Microwave Assisted Synthesis and Antimicrobial Activity of Novel Pyrrolidine Derivatives

Article abstract of DOI:10.1134/S107036322001020X

Microwave assisted synthesis of 1-acetyl-2-benzylpyrrolidine-2-carboxylic acid and its derivatives has been developed with highly encouraging yields. 2-Benzyl-tert-butylpyrrolidine-1,2-dicarboxylate is used as an initial compound in the four steps synthes

Full text of DOI:10.1134/S107036322001020X

ISSN 1070-3632, Russian Journal of General Chemistry, 2020, Vol. 90, No. 1, pp. 129–134. © Pleiades Publishing, Ltd., 2020.
Microwave Assisted Synthesis and Antimicrobial Activity
of Novel Pyrrolidine Derivatives
K. Sreekantha and Anjali Jhaa,*
a Department of Chemistry, GIS, GITAM, Rushikonda, Visakhapatnam, 530045 India
*e-mail: anjali.jhamanish@gitam.edu
Received September 25, 2018; revised January 16, 2019; accepted January 22, 2019
Abstract—Microwave assisted synthesis of 1-acetyl-2-benzylpyrrolidine-2-carboxylic acid and its derivatives has
been developed with highly encouraging yields. 2-Benzyl-tert-butylpyrrolidine-1,2-dicarboxylate is used as an
initial compound in the four steps synthesis of the target compounds. Structures of the products 5a–5h have been
confirmed by spectroscopic methods.According to antimicrobial tests 1-acetyl-2-benzylpyrrolidine-2-carboxamide
5c is determined to be the most potent product.
Keywords: novel pyrrolidine derivatives, characterization, antimicrobial activity
DOI: 10.1134/S107036322001020X
INTRODUCTION
Antibacterial activity. The bacterial activity of
compounds 5a–5h were evaluated and compared with
Chloramphenicol as standard drug. Compounds 5c
and 5f showed good antibacterial properties against
four pathogenic bacterial strains compared with other
compounds. Compound 5c exhibited inhibition about
16 μg/mL against B. subtilis and S. aureus. Compound
The pyrrolidine ring system is a structural component
of a variety of compounds with such pharmacological
activities as antimicrobial [1, 2], antitumor [3], anti-HIV
[4], and many more. For example, they are incorporated
in the molecules of antibacterial agents Tosufloxacin,
Clinafloxacin and Ceftobiprole.
In view of the above, we would like to report here
an efficient MW assisted synthesis of new pyrrolidine
derivatives as potential antimicrobial agents.
Table 1. Comparison of the conventional and microwave as-
sisted methods of synthesis of compounds 3, 4, 5a–5h
Reaction time
Yield, %
RESULTS AND DISCUSSION
Comp.
no.
microwave
synthesis,
min
microwave
synthesis,
min
conventional
synthesis, h
conventional
synthesis, h
The target pyrrolidine derivatives 5a–5h were syn-
thesized from 1-acetyl-2-benzylpyrrolidine-2-carboxylic
acid according to the conventional and MW assisted
methods (Schemes 1, 2). The latter approach turned out
to be much more efficient than the conventional approach
(Table 1). Structures of the synthesised products were
characterized by 1H and 13C NMR spectral data.
3
10
2
4
3
5
1
6
2
3
4
2
4
88
86
81
78
72
66
72
79
86
71
85
76
71
4
25
5
10
89
5a
5b
5c
5d
5e
5f
5g
5h
25
79
Antimicrobial activity. Serial dilutions of the test
compounds and reference drugs were prepared in
Müller–Hinton broth. Drugs (10 mg) were dissolved in
DMSO (1 mL). Further progressive dilutions with melted
Müller–Hinton agar were performed to obtain the required
concentrations. In primary screening 1024 and 512 μg/mL
concentrations of the synthesized drugs were taken. The
active synthesized drugs found in this primary screening
were further tested in a second set of dilution at 256, 128,
64, 32, 16, and 8 μg/mL against all microorganisms.
5
20
82
91
No active
15
No active
79
15
92
10
90
15
86
129

130
SREEKANTH, ANJALI JHA
Scheme 1. Synthetic route to 1-acetyl-2-benzylpyrrolidine-2-carboxylic acid (5).
