Pyrrolidine in Drug Discovery: A Versatile Scaffold for Novel Biologically Active Compounds

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 efficiently explore the pharmacophore space due to sp³-hybridization, (2) the contribution 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 “pseudorotation”. In this review, we report bioactive molecules with target selectivity characterized by the pyrrolidine ring and its derivatives, including pyrrolizines, pyrrolidine-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 relationship (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 synthesis and the reaction conditions, or (2) functionalization of preformed pyrrolidine rings, e.g., proline derivatives. Since one of the most significant features of the pyrrolidine ring is the stereogenicity of carbons, we highlight how the different stereoisomers and the spatial orientation of substituents can lead to a different biological profile of drug candidates, due to the different binding mode to enantioselective proteins. We believe that this work can guide medicinal chemists to the best approach in the design of new pyrrolidine compounds with different biological profiles.

1 Introduction

The development of clinically active drugs relies increasingly on the use of heterocyclic scaffolds, many of which contain nitrogen, as evidenced by the considerable number of bioactive compounds now available [1,2,3,4,5]. The introduction of heteroatomic 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 sp³-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 globular three-dimensional (3D) shape offer more opportunities to improve druggability by modifying parameters such as solubility, lipophilicity, and other ADME properties [12]. This association was also highlighted by Ritchie et al. [13] who conducted 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 surface area (PSA) values, only the pK_BHX 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.

Table 1 Comparison of molecular descriptors and parameters of the pyrrolidine nucleus with those of carbocyclic cyclopentane and aromatic pyrrole

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 projects”, and the 3D shape appears as one of the strategic goals for the design of reagents 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.

Fig. 1

Comparison of the three-dimensional (3D) shape of the non-aromatic pyrrolidine and cyclopentane rings with the aromatic pyrrole ring. Bond angles, bond lengths, and MMFF94 values were calculated using LigandScout software version 4.4 Expert

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, especially in alkaloids isolated from plants or microorganisms [17,18,19] (Fig. 2) showing different biological activities, e.g. nicotine 1 (antioxidant, anti-inflammatory, antihyperglycemic properties), scalusamides A 2 and (R)-bgugaine 3 (antimicrobial, 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, pyrrolidine and its derivatives are used widely as ligands for transition metals, organocatalysts, and effective chiral controllers in asymmetric synthesis [22,23,24].

Fig. 2

Representative structures of natural alkaloids 1–5

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 supporting the new era of green chemistry [25].

In this review, we highlight the interest of the chemistry community in pyrrolidine and its derivatives, including pyrrolizines, pyrrolidine-2-one, pyrrolidine-2,5-diones, and prolinol, incorporated in bioactive molecules with target selectivity 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 [26,27,28], 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 inactivity 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 pharmacophore 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 and stereoelectronic factors. For instance, in the case of l-proline, the endo conformer 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].

Fig. 3

Exo and endo conformers of trans- and cis-4-fluoroproline 6 and 7, respectively; trans/cis-4-fluoroproline 8 resulting from the superimposition of structures 6 and 7

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.

Fig. 4

Stereospecific pyrrolidine derivatives (R,R)-9, (S,S)-9, 11a,b, 12a,b, 13a,b, and 14

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 synthesized 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 crystallography 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 the stereospecific orientation can change the binding mode of antiestrogen benzopyran derivatives 13a,b and 14 (Fig. 4) within the hormone-binding pocket. Specifically, they showed that the orientation of 3-R-methylpyrrolidine 13a was responsible 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-diphenylpyrrolidine 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, Y_max = 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 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 balance between potency against RORγt with an EC₅₀ of 61 nM and a considerably lower PXR activity (EC₅₀ = 495, Y_max = 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 interaction 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) pyrrolidines with an unsubstituted nitrogen were weak agonists compared with tertiary amines, and (5) a para-F substituent in the 3-phenyl ring improved EC₅₀ values.

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

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 pharmacological efficacy. The following list summarizes the spatial dispositions of the pyrrolidine 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 conformation of the groups in the other positions, leading compounds able of showing full agonism in both the human and mice GRP40 receptor;

• compared with 3-S-methylpyrrolidine or an unsubstituted pyrrolidine, 3-R-methylpyrrolidine 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 selectivity towards CK1 receptors. However, the selectivity and potency for the CK1γ isoform is due to the methylene group at position 2 being involved in spontaneous intramolecular Pictet–Splenger cyclization during crystallization with the protein;

• a cis-3,4-diphenylpyrrolidine scaffold gives the molecule a “U-shape” conformation 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, leading to 3,4-cis- or 3,4-trans-substituted pyrrolidines [44].

