Synthesis and antimicrobial activity of novel thienopyrimidine linked rhodanine derivatives

Article abstract of DOI:10.1139/cjc-2018-0220

This work presents the preparation of a new series of N-(substituted phenyl)-2-(4-oxo-5-(4-(thieno[2,3-d]-pyrimidin-4-yloxy)benzylidene)-2-thioxothiazolidin-3-yl)acetamide derivatives (8a-8l). A condensation reaction of thienopyrimidin-2-thioxothiazolidin

Full text of DOI:10.1139/cjc-2018-0220

Canadian Journal of Chemistry
Synthesis and Antimicrobial Activity of Novel Thienopyrimidine Linked
Rhodanine Derivatives
Kerru Nagarajua, Surya Narayana Maddilaa, Suresh Maddilaa,b* Sreedhar
b
a
Sobhanapuram , Sreekantha B. Jonnalagadda
aSchool of Chemistry & Physics, University of KwaZulu-Natal, Westville Campus,
Chilten Hills, Private Bag 54001, Durban-4000, South Africa.
bGITAM Institute of Sciences, GITAM University,
Visakhapatnam, Andhra Pradesh, India.
*
Corresponding author
E-mail:sureshmskt@gmail.com
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Abstract:
The current work states that the preparation of a new series of N-(substituted phenyl)-2-
4-oxo-5-(4-(thieno[2,3-d]-pyrimidin-4-yloxy)benzylidene)-2-thioxothiazolidin-3-yl)acetamide
(
derivatives (8a-l). A condensation reaction of thienopyrimidin-2-thioxothiazolidin-4-one
derivative (5) with various 2-chloro-N-phenylacetamides (7a-l) was employed to afford the new
thienopyrimidine tagged rhodanine derivatives under acetone solvent in the presence of
1
potassium carbonate (K CO ). All the novel target molecules were characterized by IR, H
2
3
1
3
NMR, C NMR, and LC-MS spectral analyses and were screened for their in vitro antimicrobial
activity by using broth dilution method. Compounds 8c, 8g and 8h found to have antibacterial
potency against E. coli, B. subtilis, B. cereus and K. pneumonia with MIC values 3.25 to 6.25
µg/mL compare with the standard Gentamicin. Compounds 8c and 8f demonstrated better
antifungal potency (MICꢀ=ꢀ3.25 – 6.25 µg/mL) against A. flavus, A. niger, P. marneffei, and C.
albicans when compared with Fluconazole.
Keywords:
Synthesis;
Thieno[2,3-d]pyrimidinones;
Rhodanine;
N-phenylacetamide;
Antimicrobial activity.
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Introduction
Recently, microbial infection is one of the most complex health problems in worldwide
and facing various types of microbial (bacterial and fungal) contaminations in humans and
animals.1,2 The control of infective illnesses still scraps a significant and interesting problem
because of a mixture of influences counting an emergent infective diseases and the growing
number of multidrug resilient bacteriological, fungicidal pathogens.3-5 In spite of a huge amount
6
of antibiotics and chemotherapeutics accessible for medicinal usage, on the other hand an
occurrence of old and innovative antibiotic resistance formed in the past periods exposed a
7
considerable therapeutic necessity for new classes of antimicrobial agents. There is essential for
the finding of novel molecules endowed with antimicrobial activity, feasibly performing via
mechanisms of action, which are distinctive from those of eminent stages of bacterial proxies to
which several scientifically related pathogens are now resilient.6-8 From this point of view, it is a
vital to enhance most effective antimicrobial agents.
Although, numerous thienopyrimidine frameworks have reported with different wide-
range of pharmacological activities like antimicrobial, analgesic, anti-inflammatory11 and
9
10
1
2
antitumor. Further, these activities also inhibit various protein kinase as CK2 involved in a
1
3
specific anticancer activity. Thus, the segment displays an essential role in medicinal chemistry
always since it is a communal moiety in numerous structures of biologically active compounds
which approves a remarkable array of activities. Furthermore, rhodanine comprising hybrids
displayed several biological activities such as antidiabetic,14,15 antimicrobial16-19 and antiviral
agents20 and also medicinally important rhodanine drug Epalrestat is a clinical used for the
2
1
treatment of diabetic problems. Therefore, the improvement of such hybrid molecules with N-
phenylacetamide framework, which can readily combine with a range enzymes and receptors in
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organisms during forming hydrogen bonding interactions, has been discovered to be a reasonable
antibacterial strategy. With our continuous concern in improving various synthetic approaches
for biologically novel heterocyclic compounds22-27 and synthesis of heterocycles via one-pot,
multicomponent reactions under green conditions,28-32 we herein describing to study the
influence of thienopyrimidine moiety and 2-thioxothiazolidine-3-yl-N-phenylacetamide scaffold
combination molecules which were subsequently displayed synergistic antimicrobial effect.
