Chemical probes to study ADP-ribosylation: Synthesis and biochemical evaluation of inhibitors of the human ADP-ribosyltransferase ARTD3/PARP3

Article abstract of DOI:10.1021/jm401394u

The racemic 3-(4-oxo-3,4-dihydroquinazolin-2-yl)-N-[1-(pyridin-2-yl)ethyl] propanamide, 1, has previously been identified as a potent but unselective inhibitor of diphtheria toxin-like ADP-ribosyltransferase 3 (ARTD3). Herein we describe synthesis and evaluation of 55 compounds in this class. It was found that the stereochemistry is of great importance for both selectivity and potency and that substituents on the phenyl ring resulted in poor solubility. Certain variations at the meso position were tolerated and caused a large shift in the binding pose. Changes to the ethylene linker that connects the quinazolinone to the amide were also investigated but proved detrimental to binding. By combination of synthetic organic chemistry and structure-based design, two selective inhibitors of ARTD3 were discovered.

Full text of DOI:10.1021/jm401394u

Article
pubs.acs.org/jmc
Chemical Probes to Study ADP-Ribosylation: Synthesis
and Biochemical Evaluation of Inhibitors of the Human
ADP-Ribosyltransferase ARTD3/PARP3
Anders E. G. Lindgren,† Tobias Karlberg,‡ Torun Ekblad,‡ Sara Spjut,† Ann-Gerd Thorsell,‡
C. David Andersson,† Ton Tong Nhan,† Victor Hellsten,† Johan Weigelt,‡ Anna Linusson,†
Herwig Schuler,‡ and Mikael Elofsson*,†
̈
†Department of Chemistry, Umea University, SE-90187 Umea, Sweden
̊
̊
‡Department of Medicinal Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden
S
* Supporting Information
ABSTRACT: The racemic 3-(4-oxo-3,4-dihydroquinazolin-2-yl)-N-
[1-(pyridin-2-yl)ethyl]propanamide, 1, has previously been identified
as a potent but unselective inhibitor of diphtheria toxin-like ADP-
ribosyltransferase 3 (ARTD3). Herein we describe synthesis and
evaluation of 55 compounds in this class. It was found that the
stereochemistry is of great importance for both selectivity and
potency and that substituents on the phenyl ring resulted in poor
solubility. Certain variations at the meso position were tolerated and
caused a large shift in the binding pose. Changes to the ethylene
linker that connects the quinazolinone to the amide were also
investigated but proved detrimental to binding. By combination of
synthetic organic chemistry and structure-based design, two selective
inhibitors of ARTD3 were discovered.
difficult.1,10 Selective inhibitors would be invaluable tools to
improve our understanding of the medically important ARTD
proteins. We set out to develop small molecules, dubbed chem-
ical probes, for use as research tools to study ADP-ribosylation
and its role in cellular systems by selectively inhibiting single
family members.
Previously, a focused library consisting of 185 compounds,
many of them NAD+ mimics, was tested against 13 members of
the human ARTD family in a thermal shift assay.10 Together
with crystal structures of ARTD catalytic domain−ligand com-
plexes, this served as a powerful tool for elucidating how the
structure of a novel compound affected the binding mode,
activity and selectivity and allowed a considerably more efficient
structure-based approach.
1. INTRODUCTION
The human diphtheria toxin-like ADP-ribosyltransferase
(ARTD) family consists of 17 different proteins that all share
a conserved catalytic domain.1,2 These enzymes use nicotina-
mide adenine dinucleotide (NAD+) as a donor of ADP-ribose
units. The ARTD family can be further divided into two major
subgroups. The mono-ADP-ribose transferases (mARTs)
transfer single ADP-ribose moieties onto their targets, while
the poly-ADP-ribose polymerases (PARPs) catalyze the transfer
of ADP-ribose moieties onto growing chains of ADP-ribose on
their target proteins.3 In recognition of this, we chose to adopt
the nomenclature proposed by Hottiger et al.1 for the family of
proteins previously known as PARPs.
Although protein ADP-ribosylation was discovered more
than 40 years ago, its biological relevance is still poorly under-
stood, and most of the enzymes that catalyze ADP-ribosylation
are not well characterized.4 ADP-ribosylation plays critical roles
in several cellular processes, including cell differentiation, pro-
liferation, and maintenance of genome integrity.5,6 The two
most abundant ARTDs in human are ARTD1/PARP1 and its
closest relative ARTD2/PARP2. ARTD1, which is involved in
chromatin remodeling, DNA repair, and apoptosis,7 has been of
interest in drug discovery for over a decade, and ARTD in-
hibitors are currently undergoing clinical studies for treatment
of several forms of cancer.8,9
Herein we describe the design, synthesis and evaluation of 44
novel and 11 commercial compounds and their potency and
selectivity with respect to inhibition of ARTD3/PARP3 and
ARTD1/PARP1.
2. RESULTS AND DISCUSSION
Evaluation of the binding of 185 compounds to 13 ARTD
enzyme catalytic domains indicated that 1 (Table 1) was a
suitable starting point for a medicinal chemistry program. The
rationale for this choice was the apparently tight binding and
Currently known ARTD inhibitors are promiscuous within
the protein family, and studying individual ARTDs in vivo is
Received: July 5, 2013
Published: November 4, 2013
© 2013 American Chemical Society
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a
Table 1. Selectivity Data for 1, an ARTD3 Binder Identified by Use of a Thermal Stabilization Assay
a
Data were previously published.12 ARTD3 shares a higher degree of similarity with ARTD1 and ARTD2 than with ARTD5/tankyrase-1 and
ARTD8/PARP14.2,11
Scheme 1. Synthesis of 5a−f, 6a−m, 7a−f, 18a−f, and 19b−h
a
Table 2. Thermal Shift Data for 5a−i against Five Members of the ARTD Family
a
Data previously published.12 bCommercial compounds not synthesized as a part of this work. Racemic mixture.
c
selectivity suggested by the Tm-shift data (Table 1), paired with
straightforward synthesis of analogues that allowed a large
degree of diversity to be introduced in the last reaction step
(Scheme 1). The low molecular weight and low lipophilicity of
1 were also advantageous, as these properties tend to increase
as compounds are being further optimized.
2.1. Stereochemistry and Heteroatoms. Compound 1
was tested as a racemate. Thus, it was crucial to determine the
importance of the stereochemistry of the methyl group, and the
two enantiomers of 1 were synthesized separately according
to Scheme 1. The importance of the nitrogen in the pyridinyl
moiety was also investigated at an early stage, since a large
number of different benzylamines are readily available while
their pyridinyl analogues are scarce.13 Finally, two cyclized
analogues were synthesized in order to explore the effect of a
more rigid structure (Table 2).