C
C
O
C
O
N
O
n-BuLi, ꢀ70°C
HCl
C
N
O
O
C
C6H5CH2Br
CH3OH
O
C
O
Conventianal synthesis,
microwave synthesis
O
O
C
N
H
O
3
2
1
Acetic
anhydride
Conventianal synthesis,
microwave synthesis
NaHCO3, ethanol
C
O
C
C
OH Conventianal synthesis,
microwave synthesis
O
N
N
O
O
O
4
5
5f exhibited inhibition at ca 16 μg/mL against B. subtilis.
Products 5d and 5e exhibited moderate antibacterial
activity against all bacterial strains. Compounds 5a and
5b were determined to be of weak antibacterial activity
against S. aureus and B. subtilis.
EXPERIMENTAL
Reagents were purchased fromAcross Ltd and used as
it is. The solvents were of analytical grade and distilled
before use. NMR spectra were measured on a Bruker
400 MHz spectrometer using DMSO-d6 as a solvent and
TMS as an internal reference. The microwave assisted
syntheses were carried out using a Flexiwave–Milestone
Microwave Synthesis Platform instrument.
Antifungal activity. All the synthesized compounds
5a–5h were tested against C. Albicans. Compounds 5c
and 5f showed good antifungal properties than other com-
pounds in the series against the tested strain. Compound
5c showed inhibition at 32 μg/mL. The products 5d and
5e were moderately active.
Synthesis of 2-benzyl-1-tert-butyl-2-benzyl pyrrol-
idine-1,2-dicarboxylate (2). To a solution of compound
1 (1.6 mmol) in 20 mL of tetrahydrofuran were added
n-butyl lithium (2.0 mmol) at –70°C upon stirring in
nitrogen atmosphere, then benzyl bromide (1.5 mmol).
The mixture was stirred for 1 h at room temperature. The
resultant solution was quenched in dilute hydrochloric
acid (4N), and the product was extracted by methylene
chloride.
In the present study, different groups attaching to the
pyrrolidine ring influenced upon antimicrobial activity
of the products. Antibacterial and antifungal activities of
the tested compounds varied in the ranges 16–256 and
32–256 μg/mL, accordingly. Presence of the amino group
in the moiety resulted in the enhanced activity of the cor-
responding derivatives. The electron donating hydroxyl
and methoxy groups of pyrrolidine ring led to moderate
activity of the respective products.
Benzyl-2-benzyl pyrrolidine-2-carboxylate (3). The
compound 2 (2, 2.5 mmol) was dissolved in 15 mL of
methanol, and hydrochloric acid (1.0 mmol) was added
slowly. The reaction mixture was stirred for 10 min at
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MICROWAVE ASSISTED SYNTHESIS AND ANTIMICROBIAL ACTIVITY
131
Scheme 2. Synthetic route to the compounds 5a–5h.
C
C
CH3(CH2)2NH2
Cl
NH
Conventianal synthesis,
N
microwave synthesis
N
O
O
O
O
5b
5f
SOCl2
C
C
ROH
OR
OH
Conventianal synthesis,
microwave synthesis
N
O
N
O
O
O
5a, 5e, 5g, 5h
5
LiBH4
Conventianal synthesis
CH3(CH2)3NH2
Conventianal synthesis,
microwave synthesis
C
O
C
H
N
OH
N
N
O
O
5c
5d
R = CH3CH2 (5a), CH3 (5e), C6H5CH2 (5g), CH(CH3)2 (5h).
30–35°C in a MW oven then quenched in water, and
extracted with diethyl ether.
in water, extracted by dichloromethane, and concentrated
to half of its volume. The remaining mixture was cooled
down to 2–8°C upon stirring for 1 h. The product was
filtered off and washed with diethyl ether.
Benzyl-1-acetyl-2-benzyl pyrrolidine-2-carboxyl-
ate (4). Compound 3 was mixed with acetic anhydride
(2.0 mmol), 15 mL of methylene dichloride and sodium
carbonate (3.0 mmol). The reaction mixture was stirred
for 25 min in a MW oven, then quenched in water. The
organic layer was separated and extracted with n-hexane.
