Synthesis of substituted 5[H]phenanthridin-6-ones as potent poly(ADP-ribose)polymerase-1 (PARP1) inhibitors

Article abstract of DOI:10.1016/S0960-894X(01)00281-5

1-, 2-, 3-, 4-, 8-, or 10-Substituted 5(H)phenanthridin-6-ones were synthesized and found to be potent PARP1 inhibitors. Among the 28 compounds prepared, some showed not only low IC₅₀ values (compound 1b, 10 nM) but also desirable water solubility characteristics. These properties, which are superior to the common PARP1 inhibitors such as benzamides and isoquinolin-1-ones, are essential for potential therapeutic usage. The variety of compounds allows SAR analysis of favored substituents and substituted positions on 5(H)phenanthridin-6-one ring.

Full text of DOI:10.1016/S0960-894X(01)00281-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1687–1690
Synthesis of Substituted 5[H]Phenanthridin-6-ones as Potent
Poly(ADP-ribose)polymerase-1 (PARP1) Inhibitors
Jia-He Li,* Larisa Serdyuk, Dana V. Ferraris, Ge Xiao, Kevin L. Tays,y
Paul W. Kletzly, Weixing Li, Susan Lautar, Jie Zhang and Vincent J. Kalish
Guilford Pharmaceuticals Inc., 6611 Tributary Street, Baltimore, MD 21224, USA
Received 1 March 2001; accepted 13 April 2001
Abstract—1-, 2-, 3-, 4-, 8-, or 10-Substituted 5(H)phenanthridin-6-ones were synthesized and found to be potent PARP1 inhibitors.
Among the 28 compounds prepared, some showed not only low IC50 values (compound 1b, 10 nM) but also desirable water solu-
bility characteristics. These properties, which are superior to the common PARP1 inhibitors such as benzamides and isoquinolin-1-
ones, are essential for potential therapeutic usage. The variety of compounds allows SAR analysis of favored substituents and
substituted positions on 5(H)phenanthridin-6-one ring. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
Chemistry
Poly(ADP-ribose) polymerase1 (PARP1, EC 2.4.2.30),
also known as poly(ADP-ribose) synthetase (PARS), is
an abundant nuclear protein in most of the eukaryotic
tissues. The discovery of PARP1 involvement in ische-
mia/reperfusion injuries rekindled interest for possible
clinical use of PARP inhibitors.1 In several studies, we
demonstrated that a prototype PARP1 inhibitor, GPI
6150 (1,11b-dihydrobenzopyrano[4,3,2-de]isoquinolin-
3-one), was efficacious in reducing tissue damage in
rodent models of focal cerebral ischemia and regional
myocardial ischemia.2 A common structural feature for
the classical PARP1 inhibitors is a carboxamide
attached to an aromatic ring or a fused aromatic lactam
or imide.3 Most of these inhibitors are structurally pla-
nar and show limited solubility in both organic and
aqueous solvents. (5H)Phenanthridin-6-one (1a in Table 1)
was initially identified as a moderate PARP inhibitor.4
In vivo testing of this compound is hindered by its poor
solubility in biocompatible vehicles. It was also found to
cross inhibit heterophil arginine ADP-ribose transferase
(IC50=47 mM).4 We report here the improvement of
this class of inhibitors in potency and physicochemical
properties by installation of appropriate substituent(s)
onto 5[H]phenanthridin-6-one.
In order to incorporate specific substituents on the ring
system of 1a, several distinct synthetic pathways were
designed. Among these approaches, the most efficient
method was the one-step Smith or Schmidt reaction
using an assortment of commercially available sub-
stituted fluoren-9-ones as starting materials and sodium
azide as an insertion agent under acidic conditions5 as
illustrated in method I of Scheme 1. When a 2,7-di-
substituted fluoren-9-one was used as a starting mate-
rial, 3,8-disubstituted 5[H]phenanthridin-6-ones 1b–f
were obtained.
