Discovery of XL413, a potent and selective CDC7 inhibitor

Article abstract of DOI:10.1016/j.bmcl.2012.04.024

CDC7 is a serine/threonine kinase that has been shown to be required for the initiation and maintenance of DNA replication. Up-regulation of CDC7 is detected in multiple tumor cell lines, with inhibition of CDC7 resulting in cell cycle arrest. In this paper, we disclose the discovery of a potent and selective CDC7 inhibitor, XL413 (14), which was advanced into Phase 1 clinical trials. Starting from advanced lead 3, described in a preceding communication, we optimized the CDC7 potency and selectivity to demonstrate in vitro CDC7 dependent cell cycle arrest and in vivo tumor growth inhibition in a Colo-205 xenograft model.

Full text of DOI:10.1016/j.bmcl.2012.04.024

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3727–3731
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of XL413, a potent and selective CDC7 inhibitor
⇑
Elena S. Koltun , Amy Lew Tsuhako, David S. Brown, Naing Aay, Arlyn Arcalas, Vicky Chan,
Hongwang Du, Stefan Engst, Kim Ferguson, Maurizio Franzini, Adam Galan, Charles R. Holst, Ping Huang,
Brian Kane, Moon H. Kim, Jia Li, David Markby, Manisha Mohan, Kevin Noson, Arthur Plonowski,
Steven J. Richards, Scott Robertson, Kenneth Shaw, Gordon Stott, Thomas J. Stout, Jenny Young,
Peiwen Yu, Cristiana A. Zaharia, Wentao Zhang, Peiwen Zhou, John M. Nuss, Wei Xu, Patrick C. Kearney
Exelixis, Department of Drug Discovery, 210 East Grand, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 16 April 2012
CDC7 is a serine/threonine kinase that has been shown to be required for the initiation and maintenance
of DNA replication. Up-regulation of CDC7 is detected in multiple tumor cell lines, with inhibition of
CDC7 resulting in cell cycle arrest. In this paper, we disclose the discovery of a potent and selective
CDC7 inhibitor, XL413 (14), which was advanced into Phase 1 clinical trials. Starting from advanced lead
3, described in a preceding communication, we optimized the CDC7 potency and selectivity to demon-
strate in vitro CDC7 dependent cell cycle arrest and in vivo tumor growth inhibition in a Colo-205 xeno-
graft model.
Keywords:
CDC7 kinase inhibitor
CK2 inhibitors
Benzofuropyrimidinone
Cell cycle arrest
Ó 2012 Elsevier Ltd. All rights reserved.
CDC7 is a serine/threonine kinase that plays a critical role in the
initiation of DNA synthesis and in S phase cell cycle check point
control.1 Human CDC7 phosphorylates the mini-chromosome
maintenance protein (MCM2) leading to the unwinding of dou-
ble-stranded DNA during the G1/S transition.2 CDC7’s role in
DNA unwinding is essential for DNA replication, and subsequent
cell proliferation. In fact, inhibition of CDC7 in tumor cells with
small molecules or small interfering RNAs results in defective S
phase progression that leads to a halt in cell cycle progression
and subsequent p53-independent apoptotic cell death.3 These re-
sults, coupled with the fact that upregulation of CDC7 has been ob-
served in numerous tumor cell lines4 (colon, lung, ovary, breast,
leukemia, and prostate), make CDC7 an attractive target for cancer
therapy. Several research groups have reported potent CDC7 inhib-
itors for cancer therapy.5
1 had a similar spatial layout as (4-hydroxy-3-methylphenyl)pyri-
midinone in compound 2. Compared to 1, compound 2 had an
additional phenyl group attached to pyrimidinone ring on the
right-had side. In the preceding communication,6 we disclosed
highlights of our PIM1 program, where we combined structures 1
and 2 into a chimeric structure. Compound 3 was one of the com-
pounds that resulted from that effort; it was a potent inhibitor for
all three kinases: PIM1 (IC50 = 11 nM), CK2 (IC50 = 28 nM), CDC7
(IC50 = 7.7 nM) and had favorable ADME/PK property (e.g., mouse
PK exposures 100 mg/kg; 101 lM, 1 h; 49 lM, 4 h). Compound 3
was also shown to be efficacious in multiple tumor xenograft mod-
els, although it was not well tolerated at high doses (data not
shown). Having established that an inhibitor of three kinases
(PIM1, CK2, and CDC7 all valid cancer targets individually) with
good PK exposure has a significant in vivo efficacy, we focused
our effort on creating a selective and more cell potent CDC7 inhib-
itor. The goals were to demonstrate whether CDC7 inhibition alone
can inhibit or reverse tumor growth, and if a selective compound
would be better tolerated.
