Synthesis and evaluation of thiopyrano[3,4-c]quinoline-9-carboxamide derivatives as inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1)

Article abstract of DOI:10.1007/s00044-011-9673-6

A series of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors, 5-oxo-2,4,5,6-tetrahydro-1H-thiopyrano[3,4-c]quinoline-9-carboxamide derivatives, were successfully synthesized and their PARP-1 inhibitory activity was evaluated. These compounds were prepared from carboxylic acid 7 and the appropriate amines, and a number of the synthesized compounds were found to have significant PARP-1 activity. Among them, 9m showed potent activity in a PARP-1 enzymatic assay and cell-based assay (IC₅₀ = 0.045 μM, ED ₅₀ = 0.54 μM). Molecular modeling studies confirmed the obtained biological results. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9673-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1533–1543
DOI 10.1007/s00044-011-9673-6
ORIGINAL RESEARCH
Synthesis and evaluation of thiopyrano[3,4-c]quinoline-9-
carboxamide derivatives as inhibitors of poly(ADP-ribose)
polymerase-1 (PARP-1)
•
Chun-Ho Park Kwangwoo Chun Bo-Young Joe Jong-Hee Choi
•
•
•
•
Han-Chang Lee Il-Whea Ku Hyun Young Kim Seong-Ho Koh
•
•
•
•
•
Goang Won Cho Seung Hyun Kim Myung-Hwa Kim
Received: 15 February 2011 / Accepted: 13 May 2011 / Published online: 25 May 2011
ꢀ Springer Science+Business Media, LLC 2011
Abstract A series of poly(ADP-ribose) polymerase-1
(PARP-1) inhibitors, 5-oxo-2,4,5,6-tetrahydro-1H-thiopyr-
ano[3,4-c]quinoline-9-carboxamide derivatives, were suc-
cessfully synthesized and their PARP-1 inhibitory activity
was evaluated. These compounds were prepared from
carboxylic acid 7 and the appropriate amines, and a number
of the synthesized compounds were found to have signifi-
cant PARP-1 activity. Among them, 9m showed potent
activity in a PARP-1 enzymatic assay and cell-based assay
(IC50 = 0.045 lM, ED50 = 0.54 lM). Molecular model-
ing studies confirmed the obtained biological results.
damage (D’Amours et al., 1999; Strosznajder et al., 2005).
Depending on the cell type and the extent of DNA damage,
PARP-1 can be involved in DNA repair and neuronal death
through NAD? and ATP depletion. The function of PARP-
1 under mild to moderate DNA damage is to repair DNA
by interacting with other DNA repair effectors, such as
DNA polymerase b and DNA ligase III, and adaptor fac-
tors, such as XRCC1 (Masson et al., 1998). This pathway
occurs in cells that are exposed to certain DNA damaging
agents, such as ionizing radiation, topoisomerase inhibi-
tors, and alkylating agents (Tentori and Graziani, 2005;
Tentori et al., 2002a, b). Indeed, PARP-1 inhibitors have
been demonstrated to enhance cytotoxicity in combination
therapy with cytotoxic agents (Calabrese et al., 2003, 2004;
Miknyoczki et al., 2003; Tentori et al., 2002a, b, 2003).
Therefore, PARP-1 inhibitors could be attractive drug
candidates for the treatment of cancer. Under extensive
DNA damage, overactivation of PARP-1 is induced,
leading to NAD? and ATP depletion and necrotic cell
death (Alano et al., 2004; Ha and Snyder, 1999; Ying et al.,
2005). This pathway has been implicated in the patho-
genesis of several diseases, including stroke, myocardial
infarction, diabetes, shock, neurodegenerative disorder, and
several inflammatory processes (Tentori et al., 2002a, b;
Virag and Szabo, 2002). Thus, the development of PARP-1
inhibitor can provide remarkable therapeutic benefits in
various diseases including ischemic stroke.
Keywords Poly(ADP-ribose)polymerase ꢀ
PARP-1 inhibitor ꢀ Thiopyranoquinoline derivatives
Introduction
PARP-1 is an abundant nuclear enzyme that is responsible
for almost 97% of the poly (ADP-ribose) formation in the
brain, and its activity increased up to 500 times by DNA
C.-H. Park ꢀ K. Chun ꢀ B.-Y. Joe ꢀ J.-H. Choi ꢀ H.-C. Lee ꢀ
I.-W. Ku ꢀ M.-H. Kim (&)
Drug Discovery Laboratory, R&D Center, Jeil Pharmaceutical
Co., Ltd, 117-1, Keungok-Ri, Baekam-Myun, Cheoin-Gu,
Yongin-City, Kyunggi-Do 449-861, Republic of Korea
e-mail: mhkim@jeilpharm.co.kr
Currently, PARP inhibitors such as AG-014699,
AZD2281, ABT-888, and BSI-201 are being developed as
mono- and combination therapy for cancer in clinical studies
(Fig. 1) (Menear et al., 2008; Penning et al., 2009; Sandhu
et al., 2010). Most of the PARP-1 inhibitors compete with
NAD? and are designed to mimic nicotinamide, which forms
three hydrogen bonds to the PARP-1 enzyme. The amide
group of inhibitors is essential for binding to the catalytic
H. Y. Kim ꢀ S.-H. Koh ꢀ G. W. Cho ꢀ S. H. Kim
Department of Neurology, Hanyang University College
of Medicine, Seoul, Republic of Korea
C.-H. Park
Graduate Program in Biomaterials Science and Engineering,
Yonsei University, Seodaemun-Gu, Seoul 120-740,
Republic of Korea
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Med Chem Res (2012) 21:1533–1543
7 and derivatives of this compound were performed, and their
PARP-1 inhibitory activities were evaluated.
Results and discussion
Chemistry
The synthesis of the novel 5-oxo-2,4,5,6-tetrahydro-1
H-thiopyrano[3,4-c]quinoline-9-carboxamides 9a–z was
carried out following the synthetic pathway in Scheme 1.
4-(3,6-dihydro-2H-thiopyran-4-yl)morpholine 3 was
obtained by refluxing 4-oxothiane 1 with morpholine 2 in
the presence of catalytic p-TsOHꢀH2O and subsequently
treated without further purification. The ketamide 5, pre-
pared by reaction of the enamine 3 with ethyl 4-iso-
cyanobenzoate 4, was cyclized in 70% sulfuric acid to the
thiopyranoquinoline-9-carboxylate 6 (Khuthier et al., 1987;
Park et al., 2010; Stork et al., 1963). Hydrolysis of
the carboxylate 6 with 1 N NaOH solution gave its
corresponding carboxylic acid 7, which was optimized by
conversion of acid group to amide. 5-Oxo-2,4,5,6-tetrahy-
dro-1H-thiopyrano[3,4-c]quinoline-9-carboxamides 9a–z
were synthesized by amide coupling with aliphatic amine
or Boc-protected diamine using 1-ethyl-3-(3-dimethylami-
nopropyl) carbodiimide hydrochloride (EDCI). In particu-
lar, the aminoethyl compound 9a and piperazine compound
9j were selected and optimized to improve cellular activity.