Results and Discussions
Chemistry
The preparation of target N-(substituted phenyl)-2-(4-oxo-5-(4-(thieno[2,3-d] pyrimidin-
4
-yloxy)benzylidene)-2-thioxothiazolidin-3-yl)acetamide derivatives (8a-l) was represented in
scheme-1. Initially, the key starting material 2-aminothiophene-3-carbonitrile (1) was
synthesized using by Gewald method via multi-component condensation between elemental
Sulphur, carbonyl compound and malanonitrile in ethanol solvent, triethyl amine as a catalyst.20
Further, cyclisation of amino and cyano functionality of compound (1) on treatment with formic
acid and sulphuric acid under reflux at 110 °C resulted in the formation of thieno[2,3-d]
pyrimidin-4(3H)-one (2). Next, compound 2 reacted with phosphorous oxychloride to obtain 4-
chlorothieno[2,3-d]pyrimidine (3). Then, the reaction of compound 3 with p-hydroxy
benzaldehyde in the presence of potassium carbonate as a catalyst in acetonitrile solvent
produced the corresponding 4-(thieno[2,3-d]pyrimidin-4-yloxy)benzaldehyde (4). Subsequent
condensation of 4 using with rhodamine in the presence of sodium acetate as catalyst in acetic
acid system to furnished 5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-thioxothiazolidin-
4
-one (5). Simultaneously, another key intermediate aromatic N-phenylacetamide derivatives
(7a-l) were prepared from substituted anilines and chloro acetyl chloride in the presence of TEA
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o
as catalyst in acetone at 0 C. Finally, the title compounds, (8a-l) were synthesized by the
involving of two intermediates such as compound 5 and 7a–l in acetone in the presence of
K CO as catalyst at reflux condition. All the synthesized molecules were characterized and
2
3
analyzed by HNMR, 13CNMR, LC-MS and IR spectra. The conversion of thieno[2,3-
d]pyrimidin-4-yloxy)benzylidene)-2-thioxothiazolidin-4-one (5) to thieno[2,3-d]pyrimidin-4-
yloxy)benzylidene)-2-thioxothiazolidin-3-yl)-N-phenylacetamide derivatives (7a-l) were
1
established by the appearance of a singlet at δ 4.82-5.28 ppm due to the resonance of CH of the
2
1
N-phenylacetamide ring. H NMR spectra of compounds 8a-l exhibited a singlet around 8.72-
9
.16 ppm for pyrimidine CH proton, broad singlet range 4.82–5.26 ppm corresponding to or NH
proton and another singlet peak range at 4.82-5.26 due to CH group.
2
Biological Evaluation
All the target molecules (8a-l) were confirmed for their in vitro antimicrobial activities.
The antibacterial strains viz., Escherichia coli and Klebsiella pneumonia for Gram-negative and
Bacillus subtilis and Bacillus cereus for Gram-positive and the fungal strains Aspergillus flavus,
Aspergillus niger, Penicillium marneffei and Candida albicans were used. The minimum
inhibitory concentration (MIC) was determined by the broth dilution approach using Mueller-
Hinton nutrient broth medium and Gentamicin and Fluconazole were used as reference drugs.
The MIC (lowest concentration (highest dilution) of the compound) values were found more
potent with either less or equal as compared to standard drugs and were represented in Table 1
and Table 2.
The MIC of the assayed compounds (8a-l) against bacterial pathogens ranged from 1.56
to 100 µg/mL. Of the three compounds 8c, 8g and 8h showed most potent activity against all the
bacterial strains. The hybrid 8c having electron withdrawing 4-nitro substituted N-
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phenylacetamide was found to be the excellent activity with MIC 1.56 µg/mL against E. coli and
3
.12 µg/mL against B. subtilis, B. cereus and K. pneumonia compare with the standard
Gentamicin (MIC = 6.25 µg/mL). Although, the electron donating substituted (methoxy and
methyl) compounds 8g and 8h exhibited good antibacterial activity with MIC values of 6.25
µg/mL against B. subtilis, E. coli and K. pneumonia, respectively, with these MIC values equal
to the standard Gentamicin (MIC = 6.25 µg/mL). Further, all other reaming synthesized
derivatives (8a-b, 8d-f and 8i-l) displayed moderate to weak activity with ranging from
2
5µg/mL to 100 µg/mL or >100 µg/mL against all the tested bacterial strains.
Moreover, the antifungal activity revealed that the compounds 8c, 8e, 8f and 8l exhibited
considerable activity against all the tested fungal strains with MIC values ranging from 3.12-6.25
µg/mL due to the effect of electron withdrawing groups (NO and Cl) substituted on N-
2
phenylacetamide ring. The compounds 8a, 8d, 8g and 8k displayed comparatively good activity
against all the fungal strains with MIC 6.25-25 µg/mL. All the remaining analogues were sighted
with low effects at MIC level around 100 µg/mL, where as some were devoid of potency with
>
100 µg/mL of MIC against the mentioned panel of fungal strains.