If the two substituents at the 2,7-position on the fluo-
ren-9-one are not identical, the reaction leads to a mix-
ture of regioisomers. Compound 1b was obtained by
crystallization from a regioisomeric mixture of 3-[8-fluoro-
6(5H)phenanthridinone] sulfonic acid and of 8-[3-
fluoro-6(5H)phenanthridinone]sulfonic acid, which were
prepared by mixing 7-(2-fluoro-fluoren-9-one)sulfonic
acid and sodium azide in concentrated sulfuric acid at
25 ꢀC for 10 h followed by ice-cold water workup. The
pure isomer 3-[8-fluoro-6(5H)phenanthridinone] sulfuric
acid 1b appeared as a precipitate upon adjusting pH of
the solution to 8 and standing at 0 ꢀC for 24 h. The
structure of 1b was distinguished from its regioisomer
by observation of NOE between H-4 (d 7.68, s) and the
1
NH (d 11.9, br. s) protons in H NMR experiments.
*Corresponding author. Tel.: +1-410-631-6780; fax: +1-410-631-
6797; e-mail: lij@guilfordpharm.com
yPresent address: The R.W. Johnson Pharmaceutical Research Institute,
3210 Merryfield Row, San Diego, CA 92007, USA.
In order to enable specific substituent variation at
different positions of 1a, cross-coupling routes were
developed. Derivatives of 3-carboxyl-5[H]phenanthridin-
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00281-5

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J.-H. Li et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1687–1690
Table 1.
No.
Substitution
3-SO3H, 8-F
Method IC50 (mM)
Formulaa
1H NMRb
d
mp (ꢀC)c
300+
1b
I
I
I
0.01
0.24
3.26
C13H8FNO4S
11.9 (bs, 1H), 8.59 (dd, J=9.0, 5.1 Hz, 1H), 8.33 (d, J=8.4 Hz, 1H), 7.96
(dd, J=9.2, 2.9 Hz, 1H), 7.74 (ddd, J=9.0, 8.8, 2.9 Hz, 1H), 7.68 (s, 1H),
7.47 (d, J=8.4 Hz, 1H)
11.94 (bs, 1H), 8.55 (d, 1H, J=8.8 Hz), 8.43 (d, J=8.7 Hz, 1H), 8.24
(d, J=2.3 Hz, 1H), 7.92 (dd, J=8.8, 2.3 Hz, 1H), 7.40 (d, J=2.1 Hz, 1H),
7.32 (dd, J=8.7, 2.1 Hz, 1H)
11.05 (bs, 1H), 7.88 (d, J=8.4 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H), 7.34
(s, 1H), 7.00 (d, J=8.4 Hz, 1H), 6.46 (d, J=8.4 Hz, 1H), 6.42 (s, 1H),
5.35 (s, 2H), 5.32 (s, 2H)
12.40 (bs, 1H), 9.00 (s, 1H), 8.89 (d, J=8.4 Hz, 1H), 8.77 (d, J=8.2 Hz,
1H), 8.65 (d, J=8.4 Hz, 1H), 8.23 (s, 1H), 8.10 (d, J=8.2 Hz, 1H)
11.69 (bs, 1H), 8.54 (s, 1H), 8.44 (d, J=8.4 Hz, 1H), 8.31 (d, 1H, J=8.3
Hz, 1H), 7.99 (d, J=8.4 Hz, 1H), 7.67 (s, 1H), 7.45 (d, J=8.3 Hz, 1H)
11.87 (br s, 1H), 8.56 (d, J=8 Hz, 1H), 8.52 (d, J=8 Hz, 1H), 8.36 (d,
J=8 Hz, 1H), 8.00 (s, 1H), 7.91 (t, J=8 Hz, 1H), 7.78 (d, J=8 Hz, 1H),
7.73 (t, J=8 Hz, 1H), 3.91 (s, 3H)
1c
1d
1e
3-Cl, 8-Cl
C13H7Cl2NO
300+
.
C13H11N3O 0.2AcOH
3-NH2, 8-NH2
3-NO2, 8-NO2
300–301
I
I
35
C13H7N3O5
300+
300+
.
