Pyrrolo[3,2-b ]quinoxaline derivatives as types I1/2 and II Eph tyrosine kinase inhibitors: Structure-based design, synthesis, and in vivo validation

Article abstract of DOI:10.1021/jm5009242

The X-ray crystal structures of the catalytic domain of the EphA3 tyrosine kinase in complex with two type I inhibitors previously discovered in silico (compounds A and B) were used to design type I_1/2 and II inhibitors. Chemical synthesis of a

Full text of DOI:10.1021/jm5009242

Article
pubs.acs.org/jmc
Pyrrolo[3,2‑b]quinoxaline Derivatives as Types I1/2 and II Eph
Tyrosine Kinase Inhibitors: Structure-Based Design, Synthesis, and in
Vivo Validation
Andrea Unzue,†,§ Jing Dong,‡,§ Karine Lafleur,† Hongtao Zhao,‡ Emilie Frugier,‡ Amedeo Caflisch,*,‡
and Cristina Nevado*,†
‡
†Department of Chemistry and Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich,
Switzerland
̈
̈
S
* Supporting Information
ABSTRACT: The X-ray crystal structures of the catalytic domain of the EphA3 tyrosine kinase in complex with two type I
inhibitors previously discovered in silico (compounds A and B) were used to design type I1/2 and II inhibitors. Chemical synthesis
of about 25 derivatives culminated in the discovery of compounds 11d (type I1/2), 7b, and 7g (both of type II), which have low-
nanomolar affinity for Eph kinases in vitro and a good selectivity profile on a panel of 453 human kinases (395 nonmutant).
Surface plasmon resonance measurements show a very slow unbinding rate (1/115 min) for inhibitor 7m. Slow dissociation is
consistent with a type II binding mode in which the hydrophobic moiety (trifluoromethyl-benzene) of the inhibitor is deeply
buried in a cavity originating from the displacement of the Phe side chain of the so-called DFG motif as observed in the crystal
structure of compound 7m. The inhibitor 11d displayed good in vivo efficacy in a human breast cancer xenograft.
forming additional interactions with hydrophobic regions
adjacent to the ATP binding site are termed type I1/2 inhibitors.
Alternatively, type II inhibitors target the kinase catalytic site
but bind to the inactive conformation of the protein, thus
exploring a pocket generated upon displacement of the
phenylalanine side chains of the DFG motif.4 Type III
inhibitors, also known as allosteric inhibitors, target areas of
the kinase not related to the catalytic domain, whereas type IV
do so without competing with ATP. Higher degrees of
selectivity are to be expected with the latter two inhibitor
types.19
Recently, our groups have focused on the in silico design,
synthesis, and computational-aided optimization of potent and
selective receptor tyrosine kinase inhibitors. Successful
campaigns have yielded single-digit nanomolar EphB4 inhib-
itors whose potential antiproliferative activities have been
characterized by cellular assays.20−22 Furthermore, the
predicted binding mode could also be confirmed by X-ray
diffraction analysis of their complexes with EphA3. Given the
I. INTRODUCTION
Several protein kinases are relevant targets for the treatment of
diseases ranging from cancer, inflammation, and cardiovascular
conditions to immune related disorders.1,2 Over the past
decade, more than 13 small-molecule kinase inhibitors have
been approved by the FDA as therapeutics for various human
pathologies.3−6 In this context, receptor tyrosine kinases play a
prominent role, as they are involved in a number of biologically
relevant processes for cancer development including oncogenic
regulation, cell signal transduction, proliferation, and survival
among many others.7,8 Although irreversible inhibitors that
form covalent bonds with cysteine or other nucleophilic
residues in the ATP-binding pocket have been recently
explored,9,10 ATP competitive, noncovalent inhibitors are
much more abundant and, depending on the binding mode
with their protein target, are classified as type I−IV.11 Most
kinase inhibitor drugs are of type I, i.e., they are direct
competitors of ATP within the catalytic site of the
phosphorylated active conformation of the protein.12 However,
because of the strong similarities between the ATP binding
pocket of all human kinases, alternative approaches providing
selective binders have been sought.13−18 Small molecules
Received: June 18, 2014
Published: July 30, 2014
© 2014 American Chemical Society
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critical role of Eph receptors and (Eph)−ephrin signaling in
tumor growth and progression,23 a subset of these compounds
are currently being pursued toward preclinical development.
Here we describe a multidisciplinary campaign toward the
design of novel and potent, type I1/2 and II tyrosine kinase
inhibitors based on the crystal structure of two type I inhibitors.
