Li Petri 2021

Topics in Current Chemistry (2021) 379:34
https://doi.org/10.1007/s41061-021-00347-5
REVIEW
Pyrrolidine in Drug Discovery: A Versatile Scaffold for Novel

Biologically Active Compounds
Giovanna Li Petri1 · Maria Valeria Raimondi1 · Virginia Spanò1 · Ralph Holl2 ·

Paola Barraja1 · Alessandra Montalbano1
Received: 2 April 2021 / Accepted: 25 July 2021

© The Author(s) 2021
Abstract
The five-membered pyrrolidine ring is one of the nitrogen heterocycles used widely
by medicinal chemists to obtain compounds for the treatment of human diseases.
The great interest in this saturated scaffold is enhanced by (1) the possibility to effi-
ciently explore the pharmacophore space due to sp3-hybridization, (2) the contribu-
tion to the stereochemistry of the molecule, (3) and the increased three-dimensional
(3D) coverage due to the non-planarity of the ring—a phenomenon called “pseu-
dorotation”. In this review, we report bioactive molecules with target selectivity
characterized by the pyrrolidine ring and its derivatives, including pyrrolizines, pyr-
rolidine-2-one, pyrrolidine-2,5-diones and prolinol described in the literature from
2015 to date. After a comparison of the physicochemical parameters of pyrrolidine
with the parent aromatic pyrrole and cyclopentane, we investigate the influence of
steric factors on biological activity, also describing the structure–activity relation-
ship (SAR) of the studied compounds. To aid the reader’s approach to reading the
manuscript, we have planned the review on the basis of the synthetic strategies used:
(1) ring construction from different cyclic or acyclic precursors, reporting the syn-
thesis and the reaction conditions, or (2) functionalization of preformed pyrrolidine
rings, e.g., proline derivatives. Since one of the most significant features of the pyr-
rolidine ring is the stereogenicity of carbons, we highlight how the different stereoi-
somers and the spatial orientation of substituents can lead to a different biological
profile of drug candidates, due to the different binding mode to enantioselective pro-
teins. We believe that this work can guide medicinal chemists to the best approach in
the design of new pyrrolidine compounds with different biological profiles.
Keywords Pyrrolidine · Anticancer and antibacterial agents · Central nervous

system diseases · Antidiabetics · Anti-inflammatory and analgesic agents
* Maria Valeria Raimondi

mariavaleria.raimondi@unipa.it
Extended author information available on the last page of the article
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34 Page 2 of 46 Topics in Current Chemistry (2021) 379:34
Abbreviations
5-LOX 5-Lipoxygenase
A549 Lung cancer cell line
AChE Acetylcholinesterase
AD Alzheimer’s disease
AG α-Glucosidase
ALR2 Aldose reductase
APP Amyloid precursor protein
AR Androgen receptor
ATX Autotaxin
AZA Acetazolamide
Aβ Amyloid-β
BACE1 Beta-secretase 1
BBB Blood–brain barrier
BChE Butyrylcholinesterase
BHK Baby hamster kidney cell line
CAPAN-1 Pancreatic cancer cell line
Cas Carbonic anhydrases
CC50 Concentration required to inhibit cell proliferation by 50%
CCRF-CEM Acute lymphocytic leukemia
CNS Central nervous system
COX-1 COX-2, cyclooxygenase-1, cyclooxygenase-2
DHFR Dihydrofolate reductase
DNJ∙HCl Deoxynojirimycin chlorohydrate
DOS Diversity-oriented synthesis
DPP4 Dipeptidyl peptidase-4
α/γEC50 α/γ Half maximal effective concentration values
ED50 Median effective dose
ER Efflux ratio
ER Estrogen receptor
ES-2 Ovarian clear carcinoma cell
FAAH Fatty acid amide hydrolase
FDA Food and Drug Administration
FPA Fluorescence polarization assay
FTase Human protein farnesyltransferase
GI50 Growth inhibitory dose 50%
Glc-N-6P Glucosamine-6-phospate synthase
GLP-1 Glucagon-like peptide-1
GlyT1 Glycine transporter-1
hCA I and II Human isoenzymes I and II
HDAC2 Histone deacetylase 2
HeLa Cervix carcinoma
hGLYT1 Human glycine transporter-1
IC50 Half maximal inhibitory concentration value
K562 Chronic myeloid leukaemia cell line
KIT Tyrosine-protein kinase KIT
13
Topics in Current Chemistry (2021) 379:34 Page 3 of 46 34
MBH Morita–Baylis–Hillman
Mcl-1 Myeloid cell leukemia-1
MCF-7 Breast cancer cell line
MDA-MB-436 Breast carcinoma cell line
MES Maximal electroshock
MMP-2 -9, Matrix metalloproteinases 2, -9
NAAA N-Acylethanolamine acid amidase
NBS N-Bromosuccinimide
NCI National Cancer Institute
NCI-H522 Non-small cell lung cancer
NMDA N-Methyl-d-aspartate
NP Natural product
PARP-1-2 Poly(ADP-ribose) polymerase-1, -2
PXR Pregnane X receptor
PC-3 Prostatic cancer cell line
PDB ID Protein data bank identification
P-gp P-glycoprotein
PHB2 Prohibitin 2
PK Pharmacokinetics
PPARs Peroxisome proliferator-activated receptors
P-tau Tau protein
RORγt Retinoic acid-related orphan receptor γ
SAR Structure–activity relationship
SARMs Selective androgen receptor modulators
scPTZ Subcutaneous pentylenetetrazole
SI Selectivity index
VDCCs Voltage-gated calcium channels
VGSCs Voltage-gated sodium channels
VPA Valproic acid
1 Introduction
The development of clinically active drugs relies increasingly on the use of hetero-
cyclic scaffolds, many of which contain nitrogen, as evidenced by the considerable
number of bioactive compounds now available [1–5]. The introduction of hetero-
atomic fragments in these molecules is not a random choice, considering that they
are useful tools for modifying physicochemical parameters and obtaining the best
ADME/Tox results for drug candidates [6, 7]. Although two-dimensional (2D) flat
heteroaromatic ring scaffolds are used widely by medicinal chemists, due mainly to
their easy structural modification [8, 9], heteroatomic saturated ring systems allow
a greater chance of generating structural diversity [10]. This molecular complexity
was defined by Lovering et al. [11] through two descriptors, the s p3-hybridization
and number of chiral centers, both of which are essential for establishing the clinical
success of new bioactive molecules. Indeed, the chemical complexity and the globu-
lar three-dimensional (3D) shape offer more opportunities to improve druggability
13
by modifying parameters such as solubility, lipophilicity, and other ADME prop-

erties [12]. This association was also highlighted by Ritchie et al. [13] who con-
ducted an interesting study on a set of 19,196 molecules containing three rings with
different degrees of aromaticity. Molecules with growing hetero-aliphatic character
showed an increase in aqueous solubility compared with those comprising three
aromatic rings. Thus, each portion of the molecules can potentially influence the
balance of the physicochemical parameters, which needs to be considered when
designing compounds with an optimized pharmacokinetic (PK) profile. Table 1
summarizes a comparison of some of the molecular descriptors and parameters of
the pyrrolidine nucleus with that of carbocyclic cyclopentane and aromatic pyrrole.
As shown in Table 1, the presence of the nitrogen atom contributes to the polarity
of the molecules, producing a dipole moment (D) and a marked PSA value, which
cyclopentane lacks. Although both, pyrrole and pyrrolidine, show similar polar sur-
face area (PSA) values, only the p KBHX values (pyrrole = 0.15, pyrrolidine = 2.59)
take into account the strength of the H-bonds. The presence of nitrogen also affects
the lipophilicity of the heterocyclic rings, as evidenced by the lower LogP values of
pyrrolidine and pyrrole, and the solvent accessible surface area (SASA) FISA and
FOSA values. Also interesting is the CI_logS value of pyrrole, which is different
from the LogS, highlighting how the planarity of the aromatic ring can influence
aqueous solubility.
Over the past two decades, much effort have been made by industry to provide
the chemistry community with new sp3-enriched 3D building blocks as commercial
sources from which to produce molecules relevant from a medicinal chemistry point
of view. In this context, Goldberg et al. [14] from AstraZeneca shed light on “design
guidelines and strategic goals when designing novel reagents for drug discovery pro-
jects”, and the 3D shape appears as one of the strategic goals for the design of rea-
gents for drug discovery programs. The 3D shapes of the non-aromatic pyrrolidine
and cyclopentane rings with the aromatic pyrrole ring are compared in Fig. 1. The
different bond angles and lengths clearly suggest the flatness of the 2D structure of
pyrrole and the 3D coverage of pyrrolidine and cyclopentane.
A statistical analysis conducted by Tajabadi et al. [15] found that 70% of the
15,822 scaffolds belonging to natural products (NPs) are non-flat and represent an
interesting resource for the design of new synthetic molecules [16]. Among such
saturated ring systems, the pyrrolidine moiety is represented widely in NPs, espe-
cially in alkaloids isolated from plants or microorganisms [17–19] (Fig. 2) show-
ing different biological activities, e.g. nicotine 1 (antioxidant, anti-inflammatory,
antihyperglycemic properties), scalusamides A 2 and (R)-bgugaine 3 (antimicro-
bial, antifungal properties), 1,4-dideoxy-1,4-imino-d-ribitol 4 and aegyptolidine A 5
(anticancer properties). Therefore, it is no coincidence that the pyrrolidine nucleus is
among the most preferred scaffolds in pharmaceutical science and drug design [20],
as evidenced by the fact that it ranks first among the top five most common five-
membered non-aromatic nitrogen heterocycles, appearing in 37 drugs approved by
the United States (US) Food and Drug Administration (FDA) [21]. Moreover, pyr-
rolidine and its derivatives are used widely as ligands for transition metals, organo-
catalysts, and effective chiral controllers in asymmetric synthesis [22–24].
13
Topics in Current Chemistry
Table 1 Comparison of molecular descriptors and parameters of the pyrrolidine nucleus with those of carbocyclic cyclopentane and aromatic pyrrole
Molecule D SASA (Å2) FOSA FISA PISA DonorHB AcceptHB LogPo/w LogS CI_LogS pKBHX PSA
Cyclopentane 0.073 269.230 269.230 0 0 0 0 3.000 −2.642 −2.709 0 0

(2021) 379:34
Pyrrolidine 1.411 258.835 225.518 33.317 0 1.000 1.500 0.459 0.854 0.809 2.59 16.464
Pyrrole 2.930 236.257 0 31.512 204.745 1.000 0.500 0.750 −0.175 −0.542 0.15 13.964
D Computed dipole moment of the molecule, SASA total solvent accessible surface area using a probe with a 1.4 Å radius, FOSA hydrophobic component of the SASA
(saturated carbon and attached hydrogen); FISA, hydrophilic component of the SASA (SASA on N, O, and H on heteroatoms), PISA carbon and attached hydrogen compo-
nent of the SASA, DonorHB estimated number of hydrogen bonds donated by the solute to water molecules in aqueous solution (values are averages taken over a number
of configurations, so they can be non-integer), AcceptHB estimated number of hydrogen bonds accepted by the solute from water molecules in aqueous solution (values are
averages taken over a number of configurations, so they can be non-integer), LogPo/w predicted octanol/water partition coefficient, LogS predicted aqueous solubility (S in
mol dm−3 is the concentration of the solute in a saturated solution that is in equilibrium with the crystalline solid), CI_LogS conformation-independent predicted aqueous
solubility, pKBHX hydrogen-bond basicity scale, PSA Van der Waals surface area of polar nitrogen and oxygen atoms. Molecular descriptors and parameters were calculated
using Qikprop software (version 6.2, Schrödinger, LLC, New York, NY, 2019)
Page 5 of 46
34
13
Pyrrolidine Pyrrole Cyclopentane

Bond Length (Å)
N(1)-C(2) 1.47 1.37 -
C(1)-C(2) - - 1.53
C(2)-C(3) 1.51 1.38 1.52
C(3)-C(4) 1.51 1.42 1.52
C(4)-C(5) 1.52 1.38 1.52
C(5)-N(1) 1.47 1.37 -
C(5)-C(1) - - 1.53
Bond Angle (Å)
C(2)-N(1)-C(5) 107.63 109.33 -
C(2)-C(1)-C(5) - - 103.63
N(1)-C(2)-C(3) 105.54 108.00 -
C(1)-C(2)-C(3) - - 105.74
C(2)-C(3)-C(4) 102.26 107.34 106.30
C(3)-C(4)-C(5) 103.13 107.33 104.90
C(4)-C(5)-N(1) 106.83 108.00 -
C(4)-C(5)-C(1) - - 102.95
Fig. 1 Comparison of the three-dimensional (3D) shape of the non-aromatic pyrrolidine and cyclopen-
tane rings with the aromatic pyrrole ring. Bond angles, bond lengths, and MMFF94 values were calcu-
lated using LigandScout software version 4.4 Expert
O
HO
H
N N O
N
2
1
Scalusamides A
(S)-Nicotine
(from the cultured broth of
(from tabacco)
Penicillium citrinum)
O
Cl NH
N
N HO O
OH
O
(CH 2) 12CH 3 O
HO OH
3 4 5
(R)-bgugaine 1,4-dideoxy-1,4-imino-D-ribitol Aegyptolidine A
(from Arisarum vulgare) (from the Morus alba) (from Aspergillus aegyptiacus)
Fig. 2 Representative structures of natural alkaloids 1–5
13
The great interest in the pyrrolidine scaffold has allowed chemists to explore new
methods for its synthesis, thus bypassing conventional approaches. The application
of microwave-assisted organic synthesis (MAOS) for the synthesis of pyrrolidines
has had a strong impact, allowing synthetic efficiency to be increased, and also sup-
porting the new era of green chemistry [25].
In this review, we highlight the interest of the chemistry community in pyrro-
lidine and its derivatives, including pyrrolizines, pyrrolidine-2-one, pyrrolidine-
2,5-diones, and prolinol, incorporated in bioactive molecules with target selectiv-
ity published from 2015 to date. We have organized the review on the basis of the
synthetic approach used: (1) construction of the ring from different cyclic or acyclic
precursors, and (2) functionalization of preformed pyrrolidine rings, e.g., proline
derivatives.
2 Influence of Steric Factors on Biological Activity
Contrary to the parent aromatic compound pyrrole, which is a common scaffold of

several bioactive compounds [26–28], the great interest in synthetic pyrrolidines is
also due to the presence of up to four stereogenic carbon atoms leading to up to 16
different stereoisomers [29]. In this regard, the non-essential amino acid l-proline,
with one chiral center, is frequently employed as a building block to produce chiral
compounds, and as a catalyst for successful stereoselective synthesis [30, 31]. Since
proteins are enantioselective, the introduction of chiral centers would represent a
purposive strategy for the generation of selective ligands. Moreover, knowing the
absolute and relative configuration of chiral centers can allow the toxicity or inactiv-
ity of one of the enantiomers to be avoided, a concept regulated extensively by US
FDA guidelines on the “Development of new stereoisomeric drugs” [32]. In contrast
to the pyrrole ring, thanks to “pseudorotation”, an intrinsic property of saturated
five-membered rings, pyrrolidines gain energetically advantageous conformations
offering interesting 3D coverings [33]—a useful tool for the exploration of phar-
macophore space via diversity-oriented synthesis (DOS) [34, 35]. Overall, based
on the electronegativity of C-4 substituents, the pyrrolidine ring of proline acquires
two specific conformations called C-4 (or Cγ) -exo and -endo envelope conformers.
This means that puckering of the ring can be controlled easily through inductive
Fig. 3 Exo and endo conformers of trans- and cis-4-fluoroproline 6 and 7, respectively; trans/cis-4-fluo-
roproline 8 resulting from the superimposition of structures 6 and 7
13
and stereoelectronic factors. For instance, in the case of l-proline, the endo con-
former is preferred, whereas trans-4-fluoroproline 6 and cis-4-fluoroproline 7 favor
the exo and endo envelope conformation, respectively (Fig. 3). The overlap of trans/
cis-4-fluoroproline 8 clearly shows the different folding of the molecules induced by
the R or S configuration of the carbon bearing the fluorine atom. Conversely, C-4
alkylation of proline with a methyl or tert-butyl group is used to lock the opposite
conformation [36].
2.1 The Enantiopure Compound Shows Full Agonism Towards G‑Protein Coupled

