One-pot two-step facile synthesis of 2,3,4,5-tetra substituted dihydrooxazoles and their antimicrobial activity

Article abstract of DOI:10.1016/j.bmcl.2017.06.062

New 2,3,4,5-tetra substituted dihydrooxazoles derivatives were efficiently synthesized starting from benzaldehyde, aryl thiosemicarbazide and benzoin using designed synthetic route. Newly synthesized 2,3,4,5-tetra substituted dihydrooxazole derivatives we

Full text of DOI:10.1016/j.bmcl.2017.06.062

Accepted Manuscript
One-Pot Two-step Facile Synthesis of 2,3,4,5-Tetra Substituted dihydrooxa-
zoles and their Antimicrobial activity
Shailendra Tiwari, Poonam Pathak, Kamal Pratap Singh, Ram Sagar
PII:
S0960-894X(17)30664-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.062
BMCL 25096
Reference:
Bioorganic & Medicinal Chemistry Letters
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1 June 2017
23 June 2017
Please cite this article as: Tiwari, S., Pathak, P., Pratap Singh, K., Sagar, R., One-Pot Two-step Facile Synthesis of
2,3,4,5-Tetra Substituted dihydrooxazoles and their Antimicrobial activity, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.06.062
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One-Pot Two-step Facile Synthesis of 2,3,4,5-Tetra Substituted
dihydrooxazoles and their Antimicrobial activity
Shailendra Tiwari,a* Poonam Pathak,a Kamal Pratap Singha and Ram Sagarb
aDepartment of Chemistry, University of Allahabad, Allahabad (UP) 211002, India
bDepartment of Chemistry, Shiv Nadar University, NH-91 Dadri, GB Nagar (UP) 201314, India
Email: drshailendratiwariau@gmail.com
Abstract
New 2,3,4,5-tetra substituted dihydrooxazoles derivatives were efficiently synthesized starting
from benzaldehyde, aryl thiosemicarbazide and benzoin using designed synthetic route. Newly
synthesized 2,3,4,5-tetra substituted dihydrooxazole derivatives were screened for their
antibacterial and antifungal activities against different strains of pathogenic bacteria and fungi.
The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and
minimum fungicidal concentration (MFC) were determined for the test compounds using
positive and negative control. Compounds 4b, 4d, 4f, 4i, 4k and 4m, have shown good
antibacterial activity whereas compounds 4e, 4g, 4h, 4j, 4l and 4n have displayed better
antifungal activity.
Keywords
2,3,4,5-tetra substituted dihydrooxazoles, aryl thiosemicarbazide, benzoin, thiourea, schiff base,
antimicrobial antibacterial activity and antifungal activity.
One pot synthesis of drug-like small molecules has been interest for medicinal chemists and
chemical biologists because, this provide the important scaffolds in fewer steps and these
molecules play very important role in drug discovery processes.1 Several bacterial infections
such as diarrhea, food poisoning, rheumatic solmonellosis, extraintestinal and intestinal wall
infections are caused by gram-positive and gram-negative pathogens.2 The resistance of
pathogens bacteria towards available antibiotics is rapidly becoming a major threat to human
health world- wide.3 In addition, fungal infections continue to increase dramatically because of
growing number of immunocompromised hosts such as AIDS patients or those undergoing
1

anticancer chemotherapy and transplantation.4-6 Resistance to known antibiotics is becoming
great concern in scientific community and big challenge to develop new scaffold as biologically
active molecules. Therefore, design of new antimicrobial compounds to deal with these problems
is of prime interest.
Oxazoles are class of compounds that are believed to occur in nature from post-translational
modification of serine and threonine residue in peptides. They are the key building blocks of
natural products, pharmaceuticals and synthetic intermediates. Oxazoles have not only attracted
great interest due to their appearance as subunit of various biologically active natural products
but also because of their appearance as subunit of valuable precursors in many useful synthetic
transformations. Among the numerous heterocyclic moieties of biological and pharmacological
interests, the oxazole ring is endowed with a vital role in the manufacture of various active drugs
as brain-derived neurotropic factor induced,7 analgesic,8 trypanocidal activity,9 antimitonic
agents with pro-apoptotic activity,10 antifungal activity,11 anti-inflammatory,12 antidepressant,13
pesticidal,14 and antimicrobial activity.15
Thiourea and its derivatives have found extensive applications in the field of medicine,
agriculture and analytical chemistry. They are known to exhibit a wide variety of biological
activities such as antiviral, antibacterial, antifungal,16 The union of heterocyclic ring with
thiourea linkage often results compounds with enhanced biological performance.17
In light of the above literature and abundance on bio-potentials of oxazoles and thiourea
analogues, we designed 2,3,4,5-tetra substituted dihydrooxazoles having thiourea as one of the
appendages and were confident that these framework would provide the important structural
motifs for the discovery of new antimicrobial agents. In continuation of our research on efficient
synthesis of biologically active small molecules,18 we developed one-pot synthesis of 2,3,4,5-
tetra substituted dihydrooxazole derivatives and demonstrated their antimicrobial activity.
Chemistry
For the development of efficient and facile synthesis of new 2,3,4,5-tetra substituted
dihydrooxazole derivatives, we identified substituted benzaldehyde 1a and 4-phenyl
thiosemicarbazide 2a and benzoin as the key starting materials that can be transferred to our
2

