ThiazolidinoneâPeptide Hybrids as Dengue Virus Protease Inhibitors

Article pubs.acs.org/jmc

Thiazolidinone−Peptide Hybrids as Dengue Virus Protease Inhibitors with Antiviral Activity in Cell Culture Christoph Nitsche,† Verena N. Schreier,† Mira A. M. Behnam,† Anil Kumar,‡ Ralf Bartenschlager,‡ and Christian D. Klein*,† †

Medicinal Chemistry, Institute of Pharmacy and Molecular Biotechnology IPMB, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany ‡ Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany S Supporting Information *

ABSTRACT: The protease of dengue virus is a promising target for antiviral drug discovery. We here report a new generation of peptide−hybrid inhibitors of dengue protease that incorporate N-substituted 5-arylidenethiazolidinone heterocycles (rhodanines and thiazolidinediones) as N-terminal capping groups of the peptide moiety. The compounds were extensively characterized with respect to inhibition of various proteases, inhibition mechanisms, membrane permeability, antiviral activity, and cytotoxicity in cell culture. A sulfur/oxygen exchange in position 2 of the capping heterocycle (thiazolidinedione-capped vs rhodanine-capped peptide hybrids) has a significant effect on these properties and activities. The most promising in vitro affinities were observed for thiazolidinedione-based peptide hybrids containing hydrophobic groups with Ki values between 1.5 and 1.8 μM and competitive inhibition mechanisms. Rhodanine-capped peptide hybrids with hydrophobic substituents have, in correlation with their membrane permeability, a more pronounced antiviral activity in cell culture than the thiazolidinediones.

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INTRODUCTION Recent estimates suggest that up to 390 million dengue virus (DEN) infections occur each year, of which around 100 million become clinically apparent in about 100 countries in Latin America, Southeast Asia, Central Africa, and other regions all over the world.1−3 At the moment, no vaccines or antiviral agents are available so that mosquito control is the sole approach to reduce infections in these countries.1−4 For the development of chemotherapeutical agents, three viral enzymes are considered as promising drug targets: the DEN protease, the methyltransferase, and the RNA-dependent RNA-polymerase.4 As demonstrated for other, closely related viruses like HCV, viral proteases represent druggable targets with therapeutic relevance.5,6 Therefore, inhibitors of the DEN protease are potential drug candidates for the treatment and prevention of DEN-related diseases.7 The DEN genome consists of a single-stranded RNA, encoding for three structural and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). 4,7 The © 2013 American Chemical Society

multifunctional NS3 protein provides a protease and a helicase function, localized in different domains. The NS3 protease domain requires the cytosolic core sequence of the NS2B protein as a cofactor to form the catalytically competent complex NS2B− NS3.8,9 As in other flaviviral proteases, particularly the closely related West Nile virus protease, the cofactor was found to be partially disordered and flexible in crystal structures in the absence of a substrate or a substrate-competitive inhibitor.4,10−12 Instead of this “open” conformation, the catalytically active “closed” conformation with ordered cofactor domain is observed in crystal structures when a ligand is bound to the NS2B−NS3 construct.12−14 In this conformation, the cofactor contributes with a β-hairpin to the S2 and S3 pockets, which are crucial for substrate recognition.4 Recently, crystal structures of two inhibitors bound to the protease of DEN serotype 3 were published.14 Surprisingly, the broad-spectrum serine protease Received: June 4, 2013 Published: October 1, 2013 8389

dx.doi.org/10.1021/jm400828u | J. Med. Chem. 2013, 56, 8389−8403

Journal of Medicinal Chemistry

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Scheme 1. Synthetic Approach for the Rhodanine- and Thiazolidinedione-Based Peptide Hybrids Listed in Table 1a

a (a) KOH, EtOH, reflux; (b) NaOH, H2O or EtOH, MW 100−120 °C; (c) DMF, reflux; (d) Na2WO4, H2O2, H2O, EtOAc, rt; (e) H2O, MW 120 °C; (f) NH4OAc, AcOH, MW 180 °C; (g) piperidine, DMF; (h) Fmoc-Nle-OH, HBTU, DIPEA, DMF; (i) Fmoc-Lys(Boc)-OH, HBTU, DIPEA, DMF; (j) Fmoc-Arg(Pbf)-OH, HBTU, DIPEA, DMF; (k) HBTU, DIPEA, DMF; (l) TFA, TIPS, H2O.

been published. Low-molecular-weight inhibitors20−27 without similarity to the natural substrate of the protease have, in most cases, uncertain or low selectivity and potency but can often be considered “drug-like” with respect to permeability, stability, and ligand efficiency. Peptidic inhibitors that mimic the recognition sequence of the protease and frequently incorporate electrophilic warheads28−35 have increased potency but often carry intrinsic reactivity and pharmacokinetic liabilities due to highly basic side chains and, consequently, pronounced polarity. However, some of these inhibitors have good in vitro affinities in the lower micromolar range and below in the case of boronic acid or trifluoromethylketone as electrophilic warheads.29 Considering the successful discovery and development of HCV protease inhibitors which are now in clinical use, substrate-based peptides appear as a promising starting point to obtain clinical development candidates after several optimization cycles.4,6,36 The core challenge in the development of promising DEN protease inhibitors is to combine the high affinity and selectivity of peptide inhibitors with the drug-likeness of small-molecular compounds without any highly reactive groups. Peptide hybrids are an attractive possibility to approach this challenge. Recently,37 we described hybrids of arylcyanoacrylamides20 with short peptide sequences. These hybrids do not contain a highly reactive warhead and have a remarkable affinity against the DEN protease, with Ki-values between 5 and 10 μM for the best derivatives, but had no significant antiviral activity in cell culture.37 On the basis of this partial, initial success, we searched for alternative N-terminal cap moieties to increase the activity and to create membrane-permeable peptide hybrids with increased antiviral activity in cell culture. Herein we report Nsubstituted 5-arylidenethiazolidinone−peptide hybrids with an extensive evaluation of the thiazolidinedione and rhodanine scaffolds. These five-membered heterocycles have been previously explored as small-molecular inhibitors of the DEN protease with moderate activity.22 Rhodanines, thiazolidinediones, and related heterocycles have been discussed extensively

inhibitor aprotinin (BPTI) does not bind to the closed conformation of NS2B−NS3,4,12,14 whereas for a covalently bound tetrapeptidyl aldehyde inhibitor the closed state was observed.4,14 The closed state was also found predominantly in NMR structures of the NS2B−NS3 construct in presence of a ligand, thereby a significant conformational change from the open to the closed form was observed.15−17 More recently, NMR experiments using a NS2B−NS3 preparation without covalent linkage of the protease and the cofactor were presented. These solution structures resemble the closed conformation of NS2B− NS3 in absence and presence of an inhibitor,18 indicating that the previously observed open conformations are artifacts which arise from the non-natural NS2B−NS3 construct with covalent linkage. However, the binding of an inhibitor to alternative conformations of NS2B−NS3 may be thermodynamically favored, which would explain the high affinity of aprotinin to the enzyme and the observed crystal structure with this inhibitor.4,14 This example implies that the closed conformation is not necessarily the preferred structural target for the design and discovery of inhibitors. Instead, the open conformation or an intermediate between the open and closed conformations may also be relevant for inhibitor binding. Inhibitors that bind to these conformations of the enzyme will not display a substratecompetitive binding behavior. An additional difficulty in structure-based drug design arises from the fact that under in vivo conditions the NS3 protein is part of a larger, membraneassociated replication complex, which might not properly resemble the NS2B−NS3 protein in isolated or crystallized form.19 In particular, this caveat applies to the experimental characterization of enzyme inhibitors using the isolated, recombinant NS2B−NS3 protein. Considering these restrictions, empirical in vitro and in cellulo SAR studies of different compound classes are required to obtain potent DEN protease inhibitors and improve our understanding of their binding and inhibition modes. In the last few years, several compound series for targeting the DEN protease have 8390



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Scheme 2. Synthesis of Thiohydantoin Derivatives 43 and 44 Starting from the Corresponding Isothiocyanatesa

a

(a) Et3N, Et2O, rt; (b) NH4OAc, AcOH, MW 180 °C.