Receptor 40
Notably, Jurica et al. [37] synthesized a series of G-protein coupled receptor 40

(GRP40) agonists, like compounds (R,R)-9 and (S,S)-9, for the treatment of type 2
diabetes (Fig. 4). They demonstrated that a cis-4-CF3 substituent on the pyrrolidine
scaffold favors the pseudo-axial conformation of the groups in the other positions, as
evaluated for the acetic acid group at position 2, which is the main pharmacophore
for GRP40 agonists. By lead optimization, they observed that the enantiopure (R,R)-
9 derivative displayed full agonism, both in human GRP40 (hGRP40, 0.11 µM) and
mouse GRP40 (mGRP40, 0.054 µM) due to the different binding mode compared
to its enantiomer (S,S)-9 (hGRP40 0.49 µM and mGRP40 2.4 µM), in addition to
a better in vivo profile in lowering glucose plasma levels in mice tested in the oral
glucose tolerance test at 0.3 or 1 mg kg−1 doses, and a dual mechanism of action
including glucose-dependent insulin and GLP-1 secretion in vitro.
2.2 The New Spatial Arrangement in the Crystallized Ligand–Protein Complex

Promotes Selectivity for CK1γ
The role of steric factors was also investigated by Luxenburger et al. [38], who syn-
thesized potent and selective CK1 kinase inhibitors exhibiting a chiral pyrrolidine
scaffold, functionalizing the CK1γ inhibitor derivative 10 (Fig. 4). Interestingly,
the new enantiopure hydroxyl-functionalized pyrrolidine derivatives 11a–b (Fig. 4)
showed selectivity for CK1 in the enzyme assay against a panel of 320 different
kinases. To investigate the binding mode of the chiral moiety, X-ray crystallog-
raphy was conducted. The crystal structure showed that, due to the incorporation
of a methylene group through a spontaneously Pictet-Spengler cyclization, two
new ligands (compounds 12a,b) (Fig. 4) were formed. These compounds showed
nanomolar activity against CK1γ and CK1ε (0.011 and 0.056 µM, and 0.024 and
0.196 µM, respectively), thus suggesting that further modifications should be made
to investigate how the chiral moiety influences kinase inhibition.
2.3 The Orientation of 3‑R‑Methylpyrrolidine is Responsible for a Pure Estrogen

Receptor α Antagonist and Selective ER Degrader
In 2018, through biochemical and biophysical assays, coupled with high-resolution

X-ray crystal structures, Fanning et al. [39] provided a molecular explanation of how
13
F F
CF 3 CF 3
N N
OCH 3 OCH 3
O O
HO HO
(S,S)-9 (R,R)-9
F F
N N
O O
N N
HO OH HO OH
NH NH
O O
R
N R= NH NH
. TFA . TFA
OCH 3
11a 11b
OCH 3
H 3CO
10
11a,b
N H 3CO H 3CO
O
N N
R=
N
N N
NH
O OH OH
R OH OH
12a,b 12a 12b
OH
CH3
N N N
R=
HO O
13a 13b 14
O
Fig. 4 Stereospecific pyrrolidine derivatives (R,R)-9, (S,S)-9, 11a,b, 12a,b, 13a,b, and 14
13
the stereospecific orientation can change the binding mode of antiestrogen benzo-
pyran derivatives 13a,b and 14 (Fig. 4) within the hormone-binding pocket. Spe-
cifically, they showed that the orientation of 3-R-methylpyrrolidine 13a was respon-
sible for the compound being a pure ERα (estrogen receptor α) antagonist and a
selective ER degrader (PA-SERD) for the treatment of breast cancer, opposite to the
3-S-methylpyrrolidine 13b and the unsubstituted derivative 14. This is due to the
capability of the R-methyl group of compound 13a to increase the mobility in the
loop connecting helices 11 and 12 (H11–12 loop) of the ERα protein.
2.4 Introduction of a Cis‑3,4‑Diphenylpyrrolidine Moiety Provides a Potent

Inverse Agonist of RORγt
Recently, Jiang et al. [40] demonstrated that replacing a non-stereochemical with

a stereochemical group was beneficial for the activity of a new series of cis-3,4-di-
phenylpyrrolidine derivatives as inverse agonists of the retinoic acid-related orphan
receptor γ (RORγt)—a splice variant of the nuclear hormone receptor subfamily
RORγ involved in autoimmune diseases. The design of the new molecules started
by studying the binding conformation of bicyclic sulfonamide 15 (Fig. 5), which
showed excellent potency towards RORγt (12 nM) but suffered from undesirable
activity against pregnane X receptor (PXR, EC50 = 144 nM, Ymax = 100%), which
upregulates proteins involved in the detoxification and clearance of foreign toxic
substances from the body. In the X-ray co-crystal structure of compound 15 in
RORγt, the sulfonyl group assumes a pseudo-axial orientation with respect to the
O
S O
N
O
HO
S N
CF 3 H HO
CF 3
15
N
CN
3S
N
4S
HO O
CF 3
F3C 16
Fig. 5 Structures of retinoic acid-related orphan receptor γ (RORγt) ligands 15 and its RORγt binding
conformation (top right) [40] as well as 16
13
benzothiazine moiety, while the folding of the para-F-phenyl ring creates face-to-
face pi stacking interactions with the benzene ring of the benzothiazine moiety, as
shown in the upper panel of Fig. 5. This “U-shaped” conformation was maintained
by replacing the sulphonyl group with the cis-3,4-diphenylpyrrolidine scaffold.
Lead optimization led to piperidinyl carboxamide 16 (Fig. 5), which proved a bal-
ance between potency against RORγt with an E C50 of 61 nM and a considerably
lower PXR activity ( EC50 = 495, Ymax = 46%). Remarkably, (1) the cis-configuration
accommodates the two phenyl rings close to the face-to-face stacked arrangement,
(2) the hexafluoroisopropyl alcohol moiety establishes a hydrogen bonding interac-
tion between the hydroxy group and His479 of RORγt, (3) transposition of the C3
methyl group in C4 position leads to the loss of RORγt potency. In addition, (4) pyr-
rolidines with an unsubstituted nitrogen were weak agonists compared with tertiary
C50 values.
amines, and (5) a para-F substituent in the 3-phenyl ring improved E
2.5 Basicity and Nucleophilicity of the Pyrrolidine Nucleus
While substituents at C-4 of proline affect the puckering of the ring, substituents
at C-2 of pyrrolidine shift its basicity. In fact, the nitrogen atom of pyrrolidine, as
a secondary amine, confers basicity to the scaffold. In this context, An et al. [41]
investigated the basicity of a set of 28 pyrrolidines used as organocatalysts, showing
that particularly charged substituents have a strong effect on the basicity. Lastly, due
to its nucleophilicity, the pyrrolidine nitrogen represents a privileged position for
substitutions, with 92% of all US FDA approved pyrrolidine drugs being substituted
at the N −1 position [21].
2.6 Spatial Characteristics Influencing the Biological Activities of Pyrrolidine

Derivatives
In summary, the advantages of using the pyrrolidine scaffold in drug design are due
to the unrestricted conformation of the ring, which can be controlled and locked by
the appropriate choice of substituents. Indeed, inductive and stereoelectronic factors
influence the puckering of the pyrrolidine ring and, consequently, its pharmacologi-
cal efficacy. The following list summarizes the spatial dispositions of the pyrroli-
dine scaffold that affect the biological activity towards specific targets cited in this
chapter:
• cis-4-CF3 substituent on the pyrrolidine scaffold endorses the pseudo-axial con-

formation of the groups in the other positions, leading compounds able of show-
ing full agonism in both the human and mice GRP40 receptor;
• compared with 3-S-methylpyrrolidine or an unsubstituted pyrrolidine, 3-R-meth-
ylpyrrolidine promotes a pure ERα antagonist and selective ER degrader (PA-
SERD) for the treatment of breast cancer;
• the introduction of a chiral pyrrolidine into molecules promotes selectiv-
ity towards CK1 receptors. However, the selectivity and potency for the CK1γ
13
isoform is due to the methylene group at position 2 being involved in sponta-

neous intramolecular Pictet–Splenger cyclization during crystallization with the
protein;
• a cis-3,4-diphenylpyrrolidine scaffold gives the molecule a “U-shape” conforma-
tion that is beneficial for inverse agonistic activity on the RORγt receptor.
3 Pyrrolidine Derivatives Obtained by Ring Synthesis
3.1 Pyrrolidines Obtained by 1,3‑Dipolar Cycloadditions
A classical method for the preparation of five-membered heterocycles is the

1,3-dipolar cycloaddition [42] between a 1,3-dipole, such as a nitrone, an azide or
an azomethine ylide, with a dipolarophile, typically an olefin, both of which are
responsible for the regio- and stereoselectivity of the reaction [29, 43]. In particular,
for synthetic pyrrolidines, the 1,3-dipolar cycloaddition reaction between nitrogen-
based 1,3-dipole azomethine ylides with alkenyl dipolarophiles has been studied
extensively. As shown in Fig. 6, stereoselectivity at positions 2 and 5 (R1 and R2)
depends on the shape of the ylides, whereas stereochemistry in positions 3 and 4
correlates with the relative orientation of the substituents of the dipolarophile, lead-
ing to 3,4-cis- or 3,4-trans-substituted pyrrolidines [44].
Exploiting the 1,3-dipolar cycloaddition process between N-(methoxymethyl)-
N-(trimethylsilylmethyl)benzylamine 17 and methyl acrylate 18 (Fig. 7) under acid
conditions in the presence of trifluoroacetic acid (TFA), Min et al. [45] synthesized
a series of benzimidazole carboxamides 19a–p bearing the pyrrolidine nucleus at
position 2. In particular, they functionalized the pyrrolidine nitrogen by introducing
different aromatic rings and tested the compounds as inhibitors of poly(ADP-ribose)
Ylides:
R
R
R
+ 1 N 2
1 + 2 N R R
R N R - 3
or R
- 1 2
R R 3
W shape dipolarophile R
U shape
2,5-cis
R R 1 N 2
R R
+ 2 3
1 + R
N R R N
-
- 3
1 2 dipolarophile R
R R
S shapes 2,5-trans
Fig. 6. 1,3-Dipolar cycloadditions to stereoselectively obtain pyrrolidine derivatives
13
CONH2
O
+ a
N N
N O 7 steps
N
N
R
TMS O H
17 18 19a-p
Comp R PARP-1 PARP-2 PARP-1 PARP-2

inhibition % inhibition % IC50 [nM] IC50 [nM]
19a -CH2(CO)NH-phenyl 17.6 34.5 / /
19b -(CH2)2(CO)NH-phenyl 19.9 41.2 / /
19c -(CH2)3(CO)-phenyl 42.8 62.3 12.2 5.8
19d -CH2(CO)-phenyl -1.1 7.2 / /
19e -(CH2)3-N-isatin 22.0 74.5 / /
19f -CH2CH(OH)-phenyl 5.0 9.0 / /
19g -(CH2)2CH(OH)-phenyl -2.1 6.5 / /
19h -(CH2)3NH-phenyl 47.5 66.2 7.1 3.3
19i -(CH2)2(CO)-phenyl-4-OCH3 59.2 62.5 5.9 4.5
19j -(CH2)2(CO)-phenyl-4-Cl 65.7 65.6 3.9 4.2
19k -(CH2)2-N-isatin 6.0 37.1 / /
19l -(CH2)3-NH2 26.8 46.4 / /
19m -(CH2)2-NH2 24.8 32.8 / /
19n -(CH2)3-NH-(CO)-phenyl 19.4 39.1 / /
19o -(CH2)2-NH-(CO)-phenyl 38.4 66.2 11.1 5.7
19p -(CH2)2(CO)-phenyl 68.0 76.7 3.6 3.2
Veliparib / 63.7 78.3 5.3 1.6
Fig. 7 General synthetic scheme to benzimidazole carboxamides 19a–p. R substituents and