The structure of the synthesized thiopyranoquinoline
Fig. 1 PARP inhibitors currently in clinical studies
domain of the enzyme. In addition, the A-ring of inhibitors
binds effectively to the aryl residues, Tyr907 and Tyr896,
through a sandwiched hydrophobic p–p interaction (Ferraris,
2010; Ishida et al., 2006; Kinoshita et al., 2004; Matsumoto
et al., 2006).
We have recently reported tricyclic PARP-1 inhibitors, 9-
hydroxy-1,2-dihydro-4H-thiopyrano[3,4-c]quinoline-5(6H)-
one derivatives (Park et al., 2010). These derivatives include a
non-aromatic A-ring and fit well to the active site even though
their conformations are not flat. In this study, we describe fur-
ther modifications of 9-hydroxy-1,2-dihydro-4H-thiopyr-
ano[3,4-c] quinoline-5(6H)-ones. The PARP-1 inhibitors,
5-oxo-2,4,5,6-tetrahydro-1H-thiopyrano[3,4-c]quinoline-9-car-
boxamide derivatives, were designed and synthesized. Addi-
tionally, docking studies of thiopyranoquinoline carboxylic acid
1
compounds was established on the basis of H NMR and
mass spectral analysis.
Scheme 1 Reagents and
conditions: (a) p-TsOH•H2O,
toluene, rt; (b) (i) toluene, rt, (ii)
2 N HCl, rt; (c) 70% H2SO4, rt;
(d) 1 N NaOH, MeOH, 50ꢁC;
(e) (i) EDC, HOBt, DMF, rt, (ii)
3.7 M HCl in 1,4-dioxane, rt
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Med Chem Res (2012) 21:1533–1543
Biology
1535
Table 1 continued
Compounds
NR2R3
IC50(lM)a
ED50(lM)b
The PARP-1 inhibitory activity of all synthesized thio-
pyranoquinoline compounds 6, 7, and 9a–z was evaluated
in vitro PARP-1 enzymatic assay and cell-based assay,
which is summarized in Table 1.
9m
0.045
0.54
9n
9o
9p
9q
0.042
0.134
0.079
0.065
0.77
2.41
0.61
0.84
Table 1 Enzyme and cellular activity of the synthesized compounds
9a–z
9r
0.040
0.46
Compounds
NR2R3
IC50(lM)a
ED50(lM)b
9s
9t
0.094
0.234
2.91
1.29
6
CO2Et
COOH
0.536
0.078
0.017
–
–
7
9a
[30
9b
9c
9d
0.030
0.027
0.058
[30
[30
2.81
9u
9v
0.187
0.019
2.51
2.19
9e
9f
0.060
0.036
0.044
[30
[30
[30
9w
0.020
[30
9x
9y
0.047
0.068
0.55
0.32
9g
9h
0.166
4.02
9z
0.067
0.50
9i
9j
0.175
0.008
1.26
a
Enzymatic assays followed a commercially available protocol
(Trevigen kit, 4671-096-K) in 384 well plates. Values are the mean of
triplicate experiments
[30
b
The CHO-K1 (Chinese hamster ovary) cell line was used for cell-
based assay. Values are the mean of quadruplicate experiments
9k
9l
0.188
0.047
2.53
2.00
Generally, these compounds showed good enzymatic
activity. Some compounds (9a–c, 9e–g, and 9j), however,
displayed poor cellular activity. The polar group at the end
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Med Chem Res (2012) 21:1533–1543
of the derivative chain seems to prevent cell permeability.
Among the N-alkyl piperazine derivatives (9k–s), com-
pounds 9m, 9n, and 9p, which have a linear 3–5 carbon
chain, displayed significant activities in enzyme assays
and more potent activities in cellular assays. Additionally,
N-propyl piperazine compound 9m exhibited good water
solubility ([1 mg/ml). Compounds 9q and 9r, which have
a bulky branched carbon chain, also showed potent activity
in a PARP-1 enzymatic assay and cell-based assay. As
mentioned above, the compound with the polar group at the
end of the chain (9o) showed less activity than compound
9n. N-aryl or -arylalkyl piperazine derivatives (9t, 9u, and
9v) displayed a loss of potency in cellular activity. As
expected, the compound with the polar group (9w)
decreased cellular activity significantly.
A molecular modeling study with automated dock-
ing simulation was conducted to explore the interaction
between target (PARP-1) and the thiopyranoquinoline-
9-carboxylic acid 7. Ligand included in 1UK0 crystal was
very well docked with its protein for validation of docking
study. The amide group of the compound 7 formed
hydrogen bonds to Ser904 and Gly863 of the enzyme.
Furthermore, the hydrogen bond between the acid group of
the compound and Asp766 of the enzyme increased PARP-
1 activity (Fig. 2) compared to the carboxylate 6 which do
not formed hydrogen bond. In an attempt to comprehend
the binding conformation of N-alkyl piperazine derivatives
in the active site of the enzyme, a docking study of highly
potent inhibitor (9m) was performed, and the result showed
that the nitrogen of piperazine analogs is very important for
the hydrogen bond to Ala880 of the enzyme backbone
(Fig. 3). The docking scores were estimated binding free
Fig. 3 Docking of compound 9m in the catalytic domain of human
PARP-1 (PDB code: 1UK0) (Kinoshita et al., 2004)
energy using CHARMm implicit salvation models and
their results were -65.0, -45.6, and -6.6 kcal/mol for
compound 6, 7, and 9m, respectively. Finally, N-cycloalkyl
piperazine derivatives (9x–z) were synthesized by the same
method as described above in effort to further optimize
N-propyl piperazine compound 9m. From the docking study,
the cycloalkyl group of these derivatives was located in a
large hydrophobic pocket of the active site; these com-
pounds (9x–z) resulted in a significant increase in PARP-1
inhibitory activity. Their activities were, respectively,
IC50 = 0.047, 0.068, and 0.067 lM in an enzymatic assay,
and ED50 = 0.55, 0.32, and 0.50 lM in a cellular assay.
