Synthesis and in vitro analysis of novel dihydroxyacetophenone derivatives with antimicrobial and antitumor activities

Article abstract of DOI:10.2174/15734064113096660070

Herein we report a feasible study concerning the design, syntheses and in vitro antimicrobial and antitumoral activities of some novel compounds with dihydroxyacetophenone (DA) moiety. An efficient and general method for the preparation of diazine with dihydroxyacetophenone (DDA) skeleton under conventional thermal heating (TH), microwave (MW) and ultrasounds (US) irradiation is presented. Antimicrobial and antitumoral tests prove that some dihydroxyacetophenone compounds (the brominated derivatives BrDA 3) have a significant biological activity. It is also to be pointed out that, basically all the dihydroxyacetophenone derivatives proved to have a powerful antibacterial activity against drug resistant Gram-negative strain Pseudomonas aeruginosa ATCC 27853. Of particular interest could be the excellent antibacterial activity of our dihydroxyacetophenone compounds against drug resistant Gram-negative strain Pseudomonas aeruginosa.

Full text of DOI:10.2174/15734064113096660070

See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/258280247
Synthesis and In vitro Analysis of Novel
Dihydroxyacetophenone Derivatives with
Antimicrobial and Antitumor Activities
ARTICLE in MEDICINAL CHEMISTRY (SHĀRIQAH (UNITED ARAB EMIRATES)) · NOVEMBER 2013
Impact Factor: 1.39 · DOI: 10.2174/15734064113096660070 · Source: PubMed
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Medicinal Chemistry, 2014, 10, 000-000
1
Synthesis and In vitro Analysis of Novel Dihydroxyacetophenone
Derivatives with Antimicrobial and Antitumor Activities
Ana-Maria Zbancioc1, Anca Miron1, Cristina Tuchilus1, Pincu Rotinberg2, Cosmin-Teodor Mihai3,
Ionel I. Mangalagiu4 and Gheorghita Zbancioc4*
1 University of Medicine and Pharmacy Iasi “GrigoreT. Popa”, Str. Universitatii 16, 700115 Iasi, România; 2 National
Institute of Research and Development for Biological Sciences, branch Institute of Biological Research Iasi, Str. Lascar
3
Catargi 47, 700107, Iasi, România; "Al. I. Cuza" University of Iasi, Department of Biology, Bd. Carol I 20A, 700505,
Iasi, România; 4 "Al. I. Cuza" University of Iasi, Organic Chemistry Department, Bd. Carol 11, 700506 Iasi, Romania
Abstract: Herein we report a feasible study concerning the design, syntheses and in vitro antimicrobial and antitumoral
activities of some novel compounds with dihydroxyacetophenone (DA) moiety. An efficient and general method for the
preparation of diazine with dihydroxyacetophenone (DDA) skeleton under conventional thermal heating (TH), microwave
(MW) and ultrasounds (US) irradiation is presented. Antimicrobial and antitumoral tests prove that some dihydroxyaceto-
phenone compounds (the brominated derivatives BrDA 3) have a significant biological activity. It is also to be pointed out
that, basically all the dihydroxyacetophenone derivatives proved to have a powerful antibacterial activity against drug re-
sistant Gram-negative strain Pseudomonas aeruginosa ATCC 27853. Of particular interest could be the excellent antibac-
terial activity of our dihydroxyacetophenone compounds against drug resistant Gram-negative strain Pseudomonas aeru-
ginosa.
Keywords: Antimicrobial, Antitumoral, Dihydroxyacetophenone, Microwaves, Phthalazine, Pyridazine, Ultrasounds.
INTRODUCTION
Recently published comprehensive books [16, 17] and
papers [18, 19] indicate that MW and US irradiation is a new
trend in organic chemistry, offering a versatile and facile
pathway in a large variety of syntheses.
Infectious diseases caused by microorganisms (bacteria,
fungus, Mycobacterium tuberculosis, etc.) have substantially
increased over the last few years. This is largely due to the
development of drug resistance, particularly multidrug resis-
tance which remains a major concern in medicine, for hospi-
tals especially [1, 3]. Despite the progress achieved by mod-
ern medicinal science in cancer therapy, neoplasm remains
one of the most serious and merciless health problems of the
mankind [2]. One of the most promising strategies in cancer
therapy remains targeting DNA, designing and synthesizing
DNA alkylators agents being one of the most attractive ap-
proach in the development of potent anticancer drugs [2, 3].
As part of our ongoing research in the field of biologi-
cally active 1,2-diazine [9,12,13,20,21], we decided to syn-
thesize new dihydroxyacetophenone derivatives and diazine
with dihydroxyacetophenone skeleton derivatives in order to
combine their respective biological potentials, our attention
being focused especially on antimicrobial and anticancer
activities.
