Synthesis and cytotoxicity evaluation of 1-[3-(9H-carbazol-4-yloxy)-2- hydroxypropyl]-3-aryl-1H-pyrazole-5-carboxylic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.07.004

Several novel molecules, 1-(3′-(9H-carbazol-4-yloxy)-2′- hydroxypropyl)-3-aryl-1H-pyrazole-5-carboxylic acid derivatives 3a-g were synthesized and screened to evaluate their cytotoxicity against cancer cells in vitro. The compounds 3a-g has been prepared by the reaction of ethyl 3-aryl-1H-pyrazole-5-carboxylate with 4-oxiranylmethoxy-9H-carbazole in moderate to excellent yields. The cytotoxicity of synthesized compounds was evaluated by a SRB (sulforhodamine B) assay against cancer cell such as SK-N-SH human neuroblastoma (NB), human A549 lung carcinoma, human breast cancer MCF-7 cell lines. The results showed that seven compounds can suppress SK-N-SH tumor cancer cell growth. Among them, compound 3d was the most effective small molecule in inhibiting SK-N-SH cell growth.

Full text of DOI:10.1016/j.ejmech.2010.07.004

European Journal of Medicinal Chemistry 45 (2010) 4720e4725
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and cytotoxicity evaluation of 1-[3-(9H-carbazol-4-yloxy)-2-
hydroxypropyl]-3-aryl-1H-pyrazole-5-carboxylic acid derivatives
,
a
a
a
a
Lingaiah Nagarapu a , Hanmant K. Gaikwad , Kartheeka Sarikonda , Jhansi Mateti , Rajashaker Bantu ,
*
P.S. Raghu a, Krishna Madhuri Manda b, Shasi Vardhan Kalvendi b
a Organic Division-II, Indian Institute of Chemical Technology, Hyderabad-500607, India
b Centre for Chemical Biology, Indian Institute of Chemical Technology, Hyderabad-500607, India
a r t i c l e i n f o
a b s t r a c t
Several novel molecules, 1-(30-(9H-carbazol-4-yloxy)-20-hydroxypropyl)-3-aryl-1H-pyrazole-5-carboxy-
lic acid derivatives 3aeg were synthesized and screened to evaluate their cytotoxicity against cancer cells
in vitro. The compounds 3aeg has been prepared by the reaction of ethyl 3-aryl-1H-pyrazole-5-
carboxylate with 4-oxiranylmethoxy-9H-carbazole in moderate to excellent yields. The cytotoxicity of
synthesized compounds was evaluated by a SRB (sulforhodamine B) assay against cancer cell such as
SKeNeSH human neuroblastoma (NB), human A549 lung carcinoma, human breast cancer MCF-7 cell
lines. The results showed that seven compounds can suppress SKeNeSH tumor cancer cell growth.
Among them, compound 3d was the most effective small molecule in inhibiting SKeNeSH cell growth.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Article history:
Received 5 April 2010
Received in revised form
23 June 2010
Accepted 3 July 2010
Available online 26 September 2010
Keywords:
Cytotoxicity
Neuroblastoma cell line (SKeNeSH)
A549 cell line
MCF-7 cell line
Pyrazole-carbazole derivatives
Growth inhibition
1. Introduction
class of antimitotic agents against solid tumors [5]. But there were no
reports on the synthesis and biological evaluation of 1-[3-(9H-car-
Neuroblastoma (NB) is an embryonic malignancy of the
sympathetic nervous system arising from neuroblasts and is the
most common extracranial solid tumor in child younger than five
year of age. Neuroblastoma accounts for 7e10% of all children had
cancers, in the majority of patients older than one year of age the
disease is fatal [1]. Therefore, new therapeutic targets have been
reported that the antitumor efficacy of chemotherapeutic agents
correlated with their growth-inhibiting, differentiation-inducing or
apoptosis-inducing abilities [2]. The aim of most cancer chemo-
therapeutic drugs currently in clinical use is to kill malignant tumor
cells by inhibiting some of the mechanisms implied in cellular
division. Accordingly, the antitumor compounds developed
through this approach are cytostatic or cytotoxic. However, the
knowledge of tumor biology has exploded during the past decades
and this may pave the way for more active, targeted anticancer
drugs [3].
bazol-4-yloxy)-2-hydroxypropyl]-3-aryl-1H-pyrazole-5-carboxy-
lic acid.
In our effort to discover and develop tumor growth inhibitors
and apoptosis inducers as potential new anticancer agents, we have
identified several classes of molecules as novel tumor growth
inhibitors of varitriol derivatives [6] and novel antibacterial
C-linked imidazole derivatives [7]. In an ongoing study in our
laboratory on the design and synthesis of the small molecules, we
are interested in extending our small molecule, library to meet the
requirement of our research.
We report herein for the first time the synthesis of 1-[3-(9H-
carbazol-4-yloxy)-2-hydroxypropyl]-3-aryl-1H-pyrazole-5-carbo-
xylic acid derivatives in a single step with the aim of evaluating
their biological activities in inhibiting SKeNeSH cell growth.
