Discovery of a potent and selective protein kinase CK2 inhibitor by high-throughput docking

Article abstract of DOI:10.1021/jm030827e

To assess the potential of protein kinase CK2 as a target for developing new antitumor agents, we have undertaken a search for inhibitors of this enzyme. As part of this effort, we report here the discovery of the potent and selective CK2 inhibitor (5-oxo-5,6-dihydroindolo[1,2-a]-quinazolin-7-yl)acetic acid. We identified this inhibitor of a novel structural type by high- throughput docking of our corporate compound collection in the ATP binding site of a homology model of human CK2, using an appropriate protocol. The synthesis of the inhibitor as well as that of related analogues whose CK2 inhibitory activities give support to the binding mode proposed by the docking program is described. The results obtained suggest that virtual screening of a 3D database by molecular docking is a useful approach for lead finding provided that adapted postdocking filtering and reranking procedures are applied to the primary hit list.

Full text of DOI:10.1021/jm030827e

2656
J . Med. Chem. 2003, 46, 2656-2662
Discover y of a P oten t a n d Selective P r otein Kin a se CK2 In h ibitor by
High -Th r ou gh p u t Dock in g
Eric Vangrevelinghe,† Kaspar Zimmermann,‡ J oseph Schoepfer,† Robert Portmann,§ Doriano Fabbro,† and
Pascal Furet*,†
Oncology Research, Novartis Pharma AG, 4002 Basle, Switzerland, and Nervous System Research, Novartis Pharma AG,
CH-4002 Basle, Switzerland, and Process R&D, Novartis Pharma AG, CH-4002 Basle, Switzerland
Received February 28, 2003
To assess the potential of protein kinase CK2 as a target for developing new antitumor agents,
we have undertaken a search for inhibitors of this enzyme. As part of this effort, we report
here the discovery of the potent and selective CK2 inhibitor (5-oxo-5,6-dihydroindolo[1,2-a]-
quinazolin-7-yl)acetic acid. We identified this inhibitor of a novel structural type by high-
throughput docking of our corporate compound collection in the ATP binding site of a homology
model of human CK2, using an appropriate protocol. The synthesis of the inhibitor as well as
that of related analogues whose CK2 inhibitory activities give support to the binding mode
proposed by the docking program is described. The results obtained suggest that virtual
screening of a 3D database by molecular docking is a useful approach for lead finding provided
that adapted postdocking filtering and reranking procedures are applied to the primary hit
list.
Ch a r t 1. Previously Reported CK2 Inhibitors:
4,5,6,7-Tetrabromo-1H-benzotriazole (TBB) and
1,4-Diamino-5,8-dihydroxyanthraquinone (DAA)
In tr od u ction
Protein kinase CK2 (casein kinase II) is a ubiquitous,
constitutively active, protein kinase with the distinctive
properties of possessing hundreds of known phosphor-
ylation protein substrates and accepting both ATP and
GTP as cofactor.1 Although the precise functions of CK2
in eukaryotes are still under intense investigation, roles
in cell growth, proliferation, and survival have been
established consistent with the fact that many of its
substrates are proteins involved in signal transduction,
the regulation of the cell cycle, or apoptosis.2,3 Of
particular interest for anticancer agents research are
reports suggesting a role of CK2 in neoplastic growth.4,5
For instance, it has been reported that overexpression
of the catalytic subunit of CK2 can cause lymphoma.6
To elucidate the different cellular functions of CK2
and assess its potential as a therapeutic target in
oncology, potent and selective small molecule inhibitors
of its enzymatic activity are required. To date, two main
types of chemical inhibitors of CK2 have been de-
scribed.7 The first type consists of polyhydroxylated
aromatic compounds that include natural product de-
rivatives with anthraquinone, fluorenone, flavone, and
xanthenone core structures. Halogenated benzimidazole
and benzimitriazole compounds form the second type.
All these compounds are ATP site-directed inhibitors,
and for several of them the structural basis of inhibition
has been revealed with full atomic details by the
determination of a X-ray cocrystal structure with the
catalytic unit of Zea mays CK2.8-10 In particular, this
is the case with the halogenated benzimitriazole com-
pound TBB, a very selective CK2 inhibitor (Chart 1).11
Interestingly, despite a rather modest CK2 inhibitory
potency lying in the micromolar range, this compound
has been shown to induce apoptosis in J urkat cells.12
Compounds with increased potency would therefore
appear very promising in the study of the cellular effects
of CK2 inhibition. More potent inhibitors such as DAA
exist in the natural product class, but they are less
selective.
Considering CK2 as a possible target in our oncology
programs, we wondered whether we could identify, in
the Novartis collection of compounds, CK2 inhibitors
having the same level of selectivity as TBB but showing
higher potency. The availability of crystal structure
information on the target prompted us to envisage a
structure-based strategy for this purpose. Some recent
reports of success in the discovery of biologically active
molecules by high-throughput docking of large collec-
tions of compounds in a binding site of known struc-
ture13,14 motivated us to try this approach in our quest
for CK2 inhibitors. In this article, we describe this
virtual screening experiment targeting the ATP binding
site of CK2.
Resu lts a n d Discu ssion
Hom ology Mod elin g. Virtual screening by docking
requires an accurate three-dimensional (3D) structure
of the considered target. While several crystal structures
of the catalytic subunit of CK2 (CK2R) from Zea mays
have been published in the past years and their coor-
* Corresponding author. Tel 41 61 696 79 90, fax 41 61 696 62 46,
e-mail pascal.furet@pharma.novartis.com.
† Oncology Research, Novartis Pharma AG.
‡ Nervous System Research, Novartis Pharma AG.
§ Process R&D, Novartis Pharma AG.
10.1021/jm030827e CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/21/2003

Protein Kinase CK2 Inhibitor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2657
be thought of as pseudo-atom positions onto which
DOCK superimposes atoms of the database molecules
to generate a ligand orientation in the binding site. Only
spheres that fill the ATP binding site and more precisely
the region close to the hinge segment of CK2 have been
considered in this experiment (Figure 2). In protein
kinases, the hinge segment is the amino acid stretch
that connects the N- and C-terminal domains at the
interface of which the ATP binding site is located. The
region close to the hinge segment is the most hydro-
phobic and solvent-shielded part of the ATP pocket and
consequently inhibitors usually form their most produc-
tive interactions with amino acid residues of this region.
