Selective C-arylation of 2,5-dibromo-3-hexylthiophene via Suzuki cross coupling reaction and their pharmacological aspects

Article abstract of DOI:10.3390/molecules20035202

The present study reports the synthesis of various new derivatives based on 5-aryl-2-bromo-3-hexylthiophene with moderate-to-good yields via a palladium-catalyzed Suzuki cross-coupling reaction. This coupling method involved the reaction of 2,5-dibromo-3-

Full text of DOI:10.3390/molecules20035202

Molecules 2015, 20, 5202-5214; doi:10.3390/molecules20035202
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Selective C-Arylation of 2,5-Dibromo-3-hexylthiophene
via Suzuki Cross Coupling Reaction and Their
Pharmacological Aspects
Hafiz Mansoor Ikram 1, Nasir Rasool 1,*, Gulraiz Ahmad 1, Ghayoor Abbas Chotana 2,
Syed Ghulam Musharraf 3, Muhammad Zubair 1, Usman Ali Rana 4, Muhammad Zia-Ul-Haq 5
and Hawa Ze Jaafar 6,*
1
Department of Chemistry, Government College University Faisalabad, Faisalabad 38000, Pakistan;
E-Mails: chemistue@gmail.com (H.M.I.); gulchemist35@gmail.com (G.A.);
nr_308@hotmail.com (M.Z.)
2
Department of Chemistry, SBA School of Science & Engineering,
Lahore University of Management Sciences, Sector U, DHA, Lahore Cantt. 54792, Pakistan;
E-Mail: ghayoor.abbas@lums.edu.pk
3
International Center for Chemical and Biological Sciences, HEJ Research Institute of Chemistry,
University of Karachi, Karachi 75270, Pakistan; E-Mail: musharraf1977@yahoo.com
4
Sustainable Energy Technologies (SET) center, College of Engineering, PO-Box 800,
King Saud University, Riyadh 11421, Saudi Arabia; E-Mail: urana@ksu.edu.sa
5
The Patent Office, Karachi 74200, Pakistan; E-Mail: ahirzia@gmail.com
6
Department of Crop Science, Faculty of Agriculture, University Putra Malaysia, Serdang 43400,
Selangor, Malaysia
* Authors to whom correspondence should be addressed; E-Mails: nasirrasool@gcuf.edu.pk (N.R.);
hawazej@gmail.com (H.Z.J.); Tel.: +92-332-7491790 (N.R); Fax: +92-41-9201032 (N.R).
Academic Editor: Derek J. McPhee
Received: 4 December 2014 / Accepted: 16 February 2015 / Published: 23 March 2015
Abstract: The present study reports the synthesis of various new derivatives based on
5-aryl-2-bromo-3-hexylthiophene with moderate-to-good yields via a palladium-catalyzed
Suzuki cross-coupling reaction. This coupling method involved the reaction of
2,5-dibromo-3-hexylthiophene with several arylboronic acids in order to synthesize
corresponding thiophene derivatives under controlled and optimal reaction conditions.
The different substituents (CH3, OCH3, Cl, F etc.) present on arylboronic acids are found to
have significant electronic effects on the overall properties of new products. The synthesized

