Rational Design, Synthesis, and Biological Evaluation of 7-Azaindole Derivatives as Potent Focused Multi-Targeted Kinase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b00087

Efforts were made to improve a series of potent dual ABL/SRC inhibitors based on a 7-azaindole core with the aim of developing compounds that demonstrate a wider activity on selected oncogenic kinases. Multi-targeted kinase inhibitors (MTKIs) were then derived, focusing on kinases involved in both angiogenesis and tumorigenesis processes. Antiproliferative activity studies using different cellular models led to the discovery of a lead candidate (6z) that combined both antiangiogenic and antitumoral effects. The activity of 6z was assessed against a panel of kinases and cell lines including solid cancers and leukemia cell models to explore its potential therapeutic applications. With its potency and selectivity for oncogenic kinases, 6z was revealed to be a focused MTKI that should have a bright future in fighting a wide range of cancers.

Full text of DOI:10.1021/acs.jmedchem.6b00087

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Article
Rational Design, Synthesis, and Biological Evaluation of 7-azaindole
Derivatives as Potent Focused Multi-Targeted Kinase Inhibitors
Bénédicte Daydé-Cazals, Bénédicte Fauvel, Mathilde Singer, Clémence
Feneyrolles , Benoît Bestgen, Fanny Gassiot, Aurélia Spenlinhauer, Pierre
Warnault, Nathalie Van Hijfte, Nozha Borjini, Gwénaël Chevé, and Aziz Yasri
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00087 • Publication Date (Web): 24 Mar 2016
Downloaded from http://pubs.acs.org on March 26, 2016
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Rational Design, Synthesis, and Biological Evaluation of 7-azaindole
Derivatives as Potent Focused Multi-Targeted Kinase Inhibitors
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Bénédicte Daydé-Cazals, Bénédicte Fauvel, Mathilde Singer, Clémence Feneyrolles, Benoit
Bestgen, Fanny Gassiot, Aurélia Spenlinhauer, Pierre Warnault, Nathalie Van Hijfte, Nozha
Borjini, Gwénaël Chevé, Aziz Yasri*
OriBase Pharma, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 17383, 34189
Montpellier Cedex 4, France
ABSTRACT
Efforts were made to improve a series of potent dual ABL/SRC inhibitors based on a 7-azaindole
core with the aim of developing compounds that demonstrate a wider activity on selected
oncogenic kinases. Multi-Targeted Kinase Inhibitors (MTKIs) were then derived, focusing on
kinases involved in both angiogenesis and tumorigenesis processes. Anti-proliferative activity
studies using different cellular models led to the discovery of a lead candidate (6z) that combined
both anti-angiogenic and anti-tumoral effects. The activity of 6z was assessed against a panel of
kinases and cell lines including solid cancers and leukemia cell models to explore its potential
therapeutic applications. With its potency and selectivity for oncogenic kinases, 6z was revealed
to be a focused MTKI that should have a bright future in fighting a wide range of cancers.
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INTRODUCTION
The modulation of kinase function is an important goal in drug discovery and, more
particularly, in cancer-targeted therapies where an increased number of kinase inhibitors have
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entered the market in recent years. Two opposing concepts govern the design and development
of new kinase inhibitors in oncology. While the first strategy proposes the design of selective
molecules to increase efficacy and limit toxicity, the second strategy highlights benefits in
developing Multi-Targeted Kinase Inhibitors (MTKIs). Although almost all major human
cancers seem to harbor not a single but several concomitant dysregulations of kinase pathways,
the latest approach offers the opportunity to catch tumor progression more effectively but also to
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avoid mechanisms of resistance commonly activated by the tumor to escape targeted therapies.
Moreover, given that different types of tumors may originate from different cell types and are
frequently driven by different combinations of genetic alterations, MTKIs will have the ability
not only to be used in a wide range of cancers but will also prove to be as effective as
combinatorial therapy without the disadvantages of using multiple selective agents together
(
drug-drug interactions, difficulty in obtaining sufficient potencies against the tumor cells,
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potentiation of adverse effects, etc.). Thus, kinase inhibitor development efforts in cancer
therapy have shifted from the dogma of one molecule against one target for one disease to a
more controlled multi-targeting with the aim of addressing the genetic diversity and multiple
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driving forces of disease. The three major groups of targets in the current kinase inhibitor drug
discovery are receptor tyrosine kinases, kinases of the mitogen-activated protein kinase (MAPK)
pathway and kinases of the phosphoinositide 3-kinase (PI3K) pathway. Because these three
groups of targets are mechanistically linked, there is a compelling biological rationale for
simultaneously targeting several members of each of these groups to create therapeutic benefits.
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Using fragment-based drug design combined with medicinal chemistry approaches, we have
developed a new generation of type-II ATP-competitive MTKIs. The series was rationally
designed according to a structure-based drug design and kinase-focused approach to identify
single agents with an optimized multi-pharmacological inhibition profile. Recently, a series of
DFG-in inhibitors was first designed and evaluated against kinases of the ABL and SRC
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families. Using these DFG-in inhibitors composed of a 7-azaindole core as the initial fragment,
followed by iterative structural and kinase activity improvements, we have identified new type-II
MTKIs that are highly potent against the ABL and SRC families but also on other oncogenic
kinase members of the receptor tyrosine kinase group and the MAPK pathway. Thus, the
compounds target multiple kinase pathways involved in tumor progression, angiogenesis,
immune system modulation and metastasis. This new series presents high anti-proliferative
activity, demonstrating the inhibition of the tumor cells, the angiogenesis process and more
broadly, an effect on the tumor microenvironment against a wide range of cancer types.
