Synthesis and antimicrobial evaluation of novel ethyl 2-(2-(4-substituted)acetamido)-4-subtituted-thiazole-5-carboxylate derivatives

Article abstract of DOI:10.1016/j.bmcl.2016.06.030

A series of novel molecules containing thiazole ring structure were designed and synthesized. The structures of the synthesized compounds were elucidated and confirmed by¹H NMR,¹³C NMR, Mass spectrum and the purity was checked through HPLC analysis. Among these synthesized compounds, 3a–3i and 6a–6c were tested for their antimicrobial activity (minimum inhibitory concentration) against a series of strains of Bacillus subtilis, Staphylococcus aureus and Escherichia coli for antibacterial activity and against the strains of Candida albicans, Aspergillus flavus and Aspergillus niger for antifungal activity respectively. The results of the antimicrobial screening data revealed that most of the tested compounds showed moderate to good microbial inhibitions.

Full text of DOI:10.1016/j.bmcl.2016.06.030

Accepted Manuscript
Synthesis and antimicrobial evaluation of novel ethyl 2-(2-(4-substituted) acet-
amido)-4-subtituted-thiazole-5-carboxylate derivatives
Chandrakant D. Pawar, Aniket P. Sarkate, Kshipra S. Karnik, Sushilkumar S.
Bahekar, Dattatraya N. Pansare, Rohini N. Shelke, Chetan S. Jawale, Devanand
B. Shinde
PII:
S0960-894X(16)30641-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.030
BMCL 23983
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 May 2016
28 May 2016
10 June 2016
Please cite this article as: Pawar, C.D., Sarkate, A.P., Karnik, K.S., Bahekar, S.S., Pansare, D.N., Shelke, R.N.,
Jawale, C.S., Shinde, D.B., Synthesis and antimicrobial evaluation of novel ethyl 2-(2-(4-substituted) acetamido)-4-
subtituted-thiazole-5-carboxylate derivatives, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.06.030
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Synthesis and antimicrobial evaluation of novel ethyl 2-(2-(4-substituted)
acetamido)-4-subtituted-thiazole-5-carboxylate derivatives
Chandrakant D. Pawar,a* Aniket P. Sarkate,a Kshipra S. Karnik,a Sushilkumar S. Bahekar,b
Dattatraya N. Pansare,a Rohini N. Shelke,c Chetan S. Jawale,d Devanand B. Shindee
aDepartment of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad, 431004 (MS), India
bBond Chemicals, Aurangabad, 431004 (MS), India
cDepartment of Chemistry, Deogiri college, Station road, Aurangabad 431 005, MS, India.
dH.P.T.Arts & R.Y.K.Science College, College Road, Nashik-05 (MS), India
eShivaji University,Vidyanagar, Kolhapur- 416 004(MS), India
*Corresponding author E-mail: dbschandrakant15@rediffmail.com
Tel No.: +91 0240-2403308 Fax No.: +91 0240-2400413
Abstract
A series of novel molecules containing thiazole ring structure were designed and
synthesized. The structures of the synthesized compounds were elucidated and confirmed by 1H
13
NMR, C NMR, Mass spectrum and the purity was checked through HPLC analysis. Among
these synthesized compounds, 3a-3i and 6a-6c were tested for their antimicrobial activity
(minimum inhibitory concentration) against a series of strains of Bacillus subtilis,
Staphyllococcus aureus and Esherichia coli for antibacterial activity and against the strains of
Candida albicans, Aspergillus flavus and Aspergillus nigar for antifungal activity respectively.
The results of the antimicrobial screening data revealed that most of the tested compounds
showed moderate to good microbial inhibitions.
Keywords: Thiazole ring, Antibacterial activity, Antifungal activity.
1

