Synthesis and in vitro antitumor activity of new 4,5-dihydropyrazole derivatives

Article abstract of DOI:10.1016/j.bmc.2010.07.037

A series of 3,5-diaryl-4,5-dihydropyrazole regioisomers, and their 1-acetylated derivatives, bearing a 3,4,5-trimethoxyphenyl moiety combined with a variety of substituted phenyl rings, was synthesized and evaluated for antitumor activity. Results of the in vitro assay against a non-small cell lung carcinoma cell line (NCI-H460) showed several compounds to be endowed with cytotoxicity in micromolar to sub-micromolar range, depending on substitution pattern and position of aryl rings on 4,5-dihydropyrazole core. Potent and selective activity was also observed in the NCI 60 human cancer cell line panel. 5-(3,4,5-Trimethoxyphenyl)pyrazolines 31 and 39 were found to possess potent antiproliferative activity against SR and MDA-MB-435, with GI₅₀ inhibitory values in nanomolar range. Structure-activity relationships revealed that introduction of a (hydroxy)acetyl group at N-1 of inactive 5-(3,4,5-trimethoxyphenyl)pyrazolines, results in a clear in vitro activating effect. Compound 31 (IC₅₀ = 5.16 μM) showed inhibition of tubulin polymerization comparable to that of CA-4 (IC₅₀ = 4.92 μM).

Full text of DOI:10.1016/j.bmc.2010.07.037

Bioorganic & Medicinal Chemistry 18 (2010) 6238–6248
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro antitumor activity of new 4,5-dihydropyrazole derivatives
a
b
b
b
Cenzo Congiu a, , Valentina Onnis , Loredana Vesci , Massimo Castorina , Claudio Pisano
*
a Dipartimento di Tossicologia, Sezione di Chimica Farmaceutica, Università degli Studi di Cagliari, via Ospedale 72, Cagliari I-09124, Italy
b R&D, Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Pomezia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3,5-diaryl-4,5-dihydropyrazole regioisomers, and their 1-acetylated derivatives, bearing a
3,4,5-trimethoxyphenyl moiety combined with a variety of substituted phenyl rings, was synthesized
and evaluated for antitumor activity. Results of the in vitro assay against a non-small cell lung carcinoma
cell line (NCI-H460) showed several compounds to be endowed with cytotoxicity in micromolar to sub-
micromolar range, depending on substitution pattern and position of aryl rings on 4,5-dihydropyrazole
core. Potent and selective activity was also observed in the NCI 60 human cancer cell line panel.
5-(3,4,5-Trimethoxyphenyl)pyrazolines 31 and 39 were found to possess potent antiproliferative activity
against SR and MDA-MB-435, with GI50 inhibitory values in nanomolar range. Structure–activity relation-
ships revealed that introduction of a (hydroxy)acetyl group at N-1 of inactive 5-(3,4,5-trimethoxy-
Received 10 June 2010
Revised 12 July 2010
Accepted 15 July 2010
Available online 9 August 2010
Keywords:
Antitumor activity
Cytotoxicity
Dihydropyrazole
Tubulin
phenyl)pyrazolines, results in a clear in vitro activating effect. Compound 31 (IC50 = 5.16
inhibition of tubulin polymerization comparable to that of CA-4 (IC50 = 4.92 M).
lM) showed
l
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
cis-restricted biologically active analogs, a number of compounds
containing different 1,2-substituted heterocyclic rings as replace-
ment for the stilbene core of CA-4 have been reported.7 On the
other hand, there is a limited number of analogs where the stilbene
core of CA-4 has been replaced with 1,3-substituted heterocycles.
Examples of heterocyclic bridges include isoxazolines,8 oxadiazo-
lines,9 and pyrazoles.10 In our efforts to discover new potential
anti-cancer CA-4 based chemotherapeutic agents,11 we decided
to explore series of 3,5-diaryl-4,5-dihydropyrazole compounds
bearing a 3,4,5-trimethoxyphenyl moiety as the prerequisite for
potent cytotoxicity,6d combined with a variety of substituted phe-
nyl rings, while the ethenyl bridge in the CA-4 skeleton was re-
Combretastatins are a group of antimitotic agents derived from
the African Willow tree (Combretum caffrum) and they are power-
ful reversible inhibitors of tubulin polymerization. Among natural
combretastatins, Combretastatin A-4 (3,4,5-trimethoxy-30-hydro-
xy-40-methoxy-(Z)-stilbene, CA-4, 1) (Fig. 1) was found to be the
most potent as antiproliferative agent against a broad range of can-
cer cells including multi-drug-resistant (MDR) cell lines.1 CA-4
inhibits mitosis and microtubule assembly and is a competitive
inhibitor of colchicine binding to tubulin.2 In the recent literature
it has been reported that drugs binding to the colchicine domain
are intensively investigated as vascular-disrupting agents for can-
cer therapy.3 CA-4 itself is poorly water-soluble and several at-
tempts have been made to produce an active soluble derivative.4
A disodium phosphate derivative Combretastatin A-4 phosphate
(CA-4P, 2, Zybrestat) (Fig. 1) has shown promising results in phase
I/II cancer clinical trials.5
As microtubule inhibitor, CA-4P act as a vascular-disrupting
agent that rapidly depolymerizes microtubules of newly formed
vasculatures and subsequently shut down the blood supply to tu-
mors.6 Studies on CA-4 have established that cis-orientation of eth-
enyl bridge is essential for strong cytotoxicity. However, during
storage and administration, cis-olephine double bond is prone to
isomerize into trans-form, resulting in dramatic reduction in both
antitubulin activity and cytotoxicity. With the aim of developing
placed with
a
structurally flexible non-aromatic nitrogen
heterocycle which would augment biological solubility. Recently,
Johnson et al. developed a series of pyrazolines and a range of
N-acetylated derivatives bearing in particular a 3,4,5-trimethoxy-
phenyl group in 3-position of the heterocyclic ring.12 Among these,
a non-acetylated pyrazoline with the same substituents as CA-4
was the most active compound as both in vitro antitumor and
MeO
MeO
MeO
MeO
O
P
ONa
OMe
OMe
ONa
OH
O
OMe
OMe
2: CA-4P
1: Combretastatin A-4 (CA-4)
* Corresponding author.
E-mail address: ccongiu@unica.it (C. Congiu).
Figure 1.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.037

C. Congiu et al. / Bioorg. Med. Chem. 18 (2010) 6238–6248
6239
OMe
OMe
2. Results and discussion
2.1. Chemistry
OMe
OMe
OMe
OMe
N
H2N
O
O
N
N
3,5-Diaryl-4,5-dihydropyrazole derivatives were easily pre-
pared by the reaction sequence showed in Schemes 1–3. The start-
ing materials for the synthesis of compounds A and B were
chalcones 5, prepared through the well-established Claisen–
Schmidt condensation15 of 3,4,5-trimethoxyacetophenone with
substituted benzaldehydes in methanol solution in presence of
NaOH (Scheme 1).
N
N
O
O
3: A-105972
4: A-204197
Figure 2.
Similarly, chalcones 6, which serve as the precursors for com-
pounds C and D, were prepared by the condensation of 3,4,5-tri-
methoxybenzaldehyde with substituted acetophenones. Chalcones
5 and 6 were then reacted with hydrazine hydrate in boiling etha-
nol16 to effect the assembly of pyrazolines 7–11 in 62–92% yields,
and 12–22 in 69–88% yields, respectively (Scheme 1).
In order to evaluate the effect of substitution on the N-1 nitro-
gen atom, 1-acetylated derivatives were prepared. Firstly, upon
treatment with hydrazine hydrate in boiling acetic acid17 chal-
cones 5 and 6 were converted into 1-acetyl-3,5-diaryl-4,5-dihydro-
pyrazoles 23–27 in 56–90% yields, and 28–41 in 60–95% yields,
respectively (Scheme 2). Boc-deprotection of 40 and 41 with triflu-
microtubules disrupting agent; in contrast, the N-acetylated deriv-
ative of the same compound showed poor activity. Moreover, ear-
lier reported studies on the similar acetylated oxadiazoline series9a
demonstrated compounds 313 and 414 (Fig. 2), bearing the acetyl
moiety in position adjacent to a 3,4,5-trimethoxyphenyl group, to
be endowed with promising in vitro and in vivo antineoplastic
properties due to their ability to inhibit a variety of cancer cell
lines, including multi-drug resistant cells, and microtubules
polymerization.
In view of these findings, substitution on N-1 nitrogen atom of
the 5-(3,4,5-trimethoxyphenyl)pyrazolines should be informative
to evaluate the effect on the activity of these series of compound.
Therefore, a new series of 3-(3,4,5-trimethoxyphenyl)-4,5-dihyd-
ropyrazole A, and their 1-acetyl derivatives B were designed along
with C and D analogs, respectively, bearing a 3,4,5-trimethoxy-
phenyl ring in 5-position, for SARs studies on series of 4,5-dihydro-
pyrazole regioisomers (Fig. 3).
OMe
OMe
MeO
O
MeO
MeO
O
MeO
R
Moreover, the 1-acetyl moiety in representative D analogs was
further functionalized, that is, introduction of hydroxy or acethoxy
groups (E analogs), for further SARs studies (Fig. 3). Here we report
the synthesis of these novel 4,5-dihydropyrazole-based CA-4 ana-
logs and results of their in vitro biological activities as both antitu-
mor agents and tubulin polymerization inhibitors.
O
a
R
b
5
OMe
MeO
MeO
OMe
OMe
N
NH
R
MeO
MeO
MeO
MeO
7: R = 3-OH
8: R = 3-MeO
9: R = 4-MeO
N
N
NH
N
R
R
10: R = 4-Me2N
O
A
B
11: R = 4-Et2N
OMe
OMe
OMe
OMe
OMe
O
R
MeO
MeO
OMe
OMe
OMe
OMe
OMe
OMe
O
R
a
N
O
NH
N
N
O
6
R
R
b
C
D
OMe
OMe
OMe
OMe
OMe
NH
N
R
12: R = 4-Me
13: R = 4-F
14: R = 3-Br
15: R = 4-Br
16: R = 3-OH
18: R = 4-MeO
19: R = 4-MeS
20: R = 4-Me2N
21: R = 4-Et2N
N
N
O
R
R'
R' = OH, OCOCH3
E
22: R = 3-MeO-4-OH
17: R = 3-MeO
Scheme 1. Reagents and conditions: (a) MeOH, NaOH pellets, rt; (b) NH2NH2ꢀH2O,
Figure 3. Pyrazoline-based CA-4 analogs.