2. Results and discussion
More recently a series of phenylcarbazole molecules have been
reported asantitumoragent [4], Carbazole sulphonamides are a novel
2.1. Chemistry
Synthesis of ethyl 1-[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl]-
3-aryl-1H-pyrazole-5-carboxylic acid (3aeg) have been achieved in
moderate to excellentyieldsby thereaction between ethyl 3-aryl-1H-
* Corresponding author. Tel.: þ9140 27191509; fax: þ9140 27193198.
E-mail addresses: lnagarapuiict@yahoo.co.in, nagarapu@iict.res.in (L. Nagarapu).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.004

L. Nagarapu et al. / European Journal of Medicinal Chemistry 45 (2010) 4720e4725
4721
pyrazole-5-carboxylate (1aeg) and 4-oxiranylmethoxy-9H-carba-
3. Conclusion
zole (2) in the presence of potassium carbonate and tetrabuty-
lammonium iodide at reflux temperature in acetonitrile for 32 h
(Scheme 1). The crucial intermediate(1aeg) were obtained according
to the previously reported method [8] (Scheme 2) and 4-oxir-
anylmethoxy-9H-carbazole (2) was prepared by the reaction of
4-hydroxy carbazole (6) with epichlorohydrine (7) in the presence of
sodium hydroxide and tetrabutylammonium bromide in water at
40 ꢀC as shown in Scheme 3.
In conclusion, various substituted 1-[3-(9H-carbazol-4-yloxy)-2-
hydroxypropyl]-3-aryl-1H-pyrazole-5-carboxylic acid derivatives
(3aeg) have been prepared by the reaction of 4-oxiranylmethoxy-
9H-carbazole with ethyl 3-(4-methoxyphenyl)-1H-pyrazole-5-
carboxylate and screened in viability assays against SKeNeSH
neuroblastoma (NB), human A549 lung carcinoma, human breast
cancer MCF-7 cells in vitro. In general, most compounds showed
significantly cytotoxic effect against human SKeNeSH tumor cell
line. Compound 3a and 3d were showed significantly inhibit the
growth of SKeNeSH cells. The finding suggested that these
compounds would be very useful for further investigating the
mechanism of cell proliferation, morphology, differennation and
apoptosis in our next research project and some of them may be
a powerful drug against tumor cancer.
The structures of these compounds were confirmed from their
spectral and micro analytical data. Based on [MþþH] 458 its
molecular formula was established as C22H19NO5. The IR spectrum
of (3a) showed absorption due to eOH & eNH stretching at
3400 cmꢁ1 and acid carbonyl group at 1713 cmꢁ1 indicated that the
product formed in the above reaction is a coupled product.
Therefore the product was inferred to contain a carbazol aryl pyr-
azole carboxylic acid structure.
1H NMR spectrum (400 MHz) of (3a) recorded in DMSO-d6
exhibited signals arising due to typical chiral secondary alcohol.
The spectrum contained three multiplets, the first one appeared
4. Experimental section
4.1. Chemistry
at
d 4.16e4.26 integrating for two protons, the second one
appeared at
appeared at
also revealed the presence of one singlet at
eOCH3 group and remaining a multiplet at
d
4.49e4.58 integrating for one proton and third one
4.79e4.99 integrating for two protons. The spectrum
All commercial reagents and solvents were used as received
without further purification unless specified. The solvent were used
is dried from calcium hydride, calcium chloride for acetonitrile and
diethyl ether respectively. The reactions were monitored and Rf
value were determined using analytical thin layer chromatography
(TLC) with Merck Silica gel 60 and F-254 precoated plates (0.25-mm
thickness). Spot on the TLC plates were visualized using ultraviolet
light (254 nm). Flash column chromatography was performed with
Merck silica gel 60 (100e200 mesh). Melting points were deter-
mined in capillaries and are uncorrected. 1H NMR spectra were
recorded on Bruker DRX-300, Varian 400 and Varian-500 NMR
spectrometers. 13C NMR spectra were recorded on Bruker DRX-300.
d
d
3.81 (for 3H) due to
d
6.62e8.35 which were
assigned to the aromatic 12 protons, respectively.
2.2. Effects of the compounds on the viability of human cancer cells
The newly synthesized 1-[3-(9H-carbazol-4-yloxy)-2-hydroxy-
propyl]-3-aryl-1H-pyrazole-5-carboxylic acid derivatives (3aeg)
were evaluated for their cancer activity against the human cancer
cell line by sulforhodamine B (SRB) assay method [9,10]. The
various human tumor cell growth inhibitors potential was deter-
mined as described [11]. The inhibitory activities were presented as
micromolar concentrations of the compound that cause 50% inhi-
bition per unit of enzyme (IC50) under the assay conditions. The
data obtained from by SRB assay showed the compound 3aeg had
inhibitory effects on the growth of SKeNeSH neuroblastoma (NB),
human A549 lung carcinoma, human breast cancer MCF-7 cells and
standard doxorubicin in dosage and time dependent manner as
shown in Fig. 1a, b, c. Compounds 3d could mild inhibit the cell
Proton chemical shifts are reported in ppm (
tetramethylsilane (TMS, 0.00 or with the solvent reference rela-
tive to TMS employed as the internal standard (CDCl3, 7.26 ppm,
d) relative to internal
d
d
DMSO-d6d2.54). Infrared (IR) spectra were recorded on a Perkin
Elmer FT-IR 400 spectrometer; data are reported in wave numbers
(cmꢁ1). Mass spectra were recorded on Agilent Technologies 1100
Series (Agilent Chemistation Software).