This is why spheres located at the solvent exposed
entrance of the cavity, toward which the triphosphate
chain of ATP extends, were considered less important
and discarded to speed up the calculations.
Although the docking protocol used (see details in the
Experimental Section) has been developed internally on
several case studies and has demonstrated its ability
to rank known inhibitors for similar targets at the top
of the hit list, we decided to apply in this case all the
postprocessing filtering and reranking tasks that are
recommended to increase the hit rate in large database
docking.21 Thus, we successively applied the three
following postprocessing treatments to the primary hit
list:
• The first stage was a filtering of the hit list based
on pharmacophore information. Hydrogen bonding to
the hinge segment is a key determinant of binding
affinity to the ATP pocket of protein kinases as testified
by the presence of this interaction in almost all the
available X-ray crystal structures of inhibitor-kinase
complexes.22-24 Best scored candidates were thus ex-
amined in terms of the presence or absence of this
interaction. Compounds not forming a hydrogen bond
interaction with the hinge segment were removed.
• In a second stage, following a consensus scoring
approach, we applied to the hit list the scoring function
developed by Wang et al.25 Only the compounds best
scored by this function were retained.
• The last stage was the unavoidable visual inspection
of the remaining top ranked candidates docked in the
ATP binding site of CK2. This crucial step allowed us
to discard compounds exhibiting unfavorable interac-
tions with the binding site or adopting unrealistic
conformations as a result of the shortcomings of the
scoring functions.
F igu r e 1. Alignment of Zea mays and human CK2R se-
quences. Residues corresponding to the ATP binding site are
marked in bold type. Uppercase X symbol indicates a totally
different residue while lowercase x symbol indicates a similar
residue.
dinates made public, those of the recently determined
crystal structure of human CK215 were not publicly
available at the time of this work. However, the high
degree of conservation between the sequences of CK2R
in Homo sapiens and Zea mays allowed us to construct
an accurate 3D homology model of human CK2R, our
target, based on one of the Zea mays structures. The
amino acid identity between the human and the Zea
mays sequences is equal to 72% and reaches 82% if only
the 22 residues forming the ATP binding site are
considered. The alignment reported in Figure 1 shows
the strong similarity of the two sequences. Thus, we
expected that a homology model of human CK2R based
on this alignment would have a degree of accuracy that
made it suitable for structure-based ligand design and
molecular docking studies. We could confirm a posteriori
the accuracy of our model by comparing it to the crystal
structure of Niefind et al.15 when it became available
very recently. The root-mean-square deviation (rmsd)
between the coordinates of the main chain CR atoms of
our CK2 homology model and those of the crystal-
lographic structure calculated on the entire catalytic
kinase domain has a value of 0.92 Å. Considering only
the 22 residues forming the ATP binding site, this is
only 0.64 Å. These rmsd values are significantly lower
than the resolution of the crystallographic structure (3.1
Å), thus validating the use of our homology model for
virtual screening purpose.
Vir tu a l Scr een in g P r oced u r e. Our objective was
to screen a large subset of our corporate collection of
compounds containing around 400 000 structures. To
achieve this goal within a reasonable time frame, we
needed a fast and robust docking tool. The software
DOCK, regarded as fulfilling these criteria, was selected
for this task. DOCK, developed by Kuntz and co-
workers, has shown promise as a tool for the identifica-
tion of macromolecule ligands via database searching.16
Some successful applications of DOCK in drug discovery
research have already been claimed. These include the
identification of inhibitors for the HIV-1 protease,17
thymidylate synthase,18 influenza hemagglutinin,19 and
parasitic proteases.20
Biologica l Resu lts. Following the above procedure,
a dozen of compounds were finally selected and ordered
from the Novartis chemical archives for actual testing
in a CK2 phosphorylation inhibition assay. In the assay,
four of the selected compounds turned out to inhibit
more than 50% of the enzymatic activity of CK2 at a
concentration of 10 µM. The other eight compounds were
only marginally active, inhibiting less than 50% of the
activity of the enzyme at the same concentration. Among
the four active compounds, which belong to different
chemical classes, the most potent one, exhibiting 97%
inhibition at a concentration of 10 µM, legitimately
retained our attention. This compound, 4, is an indolo-
quinazolinone derivative synthesized at Novartis in an
unrelated medicinal chemistry project (Chart 2). Its IC50
value for CK2 inhibition was determined (Table 1). With
DOCK requires that the binding site of the target be
defined using spheres that complement the molecular
surface of the receptor. The centers of these spheres can

2658 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Vangrevelinghe et al.
F igu r e 2. Overlay of the docking site points used (yellow spheres) and the ATP analogue extracted from the crystallographic
template structure 1DAW (magenta) inside the active site of the homology model of human CK2R.
Ch a r t 2.
including the canonical hydrogen bonds to the hinge
segment are characteristic of potent protein kinase
inhibitors as revealed by numerous crystal structures,
particularly that of the anthraquinone inhibitor DAA
in complex with CK2, recently published.10 Hence, we
judged the binding mode proposed by DOCK for com-
pound 4 as quite plausible.
(5-Oxo-5,6-dihydroindolo[1,2-a]quinazolin-7-yl)acetic
Acid (4) Retrieved from the Novartis Compound
Collection
Of particular interest in the binding model is the salt
bridge formed by the 7-acetic acid group of 4 and residue
Arg 43 located on the side of the pocket opened to
solvent. This interaction drew our attention for two
reasons. First, the model suggested that it plays a
significant role in the potency of the compound because
such ionic interactions are usually very strong. Second,
we speculated that it may also play a role in the high
selectivity displayed by 4 since Arg 43 is not conserved
within the protein kinase family. For instance, in the
c-Src and c-Abl kinases, which are very poorly inhibited
by 4, this position is occupied by a valine and a histidine
residue, respectively.
