Chemical and biological evaluation of some new antipyrine derivatives with particular properties

Article abstract of DOI:10.1016/j.bioorg.2011.12.003

Starting from 4-amino-antipyrine, six new compounds were synthesized and characterized. The new compounds contain moieties with particular properties, such are ionophore (benzo-15-crown-5), fluorescent (nitrobenzofurazan), stable free radical (nitroxide),

Full text of DOI:10.1016/j.bioorg.2011.12.003

Bioorganic Chemistry 41-42 (2012) 6–12
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Chemical and biological evaluation of some new antipyrine derivatives
with particular properties
C. Remes a, A. Paun a, I. Zarafu a, M. Tudose b, M.T. Caproiu c, G. Ionita b, C. Bleotu d, L. Matei d, P. Ionita a,
⇑
a University of Bucharest, Department of Chemistry, Biochemistry and Catalysis, 90-92 Panduri, Bucharest 050663, Romania
b Institute of Physical Chemistry, 202 Spl. Independentei, Bucharest 060021, Romania
c Institute of Organic Chemistry, 202A Spl. Independentei, Bucharest 060023, Romania
d Institute of Virology, 285 M. Bravu, Bucharest 030304, Romania
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 December 2011
Available online 2 January 2012
Starting from 4-amino-antipyrine, six new compounds were synthesized and characterized. The new
compounds contain moieties with particular properties, such are ionophore (benzo-15-crown-5), fluores-
cent (nitrobenzofurazan), stable free radical (nitroxide), or other types of biological active residues, like
nitroderivatives, antipyrine or isoniazid residues. They were fully characterized by appropriate means (1H
and 13CNMR, IR, UV–Vis, fluorescence, EPR, elemental analysis) and some of their biological properties
were evaluated. Hydrophobicity (RM0, log P), total antioxidant capacity (TAC), and antimicrobial proper-
ties are also presented and discussed.
Keywords:
Antipyrine
Crown ether
NBD
Nitroxide
Isoniazid
Synthesis
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
and medicine as compounds that interrupt radical-chain oxidation
processes, causing thus a high scientific interest [10,11].
Antipyrine or phenazone (1,2-dihydro-1,5-dimethyl-2-phenyl-
3H-pyrazol-3-one) derivatives are well known compounds used
mainly as analgesic and antipyretic drugs [1]. One of the best known
antipyrine derivatives is 4-aminoantipyrine or 4-AAP (4-amino-2,3-
dimethyl-1-phenyl-3-pyrazolin-5-one), which presents a structural
similarity with metamizole, a well-known and very effective analge-
sic, anti-inflammatory and antipyretic agent [2].
Antipyrine is completely absorbed and uniformly distributed in
the body in 1 h after oral ingestion [3,4], being thus proposed as a
very suitable marker for measuring oxidative stress. The metabolic
pathwayof antipyrine hasbeen extensively studiedand showedthat
most of it is excreted in urine as 3- and 4-hydroxyantipyrine [5–7].
Protection against oxidative stress as well as prophylactic of
some diseases including cancer is important directions in medicine
and biochemistry [8,9]. Reactive oxygen species (ROS), including
free radicals, led to a decreasein the antioxidantcapacity, and, more-
over, may generate other reactive species that damage the living cell.
Oxidative stress may arise in a biological system after an increased
exposure to oxidants, so the antioxidants play a major role in pro-
tecting biological systems against such threats. Different types of
antioxidants (vitamins C and E, glutathione, lipoic acid, butylated
phenols, etc.) have been widely used in different fields of industry
Antipyrine derivatives are strong inhibitors of cycloxygenase
isoenzymes, platelet tromboxane synthesis, and prostanoids syn-
thesis [1,12]. The biological activity of these compounds has also
been attributed to its scavenging activity against reactive oxygen
and nitrogen species, as well as to the inhibition of neutrophil’s oxi-
dative burst. However, besides its well recognized benefits, antipy-
rine derivatives have been associated with potential adverse
effects characterized by leukopenia, most commonly of neutrophils,
causing neutropenia in the circulating blood (agranulocytosis). It is
worth to mention that there are studies demonstrating that this ad-
verse effect might be exaggerated [2,13]. Several congeners and
transition metal complexes of antipyrine were also biological evalu-
ated [14], being reported with antivirus and antibacterial activity
[15,16].
In this work we present the synthesis and structural character-
ization of six new compounds derived from antipyrine, together
with some of their biological evaluation data.
2. Results and discussion
2.1. Synthesis and structural characterization
The aim of our work focused on the synthesis and structural char-
acterization of some novel antipyrine derivatives, with potential
significant biological activity, starting from 4-AAP. Thus, 4-AAP
⇑
Corresponding author. Fax: +40 213121147.
