Discovery of a novel allosteric inhibitor scaffold for polyadenosine-diphosphate-ribose polymerase 14 (PARP14) macrodomain 2

Article abstract of DOI:10.1016/j.bmc.2018.03.020

The polyadenosine-diphosphate-ribose polymerase 14 (PARP14) has been implicated in DNA damage response pathways for homologous recombination. PARP14 contains three (ADP ribose binding) macrodomains (MD) whose exact contribution to overall PARP14 function in pathology remains unclear. A medium throughput screen led to the identification of N-(2(-9H-carbazol-1-yl)phenyl)acetamide (GeA-69, 1) as a novel allosteric PARP14 MD2 (second MD of PARP14) inhibitor. We herein report medicinal chemistry around this novel chemotype to afford a sub-micromolar PARP14 MD2 inhibitor. This chemical series provides a novel starting point for further development of PARP14 chemical probes.

Full text of DOI:10.1016/j.bmc.2018.03.020

Accepted Manuscript
Discovery of a novel allosteric inhibitor scaffold for polyadenosine-diphos-
phate-ribose polymerase 14 (PARP14) macrodomain 2
Moses Moustakim, Kerstin Riedel, Marion Schuller, Andrè P. Gehring, Octovia
P. Monteiro, Sarah P. Martin, Oleg Fedorov, Jag Heer, Darren J. Dixon, Jonathan
M. Elkins, Stefan Knapp, Franz Bracher, Paul E. Brennan
PII:
S0968-0896(18)30337-7
https://doi.org/10.1016/j.bmc.2018.03.020
BMC 14258
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
14 February 2018
9 March 2018
10 March 2018
Please cite this article as: Moustakim, M., Riedel, K., Schuller, M., Gehring, A.P., Monteiro, O.P., Martin, S.P.,
Fedorov, O., Heer, J., Dixon, D.J., Elkins, J.M., Knapp, S., Bracher, F., Brennan, P.E., Discovery of a novel allosteric
inhibitor scaffold for polyadenosine-diphosphate-ribose polymerase 14 (PARP14) macrodomain 2, Bioorganic &
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.03.020
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Discovery of a novel allosteric inhibitor scaffold for polyadenosine-
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Leave this area blank for abstract info.
†a,b,c
†d
a,b
d
Moses Moustakim,
Kerstin Riedel, Marion Schuller, Andrè P. Gehring,
a a,b e
a,b
c
a,f
g
*d
Octovia P. Monteiro, Sarah Martin, Oleg Federov, Jag Heer, Darren J. Dixon, Jonathan M. Elkins, Stefan Knapp, Franz Bracher, and Paul E.
a,b
Brennan
a
b
Structural Genomics Consortium, University of Oxford, ORCRB, Old Road Campus, Headington, Oxford, Oxfordshire, OX3 7DQ, UK. Target Discovery Institute, University of Oxford, NDM Research Building, Old Road Campus, Headington,
c
d
Oxford, Oxfordshire, OX3 7FZ, UK. Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, OX1 3TA, UK. Fakultät Chemie und Pharmazie, Ludwig-Maximilians-Universität München, München, 81377,
e
f
Germany. UCB Pharma Ltd, Slough, SL1 3WE, UK. Structural Genomics Consortium, Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz, Campinas, SP, 13083-886, Brazil.
g
Johann Wolfgang Goethe-University,Institute for PharmaceuticalChemistry and Buchmann Institute for Life Sciences, Frankfurt am Main, 60438, Germany.

Bioorganic
&
Medicinal
Chemistry
Discovery of a novel allosteric inhibitor scaffold for polyadenosine-diphosphate-
ribose polymerase 14 (PARP14) macrodomain 2
†
a,b,c
†d
a,b
d
a,b
Moses Moustakim,
Kerstin Riedel, Marion Schuller, Andrè P. Gehring, Octovia P. Monteiro,
e
a,b
f
c
a,g
h, i
Sarah P. Martin, Oleg Fedorov, Jag Heer, Darren J. Dixon, Jonathan M. Elkins, Stefan Knapp,
Franz Bracher, and Paul E. Brennan
*d
a,b
a
Structural Genomics Consortium, University of Oxford, ORCRB, Old Road Campus, Headington, Oxford, Oxfordshire, OX3 7DQ, UK.
Target Discovery Institute, University of Oxford, NDM Research Building, Old Road Campus, Headington, Oxford, Oxfordshire, OX3 7FZ, UK.
Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, OX1 3TA, UK.
Pharmacy Department- Center for Drug Research, Ludwig-Maximilians University, Munich, 81377, Germany.
Charles River, Chesterford Research Park, CB10 1XL, UK.
UCB Pharma Ltd, Slough, SL1 3WE, UK.
Structural Genomics Consortium, Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz, Campinas, SP, 13083-886, Brazil.
Johann Wolfgang Goethe-University,Institute for PharmaceuticalChemistry and Buchmann Institute for Life Sciences, Frankfurt am Main, 60438, Germany.