Moreover, with this multi-targeted profile, the compounds are expected not only to avoid
development of tumor resistance but also to be used as a therapeutic aid for patients developing
resistance while being treated with currently marketed kinase inhibitors.
RESULTS AND DISCUSSION
Fragment-based drug design of 7-azaindole series
Two main strategies are used to design kinase inhibitors. The strategy adopted depends on the
targeted conformation of the kinase.
As depicted in Figure 1, the type-I inhibitors are designed to interact with the kinase in its
active or DFG-in conformation. In this strategy, a cyclic core (aromatic or not) is used to interact
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with the segment linking the N-terminal to the C-terminal lobe of the kinase, therefore called the
hinge. A second fragment, usually an aromatic ring, is used to interact with a first specificity
pocket of the kinase that is separated from the hinge by a single amino acid called the
gatekeeper. As the gatekeeper presents an obstacle to the interaction with the specificity pocket
through steric hindrance, a linker is used between the aromatic ring and the cyclic ring system
interacting with the hinge to bypass this binding obstacle.
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As depicted in Figure 2, the type-II inhibitors are designed to interact and block the kinase in
its inactive, or DFG-out, conformation. As in the DFG-in strategy, the interactions with the hinge
and the specificity pocket are found in type-II inhibitors. The difference lies in the exploration of
the rest of the kinase pocket. The aromatic fragment interacting with the first hydrophobic pocket
is extended via a linker (amide or urea) by another aromatic ring holding or not holding some
substituents. This last fragment is supposed to interact with a new hydrophobic pocket formed
after the structural rearrangement of the activation loop of the kinase. This second selective
pocket is the less conserved among the kinases and allows the establishment of more interactions
between the inhibitor and the active site and therefore increases the potency and the selectivity of
the inhibitor. The design of the type-II inhibitor strategy was then selected for the present work.
Our series of compounds is composed of a 7-azaindole core that is considered an adenine
mimetic heterocycle and is predicted to dock into the purine binding site by interacting with the
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hinge. As shown in a previous study, the addition of a first aromatic ring (best with methyl-
substituted phenyl) to the 7-azaindole moiety with an aminomethyl linker allows interaction with
the first hydrophobic pocket juxtaposed to the gatekeeper existing both in the active or inactive
conformation. Accordingly, and to design selective type-II inhibitors, a second aromatic moiety
was added to the inhibitor structure to create new hydrophobic interactions with the second
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specificity pocket. A docking study predicted that such compounds should adopt the classical
binding mode of type II-inhibitors such as Imatinib (Figure 2). An example of the superposition
of our compound 6x docked to Imatinib co-crystallized ABL kinase is depicted in Figure 3 and
showed a similar type-II binding mode with at least 5 hydrogen bonds established within the
kinase active site.
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Figure 1. Classical structural scheme of type-I kinase inhibitor docked into the DFG-in
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conformation. Example of Dasatinib co-crystallized within the SRC kinase (PDB: 3G5D ). A:
Representation of the principal amino acids surrounding the ligand. B: View of the active site
surface, PBP: Purine Binding Pocket, SP I: Selectivity Pocket I.
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Figure 2. Classical structural scheme of a type-II kinase inhibitor docked into DFG-out
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conformation. Example of Imatinib co-crystallized within the SRC kinase (PDB: 2OIQ ). A:
Representation of the principal amino acids surrounding the ligand. B: View of the active site
surface, PBP: Purine Binding Pocket, SP I: Selectivity Pocket I, SP II: Selectivity Pocket II, SP
III: Selectivity Pocket III.
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Figure 3. A. Superposition of the predicted binding mode of 6x (in cyan) and Imatinib (in green)
within the active site of ABL (in grey). Potential hydrogen bonds are depicted in black dashed
lines. Docking studies were performed with ABL X-ray structure co-crystallized with Imatinib
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(
PDB: 1IEP ).
Medicinal Chemistry and Kinase Assays
8
Starting from the DFG-in series previously described, the aim was to design compounds able
to inhibit kinases belonging to the receptor tyrosine kinase family, the SRC family and the
MAPK pathway. We selected four representative kinases from the growth receptor tyrosine
kinase family (EGFR, Epidermal Growth Factor Receptor; VEGFR2, Vascular Endothelial
Growth Factor Receptor 2; FGFR2, Fibroblast Growth Factor Receptor 2; PDGFRA, Platelet-
Derived Growth Factor Receptor Alpha), the SRC kinase as a member of the SRC family and the
B-RAF kinase as one of the first kinases involved in the MAPK signaling pathway activation.
Given the role of the specificity pocket in determining type-II inhibitor selectivity, chemical
modulations were performed to explore different chemical fragments and their impact on the
activity against the six targeted kinases.