It is noticeable from the investigation of antibacterial activity results that electron
withdrawing group (nitro) and electron donating groups such as methoxy, methyl groups
irrespective of position in the N-phenylacetamide ring derivative showed strong effect against
four bacterial strains. Further, in presence of electron withdrawing groups (nitro and chloro)
substituted on N-phenylacetamide ring derivatives displayed remarkably activity against four
fungal strains.
Conclusion
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In this study, synthesis of a new series of N-(substituted phenyl)-2-(4-oxo-5-(4-
(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-thioxothiazolidin-3-yl)acetamide
derivatives
(8a-l) were described. The in vitro antimicrobial activity of these compounds were evaluated by
broth dilution method. The electron withdrawing group (nitro) compound 8c showed excellent
activity and electron donating group compounds methoxy and methyl (8g & 8h) were exhibited
good activity against all bacterial and fungal strains. The remaining compounds displayed
moderate to poor antimicrobial activity.
Experimental
Melting points were determined in open capillaries on a Mel-Temp apparatus and are
uncorrected. All the reactions were monitored by thin layer chromatography (TLC) on precoated
silica gel 60 F254 (mesh); spots were visualized with UV light. Merck silica gel (60-120 mesh)
was used for column chromatography. The IR spectra were recorded on a Perkin-Elmer BX1
‒
1 1
FTIR Spectrophotometer as KBr pellets and the wave numbers were given in cm . H NMR
1
3
(
400 MHz) and C NMR (100 MHz) spectra were recorded on a Bruker AMX 400 MHz NMR
spectrometer in CDCl /DMSO-d solution using TMS as an internal standard.The mass spectra
3
6
were recorded on Agilent 1100 LC/MSD instrument with method API-ES at 70 eV. All chemical
shifts are reported in δ (ppm) using TMS as an internal standard. Chemicals were purchased
from Sigma Aldrich and used without further purification.
Procedure for preparation of thieno[2,3-d]pyrimidin-4(3H)-one (2)
2
-Aminothiophene-3-carbonitrile (1) (6 mmol) was treated with formic acid (36 mmol)
°
for 4 h at 120 C. After exploit of reaction, the mixture was cooled and washed with water. Next,
the crude was purified by recrystallization with ethanol.
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°
-1
1
Yield: 96%; mp 186-188 C; IR (KBr) (ν /cm ): 3379, 2884, 1541; H NMR (400 MHz;
max
DMSO-d ): δ 6.86 (d, J = 8.0 Hz, 1H, thiophene-CH), 7.09 (s, 1H, NH); 7.21 (d, JHH = 8.0
6
H
HH
Hz, 1H, thiophene-CH), 8.85 (s, 1H, pyrimidine-CH), 13C NMR (100 MHz; DMSO-d ): δ
6
C
1
19.26, 129.36, 137.24, 146.19, 151.16, 162.36; LC-MS (70 eV): m/z = 153 (M+H)+.
Procedure for preparation of 4-chlorothieno[2,3-d]pyrimidine (3)
To a mixture of compound 2 (10 mmol) and phosphorus oxychloride (15 mmol) was
added and the reaction substances were refluxed for 5 h. After completion of the reaction, then
the reaction mixture was cooled to room temperature and poured into ice cold water. The
obtained crude product was separated by filtration, washed with ice cold water, dried well and
recrystallized with ethanol to obtain pure compound.
°
-1
1
Yield: 92%; mp 191-193 C; IR (KBr) (ν /cm ): 2905, 1526, 768; H NMR (400 MHz;
max
DMSO-d ): δ 6.88 (d, J = 8.0 Hz, 1H, thiophene-CH), 7.05 (d, JHH = 4.0 Hz, 1H, thiophene-
6
H
HH
1
3
CH), 9.07 (s, 1H, pyrimidine-CH); C NMR (100 MHz; DMSO-d ): δ 124.11, 132.37, 140.31,
6
C
1
47.26, 150.47, 159.91; LC-MS (70 eV): m/z = 170 (M+H)+.
General procedure for preparation of 4-(thieno[2,3-d]pyrimidin-4-yloxy)benzaldehyde (4)
A mixture of 4-chlorothieno[2,3-d]pyrimidine 3 (6 mmol) and p-hydroxy benzaldehyde (
6
mmol) dissolved in acetonitrile (15 mL) in the presence of anhydrous potassium carbonate (24
0
mmol) stirred at 40 C for 5 h. After completion of the starting compounds, then the mixture was
cooled to room temperature, poured and washed with ice cold water. Then the crude product was
dried and recrystallized with ethanol to obtain in good yields.
0
-1
1
Yield: 95%; mp 170-172 C; IR (KBr) (νmax/cm ): 2871, 1721, 1613, 1528; H NMR (400 MHz;
DMSO-d ): δ 6.97 (d, J = 8.0 Hz, 1H, thiophene-CH), 7.12 (d, JHH = 4.0 Hz, 1H, thiophene-
6
H
HH
CH), 7.36 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.64 (d, JHH = 8.0 Hz, 2H, Ar-H), 8.72 (s, 1H,
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pyrimidine-CH), 9.46 (s, 1H, CHO); 13C NMR (100 MHz; DMSO-d ): δ 119.16, 123.26,
6
C
1
2
27.28, 133.01, 141.07, 146.17, 152.46, 156.19, 158.09, 162.39, 169.71; LC-MS (70 eV): m/z =
57 (M+H)+.