E-mail address: pionita@icf.ro (P. Ionita).
0045-2068/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2011.12.003

C. Remes et al. / Bioorganic Chemistry 41-42 (2012) 6–12
7
was derivatized with different organic residues of organic, analytical
or medicinal interest, such are benzo-15-crown-5 [17], the nitroxide
stable free radical Tempo (2,2,6,6-tetramethylpiperidine-N-oxide)
[18], isoniazid (an antibacterial drug used for the treatment of tuber-
culosis) [19], and nitrobenzofurazan (a fluorescent dye) [20]. In this
way a number of six new compounds (Fig. 1) were obtained and
appropriate characterized by different means (IR, UV–Vis, 1H and
13CNMR, fluorescence, EPR spectroscopy, etc.).
Compounds 1–6 were synthesized by simple coupling reactions,
using the reactive amino group from the starting material, 4-AAP.
The amino group acts as a nuclephile towards the other reagent
used, leading finally to the desired compounds. The full practical
procedures are presented under Experimental section.
Fig. 2a. PR spectrum of 5.
To obtain compound 1, first it was tried a direct synthesis using
commercial available 4-carboxybenzo-15-crown-5 ether and
involving N,N0-dicyclohexylcarbodiimide (DCC) as coupling agent;
using this procedure, the yields in compound 1 was very poor
(5%), so a new strategy was used, converting first the carboxyl
group from 4-carboxybenzo-15-crown-5 ether into the corre-
sponding acid chloride, much more reactive towards the nucleo-
philic amino group of 4-AAP. In this way compound 1 was
obtained in satisfactory yields (50%). Compound 2 was obtained
in a single reaction step, using 4-chloro-7-nitrobenzo-2-oxa-1,3-
diazole (4-chloro-7-nitrobenzofurazan or NBD-chloride); two reac-
tion systems were tried also, the first one using chloroform as
solvent, with poor results (5%), and the second one using DMF as
solvent, with better efficiency (20%). For compound 3, it was nec-
essarily to obtain first the acid chloride from 4-cloro-3,5-dinitro-
benzoic acid, and further reaction of this with the 4-AAP afforded
both compounds 3 and 4 (yields 80% and 20%, respectively). Com-
pounds 5 and 6 are obtained from 3, with good yields (80% and
60%, respectively), also in simple coupling reactions.
by NMR, so EPR spectroscopy was employed [21]. The EPR spec-
trum consists mainly in the characteristic three lines of a nitroxide
radical with a coupling constant of 15.40 G (Fig. 2a). Fluorescence
was used to characterize compound 2 (due to the fluorescent NBD
moiety); the maximum of the emission wavelength was 440 nm
(Fig. 2b).
In order to elucidate some structural behavior of the new com-
pounds, variable temperature NMR spectra have been used for a
correct peaks attribution. An interesting case has been noticed
for compound 6. Due to the hindered free rotation induced by
the two amide groups, the hydrogen atoms H-10 and H-14 are
anizocrons; at room temperature the NMR signals are close to coa-
lescence and are noticed only by the value of the integral; rising
the temperature led to a single mediated peak in which the hydro-
gen atoms H-10 and H-14 are izocrons. For H-24 and H-25 it is no-
ticed at T = 303 K the slow motion of the molecule, so a broad
signal is noticed, and, moreover, the coupling between H-23 and
H-26 is not evidenced. However, in GCOSY spectra the relationship
between H-23 and H-26 with H-24 and H-25 is confirmed by the
off-diagonal peak.
The chemical structure of the new compounds was confirmed
by different spectral and elemental analysis. Thus, IR, UV–Vis, 1H
and 13CNMR spectrometry was used for the compounds 1–4 and
6; compound 5 is a stable free radical, and cannot be characterized
Fig. 1. Synthesis of the compounds 1–6.

8
C. Remes et al. / Bioorganic Chemistry 41-42 (2012) 6–12
Fig. 2b. Fluorescence spectrum of 2.
this method [24]. Thus, RP-TLC uses a non-polar stationary phase
Table 1
Some physical data of the compounds 1–6.
(such are silica gel impregnated with paraffin oil, silanized silica-
gel, and C18 derivatized silica gel) and a mixture of two solvents,
in which one is water (i.e. alcohol–water, acetone–water, acetoni-
trile–water, etc.). RP-TLC is widely used to measure the experimen-
tal lipophilicity (RM0) and specific hydrophobic surface (b) using
Eqs. (1) and (2). These values RM0 and b obtained as Eqs. (1) and (2)
shows are the best indicators of the lipophilicity and the specific
hydrophobic surface area of the compounds.
