Cationic surfactants based on ferrocene containing thiourea: Synthesis, self-aggregation, and antioxidant properties

Article abstract of DOI:10.1016/j.colsurfa.2020.124760

Keeping in view the significance of cationic surfactants in various fields of life since the ancient times, here we are reporting the synthesis of a new ferrocenyl thiourea derivative along with an entirely new series of ferrocene based cationic surfactan

Full text of DOI:10.1016/j.colsurfa.2020.124760

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Cationic Surfactants Based on Ferrocene Containing Thiourea:
Synthesis, Self-aggregation, and Antioxidant Properties
Mehwish Naz, Jahangeer Ali, Saira Fatima, Saira Tabassum,
Sameen Nawaz, Amin Badshah, Hongjing Dou
PII:
S0927-7757(20)30353-8
DOI:
https://doi.org/10.1016/j.colsurfa.2020.124760
COLSUA 124760
Reference:
To appear in:
Colloids and Surfaces A: Physicochemical and Engineering Aspects
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Revised Date:
Accepted Date:
12 January 2020
13 March 2020
24 March 2020
Please cite this article as: Naz M, Ali J, Fatima S, Tabassum S, Nawaz S, Badshah A, Dou H,
Cationic Surfactants Based on Ferrocene Containing Thiourea: Synthesis, Self-aggregation,
and Antioxidant Properties, Colloids and Surfaces A: Physicochemical and Engineering
Aspects (2020), doi: https://doi.org/10.1016/j.colsurfa.2020.124760
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Cationic Surfactants Based on Ferrocene Containing Thiourea:
Synthesis, Self-aggregation, and Antioxidant Properties
a
a
a
Mehwish Naz a, b, Jahangeer Ali a, Saira Fatima , Saira Tabassum , Sameen Nawaz , Amin
Badshah a,*, Hongjing Dou b,*
a Department of Chemistry, Quaid-i-Azam University Islamabad, Pakistan.
b
The State Key Laboratory of Metal Matrix Composites, School of Materials Science and
Engineering, Shanghai Jiao Tong University, Shanghai 200240, P. R. China.
a
Correspondence and requests for materials should be addressed to Amin Badshah and
Hongjing Dou b.
a Department of Chemistry, Quaid-i-Azam University Islamabad, 45320, Pakistan.
b The State Key Laboratory of Metal Matrix Composites, School of Materials Science and
Engineering, Shanghai Jiao Tong University, Shanghai 200240, P. R. China.
Tel: +92-51-9064-2131 a , +86-21-34202956 b
Email: aminbadshah@qau.edu.pk , hjdou@sjtu.edu.cn
Graphical Abstract
1

Highlights
 Chain length effects the surfactant aggregation behavior like micellization and critical
micelle concentration when studied using same head structure.
 Antioxidant efficacy mainly depend on the head structure in ferrocene based thiourea
containing cationic surfactants.
 Antioxidant activity of an already potent head group decrease with decrease in length
of added chain due to micellar effect.
 Longer chain surfactants are better candidates for major bioactivities as compared
with shorter chain length surfactants in case if chain length is having a role in
particular activity.
2

Abstract
Keeping in view the significance of cationic surfactants in various fields of life since the ancient
times, here we are reporting the synthesis of a new ferrocenyl thiourea derivative along with
an entirely new series of ferrocene based cationic surfactants in which variable alkyl chain
lengths are introduced while keeping the head structure same, thus acting as a key to
comparative study of their potential self-aggregation properties and bioactivities. Critical
micelle concentration has been determined which is a supporting evidence to the amphiphilic
nature of the synthesized compounds. Furthermore, cell cytotoxicity and three types of
antioxidant activities also helped depict the therapeutic properties of the synthesized
compounds.
Key words: Ferrocene, Thiourea, Cationic surfactants, CMC, surface tension, antioxidant
activities.
1 Introduction
Cationic surfactants with their positively charged hydrophilic head and the lipophilic tail duo
act as a viaduct between two phases, catalyzing various aquo-organic reactions[1].
Furthermore, the evolution of electrostatic associative assemblance upon interaction with
negative small molecules or surfaces makes them a desirable entity for applications in variety
of areas. The cationic surfactants acquired prime importance for the first time about 50 years
back when their inimitable antibacterial properties came to light [2, 3] and with the passing
time their consumption increased and stretched up to millions of metric tons [4]. On industrial
level cationic surfactants were regarded as quintessential work pivots because of their supreme
implication in oil recovery [5, 6], bactericidal effects [7], and sugar decolorization [8]. They
3

