Synthesis and cyclization of β-keto-enol derivatives tethered indole and pyrazole as potential antimicrobial and anticancer activity

Article abstract of DOI:10.1007/s13738-018-1362-7

Abstract: Synthesis of biologically active new indole and pyrazole derivatives has earned a substantial position in the pharmaceutical industry. The study aims to synthesize and cyclize β-keto-enol derivatives tethered indole and pyrazole as potential antimicrobial and anticancer activity. Novel derivatives of enolic keto ester 1 have been obtained through Claisen condensation of 3-acetylindole with diethyl oxalate under basic conditions. Spectral data of newly produced compounds were characterized using Fourier transform infrared, hydrogen nuclear magnetic resonance, Carbon-13 nuclear magnetic resonance, and elemental analysis. Few compounds were assessed for their antimicrobial activity, contrary to gram-positive bacterial strains, gram-negative bacterial strain, and antifungal activity that have been approved against Candida albicans. However, compound 2 showed an excellent antimicrobial activity. In vitro antitumor action of few prepared compounds in contradiction to human breast carcinoma cell line, colon cell line, and normal retina cell line was evaluated. Graphical Abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s13738-018-1362-7

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-018-1362-7
ORIGINAL PAPER
Synthesis and cyclization of β‑keto‑enol derivatives tethered indole
and pyrazole as potential antimicrobial and anticancer activity
Wesam S. Shehab1 · Gehan T. El‑Bassyouni2
Received: 23 September 2017 / Accepted: 9 April 2018
© Iranian Chemical Society 2018
Abstract
Synthesis of biologically active new indole and pyrazole derivatives has earned a substantial position in the pharmaceutical
industry. The study aims to synthesize and cyclize β-keto-enol derivatives tethered indole and pyrazole as potential antimi-
crobial and anticancer activity. Novel derivatives of enolic keto ester 1 have been obtained through Claisen condensation
of 3-acetylindole with diethyl oxalate under basic conditions. Spectral data of newly produced compounds were character-
ized using Fourier transform infrared, hydrogen nuclear magnetic resonance, Carbon-13 nuclear magnetic resonance, and
elemental analysis. Few compounds were assessed for their antimicrobial activity, contrary to gram-positive bacterial strains,
gram-negative bacterial strain, and antifungal activity that have been approved against Candida albicans. However, compound
2 showed an excellent antimicrobial activity. In vitro antitumor action of few prepared compounds in contradiction to human
breast carcinoma cell line, colon cell line, and normal retina cell line was evaluated.
Graphical Abstract
COCH3
O
NH2
OC2H5
HN
N
H
HO
O
O
N
O
N
OC2H5
OEt
N
H
N
H
EtO
O
Keywords Antimicrobial · Anticancer · Cyclization · Derivatives · Pyrazole
Introduction
pharmaceuticals, natural products, and other biologically
related compounds, or as key intermediates for synthesiz-
ing these species [5]. The synthesis of 1,3-diketones became
highly essential for chemists to be used as a starting material
to create diferent species of organic compounds [6].
Heterocycle compounds with β-keto-enol moieties were rec-
ognized as signifcant biologically active compounds. Their
medicinal chemistry advantages were steadfastly established
[1, 2]. 1, 3-Diketones are highly important intermediates
in organic synthesis [3, 4] that are widely represented in
Remarkable chemical properties of indole can motivate
chemists to synthesize a variety of indole derivatives. Such
derivatives represent many important classes in medicinal
chemistry, such as anti-HIV antagonist, anticancer, anti-
oxidant, antibacterial, antifungal, anti-allergic, and anti-
infammatory [6–9]. Pyrazoles and their derivatives display
antimicrobial [10–12], anticancer [13], and anti-infamma-
tory activities [14]. Consequently, signifcant eforts were
devoted to search for pyrazole and indole rings for decades
* Wesam S. Shehab
wesamshehab2015@gmail.com
1
Chemistry Department, Faculty of Science, Zagazig
University, Zagazig 44519, Egypt
2
Refractories, Ceramics and Building Materials Department,
National Research Centre (NRC), Dokki, Giza 12622, Egypt
Vol.:(0123456789)
1 3

Journal of the Iranian Chemical Society
[15]. Additionally, heterocycles were condensed to pyra-
zole ring as an essential source of bioactive molecules [16].
