The optimization of aminooxadiazoles as orally active inhibitors of Cdc7

Article abstract of DOI:10.1016/j.bmcl.2013.09.055

A series of aminooxadiazoles was optimized for inhibition of Cdc7. Early lead isoquinoline 1 suffered from modest cell potency (cellular IC₅₀ = 0.71 μM measuring pMCM2), low selectivity against structurally related kinases, and high IV clearance in rats (CL = 18 L/h/kg). Extensive optimization resulted in azaindole 26 (Cdc7 IC₅₀ = 1.1 nM, pMCM2 IC₅₀ = 32 nM) that demonstrated robust lowering of pMCM2 in a mouse pharmacodynamic (PD) model when dosed orally. Modifications to improve the pharmacokinetic profile of this series were guided by trapping experiments with glutathione in rat hepatocytes.

Full text of DOI:10.1016/j.bmcl.2013.09.055

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6396–6400
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The optimization of aminooxadiazoles as orally active inhibitors
of Cdc7
a
a
a
a
Paul E. Harrington a, , Matthew P. Bourbeau , Christopher Fotsch , Michael Frohn , Alexander J. Pickrell ,
Andreas Reichelt a, Kelvin Sham a, Aaron C. Siegmund a, Julie M. Bailis b, Tammy Bush c, Sonia Escobar d,
Dean Hickman e, Scott Heller c, Faye Hsieh e, Jessica N. Orf b, Minqing Rong f, Tisha San Miguel d,
Helming Tan g, Leeanne Zalameda d, John G. Allen a
⇑
a Medicinal Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
b Oncology Research, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, USA
c Oncology Research, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, USA
d Discovery Technologies, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
e Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
f Oncology Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
g Pharmaceutics, Amgen Inc., One Amgen Center Drive, 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:
A series of aminooxadiazoles was optimized for inhibition of Cdc7. Early lead isoquinoline 1 suffered from
Received 21 August 2013
Revised 16 September 2013
Accepted 18 September 2013
Available online 25 September 2013
modest cell potency (cellular IC50 = 0.71 lM measuring pMCM2), low selectivity against structurally
related kinases, and high IV clearance in rats (CL = 18 L/h/kg). Extensive optimization resulted in azain-
dole 26 (Cdc7 IC50 = 1.1 nM, pMCM2 IC50 = 32 nM) that demonstrated robust lowering of pMCM2 in a
mouse pharmacodynamic (PD) model when dosed orally. Modifications to improve the pharmacokinetic
profile of this series were guided by trapping experiments with glutathione in rat hepatocytes.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Cdc7
MCM2
Kinase inhibitor
Azaindole
Cancer
Cell division cycle 7 (Cdc7) is a serine/threonine protein kinase
that plays a pivotal role in the initiation of DNA replication.1 Dur-
ing the S phase of DNA replication, Cdc7 is activated by binding to
the regulatory subunit Dbf4.2 The Cdc7/Dbf4 complex phosphory-
lates one or more minichromosome maintenance complex (MCM2-
7) proteins leading to unwinding of double stranded DNA. The
essential role that Cdc7 plays in S phase entry and DNA replication
has been studied extensively in tumor cells. Cdc7 is overexpressed
in some human tumor cell lines, such as breast, lung, and colon
cancers.3–5 Small interfering RNA (siRNA) knockdown of Cdc7
results in p53 independent apoptotic cell death in tumor cell lines.6
In the same paper,6 siRNA knockdown of Cdc7 in normal cells was
shown to arrest growth reversibly and not cause cell death, sug-
gesting that normal tissue might be spared during cancer treat-
ment with a Cdc7 inhibitor. Furthermore, others have shown that
small molecule Cdc7 inhibitors slow the growth of human tumor
cell lines in mouse xenograft models.7–9 These results spurred
interest in the drug discovery community to develop a small mol-
ecule inhibitor of Cdc7 for use as a single agent or in combination
with chemotherapy.10–17
We recently disclosed a series of N-substituted azaindoles18 and
