N-substituted azaindoles as potent inhibitors of Cdc7 kinase

Article abstract of DOI:10.1016/j.bmcl.2013.02.007

Cdc7 kinase is responsible for the initiation and regulation of DNA replication and has been proposed as a target for cancer therapy. We have identified a class of Cdc7 inhibitors based on a substituted indole core. Synthesis of focused indole and azaindole analogs yielded potent and selective 5-azaindole Cdc7 inhibitors with improved intrinsic metabolic stability (ie 36). In parallel, quantum mechanical conformational analysis helped to rationalize SAR observations, led to a proposal of the preferred binding conformation in the absence of co-crystallography data, and allowed the design of 7-azaindole 37 as a second lead in this series.

Full text of DOI:10.1016/j.bmcl.2013.02.007

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2056–2060
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-substituted azaindoles as potent inhibitors of Cdc7 kinase
Marian C. Bryan a, James R. Falsey a, Mike Frohn b, Andreas Reichelt b, Guomin Yao b,
Michael D. Bartberger b, Julie M. Bailis c, Leeanne Zalameda d, Tisha San Miguel d,
Elizabeth M. Doherty a, John G. Allen b,
⇑
a Medicinal Chemistry Research Technologies, Therapeutic Discovery, Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA 91320, USA
b Therapeutic Discovery, Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA 91320, USA
c Oncology Research, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
d Bioassay and Profiling, Therapeutic Discovery, Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA 91320, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cdc7 kinase is responsible for the initiation and regulation of DNA replication and has been proposed as a
target for cancer therapy. We have identified a class of Cdc7 inhibitors based on a substituted indole core.
Synthesis of focused indole and azaindole analogs yielded potent and selective 5-azaindole Cdc7 inhibi-
tors with improved intrinsic metabolic stability (ie 36). In parallel, quantum mechanical conformational
analysis helped to rationalize SAR observations, led to a proposal of the preferred binding conformation in
the absence of co-crystallography data, and allowed the design of 7-azaindole 37 as a second lead in this
series.
Received 30 September 2012
Revised 28 January 2013
Accepted 1 February 2013
Available online 13 February 2013
Keywords:
Cdc7 inhibitors
Azaindole
Ó 2013 Elsevier Ltd. All rights reserved.
Cell division cycle
Conformational analysis
Cell division cycle 7 (Cdc7) is a conserved serine/threonine ki-
nase that, along with cyclin-dependent kinase (CDK), is responsible
for the initiation of DNA replication during S-phase.1–6 At replica-
tion origins, phosphorylation of minichromosome maintenance
complex (MCM) by Cdc7 leads to the unwinding of double
stranded DNA by the DNA helicase. This step is essential for initia-
tion of DNA replication in mammalian cells. In addition to initia-
tion of replication, Cdc7 is also involved in S-phase checkpoint
regulation and the maintenance of S-phase genome stability.7,8
Given its key role in DNA replication, it is unsurprising that
heightened expression of Cdc7 is found in many cancers including
leukemia, lymphoma, colorectal and gastric carcinomas, primary
breast tumor, colon and lung cancer.1,9–11 Increased Cdc7 expres-
sion also correlates with genomic instability, reduced disease-free
survival, and accelerated cell cycle progression.9 In breast cancer,
dysregulation of Cdc7 is associated with the development of an
aggressive malignant phenotype while in diffuse large cell lym-
phoma, increased Cdc7 activity is associated with a poor clinical
outcome.4,9,12 Increased interest in targeting Cdc7 has also been
driven by the potential for a wide therapeutic window. For exam-
ple, it has been found that depletion of Cdc7 leads to p53-indepen-
dent apoptosis in cancer cells,13 and to cell cycle arrest without
loss of viability in normal cells.3,4,7–9,13,14 Such alternate outcomes
may allow for treatments with limited toxicity even in self-
renewing, high-turnover tissue. Taken together these findings
suggest that Cdc7 is an attractive target for therapeutic interven-
tion in a variety of cancers.4,7,8 Others have reported the investiga-
tion of small molecule inhibitors of Cdc7,3,14–20 including a recent
disclosure by our group of a series of thiazole-based inhibitors of
Cdc7.21
A high-throughput screen of the Amgen Screening Collection
identified 6-cyanoindole 2, which had good physical properties,
was potent against Cdc7 (IC50 = 75 nM) and exhibited excellent
selectivity over functionally related CDK2 (IC50 = 16,500 nM).
