2,5-Diketopiperazine Derivatives as Potential Anti-Influenza (H5N2) Agents: Synthesis, Biological Evaluation, and Molecular Docking Study

2,5-Diketopiperazine derivatives, consisting of benzylidene and alkylidene substituents at 3 and 6 positions, have been considered as a core structure for their antiviral activities. Herein, the novel N-substituted 2,5-Diketopiperazine derivatives were successfully prepared and their antiviral activities against influenza virus were evaluated by monitoring viral propagation in embryonated chicken eggs. It was found that (3Z,6Z)-3-benzylidene-6-(2-methyl propylidene)-4-substituted-2,5-Diketopiperazines (13b–d), (3Z,6E)-3-benzylidene-6-(2-methylpropyli dene)-1-(1-ethyl pyrrolidine)-2,5-Diketopiperazine (14c), and Lansai-C exhibited negative results in influenza virus propagation at a concentration of 25 µg/mL. Additionally, molecular docking study revealed that 13b–d and 14c bound in 430-cavity of neuraminidase from H5N2 avian influenza virus and the synthesized derivatives also strongly interacted with the key amino acid residues, including Arg371, Pro326, Ile427, and Thr439.


Introduction
2,5-Diketopiperazine (2,5-DKP) is a six-membered cyclic dipeptide which is often found alone or embedded as a part of a compound in a variety of natural products from microorganisms, plants, and animals. 2,5-DKP can bind to a wide variety of receptors due to its unique structure, which includes a rigidity, chirality, and a variety of side chains. As a result, 2,5-DKP scaffolds are widely used for drug discovery [1][2][3][4][5]. Additionally, 2,5-DKP compounds exhibit many bioactivities, which include antiviral activity [6].
For example, Aplaviroc exhibited high binding affinity to chemokine co-receptor 5, and was developed for the treatment of patients with human immunodeficiency virus type 1 (HIV-1) [1]. Eutypellazine A-L isolated from fungus Eutypella sp. MCCC 3A00281 could inhibit the replication of HIV-1 with low toxicity (CC 50 > 100). Eutypellazine E showed an inhibitory effect at IC 50 3.2 + 0.4 µM. The structure-activity relationship study of these 2,5-DKP compounds showed that the thiomethyl group at C-2/C-2 and the double bond at C-6 /C-7 in Eutypellazine E remarkably enhanced the activity compared with analogs [7]. In addition, Rubrumlines D and Neoechinulin B isolated from the fungus Eurotium rubrum  The other derivatives of 2,5-DKP are Lansai C (LS-C) and Lansai D (LS-D), which can be obtained from Streptomyces sp. SUC1 isolated from the aerial roots of Ficus benjamina in our campus [10,11]. LS-C and LS-D exhibit an anti-inflammatory effect on RAW 264.7 cells [12,13]. The structures of LS-C and LS-D contain benzylidene and alkylidene substituents at 3 and 6 positions, which are similar to that of Albonoursin, except their double bond configurations. Thus, LS-C and LS-D and their antiviral activities are worth investigation. Moreover, the modification of the N-substituents of 2,5-DKPs of Albonoursin is interested, since the functionalized chains of 2,5-DKPs and their orientations can affect the binding affinity of 2,5-DKPs to the receptors and the biological activities of 2,5-DKPs compounds [14].
In this research, the novel 2,5-DKP derivatives, which were designed by emulating Albonoursin, LS-C, and LS-D scaffolds with the addition of substitution group at the position of nitrogen atoms of the 2,5-DKP ring by N-alkylation reaction, were successfully prepared. The preliminary antiviral activity test against influenza virus (H5N2) of our 2,5-DKP derivatives, LS-C, and LS-D was then performed by hemagglutination assay. In addition, a molecular docking study was conducted to investigate how the derivatives interact with amino acids in the binding pocket of neuraminidase from H5N2 avian influenza virus (H5N2, PDB ID: 5HUK). Our work could contribute to and benefit the design and development of novel 2,5-DKP derivatives as a potential antiviral drug.