EtOH, reflux, 2 h.

6240
C. Congiu et al. / Bioorg. Med. Chem. 18 (2010) 6238–6248
(HOBt), in dry MeCN, followed by treatment with potassium ace-
OMe
23: R = 3-OH
24: R = 3-MeO
25: R = 4-MeO
26: R = 4-Me2N
27: R = 4-Et2N
tate in DMSO, 1-(acethoxyacetyl)pyrazolines 44–48 were obtained
in 72–89% overall yields. Hydrolysis of 44–48 using potassium car-
bonate in aqueous methanol provided 1-(hydroxyacetyl)pyrazo-
lines 49–53 in almost quantitative yields (Scheme 3).
MeO
MeO
a
5
N
N
R
O
2.2. Pharmacology
In a primary assay, synthesized pyrazolines and their 1-acety-
lated derivatives were screened for their ability to inhibit cell
growth of a non-small cell lung carcinoma (NSLC) NCI-H460 cell
line determining the number of surviving cells with sulforhoda-
mine B test.18 Results, expressed as concentrations that inhibit
50% of cell growth (IC50), were calculated by the ALLFIT program,
and are shown in Table 1.
Among non-acetylated pyrazolines, analogs 8–11, bearing the
3,4,5-trimethoxyphenyl moiety in 3-position showed antiprolifer-
ative effect, being derivative 11 the most potent agent with IC50 va-
28: R = 4-Me
29: R = 4-F
OMe
30: R = 4-Cl
OMe
OMe
31: R = 3-Br
a
32: R = 4-Br
6
33: R = 3-OH
34: R = 3-MeO
35: R = 4-MeO
36: R = 4-MeS
37: R = 4-Me2N
38: R = 4-Et2N
39: R = 3-MeO-4-OH
40: R = 3-NH-Boc
41: R = 4-NH-Boc
42: R = 3-NH2
43: R = 4-NH2
N
N
R
O
lue of 0.30
inactive. Within 1-acetylated derivatives 23–27, only compound
27 exhibited a weak 13.8 M IC50 value, whereas regioisomers
lM; in contrast, pyrazoline regioisomers 12–22 were
l
28–33, 36–39, 42, and 43, bearing a 3,4,5-trimethoxyphenyl moi-
ety in 5-position of the 4,5-dihydropyrazole core showed growth
inhibition in micromolar to sub-micromolar range. Derivatives 31
and 39 were the most potent agents with IC50 values of 0.35 and
b
Scheme 2. Reagents and conditions: (a) NH2NH2ꢀH2O, AcOH, reflux, 3 h; (b) TFA,
CH2Cl2, rt, 16 h.
0.21 lM, respectively. Across the series of derivatives, a 4-(diethyl-
amino)phenyl moiety favorably affects the inhibitory activity
against the NCI-H460 cell line. Therefore, initial SAR study was fo-
cused on modifications of this substituent. Among non-acetylated
pyrazolines 7–11, replacement of 4-(diethylamino) group of 11
results in a progressive decrease in potency, from 10 to 7. In the
series of 1-acetylpyrazolines 28–43, introduction of a 4-methoxy-
phenyl group leads to inactive compound 35; introduction at the
same position of phenyl ring of either halogen atoms (compounds
29, 30, and 32), or methylthio (36) substituents results in weakly
active analogs. On the other hand, 4-amino- (43), 4-(dimethyl-
amino)- (37), and 4-methylphenyl (28) analogs exhibit increasing
potency in the low micromolar range. While the 3-bromo- (31)
and 3-aminophenyl (42) derivatives show sub-micromolar inhibi-
tory potency, the 3-hydroxy analog (33) is moderately active, and
the 3-methoxy analog (34) is devoid of activity. Interestingly,
introduction of a 4-hydroxy substituent in this last compound re-
sults in the most active analog (39) in the whole series. This result
can be rationalized by comparing molecular modeling of 39 and 1
(Fig. 4). Initially, compounds were constructed with standard bond
and angles, and their geometry were optimized using Spartan’08
software. The optimal structure and conformation of 39 and 1 were
further optimized using standard Hartree–Fock models. The result-
ing conformation of pyrazoline 39 is shown in Figure 4A. It is evi-
dent that the 4,5-dihydropyrazole derivative adopts a twisted
conformation similar to 1 (Fig. 4B). In particular, the phenyl ring
bearing the hydroxy and methoxy groups of 39 adopts a orienta-
tion comparable to 1, resulting in its improved ability to interact
with the biological target, although the hydroxy and methoxy sub-
stituents are in inverted position respect to CA-4.
OMe
OMe
a
12, 14, 20-22
OMe
N
N
R
O
O
O
44: R = 4-Me
45: R = 3-Br
46: R = 4-Me2N
47: R = 4-Et2N
b
48: R = 3-MeO-4-OH
OMe
OMe
OMe
N
N
R
OH
O
49: R = 4-Me
50: R = 3-Br
51: R = 4-Me2N
52: R = 4-Et2N
53: R = 3-MeO-4-OH
The effect of the functionalization of the 1-acetyl group was eval-
uated in compounds 44–53. Compounds 44–48 show a substantial
decrease in inhibition ability as compared to the parental 1-acety-
latedanalogs, likely becauseof steric hindranceimposedby theacet-
oxy group; however, hydrolysis of 44–48 to give 1-hydroxyacetyl
derivatives 49–53 restores inhibitory efficacy, as a result of both en-
hanced hydrophilicity and decreased steric hindrance.
Comprehensive examination of cytotoxicity data shows that
introduction of a (hydroxy)acetyl group at the N-1 of inactive
3-aryl-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-pyrazoles 12–
22 results in clear activating effect on their ability to inhibit cell
Scheme 3. Reagents and conditions: (a) (i) HOBt, EDC, chloroacetic acid, MeCN,
24 h, rt; (ii) potassium acetate, DMSO, 12 h, rt; (b) K2CO3, MeOH/H2O 10:1, 6 h, rt.
oroacetic acid in dichloromethane gave 42 (86%) and 43 (77%),
respectively.
Compounds 44–53, with a further functionalized acetyl moiety
at the 1-position of the pyrazoline ring, were synthesized starting
from 3-aryl-5-(3,4,5-trimethoxyphenyl)-4,5-dihydropyrazoles, as
shown in Scheme 3. Upon coupling pyrazolines 12, 14, 20–22 with
chloroacetic acid in the presence of 1-(3-dimethylaminopropyl)-3-
ethylcardodiimide hydrochloride (EDC) and hydroxybenzotriazole

C. Congiu et al. / Bioorg. Med. Chem. 18 (2010) 6238–6248
6241
Table 1
Inhibitory activity of pyrazoline 7–53
against a non-small cell lung carci-
noma NCI-H460 cell linea
Compd
IC50 SE (lM)
7
8
9
>20
11.6 0.1
3.8 0.1
1.1 0.05
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
0.30 0.01
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
13.8 0.1
1.3 0.05
12.0 0.9
11.9 1.6
0.35 0.01
6.0 0.09
2.3 0.06
>20
Figure 4. Molecular ball models of pyrazoline 39 (A) and 1 (B). Gray represents
carbon, white hydrogen, blue nitrogen, and red oxygen.
anoma, ovarian, renal, prostate, and breast.19 The compounds were
tested at five concentrations at 10-fold dilution. A 48 h continuous
drug exposure protocol was used and sulforhodamine B (SRB) pro-
tein assay was used to estimate cell growth.20 The antitumor activ-
ity of tested compounds is given by three parameters for each cell
line: pGI50 value (ꢁlog of the molar concentration of the compound
that inhibits 50% net cell growth), pTGI value (ꢁlog of the molar
concentration of the compound leading to total inhibition), and
pLC50 value (ꢁlog of the molar concentration of the compound
leading to 50% net cell death). Furthermore, a mean graph mid-
point (MG-MID) is calculated for each of the mentioned parame-
ters, giving an averaged activity parameter over all cell lines. For
the calculation of the MG-MID, insensitive cell lines are included
with the highest concentration tested.
Screening data against the NCI in vitro 60 human cancer cell pa-
nel corroborated the results described above. Representative
growth inhibitory properties of tested compounds compared to
CA-4 (1) as the reference compound are reported in Table 2. The
pGI50 MG-MID values across the series of compounds are in the
5.1–6.4 range, being 11, 31, 38, and 39 the most potent agents, with
a variety of significant cell selectivity. As compared with 1, all
tested compounds exhibit better inhibitory efficacy against the
ovarian SK-OV-3 and breast T-74D cell lines, and better or compa-
rable effect against the colon COLO 205 and HT29, and CNS SNB-75
cell lines, at both pGI50 and pTGI levels.
>20
8.0 0.1
1.8 0.06
0.98 0.03
0.21 0.005
Not tested
Not tested
0.74 0.03
2.6 0.1
3.3 0.1
>20
>20
5.0 0.2
4.5 0.1
0.69 0.02
1.1 0.02
1.3 0.01
0.95 0.05
1.85 0.07
a
Inhibitory activity was assayed
by exposure for 72 h to substances
and expressed as concentration
required to inhibit tumor cell pro-
liferation by 50% (IC50). Data are
expressed as mean SE from the
doseꢁresponse curves of at least
three independent experiments.
