Novel conformationally constrained pyrazole derivatives as potential anti-cancer agents

Article abstract of DOI:10.1515/znb-2015-0053

The synthesis of 17 novel conformationally constrained pyrazole derivatives is reported herein, along with the assessment of their anti-proliferative and antiangiogenic activities. The evaluation of their inhibitory effect on cell proliferation against He

Full text of DOI:10.1515/znb-2015-0053

Z. Naturforsch. 2015; aop
Konstantinos M. Kasiotis*, Evangelia N. Tzanetou, Dimitrios Stagos, Nikolas Fokialakis,
ENleonivKoeultsocthoenodfoororu,mDimaittriioos nKoaurleltays acndoSnerskotsrAa. Hinaroeudtoupniaynr*azole
derivatives as potential anti-cancer agents
DOI 10.1515/znb-2015-0053
Received March 26, 2015; accepted April 30, 2015
1 Introduction
Cancer represents a major public health concern that
accounts as the second most common cause of death
Abstract: The synthesis of 17 novel conformationally con-
in the USA and elsewhere, causing nearly one of every
strained pyrazole derivatives is reported herein, along
four deaths. The American Cancer Society estimates that
with the assessment of their anti-proliferative and anti-
during 2014 in the USA 1 665 540 new cancer cases will be
angiogenic activities. The evaluation of their inhibitory
diagnosed and 585 720 cancer deaths will occur [1].
effect on cell proliferation against HepG2, HeLa, and MCF-7
The main characteristic of cancer consists in the loss
cells revealed the pyrrolo[2,3-g]indazole 23 as a potent
of control of cell growth, which eventually leads to the for-
inhibitor of cell growth with IC50 values of 5 μm. Addition-
mation of tumors that frequently become life-threatening
ally, the inhibition of vascular endothelial growth factor
by obstructing vessels or organs. However, death is most
by pyrazoles 20 and 23 (30 % and 35 %, respectively) in
usually provoked by the spread of the primary tumor to
HeLa supernatant cells was evidenced. These findings
one or several sites in the body through the metastasis
highlight the usefulness of these compounds as potential
process, which makes surgical intervention almost impos-
scaffolds for the design and development of novel anti-
sible. Other types of cancers known as leukemias involve
cancer agents with pronounced anti-angiogenic activity.
the build-up of a vast number of white cells in the blood.
In an attempt to treat cancer, various molecules have
Keywords: angiogenesis; anti-cancer agents; anti-
proliferative; pyrazoles; synthesis.
been screened for the selective target of cancer cells and
the minimization of the side effects to healthy organism
compartments.
Azaheterocycles comprise an important class of
compounds that exhibit diverse biological activities,
including the battle against cancer as well. In particu-
lar, numerous synthetic small molecules from various
*Corresponding authors: Konstantinos M. Kasiotis, Laboratory
groups of azaheterocycles have been reported to influ-
of Pesticides Toxicology, Benaki Phytopathological Institute,
ence carcinogenesis and are currently in clinical trials
Department of Pesticides Control and Phytopharmacy, 8 St. Delta
[2, 3] or approved for the treatment of various cancer
types [3, 4]. Indicatively, carbazoles possessing a unique
tricyclic ring system have been permitted for the treat-
ment of cancer [5], while phenazines [6] and several pip-
erazines [7] have been found to display IC50 values in the
nanomolar range against quinone reductase inhibitors.
Recently, azoaryl sulfides were proved as selective anti-
breast cancer agents with IC50 values in the low micromo-
lar range [8, 9]. In this respect, pyrazoles that constitute
a prominent family among azaheterocycles comprise a
class of molecules with a prominent position in the anti-
cancer portfolio as having been evidenced in numerous
publications [10].
Street, Athens, Kifissia 14561, Greece, Tel.: +30-210-8180357,
Fax: +30-210-8180223, E-mail: k.kasiotis@outlook.com; and
Serkos A. Haroutounian, Department of Animal Sciences and
Aquaculture, Agricultural University of Athens, Iera odos 75,
Athens 11855, Greece, Tel.: +30-210-5294247,
Fax: +30-210-5294265, E-mail: sehar@aua.gr
Evangelia N. Tzanetou: Department of Animal Sciences and
Aquaculture, Agricultural University of Athens, Iera odos 75,
Athens 11855, Greece
Dimitrios Stagos, Eleni Koutsotheodorou and Dimitrios Kouretas:
Department of Biochemistry and Biotechnology, University of
Thessaly, 26 Ploutonos Street, 41221 Larissa, Greece
Nikolas Fokialakis: Faculty of Pharmacy, Department of
Pharmacognosy and Natural Products Chemistry, University of
Athens, Panepistimiopolis, Zografou 15771, Athens, Greece
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2ꢀ ꢀK.M. Kasiotis et al.: Novel pyrazole derivatives as anti-cancer agents
Consequently, the design and synthesis of novel pyra-
2 Results and discussion
zoles has become the subject of several research groups.
The synthesis of anti-cancer pyrazoles was recently
reviewed by Kumari et al. [11], indicating that several ana-
logues are in preclinical or initial-phase clinical trials. In
the course of our longstanding interest concerning the
development of novel bioactive heterocycles [12, 13], we
have designed and synthesized a sequence of novel bio-
active molecules containing the pyrazole residue [12–14].
Herein, we present the outcome of our vision to design
and synthesize several novel molecules containing the
pyrazole structural backbone fused with commercially
available constrained ketone substrates. Our final goal
was the assessment of their anti-proliferative and anti-
angiogenic profiles.
2.1 Chemical synthesis
2.1.1 First synthetic part
A general route to access the target pyrazole derivatives
includes the α-acylation of the corresponding 3,4-dihydro-
naphthalenon-1-one substrate and the subsequent con-
densation with a phenylhydrazine derivative. In general,
the reaction of a substituted β-dicarbonyl derivative with a
molecule of hydrazine is expected to produce a mixture of
two isomeric pyrazoles. It must be pointed out, however,
that the two reactive centers of the hydrazine molecule
may potentially react with the corresponding reaction
sites of the dicarbonyl compounds tautomers, revealing
the possible formation of eight possible isomers. Since the
outcome of the aforementioned reaction depends greatly
on the nature of both diketone substrate and the acylating
agent, we were prompted to investigate the two possible
distinct pathways thoroughly.
The first pathway involves the formylation of 6-meth-
oxy-1-tetralone and 5-methoxy-1-indanone to afford the
hydroxymethylene derivatives 1 and 8 (Scheme 1). Double
condensation of the latter with phenyl- (or 4-methoxyphe-
nyl-) hydrazine furnished predominantly the pyrazoles 4,
3, 9, while only a small amount (10 %) of the regioisomeric
pyrazole 2 was obtained (when substrate 1 was used). The
regioselectivity of this condensation may be rationalized
considering that the formyl carbons of compounds 1 and 8
react predominantly with the terminal-most nucleophilic
NH2 of phenylhydrazines to form an imine intermediate.
The latter by intramolecular ring closure provided the cor-
responding benzo[g]indazole derivatives. Subsequently,
the demethylation of compounds 2, 4, 3, 9 afforded the
desired phenolic pyrazoles 5, 7, 6, 10.
In this context, the initial design of the target com-
pounds was based on reports establishing that ring
systems containing the (E)-2-benzylidene-1-tetralones
and (E)-2-benzylidene-1-tetralone backbone exhibit
remarkable cytotoxic activity [15], initiating the use
of α-tetralones as precursors for the synthesis of bio-
active compounds [16, 17]. In another application,
6-methoxytetralone has been utilized as a substrate for
the construction of steroids [18]. It is noteworthy that
2-(1H-pyrazol-1-yl)-thiazole derivatives, which possess
a conformationally constrained scaffold, similar to the
one envisaged in this work, exhibited a potent EP1 recep-
tor antagonist profile [19]. Additionally, a noticeable
number of indanone derivatives constitute lead com-
pounds for the treatment of Alzheimer’s disease [20].
Therefore, it is apparent from bibliography that fused
pyrazoles can display pronounced biological activities,
and their further exploitation should be a constant goal
of medicinal chemists. The potential bioactivity of the
targeted compounds was preliminarily assessed by the
Cheminformatics software molinspiration [21]. The
latter has been used in numerous publications as a pre-
liminary tool that can assist rational chemical design
(see [22]). The bibliographic results and the prelimi-
nary bioactivity potential revealed by Cheminformatics
prompted us to design and synthesize molecules that
combine the indanone/tetralone and indolone moie-
ties with the pyrazole core. Thus, we herein present the
substrate-dependent/directed synthesis of various series
of novel pyrazole derivatives using as starting materi-
als three commercially available fused ketones abiding
two similar synthetic routes. Some of the final products
displayed anti-proliferative activity and possible anti-
The structure of the pyrazole products was elucidated
based on 2D-nuclear overhauser effect (NOE) NMR spec-
troscopy experiments. More specifically, the assigned
stereochemistry of compounds 4, 3, 9 is consistent with
the observed strong cross peak between 9-H (and 8-H for
compound 9) and Ar–H (for compounds 2 and 4 see cor-
relations in Fig. 1). On the contrary, for compound 2 the
observed enhancement of the aromatic protons Ar–H
corresponds to 3-H. These results were further confirmed
1
15
by gradient inverse detected long-range H– N correla-
tion experiments [23]. In this regard, for compound 2, a
angiogenic action. Additional exploitation of these com- four-bond correlation was observed between the aro-
pounds might provide agents with therapeutic activity matic proton 9-H (7.92 ppm) and the deshielded nitrogen
against various cancer malignancies.
atom (N-1) that resonates at δ ꢀ=ꢀ 285.2 ppm. It must also be
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ꢂ3
K.M. Kasiotis et al.: Novel pyrazole derivatives as anti-cancer agentsꢂ
O
R
O
O
N
N
N
N
a
b
OH
O
O
O
O
6-Methoxy-1-tetralone
1, 85 %
3, R = H, 74 %
2, 10 %
4, R = OCH3, 75 %
OH
R1
N
N
N
N
c
HO
HO
6, R1 = H, 87 %
5, 82 %
7, R1 = OH, 85 %
O
HO
O
O
N
N
N
N
OH
b
a
c
O
O
O
HO
5-Methoxy-1-indanone
8, 80 %
9, 85 %
10, 90 %
.
