Synthesis and microbiological evaluation of several benzocaine derivatives

Article abstract of DOI:10.1016/j.crci.2013.03.012

Starting from benzocaine, a well-known anaesthetic, ten derivatives were synthesized and characterized by UV-vis, IR, NMR, and elemental analysis. Most of the compounds contain residues with recognized biological activity, like nicotinic acid (vitamin B3 or PP), biotin (vitamin B7 or H), lipoic acid (thioctic acid), adamantine, as well as other residues of crown-ether type, benzofurazane, naphtylurea, di- and tri-nitrobenzene, and a nitroxide radical. The biological evaluation of the obtained compounds included hydrophobicity (lipophobicity) assay, total antioxidant and microbiological activity tests.

Full text of DOI:10.1016/j.crci.2013.03.012

C. R. Chimie 16 (2013) 665–671
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Full paper/Memoire
Synthesis and microbiological evaluation of several benzocaine
derivatives
Anca Paun a, Irina Zarafu a, Miron T. Caproiu b, Constantin Draghici b, Maria Maganu b,
Ani I. Cotar c, Mariana C. Chifiriuc c, Petre Ionita a,
*
a University of Bucharest, Department of Organic Chemistry, 90-92 Panduri, Bucharest, Romania
b Center of Organic Chemistry, 202B Spl. Independentei, Bucharest, Romania
c Department of Microbiology, Faculty of Biology, University of Bucharest, Alea Portocalelor 1-3, 60101 Bucharest, Romania
A R T I C L E I N F O
A B S T R A C T
Article history:
Starting from benzocaine, a well-known anaesthetic, ten derivatives were synthesized and
characterized by UV–vis, IR, NMR, and elemental analysis. Most of the compounds contain
residues with recognized biological activity, like nicotinic acid (vitamin B3 or PP), biotin
(vitamin B7 or H), lipoic acid (thioctic acid), adamantine, as well as other residues of
crown-ether type, benzofurazane, naphtylurea, di- and tri-nitrobenzene, and a nitroxide
radical. The biological evaluation of the obtained compounds included hydrophobicity
(lipophobicity) assay, total antioxidant and microbiological activity tests.
Received 2 February 2013
Accepted after revision 26 March 2013
Available online 6 May 2013
Keywords:
Benzocaine
Synthesis
ß 2013 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
Structure
Biological evaluation
1. Introduction
water solubility, benzocaine cannot be used in parenteral
administration. As any medicine, benzocaine has advan-
In the quest for new medicines, drug design and
synthesis play an important part. Many medicines loose
their activity due to extensive use, requesting higher doses
or the replacement of the active compound. Among
medicines, anaesthetics are a separate class of compounds,
because many of them can induce addiction and therefore,
generate drug abuse [1–4].
tages and weak issues; therefore, new derivatives contain-
ing as substructure the benzocaine moiety are always of
interest. Previous studies have demonstrated that benzo-
cain-containing local anaesthetics and novel derivatives
exhibited antimicrobial activity against different species,
either Gram-positive, or Gram-negative, or fungal strains
[6,7].
Design and synthesis of novel medicines is a quite long
and expensive process, requesting a tremendous amount
of work. Besides, the chosen chemical as a prospective
medicine has to pass a huge number of tests, in which all
the aspects of the possible chemical and biological factors
have to be carefully addressed [5].
This paper deals with the synthesis, structural char-
acterization and the microbiological activity evaluation
tests of several novel benzocaine derivatives.
2. Results and discussion
Benzocaine, an anaesthetic, is
a
simple chemical
2.1. Synthesis and structural characterization
compound that induces pain relief; it is used in topical,
dermal and mucous formulations, but, because its low
Starting from benzocaine, all the compounds 1–10
(Fig. 1) were obtained practically in a single step, coupling
benzocaine with the desired compound. Although some of
the compounds (like 1, 3, 6, 7, Fig. 1) are present in the
literature [7–11], we synthesized them to cover a broader
* Corresponding author.
E-mail address: pionita@icf.ro (P. Ionita).
1631-0748/$ – see front matter ß 2013 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.crci.2013.03.012

666
A. Paun et al. / C. R. Chimie 16 (2013) 665–671
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
4
O
O
O
3
O
HN
2
NH
HN
HN
HN
S
HN
HN
N
S
5
S
O
O
O
1
O
O
O
O
O
O
O
O
O
8
10
9
7
NO2
N
O
N
NO2
H
N
HN
O2N
HN
HN
N O
HN
H
HN
N
O
6
NO2
NO2
NO2
O
Fig. 1. Chemical structure of the synthesized compounds 1–10.
range of chemicals with possible biological activity, and
also to be compared with the new synthesized derivatives.
Thus, to obtain the desired compounds 1–5, the
coupling reactions between benzocaine and the required
carboxylic acids have been tried in three different types of
reactions, in order to get the highest yields:
solid); this is accounted for by the nitrobenzofurazan
(NBD) moiety, well known for its intense colour. Com-
pounds 7 and 8, containing nitro groups, are yellow solids,
while all the others are white-grey solids.
