Scaffold hopping identifies 6,8-disubstituted purines as novel anaplastic lymphoma kinase inhibitors

Article abstract of DOI:10.1016/j.ejpb.2017.03.011

Rearrangements of anaplastic lymphoma kinase (ALK) are associated with several cancer diseases. Due to resistance development against existing ALK-inhibitors, new, structurally unrelated inhibitors are required. By a scaffold hopping strategy, 6,8-disubstituted purines were designed as analogues of similar ALK-inhibiting thieno[3,2-d]pyrimidines. While the new title compounds indeed inhibited ALK and several ALK mutants in submicromolar concentrations, they retained poor water solubility.

Full text of DOI:10.1016/j.ejpb.2017.03.011

Accepted Manuscript
Research paper
Scaffold hopping identifies 6,8-disubstituted purines as novel anaplastic lym-
phoma kinase inhibitors
Laura Schlütke, Markus Immer, Lutz Preu, Frank Totzke, Christoph Schächtele,
Michael H.G. Kubbutat, Conrad Kunick
PII:
S0939-6411(17)30041-3
DOI:
http://dx.doi.org/10.1016/j.ejpb.2017.03.011
EJPB 12466
Reference:
European Journal of Pharmaceutics and Biophar-
maceutics
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11 January 2017
10 March 2017
13 March 2017
Please cite this article as: L. Schlütke, M. Immer, L. Preu, F. Totzke, C. Schächtele, M.H.G. Kubbutat, C. Kunick,
Scaffold hopping identifies 6,8-disubstituted purines as novel anaplastic lymphoma kinase inhibitors, European
Journal of Pharmaceutics and Biopharmaceutics (2017), doi: http://dx.doi.org/10.1016/j.ejpb.2017.03.011
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Scaffold hopping identifies 6,8-disubstituted purines as novel
anaplastic lymphoma kinase inhibitors
Laura Schlütke,a Markus Immer,a,b Lutz Preu,a Frank Totzke,c Christoph Schächtele,c
Michael H. G. Kubbutat,c Conrad Kunick*a,b
a
Technische Universität Braunschweig, Institut für Medizinische und
Pharmazeutische Chemie, Beethovenstraße 55, 38106 Braunschweig, Germany
b
Technische Universität Braunschweig, Center of Pharmaceutical Engineering
(PVZ), Franz-Liszt-Straße 35A, D-38106 Braunschweig, Germany
c
ProQinase GmbH, Breisacher Straße 117, 79106 Freiburg, Germany
*
corresponding author. E-mail address: c.kunick@tu-braunschweig.de
Abstract: Rearrangements of anaplastic lymphoma kinase (ALK) are associated with
several cancer diseases. Due to resistance development against existing ALK-
inhibitors, new, structurally unrelated inhibitors are required. By a scaffold hopping
strategy, 6,8-disubstituted purines were designed as analogues of similar ALK-
inhibiting thieno[3,2-d]pyrimidines. While the new title compounds indeed inhibited
ALK and several ALK mutants in submicromolar concentrations, they retained poor
water solubility.
Abbreviations
ALK: anaplastic lymphoma kinase; cMet, hepatocyte growth factor receptor; DIPEA,
N,N-Diisopropylethylamine; DMF, N,N-Dimethylformamide; EML4, echinoderm
microtubule-associated protein-like 4; FDA, Food and Drug Administration; gk,
gatekeeper; µW, micro wave; NSCLC, non-small cell lung cancer; pdb, protein data
bank; RT: room temperature; TEA, triethly amine; wt, wild type.
1

Introduction
In developed countries, lung cancer is top on the cancer death rate list, with non-
small cell lung cancer (NSCLC) accounting for the majority of cases [1]. As found
with other cancer diseases, in NSCLC the hyperactivity of distinct protein kinases is
often responsible for tissue hyperproliferation. In recent years, this finding has been
translated into targeted therapies employing small molecules that inhibit oncogenic
tumor-driving protein kinases [2]. The anaplastic lymphoma kinase (ALK) is a
receptor tyrosine kinase that is involved in brain development and is physiologically
expressed during embryogenesis [3]. In 2007 it was shown that an inversion within
chromosome 2p generates a fusion gene consisting of the ALK gene and parts of the
echinoderm microtubule-associated protein-like 4 (EML4) gene. This EML4-ALK
fusion was found in 6.7% of the surveyed NSCLC patients [4]. Later it was shown
that the EML4-ALK fusion occurs typically in patients with adenocarcinoma histology,
female gender, and a non-smoking history [5]. Several oncogenic variants of EML4-
ALK have been reported [6, 7], as well as oncogenic fusions of ALK with other
partner genes [8].
The EML4-ALK fusion and other ALK translocations lead to constitutive protein
kinase hyperactivity resulting in tissue proliferation, increased survival and eventually
cancer development [9]. Consequently, ALK was suggested as a target for
personalized therapy of ALK-positive NSCLC patients [4]. In 2011 the dual ALK/c-Met
receptor tyrosine kinase inhibitor crizotinib (1) was approved by the FDA [10]. In the
ALK-positive sub-group of patients, crizotinib is rather effective compared with the
standard chemotherapy first line treatment. Unfortunately, most patients acquire
resistance within the first year after start of treatment [11]. While in some cases
resistance development is due to activation of other tumor drivers [12] or to
2