General procedure for synthesis of 5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-
thioxothiazolidin-4-one (5)
A combination of compound (4) (1 mmol), rhodanine (1 mmol) and sodium acetate (3
o
mmol) in acetic acid (5 mL) was stirred and refluxed for 6 h at 90 C. The reaction mixture was
poured and washed with cold water. The resulted crude product was recrystallized by ethanol.
0
-1
1
Yield: 97%; mp 192-194 C; IR (KBr) (ν /cm ): 2852, 1701, 1620, 1530; H NMR (400 MHz;
max
DMSO-d ): δ 6.93 (d, J = 8.0 Hz, 1H, thiophene-CH), 7.09 (d, JHH = 4.0 Hz, 1H, thiophene-
6
H
HH
CH), 7.28 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.36 (s, 1H, NH), 7.59 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.81
1
3
(
s, 1H, CH), 9.08 (s, 1H, pyrimidine-CH); C NMR (100 MHz; DMSO-d ): δ 114.16, 121.86,
6 C
1
24.35, 127.48, 134.18, 138.06, 141.73, 147.24, 150.10, 154.24, 158.46, 161.20, 166.35; LC-MS
(
70 eV): m/z = 371 (M+H)+.
General procedure for preparation of thienopyrimidine-N-phenylacetamides (8a-l)
To the solution of thienopyrimidine-2-thioxothiazolidin-4-one (5) (1 mmol) in acetone
(
10 mL) solvent was added potassium carbonate (1.5 mmol) followed by substituted N-
phenylacetamides isothiocyanate (7a-l) (1 mmol). The reaction mixture was refluxed for 2 h at
8 °C after completion of the reaction, cooled to room temperature and evaporated under
7
reduced pressure. Then the resulted mixture was washed by water and dried. Further, the crude
was purified by column chromatography in good yield.
{
2-(4-Oxo-5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-thioxothiazolidin-3-yl)-N-
phenylacetamide} (8a)
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0
-1
1
Yield: 88%; mp 210-212 C; IR (KBr) (ν /cm ): 2837, 1708, 1670, 1626, 1541; H NMR (400
max
MHz; DMSO-d ): δ 4.82 (s, 2H, CH ), 6.92 (d, J = 4.0 Hz, 1H, thiophene-CH), 7.16 (bs, 1H,
6
H
2
HH
NH), 7.19 (d, JHH = 4.0 Hz, 1H, thiophene-CH), 7.30 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.41-7.67 (m,
1
3
5
H, Ar-H), 7.76 (s, 1H, CH), 8.01 (d, JHH = 8.0 Hz, 2H, Ar-H), 8.86 (s, 1H, pyrimidine-CH); C
NMR (100 MHz; DMSO-d ): δ 52.14, 109.46, 112.47, 118.52, 121.04, 124.20, 125.41, 128.40,
6
C
1
32.81, 135.86, 138.75, 140.67, 148.41, 149.63, 150.91, 155.83, 158.01, 164.32, 168.19; LC-MS
(
70 eV): m/z = 505 (M+H)+.
{
N-(4-chlorophenyl)-2-(4-oxo-5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-thioxo
thiazolidin-3-yl)acetamide} (8b)
0
-1
1
Yield: 92%; mp 194-196 C; IR (KBr) (ν /cm ): 2851, 1709, 1667, 1621, 1547; H NMR (400
max
MHz; DMSO-d ): δ 5.16 (s, 2H, CH ), 6.99 (d, J = 4.0 Hz, 1H, thiophene-CH), 7.08 (d, JHH
=
=
6
H
2
HH
4
8
2
1
1
.0 Hz, 1H, thiophene-CH), 7.22 (bs, 1H, NH), 7.48 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.62 (d, JHH
.0 Hz, 2H, Ar-H), 7.79 (s, 1H, CH), 8.16 (d, JHH = 8.0 Hz, 2H, Ar-H), 8.34 (d, JHH = 8.0 Hz,
1
3
H, Ar-H), 9.05 (s, 1H, pyrimidine-CH); C NMR (100 MHz; DMSO-d ): δ 56.48, 106.35,
6
C
14.33, 120.23, 121.25, 126.02, 127.30, 128.21, 134.02, 136.42, 137.11, 144.37, 146.08, 149.19,
51.74, 156.30, 160.27, 161.06, 167.46; LC-MS (70 eV): m/z = 539 (M+H)+.
