Tridentate bipyrazole compounds with a side-arm as a new class of antitumor agents

Article abstract of DOI:10.1007/s11164-012-0993-z

Eight tridentate bipyrazole derivatives with different side arms have been prepared in one step and with good yields. The products were screened for their cytotoxic activity against three tumor cell lines - human breast cancer cell line MDA-MB231, human prostate cancer cell line PC3, and human colorectal cell line LoVo, by use of colorimetric MTT assay. Structure-activity relationships reflected the effect of substituted drugs. Among this series, two compounds had remarkable in-vitro antiproliferative activity against the LoVo cell line with IC50 values ranging from 2.6 to 2.7 μg ml^-1. All the compounds had suitable drug-like characteristics according to Lipinski's rule.

Full text of DOI:10.1007/s11164-012-0993-z

Res Chem Intermed
DOI 10.1007/s11164-012-0993-z
Tridentate bipyrazole compounds with a side-arm
as a new class of antitumor agents
•
•
•
Fouad Malek Nihed Draoui Olivier Feron
Smaail Radi
Received: 13 October 2012 / Accepted: 19 December 2012
ꢀ Springer Science+Business Media Dordrecht 2013
Abstract Eight tridentate bipyrazole derivatives with different side arms have
been prepared in one step and with good yields. The products were screened for
their cytotoxic activity against three tumor cell lines—human breast cancer cell line
MDA-MB231, human prostate cancer cell line PC3, and human colorectal cell line
LoVo, by use of colorimetric MTT assay. Structure–activity relationships reflected
the effect of substituted drugs. Among this series, two compounds had remarkable
in-vitro antiproliferative activity against the LoVo cell line with IC50 values
ranging from 2.6 to 2.7 lg ml-1. All the compounds had suitable drug-like char-
acteristics according to Lipinski’s rule.
Keywords Bipyrazoles Á MTT assay Á Cytotoxicity Á LoVo Á MDA-MB231 Á PC3
Introduction
In 1959, the first natural pyrazole, 1-pyrazolylalanine, was isolated from
watermelon seeds. The term pyrazole was given to this class of compound by
Ludwig Knorr in 1883. Since the discovery of this natural structure, its derivatives
have been important in medicinal chemistry because of their wide range of
bioactivity, for example anti-inflammatory [1], anti-anxiety [2, 3], antipyretic [4],
F. Malek
LCOMPN-URAC 25, Facultes des Sciences, Universite Mohamed I, Oujda, Morocco
´
´
N. Draoui Á O. Feron
Angiogenesis and Cancer Research Lab, Pole of Pharmacology and Therapeutics—FATH5349,
Institute of Experimental and Clinical Research, UCL, Brussels, Belgium
S. Radi (&)
LCAE, Chemistry Department, Sciences Faculty, Mohamed the First University, Oujda, Morocco
e-mail: radi_smaail@yahoo.fr
123

F. Malek et al.
antimicrobial [5], antiviral [6], antitumor [7, 8], anticonvulsant [9], antihistaminic
[10], antidepressant [11], and antihyperglycemic [12], and as insecticides [13] and
fungicides [13]. The pyrazole structure is also found in a variety of kinase inhibitors
and it has also been found that these compounds have hypoglycemic activity; they
are also known to be inhibitors and deactivators of liver alcohol dehydrogenase and
oxidoreductases [14]. It has been shown in vivo that some pyrazole derivatives have
appreciable antihypertensive activity [15]. These compounds also have properties
such as cannabinoid hCB1 and hCB2 receptor activity, inhibition of p38 kinase, and
activity as CB1 receptor antagonists [16]. These characteristics suggest pyrazoles
would make good templates for lead-generation libraries.
In our recent work, a series of pyrazole compounds were prepared and shown to
have antitumor, antibacterial and antifungal activity [17–20]. In continuation of our
work in this field, this study was performed to investigate antitumor inhibition by
several bipyrazole units, with a lariat side, of breast cancer cell line MDA-MB231,
human prostate cancer cell line PC3, and human colorectal cell line LoVo, by use of
colorimetric MTT assay.
Results and discussion
Chemistry
Synthesis of the desired bipyrazoles 1–8 was accomplished as illustrated in
Scheme 1. The key starting materials, N-hydroxymethylpyrazoles, have already
been reported in old and recent papers [21–24]. Indeed, many heterocycles with a
ring NH group, including pyrazole, react with formaldehyde to yield addition
products of type N(CH2OH).
The target bipyrazoles were prepared, in one pot, by condensation of two
equivalents of N-hydroxymethylpyrazoles with one equivalent of amine derivatives,
under gentle conditions (room temperature, atmospheric pressure, 4–5 days), in
anhydrous acetonitrile as solvent. The reaction is very slow but is selective at room
temperature [25].
The new bipyrazole structures were characterized by spectroscopic methods (1H
NMR, IR, and MS) before the tests. Analytical and spectroscopic data for the known
Me
R
1
1
2
3
4
5
6
7
8
R1 = CH3
R2 = -CH2CH2OH
R2 = -CH2CH2OH
R2 = -CH2CH2OH
R2 = -cyclohexyl
R2 = -cyclohexyl
R2 = -(CH2)4OH
Me
OH
Me
N
ii
N
N
R1 = CO2Me
R1 = CO2Et
R1 = CO2Et
R1 = CH3
R1 = CH3
R1 = CH3
i
N
N
N
H
R
R
R
N
N
1
2
1
N
Me
R2 = -CH2CHOHCH3
R2 = -CH2CHOHCH3
R1 = CO2Et
R
1
i = Formol 35%, EtOH; ii = amine derivatives, room temperature for 4-5 days.
Scheme 1 Structure of the prepared products
123

