Design and synthesis of arylthiophene-2-carbaldehydes via suzuki-miyaura reactions and their biological evaluation

Article abstract of DOI:10.3390/molecules181214711

A series of various novel 4-arylthiophene-2-carbaldehyde compounds were synthesized in moderate to excellent yields via Suzuki-Miyaura cross-coupling with different arylboronic pinacol esters/acids. The synthesized products were screened for their antibacterial, haemolytic, antiurease, and nitric oxide (NO) scavenging capabilities and interestingly, almost all products turned out to have good activities. 3-(5-Formylthiophene- 3-yl)-5-(trifloromethyl)benzonitrile (2d) revealed excellent antibacterial activity, showing an IC50 value of 29.7 μg/mL against Pseudomonas aeruginosa, compared to the standard drug streptomycin with an IC50 value 35.2 μg/mL and was also found to be the best NO scavenger, with an IC50 value of 45.6 μg/mL. Moreover, 4-(3-chloro-4-fluorophenyl) thiophene-2-carbaldehyde (2i) exhibited a superior haemolytic action and an outstanding urease inhibition, showing an IC50 value of 27.1 μg/mL.

Full text of DOI:10.3390/molecules181214711

Molecules 2013, 18, 14711-14725; doi:10.3390/molecules181214711
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design and Synthesis of Arylthiophene-2-Carbaldehydes via
Suzuki-Miyaura Reactions and Their Biological Evaluation
Shaukat Ali 1, Nasir Rasool 1,*, Aman Ullah 2,*, Faiz-ul-Hassan Nasim 3, Asma Yaqoob 3,
Muhammad Zubair 1, Umer Rashid 4 and Muhammad Riaz 1
1
Department of Chemistry, Government College University, Faisalabad 38000, Pakistan;
E-Mails: shaukat_ali_48@yahoo.com (S.A.); zubairmkn@yahoo.com (M.Z.);
riaz_453@yahoo.com (M.R.)
2
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton,
AB T6G2P5, Canada
3
Department of Chemistry, Islamia University of Bahawalpur, Bahawalpur 63000, Pakistan;
E-Mails: faiz.nasim@hotmail.com (F.N.); asmayaqoobctn@gmail.com (A.Y.)
4
Institute of Advanced Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,
Malaysia; E-Mail: umer.rashid@yahoo.com
* Authors to whom correspondence should be addressed: E-Mails: nasirhej@yahoo.co.uk (N.R.);
amanullah@ualberta.ca (A.U.); Tel.: +92-333-661-6321 (N.R.); Fax: +92-41-920-1032 (N.R.);
Tel.: +780-492-4845 (A.U.); Fax: +780-492-4265 (A.U.).
Received: 30 September 2013; in revised form: 13 November 2013 / Accepted: 14 November 2013 /
Published: 27 November 2013
Abstract: A series of various novel 4-arylthiophene-2-carbaldehyde compounds were
synthesized in moderate to excellent yields via Suzuki-Miyaura cross-coupling with
different arylboronic pinacol esters/acids. The synthesized products were screened for their
antibacterial, haemolytic, antiurease, and nitric oxide (NO) scavenging capabilities and
interestingly, almost all products turned out to have good activities. 3-(5-Formyl-
thiophene-3-yl)-5-(trifloromethyl)benzonitrile (2d) revealed excellent antibacterial activity,
showing an IC50 value of 29.7 µg/mL against Pseudomonas aeruginosa, compared to the
standard drug streptomycin with an IC50 value 35.2 µg/mL and was also found to be the
best NO scavenger, with an IC50 value of 45.6 µg/mL. Moreover, 4-(3-chloro-4-fluoro-
phenyl)thiophene-2-carbaldehyde (2i) exhibited a superior haemolytic action and an
outstanding urease inhibition, showing an IC50 value of 27.1 µg/mL.

Molecules2013, 18
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Keywords: thiophene; Pd (0) catalyzed Suzuki coupling; antibacterial; heamolytic;
antiurease; antioxidant; activities
1. Introduction
In the recent years, Suzuki-Miyaura reactions have become one of the most powerful tools to build
carbon-carbon bonds between weakly basic heterocyclic compounds and various arylboronic
acids/esters [1,2]. These reactions have also been widely exploited in the synthesis of natural
products [3,4], and the design of the pharmaceuticals [5]. C-O, C-N, and C-S bonds can also be
constructed using these reactions due to their excellent functional group tolerance. Consequently,
Suzuki cross-coupling reactions have acquired great importance in the synthesis of substituted
aromatic compounds because these reactions are among the major and essential routes for any
synthetic organic chemist. They are a well-established family of chemical reactions very useful for the
synthesis of aryl-substituted thiophenes. Thiophene is used in advanced materials like dye-sensitized
organic solar cells and light emitting devices [6], in luminescence, electron transport, redox activity,
nonlinear optical chromism, electrochemical behavior, liquid crystalline characteristics and for its
spectroscopic properties [7–9]. Thiophene-based molecules have shown numerous biological activities
such as antitumor [10], analgesic [11], anti-inflammatory [12] and antibacterial activities [13], and as
building blocks for agrochemicals [10]. Palladium-catalyzed functionalizations of thiophene
derivatives have great importance due to the functional group compatibility, relative strength, low
toxicity, and greater versatility of organopalladium complexes. Iqbal et al. [14], Al-Adiwish et al. [15],
and Thomas et al. [16] reported that the arylthiophene derivatives bearing electron withdrawing
substituents showed excellent antibacterial activity against Gram-negative bacteria. Furthermore,
Sigmundova et al. [17] reported that arylthiophene derivatives with electron donating substituents
exhibited potent antibacterial activity against Gram-positive bacteria. Relatively less attention has been
paid to the synthesis of 4-arylthiophene-2-cabaldehydes and investigation of their biological activities,
therefore, we focused our attention on the synthesis of various of these compounds. Moreover, the
biological activities of these novel compounds were also studied to discover their potential to be used
as new pharmaceutical agents. To the best of our knowledge, the antibacterial, antiurease, heamolytic,
and antioxidant activities of these compounds have not been reported in the literature so far.
2. Results and Discussion
2.1. Chemistry
Dang and coworkers [18] reported the Suzuki cross-coupling reaction of tetrabromothiophene with
arylboronic acids to prepare the corresponding tetraarylthiophenes. Handy et al. [19] have reported
double Suzuki couplings of 4,5-dibromothiophene-2-carbaldehyde with different arylboronic acids.
Herein, we describe the application of the Suzuki cross-coupling reactions of 4-bromothiophene-2-
carbaldehyde (1) with equimolar amounts of various arylboronic acids/esters in the presence of K3PO4
and Pd(PPh3)4 to form the corresponding 4-arylthiophene-2-carbaldehydes (Scheme 1).