In NMR spectra, amino groups are shifted under the
influence of their chemical neighbours, and they appear
between 7.5 and 10.5 ppm; all other 1H and 13C NMR
values confirm the structure (see also Section 3).
Compound 6 exhibits the well-known fluorescence of
the NBD moiety, with the emission value at 500 nm;
compound 10, containing a stable free radical moiety,
shows the corresponding triplet in the ESR spectrum with a
hyperfine coupling value of 15.5 G.
ꢀ
ꢀ
coupling using dicycloxexylcarbodiimide (DCC);
coupling using N-ethoxycarbonyl-2-ethoxy-1,2-dihy-
droquinoline (EEDQ);
ꢀ
coupling using the corresponding acid chloride of the
carboxylic acid.
It was shown that the higher yields were obtained by
using the corresponding acid chloride of the carboxylic
acid; between EEDQ and DCC, in our cases EEDQ was a
better coupling agent. Compounds 6–8 were obtained by
simple and effective nucleophilic aromatic substitution,
while compounds 9 and 10 were synthesized by a coupling
reaction between an amine and an isocyanate derivative.
The general yields were between 40 and 95%.
Most of the obtained compounds required purification
by column chromatography or preparative TLC; for
analytical samples obtained in this way, structural
characterization has been performed using current spec-
troscopic methods, like IR, UV–vis, NMR, and fluorescence
and electron spin resonance (ESR), as necessarily (fluor-
2.2. Biological evaluation
2.2.1. Lipophilicity
One of the most important properties of the medicines
is their hydrophobicity or lipophilicity (usually noted log P,
where P means the partitioning coefficient), which is
correlated with the water or fat solubility, and therefore,
with the capacity of crossing the cell membrane. The
standard experimental methods used to determine the
hydrophobicity values (log P) are the n-octanol/water
repartition measurements [12] and the reverse-phase (RP)
TLC [13], the latter being employed in this study; we
escence for compound 6, containing
a benzofurazan
Table 1
moiety, and ESR for compound 10, containing a free stable
radical moiety).
UV–vis, lipophilicity and TAC values for compounds 1–10.
Comp.
lmax
RM
0
bb
Log Pc
SAc
TACd
a
b
All the analyses performed to characterize compounds
1–10 confirmed their chemical structure. Thus, in IR
spectra, intense signals are noticed for the carbonyl groups,
either from the ethyl ester moiety or from the amide or
ureido moieties (around 1700 cmꢁ1). Amino groups appear
between 3200 and 3500 cmꢁ1 as broad signals. Aliphatic
groups appear in IR at about 2900–3000 cmꢁ1, while the
aromatic ones can be observed between 3000 and
3100 cmꢁ1. Nitro groups can easily be recognized by their
values at about 1350 cmꢁ1 and 1550 cmꢁ1, while the ether
1
2
385
272
311
273
288
472
341
378
292
317
1.23
1.14
1.30
2.98
ꢁ0.36
1.51
2.42
2.39
2.91
1.57
ꢁ2.82754
ꢁ2.91535
ꢁ2.76971
ꢁ4.31451
ꢁ0.57627
ꢁ2.79154
ꢁ3.21055
ꢁ3.82341
ꢁ4.84119
ꢁ3.00695
2.50
1.23
2.92
3.49
1.70
3.05
3.28
3.23
3.74
1.69
410
603
586
372
485
515
549
580
518
607
3.2
6.4
12
3
4
0.8
8.8
10.4
8
5
6
7
8
3.2
43.2
22.3
9
10
a
nm, in methanol.
ones appear at about 1100–1200 cmꢁ1
.
b
c
Experimental values from RP–TLC.
Theoretical values.
In UV–vis spectra (Table 1), the most bathochromic
shift is noticed for compound 6 (472 nm, as an orange
d
Experimental values.

A. Paun et al. / C. R. Chimie 16 (2013) 665–671
667
4.0
calculated as well the theoretical value of log P, employing
the HyperChem 8 molecular modelling package [14].
RP–TLC requires a non-polar stationary phase (i.e. silica
gel impregnated with paraffin oil) and a mixture of two
solvents, in which one is water (i.e. acetone–water). This
method is widely used to measure the experimental
lipophilicity (RM0 ) and the specific hydrophobic surface (b)
using equations (1) and (2) [15].
3.5
3.0
2.5
2.0
1.5
1.0
ꢀ
ꢁ
1
RM ¼ log
ꢁ 1
(1)
(2)
RF
RM ¼ RM þ bC
0
The values of RM and b obtained with equations (1) and
0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
(2) are the best indicators of the lipophilicity and of the
RM0
specific hydrophobic surface area. The intercept RM is the
0
RM value of a compound extrapolated to zero organic phase
concentration in the eluent; the slope b is the change of
lipophilicity caused by a unit concentration change in the
organic phase (Table 1). A good linear correlation was
found between RM and C, characterized by high values of
the correlation coefficient R (0.90–0.99).
Fig. 2. Linear correlation between theoretical and experimental
lipophilicity values.
belonging to the following genera and species: Escherichia
coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococ-
cus aureus, and Candida albicans.