trisubstituted thiazoles19 as potent inhibitors of Cdc7. Isoquinoline
1 (Fig. 1) emerged as an early lead from a parallel effort to discover
new chemotypes. The chemical matter leading to 1 was identified
through high throughput screening of analogs from our protein
kinase B (PKB) program.20 Compounds were evaluated in an enzy-
matic assay that measured inhibition of Cdc7/Dbf4 biochemical
activity and a cellular assay in HCT-116 cells that measured phos-
phorylation of MCM2 at serine 53.21 Although 1 displayed good
potency in the Cdc7 assay21 (IC50 = 17 nM), it had several deficiencies
that were the focus of SAR efforts. First, potency in the cellular
pMCM2 assay21 (IC50 = 0.71
lM) was modest. Second, the rat IV
pharmacokinetics (PK) of 1 was very poor with an exceptionally
high clearance (CL = 18 L/h/kg) and a very short mean residence
time (MRT) of 0.2 h. We chose to first examine replacements for
the isoquinoline, without changing the aminooxadiazole core and
benzyl amine, with the goal of improving potency and PK. The
⇑
Corresponding author. Tel.: +1 805 313 5564; fax: +1 805 480 1337.
E-mail address: pharring@amgen.com (P.E. Harrington).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.055

P. E. Harrington et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6396–6400
6397
interactions and potentially improve binding with the protein. Flu-
oro isoquinoline24 2 (Table 1) had potency similar to 1, but dimin-
ished cellular activity. The lower intrinsic clearance of 2 relative to
1 in both rat and human liver microsomes (RLM/HLM) suggested
that the isoquinoline may contribute to the high clearance of 1.
This result led us to examine additional analogs that did not in-
clude an isoquinoline as the hinge binder. Although the 5,6-ring
analog thiazolopyridine 3 was less potent than 1 and 2, the 6,5-ring
compounds (4–7) proved to be more active. Indazoles 4 and 5 had
potency and selectivity profiles similar to 1, but improved micro-
somal stability in the case of 5. Azaindole 8 possessed potency
and CDK2 selectivity comparable to 1 and 5, but microsomal stabil-
ity was superior to 1 and similar to 5. Of the hinge binders exam-
ined, the azaindole provided the best balance of potency,
selectivity, and microsomal stability. Analogs 4–8 potentially offer
an additional site for H-bonding in the hinge region. Similar inter-
actions, with the surrogate protein GSK3b, were reported for struc-
turally related Cdc7 inhibitors.10
N
N
N
H
O
1
N
Rat PK IV (2 mg/kg)
Cdc7 IC50 = 0.017
pMCM2 IC50 = 0.71
CDK2 IC50 = 0.35
µM
CL = 18 L/h/kg
MRT = 0.2 h
µ
M
µ
M
Vss = 4.3 L/kg
Figure 1. Early lead 1.
benzyl amine portion would be addressed subsequently while
retaining the central aminooxadiazole core.
At the time of this work, no structures of the Cdc7 protein
bound to an inhibitor were reported.22 This complicated efforts
to design novel molecules and was especially challenging for
improving selectivity over the structurally related cyclin-dependent
kinases such as CDK1, CDK2, and CDK9.21 However, the nature of
the binding in the hinge region of kinases is well established and
the isoquinoline of 1 was assumed to bind in a similar manner.23
Thus, novel hinge binders were designed to satisfy canonical kinase
Efforts to further improve azaindole 8 focused on modifications
to the benzylic amine (Table 2). A fluoro group was used to probe
the effect of substitution on the phenyl ring. The potency of 8–11
was within 4-fold in the Cdc7 assay and 3-fold in the pMCM2
Table 1
SAR of N-benzyl 2-aminooxadiazole hinge binders
Compound
R
Cdc7 IC50
(
l
M)a
pMCM2 IC50
0.71 0.4
(l
M)a
CDK2 IC50
(l
M)a
HLM/RLM CLint
120/166
(l
L/min/mg)b
1
0.017 0.02
0.35 0.07
2
0.010 0.003
3.2 0.8
6.8
19
4
6
49/56
3
4
2.3
2
>50b
23/66
0.020 0.02
0.73 0.7
0.17 0.02
98/402
5
6
0.011 0.004
1.2 0.4
0.23 0.009
26/74
69/30
2
0.59 0.2
>50b
47
33
7
9
7
8
0.77 0.7
>50b
<14/<14
31/40
0.024 0.02
0.78 0.3
0.15 0.1
a
Unless indicated otherwise, data represent an average of at least three determinations SD.
