Figures
Abstract
Background
Polo-like kinase-1 (Plk1) plays a crucial role in cell proliferation and the inhibition of Plk1 has been considered as a potential target for specific inhibitory drugs in anti-cancer therapy. Several research groups have identified peptide-based inhibitors that target the polo-box domain (PBD) of Plk1 and bind to the protein with high affinity in in vitro assays. However, inadequate proteolytic resistance and cell permeability of the peptides hinder the development of these peptide-based inhibitors into novel therapeutic compounds.
Methodology/Principal Findings
In order to overcome the shortcomings of peptide-based inhibitors, we designed and synthesized small molecule inhibitors. Among these molecules, bg-34 exhibited a high binding affinity for Plk1-PBD and it could cross the cell membrane in its unmodified form. Furthermore, bg-34-dependent inhibition of Plk1-PBD was sufficient for inducing apoptosis in HeLa cells. Moreover, modeling studies performed on Plk1-PBD in complex with bg-34 revealed that bg-34 can interact effectively with Plk1-PBD.
Conclusion/Significance
We demonstrated that the molecule bg-34 is a potential drug candidate that exhibits anti-Plk1-PBD activity and possesses the favorable characteristics of high cell permeability and stability. We also determined that bg-34 induced apoptotic cell death by inhibiting Plk1-PBD in HeLa cells at the same concentration as PEGylated 4j peptide, which can inhibit Plk1-PBD activity 1000 times more effectively than bg-34 can in in vitro assays. This study may help to design and develop drug-like small molecule as Plk1-PBD inhibitor for better therapeutic activity.
Citation: Srinivasrao G, Park J-E, Kim S, Ahn M, Cheong C, Nam K-Y, et al. (2014) Design and Synthesis of a Cell-Permeable, Drug-Like Small Molecule Inhibitor Targeting the Polo-Box Domain of Polo-Like Kinase 1. PLoS ONE 9(9): e107432. https://doi.org/10.1371/journal.pone.0107432
Editor: Andrea Cavalli, University of Bologna & Italian Institute of Technology, Italy
Received: June 4, 2014; Accepted: August 9, 2014; Published: September 11, 2014
Copyright: © 2014 Srinivasrao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the Korea Basic Science Institute’s Research Program grant T34418 (JKB) and the Next-Generation BioGreen 21 Program (#PJ009594), RuralDevelopment Administration (NHK). K.-Y. Nam would like to thank Professor Kyoung Tai No (Yonsei Univ.) for allowing to access the computational resources in his group. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Polo-like kinases (Plks) 1–4 play critical roles in numerous cell cycle-related activities including the initiation of mitosis, chromosome segregation, centrosome maturation, bipolar-spindle formation, regulation of the anaphase-promoting complex, and execution of cytokinesis [1]–[6] whereas the Plk5 does not appear to function in cell-cycle progression. Among five human Plks, Plk1 has been studied most extensively because its activity can override spindle checkpoints and induce genetic instability and thereby promote tumorigenesis [7]–[11]. Because the overexpression of Plk1 is strongly correlated with the aggressiveness and prognosis of several cancers [9], Plk1 has been examined as a potential target for specific inhibitory drugs in anti-cancer therapy. Plk1, which is a key regulator of mitotic progression and cell division in eukaryotes, possesses an N-terminal catalytic domain and a C-terminal polo-box domain (PBD) composed of two highly homologous polo boxes (PB1 and PB2). A large body of evidence suggests that the PBD recognizes phosphoserine (pS)/phosphothreonine (pT)-containing motifs with N-proximal serine residues (S-(pT/pS)), and directs the N-terminal catalytic domain for specific subcellular locations [12]–[14], which plays crucial in cell regulating mitotic progression and proliferation [15]–[17]. Therefore, targeting the PBD rather than kinase domain could be an alternative strategy to overcome the cross-reactivity commonly associated with kinase-domain inhibitors [18], [19]. Because of its essential role in cell proliferation, there has been a high level of interest to identify the interaction between Plk1 and its centromere/kinetochore-associated binding target, that led to the discovery of polo-box-interacting protein 1 (PBIP1) and a minimal peptide, PLHSpT (Figure 1) (residues 74–78 of PBIP1) [20] and its derivatives that exhibit high anti-Plk1-PBD activity [21]–[30]. However, peptide-based drugs are widely recognized to present severe shortcoming in terms of proteolytic stability and cell permeability, and this hampers the development of the peptide drugs into novel therapeutic agents. To enhance their cell permeability, these peptide inhibitors must be modified by, for example, conjugating them with a cell-penetrating peptide or PEGylating them, but this requires complicated synthetic steps and also occasionally results in a reduction of binding affinity.
To overcome the aforementioned drawbacks, cell-permeable, drug-like small molecules that target Plk1-PBD must be developed. In addition to various PBD-binding phosphopeptides, to date, three small molecules have been identified as Plk1-PBD inhibitors: purpurogallin (PPG) [17], poloxin [15], and thymoquinone [15] (Figure 1). However, we recently demonstrated that only PPG binds to Plk1-PBD and that its binding affinity was 6-times lower than that of PLHSpT [31].
To continue our efforts aimed at discovering and developing Plk1-PBD inhibitors [29], [30], in this study, we used a structure-based approach to design a novel, potent, cell-permeable small molecule named bg-34, which exhibits a high binding affinity for Plk1-PBD; this binding affinity was 5-fold higher than that of PLHSpT and 30-fold higher than that of PPG. Furthermore, to identify bg-34 as a potential drug that targets Plk1-PBD, we examined how bg-34 inhibits Plk1 function and how the drug is taken up by HeLa cells. Moreover, to investigate the nature of bg-34 binding to Plk1-PBD, we conducted molecular-modeling studies on Plk1-PBD in complex with bg-34. To the best of our knowledge, this is the first report to describe a small molecule as a promising drug candidate designed for targeting Plk1-PBD.
Results and Discussion
Structure-guided design of small molecules
To design a drug-like small molecule that inhibits Plk1, we first closely examined the nature of Plk1-PBD binding to and complex formation with phosphopeptide ligands. Until now, seven X-ray crystal structures of human Plk1-PBD have been determined [12], [13], [15], [32], and these crystal structures have shown that the peptides bind in a similar manner within a positively charged grove located between PB1 and PB2. Specifically, analysis of the crystal structure of Plk1-PBD in complex with a ligand revealed that three binding pockets are critical to bind Plk1-PBD with high affinity: (1) SpT-dependent electrostatic binding pocket in which the SpT motif of the phosphopeptide acting as a high-affinity anchor. (2) pyrrolidine binding pocket surrounded by Trp414, Phe535, and Arg516 residues in which N-terminal Pro residue provides additional binding affinity to the Plk1 PBD through participating in a hydrogen bonding interaction between carbonyl oxygen and the guanidium moiety of Arg516 of the PBD. (3) Tyr-rich channel that is surrounded by Tyr417, Tyr481 and Tyr485 in which long chain n-alkyl phenyl group anchored on His residue of phosphopeptide provides strong binding affinity through hydrophobic interactions with the above residues (Figure S1 in File S1) [16], [30].
These observations suggest that appropriate design of non-peptide, small-molecule scaffold would provide drug-like small molecule targeting Plk1-PBD with high binding affinity. To mimic all the above interacting residues of compound 4j, we presented a benzimidazole skeleton as our scaffold which provides rigidity around the central unit. This skeleton allows silent structural features of high affinity SpT, n-alkylphenyl His motif and specificity inducing N-terminal Pro residue as in compound 4j. Moreover, its derivatives play predominant role in drug discovery and can be regarded as highly favored structures in medicinal chemistry [33].
