4-(1H-Indazol-5-yl)-6-phenylpyrimidin-2(1H)-one analogs as potent CDC7 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.07.061

A series of 4-(4-hydroxyphenyl)-6-phenylpyrimidin-2(1H)-ones were identified by HTS as inhibitors of CDC7. Molecular modeling and medicinal chemistry techniques were employed to explore the SAR for this series with a focus on removing potential metabolic liabilities and improving cellular potency.

Full text of DOI:10.1016/j.bmcl.2008.07.061

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4482–4485
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-(1H-Indazol-5-yl)-6-phenylpyrimidin-2(1H)-one analogs as potent CDC7
inhibitors
*
Cynthia M. Shafer , Mika Lindvall, Cornelia Bellamacina, Thomas G. Gesner, Asha Yabannavar, Weiping Jia,
Song Lin, Annette Walter
Oncology Division, Novartis Institutes of Biomedical Research, 4560 Horton Street, Emeryville, CA 94608, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-(4-hydroxyphenyl)-6-phenylpyrimidin-2(1H)-ones were identified by HTS as inhibitors of
CDC7. Molecular modeling and medicinal chemistry techniques were employed to explore the SAR for
this series with a focus on removing potential metabolic liabilities and improving cellular potency.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 30 May 2008
Revised 11 July 2008
Accepted 14 July 2008
Available online 17 July 2008
Keywords:
Phenol
Pyrimidone
Indazole
CDC7
Pharmacophore model
The serine/threonine kinase CDC7 plays an essential role in ini-
tiation of DNA replication in eukaryotic cells.1 After assembly of
the pre-replication complex to the replication origin, the CDC7 ki-
nase phosphorylates MCM (minichromosome maintenance) pro-
teins and allows for recruitment of CDC45 and DNA polymerase
thereby initiating DNA replication.2 CDC7 requires association with
one of its cofactors, ASK (also known as DBF4) or ASKL1 (also
known as Drf1), for kinase activation.3–6 CDC7 deficient mice die
between day 3.5 and 6.5 indicating that CDC7 is essential for early
embryonic development.7 Conditional knock-down of CDC7 in
mouse embryonic stem (ES) cell lines (CDC7À/Àtg) revealed
immediate inhibition of cell proliferation, rapid cessation of DNA
synthesis, and arrest in S phase progression.7 CDC7 has been impli-
cated in DNA damage checkpoint signaling in response to Etopo-
side treatment or DNA single strand breaks.8 A role for CDC7 in
DNA damage response is supported by the observation that CDC7
depleted mouse ES cells accumulate RAD51 foci in the nucleus.7
Deletion of CDC7 in yeast results in hypersensitivity to hydroxy-
urea treatment.9
identified, but the functional relevance of those sites remains to
be determined.3,11–13
High-throughput screening (HTS) yielded the pyrimidin-2(1H)-
one compounds (Fig. 1, 1 and 2) as potent inhibitors of CDC7. These
compounds, however, contained a phenolic moiety, which we felt
could constitute a metabolic liability for the series. Analysis of
in vitro incubation with mouse microsomes did, in fact, show a
short half-life (t1/2 4 min). Furthermore, glucuronidation was the
major metabolite as determined by CLND. In addition, these com-
pounds lacked the ability to inhibit cellular proliferation.14 We ap-
proached these issues using
a ligand-based pharmacophore
model15 to design molecules to remove the phenol, retain
in vitro potency and improve cellular inhibition.
Compound 2 (Table 1), which has a methyl group ortho to the
hydroxyl showed a 10-fold improvement in enzymatic affinity
compared to 1, which lacked the methyl group. This increase in po-
O
Recently, CDC7 has emerged as an attractive target for cancer
therapy. Depletion of CDC7 using siRNA oligonucleotides results
in induction of apoptosis in cancer cell lines while normal dermal
fibroblast cells are spared.10 Further, CDC7-mediated phosphoryla-
tion sites on MCM2, MCM4, and MCM6 in tumor cells have been
N
NH
HO
R
1 R = H
2 R = CH3 0.003 μM
0.03 μM
* Corresponding author. Tel.: +1 510 923 8086.
E-mail address: cynthia_shafer@chiron.com (C.M. Shafer).
Figure 1. CDC7 HTS phenol pyrimidin-2(1H)-one hits and IC50 values.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.061