Synthesis
The steps to synthesize tetrasubstituted 2,5-DKP derivative 5 were as follows. N-Boc-Leucine 1 and Phenylalanine methyl ester 2 were reacted to obtain dipeptide 3. Then, the The other derivatives of 2,5-DKP are Lansai C (LS-C) and Lansai D (LS-D), which can be obtained from Streptomyces sp. SUC1 isolated from the aerial roots of Ficus benjamina in our campus [10,11]. LS-C and LS-D exhibit an anti-inflammatory effect on RAW 264.7 cells [12,13]. The structures of LS-C and LS-D contain benzylidene and alkylidene substituents at 3 and 6 positions, which are similar to that of Albonoursin, except their double bond configurations. Thus, LS-C and LS-D and their antiviral activities are worth investigation. Moreover, the modification of the N-substituents of 2,5-DKPs of Albonoursin is interested, since the functionalized chains of 2,5-DKPs and their orientations can affect the binding affinity of 2,5-DKPs to the receptors and the biological activities of 2,5-DKPs compounds [14].
In this research, the novel 2,5-DKP derivatives, which were designed by emulating Albonoursin, LS-C, and LS-D scaffolds with the addition of substitution group at the position of nitrogen atoms of the 2,5-DKP ring by N-alkylation reaction, were successfully prepared. The preliminary antiviral activity test against influenza virus (H5N2) of our 2,5-DKP derivatives, LS-C, and LS-D was then performed by hemagglutination assay. In addition, a molecular docking study was conducted to investigate how the derivatives interact with amino acids in the binding pocket of neuraminidase from H5N2 avian influenza virus (H5N2, PDB ID: 5HUK). Our work could contribute to and benefit the design and development of novel 2,5-DKP derivatives as a potential antiviral drug.
1,4-Diacetyl-2,5-diketopiperazine 11 underwent aldol addition-acetyl migrationelimination cascade with Cs2CO3, alkyl halide, and various aldehydes [16] to provide highly (Z)-stereoselective products 12a-e (Scheme 2). This reaction was supported by the Zimmerman-Traxler model, which was reported by Balducci and co-workers [17]. In the case of N-benzyl derivative 12g, the cascade reaction using benzyl chloride failed to provide the desired product, probably because of the steric hindrance with the benzylidene substituent. Thus, the benzylidene derivative 12f was further benzylated to provide compound 12g. Then, the next aldol condensation was accomplished to provide trisubstituted 2,5-DKP derivatives (13a-g). However, 13a-g have low solubility in chromatographic solvent, leading to low product yields [18].
Compound 13f was a natural product from Streptomyces noursei, which was known as Albonoursin [19]. N-alkylation of compound 13f with NaH, benzyl chloride, and 1chloro-3-methyl-2-butene provided disubstituted products 14a and 14b. However, only monosubstituted product, 14c, was obtained when using the bulky 1-ethyl pyrrolidine chloride. The propylidene was then converted to E-configuration, probably because of the steric hindrance (Scheme 3). The configuration of the double bond was confirmed by NMR spectroscopy. In this research, each proton NMR spectrum of the C=CH double bond in 6-(2-methylpropylidene) was observed at 6.01-6.13 ppm, which might be the indication of (6Z)-configuration similar to the observation of Fairhurst and co-workers' work, in which the chemical shifts of proton of Zand E-isomers at the position of an alkylidene double bond (C=CH) in complex tertbutyl-3-(2-methylpropylidene)-2,5-dioxopiperazine-1-carboxylate were 6.14 and 5.42 ppm, respectively [20]. The NOEDIFF spectra of compound 12e showed that the proton on the alkylidene double bond (C=CH) only enhanced the proton of isopropyl moiety. Thus, the alkylidene double bond of compound 12e was characterized as (Z)-configuration. Another NOEDIFF analysis of compound 13d showed that 4-NH and H-11 enhanced Ar-H of benzylidene moiety and 1-N-CH 3 , respectively. Thus, compound 13d was characterized as (3Z,6Z)-3-benzylidene-6-(2-methylpropylidene)-1-methyl-2,5-diketopiperazine ( Figure 2). Other results showed steric hindrance between the proton of the aromatic ring and the carbonyl and steric repulsion between hydrogen atoms in benzylidene and the N-alkylated group, which could be caused by (3Z)-selectivity. Further, NOESY analysis was used to confirm the stereochemistry of (6E)-configuration in compound 14c. NOE enhancement from H-7 to H-18, H-19, H-21, H-22, H-23, and H-24 was observed, whereas NOE enhancement of H-8 was not found. This result indicated (6E)-configuration ( Figure 2). the proton of the aromatic ring and the carbonyl and steric repulsion between hydrogen atoms in benzylidene and the N-alkylated group, which could be caused by (3Z) selectivity. Further, NOESY analysis was used to confirm the stereochemistry of (6E) configuration in compound 14c. NOE enhancement from H-7 to H-18, H-19, H-21, H-22 H-23, and H-24 was observed, whereas NOE enhancement of H-8 was not found. This result indicated (6E)-configuration ( Figure 2).

