Modification of a promiscuous inhibitor shifts the inhibition from γ-secretase to FLT-3

Article abstract of DOI:10.1016/j.bmcl.2012.10.016

The inhibition of FLT-3 activity is an interesting target for the treatment of acute myeloid leukemia (AML). The serendipitous identification of FLT-3 inhibitors from a CK1/γ-secretase programme provided compounds with dual inhibitory activity. We analyzed the structure-activity relationship of these inhibitors and derivatized them to arrive at compounds with reduced impact on γ-secretase activity and enhanced FLT-3 inhibition.

Full text of DOI:10.1016/j.bmcl.2012.10.016

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7634–7640
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Modification of a promiscuous inhibitor shifts the inhibition from
to FLT-3
c-secretase
Ghislaine Marlyse Okala Amombo a, , Thomas Kramer a, , Fabio Lo Monte a, , Stefan Göring a,
Matthias Fach a, Steven Smith a, Stephanie Kolb a, Robert Schubenel b, Karlheinz Baumann b,
Boris Schmidt a,
⇑
a Clemens Schöpf-Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Petersenstr. 22, D-64287 Darmstadt, Germany
b F. Hoffmann-La Roche Ltd, Pharmaceutical Division, Preclinical Research CNS, Bldg. 70/345, CH-4070 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The inhibition of FLT-3 activity is an interesting target for the treatment of acute myeloid leukemia
(AML). The serendipitous identification of FLT-3 inhibitors from a CK1/ -secretase programme provided
compounds with dual inhibitory activity. We analyzed the structure–activity relationship of these inhib-
Received 14 August 2012
Revised 28 September 2012
Accepted 1 October 2012
Available online 11 October 2012
c
itors and derivatized them to arrive at compounds with reduced impact on
enhanced FLT-3 inhibition.
c-secretase activity and
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Acute myeloid leukemia
FLT-3
Kinase inhibitor
c
-Secretase
Acute myeloid leukemia (AML) is an aggressive haematological
Alzheimer’s disease we additionally profiled the compounds for ki-
nase selectivity. Serendipitously FLT-3 was part of the kinase panel
and two substances displayed significant inhibition (98% and 89%
at 10 mM) of this tyrosine kinase, Scheme 2.11
Regarding this and the fact that FLT-3 is not known to exert ef-
fects on the amyloid precursor protein metabolism we focused on
optimizing the FLT-3 inhibitory activity of 1 and reducing its mod-
malignancy with long-term survival rates of 25–70% in patients
younger than 60 years and only 5–15% in older patients.1,2 Activat-
ing mutations of FLT-3 (FMS-like tyrosine kinase-3) are abundant
molecular abnormalities found in AML.3 FLT-3 is essential for the
normal function of stem cells and the immune system and is pri-
marily expressed in immature hematopoietic cells.4 It contains
an extracellular ligand binding domain, a transmembrane domain,
and an intracellular juxtamembrane domain followed by the tyro-
sine kinase domain, which is interrupted by a kinase insert re-
gion.5,6 Internal-tandem duplications (ITDs) and tyrosine kinase
domain (TKD) point mutations are the two major classes of activat-
ing FLT-3 mutations identified in AML patients.7 FLT-3/ITD muta-
tions were estimated to occur in ꢀ23% of de novo AML.8 Several
ATP-competitive FLT-3 inhibitors have been developed for a tar-
geted therapy of this disease.9 The FLT-3 inhibitors are derived
from different structural classes and some of them displayed high
potential in preclinical and clinical trials, Scheme 1.5–10
ulation of
was to decrease the effect on
c
-secretase activity. Primary objective of our research
-secretase and to improve mean-
c
while the inhibitory activity against FLT-3. The structure of 1 guided
the variation of the structure–activity relationship (SAR) study.
The Knoevenagel condensation of indolinones and aldehydes
provides a rapid and efficient method to generate a chemical diver-
sity. The Koevenagel products were obtained from microwave irra-
diated reaction mixtures within 30 min, which was followed by
rapid purification. In the first step alkyl or aryl benzyl halides were
coupled with 4-hydroxybenzaldhydes 3 under basic conditions to
obtain a series of elongated benzaldehydes 4a–n for the subsequent
Knoevenagel condensation.11,12 A further nucleophilic substitution
of the chlorides 4k,l resulted in the tertiary amines 5a,b potential
intermediate iminium salts were hydrolysed in the aqueous work
up.11 The ether 7 was formed in a microwave heated reactor by sub-
stitution of an aromatic fluoride 6 with a 4-(4-methyl-1H-imidaz-
ole-1-yl)phenol.13 The 4-fluoro-benzaldehyde 8 was substituted
to its corresponding sulfonyl 9 and imidazol benzaldehyde 10,
Scheme 3.13,14 The phenylimidazoles 7 and 10 were prepared to
The published data suggest that FLT-3 is an attractive therapeu-
tic target for the development of kinase inhibitors for AML and
other associated diseases.11
Working on the structure–activity relationship of the indoli-
none scaffold of dual CK1/c-secretase inhibitors in the context of
⇑
Corresponding author. Tel.: +49 6151 163075; fax: +49 6151 163278.
E-mail address: Schmidt_boris@t-online.de (B. Schmidt).
These authors contributed equally to this work.
evaluate this motif, which is frequently employed on c-secretase
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.016