Ta ble 1. CK2 Inhibitory Potency of Several 7-Substituted
Indoloquinazolinone Derivatives
R
IC50 (µM)a
% inhib at 10 µM
3a
4
5
CH3
CH2COOH
CH2CH2OH
nd
0.080
nd
12
97
48
To check the importance of the 7-acetic acid group,
suggested by the model, we tested two available ana-
logues of 4 lacking an acid function to form the putative
salt bridge with Arg 43. Their chemical structures and
CK2 inhibition potencies are summarized in Table 1.
With an inhibitory activity of 12% at 10 µM, the
analogue with a 7-methyl substituent, 3a , is dramati-
cally less potent than 4. The 7-hydroxyethyl analogue
5, having an inhibitory activity of 48% at 10 µM, is also
less active than 4 but to a lesser extent. The binding
model is consistent with these results. Completely
abolishing any polar interaction with Arg 43, as in
compound 3a , is expected to be more detrimental to
potency than replacing a salt bridge interaction by a
weaker hydroxyl-guanidinium hydrogen bond as it is
done going from 4 to 5. Thus, support for the binding
hypothesis was gained from this structure-activity
relationship data.
a
The data represent averages of at least three determinations.
a value of 80 nM, we had discovered the most potent
inhibitor of the CK2 kinase ever reported. 4 showed in
addition a remarkable specificity for CK2 when assayed
for inhibition of 20 other kinases as reported in Table
2. None of the other kinases, representative of different
subfamilies, was inhibited more than 50% by 4.
The binding pose found by DOCK for this compound
in the ATP pocket of our homology model of human
CK2R is shown in Figure 3. As can be seen, the docking
program positioned 4 in the pocket in such a way that
its lactam moiety makes bidendate hydrogen bonds with
the backbone of residue Val 116 of the hinge segment.
This allows multiple hydrophobic interactions to be
made between the polycyclic core of the molecule and
residues Leu 45,Val 53, Val 66, Met 163, and Ile 174
that normally form the environment of the adenosine
part of ATP in the enzyme pocket. Such interactions
Ch em istr y. 7-Substituted indoloquinazolinones are
described only twice in the literature so far. Moore et
al.26 reported on the synthesis of the 7-phenyl derivative

Protein Kinase CK2 Inhibitor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2659
Ta ble 2. Selectivity Profile of Compound 4 in Terms of % of Inhibition of the Catalytic Activity of the Kinases at a Compound
Concentration of 10 µM
Sch em e 1
F igu r e 3. Relative binding modes of compound 4 (green) and
the ATP analogue extracted from the crystallographic template
structure 1DAW (magenta) in the human CK2R active site.
The hydrogen bonds formed with Arg 43 and Val 116 are
indicated as orange lines.
hydrolysis of the ester 3b. The hydroxyethyl derivative
5 resulted from lithium borohydride reduction of 3b.
synthesized via cyclodehydration from R,R-diphenyl-
quinazolinonemethanol. Moehrle and Seidel27 described
Con clu sion
the formation of a hexacyclic indoloquinazolinone de-
We have identified a 7-substitued indoloquinazoline
rivative obtained by condensation of anthranilic acid
compound as a novel inhibitor of protein kinase CK2
methyl ester and coumaranone. None of these methods
by virtual screening of 400 000 compounds, of which a
is suited to produce derivatives with different substit-
dozen were selected for actual testing in a biochemical
uents in position 7 of the indoloquinazolinone skeleton.
assay. The compound inhibits the enzymatic activity of
Our synthesis is based on the rearrangement of a
substituted 11-oxo-10,11-dihydro-5H-dibenzo[b,f]azepine-
10-carbonitrile into the indoloquinazolinone skeleton
CK2 with an IC50 value of 80 nM, making it the most
potent inhibitor of this enzyme ever reported. Its high
potency, associated with high selectivity, provides a
under basic conditions. No studies were undertaken to
valuable tool for the study of the biological function of
elucidate the mechanism. It is believed, however, that
CK2.
the rearrangement sequence starts by attack of the
The reported work clearly shows that large database
carbonyl group of the oxodibenzazepine by methoxide,
docking in conjunction with appropriate scoring and
leading to ring-opening. Subsequent intramolecular
filtering processes can be useful in medicinal chemistry.
nucleophilic attack of the azepine nitrogen on the
This approach has reached a maturation stage where
C-atom of the cyano group may push the cyano nitrogen
it can start contributing to the lead finding process. At
to attack the ester carbonyl leading to the tetracyclic
the time of this study, nearly one month was necessary
skeleton of the target molecules. Subsequent aromati-
to complete such a docking experiment in our laboratory
zation concludes the rearrangement. To our knowledge
settings. The Grid computing architecture recently
such a reaction is unprecedented.
developed by United Devices29 allows us to now perform
Our synthesis (see Scheme 1) of the derivatives
the same task in less than five working days using the
substituted in position 7 started by migrating the cyano
power of hundreds of desktop PC’s. High-throughput
group of 10-oxo-10,11-dihydrodibenzo[b,f]azepine-5-car-
bonitrile to position 11 under basic conditions giving 1.
Subsequently, carbon-11 can easily be alkylated, e.g.,
docking has therefore acquired the status of a routine
screening technique.
with methyl iodide to 2a or ethyl bromoacetate to 2b.
The rearrangement described above occurs upon treat-
ment with sodium methoxide in methanol yielding 3a
and 3b, respectively. This two-step alkylation-rear-
rangement sequence can be done in one-pot and is suited
for automated parallel synthesis, e.g., on a Quest
synthesizer.28 The carboxylic acid 4 was obtained by
Exp er im en ta l Section
Hom ology Mod elin g. The sequence of human CK2R was
obtained from SWISS-PROT30 (entry P19138). The Fasta
search option available in the Protein Data Bank31 helped to
identify several structural templates on which to base the
homology model. The crystallographic structure of Zea mays
CK2R32 in complex with an ATP analogue (PDB code 1DAW

2660 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Vangrevelinghe et al.
from the Protein Databank) was finally chosen as the template
because of its high resolution (2.2 Å) and the high sequence
similarity existing between the human and Zea mays enzymes
(92% sequence similarity). The sequences were aligned in the
homology module of Insight II33 using the default parameters
(scoring matrix: mutation, gap penalty: 6, and gap length
penalty: 1.65). On the basis of the resulting alignment, shown
in Figure 1, the 3D structure of human CK2R was modeled
using the WhatIf34 program with the default parameters
(PIRPSQ module, BLDPIR command). Hydrogen atoms and
Kollman united atom charges were subsequently added using
the biopolymer module of Sybyl.35 The model thus obtained
was used without further energy refinement.
the ATP pocket. Unlike many other docked compounds, they
did not show any electrostatic or hydrophobic mismatch with
the binding site and had no apparent conformational strain.