German Cancer Centre (DKFZ) and DKTK site Frankfurt/Mainz, 60590, Germany.
b
c
d
e
f
g
h
i
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The polyadenosine-diphosphate-ribose polymerase 14 (PARP14) has been implicated in DNA
damage response pathways for homologous recombination. PARP14 contains three (ADP ribose
binding) macrodomains (MD) whose exact contribution to overall PARP14 function in
pathology remains unclear. A medium throughput screen led to the identification of N-(2(-9H-
carbazol-1-yl)phenyl)acetamide (GeA-69, 1) as a novel allosteric PARP14 MD2 (second MD of
PARP14) inhibitor. We herein report medicinal chemistry around this novel chemotype to afford
a sub-micromolar PARP14 MD2 inhibitor. This chemical series provides a novel starting point
for further development of PARP14 chemical probes.
Available online
Keywords:
PARP
Poly-ADP ribsose
Macrodomain
Inhibitor Design
2
009 Elsevier Ltd. All rights reserved.
1
.
Introduction
small molecule PARP14 inhibitors have been reported and many
9
have suffered from a lack of selectivity. Most examples of
Poly-(ADP ribose) Polymerases (PARPs) are ADP-ribosyl
transferase enzymes which post-translationally modify substrate
proteins. Of at least 17 human family members of PARPs a sub-
set, referred to as mono(ADP-ribose)transferases (mARTs), are
capable of transferring on a single ADP unit to a given substrate.
10
PARP inhibitors have targeted the catalytic domain (ART) such
as a recent example by Upton and coworkers who identified
1
10e
moderately selective PARP14 inhibitors however to date no
PARP14 modulators targeting other domains such as the
2
11
macrodomains have been reported until recently.
PARP14 (ARTD8) is the largest of the mARTs and contains
multiple domains including an ADP ribose transferase domain
(
ART), a WWE domain, two (RNA binding) RRM repeats and
3
three (ADP-ribose binding) macrodomains. PARP14 was found
to be highly expressed in B-cell lymphoma and hepatocellular
carcinoma and has been associated with poor patient prognosis.
4
Furthermore PARP14 has been linked to inhibition of pro-
apoptotic kinase JNK1 which activates pyruvate kinase M2
isoform (PKM2) which in turn promotes a higher rate of
Figure 1. Initial hit PARP14 MD2 inhibitor GeA-69 (1) and sulfonamide
analogue 2.
PARP14 contains three macrodomain modules (MD1, MD2
and MD3); biophysical characterisation of macrodomain:ADP-
ribsose peptide binding was carried out revealing MD2 as the
5
glycolysis in cancer (Warburg effect) shown in some contexts to
6
be regulated by high MYC expression. Despite links with cancer
5,
7
1b, c, 7-8
pathogenesis
and inflammatory diseases,
only a few
—
——

Paul E. Brennan. Tel.: +44(0)1865 612932; e-mail: paul.brennan@sgc.ox.ac.uk, Franz Bracher. Tel. +49(0)89 218077301; e-mail:
†
franz.bracher@cup.uni-muenchen.de. These authors contributed equally to this work.

most potent ADP ribsosyl peptide binding domain and therefore
the most likely to deliver a functional effect through small
molecule inhibition (PARP14 MD1/ADP-ribose peptide K 137
D
±
7 μM, PARP14 MD2/ADP-ribose peptide K 6.8 ± 0.1 μM,
D
PARP14 MD3/ADP-ribose peptide K 15 ± 0.9 μM, Supp. Info
D
Figure 1).
An initial medium throughput screen (~50k compounds) revealed
compound GeA-69 (1) as a sub-micromolar inhibitor of PARP14
MD2 ADP-ribose binding as measured by AlphaScreen™, ITC
Figure 3. SAR studies of carbazoles GeA-69 (1) and 2.
2
-Pyridyl compound 13 and 4-pyrimidyl analogue 14 were
1
1a
and BLI. A co-crystal structure of closely related sulfonamide
derivative 2 with PARP14 MD2, which was obtained in the
course of the project revealed a unique allosteric binding mode
for this inhibitor (PDB ID 5O2D). Overlay of this structure with
bound ADP-ribose from a previously published co-crystal
obtained by regioselective nucleophilic addition of 1,9-dilithiated
carbazole (obtained in situ from 1-bromocarbazole and 4 equiv.
tert-butyllithium) to pyridine and pyrimidine, followed by
spontaneous rearomatisation during workup. The obtained
(hetero)arylcarbazoles are shown in Figure 4.
1
2
structure of PARP14 MD2 (PDB ID 3Q71) showed that
compound 2 occupied a novel pocket adjacent to the binding site
1
1a
for ADP-ribose (Figure 2A).
A
B
Scheme 1. Suzuki-Miyaura coupling of 1-bromo-9(H)-carbazole with
arylboronic acids or pinacol esters.