designed molecules in two step via Schiff’s base intermediate 3a. We initiated our synthetic
protocol with 4-methoxy benzaldehyde 1a refluxing with 4-phenyl thiosemicarbazide 2a in
ethanol in the presence of catalytic amount of acetic acid which furnished corresponding Schiff’s
bases 3a as reported in literature in good yields.19-22 The Schiff’s bases 2a were purified by
crystallization and characterized using spectroscopic data. All the NMR and IR data of the
Schiff’s bases 2a were found consistent with the reported analytical data. After obtaining
Schiff’s base 2a, it was refluxed with benzoin under various reaction conditions using different
solvents (MeOH, EtOH Toluene, THF etc) under reflux for 4-5 h, where ethanol in basic
condition was found superior with the formation of clean product 4a (TLC). Reaction mixture
was cooled to room temperature, after work-up and purification it furnished compound 4a in
very good (81%) isolated yield (Scheme 1).
S
O
H
N
H
N
H2N
+
glacial AcOH/EtOH
90 oC, 3 h, 75%
N
N
H
NH
H
S
H
MeO
MeO
Ph
2a
1a
3a
O
Ph
NH
Benzoin/EtOH/Et3N
90 oC, 5 h, 81%
N
MeO
S
N
H
4a
Scheme 1. Synthesis of 4a.
Once reaction condition was standardized, it was decided to perform same transformation in one-
pot two-step manner which may furnish the designed 2, 3, 4, 5-tetra substituted dihydrooxazole
derivatives 4a. Thus 4-methoxy benzaldehyde 1a was refluxed with 4-phenyl thiosemicarbazide
2a in ethanol in the presence of catalytic amount of acetic acid for 5 h with careful reaction
monitoring (TLC) then benzoin was added to same reaction mixture along with little excess of
o
triethyl amine. Resulting mixture was stirred continuously at high temperature (90 C) for
another 5 h which shows the formation of desired product 4a (TLC). The reaction mixture was
cooled to room temperature and after usual workup compound 4a was obtained in overall 80%
3

isolated yields (Table 1). On optimization of one-pot two-step condition a series of 4-aryl
thiosemicarbazides 2b-2n were successfully transformed into corresponding 2, 3, 4, 5-tetra
substituted dihydrooxazole derivatives 4b-4n using similar reaction protocol in good to very
good yields (Table 1). Two different benzaldehydes, viz 4-methoxy benzaldehyde and 4-hydroxy
benzaldehyde, along with seven different 4-aryl thiosemicarbazides 2a-2n were used in this
synthetic study and results are summarized in table 1.
Table 1. Synthesis of 2,3,4,5-tetra substituted dihydrooxazoles derivatives.
Ph
S
O
O
H2N
+
Ph
NH
i) glacial AcOH/EtOH
90 oC, 3-5 h
N
H
NH
N
H
R
2
R
R'
R'
ii) Benzoin/EtOH/Et3N
90 oC, 5-6 h, 70-81%
3
S
N
H
4
1a-1n
2a-2n
4a-4n
Compoun
R
R'
Yield Compoun
R
R'
Yield
%
ds
4a
4b
4c
4d
4e
4f
%
80
75
76
81
70
65
68
ds
4h
4i
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
H
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
H
79
2-OMe
4-OMe
2-Me
4-Me
2-Cl
2-OMe
4-OMe
2-Me
4-Me
2-Cl
76
74
4j
80
4k
4l
69
63
4m
4n
4-Cl
4-Cl
62
4g
The mechanism of this transformation can be postulated as follows. The hydroxyl group of
benzoin (in the presence of triethyl amine at elevated temperature) attacked on imine carbon
moving imine double bond towards nitrogen to form an unstable intermediate I (Figure 1).
Intramolecular attack of thiosemicarbazide N-atom to carbonyl carbon of benzoin formed
intermediate II which under high temperature reaction conditions lost a water molecule and
furnished stable 2,3,4,5- tetra substituted dihydrooxazoles derivatives 4a. All the compounds and
intermediates were confirmed by spectral analysis.23
4