In addition, they possessed few entropically unfavorable
rotatable bonds.
Ch em ica l Syn th esis An d Sta r tin g Ma ter ia l. Melting
points (uncorrected) were determined using a Bu¨chi 510
apparatus. MS (mass spectra) were recorded with a Micromass
Platform II using electron spray ionization (positive or negative
mode). 1H and 13C nuclear magnetic resonance spectroscopy
was performed on Brucker and Varian instruments at force
fields of 200 to 500 MHz for 1H NMR and 100 MHz for 13C
NMR. Peak assignment was performed using COSY, HSQC,
and HMBC techniques. The starting material 10-oxo-10,11-
dihydrodibenzo[b,f]azepine-5-carbonitrile (CAN: 78880-65-6,
EINECS: 2789984) is available in bulk quantities and is used
in the production synthesis of the antiepileptic drug Trileptal.40
11-Oxo-10,11-d ih yd r o-5H-d iben zo[b,f]a zep in e-10-ca r -
bon itr ile (1). Solid potassium tert-butylate (62.3 g, 544 mmol)
was added in four portions to a solution of 10-oxo-10,11-
dihydrodibenzo[b,f]azepine-5-carbonitrile (75 g, 320 mmol) in
2.5 L of 1,2-dichloroethane at rt within 2 h. Thereafter a
mixture of acetic acid (32.2 mL) and 300 mL of ice-cold water
was slowly added. The resulting suspension was stirred for
10 min, cyclohexane (650 mL) was added, and stirring con-
tinued for 1 h at 0 °C. The precipitate was collected by
filtration, washed with water (300 mL), and cyclohexane (300
mL). The crude product was recrystallized from 2.5 L of boiling
EtOH upon concentration to 1.2 L of solvent followed by slow
cooling to yield 11-oxo-10,11-dihydro-5H-dibenzo[b,f]azepine-
10-carbonitrile 1 (56.6 g, 75%) as pale-yellow crystals. mp:
195-197 °C; 1H NMR (d6-DMSO, 400 MHz) mixture of keto-
enol tautomers, therefore no peak assignment possible: δ 5.40
(s, 0.60H), 6.95-7.06 (m, 2.23H), 7.12-7.18 (m, 0.81H), 7.30-
7.45 (m, 3.38H), 7.51-7.59 (m, 1.69H), 7.80-7.85 (m(d),
0.65H), 9.84 (s, 0.65H); 13C NMR (d6-DMSO, 100 MHz) all
signals double due to keto-enol tautomerism: δ 49.41, 91.29,
116.80, 119.32, 119.73, 119.97, 120.35, 120.68, 121.07, 121.25,
121.35, 121.79, 123.25, 124.30, 125.67, 125.88, 126.45, 127.20,
128.70, 129.12, 129.57, 129.99. 130.62, 133.54, 135.34, 141.49,
147.53, 150.00, 153.17, 181.62; MS (ES, neg.) m/z 233 (M -
1), MS (ES, pos.) m/z 235 (M + 1); HR-MS m/z 233.0713 [(M
- H) calcd for C15H10N3O 233.0715]; Anal. (C15H10N2O) C, H,
N.
10-Meth yl-11-oxo-10,11-dih ydr o-5H-diben zo[b,f]azepin e-
10-ca r bon itr ile (2a ). To a cooled (5 °C) mixture of 1 (25 g,
106.7 mmol) and potassium carbonate (18 g, 130.2 mmol) in
DMF (60 mL) under nitrogen was added methyl iodide (7.8
mL, 124.9 mmol)) within 10 min. The resulting orange
suspension was stirred at 0 °C for 30 min then at rt for 2 h
before toluene (6 mL) was added dropwise, followed by water
(100 mL). Precipitation of the product was forced by cooling
to 0 °C. 10-Methyl-11-oxo-10,11-dihydro-5H-dibenzo[b,f]azepine-
10-carbonitrile 2a (14.2 g, 54%) was obtained as crude yellow-
orange crystals. mp: 174-175 °C; 1H NMR (d6-DMSO, 400
Str u ctu r e-Ba sed Da ta ba se Sea r ch . For the docking
experiment, a subset of around 400 000 chemical entities were
selected from our corporate compound collection. This subset
was created using filters to eliminate molecules that have
unwanted atoms or fragments (e.g., P, Si) and to avoid
molecules with a molecular weight inferior to 25 or superior
to 1000. Starting from the two-dimensional structures, the 3D
coordinates of all compounds were generated using Corina.36
Hydrogen atoms, protonation states and Gasteiger-Marsili37
atomic charges were then added using a Sybyl SPL macro,
and the final coordinates were stored in a multimol2 file.
Compounds were flexibly docked using the DOCK 4.01
docking software package.38 In the DOCK method the binding
site is represented geometrically as a set of overlapping
spheres39 (docking spheres) which are created using the
computer program Sphgen.38 Although a large number of
spheres may represent the 3D structure of the binding site
more accurately, in practice one has to limit the number of
spheres for computational efficiency. Restricting the covered
area to the part of the ATP binding site where kinase
inhibitors are known to make crucial interactions, 15 spheres
were finally retained. The docking protocol chosen in this work
slightly differs from those usually reported in the literature
where DOCK is used giving the standard values to the
parameters. A detailed discussion on the use of DOCK 4
can be found in ref 39. The whole molecule approach
(anchor_search option set to no) was used in combination with
a
manual matching (maximum_orientations set to 1000,
nodes_minimum set to 4, and distance_tolerance set to 0.3 Å).