Scheme 1:ꢀReagents and conditions: (a) NaH, HCOOEt, DMF, THF; (b) RC6H4NHNH2 HCl, DMF/THF 3:1, 120 °C; (c) BBr3, CH2Cl2, –78 °C.
O
The second pathway involves the synthesis of triaryl
substituted pyrazoles (Scheme 2). In this respect, the
Li enolates of the commercially available 6-methoxy-1-
tetralone and 5-methoxy-1-indanone (obtained by treat-
ment with lithium bis(trimethylsilyl)amide [LiHMDS])
were regioselectively acylated with p-anisoyl chloride to
afford triketones 11 and 16 surprisingly. The use of limited
amounts of base (up to 0.5 equivalents) in order to avoid
this overacylation was unsuccessful, since we were not
able to obtain any amount of diketone. The change of
solvent and use of toluene, as it was adapted in the second
R
2
2
1
1
N
N
N
N
9
6
3
9
6
3
8
8
4
4
7
O
7
O
5
5
2
4
Fig. 1:ꢀNOESY correlations for compounds 2 and 4.
noted that according to the allocated structure the other synthetic part (presented below), did not furnish the dik-
nitrogen atom (N-2) resonates at δ ꢀ=ꢀ 212.2 ppm and cor- etone, and surprisingly led to a decreased yield. Neverthe-
relates with the N-attached aromatic protons. For com- less, the desired novel sterically constrained pyrazoles
pound 4, a four-bond correlation was observed between were approached smoothly using the procedure described
the pyrazole proton 3-H (7.52 ppm) and the nitrogen atom by Stauffer and colleagues in 2000 [24].
that resonates at δ ꢀ=ꢀ 205.4 ppm. The latter corresponds to
In particular, the condensation of triketones (11
the shielded N-1 that also correlates with the N-attached and 16) with N-substituted hydrazine hydrochlorides
aromatic protons. Correlation between the deshielded in refluxing DMF–THF (3:1) afforded respectively the
nitrogen atom N-2 (resonating at 305.3 ppm) with the ali- benzo[g]indazoles 12, 13 and the indeno[1,2-c]pyrazoles
phatic protons 4-H (2.97 ppm) was not observed. In the 7, 8 with yields ranging from 74 % to 90 %. Subsequent
case of condensation of formate 1 with phenylhydrazine ether cleavage using BBr3 in CH2Cl2 at –78 °C provided the
hydrochloride, we encountered only the pyrazole 3 whose desired diphenols 14, 19 and triphenols 15, 20 (yields 82
conformation (same as the one of 4) was again confirmed %–91 %) as white crystalline solids. The configuration of
by nuclear overhauser effect spectroscopy (NOESY) and these novel pyrazoles was elucidated, through 2D-NOE
15
long-range 1H– N experiments.
NMR spectroscopy experiments (Fig. 2). More specifically,
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4ꢀ ꢀK.M. Kasiotis et al.: Novel pyrazole derivatives as anti-cancer agents
O
R1
R
O
O
O
O
c
a
b
N
N
N
N
O
O
O
OH
11, 66 %
O
O
HO
12, R = H, 78 %
13, R = OCH3, 77 %
14, R1 = H, 91 %
15, R1 = OH, 89 %
R1
R
O
O
O
O
O
N
N
N
N
a
c
b
OH
O
O
O
HO
O
O
19, R1 = H, 82 %
17, R = H, 88 %
18, R = OCH3, 90 %
20, R1 = OH, 90 %
16, 62 %
.
Scheme 2:ꢀReagents and conditions: (a) LHMDS, CH3C6H4COCl, THF; (b) RC6H4NHNH2 HCl, DMF/THF 3:1, 120 °C; (c) BBr3, CH2Cl2, –78 °C.
R
9
R
2.1.2 Second synthetic part
2
1
N
N
4
N
To extend the use of ketone substrates, the N-benzylated
indolone 21 was selected as starting material for the syn-
thesis of novel indazole derivatives using a modification
of a procedure published previously by our group [12].
Briefly, indolone in the presence of lithium hexamethyld-
isilazane (LHMDS) base under anhydrous conditions pro-
vided the corresponding anion, which was subsequently
condensed with p-anisoyl chloride or 3-chlorobenzothio-
phene-2-carbonyl chloride to yield the 1,3-diketone deriva-
tives. The in situ condensation of the latter with hydrazine
and its methoxyphenyl counterpart gave in one step the
desired pyrazole derivatives (compounds 24 and 22) in
moderate yields (Scheme 3). The latter was attributed to
the competing imine formation reaction of non-acylated
indolone. Finally, deprotection of indazole 22 provided
the diphenolic compound 23.
N
5
R = OCH3, H
8
O
O
4
O
O
12, 13
17, 18
Fig. 2:ꢀNOESY correlations for benzo[g]indazoles and indeno[1,2-c]
pyrazoles.
the assigned stereochemistry of compounds 17 and 18 is
consistent with the observed strong cross peak between
8-H and Ar–H. For compounds 12 and 13, the strongest
observed enhancement with the aromatic protons Ar–H
(of the C5 substituted ring) corresponds to the 4-H of the
tetralone moiety. These results were additionally con-
firmed by gradient inverse detected long-range 1H– N cor-
15
relation experiments.
In this regard, for compound 12 a four-bond correla- 2.2 Cytostatic activities
tion was observed between the aromatic proton 9-H (6.76
ppm) and the shielded nitrogen atom that resonates at 2.2.1 Assessment of inhibition of cancer and endothelial
δ ꢀ=ꢀ 203.6 ppm. Furthermore, based on the assigned struc-
ture, the other nitrogen atom resonates at δ ꢀ=ꢀ 299.7 ppm
cell proliferation
and correlates with the N-attached aromatic protons; The anti-carcinogenic activities of novel pyrazole deriv-
1
15
however, its signal is not intense (the H– N gradient atives were tested against hepatocellular (HepG2),
heteronuclear multiple quantum correlation [GHMQC] cervix (HeLa), and breast (MCF-7) cancer cell lines. The
NMR spectrum is presented as Supplementary Informa- assessed IC50 values for each cell line are depicted in
tion available online). The conformation of compound 17 Table 1. Three of compounds tested were determined as
1
15
was confirmed in a similar manner by long-range H– N the most active showing a dose-dependent inhibition of
experiments. the growth of all three cancer cell lines according to the
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K.M. Kasiotis et al.: Novel pyrazole derivatives as anti-cancer agentsꢂ
O
OH
O
O
c
b
1
1
N
4
N
N
N
N
O
Bn
2
2
NBn
NBn
4
3
3
O
HO
22, 60 %
23, 85 %
a
O
O
HN
N
Cl
O
O
Cl
Cl
1
O
b
4
2
S
Cl
S
N
N
N
S
6
N
H
Bn
Bn
a
3
Bn
5
21
24, 49 %
.
Scheme 3:ꢀReagents and conditions: (a) LHMDS, CH3OC6H4COCl, or C9H4Cl2OS, toluene; (b) CH3OC6H4NHNH2 HCl, or NH2NH2, AcOH, reflux; (c)
BBr3, CH2Cl2, –78 °C.
Table 1:ꢀCytostatic activities of synthesized compounds in
endothelial and tumor cell lines
substituted pyrazole 20 displayed on HeLa and MCF-7 cells
IC50 comparable values to those of compounds 23 and 24.
Overall, compound 23 exhibited the most potent inhibitory
activity against the growth of all tested cancer cell lines
with IC50 values of 3, 3.3, and 5 μm against HepG2, HeLa,
and MCF-7 cells, respectively.
Compoundꢁ
ꢁ
IC50 (μm)a,b
HepG2ꢁ
HeLaꢁ
MCF-7
5
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
17.0 5.1ꢃ
88.5 11.8ꢃ
34 8.8ꢃ
22.9 7.2ꢃ
ꢃ>ꢃ 100ꢃ
17 5.8ꢃ
90.0 16.9ꢃ
64.5 4.8ꢃ
63.3 12.7ꢃ
42.1 8.5ꢃ
28.1 4.9ꢃ
50.4 13.7ꢃ
5.8 3.8ꢃ
47.3 13.8
31.1 9.1
21 4.9
6
7
10
14
15
17
18
19
20
22
23
24
64.7 11.5ꢃ
70.4 12.1ꢃ
81.6 11.1ꢃ
13.4 4.3ꢃ
50.1 10.6ꢃ
20.8 5.1ꢃ
19.8 7.2ꢃ
33.2 6.5ꢃ
3.0 1.1ꢃ
60.3 9.4
82.9 14.4
75.7 9.3
24.4 5.9
33.4 10.1
19.2 6.2
13.4 3.6
19.8 5.9
5.0 2.1
2.2.2 Assessment of anti-angiogenic activity
2.2.2.1 In vitro anti-angiogenic effects using the tube
formation assay
In order to reveal the mode of anti-cancer properties for the
most potent among novel pyrazoles, we assessed their in
vitro anti-angiogenic properties using the tube formation
assay in human microvascular endothelial cells (HMEC-1).
Among the compounds tested, pyrazoles 20, 23, and 24
were determined the most potent in inhibiting the tube
formation at concentrations of 30 and 100 μm and sug-
gesting their potent anti-angiogenic properties (Table 2).
12.9 3.2ꢃ
3.3 0.9ꢃ
6.9 2.4ꢃ
7.1 3.2ꢃ
12.2 5.0
aIC50, concentration of the compound that reduces cell proliferation
by 50%.bNumber of independent experiments (n ꢃ=ꢃ 3).