MATERIALS AND METHODS
Chemistry
Pyridazine derivatives and its condensed systems phtha-
lazines, are well known scaffolds in medicinal chemistry [3,
4], possessing a large variety of biological activities such as
antihypertensive [5], diuretic [6], antiplatelet [7], cardiotonic
[8], anticancer [9], anti-HIV [10], anti-inflammatory [11],
antimicrobial (antibacterial, antifungal, anti-tuberculosis)
[12, 13], so on. Synthesis of functionalized acetophenone
derivatives is an important goal for organic synthesis, these
derivatives and particulary dihydroxyacetophenone, exhibit-
ing interesting biological properties, these including antimi-
crobial [14] and anticancer activities [15].
General Information
All the reagents and solvents employed were of the best
grade available and were used without further purification.
Melting points were determined using an electrothermal ap-
paratus (MELTEMP II) and are uncorrected. The IR spectra
were recorded on an FTIR Shimadzu Prestige 8400s spectro-
photometer. The NMR spectra were recorded on a Bruker
Avance 400 DRX spectrometer operating at 400 MHz for 1H
and 100 MHz for 13C. The following abbreviations were
used to designate chemical shift multiplicities: s = singlet, d
= doublet, t = triplet, m = multiplet, br = broad. Chemical
shifts were reported in ppm (ꢀ-scale), coupling constants (J)
in Hz. MS spectra were recorded on a Shimadzu GCMS-QP
2010. The instrument running in chemical ionization (CI)
*Address correspondence to this author at the Al. I. Cuza” University of
Iasi, Department of Organic and Biochemistry, 11 Carol I, Iasi-700506,
Romania; Tel: +40 +232201278; Fax: +40+ 232 201313;
E-mail: zbanciocg@chem.uaic.ro
1573-4064/14 $58.00+.00
© 2014 Bentham Science Publishers

2
Medicinal Chemistry, 2014, Vol. 10, No. ??
Zbancioc et al.
mode. The system was equipped with a 25 m x 0.25 mm x
0.25 μm DB-5ms capillary column. The ion source, quad-
rupole and interface temperatures were 330 °C. Helium was
used as carrier gas at constant flow (1.54 mL/min) with an
initial pressure of 90.7 kPa, while methane was used as rea-
gent gas in the mass spectrometer. The electron multiplier
voltage was set at 1750 V. Two microliters of diluted solu-
tion was injected in cold pulsed splitless mode (initial injec-
tor temperature at 250 °C). The temperature of the DB-5ms
column was programated from 50 °C, stay 2 min, then to 320
°C at a rate of 15 °C/min and finally stay 5 min to 320 °C.
from 1 and 3 position, -CH2-COOMe), 105.63 (C4, C6),
120.78 (C2), 130.36 (C5), 154.33 (C1, C3), 168.97 (2xCO
keto ester), 200.63 (CO keto); IR (KBr): ꢀ/cm-1= 3100, 3048,
3011, 2957, 2917, 1767, 1751, 1697, 1599, 1470, 1441,
1248, 1211, 1130, 1086. MS (CI) m/z(%)= 91 (7.6), 107
(5.7), 195 (5.7), 221 (9.4), 237 (34.0), 253 (20.8), 255 (15.1),
281 (Base peak, 100), 282 (15.1), 296 (M, 17.0), 297 (M+1,
11.3).
General Procedure for Syntheses of ꢀ-bromo-bis-
etherificated BrDA 3a-e Under Conventional TH, MW
and US Irradiation
The microanalyses were in satisfactory agreement with
the calculated values: C, ±0.15; H, ±0.10; N, ±0.30. For the
MW irradiation we used a 800 W STAR SYSTEM-2 mono-
mode reactor (CEM Corporation). The best results were ob-
tained when we used 20% of the full power of the magne-
tron. US assisted reactions were carried out using a Bandelin
Sonopuls GM 3200 reactor, with a nominal power of 200 W
and frequency of 20 kHz. The optimal results were obtained
when applying a pulse irradiation: 5 s pulse/ 5 s pause, 50%
from the full power of the generator. Compounds 3a and 3e
were initially investigated by us [22]; here we obtained these
compounds using a new method, under MW irradiation.
To a suspension of copper (II) bromide (10 mmol, 2.24
g) in 20 mL mixture of chloroform/ethylacetate in a 1:2 ra-
tio, DA derivatives 2a-e (5 mmol, 1.48 g) in 10 mL mixture
of chloroform/ethylacetate in a 1:2 ratio were added drop
wise, in one hour, under stirring and refluxing. The stirring
and refluxing were maintained for 300 minutes. The boiled
solution was filtered (in order to remove the copper (I) bro-
mide that formed), and the solvent was removed by reduced
pressure distillation. The crude product was crystallization
from toluene. The crude product was purified by column
chromatography on silica gel (eluted with dichloromethane).
Under MW and US irradiation, the copper (II) bromide
(10 mmol, 2.24 g), DA derivatives 2a-e (5 mmol, 1.48 g), in
15 mL solvent (chloroform/ethylacetate, 1:2 ratio) were
placed in the reaction vessel and exposed to irradiation (15
min for MW and 20 min for US). Once the heating cycle was
completed, the reaction tube was cooled to ambient tempera-
ture, removed from the reactor, and processed as indicated
for TH conditions.