Scheme 3. Synthesis of Compounds 41 and 42 via a Three Component Approacha

a

(a) H2O, rt; (b) H2O, EtOH, rt; (c) TFA, CH2Cl2, rt.

all condensation products could be separated by filtration and were used for the solid-phase peptide synthesis without any further purification steps. The geometry of the double bond is assumed to be (Z), as was previously confirmed by X-ray crystal structures for closely related analogues, which were prepared under identical conditions.22,42,43 It appears that the preferred geometry of the double bond is (Z) due to steric effects, with the sulfur atom in position 2 of the heterocycle being cis-oriented to the aromatic ring.44 The absence of (E) isomers was confirmed by LC-MS. The five-membered N-substituted heterocyclic rings were obtained in different ways, depending on the nature of the substituent (R = alkyl, benzyl, aryl) and the heterocycle itself (X = S, O). For rhodanines bearing alkyl or benzyl substituents, a microwave-assisted three component reaction in water using the amine, carbon disulfide, and chloroacetic acid was developed before.45 As this method was not adequately applicable for arylamines, an alternative method was evaluated using bis(carboxymethyl)trithiocarbonate.45 N-Alkyl- and benzylthiazolidinediones were synthesized starting with deprotonation of 2,4thiazolidinedione and isolation of the formed potassium salt, followed by nucleophilic substitution using alkyl or benzyl halides (Scheme 1).46 The 3-(p-tolyl)thiazolidine-2,4-dione derivative was synthesized by oxidation of the corresponding 3-(p-tolyl)rhodanine with sodium tungstate and hydrogen peroxide.47 3-Phenyl- and 3-benzylthiohydantoine were synthesized from the corresponding isothiocyanates and methyl glycinate (Scheme 2).48 These compounds could also be transformed into the 5-arylidene derivatives using the same Knoevenagel reaction conditions as for the other analogues. 2-[3(4-Methoxybenzyl)-4-oxo-2-thioxothiazolidin-5-yl]acetic acid as precursor for compound 41 was synthesized according to a three component reaction described in the literature (Scheme 3).49 For the synthesis of the unsaturated analogue 2-[3-(4methoxybenzyl)-4-oxo-2-thioxothiazolidin-5-ylidene]acetic acid, an alternative three component reaction was used (Scheme 3).50 This reaction was extended to the use of di-tert-butyl acetylenedicarboxylate because the resulting tert-butyl ester could easily be cleaved using TFA in the following step.

and controversially as key (or nuisance-causing) structures in medicinal chemistry.22,38−41 These compounds have a distinct binding profile to numerous targets which probably originates from a particularly high potential to form intermolecular interactions, which is not related to aggregation or other nonspecific, “promiscuous” inhibition mechanisms.22 Variations of the N-substituent at position 3 of the heterocycle are a straightforward way to obtain series of compounds with different hydrophobicity and to explore additional possibilities for intermolecular interaction. The connection to the peptide can be achieved via the aromatic 5-arylidene residue, as described before.37

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RESULTS AND DISCUSSION

Chemistry. The chemical structure of the new series of peptide hybrids is highly influenced by previously identified DEN protease inhibitors.20,22,37 Tripeptides of the sequence Arg-LysNle with C-terminally nonsubstituted amide residues were combined with N-terminal cap groups, as this had been found in a screening of peptide hybrids as the most suitable approach.37 5Arylidenerhodanines and -thiazolidinediones were chosen as Nterminal caps and combined via the aromatic ring with the peptide chain using amide bonds, as this was previously reported as an easy connection approach leading to affine compounds.37 To establish some additional intermolecular interactions and to modulate the hydrophobicity, the five-membered heterocycle was further substituted at the nitrogen atom in position 3. As a proof of principle for our design considerations, compounds with modified N-terminal caps, alternative heterocycles, and different peptide sequences were additionally synthesized. The synthesis of peptide hybrids was executed by solid-phase peptide synthesis according to the Fmoc protocol. N-Terminal cap molecules were connected to the presynthesized peptide sequences using carboxylic acid functionalities as shown in Scheme 1.37 Connection between the thiazolidinone scaffolds and the benzoic acid moiety was realized by a high-throughput Knoevenagel condensation with formylbenzoic acid under microwave-assisted conditions.22 Using acetic acid as solvent, 8391



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Table 1. Inhibitory Activity of Rhodanine- and Thiazolidinedione-Based Peptide Hybrids at the Dengue Virus Protease, West Nile Virus Protease, and Thrombind

Percent inhibition of the DEN NS2B−NS3 protease serotype 2 (enzyme: 100 nM, inhibitor 50 μM, substrate 50 μM, Km = 105 μM). bPercent inhibition of the WNV NS2B−NS3 protease (enzyme: 150 nM, inhibitor 50 μM, substrate 50 μM, Km = 212 μM). cPercent inhibition of thrombin (enzyme: 10 nM, inhibitor 25 μM, substrate 50 μM, Km = 16 μM). dThe common peptide−hybrid scaffold of the compounds is given in Scheme 1. n.i. = no inhibition. n.s. = not soluble in assay buffer at screening concentrations. a

All target compounds were tested in fluorimetric assays against the DEN and WNV proteases and thrombin. Furthermore, we included five of the most active peptide hybrids from the previously reported series of compounds (I, II, III, IV, V) for correlation and comparison purposes (Table 3). To confirm the results and to exclude false-positive results, selected (especially colored) compounds were additionally studied in an HPLCbased DEN protease assay.51 Structure−Activity Relationships. Twenty-six different substituents (including H) at the nitrogen atom in position 3 (R according to Scheme 1) were evaluated for the rhodanine and thiazolidinedione scaffolds, respectively, in the peptide hybrids shown in Table 1. The general tripeptide sequence for all compounds in Table 1 is Cap-Arg-Lys-Nle-NH2 (Scheme 1). This sequence was identified as the most active and selective one for the peptide hybrids evaluated before.37 The connection via

the arylidene system was arranged in para-substitution, also as explored before.37 All derivatives in Table 1 have only negligible activity against thrombin. Some rhodanine derivatives with hydrophobic substituents (e.g., 9a, 23a, 24a, 25a, 26a) possess notable inhibitory potency at the WNV protease, with percentage of inhibition values above 97%. However, further experiments at lower concentrations indicate that the expected IC50 values for all these compounds are above 10 μM, implicating DEN protease selectivity for these compounds. None of the thiazolidinedione derivatives reached inhibition values of 90% or higher at the WNV protease, indicating that the selectivity between the two viral targets is much higher for this compound class than for the rhodanine derivatives. The relatively high % inhibition values of some compounds in the WNV protease screen are representing a slightly lower target affinity than % inhibition values in the same range for the DEN protease screen. This is due to the difference 8392



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in Km, which is 212 μM for the WNV protease FRET substrate and 105 μM for the DEN protease FRET substrate. The high selectivity of the compounds, especially between the closely related DEN and WNV proteases, is clearly indicative of specific target−ligand recognition and makes a nonspecific or promiscuous effect of the thiazolidinedione and rhodanine heterocycles unlikely. A high activity at the DEN protease (≥97% inhibition) was observed for the peptide hybrids bearing hydrophobic substituents with a certain flexibility, e.g., butyl (7a, 7b), cyclopentyl (9a, 9b), cyclohexyl (10a, 10b), and all explored benzyl substituents (21a−25a, 21b−25b). Smaller groups with lower hydrophobicity, like hydrogen (1a, 1b), methyl (2a, 2b), or hydroxyethyl (14a, 14b) and also substituents with a lack of flexibility, like all explored aryl groups (16a, 17a, 17b, 18a, 19a, 20a), are less active. IC50 values were determined for all compounds with an inhibition of at least 97% and for selected compounds from the former series (I−V) (Table 2). Generally,