Molecules 2015, 20
thiophene molecules were studied for their haemolytic, biofilm inhibition and
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anti-thrombolytic activities, and almost all products showed potentially good properties.
The compound 2-bromo-5-(3-chloro-4-fluorophenyl)-3-hexylthiophenein particular
exhibited the highest values for haemolytic and bio-film inhibition activities among all
newly synthesized derivatives. In addition, the compound 2-bromo-3-hexyl-5-(4-
iodophenyl)thiophene also showed high anti-thrombolytic activity, suggesting the potential
medicinal applications of these newly synthesized compounds.
Keywords: Palladium(0); 2,5-dibromo-3-hexylthiophene; biofilm inhibition; hemolysis assay;
anti-thrombolytic assay
1. Introduction
In the area of synthetic organic chemistry, Suzuki cross-coupling reactions have emerged as an
efficient and remarkably effective method for carbon-carbon bond formation [1]. Hence, the coupling
reactions of weakly basic heterocyclic compounds with organoboron compounds i.e., arylboronic acids
and esters, catalyzed by palladium have been found to be very useful for the synthesis of advanced
materials, agrochemicals and pharmaceutical compounds at both the laboratory and industrial levels [2].
The Suzuki cross-coupling reaction is largely preferred over other types of coupling reactions because it
can be carried out under normal reaction conditions with a wide range of functional group tolerance, it is
unaffected by aqueous solvent, and it generates non-toxic by-products which can be separated
easily [3]. Substituted thiophene compounds are present in various natural and biologically active
compounds [4]. Thiophene based molecules have a great deal of importance and exhibit medicinal
activities such as anti-fungal, anti-inflammatory, anti-microbial, analgesic, anti-urease, anti-tubercular,
anti-depressant, BASE1 inhibitors, anti-HIV PR inhibitors and anti-breast cancer activities [4,5]. In
addition, thiophene compounds have great applications in electrochromic devices [6], non-linear
optics [7], azo dyes [4], energy storage devices [8], conductivity-based sensors, biodiagnostics,
optoelectronics systems and superconductors [4]. In order to investigate an easy and facile scheme to
make thiophene-based derivates, the aim of the present study was to synthesize different derivatives of
2,5-dibromo-3-hexylthiophene using different arylboronic acids via palladium-catalyzed Suzuki
cross-coupling reactions. In addition to the synthesis, we also investigated the biofilm inhibition,
hemolytic and anti-thrombolytic activities of these thiophene derivatives, and the results revealed
potentially high activity from these newly synthesized thiophene derivatives against several diseases.
2. Results and Discussion
2.1. Chemistry
The term “regioselectivity” refers to the greater reactivity of a carbon moiety (due to being more
electron-deficient) towards nucleophiles, whereas other carbons being reactive do not display any
response to the attacking nucleophile. This term can be applied to palladium-cataylzed cross-coupling
reactions of different substrates [9]. Furan, thiophene and pyrrole undergo electrophilic substitution

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reactions such as halogenation reactions. In these substitution reactions, the lone pair of electrons
belonging to O, N and S is donated to the ring. However, when these halogenated thiophenes undergo
palladium-catalyzed cross-coupling reactions with high oxidative addition, the arylboronate ion
preferably attacks the carbon, which is deficient in electrons due to being bonded with the halogen. It
was also noted that the transmetallation process is faster due to negatively charged arylboronate ion than
the neutral boronic acid [10]. Dang et al. [11] reported the Suzuki cross-coupling reaction involving
tetrabromothiophene and arylboronic acids to synthesize tetraarylthiophenes. Herein, we have explored
the application of Suzuki cross-coupling reactions [12] to synthesize new thiophene derivatives. To the
best of our knowledge, Suzuki cross-coupling reactions of 2,5-dibromo-3-hexylthiophene derivatives
have not been investigated so far. The reactions were carried out at 90 °C temperature to get
moderate-to-good yields of the desired products. In the current research work, Pd(PPh3)4was used as
catalyst and K3PO4 was used as a base. The Suzuki reaction of 1(1 mmol) with different arylboronic
acids (1 mmol) resulted in5-aryl-2-bromo-3-hexylthiophenes (2a–i) in moderate to good yields
(Scheme 1, Table 1). The results from the present study revealed that greater yields of products were
obtained upon using 1,4-dioxane as solvent as compared to toluene, probably due to relatively higher
solubility of arylboronic acids in 1,4-dioxane. The high boiling point of toluene makes it beneficial for
reactions carried out at high temperature, but its negative impact is poor yields of products. We found
that the yield of the final product was significantly affected by the solvent [13]. A number of different
5-aryl-2-bromo-3-hexylthiophene derivatives (2a–i) (Table 1) were synthesized by using a wide range of
arylboronic acids. Different derivatives of 2,5-dibromo-3-hexylthiophene were synthesized (Scheme 1)
and the desired products (2a–i) were attained in moderate–to-good yields when 1,4-dioxane was used
as a solvent (entries 1–9). Only moderate yields were obtained when toluene was used as solvent. The
final yields of the products were greatly influenced by the conditions such as temperature, nature of
solvent and the water content. The best solvent/water ratio was found to be 4:1 (solvent/water), as
reported by [14].
Scheme 1. Synthesis of 5-aryl-2-bromo-3-hexylthiophene (2a–i). Reagents and conditions:
1 (1 mmol), Arylboronic acids (1 mmol), K3PO4 (1.75 mmol), Pd(PPh3)4 (4 mol %),
solvent/H2O (4:1), (see Table 1), 90 °C, 12 h.