The thiazole nucleus is an important component for a huge spectrum of therapeutic
agents including anticancer, anticonvulsants, antifungal and antibacterial agents.1 This structure
has found applications in drug development for the treatment of cardiotonic, fungicidal, HIV
infection, mental retardation in children, age related and neurodegenerative brain damage
(Alzheimer’s disease, Parkinson’s disease).2 This class of heterocyclic compounds are found in
many potent biologically active molecules such as Sulfathiazol (antimicrobial drug), Ritonavir
(antiretroviral drug), Abafungin (antifungal drug) with trade name Abasol cream and
Bleomycine and Tiazofurin (antineoplastic drug).3
Furthermore, some thiazoles are used in agricultureas pesticides and plant growth
regulators. Several novel thiazole derivatives have been reported in literatures such as,
introduction of fluorine into thiazoline and synthesis of sydnonyl substituted thiazolidinone and
thiazoline derivatives.4 Thiazole ring is an important pharmacophore and its coupling with other
ringscould furnish new biologically active compounds.5
In recent times, the applications of thiazoles were found in drug development for the
treatment of allergies, hypertension, inflammation, schizophrenia, bacterial, HIV infections, and
hypnotics and more recently for the treatment of pain, as fibrinogen receptor antagonists with
antithrombotic activityand as new inhibitors of bacterial DNA gyrase B.3
Thiazole moiety has been already reported for its antimicrobial activity. Thiazole ring is
an important pharmacophore and their couplings with other ringscould furnish new biologically
active compounds. Thiazole containing compounds exhibit a wide range of biological
properties,such as antitumor, anticonvulsant,6 cardiotonic,7 IMP dehydrogenase inhibiton,8
analgesic,9 anticancer.10 It was observed that, benzotriazole and thiazole rings present in the same
molecule could be convenient models for investigation of their biological activity.5 Literature
revealed that syntheses of such thiazolyl-benzotriazole showed anti-convulsant and anti-
inflammatory activity,11 anti-tumoral activity.12 After extensive literature search, it was observed
that, thiazole coupling with a piperidine moiety would increase the chances of potent
antimicrobial activity of the thiazole containing compound.13-19 We herein report the synthesis of
new substituted thiazole derivatives (Scheme 1 and Scheme 2) with the aim of investigating their
antimicrobial activity.
Thiazoles show many biological activities so we planned to study its antimicrobial
activity along with the incorporation of substituted piperidones, as substituted piperidones shows
2

variety of biological activities. The piperidine moiety is a very important pharmacophore
because of its presence in numerous alkaloids, pharmaceuticals and diverse applications in
medicinal chemistry.20 The nucleus, as like piperidine and its derivatives are reported in
literature for varied pharmacological activities like antifungal,21 antibacterial,22 AChE
inhibitors,23 antitubercular,24 antihistaminics,25 antitumor agents26 and anticancer activity.27 The
piperidine and its derivatives are important building blocks in the synthesis of pharmaceutical
drug molecules like paroxetin, methylphenidate, raloxifene, minoxidil, risperidone and pethidine.
The biologically active alkaloids containing substituted piperidine ring systems have been
targeted by medicinal chemists, for their complete or partial synthesis.28
Our research group previously reported synthesis, characterization and antimicrobial
evaluation of derivatives of thiazole and thiazolidinone.29 Here, we wish to mention the
development and incorporation of thiazole and substituted piperidine scaffold in one framework.
The synthetic methods adopted for the preparation of the title compounds 3a-3i and 6a-6c
care depicted in the schemes presented below.
Scheme 1 Synthesis of ethyl 2-(2-(4-Substituted) acetamido)-4-subtituted-thiazole-5-carboxylate
derivatives (3a-3i)
Reagents and conditions: (a) 2,6-leutidine, DMAP, DCM, Chloroacetyl chloride at 0°C; (b) 2,6-
leutidine, DMAP added in 4-benzylpiperidine solution in THF.
3

Scheme 2 Synthesis of ethyl 2-(2-(4-Substituted) acetamido)-4-subtituted-thiazole-5-carboxylate
derivatives
Reagents and conditions: (a) 2,6-leutidine., DMAP, DCM , Chloroacetyl chloride at 0°C; (b)
2,6-leutidine, DMAP, 4-phenylpiperidine (2a) / 4-methylpiperidine (2b) / piperidine (2c) solution
in THF.
Table 1 Screening of base and solvent for synthesis of compounds 2a-2i and 5 step (a)
Entry
Base
Solvent
DMF
DMF
DMF
DMF
DMF
DCM
DCM
Time (h)
Yielda (%)
1
2
3
4
5
6
7
TEA (2 eq)
6
16
16
6
45
30
55
58
40
60
80
DIPEA(2 eq)
DMAP (2 eq)
2,6-Leutidine (2 eq)
Pyridine (2 eq)
DMAP (2 eq)
2,6-Leutidine (2 eq) and
DMAP (0.2 eq)
DIPEA(2 eq)
16
16
3
8
9
DCM
DCM
16
3
40
40
TEA (2 eq) and DMAP (0.2
eq)
10
Pyridine (2 eq)
a Isolated yield.
DCM
16
30
4