Comp. kmax (nm)a
Rf
RM0
bc
TACd
Log Pe PSAe
b
c
4-AAP 251, 281 (sh) 0.17 0.812 ꢀ1.581 92.65 0.809
52.962
102.207
123.714
147.685
186.651
183.178
201.702
1
2
3
4
5
6
261, 288 (sh) 0.08 1.471 ꢀ1.781 12.35 1.112
269, 324
277
0.46 1.280 ꢀ2.360 46.3
0.73 1.883 ꢀ1.183 22.2
0.09 2.091 ꢀ4.172 74.4
3.142
2.668
3.370
278
280
271, 358
0.75 2.585 ꢀ1.993 17.35 4.236
0.02 1.438 ꢀ4.129 63.35 1.351
In our measurements we used analytical silica gel plates
impregnated with paraffin oil, and as eluent a mixture of acetone
with water in different proportions (50–90% acetone). The method
starts with the measurement of the Rf values (see also Eqs. (1) and
(2)) using different mixtures of acetone with water. The RM values
necessary for the determination of the hydrophobicity (lipophilic-
ity) of the compounds is obtained as Eq. (1) shows. To measure the
specific hydrophobic surface area b the linear correlation between
the RM values of our compounds and the concentration of organic
solvent (C) in the eluent were calculated. The intercept RM0 is the
RM value of a compound extrapolated to zero organic phase
concentration in the eluent and the slope b is the change of lipo-
philicity caused by unit concentration change of the organic phase.
A good linear correlation was found between RM and C, charac-
terized by high values of the correlation coefficient R (0.87–0.98)
and good standard deviation values SD (0.06–0.37).
a
b
c
In methanol.
Silica gel/ethyl acetate system.
Determined lipophilicity and hydrophobic surface area.
Total antioxidant capacity measured by DPPH method.
Calculated hydrophobicity and polar surface area.
d
e
The purity of the compounds was checked by TLC. TLC can be
used also for separation in preparative purposes. In our experi-
ments the best system for separate simultaneously all the com-
pounds 1–6 and the starting material 4-AAP was the
chromatographic system made from analytical TLC plates (silica
gel) and ethyl acetate as eluent. The Rf values thus obtained are
compiled in Table 1. The most polar compounds are 1, 4 and 6. Ta-
ble 1 compiles also the UV–Vis absorption data of all the com-
pounds involved in this study.
ꢀ
ꢁ
1
RF
2.2. Biological evaluation
RM ¼ log
ꢀ 1
ð1Þ
2.2.1. Hydrophobicity (lipophilicity) and related measurements
The correlation between structure and compounds activity play
an important role in the study of some biological interaction. Among
the molecular properties the lipophilicity is important because the
biological activity is correlated in QSAR studies (QSAR – Quantitative
Structure Activity Relationships) with their capacity to cross the lip-
ophil cell membrane. Lipophilicity influences the bioavailability and
the drug absorption, drug–receptor interactions, metabolism of
molecules, as well as their toxicity [22].
For a biological evaluation, it is important for the beginning to
know the lipophilicity (or hydrophobicity) of the compounds; this
step may be achieved by experimental measurements or just by
using a computer predictor. Besides the classical methods of lipo-
philicity determination by the partition method of the compound
between an immiscible polar and a non-polar solvent pair (usually
n-octanol–water) [23], reversed phase thin layer chromatography
(RP-TLC) is often used for evaluation of the organic – water partition-
ing properties of solutes owing to the simplicity and the rapidity of
RM ¼ RM þ bC
ð2Þ
0
We can notice that the lowest RM0 values have been recorded in
the case of 4-AAP and compound 6 (the isoniazid derivative). The
highest values for RM0 have been recorded in the case of compound
5.
The values obtained using the experimental RP-TLC method
were correlated with the theoretically calculated hydrophobicity
(log P) and molecular polar surface area (PSA) (Table 1) [25]. Good
correlation of the measured RM0 values with the calculated log P
(Table 1) is obtained, as shown in Fig. 3. The correlation coefficient
R was 0.91 and the standard deviation SD was 0.37.
2.2.2. Total antioxidant capacity
It was mentioned before that antipyrine derivatives are active
compounds against reactive oxygen and nitrogen species (ROS and
RNS), and, moreover, the metabolic pathway of those compounds

C. Remes et al. / Bioorganic Chemistry 41-42 (2012) 6–12
9
As we can see in Table 1, 4-AAP has the highest value (95%), fol-
lowed by compound 4 (74%) and 6 (63%); very weak antioxidant
activity has been noticed for compound 1 (12%) and 5 (17%).