poly(adenosine 5′-diphosphate (ADP)-ribose) polymerase (PARPs) inhibition assay of compounds 19a–
p. PARP-1 and -2 inhibition (%) were evaluated at 10 nM. Reagents and conditions: a trifluoroacetic acid
(TFA), dichloromethane (DCM), 17 h, room temperature (r.t.), yield: 96%
polymerase-1 and -2 (PARP-1,-2), enzymes involved in the DNA damage repair pro-
cess, as depicted in Fig. 7.
Out of 16 compounds, 6, namely 19c,h–j,o,p, inhibited PARP-1 and -2 with I C50
values ≤10 nM. Structure–activity relationship (SAR) analysis revealed that the
length of the alkyl chain and the presence of the carbonyl group greatly influence
biological activity. Overall, the most promising derivatives were the phenyl ketone
derivatives 19c,i,j,p, rather than the N-phenylamine 19h or the N-benzamide 19o
derivatives. Comparing the activity within the entire class of phenyl ketone deriva-
tives bearing 2–4 carbon atoms in the alkyl chain (19c,d,i,j,p), compounds 19i,j,p
with the 3-carbon atom chain were the most active. Docking studies highlighted that
the phenyl ketone fragments of compounds 19j and 19p would bind the three impor-
tant amino acid residues Gly-863, Ser-904 and Glu-988 of PARP-1, instead of only
13
Gly-863 and Ser-904 as in compounds 19c and 19d. Additionally, Van der Waals or
hydrophobic interactions between the side chain of the compounds and the residues
in the active site of PARP-1 were thought to facilitate the binding of these benzimi-
dazole carboxamides. Electron donating or withdrawing groups in the para position
of the benzene ring, as in compounds 19i and 19j, caused inhibitory activity similar
to the reference compound veliparib, but with slightly higher I C50 values compared
with the unsubstituted derivative 19p. Reduction of the carbonyl group to a hydroxy
group (19f and 19g), the presence of hydrophilic amine chains (19l and 19m), or
the bulky phthalimide group (19e and 19k), were detrimental for enzymatic inhibi-
tion. Cytotoxic assays against MDA-MB-436 (breast cancer) and CAPAN-1 (pan-
creatic cancer) cell lines confirmed 19j as the most promising compound ( IC50 17.4
and 11.4 µM, respectively), followed by 19p (IC50 19.8 and 15.5 µM, respectively),
which had IC50 values much lower than those of the reference compounds olapa-
rib (IC50 30.2 and 100 µM, respectively, in MDA-MB-436 and CAPAN-1 cells) and
veliparib (IC50 100 µM in both cell lines).
The same dipolar reagent N-(methoxymethyl)-N-(trimethylsilylmethyl)ben-
zylamine 17 was reacted with the nitrovinyl substrate 20, methyl trans-4-fluorocin-
namate 21, and chalcone 22, in the presence of a catalytic amount of TFA, by Wang
et al. [46] to synthesize 3,4-disubstituted pyrrolidine sulfonamides 23a–y, 23z–ac,
and 23ad, respectively (Fig. 8). All compounds were prepared as enantiomerically
pure trans isomers and tested as selective glycine transporter-1 (GlyT1) competi-
tive inhibitors. The major role of GlyT1 is to maintain glycine concentration below
saturation level at the postsynaptic ionotropic glutaminergic N-methyl-d-aspartate
(NMDA) receptor. Since the aetiology of schizophrenia has been linked to impaired
glutamatergic neurotransmission involving the NMDA receptor, the development of
GlyT1 inhibitors may represent a putative treatment for it and other disorders associ-
ated with NMDA receptor hypofunction. The new pyrrolidine sulfonamides 23a–ad
were synthesized based on the SAR investigation conducted on the reference com-
pound 23a. The latter displayed satisfactory GlyT1 inhibitory potency in vitro, with
a Ki value of 0.198 µM, but a high efflux ratio (ER) of 8.7, indicating it as potential
substrate of P-glycoprotein (P-gp)—the most relevant efflux transporter expressed
in blood–brain barrier (BBB). The transformation of the sulfonamide moiety into
amides, carbamides, tertiary amines, and urea groups, including the pyrrolidine
nitrogen, produced inactive inhibitors of GlyT1 (structures not shown). The replace-
ment of the benzoyl group of compound 23a by aryl substituents (R2) gave excellent
outcomes, providing new potent analogues such as 23d,f–i,k,l,o–t,y,z with single or
double digit nanomolar activity. Among these, the anilines 23d,g–i,k,l,o,p,s,v,x also
exhibited lower ER values and consequently less susceptibility to P-gp efflux. Com-
parable in vitro potency and ER values were obtained when replacing the 1-meth-
ylimidazole moiety by a 1-methyl-1,2,3-triazole substituent obtaining compound
23u, which proved metabolically unstable. As reported in Fig. 8, the fluorophenyl
substituents at position 3 (R1) of the pyrrolidine sulfonamides offered better in vitro
potency and ER profile, followed by the unsubstituted phenyl ring (23b–f). Concern-
ing R2 substituents, meta-substituted derivatives showed improved biological activ-
ity. Instead, the activity of compounds with heteroaromatic substituents in position 4
(23j,m,q,r) was influenced by the number and position of the nitrogen atoms of the
13
1 2
R NH R
R NO2
N N
R
NO2 O S O
20 3
R
a 23a-y
1 2
R R
COOMe OMe
O
F N
21
N
N
a O S O
TMS O F 3
R
23z-ac
17 O
22
1 2
a R R
O
N
N O S O
3
R
23ad
Comp R1 R2 R3 ER Ki
[µM]
23a phenyl 2-Cl,3-CF3-phenyl- 1-CH3-1H-imidazol-4-yl 8.7 0.198
methanone
23b phenyl phenyl 1-CH3-1H-imidazol-4-yl 0.9 0.199
23c phenyl 2-CH3-phenyl 1-CH3-1H-imidazol-4-yl 1.3 1.51
23d phenyl 3-CH3-phenyl 1-CH3-1H-imidazol-4-yl 0.9 0.018
23e phenyl 4-CH3-phenyl 1-CH3-1H-imidazol-4-yl 0.6 1.25
23f phenyl 3-CF3-phenyl 1-CH3-1H-imidazol-4-yl - 0.008
23g 4-F-phenyl 3-CF3-phenyl 1-CH3-1H-imidazol-4-yl 0.9 0.003
23h 4-F-phenyl 3-Cl-phenyl 1-CH3-1H-imidazol-4-yl 1.1 0.003
23i 4-F-phenyl 3-CF3,4-F-phenyl 1-CH3-1H-imidazol-4-yl 0.8 0.005
23j 4-F-phenyl 5-CF3-pyridin-3-yl 1-CH3-1H-imidazol-4-yl 10 1.16
Fig. 8 General synthetic scheme to pyrrolidine sulfonamides 23a–ad. ER Efflux ratio values. Ki values
towards hGLYT1 [46]. Reagents and conditions: a TFA (catalyst), DCM, r.t., overnight
heteroaromatic, as shown in Fig. 8. Finally, the analogue 23t (R2 = indanyl) showed
the best balance between potency and ER value (Ki = 0.001 μM, ER = 1.5).
1,3-Dipolar cycloadditions between N-(methoxymethyl)-N-(trimethylsilylmethyl)
benzylamine 17 and cis- or trans-alkenyl ester derivatives 24 (Fig. 9) in TFA, were
used by Zhang et al. [47] to gain a series of 4-benzylpyrrolidine-3-carboxylic
acid derivatives as potent agonists at peroxisome proliferator-activated receptors
(PPARs). Among the new pyrrolidines, the cis-3R,4S-configured compounds 25
and 26 (Fig. 9) combine agonistic activity at PPARα and PPARγ, restoring glucose
13
23k 3-F-phenyl 3-Cl-phenyl 1-CH3-1H-imidazol-4-yl 1 0.042

23l 2-F-phenyl 3-Cl-phenyl 1-CH3-1H-imidazol-4-yl 0.9 0.021
23m 4-F-phenyl 2-CF3-pyridin-4-yl 1-CH3-1H-imidazol-4-yl 18.5 0.118
23n pyridin-3yl 3-CF3-phenyl 1-CH3-1H-imidazol-4-yl - 0.766
23o 4-F-phenyl 3-CF3-phenyl 1-CH3-1H-imidazol-4-yl 0.8 0.002
23p 4-F-phenyl 3-OCF3-phenyl 1-CH3-1H-imidazol-4-yl 0.7 0.004
23q 4-F-phenyl 4-CF3-pyridin-2-yl 1-CH3-1H-imidazol-4-yl 1.3 0.008
23r 4-F-phenyl 6-CF3-pyrimidin-4-yl 1-CH3-1H-imidazol-4-yl 7.5 0.005
23s 4-F-phenyl 3-OCF3,4-F-phenyl 1-CH3-1H-imidazol-4-yl 0.7 0.002
23t 4-F-phenyl 2,3-dihydro-1H-inden-1-yl 1-CH3-1H-imidazol-4-yl 1.5 0.001
23u 4-F-phenyl 3-CF3,4-F-phenyl 1-CH3-1H-1,2,3-triazole-4-yl 0.5 0.003
23v (R)- 3-OCF3-phenyl 1-CH3-1H-imidazol-4-yl 0.8 0.003
esahydro-2H-pyran-2-yl
23w (S)-tetrahydro-2H-furan-2yl 3-OCF3-phenyl 1-CH3-1H-imidazol-4-yl - 0.024
23x 1,3-dioxepan-2-yl 3-OCF3-phenyl 1-CH3-1H-imidazol-4-yl 0.9 0.002
23y 1,3-dioxan-2-y 3-OCF3-phenyl 1-CH3-1H-imidazol-4-yl 1.1 0.002
23z 4-F-phenyl 3-OCF3-phenoxymethyl 1-CH3-1H-imidazol-4-yl - 0.007
23aa 4-F-phenyl 3-OCF3-phenylmethanone 1-CH3-1H-imidazol-4-yl 1.3 0.029
23ab 4-F-phenyl 3-OCF3- 1-CH3-1H-imidazol-4-yl 1.2 0.060
phenylaminomethyl
23ac 4-F-phenyl 3-OCF3-phenyl- 1-CH3-1H-imidazol-4-yl 7.2 0.36
aminomethanone
23ad phenyl benzyl 1-CH3-1H-imidazol-4-yl 1.3 0.093
Fig. 8 (continued)
metabolism and ameliorating dyslipidaemia associated with type 2 diabetes. Com-

pounds 25 and 26 displayed αEC50 and γEC50 values in the low nanomolar range
(5–90 nM). In addition, compound 26 was efficient in lowering fasting glucose and
triglyceride levels in diabetic db/db mice after oral administration of a 10 mg/kg
dose once daily. SAR studies revealed that (1) the oxybenzyl pyrrolidine acid series
offered the best balance of PPARα/γ functional activities, (2) the cis-configuration
of the substituents in positions 3 and 4 of the pyrrolidine ring was preferred over the
trans orientation, and (3) N-carbamoyl and N-aryl-substituted oxybenzyl pyrrolidine
acid analogs provided potent balanced PPARα/γ dual agonists.
Conjugated systems with biological macromolecules, including carbohydrates,
lipids, proteins, and nucleic acids, require that special attention is paid to the geom-
etry of the molecules to be combined. Unlike the pyrrolidine scaffold, spiro-pyr-
rolidine molecules have rigid conformations that allow for easy incorporation into
biological macromolecules, including cholesterol. For this purpose, Periyasami
et al. [48] synthesized a small set of novel spiro-pyrrolidine/pyrrolizines by one-pot
three-component reactions. Stereo- and regioselective reactions based on 1,3-dipo-
lar cycloaddition between the dipolarophile C3-β-cholesterolacrylate 27 (Fig. 9)
under reflux in iPrOH for 2 h with azomethidine ylides, in turn generated in situ
by reaction isatin, acenaphthoquinone, or ninhydrine with the secondary amino
acids sarcosine or proline, allowed the desired compounds to be obtained as sin-
gle isomers. All compounds were evaluated for their in vitro antibacterial activity
13
X
X
a
+ N
N
Bn
TMS O 24a,b
24a: X = CO-Xc(D) (Xc-(D)) =
17 D-camphorsultam
24b: X = -COOEt
COOH
25: R = -(CO)O-phenyl
α EC50 = 5 nM; γ EC50 = 30 nM; O
26: R = 3-CF3-pyrimidine O
N
N R
αEC50 = 90 nM; γEC50 = 30 nM;
25, 26
H3C H3C
H3C
H3C H3C
CH3
isatin acenaphthaquinone
CH3
+ +
H3C sarcosine O proline H3C
b b
O
27
O O O O
ninhydrine b
+ proline
H3C
CH3 N
N N O CH3
H
O N
O
O
28 O
O
29
30
Fig. 9. 1,3-Dipolar cycloadditions to yield 4-benzylpyrrolidine-3-carboxylic acids 25 and 26, synthesis

of cholesterol-conjugated spiro-pyrrolidine/pyrrolizines 28–30, and 3D/2D interaction diagrams of com-
pound 28 with the active site of target receptor protein 1XFF [48]. Reagents and conditions: a TFA (1 M
solution in DCM); b isopropyl alcohol (iPrOH), 2 h, under reflux, yields: 76% (28), 67% (29), 63% (30)
13
against a panel of four human pathogens, including Vibrio cholerae, Proteus mira-
bilis, Micrococcus luteus, and Bacillus subtilis. The most active were compounds
28, 29, and 30 (Fig. 9), which showed good antimicrobial activity, causing zones
of growth inhibition in the performed disc diffusion assays ranging from 13.0 to
15.1 mm, at 50 µg/ml. With the aim of investigating their biological potential as
glucosamine-6-phospate synthase (Glc-N-6P) inhibitors, the authors performed
in silico molecular docking studies with compounds 28–30 in the enzyme’s active
site (PDB ID: 1XFF). The results showed significant interactions with active site
amino acids, revealing that the spiropyrrolidine moiety of compound 28 is engaged
in two H-bond interactions involving its –NH and –CO groups with those of the
aliphatic nonpolar amino acid Gly78 (Fig. 9), thus demonstrating its prominent role
in the inhibition of Glc-N-6-P synthase and thus in the antibacterial activity of the
compounds.
By a one-pot multicomponent approach, Shyamsivappan et al. [49] synthesized
phenyl/thiophene dispiro indenoquinoxaline pyrrolidine quinolone analogues, 36a–f
and 37a–f (Fig. 10), via 1,3-dipolar cycloaddition reaction of (E)-3arylidine-8-nitro-
2,3-dihydroquinolin-4(1H)-ones 33a–f, ninhydrin 31, o-phenylenediamine 32 and
benzylamine 34/thiophene methylamine 35 in MeOH under reflux for 2–3 h. All
compounds were screened for anticancer activity against MCF-7 and HeLa cells,
and only compound 37e showed good biological activity, with IC50 values of 17 and
OH H2N
OH + N
H2N NH2
O O
31 32 NH N
H
34 N H
O H
NO 2
N a
R 36a-f
N
NH2 N
O
+ S
O
NH N
H
35
N H
H
S
N R NO 2
H
NO 2 33a-f
R = H, Br, Cl, F, CH3, OCH3 37a-f
R
Fig. 10. 1,3-Dipolar cycloadditions to yield phenyl/thiophene dispiro indenoquinoxaline pyrrolidine

quinolone derivatives 36a–f and 37a–f. Reagents and conditions: a MeOH, under reflux, 2–3 h, yields:
92–98% (36a–f) and 93–96% (37a–f)
13
19 µM, respectively, comparable to those of the reference compound doxorubicin

(16 and 18 µM against MCF-7 and HeLa cells, respectively). Overall, the thiophen-
containing derivatives 37a–f showed better activity against both cell lines (IC50 in
the range of 17 and 28 µM against MCF-7, and 19 and 30 µM against HeLa) than
their respective counterparts bearing the phenyl ring 36a–f (IC50 in the range of 22
and 29 µM against MCF-7; and 26 and 37 µM against HeLa). SAR analysis indi-
cated that compounds 36e,f, and 37e,f, characterized by electron donating groups,
such as methoxy and methyl had lower IC50 values than other derivatives with elec-
tron-withdrawing groups (EWGs). Further investigation into the mechanism behind
the anticancer activity revealed that compound 37e induced apoptosis through intra-
cellular reactive oxygen species (ROS)-mediated caspase-3 activation.
3.2 Pyrrolidines Obtained by Pictet–Spengler‑Oxidative Ring Contractions
Moreover, spirocyclic motifs are emerging as an interesting feature for building

block with low-molecular weight in the drug discovery field [50]. In 2016, Hati et al.
[51] designed and synthesized a library of spiro[pyrrolidine-3,3′-oxindoles] 38a–n
(Fig. 11) as potential anti breast cancer agents with a dual activity against histone
deacetylase 2 (HDAC2) and prohibitin 2 (PHB2) enzymes. HDAC2 is responsible
for the deacetylation of lysine residues on the N-terminal part of core histones (H2A,
H2B, H3 and H4) and provides a tag for epigenetic repression, thus playing a sig-
nificant role in transcriptional regulation, cell cycle progression, and developmental
events. PHB2 acts as a mediator of transcriptional repression by nuclear hormone
receptors via recruitment of histone deacetylases and works like an ER-selective co-
regulator, potentiating the inhibitory activities of anti-estrogens and repressing the
activity of estrogens. Based on the DOS strategy, the new compounds 38a–n were
synthesized via a one-pot Pictet Spengler-oxidative ring contraction of tryptamine,
in the presence of water as reactant, mediated by stoichiometric N-bromosuccinim-
ide (NBS) as oxidant, and a catalytic amount of TFA, varying the aromatic function-
ality of the pyrrole domain. Among all compounds produced, 38d,h,i inhibited the
NH2
NH
38a: Ar = 3-CH3-phenyl
NH
a Ar 38b: Ar = 3,5-diCH3-phenyl
Ring contraction O
N Ar
38c: Ar = 2,6-diF-phenyl
N N
H H H 38d: Ar = 3-F-phenyl
Triptamyne 38a-n 38e: Ar = 3-OCH3-phenyl
1H-β-carboline
intermediate 38f: Ar = 3,4-diOCH3-phenyl
38g: Ar = 4-OCH3-phenyl
38h: Ar = 2,4,5-triOCH3-phenyl
38i: Ar = 2-F,4-OCH3-phenyl
38j: Ar = 3-CH3,4-OCH3-phenyl
38k: Ar = phenyl
38l: Ar = 2,5-diCF3-phenyl
38m: Ar = tiophen-2-yl
38n: Ar = pyridin-4-yl
Fig. 11 General synthetic scheme to spiro[pyrrolidine-3,3′-oxindoles] 38a–n. Reagents and conditions: a