The parent pyrrolo[3,2-b]quinoxaline scaffold was decorated
with characteristic functional groups present in previously
successful type II binders, thus speeding up the hit to lead
optimization campaign. The binding kinetics of the low-
nanomolar derivatives 11d (type I1/2) and 7m (type II) were
characterized by surface plasmon resonance (SPR) measure-
ments. Extensive profiling by biochemical (competition bind-
ing) and cellular assays, together with pharmacokinetic
measurements in mice resulted in the prioritization of inhibitor
11d for final validation in vivo by a human breast cancer
xenograft.
for inhibitor B due to the ethoxy-propyl substitution at the
nitrogen of the amide whose trans configuration prevents it
from acting as donor to the carbonyl group of Met696. The
lack of this hydrogen bond is consistent with the about 10-fold
weaker affinity of inhibitor B with respect to A (IC50 of 300 nM
for EphB4),24 which might also originate, at least in part, from
the different substituents of the phenyl ring in the hydrophobic
pocket, i.e., −OCH3 and −Cl in A and B, respectively.
II.2. Design of Type I1/2 and Type II Derivatives Based
on the X-ray Crystal Structure of the Type I Inhibitors A
and B. On the basis of our previous experience20,22 and earlier
reports toward the synthesis of potent type I kinase inhibitors,25
several modifications within the phenyl ring were designed in
order to fine-tune the interactions of the quinoxaline inhibitors
with the threonine gatekeeper residue (Thr693) of EphB4.
Because of the rather limited space around the phenyl group
revealed by the binding modes of A and B, the introduction of
small substituents was envisioned, including the incorporation
of a methyl and a hydroxyl group at positions 2 and 5, a
combination that had been successfully exploited in our
previous studies developing nanomolar inhibitors of EphB4.20
In addition, the binding modes of inhibitors A and B
suggested the possibility to extend the quinoxaline scaffold into
the allosteric binding site by substitution of the phenyl ring,
resulting in type II inhibitors. Our design campaign targeting
the back pocket of the kinase relied on the introduction of a
variety of substituents, some of which are present in type II
inhibitors. The so-called “tail” moiety of type II inhibitors,
located within the allosteric binding site, is characterized by a
hydrogen bond donor−acceptor pair (usually an amide or an
urea), linked to a hydrophobic substituent that occupies the
newly formed pocket in the DFG-out conformation of the
kinase.4,7,12 Amide, urea, and thiourea linkers were incorporated
onto the quinoxaline scaffold and attached to a m-CF3-phenyl
moiety present in some type II kinase inhibitors, in analogy to
AAL993,26 sorafenib,27,28 and GNF-583729 (7b−d, 7g−i, 7l,
7m, and 12n). A cyclopropyl ring, a common motif in p38α
isoform selective kinase inhibitors,30−32 was also introduced
(7a, 7f, and 7k). In an effort to increase the hydrophobic
interactions within the allosteric binding site, a 4-methyl
imidazole ring was added aiming to mimic the well-known drug
nilotinib33 (7e and 7j). In order to increase the solubility,
methyl or fluoro substituents were placed in ortho-relative
position (7c−e and 7h−j), therefore distorting the planarity of
the molecules.34 The results in Table 1 underline that, although
the identification of substituents successfully binding the
allosteric pocket based on known inhibitors was not a priori
obvious, this was nonetheless an efficient strategy to obtain
potent and selective type II kinase inhibitors in a time- and
cost-effective manner.
II. CRYSTAL STRUCTURES OF TYPE I INHIBITORS A
AND B WITH EPHA3
II.1. Docking Validation by X-ray Diffraction Analysis
of Binding Complex. Recently, we reported the discovery of
two type I EphB4 inhibitors A and B by automated docking.24
The in silico predicted binding mode of these molecules is
confirmed here by X-ray diffraction analysis of the catalytic
domain of EphA3 in complex with both A and B (Figure 1).
Figure 1. Crystal structures of the catalytic domain of the tyrosine
kinase EphA3 in complex with the high-nanomolar inhibitors A (left,
pdb code 4P4C) and B (right, pdb code 4P5Q). The ATP binding site
of the EphA3 kinase is shown in magenta ribbons, while the side
chains mentioned in the text and the inhibitors are shown by sticks
(with carbon atoms in magenta and green, respectively).