are constituents of goods ranging from body sprays to sailboat fiberglass, and also they are
frequently used in the laundry to keep the clothing softer [9].
In this study, our group has focused on the synthesis of cationic surfactants that are sulfur
based quaternary ammonium compounds (QACs) with an adjunct efficacy of ferrocene
functionality incorporated into their head group. In spite of potentially applicable
pharmacological properties pointed by some researchers, lesser polarity of ferrocene hinders
its bioavailability for in vivo applications. Hence instead of direct applications ferrocene and
its derivatives are usually implicated as a structural entity in designing various drugs.
Furthermore, due to the inertness of ferrocene towards atmospheric oxidation, its synthetic
derivatives are supposed to be very novel antioxidants, with the respective organic
functionalities increasing its bioavailability, and, enhancing the antioxidant efficacy [10].
Ferrocene based QACs are reported to possess variety of applications in biology, specifically
as disinfectants and sanitizers [11]. Also since the past few decades, scientists are trying to gain
the switchable control over the surface tension of the surfactants by introducing certain redox-
active moieties within their structure [12-14] like ferrocene [15] and azobenzene [16]. Such
reversible aggregate formation approach can be implicated in controlled drug release [17] and
release of various other enclosed species like genes and smaller molecules in response to
change in redox properties of external environment [18-20].
Moreover, ferrocenyl surfactants are also of pivot importance and keen interest in most of
studies based on the redox behavior of surfactants. These amphiphilic species comprise of a
single redox active site with slight modification of head group [21, 22], charge (cationic,
anionic, neutral) [12-37] or variation of number [38, 39] and length [29, 39] of hydrophobic
chains [40]. Now, in order to synthesize a new series of ferrocenyl surfactants, we have tried
to combine the properties of QACs along with ferrocene redox sensitivity, π-conjugation,
4

electron transfer ability, lipophilicity, lower biotoxicity and more selective cytotoxicity
towards tumor cells [12, 13].
Herein, the main aim of this study is to elaborate the effect of chain length on the surfactant
properties and behavior like self-assembly and bioactivities, which was achieved by
synthesizing a surfactant head structure and attaching different length carbon chains to it. The
synthesized surfactants were then used to carry out a comparative study of various surfactant
properties like critical micelle concentration (CMC), cell cytotoxicity and different types of
antioxidant activities like percentage DPPH inhibition, total reducing power (TRP) and total
antioxidant capacity (TAC).
2 Experimental
2.1 Materials
The chemicals used were 1-chlorooctadecane (96 %), 1-chlorohexadecane (95 %), 1-
chlorotetradecane (98 %), 4-chlorobenzoyl chloride (95%), ferrocene (98%), 4-nitroaniline
(≥99 %), potassium thiocyanate (≥99 %), hexadecyltrimethylammonium bromide (PTC
≥98 %), hydrazine, anhydrous (98 %) and hydrochloric acid (37 %) were obtained from Sigma
Aldrich. While the solvents like acetone, ethanol, diethyl ether, petroleum ether, n-hexane and
methanol obtained from Merck, Germany were dried applying standard drying methods.
2.2 Synthesis of ferrocene based cationic surfactants
For the synthesis of 1-(4-ferrocenylphenyl)-3-(4-chlorobenzoyl)thiourea 0.5 g of potassium
thiocyanate was dissolved in 40 mL of dried acetone (0.012 g/mL) and transferred to a 250 mL
round bottom flask, and 0.7 mL of 4-chlorobenzoyl chloride (0.049 g/mL) was added under
the nitrogen atmosphere. The reaction mixture was kept under stirring for 2 h at a temperature
of 50 °C. Then, 1.5 g of ferrocenyl aniline (0.037 g/mL) was added to the reaction mixture and
it was kept under stirring for further 4 h at 50 °C [41, 42]. The orange colored precipitates of
5

1-(4-ferrocenylphenyl)-3-(4-chlorobenzoyl)thiourea were obtained upon cooling in ice which
were filtered, washed, dried and recrystallized in acetone to obtain pure product. Subsequently,
this ferrocenyl thiourea precursor was used to synthesize ferrocenyl cationic surfactants of
different chain length by stirring equimolar ratio of long chain alkyl halides and ferrocenyl
thiourea in dry ethanol at 80 °C for 24 h. The progress of the reaction was kept under
monitoring by TLC at steady time intervals. After the accomplishment of the reaction, reddish
brown precipitates of the product were purified, filtered and dried [13] (Scheme-1).
2.2.1 Characterization
FT-IR spectra of the compounds were recorded using BRUKER (Tensor-37)
spectrophotometer (4000-400 cm-1) .While 1H and 13C NMR for all the synthesized compounds
were recorded using BRUKER (Advanced NMR) spectrophotometer having a 300 MHz
frequency. X-ray measurements were made on a Bruker Kappa APEXII CCD diffractometer
equipped with a graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å) source. The data
collection used ω scans, and a multi-scan absorption correction was applied. The structure was
solved by using a SHELXL-97 program and it was refined by full matrix least-squares
technique using SHELXL-97.
1-(4-ferrocenylphenyl)-3-(4-chlorobenzoyl)thiourea (F.R)
1
FT-IR :(cm-1): 3268 (N-H), 3034 (sp2 -CH), 1713 (C=O), 1263 (C=S), 483 (Fe- C5H5); H-
NMR [300 MHz (DMSO), (δ-ppm)]: 12.53 (1H, (C=S)NH, s), 11.68 (1H, (C=O)NH, s), 7.6-
13
8.01 (8H, Aromatic, m), 4.81 (2H, Cp, s), 4.37 (2H, Cp, s), 4.04 (5H, Cp, s); C-NMR: [75
MHz (DMSO), (δ-ppm)]: 178.8, 167.7, 124, 126, 129, 131, 132, 136, 138, 138.5, 69.86, 69.48,
66.8; CRYSTAL DATA: empirical formula: C24H21ClFeN2O2S (M =492.79 g/mol):
monoclinic, space group P21/c (no. 14), a = 15.9706(6) Å, b = 11.4992(3) Å, c = 11.9627(4) Å,
β = 96.016(3)°, V = 2184.84(12) Å3, Z = 4, T = 296(2) K, μ(MoKα) = 0.932 mm-1, Dcalc =
6