All docking poses were energy minimized using 100 iterations
and 10 minimization cycles. The poses were evaluated and the
ligands ranked using the energy scoring function of DOCK
which is an interaction force field score that includes van der
Waals and electrostatic terms. The use of the energy scoring
function in combination with a united atom model has proven
to give better results in a systematic assessment conducted
internally. This protocol leads to an average processing time
of 42 s per compound and per CPU on a Silicon Graphics
Origin 2000 computer possessing 8 R10000 processors. To
avoid highly flexible, very small, or very large ligands, several
filters were applied prior to docking. Compounds with more
than 12 flexible bonds were thus discarded as well as com-
pounds with less than 6 or more than 60 heavy atoms.
Compounds with a docking score inferior or equal to the
value of -35 kcal/mol were retained in a first step. This
arbitrary selection gave 12 428 compounds which were then
filtered according two different criterions. In the first stage,
only compounds presenting two hydrogen bond interactions
with the backbone of the hinge segment, either at residue Val
116 alone or simultaneously at Val 116 and Glu 114, were
retained. The binding modes of the compounds were then
evaluated using the scoring function of Wang et al.25 This
empirical scoring fuction contains a supplementary term
accounting for solvation/desolvation effects compared to the
DOCK energy function. Compounds with a score inferior to 5
according to the Wang function were discarded. The remaining
1592 compounds docked in the ATP pocket of CK2 were
visually inspected. After this inspection, 12 compounds were
selected to be experimentally tested in the CK2 inhibition
assay. These 12 compounds were judged particularly promising
because they presented high structural complementarity with
MHz) δ 1.91 (s, 3H, CH3), 6.97 (t(dd), J 2-1 ) 8.03 Hz, J 2-3
)
7.45 Hz, 1H, H-2), 7.27-7.31 (m, 1H, H-7), 7.43-7.44 (m, 3H,
H-4, H-5, H-6), 7.52 (d, J 8-7 ) 7.94 Hz, 1H, H-8), 7.57 (ddd,
J 3 ) 7.45 Hz, J 3-4 ) 7.95 Hz, J 3-1 ) 1.58 Hz, 1H, H-3), 7.75
-2
(dd, J 1-2 ) 8.03 Hz, J 1-3 ) 1.58 Hz, 1H, H-1), 10.14 (s, 1H,
H-N); 13C NMR (d6-DMSO, 100 MHz) δ 19.57 (CH3), 119.61
(C-2), 119.96 (C-1a), 120.02 (C-4), 120.26 (C-10), 121.47 (C-
9a), 121.88 (C-6), 125.42 (C-8), 126.81 (C-9), 130.34 (C-7),
131.30 (C-1), 135.17 (C-3 and CN), 140.76 (C-6a), 145.79 (C-
4a), 183.40 (C-11); MS (ES, neg.) m/z 247 (M-1); HR-MS m/z
247.0872 [(M - H) calcd for C16H12N2O 247.0871]; Anal.
(C16H12N2O) C, H, N.
7-Met h yl-6H -in d olo[1,2-a ]q u in a zolin -5-on e (3a ). So-
dium methoxide solution (20.3 mL, 112.77 mmol, 30% solution
in MeOH) was added to 2a (10 g, 40.28 mmol) in MeOH (220
mL) at rt, then immediately heated to 65 °C for 3 h. After
stirring at rt overnight, the yellow residue was heated at 60
°C, and a mixture of acetic acid (10 mL) in water (220 mL)
was added dropwise. The resulting suspension was stirred at

Protein Kinase CK2 Inhibitor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2661
0 °C for 1 h and the 7-methyl-6H-indolo[1,2-a]quinazolin-5-
one (3a ) collected by filtration (8.8 g, 88%) as yellow crystals.
mp: 298-300 °C, decomp; H NMR (d6-DMSO, 400 MHz) δ
acid. The yellow crystalline 7-(2-hydroxyethyl)-6H-indolo[1,2-
a]quinazolin-5-one (5) (1.6 g, 71%) was collected by filtration,
washed with water and ethanol, and dried in vacuo at 60 °C.
1
1
2.27 (s, 3H, CH3), 7.22 (dd, J 10-11 ) 8.28 Hz, J 10-9 ) 7.11 Hz,
1H, H-10), 7.26 (dd, J 9-10 ) 7.11 Hz, J 9-8 ) 7.53 Hz, 1H, H-9),
7.37 (dd, J 3-2 ) 7.70 Hz, J 3-4 ) 7.78 Hz, 1H, H-3), 7.51 (d,
mp: 240-249 °C, decomp.; H NMR (d6-DMSO, 500 MHz) δ
2.96 (t, J ) 6.58 Hz, 2H, CH2CH2OH), 3.61 (t, J ) 6.59 Hz,
2H, CH2OH), 4.63-4.87 (br, 1H, OH), 7.21-7.27 (m, 2H, H-9
and H-10), 7.38 (dd, J 3-2 ) 7.79 Hz, J 3-4 ) 7.79 Hz, 1H, H-3),
7.58 (dd, J 8-9 ) 7.12, J 8-10 ) 1.61, Hz, 1H, H-8), 7.85 (ddd,
J 2-3 ) 7.79 Hz, J 2-1 ) 8.46 Hz, J 2-4 ) 1.61 Hz, 1H, H-2), 8.17
J 8-9 ) 7.53 Hz, 1H, H-8), 7.84 (ddd, J 2-3 ) 7.70 Hz, J 2-1
)
8.45 Hz, J 2-4 ) 1.42 Hz, 1H, H-2), 8.18 (dd, J 4-3 ) 7.78 Hz,
J 4-2 ) 1.42 Hz, 1H, H-4), 8.25 (d, J 11-10 ) 8.28 Hz, 1H, H-11),
8.41 (d, J 1-2 ) 8.45 Hz, 1H, H-1), 11.79 (s br, 1H, H-N); 13C
NMR (d6-DMSO, 100 MHz) δ 7.53 (CH3), 90.34 (C-7), 113.78
(C-11), 115.71 (C-1), 117.29 (C-4a), 118.19 (C-8), 121.46 (C-
10), 122.68 (C-9), 123.96 (C-3), 129.59 (C-4), 130.43 (C-11a),
131.54 (C-7a), 133.50 (C-6a), 135.83 (C-2), 139.86 (C-1a), 159.64
(C-5); MS (ES, neg.) m/z 247 (M - 1), MS (ES, pos.) m/z 249
(M + 1); HR-MS m/z 249.1028 [(M + H) calcd for C16H12N2O
249.1028; Anal. (C16H12N2O) C, H, N.
