Pyrazolo[3,4-h]quinolines promising photosensitizing agents in the treatment of cancer

Article abstract of DOI:10.1016/j.ejmech.2015.08.003

A new series of pyrazolo[3,4-h]quinolines, heteroanalogues of angelicin was conveniently prepared with a broad substitution pattern. A large number of derivatives was obtained and the cellular photocytotoxicity was evaluated in vitro against 5 different h

Full text of DOI:10.1016/j.ejmech.2015.08.003

European Journal of Medicinal Chemistry 102 (2015) 334e351
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Pyrazolo[3,4-h]quinolines promising photosensitizing agents in the
treatment of cancer
a
a
a
a
ꢀ
Virginia Spano , Barbara Parrino , Anna Carbone , Alessandra Montalbano ,
Alessia Salvador b, Paola Brun c, Daniela Vedaldi b, Patrizia Diana a, Girolamo Cirrincione a,
,
*
Paola Barraja a
a
ꢀ
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF), Universita degli Studi di Palermo, Via Archirafi 32, 90123 Palermo,
Italy
b
c
ꢀ
Dipartimento di Scienze del Farmaco, Universita degli Studi di Padova, Via Marzolo 5, 35131 Padova, Italy
ꢀ
Dipartimento di Medicina Molecolare, Universita degli Studi di Padova, Via Gabelli 63, 35121 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of pyrazolo[3,4-h]quinolines, heteroanalogues of angelicin was conveniently prepared with
a broad substitution pattern. A large number of derivatives was obtained and the cellular photo-
cytotoxicity was evaluated in vitro against 5 different human tumor cell lines with GI50 values reaching
Received 6 May 2015
Received in revised form
7 July 2015
Accepted 2 August 2015
Available online 5 August 2015
the nanomolar level (14.52e0.04
mM). Selected compounds were able to photoinduce a massive cell
death with the involvement of mitochondria. Their photodamage cellular targets were proteins and
lipids and they did not cause any kind of DNA photodamage. This latter event is of considerable
importance in the modulation of long term side effects, generally associated with the use of classical
furocoumarins.
Keywords:
Pyrazolo[3,4-h]quinolines
Photodynamic therapy
Antiproliferative activity
Photosensitizing agents
Reactive oxygen species
PUVA therapy
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
in PDT. Since its initial application, PDT continues to find increasing
use in the treatment of certain types of malignancies including
Cancer drug development remains a high unmet medical need,
thus the discovery of small molecules as cancer drugs is a chal-
lenging aspect of the drug discovery [1e6].
The therapeutic properties of light have been known for thou-
sands of years, but it was only in the last century that photody-
namic therapy (PDT) was developed. PDT requires topycal or
systemic administration of a drug called photosensitizer, followed
by light activation of proper wavelength in accordance with the
absorption spectrum of the photosensitizer [7e9]. Generally, the
controlled and targeted release of ROS by photosensitizer is crucial
esophageal, head and neck, breast, prostate, bladder, skin and lung
tumors [10,11].
The use of PDT in combination with conventional therapies
[12,13] and selective photosensitizer delivery are more recent fields
of investigations [14] and a great deal of research is focused on the
development of more powerful photosensitizers that more specif-
ically target cancer cells having mitochondria and endoplasmatic
reticulum as preferential targets [15e18].
Furocoumarins are natural occurring photosensitizers which
intercalate between the base pairs of DNA “in the dark” and pho-
tobind to it upon UV-A irradiation. Among them, 8-
methoxypsoralen (8-MOP) was approved by FDA for the treat-
ment of cutaneous T-cell lymphoma and in clinical trial for the
treatment of Hodgkins and non-Hodgkins lymphomas. Consider-
able efforts have been done to develop monofunctional furo-
coumarins, which cannot crosslink with DNA, thereby reducing
indesiderable side effects [19].
Abbreviations: AO, acridine orange; BSA, bovine serum albumin; FITC, fluores-
cein isothiocyanate; DCFDA, 20,70-dichlorodihydrofluorescein diacetate; HE,
hydroethidine; JC1, 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazol-carbocya-
nine; MTT test, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
test; PI, propidium iodide; PS, phosphatidylserine; RNAase, ribonuclease; ROS,
reactive oxygen species; TBARS, thiobarbituric reactive substances.
* Corresponding author.
In this contest, the main research line dealt with the synthesis of
E-mail address: paola.barraja@unipa.it (P. Barraja).
http://dx.doi.org/10.1016/j.ejmech.2015.08.003
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

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V. Spano et al. / European Journal of Medicinal Chemistry 102 (2015) 334e351
335
angelicin, an angular furocumarin, which, for geometrical reasons,
2. Chemistry
prevents the formation of interstrand crosslinks (ISC) and its het-
eroanalogues (Chart 1), such as thioangelicins [20] and furo-
quinolinones [21].
Quinolinones are considered attractive scaffold in medicinal
chemistry because of their variety of biological activities, including
antibacterial [22], anticancer [23,24], antiviral [25] and antituber-
culosis properties [26]. However, not much is reported in literature
about their capability of interacting with light and their possible
use as photosensitizing agents.
The synthetic pathway to obtain the tricyclic system pyrazolo
[3,4-h]quinoline 10e13 is outlined in Scheme 1. On the basis of our
previous experience on pyrroloquinolinones, 1,5,6,7-tetrahydro-
4H-indazol-4-ones 8a-h would be ideal precursors for our pur-
poses, considering that they can be easily functionalized in
a po-
sition to the carbonyl to give the access to enaminoketons 9a-h.
Such type of intermediates are recognized as versatile key in-
termediates. In fact, the annular carbonyl group and the exocyclic
In our continuing studies aimed at the discovery [27e40] and
development of new potential anticancer drug candidates, we have
devoted more than ten years in the search of new photoactivating
agents [41,42] with improved photoantiproliferative activity
dissociated from side effects. In this contest, we discovered new
classes of small nitrogen heterocycles (1e5, Chart 1) [43e49].
Among sulfur derivatives, the best results were achieved with the
class of thiopyrano[2,3-e]indol-2-ones 1, which despite their
remarkable phototoxicity a significative photodamage to lipids and
proteins and photooxidation of DNA bases were observed.
Pyrroloquinolinones 3e5 demonstrated very promising features
as photosensitizing agents. In fact, in addition to an higher UV-A
dose dependent photoantiproliferative effect than the reference
drugs 8-MOP, at variance of this latter, they did not affect DNA and
mitochondria were their preferential target in the photoinduced
cell death, with high production of ROS and an extensive oxidative
damage to membrane lipids in accordance with the required
properties of photoactivable drugs. A very attractive result was that
pyrrolo[3,2-h]quinolin-2-ones 4 exert their phototoxicity without
any DNA damage (no DNA strand breaks and no DNA oxidative
damages), which is the main origin of the long term side effects of
the PUVA therapy. These findings could represent a significant
advantage for a possible use in therapy, as the main side effects of
psoralens are genotoxicity and mutagenicity. In this paper, in an
attempt to further explore the effect of the condensation of the
quinoline moiety to the pyrazole ring, extensively represented in
medicinal chemistry in the treatment of cancer [50e53], we re-
ported the synthesis, the study of the mechanism of action and the
biological activity of a new series of derivatives of the pyrazolo[3,4-
h]quinoline ring system. Some of these compounds showed much
higher activity than that of angelicin.
carbon in a position represent the two electrophilic centers, readily
available for further cyclizations with various dinucleophiles
[4,40,41,45e49,53] (Table 1).
Ketones 8a-h, were prepared by convenient modifications of the
known procedures [54e56], which led to an improvement of the
yields (60e93%) of the desired products. Such reactions started
from 2-[(dimethyl-amino)methylene]cyclohexane-1,3-dione 7, in
turn prepared from 1,3-cyclohexanedione 6 [39], and the properly
substituted hydrazines in a 1:1 ratio, heating under reflux using a
mixture of ethanol and acetic acid (2:1) as solvent, or alternatively
only acetic acid when 2-, 3- and 4-chlorophenylhydrazines are used
as nucleophiles.
Compounds 9a-h were obtained in good yield by direct intro-
duction of the enamino functionality from the corresponding ke-
tones 8a-h, with N,N-dimethylformamide dimethyl acetal
(DMFDMA), used as solvent, under conventional heating (70e90%,
Method A) or, alternatively, under microwave irradiation (PW
150 W, T 150 ꢀC, 30e45 min) using DMFDMA in a 1:10 ratio and
N,N-dimethylformamide (DMF) as solvent (95e98%, Method B). At
first, the annelation of the pyridine ring was achieved by inter-
molecular cyclization of the enaminoketones 9 using phenyl-
sulfonylacetonitrile as 1,3-dinucleophile.
This choice was dictated by our previous investigations on
pyrroloquinolinones which demonstrated the importance of the
phenylsulfonyl group in position 3 of the pyridone moiety in
modulating the photoantiproliferative activity. Thus, upon reaction
of 9a-h with phenylsulfonylacetonitrile in refluxing ethanol the
desired tricyclic compounds 10a-h were isolated in good yield
(60e88%). Preliminary phototoxicity studies indicated pyr-
azoloquinolinones 10g,h, as the most promising and thus selected
as lead compounds, for further modifications. Thus, their precursor
O
O
O
H
N
X
S
S
O
N
R
N
R
1
2
Angelicin, X = O
Thioangelicin, X = S
Furoquinolinone, X = NH
Pyrrolo[3',2':4,5]thiopyrano
[3,2-b]pyridin-2-ones
Thiopyrano[2,3-e]indol-2-ones
2
Y
O
1
N
R2
H
3
3, N-7 Pyrrolo[2,3-h]quinolin-2-ones
N
Pyrazolo[3,4-h]quinolines
9
4
9
NR
8
R1
8
4, N-8 Pyrrolo[3,2-h]quinolin-2-ones
5, N-9 Pyrrolo[3,4-h]quinolin-2-ones
Y = C=O or C-R1
N
X
N
X-X = CH2-CH2 or CH = CH
5
X
6
R7
7
Chart 1. Structures of angelicin, thioangelicin, furoquinolinone; thiopyrano[2,3-e]indol-2-ones (1); pyrrolo[30,20:4,5]thiopyrano[3,2-b]pyridin-2-ones (2); pyrrolo[2,3-h]quinolin-2-
ones (3); pyrrolo[3,2-h]quinolin-2-ones (4); pyrrolo[3,4-h]quinolin-2-ones (5); pyrazolo[3,4-h]quinolines.

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V. Spano et al. / European Journal of Medicinal Chemistry 102 (2015) 334e351
336
O
O
O
O
Me2N
N
i
N
ii
iii
N
N
R
N
R
O
O
6
7
8a-h
9a-h
a R = H; b R = Me; c R = Ph; d R = Bn;
e R = 2-Cl-C6H4; f R = 3-Cl-C6H4;
g R = 4-Cl-C6H4; h R = 4-OMe-C6H4
iv
OR2
O
O
2
1
R2
R1
N
R1
R1
R2
N
N
3
9
v
4
+
N
N
N8
X
X
5 X
N
R
N
X
N
X
X
R7
6
R
11b-l (X-X = CH2-CH2)
13c,d (X-X = CH=CH)
12b-l (X-X = CH2-CH2)
13e,f (X-X = CH=CH)
10a-j (X-X = CH2-CH2)
13a,b (X-X = CH=CH)
vi
vi
vi
(Table 1)
(Table 1)
(Table 1)
Scheme 1. Reagents and conditions: (i) DMFDMA, 96%; (ii) H2NNHR, ethanol/acetic acid (2:1) or glacial acetic acid, reflux, 2 h, 60e93%; (iii) DMFDMA, reflux, 24 h, 70e90% (Method
A), or DMFDMA, MW (PW 150 W, T 150 ꢀC), 30e45 min, 95e98% (Method B); (iv) PhSO2CH2CN or NCCH2COOEt, ethanol, reflux, 24 h, 60e88%; (v) NaH, DMF, rt, 3 h then MeI or BnBr
rt, 24 h, 22e75%; (vi) for 10g,h, 11g,h and 12g,h: DDQ, dioxane, reflux, 24 h, 50e95%.
enaminoketones 9g,h were further reacted with ethyl cyanoace-
tate. Reactions, conducted in refluxing ethanol under nitrogen at-
mosphere, led to desired tricyclic compounds 10i,j in good yield
structureeactivity relationship, the pyridine ring was further
functionalized taking advantage of ketoeenol tautomerism of the
amidic functionality, and subjected to alkylation. Reactions were
performed in DMF using NaH as the base and alkyl or arylalkyl
halides such as iodomethane and benzyl bromide, allowing the
isolation of the corresponding N-substituted (11b-l, 28e75%) and
O-substituted (12b-l, 22e64%) derivatives from the same reaction
mixtures. Tricyclic derivatives 10g,h, 11g,h and 12g,h bearing a 4-
chlorophenyl and a 4-methoxyphenyl functionality, selected ac-
cording to preliminary data on phototoxicity, were subjected to
oxidation with DDQ in dioxane under reflux, allowing the isolation
of the corresponding fully aromatic derivatives 13a-f with yields
from moderate to excellent (50e95%).
(72, 80%). Furthermore, for
a
better evaluation of the
Table 1
Pyrazolo[3,4-h]quinolines 10e13.
Cpd
R
R1
R2
XeX
Sbt
Yld(%)
10a
10b
10c
10d
10e
10f
10g
10h
10i
H
Me
Ph
Bn
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
CO2Et
CO2Et
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
CO2Et
CO2Et
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
CO2Et
CO2Et
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
H
H
H
H
H
H
H
H
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH2eCH2
CH]CH
9a
9b
9c
9d
9e
9f
9g
9h
9g
60
80
63
88
65
68
65
73
72
80
58
58
68
58
56
53
59
70
60
30
28
35
30
30
33
35
30
36
22
35
64
55
50
60
93
95
65
86
2-CleC6H4
3-CleC6H4
4-CleC6H4
4-OMeeC6H4
4-CleC6H4
4-OMeeC6H4
Me
3. Results and discussion
H
H
10j
9h
3.1. Spectrophotometric properties
11b
11c
11d
11e
11f
11g
11h
11i
Me
Me
Me
Me
Me
Me
Me
Me
Me
Bn
Bn
Me
Me
Me
Me
Me
Me
Me
Me
Me
Bn
Bn
H
10b
10c
10d
10e
10f
10g
10h
10i
10j
10g
10h
10b
10c
10d
10e
10f
10g
10h
10i
10j
10g
10h
10g
10h
11g
11h
12g
12h
Ph
Bn
The essential requirement for a photosensitizating compound is
the absorption of UVevis light. All compounds absorbed in the
UVevis range with maximum peaks in the UV-A or in the visible
light (338e406 nm) (see Table 1S in the Supporting Materials).
All compounds presented a higher molar extinction coefficient
2-CleC6H4
3-CleC6H4
4-CleC6H4
4-OMeeC6H4
4-CleC6H4
4-OMeeC6H4
4-CleC6H4
4-OMeeC6H4
Me
and
a
batochromic shift in comparison to angelicin
11j
11k
11l
(
lmax ¼ 305 nm). These effects are linked to the presence of high
electron density groups, such as phenylsulphonyl group in position
3 or phenyl group in position 7. Moreover, all compounds were
fluorescent.
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
13a
13b
13c
13d
13e
13f
Ph
Bn
2-CleC6H4
3-CleC6H4
4-CleC6H4
4-OMeeC6H4
4-CleC6H4
4-OMeeC6H4
4-CleC6H4
4-OMeeC6H4
4-CleC6H4
4-OMeeC6H4
4-CleC6H4
4-OMeeC6H4
4-CleC6H4
4-OMeeC6H4
3.2. Antiproliferative assays
The phototoxicity assays were carried out in some human tumor
cell lines by MTT test after 72 h from UV-A irradiation in the
presence of the compounds [44]. Moreover, antiproliferative ex-
periments were performed without irradiation by incubating cells
with compounds for 72 h to assess a possible cytotoxicity [35].
Results of cytotoxic experiments were shown in Table 2S of Sup-
H
CH]CH
CH]CH
CH]CH
CH]CH
Me
Me
Me
Me
porting Material, omitting compounds with GI50 > 20
lines. Some compounds reduced cellular viability even without
irradiation (GI50 16.19e1.46 M) in some cell lines. However, the
mM in all cell
CH]CH
m