III. CHARACTERIZATION OF NEW TYPE I1/2 AND II
INHIBITORS
The pyrrolo[3,2-b]quinoxaline scaffold occupies the ATP
binding site with the phenyl substituent nestled into the so-
called hydrophobic pocket. The amino substituent at position 2
of the pyrrole ring is involved in a bifurcated hydrogen bond
with the side chain hydroxyl of the Thr693 gatekeeper and the
backbone carbonyl of Glu694. Furthermore, in the structure
with inhibitor A, the amide substituent at position 3 of the
pyrrole ring is optimally oriented for two hydrogen bonds with
the backbone polar groups of Met696 so that A forms a total of
three hydrogen bonds with the backbone of the hinge region.
Only two hydrogen bonds with the same region are observed
III.1. Synthesis. The synthesis of 1H-pyrrolo[2,3-b]-
quinoxaline derivatives 6 and 7 is shown in Scheme 1.
Compound 1 was prepared according to previously reported
procedures by condensation of commercially available 2,3-
dichloroquinoxaline with malononitrile in the presence of
sodium hydride.35,36 The substitution of the chlorine at
position 3 with commercially available anilines 2a−i followed
by cyclization afforded intermediates 4a−i.37 The reaction with
synthetically prepared anilines 3a−n delivered tricyclic
intermediates 5a−n (Scheme 1).
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Table 1. EphA3/EphB4 Inhibition Data for the Synthesized Quinoxaline Derivatives
a
b
Average values of triplicate measurements. The standard deviation is smaller than 0.5 degrees. FRET-based enzymatic assay was carried out using
c
the Z-LYTE Kinase Assay Kit−Tyr 1 Peptide (Invitrogen) following the vendor instructions. Cell IC50 values were measured in a cellular
phosphorylation assay using MEF cells overexpressing EphB4 at Proqinase.
The preparation of the noncommercially available anilines
been summarized in Scheme 2. Anilines 3a−e, bearing a N-
3a−m used in the above-mentioned condensation reaction has
amide group in meta-relative position, were obtained by
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a
Scheme 1
a
Reagents and reaction conditions. (a) Protocol I: R1NH2 2a−i, 3a−d, 3f−i, 3k−l, 3n (1−4 equiv), EtOH/toluene, 1:1, 130−160 °C, 4−12 h, 30−
79%. Protocol II: R1NH2 3e, 3j, 3m (1−1.2 equiv), DMF, 80 °C, 12 h, 21−61%. (b) H2SO4, 25 °C, 30 min, 7−99%.
a
Scheme 2
a
Reagents and reaction conditions. Conditions (a) Method I: cyclopropanecarboxylic acid, TBTU, DIPEA, DMF, 25 °C, 15 h, 38%. Method II: 3-
(trifluoromethyl)benzoyl chloride, Et3N, DCM, 25 °C, 15 h, 54−99%. Method III: 3-(trifluoromethyl)benzoyl chloride, DIPEA, THF, 25 °C, 12 h,
48%. Method IV: benzoic acid, HOBt, EDC, DMF, 25 °C, 12 h, 64%. Conditions (b) for 3a, 3b, 3f, 3g, 3k and 3l, SnCl2.H2O, EtOH, 100 °C, 2 h,
53−92%; for 3c−e, 3h−j, and 3m, 10% Pd/C (10 wt %), H2, MeOH, 25 °C, 4−12 h, 44−99%. Conditions (c) Method I: amine, Et3N, DCM, 25 °C,
15 h, 50−79%. Method II: aniline, DIPEA, THF, 25 °C, 12 h, 57−78%. Method III: (i) SO2Cl, DCM, reflux, 3 h; (ii) 3-(trifluoromethyl)aniline,
DCM, 25 °C, 12 h, 77%. Conditions (d) Method I: aniline, Et3N, DCM, 25 °C, 1−3 d, 80−92%. Method II: 3-(trifluoromethyl)aniline, Et3N, THF,
25 °C, 12 h, 60%.
condensation of 3-nitro-substituted anilines 8 with the
corresponding readily available benzoyl chlorides (R3COCl)
or benzoic acids (R3COOH)38 using different protocols
(Conditions a, Methods I−IV). Reduction of the nitro group
with either SnCl2 or Pd/C and H2 (Conditions b, Supporting
Information S12) furnished the desired intermediates. Anilines
3f−j were prepared by condensation of the 3-nitro-substituted
benzoic acid or benzoyl chlorides 9 with the corresponding
anilines39 under reaction conditions c. Reduction of the nitro
group with SnCl2 or Pd/C and H2 delivered the corresponding
anilines in 30−77% overall yields. Finally, anilines 3k−m,
bearing urea moieties at meta- and para-relative positions, were
prepared by condensation of aniline 8 with the corresponding
isocyanate (for 3k−l) or by condensation of the isocyanate 10
with the respective commercially available aniline under
reaction conditions d. Reduction of the nitro group under the
conditions described above furnished the corresponding
aniline-containing ureas 3k, 3l, and 3m in 42, 85, and 41%
yields, respectively. Aniline 3n was prepared according to a
previously reported procedure.40
signal at 115−116 ppm, whereas the amino group appeared as a
37
1
broad signal at 8 to 8.5 ppm in H NMR.