Unfortunately none of these analogues (compounds 3-14)
showed any inhibition of PARP14 MD2. Only a few further
modifications of the 1-aryl substituent were performed, whereby
all new compounds contained the acetylamino moeity, which was
recognised as important for activity in this early stage of the
project.
Figure 2. A) Overlay of bound ADPR (Green sticks) (PDB ID 3Q71)
superimposed with PARP14 MD2 (cyan sheets and helices, grey loops):
compound 2 (yellow sticks) structure (PDB ID 5O2D). B) H-Bonding
displayed in co-crystal structure of PARP14 MD2 (cyan sticks): compound 2
(
yellow sticks) structure (PDB ID 5O2D).
Carbazole 2 engages PARP14 MD2 in a pocket adjacent to the
ADP-ribose binding site and the interaction is characterised by a
H-bond between the carbazole N-H and backbone carbonyl of
Pro1130 (N-O distance 2.8 Å), an H-bond between one
sulfonamide carbonyl and the backbone N-H of Ile1132 (O-N
distance 2.8 Å), and an H-bond from the sulfonamide N to a
water molecule in the binding pocket. A comparison of the two
structures rationalises inhibitory activity as carbazole 2 induces a
shift in the loop region adjacent to Pro1130 which consequently
moves into the ADP-ribose binding site (Figure 2B). Evaluation
of the co-crystal structure of carbazole 2 with PARP14 MD2 also
revealed the possibility of extending the methanesulfonamide
motif into larger substituents exploring peripheral regions of this
newly identified allosteric site.
Figure 4. 1-Aryl- and 1-heteroarylcarbazoles 3-14 from the initial compound
library. PARP14 MD2 IC50 > 50 µM for all compounds.
The aza analogue 15 was obtained from N-SEM protected 1-
bromocarbazole by Masuda borylation at C-1, directly followed
by
Suzuki-Miyaura
cross-coupling
with
4-amino-3-
bromopyridine, subsequent N-acetylation and SEM deprotection,
11a
as previously described. This compound has virtually identical
size as the active compound 1, but interestingly was found to be
completely inactive at inhibiting PARP14 MD2 presumably due
to the differences in electronics of both molecules. Consequently,
this compound could serve as a useful negative control in
biochemical experiments. The pyridyl-isomers 16 and 17 were
obtained in the same manner using 3-amino-2-chloro- and 3-
amino-4-chloropyridine in the cross-coupling reaction (Figure 5).
Furthermore, using Suzuki-Miyaura cross-coupling reactions, the
acetylaminophenyl residue was attached to position 1 (Scheme 1)
2
. Results
2
.1. Systematic SAR studies of Carbazole A-C rings
The screening hit GeA-69 (1) was part of a focused library
from the Bracher lab, originally designed for the improvement of
kinase inhibitors derived from the 1-(aminopyrimidyl)-β-
1
3
carboline alkaloid annomontine. The SAR studies on screening
hit GeA-69 (1) are described in the following compound library
generated as potential PARP14 MD2 inhibitors (Figure 3). In this
library, the β-carboline ring system was replaced by its deaza
1
5
of the β-carboline ring system in order to obtain a ring A aza-
analogue 18 and to the canthin-4-one 19 and desazacanthin-4-
1
6
one 20 ring systems in order to give analogues bearing
analogue carbazole, and
a
number of aromatic and
tetracyclic core structures (Figure 5).
heteroaromatic rings were attached to position 1 (Scheme 1)
using Suzuki-Miyaura cross coupling reactions of known 1-
1
4
bromocarbazole with commercially available or synthesised
boronic acids and esters to give compounds 3-11 (Scheme 1).

Figure 7. Analogues of GeA-69 (1) bearing substituents an N-9, as well as
dibenzofuran (26), dibenzothiophene (27), fluorenone (28), and fluorenol (29)
analogues.
Figure 5. Aza analogues of screening hit GeA-69 (1): compounds 15-18 and
analogues bearing tetracyclic core structures canthin-4-one 19, desazacanthin-
4
-one 20.
Controlled mono-acetylation of 2,2’-diaminobiphenyl with
equimolar amounts of acetic anhydride gave monoamide 30 in
moderate yield. Monoamide 30 was then used to access the seco
analogue 31 and the acridone analogue 33. Buchwald-Hartwig
arylation of the unsubstituted anilino group with iodobenzene to
give biaryl 31and with methyl 2-iodobenzoate to give biaryl 32,
respectively, was accomplished with the BINAP/Pd (dba)
An analogue of GeA-69 (1) with the acetamido group shifted
from the ortho to the meta position at the phenyl ring 21 was
prepared by Suzuki-Miyaura cross-coupling of 1-bromocarbazole
with 3-aminophenyl boronic acid, followed by N-acetylation.
