Serendipitous discovery of a prodrug of a PARP-1 inhibitor

Article abstract of DOI:10.1111/cbdd.12165

During SAR development of previously reported pyrrolocarbazole 1, a potent PARP-1 inhibitor, compound 14, was discovered serendipitously to be a prodrug of compound 1.

Full text of DOI:10.1111/cbdd.12165

Received Date : 23-Feb-2013
Revised Date : 11-Apr-2013
Accepted Date : 11-May-2013
Article type
: Research Letter
Serendipitous Discovery of a Prodrug of a PARP-1 Inhibitor
Derek Dunn, Jean Husten, Lisa D. Aimone, Mark A. Ator and
Sankar Chatterjee*
Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380-4245, USA
*Corresponding author. E-mail address: Sankar.Chatterjee24@gmail.com (S. Chatterjee)
Key words: PARP-1, oncology, pyrrolocarbazole.
Abstract. During SAR development of previously reported pyrrolocarbazole 1, a potent PARP-
1 inhibitor, compound 14 was discovered serendipitously to be a prodrug of compound 1.
Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that catalyzes the synthesis of
poly(ADP-ribose) chains from nicotinamide adenine dinucleotide (NAD+) in response to single-
strand DNA breaks as part of the DNA repair process (1). The PARP super family encompasses
several members whose biological targets are currently the focus of intense research (2 – 4).
Amongst the family members, PARP-1 has emerged as a potential therapeutic target in a variety
of indications, with early drug discovery efforts focused on the attenuation of cell death resulting
from PARP over-activation and NAD+ depletion following cerebral or cardiac ischemia (5).
More recently, PARP inhibitors have advanced to the clinic in the area of oncology; several
reports provide excellent summaries of the current status of PARP inhibitors in the clinic (6 – 8).
Previously, our laboratories reported the identification of a novel pyrrolocarbazole compound 1,
as a potent PARP-1 inhibitor (Fig. 1, IC50 36 nM) from screening of our corporate chemical
library (9, 10). Though compound 1 displayed potency, its poor drug-like characteristics,
especially its extremely low solubility in various acceptable vehicles, made it difficult to perform
in vivo experiments. To circumvent the problem, a synthetic program was initiated to identify
analogs of compound 1 having acceptable drug-like properties while maintaining the activity.
From that exploration, we communicated compound 2 (Fig. 1), 3-aminomethyl derivative of
compound 1 that was active as a chemopotentiating agent with a variety of clinically effective
chemotherapeutic reagents (11). In parallel to the above-mentioned research, a series of imide –
N (position 6) substituted analogs of compound 1 was also explored. In this Letter, a preliminary
report from this later effort is disclosed.
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'Accepted Article', doi: 10.1111/cbdd.12165
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In our synthetic program, it was observed that compound 1 could undergo preferential
regioselective alkylation, acylation and sulfonylation at the imide-NH (position 6) due to acidic
nature of this proton resulting from the electron withdrawing property of the two neighboring
carbonyl groups. Accordingly, compounds 3 – 15 were rapidly generated in a parallel fashion
(Scheme 1). Deprotonation of the imide–NH of compound 1 by a base followed by alkylation
with suitable reagents took place at room temperature to generate compounds 3 – 11.
Alternatively, the same intermediate anion reacted with the carboxyl terminus of N-protected
amino acids in the presence of coupling reagents to generate the acylated intermediates;
subsequent removal of the protecting group yielded compounds 12 – 14. Finally, sulfonylation
of the anion of compound 1 generated the target compound 15.
Target compzunds were evaluated against recombinant human poly(ADP-ribose) polymerase-1
in an in vitro enzyme assay (done in triplicate) as described previously (9 - 10). Table 1
describes the biologic activities of compounds 3 – 15 as well as the parent compound 1.
As shown in Table 1, a series of N-alkyl analogs of compound 1 in which the alkyl chain length
was varied (n = 1 – 4, compounds 3 - 11), were synthesized. Compared to parent compound 1,
they displayed poor (compounds 3 - 7 and 11) to moderate (compounds 8–10) inhibitory activity
in the enzyme assay. However, in comparison, the N-acylated and -sulfonylated products
displayed moderate (compound 13 and 15, respectively) to potent activity (compounds 12 and
14, respectively). From a molecular docking study of parent compound 1 to the catalytic domain
of chicken PARP-1, it was shown that the imide NH appears to be involved in a key interaction
with a backbone glycine (12). Thus, replacement of this hydrogen with an alkyl group in
compounds 3 - 11 might have been detrimental to productive binding. On the other hand, it was
postulated that the newly formed N – CO moiety in compounds 12 and 14 might be involved in
an additional beneficial binding imparting its potency or these compounds were reverting back
into compound 1 under the assay conditions. While compound 14 was found to be stable for at
least two hours under weakly acidic conditions (pH ~ 5), it was found to convert rapidly to
compound 1 at neutral pH. Based on its activity against the enzyme as well as desired drug-like