First-phase synthesis
We started the inhibitor synthesis by using the commercially available compounds 5-phenyl valeric acid 1 and (4-methoxyphenyl) methanamine 2 (Figure 2). The reaction of these two compounds in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI), 1-hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine (DIEA) yielded the amide compound 3. Treating the amide 3 with methyl 4-chloro-3-nitrobenzoate 4 in the presence of Pd(TFA)2, BINAP, and Cs2CO3 and then refluxing with AcOH/Fe generated the benzimidazole derivative 5, which is a key intermediate in our synthetic process [34]. The ester functional group in 5 was reduced using LiAlH4 to generate the alcohol 6. The alcohol functional group in 6 was phosphorylated by using dibenzyl-N,N-diisopropylphosphoramidite in the presence of 1H-tetrazole, and then it was oxidized using tert-butyl hydroperoxide (tBuOOH) to create the corresponding phosphoric ester 7. Finally, deprotection of benzyl groups in 7 by using TFA generated the molecule PLS5 (Figure 2) [35].
Using the aforementioned strategy, we synthesized PLS1–PLS3, in which distinct alkyl groups were tethered on the imidazole ring to target the Tyr-rich channel and the pyrrolidine-binding pocket of Plk1-PBD (Figure 3A).
Structures of PLS1–PLS3 and PLS5 (A). ELISA-based measurement of Plk1-PBD binding (B); the IC50 graph is shown (O.D., optical density).
Next, we focused on the Tyr-rich channel of Plk1-PBD, which is surrounded by Tyr417, Tyr481, and Tyr485; to target this channel, we incorporated a butylphenyl chain on the imidazole ring of PLS1, because our recent results revealed that the phenyl ring featuring four-carbon tether was efficient for reaching the Tyr-rich channel of Plk1-PBD [30]. Furthermore, to examine the effect of aromaticity by π-π stacking interactions with the Tyr-rich channel, we incorporated nonaromatic groups, which included cyclohexane in PLS2 and an alkyl chain in PLS3. Finally, we examined the pyrrolidine-binding pocket of Plk1-PBD, which is broad and shallow and is surrounded by Trp414 and Phe535 [16]. To enable binding with the pyrrolidine-binding pocket by means of π-π stacking interactions, we incorporated an aromatic (phenyl) group at N-position of bezimidazole featuring distinct carbon tethers in PLS1–PLS3 and PLS5.
Preliminary evaluation
The competitive inhibitory activity of the synthesized compounds PLS1–PLS3 and PLS5 were evaluated using an ELISA-based Plk1-PBD-inhibition assaydescribed previously [16]. The assay results revealed that PLS1–PLS3 did not show any binding affinity even at the high concentration. In contrast, PLS5, which features the same configuration as PLS1 except for the presence of a methoxy group on the phenyl ring, showed weak binding to Plk1-PBD at high concentration (Figure 3B). Based on the preliminary assay results, PLS5 became a core skeleton to further improve in binding affinity against Plk1-PBD.
Second-phase synthesis
Since PLS5 binds to Plk1-PBD weakly, we investigated to enhance binding affinity of this molecule. First, we closely examined the phospho-binding pocket of Plk1-PBD, which is critical for obtaining high-binding affinity. The phosphate group in PLS5 scaffold resembles a pSer and moreover, Elia, A. E et al. described that replacing pThr with pSer in the peptide lowered the peptide’s binding affinity for Plk1-PBD [12]. This finding suggested that adding a pThr group instead of a phosphate group (a pSer mimic) would increase the binding affinity of PLS5 for Plk1-PBD.
As stated above, to increase the binding affinity of Plk1-PBD by interacting with the electrostatic binding pocket surrounded by positively charged His538 and Lys540 residues, we synthesized bg-33 by starting from the ester 5 (Figure 4). The ester functional group in 5 was hydrolyzed using 1,4-dioxane in the presence of 4N HCl under reflux conditions; this yielded the acid derivative 8, which was used for solid-phase synthesis of the molecule bg-33 by using pThr in the presence of the coupling agents 1-O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluoro-phosphate (HBTU), HOBt, and DIEA (Figure 5). Next, to examine the effect of alkyl-chain position on the molecule’s binding affinity for Plk1-PBD, we synthesized bg-1 and bg-2 which are positional isomers of bg-33 (Figure 5). In this synthetic process, we first synthesized the acid derivative 9 by starting from methyl 3,4-diaminobenzoate 10. The diamine 10 was treated with anisaldehyde 11 in the presence of catalytic anhydrous p-TSA in DMF at 80°C, which generated the key intermediate 12 (Figure 6) [36]. The n-alkylation of 12 by using 4-phenyl butyl bromide in the presence of K2CO3 in DMF at 60°C produced the compound 13 as an isomeric mixture of 13a and 13b. The n-alkylated mixture 13 was hydrolyzed by using 1,4-dioxane in the presence of 4 N HCl under reflux conditions, which yielded the acid 9 as a non-separable isomeric mixture of 9a and 9b; from this mixture, we synthesized bg-1 and bg-2 by using Thr(PO(OBzl)OH)-OH in the presence of HBTU, HOBt, and DIEA, which served as coupling agents.
We speculate that the butylphenyl group and the methoxy phenyl group on the benzimidazole ring may block each other which hinders alkyl residues to reach the proper binding channels in Plk1-PBD effectively. In an attempt to eliminate this problem, we synthesized the mono-alkylated analogues such as bg-27 and bg-28 (Figure 5). To synthesize bg-27 and bg-28, we initially synthesized the acid derivative 14 by starting from methyl 1H-benzo[d]imidazole-5-carboxylate 15. The compound 15 was n-alkylated by using 4-phenyl butyl bromide in the presence of K2CO3 in DMF at 60°C, and this generated the compound 16 as an isomeric mixture of 16a and 16b. The ester functional group in 16 was hydrolyzed by using 1,4-dioxane in the presence of 4N HCl under reflux conditions, which yielded the acid 14 as an isomeric mixture of 14a and 14b (Figure 7); from this mixture, we synthesized bg-27 and bg-28 using Thr(PO(OBzl)OH)-OH in the presence of the coupling agents HBTU, HOBt, and DIEA.
To investigate the effect of extending the chain length between the pThr group and the benzimidazole, we synthesized bg-5, bg-6, bg-29 and bg-30 by using β-alanine. So there may be a feasibility of butylphenyl chain on the imidazole ring to reach the proper binding channel in Plk1-PBD (Figure 5).
Second-phase results of Plk1 PBD binding affinity
ELISA-based inhibition-assay results indicated that bg-33 completely lost its binding ability against Plk1-PBD because alkyl groups on imidazole ring in bg-33 may not direct in proper binding channels. In addition, the molecules bg-1 and bg-2 which are positional isomers of bg-33 also did not exhibit any binding affinity for Plk1-PBD. These results support our assumption that both alkyl groups on benzimidazole ring in bg-33 as well as bg-1 and bg-2 might be blocking each other. To investigate this problem, we examined bg-27 and bg-28 and the assay result of these two molecules did not exhibit any binding affinity for Plk1-PBD. From these results, we concluded that the alkylphenyl groups collision was not the prime factor for the failure of bg-1 and bg-2 to exhibit good binding. Next, we tested bg-5, bg-6, bg-29 and bg-30 which increase the distance between the pThr group and the benzimidazole ring. Assay results showed that bg-5 and bg-6 bind to Plk1 PBD weakly. However, bg-29 and bg-30 did not exhibit any binding activity at the tested concentration.