Two more inhibitors were prepared, first by demethylation of
6d in the presence of BBr3 under reflux to give the
corresponding phenol derivative 11d, and a second one by
condensation of the aniline 7n with m-CF3-phenyl isothiocya-
nate, to give the corresponding thiourea 12n (Scheme 3).
III.2. Biophysical Characterization. The thermodynamics
and kinetics of binding of the designed quinoxaline inhibitors
were investigated by an array of biophysical techniques
including differential scanning fluorimetry, fluorescence reso-
nance energy transfer (FRET) based enzymatic assays, and
surface plasmon resonance (SPR). Differential scanning
fluorimetry is a high throughput technique in which the
increase in thermal stability of a folded protein upon ligand
binding is detected by a fluorescent dye while measuring its
melting temperature during denaturation.41
In order to allow the binding of type II inhibitors to the
inactive form of the kinase, the protein (EphA3) was incubated
in the presence of the compounds for 1 h. The results (shown
in Table 1) highlight the differences in binding between type I,
I1/2, and II compounds, with type I being the weakest binders
(1.5−4.1 °C) and type II the most potent (with up to 16 °C
thermal shifts).
Hydrolysis of the cyano group under strong acidic conditions
furnished the desired type I and I1/2 inhibitors 6a−i and type II
inhibitors 7a−n, respectively. The presence of a cyano group in
compounds 4 and 5 was confirmed by the presence of a
characteristic IR band at around 2200 cm−1 and a 13C NMR
For type I inhibitors, the largest thermal shifts (ca. 4 °C)
were obtained for ortho-methyl (6a) and ortho-chlorine (6f)
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a
the thermal shifts described above. Compounds 6a and 6f
showed inhibitory activities higher than 66% at 1 μM (Table 1).
However, substitution at R1 by a smaller fluorine atom yielded
compound 6g with lower binding affinity (30%). Along the
same lines, bis-ortho substitutions with either methyl or
chlorine (6b,c) decreased the binding affinity (36 and 45%
inhibitory activities, respectively) probably due to unfavorable
steric effects. The presence of a hydrogen bond donor at R3
(type I1/2 inhibitors) either as a phenol (6h and 11d) or
methylene alcohol (6e) greatly improved the inhibitory activity
of the molecules thanks to the formation of hydrogen bonds
with Glu664 and Asp758 (68−105%). To our surprise, a
triazole group at the same position (6i) dramatically decreased
the binding affinity (7%).
Scheme 3
The kinetics of binding of the optimized type I1/2 (11d) and
type II urea inhibitor (7m) were investigated using SPR. Upon
titrating 11d and 7m over immobilized dephosphorylated
EphA3, dissociation constants (KD) in the low nanomolar range
were obtained (8.6 and 39.3 nM, respectively), confirming the
high affinity of the compounds (Figure 2). One of the
a
Reagents and conditions: (a) BBr3, DCM, 130 °C, 4 h, 68%; (b) m-
CF3-phenyl isothiocyanate, DMF, 25 °C, 12 h, 69%.
substituted quinoxalines, which is in agreement with previously
reported kinase inhibitors20,42−47 and could be a consequence
of restricting the accessible conformations of the phenyl ring as
we have previously reported.20 However, the ortho-fluoro
substituted inhibitor 6g or bis-ortho substituted 6b and 6c
caused a lower stabilization of the protein probably due to the
small size of fluorine or the introduction of an extra steric bulk,
respectively.
The transition from type I to type I1/2 by the presence of a
hydrogen bond donor at position R3, and therefore the
formation of hydrogen bonds with Glu664 and Asp758,
resulted in a remarkable increase in thermal shift for compound
6h (7.2 °C), which became more pronounced upon
introduction of a methyl group in the ortho-relative position
(following the trend observed in type I inhibitors) to yield 11d
with a thermal shift of 11.2 °C.