A general synthetic route to compounds 5a−f is presented
in Scheme 1. Reacting 2-aminobenzonitrile, 2a, with succinic
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anhydride in toluene gave the resulting anilide, 3a, in 96% yield.
Compound 3a was then ring-closed to the quinazolinone main
fragment 4a in 68% yield by use of urea hydrogen peroxide
(UHP) and potassium carbonate.14 Finally, 5a−f were obtained
from 4a in 53−78% yield by use of N,N′-diisopropylcarbodii-
mide (DIC) and 1-hydroxybenzotriazole (HOAt)15 together
with the appropriate amines. The amines corresponding to 5c
and 5d, (R)- and (S)-1-(pyridin-2-yl)ethanamine, were not
commercially available but were instead synthesized from
their respective alcohols by use of diphenyl phosphoryl azide
(DPPA) to produce the azides, which were then reduced to the
amines under Staudinger reduction conditions.16
The recently synthesized 5a−f and the library substances
5g−i were evaluated by thermal shift assay. In brief, protein
solutions in phosphate-buffered saline (PBS) with tris(2-
carboxyethyl)phosphine (TCEP) and SyproOrange were added
to wells containing compound solution. Thermal stability was
then measured by monitoring SyproOrange fluorescence while
heating the samples, and determining the midpoints of the
transitions. Previous investigations showed that this assay gives
a good estimate of binding affinity, adequate to indicate the
direction in which a medicinal chemistry program should be
focused.10
As shown in Table 2, the enantiomers with S as absolute con-
figuration at the meso position (5b and 5d) produced con-
siderably higher ΔTm values and showed better selectivity
toward ARTD3 compared to the R enantiomers (5a and 5c).
Importantly, removing the pyridinyl ring in favor of an
unsubstituted phenyl ring did not lead to any significant drop
in either affinity or selectivity. Rigidifying the structure, as in 5e
and 5f, diminished the importance of the stereochemistry and
decreased selectivity toward ARTD3. Also, a substituent at the
meso position (5g and 5h) was necessary in order to maintain
affinity and selectivity. Finally, the poor effect of the racemate
5i indicated that shifting the nitrogen to the para position was
unfavorable. The crystal structure of the complex of ARTD3
and 5i can provide an explanation; this nitrogen is involved in
water-mediated hydrogen bonds with the carbonyl group of
Asp291, shifting the position of the ring about 1 Å outward
from the pocket compared to 5b, resulting in a poorer fit
(Figure 1a).
2.2. Substitution Patterns. On the basis of results from
the previous compounds in combination with the recently
determined crystal structures of ARTD3 with 5b and 5d12
(Figure 1b), it was decided that the ensuing work should be
focused on phenyl rings instead of their pyridinyl counterparts.
As shown in Figure 1, the bifurcated interaction between the
amide part of the quinazolinone and Gly385 in the backbone of
ARTD3 anchors the ligand inside the nicotinamide pocket.
This interaction is the hallmark of ARTD inhibitors based on
nicotinamide mimetics. In addition to these interactions, 5a and
5b form a water-mediated hydrogen bond between the amide
group carbonyl oxygen and the Asn387 side chain. The amide
group of the R enantiomer 5c (thus also 5a) is instead rotated
away from Asn387, resulting in a missing hydrogen-bonding
opportunity with an associated loss of affinity (Figure 1c).
Additionally, crystal structures show that the pyridinyl nitrogen
in 5d does not find any favorable interactions with ARTD3 and
is thus considered nonessential. However, the nitrogen does
produce favorable interactions with ARTD1 that slightly lower
the ARTD3 selectivity of this compound.12
Figure 1. Binding of inhibitors to the cosubstrate NAD+ binding site
of ARTD3. Consensus helix αF of the regulatory domain, which makes
up one side of the distal end of the NAD+ binding pocket, is shown in
green. (a) Compounds 5b (teal, PDB ID 4GV4) and 5i (salmon, PDB
ID 4L6Z) in ARTD3. The ring structure in 5i has moved about 1 Å
outward from the pocket compared to 5b. (b) Compounds 5b (teal,
PDB ID 4GV4) and 5d (orange, PDB ID 4GV0) in ARTD3. The
conformation of the protein is highly conserved and the binding poses
of the ligands are similar. (c) Compound 5c in ARTD3 (PDB ID
4GV2). The amide carbonyl is rotated away from Asn387.
A new series of compounds (Table 3, 6a−m) was designed
and synthesized via the above procedure (Scheme 1) in order
to further explore the importance of stereochemistry and func-
tionalization at the meso position. Compounds 6a−d probed
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a
Table 3. Thermal Shift Data for 6a−r against Five Members of the ARTD Family
a
b
Commercial compounds. Racemic mixture.
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Table 4. Thermal Shift Data for 7a−k against Five Members of the ARTD Family
Scheme 2. Synthesis of 7g
the possibilities of replacing the methyl group with larger sub-
stituents, while 6e investigated how rigidifying 5g would affect
the mode of binding. Compounds 6f−j were used to further
study whether or not a substituent was needed at the meso
position and how homobenzylic amides would influence the
affinity. Finally, 6k−m, with a chlorine substituent in the C7
position of the quinazolinone scaffold, were prepared to analyze
whether lipophilic functional groups were allowed in this part
of the binding pocket. Reaction conditions (Scheme 1)
were modified accordingly to prepare the chloro-substituted li-
gands; the appropriate chloro-substituted aniline, 2b, reacted
with succinic anhydride to give 3b in 64% yield, followed by
alkali-mediated cyclization to give 4b in 75% yield. Due to
purification issues, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetra-
methyluronium hexafluorophosphate (HATU)15 replaced
DIC/HOAt in some of the amide couplings, which gave yields
of 3−96% for this series of compounds. The reactions generally
showed quantitative conversion, and no byproducts derivable
from 4b were observed regardless of the coupling reagents
used. Thus, the low yields can be attributed to the difficulty of
purifying some of these compounds.
Most of the modifications made to these compounds (Table 3)
resulted in a decrease in thermal shift compared to 5b and 5d.
One definite exception was 6j, which, at the expense of decreased
selectivity, showed the same affinity toward ARTD3 as the two
previously top-ranked compounds. These results also indicated
that the chlorine substituent was undesirable, as 6k−m showed
diminished affinity for ARTD3 and two compounds in the series
showed selectivity toward ARTD1 or ARTD2 over ARTD3.