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
RM0 = 0.36 * log P + 0.78
2.2.3. Antimicrobial evaluation
The antimicrobial activity of the investigated compounds was
tested against bacterial strains isolated from clinical specimens
as well as reference strains belonging to the Gram-positive (Staph-
ylococcus sp., Crynebacterium sp. Enterococcus faecalis) and Gram-
negative (Escherichia coli, Klebsiella oxytoca, Klebsiella pneumonia,
Pseudomonas aeruginosa, Acinetobacter baumannii, Serratia marces-
cens, Shigella sonnei, Salmonella Typhimurium, Stenotrophomonas
maltophilia, Enterobacter cloacae) species.
The qualitative screening of the susceptibility for both reference
and clinical strains was performed by an adapted diffusion tech-
nique. Our results showed that the tested compounds exhibited a
specific antimicrobial activity; thus, in all cases, the antimicrobial
properties of the new compounds were superior to that of 4-AAP.
The most active compounds have been proved to be 2, 3 and 4
against all tested Staphylococcus sp. strains, Corynebacterium sp.,
S. maltophilia, Acinetobacter baummanii, and E. faecalis (Table 2).
In order to verify if the toxic effect is due to apoptosis, a flow
cytometric assay for apoptosis that detects extracellular phospha-
tidylserine was carried out. 4-AAP and 1 induced few apoptosis
comparing with the others; the most toxic compound is 6 (see
Supplementary data for more information details).
M0
R
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
log P
Fig. 3. Correlation of the measured RM0 values with the calculated log P.
involves the formation of the hydroxyl derivatives, the same type of
compounds formed as well in reaction with ROS.
Among biological properties the total antioxidant capacity (TAC)
represents one of the most used (also known as total antioxidant
activity). There are different methods to measure TAC, and one of
the best known involves the deep colored organic stable free rad-
ical, 2,2-diphenyl-1-picrylhydrazyl (DPPH); this method is also
widely known as the DPPH method (or DPPH radical scavenging
activity) [26]. In our study TAC was obtained using Eq. (3), where
Abs0 refers to the initial absorbance of the DPPH radical (at
517 nm) and Abs30min the absorbance recorded at the same wave-
length after 30 min.
The cell cycle commitment is slightly affected by treatment with
10 lg/mL from the compounds 1–6: increases of G0/G1 in the case
of compound 5 and 6, or increases of G2/M phases in the case of com-
pound 2, 3, and 4 were noticed. On the other hand, occurrence of
apoptosis was observed in cell cycle analysis by the appearance of
one peak on the left. This is due to the fact that when DNA is frag-
mented, as in apoptotic cells, the affinity with the intercalating PI
dyeis decreased and a so-called hypodiploid peakbecomes apparent
to the left of the G0/1 peak (see also Supplementary data).
Abs0 ꢀ Abs30 min
TAC ¼
ꢁ 100
ð3Þ
Abs0
Table 2
The antimicrobial evaluation of the new compounds 1–6 expressed as bacteria growth inhibition (+), or not (ꢀ).
Compound
4-AAP
1
2
3
4
5
6
Micrococcaceae
S. aureus ATCC 25923
S. aureus ATCC 29213
S. hominis 2610
S. aureus 2669
S. aureus 2754
SCN 2672
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
+
+
+/ꢀ
SCN 3026
ꢀ
+
Enterobacteriaceae
E. coli ATCC 25922
E. coli 410
E. coli 1455
E. coli 1461
S. typhimurium ATCC 14028
K. oxytoca ATCC 700324
K. pneumoniae 3029
Enterobacter cloacae 3016
Shigella sonnei ATCC 25931
Serratia marcescens 1142
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
+
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
+
+
+
ꢀ
+
+
+
+
+
+
ꢀ
ꢀ
+/ꢀ
+
+/ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
+
+
ꢀ
ꢀ
Pseudomonadaceae
Ps. aeruginosa ATCC 27853
Ps. aeruginosa 1144
Ps. aeruginosa 1150
A. baumannii 411
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+/ꢀ
+/ꢀ
+/ꢀ
+
+/ꢀ+
+/ꢀ
+
+/ꢀ
+/ꢀ
+/ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+/ꢀ
+/ꢀ
ꢀ
+
Corynebacteriaceae
Corynebacterium sp. 1142
ꢀ
ꢀ
ꢀ
ꢀ
+
+
+
+
+
+
+
+
+
ꢀ
ꢀ
ꢀ
ꢀ
Xanthomonadaceae
Stenotrophomonas maltophilia 412
ꢀ
ꢀ
Enterococcaceae
Enterococcus faecalis ATCC 29212
+/ꢀ
+/ꢀ

10
C. Remes et al. / Bioorganic Chemistry 41-42 (2012) 6–12
In conclusion, compounds 1–6 exhibit biological properties;
at 30 mm distance. The plates were left at room temperature for
20–30 min and then incubated at 37 °C for 24 h. The positive re-
sults were read as the occurrence of an inhibition zone of microbial
growth around the spot.