General Procedure for Syntheses of Mono- and Bis-
etherificated DA 2 a-e Under Conventional TH, MW and
US Irradiation
To a mixture of dihydroyacetophenone derivatives (19
mmol, 2.88 g), potassium carbonate (38 mmol, 5.24 g) and
potassium iodide (a spatula tip) in acetonitrile (100 mL un-
der TH, 40 mL under MW and US irradiation), were added
methylchloroacetate (38 mmol, 3.35 ml), at room tempera-
ture. Under conventional TH, the solution was then refluxed
for 26 h on an oil bath. After completion of the reaction
(TLC), the mixture was cooled to room temperature, water
was added and then the compounds were extracted with
chloroform (3 x 50 ml), washed with sodium hydroxide solu-
tion (10%), dried and evaporated to afford a precipitate. The
crude product was purified by column chromatography on
silica gel (eluted with a mixture of dichloromethane / metha-
nol 99:1).
Dimethyl
2,2'-(2-(2-bromoacetyl)-1,3-phenylene)bis(oxy)
diacetate (3c)
Light brown solid (1.294 g, 69% using TH; 0.056 g, 3%
using MW; 1.576 g, 84% using US): Rf= 0.43
(CH2Cl2/CH3OH, 9.9:0.1); mp: 57-58°C; 1H NMR (400
MHz, CDCl3): ꢀppm= 3.78 (s, 6H, 2xCH3 of methyl acetate
groups from 1 and 3 positions), 4.60 (s, 2H, CH2 of bromoa-
cetyl group from 2 position), 4.67 (s, 4H, 2xCH2 of methyl
acetate groups from 1 and 3 positions), 6.50 (d, J4,5 = J6,5
=
C
8.4 Hz, 2H, H4, H6), 7.28 (t, J5,4 = J5,6 = 8.4 Hz, 1H, H5); 13
Under MW and US irradiation, the solution mixture of
reagents was placed in the reaction vessel and exposed to
irradiation (5 min for MW and 15 min for US). Once the
heating cycle was completed, the reaction tube was cooled to
ambient temperature, removed from the reactor, and proc-
essed as indicated for TH conditions.
NMR (100 MHz, CDCl3): ꢀppm= 37.51 (CH2 of bromoacetyl
group from 2 position), 52.41 (2xCH3 of methyl acetate
groups from 1 and 3 positions, -CH2-COOMe), 65.73
(2xCH2 of methyl acetate groups from 1 and 3 positions, -
CH2-COOMe), 105.94 (C4, C6, C2), 131.92 (C5), 155.86 (C1,
C3), 168.74 (2xCO keto ester), 193.42 (CO keto); IR (KBr):
ꢀ/cm-1= 3093, 2994, 2947, 2911, 1749, 1676, 1595, 1470,
1435, 1377, 1220, 1182, 1145, 1124, 613; MS (CI) m/z(%)=
237 (7.6), 253 (17.0), 255 (11.3), 281 (Base peak, 100), 282
(18.9), 315 (24.5), 317 (22.6), 374 (M-1, 11.3), 376 (M+1,
9.4).
Dimethyl
(2c)
2,2'-(2-acetyl-1,3-phenylene)bis(oxy)diacetate
Yellow solid (4.391 g, 78% using TH; 0.169 g, 3% using
MW; 0.169 g, 3% using US): Rf= 0.44 (CH2Cl2/CH3OH,
1
9.9:0.1); mp: 80-81°C; H NMR (400 MHz, DMSO): ꢀppm
=
2.45 (s, 3H, CH3 of acetyl group from 2 position), 3.69 (s,
6H, 2xCH3 of methyl acetate groups from 1 and 3 positions),
4.85 (s, 4H, 2xCH2 of methyl acetate groups from 1 and 3
positions), 6.65 (d, J4,5 = J6,5 = 8.4 Hz, 2H, H4, H6), 7.25 (t,
J5,4 = J5,6 = 8.4 Hz, 1H, H5); 13C NMR (100 MHz, DMSO):
General Procedure for Syntheses of Cycloimmonium
Salts 4a-e and 5a-e Under Conventional TH, MW and US
Irradiation
BrDA derivatives 3a-e (5 mmol, 1.88g) and diazine
compound (5 mmol) were dissolved in dry acetone (40 mL
under TH, 10 mL under MW and US irradiation). The result-
ing mixture was stirred at room temperature for 48 h. The
ꢀppm= 31.95 (CH3 of acetyl group from 2 position, COMe),
51.82 (2xCH3 of methyl acetate groups from 1 and 3 positi-
on, -CH2-COOMe), 64.88 (CH2 of methyl acetate groups

Synthesis and In vitro Analysis of Novel Dihydroxyacetophenone Derivatives
Medicinal Chemistry, 2014, Vol. 10, No. ??