Ethyl-1-acetyl-2-benzyl pyrrolidine-2-carboxylate
(5a). To the product 5 dissolved in ethanol sulphuric
acid was added slowly at 20–30°C. The reaction mixture
was treated for 25 min in a MW oven, then quenched in
ammonium bicarbonate solution, and the product was
extracted by ethyl acetate and isolated. 1H NMR, spec-
trum, δ, ppm: 1.1–1.5 t (3H, CH2–CH3), 4.2–4.6 m (2H,
CH2–CH3), 7.50–8.40 m (5H, benzene), 2.0–2.3 m (pyr-
1-Acetyl-2-benzylpyrrolidine-2-carboxylic acid (5).
Solution of compound 4 (2.0 mmol) in 15 mL of ethanol
was mixed with sodium bicarbonate. The reaction mixture
was refluxed for 10 min in a MW oven, then quenched
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SREEKANTH, ANJALI JHA
rolidine), 3.5–4.3 t (2H, pyrrolidine). 13C NMR spectrum,
δ, ppm: 14.2, 20.5, 24.2, 37.3, 46.7, 77.3, 124.7, 130.5,
132.3, 140.2, 170.6, 174.3. Found, %: C 69.71; H 7.72;
N 5.11; O 17.41. C16H21NO3. Calculated, %: C 69.79; H
7.69; N 5.09; O 17.43.
quenched in ammonium bicarbonate solution, and the
product was extracted by ethyl acetate and isolated upon
evaporation of the solvent. 1H NMR spectrum, δ, ppm:
7.30–7.60 m (5H, benzene), 1.8–2.1 m (2H, pyrrolidine),
3.5–3.6 m (2H, pyrrolidine), 3.6–3.9 s (3H, CH3–O).
13C NMR spectrum, δ, ppm: 21.5, 24.6, 37.8, 45.2, 76.1,
126.2, 129.4, 130.1, 137.7, 171.5, 172.8. Found, %: C
68.91; H 7.29; N 5.31; O 18.32. C15H19NO3. Calculated,
%: C 68.94; H 7.33; N 5.36; O 18.37.
1-Acetyl-2-benzyl pyrrolidine-2-carbonyl chloride
(5b). The mixture of compound 5 with toluene and thio-
nyl chloride was heated to 40–50°C and maintained for
5 min in a MW oven. The pure product was distilled off.
1H NMR spectrum, δ, ppm: 7.50–8.40 m (5H, benzene),
1.8–2.2 m (2H, pyrrolidine), 3.5–3.9 t (2H, pyrrolidine).
13C NMR spectrum, δ, ppm: 20.5, 24.2, 36.2, 45.3, 70.8,
124.7, 127.4, 129.4, 139.4, 177.4, 172.9. Found, %: C
63.21; H 6.02; N 5.22; O 12.01. C14H16ClNO2. Calcu-
lated, %: C 63.28; H 6.07; N 5.27; O 12.04.
1-Acetyl-2-benzyl-N-propylpyrrolidine-2-carbox-
amide (5f). To the solution of compound 5 in methylene
dichloride propyl amine was added slowly, and the mix-
ture was treated for 15 min in a MW oven, then quenched
in water. The organic layer was separated, and the product
was isolated. 1H NMR spectrum, δ, ppm: 0.8–1.1 t (3H,
CH3–CH2),1.4–1.7 m (2H, CH2–CH2–CH3), 7.30–7.60
m (5H, benzene), 1.8–2.2 m (2H, pyrrolidine), 3.5–3.7 m
(2H, pyrrolidine), 7.1–7.5 t (2H, CH2–CH2–NH), 8.0–8.3 s
(1H, NH group). 13C NMR spectrum, δ, ppm: 11.7, 24.4,
43.5; 24.8, 42.6, 46.2, 76.3, 123.4, 127.5, 128.6, 137.5,
173.3, 175.9. Found, %: C 70.77; H 8.36; N 9.73; O
11.14. C17H24N2O2. Calculated, %: C 70.80; H 8.39; N
9.71; O 11.10.