4.2. Preparation of 4-oxiranylmethoxy-9H-carbazole (2)
growth obviously at 0.1
M concentration, after 48 h of the treatment compounds 3aeg
also suppress the growth of SKeNeSH cell. At 5 M concentration
all the compound showed significantly inhibited the SKeNeSH cell
growth (Fig. 1a). Taken altogether compounds 3a and 3d are
effective compounds in suppressing SKeNeSH cell growth.
mM after 48h of the treatment. At 0.5 and
5
m
4-Hydroxy carbazole (6.0 g) was added into a round bottom
flask containing water (10 mL), epichlorohydrin (6.4 g) and tetra-
butylammonium bromide (0.6 g) were then added into the reaction
mass under stirring. Sodium hydroxide (50% aqueous) solution
(7.8 g) was slowly added in the reaction mixture at 40 ꢀC over
m
NH
O
N
O
N
O
N
K2CO3, TBAI
CH3CN, 80oC, 32 h
COOEt
+
OH
R
NH
COOH
N
H
R
2
3a-g
1a-g
R,
a = 4-OCH3
c = 4-CH3
e = 4-NO2
b=3-OCH3
d = 3-CH3
f = 3-NO2 g = 2-NO2
Scheme 1. Synthesis of 1-[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl]-3-aryl-1H-pyrazole-5-carboxylic acid (3aeg).

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L. Nagarapu et al. / European Journal of Medicinal Chemistry 45 (2010) 4720e4725
NH
N
O
O
O
COOEt
O
NH2NH2
AcOH, rt
NaOMe
COOEt
COOEt
+
Diethyl ether, rt
O
R
R
R
1a-g
5a-g
4a-g
R,
a = 4-OCH3
c = 4-CH3
e = 4-NO2
b=3-OCH3
d = 3-CH3
f = 3-NO2
g = 2-NO2
Scheme 2. Synthesis of ethyl 3-aryl-1H-pyrazole-5-carboxylate (1aeg).
a period of 2e3 h. After completion of reaction, ethyl acetate
(100 mL) and water (100 mL) were added into the reaction mass.
The lower aqueous layer was separated. The ethyl acetate layer was
washed with water to obtain neutral pH and dried over anhydrous
sodium sulphate. The ethyl acetate is distilled out under vacuum
below 50 ꢀC to about 50 mL. The reaction mass was chilled to
0e5 ꢀC and filtered and dried at 50e60 ꢀC to give the compound 2
(5.0 g, 64%), mp 167e169 ꢀC IR (KBr, v cmꢁ1): 3293 (NeH), 2926
(AreH), 1595 (C]C), 1096 (CeO). 1H NMR (300 MHz, DMSO-d6):
4.3.3. Ethyl 2,4-dioxo-4-p-tolylbutanoate (5c)
Whitish solid in a yield of 88%, mp 72e74 ꢀC, 1H NMR(300 MHz,
CDCl3):
d
1.40 (t, J ¼ 7.32 Hz, 3H, CH3), 2.44 (s, 3H, OCH3), 3.90 (s, 2H,
CH2), 4.34 (q, J ¼ 7.32 Hz, 2H, OCH2), 7.31(d, J ¼ 7.32 Hz, 2H, AreH),
7.90 (d, J ¼ 7.32 Hz, 2H, AreH), ESI-MS: m/z ¼ 257 [MþNa]þ.
4.3.4. Ethyl 2,4-dioxo-4-m-tolylbutanoate (5d)
Reddish solid in a yield of 82%, mp 72 ꢀC, 1H NMR (300 MHz,
CDCl3):
d
1.42 (t, J ¼ 7.17 Hz, 3H, CH3), 2.43 (s, 3H, OCH3), 3.92 (s, 2H,
d
2.81e2.88 (dd, J ¼ 2.45, 2.64 Hz, 1H, HA), 2.90e2.97 (dd, J ¼ 4.34,
CH2), 4.37 (q, J ¼ 7.17 Hz, 2H, OCH2), 7.30e7.40 (m, 2H, AreH),
4.91 Hz, 1H, HB), 3.50e3.57 (m, 1H, HC), 4.04e4.14 (dd, J ¼ 6.23,
6.23 Hz, 1H, HE), 4.52e4.59 (dd, J ¼ 2.26, 2.26 Hz, 1H, HD), 6.70 (d,
J ¼ 7.93 Hz, 1H, AreH), 7.05e7.20 (m, 2H, AreH), 7.25e7.39 (m, 2H,
AreH), 7.46 (d, J ¼ 7.93 Hz, 1H, AreH), 8.17(d, J ¼ 7.74 Hz,1H, AreH),
11.27(s, 1H, NH), ESI-MS: m/z ¼ 239 [Mþ].