It was well documented that Tyr-rich channel of Plk1-PBD was extremely narrow and deep, whereas the pyrrolidine-binding pocket was broad and shallow [16]. These findings indicated that small molecules must possess long tethers to interact with the Tyr-rich channel and they must be flexible to interact with the pyrrolidine-binding pocket. We surmised that in bg-33, the four-carbon tether on the phenyl group would be adequate for interacting with the Tyr-rich channel, but that the methoxy phenyl group would be flexible if we increased the distance between the phenyl and imidazole rings. Based on considering all these factors, we synthesized the molecule bg-34 (Figure 8A) by using the compound 17, the synthesis of which was started from 5-phenyl valeric acid (1) and 2-phenylethanamine 18 (Figure 9). The reaction of 1 and 18 in the presence of EDCI, HOBt, and DIEA generated the amide 19, which was treated with methyl 4-chloro-3-nitrobenzoate in the presence of Pd(TFA)2, BINAP, and Cs2CO3 and then refluxed with AcOH/Fe to generate the benzimidazole derivative 20. Hydrolysis of the ester 20 with 1,4-dioxane in the presence of 4N HCl under reflux conditions yielded the acid 17, and bg-34 was synthesized from the acid derivative 17 by using Thr(PO(OBzl)OH)-OH in the presence of HBTU, HOBt, and DIEA.
Structure of bg-34 (A) and ELISA-based measurement of Plk1-PBD binding (B); the IC50 graph is shown (O.D., optical density).
In striking contrast to the results obtained for other aforementioned molecules, our Plk1-inhibition assay results revealed that bg-34 exhibited extremely high binding affinity for Plk1-PBD and we determined its IC50 to be 4.53 µM, which is approximately 5 times more than that of PLHSpT (IC50 = 23.42 µM) (Figure 8B). These results support our hypothesis that the three functional groups and the scaffold of bg-34 together determine high-affinity binding by interacting with the phospho-binding pocket, the Tyr-rich channel, and the pyrrolidine-binding pocket of Plk1-PBD.
Relative activities of the compounds PLHSpT, PPG, PLSn (n = 1, 2, 3 & 5), Bgn (n = 1, 2, 5, 6, 27–30, 33 & 34) in the inhibition of the Plk1 PBD are summarized in Table S1 in File S1.
Molecular modeling
To enhance our understanding of the nature of bg-34 binding to Plk1-PBD, we conducted molecular-modeling studies on Plk1-PBD in complex with bg-34 (Figure 10). This modeling work also supported our hypothesis that both the benzimidazole scaffold and the three functional groups were required for strong interaction with Plk1-PBD. The model suggests that the negatively charged phosphate group of bg-34 interacts with the positively charged residues Lys540 and His538, which are located at the phospho-binding pocket. The long-chain phenyl group (butylphenyl) introduced in bg-34 entered the narrow passage of the Tyr-rich hydrophobic channel that is surrounded by Tyr485, Val415, and Tyr417 and engaged in hydrophobic interactions, and the short-chain phenyl group (ethyl phenyl) substituted in bg-34 additionally binds to the pyrrolidine-binding pocket by means of π-π stacking interactions with Trp414 and Phe535. From the modeling study of bg-34, we tried to explain inefficiency of bg-1, bg-2, bg-27 and bg-28 to show binding affinity with the Plk1 PBD. In case of bg-1 and bg-2, methoxy phenyl group could not reach the pyrrolidine binding pocket because there is no two carbon linker between methoxy phenyl and benzimidazole groups. This hypothesis was supported by increasing activity of bg-34 which has two carbon linker between phenyl group and benzimidazole group. The lost binding affinity in bg-27 and bg-28 implied that two functional groups are not enough to interact with Plk1 PBD using our benzimidazole scaffold. The above observations suggest that three functional groups are essential for achieving the effective interaction with Plk1 PBD in terms of Tyr-rich channel, pyrrolidine binding pocket and phospho binding pocket. To confirm binding mode of bg-34, we are also ongoing X-ray complex structure PBD with bg-34. We expect that X-ray complex structure will also support our hypothesis that bg-34 has mono-specificity against Plk1-PBD from closely related Plk2, and Plk3.
The model was generated using Pymol (http://www.pymol.org).
Cellular uptake
Although phosphopeptides bind to Plk1-PBD with high affinity in vitro, the peptides are inefficiently taken up by cancer cell lines. To increase the cellular uptake of inhibitors, the phosphopeptides must be conjugated with a cell-penetrating peptide or they must be PEGylated; however, these methods are time-consuming and require advanced skills, and this raises the cost of developing anti-Plk1-PBD therapeutic agents. Furthermore, these strategies occasionally cause the inhibitors to lose their Plk1-PBD-binding activity partially or completely. In light of these findings, we tested whether bg-34 is taken up by HeLa cells by performing fluorescence imaging; to examine the cellular uptake of bg-34, we conjugated it with fluorescein 5(6)-isothiocyanate (FITC) (as shown in Figure S2 in File S1) and then incubated 200 µM FITC-bg-34 with HeLa cells for 3 h.
The results of the cellular-uptake assays showed that FITC alone (control) was not taken up by HeLa cells; by contrast, the cellular uptake of FITC-bg-34 was clearly observed (Figure 11). The fluorescence distribution in HeLa cells indicated that bg-34 was localized within discrete vesicular compartments of the cells, which suggested that endocytosis was the predominant mechanism by which bg-34 entered cells. This result implied that bg-34 overcome one of the main drawback of peptide-based drugs such as cellular impermeability.
Nuclei were stained with DAPI (blue) and bg-34 was detected by means of FITC fluorescence (green) and the images were overlaid; 200× magnification.
Inhibition of Plk1-PBD function by bg-34
To investigate whether bg-34 inhibits the kinase activity of Plk1, we tested the effect of the molecule directly on cultured HeLa cells. The results showed that after treatment with bg-34, Plk1 activity was lowered to approximately 65% of the activity in control, which implies that around 35% of the activity was inhibited (Figure 12A). Furthermore, to examine whether the uptake of bg-34 by the cells is sufficient for causing apoptosis through Plk1 inhibition, we incubated cultured HeLa cells with 200 µM bg-34 for 12 h. Unexpectedly, in the absence of any carriers, bg-34 potently induced apoptotic cell death and also caused a considerable degree of necrosis in HeLa cells, whereas the control PBS treatment did not exert any inhibitory effect (Figure 12B and 12C). The most potent peptide analog available for inhibiting Plk1 is 4j (Figure 1) (IC50 = 17 nM), which must be PEGylated to make it cell permeable, but this modification drastically diminishes the ability of 4j to inhibit Plk1 (≈200 µM) in cultured HeLa cells [16]; by comparison, in the cell-based assay, the inhibitory effect of bg-34 was substantially higher (IC50 = 4.5 µM). Although less potent than 4j at inhibiting Plk1 in vitro, bg-34 exhibited superior pharmacokinetic properties, particularly enhanced membrane permeability and stability; thus, a greater fraction of the administered dose of bg-34 than of 4j was available for producing biological effects. Therefore, although both bg-34 and PEGylated 4j can inhibit Plk1, the synthesis of PEGylated 4j requires greater time and expense than bg-34 synthesis and PEGylation also occasionally leads to a reduction of the inhibitor’s binding affinity for Plk1; all of these drawbacks were overcome with the design and use of bg-34.
Inhibition of Plk1 kinase by bg-34 (A). HeLa cells were incubated with 200 µM bg-34 or with PBS and the effect of bg-34 treatment on the cancer cells was examined. Morphology of HeLa cells after incubation for 24 h with 200 µM bg-34 (B). Apoptosis in HeLa cells; apoptosis was detected by means acridine-orange staining after treatment with bg-34 for 24 h (200× magnification) (C).