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Table 1. Synthesis of 5-aryl-2-bromo-3-hexylthiophene (2a–i).
Entry
Aryl Boronic Acids
Product
Solvent/H2O (4:1)
Yield%
Dioxane
76
1
4-MeC6H4B(OH)2
Toluene
57
2
3,5-Me2C6H3B(OH)2
Dioxane
70
3
4
4-MeOC6H4B(OH)2
4-ClC6H4B(OH)2
Dioxane
Dioxane
68
77
5
4-IC6H4B(OH)2
Dioxane
69
F
Br
S
6
7
8
3,5-F2C6H3B(OH)2
3-Cl,4-FC6H3B(OH)2
4-MeSC6H4B(OH)2
Dioxane
Dioxane
Dioxane
65
68
62
F
2f
9
3-AcC6H4B(OH)2
Dioxane
72

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2.2. Pharmacological Aspects
2.2.1. Haemolytic Activity
Compared with the positive control triton X-100 standard, compounds 2a–b, 2f and 2h–i showed
moderate haemolytic activity, whereas compounds 2c and 2g exhibited the highest toxicity effects.
It was surprisingly noted that compound 2g exhibited the highest % lysis of RBCs (19.54%) against
triton X-100, which might be due to the presence of electron-withdrawing F and Cl moieties present on
the phenyl ring. Table 2 shows that compound 2e showed moderate haemolytic activity (10.22%),
probably due to the presence of a bulky iodine group at para position in the aromatic phenyl ring. The
data from Table 2 also shows that compounds 2b and 2i exhibited low toxicity effect with % lysis
values of RBC (4.54%) and (5.51%), respectively. In contrast, compound 2c showed a higher toxicity
value (19.50%). This might be due to the presence of one -OCH3 group (negative inductive effect and
positive resonance effect). Mologni et al. [15] reported that anticancer activity is usually enhanced by
the presence of electron-releasing groups (positive resonance effect) [15]. Therefore, the presence of a
-OCH3 group in compound 2c could be the possible cause of the higher % lysis of RBCs. In view of
the observed differences in the % lysis values of RBC, it is suggested that the electron withdrawing
and electron donating functional groups have an influence on the haemolytic activity of the
compounds. Ding et al. [16] reported that the compounds with the Cl functional group at the benzene
ring exhibited better and higher haemolytic activities than the compounds with -CH3 substitution at the
same position. For this reason, compound 2d (9.13%) showed better haemolytic activity value than
compound 2a (7.52%). These compounds (2a–i) showed moderate to high % lysis of RBC and can be
used as potential anticancer agents (Figure 1).
Table 2. Haemolytic activity data of 5-aryl-2-bromo-3-hexylthiophene based compounds (2a–i).
Entry
% Lysis of RBC ± SD
7.52 ± 0.042
4.54 ± 0.054
19.50 ± 0.079
9.13 ± 0.113
10.22 ± 0.084
6.55 ± 0.071
19.54 ± 0.095
5.97 ± 0.102
5.51 ± 0.089
100%
2a
2b
2c
2d
2e
2f
2g
2h
2i
Positive Control