1.498 g/cm3, 17509 reflections measured (4.374° ≤ 2 θ ≤ 54.996°), 5019 unique (Rint = 0.0323,
Rsigma = 0.0362) which were used in all calculations. The final R1 was 0.0413 (I > 2σ(I)) and
wR2 was 0.1066 (all data).
4-ferrocenyl-N-((4-chlorobenzoyl)carbamothioyl)-N-octadecylbenzenium chloride (F.R-18)
Quantities: 1-(4-ferrocenylphenyl)-3-(4-chlorobenzoyl)thiourea (0.5 g in 50 mL ethanol), 0.34
mL octadecyl chloride Yield 75%, FT-IR (cm-1): 3177 (N-H), 3034 (sp2C-H), 2921 (sp3C-
1
H)1713 (C=O), 1184 (C=S), 720 (-CH2-), 484 (Fe- C5H5); H NMR [300 MHz, DMSO, (δ-
ppm)]: 12.53 (1H, (C=O)NH, s), 11.68 (1H, (C=S, s)NH), 7.6-8.02 (8H, Ar-H, m), 4.81 (2H,
C5H5, s), 4.37 (2H, C5H5), s), 4.04 (5H, C5H5, s), 0.8-1.94 (37H, Aliphatic-H, m); 13C NMR:
[75 MHz(DMSO), (δ-ppm)]: 178.8, 167.7, 138, 137.6, 136, 131.6, 131.5, 131.1, 129.2, 129,
126.4, 124, 69.86, 69.47, 66.78, 14.43, 22.57, 26.71, 28.7, 29.18, 29.48, 31.76, 32.5
4-ferrocenyl-N-((4-chlorobenzoyl)carbamothioyl)-N-hexadecylbenzenium chloride (F.R-16)
Quantities: 1-(4-ferrocenylphenyl)-3-(4-chlorobenzoyl)thiourea (0.5 g in 50 mL ethanol), 0.3
mL hexadecyl chloride Yield 69%, FT-IR (cm-1): 3164 (N-H), 3033 (sp2C-H), 2921 (sp3C-
H)1732 (C=S), 1183 (C=O), 726 (-CH2-), 484 (Fe- C5H5); 1H NMR [300 MHz, (δ-ppm),
DMSO]: 12.53 (1H, (C=O)NH, s), 11.67 (1H, (C=S)NH, s), 7.6-8.02 (8H, Ar-H, m), 4.81 (2H,
13
C5H5, s), 4.37 (2H, C5H5, s), 4.04 (5H, C5H5, s), 0.8-1.97 (33H, Aliphatic-H, m); C NMR:
[75 MHz, (DMSO), (δ-ppm)]: 178.8, 167.7, 131, 129, 126, 124, 69.86, 69.47, 66.77, 14.43,
22.57, 26.72, 29.18, 29.49, 32.52
4-ferrocenyl-N-((4-chlorobenzoyl)carbamothioyl)-N-tetradecylbenzenium chloride (F.R-14)
Quantities: 1-(4-ferrocenylphenyl)-3-(4-chlorobenzoyl)thiourea (0.5 g in 50 mL ethanol), 0.28
mL tetradecyl chloride Yield 76%, FT-IR (cm-1): 3157 (NH), 3034 (sp2C-H), 2921 (sp3C-
1
H)1735 (C=O), 1183 (C=S), 730 (m, -CH2-) 484 (Fe- C5H5); H NMR [300 MHz (DMSO),
(δ-ppm)]: 12.55 (1H, (C=O)NH, s) 11.66 (1H, (C=S)NH), s) 7.6-8.02 (8H, Ar-H, m), 4.81 (2H,
7

