Synthesis of 6-oxopyrimidin-1(6H)-yl benzamide derivatives and evaluation of their antibacterial and cytotoxic activity

Article abstract of DOI:10.1080/00397911.2015.1135349

A series of novel 2-alkylamino and 2, 4-dialkyl amino 6-oxopyrimidin-1(6H)-yl) benzamide derivatives were prepared in good yields from a base-catalyzed ring opening of oxadiazolo[3,2-a]pyrimidin-5-one and evaluated for their antibacterial and cytotoxicity. Most of the compounds exhibited antibacterial activity. In particular, compounds 5b and 5k exhibited considerable antibiotic activity against Klebsiella pneumonia and Bacillus cereus. In addition, compounds 5g and 5i also inhibited the growth of two human tumor cell lines (A549 and H460) at micromolar concentrations.

Full text of DOI:10.1080/00397911.2015.1135349

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
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ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Synthesis of 6-Oxopyrimidin-1(6H)-yl Benzamide
Derivatives and Evaluation of Their Antibacterial
and Cytotoxic Activity
Kiran Devarasetty, Giri Tharikoppula, Tailor Sridhar, Laxminarayana
Eppakayala, Mahesh Kyasani, Premkumar Arumugam & Srinivas Pusuluri
To cite this article: Kiran Devarasetty, Giri Tharikoppula, Tailor Sridhar, Laxminarayana
Eppakayala, Mahesh Kyasani, Premkumar Arumugam & Srinivas Pusuluri (2015): Synthesis
of 6-Oxopyrimidin-1(6H)-yl Benzamide Derivatives and Evaluation of Their Antibacterial and
Cytotoxic Activity, Synthetic Communications, DOI: 10.1080/00397911.2015.1135349
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Date: 02 January 2016, At: 03:41

Synthesis of 6-oxopyrimidin-1(6H)-yl benzamide derivatives and evaluation of their
antibacterial and cytotoxic activity
Kiran Devarasetty1,2, Giri Tharikoppula1,2, Tailor Sridhar1, Laxminarayana Eppakayala3,
Mahesh Kyasani1, Premkumar Arumugam1, Srinivas Pusuluri1
1Chemistry Services Unit, GVK Biosciences Pvt. Ltd, Uppal, Hyderabad, AP, India,
2Chemistry Division, Institute of Science and Technology, JNT University, Hyderabad,
India, 3Department of Chemistry, Sreenidhi Institute of Science and Technology
(Autonomous), Ghatkesar, Hyderabad, Andhra Pradesh, India
Address Correspondence to Srinivas Pusuluri: Chemistry Services Unit, GVK
Biosciences Pvt. Ltd, Plot No. 5C, IDA, Uppal, Hyderabad 500039, AP, India.
E-mail: pusulurisrinivas@gvkbio.com
Supplemental data for this article can be accessed on the publisher’s website
Abstract
A series of novel 2-alkylamino and 2, 4-dialkyl amino 6-oxopyrimidin-1(6H)-yl)
benzamide derivatives were prepared in good yields from a base catalysed ring opening
of oxadiazolo [3, 2-a] pyrimidin-5-one and evaluated for their antibacterial and
cytotoxicity. Most of the compounds exhibited antibacterial activity. In particular,
compounds 5b and 5k exhibited considerable antibiotic activity against Klebsiella
pneumonia and Bacillus cereus. In addition, compounds 5g and 5i also inhibited the
growth of two human tumor cell lines (A549 and H460) at micromolar concentrations.
1

