Development of thioquinazolinones, allosteric Chk1 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.12.076

A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by testing compounds at high concentration. Activity was then observed at K_m for ATP and at near-physiological concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazolinone series which was optimized for potency and stability. An X-ray crystal structure for the complex of our best inhibitor bound to Chk1 was solved, indicating that it binds to an allosteric site ～13 A from the ATP binding site. Preliminary data is presented for several of these compounds.

Full text of DOI:10.1016/j.bmcl.2008.12.076

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1240–1244
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of thioquinazolinones, allosteric Chk1 kinase inhibitors
a
a
a
b
Antonella Converso a, , Timothy Hartingh , Robert M. Garbaccio , Edward Tasber , Keith Rickert ,
Mark E. Fraley a, Youwei Yan c, Constantine Kreatsoulas a, Steve Stirdivant b, Bob Drakas b, Eileen S. Walsh b,
Kelly Hamilton b, Carolyn A. Buser b, Xianzhi Mao b, Marc T. Abrams b, Stephen C. Beck b, Weikang Tao b,
Rob Lobell b, Laura Sepp-Lorenzino b, Joan Zugay-Murphy c, Vinod Sardana c,
*
Sanjeev K. Munshi c, Sylvie Marie Jezequel-Sur d, Paul D. Zuck d, George D. Hartman a
a Department of Medicinal Chemistry, Merck Research Laboratories, Merck & Co., P.O. Box 4, West Point, PA 19486, USA
b Department of Cancer Research, Merck Research Laboratories, Merck & Co., P.O. Box 4, West Point, PA 19486, USA
c Department of Structural Biology, Merck Research Laboratories, Merck & Co., P.O. Box 4, West Point, PA 19486, USA
d Department of Automated Biotechnology, Merck Research Laboratories, Merck & Co., P.O. Box 4, West Point, PA 19486, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by
testing compounds at high concentration. Activity was then observed at Km for ATP and at near-physio-
logical concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazoli-
none series which was optimized for potency and stability. An X-ray crystal structure for the complex of
our best inhibitor bound to Chk1 was solved, indicating that it binds to an allosteric site ꢀ13 Å from the
ATP binding site. Preliminary data is presented for several of these compounds.
Received 15 October 2008
Revised 16 December 2008
Accepted 17 December 2008
Available online 24 December 2008
Keywords:
Chk1
Ó 2009 Elsevier Ltd. All rights reserved.
Thioquinazolinone
Allosteric kinase inhibitor
X-ray crystal structure
Despite decades of research towards the identification of an
anticancer drug devoid of serious side-effects, DNA damaging
agents are still one of the most relied upon cancer therapeutics
in the clinic due to their efficacy. Such agents often present a lim-
ited therapeutic window and/or poor selectivity for cancer cells
over normal cells. Thus, any improvement in their efficacy is extre-
mely valuable. Recently, it has been suggested that the inhibition
of checkpoint kinase Chk1 may offer such an opportunity.
Chk1 and the tumor suppressor protein p53 protect cells that
have suffered DNA damage by arresting the cell cycle to allow
DNA repair.1–3 Importantly, 50–70% of tumor cells have defects
in their p53 DNA damage response pathway and must rely exclu-
sively on Chk1 for DNA repair and cell survival. Inhibition of
Chk1 will therefore abrogate repair from DNA damage in p53
defective tumors, ultimately resulting in premature progression
into mitosis and apoptosis. Normal cells, on the other hand, can
still repair via p53-mediated arrest. As a result, Chk1 inhibitors
should sensitize p53-deficient cancer cells to DNA damaging
agents without enhancing toxicity toward non-malignant cells.
As such, Chk1 inhibitors4 have the potential to widen the therapeu-
tic window for DNA damaging agents in p53-deficient tumors.
Chk1 is one of 518 kinases encoded by the human genome.5 Ki-
nases are ubiquitous and important for a variety of physiological as
well as pathological cellular processes.6 They are characterized by
a highly conserved ATP-binding hinge region which is the tradi-
tional site targeted to block the activity of these enzymes. An alter-
native approach has involved the identification of inhibitors that
bind outside of the catalytic cleft at an allosteric site. These alloste-
ric inhibitors are non-ATP competitive and, due to the variability—
or absence—of the allosteric site, they may offer an advantage
when trying to modulate kinases selectively.
The Chk1-targeted program at Merck has been prevalently fo-
cused on the development of ATP competitive inhibitors7 and facil-
itated by known Chk1 structural data. However, fine tuning of
leads to exclude activity towards other kinases has proven difficult.
