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

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Design of Aminopeptidase N (APN) inhibitors as Anticancer Agents Sk. Abdul Amin, Nilanjan Adhikari, and Tarun Jha J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00782 • Publication Date (Web): 09 Apr 2018 Downloaded from http://pubs.acs.org on April 9, 2018

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Journal of Medicinal Chemistry

Design of Aminopeptidase N (APN) inhibitors as Anticancer Agents Sk. Abdul Amin, Nilanjan Adhikari, Tarun Jha* Natural Science Laboratory, Division of Medicinal and Pharmaceutical Chemistry, Department of Pharmaceutical Technology, P. O. Box 17020, Jadavpur University, 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, 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. Keywords: Aminopeptidase N (APN); Moonlighting protein; APN inhibitor; Structure-activity relationship (SAR).

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1. Introduction Several types of human malignancies, including aggressive phenotypes and fast-growing tumors, express a variety of hydrolytic enzymes like 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 (γGT, 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 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 antigen on the cell surface of antigen-presenting cells (APCs), as well as cleavage of the MHC class II molecule-bound 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


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resultant invasive capacity makes APN an attractive target for the treatment of diseases including cancer. 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 structureactivity 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 are 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, fifteen clans of metallopeptidases

have been reported (Figure 1). The clan MA contains more than forty families of metallopeptidases.11



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Figure 1. Schematic representation of the fifteen clans of metallopeptidases. 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 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 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 arch4 ACS Paragon Plus Environment

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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

Figure 2. Functions of human APN/CD13. 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 tumorassociated antigen L6,23 reversion-inducing-cysteine-rich protein with kazal motifs (RECK),24



galectin-325 and galectin-4.26 Results of APN 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

Figure 3. Bestatin and its derivatives as APN inhibitors. It 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 mitogen-activated protein kinase (MAPK),


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phosphoinositide 3-kinases (PI3K)/AKT, NF-κB and subsequently, triggers gelatinase to promote migration and invasion in a variety of cancer cells (Figure 4).28

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), c-jun N-terminal kinase (JNK)].



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 coworkers.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. 4. APN as a potential therapeutic target Misregulation of APN has been found to evolve in almost all types of human malignancies.7 It includes 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).


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Figure 5. Implications of APN in different cancers.

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+, CD4+CD25+ regulatory T cells at the mRNA level and, therefore, inhibition of APN may be useful for targeting disorders namely 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 9 ACS Paragon Plus Environment


target for cerebral ischemia and neuronal degeneration.53 Apart from these abovementioned diseases, APN has also been found to take part in mediating infective viral diseases namely human cytomegalovirus (HCMV) and human coronavirus (HCoV-229E) infections.54 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 homology to related enzymes, namely aminopeptidase A,55 aminopeptidase B,56 leukotriene A4 hydrolase (LTA4H),57 thyrotropin-releasing hormonedegrading enzyme (THR-de), puromycin-sensitive aminopeptidase (PSA),58 Escherichia coli pepN, 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).


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Figure 6. Schematic seven domain hypothetical organization of human APN/CD13. [The location of the substrate/inhibitor binding site is highlighted as red dotted circle between domains V and VI]. 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 the surface area of 840 Å2 approximately. The hAPN contains the characteristic consensus motifs 352

GXMEN356 and

388

HExxHx18E411 and a thermolysin-fold.22 The Zn2+ ion is found to

coordinate with three amino acid residues of

388

HExxHx18E411 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 x 62 Å that has similarity with the porcine APN of 135 Å x 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 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.



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) are highlighted along with important amino acid residues (shown in line). 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, His388 and few bound water molecules (HOH1118, HOH1176, HOH1315, HOH1382, HOH1445, HOH1483, HOH1688, HOH1733, HOH1919) also control the binding interactions between the ligand and hAPN. The solvent accessible surface (SAS) contour also suggests that a part of the APN binding site is highly solvent exposed (Figure 8).