(29). However, this derivative did not reach the activity of the derivative containing a lysine residue (21b). A replacement of the arginine by lysine (31) and an interchange of the arginine and lysine residues (32) did also not result in high affinities. The importance of the norleucine residue is confirmed by compound 34, which is less active due to the absence of the C-terminal norleucine. Nearly all affinity is lost if the lysine residue is additionally absent (35). To confirm the para-substitution at the aromatic system as the most promising alternative, two meta-substituted derivatives were explored (37, 38) which are less active (Table 3). An alternative connection of the peptide sequence via the nitrogen atom of the rhodanine scaffold in position 3 bearing a hydrophobic arylidene moiety at position 5 did not result in highly active compounds (39, 40). The same observation was made when the aromatic connection moiety between the heterocycle in position 5 and the peptide chain was omitted (41, 42). Derivative 41 is active against thrombin with an IC50 value of 5.7 μM (Ki = 3.8 μM). Finally, thiohydantoin moieties as alternative heterocycles for rhodanines and thiazolidinediones were evaluated.22 However, the two explored derivatives (43, 44) were unconvincing (Table 3). In summary, the results shown in Table 3 underline the superiority of the Cap-Arg-Lys-Nle-NH2 sequence and a para-arylidene substituent in position 5 of the rhodanine and thiazolidinedione moieties, respectively. Six 5-arylidenerhodanines and the corresponding six 5arylidenethiazolidinedione cap molecules had only poor activity against the three assayed proteases, indicating that the tripeptide moiety is a crucial part of the inhibitors (Supporting Information Table S1). Studies on Inhibition Mechanisms. Detailed analyses with determination of the inhibition mechanism were performed for all derivatives with a DEN protease inhibition higher than 97%. Table 2 provides the Ki values for a competitive binding mode (enzyme−inhibitor complex) and Ki′ values for an uncompetitive binding mode (enzyme−substrate−inhibitor complex) and the observed inhibition mechanism. Ki and Ki′ values were determined using Dixon and Cornish-Bowden plots, respectively.52,53 A remarkable difference concerning the inhibition mechanisms of the rhodanine- (X = S) and thiazolidinedionebased (X = O) peptide hybrids was observed. The binding of five rhodanine derivatives can be described with Ki and additionally Ki′ values, resulting in a mixed inhibition model. For some of these compounds (21a, 22a, 23a) the Ki and Ki′ values are approximately in the same range, which argues for a noncompetitive (Ki = Ki′) mechanism. Only two rhodanine derivatives with aliphatic substituents bind as competitive inhibitors, with inferior Ki′ values above 10 μM. Rhodaninebased peptide hybrids 12a, 24a, and 25a display an almost uncompetitive inhibition, with negligible Ki values far above 10 μM. In summary, the inhibition mechanism of the rhodaninebased peptide hybrids is largely uncompetitive. The opposite result was found for the thiazolidinedione derivatives: all analyzed compounds have Ki values lower than 4 μM and only three compounds had Ki′ values lower than 10 μM. These three derivatives (9b, 24b, 25b) are “mixed model” inhibitors with a clear preference for a competitive inhibition mechanism. The other thiazolidinedione-based peptide hybrids can be considered as purely competitive inhibitors. Figure 1 shows the differences between the two explored compound classes for the phenylethylsubstituted derivatives 26a and 26b. The reasons for these findings are difficult to explain. One may speculate about additional interactions of the thiocarbonyl function, resulting in

Table 2. IC50, Ki, Ki′ Values and Inhibition Mechanisms for Selected Compounds at the Dengue Virus Protease (Serotype 2) No.

IC50 (μM)

Ki (μM)

Ki′ (μM)

Mechanism

7a 9a 10a 12a 21a 22a 23a 24a 25a 26a 7b 9b 10b 15b 21b 22b 23b 24b 25b 26b I II III IV V

6.2 ± 0.1 6.1 ± 0.2 6.1 ± 0.3 11.6 ± 1.2 10.9 ± 0.6 7.5 ± 1.2 7.9 ± 0.7 >10 >10 7.7 ± 0.6 3.5 ± 0.1 2.5 ± 0.1 3.4 ± 0.1 4.8 ± 0.2 2.9 ± 0.2 4.8 ± 0.4 3.3 ± 0.1 2.9 ± 0.2 3.2 ± 0.2 3.5 ± 0.2 13.3 ± 0.9 14.1 ± 0.4 5.3 ± 0.4 6.4 ± 0.2 10.5 ± 0.6

4.7 ± 1.2 3.6 ± 1.7 9.3 ± 3.4 >10 8.8 ± 3.3 6.2 ± 2.2 7.8 ± 2.3 >10 >10 13.6 ± 1.8 3.4 ± 0.7 1.8 ± 0.3 2.3 ± 0.1 3.6 ± 0.2 1.7 ± 1.0 3.1 ± 1.7 2.2 ± 0.6 1.5 ± 0.8 2.2 ± 0.8 1.8 ± 0.4 11.2 ± 4.0 14.5 ± 2.0 4.0 ± 0.5 8.2 ± 1.8 6.9 ± 2.6

>10 >10 4.4 ± 2.1 5.3 ± 2.9 5.5 ± 2.6 7.6 ± 3.2 8.3 ± 2.9 7.9 ± 3.1 8.6 ± 3.5 5.1 ± 0.3 >10 7.9 ± 2.6 >10 >10 >10 >10 >10 5.2 ± 2.3 5.4 ± 2.0 >10 >50 >50 >50 >50 >50

competitive competitive mixed uncompetitive mixed/noncompetitive mixed/noncompetitive mixed/noncompetitive uncompetitive uncompetitive mixed competitive mixed competitive competitive competitive competitive competitive mixed mixed competitive competitive competitive competitive competitive competitive

the target affinities of the thiazolidinedione derivatives (X = O) were significantly higher than that of the rhodanines (X = S) (Table 2). Within both compound classes, the cyclopentylsubstituted derivatives (9a, 9b) were most potent with an IC50 value of 6.1 μM (9a, X = S) and 2.5 μM (9b, X = O), respectively. To confirm that the sequence Cap-Arg-Lys-Nle-NH2 is the most suitable one for the peptide hybrids, 10 derivatives bearing the same highly affine N-terminal cap moiety were additionally evaluated (Table 3). For the inverse sequence Cap-D-Arg-D-LysD-Nle-NH2 (27), a significantly lower target affinity was observed. If the lysine residue is replaced by an ornithine (28), the same observation was made. High activity could be observed for a substitution of the lysine residue by an additional arginine 8393



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Table 3. Inhibitory Activity of Alternative Peptide Hybrids at the Dengue Virus Protease, West Nile Virus Protease, and Thrombin

a Percent inhibition of the DEN NS2B−NS3 protease serotype 2 (enzyme: 100 nM, inhibitor 50 μM, substrate 50 μM, Km = 105 μM). bPercent inhibition of the WNV NS2B−NS3 protease (enzyme: 150 nM, inhibitor 50 μM, substrate 50 μM, Km = 212 μM). n.i. = no inhibition. cPercent inhibition of thrombin (enzyme: 10 nM, inhibitor 25 μM, substrate 50 μM, Km = 16 μM).

