Trisubstituted and tetrasubstituted pyrazolines as a novel class of cell-growth inhibitors in tumor cells with wild type p53

Article abstract of DOI:10.1016/j.bmc.2013.09.055

Derivatives with scaffolds of 1,3,5-tri-substituted pyrazoline and 1,3,4,5-tetra-substituted pyrazoline were synthesized and tested for their inhibitory effects versus the p53^+/+ HCT116 and p53^-/- H1299 human tumor cell lines. Severa

Full text of DOI:10.1016/j.bmc.2013.09.055

Accepted Manuscript
Trisubstitutedand TetrasubstitutedPyrazolines as a Novel Class of Cell-growth
Inhibitors in Tumor Cells with Wild Type p53
Mohammad Abdel-Halim, Adam B. Keeton, Evrim Gurpinar, Bernard D. Gary,
Simon M. Vogel, Matthias Engel, Gary A. Piazza, Frank M. Boeckler, Rolf W.
Hartmann, Ashraf H. Abadi
PII:
S0968-0896(13)00835-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.09.055
BMC 11139
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
1 June 2013
13 September 2013
22 September 2013
Please cite this article as: Abdel-Halim, M., Keeton, A.B., Gurpinar, E., Gary, B.D., Vogel, S.M., Engel, M., Piazza,
G.A., Boeckler, F.M., Hartmann, R.W., Abadi, A.H., Trisubstitutedand TetrasubstitutedPyrazolines as a Novel Class
of Cell-growth Inhibitors in Tumor Cells with Wild Type p53, Bioorganic & Medicinal Chemistry (2013), doi:
http://dx.doi.org/10.1016/j.bmc.2013.09.055
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Trisubstitutedand TetrasubstitutedPyrazolines as a Novel
Class of Cell-growth Inhibitors in Tumor Cells with
Wild Type p53
1,2
3
4
3
Mohammad Abdel-Halim , Adam B. Keeton , Evrim Gurpinar , Bernard D. Gary ,
5
2
3
5
Simon M. Vogel , Matthias Engel , Gary A. Piazza , Frank M. Boeckler , Rolf W.
2
1
Hartmann and Ashraf H. Abadi
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacyand Biotechnology,
German University in Cairo, Cairo 11835, Egypt.
2
Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, D-66123
Saarbrꢀ cken, Germany.
3
Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Suite
3
029, Mobile, AL 36604
4
Department of Pharmacology and Toxicology, The University of Alabama at
Birmingham, Birmingham, AL 35294
5
Laboratory for Molecular Design and Pharmaceutical Biophysics, Department of
Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of
Tuebingen, 72074 Tuebingen, Germany.
1

Abstract
Derivatives with scaffolds of 1,3,5-tri-substituted pyrazoline and 1,3,4,5-tetra-substituted
+
/+
pyrazoline were synthesized and tested for their inhibitory effects versus the p53
-/-
HCT116 and p53 H1299 human tumor cell lines. Several compounds were active
against the two cell lines displaying IC50 values in the low micromolar range with a
+/+
clearly more pronounced effect on the p53 HCT116 cells.The compound class shows
excellent developability due to the modular synthesis, allowing independent optimization
of all three to four key substituents to improve the properties of the molecules.
Key words: pyrazoline; anticancer; p53
2

Introduction
The pro-apoptotic p53 protein is a transcription factor that responds to various stress
stimuli such as DNA damage and oxidative stress by increasing the expression of
1
proteins involved in DNA repair, cell cycle arrest and induction of apoptosis.
Consistent with its role as a tumor-suppressor, p53 is deleted or mutated in
approximately 50% of human cancers. In other tumors, p53 is retained in its wild-type
form, but its activity is effectively inhibited by the negative regulator murine double
minute 2 (Mdm2). Mdm2, also known as E3 ubiquitin-protein ligase Mdm2, is a protein
that is encoded by the MDM2 in which the human homolog is sometimes referred to as
Hdm2. Mdm2 serves to downregulate p53 levels by C-terminal ubiquitination and
subsequent proteasomal degradation. In addition, Mdm2 has been shown to inhibit p53
2-4
transcriptional activity through direct binding to the p53 transactivation domain. The
interaction between Mdm2 and p53 involves a short helix formed by residues 13-29 of
p53 and a hydrophobic cleft in Mdm2. The realization that p53 most contributing
residues to this interaction are Phe19, Trp23 and Leu26 stimulated the efforts to identify
drug like inhibitors of this protein-protein interaction. Such inhibitors are expected to
release p53 from the negative control of Mdm2, and activate the apoptotic p53 pathway
in tumors expressing wild-type p53.Whereas, tumors with mutated or deleted p53 are
5-7
expected to be less responsive. Such compounds may be useful for indications such
as colorectal cancer chemoprevention or the treatment of precancerous conditions (e.g.
adenomatous polyps) where p53 is functional but inhibited by Mdm2.
3

The prototypes for such class of inhibitors are imidazoline derivatives known as nutlinsI-
III (Figure 1). Structure activity relationships show that the cis-imidazoline represents a
useful scaffold to project functional groups similar to a helical peptide. Nutlins are
tetrasubstsituted imidazoline derivatives with two orthogonal halophenyls projecting in a
non-coplanar style relative to the core ring, other substituents are an alkoxyphenyl
substituent and a tertiary amide substituent. The alkoxyphenyl is directed towards the
Phe19 pocket, one of the halo-phenyl group sits deeply in the Trp23 pocket while the
other occupies the Leu26 pocket. Galatin and Abraham proposed a pharmacophore
model based on the crystal structure of p53 peptide in complex with Mdm2 and several
known non-peptidic small molecule inhibitors of Mdm2, with the three aryls at particular
8
distances from each other.
Later, other structurally related compounds followed including the benzodiazepinedione
IV and the spiroindole V (Figure1), all sharing at least two haloaryl groups projecting
9-11
from a core diazepindione or pyrolidine ring with nutlins.
Although several of these inhibitors exhibited cellular activity, extensive optimization of
the selectivity towards the Mdm2 binding pocket might be required, since a hydrophobic
groove is characteristic of many “hot spots” at protein-protein interaction sites; bearing
in mind that such hot spot binding pockets can possess a high conformational adaptivity
and that hydrophobic contacts are not directional and thus less specific interactions.
The chosen scaffolds for inhibitors should offer high synthetic flexibility to allow for the
12
generation of a great variety of analogues during the optimization process. Hence the
search for additional rigid scaffolds appropriate to inhibit the Mdm2-p53 interaction and
showing favorable synthetic tractability (good “developability”) is still ongoing.
4

In this report, we describe the design and synthesis of forty six novel small organic
molecules with core pyrazoline ring and two orthogonal dihalophenyl, and alkoxyphenyl
or tertiary-butyl (1-44) substituent to mimic the potential three hydrophobic residues
(
Phe19, Trp23, and Leu26) of p53. Moreover two of the compounds contain an
additional tertiary amide substituent (45, 46). As a first assay, the compounds were
tested for their growth inhibitory activity against p53 wild-type (+/+) HCT116 human
colorectal carcinoma and p53 null (-/-) H1299 human lung adenocarcinoma cell lines.
Design of compounds
Our main objective was to develop a new class of potential p53-Mdm2 antagonists with
an excellent synthetic tractability, thus providing the option to independently vary each
key moiety for the optimization of potency, selectivity and pharmacokinetic properties.
To this end, a synopsis of the hitherto published inhibitor classes led us to the design of
the tri- and tetra-substituted pyrazoline derivatives.
Thus, three sets of pyrazolines were designed with a 1,3,5-trisubstituted pyrazoline
st
scaffold. The 1 group is represented by compounds (1-29) in which the phenyls at
positions 1 and 5 are mono-substituted with a chlorine atom and the 3-phenyl is mono-
(1-27) or disubstituted (28, 29) with methoxy functions. The position of the chlorine
atoms and the methoxy group(s) was varied in a systematic way to define the optimum
substitution pattern to show the best potency and selectivity. The dimethoxy substitution
nd
was made in a later stage in compounds 28 and 29 to test the effect of the 2 methoxy
group on the activity of compound 4, the most potent growth inhibitor against the
HCT116 tumor cell line in the series.
5