KEYWORDS: Pyrimidones, oxadiazolo [3, 2-a] pyrimidin-5-ol, oxadiazolo[3,2-
a]pyrimidin-5-one, Fused Oxadiazole, Cytotoxic agents, Cancer Cell Lines.
INTRODUCTION
Pyrimidine is a prominent pharmacophore prevailing in many heterocyclic natural
products1. Pyrimidines and their derivatives have a long and distinguished history
extending from the days of their discovery as important constituents of nucleic acids (like
Uracil, Cytocine and Thymine... etc.) to their current use in the chemotherapy of AIDS.
During the last two decades, several pyrimidine derivatives were found to have wide-
spread clinical applications including chemotherapy. There are a large number of
pyrimidine based antimetabolites which act as antagonists of endogenous substrates. The
structural modification may be either on the pyrimidine ring or on the pendant sugar
group. Lamivudine2 is one of the examples which acts as an effective anti-AIDS drug
when used in combination with Zidovudine. (Figure 1)
Likewise, there are several pyrimidone-based marketed drugs such as
Uramustine,3 Tegafur,4 Fluorouracil5 (Antineoplastic), Flucytosine6 (Antifungal) and
Idoxuridine7 (Antiviral) (Figure 2). In addition, analgesic,8 anticancer,9 antioxidant,10
antibacterial,11 antiviral,12 anti-inflammatory,13 antiplatelet,14 antiproliferative15 and
antitumor16 activities were reported for pyrimidine-based derivatives.
In the course of our research devoted to the development of new class of pyrimidine and
condensed pyrimidine moieties, we have focused on the synthesis and biological
2

evaluation of several 6-oxopyrimidin-1(6H)-yl benzamide derivatives, which may show
their promising antibacterial and cytotoxic activities. Especially, condensed pyrimidine17
derivatives possessing anti-inflammatory and analgesic activities are well documented in
the literature.18 This background of pyrimidine based derivatives motivated us to
synthesize the titled derivatives for their biological evaluation.
There is an extensive work reported on nucleophilic displacement followed by
rearrangement of electron-poor five-membered systems such as 1,3,4-oxadiazoles,19
1,3,4-thiadiazoles,20 nitroimidazoles,19, 21 bis(1,3,4-thiadiazol-2-yl)-1,3,5-triazinium
halides,22 isothiazole,23 and isoxazoles.24 Pace et al.,25 studied the ANRORC
rearrangements of 1,2,4-oxadiazoles as a valid approach for the obtaining heterocycles
such as 1,2,4-triazoles,25 1,2,4-oxadiazoles,26 1,2,4-triazines,25b,27 1,2,4-oxadiazinones,28
indazoles29 and Amino-1,2,4-triazoles.25 Taking advantage of the literature precedence,
we intended to derive 2-alkylamino 6-oxopyrimidin-1(6H)-yl) benzamide derivatives
(6a-k) by nucleophilic displacement on oxadiazolo[3,2-a]pyrimidin-5-one (2).
In this paper, we report the synthesis of a new class of 2-alkylamino and 2, 4-dialkyl
amino 6-oxopyrimidin-1(6H)-yl benzamide derivatives by using base mediated ring
opening of oxadiazolo [3, 2-a] pyrimidin-5-one using DMF as solvent. The title
compounds were subjected to in vitro antibacterial, and cytotoxicity studies to examine
the relationship between structural modifications and biological activity. Compounds 5a-
k and 6a-k showed significant anti-bacterial activity in comparison to streptomycin, a
first line antibiotic drug. In addition, compounds 5g, 5i and 5k showed significant
3

cytotoxicity activity in comparison to puromycin, an amino-nucleoside antibiotic derived
from the Streptomyces alboniger bacterium.
RESULTS AND DISCUSSION
Chemistry
In this study previously reported oxadiazole,30 was converted into new 2-alkylamino and
2, 4-dialkyl amino 6-oxopyrimidin-1(6H)-yl) benzamide derivatives (5a-k and 6a-k). The
synthesis of oxadiazole (1) was achieved by reaction of ethyl 4-bromobenzoate with
hydrazine hydrate in ethanol to give hydrazide, which upon subsequent reaction with
cyanogen bromide in ethanol furnished oxadiazole (1) in 72% yield (over two steps). For
conversion of oxadiazole (1) into pyrimidin-5-one (2), the required bis(1,3,5-trichloro
phenyl) malonate reagent was prepared in 85% yield using a literature protocol by
treating malonic acid with 2,4,6-trichlorophenol in phosphorous oxychloride. Oxadiazole
(1) on reaction with bis (1,3,5-trichloro phenyl) malonate in chlorobenzene under reflux
provided 2-(4-bromophenyl)-7-hydroxy-5H-[1,3,4]oxadiazolo[3,2-a]pyrimidin-5-one (2)
in 92% yield.31 The pyrimidin-5-one 2 was subjected to chlorination using phosphorous
oxychloride to obtain chloro derivative 3 in 94% yield, which served as a synthetic
precursor for the preparation of titled derivatives.
We wanted to evaluate the feasibility of ring opening of chloro derivative 3 by a reactive
secondary amine like morpholine by screening different bases (triethyl amine, DIPEA,
and DBU) in combination with different solvent systems at different temperature
conditions. In ethanolic solvent system, room temperature reaction with TEA (Table 1,
4