Additional kinetic analyses did not indicate time-dependent inhibition modes or instability of the peptide hybrids over some hours (data not shown). To check our results, the Ki value for the most active compound 9b was confirmed by an HPLC-based assay (data not shown). Aprotinin Competition Assay. To exclude a nonspecific binding mode, a fluorescence quenching assay developed by Bodenreider et al., which can discriminate between specific and nonspecific binders, was used.54 This assay is based on the

formation of a ternary enzyme−substrate−inhibitor complex. The thiazolidinediones may have the possibility for more polar interactions using the additional carbonyl function, for example by hydrogen bonds to the enzyme, resulting in higher affinity. For the previously reported series of compounds, Ki values between 4.0 and 14.5 μM with a competitive inhibition mechanism were observed (Table 2), clearly indicating a significant in vitro affinity improvement for the thiazolidinedione-based peptide hybrids. 8394



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Figure 1. Exemplary illustration of the kinetic results for compounds 26a and 26b. Graphs (A) and (B) represent the Dixon and Cornish-Bowden plots for the rhodanine-based peptide hybrid 26a with two intersections, indicating a mixed inhibition mechanism with a Ki value of 13.6 μM and a Ki′ value of 5.1 μM, respectively. Graphs (C) and (D) represent the Dixon and Cornish-Bowden plots for the corresponding thiazolidinedione-based peptide hybrid 26b with an intersection in the Dixon plot but without any correlation of the straight lines toward an intersection in the Cornish-Bowden plot, clearly indicating a competitive inhibition mechanism for this compound with a Ki value of 1.8 μM.

Figure 2. Docking results for compound 9b (green) within the DEN protease (pdb code 3U1I). (A) Solvent-accessible surface of NS2B−NS3, with hydrophobic regions in red and hydrophilic regions in blue. The lysine residue of the ligand occupies the S1 and the arginine residue the S2 pocket, respectively. The norleucine residue is located near the S1′ pocket, and the N-terminal cap is in contact with hydrophobic areas of the protease and cofactor domains near the S3 pocket. (B) Ribbon model of the protein in the same orientation as in (A) with important side chains for the interaction with the ligand. The protease domain is colored in gray and the cofactor in blue. The figures were generated using the Chimera software.67

fluorescence will be partially restored if aprotinin, which is a highly affine protease inhibitor without any tryptophan residues, replaces the other inhibitor from the active site. This was already shown for the previously reported peptide hybrids.37 The thiazolidinedione-based peptide hybrids are perfectly applicable

intrinsic fluorescence of Trp50 of the DEN protease, which is located near the active site of the enzyme. An inhibitor that specifically binds to the protease quenches this fluorescence by FRET if a structural element of the inhibitor is able to absorb radiation in the range of the tryptophan emission. The 8395



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for this method with an absorption maximum around 330 nm so that the experiments were carried out with derivatives 21b and 26b (Supporting Information Figure S1). Both compounds show a concentration dependent nonlinear decrease of the registered fluorescence. If aprotinin was additionally added, a significant fraction of the fluorescence was restored, clearly indicating specific binding of the inhibitors at the active site. Molecular Modeling. All experiments were carried out using the protease of DEN serotype 2. No active structure of the serotype 2 protease cocrystallized with an inhibitor is available at present. An active structure that displays a “closed” conformation of the DEN protease serotype 3 with a cocrystallized tetrapeptidyl aldehyde inhibitor was recently reported by Noble et al. (pdb code: 3U1I).14 We used the 3U1I structure to obtain an idea of how the investigated peptide hybrids may interact with the target protein because 3U1I is the only available structure of the catalytically competent complex NS2B−NS3 of DEN protease. Some conformational and biochemical differences were observed for the proteases of the DEN serotypes, especially regarding the role of the cofactor domain. However, choosing the serotype 3 structure for preliminary modeling studies appears to be reasonable because the active sites are highly conserved and the substrate specificity is practically identical for all serotypes.8,11,55,56 As representative compound for the thiazolidinedione series shown in Table 1, the most active derivative 9b (R = cyclopentyl, X = O) was selected. The results are shown in Figure 2. Similar to the inhibitor−enzyme complex in the 3U1I structure, the S1 and S2 pockets are occupied by two basic side chains of 9b. This correlates with other findings, e.g., for substrate specificity of the DEN protease.55,57,58 The lysine residue is placed in the S1 and the arginine residue in the S2 pocket with the possibility of hydrogen bonds or salt bridges to Asp129 and Asp75, respectively. The norleucine residue is flexibly arranged near the S1′ pocket, interacting with a hydrophobic surface formed by two valine side chains of the NS3 domain. The N-terminal cap moiety binds near the S3 pocket, which is formed by residues of the NS3 protease and the NS2B cofactor. The arylidene moiety acts as a relatively rigid linker for the heterocycle and the hydrophobic cyclopentyl substituent, to reach a hydrophobic moiety formed by two valine side chains of the NS3 domain and a methionine and isoleucine residue of the cofactor NS2B. This could explain the remarkable preference for hydrophobic substituents connected to position 3 of the heterocycle. One may speculate about a stabilization of the “closed” conformation by these interactions of the N-terminal cap moiety with the hydrophobic residues of the protease and cofactor domains. In summary, the docking results provide some interesting information about possible interactions of the peptide hybrids in the competitive binding mode. Permeability. An often discussed restriction of peptidebased inhibitors is their lack of membrane permeability.59 Nevertheless, membrane permeability is a basic requirement for DEN protease inhibitors and should be taken into account for future drug candidates. Therefore, we estimated the passive membrane permeability behavior of the investigated compounds by an easily applicable, in vitro permeability model. The parallel artificial membrane permeability assay (PAMPA) is an established and reliable method for predicting the passive cell permeability of compounds.60 For our studies, we used advanced precoated trilayer (lipid/oil/lipid) artificial membranes. These have been described to yield permeability data in good correlation with results from cell-based Caco-2 permeability assays.61 Table 4 provides the PAMPA results for 24 analyzed

Table 4. Permeability Data of Selected Compounds in the Parallel Artificial Membrane Permeability Assay (PAMPA) No.

cAcc (μM)a

7a 9a 10a 12a 13a 18a 20a 23a 25a 26a 7b 9b 10b 15b 17b 24b 25b 26b 27 28 38 41 42 43 I II III IV V

7.1 ± 0.4 9.1 ± 1.0 7.3 ± 1.1 3.1 ± 0.3 1.9 ± 0.6 2.3 ± 0.4 3.9 ± 0.8 2.3 ± 0.4

5.5 ± 0.5 2.0 ± 0.2 7.5 ± 1.0 3.4 ± 0.4 1.8 ± 0.2 4.2 ± 0.4 3.5 ± 0.4 1.5 ± 0.1 8.8 ± 1.5

5.0 ± 0.8 3.3 ± 0.0

cDon (μM)b

Pe (10−6 cm/s)c

log Pe

164.5 ± 6.7 1.61 −5.79 147.8 ± 7.8 2.31 −5.64 150.9 ± 8.0 1.82 −5.74 133.2 ± 5.5 0.87 −6.06 164.7 ± 0.2 0.42 −6.38 141.6 ± 2.4 0.59 −6.23 142.0 ± 6.1 1.02 −5.99 143.3 ± 3.6 0.60 −6.23 insufficient solubility insufficient solubility 172.3 ± 7.1 1.18 −5.93 169.3 ± 1.7 0.44 −6.35 166.3 ± 23.2 1.69 −5.77 not permeable at detectable range 191.5 ± 7.6 0.66 −6.18 156.5 ± 6.3 0.43 −6.36 140.1 ± 16.0 1.11 −5.95 159.4 ± 0.6 0.82 −6.09 189.5 ± 4.8 0.29 −6.54 not permeable at detectable range 173.1 ± 5.5 1.90 −5.72 not permeable at detectable range not permeable at detectable range not permeable at detectable range not permeable at detectable range not permeable at detectable range 167.4 ± 1.1 1.11 −5.96 not permeable at detectable range 98.6 ± 10.8 1.26 −5.90