The general route of synthesis is described in Scheme 1. Compounds 6a-z were obtained after a
three-step synthesis from 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid methyl ester
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(
1a), commercially available. After a reductive amination with 2-methyl-5-nitrobenzaldehyde (2)
and sodium cyanoborohydride, the nitro-intermediate (3a) was reduced under hydrogen pressure
and yielded the aniline intermediate (4a). To obtain the final compounds 6a-z, the aniline (4a)
was coupled with several carboxylic acids or acyl chlorides, commercially available 5a-w or
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synthesized in house 5x-z according to procedures described in the literature.
Carboxylic
acids and acyl chlorides 5a-w are aromatic or heteroaromatic derivatives bearing hydrophobic or
hydrophilic substituents in order to create potential interactions such as π-stacking or hydrogen
bound with the second hydrophobic pocket of the kinase. The last carboxylic acids 5x-z are
fragments that were derived from marketed drugs (Imatinib, Bafetinib and Nilotinib,
respectively) well known to interact with the specificity pocket of kinases such as ABL and/or
SRC in its inactive conformation.
Scheme 1. Route of synthesis to explore the specificity pocket in the design of DFG-out type-II
a
inhibitors
H
H
N
N
O
N
O
N
OHC
+
NH2
NO2
a
NO2
R1
R1
N
H
1
1
a R =OMe
2
1
3a-b
b R =Me
1
b
H
N
O
O
N
H
N
H
O
N
c
R2
HO
Cl
R2
N
R1
N
NH2
H
R1
N
O
O
H
or
6
a-z R =OMe
R2
1
4a-b
7x-z R =Me
5a-z
1
Non commercially available carboxylic acids derived from marketed drugs
CF
3
CF3
N
N
5y
NMe
NMe2
HO C
HO C
N
2
2
HO C
2
5x
5z
N
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a
Reagents and conditions: (a) AcOH/MeOH (1/10), rt, 2 h, then NaBH CN, rt, 16 h-48 h, 82-
3
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6%; (b) H (30 bars), Pd/C 10%, DMF or MeOH/HCl aq, rt, 16 h-48 h, 96-97%; (c) carboxylic
2
acid, HATU, DIEA, rt, 12 h or acyl chloride, NEt , DMF, rt, 16 h, 4-55%.
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In this first series, a set of 26 compounds was synthesized and evaluated for their inhibition
potency for targeted kinases at a concentration of 100 nM. As summarized in Table 1, this
pharmacological assay resulted in a majority of compounds presenting at least 50% inhibition of
SRC and PDGFRA kinase activities. These two kinases were slightly impacted by the nature of
the substituent R2. Inversely, with only two compounds (6y and 6z) able to inhibit the FGFR2
kinase at more than 40% inhibition, this kinase likely adopts a more restrained type-II
conformation. For the three other targets, B-RAF, EGFR and VEGFR2, no relevant correlation
between the structure of the R of the hydrophobic groups and the inhibition percentage of these
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kinases was observed.
Otherwise, when R is a pyridine group, the nature and the position of the substituent as well
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as the position of the aromatic nitrogen atom with regard to the substitution (compounds 6m, 6n,
6o, 6p, 6q) impact the inhibition profiles of the six kinases. Whatever the nature of the
substituent (Me or CF ), the 3-pyridine group allows an increase in the number of inhibited
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kinases by comparison with compounds bearing a phenyl group or a 4-pyridine group. For
exemple, the nitrogen atom adjacent to the CF group on the 4-pyridine ring leads to a less active
3
compound (6p) whereas, with a 3-pyridine group, the compounds 6m and 6o inhibit five kinases.
Globally, these results illustrated the difficulty of rationalizing the difference in the specificity
profile observed among the 26 compounds on the targeted kinase panel. The 3-pyridine group
would seem to be the best aromatic ring in R2 position to have a MTKI profile and its
combination with Imatinib and Bafetinib moiety would be interesting to test if the chemistry
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allows that. Based on these first findings, very interesting inhibition activities were observed for
some of these compounds. Based on a value of 40% as an inhibition threshold, one compound
(6y) showed a pan high inhibition of the six kinases, and one compound (6z) exhibited a
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moderate inhibition for the six kinases. Six compounds (6m, 6n, 6o, 6s, 6u, 6x) inhibited five
kinases except for FGFR2. Moreover, four compounds (6a, 6b, 6j, 6k) inhibited four kinases and
do not inhibit EGFR and FGRF2. The rest of the compounds inhibited 3 kinases or fewer and
showed a heterogeneous inhibition profile.
These results allowed highlighting the MTKI potential of eight compounds (6m, 6n, 6o, 6s,
6u, 6x, 6y, 6z), inhibiting at least five kinases of the selected primary kinase panel.