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Figure 1. Values of haemolytic, anti-thrombolytic and biofilm inhibition activities.
2.2.2. Anti-Thrombolytic Activity
Thrombophilia and nephritic syndrome causes a common central nervous disease cerebral venous
sinus thrombosis (CVST) [17]. For the treatment of CVST, intravenous heparin and warfarin have
been explored as anti-coagulant agent, primarily due to their high efficiency [18]. At present,
thrombolytic drugs have been used as an alternative cure for this fatal disease [19]. Several
thrombolytic agents like streptokinase and urokinase are playing a vital role in the treatment of
cerebral venous sinus thrombosis [20]. The compounds 2e, 2d and 2i exhibited better thrombolytic
effects, while compounds 2a–c and 2h showed mild to moderate action when compared with the
positive control streptokinase standard. Compound 2e displayed the highest % lysis (34.27) against
streptokinase standard, which might be due to the presence of a bulky iodine group at the aryl ring. On
the other hand, the compounds 2d and 2i (Table 3) showed moderate activity values of 26.75% and
24.59%, respectively, probably due to the presence of Cl and acetyl groups at the para position of the
phenyl ring. The data from Table 3 also shows that the compounds 2a and 2h exhibited low effect with
% lysis values of 3.43% and 3.60%, respectively. Moreover, it is suggested that the presence of
electron-withdrawing groups (negative inductive effect) promote thrombolytic activity, while the
position of functional groups is also found to affects the thrombolytic activity of all newly synthesized
compounds. These compounds (2a–i) showed moderate to high % lysis and can be used as potential
thrombolytic agents (Figure 1).
Table 3. Anti-thrombolytic activity data of 5-aryl-2-bromo-3-hexylthiophene based
compounds (2a–i).
Entry
% Lysis ± SD
3.43 ± 0.022
7.58 ± 0.031
5.43 ± 0.015
26.75 ± 0.071
34.27 ± 0.019
14.54 ± 0.053
16.52 ± 0.035
3.6 ± 0.027
24.59 ± 0.046
100
2a
2b
2c
2d
2e
2f
2g
2h
2i
Positive control

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2.2.3. Biofilm Inhibition Assay
Biofilm formation by pathogenic bacteria is an increasing cause of morbidity and mortality
associated with several fatal infections. Biofilm is conglomerations of bacterial cells, which is
protected by a self-created extra polymeric substance. Biofilms are very difficult to eliminate and
cause obstinate infections [21]. The anti-biofilm activity of synthesized thiophene derivatives (2a–i)
was studied against strains of Escherichia coli. Table 4 shows the results of biofilm inhibition for all
synthesized compounds. It was observed that a higher positive charge density leads to strong
electrostatic interaction [22]. Based on the results of antimicrobial activity of the newly synthesized
compounds 2g and 2d were considerably affected by electron-withdrawing substituents and the
incorporation of electron-withdrawing groups (negative inductive effect) responsible for enhancing
activity against the test microorganism when compared with standard Rifampicin. Compounds 2d, 2e
and 2g, which contain electron-withdrawing functional groups, showed good inhibition values
(73.13%, 67.02%, and 75.66%, respectively) against E. coli. Compound 2c, which contains
electron-donating methyl groups, showed low biofilm inhibitory activity (52.12%) against E. coli.
Surprisingly, it was observed that compound 2h, which has a negative inductive and positive
resonance effect, exhibited good biofilm inhibition (71.52%) against E. coli.
Table 4. Biofilm inhibition activity data of 5-aryl-2-bromo-3-hexylthiophene based
compounds (2a–i).
Entry
% Inhibition ± SD
66.75± 0.154
65.69± 0.134
52.12± 0.165
73.13± 0.154
67.02± 0.167
51.99± 0.188
75.66± 0.176
71.52± 0.163
68.48± 0.139
97.43
2a
2b
2c
2d
2e
2f
2g
2h
2i
Positive control
3. Experimental
3.1. General
1
A Bruker Aspect AM-400 NMR instrument was used to study H-NMR and 13C-NMR
spectroscopic values in CDCl3 and CD3OD at 400/100 MHz. The chemical shift values are reported in
δ ppm and the coupling constant values in Hertz (Hz). The melting points of newly synthesized
products were obtained from a Buchi B-540 melting point apparatus. For synthesis, all analytical
chemicals were obtained from Alfa Aesar and Sigma Aldrich. EI-MS values of the products were
obtained from data system containing JMS-HX-110 spectrometer. To assist column chromatography,
both types of silica gels 70–230 mesh and 230–400 mesh were used. For proper monitoring of the