Data is the result of one determination.
b

6398
P. E. Harrington et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6396–6400
assay. The ortho-fluoro isomer 9 displayed increased selectivity
over CDK2 (17-fold selective vs 4- to 6-fold selective for 8, 10,
and 11). Pyridines 12–14 had similar Cdc7 and pMCM2 potency, al-
beit inferior to 8–11, and enhanced microsomal stability while 12
had the greatest selectivity against CDK2. Substitution at the ben-
12–14 offered improved microsomal stability, introduction of the
nitrogen heteroatom reduced Cdc7 and pMCM2 potency relative
to 8. In an effort to identify sites of metabolism within the scaffold,
compound 16 was chosen as a representative azaindole for addi-
tional studies. As shown in Figure 2, incubation of 16 with rat
hepatocytes yielded glutathione (GSH) adduct 16a as the major
metabolite. The GSH-adduct was located in the azaindole/oxadiaz-
ole region of 16a. Therefore, in addition to incorporating a pyridine
within the benzylic amine side-chain, blocking metabolism within
the azaindole by introducing a heteroatom and/or fluorination
could potentially reduce metabolism.
zylic carbon was also examined. Both
a-methyl benzyl amine
enantiomers 15 and 16 were prepared. Despite similar Cdc7 po-
tency, 16 was 7-fold more active in the pMCM2 cellular assay
and more selective against CDK2. The gem-dimethyl analog 17
was more active against CDK2 (IC50 = 7 nM) than Cdc7
(IC50 = 0.12
lM). More sterically demanding substituents at the
a
-carbon such as ethyl (18), isopropyl (19), and cyclopropyl (20)
Compounds 22–26 were designed to introduce a heteroatom
and/or fluorination into both the side-chain and azaindole groups
in order to improve metabolism and solubility. In addition to the
moderate clearance of 21, its solubility in simulated intestinal fluid
(SIF) was very low. Compounds 21–23 and 26 had promising Cdc7
cell activity and selectivity profiles against CDK1, CDK2, and CDK9;
increased potency and selectivity relative to methyl (16). The
ortho-fluoro cyclopropyl analog 21 exhibited excellent Cdc7 and
pMCM2 potency and selectivity against CDK2.
The moderate microsomal turnover of 21 translated into an
in vivo clearance (CL) of 2.0 L/h/kg (Table 4). Although pyridines
Table 2
SAR of azaindole benzylic amines
a
a
a
b
Compound
R
Cdc7 IC50
(l
M)
pMCM2 IC50
1.6 0.3
(l
M)
CDK2 IC50
(lM)
HLM/RLM CLint
(lL/min/mg)
F
9
0.035 0.03
0.58 0.3
32/46
F
10
0.010 0.009
0.58 0.3
0.69 0.4
0.061 0.01
54/71
11
12
0.025 0.02
0.17 0.2
0.098 0.02
2.5 0.5
47/58
F
N
13
2
<14/14
N
13
14
0.19 0.2
0.11 0.1
7.1
12
3
4
0.61 0.1
<14/30
<14/37
0.35 0.07
N
Me
Me
15
16
17
18
19
0.035 0.04
0.032 0.03
0.12 0.07
2.6
1
0.098 0.02
0.31 0.04
15/17
64/25
30/35
70/29
66/51
0.37 0.2
Me
Me
Et
6.2
5
0.007 0.003
1.7 0.8
0.004 0.003
0.002 0.0005
0.15 0.02
0.14 0.06
iPr
1.2 0.3
20
21
0.001 0.0006
0.0009 0.0007
0.071 0.04
1.6 0.4
56/31
59/63
F
0.020 0.009
0.24 0.06
a
Data represent an average of at least three determinations SD.