Concerning the hard work toward synthesizing biologically
active heterocyclic compounds [17–19], researchers planned
to synthesize new indole and pyrazole derivatives to study
their biological activities, Therefore, described herein are the
considered drugs, based on the β-keto-enol group embedded
with heterocyclic moieties, such as pyrazole, pyridine, and
pyrimidine using 3-acetylindole to fnd out novel biologi-
cally active compounds.
for C14H13NO4 (259.26): C, 64.86; H, 5.05; N, 5.40; found: C,
64.73; H, 4.93; N, 5.52.
3‑[2‑(1H‑indol‑3‑yl)‑2‑oxoethylidene]‑3, 4, 4a,
8a‑tetrahydro‑2H‑1, 4‑benzoxazin‑2‑one
A mixture of both ester 1 (0.39 g, 1.5 mmol) and 2-aminophe-
nol (1.7 g, 1.5 mmol) in toluene (10 ml) was refuxed for 3 h.
Subsequently, the resulting precipitate was recrystallized from
ethanol to provide compound 2. Yield 45%, m.p. 120–122 °C.
IR (KBr, v, cm−1): 3172–3150 (2NH), 1692–1662 (2C=O)
1
cm−1. H NMR (DMSO-d6, 300 MHz): δ = 7.22 (s, 1H,
CH=C), 6.81–8.02 (m, 8H, Ar–H), 8.57 (s, 1H, 2H indole),
10.65 (s, 1H, NH), 11.62 (s, 1H, NH); elemental analysis calcd
(%) for C18H14N2O3 (306.1): C, 70.58; H, 4.61; N, 9.15; found:
C, 70.50; H, 4.51; N, 9.08%.
Experimental
The newly synthesized compounds melting points were
determined on a melting point apparatus (Gallen Kamp
electric) and were kept uncorrected. ATR-Alpha spectro-
photometer was used to record IR spectra, using potassium
Ethyl 5‑(1H‑indol‑3‑yl)‑1‑phenyl‑1H‑pyrazole‑3‑car‑
boxylate
bromide (KBr) pellets in the range of [400–4000] cm−1
.
1H and 13C NMR spectra were recorded using a Bruker
A mixture of compound 1 (0.26 g, 1 mmol) and phenylhy-
drazine (0.11 g, 1 mmol) in (20 ml) ethanol was refuxed
for 8 h. The mixture was poured into iced water to cool. The
obtained solid was recrystallized from toluene. Yield 75%,
m.p. 250–252 °C. IR (KBr, v, cm−1): 3276 (NH), 1739 (C=O),
AC-300 Hz instrument. Chemical shifts for H and 13C
1
NMR spectra were expressed in δ (ppm), relative to the
tetramethylsilane (TMS) as an internal standard; however,
DMSO-d6 [(CH3)2S = O)] was used as a solvent. Elemen-
tal analyses were determined at Micro-Analytical Center,
Cairo University. Cytotoxic activity test (in vitro bioassay on
human tumor cell lines) was conducted and determined by
Bioassay-Cell Culture Laboratory, National Research Centre
(NRC), Dokki, Giza, Egypt.
1
and 1620 (C=N) cm−1. H NMR (DMSO-d6, 300 MHz):
δ=1.30 (t, 3H, J=7.6 Hz, CH3), 4.40 (q, 2H, J=7.5 Hz, CH2),
7.11 (s, 1H, pyrazole-H), 7.18–8.02 (m, 9 H, Ar–H), 8.54 (s,
1H, 2H indole), 10.60 (s, 1H, NH). 13C NMR (DMSO-d6,
75 MHz): δ=14.25, 56.82, 62.4, 111.6, 112.6, 114.6, 118.3,
119.7, 122.1, 126.3, 127.5, 128.4, 139.7, 139.8, 144.6, 151.5,
153.1, 160.2; elemental analysis calcd (%) for C20H17N3O2
(331.37): C, 72.49; H, 5.17; N, 12.68; found: C, 72.40; H,
5.15; N, 12.59.
Ethyl 2‑hydroxy‑4‑(1H‑indol‑3‑yl)‑4‑ox‑
obut‑2‑enoate
30 ml of diethyl oxalate (2.92 g, 0.02 mol) was added to a
mixture of 3-acetylindole (3.2 g, 0.02 mol) and sodium eth-
oxide (0.46 g, 0.02 mol) in ethanol. The mixture was heated
under refux for 3 h. The solvent was removed under vacuum
after cooling; then, the residue was taken up in water (100 ml)
and acidifed using concentrated HCl (1 ml). Aqueous mixture
was extracted using diethyl ether (3×50 ml). The combined
extracts were washed and dried. In order to provide com-
pound 1, the obtained solid was recrystallized from ethanol.