ArCHO, NBS, water/tetrahydrofuran (THF), TFA (cat), 0 °C to r.t., yields: 45–94%
13
growth of human breast cancer cell line MCF-7 by inducing apoptotic cell death at
low micromolar E C50 values (6.00, 4.01, and 3.53 µM, respectively). Chemical pro-
teomics indicated HDAC2 and PHB2 as potential targets of the spiro[pyrrolidine-
3,3-oxindoles] and molecular docking of the most active compound 38i with
HDAC2 (PDB ID: 4LY1) confirmed probable binding interactions. Overall, based
on their cytotoxic effects, compounds characterized by electron-donating or weak
EWGs on the phenyl ring showed higher MCF-7 cell growth inhibition rates com-
pared with compounds bearing one or two strong EWGs.
3.3 Pyrrolidines Obtained via Aminocyclizations
Polyhydroxylated pyrrolidines, known as aza-sugars, are considered metabolically

inert carbohydrates as they mimic the oxa-carbenium transition state from carbo-
hydrate processing enzymes. Due to their central role in different biological activi-
ties, they are emerging as attractive compounds for the treatment of cancer and
metabolic diseases. In 2019, using the double reductive amination reaction, Guaz-
zelli et al. [52] developed a series of polyhydroxylated pyrrolidines, belonging to
2 2
RO O RO R R
1
OR PhCH2NH2 . HCl
CHO RO N N
RO
or
L-Phe-OMe . HCl OR 1 + OR 1
OH a 1 1
OR OR OR
39: R,R = CMe2, R = H 1
OR
40: R = R1 = H D-gluco
D-galacto
41a: R,R = CMe2; R1 = H; R2 = CH2Ph
42: R,R = CMe2; R1 = H; R2 = (L)-CH(CH2Ph)COOMe 41b: R,R = CMe2; R1 = H; R2 = CH2Ph
Ph O
O O O HO
O
H
O N O N HO N
isobutyric O N
OH OH OH
hydrogenolysis anhydride OH hydrolysis
OH OH OH
OH
41a 43a 47a
44a
Ph Ph O O
H
N N N N N
OH OH OH
isobutyric
hydrolysis OH hydrolysis OH
hydrogenolysis anhydride
OH OH O OH O OH O OH OH OH
OH O O O OH
45 41b 43b 44b 47b
Ph
Ph
COOMe
O COOMe
HO
O N
HO N
OH hydrolysis
OH
OH
OH
42 46
Fig. 12 Blue panel Synthesis of diastereoisomers 41a,b and 42 obtained by reaction of 5,6-O-isopro-
pylidene-d-xylo-hexos-4-ulose 39 with benzylamine hydrochloride and l-phenylalanine methyl ester
hydrochloride, respectively. Yellow panel Synthesis of final compounds 43a,b, 44a,b, 45, 46 and 47a,b.
Reagents and conditions: a sodium cyanoborohydride (NaBH3CN), methanol (MeOH); 60 °C, 24–48 h,
yields: 37% (41a,b), 44% (42)
13
the diastereoisomeric d-glucose and d-galactose series, as dual-target inhibitors of

the enzymes α-glucosidase (AG) and aldose reductase (ALR2). Although AG repre-
sents the most important enzyme for controlling plasma sugar concentration, patho-
logical changes in nervous, renal, vascular, and ocular systems of diabetic patients
are caused by activation of ALR2. Therefore, ideal antidiabetic agents should be
able to simultaneously block the catalytic activity of AG and ALR2. Aminocycliza-
tion was conducted by reacting aldohexos-4-ulose derivatives 39 and 40 (Fig. 12)
with amines, including benzylamine, benzhydrylamine, ammonia, l- or d-phenyla-
lanine methyl ester, and sodium cyanoborohydride (NaBH3CN) as reducing agent,
to obtain mixtures of d-glucose and d-galactose diastereoisomers. Among these, the
authors isolated as pure compounds only diastereoisomers 41a,b and 42 (Fig. 12)
obtained by reaction of 5,6-O-isopropylidene-d-xylo-hexos-4-ulose 39 with ben-
zylamine hydrochloride (for compounds 41a,b) and l-phenylalanine methyl ester
hydrochloride (for compound 42). These three compounds were the starting mate-
rial for the synthesis of other derivatives. Thus, through hydrogenolysis, com-
pounds 43a,b were obtained and were, in turn, reacted with isobutyric anhydride
to give compounds 44a,b. Finally, compounds 41b, 42, and 44a,b were subjected
to hydrolysis to remove the 5,6-O-isopropylidene protecting group affording com-
pounds 45, 46, 47a,b, respectively, with enhanced hydrophilic character compared
with the parent compounds. Among all, the d-galacto derivative 43b was able to
reduce cell death and restore the physiological levels of oxidative stress, showing a
percentage of enzyme inhibition at 100 μM of 57.1% and 30.2% against ALR2 and
AG, respectively.
Recently, Li et al. [53] reported a series of (S)-pyrrolidines as CXCR4 chemokine
receptor antagonists with antimetastatic activity. The synthesis of compounds 51a–f
(Fig. 13) was carried out in a multi-step manner in which intermediate pyrrolidine
derivatives 50 were obtained by reaction of pyridin-2-yl-4-oxobutanal derivatives
48 with (R)-1-(4-methoxyphenyl)ethan-1-amine 49. Among all compounds gener-
ated, 51a with R1 = 3-CH3 showed excellent binding affinity to the CXCR4 receptor
NH2
R N
O
N N
R O
+ a 1
N R
N
O N
O
48 50 N
N
49 N
51a-f
51a: R = 3-CH3, R1 = H
51b: R = 4-CH3, R1 = H
51c: R = 5-CH3, R1 = H
51d: R = 6-CH3, R1 = H
51e: R = 3-CH3; R1 = F
51f: R = 3-CH3; R1 = CN
Fig. 13 General synthetic scheme to pyrrolidines 51a–f. Reagents and conditions: a sodium triacetoxy-
borohydride [NaBH(OAc)3], DCM, − 70 °C to r.t., overnight, yields: 21–45%
13
(IC50 = 79 nM competitively displacing fluorescent 12G5 antibody). Conversely,

by shifting the methyl group to other positions in the pyridine ring, the IC50 value
increased to 216, 278 and 2391 nM for compounds 51b, 51c and 51d, respectively.
Another feature of compound 51a was its ability to inhibit CXCL12-induced cyto-
solic calcium flux (IC50 = 0.25 nM). In order to mitigate the overall basicity of the
compounds, which can lead to issues such as hERG potassium channel inhibition,
as well as CYP enzyme inhibition and phospholipidosis, the authors introduced a
fluorine atom (compound 51e) or a cyano group (compound 51f) at the R1 position.
However, this change greatly reduced the potency of compounds 51e and 51f, by
4- and 7-fold, respectively. Interestingly, the antimetastatic behavior of compound
51a was also shown in an in vivo tumor metastasis test conducted in mice, which
received compound 51a intraperitoneally at a dose of 30 mg/kg.
3.4 Pyrrolidine‑2‑Ones Obtained by Cyclizations
The interest in polyhydroxylated pyrrolidines led Da Silva et al. [54] to search for
a short and alternative strategy to synthesize 1,4-dideoxy-1,4-imino-l-arabinitol
OH HO OH
O O CO 2 Et a,b,c
O HO
N
N N O
Boc
Boc H
Garner's aldehyde MBH adduct

52 53
d,e,f
HO OH HO OH
HO HO
N N
H H
Natural product 54
55
O CN O
O O CN
N NH N N
ClCH2 CN
N S N S
g
O O H2N
56 57
Fig. 14 General synthetic schemes to polyhydroxylated pyrrolidines 54 and pyrrolidone 57. Reagents
and conditions: a (1) O3, MeOH, − 78 °C, 40 min; (2) DMS, 40 min, − 78 °C to r.t., not isolated; b
[Zn(BH4)2], MeOH or DCM, − 20 °C, 2 h, yield: 74%; c TFA (2 equiv), DCM, 1 h, yield: 74%; d TBSCl,
imidazole, DMF, 20 h, r.t., yield: 74%; e BH3. DMS, THF, 4 h, r.t., yield: 60%; f tetra-n-butylammonium
fluoride (TBAF), THF, 12 h, r.t., yield: 99%. g triethylamine (TEA), 1,4-dioxane, 5 h, under reflux, yield:
77%
13
54 (Fig. 14). Compound 54 is a polyhydroxylated pyrrolidine that is a more potent

α-glycosidase (AG) inhibitor than its natural enantiomer compound 55 (Fig. 14). For
this reason, compound 54 is reputed to be a potential starting point for new antidia-
betic and anticancer drugs. As shown in Fig. 14, the synthetic route proposed for the
synthesis of 54 comprises six steps starting from a chiral Morita–Baylis–Hillman
(MBH) adduct 53, which in turn was prepared from Garner’s aldehyde 52. Com-
pound 54 was obtained from an intermediate pyrrolidone, which was synthesized
by a linear three-step reaction sequence involving ozonolysis of the double bond
of the MBH adduct 53, followed by a stereoselective ketone reduction using zinc
borohydride [Zn(BH4)2], concomitant N-double deprotection/O-deprotection with
TFA, and a final amidation reaction (cyclization). Finally, the pyrrolidone interme-
diate was (1) silylated to decrease water solubility, (2) reduced with borane-dimethyl
sulfide, and finally, (3) O-deprotected with tetra-n-butylammonium fluoride (TBAF)
to afford compound 54.
By intermolecular cyclization reactions between 2-cyanoacetamide derivative
56 and chloroacetonitrile, in the presence of triethylamine (TEA), under reflux in
1,4-dioxane for 5 h, Debbabi et al. [55] synthesized a series of N-ethyl-N-methyl
benzenesulfonamides. Among these, derivative 57 (Fig. 14), characterized by a
5-amino-3-cyano-2-oxopyrrolidine core, showed antiproliferative activity against
MCF-7 cells in the micromolar range (IC50 = 62.53 µM) and absence of cytotoxicity
against normal fibroblasts of baby hamster kidney cell line (BHK). By docking stud-
ies, the activity of the compound was attributed to its binding to the dihydrofolate
reductase (DHFR) (PDB ID: 4DFR).
3.5 Pyrrolidine‑2,5‑Diones Obtained by Cyclizations
Despite many years of research, it is still not clear how anticonvulsant drugs coun-
teract seizures, but it is known that many of them interact with voltage-gated sodium
channels (VGSCs) and voltage-gated calcium channels (VDCCs) in the central
nervous system (CNS). In agreement with previous studies in which pyrrolidine-
2,5-dione emerged as valuable scaffold in the treatment of epilepsy [56], in 2017,
Rybka et al. [57] synthesized a library of 1,3-disubstituted pyrrolidine-2,5-diones
59a–p (Fig. 15), obtained via cyclocondensations of dicarboxylic acids 58 with the
properly substituted 1-(2-aminoethyl)- and 1-(3-aminopropyl)-4-arylpiperazines,
at 180 °C for 1.5 h. Derivatives 59a–p were administered intraperitoneally to mice
and screened for their anticonvulsant activity by maximal electroshock (MES) and
subcutaneous pentylenetetrazole (scPTZ) seizure tests in mice. Compounds 59j,n
showed good activity in both tests (MES E D50: 88.2 mg kg−1 and 101.5 mg kg−1,
respectively; scPTZ ED50: 65.7 mg kg and 59.7 mg kg−1, respectively), indicat-
−1
ing that they are able to prevent various kinds of seizures by blocking the sodium
channel with higher affinity than phenytoin. SAR analysis revealed that the anticon-
vulsant activity is affected strongly by substituents at position 3 of the pyrrolidine-
2,5-dione scaffold. By scPTZ test, 3-benzhydryl 59a–d and 3-isopropyl 59e–h deriv-
atives showed the most favorable protection in the scPTZ test, whilst 3-methyl 59i–l
and unsubstituted 59m–p derivatives were more active in the MES test. Derivatives
13
59a: R1 = -CH-diphenyl; R2 = 3-CF3; n = 2

1
COOH R O 59b: R1 = -CH-diphenyl; R2 = 3,4-diCl; n = 2
1
R H2N
(CH 2 ) n N N
N 59c: R1 = -CH-diphenyl; R2 = 3-CF3; n = 3
COOH (CH 2 ) n N N
2 O
R
R
2 59d: R1 = -CH-diphenyl; R2 = 3,4-diCl; n = 3
58 a 59a-p 59e: R1 = -CH-diCH3; R2 = 3-CF3; n = 2
59f: R1 = -CH-diCH3; R2 = 3,4-diCl; n = 2
59g: R1 = -CH-diCH3; R2 = 3-CF3; n = 3
59h: R1 = -CH-diCH3; R2 = 3,4-diCl; n = 3
59i: R1 = CH3; R2 = 3-CF3; n = 2
59j: R1 = CH3; R2 = 3,4-diCl; n = 2
59k: R1 = CH3; R2 = 3-CF3; n = 3
59l: R1 = CH3; R2 = 3,4-diCl; n = 3
59m: R1 = H; R2 = 3-CF3; n = 2
59n: R1 = H; R2 = 3,4-diCl; n = 2
59o: R1 = H; R2 = 3-CF3; n = 3
59p: R1 = H; R2 = 3,4-diCl; n = 3
62a: R = -(CH2)-; X/R1 = O/-