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Scheme 1. Synthesis of 4-arylthiophene-2-carbaldehydes 2a–j.
O
O
H
H
S
i. Arylboronicacids/esters
S
iii. Pd( PPH3)4
ii. K2CO3
Br
iv. 90oC,12hrs
Ar
2a-j
1
The factors affecting the optimization of reactions were also studied. Thus, the effect of solvent on
the coupling reactions between 1 and various arylboronic acids/esters under thermal conditions at 85–90 °C
was evaluated (Table 1). 4-Phenylthiophene-2-carbaldehyde (2a) was obtained in good yield when 1
was coupled with phenylboronic ester in 4:1 toluene/water in the presence of 5 mol% of the Pd(0)
1
catalyst using K3PO4 as base. Its H-NMR spectrum showed a multiplet in the δ 7.3–7.5 ppm region
due to the aryl protons and two singlets at δ 7.8 ppm and δ 8.1 ppm due to the protons at the 3 and
5 positions in the thiophene ring, respectively. The aldehyde proton exhibited a single peak at δ 9.95
ppm. The mass spectrum of the product 2a exhibited a molecular ion peak at m/z 188. Moreover, the
smooth coupling of compound 1 with 3,5-trifloromethylphenylboronic ester in 4:1 dioxane/water
under heating conditions gave 4-(3,5-bis(trifloromethyl)phenyl)thiophne-2-carbaldehyde (2b) in
excellent yield. Its 1H-NMR spectrum revealed two singlets at δ 7.24 ppm and δ 7.84 ppm assigned to
the protons at positions 5 and 3 of the thiophene ring, respectively. In addition, three singlets at δ 7.99,
8.06 and δ 9.99 ppm appeared due to the 2H at positions 2 and 6 of the aryl group, 1H at position
4 and the aldehyde proton, respectively. The mass spectrum of product 2b exhibited a molecular ion
peak at m/z 323. 5-Chloro-2,2-thiophene-5-carbaldehyde (2c) was obtained in 1,4-dioxane in 66% yield.
Similarly, the cross coupling of 1 with formyltrifloromethylphenylboronic ester gave 3-(5-formylthiophene-
3-yl)-5-(trifloromethyl)benzonitrile (2d) in moderate yield after refluxing in DMF. Reaction of 1 with
p-tolylphenylboronic acid in the presence of potassium hydroxide at 90 °C yielded p-tolylphenyl-
1
thiophene-2-carbaldehyde (2e). The H-NMR spectrum of this product gave a 3H singlet for the
methyl group in the δ 2.37 ppm region and a multiplet for the phenyl ring protons in the δ 7.2–7.4 ppm
region. The thiophene protons present at positions 3 and 5 also showed singlets at δ 7.9 ppm and
δ 7.7 ppm, respectively. The singlet assigned to the aldehyde proton appeared at δ 9.94 ppm. The most
efficient coupling of 1 with 3,5-dimethylphenylboronic acid produced 4-(3,5-dimethylphenyl)thiophene-2-
1
carbaldehyde (2g) in excellent yield after heating for 12 h. Its H-NMR spectrum revealed a singlet at
δ 2.35 ppm corresponding to the 6H of the two methyls present at positions 3 and 5 in the phenyl ring
due to their similar environments. In addition, a singlet peak at δ 9.94 ppm was assigned to the
aldehyde proton. The thiophene protons at positions 3 and 5 showed singlets at δ 8.0 and δ 7.79 ppm,
respectively. The protons present at positions 2 and 6 of the aryl group gave a singlet at δ 7.2 ppm. The
proton at the 4 position of the aryl group gave a singlet at δ 6.9 ppm. The mass spectrum of product 2g
exhibited a molecular ion peak at m/z 217. 5-Methyl-2,3-bisthiophene-5-carbalehyde (2j) exhibited a
molecular ion peak at m/z 189.67. Its 1H-NMR spectrum revealed two doublets at δ 4.10 and δ 7.01 ppm
due to the protons at the 3 and 5 positions in the thiophene ring, respectively. Moreover, two singlets
appeared at δ 7.65 and δ 7.87 ppm due to the protons at positions 2 and 4 in the thiophene, respectively.

