Identification of pyrrolo[2,3-d]pyrimidines as potent HCK and FLT3-ITD dual inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.10.012

A series of novel pyrrolo[2,3-d]pyrimidines were synthesized by introducing 15 different amino acids to 7-cyclohexyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine. Compounds with potent activities against HCK and FLT3-ITD were evaluated in viabi

Full text of DOI:10.1016/j.bmcl.2017.10.012

Accepted Manuscript
Identification of pyrrolo[2,3-d]pyrimidines as potent HCK and FLT3-ITD dual
inhibitors
Yasuko Koda, Ko Kikuzato, Junko Mikuni, Akiko Tanaka, Hitomi Yuki, Teruki
Honma, Yuri Tomabechi, Mutsuko Kukimoto-Niino, Mikako Shirouzu,
Fumiyuki Shirai, Hiroo Koyama
PII:
S0960-894X(17)30986-1
https://doi.org/10.1016/j.bmcl.2017.10.012
BMCL 25341
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 August 2017
6 October 2017
7 October 2017
Please cite this article as: Koda, Y., Kikuzato, K., Mikuni, J., Tanaka, A., Yuki, H., Honma, T., Tomabechi, Y.,
Kukimoto-Niino, M., Shirouzu, M., Shirai, F., Koyama, H., Identification of pyrrolo[2,3-d]pyrimidines as potent
HCK and FLT3-ITD dual inhibitors, Bioorganic & Medicinal Chemistry Letters (2017), doi:https://doi.org/10.1016/
j.bmcl.2017.10.012
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Identification of pyrrolo[2,3-d]pyrimidines as potent HCK and FLT3-ITD dual
inhibitors
Yasuko Kodaa, Ko Kikuzatoa, Junko Mikunib, Akiko Tanakab, Hitomi Yukic, Teruki Honmac, Yuri
Tomabechib, Mutsuko Kukimoto-Niinob, Mikako Shirouzub, Fumiyuki Shiraia and Hiroo Koyamaa,*
aDrug Discovery Chemistry Platform Unit, RIKEN Center for Life Science Technologies, 2-1
Hirosawa, Wako, Saitama, 351-0198, Japan
bDrug Discovery Structural Biology Platform Unit, RIKEN Center for Life Science Technologies,
1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan
cDrug Discovery Computational Chemistry Platform Unit, RIKEN Center for Life Science
Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan
*Corresponding author: Email address: hiroo.koyama@riken.jp (Hiroo Koyama)
Key words
Acute myeloid leukemia (AML), FMS-like tyrosine kinase 3 with internal tandem duplication
mutations (FLT3-ITD), Hematopoietic cell kinase (HCK), Pyrrolo[2,3-d]pyrimidine.
Abstract
A series of novel pyrrolo[2,3-d]pyrimidines were synthesized by introducing 15 different amino
acids to 7-cyclohexyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine. Compounds
with potent activities against HCK and FLT3-ITD were evaluated in viability studies with acute
myeloid leukemia cell line MV4-11. Our structure activity relationship analyses lead to the
identification of compound 31, which exhibited potent HCK and FLT3-ITD inhibition and activity
against the MV4-11 cell line.
Acute myeloid leukemia (AML) is one of the most common and aggressive forms of acute
leukemia in adults. While approximately 40-50% of AML patients successfully enter complete
remission with initial combination chemotherapy, the majority of patients experience relapses with
poor prognosis1,2. Recent studies have revealed that overexpression and/or mutation of certain
kinases were identified among AML patients and that inhibitors of several kinases have become
drug discovery targets for the treatment of AML.3 For example, FMS-like tyrosine kinase 3 with
internal tandem duplication mutations (FLT3-ITD) is associated with karyotype AML in
approximately 30% of AML patients.4, 5 More recent studies indicated that hematopoietic cell kinase
(HCK), a non-receptor protein-tyrosine kinase belonging to the Src family, is highly expressed in
chemotherapy-resistant human AML stem cells.6 Both HCK and FLT3-ITD are associated with

irregular signaling pathways leading to uncontrolled cancer cell proliferation and differentiation in
AML. Based on the hypothesis that inhibition of both FLT3-ITD and HCK might offer a
comprehensive treatment of relapse-prone AML, we set out to find potent HCK and FLT3-ITD dual
inhibitors.
We recently reported the identification of a highly potent HCK inhibitor RK-20449.7 Herein we
would like to report our progress toward structurally-diverse HCK and FLT3-ITD dual inhibitors.