Selectively, compounds 31, 33, 38, and 39 show better inhibitory
potency than 1 against the leukemic SR cell line, with GI50 values in
nanomolar range. Compounds 31 and 39 also inhibit the MDA-MB-
435 cell line (pGI50 7.3) with potency comparable to 1 (pGI50 7.5). Sig-
nificantly, all tested compounds exhibit antiproliferative effect
against ovarian NCI/ADR-RES cell line, with pGI50 values in the 5.2–
6.7 range. This finding is important because NCI/ADR-RES, an
adriamycin-resistant cell line, expresses high levels of MDR-1 and
P-glycoprotein, and is a useful model for new drug development,
given its utility in identifying compounds subject to multi-drug
resistance.
growth. This result well correlate with those of the above mentioned
acetylated oxadiazoline series.9a Likely, the acetyl moiety in position
adjacent to a 3,4,5-trimethoxyphenyl group of a 4,5-dihydropyraz-
ole core, as in compounds 31, 38, and 39, allows the molecule to
adopt a conformation that would activate their ability to interact
with mitotic microtubules, leading to cytotoxic effect. In contrast,
the same substituents in the 1–3 positions, as in compounds
23–27, results in opposite effect, accordingly to the previous re-
ported result.12
Selected pyrazolines were submitted to the US National Cancer
Institute (NCI; Bethesda, MD), for the more extensive in vitro test-
ing against a panel of approximately 60 tumor cell lines, derived
from nine different cancer types: leukemia, lung, colon, CNS, mel-
To investigate a possible mechanism of action responsible for
the observed antiproliferative effect, active agents, and inactive
or less active congeners, were evaluated for their ability to affect
tubulin polymerization. The tubulin polymerization, performed

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C. Congiu et al. / Bioorg. Med. Chem. 18 (2010) 6238–6248
Table 2
Overview of growth inhibitory properties of compounds 9–11, 31, 33, 37–39, and 1 against the NCI human cancer cell lines, and the average pGI50 value in the full panel cell lines
screeninga
Compound
9
10
11
31
33
37
38
39
1b
c
Panel/cell line
pGI50
pTGId
pGI50
pTGI
pGI50
pTGI
pGI50
pTGI
pGI50
pTGI
pGI50
pTGI
pGI50
pTGI
pGI50
pTGI
pGI50
pTGI
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
5.2
5.7
5.6
5.3
5.5
6.0
4.0
4.9
4.0
4.6
4.7
5.4
5.8
5.8
6.4
5.5
5.8
6.6
5.2
5.3
4.1
4.9
4.9
5.1
6.7
6.6
6.4
5.9
6.4
6.6
6.2
5.7
4.9
5.3
5.4
6.0
6.5
6.6
6.9
6.5
6.8
7.4
5.7
6.3
4.9
5.8
6.2
4.0
6.0
5.8
6.2
5.3
6.0
7.0
5.3
5.3
4.2
4.0
5.3
4.0
5.6
6.0
5.8
5.5
5.5
6.2
4.0
5.2
4.0
4.0
4.8
4.0
6.5
6.9
6.2
6.6
6.8
7.2
6.0
6.3
4.0
5.5
6.3
6.2
6.5
6.8
6.4
6.4
6.7
7.3
6.0
6.3
4.0
4.0
6.1
6.3
7.0
7.5
7.5
7.0
6.8
6.9
5.3
7.2
5.7
5.4
5.2
4.1
NSCLe
EKVX
5.0
4.5
4.1
5.5
5.5
4.0
4.0
4.0
4.52
4.44
5.7
5.9
4.9
5.9
5.7
5.2
4.0
4.0
5.2
5.2
6.4
6.5
4.0
6.5
6.6
6.1
5.4
4.0
4.9
5.7
6.2
6.0
6.3
6.5
6.4
4.0
4.1
4.0
4.9
4.7
5.2
6.9
5.4
5.7
5.8
4.0
5.7
4.0
5.0
4.4
5.3
6.1
5.4
6.2
6.2
4.0
4.0
4.2
4.0
5.1
6.4
5.8
6.3
6.4
6.4
5.6
4.6
4.9
5.7
6.2
6.1
6.3
6.3
6.4
6.5
4.0
4.0
4.0
4.8
4.0
6.4
6.7
7.1
7.5
7.5
4.1
5.6
4.1
4.2
5.4
HOP-62
NCI-H322M
NCI-H460
NCI-H522
Colon
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
5.4
5.0
5.4
5.5
5.4
5.4
5.3
4.5
4.4
4.0
4.0
4.0
4.0
4.0
5.6
5.8
5.7
5.6
6.2
6.3
6.4
4.9
5.3
4.7
4.0
4.0
4.9
4.0
6.4
6.2
6.6
6.2
6.5
6.4
6.4
5.8
5.1
6.0
4.0
4.0
4.0
4.0
6.7
5.8
6.3
6.5
nt
6.2
4.6
4.0
4.5
nt
5.4
5.7
5.6
6.1
5.5
6.0
6.1
5.0
4.8
4.0
4.0
4.0
4.0
4.0
5.5
5.9
6.3
6.2
6.4
6.1
6.3
4.8
4.7
4.7
4.0
4.8
4.5
4.0
6.5
6.1
6.5
6.4
6.4
6.5
6.5
5.9
5.1
4.9
4.9
5.8
4.9
4.8
6.6
ntf
6.1
nt
5.6
7.3
7.5
7.5
6.2
7.3
7.5
4.3
5.9
6.7
5.0
5.2
6.1
4.1
6.4
6.4
6.5
6.6
6.5
4.3
4.0
4.0
4.0
4.0
KM12
SW-620
6.5
6.5
4.7
4.0
CNS
SF-268
SF-295
SF-539
SNB-75
U251
4.7
5.3
5.2
5.4
5.4
4.0
4.0
4.4
nt
5.7
6.6
6.3
6.0
6.2
4.0
6.1
5.5
4.0
4.0
6.1
7.0
6.5
6.7
6.4
nt
6.0
6.7
6.5
6.4
6.3
4.1
4.9
5.0
4.0
4.5
5.1
6.5
5.7
6.4
5.6
4.0
5.3
5.2
nt
5.6
6.8
6.0
6.1
6.2
4.0
6.2
5.3
4.0
4.0
6.1
6.7
6.5
6.6
6.4
4.7
6.2
6.2
6.2
4.9
6.2
6.7
6.5
6.6
6.4
4.0
nt
6.0
4.0
4.0
6.9
7.2
7.4
6.1
7.3
4.3
5.9
6.7
4.6
4.9
6.5
5.9
6.2
4.0
4.0
4.0
Melanoma
MALME-3M
M14
SK-MEL-2
MDA-MB-435
SK-MEL-28
SK-MEL-5
UACC-62
4.6
4.9
nt
5.5
4.4
5.4
5.1
4.0
4.0
nt
4.7
4.0
4.6
4.0
5.1
5.6
5.5
6.0
4.8
5.7
5.4
4.0
nt
4.5
nt
4.0
5.2
4.0
4.7
nt
5.8
6.8
nt
4.0
nt
4.0
6.5
nt
nt
4.3
4.2
4.1
6.6
4.2
4.9
4.7
5.6
6.1
5.2
6.6
5.3
6.2
5.6
4.0
4.0
4.0
6.1
4.0
5.3
4.0
4.0
6.3
5.1
6.7
4.8
6.2
6.2
4.0
5.6
4.0
6.2
4.0
4.9
4.0
6.7
6.4
6.3
6.8
6.1
6.4
6.5
4.6
5.5
4.4
6.4
4.7
5.4
4.9
nt
4.0
4.0
4.0
6.7
4.0
6.3
4.0
6.3
6.8
5.1
7.5
5.5
8.0
8.0
4.1
6.6
4.1
7.1
4.3
8.0
4.2
6.2
4.7
7.3
6.1
6.6
6.7
6.4
6.3
7.3
6.3
7.2
6.5
6.1
6.4
5.5
4.3
Ovarian
IGROV1
5.2
5.4
4.9
4.6
5.2
4.9
4.0
4.3
4.0
4.0
4.0
4.0
6.3
5.2
4.8
5.4
5.7
5.4
5.5
4.0
4.0
4.0
4.0
4.0
6.5
6.4
6.0
6.4
6.3
6.3
5.2
4.0
4.0
5.1
4.0
4.0
5.7
nt
6.2
6.2
6.4
6.3
4.0
nt
4.0
4.0
4.0
4.2
5.4
5.9
5.2
5.3
5.9
5.6
4.0
5.4
4.0
4.0
5.4
4.0
5.6
6.6
5.4
5.3
6.5
5.4
nt
6.1
6.4
6.2
6.4
6.7
6.4
4.5
5.0
4.6
5.3
6.3
4.8
6.3
6.6
6.4
6.2
6.6
6.5
4.0
6.2
4.0
4.0
4.0
4.0
7.2
7.4
6.6
7.4
7.2
4.5
4.2
6.8
4.1
4.4
6.4
4.0
OVCAR-3
OVCAR-4
OVCAR-8
NCI/ADR-RES
SK-OV-3
6.0
4.0
4.0
5.9
4.0
Renal
786-0
A498
CAKI-1
RXF 393
TK-10
UO-31
4.7
5.2
5.1
5.0
4.4
5.3
4.0
4.3
4.2
4.0
4.0
4.0
5.7
5.5
5.6
5.9
5.0
5.3
4.0
5.1
4.0
5.3
4.0
4.3
6.8
6.4
6.7
nt
4.4
6.1
6.3
6.1
6.1
nt
4.0
4.0
6.1
6.4
6.6
6.6
4.9
4.9
4.0
5.4
4.4
6.0
4.2
4.0
5.9
5.6
5.6
5.4
5.1
5.0
5.4
5.0
4.0
4.0
4.0
4.0
5.9
5.6
6.3
5.8
4.9
5.6
4.2
4.7
4.0
5.3
4.0
4.0
6.3
6.5
6.9
6.4
6.4
6.4
4.9
5.6
4.7
5.6
4.7
4.8
6.4
6.6
6.5
6.9
6.2
6.4
4.0
5.4
4.0
nt
4.0
4.0
7.7
7.3
7.4
6.0
6.2
6.7
4.6
6.1
4.8
4.1
5.4
4.3
Prostate
PC-3
DU-145
5.2
4.5
4.0
4.0
6.1
5.7
4.0
5.3
6.5
6.4
4.2
5.8
6.7
6.4
4.5
4.9
5.7
5.5
4.0
nt
6.3
5.7
4.0
4.9
6.5
6.4
4.9
5.7
6.6
6.5
4.0
4.9
7.4
8.0
4.0
4.0
Breast
MCF7
HS 578T
BT-549
5.6
4.8
5.1
5.4
5.4
5.1
4.6
4.0
4.0
4.0
4.2
6.4
5.6
6.1
4.7
nt
4.0
5.2
5.0
4.0
nt
6.8
6.6
6.4
nt
nt
6.2
5.8
6.2
5.3
4.0
nt
6.8
nt
nt
nt
6.7
6.2
4.8
nt
4.4
4.4
4.0
6.3
6.1
5.3
5.5
6.3
5.5
5.0
5.1
4.0
4.0
5.0
6.4
6.4
5.6
5.2
5.8
5.6
4.0
5.5
4.0
4.0
4.0
6.5
6.5
6.2
6.7
6.6
6.4
5.0
4.6
5.3
4.7
6.2
6.6
6.7
6.2
nt
6.6
6.3
4.0
6.2
4.0
nt
7.4
6.8
8.0
4.3
4.1
5.1
5.7
4.0
T-47D
MDA-MB-468
6.1
MG-MIDg
5.6
7.0
a
b
c
d
e
f
Data obtained from the NCI’s in vitro disease-oriented human tumor cells screen.
http://dtp.nci.nih.gov/dtpstandard/dwindex/index.jsp.
pGI50 is the ꢁlog of the molar concentration of the compound that causes 50% inhibition of net cell growth.
pTGI is the ꢁlog of the molar concentration of the compound leading to total inhibition of cell growth.