Compound 1, 2, and 3 all contributed to provide the direction
for our investigation (Fig. 1). Compounds 1 has been identified as
a result of HTS effort for multiple kinase targets. It possessed mod-
erate inhibitory potency for three kinases: CDC7 (IC50 = 2.0
lM),
PIM1 (IC50 = 0.53 M), and CK2 (IC50 = 1.5 M). Compound 2 was
l
l
Since access to a crystal structure of CDC7 was not feasible, a
homology model7 was built based on an in-house high resolution
co-crystal8 structure data of CK2 protein with 3 (Fig. 2). CK2 is
structurally closest analog of CDC7.5d We also hypothesized that
the two proteins were similar in ATP binding region due to parallel
SAR observed in structurally related compounds. Multiple BFP ana-
logs are potent CK2 inhibitors, which made co-crystallization with
CK2 very accessible. The orientation of the BFP core in 3 docked
(gray) in the CDC7 model is only slightly different from that bound
(blue) in the CK2 crystal structure. Among the residues that are
a potent lead compound in our CK2 inhibitor program which also
had CDC7 activity (CK2 IC50 = 19 nM, CDC7 IC50 = 1.4 nM, PIM
IC50 = 332 nM) but had poor plasma exposure in mouse PK
(100 mg/kg, po: 0.82 lM, 1 h; 0.31 lM, 4 h). It was noted that the
fused tricyclic benzofuropyrimidinone (BFP) system in compound
⇑
Corresponding author at present address: Numerate, Inc., 1150 Bayhill Drive,
San Bruno, CA 94066, USA. Tel.: +1 6507032244, +1 (650) 472 0632.
E-mail address: elena@numerate.com (E.S. Koltun).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.024

3728
E. S. Koltun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3727–3731
O
MeO
OH
MeO
OH
MeO
Br
OH
CN
O
N
a
b
c
O
N
NH
NH
CHO
Br
O
CHO
Me
HO
4
5
O
O
Br
MeO
Br
O
MeO
e,f
2
1
NH2
d
NH2
NH2
O
O
CN
Br
OH
7
6
O
NH
O
N
OH
O
N
NH2
NH
g,h
HO
O
OH
NH
MeO
3
N
Br
N
N
O
Br
Figure 1. HTS hit 1, PIM1 inhibitor 2, and crossover compound 3.
Br
8
9g
Scheme 1. Reagents and conditions: (a) Br2, AcOH; (b) NH2OH, followed by POCl3;
(c) ClCH2CONH2, K2CO3, DMF, 80 °C, 85%; (d) KOH, DMF, 85 °C, 95%; (e) ClCH2COCl,
DCE, 65 °C, 85%; (f) (S)-3-hydroxypyrrolidine, EtOH, 85 °C, 90%; (g) 1 M NaOH, EtOH,
85 °C, 90%; (h) BBr3, CH2Cl2.
different, Pro135 (CDC7) versus Glu114 (CK2) in the kinase hinge
motif is the most interesting. A conformational constraint imposed
by the Pro135 backbone forces Met134 (the gatekeeper) to be posi-
tioned closer to the compound’s binding site in CDC7. The phenyl
portion of BFP moiety resides near the hinge motif, although the
bromine is unlikely to make any specific interactions. The oxygen
of the pyrimidinone forms a hydrogen bond (1.9 Å zmCK2) with
the Lys of the conserved (Lys-Glu) salt bridge. The hydroxyl pyrrol-
idine side chain is positioned towards the G-loop and the hydroxy
moiety makes a hydrogen bond (1.6 Å zmCK2) with Asn (Asn182 in
CDC7, Asn161 in CK2). The comparison of the docked (CDC7) and
bound (zmCK2) lead compound 3, reveals few more different res-
idues around the pyrrolidine binding region, Ser181 in CDC7 is dif-
ferent from His160 in CK2, and G-loop Glu66 in CDC7 is replaced
by Arg47 in CK2.