2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-
5-Carboxanilide (XTT) cell proliferation assay (Fig. 3). 2.2.2.2 Evaluation of novel pyrazole effects against
Compound 23 was determined as the most active since it
the VEGF expression
inhibited the growth of HepG2 cells by 93 % at 20 μm and Tumor angiogenesis starts when cancerous tumor cells
MCF-7 cell growth by 48 %, 87 %, and 91 % at 20, 40, and release molecules sending signals to surrounding normal
60 μm, respectively (Fig. 3a and d). Moreover, compound host tissue and activating certain genes in the host
24 exhibited statistically significant inhibitory activ- tissue, which initiates the growth of new blood vessels.
ity against HepG2 and MCF-7 cell growth at 20, 60, and Thus, in order to reveal the molecular mechanism of
120 μm by 39 %, 50 %, and 98 %, and 34 %, 48 %, and the anti-angiogenic activity of novel pyrazoles, their
54 %, respectively (Fig. 3b and e). Finally, the triphenol effects on the vascular endothelial growth factor (VEGF)
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ꢀK.M. Kasiotis et al.: Novel pyrazole derivatives as anti-cancer agents
110
a
110
100
b
100
90
80
70
60
50
40
30
90
80
70
60
50
40
30
20
10
0
*
*
20
*
10
*
*
*
0
0
10
20
40
60
120
0
1
10
20
60
120
23 (µM)
24 (µM)
110
100
90
80
70
60
50
40
30
20
10
0
c
d
100
80
60
40
20
0
*
*
*
*
*
0
5
10
20
40
0
10
20
40
60
120
23 (µM)
20 (µM)
110
100
90
80
70
60
50
40
30
20
10
0
e
f
100
80
60
40
20
0
*
*
*
*
*
*
0
1
10
20
60
120
0
5
10
20
40
60
24 (µM)
20 (µM)
Fig. 3:ꢀEffects of compounds 20, 23, and 24 on (a), (b), and (c) HepG2 cell growth, and on (d), (e), and (f) MCF-7 cell growth. Adherent cells
proliferated in 96-well plates (104 cells per well) were incubated with different concentrations of the tested compounds for 24 h. Cell prolif-
eration was determined by XTT assay. Results were expressed as a percent of cell proliferation of control. The data shown are the mean SD
from three independent experiments carried out in triplicate; *p ꢃ<ꢃ 0.05 vs. control.
Table 2:ꢀAssessment of in vitro anti-angiogenic effects of heterocyclic
pyrazoles 20, 23, and 24 inhibited significantly the VEGF
derivatives using tube formation assay in HMEC-1.
levels in supernatant HeLa cells cultures (Fig. 4). Com-
pounds 20 and 23 were the most active, since they inhib-
Compoundsꢁ
30 μmꢁ
100 μm
ited by 30 % and 35 %, respectively, the VEGF levels at
20
23
24
ꢃ
ꢃ
ꢃ
2.5aꢃ
2.5aꢃ
2aꢃ
2a
2a
20 μm concentration.
1.5a
aScore 0, complete inhibition – no tubes; Score 1, few tubes; Score 2,
inhibition; Score 3, no inhibition, number of experiments (n ꢃ=ꢃ 3).
2.3 Cheminformatics and Bioactivity
The Cheminformatics conducted by molinspiration
predicted that compound 23 was the most active, amongst
expression – the most important pro-angiogenic mole- compounds synthesized, exhibiting activity as a G-pro-
cule – were determined in HeLa cancer cells. The respec- tein-coupled receptor (GPCR) ligand, kinase inhibitor (KI),
tive results showed that among the tested compounds, nuclear receptor ligand (NRL), and enzyme inhibitor (EI)
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K.M. Kasiotis et al.: Novel pyrazole derivatives as anti-cancer agentsꢂ ꢂ7
300
indicating that their configuration does not interplay sig-
nificantly in the bioactivity. Overall, the comparison of all
presented compounds with compound 23 proposes that
the pronounced activity of 23 might be primarily attrib-
uted to its superordinate performance in terms of enzyme
inhibition, combined with the relative high values of
GPCR and NRL. Future molecular modeling studies, espe-
cially for the diphenol 23 and pyrrolo[2,3-g]indazole 24,
will reveal the structure activity relationship modes, and
assist the structural modifications that will enhance the
cytostatic and anti-angiogenic activities.
250
200
150
100
50
0
0
10
20
40
20
23 (µM)
300
250
200
3 Conclusions
150
100
Novel conformationally rigid pyrazole derivatives with
potential cytostatic activity have been prepared following
an efficient synthetic protocol. A diphenolic substituted
pyrazole 23 had the most potent inhibitory activity against
the growth of the panel of tested cancer cell lines with IC50
values of 3, 3.3, and 5 μm in HepG2, HeLa, and MCF-7 cells,
respectively. These results render it as a potential anti-
cancer scaffold. As regards the anti-angiogenic activity
both in the tube formation assay in HMEC-1 and the inhi-
bition of VEGF, a mixed activity profile of compounds was
evidenced. Compound 24 was the most active in the tube
formation assay and compounds 20 and 23 exhibited anti-
angiogenesis action in the VEGF expression.
To further exploit these structures, in future
endeavors, targeted molecular modeling and virtual
screening approaches will be pursued in order to propose
the synthesis of additional derivatives that might
possess enhanced anti-cancer activities. The latter is an
immediate future project of our group, devoted primar-
ily to the enhancement of the activity of the promising
indazole diphenol 23 and to a lesser extent pyrrolo[2,3-
g]indazole 24 by molecular modeling guided chemical
modifications.
50
0
0
20
24 (µM)
300
250
200
150
100
50
0
0
10
20 (µM)
Fig. 4:ꢀEffects of heterocyclic derivatives 20, 23, and 24 on
VEGF expression in HeLa cells. Media conditioned by incubation
with HeLa cultures were evaluated for VEGF protein secretion
by ELISA. The data shown were the mean SD from three
independent experiments carried out in duplicate; *p ꢃ<ꢃ 0.05 vs.
control.
(Table 3). The majority of presented compounds showed
KI activity, with compounds 10, 19 and 20 projecting as
the most efficient KIs (Table 3). Compound 24 that inhib-
ited tube formation at concentrations of 30 μm exhibited
4 Experimental section
only substantial KI in molinspiration. The prognostic 4.1 General information
comparison of five- with six-membered cores (as pairs e.g.
7–10, 14–19, 15–20) showed that five-membered scaffolds All anhydrous reactions were carried out under argon
are better KIs while six-membered ones are superior NRLs. atmosphere. Solvents were dried by distillation prior
According to this approach, methoxy derivatives demon- to use. Solvent mixtures employed in chromatography
strate lower activity than their hydroxylated analogues. were reported as volume-to-volume ratios. Starting mate-
Surprisingly, and in contrast with the Cheminformatics rials were purchased from Sigma-Aldrich (analytical
prediction, compounds 5 and 7 did not differ in activity, reagent grades; St. Louis, USA) and used without further
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ꢀꢀꢀꢁ
8ꢀ ꢀK.M. Kasiotis et al.: Novel pyrazole derivatives as anti-cancer agents
Table 3:ꢀPrediction of bioactivity of compounds by molinspiration
(Cheminformatics) [21].
4.2 HPLC purification
When required, compounds were purified by semi-pre-
parative high-performance liquid chromatography (HPLC)
[column: Kromasil 100-5, C-18, (25 cm ꢀ×ꢀ 10 mm); mobile
phase CH3CN–H2O (8:2); detector: UV (λ ꢀ=ꢀ 300 nm); flow:
1.6 mL min–1; load: 5 mg per 100 μL of solution in the
mobile phase]. The HPLC system was a Hewlett Packard
1100 series instrument (Agilent Technologies, Waldbronn,
Compoundꢁ
GPCRꢁ
ICMꢁ
KIꢁ
NRLꢁ
PIꢁ
EI
23
24
22
20
19
18
17
15
14
13
12
10
9
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
0.27ꢃ –0.12ꢃ
0.04ꢃ –0.34ꢃ
0.19ꢃ –0.22ꢃ
0.19ꢃ –0.03ꢃ
0.20ꢃ –0.03ꢃ
0.09ꢃ –0.13ꢃ
0.10ꢃ –0.14ꢃ
0.22ꢃ –0.10ꢃ
0.23ꢃ –0.11ꢃ
0.12ꢃ –0.19ꢃ
0.14ꢃ –0.21ꢃ
0.23ꢃ
0.29ꢃ
0.16ꢃ
0.42ꢃ
0.43ꢃ
0.38ꢃ –0.31ꢃ
0.05ꢃ –0.38ꢃ
0.25ꢃ –0.34ꢃ
0.10ꢃ –0.15ꢃ
0.10ꢃ –0.16ꢃ
0.26
0.06
0.17
0.05
0.05
0.31ꢃ –0.03ꢃ –0.20ꢃ –0.04
0.33ꢃ –0.04ꢃ –0.21ꢃ –0.05
Germany) with
a variable wavelength UV detector,
0.18ꢃ
0.18ꢃ
0.09ꢃ
0.10ꢃ
0.44ꢃ
0.36ꢃ
0.30ꢃ
0.28ꢃ
0.04ꢃ
0.22ꢃ
0.20ꢃ
0.29ꢃ –0.28ꢃ
0.30ꢃ –0.29ꢃ
0.14ꢃ –0.31ꢃ –0.03
0.14ꢃ –0.33ꢃ –0.03
0.14ꢃ –0.33ꢃ
0.02ꢃ –0.31ꢃ –0.01
0.27ꢃ –0.49ꢃ
0.23ꢃ –0.56ꢃ
0.04ꢃ –0.51ꢃ –0.06
0.12ꢃ –0.47ꢃ –0.03
0.08ꢃ –0.58ꢃ –0.04
0.06
0.06
coupled to HP Chem Station utilizing the manufacturer’s
5.01 software package.