The attempt to correlate the experimental lipophilicity
values RM with the theoretical ones, logs P, is shown in
0
Fig. 2. It can be easily seen that there is a general trend in
the evolution of the lipophilicity values, with the exception
of compounds 2 and 10, which seem to be out of range. The
linear correlation found for all the compounds can be
Some of the tested substances exhibited moderate and
good antimicrobial activity against the tested strains. A
minimal inhibitory concentration (MIC) of 128 to 256
mg/
mL was considered as moderate, in accordance with other
written as logP ¼ 0:66RM þ 1:55 (however, R = 0.77).
studies, while a MIC value lower than 128
indicator for a good inhibitory activity.
mg/mL was an
0
2.2.2. Total antioxidant capacity
It is to be noticed that the compounds 4, 9 and 10
exhibited large spectrum of antibacterial activity
The antioxidant capacity of all the synthesized com-
pounds was measured using the well-known DPPH
method [16], in which the absorbance of a mixture of
2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) and the
compound studied was measured initially and after 30 min
at 517 nm. The total antioxidant capacity (TAC) deter-
mined using equation (3) is compiled in Table 1 [17].
a
directed against Gram-negative P. aeruginosa and Gram-
positive S. aureus strains. Three other compounds, i.e. 2, 6
and 8, exhibited only anti-pseudomonal activity (Table 2).
None of the tested compounds exhibited significant anti-
fungal activity.
Concerning the anti-biofilm activity of the tested
compounds, the microtitre assay allowed us to establish
the concentration range that inhibited the development of
microbial biofilms on the plastic walls, quantified by
measuring the absorbance of the adhered and coloured
cells at 490 nm. None of the tested compound inhibited the
E. coli biofilm development (Supplementary data, Fig. S1);
as well, all the tested compounds exhibited anti-
P. aeruginosa biofilms activity at high concentations,
Abs0 ꢁ Abs30min
TAC ¼
ꢂ 100
(3)
Abs0
The highest TAC value was obtained for compound 9,
followed by 10. These values can be related to the ureido
moiety and/or to the naphtyl or free radical moieties.
2.2.3. Microbiological evaluation
ranging from 1024 to 64
Fig. S2).
mg/mL (Supplementary data,
The antimicrobial activity [18–21] of the obtained
compounds was tested against bacterial and fungal strains
Table 2
Minimal inhibitory concentration values (
m
g/mL) of the obtained compounds.
Tested compound
Escherichia coli
Pseudomonas aeruginosa
Bacillus subtilis
Staphylococcus aureus
Candida albicans
1
2
512
1024
1024
1024
512
512
8
1024
1024
1024
1024
1024
1024
1024
1024
1024
1024
1024
1024
1024
256
512
512
3
1024
16
256
4
512
5
1024
256
512
128
8
512
1024
1024
512
6
1024
512
1024
1024
1024
256
7
8
512
1024
512
9
1024
1024
10
32
16
512

668
A. Paun et al. / C. R. Chimie 16 (2013) 665–671
The compound 10 inhibited S. aureus biofilm develop-
3.2.2. Compound 2
ment on a large range of concentrations, from 1024 to
16 mg/mL, as demonstrated by the low absorbance values
obtained for this compound (Supplementary data, Fig. S3);
compounds 2 and 3 inhibited the B. subtilis biofilm
development at the highest tested concentration of
This compound was prepared in a similar way as 1,
using biotin instead of nicotinic acid.
1H NMR(dmso-d6,
d ppm, J Hz, T = 303 K): 10.28(s, 1H,
HN-10, deuterable); 7.90(d, 2H, H-3, H-5, 8.8); 7.74(d, 2H,
H-2, H-6, 8.8); 6.46(s, 1H, H-20 or H-21, deuterable);
6.38(s, 1H, H-21 or H-20, deuterable); 4.30(dd, 1H, H-17,
3J(H18A–H17) = 4.9, 3J(H16–H17) = 7.6); 4.28(q, 2H, H-8, 7.1);
4.15(dd, 1H, H-16, 3J(H17–H16) = 7.6, 3J(H15–H16) = 4.5);
3.13(dt, 1H, H-15, 3J(H16–H15) = 4.5, 3J(H14–H15) = 8.0);
2.83(dd, 1H, H-18A, syst AB, 3J(H17–H18A) = 4.9
gemJ = 12.3); 2.59(d, 1H, H-18B, syst AB, gemJ = 12.3);
2.37(t, 2H, H-11, 7.2); 1.54ꢃ1.36(m, 4H, H-12, H-13);
1024
mg/mL (Supplementary data, Fig. S4); as well, all
the tested compounds slightly inhibited C. albicans bio-
films at higher concentrations (from 1024 to 512
(Supplementary data, Fig. S5).
