Discovery of Stereospecific PARP-1 Inhibitor Isoindolinone NMS-P515

Article abstract of DOI:10.1021/acsmedchemlett.8b00569

Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme involved in signaling and repair of DNA single strand breaks. PARP-1 employs NAD⁺ to modify substrate proteins via the attachment of poly(ADP-ribose) chains. PARP-1 is a well established targe

Full text of DOI:10.1021/acsmedchemlett.8b00569

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Letter
Discovery of Stereospecific PARP-1 Inhibitor Isoindolinone NMS-P515
Gianluca Mariano Enrico Papeo, Paolo Orsini, Nilla R. Avanzi, Daniela Borghi, Elena
Casale, Marina Ciomei, Alessandra Cirla, Viviana Desperati, Daniele Donati, Eduard
R. Felder, Arturo Galvani, Marco Guanci, Antonella Isacchi, Helena Posteri, Sonia
Rainoldi, Federico Riccardi-Sirtori, Alessandra Scolaro, and Alessia Montagnoli
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00569 • Publication Date (Web): 13 Mar 2019
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Discovery of Stereospecific PARP-1 Inhibitor Isoindolinone NMS-
P515
Gianluca Papeo,*a Paolo Orsini,a Nilla R. Avanzi,a Daniela Borghi,a Elena Casale,a Marina Ciomei,a
Alessandra Cirla,a Viviana Desperati,a Daniele Donati,a Eduard R. Felder,a Arturo Galvani,a Marco
Guanci,a Antonella Isacchi,a Helena Posteri,a Sonia Rainoldi,a Federico Riccardi-Sirtori,a Alessandra
Scolaro,a and Alessia Montagnolia
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a Oncology, Nerviano Medical Sciences S.r.l., Viale Pasteur 10, 20014 Nerviano, Milan, Italy
KEYWORDS: PARP-1, Inhibitor, isoindolinone, stereospecific, NMS-P515.
ABSTRACT: Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme involved in signaling and repair of DNA single strand
breaks. PARP-1 employs NAD+ to modify substrate proteins via the attachment of poly(ADP-ribose) chains. PARP-1 is a well
established target in oncology, as testified by the number of marketed drugs (e.g. Lynparza,™ Rubraca,™ Zejula,™ and
Talzenna™) used for the treatment of ovarian, breast and prostate tumors. Efforts in investigating uncharted region of the
previously identified isoindolinone carboxamide series delivered (S)-13 (NMS-P515), a potent inhibitor of PARP-1 both in
biochemical (Kd: 0.016 M) and cellular (IC50: 0.027 M) assays. Co-crystal structure allowed explaining NMS-P515
stereospecific inhibition of the target. After having ruled out potential loss of enantiopurity in vitro and in vivo, NMS-P515 was
synthesized in an asymmetric fashion. NMS-P515 ADME profile and its antitumor activity in a mouse xenograft cancer model
render the compound eligible for further optimization.
The genetic instability of tumors arises from their
deficiencies in fixing damaged DNA. These liabilities can be
magnified by employing drugs that further hamper cancer
DNA Damage Response (DDR) machinery.1,2 Archetype of
this approach is the use of poly(ADP-ribose) polymerase-1
(PARP-1) inhibitors. PARP-1, the main component of a 17-
membered family of proteins named ADP-Ribosyl-
Transferases Diphtheria Toxin-like (ARTDs),3,4 is a key player
of the Base Excision Repair (BER) pathway involved in DNA
single strand breaks (SSB) repair. PARP-1 employs NAD+ as
the building block to assemble, via the release of
nicotinamide, poly(ADP-ribose) chains onto several acceptor
proteins (heteromodification) including PARP-1 itself
(automodification).5 This post-translational event contributes
in signaling the presence of DNA damage, resulting in the
recruitment of proteins involved in DNA repair. Conceptually,
the inhibition of PARP-1 disables BER pathway, leading to
the accumulation of DNA double strand breaks (DSBs) whose
repair by pathways such as the Homologous Recombination
(HR) or non-homologous end joining (NHEJ), is either more
challenging or error-prone.6 The mechanism of action of such
inhibitors was postulated to potentiate DNA damaging
anticancer agents. The ensuing arrays of PARP-1 inhibitors,
initially designed to be combined with conventional
chemotherapy, finally found their way to the market (e.g.
olaparib, 1; rucaparib, 2; niraparib, 3 and talazoparib, 4, Figure
1) as monotherapy in those HR-deficient tumor (e.g.