All materials and methods used in chemistry part are
also described in a formerly published work of our group
[23, 25].
0.21ꢃ
0.02ꢃ
0.11
0.14ꢃ –0.12ꢃ
0.29ꢃ –0.13ꢃ
0.27ꢃ –0.13ꢃ
0.08ꢃ –0.03ꢃ
0.20ꢃ –0.25ꢃ
0.18ꢃ –0.27ꢃ
7
0.09
0.09
6
5
4.3 Procedures
4
3
2
0.01ꢃ –0.17ꢃ –0.02ꢃ –0.08ꢃ –0.48ꢃ –0.17
4.3.1 Synthesis of target compounds
GPCR, G-protein-coupled receptor ligand; ICM, ion channel modula-
tor; KI, kinase inhibitor; NRL, nuclear receptor ligand; PI, protease
inhibitor; EI, enzyme inhibitor.
4.3.1.1 General procedure for pyrazoles synthesis
The experimental procedure developed by Stauffer et al.
was applied for the preparation of target pyrazoles [24].
After completion of the reaction and work-up, the crude
product was purified by flash chromatography or by
purification. Analytical thin-layer chromatography (TLC) passage through a short silica gel plug eluting with an
was conducted on Merck glass plates (Darmstadt, Germany) ethyl acetate–hexane solvent system.
coated with silica gel 60 F254 and spots were visualized with
UV light or/and an alcohol solution of anisaldehyde. Flash
column chromatography was performed using Merck silica 4.3.1.2 General demethylation procedure
gel 60 (230–400 mesh ASTM).
The deprotection of the intermediate compounds was
Melting points were determined on a Büchi melting performed according to Stauffer et al. [24]. The crude phe-
point apparatus (BÜCHI Labortechnik AG, Flawil, nolic products were obtained after purification by flash
Switzerland) and are uncorrected. 1H and 2D NMR spectra chromatography and/or recrystallization from MeOH–
were recorded at 400 MHz on a Bruker DRX-400 spec- CH2Cl2 mixtures.
trometer (Billerica, MA, USA) in the indicated solvents.
The coupling constants are recorded in Hertz (Hz) and the
chemical shifts are reported in parts per million (ppm; 4.3.1.3 6-Methoxy-2-hydroxymethylen-3,4-dihydro-
δ scale) downfield from tetramethylsilane, which was
2H-naphthalen-1-one (1)
used as an internal standard (by asterisk are indicated the To a stirred solution of sodium hydride (27 mg, of 60 % dis-
overlapped peaks). Infrared spectra were obtained on a persion in oil, 1.13 mmol) in 2 mL of dry DMF under an N2
Nicolet Magna 750 (GMI-Inc, Ramsey, MN, USA), series II atmosphere, a solution of 6-methoxy-1-tetralone (0.20 g,
spectrometer.
1.13 mmol) in 2 mL of dry THF was added dropwise. After
1
15
For the H– N GHMQC spectrum, data were acquired being stirred for 4 h, ethyl formate (0.1 mL, 1.25 mmol) was
as 3072 ꢀ×ꢀ 400 data points with a total of 290 transients added and the mixture was stirred for an additional 20 h.
accumulated/t1 increment. Pulse widths were 8.55 μs for A saturated solution of NH4Cl was added to quench the
1H and 27.7 μs for 15N at powers of 0 and –3 dB. The F1 reaction mixture, and the product was extracted twice with
spectral window employed was set from 100 to 400 ppm. ethyl acetate (2 ꢀ×ꢀ 15 mL). The joint organic layers were evap-
Pulsed field gradients, gt1–gt3, had durations of 0.8 ms. orated under vacuum, and the resulting orange solid was
Gradient pairs were optimized as 70:30:50 for 15N.
chromatographed (20 % EtOAc-hexanes) to give 0.18 g of the
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K.M. Kasiotis et al.: Novel pyrazole derivatives as anti-cancer agentsꢂ ꢂ9
desired product as amorphous yellow solid in 85 % yield. M.p. δ ꢀ=ꢀ 2.73 (t, J ꢀ=ꢀ 6.8 Hz, 2H, 4-H), 2.95 (t, J ꢀ=ꢀ 6.8 Hz, 2H, 5-H),
95–96 °C (lit: 93 °C [26] and 97–100 °C [27]). – IR (KBr): 1688, 3.76 (s, 3H, CH3O), 6.53 (dd, J ꢀ=ꢀ 8.7, 2.7 Hz, 1H, 8-H), 6.75
1
3211 cm–1. – H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 2.52 (t, J ꢀ=ꢀ 6.6 (d, J ꢀ=ꢀ 8.7 Hz, 1H, 9-H), 6.84 (d, J ꢀ=ꢀ 2.7 Hz, 1H, 6-H), 7.53 (s,
Hz, 2H, 3-H), 2.85 (t, J ꢀ=ꢀ 6.6 Hz, 2H, 4-H), 3.83 (s, 3H, CH3O), 1H, 3-H), 7.41–7.49 (m, 5H, ArH) ppm. – 13C NMR (400 MHz,
6.71 (d, J ꢀ=ꢀ 2.2 Hz, 1H, 5-H), 6.84 (dd, J ꢀ=ꢀ 8.7, 2.2 Hz, 1H, 7-H), CDCl3): δ ꢀ=ꢀ 24.9, 31.2, 55.5, 117.9, 125.2, 128.8, 131.4, 137.6,
7.93 (m, 2H, Cꢀ=ꢀCH, 8-H), 14.70 (br s, 1H, OH) ppm. – 13C NMR 152.3, 163.3 ppm. – 15N NMR (400 MHz, CDCl3) δ ꢀ=ꢀ 289.7
(400 MHz, CDCl3): δ ꢀ=ꢀ 25.2, 28.4, 60.3, 114.2, 130.9, 166.7, (N-1), 214.2 (N-2) ppm. – Anal. for C18H16N2O (276.3): calcd.
168.9 (–Cꢀ=ꢀC–OH), 185.6 (Cꢀ=ꢀO) ppm. – Anal. for C12H12O3 C 78.24, H 5.84, N 10.14; found: C 78.51, H 5.74, N 10.35.
(204.2): calcd. C 70.57, H 5.92; found: C 70.44, H 6.01.
4.3.1.6 2-(4-Hydroxyphenyl)-4,5-dihydro-2H-benzo[g]
indazol-7-ol (5)
4.3.1.4 7-Methoxy-2-(4-methoxyphenyl)-4,5-dihydro-
A stirred CH2Cl2 solution of 2 (26 mg, 0.08 mmol) was
2H-benzo[g]indazole (2) and 7-methoxy-1-
deprotected using BBr3, according to the general demeth-
(4-methoxyphenyl)-4,5-dihydro-1H-benzo[g]
ylation procedure. Purification by flash chromatography
indazole (4)
(50 % EtOAc-hexanes) afforded the title compound as
Naphthalen-1-one 1 (0.3 g, 1.3 mmol) and 4-methoxyphe-
yellow crystalline solid (18 mg, 82 %). M.p. 231–233 °C. – IR
nylhydrazine hydrochloride (0.9 g, 5.2 mmol) were reacted
1
(KBr): ν ꢀ=ꢀ 3317 cm–1. – H NMR (400 MHz, [D6]acetone):
as outlined above to afford pyrazoles 2, 4 as amorphous
δ ꢀ=ꢀ 2.64 (t, J ꢀ=ꢀ 6.8 Hz, 2H, 5-H), 2.84 (t, J ꢀ=ꢀ 6.8 Hz, 2H, 4-H),
yellow solids (40 mg, 10 % and 0.32 g, 75 %, respec-
6.44 (dd, J ꢀ=ꢀ 8.5, 2.4 Hz, 1H, 8-H), 6.53 (d, J ꢀ=ꢀ 8.5 Hz, 1H,
tively) after passage through a short silica plug (20 %
9-H), 6.75 (d, J ꢀ=ꢀ 2.4 Hz, 1H, 6-H), 6.87 (d, J ꢀ=ꢀ 8.8 Hz, 2H,
EtOAc-hexanes):
ArH), 7.19 (d, J ꢀ=ꢀ 8.8 Hz, 2H, ArH), 7.47 (s, 1H, H-3), 9.52 (br
1
(2): M.p. 114–117 °C. – H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ
s, 1H, OH), 9.82 (br s, 1H, OH) ppm. – 13C NMR (400 MHz,
2.82 (t, J ꢀ=ꢀ 6.8 Hz, 2H, 4-H), 2.96 (t, J ꢀ=ꢀ 6.8 Hz, 2H, 5-H), 3.81
[D6]acetone): δ ꢀ=ꢀ 22.6, 27.5, 111.9, 115.7, 122.7, 128.5, 141.8,
(s, 3-H, CH3O), 3.83 (s, 3H, CH3O), 6.75 (d, J ꢀ=ꢀ 8.3 Hz, 1H,
152.6, 155.9 ppm. – Anal. for C17H14N2O2 (278.3): calcd. C
9-H), 6.82 (d, J ꢀ=ꢀ 2.4 Hz, 1H, 6-H), 6.86 (dd, J ꢀ=ꢀ 8.3, 2.4 Hz,
73.37, H 5.07, N 10.07; found: C 73.52, H 5.22, N 10.18.