R (%)d 15.4 23.1 22.1 32.4 17.0 28.4 27.7 27.6

12.0 14.7 14.4 3.1 21.1 28.6 19.1 4.7 10.5

14.6 49.6

a

Concentration of compound detected in the acceptor plate after 5 h of incubation. bConcentration of compound detected in the donor plate after 5 h of incubation. cPermeability of a compound calculated according to the literature.61 dMass retention of a compound calculated according to the literature.61

new peptide hybrids and five compounds from the former series. Five drugs (famotidine, amiloride, furosemide, phenytoin, and caffeine) with known permeation behavior were used as reference compounds.61 For some of the new derivatives, high mass retentions in the range of 30% were found, indicating high surface adsorptions and accumulation within the artificial membrane (Table 4). The best permeability could be observed for rhodanine derivative 9a (R = cyclopentyl, X = S) with a Pe value (permeability in 10−6 cm/s) of 2.31. By way of comparison, for caffeine and phenytoin as compounds with high membrane permeability and gastrointestinal absorption Pe values of 12.9 and 10.3 were found, respectively. Other compounds with very high membrane permeability, like ibuprofen, dilitiazem, or imipramine, have Pe values in the same range.61 This demonstrates that a value of 2.31 can be considered as a good starting point for peptide-based molecules, indicating the ability of some of the compounds to pass biological membranes and enter cells. The substituent at the heterocycle in position 3 (R) appears to have a particular influence on the permeation behavior, as compounds bearing hydrophobic aliphatic substituents in this position have a higher permeability. The rhodanine-based peptide hybrids have a higher permeability than the thiazolidinedione-based compounds. However, two of the analyzed rhodanine derivatives bearing the nonpolar chlorobenzyl (25a) and phenylethyl (26a) 8396



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Figure 3. Results of the cytotoxicity assay (firefly luciferase reporter gene) for selected compounds at very high compound concentrations.

also nontoxic at the highest dengue reporter gene assay concentration of 50 μM (Figure 3). High activity was also observed for the corresponding phenylethyl-substituted derivative 26a with EC50 = 25.6 μM (nontoxic at 50 μM). In correlation with the previously discussed results (permeability, in vitro activity), compounds with hydrophobic substituents have higher activity. Consequently, the proton- (1a, 1b), methyl- (2a), and ethanolyl-substituted (14a, 14b) derivatives possess no relevant antiviral activity (EC50 >50 μM). A tendency for higher antiviral activities (and cytotoxicity at higher concentrations) could be observed for the rhodanine derivatives compared to the thiazolidinedione derivatives, although the rhodanines are less active against the isolated protease. An explanation for this result might be the higher hydrophobicity and better permeability (in correlation with the PAMPA results) and the observed additional mode of intermolecular interaction by an uncompetitive inhibition mechanism (enzyme−substrate−inhibitor complex). Surprisingly, the ornithine congener 28, which is less active and less permeable than the corresponding lysine derivative 21b, has significantly higher antiviral activity (26.8 μM vs >50 μM). A similar observation is made for compounds 41 and 42. These two derivatives show nearly no inhibition of the DEN protease in vitro but possess antiviral activity. Because of these unexpected findings, we considered an interference of the inhibitors with the luciferase activity, which could lead to false-positive results. This possibility was studied as follows: Both luciferases were stably expressed in cell lines that were treated for a short period with the compounds. Afterward, the activities of the renilla and firefly luciferases were analyzed separately. The results are provided in Supporting Information Figure S2. An interference with both luciferases was observed for compound 42, rendering the results of the cytotoxicity and dengue virus reporter gene assays nonsignificant (Table 5, Figure 3). Other compounds display only minor interferences (Supporting Information Figure S2). To further confirm our results, six compounds (50 μM) were additionally analyzed in a dengue virus titer reduction assay that is not based on luciferase activity (plaque assay). The results are given in Figure 4 and indicate no significant reduction of the viral titer for the two compounds II and III from the previous series and for the thiazolidinedione-based peptide hybrids 25b and 28. In contrast, the two rhodanine-based peptide hybrids 10a and 25a caused a significant decrease of viral titers. Compound 25a is somewhat cytotoxic at the relevant concentration of 50 μM

substituents were insoluble in PBS buffer at the required concentration of 200 μM. A fine-tuning of hydrophobicity with respect to the two end-points permeability and solubility is therefore necessary. Antiviral Activity in Cell-Culture and Cytotoxicity. A set of 26 compounds from Table 1 and Table 3 with high, moderate, and low affinity toward the DEN protease and different molecular structures was additionally screened in an infectionbased cell culture model that utilizes a DEN serotype 2-derived reporter virus genome and the Huh-7 human hepatoma cell line. The latter stably expresses a firefly luciferase reporter gene for accurate quantification of cytotoxicity. Because no cytotoxic effects were observed in initial experiments at concentrations below 50 μM, 18 compounds (including five from the previously reported series) were selected for additional experiments at higher concentrations (Figure 3). The results of the virus reporter gene assay are summarized in Table 5. Antiviral activity was observed in a promising concentration range: The best antiviral activity in cell culture was found for the rhodanine-based peptide hybrid 10a, bearing a cyclohexyl moiety at the heterocycle, with an EC50 value of 16.7 μM. This derivative is Table 5. Results of the Dengue Virus Replication Assay for Selected Compounds No.

EC50 (μM)a

no.

EC50 (μM)a

1a 2a 7a 9a 10a 12a 13a 14a 18a 20a 23a 25a 26a

>50 >50 35.5 ± 1.1 30.3 ± 1.1 16.7 ± 1.2 48.8 ± 1.8 31.3 ± 1.3 >50 42.5 ± 1.2 35.7 ± 1.1 37.3 ± 1.1 31.5 ± 1.0 25.6 ± 1.1

1b 7b 9b 14b 21b 25b 26b 28 37 38 40 41 42b

>50 >50 44.9 ± 1.2 >50 >50 41.1 ± 1.2 37.2 ± 1.1 26.8 ± 1.3 >50 53.8 ± 1.1 >50 30.3 ± 1.2 26.0 ± 1.3

a

The half-maximal effective concentration of a compound in the dengue virus serotype 2 renilla luciferase reporter gene assay. b Compound interferes with the renilla luciferase, resulting in an unreliable assay result. 8397



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changes in the structure of the inhibitor, particularly the sulfur− oxygen exchange, cause considerably changes in the biochemical interaction profile by changing the binding mode from competitive to uncompetitive. Future work will aim to increase the target affinity further, accompanied by modifications of the peptide backbone and basic side chains to improve the pharmacokinetic properties of the compounds. It may also be considered to specifically optimize the compounds toward an uncompetitive or noncompetitive binding mode, given the higher antiviral activities of the respective inhibitors described here.