Table 1. Inhibition percentages at 100 nM for the first series of compounds
a
Percent inhibition at 100 nM
Compo
2
R
B-RAF EGFR FGFR2 PDGFRA SRC
VEGFR2
und
6
a
74%
63%
32%
35%
6%
1%
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87%
84%
85%
72%
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59%
32%
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c
24%
52%
15%
61%
99%
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Percent inhibition at 100 nM
Compo
und
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R
B-RAF EGFR FGFR2 PDGFRA SRC
VEGFR2
6d
39%
29%
16%
11%
37%
63%
39%
64%
64%
53%
7%
8%
68%
66%
47%
71%
74%
108%
105%
74%
38%
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40%
19%
9%
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f
10%
5%
6g
86%
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3%
66%
36%
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6j
14%
62%
42%
16%
5%
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93%
108%
78%
22%
42%
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Percent inhibition at 100 nM
Compo
und
2
R
B-RAF EGFR FGFR2 PDGFRA SRC
VEGFR2
6k
71%
-1%
8%
97%
65%
41%
6
l
61%
95%
2%
6%
87%
93%
58%
89%
29%
89%
6m
72%
19%
6n
85%
73%
49%
78%
-6%
82%
93%
51%
46%
73%
6o
13%
101%
6
p
q
-7%
32%
-2%
42%
42%
13%
6
50%
44%
81%
2%
-7%
1%
76%
85%
39%
73%
0%
6
r
33%
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a
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20
21
22
23
24
25
26
27
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29
30
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32
33
34
35
36
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Percent inhibition at 100 nM
Compo
und
2
R
B-RAF EGFR FGFR2 PDGFRA SRC
VEGFR2
58%
6
s
77%
69%
40%
20%
3%
4%
91%
77%
68%
34%
6t
18%
6u
65%
35%
45%
66%
-4%
-1%
87%
74%
59%
72%
59%
13%
6v
6w
51%
60%
15%
81%
4%
71%
85%
11%
93%
37%
73%
6
x
y
21%
6
92%
54%
80%
82%
75%
52%
90%
68%
96%
49%
96%
46%
6
z
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Mean of duplicate experiments. In the blue cells, the percent inhibition is superior to 40% at
1
00 nM.
In a set of pharmacomodulation experiments, the influence of the nature of the substituent on
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the 7-azaindole core was investigated. The modifications focused on 1) replacing the methyl
ester group on position 2 of the 7-azaindole core by acetyl, N-methylamide or carboxylic
functions or 2) changing the nature of the linker in position 5.
The first modifications in position 2 were introduced either by hydrolysis of the most active
compounds of the first series to afford the carboxylic derivatives 8x-z (Scheme 2-A), or by using
the 1-(5-amino-1H-pyrrolo[2,3-b]pyridin-2-yl) ethanone (1b) bearing the acetyl group in position
2
to synthesize the acetyl derivatives 7x-z according to the reaction pathway described in
Scheme 1 (R = Me). Otherwise, the methyl ester function in position 2 of the 7-azaindole was
1
replaced by its corresponding N-methylamide from 5-nitro-1H-pyrrolo[2,3-b]pyridine-2-
carboxylic acid methyl ester (9) in two steps to afford the new precursor 10. This nitro
intermediate was reduced, and the corresponding aniline was used according to the same 3-step
synthesis strategy as for the first series (reductive amination, reduction by hydrogenation,
coupling reaction), as depicted in Scheme 2-B to prepare the methylamide derivatives 12x-12y.
This second series of compounds with modifications in position 2 of the 7-azaindole core was
screened on the six main targeted kinases for their inhibition potency at 100 nM concentration
and compared to their analogs in the methylester series (Table 2).
Some differences in the inhibition profile were observed regardless of the small variations
generated at position 2 of the 7-azaindole. Whatever the nature of the R group, replacing the
2
methyl ester group by its corresponding carboxylic acid induces an important decrease of B-RAF
inhibition. Inversely, compounds with an acetyl substituent exhibit similar or better profiles than
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the ester series. These results revealed the interest in working with the methyl ester and acetyl
series in position 2 of the 7-azaindole core to optimize the series.
In parallel, several variations were performed in the position 5 of the 7-azaindole core to
modify the linker bound to the central aromatic ring and to study the impact of five new linkers
on the inhibition profile of the compounds. The chemistry investigated is depicted in Scheme 3.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Scheme 2. Synthesis pathway to modify the substituents on position 2 of the 7-azaindole core
a
Reagents and conditions: (a) KOH or LiOH, MeOH/H O (1/1), reflux, 42-76%; (b) MeNH ,
2
2
HATU, DIEA, DMF, 59%; (c) H (30 bars), Pd/C 10%, MeOH, overnight; (d) 2-methyl-5-
2
nitrobenzaldehyde 2, AcOH/MeOH (1/10), rt, 2 h then NaBH CN, rt, 16 h, overall yield for the
3
three steps: 53%; (e) carboxylic acid, HATU, DIEA, DMF, rt, 16 h, 3-7%.
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Percent inhibition at 100 nM for compounds with modifications in position 2 of the 7-
azaindole core.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Percent inhibition at 100 nM
Compo
R1
R2 B-RAF
EGFR FGFR2 PDGFRA
SRC
VEGFR2
und
6
x
MeO
MeO
MeO
Me
x
y
z
x
y
z
x
y
z
x
y
60%
92%
54%
35%
56%
55%
12%
33%
18%
86%
93%
81%
80%
82%
99%
94%
43%
96%
98%
91%
99%
101%
21%
75%
52%
5%
85%
90%
68%
96%
73%
24%
79%
66%
63%
95%
94%
93%
96%
49%
87%
91%
59%
75%
86%
69%
98%
99%
73%
96%
46%
88%
71%
24%
72%
80%
58%
93%
87%
6y
6
z
7
7
x
y
Me
58%
13%
14%
71%
49%
23%
98%
7
z
Me
8x
HO
8
y
HO
8
z
HO
12x
2y
MeHN
MeHN
1
a
Mean of duplicate experiments. In the blue cells, the percent inhibition is superior to 40% at
100 nM.