C5H5, s), 4.37 (2H, C5H5, s), 4.04 (5H, C5H5, s), 0.8-1.7 (29H, Aliphatic-H, m) ; 13C NMR: [75
MHz (DMSO), (δ-ppm)]: 178.8, 167.7, 138, 137.6, 136, 131.5, 131.1, 129, 127, 126, 124.42,
124.41, 69.86, 69.46, 66.77, 26.72, 28.70, 29.18, 29.36, 29.40, 29.48, 29.51, 31.77, 32.5
4-ferrocenyl-N-((4-chlorobenzoyl)carbamothioyl)-N-dodecylbenzenium chloride (F.R-12)
Quantities: 1-(4-ferrocenylphenyl)-3-(4-chlorobenzoyl)thiourea (0.5 g in 50 mL ethanol),
0.26 mL dodecyl chloride Yield 72%, FT-IR (cm-1): 3153 (N-H), 3032 (sp2C-H), 2923
1
(sp3C-H)1737 (C=O), 1183 (C=S), 722 (-CH2-), 484 (Fe-C5H5); H NMR [300 MHz
(DMSO), (δ-ppm)]: 12.53 (1H, (C=O)NH, s), 11.66 (1H, (C=S)NH, s), 7.6-8.02 (8H, Ar-H,
m), 4.81 (2H, C5H5, s), 4.37 (2H, C5H5, s), 4.04 (5H, C5H5, s), 0.8-1.81 (25H, Aliphatic-H,
m); 13C NMR: [75 MHz (DMSO), (δ-ppm)]: 178.8, 167.7, 131, 129, 126, 124, 69.86, 69.47,
66.78, 18.23, 22.53, 26.60, 29.31, 29.42, 29.78, 32.52
4-ferrocenyl-N-((4-chlorobenzoyl)carbamothioyl)-N-decylbenzenium chloride (F.R-10)
Quantities: 1-(4-ferrocenylphenyl)-3-(4-chlorobenzoyl)thiourea (0.5g in 50ml ethanol), 0.22
mL decyl chloride Yield 75%, FT-IR (cm-1): 3154 (NH), 3033 (sp2C-H), 2925 (sp3C-H) 1713
1
(carbonyl), 1182 (thionyl), 729 (-CH2-), 484 (Fe-C5H5); H NMR [300 MHz (DMSO), (δ-
ppm)]: 12.53 (1H, (C=O)NH, s), 11.69 (1H, (C=S)NH, s), 7.6-8.02 (8H, Ar-H, m), 4.81 (2H,
C5H5, s), 4.37 (2H, C5H5, s), 4.04 (5H, C5H5, s) 0.8-1.23 (21H, Aliphatic-H, m); 13C NMR: [75
MHz (DMSO), (δ-ppm)]: 178.8, 167.7, 138.7, 132, 131.5, 129, 126.6, 124, 69.86, 69.47, 66.76,
28.57, 29.1, 29.7, 29.81, 32.50
4-ferrocenyl-N-((4-chlorobenzoyl)carbamothioyl)-N-nonylbenzenium chloride (F.R-9)
Quantities: 1-(4-ferrocenylphenyl)-3-(4-chlorobenzoyl)thiourea (0.5g in 50 mL ethanol), 0.20
mL nonyl chloride Yield 67%, FT-IR (cm-1): 3154 (N-H),3033 (sp2C-H), 2970 (sp3C-H,) 1736
1
(carbonyl), 1229 (thionyl), 728 (-CH2-), 484 (Fe-C5H5); H NMR [300 MHz (DMSO), (δ-
ppm)]: 12.53 (1H, (C=O)NH, s), 11.66 (1H, (C=S)NH, s), 7.6-8.02 (8H, Ar-H, m), 4.81 (2H,
8