amplification of the ALK fusion gene, the majority of resistances are the result of
single point mutations of the ALK kinase domain [13], prominent examples being the
gate-keeper mutation L1196M and the C1156Y mutation [14]. The second generation
ALK inhibitors alectinib (2) [15, 16] and ceritinib (3) [11, 17] were approved recently.
Both drugs show a protein kinase inhibition selectivity pattern different to crizotinib
and also inhibit the gate-keeper mutation L1196M. The second generation ALK-
inhibitors are used for the therapy of patients who have progressed on previous
crizotinib treatment [10]. Because it can be predicted that also against these second
generation inhibitors resistances will emerge, the development of further ALK
inhibiting chemotypes is of high interest. In a recent patent application, thieno[3,2-
d]pyrimidines like compound 4a were disclosed as ALK inhibitors (IC50 4a on ALK wt
~ 1 µM) [18], which unfortunately display poor solubility (Fig. 1). Scaffold hopping is a
strategy for the discovery of structurally novel bioactive compounds starting from
known drug molecules by replacement of their core structure [19]. Predicted by the
method of Wildman and Crippen [20], the purine ring system as an isolated element
shows lower lipophilicity (clogP = 0.35) compared to the thieno[3,2-d]pyrimidine
scaffold (clogP = 1.69) present in 4a. In an attempt to develop ALK inhibitors based
on novel chemical matter with improved solubility, we designed molecules of the
series 5 by exchanging the thieno[3,2-d]pyrimidine core structure for a purine ring
system (Fig. 2). The comparative pairwise solubility prediction of 4 and 5 by the
method of Ali et al. [21] via the Swiss ADME interface (http://www.swissadme.ch)
yielded distinctly better results for the latter congeners. Since docking analyses
predicted a good fit of the designed molecules to the ATP binding pocket of the ALK
target kinase, the purine analogues 5 were synthesized and tested for inhibition of
ALK, of cancer-relevant ALK-mutants, and of related protein kinases.
3

Fig. 1 ALK-inhibitors: Crizotinib (1), Alectinib (2), Ceritinib (3), proprietary thieno[3,2-
d]pyrimidine 4a.
4

Fig. 2 Scaffold hopping: Exchange of the thieno[3,2-d]pyrimidine core structure in
4 [18] for a purine system leads to the title compounds 5.
5

Docking
To estimate the chances for successful scaffold hopping, we performed docking-
experiments with 5 in the ATP binding site of ALK. The co-crystal structure of ALK
wild type with bound ceritinib (PDB-code 4MKC)[11] was used as template for these
studies. Ceritinib (3) forms two hydrogen bonds to Met1199 (gk+3) of the hinge
region (Figure 3, A). Taking into account the binding mode of ceritinib, constraints
were implemented to provoke the formation of two hydrogen bonds between the
purine-scaffold of 5a and residues of the ALK hinge region. The docking routine
placed compound 5a into a position in which the N(9)-H group acts as hydrogen
bond donor for the carbonyl group of Met1199 (3 residues downstream of
gatekeeper; gk+3) and in which the N(3)-atom of the ligand accepts a hydrogen bond
from the NH-group of Met1199. While the 1,3,4-oxadiazole moiety is located in the
center of the binding pocket, its phenyl substituent fills the back part of the pocket
(Fig. 3, B). In contrast, the docking pose of compound 4 shows only one hydrogen
bond between the N(3)-atom of the ligand and Met1199 (Fig. 3, C). According to the
docking results, the scaffold hopping from thieno[3,2-d]pyrimidine to purine
derivatives enables the formation of an additional hydrogen bond between the ligand
and the protein kinase. Because docking of compound 5a into the ATP binding site of
the gatekeeper mutant ALK L1996M (PDB-code: 2YFX [22]) generated a similar
docking pose (Figure 3, D), it was expected that both wild type and mutant form of
ALK would be inhibited by 5a and congeners. Considering the docking pose of 5a in
ALK, we expected that introduction of an additional amino group in 2-position of the
purine core as displayed in congeners 5cc and 5dd would enable a third hydrogen
bond between ligand and hinge area. This speculation was corroborated by the
6