We started by exploring the SAR on the left-hand side of the BFP
core.9 Synthesis of bis-substituted compound 9g is shown in
Scheme 1. 2-Hydroxy-4-methoxybenzonitrile was brominated to
yield aldehyde 4. The resulting bromo-aldehyde was converted to
the corresponding bromo-nitrile 5 by reaction with hydroxylamine
followed by POCl3. Nitrile 5 was alkylated with chloroacetamide
under basic conditions to give amide 6, which underwent intramo-
lecular cyclization to yield benzofuran 7. Acylation with chloroace-
tyl chloride, followed by nucleophilic substitution with
(S)-3-hydroxypyrrolidine gave bi-cyclic amide 8. Cyclization under
basic conditions to form the BFP core, followed by deprotection of
the phenol with BBr3 gave the desired analog 9g. Compounds 3,
9a–f, 9h–i, and 10a–f were prepared via similar methods using
either commercially available nitriles or the corresponding alde-
hydes. In the case of bis-amine derivatives 10b and 10c, N0-Boc-
protected amines were used, and the parent compounds were ob-
tained by deprotection with 4N HCl in dioxane.9
In general, only few changes to the 8-bromo substituted BFP
core were tolerated (Table 1). Placing small substituents in the 6-
and 9-positions resulted in a loss of CDC7 activity (9d, 9e). The
7- and 8-positions were more amendable to substitutions, as ana-
logs with a chlorine (9b, IC50 = 2.2 nM) and a methoxy group (9c,
IC50 = 9.0 nM) in the 8-position were equipotent to lead compound
3, suggesting that a chlorine or a methoxy group could be a good
alternative to the bromine in the 8-position. 7-Hydroxy compound
9g resulted in an improvement in potency compared to the lead
compound 3. We used CK2 as a surrogate, and obtained an X-ray
structure with bound 9g. We postulated that the improvement in
potency came as a result of an interaction of the 7-hydroxy with
the backbone C@O of the Pro135 in the hinge region of CDC7
(Fig. 2). However, compound 9g did not show good exposure in a
mouse PK study (100 mg/kg (PO): 1.4 lM at 1 h, 0.15 lM at 4 h).
Docking studies showed that the left-hand side of the molecule
Table 1
SAR study in the right-hand portion of BFP10
O
6
O
OH
NH
7
N
N
8
9
R in position
8
CDC7
6
7
9
IC50 (nM)
3
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
OH
Me
OMe
Br
H
Cl
OMe
Br
Br
H
H
H
H
H
7.7
>3000
2.2
9a
9b
9c
9d
9e
9f
9g
9h
9i
9.0
1350
>3000
14
1.0
104
OMe
–OCH2O–
Figure 2. CDC7 homology model (gray) built using the zmCK2 X-ray crystal
structure (blue) as a template (PDB code 4ANM). Compound 3 (gray) docked in the
CDC7 model (gray) overlaid with 3 bound (blue) in the zmCK2 crystal structure
(blue).
Br
Br
Br
H
H
H
>3000

E. S. Koltun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3727–3731
3729
is packed tightly against the hinge region of the protein. This was
O
O
O
N
confirmed when 9i was synthesized, and found to be inactive.
Next, we turned our attention towards the modifications in the
right-hand side hydroxyl-pyrrolidine, taking advantage of the exis-
tence of non-conserved acidic residues in this region. First, we
investigated the effect of substituted N-linked pyrrolidines on
CDC7 activity and selectivity against PIM1 and CK2. We examined
the CDC7 potency and selectivity of BFP analogs made for the pre-
viously described PIM program,6 and prepared over 40 additional
analogs with the 8-bromo substitution on the left hand side. A se-
lect set of these analogs is presented in Table 2. Although some of
these analogs showed single digit nM activity against CDC7, we
were unable to find at least 10-fold selectivity among compounds
with N-linked pyrrolidine derivatives 10. Consequently, we ex-
panded our search to C-linked heterocycles and acyclic substituent
containing a basic amino group, as replacements for the hydroxyl-
pyrrolidine.
O
O
O
NH2
NH2
NH2
NH
NH
a
b,c
N
O
Br
Br
Br
R
N
Boc
11
12
13c, R = H
d
13g, R= Me
Scheme 2. Reagents and conditions: (a) N-Boc-proline, cyanuric chloride, DMA,
0 °C ? rt, 90%; (b) 4NHCl, dioxane, ꢀ100%; (c) NaOH, EtOH, 85 °C, 95%; (d) (CH2O)n,
Na(CN)BH3, DMA.