Given the impressive potency of RK-20449 (HCK IC50=0.43 nM), we decided to start our
structure-activity relationship (SAR) studies by modifying the structure of RK-20449. Since the
piperazine moiety of RK-20449 was reported to contribute to the increased binding affinity to HCK
by interacting with Asp348 of HCK, 7 we sought to find an alternative structure to piperazine with
the help of in silico docking and X-ray crystallographic studies (Fig. 1). We hoped that replacement
of the piperazine moiety with an acyclic amino group would result in interaction with different
amino acid residue(s) of the enzyme, due to its increased rotational freedom.
Figure 1. X-ray co-crystal structure of 7 with HCK (resolution: 2.85 Å unpublished). Based on this
crystal structure, derivatives incorporated with various amino acids (Ala, Phe, Pro, and Tyr
replacing Gly of 7) were assessed for hydrogen bonding potential with Asp348 and other residues in
HCK by in silico docking studies. Docking scores were summarized in Table 1.
When an acyclic version of the methyl piperazine moiety of RK-20449 was synthesized (1 and 2
in Table 1), major activity decreases were observed with HCK (IC50=226 nM and 119 nM for trans
and cis isomers, respectively) while the FLT3-ITD activities decreased only modestly (IC50=56 nM
and 44 nM, respectively). Since 1 and 2 became quite lipophilic as indicated by their cLogP values,
we decided to introduce hydrophilic substituents to attenuate the high lipophilicity.8 When a
carboxylic acid group was introduced to the aminoalkyl moiety of 1 and 2, remarkable
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improvement in the HCK activity was observed (3-6). These findings prompted us to further
explore the SAR by introducing various amino acids, first through in silico docking simulation
studies (Fig. 1), and then through actual synthesis and activity measurements. Thus, four different
amino acids (Ala, Phe, Pro, and Tyr) including their enantiomers were incorporated into the
structure of 7-cyclohexyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine. Then the
conceived compounds were subjected to docking simulation studies using the X-ray co-crystal
structure of HCK and 7, where potentials for hydrogen bond formation with Asp348 and other
residues of HCK were assessed. The results of the docking simulation studies are shown as a
docking score (kcal/mol) in Table 1, where many of the compounds were expected to have
favorable HCK binding affinities. The phenoxyphenyl moiety at the 5-position of
pyrrolo[2,3-d]pyrimidine was left unchanged since our previous SAR studies had indicated this
group was critically necessary for FLT3-ITD activity.
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Table 1. HCK and FLT3-ITD IC50 values of pyrrolo[2,3-d] pyrimidine derivatives.
IC50 (nM) b
GI50 (nM) b
Docking
score with
HCK
(kcal/mol) a
R
cLogP c
#
HCK
FLT3-ITD
MV4-11
RK-20449
-
trans
-
0.4
24
40
4.9
-
-
trans
cis
-
-
226
119
56
44
-
-
1
2
6.0
2.9
1.2
1.7
-
-
-
-
-
21
-
trans
cis
4.4
105
27
14
23
53
35
28
3
4
5
6
7
L-Leu
D-Leu
trans
cis
-
137
3.7
-
trans
98
Gly
cis
-
12
32
-
8
trans
cis
-11.27
-10.58
-11.23
-10.62
6.9
73
36
36
39
20
39
23
-
-
-
-
9
L-Ala
D-Ala
10
11
12
trans
cis
!Ala trans
-
15
33
-
1.2
0.8
13
trans
L-Ser
cis
-
-
-
-
1.8
40
18
13
17
12
>100
14
15
16
17
-
-
-
trans
D-Ser
cis
6.3
23
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trans
cis
-
-
28
25
27
-
18
19
L-Hsl
4.1
2.0
5.4
29
trans
cis
-10.93
-11.05
-11.34
-11.08
-11.30
-11.33
-11.40
-11.30
-11.36
-11.70
-11.61
-11.08
52
79
26
20
-
-
20
21
22
23
24
25
26
27
28
29
30
31
L-Pro
D-Pro
L-Phe
D-Phe
L-Tyr
D-Tyr
trans
cis
200
65
126
16
-
-
trans
cis
7.5
74
24
-
3.1
27
-
3.2
3.0
trans
cis
24
-
18
2.6
28
26
-
trans
cis
7.4
39
5.0
11
-
trans
15
-
cis
13
5.0
18
a Docking scores were determined by hydrogen bonds between each derivative and HCK residues.
