4-Phenyl-1,2,3,6-tetrahydropyridine, an excellent fragment to improve the potency of PARP-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.06.094

We have shown that a 4-phenyl-1,2,3,6-tetrahydropyridine fragment plays an important role in improving inhibitory potency against poly(ADP-ribose) polymerase-1 (PARP-1). Various benzamide analogues linked with this fragment via alkyl spacers have been prepared and evaluated. As a result, some of them have been found to be highly potent PARP-1 inhibitors.

Full text of DOI:10.1016/j.bmcl.2005.06.094

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4221–4225
4-Phenyl-1,2,3,6-tetrahydropyridine, an excellent fragment
to improve the potency of PARP-1 inhibitors
Junya Ishida,a,* Kouji Hattori,a Hirofumi Yamamoto,a Akinori Iwashita,b
Kayoko Miharab and Nobuya Matsuokab
aMedicinal Chemistry Research Laboratories, 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan
bMedicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan
Received 31 January 2002; accepted 24 June 2005
Abstract—We have shown that a 4-phenyl-1,2,3,6-tetrahydropyridine fragment plays an important role in improving inhibitory
potency against poly(ADP-ribose) polymerase-1 (PARP-1). Various benzamide analogues linked with this fragment via alkyl spac-
ers have been prepared and evaluated. As a result, some of them have been found to be highly potent PARP-1 inhibitors.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
that is essential for binding to the catalytic domain of
PARP-1. Meanwhile, 4-phenyl-1,2,3,6-tetrahydropyri-
dine plays an important role in increasing PARP-1
inhibitory potency by binding the AD site of the PARP
enzyme.6 We therefore expected utilization of 4-phenyl-
1,2,3,6-tetrahydropyridine by application to other pub-
lished benzamide analogues, such as 3,5,7,8-tetrahy-
dro-thiino[4,3-d]pyrimidine-4-one (3),7 phthalazin-
1(2H)-one (4),8 and phenanthridin-6(5H)-one (5),9 to
lead to potent PARP-1 inhibitors (Fig. 2). Although
PARP-1 inhibitors containing these scaffolds have been
published, there still remain problems with regard to
potencies, pharmacokinetics, and physical properties.
We report herein the synthesis and in vitro activity of
related derivatives linked with a 4-phenyl-1,2,3,6-tetra-
hydropyridine moiety via alkyl spacers.
Poly(ADP-ribose) polymerase-1 (PARP-1: EC 2.4.2.30)
is a chromatin-bound nuclear enzyme that plays an
important role in the process of genomic repair.1
PARP-1 is activated by DNA strand breaks and catalyz-
es the transfer of ADP-ribose units from its substrate
nicotinamide adenine dinucleotide (NAD+) to nuclear
acceptor proteins, such as histones, transcription
factors, and PARP itself. However, overactivation of
PARP caused by DNA-damaging stimuli, such as expo-
sure to reactive oxygen species could lead to depletion of
NAD+ and ATP. This depletion of ATP culminates in
cell death via a necrotic pathway.2 Recent studies,
including the availability of knockout mice, have dem-
onstrated that a lack of PARP-1 offers protection in
animal models of stroke, traumatic brain injury, and
Parkinsonꢀs disease.3 These data indicate that inhibition
of PARP-1 may prove useful for the treatment of these
diseases and consequently, a variety of PARP-1 inhibi-
tors have been reported.4 We have recently found
FR247304 (1) as a potent lead inhibitor of PARP-1
using a structure-based drug design (Fig. 1).5 This struc-
ture is characterized by two main fragments, quinazolin-
4(3H)-one and 4-phenyl-1,2,3,6-tetrahydropyridine.
Quinazolin-4(3H)-one contains a benzamido moiety
2. Chemistry
The bicyclic pyrimidin-4(3H)-one derivatives were syn-
thesized, as outlined in Scheme 1. Alkylation of com-
mercially available 4-phenyl-1,2,3,6-tetrahydropyridine
hydrochloride (6) with 4-bromobutyronitrile (7) in the
presence of triethylamine in DMF afforded 4-(4-phen-
yl-3,6-dihyro-1(2H)-pyridinyl)butanenitrile (8). This
intermediate was converted to the corresponding ami-
dine (9) by treatment with methylchloroaluminum
amide.10 Coupling of this amidine with various cyclic
b-ketoesters in the presence of potassium carbonate
afforded the target compounds (10).11
Keywords: PARP-1; Poly(ADP-ribose) polymerase; 4-phenyl-1,2,3,6-
tetrahydropyridine.
*
Corresponding author. Tel.: +81 6 6390 1143; fax: +81 6 6304
5435; e-mail: junya_ishida@po.fujisawa.co.jp
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.094