Yield 75%, m.p. 217–218 °C. IR (KBr, v, cm−1): 3443 (OH),
3172 (NH), 3083 (CH arom.), 2939, 2836 (CH aliph.), and
1751–1685 (2C=O) cm−1. 1H NMR (DMSO-d6, 300 MHz):
δ=1.29 (t, 3H, J=7.6 Hz CH3), 4.20 (q, 2H, J=7.6 Hz, CH2),
7.53 (s, 1H, olefnic (CH=C), 7.12–7.76 (m, 4H, Ar–H), 8.54
3‑(1H‑indol‑3‑yl)‑2‑phenyl‑2H‑pyridazino[3,4‑b][1,
4]benzoxazine
A mixture of compound 2 (0.31 g, 1 mmol) and phenylhy-
drazine (0.11 g, 1 mmol) in ethanol (30 ml) was refuxed
for 8 h. The mixture was cooled by pouring into iced water.
The obtained solid was recrystallized from ethanol. Yield
38%, m.p. 278–280 °C. IR (KBr, v, cm−1): 3380 (NH), 3100
1
(CH arom.), and 1618 (C=N) cm−1. H NMR (DMSO-d6,
300 MHz): δ=5.5 (s, 1H, pyridazine), 7.06–8.18 (m, 14 H,
Ar–H), 8.50 (s, 1H, 2H indole), 10.64 (s, 1H, NH); elemental
analysis calcd (%) for C24H16N4O (376.41): C, 76.58; H, 4.28;
N, 14.88; found: C, 76.49; H, 4.24; N, 14.86.
(s, 1H, 2H indole), 11.49 (s, 1H, NH), 11.91 (s, 1H, OH). 13
C
NMR (DMSO-d6, 75 MHz): δ=14.56 (CH3), 61.47 (CH2),
104.33, 111.1, 113.9, 119.01, 122.8, 126.1, 130.9, 149.3,
154.1, 162.4, 167.2, and 189.7; elemental analysis calcd (%)
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Journal of the Iranian Chemical Society
3‑Hydroxy‑1‑[5‑(1H‑indol‑3‑yl)‑1‑phenyl‑1H‑pyra‑
zol‑3‑yl]‑3‑pyridin‑3‑ylprop‑2‑en‑1‑one
General procedure for synthesis of pyrimidines 8, 9
A diketone 5 (0.82 g, 2 mmol) was added to sodium ethoxide
solution prepared from sodium metal (0.046 g, 2 mmol) and
25 ml of absolute ethanol; then, urea or thiourea [SC(NH2)2
(2 mmol)] was added. The reaction mixture was refuxed
for 16 h and left to cool. The solution was poured into
crushed ice and neutralized with diluted hydrochloric acid
(HCl). The precipitated product was collected by fltration,
washed with ethanol, and dried. Crystallization from ethanol
aforded the pyrimidine derivatives 8, 9, respectively.
10 ml of toluene was added to a suspension of sodium
(0.34 g, 1.5 mmol) and to pyrazole-3-carboxylate 3
(3.97 g, 1.2 mmol), and 20 ml of toluene was slowly
added; then, 3-acetylpyridine (1.45 g, 1.2 mmol) was
added at 0 °C in 10 ml of toluene. The attained mixture
was stirred at room temperature for 2 days. Formed pre-
cipitate was fltered, washed with toluene, and dissolved
in water; afterward, it was neutralized using acetic acid.
After extraction with CH2Cl2, the organic layer was dried
and then concentrated under vacuum. The gotten residue
was fltered and recrystallized from methanol. Yield 35%,
m.p. 132–134 °C. IR (KBr, v, cm-1): 3415 (OH), 3386
(NH), 1640 (C=O) 1618 (N=C) cm−1. 1H NMR (DMSO-
d6, 300 MHz): δ = 6.67 (s, 1H, pyrazole-H), 6.98 (s, 1H,
olefnic CH), 7.00–8.63 (m, 14 H, Ar–H), 11.60 (s, 1H,
NH), 15.32 (s, 1H, OH); elemental analysis calcd (%) for
C25H18N4O2 (406.44): C, 73.88; H, 4.46; N, 13.78; found:
C, 73.81; H, 4.43; N, 13.69.