13
C5H5, s), 4.37 (2H, C5H5, s,), 4.04 (5H, C5H5, s), 0.8-1.56 (19H, Aliphatic-H, m); C NMR:
[75 MHz (DMSO), (δ-ppm)]:178.8, 167.7, 131, 129, 126, 124 , 69.86, 69.47, 66.78, 18.23,
22.53, 26.60, 29.31, 29.42, 29.78, 32.52
4-ferrocenyl-N-((4-chlorobenzoyl)carbamothioyl)-N-octylbenzenium chloride (F.R-8)
Quantities: 1-(4-ferrocenylphenyl)-3-(4-chlorobenzoyl)thiourea (0.5 g in 50 mL ethanol), 0.16
mL octyl chloride Yield 75%, FT-IR (cm-1): 3027 (sp2C-H), 3153 (N-H), 2970 (sp3C-H), 1738
1
(carbonyl), 1261 (thionyl), 730 (-CH2-), 484 (Fe-C5H5); H NMR [300 MHz (DMSO), (δ-
ppm)]: 12.53 (1H, (C=O)NH, s), 11.67 (1H, (C=S)NH, s), 7.6-8.02 (Ar-H, m), 4.81 (2H, C5H5,
s), 4.36 (2H, C5H5 , s), 4.04 (5H, C5H5, s), 0.8-1.49 (17H, Aliphatic-H, m); 13C NMR: [75 MHz
(DMSO), (δ-ppm)]: 178.8, 167.7, 131, 129, 126, 124, 69.86, 69.47, 66.78, 14.39, 18.54, 22.4,
26.7, 28.12, 29.30, 29.43, 29.56, 32.50
2.3 Critical micelles concentration (CMC)
2.3.1 Surface tension studies
A BZY-202 surface tensiometer (automatic) was used for the determination of surface tension
with the concentrations of the surfactants ranging between 0.01-0.2 mM in ethanol/water
mixture (1:9).
2.3.2 UV-visible spectroscopy
SHIMADZU (UV-1800) UV-visible spectrophotometer was also used for the determination of
critical micelle concentration (CMC) for which the absorbance spectra were recorded by
varying the concentrations of the surfactants between 0.01-0.02 mM in ethanol/water solvent
mixture (1:9).
9

2.4 Biological evaluation (Bio-activities)
In vitro biological activities for the compounds were performed in purpose to estimate their
bioactive potential and antioxidant abilities.
2.4.1 Brine shrimp lethality assay
Cytotoxicity of the tested surfactants was scrutinized in a microtiter (96 well) plate utilizing
brine shrimp (Artemia salina) larvae as test insects [43]. Incubation of eggs of A. salina (Ocean
star, USA) was carried out within the simulated marine water (38 g/L augmented by 6 mg/L
dry yeast) at 32 °C for up to 48 h in the presence of light in a specifically designed two-section
platter. The maturated nauplii were harvested after incubation using Pasteur pipette which were
then shifted to the individual plate wells (10 nauplii per well). In the next step simulated marine
water containing 1% surfactant sample dissolved in DMSO (500, 250 and 100 µg/mL) was
added to the wells already containing brine shrimps larvae and marine water keeping the
ultimate volume of the wells up to 300 µL. Serialized concentrations of the standard drug
doxorubicin and 1 % DMSO were contained within the positive as well as negative control
wells correspondingly. Subsequently, after an incubation period of 24 h, the percentage of
deaths were calculated after counting the shrimps that were alive. LC50 (lethal concentration to
kill 50 % population) of the compounds was calculated using the table curve V-5.01 software.
2.4.2 Total antioxidant capacity determination
Total antioxidant capacity (TAC) evaluation of the titled compounds was carried out by using
the reported method [44] with few alterations. 100 μL of surfactant sample dissolved in DMSO
(4mg/mL) was added to 900 µL of the reagent aqueous solution containing 0.6 M H2SO4, 4
mM ammonium molybdate ((NH4)6Mo7O24) and 28 mM Na2PO4 (1:1:1). After 90 min
o
incubation of this reaction mixture done at 95 C it was cooled to ambient temperature. Then
the absorbance measurements were made at 695 nm using microplate reader. 100 µL DMSO
10

was used as a negative control, while ascorbic acid as a positive control for respective
calibration curve. Expression of the resultant total antioxidant capacity (TAC) was made as µg
equivalent of ascorbic acid as per milligram of dried weight (µg AAE/mg DW).
2.4.3 Total reducing power assay (TRP)
Investigation of the total reducing power (TRP) was made in accordance with the standard
protocol [44]. 100 µL of the individual sample (4 mg/mL in DMSO) was mixed with 200 µL
of 0.2 M PO4-2 buffer (pH 6.6) and 250 µL of K3Fe(CN)6 (1 % w/v).The resultant reaction
mixture was incubated for 20 min at 50 oC followed by acidification of the reaction by adding
200 µL of CCl3COOH (10 % w/v). The reaction mixture was then centrifuged at 3000 rpm for
10 min.150 µL of the liquid from supernatant floating layer was removed and mixed with 50
µL of FeCl3 (0.1 % w/v)to carry out the optical density measurement at 630 nm. The positive
control used was ascorbic acid while the expression of results of total reducing power (TRP)
was as equivalents of µg of ascorbic acid per milligrams of dried weight (µg AAE/mg DW).
2.4.4 DPPH free radical scavenging assay
Free radical scavenging assay is a common method to determine the antioxidant capacity of a
test compound in which 2,2-diphenyl picrylhydrazyl (DPPH) was used to analyze the
scavenging activity of the compounds following the standard protocol [45] with some
alterations. 10 µL of the sample solution (4 mg/mL in DMSO) was mixed with 190 µL of
DPPH solution in methanol (0.004 % w/v) and incubated for 1 h in dark. Then the microplate
reader was used to carry out the optical density measurement at 515 nm wavelength. DMSO
was used as negative whereas ascorbic acid was used as a positive control. Initially the
screening of the samples was done at 200 µg/mL and those with better inhibition activity i-e ≥
50 % were then screened further for activity at lower concentration (66.6, 22.2 and 7.41 µg/mL)
11

