.58 (d, J = 1.3 Hz, 1H); 13C NMR
(75 MHz, CDCl3) d 148.2 (C), 147.6 (C), 144.5 (C), 138.7 (CH),
131.3 (CH), 127.3 (C), 122.0 (CH), 116.2 (C), 114.1 (CH), 111.3
(CH); HRMS calcd for
281.9429.
dazo[1,2-c]pyrimidine
3 equiv),
1 (40 mg, 0.196 mmol), K2CO3 (81 mg,
2-dicyclohexylphosphino-20,40,60-triisopropylbiphenyl
(X-phos, 15 mg, 0.16 equiv), Pd2(dba)3 (14 mg, 0.08 equiv) and 3-
nitro-aniline (41 mg, 1.5 equiv) were placed. The tube was evacu-
ated and back-filled with argon (repeated three additional times).
Then, 1 mL of degassed t-BuOH was introduced. The reaction mix-
ture was allowed to stir at 100 °C for 15 h. After cooling to room
temperature and quenching with water, the aqueous layer was ex-
tracted with AcOEt (4 ꢁ 60 mL). The combined organic layers were
washed with brine (2 ꢁ 35 mL), dried over MgSO4, filtered and the
solvent was removed in vacuo. The crude product was purified by
silica gel flash chromatography (PE/AcOEt 50:50–0:100 and then
DCM/MeOH 90:10) providing the desired compound (57 mg) in
C
10H6BrClN3 [M+H]+ 281.9428 found
3.1.9. N-Phenyl-(8-bromo-benzo[4,5]imidazo[1,2-c]pyrimidin-
3-yl)amine (9)
In a Schlenk tube with a stirring bar, 8-bromo-3-chloro-
benzo[4,5]imidazo[1,2-c]pyrimidine 8 (40 mg, 0.141 mmol), K2CO3
(59 mg, 3 equiv), 2-dicyclohexylphosphino-20,40,60-triisopropylbi-
phenyl (X-phos, 11 mg, 0.16 equiv), and Pd2(dba)3 (10 mg,
0.08 equiv) were placed. The tube was evacuated and back-filled
with argon (repeated three additional times). Then, 0.9 mL of
95% yield as a yellow solid. C16H11N5O2, 305.29 g molꢀ1 1H NMR
,
(400 MHz, DMSO) d 9.92 (s, NH), 9.84 (s, 1H), 8.57 (t, J = 2.2 Hz,
1H), 8.26 (d, J = 8.0 Hz, 1H), 7.85 (dd, J = 8.1, 2.1 Hz, 1H), 7.81 (dd,
J = 8.1, 2.2 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.60 (t, J = 8.2 Hz, 1H),
7.47 (t, J = 7.7 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 6.75 (d, J = 0.8 Hz,
1H); 13C NMR (101 MHz, DMSO) d 151.7 (C), 149.5 (C), 148.3 (C),
145.5 (C), 142.0 (C), 141.5 (CH), 130.2 (CH), 127.0 (C), 126.2 (CH),
124.9 (CH), 120.3 (CH), 117.8 (CH), 115.7 (CH), 112.7 (C), 111.8
(CH), 87.6 (CH); HRMS calcd for C16H12N5O2 [M+H]+ 306.0986
found 306.0989.
t-BuOH and aniline (17 lL, 1.3 equiv) were introduced (degassed
solvents were used). The reaction mixture was allowed to stir at
75 °C for 14 h. After cooling to room temperature and dilution with
AcOEt, the mixture was filtered through a CeliteÒ pad. The solvents
of the filtrate were removed under reduced pressure. The crude
product was purified by silica gel flash chromatography (DCM/
AcOEt 100:0–95:5) to afford compound 9 in 16% yield (7.5 mg)
as a pale yellow solid. C16H11BrN4, 339.19 g molꢀ1 1H NMR
,
(400 MHz, CDCl3) d 8.97 (d, J = 1.0 Hz, 1H), 7.87 (d, J = 1.5 Hz, 1H),
7.68 (d, J = 8.5 Hz, 1H), 7.41 (t, J = 9.2 Hz, 1H), 7.38 (dd, J = 8.5,
2.2 Hz, 1H), 7.32 (d, J = 7.6 Hz, 2H), 7.19 (t, J = 7.3 Hz, 1H), 6.79
(d, J = 1.1 Hz, 1H, NH); 13C NMR (101 MHz, CDCl3) d 153.3 (C),
151.8 (C), 147.5 (C), 138.9 (CH), 138.5 (C), 129.8 (CH), 126.0 (C),
125.0 (CH), 123.8 (CH), 121.9 (CH), 121.8 (CH), 120.0 (C), 110.9
(CH), 85.0 (CH). MS (ESI) m/z 339.1, 341.0 [M+H+; 79Br, 81Br].