properties, compound 14 was subsequently advanced to additional studies.
Compounds 14 and 1 were also tested in a cell-based assay that measured their ability to
attenuate the depletion of NAD+ levels in PC12 cells following activation of PARP by DNA
damage with hydrogen peroxide (9, 10). In this assay, the superior activity of compound 14 in
comparison to compound 1 was apparent (Figure 2).
In a pharmacokinetic experiment, compound 14 was administered to rats (male, Sprague
Dawley) via intraperitoneal route (10 mg/kg, vehicle: saline, dose volume: 4 mL/kg). Compound
14 was not detected in plasma samples from early time points, but parent compound 1 was
readily detectable (Figure 3 and Table 2). Thus, it appears that the compound 14 acts as a
prodrug of parent compound 1 in vivo.
In conclusion, in this Letter, a series of imide - N substituted analogs of previously disclosed
pyrrolocarbazole compound 1 has been described. From this study, compound 14 emerged as a
lead molecule. Pharmacokinetic studies (rats) revealed that compound 14 converts to compound
1 in vivo, allowing compound 14 to be a biologic tool compound. This serendipitous discovery
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provided the impetus for additional exploration that led to a new generation inhibitor CEP–9722
that is currently in clinical trial (Phase II) for the treatment of advanced or metastatic solid
tumors.a
Acknowledgments
Authors wish to acknowledge the scientific support provided by Mr. Kurt Josef and
encouragement provided by Drs. Jeffry Vaught and John P. Mallamo during the course of this
research.
Conflict of Interest
All the authors, as the employees of Cephalon, Inc., were engaged in discovering novel PARP
inhibitors.
References
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Cianferani S., Smith S., Schreiber V., Boussin F., Dantzer F. (2011) Poly(ADP-ribose)
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associated pancreatic cancer. Anticancer Res 31: 1417 – 20.
7. Leung M., Rosen D., Fields S., Cesano A., Budman D. R. (2011) Poly(ADP-ribose)
polymerase-1 inhibition: preclinical and clinical development of synthetic lethality. Mol Med 17:
854 – 62.
8. Annunziata C. M..; O'Shaughnessy J. (2010) Poly (ADP-ribose) polymerase as a novel
therapeutic target in cancer. Clin Cancer Res 16: 4517 - 26.
9. Ator M. A., Bihovsky R., Chatterjee, S., Dunn, D., Hudkins, R. L. (2001) Multicyclic
compounds and the use thereof. US Patent 7,122,679: 1 – 74.
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10. Tao, M.; Park, C. H.; Bihovsky, R.; Wells, G. J.; Husten, J.; Ator, M. A.; Hudkins, R. L.
(2006) Synthesis and structure-activity relationships of novel poly(ADP-ribose) polymerase-1
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A., Bihovsky, R., Hudkins, R., Chatterjee, S., Klein-Szanto, A., Dionne, C., Ruggeri, B. (2003)
Chemopotentiation of Temozolomide, Irinotecan, and Cisplatin activity by CEP-6800, a
poly(ADP-ribose) polymerase inhibitor Mol Cancer Ther 2: 371 - 82.
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Note
ahttp://www.clinicaltrials.gov (accessed on February 22, 2013).
Legends for figures and scheme and titles for tables
Legends for figures
Legend for Figure 1:
Figure 1. Chemical Structures of compounds 1 and 2
Legend for Figure 2:
Figure 2. Cell-based assay for compounds 14 and 1
Legend for Figure 3:
Figure 3. Plasma levels (concentration) over time of compound 1 in rats dosed as compound 14
(ip, 10 mg/kg, vehicle: saline).
Legend for Scheme
Legend for Scheme 1:
Scheme 1. Reagents and conditions. For compounds 3 – 11: a) (i) 60% NaH in oil,
X–(CH2)n–R (X: leaving group), DMF, room temperature, 2–3 h; 60 – 80%. For compounds 12
– 14: b) (i) 60% NaH in oil, N-protected amino acids, TBTU, NMM, DMF, room temperature;
(ii) 4N HCl in dioxane, room temperature, 2 h, 60 – 65% over two steps. For compound 15: c)
60 % NaH in oil, PhSO2Cl, DMF, room temperature, 2 h, 70%.
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Titles for Tables
Title for Table 1
Table 1. Biologic Activitya of Compounds 1 and 3 – 15.
Title for Table 2.
Table 2. Pharmacokinetic data related to figure 3.
Serendipitous Discovery of a Prodrug of a PARP-1 Inhibitor
Derek Dunn, Jean Husten, Lisa D. Aimone, Mark A. Ator and
Sankar Chatterjee*
Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380-4245, USA
*Corresponding author. E-mail address: Sankar.Chatterjee24@gmail.com (S. Chatterjee)
Tables
Table 1.
R
N
O
O
N
H
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Compound
R
IC50 (nM) or
% inhibition at 10 μMb
H
36
10000
63%
1600
64%
68%
720
1
3
4
5
6
7
8
9
CH2COOH
CH2CONH2
(CH2)2OCH2Ph
(CH2)3Cl
(CH2)3-N-piperidinyl
(CH2)3OCH2Ph
(CH2)3-NEt2
819
(CH2)3OH
946
>10,000
38
10
11
12
13
14
15
(CH2)4NHCOMe
COCH2NH2 . HCl
CO(CH2)2NH2 . HCl
)-CH(NH2)(CH2)4NH2 . 2 HCl
SO2Ph
160
34
250
CO-(
S
aEnzyme: Recombinant human PARP-1
bn = 3
Table 2.
Dose: 10 mg/kg, IP
Cmax, ng/mLa
Plasma data
222 17
0.4 0.1
223 23
228 23
0.3 0.0
-
t
max, hb
AUC0 - t, ng*h / mLc
AUC0 - ∞, ng*h / mL
t1/2, h
Mean SEM, n=3
aCmax: Highest concentration achieved at the dosing regimen.
btmax: Time to achieve the highest concentration.
cAUC: Area under the curve.
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Scheme
Scheme 1.
R
N
H
N
O
O
O
O
a or b or c
N
N
H
H
3 - 15
1
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