Non-small cell lung cancer.
Not tested.
g
MG-MID = mean graph midpoint = arithmetical mean value for all tested cell lines.

C. Congiu et al. / Bioorg. Med. Chem. 18 (2010) 6238–6248
6243
Table 3
(300 MHz) in DMSO-d6 as the solvent. Chemical shifts are ex-
pressed in ppm relative to tetramethylsilane. Splitting patterns
are designated as singlet (s), doublet (d), triplet (t), quartet (q),
and multiplet (m). Infrared spectra were run on Bruker Vector 22
spectrophotometer. Silica gel thin-layer chromatography (TLC)
sheets from Fluka (silica gel precoated aluminum sheets with fluo-
rescent indicator at 254 nm) were used for TLC. Developed plates
were visualized by a Spectroline ENF 260C/F UV apparatus. Con-
centration and evaporation of the solvent after reaction or extrac-
tion were carried out on a rotary evaporator (Büchi Rotavapor)
operating at reduced pressure. Elemental analyses were carried
out with a Carlo Erba model 1106 elemental analyzer, and all val-
ues were within 0.4% of the calculated values, which indicates
>95% purity of the tested compounds.
In vitro cytotoxic effect against NCI-H460 cell line and tubulin polymerization
inhibition (TPI) of selected compounds and CA-4 (1)
Compd
IC50 SE (lM)
NCI-H460
TPIa
11
12
21
28
31
38
39
42
43
49
52
1
0.30 0.01
>20
>20
>10
>10
>10
>10
5.16 0.17
8.09 0.14
8.56 0.09
>10
10 0.2
7.72 0.17
6.14 0.11
4.92 0.2
1.3 0.05
0.35 0.01
0.98 0.03
0.21 0.005
0.74 0.03
2.6 0.1
0.69 0.02
0.95 0.05
0.032 0.002
3.2. General procedure for the preparation of 3,5-diaryl-4,5-
dihydro-1H-pyrazoles 7–22
a
Concentration of compound that inhibited tubulin polymerization by 50% (IC50
)
versus 100% polymerization (maximal attainable polymerization) of the untreated
control.
To a solution of chalcone derivative 3, 4 (1 mmol) in ethanol
(3 mL) hydrazine hydrate (0.3 mL, 6 mmol) was added. The mix-
ture was refluxed under stirring for 2 h, and then water (1 mL)
was added. The mixture was stored at 4–5 °C for 24 h, and the
formed precipitate was filtered off, washed with cold water, and al-
lowed to air dry to give pyrazolines 7–22.
by CytoDINAMIX ScreenTM (Cytoskeleton Inc., Denver, CO), was
detected by measuring the absorbance of the solution (340 nm)
for 60 min. The results show pyrazolines 31, 38, 39, 49, and 52 to
affect tubulin polymerization with inhibition potency in the same
magnitude order of that of 1 (Table 3), being compound 31
(IC50 = 5.16 lM) as potent as CA-4 (1) (IC50 = 4.92 lM). The tubulin
3.2.1. 5-(3-Hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazole (7)
assembly assay well correlates with cytotoxicity screening results,
suggesting that these new derivatives could exert their antiprolif-
erative activity through tubulin polymerization inhibition. The
lower cytotoxicity of these compounds than CA-4 indicates that
the latter compound may exert its cytotoxic effects through addi-
tional targets besides tubulin.21 Unexpectedly, the sub-micromolar
cytotoxic compounds 11 and 42 did not inhibit microtubule
Yield 80%, mp 118–120 °C. 1H NMR: d 2.95 (dd, J = 15.8, 10.2 Hz,
1H), 3.60 (m, 1H) 3.78 (s, 3H), 3.89 (s, 6H), 4.89 (t, J = 10.2 Hz, 1H),
6.76–6.90 (m, 3H), 7.02 (s, 2H), 7.24 (m, 1H), 7.38 (m, 1H), 7.56 (br
s, 1H), 9.50 (s, 1H, OH); IR (Nujol) 3453, 3283, 1590, 1566 cmꢁ1
Anal. Calcd for C18H20N2O4: C, 65.84; H, 6.14; N, 8.53. Found: C,
65.80; H, 6.16; N, 8.49.
.
assembly up to 10 lM, suggesting that their cytotoxicity may be
due to a mechanism other than antitubulin activity.
3.2.2. 5-(3-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazole (8)
In conclusion, we have synthesized and evaluated in vitro a ser-
ies of novel 4,5-dihydropyrazole-based CA-4 analogs as potential
antitumor agents. Results from the anticancer screening showed
compounds 31 and 39 to possess potent antiproliferative activity
against SR and MDA-MB-435 of the NCI 60 human cancer cell line
panel, with GI50 inhibitory values in nanomolar range. Compounds
31, 38, 39, 49, and 52 exhibited inhibition potency on tubulin poly-
merization in the same order of magnitude as CA-4. From the cur-
rent investigation, structure–activity relationships revealed that a
4-(diethylamino)phenyl moiety favorably affects the inhibitory
activity within the series of derivatives. Most importantly, intro-
duction of a (hydroxy)acetyl group at N-1 of inactive 5-(3,4,5-tri-
methoxyphenyl)pyrazolines, resulted in clear activating effect on
their ability to inhibit both cell growth and tubulin polymerization.
Based on easy synthetic approach and biological results, 3,5-
diaryl-4,5-dihydropyrazole structure constitutes a valid template
for access to potential anticancer chemotherapeutic CA-4 analogs.
In order to investigate observed structure–activity relationships
and corroborate our findings, further studies are on the way, and
the results will be reported elsewhere.
Yield 68%, mp 106–108 °C. 1H NMR: d 2.97 (dd, J = 16.4, 10.4 Hz,
1H), 3.54 (dd, J = 16.4, 10.4 Hz, 1H) 3.78 (s, 3H), 3.86 (s, 3H), 3.91 (s,
6H), 4.91 (t, J = 10.4 Hz, 1H), 6.95 (m, 1H), 7.02 (s, 2H), 7.06 (m,
1H), 7.38 (m, 1H), 7.61 (br s, 1H); IR (Nujol) 3288, 1594, 1566
cmꢁ1. Anal. Calcd for C19H22N2O4: C, 66.65; H, 6.48; N, 8.18. Found:
C, 66.61; H, 6.45; N, 8.20.
3.2.3. 5-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazole (9)
Yield 78%, mp 122–124 °C. 1H NMR: d 2.95 (dd, J = 16.3, 10.4 Hz,
1H), 3.46 (dd, J = 16.3, 10.4 Hz, 1H) 3.79 (s, 3H), 3.85 (s, 3H), 3.91 (s,
6H), 4.91 (t, J = 10.4 Hz, 1H), 6.73 (d, J = 8.4 Hz, 2H), 7.02 (m, 4H),
7.41 (d, J = 8.8 Hz, 2H), 7.50 (br s, 1H); IR (Nujol) 3335, 1591,
1565 cmꢁ1. Anal. Calcd for C19H22N2O4: C, 66.65; H, 6.48; N, 8.18.
Found: C, 66.60; H, 6.47; N, 8.14.
3.2.4. 5-[4-(Dimethylamino)phenyl]-3-(3,4,5-trimethoxyphe
nyl)-4,5-dihydro-1H-pyrazole (10)
Yield 92%, mp 142–144 °C. 1H NMR: d 2.98 (m 7H), 3.79 (s, 3H),
3.91 (m, 7H), 4.84 (t, J = 10.2 Hz, 1H), 6.82 (d, J = 8.4 Hz, 2H), 7.02 (s,
2H), 7.29 (d, J = 8.4 Hz, 2H), 7.45 (br s, 1H); IR (Nujol) 3290, 1589,
1565 cmꢁ1. Anal. Calcd for C20H25N3O3: C, 67.58; H, 7.09; N, 11.82.
Found: C, 67.54; H, 7.12; N, 11.83.
3. Experimental
3.1. Chemistry
Unless otherwise noted, all solvents, including anhydrous sol-
vents and chemicals, were purchased from Aldrich Co. and/or Alfa
Aesar, and used without further purification. Melting points were
recorded on a Stuart Scientific melting point SMP1 apparatus and
are uncorrected. Proton nuclear magnetic resonance (1H NMR)
3.2.5. 5-[4-(Diethylamino)phenyl]-3-(3,4,5-trimethoxyphenyl)-
4,5-dihydro-1H-pyrazole (11)
Yield 62%, mp 130–132 °C. 1H NMR: d 1.17 (t, J = 6.5 Hz, 6H),
2.95 (dd, J = 16.5, 10.4 Hz, 1H), 3.41 (q, J = 6.5 Hz, 4H) 3.78
(s, 3H), 3.89 (s, 6H), 4.81 (t, J = 10.4 Hz, 1H), 6.73 (d, J = 8.4 Hz,
2H), 7.02 (s, 2H), 7.25 (d, J = 8.4 Hz, 2H), 7.40 (br s, 1H); IR (Nujol)
spectra were recorded on
a Varian Unity 300 spectrometer

6244
C. Congiu et al. / Bioorg. Med. Chem. 18 (2010) 6238–6248
3222, 1592, 1564 cmꢁ1. Anal. Calcd for C22H29N3O3: C, 68.90; H,
7.62; N, 10.96. Found: C, 68.92; H, 7.60; N, 10.91.