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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. 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

352

GXMEN356 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 13 ACS Paragon Plus Environment


(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 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 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 chain containing amino acids show high APN-binding affinity. 6. APN inhibitors


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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 1 (Figure 3) for a variety of malignancies including cancers of stomach, esophagus, 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 towards 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 towards LTA4 hydrolase (IC50 = 8 nM) while exhibiting good efficacy towards 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 tri- or tetrapeptide inhibitors more strongly.68 On the basis of structural arrangements, APN inhibitors are broadly classified into the two major groups: (A) amino acid-based inhibitors and (B) non-amino 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 Streptomyces 15 ACS Paragon Plus Environment


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 antigen-presenting cells (APCs), bestatin showed the 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 analogs resulted in more potent APN inhibitors. Bestatin analog, 2 (Figure 3) was a slow binding competitive inhibitor of aminopeptidase M (EC 3.4.11.2) having an inhibition rate constant (Ki) of 1.9x10-8 M.68, 76, 77 Umezawa et al. further reported another bestatin analog namely actinonin (5) (Figure 3) having aminopeptidase M (EC 3.4.11.2) inhibition constant (Ki) of 1.7x10-7 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 at an inhibition constant of 1.9x10-8 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


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Figure 9. (A-C) Comparison between bestatin and its analogs 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). With 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 mM/Lit, bestatin: 35.70 mM/Lit). Compound 10 showed higher APN inhibition (IC50 = 7.2 µM) that was better 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 last 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 2nd 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


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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 were done by different alkyl, thioalkyl or aryl function. The t-butyl function at the urea terminal yielded an inactive molecule (IC50 > 100 µM) whereas the benzyl or phenethyl substituents resulted in moderate APN inhibition. Moreover, carbamate analogs were less effective compared to the corresponding ureido analogs. 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 anti-invasive properties. Compound 11 potentially blocked ES-2 cell invasion in a dose-dependent manner and exhibited significant activity against proliferation of ES-2 cells.



Figure 10. Structures of leucine ureido derivatives having APN inhibitory potency (11-14). Su et al. further extended their study in synthesizing another series of ureido derivative.87 The sec-butyl substitution at the α-position of the hydroxamate group yielded inactivity, whereas isobutyl substitution resulted in better efficacy. Again, phenyl analogue at this position yielded less efficacy compared to iso-butyl analogue. A phenylpropyl analogue at the terminal amide function of the urea moiety produced lower activity compared to the benzyl analogue. 20 ACS Paragon Plus Environment

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Interestingly, heterocyclic thienylmethyl substitution at the same position exhibited better APN inhibition compared to the furyl analog. 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, methyl) at the aryl group produced better efficacy than an electron withdrawing group (such as fluoro). However, m-methoxy substitution resulted in more than 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 lead 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 suggested their potential antiangiogenic and antimetastatic effects. In another study, Ma and colleagues optimized the structure of 15 which showed some potency towards 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 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).



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

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. The 2,3-dimethylbenzyl group containing 19 showed good inhibitory activity but the activity was compromised in the 2,6-dimethylphenyl containing 18. Compound 18 also showed potential antimetastatic effect (92% inhibitory rate) in a mouse hepatoma-22 (H22) pulmonary metastasis model at a dose of 80 mg/kg. Apart from the abovementioned amino acid derivatives, a number of APN inhibitors have been reported. However, these amino acid derivatives showed moderate to poor activity against APN.


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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-amino and 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).



Figure 12. (A) Structures of L-lysine-based 20; (B-C) Comparison between the L-lysine (21) and its analog (22) for their binding interaction with APN. 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 interaction with Tyr477, Glu418, His388, Arg381 and Gly352, whereas the hydroxamate group formed a bidentate chelate with the Zn2+ ion at the enzyme active site. 24 ACS Paragon Plus Environment

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Addlagatta and co-workers reported the crystal structure of APN from Escherichia 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 Llysine 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 panel of tumor cell lines (HT-60, ES-2, K-562, A-549, H7402 and PLC). 6.1.2.1. Lysine-ureido derivatives As the ureido moiety is a good bioisostere of the amide group, Su and co-workers prepared Llysine ureido derivatives to acquire more potent APN inhibitors.93 The ester and acid analogs 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 analogs (Figure 13). The iso-butyl analogue 23 was found to be the superior to the methyl, ethylthiomethyl and benzyl analogues (Figure 13).



Figure 13. The SAR of the L-lysine ureido derivatives on the template structure of 23. 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,4bromophenyl (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 L-arginine moiety enhanced APN inhibition.95 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.