2.3. Role of the Linker Region. Focus was now shifted
from the amide part of the compounds to the ethylene linker
that connects the quinazolinone to the amide. By extending this
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Scheme 3. Synthesis of Intermediate 13
Scheme 4. Synthesis of 7h,i
Scheme 5. Synthesis of 7j,k
linker by one methylene group, it appeared possible to find
interactions similar to those of 6j. Synthesis of the required
scaffold was straightforward by replacing succinic anhydride
(Scheme 1) with glutaric anhydride and letting it react with
2-aminobenzonitrile, 2a, to give 3c in 84% yield. Compound 3c
was then ring-closed by the established procedure to give 4c in
70% yield.
Forcing the ligand into a different binding pose by intro-
ducing a double bond (7g, Table 4) was expected to impair its
affinity. In order to confirm this hypothesis, the compound was
synthesized by letting 2-aminobenzonitrile 2a react with
fumaryl chloride in 66% yield. The resulting product, 8, was
then ring-closed in 36% yield to give 9 under the same con-
ditions as in the previous cases (Scheme 2). The HATU-
mediated amide couplings then proceeded in 9−81% yield to
generate the first seven compounds. The low yields for some
of the compounds were again attributed to difficulties with
purification, as quantitative conversion of the carboxylic acid
was generally observed.
Finally, the effects of a shorter linker (7h,i) and of additional
possibilities for hydrogen bond formation by replacing the
amide with a urea moiety (7j,k) were investigated. These four
compounds shared the same synthetic pathway up to
intermediate 13 (Scheme 3), after which the synthesis diverged
in two separate directions. The synthesis of intermediate 13
started with the previously developed procedure, in which 2a
reacted with methoxyacetyl chloride to give 10 in 83% yield.
Compound 10 was then ring-closed by use of UHP and
potassium carbonate to give 11 in 77% yield. Subsequent ether
cleavage by 47% HBr proceeded in 90% yield to give 12, which
was finally reacted under Appel reaction conditions with car-
bon tetrabromide and triphenylphosphine to obtain 13 in 84%
yield.17
In order to synthesize 7h,i, intermediate 13 was treated with
potassium cyanide to produce nitrile 14 in 54% yield. The ester
15 was then obtained by dissolving 14 in ethanol and adding
acetyl chloride to form the corresponding imino ether, which
upon addition of water was hydrolyzed to the desired ester in
88% yield. Compounds 7h,i were then obtained in 76−81%
yield by direct transamidation of 15 (Scheme 4). Several other,
more direct, methods of synthesizing 7h,i were also investigated.
All of these were unsuccessful, mainly due to the instability of 13
or of the carboxylic acid analogue of 15.18,19
The two urea derivatives 7j,k were synthesized by treating
intermediate 13 with sodium azide to produce the azide ana-
logue, 16, in 98% yield. The azide was then reduced under
Staudinger conditions with triphenylphosphine and water to
generate the corresponding amine, 17, in 89% yield. In the final
step, the amines were reacted with triphosgene in order to
obtain the desired isocyanates, which then reacted with 17 to
form the desired urea derivatives in 38−42% yield (Scheme 5).
The elongated linker region of compounds 7a−f (Table 4)
resulted in reduced affinity across the board. However, the
reduction was modest for all of these compounds with the
exception of the 5b-analogous 7b, which produced a
considerable drop in affinity. The thermal shift data suggested
almost completely abolished binding for 7g (Table 4), which
confirmed the hypothesis that introduction of a double bond
would force the ligand into an unfavorable binding pose with an
associated decrease in affinity. Similar results were observed for
the two compounds 7h and 7i, where the linker length was
reduced by one methylene group. Finally, introducing an
alternative hydrogen-bond acceptor by replacing the amide with
a urea moiety did not seem to generate any new beneficial
interactions, as affinity dropped considerably for 7j compared
to its amide analogue 5b.
2.4. Fine Tuning of Substituents. Modifying the linker,
changing the meso-methyl substituent to a different function-
ality, or introducing a substituent in the C7 position of the
quinazolinone all had a negative impact on the ability of ligands
to stabilize ARTD3. However, the effect of adding substituents
to the phenyl ring still had to be investigated.
The crystal structure of ARTD3 in complex with 5b (Figure 1a)
was used to investigate which substituents would be accom-
modated at different positions. The structure suggested that ortho
substituents larger than fluorine were unsuitable due to steric
clashes with the protein. Similarly, meta substituents larger than
a chlorine were also unlikely to fit inside the binding pocket
without a major change of the binding pose. The para position
would likely be able to accommodate larger substituents. How-
ever, as the thermal shift data for the two library substances 18h
and 18i suggested that large substituents in this position would
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a
Table 5. Thermal Shift Data for 18a−f against Five Members of the ARTD Family
a
b
Commercial compounds. Racemic mixture.
greatly increase the affinity for ARTD1, we decided not to
introduce substituents larger than chlorine. Synthesis of 18a−f
(Table 5) was straightforward with yields of 50−97% for the final
amide coupling step following the synthetic strategy that was
already established for our previous ARTD3 ligands (Scheme 1).
Somewhat surprisingly, all compounds with small substitu-
ents on the phenyl ring (18a−g, Table 5) produced low
thermal shifts, and binding to ARTD3 seemed almost com-
pletely abolished. This in combination with the low solubility
observed during the preparation of the compounds suggest that
they are less suited for further investigations.
2.5. Exploring α-Amino Acid Analogues. The fact that
6j, based on phenylalaninol, proved to be a highly active
compound while all other, considerably smaller modifications
of 5b decreased the affinity stirred our curiosity regarding the
binding mode of this ligand. After the crystal structure of the
complex was solved (Figure 2), it was clear that the amine part
of 6j had turned 180° compared to 5b and 5d (Figure 2),
so that the phenyl ring was now pointing out of the binding
pocket. In order to investigate whether this discovery could lead
to further improvements over 5b, a final series of compounds
was synthesized by use of α-amino acid analogues as building
blocks (Table 6). Synthesis of these compounds followed the
previously established procedures with yields of 25−84% for
the final amide coupling step (Scheme 1).
The first four members of this series of compounds (Table 6)
showed reasonable thermal stabilization of the ARTDs (19a−d)
but poor selectivity (19b−d). Compound 19e seemed promising
with similar thermal shifts as 5b. However, in a cellular system
the ester moiety would likely be converted to the corresponding
carboxylic acid, 19f, which showed poor ARTD3 affinity. The
ester moiety in 19e forces the compound to bind in a slightly
shifted position in the nicotinamide pocket; however, the
overall protein−ligand interaction seems tighter in the pocket
(Figure S1 in Supporting Information). The two ester ana-
logues, 19g and 19h, again showed modest affinity but, together
with 19a, seemed fairly selective toward ARTD3. The phenyl
ring in 19a has also turned around as in 6j, and in addition,
rigidifying the linker resulted in the phenyl ring reaching out
even further out of the pocket, as observed in the crystal
structure (Figure S1 in Supporting Information). Compound
19c binds in a similar mode as 5b but lacks the phenyl ring
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2.6. Continued Characterization of Selected Com-
pounds. During the course of this medicinal chemistry project,
the thermal shift assay was used exclusively for evaluating
the presented compounds against a series of five ARTDs. To
verify the results, an enzymatic assay was employed to further
characterize and obtain half-maximal inhibitory concentration
(IC50) values for a subset of the most promising compounds
(Table 7). The criterion for selecting these compounds was
ΔTm > 6.5 °C against ARTD3. Additional compounds for
which we had obtained crystal complex structures were also
analyzed in order to verify our conclusions about the importance
of the stereochemistry and of the methyl group.