{
N-(4-nitrophenyl)-2-(4-oxo-5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-thioxo
thiazolidin-3-yl)acetamide} (8c)
0
-1
1
Yield: 91%; mp 221-223 C; IR (KBr) (ν /cm ): 2841, 1716, 1673, 1618, 1528; H NMR (400
max
MHz; DMSO-d ): δ 5.12 (s, 2H, CH ), 6.79 (d, J = 8.0 Hz, 1H, thiophene-CH), 6.98 (d, JHH
=
=
6
H
2
HH
4
1
2
.0 Hz, 1H, thiophene-CH), 7.24 (d, JHH = 12.0 Hz, 2H, Ar-H), 7.31 (s, 1H, NH), 7.58 (d, JHH
2.0 Hz, 2H, Ar-H), 7.63 (s, 1H, CH), 7.94 (d, JHH = 8.0 Hz, 2H, Ar-H), 8.20 (d, JHH = 8.0 Hz,
1
3
H, Ar-H), 8.72 (s, 1H, pyrimidine-CH); C NMR (100 MHz; DMSO-d ): δ 54.01, 108.11,
6
C
1
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1
1
19.84, 121.16, 121.33, 125.26, 127.19, 129.72, 133.07, 136.14, 139.82, 143.22, 148.26, 149.33,
52.26, 158.26, 159.27, 162.27, 166.14; LC-MS (70 eV): m/z = 548 (M-H)-.
{
N-(2-chlorophenyl)-2-(4-oxo-5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-thioxo
thiazolidin-3-yl)acetamide} (8d)
0
-1
1
Yield: 89%; mp 232-234 C; IR (KBr) (ν /cm ): 2843, 1714, 1668, 1624, 1534; H NMR (400
max
MHz; DMSO-d ): δ 5.28 (s, 2H, CH ), 6.81 (d, J = 8.0 Hz, 1H, thiophene-CH), 6.96 (d, JHH
=
6
H
2
HH
4
4
.0 Hz, 1H, thiophene-CH), 7.29 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.42 (s, 1H, NH), 7.56-7.82 (m,
1
3
H, Ar-H), 7.86 (s, 1H, CH), 8.14 (d, JHH = 8.0 Hz, 2H, Ar-H), 8.91 (s, 1H, pyrimidine-CH); C
NMR (100 MHz; DMSO-d ): δ 50.26, 110.46, 118.70, 119.02, 124.44, 124.89, 126.31, 128.07,
6
C
1
1
29.69, 132.07, 137.06, 138.37, 140.01, 145.76, 147.03, 148.42, 150.31, 159.19, 159.74, 161.18,
65.22; LC-MS (70 eV): m/z = 539 (M+H)+.
{
N-(3-nitrophenyl)-2-(4-oxo-5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-thioxo
thiazolidin-3-yl)acetamide} (8e)
0
-1
1
Yield: 97%; mp 244-246 C; IR (KBr) (ν /cm ): 2868, 1710, 1674, 1622, 1536; H NMR (400
max
MHz; DMSO-d ): δ 5.01 (s, 2H, CH ), 6.98 (d, J = 8.0 Hz, 1H, thiophene-CH), 7.16 (d, JHH
=
6
H
2
HH
8
.0 Hz, 1H, thiophene-CH), 7.29 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.36 (s, 1H, Ar-H), 7.49 (s, 1H,
NH), 7.71 (s, 1H, CH), 7.79-7.91 (m, 3H, Ar-H), 8.09 (d, JHH = 12.0 Hz, 2H, Ar-H), 9.08 (s, 1H,
1
3
pyrimidine-CH); C NMR (100 MHz; DMSO-d ): δ 49.31, 107.19, 112.42, 118.02, 120.39,
6
C
1
1
26.48, 128.00, 128.46, 130.33, 135.18, 137.27, 139.41, 143.28, 146.19, 146.58, 149.07, 153.22,
57.42, 158.16, 160.22, 164.49; LC-MS (70 eV): m/z = 550 (M+H)+.
{
N-(2-nitrophenyl)-2-(4-oxo-5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-thioxo
thiazolidin-3-yl)acetamide} (8f)
1
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0
-1
1
Yield: 90%; mp 228-230 C; IR (KBr) (ν /cm ): 2836, 1711, 1678, 1620, 1537; H NMR (400
max
MHz; DMSO-d ): δ 5.14 (s, 2H, CH ), 6.86 (d, J = 4.0 Hz, 1H, thiophene-CH), 6.93 (d, JHH
=
6
H
2
HH
8
4
.0 Hz, 1H, thiophene-CH), 7.21 (s, 1H, NH), 7.38 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.48-7.69 (m,
1
3
H, Ar-H), 7.86 (s, 1H, CH), 8.03 (d, JHH = 8.0 Hz, 2H, Ar-H), 8.79 (s, 1H, pyrimidine-CH); C
NMR (100 MHz; DMSO-d ): δ 56.40, 109.70, 111.33, 117.48, 119.37, 123.73, 125.07, 126.32,
6
C
1
1
29.41, 131.23, 136.30, 137.83, 139.08, 141.22, 144.39, 148.14, 150.24, 158.23, 159.18, 162.27,
65.38; LC-MS (70 eV): m/z = 550 (M+H)+.
