Screening for potential antimicrobial activities of some cationic uracil biocides against wide-spreading bacterial strains

Article abstract of DOI:10.1007/s11743-010-1229-0

Eight novel uracil-based cationic surfactants containing Schiff base species were synthesized and characterized using elemental analysis, FTIR, ¹H-NMR and ¹³C-NMR spectroscopy. The surface activities of the synthesized Schiff bases and their cationic derivatives were determined based on interfacial tension measurements and partition coefficient values in water/octanol system. The synthesized Schiff bases and their cationic derivatives were evaluated as novel biocides against different bacteria and fungi strains. The results showed that the biocidal activity of the synthesized Schiff bases was considerably increased by quaternization. The influence of the cationic surfactants as biocides was increased by increasing the hydrophobic chain length and the presence of the methoxy groups. The biocidal activity was also increased by increasing the partition coefficient inwater/octanol system.The structure and surface activity/ biocidal activity of the different compounds were discussed. AOCS 2010.

Full text of DOI:10.1007/s11743-010-1229-0

J Surfact Deterg (2010) 13:503–511
DOI 10.1007/s11743-010-1229-0
ORIGINAL ARTICLE
Screening for Potential Antimicrobial Activities of Some Cationic
Uracil Biocides Against Wide-Spreading Bacterial Strains
•
Nabel A. Negm Ismail A. Aiad Salah M. Tawfik
•
Received: 25 May 2010 / Accepted: 22 July 2010 / Published online: 24 August 2010
Ó AOCS 2010
Abstract Eight novel uracil-based cationic surfactants
containing Schiff base species were synthesized and charac-
terized using elemental analysis, FTIR, 1H-NMR and
13C-NMR spectroscopy. The surface activities of the syn-
thesized Schiff bases and their cationic derivatives were
determined based on interfacial tension measurements and
partition coefficient values in water/octanol system. The
synthesized Schiff bases and their cationic derivatives were
evaluated as novel biocides against different bacteria and
fungi strains. The results showed that the biocidal activity of
the synthesized Schiff bases was considerably increased by
quaternization. The influence of the cationic surfactants as
biocides was increased by increasing the hydrophobic chain
length and the presence of the methoxy groups. The biocidal
activity was also increased by increasing the partition coeffi-
cient in water/octanol system. The structure and surface activity/
biocidal activity of the different compounds were discussed.
originates from the presence of polar groups (cationic,
anionic or zwitterionic) and the presence of organic or
hydrophobic chains. In addition to the large applications
known for surfactant including detergency [2], emulsifi-
cation [3], demulsification [4], metal flotation [5] and sol-
ubilization [6], cationic surfactants have applicability as
biocides against bacteria [7], fungi and yeast [8]. The
biocidal activity of the cationic surfactants was greatly
developed in the last 20 years due to increase of self
immunity for microorganisms toward the conventional
biocides [9–12]. Therefore, the need of new biocides is
important in the environmental and medicinal life. Differ-
ent cationic surfactants as biocides were discovered and
developed based on numerous starting materials including
simply aliphatic tertiary amines [13], heterocyclic com-
pounds and naturally occurring compounds [14]. Studies of
the cationic surfactants as biocides showed a strong rela-
tionship between their surface and biological activity.
Increasing the surface activity was accompanied in several
studies by a strong biocidal activity, and vice versa. That
was referred to action mode of these compounds as bio-
cides towards the different microorganisms. Several works
described the action mode of the cationic surfactants as
biocides [15]. In Gram positive bacteria, the adsorption
of the cationic biocides occurs in the teichoic acid layer
which characterized by the charged nature and the ability
to interact with the positively charged molecules. While
in Gram-negative bacteria, the cellular membrane is rich
in phospholipid bilayer (highly nonpolar layer) which is
consequently the target of the positively charged biocide
molecules. The adsorption of the cationic compounds at the
cellular membrane disturbs the selective permeability of
these membranes via disturbing the arrangement of the
hydrophobic core of the cellular membrane. That causes
severe disturbance for the biological reactions inside the
Keywords Uracil-Schiff base cationic surfactants ꢀ
Surface activity ꢀ Partition coefficient ꢀ Biocides ꢀ
Biological activity ꢀ Sulfate reducing bacteria
Introduction
Surfactants are compounds which have unique ability to
dissolve in polar and organic solvents [1]. That feature
Electronic supplementary material The online version of this
article (doi:10.1007/s11743-010-1229-0) contains supplementary
material, which is available to authorized users.
N. A. Negm (&) ꢀ I. A. Aiad ꢀ S. M. Tawfik
Applied Surfactant Laboratory, Petrochemicals Department,
Egyptian Petroleum Research Institute, Nasr city, Cairo, Egypt
e-mail: nabelnegm@hotmail.com
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BrCH2COOH
+
CH3(CH2)nCH2OH
cells due to the diffusion of several compounds from the
environment due to the lack of the selective permeability.
