Synthesis and biological activity of N,N-dialkylaminoalkyl-substituted bisindolyl and diphenyl pyrazolone derivatives

Article abstract of DOI:10.1016/j.bmc.2005.09.059

New compounds, structurally related to the potent protein kinase C inhibitor staurosporine, with a bisindolylpyrazolone framework and substituted on the pyrazolone nitrogens with N,N-dialkylaminoalkyl side chain, were synthesized and evaluated for growth-

Full text of DOI:10.1016/j.bmc.2005.09.059

Bioorganic & Medicinal Chemistry 14 (2006) 9–16
Synthesis and biological activity of N,N-dialkylaminoalkyl-
substituted bisindolyl and diphenyl pyrazolone derivatives
a,
a
a
b
Miguel F. Brana,a, Ana Gradillas, Angel G. Ovalles, Berta Lopez, Nuria Acero,
*
*
´
˜
Francisco Llinaresc and Dolores Munoz Mingarroa
˜
aDepartamento de Quı´mica, Bioquı´mica y Biologı´a Molecular, Facultad de Farmacia, Universidad San Pablo CEU,
Urb. Monteprı´ncipe, 28668-Boadilla del Monte, Madrid, Spain
bDepartamento Ciencias Ambientales y Recursos Naturales, Bioquı´mica y Biologı´a Molecular, Facultad de Farmacia,
Universidad San Pablo CEU, Urb. Monteprı´ncipe, 28668-Boadilla del Monte, Madrid, Spain
cDepartamento de Biologı´a Celular, Bioquı´mica y Biologı´a Molecular, Facultad de Farmacia, Universidad San Pablo CEU,
Urb. Monteprı´ncipe, 28668-Boadilla del Monte, Madrid, Spain
Received 22 April 2005; revised 20 June 2005; accepted 24 June 2005
Available online 2 November 2005
Abstract—New compounds, structurally related to the potent protein kinase C inhibitor staurosporine, with a bisindolylpyrazo-
lone framework and substituted on the pyrazolone nitrogens with N,N-dialkylaminoalkyl side chain, were synthesized and eval-
uated for growth-inhibitory properties in several human cell lines. Many showed inhibition of TNF-a production in response to
the tumor promotor TPA on HL-60 cells. The apoptotic activity on HeLa cells has been examined for several of these
compounds.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Staurosporine, an indolo[2,3-a]carbazole which was
H
R1
R2
discovered in the course of screening extracts of the
bacterium Streptomyces sp.,1 has become the ꢀleadꢁ
compound among protein kinase C (PKC) inhibitors.
PKC is a suitable a target in anticancer drug design
because its function is altered in some neoplasias,
and this dysfunction has been related to uncontrolled
proliferation.2,3
N
O
R1 = R2 = R3 = H; staurosporine
R1 = OH; R2 = R3 = H; UCN-01
R1 = R2 = H; R3 = PhCO; CGP 41251
N
N
O
N
H
CH3
OCH3
The staurosporine discovery stimulated a variety of
medicinal chemistry programmes aimed at yielding
staurosporine cogeners with higher inhibitory specificity
for PKC.4 One series of analogs was obtained by addi-
tion of substituents to the molecule (Fig. 1), e.g., addi-
tion of a hydroxyl group at the C7 position of the
lactam ring to furnish UCN-01,5 and benzoylation of
the amine in the glycoside ring to yield CGP 41251.6
Both are currently progressing through clinical evalua-
H3C
R3
R''
N
H
N
O
O
O
O
N
R
N
N
N
H
R'
(II)
(I)
O
bisindolylmaleimides
R'' = CH2CH2N(CH3)2
Keywords: Staurosporine; Bisindolopyrazolones; PKC; TNF-a;
Apoptosis.
N
*
Corresponding authors. Tel.: +34 91 3724798; fax:
3510496; e-mail addresses: mfbrana@ceu.es; gradini@ceu.es
+ 34 91
Figure 1. Staurosporine and related structures.
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.059

10
M. F. Bran˜a et al. / Bioorg. Med. Chem. 14 (2006) 9–16
tion as antitumoral agents.7,8 Another successful modifi-
cation furnished the bisindolylmaleimides. The starting
point for the preparation of these potent and selective
PKC inhibitors9 came when it was decided to break
the planarity of the aglycone structure of stauro-
sporine10,11 and when an extra carbonyl was added as
a consequence of structure–activity relationships.12
Consequently, various series of bisindolylmaleimide (I)
compounds were designed. Many of them have
appeared to be highly selective inhibitors of a broad
spectrum of PKC.13–15
On the other hand, it has been demonstrated that when
tumor necrosis factor-a (TNF-a) synthesis is stimulated
by forbol addition, a signal cascade, where PKC is in-
volved, occurs.17 The most active compounds have been
selected for the study of their regulatory activity on
TNF-a production in human promelocytic leukemia
(HL-60) cells. The apoptotic activity on human cervical
carcinoma (HeLa) cells has also been studied to com-
pare with staurosporine,
apoptosis.18,19
a
potent inducer of
In a previous work,16 we have synthesized a series of
novel bisindolylmaleimide possessing a N,N-dialkyl-
aminoalkyl side chain in their structure (II), and showed
interesting activity and selectivity on human umbilical
vein endothelial cells (HUVECs) proliferation,
Figure 1.