13f, and 13i) also emerged with enhanced selectivity over PIM
and CK2 (Table 3). Despite potent biochemical activity for 13f, this
compound only showed modest cellular potency (pMCM2
IC50 = 874 nM). In contrast, both 13b and 13i demonstrated cell
The synthesis of C-linked analogs 13b and 13g is shown in
Scheme 2. The 8-bromobenzofuran intermediate 11 was prepared
via methods described in Scheme 1, and coupled with activated N-
Boc-proline to yield intermediate 12. Boc deprotection with 4N HCl
in dioxane, followed by intramolecular cyclization under basic con-
ditions gave BFP 13c. Compound 13g was prepared by reductive
amination of compound 13c. Compounds 13a, 13c–f, 13h–j, and
14–17 were prepared in a similar fashion using common interme-
diate 11 and commercially available N-Boc-protected aminoacids.9
While a majority of the C-linked cyclic and acyclic analogs
showed potent activity against CDC7, several compounds (13b,
Table 3
C-linked Analogs10,11
O
O
NH
N
Br
R
Table 2
N-linked pyrrolidine analogs10,11
O
O
NH
N
R
Br
R =
IC50 (nM)
CDC7
CK2
PIM1
7.7
28
11
OH
3
N
N
N
pMCM2
1471
CDC7
CK2
PIM1
5.5
89
13
OH
10a
10b
pMCM2
1805
CDC7
CK2
PIM1
10
70
3.8
NH2
NH2
pMCM2
625
CDC7
CK2
207
535
10c
10d
10e
PIM1
pMCM2
6.0
—
N
N
CDC7
CK2
PIM1
3.7
92
13
pMCM2
353
CDC7
CK2
PIM1
11
512
77
362
OMe
N
pMCM2
CDC7
CK2
PIM1
13
49
142
CO2H
(rac)-
10f
N
pMCM2
>40,000

3730
E. S. Koltun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3727–3731
potencies below 100 nM in the pMCM2 cell-based assay, and thus
were considered as optimal substituents for the right hand side. In
addition to the improved selectivity versus PIM1 and CK2, these
two analogs also demonstrated good plasma exposures in mouse
PK studies dosed at 100 mg/kg (PO) (13b: 195
4 h; 13i: 82 M, 1 h; 46 M, 4 h).
lM, 1 h; 71 lM,
l
l
With the right-hand side optimized, we went back to survey the
left-hand side with the right fixed as either the (S)-pyrrolidin-2-yl-
or the (2S,4R)-4-fluoropyrrolidin-2-yl- substituted (Table 4). We
found that replacement of the 8-bromo with either the 8-chloro
or the 8-methoxy group resulted in further improvements to the
selectivity profile in both series, especially against CK2. The 8-
methoxy substituted analogs (16 and 17) were less potent in cell
assay than the 8-bromo analogs (13b and 13i). In addition, the 8-
chloro analogs (14 and 15), were more selective for CDC7. Overall,
the replacement of bromine in the 8-position of BFP core with
chlorine, and (S)-pyrrolidin-2-yl as a right hand side substituent
in 14 led to the best combination in terms of selectivity profile
(>60-fold selectivity against CK2, >10-fold selectivity against PIM,
and >300-fold selectivity against a panel of over 100 protein ki-
nases) and improved cell based potency (pMCM2 MDA-MB-231T
IC50 = 118 nM, and Colo-205 IC50 = 140 nM). Compound 14 demon-
strated excellent plasma exposures in mice [100 mg/kg (PO):
Figure 3. Flow cytometry analysis of Colo-205 cells treated with 14 (XL413). Colo-
205 cells were treated for 24 h with 1.1, 3.3, or 10
for DNA content using propidium iodide.
lM of compound 14 and analyzed
anchorage-independent growth of colo-205 in soft agar
(IC50 = 715 nM). Collectively, the cell data is consistent with the lit-
erature evidence that inhibiting CDC7 results in modified S phase
progression that subsequently leads to apoptotic cell death.3
Compound 14 demonstrated an excellent exposure profile in
full rat PK assay (dosed at 3 mg/kg: Cmax (PO) = 8.61
lM, AUC
141 lM (1 h), 81 lM (4 h)], and possessed the best PK properties
(po) = 75 M h, CL = 117 mL/h kg, Vss = 0.55 L/kg, T1/2 = 2.32 h,
l
among compounds in Table 4. Overall, compound 14 met our tar-
get property profile and was selected for further characterization.