Molecules2013, 18
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The 3H of the methyl group also showed a singlet at δ 2.37 ppm. The aldehyde proton exhibited a
singlet at δ 9.92 ppm. After the NMR and mass spectral study, these compounds were investigated for
their biological activities.
Table 1. Synthesis of 4-arylthiophene-2-carbaldehydes.
Entry
ArylBoronic Acids/Esters
Products
Solvent/H2O (4:1)
Yields%
O
CH
O
3
H
S
H C
3
B
1
Toluene
67
O
CH
H C
3
3
2a
O
CH
O
3
H C
3
H
S
B
2
3
1,4-Dioxane
1,4-Dioxane
64
70
O
CH
H C
3
3
2a
O
H
CF
S
CH
O
3
3
H C
3
B
O
CH
CF3
H C
3
CF
3
3
F C
3
2b
O
H
S
CH
O
H C
3
3
Cl
S
4
5
1,4-Dioxane
1,4-Dioxane
66
62
S
B
O
CH
Cl
H C
3
2c
3
O
CN
CF
CH
O
3
H
S
H C
3
B
CN
2d
O
CH
H C
3
3
3
F C
3
O
H
S
(HO)2B
CH
3
6
7
Toluene
64
57
CH
2e
3
O
H
S
(HO)2B
OCH
3
1,4-Dioxane
OCH
2f
3

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Table 1. Cont.
Entry
ArylBoronic Acids/Esters
Products
O
Solvent/H2O (4:1)
Yields%
H
S
CH
3
(HO)2B
8
1,4-Dioxane
71
CH
3
CH
3
H C
3
2g
O
H
S
Cl
Cl
9
1,4-Dioxane
1,4-Dioxane
1,4-Dioxane
37
40
69
(HO)2B
Cl
2h
Cl
O
H
S
F
10
11
Cl
(HO)2 B
CH
Cl
F
2i
O
H
H C
3
3
S
CH
3
S
O
B
S
O
CH
H C
3
3
CH
3
2j
2.2. Biological Evaluation
2.2.1. Antibacterial Activity
Antibacterial activity of newly synthesized compounds 2a–j was assayed against two selected
strains of Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and four strains of Gram-
negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Shigella dysenteriae, Salmonella typhi)
using the methods reported in the literature [20,21]. The plates were incubated for 24 h at 37 °C for
bacteria. The activity against the organisms were tested with solutions of 25 and 50 µg/mL
concentration and the resulting inhibition zone (IZ) was measured in mm as criterion for assessing the
antibacterial activity. The test results at the two different concentrations are presented in Tables 2 and
3 and further compared in Figure 1.
At the concentration of 25 µg/mL and 50 µg/mL, compound 2c with an IC50 value of 39.4 µg/mL
showed excellent activity against Bacillus subtilis. Compounds 2e–f, however, expressed moderate
activity against Bacillus subtilis showing IC50 values of 46.6 µg/mL, while, the compounds 2a–b, 2d,
2g–j revealed mild to moderate activity when compared to the standard streptomycin. Compound 2d
showed outstanding inhibition against Pseudomonas aeruginosa, with an IC50 value 29.7 µg/mL, whereas
compounds 2a–c, 2e–f, 2h–j exhibited mild to average activity against Pseudomonas aeruginosa.

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Table 2. Antibacterial activity of synthetic compounds at 25 µg/mL *.
Gram Positive Bacteria
Gram Negative Bacteria
Compound
S. aureus
36.6 ± 0.4
34 ± 0.01
33 ± 0.01
28 ± 0.2
B. subtilis
23 ± 0.09
P. aeruginosa
42 ± 0.02
45 ± 0.019
46 ± 0.02
44 ± 0.02
34 ± 0.04
25 ± 0.06
40 ± 0.041
42 ± 0.04
36 ± 0.16
41.5 ± 0.002
E.coli
18 ± 0.13
14 ± 0.14
32.5 ± 0.12
32 ± 0.05
9 ± 0.03
S. dysenteriae
33 ± 0.05
35 ± 0.01
35 ± 0.15
41 ± 0.05
42 ± 0.7
S. typhi
2b
2c
2d
2e
2f
33 ± 0.20
34 ± 0.05
30 ± 0.15
22.2 ± 0.14
36 ± 0.01
32.2 ± 0.11
30 ± 0.13
33 ± 0.25
29 ± 0.1
36.2 ± 0.05
28.3 ± 0.75
22.4 ± 0.13
24.4 ± 0.03
36 ± 0.08
34 ± 0.05
27 ± 0.08
2g
22.5 ± 0.11
32 ± 0.03
16 ± 0.043
26 ± 0.03
29.3 ± 0.002
32 ± 0.01
29 ± 0.19
43 ± 0.105
35 ± 0.2
2h
39.4 ± 0.05
27.8 ± 0.12
35.2 ± 0.01
15 ± 0.034
28 ± 0.03
2i
2j
36.5 ± 0.04
27.10 ± 0.01
34 ± 0.015
Streptomycin
48 ± 0.05
33 ± 0.001
* Inhibition zone (in mm); data are expressed in the form of mean ± SD.
Table 3. Antibacterial activity of synthetic compounds at 50 µg/mL *.
Gram Positive Bacteria
Gram Negative Bacteria
Compound
S. aureus
63 ± 0.18
62.4 ± 0.1
64 ± 0.11
63 ± 0.31
58 ± 0.01
61.4 ± 0.56
59 ± 0.44
59.4 ± 0.45
58 ± 0.16
65 ± 0.09
55 ± 0.03
B. subtilis
61 ± 0.14
59 ± 0.1
P. aeruginosa
67.6 ± 0.001
67 ± 0.01
E.coli
S. dysenteriae
63 ± 0.18
62.4 ± 0.1
64 ± 0.11
63 ± 0.31
58 ± 0.01
61.4 ± 0.56
59 ± 0.44
59.4 ± 0.45
58 ± 0.16
65 ± 0.09
55 ± 0.034
S. typhi
61 ± 0.13
63 ± 0.25
64 ± 0.5
2a
47 ± 0.15
38 0.18
2b
2c
60 ± 0.01
58 ± 0.35
54 ± 0.17
54 ± 0.23
56 ± 0.24
58 ± 0.22
56 ± 0.5
60 ± 0.009
67 ± 0.01
24 ± 0.17
40.5 ± 0.19
62 ± 0.01
8 ± 0.01
2d
64 ± 0.05
53 ± 0.12
63 ± 0.11
61 ± 0.16
57 ± 0.01
59.5 ± 0.25
63 ± 0.1
2e
60 ± 0.03
2f
64 ± 0.04
2g
56 ± 0.04
44.5 ± 0.19
60 ± 0.24
26 ± 0.023
66 ± 0.43
63 ± 0.002
2h
67 ± 0.021
66 ± 0.01
2i
2j
60 ± 0.05
53 ± 0.001
69 ± 0.16
Streptomycin
63 ± 0.002
53 ± 0.001
* Inhibition zone (in mm); data are expressed in the form of mean ± SD.
From the screening data, it was observed that electron withdrawing and donating groups on the aryl
ring had a substantial influence on the antibacterial activities, confirming earlier reports that on the
antibacterial activity of various aryl thiophene derivatives bearing electron donating and electron
withdrawing groups [14,16,22]. It was previously claimed that the antibacterial activity of compounds
containing electron withdrawing groups was more potent against Gram-negative bacteria. In agreement
with that statement, in our case compound 2d with CF3 and CN moieties and 2i with 3,4-dichloro
substitution on the phenyl ring showed potents result against Gram-negative bacteria. In [17], the
authors concluded that compounds with electron donating groups were more potent against
Gram-positive bacteria and showed non-significant activity against Gram-negative bacteria. In
agreement with the literature reports, compound 2g with a 3,5-dimethyl substitution on the phenyl ring
gave very poor activity, with an IC50 value of 45.16 µg/mL against Gram-negative bacteria as compared to
standard. Furthermore, 2j showed excellent antibacterial activity against Staphylococcus aureus, with an
IC50 value of 37.4 µg/mL, whereas, the compounds 2a–i exhibited mild to moderate activities against