In brief, Ni2+ chelating plates were used to capture
hexahistidine-tagged ARTD catalytic domain fragments and
recombinant histone proteins. Biotinylated NAD+ was then
added in order to initiate ADP-ribosylation. Reaction products
were detected by chemiluminescence, and the assay was run
with [NAD+] near Km and in the presence of ARTD inhibitor.
IC50 determinations were conducted with ligand concentrations
between 10 nM and 450 μM.
As expected, the analysis showed that ΔTm values provide
only a rough estimate of the potency and selectivity of a com-
pound. While a moderately strong correlation between these
two sets of results was observed for ARTD3, it was difficult to
draw any such conclusions for ARTD1. ΔTm values were also
generally lower for ARTD1 compared to ARTD3 when similar
IC50 values were considered. However, our previous con-
clusions about how the stereochemistry of 5a−f relates to the
Figure 2. Compounds 6j (yellow, PDB ID 4L7L) and 5b (teal, PDB ID
4GV4) in ARTD3. The binding pose of the amine part is shifted
compared to 5b. The bifurcated interaction between the quinazolinone
and the backbone of protein is still present, but the phenyl ring has
turned around and is reaching out of the binding pocket. The
interaction between Asn387 and the ligand also appears to be missing.
The amide group has moved ∼1 Å toward the position of the structured
water in 5b; this water is not present in the 6j crystal structure.
structure, which might explain the smaller effect on ΔTm
observed for 19c (Figure S1 in Supporting Information).
Table 6. Thermal Shift Data for 19a−h against Five Members of the ARTD Family
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a
Table 7. Effects of Selected Compounds on Catalytic Activities of ARTD3 and ARTD1
a
Data were previously published for 5a (ME0327), 5b (ME0328), 5c (ME0354), and 5d (ME0355).12 Full-length ARTD3 was used in all cases.
ARTD1 catalytic domain fragment was used for all compounds, and the IC50 determinations for 5a−d were also expanded with full-length ARTD1.12
bFull-length ARTD1. We were unable to fit a curve to the data, but an IC50 >100 μM is estimated.
c
ARTD1/ARTD3 selectivity were further reinforced by the IC50
values: ARTD3 prefers the S enantiomer while ARTD1 prefers
the R enantiomer. Out of the five α-amino acid analogues that
were characterized, only 6j showed any selectivity for ARTD3;
the remaining four, 19b−e, were unselective or ARTD1-
selective despite what their ΔTm values suggested.
mode of binding of 6a (Figure S1 in Supporting Information).
Placement of the library compounds 18h and 18i in the
structures were also almost identical to 5b, with the larger
substituents on para position of the phenyl ring reaching into
the pocket. One difference in the protein was seen; the side chain
of Met402 shifted to accommodate the larger substituents.
Several crystal structures of ARTDs in complex with selected
compounds were used throughout the study to guide selection
of compounds, together with thermal shifts and IC50 values, for
further development. Compound 6a has an ethyl group in place
of the methyl on 5b. The larger substituent did not change the
3. CONCLUDING REMARKS
The potency and ARTD3 selectivity of the parent compound,
1, was improved by systematically investigating how these two
properties were affected by modifying different structural
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20a,b, 21a, and 21b were synthesized according to procedures
described in ref 14; analytical data were in agreement with those
already published. Compound 10−12 were synthesized according to
procedures described in ref 16; analytical data were in agreement with
those already published.
features. The stereochemistry and the methyl group proved
essential since the S enantiomer (5d) is ARTD3-selective while
the R enantiomer (5c) is ARTD1-selective. Removal of the
methyl group caused the thermal shifts to decrease, as
exemplified by 5h, and ring-closing to the indane produced
ARTD1 selectivity for both enantiomers, as exemplified by 5e,f
(Table 2). Replacing this group with larger substituents also
decreased the ARTD3 thermal shifts (Table 3). However, the
pyridinyl moiety could be replaced by a phenyl group with an
associated improvement of thermal shifts and selectivity (Table 2,
compound 5d). The linker region connecting this part to the
quinazolinone scaffold was also investigated; however, none of
these modifications improved binding properties (Table 4).
Adding substituents to the phenyl ring also reduced the thermal
shifts associated with these analogues (Table 5). Finally, a
series based on α-amino acid analogues as building blocks was
synthesized and tested (Table 6), and a crystal structure
showed that these compounds adopted a different binding pose
(Figure 2). However, most of them showed poor selectivity, the
exception being 6j, which had binding properties comparable to
those of 5d.
4.2. Synthetic Procedures. 4.2.1. Procedure A: Acylation of
Aniline (Exemplified by 3c). 2-Aminobenzonitrile (2a) (1500 mg,
12.7 mmol), and glutaric anhydride (1739 mg, 15.2 mmol) were
dissolved in toluene (15 mL). The mixture was heated to 90 °C for 4 h
under nitrogen. The solid material was filtered off and washed with
1
Et2O to give 3b (2476 mg, 84%). H NMR [400 MHz, (CD3)2SO] δ
12.08 (s, 1H), 10.14 (s, 1H), 7.80 (dd, J = 7.8, 1.5 Hz, 1H), 7.68 (ddd,
J = 8.6, 7.3, 1.4 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.34 (ddd, J = 8.2,
6.6, 1.6 Hz, 1H), 2.41 (t, J = 7.5 Hz, 2H), 2.31 (t, J = 7.5 Hz, 2H),
(quin, J = 7.5 Hz, 2H); 13C NMR [100 MHz, (CD3)2SO] δ 174.1,
171.2, 140.2, 133.7, 133.1, 125.6, 125.6, 116.9, 107.6, 34.7, 32.8, 20.4.