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The key intermediates used to introduce different linkers were the 5-formyl-1H-pyrrolo[2,3-
b]pyridine-2-carboxylic acid methyl ester (14a) and the 2-acetyl-1H-pyrrolo[2,3-b]pyridine-5-
carbaldehyde (14b) obtained, respectively from the 5-cyano-1H-pyrrolo[2,3-b]pyridine-2-
carboxylic acid methyl ester (13a) and the 2-acetyl-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile
(13b). Whereas one reduction step with Raney nickel catalyst and hydrogen pressure was
sufficient in the ester series to obtain the aldehyde derivative 14a, a supplementary step of
oxidation with manganese oxide was necessary in the acetyl series to obtain 14b due to the
reduction of the acetyl group that occurred during the first reduction step. These two aldehyde
derivatives (14a and 14b) allowed access to the final compounds 25d-k with 3 different linkers
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
X-Y, either CH O, CH=CH or CH CH . To access compounds with the first cited ether linker
2
2
2
CH O, the aldehyde derivative 14a was reduced with DIBAL-H reagent to its corresponding
2
alcohol (15a). This intermediate was used in a Mitsunobu reaction with 2-methyl-5-nitrophenol.
Then, a reduction of the nitro group followed by a peptide coupling reaction with the two
carboxylic acids 5x and 5y led to the final compounds 26x and 26y. A Wittig reaction was
performed using the aldehydes 14a and 14b with 2-methyl-5-nitrobenzyltriphenylphosphonium
to generate the intermediates 17a-b with the alkene linker. These derivatives were partially or
completely reduced (step f or g, respectively) to derivatives 18a or 19a-b that were finally
coupled with the two carboxylic acids 5x and 5y to obtain the final compounds with an alkene
CH=CH (27x-y) or an alkyl linker CH CH (28x-y and 29x-y). Compounds 24x-y with the
2
2
amide linker NHC(O) were obtained from 5-amino-1H-pyrrolo[2,3-b]pyridine-2- carboxylic acid
methyl ester 1a and 5-amino-2-methyl-benzoic acid methyl ester 20 in three steps (coupling
reaction followed by nitro-reduction, then final coupling reaction). Finally, from 4-methyl-3-
nitroaniline 22, a peptide coupling reaction followed by a reduction of the nitro group and a
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
reductive amination with 14a led to the final derivative 25x with the reverse amine linker
CH NH.
2
a
Scheme 3. Synthesis pathway to modify the linker in position 5 of the 7-azaindole core
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) 14a: H (10 bars), Raney Ni, Pyridine/H O/AcOH (2/1/1),
2
2
overnight; 67%; 14b: 1) DIBAL-H, toluene, 0°C, 1.5 h, 31%, 2) MnO , THF, rt, overnight, 65%;
2
(
b) DIBAL-H, THF, 0°C, 3 h, 45%; (c) 2-methyl-5-nitrophenol, PPh , DEAD, CH Cl , rt,
3 2 2
overnight, 40%; (d) Zn, AcOEt/AcOH (2/1), 50°C, rt, 1 h 30; (e) 2-methyl-5-nitrobenzyl-
triphenylphosphonium, LiOH, MeOH, rt or reflux, overnight, 69-71% 17b; (f) Zn, AcOH/AcOEt
(
1/2), sonic bath, rt, 30 min, 56%; (g) H (10 bars), Pd/C 10%, DMF, rt, 24 h, 26%-38%; (h)
2
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8
9
HATU, DIEA, DMF, rt, w-end, 93%; (i) H (35 bars), Pd/C 10%, MeOH, rt, overnight, 81%; (j)
2
5x or 5y, DIEA, HATU, DMF, rt, overnight, 5-55%; (k) 5x, DIEA, EDCI, rt, overnight, 45%; (l)
Zn, AcOEt/AcOH (2/1), rt, 1 h 20, quant. yield; (m) 1) 14a, AcOH/MeOH (10/1), rt, 2 h, 2)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NaBH CN, rt, overnight, 17%.
3
A total of eleven new compounds (24 to 29) were synthesized bearing 5 new linkers, and their
inhibition profile was evaluated on the six targeted kinases and compared to their analogs with
the initial linker NHCH2.
Table 3. Inhibition percentages at 100 nM for compounds with modifications on position 5 of
the 7-azaindole core.
a
Percent inhibition at 100 nM
Compou
R1
X-Y
R2
B-RAF EGFR FGFR2 PDGFRA SRC VEGFR2
nd
6
x
MeO NHCH
2
2
x
y
x
y
x
x
60%
92%
71%
46%
17%
7%
81%
80%
65%
76%
63%
81%
21%
75%
8%
85%
90%
41%
94%
88%
98%
93%
96%
94%
100%
47%
58%
73%
96%
97%
93%
46%
67%
6y
MeO NHCH
2
4x
4y
MeO NHC(O)
MeO NHC(O)
2
94%
8%
25x
6x
MeO CH
NH
2
2
MeO CH
2
O
14%
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2
2
2
6y
7x
7y
8x
MeO
CH
2
O
y
x
y
x
y
x
y
x
y
15%
24%
33%
-19%
64%
35%
56%
67%
60%
93%
38%
59%
98%
97%
99%
94%
86%
83%
69%
2%
96%
88%
68%
99%
105%
96%
73%
86%
89%
87%
40%
55%
91%
101%
87%
91%
77%
97%
86%
51%
61%
98%
102%
88%
71%
92%
99%
MeO CH=CH
MeO CH=CH
38%
35%
101%
5%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MeO CH
MeO CH
CH
2
2
2
28y
2
CH
7
x
y
Me
Me
NHCH
2
7
NHCH
2
58%
18%
81%
29x
9y
Me CH
Me CH
2
2
CH
2
2
2
CH
a
Mean of duplicate experiments. In the blue cells, the percent inhibition is superior to 40% at
1
00 nM.