3
solvent was removed on a rotary evaporator and the obtained
salts were washed twice with 10 mL ether. No further purifi-
cation was required.
and Gram-negative (Escherichia coli ATCC 25922, Pseu-
domonas aeruginosa ATCC 27853) bacteria, and one yeast
strain (Candida albicans ATCC 10231).
Under MW and US irradiation, the solution of reagents
mixture was placed in the reaction vessel and exposed to
irradiation (5 min for MW and 15 min for US). Once the
heating cycle was completed, the reaction tube was cooled to
ambient temperature, removed from the reactor, and proc-
essed as indicated for TH conditions (with the difference that
dry acetone was used as washing solvent).
The in vitro anticancer activity of the synthesized com-
pounds was screened against human cervix epithelial carci-
noma cell line (HeLa) [24, 25].
Broth Micro Dilution Assay
A broth microdilution assay was used to determine the
minimum inhibitory concentrations (MIC) and the minimum
bactericidal/fungicidal concentrations (MBC/MFC) [27-29].
The compounds were dissolved in dimethyl sulfoxide at a
concentration of 10 mg/mL. 50 μL of each compound solu-
tion were mixed with 50 μL of Mueller Hinton broth for an-
tibacterial assays and 50 μL of Sabouraud broth for antifun-
gal assays and subjected to further serial two-fold dilutions
in 96-well plates. An aliquot of 2 μL of microbial suspension
(0.5 McFarland) was dispersed in each well. The plates were
incubated at 37 °C for antibacterial tests and 24 °C for anti-
fungal tests, for 24 h. The MIC value represents the lowest
concentration of compound inhibiting the visible growth of
microorganisms. The MBC/MFC values represent the lowest
concentration of the compound killing completely the mi-
croorganisms to be tested, were determined by transferring
10 μL of samples showing inhibition of visible growth on the
surface of an agar plate [28]. The subcultures were incubated
at 37 °C for antibacterial tests and 24 °C for antifungal tests
for 24 h. There were also evaluated the MIC and MBC/MFC
values of ampicillin/nystatin towards bacteria/yeast strains.
1-(2-(2,6-bis(2-methoxy-2-oxoethoxy)phenyl)-2-oxoethyl)
pyridazin-1-ium bromide (4c)
Brown solid (1.343 g, 59% using TH; 1.457 g, 64% using
MW; 1.571 g, 69% using US): Rf= 0.53 (CH2Cl2/CH3OH,
1
3:1); mp: 127-128°C; H NMR (400 MHz, CDCl3): ꢀppm
=
3.73 (s, 6H, 2xCH3 of methyl acetate groups from 10 and 14
positions), 4.99 (s, 4H, 2xCH2 of methyl acetate groups from
10 and 14 positions), 6.39 (s, 2H, CH2 from 7 position), 6.82
(t, overlapped peaks, 2H, H11, H13), 7.46 (t, J12,11 = 8.0 Hz,
J12,13 =8.4 Hz, 1H, H12), 8.77 (s, 1H, H4), 8.89 (t, J5,6 = 4.4
Hz, 1H, H5), 9.76 (d, 1H, H3), 9.94 (d, J6,5 = 4.4 Hz, 1H, H6);
13C NMR (100 MHz, CDCl3): ꢀppm= 52.07 (2xCH3 of methyl
acetate groups from 10 and 14 positions, -CH2-COOMe),
65.40 (2xCH2 of methyl acetate groups from 10 and 14 posi-
tions, -CH2-COOMe), 72.86 (CH2 from 7 position), 106.41
(C11, C13), 115.38 (C9), 133.28 (C12), 136.12 (C5), 137.66
(C4), 151.51 (C6), 154.81 (C10, C14), 155.81 (C3) 169.08 (CO
keto ester from 10 and 14 positions), 192.46 (CO keto); IR
(KBr): ꢀ/cm-1= 3095, 3032, 2969, 2949, 1753, 1690, 1597,
1470, 1437, 1423, 1253, 1219, 1130.
Cytotoxic Assay
2-(2-(2,6-bis(2-methoxy-2-oxoethoxy)phenyl)-2-oxoethyl)
phthalazin-2-ium bromide (5c)
The HeLa cells were cultured in DMEM medium (Dul-
beco’s Modified Essential Medium, Biochrom AG, Ger-
many) supplemented with 10% fetal bovine serum (Sigma,
Germany), 100 ꢁg/mL streptomycin (Biochrom AG, Ger-
many), 100 IU/mL penicillin (Biochrom AG, Germany) and
50 ꢁg/mL amphotericin B (Biochrom AG, Germany), at a
density of 5x105 cells, in a humidified 5% CO2 atmosphere
at 37°C in a Binder CB 150 incubator (Tuttlingen, Ger-
many).