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J. Ishida et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4221–4225
4-phenyltetrahydropyridine
quinazolinone
Cl
O
Cl
O
NH
NH
N
N
N
Seed compound (1)
FR247304 (2)
PARP-1 inhibition
IC50 (nM)
1200
65
Figure 1. Dramatic improvement of PARP-1 inhibitory potency by introducing 4-phenyltetrahydropyridine.
afforded 17, which was converted to the corresponding
O
O
alkyl bromide (18) by treatment with PBr3 in ethyl ace-
tate. Suzuki coupling of the bromobenzene derivatives
(18) with phenyl boronic acid gave the biphenyl product
(19). Construction of the phenanthridin-6(5H)-one core
was effected by treatment with P2O5 and POCl3 to pro-
vide 20.14 The target compound (21) was obtained by
amination of the 2-bromoalkyl phenanthridin-6(5H)-
one (20) with 4-phenyl-1,2,3,6-tetrahydropyridine
hydrochloride (6) in the presence of triethylamine.
NH
NH
N
O
N
S
NH
O
HN
N
N
NH2
O
H
3
4
5
Figure 2. Three examples of published PARP-1 inhibitors containing
3,5,7,8-tetrahydro-thiino[4,3-d]pyrimidine-4-one (3), phthalazin-
1(2H)-one (4), and phenanthridin-6(5H)-one (5) scaffolds.
The synthesis of 3-substituted phenanthridin-6(5H)-ones
is outlined in Scheme 4. The intermediate 3-carboxylic
acid (22) was prepared, according to the literature.15
Reduction of 22 using a mixed anhydride method gave
the 3-hydroxymethyl derivative (23). Selective chlorina-
tion of the hydroxylmethyl group of 23 using SOCl2
failed on account of the extremely low solubility of 23
in organic solvents. Dichlorination of both the hydroxy-
methyl and amide functionalities of 23 in refluxing
POCl3 was carried out, followed by amination with an
appropriate amine and hydrolysis of the chloropyridine
moiety under acidic conditions to give the desired
compound (24).
The synthesis of the requisite 4-substituted phthalazin-
1(2H)-one is shown in Scheme 2. The phthalazin-
1(2H)-one core was prepared, as described in the litera-
ture by Napoletano et al.12 Wittig olefination of the
phosphonium bromide (11) with benzyloxy alkanal
(12, n = 1, 2), followed by coupling with hydrazine
hydrate in EtOH, afforded the corresponding
4-benzyloxyalkylphthalazin-1(2H)-one (13). Direct bro-
mination of the benzyl ethers (13) with BBr3 in CH2Cl2
gave the bromide (14),13 which upon amination with an
appropriate amine in the presence of Et3N gave the tar-
get compounds (15). We also attempted to prepare
propyl analogue (n = 0) through the same procedure,
but bromination of 13 failed owing to decomposition
of the phthalazin-1(2H)-one nucleus.
3. Results and discussion
The PARP-1 IC50ꢀs for the new bicyclic pyrimidinone
derivatives prepared in this work are given in Table
1.16 Although the thia-containing core (10d) was expect-
ed to have the highest potency amongst these derivatives
according to literature precedent, we optimized this ali-
phatic ring to generate information on the SAR of ana-
logues bearing a 4-phenyl-1,2,3,6-tetrahydropyridine
2-Substituted phenanthridin-6(5H)-ones were synthe-
sized, as shown in Scheme 3. Monobromination at the
para position of the carbamoyl group on the phenyl
rings of 16 with bromine and sodium acetate in AcOH
i
ii
+
Br
CN
HN
NC
N
HCl
6
7
8
O
N
iii
NH
NH
X
N
N
H2N
n
9
10
Scheme 1. Reagents: (i) Diisopropylethylamine, DMF, 78%; (ii) MeAl(Cl)NH2, toluene, 76%; and (iii) b-ketoester, K2CO3, EtOH 28–57%.