The biochemical potency of 14 translates into inhibition of
CDC7 specific phosphorylation of MCM2 in cells cell lines that over
express CDC7 and phosphorylated MCM2 (data shown for MDA-
MB-231T and Colo-205 cell lines). In addition, flow cytometry
analysis of Colo-205 cells treated with 14 causes dose-dependent
accumulation of cells in the late S and G2 phases of the cell cycle,
consistent with impaired DNA synthesis (Figure ure3). Similar re-
sults were found in 27 of 31 tumor cell lines that were tested (re-
sults not shown). Prolonged treatment with compound 14 (3 days)
inhibited the cell proliferation (IC50 = 2685 nM), decreased cell via-
bility (IC50 = 2142 nM) and elicited the caspase 3/7 activity
(EC50 = 2288 nM) in Colo-205 cells.11 Profound proliferation reduc-
tion was also observed in a variety of tumor lines tested (data not
shown). In addition, compound 14 also significantly inhibited the
F = 95%). This translated into robust activity in in vivo pharmaco-
dynamic studies. In a Colo-205 xenograft model, dose dependent
target modulation was observed (Fig. 4); and a 70% inhibition of
phosphorylated MCM2 was detected even at the 3 mg/kg dose.
The ED50 is calculated to be <3 mg/kg.
Multiple-dose studies of 14 in a Colo-205 xenograft model dem-
onstrates significant anti-tumor efficacy. Tumor bearing mice were
administered 14 orally at doses of 10, 30, or 100 mg/kg once daily
(qd) for 14 days (Fig. 5). Two alternate dosing regimens were also
examined in this study: a dose of 30 mg/kg administered twice-
daily (bid) and a dose of 100 mg/kg administered every-other
day (q2d). Compound 14 was well tolerated at all the doses and
regimens examined, with no significant body weight loss observed.
Only modest tumor growth inhibition (36%) was observed for the
10 mg/kg qd dosage, but significant tumor growth inhibition
(83%) was observed at the 30 mg/kg qd dose. More impressively,
significant tumor growth regression (32%) was observed if dosed
twice-daily at 30 mg/kg. The ED50 is estimated at 13 mg/kg.
In this paper we report the discovery of XL413 (compound 14),
a potent and selective ATP competitive CDC7 inhibitor. XL413 is a
novel benzofuropyrimidinone CDC7 inhibitor with a very favorable
pharmacokinetic profile and demonstrates significant tumor
growth regression in rodent models. XL413’s tumor growth regres-
sion is linked to the arrest of tumor cells in the late S to G2 phase of
Table 4
The replacement of bromine in 13b and 13i10,11
O
O
O
O
NH
NH
N
N
R
R
A
B
HN
HN
F
Vehicle
Compound 14 (XL413), mg/kg
3
10
30
100
pMCM2 S40/41
MCM2
Actin
pMCM2 inh. %
plasma conc. µM
tumor conc. µM
-
-
-
70
11.0
3.2
77
87
86
40.1
13.1
82.2
32.1
186.0
71.4
Figure 4. Pharmacodynamic dose response of 14 (XL413) in a Colo-205 xenograft
model. Athymic nude mice bearing Colo-205 xenograft tumors were dosed orally
with 3, 10, 30, and 100 mg/kg of 14. Plasma and tissue samples were taken at 4 h
post-dose and analyzed for levels of phosphorylated MCM2 and MCM2 by Western
immunoblot analysis.
Data shown for pMCM in MDA-MB-231T cell line.

E. S. Koltun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3727–3731
3731
4. Bonte, D.; Lindvall, C.; Liu, H.; Dykema, K.; Furge, K.; Weinreich, M. Neoplasia
2008, 10, 920.
600
500
400
300
200
100
0
Vehicle:1, 10 ml/kg, PO bid
3 mg/kg, PO qd
5. (a) 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. 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. Med. Chem.
2010, 53, 7296; (b) Menichincheri, M.; Bargiotti, A.; Berthelsen, J.; Bertrand, J.