nd
In the 2 set of compounds (30-35) t-butyl group was used as a lipophilic substituent
instead of the methoxyphenyl at position 3 of the pyrazoline. Besides, several lipophilic
groups were tied to position 5 like naphthyl, biphenyl, 2, 4- dichlorophenyl, 4-
chlorophenyl and 4-bromophenyl. In the third set (36-44), haloaryl and carboaryls at
positions 1 and 5 of pyrazoline are employed similar to the first and second sets.
However, the 2-ethoxy phenyl part reported in nutlin-2 is used at position 3. In the last 2
th
compounds (45-46), we introduced a 4 substitution at position 4 of the pyrazoline with
morpholinocarbonyl group to test the effect of introducing a tertiary amide upon activity
and to improve the water solubility.
To check the validity of our design, a three-dimensional overlay of the energy minimized
forms of the 1,3.5-triphenylpyrazoline scaffold (for which different substitution patterns
are tried in compounds (1-29) and compound 45 with nutlin-2 from PDB 1RV1 was
done. It can be seen that all the substituents relevant to the binding of nutlin-2 to Mdm-2
overlapped well with the corresponding moieties of our compounds (Figure 2).
Results and Discussion
Chemistry
The synthesis of the desired pyrazolines was carried out in two steps. In the first step
(
Scheme1), a Claisen-Schmidt condensation was carried out between aromatic
aldehydes and pinacolone or acetophenone analogues, using 10% aq. KOH as a
catalyst in methanol, in order to afford the required enones. In the second step, these
enones were reacted to give the desired pyrazolines using the previously reported
13
regioselective synthesis of 1,3,5-triarylpyrazolines. This was accomplished via enone-
6

arylhydrazine hydrochloride condensation reaction under an inert atmosphere using
o
DMF as solvent while heating for 5 hours at 85 C. To introduce the morpholinocarbonyl
group at position 4 of the respective pyrazoline derivative (34, 36), deprotonation was
o
carried out by LDA at -78 C to create the anion which reacted with morpholinecarbonyl
13
chloride to give the desired product (Scheme1).
1
13
The structure and purity of the compounds were elucidated by H- , C-NMR, and
LC/MS. The successful formation of the pyrazoline has been confirmed with the
appearance of doublet of doublet signals at about 3.0 and 4.0 PPM due to the non-
magnetically equivalent C-4 methylene protons and a doublet of doublet at around 5.0-
6
.0 PPM due to the angular proton at C-5. A downfield shift of the later proton seems
dependent on the presence of an ortho chlorine substituent at the N-1 phenyl.
Since most of the compounds bear chlorine or bromine substituent, they all showed M ,
+
+
+
M +2 and M +4 peaks due to the isotopic nature of the respective halogen.
Biological Activity
To evaluate whether our new scaffolds are active in cells and in addition show
indications of the desired mechanism of action, we screened all of the synthesized
pyrazolines for their ability to inhibit the growth of HCT116 and H1299 cell lines at 20
µM (higher concentrations could not be used to avoid solubility problems of some
compounds). IC50 values were determined in concentration-response studies for
compounds which showed more than 50 % inhibition in the single dose screen. H1299
is a human non-small cell lung carcinoma cell line derived from the lymph node; which
has a homozygous partial deletion of the TP53 gene and as a result, does not express
7

the p53 protein.On the other hand, HCT116 cells are human colorectal carcinoma cells
with wild-type p53 that are widely used to study the effect of small molecule Mdm2
antagonists.
Nutlin-3 was used as a positive control to benchmark the activity of the new derivatives.
Mdm2 antagonists are expected to show greater potency to inhibit the growth of
HCT116 cells compared to H1299 cells, as was observed for nutlin-3 (showing IC50
values of 1.6 µM vs. 14.7 µM, respectively).The results are shown in Table 1.
Structure activity relationship. Generally, active cell-growth inhibitors showed
preferential inhibition of HCT116 cells containing wild-type p53 in a behavior analogous
to nutlin-3 (compounds 4, 16, 22, 23, 30, 33, 35 and 45). Compounds 32 and 46 were
the only exceptions and were found to display almost equivalent growth inhibition in
both tumor cell lines.
In the first set of compounds (1-27), the activity was sharply dependent on the halogen
substitution pattern at the 1- and 5-phenyls and several clear relationships between
structure and cell-growth inhibition could be observed for the most favorable
substitutions.
At the 1-phenyl moiety, a chlorine substituent in ortho position was a consistent feature
in three of the most potent growth inhibitors (4, 16 and 22). Compound 23 the meta-
chloro analogue of 22 showed comparable activities. On the other hand, none of the
para-chloro derivatives showed significant activity. Similar to the 1-phenyl, substitution
with 4-chloro at the phenyl ring in position 5 did not lead to active compounds, while
shifting the chlorine to meta position was optimal for the activity, as demonstrated by 3
8

st
of the most potent member of the 1 set bearing a meta-chloro substituent at their 5-
phenyl moiety (4, 22, 23). Nevertheless, ortho-chloro at the 1-phenyl could still give
potent compounds (16).
Concerning the methoxy substitution at the 3-phenyl, it was tolerated at all positions
with high potency whether ortho (compounds 22 and 23), meta (compound 16) or para
(4) indicating that this moiety does not significantly affect potency. However, in
compound 4, the most potent growth inhibitor against both cell lines, introducing a
second methoxy either at meta or ortho positions was deleterious to the activity as
exemplified by compounds 28 and 29.
For the second set of compounds, the 3-t-butyl derivatives, four out of the six analogues
showed growth inhibitory activity, indicating that t-Bu group could act as a possible
replacement for the methoxyphenyl in the first set of compounds. However, none of the
compounds were more potent than the triphenylpyrazolines in the first set. Compound
3
2 with bis-1,5-(4-chlorophenyl) moieties showed marginal activity towards both tumor
cell lines. Replacement of the 4-chlorophenyl at position 5 of the pyrazoline in
compound 32 by a 2-naphthyl (compound 30) caused a slight improvement in activity
towards HCT116 cells only, while the biphenyl moiety in compound 31 seemed too
bulky to be tolerated.
On the other hand, adding another chlorine at position 3 of the 5-phenyl (33) or
replacing the 4-Cl at the 1-phenyl by 4-Br (35) caused a 3-4 fold increase in the
inhibitory activity towards HCT116 cell line with slight or no improvement in the activity
towards H1299 cell line. Surprisingly, replacement of the two chlorine atoms in
compound 32 by bromine atoms was inferior for activity (34).
9

Unexpectedly, adapting the 2-ethoxyphenyl at the 3 position of the pyrazoline abolished
the activity (compounds36-44). This negative result might be due to the sterical
requirements of the ethoxy group that are not provided by the potential receptor pocket.
Interestingly, introducing morpholinocarbonyl at position 4 of compound 36 (compound
4
6) recovered some of the potency on both cell lines equally. This might be due to a
direct interaction achieved by the introduced group, or its effect on the relative
orientation and conformation of the other substituents arising from the pyrazoline core.
In addition, the morpholinocarbonyl group is expected to have a profound effect in
enhancing the hydrophilic characteristics, which might improve the performance of the
th
compound in the assay conditions. Similarly, the
4
substitution with
morpholinocarbonyl group in compound 32 (compound 45) has increased the
selectivity, with potency improved by more than 4 fold, against HCT116 cell line.
In summary, the 1,3,5-trisubstituted and 1,3,4,5-tetrasubstituted pyrazoline scaffolds
yielded potent inhibitors of the colorectal cancer line HCT116 in the low micro molar
range, showing a good SAR with clear dependence on the halogen substitution pattern
at the 1- and 5- phenyl rings as described herein. Many compounds showed a
preferential inhibition pattern similar to nutlin-3 when comparing HCT116 and H1299
cell lines, proposing Mdm2 as a potential molecular target. Thus, we tested first whether
the compounds were able to displace a fluorescence-labeled peptide (derived from p53)
in a concentration-dependent manner from its complex with Mdm2 or MdmX,
respectively. Successful displacement leads to a substantial decrease in fluorescence
anisotropy reported as a relative value (%FA) in comparison to lithocholic acid as a
positive control leading to a complete displacement of the fluorescence-labeled peptide
1
0