Conclusions
We have demonstrated that bg-34 is a promising drug-like small molecule that can bind to Plk1-PBD with high affinity and, moreover, can be efficiently taken up by cells even when it is in an unmodified state. We have also demonstrated that bg-34-dependent inhibition of Plk1 activity is sufficient for inducing apoptosis in HeLa cells. Furthermore, the results of molecular-modeling studies performed on Plk1-PBD in complex with bg-34 support our hypothesis that the scaffold used in the compound is suitable for enabling its effective binding to Plk1-PBD. This study could thus help guide the design and development of drug-like small molecules that function as Plk1-PBD-specific inhibitors and exhibit high therapeutic activity.
Materials and Methods
All reactions requiring anhydrous conditions were conducted in flame-dried reaction vessels under a positive pressure of Argon. Reagents were obtained commercially (Sigma-Aldrich or TCI) and used without further purification. For the chromatography analysis, high-performance liquid chromatography (HPLC) grade solvents such as hexane, ethyl acetate, methylene chloride, and methanol were used. Thin layer chromatography (TLC) was performed on analytical Merck silica gel 60 F254 and flash chromatography was performed on Merck silica gel 60 (230–400 mesh). Proton, carbon-13 and phosphorus-31 NMR spectra were acquired on a Bruker Avance-300 MHz spectrometer operating at 300, 75 and 121 MHz, respectively. 1H NMR data are expressed as chemical shifts in ppm followed by multiplicity (s–singlet; d–doublet; t–triplet; q–quartet; m–multiplet), number of proton(s), and coupling constant(s) J (Hz). 13C NMR chemical shifts are expressed in ppm. The molecular masses of purified compounds were determined using matrix assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Shimadzu, Japan). Reverse-phase HPLC analysis (RP-HPLC) was carried out at 230 nm on an Agilent HPLC system equipped with a C18 analytical column (25010 mm, 5 micron). Two different linear gradients of 0.05% aq. TFA (eluent A) and 0.05% TFA in CH3CN (eluent B) were used with a flow rate of 1.5 mL/min at 25°C.
Synthesis of N-(4-methoxybenzyl)-5-phenylpentanamide 3
5-phenylpentanoic acid 1 (1 g, 5.6 mmol) and methoxybenzylamine 2 (0.73 mL, 5.6 mmol) were stirred in the presence of coupling agents EDCI (1.12 g, 6.2 mmol) HOBt (0.84 g, 6.2 mmol) and DIEA (2.34 mL, 16.8 mmol) for 1 h in dry DCM, After completion of the reaction, mixture was quenched with H2O (5 mL) and extracted with DCM (250 mL). The combined organic layers were washed with saturated citric acid solution (20 mL), saturated NaHCO3 solution (20 mL) and brine. The organic phase was dried over anhydrous Na2SO4 and the solvent was evaporated. The resulted residue was purified by silica gel column chromatography using (ethyl acetate/hexane = 2∶3) yielded amide 3 (1.95 g, 90%) as liquid.
Spectral data.
1H NMR (CDCl3, 300 MHz): δ 7.29–7.23 (m, 2H), 7.20–7.13 (m, 5H), 6.84 (d, 1H, J = 6.0 Hz), 5.73–5.67 (br s, 1H), 4.34 (d, 2H, J = 6.0 Hz), 3.78 (s, 3H), 2.61 (d, 2H, J = 5.7 Hz), 2.19 (d, 2H, J = 5.4 Hz), 1.73–1.60 (m, 4H).; 13C NMR (CDCl3, 75 MHz): 172.8, 158.8, 142.1, 130.6, 129.0, 128.3, 128.2, 125.7, 113.9, 55.2, 42.8, 36.3, 35.6, 31.0, 25.4.
Synthesis of Methyl 1-(4-methoxybenzyl)-2-(4-phenylbutyl)-1H-benzo[d]imidazole-5-carboxylate 5
The compound 3 was reacted with methyl 4-chloro-3-nitrobenzoate (0.2 g, 0.93 mmol), (0.55 g, 1.12 mmol), in the presence of palladium trifluoroacetate (30 mg, 0.04 mmol), (R)-BINAP (56 mg, 0.04 mmol), and cesium carbonate (493 mg, 0.7 mmol) in dry toluene. The mixture was heated to 80°C under argon atmosphere and stirred for 12 h. After completion, reaction mixture was quenched with water (3 mL) and filtered through a pad of Celite. The filterate was washed with ethyl acetate (3×6 mL) and the collected organic layer was dried and evaporated. The resulted residue was dissolved in glacial acetic acid (20 mL) and was heated to reflux for 1 h in the presence of iron powder (0.63 g). The acetic acid was removed under vacuum, and the residue was suspended in saturated NaHCO3 and extracted with ethyl acetate (250 mL). The combined organic layer was washed with brine and dried over anhydrous Na2SO4. The resulted crude residue was purified using silica gel column chromatography (ethyl acetate/hexane = 2∶3) to yield the desired product 5 (0.23 g, 60% for two steps) as liquid.
Spectral data.
1H NMR (CDCl3, 300 MHz): δ 8.46 (s, 1H); 7.93 (d, 1H, J = 6.0 Hz); 7.27–7.22 (m, 3H); 7.18–7.11 (m, 3H); 6.94 (d, 1H, J = 9.0 Hz), 6.81 (d, 1H, J = 6.0 Hz), 5.24 (s, 2H), 3.92 (s, 3H), 3.76 (s, 3H), 2.86 (t, 2H, J = 6.0 Hz), 2.62 (t, 2H, J = 6.0 Hz), 1.92–1.84 (m, 2H); 1.76–1.68 (m, 2H); 13C NMR (CDCl3, 75 MHz): 167.6, 159.3, 157.0, 142.2, 142.0, 138.6, 128.3, 128.2, 127.4, 127.3, 125.7, 124.2, 124.0, 121.5, 114.4, 109.2, 55.2, 52.0, 46.6, 35.5, 31.0, 27.5, 27.0.
Synthesis of (1-(4-methoxybenzyl)-2-(4-phenylbutyl)-1H-benzo[d]imidazol-5-yl)methanol 6
LiAlH4 (10 mg, 0.24 mmol) in dry THF (10 mL) was added to the solution of compound 5 (0.2 g, 0.48 mmol) in THF (15 mL) at 0°C. The resulted reaction mixture was brought to room temperature and stirred for 1 h. After completion, reaction mixture was quenched with water and 15% NaOH solution at 0°C. The resultant white precipitate was filtered and washed with ethyl acetate (20 mL). The filtrate was concentrated by vacuo and purified by silica gel column chromatography (MeOH/DCM = 1∶49) to afford alcohol 6 (0.17 g, 90%) as a liquid.
Spectral data.
1H NMR (CDCl3, 300 MHz): δ 7.69 (s, 1H), 7.23 (t, 3H, J = 6.0 Hz), 7.13 (dd, 4H, J = 6 and 15 Hz), 6.92 (d, 2H, J = 6 Hz), 6.79 (d, 2H, J = 6 Hz), 5.20 (s, 2H), 4.74 (s, 2H), 3.74 (s, 3H), 2.83 (t, 2H, J = 6.0 Hz), 2.59 (t, 2H, J = 6.0 Hz), 1.84 (q, 2H, J = 6.0), 1.69 (q, 2H, J = 6.0).; 13C NMR (CDCl3, 75 MHz): 159.2, 155.6, 142.5, 142.0, 135.3, 134.8, 128.3, 128.2, 127.9, 127.4, 125.7, 122.0, 118.0, 114.3, 109.5, 65.6, 55.2, 46.5, 35.5, 31.0, 27.4, 27.2.