C13H7NNa2O7S2 1.3H2O
1f
3-SO3Na, 8-SO3Na
3-COOMe
>100
0.184
5a1
II
C15H11NO3
276–279
5a2
5b1
5b2
5c1
8-F, 3-COOMe
3-COONa
II
II
II
II
0.037
0.057
0.021
0.035
C15H10FNO3
11.97 (bs, 1H), 8.63 (m, 1H), 8.47 (d, J=8 Hz, 1H), 7.97 (m, 2H), 7.77
(m, 2H), 3.90 (s, 3H)
7.66 (d, J=8.0 Hz, 1H), 7.54 (d, J=8.0 Hz, 1H), 7.45 (d, J=8.0 Hz, 1H),
7.40 (t, J=8.0 Hz, 1H), 7.26 (m, 2H), 7.01 (s, 1H)
7.38 (m, 1H), 7.20 (m, 2H), 7.11 (d, J=8.0 Hz, 1H), 7.02 (t, J=8.0 Hz,
1H), 6.95 (s, 1H)
11.82 (bs, 1H), 8.58 (d, J=8.0 Hz, 1H), 8.54 (t, J=8.0 Hz, 1H), 8.49
(d, J=8.0 Hz, 1H), 8.34 (d, J=8.0 Hz, 1H), 7.89 (t, J=8.0 Hz, 1H),
7.83 (s, 1H), 7.70 (m, 2H), 3.58 (m, 5H), 3.40 (m, 2H), 3.31 (m, 5H)
11.98 (bs, 1H), 8.67 (m, 1H), 8.55 (t, J=8.0 Hz, 1H), 8.47 (d, J=12 Hz,
1H), 7.99 (d, J=8.0Hz, 1H), 7.84 (s, 1H), 7.79 (t, J=8.0 Hz, 1H), 7.70
(d, J=8.0 Hz, 1H), 3.59 (m, 5H), 3.41 (m, 2H), 2.45 (m, 5H)
11.97 (bs, 1H), 8.70 (m, 2H), 8.47 (d, J=8.0 Hz, 1H), 8.00 (d, J=8.0 Hz,
1H), 7.84 (s, 1H), 7.75 (t, J=8.0 Hz, 1H), 7.68 (d, J=8.0 Hz, 1H), 7.30
(m, 5H), 3.53 (t, J=8.0 Hz, 2H), 2.88 (t, J=8.0 Hz, 2H)
11.85 (bs, 1H), 8.39 (s, J=7.9 Hz, 1H), 8.37 (s, J=8.1 Hz, 1H), 7.83
(t, J=7.3 Hz, 1H), 7.69 (t, J=7.9 Hz, 1H), 7.54 (t, J=8.1 Hz, 1H), 7.46
(d, J=8.1 Hz, 1H), 7.27 (d, J=7.3 Hz, 1H)
280–300
300+
.
C14H9NO3 1Na
.
14H8FNO3 1Na
8-F, 3-COONa
C
300+
C20H21N3O3
264–267d
5c2
5c3
9a
II
II
0.014
0.15
2.01
C20H20FN3O3
C22H17FN2O2
C14H9NO3
285–290
293–298
300+
1-COOH
III
9b
9c
4-NH2
4-NO2
III
III
0.312
6.1
C13H10N2O
10.69 (bs, 1H), 8.42 (d, J=7.8, 1H), 8.29 (d, J=8.2 Hz, 1H), 7.77 (m, 1H), 263–268
7.57 (m, 2H), 7.04 (t, J=7.8 Hz, 1H), 6.86 (d, J=7.4 Hz, 1H), 5.09 (s, 2H)
C13H8N2O3 0.4.9H2O
11.32 (bs, 1H), 8.50 (d, J=8.0 Hz, 1H), 8.46 (d, J=8.0 Hz, 1H), 8.40 (d,
J=8.3 Hz, 1H), 8.22 (d, J=8.3 Hz, 1H), 7.80 (t, J=7.7 Hz, 1H), 7.63 (t,
J=7.7 Hz, 1H), 7.32 (t, J=8.1 Hz, 1H)
263–264
9d
9e
10-Me, 3-F
10-COOH
III
III
0.092
C14H10FNO
11.81 (bs, 1H), 8.49 (m, 1H), 8.29 (d, J=7.8 Hz, 1H), 7.73 (d, J=6.7 Hz,
1H), 7.54 (t, J=7.6 Hz, 1H), 7.17 (d, J=7.1 Hz, 1H), 7.11 (t, J=8.5 Hz,
1H), 2.91 (s, 3H)
11.50 (bs, 1H), 8.52 (d, J=8.0 Hz, 1H), 8.12 (d, J=8.2 Hz, 1H), 7.91 (d,
J=7.2 Hz, 1H), 7.84 (d, J=8.3 Hz, 1H), 7.34 (t, J=7.7 Hz, 1H), 7.67 (t,
1H), 7.33 (t, J=7.7 Hz, 1H)
300+
>60
272–275e
.