3.1.7. N-(3-Methoxyphenyl)-benzo[4,5]imidazo[1,2-
c]pyrimidin-3-ylamine (7)
In a Schlenk tube with a stirring bar, 3-chlorobenzo[4,5]imi-
dazo[1,2-c]pyrimidine
3 equiv),
1 (40 mg, 0.196 mmol), K2CO3 (81 mg,
2-dicyclohexylphosphino-20,40,60-triisopropylbiphenyl
(X-phos, 15 mg, 0.16 equiv), and Pd2(dba)3 (14 mg, 0.08 equiv)
were placed. The tube was evacuated and back-filled with argon
(repeated three additional times). Then, 1 mL of degassed t-BuOH
3.1.10. 8-Bromo-3-(4-methoxyphenyl)-benzo[4,5]imidazo[1,2-
c]pyrimidine (10)
and m-anisidine (33
l
L, 1.5 equiv) were introduced. The reaction
In a Schlenk tube, under inert atmosphere, Pd(Ph3)4 (16 mg,
0.1 equiv), K2CO3 (59 mg, 3 equiv) and 4-methoxyphenylboronic
acid (26 mg, 1.2 equiv) were added to a 0.05 M suspension of
mixture was allowed to stir at 100 °C for 15 h. After cooling to
room temperature and quenching with water, the aqueous layer
was extracted with DCM (4 ꢁ 30 mL). The combined organic layers
were washed with brine (2 ꢁ 35 mL), dried over MgSO4, filtered
and the solvent was removed under reduced pressure. The crude
product was purified by silica gel flash chromatography (PE/AcOEt
50:50–20:80) providing the desired compound (37 mg) in 65%
8-bromo-3-chlorobenzo[4,5]imidazo[1,2-c]pyrimidine
8 (40 mg,
0.141 mmol) in 1,4-dioxane/H2O 5:1 mixture. This mixture was
stirred at 70 °C for 16 h. After cooling to rt and diluting with AcOEt,
the mixture was filtered through a CeliteÒ pad. The solvents were
removed in vacuo. The crude product was purified by silica gel
flash chromatography (DCM/AcOEt 100:0–90:10, then DCM/MeOH
98:2) to afford compound 10 in 9% yield (4.5 mg) as a pale yellow
yield as a pale yellow solid. C17H14N4O, 290.32 g molꢀ1 1H NMR
,
(300 MHz, CDCl3) d 9.00 (s, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.75 (d,
J = 8.1 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.33–7.22 (m, 2H), 6.95–
6.83 (m, 4H), 6.70 (d, J = 8.2 Hz, 1H), 3.81 (s, OMe); 13C NMR
(75 MHz, CDCl3) d 160.8 (C), 152.6 (C), 150.8 (C), 146.2 (C), 140.1
(C), 139.0 (CH), 130.5 (CH), 127.0 (C), 126.7 (CH), 120.9 (CH),
119.0 (CH), 113.7 (CH), 110.0 (CH), 109.9 (CH), 107.3 (CH), 85.7
(CH), 55.5 (CH3); HRMS calcd for C17H14N4O [M+H]+ 291.1240
found 291.1241.
solid. C17H12BrN3O, 354.20 g molꢀ1 1H NMR (300 MHz, CDCl3) d
,
9.30 (d, J = 1.4 Hz, 1H), 8.17 (d, J = 1.7 Hz, 1H), 8.09 (d, J = 8.9 Hz,
2H), 7.87 (d, J = 1.0 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.70 (dd,
J = 8.7, 1.8 Hz, 1H), 7.05 (d, J = 8.9 Hz, 2H), 3.90 (s, 3H); MS (ESI)
m/z 354.2, 356.2 [M+H+; 79Br, 81Br].