1H, 8-H), 6.98 (d, J ꢀ=ꢀ 8.8 Hz, 2H, ArH), 7.62 (s, 1H, 3-H), 7.65
(d, J ꢀ=ꢀ 9.3 Hz, 2H, ArH) ppm. – 13C NMR (400 MHz, CDCl3):
δ ꢀ=ꢀ 24.8, 31.2, 55.9, 114.3, 120.4, 131.5, 136.8, 150.9, 155.8
4.3.1.7 2-Phenyl-4,5-dihydro-2H-benzo[g]indazol-7-ol (6)
ppm. – 15N NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 285.2 (N-1), 212.2
A stirred CH2Cl2 solution of 3 (0.2 g, 0.72 mmol) was depro-
(N-2) ppm. – Anal. for C19H18N2O2 (306.4): calcd. C 74.49, H
tected using BBr3, according to the general demethylation
5.92, N 9.14; found: C, 74.35, H 5.76, N, 9.25.
procedure. Purification by flash chromatography (40 %
(4): M.p. 105–106 °C. – 1H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ
EtOAc-hexanes) afforded the title compound as white
2.76 (t, J ꢀ=ꢀ 6.9 Hz, 2H, 4-H), 2.97 (t, J ꢀ=ꢀ 6.9 Hz, 2H, 5-H),
crystalline solid (0.13 g, 87 %). M.p. 229–231 °C. – IR (KBr):
3.79 (s, 3H, CH3O), 3.89 (s, 3H, CH3O), 6.55 (dd, J ꢀ=ꢀ 8.6, 2.4
1
ν ꢀ=ꢀ 3317 cm–1. – H NMR (400 MHz, [D6]acetone): δ ꢀ=ꢀ 2.63
Hz, 1-H, 8-H), 6.76 (d, J ꢀ=ꢀ 8.6 Hz, 1H, 9-H), 6.87 (d, J ꢀ=ꢀ 2.4
(t, J ꢀ=ꢀ 6.8 Hz, 2H, H-5), 2.84 (t, J ꢀ=ꢀ 6.8 Hz, 2H, H-4), 6.39
Hz, 1H, 6-H), 6.99 (d, J ꢀ=ꢀ 8.8 Hz, 2H, ArH), 7. 44 (d, J ꢀ=ꢀ 8.8
(dd, J ꢀ=ꢀ 8.7, 2.2 Hz, 1H, H-8), 6.49 (d, J ꢀ=ꢀ 8.7 Hz, 1H, H-9),
Hz, 2H, ArH), 7.52 (s, 1H, 3-H) ppm. – 13C NMR (400 MHz,
6.74 (d, J ꢀ=ꢀ 2.3 Hz, 1H, 6-H), 7.45–7.54 (m, 5H, ArH), 7.58 (s,
CDCl3): δ ꢀ=ꢀ 24.5, 30.5, 55.6, 114.4, 122.8, 131.6, 136.7, 149.2,
1H, 3-H), 9.53 (br s, 1H, OH) ppm. – 13C NMR (400 MHz,
159.5 ppm. – 15N NMR (400 MHz, CDCl3): δ, 205.4 (N-1),
[D6]acetone): δ ꢀ=ꢀ 24.5, 29.4, 111.9, 115.8, 122.4, 125.5, 128.6,
305.3 (N-2) ppm. – Anal. for C19H18N2O2 (306.4): calcd. C
143.8, 154.3, 162.7 ppm. – Anal. for C17H14N2O (262.3): calcd.
74.49, H 5.92, N 9.14; found: C, 74.31, H 5.76, N 9.37.
C 77.84, H 5.38, N 10.68; found: C 77.70, H 5.51, N 10.41.
4.3.1.5 7-Methoxy-2-phenyl-4,5-dihydro-2H-benzo[g]
4.3.1.8 4,5-Dihydro-1-(4-hydroxyphenyl)-1-benzo[g]
indazol-7-ol (7)
indazole (3)
Naphthalen-1-one 1 (0.3 g, 1.29 mmol) and phenylhydra- A stirred CH2Cl2 solution of 4 (0.26 g, 0.85 mmol) was
zine hydrochloride (0.93 g, 6.45 mmol) were reacted as out- deprotected using BBr3, according to the general demeth-
lined above to afford 3 as amorphous yellow solid (0.25 g, ylation procedure. Purification by flash chromatography
74 %) after passage over a short silica plug (20 % EtOAc- (60 % EtOAc-hexane) afforded the title compound as white
1
hexanes). M.p. 110–113 °C. – H NMR (400 MHz, CDCl3): crystalline solid (0.19 g, 85 %). M.p. 259–261 °C. – IR (KBr):
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ν ꢀ=ꢀ 3401 cm–1. – 1H NMR (400 MHz, [D6]acetone): δ ꢀ=ꢀ 2.62 yellow crystalline solid (32 mg, 90 %). M.p. 199–201 °C. –
(t, J ꢀ=ꢀ 6.8 Hz, 2H, 5-H), 2.82 (t, J ꢀ=ꢀ 6.8 Hz, 2H, 4-H), 6.42 IR (KBr): ν ꢀ=ꢀ 3183 cm–1. – 1H NMR (400 MHz, [D6]acetone):
(dd, J ꢀ=ꢀ 8.5, 2.4 Hz, 1H, 8-H), 6.51 (d, J ꢀ=ꢀ 8.5 Hz, 1H, 9-H), δ ꢀ=ꢀ 3.06 (s, 2H, H-4), 6.76 (dd, J ꢀ=ꢀ 8.5, 2.1 Hz, 1H, H-8), 7.03
6.72 (d, J ꢀ=ꢀ 2.4 Hz, 1H, 6-H), 6.88 (d, J ꢀ=ꢀ 8.8 Hz, 2H, ArH), (d, J ꢀ=ꢀ 8.5 Hz, 2H, ArH), 7.06 (d, J ꢀ=ꢀ 2.1 Hz, 1H, H-5), 7.28
7.20 (d, J ꢀ=ꢀ 8.8 Hz, 2H, ArH), 7.44 (s, 1H, 3-H), 9.53 (br s, (d, J ꢀ=ꢀ 8.5 Hz, 1H, H-8), 7.49 (s, 1H, H-3), 7.52 (d, J ꢀ=ꢀ 8.5 Hz,
1H, OH), 9.83 (br s, 1H, OH) ppm. – 13C NMR (400 MHz, 2H, ArH), 8.58 (s, 1H, OH), 8.83 (s, 1H, OH) ppm. – 13C NMR
[D6]acetone): δ ꢀ=ꢀ 24.8, 29.6, 111.6, 116.6, 123.8, 129.2, 143.8, (400 MHz, [D6]acetone): δ ꢀ=ꢀ 35.9, 111.2, 116.4, 123.9, 129.2,
147.7, 159.7 ppm. – Anal. for C17H14N2O2 (278.3): calcd. C 140.4, 147.5, 159.5 ppm. – Anal. for C16H12N2O2 (264.3): calcd.
73.37, H 5.07, N 10.07; found: C 73.50, H 5.16, N 9.91.
C 72.22, H 4.58, N 10.60; found: C 72.10, H 4.49, N 10.76.
4.3.1.9 2-Hydroxymethylen-5-methoxy-indan-1-one (8)
Compound 8 was synthesized from 5-methoxy-1-indanone
4.3.1.12 6-Methoxy-2,2-di-(4-methoxybenzoyl)-3,
4-dihydro-2H-naphthalen-1-one (11)
following the same process used for the preparation of 6-Methoxy-1-tetralone (0.09 g, 0.51 mmol) in 5 mL THF was
compound 1. After completion of the reaction and subse- added dropwise to a stirred solution of 1 m LiHMDS (0.5 mL,
quent work-up, the resulting orange solid was chromato- 0.51 mmol) in THF over a period of 30 min. The resulting
graphed (ethyl acetate-hexane 4:1, v/v) to give 94 mg of the solution was stirred for a further 15 min prior to the addi-
desired product as pale-yellow solid in 80 % yield. M.p. tion of p-anisoyl chloride in THF. The reaction blend was
135–136 °C (lit: 138–138.5 °C [28]). – IR (KBr): ν ꢀ=ꢀ 1692, stirred for 15 min and subsequently extracted using ethyl
3301 cm–1. – 1H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 3.45 (s, 2H, 3-H), acetate (3 ꢀ×ꢀ 30 mL). The combined organic solvents were
3.80 (s, 3H, CH3O), 6.83–6.89 (m, 2H, 4-H, 6-H), 7.31 (s, 1H, dried over MgSO4 and concentrated in vacuo. Purification
Cꢀ=ꢀCH), 7.68 (d, J ꢀ=ꢀ 8.3 Hz, 1H, 7-H), 9.82 (br s, 1H, OH). – was achieved using silica gel chromatography. Elution with
13C NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 30.4, 58.4, 110.8, 127.1, 145.3, 10 % ethyl acetate in hexane yielded 80 mg (66 %) of the
165.2, 170.3 (–Cꢀ=ꢀC–OH), 188.6 (Cꢀ=ꢀO) ppm. – C11H10O3 desired product as a pale-yellowish solid yield while 25 %
(190.2): calcd. C 69.46, H 5.30; found: C 69.34, H 5.19.
of the unreacted tetralone was recovered. M.p. 133–134 °C. –
IR (KBr): ν ꢀ=ꢀ 1702 cm–1. – 1H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 2.69
(t, J ꢀ=ꢀ 6.6 Hz, 2H, 3-H), 2.88 (t, J ꢀ=ꢀ 6.6 Hz, 2H, 4-H), 3.60 (s,
3H, CH3O), 3.80 (s, 3H, CH3O), 3.82 (s, 3H, CH3O), 6.53–6.71
(m, 6H, ArH), 7.01 (d, J ꢀ=ꢀ 8.3 Hz, 1H, ArH), 7.57–7.67 (m, 4H,
4.3.1.10 6-Methoxy-2-(4-methoxyphenyl)-2,
4-dihydro-2H-indeno[1,2-c]pyrazole (9)
Indan-1-one 8 (30 mg, 0.16 mmol) was reacted with ArH) ppm. – 13C NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 26.2, 34.4, 62.1,
4-methoxyphenylhydrazine hydrochloride (0.14 g, 0.8 112.4, 116.5, 122.1, 125.8, 130, 162, 189, 198 ppm. – Anal. for
mmol) according to the general procedure to afford 9 as C27H24O6 (444.5): calcd. C 72.96, H 5.44; found C 72.77, H 5.69.
pale-yellow crystalline solid (41 mg, 85 %) after purifica-
tion by flash chromatography (20 % EtOAc-hexanes). M.p.