2. Results and discussion
2.1. Chemistry
Compounds listed in Table 1 were obtained by the
methods outlined in Schemes 1, 2. The synthesis of
the pyrazolone-based compounds 4–6 required the
preparation of the corresponding b-ketoesters 3 as
starting material. Compound 3a was prepared accord-
ing to the procedure described in the literature.20 The
compounds 3b and 3c were prepared in the same pro-
cedure by reaction of ethyl-(1-methyl-1H-indol-3-yl)ac-
etate 1 and the appropriate acyl chloride 2. It was
found that the yield was about 60% when the base
was generated at À40 °C and when the reaction time
was extended overnight.
In the course of structure–activity studies on PKC inhib-
itors as antitumoral agents and based on our previous
findings, we have been engaged in new structural modi-
fication. We report here the synthesis of the compounds
listed in Table 1, in which different side chains have been
attached to a pyrazolone nucleous which permits to ob-
tain three different regioisomers, we wished to determine
if the additional nitrogen and the side chains contribute
to the biological activity.
Table 1. Growth inhibitory properties for pyrazolone derivatives 4, 5 and 6
Y
N
O
X
N
R2
R3
Compound
R2
R3
X
Y
IC50 (lM)
HT-29
HeLa
PC-3
4a
4b
4c
5a
5b
5c
5e
6a
6b
6d
6e
Ph
1-Me-1H-indol-3-yl
Ph
1-Me-1H-indol-3-yl
H
H
H
H
H
H
H
H
>50
23.5
>50
>50
>50
>50
14.4
>50
27.5
21.0
48.5
>50
22.2
>50
>50
>50
>50
12.8
>50
>50
17.4
16.1
>50
11.3
>50
>50
>50
>50
46.7
35.2
43.2
35.2
>50
H
2-Cl-1-Me-1H-indol-3-yl
Ph
1-Me-1H-indol-3-yl
Ph
H
(CH2)2N(CH3)2
Ph
Ph
Ph
Ph
(CH2)2N(CH2CH3)2
(CH2)3N(CH3)2
(CH2)2N(CH2CH3)2
1-Me-1H-indol-3-yl
Ph
Ph
1-Me-1H-indol-3-yl
Ph
Ph
(CH2)2N(CH3)2
H
H
H
H
(CH2)2N(CH2CH3)2
(CH2)2N(CH3)2
(CH2)2N(CH2CH3)2
1-Me-1H-indol-3-yl
1-Me-1H-indol-3-yl
1-Me-1H-indol-3-yl
1-Me-1H-indol-3-yl
COOEt
O
O
COOEt
Cl
R1
a
+
R1
N
N
N
N
CH3
CH3
CH3
CH3
2b: R1 = H
2c: R1 = Cl
3b: R1 = H
3c: R1 = Cl
1
Scheme 1. Synthesis of 3. Reagents and condition: (a) BuLi/THF, diisopropylamine, À78 °C.

M. F. Bran˜a et al. / Bioorg. Med. Chem. 14 (2006) 9–16
11
H
N
O
COOEt
R3
a
O
HN
R2
R2
R3
3a: R2 = R3 = Ph
4a: R2 = R3 = Ph
3b: R2 = R3 = 1-methyl-1H-indol-3-yl
3c: R2 = 2-chloro-1-methyl-1H-indol-3-yl
R3 = 1-methyl-1H-indol-3-yl
4b: R2 = R3 = 1-methyl-1H-indol-3-yl
4c: R2 = 2-chloro-1-methyl-1H-indol-3-yl
R3 = 1-methyl-1H-indol-3-yl
5a: R2 = R3 = Ph
R
R
n = 2, R = Me
N
5b: R2 = R3 = Ph
(CH2)n
n = 2, R = Et
N
O
5c: R2 = R3 = Ph
HN
R2
n = 3, R = Me
5d: R2 = R3 = 1-methyl-1H-indol-3-yl
n = 2, R = Me
O
COOEt
R3
R3
b
5e: R2 = R3 = 1-methyl-1H-indol-3-yl
n = 2, R = Et
R2
5f: R2 = R3 = 1-methyl-1H-indol-3-yl
n = 3, R = Me
3a: R2 = R3 = Ph
3b: R2 = R3 = 1-methyl-1H-indol-3-yl
H
R
R
N
O
N
(CH2)n
N
6a: R2 = R3 = Ph; n = 2, R = Me
6b: R2 = R3 = Ph; n = 2, R = Et
R2
R3
6d: R2 = R3 = 1-methyl-1H-indol-3-yl; n = 2, R = Me
6e: R2 = R3 = 1-methyl-1H-indol-3-yl; n = 2, R = Et
Scheme 2. Synthesis of 4–6. Reagents and conditions: (a) Camphoric acid/EtOH, NH2–NH2, reflux, (b) camphoric acid/EtOH, H2N-HN-(CH2)n
N-(R)2, reflux.
N
In order to prepare 2b, 1-methyl-1H-indole-3-carbox-
N
H
ylic acid was refluxed with fresh distilled SOCl2. How-
ever, the reaction led to the unexpected compound 2c.
The APCI-MS of corresponding acid of 2c gave the
characteristic isotope pattern from chloro, indicating
its presence, and the substitution in the 2 position
H
H
H
H
N
O
N
No nOe
nOe
5%
N
O
H
HN
H
H
H
H
1
H
was confirmed by H NMR spectrum. Therefore, we
decided to carry out the reaction under milder condi-
tions with SOC12 at room temperature and DMF
to yield 2b, which was used without further
purification.
6a
5a
Figure 2. The NOE correlations used for the structural assignment of
compounds 5a and 6a.
Thus, treatment of 3 with the appropriate hydrazine in
ethanol at reflux in the presence of camphoric acid14
yielded the corresponding pyrazolones 4–6, Scheme 2.