Additionally, the complete acetylaminophenyl residue was
shifted from C-1 to N-9, whereby in one example a rigid isomer
2
3
catalyst system. Ester 32 was hydrolysed to give the
corresponding carboxylic acid, which was converted into the
acridone 33 by polyphosphoric acid-mediated intramolecular
2
2 was obtained, and in the other, by means of a methylene
spacer, a product 23 in which by appropriate rotation both the
phenyl and the acetamido group can adopt positions that are very
similar to those these groups have in the lead structure GeA-69
2
0
acylation (Scheme 2).
(
2
1). Compound 22 was obtained by N-arylation of carbazole with
-fluoro-1-nitrobenzene, subsequent reduction of the nitro
1
7
group, and N-acetylation. N-Benzyl analogue 23 was prepared in
an analogous manner via N-alkylation of carbazole with 3-
nitrobenzyl chloride (Figure 6).
Scheme 2. Synthesis of seco analogue 31 and acridone analogue 33.
Figure 6. Analogues of GeA-69 (1) with the acetylaminophenyl residue
shifted to other positions.
Further, a series of modifications of ring C was performed.
As modifications of the central pyrrole ring (ring B) of GeA-
9 (1) N-methyl and N-benzyl analogues 24 and 25 were
Ring-substituted analogues 37-39 were obtained in two steps
2
1
6
from readily available 1,2,3,4-tetrahydrocarbazol-1-ones 34-36
prepared starting from corresponding N-substituted 1-
bromocarbazoles via Suzuki-Miyaura cross-coupling with 2-
aminophenylboronic acid and subsequent N-acetylation.
Dibenzofuran analogue 26 and dibenzothiophene analogue 27
were obtained in a similar manner from commercially available
in two steps. Treatment of the ketones with POBr in anisole
3
gave
the
corresponding
1-bromocarbazoles
under
bromination/dehydrogenation conditions in moderate to poor
yields. Subsequent standard Suzuki-Miyaura cross-coupling gave
the desired arylcarbazoles 37-39 (Scheme 3).
4
-bromodibenzofuran and known 4-iododibenzothiophene
18
(
Figure 6). These experiments were performed before we
obtained the crystal structure of PARP14 MD2 with inhibitor 2,
which demonstrated the relevance of the pyrrole NH-group
(
Figure 2).
In order to replace the NH group of ring C with either an
alternative hydrogen bond donor (hydroxy group) or a hydrogen
1
9
bond acceptor (carbonyl group), known 1-iodofluorenone was
coupled in the established manner to give the 1-arylfluorenone 28
which was easily reduced to the racemic fluorenol 29 with
sodium borohydride (Figure 7).
Scheme 3: Synthesis of analogues of of GeA-69 (1) bearing additional
substituents at ring C.
8
-Aza analogue 43 was obtained by a series of three
22
consecutive Pd-catalyzed coupling reactions. Chemoselective
Buchwald-Hartwig amination of 1-bromo-2-iodobenzene with 2-
amino-3-bromopyridine 40 using XantPhos as a ligand gave
phenylaminopyridine 41, which was cyclised to 8-bromo-α-
carboline 42 using CyJohnPhos in an intramolecular Heck

Scheme 4. Synthesis of an 8-aza analogue 43 of GeA-69 (1).
coupling. Finally, the acetylaminophenyl residue was
introduced in a standard Suzuki-Miyaura cross-coupling (Scheme
4
).
Analogue 44 bearing a partially hydrogenated A-ring was
obtained from the corresponding brominated
2
3
tetrahydrocarbazole via Suzuki-Miyaura cross-coupling. A
truncated analogue, the 7-aryl-3-isopropylindole 45, in which
ring C is replaced by an isopropyl group, was obtained by
Suzuki-Miyaura cross-coupling of the respective 7-bromoindole.
The 6-aza-5,6,7,8-tetrahydro analogue 47 was prepared in a
similar manner from known intermediate 46. Improved yields
were obtained, if the secondary amine was protected with the Boc
group prior to the cross-coupling reaction (Scheme 5).
Scheme 6. Variations of the acetamide group (thioamide 52, reduced N-
ethylamine 53, N-ethyl analogue 54, urea analogue 49). Synthesis of the
proposed amide bioisoster 51 from aniline 48.
2
4
A screening of the above presented compounds on PARP14
MD2 clearly demonstrated that lead structure GeA-69 (1) is very
sensitive to structural modifications. Carbazoles bearing
(hetero)aromatic residues different from the acetylaminophenyl
residue of GeA-69 (1) (Figure 4) were found to be inactive.
Analogues with almost identical shape albeit very different
electronically (aza analogues in the rings A, C and D) are
completely or virtually (β-carboline 18, IC50 30 μM) inactive.
Any changes in the central pyrrole ring (ring B) eliminated
inhibitory activity as well. The NH group was found to be
essential, it can not be replaced by another hydrogen bond donor,
as demonstrated by the inactive fluorenol analogue, 29.