O 62b: R = -(CH2)2-; X/R1 = O/-
S
62c: R = -(CH2)2-; X/R1 = N/4-F-Ph
N
1 1 62d: R = -(CH2)2-; X/R1 = N/3-CF3-Ph
X
R R N X R
N O
62e: R = -(CH2)2-; X/R1 = N/3-Cl-Ph
H2N R
62f: R = -(CH2)2-; X/R1 = N/4-Cl-Ph
a 62a-g
62g: R = -(CH2)2-; X/R1 = N/3,4-diCl-Ph
COOH
S
H2N O
COOH
60 OH
a
O 63a: X/R1 = O/-
1 O
S HN X R
S 63b: X/R1 = N/4-F-Ph
N
N 63c: X/R1 = N/3-CF3-Ph
COOH 1
N X R 63d: X/R1 = N/2-Cl-Ph
O O
O
61 63e: X/R1 = N/3-Cl-Ph
63a-h 63f: X/R1 = N/4-Cl-Ph
63g: X/R1 = N/2,3-diCl-Ph
63h: X/R1 = N/3,4-diCl-Ph
Fig. 15 General synthetic routes to pyrrolidine-2,5-diones 59a–p, 62a–g, and 63a–h. Reagents and con-
ditions: a 180 °C, 1.5 h., yields: 60–82% (59a–p), 70% (61), 54–86 (62a–g)
with a phenylpiperazine moiety bearing a 3-trifluoromethyl group were most active

in the MES test, whilst 3,4-dichlorophenylpiperazines were active in both the MES
and scPTZ tests. Finally, the increase in length of the alkyl chain resulted in deriva-
tives that displayed quick onset and long-lasting anticonvulsant activity. A similar
class of anticonvulsant and antinociceptive agents was also synthesized recently by
Góra et al. [58], who designed hybrid derivatives of pyrrolidine-2,5-dione with the
thiophene ring, compounds 62a–g and 63a–h (Fig. 15). While compounds 62a–g
were obtained by the reaction of 2-(3-methylthiophen-2-yl)succinic acid 60 with
aminoalkylmorpholine or 1-(3-aminopropyl)-4-phenylpiperazine in one step, deriva-
tives 63a–h were obtained in two steps by the reaction of 2-(3-methylthiophen-2-yl)
succinic acid 60 with aminoacetic acid to give intermediates 61, which, in turn,
underwent the coupling reaction. The best anticonvulsant activity was observed with
compound 62b, which displayed an ED50 value of 62.14 mg kg−1 in the MES test,
compared with references ethosuximide (ED50 > 500 mg kg−1) and valproic acid
(VPA) (ED50 252.7 mg kg−1), and 75.59 mg kg−1 in the psychomotor seizure (6 Hz)
test, compared with references ethosuximide ( ED50 221.7 mg kg−1) and VPA (ED50
13
130.6 mg kg−1). In addition, four of the tested compounds (62a,b,d,g) revealed

peripheral analgesic activity in the writhing test. In particular, compound 62g was
active at a dose of 30 mg/kg, similar to aspirin at the same dose. Compounds 62d
and 62g also showed central analgesic activity in the hot-plate test at a dose of
30 mg kg−1. SAR analysis revealed that the acetamide moiety can extend anticon-
vulsant activity in both the MES and scPTZ tests.
Later, Obniska et al. [59] expanded the same class of pyrrolidine-2,5-diones by
substituting the thiophene ring with an unsubstituted phenyl moiety. The new com-
pounds 66a–m (Fig. 16) were obtained by the condensation of 2-methyl-2-phenyl
succinic acid 64 with 3-aminopropanoic acid to give the intermediate derivatives
O N O
O
H2N O
HN N
OH O N
N R O
OH
OH
a
OH N
O O
R
64 65
66a-m
66a: R = H
66b: R = 2-F
66c: R = 4-F
66d: R = 2-Cl
66e:R = 3-Cl
66f:R = 4-Cl
66g: R = 2,3-diCl
66h:R = 3,4-diCl
66i:R = 3-CF3
66j:R = 2-CH3
66k:R = 3-CH3
66l:R = 2-OCH3
66m:R = 3-OCH3
O H2N O R Series I
R OH
OH 1
O 69a: R,R1 = -Ph; R2 = 2-F
R
R
1
OH a N O 69b: R,R1 = -Ph; R2 = 4-F
O O 69c: R,R1 = -Ph; R2 = 3-CF3
OH
67 68 69d: R,R1 = -Ph; R2 = 2-Cl
69e: R,R1 = -Ph; R2 = 3-Cl
69f: R,R1 = -Ph; R2 = 4-Cl
69g: R,R1 = -Ph; R2 = 2,3-diCl
HN N
R2 69h: R,R1 = -Ph; R2 = 3,4-diCl
O Series II
69i: R = -C2H5; R1 = -CH3; R2 = 2-F
N R2
1 69j: R = -C2H5; R1 = -CH3; R2 = 4-F
R N N
69k: R = -C2H5; R1 = -CH3; R2 = 3-CF3
O O
R
69l: R = -C2H5; R1 = -CH3; R2 = 2-Cl
69a-p 69m: R = -C2H5; R1 = -CH3; R2 = 3-Cl
69n: R = -C2H5; R1 = -CH3; R2 = 4-Cl
69o: R = -C2H5; R1 = -CH3; R2 = 2,3-diCl
69p: R = -C2H5; R1 = -CH3; R2 = 3,4-diCl
Fig. 16 General synthetic routes to pyrrolidine-2,5-diones 66a–m and 69a–p. Reagents and conditions: a
180 °C, 1 h, yields: 68% (65), 70% (68, R, R 1 = phenyl), 36% (68, R = methyl, R1 = ethyl)
13
65, at 180 °C for 1 h. This structural change did not improve the pharmacologi-
cal activity compared with the previously mentioned compound 62b. However, the
activity of the most promising derivatives 66b,c,h was better than the reference
VPA and ethosuximide, both in the MES test with E D50 values of 78.3, 83.51 and
97.67 mg kg−1, respectively, and in the scPTZ test, in which only compound 66b
was active ( ED50 114.15 mg kg−1). Finally, in 2021, Góra et al. [60] studied the anti-
convulsant properties of two new series of pyrrolidine-2,5-dione-acetamides 69a–p
(Fig. 16), exhibiting a benzhydryl or sec-butyl group in position 3 of the pyrrolidine
ring. The compounds were synthesized via intermediates 68, which were obtained
by the reaction of succinic acid derivatives 67 with aminoacetic acid at 180 °C for
1 h. Among the tested compounds, derivative 69k showed the best E D50 values of
80.38 mg kg−1 in the MES and 108.80 mg kg−1 in the 6 Hz tests, emerging as more
effective than VPA. Summarizing the SAR analysis of this class of compounds, the
activity appeared to be influenced by the substituent at position 3 of the pyrrolidine-
2,5-dione ring, as well as the type of phenylpiperazine attached to the acetamide
fragment. In particular, the non-aromatic substituent (sec-butyl) in position 3 of the
pyrrolidine-2,5-dione ring and the 3-trifluoromethylphenylpiperazine fragment posi-
tively affect the anticonvulsant activity as for compound 69k. Slightly less active
than compound 69k in the 6 Hz test was its 2-chlorophenylpiperazine analogue
69l with a 1.2 fold higher ED50 value. On the other hand, the introduction of the
benzhydryl group in position 3 of the pyrrolidine-2,5-dione ring and a 4-chloro-
or 2,3-dichlorophenylpiperazine fragment (compounds 69f and 69g) increased the
activity in the scPTZ test (data not shown).
O
O O 71a: R = 4-C2H5-phenyl
Cl
OH 71b: R = 2,4,6-triCH3-phenyl
R-NH2
O
NH
a N R 71c: R = naphthalen-1-yl
R
71d: R = 4-CF3-quinolin-5-yl
O O O
71e: R = 4-benzoic acid
70 71a-g 71f: R = 3-benzoic acid
71g: R = 4-CN-phenyl
O O X
R
O O
N S NH2
O
+ X
b
O
O
N S NH2
O R
O
72 73
74a-e O
74a: X = N; R = -H
74b: X = -CH; R = -H
74c: X = -CH; R = -OCH3
74d: X = -CH; R = -Cl
74e: X = -CH; R = -CH3
Fig. 17 General synthesis of pyrrolidine-2,5-diones 71a–g and 74a–e. Reagents and conditions: a SOCl2,
under reflux, 5–8 h, yields: 83–96%; b OtBU-l-threonine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),
chloroform (CHCl3), r.t., yields: 51.3–63.2%
13
Pyrrolidine-2,5-dione is a versatile scaffold, as demonstrated by Oktay et al.

[61] who prepared a series of 3-chloro-1-aryl pyrrolidine-2,5-diones evaluated
for their inhibitory activity on the human physiologically relevant carbonic
anhydrase (CA) isoenzymes hCA I and hCA II. Both isoenzymes are involved
in several diseases, such as retinal and cerebral edema, glaucoma, and epi-
lepsy. Therefore, their inhibition could be useful to counteract these issues.
The synthetic route to obtain the 3-chloro-N-aryl pyrrolidine-2,5-dione deriva-
tives 71a–g (Fig. 17) started with the reaction between maleic anhydride and
aromatic amines, with consequent opening of the ring to yield (Z)-4-oxo-4-
(arylamino)but-2-enoic acid 70, which, in turn afforded compounds 71a–g by
reaction with thionyl chloride (SOCl2) under reflux. All 3-chloro-1-aryl pyrro-
lidine-2,5-diones, except compound 71e, were able to inhibit hCA I (Ki in the
range of 23.27–36.83 nM) and hCA II (Ki in the range of 10.64 and 23.34 nM)
with higher or comparable activity than the reference hCA inhibitor acetazola-
mide (AZA) (hCA I: Ki = 34.70 nM; hCA II: Ki = 31.93 nM). The most active
compounds were derivatives 71d (hCA I: Ki = 23.27 nM) and 71c (hCA II:
Ki = 10.64 nM), both decorated at the pyrrolidine-2,5-dione nitrogen atom with
bicyclic scaffolds, such as quinoline and naphthalene.
As reported in the literature, cyclic imides, such as pyrrolidine-2,5-diones,
possess interesting pharmacological properties due to the ability of the imide
group to facilitate the crossing of biological membranes. Recently, Jan et al.
[62] synthesized a series of pyrrolidine-2,5-dione derivatives 74a–e (Fig. 17) as
multitarget anti-inflammatory agents by applying a synthetic strategy based on
Michael additions of ketones 73 to N-substituted maleimide 72, at room tem-
perature in the presence of OtBU-l-threonine and 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU), using a self-assembled three-component system as organocatalyst.
Biological activity was determined by in vitro assays, such as cyclooxygenase-1
(COX-1), cyclooxygenase-2 (COX-2), and 5-lipoxygenase (5-LOX), albu-
min denaturation and anti-protease assays, and in vivo in mice. In the in vitro
assays, all compounds showed marked COX-2 inhibition compared with the ref-
erence diclofenac (IC50 = 10.05 µM) with IC50 values in the range of 0.98 and
8.94 µM. The most potent compound was 74e, which showed selectivity for
COX-2 over COX-1, with a selectivity index value (SI) [SI = IC50 (COX-1)/IC50
(COX-2)] of 31.5. In contrast, the SI values of compounds 74a–d were 4.88,
11.5, 18.7 and 10.9, respectively. Furthermore, aryl ketone derivatives 74a and
74e showed excellent inhibition of human 5-LOX with IC50 values of 0.81 and
0.86 µM, respectively, which were slightly higher than the standard drug zileu-
ton (IC50 = 0.63 µM). Finally, compound 74a also induced albumin denaturation
(IC50 = 5.36) and protease inhibition (IC50 = 13.39 µM). The in vivo test with
the most promising compounds 74a and 74e revealed anti-inflammatory activ-
ity, which was ascertained with various mediators like histamine, bradykinin,
prostaglandin and leukotriene. In addition, the same compounds showed safety
in an acute toxicity study, in which the lethal dose (LD50) of both compounds
in the experimental mice was approximately 1000 mg/kg. The SAR analysis
revealed that the para-substituent on the phenyl ketone influences the biologi-
cal activity. However, the replacement of the aryl ketone at position 3 of the
13
NHCOCH 2 Cl
CN
CN
NHR 78: R = -CO-phenyl
CN 79: R = -SO2-4-CH3-phenyl
N N
Br
a O O
80: R = 77-CO-CO-77
75 HN HN
81: R = -CO-CH(CH3)-phenyl-4-CH2CH(CH3)2
CN
CN
N
H Br Br
76 77: R = H
CN CN
CN
H NH NH
N
O Cl N
N N
N O O
O O N
N CH3
HN HN
O
Br Br Br
83 82 84
Fig. 18 General synthetic scheme to pyrrolizine carboxamide derivatives 77–84. Reagents and condi-
tions: a K2CO3, acetone, under reflux, 24 h, yields: 68% (77)
pyrrolidine-2,5-dione nucleus with oxocycloalkyl/oxoalkyl groups (structure not

shown) was detrimental to the anti-inflammatory properties.
3.6 Pyrrolizines Obtained by Cyclizations
In 2016, Gouda et al. [63] designed and synthetized a new series of pyrrolizine
carboxamides 77–84 (Fig. 18) as dual cyclooxygenase (COX) and 5-LOX inhibi-
tors with safer gastric profile. Pyrrolizine 77 was synthesized via the intramo-
lecular cyclization of an intermediate obtained by reacting N-(4-bromophenyl)-
2-chloroacetamide 75 with 2-(pyrrolidin-2-ylidene)malononitrile 76 under reflux
in acetone for 24 h, and was used as starting material for the synthesis of deriva-
tives 78–84. All pyrrolizines were assayed for anti-inflammatory activity and
showed IC50 values of 2.45–5.69 µM and 0.85–3.44 µM for COX-1 and COX-2,
respectively. Furthermore, compound 77 exhibited higher anti-inflammatory and
analgesic activities compared with ibuprofen. Upon N H2-acylation, its analogues,
such as the 2-chloroacetyl (82) and the benzoyl derivative (78) were obtained.
Introduction of the 4-tolylsulfonyl moiety into compound 79 further improved
both biological activities. Conversely, the hybrid compound 81, composed of the
pyrrolizine precursor 77 and ibuprofen, did not show antiinflammatory activity
and only a weak analgesic effect. 4-Methylpiperazine derivative 84, the diazepine
derivative 83, and the dimer 80 showed lower anti-inflammatory and analgesic
activities. Nevertheless, compounds 78 and 81 revealed better safety profiles than
ibuprofen in acute ulcerogenicity and histopathological studies. Docking studies
into COX-1 (PDB ID: 1EQG), COX-2 (PDB ID: 1CX2) and 5-LOX (PDB ID:
13
3O8Y, 3V99) showed that compound 79 fits well into the active sites of COX-1
and COX-2, whereas compound 79 exhibits the highest binding affinity for
5-LOX.
4 Pyrrolidine Derivatives from Commercial Building Blocks
4.1 Pyrrolidines from Proline
The interest in the pyrrolidine nucleus as skeleton of molecules with biological

potential is endorsed by its similarity to the non-essential l-proline amino acid,
which is used to obtain molecules with a specific stereochemistry. In 2017, Pan-
nala et al. [64] developed a metal- and catalyst-free three-component decarboxyla-
tive coupling reaction of proline, aldehydes and 4-hydroxycarbazole to access pyr-
rolidinyl-carbazole derivatives 85a–p (Fig. 19) with antiproliferative and antioxidant
activities. The antiproliferative activity was evaluated by in vitro assays on three dif-
ferent cancer cell lines (MCF-7, MDA-MB-231, and lung cancer cells A549) and
the results were compared with those obtained with the positive control doxorubicin.
Overall, compounds 85b,c,k,p showed the best cytotoxic activity against MCF-7
cells (IC50 8.3–16.4 µM), whereas compound 85p was also able to reduce the growth
85a: R = phenyl
85b: R = 4-F-phenyl
O
85c: R = 4-Cl-phenyl
85d: R = 4-Br-phenyl
R NH N
85e: R = 4-CN-phenyl
OH 85f: R = 4-CF3-phenyl NC
85g: R = 3-OCH3-phenyl
N 86a-d
85h: R = 3-OCH2CH3,4-OH-phenyl
R N 85i: R = 3,4-diOCH3-phenyl 86a: R = piperidin-1-yl
H
85j: R = 4-benzyloxy,3-OCH3-phenyl 86b: R = 4-C2H5-piperazin-1-yl
85a-p
85k: R = 2,4,6-triF-phenyl 86c: R = 4-benzyl-piperazin-1-yl
85l: R = 2,4,6-triOCH3-phenyl 86d: R= 4-(4-OCH3-benzyl)-piperazin-1-yl
85m: R = pyridin-2-yl
85n: R = pyridin-3-yl
85o: R = napht-1-yl
85p: R = -CH2-CH2-CH3
88a: R1 = phenyl; R2 = -CH2-phenyl; R3 = -NHSO2-phenyl