entry 1) and reflux condition with DIPEA (entry 2) gave poor yields (entry 1-2). DCM
solvent system with DBU provided moderate yield (entry 3), whereas in DMF solvent
system, room temperature reaction with K2CO3 (entry 4) gave good yields in a relatively
shorter time (2 h).
Based on optimized conditions (Table 1, entry 4) for ring opening reaction of chloro
derivative (3) with morpholine (4a), similar reaction was attempted with different amines
(4b-k) (Table 2).
In addition, ring opening of chloro derivative (3) was attempted with excess amines (4a-
k) in DMSO at 90 oC for 16 h to get 2, 4-dialkyl amino 6-oxopyrimidin-1(6H)-yl)
benzamide derivatives (6a-k) (Table 3).
BIOLOGICAL ACTIVITY
Antibacterial Activity
The antibacterial activity of compounds 5a-i and 6a-i were assessed and compared
against Streptomycin using agar well diffusion method.32 The antibacterial activity was
assayed by measuring the diameter of the inhibition zone. About 1 mg ml-1 of the test
compounds (5a-k & 6a-k) and 1 mg ml-1 of Streptomycin (control) were dispensed into
the wells. The plates were incubated at 37 °C for 24 h. The sensitivity of the test
organisms to the compounds (5a-k & 6a-k) was determined by measuring the diameters
of the zone of inhibition surrounding the wells. The diameters of the zones of inhibition
were measured with a ruler and indicated in Table – 4.
5

Cytotoxicity Assay
The synthesized compounds were tested for cytotoxicity by measuring their effect on the
percentage viability of two different cell lines, adenocarcinomic human alveolar basal
epithelial cells (A549) and human lung cancer cells (H460) by applying the XTT
reagent.33 Compounds were tested over a range of concentrations from 1mM to 0.051
μM, and the calculated IC50 values i.e. the concentration (μM) of compounds able to
cause 50% of cell death with respect to the control culture, are reported in Table 5.
Result shows that few compounds inhibited the growth of human cancer cell lines with
IC50 values in the micromolar range. Compound 5g with longer and bulky side chain
showed significant activity (IC50 0.4µM) against A549 cell line, lipophilic group
containing 5i and 5k showed moderate inhibition against A549 cell line, while 5k, 5g &
5i showed moderate activity in H460 cell line compared to puromycin. Compounds with
apolar and shorter side chains were less active (IC50 about 10 fold higher).
From the Cytotoxic Activity results of 2-alkylamino 6-oxopyrimidin-1(6H)-yl-
benzamide derivatives (5) on Human Lung carcinoma Cell Sublines, it was observed that
the better results were shown by 5g and 5i. Thus the compounds 5g and 5i were assayed
for cytotoxic activity against non tumoral mammalian cell lines MDCK and Vero cells.
Compound 5g has shown IC50 value of 29.4 µM on MDCK and 38.3 µM on Vero cells
when compared with standard Puromycin having IC50 value of 0.29 µM for MDCK and
0.57 µM for Vero cells, whereas 5i did not show any cytotoxicity on both the tested cell
lines. The results were analyzed using the Graph Pad Prism4 program (Figure 1).
6