mg/mL)
3. Experimental
3.1. Apparatus and materials
1.31(t, 3H, H-9, 7.1). 13C NMR(dmso-d6,
d ppm, T = 303 K):
171.80(C-10); 165.33(C-7); 162.72(C-22); 143.65(C-13);
143.78(C-12); 141.03(C-1); 130.15(C-3, C-5); 123.91(C-4);
118.33(C-2, C-6); 61.04(C-17); 60.36(C-8); 59.21(C-16);
55.35(C-15); 39.83(C-18); 36.29(C-11); 28.19(C-12);
28.07(C-13); 24.93(C-14); 14.19(C-9). Elemental analysis:
All the chemicals, materials and solvents were pur-
chased from Sigma–Aldrich, Alfa Aesar or Chimopar, and
used as received. UV–vis spectra were recorded in
methanol at ambient temperature, on an UVD-3500 double
bean spectrometer, using quartz cell with 1-cm path
length. IR spectra were recorded with a Bruker Vertex 70
spectrometer (as solid samples, ATR); 1H- and 13C NMR
spectra were recorded with a Varian Inova-400 spectro-
meter (at selected temperatures, in deuterated solvents
CDCl3 and DMSO-d6, isotopic purity 99.9%). ESR spectra
were recorded with a Jeol JES-FA 100 spectrometer in DCM
as a solvent and at room temperature.
calculated
for
C19H25N3O4S
(M = 391): C = 58.29;
H = 6.44; N = 10.73; found: C = 58.29; H = 6.39; N = 10.70.
IR (ATR, cmꢁ1): 3249.80; 2928.34; 2858.72; 2361.27;
1692.16; 1596.46; 1529.39; 1462.28; 1407.37; 1366.57;
1308.09; 1275.16; 1171.99; 1103.10; 1019.92; 856.04;
767.83; 694.75; 615.00. Rf = 0.42 (DCM:methanol = 9:1).
3.2.3. Compound 3
To 1 mmol of lipoic acid, dissolved in 25 mL of DCM,
were added 1 mmol of benzocaine and 1.2 mmol of EEDQ;
after 2 days, 75 mL of DCM were added, and the organic
phase extracted with 100 mL of diluted aqueous hydro-
chloric acid (10%), followed by aqueous sodium hydrogen
carbonate (3%), and then dried using anhydrous sodium
sulphate. The removal of the DCM affords the crude
compound, which can be purified on preparative silica TLC
plates, using DCM as eluent.
3.2. Synthesis
3.2.1. Compound 1
To 2 mmol of nicotinic acid suspended in 30 mL of 1,2-
dichloroethane were added two drops of DMF and 2 mL of
thionyl chloride, and the mixture was refluxed for 2 h; the
solvent and excess thionyl chloride were removed under
vacuum, and to the residue were added 50 mL of DCM,
2 mmol of benzocaine, and 5 mL of thiethylamine; the next
day, 50 mL of DCM were added, and the organic phase
extracted with 100 mL of diluted aqueous hydrochloric
acid (10%), followed by aqueous sodium hydrogen
carbonate (3%), and then dried using anhydrous sodium
sulphate. The removal of the DCM affords the crude
compound, which can be purified on preparative silica TLC
plates, using DCM as an eluent.
1H NMR(CDCl3,
d ppm, J Hz, T = 303 K): 8.19(s, 1H, HN,
deuterable); 7.98(d, 2H, H-3, H-5, 8.8); 7.64(d, 2H, H-2, H-6,
8.8); 4.35(q, 2H, H-8, 7.1); 3.53(qv, 1H, H-15, 7.1); 3.12(m,
2H, H-17); 2.45(m, 1H, H-16A, syst. AB); 2.40(t, 2H, H-11,
7.7); 1.89(m, 1H, H-16B); 1.56ꢃ1.48(m, 4H, H-12, H-13);
1.38(t, 3H, H-9, 7.1). 13C NMR(CDCl3,
d ppm, T = 303 K):
171.77(C-10); 166.31(C-7); 142.33(C-1); 130.71(C-3, C-5);
127.94(C-4); 118.91(C-2, C-6); 60.94(C-8); 56.37(C-15);
1H NMR(CDCl3,
d
ppm, J Hz, T = 303 K): 9.11(bs, 1H, H-
40.23(C-17);
28.83(CH2); 25.14(CH2); 14.35(C-9). Elemental analysis:
calculated for C17H23NO3S2 (M = 353): C = 57.76;
38.48(CH2);
37.39(CH2);
34.60(CH2);
15); 8.78(bs, 1H, HN, deuterable); 8.73(bs, 1H, H-14);
8.21(d, 1H, H-12, 7.8); 8.02(d, 2H, H-3, H-5, 8.6); 7.75(d,
2H, H-2, H-6, 8.6); 7.40(d, 1H, H-13, 7.8); 4.36(q, 2H, H-8,
7.1); 1.39(t, 3H, H-9, 7.1). 13C NMR(CDCl3,
H = 6.56; N = 3.96; found: C = 57.81; H = 6.56; N = 3.92. IR
(ATR, cmꢁ1): 3310.45; 2975.64; 2940.72; 2925.04; 2858.51;
1704.06; 1661.41; 1608.81; 1591.95; 1520.57; 1463.04;
1404.02; 1363.57; 1334.80; 1306.00; 1275.06; 1256.19;
1170.93; 1105.86; 1039.77; 1018.42; 960.88; 852.69;
767.94; 730.37; 692.57; 579.63; 498.96. Rf = 0.53
(DCM:ethyl acetate = 9:1).
d ppm,
T = 303 K): 166.20(C-7); 164.30(C-10); 152.37(bs, C-15);
148.02(bs, C-14); 141.98(C-1); 135.87(C-12); 131.56(C-
11); 130.79(C-3, C-5); 126.52(C-4); 123.86(C-13);
119.68(C-2, C-6); 61.04(C-8); 14.33(C-9). Elemental ana-
lysis: calculated for C15H14N2O3 (M = 270): C = 66.66;
H = 5.22; N = 10.36; found: C = 66.62; H = 5.25; N = 10.29.