Also, the presence of the halogen atoms as counter ions
increases the potency when penetrated into the cells. The
action mode of the cationic biocides is seemed to be
identical in case of fungi and bacteria [16]. There are no
details on the synthesis of uracil cationic surfactants in the
surfactant synthesis field and no publications dealing with
the biocidal activity of uracil-based surfactants. Hence, in the
present study, we synthesized eight novel uracil-based sur-
factants and characterized those using different tools to elu-
cidate their chemical structures and evaluated as biocides
against different strains of bacteria and fungi, in addition to
the sulfate reducing bacteria. The relation between the bio-
cidal activity and the surface activity was discussed.
100 mL xylene
p-toluene sulfonic acid (0.1 wt% )
O
C
+ H2O
CH2(CH2)n CH3
Br CH2
O
n = 10 (L), 12 (M), 14 (C) and 16 (S)
Alkyl bromoacetate esters
Scheme 1 Synthesis of the alkyl bromoacetate esters (L, M, C, S)
Synthesis of N-Benzylidene-5-amino-2,4-dihydroxy
Pyrimidine (UA) and N-(4-Methoxy-benzylidene)-
5-amino-2,4-dihydroxypyrimidine (UB)
Samples (0.1 mol) of 5-amino-2,4-dihydroxypyrimidine
(U) and benzaldehyde (A) or anisaldehyde (B) were
refluxed individually in 100 mL of iso-propanol as a sol-
vent for 24 h in the presence of (0.01% wt) of p-toluene
sulfonic acid as a catalyst. The reaction mixture was left to
cool in the dark overnight and then filtered. The products
were recrystallized twice from iso-propanol, washed with
water and dried in vacuum oven at 80 °C to afford pale
yellow crystals (UA, UB), yield 93%, and mp more than
280 °C, Scheme 2.
Experimental
Materials
Bromoacetic acid, dodecyl alcohol, tetradecyl alcohol,
hexadecyl alcohol, octadecyl alcohol, benzaldehyde, anis-
aldehyde, cetyltrimethyl ammonium bromide were products
of Aldrich Chemical, Germany (purity [ 99%). p-Toluene
sulphonic acid was a product of Merck, Germany (purity
99.8%). 5-Amino-2,4-dihydroxypyrimidine, piperidine were
products of Fluka, Switzerland (purity 99.6%). Xylene,
iso-propanol and paraffin oil were products of ADWIC,
Egypt (purity [ 98%).
Synthesis of 5-(Benzylideneamino)-2,4-dihydroxy-1,3-
dialkylpyrimidinediium Dibromide and 2,4-Dihydroxy-5-
[(4-methoxybenzylidene)amino]-1,3-dialkyl
Pyrimidinediium Dibromide
Synthesis
Compounds UA or UB (0.05 mol) were refluxed individ-
ually with the prepared fatty esters of the bromoacetic acid
(0.1 mol) in the presence of 150 mL of iso-propanol as a
solvent and few drops of piperidine as a catalyst for 48 h.
The reaction mixture was left in the dark for a week to
Synthesis of the Fatty Alcohol Ester of Bromoacetic Acid
Bromoacetic acid (0.1 mol) and dodecyl, tetradecyl,
hexadecyl and octadecyl alcohol (0.1 mol) were esterified
individually in xylene as a solvent and in the presence of
0.1% p-toluene sulphonic acid as catalyst. The mixture was
refluxed until the theoretical amount of water (1.8 mL) was
removed. After removal of the solvent under vacuum using
a rotary evaporator, the catalyst was extracted from the
reaction medium using petroleum ether. Subsequent puri-
fication was done using vacuum distillation to remove the
excess and unreacted and residual materials. The fatty
alcohol esters of bromoacetic acid produced were: dodecyl
bromoacetate (L), tetradecyl bromoacetate (M), hexadecyl
bromoacetate (C) and octadecyl bromoacetate (S) with the
average yield of 90–92% [17–19], Scheme 1. Vacuum
distillation was performed at 15 mm Hg and the removed
fractions were as follows: xylene (32.7 °C), dodecyl alco-
hol (128 °C), tetradecyl alcohol (132 °C), hexadecyl
alcohol (202 °C) and octadecyl alcohol (210 °C).
OH
H
NH2
N
O
C
R
+
HO
N
N
Reflux in
iso-propanol
for 24 h
OH
N
H
C
R
N
HO
R = H: 5-(benzylideneamino)pyrimidine-2,4-diol (UA).
