Synthesis and evaluation of pyrido-thieno-pyrimidines as potent and selective Cdc7 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.11.093

Cdc7 kinase plays a critical role in the regulation of DNA replication in eukaryotic cells and has been proposed as a target for cancer therapy. We have identified a class of Cdc7/Dbf4 inhibitors with a pyrido-thieno-pyrimidine core structure. Synthesis o

Full text of DOI:10.1016/j.bmcl.2008.11.093

Bioorganic & Medicinal Chemistry Letters 19 (2009) 319–323
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of pyrido-thieno-pyrimidines as potent
and selective Cdc7 kinase inhibitors q
b
b
b
c
Chunlin Zhao a, , Christian Tovar , Xuefeng Yin , Qui Xu , Ivan T. Todorov
*
a
Lyubomir T. Vassilev b, , Li Chen
*
a Discovery Chemistry, Research Center, Roche, Nutley, NJ 07110, USA
b Discovery Oncology, Research Center, Roche, Nutley, NJ 07110, USA
c Gonda Diabetes Center, Beckman Research Institute of City of Hope, 1500 East Duarte Road, Duarte, CA 91010, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cdc7 kinase plays a critical role in the regulation of DNA replication in eukaryotic cells and has been pro-
posed as a target for cancer therapy. We have identified a class of Cdc7/Dbf4 inhibitors with a pyrido-thi-
eno-pyrimidine core structure. Synthesis of a focused pyrido-thieno-pyrimidine library yielded potent
and selective Cdc7 inhibitors with antiproliferative activity against cancer cells in vitro. Their synthesis
and SAR data are presented herein.
Received 12 September 2008
Revised 20 November 2008
Accepted 24 November 2008
Available online 27 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Cdc 7 inhibitors
Anti-cancer agent
Cdc7 is a conserved serine/threonine kinase that plays an
important role in initiation of DNA replication in eukaryotic
cells.1–5 It is expressed continuously during the cell cycle but acti-
vated during late-G1 and S phase by binding with its subunit Dbf4.
Cell cycle-dependent expression of Dbf4 allows for a precise con-
trol of Cdc7 activity. Among the most important known substrates
of Cdc7/Dbf4 are minichromosome maintenance proteins (MCM2-
7) representing essential components of the origin initiation com-
plex (ORC). Phosphorylation of one or more MCM proteins is
thought to be a critical step in the initiation of DNA replication
in eukaryotes.6–8 The role of Cdc7 in this process has been sup-
ported by the observation that knockdown of its expression causes
an abortive S phase, followed by cell death.9 These results and the
fact that Cdc7 has been found overexpressed in human cancer cell
lines and tumor tissue samples10 have suggested that Cdc7 could
be a target for cancer therapy. Here, we report the synthesis and
evaluation of pyrido-thieno-pyrimidines (Fig. 1) as potent and
selective Cdc7 inhibitors.11
structure. A focused library was synthesized using microwave-as-
sisted chemistry outlined in Scheme 1.
Treatment of 2-chloro-nicotinonitrile 1 with sodium sulfide
generated 2-mercapto-nicotinonitrile 2, which was reacted with
2-chloro-acetamide to give 3-amino-thieno-[2,3-b]-pyridine-2-
carboxylic acid amide 3. Compound 4 was made by reacting 3 with
trimethyl orthoformate with the catalysis of acid. Chlorination of 4
with phosphorus oxychloride followed by substitution of chlorine
with amine NHR4R5 gave the desired product 6 in Table 2.12
Micro-wave radiation has been used in all steps of the synthesis
except the chlorination. All reactions were clean and no chroma-
tography was needed for purifying intermediates.
Table 1 lists the effect of bases on the formation of intermediate
3. The IR spectrum of compound B revealed a typical CN absorption
at 2220 cmꢀ1, which is consistent with the open-chain structure.
To compare the results of microwave-assisted reactions with
the conventional synthetic protocol, two conventional reactions
were run using the same stoichiometries as reactions I and II (Table
1) and refluxing with regular oil-bath heating. The microwave-as-
A high-throughput screen of Roche chemical library for inhibi-
tors of Cdc7/Dbf4 identified 6h (Fig. 1, R1 = CH3, R2 = H; R3 = CH3,
R4 = H, and R5 = H) as a lead compound with IC50 of 400 nM against
Cdc7/Dbf4 and over 10-fold selectivity over the cyclin-dependent
kinases (CDK1, CDK2, and CDK4), the closest human kinases in
3
R
2
R
N
N
N
R
1
R
N
S
q
See Ref. 1.
* Corresponding authors. Tel.: +1 9732354606; fax: +1 9732356389 (C.Z.); tel.: +1
9732358106; fax: +1 9732356185 (L.T.V.).
E-mail addresses: chunlin.zhao@roche.com, czhao000@aol.com (C. Zhao),
lyubomir.vassilev@roche.com (L.T. Vassilev).
5
4
R
Figure 1. General structure of pyrido-thieno-pyrimidines.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.093