3.2.14. 3-[4-(Dimethylamino)phenyl]-5-(3,4,5-trimethoxyphen
yl)-4,5-dihydro-1H-pyrazole (20)
Yield 81%, mp 140–142 °C. 1H NMR: d 2.88 (dd, J = 16.6, 10.2 Hz,
1H), 3.04 (s, 6H), 3.76 (s, 3H), 3.85 (s, 6H), 3.94 (m, 1H), 4.84 (t,
J = 10.2 Hz, 1H), 6.74 (d, J = 8.2 Hz, 2H), 6.82 (s, 2H), 7.50 (d,
J = 8.2 Hz, 2H); IR (Nujol) 3291, 1610, 1591 cmꢁ1. Anal. Calcd for
3.2.6. 3-(4-Methylphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazole (12)
Yield82%,mp116–118 °C.1HNMR:d2.43(s, 3H),2.94(dd,J = 16.2,
11.2 Hz, 1H), 3.75(s, 3H), 3.87 (s, 6H), 3.94 (m, 1H), 4.88(t, J = 11.2 Hz,
1H), 6.82 (s, 2H), 7.30 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H);IR(Nu-
jol) 3264, 1590 cmꢁ1. Anal. Calcd for C19H22N2O3: C, 69.92; H, 6.79; N,
8.58. Found: C, 69.90; H, 6.81; N, 8.55.
C20H25N3O3: C, 67.58; H, 7.09; N, 11.82. Found: C, 67.60; H, 7.12;
N, 11.77.
3.2.15. 3-[4-(Diethylamino)phenyl]-5-(3,4,5-trimethoxyphenyl)
-4,5-dihydro-1H-pyrazole (21)
3.2.7. 3-(4-Fluorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazole (13)
Yield 76%, mp 138–140 °C. 1H NMR: d 1.19 (t, J = 7.0 Hz, 6H),
2.84 (m, 1H), 3.44 (d, J = 7.0 Hz, 4H), 3.74 (s, 3H), 3.86 (s, 7H),
4.81 (t, J = 9.8 Hz, 1H), 6.75 (d, J = 8.4 Hz, 2H), 6.81 (s, 2H), 7.53
(d, J = 8.4 Hz, 2H); IR (Nujol) 3315, 1610, 1590 cmꢁ1. Anal. Calcd
for C22H29N3O3: C, 68.90; H, 7.62; N, 10.96. Found: C, 68.87; H,
7.60; N, 10.98.
Yield 74%, mp 128–130 °C. 1H NMR: d 3.00 (dd, J = 16.2, 10.6 Hz,
1H), 3.75 (s, 3H), 3.86 (s, 6H), 3.94 (m, 1H), 4.88 (t, J = 10.6 Hz, 1H),
6.87 (s, 2H), 7.40 (m, 2H), 7.92 (m, 2H); IR (Nujol) 3274, 1593 cmꢁ1
Anal. Calcd for C18H19FN2O3: C, 65.44; H, 5.80; N, 8.48. Found: C,
65.41; H, 5.81; N, 8.51.
.
3.2.16. 3-(4-Hydroxy-3-methoxy-phenyl)-5-(3,4,5-trimethoxyp
henyl)-4,5-dihydro-1H-pyrazole (22)
3.2.8. 3-(3-Bromophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazole (14)
Yield 84%, mp 146–148 °C. 1H NMR: d 2.90 (m, 1H), 3.75 (s, 3H),
3.86 (s, 6H), 3.94 (s, 3H), 4.86 (t, J = 10.0 Hz, 1H), 6.74 (d, J = 8.2 Hz,
2H), 6.82 (s, 2H), 6.90–7.36 (m, 2H), 7.45 (s, 1H), 9.80 (s, 1H); IR
(Nujol) 3346, 3328, 1592 cmꢁ1. Anal. Calcd for C19H22N2O5: C,
63.68; H, 6.19; N, 7.82. Found: C, 63.65; H, 6.16; N, 7.80.
Yield 77%, mp 142–144 °C. 1H NMR: d 2.98 (dd, J = 16.8, 10.8 Hz,
1H), 3.75 (s, 3H), 3.88 (s, 6H), 3.95 (m, 1H), 4.90 (t, J = 10.8 Hz, 1H),
6.88 (s, 2H), 7.48–7.88 (m, 3H), 8.02 (s, 1H); IR (Nujol) 3354,
1593 cmꢁ1. Anal. Calcd for C18H19BrN2O3: C, 55.26; H, 4.89; N,
7.16. Found: C, 55.27; H, 4.90; N, 7.14.
3.3. General procedure for the preparation of 1-acetyl-3,5-
3.2.9. 3-(4-Bromophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazole (15)
diaryl-4,5-dihydro-1H-pyrazoles 23-41
Yield 86%, mp 138–140 °C. 1H NMR: d 2.98 (dd, J = 16.4, 11.0 Hz,
1H), 3.76 (s, 3H), 3.85 (s, 6H), 3.96 (m, 1H), 4.90 (t, J = 11.0 Hz, 1H),
6.88 (s, 2H), 7.48 (d, J = 8.2 Hz, 2H), 7.86 (d, J = 8.2 Hz, 2H); IR (Nu-
jol) 3336, 1591 cmꢁ1. Anal. Calcd for C18H19BrN2O3: C, 55.26; H,
4.89; N, 7.16. Found: C, 55.25; H, 4.87; N, 7.15.
To a solution of chalcone derivative 3, 4 (1 mmol) in acetic acid
(3 mL) hydrazine hydrate (0.3 mL, 6 mmol) was added. The mix-
ture was refluxed under stirring for 3 h, and then poured onto
crushed ice. The precipitate was filtered off, washed with cold
water, and crystallized from methanol to give pyrazolines 23–41.
3.2.10. 3-(3-Hydroxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazole (16)
3.3.1. 1-Acetyl-5-(3-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)
-4,5-dihydro-1H-pyrazole (23)
Yield 80%, mp 127–129 °C. 1H NMR: d 3.00 (dd, J = 16.4, 10.6 Hz,
1H), 3.76 (s, 3H), 3.86 (s, 6H), 3.94 (m, 1H), 4.88 (t, J = 10.6 Hz, 1H),
6.85 (s, 2H), 7.04 (s, 1H), 7.12–7.36 (m, 3H), 7.48 (br s, 1H), 9.82 (s,
1H); IR (Nujol) 3368, 3311, 1592 cmꢁ1. Anal. Calcd for C18H20N2O4:
C, 65.84; H, 6.14; N, 8.53. Found: C, 65.81; H, 6.15; N, 8.50.
Yield 65%, mp 116–118 °C. 1H NMR: d 2.42 (s, 3H), 2.96 (m, 1H),
3.76 (s, 3H), 3.85 (s, 6H), 3.93 (s, 3H), 5.58 (m, 1H), 6.66–7.00 (m,
3H), 7.18 (s, 2H), 7.36 (s, 1H), 9.58 (br s, 1H); IR (Nujol) 3421,
1660, 1588 cmꢁ1. Anal. Calcd for C20H22N2O5: C, 64.85; H, 5.99;
N, 7.56. Found: C, 64.81; H, 6.03; N, 7.55.
3.2.11. 3-(3-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazole (17)
3.3.2. 1-Acetyl-5-(3-methoxyphenyl)-3-(3,4,5-trimethoxyphen
yl)-4,5-dihydro-1H-pyrazole (24)
Yield 68%, mp 124–126 °C. 1H NMR: d 2.98 (dd, J = 16.5, 10.8 Hz,
1H), 3.76 (s, 3H), 3.85 (s, 3H), 3.90 (s, 6H), 3.95 (m, 1H), 4.88 (t,
J = 10.8 Hz, 1H), 6.86 (s, 2H), 7.04 (s, 1H), 7.10–7.40 (m, 3H), 7.50
(br s, 1H); IR (Nujol) 3330, 1591 cmꢁ1. Anal. Calcd for C19H22N2O4:
C, 66.65; H, 6.48; N, 8.18. Found: C, 66.60; H, 6.46; N, 8.13.
Yield 63%, mp 120–122 °C. 1H NMR: d 2.43 (s, 3H), 3.02 (m, 1H),
3.75 (s, 3H), 3.86 (s, 6H), 3.90 (s, 3H), 3.93 (dd, J = 10.4, 17.0 Hz,
1H), 5.58 (dd, J = 4.8, 10.4 Hz, 1H), 6.80–7.06 (m, 3H), 7.18 (s,
2H), 7.96 (s, 1H); IR (Nujol) cmꢁ1. Anal. Calcd for C21H24N2O5: C,
65.61; H, 6.29; N, 7.29. Found: C, 65.64; H, 6.30; N, 7.31.
3.2.12. 3-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazole (18)
3.3.3. 1-Acetyl-5-(4-methoxyphenyl)-3-(3,4,5-trimethoxyphen
yl)-4,5-dihydro-1H-pyrazole (25)
Yield82%, mp120–122 °C. 1HNMR:d2.96(dd, J = 16.5, 10.8 Hz, 1H),
3.74 (s, 3H), 3.88 (s, 6H), 3.90 (s, 3H), 3.95 (m, 1H), 4.90 (t, J = 10.8 Hz,
1H), 6.84 (s, 2H), 7.26 (d, J = 7.8 Hz, 2H), 7.50 (br s, 1H), 7.78 (d,
Yield 56%, mp 134–136 °C. 1H NMR: d 2.42 (s, 3H, Me), 3.00 (dd,
J = 4.2, 17.0 Hz, 1H), 3.82 (s, 3H), 3.84 (s, 6H), 3.90 (s, 3H), 5.55 (dd,
J = 4.2, 11.0 Hz, 1H), 7.18 (s, 2H), 7.26 (d, J = 7.6 Hz, 2H), 7.78 (d,
J = 7.6 Hz, 2H); IR (Nujol) 1660, 1620, 1589 cmꢁ1. Anal. Calcd for
J = 7.8 Hz, 2H); IR (Nujol) 3336, 1592 cmꢁ1
.