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V. Spano et al. / European Journal of Medicinal Chemistry 102 (2015) 334e351
337
cytotoxicity of this class of compounds was very fragmentary and
no compounds was active in more than two cell lines. Data of the
phototoxicity experiments were reported in Table 2; compounds
a more significative decrease in cell viability.
For the 3-ethoxycarbonyl pyrazolo[3,4-h]quinolines, a decrease
in the phototoxicity was observed in comparison with the 3-
with GI50 > 20
m
M in all cell lines were omitted.
phenylsulfonyl series: compare 12g (GI50 1.69e0.43
(GI50 1.52e0.53 M) with the corresponding 12i (GI50
2.30e1.13 M) and 12j (GI50 6.43e4.30 M), inactive on the
adenocarcinoma cell line MCF-7.
mM) and 12h
Among the dihydro derivatives, compounds 12b-l having a 2-
methoxy group were generally more phototoxic (at the highest
UV-A dose: GI50 12.68e0.43
m
m
m
mM) than those bearing the carbonyl
group in the same position,11e-i,k,l (GI50 13.06e2.44
exceptions were 12b, selectively active in leukemic cell lines, and
12k, which was devoid of activity.
Among the 2-methoxy substituted quinoline structures, the best
results were achieved for derivative 12g (GI50 1.69e0.43
m
M). The only
Among the fully aromatic derivatives 13a-f, different photo-
toxicity trends were observed.
In fact, the 2-methoxy pyrazolo[3,4-h]quinoline 13e,f, analogs of
the most active compounds among the dihydro derivative 12g,h,
maintained a remarkable phototoxicity: they reached the nano-
mM) and
12h (GI50 1.52e0.53 M) bearing a 4-chlorophenyl group and a 4-
m
molar level, with range of GI50 values of 3.23e0.71
of 0.76e0.09 M for 13f, respectively.
mM for 13e, and
methoxyphenyl group respectively at the pyrazole moiety. Com-
pounds 12g and 12h showed an antiproliferative effect at sub-
micromolar level at the highest UV-A dose. It is noticeable that
these compounds were more potent than the reference drug
angelicin against 3 out of 5 cell lines. Within the series of amidic
compounds 10 and 11, the NH derivatives 10a-j showed poor ac-
tivity, with the exception of 10e, which showed micromolar GI50
values on all tested cell lines at the highest dose UV-A (GI50
m
The amidic derivatives 13a,b, were inactive in analogy to the
dihydro derivatives 10g,h after irradiation. Once again, the intro-
duction of a methyl group in the amidic nitrogen led to an increase
of phototoxicity: in fact compounds 13c and 13d had potent pho-
toantiproliferative effect with GI50 values from submicromolar to
nanomolar level (0.31e0.04 mM for 13c and 0.44e0.08 mM for 13d),
demonstrating a remarkable increase of growth inhibitory effect if
10.4e2.76
mM). The N-methyl substitution led to an increase of
compared with their dihydro analogs 11g,h.
activity, even if compounds bearing the same decoration at the
pyrazole moiety were generally less potent than their analogs of
the 2-methoxy series (compare 12g with 11g, and 12h with 11h)
with decreased activity either in leukemic cell lines Jurkat and HL-
60 and in solid tumors MCF-7, A-549 and LoVo. The presence of a
very hydrophobic group in the 7 position seemed to improve the
phototoxicity. In fact, compounds bearing an idrogen or a methyl
group in the pyrazole nitrogen were inactive, whereas compounds
with a phenyl, a benzyl, a chlorophenyl or methoxyphenyl showed
Thus,
7-(4-chlorophenyl)-1-methyl-3-(phenylsulfonyl)-1,7-
dihydro-2H-pyrazolo[3,4-h]quinolin-2-one (13c) and 7-(4-
methoxyphenyl)-1-methyl-3-(phenylsulfonyl)-1,7-dihydro-2H-
pyrazolo[3,4-h]quinolin-2-one (13d) were the most potent com-
pounds of the entire series, with inhibitory capacity on tumor
growth in some cases of an order of magnitude higher than that of
angelicin. In particular, considering the MCF-7 and LoVo cell lines at
the UV-A dose of 3.75 J/cm2, 13c was 8 and 27 times, respectively,
more potent than angelicin as well as 13d which was 10 and 12
Table 2
Photocytotoxicity GI50 (m
M)a of pyrazolo[3,4-h]quinolines 10e13 in human tumor cell lines after 72 h.
JURKAT
HL-60
MCF-7
A-549
LoVo
2.5 J/cm2
1.25 J/cm2
2.5 J/cm2
1.25 J/cm2
3.75 J/cm2
2.5 J/cm2
3.75 J/cm2
2.5 J/cm2
3.75 J/cm2
2.5 J/cm2
Ang
10c
10d
10e
10f
10i
11c
11d
11e
11f
11g
11h
11i
1.00 0.20b
>20
6.88 0.94
3.95 0.34
>20
n.d.
>20
>20
11.27 1.46
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
3.65 0.47
2.48 0.48
1.93 0.14
1.95 0.35
0.72 0.11
1.15 0.18
4.47 0.72
9.93 1.31
>20
1.20 0.10
>20
5.06 0.63
2.76 0.40
>20
n.d.
>20
>20
11.48 1.69
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
2.83 0.31
3.83 0.57
3.78 0.53
3.42 0.46
4.03 1.19
5.29 1.21
5.38 1.01
7.71 1.41
>20
1.50 0.2
>20
>20
10.4 0.60
>20
9.85 0.93
>20
4.42 0.53
>20
>20
12.99 1.48
>20
7.32 0.98
>20
>20
>20
12.67 1.74
>20
>20
>20
>20
>20
>20
2.47 0.34
4.00 0.51
3.13 0.35
5.15 1.10
7.09 2.02
2.53 0.24
>20
n.d.
14.06 0.94
>20
8.97 0.95
9.53 1.18
>20
>20
>20
13.02 1.27
8.40 1.43
>20
>20
>20
6.64 1.12
>20
>20
2.89 0.32
5.74 1.04
4.80 0.50
6.61 0.85
1.49 0.35
1.52 0.15
2.30 0.31
4.67 0.56
5.74 0.96
4.26 0.67
0.08 0.03
0.08 0.03
0.75 0.21
0.09 0.03
n.d.
>20
>20
13.33 2.20
14.14 1.51
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
7.56 0.69
10.37 1.48
14.32 2.66
13.33 3.48
5.28 1.03
4.02 0.44
3.68 0.61
6.99 0.74
>20
5.39 0.93
0.26 0.25
0.27 0.01
3.09 1.15
0.35 0.05
1.11 0.41
>20
14.52 2.43
10.14 1.09
>20
4.00 0.40
>20
>20
>20
>20
>20
>20
>20
>20
13.12 1.05
>20
>20
>20
11.08 1.24
>20
>20
>20
>20
>20
>20
6.99 1.15
5.67 0.93
5.21 0.84
2.81 0.41
3.01 0.60
6.68 0.42
>20
6.79 0.54
4.82 0.24
2.44 0.24
2.88 0.42
7.65 0.55
7.67 1.10
7.17 1.44
>20
9.79 1.46
0.79 0.06
1.30 0.16
0.81 0.15
0.53 0.14
0.48 0.10
0.72 0.12
1.71 0.27
4.69 0.67
9.09 1.38
>20
>20
13.04 2.20
13.06 1.85
7.54 1.17
11.11 2.40
>20
12.62 1.04
9.01 1.19
>20
>20
>20
9.89 1.45
>20
>20
1.84 0.19
3.05 0.48
1.59 0.12
2.52 0.40
1.69 0.18
1.18 0.12
>20
>20
>20
>20
0.17 0.03
0.14 0.03
3.23 0.46
0.59 0.07
>20
11k
11l
4.97 1.45
>20
3.72 0.91
4.12 1.19
>20
12b
12c
12d
12e
12f
12g
12h
12i
12.68 1.58
0.71 0.13
1.40 0.15
0.90 0.13
1.34 0.27
0.43 0.13
0.64 0.15
1.81 0.35
6.43 0.55
>20
>20
1.10 0.20
1.41 0.25
1.78 0.36
2.99 0.87
0.73 0.15
0.53 0.11
1.13 0.41
4.30 0.52
2.76 0.84
>20
3.14 0.30
5.32 0.30
5.73 0.51
9.84 1.14
5.53 1.00
3.56 0.63
2.79 0.47
5.81 0.32
>20
12j
12l
>20
>20
>20
0.33 0.06
0.29 0.03
5.36 0.73
1.23 0.14
13b
13c
13d
13e
13f
>20
>20
>20
>20
0.10 0.02
0.25 0.04
0.96 0.20
0.54 0.11
0.20 0.06
0.58 0.15
4.23 1.15
1.50 0.40
0.31 0.02
0.44 0.03
1.20 0.14
0.76 0.12
0.39 0.05
0.61 0.08
2.16 0.24
0.99 0.07
0.04 0.02
0.09 0.02
0.71 0.15
0.16 0.03
0.16 0.03
0.19 0.03
1.43 0.30
0.42 0.02
a
GI50 ¼ the concentration that causes the 50% growth inhibition.
b
Data are expressed as mean S.E.M. of at least four different independent experiments.

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338
times more potent.
phototoxicity of 12h was mainly reduced in the presence of scav-
engers for free or hydroxyl scavangers like many other photo-
chemotherapics synthesized by some of us [44,46]. For the other
compounds, singlet oxygen could be implicated in their phototox-
icity since NaN3 or DABCO showed a protective effect.
Anyway, the most effective scavengers have often lipophilic
features, so they probably have the same localization of
pyrazoloquinolines.
3.2.1. Colony formation assay
The survival ability of A-549 cells after irradiation with selected
pyrazoloquinolinones (12g, 12h, 13c and 13d, which were chosen
among the most active compounds) was evaluated by colony for-
mation assay [6]. Cells were irradiated and cellular medium was
periodically changed until colony formation was assessed by optical
microscopy. Colony formation was clearly inhibited by all com-
pounds in a concentration-dependent manner (Fig. 1). The level of
colony inhibition was correlated with the phototoxicity data of the
previous experiments: in fact, 13c and 13d were able to cause a
reduction in colony growth at lower concentrations than 12g and
12h.
3.3. Determination of reactive oxygen species
Production of reactive oxygen species is one of the mechanisms
underlying drug induced photosensitization damage. Since some
scavengers led to an increase in cell viability, the determination of
some oxygen reactive species, such as singlet oxygen and super-
oxide anion, was assessed in vitro in aqueous solution after UV-A
irradiation as described in Ref. [58]. Superoxide formation can be
linked to type I photosensization mechanism, which involves free
radical intermediates and ground state oxygen; while formation of
singlet oxygen by energy transfer can be observed in type II [59].
Compounds 13c and 13d efficiently generated singlet oxygen
(Fig. 2B; RNO bleaching) and superoxide anion (Fig. 2C; increase in
absorbance at 560 nm) under UV-A irradiation, while the produc-
tion of ROS of the other two compounds was very low. In particular,
13c and 13d generated comparable levels of superoxide anion and
higher levels of singlet oxygen than the angelicin reference com-
pound [60], suggesting an involvement of both oxygen-dependent
photosensization mechanisms.
3.2.2. Effect of antioxidant compounds
With the purpose of evaluating which reactive species were
involved in the mechanism(s) of phototoxicity, experiments irradi-
ating Jurkat cells with 12g, 12h, 13c and 13d in the presence of
different scavengers were performed, as previously reported [57]. We
used the free radical scavengers tocopherol acetate (vitamin E) and
glutathione (GSH), the singlet oxygen scavengers sodium azide
(NaN3) and 1,4-diazabicyclo[2.2.2]octane (DABCO), and the hydroxyl
radical scavengers dimethylthiourea (DMTU) and mannitol (MANN).
a-Tocopherol was successful in protecting cells from pyr-
azoloquinolines injuries (Fig. 2A); since vitamin E is a lipophilic
vitamin which can protect membranes, we can hypothesize the
involvement of the plasma membrane structure in the mechanism
of action of these compounds.
The other scavengers exerted their action in a different manner
for each molecule: for example, for 12g and 12h, the lipophylic
scavangers increased the cellular viability, while all scavangers led
to an increase of cellular survival in the presence of 13c. The
3.4. Cell death evaluation
To gain a better understanding of the cell death mechanisms
that underlined the antiproliferative activities of this series of
Fig. 1. Colony formation assays were performed as described in the Experimental Section. A) The images showed representative wells of A-549 colonies stained with crystal violet
after irradiation with the indicated concentrations of 12g or 13d. B) Results were represented as percent colony formation relative to untreated cells (C ¼ 100%); error bars represent
the mean SEM of three independent experiments. C ¼ not irradiated control; IC ¼ irradiated control.

Fig. 2. A) Effect of different scavengers on the phototoxicity induced by compounds in Jurkat cells. Cell viability was assayed by MTT test after 72 h from the irradiation (2.5 J/cm2) in
the presence of the tested compounds (0.5 M 12g and 12h; 0.2 M 13c and 13d) and vitamin E (60 M), NaN3 (10 mM), DABCO (1 mM), GSH (1 mM), DMTU (1 mM) and Man
m
m
m
(10 mM). B) RNO bleaching induced by singlet oxygen and C) increase in absorbance at 560 nm of NBT reduced by superoxide anion produced compounds under increasing UV-A
doses. A sample without compounds was used as control.