(dd, J 4-3 ) 7.79 Hz, J 4-2 ) 1.48 Hz, 1H, H-4), 8.26 (d, J 11-10
)
7.65 Hz, 1H, H-11), 8.41 (d, J 1-2 ) 8.47 Hz, 1H, H-1), 11.66-
11.82 (s br, 1H, H-N); 13C NMR (d6-DMSO, 125 MHz) δ 25.34
(CH2), 60.85 (CH2OH), 92.40 (C-7), 113.07 (C-11), 115.09 (C-
1), 116.38 (C-4a), 117.73 (C-8), 120.70 (C-10), 121.91 (C-9),
123.30 (C-3), 128.76 (C-4), 129.67 (C-11a), 130.33 (C-7a), 132.67
(C-6a), 135.19 (C-2), 138.95 (C-1a), 158.50 (CONH); MS (ES,
neg.) m/z 277 (M-1), MS (ES, pos.) m/z 279 (M + 1); HRMS
m/z 277.0974 [(M-H) calcd for C17H14N2O2 279.0977; Anal.
(C17H14N2O2) C, H, N.
(10-Cyan o-11-oxo-10,11-dih ydr o-5H-diben zo[b,f]azepin -
10-yl)a cetic Acid Eth yl Ester (2b). The ethyl acetate
derivative 2b was synthesized in analogy to 2a from 1 and
ethyl bromoacetate. The resulting crude orange oil was directly
used for the following conversion into 3b. MS (ES, pos.) m/z
321 (M + 1).
Kin a se In h ibition Assa ys. The CK2 inhibition assay used
is described in ref 41. Briefly, one looks at the ability of a
compound to inhibit the phosphorylation of a peptide substrate
(RRRADDSDDDDD) by native CK2 purified from rat liver.
Human (SWISS-PROT entry P19138) and rat CK2R (SWISS-
PROT entry P19139) have 100% sequence identity. Therefore,
similar compound potencies as in the rat enzyme assay would
be expected in a human enzyme assay. This has been checked
for some inhibitors11 and has proven to be the case. The
selectivity kinase inhibition assays reported in Table 2 were
performed under conditions optimized for each kinase and with
ATP concentrations similar to the Km of the respective enzyme
toward ATP: 8.0 µM (KDR, Flt-1, FGFR-1, Tek), 1.0 µM
(PDGFR-â, c-Kit, c-Met), 13 µM (Flt-4), 5 µM (c-abl), 2.0 µM
(Her-1, Her-2), 20 µM (c-Src), 30 µM (IGF-1R), 7.5 µM (CDK1),
and 50 µM (PKA). For tyrosine kinases, filter binding assays
using recombinant GST-fused kinase domains of the receptors
expressed in baculovirus and purified over glutathione
sepharose were employed. [33 P]-ATP was used as the phos-
phate donor, and the polyGluTyr (4:1) peptide was used as
the acceptor. The CDK1 and PKA assays are described in refs
42 and 43, respectively.
(5-Oxo-5,6-d ih yd r oin d olo[1,2-a ]q u in a zolin -7-yl)a ce-
tic Acid Meth yl Ester (3b). The methyl ester 3b was
synthesized from 2b (14.7 g, 45.89 mmol) in analogy to the
procedure used for 3a . Transesterification occurred from ethyl
to methyl ester. (5-Oxo-5,6-dihydroindolo[1,2-a]quinazolin-7-
yl)acetic acid methyl ester (3b) (9.4 g, 67%) was obtained as
yellow crystals. mp: 245-252 °C, decomp; 1H NMR (d6-DMSO,
500 MHz) δ 3.62 (s, 3H, OCH3), 3.94 (s, 2H, CH2), 7.22-7.27
(m, 2H, H9 and H-10), 7.41 (dd, J 3-2 ) 7.4 Hz, J 3-4 ) 7.61 Hz,
1H, H-3), 7.48-7.50 (m, 1H, H-8), 7.87 (ddd, J 2-1 ) 8.44 Hz,
J 2-3 ) 7.53 Hz, J 2-4 ) 1.30 Hz, 1H, H-2), 8.20 (dd, J 4-3 ) 7.70
Hz, J 4-2 ) 1.26 Hz, 1H, H-4), 8.25 (m, 1H, H-11), 8.45 (d, J 1-2
) 8.45 Hz, 1H, H-1), 11.93 (s, 1H, H-N); 13C NMR (d6-DMSO,
125 MHz) δ 27.27 (CH2), 51.74 (OCH3), 87.77 (C-7), 113.16 (C-
11), 115.18 (C-1), 116.34 (C-4a), 117.55 (C-8), 120.99 (C-10),
122.16 (C-9), 123.60 (C-3), 128.76 (C-4), 129.60 and 129.89 (C-
7a and C-11a), 133.50 (C-6a), 135.30 (C-2), 138.76 (C-1a),
158.57 (CONH), 171.51 (COO); MS (ES, neg.) m/z 305 (M -
1), MS (ES, pos.) m/z 307 (M + 1); HR-MS m/z 307.1085 [(M
+ H) calcd for C18H14N2O3 307.1083; Anal. (C18H14N2O3) C, H,
N.
Ack n ow led gm en t. The authors wish to acknowl-
edge Corinne Marx and Simon Pfister for technical
support.
(5-Oxo-5,6-d ih yd r oin d olo[1,2-a ]q u in a zolin -7-yl)a ce-
tic Acid (4). A slurry of 3b (5.0 g, 16.32 mmol) in DMSO (50
mL) was treated with 1 N aqueous sodium hydroxyde solution
(45.7 mL, 45.7 mmol) at rt. A clear solution was observed
followed by precipitation of a yellow solid. The mixture was
diluted with water (150 mL) and cooled to 5 °C, and acetic
acid (20 mL) was added. After 45 min, (5-oxo-5,6-dihydroin-
dolo[1,2-a]quinazolin-7-yl)acetic acid 4 was obtained by filtra-
tion as yellow crystals (4.54 g, 95%). mp: 234-236 °C, decomp.;
1H NMR (d6-DMSO, 500 MHz) δ 3.85 (s, 2H, CH2), 7.22-7.30
(m, 2H, H-9 and H-10), 7.42 (dd, J 3-2 ) 7.39 Hz, J 3-4 ) 7.79
Hz, 1H, H-3), 7.49-7.55 (m, 1H, H-8), 7.88 (ddd, J 2-1 ) 8.46
Refer en ces
(1) Pinna, L. A. Protein kinase CK2: a challenge to canons. J . Cell
Sci. 2002, 115, 3873-3878.