3.1.11. 7-Bromo-4-chlorobenzo[4,5]imidazo[1,2-a]pyrazine (11)
Under an inert atmosphere and in a 10 mL round-bottom flask,
4-chlorobenzo[4,5]imidazo[1,2-a]pyrazine 2 (40 mg, 0.196 mmol)
3.1.8. 8-Bromo-3-chloro-benzo[4,5]imidazo[1,2-c]pyrimidine
(8)
was suspended in 1 mL of anhydrous DCM. Bromine (20 lL,
Under an inert atmosphere and in a 25 mL round-bottom
2 equiv) was added dropwise and the mixture as allowed to stir
for 16 h at room temperature. Then, the reaction was quenched
with a water/saturated aqueous Na2S2O3 1:1 mixture. The aqueous
flask, 3-chlorobenzo[4,5]imidazo[1,2-c]pyrimidine
1
(138 mg,
0.677 mmol) was suspended in 7 mL of anhydrous THF. N-Bromo-

1310
S. Tardy et al. / Bioorg. Med. Chem. 22 (2014) 1303–1312
layer was extracted with DCM (4 ꢁ 20 mL). The combined organic
layers were washed with brine, dried over MgSO4, filtered and the
solvent was removed in vacuo. The crude product was purified by
silica gel flash chromatography (DCM/AcOEt 95:5) to afford the
brominated compound (30 mg) in 54% yield as a white solid. C10H5-
cuo. The crude product was purified by silica gel flash chromatogra-
phy (DCM/AcOEt 75:25–60:40), followed by a trituration in MeOH
and filtration. The desired product was the solid obtained by tritur-
ation in 86% yield (96 mg) as a pale yellow solid. C17H12ClN3O,
309.75 g molꢀ1 1H NMR (400 MHz, CDCl3) d 9.26 (s, 1H), 7.95
,
BrClN3, 282.52 g molꢀ1
,
1H NMR (500 MHz, DMSO) d 9.29 (s, 1H),
(d, J = 8.9 Hz, 1H), 7.78 (s, 1H), 7.52 (d, J = 8.7 Hz, 2H), 7.50 (dd,
J = 8.6, 2.2 Hz, 1H), 7.16 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 1.7 Hz, 1H),
3.97 (s, 3H); 13C NMR (101 MHz, CDCl3) d 161.7 (C), 144.3 (CH),
143.6 (C), 143.4 (C), 133.8 (C), 130.7 (CH), 128.7 (C), 128.3 (CH),
128.2 (C), 127.7 (CH), 122.7 (C), 122.3 (CH), 115.1 (CH), 115.0
(CH), 55.7 (CH3). MS (ESI) m/z = 310.2 [M+H]+, 641.1 [2M+Na]+.
8.80 (d, J = 1.8 Hz, 1H), 8.18 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.84
(dd, J = 8.9, 1.8 Hz, 1H); 13C NMR (126 MHz, DMSO) d 143.6 (C),
143.1 (CH), 142.8 (C), 130.2 (CH), 129.1 (C), 126.6 (CH), 125.7 (C),
122.6 (CH), 118.1 (CH), 115.3 (C); MS (ESI) m/z 282, 284 [M+H+;
79Br, 81Br].