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Figure 14. Structural modifications of 24 resulted in better active APN inhibitors (25-27). 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 electron-withdrawing 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,98 Yang et 27 ACS Paragon Plus Environment


al. synthesized a variety of L-isoserine derivatives as APN inhibitors.99 Interestingly, these carboxylates ester-containing L-isoserine molecule showed comparatively poor APN inhibitory activity compared to their corresponding carboxylic acid analogues. Moreover, the iso-propyl moiety directed towards 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 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).

Figure 15. Developments of 29 from 28 and 1.


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Zhang et al. investigated a new series of 3-amino-2-hydroxyl-3-phenylpropionic acid analogs 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 = 2,12,970 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. having promising APN inhibitory activity.101 The best active compound of this series 30 (Figure 16A) possessed good APN inhibitory activity (IC50 = 2510 nM).



Figure 16. (A) Development of 30 from 28, (B-C) Structure of APN inhibitors (31-32) and their APN inhibitory activity. 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 towards 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 N2phenylacetyl-L-isoglutamines.104-105 These compounds, inspired by antineoplastin,106 displayed antitumor activity and showed moderate APN inhibition. Meanwhile, the same research group again designed some L-isoglutamine deriavties and this time the activity profile was increased upon modification of ZBG.107 Hydroxamate-based derivative (32) (Figure 16C) showed better APN inhibitory property compared to the corresponding non-hydroxamate analogs. Moreover, 32 showed good antiproliferative effect against HL-60 tumor cell line. 6.1.7. Miscellaneous amino acid-based inhibitors Gredicak et al. reported a structurally distinct group of compounds (enediyne-peptide conjugates) as APN inhibitors.108 In the enediyne-peptide conjugates, the 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 his colleagues developed some substrate-based cyclic peptide inhibitors of 30 ACS Paragon Plus Environment

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APN.67 The lead peptide inhibitor cycLHSPW showed outstanding specificity towards 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 acid, 3-mino-2-tetralone, cyclic imide, pyrrolidine, AHPA and indoline-2, 3-dione were also found to impart promising APN inhibitory effects. These non-amino acid-based APN inhibitors are as follows: 6.2.1. Mercaptan derivatives Mercaptan derivatives such as L-leucinthiol (33, Figure 17A) exhibited competitive leucine aminopeptidase (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.



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

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 analogs were less potent APN inhibitors. However, the corresponding thiol analog enhanced the activity by a minimum of 2500-fold over these former analogs. 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 cyclohexylmethyl) reduced the activity but all of them displayed activity at nM concentrations (IC50 ≤ 130 nM). In addition, decreasing the length


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of the phenyl group slightly increased the activity compared to the benzyl analog (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, both 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 analog 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). 2Amino-4-methylsulfonyl butanethiol (PC18, 37), a methionine thiol analog, emerged as APN active compound having IC50 values of 11 nM. Réaux et al. further investigated the in vitro selectivity of 37 towards three aminopeptidases including APN, aminopeptidase B (APB) and aminopeptidase A (APA).111 Compound 37 (Figure 17C) was found to be 125-fold and 2,150-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 half-life 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 towards APA over APN. Only 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 towards APA (Ki = 13 nM). While keeping the sulfonate ethyl group as the P1 substituent, modifying the amino acid residue towards the S1´ and S2´ sites resulted in highly potent and selective APA inhibitors.112

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


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Chauvel et al. synthesized some β-aminothiols and screened these against APA and APN.113 Most of these compounds were good APN inhibitors.113 The carboxylic acid analog 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 moiety of 39 (APN Ki = 15500 nM) and 40 (APN Ki = 330 nM) was replaced with a hydroxamic acid, exemplified by 41 (APN Ki = 37 nM), or a thiol, compound 42 (APN Ki = 32nM), 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 towards 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 towards 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)-analog drastically reduced APN inhibition but was selective towards 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 β to 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 inhibitor over APA. Moreover, replacement of the phenyl moiety of the p-carboxymethylphenyl analog 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 1butaneboronic 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).