7.72e7.81 (m, 2H, AreH), ESI-MS: m/z ¼ 257 [MþNa]þ
4.3.5. Ethyl 4-(4-nitrophenyl)-2,4-dioxobutanoate (5e)
Yellow solid in a yield of 89%, mp 72e74 ꢀC, 1H NMR (300 MHz,
CDCl3):
d
1.44 (t, J ¼ 7.17 Hz, 3H, CH3), 4.40 (q, J ¼ 7.17 Hz, 2H, OCH2),
7.06 (s, CH2), 8.35 (d, J ¼ 8.30 Hz, 2H, AreH), 3.90 (s, 2H, CH2), ESI-
MS:m/z ¼ 288 [MþNa]þ.
4.3. General procedure for the synthesis of ethyl 2,4-dioxo-
4-arylbutanoate (5aeg)
4.3.6. Ethyl 4-(3-nitrophenyl)-2,4-dioxobutanoate (5f)
Yellow solid in a yield of 87%, mp 82 ꢀC, 1H NMR(300 MHz,
CDCl3):
d
1.45 (t, J ¼ 7.17 Hz, 3H, CH3), 3.97 (s, 2H, CH2), 4.42 (q,
A mixture of a freshly prepared NaOCH3 (0.86 g, 0.016 mol) and
diethyl oxalate (1.95 g, 0.013mol) was dissolved in diethyl ether
(50 mL) and stirred at room temperature under nitrogen atmo-
sphere. Followed by to this solution was added a 4-methoxy-
acetophenone 4a (2.0 g, 0.013mol) in diethyl ether (10 mL). A yellow
precipitate immediately formed and the reactionwas stirred for 12 h
at room temperature. The precipitate was filtered and dissolved in
water (100 mL) and the solution was adjusted to pH 3 with acetic
acid. The resulting precipitate was filtered and dried to give 3.0 g
(92%) of ethyl 2,4-dioxo-4-(methoxyphenyl)butanoate (5a).
J ¼ 7.17 Hz, 2H, OCH2), 7.72 (t, J ¼ 7.93 Hz, 1H, AreH), 8.32 (d,
J ¼ 8.30 Hz, 1H, AreH), 8.45 (d, J ¼ 8.30, Hz, 1H, AreH), 8.81(s, 1H,
AreH); ESI-MS:m/z ¼ 288 [MþNa]þ
4.3.7. Ethyl 4-(2-nitrophenyl)-2,4-dioxobutanoate (5g)
Yellow oil in a yield of 86%, 1H NMR(300 MHz, CDCl3):
d 1.40 (t,
J ¼ 7.17 Hz, 3H, CH3), 3.92 (s, 2H, CH2), 4.36 (q, J ¼ 7.17 Hz, 2H,
OCH2), 7.59 (dd, J ¼ 7.17, 5.66 Hz, 1H, AreH), 7.67 (dd, J ¼ 7.36,
4.53 Hz, 1H, AreH), 7.73 (d, J ¼ 7.36 Hz, 1H, AreH), 8.04 (d,
J ¼ 7.74 Hz, 1H, AreH), ESI-MS: m/z ¼ 266 [MþH]þ.
4.3.1. Ethyl 4-(4-methoxyphenyl)-2,4-dioxobutanoate (5a)
Yellow solid in a yield of 92%, mp 56 ꢀC, 1H NMR (300 MHz,
4.4. General procedure for the synthesis of ethyl aryl pyrazole
CDCl3):
d
1.42 (t, J ¼ 7.17 Hz, 3H, CH3), 3.88 (s, 3H, OCH3), 3.92 (s, 2H,
carboxylates (1aeg)
CH2), 4.37 (q, J ¼ 7.17 Hz, 2H, OCH2), 6.94(d, J ¼ 9.06 Hz, 2H, AreH),
7.97 (d, J ¼ 9.06 Hz, 2H, AreH), ESI-MS: m/z ¼ 273 [MþNa]þ.
A mixture 5a (2.0 g, 0.008 mol) and acetic acid (30 mL) was
stirred at room temperature. To this solution was added hydrazine
(4.0 mL, 0.08 mol) and the reaction mixture was stirred overnight.
The precipitate was filtered, washed with hexane and dried to give
following ethyl ester.
4.3.2. Ethyl 4-(3-methoxyphenyl)-2,4-dioxobutanoate (5b)
Whitish solid in a yield of 89%, mp 72 ꢀC, 1H NMR(300 MHz,
CDCl3):
d
1.43 (t, J ¼ 7.17 Hz, 3H, CH3), 3.88 (s, 3H, OCH3), 3.94 (s, 2H,
CH2), 4.38 (q, J ¼ 7.17 Hz, 2H, OCH2), 7.10 (d, J ¼ 8.12 Hz, 2H, AreH),
7.37 (t, J ¼ 7.93, Hz, 1H, AreH), MS: m/z ¼ 251 [MþH]þ.