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reaction, the Merck silica gel 60 PF254 TLC cards were used. The new compounds were investigated
by UV lamp of wavelength 254 to 365 nm.
3.2. General Procedure for the Synthesis of 5-Aryl-2-Bromo-3-Hexylthiophene (2a–i)
The 2,5-dibromo-3-hexylthiophene (1 mmol) and 4 mol % Pd(PPh3)4 were added in an organic
solvent 1,4-dioxane (2 mmol) under inert (argon) atmosphere. The mixture was stirred in a Schlenk
flask at 25 °C for a duration of 30 min. After stirring, the arylboronic acids (1 mmol) and K3PO4
(1.75 mmol) were added along with water (0.5 mL) under the same atmosphere. The solution was
stirred again at 90 °C for a duration of 12 h. After stirring for 12 h, the reaction mixture was cooled to
room temperature. The organic layer in the reaction mixture was separated by using CH3COOC2H5
and desiccated above MgSO4. After that, the excess solvent was evaporated by applying reduced
pressure on a rotary evaporator. The crude product thus obtained, and was purified using column
chromatography. In chromatography, ethyl acetate and n-hexane were used in equal ratios to acquire
the final compounds. The end products were characterized by different spectroscopic techniques [23].
3.3. Characterization Data
1
2-Bromo-3-hexyl-5-(4-methylphenyl)thiophene (2a). Mp: 170–173 °C; H-NMR (CDCl3 + CD3OD):
δ = 7.70 (d, J = 8.05 Hz, 2H-aryl), 7.30 (d, J = 8.05 Hz, 2H-aryl), 2.35 (s, 3H-CH3), 6.80
(s, 1H-Thiophene), 2.65 (t, J = 7.83 Hz, 2H-Methylene), 1.35 (m, 6H-Methylene), 1.30
13
(m, 2H-Methylene), 0.85 (t, J = 6.08 Hz, 3H-CH3). C-NMR (CDCl3 + CD3OD): δ = 14.23, 21.11,
22.94, 27.50, 29.01, 31.11, 31.98, 110.10, 125.72, 126.80, 129.70, 130.87, 131.90, 141.43, 142.10.
EIMS (m/z + ion mode): 337.32: [M-Br]+ = 257.42: [M-C6H13]+ = 172.42: [M-Thiophene]+ = 91.42:
[M-CH3]+ = 76.15. Anal. Calcd. for C17H21BrS (337.32): C, 60.53; H, 6.28. Found: C, 60.41; H, 6.17%.
2-Bromo-3-hexyl-5-(3,5-dimethylphenyl)thiophene (2b). Mp: 175–177 °C; 1H-NMR (CDCl3 + CD3OD):
δ = 7.60 (s, 2H-aryl), 7.35 (s, 1H-aryl), 2.35 (s, 6H-2CH3), 6.90 (s, 1H-Thiophene), 2.62 (t,
J = 7.61 Hz, 2H-Methylene), 1.32 (m, 6H-Methylene), 1.28 (m, 2H-Methylene), 0.88 (t, J = 6.20 Hz,
13
3H-CH3). C-NMR (CDCl3 + CD3OD): δ = 14.21, 21.98, 22.89, 27.38, 28.99, 31.19, 32.01, 110.15,
127.13, 127.51, 130.99, 133.59, 138.90, 141.40, 142.05. EIMS (m/z, + ion mode): 351.34:
[M-Br]+ = 271.44: [M-C6H13]+ = 186.44: [M-Thiophene]+ = 105.44: [M-2CH3]+ = 75.40. Anal. Calcd.
for C18H23BrS (351.34): C, 61.53; H, 6.60. Found: C, 61.43; H, 6.51%.
1
2-Bromo-3-hexyl-5-(4-methoxyphenyl)thiophene (2c). Mp: 174 °C; H-NMR (CDCl3 + CD3OD):
δ = 7.50 (d, J = 8.28 Hz, 2H-aryl), 7.05 (d, J = 6.18 Hz, 2H-aryl), 6.85 (s, 1H-Thiophene), 3.83
(s, 3H-OCH3), 2.60 (t, J = 7.71 Hz, 2H-Methylene), 1.33 (m, 6H-Methylene), 1.28 (m, 2H-Methylene),
13
0.90 (t, J = 6.23 Hz, 3H-CH3). C-NMR (CDCl3 + CD3OD): δ = 14.15, 22.84, 27.49, 29.07, 31.15,
31.85, 55.89, 110.13, 114.71, 126.08, 127.11, 128.61, 141.28, 142.06. 160.67. EIMS (m/z, + ion mode):
353.32: [M-Br]+ = 273.42: [M-C6H13]+ = 188.42: [M-Thiophene]+ = 107.42: [M-OCH3]+ = 76.20.
Anal. Calcd. for C17H21BrOS (353.32): C, 57.79; H, 5.99. Found: C, 57.68; H, 5.91%.
1
2-Bromo-3-hexyl-5-(4-chlorophenyl)thiophene (2d). Mp: 175 °C; H-NMR (CDCl3 + CD3OD):
δ = 7.60 (d, J = 7.91 Hz, 2H-aryl), 7.75 (d, J = 6.01 Hz, 2H-aryl), 6.88 (s, 1H-Thiophene), 2.63