Although CDK2 itself is dispensable in cancer cell progression,
CDK2 selectivity serves as a marker for off-target effects such as
CDK1 activity which could lead to increased toxicity towards nor-
mal cells.22,23 Efforts to establish the structure–activity relation-
ship (SAR) around the indole core of 2 were initiated.
The core, N1, C3 and C6 substitution were rapidly explored fol-
lowing the concise syntheses shown in Scheme 1. Nucleophilic aro-
matic substitution (SNAr) of the commercially available indole
amines (1) under basic conditions provided N1-substituted indole
analogs 2 through 7, 9 through 24, and 37. Alternately, copper-cat-
alyzed N-arylation of 1H-indole-6-carbonitrile with 4-chloropyr-
imidine was required for the generation of pyrimidine 8. The
indole core was further differentiated at position C3. Molecules
26 through 36 were generated from 1b in three steps. Iodination
under basic conditions provided iodide 25 regiospecifically. Aryla-
tion using the SNAr conditions described above followed by Suzuki
⇑
Corresponding author.
E-mail address: johallen@amgen.com (J.G. Allen).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.007

M. C. Bryan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2056–2060
2057
4
CN
5
a
or
or
or
N
7
CN
CN
R2
R3
N
N
N
N
N
H
R1
1
2-7
9-16
NH2
17
NH2
18-24
N
N
N
N
N
N
NH2
N
N
or
N
37
NH2
N
b
N
CN
N
H
CN
N
8
1a
1b
N
I
R5
N
c
a,d
N
N
R4
N
R4
N
R4
N
H
H
25
N
26 36
-
N
NH2
Scheme 1. General synthesis of substituted indoles and azaindoles. Reagents and conditions: (a) R1Cl, base (Cs2CO3, K2CO3 or NaH), DMSO,
D
(2–92%); (b) CuI,
4-chloropyrimidine, K3PO4, (1R,2R)-4-cyclohexene-1,2-diamine (10%); (c) I2, KOH (87%); (d) Pd(PPh3)4, K2CO3, R5B(OH)2, toluene:EtOH:H2O (6:3:2), 100 °C (5–49%).
coupling of the iodide with various boronic acids provided the in-
doles functionalized at N1, C3 and C6. These routes allowed three
regions of interest to be modified in parallel. With the substituted
indoles in hand, we examined their potency against Cdc7 and
selectivity over CDK2.
Biological testing of indole core analogs indicated that minimal
modification to the aminopyrimidine was tolerated (Table 1).
Methylation of the primary amine (3, IC50 = 0.025 lM) showed
modest improvement in Cdc7 potency without significantly
impacting selectivity over CDK2 (IC50 = 2.7 M). Removal of one
l
of the two pyrimidine nitrogens (4, 5) was poorly tolerated, as
was rearrangement of the pyrimidine ring (6, 7), although it was
noted that inhibition of CDK2 was reduced. Significant loss in po-
tency for Cdc7 was also seen with removal of the primary amine
(8). These findings highlighted the importance of R1, presumably
binding to the hinge region of the protein, for potency and possibly
for selectivity. Our attention then turned to substitution of the C6
position of the indole.
Removal of the nitrile to give unsubstituted indole 9 resulted in
a >50Â loss in potency relative to 2 (Table 2). Halides such as F, Cl,
and Br were well tolerated at C6 with chloride 11 equipotent to ni-
Table 1
Hinge binder SAR
No.
R1
Cdc7 SDa
(l
M)
CDK2 SDa
16.5 3.3
(
lM)
trile 2 (IC50 = 0.066 lM), though an order of magnitude less selec-
tive for CDK2 (220Â vs 56Â). Nitroindole 13 was also equipotent
with nitrile 2 and highly selective against CDK2 but was not pur-
sued due to potential metabolic liabilities. Substitution of C6 with
either a trifluoromethyl (14) or a hydroxyl (15) resulted in low
micromolar activity for Cdc7 while substituting alkyne (16) for ni-
trile (2) resulted in a compound that was an order of magnitude
less potent. The regioisomer of 2, C5 nitrile 17, was also signifi-
cantly less active.
N
N
2
0.075 0.065
0.025 0.026
N
NH2
3
2.7 1.5
N
N
H
Although compound 2 had good potency against Cdc7, it was
found to have poor microsomal stability, particularly in human li-
ver microsomes. Introduction of nitrogen to the phenyl ring core
N
4
5
6
7
8
10.4 2.7
9.0 2.7
2.7 1.7
8.8 5.5
1.5 0.4
45 21
39 21
>83b
NH2
NH2
NH2
NH2
Table 2
N
R2 indole SAR
R2
Cdc7 SDa
(
lM)
CDK2 SDa
(lM)
N
N
No.