4.4.1. Ethyl 3-(4-methoxyphenyl)-1H-pyrazole-5-carboxylate (1a)
Yellow solid in a yield of 94%, mp 146e148 ꢀC, IR (KBr, cmꢁ1):
3141 (NeH), 2925 (AreH), 1725 (C]O), 1H NMR (400 MHz, DMSO-
d6):
d
1.41 (t, J ¼ 7.17 Hz, 3H, CH3), 3.83(s, 3H, OCH3), 4.36 (q,
O
O
HO
J ¼ 7.16 Hz, 2H, OCH2CH3), 6.90 (d, J ¼ 8.05 Hz, 2H, AreH), 7.57 (s,
1H, AreH), 7.66 (bs, 2H, AreH), ESI-MS: m/z ¼ 269 [MþNa]þ.
O
NaOH, TBAB
Water, 40oC
Cl
+
N
H
N
4.4.2. Ethyl 3-(3-methoxyphenyl)-1H-pyrazole-5-carboxylate (1b)
Yellow solid in a yield of 83%, mp 154 ꢀC, IR (KBr, cmꢁ1): 3141
(NeH), 2952 (AreH), 1729 (C]O), 1H NMR (500 MHz, DMSO-d6):
H
2
7
6
Scheme 3. Synthesis of 4-oxiranylmethoxy-9H-carbazole (2).
d
1.41 (t, J ¼ 7.19 Hz, 3H, CH3), 3.84 (s, 3H, OCH3), 4.35 (q, J ¼ 7.19 Hz,

L. Nagarapu et al. / European Journal of Medicinal Chemistry 45 (2010) 4720e4725
4723
Fig. 1. (a) Analysis of the cytotoxic effect of various derivatives in a human NB cell line (SKeNeSH). SKeNeSH) NB cells were treated with various concentrations (0.1, 1 and 5 mM) of
3aeg for 48 h. A viability assay was carried out. Experiments were performed in triplicate; data are expressed as means of the triplicate determinations of a representative
experiment in % cell viability of untreated cells [100%]. (b) Analysis of the cytotoxic effect of various derivatives in a human A549 lung carcinoma cell line. A549 cells were treated
with various concentrations (0.1, 1 and 5
triplicate determinations of a representative experiment in % cell viability of untreated cells [100%]. (c) Analysis of the cytotoxic effect of various derivatives in a human breast cancer
MCF-7 cell line. MCF-7 cells were treated with various concentrations (0.1, 1 and 5 M) of 3aeg for 48 h. A viability assay was carried out. Experiments were performed in triplicate;
data are expressed as means of the triplicate determinations of a representative experiment in % cell viability of untreated cells [100%].
mM) of 3aeg for 48 h. A viability assay was carried out. Experiments were performed in triplicate; data are expressed as means of the
m
2H, OCH2CH3), 6.82 (d, J ¼ 7.19 Hz, 1H, AreH), 7.00 (d, J ¼ 5.13 Hz,
1H, AreH), 7.29 (m, 3H, AreH), ESI-MS: m/z ¼ 247 [MþH]þ.
2H, OCH2CH3), 6.82 (d, J ¼ 7.16 Hz, 1H, AreH), 7.57 (s, 1H, AreH),
7.60 (b, J ¼ 5.13 Hz, 1H, AreH), 7.80 (m, 3H, AreH), ESI-MS: m/
z ¼ 231 [MþH]þ.
4.4.3. Ethyl 3-p-tolyl-1H-pyrazole-5-carboxylate (1c)
Whitish solid in a yield of 82%, mp 202 ꢀC, IR (KBr, cmꢁ1): 3220
4.4.5. Ethyl 3-(4-nitrophenyl)-1H-pyrazole-5-carboxylate (1e)
Yellow solid in a yield of 98%, mp 210 ꢀC, IR (KBr, cmꢁ1), 3139
(NeH), 2928 (AreH), 1733(C]O), 1H NMR (400 MHz, DMSO-d6):
(NeH), 3005 (AreH), 1725 (C]O), 1H NMR (400 MHz, DMSO-d6):
d
1.41 (t, J ¼ 7.16 Hz, 3H, CH3), 2.37 (s, 3H, CH3), 4.35 (q, J ¼ 7.16 Hz,
2H, OCH2CH3), 7.18 (d, J ¼ 8.05 Hz, 2H, AreH), 6.95 (s, 1H, AreH),
d
1.42 (t, J ¼ 7.17 Hz, 3H, CH3); 4.38(q, J ¼ 7.17 Hz, 2H, OCH2CH3), 7.19
7.62 (b, J ¼ 8.05, 2H, AreH), ESI-MS: m/z ¼ 231 [MþH]þ.
(s, 1H, AreH), 8.02 (d, J ¼ 8.12 Hz, 2H, AreH), 8.25 (d, J ¼ 8.30 Hz,
2H, AreH), ESI-MS: m/z ¼ 284 [MþNa]þ.
