Discovery of novel thieno[2,3-d]pyrimidin-4-yl hydrazone-based inhibitors of Cyclin D1-CDK4: Synthesis, biological evaluation, and structure-activity relationships

Article abstract of DOI:10.1016/j.bmcl.2008.11.090

The synthesis and evaluation of new analogues of thieno[2,3-d]pyrimidin-4-yl hydrazones are described. 2-Pyrdinecarboxaldehyde [6-(tert-butyl)thieno[2,3-d]pyrimidine-4-yl]hydrazone derivatives have been identified as cyclin-dependent kinase 4 (CDK4) inhibitors. The potency, selectivity profile, and structure-activity relationship of this series of compounds are discussed.

Full text of DOI:10.1016/j.bmcl.2008.11.090

Bioorganic & Medicinal Chemistry Letters 19 (2009) 305–308
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel thieno[2,3-d]pyrimidin-4-yl hydrazone-based
inhibitors of Cyclin D1-CDK4: Synthesis, biological evaluation,
and structure–activity relationships
*
Takao Horiuchi , Jun Chiba, Kouichi Uoto, Tsunehiko Soga
Medicinal Chemistry Research Laboratory II, Daiichi Sankyo Co. Ltd., 16-13, Kita-Kasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and evaluation of new analogues of thieno[2,3-d]pyrimidin-4-yl hydrazones are described.
2-Pyrdinecarboxaldehyde [6-(tert-butyl)thieno[2,3-d]pyrimidine-4-yl]hydrazone derivatives have been
identified as cyclin-dependent kinase 4 (CDK4) inhibitors. The potency, selectivity profile, and struc-
ture–activity relationship of this series of compounds are discussed.
Received 9 October 2008
Revised 20 November 2008
Accepted 24 November 2008
Available online 27 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Cyclin D1/CDK4 inhibitor
Thieno[2,3-d]pyrimidin-4-yl hydrazone
The cell cycle of eukaryotic cells is regulated by cyclin-dependent
kinases (CDKs). CDKs are a family of serine/threonine kinases which
play a key role in the growth, development, proliferation, and death
of eukaryotic cells.1–3 CDKs, along with their regulatory subunit, the
cyclins, are responsible for coordinating the events by which cells
progress through the cell cycle and they become active at specific
phases: G1, S, G2 and M.4–6 In the G1 phase, as cells respond to the
presence of mitogens, the cyclin D1 increases to trigger the activa-
tion of CDK4/6 in early G1 and then cyclin E/CDK2 activates.7 These
kinasesphosphorylatetheretinoblastoma tumorsuppressorprotein
(Rb). The hyperphosphorylated Rb causes the release of members of
the transcription factors, E2F proteins. E2F leads to transcriptional
activation of gene expression that results in entry into the S phase
of the cell cycle. It is noteworthy that CDK4 restricts the passage only
through G1 phase, whereas CDK2 controls the passage through not
only G1 but also S phase with cyclin A.7–9
In normal cells, CDK4/6 activities are negatively regulated by
the tumor suppressor p16, a cyclin-dependent kinase inhibitor of
the INK4 family, and the activity of CDK2 is negatively regulated
by CKIs of the Cip/Kip family.10 But many tumors have been re-
ported to contain mutations, deletions or silencing of the p16 or
the Rb gene.11,12 Moreover, mutations in CDKs and abnormal
expressions of their regulators have been found in a large percent-
age of melanoma patients.13 From these findings, the deregulation
of the Rb pathway, and CDK4, is important in cancer progression.
Recently, Wyeth Research group14 demonstrated that inhibition
of endogenous cyclin D1 or CDK4 expression results in
hypophosphorylation of Rb and accumulation of cells in the G1
phase. In addition, Malumbres15 has reported that knockdown of
CDK4 in mammary tumor cells prevents tumor formation. These
results suggest that selective inhibition of CDK4 may restore nor-
mal cell activity and could be a more valuable approach to cancer
therapy than that of CDK2, especially for those who have lost the
INK4 family, such as p16.
A number of groups have identified CDK inhibitors.16–18 Flavo-
piridol, seliciclib and several small-molecule CDK inhibitors have
been developed and advanced to clinical trial. But it is still the case
that more CDK2 inhibitors have been reported than CDK4 inhibi-
tors and PD033299119 is known to be a selective CDK4 inhibitor
under active development.
