Synthesis and characterization of amino acid substituted sunitinib analogues for the treatment of AML

Article abstract of DOI:10.1016/j.bmcl.2018.06.026

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Sunitinib, a multikinase inhibitor, was the first Fms-like tyrosine kinase 3 (FLT3) inhibitor clinically used against AML. Off-target effects are a major concern for multikinase inhibitors. As targeted delivery may reduce such undesired side effects, our goal was to develop novel amino acid substituted derivatives of sunitinib which are potent candidates to be used conjugated with antibodies and peptides. In the current paper we present the synthesis, physicochemical and in vitro characterization of sixty two Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutant kinase inhibitors, bearing amino acid moieties, fit to be conjugated with peptide-based delivery systems via their carboxyl group. We determined the solubility, pK_a, CHI and LogP values of the compounds along with their inhibition potential against FLT3-ITD mutant kinase and on MV4-11 cell line. The ester derivatives of the compounds inhibit the growth of the MV4-11 leukemia cell line at submicromolar concentration.

Full text of DOI:10.1016/j.bmcl.2018.06.026

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Accepted Manuscript
Synthesis and characterization of amino acid substituted sunitinib analogues for
the treatment of AML
Zoltán Nemes, Krisztina Takács-Novák, Gergely Völgyi, Klara Valko, Szabolcs
Béni, Zoltán Horváth, Bálint Szokol, Nóra Breza, Judit Dobos, Csaba Szántai-
Kis, Eszter Illyés, Sándor Boros, Robbert Jan Kok, László Őrfi
PII:
S0960-894X(18)30515-8
https://doi.org/10.1016/j.bmcl.2018.06.026
BMCL 25907
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 April 2018
7 June 2018
14 June 2018
Please cite this article as: Nemes, Z., Takács-Novák, K., Völgyi, G., Valko, K., Béni, S., Horváth, Z., Szokol, B.,
Breza, N., Dobos, J., Szántai-Kis, C., Illyés, E., Boros, S., Jan Kok, R., Őrfi, L., Synthesis and characterization of
amino acid substituted sunitinib analogues for the treatment of AML, Bioorganic & Medicinal Chemistry Letters
(2018), doi: https://doi.org/10.1016/j.bmcl.2018.06.026
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Bioorganic & Medicinal Chemistry Letters
Synthesis and characterization of amino acid substituted sunitinib analogues for
the treatment of AML
Zoltán Nemesa, d, Krisztina Takács-Nováka, Gergely Völgyia, Klara Valkob, Szabolcs Bénia,c, Zoltán
Horváthd, Bálint Szokold, Nóra Brezad, Judit Dobosd, Csaba Szántai-Kisd, Eszter Illyésd, Sándor Borosd,
Robbert Jan Koke, László Őrfia, d*
a Department of Pharmaceutical Chemistry, Semmelweis University, Hőgyes Endre street 9, Budapest H-1092, Hungary
b Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom
c Department of Pharmacognosy, Semmelweis University, Üllői street 26, Budapest H-1085, Hungary
d Vichem Chemie Ltd, Herman Ottó street 15, Budapest H-1022, Hungary
e Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Universiteitsweg 99, Utrecht 3584 CG, The
Netherlands
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Sunitinib, a
multikinase inhibitor, was the first Fms-like tyrosine kinase 3 (FLT3) inhibitor clinically used
against AML. Off-target effects are a major concern for multikinase inhibitors. As targeted
delivery may reduce such undesired side effects, our goal was to develop novel amino acid
substituted derivatives of sunitinib which are potent candidates to be used conjugated with
antibodies and peptides. In the current paper we present the synthesis, physicochemical and in
vitro characterization of sixty two Fms-like tyrosine kinase 3-internal tandem duplication
(FLT3-ITD) mutant kinase inhibitors, bearing amino acid moieties, fit to be conjugated with
peptide-based delivery systems via their carboxyl group. We determined the solubility, pKa, CHI
and LogP values of the compounds along with their inhibition potential against FLT3-ITD
mutant kinase and on MV4-11 cell line. The ester derivatives of the compounds inhibit the
growth of the MV4-11 leukemia cell line at submicromolar concentration.