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340
compounds, we performed some flow cytofluorimetric and
immunoblotting experiments with some of the most active com-
pounds: 12g, 12h, 13c and 13d. Cell death by apoptosis is desirable
for a chemotherapic drug, since the apoptotic process does not
cause inflammatory reaction as there is no release of intracellular
components like in the necrotic death [60]. First, the typical
apoptotic loss of plasmatic membrane asymmetry was evaluated by
flow cytometry. In this cytofluorimetric analysis, two fluorophores
were used 24 h after irradiation of Jurkat cells with the selected
compounds: PI (propidium iodide), which stains DNA and is
permeable only to dead cells, and fluorescent immunolabeling of
the protein Annexin-V-FITC, which binds to phosphatidylserine
(PS). PS is a phospholipid that flips from the inner to the outer
leaflet of the plasma membrane during apoptosis. Positive staining
with Annexin-V correlates with the loss of plasma membrane
asymmetry but precedes the complete loss of membrane integrity
that accompanies the later stages of in vitro cell death. In contrast,
PI can only enter cells after the loss of their membrane integrity.
Thus, dual staining with Annexin-V and PI allows clearly to
discriminate between unaffected cells (Annexin-Vꢁ/PIꢁ), early
apoptotic cells (Annexin-Vþ/PIꢁ), late apoptotic cells (Annexin-
Vþ/PIþ), and necrotic cells (Annexin-Vꢁ/PIþ) [61].
case of compounds 13c and 13d (Fig. 3B). Our hypothesis was that
12g and 12h induced cell death mainly by necrosis while 13c and
13d mainly by apoptosis.
3.4.1. Mitochondrial and lysosomal integrity assessment
To better understand the mode of photoinduced cellular death
by pyrazoloquinolines, some flow cytometric tests which assessed
the involvement of some intracellular organelles were performed.
The complex role of mitochondria in apoptosis was identified by
numerous biochemical studies that pointed out that many mito-
chondrial proteins are able to directly activate cellular apoptotic
programs [63]. Two different tests were carried out to check
mitochondrial dysfunction: variations in mitochondrial membrane
potential (DJM) and reactive oxygen species (ROS) generation.
Mitochondrial membrane depolarization and the decrease of
mitochondrial potential can be assessed by flow cytometry using
JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzylimidazolil-car-
bocianine chloride) as a probe. JC-1 is a lypophylic fluorescent
cation that is able to enter mitochondria and to change its fluo-
rescent properties according to mitochodrial potential. In fact, it
forms orange fluorescence aggregates with normal mitochondrial
potential, whereas when the mitochondrial membrane is depolar-
ized (low DJM), JC-1 forms monomers which emit at 530 nm [64].
For this test, Jurkat cells were irradiated (2.5 J/cm2) in the presence
After 24 h from irradiation, all compounds were able to cause an
important drop in alive cells in comparison to irradiated control
and a clear increase in Annexin-Vþ/PI þ cells (Fig. 3A).
of 1 or 2 mM 12g, 12h, 13c and 13d. After 24 h from irradiation,
Caspases activation, which occurs at relatively early stages of
apoptosis, is also considered a hallmark of this mode of cell death
[62]; thus, an immunoblotting test was carried out to verify a
possible activation of caspase 3 by compounds. Caspase 3 is acti-
vated after the cleavage of active portion of pro-caspase 3 by other
caspases. The levels of cleaved caspase 3 were evaluated by
immunoblotting in cellular lisates after 24 h from irradiation. The
level of cleaved caspase was very low when irradiation was carried
out in the presence of 12g and 12h while it was much higher in the
mitochondrial depolarization occurred for all compounds in
different extents, however, 13c was able to photoinduce a higher
drop in mitochondrial potential (Fig. 4A).
The reduction of DJM, concomitant with uncoupling of mito-
chondrial electron chain transport and ATP synthesis, causes
increased generation of ROS, which, in turn, are important media-
tors of apoptosis [65]. The ROS production was assessed by flow
cytometry after 24 h from irradiation of Jurkat cells in the presence
of the tested compounds. Two probes were used: one for the
detection of superoxide anion and the other for the detection of
hydrogen peroxide [66]. A clear increase in ROS production was
observed when Jurkat cells were irradiated in the presence of 13c
and 13d, while no significant increase was assessed with 12g and
12h. In conclusion, mitochondria were deeply involved only in cell
death photoinduced by 13c and 13d (Fig. 4B).
Other cellular organelles such as lysosomes can be involved in
the ordered propagation of apoptotic events [67]. To evaluate the
involvement of lysosomes in cell death induction, the acridine or-
ange (AO) re-uptake cytofluorimetric test was carried out as pre-
viously reported [68]. Cells irradiated in the presence of 12g and
12h did not show an important involvement of lysosomes in cell
death mechanism, while lysosomes were clearly damaged after
irradiation in the presence of 13c and 13d (Fig. 4C).
3.5. DNA binding and photodamage
Since DNA is the major target of furocoumarins for their pho-
tocytotoxicity [69], the binding of the test compounds to this
macromolecule was investigated. UVevis and fluorimetric titration
with DNA did not show any variation of the absorption spectra or
the presence of isosbestic points indicative of the formation of a
complex between the test compounds and the DNA. Furthermore, a
poor fluorescence quenching of the emission of the free drug in the
presence of DNA suggested that the title compounds were loosely
bound to DNA (data not shown). Even if the binding to DNA was
very low, a possible photodamage to this macromolecule was also
investigated. In particular, the photoinduction of frank strand
breaks was evaluated irradiating a supercoiled double stranded
plasmid (pBR322) in the presence of the tested compounds. The
formation of open circular (OC) or linear (L) form from supercoiled
Fig. 3. A) Exposure of PS and analysis of PI staining 24 h after irradiation (2.5 J/cm2) of
Jurkat cells treated with compounds at 1 or 2 mM. Cells were stained with Annexin
VeFITC and PI and analyzed by flow cytometry; the percentage values represent the
mean of three independent experiments. B) Western blot analysis of caspase 3 and
cleaved caspase 3 in Jurkat cell extracts after 24 h from irradiation (2.5 J/cm2) in the
absence or in the presence of 1
mM compounds. IC ¼ irradiated control.

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V. Spano et al. / European Journal of Medicinal Chemistry 102 (2015) 334e351
341
pirimidine damages, respectively [70,71]. For this test, solutions of
pBR322 were irradiated (UV-A dose: 7.5 J/cm2) in the presence or
absence of compounds 12g, 12h, 13c and 13d in rate [compound]/
[DNA] ¼ 3/1 (See Fig. 1S).
Gel analysis showed that compounds were not able to induce
frank strand breaks after UV-A irradiation; moreover, no oxidative
damages were assessed in DNA bases. All together these data
suggested that DNA was not the target of photodamage of these
compounds.
3.6. Protein photodamage
A possible protein photodamage was also evaluated since these
macromolecules represent the 68% of cellular dry weight and may
be an important target for various photosensitizing agents [72].
Moreover, proteins present endogen chromophores within their
structure, such as aromatic aminoacids or prosthetic groups (flavins
and heme), or may bind exogenous chromophores which can be
easily assessed for possible photoinduced damages or changes.
Proteins can easily react with ROS which can induce photo-
oxidative damage causing for example, loss in activity, fragmenta-
tion, aggregation, denaturation, modifications in hydrophobicity or
structure [73]. In order to investigate if proteins were photo-
damaged by the tested compounds, solutions containing bovine
serum albumin (BSA) or ribonuclease A (RNAseA), as models, and
pyrazoloquinolines 12g, 12h, 13c and 13d were irradiated several
times. The protein photodamage was first assessed by monitoring
the protein fluorescence due to tryptophan (Trp) in BSA sample and
tyrosine (Tyr) in RNAse one. Then, the degree of oxidative modifi-
cations was measured by monitoring the carbonyl content. Results
were reported in Fig. 5 and demonstrated that the amount of the
aromatic aminoacid Trp was reduced in UV-A dependent mode for
all compounds with the exception of 13d (Fig. 5A). No significant
decrease in Tyr fluorescence was observed if RNAase was irradiated
in the presence of the tested compounds (Fig. 5B). The carbonyl
content in BSA increased for all compounds after UV-A irradiation;
however, 13c and 13d were able to photoinduce higher carbonyl
formation rate. Pyrazoloquinazolines caused an oxidative damage
in model proteins even if not all the aminoacids could be involved
and these macromolecules could be an intracellular target of these
compounds.
3.7. Lipid peroxidation
Pyrazoloquinolines are hydrophobic molecules, thus they
probably localize in hydrophobic cellular structures such as cellular
membranes, which could be easy target of photodamage induced
by the tested compounds. The thiobarbituric acid (TBA) assay was
used to determine whether lipid peroxidation occurred upon irra-
diation of Jurkat cells incubated in the presence of test compounds.
TBA reacts with one of the final product of lipid peroxidation, such
as malondialdehyde [74]. The TBA test was performed on the su-
pernatant medium and in the cell homogenates 24 h after irradi-
ation. Thiobarbituric reactive substances (TBARS) were significantly
produced when the cells were exposed to the compounds and UV-A
both in the supernatant and in the cell extract (Fig. 6). Thus, lipids
could be an important target of compound photodamage.
Fig. 4. Mitochondrial (A and B) and lysosomal (C) involvement in photo-induced cell
death. A) Percentage of cells with loss of mitochondrial membrane potential (DJ
measured by JC-1 staining 24 h after irradiation (2.5 J/cm2) of Jurkat cells treated with
compounds at 1 or 2 M; cells were stained with JC-1 and analyzed by fluorescence-
activated cell sorting (FACS). B) Production of ROS in Jurkat cells 24 h after irradiation
(2.5 J/cm2) and treatment with compounds at 1 or 2
M; cells were stained with HE or
)
m
m
DCFDA and analyzed by FACS. C) Percentages of cells stained with AO were analyzed by
flow cytometry 24 h after irradiation (2.5 J/cm2) in the presence of compounds at 1 or
2
m
M. Values represent the mean
SEM of three independent experiments.
4. Conclusion
IC ¼ irradiated cells without compound.
A new series of pyrazolo[3,4-h]quinolines has been successfully
prepared by annelation of the pyridine ring on the indazole moiety.
The synthetic pathway presented herein was never described
before in the pyrazole series and proved to be versatile allowing the
preparation of a large number of derivatives. Some of them showed
(SC) DNA was monitored by separating them by agarose electro-
phoresis. Moreover, oxidative damages were assessed incubating
the irradiated mixtures (pBR322 þ tested compounds) with repair
enzymes such as Fpg and Endo III, which recognized purine and

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V. Spano et al. / European Journal of Medicinal Chemistry 102 (2015) 334e351
342
Fig. 6. Lipid peroxidation induced in Jurkat cells 24 h after irradiation (2.5 J/cm2) in the
presence of compounds at 2 M. The TBA test was performed on the supernatant
m
medium and in the intracellular lisates. Values represent the mean
SEM of three
independent experiments. IC ¼ irradiated cells without compound.
potent inhibitory growth effect under UV light irradiation, being
from 8 to 27 times more active than the reference drug. Evaluation
of SAR of a number of compounds indicated that the presence of 4-
chlorophenyl or a 4-methoxyphenyl substituent in the pyrazolo
[3,4-h]quinolines structure is crucial to express the best activity.
We deeply analyzed the photodamage induced by four among
the most phototoxic compounds: in particular, two of them, 12g
and 12h were dihydro derivatives while 13c and 13d presented the
lowest GI50 values and were fully aromatic molecules. They showed
some differences in their mode of inducing cellular death: in fact,
the dihydro derivatives mainly caused necrosis with a minimal
involvement of mitochondria and lysosomes, whereas 13c and 13d
were associated with apoptosis with a large mitochondrial and
lysosomal involvement. Interestingly, these latter generated com-
parable levels of superoxide anion and higher levels of singlet ox-
ygen than the angelicin reference compound, suggesting an
involvement of both oxygen-dependent photosensization mecha-
nisms (type I and type II).
We hypothesized that lipids were the main target of pyr-
azoloquinoline photodamage as these compounds were able to
induce high degrees of lipid peroxidation and their phototoxicity
was really decreased when cellular irradiation was carried in the
presence of vitamin E. Interestingly, DNA seemed not to be involved
in pyrazoloquinoline photodamage which is crucial and a clear
advantage, possibly preventing some typical side effects of furo-
cumarins such as cancerogenesis and mutagenesis.
In conclusion, compounds 12g, 12h, 13c and 13d can be recog-
nized as the lead compounds of the class of pyrazolo[3,4-h]quino-
lines and could represent the seeds for further developments
directed to the improvement of water solubility. Moreover, the
design of new bathochromically-shifted derivatives in their ab-
sorption spectrum which could represent a very appealing target.
This class of compounds have been already covered by a patent [75]
and a clinical development is desirable.
Fig. 5. Photosensitized protein modification by pyrazoloquinolines. BSA and RNAse
solutions were irradiated at different UV-A doses in the presence or in the absence of
5. Experimental
compounds (10
mM). Protein photomodification was evaluated fluorimetrically by
monitoring the BSA Trp fluorescence (A) or RNAse Tyr fluorescence (B) or spectro-
photometrically by monitoring the carbonyl content in BSA after derivatization with
2,4-dinitrophenylhydrazine (DNPH) (C).
5.1. Synthesis and characterization
All melting points were taken on a Buchi-Tottoli capillary
apparatus; IR spectra were determined, in CHBr3, with a Jasco FT/IR

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V. Spano et al. / European Journal of Medicinal Chemistry 102 (2015) 334e351
343
5300 spectrophotometer; 1H and 13C NMR spectra were measured
in DMSO-d6 or CDCl3 solutions, at 200 and 50 MHz respectively,
using a Bruker Avance II series 200 MHz spectrometer. Column
chromatography was performed with Merck silica gel 230e400
Mesh ASTM or with a SEPACOR chromatography apparatus BÜCHI.
Elemental analyses (C, H, N) were within 0.4% of the theoretical
values.
ppm):
d
2.03e2.12 (m, 2H, CH2), 2.45 (t, J ¼ 6.1 Hz, 2H, CH2), 2.66 (t,
J ¼ 6.1 Hz, 2H, CH2), 7.56e7.79 (m, 4H, Ar), 8.07 (s, 1H, H-3); 13C
NMR (DMSO-d6, 50 MHz, ppm): d 21.0 (t), 22.9 (t), 37.5 (t), 119.0 (s),
128.5 (d), 129.6 (d), 130.4 (d), 130.4 (s), 131.6 (d), 135.4 (s), 137.7 (d),
151.9 (s), 192.4 (s). Anal calcd for C13H11ClN2O (246.69): C, 63.29; H,
4.49; N, 11.36. Found: C, 63.37; H, 4.35; N, 11.51.
Compound 7 was prepared according to procedure described
elsewhere [39].
5.1.1.6. 1-(3-Chlorophenyl)-1,5,6,7-tetrahydro-4H-indazol-4-one (8f).
This compound was obtained from reaction of
chlorophenylhydrazine. Brown solid; yield: 93%; m.p. 92e93 ꢀC;
IR cmꢁ1 1664 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
7 with 3-
5.1.1. General procedure for the preparation of 1-substituted 1,5,6,7-
tetrahydro-4H-indazol-4-ones(8a-h)
:
d
2.05e2.11 (m, 2H, CH2), 2.45 (t, J ¼ 6.2 Hz, 2H, CH2), 3.05 (t,
To a solution of compound 7 (3 g, 18 mmol) in a mixture of
ethanol and acetic acid (2:1) (48 mL) or, for the synthesis of com-
pounds 8 e-g, only glacial acetic acid (30 mL) the suitable hydrazine
(18 mmol) was added and the reaction mixture was heated under
reflux for 2 h. Then, the reaction mixture was poured onto crushed
ice and the solid was filtered off, dried and purified by chroma-
tography (DCM/AcOEt 9:1) as eluent.
J ¼ 6.2 Hz, 2H, CH2), 7.54e7.73 (m, 4H, Ar), 8.07 (s, 1H, H-3); 13C
NMR (DMSO-d6, 50 MHz, ppm): d 22.4 (t), 23.0 (t), 37.4 (t), 120.2 (s),
122.0 (d), 123.2 (d), 128.0 (d), 131.2 (d), 133.7 (s), 138.2 (d), 139.6 (s),
150.3 (s), 192.6 (s). Anal calcd for C13H11ClN2O (246.69): C, 63.29; H,
4.49; N, 11.36. Found: C, 63.41; H, 4.32; N, 11.55.
5.1.1.7. 1-(4-Chlorophenyl)-1,5,6,7-tetrahydro-4H-indazol-4-one
(8g). This compound was obtained from reaction of 7 with 4-
chlorophenylhydrazine. Brown solid; yield: 74%; m.p. 133e134 ꢀC;
5.1.1.1. 1,5,6,7-Tetrahydro-4H-indazol-4-one (8a). This compound
was obtained from reaction of 7 with hydrazine hydrate. Yellow
solid; yield: 67%; m.p. 165e166 ꢀC; IR cmꢁ1: 3186 (NH), 1647 (CO);
IR cmꢁ1
:
1670 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
2.01e2.13 (m, 2H, CH2), 2.45 (t, J ¼ 6.1 Hz, 2H, CH2), 3.02 (t,
1H NMR (DMSO-d6, 200 MHz, ppm):
d
1.96e2.08 (m, 2H, CH2), 2.37
(t, J ¼ 6.1 Hz, 2H, CH2), 2.81 (t, J ¼ 6.1 Hz, 2H, CH2), 7.91 (s, 1H, H-3),
13.21 (s, 1H, NH); 13C NMR (DMSO-d6, 50 MHz, ppm):
21.1 (t), 23.3
J ¼ 6.1 Hz, 2H, CH2), 7.63 (d, J ¼ 9.3 Hz, 2H, H-300 and H-500), 7.68 (d,
J ¼ 9.3 Hz, 2H, H-200 and H-600), 8.07 (s,1H, H-3); 13C NMR (DMSO-d6,
d
50 MHz, ppm):
d
22.4 (t), 23.0 (t), 37.4 (t), 120.1 (s), 125.0 (2 ꢂ d),
(t), 38.1 (t), 117.8 (d), 133.8 (s), 155.2 (s), 192.9 (s). Anal calcd for
C7H8N2O (136.15): C, 61.75; H, 5.92; N, 20.58. Found: C, 61.88; H,
6.11; N, 20.39.
129.4 (2 ꢂ d), 132.4 (s), 137.2 (d), 138.0 (s), 150.1 (s), 192.6 (s). Anal
calcd for C13H11ClN2O (246.69): C, 63.29; H, 4.49; N, 11.36. Found: C,
63.18; H, 4.63; N, 11.46.
5.1.1.2. 1-Methyl-1,5,6,7-tetrahydro-4H-indazol-4-one
(8b).
5.1.1.8. 1-(4-Methoxyphenyl)-1,5,6,7-tetrahydro-4H-indazol-4-one
(8h). This compound was obtained from reaction of 7 with 4-
methoxyphenylhydrazine. Brown solid; yield: 70%; m.p.
153e154 ꢀC; IR cmꢁ1: 1668 (CO); 1H NMR (DMSO-d6, 200 MHz,
This compound was obtained from reaction of 7 with methylhy-
drazine. Brown solid; yield: 60%; m.p. 95e96 ꢀC; IR cmꢁ1: 1668
(CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
2.01e2.07 (m, 2H, CH2),
2.35 (t, J ¼ 6.2 Hz, 2H, CH2), 2.84 (t, J ¼ 6.2 Hz, 2H, CH2), 3.77 (s, 3H,
CH3), 7.74 (s,1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm):
20.3 (t),
ppm):
d
2.02e2.11 (m, 2H, CH2), 2.43 (t, J ¼ 6.1 Hz, 2H, CH2), 2.94 (t,
d
J ¼ 6.1 Hz, 2H, CH2), 3.83 (s, 3H, CH3), 7.10 (d, J ¼ 9.0 Hz, 2H, H-300
and H-500), 7.53 (d, J ¼ 9.0 Hz, 2H, H-200 and H-600), 8.01 (s, 1H, H-3);
27.7 (t), 35.9 (q), 37.4 (t), 118.5 (s), 135.8 (d), 149.9 (s), 192.1 (s). Anal.
C8H10N2O (150.18): C, 63.98; H, 6.71 N, 18.65. Found: C, 64.10; H,
6.55; N, 18.80.
13C NMR (DMSO-d6, 50 MHz, ppm):
d 22.2 (t), 23.0 (t), 37.4 (t), 57.4
(q),114.4 (2 ꢂ d),119.6 (s), 125.0 (2 ꢂ d),131.4 (d),137.3 (s), 149.6 (s),
158.8 (s), 192.5 (s). Anal calcd for C14H14N2O2 (242.27): C, 69.41; H,
5.82; N, 11.56. Found: C, 69.56; H, 5.71; N, 11.39.
5.1.1.3. 1-Phenyl-1,5,6,7-tetrahydro-4H-indazol-4-one
(8c). This
compound was obtained from reaction of 7 with phenylhydrazine.
Brown solid; yield: 65%; m.p. 137e138 ꢀC; IR cmꢁ1: 1664 (CO); 1H
5.1.2. General procedure for the preparation of 1-substituted 5-
[(dimethylamino)methylidene]-1,5,6,7-tetrahydro-4H-indazol-4-
ones (9a-h)
A solution of the suitable ketone 8a-h (5.3 mmol) in DMFDMA
(8 mL) was heated at reflux for 24 h (Method A). Then the reaction
mixture was cooled and the solvent was removed under reduced
pressure. The crude was stirred in diethyl ether.
NMR (DMSO-d6, 200 MHz, ppm):
J ¼ 6.1 Hz, 2H, CH2), 3.01 (t, J ¼ 6.1 Hz, 2H, CH2), 7.47e7.66 (m, 5H,
Ar), 8.05 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm):
22.4 (t),
d 2.03e2.13 (m, 2H, CH2), 2.45 (t,
d
23.1 (t), 37.4 (t), 119.9 (s), 123.4 (2 ꢂ d), 128.4 (d), 129.4 (2 ꢂ d), 137.7
(d), 138.4 (s), 149.9 (s), 192.6 (s). Anal calcd for C13H12N2O (212.25):
C, 73.56; H, 5.70; N, 13.20. Found: C, 73.68; H, 5.59; N, 13.48.
Alternatively to
a solution of the suitable ketone 8a-h
5.1.1.4. 1-Benzyl-1,5,6,7-tetrahydro-4H-indazol-4-one
(8d). This
(1.5 mmol) in anhydrous DMF (2 mL), DMFDMA (2 mL, 15 mmol)
was added and the mixture was stirred under microwave irradia-
tion (Method B). (MW conditions: Power 150 W; Time 30e45 min;
Temperature (max) 150 ꢀC). The reaction mixture was poured onto
crushed ice.
Enaminoketones having a solid state, were filtered off, washed
with diethyl ether and dried. The oily ones were decanted, solu-
bilized in dichloromethane and the organic layer was dried
(Na2SO4) and the solvent was removed under reduced pressure.
compound was obtained from reaction of 7 with benzylhydrazine.
Brown oil; yield: 63%; IR cmꢁ1: 1653 (CO); 1H NMR (DMSO-d6,
200 MHz, ppm):
CH2), 2.85 (t, J ¼ 6.2 Hz, 2H, CH2), 5.36 (s, 2H, CH2), 7.19e7.40 (m,
5H, Ar), 7.82 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm):
20.6
d
1.97e2.09 (m, 2H, CH2), 2.36 (t, J ¼ 6.2 Hz, 2H,
d
(t), 22.8 (t), 37.4 (t), 52.2 (t), 118.8 (s), 127.4 (2 ꢂ d), 127.7 (d), 128.6
(2 ꢂ d), 136.4 (s), 136.5 (d), 149.9 (s), 192.2 (s). Anal calcd for
C
14H14N2O (226.27): C, 74.31; H, 6.24; N, 12.38. Found: C, 74.18; H,
6.36; N, 12.22.
5.1.2.1. 5-[(Dimethylamino)methylidene]-1,5,6,7-tetrahydro-4H-
indazol-4-one (9a). This compound was obtained from 8a. Brown
solid; yield: 90% (Method A), 98% (Method B); m.p. 131e132 ꢀC; IR
cmꢁ1: 3116 (NH), 1668 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
5.1.1.5. 1-(2-Chlorophenyl)-1,5,6,7-tetrahydro-4H-indazol-4-one
(8e). This compound was obtained from reaction of 7 with 2-
chlorophenylhydrazine. Pale brown solid; yield: 62%; m.p.
126e127 ꢀC; IR cmꢁ1: 1672 (CO); 1H NMR (DMSO-d6, 200 MHz,
d
2.31e2.39 (m, 4H, 2 ꢂ CH2), 3.77 (s, 3H, CH3), 3.83 (s, 3H, CH3), 7.74