results of docking 5cc into the ATP binding pocket of ALK (PDB: 4MKC). Hence, we
also prepared these compounds in the hope to improve the ALK inhibitory activity.
Furthermore, we synthesized the analogues 14a-c to evaluate the contribution of the
8-aryl substituent of the title compounds for binding to and for inhibition of ALK.
7

B
A
Met1199
Met1199
C
D
Met1199
Met1199
Fig. 3 Results of docking studies. 3A: co-crystal structure of Ceritinib (3, blue) and
ALK wt (4MKC [11], green); 3B: docking-pose of 5a (magenta) in the ATP binding
site of ALK wt (4MKC, green); 3C: docking-pose of 4 (gray) in the ATP binding site of
ALK wt (4MKC, green); 3D: docking-pose of 5a (magenta) in the ATP binding site of
ALK L1196M (2YFX [22], gray). Docking was carried out with GOLD [23] (refer to
supporting information for details).
8

Synthesis
The central step of the synthesis of the title compounds 5 consisted in a nucleophilic
substitution reaction of the 8-aryl-6-chloropurines 8 with various 1,3,4-oxadiazole-2-
thioles 9. The starting materials 8 were synthesized according to the method
developed by Legraverend et al.[24, 25] by ring closure reaction of 6-
chloropyrimidine-4,5-diamine (7) with carboxylic acid chlorides 6 in POCl3 in the
presence of NH4Cl. The 1,3,4-oxadiazole-2-thioles 9 were prepared by ring closure
reaction of hydrazides with CS2 by literature procedures [26-29]. The synthesis of the
2-aminopurine derivatives 5cc, 5dd started from 2-amino-8-bromo-1H-purin-6(9H)-
one (10) which yielded 2-amino-8-(4-fluorophenyl)-1H-purin-6(9H)-one (11) by Suzuki
reaction with 4-fluorophenylboronic acid in a microwave-assisted reaction. Treatment
of 11 with POCl3 catalyzed by N,N-dimethylaniline led to the 6-chloro derivative 12
[30-32]. Subsequently, the 1,3,4-oxadiazole-2-thioles 9 and the purine derivatives 8
or 12, respectively, were reacted yielding the title compounds 5a-5dd. Under
conventional conditions, these nucleophilic substitution reactions required up to 40 h
in refluxing propan-2-ol or butan-1-ol. Assisted by microwave irradiation, similar
reactions in DMF needed only 20-30 min (scheme 1). In an analogous process, the
commercially available 2-amino-6-chloropurine (13) reacted with 1,3,4-oxadiazole-2-
thioles 9 furnishing the 8-unsubstituted congeners 14 (scheme 2).
9

Scheme 1 Synthesis of compounds 5: (i) POCl3, NH4Cl, 100 °C, 15 – 24 h, yield: 20
– 59%; (ii) TEA or DIPEA, propan-2-ol or butan-1-ol, reflux, 15-40 h, 18 – 65%; (iii)
TEA or DIPEA, µW, DMF, 120 °C, 20 - 30 min, 10-66%; (iv) 4-fluorophenylboronic
acid, K2CO3, Pd(PPh3)4, dioxane/H2O 3:1, µW, 130 °C, 1 h, yield: 31%; (v) POCl3,
N,N-dimethylaniline, reflux, 2 h, yield: 26%. For residues R1 and R2 refer to table 1.
10

Scheme 2 Synthesis of compounds 14a-c: (i) DMF, TEA, 120 °C, µW, 30 min, 61–
74%. For residues R2 refer to table 2.
11