2
9
CN
H
F
Cl
Br
NO2
CF3
OH
CCH
—
0.075 0.065
4.7 1.1
16.5 3.3
28.5 8.2
34 21
3.7 1.7
8.2 4.3
>30.6
17 11
5.0 1.2
10 2.0
>29.1
N
10
11
12
13
14
15
16
17
0.156 0.039
0.066 0.014
0.35 0.20
0.082 0.033
1.1 0.8
3.3 1.4
0.76 0.26
8.5 5.2
N
>29
>83b
N
N
a
Values are the mean of at least three determinations.
Values are the result of one determination.
b
a
Values are the mean of at least three determinations.

2058
M. C. Bryan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2056–2060
Table 4
was investigated in order to address potential metabolic instabil-
ity.24 As shown in Table 3, indole 9 exhibited high intrinsic clear-
ance in both human and rat liver microsomes (HLM/RLM),
similarly to 2. Insertion of nitrogen at position 7 (18) was poorly
tolerated by Cdc7 and did not improve metabolic stability. 6-Aza-
indole 19 and 5-azaindole 20 showed equal improvements in Cdc7
potency relative to indole 9 and similar intrinsic clearance, though
R5 5-azaindole SAR
R5
O
Ar
N
NH2
N
S
N
N
N
25
20 26 35
,
-
affinities for CDK2 differed (IC50 = 45.1 lM vs IC50 = 3.7 lM, respec-
N
N
tively). The final heterocycle tested in this series, 4-azaindole 21,
was modestly less potent but exhibited a 6Â improvement in met-
abolic stability relative to indole 9 as measured by RLM.
NH2
NH2
No.
R5
Cdc7 SDa
(
l
M)
CDK2 SDa
(lM)
Efforts to improve the potency of the azaindoles through rein-
troduction of C6 substitution while maintaining the improved met-
abolic stability from the 5-nitrogen were then attempted. C6 nitrile
22 did not improve potency compared to unsubstituted azaindole
20, although metabolic stability was similar. C6 Chloride 23 was
3Â more potent for Cdc7 and selectivity over CDK2 increased from
3Â to >100Â while modestly impacting intrinsic clearance. Nitrile
substitution of 4-azaindole 21 to yield 24 did not significantly im-
prove potency. 5-Azaindole compounds 20, 22 and 23 provided an
attractive balance of potency, selectivity and metabolic stability
relative to 9.
20
26
27
28
29
30
31
32
33
34
35
H
Ph
0.98 0.65
3.7 0.5
0.104 0.005
0.16 0.05
0.25 0.09
0.58 0.29
0.12 0.01
0.16 0.03
0.011 0.002
0.16 0.02
0.33 0.23
0.007 0.008
0.31 0.03
1.1 0.29
0.16 0.06
0.55 0.05
0.10 0.05
0.16 0.02
0.11 0.01
>83b
3-Pyridyl
4-Pyridyl
3-Pyrazole
3-Furan
4-(3,6-Dihydro-2H-pyran)
2-Chlorophenyl
3-Chlorophenyl
4-Chlorophenyl
3-(2-Chloropyridyl)
0.06 0.13
0.31 0.04
a
Values are the mean of at least three determinations.
Value is the mean of one determination.
b
In order to recover potency in the more metabolically stable
5-azaindole compounds, we considered the SAR of the related thi-
azole series.21 Overlaying thiazole carboxamide 25 with azaindole
(Table 4) suggested C3 aryl substitution would be tolerated.
Accordingly, the SAR surrounding C3 was investigated starting
with 5-azaindole 20 (Table 4). Phenyl substitution of C3 (26) gave
a 9Â improvement in potency for Cdc7 relative to 5-azaindole 20.