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(s, 1H, CH), 7.96 (s, 1H, H-3), 13.23 (s, 1H, NH); 13C NMR (DMSO-d6,
50 MHz, ppm):
135.9 (d), 149.9 (s), 156.3 (s), 192.2 (s). Anal calcd for C10H13N3O
(191.23): C, 62.81; H, 6.85; N, 21.97. Found: C, 62.99; H, 6.72; N,
22.06.
cmꢁ1: 1648 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d 2.89e3.01
d
22.2 (t), 23.3 (t), 37.4 (2 ꢂ q), 118.4 (s), 130.7 (d),
(m, 4H, 2 ꢂ CH2), 3.08 (s, 6H, 2 ꢂ CH3), 7.37 (s, 1H, CH), 7.58 (d,
J ¼ 8.5 Hz, 2H, H-300and H-500), 7.62 (d, J ¼ 8.5 Hz, 2H, H-200 and H-
600), 7.94 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm):
d 22.2 (t),
23.3 (t), 43.2 (2 ꢂ q), 100.5 (s), 121.8 (s), 124.7 (2 ꢂ d), 129.3 (2 ꢂ d),
132.6 (s), 138.4 (d), 147.8 (d), 149.4 (s), 156.6 (s), 181.4 (s). Anal calcd
for C16H16ClN3O (301.77): C, 63.68; H, 5.34; N, 13.92. Found: C,
63.55; H, 5.23; N, 14.11.
5.1.2.2. 5-[(Dimethylamino)methylidene]-1-methyl-1,5,6,7-
tetrahydro-4H-indazol-4-one (9b). This compound was obtained
from 8b. Brown oil; yield: 77% (Method A), 96% (Method B); IR
cmꢁ1: 1639 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
2.74 (t,
5.1.2.8. 5-[(Dimethylamino)methylidene]-1-(4-metoxyphenyl)-
1,5,6,7-tetrahydro-4H-indazol-4-one (9h). This compound was ob-
tained from 8h. Brown solid; yield: 71% (Method A), 96% (Method
B); m.p. 151e152 ꢀC; IR cmꢁ1: 1666 (CO); 1H NMR (DMSO-d6,
J ¼ 6.4 Hz, 2H, CH2), 2.96 (t, J ¼ 6.4 Hz, 2H, CH2), 3.05 (s, 6H, 2 ꢂ
CH3), 3.74 (s, 3H, CH3), 7.29 (s, 1H, CH), 7.61 (s, 1H, H-3); 13C NMR
(DMSO-d6, 50 MHz, ppm):
99.9 (s), 118.5 (s), 135.1 (d), 145.6 (s), 146.5 (d), 180.5 (s). Anal.
11H15N3O (205.26): C, 64.37; H, 7.37; N, 20.47. Found: C, 64.25; H,
d
19.2 (t), 22.0 (t), 34.6 (q), 42.1 (2 ꢂ q),
200 MHz, ppm):
d
2.87e2.95 (m, 4H, 2 ꢂ CH2), 3.07 (s, 6H, 2 ꢂ CH3),
C
3.82 (s, 3H, CH3), 7.09 (d, J ¼ 8.7 Hz, 2H, H-300 and H-500), 7.36 (s, 1H,
7.51; N, 20.58.
CH), 7.50 (d, J ¼ 8.7 Hz, 2H, H-200 and H-600), 7.88 (s, 1H, H-3); 13C
NMR (DMSO-d6, 50 MHz, ppm):
d
22.0 (t), 23.3 (t), 43.2 (2 ꢂ q), 55.4
5.1.2.3. 5-[(Dimethylamino)methylidene]-1-phenyl-1,5,6,7-
tetrahydro-4H-indazol-4-one (9c). This compound was obtained
from 8c. Brown oil; yield: 80% (Method A), 98% (Method B); IR
(q),100.8 (s),114.4 (2 ꢂ d),120.7 (s),124.8 (2 ꢂ d),131.7 (d),137.7 (s),
146.1 (d), 147.6 (s), 158.5 (s), 181.5 (s). Anal calcd for C17H19N3O2
(297.35): C, 68.67; H, 6.44; N, 14.13. Found: C, 68.91; H, 6.19; N,
14.32.
cmꢁ1: 1657 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
2.80e2.95
(s, 4H, 2 ꢂ CH2), 3.07 (s, 6H, 2 ꢂ CH3), 7.37 (s, 1H, CH), 7.55e7.58 (m,
5H, Ar), 7.92 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm):
22.3
d
5.1.3. General procedure for the preparation of 3,7-substituted
1,5,6,7-tetrahydro-2H-pyrazolo[3,4-h]quinolin-2-ones (10a-j)
To a solution of the suitable enaminoketons 9a-h (20 mmol) in
anhydrous ethanol (30 mL), a solution of the proper cyano-
methylene compound (20 mmol) in anhydrous ethanol (30 mL)
was added dropwise under nitrogen atmosphere. Then the reaction
mixture was heated at reflux for 24 h. Upon cooling, a solid sepa-
rated which was filtered, dried (Na2SO4) and recrystallized from
ethanol.
(t), 23.3 (t), 43.2 (2 ꢂ q), 100.7 (s), 121.2 (s), 123.2 (2 ꢂ d), 127.6 (d),
129.3 (2 ꢂ d), 138.1 (d), 138.6 (s), 146.3 (s), 147.7 (d), 181.5 (s). Anal
calcd for C16H17N3O (267.33): C, 71.89; H, 6.41; N, 15.72. Found: C,
71.78; H, 6.60; N, 15.54.
5.1.2.4. 1-Benzyl-5-[(dimethylamino)methylidene]-1,5,6,7-
tetrahydro-4H-indazol-4-one (9d). This compound was obtained
from 8d. Brown oil; yield: 82% (Method A), 95% (Method B); IR
cmꢁ1: 1668 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d 2.73e2.93
(m, 4H, 2 ꢂ CH2), 2.89 (s, 6H, 2 ꢂ CH3), 5.36 (s, 2H, CH2), 7.13e7.36
5.1.3.1. 3-(Phenylsulfonyl)-1,5,6,7-tetrahydro-2H-pyrazolo[3,4-h]qui-
nolin-2-one (10a). This compound was obtained from reaction of
9a with phenylsulfonylacetonitrile. White solid; yield: 60%; m.p.
>410 ꢀC; IR cmꢁ1: 3110 (NH), 3078 (NH), 1653 (CO); 1H NMR
(m, 5H, Ar), 7.79 (s, 1H, CH), 7.84 (s, 1H, H-3); 13C NMR (DMSO-d6,
50 MHz, ppm):
d
22.9 (t), 27.3 (t), 40.7 (2 ꢂ q), 61.7 (t), 108.5 (s),
117.5 (s), 127.4 (2 ꢂ d), 128.3 (d), 128.7 (2 ꢂ d), 131.9 (s), 136.5 (d),
146.7 (d), 162.3 (s), 180.8 (s). Anal calcd for C17H19N3O (281.35): C,
72.57; H, 6.81; N, 14.94. Found: C, 72.33; H, 7.02; N, 14.99.
(DMSO-d6, 200 MHz, ppm):
d
2.75e2.91 (m, 4H, 2 ꢂ CH2), 7.55e7.67
(m, 3H, H-30, H-40 and H-50), 7.96 (dd, J ¼ 7.1, 1.5 Hz, 2H, H-20 and H-
60), 8.06 (s, 1H, H-9), 8.20 (s, 1H, H-4), 12.60 (s, 1H, NH), 13.17 (s, 1H,
5.1.2.5. 1-(2-Chlorophenyl)-5-[(dimethylamino)methylidene]-1,5,6,7-
tetrahydro-4H-indazol-4-one (9e). This compound was obtained
from 8e. Brown solid; yield: 84% (Method A), 98% (Method B); m.p.
147e148 ꢀC; IR cmꢁ1: 1637 (CO); 1H NMR (DMSO-d6, 200 MHz,
NH); 13C NMR (DMSO-d6, 50 MHz, ppm):
d 20.7 (t), 25.5 (t),110.1 (s),
121.9 (s), 127.8 (2 ꢂ d), 128.7 (2 ꢂ d), 129.3 (d), 133.0 (d), 140.7 (s),
143.9 (d), 144.7 (s), 145.2 (s), 151.6 (s), 157.2 (s). Anal. C16H13N3O3S
(327.36): C, 58.70; H, 4.00, N, 12.84. Found: C, 58.85; H, 4.33, N,
12.68.
ppm):
3.06 (s, 6H, 2 ꢂ CH3), 7.38 (s, 1H, CH), 7.54e7.76 (m, 4H, Ar), 7.92 (s,
1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm):
21.0 (t), 23.1 (t), 43.2
d
2.58 (t, J ¼ 6.6 Hz, 2H, CH2), 2.96 (t, J ¼ 6.6 Hz, 2H, CH2),
d
5.1.3.2. 7-Methyl-3-(phenylsulfonyl)-1,5,6,7-tetrahydro-2H-pyrazolo
[3,4-h]quinolin-2-one (10b). This compound was obtained from
reaction of 9b with phenylsulfonylacetonitrile. Yellow solid; yield:
80%; m.p. 380e382 ꢀC; IR cmꢁ1: 3157 (NH), 1631 (CO); 1H NMR
(2 ꢂ q), 100.6 (s), 120.1 (s), 128.3 (d), 129.6 (d), 130.3 (d), 130.4 (s),
131.2 (d), 135.7 (s), 138.1 (d), 148.0 (d), 148.3 (s), 181.3 (s). Anal calcd
for C16H16ClN3O (301.77): C, 63.68; H, 5.34; N, 13.92. Found: C,
63.80; H, 5.43; N, 14.18.
(DMSO-d6, 200 MHz, ppm):
d 2.49e2.51 (m, 2H, CH2), 2.90 (s, 3H,
CH3), 2.66e2.78 (m, 2H, CH2), 7.53e7.69 (m, 3H, H-30, H-40 and H-
50), 7.06 (dd, J ¼ 7.5, 1.4 Hz, 2H, H-20 and H-60), 8.05 (1H, s, H-9), 8.17
(1H, s, H-4), 12.56 (1H, s, NH); 13C NMR (DMSO-d6, 50 MHz, ppm):
5.1.2.6. 1-(3-Chlorophenyl)-5-[(dimethylamino)methylidene]-1,5,6,7-
tetrahydro-4H-indazol-4-one (9f). This compound was obtained
from 8f. Brown oil; yield: 70% (Method A), 95% (Method B); IR
d
19.0 (t), 24.9 (t), 35.9 (q), 107.3 (s), 110.7 (s), 121.1 (s), 127.8 (2 ꢂ d),
cmꢁ1: 1639 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
2.91e3.03
128.7 (2 ꢂ d), 133.0 (d), 135.3 (d), 140.9 (s), 143.6 (d), 143.8 (s), 144.6
(s), 157.2 (s). Anal. C17H15N3O3S (341.38): C, 59.81; H, 4.43 N, 12.31.
Found: C, 60.05; H, 4.25; N, 12.40.
(m, 4H, 2 ꢂ CH2), 3.08 (s, 6H, 2 ꢂ CH3), 7.37 (s, 1H, CH), 7.53e7.69
(m, 4H, Ar), 7.95 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm):
d
22.2 (t), 23.3 (t), 43.2 (2 ꢂ q), 100.5 (s), 121.5 (s), 121.7 (d), 122.8
(d), 127.5 (d), 131.1 (d), 133.6 (s), 138.6 (d), 139.8 (s), 146.7 (s), 147.8
(d), 181.4 (s). Anal calcd for C16H16ClN3O (301.77): C, 63.68; H, 5.34;
N, 13.92. Found: C, 63.77; H, 5.21; N, 13.96.
5.1.3.3. 7-Phenyl-3-(phenylsulfonyl)-1,5,6,7-tetrahydro-2H-pyrazolo
[3,4-h]quinolin-2-one (10c). This compound was obtained from
reaction of 9c with phenylsulfonylacetonitrile. Brown solid; yield:
63%; m.p. 392e393 ꢀC; IR cmꢁ1: 3105 (NH), 1643 (CO); 1H NMR
5.1.2.7. 1-(4-Chlorophenyl)-5-[(dimethylamino)methylidene]-1,5,6,7-
tetrahydro-4H-indazol-4-one (9g). This compound was obtained
from 8g. Brown oil; yield: 70% (Method A), 95% (Method B); IR
(DMSO-d6, 200 MHz, ppm):
d
2.92 (t, J ¼ 7.2 Hz, 2H, CH2), 3.06 (t,
J ¼ 7.2 Hz, 2H, CH2), 7.43e7.71 (m, 8H, Ar), 7.99 (dd, J ¼ 6.6, 1.5 Hz,
2H, H-20 and H-60), 8.24 (s, 1H, H-9), 8.35 (s, 1H, H-4), 12.73 (s, 1H,

