Application of Chiral Piperidine Scaffolds in Drug Design

Chiral piperidine scaffolds are prevalent as the common cores of a large number of active pharmaceuticals in medical chemistry. This review outlined the diversity of chiral piperidine scaffolds in recently approved drugs, and also covers the scaffolds in leads and drug candidates. The significance of chiral piperidine scaffolds in drug design is also discussed in this article. With the introduction of chiral piperidine scaffolds into small molecules, the exploration of drug-like molecules can be benefitted from the following aspect: (1) modulating the physicochemical properties; (2) enhancing the biological activities and selectivity; (3) improving pharmacokinetic properties; and (4) reducing the cardiac hERG toxicity. Given above, chiral piperidine-based discovery of small molecules will be a promising strategy to enrich our molecules' library to fight against diseases.


Introduction
Chiral drugs has attracted more and more attention due to their perfect adaptability to protein-binding sites. The chiral state of a molecule can be achieved by introducing a chiral center to the structure, and owing to this fact, their druggability may be greatly influenced. 1 Piperidine, as a sixmembered nitrogenous heterocyclic ring, is widely present in the structure of approved drugs. 2 Thus, the study of stereochemistry of piperidine scaffolds has attracted a great deal of interest since the late last century, and has become a hot topic recently. 3 According to the U.S. Food and Drug Administration data, there are 245 drugs approved from 2015 to June 2020, including small molecules and macromolecules. Among them, the number of chiral piperidinecontaining drugs is nine, including avycaz (1), cotellic (2), varubi (3), zejula (4), daurismo (5), galafold (6), akynzeo (7), ubrelvy (8), and recarbrio (9) (►Table 1, ►Fig. 1). 4 Although the introduction of chiral centers in piperidine scaffolds is often associated with increased number of synthetic steps and unusual reaction procedures, and ultimately leads to an increase in synthesis effort, 5,6 medical researchers believe that the increased expenditure is worth because of the expected favorable effects on physicochemical properties, potency, selectivity, and pharmacokinetic (PK) profile that may be induced by the chiral piperidine scaffolds. Thus, this review aimed to provide a broad perspective on the latest advances of chiral piperidine scaffolds in medicinal chemistry. The study would assist drug discoverers to rationally design molecules for various diseases.

Application of Chiral Piperidine Scaffolds in Drug Design
Modulating the Physicochemical Properties Physicochemical properties of a compound can be predicted by the parameters of pK a , logD, and logP, and methods for improving the physicochemical properties of drugs include introducing hydrophilic or lipophilic groups, changing charge state and their spatial configurations to form intermolecular or intramolecular forces, etc. 7,8 The piperidine ring originally is a kind of groups with properties of both hydrophilicity and lipophilicity. The introduction of chiral centers in the piperidine ring, changing the position of a substituent, or introducing another substituent may alter its physicochemical properties effectively.