We have optimized condition for the preparation of our substituted products by varying
different bases, varying solvents and reaction time. We presented optimization conditions for
both the schemes of step (a) and (b) in Tables 1 and Tables 2 respectively. For the step (a), In
the base TEA with DMF solvent gives the corresponding product in a 45% yield, which was the
worst among these solvents (Table 1, entry 1). Nevertheless, all of these yields were generally
low before further optimizations. To increase the efficiency of the condensation reaction, the
effects of different solvents were investigated (Table 1, entries 1-10). DCM exhibited the best
performance of the solvent; the product yield was 80%, reaction completion time only 3 h (Table
1, entries 7). The DMF gave lower yields as a solvent with used bases (Table 1, entries 1-3, and
5). But interestingly in the base 2,6-Leutidine the product yield was 58%, All the reactions were
carried out with various amounts of each bases in 1 mL of solvent. Among these reactions same
amounts of the solvent, 1 mL of DCM turned out to be the best choice with 2,6-Leutidine (2 eq)
and DMAP (0.2 eq) gives yield of 80% (Table 1, entry 7). We like to mention here in DCM as a
solvent and with 2,6-Leutidine (2 eq) and DMAP (0.2 eq) were the best choice and less time
required for reaction completion. We decided to carry out the further reaction in DCM.
Table 2 Screening of base and solvent for synthesis of compounds 3a-3i and 6a-6c step (b)
Entry
Base
Solvent
Time (h)
Yielda (%)
1
2,6-Leutidine (2 eq) and DMAP (0.2
THF
6
90
eq)
2
3
4
2,6-Leutidine (2 eq) and DMAP (0.2
DMF
16
16
16
70
30
40
eq)
2,6-Leutidine (2 eq) and DMAP (0.2
DCM
eq)
2,6-Leutidine (2 eq) and DMAP (0.2
eq)
Acetone
5
6
7
8
Pyridine (2 eq)
DMF
DCM
DCM
DCM
16
16
16
12
30
40
40
40
DMAP (2 eq)
DIPEA(2 eq)
TEA (2 eq) and DMAP (0.2 eq)
a Isolated yield.
5

For the step (b), we have again used different bases and solvents and finally optimized
one condition that gives good yield along with reduced reaction time (Table 2, entry 1) without
purification and crude used further for next reactions.
It was observed from the that 2,6- leutidine (2eq) and DMAP (0.2eq) as a base along with
THF as the solvent gave the highest yield of 90% and the time required for the reaction was also
reduced to 6 h (Table 2, entry 1), hence it was chosen as the base and the solvent for the
synthesis of step (b). Hence we decided to carry out the further reaction in THF as a solvent and
2,6- leutidine (2eq), DMAP (0.2eq) as a base. All synthesized compounds of ethyl 2-(2-(4-
substituted) acetamido)-4-subtituted-thiazole-5-carboxylate derivatives (3a-3i and 6a-6c)30 are
shown in Table 3.
All the synthesized compounds were screened for in vitro antimicrobial activity. The
antibacterial activity was evaluated against different bacterial strains such as Staphylococcus
aureus (NCIM-2901), Bacillus subtilus (NCIM-2063) and Escherichia coli (NCIM-2256).
Minimum inhibitory concentration (MIC, µg/mL) of antibacterial activity was determined using
broth dilution methodas per CLSI guidelines.31-36 Levofloxacin was used as a standard drug for
Table 3 Synthesis of ethyl 2-(2-(4-Substituted) acetamido)-4-subtituted-thiazole-5-Carboxylate
Derivatives
Compound Reactant (Step a)
Reactant (Step b)
4-benzylpiperidine
4-benzylpiperidine
Melting
point (°C)
143
Yield
(%)
82
Ethyl-2-amino-4-
3a
3b
3c
3d
methylthiazole-5-carboxylate
Ethyl-2-aminothiazole-5-
carboxylate
118
153
117
90
90
90
Ethyl-2-amino-4 ethylthiazole- 4-benzylpiperidine
5-carboxylate
Methyl-2-
4-benzylpiperidine
aminomethylthiazole-5-
carboxylate
Methyl-2-amino-4-
4-benzylpiperidine
113
90
3e
6