4.4.4. Ethyl 3-m-tolyl-1H-pyrazole-5-carboxylate (1d)
Yellow solid in a yield of 78%, mp 148 ꢀC, IR (KBr, cmꢁ1): 3236
4.4.6. Ethyl 3-(3-nitrophenyl)-1H-pyrazole-5-carboxylate (1f)
Yellow solid in a yield of 96%, mp 146 ꢀC, IR (KBr, cmꢁ1) 3304
(NeH), 2984 (AreH), 1700 (C]O), 1H NMR (300 MHz, DMSO-d6):
(NeH), 2947 (AreH), 1691 (C]O), 1H NMR (300 MHz, DMSO-d6):
d
1.41 (t, J ¼ 7.16 Hz, 3H, CH3), 3.37 (s, 3H, CH3), 4.35 (q, J ¼ 7.16 Hz,

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L. Nagarapu et al. / European Journal of Medicinal Chemistry 45 (2010) 4720e4725
d
1.40 (t, J ¼ 7.08 Hz, 3H, CH3), 4.36 (q, J ¼ 7.08 Hz, 2H, OCH2CH3),
4.5.4. 1-[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl]-3-m-tolyl-1H-
7.32 (s, 1H, AreH), 7.69 (t, J ¼ 6.51 Hz,1H, AreH), 8.15 (d, J ¼ 7.36 Hz,
1H, AreH), 8.24 (d, J ¼ 7.36 Hz, 1H, AreH), 8.66 (s, 1H, AreH), ESI-
MS: m/z ¼ 262 [MþH]þ.
pyrazole-5-carboxylic acid (3d)
Brown solid in a yield of 76%, mp 150 ꢀC, IR (KBr, v cmꢁ1): 3441
(OeH, NeH), 2925 (AreH), 1634 (C]O), 1H NMR (300 MHz, DMSO-
d6):
d
2.78 (s, 1H, OH), 2.39 (s, 3H, CH3), 4.24 (d, J ¼ 4.91 Hz, 2H,
4.4.7. Ethyl 3-(2-nitrophenyl)-1H-pyrazole-5-carboxylate (1g)
Yellow solid in a yield of 98%, mp 106 ꢀC, IR (KBr, cmꢁ1): 3276
(NeH), 2992 (AreH), 1705 (C]O), 1H NMR (400 MHz, DMSO-d6):
CH2), 4.53e4.63 (m, 1H, CH), 4.92e4.99 (m, 2H, CH2), 6.58 (d,
J ¼ 7.74 Hz, 1H, AreH), 7.01e7.15 (m, 3H, AreH), 7.18e7.31 (m, 3H,
AreH), 7.40 (d, J ¼ 8.12 Hz, 1H, AreH), 7.55 (d, J ¼ 7.74 Hz, 1H,
AreH), 7.60 (s, 1H, AreH), 7.82 (s, 1H, AreH), 8.35 (d, J ¼ 7.74 Hz, 1H,
d
1.41 (t, J ¼ 7.17 Hz, 3H, CH3), 4.36 (q, J ¼ 7.17 Hz, 2H, OCH2CH3), 6.88
(s, 1H, AreH), 7.50 (t, J ¼ 7.64 Hz, 1H, AreH), 7.62 (t, J ¼ 7.45 Hz, 1H,
AreH), 10.79 (s, 1H, NH), 13C NMR (DMSO-d6, 75 MHz)
d: 21.14,
AreH), 7.72 (d, J ¼ 7.17 Hz, 2H, AreH), ESI-MS: m/z ¼ 262 [MþH]þ
54.04, 68.94, 70.06,100.48, 104.14, 107.75, 110.43, 111.74, 118.81,
121.83, 122.50, 122.96, 124.71, 125.82, 126.60, 128.67, 132.45, 136.26,
137.98, 139.02, 141.24, 149.08, 154.93, 161.27; ESI-MS: m/z ¼ 464
[MþNa]þ
4.5. General procedure for the synthesis 1-[3-(9H-carbazol-4-
yloxy)-2-hydroxypropyl]-3-aryl-1H-pyrazole-5-carboxylic acid
(3aeg)
4.5.5. 1-[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl]-
A mixture of 5-(4-methoxyphenyl)-2H-pyrazole-3-carboxylic
acid ethyl ester 1a (103 mg, 0.42 mmol), 4-oxiranylmethoxy-9H-
carbazole (2, 100 mg, 0.42 mmol), potassium carbonate (116 mg,
0.82) and tetrabutylammonium iodide (2 mg) were heated in
acetonitrile (10 mL) at 80 ꢀC for 32 h under nitrogen atmosphere.
The reaction mass were extracted with ethyl acetate. The organic
layer dried over sodium sulphate and concentrated crude product
obtained was purified by column chromatography over silica gel to
give following compounds.
