Design of Aminopeptidase N Inhibitors as Anti-cancer Agents - Journal

Perspective Cite This: J. Med. Chem. 2018, 61, 6468−6490

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Design of Aminopeptidase N Inhibitors as Anti-cancer Agents Sk. Abdul Amin, Nilanjan Adhikari, and Tarun Jha*

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Natural Science Laboratory, Division of Medicinal and Pharmaceutical Chemistry, Department of Pharmaceutical Technology, Jadavpur University, P.O. Box 17020, Kolkata 700032, West Bengal, India ABSTRACT: Aminopeptidase N (APN) is an important metalloenzyme. It regulates multivariate cellular functions by different mechanisms such as enzymatic cleavage of peptides. This may play a role in endocytosis and regulate signal transduction. APN, a member of the M1 zinc metallopeptidase family, plays crucial roles in a variety of functions such as migration and invasion, and angiogenesis and metastasis of tumor cells. Therefore, APN inhibitors may be useful for the treatment of cancer. In this Perspective, structure−activity relationships of APN inhibitors are discussed to get an idea of possible lead candidates. APN inhibitors should possess an aryl hydrophobic function along with a zinc binding group attached to the hydrophobic group(s) to achieve high potency. This and other design aspects of APN inhibitors are discussed in this Perspective.

1. INTRODUCTION Several types of human malignancies, including aggressive phenotypes and fast-growing tumors, express a variety of hydrolytic enzymes such as esterases, proteases, and peptidases. Aminopeptidase N (APN; EC 3.4.11.2) is one of the most-studied extracellular exopeptidases. It cleaves an amino acid residue or a peptide chain from the N-terminus of a substrate.1−11 APN is identical to the human lymphocyte surface cluster of differentiation CD13 antigen, as observed from the comparison between the sequences of cloned complementary DNA (cDNA).6−8 Unlike matrix metalloproteinases (MMPs), the activity of APN is similar to that of dipeptidyl peptidase IV (DPPIV, CD26), neutral endopeptidase (NEP, CD10), and serum γ-glutamyl transpeptidase (γ-GT, CD224).3,9 APN is also known as alanine aminopeptidase, particle-bound aminopeptidase, microsomal aminopeptidase, aminopeptidase M, peptidase E, and pseudo leucine aminopeptidase.10,11 Aminopeptidase N is a ubiquitous transmembrane ectoenzyme. It is found mainly in the liver and brush border of the kidneys, small intestine, and placenta. APN takes part in the terminal degradation of peptides present in the intestinal brush border. It is also involved in the inactivation of the neuropeptide hormones endorphins and enkephalins. Angiotensin III (AngIII), kinins, and chemotactic peptides (MCP-1) are also substrates of APN. In addition, APN/CD13 has been reported to be involved in the processing of antigens on the cell surface of antigen-presenting cells (APCs), as well as cleavage of the MHC class II moleculebound peptides.12−14 APN is also involved in a number of processes such as cell survival, cell migration, angiogenesis, blood pressure regulation, and viral uptake.5 It is now established that tumor cell invasion is enhanced upon APN expression. A strong correlation between the level of APN expression of a cell and its resultant invasive capacity makes APN an attractive target for the treatment of diseases including cancer. © 2018 American Chemical Society

Previous reviews highlight various biological aspects of APN and its inhibitors.1−5,15−20 In this Perspective, we focus on the development of lead inhibitors, with special emphasis on structure− activity relationships (SARs) that may lead to increased inhibitory potency. Moreover, recent advances, including X-ray co-crystal structures of human APN with bound inhibitors, are highlighted to allow comparison to previously reported inhibitors. The importance of different hydrophobic groups and zinc binding groups (ZBGs), as well as their relationships, is emphasized for lead APN inhibitors. The shape and flexibility of those molecules that are improved APN inhibitors are also explored. This Perspective may provide a stepping stone for the future development of anticancer drugs.

2. APN/CD13: AN EMERGING MEMBER OF M1 ZINC METALLOPEPTIDASES FAMILY According to the International Union of Biochemistry and Molecular Biology (IUBMB),10 peptidases are recognized in two sets of sub-subclasses, namely exopeptidases (EC 3.4.11−EC 3.4.19) and endopeptidases (EC 3.4.21−EC 3.4.24 and EC 3.4.99).10 Aminopeptidases (EC 3.4.11) belong to the sub-subclass exopeptidases. These act near the N-terminus of polypeptide chains. All of these peptidases are clustered into “clans”. The “clan” contains families of peptidases sharing a single evolutionary origin.1,11 To date, 15 clans of metallopeptidases have been reported (Figure 1). The clan MA contains more than 40 families of metallopeptidases.11 3. APN/CD13: A MOONLIGHTING PROTEIN APN/CD13 is recognized to be involved in multiple functions. These include a variety of mechanisms that elicit different biological effects. APN may act through versatile mechanisms such Received: May 29, 2017 Published: April 9, 2018 6468

DOI: 10.1021/acs.jmedchem.7b00782 J. Med. Chem. 2018, 61, 6468−6490

Journal of Medicinal Chemistry

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gene silencing by small interfering RNA demonstrated the possible mechanisms of APN in activating signaling pathways and gelatinase (MMP-2 and -9) activity. APN positively contributes to the invasion of human osteosarcomas enhanced by interleukin-6 (IL-6) and soluble IL-6 receptor (SIL-6R).27 IL-6-induced invasion of osteosarcoma was found to be reduced by the APN inhibitor bestatin (1, Figure 3).27 This is supported by the observations of Liang et al., where the correlation between enzymatic and signaling functions of APN is noticed in osteosarcoma invasion.28 APN plays a major role in modulating the signaling pathways through activating mitogenactivated protein kinase (MAPK), phosphoinositide 3-kinases (PI3K)/AKT, and NF-κB and subsequently triggerng gelatinase to promote migration and invasion in a variety of cancer cells (Figure 4).28 Previously, Liao and co-workers showed that gallic acid potentially inhibited the migration and invasion of human osteosarcoma U-2 OS cells through downregulation of mitogen-activated protein kinase (MAPK), protein kinase B (PKB), and protein kinase C (PKC) signaling pathways.29 These subsequently suppress the expression of gelatinases. Various markers including receptors for vascular growth factors, integrins αvβ3 and αvβ5, as well as matrix metalloproteases are found to be expressed in angiogenic blood vessels. During tissue angiogenesis, APN may facilitate endothelial cellular invasion.30 The interplay between the enzymatic and signaling activities of APN involves complex phenomena like invasion and angiogenesis. In addition, the role of APN in the metabolism of Ang III was investigated by Zini and co-workers.31 It is evidenced that the Ras-MAP kinase pathway plays a critical role in the signal transduction pathway and results in cell growth and differentiation. Santos et al. speculated about the relevance of APN/CD13-linking phosphorylation of mitogen-activated protein (MAP) kinases and increased [Ca2+]i efflux in the cells.12 Therefore, APN/CD13 may be involved in signal transduction via peptide hydrolysis.

Figure 1. Schematic representation of the 15 clans of metallopeptidases.

as (i) an enzyme for peptide cleavage, (ii) a receptor in endocytosis, and (iii) a signaling molecule in signal transduction (Figure 2). Therefore, APN/CD13 may be termed as a “moonlighting protein”.5,21

4. APN AS A POTENTIAL THERAPEUTIC TARGET Misregulation of APN has been found to evolve in almost all types of human malignancies,7 including breast cancer,32,33 cervical cancer,34 ovarian cancer,35,36 prostate cancer,37 non-small-cell lung cancer (NSCLC),38,39 liver cancer,40 colon cancer,41 scirrhous gastric cancer,42 pancreatic cancer,43,44 renal cell carcinoma (RCC),45 hepatocellular carcinoma (HCC),46 head and neck squamous cell carcinoma (SCC),47 melanoma,48 osteosarcoma,27 and thyroid cancer49 (Figure 5). Moreover, APN has been implicated in the degradation of the extracellular matrix along with immunomodulatory peptides, cytokines, neuropeptides, and angiotensins.50 Overexpression of APN has been observed in a number of T cells including CD4+, CD8+, and CD4+CD25+ regulatory T cells at the mRNA level; therefore, inhibition of APN may be useful for targeting disorders, specifically autoimmune diseases of the central nervous system (CNS), including experimental autoimmune encephalomyelitis (EAE).50 Consequently, it also has utility as a target for diseases with imbalanced T cell response, such as inflammatory bowel disease (IBD).51 Again, overactivity of APN has also been found in collagen vascular diseases (CVDs) along with systemic sclerosis, rheumatoid arthritis (RA), dermatomyositis, and systemic lupus erythematosus (SLE).52 Not only that, APN overexpression has also been established for diabetic nephropathy and renal damage.52 Therefore, targeting APN is an important therapeutic approach that may be utilized for the treatment of these diseases. Nevertheless, APN is also a potential therapeutic

Figure 2. Functions of human APN/CD13.