The N,N-dialkylaminoalkylhydrazines employed were
obtained by reaction of the corresponding N,N-di-
alkylminoalkyl chlorides with excess of hydrazine
monohydrate.21 The condensation reaction could lead
to regioisomers 5 and 6. Both isomers could be separated
by chromatography. Only when N,N-dimethyl-
aminopropylhydrazine was used, 5c and 5f were ob-
tained as unique compounds. The structure of the
corresponding isomers was confirmed by NOE experi-
ments, considering 1H chemical shifts (positive NOE
on signals of protons due to aromatic system upon irra-
diation of the CH2 resonance with compounds 5a and
6a), as described in Figure 2.
But the unequivocal result was given by X-ray crystallo-
graphic analysis,22 Figures 3 and 4. A complete charac-
terization of the compounds 5d and 5f was performed
but disappointingly these compounds darkened with
time and they were not sufficiently stable for further
experimental cytotoxic activity.
Figure 3. X-ray crystallographic analysis of 5a.

12
M. F. Bran˜a et al. / Bioorg. Med. Chem. 14 (2006) 9–16
kylaminoalkylpyrazolone derivatives, so 3-alcoxy-pyra-
zol turned out to be the only product. The structure of
all novel compounds was confirmed by mainly NMR
spectroscopic methods and by X-ray crystallographic
analysis, Figure 5.
3. Biological activity
In vitro cytotoxic potencies of the novel compounds 4–7
against human colon adenocarcinoma cell line (HT29),
human cervical carcinoma (HeLa) and human prostate
carcinoma (PC-3) are reported in Tables 1 and 2.
Although none of them were as active as staurosporine,
where IC50 values are in the nanomolar range as de-
scribed in the literature,24 these results indicate that
some of the bisindole series have shown a moderate anti-
proliferative activity. Neither the introduction of side
chains attached to the pyrazolone nitrogens nor the car-
bonyl group have significant effect on the activity.
According to the IC50 observed, only 5e, 6d, 6e, 7a
and 7b were selected for proposed further biological
evaluation.
Figure 4. X-ray crystallographic analysis of 6a.
An alternative, selective approach to N-substitution
products was also possible via the reaction of 4a and
4b with N,N-dimethylaminoethanol under Mitsunobu
conditions23 shown in Scheme 3. This synthetic route
did not permit access to the corresponding N,N-dial-
The study of tumor promotion in rodent carcinogenesis
using chemical tumor promoters has revealed various
tumor promotion pathways, such as the 12-o-tetradeca-
noyl phorbol-13-acetate (TPA) pathway mediated
through activation of protein kinase C.25 It has also pre-
viously demonstrated that the treatment of human HL-
60 promyelocytic leukemia cells with TPA is associated
with induction of TNF-a.26
CH3
H
N
N
N
O
CH3
O
HN
R2
a
HN
R2
R3
R3
The study reported here has examined the effects on
TNF-a promotion of those compounds, among which
IC50 was promising in suggesting a link between the
TPA pathway and endogenous tumor promotion path-
way of TNF-a in HL-60 cells, which have been widely
used as a model cell system for studying the biological
roles of PKC.
4a: R2 = R3 = Ph
4b: R2 = R3 = 1-Me-1H-indol-3-yl
7a: R2 = R3 = Ph
7b: R2 = R3 = 1-Me-1H-indol-3-yl
Scheme 3. Synthesis of 7. Reagents and conditions: (a) N,N-dimeth-
ylaminoethanol, Ph3P, DIAD/THF, 0 °C.
All selected compounds significantly reduce TNF-a pro-
duction by TPA-stimulated HL-60 cells, Table 3. The
amount of TNF-a produced in the presence of TPA
alone was defined as 100%.27
In HeLa apoptosis assay,28 two concentrations of each
compound, a positive control (staurosporine, 20 lM)
and a blank (phosphate saline buffer, pH 7.4), were
used. Typical apoptotic bodies were observed 24 h after
cells were treated with the selected compounds at 50 and
100 (lM). Compounds 5e, 6e and 7b exhibit a consider-
able apoptotic capacity, Table 4.
4. Conclusions
In summary, different regioisomers, from diphenyl and
bisindolyl, have been synthesized and completely char-
acterized, possessing a dialkylaminoalkyl side chain in
the pyrazolone nucleus. We explored the SAR for these
new series of bisindolyl and diphenyl pyrazolone and the
results obtained indicate that all the compounds possess
Figure 5. X-ray crystallographic analysis of 7a.

M. F. Bran˜a et al. / Bioorg. Med. Chem. 14 (2006) 9–16
Table 2. Growth inhibitory properties for 3-alcoxy pyrazole derivatives 7
13
CH3
CH3
N
N
O
HN
R2
R3
Compound
R2
R3
IC50 (lM)
HT-29
HeLa
PC-3
7a
7b
Ph
1-Me-1H-indol-3-yl
Ph
1-Me-1H-indol-3-yl
>50
21.8
19.1
23.8
21.5
22.35
Table 3. TNF-a production
red (IR) spectra were recorded with a Perkin-Elmer
1330 infrared spectrophotometer. All 1H NMR and
13C NMR spectra were recorded on a Brucker AM
300 MHz spectrometer in CDC13, with tetramethylsi-
lane as internal standard. Chemical shifts are reported
in ppm, the following abbreviations are used: singlet
(s), doublet (d), triplet (t), quadruplet (c), quintuplet
(q), multiplet (m). Elemental analyses were determined
by the Microanalyses Service of the Complutense Uni-
versity of Madrid with an elemental Perkin Elmer
2400 CHN. Electrospray impact mass spectra (ESIMS)
and atmosphere pressure chemical ionization mass spec-
tra (APCI) were recorded on Brucker Daltonics ES-
QUIRE 3000. Melting points were obtained using a
Stuart Scientific SMP3 apparatus and are uncorrected.