3.1.12. 4,7-Bis-[4-(4-methyl-piperazin-1-yl)-phenyl]-
benzo[4,5]imidazo[1,2-a]pyrazine (12)
3.1.15. 7-Chloro-4-(E)-styryl-benzo[4,5]imidazo[1,2-a]pyrazine
(15)
In a Schlenk tube, under inert atmosphere, Pd(Ph3)4 (10 mg,
0.1 equiv), K2CO3 (50 mg, 4 equiv) and 4-(4-methylpiperazin-
1-yl)phenylboronic acid pinacol ester (81 mg, 3 equiv) were added
In a Schlenk tube, under inert atmosphere, Pd(Ph3)4 (24 mg,
0.1 equiv), K2CO3 (85 mg, 3 equiv) and (E)-styrylboronic acid
(39 mg, 1.3 equiv) were added to a 0.04 M suspension of 4,7-dic-
hlorobenzo[4,5]imidazo[1,2-a]pyrazine 13 (49 mg, 0.206 mmol) in
THF/H2O 5:1 mixture (5 mL). This mixture was stirred at 70 °C for
16 h. After cooling to rt and diluting with AcOEt, the mixture was
filtered through a CeliteÒ pad. The solvents were removed under re-
duced pressure. The crude product was purified by silica gel flash
chromatography (DCM/AcOEt 90:10–70:30), followed by a tritur-
ation in MeOH and filtration. The desired product was the solid ob-
tained by trituration in 67% yield (42.5 mg) as a yellow solid.
to
a 0.05 M suspension of 8-bromo-3-chlorobenzo[4,5]imi-
dazo[1,2-c]pyrimidine 11 (25.4 mg, 0.0899 mmol) in 1,4-dioxane/
H2O 5:1 mixture. This mixture was stirred at 100 °C for 17 h. After
cooling to rt and diluting with AcOEt, the mixture was filtered
through a CeliteÒ pad. The solvents were removed in vacuo. The
crude product was purified by silica gel flash chromatography
(DCM/MeOH 95:5–85:15) to afford compound 12 in 77% yield
(35.9 mg) as a pale yellow solid. C32H35N7, 517.67 g molꢀ1 1H
,
NMR (400 MHz, CDCl3) d 9.21 (s, 1H), 8.00 (d, J = 8.7 Hz, 1H),
7.80–7.73 (m, 2H), 7.49 (d, J = 8.6 Hz, 2H), 7.31 (d, J = 8.7 Hz, 2H),
7.13 (d, J = 0.8 Hz, 1H), 7.10 (d, J = 8.7 Hz, 2H), 6.91 (d, J = 8.7 Hz,
2H), 3.43–3.35 (m, 4H), 3.28–3.20 (m, 4H), 2.67–2.61 (m, 4H),
2.61–2.57 (m, 4H), 2.40 (s, 3H), 2.37 (s, 3H); 13C NMR (101 MHz,
CDCl3) d 152.7 (C), 150.5 (C), 143.9 (C), 143.7 (CH), 143.5 (C),
135.3 (C), 134.3 (C), 131.8 (C), 130.4 (CH), 129.1 (s), 127.8 (CH),
127.7 (CH), 126.0 (CH), 121.1 (CH), 121.1 (s), 116.1 (CH), 115.4
(CH), 112.4 (CH), 55.1 (CH2), 55.0 (CH2), 48.8 (CH2), 48.3 (CH2),
46.3 (CH2), 46.2 (CH2); HRMS calcd for C32H36N7 [M+H]+
518.3027 found 518.3010.
C ,
18H12ClN3, 305.76 g molꢀ1 1H NMR (500 MHz, CDCl3) d 9.17 (s,
1H), 8.05 (d, J = 1.7 Hz, 1H), 8.02 (s, 1H), 7.96 (d, J = 8.9 Hz, 1H),
7.65 (d, J = 7.2 Hz, 2H), 7.55 (dd, J = 8.9, 1.9 Hz, 1H), 7.50 (d,
J = 15.1 Hz, 1H), 7.49 (t, J = 7.3 Hz, 2H), 7.43 (t, J = 7.3 Hz, 1H), 7.36
(d, J = 15.9 Hz, 1H). 13C NMR (126 MHz, CDCl3) d 143.8 (CH), 143.4
(C), 143.1 (C), 137.6 (CH), 135.2 (C), 133.3 (C), 130.0 (CH), 129.4
(CH), 129.0 (C), 128.9 (C), 127.7 (CH), 127.6 (CH), 126.1 (CH),
122.4 (CH), 117.0 (CH), 114.7 (CH); MS (ESI) m/z = 306.2 [M+H]+.