Virus Propagation Inhibition Assay
The efficacy of LS-C, LS-D, and 2,5-DKP derivatives to influenza virus (H5N2) prop agation inhibition was evaluated at various concentrations in embryonated chicken eggs A hemagglutination test was performed to estimate virus propagation. The summary re sults are displayed in Table 1. The negative control (PBS) showed the ratio of the last di lution at 1:3072, indicating complete agglutination. On the other hand, 1-adamantanamine hydrochloride and oseltamivir carboxylate were used as the positive controls. The results show that they exhibited the virus inhibition at 12.5 µg/mL while all of our tested com pounds could not inhibit the virus propagation at a concentration of 12.5 µg/mL. LS-C showed the virus inhibition at concentration of 25 µg/mL, whereas LS-D could not inhibit virus propagation even at a high concentration (100 µg/mL). The result indi cated that N-OH group of LS-C were essential for the antiviral activity. Moreover, com pound 13d, containing opposite double bond configuration to LS-D, showed an inhibition against influenza virus (H5N2) propagation at a concentration of 25 µg/mL, suggesting that the double bond in Z-configuration might enhance the activity of the compound. Ad ditionally, either the unprotected nitrogen or protected nitrogen with alkyl substituents such as allyl and 3-methyl-2-butene adjacent to (Z)-benzylidene inhibited virus propaga tion at a concentration of 25 µg/mL, as was observed in compounds 13b-d and compound 14c. In contrast, the benzyl substituent 13g had a different impact on the antiviral activity thus there should not be any substitution on nitrogen adjacent to isopropylidene, for the

Virus Propagation Inhibition Assay
The efficacy of LS-C, LS-D, and 2,5-DKP derivatives to influenza virus (H5N2) propagation inhibition was evaluated at various concentrations in embryonated chicken eggs. A hemagglutination test was performed to estimate virus propagation. The summary results are displayed in Table 1. The negative control (PBS) showed the ratio of the last dilution at 1:3072, indicating complete agglutination. On the other hand, 1-adamantanamine hydrochloride and oseltamivir carboxylate were used as the positive controls. The results show that they exhibited the virus inhibition at 12.5 µg/mL while all of our tested compounds could not inhibit the virus propagation at a concentration of 12.5 µg/mL.
LS-C showed the virus inhibition at concentration of 25 µg/mL, whereas LS-D could not inhibit virus propagation even at a high concentration (100 µg/mL). The result indicated that N-OH group of LS-C were essential for the antiviral activity. Moreover, compound 13d, containing opposite double bond configuration to LS-D, showed an inhibition against influenza virus (H5N2) propagation at a concentration of 25 µg/mL, suggesting that the double bond in Z-configuration might enhance the activity of the compound. Additionally, either the unprotected nitrogen or protected nitrogen with alkyl substituents such as allyl and 3-methyl-2-butene adjacent to (Z)-benzylidene inhibited virus propagation at a concentration of 25 µg/mL, as was observed in compounds 13b-d and compound 14c. In contrast, the benzyl substituent 13g had a different impact on the antiviral activity, thus there should not be any substitution on nitrogen adjacent to isopropylidene, for the isopropylidene is in Z-configuration in order to maintain the antiviral activity. The presence of double bonds was also important for the antiviral activity in compounds 6-9.
In addition, the cytotoxicity of LS-C, LS-D, 13b-d, and 14c, which exhibited the virus inhibition at a concentration of 25 µg/mL, were investigated against Rhesus monkey kidney epithelial cells (LLC-MK2 cell lines). It was found that their cytotoxicity activities (IC 50 ) against LLC-MK2 were 287.65 to 507.84 µg/mL, indicating that cytotoxicities of these compounds were negligible to the normal cells.