Brown solid, (1.617 g, 64% using TH; 1.920 g, 76% u-
sing MW; 1.996 g, 79% using US): Rf= 0.67
(CH2Cl2/CH3OH, 3:1); mp: 130-131°C; 1H NMR (400 MHz,
CDCl3): ꢀppm= 3.74 (s, 6H, 2xCH3 of methyl acetate groups
from 12 and 16 positions), 5.02 (s, 2H, CH2 of methyl aceta-
te groups from 12 and 16 positions), 6.37 (s, 2H, CH2 from 9
position), 6.84 (d, J13,14 = J15,14 = 7.6 Hz overlapped peaks,
2H, H13, H15), 7.46 (t, J14,13 = J14,15 = 7.6 Hz, 1H, H14), 8.52
(d, J5,4 = 6.8 Hz, 1H, H5), 8.62 (d, J6,7 = 6.8 Hz, 1H, H6),
8.68 (d, J4,5 = 6.8 Hz, 1H, H4), 8.81 (d, J7,6 = 6.8 Hz, 1H,
H7), 10.23 (s, 1H, H3), 10.80 (s, 1H, H8); 13C NMR (100
MHz, CDCl3): ꢀppm= 52.00 (CH3 of methyl acetate groups
from 12 and 16 positions, -CH2-COOMe), 65.34 (CH2 of
methyl acetate groups from 12 and 16 positions, -CH2-
COOMe), 71.60 (CH2 from 9 position), 106.28 (C13, C15),
115.60 (C11), 127.22 (C4), 128.54 (C7), 130.74 (C14), 133.06
(C3a), 136.05 (C7a), 136.54 (C5), 139.89 (C6), 153.14 (C8),
154.88 (C3),155.72 (C12, C16) 169.04 (CO keto ester from 12
and 16 positions), 192.80 (CO keto); IR (KBr): ꢀ/cm-1: 3080,
3056, 2955, 2936, 1751, 1687, 1597, 1470, 1437, 1395,
1281, 1219, 1130, 1080.
The cells monolayer was further removed with 0.25%
trypsin and 0.02% EDTA (ethylenediaminetetraacetic acid,
Biochrom AG, Germany) and centrifuged in a Sigma - Sarto-
rius (Gottingen, Germany) centrifuge at 1800 rpm for 2 min.
The sediment of the cells was then suspended in the normal
medium (DMEM medium). Volumes of 2 mL of suspension
(1x105 cells) were inoculated in the experimental tubes
which were kept in the same conditions mentioned above.
After 24 h the medium was discarded and replaced either
with a normal one (control cultures) or with one containing
the synthesized compounds at different concentrations. At
least four concentrations were used for each compound: 200,
300, 400 and 500 ꢁg/mL (the brominated compounds were
also tested in lower concentrations at 25, 50 and 100 ꢁg/mL).
After 48 h the medium was discarded from the test tubes; the
cells layer was washed with PBS (phosphate buffer saline)
and then subjected to Lowry method modified by Oyama in
order to evaluate the total protein content [30]. The test tubes
containing HeLa cells were treated with 2% sodium carbon-
ate, 0.1 N sodium hydroxide, 0.02% potassium tartrate and
Biological Activity
The synthesized compounds were tested for their in vitro
antimicrobial activity against six different strains Gram-
positive (Staphylococcus aureus ATCC 25923, Sarcina lutea
ATCC 9341, Bacillus cereus ATCC 14579, Bacillus subtilis)

4
Medicinal Chemistry, 2014, Vol. 10, No. ??
Zbancioc et al.
O
O
N
N
O
Cl
O
O
O
n
II
Cl
I
n
Anticancer
Antimicrobial
X
Br
O
N
N
N
O
N
O
O
COOR
COOR
COOR
O
O
X= H, Br
COOR
Scheme 1. Design in the class of diazine with dihydroxyacetophenone skeleton derivatives.
HO
COOMe
OH
+
O
O
O
O
CH2
CH2
O
K2CO3/KI
C
C
Cl CH2 COOMe
+
C
CH2
H3C
H3C
TH,
O
OH
COOMe
H3C
COOMe
MW/US irradiation
DA
2a-e
2CuBr2
TH,
2,4-dihydroxyacetophenone - 1a
2,5-dihydroxyacetophenone - 1b
2,6-dihydroxyacetophenone - 1c
3,4-dihydroxyacetophenone - 1d
3,5-dihydroxyacetophenone - 1e
2,4-dihydroxy - 2f
2,5-dihydroxy - 2g
2,6-dihydroxy - 2h
MW/US irradiation
N
COOMe
Br
O
O
N
N
N
COOMe
CH2
CH2
O
O
O
CH2
DDA
C
O
CH2
CH2
C
COOMe
Br CH2
COOMe
TH,
4a-e
BrDA
3a-e
MW/US irradiation
N
N
COOMe
Br
O
CH2
CH2
CH2
C
O
O
DDA
COOMe
5a-e
Scheme 2. Reaction pathway for preparation of DA, BrDA and DDA.