(
0 %FA). Protein expression and purification, as well as the setup of the FP assay were
14
performed as described recently. To facilitate measurements despite solubility issues
of various compounds, we have been using 10% v/v DMSO for all experiments. Under
these conditions compounds used as positive controls did not indicate deviations from
standard conditions. For none of the compounds identified as hits in cell lines a
reduction to at least 50%FA was found when titrating from 5µM up to concentrations of
1
mM. Among the compounds with inhibitory potency in cell lines, 4, 15, 16, 22, 23, and
6 showed intrinsic fluorescence possibly interfering with the assay. Still, only for 16 an
4
irregular FP curve was retrieved showing increase of fluorescence instead of the
expected decrease. Compounds 30, 32, 33, 35, and 45 showed no intrinsic
fluorescence, but also no decrease beyond 70% relative fluorescence anisotropy for
both Mdm2 and MdmX. Of course, all these results can be biased by solubility issues of
the compounds under the used assay conditions. However, clearly none of the
compounds showed binding to Mdm2 or MdmX similar to the positive controls.
In a further attempt to investigate the potential relationship between the differential
tumor cell cytotoxicity and p53 modulation, we performed a p53 translocation assay
using immunocytochemical staining and high content screening. The results are
presented in Table 2; the compounds were selected according to the highest
IC (H1299)/IC (HCT116) ratio found with different substituents (16, 22 and 35), and
50
50
three control compounds were included: compound 8 which had shown weak cell
growth inhibition at 20 µM, compound 46, which inhibited both cell lines with equal
potency, and nutlin-3 as a positive control. In HCT116 cells, a slightly increased p53
nuclear/cytosolic ratio was noted in some experiments after treatment with 16 at 10µM,
1
1

and with 8 at 3.3 µM (Table 2), however, this tendency did not reach statistical
significance. Furthermore, no effect could be observed anymore at 30 µM
concentrations for 16 (not shown) and for 8 at 10 µM. The high lipophilicity and poor
solubility of the analyzed compounds might generally hamper their analysis in cell-
based functional assays. For the same reason, compound 8 might have failed to show
potent inhibition during the initial screening performed at 20 µM. At least the cell growth
inhibition was stronger with HCT116 cells (33%) than with H1299 cells (10%) at this
concentration, in line with the general trend.
The control compound 46 did not show any effect, as would have been expected,
however, compounds 22 and 35 also failed to induce p53 translocation to the nucleus
(
Table 2). Altogether, our data did not confirm that the p53/Mdm2 interaction is targeted
by the compounds in HCT116 cells. It may be speculated that other or additional targets
-/-
play a major role. Of note, nutlin-3 is still suppressing cell growth of the p53 H1299 cell
line – albeit 9-fold less potent than the p53 wild-type cell line– suggesting that it might
address further target proteins as well.
Thus, it remains unclear through which mode of action the encouraging results in the
cell lines can be explained. Substantial modifications to improve polarity and solubility
characteristics should be useful to elucidate the involved pathways.
1
2

EXPERIMENTAL SECTION
Chemistry: Solvents and reagents were obtained from commercial suppliers and used
1
13
as received. A Bruker DRX 500 spectrometer was used to obtain H NMR and C NMR
spectra. The chemical shifts are referenced to the residual protonated solvent signals or
TMS was used as a reference. At least 95% purity in all the tested compounds (table 1)
was obtained by means of HPLC coupled with mass spectrometry. Mass spectra
(
HPLC-ESI-MS) were obtained using a TSQ quantum (Thermo Electron Corporation)
instrument prepared with a triple quadrupole mass detector (Thermo Finnigan) and an
ESI source. All samples were inserted using an autosampler (Surveyor, Thermo
Finnigan) by an injection volume of 10 μL. The MS detection was determined using a
source CID of 10 V and carried out at a spray voltage of 4.2 kV, a nitrogen sheath gas
5
o
pressure of 4.0 X 10 Pa, a capillary temperature of 400 C , a capillary voltage of 35 V
5
and an auxiliary gas pressure of 1.0 X 10 Pa. The stationary phase used was an RP
C18 NUCLEODUR 100-3 (125 X 3 mm) column (Macherey-Nagel). The solvent system
consisted of water containing 0.1% TFA (A) and 0.1% TFA in acetonitrile (B). HPLC-
Method: flow rate 400 μL/min. The percentage of B started at an initial of 5%, was
increased up to 100% during 16 min, kept at 100% for 2 min, and flushed back to 5% in
2
min. Melting points were determined using a Mettler FP1 melting point apparatus and
are uncorrected.
General procedure for enone synthesis.
To a an ice-cooled solution of the appropriate ketone (10 mmol) in MeOH (50 mL) 10 %
KOH aq. solution (30 ml) was added, followed by gradual addition of the corresponding
aryl aldehyde (10 mmol). The mixture was left to attain room temperature and stirred
1
3

overnight. The solid product was filtered and washed three times with MeOH/H O
2
mixture (5:3) and left to dry. In case of oily products the reaction mixture was extracted
by CH Cl (4 x 20 mL) and the combined organic layers were washed with water,
2
2
filtered over anhydrous MgSO , evaporated under reduced pressure and used without
4
further purification.
(E)-3-(4-chlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (C1).
Synthesized according to the general procedure for enone synthesis using 1-(4-
methoxyphenyl)ethanone and 4-chlorobenzaldehyde; yellow solid; yield: 2.61 g (96 %);
15
mp 125-126 °C.
E)-3-(3-chlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (C2).
(
Synthesized according to the general procedure for enone synthesis using 1-(4-
methoxyphenyl)ethanone and 3-chlorobenzaldehyde; yellow solid; yield: 2.32 g (85 %);
16
mp 97-99 °C.
E)-3-(2-chlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (C3).
(
Synthesized according to the general procedure for enone synthesis using 1-(4-
methoxyphenyl)ethanone and 2-chlorobenzaldehyde; yellow solid; yield: 2.48 g (91 %);
17
mp 79-80 °C.
E)-3-(4-chlorophenyl)-1-(3-methoxyphenyl)prop-2-en-1-one (C4).
Synthesized according to the general procedure for enone synthesis using 1-(3-
(
18
methoxyphenyl)ethanone and 4-chlorobenzaldehyde; yellow oil; yield: 2.21 g (81 %).
E)-3-(3-chlorophenyl)-1-(3-methoxyphenyl)prop-2-en-1-one (C5).
Synthesized according to the general procedure for enone synthesis using 1-(3-
(
19
methoxyphenyl)ethanone and 3-chlorobenzaldehyde; yellow oil; yield: 2.4 g (88 %).
1
4