Keywords:
FLT3-ITD kinase inhibitior
acute myeloid leukemia
Chromatographic Hydrophobicity Index
shake-flask partition coefficient determination
peptide based targeted delivery
2009 Elsevier Ltd. All rights reserved.
CH3
 Corresponding author. e-mail: orfi.laszlo@pharma.semmelweis-
N
univ.hu
O
CH3
Sunitinib
H3
C
X2
NH
O
X1: -F X2:
X1: -H X2:
OH
CH3
N
H
Acute myeloid leukemia (AML) is the most common type of
leukemia in adults. In 2015 >20,000 estimated new cases and
>10,000 estimated deaths, related to AML, were reported, solely
in the US1.
X1
Orantinib
17864
O
N
H
O
N
X1: -F X2:
NH
To decrease the dose-limiting toxicities and side effects of
sunitinib7 targeted delivery approaches may be applied.
Previously we have presented the possibility of targeting cells
with a sunitinib analogue – 17864 (Figure 1), designed for
platinum-based targeted delivery8, where the N1,N1-
diethylethane-1,2-diamine side chain of sunitinib was replaced by
1-(pyridin-4-yl)methanamine. To circumvent the use of platinum,
we considered possibilities of implementing different linkage
systems with other modified, sunitinib analogue kinase
inhibitors.
Sunitinib (Figure 1), a multikinase inhibitor approved by the
US Food and Drug Administration for the treatment of renal cell
carcinoma and gastrointestinal stromal tumor inhibits over 200
kinases with Ki <1 μM2, including the internal tandem
duplication (ITD) mutant of the Fms-like tyrosine kinase 3
(FLT3) kinase3. Sunitinib was the first FLT3 kinase inhibitor
clinically used against AML4-6.
Figure 1. Structure of sunitinib, orantinib and 17864, an
analogue of sunitinib used for targeted delivery.
It was shown that methotrexate (MTX) – antibody conjugates,
linked via the carboxyl group of MTX through variable peptide
chains or attached directly to antibodies, can be taken up actively
by cells, and following lysosomal degradation of the conjugates

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active metabolite(s) are released9,10. In case of naproxen (NAP) –
lysozyme (LZM), a low molecular weight protein (LMWP)
conjugate, it was determined that the amino acid directly coupled
to the NAP remained attached to NAP after lysosomal
degradation11. Hence, if a compound is linked to peptide-based
delivery systems, such as antibodies or even RGD peptides,
which are taken up actively by the cell12,13, the conjugate will go
through lysosomal degradation and the released active compound
(RAC) will be the drug-amino acid derivative.
Scheme 2. Left: synthesis of compounds 6a and 6b via
Knoevenagel condensation. Right: Series of 1H NMR spectra
highlighting the 1H-NMR enantiomeric purity characterization
of compounds 6a and 6b using β-cyclodextrin (CD) as chiral
selector.
HDO
CD
6a&b+CD
O
O
6b+CD
H3C
NH
O
OH
O
6a+CD
H3C
NH
OH
CH3
N
H
i
6b
6a
O
+
CH3
O
N
H
N
H
N
H
O
In this paper we present the synthesis and physicochemical
characterization of amino acid-substituted sunitinib analogue
RAC candidates (Table 1) and their full ester derivatives (Table
2) that inhibit the MV4-11 leukemia cell line. The RAC
candidates are fit to be conjugated with peptide-based delivery
systems via their carboxyl group(s) and expected to be released
unaffected after lysosomal degradation from the proper peptide-
based conjugate. During this study, the main goal was to
determine whether R1 – oxindole or R2 – pyrrole, amino acid
substitution of the RAC candidates is superior. The full ester
derivatives were synthesized to aid the characterization of the
RAC candidates.
2-oxindole
5a-b
6a-b
Reagents and conditions: (i) piperidine, MeOH, 60-70°C, 4h. Zoomed spectra
show the chemical shifts of the alpha (methane) proton of the phenylalanine
moiety: 6a (1), 6b (2), 6a with β-cyclodextrin (3), 6b with β-cyclodextrin (4),
and a 1:1 mixture of 6a and 6b with β-cyclodextrine (5).
2-Oxoindoline-5-carboxylic acid 7, the starting material of 2-
oxindole derivatives (Scheme 3), was synthesized according to
literature procedures in three steps16. Methyl 2-oxoindoline-5-
carboxylate 8 was prepared with thionyl chloride in MeOH,
yielding 71%. 9-11 L- and D-5-amino acid-2-oxindole
compounds were synthesized with the same reaction conditions
as 3-5 L- and D-3-amino acid-pyrrole-aldehydes, with the
difference that DMF was used as solvent for the amidation step.