Surprisingly, the dibenzothiophene analogue 27 showed
considerable inhibition (IC50 2.5 μM), whereas the dibenzofuran,
2
6 and the acridone, 33 were inactive. The same holds for the
Scheme 5. Analogues of GeA-69 (1) with partially hydrogenated or truncated
ring A.
(deaza)compounds having tetracyclic canthin-4-one backbones
(canthin-4-one 19, deazacanthin-4-one 20). The seco analogue of
GeA-69 (1), biaryl 31, was completely inactive, demonstrating
that not only the presence of the functional groups of the lead
structure, but also their fixation by the carbazole backbone is
most important.
The tetrahydro-analogue 44 showed only a slight loss in
activity (IC50 1.1 μM) compared to GeA-69 (1), whereas its 6-aza
analogue 47 bearing a polar aliphatic amino group in ring A, was
inactive. Lipophilic chlorine substituents at ring A (compounds
37-38) were fairly tolerated (IC50 1.4 and 3.0 μM), but the 6-
methoxy analogue 39 was inactive. These observations can be
rationalised by the hydrophobic environment in the binding
region of ring A consisting of residues V1032, V1092, M1108,
I111, I1112, F1129, I1132 (Figure 2).
Removal of the N-acetyl residue from GeA-69 (1), conversion
of the acetamide into a tertiary amide 54 or into the proposed
trifluoroalkyl bioisoster 51, as well as reduction of the amide
moiety to an amine 53 resulted in complete loss of activity, the
thioamide 52 was an order of magnitude less active (IC50 10.5
μM) than GeA-69 (1).
Finally, modifications of the acetamido group located at the 1-
phenyl substituent were performed. Aminophenyl intermediate
4
8 was further converted into the urea analogue 49 by treatment
with tert-butyl isocyanate (Scheme 6). Since α-
trifluoroethylamines are known as bioisosteres of amide groups
2
5
from peptide chemistry, we also prepared compound 51 for
SAR studies. Intermediate 48 was thus converted into 1,1,1-
trifluoropropan-2-imine 50 by Pd-catalysed cross-coupling with
2
6
2
-bromo-3,3,3-trifluoro-1-propene; subsequent reduction with
sodium borohydride gave the racemic target compound 51.
Treatment of GeA-69 (1) with Lawesson’s reagent gave the
thioamide analogue 52. Reduction of the amide group in 1 with
borane-disulfide yielded the N-ethyl analogue 53, which in turn
could be N-acetylated to give the N-ethyl acetamide 54.
In conclusion, these data confirm a very narrow structure-
activity relationship for rings A-C (Figure 3), and for further
optimisation of the screening hit GeA-69 (1) only modifications
of either the N-acyl residue or ring D were deemed promising.
2
.2. SAR studies of carbazole ring D and N-acyl residues.

Initial construction of the carbazole series was performed
using 1-bromo-9H-carbazole and a series of pinacol boronic
esters which were coupled under standard Suzuki-Miyaura
conditions, furnishing biaryl products in moderate to good yields
further development as they enabled rapid access to diversity and
provide a suitable vector for binding pocket exploration. A
number of hetero- and substituted- aromatics were appended onto
the biarylcore (examples 83-108, Table 2). Moderately flat SAR
was observed for both 2- and 4- substituted phenylacetyl and
phenylmethanesulfonamide groups. It was found that
(Scheme 1). A number of these compounds were then converted
to the corresponding acetamides or methanesulfonamides and
profiled for their binding activity with PARP14 MD2. Whilst
binding activity was not improved, additional substituents on ring
D such as methyl, fluoro and cyano were tolerated maintaining
single digit μM activity (compounds 55-57, Table 1). As
previously observed a comparison of these compounds with the
inactive non-acetylated and non-sulfonylated anilines (eg
compounds 59-61, Table 1) showed the requirement of this group
for binding activity .
Further modification of biaryl-amine 48 to the corresponding
amides or sulfonamides (Scheme 7) was carried out. The
corresponding amides and sulfonamides 62-108 were then
profiled for their PARP14 MD2 binding affinity (Table 1 and
Table 2).
introduction
of
a
3-cyano
substituent
in
the
phenylmethanesulfonamide series provided a slight improvement
in binding activity compared with GeA-69 (1). Carbazole 108
displays sub-micromolar activity for PARP14 MD2 (IC50 660 ±
30 nM). Notably, by comparison the corresponding 3-
cyanophenylacetamide 107 displays diminished binding activity
relative to sulfonamide 108, potentially due to the greater
tolerance of the sulfonamide to maintaining H-bond acceptor
interactions as shown in the PARP14 MD2:compound 2 co-
crystal structure (Figure 2B). The 3-cyanobenzyl group of
compound 108 may make interactions with adjacent hydrophobic
residues M1108, L1137 and F1144. Although we were unable to
obtain a crystal structure of compound 108 to confirm these
interactions, we performed docking studies to examine possible
binding modes of the larger compound compared to compound 2.