BRCA1/2-mutated) phenotypes that cannot withstand a DNA
damage overload.7,8 To our knowledge, all the clinical and pre-
clinical PARP-1 inhibitors under development are NAD+-
competitive nicotinamide mimics, featuring a lactam ring or a
primary amide conformationally locked through an
intramolecular hydrogen bond with an adjacent heteroatom.9,10
O
O
H
F
NH
O
N
NH
N
N
N
N
O
NH2
O
F
N
NH
N
HN
N
N
F
N
H
N
F
O
NHMe
1 Olaparib
2 Rucaparib
3 Niraparib
4 Talazoparib
O
NH2
O
NH2
O
NH2
O
O
O
4
3
F
2 N
NH
N
N
N
N
N
F
1
F
5
6
7
Figure 1. Marketed PARP-1 inhibitors (1-4) and
isoindolinone carboxamide derivatives (5-7).
Interestingly, molecules with such an apparent architectural
resemblance and similar on-target biochemical potency
possess however striking differences in term of ARTDs
selectivity profile,11 and cellular and in vivo activity. A
number of hypotheses (e.g. different off-target activity,
trapping effects) have been invoked to account for the
observed dissimilarities.12
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One of the most valuable and innovative series of PARP-1 Insert Table 1
Page 2 of 13
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inhibitors in pre-clinical development is represented by the
isoindolinone carboxamides chemical class. This series
emerged from both an HTS campaign on our proprietary
chemical collection (e.g. NMS-P118, 5, Figure 1),13 and a
structure-based drug design approach performed in other
laboratories (e.g. 6, Figure 1).14 Aiming at improving the
binding efficiency as well as the drug-like properties of this
class, we focused on the introduction of a methyl substituent15
in position 1 of the bicyclic core (see Figure 1 for numbering
system). Planned compounds were considered eligible for
further progression based on concurrent thresholds of
The introduction of a fluorine atom meta to the primary
carboxamide, which usually reinforces the cellular potency of
PARP inhibitors (e.g. 2, 4, 5), was also explored within this
series (Scheme 2). Thus, by subjecting previously described
methyl ester (14)13 to benzylic bromination, followed by a
one-pot nucleophilic displacement-ring closure sequence,
intermediate isoindolinone (15) was obtained. C1 methylation
using NaHMDS as the base and subsequent cyanation afforded
nitrile (17), whose functional group manipulation resulted in
compound ()-18.
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biochemical activity (KD
< 0.1 M in fluorescence
polarization (FP) displacement assay16 against PARP-1) and
cellular inhibition of poly(ADP-ribose) synthesis (PAR assay,
IC50 < 0.1 M). Capitalizing on our acquired knowledge of the
Scheme 2. Synthesis of compound (±)-18a
series,13
()-1-methyl-3-oxo-2-(piperidin-4-yl)-2,3-dihydro-
I
O
I
I
O
O
1H-isoindole-4-carboxamide (7, PARP-1 KD: 0.04 M; PAR
assay IC50: 0.256 M; Figure 1) was adopted as the starting
point of our structure-activity relationship investigation. A
rapid interrogation of the chemical space departing from the
piperidine nitrogen atom was then performed. The planned
derivatives, as exemplified by the preparation of compound
()-13 (Scheme 1), were chemically assembled starting from
2-acetylfuran by using an intramolecular Diels-Alder as the
key step.
OMe
CN
a
b
N
N
N
N
F
F
F
14
15
16
O
NH2
O
O
c
d
N
N
N
N
F
F
17
(±)-18
aReagents and conditions: (a) NBS, Bz2O2, methyl pivalate, reflux then
1-cyclohexyl-piperidine-4-ylamine, TEA, acetonitrile, reflux, 40% (b)
MeI, NaHMDS 1M in THF, rt, 90 minutes; (c) CuCN, DMF, reflux, 90
minutes, 54%; (d) 37% HCl, 100 °C then HOBt.NH3, EDCI.HCl, DIPEA,
DMF, rt, 38%.
Scheme 1. Synthesis of compound (±)-13a
O
OH
H
O
N
a
b
c
O
O
O
N
O
O
O
N
N
O
Unfortunately, fluoro-isoindolinone (±)-18 (PARP-1 KD:
0.06 M; PAR assay IC50: 0.64 M) proved to be less
attractive than its corresponding des-fluoro analogue.