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Figure 19. (A) Structures of boronic acid derivatives 47-50 active against cytosolic and microsomal leucine aminopeptidases; (B) Structures of 51, 52. Farsa et al. compared the APN inhibitory activity of two different series of compounds namely 4phenoxy-[1,3,2]dioxaborolan-2-ol containing compound 51 and 3-phenoxy-1,2-propandiol containing 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 the year 1987, Giannousis and Bartlett reported a group of phosphorous amino acids and dipeptide analogs 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.



Figure 20. Structures of phosphonic acid derivatives (53-57) as APN inhibitors.

Aminophosphinic acid derivatives bearing a hydrophobic side chain were synthesized as APN inhibitors.121 The hydrophobic side chain interacts with the S1 pocket of APN. Moreover, the coupling of the α-aminophosphinic acids with the dipeptide Phe-Phe analog may engage the S1' and S2' pockets of APN enzyme. This study resulted in new "phosphino" peptides designed as transition state analogs 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 towards 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 towards LAP. However, 56


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(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 αaminophosphonates directed towards the S1 pocket of leucine aminopeptidase (LAP).123 An increase in LAP and APN inhibitory activities were observed for compounds having increased side chain 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 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 towards aminopeptidase and designed 59 as a lead molecule (Figure 21).124 Modifying the formerly mentioned 59124 (Figure 21), Grzywa and co-workers designed a new class of α-aminoalkyl-(N-substituted)thiocarbamoyl-phosphinates and found some compounds as 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.



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

Weglarz-Tomczak et al. reported a series of 1, 2-diaminoethylphosphonic acids as promising inhibitors of N. meningitidis APN (NmAPN), porcine APN (SsAPN) and human APN (HsAPN or hAPN).126 The activity of these compounds was dependent on the substituents 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 analog 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 towards SsAPN and HsAPN whereas amide substitution 40 ACS Paragon Plus Environment

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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 towards HsAPN. Moreover, p-methoxybenzyl or pmethoxyphenethyl substitution displayed potent and selective HsAPN inhibition whereas phenethyl or 4-aminobenzyl substitution reduced HsAPN inhibition and selectivity several fold. Piperidinyl or morpholinyl analogs 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).

Figure 22. Structures of phosphonic acid derivatives (61-62) as APN inhibitors. 41 ACS Paragon Plus Environment


The α-amino group formed a hydrogen-bonding interaction 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 analogs were also investigated.126 Here also, the cyclic aliphatic analogs (such as c-hexyl and c-heptyl) yielded higher potency and selectivity towards HsAPN compared to the corresponding linear or branched alkyl functions substituted at the terminal amide moiety. Furthermore, the pmethoxyphenethyl 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 Nonpeptidic 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 100fold higher inhibition than 1. These compounds 63 and 64 were very selective towards 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 leucine aminopeptidase (LAP: EC 3.4.11.1).


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



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

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 as a lead candidate for further optimization by chemical modification and derivatization. Bioisosteric modification of 67 resulted in 68 as a compound 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 44 ACS Paragon Plus Environment

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with a Ki value of 70 nM while the corresponding 1-bromo-4-phenyl derivative 71 inhibited APN with a Ki value of 60 pM without compromising 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); 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 aminopeptidases 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 ‘twozinc’ aminopeptidase inhibitory activity.133 The thiophenyl derivative 72 (Figure 24) exhibited good APN inhibitory potency (Ki = 60 nM). The binding pose of 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).



Figure 25. Binding pose of the hydrated form of thiophenyl substituted moiety. 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 di-nitro substituents at the 2′- and 3-positions resulted in the best APN inhibitor of this series (Figure 26).


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Figure 26. Structure of flavone-based APN inhibitor 73 and structure of 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 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), naturally47 ACS Paragon Plus Environment


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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 phenyl ring 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).

Figure 27. (A) Structure of cyclic imide scaffold containing 75 along with SAR of cyclic imide derivatives, (B) Modification of 76 resulted in better active APN inhibitor (77). 48 ACS Paragon Plus Environment

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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 piperidinedione-N-acetamide compound and reported some cyclic imide peptidomimetics having potency towards 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 1 (inhibitory rate 45%). In the human ovarian carcinoma cell line ES-2 bearing nude mice, 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 2nd and 4th methyl groups of 2,4dimethylaniline derivative projects into the S1′ and S2′ pockets of APN, respectively (Figure 28).