3.1.16. N-(4-[4-(4-Methoxyphenyl)-benzo[4,5]imidazo[1,2-
a]pyrazin-7-yl]phenyl)acetamide (16)
In a Schlenk tube with a stirring bar, 7-chloro-4-(4-methoxy-
phenyl)benzo[4,5]imidazo[1,2-a]pyrazine 14 (36 mg, 0.116 mmol),
K3PO4 (74 mg, 3 equiv), (4-acetamidophenyl)boronic acid (41 mg,
2 equiv), 2-dicyclohexylphosphino-20,60-dimethoxybiphenyl (S-
phos, 9.5 mg, 0.2 equiv) and Pd(OAc)2 (2.6 mg, 0.1 equiv) were
placed. The tube was evacuated and back-filled with argon (re-
peated three additional times). Then, 2.3 mL of degassed n-butanol
was introduced to obtain a 0.05 M suspension. The reaction mix-
ture was allowed to stir at 100 °C for 16 h. After cooling to room
temperature and dilution with AcOEt, the mixture was filtered
through a CeliteÒ pad. The solvents of the filtrate were removed
in vacuo. The crude product was purified by silica gel flash chroma-
tography (DCM/MeOH 98:2–97:3) to provide the desired product
3.1.13. 4,7-Dichlorobenzo[4,5]imidazo[1,2-a]pyrazine (13)
Under an inert atmosphere and in a 25 mL round-bottom flask,
4-chlorobenzo[4,5]imidazo[1,2-a]pyrazine
2
(82.5 mg,
0.405 mmol) was dissolved in 4 mL of anhydrous DMF. N-Chloro-
succinimide (86 mg, 1.6 equiv) was added and the mixture was
stirred for 21 h at room temperature. Then, the reaction was
quenched with water (30 mL), saturated NaHCO3 solution
(10 mL), and brine (10 mL). The aqueous layer was extracted with
AcOEt (4 ꢁ 15 mL). The combined organic layers were washed with
a water/brine 1:1 mixture (3 ꢁ 30 mL), dried over MgSO4, filtered
and the solvent was removed under reduced pressure. The crude
product was purified by silica gel flash chromatography (DCM/
AcOEt 95:5–85:15) to provide the di-halogenated compound
in 80% yield (38 mg) as
a pale yellow solid. C25H20N4O2,
(53 mg) in 54% yield as a white solid. C10H5Cl2N3, 238.07 g molꢀ1
,
408.45 g molꢀ1 1H NMR (400 MHz, MeOD) d 9.17 (s, 1H), 7.98 (d,
,
1H NMR (500 MHz, CDCl3) d 9.16 (s, 1H), 8.54 (d, J = 1.9 Hz, 1H),
7.96 (d, J = 8.9 Hz, 1H), 7.94 (s, 1H), 7.60 (dd, J = 8.9, 2.0 Hz, 1H);
13C NMR (126 MHz, CDCl3) d 143.9 (C), 143.4 (CH), 143.0 (C),
129.4 (C), 128.9 (C), 128.5 (CH), 127.0 (CH), 125.6 (C), 122.4 (CH),
115.4 (CH); MS (ESI) m/z = 238.2 [M+H]+.
J = 8.7 Hz, 1H), 7.81 (dd, J = 10.1 Hz, J = 1.5 Hz, 1H), 7.80 (s, 1H),
7.59 (s, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 8.6 Hz, 2H), 7.31
(d, J = 8.6 Hz, 2H), 7.20 (d, J = 8.7 Hz, 2H), 7.03 (s, 1H), 3.95 (s,
3H), 2.14 (s, 3H); 13C NMR (101 MHz, MeOD) d 171.0 (C), 162.6
(C), 144.0 (C), 143.8 (C), 143.6 (CH), 138.7 (C), 136.8 (C), 136.6
(C), 135.3 (C), 131.5 (CH), 129.5 (C), 128.2 (CH), 128.0 (CH), 127.4
(CH), 123.4 (C), 121.2 (CH), 121.0 (CH), 115.5 (CH), 113.6 (CH),
56.0 (CH3), 23.9 (CH3); HRMS calcd for C25H21N4O2 [M+H]+
409.1659 found 409.1656.