4.2.2. Procedure B: Cyclization of 2-Cyanoanilide (Exemplified by
4c). Compound 3c (2316 mg, 9.97 mmol), K2CO3 (2756 mg, 19.94
mmol), and UHP (2815 mg, 29.94 mmol) were dissolved in acetone/
H2O 1:1 (100 mL). The mixture was heated to 80 °C for 22 h. K2CO3
(2756 mg, 19.94 mmol) and UHP (2815 mg, 29.94 mmol) were
added and the mixture was heated to 80 °C overnight. pH was
adjusted to ∼4 with 6 M HCl. The solid material was filtered off and
1
washed with MeOH and DCM to give 4c (1626 mg, 70%). H NMR
A thermal shift assay was used throughout for compound
profiling. However, in order to verify our conclusions, a subset
of the best ARTD3 ligands was characterized with IC50 values
determined by an enzymatic activity assay. Using these assays,
we successfully identified two compounds, 5b (IC50 = 0.9 μM
[400 MHz, (CD3)2SO] δ 12.16 (s, 1H), 12.09 (s, 1H), 8.07 (dd,
J = 7.9, 1.6 Hz, 1H), 7.76 (ddd, J = 8.2, 7.2, 1.6 Hz, 1H), 7.60 (d, J =
8.1 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 2.64 (t, J = 7.5 Hz, 2H), 2.32 (t,
J = 7.4 Hz, 2H), 1.96 (quin, J = 7.5 Hz, 2H); 13C NMR [100 MHz,
(CD3)2SO] δ 174.0, 161.7, 156.8, 148.8, 134.2, 126.8, 127.0, 125.7,
120.9, 33.5, 32.8, 21.8.
against ARTD3 and 3.70 μM against ARTD1) and 6j (IC50
=
1.0 μM against ARTD3 and 6.46 μM against ARTD1), as
potent and selective inhibitors of ARTD3. These compounds
also maintain the advantageous low molecular weight and per-
meability of the parent compound, 1. The IC50 data also
verified the intriguing relationship between stereochemistry and
selectivity, where 5b and 5d are ARTD3-selective while their
enantiomers 5a and 5c are ARTD1-selective. Finally, one of
these compounds, 5b, was characterized in cellular assays,
proving that it is a potent and selective inhibitor of ARTD3
in vivo. No ARTD1 inhibitory effects were observed despite the
modest selectivity suggested by the IC50 values.12
While this compound shares structural features with several
previously reported ARTD inhibitors, it is important to note
that most of these previous compounds have been selective for
ARTD1, -2, -5, or -6 and mostly lack chiral centers, which
according to our study are important in order to differentiate
between different ARTDs.20−27
4.2.3. Procedure C: Amide Coupling of α-Methylbenzylamine
(Exemplified by 18b). Compound 4a (40 mg, 0.183 mmol), (S)-4-
chloro-α-methylbenzylamine (34.2 mg, 0.22 mmol), HATU (83.6 mg,
0.22 mmol), and TEA (61 μL, 0.44 mmol) were dissolved in DMF
(1 mL), and the mixture was stirred at room temperature overnight.
The solid residue was filtered off to give (S)-N-[1-(4-chlorophenyl)-
ethyl]-3-(4-oxo-3H-quinazolin-2-yl)propanamide, 18b (63 mg, 97%).
1H NMR [400 MHz, (CD3)2SO] δ 12.12 (s, 1H), 8.44 (d, J = 7.8 Hz,
1H), 8.07 (dt, J = 7.9 Hz, 1H), 7.78 (t, J = 7.7 Hz, 1H), 7.55 (d, J = 8.1
Hz, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.27 (q, J = 8.7 Hz, 4H), 4.87 (quin,
J = 7.2 Hz, 1H), 2.91−2.77 (m, 2H), 2.75−2.58 (m, 2H), 1.31 (d, J =
7.0 Hz, 3H); 13C NMR [100 MHz, (CD3)2SO] δ 170.2, 161.6, 156.7,
148.7, 143.9, 134.2, 130.9, 128.0 (2C), 127.7 (2C), 126.7, 125.9, 125.7,
120.9, 47.3, 31.3, 29.6, 22.3.
4.2.4. Procedure D: Appel Reaction (Exemplified by 13).
Triphenylphosphine (152 mg, 0.58 mmol) was added to a mixture
of 12 and CBr4 (193 mg, 0.58 mmol) in DCM (8 mL) at 0 °C. The
reaction was stirred at 0 °C for 1 h, and then triphenylphosphine
(152 mg, 0.58 mmol) and CBr4 (193 mg, 0.58 mmol) were added.
The mixture was stirred at 0 °C for 1 h and then at room temperature
for 20 h. Purification by column chromatography on silica gel (DCM/
MeOH 96:4) followed by careful trituration with DCM to remove
PPh3O gave 2-(bromomethyl)quinazolin-4(3H)-one, 13 (196 mg,
84%). 1H NMR [400 MHz, (CD3)2SO3] δ 12.55 (s, 1H), 8.11 (dd, J =
7.9, 1.5 Hz, 1H), 7.82 (ddd, J = 8.0, 7.2, 1.6 Hz, 1H), 7.66 (d, J = 8.2
Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 4.40 (s, 2H); 13C NMR [100 MHz,
(CD3)2SO3] δ 161.5, 152.8, 148.3, 134.6, 127.2 (2C), 125.8, 121.1, 29.7.
4.2.5. Procedure E: Transformation of Nitrile to Ester via the
Corresponding Imino Ether (Exemplified by 15). Compound 14
(55 mg, 0.30 mmol) was dissolved in a mixture of THF (0.3 mL) and
ethanol (2.2 mL). AcCl (1.4 mL, 19.30 mmol) was added dropwise at
0 °C. The reaction was stirred at room temperature for 6 h and then
concentrated under reduced pressure before water (1 mL) was added.
The mixture was stirred at room temperature for 30 min and then
concentrated and purified by column chromatography on silica gel
(DCM/MeOH 99:1) to give ethyl 2-(4-oxo-3H-quinazolin-2-yl)-
4. EXPERIMENTAL SECTION
4.1. General Chemical Procedures. LC-MS analysis was carried
out on a Waters LC system equipped with an Xterra MS C18 18.5 μm
4.6 × 50 mm column and an eluent system consisting of MeCN in
water, both of which contained 0.2% formic acid. Detection was
performed at 214 and 254 nm. Mass spectra were obtained by use of a
Waters micromass ZG 2000, using both positive and negative elec-
trospray ionization. 1H NMR and 13C NMR spectra were recorded on
a Bruker DRX-400 spectrometer in CDCl3 solution [residual CHCl3
(δH 7.26 ppm, δC 77.16 ppm) as internal standard] or in (CD3)2SO
solution [residual (CH3)2SO (δH 2.50 ppm, δC 39.52 ppm) as internal
standard]. Optical rotations were measured on a Perkin-Elmer
polarimeter 343 at 20 °C. A Biotage Initiator 400W was used for
microwave heating. All target compounds were ≥95% pure according
to LC-MS UV traces. Chiral HPLC was performed on a Beckman
system equipped with a Pirkle Covalent (S,S) Whelk-O 1 10/100
Krom FEC 25 cm × 4.6 mm column or a Supelco Astec Chirobiotic T
25 cm × 4.6 mm, 7 μm column and an eluent system consisting of
2-propanol and hexane in a 1:1 ratio. Detection was performed at
254 nm with a System Gold 166 detector. Compounds 3a, 4a, 5a−d, ,
1
acetate, 15 (61 mg, 88%). H NMR (400 MHz, CDCl3) δ 11.02 (s,
1H), 8.28 (d, J = 7.8 Hz, 1H), 7.82−7.69 (m, 2H), 7.51 (t, J = 7.5 Hz,
1H), 4.27 (q, J = 7.2 Hz, 2H), 3.90 (s, 2H), 1.31 (t, J = 7.2 Hz, 3H);
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13C NMR (100 MHz, CDCl3) δ 168.5, 162.1, 149.7, 147.9, 135.2,
127.5, 126.9, 126.7, 121.1, 62.4, 40.2, 14.2.