Molecular Docking Study
The molecular docking is a computer-aided procedure that generates ligand with different orientations and conformation and predicts the best match between ligand and protein target, where the lower the binding energy of a complex indicates that the complex is more stable [21]. Herein, molecular docking approach is employed not only to visualize how our compounds interact with neuraminidase from H5N2 avian influenza virus, which was the well-known drug target to prevent the spread of influenza infection [22], but also to gain valuable guidance at a molecular level for the development of new 2,5-DKP derivatives as the antiviral drugs.
The comparison of binding positions of our selected compounds with virus propagation inhibitor (LS-C and compounds 7, 9, 13a-13d, and 14c) in the active site of H5N2 was displayed in Figure 3a. The binding energies, amino acid interaction, along with hydrogen bond length of our compounds bound in the active site of receptors, were also demonstrated in Table 2. As can be seen in Figure 3b and Table 2, oseltamivir carboxylate, zanamivir, and peramivir fit and interact with amino acids in the sialic acid cavity of H5N2 in similar manner to the observation in the crystal structures of neuraminidase-inhibitor complexes [29][30][31], which validates our docking method. Moreover, compound 7 binds in the front of sialic acid cavity of H5N2 and forms hydrogen bond with ASN249 due to its large structure containing three benzyl moieties.
neuraminidase from H5N2 avian influenza virus, and their docking results were compared with our potential antiviral drugs (LS-C and compounds 7, 9, 13a-13d, and 14c). The initial structures of oseltamivir carboxylate, zanamivir, and peramivir were taken from crystal structures of neuraminidase-inhibitor complexes PDB ID: 2HU4, 3CKZ, and 3K39, respectively.
The comparison of binding positions of our selected compounds with virus propagation inhibitor (LS-C and compounds 7, 9, 13a-13d, and 14c) in the active site of H5N2 was displayed in Figure 3a. The binding energies, amino acid interaction, along with hydrogen bond length of our compounds bound in the active site of receptors, were also demonstrated in Table 2. As can be seen in Figure 3b and Table 2, oseltamivir carboxylate, zanamivir, and peramivir fit and interact with amino acids in the sialic acid cavity of H5N2 in similar manner to the observation in the crystal structures of neuraminidaseinhibitor complexes [29][30][31], which validates our docking method. Moreover, compound 7 binds in the front of sialic acid cavity of H5N2 and forms hydrogen bond with ASN249 due to its large structure containing three benzyl moieties.    On the other hand, the LS-C and compound 9, 13a-13d, and 14c, are located in a hydrophobic 430-cavity of H5N2, as shown in Figure 3c. The interaction between these compounds and key amino acid residue, PRO326, ILE427, and THR439, are also observed. Moreover, their carbonyl groups on 2,5-DKP scaffold strongly interact with ARG371, which is one of the arginine triad residues (ARG118-ARG292-ARG371), indicating the important role of 2,5-DKP scaffold for H5N2-binding and H5N2 inhibition. Hydrogen bond interactions between some of our compounds (13c, 13d, and 14c) are represented in Figure 4. In comparison to binding energy, our compounds have higher binding energy (−102.25 On the other hand, the LS-C and compound 9, 13a-13d, and 14c, are located in a hydrophobic 430-cavity of H5N2, as shown in Figure 3c. The interaction between these compounds and key amino acid residue, PRO326, ILE427, and THR439, are also observed. Moreover, their carbonyl groups on 2,5-DKP scaffold strongly interact with ARG371, which is one of the arginine triad residues (ARG118-ARG292-ARG371), indicating the important role of 2,5-DKP scaffold for H5N2-binding and H5N2 inhibition. Hydrogen bond interactions between some of our compounds (13c, 13d, and 14c) are represented in