3.1.14. 7-Chloro-4-(4-methoxyphenyl)-benzo[4,5]imidazo[1,2-
a]pyrazine (14)
In a Schlenk tube, under inert atmosphere, Pd(Ph3)4 (42 mg,
0.1 equiv), K2CO3 (150 mg, 3 equiv) and 4-methoxyphenyl boronic
acid (66 mg, 1.2 equiv) were added to a 0.04 M suspension of 4,7-
dichlorobenzo[4,5]imidazo[1,2-a]pyrazine 13 (86 mg, 0.36 mmol)
in THF/H2O 5:1 mixture (9 mL). This mixture was stirred at 70 °C
for 16 h. After cooling to rt and diluting with AcOEt, the mixture
was filtered through a CeliteÒ pad. The solvents were removed in va-
3.1.17. 4-(4-Methoxyphenyl)-7-[4-(4-methyl-piperazin-1-yl)-
phenyl]benzo[4,5]imidazo[1,2-a]pyrazine (17)
In a Schlenk tube with a stirring bar, 7-chloro-4-(4-methoxy-
phenyl)benzo[4,5]imidazo[1,2-a]pyrazine 14 (35 mg, 0.113 mmol),
K3PO4 (74 mg, 3 equiv), 4-(4-methylpiperazin-1-yl)phenylboronic

S. Tardy et al. / Bioorg. Med. Chem. 22 (2014) 1303–1312
1311
acid pinacol ester (68 mg, 2 equiv), 2-dicyclohexylphosphino-20,60-
dimethoxybiphenyl (S-Phos, 9.5 mg, 0.2 equiv) and Pd(OAc)2
(2.6 mg, 0.1 equiv) were placed. The tube was evacuated and
back-filled with argon (repeated three additional times). Then,
2.3 mL of degassed n-butanol were introduced to obtain a 0.05 M
suspension. The reaction mixture was allowed to stir at 100 °C
for 14 h. After cooling to room temperature and dilution with
AcOEt, the mixture was filtered through a CeliteÒ pad. The solvents
of the filtrate were removed under reduced pressure. The crude
product was purified by silica gel flash chromatography (DCM/
MeOH 98:2–92:8) to provide the desired product in 40% yield
ation in MeOH and filtration. The desired compound was the solid
obtained by trituration in 41% yield (17 mg) as a yellow solid.
C
25H19N3O, 377.44 g molꢀ1 1H NMR (500 MHz, CDCl3) d 9.20 (s,
,
1H), 8.25 (s, 1H), 8.09 (d, J = 8.7 Hz, 1H), 8.04 (s, 1H), 7.84 (dd,
J = 8.7, 1.4 Hz, 1H), 7.69 (d, J = 16.1 Hz, 1H), 7.66 (d, J = 7.2 Hz,
2H), 7.56 (d, J = 8.7 Hz, 2H), 7.48 (t, J = 7.3 Hz, 2H), 7.45–7.41 (m,
1H), 7.38 (d, J = 16.1 Hz, 1H), 6.99 (d, J = 8.7 Hz, 2H), 3.86 (s, 3H);
13C NMR (126 MHz, CDCl3) d 159.5 (C), 144.1 (C), 143.7 (CH),
143.1 (C), 137.0 (CH), 136.8 (C), 135.5 (C), 133.7 (C), 133.5 (C),
129.8 (CH), 129.6 (C), 129.4 (CH), 128.6 (CH), 127.5 (CH), 126.8
(CH), 125.9 (CH), 121.6 (CH), 117.8 (CH), 114.7 (CH), 112.5 (CH),
55.5 (CH3); HRMS calcd for C25H20N3O [M+H]+ 378.1601 found
378.1588.