95–96 °C. – 1H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 3.52 (s, 2H, 4-H), 4.3.1.13 7-Methoxy-3-(4-methoxyphenyl)-2-phenyl-4,
3.80 (s, 3H, CH3O), 3.81 (s, 3H, CH3O), 6.77 (dd, J ꢀ=ꢀ 8.3, 2.2
5-dihydro-2H-benzo[g]indazole (12)
Hz, 1H, 7-H), 7.03 (d, J ꢀ=ꢀ 8.7 Hz, 2H, ArH), 7.06 (d, J ꢀ=ꢀ 2.2 Hz, The triketone 11 (0.10 g, 0.22 mmol) was reacted with phe-
1H, 5-H), 7.31 (d, J ꢀ=ꢀ 8.3 Hz, 1H, 8-H), 7.56 (s, 1H, 3-H), 7.58 nylhydrazine hydrochloride (0.13 g, 0.88 mmol) according
(d, J ꢀ=ꢀ 8.7 Hz, 2H, ArH) ppm. – 13C NMR (400 MHz, CDCl3): to the general procedure above. Upon purification by flash
δ ꢀ=ꢀ 24.5, 30.5, 58.9, 116.4, 122.5, 131.8, 136.6, 142.5, 149.2, chromatography (30 % EtOAc-hexanes) the title compound
164.5 ppm. – 15N NMR (CDCl3): δ ꢀ=ꢀ 203.9 (N-1), 304.1 (N-2) was obtained as amorphous orange solid (50 mg, 78 %).
1
ppm. – Anal. for C19H18N2O2 (292.3): calcd. C 73.95, H 5.52, N M.p. 161–163 °C. – H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 2.95 (m,
9.58; found: C 74.01, H 5.59, N 9.45.
2H, 5-H), 3.01 (m, 2H, 4-H), 3.78 (s, 3H, CH3O), 3.82 (s, 3H,
CH3O), 6.54 (dd, J ꢀ=ꢀ 8.6, 2.1 Hz, 1H, 8-H), 6.76 (d, J ꢀ=ꢀ 8.6 Hz,
1H, 9-H), 6.87 (d, J ꢀ=ꢀ 2.1 Hz, 1H, 6-H), 6.98 (d, J ꢀ=ꢀ 8.6 Hz,
2H, ArH), 7.43–7.52 (m, 3H, ArH), 7.57 (m, 2H, ArH), 7.70 (d,
J ꢀ=ꢀ 8.6 Hz, 2H, ArH) ppm. – 13C NMR (400 MHz, CDCl3):
4.3.1.11 2-(4-Hydroxyphenyl)-2,4-dihydro-indeno[1,2-c]
pyrazol-6-ol (10)
A stirred CH2Cl2 solution of 9 (40 mg, 0.14 mmol) was δ ꢀ=ꢀ 23.7, 30.4, 57.5, 115.9, 122.2, 128.2, 129.3, 135.8, 138.5,
deprotected using BBr3, according to the general demeth- 151.8, 160.7 ppm. – 15N NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 203.6
ylation procedure. Purification by flash chromatography (N-1), 299.7 (N-2) ppm. – C25H22N2O2 (382.5): calcd. C 78.51,
(50 % EtOAc-hexane) afforded the title compound as H 5.80, N 7.32; found: C 78.22, H 5.64, N 7.42.
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4.3.1.14 7-Methoxy-2,3-di-(4-methoxyphenyl)
-4,5-dihydro-2H-benzo[g]indazole (13)
152, 151, 138, 135, 129, 128, 122, 115, 28, 23 ppm. – Anal. for
C23H18N2O3 (370.4): C 74.58, H 4.90, N 7.56; Found: C 74.35,
Triketone 11 (0.10 g, 0.22 mmol) and 4-methoxyphenylhy- H 5.04, N 7.67.
drazine hydrochloride (0.16 g, 0.37 mmol) were reacted as
outline above to afford 13 as amorphous yellow solid after
4.3.1.17 5-Methoxy-2,2-di-(4-methoxybenzoyl)-indan-
flash chromatographic purification (77 %). M.p. 141–143 °C.
1-one (16)
1
– H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 2.95–3.01 (m, 4H, 5-H,
Compound 16 was synthesized from 5-methoxy-1-indanone
following the same procedure used for the preparation of
compound 11. Purification was achieved using silica gel
chromatography. Elution with 30 % ethyl acetate in hexane
furnished 0.13 g of the desired product as a pale-yellowish
solid in 62 % yield while 29 % of the unreacted indanone
precursor was recovered. M.p. 121–122 °C. – IR (KBr): ν ꢀ=ꢀ
1742, 1737 cm–1. – 1H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 3.87 (s, 3H,
CH3O), 3.89 (s, 3H, CH3O), 3.91 (s, 3H, CH3O), 4.05 (s, 2H,
3-H), 6.64 (dd, J ꢀ=ꢀ 8.8, 2.1 Hz, 1H, 6-H), 6.85 (d, J ꢀ=ꢀ 2.1 Hz,
1H, 4-H), 6.91 (d, J ꢀ=ꢀ 9.0 Hz, 2H, ArH), 6.97 (d, J ꢀ=ꢀ 9.0 Hz,
2H, ArH), 7.00 (d, J ꢀ=ꢀ 9.0 Hz, 2H, ArH), 7.72 (d, J ꢀ=ꢀ 9.0 Hz,
2H, ArH), 8.23 (d, J ꢀ=ꢀ 8.8 Hz, 1H, H-7) ppm. – 13C NMR (400
MHz, CDCl3): δ ꢀ=ꢀ 25.2, 62.6, 112.4, 116.2, 122.5, 125.7, 130.3,
155.5, 162.8, 192.1, 198.9 ppm. – Anal. for C26H22O6 (430.5):
calcd. C 72.55, H, 5.15; found: C 72.31, H 5.01.
4-H), 3.80 (s, 3H, CH3O), 3.84 (s, 3H, CH3O), 3.87 (s, 3H,
CH3O), 6.56 (dd, J ꢀ=ꢀ 8.5, 2.5 Hz, 1H, 8-H), 6.75 (d, J ꢀ=ꢀ 8.5
Hz, 1H, 9-H), 6.87 (d, J ꢀ=ꢀ 2.5 Hz, 1H, H-6), 6.98–7.02 (m, 4H,
ArH), 7.57 (d, J ꢀ=ꢀ 8.7 Hz, 2H, ArH), 7.71 (d, J ꢀ=ꢀ 8.5 Hz, 2H,
ArH) ppm. – 13C NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 23.4, 30.3, 57.1,
115.1, 122.5, 128.2, 129.3, 135.2, 138.9, 151.2, 157.8, 166.4 ppm.
–
15N NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 202.1 (N-1), 294.5 (N-2)
ppm. – Anal. for C26H24N2O3 (412.5): calcd. C 75.71, H 5.86, N
6.79; found: C 75.84, H 5.73, N 6.63.
4.3.1.15 3-(4-Hydroxyphenyl)-1-phenyl-4,5-dihydro-
2H-benzo[g]indazol-7-ol (14)
A stirred CH2Cl2 solution of 12 (23 mg, 0.06 mmol) was
deprotected using BBr3, according to the general demeth-
ylation procedure. Purification by flash chromatography
(50 % EtOAc-hexanes) afforded the title compound as
white crystalline solid (16 mg, 91 %). M.p. 165–166 °C. – 4.3.1.18 6-Methoxy-3-(4-methoxyphenyl)-1-phenyl
1
IR (KBr): ν ꢀ=ꢀ 3201 cm–1. – H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ
-1,4-dihydro-indeno[1,2-c]pyrazole (17)
2.84–2.90 (m, 4H, 4-H, 5-H), 6.46 (dd, J ꢀ=ꢀ 8.6, 2.4 Hz, 1H, Triketone 16 (50 mg, 0.12 mmol) and phenylhydrazine
8-H), 6.64 (d, J ꢀ=ꢀ 8.6 Hz, 1H, 9-H), 6.84 (d, J ꢀ=ꢀ 2.4 Hz, 1H, hydrochloride (0.16 g, 0.37 mmol) were reacted as outlined
6-H), 6.89 (d, J ꢀ=ꢀ 8.6 Hz, 2H, ArH), 7.41–7.46 (m, 1H, ArH), above to afford 17 as amorphous yellow solid after flash
7.48–7.53 (m, 3H, ArH), 7.59 (d, J ꢀ=ꢀ 9 Hz, 2H, ArH), 7.60–7.63 chromatographic purification (88 %). M.p. 149–150 °C. –
(m, 1H, ArH), 8.48 (bs, 1H, OH), 8.52 (bs, 1H, OH) ppm. 1H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 3.81 (s, 2H, 4-H), 3.83 (s, 3H,
13
– C NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 23.8, 30.5, 115.1, 122.1, CH3O), 3.88 (s, 3H, CH3O), 6.83 (dd, J ꢀ=ꢀ 8.5, 2.3 Hz, 1H, 7-H),
128.9, 129.6, 135.7, 138.9, 148.9, 150.2, 157.4 ppm. – Anal. for 6.99 (d, J ꢀ=ꢀ 8.7 Hz, 2H, ArH), 7.13 (d, J ꢀ=ꢀ 2.3 Hz, 1H, 5-H),
C23H18N2O2 (354.4): calcd. C 77.95, H 5.12, N 7.90; found: C 7.43 (d, J ꢀ=ꢀ 8.7 Hz, 2H, ArH), 7.54 (d, J ꢀ=ꢀ 8.7 Hz, 1H, 8-H),
77.78, H 5.01, N 7.73.