General Experimental Procedures
All reactions sensitive to air or moisture were carried out under anhydrous conditions, unless otherwise stated. Solvents and reagents were used without further purification. The reagents were purchased from Sigma-Aldrich (Darmstadt, Germany), Tokyo Chemical Industry (Tokyo, Japan), and Fluka Chemical (Buchs, Switzerland) Companies. 1 H-and 13 C-NMR spectra were measured in CDCl3 or DMSO-d6 by a Bruker Avance 300 spectrometer (Bruker, Massachusetts, USA; 300 MHz for 1 H, 75 MHz for 13 C). Melting points were measured by using a Stuart Scientific SMP 2 melting point apparatus (Cole-Parmer Ltd., Staffordshire, UK). Mass spectra were measured by using micrOTOF (Bruker, Billerica, MA, USA). The reactions were monitored by thin-layer chromatography (TLC) and by using an aluminum sheet pre-coated with silica gel 60 F254 (Merck, Darmstadt, Germany). Column chromatography was conducted on Merck Kieselgel 60 (Merck, Darmstadt, Germany). (N-tert-butoxycarbonyl)-L-leucine 1 and L-Phenylalanine methyl ester hydrochloride 2 were prepared in accordance with the previous literatures [32,33]. Lansai A, Lansai B, LS-C, and LS-D were isolated, purified, and identified as previously described [10,11]. The 1 H-NMR, 13 C-NMR, HRMS spectra of the synthesized compounds were recorded, as shown in the Supplementary Materials.

General Experimental Procedures
All reactions sensitive to air or moisture were carried out under anhydrous conditions, unless otherwise stated. Solvents and reagents were used without further purification. The reagents were purchased from Sigma-Aldrich (Darmstadt, Germany), Tokyo Chemical Industry (Tokyo, Japan), and Fluka Chemical (Buchs, Switzerland) Companies. 1 H-and 13 C-NMR spectra were measured in CDCl 3 or DMSO-d 6 by a Bruker Avance 300 spectrometer (Bruker, Massachusetts, USA; 300 MHz for 1 H, 75 MHz for 13 C). Melting points were measured by using a Stuart Scientific SMP 2 melting point apparatus (Cole-Parmer Ltd., Staffordshire, UK). Mass spectra were measured by using micrOTOF (Bruker, Billerica, MA, USA). The reactions were monitored by thin-layer chromatography (TLC) and by using an aluminum sheet pre-coated with silica gel 60 F 254 (Merck, Darmstadt, Germany). Column chromatography was conducted on Merck Kieselgel 60 (Merck, Darmstadt, Germany). (Ntert-butoxycarbonyl)-L-leucine 1 and L-Phenylalanine methyl ester hydrochloride 2 were prepared in accordance with the previous literatures [32,33]. Lansai A, Lansai B, LS-C, and LS-D were isolated, purified, and identified as previously described [10,11]. The 1 H-NMR, 13 C-NMR, HRMS spectra of the synthesized compounds were recorded, as shown in the Supplementary Materials.