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NH); 13C NMR (DMSO-d6, 50 MHz, ppm):
d
20.6 (t), 25.2 (t), 108.0
J ¼ 8.7 Hz, 2H, H-200 and H-600), 7.58e7.67 (m, 3H, Ar), 7.98 (d, J ¼ 7.4,
(s), 112.5 (s), 122.1 (s), 123.4 (2 ꢂ d), 127.8 (2 ꢂ d), 128.1 (d), 128.7
(2 ꢂ d), 129.4 (2 ꢂ d), 133.1 (d), 137.1 (d), 138.2 (s), 140.7 (s), 143.8
(d), 144.2 (s), 144.6 (s), 157.2 (s). Anal. C22H17N3O3S (403.45): C,
65.49; H, 4.25; N, 10.42. Found: C, 65.55; H, 4.09; N, 10.31.
1.6 Hz, 2H, H-20 and H-60), 8.19 (s, 1H, H-9), 8.28 (s, 1H, H-4), 12.59
(s, 1H, NH); 13C NMR (DMSO-d6, 50 MHz, ppm):
d 20.4 (t), 25.3 (t),
55.5 (q), 113.5 (s), 114.4 (2 ꢂ d), 124.8 (s), 125.0 (2 ꢂ d), 125.2 (d),
127.8 (2 ꢂ d), 128.7 (2 ꢂ d), 131.3 (s), 132.9 (s), 136.7 (d), 140.9 (s),
143.2 (s), 143.9 (d), 156.7 (s), 157.8 (s), 158.8 (s). Anal. C23H19N3O4S
(433.48): C, 63.73; H, 4.42; N, 9.69. Found: C, 63.92; H, 4.38; N, 9.81.
5.1.3.4. 7-Benzyl-3-(phenylsulfonyl)-1,5,6,7-tetrahydro-2H-pyrazolo
[3,4-h]quinolin-2-one (10d). This compound was obtained from
reaction of 9d with phenylsulfonylacetonitrile. White solid; yield:
88%; m.p. 285e287 ꢀC; IR cmꢁ1: 3089 (NH), 1631 (CO); 1H NMR
5.1.3.9. Ethyl 7-(4-chlorophenyl)-2-oxo-2,5,6,7-tetrahydro-1H-pyr-
azolo[3,4-h]quinolin-3-carboxylate (10i). This compound was ob-
tained from reaction of 9g with ethyl cyanoacetate. Yellow solid;
yield: 72%; m.p. 271e272 ꢀC; IR cmꢁ1: 3409 (NH), 1690 (CO), 1645
(DMSO-d6, 200 MHz, ppm):
d
2.89e2.94 (m, 4H, 2 ꢂ CH2), 5.37 (s,
2H, CH2), 7.16 (dd, J ¼ 7.5, 2.0 Hz, 2H, Ar), 7.28e7.35 (m, 3H, Ar),
7.53e7.66 (m, 3H, Ar), 7.96 (dd, J ¼ 8.0, 1.7 Hz, 2H, H-20 and H-60),
8.14 (s, 1H, H-9), 8.18 (s, 1H, H-4), 12.62 (s, 1H, NH); 13C NMR
(CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
CH3), 2.86 (t, J ¼ 7.0 Hz, 2H, CH2), 3.05 (t, J ¼ 7.0 Hz, 2H, CH2), 4.21 (q,
J ¼ 7.1 Hz, 2H, CH2), 7.55e7.73 (m, 4H, Ar), 8.03 (s, 1H, H-9), 8.35 (s,
1H, H-4), 12.38 (s, 1H, NH); 13C NMR (DMSO-d6, 50 MHz, ppm):
d
1.27 (t, J ¼ 7.1 Hz, 3H,
(DMSO-d6, 50 MHz, ppm): d 19.3 (t), 25.0 (t), 52.3 (t), 107.3 (s), 111.1
(s), 121.4 (s), 127.4 (2 ꢂ d), 127.6 (d), 127.7 (2 ꢂ d), 128.5 (2 ꢂ d),
128.6 (2 ꢂ d), 133.0 (d), 135.9 (s), 136.4 (s), 140.9 (d), 143.7 (d), 144.6
(s), 148.2 (s), 157.2 (s). Anal. C23H19N3O3S (417.48): C, 66.17; H, 4.59;
N, 10.07. Found: C, 66.34; H, 4.77; N, 10.21.
d
14.3 (q), 20.9 (t), 25.6 (t), 59.7 (t), 109.1 (d), 125.1 (2 ꢂ d), 128.1 (s),
129.4 (2 ꢂ d), 132.6 (s), 135.4 (s), 136.5 (s), 137.2 (d), 144.1 (s), 147.4
(s), 152.1 (s), 156.3 (s), 162.6 (s). Anal calcd for C19H16ClN3O3
(369.80): C, 61.71; H, 4.36; N, 11.36. Found: C, 61.86; H, 4.16; N,
11.55.
5.1.3.5. 7-(2-Chlorophenyl)-3-(phenylsulfonyl)-1,5,6,7-tetrahydro-
2H-pyrazolo[3,4-h]quinolin-2-one (10e). This compound was ob-
tained from reaction of 9e with phenylsulfonylacetonitrile. White
solid; yield: 65%; m.p. 349e350 ꢀC; IR cmꢁ1: 3238 (NH), 1637 (CO);
5.1.3.10. Ethyl
7-(4-methoxyphenyl)-2-oxo-2,5,6,7-tetrahydro-1H-
pyrazolo[3,4-h]quinolin-3-carboxylate (10j). This compound was
obtained from reaction of 9h with ethyl cyanoacetate. Yellow solid;
yield: 80%; m.p. 275e276 ꢀC; IR cmꢁ1: 3414 (NH), 1685 (CO), 1644
1H NMR (DMSO-d6, 200 MHz, ppm):
d
2.73 (t, J ¼ 7.6 Hz, 2H, CH2),
2.92 (t, J ¼ 7.6 Hz, 2H, CH2), 7.52e7.78 (m, 7H, Ar), 7.98 (dd, J ¼ 8.0,
1.7 Hz, 2H, H-20 and H-60), 8.23 (s, 1H, H-9), 8.35 (s, 1H, H-4), 12.80
(CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
d
1.27 (t, J ¼ 7.1 Hz, 3H,
(s, 1H, NH); 13C NMR (DMSO-d6, 50 MHz, ppm):
d
19.8 (t), 24.9 (t),
CH3), 2.85 (t, J ¼ 6.7 Hz, 2H, CH2), 2.98 (t, J ¼ 6.7 Hz, 2H, CH2), 3.83
(s, 3H, CH3), 4.21 (q, J ¼ 7.1 Hz, 2H, CH2), 7.10 (d, J ¼ 8.9 Hz, 2H, H-300
and H-500), 7.51 (d, J ¼ 8.9 Hz, 2H, H-200 and H-6”), 8.03 (s, 1H, H-9),
8.31 (s, 1H, H-4), 12.11 (s, 1H, NH); 13C NMR (DMSO-d6, 50 MHz,
111.4 (s), 122.2 (s), 127.8 (2 ꢂ d), 128.5 (d), 128.9 (2 ꢂ d), 129.5 (d),
130.4 (d), 130.5 (s), 131.7 (d), 133.1 (d), 135.3 (s), 137.1 (d), 138.2 (s),
140.8 (s), 143.9 (d),144.3 (s),146.2 (s), 157.2 (s). Anal. C22H16ClN3O3S
(437.90): C, 60.34; H, 3.68, N, 9.60. Found: C, 60.48; H, 3.39, N, 9.78.
ppm):
d 14.3 (q), 20.5 (t), 21.4 (t), 55.5 (q), 59.8 (t), 105.9 (s), 114.4
(2 ꢂ d),125.0 (2 ꢂ d),131.3 (s),133.1 (s),136.6 (d),139.0 (s),142.4 (s),
5.1.3.6. 7-(3-Chlorophenyl)-3-(phenylsulfonyl)-1,5,6,7-tetrahydro-
2H-pyrazolo[3,4-h]quinolin-2-one (10f). This compound was ob-
tained from reaction of 9f with phenylsulfonylacetonitrile. Gray
solid; yield: 68%; m.p. 379e381 ꢀC; IR cmꢁ1: 3103 (NH), 1649 (CO);
143.6 (d), 155.3 (s), 158.7 (s), 159.9 (s), 165.2 (s). Anal calcd for
C20H19N3O4 (365.38): C, 65.74; H, 5.24; N, 11.50. Found: C, 65.93; H,
5.11; N, 11.65.
1H NMR (DMSO-d6, 200 MHz, ppm):
d
2.93 (t, J ¼ 6.8 Hz, 2H, CH2),
5.1.4. General procedure for the preparation of 3,7-disubstituted 1-
methyl-1,5,6,7-tetrahydro-2H-pyrazolo[3,4-h]quinolin-2-ones (11b-
l) and 2-methoxy-7-methyl-6,7-dihydro-5H-pyrazolo[3,4-h]
quinolines (12b-l)
3.09 (t, J ¼ 6.8 Hz, 2H, CH2), 7.59e7.71 (m, 7H, Ar), 7.98 (d, J ¼ 6.8 Hz,
2H, H-20 and H-60), 8.25 (s, 1H, H-9), 8.37 (s, 1H, H-4), 12.76 (s, 1H,
NH); 13C NMR (DMSO-d6, 50 MHz, ppm):
d 22.5 (t), 25.1 (t), 122.1
(d), 123.2 (d), 127.9 (2 ꢂ d), 128.1 (d), 128.7 (2 ꢂ d), 131.1 (d), 133.1
(d), 133.7 (s), 135.5 (s), 136.0 (s), 137.5 (d), 139.3 (s), 140.7 (s), 142.9
(s), 143.8 (d), 144.6 (s), 151.4 (s), 157.2 (s). Anal. C22H16ClN3O3S
(437.90): C, 60.34; H, 3.68; N, 9.60. Found: C, 60.18; H, 3.77; N, 9.79.
To a solution of the suitable pyrazoloquinolinone 10b-l (2 mmol)
in anhydrous DMF (8 mL), a stoichiometric amount of NaH was
added. The reaction mixture was stirred for 3 h at room tempera-
ture and then the appropriate halide (2 mmol) was added. After
24 h, it was poured onto crushed ice and the precipitate was filtered
off, dried and purified by chromatography (DCM/AcOEt 95:5) to
obtain N-methyl derivatives 11b-l and O-methyl derivatives 12b-l.
5.1.3.7. 7-(4-Chlorophenyl)-3-(phenylsulfonyl)-1,5,6,7-tetrahydro-
2H-pyrazolo[3,4-h]quinolin-2-one (10g). This compound was ob-
tained from reaction of 9g with phenylsulfonylacetonitrile. Yellow
solid; yield: 65%; m.p. >410 ꢀC; IR cmꢁ1: 3086 (NH), 1649 (CO); 1H
5.1.4.1. 1,7-Dimethyl-3-(phenylsulfonyl)-1,5,6,7-tetrahydro-2H-pyr-
azolo[3,4-h]quinolin-2-one (11b). This compound was obtained
from reaction of 10b with iodomethane. Yellow solid; yield: 58%;
m.p. 264e266 ꢀC; IR cmꢁ1: 1649 (CO); 1H NMR (DMSO-d6,
NMR (DMSO-d6, 200 MHz, ppm):
d
2.92e3.07 (m, 4H, 2 ꢂ CH2), 7.10
(d, J ¼ 7.4 Hz, 2H, H-30 and H-50), 7.55e7.68 (m, 5H, Ar), 7.98 (dd,
J ¼ 7.4, 1.7 Hz, 2H, H-20 and H-60), 8.25 (s, 1H, H-9), 8.35 (s, 1H, H-4),
12.75 (s,1H, NH); 13C NMR (DMSO-d6, 50 MHz, ppm):
d
20.5 (t), 25.1
200 MHz, ppm): d 2.91e2.98 (m, 2H, CH2), 3.04e3.11 (m, 2H, CH2),
(t), 122.3 (s), 125.1 (2 ꢂ d), 125.9 (s), 127.9 (2 ꢂ d), 128.7 (2 ꢂ d),
129.4 (2 ꢂ d), 131.7 (s), 132.5 (s), 133.1 (d), 137.0 (s), 137.4 (d), 140.7
(s), 157.6 (d), 144.4 (s), 144.5 (s), 157.3 (s). Anal. C22H16ClN3O3S
(437.90): C, 60.34; H, 3.68; N, 9.60. Found: C, 60.52; H, 3.55; N, 9.40.
3.79 (s, 3H, CH3), 3.86 (s, 3H, CH3), 7.57e7.74 (m, 3H, H-30, H-40 and
H-50), 7.79 (s, 1H, H-9), 7.92 (dd, J ¼ 7.3, 1.4 Hz, 2H, H-20 and H-60),
8.17 (s, 1H, H-4); 13C NMR (DMSO-d6, 50 MHz, ppm):
d 18.8 (t), 26.1
(t), 35.9 (q), 53.6 (q), 116.7 (s), 117.8 (s), 120.7 (s), 127.7 (2 ꢂ d), 129.0
(2 ꢂ d), 133.5 (d), 134.7 (d), 137.8 (d), 140.7 (s), 144.0 (s), 153.4 (s),
158.2 (s). Anal. C18H17N3O3S (355.41): C, 60.83; H, 4.82; N, 11.82.
Found: C, 60.98; H, 4.64; N, 12.18.
5.1.3.8. 7-(4-Methoxyphenyl)-3-(phenylsulfonyl)-1,5,6,7-tetrahydro-
2H-pyrazolo[3,4-h]quinolin-2-one (10h). This compound was ob-
tained from reaction of 9h with phenylsulfonylacetonitrile. Yellow
solid; yield: 73%; m.p. 387e389 ꢀC; IR cmꢁ1: 3078 (NH), 1645 (CO);
5.1.4.2. 1-Methyl-7-phenyl-3-(phenylsulfonyl)-1,5,6,7-tetrahydro-
2H-pyrazolo[3,4-h]quinolin-2-one (11c). This compound was ob-
tained from reaction of 10c with iodomethane. Yellow solid; yield:
1H NMR (DMSO-d6, 200 MHz, ppm):
d
2.90e2.99 (m, 4H, 2 ꢂ CH2),
3.82 (s, 3H, CH3), 7.10 (d, J ¼ 8.7 Hz, 2H, H-300 and H-500), 7.50 (d,