Results and discussion
The title compounds 5 and their 8-unsubstituted analogues 14 were evaluated for
inhibitory activity against wild type ALK, ALK mutants, ALK fusion gene products and
other tumor relevant protein kinases in a radiometric protein kinase assay. For means
of comparison, crizotinib (1) was tested as a standard ALK inhibitor (Tables 1, 2, and
3; for complete data refer to tables S1 and S2 in the supporting information). The
results revealed that the scaffold hopping was successful. Four of the new title
compounds (5b, 5h, 5l, 5u) displayed IC50-values towards ALK wt in submicromolar
concentrations, with compound 5l exhibiting the strongest inhibition (IC50 = 457 nM).
Structurally closely related compounds showed slightly decreased ALK inhibition in
single digit micromolar concentrations. All new compounds were clearly inferior
compared to crizotinib which inhibited ALK with an IC50 value of 21 nM in our assay.
The test outcome allows a preliminary statement regarding structure activity
relationships in the new family of ALK inhibitors. It is obvious from the results
collected in Table 1 that substitution of the oxadiazole moiety by aliphatic instead of
aromatic substituents R2 (5q, 5r, 5s, 5aa) leads to decrease of ALK wild type
inhibition potency by two orders of magnitude. Similarly, substitution at this position
by heteroaromatic residues, e.g. pyridin-4-yl (5g, 5m, 5y) or furan-2-yl (5bb) impaired
ALK inhibitory potency. In contrast to our expectation and the results of our docking
studies, the addition of a 2-amino group at the purine ring (5cc, 5dd) failed to
optimize the potency, but led to loss of activity. The analogues 14a-c lacking the 8-
aryl substitution were even less potent compared to 5cc and 5dd (Table 2). Most of
the new compounds 5 and 14 maintained activity upon testing against the important
gatekeeper mutant ALK L1196M, displaying an advantage over crizotinib which loses
potency by one order of magnitude on the indicated mutant. While crizotinib is a well-
12

known inhibitor of the cancer-relevant kinase c-Met [33, 34], the new compounds
reported here showed, if any, only marginal inhibition of c-Met. A set of selected
compounds from series 5 and 14 were also tested on various additional relevant ALK
mutants and fusion proteins (refer to collected data in tables S1-S3 in the supporting
information); some results for 5l and 5u are shown in Table 3. Crizotinib inhibited all
ALK mutants with comparable potency, with the exception of the gatekeeper mutant
L1196M. In contrast, 5l performed less consistent, inhibiting two of the mutants (ALK
C1156Y and ALK F1174L) in three- to fourfold higher concentrations. Compound 5u
turned out to be most robust with regard to ALK mutations, since all tested mutants
were inhibited in concentrations between 0.8 and 1.4 µM.
13

Table 1: Protein kinase inhibition by compounds 5 (IC50, [µM]) a
ALK
entry
R1
R2
R3
ALK wt
cMet
L1196M
0.233
4.86
n.a.b
F
n.a. b
Phenyl
n.a. b
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.021
6.01
0.816
1.95
4.98
6.34
5.18
> 100
0.961
1.90
4.81
4.80
0.457
9.64
2.08
2.06
2.99
51.7
31.4
> 100
5.95
0.857
0.104
33.6
1
5a
5b
5c
5d
5e
5f
F
4-Cl-Phenyl
4-CF3-Phenyl
4-OCH3-Phenyl
2-Cl-Phenyl
2,4-di-Cl-Phenyl
4-Pyridyl
0.822
4.80
6.89
F
22.8
F
4.94
> 100
> 100
> 100
> 100
66.7
F
7.12
F
3.58
5g
5h
5i
F
> 100
1.17
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
4-Cl-Phenyl
4-CF3-Phenyl
4-OCH3-Phenyl
2-Cl-Phenyl
2,4-di-Cl-Phenyl
4-Pyridyl
1.86
> 100
> 100
> 100
> 100
> 100
33.3
5j
4.95
5k
5l
5.08
0.875
9.40
5m
5n
5o
5p
5q
5r
4-CH3-Phenyl
2-CH3-Phenyl
4-OH-Phenyl
Benzyl
1.95
1.81
> 100
> 100
> 100
75.5
3.04
30.9
tert-Butyl
41.0
5s
5t
Morpholinomethyl
4-Cl-Phenyl
4-CF3-Phenyl
> 100
7.00
> 100
> 100
> 100
5u
H
0.963
14