The eukaryotic cell culture used in our assays were represented
by HCT8 (CCL-244) cells. The cells were maintained as an adherent
cell line in Dulbecco’s Modified Essential Medium DMEM (Sigma)
supplemented with 10% foetal calf serum (Sigma) at 37 °C, 5%
CO2, in a humid atmosphere.
depending on their structure, they have different antioxidant and
antimicrobial activity. Compound 4, containing twice the antipy-
rine moiety, has a good antioxidant and antimicrobial behavior,
and a low toxicity. Further studies on these or on similar com-
pounds may bring some new significant information.
3. Materials and methods
3.1. Apparatus and chemicals
3.5.1. Cell viability assay
To prepare cells for plating into 96-well assay plates, adherent
cells were detached, centrifuged, suspended in fresh medium,
counted by tripan blue exclusion and adjusted to 5 ꢁ 104 cells/
Starting materials (chemicals, TLC plates) were achieved from
Sigma–Aldrich and used as received. Solvents were purchased from
Chimopar and used as received. IR spectra were recorded on a Bru-
ker Vertex 70 spectrometer (solid sample, ATR); 1H and 13CNMR
spectra were recorded on a Varian Inova-400 spectrometer (at se-
lected temperatures, in deuterated solvents CDCl3 and DMSO-d6,
isotopic purity 99.9%); EPR spectra were recorded on a Jeol JES-
FA100 spectrometer (typical settings used for the EPR measure-
ments: concentration of the sample 10ꢀ4 M, number of scans 1,
centre field 3360 G, sweep field 100 G, frequency 9.42 GHz, power
1 mW, sweep time 60 s, time constant 0.1 s, modulation frequency
100 kHz, gain 100, and modulation width 1 G); UV–Vis spectra
were recorded on a UVD-3500 spectrometer, in methanol at ambi-
ent temperature.
mL. Cells were dispensed into assay plates (100 lL/well) and
briefly equilibrated at 37 °C, in 5% CO2 prior to addition of the com-
pounds 1–6. The compounds were initially dissolved into DMSO to
prepare a concentrated stock solution and additional dilutions
were prepared in culture medium. Different dilutions were added
to cell culture in 96-well assay plates. After 24 h, tetrazolium
reduction was measured using Cell Titer 96 Aqueous One Solution
Cell Proliferation assay (Promega Corporation, USA, a colorimetric
method). 20 lL of CellTiter Solution were directly added to each
well and plates were incubated to 37 °C, in 5% CO2 for 1–4 h, to al-
low viable cells to convert tetrazolium into formazan. Plates were
shaken for at least 10 min prior to determining absorbance at
490 nm. Samples were run in triplicate, and each experiment was
repeated three times.
3.2. RP-TLC measurements
Chromatography was performed on 20 ꢁ 10 cm silica gel pre-
coated plates. The plates were impregnated by overnight predevel-
opment in n-hexane-paraffin oil (95:5 v/v), then dried and kept in
desiccators until used. Solutions of the compounds 1–6 were pre-
pared in DCM and spotted on the plates. The plates were then
developed in a tank chamber at room temperature using different
mixtures of water and acetone (aqueous acetone solutions contain-
ing between 30% and 70% acetone). The Rf values thus obtained
were used in Eqs. (1) and (2) for the determination of the lipophil-
icity and the hydrophobic surface area.
3.5.2. Apoptosis detection
In order to establish the treatment effect and to discriminate
between intact and apoptotic HCT8 cells we used Annexin V-FITC
Apoptosis Detection Kit I (BD Bioscience Pharmingen, USA), accord-
ing to manufacturer protocol. Briefly, 5 ꢁ 105 cells were seeded in
25 cm2 flask and treated with 100
l
g/mL of the compounds 1–6
L of binding buf-
fer (10 mM of HEPES/NaOH, pH value of 7.4, 140 mM of NaCl, and
2.5 mM of CaCl2), and stained with 5 L Annexin V-FITC and 5 lL
for 24 h. The total cells were resuspended in 100
l
l
propidium iodide for 10 min in dark. At least 10,000 events from
each sample were acquired using a Beckman Coulter flow cytome-
ter. The percentage of treatment affected cells was determined by
subtracting the percentage of apoptotic/necrotic cells in the un-
treated population from percentage of apoptotic cells in the treated
population.
3.3. TAC measurements
A DPPH solution in methanol was prepared at a 2 ꢁ 10ꢀ4 M con-
centration. Solution of 4-AAP and compounds 1–6 were prepared
also in methanol at a 0.1 mg/mL concentration. To 1.8 mL DPPH
solution was added 0.2 mL solution of each compound, and the
mixture kept in dark for 30 min, followed by absorbance measure-
ment at 517 nm. The blank mixture was obtained from 1.8 mL
solution of DPPH and 0.2 mL of methanol. TAC values were deter-
mined according to Eq. (3).