(2) Litchfield, D. W. Protein kinase CK2: structure, regulation and
role in cellular decisions of life and death. Biochem. J . 2003,
369, 1-15.
(3) Ahmed, K.; Gerber, D. A.; Cochet, C. J oining the cell survival
squad: an emerging role for protein kinase CK2. Trends Cell
Biol. 2002, 12, 226-230.
(4) Tawfic, S.; Yu, S.; Wang, H.; Faust, R.; Davis, A.; Ahmed, K.
Protein kinase CK2 signal in neoplasia. Histol. Histopathol.
2001, 16, 573-582.
(5) Guerra, B.; Issinger, O. G. Protein kinase CK2 and its role in
cellular proliferation, development and pathology. Electrophore-
sis 1999, 20, 391-408.
(6) Seldin, D. C.; Leder, P. Casein kinase 2R-induced murine
lymphoma: relation to Teilleriosis in cattle. Science. 1995, 267,
894-897.
(7) Sarno, S.; Moro, S.; Meggio, F.; Zagotto, G.; Dal Ben, D.;
Ghisellini, P.; Battistutta, R.; Zanotti, G.; Pinna, L. A. Toward
the rational design of protein kinase casein kinase-2 inhibitors.
Pharmacol. Ther. 2002, 93, 159-168.
(8) Battistutta, R.; De Moliner, E.; Sarno, S.; Zanotti, G.; Pinna, L.
A. Structural features underlying selective inhibition of protein
kinase CK2 by ATP site-directed tetrabromo-2-benzotriazole.
Protein Sci. 2001, 10, 2200-2206.
Hz, J 2-3 ) 7.39 Hz, J 2-4 ) 1.75 Hz, 1H, H-2), 8.20 (dd, J 4-3
)
7.79 Hz, J 4-2 ) 1.61 Hz, 1H, H-4), 8.29 (dd, J 11-10 ) 8.46 Hz,
J 11-9 ) 2.15 Hz, 1H, H-11), 8.47 (d, J 1-2 ) 8.46 Hz, 1H, H-1),
11.92 (s br, 1H, COOH), 12.28 (s br, 1H, NH); 13C NMR (d6-
DMSO, 125 MHz) δ 27.64 (CH2), 87.59 (C-7), 113.13 (C-11),
115.15 (C-1), 116.34 (C-4a), 117.59 (C-8), 120.91 (C-10), 122.10
(C-9), 123.54 (C-3), 128.77 (C-4), 129.60 (C-11a), 130.05 (C-
7a), 133.35 (C-6a), 135.30 (C-2), 138.82 (C-1a), 158.57 (CONH),
172.60 (COOH); MS (ES, neg.) m/z 291 (M - 1), MS (ES, pos.)
m/z 293 (M + 1); HR-MS m/z 293.0928 [(M + H) calcd for
C
17H12N2O3 293.0926; Anal. (C17H12N2O3) C, H, N.
7-(2-Hyd r oxyet h yl)-6H-in d olo[1,2-a ]q u in a zolin -5-on e
(5). A THF solution (25 mL) of lithium borohydride (0.62 g,
28.56 mmol) was added dropwise to a suspension of 3b (2.5 g,
8.16 mmol) in THF (40 mL) at rt. After the exothermic reaction
had ceased the mixture was heated at 75 °C for 18 h. At 10
°C, ethanol (100 mL) and water (20 mL) were added, further
chilled to 0 °C, and acidified to pH ) 7 with 2 N hydrochloric
(9) Battistutta, R.; Sarno, S.; De Moliner, E.; Papinutto, E.; Zanotti,
G.; Pinna, L. A. The replacement of ATP by the competitive
inhibitor emodin induces conformational modifications in the
catalytic site of protein kinase CK2. J . Biol. Chem. 2000, 275,
29618-29622.

2662 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Vangrevelinghe et al.
(10) De Moliner, E.; Moro, S.; Sarno, S.; Zagotto, G.; Zanotti, G.;
Pinna, L. A.; Battistutta, R. Inhibition of protein kinase CK2
by anthraquinone-related compounds. A structural insight. J .
Biol. Chem. 2003, 278, 1831-1836.
(11) Sarno, S.; Reddy, H.; Meggio, F.; Ruzzene, M.; Davies, S. P.;
Donella-Deana, A.; Shugar, D.; Pinna, L. A. Selectivity of 4,5,6,7-
tetrabromobenzotriazole, an ATP site-directed inhibitor of pro-
tein kinase CK2 (‘casein kinase-2’). FEBS Lett. 2001, 496, 44-
48.
(12) Ruzzene, M.; Penzo, D.; Pinna, L. A. Protein kinase CK2
inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) induces apop-
tosis and caspase-dependent degradation of haematopoietic
lineage cell-specific protein 1 (HS1) in J urkat cells. Biochem. J .
2002, 364, 41-47.
(13) Shoichet, B. K.; McGovern, S. L.; Wei, B.; Irwin, J . J . Lead
discovery using molecular docking. Curr. Opin. Chem. Biol.
2002, 6, 439-446.
(14) Schneider, G.; Bo¨hm, H.-J . Virtual screening and fast automated
docking methods. Drug Discov. Today 2002, 7, 64-70.
(15) Niefind, K.; Guerra, B.; Ermakowa, I.; Issinger, O. G. Crystal
structure of human protein kinase CK2: insights into basic
properties of the CK2 holoenzyme. EMBO J . 2001, 20, 5320-
5331.
(16) Kuntz, I. D.; Meng, E. C.; Shoichet, B. K. Structure-based
molecular design [Review]. Acc. Chem. Res. 1994, 27, 117-123.
(17) Filikov, A. V.; J ames, T. L. Structure-based design of ligands
for protein basic domains: application to the HIV-1 Tat protein.