O
8
9
O
OH
O
OH
O
NH2
O
O
O
d
e, f
g
O
O
Physicochemical and ADME profiling of ()-13
demonstrated that the compound is moderately soluble (140
M) at neutral pH, it is highly permeable with an excellent
metabolic stability in vitro, and has a high plasma protein
binding (see Supporting Information, Table 1). Compound ()-
13 modestly inhibited two P450 cytochrome isoforms (CYP-
2D6 IC50: 0.60 µM; CYP-2B6 IC50: 4.90 µM) out of eight
tested. The ensuing evaluation of IV/OS pharmacokinetic
parameters in mice further corroborated the drug-like
properties of ()-13, as it is slowly cleared from the body, with
high exposure and complete oral bioavailability (Table 2).
N
NH
N
N
N
NH
HCl
HCl
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11
12
O
NH2
O
N
N
(±)-13
aReagents and conditions: (a) 4-amino-piperidine-1-carboxylic acid
tert-butyl ester, 1M TiCl4 in DCM, TEA, DCM, reflux, then NaCNBH3,
MeOH, rt, 80%; (b) maleic anhydride, toluene, reflux, 6h, 80%; (c) 37%
HCl, reflux, 70%; (d) di-tert-butyl dicarbonate, K2CO3, pyridine, MeOH,
rt, 78%; (e) HOBt.NH3, EDCI.HCl, DIPEA, DMF, rt, 75%; (f) 4M HCl in
dioxane, 50 °C; (g) cyclohexanone, NaCNBH3, NaOAc, DCM / MeOH, rt,
76%.
Insert Table 2
Following the initial pre-clinical profiling of compound ()-
13, we next scrutinized the contribution of each separated
enantiomer to the overall bioactivity picture of the racemate.
Chiral HPLC on a preparative scale allowed to separate up to 2
g of ()-13 with 93% recovery and  99.5 : 0.5 enantiomeric
ratio for each enantiomer. X-ray co-crystal structure of (S)-13
and (R)-13 with chicken PARP-1 catalytic domain (both
solved at 2.1 Å; Figure 2) permitted the unambiguous
attribution of their absolute stereochemistry.
Noteworthy, the cycloaddition delivered a single exo-
diastereomer17,18 (9) with the methyl substituent opposite to
the ethereal bridge. Diversification by means of a reductive
amination reaction was performed at the very end of the
synthesis, by employing differently substituted aromatic,
heteroaromatic, aliphatic aldehydes and ketones. This
exploration allowed us to select the cyclohexyl- derivative as
the most compelling PARP-1 inhibitor of the series (()-13,
PARP-1 KD < 0.03 M16). Furthermore, compound ()-13 is
selective amongst the ARTDs tested (PARP-2, -3 and
tankyrase-1 (TNKS-1) KD > 10 M) and potently abrogate
PAR synthesis in cells (IC50: 0.050 M) (Table 1).
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judging the maintenance of the stereochemical integrity of
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NMS-P515 in different conditions was planned. Thus,
exposure of NMS-P515 to a wide pH range (1.5, 7.4 and 10) at
room temperature for up to 24 hours did not result in any
erosion of its enantiopurity. Similarly, NMS-P515 stability in
human plasma, was evaluated after incubation at 37 °C for up
to 24 hours, again with no apparent detection of (R)-13.
Finally, to investigate enantiomer integrity in vivo, mice were
treated with a single dose (100 mg/kg, OS) of NMS-P515 and
blood samples were withdrawn after 45 minutes
(corresponding to nearly the Tmax value of ()-13 as
measured in the PK experiments). Also in this case, chiral
HPLC analysis showed complete preservation of NMS-P515
enantiopurity.
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Detailed physicochemical and ADME profiling of NMS-
P515 are reported in Supporting Information, Table 1. NMS-
P515 is more soluble at neutral pH (> 225 M) than its
corresponding racemate, its plasma protein binding is slightly
lower and it does not inhibit any of the P450 cytochrome
isoforms against which it was tested (CYPs IC50 > 31 M).