For this reason we prioritized the identification of allosteric leads
and developed a screening strategy that could be useful towards
the future discovery of non-ATP competitive kinase inhibitors. To
this end, a multi-step homogenous time-resolved fluorescence
(HTRF) assay suitable for high throughput screen was imple-
mented in 1536-well plate format.8
Under the assumption that allosteric leads may have low affinity,
we chose to screen at high compound concentration (50 lM). Hits
werethenevaluatedinthepresenceof ATPatKm andatphysiological
ATP concentration (0.1 and 2.0 mM) in order to eliminate ATP-com-
* Corresponding author. Tel.: +1 215 652 4856; fax: +1 215 652 7310.
E-mail address: antonella_converso@merck.com (A. Converso).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.076

A. Converso et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1240–1244
1241
Table 1
O
4
Chk1 inhibition data for thioquinazolinone amides 1–14
5
8
6
7
N
Cl
O
2
O
N
N
S
NH2
N
N
S
R4
N
1
O
R5
Figure 1. Chk1 inhibitor lead 1.
O
petitive compounds: the potency of allosteric inhibitors should be
insensitive to ATP concentration. A single lead, thioquinazolinone
1 (see Fig. 1), was identified from this effort with an IC50 of 17 and
Compound
NR4R5
SL Chk1 IC50 0.1 mM ATP (lM)
O
1
17
N
24
l
M at 0.1 and 2.0 mM ATP concentration, respectively.
NH2
Thioquinazolinone 1 constituted the starting point for our lead
optimization effort. Analogs of 1 were prepared using the general
reaction sequence shown in Scheme 1.9
Anthranilic acid 2 was reacted with aromatic and aliphatic iso-
thiocyanates to give mercapto-quinazolinones 3 in 80–95% yield.
Alkylation of the thiol in 3 with a variety of a-bromo acids afforded
4, which was then coupled with a variety of amines to yield thio-
6
7
Max inh. 62% @ 100
l
M
N
H
N
>100
>100
>100
8
quinazolinone amides 5.
N
H
Medicinal chemistry efforts began by varying the R4 and R5
groups on the terminal amide, which was conveniently introduced
at the end of the synthesis for this series. Analogues synthesized
demonstrated tight SAR (Table 1). It was generally found that a
fully substituted amide is necessary for potency. Ethyl esters were
inactive against Chk111 (data not shown), and so were primary
amides (compounds 7–10). Piperazines and piperidines enhanced
potency with compounds 11 and 13 providing the greatest potency
H
N
9
H
N
10
11
12
13
14
Max inh. 3% @100
lM
3.3
6.0
3.8
4.5
N
N
N
S
with IC50 of 3 lM. Diphenylpiperidine 14, while moderately potent,
showed poor solubility, and sulfonylamine 12 suffered from stabil-
O
O
ity issues. Neither was pursued further.
We then investigated the R3 group on the mercaptoquinazoli-
none carboxylic acid. To this end, the aromatic thiol was reacted
N
N
NH
with a variety of
a-bromo acids, followed by coupling to piperi-
dine-4-carboxamide (Table 2).
Ph
Ph
The aromatic analogue 16 derived from
a-bromophenylacetic
acid presented similar activity as the original lead giving an IC50
of 21 M. Branching at the b-position renders the corresponding
thioquinazolinone inactive (15), while -branching as in com-
l
c
pound 17 gave a sixfold improvement in potency. However, the
combination of the best amide in 13 with this R3 group did not lead
to further potency gains. Interestingly, we observed acid-instability
issues when we removed the R substituent altogether: the final
amide coupling appeared to be successful, but removal of the
acidic solvent mixture after reverse-phase HPLC separation gave
complete and clean decomposition to carboxylic acid 4 even at
room temperature.
We next turned our attention to the aromatic ring on the
thioquinazolinone core and the internal thioquinazolinone amide
substituent R2 introduced in the first synthetic step. Coupling of
anthranilic acid with a variety of isothiocyanates allows variation
of the R2 group as reported in Table 3. Again, SAR proved to be
Br
OH
O
O
N
R3
R1
R1
R2
EtOH
R2
O
OH
NH2
N
N C S
75 oC
Et3N, DMF
60 oC
40-90%
SH
2
3
5
80-95%
O
N
O
R1
R1
R2
S
R2
N
N
HNR4R5
R4
N
N
S
PyBOP, Et3N,
DMF, rt
OH
R3
R3
R5
4
50-92%
O
O
Scheme 1. Synthesis of thioquinazolinones. Detailed synthetic procedures can be found in Ref. 10.