G. M. O. Amombo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7634–7640
7635
Scheme 1. FLT-3 inhibitors evaluated in preclinical or clinical trials.5–7,9,10
temperature by the LANCE detection method.24 All experiments
were carried out as technical replicates, the average of two such
replicates is listed in Table 1.
The results are expressed as a percent of control (Staurosporine)
specific activity ((measured specific activity/control specific activ-
ity) Â 100) obtained in the presence of the test compounds
(10 lM), Table 1. The previously described liquid phase electro-
chemiluminescence (LPECL) assay was used to measure the Ab42
isoform to evaluate the compounds for their potency in inhibiting
c
-secretase activity, Table 1.23
To confirm the FLT-3 inhibition-ratio observed at 10 lM con-
Scheme 2. Screening hits of potential FLT-3 inhibibitors in a kinase panel of 43
kinases.11
centration we determined the IC50 values of compounds 16e,j,l,n.
The four compounds showed potent inhibition of FLT-3 activity:
IC50 = 4.1 nM for 16e, IC50 = 14.0 nM for 16j, IC50 = 29.0 nM for
16l and IC50 = 17.0 nM for 16n, which exceeded the IC50 in the
Ab42 generation assay by 3 powers of 10. These activities indicated
modulators.15 The opportunity to obtain fluorescent derivatives
was addressed by two derivatives: (A) a two step synthesis resulted
in the azobenzene derivative 12, the 4-aminobenzaldehyde 11 was
converted to its diazonium derivative via diazotation and the final
azobenzene 12 was formed by an azo coupling reaction.16 (B) The
9-methyl-9H-carbazole 13 was converted to the bulky aldehyde
14 in a two step procedure under Vilsmeier–Haack conditions,
Scheme 3.17
Commercial aldehydes and the previously synthesized benzal-
dehydes (Scheme 3) were coupled with the indolinones 15a,b un-
der microwave supported Knoevenagel conditions, Scheme 4.11
The resulting products 16a–x were obtained in yields up to 97%,
Table 1. This class of compounds was shown to isomerize within
2 days in methanol, thus no attempts were made to separate the
isomers.11 A further reduction of compound 16e resulted in 77%
yield of product 16y, Scheme 5a. Finally, the product 16i was deriv-
atized to obtain the tetrazole 16z in a moderate yield of 62%,
Scheme 5b.18
that we have differentiated FLT-3 inhibition from
inhibition.
c-secretase
The most potent
imidazole, which is frequently encountered in
c
-secretase inhibitors 16s,v feature a phenyl-
-secretase modula-
c
tors. However, both compounds display reduced FLT-3 inhibition.
As indolinones are well known as kinase inhibitors25,26 we deter-
mined the broader selectivity of our most active FLT-3 inhibitor
16e. The selectivity of compound 16e was evaluated at a concen-
tration of 1 lM against 50 human protein kinases, Figure 1. Most
of the 50 kinases in this panel showed a residual activity higher
than 80%, whereas FLT-3 displayed a residual activity of only
14.7%. The only kinases, which were also significantly inhibited
by this compound, were the Ser/Thr kinase HGK (MAP4K4) and
the Tyr kinase JAK3.
In addition to the H4 APP#9 cell-based toxicity assay, we eval-
uated the compounds in a zebrafish embryo phenotype assay,
which enabled toxicity determination in whole organisms.27 The
embryos were collected and maintained in E2 medium at 28 °C.
Compound 16e was added 4–5 hpf (hours post fertilization) and
the phenotypes were compared after 48 hpf. Compound 16e causes
Human recombinant FLT-3 was used in the FLT-3 in vitro kinase
assay to ascertain the inhibitory activity. The concentration of the
phosphorylated substrate peptide phospho-Ulight-CAGAGAIETDK
EYYTVKD (Starting unphosphorylated peptide concentration:
100 nM) was determined after 90 min incubation time at room