glycerol, 0.2 M sodium chloride, and 1−2 mM compound, and then
frozen in liquid nitrogen. Diffraction data were collected on the frozen
crystals on synchrotron beamlines I04 at Diamond (Oxfordshire, U.K.)
and on BL14.1 at Bessy (Berlin, Germany). All data were indexed and
integrated by use of XDS software.28 Manual model building was done
with Coot29 and refinement with Refmac530 or Buster.31 The refine-
ment progress was monitored by decreasing R and Rfree values.
Statistics for data collection, processing, and refinement can be are
given in Table 8.
4.2.6. Procedure F: Transamidation by Use of α-Methylbenzyl-
amine (Exemplified by 7h). Compound 15 (15 mg, 0.065 mmol) and
(R)-(+)-1-phenylethylamine (0.5 mL, 3.91 mmol) were stirred at 45
°C under nitrogen overnight. The precipitate was filtered off, washed
with EtOAc, and dried in vacuo to give (R)-2-(4-oxo-3H-quinazolin-2-
yl)-N-(1-phenylethyl)acetamide, 7h (16 mg, 81%). 1H NMR [400
MHz, (CD3)2SO] δ 12.12 (s, 1H), 8.64 (d, J = 7.9 Hz, 1H), 8.09 (dd,
J = 7.9, 1.5 Hz, 1H), 7.79 (ddd, J = 8.2, 7.1, 1.6 Hz, 1H), 7.59 (d, J =
8.1 Hz, 1H), 7.48 (ddd, J = 8.0, 7.2, 0.9 Hz, 1H), 7.40−7.29 (m, 4H),
7.26−7.20 (m, 1H), 4.93 (quin, J = 7.2 Hz, 1H), 3.59 (s, 1H), 1.39 (d,
J = 7.0 Hz, 3H); 13C NMR [100 MHz, (CD3)2SO] δ 165.9, 161.6,
152.6, 148.7, 144.3, 134.2, 128.2 (2C), 126.7 (2C), 126.1, 126.0 (2C),
125.7, 120.9, 48.2, 41.9, 22.5.
4.6. Enzymatic Activity Assay. ADP-ribosyltransferase activity
was measured as described in detail before.14
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional text and 54 figures, showing binding of inhibitors 6a,
18h, 18i, 19a, 19c, and 19e to ARTD3 and copies of NMR
spectra and detailed experimental procedures for compounds
3b,c, 4b,c, 5e,f, 6a−m, 7a−k, 8, 9, 13−17, 18a−f, 19a−h, 22,
and 23. This material is available free of charge via the Internet
at http://pubs.acs.org.
4.2.7. Procedure G: SN2 Reaction at Benzylic Position
(Exemplified by 16). Compound 13 (730 mg, 3.05 mmol) and
NaN3 (238 mg, 3.66 mmol) in acetone/water 2:1 (40 mL) was divided
into two 10−20 mL vials and heated to 70 °C for 20 min by
microwave irradiation. The solution was diluted with DCM and
washed with brine. The organic phase was dried (Na2SO4), filtered,
concentrated, and purified by column chromatography on silica gel
(DCM/MeOH 98:2) gave 2-(azidomethyl)quinazolin-4(3H)-one, 16
Accession Codes
1
Coordinates and structure factors for the crystal structures have
been deposited in the Protein Data Bank under Accession
Codes 4L7L (6j), 4L6Z (5i), 4L7N (18h), 4L7O (18i), 4L70
(6a), 4L7U (19e), 4L7R (19c), and 4L7P (19a).
(600 mg, 97%). H NMR [400 MHz, (CD3)2SO3] δ 12.39 (s, 1H),
8.11 (dd, J = 7.9, 1.5 Hz, 1H), 7.83 (ddd, J = 8.2, 7.2, 1.6 Hz, 1H), 7.67
(d, J = 8.2 Hz, 1H), 7.53 (ddd, J = 8.0, 7.1, 0.9 Hz, 1H), 4.38 (s, 2H);
13C NMR [100 MHz, (CD3)2SO3] δ 161.4, 152.3, 148.2, 134.5, 127.1,
126.8, 125.8, 121.3, 50.9.
4.2.8. Procedure H: Staudinger Reduction (Exemplified by 17).
Compound 16 (400 mg, 1.98 mmol) and triphenylphosphine
(623 mg, 2.37 mmol) were dissolved in THF (10 mL), and H2O
(0.53 mL, 29.65 mmol) was added. The mixture was stirred at room
temperature under nitrogen for 19 h and then concentrated and
purified by column chromatography on silica gel (DCM/MeOH 9:1)
to give 2-(aminomethyl)quinazolin-4(3H)-one, 17 (309 mg, 89%). 1H
NMR [400 MHz, (CD3)2SO3] δ 8.10 (dd, J = 7.9, 1.3 Hz, 1H), 7.78
(ddd, J = 8.2, 7.1, 1.5 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.47 (ddd, J =
8.4, 7.4, 0.9 Hz, 1H), 5.43 (s, 2H), 3.65 (s, 2H); 13C NMR [100 MHz,
(CD3)2SO3] δ 161.7, 158.5, 148.7, 134.3, 126.7, 126.0, 125.8, 121.1,
44.7.
AUTHOR INFORMATION
Corresponding Author
*Phone +46-90-7869328; fax +46-90-7867655; email mikael.
elofsson@chem.umu.se.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the beamline staff at the Helmholtz-Zentrum Berlin
(HZB) at BESSY II (Berlin, Germany) and the Diamond Light
Source (Didcot, U.K.) for excellent support, and Martin Moche
(Protein Science Facility, Karolinska Institutet, Stockholm,
Sweden) for assistance with synchrotron data collection. This
work was financed by the Swedish Foundation for Strategic
Research (RBc08-0014). H.S. thanks the Structural Genomics
Consortium for additional support.