TPAa
Compound
%
5e
6d
6e
7a
7b
34.90
41.47
18.98
41.40
28.12
a TPA, 12-o-tetradecanoylphorbol. The amount of TNF-a in the
presence of TPA alone was defined as 100%.
Table 4. Apoptotic cells percentage
Compound
100 lM
54.7*
9.45
45.6*
28.56*
42.8*
50 lM
23.87*
9.76
25.76*
11.15
36.6*
5e
6d
5.2. 1-Methyl-1H-indole-3-carbonyl chloride (2b)
6e
7a
To a solution of 1 mmol of 1-methyl-1H-indole-3-car-
boxylic acid with in 20 ml of fresh distilled SOCl2, 3
drops of DMF were added, and the mixture was stirred
for 20 h at room temperature. Then, the reaction mix-
ture was evaporated in vacuo. The crude product was
used without further purification.
7b
Control
Staurosporine
8.65
63.2*
Each result is the average of three recounts.
* Significantly different (p < 0.05).
a moderate antiproliferative activity in the micromolar
range, the bisindole series were more potent than the
diphenyl ones. These compounds could act as PKC
inhibitors. Although PKC is involved in the assays stud-
ied (TNF-a production and apoptosis), it could not be
confirmed that these compounds express their cytotoxic-
ity via PKC inhibition. However, it should definitely be
noted in future studies. The differential responses
to individual inhibitors of PKC may be related to
the multiple PKC isoforms and inhibitory and selectivity
properties toward PKC isoforms are ongoing.
5.3. 2-Chloro-1-methyl-1H-indole-3-carbonyl chloride
(2c)
A mixture of 1 mmol of l-methyl-1H-indole-3-carboxylic
acid in 20 ml of fresh distilled SOCl2, was refluxed under
stirring for 20 h. Then, the reaction mixture was evapo-
rated in vacuo. The crude product was used without
further purification.
5.4. General procedure for the preparation of b-ketoesters
(3)
BuLi (2.2 mmol) was added to a solution of diisopropyl-
amine (2.6 mmol) in THF (4 ml) under argon at À40 °C
over 30 min with stirring. The solution was cooled at
À78 °C and compound 1 (1 mmol) was added. After 1
h, the corresponding acyl halide 2 (1.2 mmol) was added
dropwise, and stirred till room temperature.
5. Experimental section
5.1. General methods
Thin-layer chromatography (TLC) was performed on E.
Merck AL silicagel 60F254 plates and visualized under
UV light. Flash column chromatography was conducted
using E. Merck silica gel 60 (0.040–0.063 mm). Solvents
employed were A (hexane/ethyl acetate, 1:1), B (ethyl
acetate/methanol, 4:1), C (gradient of ethyl acetate/etha-
nol, 9:1 to ethyl acetate/ethanol/NH3, 9:0.5:0.5). Infra-
5.5. Ethyl 2,3-bis-(1-methyl-1H-indol-3-yl)-3-oxopropan-
oate (3b)
3 g (13.81 mmol) of 1 and 3.28 g (17 mmol) of 2b were
used. Once the reaction was finished, the solvent was

14
M. F. Bran˜a et al. / Bioorg. Med. Chem. 14 (2006) 9–16
1
evaporated in vacuo, and the crude product was dis-
solved in AcOEt, washed with 10% sodium bicarbonate
solution, water and saturated sodium chloride solution,
dried (MgSO4) and evaporated. The product was puri-
fied by flash chromatography over silica gel using chro-
matography solvent A; yield 52%; mp 132–135 °C (from
3400, 3340 cmÀ1; H NMR d: 3.61 (s, 3H, CH3), 3.77
(s, 3H, CH3), 6.97 (m, 2H, ArH), 7.13–7.23 (m, 3H,
ArH), 7.26–7.32 (m, 3H, ArH), 7.38 (d, 1H, J = 8.25,
ArH), 7.65 (d, 1H, J = 7.68, ArH); 13C NMR d: 32.47,
32.59, 96.67, 104.62, 105.00, 108.80, 109.30, 118.58,
119.88, 119.97, 120.67, 121.09, 121.90, 125.36, 127.36,
127.03, 128.34, 128.92, 136.45, 136.75, 162.12; Anal.
C21H18N4O requires C, 73.67; H, 5.30; N, 16.36; found
C, 73.55; H, 5.44; N, 16.58; ESIMS m/z 343.4 (M+).
1
EtOH); IR (KBr) 1740, 1650 cmÀ1; H NMR d: 1.27 (t,
3H, J = 7.14, CH3), 3.76 (s, 3H, CH3), 3.78 (s, 3H, CH3),
4.24 (c, 2H, J = 7.14, CH2), 5.70 (s, 1H, CH), 7.16–7.27
(m, 3H, ArH), 7.30–7.33 (m, 5H, ArH), 7.70 (d, 1H,
J = 7.14, ArH), 7.82 (s, 1H, ArH); 13C NMR d: 14.10,
32.75, 33.42, 53.22, 61.43, 107.46, 109.40, 114.82,
118.32, 119.32, 121.59, 122.61, 122.71, 123.45, 123.67,
126.67, 127.08, 128.82, 136.08, 136.55, 137.23, 169.71,
187.97; Anal. C23H22N2O3 requires C, 73.78; H, 5.92;
N, 7.48; found C, 73.70; H, 6.07; N, 7.16; APCIMS
m/z 375.3 (M + H).