A.; Bossi, R.; Ciavolella, A.; Cirla, A.; Cristiani, C.; Croci, V.; D’Alessio, R.; Fasolini,
M.; Fiorentini, F.; Forte, B.; Isacchi, A.; Martina, K.; Molinari, A.; Montagnoli, A.;
Orsini, P.; Orzi, F.; Pesenti, E.; Pezzetta, D.; Pillan, A.; Poggesi, I.; Roletto, F.;
Scolaro, A.; Tato, M.; Tibolla, M.; Valsasina, B.; Varasi, M.; Volpi, D.;
Santocanale, C.; Vanotti, E. J. Med. Chem. 2009, 52, 293; (c) Zhao, C.; Tovar, C.;
Yin, X.; Xu, Q.; Todorov, I. T.; Vassilev, L. T.; Chen, L. Bioorg. Med. Chem. Lett.
2009, 19, 319; (d) Vanotti, E.; Amici, R.; Bargiotti, A.; Barthelsen, J.; Bosotti, R.;
Ciavolella, A.; Cirla, A.; Cristiani, C.; D’Alessio, R.; Forte, B.; Isacchi, A.; Martina,
K.; Molinari, A.; Menichincheri, M.; Montagnoli, A.; Orsini, P.; Pillan, A.; Roletto,
F.; Scolaro, A.; Tibolla, M.; Valsasina, B.; Varasi, M.; Volpi, D.; Santocanale, C. J.
Med. Chem. 2008, 51, 486; (e) Shafer, C. M.; Lindvall, M.; Bellamacina, C.;
Gesner, T. G.; Yabannavar, A.; Jia, W.; Lin, S.; Walter, A. Bioorg. Med. Chem. Lett.
2008, 18, 4482.
6. Tsuhako, A.L.; Brown, D.S.; Koltun, E.S.; Aay, A.; Arcalas, A.; Chan, V.; Du, H.;
Engst, S.; Franzini, F.; Galan, A.; Huang, P.; Johnston, S.; Kane, B.; Kim, M.H.;
Stott, G.; Stout, T.J.; Yu, P.;Zaharia, Z.A.; Zhang, W.; Zhou, P.; Nuss, J.M.;
Kearney, P.C.; Xu, W. Bioorg. Med. Chem. Lett. 2012. http://dx.doi.org/10.1016/
j.bmcl.2012.04.025.
7. The CDC7 homology model was built using MOE 2009 (Chemical Computing
Group, Inc.).
8. (a) Niefind, K.; Guerra, B.; Ermakowa, I.; Issinger, O. EMBO J. 2001, 20, 5320; (b)
Niefind, K.; Guerra, B.; Pinna, L.; Issinger, O.; Schomburg, D. EMBO J 1998, 17,
2451. The X-ray crystal structure coordinates of 3 in CK2 have been deposited
in the Protein Data Bank (PDB code 4ANM).
10 mg/kg, PO qd
30 mg/kg, PO qd
100 mg/kg, PO qd
30 mg/kg, PO bid
100 mg/kg, PO q2d
0
5
10
15
20
25
Days Post Implantation
Regression BW loss
Dose
skips
TGI (%)
(%)
(%)
-0.5
3 mg/kg qd
10 mg/kg qd
30 mg/kg qd
100 mg/kg qd
30 mg/kg bid
100 mg/kg q2d
8.7 (ns)
36 (ns)
83
1
1
5
7
6
5
-3.4
-8.3
-4.7
-6.4
-4.5
>100
>100
>100
70
32
21
9. Detailed experimentals for this manuscript can be found in the following
patent: Brown, S. D.; Du, H.; Franzini, M.; Galan, A. A.; Huang, P.; Kearney, P.C.;
Kim, M. H.; Koltun, E. S.; Richards, S. J.; Tsuhako, A.L.; Zaharia, C.A. WO
2009086264 A1 2009.
Figure 5. Colo-205 nude mouse xenograft study with 14 (XL413).