3-(4-nitrophenyl)-1H-pyrazole-5-carboxylic acid (3e)
Yellow solid in a yield of 75%, mp 145 ꢀC, IR (KBr, v cmꢁ1): 3407
(OeH, NeH), 2928(AreH), 1707 (C]O), 1H NMR (400 MHz, DMSO-
d6): d2.79 (s, 1H, OH), 4.16 (dd, 2H, CH2), 4.45 (m, 1H, CH), 4.93 (dd,
2H, CH2), 6.56 (d, J ¼ 8.0 Hz, 1H, AreH), 7.02 (d, J ¼ 8.05 Hz, 1H,
AreH), 7.12 (m, 2H, AreH), 7.19 (t, J ¼ 8.05, 1H, AreH), 7.25 (t,
J ¼ 8.05 Hz, 1H, AreH), 7.37 (d, J ¼ 8.05 Hz, 1H, AreH), 7.92
(d, J ¼ 8.79 Hz, 2H, AreH), 8.14 d, J ¼ 8.79 Hz, 2H, AreH), 8.31 (d,
J ¼ 8.05 Hz, 1H, AreH), 10.79 (s, 1H, NH), 13C NMR (DMSO-d6,
75 MHz) d: 54.12, 69.38, 70.04, 100.42, 104.00, 107.53, 110.36, 11.65,
4.5.1. 1-[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl]-3-
118.69, 121.73, 122.73, 124.07, 124.56, 125.78, 126.46, 138.97, 139.60,
141.15, 142.04, 146.18, 146.32, 154.85, 162.56, ESI-MS: m/z ¼ 495
[M þ þNa]þ.
(4-methoxyphenyl)-1H-pyrazole-5-carboxylic acid (3a)
Brown solid in a yield of 67%, mp 140e142 ꢀC, IR (KBr, v cmꢁ1):
3400 (OeH, NeH), 2931 (AreH), 1713 (C]O), 1H NMR (400 MHz,
DMSO-d6):
d
2.74 s, 1H, OH), 3.81 (s, 3H, OCH3), 4.16e4.26 (m, 2H,
4.5.6. 1-[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl]-
CH2), 4.49e4.58 (m, 1H, CH), 4.79e4.99 (m, 2H, CH2), 6.62 (d,
J ¼ 8.05 Hz,1H, AreH), 6.93 (d, J ¼ 8.95 Hz, 2H, AreH), 7.04e7.15 (m,
3H, AreH), 7.21e7.35 (m, 2H, AreH), 7.42 (d, J ¼ 7.16 Hz, 1H, AreH),
7.73 (d, J ¼ 8.95 Hz, 2H, AreH), 8.35 (d, J ¼ 8.05 Hz, 1H, AreH), 11.10
3-(3-nitrophenyl)-1H-pyrazole-5-carboxylic acid (3f)
Pale yellow solid in a yield of 73%, mp 138 ꢀC, IR (KBr, v cmꢁ1):
3048 (OeH, NeH), 2928 (AreH), 1712 (C]O); 1H NMR (400 MHz,
DMSO-d6):
d
2.81 (s, 1H, OH); 4.28 (d, J ¼ 4.72 Hz, 2H, CH2), 5.04 (d,
(s, 1H, NH), 13C NMR (DMSO-d6, 75 MHz)
d
: 53.79, 55.03, 68.67,
J ¼ 5.85 Hz, 2H, CH2), 4.62 (m, 1H, CH), 6.59 (d, J ¼ 7.93 Hz, 1H,
AreH), 7.03 (d, J ¼ 7.93 Hz, 1H, AreH), 7.12 (t, J ¼ 7.46 Hz,1H, AreH),
7.24 (m, 3H, AreH), 7.38 (d, J ¼ 8.12 Hz, 1H, AreH), 7.57 (t,
J ¼ 7.83 Hz, 2H, AreH), 8.12 (d, J ¼ 7.93 Hz, 1H, AreH), 8.34 (d,
J ¼ 7.74 Hz, 1H, AreH), 8.61 (s, 1H, AreH), 10.47 (s, 1H, NH), 13C NMR
70.01, 100.28, 103.91, 107.17, 110.19, 111.56, 114.01, 118.57, 121.61,
122.75, 124.47, 125.02, 126.37, 135.33, 138.84, 141.05, 148.80, 154.76,
158.97,160.86, ESI-MS: m/z ¼ 458 [MþH]þ.
4.5.2. 1-[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl]-3-
(DMSO-d6, 75 MHz) d: 54.28, 68.77, 70.02, 100.30, 103.97, 108.34,
(3-methoxyphenyl)-1H-pyrazole-5-carboxylic acid (3b)
110.25, 111.58, 118.59, 119.17, 121.65, 122.32, 122.79, 124.50, 126.40,
130.37, 131.50, 134.19, 137.05, 138.88, 141.08, 146.69, 148.26, 154.74,
160.99, ESI-MS: m/z ¼ 495 [MþNa].