Synthesis of dibenzyl (1-phenethyl-2-(4-phenylbutyl)-1H-benzo[d]imidazol-5-yl) methyl phosphate 7
The compound 6 (0.15 g, 0.39 mmol) was reacted with dibenzyl N,N-diisopropylphosphoramidite (0.39 mL, 1.17 mmol) in the presence of 1H-tetrazole (4.3 mL of 0.45 M in acetonitrile, 1.95 mmol) in dry DMF (5 mL) at 0°C. The reaction mixture warmed to room temperature and stirred for 3 h. After completion, reaction mixture was cooled to 0°C and slowly added tBuOOH (0.4 mL of 5–6 M solution in n-decane, 2.34 mmol,), and stirring was continued for 1 h at 0°C. The residue was taken in ethyl acetate (50 mL), washed with saturated NaHCO3 and brine. The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. The resulted crude residue was purified by using silica gel column chromatography (ethyl acetate/hexane = 2∶3) gave phosphoric ester 7 (0.20 g, 82% for two steps) as a liquid.
Spectral data.
1H NMR (CD3OD, 300 MHz): δ 7.84 (d, 2H, J = 6.0 Hz), 7.78 (d, 1H, J = 6.3 Hz), 7.69 (d, 1H, J = 6.6 Hz), 7.60 (d, 1H, J = 6.3 Hz), 7.55 (d, 1H, J = 6.6 Hz), 7.27–7.21 (m, 4H), 7.19–7.10 (m, 8H), 6.98 (d, 1H, J = 6.0 Hz), 6.90 (d, 1H, J = 6.3 Hz), 5.60 (s, 2H), 5.11 (t, 4H, J = 5.4 Hz), 4.67 (t, 2H, J = 4.8 Hz), 3.26–3.16 (m, 4H), 2.77 (t, 4H, J = 6.0 Hz), 2.66–2.59 (m, 4H).
Synthesis of (1-phenethyl-2-(4-phenylbutyl)-1H-benzo[d]imidazol-5-yl)methyl dihydrogen phosphate PLS5
The compound 7 (40 mg, 0.062 mmol) was treated with TFA (2 mL) in the presence of TIPS (0.5 mL) at 0°C. The reaction mixture was warmed to room temperature and stirred for 2 h. After completion, TFA was removed under vacuum and the resulted residue was dissolved in DCM (20 mL) and washed with saturated NaHCO3, H2O and brine. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuo, afforded crude product of PLS5 (26 mg, 90%). The crude residue was purified by using HPLC gave the pure product PLS5 as amorphous solid.
Spectral data.
1H NMR (CD3OD, 300 MHz): δ 7.85 (s, 1H); 7.69 (d, 1H, J = 6.3 Hz), 7.54 (d, 1H, J = 6.3 Hz), 7.24–7.19 (m, 2H), 7.18–7.10 (m, 5H), 6.90 (d, 1H, J = 6.6 Hz); 5.60 (s, 2H), 5.11 (d, 2H, J = 5.4 Hz), 3.76 (s, 3H), 3.23 (t, 2H, J = 5.4 Hz), 2.61 (t, 2H, J = 5.7 Hz), 1.85–1.68 (m, 4H).; 13C NMR (CD3OD, 75 MHz): 161.5, 155.8, 142.8, 138.9, 133.1, 132.1, 129.6, 129.4, 127.1, 127.0, 126.5, 115.7, 113.9, 113.6, 67.7, 55.8, 36.1, 31.8, 27.3, 26.6.; 31P NMR (121 MHz, CD3OD): δ 0.48.
RP-HPLC Rt = 19.52 (linear gradient, 5–95% B, 30 min, 2.0 mL/min) and % of purity = 97.5; MS (MALDI-TOF) m/z = 481.27 [M+H] (calculated m/z = 480.18).
Synthesis of (1-benzyl-2-(4-phenylbutyl)-1H-benzo[d]imidazol-5-yl)methyl dihydrogen phosphate PLS1
Compound PLS1 was synthesized using general procedure followed for the synthesis of PLS5.
Spectral data.
1H NMR (CD3OD, 300 MHz): δ 7.82–7.60 (m, 2H), 7.26 (d, 1H, J = 6.0 Hz); 7.20–7.15 (m, 8H), 6.92 (dd, 2H, J = 3.0 and 6.0 Hz), 5.14 (d, 2H, J = 6.0 Hz), 4.67 (t, 2H, J = 6.0 Hz), 3.19 (t, 2H, J = 6.0 Hz), 2.66–2.58 (m, 4H), 1.63–1.53 (m, 4H). 13C NMR (CD3OD, 75 MHz): 155.6, 142.8, 138.8, 138.7, 138.3, 132.4, 131.8, 130.1, 129.5, 129.4, 128.6, 127.1, 126.6, 113.9, 113.5, 67.9, 36.1, 35.7, 31.9, 26.7, 26.0.; 31P NMR (121 MHz, CD3OD): δ 0.43.
RP-HPLC Rt = 20.23 (linear gradient, 5–95% B, 30 min, 2.0 mL/min) and % of purity = 98.2, MS (MALDI-TOF) m/z = 451 [M+H] (calculated m/z = 450.17).
Synthesis of (2-(4-cyclohexylbutyl)-1-phenethyl-1H-benzo[d]imidazol-5-yl)methyl dihydrogen phosphate PLS2
Compound PLS2 was synthesized using general procedure followed for the synthesis of PLS5.
Spectral data.
1H NMR (CD3OD, 300 MHz); δ 7.85 (s, 1H), 7.82 (d, 1H, J = 3.0 Hz), 7.64 (d, 1H, J = 6.0 Hz). 7.24–7.12 (m, 3H), 6.96–6.94 (m, 2H), 5.16 (d, 1H, J = 6.0 Hz), 4.71 (t, 2H, J = 6.0 Hz), 3.23 (t, 2H, J = 6.0 Hz), 2.61 (t, 2H, J = 6.0 Hz), 1.72–1.51 (m, 5H), 1.32–1.27 (m, 2H), 1.23–1.14, (m, 8H), 0.93–0.87 (m, 2H). 13C NMR (CD3OD, 75 MHz); 155.9, 138.6, 138.5, 138.4, 132.5, 131.8, 130.1, 128.6, 126.7, 114.0, 113.5, 68.1, 68.0, 38.7, 37.9, 35.7, 34.5, 27.7, 27.5, 27.4, 26.1. 31P NMR (121 MHz, CD3OD); δ 0.21.
RP-HPLC Rt = 22.22 (linear gradient, 5–95% B, 30 min, 2.0 mL/min) and % of purity = 96.8; MS (MALDI-TOF) m/z = 471.33 [M+H] (calculated m/z = 470.23).
Synthesis of (2-heptyl-1-(4-phenylbutyl)-1H-benzo[d]imidazol-5-yl)methyl dihydrogen phosphate PLS3
Compound PLS3 was synthesized using general procedure followed for the synthesis of PLS5.
Spectral data.
1H NMR (CD3OD, 300 MHz); δ 7.81 (s, 1H), 7.23 (d, 1H, J = 6.3 Hz), 7.57 (d, 1H, J = 6.0 Hz), 7.21–7.20 (m, 2H), 7.14 (d, 3H, J = 6.0 Hz), 5.11 (d, 2H, J = 6.0 Hz), 4.42 (t, 2H, J = 6.0 Hz), 3.12 (t, 2H, J = 6.0 Hz), 2.67 (t, 2H, J = 6.0 Hz), 1.90–1.73 (m, 6H), 1.45–1.29 (m, 8H), 0.88 (t, 2H, J = 6.0 Hz). 13C NMR (CD3OD, 75 MHz); 154.0, 141.3, 137.4, 131.3, 130.5, 128.1, 128.0, 125.7, 125.0, 112.1, 66.3, 44.7, 34.6, 31.3, 28.8, 28.5, 28.2, 27.7, 26.4, 24.9, 22.2, 14.0. 31P NMR (121 MHz, CD3OD).δ 0.45.