5.9. 5-(2-Chloro-1-methyl-1H-indol-3-yl)-4-(1-methyl-
1H-indol-3-yl)-1,2-dihydropyrazol-3-one (4c)
1.494 g of compound 3c (3.65 mmol), 548 mg
(10.95 mmol) of hydrazine monohydrate and 730 mg
(3.65 mmol) of camphoric acid were used. In this case,
the solid obtained was filtered in vacuo. Yield 59%;
mp 272–275 °C (from DMF); IR (KBr) 3500,
1
5.6. Ethyl 3-(2-chloro-1-methyl-1H-indol-3-yl)-2-
(1-methyl-1H-indol-3-yl)-3-oxopropanoate (3c)
3380 cm1; H NMR d: 3.83 (s, 3H, CH3), 4.24 (s, 3H,
CH3), 5.72 (s, 2H, 2NH), 6.97 (t, 1H, J = 7.95, ArH),
7.12–7.31 (m, 5H, ArH), 7.45 (d, 1H, J = 7.95, ArH),
7.52 (d, 1H, J = 7.95, ArH); 7.91 (d, 1H, J = 7.32,
ArH); 13C NMR d: 29.96, 32.55, 100.01, 100.09,
102.26, 104.01, 109.42, 118.70, 119.12, 119.69, 121.21,
121.44, 122.82, 124.69, 126.47, 126.58, 128.22, 135.48,
136.51, 137.68, 155.27; Anal. C24H24ClN5O2.C3H7NO
requires C, 64.07; H, 5.38; N, 15.57; found C, 69.44;
H, 5.36; N, 15.44; ESIMS m/z 377.1 (M+).
2.17 g (10 mmol) of 1 and 2.59 g (11.41 mmol) of 2c
were used. Once the reaction was completed, the solid
obtained was filtered in vacuo, dissolved in CHCl3,
washed with 10% NH4Cl, water and saturated sodium
chloride solution, dried (MgSO4) and evaporated. The
crude product was washed with AcOEt and filtered in
vacuo. Yield 32%; mp 167–169 °C; IR (KBr) 1750,
1650 cmÀ1 1H NMR d: 1.25 (t, 3H, J = 7.14, CH3),
;
3.71 (s, 3H, CH3), 3.72 (s, 3H, CH3), 4.24 (c, 2H,
J = 7.14, CH2), 6.16 (s, 1H, CH), 7.13–7.22 (m, 3H,
ArH), 7.25–7.37 (m, 4H, ArH), 7.68 (d, 1H, J = 7.71,
ArH), 8.44 (d, 1H, J = 5.55, ArH); 13C NMR d: 14.10,
30.17, 32.70, 53.37, 61.32, 106.84, 109.28, 111.71,
118.81, 119.27, 121.57, 122.07, 123.07, 123.61, 126.16,
127.53, 128.74, 128.78, 131.56, 135.40, 136.70, 169.69,
188.40; Anal. C23H21ClN2O3 requires C, 67.56; H,
5.18; N, 6.85; found C, 67.27; H, 5.17, N, 6.99; APCIMS
m/z 409.1 (M + H).
5.10. 2-(2-Dimethylaminoethyl)-4,5-diphenyl-1,2-di-
hydropyrazol-3-one (5a)
1 g of compound 3a (3.72 mmol), 1.15 g of N,N-dim-
ethylaminoethylhydrazine (11.16 mmol) and 746 mg of
acid camphoric (3.72 mmol) were used. Chromatogra-
phy solvent B; yield 20%; mp 195–198 °C (from
1
EtOH); IR (KBr) 3540, 3340 cmÀ1; H NMR d: 2.60
(s, 6H, 2CH3), 3.06 (t, 2H, J = 4.41, CH2), 4.30 (t,
2H, J = 4.41, CH2), 7.13 (t, 1H, J = 6.60, ArH), 7.22–
7.29 (m, 7H, ArH), 7.46 (m, 2H, ArH); 13C NMR d:
43.30, 45.76, 58.77, 100.43, 124.80, 127.28, 127.76,
127.86, 128.52, 133.27, 133.28, 133.83, 148.19, 155.28;
Anal. C19H21N3O requires C, 74.24; H, 6.89; N,
13.67; found C, 73.98; H, 6.85; N, 13.51; ESIMS m/z
308.0 (M+).
5.7. General procedure for the preparation of pyrazolones
(4–6)
Camphoric acid (1 mmol) and the corresponding hydra-
zine (1 mmol) was added to a suspension of the appro-
priate b-ketoesther (3) (1 mmol) in EtOH (15 ml).
After heating the mixture under reflux for 2 h, an excess
of hydrazine (2 mmol) was added, and the solution stir-
red and heated under reflux for 24 h. The mixture was
cooled, the solvent was evaporated in vacuo, and the
crude product was dissolved in AcOEt, washed with
10% sodium bicarbonate solution, water and saturated
sodium chloride solution, dried (MgSO4), and evaporat-
ed. The products were purified by flash chromatography
over silica gel.