The pharmacomodulations with the reverse amine linker (CH NH) or the alkene (CH=CH)
2
linker induce an important decrease of the activity on the targeted kinases, excluding PDGFRA
for which the modifications that were used slightly affect the inhibition of this kinase. For the
other new tested linkers (NHC(O), CH O or CH CH ), similar MTKI profiles were obtained on
2
2
2
the targeted kinases when compared with compounds bearing the initial linker NHCH except for
2
B-RAF. Indeed, B-RAF kinase inhibition was the most negatively affected by the nature of the
linker (decrease of the activity for 8 compounds over the 11 new ones). The best inhibition
profiles were obtained for compounds bearing linkers NHCH and CH CH This new series of
2
2
2.
variations on position 5 of the 7-azaindole allowed highlighting the MTKI potency of six new
compounds: 24x, 24y, 26y, 28y, 29x, 29y. These derivatives inhibited at least 5 of 6 targeted
kinases with at least a 40% inhibition at 100 nM as the compounds 6x, 6y, 7y.
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Cellular Anti-Proliferative Effects of Compounds
The six targeted kinases are members of different signaling pathways involved in tumor
growth and progression, including angiogenesis and metastasis processes. To validate the dual
mechanism of action of the nineteen best compounds inhibiting at least 5 of 6 targeted kinases
developed to date, their anti-proliferative potency was evaluated in a first screening experiment
on human primary endothelial cells (HUVEC; Human Umbilical Vein Endothelial Cells) to
prove the antiangiogenic effect and on a hepatocellular carcinoma cell line (HepG2) for the anti-
tumoral activity of the compounds. In parallel, Sorafenib, a marketed MTKI approved by the
FDA in the treatment of liver cancer, was incubated with cells and used as a reference
compound. While presenting a good profile of kinase inhibition, 8y and 8z were not evaluated in
cellular assays. We previously observed a lack of anti-proliferative activity for other DFG-out
inhibitors bearing, as compounds 8y and 8z, a carboxylic acid in position 2 of the 7-azaindole.
The EC50 on cells for different carboxylic compounds of this series could not have been
calculated because no inhibition of proliferation was observed even at the highest evaluated
concentrations of 3 µM, indicating an EC50 at least 10-fold the EC50 of corresponding
methylester compounds. Consequently, for this series, the presence of the carboxylic acid would
avoid cell membrane penetration and thus would not allow such compounds to demonstrate anti-
proliferative activity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4. Anti-proliferative effects of compounds represented by their EC50 values on the
HUVEC primary cells and the HepG2 cancer cell line.
EC50 (nM)
a
Ref
6m
6n
6o
6s
6u
6x
6y
6z
34
38
7y
12x
12y
24x
24y
26y
28y
29x
29y
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HUVEC 718 >1000 986
390
298
199 >1000 689
97 >1000 83 >1000 631
426
171
118
163
HepG2 302
510 >1000 963 >1000 >1000 >1000 673
275 >1000 179 >1000 977 >1000 >1000 >1000 248
a
Reference: Sorafenib.
While three compounds (6x, 12x and 24x) have failed to demonstrate any activity on HUVEC
and HepG2 (concentration of compound superior to 1 µM is needed to obtain a 50% inhibition of
the cell proliferation), the other compounds exhibit good to excellent activity on at least one cell
type as described in Table 4. With seven compounds showing both anti-proliferative and anti-
angiogenic activities, four of the compounds out-performed Sorafenib (6z, 7y, 12y, 29y). These
four new leads were subjected to a second cellular screening approach aiming to confirm and
compare them in terms of anti-proliferative activities on another type of endothelial cells
(HRMEC; Human Retinal Microvascular Endothelial Cells) and two other human cancer cell
lines (A549, a non-small cell lung cancer cell line and HT29, a colorectal cancer cell line).
Erlotinib and Regorafenib were selected as reference compounds for lung and colorectal cancer
cell lines, respectively, and demonstrated weak anti-proliferative activities. These new assays
confirmed previous findings revealing new anti-proliferative effects on lung and colorectal cell
lines. The compound 6z showed the best anti-proliferative and anti-angiogenic effects with EC50
lower than 70 nM on each cell type (Table 4 and 5). Demonstrating high in cellulo anti-
angiogenic and anti-tumoral potency, 6z was thus selected as our most promising lead compound
for further in vitro development.
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Table 5. Anti-proliferative effects of compounds represented by their EC50 values on the
HRMEC primary cells and the A549 and HT29 cancer cell lines.
EC (nM)
5
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
z
7y 12y 29y Sorafenib Erlotinib Regorafenib
A549
64 204 495 293
37 150 227 303
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
HT29
a
HRMEC 62 179 81 304
nd
a
nd: not determined.