Pharmacokinetic properties and microsomal stabilities
of selected PARP-1 inhibitors are displayed in Tables 2
and 3, respectively. Compound 9m displayed high clear-
ance and moderate half-time and showed good metabolic
stability, with a 41.7 min half-life in the human liver
microsomal stability test compared to those of compounds
9q and 9x.
In conclusion, we have described a straightforward
synthesis, docking study and biological evaluation of
Table 2 Rat PK profile of 9m (5 mg/kg), n = 3
Compound
9m
5
Dose (mg/kg)
AUC0-inf (h ng/ml)
CL (l/h/kg)
t1/2 (h)
1303.1
3.8
1.4
Fig. 2 Docking of compound 7 in the catalytic domain of human
PARP-1 (PDB code: 1UK0) (Kinoshita et al., 2004)
Values were detected by LC/MS/MS after intravenous administration
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1537
Ethyl 4-isocyanatobenzoate 4 (6.6 g, 34.43 mmol) in
toluene (30 ml) was added dropwise to a stirred solution of
unstable enamine 3 in toluene (20 ml) at room temperature,
and the stirring was continued at room temperature over-
night. Then 2 N HCl (8.7 ml, 17.21 mmol) was added, and
the resulting mixture was stirred at room temperature for
1 day. The mixture was cautiously neutralized with 2 N
NaOH, and the precipitate was filtered. The filtrate was
taken up with water and extracted with dichloromethane.
The organic layer was washed with brine, dried over
Na2SO4, filtered and concentrated. The residue was then
purified by flash column chromatography to afford the title
compound 5 as a white solid (2.8 g, 53%, 2 step).
1H NMR (400 MHz, DMSO-d6): d 9.39 (s, 1H, –C(O)NH–),
8.01 (d, J = 8.4 Hz, 2H, Ar-H), 7.63 (d, J = 8.4 Hz, 2H,
Ar-H), 4.35 (qt, J = 7.2 Hz, 2H, –OCH2CH3), 3.69 (dd,
J = 10.4 Hz, J = 4.8 Hz, 1H, thiopyran), 3.47–3.40 (m,
1H, thiopyran), 3.22 (dd, J = 10.4 Hz, 1H, thiopyran),
3.06–2.98 (m, 2H, thiopyran), 2.91–2.80 (m, 2H, thiopy-
ran), 1.38 (t, J = 7.2 Hz, 3H, –OCH2CH3); MS (ESI?) m/
z: [M?H]? 308.1.
Table 3 Human liver microsomal stability of selected compounds
Drug
t1/2 (min)
Buspirone
4.0
41.7
15.9
14.5
9m
9q
9x
Microsomal activity was detected at 0, 15, 45, and 80 min by LC/MS/
MS. The incubation temperature was 37ꢁC. Values are the mean of
triplicate experiments. Buspirone was used as a reference
5-oxo-2,4,5,6-tetrahydro-1H-thiopyrano[3,4-c]quinoline-
9-carboxamide derivatives as potent PARP-1 inhibitors.
Our studies indicate that compound 9m was found to be a
potent PARP-1 inhibitor, showing IC50 = 0.045 lM and
ED50 = 0.54 lM in enzyme and cellular activity, respec-
tively. Moreover, compound 9m showed good metabolic
stability and aqueous solubility. These findings suggest that
a potent PARP-1 inhibitor 9m could be a useful therapeutic
candidate for cancers and ischemic stroke.
Experimental section
Ethyl 5-oxo-2,4,5,6-tetrahydro-1H-thiopyrano
[3,4-c]quinoline-9-carboxylate (6)
Chemistry
Ketamide 5 (1.0 g, 3.25 mmol) was suspended in 15 ml of
70% H2SO4 and the resulting mixture was stirred at room
temperature for 1 day. The reaction mixture was poured
into ice water and stirred for 1 h. The precipitate was fil-
tered, washed with water, and dried in vacuo to afford the
title compound 6 as a brown solid (710 mg, 74%).
Melting points were determined on a Stuart SMP3 melting
point apparatus and are uncorrected. 1H NMR spectra were
recorded at 400 MHz on a Varian 400 Mercury plus
spectrometer; chemical shifts are reported in d (ppm) units
relative to the internal reference tetramethylsilane (TMS).
Mass spectra were performed on an Applied Biosystems
API 2000 mass spectrometer and Agilent 1200 series LC
system. All compounds were routinely checked by TLC
1
M.p.: 235ꢁC; H NMR (400 MHz, DMSO-d6): d 12.15
(s, 1H, –C(O)NH–), 8.29 (s, 1H, Ar-H), 8.02 (d,
J = 8.4 Hz, 1H, Ar-H), 7.36 (d, J = 8.4 Hz, 1H, Ar-H),
4.32 (qt, J = 7.2 Hz, 2H, –OCH2CH3), 3.59 (s, 2H, thio-
pyran), 3.15 (d, J = 4.8 Hz, 2H, thiopyran), 2.93 (t,
J = 5.6 Hz, 2H, thiopyran), 1.32 (t, J = 7.2 Hz, 3H,
–OCH2CH3); MS (ESI?) m/z: [M?H]? 290.1.
1
and H NMR. TLC was performed on glass-backed silica
gel plates (Merck, DC Kieselgel 60 F254). Flash chroma-
tography was carried out on E. Merck Kieselgel 60 silica
gel (230–400 mesh). All chemicals were obtained com-
mercially. All solvents were reagent-grade and were used
without further purification, unless otherwise stated.
5-Oxo-2,4,5,6-tetrahydro-1H-thiopyrano[3,4-c]quinoline-
9-carboxylic acid (7)
Ethyl 4-(4-oxotetrahydro-2H-thiopyran-
3-carboxamido)benzoate (5)
To a stirred solution of thiopyrano[3,4-c]quinoline-9-car-
boxylate 6 (680 mg, 2.35 mmol) in MeOH (12 ml) was
added aqueous 1 N NaOH solution (11.75 ml). The mix-
ture was then heated to 50ꢁC and stirred for 5 h. After
completion of the reaction, the cooled mixture was poured
on water and extracted with ethyl acetate. The aqueous
layer was then acidified to pH 2 with 2 N HCl. The pre-
cipitate was filtered and washed with water, yielding the
title compound 7 as a light yellow solid (420 mg, 68%).