We report herein the synthesis of novel analogues of thie-
no[2,3-d]pyrimidin-4-yl hydrazones and their inhibitory activities
for CDK4 with selectivity against CDK2. We also describe their
cytotoxic potential against human cancer cell lines to verify our
hypothesis that selective CDK4 inhibitor will provide an effective
treatment for the inhibition of tumor growth.
S
N
HN
4
N
6
S
N
1a
* Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5696 8772.
E-mail address: horiuchi.takao.pi@daiichisankyo.co.jp (T. Horiuchi).
Figure 1. A compound identified by in-house high-throughput screening.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.090

306
T. Horiuchi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 305–308
High-throughput screening efforts with the Daiichi Sankyo
compound collection revealed that thieno[2,3-d] pyrimidin-4-yl
hydrazone 1a (Fig. 1) was CDK4 (IC50 = 0.75 g/mL) inhibitor pos-
sessing modest selectivity against CDK2 (IC50 = 1.10 g/mL) activ-
2a: R1=Et, R2=H
2b: R1=Me, R2=H
-Pr, R2=H
R2
CO2Me
NH2
O
2c: R1=
i
a
b
R1
2d: R1=t-Bu, R2=H
2e: R1=Bn, R2=H
2f : R1=Me, R2=Et
2g: R1R2= -(CH2)4-
l
R2
R1
S
l
2a-g
ity (Table 1). This finding prompted us to initiate our study to
3a-g
S
explore the activity of this class of compounds.
NH2
N
R2
R2
O
HN
N
N
Our goal in this study was to improve the potency and clarify
the SAR of HTS hit compounds. To get a starting exploration of
structural requirements, substituted analogues of thieno[2,3-
d]pyrimidin-4-yl hydrazones were prepared and tested in terms
of CDK4 and CDK2 activity.
R2
HN
N
c, d
e
NH
R1
R1
N
R1
S
S
N
S
5a-g
5h: R1=H, R2=Me
4a-g
1a-h
Preparation of thieno[2,3-d]pyrimidin-4-yl hydrazones 1a–h
was accomplished using a general synthetic route, as shown in
Scheme 1. Thiophene intermediates 3a–g were synthesized from
commercially available aldehydes 2a–e or ketones 2f,g with
methyl cyanoacetate in the presence of sulfur using the general
method of Tinney et al.20 Cyclization of 3a–g with formamide ob-
tained thieno[2,3-d]pyrimidin-4-ones 4a–g. Hydrazines 5a–g were
prepared from chlorination of the carbonyl group at the C-4 posi-
tion of 4a–g with phosphorus oxychloride, followed by treatment
with hydrazine monohydrate in ethanol under reflux.21 Hydrazine
5h is commercially available. Finally, hydrazones 1a–h were pro-
duced by a classical condensation reaction with 5a–h and 2-thio-
phenecarboxaldehyde in benzene.
The C-2 substituent compounds 1i,j were prepared as shown in
Scheme 2. Following the procedure reported by Gewald et al.,22
intermediate 6 was prepared from n-butyraldehyde, 2-cyanoacet-
amide and sulfur in DMF. The resulting thiophene 6 was treated with
acetic anhydride to obtain 7a. Cyclization of 7a with sodium ethox-
ide afforded thieno[2,3-d]pyrimidin-4-one 4i. On the other hand, 6
was treated with benzoyl chloride to obtain 7b, and 4j was prepared
by cyclization of 7b with aqueous sodium hydroxide under reflux.
The C-4 substituent compounds, ethylidene hydrazone derivative
1k and ring-closed compound 1l, were also synthesized from 5c.
Hydrazine 5c was treated with 2-acetylthiophene to give 1k. Then,
following a procedure foundin the literature,23 1k was lithiated with
n-butyl lithium, acylated and cyclized with ethyl formate, and final-
ly treated with aqueous HCl to afford pyrazole 1l.
Scheme 1. Reagents and conditions: (a) methyl cyanoacetate, S8, Et3N, DMF; (b)
HCONH2, 210 °C, 7–84% from 2a–g; (c) POCl3, 110 °C; (d) NH2NH2Á1H2O, EtOH,
reflux, 33–57% from 4a–g; (e) 2-thiophene-carboxaldehyde, benzene, reflux, 9–92%.