1242
A. Converso et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1240–1244
Table 4
Table 2
Effect of R3 on Chk1 potency of thioquinazolinones
Effect of thioquinazolinone ring substitution on potency
O
O
N
N
Cl
O
N
Cl
O
R1
N
S
NH2
N
S
NH2
R3
N
O
O
Compound
R3
SL Chk1 IC50 0.1 mM ATP (
17
lM)
Compound
R1
SL Chk1 IC50 0.1 mM ATP (
lM)
1
26
27
28
29
30
31
5-Me
6-Me
7-Me
8-Me
8-OH
8-OMe
>100
Max inh. 30% @ 100 lM
15
16
17
>100
21
28
17
20
21
2.8
Substituents R1 on the aromatic region of the thioquinazolinone
were then examined (Table 4). Introduction of a methyl group at
positions 5 and 6 was not tolerated, while methyl substitution at
positions 7 and 8 had little to no effect on the IC50. A wide range
of different substituents at C7 and C8 (data not shown) was there-
fore studied, in the hopes of increasing potency. While potency de-
creased following substitution at C7, substitution at the 8-position
had virtually no effect.
While investigating the SAR for the series, we also focused on
addressing its stability issues. We observed thioquinazolinone
decomposition in three specific instances: (1) for certain amides;
(2) when lacking R3 substitution on the sulfur-containing side
chain, and (3) in the presence of electron rich R2 groups. In all cases
we obtained clean conversion to the starting carboxylic acid 4.
Examination of the experimental data led us to propose that the
N1 lone pair in the thioquinazolinone is involved in intramolecular
amide decomposition as depicted in Scheme 2.
Formation of charged intermediate 33 is consistent with our
observations for a couple of reasons. First, when R3 = H, the carbonyl
carbon is less hindered towards nucleophilic attack. Secondly, an al-
kyl rather than aryl R2 group renders the thioquinazolinone more
electron rich so that the N1 lone pair is more nucleophilic, resulting
in accelerated decomposition. In further support of the proposed
mechanism, we were able to trap 34 with isopropanol, which pro-
vided the corresponding isopropyl ester.
tight: the aromatic residue was necessary for activity, with all ali-
phatic thioquinazolinones synthesized showing low activity and
poor stability, rapidly decomposing to carboxylic acid 4 in the pres-
ence of diluted aqueous TFA (compounds 18–20). Substitution on
the aryl ring in R2 was then examined and a chlorine scan showed
that the starting meta position was optimal for activity (com-
pounds 21–22). Introduction of heterocycles either attached di-
rectly to the amide nitrogen (23 and 24) or separated by a
methylene unit (25) caused a loss in potency.
Table 3
Effect of R2 groups on Chk1 potency of thioquinazolinones
O
R2
N
O
N
S
NH2
N
O
Compound
R2
SL Chek1 IC50 0.1 mM ATP (lM)
18
Max inh. 17% @ 100
Max inh. 20% @ 100
Max inh. 48% @ 100
41
lM
lM
lM
Our previous work at C8 of the thioquinazolinone ring showed
that potency in this series is independent of C8 substitution. At the
19
20
21
1
R5
N H
R
O
N
O
Me
R2
S
R2
S
N
N
4
Cl
R4
N
N
5
R3
R3
R5
R
N
17
32
33
O
R4
Cl
Cl
O H
22
23
24
25
>100
O
N
O
R2
R2
N
N
Max inh. 35% @ 100
Max inh. 20% @ 100
Max inh. 26% @ 100
lM
lM
lM
N
S
N
S
N
O
R3
R3
O
34
35
Cl
O
RO
N
Scheme 2. Proposed mechanism for the decomposition of thioquinazolinones.

A. Converso et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1240–1244
1243
O
N
O
R2
S
R2
N
N
R4
N
N
S
OH
R3
R5
R3
O
O
36
37
Scheme 3. Effect of C8 substituent on the decomposition of 36.
O
N
Cl
N
S
N
N
H
O
38
Figure 2. Active diastereomer of thioquinazolinone mixture 13. IC50 = 1.3 lM.
same time, a substituent at C8 has the maximum impact on the
steric environment of the nucleophilic N1 nitrogen. If our hypoth-
esis for the observed decomposition is correct, shielding of the N1
lone pair by the introduction of a vicinal methyl should impart
robustness to the central core by preventing intramolecular nucle-
ophilic attack (Scheme 3). Our hypothesis was nicely confirmed by
stability studies performed on 36, the 8-Me analogue of compound
13, which, unlike 13 did not decompose in concentrated aqueous
TFA even after several days.