(E)-3-(2-chlorophenyl)-1-(3-methoxyphenyl)prop-2-en-1-one (C6).
Synthesized according to the general procedure for enone synthesis using 1-(3-
methoxyphenyl)ethanone and 2-chlorobenzaldehyde; yellow solid; yield: 2.56 g (94 %);
20
mp117-117 °C.
E)-3-(4-chlorophenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (C7).
Synthesized according to the general procedure for enone synthesis using 1-(2-
(
21
methoxyphenyl)ethanone and 4-chlorobenzaldehyde; yellow oil; yield: 2.51 g (92 %).
E)-3-(3-chlorophenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (C8).
(
Synthesized according to the general procedure for enone synthesis using 1-(2-
methoxyphenyl)ethanone and 3-chlorobenzaldehyde; yellow solid; yield: 2.37 g (87 %);
1
mp96-98 °C; H NMR (500 MHz, CDCl ) δ 7.48 (dd, J = 7.6, 1.8 Hz, 1H), 7.40 (dd, J =
3
8
.8, 6.9 Hz, 2H), 7.33 (ddd, J = 8.4, 7.4, 1.8 Hz, 1H), 7.30 (dt, J = 7.1, 1.6 Hz, 1H), 7.25
13
–
7.16 (m, 3H), 6.90 – 6.87 (m, 1H), 6.85 (d, J = 15.8 Hz, 1H), 3.76 (s, 3H); C NMR
(
125 MHz, CDCl ) δ 192.41, 158.24, 141.15, 137.08, 134.86, 133.16, 130.45, 130.08,
3
1
29.98, 129.01, 128.25, 127.93, 126.60, 120.82, 111.67, 55.79.
(E)-3-(2-chlorophenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (C9).
Synthesized according to the general procedure for enone synthesis using 1-(2-
methoxyphenyl)ethanone and 2-chlorobenzaldehyde; yellow solid; yield: 2.29 g (84 %);
22
mp 60-62 °C.
E)-3-(3-chlorophenyl)-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (C10).
(
Synthesized according to the general procedure for enone synthesis using 1-(3,4-
dimethoxyphenyl)ethanone and 3-chlorobenzaldehyde; yellow solid; yield: 2.75 g (91
1
%
); mp 110-111 °C; H NMR (500 MHz, CDCl ) δ 7.60 (d, J = 15.6 Hz, 1H), 7.58 – 7.53
3
1
5

(
m, 1H), 7.53 – 7.49 (m, 2H), 7.42 (d, J = 15.6 Hz, 1H), 7.37 (ddd, J = 6.4, 2.3, 1.5 Hz,
13
1
H), 7.27 – 7.20 (m, 2H), 6.83 – 6.78 (m, 1H), 3.85 (d, J = 0.8 Hz, 6H); C NMR (125
MHz, CDCl ) δ 188.12, 153.50, 149.36, 142.17, 136.95, 134.93, 131.06, 130.15,
3
1
30.11, 127.77, 126.73, 123.13, 122.91, 110.80, 110.01, 56.10, 56.06.
E)-3-(3-chlorophenyl)-1-(2,4-dimethoxyphenyl)prop-2-en-1-one (C11).
Synthesized according to the general procedure for enone synthesis using 1-(2,4-
(
23
dimethoxyphenyl)ethanone and 3-chlorobenzaldehyde; yellow oil; yield: 2.66 g (88%).
E)-4,4-dimethyl-1-(naphthalen-2-yl)pent-1-en-3-one (C12).
Synthesized according to the general procedure for enone synthesis using pinacolone
(
24
and 2-naphthaldehyde; yellow solid; yield: 2.24 g (94 %); mp 117-119 °C.
E)-1-([1,1'-biphenyl]-4-yl)-4,4-dimethylpent-1-en-3-one (C13).
Synthesized according to the general procedure for enone synthesis using pinacolone
(
25
and [1,1'-biphenyl]-4-carbaldehyde; yellow solid; yield: 2.3 g (87 %); mp 128-130 °C.
E)-1-(4-chlorophenyl)-4,4-dimethylpent-1-en-3-one (C14).
Synthesized according to the generalprocedure for enone synthesis pinacolone and 4-
(
26
chlorobenzaldehyde; white solid; yield: 2.11 g (95 %); mp 85-87 °C.
E)-1-(3,4-dichlorophenyl)-4,4-dimethylpent-1-en-3-one (C15).
Synthesized according to the general procedure for enone synthesis using pinacolone
(
27
and 3,4-dichlorobenzaldehyde; yellow solid; yield: 2.08 g (81 %); mp 94-96°C.
E)-1-(4-bromophenyl)-4,4-dimethylpent-1-en-3-one (C16).
Synthesized according to the general procedure for enone synthesis using pinacolone
(
26
and 4-bromobenzaldehyde; yellow solid; yield: 2.48 g (93 %); mp102-103 °C.
E)-3-(4-chlorophenyl)-1-(2-ethoxyphenyl)prop-2-en-1-one (C17).
(
1
6

Synthesized according to the general procedure for enone synthesis using 1-(2-
1
ethoxyphenyl)ethanone and 4-chlorobenzaldehyde; yellow oil; yield: 2.32 g (81 %); H
NMR (500 MHz, CDCl ) δ 7.54 (dd, J = 7.6, 1.8 Hz, 1H), 7.45 (d, J = 15.9 Hz, 1H), 7.40
3
–
7.36 (m, 2H), 7.36 – 7.30 (m, 2H), 7.26 – 7.20 (m, 2H), 6.90 (td, J = 7.5, 0.9 Hz, 1H),
13
6
.83 (dd, J = 11.9, 4.4 Hz, 1H), 4.00 (q, J = 7.0 Hz, 2H), 1.29 (t, J = 7.0 Hz, 3H); C
NMR (125 MHz, CDCl ) δ 192.33, 157.76, 140.79, 135.91, 133.83, 133.18, 130.63,
3
1
29.35, 129.14, 127.71, 120.77, 116.32, 112.62, 64.25, 14.84.
(E)-3-(4-bromophenyl)-1-(2-ethoxyphenyl)prop-2-en-1-one (C18).
Synthesized according to the general procedure for enone synthesis using 1-(2-
1
ethoxyphenyl)ethanone and 4-bromobenzaldehyde; yellow oil; yield: 2.84 g (86 %); H
NMR (500 MHz, CDCl ) δ 7.73 – 7.68 (m, 1H), 7.60 (d, J = 15.9 Hz, 1H), 7.57 – 7.53
3
(
m, 3H), 7.51 – 7.45 (m, 3H), 7.06 (td, J = 7.5, 0.8 Hz, 1H), 6.99 (dd, J = 12.8, 6.3 Hz,
13
1
H), 4.17 (q, J = 7.0 Hz, 2H), 1.45 (t, J = 7.0 Hz, 3H); C NMR (125 MHz, CDCl3)
δ192.29, 157.77, 140.80, 134.25, 133.20, 132.09, 130.63, 129.57, 129.09, 127.79,
24.22, 120.76, 112.60, 64.23, 14.83.
E)-3-(5-bromothiophen-2-yl)-1-(2-ethoxyphenyl)prop-2-en-1-one
1
(
(C19).
Synthesized according to the general procedure for enone synthesis using 1-(2-
ethoxyphenyl)ethanone and 5-bromothiophene-2-carbaldehyde; yellow oil; yield: 3 g (89
1
%
); H NMR (500 MHz, CDCl ) δ 7.91 (dt, J = 4.2, 2.1 Hz, 1H), 7.87 (d, J = 15.4 Hz,
3
1
4
1
1
H), 7.70 – 7.64 (m, 1H), 7.51 – 7.47 (m, 1H), 7.26 – 7.21 (m, 3H), 7.19 – 7.16 (m, 1H),
13
.38 – 4.32 (m, 2H), 1.69 (t, J = 7.0 Hz, 3H); C NMR (125 MHz, CDCl ) δ 191.28,
3
57.94, 142.56, 133.71, 133.35, 131.62, 131.20, 130.79, 128.78, 126.56, 120.75,
15.53, 112.54, 64.25, 14.89.
1
7