1H NMR (400 MHz, DMSO-d6): d 12.94 (br, 1H,
–C(O)OH), 12.14 (s, 1H, –C(O)NH–), 9.30 (s, 1H, Ar-H),
To a stirred solution of 4-oxothiane 1 (2.0 g, 17.21 mmol)
in toluene (20 ml) were added morpholine 2 (2.25 ml,
25.82 mmol) and catalytic p-TsOHꢀH2O (160 mg,
0.86 mmol), and the resulting mixture was refluxed for
1 day with a Dean-Stark trap. After completion, the mix-
ture was cooled to room temperature and evaporated under
reduced pressure. The residue was concentrated in vacuo to
afford the unstable enamine 3, which was used without
further purification.
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Med Chem Res (2012) 21:1533–1543
N-(4-Aminobutyl)-5-oxo-2,4,5,6-tetrahydro-
1H-thiopyrano[3,4-c]quinoline-9-carboxamide (9c)
8.02 (d, J = 8.0 Hz, 1H, Ar-H), 7.37 (d, J = 8.0 Hz, 1H,
Ar-H), 3.61 (s, 2H, thiopyran), 3.18 (t, J = 5.6 Hz, 2H,
thiopyran), 2.95 (t, J = 6.0 Hz, 2H, thiopyran); MS
(ESI?) m/z: [M?H]? 262.1.
Light yellow solid; yield: 13%; m.p.: 104ꢁC; 1H NMR
(400 MHz, DMSO-d6):
d 12.05(s, 1H, –C(O)NH–),
8.72(t, J = 6.4 Hz, 1H, Ar-C(O)NH–), 8.27(s, 1H, Ar-H),
7.97(d, J = 7.6 Hz, 1H, Ar-H), 7.90(br, 2H, –NH2),
7.34(d, J = 8.0 Hz, 1H, Ar-H), 3.51(s, 2H, thiopyran),
3.30(d, J = 5.2 Hz, 2H, –(CH2)4–N), 3.20(d, J = 5.2 Hz,
2H, thiopyran), 2.97(t, J = 6.0 Hz, 2H, thiopyran), 2.80(d,
J = 5.2 Hz, 2H, –(CH2)4–N), 1.59(m, 4H, –(CH2)4–N);
MS (ESI?) m/z: [M?H]? 332.2.
General procedure for the synthesis of 5-oxo-2,4,5,
6-tetrahydro-1H-thiopyrano[3,4-c]quinoline-
9-carboxamide hydrochloride derivatives (9a–z)
To a stirred solution of thiopyrano[3,4-c]quinoline-9-car-
boxylic acid 7 (40 mg, 0.15 mmol) in DMF (3 ml) were
added N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride (EDCI, 32 mg, 0.17 mmol), hydroxybenzo-
triazole (HOBt, 23 mg, 0.17 mmol), and an appropriate
amine compound (0.17 mmol). The resulting mixture was
stirred at room temperature for 8–12 h and poured into
saturated aqueous NaHCO3 solution. The mixture was
extracted with ethyl acetate, washed with brine, dried over
Na2SO4, and concentrated to dryness. The residue was then
purified by flash column chromatography to afford thio-
pyrano[3,4-c]quinoline-9-carboxamide as a free base.
3.7 M HCl in 1,4-dioxane (30 equiv.) was added to a
stirred solution of thiopyrano[3,4-c]quinoline-9-carbox-
amide in 1,4-dioxane (3–5 ml) at 0ꢁC. After stirring for
1–10 h, the reaction mixture was concentrated in vacuo to
give the title compound 9.
5-Oxo-N-[2-(piperidin-1-yl)ethyl]-2,4,5,6-tetrahydro-
1H-thiopyrano[3,4-c]quinoline-9-carboxamide (9d)
1
White solid; yield: 14%; m.p.: 268ꢁC (decomp.); H NMR
(400 MHz, DMSO-d6): d 12.05 (s, 1H, –C(O)NH–), 10.20
(br, 1H, HCl salt), 9.09 (t, J = 5.4 Hz, 1H, Ar-C(O)NH–),
8.34 (s, 1H, Ar-H), 8.00 (d, J = 8.8 Hz, 1H, Ar-H), 7.33
(d, J = 8.0 Hz, 1H, Ar-H), 3.68 (qt, J = 5.6 Hz, 2H,
–NH–CH2–), 3.58 (s, 2H, thiopyran), 3.54–3.50 (m, 2H,
–CH2–piperidine), 3.21–3.20 (m, 4H, CH2, thiopyran and
CH2, piperidine), 2.94 (t, J = 6.0 Hz, 2H, thiopyran),
2.91–2.84 (m, 2H, piperidine), 1.77 (m, 4H, piperidine),
1.69–1.66 (m, 1H, piperidine), 1.39–1.34 (m, 1H, piperi-
dine); MS (ESI?) m/z: [M?H]? 372.2.
The physical and analytical data of the synthesized title
compounds are given, as follows:
N-(2-Morpholinoethyl)-5-oxo-2,4,5,6-tetrahydro-
1H-thiopyrano[3,4-c]quinoline-9-carboxamide (9e)
N-(2-Aminoethyl)-5-oxo-2,4,5,6-tetrahydro-
1H-thiopyrano[3,4-c]quinoline-9-carboxamide (9a)
Yellow solid; yield: 31%; m.p.: 253ꢁC (decomp.); 1H NMR
(400 MHz, DMSO-d6): d 12.07 (s, 1H, –C(O)NH–), 10.74
(br, 1H, HCl salt), 9.05 (br, 1H, Ar-C(O)NH–), 8.35 (s, 1H,
Ar-H), 8.01 (d, J = 8.8 Hz, 1H, Ar-H), 7.35 (d,
J = 8.0 Hz, 1H, Ar-H), 3.99–3.96 (m, 2H, –NH–CH2–),
3.82–3.76 (m, 2H, –CH2–morpholine), 3.71–3.70 (m, 2H,
morpholine), 3.60 (s, 2H, thiopyran), 3.45 (m, 2H, mor-
pholine), 3.32–3.31 (m, 2H, morpholine), 3.22 (m, 2H,
thiopyran), 3.13–3.10 (m, 2H, morpholine), 2.96 (t,
J = 5.2 Hz, 2H, thiopyran); MS (ESI?) m/z: [M?H]?
374.2.
Yellow solid; yield: 26%; m.p.: 289ꢁC (decomp.); 1H NMR
(400 MHz, DMSO-d6): d 12.07 (s, 1H, –C(O)NH–), 8.97
(br, 1H, Ar-C(O)NH–), 8.32 (s, 1H, Ar-H), 8.01–7.99 (m,
4H, Ar-H, -NH2ꢀHCl), 7.34 (d, J = 8.4 Hz, 1H, Ar-H),
3.61 (s, 2H, thiopyran), 3.56–3.51 (m, 2H, –(CH2)2N), 3.21
(m, 2H, thiopyran), 3.00–2.95 (m, 4H, thiopyran,
–(CH2)2N); MS (ESI?) m/z: [M?H]? 304.1.