1-Acetyl-2-benzyl-n-butylpyrrolidine-2-carbox-
amide (5c). To the mixture of compound 5, methylene
dichloride and 5-methoxy-2-iodophenylboronic was
added butyl amine. The mixture was treated for 15 min
in a MW oven, then quenched in water. The organic
layer was separated to give the desired product. 1H NMR
spectrum, δ, ppm: 0.7–2.0 t (3H, CH3–CH2); 1.2–1.4 m
(2H, CH2–CH2–CH3); 1.4–1.8 m (2H, CH2–CH2–CH2);
3.0–3.4 t (2H, CH2–CH2); 7.40–7.9 m (5H, benzene),
1.9–2.2 m (2H, pyrrolidine), 3.5–3.8 t (2H, pyrrolidine),
7.6–8.3 s (1H, NH). 13C NMR spectrum, δ, ppm: 14.4,
21.6, 33.8, 41.4; 25.2, 38.3, 47.2, 78.5, 125.1, 129.2,
131.9, 136.4, 171.2, 174.5. Found, %: C 71.47; H 8.65;
N 9.23; O 10.55. C18H26N2O2. Calculated, %: C 71.49;
H 8.67; N 9.26; O 10.58.
Benzyl 1-acetyl-2-benzylpyrrolidine-2-carboxylate
(5g). To the mixture of compound 5 with benzyl alcohol
was added sulphuric acid upon stirring at 20–30°C. The
reaction mixture was treated for 10 min in a MW oven,
then quenched in ammonium bicarbonate solution. The
product was extracted by ethyl acetate and isolated. 1H
NMR spectrum, δ, ppm: 7.20–7.90 m (10H, benzene),
1.8–2.2 m (2H, pyrrolidine), 3.5–3.9 m (2H, pyrrolidine),
5.2–5.6 s (2H, O–CH2–Ph). 13C NMR spectrum, δ, ppm:
25.6, 36.3, 47.2, 77.8, 67.5, 123.2, 128.3, 130.4, 139.9,
173.8.4, 171.2. Found, %: C 74.71; H 6.84; N 4.12; O
14.21. C21H23NO3. Calculated, %: C 74.75; H 6.87; N
4.15; O 14.23.
1-[2-Benzyl-2-(hydroxymethyl)pyrrolidine-1-yl]-
ethanone (5d). To the solution of compound 5 in THF
at 2–8°C were added BuLi and lithium borohydride.
The mixture was stirred for 2 h then quenched in water,
the product was extracted by methylene dichloride and
isolated upon evaporation of the solvent. 1H NMR spec-
trum, δ, ppm: 17.30–7.80 m (5H, benzene), 1.8–2.1 m
(2H, pyrrolidine), 3.5–3.8 m (2H, pyrrolidine), 3.6–3.9 s
(2H, CH2–OH), 3.2.3.6 s (1H, OH). 13C NMR spectrum,
δ, ppm: 23.2, 19.5, 36.4, 46.5, 71.5, 124.1, 124.4, 130.8,
138.3, 171.2. Found, %: C 72.01; H 8.16; N 5.97; O
13.65. C14H19NO2. Calculated, %: C 72.07; H 8.21; N
6.00; O 13.72.
Isopropyl 1-acetyl-2-benzylpyrrolidine-2-carboxyl-
ate (5h). To the solution of compound 5 in isopropyl al-
cohol sulphuric acid was added upon stirring at 20–30°C.
The reaction mixture was treated for 15 min in a MW
oven then quenched in ammonium bicarbonate solution,
the product was extracted by ethyl acetate and isolated.
1H NMR spectrum, δ, ppm: 1.2–1.5 d [6H, (CH3)2CH];
4.7–5.2 m [1H, (CH3)2CH]; 7.30–7.70 m (5H, benzene),
1.8–2.1 m (2H, pyrrolidine), 3.5–3.7 m (2H, pyrrolidine),
2.0–2.4 s (3H, O=CH3). 13C NMR spectrum, δ, ppm:
21.9; 69.4; 21.2, 24.6, 38.4, 47.5, 76.2, 124.6, 127.8,
128.3, 137.2, 171.8. Found, %: C 70.53; H,8.04; N,4.81;
Methyl-1-acetyl-2-benzyl pyrrolidine-2-carbox-
ylate (5e). To the solution of compound 5 in methanol
was added sulphuric acid upon stirring at 20–30°C. Then
the reaction mixture was treated for 15 min in a MW
oven. Upon completion of the process the mixture was
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MICROWAVE ASSISTED SYNTHESIS AND ANTIMICROBIAL ACTIVITY
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Table 2. Antimicrobial activity (MIC values) of the synthesized compounds 5a–5h
MIC, μg/mL
gram positive bacteria
Compound
gram negative bacteria
fungus
E. Coli
128
256
32
P. aeruginosa
S. aureus
256
512
16
B. subtilis
128
256
16
C. albicans
5a
64
128
64
64
32
32
64
256
16
–
256
128
32
5b
5c
5d
32
128
128
16
128
64
64
5e
64
128
64
5f
32
32
5g
128
64
128
32
128
64
256
128
–
5h
Chloramphenicol
Ketoconazole
16
16
16
–
–
–
16
O,16.56. C17H23NO3. Calculated, %: C 70.56; H 8.01; N
4.84; O 16.59.