C14H9NO3 1.1H2O
10a1 10-Me, 3-CF3, 2-NO2
III
III
III
0.136
0.156
0.18
C15H9F3N2O3
C13H8N2O3
C13H10N2O
12.37 (bs, 1H), 9.22 (s, 1H), 8.32 (d, J=8.0 Hz, 1H), 7.90 (s, 1H), 7.84 (d,
J=8.0 Hz, 1H), 7.70 (t, J=8.0 Hz, 1H), 2.98 (s, 3H)
300+
10a2
10b1
2-NO2
2-NH2
12.2 (bs, 1H), 9.21 (s, 1H), 8.69 (d, J=8.0 Hz, 1H), 8.37–8.34 (m, 2H),
7.94 (t, J=8.0 Hz, 1H), 7.75 (t, J=8.0 Hz, 1H), 7.52 (d, J=9.0 Hz, 1H)
11.34 (bs, 1H), 8.30 (d, J=7.6 Hz, 1H), 8.22 (d, J=8.0 Hz, 1H), 7.82 (t,
J=7.2 Hz, 1H), 7.60 (t, J=7.2 Hz, 1H), 7.48 (s, 1H), 7.11 (d, J=8.4 Hz,
1H), 6.82 (d, J=8.0 Hz, 1H), 5.02 (s, 2H)
381–382
284–285
.
C13H11N3O 0.4H2O
10b2
3-NH2, 2-NH2
III
III
III
0.18
0.044
0.03
11.15 (bs, 1H), 8.18 (d, J=7.6 Hz, 1H), 7.99 (d, J=8.2 Hz, 1H), 7.69 (t,
J=7.0 Hz, 1H), 7.38–7.35 (m, 2H), 6.48 (s, 1H), 5.19 (s, 2H), 4.50 (s, 2H)
11.46 (bs, 1H), 8.30 (d, J=8.0 Hz, 1H), 8.00 (s, 1H), 7.71 (d, J=8.0 Hz,
1H), 7.57 (t, J=8.0 Hz, 1H), 7.46 (s, 1H), 5.44 (s, 2H), 2.92 (s, 3H)
11.76 (bs, 1H), 11.03 (bs, 1H), 8.68 (s, 1H), 8.34 (d, J=8.0 Hz, 1H), 8.27
(d, J=8.0 Hz, 1H), 7.91 (t, J=8.0 Hz, 1H), 7.65 (m, 2H), 7.37 (d, J=8.0
Hz, 1H), 4.41 (s, 2H), 3.34 (s, 9H)
300+
300+
10b3 10-Me, 3-CF3, 2-NH2
C15H11F3N2O
.
C18H20ClN3O2 0.7H2O
10c1
269–272
.