to carry out calculation of their LC50 values. Following formula was used to calculate the
percentage inhibition.
퐴푏푠
% inhibition of the sample under test = Percent scavenging activity = (1 −
⁄
) × 100
퐴푏푐
Where,
Abs = λmax (DPPH solution + sample)
Abc = λmax negative control (reagent + solvent)
The calculation of LC50 was carried out by applying the 2D Version-4 Table curve software.
3
Results and discussion
Synthetic strategy for the preparation of the surfactants is outlined in Scheme-1. Ferrocene
functionalized cationic surfactants were synthesized through a three step mechanism. In the
first step, ferrocenyl aniline was synthesized by treating 4-nitroaniline with sodium nitrite in
the presence of HCl, and then reacting the resultant product with ferrocene in the presence of
phase transfer catalyst to obtain nitrophenyl ferrocene. The nitrophenyl ferrocene precursor
was then finally reduced to ferrocenyl aniline in the presence of hydrazine and palladium
charcoal [12, 40] . In the second step, 1-(4-ferrocenylphenyl)-3-(4-chlorobenzoyl)thiourea
derivative was synthesized by reacting 4-chlorobenzoyl chloride with potassium thiocyanate
which was further treated with ferrocenyl aniline to yield the product. In the third step, the
resultant product (F.R) was treated with different chain length alkyl chlorides to prepare
respective ferrocenyl cationic surfactants comprising different chain length.
Characterization of the synthesized compounds was carried out through single crystal XRD,
FT-IR, 1H and 13C-NMR spectroscopy.
3.1 Spectroscopic analysis
12

The proton NMR spectrum of ferrocenyl thiourea (F.R) shows two characteristic signals for
N-H protons at 12.5 ppm and 11.68 ppm confirming the formation of thiourea. The signals for
aromatic protons appear in the region between 7.57-8.02 ppm. The ferrocene signals lie in the
region between 4.04-4.81 ppm. (Figure S1a). In case of ferrocenyl cationic surfactants
containing aliphatic carbon chain the appearance of additional alkyl chain signals at 0.8-1.9
ppm and 3.5-3.9 ppm confirm the attachment of the long chain at N-H of thiourea adjacent to
the aromatic ring. There is also a slight shift in the value of two N-H peaks lying at 11.66 ppm
and 12.54 ppm respectively which might be due to the influence of the attached chain (Figure
S2a).
In case of 13C NMR spectra of ferrocenyl thiourea, the appearance of two extra signals at 168
ppm and 167 ppm that correspond to the carbonyl and thionyl group carbon atoms respectively,
confirmed the thiourea formation. The signals in the region of 124-138 ppm are assigned to
aromatic carbon atom. The region between 66.78-84.58 ppm corresponds to the Cp ring carbon
atoms of ferrocenyl moiety. (Figure S1b). 13C NMR of ferrocenyl cationic surfactants show a
bulk additional signals of long chain carbons in the region of 14-33 ppm that correspond to the
aliphatic C-atoms. The ferrocene carbons give four signals in the region of 66-85 ppm.
Chemical shift values of all the aromatic ring carbons lie in the region of 124-139 ppm. Signals
at 178.7 ppm and 167.6 ppm are specified for carbonyl and thionyl carbon atoms respectively
(Figure S2b).
FT-IR studies depict that the N-H band appears in the range of 3150-3270 cm-1 and sp2 (C-H)
band emanates at about 3027-3034 cm-1. The region between 2910-2970 cm-1 is asssigned to
sp3 (C-H), while the corresponding carbonyl (C=O) and thionyl (C=S) signals lie in the region
of 1710-1740 cm-1 and 1180-1270 cm-1 respectively. The intensity of carbonyl band appears to
be low due to the H-bonding between the carbonyl group oxygen atom and the H-atom of N-
H group next to the thionyl moiety. While the characteristic band for long-chain methylene (-
13