0.5% copper sulphate. After mixing, the absorbance of each
test tube was measured at ꢀ= 660 nm. The results were ex-
pressed as IC50 values representing the concentration of the
drug inducing a 50% inhibition of cell growth. Each experi-
ment was performed five times. 5-Fluoro-uracil, etoposide
were used as the references drug.
In according with our goal, we have synthesized three
new types of dihydroxyacetophenone: bis-etherificated dihy-
droxyacetophenone (DA, 2a-e), ꢁ-brominated dihydroxyace-
tophenone (BrDA, 3a-e) and diazine with dihydroxyaceto-
phenone skeleton derivatives (DDA, 4a-e and 5a-e). The
strategies adopted for the construction of our dihydroxyace-
tophenone derivatives are depicted in (Scheme 2).
RESULTS AND DISCUSSION
In the first step, using conventional TH, we obtained the
expected 2,4-; 2,5-; 2,6-; 3,4- and 3,5- bis-etherificated DA,
2a-e, in good yields (between 78-87%, Table 1). Unexpect-
edly, in the case of dihydroxyacetophenone bearing a hy-
droxyl group in the 2nd the position (2,4-; 2,5-; 2,6- DA), we
also obtained traces of mono-etherificated DA, 2f-h, regard-
less of the conditions that were employed (Table 1).
Recently, we reported successful results on the identifica-
tion of new antimicrobial (type I) [12-14] and anticancer
(type II) [9,21] compounds, which contain a diazine or an
acetophenone skeleton as pharmacophoric moiety. As a con-
sequence, in an attempt to increase the potential of pharma-
cophoric models, we decided to combine their respective
biological potentials. In this respect a bis-etherificated DA
unit was grafted onto a diazine heterocycles, (Scheme 1).
As we may notice from (Table 1), under conventional TH
the reaction pathways had some major disadvantages, includ-
ing moderate selectivity, long reaction time (1560 min), etc.
Having in view the above considerations, we decided to per-
form these reactions using US and MW irradiation. For the
MW irradiation we used a 800 W STAR SYSTEM-2 mono-
mode reactor (CEM Corporation).
The bis-etherificated DA would probably act as bis-
acylation units, analogous to bis-alkylating units from nitro-
gen mustards anticancer derivatives. As a whole, the entire
molecule of DDA could exert both antimicrobial and anti-
cancer activity.

Synthesis and In vitro Analysis of Novel Dihydroxyacetophenone Derivatives
Medicinal Chemistry, 2014, Vol. 10, No. ??
5
Table 1. Synthesis of DA Derivatives Under Conventional TH, MW and US Irradiation
MW
Reaction Time (min)
US
Conventional TH
Comp.
Yield (%)
Reaction Time (min)
Yield (%)
Reaction Time (min)
Yield (%)
traces
traces
traces
92
15
15
15
15
15
15
15
15
traces
traces
traces
93
1560
1560
1560
1560
1560
1560
1560
1560
83
83
2a
2b
2c
2d
2e
2f
5
5
5
5
5
5
5
5
78
84
95
96
87
90
90
traces
traces
traces
89
2g
2h
87
50
87
starting materials remained. In the quaternization case the
data presented in (Table 2), leads to the conclusion that MW
and US irradiation induced again a remarkable acceleration
of the reactions and, in some cases, the yields were higher.
The US assisted reactions were carried out using a Sono-
plus GM 3200 reactor (Bandelin) with a frequency of 20
KHz and a nominal power of 200 W. As indicated in (Table
1), under MW and US irradiation, the reactions occured dif-
ferently in accordance with the relative position of the two
hydroxyl groups on the acetophenone ring. When the hy-
droxyl groups were in 3,4- and 3,5- positions, the reactions
occured selective, the bis-etherificated DA 2d,e, being ob-
tained with better yields compared with TH. When the hy-
droxyl groups were in 2,4-, 2,5-, and 2,6- positions, the
mono-etherificated compounds (DA 2f-h) were obtained as
major products (in good to moderate yields) while the bis-
etherificated compounds (DA 2a-c) were obtained only in
traces. We may also notice from (Table 1) that, in all cases,
under MW and US irradiation, the reaction time decreased
considerably from hours to minutes.
A preliminary antimicrobial screening was performed by
agar diffusion assay [23] using nutrient agar medium (Muel-
ler Hinton agar for antibacterial tests and Sabouraud agar for
antifungal tests). The most active compounds were subjected
to a broth micro dilution assay in order to determinate the
minimum inhibitory concentrations (MIC) and the minimum
bactericidal/fungicidal concentrations (MBC/MFC) [27-29]
(Table 3).
Compound 3e showed the highest effects against all
tested bacteria and yeast strains. It exhibited bactericidal
effects against Pseudomonas aeruginosa ATCC 27853, Es-
cherichia coli ATCC 25922 and Bacillus subtilis with MIC
and MBC values of 0.625 mg/mL. The other Gram-positive
bacteria strains (Staphylococcus aureus ATCC 25923, Sar-
cina lutea ATCC 9341, Bacillus cereus ATCC 14579) were
more susceptible to compound 3e, with MIC values of 0.31
mg/mL.