4‑[5‑(1H‑indol‑3‑yl)‑1‑phenyl‑1H‑pyra‑
zol‑3‑yl]‑6‑pyridin‑3‑ylpyrimidine‑2(1H)‑one
Yield 68%, m.p. 218–220 °C. IR (KBr, v, cm−1): 3238, 3170
(2NH), 1669 (C=O), 1633 (C=N) cm−1; 1H NMR (DMSO-
d6, 300 MHz): δ = 5.46 [s, 1H, pyrimidine C(5)], 6.63 (s,
1H, CH pyrazole), 7.00–8.63 (m, 14 H, Ar–H), 9.94 (s, 1H,
pyrimidine NH), 12.07 (s, 1H, indole NH) ppm. Anal. Calcd
for C26H18N6O (430.46): C, 72.55; H, 4.21; N, 19.52; found:
C, 72.54; H, 4.13; N, 19.44%.
3‑[1‑Phenyl‑3‑(3‑pyridin‑3‑ylisoxazol‑5‑yl)‑1H‑pyra‑
zol‑5‑yl]‑1H‑indole
4‑[5‑(1H‑indol‑3‑yl)‑1‑phenyl‑1H‑pyra‑
zol‑3‑yl]‑6‑pyridin‑3‑ylpyrimidine‑2(1H)‑thione
A mixture of diketone 5 (0.41 g, 1 mmol), hydroxylamine
hydrochloride (0.07 g, 1 mmol), and anhydrous potassium
carbonate (0.14 g, 1 mmol), in 20 ml ethanol, was refuxed
for 8 h. The mixture was poured into cold water, and the
solid product was fltered of, washed with water, dried, and
crystallized from ethanol to aford isoxazole derivative 6.
Yield 64%, m.p. 140–142 °C. IR (KBr, v, cm-1): 3386 (NH),
1620 (C=N) cm−1; 1H NMR (DMSO-d6, 300 MHz): δ=6.32
(s, 1H, CH pyrazole), 7.00–8.63 (m, 15 H, Ar–H), 11.61 (s,
1H, NH). Anal. Calcd for C25H17N5O (403.44): C, 74.43; H,
4.25; N, 17.36; found: C, 74.35; H, 4.20; N, 17.29%.
Yield 64%, m.p. 214–216 °C. IR (KBr, v, cm−1): 3238–3170
(2NH), 1623 (C=N),1256 (C=S) cm−1; 1H NMR (DMSO-
d6, 300 MHz): δ = 5.60 [s, 1H, pyrimidine C(5)], 6.60 (s,
1H, CH pyrazole), 7.00–8.63 (m, 14 H, Ar–H), 9.90 (s,
1H, pyrimidine NH), 11.27 (s, 1H, indole NH) ppm. Anal.
Calcd for C26H18N6S (446.53): C, 69.94; H, 4.06; N, 18.82;
S, 7.18; found: C, 69.90; H, 4.05; N, 18.71; S, 7.10%.
Antimicrobial activity
A modifed Kirby‐Bauer disc difusion method was used
to determine antimicrobial activity of some prepared com-
pounds [21]. 100 μl of the test bacteria/fungi was grown
in a 10 ml of fresh media, until the cell concentration was
standardized to approximately 1×108 (cells/ml) for bacte-
ria and 1×105 (cells/ml) for fungi [22]. The antimicrobial
resistance intimidates the activities of science and medicine,
as it deactivates the conventional antimicrobial therapeutics
[23]. The microbial suspension of 100 μl was spread onto the
agar plates, corresponding to the broth in which they were
maintained. Isolated colonies of each organism that might
be playing a pathogenic role were selected from primary
agar plates and tested for susceptibility by disc difusion
method [24–28].
5‑(1H‑indol‑3‑yl)‑1‑phenyl‑5′‑pyridin‑3‑yl‑1H, 2′H‑3,
3′‑bipyrazole
Hydrazine hydrate (0.01 g, 2 mmol) has been added to a
solution of diketone 5 (0.82 g, 2 mmol) in 30 ml of etha-
nol. The mixture was refuxed for 6 h and left to cool; the
formed solid product was fltered of, dried, and then crystal-
lized from ethanol to provide compound 7. Yield 62%, m.p.