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EXPERIMENTAL SECTION

Expression and Purification of the Viral Proteases. The DEN (serotype 2) and WNV NS2B−NS3 protease constructs were described before.58,62 Transformation of the pET28a plasmid (Novagen, Germany), expression in Escherichia coli BL 21 λ (DE3) cells, and purification by nickel affinity chromatography was done according to the protocol described by Steuer et al.21,63 For both viral enzymes, the cofactor is covalently connected to the protease domain by a glycineserine linker (GGGGSGGGG). Dengue and West Nile Virus Protease Assays. The DEN and WNV protease assays were performed as described previously.20,37,51,63 In short, continuous enzymatic assays were performed in black 96-well V-bottom plates (Greiner Bio-One, Germany) and monitored using a BMG Labtech Fluostar OPTIMA microtiter fluorescence plate reader at an excitation wavelength of 320 nm and a monitored emission wavelength of 405 nm. The inhibitors (final concentration 50 μM, from 10 mM stock solutions in DMSO) were preincubated for 15 min with the DEN protease (100 nM) or WNV protease (150 nM), respectively. Afterward, the reaction was initiated by the addition of the substrate to a final concentration of 50 μM. FRET substrates Abz-NleLys-Arg-Arg-Ser-3-(NO2)Tyr and Abz-Gly-Leu-Lys-Arg-Gly-Gly-3(NO2)Tyr were used for DEN and WNV protease, respectively. The activity of the enzyme was determined as the slope per second (RFU/s) and monitored for 15 min. Determinations of percentage inhibition were calculated in relation to a positive control (without inhibitor) and performed in triplicate. For the HPLC-based DEN protease assay 10 μL of 4% TFA were added to every well after 15 min of enzymatic reaction, and the samples were analyzed by a Jasco HPLC system with an FP-2020 plus fluorescence detector (excitation, 320 nm; emission, 405 nm) with an RP-18 column Phenomenex Luna C18(2) (5 μm, 150 mm × 3 mm) using the following conditions: flow rate, 1.0 mL/min; eluent A, water (0.1% TFA); eluent B, acetonitrile (0.1% TFA); gradient, 10% B (1 min), 55% B (9.5 min), 95% B (9.6 min), 10% B (12.6 min), 10% B (15 min).51 Thrombin Assay. The thrombin assay was performed as a continuous fluorimetric assay in black 96 well V-bottom plates (Greiner Bio-One, Germany), using a BMG Labtech Fluostar OPTIMA microtiter fluorescence plate reader and operating at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The inhibitors (final concentration 25 μM, from 10 mM stock solutions in DMSO) were preincubated with thrombin (10 nM) for 15 min. The cleavage reaction was initiated by addition of the Boc-Val-Pro-Arg-AMC substrate (Bachem, Germany) at a final concentration of 50 μM. The assay buffer was used according to the literature as 50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.05% Tween 20.64 The activity of thrombin was determined as the slope per second (RFU/s) and monitored for 10 min. Determinations of percentage inhibition were calculated in relation to a positive control (without inhibitor) and performed in triplicate. Determination of Ki, Ki′, and IC50 Values. The assay conditions were generally used as described before. The assays were performed using different inhibitor (0, 0.5, 1, 2, 3, 4, 5, 6 μM) and substrate concentrations (50, 100, 150, 200 μM) in triplicate. Compounds from the former series of peptide hybrids were assayed at concentrations of 5, 10, 15, 20, 30, 40, and 50 μM. Observed values were adjusted using correction factors due to the inner filter effect of the FRET substrate.51,65

Figure 4. Results of the virus titer reduction assay for selected compounds at a concentration of 50 μM. The peptide hybrids from previous series (II and III) and the thiazolidinedione-based peptide hybrids (25b and 38) cause only minor inhibition of viral replication, whereas the rhodanine-based peptide hybrids (10a and 25a) have significant antiviral effects.

(Figure 3), which might contribute to the more pronounced reduction of viral replication compared to 10a. The latter compound has a cytotoxic effect at concentrations above 100 μM and causes a 2/3 reduction of viral replication at a concentration of 50 μM. These results indicate an advantageous antiviral/ cytotoxic activity ratio for rhodanine-based peptide hybrids bearing hydrophobic aliphatic moieties (e.g., 10a) in comparison to benzyl substituted analogues such as 25a.

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CONCLUSIONS A promising new generation of peptide hybrids as DEN protease inhibitors was explored and extensively analyzed toward their possible biochemical and biological potential of interaction. It was confirmed that two basic side chains (arginine and lysine) are sufficient to realize high target affinity and selectivity with Ki values below 2 μM. It is also evident that reactive, electrophilic moieties which form covalent bonds to the catalytic serine are not essential to obtain high affinities. From the structure−activity relationships of our previous and current works the following consequences for DEN protease peptide hybrid inhibitors can be drawn: The connection between the thiazolidinone heterocycle and the peptide should be accomplished by relatively rigid arylidene moieties using para-substitution. The Cap-Arg-LysNle-NH2 motif appears to be optimal for high target affinity. The character of the terminal substituent at the nitrogen atom of the thiazolidinone heterocycle in position 3 is very important for the target affinity, the permeability, and the antiviral activity in cell culture. More hydrophobic substituents, with a tendency toward aliphatic groups, have increased membrane permeability and higher in vitro and in cellulo activities. The oxygen vs sulfur exchange in position 2 of the 4-thiazolidinone scaffold has a significant influence on various properties of the compounds: The rhodanine (X = S) derivatives are somewhat less potent at the isolated enzyme (IC50 never below 6 μM), show a mixed inhibition mode with significant uncompetitive contribution, are slightly better permeable, and possess higher activity in cell culture. The thiazolidinedione (X = O) derivatives are predominantly substrate-competitive inhibitors with higher in vitro affinities (Ki up to 1.5 μM) but inferior permeation and antiviral activity. A striking observation is the fact that minor 8398



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IC50 values were calculated for a substrate concentration of 50 μM using Prism 5.0 (GraphPad Software, Inc.). For Ki value determination data were plotted 1/v (reciprocal enzymatic activity) against inhibitor concentration (Dixon plot) for the four different measured substrate concentrations. For Ki′ value determination, the data were plotted S/v (substrate concentration divided by enzymatic activity) against inhibitor concentration (Cornish-Bowden plot) for the four measured substrate concentrations.52,53 Aprotinin Competition Assay. This tryptophan quenching assay was performed as described by Bodenreider et al. before.54 In short, the DEN protease (4 μM) was incubated with inhibitors at different concentrations (0, 5, 10, 20, 30, 50 μM) using the assay buffer described before.63 Additionally, the protease was incubated with the inhibitor (50 μM) and aprotinin (10 μM) together. As negative control, the autofluorescence of the enzyme and aprotinin without inhibitor at the same concentrations was additionally determined. The fluorescence of the assay samples was monitored on a Tecan Safire II instrument using an excitation wavelength of 280 nm and an emission wavelength of 330 nm. All determinations were performed in triplicate. Docking. The calculations were performed on an Intel(R) Core(TM)2 Quad CPU Q9450 @ 2.66 GHz running open SuSE 11.0, using GOLD 5.1 and its graphical interface Hermes 1.4.66 The crystal structure of the NS2B−NS3 protease of DEN serotype 3 in complex with a tetrapeptidyl aldehyde was extracted from the dimer structure 3U1I.14 All waters and sulfate ions were removed, and the structure was further prepared using Dock Prep (Chimera).67 All hydrogens were added and the tetrapeptidyl aldehyde ligand was extracted from the structure. The binding site was defined in a radius of 10 Å around the extracted ligand and automatic cavity detection was used. Ten different solutions were calculated for every ligand, and the docking results with the highest GOLD scores were aligned and visualized using Chimera.67 Parallel Artificial Membrane Permeability Assay (PAMPA). Concentration determinations were carried out on a Tecan Safire II instrument using 96 well U-bottom polystyrene plates (Greiner BioOne, Germany) and alternatively a Jasco HPLC system with a single wavelength UV detector and an RP-18 column (ReproSil-Pur-ODS, Dr. Maisch GmbH, Germany, 3 μm, 50 mm × 2 mm), using acetonitrile (0.1% TFA) and water (0.1% TFA) with a standard linear gradient. For all experiments, phosphate buffered saline (PBS), which was purchased from Sigma-Aldrich (Germany), was used, resulting in 0.01 M phosphate buffer, 0.0027 M potassium chloride, and 0.137 M sodium chloride at pH 7.4. Calibration curves were generated for all compounds including the references (famotidine, amiloride, furosemide, phenytoin, caffeine) at 10, 25, 50, 100, 150, and 200 μM using the HPLC-UV system (detection at 254 nm) and the UV plate reader (at 330 and 390 nm), respectively. A detection wavelength of 254 nm was used for famotidine, amiloride, furosemide, phenytoin, caffeine, 41 and 42, I, II, IV, and V, 330 nm for compounds 7a, 9a, 10a, 7b, 9b, 10b, 15b, 17b, 24b, 25b, 26b, 27, 28, 38, and III, and 390 nm for 12a, 13a, 18a, 20a, 23a, and 43, respectively. All correlation coefficients (R2) were found to be at least 0.99 for the calibration curves. The precoated PAMPA plate system was purchased from BD Bioscience (Germany) and stored at −20 °C till use. Before use, the plates were warmed to room temperature for 1 h. Then 300 μL of the 200 μM compound solutions in PBS (final concentration of DMSO: 2%) were dispensed in triplicate into the donor plate. Then 200 μL of PBS buffer were added to all wells of the acceptor plate, which was then carefully placed on the donor plate. The plate system was covered and incubated at room temperature for 5 h. Afterward, the plates were separated and the solutions of donor and acceptor plates were transferred into 96 well polystyrene U-bottom plates (Greiner Bio-One, Germany) and analyzed by UV absorption using the plate reader and the HPLC system, respectively. The concentrations of the compounds in donor and acceptor plates were determined using the previously generated calibration curves. Calculations of the permeability (Pe) and the mass retention (R) were done according to the literature.61 Dengue Virus Replication and Cytotoxicity Assay in Huh-7 Cells by using Renilla and Firefly Luciferase Reporter Genes. The inhibitors were reconstituted as 10 mM stock solutions in DMSO,