R = OCH3: 5-[(4-methoxybenzylidene)amino]pyrimidine-2,4-diol (UB).
Scheme 2 Synthesis of pyrimidine Schiff bases UA and UB
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505
complete the precipitation of the desired cationic salt. The
products were then filtered off and recrystallized twice
from iso-propanol to afford the different quaternary uracil
surfactants (UAL, UAM, UAC, UAS, UBL, UBM, UBC
and UBS), yield 90% and mp [ 280 °C, Scheme 3.
adjusted to touch the solution surface. Light paraffin
(15 mL) oil was then added smoothly on the surface and
the system was left to equilibrate for 2 h. The readings
were recorded at the point which the ring detached from the
aqueous to the organic layer (paraffin oil). The ring was
removed and washed with acetone followed by distilled
water and the measurements repeated three times for each
surfactant. The interfacial tension values were taken as the
mean values of three readings [20].
Measurements
Instrumentation
Melting points were determined by a Fisher-Johns melting
point apparatus, USA. Elemental analyses were measured
in the Microanalytical Center, Cairo University using a
Vario Elementar Analyzer (Germany) using the classical
Pregl Dumas method, where the burning gas is oxygen and
helium as the transporter gas. The UV spectra were
recorded by a Jenway 6050 UV/Vis spectrophotometer,
England. FTIR spectra recorded in the 4,000–400 cm-1
region with a Perkin Elmer FTIR System (Genesis Fourier
Transformer FTIRTM) using KBr disks. 1NMR spectra
recorded on (Varian NMR-300, Mercury 300 MHz) spec-
trometers (France) in CDCl3 solvent and TMS as the
internal standard and the chemical shifts reported in d
(ppm). Interfacial tension measurements were measured by
a platinum ring Tensiometer K6 from Kru¨ss, Germany.
Partition Coefficient Measurements
The partition coefficient values of the prepared compounds
were determined between octanol and water using the
Shake Flask method [21]. 0.1 gm of different uracil
derivatives was shaken continuously in a mixture of water
and octanol (50 mL/50 mL) for 2 h and allowed to equil-
ibrate for 24 h. Then, the content of the surfactant in each
phase (water and octanol) was determined (using UV
spectra at 280 nm). The experiment was performed three
times for each surfactant and the given value is the average
of the three replicates. The partition coefficient values were
calculated according to Eq. 1:
Â
à Â
Ã
Log P ¼ Log Sorganic = Saqueous
ð1Þ
where, P partition coefficient, Sorganic is the concentration
of the surfactant in the octanol layer, and Saqueous is the
concentration of the surfactant in the water layer.
Interfacial Tension Measurements
The data of Log P were confirmed using advanced
software ACD labs [22]. This program is a result of
the computational chemistry, and allows calculating
Log P values by using the chemical structures of the
Interfacial tension was measured between two layers of
surfactant solution and light paraffin oil at 25.0 0.1 °C.
The surfactant solution (15 mL of 0.1% wt) was added
firstly into the Teflon holder and the platinum ring was
Scheme 3 Synthesis of
pyrimidine Schiff base cationic
surfactants (UAL, UBL, UAM,
UBM, UAC, UBC, UAS and
UBS)
OH
N
H
C
O
R
N
Br CH2
C
O
CH2(CH2)n CH3
N
+
HO
Reflux
2 drops of piperidine
OH
H
2+
R
O
C
H3C (CH2)n CH2
O
CH2
HO
N
C
N
2Br-
N
H2C
C O CH2(CH2)n CH3
O
R=H: 5-(benzylidene amino)-2,4-dihydroxy-1,3-dialkyl pyrimidinediium dibromide
R=OCH3: 2,4-dihydroxy-5-[(4-methoxybenzylidene)amino]-1,3-dialkyl
pyrimidinediium dibromide.
n= 10 (dodecyl-): UAL, UBL, n= 12 (tetradecyl-): UAM, UBM, n= 14 (hexadecyl-): UAC, UBC
n= 16 (octadecyl-): UAS, UBS.
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compounds and some properties, e.g., molecular weight,
heteroatoms, side chains and polarity of the compound.
microorganisms. The plates were left on a leveled surface,
incubated for 24 h at 30 °C for bacteria and then
the diameters of the inhibition zones were measured. The
inhibition zone formed by these compounds against the
particular test microorganisms, determined the biocidal
activity of the synthetic compounds. The mean value for
three replicates was used to calculate the zone of growth
inhibition of each sample.