Curiously, APN takes part in cholesterol crystallization and its uptake. However, mammalian APN has been reported to serve as one of the important receptors for canine enteric coronavirus (CCoV), feline enteric coronavirus (FCoV), porcine transmissible gastroenteritis virus (TGEV), and human respiratory coronavirus 229E (HCoV229E).5,14 Human APN (hAPN) is a type-2 membrane glycoprotein. It contains an N-terminal membrane anchor that may lead to an arch-like arrangement on the cell surface.22 Due to this arch-like architecture, each monomer of hAPN can form an open and a closed conformation, as suggested by Wong et al.22 Conversion from the open or open dimer to the closed or closed dimer may result in a large structural change.22 These conformational variations may underlie the basis of the signal transduction events associated with APN. The signaling capacity of APN/CD13 depends on APN-associated “auxiliary” proteins5,17 like reversion-inducing cysteine-rich protein with tumor-associated antigen L6,23 reversion-inducing cysteine-rich protein with kazal motifs (RECK),24 galectin-3,25 and galectin-4.26 Results of APN 6469



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Figure 3. Bestatin and its derivatives as APN inhibitors.

Figure 4. Probable signaling pathways activated by APN while binding to a specific substrate (red star) that mediates expression of gelatinase (MMP-2 and -9) and finally promotes migration and invasion of cancer cells. Focal adhesion kinase (FAK), growth factor receptor-bound protein 2 (GRB2), phosphoinositide 3-kinases (PI3K), protein kinase kinase 3 (MKK3), extracellular signal-regulated kinases (ERK), and c-jun N-terminal kinase (JNK).

target for cerebral ischemia and neuronal degeneration.53 Apart from these above-mentioned diseases, APN has also been found to take part in mediating infective viral diseases, namely human cytomegalovirus (HCMV) and human coronavirus (HCoV229E) infections.54

homology to related enzymes, namely aminopeptidase A,55 aminopeptidase B,56 leukotriene A4 hydrolase (LTA4H),57 thyrotropinreleasing hormone-degrading enzyme (THR-de), puromycinsensitive aminopeptidase (PSA),58 Escherichia coli PepN, and rat vesicle protein Vp 165.55−58 The GAMEN and HExxHx18E sequence motifs are the major characteristic features of the M1 zinc metallopeptidases family.59−65 APN is anchored in the plasma membrane with a short N-terminal cytoplasmic domain along with a transmembrane domain (Figure 6).

5. STRUCTURAL ASPECTS OF APN APN is a member of the M1 zinc metallopeptidase family [clan MA ≫ subclan MA (E) ≫ family M1]. It shows structural 6470



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whereas the leucine residue directs toward the S1′ pocket of hAPN (Figure 7).22 Moreover, the carboxylic acid group associated with the leucine side chain coordinates the catalytic Zn2+ ion. The carbonyl function of the carboxylic acid group of 1 forms a hydrogen-bonding interaction with the p-hydroxy group of Tyr477. In addition, the isobutyl group adjacent to the carboxylic acid group makes a π-alkyl interaction with the phenyl ring of Tyr477. Another π-interaction is noticed between the phenyl ring of 1 and Ser469. Moreover, catalytic amino acid residues such as Ala353, Val385, and His388 and a few bound water molecules (HOH1118, HOH1176, HOH1315, HOH1382, HOH1445, HOH1483, HOH1688, HOH1733, HOH1919) also control the binding interactions between the ligand and hAPN. The solventaccessible surface (SAS) contour also suggests that a part of the APN binding site is highly solvent exposed (Figure 8). Therefore, water-mediated interactions play crucial roles in the ligand−receptor interaction. The α-hydroxyl function and carbonyl oxygen atom of 1 form water-mediated interactions with hAPN. However, in other complexes,61,63,64 the carbonyl oxygen atom and the α-hydroxyl function are found to coordinate the catalytic zinc ion. In the AngIV complexed with the APN enzyme, the carbonyl oxygen atom of the N-terminal valine residue accepts a hydrogen bond from Tyr477. In addition, two glutamic acid residues (Glu355 of 352GXMEN356 and Glu411 of 388HExxHx18E411) and one glutamine residue (Gln213) are involved in a hydrogen-bonding interaction with the α-amino acid of valine residue for the stabilization of substrate binding. The valine, tyrosine, and isoleucine residues of the substrate (AngIV) are found to insert deeply into the APN active pockets. Moreover, the side chain of valine is surrounded by a hydrophobic loop formed by Phe472, Met354, Ala351, Gln211, and Gln213. The amide nitrogen and carbonyl oxygen atoms of Tyr residue (P1′) formed hydrogen-bonding

Figure 5. Implications of APN in different cancers.

In 2012, Wong et al. reported X-ray crystal structures of human aminopeptidase N (hAPN, a 967-residue type-2 membrane glycoprotein) complexed with peptidomimetic inhibitors including bestatin (1) and amastatin (2) (Figure 3) as well as the peptide hormone angiotensin IV.22 Each monomeric ectodomain of hAPN possesses a seven-domain organization (domains I−VII) that is characteristic of M1 metallopeptidases. During interactions, each of the monomeric units covers a surface area of approximately 840 Å2. The hAPN contains the characteristic consensus motifs 352 GXMEN356 and 388HExxHx18E411 and a thermolysin fold.22 The Zn2+ ion is found to coordinate with three amino acid residues of the 388HExxHx18E411 motif, i.e., His388, His392, and Glu411 of the native enzyme. The orientation of the dimer structure of hAPN covers an area of 131 Å × 62 Å that has similarity with the porcine APN of 135 Å × 55 Å.66 From the hAPN X-ray crystal structure in complex with 1 (PDB: 4FYR), it is observed that the phenylalanine residue of 1 is inserted deeply into the S1 subsite,

Figure 6. Schematic seven-domain hypothetical organization of human APN/CD13. The location of the substrate/inhibitor binding site is highlighted as a red dotted circle between domains V and VI. 6471



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Figure 7. Bestatin (1)-bound X-ray crystal structure of human APN (PDB: 4FYR) [bestatin (stick) with metal zinc ion (dark blue ball)]. In the inset, structure-based hydrophobic contour at the hAPN active site (PDB: 4FYR), mapped with 1 (in stick), is highlighted along with important amino acid residues (shown in lines).

polar interactions. Consequently, polar side chains show additional binding interactions. Generally, the APN-binding affinity of the substrate or inhibitor is correlated with the extent of hydrophobic interactions. Moreover, nonpolar side chains containing amino acids show high APN-binding affinity.

6. APN INHIBITORS A broad range of specificity was observed among N-terminal amino acid (P1) of the peptide substrates of M1 zinc metallopeptidase family members.22,68 A number of clinical assessments were conducted with APN inhibitor bestatin (1, Figure 3) for a variety of malignancies, including cancers of the stomach, esophagus, and lung, melanoma, lymphomas, and leukemias.7,69 Effective results were also obtained in clinical studies upon treatment with bestatin for NSCLC and squamous cell carcinoma.7 Recently, it has been successfully studied in phase 2 clinical trials for the treatment of lymphedema.70,71 Tosedostat/CHR-2797 (3) (Figure 3) has undergone extensive phase 1 and phase 2 clinical evaluation for the treatment of acute myeloid leukemia, pancreatic cancer, and NSCLC along with advanced solid tumors.72,73 It was found to be 300-fold more potent than bestatin and showed potent inhibition toward leucine aminopeptidase (LAP) (IC50 = 100 nM) with 2-fold selectivity over APN (IC50 = 220 nM). The active form of tosedostat after hydrolysis (CHR-79888) (4, Figure 3) became potent and selective toward LTA4 hydrolase (IC50 = 8 nM) while exhibiting good efficacy toward APN (IC50 = 190 nM) and LAP (IC50 = 30 nM).73 Distinguishable differences in the binding pattern of cytosolic and membrane-bound aminopeptidase have also been documented. The cytosolic aminopeptidase LAP binds to dipeptide inhibitors strongly compared to the tri- or tetrapeptide inhibitors, whereas APN, a membrane-bound aminopeptidase, coordinates the trior tetrapeptide inhibitors more strongly.68 On the basis of structural arrangements, APN inhibitors are broadly classified into two major groups: (A) amino acid-based inhibitors and (B) nonamino acid-based inhibitors. 6.1. Amino Acid-Based Inhibitors. 6.1.1. L-Leucine Derivatives. The basic research on the aminopeptidase inhibitors was started during the 1970s. Umezawa et al. isolated 1 [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine] from

Figure 8. Structure-based solvent-accessible surface (SAS) contour at the hAPN active site (PDB: 4FYR), mapped with 1 (in stick). APN subsites and important catalytic amino acid residues are highlighted for better understanding.

interactions with Ala353 and Gly352, respectively. Apart from the π-stacking interaction with His388 and Val385, the hydroxyl group of Tyr forms a water-mediated hydrogen bond with Glu418. It is observed from the hAPN inhibitor amastatin− enzyme complex that the α-hydroxy function of 2 coordinates the catalytic Zn2+ ion. The leucine residue of 2 exhibits similar interaction compared to the valine residue of AngIV at the S1 site. Moreover, the valine moiety of 2 exhibits an interaction with the backbone residues similar to the Tyr residue of AngIV at the S1′ site. Indeed, recent crystal structures of APN complexed with various amino acids demonstrated the molecular basis for the P1 substrate specificity.67 Joshi and co-workers demonstrated the key interactions that dictate the substrate specificity.67 The S1 pocket contains hydrophobic walls (Met349, Phe467, and Phe472) with an open-ended architecture that allows hydrophobic P1 residues for specificity (Figure 7). Meanwhile, the positively charged side chains containing amino acids (lysine, arginine) may accommodate well into the S1 pocket. These side chains interact with Met349, Phe467, and Phe472 in the S1 pocket to form hydrophobic and 6472