N-(3-Aminopropyl)-5-oxo-2,4,5,6-tetrahydro-
1H-thiopyrano[3,4-c]quinoline-9-carboxamide (9b)
5-Oxo-N-[2-(piperazin-1-yl)ethyl]-2,4,5,6-tetrahydro-
1H-thiopyrano[3,4-c]quinoline-9-carboxamide (9f)
Light yellow solid; yield: 40%; m.p.: 286ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 12.07 (s, 1H, –C(O)NH–), 8.83
(br, 1H, Ar-C(O)NH–), 8.28 (s, 1H, Ar-H), 7.98 (d,
J = 8.4 Hz, 1H, Ar-H), 7.87 (br, 2H, –NH2), 7.34 (d,
J = 8.4 Hz, 1H, Ar-H), 3.61 (s, 2H, thiopyran), 3.56 (s,
2H, thiopyran), 3.21 (t, J = 5.6 Hz, 2H, –(CH2)3–N), 2.97
(t, J = 6.0 Hz, 2H, thiopyran), 2.85 (q, J = 6.0 Hz, 2H,
–(CH2)3–N), 1.82 (t, J = 6.8 Hz, 2H, –(CH2)3–N); MS
(ESI?) m/z: [M?H]? 318.1.
Yellow solid; yield: 23%; m.p.: 240ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 12.08 (s, 1H, –C(O)NH–), 11.47
(br, 1H, HCl salt), 9.50 (br, 2H, piperazine NHꢀHCl salt),
8.99 (br, 1H, Ar-C(O)NH–), 8.35 (s, 1H, Ar-H), 8.02 (d,
J = 8.0 Hz, 1H, Ar-H), 7.34 (d, J = 8.8 Hz, 1H, Ar-H),
3.81–3.38 (m, 14H, CH2, thiopyran and 4CH2, piperazine
and 2CH2, –(CH2)2–piperazine), 3.22 (m, 2H, thiopyran),
123

Med Chem Res (2012) 21:1533–1543
1539
2.96 (t, J = 5.6 Hz, 2H, thiopyran); MS (ESI?) m/z:
[M?H]? 373.2.
1H, Ar-H), 7.37 (d, J = 8.4 Hz, 1H, Ar-H), 3.70–3.60 (m,
6H, CH2, thiopyran and 2CH2, piperazine), 3.16–3.15 (m,
6H, CH2, thiopyran and 2CH2, piperazine), 2.96–2.93 (m,
2H, thiopyran); MS (ESI?) m/z: [M?H]? 330.2.
5-Oxo-N-{2-[4-(pyridin-4-yl)piperazin-1-yl]ethyl}-2,4,5,
6-tetrahydro-1H-thiopyrano[3,4-c]quinoline-
9-carboxamide (9g)
9-(4-Methylpiperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9k)
Yellow solid; yield: 20%; m.p.: 252ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 12.08 (s, 1H, –C(O)NH–), 11.36
(br, 1H, Ar-C(O)NH–), 9.11 (br, 1H, HCl salt), 8.38–8.36
(m, 3H, CH, Ar-H, 2CH, pyridine), 8.04 (d, J = 8.8 Hz,
1H, Ar-H), 7.37–7.30 (m, 3H, CH, Ar-H and 2CH, pyri-
dine), 4.52–4.44 (m, 2H, -NH–CH2-), 3.84–3.61 (m, 8H,
CH2, thiopyran and 2CH2, piperazine and CH2, –CH2–
piperazine), 3.37–3.35 (m, 2H, thiopyran), 3.27–3.18 (m,
4H, piperazine), 2.96 (t, J = 5.2 Hz, 2H, thiopyran); MS
(ESI?) m/z: [M?H]? 450.2.
Yellow solid; yield: 36%; m.p.: 327ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 12.05 (s, 1H, –C(O)NH–), 10.75
(br, 1H, HCl salt), 7.80 (s, 1H, Ar-H), 7.56 (d, J = 8.4 Hz,
1H, Ar-H), 7.38 (d, J = 8.4 Hz, 1H, Ar-H), 3.61 (s, 2H,
thiopyran), 3.40 (m, 4H, piperazine) 3.16–3.06 (m, 6H,
CH2, thiopyran, and 2CH2, piperazine), 2.96–2.93 (m, 2H,
thiopyran), 2.78 (s, 3H, –N–CH3); MS (ESI?) m/z:
[M?H]? 344.2.
9-(4-Ethylpiperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9l)
5-Oxo-N-[2-(4-phenylpiperazin-1-yl)ethyl]-2,4,5,
6-tetrahydro-1H-thiopyrano[3,4-c]quinoline-
9-carboxamide (9h)
Brown solid; yield: 55%; m.p.: 280ꢁC; 1H NMR (400 MHz,
DMSO-d6): d 12.05 (s, 1H, –C(O)NH–), 10.61 (br, 1H, HCl
salt), 7.81 (s, 1H, Ar-H), 7.56 (d, J = 8.4 Hz, 1H, Ar-H),
7.38 (d, J = 8.4 Hz, 1H, Ar-H), 3.61 (s, 2H, thiopyran),
3.57–3.22 (m, 4H, piperazine), 3.15–3.11 (m, 6H, CH2,
thiopyran and 2CH2, piperazine), 3.05–2.93 (m, 4H, CH2,
thiopyran and CH2, –N–CH2CH3), 1.24 (t, J = 6.7 Hz, 3H,
–N–CH2CH3); MS (ESI?) m/z: [M?H]? 358.2.
Yellow solid; yield: 18%; m.p.: 261ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 12.06 (s, 1H, –C(O)NH–), 11.04
(s, 1H, HCl salt), 9.16 (t, J = 4.4 Hz, 1H, Ar-C(O)NH–),
8.38 (s, 1H, Ar-H), 8.02 (d, J = 8.4 Hz, 1H, Ar-H), 7.34
(dd, J = 8.4 Hz, J = 2.0 Hz, 1H, Ar-H), 7.23 (dd,
J = 14.0 Hz, J = 6.8 Hz, 2H, Ar-H), 6.99 (d, J = 8.0 Hz,
2H, Ar-H), 6.84 (t, J = 8.0 Hz, 1H, Ar-H), 3.82–3.49 (m,
6H, CH2, thiopyran and 2CH2, piperazine), 3.37 (d,
J = 4.0 Hz, 2H, –NH–CH2–), 3.22–3.17 (m, 8H, CH2,
thiopyran and 2CH2, piperazine and CH2, –CH2–pipera-
zine), 2.93 (t, J = 4.8 Hz, 2H, thiopyran); MS (ESI?) m/z:
[M?H]? 449.2.