Molecules2013, 18
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Staphylococcus aureus when compared to the standard. In addition, 2e and 2j were found to be active
against Escherichia coli, with IC50 values of 40 µg/mL. Nevertheless, 2a–d, 2f–i were found to be inactive
with zero IC50 value against Escherichia coli. Compound 2j with an IC50 value of 37.4 µg/mL showed
excellent activity against Salmonella typhi. Compound 2f with a 4-methoxyphenyl ring was found to be the
most active compound against the Gram-negative bacterium Salmonella typhi, with an IC50 value
37.9 µg/mL, but the main contribution to the activity probably came from the oxygen moiety of the
methoxy group on the phenyl ring.
Figure 1. IC50 values of antibacterial activity.
2.2.2. Antiurease Activity
Antiurease activity testing was carried out in accordance with the literature protocol [23] by using
thiourease, with an IC50 value of 27.5 µg/mL, as the standard inhibitor. The synthesized compounds
2a–j were examined for their urease inhibition activity at 25 µg/mL and 50 µg/mL concentration
(Table 4, Figure 2). It was considered that all these compounds could have the ability to bind with the
enzyme’s active site, whereby the enzyme stops catalyzing hydrolysis and the enzyme activity is
inhibited. All synthesized compounds showed moderate to excellent urease inhibition. The compounds
2a and 2i proved to be the most potent urease inhibitor, with IC50 values of 27.9 µg/mL and 27.1 µg/mL,
respectively. These values were comparable to the 27.5 µg/mL value of the thiourea standard.
Molecules 2b–h exhibited comparatively moderate antiurease activities. It was observed that
compound 2j was inactive against urease enzyme, with an IC50 value of 0 µg/mL. Noteworthy,
compound 2e gave good urease inhibition, with an IC50 value of 29.2 µg/mL at 50 µg/mL. Notably, we
observed that various functional groups present on aryl group were responsible for variable urease
inhibitions. The compound 2i with 3-chloro, 4-floro groups lead to enhanced urease inhibition, while
the compounds 2b, 2f with electron donating groups exhibited less urease inhibition, although,
compound 2d with electron withdrawing CN functional group showed surprisingly poor urease
inhibiting action.

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Table 4. Antiurease activity of synthesized compounds.
%age Activity
%age Activity
Entry
IC50 (µg/mL)
at 25 μg
at 50 μg
2a
45 ± 0.0162
35 ± 0.014
86.9 ± 0.012
87.0 ± 0.084
87.8 ± 0.006
88.7 ± 0.005
76.5 ± 0.05
80.2 ± 0.0017
87.2 ± 0.5
27.9
32.2
28.4
31.2
29.2
30.0
29.1
28.5
27.1
Zero
27.5
2b
2c
44 ± 0.0042
37 ± 0.0042
44.5 ± 0.057
42.3 ± 0.0007
42.5 ± 0.0070
44 ± 0.084
2d
2e
2f
2g
2h
86.4 ± 0.44
90.6 ± 0.05
9.7 ± 0.2
2i
2j
46.2 ± 0.0042
2 ± 0.219
Thiourea
47 ± 0.007
77 ± 0.015
Figure 2. IC50 values of anti-urease activity.
2.2.3. Nitric Oxide (NO) Scavenging Assay
Nitric oxide (NO) is a free radical causing inflammatory disorders and cancer in the human body
when sodium nitroprusside decomposes to produce nitric oxide [24,25]. The antioxidant activity of
various thiophene derivatives has been reported in the literature and it has also been observed that
compounds with high antioxidant activity served as antitumor agents [26]. The newly synthesized
compounds reacted directly with nitric oxide and inhibited nitrite formation in an excellent way. The
compounds 2a–j were evaluated for their nitric oxide scavenging activity according to the literature
protocol [27], at concentrations of 25, 50 and 100 µg/mL, respectively, and ascorbic acid (vitamin C)
was used as standard. Ascorbic acid showed a 40% ± 0.038% NO scavenging activity at 25 µg/mL,
61 ± 0.004 at 50 µg/mL and 77 ± 0.001 at 100 µg/mL concentrations, respectively (Table 5).