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Streptomyces olivoreticuli (MD976-C7).74 The structure of 1 (Figure 3) resembles the naturally occurring dipeptide Phe-Leu substrate. Bestatin (Ubenimex) stimulated numerous cells to secrete cytokines and activated macrophages. The mitogenic effects of bestatin on T cells were also reported. APN is one of the major bestatin-sensitive targets, and 1 inhibits APN.75 On the immune cell surface, it might prevent cytokine inactivation. On the surface of APCs, bestatin showed immunogenic potential by preventing epitope trimming. It also arrested tumor cell growth and differentiation by modulating the role of signal transduction mechanisms of APN.75 As a consequence of the slow binding process, increasing the peptide chain length of bestatin analogues resulted in more potent APN inhibitors. Bestatin analogue 2 (Figure 3) was a slowbinding competitive inhibitor of aminopeptidase M (EC 3.4.11.2), having an inhibition rate constant (Ki) of 1.9 × 10−8 M.68,76,77 Umezawa et al. further reported another bestatin analogue, namely actinonin (5, Figure 3), having an aminopeptidase M (EC 3.4.11.2) inhibition constant (Ki) of 1.7 × 10−7 M.78 This molecule was found to be a competitive inhibitor of aminopeptidase M (EC 3.4.11.2: IC50 = 400 nM). Moreover, probestin (6, Figure 3), isolated from Streptomyces azureus MH663-2F6, inhibited aminopeptidase M with an inhibition constant of 1.9 × 10−8 M.79,80 Valistatin (7, Figure 3) also inhibited aminopeptidase enzyme. Apart from these aminopeptidase inhibitors, a new compound, namely 3-amino-2-hydroxy-4-phenylbutanoylvalylisoleucine (8, Figure 3), was found in Streptomyces parvus strain HCCB10043.81 This molecule showed greater APN inhibitory activity. LYP (9, Figure 9), an ester derivative of 1, was found to be an effective APN inhibitor, as evidenced by in vivo and in vitro experiments conducted by Luan et al.82

With the aim of improving the water solubility of 1, Luan et al. elongated the bestatin structure at the C-terminal position by incorporating the L-serine benzylamide function.83 The designed compound LYP3 (10, Figure 9) possessed more than 2-fold improved solubility compared to bestatin in PBS (LYP3: 85.17 mmol/L, bestatin: 35.70 mmol/L). Compound 10 showed higher APN inhibition (IC50 = 7.2 μM) than 1 (IC50 = 14.9 μM). The hydroxyl functionality of the L-serine moiety may be involved in the formation of a hydrogen-bonding interaction with an amino acid residue at the S2′ pocket of APN (Figure 9). Interestingly, compound 10 exhibited better antiproliferative activity against higher APN-expressing tumor cell lines (ES-2: IC50 = 12.6 μM and A-549: IC50 = 36 μM) compared to tumor cell lines with lower APN expression (MDA-MB 231 IC50 = 260 μM). Compared to 1, compound 9 more effectively suppressed APN activity on the surface of ES-2 cells.84 There has been considerable interest in the optimization of 1 over the past three decades. Chen et al. interestingly elaborated the progress in the development of bestatin derivatives with special emphasis on structural optimization.85 From the overall SAR study of these bestatin derivatives, some crucial observations may be inferred: (i) the β-amino α-hydroxy amide motif is crucial for binding with aminopeptidases, (ii) the S-configuration of the carbon atom at the second position of bestatin is important, (iii) addition of amino acid or peptide to the Leu residue of bestatin may improve the inhibitory activity, and (iv) benzyl group replacement may alter the inhibitory properties. 6.1.1.1. Leucine-Ureido Derivatives. Su and co-workers reported some ureido derivatives having APN inhibitory activity.86 Substitution at the α-position of the hydroxamate group and the terminal amide nitrogen atom of the urea moiety was done by using different alkyl, thioalkyl, or aryl functions. The tert-butyl function at the urea terminal yielded an inactive molecule (IC50 > 100 μM), whereas benzyl or phenethyl substituents resulted in moderate APN inhibition. Moreover, carbamate analogues were less effective compared to the corresponding ureido analogues. The iso-butyl, sec-butyl, or methylthioethyl substitution at the α-position of the hydroxamate moiety produced better APN inhibitory activity compared to the smaller methyl and iso-propyl substitutions. However, benzyl substitution at this position yielded a potent APN inhibitor, 11 (Figure 10). It was investigated for in vitro ES-2 cell proliferation along with anti-migratory and antiinvasive properties. Compound 11 potentially blocked ES-2 cell invasion in a dose-dependent manner and exhibited significant activity against proliferation of ES-2 cells. Su et al. further extended their study by synthesizing another series of ureido derivatives.87 The sec-butyl substitution at the α-position of the hydroxamate group yielded inactivity, whereas iso-butyl substitution resulted in better efficacy. Again, a phenyl analogue at this position yielded less efficacy compared to an iso-butyl analogue. A phenylpropyl analogue at the terminal amide function of the urea moiety produced lower activity compared to the benzyl analogue. Interestingly, heterocyclic thienylmethyl substitution at the same position exhibited better APN inhibition compared to the furyl analogue. In addition, α-naphthyl and α-naphthylmethyl substitutions yielded potent APN inhibitors (IC50 = 99 nM and 50 nM, respectively), whereas bigger biphenyl substitution resulted in a drastic loss of APN inhibition. However, a substituted phenyl or benzyl group at the urea terminal also resulted in better APN inhibition. Electron-donating functions (such as methoxy and methyl) at the aryl group produced better efficacy than an electron-withdrawing group (such as fluoro). However, m-methoxy substitution resulted in more than

Figure 9. Comparison between bestatin and its analogues for their binding interaction with APN.

This compound showed better water solubility compared to 1. Compound 9 was accommodated well into the bigger S2′ pocket of APN. Moreover, the tertiary amino moiety formed a hydrogenbonding interaction with Arg825 at the APN active site (Figure 9). 6473



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ring of 15 with a benzyl moiety resulted in comparable activity. The o-bromophenyl analogue 16 possessed the highest APN inhibitory activity (IC50 = 93 nM) among these compounds and was more active than the corresponding o-bromobenzyl compound 17 (IC50 = 130 nM) (Figure 11). In another study, some leucine ureido derivatives were designed and synthesized by Ma et al.89 Compound 18 (Figure 11) was found to be the most active APN inhibitor of this series, having anti-invasive activity as evidenced by the Matrigel invasion assay carried out against ES-2 cells. Compound 19, containing the 2,3-dimethylbenzyl group, showed good inhibitory activity, but the activity was compromised in the 2,6-dimethylphenyl-containing 18. Compound 18 also showed potential anti-metastatic effect (92% inhibitory rate) in a mouse hepatoma-22 (H22) pulmonary metastasis model at a dose of 80 mg/kg. Apart from the above-mentioned amino acid derivatives, a number of APN inhibitors have been reported. However, these amino acid derivatives showed moderate to poor activity against APN. In order to provide a better understanding of the SAR, a short description of each of the classes is provided. 6.1.2. L-Lysine Derivatives. Wang et al. designed some α-aminoand N6-amino-substituted L-lysine-based MMP-2 and APN inhibitors.90,91 These molecules showed better MMP-2 inhibition compared to APN. Compound 20 exhibited the highest APN inhibition in this series of compounds along with moderate antiproliferative activity against a panel of cancer cell lines, including HL-60, ES-2, A-549, H7402, and PLC (Figure 12A). The molecular docking study suggested that the phenyl group of 20 interacted with Phe472 through π−π stacking interaction. The sulfonyl function was suggested to form hydrogen-bonding interactions with Tyr477, Glu418, His388, Arg381, and Gly352, whereas the hydroxamate group formed a bidentate chelate with the Zn2+ ion at the enzyme active site. Addlagatta and co-workers reported the crystal structure of APN from E. coli (ePepN) complexed with L-lysine (21, Figure 12B).62 Compound 21 occupied the S1 pocket of APN but could not stretch in the S1′ pocket. In order to address this concern, Wang et al. modified the L-lysine part and designed some L-lysine-based APN inhibitors.92 These molecules were found to occupy hydrophobic S1 and S1′ pockets concurrently (Figure 12C), similar to 1. One compound, 22 (APN IC50 = 9600 nM), displayed a more or less similar cytotoxicity profile compared to 1 when tested in a

Figure 10. Structures of leucine ureido derivatives having APN inhibitory potency, 11−14.