CH2-) for the surfactants lie in the range of 720-730 cm-1 and the peaks for Fe-Cp lie at 483
cm-1 for thiourea and at 484 cm-1 for the ferrocene-based cationic surfactants (Figure S3, Figure
S4) [46, 47].
3.2 Single crystal XRD studies
The crystallization of ferrocenyl thiourea monohydrate precursor in acetone yielded orange
colored small sized crystals. The crystal data shows that the crystal has monoclinic system with
space group P21/c (Table-1). The crystal structure diagram with numbering scheme (Figure-
1a) shows that the ferrocene Cp rings A(C15-C19) and B( C20-C24) are fully eclipsed.
Whereas A(C15-19) and D(C3-C8) are more or less planar to each other while C (C9-C14) is
slightly out of plane. Ferrocene moieties lie trans to each other according to the crystal packing
along the b-axis, Moreover, according to the packing along c-axis they lie perpendicular to
each other (Figure-1c,d). Figure 1-b shows the molecular packing with a cube exhibiting
minimal repulsion. Supramolecular interactions can also be seen within the crystal structure;
and water molecule is also incorporated with thiourea structure through intermolecular H-
bonding between the oxygen atom of the water molecule and the H-atom of one of the N-H
group. Intramolecular H-bonding between the oxygen atom of the carbonyl group (C=O) and
the H-atom of the N-H group (next to the thionyl C=S group) is also present within the structure
(Figure-1a).
3.3 Critical micelle concentration (CMC) determination
Surface tension studies were performed in order to determine the CMC points. Plot of surface
tension (γ) as a function of concentration of synthesized series of new ferrocene based cationic
surfactants are shown in Figure-2. A linear decrease in the surface tension value with each
successive addition of surfactant was observed and onset of micellization that trend deviates.
14

The point of deviation in plots of γ versus concentration gave the critical micelles concentration
in the ethanol water mixture (1:9). The plot depicts the clear indication of earlier micellization
in case of new ferrocene based cationic surfactants having longer alkyl chain length and follow
the trend F.R-8 > F.R-9 > F.R-10 > F.R-12 > F.R-14 > F.R-16 > F.R-18. (Table S1, Figure
2).
UV-visible spectroscopic studies were also employed to verify the critical micelle
concentration values of the synthesized compounds in ethanol-water mixture (1:9). For this
purpose, absorbance versus concentration profile was recorded, which obeying Beer-Lambert
law exhibited sharp rise in the beginning but at a certain point a slowdown in the absorbance
increment rate was obtained for the equal increase in concentration. The hinderance in
absorbance can be attributed to the formation molecular aggregates that prevent the UV-
radiations to penetrate the inner micellar structures. The data thus obtained was used to plot
absorbance versus concentration curves for the respective surfactants (Figure-3). The points of
inflection in the graphs indicated the micelle formation for particular surfactant at that
concentration and thus labeled as CMC. The CMC values as calculated by UV absorbance
method are in accordance with the values already determined through surface tension method
(Table S1). It was concluded from the CMC data of the seven surfactants that decreasing alkyl
chain length has a direct impact on micellization. Since, the process of aggregation is more
facile when compounds have higher hydrophobicity therefore the surfactants with longer chain
formed micelles at comparatively lower concentration and CMC ultimately increased with a
decrease in chain length. [48].
3.4 Bio-activities
The cytotoxic activity against the brine shrimp larvae was performed using three different
sample concentrations, out of which 500 µg/mL and 250 µg/mL were high enough to kill all
the brine shrimp larvae population, so the results could not have been utilized for a comparative
15

cytotoxicity effect of surfactants. For this purpose another lower sample concentration set was
employed which produced desired results pretty enough for comparative study of the
cytotoxicity effect, and longer chain compounds like F.R-18 and F.R-16 cytotoxicity was
comparable to that of the reference drug doxorubicin.
By considering the impact of alkyl chain hydrophobicity, the molecules comprising longer
alkyl chains were expectedly more toxic, which is also reported in the previous studies. But the
long chain is not the only factor, cationic head group also has some contribution in this
cytotoxic activity mechanism, due to the presence of ferrocene and thionyl functionalities. This
can be seen from the cytotoxic activity of ferrocenyl thiourea that is not containing any long
chain but still shows remarkable cytotoxicity. Upon addition of long chain to the ferrocenyl
thiourea cytotoxic activity is enhanced, which can be attributed to the long chain as well as the
positive charge of the head group. All the surfactants showed greater cytotoxic activity than
their parent ferrocenyl thiourea but on mutual comparison a descending order decrease in the
activity was observed with the subtraction of each ethyl moiety. This respective phenomena
can be ultimately accredited to the chain length effect. [49]. (Figure-4, Table S2). The LC50
values showed a variable trend with F.R-14, F.R-9 and F.R-8 showing highest LC50 values.
Antioxidant activities of the compounds were tested through DPPH inhibition assay, TRP assay
and TAC assay. The results obtained through all the three experiments showed approximately
similar antioxidant activity trend as observed through cytotoxicity assay, with the longer chain
surfactants showing comparatively greater cytotoxicity and an overall decreasing activity trend
was observed with the decrease in chain length. Herein the ferrocenyl thiourea (F.R) parent
group activity in the three graphs ( Figure-5, S5 Table S3) depicts that the main contributor
towards the antioxidant activity is probably the surfactant head group comprising ferrocene
antioxidant potential which is overall enhanced by the additional head group alkylation by
enhancing its bioavailability. Upon addition of the hydrophobic chain the antioxidant activity
16