Data is the result of one determination.
b

P. E. Harrington et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6396–6400
6399
O
O
NH2
CCl3
N
H
HN
HN
HN
HN
a-c
d
e
f
N
N
N
N
Br
F
F
F
27
28
29
30
F
N
NH
N
N
N
O
NH
O
O
HN
N
HN
g
N
F
F
31
26
O
S
O
S
Me
Me
Me
Me
N
HN
NH2·HCl
F
Me
Me
CHO
F
h
i
j
N
F
N
F
N
N
32
33
34
35
Scheme 1. Synthesis of 26. (a) NaH, TIPSCl, THF, rt, 91% yield; (b) n-BuLi, (PhSO2)2NF, -78 °C to rt, 59% yield; (c) TBAF, THF, rt, 98% yield; (d) AlCl3, ClCOCCl3, CH2Cl2, 40 °C; (e)
H2NNH2, MeOH, 60 °C, 80% yield (2 steps); (f) CDI, iPr2NEt, DMF, 60 °C, 41% yield; (g) 35, BOP (benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophos-
phate), iPr2NEt, DMF, rt, 41% yield; (h) (S)-(-)-2-methyl-2-propane-sulfinamide, Ti(OEt)4, THF, rt, 95% yield; (i) cyclopropylmagnesium bromide, CH2Cl2, -78 °C, 68% yield; (j)
HCl, MeOH, rt, 95% yield.
26 were determined and despite encouraging microsomal stability
Me
Me
(Table 3), only 26 had CL < 2 L/h/kg along with acceptable oral bio-
availability (Table 4). The pMCM2 potency for 21–23 and 26 were
within 3-fold of one another, and the cellular viability in HCT-116
cells21 was within 6-fold, therefore these parameters were not sig-
nificant differentiators. All four compounds were dosed orally in a
mouse pharmacodynamic (PD) model21 at 100 mg/kg. The levels of
pMCM2 and associated free drug concentrations were measured at
6 and 16 h. All four compounds lowered pMCM2 at 6 and 16 h rel-
ative to the vehicle control group. Both 23 and 26 sustained greater
levels of pMCM2 reduction and unbound plasma concentrations at
16 h than 21 and 22.25
N
N
NH
NH
N
N
F
F
O
O
Rat hepatocytes
16
16a
GSH
HN
HN
N
N
Major Metabolite
Figure 2. Rat hepatocyte and trapping results with 16.
The synthesis of 26 is shown in Scheme 1. A similar route was
used to prepare the analogs in Tables 1–3. Commercially available
5-bromoazaindole 27 was converted to fluoro intermediate 28 via
a three-step protection-lithiation-fluorination sequence. Acylation
of 28 with trichloroacetyl chloride and AlCl3 provided 29 which
therefore, these were evaluated in additional in vitro and in vivo
studies.
The addition of a nitrogen atom increased the solubility of ana-
logs 22, 23, and 26 relative to 21 (Table 4). The rat PK of 21–23 and
Table 3
SAR of a-cyclopropyl azaindole analogs
Compound
A
B
X
Y
Cdc7 IC50
(l
M)a
pMCM2 IC50
(l
M)a CDK2 IC50
(l
M)a CDK1 IC50
(l
M)a CDK9 IC50
(l
M)a HLM/RLM CLint L/min/mg)c
(l
21
22
23
24
25
26
CH CH CH CH 0.00085 0.0007 0.020 0.009
0.24 0.06
0.49 0.03
1.9 0.4
1.6 0.6
7.3b 0.5
>50c
0.031 0.02
0.12 0.08
0.94 0.8
0.88 0.8
2.4 0.5
59/63
21/80
27/45
<14/34
<14/413
<14/20
N
CH
CH CH CH 0.00069 0.0004 0.051 0.01
CH CH 0.0018 0.0009 0.060 0.03
N
CH CH CH
N
CH
0.0015 0.0005
0.0039 0.002
0.0011 0.0005 0.032 0.01
0.13 0.05
0.25 0.2
0.75 0.5
>50c
N
N
CH CF
CH CH CF
4.0
3
>50c
0.67 0.2
6.6b
1
0.46 0.3
a
b
c
Unless indicated otherwise, data represent an average of at least three determinations.