2-fold better efficacy than the corresponding ortho and para counterparts. In addition, replacement of the terminal amide functions of the urea moiety with ether or methylene functions led to inactivity. The three compounds 12−14 exhibiting APN inhibitory potency at nanomolar concentrations also showed anti-invasive properties against ES-2 cells in vitro without obvious cytotoxicity (Figure 10).87 Interestingly, a significant reduction of the B16BL6 pulmonary colonies was noticed upon administration of 12−14, suggesting their potential anti-angiogenic and anti-metastatic effects. In another study, Ma and colleagues optimized the structure of 15, which showed some potency toward APN inhibition (Figure 11).88 All of these designed compounds displayed more or less similar efficacy with the lead compound 15, although these molecules were inactive against histone deacetylase (HDAC). Fruitful results were obtained when the terminal phenyl moiety of 15 was substituted with a halogen at the ortho position. Replacing the phenyl

Figure 11. Structural modification of 15 resulted in more active APN inhibitors. 6474



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6.1.2.1. Lysine-Ureido Derivatives. As the ureido moiety is a good bioisostere of the amide group, Su and co-workers prepared L-lysine ureido derivatives to acquire more potent APN inhibitors.93 The ester and acid analogues were inactive against APN (IC50 > 50 μM), whereas the corresponding hydroxamates were comparatively better APN inhibitors (IC50 < 25 μM). As far as the SAR of these compounds was concerned, only hydroxamate-based L-lysine ureido derivatives showed better APN inhibitory activity compared to the corresponding carboxylic acids and their ester analogues (Figure 13). The iso-butyl analogue 23 was found to be superior to the methyl, ethylthiomethyl, and benzyl analogues (Figure 13). 6.1.3. L-Arginine Derivatives. A series of L-arginine derivatives were synthesized and evaluated for APN inhibition by Mou et al.94 Compounds having 2,4-dichlorophenyl (24: APN IC50 = 5300 nM) and 3-nitro-4-bromophenyl (25: APN IC50 = 5100 nM) functionality exhibited 66-fold and 46-fold APN selectivity over MMP-2, respectively (Figure 14). Subsequently, the introduction of bi- and tripeptides into the 95 L-arginine moiety enhanced APN inhibition. Due to the elongation of the side chain length, these bi- and tripeptides exhibited better APN inhibition compared to the previously reported compounds.94 Two aromatic rings of the active compounds 26 (APN IC50 = 4200 nM) and 27 (APN IC50 = 4300 nM) occupied the S1′ and S2′ hydrophobic pockets at the APN active site (Figure 14). The hydroxyl and amide functionalities of the hydroxamate moiety interacted with the carbonyl group of Ala262 and the carboxyl function of Glu264, respectively. In a recent study, Mou and co-workers designed and synthesized a series of L-arginine-based APN inhibitors.96 The SAR data suggested that the phenyl group with two electron-withdrawing substituents at the carboxamide moiety resulted in better efficacy compared to the corresponding compounds with single electronwithdrawing substituents. Moreover, a bulky naphthyl group at the same position was tolerated for APN inhibition.96 6.1.4. L-Aspartic Acid Derivatives. Liu et al. initially selected L-aspartic acid and further introduced a cinnamic acid moiety into the L-aspartic acid scaffold to design new APN inhibitors.97 Most of these N-cinnamoyl-L-aspartic acid derivatives were moderate APN inhibitors compared to MMP-2. 6.1.5. L-Isoserine Derivatives. More than four decades ago, Nishizawa and co-workers demonstrated that L-isoserine-L-leucine dipeptide might be a moderate inhibitor of aminopeptidase B.98 Inspired by their work, Yang et al. synthesized a variety of L-isoserine

Figure 12. (A) Structures of L-lysine-based 20. (B,C) Comparison between the L-lysine (21) and its analogue (22) for their binding interaction with APN.

panel of tumor cell lines (HT-60, ES-2, K-562, A-549, H7402, and PLC).

Figure 13. SAR of the L-lysine ureido derivatives on the template structure of 23.

Figure 14. Structural modifications of 24 resulted in better active APN inhibitors, 25−27. 6475



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derivatives as APN inhibitors.99 Interestingly, these carboxylate ester-containing L-isoserine molecules showed poor APN inhibitory activity compared to their corresponding carboxylic acid analogues. Moreover, the iso-propyl moiety directed toward the S2′ pocket appeared to be important for APN inhibition based on the SAR observations. The most potent compound, 28 (APN IC50 = 12 200 nM) (Figure 15), exhibited lower antiproliferative

Figure 16. (A) Development of 30 from 28. (B,C) Structure of APN inhibitors 31 and 32 and their APN inhibitory activity.

compared to the corresponding non-hydroxamate analogues. Moreover, 32 showed good antiproliferative effect against HL-60 tumor cell line. 6.1.7. Miscellaneous Amino Acid-Based Inhibitors. Gredičak et al. reported a structurally distinct group of compounds (enediyne−peptide conjugates) as APN inhibitors.108 In the enediyne−peptide conjugates, hydrophobic amino acids (such as alanine, valine, leucine, and phenylalanine) were taken into account as the carrier of enediyne function. The enediyne scaffold was embedded into a 12-membered-ring structure. The amino group was attached to the lysine residue through a 5-aminopentanoic acid linker function. Recently, Joshi and colleagues developed some substrate-based cyclic peptide inhibitors of APN.67 The lead peptide inhibitor cycLHSPW showed outstanding specificity toward APN over other cancer-associated proteases, namely aminopeptidase A, chymotrypsin, KLK7, trypsin, PSA, and exopeptidase FAP. The cycLHSPW also decreased tumor growth in PC3 and DU145 xenografts. 6.2. Non-Amino Acid Inhibitors. Non-amino acid-based compounds have also been found to exert potent APN inhibition. A number of non-amino acid APN inhibitors based on different scaffolds or moieties, such as mercaptan, boronic acid, phosphonic and phosphinic acids, 3-amino-2-tetralone, cyclic imide, pyrrolidine, AHPA, and indoline-2,3-dione, were also found to impart promising APN inhibitory effects. 6.2.1. Mercaptan Derivatives. Mercaptan derivatives such as L-leucinethiol (33, Figure 17A) exhibited competitive LAP inhibitory activity (Ki = 22 nM).109 A leucinethiol molecule incorporating a hydrophobic iso-butyl chain may be equivalent to the α-amino moiety of the specific substrates of APN. Fournie-Zaluski and co-workers modified the leucinethiol derivative 33 and reported a number of potent aminopeptidase inhibitors.110 They reported some L-phenylalanine- and thiol-based compounds and screened these against APN purified from hog kidney using the substrate [3H]Leu-enkephalin. Among these compounds, β-aminothiols showed effective APN inhibition. The phenylalanine-based carboxylic acid, phosphonic acid, and hydroxamic acid analogues were less potent APN inhibitors. However, the corresponding thiol analogue enhanced the activity by a minimum of 2500-fold over these former analogues. Increasing the bulky hydrophobic aromatic group at the lateral chain of the β-aminothiols resulted in lower APN inhibition. The flexibility with different aryl and cycloalkyl functions (such as p-methoxybenzyl, β-naphthylmethyl, benzyloxymethyl, benzyloxythiomethyl, and

Figure 15. Development of 29 from 28 and 1.

activity compared to 1 against a human promyelocytic leukemia cell line (HL-60 IC50 = 170 nM) and against a human ovarian carcinoma cell line (SKOV-3 IC50 = 380 nM). Zhang et al. investigated a new series of 3-amino-2-hydroxyl-3phenylpropionic acid analogues derived from the key scaffolds of a lead molecule 28 and 1.100 Compounds without a hydroxamate functionality at the lateral position exhibited poor APN as well as MMP-2 inhibitory activity. 3-Amino-2-hydroxyl-3-phenylpropionic acid derivatives with hydroxamate substitution were found to be pivotal for inhibition of these metalloenzymes. Compound 29 (Figure 15) showed more than 200-fold APN selectivity over MMP-2 inhibition (APN IC50 = 1260 nM, MMP-2 IC50 = 212 970 nM) and also possessed an almost equal antiproliferative effect against human ovarian carcinoma cell line ES-2 compared to 1. Moreover, two series of L-isoserine tripeptides were reported by Pan et al. to have promising APN inhibitory activity.101 The best active compound of this series, 30 (Figure 16A), possessed good APN inhibitory activity (IC50 = 2510 nM). 6.1.6. L-Isoglutamine Derivatives. Wang and co-workers previously observed that L-isoglutamine derivatives exhibited antitumor activity and showed moderate APN inhibition.102 In order to improve the biological activity toward metalloenzyme inhibition, Li et al. cyclized the L-isoglutamine derivatives by fusing the amide nitrogen atom with the carboxylic acid moiety.103 These molecules showed comparatively better MMP-2 inhibitory activity than APN. However, one of the molecules, compound 31 (Figure 16B), displayed good APN inhibitory activity (IC50 = 5200 nM). In a another study, Li et al. synthesized and performed in vitro metalloenzyme inhibition assays with peptidomimetic N2-phenylacetyl-L-isoglutamines.104,105 These compounds, inspired by antineoplastin,106 displayed anti-tumor activity and showed moderate APN inhibition. Meanwhile, the same research group again designed some L-isoglutamine derivatives, and this time the activity profile was increased upon modification of ZBG.107 Hydroxamate-based derivative 32 (Figure 16C) showed better APN inhibitory properties 6476



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Figure 17. (A) Structure of L-leucinethiol (33). (B) SAR of leucinethiol-based APN inhibitors. (C) Developing cascade involved in the designing of better active APN inhibitor PC18 (37).