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(t, J = 7.69 Hz, 2H-Methylene), 1.31 (m, 6H-Methylene), 1.27 (m, 2H-Methylene), 0.86 (t, J = 6.16 Hz,
3H-CH3). 13C-NMR (CDCl3 + CD3OD): δ = 14.12, 22.91, 27.47, 28.93, 31.24, 31.94, 110.03, 126.93,
128.88, 129.41, 131.78, 134.43, 141.33, 142.17. EIMS (m/z, + ion mode): 357.74: [M-Br]+ = 277.84:
[M-C6H13]+ = 192.80: [M-Thiophene]+ = 111.80: [M-Cl]+ = 76.30. Anal. Calcd. for C16H18BrClS
(357.74): C, 53.72; H, 5.07. Found: C, 53.67; H, 5.01%.
2-Bromo-3-hexyl-5-(4-iodophenyl)thiophene (2e). Mp: 179 °C; 1H-NMR (CDCl3 + CD3OD): δ = 7.62
(d, J = 8.18 Hz, 2H-aryl), 8.05 (d, J = 6.25 Hz, 2H-aryl), 6.82 (s, 1H-Thiophene), 2.65 (t, J = 7.58 Hz,
2H-Methylene), 1.35 (m, 6H-Methylene), 1.29 (m, 2H-Methylene), 0.89 (t, J = 6.17 Hz, 3H-CH3).
13C-NMR (CDCl3 + CD3OD): δ = 14.08, 22.73, 27.49, 28.81, 31.17, 32.01, 94.37, 110.07, 127.12,
129.14, 132.67, 138.19, 141.31, 142.01. EIMS (m/z, + ion mode): 449.19: [M-Br]+ = 369.29:
[M-C6H13]+ = 284.20: [M-Thiophene]+ = 203.20: [M-I]+ = 76.32. Anal. Calcd. for C16H18BrIS
(449.19): C, 42.78; H, 4.04. Found: C, 42.69; H, 3.99%.
1
2-Bromo-5-(3,5-difluorophenyl)-3-hexylthiophene (2f). Mp: 171–173 °C; H-NMR (CDCl3 + CD3OD):
δ = 7.35 (s, 2H-aryl), 6.85 (s, 1H-aryl), 6.75 (s, 1H-Thiophene), 2.60 (t, J = 7.74 Hz, 2H-Methylene),
1.33 (m, 6H-Methylene), 1.27 (m, 2H-Methylene), 0.90 (t, J = 6.19 Hz, 3H-CH3). 13C-NMR
(CDCl3 + CD3OD): δ = 14.19, 22.79, 27.57, 28.92, 31.18, 31.95, 103.76, 109.16, 111.78, 126.90, 136.73,
140.87, 143.01, 167.02. EIMS (m/z, + ion mode): 359.27: [M-Br]+ = 279.37: [M-C6H13]+ = 194.34:
[M-Thiophene]+ = 113.34: [M-2F]+ = 75.35. Anal. Calcd. for C16H17BrF2S (359.27): C, 53.49; H, 4.77.
Found: C, 53.37; H, 4.68%.
2-Bromo-5-(3-chloro-4-fluorophenyl)-3-hexylthiophene (2g). Mp: 174 °C; 1H-NMR (CDCl3 + CD3OD):
δ = 8.05 (s, 1H-aryl), 7.70 (d, J = 7.95 Hz, 1H-aryl), 7.25 (d, J = 6.37 Hz, 1H-aryl), 6.85
(s, 1H-Thiophene), 2.65 (t, J = 7.68 Hz, 2H-Methylene), 1.37 (m, 6H-Methylene), 1.30 (m,
2H-Methylene), 0.85 (t, J = 6.01 Hz, 3H-CH3). 13C-NMR (CDCl3 + CD3OD): δ = 14.13, 21.99, 27.12,
28.10, 31.01, 31.56, 110.47, 117.67, 121.52, 127.10, 127.98, 129.87, 130.97, 141.56, 142.77, 158.98.
EIMS (m/z, +ion mode): 375.73: [M-Br]+= 295.83: [M-C6H13]+ = 210.83: [M-Thiophene]+ = 129.80:
[M-F]+ = 110.83: [M-Cl]+ = 75.20. Anal. Calcd. for C16H17BrClFS (375.53): C, 51.15; H, 4.56. Found:
C, 51.09; H, 4.53%.
1
2-Bromo-3-hexyl-5-(4-(methylthio)phenyl)thiophene (2h). Mp: 175 °C; H-NMR (CDCl3 + CD3OD):
δ = 7.75 (d, J = 8.15 Hz, 2H-aryl), 7.50 (d, J = 6.18 Hz, 2H-aryl), 6.88 (s, 1H-Thiophene) 2.55
(s, 3H-SCH3), 2.63 (t, J = 7.56 Hz, 2H-Methylene), 1.32 (m, 6H-Methylene), 1.28 (m, 2H-Methylene),
13
0.89 (t, J = 6.16 Hz, 3H-CH3). C-NMR (CDCl3 + CD3OD): δ = 14.04, 14.97, 23.03, 27.23, 28.58,
31.43, 32.17, 109.98, 126.88, 127.05, 128.12, 130.49, 139.65, 141.70, 142.35. EIMS (m/z, +ion mode):
369.38: [M-Br]+ = 289.40: [M-C6H13]+ = 204.40: [M-Thiophene]+ = 123.41: [M-SCH3]+ = 76.35. Anal.
Calcd. for C17H21BrS2 (369.38):C, 55.28; H, 5.73. Found: C, 55.15; H, 5.64%.
1
2-Bromo-3-hexyl-5-(3-acetylphenyl)thiophene (2i). Mp: 172 °C; H-NMR (CDCl3 + CD3OD):
δ = 8.85 (s, 1H-aryl), 8.05 (d, J = 7.65 Hz, 1H-aryl), 7.65 (t, J = 7.90 Hz, 1H-aryl), 7.95 (d, J = 7.43
Hz, 1H-aryl), 6.83 (s, 1H-Thiophene), 2.52 (s, 3H-CH3), 2.67 (t, J = 7.59 Hz, 2H-Methylene), 1.35
(m, 6H-Methylene), 1.30 (m, 2H-Methylene), 0.92 (t, J = 6.08 Hz, 3H-CH3). 13C-NMR