3-Pyridyl 27 and 4-pyridyl 28 had comparable potency to phenyl
26, and 27 showed a small improvement in CDK2 selectivity. 3-
Pyrazole 29 was only slightly more potent than the unsubstituted
indole with complete loss of CDK2 selectivity. 3-Furan 30 and
4-(3,6-dihydro-2H-pyran) 31 gave comparable activity to C3-phenyl
azaindole 26 but again lacked selectivity over CDK2. The first
significant improvement in potency over compound 26 was seen
when chlorinated phenyl rings were introduced. 2-Chlorophenyl
only moderate selectivity (44Â) but was very potent
(IC50 = 0.007 M). Both molecules had low to moderate intrinsic
clearance in HLM/RLM assays. Combination of these SAR features
gave pyridinyl azaindole 36 (Table 5), which was slightly less po-
tent for Cdc7 than 35 (IC50 = 0.030 lM), but was highly selective
over CDK2 (>2700Â), and maintained low intrinsic clearance. Also
shown in Table 5 is cell-based potency data determined in HCT116
cells for the phosphorylation of the proximal substrate MCM2.21
These data indicate a fairly consistent 100Â loss in potency for
these compounds in the cellular assay versus the biochemical
assay.
l
Quantum mechanical conformational analysis of the com-
pounds in Table 6 provides a clear rationale for the observed SAR
trends in this series. It was initially speculated that the loss in po-
tency of 18 versus 9 may have been due to the hinge binder pyrim-
idine being oriented out of the preferred binding conformation due
to intramolecular azaindole N/pyrimidine N repulsion. Figure 1 de-
picts the predicted (B3LYP/6-31GÃ) dihedral profile about the
rotatable C–N bond of 2, 5, 9 and 18. Compounds 2 and 9 exist
as a pair of near-coplanar minima (ca. ꢀ20° and ꢀ160°, syn and
32 (IC50 = 0.011 lM) improved activity an order of magnitude com-
pared to phenyl 26, while chloro substitution at C3 (33) and C4
(34) of the appended phenyl ring were less impactful. Interestingly,
compound 33 showed excellent selectivity over CDK2 relative to
26 (>500Â vs 3Â). 3-(2-Chloropyridyl) (35), was equipotent to
chlorophenyl 32 with improved selectivity relative to 27 (44Â vs
7Â).
We next attempted to combine SAR findings around the pyrim-
idine, indole phenyl ring, and C3 in order to identify molecules
with improved potency, selectivity, and metabolic stability. Azain-
anti, respectively) of similar energy (
DE = 0.3 kcal/mol) Conversely,
18 is predicted to exist only in anti (180°) orientation, due to N–N
dole 24 had modest potency for Cdc7 (IC50 = 0.36
excellent selectivity over CDK2 (>100Â). Chloropyridine 35 had
l
M) but had
repulsion rendering its syn-like form thermally inaccessible (DE
>6 kcal/mol at the ꢀ30° local minimum).25 Comparison of the po-
tency of 18 versus 9 suggests the anti binding mode is disfavored
by approximately 5Â.
The relatively weaker activity of 5 is also satisfactorily ex-
plained on the basis of conformational effects. Due to C–H/C–H
repulsion, 5 exists as a pair of nonplanar (ca. 40° and 140°) minima,
with adoption of either planar conformation disfavored on the or-
der of 4 kcal/mol. This may in part explain the >100Â loss in po-
tency for 5 versus 2.
In an effort parallel to the SAR work above, the singleton com-
pound, 7-azaindole 37 (also shown in Fig. 1) was designed based
not only on its predicted planar conformation such as for 2 and
9, but with the added advantage of existing solely in the syn form,
thus doubling the population of the more potently binding con-
former. The 8Â improvement of 37 over 2 suggests the pyrimidine
N-1 forms additional favorable interactions with the protein com-
pared to 2. Although functional group contacts with the protein
cannot be ignored, it can be inferred from the potency differences
Table 3
Nitrogen heterocycle SAR
No.
N
R3
Cdc7 SDa
(l
M) CDK2 SDa M) HLMc RLMc
(l
2
9
Indole CN
0.075 0.065
4.7 1.1
16.5 3.3
28.5 8.2
42 18
45.1 3.8
3.7 0.5
21 7.4
>44
430
387
338
134
62
60
94
104
108
124
375
282
64
101
50
49
134
64
Indole R2 = H
18
19
20
21
22
23
24
7-N
6-N
5-N
4-N
5-N
5-N
4-N
H
—
H
H
CN
Cl
CN
26.7 4.9
0.93 0.82
0.98 0.65
12.6 2.8
1.24 0.28
0.36 0.23
5.3 1.3
38 12
>83b
a
b
c
Values are the mean of at least three determinations.
Value is the result of one determination.
ll/min/mg.

M. C. Bryan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2056–2060
2059
M)
Table 5
Comparison of enzyme and cellular potency, selectivity, and metabolic stability of compounds 24, 35, and 36
No.