RP-HPLC Rt = 19.82 (linear gradient, 5–95% B, 30 min, 2.0 mL/min), and % of purity = 97.2; MS (MALDI-TOF) m/z = 459.38 [M+H] (calculated m/z = 458.23).
Synthesis of 1-(4-methoxybenzyl)-2-(4-phenylbutyl)-1H-benzo[d]imidazole-5-carboxylic acid 8
Compound 5 (0.2 g, 0.48 mmol) was treated with 4N HCl (5 mL) and refluxed for 2 h. After completion, the solvent was removed under vacuo and the resulted residue was dissolved in ethyl acetate (20 mL) and washed with saturated NaHCO3, H2O and brine. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuo. The crude residue was purified using silica gel column chromatography (MeOH/DCM = 1∶19) to afford 8 (0.15 g, 80%) as a white solid.
Spectral data.
1H NMR (CDCl3, 300 MHz): δ 8.42 (s, 1H); 7.91 (d, 1H, J = 6.0 Hz); 7.24–7.22 (m, 3H); 7.15–7.11 (m, 3H); 6.84 (d, 1H, J = 9.0 Hz), 6.71 (d, 1H, J = 6.0 Hz), 5.21 (s, 2H), 3.73 (s, 3H), 2.68 (t, 2H, J = 6.0 Hz), 2.52 (t, 2H, J = 6.0 Hz), 1.90–1.84 (m, 2H); 1.76–1.68 (m, 2H); 13C NMR (CDCl3, 75 MHz): 166.6, 159.1, 156.8, 141.5, 142.2, 138.6, 128.1, 127.2, 127.1, 125.7, 124.1, 124.0, 120.7, 114.2, 109.0, 54.7, 51.8, 45.9, 35.6, 31.3, 26.9, 26.5.
Synthesis of methyl 2-(4-methoxyphenyl)-1H-benzo[d]imidazole-5-carboxylate 12
Methyl 3,4-diaminobenzoate (0.61 g, 3.67 mmol) 10 was added to anisaldehyde 11 (0.45 mL, 3.67 mmol) in the presence of anhydrous p-TsOH (0.13 g, 0.73 mmol) in DMF (2 mL). The resulted reaction mixture was heated to 80°C and stirred for 1 h. The reaction mixture was cooled to 0°C and added saturated Na2CO3 solution dropwise with vigorous stirring. Then the mixture was treated with ethyl acetate (20 mL) and washed with H2O, brine and dried over anhydrous Na2SO4. The resulted organic layer was evaporated under vacuum to yield the crude product, which was purified using silica gel column chromatography (MeOH/DCM, 1∶49) afforded desired product 12 (0.83 g, 80%) as liquid.
Synthesis of methyl 2-(4-methoxyphenyl)-1-(4-phenylbutyl)-1H-benzo[d]imidazole-5-carboxylate 13
The compound 12 (0.5 g, 1.77 mmol) was reacted with,4-phenylbutylbromide (0.31 mL, 1.77 mmol) in anhydrous DMF (10 mL) and K2CO3 (0.49 g, 3.54 mmol) was slowly added at 0°C. The mixture was warmed to 60°C and stirred for 2 h. After completion, reaction mixture was quenched with saturated NH4Cl solution and extracted with ethyl acetate (2×50 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The obtained residue was purified over silica gel column chromatography (ethyl acetate/hexane = 2∶3) gave non separable isomeric mixture 13 (0.67 g, 92%) as a colorless liquid.
Spectral data for isomers.
1H NMR (CDCl3, 300 MHz): δ 8.49 (d, 1H, 0.6 Hz), 8.12 (d, 1H, 0.6 Hz), 8.01 (t, 1H, J = 1.5 Hz), 7.99 (t, 1H, J = 1.5 Hz), 7.79 (d, 1H, J = 6.3 Hz), 7.61 (dd, 4H, J = 4.0 and 6.3 Hz), 7.34 (d, 1H, J = 6.3 Hz), 7.28–7.23 (m, 5H), 7.20 (dd, 4H, J = 1.2 and 2.4 Hz), 7.18 (d, 1H, J = 2.4 Hz), 7.10–7.05 (m, 4H), 7.03 (d, 1H, J = 6.6 Hz), 4.30–4.20 (m, 4H), 3.97 (s, 3H), 3.95 (s, 3H), 3.89 (s, 6H), 2.57 (t, 1H, J = 5.4 Hz), 1.90–1.80 (m, 4H), 1.65–1.55. 13C NMR (CDCl3, 75 MHz); 167.7, 161.0, 156.3, 155.3, 146.6, 142.6, 141.3, 141.2, 138.8, 135.3, 130.7, 128.4, 128.3, 126.0, 124.2, 124.1, 122.3, 122.0, 114.3, 112.2, 109.3, 55.4, 52.1, 44.8, 35.0, 29.2, 29.0, 28.1.
Synthesis of 2-(4-methoxyphenyl)-1-(4-phenylbutyl)-1H-benzo[d]imidazole-5-carboxylic acid 9
4N HCl (10 mL) was treated with compound 13 (0.6 g, 1.45 mmol and refluxed for 2 h. The reaction mixture was concentrated under vacuum, and the residue was dissolved in ethyl acetate (2×100 mL) and washed with saturated NaHCO3 and brine. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The obtained crude residue was purified using silica gel column chromatography (MeOH/DCM, from 1∶20 to 1∶5) to yield the mixture of isomers 9a and 9b (0.5 g, 86%) as liquid.
Spectral data.
1H NMR (CDCl3, 300 MHz); δ 7.86 (d, 1H, 6.0 Hz), 7.70 (t, 1H, 3.0 Hz), 7.60 (dd, 2H, J = 6.0 and 9.0 Hz), 7.22 (dd, 1H, J = 3.0 and 6.0 Hz), 7.16–7.14 (m, 2H), 7.12–7.10 (m, 3H), 7.03 (d, 1H, J = 6.0 Hz), 4.38–4.31 (m, 2H), 3.86 (s, 3H), 2.05 (t, 1H, J = 6.0 Hz), 1.71–1.66 (m, 2H), 1.48–1.45 (m, 2H). 13C NMR (CDCl3, 75 MHz); 169.1, 160.3, 154.6, 144.7, 142.1, 141.5, 137.9, 135.2, 1238.2, 128.1, 125.7, 123.6, 123.4, 122.5, 120.3, 117.8, 114.2, 112.0, 109.8, 55.3, 44.0, 34.2, 28.7, 27.7.
Synthesis of methyl 1-(4-phenylbutyl)-1H-benzo[d]imidazole-5-carboxylate 16
Compound 15 (0.5 g, 2.84 mmol) was reacted with 4-phenylbutylbromide (0.5 mL, 2.84 mmol) in anhydrous DMF (10 mL) and K2CO3 (0.79 g, 5.68 mmol) was added slowly at 0°C. The resulting reaction mixture was warmed to 60°C and stirred for 2 h. After completion, reaction mixture was quenched with saturated NH4Cl, and extracted with ethyl acetate (250 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The crude residue was purified over silica gel column chromatography (MeOH/DCM = 1∶49) to yield the mixture of isomers 16 (0.79 g, 90%) as a colorless liquid.
Spectral data for isomers.