7.56 (d, J ꢀ=ꢀ 8.8 Hz, 1H, ArH), 7.78 (d, J ꢀ=ꢀ 8.8 Hz, 2H, ArH),
7.90 (d, J ꢀ=ꢀ 8.8 Hz, 2H, ArH) ppm. – 13C NMR (400 MHz,
CDCl3): δ ꢀ=ꢀ 28.5, 57.8, 115.3, 122.6, 125.5, 129.9, 135.3, 138.8,
152.2, 163.5 ppm. – 15N NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 202.8
(N-1), 301.7 (N-2) ppm. – Anal. for C24H20N2O2 (368.4): calcd.
4.3.1.16 2,3-Di-(4-hydroxyphenyl)-4,5-dihydro-
2H-benzo[g]indazol-7-ol (15)
A stirred CH2Cl2 solution of 13 (23 mg, 0.06 mmol) was C 78.24, H 5.47, N 7.60; found: C 78.49, H 5.33, N 7.71.
deprotected using BBr3, according to the general demeth-
ylation procedure. Purification by flash chromatography
(50 % EtOAc-hexanes) afforded the title compound as 4.3.1.19 6-Methoxy-1,3-bis(4-methoxyphenyl)
white crystalline solid (16 mg, 89 %). M.p. 301–303 °C. –
-1,4-dihydro-indeno[1,2-c]pyrazole (18)
IR (KBr): ν ꢀ=ꢀ 3470, 3501 cm–1. – H NMR (400 MHz, [D6] Triketone 16 (70 mg, 0.16 mmol) was reacted with 4-meth-
acetone): δ ꢀ=ꢀ 2.86–2.96 (m, 4H, 4-H, 5-H), 6.45 (dd, J ꢀ=ꢀ 8.5, oxyphenylhydrazine hydrochloride (0.12 g, 0.64 mmol)
2.4 Hz, 1H, 8-H), 6.70 (d, J ꢀ=ꢀ 8.5 Hz, 1H, 9-H), 6.85 (d, J ꢀ=ꢀ according to the general procedure to afford 18 as amor-
2.4 Hz, 1H, 6-H), 6.92 (d, J ꢀ=ꢀ 8.9 Hz, 2H, ArH), 7.00 (d, J ꢀ=ꢀ phous pale-yellow solid (30 mg, 90 %) after purification
8.5 Hz, 2H, ArH), 7.32 (d, J ꢀ=ꢀ 8.9 Hz, 2H, ArH), 7.62 (d, J ꢀ=ꢀ by flash chromatography (30 % EtOAc-hexanes). M.p. 157–
1
1
8.5 Hz, 2H, ArH), 8.50 (s, 1H, OH), 8.53 (s, 1H, OH), 8.86 (s, 159 °C. – H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 3.81 (s, 2H, 4-H),
1H, OH) ppm. – 13C NMR (400 MHz, [D6]acetone): δ ꢀ=ꢀ 155, 3.83 (s, 3H, CH3O), 3.85 (s, 3H, CH3O), 3.89 (s, 3H, CH3O),
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6.79 (dd, J ꢀ=ꢀ 8.8, 2.4 Hz, 1H, 7-H), 6.98 (d, J ꢀ=ꢀ 8.4 Hz, 2H, a syringe in one-pot under stirring. After approximately
ArH), 7.05 (d, J ꢀ=ꢀ 8.8 Hz, 2H, ArH), 7.12 (d, J ꢀ=ꢀ 2.4 Hz, 1H, 1 min to allow the formation of the anion, p-anisoyl chlo-
5-H), 7.35 (d, J ꢀ=ꢀ 8.8 Hz, 1H, 8-H), 7.65 (d, J ꢀ=ꢀ 8.8 Hz, 2H, ride (0.15 mL, 1.11 mmol) was added. The ice bath was
ArH), 7.88 (d, J ꢀ=ꢀ 8.8 Hz, 2H, ArH). – 13C NMR (400 MHz, removed, and after 1 min, 2 mL of AcOH was added. The
CDCl3): δ ꢀ=ꢀ 26.8, 55.4, 115.7, 122.7, 124.2, 128.9, 135.3, 138.4, reaction was stirred for an additional 2 min, and consecu-
152.6, 157.4, 163.5 ppm. – 15N NMR (CDCl3): δ ꢀ=ꢀ 201.9 (N-1), tively EtOH (10 mL), THF (5 mL), and an excess of 4-meth-
302.4 (N-2) ppm. – Anal. for C25H22N2O3 (398.5): calcd. C oxyphenylhydrazine hydrochloride (1.73 g, 10 mmol) were
75.36, H 5.57, N 7.03; found: C 75.55, H 5.39, N 7.15.
added. The reaction mixture was refluxed for 15 min and
completion of the reaction was verified by TLC. Then,
the resulting solution was added to 5 mL of 1.0 m NaOH
and extracted twice with EtOAc (2 ꢀ×ꢀ 20 mL). The com-
bined organic fractions were washed with brine, dried
4.3.1.20 1,4-Dihydro-3-(4-hydroxyphenyl)-
1-phenylindeno[1,2-c]pyrazol-6-ol (19)
A stirred CH2Cl2 solution of 17 (35 mg, 0.09 mmol) was over Na2SO4, and evaporated under reduced pressure.
deprotected using BBr3, according to the general demeth- The resulting residue was purified by flash column chro-
ylation procedure. Purification by flash chromatography matography using a mixture of n-hexane–EtOAc (8:2) to
(40 % EtOAc-hexanes) afforded 26 mg of the title compound provide compound 22 (0.61 g, 60 %) as pale-pink crystal-
as white crystalline solid in 82 % yield. M.p. 211–212 °C. – line needles. M.p. 161 °C. – 1H NMR (400 MHz, CDCl3): δ ꢀ=ꢀ
IR (KBr): ν ꢀ=ꢀ 3307 cm–1. – 1H NMR (400 MHz, [D6]acetone): 2.79 (t, J ꢀ=ꢀ 8.0 Hz, 2H, 3-H), 3.04 (t, J ꢀ=ꢀ 8.3 Hz, 2H, 4-H), 3.85
δ ꢀ=ꢀ 3.81 (s, 1H, 4-H), 6.81 (dd, J ꢀ=ꢀ 8.2, 2.3 Hz, 1H, 7-H), 6.95 (s, 3H, CH3O), 3.89 (s, 3H, CH3O), 5.08 (s, 2H, NCH2Bz), 5.81
(d, J ꢀ=ꢀ 8.5 Hz, 2H ArH), 7.14 (d, J ꢀ=ꢀ 2.3 Hz, 1H, 5-H), 7.42 (d, (d, J ꢀ=ꢀ 2.9 Hz, 1H, pyrrole), 6.51 (d, J ꢀ=ꢀ 2.9 Hz, 1H, pyrrole),
J ꢀ=ꢀ 8.5 Hz, 1H, ArH), 7.45 (d, J ꢀ=ꢀ 7.6 Hz, 1H, ArH), 7.62 (d, J ꢀ=ꢀ 6.97 (d, J ꢀ=ꢀ 8.9 Hz, 2H, ArH), 7.01 (d, J ꢀ=ꢀ 8.9 Hz, 2H, ArH),
8.2 Hz, 1H, 8-H), 7.65 (d, J ꢀ=ꢀ 7.6 Hz, 1H, ArH), 7.82 (d, J ꢀ=ꢀ 7.9 7.06 (d, J ꢀ=ꢀ 7.2 Hz, 2H, ArH), 7.26–7.33 (m, 3H, ArH), 7.58 (d,
Hz, 2H, ArH), 7.85 (d, J ꢀ=ꢀ 8.5 Hz, 2H, ArH), 8.52 (s, 1H, OH), J ꢀ=ꢀ 8.9 Hz, 2H, ArH), 7.69 (d, J ꢀ=ꢀ 8.9 Hz, 2H, ArH) ppm. –
8.60 (s, 1H, OH) ppm. – 13C NMR (400 MHz, [D6]acetone): 13C NMR (400 MHz, CDCl3): δ ꢀ=ꢀ 22.8, 54.8, 95.4, 115.6, 119.2,
δ ꢀ=ꢀ 29.5, 115.4, 123.8, 128.4, 129.6, 135.3, 138.3, 148.8, 151.9, 127.1, 132.5, 134.7, 146.9, 160.9 ppm. – Anal. for C30H27N3O2
157.1 ppm. – Anal. for C22H16N2O2 (340.4): C 77.63, H 4.74, N (461.6): calcd. C, 78.07, H, 5.90, N, 9.10; found: C, 77.72, H,
8.23; found: C 77.75, H 4.86, N 8.39.
5.72, N, 9.28.