Fig. 6.

1,3-Dipolar cycloadditions to stereoselectively obtain pyrrolidine derivatives

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) polymerase-1 and -2 (PARP-1,-2), enzymes involved in the DNA damage repair process, as depicted in Fig. 7.

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%

Out of 16 compounds, 6, namely 19c,h–j,o,p, inhibited PARP-1 and -2 with IC₅₀ 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 derivatives 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 important amino acid residues Gly-863, Ser-904 and Glu-988 of PARP-1, instead of only 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 benzimidazole 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 IC₅₀ 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 inhibition. Cytotoxic assays against MDA-MB-436 (breast cancer) and CAPAN-1 (pancreatic 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 olaparib (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)benzylamine 17 was reacted with the nitrovinyl substrate 20, methyl trans-4-fluorocinnamate 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) competitive 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 associated with NMDA receptor hypofunction. The new pyrrolidine sulfonamides 23a–ad were synthesized based on the SAR investigation conducted on the reference compound 23a. The latter displayed satisfactory GlyT1 inhibitory potency in vitro, with a K_i 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 replacement 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. Comparable in vitro potency and ER values were obtained when replacing the 1-methylimidazole 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). Concerning R² substituents, meta-substituted derivatives showed improved biological activity. 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 heteroaromatic, as shown in Fig. 8. Finally, the analogue 23t (R² = indanyl) showed the best balance between potency and ER value (K_i = 0.001 μM, ER = 1.5).

Fig. 8

General synthetic scheme to pyrrolidine sulfonamides 23a–ad. ER Efflux ratio values. K_i values towards hGLYT1 [46]. Reagents and conditions: a TFA (catalyst), DCM, r.t., overnight

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 metabolism and ameliorating dyslipidaemia associated with type 2 diabetes. Compounds 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.

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 compound 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)

Conjugated systems with biological macromolecules, including carbohydrates, lipids, proteins, and nucleic acids, require that special attention is paid to the geometry of the molecules to be combined. Unlike the pyrrolidine scaffold, spiro-pyrrolidine 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-dipolar 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 single isomers. All compounds were evaluated for their in vitro antibacterial activity against a panel of four human pathogens, including Vibrio cholerae, Proteus mirabilis, 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 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 indicated 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 electron-withdrawing groups (EWGs). Further investigation into the mechanism behind the anticancer activity revealed that compound 37e induced apoptosis through intracellular reactive oxygen species (ROS)-mediated caspase-3 activation.

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)

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 significant 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-bromosuccinimide (NBS) as oxidant, and a catalytic amount of TFA, varying the aromatic functionality of the pyrrole domain. Among all compounds produced, 38d,h,i inhibited the growth of human breast cancer cell line MCF-7 by inducing apoptotic cell death at low micromolar EC₅₀ values (6.00, 4.01, and 3.53 µM, respectively). Chemical proteomics 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 compared with compounds bearing one or two strong EWGs.

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%

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 carbohydrate processing enzymes. Due to their central role in different biological activities, they are emerging as attractive compounds for the treatment of cancer and metabolic diseases. In 2019, using the double reductive amination reaction, Guazzelli et al. [52] developed a series of polyhydroxylated pyrrolidines, belonging to the diastereoisomeric d-glucose and d-galactose series, as dual-target inhibitors of the enzymes α-glucosidase (AG) and aldose reductase (ALR2). Although AG represents the most important enzyme for controlling plasma sugar concentration, pathological 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. Aminocyclization was conducted by reacting aldohexos-4-ulose derivatives 39 and 40 (Fig. 12) with amines, including benzylamine, benzhydrylamine, ammonia, l- or d-phenylalanine 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 benzylamine hydrochloride (for compounds 41a,b) and l-phenylalanine methyl ester hydrochloride (for compound 42). These three compounds were the starting material for the synthesis of other derivatives. Thus, through hydrogenolysis, compounds 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 compounds 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.

Fig. 12

Blue panel Synthesis of diastereoisomers 41a,b and 42 obtained by reaction of 5,6-O-isopropylidene-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)

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 generated, 51a with R¹ = 3-CH₃ showed excellent binding affinity to the CXCR4 receptor (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 cytosolic 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.