In 2012, Ndungu et al reported a series of measles virus RNA-dependent RNA polymerase (MeV-RdRp) inhibitors. 8 In their earlier work, they identified 10 as a potent and selective MeV-RdRp inhibitor, which exhibited an excellent activity Imipenem; inhibit cell-wall synthesis, cilastatin; inhibitor of renal dehydropeptidase, and relebactam; β-lactamase inhibitor (EC 50 ¼ 14 nmol/L) but suffered from poor water solubility and low oral bioavailability. Further structure-activity relationship (SAR) studies showed that introducing a substituent at the 2position of the piperidine ring could effectively enhance the aqueous solubility of this series of compounds. Then, optimization of in vivo potency and aqueous solubility of compound 10 led to the discovery of compound 11, which showed an improved aqueous solubility of 60 μg/mL and a maintained MeV-RdRp inhibition (EC 50 ¼ 60 nmol/L) (►Fig. 2). SUCNR1 (succinate receptor 1, initially named GPR91) is a G-protein-coupled receptor, and identifies succinate as its endogenous ligand. 9 Succinate is responsible for ATP formation and energy supply, 10 and is normally located in the mitochondria, but under certain pathological conditions, it acts as a signaling molecule, and is recognized as a danger signal by SUCNR1. 10,11 In the process of exploring a new class of SUCNR1 inhibitors, Velcicky et al discovered that both 4piperidyl analog (12) and 3-piperidyl analog (13) had a good SUCNR1 inhibition. 12 However, the logD (pH 7.4) values of the two compounds varied a lot (►Table 2), and compound 13 showed quite remarkable increase in the permeability and lipophilicity when compared with 12. Further research   revealed that the two compounds populated remarkably different shapes in an aqueous solution (►Fig. 3). 13 Compound 12 preferred an elongated conformation, while compound 13 was folded with an intramolecular salt bridge formed between the piperidine and the carboxylic acid moieties. 12

Influence on the Biological Activity
Improving the Biological Activity Chiral centers are widely found in approved drugs and drug candidates, and have shown unique privileges in enhancing drug efficacy. Due to the asymmetry of organisms, the biological properties of chiral molecules are more abundant than achiral molecules. 14 Currently, the study of stereochemistry of the piperidine scaffold has fascinated chemists because of its profound effect on biological activity of a drug. 15 As is shown in a wide range of research studies, introducing a chiral center in the piperidine ring can not only provide researchers with more chiral piperidine-containing compounds, which is essential to improve the activity of the drugs, but also make the compound have more configurational isomers to fit in the cavum of protein. 15 The mitogen-activated protein kinase (MAPK) signaling pathway has four branches. Among which, the extracellular regulated protein kinases (ERK) pathway plays an important role in the development of tumors. 16 In this pathway, there are three important target proteins, including Ras, Raf, and mitogen-activated protein (MEK). Many research studies suggested that inhibition of MEK may suppress the activation of ERK effectively, which is essential to the blockage of this pathway. 16,17 In the process of identifying a new series of MEK1/2 inhibitors, the imidazoquinoline core was considered, and Poddutoori et al discovered 1-(piperidin-4-yl)-1Himidazo [4,5-c]quinoline (14) as a high-throughput screening hit, which had an acceptable MEK1/2 inhibition (EC 50 ¼ 400 nmol/L). 18 The structure-based design led to the discovery of analogue 15, whose MEK1/2 inhibition is eightfold higher than 14. Further optimization focused on the modification of the piperidine ring of 15, with a fluorine introduced at the 3position of the piperidine ring, to give chiral 16, which exhibited a further improved potency (EC 50 ¼ 5 nmol/L) and aqueous solubility (logP ¼ 1.6) as well as high oral bioavailability (F% ¼ 65) (►Fig. 4). The crystal structure of MEK1 in complex with 16 revealed that the 3-substitued piperidine side chain can fit into the cavum of MEK easily, which is essential to increase the drug potency (►Fig. 5).
In exploration of a new series of CVF-Bb (Cobra Venom Factor binds factor B) inhibitors, Mainolfi et al discovered the significance of R groups on the potency of CVF-Bb inhibition of a compound (►Table 3). 19 When the R group is a morpholine ring or a piperidine ring with no chiral center, the potency was low. Taking compound 18 as an example, it had an IC 50 value of 50 μmol/L; however, when a phenyl was introduced at 2-position of the piperidine ring of 18, compound 19 was obtained, with a great increase in CVF-Bb inhibitory potency (IC 50 ¼ 5.9 μmol/L). Further optimization led to the discovery of 20 and 21, whose potencies are approximately 170-fold higher than 19. It is noteworthy that the introduction of an ethoxy group at the 4-position of the piperidine ring provided 21 with approximately twofold potency higher than 20, which also proved the good effect of introducing a chiral center in the piperidine ring. Then, the replacement of methyl with a methoxy led to

22
, whose potency was further increased and PK properties are good (►Fig. 6).