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346
58%; m.p. 289e291 ꢀC; IR cmꢁ1: 1649 (CO); 1H NMR (DMSO-d6,
solid; yield: 59%; m.p. 236e237 ꢀC; IR cmꢁ1: 1653 (CO); 1H NMR
200 MHz, ppm):
d
2.89e3.01 (m, 4H, CH2), 3.73 (s, 3H, CH3),
(DMSO-d6, 200 MHz, ppm):
d
2.84e2.94 (m, 4H, 2 ꢂ CH2), 3.73 (s,
7.48e7.70 (m, 8H, Ar), 7.98 (d, J ¼ 7.1 Hz, 2H, H-20 and H-60), 8.26 (s,
3H, CH3), 3.83 (s, 3H, CH3), 7.12 (d, J ¼ 8.9 Hz, 2H, H-300 and H-500),
7.52 (d, J ¼ 8.9 Hz, 2H, H-200 and H-600), 7.58e7.67 (m, 3H, Ar), 7.98
(dd, J ¼ 7.4, 1.6 Hz, 2H, H-20 and H-60), 8.25 (s, 1H, H-9), 8.30 (s, 1H,
1H, H-9), 8.16 (s, 1H, H-4); 13C NMR (DMSO-d6, 50 MHz, ppm):
d
20.5 (t), 26.9 (t), 33.4 (q), 109.8 (s), 113.1 (s), 121.1 (s), 123.7 (2 ꢂ d),
128.1 (2 ꢂ d), 128.3 (d), 128.7 (2 ꢂ d), 129.4 (2 ꢂ d), 133.1 (d), 137.8
H-4); 13C NMR (DMSO-d6, 50 MHz, ppm):
d 20.3 (t), 33.4 (t), 38.2
(s), 139.4 (d), 140.5 (s), 141.5 (d), 145.2 (s), 147.2 (s), 156.5 (s). Anal.
(q), 55.1 (q), 109.7 (s), 112.6 (s), 114.5 (2 ꢂ d), 120.8 (s), 125.3 (2 ꢂ d),
128.1 (2 ꢂ d), 128.7 (2 ꢂ d), 130.9 (s), 133.1 (d), 139.0 (d), 140.5 (s),
141.5 (d), 145.1 (s), 147.4 (s), 156.5 (s), 159.0 (s). Anal. C24H21N3O4S
(447.51): C, 64.41; H, 4.73; N, 9.39. Found: C, 64.23; H, 4.99; N, 9.10.
C
23H19N3O3S (417.48): C, 66.17; H, 4.59; N, 10.07. Found: C, 66.31; H,
4.48; N, 9.97.
5.1.4.3. 7-Benzyl-1-methyl-3-(phenylsulfonyl)-1,5,6,7-tetrahydro-
2H-pyrazolo[3,4-h]quinolin-2-one (11d). This compound was ob-
tained from reaction of 10d with iodomethane. Gray solid; yield:
68%; m.p. 204e206 ꢀC; IR cmꢁ1: 1643 (CO); 1H NMR (CDCl3,
5.1.4.8. Ethyl 7-(4-chlorophenyl)-1-methyl-2-oxo-2,5,6,7-tetrahydro-
1H-pyrazolo[3,4-h]quinoline-3-carboxylate (11i). This compound
was obtained from reaction of 10i with iodomethane. Yellow solid;
yield: 70%; m.p. 228e229 ꢀC; IR cmꢁ1: 1679 (CO), 1656 (CO); 1H
200 MHz, ppm):
d
2.86e2.92 (m, 4H, 2 ꢂ CH2), 3.67 (s, 3H, CH3),
5.44 (s, 3H, CH2), 7.19e7.35 (m, 5H, Ar), 7.53e7.69 (m, 3H, Ar), 7.98
NMR (CDCl3, 200 MHz, ppm):
d
1.40 (t, J ¼ 7.1 Hz, 3H, CH3), 2.84 (t,
(d, J ¼ 7.0 Hz, 2H, H-20 and H-60), 8.12 (s, 1H, H-9), 8.21 (s, 1H, H-4);
J ¼ 7.4 Hz, 2H, CH2), 2.98 (t, J ¼ 7.4 Hz, 2H, CH2), 3.93 (s, 3H, CH3),
4.37 (q, J ¼ 7.1 Hz, 2H, CH2), 7.44 (d, J ¼ 8.9 Hz, 2H, H-300 and H-500),
7.53 (d, J ¼ 8.9 Hz, 2H, H-200 and H-600), 8.06 (s, 1H, H-9), 8.10 (s, 1H,
13C NMR (CDCl3, 50 MHz, ppm):
d 19.3 (t), 26.8 (t), 33.2 (q), 52.3 (t),
109.2 (s), 111.7 (s), 120.3 (s), 127.4 (2 ꢂ d), 127.7 (d), 128.1 (2 ꢂ d),
128.6 (2 ꢂ d), 128.7 (2 ꢂ d), 133.0 (d), 136.3 (s), 138.3 (d), 140.5 (s),
141.5 (d),145.7 (s), 147.6 (s), 156.4 (s). Anal. C24H21N3O3S (431.51): C,
66.80; H, 4.91; N, 9.74. Found: C, 66.65; H, 5.04; N, 9.55.
H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 14.4 (q), 21.3 (t), 27.9 (t),
34.2 (q), 61.0 (t),109.8 (s),114.2 (s),114.3 (s),125.1 (2 ꢂ d),129.7 (2 ꢂ
d), 134.5 (s), 136.6 (s), 138.7 (d), 144.1 (s), 144.3 (d), 145.1 (s), 159.9
(s), 165.9 (s). Anal calcd for C20H18ClN3O3 (383.83): C, 62.58; H,
4.73; N, 10.95. Found: C, 62.71; H, 4.55; N, 11.19.
5.1.4.4. 7-(2-Chlorophenyl)-1-methyl-3-(phenylsulfonyl)-1,5,6,7-
tetrahydro-2H-pyrazolo[3,4-h]quinolin-2-one (11e). This compound
was obtained from reaction of 10e with iodomethanecome. Yellow
solid; yield: 58%; m.p. 213e214 ꢀC; IR cmꢁ1: 1649 (CO); 1H NMR
5.1.4.9. Ethyl 7-(4-methoxyphenyl)-1-methyl-2-oxo-2,5,6,7-
tetrahydro-1H-pyrazolo[3,4-h]quinoline-3-carboxylate
This compound was obtained from reaction of 10j with iodo-
methane. Dark yellow solid; yield: 60%; m.p. 217e218 ꢀC; IR cmꢁ1
1652 (CO), 1684 (CO); 1H NMR (CDCl3, 200 MHz, ppm):
1.40 (t,
(11j).
(DMSO-d6, 200 MHz, ppm):
d
2.69 (t, J ¼ 7.6 Hz, 2H, CH2), 2.91 (t,
J ¼ 7.6 Hz, 2H, CH2), 3.75 (s, 3H, CH3), 7.54e7.80 (m, 7H, Ar), 7.98 (d,
:
J ¼ 6.6 Hz, 2H, H-20 and H-60), 8.25 (s, 1H, H-9), 8.37 (s, 1H, H-4); 13C
d
NMR (DMSO-d6, 50 MHz, ppm):
d
19.9 (t), 26.7 (t), 33.4 (q),109.5 (s),
J ¼ 7.1 Hz, 3H, CH3), 2.78e2.98 (m, 4H, 2 ꢂ CH2), 3.88 (s, 3H, CH3),
3.93 (s, 3H, CH3), 4.38 (q, J ¼ 7.1 Hz, 2H, CH2), 7.03 (d, J ¼ 9.0 Hz, 2H,
H-300 and H-500), 7.40 (d, J ¼ 9.0 Hz, 2H, H-200 and H-600), 8.03 (s, 1H,
112.1 (s), 121.1 (s), 128.1 (2 ꢂ d), 128.5 (d), 128.7 (2 ꢂ d), 129.6 (d),
130.3 (s), 130.4 (d), 131.7 (d), 133.1 (d), 135.1 (s), 139.3 (d), 140.5 (s),
141.7 (d), 147.2 (s), 147.3 (s), 156.4 (s). Anal. C23H18ClN3O3S (451.93):
C, 61.13; H, 4.01; N, 9.30. Found: C, 61.34; H, 3.80; N, 9.42.
H-9), 8.10 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 14.4 (q),
22.1 (t), 27.9 (t), 34.1 (q), 55.7 (q), 60.9 (t), 109.8 (s), 113.5 (s), 113.8
(s),114.6 (2 ꢂ d),125.4 (2 ꢂ d),131.2 (s),138.1 (d),144.1 (s),144.3 (d),
145.5 (s), 159.7 (s), 160.0 (s), 166.0 (s). Anal calcd for C21H21N3O4
(379.41): C, 66.48; H, 5.58; N, 11.08. Found: C, 66.59; H, 5.67; N,
10.95.
5.1.4.5. 7-(3-Chlorophenyl)-1-methyl-3-(phenylsulfonyl)-1,5,6,7-
tetrahydro-2H-pyrazolo[3,4-h]quinolin-2-one (11f). This compound
was obtained from reaction of 10f with iodomethane. Yellow solid;
yield: 56%; m.p. 245e247 ꢀC; IR cmꢁ1: 1651 (CO); 1H NMR (DMSO-
d6, 200 MHz, ppm):
d
2.90 (t, J ¼ 7.3 Hz, 2H, CH2), 3.04 (t, J ¼ 7.3 Hz,
5.1.4.10. 1-Benzyl-7-(4-chlorophenyl)-3-(phenylsulfonyl)-1,5,6,7-
tetrahydro-2H-pyrazolo[3,4-h]quinolin-2-one (11k). This compound
was obtained from reaction of 10g with benzyl bromide. Light
yellow solid; yield: 30%; m.p. 151e152 ꢀC; IR cmꢁ1: 1653 (CO); 1H
2H, CH2), 3.73 (s, 3H, CH3), 7.54e7.73 (m, 7H, Ar), 7.99 (d, J ¼ 8.3, 2H,
H-20 and H-60), 8.27 (s, 1H, H-9), 8.38 (s, 1H, H-4); 13C NMR (DMSO-
d6, 50 MHz, ppm):
d 20.4 (t), 26.9 (t), 33.5 (q), 110.0 (s), 113.4 (s),
121.3 (s), 122.4 (d), 123.5 (d), 128.2 (2 ꢂ d), 128.3 (d), 128.7 (2 ꢂ d),
131.1 (d), 133.1 (d), 133.7 (s), 139.0 (s), 139.7 (d), 140.4 (s), 141.6 (d),
145.6 (s), 147.0 (s), 156.5 (s). Anal. C23H18ClN3O3S (451.93): C, 61.13;
H, 4.01; N, 9.30. Found: C, 60.97; H, 4.33; N, 9.17.
NMR (CDCl3, 200 MHz, ppm):
2H, CH2), 7.23e7.56 (m, 12H, Ar), 7.87 (d, J ¼ 7.2 Hz, 2H, Ar), 8.08 (s,
1H, H-9), 8.23 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
21.3
(t), 27.3 (t), 68.6 (t), 119.4 (s), 119.8 (s), 120.4 (s), 124.6 (2 ꢂ d), 128.0
(d), 128.3 (2 ꢂ d), 128.4 (2 ꢂ d), 128.5 (2 ꢂ d), 128.6 (2 ꢂ d), 129.5
(2 ꢂ d), 132.9 (d), 133.7 (s), 136.3 (s), 137.4 (s), 137.6 (d), 138.0 (d),
140.8 (s), 142.6 (s), 152.9 (s), 158.5 (s). Anal calcd for C29H22ClN3O3S
(528.02): C, 65.97; H, 4.20; N, 7.96. Found: C, 65.81; H, 4.02; N, 8.17.
d
2.99e3.11 (m, 4H, 2 ꢂ CH2), 5.44 (s,
d
5.1.4.6. 7-(4-Chlorophenyl)-1-methyl-3-(phenylsulfonyl)-1,5,6,7-
tetrahydro-2H-pyrazolo[3,4-h]quinolin-2-one (11g). This compound
was obtained from reaction of 10g with iodomethane. Pale yellow
solid; yield: 53%; m.p. 221e222 ꢀC; IR cmꢁ1: 1653 (CO); 1H NMR
(CDCl3, 200 MHz, ppm):
d
2.89e3.02 (m, 4H, CH2), 3.73 (s, 3H, CH3),
5.1.4.11. 1-Benzyl-7-(4-methoxyphenyl)-3-(phenylsulfonyl)-1,5,6,7-
tetrahydro-2H-pyrazolo[3,4-h]quinolin-2-one (11l). This compound
was obtained from reaction of 10h with benzyl bromide. Brown
solid; yield: 28%; m.p. 134e135 ꢀC; IR cmꢁ1: 1657 (CO); 1H NMR
7.54e7.70 (m, 7H, Ar), 7.98 (dd, J ¼ 8.0, 1.7 Hz, 2H, H-20 and H-60),
8.27 (s, 1H, H-9), 8.37 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d
20.4 (t), 26.9 (t), 33.4 (q),109.9 (s), 113.3 (s), 121.2 (s), 125.4 (2 ꢂ d),
128.2 (2 ꢂ d), 128.7 (2 ꢂ d), 129.5 (2 ꢂ d), 132.8 (s), 133.1 (d), 136.7
(CDCl3, 200 MHz, ppm):
d
2.89e3.05 (m, 4H, 2 ꢂ CH2), 3.86 (s, 3H,
(s), 139.6 (d), 140.4 (s), 141.6 (d), 145.4 (s), 147.1 (s), 156.5 (s). Anal.
CH3), 5.53 (s, 2H, CH2), 6.98e7.04 (m, 4H, Ar), 7.26e7.33 (m, 4H, Ar),
C
23H18ClN3O3S (451.93): C, 61.13; H, 4.01; N, 9.30. Found: C, 61.22;
7.46e7.75 (m, 5H, Ar and H-9), 8.15 (d, J ¼ 6.7 Hz, 2H, H-20 and H-
H, 3.96; N, 9.45.
60), 8.38 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 21.0 (t), 28.3
(t), 55.7 (q), 71.8 (t), 109.9 (s), 112.5 (s), 114.6 (2 ꢂ d), 123.5 (s), 125.3
(2 ꢂ d),127.5 (2 ꢂ d),128.6 (2 ꢂ d),128.9 (2 ꢂ d),129.2 (2 ꢂ d),130.9
(d), 131.0 (s), 133.1 (d), 135.0 (s), 138.3 (d), 140.3 (s), 142.5 (d), 144.5
(s), 147.5 (s), 157.5 (s), 159.7 (s). Anal calcd for C30H25N3O4S
5.1.4.7. 7-(4-Methoxyphenyl)-1-methyl-3-(phenylsulfonyl)-1,5,6,7-
tetrahydro-2H-pyrazolo[3,4-h]quinolin-2-one (11h). This compound
was obtained from reaction of 10h with iodomethane. Light green