The A549 and H460 cell lines were maintained in Ham’s F12k and RPMI Medium
supplemented with 10% FBS and 1% antibiotics (100 U/mL penicillin, 100 µg/mL
streptomycin) respectively. The cells were grown at 37 °C in a humidified incubator with
5% CO2. Cells were sub-cultured by treating with cell dissociation buffer. Both cell lines
were seeded in a 96-well microtitre plate at a concentration of 7500 cells per well. Cells
were allowed to attach for 24 h at 37 °C, 5% CO2. The cells were exposed to the test
compounds and positive drug control, Puromycin (SIGMA). The microtitre plate was
incubated for further 72 h and thereafter cytotoxicity was measured using the XTT
(Invitrogen) reagent (1mg/ml XTT and 7.7µg/ml Phenazine methosulfate in PBS). The
plates were incubated for 2 to 4 h and absorbance was read at 450 nm using a Spectra
Max Multi-Mode Microplate Reader (Molecular Devices). The results were analysed
using the Graph Pad Prism4 program.
Experimental Section:
General experimental details: Analytical grade solvents and commercially available
reagents were used without further purification. The column chromatography was carried
over silica gel (60-120 mesh), purchased from Sisco Research Laboratories Pvt Ltd.
Melting points were determined in open capillaries in electrical melting point apparatus
and are uncorrected. 1H NMR and 13CNMR spectra were recorded on 400 MHz Varian
spectrometer in DMSO-d6 or CDCl3 using tetramethylsilane (TMS) as an internal
standard. Chemical shifts are given in δ relative to TMS, the coupling constants are given
in Hz. IR spectra in KBr disk were recorded from 4000 to 400 cm-1 on Avatar 330 FT-IR
spectrometer equipped with DTGS detector. Mass spectra were recorded using Agilent
7

1100 MSD spectrometer in electro spray mode. The starting compound 1 was prepared
by previously reported direct cyclization of 4-bromobenzohydrazide and Cyanogen
bromide
General procedure for the preparation of 5- substituted 2-(4-bromophenyl)-7-
chloro-5H-[1,3,4]oxadiazolo[3,2-a] pyrimidin-5-ol (5a–i). The mixture of 2-(4-
bromophenyl)-7-chloro-5H-[1,3,4]oxadiazolo[3,2-a]pyrimidin-5-one 3 (0.5 g, 1.53
mmol), potassium carbonate (0.63 g, 4.59 mmol) and the appropriate amine 4 (1.53
mmol) in dimethylformamide (5 mL) was stirred at room temperature for 2-16 h. The
completion of the reaction was indicated by TLC and the solvent was evaporated and
residue was poured into water, precipitate formed was filtered off, washed with water and
then purified by flash column chromatography using hexane and ethyl acetate to give the
corresponding oxadiazolo[3,2-a]pyrimidin-5-ol derivative (5).
General procedure for the preparation of 5,7-disubstituted 2-(4-bromophenyl)-5H-
[1,3,4]oxadiazolo[3,2-a]pyrimidin-5-ol (6a–k). The mixture of 2-(4-bromophenyl)-7-
chloro-5H-[1,3,4]oxadiazolo[3,2-a]pyrimidin-5-one 3 (0.5 g, 1.53 mmol) and the
appropriate Amine (4) (7.65 mmol) in dimethylsulfoxide (5 mL) was stirred at 90 °C for
16 h. The completion of the reaction was indicated by TLC and the reaction mixture was
poured into water, precipitate formed was filtered off, washed with water and then
purified by flash column chromatography using Hexane and Ethyl acetate to give the
corresponding oxadiazolo[3,2-a]pyrimidin-5-ol derivative (6).
8