P53 is a kind of tumor suppressor protein that maintains the integrity of the genome in a cell. 20 Human double minute 2 (HDM2) is a ubiquitin protein ligase that negatively regu-lates p53 and lessens its transcriptional activity as well as promotes p53 protein degradation. 20,21 Disrupting the HDM2-p53 protein-protein interaction (PPI) with a small molecule has been recognized as a potential way in cancer therapies. 22 In 2014, Ma et al reported the discovery of a new series of HDM2-p53 PPI inhibitors. 21 Structure-based design led to the discovery of 23 that had a piperidine-containing core and an acceptable potency (IC 50 ¼ 169 nmol/L). Further optimization of 23 led to compound 24, which was a potent HDM2-p53 PPI inhibitor (IC 50 ¼ 41 nmol/L) with an allyl at the 2-position of its piperidine ring. Compared with 23, compound 24 showed an improved inhibition toward HDM2-p53 PPI and a favorable toxicity window between wt-p53 and mutant-p53 cell lines (►Fig. 7).
In 2017, Rutaganira et al reported the discovery of a new series of calcium-dependent protein kinase 1 (CDPK1) inhibitors. 23 Among which, compound 27 showed the best potency with an IC 50 value of 10.9 nmol/L (compared with 25 and 26, whose IC 50 values are 14.15 and 77.5 nmol/L), suggesting an important role of two fluorine atoms at 3-position of the piperidine ring (►Table 4). Interestingly, 27 produced a chiral center at the 4-position of the piperidine ring. The skeleton of 27 fits well into the cavum (►Fig. 8a), observed by the crystal structure of CDPK1 in complex with 27 (PDB code: 5W9E), and the introduction of a chiral center made the nitrogen of the piperidine ring form two salt bridges with the GLU135 and GLU178 residues of CDPK1 (►Fig. 8b).
Glucagon-like peptide-1 receptor (GLP-1R) is the most important incretin in type-2 diabetes therapy. GLP-1R enhances the release of glucose-dependent insulin and suppresses the apoptosis of islet β cells. 24 However, the half-life    time of this native peptide in blood is very low (1 À 2 minutes) as it can be mainly rapidly degraded by circulating dipeptidyl peptidase (DPP-4). 25 Therefore, it is of great importance to develop a potent GLP-1R agonist. In 2021, Decara et al discovered a new series of GLP-1R agonists that contain a 1,2,4-oxadiazole core and a substituted piperidine ring. 25 Among which, the position of the substituent on the piperidine ring mattered much to their activity. When a substituent is present at 4-position of the piperidine ring (28), a poor GLP-1 potentiation (E max ¼ 24%) was achieved. When the substituent is morpholine-1-methyl and at 3-position of the piperidine ring (31), the GLP-1 potentiation of the compound was increased to 60% (►Table 5).
In 2016, Humphreys et al reported the discovery of a series of p300/CBP and p300/CBP associated factor (PCAF) inhibitors that had a pyridazin-3(2H)-one core, among which, the type of R groups was of great significance to their   activities. 26 When the R group was a pyrrolidine (32) or a 4substituted piperidine (33), the potencies of compounds were low. But when the substitution site in the piperidine ring changed from 4 to 3 (34), the potencies of compounds improved a lot (►Table 6). Further SAR research studies discovered that introducing a phenyl at 5-position of the piperidine ring can further increase its activity, which led to 35, a potent PCAF inhibitor with a pIC 50 value of 7.4 and exceptional selectivity over the bromodomain and extraterminal domain (BET) bromodomain family (►Fig. 9).
In 2019, Huang et al discovered a series of PCAF inhibitors that had a similar structure to that of Humphreys et al. 27 The results also proved that the introduction of a chiral center in the piperidine ring improves the potencies of the series of compounds (►Table 7). As shown in ►Table 7, when the R group was a symmetric piperidine-containing structure (36, 37, and 38), the K D values of the compounds were above 200 nmol/L. But when the substituent was at the 3-and 5position of the piperidine ring (39), the K D value increased to approximately 150 nmol/L. Further optimization led to 40, a potent PCAF inhibitor that had similar structure to 39 and a further increased potency (►Fig. 10).