320
C. Zhao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 319–323
R3
R3
N
R3
N
R2
N
O
R2
N
R2
a
b
1
R
N
1
R
1
R
S
N
Cl
N
S
3
1
2
c
R3
R3
N
R3
N
R2
N
R2
N
R2
N
O
e
d
1
R
N
N
1
R
N
1
R
N
S
S
S
N
R4
5
R
Cl
5
4
6
Scheme 1. Reagents and conditions: (a) Na2S, t-Butanol, microwave, 150 °C, 20 min; (b) chloroacetamide, K2CO3/EtOH, microwave, 120 °C, 10 min; (c) HC(OCH3)3, CH3COOH,
microwave, 150 °C, 10 min; (d) POCl3, refluxing; (e) HNR4R5 microwave, 120 °C, 20 min (when R4 and R5 are H, the reaction was run in a sealed tube at 80 °C overnight).
Table 1
Effect of base to the formation of thieno ring
N
N
NH2
NH2
NH2
Cl
+
or
NH2
N
S
O
N
SH
N
S
O
O
B
A
Reaction No.
Base used
Product type: A or B
Reaction conditions (microwave)
Conversion ratea (%)
I
II
III
IV
K2CO3/EtOH
NaOMe/MeOH
NaOAc/EtOH
NaOH/MeOH
A
A
B
A
120 °C/10 min
120 °C/10 min
120 °C/10 min
120 °C/10 min
100
100
100
100
a
Determined by the LC–MS results of reaction mixtures.
sisted protocol offered one clear advantage, much shorter reaction
time. While reactions I and II were completed within 10 min with
micro-wave heating, their counterparts took more than 3 h to com-
plete under regular heating conditions.13
To carry the reaction from 3 to 4, both p-toluenesulfonic acid
and acetic acid were used as acid catalysts, respectively (Scheme
2). While acetic acid produced the desired product, the p-toluene-
sulfonic acid-catalyzed reaction yielded a dark-colored solution
with unclear LC–MS profile.
Synthesis of 2-methylated analog can be achieved using the
same method. Cooking 3h with neat acetic anhydride yielded clean
result (Scheme 3).
Although the crude product of chloride 5 (Scheme 1) was used
for the next reaction without chromatography, the quick wash-up
H
NH2
N
N
O
O
+
NH2
O
N
S
N
S
O
O
4h
3h
Scheme 2. Building pyrimidinone ring by acid catalysis.
NH2
NH2
N
O
O
O
+
N
O
N
S
N
S
O
Scheme 3. Synthesize 2-methyl pyrido-thieno-pyrimidinone analog.

C. Zhao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 319–323
321
N
N
N
O
2M NH3 in MeOH
N
+
N
N
S
N
N
S
N
S
Cl
N
5h
H
H
6h
Scheme 4. Methanol as solvent caused the formation of by-product.
Table 2
Inhibition of Cdc7/Dbf4 activity in vitro
R3
R2
R1
N
N
N
N
S
5
R
R4
Compound
R1
R2
R3
R4
R5
Cdc7 (IC50) (nM)
Ki (nM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
Me
Me
–(CH2)4–
–(CH2)3–
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
5
10
20
20
30
40
50
400
0.5
1
2
2
3
4
5
40
Et
Me
Me
Me
–(CH2)4–
–(CH2)3–
Me
Me
Me
Allyl
Me
Me
Me
Me
Me
CF3
H
H
H
Me
Me
Me
Me
Me
Cyclopropyl
H
Me
H
Me
Me
Me
H
Benzyl
H
Ph
2000
4000
9000
10,000
12,000
>30,000
>30,000
>30,000
200
400
900
1000
1200
>3000
>3000
>3000
Et-O-C@O
H
H
H
H
H
Me
Me
Ph
of residual phosphorus oxychloride and its by-product with cold
aqueous sodium bicarbonate and water was critical for the success
of reaction e.
For the transformation of compound 5 to 6, the lower alcohols
such as ethanol or isopropanol were used as solvents. It was found
that methanol competed with ammonia for the replacement of
chlorine, with the ratio of product to by-product about 1:1 for
the following reaction (Scheme 4).
and emerging structure–activity (SAR) relationship for compound
6.
The following trends have emerged from the data in Table 2:
using 6h as a reference compound, adding small aliphatic groups
at R2 position dramatically increased the potency (compounds
6a–g). Bigger groups such as benzyl at R2 position decreased activ-
ity approximately 5-fold (6i). Unsubstituted pyrido showed a sub-
stantially lower activity (6m), suggesting that there might be a
small hydrophobic cavity within Cdc7/Dbf4 complex interacting
with this part of the molecule. Any substitution on the R4 and R5
positions led to loss of activity, which indicates a possible role of
The test compounds were evaluated for inhibition of Cdc7/Dbf4
enzyme activity using a previously published Homogeneous Time-
Resolved Fluorescence (HTRF) assay.14 Table 2 shows the activity
0.7
Table 3
Compound 6d was tested for activity against a panel of kinases using the IMAP assay
platform
0.6
N
N
0.5
N
S
Kinase
Ki (nM)
N
0.4
Cdc7/Dbf4
CDK1/Cyclin B
CDK2/Cyclin E
CDK4/Cyclin D
AKT
PKA
PKC-a
PKC-d
ERK2
2
>5000
>5000
>5000
>5000
1100
>5000
>5000
>5000
>5000
>5000
>5000
0.3
0.2
0.1
0
0
2
4
6
8
10 12
ATP Concentration (μM)
SGK
Fyn
EphB3
Figure 2. Pyrido-thieno-pyrimidines are ATP-competitive inhibitors. The IC50 of
compound 6h was determined as a function of the ATP concentration.