5.11. 1-(2-Dimethylaminoethyl)-4,5-diphenyl-2H-pyrazol-
3-one (6a)
1 g of compound 3a (3.72 mmol), 1.15 g of N,N-dim-
ethylaminoethylhydrazine (11.16 mmol) and 746 mg of
acid camphoric (3.72 mmol) were used. Chromatogra-
phy solvent B; yield 10%; mp 178–180 °C (from ace-
1
tone); IR (KBr) 3500, 3380 cmÀ1; H NMR d: 2.17 (s,
6H, 2CH3), 2.73 (t, 2H, J = 7.14, CH2), 3.97 (t, 2H,
J = 7.14, CH2), 7.09 (d, 1H, J = 7.14, ArH), 7.17 (t,
2H, J = 7.68, ArH), 7.28 (t, 2H, J = 7.14, ArH), 7.33
(m, 2H, ArH), 7.43 (m, 3H, ArH); 13C NMR d: 45.43,
46.30, 58.62, 66.80, 97.06, 104.72, 125.30, 127.93,
128.14, 128.95, 130.41, 132.05, 141.95, 159.44; Anal.
C19H21N3O.H2O requires C, 70.13; H, 7.12; N, 12.91;
found C, 70.49; H, 6.73; N, 12.74; ESIMS m/z 308.0
(M+).
5.8. 4,5-Bis-(1-methyl-1H-indol-3-yl)-1,2-dihydropyrazol-
3-one (4b)
0.5 g of compound 3b (1.33 mmol), 266 mg of hydrazine
monohydrate (5.32 mmol) and 268 mg of acid cam-
phoric (1.33 mmol) were used. Chromatography solvent
A; yield 80%; mp 178–181 °C (from EtOH); IR (KBr)

M. F. Bran˜a et al. / Bioorg. Med. Chem. 14 (2006) 9–16
15
5.12. 2-(2-Diethylaminoethyl)-4,5-diphenyl-1H-pyrazol-3-
one (5b)
6.91 (m, 2H, ArH), 7.03 (s, 1H, ArH), 7.11–7.13 (m,
2H, ArH), 7.20–7.37 (m, 4H, ArH), 7.44 (d,
1H,J = 7.71, ArH); 13C NMR d: 32.35, 32.64, 44.80,
46.26, 58.30, 100.23, 103.67, 105.22, 108.59, 109.37,
118.40, 119.45, 120.02, 120.61, 120.89, 121.94, 127.07,
127.16, 127.67, 129.47, 136.47, 136.50, 137.17, 140.98;
Anal. C25H27N5O requires C, 72.61; H, 6.58; N, 16.94;
found C, 72.74; H, 6.55; N, 16.73; ESIMS m/z
414.1(M+).
One gram of compound 3a (3.72 mmol), 1.46 g of N,N-
diethylaminoethylhydrazine (11.16 mmol) and 746 mg
of acid camphoric (3.72 mmol) were used. Chromatog-
raphy solvent B; yield 12%; mp 160–163 °C (from
1
EtOH); IR (KBr) 3520, 3380 cmÀ1; H NMR d: 1.25
(t, 6H, J = 7.14, 2CH3), 2.96 (c, 4H, J = 7.14, 2CH2),
3.14 (t, 2H, J = 4.95, CH2), 4.33 (t, 2H, J = 4.95,
CH2), 7.11 (d, 1H, J = 7.14, ArH), 7.22–7.33 (m, 7H,
ArH), 7.42-7.44 (m, 2H, ArH); 13C NMR d: 9.96,
46.49, 47.73, 53.83, 100.49, 124.95, 127.20, 128.00,
128.06, 128.80, 133.73, 134.67, 148.22, 153.97; Anal.
C21H25N3O requires C, 75.19; H, 7.51; N, 12.53; found
C, 74.85; H, 7.19; N, 12.66; ESIMS m/z 336.1 (M+).
5.16. 1-(2-Diethylaminoethyl)-4,5-bis-(1-methyl-1H-
indol-3-yl)- 2H-pyrazol-3-one (6e)
One gram of compound 3c (2.67 mmol), 1.05 g of N,N-
diethylaminoethylhydrazine (8.01 mmol) and 535 mg of
acid camphoric (2.67 mmol) were used. Chromatogra-
phy solvent B; yield 9%; mp 195–197 °C (from EtOH);
1
5.13. 1-(2-Diethylaminoethyl)-4,5-diphenyl-2H-pyrazol-3-
one (6b)
IR (KBr) 3520, 3360 cmÀ1; H NMR d: 0.82 (t, 6H,
J = 7.14, 2CH3), 2.38 (c, 4H, J = 7.14, 2CH2), 2.84 (t,
2H, J = 7.71, CH2), 3.61 (s, 3H, CH3), 3.72 (s, 3H,
CH3), 3.97 (t, 2H, J = 7.71, CH2), 6.85–6.88 (m, 2H,
ArH), 7.06–7.11 (m, 3H, ArH), 7.18 (d, 1H, J = 8.25,
ArH), 7.23 (d, 1H, J = 5.49, ArH), 7.31 (d, 1H,
J = 8.25, ArH), 7.45 (d, 1H, J = 7.71, ArH), 7.46 (d,
1H, J = 8.25, ArH); 13C NMR d: 11.72, 32.59, 32.62,
32.91, 47.15, 47.46, 52.48, 99.91, 104.51, 106.05,
108.59, 109.32, 118.49, 120.07, 120.13, 120.92, 121.47,
122.01, 127.33, 127.60, 129.51, 136.02, 136.65, 136.70,
160.08; Anal. C27H31N5O requires C, 73.44; H, 7.08;
N, 15.86; found C, 73.76; H, 7.00; N, 15.80; ESIMS
m/z 442.16 (M+).