Simple minimisation of compound 108 in PARP14 MD2 is
unable to find a binding pose due to clashes between the larger 3-
cyanophenyl group and the protein. To account for potential side
chain rotations that would be necessary to accommodate this
group, we performed SCARE docking (SCan Alanines and
Scheme 7. Synthesis of amide and sulfonamide derivatives of aniline 48.
2
7
Refine) using ICM.
The optimised pose for compound 108
shows a rotation of the side chain of F1144 to open up space so
that the 3-cyanophenyl group can make interactions with M1108
and L1137 in addition to a pi-stacking interaction with F1144
Compounds were profiled for binding activity with PARP14
MD2 through a competitive (AlphaScreen™) binding assay
measuring the displacement of ADP-ribose peptide from
1
1a
(
Figure 8). However, it is not obvious from this docking study
PARP14 MD2.
Promising compounds were additionally
why the 3-cyanophenyl group would be preferred to other
hydrophobic groups such as in compounds 79 and 83-107.
profiled by biophysical assays such as Bio-Layer Interferometry
1
1a
or Isothermal Titration Calorimetry as previously described.
Sub-micromolar PARP14 MD2 affinity of carbazole 108 was
also confirmed by BioLayer Interferometry (BLI) providing a
As previously described the parent carbazole GeA-69 (1) was
profiled for its broader selectivity over 12 other human
macrodomains, showing exquisite selectivity for MD2 of
calculated K of 550 nM ± 220. Whilst lead compound 108 is
D
1
1a
larger and a less ligand efficient inhibitor of PARP14 MD2 than
original hit compound 1, owing to the more tolerant SAR around
it represents an attractive chemical starting point for future
development. Additional examples similar to compound 108 (see
SI, compounds 109-116) have been explored and work to
improve the binding activity and physicochemical properties of
this lead molecule will be reported in due course.
PARP14. Furthermore a representative selectivity screen of 46
kinases in a Differential Scanning Calorimetry assay did not
reveal any significant activity of carbazole GeA-69 (1) at 10
1
1a
μM.
3
. Discussion
The binding activities of synthesised PARP14 MD2 inhibitors
are summarised in Table 1 and Table 2. Despite comprehensive
SAR studies of the A-C rings of this carbazole series, no points
for the development of more potent ligands were discovered, a
number of derivatives were synthesised functionalising ring D
(
Figure 3). Only small additional substituents to the ring were
tolerated (e.g. compounds 55-57, Table 1). Interestingly,
elaboration of the sulfonamide in compound 2 into the
homologated ethane-, propane and butane-sulfonamides
analogues (compounds 62-64, Table 1) furnished equipotent
compounds. Further elaboration of the acetamide in GeA-69 (1)
mostly retained single digit μM binding activity (eg compounds
6
6,67). Interestingly the n-pentanoyl analogue 68 was seemingly
inactive, which may be due the entropic penalty associated with
longer alkyl substituents or a steric clash with the protein.
However, guided by the apparent tolerance of some larger
substituents in place of the acetamide in GeA-69 (1) and
methanesulfonamide in compound 2, the 2-phenylacetamide and
phenylmethanesulfonamide of compounds 78 and 79 (IC50 7.6 ±
Figure 8 Flexible side-chain docking studies of carbazole108 with PARP14
MD2 (from PDB ID 5O2D) reveal new potential hydrophobic interactions
with M1108, L1137 and F1144 after rotation of F1144 (black arrow,
conformation in 5O2D shown in green sticks) to accommodate the 3-
cyanophenyl
group..
0
.3 and 3.6 ± 0.3 μM respectively, Table 1) were chosen for

Table 1. Binding affinity characterisation data of carbazole series for PARP14 MD2
R
R’
IC50 (μM)
K
D
(μM)
R
X
IC50 (μM)
1
(GeA-69)
-NHAc
H
H
0.72 ± 0.04
0.9 ± 0.09
>50
0.86 ± 0.04
2.1 ± 0.1
n.d.
66
67
68
69
-Et
C
C
1.0 ± 0.03
0.9 ± 0.04
>50
2
-NHSO
2
Me
n-Pr
n-Bu
1
4
7
9
-NHAc
3-aza
H
C
-NHC(O)NH-tBu
7.2 ± 1.4
n.d.
-CH
2
CH
2
OMe
SO
8.6 ± 0.4
5
-Methylisoxazo-
5
2
-NHC(S)CH
3
H
10.5 ± 0.4
n.d.
70
SO
>50
4
-yl
5
5
5
5
5
5
5
6
6
6
6
6
6
3
4
5
6
7
8
9
0
1
2
3
4
5
-NHEt
H
>50
>50
n.d.
n.d.
71
72
73
74
75
76
77
78
79
80
81
82
-NMe
2
SO
SO
C
2.5 ± 0.1
>50
-NEtAc
H
Ph
-NHSO
2
2
Me
Me
4-Me
4-CN
6-Me
1.1 ± 0.1
n.d.
-CF
3
1.1 ± 0.07
1.2 ± 0.03
>50
a
-NHSO
n.d.