3.5.3. Cell cycle distribution
5 ꢁ 105 cells were plated in 25 cm2 flasks and treated for 40 h
with 10 lg/mL from each compounds 1–6. After treatment period
cells were taken from the substrate, fixed in 70% cold ethanol for
at least 30 min at ꢀ20 °C, washed twice in PBS, and then incubated
15 min, at 37 °C, with RNAse A (10 mg/mL), and 1 h with propidi-
um iodide (10 mg/mL). After staining of cells with propidium io-
dide the acquisition was done using Epics Beckman Coulter flow
cytometer. Data were analysed using Beckman Coulter XLM soft-
ware and expressed as fractions of cells in the different cycle
phases.
3.4. Computed Log P and PSA data
The Log P and PSA values were obtained using the Molinspira-
tion online property calculator, based on groups contribution; the
web-interactive program allows an easy calculation of molecular
properties, as well as generation of data tables which may be used
for structure–activity QSAR studies [25].
3.6. Synthesis
3.5. Antimicrobial evaluation
3.6.1. Compound 1: Method 1
To 203 mg 4-AAP (1 mmol) dissolved in 50 mL DCM were added
312 mg (1 mmol) 4-carboxybenzo-15-crown-5 ether, followed by
230 mg dicyclocarbodiimide (DCC, 1.1 mmol), and the mixture left
at room temperature for 3 days and filtered off. The organic phase
was extracted twice with 50 mL acid aqueous hydrochloric acid
(1 M), and then twice with 50 mL aqueous sodium hydrogen car-
The qualitative screening was performed by an adapted disk dif-
fusion method. Briefly, Petri dishes with Mueller Hinton medium
were seeded with 0.5 McFarland density bacterial inocula as for
the classical antibiotic susceptibility testing disk diffusion method.
5 lL of 1 mg/mL compounds were spotted on the seeded medium,

C. Remes et al. / Bioorganic Chemistry 41-42 (2012) 6–12
11
bonate (3%), followed by 50 mL water. The organic phase was then
dried on anhydrous sodium sulfate, filtered off, and the solvent re-
moved using a rotavap. The components of the residue were sepa-
rated using preparate TLC, showing only traces of the desired
compound (ꢂ5%).
13CNMR(CDCl3, d ppm, T = 303 K): 161.45(C-5); 150.76(C-15);
144.17(Cq); 143.84(Cq); 143.20(Cq); 135.33(C-13); 133.94(Cq);
129.77(Cq); 128.64(C-17, C-19); 128.13(C-18); 125.40(C-16, C-
20); 107.52(C-3); 101.93(C-14); 35.52(C-6); 11.03(C-3).
FT-IR(ATR in solid,
2856 m; 1642; 1584; 1533; 1493; 1442; 1404; 1298; 1153;
m
cmꢀ1): 3463; 3175; 3115; 3051; 2925;
1033; 999; 906; 714.
3.6.2. Method 2
Fluorescence (methanol, kmax emission nm): 440.
UV–Vis (methanol, kmax nm): 324.
Rf (silicagel, ethyl acetate): 0.46.
312 mg (1 mmol) 4-carboxybenzo-15-crown-5 ether were dis-
solved in 25 mL dichloretane, and 0.5 mL thionyl chloride and
one drop of DMF were added. The mixture was refluxed for 0.5 h,
and then the solvent and the excess of thionyl chloride were re-
moved in vacuo using a rotavap, yielding quantitatively the corre-
sponding acid chloride. The acid chloride was dissolved in 25 mL
DCM, and 203 mg 4-AAP (1 mmol) dissolved in 25 mL DCM were
added, followed by 1 mL triethylamine. The reaction mixture was
left overnight at room temperature, and the next day was extracted
twice with 50 mL aqueous hydrochloric acid (1 M), and then twice
with 50 mL aqueous sodium hydrogen carbonate (3%), followed by
50 mL water. The organic phase was then dried on anhydrous so-
dium sulfate, filtered off, and the solvent removed using a rotavap.
The residue was chromatographied using a silica gel column (or
preparative TLC plates) and ethyl acetate as eluent. Yields: ꢂ50%.
Elemental analysis: C26H31N3O7, M = 497; calc.: C = 62.77%,
H = 6.28%, N = 8.45%; found: C = 62.97%, H = 6.33%, N = 8.41%.
1HNMR(CDCl3, d ppm, J Hz): 8.87(bs, 1H, H-8); 7.56–7.21(m,
7H, H3, H-5, H-17–H-21); 6.73(bd, 1H, H-6, 8.0); 4.07(m, 4H, H-
22, H-23); 3.92–3.66(m, 12H, H-CE); 3.05(s, 3H, H-15); 2.20(s,
3H, H-14).