One gram of compound 3a (3.72 mmol), 1.46 g of N,N-
diethylaminoethylhydrazine (11.16 mmol) and 746 mg
of acid camphoric (3.72 mmol) were used. Chromatog-
raphy solvent B; yield 4%; mp 157–159 °C (from ace-
1
tone); IR (KBr) 3500, 3380 cmÀ1; H NMR d: 0.95 (t,
6H, J = 7.14, 2CH3), 2.50 (c, 4H, J = 7.14, 2CH2), 2.82
(t, 2H, J = 7.69, CH2), 3.97 (t, 2H, J = 7.69, CH2),
7.09 (d, 1H, J = 6,57, ArH), 7.18 (t, 2H, J = 7.69,
ArH), 7.25 (t, 2H, ArH), 7.33 (m, 2H, ArH), 7.44 (m,
3H, ArH); 13C NMR d: 11.75, 46.73, 47.66, 52.21,
125.33, 127.91, 128.11, 128.84, 128.99, 130.20, 132.00,
141.90, 159.39; Anal. C21H25N3OÆH2O requires C,
71.36; H, 7.70; N, 11.89; found C, 71.09; H, 7.31; N,
11.56; ESIMS m/z 336.1 (M+).
5.17. 2-(2-Diethylaminoethyl)-4,5-bis-(1-methyl-1H-
indol-3-yl)-2H-pyrazol-3-one (5e)
5.14. 2-(3-Dimethylaminopropyl)-4,5-diphenyl-1H-pyra-
zol-3-one (5c)
One gram compound 3c (2.67 mmol), 1.05 mg of N,N-
diethylaminoethylhydrazine (8.01 mmol) and 535 mg of
acid camphoric (2.67 mmol) were used. Chromatogra-
phy solvent B; recrystallized from AcOEt/Hexane; yield
5%; IR (KBr) 3500, 3370 cmÀ1; 1H NMR d: 1.15 (t, 6H,
J = 7.14, 2CH3), 2.76 (c, 4H, J = 7.14, CH2), 2.97 (dd,
2H, J = 4.41, J = 4.92, CH2), 3.52 (s, 3H, CH3), 3.77
(s, 3H, CH3), 4.37 (dd, 2H, J = 4.92, J = 4.41, CH2),
6.83 (s, 1H, ArH), 6.69 (t, 1H, J = 7.14, ArH), 7.06–
7.25 (m, 5H, ArH), 7.30 (d, 1H, J = 8.25, ArH), 7.40
(d, 1H, J = 7.68, ArH), 8.20 (d, 1H,J = 8.25, ArH);
13C NMR d: 10.32, 11.95, 32.66, 32.79, 46.65, 47.30,
54.02, 107.23, 108.68, 108.87, 109.47, 119.40, 119.52,
121.40, 122.22, 126.55, 128.03, 128.13, 136.61, 137.04,
145.17; ESIMS m/z 442.16 (M+).
One gram of compound 3a (3.72 mmol), 1.3 g of 3-(N,N-
dimethylamino)propylhydrazine (11.16 mmol) and
746 mg of acid camphoric (3.72 mmol) were used. Chro-
matography solvent B; yield 16%; mp 174–176 °C (from
1
EtOH); IR (KBr) 3520, 3400 cm1; H NMR d: 2.10 (q,
2H, J = 6.06, CH2), 2.36 (s, 6H, 2CH3), 2.51 (t, 2H,
J = 6.06, CH2), 4.18 (t, 2H, J = 6.06, CH2), 7.12–7.17
(m, 1H, ArH), 7.23–7.32 (m, 5H, ArH), 7.35–7.38 (m,
2H, ArH), 7.48–7.52 (m, 2H, ArH); 13C NMR d:
26.26, 43.03, 43.39, 54.63,100.41, 124.79, 127.08,
127.89, 128.02, 128.08, 128.74, 133.92, 134.82, 148.14,
153.27; Anal. C20H23N3O requires C, 74.24; H, 7.21;
N, 13.07; found C, 74.06; H, 7.18; N, 12.75; ESIMS
m/z 322.1 (M+).
5.18. General procedure for the preparation of 3-alcoxy-
pyrazoles (7a–7c)
5.15. 1-(2-Dimethylaminoethyl)-4,5-bis-(1-methyl-1H-
indol-3-yl)-2H-pyrazol-3-one (6d)
To a solution of 1 mmol of the corresponding pyrazo-
lone (4a–4b), 1.5 mmol of Ph3P, and 1.25 mmol of
N,N-dimethylamino ethanol in 20 ml of THF, 1.5 mmol
of diisopropylazodicarboxilate was added slowly, and
the mixture was stirred for 20 h at room temperature.
Then, 1 ml of MeOH was added, the mixture was
poured onto 20 ml of water and was then exhaustively
extracted with ether. The combined organic phases were
washed with 2 M NaOH, water (several times), and
One gram of compound 3b (2.67 mmol), 826 mg of N,N-
dimethylaminoethylhydrazine (8.01 mmol) and 535 mg
of acid camphoric (2.67 mmol) were used. Chromatog-
raphy solvent B; yield 9%; mp 258–261 °C (from EtOH);
IR (KBr) 3500, 3360 cm1; 1H NMR d: 2.13 (s, 6H,
2CH3), 2.71 (t, 2H, J = 7.71, CH2), 3.66 (s, 3H, CH3),
3.73 (s, 3H, CH3), 3.98 (t, 2H, J = 7.71, CH2), 6.87–

16
M. F. Bran˜a et al. / Bioorg. Med. Chem. 14 (2006) 9–16
6. Meyer, T.; Regenass, U.; Fabbro, D.; Alteri, E.; Ro¨sel, J.;
Muller, M.; Caravatti, G.; Matter, A. Int. J. Cancer 1989,
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vacuo.