Antimicrobial Studies
The synthesized Schiff bases (UA, UB) and their diqua-
ternary derivatives (UAL, UAM, UAC, UAS, UBL, UBM,
UBC and UBS) were screened for their antimicrobial
activity against bacteria and fungi using the well-diffusion
method [23, 24]. Cetyl trimethyl ammonium bromide
(CTAB) was taken as a reference [25]. The mean value
obtained for three individual replicates was used to calcu-
late the zone of growth inhibition of each sample. The
results were compared with a similar run of CTAB, while
Grisofluvine was taken as an antifungal reference.
Microorganism
The antibacterial activity of the synthesized compounds
was tested against Staphylococcus aureus (NCTC-7447)
(SA), Pseudomonas aeruginosa (ATCC-13311) (PA),
Bacillus subtilis (NCIB-3610) (BS), Escherichia coli
(NCTC-10416) (EC) and Desulfomonas pigra (SRB).
While, the antifungal activity was tested against Aspergil-
lus niger (Ferm-BAM-C-21) (AN) and Aspergillus flavus
(Ferm-BAM-C-22) (AF). The bacterial and fungal strains
were isolated and incubated in the Center of Microbiology
and Fermentation (Frem-BAM), Faculty of Science, Cairo
University.
Growing of Microorganisms
The bacterial and fungal strains were cultured according to
standards of the National Committee for Clinical Labora-
tory Standards (NCCLS) [24]. The bacterial species were
grown on nutrient agar, while fungi molds were grown on
Czapek’s dox agar. The following media were used in the
determination of the antimicrobial activities of the syn-
thesized biocides. For bacteria, the nutrient agar medium
started with beef extract (3.0 g/L), peptone (5.0 g/L),
sodium chloride (5.0 g/L), agar (20.0 g/L), then was
brought to a volume of one liter with distilled water, heated
until the mixture was boiling, and sterilized in an auto-
clave. For fungi, the dox agar medium consisted of sucrose
(20.0 g/L), sodium nitrate (2.0 g/L), magnesium sulfate
(0.5 g/L), potassium chloride (0.5 g/L), ferrous sulfate
(0.01 g/L), agar (20.0 g/L) which was brought to a volume
of one liter, heated until it was boiling, and sterilized in an
autoclave.
Results and Discussion
Structure
The purity of the synthesized Schiff bases and their cat-
ionic derivatives was established by the elemental analyses
data as shown in Table 1. Data in Table 1 revealed that the
synthesized surfactants are highly pure.
In order to elucidate the chemical structures of the novel
1
synthesized surfactants, IR, H-NMR and 13C-NMR spec-
troscopy were performed.
The bacteria and fungi were kept on nutrient agar media
and showed no inhibition zones. The agar media were
inoculated with different test microorganisms. After 24 h
of incubation at 30 °C for bacteria and 48 h of incubation
at 28 °C for fungi, the diameter of the inhibition zones
(mm) was measured.
Infrared spectroscopic analyses of the synthesized Schiff
bases UA and UB determine the function groups presented
in their chemical structures. The IR spectra of the synthe-
sized Schiff bases (UA and UB) showed the disappearance
of the characteristic (N–H) absorption band at 3200 cm-1
and the appearance of 1663 cm-1 band which characterizes
the azomethine group (–CH=N–), that proves the occur-
rence of coupling reaction. The absorption bands of the
phenyl groups were appeared in the range of 826–
903 cm-1. UB derivative showed a characteristic absorp-
tion band at 1251–1254 cm-1 corresponded to the methoxy
groups attached to the phenyl ring. The IR spectra of the
cationic derivatives (UAL, UAM, UAC, UAS, UBL, UBM,
UBC and UBS) showed sharp absorption band at
3040 cm-1 corresponded to the quaternary nitrogen atom
(N?). The sharp absorption band at 3404–3407 cm-1
indicates the presence of the hydroxyl groups attached to
the pyrimidine ring.
Measurements of Resistance and Susceptibility
For preparation of discs and inoculation, 1.0 mL of inocula
was added to 50 mL of agar media (40 °C) and mixed. The
agar was poured into 120-mm Petri dishes and allowed to
cool to room temperature. Wells (6 mm in diameter) were
cut in the agar plates using proper sterile tubes. Then, wells
were filled up to the surface of agar with 0.1 mL solution
of the synthesized compounds consisting of 1 mg tested
biocides in 1 mL of dimethyl formamide (DMF), pro-
vided that DMF has no effect on the growth of the
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507
Table 1 Elemental analyses of the synthesized compounds
Compound*
Chemical
structure
Molecular weight
(gm mol-1
Elemental analysis (wt%)**
)
C%
H%
Br%
N%
Calc.
Found
Calc.
Found
Calc.
Found
Calc.