The NMS-P515 pharmacokinetic profile mostly overlaps with
the one of ()-13, however with some noteworthy differences
(Table 2). Thus, NMS-P515 clearance and volume of
distribution were nearly twice the one of the racemate. On the
contrary, the exposure of NMS-P515 resulted lower than that
of ()-13, with an oral bioavailability of 78%. NMS-P515
further progression as a lead compound was however
challenged by the lack of an efficient and scalable asymmetric
synthesis, which is mandatory to overcome the limitations of a
costly and time consuming chiral HPLC separation. After a
number of unfruitful attempts,19 the engagement of the
Figure 2. Co-crystal structure of (S)-13 (NMS-P515, blue,
top left) and (R)-13 (green, top right) with chicken PARP-1
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CD and their overlapping (bottom).
Thus, based on chiral HPLC retention time (see Supporting
Information, Figure 1), the first eluting peak was demonstrated
to be the (S)-13 enantiomer (followed by the (R)-13 peak). As
soon as the enantiomers were tested, it became clear that (S)-
13 (NMS-P515) solely contributes to the overall biochemical
potency and the cellular activity (PARP-1 KD < 0.03 M;16
PAR assay IC50: 0.027 M) showed by ()-13 (Table 1). To
further substantiate FP displacement data, both (S)-13 and (R)-
13 were also tested against PARP-1 catalytic domain using
Surface Plasmon Resonance (SPR) binding assay (Table 1).16
As this technique does not carry any sensitivity limit, evidence
of the different behavior between enantiomers emerged even
more strikingly ((S)-13, PARP-1 Kd: 0.016 M; (R)-13,
PARP-1 Kd: 1.76 M). Same differences were also
appreciated at the cellular level (Table 1 and Figure 3A).
Similarly to ()-13, also NMS-P515 was selective against
PARP-2, -3 and TNKS-1 with a recorded KD > 10 M on each
enzyme (Table 1). The identification of NMS-P515 as a
stereoselective PARP-1 inhibitor comes along with the effort
in rationalizing its superior activity compared to (R)-13.
Having successfully gathered PARP-1 co-crystal data for both
enantiomers, a structure-based hypothesis was formulated. As
expected,13,14 the isoindolinone core of both NMS-P515 and
(R)-13 is anchored to the nicotinamide pocket of PARP-1 by
the classical hydrogen-bond triad with Gly863 and Ser904 and
by a π-π interaction with the phenyl ring of Tyr907. The 1-
cyclohexyl-4-piperidinyl substituent, which departs from the
bicyclic scaffold, extends outside the nicotinamide site and
fills a cleft defined by the D-loop and the helix 5 of the helical
bundle domain. However, NMS-P515 and (R)-13 overall
orientations are slightly different (Figure 2). NMS-P515 C1
methyl nicely adapts to the binding site. Differently, the C1
methyl in (R)-13 points toward Tyr896, forcing the whole
molecule to rotate about 10° outward from the pocket to avoid
any steric clash. The net result is a poorer binding site fit for
(R)-13 compared to NMS-P515.
previously described20 (S)-1-furan-2-yl-ethylamine in
a
reductive amination reaction with 1-cyclohexyl-piperidin-4-
one was key for the proper installation of the desired
stereocenter (Scheme 3). The resulting (S)-19 proved to be
optically pure (> 99.5 : 0.5 enantiomeric ratio by chiral HPLC,
using ad hoc prepared ()-19 as standard). Diels-Alder
reaction, followed by functional groups manipulation, resulted
in the asymmetric synthesis of (S)-13 (NMS-P515), with a
21% overall yield from (S)-1-furan-2-yl-ethylamine and a 95.5
: 4.5 enatiomeric ratio.
Scheme 3. Asymmetric Synthesis of NMS-P515a
O
OH
H
O
N
a
b
N
O
O
O
N
N
NH2
19
e.r. % > 99%
20
O
OH
O
NH2
O
O
c
d
N
N
N
N
NMS-P515
(S)-13
21
e.r.: 95.5 : 4.5
aReagents and conditions: (a) 1-cyclohexyl-piperidin-4-one, 1M TiCl4,
DCM, then NaCNBH3, MeOH, 52%; (b) maleic anhydride, toluene, 110
°C; (c) 37% HCl, 100 °C; (d) HOBt.NH3, EDCI.HCl, Et3N, THF, 40%.