{
N-(4-methoxyphenyl)-2-(4-oxo-5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-thioxo
thiazolidin-3-yl)acetamide} (8g)
0
-1
1
Yield: 87%; mp 236-238 C; IR (KBr) (ν /cm ): 2847, 1713, 1676, 1619, 1540; H NMR (400
max
MHz; DMSO-d ): δ 3.96 (s, 3H, OCH ), 5.18 (s, 2H, CH ), 6.91 (d, J = 8.0 Hz, 1H,
6
H
3
2
HH
thiophene-CH), 7.12 (d, JHH = 8.0 Hz, 1H, thiophene-CH), 7.24 (d, JHH = 8.0 Hz, 2H, Ar-H),
7
1
.32 (s, 1H, NH), 7.41 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.69 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.81 (s,
1
3
H, CH), 8.12 (d, JHH = 8.0 Hz, 2H, Ar-H), 8.91 (s, 1H, pyrimidine-CH); C NMR (100 MHz;
DMSO-d ): δ 54.30, 55.14, 108.20, 110.57, 118.30, 120.26, 124.22, 125.21, 129.27, 130.36,
6
C
1
34.75, 139.46, 140.28, 143.03, 146.28, 150.21, 156.07, 158.43, 159.09, 163.26; LC-MS (70
eV): m/z = 535 (M+H)+.
{
2-(4-Oxo-5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-thioxothiazolidin-3-yl)-N-(p-
tolyl)acetamide} (8h)
0
-1
1
Yield: 85%; mp 198-200 C; IR (KBr) (ν /cm ): 2878, 1711, 1668, 1612, 1539; H NMR (400
max
MHz; DMSO-d ): δ 2.18 (s, 3H, CH ), 5.26 (s, 2H, CH ), 6.86 (d, J = 4.0 Hz, 1H, thiophene-
6
H
3
2
HH
CH), 6.98 (d, JHH = 8.0 Hz, 1H, thiophene-CH), 7.19 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.29 (d, JHH
=
8
.0 Hz, 2H, Ar-H), 7.42 (s, 1H, NH), 7.76 (s, 1H, CH), 7.89 (d, JHH = 8.0 Hz, 2H, Ar-H), 8.06
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1
3
(
d, JHH = 8.0 Hz, 2H, Ar-H), 8.86 (s, 1H, pyrimidine-CH); C NMR (100 MHz; DMSO-d ): δ
6 C
2
1
6.38, 54.26, 110.14, 116.26, 119.43, 123.31, 124.10, 127.73, 129.30, 133.41, 138.09, 140.32,
40.96, 145.53, 148.63, 152.48, 155.27, 159.39, 160.28, 166.37; LC-MS (70 eV): m/z = 519
(
M+H)+.
N-(4-bromophenyl)-2-(4-oxo-5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-thioxo
{
thiazolidin-3-yl)acetamide} (8i)
0
-1
1
Yield: 84%; mp 242-244 C; IR (KBr) (ν /cm ): 2868, 1714, 1673, 1609, 1552; H NMR (400
max
MHz; DMSO-d ): δ 5.07 (s, 2H, CH ), 6.82 (d, J = 8.0 Hz, 1H, thiophene-CH), 6.97 (d, JHH
=
6
H
2
HH
4
7
.0 Hz, 1H, thiophene-CH), 7.23 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.31 (d, JHH = 8.0 Hz, 2H, Ar-H),
.38 (s, 1H, NH), 7.62 (s, 1H, CH), 7.93 (d, JHH = 8.0 Hz, 2H, Ar-H), 8.27 (d, JHH = 8.0 Hz, 2H,
13
Ar-H), 8.97 (s, 1H, pyrimidine-CH); C NMR (100 MHz; DMSO-d ): δ 49.35, 112.35, 114.68,
6
C
1
1
17.27, 119.48, 120.26, 126.32, 128.41, 130.38, 137.26, 139.30, 142.37, 146.23, 148.67, 150.37,
56.30, 159.87, 160.39, 165.37; LC-MS (70 eV): m/z = 582 (M+H)+.
{
2-(4-Oxo-5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-thioxothiazolidin-3-yl)-N-(o-
tolyl)acetamide} (8j)
0
-1
1
Yield: 90%; mp 238-240 C; IR (KBr) (ν /cm ): 2851, 1709, 1670, 1618, 1548; H NMR (400
max
MHz; DMSO-d ): δ 2.26 (s, 3H, CH ), 5.19 (s, 2H, CH ), 6.88 (d, J = 8.0 Hz, 1H, thiophene-
6
H
3
2
HH
CH), 6.95 (d, JHH = 8.0 Hz, 1H, thiophene-CH), 7.26 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.30 (d, JHH
=
8
.0 Hz, 2H, Ar-H), 7.38 (s, 1H, NH), 7.52 (dd, JHH = 4.0 Hz and 8.0 Hz, 2H, Ar-H), 7.73 (s, 1H,
1
3
CH), 7.98 (m, 2H, Ar-H), 8.76 (s, 1H, pyrimidine-CH); C NMR (100 MHz; DMSO-d ): δ
6
C
2
1
3.41, 53.43, 108.39, 113.32, 117.43, 119.19, 120.23, 124.47, 127.36, 129.47, 131.32, 136.49,
39.57, 140.08, 142.97, 143.28, 149.37, 150.33, 154.37, 159.41, 164.35, 167.86; LC-MS (70
eV): m/z = 517 (M-H)-.