7636
G. M. O. Amombo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7634–7640
Scheme 4. Synthesis of indolinone derivatives under microwave conditions
(R2 = phenyl moieties).11,19–22
revealed potential inhibitor-enzyme interactions, Figure 3.8 In
both cases the docking result indicates four potential interac-
tions. The indolinone motif interacts edge-to-face with the
Phe830 of the DFG motif. In addition, the indolinone fits to the
FLT-3 hinge region by face-to-face interplay with Phe691 and
the hydrogen bonding with Lys644.8 The phenyl group of the
inhibitors interacts with Met665.28 In addition to these interac-
tions, the rigidity of the bridge between the indolinone and
the phenyl moiety may contribute to potent FLT-3 inhibition.
Therefore, we synthesized compounds 16e and 16y, which differ
in orientation and rotational flexibility of the aryl substituent.
The FLT-3 in vitro assay results of compounds 16e (FLT-3 inhibi-
tion-ratio = 100%) and 16y (FLT-3 inhibition-ratio = 47%) revealed
a twofold increased FLT-3 inhibition-ratio for 16e and exemplify-
ing the influence of rigidity. A FLT-3 inhibition comparison of all
compounds revealed that an electron donor motif is needed at
the end of the elongated, variable alkyl chain. For example com-
pound 16c, which lacks this motif, showed a decreased inhibi-
tion activity compared to 16d,i. This may be explained by a
polar area formed by Glu573 and Gln577, which is in close vicin-
ity. A comparison of the FLT-3 inhibition results and the docking
studies offers the possible suggestion that the benzylidene ind-
olinone moiety may occupy the entrance of the ATP-binding site
(compound 16a,b,k) and the elongated alkyl chain ranging from
C2 (16e) to C4 (16h) could act as a flexible ‘anchor’ in the inner
side of the ATP-binding pocket, for example, 16d–j. Furthermore,
a comparison of the FLT-3 inhibition results of Table 1 revealed
that bulky residues at the phenyl moiety lead to a decreased
inhibitory activity.
The inhibitory activity of the synthesized compounds in the
c
-secretase assay cannot be correlated to the FLT-3 inhibition-ra-
tio, Figure 4. In the case of compounds 16i (FLT-3 inhibition-ra-
tio = 96%; IC50 (Ab42) > 40 M) and 16n (FLT-3 inhibition-
ratio = 87%; IC50 (Ab42) > 160 M) we expected the same result,
but surprisingly, they were not active against the -secretase,
l
l
c
Table 1 and Fig. 4. In comparison with other compounds, for
example, 16d,l and 16p,r, the nitrile and alkyl chain combination
is important for this selectivity.
Conclusion: we have identified several potent FLT-3 inhibitors
based on the scaffold of the screening hit 1, which display neg-
lible activity on
c-secretase inhibition. The most active com-
pound 16e did not display significant toxicity in H4 APP#9
cells and the zebrafish embryo phenotype assay. This lack of
apparent toxicity may be due to lack of permeation, yet perme-
ation is indicated by the activity in the Ab42 generation assay.
The combination of FLT-3 in vitro results and docking studies re-
vealed likely enzyme-inhibitor interactions with the amino acids
Lys644, Met665, Phe691 and Phe830. Further improvement of
these FLT-3 inhibitors will focus on the reduction of the logP
to enhance to pharmacokinetic properties. Our most active FLT-
3 inhibitor 16e exhibits a ClogP value of 5.28, Table 1, which
implies impaired solubility.
Scheme 3. Aldehyde syntheses (R1 = H, OMe; R2 = H, OMe; R3 = alkyl/benzyl
moieties).11–14,16,17
a development delay at 20 lM, Figure 2. Compared to the control,
zebrafish embryos treated with 16e were still covered by the cho-
rion. Nevertheless, they did not reveal other abnormalities. The
zebrafish embryo assay did not reveal lethality and peculiarities
at a concentration below 20 lM of 16e.
The docking studies (Molegro Virtual Docker 5) of the FLT-3
crystal structure (PDB: 1RJB) and compounds 16e and 16n