CONHCOMe
d
b
c
4i
4j
NHCOMe
CONH2
NHCOPh
Et
Et
CONH2
NH2
S
a
7a
CHO
Et
S
e
2a
6
S
7b
S
NH2
N
R3
O
HN
N
N
N
f, g
h
HN
N
Et
Et
R3
S
N
H
S
Et
S
N
R3
S
4i: R3
=Me
5i,j
1i,j
4j: R3=Ph
S
NH2
N
j, k
N
i
HN
N
N
HN
N
N
N
S
N
S
S
N
5c
1k
1l
Scheme 2. Reagents and conditions: (a) 2-cyanoacetamide, S8, Et3N, DMF, 62%; (b)
Ac2O, reflux, 24%; (c) BzCl, Et3N, benzene, reflux, quant.; (d) NaOEt, EtOH, reflux,
57%; (e) NaOH aq., reflux, 25%; (f) POCl3, 110 °C; (g) NH2NH2Á1H2O, EtOH, reflux, 2
steps 97% (5i), 60% (5j); (h) 2-thiophene-carboxaldehyde, benzene, reflux, 65% (1i),
56% (1j); (i) 2-acetylthiophene, benzene, reflux, 34%; (j) n-BuLi, HCO2Et; (k) HCl aq.,
reflux, 80% from 1k.
Table 1 summarizes the CDK4/CDK2 inhibitory activity of thie-
no[2,3-d]pyrimidin-4-yl hydrazones containing HTS hit compound
1a. Compounds 1c,d with isopropyl or tert-butyl group at the C-6
(R1) position had more potent CDK4 inhibitory activities
Table 1
Enzyme inhibition of substituted thieno[2,3-d]pyrimidines (IC50
l
g/mL) 1a–l against CDK4 and CDK2
R5
R4
S
N
N
R2
N
R1
R3
S
N
R1
R2
R3
R4
R5
CDK4 IC50
(lg/mL)
CDK2 IC50 (lg/mL)
a
a
Compound
1a
1b
1c
1d
1e
1f
1g
1h
1i
Et
H
H
H
H
H
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
0.75
1.90
0.05
0.12
>20.0
16.00
>20.0
>20.0
>20.0
>20.0
2.80
1.10
1.70
0.35
0.56
Me
i-Pr
t-Bu
Bn
6.50
Me
12.00
>20.0
>20.0
>20.0
>20.0
8.90
–(CH2)4–
H
Et
Et
i-Pr
i-Pr
Me
H
H
H
H
Me
Ph
H
1j
1k
1l
H
–CH@CH–
>20.0
>20.0
a
Concentration (lg/mL) needed to inhibit Rb phosphorylation by 50%, as determined from the dose-response curve. Values are the means of at least two determinations.

T. Horiuchi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 305–308
307
R6
(IC50 = 0.05 and 0.12
against CDK2 (5- to 7-fold). However, the benzyl compound 1e
showed no inhibitory activity up to 20 g/mL. Compounds that al-
lg/mL, respectively) than 1a with selectivity
NH2
N
N
N
HN
N
HN
N
l
a, b
kyl groups were introduced to the C-5 (R2) position, as exemplified
in 1f and 1h, or fused to cyclohexane ring as in 1g, showed only
weak or no inhibitory activity. Substitution at the C-2 (R3) position
was found to be inactivated to CDK4 activity, as in 1i,j. Introduction
of methyl group into the R5 position as in 1k lead to significant de-
crease in potency. To elucidate the effect of the hydrazone part,
pyrazole 1l was synthesized as a fused ring compound. But no
R1
R1
S
S
5a,c,d
8-23
N
NH2
N
N
N
N
Cl
N
N
N
N
N
b
c
N
S
S
S
inhibitory activity up to 20 lg/mL was observed. In addition, com-
pounds 1a–l have extremely poor aqueous solubility, which is
24
4d
5k
Scheme 3. Reagents and conditions: (a) aldehyde, benzene or toluene, reflux; (b)
deprotection if necessary, 41–95% from 5a,c,d; (c) MeNHNH2, EtOH, reflux, 79%; (d)
6-[(dimethylamino)methyl]-pyridine-2-carbaldehyde, EtOH, reflux, 73%.
attributed to their highly lipophilic profiles. For example, the solu-
bility of 1c in water was below 0.1 lg/mL.