CONCLUSION
In conclusion, we have synthesized the 2-alkylamino 6-oxopyrimidin-1(6H)-yl)
benzamide (5a-k) and 2, 4-dialkyl amino 6-oxopyrimidin-1(6H)-yl) benzamide
derivatives (6a-k) from key intermediate 3. The compounds 5a-k and 6a-k exhibited very
good anti-bacterial activity against both Gram positive and Gram negative bacteria in
comparison to Streptomycin, a first line antibiotic drug (Table 4). Compounds 5g, 5i and
5k showed potent cytotoxicity activity. The structure and biological activity relationship
of title compounds showed that the presence of Pyrimidone nuclei and amino guanidine
skeleton, as well as biologically active Aminoethyl cyclohexane, morpholine, piperidine,
3-aminopyrrolidine, Dimethyl amine, 2,2,2-trifluoroethylamine, groups attached to the
pyrimidone ring are responsible for good antibacterial activity and cytotoxic activity.
Based on these results, new compounds are being synthesized by keeping pyridone nuclei
with suitable substituents for their cytotoxicity activity which will be reported in due
course.
ACKNOWLEDGEMENTS
The authors are grateful to the management of GVK Biosciences Private Limited, India
for financial and analytical support during this program. Special thanks to JNTU,
Hyderabad, India for continuous encouragement.
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13

Table 1. Optimization of ring openinga reaction
Entry Base
Solvent
EtOH
EtOH
DCM
DMF
Time
20 h, 16 hc
16 h
Temp (oC)
RT
Yieldsb (%)
1
2
3
4
TEA
10
DIEA
DBU
K2CO3
Reflux
RT
22
16 h
47.2
78
2 h
RT
14

Table 2. Synthesis of 2-alkylamino 6-oxopyrimidin-1(6H)-yl-benzamide derivatives using
Scheme-2.
15

Table 3. Synthesis of 2, 4-dialkyl amino 6-oxopyrimidin-1(6H)-yl) benzamide derivatives (6)
16

Table 4. Antimicrobial activity of the synthesized compounds 5a-i & 6a-i.c
Diameter of inhibition (mm)
Klebsiella
Escheric Proteus
hia coli vulgaris
Salmonella
paratyphi
Staphylococc Bacillus
pneumoni
a
Compound
us aureus
cereus
7
3
2
2
2
2
2
3
5
1
7
3
2
1
1
5
4
2
5
2
2
8
2
8
4
5
3
4
3
2
7
2
2
7
2
4
2
4
7
5
3
6
5
2
5
7
6
5
3
4
2
2
4
2
15
9
1
3
2
3
4
4
2
4
2
5
4
3
8
6
4
6
6
4
3
2
1
7
7
3
3
4
4
3
4
3
4
2
5
6
5a
14
11
8
14
12
10
8
5b
5c
5d
2
5e
2
8
5f
2
2
5g
10
6
5
5h
6
5i
12
13
10
4
10
10
10
5
5j
5k
6a
6b
5
5
6c
4
8
6d
10
6
8
6e
10
10
9
6f
7
6g
12
10
9
6h
11
7
6i
6j
10
6
6k
Streptomyc
6
8
10
20
10
22
in (1 mg /1
17

Table 5. Cytotoxic Activity of 5a-i & 6a-i Against Human Lung carcinoma Cell
Sublines.
Compound
A549
H460
Compound A549
H460
>100
88
91
>100
95
94
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
93
>100
>100
>100
92
>100
>100
84
>100
>100
91
>100
>100
88
>100
0.4
94
>100
84
95
23
88
5g
5h
5i
6g
6h
6i
>100
82
>100
>100
>100
15
>100
92
>100
>100
>100
94
>100
14
>100
86
5j
6j
5k
6k
0.387
0.281
Puromycin
(1mg/1ml)
18

Scheme 1. Synthesis of key intermediate 3
19

Scheme-2. Synthesis of 2-alkylamino 6-oxopyrimidin-1(6H)-yl) benzamide derivative
(5a)
20

Scheme 3: Synthesis of 2, 4-dialkyl amino 6-oxopyrimidin-1(6H)-yl) benzamide
derivatives (6a-k).
21

Figure 1. Structures of Lamivudine and Zidovudine
22

Figure 2. Biologically active pyrimidone based drugs
23

Figure 3: A & B: Inhibition of Compounds 5g, 5h, 5i and 5k against adenocarcinomin
human alveolar basal epithelial cells (A549 and H460 cells) at 37 °C and 5 % CO2. C &
D: Inhibition of Compounds 5g and 5i against non tumoral mammalian cell lines (MDCK
and Vero cells) at 37 °C and 5 % CO2.
24