Figure 18. (A) Structure of pseudotripeptide thiol-containing 38. (B) SAR of β-aminothiols having promising APN inhibitory activity. (C) Comparison of some β-aminothiols, 39−46.

was found to be 125-fold and 2150-fold less active against APB (Ki = 1000 nM) and APA (Ki = 17 200 nM), respectively than APN (Ki = 8 nM). Results of the in vivo activity of 37 tested in mice brain were very encouraging. This APN inhibitor induced [3H]-AngIII (radioactive angiotensin III) accumulation and also increased its half-life. Interestingly, 37 increased the release of vasopressin in a dose-dependent manner by enhancing the halflife of endogenous AngIII in the mouse brain. After administration of 37 at a dose of 100 μg, a more than 220% increase in vasopressin release was observed. David et al. designed a series of pseudotripeptidic thiol compounds having potency against APA and APN.112 Initially, the P1 substituents of these molecules were varied, taking into account isoleucine or tyrosine as P1′ substituents and aspartic acid as the P2′ substituent. All of these compounds resulted in higher potency and selectivity toward APA over APN. The (S)-isoleucine derivative 38 (Figure 18A), having a carboxyethyl group as a P1 substituent, showed the highest APN inhibition (Ki = 120 nM) of this series, but it was 9-fold more selective toward APA (Ki = 13 nM). While keeping the sulfonate ethyl group as the P1 substituent, modifying the amino acid residue toward the S1′ and S2′ sites resulted in highly potent and selective APA inhibitors.112

cyclohexylmethyl) reduced the activity, but all of them displayed activity at nanomolar concentrations (IC50 ≤ 130 nM). In addition, decreasing the length of the phenyl group slightly increased the activity compared to the benzyl analogue (Figure 17B). The S1 pocket of APN is deep though it is not very large. Therefore, side chains of these inhibitors should be moderate in size to fit the pocket optimally. The inhibitory potencies of these β-aminothiols may be dependent on the size along with hydrophobicity of the lateral chain. Derivatives bearing aliphatic side chains, compounds 34−37, were found to be slightly more active than compounds containing aromatic or cyclic moieties (Figure 17C). Interestingly, benzyl substitution at the amine terminal as well as the thiol function drastically reduced APN inhibition. Moreover, the benzyl side chain of the phenylalanine-based thiol analogue was modified to achieve higher APN inhibitory potency. Furthermore, chain elongation resulted in comparatively better APN inhibition (compound 34 vs 36, compound 35 vs 37, Figure 17C). 2-Amino4-methylsulfonyl butanethiol (PC18, 37), a methionine thiol analogue, emerged as an APN active compound having IC50 = 11 nM. Réaux et al. further investigated the in vitro selectivity of 37 toward three aminopeptidases, i.e., APN, aminopeptidase B (APB), and aminopeptidase A (APA).111 Compound 37 (Figure 17C) 6477



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Chauvel et al. synthesized some β-aminothiols and screened them against APA and APN.113 Most of these compounds were good APN inhibitors.113 The carboxylic acid analogue with trimethylene units was a highly potent APN inhibitor, whereas increasing the alkyl chain length between the carboxylic acid and β-amino function retained potent APN inhibition (Figure 18B). However, branching in the alkyl chain reduced both APN and APA inhibitory potency. An interesting observation from the work of Chauvel and co-workers was that APN inhibitory activity increased if the carboxylate moieties of 39 (APN Ki = 15500 nM) and 40 (APN Ki = 330 nM) were replaced with a hydroxamic acid, exemplified by 41 (APN Ki = 37 nM), or a thiol, compound 42 (APN Ki = 32 nM), as depicted in Figure 18C.113 Moreover, compounds 41 and 42 showed more than 50-fold APN selectivity over APA. However, the corresponding sulfone and phosphonic acid derivatives exhibited higher potency and selectivity toward APA over APN. Therefore, the decrease in the value of pKa of the side-chain component of these derivatives may be positively correlated with the binding affinity toward APN. In a continuation of their earlier observation, Chauvel et al. further reported some thiol-based inhibitors for both APN and APA.114 The (S)-Glu-thiol was a highly potent but nonselective APN inhibitor, whereas the corresponding (R)-analogue drastically reduced APN inhibition but was selective toward APA. Methyl substitution at the amine group of the (S)-Glu-thiol derivative 43 reduced APA inhibition by 5-fold and exhibited a high degree of selectivity over APN (Figure 18C). Displacement of the sulfhydryl group from the β to the γ position also yielded the highly selective APA inhibitor 44. Compound 45, having a p-benzylic acid moiety at the side chain, resulted in moderately active and nonselective APN inhibition over APA. Moreover, replacement of the phenyl moiety of the p-carboxymethylphenyl analogue with a cyclohexyl group resulted in the most potent APN inhibitor (46: IC50 = 35 nM) of this series, with 80-fold selectivity over APA (Figure 18C). 6.2.2. Boronic Acid Derivatives. Aliphatic boronic acid derivatives have been proposed to act as transition-state analogues.115 1-Butaneboronic acid significantly increased the uptake of one g-atom of the Zn2+ ion from metal ion buffer.116 Some α-aminoboronic acid derivatives were synthesized by Shenvi et al.117 These compounds, 47−50, exhibited good time-dependent inhibition against cytosolic and microsomal leucine aminopeptidases (Figure 19A). Farsa et al. compared the APN inhibitory activity of two different series of compounds, namely 4-phenoxy-[1,3,2]dioxaborolan2-ol-containing compound 51 and 3-phenoxy-1,2-propandiolcontaining compound 52.118 The former showed comparatively lower APN inhibition (Figure 19B). 6.2.3. Phosphonic and Phosphinic Acid Derivatives. Due to the tetrahedral shape of the phosphonate or phosphinate groups, phosphonic acids may offer some important structural and electronic features to bind to aminopeptidases. The phosphonate or phosphinate groups serve as weak zinc binder moieties. In 1987, Giannousis and Bartlett reported a group of phosphorus amino acids and dipeptide analogues having LAP inhibitory activity.119 Inspired by their work, Lejczak and colleague synthesized some aminophosphonate derivatives having inhibitory activity against cytosolic (EC 3.4.11.1) and microsomal (EC 3.4.11.2) aminopeptidases.120 Compound 53 (Figure 20) showed the most potent microsomal aminopeptidase inhibitory activity (Ki = 870 nM) through a time-dependent mechanism. Aminophosphinic acid derivatives bearing a hydrophobic side chain were synthesized as APN inhibitors.121 The hydrophobic

Figure 19. (A) Structures of boronic acid derivatives 47−50 active against cytosolic and microsomal leucine aminopeptidases. (B) Structures of 51 and 52.

Figure 20. Structures of phosphonic acid derivatives 53−57 as APN inhibitors.

side chain interacts with the S1 pocket of APN. Moreover, the coupling of the α-aminophosphinic acids with the dipeptide PhePhe analogue may engage the S1′ and S2′ pockets of APN enzyme. This study resulted in new “phosphino” peptides designed as transition-state analogues that may interact with the S1, S1′, and S2′ subsites of the APN, showing good inhibitory activities. Compounds 54 and 55 showed significant inhibitory activities and selectivities toward APN over APA and APB (Figure 20). Grembecka et al. reported some phosphinate dipeptide derivatives as promising inhibitors of APN and LAP.122 Most of these compounds showed better affinities toward LAP. However, 56 (Figure 20) was the best APN inhibitor (Ki = 36 nM) of this series, with 2-fold selectivity over LAP (Ki = 67 nM). Drag and co-workers optimized the shape of the side chain of structurally diverse α-aminophosphonates directed toward the S1 pocket of LAP.123 Increases in LAP and APN inhibitory activities were observed for compounds having increased sidechain length. The optimized P1 side chain of three carbon atoms separating the aromatic group and aminoalkylphosphonate functionality may be required for binding at the S1 pocket of APN. Compound 57 (Figure 20) showed the most potent APN inhibition among these compounds.123 6478



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Motivated from the earlier work119 (LAP inhibitor 58: Ki = 56 000 nM), Drag et al. modified the LAP inhibitor 58 to improve the binding affinity toward aminopeptidase and designed 59 as a lead molecule (Figure 21).124

Figure 22. Structures of phosphonic acid derivatives 61 and 62 as APN inhibitors.

also, the cyclic aliphatic analogues (such as c-hexyl and c-heptyl) yielded higher potency and selectivity toward HsAPN compared to the corresponding linear or branched alkyl functions substituted at the terminal amide moiety. Furthermore, the p-methoxyphenethyl analogue 62 afforded potent HsAPN inhibition (Ki = 78 nM) with a high degree of selectivity over NmAPN, SsAPN, and LAP (Figure 22). 6.2.4. 3-Amino-2-tetralone Derivatives. Non-peptidic APN inhibitors like 3-amino-2-tetralone derivatives competitively inhibited membrane-bound Zn2+-dependent aminopeptidase (EC 3.4.11.2) that was isolated from porcine kidney.127 Compounds 63 and 64 (Figure 23) were reported as equipotent to 2 and showed

Figure 21. Structures of phosphonic acid-based LAP inhibitor 58 and promising phosphonic acid-based APN inhibitors 59 and 60.