(E)-1-(2-ethoxyphenyl)-3-(naphthalen-2-yl)prop-2-en-1-one (C20).
Synthesized according to the general procedure for enone synthesis using 1-(2-
ethoxyphenyl)ethanone and 2-naphthaldehyde; yellow solid; yield: 2.74 g (91 %); mp
28
8
9-91 °C.
(E)-3-([1,1'-biphenyl]-4-yl)-1-(2-ethoxyphenyl)prop-2-en-1-one (C21).
Synthesized according to the general procedure for enone synthesis using 1-(2-
ethoxyphenyl)ethanone and [1,1'-biphenyl]-4-carbaldehyde; yellow solid; yield: 3.08 g
1
(
94 %); mp98-99 °C; H NMR (500 MHz, CDCl ) δ 7.48 – 7.45 (m, 3H), 7.44 (d, J = 2.4
3
Hz, 2H), 7.43 – 7.39 (m, 4H), 7.31 (d, J = 15.9 Hz, 1H), 7.27 – 7.21 (m, 2H), 7.15 (ddt, J
=
=
1
1
5.1, 3.9, 1.9 Hz, 1H), 6.82 (td, J = 7.5, 0.9 Hz, 1H), 6.77 (d, J = 8.3 Hz, 1H), 3.94 (q, J
13
7.0 Hz, 2H), 1.23 (t, J = 7.0 Hz, 3H); C NMR (125 MHz, CDCl ) δ 192.69, 157.71,
3
42.86, 142.08, 140.23, 134.27, 132.97, 130.58, 129.43, 128.87, 128.77, 127.78,
27.52, 127.14, 127.01, 120.74, 112.65, 64.29, 14.87.
(E)-3-(3,4-dichlorophenyl)-1-(2-ethoxyphenyl)prop-2-en-1-one (C22).
Synthesized according to the general procedure for enone synthesis using 1-(2-
ethoxyphenyl)ethanone and 3,4-dichlorobenzaldehyde; yellow solid; yield: 2.53 g (79
1
%
);mp80-82 °C; H NMR (500 MHz, CDCl ) δ 7.47 (dd, J = 7.7, 1.8 Hz, 1H), 7.44 (d, J =
3
2
=
3
1
.0 Hz, 1H), 7.30 – 7.26 (m, 2H), 7.26 – 7.23 (m, 2H), 7.20 – 7.16 (m, 1H), 6.81 (ddd, J
4.9, 3.3, 1.0 Hz, 1H), 6.78 – 6.75 (m, 1H), 3.93 (q, J = 7.0 Hz, 2H), 1.22 (t, J = 7.0 Hz,
13
H); C NMR (125 MHz, CDCl ) δ 191.87, 157.90, 139.16, 135.49, 133.83, 133.47,
3
33.19, 130.86, 130.74, 130.34, 129.63, 128.82, 127.20, 120.83, 112.61, 64.27, 14.85.
1
8

(E)-3-(2,4-dichlorophenyl)-1-(2-ethoxyphenyl)prop-2-en-1-one (C23).
Synthesized according to the general procedure for enone synthesis using 1-(2-
ethoxyphenyl)ethanone and 2,4-dichlorobenzaldehyde; yellow solid; yield: 2.79g (87
1
%
);mp75-77 °C; H NMR (500 MHz, CDCl ) δ 7.72 (d, J = 15.9 Hz, 1H), 7.45 (dt, J = 8.5,
3
4
6
3
1
.3 Hz, 1H), 7.42 – 7.37 (m, 1H), 7.27 – 7.20 (m, 3H), 7.04 (dd, J = 8.3, 1.5 Hz, 1H),
.84 – 6.78 (m, 1H), 6.74 (t, J = 9.5 Hz, 1H), 3.94 – 3.88 (m, 2H), 1.19 (t, J = 7.0 Hz,
13
H); C NMR (125 MHz, CDCl ) δ 192.12, 157.80, 136.75, 135.98, 135.90, 133.36,
3
32.21, 130.74, 130.04, 129.88, 128.88, 128.22, 127.47, 120.82, 112.57, 64.25, 14.84.
(E)-1-(2-ethoxyphenyl)-3-(4-fluorophenyl)prop-2-en-1-one (C24).
Synthesized according to the general procedure for enone synthesis using 1-(2-
1
ethoxyphenyl)ethanone and 4-fluorobenzaldehyde; yellow oil; yield: 2.51 g (93%); H
NMR (500 MHz, CDCl ) δ 7.82 (dt, J = 5.8, 2.9 Hz, 1H), 7.78 – 7.69 (m, 3H), 7.63 – 7.59
3
(
m, 1H), 7.57 (d, J = 15.5 Hz, 1H), 7.27 – 7.21 (m, 2H), 7.21 – 7.16 (m, 1H), 7.14 (dd, J
13
=
5.8, 2.5 Hz, 1H), 4.29 (dd, J = 9.2, 4.8 Hz, 2H), 1.58 (t, J = 7.0 Hz, 3H); C NMR (125
MHz, CDCl ) δ 192.53, 164.79, 162.79, 157.68, 141.15, 133.06, 131.51, 131.49,
3
1
5
30.54, 130.08, 130.01, 129.21, 127.00, 126.98, 120.72, 116.07, 115.89, 112.61, 64.23,
0.75, 14.80.
General procedure for pyrazoline synthesis.
A mixture of the enone derivative (2 mmol) and the corresponding phenylhydrazine
hydrochloride (3 mmol) in 15 mL of anhydrous DMF was heated to 85 °C for 5 hours
under argon atmosphere. The reaction solution was cooled to room temperature and
partitioned between 50 mL of diethyl ether and 20 mL of water. The organic layer was
separated and washed with three 20 mL-portions of water. The aqueous layers were
1
9

combined and extracted with three 20 mL-portions of diethyl ether. The organic layers
were combined, dried over anhydrous Na SO and concentrated in vacuo. The residue
2
4
was purified using column chromatography or used in the next step without further
purification.
1
-(2-chlorophenyl)-5-(4-chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazole (1).
The title compound was prepared by reaction of (E)-3-(4-chlorophenyl)-1-(4-
methoxyphenyl)prop-2-en-1-one(C1) and 2-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; light brown solid; yield:
o
1
0
.35 g (45%); mp162-163 C; H NMR (500 MHz, CDCl ) δ 7.73 – 7.68 (m, 2H), 7.40
3
(
dd, J = 12.5, 6.9 Hz, 1H), 7.19 (dt, J = 9.4, 4.7 Hz, 1H), 7.12 – 7.05 (m, 5H), 6.97 –
.92 (m, 2H), 6.88 – 6.83 (m, 1H), 5.79 (dd, J = 11.3, 4.6 Hz, 1H), 3.85 (s, 3H), 3.75 (dt,
6
13
J = 28.6, 14.3 Hz, 1H), 3.32 (dd, J = 16.8, 4.6 Hz, 1H); C NMR (125 MHz, CDCl ) δ
3
1
1
49.96, 142.94, 139.56, 133.39,131.12, 130.22, 128.62, 128.14, 127.54, 127.09,
25.18, 124.59, 124.06, 123.54, 114.11, 65.51, 55.38, 42.64; MS (ESI):m/z= 397.05
+
(
M+H) .
1
-(3-chlorophenyl)-5-(4-chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazole (2).
The title compound was prepared by reaction of (E)-3-(4-chlorophenyl)-1-(4-
methoxyphenyl)prop-2-en-1-one(C1) and 3-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; brown solid; yield: 0.28 g
o
1
(
36%); mp121-122 C; H NMR (500 MHz, CDCl ) δ 7.67 – 7.62 (m, 2H), 7.32 – 7.28 (m,
3
2
H), 7.24 – 7.19 (m, 2H), 7.15 (t, J = 2.1 Hz, 1H), 7.04 (t, J = 8.1 Hz, 1H), 6.93 – 6.89
2
0