4.3.1.21 1,3-Bis-(4-Hydroxyphenyl)-1,4-dihydro-
indeno[1,2-c]pyrazol-6-ol (20)
4.3.1.23 4,4′-(6-Benzyl-4,5-dihydropyrrolo[2,3-g]
indazole-1,3(6H)-diyl)diphenol (23)
A stirred CH2Cl2 solution of 18 (19 mg, 0.05 mmol) was
deprotected using BBr3, according to the general demeth- Compound 23 was prepared from compound 22, follow-
ylation procedure. Purification by flash chromatography ing the general demethylation protocol (85 % yield, pale-
1
(40 % EtOAc-hexanes) afforded the title compound as yellow crystals). M.p. 190 °C. – H NMR (400 MHz, [D6]
white crystalline solid (15 mg, 90 %). M.p. 279–281 °C. – acetone): δ ꢀ=ꢀ 2.80 (t, J ꢀ=ꢀ 8.0 Hz, 2H, H-3), 2.99 (t, J ꢀ=ꢀ 8.0
1
IR: ν ꢀ=ꢀ 3245 cm–1. – H NMR (400 MHz, [D6]acetone): δ ꢀ=ꢀ Hz, 2H, H-4), 5.21 (s, 2H, NCH2Bz), 5.78 (d, J ꢀ=ꢀ 2.8 Hz, 1H,
3.81 (s, 1H, 4-H), 6.79 (dd, J ꢀ=ꢀ 8.2, 2.1 Hz, 1H, 7-H), 6.93 (d, pyrrole), 6.68 (d, J ꢀ=ꢀ 2.6 Hz, 1H, pyrrole), 6.89 (d, J ꢀ=ꢀ 8.6
J ꢀ=ꢀ 8.5 Hz, 2H, ArH), 7.07 (d, J ꢀ=ꢀ 8.8 Hz, 2H, ArH), 7.12 (d, Hz, 2H, ArH), 6.99 (d, J ꢀ=ꢀ 8.5 Hz, 2H, ArH), 7.15 (d, J ꢀ=ꢀ 7.5
J ꢀ=ꢀ 2.1 Hz, 1H, 5-H), 7.31 (d, J ꢀ=ꢀ 8.2 Hz, 1H, 8-H), 7.59 (d, J ꢀ=ꢀ Hz, 2H, ArH), 7.26–7.34 (m, 3H, ArH), 7.44 (d, J ꢀ=ꢀ 8.6 Hz, 2H,
8.5 Hz, 2H, ArH), 7.80 (d, J ꢀ=ꢀ 8.5 Hz, 2H, ArH), 8.50 (s, 1H, ArH), 7.60 (d, J ꢀ=ꢀ 8.6 Hz, 2H, ArH), 8.40 (s, 1H, OH), 8.75 (s,
OH), 8.53 (s, 1H, OH), 8.78 (s, 1H, OH) ppm. – 13C NMR (400 1H, OH) ppm. – 13C NMR (400 MHz, [D6]acetone): δ ꢀ=ꢀ 21.2,
MHz, [D6]acetone): δ ꢀ=ꢀ 29.9, 115.5, 118.4, 129.4, 135.7, 138.8, 54.5, 95.6, 115.1, 118.8, 127.4, 132.2, 134.3, 144.9, 155.5 ppm.
148.9, 151.2, 158.5 ppm. – Anal. for C22H16N2O3 (356.4): calcd. – Anal. for C28H23N3O2 (433.5): calcd. C 77.58, H 5.35, N 9.69;
C 74.15, H 4.53, N 7.86; found: C 74.38, H 4.68, N 7.97.
found: C 78.34, H 5.22, N 10.18.
4.3.1.22 6-Benzyl-1,3-bis(4-methoxyphenyl)-1,4,5,
6-tetrahydropyrrolo[2,3-g]indazole (22)
4.3.1.24 6-Benzyl-3-(3-chlorobenzo[b]thiophen-2-yl)-
2,4,5,6-tetrahydropyrrolo[2,3-g]indazole (24)
Compound 21 (0.5 g, 2.2 mmol) was dissolved in 7 mL
of anhydrous toluene, cooled to 0 °C under argon and Compound 24 was prepared following the same proce-
LiHMDS (2.3 mL, 1.0 m in THF, 2.3 mmol) and added via dure presented for the synthesis of compound 22. Initially
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3-chlorobenzo[b]thiophene-2-carbonyl chloride was Data were calculated as a percentage of inhibition by the
reacted with compound 21 0.5 g (2.2 mmol), and subse- following formula:
quently condensed with hydrazine. The end product 24
Inhibition(%) = [(O.D.control − O.D.sample ) /O.D.control ] × 100
(0.44 g, 49 % yield) was obtained in the form of yellow
crystals. M.p. 144–146 °C. – 1H NMR (400 MHz, [D6] where O.D.control and O.D.sample indicate the optical density
acetone): δ ꢀ=ꢀ 2.80 (t, J ꢀ=ꢀ 8.0 Hz, 2H, 3-H), 2.97 (t, J ꢀ=ꢀ 8.5 of the negative control and the tested substances, respec-
Hz, 2H, 4-H), 5.22 (s, 2H, –CH2Bz), 6.38 (d, J ꢀ=ꢀ 2.6 Hz, 1H, tively. The concentration of the tested compounds caused
1-Hpyrrole), 6.82 (d, J ꢀ=ꢀ 2.8 Hz, 1H, 2-Hpyrrole), 7.15 (d, J ꢀ=ꢀ 7.5 Hz, 50 % cellular proliferation inhibition of cancer cells (IC50),
2H, ArH), 7.36 (m, 3H, ArH), 7.49 (t, J ꢀ=ꢀ 8.0 Hz, 1H, 5-H), which was calculated thereafter from the graph plotted
7.55 (t, J ꢀ=ꢀ 8.0 Hz, 1H, 6-H), 7.87 (d, J ꢀ=ꢀ 7.7 Hz, 1H, 4-H), 7.98 percentage inhibition against extract concentration. All
(d, J ꢀ=ꢀ 8.0 Hz, 1H, 3-H). – 13C NMR (400 MHz, [D6]acetone): experiments were carried out in triplicate and at least on
δ ꢀ=ꢀ 18.9, 22.8, 54.9, 98.6, 116.8, 120.9, 125.1, 128.9, 134.1, three separate occasions.
140.9 ppm. – Anal. for C24H18ClN3S (415.9): calcd. C 69.30,
H 4.36, Cl 8.52, N 10.10, S 7.71; found: C 71.03, H, 4.16, Cl,
4.6 Assessment of VEGF expression levels
8.75, N 9.76, S 7.61.
The human VEGF Quantakine ELISA kit (R&D Systems, MN,
USA) was used for the quantitative determination of VEGF
in culture supernates of HeLa cells. Cells were treated with
4.4 Cell cultures and reagents
vehicle control (1 % dimethyl sulfoxide [DMSO] in the cell
The human liver cancer HepG2 cell was donated by
culture medium) or different concentrations of tested com-
Dr. P. Liakos (University of Thessaly, Greece), and MCF-7 and
pounds for 24 h. After treatment, cell culture supernatants
cervix cancer HeLa cells were obtained from Dr. A.-M. Psarra
were collected, and subjected to VEGF ELISA according to
(University of Thessaly, Greece). All cells were cultured in
the manufacturer’s protocol. Absorbance was measured
standard Dulbecco’s modified Eagle’s medium (DMEM,
using an automatic microplate reader BioTek ELx800
Gibko, UK), containing 10 % (v/v) fetal bovine serum, 2 mm
(Winooski, VT, USA) at 450 and 540 nm. The concentration
l-glutamine (Gibko, UK), 100 units per mL of penicillin, and
of VEGF-A in each sample was calculated as in the pub-
100 units per mL of streptomycin (Gibko, UK) in plastic dis-
lished procedure by Myers et al. [29].
posable tissue culture flasks at 37 °C in 5 % CO2.
4.5 XTT assay for cell proliferation inhibition 4.7 Tube formation assay
Cell proliferation inhibition was assessed using the XTT Briefly, 150 μL of an extracellular matrix solution
assay kit (Roche, Germany). Briefly, cell lines were sub- (MatrigelTM, BD Biosciences, NJ, USA) was added to each
cultured into a 96-well plate with 1 ꢀ×ꢀ 104 cells per well well of a six-well plate and allowed to solidify for at least
for HepG2 and MCF-7 cells and 5 ꢀ×ꢀ 103 cells per well for 30 min at 37 °C. Afterward, HMEC-1 were plated (1 ꢀ×ꢀ 105
HeLa cells in the DMEM. After 24 h of incubation, the cells cells per well) on the surface of the matrigel and treated
were treated with increasing concentrations (1–240 μm) with tested compounds (30 and 100 μm) or vehicle. Cells
of tested compounds in the serum-free DMEM for 24 h. were incubated for 16 h, and then the effect of tested
A volume of 50 μL of XTT test solution, which was pre- compounds on tubular morphogenesis was documented
pared by mixing 50 μL of XTT-labeling reagent with 1 μL microscopically and photographed. Each experiment was
of electron coupling reagent, was then added to each repeated minimum three times.
well. After 4 h of incubation, absorbance was measured at
450 nm and also at 690 nm as a reference wavelength in
a BioTek ELx800 microplate reader (Winooski, VT, USA). 4.8 Statistical analysis
The serum-free DMEM was used as a negative control.
Also, the absorbance of each tested compound concentra- All results are expressed as mean SD (n ꢀ=ꢀ 3). For statis-
tion alone in the serum-free DMEM and XTT test solution tical analysis, one-way ANOVA was applied followed by
was tested at 450 nm. The absorbance values shown by Dunnett’s test for multiple pairwise comparisons. Spear-
the tested compounds alone were subtracted from those man’s correlation analysis examined dose response rela-
derived from cancer cell treatment with grape extracts. tionships. Differences were considered significant when
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14ꢀ ꢀK.M. Kasiotis et al.: Novel pyrazole derivatives as anti-cancer agents
[9] I. M. Yonova, C. A. Osborne, N. S. Morrissette, E. R. Jarvo,
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two-dimensional NMR spectra are presented in order to
facilitate the reading of this manuscript (http://dx.doi.
org/10.1515/znb-2015-0053).
Acknowledgments: This work was supported by grants
from Thalis project with acronym “SERMENCO” entitled
“Development of Selective Estrogen Receptor Modulators
as Agents against Menopause.” This research project was
co-funded by the Greek Operational Programme “Educa-
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Supplemental Material: The online version of this article
(DOI: 10.1515/znb-2015-0053) offers supplementary material,
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Z. Naturforsch.ꢂ
ꢂ2015 | Volume x | Issue x(b)
Graphical synopsis
Konstantinos M. Kasiotis,
Evangelia N. Tzanetou, Dimitrios
Stagos, Nikolas Fokialakis,
Eleni Koutsotheodorou, Dimitrios
Kouretas and Serkos A. Haroutounian
Novel conformationally constrained
pyrazole derivatives as potential anti-
cancer agents
DOI 10.1515/znb-2015-0053
Z. Naturforsch. 2015; x(x)b: xxx–xxx
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