During the amidation of 7 in DMF, dimethyl amide side product
formation was not detected. The reactions resulted in acceptable
yields: (37-65% for compounds 9-10 and 77-75% for compounds
11).
Synthesis and characterization of the compounds
A total of sixty-two sunitinib analogues were synthesized (for
details see supporting material) from the appropriate pyrrole-
aldehydes 1-5 (Scheme 1), via Knoevenagel condensation14
(Scheme 2) with the appropriate 2-oxindoles 7-11 (Scheme 3).
Optical isomers were sorted into twenty-two groups (6, 12-32)
and each individual compound is represented by a group number
and an appropriate letter of a, b, c and d (Table 1 and 2).
Scheme 3. Synthesis of 2-oxindole derivatives 7-11.
O
O
H3C
HO
O
i
O
O
N
H
N
H
Scheme 1. Synthesis of the pyrrole derivatives (1-5).
R
7
R
8
ii
O
O
O
O
O
O
R
CH3
R
O
O
H3
C
H3C
H3
C
H3
C
O
NH
OH
NH
O
OH
O
CH3
iii
O
i
ii
HO
H3C
NH
NH
iii
O
CH3
CH3
CH3
CH3
O
N
H
N
H
O
N
H
N
H
N
H
N
H
O
O
O
O
1
2
R:
9a-b
R:
11a-b
R:
5a-b
R:
3a-b
O
O
H3C
O
O
H3C
R:
10a-b
R:
4a-b
Reagents and conditions: (i): SOCl2, MeOH, reflux, 2h; (ii): HBTU, DIPEA,
DMF, L- or D- H-Phe-OMe*HCl or H-Glu(OMe)-OMe*HCl, 25°C, 16h;
(iii): KOH, H2O 25°C, 1.5h.
Reagents and conditions: (i) KOH, H2O reflux, 4h; (ii) HBTU, DIPEA,
DMSO, L- or D- H-Phe-OMe*HCl or H-Glu(OMe)-OMe*HCl, 25°C, 16h;
(iii): KOH, H2O 25°C, 1.5h.
High throughput and semi-high throughput methods were used
for the preliminary characterization of the compounds (for details
see supporting material). RP-HPLC method, reported
previously17,18, was used to rank the compound solubility,
determined from DMSO stock solutions. During the experiments
DMSO at final concentration was 1.2%. This method gives only
approximate kinetic solubility values, but it is fast, based on the
results it can be easily determined whether a compound is
soluble, slightly soluble or insoluble at a given pH, and these
results are crucial in designing other physical-chemical or
biological experiments. In addition to measurements at pH = 7.4,
the solubility values were also determined at pH = 5.5, the pH of
the lysosomes19. To characterize the compound lipophilicity, we
determined the pH - Chromatographic Hydrophobicity Index
(CHI) profile of each compound20, at starting pHs = 10.5 – fully
deprotonated, 7.4 – physiological, 5.5 – lysosomal and 1.2 – fully
protonated. The measurements were performed with RP-HPLC
using buffer/ACN gradient elution. Each compound was tested
for FLT3 ITD inhibition at two concentrations (12.5 µM and 1.25
µM) with fluorescence polarization-based Immobilized Metal
Assay for Phosphochemicals (IMAP™) kinase assay from
Molecular Devices (CA, USA).
Ethyl 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylate 1 and
5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid were
2
synthesized as described in the literature15. 2 was reacted with L-
and D-phenylalanine methyl ester hydrochloride (H-Phe-
OMe*HCl) and glutamic acid dimethyl ester hydrochloride (H-
Glu(OMe)-OMe*HCl) to synthesize 3a, 3b, 4a and 4b, in DMSO
using HBTU as coupling agent and DIPEA as base, resulting in
acceptable yields (43-49%). DMSO was used instead of DMF to
avoid the formation of the dimethyl amide side product of 2.
Similarly to the ester hydrolysis of 1, KOH/water was used for
the synthesis of 5a and 5b as well, yielding (89-90%), although
the temperature was kept at room temperature to avoid
racemisation. Racemisation was ruled out by 1H-NMR
characterization, using β-cyclodextrin as the chiral selector
(Scheme 2), of group 6 containing compounds 6a and 6b,
synthesized from 5a and 5b via Knoevenagel condensation with
2-oxindole. Optical purity of compounds 6a and 6b were
determined by measuring the chemical shifts of the alpha
methane (-CH) protons of the Phe moieties (for details see
supporting material).