To further improve the CDK4 inhibitory activity and physical
profile, our effort was focused on determining the effect of the thi-
ophene ring at the C-4 (R6) position. Alkyl or aryl groups were
introduced instead of thiophene ring in combination with ethyl,
isopropyl or tert-butyl group at the C-6 (R1) position, as shown in
Table 2.
and 32.4-fold, respectively). In particular, the 6-[(dimethyl-
amino)methyl]pyridine compound 21 was significantly improved
in its CDK4 inhibitory activity (IC50 = 0.056
lg/mL) and also
improved in its solubility in water to 44 g/mL. In contrast, the
l
As shown in Scheme 3, C-4 substituent analogues 8–23, focused
on the aryl moiety at the hydrazone, were synthesized using se-
lected hydrazines and appropriate aldehydes instead of 2-thio-
phenecarboxaldehyde. In addition, methylhydrazone 24 was
prepared, from the intermediate chloride 4d and methyl hydrazine.
Alkyl compounds 8 and 9 were less active than the parent com-
pound 1a. Heteroaryl compounds 10–16 had moderate CDK4
inhibitory activity, but most of them lost their selectivity against
CDK2 and only 2-pyridinyl derivatives 10 and 15 showed some
selectivity (3.5- and 3.1-fold, respectively). When substituent
groups were introduced on the pyridine ring, better selectivity
was observed with 5- and 6-methyl-2-pyridine derivatives 17
and 18 (13.6- and 13.3-fold, respectively). Our interest was focused
on introducing substituents on the pyridine ring to increase the
aqueous solubility. As a result, we found that the series of
6-aminomethyl derivatives 19–22 relatively maintained their
inhibitory activity, and 20–22 had good selectivity (28.4-, 25.0-,
6-hydroxymethyl compound 23 was less potent than 21. More-
over, when the NH proton of the hydrazones was converted to a
methyl group as compound 24 (Scheme 3), the resulting analogue
did not show any inhibitory activity. This result suggests that the
NH proton of the hydrazone part is essential for CDK4 inhibitory
activity.
The derivatives were also tested for their antiproliferative activ-
ities in tumor cell lines. Compounds 10, 15, 17, 19 and 23 had po-
tent antiproliferative activities in human colon carcinoma
(HCT116) and human lung carcinoma (PC6) cell lines with IC50
s
ranging from 0.003 to 0.059 g/mL, whereas compound 21 with
l
the strongest CDK4 inhibitory activity showed less potent antipro-
liferative activity.
Further investigations of the antiproliferative effects were car-
ried with 1a, 15, 20 and 21 against various tumor cell lines. Com-
pound 15 had very potent antiproliferative activity in all the cell
lines, as shown in Table 3.
Table 2
Enzymatic and cellular activity for substituted thieno[2,3-d]pyrimidines
R6
N
HN
N
R1
S
N
R1
R6
CDK4 IC50
(
lg/mL)
CDK2 IC50
(l
g/mL)
HCT116 IC50
(lg/mL)
PC6 IC50 (lg/mL)
a
a
b
b
Compound
1a
8
9
Et
Et
Et
Thiophenyl
t-Bu
Cyclopropyl
2-Pyridinyl
3-Pyridinyl
4-Pyridinyl
Phenyl
2-Franyl
0.75
4.40
3.20
0.88
0.55
0.24
0.47
0.39
0.90
0.50
0.88
0.52
0.59
0.25
0.056
0.17
0.35
1.10
3.80
4.10
3.10
0.70
0.33
0.88
0.55
2.80
0.56
12.00
6.90
3.60
7.10
1.40
5.50
2.10
1.610
1.660
NTc
0.566
1.550
NTc
10
11
12
13
14
15
16
17
18
19
20
21
22
23
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
0.010
0.003
0.889
>10.0
0.651
3.780
0.009
1.450
0.009
0.258
0.036
0.189
0.381
0.261
0.034
NTc
NTc
NTc
NTc
2-Pyridinyl
3-Pyridinyl
0.014
NTc
5-Methyl-2-pyridinyl
6-Methyl-2-pyridinyl
6-Aminomethyl-2-pyridinyl
6-[(Methylamino)methyl]-2-pyridinyl
6-[(Dimethylamino)methyl]-2-pyridinyl
6-(Morpholin-4-ylmethyl)-2-pyridinyl
6-Hydroxymethyl-2-pyridinyl
0.020
0.268
0.059
0.185
0.429
0.363
0.039
a
Concentration (
l
g/mL) needed to inhibit Rb phosphorylation by 50%, as determined from the dose–response curve. Values are the means of at least two determinations.