3.6.5. Compounds 3 and 4
To 450 mg 4-AAP (2.2 mmoli) dissolved in 100 mL DCM were
added 530 mg (2 mmoli) 4-chloro-3,5-dinitrobenzoic acid and
2 mL triethylamine, and the mixture left at room temperature.
Next day the organic phase was extracted twice with 100 mL aque-
ous hydrochloric acid (1 M), and then twice with 100 mL aqueous
sodium hydrogen carbonate (3%), followed by 100 mL water. The
organic phase was then dried on anhydrous sodium sulfate, filtered
off, and the solvent removed using a rotavap. The residue was
chromatographied using a silica gel column (or preparative TLC
plates) and ethyl acetate as eluent. Yields: 3 ꢂ80%; 4 ꢂ20%.
3.6.6. Compound 3
Elemental analysis: C18H14ClN5O6, M = 431.5; calc.: C = 50.07%,
H = 3.27%, N = 16.22%; found: C = 50.10%, H = 3.25%, N = 16.09%.
1HNMR(DMSO-D6, d ppm, J Hz): 10.19(s, 1H, HN, deuterable);
8.90(s, 2H, H-10, H-14); 7.52(dd, 2H, H-17, H-19, 7.4, 8.4);
7.38(dd, 2H, H-16, H-20, 1.3, 8.4); 7.35(tt, 1H, H-18, 1.3, 7.4);
3.13(s, 3H, H-6); 2.21(s, 3H, H-3).
13CNMR(CDCl3, d ppm): 165.93(C-7); 162.19(C-13); 151.93(C-16);
150.19(Cq); 148.48(Cq); 134.47(Cq); 129.38(C-18, C-20); 126.43(C-
19); 124.80(C-17, C-21); 123.69(C-9); 121.51(C-5); 112.38(C-6);
13CNMR(DMSO-D6,
d
ppm): 161.37(C-5); 161.17(C-8);
152.43(C-15); 148.75(C-11, C-13); 134.89(C-12); 129.15(C-17, C-
19); 127.65(C-10, C-14); 126.55(C-18); 123.91(C-16, C-20);
121.72(C = 4); 106.16(C-3); 35.77(C-6); 11.06(C-7).
113.02(C-3);
69.24(CH2AO); 68.65(CH2AO); 35.96(C-15); 12.43(C-10).
IR(ATR in solid,
cmꢀ1): 3454; 3272, 2925, 2870, 1645, 1591,
108.72(C-10);
71.04(CH2AO);
70.36(CH2AO);
m
IR(ATR in solid,
m
cmꢀ1): 3180, 3069, 2930, 2851, 1731, 1672,
1494, 1452, 1297, 1266, 1213, 1128, 1048, 935, 761, 586.
1621, 1538, 1489, 1449, 1347, 1297, 1239, 1060, 918, 694, 586.
UV–Vis (methanol, kmax nm): 261.
Rf (silicagel, ethyl acetate): 0.08.
UV–Vis (methanol, kmax nm): 277.
Rf (silicagel, ethyl acetate): 0.73.
3.6.3. Compound 2: Method 1
3.6.7. Compound 4
To 225 mg 4-AAP (1.1 mmol) dissolved in 75 mL chloroform
were added 200 mg (1 mmol) 7-nitro-4-chlorobenzofurazan and
1 mL triethylamine, and the reaction mixture left overnight at
room temperature. Next day the organic phase was extracted twice
with 75 mL aqueous hydrochloric acid (1 M), followed twice by
75 mL aqueous sodium hydrogencarbonate (3%), and then by
75 mL water. The organic phase was then dried over anhydrous so-
dium sulfate, filtered off and the solvent removed under vacuo. TLC
on silica gel plates and ethyl acetate as eluent showed the desired
compound only in small amount (ꢂ5%) together with many other
unidentified compounds.
Elemental analysis: C29H26N8O7, M = 598; calc.: C = 58.19%,
H = 4.38%, N = 18.72%; found: C = 58.11%, H = 4.50%, N = 18.79%.
1HNMR(CDCl3, d ppm, J Hz): 8.70(s, 2H, H-10, H-14); 7.51–
7.28(m, 10H, H-16–H-20, H-27–H-31, H-arom); 3.12(s, 3H, H-6);
3.10(3H, H-25); 2.30(s, 3H, H-7); 2.10(s, 3H, H-24).