C19H22ClN3O2 1.5H2O
10c2
10c3
11a1
III
III
III
0.195
0.033
0.03
11.72 (bs, 1H), 10.30 (bs, 1H), 8.34 (m, 3H), 7.86 (t, J=8.0 Hz, 1H),
7.65 (t, J=8.0 Hz, 1H), 7.23 (s, 1H), 4.43 (s, 2H), 3.32 (s, 9H),
2.31 (s, 3H)
11.98 (bs, 1H), 10.02 (s, 1H), 8.54 (s, 1H), 8.34 (d, J=8.0 Hz, 1H),
7.70 (m, 2H), 7.66 (t, J=8.0 Hz, 1H),
4.38 (s, 2H)
12.1 (bs,1H), 9.65 (bs, 1H), 8.79 (s, 1H), 8.56 (d, J=8.1 Hz, 1H), 8.36
(d, J=7.2 Hz, 1H), 7.93 (m, 2H), 7.75 (t, J=7.5 Hz, 1H), 7.54 (d, J=8.7
Hz, 1H), 3.41 (d, J=12.2 Hz, 2H), 3.14 (m, 4H), 2.89 (t, 2H), 1.71 (m, 6H)
300+
300+
C17H12ClF3N2O2
.
C20H24ClN3O3S 0.4H2O
262–267
(continued)

J.-H. Li et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1687–1690
1689
Table 1 (continued)
No.
Substitution
Method IC50 (mM)
Formulaa
1H NMRb
d
mp (ꢀC)c
275–279
.
C21H27N3O6S2 0.5H2O
11a2
III
0.036
12.10 (bs, 1H), 9.30 (bs, 1H), 8.75 (s, 1H), 8.54 (d, J=8.0 Hz, 1H), 8.36
(d, J=8.0 Hz, 1H), 7.94 (m, 2H), 7.75 (t, J=8.0 Hz, 1H), 7.55 (d, J=8.0
Hz, 1H), 3.43 (d, J=12.0 Hz, 2H), 3.14 (s, 4H), 2.91 (m, 2H), 2.30 (s, 3H),
1.7 (m, 3H)
.
C27H22N2O3S 0.6 H2O
11a3
III
14
12.02 (bs, 1H), 8.69 (bs, 1H), 8.45 (d, J=8.0 Hz, 1H), 8.34 (d, J=8.0 Hz,
1H), 7.93 (t, J=7.6 Hz, 1H), 7.84 (d, J=8.6 Hz, 1H), 7.73 (m, 2H), 7.46
(d, J=8.6 Hz, 1H), 7.24 (m, 8H), 7.13 (m, 2H), 5.76 (s, 1H), 4.11 (t, J=7.7
Hz, 1H), 3.41 (d, J=7.7 Hz, 2H)
265–268
aBased on elemental analyses experiments.
bMeasured on a Bruker Avance-400 MHzNMR spectrometer using trimethylsilane as an internal standard and DMSO- d6 as solvent.
cMeasured on a Thomas-Hoover melting point apparatus, uncorrected.
dLiterature:10a 227–228 ꢀC.
eLiterature:10b 352.5–353 ꢀC.
6-ones 5 were synthesized as outlined in method II of
Scheme 1. Commercially available methyl-2-iodo (or
bromo6) benzoate 2 reacted with methyl-3-nitro-4-iodo
(or bromo7) benzoate 3 in an Ullmann coupling to
afford biphenyl diester derivatives 4 in 42–50% yield.
Reduction of the biphenyl derivative 4 using hydrazine
and Raney nickel efficiently produced 3-methyl
5[H]phenanthridin-6-one carboxylate 5a, which was
subsequently hydrolyzed with base to afford 5[H]phenan-
thridin-6-one-3-carboxylic acid 5b. Amidation of this
acid using standard EDC coupling conditions with an
appropriate amino compound yielded carboxamides 5c.