As part of our medicinal chemistry efforts, we separated 13
(IC50 = 3
the (S,S) diastereomer 38 (Fig. 2) binds to Chk1 with an IC50 of
1.3 M, with all the others being inactive in our assay.
lM) into its four diastereomers and we found that only
l
Figure 4. Two perspectives of 38 (gold) bound to Chk1. Direct hydrogen bonding
contacts to the carboxylic acid of Glu205 and the backbone amide nitrogen of
Leu206 are shown in white dashes. Water mediated hydrogen bond to Glu134 is
shown as a yellow dash. The p-chlorphenyl quinazolinone occupies a hydrophobic
pocket formed by Phe93, Ile96, Pro133, and Leu206, forming a p–p interaction.
Satisfyingly, we were also able to obtain a crystal structure of
diastereomerically pure 38 bound to a truncated version of
Chk113 (Fig. 3).
The structure clearly confirms 38 as an allosteric inhibitor,
binding ꢀ13 Å from ATP and its pocket in a shallow hydrophobic
region on the surface of the enzyme. The spiropiperidine amine
is engaged in two direct hydrogen bonding contacts with the car-
boxylic acid of Glu205 and the backbone amide nitrogen of
Leu206 (white dashes in Fig. 4). The thioquinazolinone carbonyl
oxygen participates in
a water mediated hydrogen bond to
Glu134 (yellow dash in Fig. 4).
The crystal structure is consistent with the medicinal chemistry
results obtained in our series. The aromatic ring at R2 slides into a
very narrow hydrophobic cleft that wraps it tightly. Serendipi-
tously, the initial meta-chloro substitution on R2 appears optimal
in terms of size and lipophilicity.
The lack of sensitivity to substitution at the C-8 position of the
thioquinazolinone can now be rationalized by the fact that 38
binds on the surface of the enzyme, and that the thioquinazolinone
aromatic core overlaps with a very hydrophobic region on it. Any
small substituent on the thioquinazolinone ring is out of reach of
hydrogen bond acceptors or donors on the enzyme that could sta-
bilize its interaction with 38.
Figure 3. Compound 38 (gold) bound to Chk1. ATP (semitransparent gray) from
1PHK12 used to orient the reader.

1244
A. Converso et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1240–1244
Steen, J. T.; Yang, F. PCT Int. Appl. WO 2006086255, 2006.; (d) Brnardic, E. J.;
As the crystal structure for 38 reveals, the thioquinazolinones
Fraley, M. E.; Garbaccio, R. M.; Steen, J. T. PCT Int. Appl. WO 2006074281, 2006.;
(e) Fraley, M. E.; Steen, J. T.; Brnardic, E. J.; Arrington, K. L.; Spencer, K. L.;
Hanney, B. A.; Kim, Y.; Hartman, G. D.; Stirdivant, S. M.; Drakas, B. A.; Rickert,
K.; Walsh, E. S.; Hamilton, K.; Buser, C. A.; Hardwick, J.; Tao, W.; Beck, S. C.;
Mao, X.; Lobell, R. B.; Sepp-Lorenzino, L.; Yan, Y.; Ikuta, M.; Munshi, S. K.; Kuo,
L. C.; Kreatsoulas, C. Bioorg. Med. Chem. Lett. 2006, 23, 6049; (f) Huang, S.;
Garbaccio, R. M.; Fraley, M. E.; Steen, J.; Kreatsoulas, C.; Hartman, G.; Stirdivant,
S.; Drakas, B.; Rickert, K.; Walsh, E.; Hamilton, K.; Buser, C. A.; Hardwick, J.;
Mao, X.; Abrams, M.; Beck, S.; Tao, W.; Lobell, R.; Sepp-Lorenzino, L.; Yan, Y.;
Ikuta, M.; Murphy, J.; Sardana, V.; Munshi, S.; Kuo, L.; Reilly, M.; Mahan, E.
Bioorg. Med. Chem. Lett. 2006, 22, 5907.
bind to the enzyme in a highly solvent-exposed fashion. The neces-
sary desolvation energy may not be well compensated by these
limited interactions with the enzyme and might explain the low
potencies shown by this series.