4.2.9. Procedure I: Urea Derivative Synthesis Using Triphosgene
(Exemplified by 7j). (S)-(−)-1-Phenylethylamine (26 μL, 0.206
mmol) was dissolved in EtOAc (1 mL), and triphosgene (30.5 mg,
0.103 mmol) was added. The solution was heated to 80 °C for 4 h in a
sealed vial. The solvent was evaporated and THF (1 mL) was added,
followed by 17 (30 mg, 0.17 mmol). The mixture was stirred at room
temperature for 3 h and then concentrated and purified by column
chromatography on silica gel (DCM/MeOH 95:5) to give (S)-1-[(4-
oxo-3H-quinazolin-2-yl)methyl]-3-(1-phenylethyl)urea, 7j (21 mg,
ABBREVIATIONS USED
■
1
38%). H NMR [400 MHz, (CD3)2SO] δ 12.13 (s, 1H), 8.09 (dd,
Ac, acetate; ADP, adenosine diphosphate; ARTD, diphtheria
toxin-like ADP-ribosyltransferases; DCM, dichloromethane;
DIC, N,N′-diisopropylcarbodiimide; DMF, N,N′-dimethylfor-
mamide; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic
acid; DPPA, diphenyl phosphoryl azide; EC50, effective
concentration 50%; Et, ethyl; HATU, O-(7-azabenzotriazol-1-
yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate;
HOAt, 1-hydroxybenzotriazole; LC-MS, liquid chromatography
mass spectrometry; mART, mono-ADP-ribose transferase; Me,
methyl; NAD+, nicotinamide adenine dinucleotide; PARP,
poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline;
Ph, phenyl; TCEP, tris(2-carboxyethyl)phosphine; TEA,
triethylamine; THF, tetrahydrofuran; Tm, midpoint of tran-
sition; UHP, urea hydrogen peroxide
J = 7.9, 1.6 Hz, 1H), 7.80 (ddd, J = 8.4, 6.7, 1.5 Hz, 1H), 7.61 (d, J =
8.2 Hz, 1H), 7.48 (ddd, J = 8.1, 7.1, 0.9 Hz, 1H), 7.34−7.26 (m, 4H),
7.23−7.16 (m, 1H), 6.89 (d, J = 8.1 Hz, 1H), 6.37 (t, J = 5.7 Hz, 1H),
4.76 (quin, J = 7.4 Hz, 1H), 4.18 (d, J = 5.7 Hz, 2H), 1.34 (d, J = 7.0
Hz, 3H); 13C NMR [100 MHz, (CD3)2SO] δ 161.4, 157.3, 155.3,
148.5, 145.6, 134.4, 128.2 (2C), 126.8, 126.4, 126.2, 125.8 (2C), 125.7,
121.2, 48.7, 42.2, 23.3.
4.3. Thermal Shift Assay. Thermal shift assays were carried out
with ARTD catalytic domain fragments as described in detail before.12
The results are expressed as ΔTm in degrees Celsius, indicating the
shift in protein melting temperature upon ligand binding. Values
reported are means SD of 2−3 independent determinations, each
with three or more replicates.
4.4. Protein Crystallization. Crystals of ARTD3 complexes with
compounds 5i, 6a, 6j, 18h, 18i, 19a, 19c, and 19e were obtained by
the sitting drop vapor diffusion method in 96-well plates (Corning),
similarly as previously described.14
4.5. Data Collection, Structure Solution, and Refinement.
Crystals were briefly transferred to a cryo solution consisting of 1.7−
2.0 M DL-malic acid, 0.1 M bis-tris-propane, pH 7.0, 20−25% (w/v)
REFERENCES
■
(1) Hottiger, M. O.; Hassa, P. O.; Luscher, B.; Schuler, H.; Koch-
Nolte, F. Toward a unified nomenclature for mammalian ADP-
ribosyltransferases. Trends Biochem. Sci. 2010, 35, 208−219.
̈
̈
9567
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Article
(2) Karlberg, T.; Langelier, M. F.; Pascal, J. M.; Schuler, H. Structural
Mihara, K.; Matsuoka, N.; Hattori, K. Discovery of potent and
selective PARP-1 and PARP-2 inhibitors: SBDD analysis via a
combination of X-ray structural study and homology modeling. Bioorg.
Med. Chem. 2006, 14, 1378−1390.
(23) Iwashita, A.; Hattori, K.; Yamamoto, H.; Ishida, J.; Kido, Y.;
Kamijo, K.; Murano, K.; Miyake, H.; Kinoshita, T.; Warizaya, M.;
Ohkubo, M.; Matsuoka, N.; Mutoh, S. Discovery of quinazolinone and
quinoxaline derivatives as potent and selective poly(ADP-ribose)
polymerase-1/2 inhibitors. FEBS Lett. 2005, 579, 1389−1393.
(24) Shulz, M. D.; Cheung, A. K.; Kirby, C. A.; Firestone, B.; Fan, J.;
Chen, C. H.; Chen, Z.; Chin, D. N.; DiPietro, L.; Fazal, A.; Fend, Y.;
Fortin, P. D.; Gould, T.; Lagu, B.; Lei, H.; Lenoir, F.; Majumdar, D.;
Ochala, E.; Palermo, M. G.; Pham, L.; Pu, M.; Smith, T.; Stams, T.;
̈
biology of the writers, readers, and erasers in mono- and poly(ADP-
ribose) mediated signaling. Mol. Aspects Med. 2013, 3410881108.
(3) Kim, M. Y.; Zhang, T.; Kraus, W. L. Poly(ADP-ribosyl)ation by
PARP-1: ‘PAR-laying’ NAD+ into a nuclear signal. Genes Dev. 2005, 19,
1951−1967.
(4) Gibson, B. A.; Kraus, W. L. New insights into the molecular and
cellular functions of poly(ADP-ribose) and PARPs. Nat. Rev. Mol. Cell.
Biol. 2012, 13, 411−424.
(5) Burkle, A. Poly(ADP-ribose). The most elaborate metabolite of
̈
NAD+. FEBS J. 2005, 272, 4576−4589.
(6) Schreiber, V.; Dantzer, F.; Ame, J. C.; de Murcia, G. Poly(ADP-
ribose): novel functions for an old molecule. Nat. Rev. Mol. Cell Biol.
2006, 7, 517−528.