Anal. Calcd for
C19H22N2O4: C, 66.65; H, 6.48; N, 8.18. Found: C, 66.61; H, 6.45; N, 8.15.
C21H24N2O5: C, 65.61; H, 6.29; N, 7.29. Found: C, 65.60; H, 6.27; N, 7.26.
3.2.13. 3-(4-Methylthiophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazole (19)
3.3.4. 1-Acetyl-5-[4-(dimethylamino)phenyl]-3-(3,4,5-
trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (26)
Yield88%, mp145–147 °C. 1HNMR:d2.95 (dd, J = 16.4, 10.4 Hz, 1H),
3.45 (s, 3H), 3.76 (s, 3H), 3.88 (s, 6H), 3.92 (m, 1H), 4.90 (t, J = 10.4 Hz,
1H), 6.83 (s, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H); IR (Nu-
jol) 3331, 1591 cmꢁ1. Anal. Calcd for C19H22N2O3S: C, 63.66; H, 6.19; N,
7.81. Found: C, 63.62; H, 6.21; N, 7.80.
Yield 90%, mp 148–150 °C. 1H NMR: d 2.40 (s, 3H), 2.96 (s, 6H),
3.30 (m, 1H), 3.82 (s, 3H), 3.90 (s, 3H) 3.94 (s, 6H), 5.56 (m, 1H),
6.78 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 7.17 (s, 2H); IR (Nu-
jol) 1661, 1620, 1572 cmꢁ1. Anal. Calcd for C22H27N3O4: C, 66.48; H,
6.85; N, 10.57. Found: C, 66.45; H, 6.86; N, 10.55.

C. Congiu et al. / Bioorg. Med. Chem. 18 (2010) 6238–6248
6245
3.3.5. 1-Acetyl-5-[4-(diethylamino)phenyl]-3-(3,4,5-trimethoxy
phenyl)-4,5-dihydro-1H-pyrazole (27)
(m, 1H), 6.54 (s, 2H), 7.00 (m, 1H), 7.21–7.38 (m, 3H); IR (Nujol)
1663, 1589 cmꢁ1. Anal. Calcd for C21H24N2O5: C, 65.61; H, 6.29;
N, 7.29. Found: C, 65.60; H, 6.31; N, 7.26.
Yield 88%, mp 143–145 °C. 1H NMR: d 1.16 (t, J = 6.9 Hz, 6H),
2.40 (s, 3H), 3.30 (dd, J = 4.0, 18.0 Hz, 1H), 3.40 (d, J = 6.9 Hz, 4H),
3.82 (s, 3H), 3.88 (dd, J = 11.5, 18.0 Hz, 1H), 3.94 (s, 6H), 5.52 (dd,
J = 4.0, 11.5 Hz, 1H), 6.70 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.4 Hz,
2H), 7.17 (s, 2H); IR (Nujol) 1657, 1612, 1571 cmꢁ1. Anal. Calcd
for C24H31N3O4: C, 67.74; H, 7.34; N, 9.87. Found: C, 67.71; H,
7.35; N, 9.84.
3.3.13. 1-Acetyl-3-(4-methoxyphenyl)-5-(3,4,5-trimethoxy-
phenyl)-4,5-dihydro-1H-pyrazole (35)
Yield 60%, mp 102–104 °C. 1H NMR: d 2.43 (s, 3H, Me), 3.22 (dd,
J = 5.0, 17.0 Hz, 1H), 3.73 (s, 3H), 3.83 (s, 6H), 3.92 (dd, J = 11.2,
17.0 Hz, 1H), 5.58 (dd, J = 5.0, 11.2 Hz, 1H), 6.57 (s, 2H), 7.46
(d, J = 6.4 Hz, 2H), 7.80 (d, J = 6.1 Hz, 2H); IR (Nujol) 1660,
1590 cmꢁ1. Anal. Calcd for C21H24N2O5: C, 65.61; H, 6.29; N, 7.29.
Found: C, 65.63; H, 6.28; N, 7.31.
3.3.6. 1-Acetyl-3-(4-methylphenyl)-5-(3,4,5-trimethoxyphenyl)-
4,5-dihydro-1H-pyrazole (28)
Yield 71%, mp 92–94 °C. 1H NMR: d 2.43 (s, 3H), 2.45 (s, 3H),
3.24 (dd, J = 4.6, 17.0 Hz, 1H), 3.73 (s, 3H), 3.83 (s, 6H), 3.88 (dd,
J = 10.8, 17.0 Hz, 1H), 5.58 (dd, J = 4.6, 10.8 Hz, 1H), 6.56 (s, 2H),
7.38 (d, J = 6.0 Hz, 2H), 7.78 (d, J = 6.0 Hz, 2H); IR (Nujol) 1660,
1593 cmꢁ1. Anal. Calcd for C21H24N2O4: C, 68.46; H, 6.57; N, 7.60.
Found: C, 68.42; H, 6.55; N, 7.61.
3.3.14. 1-Acetyl-3-(4-methylthiophenyl)-5-(3,4,5-trimethoxy-
phenyl)-4,5-dihydro-1H-pyrazole (36)
Yield 77%, mp 149–151 °C. 1H NMR: d 2.43 (s, 3H), 3.24 (dd,
J = 4.8, 16.8 Hz, 1H), 3.73 (s, 3H), 3.83 (s, 6H), 3.90 (dd, J = 10.8,
16.8 Hz, 1H), 5.58 (dd, J = 4.8, 10.8 Hz, 1H), 6.56 (s, 2H), 7.42 (d,
J = 6.1 Hz, 2H), 7.81 (d, J = 6.1 Hz, 2H); IR (Nujol) 1654, 1592 cm
ꢁ1. Anal. Calcd for C21H24N2O4S: C, 62.98; H, 6.04; N, 6.99. Found:
C, 62.98; H, 6.04; N, 7.03.
3.3.7. 1-Acetyl-3-(4-fluorophenyl)-5-(3,4,5-trimethoxyphenyl)-
4,5-dihydro-1H-pyrazole (29)
Yield78%, mp135–137 °C. 1H NMR:d 2.44 (s, 3H), 3.31 (dd, J = 5.0,
17.0 Hz, 1H), 3.73 (s, 3H), 3.89 (s, 6H), 3.94 (dd, J = 10.6, 17.0 Hz, 1H),
5.60 (dd, J = 5.0, 10.6 Hz, 1H), 6.57 (s, 2H), 7.42 (m, 2H), 7.95 (m, 2H);
IR (Nujol) 1650, 1594 cmꢁ1. Anal. Calcd for C20H21FN2O4: C, 64.51; H,
5.68; N, 7.52. Found: C, 64.50; H, 5.66; N, 7.50.
3.3.15. 1-Acetyl-3-[4-(dimethylamino)phenyl]-5-(3,4,5-trimetho-
xyphenyl)-4,5-dihydro-1H-pyrazole (37)
Yield 88%, mp 143–145 °C. 1H NMR: d 2.45 (s, 3H), 3.08 (s, 6H),
3.21 (dd, J = 4.8, 17.4 Hz, 1H), 3.74 (s, 3H), 3.83 (s, 6H), 3.90 (m,
1H), 5.54 (dd, J = 4.8, 10.4 Hz, 1H), 6.55 (s, 2H), 6.85 (d, J = 6.4 Hz,
2H), 7.70 (d, J = 6.4 Hz, 2H); IR (Nujol) 1642, 1606 cmꢁ1. Anal. Calcd
for C22H27N3O4: C, 66.48; H, 6.85; N, 10.57. Found: C, 66.44; H, 6.87;
N, 10.55.
3.3.8. 1-Acetyl-3-(4-chlorophenyl)-5-(3,4,5-trimethoxyphenyl)-
4,5-dihydro-1H-pyrazole (30)
Yield 95%, mp 147–149 °C. 1H NMR: d 2.44 (s, 3H), 3.28 (dd,
J = 4.8, 16.8 Hz, 1H), 3.74 (s, 3H), 3.84 (s, 6H), 3.94 (dd, J = 10.8,
16.8 Hz, 1H), 5.59 (dd, J = 4.8, 10.8 Hz, 1H), 6.58 (s, 2H), 7.65 (d,
3.3.16. 1-Acety-3-[4-(diethylamino)phenyl]-5-(3,4,5-trimethoxy-
phenyl)-4,5-dihydro-1H-pyrazole (38)
J = 7.7 Hz, 2H), 7.91 (d, J = 7.7 Hz, 2H); IR (Nujol) 1667, 1592 cmꢁ1
.
Anal. Calcd for C20H21ClN2O4: C, 61.78; H, 5.44; N, 7.20. Found: C,
61.75; H, 5.41; N, 7.24.
Yield 82%, mp 138–140 °C. 1H NMR: d 1.21 (t, J = 6.5 Hz, 6H),
2.45 (s, 3H), 3.17 (dd, J = 5.0, 17.6 Hz, 1H), 3.48 (d, J = 6.5 Hz, 4H),
3.74 (s, 3H), 3.84 (s, 6H), 5.53 (dd, J = 5.0, 11.0 Hz, 1H), 6.55 (s,
2H), 6.80 (d, J = 8.8 Hz, 2H), 7.67 (d, J = 8.8 Hz, 2H); IR (Nujol)
1655, 1607 cmꢁ1. Anal. Calcd for C24H31N3O4: C, 67.74; H, 7.34;
N, 9.87. Found: C, 67.70; H, 7.33; N, 9.84.