Thioquinazolinone 38 and its active analogues present a mark-
edly different selectivity profile compared to ATP-competitive
Chk1 inhibitors previously synthesized in our laboratories. A more
in-depth discussion of this aspect of binding, together with full bio-
chemical characterization of the complex inhibition mode of this
series, will be reported in a separate publication.
In summary, a series of thioquinazolinone allosteric Chk1 inhibi-
tors were discovered by an appropriately targeted HTS campaign
designed for the identification of weak, non-ATP competitive leads.
An efficient synthetic route has been developed to facilitate the SAR
studies. We identified stability issues with some members of this ser-
ies, a mechanism for the decomposition and a way to prevent it. Addi-
tional efforts have resulted in the solution of the first crystal structure
of an inhibitor bound to the allosteric site of the Chk1 enzyme.
8. In the first step, Chk1 enzyme was incubated with ATP and biotin-labeled
GSK3
anti-phospho-GSK3
and streptavidin (SA) linked to XL665 (SA-XL665). The Eu-Ab antibody
recognizes the phosphoserine residue of GSK3 peptide, whereas SA-XL665
binds to the biotin at the N-terminus of the peptide. This ternary complex
a
peptide. In the second step the sample was treated with monoclonal
a
peptide antibody labeled with europium (Eu3+) (Eu-Ab)
a
(biotin-phosphorylated GSK3a/Eu-Ab/SA-XL665) was then detected by
excitation of Eu3+ at 337 nm, fluorescence resonance energy transfer (FRET)
from Eu3+ to XL665, and fluorescence emission from XL665 at 665 nm. In this
assay, an inhibitor of Chk1 enzyme could be detected by
fluorescence.
a decrease of
9. Bhargava, P. R.; Ram, P. Bull Chem. Soc. Jpn. 1967, 38, 342.
10. Representative experimental procedure: 3-Alkyl-2-sulfanylquinazolin-4(3H)-
one 3: Anthranilic acid 2 was dissolved in EtOH (0.5 M) and heated to 75 °C.
The isothiocyanate (2 equiv) was added in 0.5 equiv portions over the course of
8 h until disappearance of anthranilic acid. The mixture was cooled, and the
slurry was filtered and washed with cold ethanol to afford pure 2-
Supplementary data
mercaptoquinazolin-4(3H)-one
sulfanylquinazolin-4(3H)-one 3,
3.
Thioquinazolinone
5:
3-Alkyl-2-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.12.076.
a-bromoacid (1.3 equiv) and triethylamine
(6 equiv) were dissolved in anhydrous DMF (0.5 M) and stirred at 75 °C until
LC–MS indicated disappearance of starting material to yield 3-alkyl-2-[(3-
methyl-4-oxo-3,4-dihydroquinazolin-2-yl)sulfanyl] acid 4. The reaction
mixture was cooled, and triethylamine (3 equiv), the amine (2 equiv), and
PyBOP (2 equiv) were added. Once LC–MS showed that the reaction was
complete, the reaction mixture was filtered and purified with reverse-phase
HPLC (H2O/CH3CN gradient w/0.1% TFA present) to yield pure product 5.
11. Chk1 inhibitory activity was measured using a homogenous time-resolved
fluorescence assay which measures phosphorylation of a biotinylated GSK-3
peptide as described in Barnett et al. Biochem. J. 2005, 385, 399. For the
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construct,
a naturally occurring exon 10 splice variant of human Chk1
described in patent application US20050266469(A1), containing primarily
the kinase domain, was expressed in baculovirus with a C-terminal 6-histidine
tag. This protein was purified on a Ni affinity column and used as is for kinetic
assays, or purified further on Heparin and SEC columns for crystallography. The
Chk1 concentration was 0.5 nM and ATP was used at 0.1 mM. IC50 values are
reported as the averages of at least two independent determinations; standard
deviations are within 25–50% of IC50 values.
12. Compound 38 was diffused into pre-formed apo Chk1 kinase domain crystals
by the soaking method. The X-ray diffraction data were collected from these
Chk1 inhibitor complex crystals to 1.9 Å, resolution with Rsym
= 0.066 and
completeness = 98%, respectively. The complex structures were refined to an R-
factor of 0.182. The chlorobenzene ring of compound 38 has two
conformations at 70% and 30% occupancies, respectively. The detailed X-ray
diffraction data and refinement statistics are listed under PDB code 3F9N at the
protein data bank. The crystallographic parameters are reported in the
supporting information for this article.
13. Owen, D. J.; Noble, M. E.; Garman, E. F.; Papageorgiou, A. C.; Johnson, L. N.
Structure 1995, 3, 467.