In the next step we have synthesized the ꢀ-bromo-bis-
etherificated BrDA 3a-e, (Scheme 2). As bromination
method we chose a procedure previously reported by us [22],
the bromination in heterogeneous catalysis, taking into con-
sideration the advantages of better selectivity, better yields
and less polluted setup procedure. Using this method, the
desired ꢀ-bromo-bis-etherificated BrDA 3a-e were synthe-
sized by bromination of DA derivatives 2a-e with copper (II)
bromide. In the final stage, by quaternization of 1,2-diazine
heterocycles (pyridazine and phthalazine) with the ꢀ-
brominated BrDA 3a-e, we obtained the corresponding diaz-
ine with dihydroxyacetophenone skeleton derivatives, 4a-e
and 5a-e, (Scheme 2). In the (Table 2) are listed the opti-
mized conditions used for bromination and quaternization,
under MW, US and conventional TH.
It is worthy to note that all the brominated compounds
showed good antibacterial effects. If we compare the effects
of non-brominated derivatives 2a, 2c and 2g, with those of
the brominated analogues 3a, 3c and 3b, we may notice a
considerable increase in antibacterial activity for the bromi-
nated compounds, the influence of bromine atom being cer-
tain.
In the case of diazine with dihydroxyacetophenone skele-
ton derivatives, an unexpected behaviour occurred. Litera-
ture data mentions that diazine salts with acetophenone
skeleton possess a very good antimicrobial activity [10-12].
As far, for our derivatives, the antibacterial and antifungal
effects were lower in comparison to those of the brominated
analogues.
As indicated in (Table 2), under conventional TH, the
bromination occured highly selective (no nuclear bromina-
tion was observed) but the yields were moderate. In the case
of US irradiation, the yields were higher, by an average of
10-15% and the reaction time decreases from hours to min-
utes. Unexpectedly, under MW irradiation, regardless of the
conditions we employed, there were obtained only traces of
the desired brominated compounds 3a-e, and the unreacted
All tested compounds showed a good antifungal activity
against Candida albicans ATCC 10231.

6
Medicinal Chemistry, 2014, Vol. 10, No. ??
Zbancioc et al.
Table 2. Synthesis of ꢀ-bromo-bis-etherificated BrDA (3a-e) and DDA (4a-e and 5a-e), Under Conventional TH, MW and US
Irradiation
MW
Reaction Time (min)
US
Reaction Time (min)
Conventional TH
Comp.
Yield (%)
Yield (%)
Reaction Time (min)
Yield (%)
15
15
15
15
15
5
traces
traces
traces
traces
traces
81
20
20
20
20
20
15
15
15
15
15
15
15
15
15
15
86
88
84
83
80
79
70
69
73
82
86
74
79
87
89
300
300
71
75
69
76
65
69
61
59
66
64
76
65
64
77
79
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
300
300
300
2880
2880
2880
2880
2880
2880
2880
2880
2880
2880
5
71
5
64
5
73
5
79
5
85
5
77
5
76
5
85
5
85
Table 3. Antibacterial and Antifungal Activities of the Tested Compounds 2-5
S. Lutea ATCC
B. Cereus
E. Coli ATCC
25922
P. Aeruginosa
C. Albicans
S. Aureus
B. Subtilis
Strain
Comp.
9341
ATCC 14579
ATCC 27853
ATCC 10231
ATCC 25923
MIC
2.5
MBC
5
MIC
0.62
0.62
0.62
0.62
0.62
0.62
0.62
0.31
0.62
0.62
0.15
-
MBC
1.25
1.25
1.25
2.5
MIC
1.25
2.5
MBC
5
MIC
MBC
2.5
10
MIC
MBC
1.25
5
MIC
1.25
2.5
MBC
2.5
5
MIC
1.25
1.25
1.25
1.25
1.25
2.5
MFC
2.5
5
2aa
2ca
1.25
10
1.25
1.25
1.25
1.25
1.25
1.25
1.25
0.62
2.5
1.25
1.25
0.62
1.25
1.25
1.25
0.31
1.25
2.5
2.5
2.5
2.5
2.5
2.5
2.5
0.62
2.5
5
5
2ga
1.25
1.25
0.62
2.5
2.5
2.5
1.25
5
1.25
1.25
1.25
1.25
0.62
0.62
10
2.5
2.5
2.5
2.5
1.25
0.62
10
2.5
2.5
2.5
2.5
2.5
0.62
5
2.5
10
5
2.5
2.5
2.5
5
3aa
2.5
3ba
2.5
2.5
5
3ca
1.25
1.25
0.62
1.25
1.25
0.31
-
2.5
5
3da
0.62
0.31
1.25
1.25
1.25
0.62
2.5
5
2.5
5
1.25
1.25
2.5
2.5
2.5
5
3ea
0.62
2.5
0.62
5
4ea
5ca
2.5
5
2.5
5
2.5
10
1.25
-
5
c
c
c
c
Ampicillinb
Nystatinb
0.25
0.5
-
-
0.15
-
0.62
-
0.31
-
0.62
-
-
-
-
-
-
-
-
-
-
1
2
aconcentration expressed in mg/mL
bconcentration expressed in ꢀg/mL
cno zone inhibition

Synthesis and In vitro Analysis of Novel Dihydroxyacetophenone Derivatives
Medicinal Chemistry, 2014, Vol. 10, No. ??