Fig. 13

General synthetic scheme to pyrrolidines 51a–f. Reagents and conditions: a sodium triacetoxyborohydride [NaBH(OAc)₃], DCM, − 70 °C to r.t., overnight, yields: 21–45%

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 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 antidiabetic 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. Compound 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 intermediate 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.

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%

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 studies, 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 counteract 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 ED₅₀: 88.2 mg kg⁻¹ and 101.5 mg kg⁻¹, respectively; scPTZ ED₅₀: 65.7 mg kg⁻¹ and 59.7 mg kg⁻¹, respectively), indicating 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 anticonvulsant 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 derivatives 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 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 derivatives 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, derivatives 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 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 anticonvulsant activity in both the MES and scPTZ tests.

Fig. 15

General synthetic routes to pyrrolidine-2,5-diones 59a–p, 62a–g, and 63a–h. Reagents and conditions: a 180 °C, 1.5 h., yields: 60–82% (59a–p), 70% (61), 54–86 (62a–g)

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 compounds 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 65, at 180 °C for 1 h. This structural change did not improve the pharmacological 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 ED₅₀ 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 anticonvulsant 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 ED₅₀ 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 positively 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).

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, R¹ = phenyl), 36% (68, R = methyl, R¹ = ethyl)

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 epilepsy. Therefore, their inhibition could be useful to counteract these issues. The synthetic route to obtain the 3-chloro-N-aryl pyrrolidine-2,5-dione derivatives 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 pyrrolidine-2,5-diones, except compound 71e, were able to inhibit hCA I (K_i in the range of 23.27–36.83 nM) and hCA II (K_i in the range of 10.64 and 23.34 nM) with higher or comparable activity than the reference hCA inhibitor acetazolamide (AZA) (hCA I: K_i = 34.70 nM; hCA II: K_i = 31.93 nM). The most active compounds were derivatives 71d (hCA I: K_i = 23.27 nM) and 71c (hCA II: K_i = 10.64 nM), both decorated at the pyrrolidine-2,5-dione nitrogen atom with bicyclic scaffolds, such as quinoline and naphthalene.

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%

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 temperature 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), albumin 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 reference 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 zileuton (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 activity, 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 biological activity. However, the replacement of the aryl ketone at position 3 of the 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 inhibitors with safer gastric profile. Pyrrolizine 77 was synthesized via the intramolecular 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 derivatives 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 NH₂-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: 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.

Fig. 18

General synthetic scheme to pyrrolizine carboxamide derivatives 77–84. Reagents and conditions: a K₂CO₃, acetone, under reflux, 24 h, yields: 68% (77)

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, Pannala et al. [64] developed a metal- and catalyst-free three-component decarboxylative coupling reaction of proline, aldehydes and 4-hydroxycarbazole to access pyrrolidinyl-carbazole derivatives 85a–p (Fig. 19) with antiproliferative and antioxidant activities. The antiproliferative activity was evaluated by in vitro assays on three different 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 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 trifluoromethyl group (85f) on the phenyl ring participate in hydrophobic binding interactions 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.

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

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 antidiabetic 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 inactivate 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 (K_i = 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, aromatic or heteroaromatic rings at R², and different benzenesulfonamides at R³. Compounds 88c and 88f showed the best binding affinities towards Mcl-1, with K_i values of 0.94 and 0.53 µM, respectively, slightly higher than the positive control gossypol (K_i = 0.39 µM) but very much lower than compound 87. The replacement of phenyl or 4-bromophenyl at R² with 3-indolyl and p-hydroxyphenyl (88i,j,p) was unfavorable 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 (K_i > 10 µM). Moreover, compound 88f exhibited good antiproliferative activities against MDA-MB-231, PC-3 (prostate 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 responsible 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 natural amino acid S-proline, the naturally derived S-pyroglutamic acid ((2S)-5-oxopyrrolidine-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 containing a benzyl-ether type substituent, which bears carboxylate, methyl ester, sulfonamide, 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 the ATX enzyme, whereas only one out of six compounds—the carboxylic acid derivative 89d—was the least active (IC₅₀ 800 nM).