J. Ishida et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4221–4225
4223
O
O
NH
ii
i
OHC
OBn
N
O
+
Br-
PPh3
OBn
n
+
n
11
12a (n = 1)
12b (n = 2)
13a (n = 1)
13b (n = 2)
O
O
NH
N
NH
iii
N
R1
N
Br
R2
n
n
14a (n = 1)
14b (n = 2)
R1
N
15a (n = 1)
15b (n = 2)
=
R2
R2
N
N
R1
N
O
=
15c (n = 1)
Scheme 2. Reagents: (i) (a) Et3N, THF, (b) NH2NH2ÆH2O, EtOH, 13a = 51%, 13b = 40%; (ii) BBr3, CH2Cl2, 14a = 58%, 14b = 75%; and (iii) 6 or
morpholine, Et3N, DMF, 15a = 48%, 15b = 27%, 15c = 25%.
NHCO2Et
NHCO2Et
Br
NHCO2Et
Br
ii
i
HO
HO
Br
n
n
n
16a (n = 1)
16b (n = 2)
17a (n = 1)
17b (n = 2)
18a (n = 1)
18b (n = 2)
O
O
NH
NH
NHCO2Et
iii
iv
v
Br
n
N
n
Br
n
19a (n = 1)
19b (n = 2)
20a (n = 1)
20b (n = 2)
21a (n = 1)
21b (n = 2)
Scheme 3. Reagents: (i) Br2, NaOAc, AcOH, 17a = 92%, 17b = 100%; (ii) PBr3, AcOEt, 18a = 66%, 18b = 62%; (iii) phenylboronic acid, Pd(PPh3)4,
DME, 2 M Na2CO3 (aq), 19a = 100%, 19b = 87%; (iv) (a) P2O5, POCl3, 20a = 74%, 20b = 79%; and (v) 6, Et3N, DMF, 21a = 45%, 21b = 29%.
moiety. Optimization of the ring size fused with the pyri-
midinone showed that a six-membered ring (10b) result-
ed in having the highest potency among the three
different sizes of aliphatic rings prepared (20 nM). As
expected, the thia analogue (10d) had the highest potency
among these derivatives (8.9 nM). According to our as-
say protocol, known compound (3), which has no 4-
phenyl-1,2,3,6-tetrahyropyridine, had an IC50 value of
only 498 nM therefore, we could demonstrate that intro-
duction of a 4-phenyl-1,2,3,6-tetrahyropyridine moiety
potentiates PARP-1 inhibition drastically. It is notewor-
thy that replacement of the carbon atom at the 6 position
of the tetrahydroquinazolinone (10b) by a sulfur atom
(10d) led to an approximately 2 times increase in poten-
cy, whilst a similar substitution with nitrogen or oxygen
(10e, 10f) reduced activity 1 or 2 orders of magnitude.
The decrease in potency of 10e and 10f may be attributed
to the hydrophilic nitrogen or oxygen interacting electro-
statically with hydrophobic residues of the PARP en-
zyme around this region. The addition of methyl
groups to the cyclohexene moiety resulted in a dramatic
loss of potency (10g, 10h), presumably on account the
planar pocket formed by two parallel tyrosine residues
(Tyr 896 and Tyr 907) could not accommodate a cyclo-
hexene moiety having bulky methyl groups efficiently.
The activity of 4-substituted phthalazin-1(2H)-one
derivatives is given in Table 2. Compound 15a linked