7
The in vitro anticancer activity of the synthesized com-
pounds was evaluated and screened against human cervix
epithelial carcinoma cell line (HeLa) [24, 25]. At least four
concentrations were tested for each compound: 200, 300,
400 and 500 ꢀg/mL. In order to calculate IC50 values, lower
concentrations of brominated compounds were tested. The
results were expressed as IC50, which is the concentration of
the drugs inducing a 50% inhibition of cell growth. Each
experiment was performed five times. 5-Fluorouracil and
Etoposide were used as the references drugs. (Table 4) sum-
marizes the antitumoral activity of the compounds expressed
as IC50.
The in vitro evaluation revealed that some of the tested
compounds showed high or moderate antitumoral activity.
The brominated compounds 3a-e were proved to be the most
active against the tested HeLa cells, while the non-
bromurated DA 2 and the salts 4 and 5 do not have or have
only a moderate cytotoxic activity (IC50 between >500 to
roughly 300 μg/mL). As for the antimicrobial assay, these
results suggest a certain influence of bromine atom concern-
ing anticancer activity.
Table 4. In vitro Cytotoxicity of the Synthesized Compounds
Against HeLa Cancer Cell Lines
Compound
Cytotoxicity (IC50a, μg/mL)
2a
2b
2c
2d
2e
2f
364.3±17.5
380.8±16.7
442.4±27.7
380.6±15.3
486.7±18.0
412.8±15.2
462.6±25.5
221.7±48.8
41.5±1.3
101.8±9.1
30.9±7.23
64.6±8.0
203.4±11.6
>500b
We may notice that the brominated series the antitumoral
activity decreases in order 3c (IC50 30.9 μg/mL, 2,6-
disubstituted BrDA)
disubstituted BrDA)
>
>
3a (IC50 41.5 μg/mL, 2,4-
3e (IC50 64.6 μg/mL, 3,5-
disubstituted BrDA) > 3b (IC50 101.8 μg/mL, 2,5-
disubstituted BrDA) > 3d (IC50 203.4 μg/mL, 3,4-
disubstituted BrDA).
These results suggest that the positions of O-methyl ace-
tate substituents onto phenyl ring have also importance, a
location into 2 or 3 positions onto phenyl ring being favour-
able for activity, (Scheme 3).
2g
2h
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
O
CH2 COOMe
O
O
or
C
C
Br CH2
H2C Br
2
3
O
CH2 COOMe
Scheme 3. Brominated DA: positions onto phenyl ring favourable
for activity.
CONCLUSION
In conclusion, we reported herein an efficient and general
method for preparation of new diazine with dihydroxyaceto-
phenone skeleton under conventional TH, MW and US irra-
diation. MW and US irradiation induce a remarkable accel-
eration for reactions and, in some cases, the yields were
higher. The in vitro antimicrobial and anticancer activities of
the DA, BrDA and DDA compounds were evaluated. The
tested compounds have a significant antibacterial activity
against Gram-positive strain Bacillus cereus ATCC 14579
and, more important, against drug resistant Gram-negative
strain Pseudomonas aeruginosa ATCC 27853. The bromi-
nated derivatives BrDA have the most powerful antibacterial
activity, the influence of bromine atom being certain. All the
tested compounds have a moderate antifungal activity. The
in vitro antitumoral evaluation revealed that some of the
tested compounds show high or moderate anticancer activity.
The brominated derivatives BrDA proved to be the most
active against tested HeLa cells, suggesting again (as for the
antimicrobial assay) a certain influence of bromine atom
concerning anticancer. In the brominated BrDA series, the
position of substituents onto phenyl ring seems to be impor-
tant concerning anticancer activity.
>500b
>500b
>500b
>500b
459.3±5.5
>500b
343.1±10.2
432.0±7.3
468.9±13.7
17.2±3.87
19.3±2.1
5-Fluorouracil
Etoposide
aIC50 value: the average of three IC50 values ± SD
bCompounds with IC50 > 500 μg/mL were considered to be inactive.

8
Medicinal Chemistry, 2014, Vol. 10, No. ??
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CONFLICT OF INTEREST
The authors confirm that this article content has no con-
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ACKNOWLEDGEMENTS
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Received: April 19, 2013
Revised: October 03, 2013
Accepted: October 26, 2013