Type II compounds bearing an amide linker followed by a m-
CF3-phenyl group caused a similar stabilization effect in the
protein as the type I1/2 inhibitor 11d. As expected from
previous results with type I and I1/2 compounds, the addition of
a methyl (7c and 7h) or fluorine (7d and 7i) substituent in R1
lead to higher thermal shifts. Interestingly, 3-amides (7g−i)
triggered a higher stabilization of the kinase than 1-amides
(7b−d), which could indicate the formation of a more favorable
hydrogen bond with Glu664. The presence of imidazoles within
the allosteric binding site led to compounds 7e and 7j, which
showed the most promising thermal shifts (16 °C). Urea (7m)
or thiourea (12n) linkers located in para-relative position
retained or even enhanced (in the case of urea 7m) the binding
affinity, whereas compound 7l, bearing the urea in meta-relative
position barely presented any thermal shift, suggesting a
disruption or a nonfavorable hydrogen bond interaction with
Glu664. The replacement of the m-CF3-phenyl group by a
cyclopropyl ring proved to be detrimental in all cases, and no
thermal shift was observed for products 7a, 7f, and 7k.
The inhibitory activities of type I and type I1/2 inhibitors
were further evaluated on an enzymatic assay based on
fluorescence resonance transfer (FRET) at a single concen-
tration (1 μM, Table 1, column 8). The results were in line with
Figure 2. SPR analysis of the binding of 11d (A) and 7m (B) to
EphA3. A 3-fold serial dilution of the compounds was made starting
from 200 nM (for 11d) and 10 μM (for 7m) in duplicate. The
theoretical global fit to a 1:1 interaction model is shown in straight
lines.
advantageous characteristics of type II inhibitors over type I or
I1/2 is their slow dissociation rate from the target protein,48−50
as demonstrated by the remarkably slow koff measured for 7m
(1.45 × 10−4 s−1) in comparison to the type I1/2 11d (1.52 ×
10−3 s−1). The slow koff of 7m corresponds to a residence time
of 115 min, a value that compares positively with that of
marketed drugs such as imatinib (28 min for dephosphorylated
ABL), nilotinib (202 and 205 min for dephosphorylated and
phosphorylated ABL, respectively), and dasatinib (15 and 4
min).51 The long residence time of type II inhibitors is
considered to be beneficial for drug efficacy and selectivity in
vivo due to the high concentration of the drug near the
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target,50,52 as described for the EGFR-specific inhibitor
pyrrolo[3,2-b]quinoxaline scaffold occupies the ATP binding
site and is involved in the same hydrogen bonds with the hinge
region as the type I inhibitor A (Figure 1). In addition, the urea
linker of inhibitor 7m acts as hydrogen bond acceptor from the
Ser757 side chain and the amide backbone of Asp758, and
hydrogen bond donor to the side chain of Glu664. The m-CF3-
phenyl moiety is nestled in the hydrophobic pocket, which
originates from the displacement of the Phe side chain of the
DFG motif. Thus, the type II binding mode of compound 7m
validates our design based on the crystal structures of the
complexes with the type I inhibitors (vide supra, section II.2).
lapatinib.53
IV. VALIDATION OF TYPE II BINDING BY X-RAY
CRYSTAL STRUCTURE DETERMINATION
The crystal structure of the catalytic domain of the EphA3
kinase in complex with inhibitor 7m (solved at 2.0 Å resolution,
Supporting Information S34) confirms a type II binding mode,
i.e., with the DFG-out conformation (Figure 3). The
V. SELECTIVITY AND CELLULAR ACTIVITY
V.1. Selectivity Profiles from Biochemical Assays. The
selectivity profile of inhibitors 11d, 7b, and 7g was determined
by an in vitro competition binding assay using recombinant
kinases (KINOMEscan at DiscoveRx).54 It is important to note
that this assay reports on binding affinity and does not require
ATP. The selectivity panel consisted of 453 human kinases, 58
of which were disease related mutant kinases (mainly of ABL1,
EGFR, and PIK3CA). Single dose measurements were carried
out at 1 μM concentration of the inhibitor.
Compounds 11d, 7b, and 7g present a very similar selectivity
profile (Figure 4); in particular, strong binding is only observed
for tyrosine kinases with threonine as a gatekeeper residue, e.g.,
ABL1/2, BRAF, DDR1, EphA/B (all but EphA7, which has a
Ile gatekeeper), KIT, LCK, SRC, and YES. The latter data
suggest that most (or even all) tyrosine kinases with a Thr
gatekeeper can assume the DFG-out conformation. Quantita-
Figure 3. Crystal structures of the catalytic domain of the tyrosine
kinase EphA3 in complex with the low-nanomolar inhibitor 7m (left,
pdb code 4P5Z) and superposition of the three inhibitors A, B, and
7m based on structural alignment of the Cα atoms of the EphA3 kinase
domain (right). The ATP binding site of the EphA3 kinase is shown in
magenta ribbons, while the side chains mentioned in the text and the
inhibitors are shown by sticks.