88b: R1 = phenyl; R2 = -CH2-phenyl; R3 = -NHSO2-(4-CH3)-phenyl
88c: R1 = phenyl; R2 = -CH2-phenyl; R3 = -NHSO2-(3NO2,4Cl)-phenyl
O O
O NH
88d: R1 = phenyl; R2 = -CH2-(4-Br)-phenyl; R3 = -NHSO2-phenyl
3
OH
O
R 88e: R1 = phenyl; R2 = -CH2-(4-Br)-phenyl; R3 = -NHSO2-(4-CH3)-phenyl
H3 C 2
O NH R
N
88f: R1 = phenyl; R2 = -CH2-(4-Br)-phenyl; R3 = -NHSO2-(3NO2,4Cl)-phenyl
O O
H3 C H3 C
CH3
88g: R1 = phenyl; R2 = -CH2-(4-Br)-phenyl; R3 = -NHSO2-(4-NO2)-phenyl
N
O O 88h: R1 = phenyl; R2 = -CH2-(4-Br)-phenyl; R3 = -NHSO2-(4-Cl)-phenyl
H3 C
CH3
O
88i: R1 = phenyl; R2 = -CH2-1H-indole-3-yl; R3 = -NHSO2-phenyl
88j: R1 = phenyl; R2 = -CH2-1H-indole-3-yl; R3 = -NHSO2-(3NO2,4Cl)-phenyl
1 88k: R1 = 4-CH3-phenyl; R2 = -CH2-phenyl; R3 = -NHSO2-phenyl
R
87 88l: R1 = 4-CH3-phenyl; R2 = -CH2-phenyl; R3 = -NHSO2-(4-CH3)-phenyl

88a-p 88m: R1 = 4-CH3-phenyl; R2 = -CH2-phenyl; R3 = -NHSO2-(3NO2,4Cl)-phenyl
88n: R1 = 4-CH3-phenyl; R2 = -CH2-(4-Br)-phenyl; R3 = -NHSO2-(3NO2,4Cl)-phenyl
88o: R1 = 4-Cl-phenyl; R2 = -CH2-(4-Br)-phenyl; R3 = -NHSO2-(3NO2,4Cl)-phenyl
88q: R1 = phenyl; R2 = -CH2-(4-OH)-phenyl; R3 = -NHSO2-(3NO2,4Cl)-phenyl
Fig. 19 Molecular structures of pyrrolidinyl-carbazole derivatives 85a–p, pyrrolidine-2-carbonitriles

86a–d, Mcl-1 inhibitor 87, and pyrrolidine-1-carboxylates 88a–p
13
of A549 with an IC50 value of 15.7 µM. Furthermore, compounds 85b,f,h,j,m

showed higher radical scavenging abilities than ascorbic acid (IC50 40.9 µM), with
the best results displayed by compound 85 h (IC50 = 27 µM) due to the presence of
the hydroxy group on the phenyl ring. Docking analysis in the colchicine binding
site (PDB ID: 1SA0) indicated that a methoxy-benzyloxy moiety (85j) and a trifluo-
romethyl group (85f) on the phenyl ring participate in hydrophobic binding interac-
tions with several amino acid residues. Halogenated compounds 85b–d,k displayed
similar binding modes in the tubulin active site. Finally, a bulky naphthyl ring (85o)
reduced the binding affinity.
The use of proline for the synthesis of pyrrolidine derivatives was also a strategic
path for Nabil Aboul-Enein et al. [65], who synthesized a small class of antidia-
betic compounds. The (S)-1-[(cyclohexylmethyl)glycyl]pyrrolidine-2-carbonitriles
86a–d obtained (Fig. 19) were biologically evaluated for inhibitory activity toward
dipeptidyl peptidase-4 (DPP4), a serine exopeptidase belonging to the S9B protein
family, which cuts X-proline dipeptides from the N-terminus of polypeptides, such
as chemokines, neuropeptides, and peptide hormones. As DPP4 is known to inac-
tivate the incretin glucagon-like peptide-1 (GLP-1), the discovery of new DPP4
inhibitors is considered an indirect approach to increase GLP-1 levels and thus to
manage diabetes mellitus type 2. The starting point for the synthesis of derivatives
86a–d was (2S)-1-(chloroacetyl)pyrrolidine-2-carbonitrile, which could be obtained
from S-proline via a chloroacetylation followed by an amidation of its carboxylate
group and a final dehydration. The ability of compounds 86a–d to inhibit the DPP4
enzyme was studied in diabetic mice that received the compounds orally at a dose
of 100 mg/kg and the results were compared with those of the control group that
received vildagliptin at the same dose. Serum DPP4 inhibition was evaluated 3 h
after treatment. Among all, compounds 86b,c inhibited DPP4 activity better (153%
and 138%, respectively) than the control group (114%). In contrast, replacement
of the piperazine ring with piperidine (86a) or (4-methoxybenzyl)piperazine (86d)
reduced DPP4 inhibition to 85% and 105%, respectively. Docking studies confirmed
a good binding affinity of compounds 86b,c in the active site of DPP4 (PDB ID:
3W2T) in agreement with their anti-diabetic activity.
Starting from (2S,4R)-4-hydroxyproline, Wan et al. [66] designed and synthesized
a new series of pyrrolidine derivatives based on compound 87, which was described
as potent inhibitor of myeloid cell leukemia-1 (Mcl-1) protein (Ki = 8.4 µM)
(Fig. 19). Using a fluorescence polarization assay (FPA), these authors observed that
the most potent compounds 88a–p were characterized by phenyl groups at R1, aro-
matic or heteroaromatic rings at R 2, and different benzenesulfonamides at R3. Com-
pounds 88c and 88f showed the best binding affinities towards Mcl-1, with Ki values
of 0.94 and 0.53 µM, respectively, slightly higher than the positive control gossypol
(Ki = 0.39 µM) but very much lower than compound 87. The replacement of phenyl
or 4-bromophenyl at R 2 with 3-indolyl and p-hydroxyphenyl (88i,j,p) was unfavora-
ble for the biological activity. Finally, the replacement of the benzensulfonamide
moiety with a hydroxy group and the introduction of linear chains or substituted
phenyls instead of the 4-bromobenzyl group (structures not shown) resulted in the
loss of binding affinity toward the Mcl-1 protein (Ki > 10 µM). Moreover, compound
88f exhibited good antiproliferative activities against MDA-MB-231, PC-3 (prostate
13
O
O
NH
NH
O 1 O O
N R
N B
O
O 89a-e O 90
NH O
OH
89c: R = -F; R =
1
89a: R = -H; R = 1
B
O O
O
89b: R = -H; R1 = B 89d: R = -F; R1 = -COOH
O
89e: R = -F; R1 = -B(OH)2
91a: R = -H; R1 = -H
91b: R = -H; R1 = -NO2
N 91c: R = -H; R1 = -Cl
CONHR
91d: R = -ethyl; R1 = -H
NO 2
91e: R = -ethyl; R1 = -NO2
R
1 91f: R = -ethyl; R1 = -Cl
91g: R = -propyl; R1 = -H
91h: R = -propyl; R1 = -NO2
91a-k
91i: R = -phenyl; R1 = -H
91j: R = -phenyl; R1 = -NO2
91k: R = -phenyl; R1 = -Cl
Fig. 20 Molecular structures of pyroglutamic acid derivatives 89a–e and 90 and N-(2′-nitrophenyl)pyrro-
lidine-2-carboxamides 91a–k
cell cancer), and K562 (chronic myeloid leukaemia) cell lines with IC50 values of
13.6, 10.7, and 23.0 µM, respectively.
In 2020, Gerokonstantis et al. [67] synthesized compounds 89a–e and 90
(Fig. 20), which are potent inhibitors of autotaxin (ATX), a glycoprotein respon-
sible for the hydrolysis of lysophosphatidylcholine (LPC) into bioactive lipid
lysophosphatidic acid (LPA), whose upregulation is involved in pathological
inflammatory conditions. The main scaffolds of the new derivatives were the nat-
ural amino acid S-proline, the naturally derived S-pyroglutamic acid ((2S)-5-ox-
opyrrolidine-2-carboxylic acid), and their enantiomers. In both cases, the new
derivatives possess a benzyl type substituent at the pyrrolidine nitrogen, while the
carboxylic group at position 5 is coupled to side chain via an amide bond contain-
ing a benzyl-ether type substituent, which bears carboxylate, methyl ester, sul-
fonamide, boronic ester, imidazole, hydroxamate, tetrazole, triazol, pyridine and
boronic acid moieties in para-position (structures not shown). The in vitro assay
showed that the pyroglutamic acid derivatives, including hydroxamic acid 89a
(IC50 700 nM) and boronic acid derivatives 89b (IC50 50 nM), 89c (IC50 120 nM),
90 (IC50 180 nM), and 89e (IC50 35 nM) were the most active compounds against
13
the ATX enzyme, whereas only one out of six compounds—the carboxylic acid
derivative 89d—was the least active (IC50 800 nM).
Antimicrobial peptides (AMPs) are small peptides with a wide range of inhibi-
tory effects against bacteria and other pathogens. Pyrrhocoricin, apidaecin and
drosocin are some examples of proline-rich antibacterial peptide family members
able to bind the bacterial DnaK protein. In 2020, Odusami et al. [68] reported
the synthesis of novel N-(2′-nitrophenyl)pyrrolidine-2-carboxamides 91a–k
(Fig. 20) with the aim of searching for amino acid analogues with antibacterial
properties capable of mimicking antimicrobial peptides. The main structural fea-
tures of compounds 91a–k were the simultaneous presence of (1) a hydropho-
bic group provided by the phenyl ring, and (2) a cationic charge given by the
amino group after its protonation, (3) different N′-substituents introduced to study
the effect of conformational flexibility on the antimicrobial activity. The activ-
ity was evaluated both against Gram-positive [Bacillus subtilis (ATCC 19659),
Enterococcus faecalis (ATCC 14506), Mycobacterium smegmatis (ATCC 14468),
Staphylococcus epidermidis (ATCC 12228) and Staphylococcus aureus (ATCC
25923)] and Gram-negative [Enterobacter cloacae (ATCC 13047), Escherichia
coli (ATCC 25922), Proteus vulgaris (ATCC 33420), Klebsiella oxytoca (ATCC
8724) and Proteus mirabilis (ATCC 7002)] bacterial strains, and the results were
compared with those of reference compounds streptomycin and nalidixic acid.
As assessed by the minimum inhibitory concentration (MIC) values, all the car-
boxamides 91a–k were more potent against S. aureus and E. cloacae than the
standard streptomycin (256 µg/ml and > 512 µg/ml, respectively), with com-
pounds 91b (15.6 µg/ml), 91c and 91k (62.5 µg/ml for both) being most potent
against S. aureus, and compounds 91c (62.5 µg/ml) and 91j (31.3 µg/ml) being
most potent against E. cloacae. Furthermore, the MIC values of compounds
91a–k were lower than those found with nalidixic acid (≥ 500 µg/ml) against E.
faecalis, M. smegmatis, E. coli, and P. vulgaris (MIC ≤ 250 µg/ml). Conversely,
for other strains, the MICs of the carboxamides were higher than the ones of the
reference compounds. SAR investigation showed that, in consideration of the N′-
substituents, antibacterial activity increased in the order: N′-Et (91d–f) < N′-H
(91a–c) < N′-Pr (91g,h) < N′-Ph (91i–k), whereas with the 4′-phenyl substitu-
ents, the activity increased in the order: 4′-PhH (91a,d,g,i) < 4′-PhCl (91c and
91f) < 4′-PhNO2 (91b,e,h) except for the N′-phenyl carboxamides 91i–91k, where
91k (4′-Cl) > 91j (4′-NO2) > 91i (4′-H).
4.2 Derivatives from Other Preformed Pyrrolidine Scaffolds
Given the interest of the scientific community in the pyrrolidine nucleus, many
chemical industries have synthesized variously substituted pyrrolidines as build-
ing blocks for new drugs. Among these, pyrrolidine, pyrrolidin-2-one, pyrrolidine-
2,5-dione, and prolinol scaffolds are very useful preformed rings for the synthesis of
new bioactive compounds.
Beta-secretase 1 (BACE1) is the enzyme responsible for the proteolytic pro-
cessing of the amyloid precursor protein (APP), which leads to the generation of
13
93-94a: R = -H
93-94b: R = -2,3-diOCH3
F 93-94c: R = -3,4,5-triOCH3
Boc 93-94d: R = -4-OH
HN
N 93-94e: R = -2-OH
N R R
93-94f: R = -2,4-diOH
N N N
93-94g: R = -3-CH3
H 93-94h: R = -4-CH3
O
X
N N 93-94i: R = -4-NO2
H
93-94j: R = -4-CF3
93a-o 94a-o
92a: X = CO 93-94k: R = -4-OCF3
92b: X = CH2 93-94l: R = -4-Cl
93-94m: R = -4-F
93-94n: R = -4-Br
93-94o: R = -2,4-diF
N
S
O S
O N
95a-d
CN CN
H3C
N
N S
H2 N N
F
O O O
CN
95a 95b 95c 95d
O O
- -
R O + R O +
N N
N O P O N O P O
O O
96 97a,b
-
- O +
O + N
N O P O
O P O
O O
O
N O N
R R
98 99a,b
( )11 ( )7
96, 97b, 99b R = 97a, 98, 99a R = ( )14
Fig. 21 Molecular structures of pyrrolidine derivatives 92a,b, Schiff bases 93a–o and their reduced
counterparts 94a-o, benzenesulfonylpyrrolidines 95a–d, and pyrrolidine-based 3-deoxysphingomyelins
96, 97a,b, 98, 99a,b
amyloid-β (Aβ) peptides. The aggregation of Aβ in the brain of patients is respon-