O
H
N
S
N
Ph
OH
Ph
H
Ph
HN
N
S
O
Ph +
MeO
Benzoin
H
HN
O
HN
H
3a
MeO
I
Ph
HO
Ph
Ph
H
H
N
O
S
N
H
Ph
O
N
HN
NH
- H2O
MeO
MeO
S
N
H
II
4a
Figure 1. Proposed Mechanism
Biology
The synthesized pure compounds were screened for antibacterial and antifungal activities
adopting standard protocols.24 The antibacterial activity, of prepared final pure compounds 4a-4n
was performed against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Klebsiella
pneumoniae using Ciprofloxacin as positive and DMSO as negative control. Minimum inhibitory
concentration (MIC) and minimum bactericidal concentration (MBC) were determined and the
activity was reported in μg/mL. The nutrient broth, which contained logarithmic serially two fold
diluted amount of test compounds and controls were inoculated with approximately 5×105
c.f.u/mL of activity dividing bacteria cells. The cultures were incubated for 24 h at 37 ºC and the
growth was monitored visually and spectrophotometrically. The antibacterial results are
summarized in Table 2, only for those compounds which were found active against any tested
strain of bacteria. It is inferred from Table 2 that compounds 4b, 4d and 4m showed moderate
activity against gram-positive bacteria S. aureus and B. subtilis where as 4d, 4i and 4k were
active against gram-negative K. pneumoniae ranging from 30-50 μg/mL concentration and only
one compound 4f was found active against E. coli 25 μg/mL concentration (Table 2). MBC/MIC
ratio of all active compounds is ranging from 2.0 to 3.3 suggesting these compounds are
bactericidal not bacteriostatic. Antimicrobial agent is considered bacteriostatic when the minimal
5

MBC/MIC ratio is greater than or equal to 8 whereas it is considered bactericidal if MBC/MIC
ratio is less than or equal to 4.
TABLE 2. Antibacterial activity of 2,3,4,5- tetra substituted dihydrooxazoles derivatives
Gram-positive bacteria
S. aureus B. subtilis
MBC MIC MBC
Gram-negative bacteria
E. coli K. pneumonia
MBC
Compd
MIC
40
MIC
n.a.
n.a.
25
MIC
n.a.
25
MBC
n.a.
50
100
100
n.a.
n.a.
n.a.
50
30
50
n.a.
n.a.
50
4b
4d
4f
30
20
50
n.a.
n.a.
n.a.
30
n.a.
n.a.
n.a.
40
n.a.
n.a.
n.a.
100
25
n.a.
25
n.a.
50
n.a.
n.a.
n.a.
6.25
n.a.
n.a.
n.a.
n.a.
25
4i
50
100
n.a.
10.25
n.a.
4k
4m
n.a.
6.25
n.a.
Ciprofloxacin 6.5
DMSO n.a.
12.5
n.a.
10.0
n.a.
n.a.
n.a.
MIC (μg/mL), minimum inhibitory concentration i.e., the lowest concentration of the compound to inhibit growth of
bacteria completely: MBC (μg/mL) minimum bacterial concentration i.e., the lowest concentration of the compound
for killing the bacteria completely; MBC/MIC ratio are against- S. aureus (4b 2.5, 4d 3.3, 4m 1.6), B. subtilis (4b
1.6, 4d 2.5, 4m 2.5), E. coli (4f 2.0), and K. pneumonia aureus (4d 2.0, 4i 2.0, 4k 2.0), n.a.-NO activity detected.
The antifungal activity, of the prepared pure compounds was performed against Tilletia indica,
Trichoderma, P. cubensis, S. fuliginea and P. infestans using Griseofulvin as positive and
DMSO as negative control. Minimum inhibitory concentration (MIC) was determined and
reported in μg/mL. Antifungal activity was carried out through disk diffusion method.25 All
fungal cultures were routinely maintained on sabouraud dextrose agar (SDA) and incubated at 28
oC. The antifungal activities are summarized in Table 3, only for those compounds which were
found active against any of these strains of fungi. It is inferred from Table 3 that compounds, 4e,
4g, 4h, 4j, 4l and 4n showed antifungal activity against T. indica, Trichoderma, P. cubensis, S.
fuliginea and P. infestans strain of fungi ranging from 10-20 μg/mL in concentration which is
comparable to Griseofulvin even superior in cases of 4g, 4h and 4l.
6