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Table 5. NO scavenging activity of synthesized compounds.
% Activity
at 25 µg
12 ± 0.037
10 ± 0.02
18 ± 0.007
12.0 ± 0.025
−15 ± 0.04
5 ± 0.005
13 ± 0.002
13 ± 0.002
12 ± 0.05
% Activity
at 50 µg
% Activity
at 100 µg
IC50
Compound
µg/mL
92.5
66.6
83.3
45.6
NA
NA
NA
NA
2a
2b
2c
2d
2e
2f
2g
2h
33 ± 0.0035
45 ± 0.001
40 ± 0.038
58 ± 0.010
−12 ± 0.037
10 ± 0.028
28 ± 0.007
20 ± 0.025
57 ± 0.0021
50 ± 0.024
61 ± 0.004
48 ± 0.049
60 ± 0.021
55 ± 0.040
73 ± 0.0028
3 ± 0.0014
25 ± 0.005
43 ± 0.002
35 ± 0.077
72 ± 0.009
65 ± 0.013
77 ± 0.001
2i
2j
46.1
49.3
56.33
15 ± 0.021
40 ± 0.038
Ascorbic Acid
The compounds 2d and 2i when systematically analyzed were found to be the most active in NO
scavenging, with percent activities of 58 ± 0.010 and 57 ± 0.0021 at 50 µg/mL and IC50 values of
45.6 µg/mL and 46.1 µg/mL, respectively. Compound 2j expressed comparatively excellent activity
showing a NO scavenging percentage 50 ± 0.024 at 50 µg/mL and 65 ± 0.013 at 100 µg/mL and an IC50
value of 49.3 µg/mL. It was noted that compound 2b also exhibited a significant NO scavenging activity,
with percentages of 45 ± 0.001 at 50 µg/mL and 60 ± 0.021 at 100 µg/mL and an IC50 value of 66.6 µg/mL.
Moderate NO scavenging percentages of 33 ± 0.0035 and 40 ± 0.038 at 50 µg/mL were observed for
2a and 2c, with IC50 values 92.5 µg/mL and 83.3 µg/ML, respectively, whereas, the compounds 2e–h
were found to be inactive for NO scavenging. Based on above results, we conclude that different
functional groups present on 4-arylthiophene-2-carbaldehyde derivatives 2a–j had substantial effects on
the percent NO scavenging activities. It was surprisingly observed that compound 2i with 3-chloro,
4-fluoro moiety showed a higher percentage of NO scavenging.
2.2.4. Haemolytic Activity
The cytotoxicity to blood lymphocytes, thymocytes and spleen cells of various thiophene
derivatives has already been reported [28]. The newly synthesized compounds 2a–j were also studied
for haemolytic activity by the method used by Sharma and Powell [29,30]. The compounds 2f and 2i
exhibited the highest haemolytic action, but less than the positive control (Triton-X 100), which
showed the maximum percentage of lysis (99.824 ± 0.536). Compounds 2d and 2e displayed the
lowest haemolytic property, but higher than the negative control PBS which showed zero percentage
lysis (Table 6). Compounds 2a–c, 2g–h, 2j demonstrated moderate haemolytic activity. The compounds
which showed the highest haemolytic activity might be considered as potential antitumor agents.
Table 6. Haemolytic activity of synthetic compounds 2a–j.
Entry
2a
2b
2c
2d
Average ± SD
3.947 ± 0.078
5.003 ± 0.078
3.934 ± 0.095
3.306 ± 0.058
3.356 ± 0.075
Entry
2f
2g
2h
2i
2j
Average ± SD
6.083 ± 0.143
4.525 ± 0.075
3.487 ± 0.054
9.779 ± 0.095
4.135 ± 0.095
2e
Standard
99.824 ± 0.536