(
m, 2H), 6.72 (dddd, J = 8.2, 4.6, 2.1, 0.8 Hz, 2H), 5.17 (dd, J = 12.2, 6.7 Hz, 1H), 3.85
1
3
–
1
1
1
3.76 (m, 4H), 3.07 (dd, J = 17.0, 6.7 Hz, 1H); C NMR (125 MHz, CDCl ) δ 160.50,
3
47.71, 145.82, 140.62, 134.82, 133.50, 129.88, 129.43, 127.43, 127.25, 124.90,
+
18.78, 114.11, 113.39, 110.99, 63.53, 55.36, 43.78; MS (ESI): m/z = 397.02 (M+H) .
,5-bis(4-chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole (3).
The title compound was prepared by reaction of (E)-3-(4-chlorophenyl)-1-(4-
methoxyphenyl)prop-2-en-1-one(C1) and 4-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; beige solid; yield: 0.4 g
o
1
(
51%); mp111-112 C; H NMR (500 MHz, CDCl ) δ 7.67 – 7.60 (m, 2H), 7.30 – 7.26 (m,
3
2
H), 7.20 (dd, J = 11.0, 7.2 Hz, 2H), 7.13 – 7.07 (m, 2H), 6.94 – 6.87 (m, 4H), 5.15 (dd,
13
J = 12.1, 7.1 Hz, 1H), 3.84 – 3.75 (m, 4H), 3.07 (dd, J = 17.0, 7.1 Hz, 1H); C NMR
125 MHz, CDCl ) δ 160.67, 147.63, 143.74, 140.95, 133.74, 130.22, 129.06, 127.58,
(
3
1
27.56, 125.27, 124.09, 114.64, 114.35, 64.09, 55.61, 44.08; MS (ESI):m/z = 397.01
+
(
M+H) .
1
-(2-chlorophenyl)-5-(3-chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazole (4).
The title compound was prepared by reaction of (E)-3-(3-chlorophenyl)-1-(4-
methoxyphenyl)prop-2-en-1-one (C2) and 2-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; yellow solid; yield: 0.37 g
o
1
(
47%); mp130-131 C; H NMR (500 MHz, CDCl ) δ 7.73 – 7.65 (m, 2H), 7.41 (dt, J =
3
6
7
1
.4, 3.2 Hz, 1H), 7.20 (dd, J = 8.0, 1.4 Hz, 1H), 7.16 (dd, J = 2.6, 1.4 Hz, 1H), 7.12 –
.01 (m, 4H), 6.96 – 6.90 (m, 2H), 6.85 (ddd, J = 7.9, 7.5, 1.6 Hz, 1H), 5.76 (dd, J =
1.3, 4.8 Hz, 1H), 3.84 (s, 3H), 3.76 (dd, J = 16.8, 11.3 Hz, 1H), 3.32 (dd, J = 16.8, 4.8
2
1

13
Hz, 1H); C NMR (125 MHz, CDCl ) δ 160.77, 150.19, 143.33, 143.14, 134.48, 130.02,
3
1
1
1
28.11, 127.82, 127.33, 127.14, 125.38, 125.14, 124.97, 124.36, 123.80, 114.41,
+
14.36, 65.94, 55.63, 42.93; MS (ESI): m/z = 397.06 (M+H) .
,5-bis(3-chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole (5).
The title compound was prepared by reaction of (E)-3-(3-chlorophenyl)-1-(4-
methoxyphenyl)prop-2-en-1-one(C2) and 3-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; brown solid; yield: 0.43 g
o
1
(
(
(
(
55%); mp146-147 C; H NMR (500 MHz, CDCl ) δ 7.66 – 7.60 (m, 2H), 7.30 – 7.26
3
m, 1H), 7.26 – 7.21 (m, 2H), 7.18 – 7.12 (m, 2H), 7.03 (t, J = 8.1 Hz, 1H), 6.93 – 6.86
m, 2H), 6.71 (tdd, J = 8.6, 2.1, 0.8 Hz, 2H), 5.14 (dd, J = 12.2, 6.8 Hz, 1H), 3.85 – 3.79
13
m, 4H), 3.08 (dd, J = 17.1, 6.8 Hz, 1H); C NMR (125 MHz, CDCl ) δ 160.52, 147.71,
3
1
1
45.84, 144.28, 135.12, 134.84, 130.58, 129.90, 128.03, 127.45, 125.97, 124.85,
23.95, 118.85, 114.10, 113.41, 110.95, 63.67, 55.36, 43.78; MS (ESI):m/z = 397.05
+
(
M+H) .
5
-(3-chlorophenyl)-1-(4-chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazole (6).
The title compound was prepared by reaction of (E)-3-(3-chlorophenyl)-1-(4-
methoxyphenyl)prop-2-en-1-one(C2) and 4-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; brown solid ; yield: 0.31 g
o
1
(
(
39%); mp 106-108 C; H NMR (500 MHz, CDCl ) δ, 7.68 – 7.61 (m, 2H), 7.35 – 7.29
3
m, 2H), 7.27 – 7.24 (m, 1H), 7.17 (dt, J = 6.8, 1.9 Hz, 1H), 7.14 – 7.09 (m, 2H), 6.95 –
6
1
.93 (m, 1H), 6.92 (q, J = 2.1 Hz, 2H), 6.91 – 6.90 (m, 1H), 5.14 (dd, J = 12.2, 7.2 Hz,
13
H), 3.85 – 3.78 (m, 4H), 3.10 (dd, J = 17.1, 7.2 Hz, 1H); C NMR (125 MHz, CDCl ) δ
3
2
2

1
1
60.44, 147.39, 144.38, 143.50, 135.10, 130.58, 129.28, 128.84, 128.00, 127.36,
26.06, 124.01, 123.89, 114.38, 114.11, 63.96, 55.37, 43.83; MS (ESI): m/z = 396.99
+
(
M+H) .
1
,5-bis(2-chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole (7).
The title compound was prepared by reaction of (E)-3-(2-chlorophenyl)-1-(4-
methoxyphenyl)prop-2-en-1-one(C3) and 2-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; yellowish white solid; yield:
o
1
0
.5 g (63%); mp142-143 C; H NMR (500 MHz, CDCl ) δ 7.72 – 7.65 (m, 2H), 7.44 (dd,
3
J = 8.2, 1.5 Hz, 1H), 7.32 (ddd, J = 6.9, 4.9, 1.9 Hz, 2H), 7.27 – 7.24 (m, 1H), 7.12
(
dtdd, J = 14.7, 8.8, 7.4, 1.4 Hz, 3H), 6.93 – 6.86 (m, 3H), 6.15 (dd, J = 11.3, 6.4 Hz,
1
3
1
1
1
H), 3.86 – 3.81 (m, 4H), 3.09 (dd, J = 16.6, 6.4 Hz, 1H); C NMR (125 MHz, CDCl ) δ
3
60.49, 149.65, 142.87, 139.05, 132.08, 130.96, 129.66, 128.54, 127.91, 127.59,
27.10, 127.06, 125.19, 124.49, 123.40, 121.59, 114.04, 63.01, 55.36, 42.02; MS (ESI):
+
m/z = 397.07 (M+H) .
5
-(2-chlorophenyl)-1-(3-chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazole (8).
The title compound was prepared by reaction of (E)-3-(2-chlorophenyl)-1-(4-
methoxyphenyl)prop-2-en-1-one(C3) and 3-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; beige solid; yield: 0.23 g
o
1
(
29%); mp148-149 C; H NMR (500 MHz, CDCl ) δ 7.76 – 7.71 (m, 2H), 7.51 (dddd, J =
3
6
.9, 6.5, 1.2, 0.5 Hz, 1H), 7.31 – 7.20 (m, 4H), 7.11 (dt, J = 5.5, 4.8 Hz, 1H), 7.01 – 6.95
m, 2H), 6.82 – 6.77 (m, 1H), 6.71 (ddd, J = 8.3, 2.2, 0.9 Hz, 1H), 5.65 (dd, J = 12.2, 6.4
Hz, 1H), 4.01 (dd, J = 17.2, 12.2 Hz, 1H), 3.89 (s, 3H), 3.11 (dd, J = 17.2, 6.4 Hz, 1H);
(
2
3