of the long chain surfactants remains the same but as the chain length decreases the antioxidant
activity decreases rapidly. This phenomena can be attributed to the micellar effect since the
concentration of the surfactant samples for antioxidant activity was very much higher than their
individual CMC values. At such higher concentration the long rate of micelle degradation was
higher as compared with micelle formation in case of longer chain surfactants, leading to higher
monomer to micelle ratio. while in case of shorter chain surfactants there were enough micelles
to overall masking the bioavailability of the head group.[48-51]
5
Conclusions
Successful synthesis of ferrocene containing thiourea based cationic surfactants homologous
series has been carried out, with complete characterization of the compounds using different
techniques. The studies showed that the alkyl chain length effects the surfactant overall
properties like self-aggregation and biological properties. Furthermore it is also concluded that
longer chain length surfactants are more bio-applicable as compared with the shorter chain
length surfactants. Antioxidant potential of the surfactants was solely a property of head
structure containing ferrocene, which ultimately showed a decline with the reduction of the
added chain length. These experimental studies can be extended to carry out further analytical
comparison of more surfactant properties. Head structure, experimental conditions and solvent
compositions can also be varied to carry out more detailed studies.
Supplementary data
The crystallographic structural data for ferrocene containing thiourea compound being reported
in this paper is submitted in the Cambridge Crystallographic data center with supplementary
publication number CCDC-1909730. For which the crystallographic data copies will be
accessible at deposit@ccdc.cam.ac.uk.
17

Credit Author Statement:
The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript.
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
Acknowledgements
We are grateful to the financial support from the National Natural Science Foundation of China
(No. 21871180), the Program for Professor of Special Appointment (Eastern Scholar) at
Shanghai Institutions of Higher Learning (No. SHDP201802), the “Shuguang Program”
supported by the Shanghai Education Development Foundation and the Shanghai Municipal
Education Commission (No. 17SG12), the Science and Technology Commission of Shanghai
Municipality (Nos. 18520710300 and 17ZR1404100), and Biomedical Interdisciplinary
Research Foundation of SJTU and Pakistan Academy of Sciences, Pakistan.
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Scheme 1: Synthetic scheme for ferrocenyl thiourea cationic surfactants.
21

Step 1
O2N
HCl
NaNO2
90 min
P.T.C
O2N
N NCl
NH2
O2N
Fe
Diethyl
Ether
Fe
Pd Charcoal
24hrs reflux
Hydrazine
H2N
Step 2
O
C
O
Acetone
+
KSCN
Cl
Cl
C N C S
Room
Temperatu
re
Fe
12-15hrs
Acetone
O
S
Cl
72Hours
C N C N
R Cl
H
H
Step 3
Fe
Ethanol
O
S R
R= C18H37, C16H33, C14H29,
C12H25, C10H21, C9H19,
C8H17
Cl
C N C N
Cl
H
H
Fe
22

Figure 1: 1-(4-ferrocenylphenyl)-3-(4-chlorobenzoyl)thiourea (F.R) (a) molecular structure,
(b) molecular packing within the unit cell, (c) molecular packing along b-axis, (d) molecular
packing along c-axis.
Figure 2: Surface tension versus concentration graph showing chain length dependency of
CMC.
23

Figure 3: Absorbance versus concentration plots for the surfactants CMC determination.
Solvent used was ethanol/water mixture (1:9)
24

Figure 4: Cytotoxicity profiles of Brine shrimp larvae incubated with synthesized surfactants
and the reference drug Doxorubicin (100µg/mL).
25

Figure 5: Antioxidant activity of ferrocene based cationic surfactants versus their head group
molecule (a) DPPH free radical scavenging assay, (b) Total reducing power assay (TRP), (c)
Total antioxidant capacity assay (TAC).
Table 1: Crystallographic and refinement details of F.R.
Identification code
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
F.R
C24H21ClFeN2O2S
492.79
296(2)
Monoclinic
P21/c
a/Å
15.9706(6)
26

b/Å
11.4992(3)
c/Å
α/°
11.9627(4)
90
β/°
96.016(3)
γ/°
90
Volume/Å3
2184.84(12)
Z
4
1.498
ρcalcg/cm3
μ/mm-1
0.932
F(000)
1016.0
Crystal size/mm3
0.51 × 0.17 × 0.14
MoKα (λ = 0.71073)
4.374 to 54.996
-20 ≤ h ≤ 14, -14 ≤ k ≤ 11, -15 ≤ l ≤ 15
17509
Radiation
2θ range for data collection (°)
Index ranges
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indexes [I>=2σ (I)]
Final R indexes [all data]
Largest diff. peak/hole / e Å-3
5019 [Rint = 0.0323, Rsigma = 0.0362]
5019/3/286
1.026
R1 = 0.0413, wR2 = 0.0945
R1 = 0.0637, wR2 = 0.1066
0.34/-0.33
27