Cellular senescence exists widely in living beings, which has recently been considered as a potent strategy to suppress cancer progression. 28,29 In 2020, Oh et al reported the discovery of a new class of senescence-inducing small mol-ecules with antimelanoma activities in vitro. 30 Highthroughput screening and high-content screening led to the identification of compound 44, a senescence inducer with good antimelanoma activities in vitro. SAR research studies suggested a great significance of the 1,3-substituent piperidine moiety in improving the potency of the compound. When the 1,3-disubstituent on the piperidine moiety was replaced by an azetidine (41), pyrrolidine (42), or symmetric piperidine (43) moiety, the activities of the compounds decreased a lot (►Table 8). Further modification focused on the replacement of the thiazole moiety, which led to the identification of 45 (►Fig. 11), a potent cellular senescence inducer with remarkable antimelanoma activity in vitro (EC 50 ¼ 40 nmol/L, IC 50 ¼ 30 nmol/L).
In 2014, Basarab et al reported a series of pyrrolamide topoisomerase II inhibitors. 31 Among them, compound 46 had a (piperidin-1-yl)thiazole-5-carboxylic acid moiety, and displayed DNA gyrase inhibitory potency and antibacterial activity toward gram-positive pathogens. Compound 46 functions by inhibiting the type II bacterial topoisomerases, DNA gyrase, and topoisomerase IV (Topo IV). To further   improve its activity against topoisomerase II of bacteria, a methoxy group was introduced at 3-position of the piperidine ring to obtain 47, which showed an improved inhibition to a wide range of gram-positive and gram-negative bacteria (►Table 9). Further modification was focused on the 4position of the thiazole ring. For example, the introduction of (S)-((1-methoxypropan-2-yl)carbamoyl)thiazole-5-carboxylic acid unit led to 48, which was proved to exhibit a further improved activity against bacterial growth.

Regulating the Target Selectivity of Drug Molecules
In 2018, Karlsson et al identified a series of new fibrinolysis inhibitors. 32 They found that introducing a chiral center in the piperidine ring of the series of compounds can effectively increase their selectivity over GABAa (►Table 10). Structurebased virtual screen led to the piperidinyl-substituted isoxazol-3-ol 49, a fibrinolysis inhibitor with high potency but low selectivity over GABAa. Further research led to 50 that had a methyl at the 2-position of the piperidine ring. As is shown in ►Table 10, the potency of 50 decreased but the selectivity over GABAa remarkably increased. Then, the methyl group at 2-position of the piperidine ring was replaced by a neopentyl to give 51, which had a further increased potency, yet maintained selectivity over GABAa.
In 2019, Shen et al reported the identification of a new series of EGFR L858R/T790M/C797S inhibitors. 33 They discovered that the type of R groups was of great significance to their selectivity over EGFR WT . When the R group is a cyclohexanamine-containing moiety (52 and 53) or a symmetrical piperidine moiety (54), the selectivity of this series of compounds was low. But when the site of substitution was changed from 4-position to 3-position, their selectivity over EGFR WT increased remarkably (55,►Table 11). At last, a fluorine substituent was introduced at 3-position of the phenyl and its potency was further increased (56, ►Fig. 12).