Molecules2013, 18
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3. Experimental
3.1. General
Melting points were determined using a Buchi Melting Point B-540 apparatus. All reagents were
purchased from Alfa Aesar Chemical Co. (St. Parkridge Ward Hill, MA, USA) and Sigma Aldrich
1
13
Chemical Co. (St. Louis, MO, USA). H-NMR and C-NMR spectra were measured in CDCl3 and
CD3OD on a Bruker Aspect AM-400 instrument at 400/100 MHz The chemical shift was given in δ in ppm
and coupling constant in Hz. EI-MS spectra were recorded on a JMS-HX-110 spectrometer with a data
system. For column chromatography technique, silica gel (70–230 mesh) and silica gel (230–400 mesh)
were used. The reactions were monitored on TLC, using Merck silica gel 60 PF254 cards. The
compounds were visualized by UV lamp (254–365 nm).
3.2. General Procedure of Synthesis of 4-Arylthiophene-2-Carbaldehyde
To 4-bromothiophene-2-carbaldehdyde (1, 0.704 mmol) 5% mol Pd(PPh3)4 was added in 1,4-dioxane
under argon atmosphere. The reaction mixture was stirred at room temperature for 30 min. Arylboronic
esters/acids (0.774 mmol), and potassium phosphate (1.409 mmol) were added along with water (1.5 mL)
under an argon atmosphere. The solution was stirred at 90 °C for 12 h and then cooled to room
temperature. The organic layer was extracted using ethyl acetate, decanted and dried over magnesium
sulphate. Then the solvent was removed under reduced pressure. The crude residue obtained was
purified by column chromatography using n-hexane and ethyl acetate in 1:1 ratio to obtain the desired
products, which were characterized by spectroscopic techniques.
3.3. Characterization Data
1
4-Phenylthiophene-2-carbaldehyde (2a). Mp: 57–59 °C; H-NMR (CDCl3 + CD3OD): δ = 9.95 (s, 1H),
8.01 (s, 1H-thiophene), 7.83 (s, 1H-thiophene), 7.5 (d, J = 1.5 Hz, 1H-aryl), 7.44–7.39 (m, 2H-aryl),
7.36–7.31 (m, 2H-aryl). 13C-NMR (CDCl3 + CD3OD): δ = 126.30, 127.19, 128.09, 128.77, 129.06,
129.63, 130.86, 134.34, 134.73, 143.64, 144.37, 183.01; EIMS (m/z +ion mode): 188 [M‒H+];
[M‒CHO]+ = 160.01; [M‒C7H6]+ = 102.1; [M‒phenyl]+ = 115.1. Anal. Calcd. for C11H8OS (188.03):
C, 70.18; H, 4.28; O, 8.50; S, 17.03. Found: C, 70.22; H, 4.26; O, 8.55; S, 17.12%.
1
4-(3,5-Bis(trifloromethyl)phenyl)thiophene-2-carbaldehyde (2b). Mp: 95–97 °C; H-NMR (CDCl3 +
CD3OD): δ = 9.99 (s, 1H), 8.06 (s, 1H-aryl), 7.99 (s, 2H-aryl), 7.84 (s, 1H-thiophene), 7.24 (s,
1H-thiophene). 13C-NMR (CDCl3 + CD3OD): δ = 121.57, 126.32, 131.31, 132.37, 132.81, 133.76, 136.42,
140.44, 145.39, 182.56; EIMS (m/z +ion mode): 323 [M‒H+]; [M‒2C]+ = 226.0; [M‒O]+ = 207;
[M‒F2,C7H6O]+ = 182.1; [M‒Ph‒3-CF3‒5-CF3]+ = 113. Anal. Calcd. for C13H6F6OS (324.00): C, 48.16; H,
1.87; F, 35.16; O, 4.93; S, 9.89. Found: C, 48.22; H, 1.93; F, 35.05; O, 4.81; S, 9.78%.
1
5-Chloro-2,2-bisthiophene-5-carbaldehyde (2c). Mp: 229–231 °C; H-NMR (CDCl3 + CD3OD):
δ = 9.84 (s, 1H), 8.00 (d, J = 1.1 Hz, 1H-thiophene), 7.83 (d, J = 1.2 Hz, 1H-thiophene), 7.51 (d,
13
J = 6.9 Hz, 1H-thiophene), 6.7 (d, J = 3.9 Hz, 1H-thiophene). C-NMR (CDCl3 + CD3OD): δ = 123.4,
125.2, 126.3, 127.5, 136.7, 138.1, 140.2, 143.4, 182.4; EIMS (m/z +ion mode): 229 [M‒H+];