Modifying the formerly mentioned 59124 (Figure 21), Grzywa and co-workers designed a new class of α-aminoalkyl-(N-substituted)thiocarbamoylphosphinates and found some compounds to be promising APN inhibitors.125 Compound 60 (Figure 21) was the best APN inhibitor among this series of compounds (IC50 = 250 nM). This study suggested the additive effect of the optimal substituents at both S1 and S1′ pockets of APN (Figure 21) for higher inhibition. Weglarz-Tomczak et al. reported a series of 1, 2-diaminoethylphosphonic acids as promising inhibitors of Neisseria meningitidis APN (NmAPN), porcine APN (SsAPN), and human APN (HsAPN or hAPN).126 The activity of these compounds was dependent on the substituent at the terminal amino function. Increasing the length of the alkyl chain from methyl to n-propyl to n-hexyl may increase APN inhibitory activity. Surprisingly, the unsubstituted analogue was observed to exert the highest NmAPN inhibition. Branching in the alkyl function (such as iso-butyl) was not favored for APN inhibitory activity. Alkylamine or alkylamide substitution was also favorable for APN inhibition. It was interesting to note that cycloalkyl substitution (c-hexyl and c-heptyl) exhibited a higher degree of inhibition toward SsAPN and HsAPN, whereas amide substitution at the c-hexyl function drastically reduced HsAPN inhibitory activity. Tetrahydrofurylmethyl substitution yielded a highly potent and selective HsAPN inhibitor over other APNs and LAP. Piperidinyl-4-ylmethyl and piperazinylethyl substitutions reduced HsAPN inhibitory activity several-fold, but these were also selective HsAPN inhibitors. Benzyl or α-methylbenzyl substitution resulted in selectivity toward HsAPN. Moreover, p-methoxybenzyl or p-methoxyphenethyl substitution displayed potent and selective HsAPN inhibition, whereas phenethyl or 4-aminobenzyl substitution reduced HsAPN inhibition and selectivity several-fold. Piperidinyl or morpholinyl analogues drastically reduced HsAPN inhibition. A docking study with the most active HsAPN inhibitor 61 (Ki = 65 nM) to the enzyme active site showed that the two hydroxyl groups of the phosphinic acid coordinated to Zn2+ ion in a bidentate fashion (Figure 22). The α-amino group formed hydrogen-bonding interactions with Gln213, Glu355, and Glu414. Indeed, the 4-methoxyphenyl group filled the S1 pocket, which is lined with residues Glu211, Phe349, Asn350, and Ala351. Moreover, some 1,2-diaminoethylphosphinic dipeptide analogues were also investigated.126 Here

Figure 23. Structures of 3-amino-2-tetralone derivatives as promising APN inhibitors 63−65.

100-fold higher inhibition than 1. These compounds 63 and 64 were very selective toward AP-M (EC 3.4.11.2) with respect to aminopeptidase-A (EC 3.4.11.7) and aminopeptidase-B (EC 3.4.11.6). However, at high concentrations, these derivatives poorly inhibited cytosolic LAP (EC 3.4.11.1). Albrecht et al. reported a series of 3-amino-2-tetralones having a Ki value in the lower micromolar range against “single zinc” aminopeptidases (those aminopeptidases having one zinc ion in their active site), especially APN and leukotriene A4 hydrolase (LTA4H: EC 3.3.2.6).128 Compound 65 (Figure 23) showed the best APN inhibition (Ki = 4000 nM). It was noted that the 6-amino substitution resulted in inactive 66 (Figure 24). 6479



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Figure 24. Structures of 3-amino-2-tetralone derivatives 66−72 as promising APN inhibitors.

In another study, Albrecht and co-workers tried to improve the stability profile of aminotetralone derivatives and synthesized some amino-benzosuberone derivatives as selective and potent APN inhibitors.129 These amino-benzosuberones showed improved stability. Compound 67 (Figure 24) may be a lead candidate for further optimization by chemical modification and derivatization. Bioisosteric modification of 67 resulted in 68 with a similar level of inhibitory activity (Figure 24).130 Maiereanu et al. further modified the structure of aminobenzosuberone 67 and synthesized new compounds.131 Compound 69, with a 1-phenyl-4-bromo moiety, exhibited APN inhibitory activity with a Ki value of 70 nM, while the corresponding 1-bromo4-phenyl derivative 71 inhibited APN with a Ki value of 60 pM without compromising the APN selectivity profile over “two-zinc” aminopeptidases [those aminopeptidases containing two zinc ions in their active site; for example: LAP (EC 3.4.11.1) and APaero (EC 3.4.11.10)]. These 3-amino-2-benzosuberone derivatives bind as transition-state mimics to highly conserved substrate recognition sites and, thus, exhibit outstanding M1 aminopeptidase selectivity. However, diverse substituents on the 3-amino-2-benzosuberone scaffold influence the complementarity to the active-site environment among this family of enzymes. Indeed, the chemical architectures and dynamics of the active site of APN were recently reported by Peng et al.132 Their studies gave rise to high-resolution X-ray structures of E. coli APN (PepN) in complex with 67, 70, and 71 (Figure 24).132 A new series of 7-aminobenzocyclohepten-6-ones were reported for their “single-zinc” and “two-zinc” aminopeptidase inhibitory activity.133 The thiophenyl derivative 72 (Figure 24) exhibited good APN inhibitory potency (Ki = 60 nM). The binding pose of the hydrated form of the thiophenyl-substituted moiety may be marginally shifted such that it may lead to an overall rotation of the suberone scaffold. The primary amine may form a strong electrostatic interaction with the conserved glutamate residues (GAMEN site) of the “single-zinc” metalloprotease family (Figure 25). 6.2.5. Flavone Derivatives. A series of flavone-8-acetic acids were synthesized and evaluated for their reversible aminopeptidase inhibitory property by Bauvois et al.134 Compound 73, containing dinitro substituents at the 2′- and 3-positions, was the best APN inhibitor of this series (Figure 26).

Figure 25. Binding pose of the hydrated form of thiophenyl-substituted moiety.

Figure 26. Structures of APN inhibitors 73 and 74.

6.2.6. Pyrrolidine Derivatives. A series of cinnamoyl pyrrolidines were designed and synthesized by Zhang et al.135 These molecules were evaluated against gelatinase A (MMP-2) and APN. Compound 74 (Figure 26) showed the most potent APN inhibition (IC50 = 75.2 nM) as well as MMP-2 inhibition (IC50 = 5.2 nM). The carboxylic acid ester moiety may serve as a good metal chelator for the MMP-2 enzyme (the ester group interacted with the Zn2+ ion at a distance of 1.21 Å), but it was unable to chelate the Zn2+ ion of APN derived from the homology model of human leukotriene A4 hydrolase (LTA4H). Moreover, some pyrrolidine derivatives synthesized by Cheng et al. showed selective MMP-2 inhibition compared to APN.136 Indeed, a new series of pyrrolidine-containing compounds were reported by 6480



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Zhang et al.137 Here also, most of these derivatives were found to be potent MMP-2 inhibitors with poor activity against APN. 6.2.7. Cyclic Imides. A series of N-phenyl cyclic imide derivatives were reported by Miyachi and co-workers.138 Some of these compounds were found to be more active than 1 and actinonin (5, Figure 3), naturally occurring APN inhibitors. APN inhibitory activity was increased when a five-membered ring was converted to a six-membered ring system. For N-(2,6-disubstituted phenyl)homophthalimides, APN inhibitory activity was increased if the dimethylphenyl group was replaced with a 2,6-diisopropylphenyl moiety, as evidenced by the SAR study. The unsubstituted phenylring-containing compound showed poor APN inhibitory activity. Therefore, a specific steric interaction is required for tight binding of these compounds to APN. Compounds containing a 2,6-diethylphenylhomophthalimide moiety (75, Figure 27A) showed more potent APN inhibitory activity than 5. It may be the superior lead

compound for the development of low-molecular-weight nonpeptide APN inhibitors (Figure 27A). Li and co-workers synthesized cyclic imide-containing peptidomimetics having good APN inhibitory activities.139 Hydroxamate derivatives showed better APN inhibition. Compound 76 (Figure 27B) exhibited the most potent inhibitory property (IC50 = 3100 nM). Li et al. further modified the piperidinedioneN-acetamide compound and reported some cyclic imide peptidomimetics having potency toward APN inhibitory activity.140 The benzyl derivative 77 (IC50 = 1800 nM) showed the most potent APN inhibition (Figure 27B). Compound 77 also exhibited good H22 cell inhibition, which was quite higher than that of 1 (inhibitory rate 45%). In nude mice bearing the human ovarian carcinoma cell line ES-2, 77 showed 30% tumor growth inhibition. In another study, Zhang et al. modified piperidinedione derivatives by replacing the hydroxamate moiety of 77 with different amino acids and their benzyl esters, but APN inhibitory activity was not improved significantly.141 However, 78 (Figure 28) showed promising APN inhibition (IC50 = 5000 nM) and exhibited antiproliferative effect against HL-60 cell line (IC50 = 1970 nM). The carbonyl group of the L-Phe moiety interacts with Zn2+, and the phenyl ring of the L-Phe moiety enters into the S1 pocket of APN (Figure 28). The second and fourth methyl groups of 2,4-dimethylaniline derivative projects into the S1′ and S2′ pockets of APN, respectively (Figure 28). Incorporation of an ether function between the carbonyl and hydroxamate functions of 77 yielded a cyclic imide peptidomimetic CIP-13F (79, Figure 28). Compound 79 significantly arrested APN activity on the surface of human ovarian carcinoma (OVCA) ES-2 cells.142 This compound showed anti-migratory property against ES-2 cells and also induced ES-2 cell apoptosis. In mice, 79 significantly delayed the growth of ES-2 xenograft after 2 weeks. On the other hand, replacement of the six-membered heteroaryl ring of 77 with a five-membered heteroaryl ring increased APN inhibition (Figure 28).143 Compound 80, a 2,5-pyrrolidinedione peptidomimetic derivative, showed better APN inhibitory activity (IC50 = 1000 nM) compared to 77 (Figure 28). Compound 80 not only exhibited significant APN inhibition but also showed potential in vitro and in vivo antiproliferative activity.