Hydrogen bonds whose energy magnitude is >2 kcal/mol were detected and calculated by
Molecular Operating Environment (MOE), (ver. 2016.0802, Chemical Computing Group Inc.,
Montreal, QC, Canada, 2016.)
b Data represents an average of at least two separate determinations
c Dassault Systemes BIOVIA, BIOVIA Pipeline Pilot, Version 9.0.2, San Diego: Dassault Systemes,
2013.
The actual synthesis was carried out as described in Scheme 1. First, a commercially available
pyrrolopyrimidine 32 was iodinated to give 33. Then the cyclohexanone and the phenoxyphenyl
moieties were successively introduced to 33 by the Mitsunobu reaction and the Suzuki coupling to
yield 35. 9 Introduction of the amino group, deprotection of the ketal group, and reductive amination
with corresponding amino acid esters followed by ester hydrolysis furnished the desired products 3
to 31. Compounds 1 and 2 were synthesized from 35 in a similar fashion employing isoamylamine
instead of an amino acid ester.9 The cis and trans isomers of the 1,4-positions of the cyclohexyl
group were formed during the reductive amination step and chromatographically separated. The cis
isomers were obtained as major products in most cases.
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Scheme 1. Reagents and conditions: (a) NIS, DMF, rt, 95%; (b) DIAD, PPh3, THF, 0-4° C to rt,
53 %; (c) Pd(PPh3)4, sat-Na2CO3, DME, 80° C, >95%; (d) NH4OH, dioxane, 120° C, 96%; (e)
6N-HCl, acetone, 40° C, 88%; (f) amino acid esters, NaBH(OAc)3, DCM, rt, cis and trans
ratio=3:1; (g) 2M NaOH, EtOH, 70° C.
Enzymatic activities10 against HCK and FLT3-ITD of the newly synthesized compounds 1-31 are
summarized in Table 1. It is interesting to note that the HCK activities are generally sensitive to the
cis/trans conformation of the cyclohexane ring, with the trans isomers showing more potent
activities in most cases, while the FLT3-ITD activities are less sensitive. It appears that the results
of the docking studies with HCK are generally consistent with the cis and trans isomers’ activities,
while activities between compounds with different amino acids are not. Some of the differences in
HCK activity between the cis and trans isomers can be explained by X-ray co-crystal structure
analyses; the amino group of the trans isomers 3 and 5 interacts with the backbone carbonyl of
Asp348, whereas the amino group of the corresponding cis isomers 4 and 6 does not interact with
the enzyme (Fig. 2). The crystal structure of HCK and 31 revealed an interesting binding motif
where the amino group and the tyrosine carbonyl group of 31 interact with the backbone carbonyl
of Ala390 via a water molecule, and that the phenol hydroxyl group of 31 interacts with the
backbone carbonyl of Gly279 (Fig. 3). Introduction of amino acids with aromatic substituents
such as phenylalanine and tyrosine improved the FLT3-ITD activities (24-31).
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Figure 2. Crystal structures of 3-6 bound to HCK (grey area) in stick model. The nitrogen (blue),
oxygen (red), sulfur (yellow) atoms and hydrogen bonds (dotted lines) were shown. (a) 3 (green,
PDB ID: 5H0B) and 4 (magenta, PDB ID: 5H0A); (b) 5 (green, PDB ID: 5H0D) and 6 (magenta,
PDB ID: 5H0C).
Figure 3. (a) Crystal structure of 31 bound to HCK (grey area) in stick model (PDB ID: 5H0F). The
carbon atoms (grey), water oxygen atom (red ball) and hydrogen bonds (dotted lines) were shown.
(b) An alternative view (45° rotation of (a)).
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Compounds with good enzymatic activities were tested in the AML-derived MV4-11 cell viability
studies. Based on the structural diversity and potent activities against HCK and FLT3-ITD,
compounds 3, 7, 14, 19, 27 and 31 were chosen for the study. The results of the study are
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summarized in Table 1. While compounds 3, 19, 27 and 31 exhibited good anti-leukemic activities,
compounds 7 and 14 showed only weak activities despite their good kinase activities. This apparent
discrepancy may be attributed to cell membrane penetration potential. As indicated by their cLogP
values, relatively low lipophilicity of compounds 7 and 14 may make cell penetration difficult.