cyclopropylthiazole-5-
carboxylate
Ethyl-2-amino-4-
4-benzylpiperidine
153
127
89
88
3f
phenylthiazole-5-carboxylate
1-(2-amino-4-methylthiazol-5- 4-benzylpiperidine
yl)ethanone
3g
4,5-dimethylthiazol-2-amine
4-methylthiazol-2-amine
Ethyl-2-amino-4-
4-benzylpiperidine
4-benzylpiperidine
4-phenylpiperidine
118
108
173
87
92
92
3h
3i
6a
methylthiazole-5-carboxylate
Ethyl-2-amino-4-
4-methylpiperidine
piperidine
147
133
90
86
6b
6c
methylthiazole-5-carboxylate
Ethyl-2-amino-4-
methylthiazole-5-carboxylate
Table 4.Antimicrobial activity of the synthesized compounds (3a-3i and 6a-6c)
Compound
MIC valuesa (µg/ml)
B. subtilis
E. coli
50
S. aureus C. Albicans
A. Flavus
50
A. Niger
50
35
29
40
35
29
25
35
28
35
28
35
45
70
28
75
25
3a
3b
3c
3d
3e
3f
29
12.5
100
50
12.5
100
50
100
60
100
50
75
100
25
29
28
12.5
25
12.5
25
29
28
50
60
50
100
25
50
50
3g
3h
3i
29
30
12.5
50
12.5
50
50
70
75
6a
6b
6c
29
30
25
12.5
50
25
50
70
75
50
60
100
75
100
100
7

Levofloxacin
Fluconazole
Miconazole
29
-
29
-
28
-
-
-
-
40
25
25
-
-
-
12.5
12.5
12.5
aValues are the average of three readings.
the comparison of antibacterial activity (Table 4). Fluconazole and miconazole were used as
standard drugs for the comparison of antifungal activity. Dimethyl sulfoxide was used as solvent
control. From the antimicrobial data, it is observed that all the newly synthesized compounds
shows good to moderate level of antibacterial and antifungal activity (Table 4). The
antimicrobial activity data reveals that compounds (3b, 3e, 3f, 3h and 6a), are found to be most
active and potent as antimicrobial agents among the series.
The antimicrobial activity data reveals that among the synthesized compounds 3b, 3e, 3f, 3h
and 6a are found to be most active and potent antimicrobial agents among the series when
compared with the standard. The compound 3c as well as 6c containing the ethyl and methyl
moiety on the thiazole ring and no substitution on the piperidine ring showed reduced
antimicrobial activity. The compounds 3a, 3d and 3g containing methyl as well as hydrogen
moiety on the thiazole ring showed intermediate antibacterial activity. The same compounds 3b,
3e, 3f, 3h and 6a showed higher antifungal activity when tested on the fungal strains. The
structure-activity relationship of the series can be explained as follows:
 Effect of alkyl group substitution on the thiazole ring (R2): The molecules gave increased
antimicrobial activity due to the presence of methyl as well as hydrogen group on
thiazole ring. The activity was further found to be increased when there was the presence
of more bulky groups on the ring like cyclopropyl and phenyl.
 Effect of ester group substitution on the thiazole ring (R1): The activity was found to be
increased from good to potent when ethyl ester group was replaced by methyl ester on the
ring.
 Effect of substitution on the piperidine ring: The activity was observed only when there
was the presence of bulky groups on the piperidne ring like benzyl and phenyl. The
activity decreased when less bulky groups were substituted on the ring.
8