pling followed by intramolecular Friedel–Crafts acyla-
tion using a biphenylisocyanate as an intermediate as
described in method III of Scheme 1. The cross-coupling
of 5-substituted 2-nitro bromobenzene derivative 6 and
phenylboric acid compounds 7 provided the biphenyl
product 8 in reasonable yield of 40–60%. The nitro
group was easily reduced to the amino functionality 8b
by either treatment with hydrazine and Raney nickel or
typical hydrogenation methods. The desired compound
9 was formed in good yield through intramolecular
Friedel–Crafts acylation of isocyano intermediate 8c,
generated in situ by condensing the amino group with
phosgene.8
Substituted 5[H]phenanthridin-6-ones at 1-, 2-, 3-, 4-, or
10-position were synthesized through Suzuki cross-cou-
Nitration of the resulting 3- or 10-substituted 5[H]phe-
nanthridin-6-ones 9 at the 2-position followed by
hydrogenation provided 2-amino-5[H]phenanthridin-6-
ones 10b. Using the 2-amino group as a linker, desired
products of carboxamides 10c were prepared readily by
reaction of 10b with carboxylic acid halide derivatives in
high yields. Monosulfonation of commercially available
6(5H)-phenanthridinone 1a using neat chlorosulfonic
acid gave sulfonyl chloride compound 11 in good yield.
Amidation of compound 11 using primary amines
afforded target sulfonamide compounds 11a1–3. Thus,
about 30 compounds were made using the methods
described in Scheme 1. Some of these containing a basic
amine or carboxylic acid group were converted to either
hydrochloride or methansufonic acid salts or sodium
salt, respectively. All of the salts showed good solubility
in water. For example, both hydrochloride or methan-
sufonic acid salts (entry 11a1 and 11a2, in Table 1) pre-
pared from the piperidine derivative have solubility in
water greater than 10 mg/mL.
Biological Evaluation and Discussion
In the absence of any detailed structure–activity rela-
tionship (SAR) information about substituted 5[H]phe-
nanthridin-6-ones, optimization of the tricyclic ring was
accomplished by systematically varying the substituents
at 1-, 2-, 3-, 4-, 8-, and 10-positions. All compounds
Scheme 1. Reagents and conditions: (a) NaN3, H2SO4, 25 ꢀC, 3 h; (b)
Cu, neat, 200 ꢀC, 8 h; (c) NH2NH2, Raney nickel, MeOH; (d) 1 N
NaOH; (e) R-NH2, EDC, DMAP in DCM; (f) Na2CO3, Pd(PPh3)4,
Et3N in DME, reflux, 20 h; (g) phosgene in toluene, reflux; (h)
Et2OBF3, 0 ꢀC; (i) HNO3, HOAc, reflux; (j) R-COCl, Et3N; (k) HOS-
O2Cl neat, 0 ꢀC; (l) R-NH2/Et3N in DCM.
1
were fully characterized by H NMR, elemental analy-
sis, and melting point. The IC50’s were determined from
full dose–response curves.9 The structures, inhibition
values and physicochemical properties of 5[H]phenan-
thridin-6-ones are given in Table 1. The SAR of these

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J.-H. Li et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1687–1690
compounds shows that most substituted 5[H]phenan-
thridin-6-ones are more potent inhibitors than the ‘parent’
1a. Only about a quarter of the substituted compounds
have a decreased potency due to unfavorable substitution
position or structural character.
lity dramatically in the acid or salt form. For example,
three of the most potent inhibitors (1b, 10c1, and 11a2)
exhibited excellent water solubility properties with IC50
values as low as 30 nM.
Substitution at 2- and 3-positions
Conclusion
A brief examination of the top 10 potent inhibitors
confirmed that the 2- or 3-positions were preferred
attachment points for substitution. Substitutions at
these positions generally resulted in submicromolar
inhibitors, with many of the potent ones at low nano-
molar range, suggesting beneficial interactions between
the substituent and the enzyme. The electronic nature of
2- or 3-substituents appears insignificant to affect the
inhibition. This was exemplified by the electron-with-
drawing 2-nitro compound 10a2 and electron-donating
2-amino compounds 10b1 and 2,3-diamino 10b2. All of
these three compounds showed values of IC50’s at ca.
0.17 mM and about 3-fold better than the ‘parent’ 1a.
Compounds with large aliphatic substituents (5c1, 5c3,
11a1) show very high potency, indicating that the
volume tolerance at the direction is sufficiently large to
accommodate most substituents. However, there is a
limit defined by the pocket, as in the case of the 3-sub-
stituted compound (11a3, IC50 14 mM), which has a
gem-diphenyl group at the end of carbon chain and is
ca. 30-fold less active than the core 1a.