Figure 27. (A) Structure of cyclic imide scaffold-containing 75 along with SAR of cyclic imide derivatives. (B) Modification of 76 resulted in the more active APN inhibitor 77.

Figure 28. Structural modifications of 77 resulting in 78−80. 6481



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HL-60 cell proliferation. Both (2S,3R)-AHPA and (2RS,3S)AHPA may be accommodated in the active site of APN, as suggested by the docking study. Therefore, the configuration of the AHPA derivatives was not positively correlated with the enzyme binding. 6.2.10. Chloramphenicol Amine Derivatives. A series of chloramphenicol amine derivatives (Figure 31) having APN

This compound was a better inhibitor of HL-60 cell proliferation (IC50 = 149 μM) than the known APN inhibitor 1 (IC50 = 245 μM), as observed in MTT assay. 6.2.8. β-Dicarbonyl Derivatives. Curcumin (81, Figure 29), a phenolic derivative (two vinylguaiacol motifs attached through a

Figure 29. Structures of 81 and β-dicarbonyl derivative 82 as APN inhibitors.

β-diketone unit) isolated from Curcuma longa, was found to be an irreversible and non-competitive inhibitor of APN.144 In a dosedependent manner, curcumin arrested the tumor invasion of APN-positive cells. To identify more potent and selective APN inhibitors, Ma et al. modified 81 to synthesize a series of β-dicarbonyl compounds.145 The APN inhibitory properties of these derivatives were comparable to those of 1 and 81. These compounds were also investigated for HDAC inhibition, but the results were not encouraging. Compound 82 (Figure 29) exhibited the most potent APN inhibition (IC50 = 1410 nM) of this series and showed better activity than 1 and 81. 6.2.9. β-Amino-α-hydroxyl-phenylbutanoic Acid (AHPA) Derivatives. Shang and co-workers designed new AHPA-based analogues.146,147 Some of these compounds (83, 84) showed promising and selective APN inhibition over MMP-2 (Figure 30A). Chen et al. reported another series of (2RS, 3S)-AHPA derivatives having APN inhibitory activity.148 The SAR study suggested that an aryl group at the R position was unfavorable compared to alkyl groups (Figure 30B). Cyclization between the amide nitrogen atom and R groups was not tolerated for APN inhibition (Figure 30B). Compound 85 (Figure 30B) exhibited better inhibitory potency compared to 1 against both APN and

Figure 31. Structural modifications of 86 resulted in comparatively better APN inhibitor 87.

inhibitory activity was reported.149 (2R,3S)-2-Amino-3-hydroxy3-(4-nitrophenyl)propanoic acid (AHNPA) dipeptide derivative 86 exhibited the most potent APN inhibitory activity among these compounds (Figure 31). The nitrophenyl group of 86 protruded into the S1 pocket and formed hydrophobic interactions. The terminal phenyl ring inserted well into the S2′ pocket, and another phenyl group interacted with the S1′ pocket (Figure 31). Compound 86 also showed promising cell-based inhibitory activity against the HL-60 cell line and may be considered as a lead molecule. Further modification of chloramphenicol amine was investigated by Jia and co-workers for designing a series of compounds having (1S,2S)-2,3-diamino-1-(4-nitrophenyl)propan-1-ol (DANP) as

Figure 30. (A) Structures of 83 and 84. (B) SAR of (2RS, 3S)-AHPA derivatives having APN inhibitory activity along with the structure of 85. 6482



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the key scaffold (Figure 31).150 The enzymatic assay of these compounds showed moderate and selective APN inhibition over MMP-2. It was interesting to note that compound 87 (Figure 31) exhibited the highest inhibition in both the enzymatic (APN IC50 = 6100 nM) and cell-based assays (HL-60 IC50 = 1340 nM). The molecular docking study of compound 87 revealed that the phenyl function of the phenylalanine moiety projected into the S1 subsite, while the aniline and leucine residues inserted into the S1′ and S2′ subsites of APN, respectively (Figure 31).150 6.2.11. Indoline-2,3-dione Derivatives. A series of indoline2,3-dione-containing APN inhibitors were reported by Jin et al.151 In the preliminary study, compound 88 (Figure 32) emerged as a

heterocyclic counterparts (Figure 32). However, the six-membered heterocyclic derivatives displayed poor APN inhibitory activity. Therefore, the spatial arrangement of the spiroketal scaffold may be important for a hydrophobic interaction with amino acid residue Met260 of APN. The hydrophobic backbone motif of 5′-chlorospiro[1,3-dioxepane-2,3′-indolin]-2′-one molecule protruded deep into the S1 pocket of APN, and the hydroxamate residue may serve as the zinc binder. Most of these molecules were indicative in HDAC assays. However, 90 (Figure 32) possessed good HDAC inhibitory activity (IC50 = 330 nM) but showed comparatively poor APN inhibition (IC50 = 6660 nM), as this molecule has an elongated linker and thus may bind to HDAC more efficiently.151 6.2.12. 1,2,3,4-Tetrahydroisoquinoline-3-carboxylic Acid Derivatives. Encouraged by results with the cyclic imide derivative 77 (APN IC50 = 1800 nM), Zhang et al. designed the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivative 91 by cyclizing the amino group with the phenyl ring of the former compound (Figure 33).154 The APN inhibitory activity of this

Figure 32. Structures of indoline-2,3-dione-containing APN inhibitors 88−90.

potent APN inhibitor (IC50 = 360 nM). They optimized the structure of 88 and designed a number of compounds having APN inhibitory activities. This study gave rise to the development of 89 (IC50 = 74 nM), which was more active compared to the former one (Figure 32).151 Compound 89 exhibited significant inhibition against cancer cell proliferation in the human ovarian carcinoma (OVCA) cell lines ES-2 (IC50 = 43 800 nM) and 3AO (IC50 = 70 200 nM). This molecule also displayed good inhibitory activities against A549 (inhibition = 42% at 160 μM dose) and HL60 (inhibition = 48% at 160 μM dose), which were better than 1. Additionally, 87 showed anti-migratory (inhibition of 56% and 77% in ES-2 cells and 3AO cells, respectively, at 15 μM dose) and anti-invasive (inhibition 76% and 67% in ES-2 cells and 3AO cells, respectively at 15 μM dose) properties. Moreover, 89 possessed good biocompatibility in H22 xenograft mice model and showed better anti-tumor efficacy than 1.152,153 From the SAR observations, it may be inferred that electron-withdrawing bromo or chloro substituents were favorable in the R position for APN inhibition, while the alkyl group yielded comparatively poor results (Figure 32). Chlorine substitution at the R position resulted in better APN inhibitory ability compared to the corresponding brominated and fluorinated analogues. Probably due to the stronger electron-withdrawing effect of the fluorine, the energy of the π−π interaction with Tyr376 of APN was reduced. Therefore, electrical properties and spatial structures of these compounds were responsible for better APN inhibition. In addition, five- to sevenmembered spiro rings at the third position of the indolin-2-one nucleus influenced the APN inhibitory activities. The compounds containing a seven-membered heterocyclic ring exhibited the highest APN inhibitory potency, followed by the five-membered

Figure 33. Structural modifications of 77 resulted in comparatively poor APN inhibitor 91, but further modification of 91 resulted in promising APN inhibitor 92.

molecule was compromised due to the cyclization of the amino group. Probably, the free amino group is important for APN interaction (77 vs 91). Subsequently, replacing the cyclic imide scaffold of 91 with amino acids or dipeptides enhanced APN inhibitory activity. For example, the phenylglycine-containing derivative 92 (IC50 = 6280 nM) exhibited the most potent APN inhibition among these compounds (Figure 33). Moreover, 92 was more selective toward exopeptidase APN compared to endopeptidase MMP-2.154 6.2.13. Miscellaneous. Li and co-workers designed and synthesized a series of quinoxalinone peptidomimetic derivatives.155 These compounds showed significant MMP-2 selectivity over APN. A docking study with FLEX-X flexible-dock program suggested that one of the most potent MMP-2 inhibitors was unable to form proper binding interactions with the active site of APN (S1′ and S2′ pockets).155 Indeed, quinoxalinone and spiro heterocyclic-derived hydrazide peptidomimetics were reported by Yang et al.156 These compounds exhibited selective gelatinase inhibition over APN. These compounds showed moderate to poor APN binding affinity, probably due to their structural orientation that may be unable to occupy the two active subdomains (S1 and S1′ pockets) of APN. 6483



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good biodistribution was observed, with inhibition of APN activity in the brain, liver, and kidney demonstrated. Surprisingly, 95−97 interacted with the S1−S5′ subsites without engaging the zinc atom (Figure 34). The improved APN selectivity of compounds may be facilitated by targeting Ala351, Arg442, Ala474, Phe896, and Asn900 amino acid residues of human APN.