In conclusion, we have demonstrated that potent HCK and FLT3-ITD inhibition activities can be
incorporated in a series of novel pyrrolo[2,3-d]pyrimidines with the help of in silico docking and
X-ray crystallographic studies. Among the compounds prepared, compound 31 showed potent
activity in the AML-derived MV4-11 cell viability study. Some of the HCK binding motifs revealed
by the crystallographic studies may provide valuable information for the development of potent
HCK and FLT3-ITD dual inhibitors.
Acknowledgments
We would like to thank Drs. Fumihiko Ishikawa and Yoriko Saito of RIKEN Center for integrative
medical sciences and Dr. Takehiro Fukami of RIKEN Program for drug discovery and medical
technology platforms for helpful discussions, and Dr. Hiroaki Ohtake of Nard Institute for technical
support.
References and notes
1. Kantarjian H.; O'Brien, S.; Cortes, J.; Giles, F.; Faderl, S.; Jabbour, E.; Garcia-Manero, G.;
Wierda, W.; Pierce, S.; Shan, J.; Estey, E. Cancer 2006, 106, 1090.
2. Patel, J. P.; Gonen, M.; Figueroa, M. E.; Fernandez, H.; Sun, Z.; Racevskis, J.; Van Vlierberghe,
P.; Dolgalev, I.; Thomas, S.; Aminova, O.; Huberman, K.; Cheng, J.; Viale, A.; Socci, N. D.;
Heguy, A.; Cherry, A.; Vance, G.; Higgins, R. R.; Ketterling, R. P.; Gallagher, R. E.; Litzow,
M.; van den Brink, M. R.; Lazarus, H. M.; Rowe, J. M.; Luger, S.; Ferrando, A.; Paietta, E.;
Tallman, M. S.; Melnick, A.; Abdel-Wahab, O.; Levine, R. L. N. Engl. J. Med. 2012, 366, 1079.
3. Myers, S. H.; Brunton, V. G.; Unciti-Brocera, A. J. Med. Chem. 2015, 59, 3693.
4. Chatain, N.; Ziegler, P.; Fahrenkamp, D.; Jost, E.; Moriggl, R.; Schmitz-Van de Leur, H.;
Muller-Newen, G. Oncogene 2013, 32, 3587.
5. Nogami, A.; Oshikawa, G.; Okada, K.; Fukutake, S.; Umezawa, Y.; Nagao, T.; Kurosu, T.;
Miura, O. Oncotarget 2015, 6, 9189.
6. Saito, Y.; Uchida, N.; Tanaka, S.; Suzuki, N.; Tomizawa-Murasawa, M.; Sone, A.; Najima, Y.;
Takagi, S.; Aoki, Y.; Wake, A.; Taniguchi, S.; Shultz, L. D.; Ishikawa, F. Nat. Biotechnol. 2010,
28, 275.
7. Saito, Y.; Yuki, H.; Kuratani, M.; Hashizume, Y.; Takagi, S.; Honma, T.; Tanaka, A.; Shirouzu,
M.; Mikuni, J.; Handa, N.; Ogahara, I.; Sone, A.; Najima, Y.; Tomabechi, Y.; Wakiyama, M.;
Uchida, N.; Tomizawa-Murasawa, M.; Kaneko, A.; Tanaka, S.; Suzuki, N.; Kajita, H.; Aoki, Y.;
Ohara, O.; Shultz, L. D.; Fukami, T.; Goto, T.; Taniguchi, S.; Yokoyama, S.; Ishikawa, F. Sci.
Transl. Med. 2013, 5, 181ra52.
8. Lipinski, C.A., Lombardo, F., Dominy, B.W., and Feeney, P.J. Adv. Drug Deliv. Rev. 2001, 46,
3–26.
9. Calderwood, D.J.; Johnston, D.N.; Munschauer, R.; Rafferty, P.; Bioorg. Med. Chem. Lett. 2002,
12, 1683.
10. Tsuganezawa, K.; Watanabe, H.; Parker, L.; Yuki, H.; Taruya, S.; Nakagawa, Y.; Kamei, D.;
ꢀ

Mori, M.; Ogawa, N.; Tomabechi, Y.; Handa, N.; Honma, T.; Yokoyama, S.; Kojima, H.; Okabe,
T.; Nagano, T.; Tanaka, A. J Mol Biol 2012, 417, 240.
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Identification of HCK and FLT3-ITD dual inhibitors
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