Figure 4. Selectivity profiles of compound 11d (left), 7b (center), and 7g (right) tested on a panel of 395 nonmutant (top) and 58 mutant (bottom)
kinases at DiscoveRx. Measurements were performed at a concentration of 1 μM of the inhibitor. The affinity is defined with respect to a DMSO
control. The dendrogram was obtained from KinomeScan using the KinomeTree software.
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tively, each of the three inhibitors 11d, 7b, and 7g binds with an
affinity 10-fold (100-fold) higher than the DMSO negative
control to only about 10% (5%) of the 395 wild-type kinases
tested. Interestingly, the selectivity profiles of the type I1/2
(11d) and II (7b and 7g) quinoxaline-based inhibitors is very
similar to the one of our previously reported type I and I1/2
xanthine-based inhibitors (compounds 40 and 3, respectively,
in ref 22), which is due, at least in part, to the use of an Eph
tyrosine kinase (EphB4) as primary target for the in silico
screening and optimization.
Table 2. Antiproliferative Activity against NCI Tumor Cell
Lines
a
MDA
compd -MB-231
K-562 A498
59.3 50.3
36.4 90.5
HT29
KM12
HOP-92
6d
6e
6i
44.9
50.9 64.5
5.88 4.59
13.4 29.4
11d
7b
2.64
3.09
1.05
1.52
1.55
3.98
(4.03)
0.49
4.65
(3.91)
V.2. Cellular Assays. The most potent inhibitors obtained
in the optimization campaign were further tested in cell-based
assays. Cellular phosphorylation assays on MEF cells trans-
fected with myc-tagged human EphB4 revealed a comparable
tendency to the one observed in the enzymatic assay (Table 1,
column 9). The type I inhibitors (6a−d) displayed cellular IC50
values in the 230−4400 nM range, with the ortho-methyl
substituted derivative 6a as the most potent member of this
series. The type I1/2 inhibitor 11d and type II compounds
bearing amide linkers and a m-CF3-phenyl group (7b−d and
7g−i) displayed levels of inhibitory activity in the low
nanomolar range (6−24 nM), thus being the most promising
molecules of the optimization campaign. In agreement with
thermal shift experiments, the presence of a urea (7m) and
specially the thiourea linker (12n) decreased the potency of the
compounds (89 and 560 nM, respectively). Interestingly, the
imidazole substituted compounds (7e and 7j) proved to be the
weakest type II inhibitors (170 and 270 nM, respectively) in
contrast to the high thermal shifts obtained (16 °C) in the
differential scanning fluorimetry measurements, pointing
toward potential cell permeability or efflux issues.
EphB4 overexpression has been linked to several types of
cancer, including breast,55 colon, 56 and ovarian.57 Compounds
11d and 7m were screened against the NCI-60 cancer cell line
panel (Supporting Information S3 and S4) displaying
antiproliferative activities against leukemia (K-562), lung
(HOP-92), colon (HT-29), renal (A498), and breast cancer
cells (MDA-MB-231 and HS 578T) in the low nanomolar
range. Driven by these results, the most promising inhibitors of
our optimization campaign were tested in-house against the
above-mentioned NCI cancer cell lines (Table 2). The
leukemia K-562 cell line was particularly sensitive toward the
optimized type II quinoxaline inhibitors, especially in the case
of 3-amide compounds 7h and 7i, which showed remarkably
low GI50 values (36 and 81 nM, respectively). Interestingly,
similar levels of potency were found for imidazole substituted
compounds 7e and 7j, which seemed to be among the weakest
type II binders in the cellular phosphorylation assays, possibly
indicating other targets than Eph for these molecules. In
addition, the potential of 11d (the most potent compound on
cellular phosphorylation assays with an IC50 of 6 nM) to inhibit
the growth of patient-derived tumor cell lines was studied using
a propidium iodide-based proliferation assay and dasatinib as a
reference (Oncotest, Table 3). Cell lines included colon, lung,
kidney, pancreatic, prostate, and stomach cancer cells. Whereas
dasatinib presented double-digit nanomolar activities against
RXF 393NL, LXFA 983L, and PRXF DU145, 11d exhibited
low micromolar GI50 values, with RXF 393NL being the most
sensitive cell line.