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347
(523.60): C, 68.82; H, 4.81; N, 8.03. Found: C, 68.76; H, 4.99; N, 8.16.
5.1.4.17. 7-(4-Chlorophenyl)-2-methoxy-3-(phenylsulfonyl)-6,7-
dihydro-5H-pyrazolo[3,4-h]quinoline (12g). This compound was
obtained from reaction of 10g with iodomethane. White solid;
yield: 30%; m.p. 257e259 ꢀC; 1H NMR (CDCl3, 200 MHz, ppm):
5.1.4.12. 2-Methoxy-7-methyl-3-(phenylsulfonyl)-6,7-dihydro-5H-
pyrazolo[3,4-h]quinoline (12b). This compound was obtained from
reaction of 10b with iodomethane. White solid; yield: 35%; m.p.
d
2.98e3.09 (m, 4H, 2 ꢂ CH2), 3.90 (s, 3H, CH3), 7.58e7.65 (m, 7H,
Ar), 7.94 (dd, J ¼ 7.6,1.7 Hz, 2H, H-20 and H-60), 8.13 (s, 1H, H-9), 8.25
199e201 ꢀC; 1H NMR (DMSO-d6, 200 MHz, ppm):
d 2.80e2.92 (m,
(s, 1H, H-4); 13C NMR (DMSO-d6, 50 MHz, ppm):
d 20.4 (t), 26.3 (t),
4H, 2 ꢂ CH2), 3.66 (s, 3H, CH3), 3.83 (s, 3H, CH3), 7.52e7.69 (m, 3H,
H-30, H-40 and H-50), 7.96 (dd, J ¼ 7.3, 1.4 Hz, 2H, H-20 and H-60), 8.04
(s, 1H, H-9), 8.21 (s, 1H, H-4); 13C NMR (DMSO-d6, 50 MHz, ppm):
53.8 (q), 118.7 (s), 118.8 (s), 121.3 (s), 124.8 (2 ꢂ d), 127.8 (2 ꢂ d),
129.1 (2 ꢂ d), 129.4 (2 ꢂ d), 133.1 (s), 133.6 (d), 137.1 (d), 137.4 (s),
138.0 (d), 140.6 (s), 143.8 (s), 152.6 (s), 158.3 (s). Anal.
d
19.0 (t), 26.7 (t), 33.2 (q), 36.2 (q),109.1 (s),111.3 (s),120.1 (s),128.1
C23H18ClN3O3S (451.93): C, 61.13; H, 4.01; N, 9.30. Found: C, 60.88;
(2 ꢂ d), 128.6 (2 ꢂ d), 133.1 (d), 137.7 (d), 140.6 (s), 141.5 (d), 145.8
(s), 147.8 (s), 156.4 (s). Anal. C18H17N3O3S (355.41): C, 60.83; H, 4.82;
N, 11.82. Found: C, 60.95; H, 4.60; N, 12.10.
H, 4.27; N, 9.13.
5.1.4.18. 2-Methoxy-7-(4-methoxyphenyl)-3-(phenylsulfonyl)-6,7-
dihydro-5H-pyrazolo[3,4-h]quinoline (12h). This compound was
obtained from reaction of 10h with iodomethane. White solid;
yield: 36%; m.p. 186e187 ꢀC; 1H NMR (CDCl3, 200 MHz, ppm):
5.1.4.13. 2-Methoxy-7-phenyl-3-(phenylsulfonyl)-6,7-dihydro-5H-
pyrazolo[3,4-h]quinoline (12c). This compound was obtained from
reaction of 10c with iodomethanecome. White solid; yield: 30%;
d
2.98e3.03 (m, 4H, 2 ꢂ CH2), 3.87 (s, 3H, CH3), 3.98 (s, 3H, CH3),
m.p. 197e199 ꢀC; 1H NMR (DMSO-d6, 200 MHz, ppm):
d 2.98e3.11
7.00 (d, J ¼ 8.8 Hz, 2H, H-300 and H-500), 7.40 (d, J ¼ 8.8 Hz, 2H, H-200
and H-600), 7.50e7.55 (m, 3H, H-30, H-40 and H-50), 7.98 (dd, J ¼ 7.6,
1.7 Hz, 2H, H-20 and H-60), 8.06 (s, 1H, H-9), 8.19 (s, 1H, H-4); 13C
(m, 4H, 2 ꢂ CH2), 3.90 (s, 3H, CH3), 7.46e7.72 (m, 8H, Ar), 7.96 (dd,
J ¼ 7.4, 1.6 Hz, 2H, H-20 and H-60), 8.11 (s, 1H, H-9), 8.25 (s, 1H, H-4);
13C NMR (DMSO-d6, 50 MHz, ppm):
d 20.5 (t), 26.3 (t), 53.7 (q),118.5
NMR (CDCl3, 50 MHz, ppm):
d 21.1 (t), 27.4 (t), 54.0 (q), 55.6 (q),
(s),118.6 (s),121.3 (s), 123.1 (2 ꢂ d),127.8 (2 ꢂ d),129.1 (2 ꢂ d), 129.3
(d), 129.4 (2 ꢂ d), 133.5 (d), 136.8 (d), 137.9 (d), 138.5 (s), 140.5 (s),
145.7 (s), 147.6 (s), 156.4 (s). Anal. C23H19N3O3S (417.48): C, 66.17; H,
4.59; N, 10.07. Found: C, 66.02; H, 4.68; N, 10.23.
114.5 (2 ꢂ d), 119.0 (s), 119.1 (s), 120.3 (s), 125.0 (2 ꢂ d), 128.4 (2 ꢂ
d), 128.6 (2 ꢂ d), 132.0 (s), 133.1 (d), 136.9 (d), 137.9 (d), 141.0 (s),
142.6 (s), 153.4 (s), 130.3 (s), 159.2 (s). Anal. C24H21N3O4S (447.51):
C, 64.41; H, 4.73; N, 9.39. Found: C, 64.54; H, 4.60; N, 9.47.
5.1.4.19. Ethyl 7-(4-chlorophenyl)-2-methoxy-6,7-dihydro-5H-pyr-
azolo[3,4-h]quinoline-3-carboxylate (12i). This compound was ob-
tained from reaction of 10i with iodomethane. White solid; yield:
22%; m.p. 156e157 ꢀC; IR cmꢁ1: 1710 (CO); 1H NMR (CDCl3,
5.1.4.14. 7-Benzyl-2-methoxy-3-(phenylsulfonyl)-6,7-dihydro-5H-
pyrazolo[3,4-h]quinoline (12d). This compound was obtained from
reaction of 10d with iodomethane. White solid; yield: 30%; m.p.
123e124 ꢀC; 1H NMR (CDCl3, 200 MHz, ppm):
d 2.90e3.10 (m, 4H,
200 MHz, ppm):
d
1.39 (t, J ¼ 7.1 Hz, 3H, CH3), 2.95e3.10 (m, 4H, 2 ꢂ
2 ꢂ CH2), 3.86 (s, 3H, CH3), 5.38 (s, 3H, CH2), 7.17e7.34 (m, 5H, Ar),
7.57e7.70 (m, 3H, Ar), 7.87e7.93 (m, 3H, H-20 and H-60, H-9), 8.16 (s,
CH2), 4.12 (s, 3H, CH3), 4.36 (q, J ¼ 7.1 Hz, 2H, CH2), 7.42e7.50 (m,
4H, Ar), 8.02 (s, 1H, H-9), 8.15 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz,
1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 19.1 (t), 26.1 (t), 52.2 (t),
ppm): d 14.4 (q), 21.5 (t), 27.3 (t), 54.1 (q), 60.9 (t),109.8 (s), 119.7 (s),
53.6 (q), 117.2 (s), 118.0 (s), 120.8 (s), 127.2 (2 ꢂ d), 127.6 (d), 127.8
(2 ꢂ d), 128.6 (2 ꢂ d), 129.0 (2 ꢂ d), 133.5 (d), 135.4 (d), 136.8 (s),
137.9 (d), 140.7 (s), 144.1 (s), 153.1 (s), 158.3 (s). Anal. C24H21N3O3S
(431.51): C, 66.80; H, 4.91; N, 9.74. Found: C, 66.99; H, 4.75; N, 9.88.
120.3 (s), 124.6 (2 ꢂ d), 129.5 (2 ꢂ d), 133.5 (s), 137.5 (d), 137.6 (s),
140.2 (d), 142.3 (s), 151.3 (s), 162.0 (s), 165.1 (s). Anal calcd for
C20H18ClN3O3 (383.83): C, 62.58; H, 4.73; N, 10.95. Found: C, 62.37;
H, 4.65; N, 11.07.
5.1.4.15. 7-(2-Chlorophenyl)-2-methoxy-3-(phenylsulfonyl)-6,7-
dihydro-5H-pyrazolo[3,4-h]quinoline (12e). This compound was
obtained from reaction of 10e with iodomethane. White solid;
yield: 33%; m.p. 187e188 ꢀC; 1H NMR (DMSO-d6, 200 MHz, ppm):
5.1.4.20. Ethyl
2-methoxy-7-(4-methoxyphenyl)-6,7-dihydro-5H-
pyrazolo[3,4-h]quinoline-3-carboxylate (12j). This compound was
obtained from reaction of 10j with iodomethane. Pale brown solid;
yield: 35%; m.p. 154e155 ꢀC; IR cmꢁ1: 1711 (CO); 1H NMR (CDCl3,
d
2.77 (t, J ¼ 7.8 Hz, 2H, CH2), 3.10 (t, J ¼ 7.8 Hz, 2H, CH2), 3.91 (s, 3H,
200 MHz, ppm):
d
1.39 (t, J ¼ 7.1 Hz, 3H, CH3), 2.91e3.09 (m, 4H, 2 ꢂ
CH3), 7.56e7.77 (m, 7H, Ar), 7.92e7.97 (m, 2H, H-20 and H-60), 8.11
CH2), 3.87 (s, 3H, CH3), 4.12 (s, 3H, CH3), 4.36 (q, J ¼ 7.1 Hz, 2H, CH2),
7.01 (d, J ¼ 8.9 Hz, 2H, H-300 and H-500), 7.41 (d, J ¼ 8.9 Hz, 2H, H-200
and H-600), 8.01 (s, 1H, H-9), 8.12 (s, 1H, H-4); 13C NMR (CDCl3,
(s, 1H, H-9), 8.23 (s, 1H, H-4); 13C NMR (DMSO-d6, 50 MHz, ppm):
d
19.6 (t), 26.1 (t), 53.8 (q), 117.6 (s), 118.6 (s), 121.1 (s), 122.2 (s),
127.8 (2 ꢂ d), 128.4 (d), 129.1 (2 ꢂ d), 129.5 (d), 130.4 (d), 131.4 (d),
133.5 (d), 135.6 (s), 136.7 (d), 138.1 (d), 140.6 (s), 145.6 (s), 152.8 (s),
158.4 (s). Anal. C23H18ClN3O3S (451.93): C, 61.13; H, 4.01; N, 9.30.
Found: C, 61.25; H, 3.83; N, 9.05.
50 MHz, ppm): d 14.4 (q), 22.3 (t), 27.3 (t), 54.1 (q), 55.6 (q), 60.8 (t),
109.4 (s), 114.4 (2 ꢂ d), 119.6 (s), 119.8 (s), 125.0 (2 ꢂ d), 130.9 (s),
132.2 (s), 136.8 (d), 140.2 (d), 142.4 (s), 159.1 (s), 162.0 (s), 165.1 (s).
Anal calcd for C21H21N3O4 (379.41): C, 66.48; H, 5.58; N, 11.08.
Found: C, 66.37; H, 5.72; N, 10.88.
5.1.4.16. 7-(3-Chlorophenyl)-2-methoxy-3-(phenylsulfonyl)-6,7-
dihydro-5H-pyrazolo[3,4-h]quinoline (12f). This compound was
obtained from reaction of 10f with iodomethane. Yellow solid;
yield: 35%; m.p. 220e222 ꢀC; 1H NMR (DMSO-d6, 200 MHz, ppm):
5.1.4.21. 2-(Benzyloxy)-7-(4-chlorophenyl)-3-(phenylsulfonyl)-6,7-
dihydro-5H-pyrazolo[3,4-h]quinoline (12k). This compound was
obtained from reaction of 10g with benzyl bromide. White solid;
yield: 64%; m.p. 216e217 ꢀC; 1H NMR (CDCl3, 200 MHz, ppm):
d
3.12e3.15 (m, 4H, 2 ꢂ CH2), 3.90 (s, 3H, CH3), 7.54e7.72 (m, 7H,
Ar), 7.93e7.97 (m, 2H, H-20 and H-60), 8.14 (s, 1H, H-9), 8.25 (s, 1H,
H-4); 13C NMR (DMSO-d6, 50 MHz, ppm):
20.4 (t), 26.2 (t), 53.8
d
2.99e3.11 (m, 4H, 2 ꢂ CH2), 5.44 (s, 2H, CH2), 7.23e7.56 (m, 12H,
d
Ar), 7.87 (d, J ¼ 7.2 Hz, 2H, H-20 and H-60), 8.08 (s, 1H, H-9), 8.23 (s,
(q), 118.8 (s), 119.0 (s), 121.4 (s), 121.8 (d), 122.9 (d), 127.6 (d), 127.8
(2 ꢂ d), 129.1 (2 ꢂ d), 131.1 (d), 133.6 (d), 133.7 (s), 137.3 (d), 138.0
(s), 139.7 (d), 140.6 (s), 144.0 (s), 152.6 (s), 158.3 (s). Anal.
1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 21.3 (t), 27.3 (t), 68.6 (t),
119.4 (s), 119.8 (s), 120.4 (s), 124.6 (2 ꢂ d), 128.0 (d), 128.3 (2 ꢂ d),
128.4 (2 ꢂ d), 128.5 (2 ꢂ d), 128.6 (2 ꢂ d), 129.5 (2 ꢂ d), 132.9 (d),
133.7 (s), 136.3 (s), 137.4 (s), 137.6 (d), 138.0 (d), 140.8 (s), 142.6 (s),
152.9 (s), 158.5 (s). Anal calcd for C29H22ClN3O3S (528.02): C, 65.97;
C
23H18ClN3O3S (451.93): C, 61.13; H, 4.01; N, 9.30. Found: C, 61.01;
H, 3.84; N, 9.55.