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Canadian Journal of Chemistry
{
N-(2-methoxyphenyl)-2-(4-oxo-5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-
thioxothiazolidin-3-yl)acetamide} (8k)
0
-1
1
Yield: 86%; mp 218-220 C; IR (KBr) (ν /cm ): 2856, 1710, 1672, 1621, 1540; H NMR (400
max
MHz; DMSO-d ): δ 3.86 (s, 3H, OCH ), 5.20 (s, 2H, CH ), 6.83 (d, J = 4.0 Hz, 1H,
6
H
3
2
HH
thiophene-CH), 6.96 (d, JHH = 8.0 Hz, 1H, thiophene-CH), 7.20 (d, JHH = 8.0 Hz, 2H, Ar-H),
7
8
1
1
.29 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.43 (s, 1H, NH), 7.56-7.78 (m, 4H, Ar-H), 7.86 (s, 1H, CH),
1
3
.89 (s, 1H, pyrimidine-CH); C NMR (100 MHz; DMSO-d ): δ 51.41, 58.48, 108.87, 114.23,
6
C
16.24, 119.75, 121.28, 124.67, 128.36, 129.07, 133.22, 136.58, 139.14, 140.73, 143.79, 146.24,
49.09, 151.27, 155.70, 158.37, 165.41, 168.71; LC-MS (70 eV): m/z = 535 (M+H)+.
{
N-(3,4-dichlorophenyl)-2-(4-oxo-5-(4-(thieno[2,3-d]pyrimidin-4-yloxy)benzylidene)-2-
thioxothiazolidin-3-yl)acetamide} (8l)
0
-1
1
Yield: 91%; mp 223-225 C; IR (KBr) (ν /cm ): 2891, 1710, 1668, 1608, 1536; H NMR (400
max
MHz; DMSO-d ): δ 5.16 (s, 2H, CH ), 6.96 (d, J = 4.0 Hz, 1H, thiophene-CH), 7.09 (d, JHH
=
=
6
H
2
HH
8
8
9
1
1
.0 Hz, 1H, thiophene-CH), 7.31 (d, JHH = 8.0 Hz, 2H, Ar-H), 7.46 (s, 1H, NH), 7.58 (d, JHH
.0 Hz, 2H, Ar-H), 7.69 (s, 1H, CH), 7.81 (s, 1H, Ar-H), 8.23 (d, JHH = 12.0 Hz, 2H, Ar-H),
1
3
.16 (s, 1H, pyrimidine-CH); C NMR (100 MHz; DMSO-d ): δ 56.19, 109.26, 115.18, 118.40,
6
C
19.38, 120.46, 123.22, 127.81, 130.18, 133.37, 135.73, 138.22, 141.71, 147.20, 149.20, 150.09,
54.70, 157.21, 158.09, 162.28, 166.34; LC-MS (70 eV): m/z = 572 (M+H)+.
Biological Evaluation
Antibacterial and antifungal assays
All the synthesized compounds were assessed for their antimicrobial (antibacterial and
antifungal) activities. For antibacterial studies were screened by the in vitro minimum inhibitory
concentrations (MICs) of the target compounds using with NCCLS broth dilution method in 96-
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well micro test plates conferring to the National Committee for Clinical Laboratory
Standards.33,34 For antifungal tests were evaluated by MIC approach using with serial plate
dilution method35,36 and Gentamicin and Fluconazole were used as standard drugs.
Antibacterial assay
The synthesized target molecules were assessed for their antibacterial activity against
Escherichia coli and Klebsiella pneumonia as Gram-negative Bacteria and Bacillus cereus and
Bacillus subtilis as Gram-positive bacteria. The strain suspension was attuned with hygienic
5
saline to a concentration of 1.0×10 c.f.u/mL. The tested molecules were dissolved using with
DMSO (10 µL). It was added carefully in duplicate onto the surface of the pre immunized plate
and allowed to dry before incubation. Further, the solvent to synthesis the standard solutions and
also standard drugs were synthesized using with Mueller Hinton broth method by serial dilution
to afford the various prerequisite concentrations such as 1.56, 3.12, 6.25, 12.5, 25, 50 and 100
°
µg/mL. All the dilutions were incubated for 24 h at 37 C. To endorse that the solvent had no
effect on bacterial growth and a control test was exhibited with test standard accompanied with
DMSO at the same dilutions as used in the experiment. The MIC was perceived by detecting the
lowermost concentration of the test compounds at which there was no visible progress in the
presence of Gentamicin as standard.