5.2 ± 1.7
1.6 ± 0.7
n.d.
-Cyclopropyl
-Cyclohexyl
-2-furyl
C
-NHAc
-NHSO Me
-NH
1.7 ± 0.1
>50
C
2
5-CF
3
C
>50
2
2
2
4-Me
>50
n.d.
-Ph
C
1.9 ± 0.07
7.6 ± 0.3
3.6 ± 0.3
>50
-NH
-NH
5-CF
6-Me
H
3
>50
n.d.
-CH
-CH
2
2
Ph
Ph
C
>50
n.d.
SO
C
-NHSO
-NHSO n-Pr
-NHSO n-Bu
-NHC(O)CH NMe
2
Et
1.2 ± 0.03
2.9 ± 0.1
3.3 ± 0.1
5.5 ± 0.6
n.d.
2-OMe-Ph
3-OMe-Ph
4-OMe-Ph
2
H
n.d.
C
12.7 ± 1.5
9.0 ± 1.4
2
H
n.d.
C
2
2
H
n.d.
a
Data was not successfully obtained due to solubility issues in the AlphaScreen assay with this example.
Table 2. Binding affinity characterisation data of carbazole series for PARP14 MD2
a
a
R/Het
X
IC50 (μM)
K
D
(μM)
R/Het
X
IC50 (μM)
a
8
8
8
3
4
5
3-aza
3-aza-4-Me
3, 6-aza
C
C
C
1.1
1.0
1.5
96
97
98
4-Cl
C
SO
C
6.2 ± 0.6
8.1 ± 0.7
4.3 ± 0.3
a
a
2.7
.9
4-CF
3
2.1 ± 0.1
3,4-OMe
3
3
,4-
8
8
6
7
4-aza-3-CN
3-aza-4-CN
C
C
2.4 ± 0.2
3.5 ± 0.2
99
C
6.9 ± 0.6
n.d.
n.d.
dioxole
100
2-F-5-CN
SO
1.2 ± 0.0
(K 1.3 ± 0.51)
D
8
8
9
9
9
9
9
8
9
0
1
2
3
4
3-aza-4-OH
2-F
C
SO
C
6.6 ± 0.4
2.4 ± 0.1
2.8 ± 0.2
4.4 ± 0.3
8.7 ± 1.2
>50
101
102
103
104
105
106
107
2,5-Me
3,4-Cl
3-OMe
3-F
C
C
44.6 ± 10.4
6.3 ± 0.6
6.6 ± 0.5
4.2 ± 0.3
1.4 ± 0.1
7.1 ± 0.7
2.1 ± 0.1
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
2-F
C
2-Cl
C
C
4-Me
4-F
C
3-F
SO
C
C
3-CF
3
4-OMe
C
6.2 ± 0.6
3-CN
C
0
.66 ± 0.03
9
5
4-CN
SO
6.2 ± 0.6
108
3-CN
SO
n.d.
(K
D
0.55 ± 0.22)
a
D
No error of fit obtained for these K values. n.d. denotes not determined.

4
.
Yamashita, T.; Ji, J.; Budhu, A.; Forgues, M.; Yang, W.;
4
. Summary
Wang, H.-Y.; Jia, H.; Ye, Q.; Qin, L.-X.; Wauthier, E.; Reid, L. M.;
Minato, H.; Honda, M.; Kaneko, S.; Tang, Z.-Y.; Wang, X. W.,
EpCAM-positive hepatocellular carcinoma cells are tumor-initiating
cells with stem/progenitor cell features. Gastroenterology 2009, 136,
1
5
G.; Dyson, J.; Del Rio, A.; D’Santos, C.; Williams, R.; Chokshi, S.;
Anders, R. A.; Bubici, C.; Papa, S., PARP14 promotes the Warburg
effect in hepatocellular carcinoma by inhibiting JNK1-dependent
PKM2 phosphorylation and activation. Nat. Commun. 2015, 6, 7882.
We herein report the development of a novel class of allosteric
modulators of the second macrodomain of PARP14. Initial
identification of carbazole GeA-69 (1) as a submicromolar
inhibitor of PARP14 MD2 was made following a medium
012-1024.
Iansante, V.; Choy, P. M.; Fung, S. W.; Liu, Y.; Chai, J.-
.
1
1a
throughput screen.
Inhibitory activity can be rationalised
through a PARP14 MD2 co-crystal of a similar derivative,
sulfonamide 2 (PDB ID 5O2D). Investigation into this carbazole
series was then made revealing new opportunities for ligand
elaboration. Systematic analysis of SAR demonstrated a very
narrow structure activity relationship for rings A-C (carbazole
scaffold), and for further optimisation of the screening hit 1 only
modifications of either the N-acyl residue or ring D showed
promise. A number of carbazole containing compounds were
tolerated in this newly identified allosteric site of PARP14 MD2
including a 3–cyano substituted phenylmethanesulfonamide 108.