studied. The antibiotic Chloramphenicol (16 μg/mL) and
Ketoconazole (16 μg/mL) are used as reference antibacte-
rial and antifungal agents, respectively for comparison.
Antimicrobial activity. The standard microbial strains
were procured from National Collection of Industrial
Microorganisms (NCIM), National Chemical Laboratory,
Pune, India. The antimicrobial activity of the synthesized
compounds was determined by broth micro dilution
method as recommended by the NCCLS [5, 6]. The
compounds synthesized were evaluated against gram
positive bacteria Bacillus subtilis (NCIM 2063), Staphy-
lococcus aureus (NCIM 2079), gram negative bacteria
Pseudomonas aeruginosa (NCIM 2036), Escherichia
coli (NCIM 2065), and fungal species Candida albicans
(NCIM 3102) (Table 2).
Antimycotic assay. The yeasts were maintained in Sab-
ouraud dextrose broth after incubation for 24 h at 25±1°C.
Testing was carried out in Sabouraud dextrose broth at pH
7.4 and the 2-fold serial dilution technique was applied.
The microorganisms were grown overnight and the final
inoculum size was 104 CFU/mL.Aset of tubes containing
only inoculated broth was kept as controls. After incuba-
tion for 48 h at 25±1°C for the antifungal assay, the last
tube with no growth of yeast was recorded to represent
MIC expressed in μg/mL. Each experiment for antimy-
cotic activity and antibacterial assay was replicated twice
in order to define the MIC values.
Antibacterial assay. The cultures were obtained in
Müller–Hinton broth for all the bacteria after 24 h of
incubation at 37±1°C. Testing was carried out at pH 7.4,
and the 2-fold serial dilution technique was applied. The
microorganisms were grown overnight and the final in-
oculum size was 105 CFU/mL. A set of tubes containing
only inoculated broth was kept as control. After incuba-
tion for 24 h at 37±1°C, the last tube with no growth of
microorganism was recorded to represent MIC expressed
in μg/mL. To ensure that the solvent had no effect on the
bacterial growth, a control was performed with the test
medium supplemented with DMSO at the same dilutions
as used in the experiments and it was observed that DMSO
had no effect on the microorganisms in the concentrations
CONCLUSIONS
A series of novel 1-acetyl-2-benzylpyrrolidine-
2-carboxylic acid derivatives has been synthesized by
conventional and microwave technology. The structures
of products have been confirmed by spectral analysis.
The microwave assisted synthesis is the more efficient
approach to the desired compounds. The results of pre-
liminary bioassays indicate that a number of the products
exhibit antibacterial activity against gram-positive and
gram-negative bacteria. Modification of the pyrrolidine
ring of the parent compounds allows to develop more
active derivatives.
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SREEKANTH, ANJALI JHA
3. Li, X., Li, Y., and Xu, W., Bioorg. Med. Chem., 2005,
ACKNOWLEDGMENTS
vol. 14, p. 1287.
https://doi.org/10.1016/j.bmc.2005.09.031
We thank the Department of Science and Technology,
India under DST-FIST Program 2013 with grant no. SR/FST/
CSI-252/2013.The authors are thankful to the GITAM for
providing the necessary facility to do this work.
4. Imamura, S., Ishihara, Y., Hattori, T., Kurasawa, O.,
Matsushita, Y., and Sugihara, Y., Chem. Pharm. Bull.,
2004, vol. 52, no. 1, p. 63.
CONFLICT OF INTEREST
No conflict of interest was declared by the authors.
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