1H NMR (CDCl3, 300 MHz); δ 8.53 (s, 1H), 8.10 (t, 2H, J = 6.0 Hz), 7.34–7.29 (m, 3H), 7.16–7.14 (m, 3H), 4.23–3.94 (m, 2H), 2.68–2.63 (m, 2H), 2.30–2.20 (m, 2H). 13C NMR (CDCl3, 75 MHz): 167.7, 147.3, 144.6, 139.9, 133.4, 128.7, 126.5, 124.8, 123.5, 122.8, 120.1, 112.1, 52.2, 44.5, 32.6, 30.9.
Synthesis of 1-(4-phenylbutyl)-1H-benzo[d]imidazole-5-carboxylic acid 14
4N HCl (10 mL) was added to compound 16 (0.7 g, 2.27 mmol) in 1,4-dioxane (5 mL) and refluxed for 2 h. The reaction mixture was concentrated under vacuum and dissolved in ethyl acetate (20 mL). The mixture was washed with saturated NaHCO3, brine and dried over anhydrous Na2SO4. The combined organic layer was concentrated under vacuum. The crude residue was purified using silica gel column chromatography (MeOH/DCM, from 1∶20 to 1∶5) to yield the mixture of isomers 14a and14b (0.56 g, 84%) as liquid.
Spectral data for isomers.
1H NMR (CDCl3, 300 MHz); δ 8.51 (s, 1H), 8.12 (t, 2H, J = 6.0 Hz), 7.32–7.21 (m, 3H), 7.17–7.13 (m, 3H), 4.21–3.93 (m, 2H), 2.65–2.61 (m, 2H), 2.32–2.23 (m, 2H). 13C NMR (CDCl3, 75 MHz): 166.7, 147.1, 144.2, 138.3, 134.6, 126.9, 125.6, 124.9, 123.5, 121.3, 111.1, 51.1, 45.8, 33.4, 29.6.
Synthesis of N-phenethyl-5-phenylpentanamide 19
5-phenylpentanoic acid (1 g, 5.6 mmol) was reacted with 2-phenylethanamine (0.65 mL, 5.6 mmol) in the presence of coupling agents EDCI (1.12 g, 6.2 mmol), HOBt (0.84 g, 6.2 mmol) and Et3N (2.34 mL, 16.8 mmol) in dry DCM (30 mL) and stirred for 4 h. The reaction mixture was quenched with water and washed with saturated citric acid, NaHCO3 and brine. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The crude residue was purified using silica gel column chromatography (ethyl acetate/hexane = 3∶7) to yield the amide 19 (1.42 g, 90% for two steps) as liquid.
Spectral data.
1H NMR (CDCl3, 300 MHz); δ 7.32–7.21 (m, 5H), 7.21–7.13 (m, 5H), 5.37 (br s, 1H), 3.51 (q, 2H, J = 5.1 Hz), 2.80 (t, 2H, J = 5.4 Hz), 2.61 (t, 2H, J = 5.4 Hz); 2.13 (t, 2H, J = 5.1 Hz); 1.66–1.54 (m, 4H). 13C NMR (CDCl3, 75 MHz); 172.7, 142.2, 138.9, 128.7, 128.6, 128.4, 128.3, 126.5, 125.7, 40.4, 36.6, 35.7, 35.6, 31.0, 25.3.
Synthesis of methyl 1-phenethyl-2-(4-phenylbutyl)-1H-benzo[d]imidazole-5-carboxylate 20
The compound 19 (0.55 g, 1.12 mmol) was reacted with methyl 4-chloro-3-nitrobenzoate (0.20 g, 0.93 mmol), in the presence of palladium trifluoroacetate (30 mg, 0.04 mmol), (R)-BINAP (56 mg, 0.04 mmol), and cesium carbonate (493 mg, 0.7 mmol) in dry toluene (10 mL). The reaction mixture was heated to 80°C under argon atmosphere and stirred for 12 h. After completion, reaction mixture was quenched with water (3 mL) and filtered through a pad of Celite. The filterate was washed with ethyl acetate (3×6 mL) and the collected organic layers was dried and evaporated. The resulted residue was dissolved in glacial acetic acid (20 mL) and was heated to reflux for 1 h in the presence of iron powder (0.63 g). The acetic acid was removed under vacuum, and the residue was suspended in saturated NaHCO3 and extracted with ethyl acetate (250 mL). The combined organic layers washed with brine and dried over anhydrous Na2SO4 and evaporated. The resulted crude residue was purified using silica gel column chromatography (ethyl acetate/hexane = 2∶3) to afford the desired product 20 (0.23 g, 60% for two steps) as a liquid.
Spectral data.
1H NMR (CDCl3, 300 MHz); δ 8.37 (s, 1H), 7.90 (dd, 1H, J = 1.2 and 8.4 Hz), 7.24–7.14 (m, 6H), 7.12–7.05 (m, 3H), 6.89–6.83 (m, 2H), 4.24 (t, 2H, J = 6.9 Hz), 3.88 (s, 3H), 2.98 (t, 2H, J = 6.9 Hz), 2.56 (t, 2H, J = 7.5 Hz), 2.41 (t, 2H, J = 7.5 Hz), 1.77–1.67 (m, 2H), 1.64–1.54 (m, 2H).
Synthesis of 1-phenethyl-2-(4-phenylbutyl)-1H-benzo[d]imidazole-5-carboxylic acid 17
4N HCl (10 mL) was added to compound 20 (0.7 g, 2.27 mmol) in 1,4-dioxane (5 mL) and refluxed for 2 h. The reaction mixture was concentrated under vacuum and dissolved in ethyl acetate (20 mL). The mixture was washed with saturated NaHCO3, brine and dried over anhydrous Na2SO4. The combined organic layer was concentrated under vacuum. The crude residue was purified using silica gel column chromatography (MeOH/DCM, from 1∶20 to 1∶5) to yield the desired product 17 (0.15 g, 80%) as a white solid.
Spectral data.
1H NMR (CDCl3, 300 MHz); δ 8.69 (s, 1H), 8.10 (d, 1H, J = 5.4 Hz), 7.32–7.20 (m, 6H), 7.15 (d, 2H, J = 5.7 Hz), 6.94 (d, 2H, J = 4.5 Hz), 4.30 (t, 2H, J = 4.5 Hz), 3.04 (t, 2H, J = 4.5 Hz), 2.69–2.59 (m, 4H), 1.85–1.75 (m, 2H), 1.74–1.64 (m, 2H).; 13C NMR (CDCl3, 75 MHz): 171.0, 157.0, 142.1, 141.3, 137.6, 137.3, 128.9, 128.7, 128.4, 128.3, 127.2, 125.7, 124.5, 121.8, 109.0, 45.6, 35.9, 35.5, 31.0, 27.2, 26.7.
General procedures for the synthesis of bg-33, bg-1, bg-2, bg-5, bg-6, bg-27, bg-28, bg-29, bg-30 and bg-34 from acid derivatives.
The molecules bg-33, bg-1, bg-2, bg-5, bg-6, bg-27, bg-28, bg-29, bg-30 and bg-34 were synthesized using the standard Fmoc-based, solid-phase peptide synthesis (SPPS) method using Rink amide resin, with an initial loading of 0.61 mmol/g, unless otherwise noted. Fmoc-Thr(PO(OBzl)OH)-OH and Fmoc-β-Ala-OH amino acids were purchased from Novabiochem. Resins were swollen in N,N-dimethylformamide (DMF) for 45 min prior to synthesis. For the bg-33, bg-1, bg-2, bg-27, bg-28, and bg-34 synthesis, only Thr(PO(OBzl)OH)-OH (5.0 eq.) was activated by treatment with HBTU (5.0 eq.) and HOBt (5.0 eq.) in DMF (2 mL) for 2 min. This solution was added to the free amine on resin, along with N,N-diisopropylethylamine (10.0 eq.), and the coupling reaction was allowed to proceed for 1 h with vortex stirring. After washing with DMF, Fmoc deprotection was achieved with 20% piperidine in DMF (1×10 min, 2×3 min). The resin was washed once again, and added synthesized acid derivatives in the presence of HBTU (5.0 eq.) and HOBt (5.0 eq.) along with N,N-diisopropylethylamine (10.0 eq.) continued vortex stirring 6 h. For the synthesis bg-5, bg-6, bg-29 and bg-30 initially coupled with Thr(PO(OBzl)OH)-OH followed by Fmoc-β-Ala-OH and then synthesized acid derivative were coupled. Finally, the resin was washed sequentially with DMF, methanol, dichloromethane, and ether, and then dried under a vacuum. Cleavage of the resin with TFA, TIPS and H2O afforded the molecules, which were purified by HPLC.