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[M‒CHO]+ = 201.00; [M‒O]+ = 211.00; [M‒thiophene]+ = 147.17. Anal. Calcd. for C9H5OS2 (192.98):
C, 55.92; H, 2.61; O, 8.28; S, 33.18. Found: C, 55.97; H, 2.65; O, 8.32; S, 33.13%.
1
3-(5-Formylthiophene-3-yl)-5-(trifloromethyl)benzonitrile (2d). Mp: 252–254 °C; H-NMR (CDCl3 +
CD3OD): δ = 9.97 (s, 1H), 8.08 (s, 1H-aAryl), 8.03 (s, 1H-aryl), 7.61–7.53 (m, 2H-thiophene, 1H-aryl).
13C-NMR (CDCl3 + CD3OD): δ = 22.69, 116.97, 127.09, 129.65, 131.54, 132.79, 133.40, 134.69, 137.95,
143.65, 144.30, 182.98; EIMS (m/z +ion mode): 280.58 [M‒H+]; [M‒CN]+ = 256; [M‒F2]+ = 243.91;
[M‒thiophene]+ = 198.70. Anal. Calcd. for C13H6F3NOS (281.05): C, 55.52; H, 2.15; F, 20.26; N,
4.98; O, 5.69; S, 11.40. Found: C, 55.57; H, 2.19; F, 20.21; O, 5.64; S, 11.48%.
1
4-p-Tolylthiophene-2-carbaldehyde (2e). Mp: 58–60 °C; H-NMR (CDCl3 + CD3OD): δ = 9.94 (s,
1H), 7.99 (s, 1H-thiophene), 7.791 (s, 1H-thiophene), 7.4 (d, J = 7.5 Hz, 1H-aryl), 7.2 (d, J = 7.8 Hz,
1H aryl), 2.37 (s, 3H). 13C-NMR (CDCl3 + CD3OD): δ = 22.67, 126.18, 129.06, 129.74, 131.58,
134.69, 137.95, 143.65, 144.30, 182.98; EIMS (m/z +ion mode): 202.01 [M‒H+]; [M‒CH3]+ = 187.05;
[M‒CHO]+ = 173.98; [M‒Ph]+ = 111.76; [M‒thiophenecarbaldehyde]+ = 77.58. Anal. Calcd. for C12H10OS
(202.01): C, 17.25; H, 4.98; O, 7.91; S, 15.85. Found: C, 17.28; H, 4.92; O, 7.93; S, 15.82%.
1
4-(4-Methoxyphenyl)thiophene-2-carbaldehyde (2f). Mp: 68–70 °C; H-NMR (CDCl3 + CD3OD):
δ = 9.84 (s, 1H), 8.16 (s, 1H-thiophene), 8.06 (s, 1H-thiophene), 7.68 (m, 2H-aryl), 7.05 (d, J = 8.4 Hz,
2H-aryl), 3.83 (s, 3H-OCH3). 13C-NMR (CDCl3 + CD3OD): δ = 55.38, 114.48, 127.72, 128.63, 132.18,
134.58, 143.38, 144.28, 159.57, 183.01; EIMS (m/z +ion mode): 217.86 [M‒H+]; [M‒CHO]+ = 190.56;
[M‒OCH3]+ = 188.03; [M‒Ph]+ = 141.76; [M‒thiophenecarbaldehyde]+ = 108.07. Anal. Calcd. for
C12H10O2S (218.02): C, 66.03; H, 4.62; O, 14.66; S, 14.69. Found: C, 66.12; H, 4.66; O, 14.59; S, 14.72%.
1
4-(3,5-Dimethylphenyl)thiophene-2-carbaldehyde (2g). Mp: 71–73 °C; H-NMR (CDCl3 +CD3OD):
δ = 9.94 (s, 1H), 8.00 (s, 1H-thiophene), 7.79 (s, 1H-thiophene), 7.18 (s, 2H-aryl), 6.98 (s, 1H-aryl), 2.35
(s, 6H, 2CH3). 13C-NMR (CDCl3 + CD3OD): δ = 21.35, 124.24, 129.47, 129.70, 134.28, 134.98,
138.66, 143.91, 144.23, 183.01; EIMS (m/z +ion mode): 217 [M‒H+]; [M‒CHO]+ = 189.00;
[M‒2CH3]+ = 189.00. Anal. Calcd. for C13H12OS (216.06): C, 72.19; H, 5.59; O, 7.40; S, 14.82.
Found: C, 72.24; H, 5.62; O, 7.38; S, 14.85%.
1
4-(3,4-Dichlorophenyl)thiophene-2-carbaldehyde (2h). Mp: 158–160 °C; H-NMR (CDCl3 + CD3OD):
δ = 9.95 (s, 1H), 7.94 (s, 1H-thiophene), 7.79 (s, 1H-thiophene), 7.62 (s, 1H-Aryl), 7.59 (d, J = 2.0 Hz,
13
1H-Aryl), 7.42 (d, J = 2.4 Hz, 1H-Aryl). C-NMR (CDCl3 + CD3OD): δ = 117.32, 121.87, 126.10,
129.91, 131.74, 134.15, 141.31, 144.81, 156.63, 159.12, 182.72; EIMS (m/z +ion mode): 256 [M‒H+];
[M‒CO]+ = 229.17.00; [M‒thiophene]+ = 85.00. Anal. Calcd. for C11H6Cl2OS (255.96): C, 51.38; H,
2.35; Cl, 27.58; O, 6.22; S, 12.47. Found: C, 51.40; H, 2.38; Cl, 27.63; O, 6.18; S, 12.51%.
4-(3-Chloro-4-fluorophenyl)thiophene-2-carbaldehyde (2i). Mp: 108–110 °C; 1H-NMR (CDCl3 + CD3OD):
δ = 9.95 (s, 1H), 7.83 (s, 1H-thiophene), 7.50 (s, 1H-thiophene), 7.48 (s, 1H-aryl), 7.41 (d, J = 2.0 Hz,
13
1H-Aryl), 7.39 (d, J = 2.0 Hz, 1H-aryl). C-NMR (CDCl3 + CD3OD): δ = 117.20, 126.07, 128.53,
129.90, 131.73, 134.13, 141.31, 144.81, 156.63, 159.12, 182.71; EIMS (m/z +ion mode): 240.25 [M‒H+];