IR (ATR, cmꢁ1): 3497.09; 3315.39; 3188.23; 3010.44;
2975.56; 2937.49; 2874.14; 1683.34; 1596.87; 1535.01;
1509.94; 1486.04; 1453.11; 1422.82; 1406.38; 1388.43;
1361.21; 1306.92; 1274.58; 1244.91; 1169.54; 1102.34;
1030.12; 1010.88; 856.53; 829.99; 769.22; 698.76; 499.91.
Rf = 0.27 (DCM:ethyl acetate = 1:1).
3.2.4. Compound 4
This compound was prepared in a similar way as 1,
using 1-adamatane carboxylic acid instead of nicotinic
acid.
1H NMR(CDCl3,
d
ppm, J Hz, T = 303 K): 7.49(s, 1H, HN,
deuterable); 8.00(d, 2H, H-3, H-5, 8.6); 7.64(d, 2H, H-2,

A. Paun et al. / C. R. Chimie 16 (2013) 665–671
669
H-6, 8.6); 4.36(q, 2H, H-8, 7.1); 2.06(m, 3H, H-15, H-16, H-
17); 1.98(m, 6H, H-12, H-13, H-14); 1.74(m, 6H, H-18, H-
845.14; 809.56; 773.15; 755.76; 734.11; 699.49; 685.80;
620.58; 596.36; 511.27. Rf = 0.75 (DCM:ethyl acet-
ate = 9:1), Rf = 0.22 (DCM).
19, H-20); 1.39(t, 3H, H-9, 7.1). 13C NMR(CDCl3,
d ppm,
T = 303 K): 176.29(C-7); 171.19(C-10); 142.23(C-1);
130.73(C-3, C-5); 125.79(C-4); 118.94(C-2, C-6);
60.83(C-8); 41.75(C-11); 39.23(C-12, C-14, C-18);
36.23(C-18, C-19, C-20); 27.69(C-15, C-16, C-17);
14.36(C-9). Elemental analysis: calculated for C20H25NO3
(M = 327): C = 73.37; H = 7.70; N = 4.28; found: C = 73.32;
H = 7.70; N = 4.27. IR (ATR, cmꢁ1): 3295.38; 2901.47;
2850.13; 2677.73; 1804.74; 1716.19; 1653.48; 1593.49;
1519.16; 1474.05; 1450.01; 1405.24; 1307.43; 1275.51;
1249.83; 1172.45; 1153.04; 1100.01; 995.86; 973.29;
935.36; 854.79; 766.00; 680.77; 617.42; 504.04. Rf = 0.35
(DCM).
3.2.7. Compound 7
This compound was prepared in a similar way as 6,
using 2,4-dinitro-1-fluorobenzene instead of NBD-chlor-
ide, and keeping the reaction on for 2 days. The crude
compound can be purified on a silica column, using DCM as
an eluent.
1H NMR(CDCl3,
d ppm, J Hz, T = 303 K): 10.04(s, 1H, HN,
deuterable); 9.19(d, 1H, H-12, 2.5); 8.24(dd, 1H, H-14, 2.5,
9.2); 8.17(d, 2H, H-3, H-5, 8.5); 7.39(d, 2H, H-2, H-6, 8.5);
7.35(d, 1H, H-15, 9.2); 4.42(q, 2H, H-8, 7.1); 1.43(t, 3H, H-9,
7.1). 13C NMR(CDCl3,
d ppm, T = 303 K): 165.50(C-7);
145.72(C-10); 141.01(C-1); 138.26(C-13); 132.13(C-11);
131.66(C-3, C-5); 130.06(C-14); 129.08(C-4); 124.02(C-
15); 123.91(C-2, C-6); 116.30(C-12); 61.40(C-8); 14.35(C-
9). Elemental analysis: calculated for C15H13N3O6
(M = 331): C = 54.38; H = 3.96; N = 12.68; found: C = 54.35;
H = 3.95; N = 12.65. IR (ATR, cmꢁ1): 3321.81; 3113.80;
3090.06; 2991.76; 2924.50; 2854.23; 1704.04; 1621.19;
1589.76; 1514.12; 1441.72; 1422.00; 1364.52; 1336.07;
1262.69; 1221.81; 1175.14; 1127.45; 1060.21; 1017.59;
923.68; 868.81; 848.56; 828.10; 763.38; 736.91; 701.00;
686.09; 587.14; 505.14. Rf = 0.6 (DCM).
3.2.5. Compound 5
This compound was prepared in a similar way as 1,
using 4-carboxybenzo-15-crown-5 acid instead of nico-
tinic acid.