7c
3.88
1.44
0.73
0.37
2.50 5.87
1.88 1.80
0.80
2.16
4.23
2.06
7d
(9.43)
7e
7g
7h
7i
1.93
10.8
1.32
2.69
3.05
10.3
9.63
0.030
0.820
0.036
0.081
0.029
5.45
4.01 13.4
10.6 23.1
2.07 2.82
2.44 2.97
5.71 4.36
10.8 18.0
8.73 16.3
2.54
2.78
1.57
1.87
2.84
0.67
2.52
5.12
5.13
1.92
2.12
3.01
10.0
14.25
7j
7m
12n
5.07
a
GI50 values were determined using resazurin reduction after 2−3 days
of incubation with the corresponding compound. GI50 values are given
in micromolar concentrations (μM) as the mean of at least three
independent experiments. Variability around the mean value was <50%
unless otherwise indicated by an SE value in parentheses.
Table 3. Antiproliferative Activity against Patient Derived
Tumor Cell Lines
a
compd
11d
dasatinib
RXF 393NL
CXF 1103L
LXFA 983L
GXF 251L
0.725
3.83
2.22
8.01
2.92
2.92
0.0217
4.36
0.0565
2.25
PAXF 1657L
PRXF DU145
0.121
0.0623
a
GI50 values were determined at Oncotest using a modified propidium
iodide assay. Measurements were performed after 4 days of incubation
with 11d and dasatinib. GI50 values are given in micromolar
concentrations (μM).
us to examine the efficacy of 11d on human endothelial cell
sprouting in a spheroid based cellular angiogenesis assay
(ProQinase, Supporting Information S36).60 Compound 11d
was able to successfully inhibit VEGF-A induced HUVEC
(primary human umbilical vein endothelial cells) sprouting in a
dose dependent manner with an IC50 value of 1.5 μM.
VI. IN VIVO DATA
Three of the most promising compounds from these series (7b,
7g, and 11d) were selected for evaluation of pharmacokinetic
properties in 20−30 g male CD-1 (ICR) mice on intravenous
(IV) and oral (PO) administration. Low to moderate oral
bioavailability of tested compounds in mice was observed, with
compounds 11d and 7g giving the highest values (Table 4).
Promising cellular efficacy and pharmacokinetic properties
incited the subsequent evaluation of compound 11d in a
xenograft mouse model with a tumor derived from the MDA-
MB-231 cell line. High compound clearance (Cl) and moderate
half-life (t1/2) values determined in the pharmacokinetic study
motivated a twice-daily dosing regime totaling 100 mg/kg/day
of compound 11d over 21 days. Median tumor volume
The implication of EphB4-ephrinB2 signaling in sprouting
angiogenesis and blood vessel maturation58 and the inhibition
of vascular endothelial growth factor (VEGFR)-driven angio-
genesis by the selective EphB4 inhibitor NVP-BHG712,59 led
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firmed the type I binding mode obtained previously by
automatic docking (Figure 1). This structural information was
used to design type I1/2 and type II derivatives by taking
advantage of the existing knowledge on privileged chemical
motifs, i.e., hydroxyl group in meta position of the phenyl ring
(for type I1/2) and hydrophobic moieties connected to the
phenyl ring by amide or urea linkers (on type II). Chemical
synthesis of ca. 25 derivatives (Table 1) culminated in several
low nanomolar inhibitors with a good selectivity profile (Figure
4). The X-ray crystal structure of the EphA3 kinase in the
complex with the inhibitor 7m (Figure 3) provided the final
validation of the structure-based design; in particular, the DFG-
out conformation confirmed the type II binding. Moreover, the
slow kinetics of unbinding of compound 7m (measured by
SPR, Figure 2) is congruent with the type II binding mode.
Three interesting observations emerge from this study. First, it
is possible to “elongate” a type I1/2 into a type II inhibitor by
introducing an amide or urea linked to a bulky hydrophobic
group. These type II linkers are involved in the same hydrogen
bonds as the type I1/2 bearing a hydroxyl group in the same
position, while the hydrophobic moiety occupies the pocket
resulting from the displacement of the Phe side chain of the
DFG motif. The similar selectivity profiles of type I1/2 and type
II inhibitors indicate that mainly the moiety in contact with the
gatekeeper’s side chain and hinge region determines specificity.
Finally, in vivo assays (mice xenografted with human breast
cancer) confirmed the cytostatic activity of one of our inhibitors
(11d), which makes this type I1/2 compound a candidate lead
for further preclinical development.