The partial erosion of the stereochemical integrity during
the synthesis of (S)-13 was ascribed to the harsh reaction
conditions involved in the dehydration-aromatization step. The
As the pre-clinical progress of enantiopure synthetic
materials brings about a higher cost of goods compared to the
corresponding racemate, a number of experiments aimed at
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batch of NMS-P515 produced through asymmetric synthesis that render this PARP-1 stereospecific inhibitor eligible for
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2
3
4
5
6
7
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recapitulates the biochemical and cellular activity of (S)-13
arising from chiral HPLC separation (data not shown). NMS-
P515 was then subjected to in vivo efficacy studies, both as
monotherapy and in combination, on subcutaneously
implanted Capan-1 pancreatic (BRCA2-mutated) mouse
xenografts. Thus, administration of NMS-P515 for 12 days
(once a day, oral dose of 80 mg/kg21) as a methocel suspension
clearly reduced the tumor growth (maximal tumor growth
inhibition observed: 48%, maximum body weight loss: 6%;
Figure 3B). An even more pronounced effect (maximal tumor
growth inhibition: 79%, maximum body weight loss: 17%;
Figure 3B), was observed when animals were treated with
NMS-P515 (12 days, once a day oral dose of 80 mg/kg) in
combination with temozolomide, a DNA damaging drug (5
days starting from day 3, once a day IV dose of 62.5 mg/kg).
For comparison, same dose of temozolomide, administered as
single agent, resulted in maximal tumor growth inhibition of
46%, with a maximum body weight loss of 8% (Figure 3B).
further optimization.
Supporting Information
Synthetic procedures and analytical characterization for all new
compounds described. Experimental details for analytical and
preparative enantiomers separation, evaluation of NMS-P515
stereochemical erosion in vitro and in vivo, X-ray
crystallography, ADME in vitro data, biochemical and cellular
assays. Detailed description of preliminary pharmacokinetic and
in vivo efficacy studies (file type: PDF).
9
The Supporting Information is available free of charge on the
ACS Publications website.
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Accession Codes
Reported structures have been deposited to the Protein
Data Bank (PDB) with accession codes: 618M and 618T.
AUTHOR INFORMATION
Corresponding Author
*Phone: +39-0331581537. Fax: +39-0331581347. E-mail:
gianluca.papeo@nervianoms.com.
ORCID
Gianluca Papeo: 0000-0002-4773-4437
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Present Addresses
 For V. D.: Bracco Imaging S.p.A., via Caduti di Marcinelle, 13
- 20134 - Milano, Italy.
For M. G.: Helsinn Healthcare SA, via Pian Scairolo 9 – 6912
Pazzallo-Lugano – Switzerland.
For F. R.-S.: Quantitative Pharmacology and Drug Disposition
(QPD) - NBE Bioanalytics Unit MERCK Bioindustry Park
I-10010 Colleretto Giacosa (TO), Italy.
For A. S.: Fresenius Kabi Business Unit API, piazza Maestri del
Lavoro, 7 - 20063 - Cernusco sul Naviglio – Milano, Italy.
Figure 3. A) ArrayScan Images of HeLa cells treated with
dimethylsulfoxide (DMSO, control), and same concentration
(0.37 M) of: (±)-13, NMS-P515 and (R)-13 followed by
hydrogen peroxide-induced PAR formation (blue: Hoechst
(DNA); green: antibody anti-PAR). B) Tumor growth
inhibition of Capan-1 pancreatic cancer xenograft model
(control: blue line, NMS-P515: purple line; temozolomide:
green line; NMS-P515 + temozolomide: red line).
Notes
All procedures adopted for housing and handling of animals were
in strict compliance with Italian and European guidelines for
Laboratory Animal Welfare and the protocols were approved by
IRB.
The Authors declare no competing financial interest.
ACKNOWLEDGMENT
We gratefully acknowledge the technical skill and support of Rita
Perego (Protein Production), Emiliana Corti and Rosita Lupi
(Assay Development), Francesco Sola and Ivan N. Fraietta
(Cellular Biology), Barbara Forte and Mikhail Y. Krasavin
(Medicinal Chemistry) and Fabio Zuccotto (Molecular Modeling).