13
C NMR (125 MHz, CDCl ) δ 160.49, 148.10, 145.63, 138.76, 134.90, 131.79, 129.96,
3
1
5
5
29.94, 128.92, 127.66, 127.45, 127.18, 124.98, 118.68, 114.07, 113.23, 110.71, 60.96,
+
5.35, 42.28; MS (ESI): m/z = 397.07 (M+H) .
-(2-chlorophenyl)-1-(4-chlorophenyl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-
pyrazole (9).
The title compound was prepared by reaction of (E)-3-(2-chlorophenyl)-1-(4-
methoxyphenyl)prop-2-en-1-one(C3) and 4-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; yellow solid; yield: 0.35 g
o
1
(
44%); mp149-151 C; H NMR (500 MHz, CDCl ) δ 7.67 – 7.63 (m, 1H), 7.45 – 7.43 (m,
3
1
6
3
1
1
1
1
H), 7.28 – 7.24 (m, 2H), 7.23 – 7.16 (m, 2H), 7.15 – 7.10 (m, 2H), 7.01 – 6.96 (m, 1H),
.93 – 6.86 (m, 3H), 5.57 (dd, J = 12.2, 6.7 Hz, 1H), 3.95 (dd, J = 17.1, 12.2 Hz, 1H),
13
.83 (s, 3H), 3.03 (dd, J = 17.2, 6.7 Hz, 1H); C NMR (125 MHz, CDCl ) δ 160.41,
3
47.74, 146.71, 143.28, 138.88, 129.95, 129.04, 128.89, 127.66, 127.36, 125.32,
13
25.09, 123.68, 114.13, 114.07, 61.20, 55.37, 42.28; C NMR (125 MHz, CDCl ) δ
3
56.78, 148.61, 142.39, 131.18, 130.72, 130.70, 130.31, 130.12, 129.95, 127.73,
27.34, 127.23, 124.11, 123.46, 115.11, 65.59, 54.88, 44.54; MS (ESI): m/z = 397
+
(
M+H) .
1
-(2-chlorophenyl)-5-(4-chlorophenyl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-
pyrazole (10).
The title compound was prepared by reaction of (E)-3-(4-chlorophenyl)-1-(3-
methoxyphenyl)prop-2-en-1-one(C4) and 2-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; brown solid; yield: 0.24 g
o
1
(
30%); mp112-113 C; H NMR (500 MHz, CDCl ) δ 7.43 (dd, J = 8.1, 1.5 Hz, 1H), 7.39
3
2
4

(
dd, J = 2.5, 1.5 Hz, 1H), 7.36 – 7.31 (m, 1H), 7.30 (dt, J = 7.6, 1.3 Hz, 1H), 7.21 (dd, J
=
=
1
1
1
3
1
8.0, 1.4 Hz, 1H), 7.15 – 7.07 (m, 5H), 6.94 (ddd, J = 8.0, 2.6, 1.2 Hz, 1H), 6.89 (ddd, J
7.9, 7.4, 1.6 Hz, 1H), 5.85 (dd, J = 11.5, 4.7 Hz, 1H), 3.87 (s, 3H), 3.79 (dd, J = 16.9,
13
1.5 Hz, 1H), 3.36 (dd, J = 16.9, 4.7 Hz, 1H); C NMR (125 MHz, CDCl ) δ 159.78,
3
49.77, 142.52, 139.37, 133.74, 133.46, 130.22, 129.63, 128.64, 128.10, 127.09,
24.66, 124.31, 123.74, 118.69, 115.23, 110.73, 65.62, 55.34, 42.45.; MS (ESI): m/z =
+
97.05(M+H) .
-(3-chlorophenyl)-5-(4-chlorophenyl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-
pyrazole (11).
The title compound was prepared by reaction of (E)-3-(4-chlorophenyl)-1-(3-
methoxyphenyl)prop-2-en-1-one (C4) and 3-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; brown solid; yield: 0.46 g
o
1
(
58%); mp91.6-93 C; H NMR (500 MHz, CDCl ) δ 7.27 – 7.22 (m, 4H), 7.17 (ddt, J =
3
9
1
3
.0, 4.4, 3.2 Hz, 3H), 7.12 (t, J = 2.1 Hz, 1H), 6.99 (t, J = 8.1 Hz, 1H), 6.87 – 6.83 (m,
H), 6.69 (dddd, J = 8.0, 5.9, 2.1, 0.9 Hz, 2H), 5.16 (dd, J = 12.3, 6.7 Hz, 1H), 3.81 (s,
13
H), 3.77 (dd, J = 17.1, 12.3 Hz, 1H), 3.03 (dd, J = 17.1, 6.7 Hz, 1H); C NMR (125
MHz, CDCl ) δ 160.00, 147.84, 145.65, 140.62, 135.08, 133.82, 133.71, 130.15,
3
1
4
1
29.88, 129.70, 127.43, 119.37, 118.83, 115.38, 113.76, 111.37, 111.03, 63.79, 55.60,
+
3.84; MS (ESI): m/z = 397.06 (M+H) .
,5-bis(4-chlorophenyl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-pyrazole (12).
The title compound was prepared by reaction of (E)-3-(4-chlorophenyl)-1-(3-
methoxyphenyl)prop-2-en-1-one(C4) and 4-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; light brown solid; yield:
2
5

o
1
0
7
1
1
1
4
1
.29 g (37%); 156-158 C; H NMR (500 MHz, CDCl ) δ 7.34 – 7.28 (m, 4H), 7.26 –
3
.19 (m, 3H), 7.16 – 7.10 (m, 2H), 6.98 – 6.94 (m, 2H), 6.91 (ddd, J = 8.2, 2.6, 1.0 Hz,
H), 5.22 (dd, J = 12.3, 7.0 Hz, 1H), 3.89 – 3.80 (m, 4H), 3.11 (dd, J = 17.1, 7.0 Hz,
13
H); C NMR (125 MHz, CDCl ) δ 160.05, 147.50, 143.35, 140.73, 133.86, 133.83,
3
29.88, 129.69, 129.10, 127.52, 124.47, 118.77, 115.27, 114.79, 111.04, 64.14, 55.59,
+
3.91; MS (ESI): m/z = 397.05 (M+H) .
-(2-chlorophenyl)-5-(3-chlorophenyl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-
pyrazole (13).
The title compound was prepared by reaction of (E)-3-(3-chlorophenyl)-1-(3-
methoxyphenyl)prop-2-en-1-one (C5) and 2-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; brown solid; yield: 0.41 g
o
1
(
52%); mp99.8 C; H NMR (500 MHz, CDCl ) δ 7.57 (dd, J = 8.1, 1.5 Hz, 1H), 7.53 –
3
7
1
.50 (m, 1H), 7.43 (ddd, J = 9.0, 7.7, 4.6 Hz, 2H), 7.35 (dt, J = 6.1, 3.1 Hz, 1H), 7.31 (s,
H), 7.27 – 7.19 (m, 4H), 7.08 (ddd, J = 8.0, 2.6, 1.1 Hz, 1H), 7.03 (ddd, J = 8.9, 5.9, 1.6
Hz, 1H), 5.94 (dd, J = 11.5, 4.9 Hz, 1H), 4.01 (s, 3H), 3.93 (dd, J = 16.9, 11.5 Hz, 1H),
1
3
3
1
1
3
1
.50 (dd, J = 16.9, 4.9 Hz, 1H); C NMR (125 MHz, CDCl ) δ 159.82, 149.77, 142.90,
3
42.49, 134.28, 133.71, 130.30, 129.81, 129.63, 127.96, 127.09, 126.87, 124.88,
24.84, 124.39, 123.79, 118.74, 115.30, 110.80, 65.83, 55.37, 42.53; MS (ESI): m/z =
+
97.08 (M+H) .
,5-bis(3-chlorophenyl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-pyrazole (14).
The title compound was prepared by reaction of (E)-3-(3-chlorophenyl)-1-(3-
methoxyphenyl)prop-2-en-1-one(C5) and 3-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; beige solid; yield: 0.52 g
2
6