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(CDCl3 + CD3OD): δ = 14.08, 22.81, 26.78, 27.31, 28.59, 31.28, 32.12, 110.17, 126.42, 127.24,
129.04, 129.47, 131.07, 133.73, 137.63, 141.13, 142.30, 198.01. EIMS (m/z, +ion mode): 365.33:
[M-Br]+ = 285.43: [M-C6H13]+ = 200.41: [M-Thiophene]+ = 119.41: [M-Ac]+ = 76.30. Anal. Calcd. for
C18H21BrOS (365.33): C, 59.18; H, 5.79. Found: C, 59.07; H, 5.73%.
3.4. Haemolytic Activity
The haemolytic activity of newly synthesized compounds was studied using the previously reported
method [24]. Ethyl acetate was used to make the solution of newly synthesized compounds under
study. Approximately 3 mL of newly attained heparinized human blood was gently mixed, poured into
a 15 mL sterile Falcon tube and centrifuged at 850 rpm for the period of five minutes. The supernatant
was decanted off and the red blood cells were washed three times with ice-cold (4 °C) disinfected
isotonic phosphate buffered saline (PBS) pH 7.4 (5 mL) and kept for half an hour at room temperature.
The cells were kept in 20 mL ice-cold PBS buffer. Erythrocytes were calculated by heamacytometer,
maintained 7.068 × 108 cell per mL and diluted with phosphate buffer saline. About 180 µL
suspension of red blood cells was added in the eppendorfs alongwith 20 µL of sample solutions. The
tubes were incubated at 37 °C for aperiod of 35 min. After incubation, the tubes were agitated again
for 5 min. at 1310 rpm after placing the tubes on ice for the same time. After centrifugation was
completed, 100 µL supernatant was diluted with 900 µL ice-cold phosphate buffer saline and samples
were placed on ice. About 200 µL solution of each sample was added into 96-well plastic tissue
culture plates. 0.1% triton X-100 was used as a positive control for every evaluation, and as a negative
control, phosphate buffer saline was used. The absorbance of each sample was observed at 576 nm by
Quant instrument.
3.5. Biofilm Inhibition Assay
The biofilm inhibition activity of newly synthesized compounds was studied using the method
reported in [25]. One hundred micro liters of nutrient broth (Oxoid, UK) alongwith 100 μL sample
solutions (2a–i) and 20 μL of bacterial suspension were kept on the wells of sterile 96-well plastic
tissue culture plates. Negative control wells involved only nutrient broth. For 24 h, the plates were
incubated at 37 °C after covering. The sterile phosphate buffer (220 μL) was used to wash the content of
each well thrice. The non-adherent bacteria were eliminated by shaking the plates vigorously. 220 μL
methanol solution with the concentration of 99% was used to fix the remaining bacteria, and after
15 min the empty plates were left to dry. The dried plates were stained with 50% crystal violet
(220 mL) for aperiod of 5 min. The plates were washed with tap water for the purpose of removing
excess stain. After drying the plates, the dye bound to the adherent cells was resolublized with 220 µL
glacial acetic acid 33% per well. The OD of each well was determined at 630 nm by using the
micro-plate reader (BioTek, Winooski, VT, USA). The following formula was used to calculate the
bacterial growth inhibition (INH%):
100 - (OD
× 100)
630 sample
INH% =
OD 630 control