1H NMR(CDCl3,
d ppm, J Hz, T = 303 K): 8.97(bs, 1H, NH,
deuterable); 8.00 (d, 2H, H-3, H-5, 8.6); 7.88(d, 2H, H-2, H-
6, 8.6); 7.75(dd, 1H, H-16, 1.8, 8.4); 7.59(d, 1H, H-12, 1.8);
6.89(d, 1H, H-15, 8.4); 4.36(q, 2H, H-8, 7.1); 4.20(m, 4H, H-
20, H-27, syst. A2B2); 3.92(m, 4H, H-21, H-26, syst. A2B2);
3.76(bs, 8H, H-22 ꢃ H-25);1.39(t, 3H, H-9, 7.1). 13C
NMR(CDCl3, T = 303 K,
d ppm): 166.31(C-7); 165.59(C-
10); 162.40(Cq); 154.51(Cq); 142.86(C-1);121.37(C-11);
125.59(C-4); 130.61(C-3, C-5); 125.45(C-2, C-6); 119.41(C-
3.2.8. Compound 8
This compound was prepared in a similar way as 6,
using 2,4,6-trinitro-1-chlorobenzene instead of NBD-
chloride; no purification was required.
16);
114.99(C-15);
112.02(C-12);
71.03(CH2-CE);
70.30(CH2-CE); 70.19(CH2-CE); 69.21(CH2-CE); 60.80(C-
8); 14.38(C-9). Elemental analysis: calculated for
1H NMR(CDCl3,
d ppm, J Hz, T = 303 K): 10.26(s, 1H, HN,
C
24H29NO8 (M = 459); C = 62.73; H = 6.36; N = 3.05; found:
deuterable); 9.13(s, 2H, H-12, H-14); 8.06(d, 2H, H-3, H-5,
8.6); 7.11(d, 2H, H-2, H-6, 8.6); 4.38(q, 2H, H-8, 7.1); 1.40(t,
C = 62.68; H = 6.33; N = 3.01. IR (ATR, cmꢁ1): 3504.96;
2928.12; 2870.04; 1770.45; 1708.44; 1669.36; 1596.33;
1510.90; 1451.97; 1427.62; 1347.25; 1321.39; 1267.64;
1211.39; 1174.64; 1127.40; 1029.74; 935.32; 872.12;
770.84; 755.34; 728.62; 697.93; 610.64. Rf = 0.47
(DCM:methanol = 9:1).
3H, H-9, 7.1). 13C NMR(CDCl3,
d ppm, T = 303 K): 165.23(C-
7); 141.22(C-1); 138.50(C-13); 138.06(C-11, C-15);
131.58(C-3, C-5); 130.79(C-10); 129.30(C-4); 127.24(C-12,
C-14); 120.02(C-2, C-6); 61.36(C-8); 14.31(C-9). Elemental
analysis: calculated for C15H12N4O8 (M = 376): C = 47.88;
H = 3.21; N = 14.89; found: C = 47.81; H = 3.21; N = 14.83. IR
(ATR, cmꢁ1): 3289.61; 3096.11; 3058.85; 2980.35; 2936.26;
1705.89; 1621.42; 1593.59; 1554.38; 1517.55; 1463.78;
1433.04; 1418.44; 1356.76; 1333.19; 1310.27; 1279.88;
1170.87; 1118.19; 1103.67; 1088.96; 1019.06; 932.82;
869.91; 825.26; 786.08; 766.68; 736.79; 722.96; 699.51;
675.01; 574.23; 485.41. Rf = 0.58 (DCM).
3.2.6. Compound 6
To 1 mmol of benzocaine dissolved in 7 mL of DMF were
added 1 mmol of NBD-chloride and 2 mmol of powdered
sodium hydrogencarbonate; the next day, the mixture was
poured in about 100 mL of cold water, and the precipitate
filtered off and dried, affording the crude compound, which
can be purified on a silica column, using DCM as an eluent.
1H NMR(CDCl3,
d
ppm, J Hz, T = 303 K): 8.50(d, 1H, H-14,
3.2.9. Compound 9
8.6); 8.19(d, 2H, H-3, H-5, 8.6); 7.95(s, 1H, HN, deuterable);
7.48(d, 2H, H-2, H-6, 8.6); 6.95(d, 1H, H-15, 8.6); 4.42(q,
To 1 mmol of benzocaine dissolved in 50 mL of DCM
was added 1 mmol of 1-naphtylisocyanate; after 1 week,
50 mL of DCM were added, and the organic phase was
extracted with 100 mL of diluted aqueous hydrochloric
acid (10%), followed by aqueous sodium hydrogen
carbonate (3%), and then dried using anhydrous sodium
sulphate. The removal of the DCM affords the crude
compound, which can be purified on preparative silica TLC
plates, using DCM as an eluent.