Figure 28. Structural modifications of 77 resulted in 78-80. 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 the APN activity on the surface of human ovarian carcinoma (OVCA) ES-2 cells.142 This compound showed antimigratory property against ES-2 cell 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 6-membered heteroaryl ring of 77 with a 5-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


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potential in vitro and in vivo antiproliferative activity. 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 βdiketone unit) isolated from Curcuma longa, was found to be an irreversible and non-competitive inhibitor of APN.144 In a dose-dependent manner, curcumin arrested the tumor invasion of APNpositive cells.

Figure 29. Structure of 81 and β-dicarbonyl derivative 82 as APN inhibitors. 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 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 analogs.146-147 Some of these compounds (83, 84) showed promising and selective APN inhibition over MMP-2 (Figure 30A).

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. 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 HL-60 cell proliferation. Both (2S, 3R)-AHPA and (2RS, 3S)-AHPA may be accommodated in the active site of APN as


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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 inhibitory activity was reported.149 (2R,3S)-2-amino-3-hydroxy-3-(4-nitrophenyl)propanoic acid (AHNPA)-dipeptide derivative (86) exhibited the most potent APN inhibitory activity among these compounds (Figure 31).

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



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 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 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 both in the enzymatic (APN IC50 = 6100 nM) and cell-based assay (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 residue inserted into the S1′ and S2′ subsites of APN, respectively (Figure 31).150 6.2.11. Indoline-2,3-dione derivatives A series of indoline-2,3-dione containing APN inhibitors were reported by Jin et al.151 In the preliminary study, compound 88 (Figure 32) emerged as a potent APN inhibitor (IC50 = 360 nM). They have 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 line ES-2 (IC50 = 43,800 nM) and 3AO (IC50 = 70,200 nM). This molecule also displayed good inhibitory activity against A549 (inhibition = 42% at 160 µM dose), HL60 (inhibition = 48% at 160 µM dose) which were better than 1. 54 ACS Paragon Plus Environment

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Figure 32. Structures of indoline-2,3-dione containing APN inhibitors 88-90. 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 antitumor 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 seven-membered spiro rings at the 3rd position of indolin-2-one nucleus influenced the APN inhibitory activities. The seven-membered heterocyclic ring containing compounds exhibited the highest APN inhibitory potency followed by the five-membered 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, it may bind to HDAC more efficiently.151 6.2.12. 1,2,3,4-tetrahydroisoquinoline-3-carboxilic acid derivatives Encouraged by results with the cyclic imide derivative 77 (APN IC50 = 1800 nM), Zhang et al. designed 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivative 91 by cyclizing the amino group with the phenyl ring of the former one (Figure 33).154 The APN inhibitory activity of this 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


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inhibitor among these compounds (Figure 33). Moreover, 92 was more selective towards exopeptidase APN compared to endopeptidases MMP-2.154

Figure 33. Structural modifications of 77 resulted in comparatively poor APN inhibitor 91 but further modification of 91 resulted in promising APN inhibitor 92. 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 57 ACS Paragon Plus Environment


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. Flipo and co-workers reported some quinoline-based zinc metalloaminopeptidase inhibitors.157 Most of these compounds showed potency towards PfA-M1 (Plasmodium falciparum APN) over mammalian APN. Compound 93 (Figure 34) was the most potent APN inhibitor (IC50 = 28 nM) having 30-fold selectivity over PfA-M1.

Figure 34. Structures of APN inhibitors 93-97. 58 ACS Paragon Plus Environment

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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 towards 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 towards PfA-M1 (IC50 = 45 nM). Very recently, Pascual and colleagues identified three non-competitive 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, good biodistribution was observed, with inhibition of APN activity in the brain, liver and kidney demonstrated. Surprisingly, 95-97 interacted with the S1 to S5´ subsites without engaging the zinc atom (Figure 34). The improved APN selectivity of compounds may be facilitated by targeting Ala351, Arg442, Ala474, Phe-896 and Asn900 amino acid residues of human APN. 7. Future perspectives A malignant cell passes through numerous connective tissue barriers in the metastatic cascade. Under these circumstances, the degradation of extracellular matrix (ECM) by several classes of enzymes such as serine proteinases, matrix metalloproteinases and aminopeptidases are 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 and 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-peptideic small molecular inhibitors to monoclonal antibodies. The 59 ACS Paragon Plus Environment


neovasculature binding properties of tumor-homing cyclic peptide asparagine-glycine-arginine (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. Depending on the observations highlighted here, APN inhibitors possess common structural features associated with higher inhibitory activity. Numerous structural comparisons have allowed interpretation of 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 indoline2, 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. A ZBG such as a thiol, a boronic acid or a phosphonate are comparable to hydroxamate for APN inhibition. Indeed, rigid small molecules exhibit better efficacy than bigger molecules. Reports suggested that the 3-amino-2benzosuberones show subnanomolar 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,