sible for the onset of Alzheimer’s disease (AD). Therefore, the development of
treatments towards BACE1 could be a good strategy to fight this devastating neuro-
degenerative disease. In 2016, De Tran et al. [69] synthesized a library of (3S,4S)-
4-aminopyrrolidine-3-ol derivatives 92a,b (Fig. 21) as potential anti-AD agents with
a target selectivity toward BACE1. An in vitro inhibition assay of BACE1 showed
that compound 92a was the most active, with an IC50 value of 0.05 µM. However,
when replacing the carbonyl group of 92a with a methylene unit as in the compound
13
92b, the inhibition of BACE1 was approximately two times lower (IC50 = 0.12 µM).
Unexpectedly, the opposite was observed in the cell-based assay ,where compound
92b was more effective than compound 92a, most probably due to a difference in
cell permeability (IC50 values 1.7 versus 40 µM, respectively). Boc-deprotected
derivatives having the pyrrolidine nitrogen atom unsubstituted or substituted with
alkyl, acyl or sulfonyl groups were less active (structures not shown). A molecular
docking study confirmed interactions with the BACE1 active site.
Novel compounds for the treatment of Alzheimer’s disease were also investi-
gated by Choubey et al. [70], who synthesized multitargeted molecular hybrids of
N-benzyl pyrrolidine derivatives, like imines 93a–o and their reduced counterpart
94a–o (Fig. 21). All compounds were studied for their inhibitory activity towards
of cholinesterases (AChE and BChE) and BACE-1 in order to evaluate their mul-
titarget profile. Overall, derivatives 94a–o inhibited both cholinesterases as well as
BACE-1 with greater potency than Schiff bases 93a–o. SAR studies revealed that
electron donating groups (EDGs) at the terminal phenyl ring elicit less potency
with respect to acetylcholinesterase (AChE) inhibition than EWGs. Thus, deriv-
atives bearing an EWG exhibited IC50 values of less than 1 µM, as observed for
compounds 94k (0.058 µM) and 94o (0.069 µM), which were found to be almost
as active as the reference donepezil (0.042 µM). A similar trend was observed for
butyrylcholinesterase (BChE) inhibition. All the Schiff bases, with the exception of
the compound 93j containing a 4-CF3 group, showed a moderate inhibitory effect,
whereas, among compounds 94a–o, substitution with EWGs gave excellent BACE-1
inhibitors. Only in a few cases, such as for compounds 93j,k,o, and 94c,d,f,i, was
the intended multitarget profile not observed.
Anticancer activity of pyrrolidine derivatives was studied by Bashandy et al.
[71], who synthetized a new series of compounds characterized by the presence of
a benzenesulfonylpyrrolidine moiety bearing a variously substituted 1,3-thiazole
ring in position 4 of the phenyl ring. All compounds were tested for their in vitro
antiproliferative activity against MCF-7 cells. Only a few compounds, namely
95a (benzo[4,5]imidazo[1,2-a]pyridine), 95b ((4-oxo-3-phenyl-1,3-thiazolidin-
2-ylidene)ethanenitrile), 95c ((4-fluorophenyl)-acrylonitrile), and 95d (benzo[f]
chromen-3-one) (Fig. 21), exhibited a slight improved activity compared to doxo-
rubicin (IC50 = 68.6 μM), with IC50 values of 49.11, 48.01, 49.78 and 49.27 μM,
respectively. In addition, molecular docking studies confirmed their ability to bind
the DHFR active site (PDB ID: 4DFR).
In 2019, Hassan et al. [72] synthesized a series of pyrrolidine-based 3-deox-
ysphingomyelin analogues carrying various acyl chains (palmitoyl, palmitoleoyl,
oleoyl, erucoyl, linoleoyl, and α-linolenoyl) at the pyrrolidine nitrogen atom and
evaluated the compounds as antitumor agents against a panel of cancer cell lines
including breast, non-small-cell lung, liver, and skin cancers. The most promising
compounds were characterized by erucoyl (96, 97b, 99b) or palmitoyl (97a, 98,
99a) chains (Fig. 21). The best results were obtained for MCF-7 cells, which were
more sensitive to the treatment with compounds 96, 97b, and 99b, eliciting G I50
values at the micromolar level (15.7–24.8 µM). This effect was also confirmed in
their study on the inhibition of Akt phosphorylation. In fact, compounds 97a, 98,
and 99a bearing identical acyl chains but exhibiting different stereochemistry, were
13
H H Me OH
N OH N
O OH
O
O N Me
O -
O O 2
O S N N R
4 O -
N
+ O
O N 4
O N
5 O + 1
H 5 N R
N H
100a,b O
101a,b O CN
N
100a: 5-benzofuroxan 101a: 5-benzofuroxan 102a-c
100b: 4-benzofuroxan 101b: 4-benzofuroxan 102a: R1 = Cl; R2 = CH3
102b: R1 = Cl; R2 = H
102c: R1 = F; R2 = CH3
CHO N N
N Cl
N
O N O O
O R
N N N N
N N N N N N N N
N N N N N N N N N N N N
O O O O
104a,b
103 104a: R = 2,6-diCl-phenyl 105 106
104b: R = H
N
O
R
N
109a: R = -C6H11; 109b: R = -OC6H11
107 O
N
O
N
110
O
108 N
111
Fig. 22 Molecular structures of hybrid benzofuroxan-based pyrrolidine hydroxamates 100a,b and

101a,b, pyrrolidine benzonitriles 102a–c, hybrid pyrrolidine derivatives 103, 104a,b, 105, 106, and pyr-
rolidine amides 107, 108, 109a,b, 110, 111
equally active, with G

I50 values of 21.1, 26.4, and 32.5 µM, respectively, confirming
the stereochemistry as irrelevant for activity. Molecular docking studies established
that the interaction with Akt (PDB ID: 3O96) may be the predominant mechanism
of action of the 3-deoxysphingomyelin analogues tested.
In 2018, Zhang et al. [73] synthesized new hybrid benzofuroxan-based pyrro-
lidine hydroxamates carrying on the pyrrolidine nitrogen atom two different sub-
stituents: a 3-phenoxybenzenesulfonyl (100a,b) or a (3,4-dimethoxyphenyl)prop-2-
enoly (101a,b) moiety (Fig. 22). All compounds elicited antiproliferative activity
against several tumor cell lines, including A549, ES-2 (ovarian clear carcinoma
cell), HeLa (cervix carcinoma), K562, MCF-7, and MDA-MB-231 (IC50 values
of 3.56–25.64 µM), as well as NO-releasing capability (25.51–34.43 μM/l). The
13
anticancer activity was ascribed to the inhibition of matrix metalloproteinases 2 and

9 (MMP-2 and MMP-9), as indicated by the reduced proteolytic activity after their
isolation from treated cells. Among the phenoxybenzenesulfonamides, the 5-ben-
zofuroxan 100a showed higher MMP-2 and -9 inhibitory activity (IC50 values of
102 and 162 nM, respectively) than the 4-benzofuroxan analog 100b (IC50 values of
182 and 242 nM, respectively), whereas the (3,4-dimethoxyphenyl)prop-2-enamide-
5-benzofuroxan (101a) and the 4-benzofuroxan (101b) were less effective (IC50 val-
ues of 345–524 nM). Docking studies carried out in the active site of MMP-2 (PDB
ID: 1HOV) with compound 101a highlighted the ability of the hydroxamate group
to chelate the catalytic zinc ion and both arylsulfonyl and benzofuroxan groups to
create hydrogen bonds with amino acid residues.
Although polyhydroxylated pyrrolidines are important scaffolds of molecules for
the treatment of metabolic diseases [52, 54], hydroxy groups could be subject to
oxidation and conjugation reactions that would make the molecules metabolically
unstable. In this regards, with the aim of modifying the pharmacokinetic profile,
Asano et al. [74] synthesized 4-(pyrrolidin-1-yl)benzonitrile derivatives 102a–c
(Fig. 22) as selective androgen receptor modulators (SARMs) by optimizing the
structure of previously reported 1-(4-cyano-1-naphthyl)-2,3-disubstituted pyrroli-
dine derivatives [75]. In particular, they introduced a methyl group at C-3 of the
pyrrolidine ring that, due to its steric hindrance, should prevent metabolic instabil-
ity, conferring better pharmacokinetic (PK) profiles than the parent 3-hydroxy com-
pounds. Compounds 102b,c had low cLogP values (< 3), showed better metabolic
stability, and retained potent androgen receptor (AR) agonistic activity. In addi-
tion, 2-chlorobenzonitrile 102b and 3-fluoro-2-methylbenzonitrile 102c derivatives
showed good bioavailability values (53.4 and 46.3%, respectively) and strong ana-
bolic activity in levator ani muscle (> 300%), which was dose-dependent as dem-
onstrated by in vivo studies on rats after oral administration. The X-ray co-crystal
structure of 102c bound to the AR LBD (PDB: 5T8J) highlighted a binding mode
almost identical to that of the previously studied (2S,3S)-2-methyl-3-hydroxylpyrro-
lidine-2-chloro-3-methylbenzonitrile [75].
In 2018, Kaur et al. [76] designed and synthesized a library of hybrid mol-
ecules by combining triazine-indole with morpholine/piperidine/pyrrolidine and
pyrazole/pyrimidine/oxindole moieties, as novel anti-inflammatory agents. All
compounds were tested for inhibition of COX-1 and COX-2 and the results were
compared with that of diclofenac, a COXs non-selective inhibitor, and celecoxib,
a potent and selective COX-2 inhibitor. Of all compounds tested, pyrrolidine
derivatives 103,104a,b,105,106 (Fig. 22) inhibited COX-2 with IC50 values in
the range of 1–8 µM. The most potent was the compound 106, characterized by a
1-(3-chlorophenyl)-3-methyl-2-pyrazolin-5-one moiety, with an I C50 value of 1 µM.
The SI ratio (IC50 COX-1/IC50 COX-2) revealed compound 106 as selective for
COX-2 (SI = 7), compared with compounds 106,104a,b, and 105, which were non-
selective. In addition, docking studies highlighted a well-docked pose in the COX-2
active site showing H-bond and hydrophobic interactions. Surprisingly, compound
106 also decreased formalin-induced analgesia by 69%.
N-Acylethanolamine acid amidase (NAAA) is a lysosomal hydrolase that cata-
lyzes the degradation of N-acylethanolamines into fatty acids and ethanolamine in
13
animal tissues. Its inhibition is a therapeutic tool in several pathophysiological con-

ditions, such as inflammation and immune disorders, as well as pain. In 2019, Zhou
et al. [77] synthesized a new series of pyrrolidine amide derivatives as antiinflamma-
tory agents and tested their selectivity towards NAAA and fatty acid amide hydro-
lase (FAAH), using rat NAAA (rNAAA) and rat FAAH (rFAAH) as animal model.
The starting idea was to modify the linker chain and the terminal phenyl group
of compounds 107 and 108 (Fig. 22), which were previously described as potent
NAAA inhibitors (IC50 = 12.8 and 2.1 µM, respectively). The most active com-
pounds were 109a, (IC50 = 0.5 µM) 109b (IC50 = 0.7 µM), 110 (IC50 = 0.48 µM), and
111 (IC50 = 1.5 µM) (Fig. 22) showing good NAAA inhibitory effects. Conversely,
no relevant activity towards FAAH was found. Studies on the chemical space on
compounds 107 and 108 indicated that the activity was lost upon substitution with
CH3, Cl, and F in position 2 of the terminal phenyl ring of parent 107. Instead, an
improvement of activity was observed upon introduction of the same substituents
at positions 3 or 4 ( IC50 3.7–9.6 µM), except for the 4-Cl derivative ( IC50 34.5 µM)
(structures not shown). The same modifications on the terminal phenyl ring of com-
pound 108 were detrimental for activity, most probably due to the conformationally
restricted chain. Isosteric replacements of the distal phenyl ring with aromatic moie-
ties such as 2-pyridyl, 3-pyridyl, 2-thienyl, and 3-thienyl, did not improve potency.
This is in contrast to the introduction of a cyclohexyl ring as in compounds 109a
O
5
R O
X N N 4
R
N
N N O
R 3 1
R R 6
R
112-113a,b R
2
X: S; 112a: R = 4-OCH3; 112b: R = 3-CF3 115a-o

X: O; 113a: R = 4-OCH3; 113b: R = 3-CF3 115a: R1,4,5 = Br; R2,3 = OH; R6= H
115b: R1 = H; R2,3 = OH; R4,5 = Br; R6= COOH
R
1
115c: R1 = H; R2,3 = OH; R4,5 = Br; R6= COOCH3
R2 115d: R1,4,5 = Br; R2,3 = OCH3; R6= H
R 115e: R1 = H; R2,3 = OCH3; R4,5 = Br; R6= COOH
HN 115f: R1 = H; R2,3 = OCH3; R4,5 = Br; R6= COOCH3
115g: R1,6 = H; R2,3 = OCH3; R4,5 = Br
O
N
115h: R1,5,6 = H; R2,3 = OCH3; R4 = Br
O
115i: R1,4,6 = H; R2,3 = OCH3; R5 = Br
115j: R1,2,4,5,6 = H; R3 = OCH3
114a-e
114a: R = OH; R1 = Cl; R2 = 5-Cl 115k: R1,4,5,6 = H; R2 = Br; R3 = OCH3
1143b: R = H; R1 = OCH3; R2 = 4-OH 115l: R1,6 = H; R2,3 = OH; R4,5 = Br
114c: R = H; R1 = H; R2 = 4-OCH3 115m: R1,5,6 = H; R2,3 = OH; R4 = Br
114d: R = OH; R1 = OCH3; R2 = H 115n: R1,4,6 = H; R2,3 = OH; R5 = Br
114e: R = OH; R1 = H; R2 = 5-OCH3 115o: R1,2,4,5,6 = H; R3 = OH
Fig. 23 Molecular structures of triazine-pyrrolidine-2-thiones 112a,b and pyrrolidin-2-ones 113a,b, pyr-

rolidin-2-ones 114a–e, and pyrrolidin-2-ones 115a–o
13
and 109b, highlighting that (hetero)aromatic rings are less tolerated in the hydro-
phobic pocket of NAAA than aliphatic rings. Regarding the carbon chain between
the pyrrolidine and the phenyl ring, the authors showed that flexible linkers led to
a progressive increase in potency, except for compound 111, which was well suited
to the hydrophobic pocket of NAAA. Molecular docking studies (PDB ID:6DY2)
suggested that 111 may inhibit NAAA via a reversible and competitive inhibition
mechanism.
As analogues of isoxazole compounds, in 2019 Lucescu et al. [78] synthesized
and tested triazine-pyrrolidine-2-thiones 112a,b (Fig. 23) on the human protein
farnesyltransferase (FTase). This protein catalyses the addition of a C15-farnesyl
lipid group to the cysteine residue located in the carboxy-terminal tetrapeptide motif
of a variety of important substrate proteins playing an important role in malignant
transformations including proliferation, apoptosis, angiogenesis, and metastasis.
Compound 112a showed good inhibitory activity toward the FTase enzyme with an
IC50 of 3.82 µM, which was approximately 11-fold lower than that of compound
112b (IC50 = 41.06 µM), highlighting the importance of the EWG on the phenyl
ring. Interestingly, these two derivatives showed much better activity compared with
the analogue in which pyrrolidine was replaced by the aromatic oxazole ring (struc-
ture not shown). In addition, the replacement of pyrrolidine-2-thione with pyrroli-
din-2-one 113a,b (Fig. 23) did not affect activity toward the FTase protein.
The pyrrolidine-2-one scaffold is a structural feature recurrent in antitumor
agents, as demonstrated by Kumar et al. [79] who synthesized the novel derivatives
114a–e (Fig. 23) by the reaction of substituted salicylaldehydes and 2-(2-oxopyr-
rolidin-1-yl)acetamide, leading to the respective Schiff base intermediates which
were, in turn, reduced with sodium borohydride. Among all, compounds 114d and
114e showed the highest growth inhibition percentage against CCRF-CEM (acute
lymphocytic leukemia) (114d = 73.15%) and NCI-H522 (non-small cell lung can-
cer) (114e = 41.8%) of the National Cancer Institute (NCI) panel. SAR analysis
revealed that EWGs at positions 3 or 5 of the phenyl ring were unfavorable for anti-
proliferative activity compared with electron-donating methoxy groups. Hydroxy
group introduction at position 2 (114d and 114e) led to an increase in antiprolif-
erative activity compared with compounds 114b and 114c. Docking analyses sug-
gested binding to the podophyllotoxin pocket of the protein gamma tubulin (PDB
ID: 1SA1) as a potential mechanism of action underlying the anticancer activity.
The versatility of the pyrrolidine-2-one scaffold was demonstrated by Rezai et al.
[80], who synthesized novel N-benzyl-2-pyrrolidone derivatives 115a–o (Fig. 23)
as antioxidants and inhibitors of AChE and BChE. AChE is an ubiquitous enzyme
of the serine hydrolases class responsible for hydrolyzing the neurotransmitter ace-
tylcholine, together with the homologous BChE. AD is the most common form
of dementia, characterized by loss of short-term memory, spatial disorientation,
progressive loss of cognitive function, decreased intellect, and some other minor
expressions. A common feature of the AD is the presence of AChE, which is usually
related to the Aβ plaques and neurofibrillary tangles in the patient’s brain. Recent
findings suggested that both Aβ and abnormally hyperphosphorylated tau protein
(P-tau) may influence the AChE expression with the development of a vicious cycle
of Aβ and P-tau dysregulation. In this context, AChE and BChE inhibitors can
13
2
R O
1 O
R
S
O
R
N N
O
N
O 117 OH
N
R
O
116a-m
116a: R = -4-CH3 ; R1 = -H; R2 = -H
116b: R = -4-CH3 ; R1 = -F; R2 = -H
N
116c: R = -4-CH3 ; R1 = -F; R2 = -F
OH
116d: R = -2-furyl in place of phenyl; R1 = -F; R2 = -F
116e: R = -2-Cl ; R1 = -H; R2 = -H 118: R = -decyl
116f: R = -H; R1 = -H; R2 = -H
116g: R = -4-F ; R1 = -H; R2 = -H
116h: R = -H ; R1 = -F; R2 = -H
116i: R = -thiophen-2-yl in place of phenyl ; R1 = -H; R2 = -H
116j: R = -2-furyl in place of phenyl ; R1 = -H; R2 = -H
116k: R = -2-furyl in place of phenyl ; R1 = -F; R2 = -H
116l: R = -3,4-diOCH3; R1 = -F; R2 = -F
116m: R = -3,4diCl ; R1 = -H; R2 = -H
O O
N N
HN HN
1
Ph
R N N 1 N
R N
COOEt
- + H
O Na NH
- + N COOEt
N P O Na N P
O O
Ph
1 1 9- 1 20 a , b 12 1 - 12 2 a, b
(R)-119a: R1 = -NH2 (R)-121a: R1 = -NH2
(S)-120a: R1 = -NH2 (S)-122a: R1 = -NH2
(R)-119b: R1 = -H (R)-121b: R1 = -H
(S)-120b: R1 = -H (S)-122b: R1 = -H
O
O O
N
HN N N
HN HN
H2N N N
H2N N N R N N
NH
N N
N
123 P(O)(OH) 2 P(O)(OH) 2
O
124 O P(O)(OH) 2
(HO) 2 (O)P
125a-b
125a: R = NH2
125b: R = -H
Fig. 24 Molecular structures of hybrid pyrrolidine-2,5-diones 116a–m, 2-(hydroxymethyl)pyrrolidines