3.3.9. 1-Acetyl-3-(3-bromophenyl)-5-(3,4,5-trimethoxyphenyl)-
4,5-dihydro-1H-pyrazole (31)
Yield 64%, mp 152–154 °C. 1H NMR: d 2.45 (s, 3H), 3.31 (dd,
J = 5.2, 17.2 Hz, 1H), 3.74 (s, 3H), 3.85 (s, 6H), 3.93 (dd, J = 10.2,
17.6 Hz, 1H), 5.60 (dd, J = 5.0, 10.2 Hz, 1H), 6.58 (s, 2H), 7.52–
7.91 (m, 3H), 8.06 (s, 1H); IR (Nujol) 1656, 1593 cmꢁ1. Anal. Calcd
for C20H21BrN2O4: C, 55.44; H, 4.89; N, 6.47. Found: C, 55.46; H,
4.87; N, 6.44.
3.3.17. 1-Acetyl-3-(4-hydroxy-3-methoxyphenyl)-5-(3,4,5-tri-
methoxyphenyl)-4,5-dihydro-1H-pyrazole (39)
Yield 84%, mp 164–166 °C. 1H NMR: d 2.43 (s, 3H), 3.24 (dd,
J = 5.0, 17.2 Hz, 1H), 3.73 (s, 3H), 3.84 (s, 6H), 3.93 (s, 3H), 5.58
(dd, J = 5.0, 11.2 Hz, 1H), 6.56 (s, 2H), 6.94 (d, J = 8.4 Hz, 1H), 7.39
(m, 1H), 7.43 (s, 1H), 9.79 (br s, 1H); IR (Nujol) 3082, 1629,
1595 cmꢁ1. Anal. Calcd for C21H24N2O6: C, 62.99; H, 6.04; N, 7.00.
Found: C, 63.03; H, 6.02; N, 7.01.
3.3.10. 1-Acetyl-3-(4-bromophenyl)-5-(3,4,5-trimethoxyphenyl)-
4,5-dihydro-1H-pyrazole (32)
Yield 91%, mp 138–140 °C. 1H NMR: d 2.44 (s, 3H), 3.27 (dd,
J = 4.6, 16.8 Hz, 1H), 3.74 (s, 3H), 3.84 (s, 6H), 3.93 (dd, J = 10.6,
16.8 Hz, 1H), 5.60 (dd, J = 4.6, 10.6 Hz, 1H), 6.57 (s, 2H), 7.78
(d, J = 6.0 Hz, 2H), 7.84 (d, J = 6.0 Hz, 2H); IR (Nujol) 1667,
1592 cmꢁ1. Anal. Calcd for C20H21BrN2O4: C, 55.44; H, 4.89; N,
6.47. Found: C, 55.43; H, 4.90; N, 6.43.
3.3.18. 1-Acetyl-3-[3-[(tert-buthoxycarbonyl)amino]phenyl]-5-
(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (40)
Yield 82%, mp 126–128 °C. 1H NMR: d 1.57 (s, 9H), 2.44 (s,
3H), 3.29 (m, 1H), 3.73 (s, 3H), 3.84 (s, 6H), 3.93 (m, 1H), 5.65
(m, 1H), 6.56 (s, 2H), 6.80–7.10 (m, 2H), 7.20 (s, 1H), 7.29 (m,
1H), 9.74 (s, 1H); IR (Nujol) 1728, 1660, 1589 cmꢁ1. Anal. Calcd
for C25H31N3O6: C, 63.95; H, 6.65; N, 8.95. Found: C, 63.92; H,
6.66; N, 8.99.
3.3.11. 1-Acetyl-3-(3-hydroxyphenyl)-5-(3,4,5-trimethoxyphenyl)-
4,5-dihydro-1H-pyrazole (33)
Yield76%, mp141–143 °C. 1H NMR:d 2.43 (s, 3H), 3.20(dd, J = 4.6,
16.8 Hz, 1H), 3.73 (s, 3H), 3.84 (s, 6H), 3.90 (m, 1H), 5.58 (m, 1H), 6.56
(s, 2H), 6.97 (m, 1H), 7.27–7.40 (m, 3H), 9.84 (s, 1H); IR (Nujol) 3245,
1632, 1593 cmꢁ1. Anal. Calcd for C20H22N2O5: C, 64.85; H, 5.99; N,
7.56. Found: C, 64.81; H, 5.96; N, 7.58.
3.3.19. 1-Acetyl-3-[4-[(tert-buthoxycarbonyl)amino]phenyl]-5-
(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (41)
Yield 73%, mp115–117 °C. 1H NMR:d 1.59 (s, 9H), 2.43 (s, 3H), 3.25
(m, 1H), 3.73 (s, 3H), 3.83 (s, 6H), 3.92 (m, 1H), 5.65 (m, 1H), 6.55 (s,
2H), 7.67 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.4 Hz, 2H), 9.71 (s, 1H); IR
(Nujol) 3360, 1724, 1658, 1592 cmꢁ1. Anal. Calcd for C25H31N3O6: C,
63.95; H, 6.65; N, 8.95. Found: C, 63.96; H, 6.62; N, 8.98.
3.3.12. 1-Acetyl-3-(3-methoxyphenyl)-5-(3,4,5-trimethoxyphe
nyl)-4,5-dihydro-1H-pyrazole (34)
Yield 71%, mp 115–117 °C. 1H NMR: d 2.45 (s, 3H), 3.20 (dd,
J = 4.8, 17.0 Hz, 1H), 3.74 (s, 3H), 3.84 (m, 7H), 3.98 (s, 3H), 5.56

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C. Congiu et al. / Bioorg. Med. Chem. 18 (2010) 6238–6248
3.4. General procedure for Boc-deprotection of 40 and 41
3.5.3. 1-(Acetoxyacetyl)-3-[4-(dimethylamino)phenyl]-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (46)
To a solution of pyrazoline 40, 41 (0.6 mmol) in dichlorometh-
ane (5 mL) trifluoroacetic acid (2 mL) was added. The mixture
was stirred at room temperature for 16 h, and then evaporated to
dryness in vacuo. To the residue water (15 mL) was added and
the solution treated with 28% ammonia solution to pH 9–10. The
formed solid was filtered off, air died, and crystallized from iso-
propyl ether-methanol 1:1 to give 42, 43.
Following the general procedure, the title compound was ob-
tained from 20. Yield 89%; mp 132–134 °C (from methanol). 1H
NMR: d 2.19 (s, 3H), 3.08 (s, 6H), 3.19 (dd, J = 4.4, 16.4 Hz,
1H), 3.74 (s, 3H), 3.86 (s, 6H), 3.90 (m, 1H), 5.07 (d,
J = 12.4 Hz, 1H), 5.35 (d, J = 12.4 Hz, 1H), 5.54 (dd, J = 4.4,
11.0 Hz, 1H), 6.58 (s, 2H), 6.85 (d, J = 9.0 Hz, 2H), 7.71 (d,
J = 9.0 Hz, 2H); IR (Nujol) 1740, 1676, 1610 cmꢁ1. Anal. Calcd
for C24H29N3O6: C, 63.28; H, 6.42; N, 9.22. Found: C, 63.24; H,
6.46; N, 9.26.
3.4.1. 1-Acetyl-3-(3-aminophenyl)-5-(3,4,5-trimethoxyphenyl)-
4,5-dihydro-1H-pyrazole (42)
Yield 84%; mp 122–124 °C. 1H NMR: d 2.42 (s, 3H), 3.14 (dd,
J = 5.0, 17.4 Hz, 1H), 3.74 (s, 3H), 3.84 (s, 6H), 3.90 (m, 1H), 5.56
(dd, J = 5.0, 10.8 Hz, 1H), 5.35 (s, 2H), 6.55 (s, 2H), 6.77 (m, 1H),
6.98 (m, 1H), 7.17 (s, 1H), 7.20 (m, 1H); IR (Nujol) 3432, 3349,
1644, 1594 cmꢁ1. Anal. Calcd for C20H23N3O4: C, 65.03; H, 6.28;
N, 11.37. Found: C, 65.00; H, 6.29; N, 11.40.
3.5.4. 1-(Acetoxyacetyl)-3-[4-(diethylamino)phenyl]- 5-(3,4,5-
trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (47)
Following the general procedure, the title compound was ob-
tained from 21. Yield 83%; mp 122–124 °C (from ethanol). 1H
NMR: d 1.20 (t, J = 6.8 Hz, 6H), 2.42 (s, 3H), 3.21 (dd, J = 5.0,
17.0 Hz, 1H), 3.46 (d, J = 6.8 Hz, 2H), 3.74 (s, 3H), 3.85 (s, 6H),
3.90 (m, 1H), 5.04 (d, J = 12.8 Hz, 1H), 5.32 (d, J = 12.8 Hz, 1H),
5.55 (dd, J = 5.0, 10.8 Hz, 1H), 6.56 (s, 2H), 6.78 (d, J = 8.4 Hz, 2H),
7.65 (d, J = 8.4 Hz, 2H); IR (Nujol) 1747, 1666, 1607 cmꢁ1. Anal.
Calcd for C26H33N3O6: C, 64.58; H, 6.88; N, 8.69. Found: C, 64.54;
H, 6.85; N, 8.73.
3.4.2. 1-Acetyl-3-(4-aminophenyl)-5-(3,4,5-trimethoxyphenyl)-
4,5-dihydro-1H-pyrazole (43)
Yield 76%; mp 108–110 °C. 1H NMR: d 2.39 (s, 3H), 3.14 (dd,
J = 4.8, 17.0 Hz, 1H), 3.73 (s, 3H), 3.83 (s, 6H), 3.90 (m, 1H), 5.52
(dd, J = 4.8, 11.0 Hz, 1H), 5.76 (s, 2H), 6.54 (s, 2H), 6.70 (d,
J = 8.0 Hz, 2H), 7.56 (d, J = 6.4 Hz, 2H); IR (Nujol) 3433, 3353,
1631, 1607 cmꢁ1. Anal. Calcd for C20H23N3O4: C, 65.03; H, 6.28;
N, 11.37. Found: C, 65.06; H, 6.32; N, 11.41.