Tridentate bipyrazole compounds with a side-arm
compounds were in agreement with those reported previously by our laboratory
[26–31].
Biological assays
Compounds 1–8 described in this manuscript were tested for their activity against
three human cancer cell lines by use of MTT tests. These cancer cell lines cover
three histological cancer types—breast (MDA-MB231), prostate (PC3), and
colorectal (LoVo).
The results are shown in Table 1.
As shown in our recent previous work, several of the monopyrazole derivatives
tested had no interesting effect against any of the three cancer cell lines examined
[17]. However, products herein containing two pyrazole rings had different anti-
proliferative activity against a variety of cancer cell lines, as evident from the range
of IC50 values (Table 1). Among the bipyrazoles, compound 6 and 7 consistently
had higher anti-proliferative effects against the colorectal cell line (LoVo). The
IC50 values were 2.6 and 2.7 lg ml-1 respectively.
The bipyrazole derivatives have two regions for SAR evaluation; the greater
potency of compounds 6 and 7 could be attributed most probably to the side arm
with the alcohol donor atom; it is known that a particular substituent may modify
activity relative to the parent compound by virtue of resulting changes in
hydrophobic, electronic, and steric effects [32]. As another characteristic, hydrogen-
bonding substituent properties were determined by a simple approach taking into
account the topological distances of the hydrogen bond donor and acceptor centers
from the substituent root [33].
In another variation, we alternated methyl and carboxylate groups at the 3-position
of the pyrazole region. The result indicated the importance of the methyl group in the
3-position for the three cancer cell lines examined; indicating methyl might be a good
substituent for modification. These results are in agreement with previous reports [17].
Table 1 IC50 values of compounds 1–8 against breast, prostate and colorectal cancer cell lines as
determined by MTT assay using Normoxie condition
Compound
MDA-MB231
PC3
LoVo
IC50
(lg ml-1
IC50
(lM)
IC50
(lg ml-1
IC50
(lM)
IC50
(lg ml-1
IC50
(lM)
)
)
)
1
2
3
4
5
6
7
8
20.8
27.7
N/A
32.2
N/A
20.6
21.3
28.5
75
8.5
30.9
36
6.3
6.0
31.2
9.6
5.9
2.6
2.7
9.9
22.7
16.6
79.5
22.4
18.9
8.5
76
13.1
N/A
15.9
21.9
11.4
8.6
N/A
74.7
N/A
67.5
73.4
69.9
N/A
37
69.5
37.3
29.5
51.5
9.3
21.0
24.3
N/A means non applicable because IC50 [ 100 lg ml-1
123

F. Malek et al.
The results obtained clearly indicate that the series of bipyrazoles discussed here
are moderately active toward the three cancer cell lines. Products 6 and 7 were most
active against the colorectal cell line (LoVo). We conclude that introduction of
different side arms can contribute favorably to antitumor activity; the activity could
also be related to different phenomena, for example complexation capacity, which is
easy in tripodal compounds.
The properties formulated in Lipinski’s rule of five were also considered. Both
active compounds 6 and 7 fulfil criteria for becoming orally active drugs
(Table 2).The rule states that, in general, an orally active drug violates no more
than one of the following criteria [34]:
•
no more than five hydrogen-bond donors (nitrogen or oxygen atoms with one or
more hydrogen atoms)
•
•
•
no more than ten hydrogen-bond acceptors (nitrogen or oxygen atoms)
a molecular mass \500 Da
an octanol–water partition coefficient log P not [5.
Experimental
Chemistry
¨
Melting points were determined by using a Buchı 510 m.p. apparatus. NMR spectra
were obtained with a Bruker AC 300 spectrometer. Infrared spectra were recorded
on a Perkin–Elmer 1310 spectrophotometer. Molecular weights were determined on
a Jeol JMS DX-300 mass spectrometer.
General procedure
The bipyrazole products were prepared by addition of primary amine derivatives
(5 mmol) to a solution of the substituted hydroxymethylpyrazole (10 mmol) in
acetonitrile (25 ml). The mixture was stirred at room temperature for 4–5 days. The
crude material was evaporated, washed with water and CH2Cl2, and purified by
silica gel column flash-chromatography to give the target product.
Table 2 Product properties with Lipinski’s rule of five
Lipinski’s rule of five
Compound 6
Compound 7
Hydrogen bond donors B 5
Hydrogen bond acceptors B 10
Molecular mass B 500 g mol-1
Coefficient log P B 5
1
1
6
6
305.40
291.39
2.51 0.56
3.18 0.57
123