¨
43, 851.
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11. Toullec, D.; Pianetti, P.; Coste, H.; Bellevergue, P.;
Grand-Perret, T.; Ajakane, M.; Baudet, V.; Boissin, P.;
Boursier, E.; Loriolle, F. J. Biol. Chem. 1991, 266, 15771.
12. Prudhomme, M. Curr. Pharm. Des. 1997, 3, 265.
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5.19. N-[2-(4,5-Diphenyl-1H-pyrazol-3-yloxy)ethyl]
dimethylamine (7a)
500 mg (2.11 mmol) of 4a was used. Chromatography
solvent C; yield 97%; mp 138–141 °C (from benzene/hex-
1
ane); IR (KBr) 3500, 3350 cmÀ1; H NMR d: 2.27 (s,
6H, 2CH3), 2.72 (t, 2H, J = 6.06, CH2), 4.39 (t, 2H,
J = 6.06, CH2), 7.17 (d, 1H, J = 6.06, ArH), 7.24 (t,
2H, J = 7.74, ArH), 7.32 (m, 7H, ArH);13C NMR d:
45.70, 58.09, 67.00, 97.06, 104.19, 125.89, 127.05,
127.76, 128.03, 128.24, 128.45, 128.66, 129.09, 130.44,
131.64, 141.25, 161.06; Anal. C19H21N3O requires C,
74.24; H, 6.89; N, 13.67; found C, 74.17; H, 6.98; N,
13.63; APCIMS m/z 308.1 (M + H).
5.20. N-{2-[4,5-Bis-(1-methyl-1H-indol-3-yl)-1H-pyrazol-
3-yloxy]ethyl}dimethylamine (7b)
´
14. Jirousek, M. R.; Gillig, J. R.; Gonzalez, C. M.; Heath, W.
F.; McDonald, J. H.; Neel, D. A.; Rito, C. J.; Singh, U.;
Stramm, L. E.; Melikian-Badalian, A.; Baevsky, M.;
Ballas, L. M.; Hall, S. E.; Winneroski, L. L.; Faul, M.
M. J. Med. Chem. 1996, 39, 2664.
573 mg (1.67 mmol) of 4b was used. Chromatography
solvent C; yield 27%; mp 176–178 °C (from toluene);
1
IR (KBr) 3520, 3440 cmÀ1; H NMR d: 2.29 (s, 6H,
15. Kleinschroth, J.; Harteinstein, J.; Rudolph, C.; Scha¨ch-
tele, C. J. Bioorg. Med. Chem. Lett. 1993, 3, 1959.
16. Bran˜a, M. F.; An˜orbe, L.; Tarrason, G.; Mitjans, F.;
Piulats, J. Bioorg. Med. Chem. Lett. 2001, 11, 2701.
17. Arnott, C. H.; Scott, K. A.; Moore, R. J.; Hewer, A.;
Phillips, D. H.; Parker, P.; Balkwill, F. R.; Owens, D. M.
Oncogene 2002, 21, 4728.
18. Ferraris, C.; Cooklis, M.; Polakowska, R. R.; Haake, A.
R. Exp. Cell Res. 1997, 234, 37.
19. Sachsenmaier, C. Onkologie 2001, 24, 346.
20. Leping, L. I. Int. Patent Appl. WO01/82930, 2001.
21. Elslager, E. F.; Weinstein, E. A.; Worth, D. F. J. Med.
Chem. 1964, 7, 493.
2CH3), 2.73 (t, 2H, J = 6.06, CH2), 3.63 (s, 3H, CH3),
3.75 (s, 3H, CH3), 4.41 (t, 2H, J = 6.06, CH2), 6.93-
6.95 (m, 2H, ArH), 7.06 (s, 1H, ArH), 7.13–7.17 (t,
2H, ArH), 7.25.7.34 (m, 4H, ArH), 7.65 (d, 1H,
J=7.14, ArH); 13C NMR d: 32.73, 32.82, 45.80, 58.19,
66.80, 97.06, 105.06, 105.32, 108.88, 109.55, 118.64,
119.80, 120.21, 121.31, 122.08, 125.24, 125.84, 127.07,
128.16, 128.36, 128.98, 136.61, 136.81, 161.81; Anal.
C25H27N5O requires C, 72.61; H, 6.58; N, 16.94; found
C, 72.52; H, 6.63; N, 16.58; APCIMS m/z 414.0
(M + H).
22. Crystals of compounds 5a, 6a, 6d and 7a were grown from
EtOH, CHC13, EtOH and benzene/hexane (1:1), respec-
tively. Crystal growth was attempted under different
conditions but the quality of the crystals obtained was
poor in all cases. Therefore, the data obtained using a
Nonius Mach 3 diffractometer, equipped with monchro-
matic Mo(Ka1) radiation, were not good enough to
perform a detailed structure determination and refine-
ment. However, the molecular connectivity and topology
determined by XRD confirm those obtained by other
techniques.
Acknowledgments
We are grateful to the Universidad San Pablo CEU
(Grant No. U.S.P 20/03) for financial support. A. G.
Ovalles acknowledges to Universidad San Pablo CEU
for a predoctoral fellowship. We also thank Dr. A. Ulis-
´
es and J. Garcıa Lanz for the X-ray crystal structure and
to Linda Hamalainen for linguistic assistance.
23. Chiba, P.; Holzer, W.; Landau, M.; Bechmann, G.;
Lorenz, K.; Plagens, B.; Hitzler, M.; Richter, E.; Ecker,
G. J. Med. Chem. 1998, 41, 4001.
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