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3.6. Anti-Thrombolytic Activity
The anti-thrombolyticactivity of newly synthesized products was studied using the previously
reported method [26].The venous blood samples were taken from healthy human volunteers and
shifted to sterile eppendorf tubes/microcentrifuge tubes. Five hundred microliters of blood containing
microcentrifuge tubes were incubated for the period of 45 min at 37 °C temperature to form clots.
After clot formation, the microcentrifuge tubes, without serum which was discarded earlier, were filled
with 100 μL solution of test samples (2a–i). After this addition, all eppendorf tubes were again
incubated for 90 min at 37 °C and observed. For standard clot lysis agent, the thrombolytic drug
streptokinase was used while in case of negative thrombolytic control, and water was used in each
assay. The conclusions for clot lysis activity were determined as percentages.
4. Conclusions
The present study reports the Suzuki-Miyaura cross-coupling reaction for the synthesis of a series
of 5-aryl-2-bromo-3-hexylthiophene (2a–i). TLC was used to examine the progress of reaction and
purity of all new products, and their structure elucidation was confirmed by spectroscopic techniques.
The detailed investigation shows that aryl rings possessing different electron-withdrawing and
electron-donating groups have appreciable effects on the haemolytic, biofilm inhibition and
anti-thrombolytic activities of the newly synthesized compounds. Excellent haemolytic, biofilm
inhibition and anti-thrombolytic actions were exhibited by compounds with electron-withdrawing
groups on the aryl ring. The positions and natures of functional groups on aryl rings were compared with
each other to investigate the efficiency of various 5-aryl-2-bromo-3-hexylthiophene (2a–i) derivatives.
The compound 2-bromo-5-(3-chloro-4-fluorophenyl)-3-hexylthiophene (2g) was found to be most
efficient and active against bacteria and revealed superior haemolytic as well as biofilm inhibition
activities, while other compounds showed mild to moderate activities. In addition, 2-bromo-3-hexyl-5-
(4-iodophenyl)thiophene (2e) also exhibited highest anti-thrombolytic activity, indicating the
usefulness of these newly synthesized compounds for medicinal applications.
Acknowledgments
The present data is part of the M.Phil thesis research work of Hafiz Mansoor Ikram. The authors
gratefully acknowledge the financial support of the HEC (HEC Project No. 20-1465/R&D/09/5458).
The authors would also like to extend their sincere appreciation to the Deanship of Scientific Research
(DSR) at King Saud University for its funding of this research through the Research Group Project no
RGP-VPP-345
Author Contributions
H.M.I, N.R, G.A, G.A.C, S.G.M, MZ, and U.A.R, made a significant contribution toexperiment
design, acquisition of data, analysis and drafting of the manuscript. M.ZU.H and H.Z.J. have made a
substantial contribution to interpretation of data, drafting and carefully Revising the manuscript for
intellectual content. All authors read and approved the final manuscript.

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Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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