Improving PK Properties of Drug Molecules
Absorption, distribution, metabolism, and excretion properties are key indexes evaluating the druggability of drugs.   Piperidine rings, especially chiral piperidine rings, are usually introduced to improve PK properties (e.g., half-life, clearance, bioavailability) of a drug, and thus a common structural unit existing in drugs. 34 Bruton's tyrosine kinase (BTK), a member of the Tec family of nonreceptor cytoplasmic tyrosine kinases, plays an essen-tial role in B cell receptor signaling pathways and Fcγ receptor signaling in leukocytes. 35 BTK participated in the regulating of pathways that contribute to rheumatoid arthritis (RA), a multifactorial autoimmune disease. 36 In 2019, Watterson et al reported the discovery of Branebrutinib (60, ►Fig. 13), which is an irreversible BTK inhibitor, and currently in phase II clinical trials for the treatment of RA. 37 It is derived from the earlier described reversible BTK inhibitor BMS-986142 (57, ►Fig. 13) whose quinazolinedione ring system provides two bridging hydrogen bonds with Cys481 and Gln412 through two ordered water molecules. 37 To get a covalent, irreversible BTK inhibitor, they first replaced the quinazolinedione ring with a simple acrylamide acceptor and simplified the hexahydro carbazole core to the 2,3dimethylindole ring system to achieve compound 58 (BTK IC 50 ¼ 0.17 nmol/L, Romas cell IC 50 ¼ 16 nmol/L; ►Fig. 13). However, 58 had a poor plasma PK in a mouse model, and this may be possibly due to the high acrylamide reactivity of the compound with thiols such as glutathione. Thus, alternation of accepters makes less contribution to resolving PK issue while sustaining the desirable potency. Therefore, the Nphenylacrylamide linker should be changed. Considering the balance between potency, electrophile reactivity, and PK profile, an S-3-piperidine appended with a but-2-ynamide accepter was introduced into the compound and gave 59 (►Fig. 13), a covalent BTK inhibitor with acceptable plasma concentrations in vivo but decreased potency (BTK IC 50 ¼ 0.52 nmol/L, Romas cell IC 50 ¼ 40 nmol/L). At last, a fluoro was introduced at the C5-position of the dimethylindole core and gave branebrutinib (60), which demonstrated improved potency (BTK IC 50 ¼ 0.1 nmol/L, Ramos cell IC 50 ¼ 7.2 nmol/L) and BTK inactivation rate (3.5 Â 10 À4 nmol/L À1 ·min À1 ). The cocrystal structure of 60 binding to BTK showed that its chiral piperidine linker fit into the cavum of BTK well and the but-2-ynamide accepter formed a covalent bond with Cys481 (►Fig. 14). Further evaluation showed that the fluorine-substituted analogue 60 had a desirable safety and tolerability profile (►Table 12), and finally, 60 was advanced for clinical evaluation.
Checkpoint kinase 1 (CHK1) is a kind of Ser/Thr protein kinases that mediate the cellular response to DNA damage. 38 CHK1 inhibitors have been identified as potential cancer   therapeutics, and had gained substantial interest from both academia and industry. In 2017, Yang et al reported the discovery of a series of CHK1 inhibitors. 38 In their previous work, the compound (61) with a 7-carboxamide thienopyridine core and a piperidine side chain was explored, which had a good potency but suffered from short half-life. Further SAR experiments showed that introducing a methyl at 2position of the piperidine ring can effectively prolong its half-life. This led to the discovery of 62, a potent CHK1 inhibitor with good potency and PK properties both in vitro and in vivo (►Table 13).
In the process of identifying a new series of plateletderived growth factor receptor (PDGFR) inhibitors, Hicken et al reported the discovery of 64, a piperidine-containing PDGFR inhibitor with excellent potency and PK properties. 39 Structure-based design led to the discovery of 63, which had satisfied potency and selectivity. However, 63 suffers from poor oral exposure and bioavailability. Further SAR study of 63 revealed that introducing a fluorine substituent at 3position of the piperidine ring can effectively improve the PK properties of this series of compounds, which led to the discovery of 64. Compared with 63 which has a symmetrical piperidine ring, 64 has an asymmetric piperidine ring with a fluorine substituent at the 3-position which enables its good oral exposure and bioavailability (►Table 14).
Reducing hERG Toxicity hERG, the abbreviation of "human-ether-a-go-go-related gene," is a gene encoding a cardiac potassium channel whose protein product is the inner pore-forming part of a key membrane-bound potassium channel in myocardial tissue. 40 When the compound binds to the hERG potassium channel, the outflow of potassium ions is blocked and the  repolarization time of cardiomyocytes is prolonged, which may induce a fatal risk of torsades de pointes. 41,42 With a wider and deeper knowledge of interaction between hERG inhibitors and their action site, possible strate-gies for decreasing drugs' hERG affinity have been focused, which include adjusting drug flexibility, 43 changing the charge state, 44 decreasing the pK a , 45 and decreasing the lipophilicity. 45 Originally, the piperidine ring is a kind of structure with high lipophilicity, and usually has higher hERG inhibition than other groups. 46 Introducing a chiral center into the piperidine ring, either changing the position of a substituent or introducing another substituent, would help in decreasing hERG affinity, because by which, the spatial configuration of the compound may be changed, in parallel with a decreased pK a , increased hydrophilicity, as well as modified polarity.