322
C. Zhao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 319–323
predominant form of MCM2 in the control cell that transitioned
100
75
50
25
0
N
through S phase in the absence of Cdc7 inhibitor (Fig. 4). This indi-
cated that compound 6d penetrated the cells and inhibited intra-
cellular Cdc7 activity. The MTT18 assay for cell viability detected
a dose-dependent antiproliferative activity in three human colon
cancer cell lines (HCT116, RKO, and SW480) in vitro with IC50 in
N
N
N
N
S
S
N
N
BSA
0.2
the 6- to 10-lM range (Fig. 5). The relatively low Km for ATP of
the Cdc7/Dbf4 complex (ꢁ500 nM)14 is most likely the reason for
the modest cellular potency of 6d that has to compete with milli-
molar intracellular ATP concentrations.
mg/ml
2.0 mg/ml
During the preparation of this letter, two different classes of
ATP-competitive Cdc7 inhibitors, indazoles19 and pyrrolopyridi-
nones,20 have been reported. The most potent compounds in these
series have shown single nanomolar Ki values close to the Ki of the
most potent members of the pyrido-thieno-pyrimidines described
here.
0.001
0.01
0.1
1
10
Concentration (µM)
Figure 3. IC50 of compound 6d was determined in the presence of 0.2 and 2 mg/mL
of bovine serum albumin.
Pyrido-thieno-pyrimidine scaffold has relatively low molecular
weight (<300) and good permeability (Caco-2 > 200 ꢂ 10ꢀ7 cm/s).
However, it showed limited solubility (<30 lg/mL at pH 6.5) and
relatively high clearance in human liver microsomes (HLM) prepa-
rations in vitro. The poor solubility may also contribute to the
moderate cellular activity. Interestingly, the intermediate 3h was
much more stable than its final product 6h in HLM and the solubil-
ity of 3h was about 2-fold higher than 6h, suggesting that the
pyrimidine part of pyrido-thieno-pyrimidine is prone to metabo-
lism and the stacking effect of the highly conjugated system in pyr-
ido-thieno-pyrimidines is detrimental to the solubility.
In conclusion, we have developed an efficient method for the
synthesis of pyrido-thieno-pyrimidines. These analogs are potent
and selective inhibitors of Cdc7/Dbf4 kinase in vitro. The represen-
tative compounds from these series can penetrate cultured cancer
cells, inhibit MCM2 phosphorylation and show a moderate antipro-
liferative effect in cancer cells. Further improvement of potency
and optimization for solubility and metabolic stability is needed
to address the potential of Cdc7 as a cancer target in vivo.
1
2
3
MCM2
P-MCM2
Figure 4. Inhibition of MCM2 phosphorylation in HCT 116 cancer cells. (1) G1 cells
(control); (2) G1 cells + 20 M compound 6d; (3). G1 cells + DMSO (18 h, control).
l
the nitrogen proton as a H-bond donor. Interestingly, the interme-
diates (3a–p) showed similar inhibitory activity as the final prod-
ucts in the 1- to 20-lM range.
Compounds 6h and 6d were evaluated further. The ATP-depen-
dence of 6h activity indicated that these series of Cdc7 inhibitors
are ATP-competitive (Fig. 2).
Compound 6d demonstrated >500-fold selectivity15 in vitro
against a panel of 11 diverse human kinases including some of
the cyclin-dependent kinases, CDK1, CDK2, and CDK4 (Table 3).
This compound showed practically identical activity in the pres-
ence of 0.2 and 2.0 mg/mL bovine serum albumin suggesting that
non-specific protein binding may not be a problem for this class
of molecules (Fig. 3).
MCM2 is one of the important cellular targets of Cdc7/Dbf4.
Therefore, compound 6d was tested for inhibition of MCM2 phos-
phorylation in proliferating HT116 colon cancer cells (Fig. 4). Since
Cdc7/Dbf4 complex phosphorylates MCM2 during S phase, cells
were pre-synchronized in G1 phase before entry into S phase by
mitotic shake-off.16 They were then incubated in the presence of
Acknowledgments
We thank Dr. Kin-Chun Luk for critically reviewing this article.
References and notes
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100
8.3
HCT116
6.6
RKO
80
9.6
SW480
60
N
N
40
N
S
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Figure 5. Antiproliferative activity of compound 6d in HCT 116, RKO, and SW480
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