5v
5w
5x
H
H
H
H
H
H
H
F
4-OCH3-Phenyl
2-Cl-Phenyl
2,4-di-Cl-Phenyl
4-Pyridyl
H
H
4.29
8.81
1.60
26.6
10.5
75.5
22.5
13.5
13.0
3.12
6.04
1.93
51.1
7.16
54.6
18.0
12.1
4.58
> 100
81.1
H
> 100
> 100
> 100
> 100
> 100
21.7
5y
H
5z
4-CH3-Phenyl
Benzyl
H
5aa
5bb
5cc
5dd
H
2-Furyl
H
4-Cl-Phenyl
2,4-di-Cl-Phenyl
NH2
NH2
F
> 100
a Z’-factors [35] for high and low controls ≥0.54. b n.a. = not applicable
15

Table 2: Protein kinase inhibition by compounds 14 (IC50, [µM]) a
ALK
entry
R2
ALK wt
cMet
L1196M
13.1
14a
14b
14c
4-Cl-Ph
4-CF3-Ph
2-Cl-Ph
21.1
21.4
18.6
> 100
> 100
> 100
16.0
17.2
a Z’-factors[35] for high and low controls ≥0.54.
16

Table 3: Comparison of ALK mutant selectivity profile of crizotinib with 8-aryl-6-
(1,3,4-oxadiazol-2-ylthio)purines 5l and 5u (IC50, [µM]) a
Protein kinase
ALK wt
Crizotinib (1)
5l
5u
0.021
0.457
0.875
1.73
0.857
0.963
1.11
ALK L1196M
ALK C1156Y
ALK F1174L
ALK F1174S
ALK R1275Q
NPM1- ALK
0.233
0.033
0.035
2.30
1.13
0.039
0.021
0.034
0.054
n.d.b
1.36
0.332
0.322
0.313
0.977
0.965
0.961
NPM1- ALK/F1174L
a Z’-factors[35] for high and low controls ≥0.54. b n.d.=not determined
17

Despite the integration of the purine scaffold, the new compounds presented here
still display a high degree of lipophilicity and a low sp3/sp2 ratio, both unfavorable
features for water solubility of organic molecules. Indeed, none of four representative
test compounds (5b, 5f, 5cc, 5dd) which were subjected to thermodynamic solubility
determination (aqueous phosphate buffer, pH 7.4) produced in the HPLC runs
signals exceeding the limit of quantification (4 µM). Additional molecular
modifications aiming at increasing water solubility are therefore essential for a further
rational development process in this class of compounds.
Conclusion
Starting from thieno[3,2-d]pyrimidine derivatives 4, 6,8-disubstituted purine
derivatives 5 were developed as a class of new ALK inhibitors, demonstrating a vivid
example for the efficiency of the scaffold hopping approach. Four of the new
compounds exhibited ALK inhibitory activity in the submicromolar concentration
range. Compound 5l proved to be the most potent ALK inhibitor in the series (IC50 =
457 nM). Preliminary data regarding structure activity relationships in the series 5
show that aromatic substituents both at position 8 of the purine system and at
position 2 of the 1,3,4-oxadiazole ring are important for kinase inhibition potency. In
contrast to the approved ALK inhibitor crizotinib, 5 are able to maintain inhibition
potency for of the gatekeeper mutant ALK L1196M. Docking studies suggest that 5
bind to the ATP binding pocket of ALK, where they are rendered in position by a pair
of hydrogens bonds between the purine scaffold and Met1199 (gk+3) of the host
protein. Inspired by the inhibitor poses produced through molecular docking,
derivatives 5cc and 5dd were prepared which bear an additional amino group at
18

position 2 of the purine ring as anchoring point for a third hydrogen bond to the hinge
region of the kinase. This strategy was not successful, for both compounds showed
decreased potency compared to the corresponding unsubstituted congeners 5b and
5f. The derivatives 5 presented here still display only poor water solubility. Additional
molecular modifications are therefore ongoing to optimize these properties, as well as
to investigate the cellular activities and the pharmacokinetic properties in the
compound class.
Acknowledgements
L.S. was supported by a fellowship from the Cusanuswerk, Bonn, Germany. M.I. was
supported by a stipend in the framework of the joint project „SynFoBiA“ (Neuartige
Synthese- und Formulierungsverfahren für schwerlösliche Arzneistoffe und
empfindliche Biopharmazeutika) funded by the state of Lower Saxony (Germany).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the only version, at
http://dx.doi.org/XXXXXXXXXXXXXXX
19

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Scaffold hopping identifies novel 6,8-disubstituted purines as anaplastic
lymphoma kinase inhibitors
Laura Schlütke, Markus Immer, Lutz Preu, Frank Totzke, Christoph Schächtele,
Michael H. G. Kubbutat, Conrad Kunick
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