Tridentate bipyrazole compounds with a side-arm
1-{Bis[(3-ethylcarboxylate-5-methyl-1H-pyrazol-1-yl)methyl]amino}propan-2-ol (8)
Yield 88 %. IR (KBr, cm-1): 3340 (mO–H), 2880 (m=C–H), 2800 (mC–H), 1710 (mC=O),
1440 (mC=C), 1H NMR (300 MHz, CDCl3) d ppm: 1.12 (d, 3H, CH3–CHOH–CH2N,
J = 6.0 Hz), 1.40 (t, 6H, –O–CH2–CH3, J = 6.9 Hz), 2.25 (m, 2H, –CHOH–
CH2N), 2.30 (s, 6H, pyrazole–CH3), 3.40 (br s, 1H, –CHOH–), 3.76 (m, 1H,
–CHOH–). 4.48 (q, 4H, –O–CH2–CH3, J = 6.9 Hz), 5.40 (s, 4H, N–CH2–N), 5.68
(s, 2H, PzH). m/z (M?): 407.46.
MTT cell-viability assay
Prepared compounds were screened against three tumor cell lines at the
Angiogenesis and Cancer Research Laboratory, Institute of Experimental and
Clinical Research (UCL, Brussels, Belgium).
MTT tests were performed to rapidly, i.e. within 3 days, measure the effect of
compounds on overall cell growth. The test measures the number of metabolically
active living cells that are able to transform the yellow product 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (herein referred to as MTT) into the
blue product formazan dye by mitochondrial reduction. The amount of formazan
obtained at the end of the experiment, measured by means of a spectrophotometer
(Plate Reader Victor 94 Microplate reader (Perkin Elmer), is directly proportional
to the number of living cells.
To perform the assay, cells were grown in 96-well microplates with a flat bottom
after addition of 100 ll cell suspension per well with 4,500 cells/well. Cell lines
were seeded in Glutamax-containing RPMI supplemented with 10 % foetal bovine
serum (PAA) and 1 % penicillin ? streptomycin mix (Invitrogen).
The detailed experimental procedure was as follows. After incubation for 24 h at
37 ꢁC, the culture medium was replaced with 100 ll fresh medium in which the
tested compounds had previously been dissolved at concentrations of 10-8
5 9 10-8, 10-7, 5 9 10-7, 10-6, 5 9 10-6, 10-5, 5 9 10-5, and 10-4 g ml-1
,
.
Each experiment was performed in sextuplicate. Cells were then incubated at 37 ꢁC
in a Binder incubator under a 5 % CO2-containing humidified atmosphere.
After incubationfor 72 h, with or without the compound beingtested, the medium was
replaced with 100 ll HBSS (without phenol red) containing MTT at a concentration of
1 mg/ml. The micro-wells were subsequently incubated for 3 h at 37 ꢁC thencentrifuged
at 1,300 rpm for 10 min. Medium was removed and formazan crystals formed were
dissolved in 100 ll DMSO. The micro-wells were shaken for 5 min and read on a
spectrophotometer at a wavelength of 570 nm (maximum absorbance of formazan).
For each experimental condition, the mean optical density was calculated, enabling
determination of the percentage of living cells in comparison with the control.
Calculation of lipophilicity
Log P, the logarithm of the n-octanol–water partition coefficient, was calculated by
use of the software ACD/ChemSketch ver. 12.01 (ACD/log P) and a variety of
approaches available online.
123

F. Malek et al.
Conclusion
Eight bipyrazole-derivative drugs were successfully prepared in one step from
readily accessible starting materials. In-vitro test results against three human cancer
cell lines, breast cancer MDA-MB231, prostate cancer PC3, and colorectal cancer
LoVo, were indicative of substantial antitumor activity, especially for products 6
and 7 against the colorectal cancer cell line (LoVo). Based on Lipinski’s rule of five
these products may be suitable candidates for orally active drugs.
Acknowledgments This work was supported by the CUD (Commission Universitaire pour le
´
Developpement, Belgium) within the framework of the P3 program.
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