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348
H, 4.20; N, 7.96. Found: C, 66.08; H, 4.02; N, 7.85.
was obtained from reaction of 11h. Pale yellow solid; yield: 95%;
m.p. 232e233 ꢀC; IR cmꢁ1: 1657 (CO); 1H NMR (CDCl3, 200 MHz,
5.1.4.22. 2-(Benzyloxy)-7-(4-methoxyphenyl)-3-(phenylsulfonyl)-
6,7-dihydro-5H-pyrazolo[3,4-h]quinoline (12l). This compound was
obtained from reaction of 10h with benzyl bromide. Pale green
solid; yield: 55%; m.p.188e189 ꢀC; 1H NMR (CDCl3, 200 MHz, ppm):
ppm):
d
3.91 (s, 3H, CH3), 4.10 (s, 3H, CH3), 7.44 (d, J ¼ 8.9 Hz, 2H, H-
300 and H-500), 7.45e7.68 (m, 7H, Ar), 8.18 (dd, J ¼ 8.1, 1.6 Hz, 2H, H-20
and H-60), 8.60 (s, 1H, H-9), 8.90 (s, 1H, H-4); 13C NMR (CDCl3,
50 MHz, ppm): d 33.7 (q), 55.7 (q),107.2 (d), 112.0 (s), 113.6 (s), 114.9
d
3.01e3.05 (m, 4H, 2 ꢂ CH2), 3.87 (s, 3H, CH3), 5.44 (s, 2H, CH2),
(2 ꢂ d), 125.8 (2 ꢂ d), 127.2 (s), 128.7 (2 ꢂ d), 129.1 (2 ꢂ d), 129.9 (d),
131.6 (s), 133.5 (d), 135.9 (d), 139.4 (s), 139.7 (s), 142.4 (s), 143.6 (d),
157.4 (s), 159.7 (s). Anal calcd for C24H19N3O4S (445.49): C, 64.71; H,
4.30; N, 9.43. Found: C, 64.56; H, 4.24; N, 9.60.
7.01 (d, J ¼ 9.0 Hz, 2H, H-300 and H-500), 7.24e7.55 (m, 10H, Ar), 7.87
(d, J ¼ 7.2 Hz, 2H, H-20 and H-60), 8.06 (s, 1H, H-9), 8.21 (s, 1H, H-4);
13C NMR (CDCl3, 50 MHz, ppm):
d 22.1 (t), 27.4 (t), 55.6 (q), 68.5 (t),
114.5 (2 ꢂ d), 119.0 (s), 119.1 (s), 120.5 (s), 125.0 (2 ꢂ d), 127.9 (d),
128.3 (2 ꢂ d), 128.4 (2 ꢂ d), 128.5 (2 ꢂ d), 128.6 (2 ꢂ d), 132.1 (s),
132.9 (d), 136.4 (s), 137.0 (d), 138.0 (d), 140.9 (s), 142.6 (s), 153.3 (s),
158.4 (s), 159.2 (s). Anal calcd for C30H25N3O4S (523.60): C, 68.82; H,
4.81; N, 8.03. Found: C, 68.94; H, 4.69; N, 8.32.
5.1.5.5. 7-(4-Chlorophenyl)- 2-methoxy-3-(phenylsulfonyl)-7H-pyr-
azolo[3,4-h]quinoline (13e). This compound was obtained from
reaction of 12g. White solid; yield: 65%; m.p. 245e246 ꢀC; 1H NMR
(CDCl3, 200 MHz, ppm):
d 4.14 (s, 3H, CH3), 7.48e7.84 (m, 9H, Ar),
8.07 (d, J ¼ 6.8 Hz, 2H, H-20 and H-60), 8.71 (s, 1H, H-9), 9.00 (s, 1H,
5.1.5. General procedure for the preparation of 1,7-dihydro-2H-
pyrazolo[3,4-h]quinolin-2-ones (13a-f)
H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 55.3 (q), 110.2 (d), 119.1 (s),
121.0 (s), 122.9 (s), 124.6 (2 ꢂ d), 128.5 (d), 128.7 (2 ꢂ d), 128.8 (2 ꢂ
d), 129.8 (2 ꢂ d), 133.4 (s), 133.5 (d), 136.1 (d), 138.0 (s), 140.4 (s),
140.6 (s), 141.0 (d), 145.0 (s), 158.4 (s). Anal calcd for C23H16ClN3O3S
(449.91): C, 61.40; H, 3.58; N, 9.34. Found: C, 61.58; H, 3.32; N, 9.61.
To a solution of the proper 1,5,6,7-tetrahydro-2H-pyrazolo[3,4-
h]quinoline 10g,h, 11g,h and 12g,h (30 mmol) in anhydrous
dioxane (100 mL), DDQ was added (27.24 g, 120 mmol). The reac-
tion mixture was heated under reflux for 24 h. Then the solvent was
removed under reduced pressure and the crude was purified by
chromatography with a SEPACORE BÜCHI (DCM/AcOEt 95:5).
5.1.5.6. 2-Methoxy-7-(4-methoxyphenyl)-3-(phenylsulfonyl)-7H-
pyrazolo[3,4-h]quinoline (13f). This compound was obtained from
reaction of 12h. Pale yellow solid; yield: 86%; m.p. 225e226 ꢀC; 1H
5.1.5.1. 7-(4-Chlorophenyl)-3-(phenylsulfonyl)-1,7-dihydro-2H-pyr-
azolo[3,4-h]quinolin-2-one (13a). This compound was obtained
from reaction of 10g. Brown solid; yield: 50%; m.p. 309e310 ꢀC; IR
cmꢁ1: 3240 (NH), 1653 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm):
NMR (CDCl3, 200 MHz, ppm): d 3.91 (s, 3H, CH3), 4.14 (s, 3H, CH3),
7.09 (d, J ¼ 9.0 Hz, 2H, H-300 and H-500), 7.52e7.67 (m, 6H, Ar), 7.77
(d, J ¼ 9.0 Hz, 1H, Ar), 8.04e8.09 (m, 2H, H-20 and H-60), 8.68 (s, 1H,
H-9), 9.00 (s, 1H, H-4); 13C NMR (CDCl3, 50 MHz, ppm):
d 54.3 (q),
d
7.63e7.84 (m, 8H, Ar), 8.02e8.06 (m, 3H, Ar), 8.97 (s,1H, H-9), 9.09
55.7 (q), 110.5 (d), 114.8 (2 ꢂ d), 118.9 (s), 121.4 (s), 122.4 (s), 125.2
(2 ꢂ d), 128.0 (d), 128.7 (2 ꢂ d), 128.8 (2 ꢂ d), 132.5 (s), 133.4 (d),
135.3 (d), 140.5 (s), 140.9 (s), 141.0 (d), 145.1 (s), 158.3 (s), 159.2 (s).
Anal calcd for C24H19N3O4S (445.49): C, 64.71; H, 4.30; N, 9.43.
Found: C, 64.95; H, 4.08; N, 9.57.
(s, 1H, H-4), 13.09 (s, 1H, NH); 13C NMR (DMSO-d6, 50 MHz, ppm):
d
101.4 (s), 106.8 (d), 113.8 (s), 124.8 (2 ꢂ d), 128.2 (2 ꢂ d), 128.9 (2 ꢂ
d), 129.2 (s), 129.7 (2 ꢂ d), 132.0 (s), 133.6 (d), 135.7 (d), 137.6 (s),
139.8 (s), 140.6 (s), 145.1 (d), 150.8 (s), 151.8 (d), 157.1 (s). Anal calcd
for C22H14ClN3O3S (435.88): C, 60.62; H, 3.24; N, 9.64. Found: C,
60.87; H, 3.11; N, 9.88.
5.2. Biology
5.1.5.2. 7-(4-Methoxyphenyl)-3-(phenylsulfonyl)-1,7-dihydro-2H-
pyrazolo[3,4-h]quinolin-2-one (13b). This compound was obtained
from reaction of 10h. Dark yellow solid; yield: 60%; m.p.
373e374 ꢀC; IR cmꢁ1: 3248 (NH), 1649 (CO); 1H NMR (DMSO-d6,
5.2.1. Reagents
If not otherwise indicated, all chemicals were purchased from
SigmaeAldrich (Milan, Italy).
200 MHz, ppm):
d
3.86 (s, 3H, CH3), 7.17 (d, J ¼ 9.0 Hz, 2H, H-300 and
5.2.2. Irradiation procedure
H-500), 7.50 (d, J ¼ 9.0 Hz, 2H, H-200 and H-600), 7.58e7.72 (m, 4H, Ar),
HPW 125 Philips lamps (Wood's lamps), emitting mainly at
7.95e8.06 (m, 3H, H-30, H-40 and H-50), 8.91 (s, 1H, H-9), 9.07 (s, 1H,
l
¼ 365 nm, were used for irradiation experiments. The spectral
H-4), 13.07 (s, 1H, NH); 13C NMR (DMSO-d6, 50 MHz, ppm):
d
55.5
irradiance of the source was 4.0 mW/cm2 as measured by a radi-
ometer (ColeeParmer Instrument Company, Niles, IL, USA) equip-
ped with a 365-CX sensor.
(q),106.7 (d),110.8 (s), 112.2 (s), 114.8 (2 ꢂ d),125.0 (2 ꢂ d),127.2 (s),
128.2 (2 ꢂ d), 128.9 (2 ꢂ d), 129.4 (d), 131.7 (s), 133.6 (d), 135.0 (d),
136.8 (s), 139.9 (s), 140.8 (s), 145.2 (d), 157.1 (s), 158.7 (s). Anal calcd
for C23H17N3O4S (431.46): C, 64.03; H, 3.97; N, 9.74. Found: C, 64.22;
H, 4.08; N, 9.63.
5.2.2.1. Spectrophotometric
measurements. UV/Vis
absorption
spectra were recorded on a PerkinElmer Lambda 15 spectropho-
tometer, and emission spectra were recorded on a PerkinElmer LS-
50B fluorimeter.
5.1.5.3. 7-(4-Chlorophenyl)-1-methyl-3-(phenylsulfonyl)-1,7-
dihydro-2H-pyrazolo[3,4-h]quinolin-2-one (13c). This compound
was obtained from reaction of 11g. White solid; yield: 93%; m.p.
264e265 ꢀC; IR cmꢁ1: 1731 (CO); 1H NMR (CDCl3, 200 MHz, ppm):
5.2.3. Cell lines
Jurkat (human T-cell leukemia) and HL-60 (human promyelo-
cytic leukemia) cells were cultured in RPMI-1640 medium; LoVo
(human intestinal adenocarcinoma) cells were grown in Ham's F-
12 medium, and MCF-7 (human breast adenocarcinoma) and A-549
(human lung carcinoma) cells were cultured in DMEM. All media
were supplemented with penicillin G (115 U/mL), streptomycin
(115 mg/mL), and 10% fetal bovine serum (Invitrogen, Milan, Italy).
d
4.10 (s, 3H, CH3), 7.50e7.73 (m, 9H, Ar), 8.19 (dd, J ¼ 8.1, 1.5 Hz, 2H,
H-20 and H-60), 8.63 (s, 1H, H-9), 8.90 (s, 1H, H-4); 13C NMR (CDCl3,
50 MHz, ppm):
d
33.7 (q), 106.8 (d), 112.3 (s), 114.0 (s), 125.3 (2 ꢂ d),
127.7 (s), 128.7 (2 ꢂ d), 129.2 (2 ꢂ d), 130.0 (2 ꢂ d), 130.4 (d), 133.5
(d), 134.2 (s), 136.5 (d), 137.2 (s), 139.3 (s), 139.6 (s), 142.1 (s), 143.5
(d), 157.3 (s). Anal calcd for C23H16ClN3O3S (449.91): C, 61.40; H,
3.58; N, 9.34. Found: C, 61.27; H, 3.45; N, 9.49.
5.2.3.1. Cellular
antiproliferative
tests
and
phototoxicity.
5.1.5.4. 7-(4-Methoxyphenyl)-1-methyl-3-(phenylsulfonyl)-1,7-
dihydro-2H-pyrazolo[3,4-h]quinolin-2-one (13d). This compound
Individual wells of a 96-well tissue culture microtiter plate were
inoculated with 100 mL complete medium containing 5 ꢂ 103 cells.

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349
Plates were harvested at 37 ꢀC in a humidified incubator (5% CO2)
for 24 h prior to cell viability experiments. Drugs were dissolved in
DMSO and were then diluted with Hank's balanced salt solution
(HBSS, pH 7.2) for phototoxicity experiments or in the appropriate
complete medium for the cytotoxicity assays. In cytotoxicity tests
5.2.7. Assessment of mitochondrial changes
The mitochondrial membrane potential was measured with the
lipophilic cation 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimi-
dazolcarbocyanine (JC-1, Molecular Probes, USA), as described
elsewhere [63]. Briefly, 24 h post-irradiation, Jurkat cells were
collected by centrifugation and resuspended in HBSS containing JC-
after medium removal, 100
tions were added to each well and incubated at 37 ꢀC for 72 h. In
phototoxicity experiments after medium removal, 100 L drug so-
mL drug solution at various concentra-
1 at 1
m
M. Cells were then incubated at 37 ꢀC for 10 min, and
m
fluorescence was examined at 530 and 590 nm. The production of
ROS was measured by flow cytometry using the fluorescence
probes HE and DCFDA (both from Molecular Probes, USA); 24 h
after irradiation, Jurkat cells were incubated in HBSS containing HE
lution were added into each well and incubated at 37 ꢀC for 30 min
and then irradiated (1.25, 2.5 and 3.75 J/cm2). After irradiation, the
drug solution was replaced by cell culture medium, and plates were
incubated for 72 h. In both cases, cell viability was assayed by the
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
(MTT) test [76]. Analogous phototoxicity experiments were carried
out in the presence of different scavengers.
or DCFDA at concentrations of 2.5 and 5 mM, respectively. Cells were
then incubated at 37 ꢀC for 30 min, centrifuged, and resuspended in
HBSS. The fluorescence was recorded directly with the flow cy-
tometer by using excitation at
and 530 nm for HE and DCFDA, respectively [66].
l
¼ 488 nm and emission at
l
¼ 585
5.2.3.2. Colony formation assay. 10 ꢂ 103 A-549 cells were plated in
dishes. After 24 h, cells were irradiated (2.5 J/cm2) in the presence
of the tested compounds at different concentrations. After irradi-
ation, medium was replaced and cells were cultured for 1e2 weeks
until well-defined colonies had formed (replacing culture medium
every 2e3 days). Cells were stained with crystal violet as described
in Ref. [6] and colonies of ꢃ50 cells were then counted visually.
Data represented the average of the three values and error bars
represented the SEM.
5.2.8. Lysosomal stability assessment
Jurkat cells were assessed for lysosomal stability using the ac-
ridine orange (AO) uptake method [68]. After 24 h from the irra-
diation in the presence of test compounds, cells were stained with
AO at 5 mg/mL at 37 ꢀC for 15 min, washed, and then analyzed by
flow cytometry using excitation at
emission at
¼ 546 nm.
l
¼ 488 nm and detecting the
l
5.2.9. DNA frank strand breaks
5.2.4. Generation of active oxygen species
pBR322 DNA samples (100 ng) dissolved in phosphate buffer
10 mM pH 7.2 were irradiated (UV-A dose: 7.5 J/cm2) in the pres-
ence of the tested compound at [Compound]/[DNA] ratio ¼ 3/1.
After irradiation, two aliquots of sample were incubated at 37 ꢀC
with Fpg (formamydo pyrimidin glycosilase) and Endo III (Endo-
nuclease III), respectively. The samples were loaded on 1% agarose
gel and the run was carried out in TAE buffer (0.04 M Triseacetate,
1 mM EDTA) at 70 V for 4 h. The quantification of DNA bands was
carried out as described previously [71].
Samples containing the test compounds (2.2 ꢂ 10ꢁ5 M), RNO
(N,N-dimethyl-p-nitrosoaniline) (4
ꢂ
10ꢁ5 M) and imidazole
(4 ꢂ 10ꢁ5 M) in phosphate buffer (pH 7.3) were irradiated with
increasing UV-A doses and their absorbance at 440 nm was then
measured for the determination of singlet oxygen. Data were
expressed as percentage of RNO bleaching. Samples containing the
tested compounds (10ꢁ5 M) and nitroblue tetrazolium
(1.6 ꢂ 10ꢁ4 M) in carbonate buffer (pH 10) were irradiated with
increasing UV-A doses. Their absorbance at 560 nm was measured
to monitor superoxide anion formation [77].
5.2.10. Photoreaction of isolated proteins
Solutions of bovine serum albumin (BSA) (0.5 mg/mL) in phos-
phate buffer 10 mM were irradiated in the presence of the test
compounds (10 mM) for various time in a quartz cuvette. Solutions
5.2.5. Externalization of phosphatidylserine
Surface exposure of phosphatidylserine (PS) by apoptotic cells
was measured with a BD FACS Calibur flow cytometer by adding
Annexin VeFITC to cells according to the manufacturer's in-
structions (Annexin V Fluos, Roche Diagnostics, Indianapolis, IN,
USA). Cells were simultaneously stained with PI. Excitation was set
of ribonuclease A (RNAseA), 0.5 mg/mL in phosphate buffer 10 mM
were irradiated in the presence of the test compounds for various
time in a quartz cuvette. At each time, the tryptophan (Trp) or
tyrosine (Tyr) content was followed by monitoring the character-
istic Trp or Tyr fluorescence. In further experiments, the degree of
protein oxidation was monitored spectrophotometrically by
derivatization with 2,4-dinitrophenylhydrazine (DNPH) as
described in [77].
at
[61].
l
¼ 488 nm, and the emission filters were at ¼ 525 and 585 nm
l
5.2.6. Western blot analysis
Jurkat cells were irradiated (2.5 J/cm2) in the presence of the
tested compounds (1 mM), after 24 h, were centrifuged, and washed
5.2.11. Lipid peroxidation
two times with ice cold phosphate-buffered saline (PBS). The pellet
was resuspended in lysis buffer [35]. After the cells were lysed on
ice for 20 min, lysates were centrifuged at 10,000 g at 4 ꢀC for
20 min. The protein concentration in the supernatant was deter-
For these experiments, Jurkat cells were seeded at the concen-
tration of 50.000 cells/mL in 24-well tissue culture microtiter plates
and then were irradiated in HBSS in the presence of the test com-
pounds. Lipid peroxidation was assessed following the method of
Morliere et al. [74]. A standard curve of 1,1,3,3-tetraethoxypropane
was used to quantify the amount of produced malonaldehyde. Data
were expressed in terms of nanomoles of TBARS normalized to the
total protein content in an aliquot of the cell extract.
mined. Equal amounts of protein (20
mg) were resolved using
8e16% gradient polyacrylamide precast gels (Thermo Scientific)
and transferred on a nitrocellulose Hybond-p membrane (GE
Healthcare). Membranes were blocked with 5% skim milk powder
in Tween-PBS for at least 2 h. Membranes were incubated with
primary antibodies against caspase-3 and
b-actin (all rabbit,
1:1000, Cell Signaling), for 2 h. Membranes were next incubated
with peroxidase-labeled goat antirabbit IgG (1:3000, Cell Signaling)
for 60 min. All membranes were visualized using ECL Advance (GE
Healthcare) and exposed to Hyperfilm MP (GE Healthcare).
Acknowledgments
This work was financially supported by Ministero dell'Istruzione
ꢀ
dell'Universita e della Ricerca.

ꢀ
350
V. Spano et al. / European Journal of Medicinal Chemistry 102 (2015) 334e351
them (and conventional quinolones) for prevention and/or treatment of in-
fectious diseases, PCT Int. Appl. WO 2014171527, Chem. Abstr. 161 (2014)
647104.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.08.003.
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