Using several synthetic approaches, we have prepared 1-,
2-, 3-, 4-, 8-, or 10-substituted 5[H]phenanthridin-6-
ones. The attachment of two optimal substituents onto
the core skeleton resulted in the water-soluble com-
pound, 3(10-fluoro-5[H]phenanthridin-6-one) sulfonyl
acid 1b, which exhibits an IC50 value of 10 nM and 52-
fold greater inhibition activity as compared to the core
structure of 5[H]phenanthridin-6-one 1a. Thus, by
incorporation of substitution with a suitable acidic or
basic residue onto the ring system, we have observed
improvements of potency in vitro, along with obvious
effects on physicochemical characteristics such as water
solubility. In vivo data of some of these compounds will
be disclosed in future publication.
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32, 4525.
6. Pan, H.-L.; Fletcher, T. L. J. Med. Chem. 1965, 8, 491.
7. Ling, C. C. K.; Harris, M. H. J. Chem. Soc. 1964, 1825.
8. Balazs, L.; Nyerges, M.; Kadas, I.; Toke, L. Synthesis 1995,
11, 1373.
Substitution at 8-position
Looking at 3,8-disubstituted compounds (1b–f), 8-fluoro
is clearly the best among those whereas the sulfonyl
worst. The potency increase appears to follow the trend
of F>Cl>H>NH2>NO2>SO3Na, with the 8-sulfonic
acid compound 1f being completely deactivated.
Because 3-substitution generally enhances the potency
as discussed above, 8-substitution may play a dominant
role in the series. In general, bulky 8-substitution (i.e.,
compare 1f to 1b) dramatically diminished the inhibi-
tion of PARP1. In contrast, a smaller halogen, such as
fluorine, potentiates the in vitro efficacy.
Substitution at 1, 4, or 10 positions
9. PARP1 inhibition assay: Purified recombinant human
PARP from Trevigan (Gaithersburg, MD, USA) was used to
determine the IC50 value of a PARP inhibitor. The PARP
enzyme assay is set up on ice in a volume of 100 mL consisting
of 50 mM Tris–HCl (pH 8.0), 2 mM MgCl2, 30 mg/mL of
DNase I activated herring sperm DNA (Sigma, MO, USA), 30
mM [3H]nicotinamide adenine dinucleotide (67 mCi/mmol), 75
mg/mL PARP enzyme, and various concentrations of the
compounds to be tested. The reaction is initiated by incubat-
ing the mixture at 25 ꢀC. After 15 min of incubation, the
reaction is terminated by adding 500 mL of ice cold 20% (w/v)
trichloroacetic acid. The precipitate formed is transferred onto
a glass fiber filter (Packard Unifilter-GF/B) and washed three
times with ethanol. After the filter is dried, the radioactivity is
determined by scintillation counting.
Limited numbers of compounds were prepared in each
of these series. But with an IC50 value of 2 mM and 4-
fold worse than the core structure, 1-carboxylic acid 9a
apparently obstructed the interaction with PARP. Inhi-
bition trend of 4-substituted compounds appeared 4-
amino 9b>1a>4-nitro 9c. In the 10-substitution series,
the methyl group was beneficial (9d, 0.092 mM) whereas
10-carboxylic acid showed little activity (9e, >60 mM),
suggesting that unlike 3-substituted benzamides, the 10-
position (corresponding to 3-position of benzamide) is
quite sensitive to substitutional modification in PARP1
inhibition.
In summary, introduction of either an acidic or amino-
containing side chain at the 2- and/or 3-position not
only enhanced potency by more than 20-fold as com-
pared to the core 1a, but also increases aqueous solubi-
10. (a) Cerbai, G.; Turbanti, L.; Baldacci, G. P.; Tellini, N.
Farmaco, Ed. Sci. 1972, 27, 939. (b) Migachev, G. I.; Ere-
menko, L. V.; Urman, Ya. G.; Bulai, A. Kh.; Dyumaev, K. M.
Zh. Org. Khim. 1979, 15, 1491.