Flipo and co-workers reported some quinoline-based zinc metalloaminopeptidase inhibitors.157 Most of these compounds showed potency toward PfA-M1 (Plasmodium falciparum APN) over mammalian APN. Compound 93 (Figure 34) was the most

7. FUTURE PERSPECTIVES A malignant cell passes through numerous connective tissue barriers in the metastatic cascade. Under these circumstances, the degradation of extracellular matrix by several classes of enzymes, such as serine proteinases, matrix metalloproteinases, and aminopeptidases, is crucial for the tumor cell. APN, an important transmembrane zinc-dependent metalloprotease enzyme, has been found to play crucial roles in controlling numerous functions, including migration, invasion, angiogenesis, and metastatic events of cancer cells. APN expression is positively correlated with invasiveness of tumor cells. Therefore, APN may be considered as a potential target for the design of inhibitors of cancer cell metastasis. Many academic and commercial laboratories have developed a diverse set of APN-targeted molecules, ranging from synthetic peptidomimetic or non-peptidic small-molecular inhibitors to monoclonal antibodies. The neovasculature binding properties of tumor-homing cyclic peptide asparagine-glycinearginine (cycNGR) may depend on the interaction with APN/ CD13. The NGR motif mimics extracellular matrix proteins that APN is known to bind but not degrade.67 However, NGR is rapidly converted to isoaspartate-glycine-arginine (isoDGR) by asparagine deamidation. This phenomenon led to a decreased specificity and resulted in potential off-target effects. Therefore, it is of importance to design low-molecular-weight APN-targeted therapeutics that are specific and metabolically stable. In this Perspective, key advances in the fields of APN inhibition have been highlighted and discussed extensively. Based on the observations highlighted here, APN inhibitors possess common structural features associated with higher inhibitory activity. Numerous structural comparisons have allowed interpretation of

Figure 34. Structures of APN inhibitors 93−97.

potent APN inhibitor (IC50 = 28 nM), having 30-fold selectivity over PfA-M1. Flipo et al. further designed some hydroxamate-based APN inhibitors, but these were less potent mammalian APN inhibitors compared to their earlier reported inhibitors.157,158 However, these compounds were non-selective toward mammalian APN over PfA-M1. Compound 94 (Figure 34) was the most active APN inhibitor (IC50 = 443 nM) of this series, although this molecule was more selective toward PfA-M1 (IC50 = 45 nM). Very recently, Pascual and colleagues identified three noncompetitive APN inhibitors (α > 1) by performing a virtual screening of 14 400 bidimensional compounds from the Maybridge HitFinder Library.159 After intraperitoneal administration of 95−97 to rats,

Figure 35. Highly potent APN inhibitors with important scaffolds. 6484



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the SAR observed for highly active APN inhibitors. Compounds possessing scaffolds with specific structural features have been found to be important for the expression of potent APN inhibitory property. These scaffolds include leucine ureido function, leucinethiol, 3-amino-2-tetralone, pyrrolidine, cyclic imide, AHPA, and indoline-2,3-dione moieties (Figure 35). Keeping in mind of these structures, newer, better active APN inhibitors may be designed in future. Generally, a hydrophobic group followed by a ZBG attached to another hydrophobic group(s) is required for maintaining APN inhibitory activity. ZBGs such as thiol, boronic acid, and phosphonate are comparable to hydroxamate for APN inhibition. Indeed, rigid small molecules exhibit better efficacy than bigger molecules. Reports suggested that the 3-amino-2-benzouberones show sub-nanomolar binding constants with very significant ligand efficiencies. These are highly specific for M1 aminopeptidases. Very recently, Peng and co-workers reported the high-resolution X-ray structures of E. coli aminopeptidase N (PepN) in complex with 67, 70, and 71.132 This has allowed a better understanding of the recognition of these low-molecularweight derivatives by M1 aminopeptidases and thus opens a new vista for the design of APN inhibitors. Last but not least, while the past decade has witnessed considerable progress in the design of APN inhibitors, few have demonstrated clinical efficacy. Nevertheless, there is still a need to design more potent and specific APN inhibitors that would exhibit fewer adverse reactions. Our laboratory has been pursuing the design of metalloprotease inhibitors, including APN, for the past decade, and we have rationally designed some APN inhibitors that are currently undergoing extended evaluation.160 We hope that our efforts will contribute to the design of more compelling APN-inhibiting lead molecules and that, in this Perspective, we have emphasized the importance of pursuing more potent and selective APN inhibitors.

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Commission (UGC), New Delhi, and is pursuing research in the Department of Pharmaceutical Technology, Jadavpur University, under the guidance of Tarun Jha. His research area includes design and synthesis of anti-cancer small molecules. He has published 56 research/ review articles in different reputed peer-reviewed journals and five book chapters. Tarun Jha, a faculty member of the Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India, has supervised 14 Ph.D. students and guided eight research projects funded by different organizations. He has published more than 110 research articles in different reputed peer-reviewed journals. His research area includes design and synthesis of anti-cancer small molecules. Prof. Jha is a member of the Academic Advisory Committee of National Board of Accreditation (NBA), New Delhi, India.

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ACKNOWLEDGMENTS N.A. is grateful to University Grants Commission (UGC), New Delhi, India, for providing the Rajiv Gandhi National Fellowship. T.J. is thankful for financial support from the University with Potential for Excellence (UPE) Phase II Program of UGC, New Delhi, to Jadavpur University, Kolkata, India. We are thankful to the authority of Jadavpur University, Kolkata, India, for providing research facilities. Dr. Balaram Ghosh of BITS-Pilani, Hyderabad Campus, India; Dr. Shovanlal Gayen of Dr. Harisingh Gour University, Sagar, India; and Dr. Partha Sarathi Panda of Dr. B. C. Roy College of Pharmacy and Allied Health Sciences, Durgapur, India, are gratefully acknowledged for their critical reading and corrections of the manuscript. We thankfully acknowledge Mr. Sandip Kumar Baidya, Mr. Suvankar Banerjee, and Mr. Saptarshi Sanyal of Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India, for their support. We sincerely appreciate the comments of learned reviewers and the Editor which helped to modify the manuscript in its current form. We would like to express sincere gratitude to the Editor of this Journal for providing editorial and bibliographic assistance.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +9133-2457 2495 (O). +91-9433187443 (M).

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ORCID

ABBREVIATIONS USED γ-GT, γ-glutamyl transpeptidase; AngIII, angiotensin III; APA, aminopeptidase A; APB, aminopeptidase B; APCs, antigenpresenting cells; APN, aminopeptidase N; CcoV, canine enteric coronavirus; cDNA, complementary DNA; DPPIV, dipeptidyl peptidase IV; GRB2, growth factor receptor-bound protein 2; EC number, Enzyme Commission number; ERK, extracellular signal-regulated kinases; FAK, focal adhesion kinase; FcoV, feline enteric coronavirus; hAPN, human APN; HCoV229E, human respiratory coronavirus 229E; HDAC, histone deacetylase; IL-6, interleukin-6; IUBMB, International Union of Biochemistry and Molecular Biology; JNK, c-jun N-terminal kinase; LTA4H, leukotriene A4 hydrolase; LAP, leucine aminopeptidase; MAPK, mitogen-activated protein kinase; MMPs, matrix metalloproteinases; MKK3, mitogen-activated protein kinase kinase 3; NEP, neutral endopeptidase; NSCLC, non-small-cell lung cancer; PDB, Protein Data Bank; PI3K, phosphoinositide 3-kinases; PSA, puromycin-sensitive aminopeptidase; RECK, reversion-inducing cysteine-rich protein with kazal motifs; SAR, structure−activity relationship; SAS, solventaccessible surface; SIL-6R, soluble IL-6 receptor; TGEV, transmissible gastroenteritis virus; THR-de, thyrotropin-releasing hormone-degrading enzyme; ZBG, zinc binding group

Sk. Abdul Amin: 0000-0003-4799-7322 Nilanjan Adhikari: 0000-0001-5523-7716 Tarun Jha: 0000-0002-9996-738X Notes

The authors declare no competing financial interest. Biographies Sk. Abdul Amin is a research scholar in the Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India, under the guidance of Tarun Jha. He completed his Master of Pharmacy (M. Pharm.) in 2016 from Dr. Harisingh Gour University, Sagar, India. His research area includes design and synthesis of small molecules with anti-cancer properties, computational chemical biology, and large-scale structure−activity relationship analysis. Currently, he is working on metalloenzyme inhibitors. Mr. Amin has published 33 research/review articles in different reputed peer-reviewed journals and a book chapter. He enjoys a good conversation on science, contemporary art, books, film, and music. Nilanjan Adhikari is a researcher in the Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India. He has completed his B. Pharm. (2007) and M. Pharm. (2009) degrees from Jadavpur University, Kolkata. He is a research fellow of the University Grants 6485



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