Virus Propagation Inhibition Assay
Virus propagation inhibition assays were examined by embryonated chicken egg inoculation. One hundred microliters of LS-C, LS-D, and 2,5-DKP derivatives at various concentrations (25,50, and 100 µg mL −1 ) in 0.1 M PBS (pH 7.2) (from DMSO stock) were incubated with 100 µL of virus suspension at 37 • C for 30 min. The mixture (100 µL) was inoculated into each embryonated chicken egg and incubated at 37 • C for 4 days. Virus dissolved in saline solution was used as a negative control. The allantoic fluid was investigated by Hemagglutination assay [40].

Hemagglutination Assay (HA)
Fresh chicken blood was taken in Alsever's solution and centrifuged at 2000 rpm for 5 min. The chicken red blood cells (RBCs) were separated from the supernatant and washed with PBS until the supernatant was cleared. The 2.5% suspension of RBCs was prepared by mixing with PBS. Later, the allantoic fluid 100 µL, which was harvested from inoculated embryonate chicken eggs, was mixed with PBS 50 µL and diluted with 2-fold serial dilution in PBS from 1:1 to 1:3072 in a 96-well micro titer plate. 1-Adamantanamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) used a positive control, and PBS used as negative control, were included on micro plates. The 2.5% RBCs suspension 100 µL was added and mixed to every well. The microplate was kept at 4 • C for 45-60 min. Negative results displayed of the RBCs precipitation by gravity to the bottom of the well demonstrated absence of the virus, while positive results displayed the formation of a diffuse mat on the bottom of the well, leading to a lattice formation of RBCs and the virus. The HA titers were reported as the end point of the virus's last dilution, showing complete agglutination. The experiments were performed in triplicate.

Cytotoxicity
Cytotoxicity was evaluated against normal cell line Rhesus monkey kidney epithelial cells LLC-MK2 (Korean Cell Line Bank, KCLB) by using a MTT assay [41]. A stock solution of LS-C, LS-D, and 2,5-DKP derivatives was prepared at the concentration of 5000 µg/mL in EtOH. The stock solution was further diluted to 128 µg/mL prior to use and added to the first wells. Two-fold serial dilutions were employed to obatin a by culture medium with final concentration of 1 µg/mL. Cells were seeded at a density of 5 × 10 4 cells/well and incubated for 16 h in a 96 well plate, followed by the treatment with the test compounds. 2.5% DMSO was used as a control culture. After 24 h, LLC-MK2 cells were incubated with MTT (500 µg/mL) for 4 h. DMSO was then added to dissolve the formation of blue formazan crystals. Finally, optical density at 450 nm was determined by a microplate reader.

Molecular Docking Study
Molecular docking study was performed using iGEMDOCK (Generic Evolutionary Method for Molecular DOCKing) v.2.1 (Institute of Bioinformatics National Chiao-Tung University, Hsinchu, Taiwan) to investigate the possible binding between neuraminidase from H5N2 avian influenza virus (H5N2, PDB ID: 5HUK) and our compounds (LS-C, compound 7, 9, 13a-13d, and 14c) to explore the capabilities of the compounds as virus propagation inhibitors in comparison to oseltamivir carboxylate, zanamivir, and peramivir. The accurate docking (very slow) with population size (N = 800), 80 generation, and 10 solutions were applied for the docking of each ligand against protein. The docking pose with the lowest binding energy value for each ligand-protein complex was then analyzed and imaged using BIOVIA Discovery Studio Visualizer [24].

Conclusions
2,5-Diketopiperazine derivatives have been successfully prepared by addition of benzylidene and isopropylidene substituents based on the structure of Albonoursin. Using hemagglutination assay, their antiviral activity against influenza virus (H5N2) was investigated and compared with those of the natural compounds with a similar structure (Lansai C and D). The results showed that LS-C and compounds 13b-d and 14c exhibited antiviral activity against influenza virus (H5N2) at a concentration of 25 µg/mL. Furthermore, the molecular docking study showed that these compounds could bind and strongly interact to key amino acid residues in 430-cavity of neuraminidase from H5N2 avian influenza virus.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.