́
Tomlinson, R. C.; Toure, B. B.; Visser, M.; Wang, R. M.; Waters, N. J.;
Shao, W. Identification of NVP-TNKS656: The use of structure-
efficiency relationships to generate a highly potent, selective, and orally
active tankyrase inhibitor. J. Med. Chem. 2013, 56, 6495−6511.
(25) Pellicciari, R.; Camaioni, E.; Costantino, G.; Formentini, L.;
Sabbatini, P.; Venturoni, F.; Eren, G.; Bellocchi, D.; Chiarugi, A.;
Moroni, F. On the way to selective PARP-2 inhibitors. Design,
synthesis, and preliminary evaluation of a series of isoquinolinone
derivatives. ChemMedChem 2008, 3, 914−923.
(26) Perkins, E.; Sun, D.; Nguyen, A.; Tulac, S.; Francesco, M.;
Tavana, H.; Nguyen, H.; Tugendreich, S.; Barthmaier, P.; Couto, J.;
Yeh, E.; Thode, S.; Jarnagin, K.; Jain, A.; Morgans, D.; Melese, T.
Novel inhibitors of poly(ADP-ribose) polymerase/PARP1 and PARP2
identified using a cell-based screen in yeast. Cancer Res. 2001, 61,
4175−4183.
(7) Hassa, P. O.; Hottiger, M. O. The diverse biological roles of
mammalian PARPs, a small but powerful family of poly-ADP-ribose
polymerases. Front. Biosci. 2008, 13, 3046−3082.
(8) Underhill, C.; Toulmonde, M.; Bonnefoi, H. A review of PARP
inhibitors: from bench to bedside. Ann. Oncol. 2011, 22, 268−279.
(9) Ekblad, T.; Camaioni, E.; Schuler, H.; Macchiarulo, A. PARP
̈
inhibitors: polypharmacology vs selective inhibition. FEBS J. 2012,
280, 3563−3575.
(10) Wahlberg, E.; Karlberg, T.; Kouznetsova, E.; Markova, N.;
̈
Macchiarulo, A.; Thorsell, A.; Pol, E.; Frostell, Å.; Ekblad, T.; Oncu,
̈
D.; Kull, B.; Robertson, G.; Pellicciari, R.; Schuler, H.; Weigelt, J.
Family wide chemical profiling and structural analysis of PARP and
̈
Tankyrase inhibitors. Nat. Biotechnol. 2012, 30, 283−288.
(11) Lehtio, L.; Jemth, A. S.; Collins, R.; Loseva, O.; Johansson, A.;
̈
(27) Hattori, K.; Ishida, J.; Iwashita, A.; Kamijo, K.; Kido, Y.;
Matsuoka, N.; Miyake, H.; Nakanishi, I.; Ohkuno, M.; Yamamoto, H.;
Yamazaki, S. Quinazolinone derivatives. Patent Application PCT/
JP2001/010601, 2002.
Markova, N.; Hammarstrom, M.; Flores, A.; Holmberg-Schiavone, L.;
̈
Weigelt, J.; Helleday, T.; Schuler, H.; Karlberg, T. Structural basis for
̈
inhibitor specificity in human poly(ADP-ribose) polymerase-3. J. Med.
Chem. 2009, 52, 3108−3111.
(12) Lindgren, A. E. G.; Karlberg, T.; Thorsell, A.-G.; Hesse, M.;
Spjut, S.; Ekblad, T.; Andersson, C. D.; Pinto, A. F.; Weigelt, J.;
(28) Kabsch, W. XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr.
2010, 66, 125−132.
(29) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and
development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010,
66, 486−501.
Hottiger, M. O.; Linusson, A.; Elofsson, M.; Schuler, H. A PARP
̈
inhibitor with selectivity toward ADP-ribosyltransferase ARTD3/
PARP3. ACS Chem. Biol. 2013, 8, 1698−1703.
́
(30) Murshudov, G. N.; Skubak, P.; Lebedev, A. A.; Pannu, N. S.;
(13) Several of the major retailers of fine chemicals were investigated.
(14) Bavetsias, V. A facile route to quinazolin-4(3H)-ones
functionalised at the 2-position. Synth. Commun. 1998, 28, 4547−
4559.
Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A.
REFMAC5 for the refinement of macromolecular crystal structures.
Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 355−367.
(31) Bricogne, G.; Blanc, E.; Brandl, M.; Flensburg, C.; Keller, P.;
Paciorek, W.; Roversi, P.; Sharff, A.; Smart, O. S.; Vonrhein, C.;
Womack, T. O. BUSTER, version 2.11.5; Global Phasing Ltd.,
Cambridge, U.K., 2011.
(15) Valeur, E.; Bradley, M. Amide bond formation: beyond the myth
of coupling reagents. Chem. Soc. Rev. 2009, 38, 606−631.
̈
(16) Staudinger, H.; Meyer, J. Uber neue organische Phosphorver-
bindungen III. Phosphinmethylenderivate und Phosphinimine. Helv.
Chim. Acta 1919, 2, 635−646.
(17) Appel, R. Tertiary phosphane/tetrachloromethane, a versatile
reagent for chlorination, dehydration, and P-N linkage. Angew. Chem.,
Int. Ed. 1975, 14, 801−811.
(18) Xu, W.; Fu, H. Amino acids as the nitrogen-containing motifs in
copper-catalyzed domino synthesis of N-heterocycles. J. Org. Chem.
2011, 76, 3846−3852.
(19) Other methods that were attempted: reaction of 2a with
ethyl(chloroformyl) acetate followed by cyclization to give 15 in two
steps; palladium-catalyzed aminocarbonylation of 13; acidic hydrolysis
of 14 to the corresponding carboxylic acid; and CuBr-catalyzed
synthesis of the carboxylic acid analogue of 15 starting from 2-
bromobenzamide and L-aspartic acid. All of these worked poorly or not
at all.
(20) Bregman, H.; Gunaydin, H.; Gu, Y.; Schneider, S.; Wilson, C.;
DiMauro, E. F.; Huang, X. Discovery of a class of novel tankyrase
inhibitors that bind to both the nicotinamide pocket and the induced
pocket. J. Med. Chem. 2013, 56, 1341−1345.
(21) Hattori, K.; Kido, Y.; Yamamoto, H.; Ishida, J.; Iwashita, A.;
Mihara, K. Rational design of conformationally restricted quinazoli-
none inhibitors of poly(ADP-ribose)polymerase. Bioorg. Med. Chem.
Lett. 2007, 17, 5577−5581.
(22) Ishida, J.; Yamamoto, H.; Kido, Y.; Kamijo, K.; Murano, K.;
Miyake, H.; Ohkubo, M.; Kinoshita, T.; Warizaya, M.; Iwashita, A.;
9568
dx.doi.org/10.1021/jm401394u | J. Med. Chem. 2013, 56, 9556−9568