246–248 °C. IR (KBr, v, cm−1): 3281–3221 (2NH), 1619
(C=N) cm-1; 1H NMR (DMSO-d6, 300 MHz): δ=6.32 (s,
1H, CH pyrazole), 6.63 (s, 1H, CH pyrazole). 7.00–8.63
(m, 14 H, Ar–H), 11.61 (s, 1H, NH), 13.70 (s, 1H, pyrazole
NH). Anal. Calcd for C25H18N6 (402.45): C, 74.61; H, 4.51;
N, 20.88; found: C, 74.53; H, 4.48; N, 20.86%.
Powder samples coded 1, 2, and 3 were dissolved in dime-
thyl sulfoxide [DMSO, (CH3)2SO] to reach a fnal concentra-
tion of 10 (mg/ml). On the other hand, liquid samples coded
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Journal of the Iranian Chemical Society
HO
O
4 and 5 were dissolved in methanol (CH3OH). 50 ml of the
bioassay medium (nutrient agar) seeded with 100 μl of cell
suspension contained [1.5×108 CFU/ml; 0.5 mac fr land]
of the 24-hour-old test organisms. 5 ml well diameter was
cut into the agar medium using a sterilized cork borer, and
then 50 μl of the samples was poured into the wells, while
flter discs were cut for the samples coded 4 and 5; however,
flter discs (doublet) were saturated with 20 and 50 μl of
each sample separately and then placed onto the surface of
each seeded plate with the tested organisms. It was then left
in the refrigerator for 1 h, followed by incubation at 37 °C
for 24 h. Subsequently, the diameter of inhibition zones was
measured in mm.
O
O
O
O
O
COCH3
EtONa
OEt
OC2H5
OC2H5
+
EtO
N
H
N
H
1
N
H
O
1a
Scheme 1 Synthesis of Enolic Keto Ester 1
O
NH2
HN
OC2H5
OH
HO
O
O
N
Ar
O
O
NH2
N
O
OC2H5
Ar
N
H
N
H
1
2
3
NH2
HN
N
Ar
Ph
Anticancer activity
N
Ar =
O
N
N
Cell culture
4
H
Cells were obtained from Bioassay-Cell Culture Laboratory,
National Research Centre, Dokki, Giza 12622, Egypt. Cells
were grown in RPMI-1640 medium [Roswell Park Memorial
Institute-1640 medium]. The samples were improved using
10% heat, inactivated fetal bovine serum (FBS), 50 units/ml
of penicillin, and 50 mg/ml of streptomycin. It was preserved
in a humidifed atmosphere encompassing 5% CO2. The cells
were sustained as monolayer culture by serial subculturing.
Cell culture reagents were obtained from Lonza (Basel,
Switzerland). The anticancer activity of tested compounds
was evaluated against MCF-7 cells (breast cancer), HCT116
[colon cell line], and RPE1 [normal retina cell line] [27].
The half maximal inhibitory concentration (IC50) values
were obtained by the analysis of obtained data using the
IBM SPSS software [28].
Scheme 2 Synthesis of Benzoxain and Pyrazole derivatives
5-(1H-indol-3-yl)-1-phenyl-1H-pyrazole-3-carboxylate 3.
Spectral data of compound 2 were confrmed by IR and 1H
NMR. The IR data revealed the disappearance of the OH
group band, accompanied with the appearance of new band
in the range of 3172–3150 (cm−1) due to the benzoxazinone
NH. These outcomes were confrmed by the data given
from the 1H NMR that elucidated the fading of triplet and
quartet of ethyl group (C2H5) accompanied with the pres-
ence of the amine (NH) signal for benzoxazinone NH at
10.65 ppm. IR spectra of compound 3 showed the existence
of bands at 1739 cm−1, targeting the C=O and at 1620 cm−1
for C=N; In contrast, no band of OH group was identifed.
1H NMR spectra of similar compound revealed the presence
of triplet at 1.30 ppm, assigned to CH3 ester and a quartet
at 4.40 ppm owing to the CH2 ester. The heterocyclization
of benzoxazinone 2 using phenyl hydrazine as bidentate
nucleophile ensued 2-phenylpyridazino[3,4-b] benzoxazine
4 [1, 4]. The structure of compound 4 was confrmed via
IR spectra, which exhibited the disappearance of the C=O
absorption bands, and in 1H NMR, the proton at position 4 in
dihydropyridazine ring appeared in olefnic region 5.5 ppm
(Scheme 2).