Found
L
C14H27O2Br
307.27
335.33
363.38
391.43
215.22
245.29
829.75
857.81
885.86
913.92
859.78
887.83
915.89
943.94
54.72
57.31
59.5
54.34
56.91
59.08
60.94
60.96
58.36
56.06
57.01
57.89
58.73
55.49
56.42
57.3
8.86
9.32
9.71
10.04
4.22
4.52
7.65
7.87
8.08
8.27
7.62
7.83
8.03
8.22
8.8
26
25.82
23.66
21.84
20.27
–
–
–
M
C
C
C
C
C
C
C
C
C
C
C
C
C
16H31O2Br
9.25
9.64
9.97
4.29
4.49
7.6
23.83
21.99
20.41
–
–
–
C
18H35O2Br
–
–
S
20H39O2Br
61.37
61.39
58.77
56.45
57.41
58.3
–
–
UA
11H9N3O2
19.53
17.13
5.06
4.9
19.39
17.01
5.03
4.86
4.71
4.57
4.85
4.7
UB
12H11N3O3
–
–
UAL
UAM
UAC
UAS
UBL
UBM
UBC
UBS
25H36O4N3Br
27H40O4N3Br
29H44O4N3Br
31H48O4N3Br
26H38O5N3Br
28H42O5N3Br
30H46O5N3Br
32H50O5N3Br
19.26
18.63
18.04
17.49
18.59
18
19.12
18.5
7.82
8.02
8.21
7.57
7.78
7.98
8.1
17.91
17.36
18.46
17.87
17.33
16.81
4.74
4.6
59.14
55.88
56.82
57.7
4.89
4.73
4.59
4.45
17.43
16.93
4.56
4.42
58.53
58.12
* L, M, C, S: dodecyl bromoacetate, tetradecyl bromoacetate, hexadecyl bromoacetate, octadecyl bromoacetate; UA, UB: uracil benzaldehyde
and uracil anisaldehyde Schiff bases; UAL, UAM, UAC, UAS: uracil benzaldehyde quaternized by L, M, C, S; UAL, UAM, UAC, UAS: uracil
anisaldehyde quaternized by L, M, C, S (See Experimental section for more details)
** Data are average of three replicates with a mean error of 7%
1
The H-NMR spectra of the synthesized Schiff bases
UA and UB showed the following signals: 3.38 ppm
(s, 1H, –CH=N–), 6.20 ppm (s, 1H, –CH– pyrimidine ring),
10.1 ppm (s, 1H, –OH) disappeared by the deuteration
and a signal at 2.5 ppm (s, 3H, –OCH3) in compound
(UB).
The above spectral data revealed that the chemical
structures of UA, UB, UAM, UAC, UAS, UBM, UBC and
UBS are as represented in Schemes 1 and 2.
Biological Activity of the Synthesized Cationic Schiff
Base Biocides
1H-NMR spectra of UAL as representative sample for
the synthesized surfactants (additional spectra for 1H-NMR
found in the supplementary data) showed the following
signals: 0.98 ppm (t, 6H, CH3), 1.31 ppm (m, 40H, CH2),
2.4 ppm (s, 4H, CO–CH2–N?), 4.12 ppm (t, 4H, CH2–O),
5.1 ppm (s, 2H, OH) disappeared be deuteration, 7.7 ppm
(m, 5H, phenyl group), 8.4 ppm (s, 1H, CH=N–). Addi-
tional signal was appeared at 3.85 ppm (s, 3H, O–CH3) for
UBL. The different uracil cationic derivatives (UAM,
UAC, UAS, UBM, UBC and UBS) showed identical sig-
nals as represented above, but the signal at 1.31 ppm was
integral of the number of methylene groups in these
derivatives.
13C-NMR spectra for UAL as representative sample for
the synthesized surfactants (additional spectra for 1H-NMR
found in the supplementary data) showed the following
signals: 14.5 ppm (CH3), 30.0 ppm (CH2), 50 ppm (CH2–
N?), 65 ppm (CH2–O), 129 ppm (ortho-, meta- and para-
carbons of the phenyl group), 160 ppm (N=CH–), 171 ppm
(–C=O). An additional signal appeared for compound UBL
at 55.9 ppm (–OCH3).
Antimicrobial Activity–Chemical Structure Relationship
The synthesized uracil Schiff bases UA and UB (parent
Schiff bases) were evaluated as antimicrobial agents
against various pathogenic Gram positive and Gram neg-
ative bacteria and fungi. The antimicrobial activity was
expressed in terms of inhibition zone diameter, Table (2).
The antimicrobial activities ranged between 10 and 13 mm
compared to the standard used (CTAB) 12.5 mm. The
biocidal activities of these compounds strongly related to
the presence of the heteroatoms and the azomethine groups
in their chemical structures. Table 2 revealed that the
uracil-anisaldehyde Schiff base (UB) showed relatively
higher antimicrobial activities than uracil-benzaldehyde
Schiff base (UA). That could be attributed to the higher
adsorption tendency of the oxygen atom in the methoxy
groups (–O–CH3) due to the lone pair of electrons of
oxygen atom [26, 27].