9-(4-Propylpiperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9m)
Yellow solid; yield: 56%; m.p.: 298ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 12.06 (s, 1H, –C(O)NH–), 11.03
(br, 1H, HCl salt), 7.81 (s, 1H, Ar-H), 7.57 (d, J = 8.3 Hz,
1H, Ar-H), 7.38 (d, J = 8.3 Hz, 1H, Ar-H), 3.61 (s, 2H,
thiopyran), 3.60–2.93 (m, 14H, 2CH2, thiopyran and 4CH2,
piperazine and CH2, –N–(CH2)2CH3), 1.70 (m, 2H, –N–
(CH2)2CH3), 0.91 (t, J = 7.3 Hz, 3H, –N–(CH2)2CH3); MS
(ESI?) m/z: [M?H]? 372.2.
9-(Piperidine-1-carbonyl)-4,6-dihydro-1H-thiopyrano
[3,4-c]quinolin-5(2H)-one (9i)
Yellow solid; yield: 57%; m.p.: 241ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 11.88 (s, 1H, –C(O)NH–), 7.67
(s, 1H. Ar-H), 7.37 (d, J = 8.0 Hz, 1H, Ar-H), 7.30 (d,
J = 8.4 Hz, 1H, Ar-H), 3.69(s, 2H, thiopyran), 3.67–3.61
(m, 2H, piperidine), 3.29–3.28 (m, 2H, piperidine), 3.10 (t,
J = 5.8 Hz, 2H, thiopyran), 2.87 (t, J = 5.8 Hz, 2H, thi-
opyran), 1.59 (m, 4H, piperidine), 1.45 (m, 2H, piperidine);
MS (ESI?) m/z: [M?H]? 329.2.
9-(4-Butylpiperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9n)
Yellow solid; yield: 51%; m.p.: 240ꢁC (decomp.); 1H NMR
(400 MHz, DMSO-d6): d 12.05 (s, 1H, –C(O)NH–), 11.13
(br, 1H, HCl salt), 7.81 (s, 1H, Ar-H), 7.57 (d, J = 8.3 Hz,
1H, Ar-H), 7.37 (d, J = 8.3 Hz, 1H, Ar-H), 3.61 (s, 2H,
thiopyran), 3.60–2.93 (m, 14H, 2CH2, thiopyran and 4CH2,
piperazine and CH2, –N–(CH2)3CH3), 1.68 (m, 2H, –N–
(CH2)3CH3), 1.32 (m, 2H, –N–(CH2)3CH3), 0.90 (t,
J = 7.3 Hz, 3H, –N–(CH2)3CH3); MS (ESI?) m/z:
[M?H]? 386.2.
9-(Piperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9j)
Yellow solid; yield: 63%; m.p.: 230ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 12.04 (s, 1H, –C(O)NH–), 9.24
(br, 1H, HCl salt), 7.82 (s, 1H, Ar-H), 7.56 (d, J = 8.0 Hz,
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Med Chem Res (2012) 21:1533–1543
9-(4-(4,4,4-Trifluorobutyl)piperazine-1-carbonyl)-4,
6-dihydro-1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9o)
9-(4-Hexylpiperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9s)
Yellow solid; yield: 72%; m.p.: 285ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 12.05 (s, 1H, –C(O)NH–), 11.38
(br, 1H, HCl salt), 7.81 (s, 1H, Ar-H), 7.58 (d, J = 8.3 Hz,
1H, Ar-H), 7.38 (d, J = 8.3 Hz, 1H, Ar-H), 3.61 (s, 2H,
thiopyran), 3.54–2.93 (m, 14H, 2CH2, thiopyran and 4CH2,
piperazine and CH2, –N–(CH2)3CF3), 2.41 (m, 2H, –N–
(CH2)3CF3), 1.97 (m, 2H, –N–(CH2)3CF3); MS (ESI?) m/z:
[M?H]? 440.2.
Yellow solid; yield: 47%; m.p.: 170ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 12.05 (s, 1H, –C(O)NH–), 10.84
(br, 1H, HCl salt), 7.80 (s, 1H, Ar-H), 7.56 (d, J = 8.4 Hz,
1H, Ar-H), 7.38 (d, J = 8.4 Hz, 1H, Ar-H), 3.61 (s, 2H,
thiopyran), 3.56–3.22 (m, 4H, piperazine), 3.15–3.06 (m,
2H, thiopyran), 3.04–2.96 (m, 4H, piperazine), 2.95–2.93
(m, 2H, thiopyran), 1.68–1.60 (m, 2H, –N–(CH2)5–CH3),
1.28–1.21 (m, 8H, –N–(CH2)5–CH3), 0.87–0.85 (m, 3H,
–N–(CH2)5–CH3); MS (ESI?) m/z: [M?H]? 414.2.
9-(4-Pentylpiperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9p)
9-(4-Phenylpiperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9t)
Yellow solid; yield: 59%; m.p.: 305ꢁC (decomp.); 1H NMR
(400 MHz, DMSO-d6): d 12.01 (s, 1H, –C(O)NH–), 11.10
(br, 1H, HCl salt), 7.79 (s, 1H, Ar-H), 7.57 (d, J = 8.4 Hz,
1H, Ar-H), 7.36 (d, J = 8.4 Hz, 1H, Ar-H), 3.60 (s, 2H,
thiopyran), 3.59–2.91 (m, 14H, 2CH2, thiopyran and 4CH2,
Dark brown solid; yield: 92%; m.p.: 258ꢁC; 1H NMR
(400 MHz, DMSO-d6); d 12.04 (s, 1H, –C(O)NH–), 7.84 (s,
1H, Ar-H), 7.61(d, J = 8.0 Hz, 1H, Ar-H), 7.47–7.37(m, 5H,
piperazine-Ar-H), 7.17 (m, 1H, Ar-H), 3.95–3.82 (m, 4H,
piperazine), 3.60 (s, 2H, thiopyran), 3.43–3.42 (m, 4H,
piperazine), 3.16 (t, J = 5.2 Hz, 2H, thiopyran), 2.94 (t,
J = 5.6 Hz, 2H, thiopyran); MS(ESI?) m/z:[M?H]? 406.2.
piperazine and CH2, –N–(CH2) CH3), 1.69 (m, 2H, –N–
4
(CH2) 4CH3), 1.28 (m, 4H, 2CH2, –N–(CH2) 4CH3), 0.87 (t,
J = 7.0 Hz, 3H, –N–(CH2) 4CH3); MS (ESI?) m/z:
[M?H]? 400.2.