Structure
Cdc7a SD(
lM)
CDK2a SD(
l
M)
HLMc
104
RLMc
134
pMCM2 SDa
(l
N
N
N
Cl
24
0.36 0.23
38 12
>50b
N
NH2
N
Cl
N
35
0.007 0.008
0.31 0.04
21
72
0.85 0.27
N
N
N
NH2
N
Cl
N
36
0.03 0.02
>83b
30
102
2.3 3.1
N
Cl
N
N
NH2
a
b
c
Values are the mean of at least three determinations.
Value is the result of one determination.
ll/min/mg.
Table 6
Comparison compounds for conformational analysis
No.
Cdc7 SDa
(l
M)
CDK2 SDa
(l
M)
HLMc
RLMc
pMCM2 SDa
(l
M)
2
5
9
18
37
0.075 0.065
9.0 2.7
4.7 1.1
16.5 3.3
39 21
28.5 8.2
42 18
>83b
430
201
387
338
34
124
122
375
282
46
5.0 2.9
>50b
>50b
26.7 4.9
>50b
0.009 0.003b
1.37 0.17b
a
b
c
Values are the mean of at least three determinations.
Value is the mean of two determinations.
ll/min/mg.
observed for 9 versus 18, 2 versus 5 and 37 versus 18, along with
the conformational profile depicted in Figure 1 that this class of in-
dole and azaindole compounds binds potently to Cdc7 in an
approximately coplanar syn, C-shaped orientation. Gratifyingly, in
addition to good potency, 37 also possessed excellent CDK2 selec-
tivity (cf. 6) with low microsomal clearance. The reason for the
excellent CDK2 selectivity of 37 was not determined. Similar to
the compounds in Table 5, 37 had a significant loss in potency in
the cellular assay (ꢀ150Â).
In summary, structure–activity relationship studies around ini-
tial hit 2 defined the scope of the SAR with respect to potency for
Cdc7 and selectivity over functionally related CDK2. Introduction
of heteroatoms in the indole core provided azaindoles that showed
increased intrinsic metabolic stability, although with a loss of Cdc7
potency. Potency was recovered via modifications to C3 and C6 of
the azaindole core while metabolic stability was maintained. An
alternate solution to the initial loss of potency of the azaindoles
was discovered following conformational analysis of the SAR. This
study also allowed us to propose the preferred binding orientation
of this class of compounds in the absence of a Cdc7 crystal struc-
ture, and highlights the use of rational conformational control as
an avenue for increasing activity independent of protein contacts.
Although 5-azaindole (36) and 7-azaindole (37) achieved good
metabolic stability and potency for Cdc7, and excellent selectivity
Figure 1. Dihedral profile for compounds 2, 5, 9, 18 and 37, with 0° (syn)
conformations depicted to the right.
over CDK2, cellular potency remained poor and was similar to
screening compound 2.
Acknowledgments
The authors wish to thank Andrew Tasker for assistance in
manuscript preparation and Zhihong Li for the preparation of

2060
M. C. Bryan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2056–2060
15. Ermoli, A.; Bargiotti, A.; Brasca, M. G.; Ciavolella, A.; Colombo, N.; Fachin, G.;
Isacchi, A.; Menichincheri, M.; Molinari, A.; Montagnoli, A.; Pillan, A.; Rainoldi,
S.; Sirtori, F. R.; Sola, F.; Thieffine, S.; Tibolla, M.; Valsasina, B.; Volpi, D.;
Santocanale, C.; Vanotti, E. J. Med. Chem. 2009, 52, 4380.
compound 3, Kexue Li for the preparation of compound 13, and
Ben Fisher for the preparation of compound 15.
16. Menichincheri, M.; Albanese, C.; Alli, C.; Ballinari, D.; Bargiotti, A.; Caldarelli,
M.; Ciavolella, A.; Cirla, A.; Colombo, M.; Colotta, F.; Croci, V.; D’Alessio, R.;
D’Anello, M.; Ermoli, A.; Fiorentini, F.; Forte, B.; Galvani, A.; Giordano, P.;
Isacchi, A.; Martina, K.; Molinari, A.; Moll, J. R. K.; Montagnoli, A.; Orsini, P.;
Orzi, F.; Pesenti, E.; Pillan, A.; Roletto, F.; Scolaro, A.; Tatò, M.; Tibolla, M.;
Valsasina, B.; Varasi, M.; Vianello, P.; Volpi, D.; Santocanale, C.; Vanotti, E. J.
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