Analytical HPLC conditions
The above synthesized molecules bg-33, bg-1, bg-2, bg5, bg-6, bg-2, bg-28, bg-29, bg-30 and bg-34 were purified to a minimum purity of 97% using a phenomenex column (C18, 250×10 mm, 5 micron), with a linear gradient from 5% aqueous acetonitrile (0.05% trifluoroacetic acid) to 95% acetonitrile (0.05% trifluoroacetic acid), over 35 min at a flow rate of 2.0 mL/min and detection at 230 nm (Table S2 in File S1).
ELISA-based PBD-binding inhibition assay
A biotinylated p-T78 (DPPLHSpTAIYADEE-NH2) peptide was diluted with coating solution (KPL, Inc., Gaithersburg, MD) to the final concentration of 0.3 µM, and then 50 µL of the resulting solution was immobilized onto a 96-well, streptavidincoated plate (Nalgene Nunc, Rochester, NY). The wells were washed once with PBS + 0.05% Tween 20 (PBST), and incubated with 200 µL of PBS + 1% BSA (blocking buffer) for 1 h to prevent the unoccupied sites. Mitotic 293A lysates expressing HA-EGFP-Plk1 were prepared in the TBSN buffer (60 µg total lysates in 100 µL), applied onto the biotinylated peptide-coated ELISA wells immediately after mixing with the indicated amount of the competitor peptides, and then incubated with constant rocking for 1 h at 25°C. Next, the plates were washed four times with PBST, and to detect the binds HA-EGFP-Plk1, the plates were incubated for 2 h with 100 µL/well of anti-HA antibody at a concentration of 0.5 µg/mL in the blocking buffer, and then the plates were washed five times. Furthermore, the plates were incubated with 100 µL/well of secondary antibody at a 1∶1,000 dilution in the blocking buffer. Afterward, the plates were washed five times with PBST and incubated with 100 µL/well of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Sigma, St. Louis, MO) until a desired absorbance was achieved. The reactions were terminated by the addition of 1 N H2SO4, and the optical densities were measured at 450 nm using an ELISA plate reader (Molecular Device, Sunnyvale, CA). Data are shown in Figures 3B and 6B.
Molecular Modeling
All calculations were performed using Discover 2.98/InsightII with CVFF force field as described previously.37 The crystal structure of PBD of Plk1 having a pThr mimetic-containing C6H5(CH2)8− group binds was used as the computational model (3RQ7.pdb). The synthetic peptide inhibitor bg-34 was built from the coordinates of this pThr mimetic-containing C6H5(CH2)8− group as found in the crystal structure using the Builder module in Insight II. The computational complex model was solvated using a solvent sphere of water extending 30.0 Å around the phosphate atom of pThr. The system was initially minimized using 1000 steps of steepest decent and 3000 steps of conjugated gradient with a 14.0 Å nonbonded cutoff distance.
Cellular uptake of bg-34
For evaluation of cellular uptake of bg-34, we tested with human cancer cell line, HeLa (human epithelial carcinoma cell line) that it was purchased from American Type Culture Collection (ATCC). HeLa cells were growth in Dulbecco's Modified Eagle Medium (DMEM, GIFCO BRL, Gaithersburg, MD, USA) that had been supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, Gran Island, NY, USA), 100 U/mL of penicillin, and 100 µg/mL of streptomycin (Gibco) in 5% CO2 humidified air at 37°C. The uptake ability of Bg-34 on cancer cell lines was performed with FITC conjugated bg-34. 2×104 cells/well of HeLa cells were prepared and cultured into 24-well polystyrene plates for 24 h. The cells were incubated for 3 h with 200 µM of FITC conjugated bg-34, then washed tree times with PBS, and mounted with mount solution (VECTOR Shield. VECTOR corp., USA). Cellular uptake of Bg-34 was imaged on a fluorescence microscope (OLYMPUS IX81, Olympus Inc, Japan). Images were processed using Image analysis program software (Metamorph, Molecular Devices Inc., USA).
Morphological change by treatment of bg-34
2×104 cells/well of HeLa cells were cultured into 24-well plates for 24 h and incubated for 24 h after treatment with 300 µM of bg-34. The morphological change of HeLa cells by Bg-34 was showed after fixation with 4% paraformaldehyde (PFA, Biosesang Inc., Seongnam, Republic of Korea). The morphological change was imaged on a microscope (OLYMPUS IX81, Olympus Inc, Japan), and its images were processed using image analysis program software (Metamorph, Molecular Devices Inc., USA).
Apoptotic Effect
To confirm the apoptotic effect by treatment of bg-34, the compound was added in Hela cells with 300 µM concentration, and incubated for 24 h. For staining, cover-slip was fixed with 4% PFA and was stained with 100 µg/mL of Acridine orange/ethidium bromide solution (Sigma Aldrich, St. Louis, USA) for 15 min at 37°C. The cover-slips were washed tree times with PBS. The result of staining was imaged on a fluorescence microscope using a blue filter. Images were processed using Image analysis program.
PLK-1 kinase Inhibition Test
The kinase inhibition effect of PLK-1 by treatment of bg-34 was studied using the CycLex Polo-like kinase Assay/Inhibitor Screening Kit (MBL Inc., Japan). 2.5×105 cells/well of HeLa cells were cultured in 6-well in media with 300 µM of bg-34 for 12 h. The 30 µg/100 µL of total proteins were purified from cancer cells were mixed with kinase reaction buffer, was incubated at 30°C for 30 min. For detection of kinase activity of PLK-1, samples were incubated for 1 h with anti-phospho-serine/threonine polyclonal antibody PPT-07 solution, for 1 h with HRP-conjugated Anti-rabbit IgG, and for 10 min with substrate reagent solution. Finally, stop solution was added into each well. Measure absorbance of each well was read using a spectrophotometric plate reader at 450 nm/560 nm.
Supporting Information
File S1.
Contains the files: Figure S1 Modeling structure of the Plk1-PBD, which shows the presence of the phosphate binding pocket, pyrrolidine binding pocket and Try rich hydrophobic channel. Figure S2 Solid phase synthesis of FITC conjugated derivative of bg-34. Table S1 Inhibitory activity of Plk1 PBD by compounds PLHSpT, PPG, PLS1-PLS3, PLS5, Bg-1, Bg-2, bg-27- Bg-30, Bg-33 and Bg-34. Table S2 HPLC retention time, % of purity and MALDI-TOF mass values of bg-33, bg-1, bg-2, bg5, bg-6, bg-2, bg-28, bg-29, bg-30 and bg-34 molecules.
https://doi.org/10.1371/journal.pone.0107432.s001
(DOC)
Author Contributions
Conceived and designed the experiments: JKB ER N-HK KSL. Performed the experiments: GS J-EP SK. Analyzed the data: MA EH PG. Contributed reagents/materials/analysis tools: SYS K-YN. Contributed to the writing of the manuscript: CC JKB.
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