2H, H-8, 7.1); 1.43(t, 3H, H-9, 7.1). 13C NMR(CDCl3,
d ppm,
T = 303 K): 165.49(C-7); 145.07(C-13); 143.78(C-12);
141.03(C-1); 139.36(C-11); 135.42(C-14); 131.69(C-3, C-
5); 128.45(C-4); 127.01(Cq); 121.79(C-2, C-6); 102.20(C-
15); 61.37(C-8); 14.35(C-9). Elemental analysis: calculated
for C15H12N4O5 (M = 328): C = 54.88; H = 3.68; N = 17.07;
found: C = 54.92; H = 3.68; N = 17.05. IR (ATR, cmꢁ1):
3278.64; 3097.08; 3050.33; 2985.58; 2922.98; 2853.09;
1702.40; 1626.82; 1597.56; 1554.40; 1505.53; 1446.94;
1417.18; 1397.11; 1366.16; 1308.57; 1278.61; 1184.36;
1162.47; 1091.43; 1034.79; 1016.76; 993.95; 898.26;
1H NMR(dmso-d6,
d ppm, J Hz, T = 303 K): 10.21(s, 1H,
HN, deuterable); 9.33(s, 1H, HN, deuterable); 8.34(d, 1H,
H-19, 8.0); 8.04(dd, 1H, H-12, 1.4, 8.0); 7.92(m, 1H, H-16);
7.91(d, 2H, H-3, H-5, 8.8); 7.68(d, 2H, H-2, H-6, 8.8);

670
A. Paun et al. / C. R. Chimie 16 (2013) 665–671
7.66(dd, 1H, H-14, 1.4, 8.0); 7.50ꢃ7.63(m, 2H, H-17, H-18);
nephelometric standard, obtained from 15–18 h bacterial
cultures developed on solid media and stock solutions of
the new compounds of 1024 mg/mL concentration. For the
7.49(t, 1H, H-13, 8.0); 4.29(q, 2H, H-8, 7.1); 1.32(t, 3H, H-9,
7.1). 13C NMR(dmso-d6,
d
ppm, T = 303 K): 165.42(C-7);
152.75(C-10); 144.52(C-11); 134.01(C-1); 133.63(C-15);
130.32(C-3, C-5); 128.24(CH-13); 125.92(C-4); 125.84(CH-
naft); 125.72(CH-naft); 125.66(CH-naft); 123.08(CH-naft);
122.52(C-20); 121.69(CH-19); 117.43(CH-12); 117.02(C-2,
C-6); 60.19(C-8); 14.16(C-9). Elemental analysis:
calculated for C20H18N2O3 (M = 334): C = 71.84; H = 5.43;
N = 8.38; found: C = 71.86; H = 5.43; N = 8.39. IR (ATR,
cmꢁ1): 3338.90; 3266.42; 2983.45; 1703.76; 1646.35;
1595.33; 1546.53; 1490.58; 1442.94; 1406.26; 1364.23;
1343.54; 1321.97; 1271.08; 1239.46; 1215.79; 1174.10;
1101.12; 856.57; 786.03; 763.81; 729.40; 695.14; 554.51.
Rf = 0.45 (DCM:ethyl acetate = 9:1).
MIC assay, serial binary dilutions were performed in
nutrient broth distributed in 96-multiwell plates, further
inoculated with a standard inoculum of the microbial
strains. The plates were incubated at 37 8C for 24 h.
The MIC was read by wells’ observation: in the first
wells, containing high concentrations of compounds, the
culture growth was not visible, the microbial cells being
killed or inhibited by the tested compound. At lower
concentrations of the tested compounds, the microbial
culture became visible. The lowest concentration, which
inhibited the visible microbial growth, was considered the
MIC value for the tested compound. In the next wells,
including the standard culture growth control wells, the
medium become muddy as a result of the microbial
growth. In the sterility control wells series, the medium
had to remain clear. From the last well without any visible
microbial growth and from the first one with a microbial
growth, Gram stained smears were performed for the
results confirmation. For the evaluation of the influence of
different complex concentrations on the ability of the
tested bacterial strains to colonize the inert substratum, a
very simple microtitre plate method was used. In this
purpose, the microplates used for the MIC assay were
emptied, washed three times by PBS (phosphate buffered
saline). The biofilm formed on the plastic wells wall was
fixed for 5 min with cold methanol, coloured for 15 min by
violet crystal solution and resuspended by a 33% acetic acid
solution. Cell density was measured by reading the optical
density of the coloured solution at 490 nm using an ELISA
reader (Apollo LB 911).
3.2.10. Compound 10
To 0.5 mmol of benzocaine dissolved in THF was added
0.51 mmol of 4-isocyanto-TEMPO dissolved in a small
amount of THF, and then, the mixture was stirred for about
1 day. A cold solution of aqueous hydrochloric acid (1 M)
was added and after 15 min, the mixture was extracted
with DCM, the organic layer separated, dried over
anhydrous sodium sulphate, and the solvent removed
under vacuum, affording the crude product, which was
purified by preparative TLC.
Elemental analysis: Calculated for C19H28N3O4
(M = 362): C = 62.96; H = 7.79; N = 11.59; found: C = 62.81;
H = 7.77; N = 11.33. IR (ATR, cmꢁ1): 3387.39; 3291.98;
3083.97; 2960.59; 2928.49; 1623.34; 1592.31; 1502.03;
1420.68; 1354.88; 1271.08; 1172.59; 1115.29; 1072.42;
935.52; 905.70; 870.15; 814.40; 729.78; 657.64; 514.68.