Selectivity profile of 6z
To assess the kinase selectivity profile of 6z, a broad panel kinase screening was conducted
using a biochemical enzymatic assay by evaluating the ability of the compound to inhibit kinases
at a 100 nM concentration (Table 6). In these studies, 6z was found to be a multi-targeted
inhibitor with inhibition superior to 40% against 34 kinases of 104 kinases tested. The main
kinases targeted by 6z are important clinical targets in a variety of malignancies and support the
therapeutic potential of 6z as a broad anti-tumor agent. Kinases such as the Aurora kinase family
or the Polo-like kinase 1 (PLK1) whose inhibition may have negative and particularly adverse
effects (i.e., bone marrow suppression and neutropenia) were not inhibited by the compound.
Moreover, 6z is active on mutated kinases such as different ABL mutants involved in leukemia
resistance, the well-known B-RAF mutant involved in melanoma and the mutation T790M of
EGFR that is involved in the resistance of cells to anti-EGFR therapies in lung cancer. An IC50
G250E
Y253F
E255K
inferior or close to 50 nM was observed on ABL WT, ABL
, ABL
, ABL
and SRC,
historically the first kinases targeted by this compound. As an MTKI, 6z presents IC50 values
V600E
less than 50 nM against at least EGFR WT, LYN A, HCK, PDGFRA, YES, and B-RAF
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(
Table 7). These results confirm that our approach allows the selective design of MTKI
compounds targeting only oncogenic kinases.
Table 6. Kinase inhibition profile of 6z for a broad panel of kinases
Percent inhibition values of the kinase activity at 100 nM
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
% < 40
FGFR1
40 ≤ % > 80
c-SRC
ARG
% ≥ 80
T315I
ABL
PI3Kα
PIM1
PKA
ABL WT
E255K
AKT2
GSK3b
IGF-1R
IKK beta
IKK alpha
INSRR
ITK
ABL
G250E
AMPK
Aurora A
B-RAF
BTK
ABL
Y253F
PKCa
PKCd
PKCe
PKCg
PKCθ
PKD2
PLK1
ROCKII
ROS1
SYK
ABL
Aurora B
CSK
BLK
BMX
T790M
Aurora C
EGFR
AXL
EPHA1
EPHA2
EPHA4
EPHB4
ERBB4
FGFR2
FLT3
EGFR WT
EPHB2
FGR
CaMKII d
CDK1/Cyclin B
CDK2/Cyclin A
CDK5/p25
CDK7/CyclinH/MNAT1
Cdk9/cyclin T1
CHK2
JAK2
JAK3
MEK1
HCK
ERK2
LCK
MAPKAPK2
MAPKAPK5
MATK
MST1R
mTOR
LYN A
YES
V600E
TEC
CSF1R
FRK
B-RAF
CK1
TIE2
c-KIT
TRKA
TSSK2
TYK1
FYN
c-MER
MLCK
p38 alpha
PDGFRA
c-MET
NEK2
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DMPK
ERBB2
FAK
NUAK1
p70S6K
PAK2
TYK2
TYRO3
VEGFR1
VEGFR3
ZAP70
RET
VEGFR2
10
11
12
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46
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48
49
50
51
52
53
54
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57
58
59
60
FER
PDGFRb
PDK1
FES
PHKG2
Table 7: IC50 value of 6z on selected kinases inhibited with percent inhibition superior to 40%
at 100 nM
Kinases
IC50 (nM) Kinases
IC50 (nM)
ABL WT
6
PDGFRA
50
51
V600E
Y253F
B-RAF
EGFR WT
HCK
10
15
22
37
40
44
46
ABL
FRK
LCK
62
69
E255K
LYN A
c-SRC
YES
ABL
73
T790M
EGFR
FYN
112
131
G250E
ABL
Broad anti-proliferative effects of 6z
Finally, we evaluated the therapeutic potential of 6z against different types of cancers. The
anti-proliferative effect of 6z was compared to different reference compounds that are currently
approved agents in the indication of interest or under evaluation in clinical trial phases. With the
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MTKI profile of 6z and its ability to inhibit clinically validated oncogenic kinases in almost all if
not all types of cancer, we used either sensitive cancer cell lines or resistant/aggressive cells. The
cells that were considered as sensitive are the BxPC-3 and Caki-2 cell lines which, respectively,
represent human pancreatic and kidney cancer and the murine BaF3 WT leukemia-derived cell
line expressing the human BCR-ABL wild-type fusion protein that is used as a model for the
Philadelphia chromosome (Ph)-positive leukemias. PC-3 (human non-hormone-dependent
prostate cancer cell line), MDA-MB-231 (triple-negative breast cancer cell lines), NCI-H1975
(non-small cell lung cancer cell line harboring the genetic modification of EGFR leading to the
expression of a protein with L858R and T790M double mutations) and BaF3 T315I (the murine
leukemia-derived cell line expressing the well-known mutated form of BCR-Abl fusion protein)
were selected as representative of resistant and/or aggressive cancer cells.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 8: Anti-proliferative effects of 6z in comparison with reference compounds represented by
their EC50 values on different cancer cell lines.
a
EC (nM)
5
0
MDA-
MB-231
158
NCI-
H1975
104
BaF3
WT
BaF3
T315I
1.8
PC-3
Caki-2
BxPC-3
6
z
152
232
22
55
35
33
0.04
0.04
Dasatinib
Erlotinib
Sunitinib
Ponatinib
Imatinib
Sorafenib
44
173
>1000
nd
a
>1000
>1000
>1000
nd
>1000
>1000
653
>1000
>1000
483
>1000
>1000
544
>1000
>1000
>1000
nd
nd
nd
0.05
34
>1000
0.3
nd
nd
nd
>1000
nd
nd
>1000
>1000
>1000
>1000
nd
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Afatinib
nd
nd
>1000
>1000
116
nd
nd
a
nd: not determined. In the grey cells, marketed references currently approved in the
indication.