G. M. O. Amombo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7634–7640
7637
Table 1
Synthesized indolinones, their chemical properties and in vitro assay results23,24
Compound
R1
R2
FLT-3 inhibition -ratioa (%)
IC50 of A42 LPECL assayb
n.t.f
(l
M)
Toxicity (
l
M)c
Yield (%)
ClogPd
Staurosporinee
Sunitinibe
—
—
—
—
100
100
n.t.
—
—
4.19
3.00
f
f
f
n.t.
n.t.
1
Cl
98
4.7
—
Y (40)
—
—
5.04
2
Cl
89
—
3.50
16a
16b
16c
16d
16e
16f
Cl
Cl
Cl
Cl
Cl
H
94
85
79
99
100
97
47.6
>40
>40
7.6
N (40)
N (40)
N (40)
N (40)
Y (40)
—
28
88
83
65
55
95
3.53
4.12
5.71
4.22
5.28
4.31
17.7
—
16g
Cl
99
—
—
80
5.63
16h
16i
16j
Cl
Cl
Cl
99
96
96
—
—
64
90
84
5.56
3.94
4.24
>40
11.3
Y (40)
N (40)
16k
16l
Cl
Cl
Cl
97
98
99
15.0
11.5
11.5
N (40)
Y (40)
Y (40)
97
31
42
5.10
3.96
5.02
16m
16n
Cl
87
>160
N (160)
67
3.68
(continued on next page)

7638
G. M. O. Amombo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7634–7640
Table 1 (continued)
Compound
R1
Cl
R2
FLT-3 inhibition -ratioa (%)
IC50 of A42 LPECL assayb
(l
M)
Toxicity (
l
M)c
Yield (%)
71
ClogPd
16o
16p
100
10.0
N (40)
4.31
Cl
Cl
75
58
27.3
38.8
N (40)
N (40)
93
70
5.32
5.86
16q
16r
16s
Cl
Cl
77
83
>40
5.4
N (40)
Y (10)
48
41
5.32
5.88
16t
H
77
48
>80
7.3
N (80)
Y (20)
9
5.55
2.49
16u
Cl
85
16v
Cl
74
6.7
Y (80)
55
4.25
16w
Cl
H
69
78
19.8
27.6
N (80)
Y (80)
17
15
6.04
5.07
16x
16y
H
47
83
—
—
77
62
3.87
3.76
16z
Cl
17.7
Y (40)
a
Percent of control (Staurosporine) specific activity ((measured specific activity/control specific activity) Â 100); activity at a concentration of 10
lM.
b
c
d
e
f
Ab liquid phase electrochemiluminescence (LPECL) assay, H4 APP#9 cells.
Determined in H4-cells, Y = Yes, N = No.
Calculated by ChemDraw Ultra (9.0.1).
Control.
n.t. = Not tested.

G. M. O. Amombo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7634–7640
7639
Scheme 5. Post-Knoevenagel modifications: (a) Reduction with H2 and Pd/C; (b) Tetrazole synthesis under microwave conditions.18
Figure 1. Screening of compound 16e against a panel of human protein kinases. Each bar represents the activity of one individual protein kinase (determination method:
percent of control (Staurosporine) specific activity [(measured specific activity/control specific activity) Â 100]). Compound 16e was tested at a concentration of 1
lM against
50 protein kinases. See the Supplementary data for more details.24
A
B
C
Figure 2. Exposure of zebrafish embryos to (A) 20 lM of 16e, (B) 5 lM of 16e, (C) control. The embryos were collected and maintained in E2 medium at 28 °C. Compound 16e
was added 4–5 hpf (hours post fertilization) and the phenotypes were compared after 48 hpf.

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G. M. O. Amombo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7634–7640
Figure 3. Docking of compound 16e (left) and 16n (right) into the PDB crystal structure 1RJB of FLT-3; important interactions are highlighted; Software: Molegro Virtual
Docker 5.
Figure 4. Correlation plot of FLT-3 activity inhibition and Ab42 inhibition (IC50); avalues at 40
l
M are determined as >40, see Table 1; bdetermination method: percent of
control (Staurosporine) specific activity [(measured specific activity/control specific activity) Â 100]).
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.10.
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