10. Biochemical assay: N-terminally Myc-tagged human CDC7 (amino acids: E2–
L574) and N-terminally His-tagged human ASK (N2–F674) were co-expressed
the cell cycle, a cell phenotype indicative of CDC7 inhibition. In
addition, XL413 is relatively CYP clean (CYP3A4, 2C9, 2D6, 2C19
in E. coli and purified using nickel affinity chromatography. Human CK2
a
isoform A (R8–R333) and full-length b subunits were expressed separately as
N-terminally MBP-tagged proteins in E. coli and purified using amylose
Sepharose chromatography. The purified subunits were reconstituted to form
IC50 >50
l
M, CYP1A2 IC50 = 6.9
l
M) among the major isoforms
M). The attrac-
tested; and was inactive against hERG (IC50 > 30
l
the tetrameric
a2b2 CK2 holoenzyme. Human PIM1 (E32–D292) was
tive profile of XL413 resulted in its selection for preclinical devel-
opment and subsequent advancement into Phase 1 clinical trials.
expressed as N-terminally His-tagged proteins in E. coli and purified using
nickel affinity chromatography. Protein concentration was determined by the
Bradford assay and identification was confirmed by trypsin digestion and mass
spectrometry. Kinase activity and compound inhibition were determined using
the luciferase-luciferin-coupled chemiluminescence assay and measured as
the percentage of ATP utilized following the kinase reaction in a 384-well
format as described previously The final CDC7 kinase assay condition was 6 nM
CDC7/ASK, 1 lM ATP, 50 mM Hepes pH 7.4, 10 mM MgCl2, 0.02% BSA, 0.02%
brij 35, 0.02% tween 20 and 1 mM DTT. It is worthy to note that the CDC7/ASK
protein exhibited substrate-independent ATP utilization. The final PIM1 kinase
Acknowledgments
The authors greatly appreciate contributions from Jason Chew,
Leanne Goon, Stuart Johnson, Eun Ok Kim, Iris Ngan, Yongchang
Shi, Scott Detmer, Richard Venture, Rui Lin, Nicole Miller, Tim Heu-
er, Douglas Den Otter and the departments of Exelixis Genome Bio-
chemistry, New Lead Discovery, Cell facility, and Compound
Repository.
assay condition was 2.4 nM PIM1, 0.5
(AKRRRLSA), 20 mM Hepes pH 7.4, 10 mM MgCl2, 0.03% Triton, and 1 mM DTT.
The final CK2 kinase assay condition was 4 nM CK2 holoenzyme, 2 M ATP,
M casein, 20 mM Hepes pH 7.5 10 mM MgCl2, 0.03% Triton, 1 mM DTT and
lM ATP, 10 lM peptide substrate
l
2
l
0.1 mM NaVO3. All kinase reactions were incubated at room temperature for
1–2 h. Munagala, N. Nguyen S., Lam W., Lee J., Joly A., McMillan K. and Zhang
W. Assay Drug Dev Technol., 2007, 5, 65–73.
References and notes
11. Cell
assays:
Endogenous
MCM2
phosphorylation
fixed
cell
1. For general reviews see: (a) Sawa, M.; Masai, H. Drug Des. Dev. Ther. 2008, 2,
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13, 2111; (d) Snaith, H. A.; Brown, G. W.; Forburg, S. L. Mol. Cell Biol. 2000, 20,
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Mol. Biol. Cell 2001, 12, 1257; (f) Fung, A. D.; Ou, J.; Bueler, S.; Brown, G. W. Mol.
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2770; (i) Montagnoli, A.; Moll, J.; Colotta, F. Clin. Cancer Res. 2010, 16, 4503.
3. (a) Montagnoli, A.; Tenca, P.; Sola, F.; Carpani, D.; Brotherton, D.; Albanese, C.;
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immunofluorescence assay was conducted with MDA-MB-231T cells (from
ATCC) seeded at 1.0 ꢁ 104 cells/well onto 96-well plates in complete DMEM
containing 10% FBS. Cells were treated serial dilutions of test compound in 3%
DMSO and incubated for 4 h. Cells were then fixed for 20 min treated with
Triton X-100 in PBS for 5 min, followed by overnight incubation with pMCM2
(S53) antibody (#AN3011, custom produced for Exelixis by Anaspec, Inc.).
pMCM2 (S53) was read on a Cellomics Arrayscan after incubation with Alexa
Fluor 546 Goat anti-rabbit IgG (H+L) (Cat#A11010, Invitrogen). IC50 values
were determined based on pMCM2 (S53) intensity with compound treatment
versus pMCM2 (S53) intensity with DMSO treatment alone. The cell
proliferation was measured by BrdU incorporation assay, viability was
assayed by Cell Titer–Glo kits, and the apoptosis assay was measured by
Apo-ONE Homogeneous Caspase-3/7 Assay kit (Promega).