TABLE 3. Antifungal activity of 2,3,4,5-tetra substituted dihydrooxazoles derivatives.
Compounds
Fungal species and MIC (μg/ml)
T. indica
20
Trichoderma
P. cubensis
S. fuliginea
P. infestans
16
12
10
12
14
16
18
n.a.
12
14
10
16
12
14
16
n.a.
16
12
10
14
10
18
18
n.a.
14
16
10
16
14
16
20
n.a.
4e
18
4g
12
4h
14
4j
4l
16
18
4n
Griseofulvin
DMSO
20
n.a.
MIC(μg/ml), minimum inhibitory concentration i.e. the lowest concentration of the compound to
inhibit the growth of fungi. n.a.(NO activity detected).
The structure- activity relationship (SAR) of the tested compounds for antibacterial activity can
be summarized as follows: i) In the series of the 2,3,4,5-tetra substituted dihydrooxazoles
derivatives the compounds prepared from o-substituted aryl thiosemicarbazide in this series has
shown better antibacterial activity than the p-substituted groups. ii) Presence of thiourea linkage
at position 3 with substituted at phenyl ring is an important scaffold for better antibacterial
activity. iii) Most of the antibacterial compounds have MBC/MIC ratio below 4, it means they
are bactericidal not bacteriostatic. Antifungal activity of these compounds was similar or even
better in some cases as compare to Griseofulvin, a known antifungal agent. The possible
mechanism for the antibacterial activity of examined compounds is not known at the moment
and investigations are being done to investigate the mechanism of antibacterial action, and to
synthesize more effective compounds.
Conclusion
In summary, an efficient, clean and convenient one-pot three components synthesis of 2,3,4,5-
tetra substituted dihydrooxazole derivatives has been developed. The procedure offers several
advantages including simple reaction conditions and simple experimental and product isolation
procedures. Moreover, the synthesis of dihydrooxazole derivatives were designed through the
7

recombination of cheap and readily available starting materials, benzoin, 4-substituted
benzaldehyde and aryl thiosemicarbazides. A preliminary anti-bacterial and anti-fungal
screenings were performed on these compounds which revealed that newly generated
compounds possess antimicrobial activity and these can be elaborated as potential antimicrobial
agents.
Acknowledgements
We are thankful to the Head, Department of Chemistry, University of Allahabad, Allahabad for
necessary laboratory facilities. The authors would also like to thank SAIF, Punjab University,
Chandigarh and SAIF, Central Drug Research Institute, Lucknow, for spectral and analytical
data and centre of biotechnology, University of Allahabad, Allahabad for antimicrobial and
antifungal activity data.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://.
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1
Str), 3019 (=C–H Str), 1602 (C=C Str), 1156 (>C=S Str ); H NMR (400 MHz, DMSO-d6):
δ 9.46 (s, 1H, N-H), 8.00 (d, 2H, J = 7.5 Hz, ArH), 7.94 (d, 2H, J = 7.5 Hz, ArH), 7.76 (t,
1H, J = 7.5 Hz, ArH); 7.62-7.51 (m, 3H, ArH), 7.45-7.41 (m, 4H, ArH), 7.37-7.20 (m, 4H,
ArH), 6.90-6.86 (m, 2H, ArH), 6.08 (s, 1H, CH), 3.72 (s, 3H, OCH3); 13C NMR (100 MHz,
9

DMSO-d6): δ 194.6 (C=S), 156.4 (ArqC), 139.6, 135.2, 134.6, 133.0, 132.2, 132.1, 129.4,
129.2, 128.7, 128.4, 128.3, 127.5, 127.1, 126.0, 118.3, 113.4, 75.7 (CH), 55.0 (OCH3); FAB
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C29H25N3O2S: C, 72.63; H, 5.25; N, 8.76. Found: C, 72.57; H, 5.18; N, 8.66.
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PP 1-25.
10

Graphical Abstract
Ph
S
O
O
H2N
Ph
NH
i) glacial AcOH/EtOH
90 oC, 3-5 h
N
H
NH
N
H
R
+
2
R
R'
R'
ii) Benzoin/EtOH/Et3N
90 oC, 5-6 h, 62-81%
3
S
N
H
4
1a-1n
2a-2n
4a-4n
MIC = 20-50 mg/mL against S. aureus, B. subtitles, E. coli and K. Pneumoniae,
= 10-20 mg/mL against Tilletia indica, Trichoderma, P. cubensis, S. fuliginea and P. infestans