Synthesis of some pyrazole derivatives and preliminary investigation of their affinity binding to P-glycoprotein

Article abstract of DOI:10.1016/j.bmcl.2005.05.067

A series of substituted pyrazolines were synthesized and evaluated for their anticancer activity and for their ability to inhibit P-glycoprotein- mediated multidrug resistance by direct binding to a purified protein domain containing an ATP-binding site and a modulator interacting region. Compounds 2a and e have been found to bind to P-glycoprotein with greater affinity.

Full text of DOI:10.1016/j.bmcl.2005.05.067

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4632–4635
Synthesis of some pyrazole derivatives and preliminary
investigation of their affinity binding to P-glycoprotein
Fedele Manna,a Franco Chimenti,a Rossella Fioravanti,a,* Adriana Bolasco,a
Daniela Secci,a Paola Chimenti,a Cristiano Ferlinib and Giovanni Scambiab
a
`
Dip. di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive. Universita di Roma ‘‘La Sapienza.’’
P.le Aldo Moro, 5, 00185 Rome, Italy
b
`
Dip. di Ostetricia e Ginecologia, Universita Cattolica del Sacro Cuore, Largo Agostino Gemelli, 8 00168 Rome, Italy
Received 15 March 2005; revised 11 May 2005; accepted 17 May 2005
Available online 15 August 2005
Abstract—A series of substituted pyrazolines were synthesized and evaluated for their anticancer activity and for their ability
to inhibit P-glycoprotein-mediated multidrug resistance by direct binding to a purified protein domain containing an ATP-binding
site and a modulator interacting region. Compounds 2a and e have been found to bind to P-glycoprotein with greater affinity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The identification of novel structures that can be poten-
tially useful in designing new, potent selective, and less
toxic anticancer agents is still a major challenge to
medicinal chemistry researchers.
As reported in the literature,1 1,3-diaryl-2-propen-1-
ones or chalcones 1 (see Chart 1) are a class of antican-
cer agents that have shown promising therapeutic effica-
cy in the management of human cancers. Several
chalcones are cytotoxic toward a number of different tu-
mor lines. In particular, they inhibit the proliferation of
both established and primary ovarian cells. Moreover,
recent studies have shown that chalcones also induce
apoptosis in a variety of cell types, including breast can-
cer.2,3 Chalcones have shown preferential reactivity
toward thiols4 in contrast to amino and hydroxyl
Chart 1. 1, 1,3-Diaryl-2-propen-1-ones (chalcones); 2, 1-acetyl-3,5-
diphenyl-4,5-dihydro-(1H)-pyrazole; 3, 4-hydroxypyrazole C-glycoside
(pyrazofurin).
groups. Therefore, because interactions with nucleic
acids may be absent, problems of mutagenicity and car-
cinogenicity that have been associated with certain
alkylating agents used in cancer chemotherapy could
be eliminated. It is important to note that the resistance
of human malignancy to chemotherapeutic agents re-
mains a major obstacle to an efficient cancer therapy.
The induction of multiple drug resistance (MDR) is
the phenomenon in which the exposure of tumor cells
to a single cytotoxic agent results in cross-resistance to
the other structurally unrelated classes of cytotoxic
agents. Moreover, on account of MDR many bacteria
that cause infections are becoming increasingly difficult
to treat.5–7 The multidrug resistance of cancer cells is of-
ten associated with an accelerated efflux of the chemo-
therapeutic agent by an ATP-dependent process. A
family of proteins is responsible for this efflux. In partic-
ular, the over-expression of P-glycoprotein (P-gp), an
ABC-type plasma membrane transporter that rejects
Keywords: Anticancer; Pyrazoline; P-glycoprotein.
*
Corresponding author. Tel.: +39 06 49913975; fax: +39 06
49913763; e-mail: rossella.fioravanti@uniroma.it
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.067