J . Comput.-Aided Mol. Des. 1998, 12, 229-240.
(18) Shoichet, B. K.; Stroud, R. M.; Santi, D. V.; Kuntz, I. D.; Perry,
K. M. Structure-based discovery of inhibitors of thymidylate
synthase. Science 1993, 259, 1445-1450.
(19) Bodian, D. L.; Yamasaki, R. B.; Buswell, R. L.; Stearns, J . F.;
White, J . M.; Kuntz, I. D. Inhibition of the fusion-inducing
conformational change of influenza hemagglutinin by benzo-
quinones and hydroquinones. Biochemistry 1993, 32, 2967-2978.
(20) Ring, C. S.; Sun, E.; McKerrow, J . H.; Lee, G. K.; Rosenthal, P.
J .; Kuntz, I. D.; Cohen, F. E. Structure-based inhibitor design
by using protein models for the development of antiparasitic
agents. Proc. Natl. Acad. Sci. 1993, 90, 3583-3587.
(21) Boehm, H.-J .; Stahl, M. The use of scoring functions in drug
discovery applications. In Reviews in Computational Chemistry;
Lipkowitz K. B., Boyd, D. B., Eds.; Wiley-VCH: New York, 2002;
Vol. 18, pp 41-87.
(22) Toledo, L. M.; Lydon, N. B.; Elbaum, D. The Structure-based
design of ATP-site directed protein kinase inhibitors. Curr. Med.
Chem. 1999, 6, 775-805.
(23) Traxler, P.; Furet, P. Strategies toward the design of novel and
selective protein tyrosine kinase inhibitors. Pharmacol. Ther.
1999, 82, 195-206.
(24) Furet, P.; Meyer, T.; Strauss, A.; Raccuglia, S.; Rondeau, J . M.
Structure-based design and protein X-ray analysis of a protein
kinase inhibitor. Bioorg. Med. Chem. Lett. 2002, 12, 221-224.
(25) Wang, R.; Liu, L.; Lai, L.; Tang, Y. SCORE: A New Empirical
Method for Structure-Based Estimation of Binding Affinities.
J . Mol. Model. 1998, 4, 379-394.
(26) Moore, J . A.; Sutherland, G. J .; Sowerby, R.; Kelly, E. G.;
Palermo, S.; Webster, W. Reactions of anthranilamide and
o-aminoacetophenone with benzil and benzoin. J . Org. Chem.
1969, 34, 887-892.
(27) Moehrle, H.; Seidel, Ch. M. Quinolinones as starting products
for pentacyclic heterocycles. Arch. Pharm. (Weinheim, Ger.)
1974, 307, 785-791.
(28) described in lit.: Zimmermann, K.; Portmann, R.; Rigel, D. F.
Preparation of indoloquinazolinones as PARP inhibitors. PCT
Int. Appl. WO 0206284 A1, 2001.
(29) United Devices, Austin, TX (see website: http://www.ud.com).
(30) Bairoch, A.; Boeckmann, B. The SWISS-PROT protein sequence
data bank: current status. Nucleic Acids Res. 1994, 22, 3578-
3580.
(31) Bernstein, F. C.; Koetzle, T. F.; Williams, G. J . B.; Meyer, E. F.;
Brice, M. D.; Rodgers, J . R.; Kennard, O.; Shimanouchi, T.;
Tasumi, M. The Protein Data Bank: a Computer-based Archival
File for Macromolecular Structures. J . Mol. Biol. 1977, 112,
535-542.
(32) Niefind, K.; Putter, M.; Guerra, B.; Issinger, O. G.; Schomburg,
D. GTP plus water mimic ATP in the active site of protein kinase
CK2. Nat. Struct. Biol. 1999, 12, 1100-1103.
(33) Insight II, Accelrys, San Diego, CA.
(34) Vriend, G.; WHAT IF: A molecular modeling and drug design
program. J . Mol. Graph. 1990, 8, 52-56.
(35) Tripos Inc. Sybyl 6.6. St. Louis MO, 2001.
(36) Gasteiger, J .; Rudolph, C.; Sadowski, J . Automatic generation
of 3D atomic coordinates for organic molecules. Tetrahedron
Comput. Methodol. 1990, 3, 537-547.
(37) Gasteiger, J .; Marsili, M. Iterative Partial Equalization of Orbital
Electronegativity - A Rapid Access to Atomic Charges. Tetra-
hedron 1980, 36, 3219-3228.
(38) Kuntz, I. D.; Blaney, J . M.; Oatley, S. J .; Langridge, R. T.; Ferrin,
T. E. A geometric approach to macromolecule-ligand interactions.
J . Mol. Biol. 1982, 161, 269-288.
(39) Ewing, T. J . A.; Kuntz, I. D. Critical evaluation of search
algorithms for automated molecular docking and database
screening. J . Comput. Chem. 1997, 18, 1175-1189.
(40) described in lit.: Citterio, A.; Breviglieri, G.; Bruno, G. Eur. Pat.
Appl. (2001), EP 1127877. A2 and Aufderhaar, E. Eur. Pat. Appl.
EP 28028, 1981.
(41) Meggio, F.; Donella Deana, A.; Ruzzene, M.; Brunati, A. M.;
Cesaro, L.; Guerra, B.; Meyer, T.; Mett, H.; Fabbro, D.; Furet,
P.; Dobrowolska, G.; Pinna, L. A. Different susceptibility of
protein kinases to staurosporine inhibition. Kinetic studies and
molecular bases for the resistance of protein kinase CK2. Eur.
J . Biochem. 1995, 234, 317-322.
(42) Rialet, V.; Meijer, L. A New Screening Test for Antimitotic
compounds affinity-immobilized on p13suc1 -coated microtitra-
tion plates. Anticancer Res. 1991, 11, 1581-1590.
(43) Meyer, T.; Regenass, U.; Fabbro, D.; Alteri, E.; Roesel, J .; Muller,
M.; Caravatti, G.; Matter, A. A derivative of staurosporine (CGP
41 251) shows selectivity for protein kinase C inhibition and in
vitro anti-proliferative as well as in vivo antitumor activity. Int.
J . Cancer 1989, 43, 851-856.
J M030827E