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[M‒CHO]+ = 213.08; [M‒F]+ = 195.08. Anal. Calcd. for C11H6ClFOS (239.98): C, 54.89; H, 2.51; Cl,
14.73; F, 7.89 O, 6.65; S, 13.32. Found: C, 54.93; H, 2.51; Cl, 14.69; F, 7.91; O, 6.59; S, 13.36S%.
1
5-Methyl-2,3-bisthiophene-5-carbaldehyde (2j). Mp: 218–220 °C; H-NMR (CDCl3 + CD3OD): δ = 9.92
(s, 1H), 7.87 (s, 1H-thiophene), 7.65 (s, 1H-thiophene), 7.01 (d, J = 3.2 Hz, 1H-thiophene), 4.10 (d,
13
J = 6.8 Hz, 1H-thiophene), 2.02 (s, 3H-methyl). C-NMR (CDCl3 + CD3OD): δ = 15.34, 21.02, 22.67,
29.68, 60.38, 123.92, 126.05, 127.70, 133.91, 182.81; EIMS (m/z +ion mode): 189.67 [M‒H+];
[M‒CH3]+ = 175.87; [M‒CO]+ = 161.67; [M-thiophene]+ = 107.75. Anal. Calcd. for C10H8OS2
(208.02): C, 57.66; H, 3.37; O, 7.68; S, 30.79. Found: C, 57.61; H, 3.42; O, 7.64; S, 30.82%.
3.4. General Antibacterial Activity Assay Procedure
The newly synthesized compounds 2a–j were screened for their antibacterial activity through
microplate reader-based high throughput screening method [31] against four different strains of
Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Shigella dysenteriae, Salmonella
typhi) and two strains of Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis). These
strains were obtained from the Agha Khan University, Karachi, Pakistan. The standard antibiotic discs
were purchased from the manufacturer (Oxoid Ltd, Basingstoke, UK). The synthesized compounds
were dissolved in a solvent to get solutions of known concentration (5 µg/mL and 10 µg/mL). Using
Whatman filter paper (No. 44), two discs of 5 mm diameter and 0.65 mm thickness were cut and
sterilized in a hot air oven. In order to prepare sample discs of different concentrations, known
volumes of compounds solutions were applied on the discs, air dried and used. The standard antibiotic
discs were also prepared in the same way and used as positive control. Similarly, a disc prepared by
applying only solvent was used as negative control. The glass apparatus used for the antibacterial
activity was washed with plenty of water and sterilized in a hot air oven at 137 °C for 30 min. The
sterilized nutrient agar medium was poured under aseptic conditions into sterile glass petri
dishes/plates. For the maintenance of sub-cultures inoculation from stock cultures into nutrient broth
medium poured either in test tubes or flasks was done and cultures were grown over night for 16 h at
37 °C on shaker. After inoculation petri plates were kept at room temperature for 30 min so that the poured
medium is adsorbed. All the discs, including test sample, positive and negative controls were placed on
inoculated nutrient agar medium solidified in petri dishes with the help of sterile forceps and the petri plates
were incubated at 37 °C ± 2 °C for 24 h. Zone of inhibition was measured in mm using an ordinary ruler.
3.5. Antiurease Activity Assay Procedure
The reaction mixtures comprising sample solution (2 µL), phosphate buffer (17 µL) and sodium
nitroprusside (20 µL) were incubated at 37 °C for 2 h in well plates. Then Griess reagent (20 µL, 0.3%
sulphanilic acid in glacial acetic acid + 0.1% naphthylethylenediamine (NED): equal volumes of both
solutions mixed just before use) was added to each well plate and the mixture kept at room temperature
for 20 min to develop the color. The absorbance was measured at 528 nm by using an ELISA reader.
The results were processed using Gen 5 software. All the reactions were performed in triplicate.
Positive control (containing known antioxidant) and control (containing all samples) were run in

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parallel. Negative control was used to calibrate the instrument. The % age antioxidant activity was
determined by using the following formula:
% antioxidant activity = [(Absorbance of control − absorbance of sample)/(Absorbance of control)] × 100
3.6. Haemolytic Activity Assay Procedure
The newly synthesized compounds 2a–j were studied for haemolytic activity [32]. Three mL of
freshly obtained human blood was placed in heparinized tubes to avoid coagulation, gently mixed and
poured into a sterile 15 mL Falcon tube and centrifuged for 5 min at 1,000 ×g. The supernatant was
discarded and red blood cells were washed three times with chilled (4 °C) sterile isotonic phosphate
buffer saline pH 7.4 (5 mL). Erythrocytes (108 cells/mL) were maintained for each assay by dilution
with phosphate buffer saline. Erythrocytes were counted on a heamacytometer. Twenty µL of each
compound was mixed with red blood cells suspension (180 μL) separately. The samples were
incubated at 37 °C for 35 min. The samples were placed on ice for 5 min and then agitated at 1,000 ×g
for 5 min. Supernatant (100 µL) were taken from each tube and diluted 10 times with chilled (4 °C)
phosphate buffered saline. Triton X-100 (0.1% v/v) was taken as a positive control and PBS was negative
control and then subjected to the same process. The absorbance was noted at 576 nm by using a Quant
instrument (BioTek, Winooski, VT, USA). The percentage RBSs lyses for each sample was calculated.
4. Conclusions
Suzuki-Miyaura reactions were successfully exploited to synthesize a series of 4-arylthiophene-2-
carbaldehyde 2a–j. The reaction progress and purity of all synthesized compounds were monitored by
TLC and products were characterized and their structure confirmed by NMR, elemental analysis, mass
spectroscopy and melting points. It was observed from the results of antibacterial activity, heamolytic
activity, urease inhibition and NO scavenger property that both electron withdrawing and electron
donating groups on aryl ring substantially influenced the activities. The compounds possessing electron
withdrawing groups on aryl ring exhibited potent antibacterial action against Gram-negative bacteria and
those with electron donating group on aryl ring showed excellent antibacterial activity against
Gram-positive bacteria. To evaluate the efficiency of the various 4-arylthiophene-2-cabaldehyde
derivatives, we compared different functional groups present at various positions on aryl ring.
Compound 2d emerged as the most active against Gram-negative bacteria and was also found to be an
outstanding NO scavenger, while the remaining compounds also exhibited mild to moderate activities
when compared to the standards. The antiurease and the heamolytic screening results indicated that
compound 2i was an excellent antiurease and heamolytic agent, while the other compounds showed
mild to significant activities as compared to the standards employed.
Acknowledgments
The present data is the part of Ph.D thesis research work of Shaukat Ali. The authors gratefully
acknowledge the financial support by HEC and HEJ, Institute of Chemistry, University of Karachi for
providing access to scientific instruments.

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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 available from the authors.
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