Results and discussion
Chemistry
Claisen reaction between 3-acetylindole and diethyl oxalate
furnished β-diketones 1 that mainly presents thermodynamic
enol form (stabilized by conjugation addition to intramo-
lecular hydrogen bond to enol). Spectral data of compound
1 were confrmed by elemental analyses, IR, 1H NMR, and
13C NMR. The IR spectra showed the existence of charac-
teristic bands for OH at 3443 (cm−1) and 2C=O in the range
of 1751–1685 (cm−1) (Scheme 1).
Base-mediated intermolecular condensation of
3-acetylpyridine and ethyl heterocyclic-3-carboxylate 3
produced the stabilized enolic form 3-hydroxy-1-[5-(1H-
indol-3-yl)-1-phenyl-1H-pyrazol-3-yl]-3-pyridin-3-ylprop-
2-en-1-one 5. Interconversion of β-keto-enol was determined
using 1H NMR integration of signals from the enol=C–H
and the ketone CH2. Certainly, the parent β-diketones occur
entirely in the enol form, and only a trace of the keto form
was seen about 4 ppm. Synthesis started with the formation
of a ketone enolate nucleophile in cool conditions at zero
Cyclization of O-aminophenol with 2-hydroxy-4-(1H-
indol-3-yl)-4-oxobut-2-enoic acid 1 gave the benzox-
azinone 2. Compound 1 underwent pyrazole cyclization
by nucleophilic attack of phenyl hydrazine directed to the
electrophilic center of compound 1. This process was fol-
lowed by intramolecular cyclodehydration to give ethyl
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Journal of the Iranian Chemical Society
celsius after the addition of proper heterocyclic carboxy-
late. Obtained mixture was continuously stirred for 2 days at
room temperature. The formed enolate initially experienced
nucleophilic attack at the ester carbonyl to yield tetrahedral
intermediate. In order to produce a stabilized enolate ion
product (C) as a precipitated salt, the expulsion of ethoxide
ion from the unstable tetrahedral intermediate of the initial
Claisen yielded a β-diketone. Acidic alpha proton from the
β-diketone was consequently removed. The precipitate was
fltered, washed with toluene, dissolved in water, and then
neutralized using acetic acid (CH3COOH) to give the title
products with an acceptable yield (Schemes 3 and 4).
The reactivity of diketone 5 toward nitrogen nucleophiles
is a suitable functionality for further heterocyclization.
Therefore, compound 5 reaction with hydroxylamine hydro-
chloride in refuxing ethanol furnished isoxazole derivative
6. Meanwhile, the reaction of 5 with hydrazine hydrate gave
rise to the pyrazole derivatives 7 in an upright yield. IR
spectra of compounds 6 and 7 revealed the disappearance
of the two carbonyl groups with the appearance of the NH
band in case of compound 7 within the range of 3281–3221
(cm−1), while 1H NMR spectra was recorded for isoxazole
ring 6 signal at aromatic region with the appearance of a new
NH signal due to pyrazole ring of compound 7 at 13.70 ppm.
An essential principle for the synthesis of pyrimidines is
the reaction of 1, 3-dicarbonyl compound with urea and/or
thiour [20]. Consequently, pyrimidine cyclization of dicar-
bonyl compound 5 was accomplished by the reaction with
urea and/or thiourea in the presence of sodium ethoxide
(C2H5ONa) that provided the pyrimidine derivatives 8, 9,
respectively (Scheme 5). The structure of compound 8 was
confrmed by IR. It revealed bands at 3238, 3170 cm−1 for
the 2NH groups and a band at 1669 cm−1 typical for the
C=O, respectively. 1H NMR displayed two singlets at δ 9.94
for pyrimidine NH and δ 12.07 for indole NH by ppm. On
the other hand, the IR of pyrimidine derivatives 9 exhibited
a band at 1256 cm−1 owing to C=S while 1H NMR revealed
a signal of pyrimidine NH at δ 9.90.
Scheme 4 A plausible mechanistc pathway to explain the formation
of Diketone 5
of strains of bacteria and fungi. This approach used gram-
positive strains (Staphylococcus aureus and Streptococcus
pyogenes) and gram-negative strains (Salmonella enterica
and Klebsiella pneumonia). Besides, Candida albicans was
used as dimorphic fungal strain. Results of antimicrobial
activity are listed in Table 1.