Carbazole 108 displays submicromolar activity binding to
PARP14 MD2 by AlphaScreen (IC50 0.66 μM) which was also
6.
Mushtaq, M.; Darekar, S.; Klein, G.; Kashuba, E.,
Different Mechanisms of Regulation of the Warburg Effect in
Lymphoblastoid and Burkitt Lymphoma Cells. PLOS ONE 2015, 10
(8), e0136142.
7
.
(a) Barbarulo, A.; Iansante, V.; Chaidos, A.; Naresh, K.;
Rahemtulla, A.; Franzoso, G.; Karadimitris, A.; Haskard, D. O.;
Papa, S.; Bubici, C., Poly(ADP-ribose) polymerase family member
1
signal in multiple myeloma. Oncogene 2013, 32, 4231-4242; (b)
Cho, S. H.; Ahn, A. K.; Bhargava, P.; Lee, C.-H.; Eischen, C. M.;
McGuinness, O.; Boothby, M., Glycolytic rate and lymphomagenesis
depend on PARP14, an ADP ribosyltransferase of the B aggressive
lymphoma (BAL) family. Proc. Natl. Acad. Sci. U S A 2011, 108
(38), 15972-15977.
4 (PARP14) is a novel effector of the JNK2-dependent pro-survival
confirmed by BLI (K 0.55 μM). This lead molecule along with
D
others in this series are useful chemical starting points in the
development of chemical probes for this poorly understood
epigenetic target.
8
.
Han, W.; Li, X.; Fu, X., The macro domain protein family:
Acknowledgments
Structure, functions, and their potential therapeutic implications.
Mutat. Res., Rev. Mutat. Res. 2011, 727, 86-103.
The SGC is a registered charity (number 1097737) that receives
funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim,
Canada Foundation for Innovation, Eshelman Institute for
Innovation, Genome Canada, Innovative Medicines Initiative
9.
(a) Sonnenblick, A.; de Azambuja, E.; Azim, H. A., Jr.;
Piccart, M., An update on PARP inhibitors-moving to the adjuvant
setting. Nat. Rev. Clin. Oncol. 2015, 12, 27-41; (b) Lord, C. J.;
Ashworth, A., PARP inhibitors: Synthetic lethality in the clinic.
Science 2017, 355, 1152-1158.
(EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck
1
0.
(a) Peng, B.; Thorsell, A.-G.; Karlberg, T.; Schüler, H.;
KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario
Ministry of Economic Development and Innovation, Pfizer, São
Paulo Research Foundation-FAPESP, Takeda, and Wellcome
Yao, S. Q., Small Molecule Microarray Based Discovery of PARP14
Inhibitors. Angew. Chem. Int. Ed. 2017, 56 (1), 248-253; (b)
Andersson, C. D.; Karlberg, T.; Ekblad, T.; Lindgren, A. E. G.;
Thorsell, A.-G.; Spjut, S.; Uciechowska, U.; Niemiec, M. S.;
Wittung-Stafshede, P.; Weigelt, J.; Elofsson, M.; Schüler, H.;
Linusson, A., Discovery of Ligands for ADP-Ribosyltransferases via
Docking-Based Virtual Screening. J. Med. Chem. 2012, 55 (17),
[
106169/ZZ14/Z]. M.M. is grateful to the EPSRC Centre for
Doctoral Training in Synthesis for Biology and Medicine
EP/L015838/1) for a studentship, generously supported by
(
AstraZeneca, Diamond Light Source, Defence Science and
Technology Laboratory, Evotec, GlaxoSmithKline, Janssen,
Novartis, Pfizer, Syngenta, Takeda, UCB and Vertex. We thank
Carina Glas and Britta Hettich for support in chemical synthesis.
7
706-7718; (c) Wang, P.; Li, J.; Jiang, X.; Liu, Z.; Ye, N.; Xu, Y.;
Yang, G.; Xu, Y.; Zhang, A., Palladium-catalyzed N-arylation of 2-
aminobenzothiazole-4-carboxylates/carboxamides: facile synthesis
of PARP14 inhibitors. Tetrahedron 2014, 70 (35), 5666-5673; (d)
Ekblad, T.; Lindgren, A. E. G.; Andersson, C. D.; Caraballo, R.;
Thorsell, A.-G.; Karlberg, T.; Spjut, S.; Linusson, A.; Schüler, H.;
Elofsson, M., Towards small molecule inhibitors of mono-ADP-
ribosyltransferases. Eur. J. Med. Chem. 2015, 95, 546-551; (e)
Upton, K.; Meyers, M.; Thorsell, A.-G.; Karlberg, T.; Holechek, J.;
Lease, R.; Schey, G.; Wolf, E.; Lucente, A.; Schüler, H.; Ferraris, D.,
Design and synthesis of potent inhibitors of the mono(ADP-
ribosyl)transferase, PARP14. Bioorg. Med. Chem. Lett. 2017, 27
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