9-(4-Benzylpiperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9u)
9-(4-Isopentylpiperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9q)
Yellow solid; yield: 55%; m.p.: 222ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 12.05 (s, 1H, –C(O)NH–), 10.82
(br, 1H, HCl salt), 7.79 (s, 1H, Ar-H), 7.57–7.47 (m, 6H,
CH, Ar-H and 5CH, benzl), 7.37 (d, J = 8.4 Hz, 1H,
Ar-H), 3.61 (s, 2H, CH2, benzyl), 3.56–3.31 (m, 8H, CH2,
thiopyran and 3CH2, piperazine), 3.16–3.13 (m, 4H, CH2,
thiopyran and CH2, piperazine), 2.95 (t, J = 5.8 Hz, 2H,
thiopyran); MS (ESI?) m/z: [M?H]? 420.2.
Yellow solid; yield: 43%; m.p.: 266ꢁC (decomp.); 1H NMR
(400 MHz, DMSO-d6): d 12.05 (s, 1H, –C(O)NH–), 11.56
(br, 1H, HCl salt), 7.80 (s, 1H, Ar-H), 7.57 (d, J = 8.3 Hz,
1H, Ar-H), 7.39 (d, J = 8.3 Hz, 1H, Ar-H), 3.60 (s, 2H,
thiopyran), 3.60–3.40 (m, 6H, CH2, thiopyran and 2CH2,
piperazine), 3.15–2.92 (m, 8H, CH2, thiopyran, 2CH2,
piperazine, CH2, –N–(CH2)2CH(CH3)2), 1.61(m, 3H, –N–
(CH2)2CH(CH3)2), 0.88 (d, J = 5.9 Hz, 6H, –N–
(CH2)2CH(CH3)2); MS (ESI?) m/z: [M?H]? 400.2.
9-(4-Phenethylpiperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9v)
Yellow solid; yield: 31%; m.p.: 179ꢁC; 1H NMR (400 MHz,
DMSO-d6): d 12.07 (s, 1H, –C(O)NH–), 11.62(br, 1H, HCl
salt), 7.82 (s, 1H, Ar-H), 7.58 (d, J = 8.4 Hz, 1H, Ar-H),
7.40–7.21 (m, 6H, CH, Ar-H and 5CH, piperazine-Ar-H),
3.68–3.42 (m, 8H, CH2, thiopyran and 2CH2, piperazine and
CH2, –N–CH2CH2), 3.35–3.28 (m, 2H, –N–CH2CH2), 3.20–
3.07 (m, 6H, CH2, thiopyran and 2CH2, piperazine), 2.95 (t,
J = 5.6 Hz, 2H, thiopyran); MS(ESI?) m/z:[M?H]? 434.2.
9-[4-(Pentan-2-yl)piperazine-1-carbonyl]-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9r)
Brown solid; yield: 66%; m.p.: 272ꢁC; 1H NMR (400 MHz,
DMSO-d6): d 12.04 (s, 1H, –C(O)NH–), 10.29 (br, 1H, HCl
salt), 7.83 (s, 1H, Ar-H), 7.60 (d, J = 8.4 Hz, 1H, Ar-H),
7.38 (d, J = 8.4 Hz, 1H, Ar-H), 3.61 (s, 2H, thiopyran),
3.39–3.34 (m, 6H, piperazine), 3.21–3.13 (m, 4H, CH2, thi-
opyran and CH2, piperazine), 2.97–2.95 (m, 2H, thiopyran),
1.84–1.17 (m, 1H, –N–CHCH3(CH2)2CH3), 1.47–1.31
(m, 2H, –N–CHCH3(CH2)2CH3), 1.28–1.12 (m, 5H, –N–
CHCH3(CH2)2CH3), 0.94–0.90 (m, 3H, –N–CHCH3(CH2)2
CH3); MS (ESI?) m/z: [M?H]? 400.2.
9-[4-(Pyridin-4-yl)piperazine-1-carbonyl]-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9w)
White solid; yield: 56%; m.p.: 302ꢁC; 1H NMR (400 MHz,
DMSO-d6): d 13.71 (br, 1H, HCl salt), 12.06 (s, 1H,
123

Med Chem Res (2012) 21:1533–1543
1541
–C(O)NH–), 8.29 (d, J = 6.4 Hz, 2H, pyridine), 7.84 (s,
1H, Ar-H), 7.60 (d, J = 8.4 Hz, 1H, Ar-H), 7.38 (d,
J = 8.4 Hz, 1H, Ar-H), 7.18 (m, 2H, pyridine), 3.77 (m,
8H, piperazine), 3.61 (s, 2H, thiopyran), 3.16–3.14 (m, 2H,
thiopyran), 2.94 (t, J = 5.2 Hz, 2H, thiopyran); MS
(ESI?) m/z: [M?H]? 407.1.
docking of PARP-1 inhibitors into the active site of human
PARP-1 was performed. The crystal structure of human
PARP-1 (pdb code: 1UK0) obtained from the Protein Data
Bank was refined to remove water molecules and to add
hydrogen atoms to the whole enzyme at a pH of 7.0.
The ligands were optimized using a forcefield function,
CHARMm. Ten poses were docked for each ligand.
Among the docked conformations, the over-fitted confor-
mation was eliminated.
9-(4-Cyclopentylpiperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9x)
1
White solid; yield: 26%; m.p.: 245ꢁC (decomp.); H NMR
PARP-1 enzyme inhibitory activity
(400 MHz, DMSO-d6): d 12.06 (s, 1H, –C(O)NH–), 11.68
(s, 1H, HCl salt), 7.82 (s, 1H, Ar-H), 7.58 (d, J = 8.8 Hz,
1H, Ar-H), 7.37 (d, J = 8.0 Hz, 1H, Ar-H), 3.60 (s, 2H,
thiopyran), 3.45 (m, 5H, 2CH2, piperazine and CH,
cyclopentane), 3.12–3.05 (m, J = 8.0 Hz, 6H, CH2, thio-
pyran and 2CH2, piperazine), 2.94 (t, J = 5.6 Hz, 2H,
thiopyran), 1.98 (t, J = 2.2 Hz, 2H, cyclopentane), 1.83
(br, 2H, cyclopentane), 1.71 (br, 2H, cyclopentane), 1.53 (t,
J = 5.6 Hz, 2H, cyclopentane); MS (ESI?) m/z: [M?H]?
398.2.