On quaternization of the uracil-Schiff bases (UA, UB)
by the synthesized bromoesters (L, M, S, C), the biocidal
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Table 2 Antimicrobial activity of 5-(benzylidene amino)-2,4-dihydroxy-1,3-dialkyl pyrimidinediium dibromide and 2,4-dihydroxy-5-[(4-
methoxybenzylidene)amino]-1,3-dialkyl pyrimidinediium dibromide biocides against pathogenic bacteria and fungi
Surfactant*
Inhibition zone diameter (mm/mg sample)**
Bacillus
subtilis
Staphylococcus
aureus
Escherichia
coli
Pseudomonas
aeruginosa
Candida
albicans
Aspergillus
niger
Standard
UA
12.3
12
13
13
13
14
17
11
14
16
19
12.3
11
13
14
15
16
18
13
16
16
19
12.3
12
12.3
11
12
10
11
13
15
11
14
16
19
12.3
10
11.6
10
10
12
13
13
15
12
14
17
19
UB
12.5
13
11.0
13
UAL
UAM
UAC
UAS
UBL
UBM
UBC
UBS
13
13
17
13
16
14
12
11
12
12
15
14
16
20
* UA, UB: uracil benzaldehyde and uracil anisaldehyde Schiff bases; UAL, UAM, UAC, UAS: uracil benzaldehyde quaternized by L, M, C, S;
UAL, UAM, UAC, UAS: uracil anisaldehyde quaternized by L, M, C, S (See Experimental section for more details)
** Data are averages of three replicates with an error ratio of 5%
activity of the produced cationic surfactants was changed
considerably. The antimicrobial activities of the cationic
uracil-based surfactants (UAL, UAM, UAC, UAS, UBL,
UBM, UBC and UBS) ranged between 13 and 19 mm
which are relatively high compared to the parent Schiff
bases (UA, UB) and the standard (CTAB) used.
Table 3 Interfacial tension, emulsion stability and partition coeffi-
cient values of the uracil derivatives at 25 °C
Surfactants*
Interfacial
tension (mN m-1
Log P
)
Theor.**
Exp.***
UA
–
–
-1.55
-1.43
-1.49
-1.39
The high efficacy of the quaternary derivatives is basi-
cally related to the presence of the two positively charged
nitrogen atoms [28]. The biocidal activities produced for
the targeted microorganisms with the conventional diqua-
ternary cationic biocides [29–31] ranged between 13 and
15 mm. Comparing the potency of the conventional and the
synthesized cationic uracil-Schiff base biocides revealed
the higher efficacy of the later than the former which
resulted due to the influence of the heteroatoms and the
azomethine groups.
UB
UAL
UAM
UAC
UAS
UBL
UBM
UBC
UBS
20
14
9
-17.08
-19.21
-21.33
-23.46
-17.21
-19.33
-21.46
-23.58
-16.81
-19.00
-21.10
-23.22
-17.19
-19.12
-21.21
-23.35
6
22
18
10
5
Data are means of three replicates with an average error of 8%
Table 2 represents the effect of hydrophobic chain length
on the antimicrobial activities of the synthesized Schiff
bases. It is clear that the hydrophobic chain length has an
increment role on the biological activities of the cationic
compounds. Increasing the hydrophobic chain length from
dodecyl to octadecyl chain increases the biological activity
considerably. The maximum inhibition efficiency was cor-
responding to the octadecyl derivatives (UAS, UBS), while
the lowest was obtained by using dodecyl derivatives (UAL,
UBL). That was attributed to the adsorption tendency of the
different uracil-Schiff base cationic derivatives at the
interfaces as reflected from the surface activity data,
Table 3. Longer chain length decreases the interfacial ten-
sion values which consequently indicate the adsorption of
the molecules at the interfaces. On the contrary, surfactants
* UA, UB: uracil benzaldehyde and uracil anisaldehyde Schiff bases;
UAL, UAM, UAC, UAS: uracil benzaldehyde quaternized by L, M,
C, S; UAL, UAM, UAC, UAS: uracil anisaldehyde quaternized by L,
M, C, S (See Experimental section for more details)
** Data obtained for the shake flask method according to Ref. [21]
*** Software obtained data as represented in Ref. [22]
having shorter hydrophobic chains had higher interfacial
tension values which represented their lower tendency
towards adsorption at the interfaces.
Partition Coefficient—Antimicrobial Activity Relationship
The partition coefficient (P) is defined as the ratio of
unionized compound between two solutions. The partition
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J Surfact Deterg (2010) 13:503–511
509
coefficient can be calculated using the following logarithm
relation:
molecules increases the penetration through the cellular
membrane, as a result, the biocidal activities increased
gradually [16].