13CNMR(CDCl3, d ppm, T = 303 K): 162.09(C-5); 161.12(C-8);
160.72(C-23); 150.73(C-15 or C-26); 150.66(C-26 or C-15);
150.18(C-11 or C-13); 149.37(C-11 or C-13); 139.51(Cq);
138.80(Cq); 134.49(C-12); 133.94(C-26); 121.84(C-4, C-21);
129.48(C-28, C-30); 129.25(C-17, C-19); 127.83(C-10, C-14);
127.37(C-29); 127.34(C-18); 125.71(C-27, C-31); 124.83(C-16, C-
20); 109.16(C-22); 107.83(C-3); 35.88(C-6); 35.58(C-25);
11.78(C-24); 10.73(C-7).
3.6.4. Method 2
To 225 mg 4-aminoantipyrine (1.1 mmol) dissolved in 10 mL
DMF were added 200 mg (1 mmol) 7-nitro-4-chlorobenzofurazan
and 200 mg solid sodium hydrogencarbonate, and the mixture stir-
red at room temperature for 6 h. 100 mL of cold water were added
to the previous mixture, and the solid formed was filtered off and
dried. The residue thus obtained was purified on preparative TLC,
using silica gel plates and ethyl acetate as eluent. Yields: ꢂ20%.
Elemental analysis: C17H14N6O4, M = 366; calc.: C = 55.74%,
H = 3.85%, N = 22.94%; found: C = 55.61%, H = 3.85%, N = 22.78%.
1HNMR(CDCl3, d ppm, J Hz): 8.92(bs, 1H, H-8 deuterable);
8.25(d, 1H, H-13, 8.6); 7.53(t, 2H, H-17, H-19, 7.6); 7.44(d, 2H, H-
16, H-20, 7.6); 7.40(t, 1H, H-18, 7.6); 6.13(d, 1H, H-14, 8.6);
3.26(s, 3H, H-6); 2.21(s, 3H, H-7).
FT-IR(ATR in solid, m
cmꢀ1): 3435; 3221; 2994; 2927; 1620;
1528; 1487; 1453; 1418; 1345; 1274; 1139; 1103; 1062; 1024;
921; 756; 720; 638.
UV–Vis (methanol, kmax nm): 278.
Rf (silicagel, ethyl acetate): 0.09.
Compound 4 may be obtained also in good yields (80%) from 3
in reaction with 4-AAP in the presence of triethylamine, following
a similar procedure.
3.6.8. Compound 5
To 215 mg compound 3 (0.5 mmol) dissolved in 50 mL DCM
were added 120 mg (0.75 mmol) 4-aminotempo and 1 mL triethyl-
amine, and the reaction mixture left for 3 days at room tempera-

12
C. Remes et al. / Bioorganic Chemistry 41-42 (2012) 6–12
ture. The organic phase was then extracted twice with 50 mL aque-
ous hydrochloric acid (1 M), and then twice with 50 mL aqueous
sodium hydrogen carbonate (3%), followed by 50 mL water. The or-
ganic phase was then dried on anhydrous sodium sulfate, filtered
off, and the solvent removed using a rotavap. The residue was
chromatographied using a silica gel column (or preparative TLC
plates) and ethyl acetate/DCM 1/1 (v/v) as eluent. Yields: ꢂ80%.
Elemental analysis: C27H32N7O7, M = 566; calc.: C = 57.24%,
H = 5.69%, N = 17.30%; found: C = 57.41%, H = 5.85%, N = 17.18%.
EPR (DCM, Gauss): 15.40.
Acknowledgment
This work was partially supported by a Grant of the Romanian
National Authority for Scientific Research, CNCS – UEFISCDI, pro-
ject number PN-II-ID-PCE-2011-3-0408.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bioorg.2011.12.003.
IR(ATR in solid,
m
cmꢀ1): 3329; 2974; 2921; 2853; 1624; 1527;
1492; 1457; 1353; 1277; 1180; 1104; 920; 693; 586; 500.
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To 215 mg compound 3 (0.5 mmol) dissolved in 50 mL DCM
were added 137 mg (1 mmol) isoniazid and 10 mL triethylamine.
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13CNMR(DMSO-D6,
d
ppm): 171.88(C-21); 162.37(C-5);
161.76(C-8); 152.64(C-15); 149.40(C-24, C-25); 149.34(C-22);
144.18(Cq); 133.89(Cq); 133.55(C-4); 129.73(C-10, C-14);
129.05(C-17, C-19); 126.23(C-18); 123.58(C-16, C-20); 121.15(C-
23, C-26); 116.71(Cq); 107.32(C-3); 35.92(C-6); 11.03(C-7).
FT-IR(ATR in solid,
2853; 1623; 1595; 1528; 1490vs; 1407; 1362; 1281; 1221;
m
cmꢀ1): 3391; 3258; 3072; 2965; 2922;
1180; 1144; 1103; 1066; 749.
UV–Vis (methanol, kmax nm): 358.
Rf (silicagel, ethyl acetate): 0.02.
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