(20 mg) as a light orange solid. C28H27N5O, 449.55 g molꢀ1 1H
,
NMR (400 MHz, CDCl3) d 9.25 (s, 1H), 8.03 (d, J = 8.7 Hz, 1H), 7.78
(dd, J = 8.1, 2.2 Hz, 1H), 7.77 (s, 1H), 7.57 (d, J = 8.7 Hz, 2H), 7.31
(d, J = 8.7 Hz, 2H), 7.16 (d, J = 8.7 Hz, 2H), 7.08 (d, J = 1.1 Hz, 1H),
6.94 (d, J = 8.8 Hz, 2H), 3.95 (s, 3H), 3.31–3.20 (m, 4H), 2.67–2.56
(m, 4H), 2.38 (s, 3H); 13C NMR (101 MHz, CDCl3) d 161.6 (C),
150.5 (C), 144.1 (CH), 143.9 (C), 143.4 (C), 135.8 (C), 133.9 (C),
131.9 (C), 130.9 (CH), 129.1 (C), 128.0 (CH), 127.9 (CH), 126.3
(CH), 123.4 (C), 121.3 (CH), 116.2 (CH), 114.8 (CH), 112.3 (CH),
55.8 (CH3), 55.0 (CH2), 48.7 (CH2), 46.1 (CH3); HRMS calcd for
3.2. Biological assays
Recombinant wild-type and L1196M mutant ALK catalytic do-
mains were expressed in the Baculovirus system, purified by affin-
ity chromatography and used in the ELISA-based colorimetric
kinase assay as described previously.15 Cell growth inhibition
was tested in a [3H]-thymidine incorporation assay, as described.18
Kinase activity or cell-associated radioactivity were plotted as a
function of inhibitor concentration, using GraphPad software. The
concentration that causes 50% enzyme inhibition compared to
the DMSO-treated control was recorded (IC50). Detailed procedures
are provided in the Supplementary material.
C
28H28N5O [M+H]+ 450.2288 found 450.2270.
3.1.18. 4-(4-Methoxyphenyl)-7-(E)-styryl-
benzo[4,5]imidazo[1,2-a]pyrazine (18)
In a Schlenk tube with a stirring bar, 7-chloro-4-(4-methoxy-
phenyl)benzo[4,5]imidazo[1,2-a]pyrazine 14 (32 mg, 0.103 mmol),
K3PO4 (66 mg, 3 equiv), (E)-styrylboronic acid (30.5 mg, 2 equiv), 2-
dicyclohexylphosphino-20,60-dimethoxybiphenyl (S-Phos, 8.5 mg,
0.2 equiv) and Pd(OAc)2 (2.3 mg, 0.1 equiv) were placed. The tube
was evacuated and back-filled with argon (repeated three addi-
tional times). Then, 2.2 mL of degassed n-butanol was introduced
to obtain a 0.05 M suspension. The reaction mixture was allowed
to stir at 100 °C for 14 h. After cooling to room temperature and
dilution with AcOEt, the mixture was filtered through a CeliteÒ
pad. The solvents of the filtrate were removed in vacuo. The crude
product was purified by silica gel flash chromatography (DCM/
MeOH 98:2), followed by a trituration in MeOH and filtration. The
desired product was the solid obtained by trituration in 52% yield
3.3. Molecular docking
The 3D-coordinates of the wild type human Anaplastic Lym-
phoma Kinase (ALK) catalytic domain in complex with the inhibi-
tor NVP-TAE684 was retrieved from the Protein Data Bank (PDB
2XB7)16 and energetically minimized in the internal coordinate
space with Molsoft ICM v3.7-3d.19 Molecular docking was per-
formed using a ICM-VLS (Molsoft ICM) protocol validated by
assessing that redocking of NVP-TAE684 into the X-ray structure
(PDB 2XB7) devoid of ligand reproduces the experimentally deter-
mined binding mode. This protocol includes that the binding site is
represented by five types of interaction potentials: (i) van der
Waals potential for a hydrogen atom probe; (ii) van der Waals po-
tential for a heavy-atom probe (generic carbon of 1.7 Å radius); (iii)
optimized electrostatic term; (iv) hydrophobic terms; and (v) loan-
pair-based potential, which reflects directional preferences in