F. Manna et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4632–4635
4633
chemotherapeutic drugs out of the cell using ATP
hydrolysis as an energy source.8 On account of the del-
eterious effect of P-glycoprotein on chemotherapeutic
efficiency, compounds that modify its function have a
potential by clinical value. Recently, several inhibitors
of P-glycoprotein-mediated drug efflux have been
identified. These so-called MDR modulators lead to
resensitization of multidrug resistant tumor cells to che-
motherapeutic agents. They include a variety of struc-
tural classes, such as calcium channel blockers (such as
verapamil, anthracyclines, vinca alkaloids, taxanes,
and epipodophyllotoxins) and immunosuppressors
(such as cyclosporine A that usually acts by competing
with cytotoxic drugs to bind to P-glycoprotein).9,10
iv
It is therefore of high priority to develop cytotoxic
agents that show good anticancer activity and can inhib-
it P-gp activity. Since chalcones 1 (see Chart 1) are inter-
mediates in the synthesis of pyrazoline, pursuing our
studies11,12 on these moieties we have synthesized and
evaluated the in vitro activity of derivatives 2 (see Chart
1) to investigate and evaluate their in vitro anticancer
activity, following literature reports on the discovery
of natural antibiotic pyrazofurin 3 (see Chart 1).13
Scheme 2. Reagents and condition: (i) PPTS, CH2Cl2; (ii) Ba(OH)2,
EtOH, 30 ꢁC; (iii) HCl 3 N, EtOH; (iv) N2H4, CH3COOH, refluxed.
Chalcones 1a–g were obtained by the direct Claisen–
Schmidt procedure, according to which the condensa-
tion of benzaldehyde derivatives with the substituted
acetophenone is effected using barium hydroxide as a
catalyst in ethanol.14b
Pyrazofurin has received considerable attention as a re-
sult of its many biological effects, including potent anti-
microbial and broad spectrum antiviral activities against
many DNA and RNA viruses. It has been reported that
pyrazofurin has been evaluated against several cell lines.
Consequently, it has only been used clinically as an anti-
cancer agent. Lastly, we investigated the ability of deriv-
atives 2 to bind P-glycoprotein, since, to our knowledge,
there are no reports investigating the antitumor activity
of pyrazolines on multidrug resistant (MDR) cells
or their potential to modulate the protein activity of
P-glycoprotein (P-gp).
The hydroxyl groups present in derivatives 1h–u were
protected with 3,4-dihydro-a-pyrane and then hydro-
lyzed.14c Structural assignment for compounds is based
1
on UV and H NMR, and is according to the literature
data. The chemical and physical data are reported in
Table 1. The microanalyses of derivatives 2a–u are
reported in Table 2.
All compounds 2a–u were tested to evaluate their cyto-
toxicity against human tumor cell lines. Pyrazoline
derivatives were dissolved in DMSO (stock solution,
Pyrazolines 2a–u were prepared, according to the
one-pot synthesis methods shown in Schemes 1 and 2.
Starting from chalcone 1, the 1-acetyl-3,5-diphenyl-4,5-
dihydro-(1H)-pyrazole derivatives 2a–u were obtained
by the addition of hydrazine hydrate to acetic acid.14a
Table 1. Chemical and physical data of compounds 2a–u
Compound
R
R0
Yield (%)
Mp (ꢁC)
2a
2b
2c
2d
2e
2f
H
H
64
44
54
43
53
49
52
91
74
68
50
68
80
92
62
67
71
84
71
81
89
122–124
110–112
78–80
H
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
H
H
H
110–111
157–159
162–164
105–107
170–173
183–186
157–159
195–198
154–156
146–149
184–187
126–128
130–133
160–161
185–188
140–143
154–156
149–151
H
H
2g
2h
2i
H
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
2,4-OH
2,4-OH
2,4-OH
2,4-OH
2,4-OH
2,4-OH
2,4-OH
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
H
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
2t
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
Scheme 1. Reagents and conditions: (i) Ba(OH)2, EtOH, 25 ꢁC;
(ii) N2H4, CH3COOH, refluxed.
2u