Table 4. Pharmacokinetic Properties in Mice
11d
7b
7g
compd
iv
po
iv
po
iv
po
dose (mg/kg)
Cl (mL/min/kg)
Vss (L/kg)
1
5
1
5
1
5
42
32
31
1.6
1.7
392
2.2
1.2
506
2.2
1.1
533
t1/2 (h)
1.7
493
25
5.0
263
10
2.8
803
30
AUClast (h·ng/mL)
F (%)
progression over time, starting from 108 mm3, of both
treatment and control cohorts is given in Figure 5.
In this study, median treatment-group tumor volume
remained essentially stable throughout the treatment period,
achieving a median tumor volume of 126 mm3 at day 19 as
opposed to the control group whose median tumor reached
650 mm3 in the same period. Tumor growth inhibition (%TGI)
was statistically significant (Mann−Whitney U = 0, P ≤ 0.001,
two-tailed) and quantified at 81% relative to the control group.
Mean body weight of the treatment cohort decreased up to
16.3% of the initial mean body weight of this cohort during the
treatment period. Treatment with compound 11d provides a
significant limitation in tumor progression over the control,
suggesting that further studies of such xenograft model at lower
doses of compound 11d might provide tumor volume control
with lessened weight loss. The further evaluation of compounds
11d on mouse models of K-562 leukemia is underway.
VII. CONCLUSIONS
The X-ray crystal structures of the EphA3 kinase in complex
with two high-nanomolar inhibitors based on the 2-amino-1-
phenyl-pyrrolo[3,2-b]quinoxaline-3-carboxamide scaffold con-
Figure 5. In vivo antitumor activity of compound 11d in MDA-MB-231 nude mice xenografts. The mice received by gavage twice-daily 50 mg/kg of
compound 11d (red) or vehicle control (blue). Each data point is the median or mean of a cohort of 9 animals. Error bars show standard deviations
of the mean.
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ASSOCIATED CONTENT
* Supporting Information
General procedures for the synthesis and characterization,
biological evaluation of all reported compounds, and X-ray
crystal structure refinement data. This material is available free
of charge via the Internet at http://pubs.acs.org.
■
S
Accession Codes
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Moro, S. ATP non-competitive Ser/Thr kinase inhibitors as potential
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selective protein tyrosine kinase inhibitors. Pharmacol. Ther. 1999, 82,
195−206.
PDB codes for EphA3 in complex with the inhibitors A, B, and
7m are 4P4C, 4P5Q, and 4P5Z, respectively.
AUTHOR INFORMATION
Corresponding Authors
*(C.N.) E-mail: cristina.nevado@chem.uzh.ch. Phone: (41)
■
446353945. Fax: (41) 446353948.
(14) Huang, D.; Zhou, T.; Lafleur, K.; Nevado, C.; Caflisch, A.
Kinase selectivity potential for inhibitors targeting the ATP binding
site: a network analysis. Bioinformatics 2010, 26, 198−204.
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Schneider, K. Assessment of chemical coverage of kinome space and
its implications for kinase drug discovery. J. Med. Chem. 2008, 51,
7898−7914.
*(A.C.) E-mail: caflisch@bioc.uzh.ch.
Author Contributions
§A.U. and J.D. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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Structure-based optimization of potent and selective inhibitors of the
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ACKNOWLEDGMENTS
■
We thank Dr. Danzhi Huang for interesting discussions. We
also thank Prof. Sirano Dhe-Paganon for providing the plasmid
used for EphA3 expression. The authors would like to thank the
Swiss National Science Foundation and the Swiss Cancer
League (Krebsliga) for financial support.
ABBREVIATIONS USED
■
Eph, erythropoietin-producing human hepatocellular carcinoma
receptor; DFG, aspartate-phenylalanine-glycine; ATP, adeno-
sine triphosphate; SPR, surface plasmon resonance; DMF,
dimethylformamide; TBTU, N,N,N′,N′-tetramethyl-O-(benzo-
triazol-1-yl)uronium tetrafluoroborate; DIPEA, diisoproylethyl-
amine; DCM, dichloromethane; THF, tetrahydrofuran; HOBt,
hydroxybenzotriazole; EDC, N-(3-(dimethylamino)propyl)-N′-
ethylcarbodiimide; FRET, fluorescence-resonance energy trans-
fer; Abl, abelson murine leukemia viral oncogene homologue;
EGFR, epidermal growth factor receptor; DDR, discoidin
domain receptor; Lck, lymphocyte-specific kinase; DMSO,
dimethyl sulfoxide; MEF, mouse embryonic fibroblasts; VEGF,
vascular endothelial growth factor; HUVEC, human umbilical
vein endothelial cells
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