hydrogen bonding. The energy terms are based on the Merck
Molecular Force Field (MMRF) to account for solvation free energy
and entropic contribution.20 Modified inter-molecular terms such
as soft van der Waals and hydrogen-bonding, as well as a hydro-
phobic, term are added. Conformational sampling is based on the
biased probability Monte Carlo (BPMC) procedure, which randomly
selects a conformation in the internal coordinate space and then
makes a step to a new random position independent of the previ-
ous one, but according to a predefined continuous probability dis-
tribution. It has also been shown that after each random step, full
local minimization greatly improves the efficiency of the proce-
dure. In the ICM-VLS (Molsoft ICM) screening procedure, the ligand
scoring is optimized to obtain maximal separation between the
binders and non-binders. Each compound is assigned a score
according to its fit within the receptor; this ICM score accounts
for continuum and discreet electrostatics, hydrophobicity and en-
tropy parameters.20,21
(20.2 mg) as a yellow solid. C25H19N3O, 377.44 g molꢀ1 1H NMR
,
(500 MHz, CDCl3) d 9.25 (s, 1H), 7.99 (d, J = 8.7 Hz, 1H), 7.79 (s,
1H), 7.76 (dd, J = 8.7, 1.5 Hz, 1H), 7.57 (d, J = 8.7 Hz, 2H), 7.45 (d,
J = 7.3 Hz, 2H), 7.35 (t, J = 7.6 Hz, 2H), 7.28–7.24 (m, 1H), 7.20 (d,
J = 8.7 Hz, 2H), 6.99 (d, J = 16.1 Hz, 1H), 6.97 (d, J = 0.6 Hz, 1H),
6.88 (d, J = 16.3 Hz, 1H), 4.00 (s, 3H); 13C NMR (126 MHz, CDCl3) d
161.65 (C), 144.82 (C), 144.18 (CH), 143.65 (C), 137.20 (C), 133.90
(C), 132.37 (C), 130.94 (CH), 128.99 (CH), 128.98 (C), 128.86 (CH),
128.68 (CH), 128.16 (CH), 127.90 (CH), 126.57 (CH), 125.58 (CH),
123.40 (C), 121.40 (CH), 114.87 (CH), 113.04 (CH), 55.78 (CH3);
HRMS calcd for C25H20N3O [M+H]+ 378.1601 found 378.1613.
3.1.19. 4-(E)-Styryl-7-(4-methoxyphenyl)-
benzo[4,5]imidazo[1,2-a]pyrazine (19)
In
a Schlenk tube with a stirring bar, 7-chloro-4-(E)-
styryl-benzo[4,5]imidazo[1,2-a]pyrazine 15 (33 mg, 0.108 mmol),
K3PO4 (69 mg, 3 equiv), 4-methoxyphenyl boronic acid (33 mg,
2 equiv), 2-dicyclohexylphosphino-20,60-dimethoxybiphenyl (S-phos,
8.9 mg, 0.2 equiv) and Pd(OAc)2 (2.4 mg, 0.1 equiv) were placed.
The tube was evacuated and back-filled with argon (repeated three
additional times). Then, 2.2 mL of degassed n-butanol were
introduced to obtain a 0.05 M suspension. The reaction mixture
was allowed to stir at 100 °C for 14 h. After cooling to room tem-
perature and dilution with AcOEt, the mixture was filtered through
a CeliteÒ pad. The solvents of the filtrate were removed under
reduced pressure. The crude product was purified by silica gel flash
chromatography (DCM/MeOH 99:1–98:2), followed by a tritur-
Acknowledgments
The authors thank «Lyon Sciences Transfert» for financial sup-
port (S.T.). Financial support from the European Union (Contract
N° LSHB-CT-2004-503467) and from Lombardy regional govern-

1312
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Supplementary data
Supplementary data (general experimental procedures, bio-
chemical assay procedures, inhibition data at different ATP concen-
trations, and criteria of purity (1H and 13C NMR spectra, melting
points, and HPLC chromatograms)) associated with this article
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