Brown solid in a yield of 90%, mp 142 ꢀC, IR (KBr, v cmꢁ1): 3407
(OeH, NeH), 2932 (AreH), 1668 (C]O), 1H NMR (300 MHz, DMSO-
d6):
d
2.79 (s,1H, OH), 3.79 (s, 3H, OCH3), 4.24 (d, J ¼ 5.09 Hz, 2H, CH2),
4.56 (m, 1H, CH), 4.99 (m, 2H, CH2), 6.59 (d, J ¼ 7.93 Hz, 1H, AreH),
6.79 (d, J ¼ 7.55 Hz, 1H, AreH), 7.07 (m, 3H, AreH), 7.24 (m, 2H,
AreH), 7.42 (d, J ¼ 7.16 Hz,1H, AreH), 7.32 (t, J ¼ 6.98 Hz, 2H, AreH),
7.40 (d, J ¼ 7.93 Hz,1H, AreH), 7.82 (s,1H, AreH), 8.36 (d, J ¼ 7.74 Hz,
1H, AreH), 10.79 (s, 1H, NH), ESI-MS: m/z ¼ 480[MþNa]þ.
4.5.7. 1-[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl]-
3-(3-nitrophenyl)-1H-pyrazole-5-carboxylic acid (3g)
Pale yellow solid in a yield of 76%, mp 139 ꢀC, IR (KBr, v cmꢁ1):
3416 (OeH, NeH), 2928 (AreH), 1704 (C]O), 1H NMR (400 MHz,
DMSO-d6):
d
2.80 (s, 1H, OH), 4.22 (d, J ¼ 5.09 Hz, 2H, CH2), 4.56 (m,
1H, CH), 4.97 (d, J ¼ 5.66 Hz, 2H, CH2), 6.60 (d, J ¼ 7.74 Hz, 1H,
AreH), 6.96 (s, 1H, AreH), 7.04 (d, J ¼ 8.12 Hz, 1H, AreH), 7.12 (t,
J ¼ 7.17 Hz, 1H, AreH), 7.19e7.31 (m, 2H, AreH), 7.39 (d, J ¼ 7.93 Hz,
1H, AreH), 7.49 (t, J ¼ 7.64 Hz, 1H, AreH), 7.72(t, J ¼ 7.74 Hz, 2H,
AreH), 8.32 (d, J ¼ 7.74 Hz, 1H, AreH), 10.69 (s, 1H, NH), 13C NMR
4.5.3. 1-[3-(9H-carbazol-4-yloxy)-2-hydroxypropyl]-3-p-tolyl-1H-
pyrazole-5-carboxylic acid (3c)
Light brown solid in a yield of 78%, mp 154 ꢀC, IR (KBr, v cmꢁ1):
3410 (OeH, NeH), 2926 (AreH), 1709 (C]O), 1H NMR (500 MHz,
DMSO-d6):
d
2.73 (s, 1H, OH), 2.29 (s, 3H, CH3), 4.14e4.23 (m, 2H,
(DMSO-d6, 75 MHz) d: 54.16, 68.83, 69.99, 100.53, 104.14, 109.58,
CH2), 4.46e4.55 (m, 1H, CH), 4.83e4.99 (m, 2H, CH2), 6.51 (d,
J ¼ 9.25 Hz,1H, AreH), 6.97 (d, J ¼ 8.22 Hz,1H, AreH), 6.99e7.14 (m,
3H, AreH), 7.21(t, J ¼ 8.22 Hz, 1H, AreH), 7.32 (d, J ¼ 8.22 Hz, 2H,
AreH), 7.58 (d, J ¼ 8.22 Hz, 2H, AreH), 7.62 (s, 1H, AreH), 8.29 (d,
J ¼ 8 Hz, 1H, AreH), 10.55 (s, 1H, NH), 13C NMR (DMSO-d6, 75 MHz)
110.39, 111.71, 118.83, 121.80, 122.94, 123.74, 124.71, 125.38, 126.60,
129.40, 130.38, 132.36, 136.49, 138.99, 141.21, 144.76, 148.62, 154.85,
160.90, ESI-MS: m/z ¼ 495 [MþNa]þ.
4.6. In vitro cytotoxic activity evaluation by SRB assay
d
: 20.82, 53.87, 69.18, 70.07, 100.35, 104.02, 106.67, 110.34, 111.65,
118.66, 121.74, 122.82. 124.56, 125.06, 126.46, 129.25, 130.06, 136.93,
138.47, 138.96, 141.15, 148.65, 154.85, 162.06, ESI-MS: m/z ¼ 464
[MþNa]þ.
The newly obtained derivatives were evaluated for cytotoxic
activity to cancer cell lines by using the sulforhodamine B (assay)
method [8,9]. The cells were grown in minimal essential medium

L. Nagarapu et al. / European Journal of Medicinal Chemistry 45 (2010) 4720e4725
4725
supplemented with 10% fetal bovine serum (Invitrogen), 100 U/ml
Penicillin (Invitrogen) and 100 g/ml Streptomycin (Invirtogen).
The cells were seeded in 24-well microplates and treated with the
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Authors thank the Director and Head, Organic Chemistry Divi-
sion-II, IICT for their support. HKG thank CSIR, New Delhi for
financial assistance.