Dose–response curves were determined at ten concentrations. The IC50 values are the concentrations needed to inhibit cell growth by 50%, as determined from these
curves.
b
c
NT = not tested.

308
T. Horiuchi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 305–308
Table 3
Antiproliferative effects of 1a, 15, 20 and 21 (IC50: ng/mL)a against various tumor cell lines
Compound
WiDrb
DLD-1b
HCT116b/ Tere-1c
HCT116b/ SN2-3d
MKN28e
MDA-MB-231f
MDA-MB-468f
BL-6g
P388h
1a
15
20
21
1360
19
263
889
1120
37
221
506
1210
7
290
973
1340
5
269
755
2860
49
686
1390
1870
19
279
919
48
8
159
315
2440
45
379
1090
1010
6
131
370
a
The IC50 values are the concentrations needed to inhibit cell growth by 50% by MTT assay.
b
c
d
e
f
Human colon cancer.
Docetaxal-resistant cell established in-house.
SN-38-resistant cell established in-house.
Human gastric cancer.
Human breast cancer.
Murine melanoma.
g
h
Murine leukemia.
Table 4
15 is efficacious in xenograft models of human colon carcinoma.
These results provide valuable information for the design of
CDK4 inhibitor. Further work will be reported on the improvement
of CDK4 selectivity and the physical profile in the near future.
Antitumor effects of 15 against HCT116 solid tumors
Route
dose
(mg/kg)
Administration
schedule
IRTVmaxa
(%)
BWLmaxb
(%)
D/Nc
i.v.
p.o.
200.0
200.0
qdx4
qdx4
67.9
54.1
22.6
4.6
0/5
0/5
Acknowledgments
a
Maximum Inhibition Rate of Tumor volume.
Maximum Body Weight Loss.
Number of mice dead from toxicity/Number of mice used.
b
c
We thank members of Biological Research Laboratories
IV for their biological assays and the analysis group of Daiichi
Sankyo R&D Associe Co., Ltd. for their analytical and spectral
determinations.
In the G1-S transition of the cell cycle, inhibition of cellular
CDK4 activity will result in cell cycle arrest at the G1 phase. To
investigate whether these compounds will induce a G1 cell cycle
arrest, we evaluated the effects of selected compounds 15 and 21
by cell cycle analysis in tumor cells. A cell cycle distribution study
was performed by treating HCT116 cells with various concentra-
tions of compound 15. Cells were collected after 16 hr treatment
of the compounds and the DNA content of the cells was assessed
by flow cytometry analysis.24 After treating the tumor cell with
15, a significant accumulation of the G1 population (64%) was ob-
served and there were decreases in the S and G2/M populations at
a concentration of 500 ng/mL. Compound 21 caused an increase in
the G1 population in a dose-dependent manner, too. This observa-
tion of G1 cell cycle arrest is consistent with its cyclin D1/CDK4
inhibitory activity.
Finally, compound 15 caused tumor growth delay in the xeno-
graft model of human colon carcinoma HCT116 cell. Taking into ac-
count the poor solubility of 15, hydrochloride salt dissolved with
40% Captisol25 was used. A reduction in colon tumor growth of
67.9% was observed using i.v. administration of 15 when given at
a dose of 200 mg/kg once a day for 4 days continuously. The anti-
tumor effect was also found with a 54.1% reduction after oral
administration at a similar dose without any serious toxicity, as
shown in Table 4.
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In conclusion,
a
series of thieno[2,3-d]pyrimidin-4-yl
hydrazones was synthesized and evaluated, and several 2-pyrdin-
ecarboxaldehyde [6-(tert-butyl)thieno [2,3-d]pyrimidine-4-yl]-
hydrazones were shown to be potent, selective inhibitors of
CDK4 with improved physical profiles. In addition, these com-
pounds have antiproliferative activities and act as cytotoxic agents
with the ability to prevent cell progression. Moreover, compound
22. Schinke, E.; Gewald, K.; Böttcher, H. Chem. Ber. 1966, 99, 94.
23. Beam, C. F.; Sandifer, R. M.; Foote, R. S.; Hauser, C. R. Synth. Commun. 1976, 6, 5.
24. Becton Dickinson FACS Calibur flow cytometer was used.
25. Solubilizing agent of CyDex Pharmaceuticals, Inc. was used.