Antifungal assay
For antifungal evaluating, the target molecules and Fluconazole (reference drug) solution
was synthesized in DMSO to make an efficient various concentrations (3.12, 6.25, 12.5, 25.0,
5
0.0 and 100 µg/mL). Isolated fungal strains such as Aspergillus flavus, Aspergillus niger,
Penicillium marneffei and Candida albicans were choosen and also Sabouraud dextrose agar was
prepared at pH 5.7. A loopful of specific fungal strain was taken into saline (3 mL) to make a
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Page 15 of 22
Canadian Journal of Chemistry
suspension of following species. Next, 20 mL of agar media were poured into each Petri dish.
Then, excess suspension was transferred and petri dishes were dried by placing in an incubator at
0
3
7 C for 1 h. An agar sock was utilized to prepare wells and MICs of the test molecules in
dimethylsulfoxide (DMSO) were added into each marked well. A control was prepared for the
plates in the same way using solvent DMSO (10 µL). All the dishes were made in triplicate and
°
preserved for 3-4 days at 37 C. The activity was measured by calculating the MIC values.
Acknowledgements
The authors are thankful to the authorities of the Department of Chemistry, GITAM
Institute of Sciences, GITAM University, Visakhapatnam. India and School of Chemistry,
University of KwaZulu-Natal, Westville Campus, Durban, South Africa for providing facilities.
References
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11] Bekhit, A.A.; Farghaly, A.M.; Shafik, R.M.; Elsemary, M.M.A.; Bekhit, A.A.; Guemei,
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Pharmaceu. Sci., 2015, 1, 33-41.
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[
[
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[
[
[
[
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6
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Scheme 1: Synthesis of thienopyrimidin-2-thioxothiazolidin-3-yl)acetamide derivatives (8a-l)
CHO
O
Cl
N
O
N
CN
HO
CHO
(i)
NH
(ii)
N
N
S
NH2
S
N
(iii)
S
S
1
3
4
2
O
O
H
N
NH
S
N
H
S
S
N
O
N
O
O
O
S
S
R
7
a-l
S
N
N
8a-l
(
vi)
(iv)
S
S
N
5
O
Cl
NH2
HN
Cl
(v)
Cl
R
O
R
7
a-l
6a-l
R = H, 4-Cl, 4-NO , 2-Cl, 3-NO , 2-NO , 4-OCH ,
2
2
2
3
4
-CH , 4-Br, 2-CH ,2-OCH , 3,4-Cl
3 3 3
Compound
7c
7d
7e
7f
7g
7j
7k
7l
7a
7b
7h
7i
R
H
4-Cl 4-NO2 2-Cl
3-NO2 2-NO2 4-OCH3 4-CH3 4-Br
2-CH3 2-OCH3 3,4-Cl
Reagents and conditions:
°
(
(
(
(
(
(
i) HCOOH, 4 h, Reflux at 120 C.
ii) POCl , 5 h, Reflux.
3
iii) K CO , Acetonitrile, 5 h, 30-40 °C;
2
3
o
iv) NaOAc, AcOH, 6 h, Reflux at 90 C.
v) Et N, Acetone, 0-10 °C
3
vi) Acetone, K CO , 3 h, Reflux.
2
3
2
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Table 1: Antibacterial activities of the title compounds (8a-l)
Compound
MIC in µg/mL
Gram-possitive Gram-negative
B. cereus B. subtilis E. coli K. pneumonia
8
a
12.5
25
12.5
25
25
25
12.5
25
8
b
8
c
3.12
25
1.56
100
12.5
25
1.56
50
3.12
‒
8
d
8
e
6.25
6.25
6.25
3.12
50
12.5
12.5
6.25
6.25
25
6.25
12.5
6.25
6.25
25
8
f
8
g
12.5
6.25
‒
8
h
8
i
8
j
‒
50
100
50
‒
8
k
50
100
100
6.25
50
8
l
50
‒
50
Gentamicin a
6.25
6.25
6.25
‒
Indicates bacteria is resistant to the compounds at >100 µg/mL
aGentamicin was used as a standard
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Canadian Journal of Chemistry
Table 2: Antifungal activities of the title compounds (8a-1)
Compound
MIC in µg/mL
A. flavus A. niger P. marneffei C. albicans
8
a
12.5
‒
25
25
25
25
12.5
50
8
b
8
c
3.12
12.5
6.25
3.12
6.25
25
3.12
12.5
12.5
3.12
6.25
‒
3.12
6.25
6.25
6.25
12.5
25
3.12
12.5
6.25
3.12
6.25
50
8
d
8
e
8
f
8
g
8
h
8
i
50
25
100
‒
‒
8
j
25
‒
50
8
k
25
12.5
6.25
3.12
25
25
8
l
6.25
6.25
12.5
6.25
6.25
6.25
Fluconazolea
‒
Indicates fungus is resistant to the compounds at >100 µg/mL
aFluconazole was used as a standard
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