In summary, mindful exploration of the C1 methylated
isoindolinone, a privileged scaffold within the realm of pre-
clinical PARP-1 inhibitors, delivered NMS-P515. This
compound potently and selectively inhibits the target enzyme,
with a clear mechanism of action in cells. After a thorough
assessment of the preservation of its stereochemical integrity,
both in vitro and in vivo, NMS-P515 was deeply profiled in
terms of physicochemical and pharmacokinetic properties. The
ensuing (scalable) asymmetric synthesis of NMS-P515
overcomes the limitations associated with preparative chiral
HPLC separation. NMS-P515 lead-like performance was
reinforced by preliminary antitumor efficacy studies in vivo
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PARadoxes. Mol. Aspects Med. 2013, 34, 1046-1065.
a]quinoline-10-carboxylic acids. Synthesis 2005, 1859-1875.
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(19) Di Mola, A.; Palombi, L.; Massa, A. An overview of asymmetric
synthesis of 3-substituted isoindolinones. In Targets in Heterocyclic
Systems, 18th ed.; Attanasi, O. A.; Noto, R.; Spinelli, D., Eds.; Italian
Society of Chemistry: Rome, Italy, 2014; pp 113–140.
(6) Lord, C. J.; Ashworth, A. PARP inhibitors: Synthetic lethality in
the clinic. Science, 2017, 355, 1152-1158.
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(7) Ashworth, A.; Lord, C. J. Synthetic lethal therapies for cancer:
what’s next after PARP inhibitors? Nature Rev. 2018, 15, 564-576.
(20) Alvaro, G.; Martelli, G.; Savoia, D.; Zoffoli, A. Synthesis of (S)-
and (R)-1-(2-furyl)alkylamines and (S)- and (R)--amino acids
through the addition of organometallic reagents to imines derived
from (S)-valinol. Synthesis 1998, 1773-1777.
(8) Ringley, J. T.; Moore, D. C.; Patel, J.; Rose, M. S. Poly (ADP-
ribose) polymerase inhibitors in the management of ovarian cancer: A
drug class review. P&T, 2018, 43, 549-556.
(21) Such an aggressive BRCA-2 mutated pancreatic tumor model
requires high doses of PARP-1 inhibitors (approaching the maximum
tolerated one) to show efficacy. For (±)-13, maximum tolerated dose
in this model proved to be 100 mg/kg (Data not shown). As
enantiomer NMS-P515 is the actual responsible for the target
inhibition observed with (±)-13, a theoretical efficacious dose of 50
mg/kg can thus be envisioned. However, being NMS-P515 oral
bioavailability and exposure level lower than its racemate (see Table
2), an intermediate dose (80 mg/kg) was selected for efficacy studies.
(9) Wang, Y.-Q.; Wang, P.-Y.; Wang, Y.-T.; Yang, G.-F.; Zhang, A.;
Miao Z.-H. An Update on Poly(ADP-ribose)polymerase-1 (PARP-1)
Inhibitors: Opportunities and Challenges in Cancer Therapy. J. Med.
Chem. 2016, 59, 9575-9598.
(10) Kirby, I. T.; Cohen, M. S. Small-molecule inhibitors of PARPs:
From tools for investigating ADP-Ribosylation to therapeutics. Curr.
Top. Microbiol. Immunol. 2018, doi: 10.1007/82_2018_137. [Epub
ahead of print].
(11) Thorsell, A.-G.; Ekblad, T.; Karlberg, T.; Löw, M.; Pinto, A. F.;
Trésaugues, L.; Moche, M.; Cohen, M. S.; Schüler, H. Structural
Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase
(PARP) and Tankyrase Inhibitors. J. Med. Chem. 2017, 60, 1261-
1271.
(12) Pommier, Y.; O’Connor, M. J.; de Bono, J. Laying a trap to kill
cancer cells: PARP inhibitors and their mechanism of action. Sci.
Transl. Med. 2016, 8, 362ps17.
(13) Papeo, G.; Posteri, H.; Borghi, D.; Busel, A. A.; Caprera, F.;
Casale, E.; Ciomei, M.; Cirla, A.; Corti, E.; D’Anello, M.; Fasolini,
M.; Forte, B.; Galvani, A.; Isacchi, A.; Khvat, A.; Krasavin, M. Y.;
Lupi, R.; Orsini, P.; Perego, R.; Pesenti, E.; Pezzetta, D.; Rainoldi, S.;
Riccardi-Sirtori, F.; Scolaro, A.; Sola, F.; Zuccotto, F.; Felder, E. R.;
Donati,
D.;
Montagnoli,
A.