o
1
(
66%); mp80-82 C; H NMR (500 MHz, CDCl ) δ 7.36 – 7.30 (m, 2H), 7.29 (dd, J = 4.9,
3
2
=
3
1
1
4
5
.9 Hz, 2H), 7.27 – 7.25 (m, 2H), 7.19 (t, J = 2.1 Hz, 1H), 7.18 – 7.14 (m, 1H), 7.06 (t, J
8.1 Hz, 1H), 6.94 – 6.89 (m, 1H), 6.79 – 6.71 (m, 2H), 5.21 (dd, J = 12.4, 6.7 Hz, 1H),
13
.88 – 3.80 (m, 4H), 3.12 (dd, J = 17.2, 6.7 Hz, 1H); C NMR (125 MHz, CDCl ) δ
3
59.80, 147.64, 145.46, 144.06, 135.18, 134.89, 133.45, 130.64, 129.95, 129.65,
28.14, 125.94, 123.92, 119.23, 118.65, 115.25, 113.57, 111.12, 110.83, 63.72, 55.39,
+
3.64; MS (ESI): m/z = 397.04 (M+H) .
-(3-chlorophenyl)-1-(4-chlorophenyl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-
pyrazole (15).
The title compound was prepared by reaction of (E)-3-(3-chlorophenyl)-1-(3-
methoxyphenyl)prop-2-en-1-one (C5) and 4-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; beige solid; yield: 0.21 g
o
1
(
27%); mp137-139 C; H NMR (500 MHz, CDCl ) δ 7.28 (ddd, J = 8.0, 5.7, 2.9 Hz, 3H),
3
7
–
–
1
1
3
1
.26 – 7.23 (m, 2H), 7.22 – 7.19 (m, 1H), 7.16 – 7.13 (m, 1H), 7.13 – 7.09 (m, 2H), 6.96
6.92 (m, 2H), 6.89 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 5.18 (dd, J = 12.4, 7.1 Hz, 1H), 3.86
13
3.78 (m, 4H), 3.10 (dd, J = 17.1, 7.1 Hz, 1H); C NMR (125 MHz, CDCl ) δ 159.80,
3
47.26, 144.15, 143.10, 135.15, 133.56, 130.61, 129.63, 128.88, 128.08, 126.02,
24.27, 123.96, 118.54, 115.08, 114.52, 110.78, 64.00, 55.34, 43.66; MS (ESI): m/z =
+
97 (M+H) .
,5-bis(2-chlorophenyl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-pyrazole (16).
The title compound was prepared by reaction of (E)-3-(2-chlorophenyl)-1-(3-
methoxyphenyl)prop-2-en-1-one (C6) and 2-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; light brown solid; yield:
2
7

o
1
0
1
2
3
1
1
.27 g (34%); mp122-124 C; H NMR (500 MHz, CDCl ) δ 7.44 (dd, J = 8.2, 1.5 Hz,
3
H), 7.32 (ddd, J = 9.2, 5.0, 1.4 Hz, 2H), 7.28 (tt, J = 4.2, 2.2 Hz, 2H), 7.26 – 7.23 (m,
H), 7.16 – 7.05 (m, 3H), 6.91 – 6.85 (m, 2H), 6.20 (dd, J = 11.5, 6.5 Hz, 1H), 3.89 –
13
.83 (m, 4H), 3.11 (dd, J = 16.7, 6.6 Hz, 1H); C NMR (125 MHz, CDCl ) δ 159.77,
3
49.31, 142.44, 138.82, 133.83, 132.11, 130.98, 129.72, 129.56, 128.62, 127.89,
27.10, 127.08, 124.60, 123.67, 121.87, 118.73, 115.28, 110.76, 63.14, 55.35, 41.90;
+
MS (ESI): m/z = 397.06 (M+H) .
5
-(2-chlorophenyl)-1-(3-chlorophenyl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-
pyrazole(17).
The title compound was prepared by reaction of (E)-3-(2-chlorophenyl)-1-(3-
methoxyphenyl)prop-2-en-1-one (C6) and 3-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; brown solid; yield: 0.38 g
o
1
(
47.5%); mp153-154 C; H NMR (500 MHz, CDCl ) δ 7.52 (t, J = 7.4 Hz, 1H), 7.41 –
3
7
.39 (m, 1H), 7.37 (t, J = 7.9 Hz, 1H), 7.34 – 7.28 (m, 2H), 7.28 – 7.26 (m, 1H), 7.24 (d,
J = 4.2 Hz, 2H), 7.15 (dd, J = 14.0, 6.0 Hz, 1H), 7.00 – 6.94 (m, 1H), 6.87 – 6.81 (m,
1
1
1
1
H), 6.77 – 6.72 (m, 1H), 5.71 (dd, J = 12.3, 6.3 Hz, 1H), 4.03 (dd, J = 17.3, 12.3 Hz,
13
H), 3.94 (s, 3H), 3.13 (dd, J = 17.3, 6.3 Hz, 1H); C NMR (125 MHz, CDCl ) δ 159.79,
3
48.04, 145.28, 138.56, 134.94, 133.57, 131.77, 130.02, 129.98, 129.61, 129.01,
27.69, 127.12, 119.07, 118.66, 115.23, 113.40, 110.89, 110.78, 61.03, 55.38, 42.17;
+
MS (ESI): m/z = 397.04 (M+H) .
5
-(2-chlorophenyl)-1-(4-chlorophenyl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-
pyrazole (18).
2
8

The title compound was prepared by reaction of (E)-3-(2-chlorophenyl)-1-(3-
methoxyphenyl)prop-2-en-1-one (C6) and 4-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; brown solid; yield: 0.43 g
o
1
(
54%); mp 98-99 C; H NMR (500 MHz, CDCl ) δ 7.58 – 7.53 (m, 1H), 7.42 (dt, J = 4.2,
3
2
7
1
.1 Hz, 1H), 7.40 (t, J = 7.9 Hz, 1H), 7.38 – 7.35 (m, 1H), 7.35 – 7.32 (m, 1H), 7.29 –
.22 (m, 4H), 7.05 – 6.98 (m, 3H), 5.73 (dd, J = 12.4, 6.6 Hz, 1H), 4.07 (dd, J = 17.2,
13
2.4 Hz, 1H), 3.97 (s, 3H), 3.16 (dd, J = 17.2, 6.6 Hz, 1H); C NMR (125 MHz, CDCl )
3
δ 160.04, 147.87, 143.16, 138.92, 133.93, 132.05, 130.25, 129.85, 129.23, 129.17,
1
27.92, 127.46, 124.32, 118.81, 115.33, 114.53, 110.98, 61.48, 55.60, 42.41; MS (ESI):
+
m/z = 397.02 (M+H) .
1
-(2-chlorophenyl)-5-(4-chlorophenyl)-3-(2-methoxyphenyl)-4,5-dihydro-1H-
pyrazole (19).
The title compound was prepared by reaction of (E)-3-(4-chlorophenyl)-1-(2-
methoxyphenyl)prop-2-en-1-one (C7) and 2-chlorophenylhydrazine hydrochloride
according to the general procedure for pyrazoline synthesis; beige solid; yield: 0.33 g
o
1
(
41%); mp103-105 C; H NMR (500 MHz, DMSO) δ 7.82 (dd, J = 7.7, 1.8 Hz, 1H), 7.44
–
6
1
7.35 (m, 2H), 7.31 – 7.17 (m, 5H), 7.17 – 7.07 (m, 2H), 7.02 (td, J = 7.5, 1.1 Hz, 1H),
.93 (ddd, J = 7.9, 7.3, 1.6 Hz, 1H), 5.76 (dd, J = 11.1, 5.6 Hz, 1H), 3.89 (dd, J = 17.8,
1.2 Hz, 1H), 3.82 (s, 3H), 3.38 (dd, J = 17.7, 5.6 Hz, 1H) ; MS (ESI): m/z = 397.08
+
(
M+H) .
1
-(3-chlorophenyl)-5-(4-chlorophenyl)-3-(2-methoxyphenyl)-4,5-dihydro-1H-
pyrazole (20).
2
9