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71.132 This has allowed a better understanding of the recognition of these low molecular weight derivatives by M1 aminopeptidases and thus, opens a new vista for the design of APN inhibitors.

Figure 35. Highly potent APN inhibitors with important scaffolds. Last but not least, while the last 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 last 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 61 ACS Paragon Plus Environment


APN-inhibiting lead molecules and in this Perspective, we have emphasized the importance of pursuing more potent and selective APN inhibitors. AUTHOR INFORMATION Corresponding Author * Phone: +9133-2457 2495 (O). +91-9433187443 (M). E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. 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 has completed his Master of Pharmacy (M. Pharm.) in the year 2016 from Dr. Harisingh Gour University, Sagar, India. His research area includes designing and synthesis of small molecules with the anticancer property, computational chemical biology and large-scale structure-activity relationship analysis. Currently, he is working on metalloenzyme inhibitors. Mr. Amin has published thirty three research/review articles in the different reputed peer-reviewed journals and a book chapter. He enjoys a good conversation on science, contemporary art, books, film & music.


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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 University Grants Commission (UGC), New Delhi and is pursuing research in Department of Pharmaceutical Technology, Jadavpur University, under the guidance of Tarun Jha. His research area includes designing and synthesis of anticancer small molecules. He has published fifty six research/review articles in different reputed peer-reviewed journals and five book chapters. Tarun Jha, a faculty member of Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India has supervised fourteen Ph.D. students and guided eight research projects funded by different organizations. He has published more than one hundred and ten research articles in different reputed peer-reviewed journals. His research area includes designing and synthesis of anticancer small molecules. Prof. Jha is one of the members of Academic Advisory Committee of National Board of Accreditation (NBA), New Delhi, India. ACKNOWLEDGMENT NA is grateful to University Grants Commission (UGC), New Delhi, India for providing Rajiv Gandhi National Fellowship. TJ is thankful for the financial support from 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; 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 ‘Journal of Medicinal Chemistry’ for providing editorial and bibliographic assistance. ABBREVIATIONS USED γ-GT, γ-glutamyl transpeptidase; AngIII,

angiotensin III; APA, aminopeptidase A; APB,

aminopeptidase B; APCs, antigen-presenting 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; 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 kinase3; NEP, neutral endopeptidase; PDB, protein data bank; PI3K, phosphoinositide 3-kinases; PSA, puromycin-sensitive aminopeptidase; RECK, reversioninducing-cysteine-rich protein with kazal motifs; SARs, structure-activity relationships; SAS, solvent accessible surface; SIL-6R, soluble IL-6 receptor; TGEV, transmissible gastroenteritis virus; THR-de, thyrotropin-releasing hormone-degrading enzyme; ZBG, zinc binding group. References


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Table of Contents (TOC) Graphic


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Figure 1. Schematic representation of the fifteen clans of metallopeptidases. 132x132mm (96 x 96 DPI)

ACS Paragon Plus Environment


Figure 2. Functions of human APN/CD13. 243x208mm (96 x 96 DPI)


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Figure 3. Bestatin and its derivatives as APN inhibitors. 271x201mm (300 x 300 DPI)



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 3kinases (PI3K), protein kinase kinase 3 (MKK3), extracellular signal-regulated kinases (ERK), c-jun Nterminal kinase (JNK)]. 311x270mm (96 x 96 DPI)


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Figure 5. Implications of APN in different cancers. 212x138mm (96 x 96 DPI)



Figure 6. Schematic seven domain hypothetical organization of human APN/CD13. [The location of the substrate/inhibitor binding site is highlighted as red dotted circle between domains V and VI]. 262x252mm (96 x 96 DPI)