Discovery
of
2-[1-(4,4-
difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-
isoindole-4-carboxamide (NMS-P118): a Potent, Orally Available and
Highly Selective PARP-1 Inhibitor for Cancer Therapy. J. Med.
Chem. 2015, 58, 6875-6898.
(14) Gandhi, V. B.; Luo, Y.; Liu, X.; Shi, Y.; Klinghofer, V.;
Johnson, E. F.; Park, C.; Giranda, V. L.; Penning, T. D.; Zhu, G.-D.
Discovery and SAR of substituted 3-oxoisoindoline-4-carboxamides
as potent inhibitors of poly(ADP-ribose) polymerase (PARP) for the
treatment of cancer. Bioorg. Med. Chem. Lett. 2010, 20, 1023–1026.
(15) Sun, S.; Fu, J. Methyl-Containing Pharmaceuticals: Methylation
in Drug Design. Bioorg. Med. Chem. Lett. 2018, 28, 3283-3289.
(16) Papeo, G.; Avanzi, N.; Bettoni, S.; Leone, A.; Paolucci, M.;
Perego, R.; Quartieri, F.; Riccardi-Sirtori, F.; Thieffine, S.;
Montagnoli, A.; Lupi R. Insights into PARP inhibitors selectivity
using fluorescence polarization and surface plasmon resonance
binding assays. J. Biomol. Screen. 2014, 19, 1212-1219.
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Table 1. Biochemical and cellular data of compounds (±)-13 and the separated enantiomers.
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Compound
Biochemical Assay
Enzyme
Type
(±)-13
 0.03a
> 10
> 10
> 10
-
NMS-P515
 0.03a
> 10
(R)-13
PARP-1
PARP-2
PARP-3
TNKS -1
PARP-1
1.32
-
10
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FP (KD, M)
> 10
-
-
> 10
0.016
1.76
SPR (KD, M)
Cellular Assay Type
PAR (IC50, M)
Compound
NMS-P515
0.027
Cell Line
(±)-13
(R)-13
HeLa
0.050
0.162
a Assay Sensitivity Limit.
Table 2. Preliminary pharmacokinetic parameters of compounds (±)-13 and NMS-P515.
PK dataa iv
PK dataa per os
Cpd 
doseb:
(mg/kg)
Cmax
(µM)
CL
Vss
(L/kg)
dosec:
(mg/kg)
Cmax
(µM)
Fd
(%)
AUC
AUC
t1/2 (h)
t1/2 (h)
(µM.h)
(µM.h)
(mL/min/kg)
2.5 
1.8 
2.5 
0.4
13.5 
3.1 
0.7
(±)-13
5
10
10
100
6.3  0.0
8.2  0.8
32.6  0.8
53.4  8.0
3.6  0.4
6.8  1.0
0.3
0.1
1.72
NMS-
P515
4.9 
0.6
2.1 
0.4
1.4 
0.2
4.9 
0.1
2.6 
0.9
10
78.3
a Harlan Nu/Nu Mice; n = 3 animals per study.
b Dosed iv (intravenous administration): 50% PEG 400 in glucosate for (±)-13 and in situ hydrochloride salt, 1% Tween 80 in
dextrose for NMS-P515.
c Dosed per os (oral administration): 0.5% methocel.
d Bioavailability.
TOC Graphic
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Figure 1. Marketed PARP-1 inhibitors (1-4) and isoindoli-none carboxamide derivatives (5-7).
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Figure 2. Co-crystal structure of (S)-13 (NMS-P515, blue, top left) and (R)-13 (green, top right) with
chicken PARP-1 CD and their overlapping (bottom).
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Figure 3. A) ArrayScan Images of HeLa cells treated with dimethylsulfoxide (DMSO, control), and same
concentration (0.37 μM) of: (±)-13, NMS-P515 and (R)-13 followed by hydrogen peroxide-induced PAR
formation (blue: Hoechst (DNA); green: antibody anti-PAR). B) Tumor growth inhibi-tion of Capan-1
pancreatic cancer xenograft model (control: blue line, NMS-P515: purple line; temozolomide: green line;
NMS-P515 + temozolomide: red line).
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a
Reagents and conditions: (a) 4-amino-piperidine-1-carboxylic acid tert-butyl ester, 1M TiCl in DCM, TEA,
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