4224
J. Ishida et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4221–4225
O
O
Cl
NH
ii (a)
i
NH
N
OH
Cl
CO2H
23
22
Cl
O
HCl
ii (b)
ii (c)
N
NH
R1
N
R1
N
R2
R2
R1
N
=
=
24a
24b
R2
R2
N
N
R1
N
O
Scheme 4. Reagents: (i) (a) i-BuOCOCl, Et3N, THF, (b) NaBH4, THF, H2O, 60%; (ii) (a) POCl3, (b) 6 or morpholine, Et3N, DMF, (c) 4 N HCl (aq),
EtOH, 24a = 68%, 24b = 87%.
Table 1. PARP-1 inhibition of pyrimidin-4(3H)-one derivatives
Table 2. PARP-1 inhibition of phthalazin-1(2H)-one derivatives
O
O
NH
NH
X
N
N
N
R1
N
n
R2
Compound
n
X
IC50 (nM)
n
10a
10b
10c
10d
10e
10f
10g
10h
10i
3
1
2
3
2
2
2
2
2
2
CH2
CH2
CH2
S
249
20
Compound
n
IC50 (nM)
R1
N
495
R2
8.9
NH
O
8600
200
15a
1
64
NMe
(Me)CH
(Me)2C
6100
274
N
N
>10,000
467
15b
15c
2
1
119
950
O
with phenyltetrahydropyridine via four methylene units,
having an IC50 value of 64 nM, was twofold more po-
tent than the five carbon analogue 15b (119 nM). To val-
idate the effect of a 4-phenyl-1,2,3,6-tetrahyropyridine,
the morpholine analogue (15c) was estimated, revealing
that absence of a phenyl ring led to a severe loss of
PARP-1 inhibitory potency (950 nM).
an IC50 value of 110 nM , which was relatively potent,
regardless of the absence of a 4-phenyl-1,2,3,6-tetrahy-
dropyridine. This result implied that the SAR of phenan-
thridin-6(5H)-one derivatives was different from the
previous two cases therefore; the morpholine analogue
(24c) was prepared to verify the role of a 4-phenyl-
1,2,3,6-tetrahydropyridine. Unexpectedly, 24c had very
high potency against PARP-1 (23 nM). This is presum-
ably because conformation around the nitrogen atom
was fixed effectively to interact with the residue of the
PARP-1 enzyme in the case of 24c. In other words, in
the case of phenanthridin-6(5H)-one derivatives, the
fused benzene ring connected with an alkylamino chain
may play a crucial role in fixing the nitrogen atom to
have high affinity. Meanwhile, in the case of bicyclic pyri-
The activity of phenanthridin-6(5H)-one derivatives is
given in Table 3. In the case of 2-substituted derivatives,
the compound bearing a propyl linker (21a) had more
potency (46 nM) than the butyl analogue (21b,
119 nM). The 3-substituted derivative (24), however,
had the highest activity (12 nM) against PARP-1 within
this series, revealing that substitution at the 3-position is
the most favorable for improving PARP-1 inhibitory
potency. On the other hand, known compound 5 had

J. Ishida et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4221–4225
Table 3. PARP-1 inhibition of phenanthridin-6(5H)-one derivatives
4225
O
NH
R1
R2
Compound
IC50 (nM)
21a
R1 =
R2 = H
46
N
21b
24a
R1 =
R2 = H
R2 =
119
16
N
R1 = H
N
N
O
24b
5
R1 = H
R2 =
23
110
3. (a) Laplaca, M. C.; Zhang, J.; Raghupathi, R.; Li, J. H.;
Smith, F.; Bareyre, F. M.; Snyder, S. H.; Graham, D. I.;
Mclntosh, T. K. J. Neurotrauma 2001, 18(4), 369; (b)
Adbelkarim, G. E.; Harms, C.; Katchanov, J.; Dirnagl,
midinone and phthalazin-1(2H)-one derivatives, the
phenyl ring of the 4-phenyl-1,2,3,6-tetrahydropyridine
moiety may permit this vital fixation of the nitrogen
atom by efficiently binding the AD site.
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In conclusion, we have demonstrated that 4-phenyl-
1,2,3,6-tetrahydropyridine is an excellent fragment to im-
prove PARP-1 inhibitory potency by connecting it via
alkyl spacers to published scaffolds, such as pyrimidine-
4-one, phenanthridin-6(5H)-one, and phthalazin-1(2H)-
one. We have obtained highly potent PARP-1 inhibitors
10b, 10d, 15a, 21a, and 24a. This utilization of 4-phenyl-
tetrahydropyridine could be extended to other published
PARP-1 inhibitors containing scaffolds that mimic benz-
amide. Detailed SAR, pharmacokinetic, and in vivo data
for these derivatives will be reported in due course.
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Acknowledgment
We thank Dr. David Barrett for his critical reading of
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