C. M. Shafer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4482–4485
4483
Table 1
Structure–activity relationship of the pyrimidin-2(1H)-one series for CDC7
O
N
NH
Ar
R
Compound
Ar
R
CDC7 DBF4 IC50
0.03
(lM)
Compound
Ar
R
CDC7 DBF4 IC50
0.006
(lM)
1
—
8
3-OBn
HN
N
HO
2
3
4
5
6
7
—
0.003
0.01
0.01
0.15
0.005
0.11
9
4-OBn
4-Et
0.35
0.10
0.04
>13
HO
HN
N
CH3
—
10
11
12
13
14
HN
N
HN
N
2-F
3-F
3-Cl
3-CF3
N
NH
4-
HN
N
HN
N
3-Cl
HN
N
N
N
2,4-OCH3
2,4-OCH3
1.0
HN
N
NH2
N
N
7.3
HN
N
H3C
CH3
Figure 2. Overlap of 2 (gray) and 6 (light brown) in their phenol and indazole
moieties, respectively.
Figure 3. The binding site in a crystal structure of 2 in PIM-1 (PDB ID: 3DCV).

4484
C. M. Shafer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4482–4485
O
O
N
NH
a) Urea, i-PrOH, HCl
a
b
Ar
H
O
b) Urea, 80 ºC
O
Ar
R
R
O
O
piperidine, acetic
acid, 0-130 ºC
NaOEt, EtOH,
Urea, 80 ºC
O
N
NH
+
Ar
R
R
Ar
H
Ar
R
Scheme 1. Synthetic methods for pyrimidin-2(1H)-one series.
tency may be partially due to the fact that the ortho-methyl group
induces a preference for a single hydroxyl group conformation and
preorganizes the group for binding to CDC7, presumably in the
hinge region. Quantum mechanical conformational analysis sug-
gests that the hydroxyl hydrogen preferably points away from
the methyl group. In evaluating this scaffold with the intention
of replacing the hydroxyl group with another group which would
make similar interactions and have less propensity for rapid
metabolism, the indazole moiety was suggested as a phenol isoste-
re. Modeling confirmed that the indazole of 6 overlaid well with
the phenol (2, Fig. 2), and the required tautomer was preferred
by quantum mechanical modeling.16 Furthermore, the preferred
indazole tautomer and preferred phenol conformation of 2 pro-
vided overlap of the hydrogen bond donors groups.
The indazole analog 3 was then synthesized and found to retain
similar affinity as the phenols (1 and 2). A small set of analogs with
a 4-indazole moiety was then synthesized with varying substitu-
ents on the 6-phenyl ring (compounds 4–11 in Table 1). Compound
4, which has a fluorine at the ortho position of the phenyl ring,
exhibited identical potency as unsubstituted analog 3 while substi-
tution at the meta position with a fluorine (5) or a trifluoromethyl
group (7) led to a significant decrease in in vitro activity. Interest-
ingly though, a meta-chloro substituent (6) did not lead to a reduc-
tion in enzymatic potency. Finally, the large, hydrophobic 3-
benzyloxy analog (8) was also potent against CDC7. The corre-
sponding para-substituted benzyloxy analog (9), however, led to
a nearly 60-fold decrease in CDC7 affinity compared to 8. A hydro-
phobic group such as ethyl at the para position (10) was also less
potent while the 4-piperazinyl analog (11) gave an IC50 of
conformation of the compound, where the phenol and pyrimidi-
none are coplanar while the pendant phenyl is out of plane relative
to the rest of the molecule, were predicted. These details are com-
patible with the CDC7 binding site shape and with interactions
generally observed in kinases.
Although the 4-(1H-indazol-5-yl)-6-phenylpyrimidin-2(1H)-
one analogs showed striking affinity for CDC7 in vitro, only com-
pound 6 inhibited cellular growth (A549 lung carcinoma GI50
0.6 lM). Evaluation of this series in CaCO-2 cells indicates both
permeability and efflux as issues for this series. Compound 6, the
only compound to show inhibition of cellular growth, exhibited
the highest permeability and lowest efflux ratio (BA/AB) for the
series.18 In addition, the efflux ratios for the series of compounds
correlated well with the cellular potency observed. Furthermore,
assessment of microsomal stability indicates that the indazole
moiety did not improve the half-life for this series indicating that
another portion of the molecule is also prone to metabolism.
The pyrimidin-2(1H)-one analogs described herein, were syn-
thesized either by a modified Biginelli reaction19 (Scheme 1a) or
by condensation of a chalcone with urea under basic conditions20
(Scheme 1b). Despite the moderate yields described in the litera-
ture, the yields obtained for the pyrimidin-2(1H)-ones presented
in this letter were typically less than 5% regardless of the method
used.
In conclusion, from the initial HTS hits, the 4-(1H-indazol-5-yl)-
6-phenylpyrimidin-2(1H)-ones, a novel series of compounds po-
tent against the kinase CDC7, were designed and synthesized.
While cellular potency was obtained in only one example, further
modifications could be envisioned which might increase cell per-
meability and thus improve cellular proliferation.
0.04 lM against CDC7 suggesting that the para position groups ex-
tend to a hydrophilic part of the protein or to solvent.
Finally, to test the validity of the ligand-based pharmacophore
model, several pyridine analogs were synthesized where the phe-
nol was replaced by a pyridine or substituted pyridine as in com-
pounds 12–14. Modeling suggested that the pyridine nitrogen
acceptor would be one bond length too short for optimal hydrogen
binding to the hinge. The in vitro data supported this hypothesis as
the unsubstituted pyridine (12) did not inhibit CDC7, and the
dimethylaminopyridine 14 exhibited poor affinity. In addition,
the aminopyridine (13) gave slight inhibition suggesting the neces-
sity of a hydrogen bond donor.
Acknowledgments
The authors thank Weiping Jia, Elaine Ginn, and Li Zhang for
their analytical support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.07.061.
Subsequently, a crystal structure of 2 in complex with PIM-1
(Proviral Insertion of Maloney kinase)17 was obtained (Fig. 3).
While the details of binding interactions are likely to be different
in PIM-1 than in CDC7—as 2 is 50-fold less potent against PIM-1
than CDC7, and the indazole analogs of 2 are even weaker inhibi-
tors of PIM-1—the PIM-1 co-crystal structure supported the role
of the phenol as the hinge binding group. The co-crystal structure
also supported the details used in the modeling in that the pre-
ferred pyrimidinone tautomer as drawn in Figure 2 and overall
References and notes
1. Jiang, W.; McDonald, D.; Hope, T. J.; Hunter, T. EMBO J. 1999, 18, 5703.
2. Kim, J. M.; Yamada, M.; Masai, H. Mutat. Res. 2003, 532, 29.
3. Masai, H.; Taniyama, C.; Ogino, K.; Matsui, E.; Kakusho, N.; Matsumoto, S.; Kim,
J.-M.; Ishii, A.; Tanaka, T.; Kobayashi, T.; Tamai, K.; Ohtani, K.; Arai, K.-I. J. Biol.
Chem. 2006, 281, 39249.
4. Sato, N.; Sato, M.; Nakayama, M.; Saitoh, R.; Arai, K.-I.; Masai, H. Genes Cells
2003, 8, 451.
5. Montagnoli, A.; Bosotti, R.; Villa, F.; Rialland, M.; Brotherton, D.; Mercurio, C.;
Berthelsen, J.; Santocanale, C. EMBO J. 2002, 21, 3171.