In conclusion by using this methodology, substituted thiazole-5-carboxylate derivatives
were synthesized in gram scale. All the compounds are purified through column chromatography
by using different proportions and ethyl acetate and hexane. We have developed simple and
continent method for the synthesis of some novel substituted thiazole derivatives through a
reaction of substituted thiazole carboxylates and piperidines by simple reaction steps. No costly
reagents are required, no any pre-purification is needed and all the compounds synthesized were
obtained in good yields. We developed methodologies for the synthesis of substituted thiazoles
on a laboratory scale. The mild reaction conditions, shorter reaction time and promising
antimicrobial activity of the compounds (3b, 3e, 3f, 3h and 6a) compared to the standard are the
advantage of the present method.
Acknowledgements
The authors are thankful to the Head, Department of ChemicalTechnology, Dr.
Babasaheb Ambedkar Marathwada University, Aurangabad 431004, MS, India for providing the
laboratory facility.
References and notes
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30. General experimental procedure for synthesis of compound 3a-3i:
Step (a): To a stirred solution of Amino-methylthiazole-5-carboxylate (1 eq) in DCM (10
times) was added 2,6-Leutidine (2 eq) and DMAP (0.2 eq) the mixture was stirred at rt for
15 min. RM was cooled to 0⁰C and Chloroacetyl chloride CAC (1.2 eq) was added drop
wise followed by stirring at rt for 3 h. The reaction was monitored by TLC and LCMS,
after completion of reaction evaporated reaction mass under reduced pressure to obtain
crude gummy material. The RM was diluted with water (10 times of reaction) and
extracted with DCM twice. The organic layer was washed with saturated sodium
bicarbonate solution, and brine solution, organic layer was evaporated under reduced
pressure to get desired product. The product was confirmed by LCMS and used as such for
next step.
Step (b): To a stirred solution of substituted 4-benzyl piperidine (0.4 g, 2.29 mmol ) in dry
THF (10 ml) was added 2,6-leutidine (0.4 g, 3.81 mmol ) and DMAP (0.046 g, 0.38 mmol
) at room temperature and then the above obtained product was sequentially added to the
formed reaction mixture (0.5 g, 1.90 mmol ). The reaction mixture was stirred at rt for 6 h
TLC shows starting material was consumed. The evaporated reaction mass was reduced
under pressure to obtain crude compound as gummy material. This was washed with 20 %
EtoAc:Hexane (20 ml ) and 20% Acetone:Hexane (20 ml) to obtain solid compounds
which are finally washed with 100% diethyl ether ( 10 ml )and 100 % pentane ( 10 ml ) to
obtain crude compound as solids. Purification of crude compound done by silica gel (100-
200 mesh) column chromatography by using 50% EtoAc: Hexane to obtain white solid
compound.
General experimental procedures for synthesis of compound 6a-6c:
Step (a): To a stirred solution of Ethyl 2-amino-4-methylthiazole-5-carboxylate (1 eq) in
DCM (10 times ) was added 2,6-Leutidine (2 eq) and DMAP (0.2 eq) the mixture was
stirred at rt for 15 min. RM was cooled to 0⁰C and Chloroacetyl chloride CAC (1.2 eq) was
11

added drop wise followed by stirring at rt for 3 h.Reaction was monitored by TLC and
LCMS , after completion of reaction evaporated reaction mass under reduced pressure to
obtain crude gummy material. RM was diluted with water (10 times of reaction) and
extracted with DCM twice. Washed organic layer with saturated sodium bicarbonate
solution, and brine solution, organic layer evaporated under reduced pressure to get desired
product. The product was confirmed by LCMS and used as such for next step.
Step (b): To a stirred solution of 4-phenylpiperidine/ 4-methylpiperidine/ piperidine (1.2eq)
in dry THF (80 mL) was added 2,6-leutidine (2 eq) and DMAP (0.2 eq ) at room
temperature and then the above obtained product was sequentially added to the formed
reaction mixture (1 eq). The reaction mixture was stirred at rt for 6 h. TLC shows starting
material was consumed. The evaporated reaction mass was reduced under pressure to
obtain crude compound as gummy material. This was washed with 20 % EtoAc:Hexane
and 20% Acetone:Hexane to obtain solid compounds which are finally washed with 100%
diethyl ether and 100 % pentane to obtain crude compounds as solids. All obtained
compounds are purified by column chromatography by using 30-70: EtoAc-Hexane to
obtain all compounds as white solids.
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Graphical Abstract
Synthesis and antimicrobial evaluation of novel ethyl 2-(2-(4-substituted)
acetamido)-4-subtituted-thiazole-5-carboxylate derivatives
Chandrakant D. Pawar, Aniket P. Sarkate, Kshipra S. Karnik, Sushilkumar S. Bahekar,
Dattatraya N. Pansare, Rohini N. Shelke, Chetan S. Jawale, Devanand B. Shinde
13