4634
F. Manna et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4632–4635
Table 2. Table of microanalyses of derivatives 2a–u
data obtained in the three experiments with the sigmoid-
Emax model using non-linear regression, weighted by the
reciprocal of the concentration effect.15 All tested com-
pounds showed non-significant anticancer activity at
IC50 values higher than 10,000 nM.
Compound
C%
H%
N%
2a
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
Calcd
Found
84.53
85.50
84.58
84.57
84.58
84.60
84.58
84.55
80.46
80.49
80.46
80.46
80.46
80.44
80.23
80.20
80.46
80.49
80.46
80.45
80.46
80.48
76.72
76.75
76.72
76.70
76.72
76.69
76.34
76.34
76.72
76.70
76.72
76.75
76.72
76.71
73.32
73.30
73.32
73.31
73.32
73.35
6.08
6.10
6.45
6.44
6.45
6.44
6.45
6.47
6.14
6.13
6.14
6.15
6.14
6.15
5.77
5.80
6.14
6.12
6.14
6.12
6.14
6.13
5.85
5.81
5.85
5.83
5.85
5.86
5.49
5.45
5.85
5.82
5.85
5.90
5.85
5.84
5.59
5.57
5.59
5.61
5.59
5.58
9.39
9.40
8.97
8.99
8.97
8.99
8.97
8.98
8.53
8.55
8.53
8.52
8.53
8.55
8.91
8.92
8.53
8.55
8.53
8.56
8.53
8.54
8.13
8.15
8.13
8.16
8.13
8.16
8.48
8.51
8.13
8.19
8.13
8.10
8.13
8.16
7.77
7.79
7.77
7.80
7.77
7.75
2b
2c
2d
2e
2f
All compounds 2a–u were also tested to investigate their
affinity binding to P-glycoprotein. A Rhodamine 123
(Rh 123) fluorescent probe (molecular probes) was used
to measure the functionality of the P-gp efflux pump
according to the protocol of the National Cancer Insti-
tute Drug Screen16, except for a minor modification.
Briefly, MCF-7 ADRr cells were loaded at 37 ꢁC with
0.5 lg/ml of the dye in PBS supplemented by 0.2%
BSA. After 15 min, the cells were transferred onto ice
and washed twice to remove free Rh 123 from the medi-
um. After washing, 10 lM of the potential P-gp inhibi-
tors was added and the cells were kept at 37 ꢁC for
30–120 min. The positive control was Quercetin, a
well-known inhibitor of the P-gp function. An aliquot
of cells was maintained on ice to prevent dye efflux (con-
trol at 4 ꢁC), and maximal efflux was effected by adding
the vehicle DMSO 0.1% and permitting efflux at 37 ꢁC.
As regards flow cytometric acquisition, a minimum of
20,000 cells were acquired using an Epics-XL flow
cytometer with standard collection filters and electron-
ics. The mean channel of Rh 123 fluorescence was calcu-
lated for each condition and time point. The ratio of
mean channel between control at 37 ꢁC and control at
4 ꢁC was considered as the control dye efflux. Similarly,
the mean channel of Rh 123 fluorescence of treated cells
was divided by the control at 4 ꢁC. This ratio was divid-
ed by the control dye efflux to establish the potency of P-
gp inhibition. The results from two independent experi-
ments were averaged.
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
2t
The in vitro results of tests of P-gp inhibition are report-
ed in Figure 1. The data representation of derivatives, 2l,
o, p, t, and u, is not reported, because they are totally
inactive (0%) at a concentration value of 10 lM. Figure
1 shows the percentage of inhibition of Quercetin used
as control (100%, dashed line). Derivatives 2b, c, d, g,
i, j, k, m, and q are comparable to or less active than
the control (6100%); derivatives 2f, h, n, and r show a
2u
10 mM) and used within 7 days. The final DMSO con-
centration never exceeded 0.1% (v/v) either in controls
or in treated samples.
The cytotoxic activities of the synthesized compounds
were evaluated in vitro against A2780, A2780-CIS,
and ECACC.
To perform growth experiments, cells were seeded
(20,000 cells/well) in 96-well flat-bottomed plates (Cul-
tureplates, Perkin-Elmer Life Science). After 24 h, the
media were replaced, and after one wash those contain-
ing the drugs were added. Three independent experi-
ments were performed in quadruplicate. After 72 h of
culture in the presence of the tested compounds, the
plates were harvested and the number of viable cells
was estimated by dosing ATP using the ATPlite kit (Per-
kin-Elmer Life Science). The kit was employed following
manufacturerÕs suggestions. For each drug/cell line, a
dose–response curve was plotted and IC50 values were
then calculated by fitting the concentration–effect curve
Figure 1. Percentage of P-glycoprotein inhibition of derivatives 2a–k,
m, n, q, r, and s in comparison to Quercetin (P-gp inhibition 100%
dashed line).