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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) are highlighted along with important amino acid residues (shown in line). 349x163mm (96 x 96 DPI)



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. 187x139mm (96 x 96 DPI)


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Figure 9. (A-C) Comparison between bestatin and its analogs for their binding interaction with APN. 174x262mm (300 x 300 DPI)



Figure 10. Structures of leucine ureido derivatives having APN inhibitory potency (11-14). 199x234mm (300 x 300 DPI)


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Figure 11. Structural modification of 15 resulted in better active APN inhibitors. 292x196mm (300 x 300 DPI)



Figure 12. (A) Structures of L-lysine-based 20; (B-C) Comparison between the L-lysine (21) and its analog (22) for their binding interaction with APN. 165x242mm (300 x 300 DPI)


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Figure 13. The SAR of the L-lysine ureido derivatives on the template structure of 23. 160x98mm (300 x 300 DPI)



Figure 14. Structural modifications of 24 resulted in better active APN inhibitors (25-27). 293x178mm (300 x 300 DPI)


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Figure 15. Developments of 29 from 28 and 1. 217x165mm (300 x 300 DPI)



Figure 16. (A) Development of 30 from 28, (B-C) Structure of APN inhibitors (31-32) and their APN inhibitory activity. 256x186mm (300 x 300 DPI)


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Figure 17. (A) Structure of L-leucinthiol (33), (B) SAR of leucinethiol-based APN inhibitors, (C) the developing cascade involved in the designing of better active APN inhibitor PC18 (37). 271x133mm (300 x 300 DPI)



Figure 18. (A) Structure of pseudotripeptide thiol containing 38; (B) SAR of β-aminothiols having promising APN inhibitors; (C) Comparison of some β-aminothiols (39-46). 295x209mm (300 x 300 DPI)


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Figure 19. (A) Structures of boronic acid derivatives 47-50 active against cytosolic and microsomal leucine aminopeptidases; (B) Structures of 51, 52. 157x217mm (300 x 300 DPI)



Figure 20. Structures of phosphonic acid derivatives (53-57) as APN inhibitors. 261x163mm (300 x 300 DPI)


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Figure 21. Structures of phosphonic acid-based LAP inhibitor 58 and promising phosphonic acid-based APN inhibitors (59 and 60). 220x141mm (300 x 300 DPI)



Figure 22. Structures of phosphonic acid derivatives (61-62) as APN inhibitors. 218x175mm (300 x 300 DPI)


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Figure 23. Structures of 3-amino-2-tetralone derivatives as promising APN inhibitors 63-65. 166x171mm (300 x 300 DPI)



Figure 24. Structures of 3-amino-2-tetralone derivatives as promising APN inhibitors (66-72). 272x194mm (300 x 300 DPI)


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Figure 25. Binding pose of the hydrated form of thiophenyl substituted moiety. 105x98mm (300 x 300 DPI)



Figure 26. Structure of flavone-based APN inhibitor 73 and structure of 74. 194x92mm (300 x 300 DPI)


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Figure 27. (A) Structure of cyclic imide scaffold containing 75 along with SAR of cyclic imide derivatives, (B) Modification of 76 resulted in better active APN inhibitor (77). 217x172mm (300 x 300 DPI)



Figure 28. Structural modifications of 77 resulted in 78-80. 248x185mm (300 x 300 DPI)


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Figure 29. Structure of 81 and β-dicarbonyl derivative 82 as APN inhibitors. 149x121mm (300 x 300 DPI)



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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. 265x149mm (300 x 300 DPI)


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Figure 31. Structural modifications of 86 resulted in comparatively better APN inhibitor 87. 157x188mm (300 x 300 DPI)



Figure 32. Structures of indoline-2,3-dione containing APN inhibitors 88-90. 208x175mm (300 x 300 DPI)


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Figure 33. Structural modifications of 77 resulted in comparatively poor APN inhibitor 91 but further modification of 91 resulted in promising APN inhibitor 92. 241x196mm (300 x 300 DPI)



Figure 34. Structures of APN inhibitors 93-97. 284x206mm (300 x 300 DPI)


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Figure 35. Highly potent APN inhibitors with important scaffolds. 298x253mm (96 x 96 DPI)


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

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