The IC50 of PARP inhibitor compounds was determined
using Trevigen PARP inhibition kit (Gaithersburg, USA).
The assay was performed in 384-well, small volume mi-
croplates following a modified previously reported method
(Lee et al., 2005), as follows.
The PARP-1 enzyme assay was set up in a volume of
12 ll. Wells were coated with diluted histones and incu-
bated at 25ꢁC for 2 h. After the plates were washed four
times with PBS, all the liquid was removed following each
wash by tapping the plate onto paper towels. To block the
nonspecific signal, the wells were blocked by adding the
Strep-diluent and incubated at 25ꢁC for 1 h. The plates
were washed four times with PBS, and serial dilutions of
compound were added. The diluted PARP-1 enzyme was
then added to each well (0.12 unit/well), except for the
negative control wells, and combined with a 29 PARP
cocktail. The reaction was allowed to proceed for 30 min at
25ꢁC. After washing four times with PBS, streptavidin-
linked peroxidase was added to detect the extent of ribo-
sylation, and the plate was incubated at 37ꢁC for 30 min.
The plates were then washed four times with PBS, then the
TACS-Sapphire substrate was added and the color allowed
to develop for 10 min. Finally, the reaction was stopped by
adding 0.2 N HCl, and the optical densities were read at
450 nm using a Wallac EnVisionTM plate reader (Perkin-
Elmer Oy, Turku, Finland). All the data points were
determined in triplicate and the data were analyzed using
SigmaPlot 10 (Systat Software Inc., USA).
9-(4-Cyclohexylpiperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9y)
Yellow solid; yield: 31%; m.p.: 243ꢁC; 1H NMR
(400 MHz, DMSO-d6): d 12.01 (s, 1H, –C(O)NH–), 10.71
(s, 1H, HCl salt), 7.81 (s, 1H, Ar-H), 7.57 (dd, J = 2.0 Hz,
J = 7.4 Hz, 1H, Ar-H), 7.35 (d, J = 8.80 Hz, 1H, Ar-H),
3.60 (s, 2H, thiopyran), 3.42 (br, 5H, 2CH2, piperazine and
CH, cyclohexane), 3.12 (m, 6H, CH2, thiopyran and 2CH2,
piperazine), 2.93 (t, J = 5.8 Hz, 2H, thiopyran), 2.08–1.09
(m, 10H, cyclohexane); MS (ESI?) m/z: [M?H]? 412.2.
9-(4-Cycloheptylpiperazine-1-carbonyl)-4,6-dihydro-
1H-thiopyrano[3,4-c]quinolin-5(2H)-one (9z)
Pale yellow solid; yield: 31%; m.p.: 221ꢁC; 1H
NMR(400 MHz, DMSO-d6): d 12.00 (s, 1H, –C(O)NH–),
11.00 (s, 1H, HCl salt), 7.81 (s, 1H, Ar-H), 7.58 (d,
J = 8.4 Hz, 1H, Ar-H), 7.35 (d, J = 8.4 Hz, 1H, Ar-H),
3.61 (s, 2H, thiopyran), 3.35 (br, 5H, 2CH2, piperazine and
CH, cycloheptane), 3.13 (m, 6H, CH2, thiopyran and 2CH2,
piperazine), 2.93 (t, J = 5.6 Hz, 2H, thiopyran), 2.11 (m,
2H, cycloheptane), 1.69–1.50 (m, 4H, cycloheptane), 1.49–
1.40 (m, 6H, cycloheptane); MS (ESI?) m/z: [M?H]?
426.2.
Intracellular PARP inhibitory activity
This protocol describes a real-time assay to assess an
imbalance of DNA single-strand break repair by indirectly
measuring PARP activation through the depletion of
intracellular NAD(P)H (Nakamura et al., 2003). Chinese
hamster ovary cells (CHO-K1) were cultured in RPMI
media supplemented with 10% fetal bovine serum (FBS).
The cultured CHO-K1 cells were seeded at a density of
2.9 9 103 cells/well in 96-well plate and cultured at 37ꢁC
and 5% CO2 for 16 h. The cells were then treated with
various concentrations of the synthesized compounds and
Docking study
All the molecular docking studies were performed using
the docking software LigandFit module of Discovery Stu-
dio 2.5 (Accelrys, San Diego, CA). In this study, the
123

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Med Chem Res (2012) 21:1533–1543
incubated at 37ꢁC for 2 h. DNA damage was induced using
1.5 mM MMS (methyl methanesulfonate), and the cells
were simultaneously treated with a CCK-8 (Cell Count
Kit-8) solution (DOJINDO, CK01-13) for colorimetric
assay. At 4 h after the treatment with MMS, the amount of
NAD(P)H secreted into the culture media was quantified
using a Wallac EnVisionTM plate reader at 450 nm. The
results obtained according to various concentrations of the
compounds are the average values obtained from four
wells, and the results were calculated by regression
analysis.
(Column: Waters Xterra MS C18 (2.1 9 50 mm, 3.5 lm);
Mobile phase: acetonitrile/DI water/0.1% formic acid; Ion
source: turbo ion spray). Analytical data were processed
using AnalystTM 1.4.2. (Applied Biosystems, Concord,
Canada). Pharmacokinetic parameters were obtained by
non-compartmental analysis of the plasma concentration–
time profiles using PK Solution 2.0 (Summit Research
Services, Montrose, CO, USA).
Acknowledgments This study was supported by a grant from the
Korea Healthcare Technology R&D Project, Ministry for Health,
Welfare & Family Affairs, Republic of Korea (A100453-1001-
0000100). We thank Professor Gyoonhee Han (Department of Bio-
technology, Yonsei University, Republic of Korea) for his assistance
with the docking study.
Human liver microsomal stability
All incubations were conducted in triplicate at 37ꢁC in
micro-tubes. Incubation mixtures consisted of human liver
microsomes (1 mg/ml), NADPH (1 mM) and PARP-1
inhibitor (9m, 9q, or 9s; 1 lM) in 0.1 M potassium phos-
phate buffer (pH 7.4). The micro-tubes containing PARP-1
inhibitor (9m, 9q, or 9s) and b-NADPH were pre-incubated
for 5 min at 37ꢁC. The reaction was terminated at 0, 15, 45,
and 80 min by the addition of ice-cold acetonitrile con-
taining carbamazepine as the internal standard. The sample
was centrifuged at 3,000 rpm for 10 min and the super-
natant was analyzed using an API2000 LC/MS/MS system
(Applied Biosystems, Concord, Canada) in positive MRM
mode. LC/MS/MS data were analyzed by Analyst 1.4.2
(Applied Biosystems, Concord, Canada).
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