3.5.5. 1-(Acetoxyacetyl)-3-(4-hydroxy-3-methoxyphenyl)-5-
(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (48)
Following the general procedure, the title compound was ob-
tained from 22. Yield 72%; mp 158–160 °C (from MeCN). 1H
NMR: d 2.20 (s, 3H), 3.08 (s, 6H), 3.28 (m, 1H), 3.73 (s, 3H),
3.90 (m, 7H), 5.06 (d, J = 16.0 Hz, 1H), 5.36 (d, J = 16.0 Hz, 1H),
5.58 (m, 1H), 6.57 (s, 2H), 6.88–7.36 (m, 3H), 7.43 (s, 1H), 9.80
(br s, 1H, OH); IR (Nujol) 3108, 1748, 1681, 1594 cmꢁ1. Anal.
Calcd for C23H26N3O8: C, 60.26; H, 5.72; N, 6.11. Found: C,
60.23; H, 5.74; N, 6.09.
3.5. General procedure for the preparation of 1-(Acetoxyacetyl)-
4,5-dihydro-1H-pyrazoles 44–48
3.5.1. 1-(Acetoxyacetyl)-3-(4-methylphenyl)-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (44)
To a solution of chloroacetic acid (0.19 g, 2 mmol) in 5 mL of anhy-
drousMeCN, HOBt(0.27 g, 2 mmol)and EDC(0.43 g, 2.2 mmol)were
added. The mixture was stirred for 30 min at room temperature,
then pyrazoline 12 (0.65 g, 2 mmol) was added, and the stirring con-
tinued for additional 18 h. After evaporation of the solvent in vacuo,
0.1 M aqueous hydrochloric acid (30 mL) was added and the mixture
extracted with ethyl acetate (3 ꢂ 5 mL). The combined ethyl acetate
layers were back-extracted with saturated sodium bicarbonate
(1 ꢂ 5 mL) and brine (1 ꢂ 5 mL), dried over MgSO4, filtered, and con-
centrated in vacuo. The residue was dissolved in 5 mL of DMSO, then
potassium acetate (0.20 g, 2 mmol) was added, and the mixture stir-
red at room temperature for 12 h. The reaction mixture was poured
into 50 mL of ice-water and the formed solid filtered off, air dried,
and crystallized from iso-propyl ether-methanol 1:1 to give 44. Yield
80%; mp 156–158 °C. 1H NMR: d 2.20 (s, 3H), 2.46 (s, 3H), 3.31 (dd,
J = 4.8, 16.8 Hz, 1H), 3.74 (s, 3H), 3.86 (s, 6H), 3.90 (m, 1H), 5.10 (d,
J = 15.8 Hz, 1H), 5.37 (d, J = 15.8 Hz, 1H), 5.61 (dd, J = 4.8, 10.8 Hz,
1H), 6.60 (s, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.82 (d, J = 8.0 Hz, 2H); IR
(Nujol) 1743, 1678, 1593 cmꢁ1. Anal. Calcd for C23H26N2O6: C,
64.78; H, 6.15; N, 6.57. Found: C, 64.86; H, 6.10; N, 6.60.
3.6. General procedure for the preparation of 1-(hydroxy
acetyl)-4,5-dihydro-1H-pyrazoles 49–53
To a solution of 44–48 (0.5 mmol) in methanol (10 mL), a solu-
tion of potassium carbonate (0.35 g, 2.5 mmol) in water (1 mL) was
added. The mixture was stirred at room temperature for 4 h, then
diluted with water (30 mL), and extracted with dichloromethane
(3 ꢂ 10 mL). The combined organic layers were back-extracted
with brine (1 ꢂ 10 mL), dried over MgSO4, filtered, and concen-
trated in vacuo. Crystallization from MeCN gave 49–53.
3.6.1. 1-(Hydroxyacetyl)-3-(4-methylphenyl)-5-(3,4,5-trime
thoxyphenyl)-4,5-dihydro-1H-pyrazole (49)
Following the general procedure, the title compound was ob-
tained from 44. Yield 94%; mp 106–108 °C. 1H NMR: d 2.44 (s,
3H), 3.21 (m, 1H), 3.73 (s, 3H), 3.84 (s, 6H), 3.93 (m, 1H), 4.57
(m, 2H), 5.05 (m, 1H), 5.59 (dd, J = 4.8, 11.2 Hz, 1H), 6.57 (s, 2H),
7.37 (d, J = 7.2 Hz, 2H), 7.76 (d, J = 7.2 Hz, 2H); IR (Nujol) 3480,
1652, 1594 cmꢁ1. Anal. Calcd for C21H24N2O5: C, 65.61; H, 6.29;
N, 7.29. Found: C, 65.60; H, 6.25; N, 7.34.
3.5.2. 1-(Acetoxyacetyl)-3-(3-bromophenyl)-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (45)
3.6.2. 3-(3-Bromophenyl)-1-(hydroxyacetyl)-5-(3,4,5-trime
thoxyphenyl)-4,5-dihydro-1H-pyrazole (50)
Following the general procedure, the title compound was ob-
tained from 14. Yield 78%; mp 146–148 °C (from methanol). 1H
NMR: d 2.42 (s, 3H), 3.26 (dd, J = 5.2, 17.4 Hz, 1H), 3.73 (s, 3H),
3.83 (s, 6H), 3.94 (dd, J = 10.2, 17.4 Hz, 1H), 5.04 (d, J = 13.0 Hz,
1H), 5.10 (d, J = 13.0 Hz, 1H), 5.58 (dd, J = 5.2, 10.2 Hz, 1H), 6.56
(s, 2H), 7.40–7.82 (m, 3H), 8.08 (s, 1H); IR (Nujol) 1747, 1681,
1593 cmꢁ1. Anal. Calcd for C22H23BrN2O6: C, 53.78; H, 4.72; N,
5.70. Found: C, 53.74; H, 4.75; N, 5.68.
Following the general procedure, the title compound was ob-
tained from 45. Yield 93%; mp 134–136 °C. 1H NMR: d 3.28 (m,
1H), 3.74 (s, 3H), 3.85 (s, 6H), 3.94 (m, 1H), 4.61 (m, 1H), 5.05
(br s, 1H), 5.65 (m, 1H), 6.60 (s, 2H), 7.45–7.78 (m, 3H), 8.06
(s, 1H); IR (Nujol) 3445, 1661, 1592 cmꢁ1
C
.
Anal. Calcd for
20H21BrN2O5: C, 53.47; H, 4.71; N, 6.23. Found: C, 53.44; H,
4.70; N, 6.18.

C. Congiu et al. / Bioorg. Med. Chem. 18 (2010) 6238–6248
6247
3.6.3. 3-[4-(Dimethylamino)phenyl]-1-(hydroxyacetyl)-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (51)
a series of standard 96-well microtrite plates in 100 mL of med-
ium.22,23 Density of inoculum depends on the type of tumor cell
and from its growth characteristics. These cells are then preincu-
bated on the microtrite plate for 24 h before adding the com-
pounds. These were tested in DMSO solution at five different
concentrations (10ꢁ4, 10ꢁ5, 10ꢁ6, 10ꢁ7, and 10ꢁ8 M). After an incu-
bation of the chemical agent for 48 h with the tumor cell lines, a
sulforhodamine B (SRB) protein assay was used to estimate cell
viability or growth. The cytotoxic effects are evaluated and the as-
say results and dose–response parameters were calculated as pre-
viously described.24
Following the general procedure, the title compound was ob-
tained from 46. Yield 95%; mp 118–120 °C. 1H NMR: d 3.08 (s,
6H), 3.20 (dd, J = 4.8, 17.2 Hz, 1H), 3.74 (s, 3H), 3.84 (s, 6H), 3.92
(dd, J = 10.8, 17.2 Hz, 1H), 4.56 (d q, J = 6.3, 15.4 Hz, 2H), 4.87 (t,
J = 6.3 Hz, 1H), 5.55 (dd, J = 4.8, 10.8 Hz, 1H), 6.58 (s, 2H), 6.84 (d,
J = 8.8 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H); IR (Nujol) 3431, 1645,
1607 cmꢁ1. Anal. Calcd for C22H27N3O5: C, 63.91; H, 6.58; N,
10.16. Found: C, 63.93; H, 6.54; N, 10.12.
3.6.4. 3-[4-(Diethylamino)phenyl]-1-(hydroxyacetyl)-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (52)
Acknowledgments
Following the general procedure, the title compound was ob-
tained from 47. Yield 92%; mp 110–112 °C. 1H NMR: d Anal. Calcd
for (C24H31N3O5 441.53) C, H, N. 1.21 (t, J = 6.8 Hz, 6H), 3.20 (dd,
J = 4.6, 17.0 Hz, 1H), 3.58 (d, J = 6.8 Hz, 2H), 3.74 (s, 3H), 3.88 (m,
7H), 4.56 (d q, J = 6.2, 16.3 Hz, 2H), 4.87 (t, J = 6.2 Hz, 1H), 5.53
(dd, J = 4.6, 10.8 Hz, 1H), 6.58 (s, 2H), 6.79 (d, J = 8.7 Hz, 2H), 7.67
(d, J = 8.7 Hz, 2H); IR (Nujol) 3440, 1648, 1600 cmꢁ1. Anal. Calcd
for C24H31N3O5: C, 65.29; H, 7.05; N, 9.52. Found: C, 65.32; H,
7.02; N, 9.47.
The authors thank the Developmental Therapeutics Program of
the National Cancer Institute, Bethesda, MD (USA), for providing
the in vitro antitumor screening data. The authors are indebted
to Mrs. M. Cati and Mr. S. Zavatto for expert technical assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.037. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
3.6.5. 1-(Hydroxyacetyl)-3-(4-hydroxy-3-methoxyphenyl)-5-
(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (53)
Following the general procedure, the title compound was ob-
tained from 48. Yield 93%; mp 128–130 °C. 1H NMR: d 3.24 (m,
1H), 3.58 (d, J = 6.8 Hz, 2H), 3.74 (s, 3H), 3.85 (s, 3H), 3.94 (m,
4H), 4.58 (m, 1H), 4.87 (m, 1H), 5.53 (m, 1H), 6.58 (s, 2H), 6.90–
7.34 (m, 3H), 7.42 (s, 1H), 9.84 (br s, 1H); IR (Nujol) 3446, 3144,
1650, 1594 cmꢁ1. Anal. Calcd for C21H24N3O7: C, 60.57; H, 5.80;
N, 6.73. Found: C, 60.55; H, 5.77; N, 6.70.
References and notes
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