Uprosertib (65, also known as GSK-795) is a kind of protein kinase B (Akt) inhibitor identified by GlaxoSmithKline, and showed activities toward Akt1/2/3. 47 In 2019, Dong et al reported a new series of Akt1 inhibitors, which were based on the structure of uprosertib, and additionally introduced a piperidine moiety. 48 The connection between the primary amine moiety and the α-position of phenyl led to 66, a potent Akt1 inhibitor with an IC 50 value of 3 nmol/L but low hERG selectivity. Further optimization was focused on the  modification of its piperidine moiety, which led to 67, an Akt1 inhibitor with even better potency and a higher hERG selectivity (►Fig. 15).
In 2012, Reck et al reported a series of aminopiperidinecontaining compounds targeting bacterial type II topoisomerases (DNA gyrase and topo-isomerase IV). 49 In their work, the quinolinone carbonitrile derivative 68 was found to have a potent antibacterial activity, but suffered from hERG inhibition (hERG IC 50 ¼ 44 μmol/L) and QT prolongation in vivo. Introducing electron-withdrawing substituents such as hydroxy, methoxy, and fluoro groups at 3-position of the piperidine moiety could reduce the pK a value of this series of compounds, and led to the subsequent hERG inhibition through reduction of binding affinity. Compound 69, a cis-fluoro-substituted analogue, exhibited an improved selectivity over hERG (IC 50 ¼ 233 μmol/L) and a maintained potency toward gram-positive organisms (►Fig. 16). With good inhibition to bacterial growth and high hERG selectivi-ty, 69 was selected as the candidate compound for a deeper preclinical study.
Renin, an aspartic protease, cleaves angiotensinogen to release inactive peptide angiotensin I (Ang-I) and controls the first and rate-limiting step of the renin-angiotensinaldosterone system. 50 Blockade of renin has been considered as a useful method to treat hypertension and to protect from end-organ damage. 51 In 2014, Ehara et al identified a new class of renin inhibitors. 52 Compound 70, a racemic cisconfigured 3,5-disubstituted piperidine with weak renin inhibition, was discovered from a piperidine-based combinatorial library through high-throughput screens. Guided by structure-based design, researchers discovered 71, whose activity was improved a lot but the hERG selectivity was low. Further modification was focused on improving hERG selectivity of this series of compounds. By introducing an (R)-configured hydroxyl at 4-position of the piperidine ring, 72 showed a higher renin inhibition and, most importantly, a good hERG selectivity with a hERG IC 50 value of above 30 μmol/L (►Fig. 17).

Conclusion
Chiral piperidine scaffolds can be used to alter structure patterns with normal orientation, and have fascinated chemists for exploring desired molecules in medicinal chemistry. Due to distinctive three dimensionality that chiral centers impart, chiral piperidine scaffolds usually exhibit good adaptation to the binding site of the protein, resulting in the enhancement in activity and selectivity as well as fewer off-target effects. 53 Besides, π-π stacking interaction of a molecule may be reduced by the introduction of a chiral piperidine ring, thus, drug solubility and PK properties will be improved. 54 Furthermore, Fig. 15 The influence introducing chiral center on piperidine ring on hERG selectivity of Akt1 inhibitors. Fig. 16 The influence of introducing chiral center on piperidine ring on hERG selectivity of topoisomerase II inhibitors. chiral piperidine rings were also associated with good hERG selectivity, and this may be due to the modification of polarity and lipophilicity of a molecule. 55 Taken together, chiral piperidine rings are a powerful tool for medicinal chemists to expand novel structures in the current drug discovery.