Â
ꢀ
Ã
Log P ¼ Log ðsoluteÞoctanol ðsoluteÞwater
where (solute)octanol is the concentration of the dissolved
part in the nonpolar layer (octanol) and (solute)water is the
concentration of the dissolved part in the polar layer
(water). The values of the partition coefficients of the
synthesized uracil derivatives in the water/octanol system
were measured and listed in Table 3.
Biocidal Activity of the Synthesized Biocides Against
Sulfate Reducing Bacteria (SRB)
As a result of the numerous problems caused by sulfate
reducing bacteria in oil and gas production operations,
aggressive measures have been taken to monitor and con-
trol bacterial populations. Measurements of bacterial
growth are not usually considered until after corrosion
failures point due to microbial induced corrosion (MIC).
By the time MIC is discovered, extensive and costly
damage to the operating systems has often already occur-
red. Monitoring is often conducted in sweet systems with
no hint of bacterial contamination to ensure that bacterial
populations are under control and that operating costs and
risks (health, safety, environmental, and mechanical fail-
ures) cannot be lowered by initiating an effective biocide
program [33].
The role of diffusion of biologically active compounds
in the nonpolar medium is similar to the penetration of
these compounds through the biological membranes. Pro-
teins and sugar polymers do not permeate at all; in contrast,
water and alcohols permeate most membranes in less than a
second. This variation is caused by the phospholipids
bilayers which give the membrane its characteristic per-
meability. Partition coefficients are useful for estimating
distribution and diffusion of compounds within the cell
membrane. Hydrophobic compounds with high partition
coefficients are preferentially distributed to hydrophobic
compartments (nonpolar or organic) such as phospholipids
bilayers of cells while hydrophilic compounds with low
partition coefficients preferentially are found in hydrophilic
compartments such as polysaccharide layers.
As a vital application of the synthesized biocides in the
petroleum engineering life, the synthesized cationic sur-
factants were evaluated as biocides against sulfate reducing
bacteria (Desulfomonas pigra) contaminating oil fields.
The biocidal activity results of the uracil-Schiff bases
surfactants showed imperative future due to their relatively
high efficiency. The inhibiting efficiencies of the tested
cationic surfactants in terms of inhibition zone diameter
(mm) ranged between 16 and 25 mm, which are consid-
erably higher results relative to the blank used (CTAB),
Fig. 2.
The logarithms of the partition coefficients of the syn-
thesized parent Schiff bases (UA, UB) ranged between
-1.49 and -1.39, respectively. That indicates their high
hydrophilic characters and consequently low penetration
tendency through the cellular membrane; as a result, the
antimicrobial activities get low [32]. While, quaternization
increases considerably the log P values of the parent Schiff
bases (UA, UB). Increasing the carbon content (hydro-
phobic chain length) of the uracil-Schiff base cationic
biocides increases the partition coefficient values and
consequently the hydrophobicity of the molecules, Fig. 1.
The increase of the hydrophobicity of the different biocide
30
25
20
15
10
5
30
25
20
15
10
5
0
0
12
14
16
18
12
14
16
18
Number of methylene groups
Number of methylene groups
Fig. 1 Effect of hydrophobic chain length on the partition coefficient
values (log P) of the synthesized cationic biocides, white: UA
cationics; dark: UB cationics
Fig. 2 Effect of chain length on the biocidal activity of the
synthesized uracil-based surfactants against sulfate reducing bacteria,
white: UA cationics; dark: UB cationics
123

510
J Surfact Deterg (2010) 13:503–511
7. Negm NA, Zaki MF, Salem MAI (2010) Cationic Schiff base
amphiphiles and their metal complexes: surface and biocidal
activities against bacteria and fungi. Colloids Surf B 77:96–103
8. Negm NA, Mohamed AS (2008) Synthesis, characterization and
biological activity of sugar-based Gemini cationic amphiphiles.
J Surf Deterg 11:215–221
9. Erskine PT, Newbold R, Roper J, Coker A (1999) The Schiff base
complex of yeast 5-aminolaevulinic acid dehydratase with laev-
ulinic acid. Protein Sci 8:1250–1257
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some Thieno [2, 3-d] Pyrimidine nucleosides. Nucleotides
25:17–25
11. Young ME, Alakomi HL, Fortune I, Gorbushina AA, Krumbein
WE, Maxwell I, McCullagh C, Robertson P, Saarela M, Valero J,
Vendrell M (2008) Development of a biocidal treatment regime
to inhibit biological growths on cultural heritage: BIODAM.