Fig. 20

Molecular structures of pyroglutamic acid derivatives 89a–e and 90 and N-(2′-nitrophenyl)pyrrolidine-2-carboxamides 91a–k

Antimicrobial peptides (AMPs) are small peptides with a wide range of inhibitory 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 features of compounds 91a–k were the simultaneous presence of (1) a hydrophobic 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 activity 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 carboxamides 91a–k were more potent against S. aureus and E. cloacae than the standard streptomycin (256 µg/ml and > 512 µg/ml, respectively), with compounds 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 substituents, 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 building 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 processing of the amyloid precursor protein (APP), which leads to the generation of amyloid-β (Aβ) peptides. The aggregation of Aβ in the brain of patients is responsible for the onset of Alzheimer’s disease (AD). Therefore, the development of treatments towards BACE1 could be a good strategy to fight this devastating neurodegenerative 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 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.

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

Novel compounds for the treatment of Alzheimer’s disease were also investigated 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 multitarget 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, derivatives 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 doxorubicin (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-deoxysphingomyelin 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 GI₅₀ 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 equally active, with GI₅₀ 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 pyrrolidine hydroxamates carrying on the pyrrolidine nitrogen atom two different substituents: 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 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-benzofuroxan 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 values 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.

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 pyrrolidine amides 107, 108, 109a,b, 110, 111

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 pyrrolidine 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 instability, conferring better pharmacokinetic (PK) profiles than the parent 3-hydroxy compounds. Compounds 102b,c had low cLogP values (< 3), showed better metabolic stability, and retained potent androgen receptor (AR) agonistic activity. In addition, 2-chlorobenzonitrile 102b and 3-fluoro-2-methylbenzonitrile 102c derivatives showed good bioavailability values (53.4 and 46.3%, respectively) and strong anabolic activity in levator ani muscle (> 300%), which was dose-dependent as demonstrated 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-hydroxylpyrrolidine-2-chloro-3-methylbenzonitrile [75].

In 2018, Kaur et al. [76] designed and synthesized a library of hybrid molecules 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 IC₅₀ 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 catalyzes the degradation of N-acylethanolamines into fatty acids and ethanolamine in animal tissues. Its inhibition is a therapeutic tool in several pathophysiological conditions, 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 antiinflammatory agents and tested their selectivity towards NAAA and fatty acid amide hydrolase (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 compounds 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 compound 108 were detrimental for activity, most probably due to the conformationally restricted chain. Isosteric replacements of the distal phenyl ring with aromatic moieties 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 and 109b, highlighting that (hetero)aromatic rings are less tolerated in the hydrophobic 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 (structure not shown). In addition, the replacement of pyrrolidine-2-thione with pyrrolidin-2-one 113a,b (Fig. 23) did not affect activity toward the FTase protein.

Fig. 23

Molecular structures of triazine-pyrrolidine-2-thiones 112a,b and pyrrolidin-2-ones 113a,b, pyrrolidin-2-ones 114a–e, and pyrrolidin-2-ones 115a–o

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-oxopyrrolidin-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 cancer) (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 antiproliferative activity compared with electron-donating methoxy groups. Hydroxy group introduction at position 2 (114d and 114e) led to an increase in antiproliferative activity compared with compounds 114b and 114c. Docking analyses suggested 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 acetylcholine, 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 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 K_i values in the range of 2.60 and 16.36 nM. The most powerful AChE inhibitor was compound 115f with a K_i value of 2.60 nM. Furthermore, all compounds inhibited BChE with K_i values in the range of 13.10 and 54.47 nM. Measuring the DPPH radical scavenging activity, compounds 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 pioglitazone (IC₅₀ 40.3 µM). Further studies with compound 116b demonstrated its ability 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.

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

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 investigation of potent and selective inhibitors previously reported in the literature (compound 117, SphK1-selective inhibitor) (Fig. 24). These enzymes are involved in the production of sphingosine 1-phosphate (S1P) from sphingosine and ATP, in a signalling 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 moiety changes the selectivity for SphK1 and SphK2. In this regard, the authors introduced 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 K_i = 0.679 μM, SphK2 K_i = 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 improvement 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 chiral compounds in which a pyrrolidine ring is incorporated in the linker connecting the 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-guanine-(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 K_i values in the range between 9 and > 50 mM for human HGPRT, 5–44 mM for PfHGXPRT and between 20 and > 50 mM for PvHGPRT, 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 membered-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 bisphosphoramidate 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 general overview of recent research concerning this scaffold, offering quick identification of the best synthetic route to apply. From this work, pyrrolidine emerges as a versatile scaffold found in molecules that exhibit a broad spectrum of biological activities. Pyrrolidines are useful to build compounds for fighting cancer and microbial infections, 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.