117 and 118, pyrrolidine phosphonates 119,120a,b, and phosphoramidate prodrugs 121,122a,b
improve the cholinergic transmission, but with modest and temporary therapeutic
effects. All compounds synthesized by the authors were able to inhibit the AChE
enzyme at low nanomolar concentrations, with Ki values in the range of 2.60 and
13
16.36 nM. The most powerful AChE inhibitor was compound 115f with a Ki value
of 2.60 nM. Furthermore, all compounds inhibited BChE with Ki values in the range
of 13.10 and 54.47 nM. Measuring the DPPH radical scavenging activity, com-
pounds 115b,c,l–o showed interesting antioxidant activity with half maximal radical
scavenging concentrations (IC50 µg/ml) in the range of 4.71 and 53.30. The radical
scavenger activity is affected by the phenolic fraction and varies according to the
number and position of the hydroxy groups.
To escape the problem of drug resistance, Tilekar et al. [81] recently published
new pyrazoline-substituted pyrrolidine-2,5-dione hybrids 116a–m (Fig. 24), with
anticancer activity against MCF7, HT29, and K562 cancer cells. Compounds
116b,f,g, showed nanomolar activity against MCF7 with IC50 values in the range
of 0.42 and 0.78 µM. Compounds 116b and 116m showed also activity in the sub-
micromolar range against HT29 cells ( IC50 0.92 µM and 0.39 µM, respectively). The
cytotoxicity assay against K562 revealed that compounds 116a and 116g (IC50 24.74
and 31.56 µM, respectively) were more potent than the reference compound piogl-
itazone (IC50 40.3 µM). Further studies with compound 116b demonstrated its abil-
ity to reduce the cell population in the G2/M phase and increase the cell population
in the G0/G1 phase, as well as inhibition of the anti-apoptotic protein Bcl-2 in a
dose-dependent manner.
To obtain potent dual sphingosine kinase 1/2 (SphK1/SphK2) inhibitors, Li et al.
[82] synthesized a series of 2-(hydroxymethyl)pyrrolidines, based on SAR investi-
gation of potent and selective inhibitors previously reported in the literature (com-
pound 117, SphK1-selective inhibitor) (Fig. 24). These enzymes are involved in the
production of sphingosine 1-phosphate (S1P) from sphingosine and ATP, in a sig-
nalling pathway that is involved in cancer progression and immune cell chemiotaxis.
While the 2-(hydroxymethyl)pyrrolidine scaffold is essential for hydrogen bonding
with Asp178 in the binding pocket of SphK1, alteration of the aryl sulphonyl moi-
ety changes the selectivity for SphK1 and SphK2. In this regard, the authors intro-
duced various lipophilic substituents (i.e., alkyl, aryl, alkoxy, etc.) to the scaffold
of compound 117 in place of the aryl sulphonyl moiety to mimic the sphingosine
substrate of SphK1 and SphK2. The dodecyl analogue 118 proved to be the most
potent dual SphK1/SphK2 inhibitor (SphK1 Ki = 0.679 μM, SphK2 Ki = 0.951 μM)
compared with derivatives with a shorter alkyl tail. On the other hand, the diaryl
ether, alkoxy, alkenyl, alkynyl analogues (structures not shown) showed no improve-
ment in biological activity. Molecular docking studies highlighted that compound
118 fitted in the Sph binding pocket of SphK1 establishing hydrogen bonding of
the 2-(hydroxymethyl)pyrrolidine moiety. Specifically, (1) the primary alcohol
hydrogen bonded with Ser168 and (2) the tertiary nitrogen hydrogen bonded with
Asp178. Similarly, hydrogen bonds between the pyrrolidine nitrogen and Asp308
as well as the hydroxyl group and Ser298 were observed in the homology model of
SphK2 docked with 118. The aliphatic dodecyl alkyl tail of compound 118 acquires
a specific “J-shape”, generating hydrophobic interactions within the binding pocket
of the enzyme.
Recently, Frydrych et al. [83] used commercially available d- and l-prolinol as
the starting material for the synthesis of novel antimalarial agents, yielding new chi-
ral compounds in which a pyrrolidine ring is incorporated in the linker connecting the
13
purine base to the phosphonate group(s) (119a,b and 120a,b) (Fig. 24). Compounds
119a,b and 120a,b were evaluated as inhibitors of the plasmodial hypoxanthine-gua-
nine-(xanthine)-phosphoribosyltransferase [HG(X)PRT] of Plasmodium falciparum,
P. vivax (HGPRT) and human HGPRT, and the results were compared with those of
the previously published inhibitors 123, 124, 125a,b (Fig. 24). The biological results
did not show an improvement in activity over compounds 123, 124, 125a,b. In fact,
the new nucleotide analogues had Ki values in the range between 9 and > 50 mM for
human HGPRT, 5–44 mM for PfHGXPRT and between 20 and > 50 mM for PvHG-
PRT, despite the increased flexibility achieved by the implementation of a CH2 group
between the nucleobase and the pyrrolidine ring, which allows a free rotation. The
same results were obtained when replacing the pyrrolidine nucleus with a six mem-
bered-ring, such as piperidine or piperazine (structures not showed). However, the
phosphoramidate prodrugs 121a,b and 122a,b (Fig. 24), exhibited good antimalarial
activity in a P. falciparum-infected human erythrocyte assay. In particular, the bisphos-
phoramidate prodrug 122a was potent ( IC50 = 2.5 µM) against the chloroquine resistant
P. falciparum W2 strain, with low cytotoxicity in human hepatocellular liver carcinoma
(HepG2) and normal human dermal fibroblasts (NHDF) cell lines at a concentration of
100 µM.
5 Concluding Remarks
The present review is intended to provide significant support to medicinal chemists

in the discovery of new biologically active pyrrolidine derivatives, providing a gen-
eral overview of recent research concerning this scaffold, offering quick identification
of the best synthetic route to apply. From this work, pyrrolidine emerges as a versa-
tile scaffold found in molecules that exhibit a broad spectrum of biological activities.
Pyrrolidines are useful to build compounds for fighting cancer and microbial infec-
tions, for metabolic diseases, as agents active in the CNS and for neurodegenerative
diseases and immune disorders. Since three-dimensionality is an essential element
of ligand–target interactions, the stereocenters present in pyrrolidine scaffolds would
allow medicinal chemists to develop molecules with the most suitable configurations
to fit into the ligand binding site of a target protein. In order to discover structurally
novel compounds, the scaffold hopping strategy could be applied to the pyrrolidine
core while maintaining its stereochemistry, together with exploration of the SAR of
the most active pyrrolidine derivatives. We believe that the chemical versatility of the
pyrrolidine nucleus and its ability to generate structural diversity could be essential to
establish the clinical success of new bioactive molecules.
Funding Open access funding provided by Università degli Studi di Palermo within the CRUI-CARE
Agreement. No funding was received to assist with the preparation of this manuscript.
Declarations
Conflict of Interest The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
13
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ses/by/4.0/.
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Authors and Affiliations
Giovanna Li Petri1 · Maria Valeria Raimondi1 · Virginia Spanò1 · Ralph Holl2 ·

Paola Barraja1 · Alessandra Montalbano1
1
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies
(STEBICEF), University of Palermo, Via Archirafi 32, 90123 Palermo, Italy
2
Department of Chemistry, Institute of Organic Chemistry, University of Hamburg,
Martin‑Luther‑King‑Platz 6, 20146 Hamburg, Germany
13

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Topics in Current Chemistry (2021) 379:34

Pyrrolidine in Drug Discovery: A Versatile Scaffold for Novel

Giovanna Li Petri1 · Maria Valeria Raimondi1 · Virginia Spanò1 · Ralph Holl2 ·

Received: 2 April 2021 / Accepted: 25 July 2021

Keywords Pyrrolidine · Anticancer and antibacterial agents · Central nervous

* Maria Valeria Raimondi

by modifying parameters such as solubility, lipophilicity, and other ADME prop-

Cyclopentane 0.073 269.230 269.230 0 0 0 0 3.000 −2.642 −2.709 0 0

Pyrrolidine Pyrrole Cyclopentane

Fig. 2 Representative structures of natural alkaloids 1–5

2 Influence of Steric Factors on Biological Activity

Contrary to the parent aromatic compound pyrrole, which is a common scaffold of

2.1 The Enantiopure Compound Shows Full Agonism Towards G‑Protein Coupled

Notably, Jurica et al. [37] synthesized a series of G-protein coupled receptor 40

2.2 The New Spatial Arrangement in the Crystallized Ligand–Protein Complex

2.3 The Orientation of 3‑R‑Methylpyrrolidine is Responsible for a Pure Estrogen

In 2018, through biochemical and biophysical assays, coupled with high-resolution

2.4 Introduction of a Cis‑3,4‑Diphenylpyrrolidine Moiety Provides a Potent

Recently, Jiang et al. [40] demonstrated that replacing a non-stereochemical with

2.5 Basicity and Nucleophilicity of the Pyrrolidine Nucleus

2.6 Spatial Characteristics Influencing the Biological Activities of Pyrrolidine

• cis-4-CF3 substituent on the pyrrolidine scaffold endorses the pseudo-axial con-

isoform is due to the methylene group at position 2 being involved in sponta-

3 Pyrrolidine Derivatives Obtained by Ring Synthesis

3.1 Pyrrolidines Obtained by 1,3‑Dipolar Cycloadditions

A classical method for the preparation of five-membered heterocycles is the

Fig. 6. 1,3-Dipolar cycloadditions to stereoselectively obtain pyrrolidine derivatives

Comp R PARP-1 PARP-2 PARP-1 PARP-2

Fig. 7 General synthetic scheme to benzimidazole carboxamides 19a–p. R substituents and

23k 3-F-phenyl 3-Cl-phenyl 1-CH3-1H-imidazol-4-yl 1 0.042

metabolism and ameliorating dyslipidaemia associated with type 2 diabetes. Com-

Fig. 9. 1,3-Dipolar cycloadditions to yield 4-benzylpyrrolidine-3-carboxylic acids 25 and 26, synthesis

Fig. 10. 1,3-Dipolar cycloadditions to yield phenyl/thiophene dispiro indenoquinoxaline pyrrolidine

19 µM, respectively, comparable to those of the reference compound doxorubicin

3.2 Pyrrolidines Obtained by Pictet–Spengler‑Oxidative Ring Contractions

Moreover, spirocyclic motifs are emerging as an interesting feature for building

Fig. 11 General synthetic scheme to spiro[pyrrolidine-3,3′-oxindoles] 38a–n. Reagents and conditions: a

3.3 Pyrrolidines Obtained via Aminocyclizations

Polyhydroxylated pyrrolidines, known as aza-sugars, are considered metabolically

the diastereoisomeric d-glucose and d-galactose series, as dual-target inhibitors of

­(IC50 = 79 nM competitively displacing fluorescent 12G5 antibody). Conversely,

3.4 Pyrrolidine‑2‑Ones Obtained by Cyclizations

Garner's aldehyde MBH adduct

54 (Fig. 14). Compound 54 is a polyhydroxylated pyrrolidine that is a more potent

3.5 Pyrrolidine‑2,5‑Diones Obtained by Cyclizations

59a: R1 = -CH-diphenyl; R2 = 3-CF3; n = 2

62a: R = -(CH2)-; X/R1 = O/-

with a phenylpiperazine moiety bearing a 3-trifluoromethyl group were most active

130.6 mg ­kg−1). In addition, four of the tested compounds (62a,b,d,g) revealed

R2 69h: R,R1 = -Ph; R2 = 3,4-diCl

Pyrrolidine-2,5-dione is a versatile scaffold, as demonstrated by Oktay et al.

pyrrolidine-2,5-dione nucleus with oxocycloalkyl/oxoalkyl groups (structure not

3.6 Pyrrolizines Obtained by Cyclizations

4 Pyrrolidine Derivatives from Commercial Building Blocks

4.1 Pyrrolidines from Proline

The interest in the pyrrolidine nucleus as skeleton of molecules with biological

88a: R1 = phenyl; R2 = -CH2-phenyl; R3 = -NHSO2-phenyl

87 88l: R1 = 4-CH3-phenyl; R2 = -CH2-phenyl; R3 = -NHSO2-(4-CH3)-phenyl

Fig. 19 Molecular structures of pyrrolidinyl-carbazole derivatives 85a–p, pyrrolidine-2-carbonitriles

of A549 with an I­C50 value of 15.7 µM. Furthermore, compounds 85b,f,h,j,m

2 Influence of Steric Factors on Biological Activity

2.1 The Enantiopure Compound Shows Full Agonism Towards G‑Protein Coupled

2.2 The New Spatial Arrangement in the Crystallized Ligand–Protein Complex

2.3 The Orientation of 3‑R‑Methylpyrrolidine is Responsible for a Pure Estrogen

2.4 Introduction of a Cis‑3,4‑Diphenylpyrrolidine Moiety Provides a Potent

2.5 Basicity and Nucleophilicity of the Pyrrolidine Nucleus

2.6 Spatial Characteristics Influencing the Biological Activities of Pyrrolidine

3 Pyrrolidine Derivatives Obtained by Ring Synthesis

3.1 Pyrrolidines Obtained by 1,3‑Dipolar Cycloadditions

3.2 Pyrrolidines Obtained by Pictet–Spengler‑Oxidative Ring Contractions

3.3 Pyrrolidines Obtained via Aminocyclizations

(IC50 = 79 nM competitively displacing fluorescent 12G5 antibody). Conversely,

3.4 Pyrrolidine‑2‑Ones Obtained by Cyclizations

3.5 Pyrrolidine‑2,5‑Diones Obtained by Cyclizations

130.6 mg kg−1). In addition, four of the tested compounds (62a,b,d,g) revealed

3.6 Pyrrolizines Obtained by Cyclizations

4 Pyrrolidine Derivatives from Commercial Building Blocks

4.1 Pyrrolidines from Proline

of A549 with an IC50 value of 15.7 µM. Furthermore, compounds 85b,f,h,j,m

4.2 Derivatives from Other Preformed Pyrrolidine Scaffolds