Data is the result of two determinations.
Data is the result of one determination SD.

6400
P. E. Harrington et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6396–6400
Table 4
In vivo PK and PD data for 21-23 and 26.
Compound SIF Solubility
(mg/mL)
Rat PK
Cell Viability
IC50 (lM)
Mouse PK/PD 100 mg/kgb
a
IV/PO Dose
(mg/kg)
CL (L/h/ MRT
Vss (L/
kg)
%F
% pMCM2 Remaining
at 6/16 hd
Unbound Plasma Concentration at
6/16 hc
M)
kg)
(h)
(l
21
22
23
26
0.005
0.20
0.20
0.16
2/5
1/5
2/2
1/5
2.0
4.5
4.5
1.7
2.7
1.3
1.4
1.9
5.3
5.9
6.1
3.3
35 0.88
41 1.8
14 5.7
50 2.4
38/46
40/41
62/24
46/30
0.15/0.06
1.3/0.07
2.0/0.45
0.61/0.30
a
b
c
HCT-116 cells, 4 d.
NCR/nude female mice, n = 3 animals,% pMCM2 remaining is relative to vehicle control group.
Calculated from total concentration and percent free fraction in mouse plasma.
All values were statistically significant (p < 0.05) by one-way analysis of variance (ANOVA) followed by Dunnett’s test.
d
Orsini, P.; Orzi, F.; Pesenti, E.; Pezzetta, D.; Pillan, A.; Poggesi, I.; Roletto, F.;
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Santocanale, C.; Vanotti, E. J. Med. Chem. 2009, 52, 293.
was converted to acyl hydrazine 30 in 80% overall yield. Acyl
hydrazine 30 was converted to the key intermediate 31 with CDI
in modest yield. The oxadiazole ring in 26 was formed by BOP-
mediated coupling of 35 with 31.26 Amine 35 was prepared as
shown in Scheme 1 using the Ellman sulfinamide chemistry.27,28
Aldehyde 32 was condensed with the S enantiomer of the tert-
butanesulfinamide to give 33 in excellent yield. Addition of cyclo-
propylmagnesium bromide gave a 7:3 ratio of diastereomers and
isomer 34 was isolated in 68% yield. The sulfinamide was then
cleaved with HCl to give 35.
In summary, we have extensively optimized a series of ami-
nooxadiazoles for potency, selectivity, and in vivo PK starting from
lead isoquinoline 1. The isoquinoline hinge binder in 1 was re-
placed with an azaindole which improved microsomal stability
without compromising the in vitro potency and selectivity profile.
Modifying the benzylic amine portion enhanced the in vitro po-
tency and selectivity. Trapping experiments with glutathione in
rat hepatocytes provided insight for structural modifications with-
in the azaindole to potentially improve metabolic stability. Azain-
dole 26 (Cdc7 IC50 = 1.1 nM, pMCM2 IC50 = 32 nM) demonstrated
robust lowering of pMCM2 at both 6 and 16 h. Additional details
of the in vivo efficacy of 26 will be reported in a future publication.
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Acknowledgements
The authors wish to thank Andrew Tasker for assistance in
manuscript preparation.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.09.
055.
References and notes
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25. The PD effect for 23 and 26 was greater at 16 h than 6 h despite lower
concentrations at 16 h. This is consistent with the cell cycle dependency of the
target. Inhibition of MCM2 phosphorylation is time-dependent and cells must
cycle through S phase for Cdc7 to be inhibited.
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