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During the extended characterization, nine compounds were
selected to determine their LogD/LogP values with the classical
shake-flask (SF) method, as described previously21. To gain
LogP values from the measured LogD data, the acidity of the
four different carboxyl moieties present in the investigated
compounds was measured, by automated UV/pH titration as
described earlier22 with MeOH as co-solvent23. Furthermore, as
part of the extended characterization, twenty six compounds were
selected to determine their IC50 values on MV4-11 leukemia cell
line.
The approximate solubility results (Table 1 and 2) showed that
the di-carboxyl compounds 12 and 14 have the highest solubility.
Poor solubility of compounds 13 and orantinib at pH = 5.5
suggested that aromatic, pyrrole-3-carboxylic acid (R2-COOH)
and aliphatic propionic acid are weaker acids than the
phenylalanine carboxyl group (Phe-COOH). This was also
supported by the comparison of the compound pairs 15-16 and
17-18. Not surprisingly, comparison of the amino acid-ester
substituents revealed that– the more hydrophilic Glu(OMe)-OMe
esters 23-24 are more soluble than the aromatic Phe-OMe esters
21-22. The di/tri-ester derivatives 25-32 were not soluble either
at pH = 7.4 or at pH = 5.5.
Preliminary physicochemical and biochemical characterization
Solubility
Table 1. Preliminary characterization of the RAC candidate, di-acid 12-14 and mono-acid 15-24, compounds.
O
R2
H3C
O
CH3
N
R1
H
O
N
H
Chirality
Sol. (µM)* at pH =
CHI* at pH =
10.5 7.4 5.5 1.2 12.5 µM
FLT3-ITD inh. %**
Group
R1
R2
ID
R1
R2
7.4
5.5
1.25 µM
HOOC
-
-
L
D
≥100.0
≥100.0
100.0
87.0
12a
12b
-OH
≥120
~110
24
24
29
32
35
41
61
63
12
NH
HN
L
D
-
-
≥100.0
≥100.0
97.4
87.1
13a
13b
-OH
≥120
≥120
<5
13
14
COOH
COOH
L
L
D
D
L
D
L
≥100.0
≥100.0
≥100.0
≥100.1
98.7
103.9
74.2
14a
14b
14c
14d
HOOC
HN
~110
36
39
40
70
NH
D
76.8
MeOOC
-
-
L
D
98.7
≥100.0
85.8
97.4
15a
15b
-OH
~100
~20
<5
<1
49
47
35
34
58
51
52
56
38
42
60
53
59
68
45
54
61
54
73
73
58
59
86
76
15
16
17
18
19
20
NH
HN
L
D
-
-
71.6
27.5
69.0
76.7
16a
16b
-OH
COOMe
MeOOC
COOMe
-
-
L
D
≥100.0
≥100.0
97.4
93.5
17a
17b
-OH
≥120
~100
~20
~30
<5
NH
HN
L
D
-
-
28.8
12.1
58.7
80.6
18a
18b
-OH
MeOOC
COOMe
COOH
HN
L
D
-
-
≥100.0
96.2
90.9
47.0
19a
19b
-OEt
<5
HOOC
-
-
L
D
≥100.0
≥100.0
90.9
91.0
20a
20b
-OMe
≥120
<5
NH
NH
L
L
D
D
L
L
D
D
L
L
D
D
L
L
D
D
L
D
L
D
L
D
L
D
L
D
L
D
L
D
L
D
≥100.0
≥100.0
94.8
94.8
93.5
18.6
66.4
58.6
89.6
74.2
28.9
98.7
90.9
74.2
39.2
58.7
70.3
67.7
73.0
21a
21b
21c
21d
22a
22b
22c
22d
23a
23b
23c
23d
24a
24b
24c
24d
MeOOC
HN
~50
~70
~20
~30
~60
~30
58
57
47
46
60
59
50
49
60
60
50
50
81
80
68
67
21
22
23
24
COOH
COOMe
COOH
≥100.0
≥100.0
≥100.0
≥100.0
≥100.0
≥100.0
≥100.0
≥100.0
≥100.0
≥100.1
≥100.0
≥100.0
≥100.0
HOOC
MeOOC
HOOC
HN
HN
HN
NH
NH
NH
COOMe
≥120
~100
MeOOC
COOMe
-
-
-
-
-
-
≥120
≥120
~5
42
98
47
57
59
55
75
51
≥100.0
76.7
92.3
orantinib
sunitinib
≥120
94.8
*The compounds solubility and Chromatographic Hydrophobicity Index were determined by HPLC and indicated as the average of the compound group.