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EC50 values presented in Table 8 demonstrate that 6z is highly cytotoxic with similar or even
better potency than reference compounds against a large panel of cancer cell types even if
aggressive or resistant.
Pharmacokinetic profile of 6z in mice
The pharmacokinetic (PK) parameters of 6z were assessed in mice following intravenous (IV)
and oral (PO) administrations as described in Table 9. After IV administration at a dose of 2
mg/kg, the maximum concentration (Cmax) reached 853 ng/mL and the area under the curve
(AUC ) was 558 ng/mL*h. After oral administration at 20 mg/kg, the C reached 610 ng/mL
0-∞ max
(1.1 µM) at 0.5 h post-dosing and the AUC0-∞ was 3029 ng/mL*h (5.4 µM*h).
These results demonstrate good PK properties of 6z in mice with high oral bioavailability (close
to 47%) and a sustained plasma concentration exceeding in vitro EC during at least 8 hours
5
0
after oral dosing. These data indicate a favorable pharmacokinetic profile of 6z in preparation for
efficacy studies in mice.
Table 9: Pharmacokinetic profile of 6z in mice after IV and PO dosing
Administration
Dose
(mg/kg)
2
Cmax
(ng/mL)
853
Tmax
(h)
AUC0-∞
(ng/mL*h)
558
F
route
IV
(%)
a
0.167
0.500
N/A
47
6z
PO
20
610
3029
a
N/A: not applicable
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in vitro hERG patch clamp assay
Blockade of the cardiac ion channel coded by human ether-à-gogo-related gene (hERG) can
lead to cardiac arrhythmia, which has become a major concern in drug discovery and
development, and mainly in the field of kinase inhibitors. The inhibitory effect of 6z on the
hERG channel was assessed using conventional patch clamp experiments. With very modest
inhibition of hERG channel (26% inhibition at a 30µM concentration), the compound 6z bears a
minor risk of potentially problematic cardiac side-effects.
0
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0
in vitro cytotoxicity effect of 6z on human activated and resting PBMC
1
6,17
Several in vitro methods have been suggested to predict drug-induced haematotoxicity.
We
used primary cells (PBMC) isolated from peripheral blood from two healthy donors to assess and
detect drug sensitivity in a short-term assay. Cells were exposed to increasing concentrations of
6z or Dasatinib. Dasatinib was used as reference because it is the most similar compound to 6z in
term of anti-proliferative activity as observed on cancer cell lines (Table 8) and its
1
8
immunosuppressive activity was demonstrated in patients. When PBMC were stimulated with
phytohemagglutinin (PHA) supplemented with IL-2 (PHA/IL-2), mitogen/cytokine combination
used widely to induce cell division, the EC of 6z and Dasatinib were close to 4.8 µM and
50
2
.4µM respectively for donor 1 and close to 1.2 µM and 1.6 µM respectively for donor 2 (Figure
, left). No EC could be determined for both compounds on resting human PBMC due to the
4
5
0
plateau shaped dose-response curves (Figure 4, right). Human cancer cell lines showed higher
sensitivity in terms of EC to 6z than human PBMC indicating that even if 6z might induce
50
immunosuppression in clinic, this should be manageable and there could be a therapeutic
window for 6z in a broad range of cancer types. Nevertheless, these results suggest that a
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compound optimization stage, most probably by re-designing 6z to selectively avoid inhibition
of LCK, could still improve its safety profile to prevent potential side effects and
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immunosuppressive complications.
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PHA/IL-2 activated PBMC
Resting PBMC
1
20
120
100
80
60
40
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0
100
80
6
0
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6
z Donor 1
6z Donor 2
2
0
Dasatinib Donor 1
Dasatinib Donor 2
0
-8
-7
-6
-5
-8
-7
-6
-5
Log [compound (M)]
Log [compound (M)]
Figure 4. in vitro evaluation of cytotoxic effect of 6z on activated (PHA/IL-2) or resting human
PBMC from 2 healthy donors (error bars represent SD).
CONCLUSIONS
Using structure-based drug design combined with medicinal chemistry approaches, we have
designed and synthesized a new and potent 7-azaindole-based chemical series showing a multi-
target kinase inhibition profile. The compounds from the new series are all type-II inhibitors and
are follow-up series from the study of dual ABL and SRC kinase type-I inhibitors we published
8
previously.
A library of more than forty type-II kinase inhibitors was synthesized in 3 to 6 chemical steps
from commercially available 7-azaindole scaffolds.
A step wise medicinal chemistry was used to perform pharmacomodulations around the 7-
azaindole core and to increase the potency of compounds to obtain a multi-target kinase
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