Rf = 0.25 (DCM:ethyl acetate = 9:1).
3.3. Lipophilicity measurements
4. Conclusion
Analytical silica gel plates, impregnated overnight with
5% paraffin oil in heptane, were allowed to dry at ambient
temperature and then used. The eluent was a mixture of
acetone with water in different proportions (40–80%
acetone). The RM values necessary for the determination
of the hydrophobicity are obtained as shown in equation
(1). The specific hydrophobic surface area b was obtained
using the linear correlation between the RM values and the
concentration of organic solvent (C) in the eluent. The
Ten compounds derived from benzocaine were synthe-
sized and their corresponding structure characterized by
specific means. The biological assays have demonstrated
that the substances 9 and 10 have the potential to be used
as large spectrum antimicrobial agents, being active both
on Gram-negative P. aeruginosa and the Gram-positive
S. aureus. Four of the tested compounds, 2, 4, 9, and 10,
proved to be very active, exhibiting low MIC values on
P. aeruginosa strains. The tested substances also exhibited
an inhibitory activity on the ability of microbial strains to
develop biofilms, the intensity of this effect being
dependent on the tested microbial strains and the
concentration of the tested substances.
calculated log
P values were obtained using the
HyperChem 8 molecular modelling package.
3.4. TAC measurements
These measurements were performed in methanol at
ambient temperature, the final mixture containing DPPH
at a 1.5 ꢂ 10ꢁ4 M concentration and one of the compounds
1–10 at a 0.2 mg/mL concentration. Each mixture was kept
for 30 min, followed by absorbance measurement at
517 nm. The TAC values were obtained using equation (3).
Acknowledgements
This work was partially supported by a Grant of the
Romanian National Authority for Scientific Research, CNCS–
UEFISCDI, project number PN-II-ID-PCE-2011-3-0408.
Appendix A. Supplementary data
3.5. Microbiological activity testing
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
crci.2013.03.012.
In our experiments, we used bacterial suspensions of
1.5 ꢂ 108 CFU/mL density, corresponding to 0.5 McFarland

A. Paun et al. / C. R. Chimie 16 (2013) 665–671
[11] B. Linke, J. Prak. Chem. 91 (1915) 202.
671
References
[12] C. Hansch, A. Leo, S.B. Mekapati, A. Kurup, Bioorg. Med. Chem. 12 (2004)
3391.
[13] G. Ionita, T. Constantinescu, P. Ionita, J. Planar Chromatogr. - Mod. TLC
11 (1998) 141.
[1] G.R. Strichartz, V. Sanchez, R. Arthue, R. Chafetz, D. Martin, Anesth.
Analg. 71 (1990) 158.
[2] L.M.A. Pinto, D.K. Yokaichiya, L.F. Fraceto, E. de Paula, Biophys. Chem. 87
[14] www.hyper.com.
(2000) 213.
[15] Y. Darwish, T. Cserhati, E. Forgacs, J. Chromatogr. A 668 (1994) 485.
[16] C. Remes, A. Paun, I. Zarafu, M. Tudose, M.T. Caproiu, G. Ionita, C. Bleotu,
L. Matei, P. Ionita, Bioorg. Chem. 41–42 (2012) 6.
[17] R.L. Prior, X. Wu, K. Schaich, J. Agric. Food Chem. 53 (2010) 4290.
[18] P.B. Mohite, R.B. Pandhare, S.G. Khanage, Biointerface Res. Appl. Chem.
2 (2012) 258.
[19] P.A. Pavaskar, S.S.V. Patil, I. Furtado, A.V. Salker, Biointerface Res. Appl.
Chem. 2 (2011) 374.
[20] G.M. Niculescu, C. Draghici, M.C. Chifiriuc, L. Marutescu, C. Bleotu, A.V.
Missir, Med. Chem. Res. 21 (2012) 308.
[3] P.J. Kuzma, M.D. Kline, M.D. Calkins, P.S. Staats, Reg. Anesth. 22 (1997)
543.
[4] H.-Y. Yu, P. Sun, W.-Y. Hou, Int. J. Pharm 176 (1998) 133.
[5] S.R. Prasad, T. Saraswathy, V. Niraimathi, B. Indhumathi, Int. J. Pharm.
Pharmac. Sci. 4 (2012) 285.
[6] M.E. Morrow, C.W. Berry, Anesth. Prog. 35 (1988) 9.
[7] Z. Pongratz, Monatsh. Chem. 88 (1957) 330.
[8] L.J. Reed, M. Koike, M.E. Levitch, F.R. Leach, J. Biol. Chem. 232 (1958)
143.
[9] I.-H. Kim, Y.-K. Park, B.D. Hammock, K. Nishi, J. Med. Chem. 54 (2011)
1752.
[10] J.-C. Halle, P. Soulie, M.-J. Pouet, M. Mokhtari, Can. J. Chem. 75 (1997)
1240.
[21] R. Olar, M. Badea, D. Marinescu, M.C. Chifiriuc, C. Bleotu, M.N. Grecu, E.
Iorgulescu, M. Bucur, V. Lazar, A. Finaru, Eur. J. Med. Chem. 45 (2010)
2868.