All of the tested compounds showed no antimicrobial
efect on S. pyogenes. Low antimicrobial activity was exhib-
ited on the S. enterica and K. pneumonia with inhibition
zone diameter (IZD) in the range of (11–23 mm). On the
other hand, the unassertive efect on C. albicans verifed an
inhibition zone diameter range (6–34 mm). Efcient antimi-
crobial activity presented gram-positive bacteria (S. aureus)
with inhibition zone of a diameter ranging from (12 to
60 mm). Accordingly, it could be concluded that compound
2 possessed good antibacterial activity as well as antifungal
activity in comparison with other compounds (Figs. 1, 2).
Thus, compound 2 can be used as a comprehensive spectrum
antibacterial agent.
Anticancer activity test
Anticancer activity test was held against human breast carci-
noma cell line (MCF7), HCT116 [colon cell line], and RPE1
[normal retina cell line], using positive control [adriamycin
(doxorubicin)]. Compound 2 being the most potent com-
pound concerning the antibacterial and antifungal activity
has been chosen in parallel with compounds 1 and 5.
Screening of antimicrobial activity
Five synthesized compounds were assessed by screening the
in vitro growth inhibitory activity contrary to the variety
O
O
NH
N
Ph
O
N
N
N
N
N
Ph
N
N
Ar
N
HONH2.HCl
K2CO3
NH2NH2
Ar
N
N
O
OH
O
O
N
Ar
Ph
O
N
O
N
6
5
7
O
O
N
Na/Toluene
Stirring r.t
S
N
+
H2N NH2
N
Ar
O
N
H2N NH2
Ar
Ar
N
N
S
Ph
N
NH
Ph
Ph
Diketone
N
NH
3
5
(Keto-enol)
Ar
N
Ar
N
Ar =
N
Ar =
N
N
Ph
N
N
N
H
Ph
H
8
9
Scheme 5 Synthesis of isoxazole, pyrazole and pyrimidine deriva-
Scheme 3 Formation of Diketone 5
tives
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Journal of the Iranian Chemical Society
Table 1 Outcomes of
Sample code
Salmonella
enterica
Klebsiella pneu- Streptococcus
Staphylococcus Candida
antimicrobial activity
moniae
pyogenes
aureus
albicans
Compound 1
Compound 2
Compound 3
Compound 4
20 µL
Nil
23
Nil
11
Nil
Nil
Nil
Nil
Nil
12
60
16
Nil
34
Nil
Nil
Nil
Nil
Nil
Nil
Nil
19
26
6
12
50 µL
Compound 5
20 µL
50 µL
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
12
17
Nil
Nil
Nil
Nil
DMSO (blank)
Table 2 In vitro cytotoxic activity IC50 at 100 ppm
Sample code
MCF7
HCT116
RPE1
IC50 (µg/ml) (%)
IC50 (µg/ IC50 (µg/ml) (%)
ml) (%)
1
2
5
59.4
100
11.2
3
5.6
100
2.8
1
83.3
100
27.5
5
DMSO
Negative control
0
0
0
Sample concentration ranged between 100 and 0.78 (µg/
ml), using a colorimetric assay for assessing cell metabolic
activity (MTT assays). IC50 is the half maximal inhibitory
concentration as compared with the control structures in
the absence of an inhibitor (Table 2).
Fig. 1 Antibacterial activity
Conclusion
This study presented a comprehensive assessment to
provide the description of Claisen reaction taking place
between 3-acetylindole and diethyl oxalate. Our study
highlights the efect of 1, 3-dicarbonyl compound deriva-
tives, linked to the indole moiety. It has been delineated
that these compounds, particularly compound 2, have a
promising infuence on antitumor and anticancer impact.
Acknowledgements The authors are very thankful to all the associ-
ated personnel in any reference that contributed in/for the purpose of
this research.
Fig. 2 Antifungal activity
1 3

Journal of the Iranian Chemical Society
14. S.M. El-Moghazy, F.F. Barsoum, H.M. Abdel-Rahman, A.A. Mar-
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311–340 (2014)
Compliance with ethical standards
Conflict of interest All the authors declare that they have no confict
of interest.
16. D. Rafa, B. Maggio, M.V. Raimondi, S. Cascioferro, F. Plescia,
G. Cancemi, G. Daidone, Recent advanced in bioactive systems
containing pyrazole fused with a fve membered heterocycle. Eur.
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