F. Manna et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4632–4635
4635
10. Chou, T. C.; Depew, K. M.; Zheng, Y. H.; Safer, M. L.;
Chan, D.; Helfrich, B.; Zatorska, D.; Zatorski, A.;
Bornmann, W.; Danishefsky, S. J. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 8369.
better binding affinity to P-glycoprotein than the control
(>100%). The most active derivatives are 2a, e, and s,
which are three (2s) or four times (2a and e) more potent
than Quercetin. Considering the data in Figure 1 of this
preliminary study, the following point on the structure–
activity relationship needs to be highlighted. The highest
affinity to P-gp was observed with compounds 2a and e,
where ring A remains unsubstituted, and which appears
to be the most important prerequisite for affinity when
comparing with other compounds. The presence of an
R0 substituent, such as hydrogen or 2-OCH3 group, in
ring B also appears to be an important factor. In fact,
when R = H in ring A, the presence of a methyl group
and of a 3- or 4-OCH3 group in R0 gives compounds
with no (2b–d, g) or non-significant affinity (2f). The
only exception was that observed with compound 2s,
which bears 2,4-OH in ring A and a 2-OCH3 group in
ring B, and whose affinity to P-gp is one time lower than
those of 2a and e. All compounds with a 4-OH or 2,4-
OH in ring A (2h–n) showed no or very weak affinity
to P-gp, except for the above-mentioned compound 2s.
11. Manna, F.; Chimenti, F.; Bolasco, A.; Secci, D.; Bizzarri,
´
B.; Befani, O.; Turini, P.; Mondovı, B.; Alcaro, S.; Tafi, A.
Bioorg. Med. Chem. Lett. 2002, 12, 3629.
12. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.;
Granese, A.; Befani, O.; Turini, P.; Alcaro, S.; Ortuso,
F. Bioorg. Med. Chem. Lett. 2004, 14, 3697.
13. Rostom, S. A. F.; Shalaby, M. A.; El Demellawy, M. A.
Eur. J. Med. Chem. 2003, 38, 959.
14. (a) A solution of chalcones 1a–u (0.03 mol) in 30 mL of
acetic acid was added dropwise to a solution of hydrazine
hydrate (0.06 mol) in 10 mL absolute ethanol and stirred
with refluxing for 24 h. The mixture was then poured into
ice to obtain the crude products 2a–u, which were
crystallized from ethanol; (b) Barium hydroxide octahy-
drate (0.03 mol) was added to a solution of the appropri-
ate acetophenone (0.03 mol) and the appropriate aryl
aldehyde (0.03 mol) in 96% ethanol. The suspension was
stirred overnight at room temperature. The reaction
mixture was then poured into ice and extracted with
chloroform after neutralization. The organic layer was
washed with water (3· 20 mL), dried (Na2SO4), and
concentrated in vacuo to obtain the crude product.
Chalcones 1a–g were crystallized from methanol; (c) A
solution of 3,4-dihydro-a-pyran in methylene chloride
(30 mL) was added dropwise to a suspension of suitable
acetophenone (0.03 mol) and pyridinium p-toluene sulfo-
nate (PPTS) (0.015 mol) in methylene chloride (20 mL),
and stirred at room temperature for one night. The
reaction mixture was then poured into ice and extracted
with chloroform (4· 20 mL). The organic layer was
washed with water (3· 20 mL), dried (Na2SO4), and
concentrated in vacuo to obtain the protected acetophe-
none. Barium hydroxide octahydrate (0.03 mol) was
added to a solution of 4-hydroxy-4-(tetrahydropyran-2-
yloxy) acetophenone (0.03 mol) or 2,4-dihydroxy-4-(tetra-
hydropyran-2yloxy)acetophenone (0.03 mol) and appro-
priate benzaldehyde (0.03 mol) in dry ethanol and stirred
for 24 h at 30 ꢁC. The reaction mixture was then poured
into ice and extracted with chloroform after neutraliza-
tion. The organic layer was washed with water (3· 20 mL),
dried (Na2SO4), and concentrated in vacuo to obtain the
crude 4-hydroxy-4-(tetrahydropyran-2-yloxy) chalcone or
2,4-dihydroxy-4-(tetrahydropyran-2-yloxy) chalcone. The
crude products were suspended in ethanol and HCl 3 N
(15 mL) was added. The reaction suspension was stirred
for one night at room temperature, then poured into ice,
neutralized with Na2CO3, and extracted with chloroform
(3· 30 mL). The organic layer was dried over sodium
sulfate and evaporated in vacuo to give the crude product.
Chalcones 1h–u were crystallized from methanol.
Acknowledgments
This work is supported by a grant from MURST 60%.
We also acknowledge Mr. Anton Gerada, a professional
translator, Fellow of the Institute of Translation and
Interpreting of London and Member of AIIC (Associa-
ˆ
tion Internationale des Interpretes de Conferences—
Geneva) for revising the manuscript.
´
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