Environ Geol 56:631–641
12. Cerchiaro G, Ferreira AM, Teixeira AB, Magalhaes HM (2006)
Synthesis and crystal structure of 2, 4-dihydro-4-[(5-hydroxy-3-
methyl-1-phenyl-1H-pyrazol-4-yl)imino]-5-methyl-2-phenyl-3H-
pyrazol-3-one and its copper complexes. Polyhedron 25:
2055–2056
13. Finlay JA (1996) The potential of alkyl amines as antifouling
biocides: toxicity and structure activity relationships. Biofouling
9:257–268
Inspection of the antimicrobial activity of the synthe-
sized cationic Schiff bases revealed that increasing the
hydrophobic chain length and introducing the methoxy
groups in their chemical structures of the synthesized
uracil-based surfactants considerably increased their bio-
cidal activity. The most efficient biocidal activity was
monitored for the anisaldehyde derivatives: 2,4-dihydroxy-
6-[(4-methoxybenzylidene)amino]-1,3-dialkyl pyrimidinedi-
ium dibromide in the sequence of increasing the hydrophobic
chain length as follows:
UBL [ UBM [ UBC [ UBS.
This can be related to the chemical structures of these
derivatives which contain two positively charged centers
on the nitrogen atoms and also to the increasing tendency
of the (-OCH3) group towards adsorption at the biological
membrane. The promising inhibition efficiency of these
compounds against the sulfate reducing bacteria facilitates
them to be applicable in the petroleum field as new
categories of SRB biocides.
14. MAl-Thebeiti MS (2001) Synthesis of some new derivatives of
thiazolo-3, 2-pyrimidine-3, 5, 7(2H)-trione of potential biological
activity. Boll Chim Farm 140:221–223
Conclusion
15. Negm NA, Morsy SM, Said MM (2005) Biocidal activity of
some mannich base cationic derivatives. Bioorg Med Chem
13:5921–5926
16. Negm NA, Morsy SM, Said MM (2010) Pyrazole derived cat-
ionic surfactants and their tin and copper complexes: synthesis,
surface activity, antibacterial and antifungal efficacy, J Surf
Deterg (published on the web). doi:10.1007/s11743-009-1178-7
17. Negm NA (2002) Surface activities and electrical properties of
long chain diquaternary bola-form amphiphiles. Egypt J Chem
45:483–499
1. Uracil Schiff bases showed moderate antimicrobial
activities near the conventional cationic biocides.
2. Quaternization of the uracil derivatives considerably
increases their biocidal activities.
3. Increasing the hydrophobic chain length and the
partition coefficients of the uracil derivatives increases
their biocidal activities.
18. Cukurovali A, Yilmaz I, Gur S, Kazaz C (2006) Synthesis,
antibacterial and antifungal activity of some new thiazolylhyd-
razone derivatives containing 3-substituted cyclobutane ring.
J Medic Chem 41:201–207
4. The growth of sulfate reducing bacteria is inhibited in
presence of the targeted biocides.
19. Negm NA, Aiad IA (2007) Synthesis and characterization of
multifunctional surfactants in oil-field protection applications.
J Surf Deterg 8:87–92
20. Negm NA, Mahmoud SA (2003) Effect of structure on the
physicochemical properties of nonionic phosphate amphiphiles.
Egypt J Petrol 12:11–20
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quaternary ammonium surfactants. He spent 6 month as a research
fellow at the Max Planck Institute of Colloids and Surfaces, Golm,
Germany. He is currently an assistant professor in the Petrochemicals
Department of the Egyptian Petroleum Research Institute. He is
interested in several surfactants applications, including: detergency,
emulsification, solubilization, corrosion inhibition of metals, and oil
field chemicals. Also, he is interested in the biological activity of
surfactants and their metal complexes towards bacteria, fungi and
yeast. His recent R & D was focused on mixed surfactants and mixed
surfactant- protein systems.
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rosion inhibition efficiency and surface activity of benzothiazol-
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Ismail A. Aiad received his Ph.D. in physical chemistry from the
faculty of Science, Zagazig University. He is a professor of applied
chemistry in the Surfactant Laboratory of the Egyptian Petroleum
Research Institute.
32. Badawi AM, Hafiz AA (2007) Synthesis and immunomodulatory
activity of some novel amino acid germinates. J Iranian Chem
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Salah M. Tawfik is a research assistant in the petrochemicals
department, Egyptian Petroleum Research Institute. His M.Sc. thesis
in 2010 at Helwan University was focused on the synthesis and
application of novel surfactants containing heterocyclic rings and
their application as corrosion inhibitors and biocides.
Author Biographies
Nabel A. Negm received his Ph.D. from Ain Shams University in
2000 with a dissertation on the synthesis and evaluation of some
123