aliquoted and stored at −20 °C until use. For testing at higher concentrations (100, 200, and 400 μM), the stocks were reconstituted at 40 mM in DMSO. Stock solutions were 40 mM in DMSO. The stock solutions were freshly diluted before each experiment in DMEM (Dulbecco’s Modified Minimal Essential Medium (GIBCO, Invitrogen) containing 2 mM L-glutamine (GIBCO, Invitrogen), 1× nonessential amino acids (GIBCO, Invitrogen), 100 μg/mL penicillin (GIBCO, Invitrogen), 100 μg/mL streptomycin (GIBCO, Invitrogen), and 10% fetal calf serum (heat inactivated at 56 °C for 30 min (GIB-CO, Invitrogen)) in 2-fold dilution series to prepare solutions of adequate concentration, which were used later as 4× stock solutions. Huh-7 cells stably expressing a firefly luciferase reporter gene were seeded at 5000 cells/well into white clear-bottom 96 well plates (Greiner BioOne) in a volume of 100 μL DMEM.68 To reduce the effects due to medium evaporation, the outer row of wells were filled with medium but were not used for the experiment. After overnight incubation of the cells in a 37 °C incubator, 50 μL of 4× compound stocks in DMEM were added to the cells at various final concentrations (50, 25, 12.5, 3.13, and 1.56 μM for the virus replication assay and additionally 100, 200, and 400 μM for the determination of cytotoxicity). Four hours later, additional 50 μL of DMEM or DMEM containing dengue 2 renilla luciferase reporter virus at a multiplicity of infection of 10 were added to the cells.68 The cytotoxicity was measured using compound treated cells which were uninfected but incubated with the compound for similar time as infected cells. The cells were incubated at 37 °C for further 48 h and harvested by removing the media, washing 1× with PBS and adding 50 μL of luciferase lysis buffer (1% Triton X-100, 25 mM glycylglycine, 15 mM magnesium sulfate, 4 mM EGTA, and 1 mM DTT, pH 7.8) and kept frozen at −20 °C until measurement. The luciferase reporter activity was measured using a Mithras LB940 plate luminometer (Berthold Technologies). The plates were thawed and kept at 4 °C until measurement. The cytotoxicity was measured by firefly reporter assay by adding 75 μL of assay buffer (25 mM glycylglycine, 15 mM magnesium sulfate, 4 mM EGTA, 1 mM DTT, 2 mM ATP, and 15 mM potassium phosphate, pH 7.8), containing 70 μM luciferin to each well and measuring the luminescence for one second/ well in the plate luminometer. Virus replication was estimated by renilla luciferase reporter activity by the addition of 50 μL of assay buffer (25 mM glycylglycine, 15 mM magnesium sulfate, 4 mM EGTA, and 15 mM potassium phosphate, pH 7.8), containing 5 μg/mL coelenterazine (PJK), and the luminescence was measured for one second/well in the plate luminometer. For each inhibitor concentration, the viral replication was measured in at least four independent wells, and the replication was expressed as percentage in relation to the control (DMSO-treated) cells. The cytotoxicity for each concentration was measured as the mean of three independent wells and expressed as a percentage of the mean of the DMSO control. The EC50 values were estimated using Prism 5.0 (GraphPad Software, Inc.) using nonlinear regression analysis. Compounds with EC50 values higher than the highest concentration tested (50 μM) were reported as >50 μM. Virus Reduction Assay (Plaque Assay). Huh-7 cells were seeded into 12-well plates (1.5 × 105 cells/well) in 1 mL complete DMEM. After overnight incubation of the cells at 37 °C, medium was replaced with DMEM containing appropriate concentration of the assayed compound. Four hours later, the cells were infected for one hour with dengue reporter virus (MOI of 5) in the presence of the compound in a volume of 200 μL. Later the medium was replaced with DMEM containing appropriate concentration of the test compound, and 48 h later the supernatant was harvested, filtered through a 0.4 μm filter, and stored at −80 °C. The virus titer was determined by plaque assay on Vero cells. The values are expressed as the mean of three independent experiments with standard deviation. Chemistry. All chemicals for the precursor synthesis were obtained from Sigma-Aldrich (Germany) and Alfa Aesar, Johnson Matthey (Germany) and were of analytical grade. No further purification steps were performed unless indicated. All solvents were used as obtained from the commercial sources. The protected amino acids were purchased from Orpegen (Germany), Novabiochem, Merck (Germany), and Sigma-Aldrich (Germany). HBTU and Rink amide resin 8399