C. M. Shafer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4482–4485
4485
6. Yoshizawa-Sugata, N.; Ishii, A.; Taniyama, C.; Matsui, E.; Arai, K.-I.; Masai, H. J.
14. GI50 data in the A549 human lung carcinoma cell line is >10
compounds 1 and 2.
l
M for both
Biol. Chem. 2005, 280, 13062.
7. Kim, J. M.; Nakao, K.; Nakamura, K.; Saito, I.; Katsuki, M.; Arai, K.-I.; Masai, H.
EMBO J. 2002, 21, 2168.
8. Costanzo, V.; Shechter, D.; Lupardus, P. J.; Cimprich, K. A.; Gottesman, M.;
Gautier, J. Mol. Cell 2003, 11, 203.
9. Weinreich, M.; Stillman, B. EMBO J. 1999, 18, 5334.
10. Montagnoli, A.; Tenca, P.; Sola, F.; Carpani, D.; Brotherton, D.; Albanese, C.;
Santocanale, C. Cancer Res. 2004, 64, 7110.
15. There is no reported CDC7 crystal structure, and the closest kinases have low
homology of 30% for the purpose of building homology models for docking.
16. Jaguar version 6.0 (Schrodinger, LLC, Portland, Oregon, 2005) was used for
geometry optimization at the B3LYP/6-31G* level of theory in the presence of
Jaguar’s continuum water model.
17. Meeker, T. C.; Nagarajan, L.; ar-Rushdi, A.; Croce, C. M. J. Cell. Biochem. 1987, 35,
105.
11. Montagnoli, A.; Valsasina, B.; Brotherton, D.; Troiani, S.; Rainoldi, S.; Tenca, P.;
Molinari, A.; Santocanale, C. J. Biol. Chem. 2006, 281, 10281.
12. Tsuji, T.; Ficarro, S. B.; Jiang, W. Mol. Biol. Cell 2006, 17, 4459.
13. Sheu, Y.-J.; Stillman, B. Mol. Cell 2006, 24, 101.
18. Unpublished data.
19. Sedova, V. F.; Shkurko, O. P. Chem. Heterocycl. Compd. 2004, 40, 194.
20. Sabri, S. S.; Hussein, A. Q.; Al-Hajjar, F. H. J. Chem. Eng. Data 1985, 30,
512.