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(capacity 0.63 mmol/g) were purchased from Iris Biotech (Germany). NMR spectra were recorded on Varian NMR instruments at 300 or 500 MHz, 300 K in CDCl3, acetone-d6, or DMSO-d6. Chemical shifts (δ) are given in parts per million (ppm). Residuals of nondeuterated solvents were used as internal standard. Coupling constants (J) are given in hertz (Hz). Mass spectra were measured on Finnigan MAT 8200 (EI) and Bruker micrOTOF-Q II (HR-ESI) instruments. Combustion elemental analysis was performed by double determination using a Foss Heraeus Vario EL analyzer. Flash chromatography was performed on a Biotage Isolera One purification system using silica gel (0.060−0.200 mm) cartridges (KP-Sil) and UV monitoring at 254 and 280 nm. The reaction progress was determined by thin layer chromatography on Merck silica gel plates 60 F254 (UV detection). Microwave synthesis was done using a Monowave 300 synthesis reactor with IR temperature sensor from Anton Paar. Purity of the compounds used in biological assays was determined by LC-MS using an Agilent 1200 HPLC system with a MWD detector combined with a Bruker micrOTOF-Q II instrument on an RP-18 column (ReproSil-Pur-ODS-3, Dr. Maisch GmbH, Germany, 3 μm, 50 mm × 2 mm). Procedure A: Synthesis of Peptide Hybrids and Peptidic Assay Substrates. Peptidic DEN and WNV protease assay substrates and Nterminally capped peptide hybrids were synthesized as described before.37 In short, the N-terminal cap (3 equiv) as unprotected carboxylic acid was coupled to the desired peptide sequence using the Fmoc protocol. After cleavage from the resin and precipitation in cold diethyl ether, all crude peptides were purified by preparative HPLC on an Ä KTA Purifier, GE Healthcare (Germany), with an RP-18 pre and main column (Rephospher, Dr. Maisch GmbH, Germany, C18-DE, 5 μm, 30 mm × 16 mm and 120 mm × 16 mm). After freeze-drying, the peptides were characterized by HR-ESI and purity was determined by LC-MS. All evaluated peptide hybrids were obtained with a purity of at least 95% unless indicated otherwise. Procedure B: Synthesis of 3-Alkyl- and 3-Benzylrhodanines with Exemplary Compound Data. A suspension of amine (10 mmol), sodium hydroxide (22 mmol), and carbon disulfide (11 mmol) in water or ethanol (10 mL) was reacted in a microwave reactor for 5 min at 100 °C. After automated cooling to 40 °C, chloroacetic acid (11 mmol) was added and the mixture was reacted again at 100 °C for 5 min. After cooling (40 °C), concentrated hydrochloric acid (3 mL) was added and the reaction was finished at 120 °C for 30 min. The crude product was extracted with ethyl acetate (if ethanol was used as solvent, water was added before extraction) and purified by flash chromatography (cyclohexane/ethyl acetate). When glycine or phenylalanine were used as amine component, the first reaction step was carried out at room temperature for 16 h, followed by the second step for additional 3 h and a final refluxing in aqueous hydrochloric acid for another 16 h. After extraction, these two compounds were used as crude products without further purification. 3-Cyclopentyl-2-thioxo-1,3-thiazolidin-4-one. Colorless solid (59% yield). 1H NMR (300 MHz, CDCl3): δ = 1.53−1.67 (m, 2H), 1.80−2.00 (m, 4H), 2.03−2.17 (m, 2H), 3.81 (s, 2H), 5.31 (quint, J = 8.7 Hz, 1H) ppm. 13C NMR (75 MHz, CDCl3): δ = 25.5, 27.5, 34.2, 57.8, 173.8, 202.5 ppm. MS (EI, 70 eV), m/z (%): 201.0 (76) [M+]. Anal. Calcd for C8H11NOS2: C, 47.73; H, 5.51; N, 6.96. Found: C, 47.73; H, 5.58; N, 6.97. 3-(4-Chlorobenzyl)-2-thioxo-1,3-thiazolidin-4-one. Pale-yellow solid (80% yield). 1H NMR (300 MHz, CDCl3): δ = 3.98 (s, 2H), 5.13 (s, 2H), 7.27 (m, 2H), 7.38 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3): δ = 35.6, 46.9, 128.7, 130.6, 133.1, 134.2, 173.7, 200.8 ppm. MS (EI, 70 eV): m/z (%), 256.8 (85) [M+]. Anal. Calcd for C10H8ClNOS2: C, 46.60; H, 3.13; N, 5.43. Found: C, 46.48; H, 3.43; N, 5.23. 3-(2-Phenylethyl)-2-thioxo-1,3-thiazolidin-4-one. Pale-yellow solid (65% yield). 1H NMR (300 MHz, CDCl3): δ = 2.93 (m, 2H), 3.92 (s, 2H), 4.19 (m, 2H), 7.20−7.33 (m, 5H) ppm. 13C NMR (75 MHz, CDCl3): δ = 32.6, 35.2, 45.7, 126.8, 128.5, 128.9, 137.3, 173.4, 200.9 ppm. MS (EI, 70 eV): m/z (%), 236.9 (76) [M+]. Anal. Calcd for C11H11NOS2: C, 55.67; H, 4.67; N, 5.90. Found: C, 55.67; H, 4.82; N, 5.88. Procedure C: Synthesis of 3-Arylrhodanines with Exemplary Compound Data. A suspension of amine (5 mmol) and bis-

(carboxymethyl)trithiocarbonate (5.5 mmol) in water was reacted in a microwave reactor at 160 °C for 15 min. After automated cooling to 40 °C, the crude product was extracted with ethyl acetate and purified by flash chromatography (cyclohexane/ethyl acetate). For methyl 2aminohexanoate and ethanolamine as amine components, this procedure was also used instead of procedure B. 3-Phenyl-2-thioxo-1,3-thiazolidin-4-one. Pale-yellow solid (32% yield). 1H NMR (300 MHz, CDCl3): δ = 4.18 (s, 2H), 7.19 (m, 2H), 7.52 (m, 3H) ppm. 13C NMR (75 MHz, CDCl3): δ = 36.3, 128.3, 129.6, 129.8, 134.9, 173.4, 201.1 ppm. MS (EI, 70 eV): m/z (%), 208.9 (100) [M+]. Anal. Calcd for C9H7NOS2: C, 51.65; H, 3.37; N, 6.69. Found: C, 50.51; H, 3.42; N, 6.33. 3-(4-Methoxyphenyl)-2-thioxo-1,3-thiazolidin-4-one. Pale-yellow solid (39% yield). 1H NMR (300 MHz, CDCl3): δ = 3.84 (s, 3H), 4.16 (s, 2H), 7.02 (m, 2H), 7.10 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3): δ = 36.2, 55.5, 114.9, 127.2, 129.4, 160.3, 173.5, 201.5 ppm. MS (EI, 70 eV): m/z (%), 238.9 (100) [M+]. Anal. Calcd for C10H9NO2S2: C, 50.19; H, 3.79; N, 5.85. Found: C, 50.24; H, 3.73; N, 5.82. 3-(2-Hydroxyethyl)-2-thioxo-1,3-thiazolidin-4-one. Pale-yellow oil (17% yield). 1H NMR (300 MHz, CDCl3): δ = 3.90 (t, J = 5.5 Hz, 2H), 4.01 (s, 2H), 4.25 (t, J = 5.5 Hz, 2H) ppm. 13C NMR (75 MHz, CDCl3): δ = 35.4, 46.6, 59.9, 174.6, 201.9 ppm. MS (EI, 70 eV): m/z (%), 177.0 (60) [M+]. Anal. Calcd for C5H7NO2S2: C, 33.88; H, 3.98; N, 7.90. Found: C, 33.29; H, 4.12; N, 7.20. Procedure D: Synthesis of 3-Alkyl- and 3-Benzylthiazolidine-2,4diones and Precursor with Exemplary Compound Data. To a refluxing solution of thiazolidine-2,4-dione (43 mmol) in ethanol (25 mL) was added a hot solution of potassium hydroxide (45 mmol) in ethanol (25 mL). After additional refluxing for 30 min, the mixture was cooled to 0 °C and the precipitate was filtered and washed with cold ethanol. The obtained potassium 2,4-dioxothiazolidin-3-ide (5 mmol) was refluxed with alkyl or benzyl halides (5.5 mmol) in DMF (15 mL) for 3−4 h. After cooling to room temperature and addition of water (40 mL), the crude product was extracted with ethyl acetate, washed with brine, and purified by flash chromatography (cyclohexane/ethyl acetate). 3-Methylthiazolidine-2,4-dione. Pale-yellow solid (89% yield). 1H NMR (300 MHz, CDCl3): δ = 3.12 (s, 3H), 3.95 (s, 2H) ppm. HRMS (ESI): m/z [M + Na]+ calcd for C4H5NNaO2S, 153.9933; found, 153.9943. 3-Butylthiazolidine-2,4-dione. Pale-yellow oil (71% yield). 1H NMR (300 MHz, CDCl3): δ = 0.93 (t, J = 7.3 Hz, 3H), 1.33 (m, 2H), 1.58 (m, 2H), 3.62 (m, 2H), 3.93 (s, 2H) ppm. HRMS (ESI): m/z [M + Na]+ calcd for C7H11NNaO2S, 196.0403; found, 196.0428. 3-(4-Methylbenzyl)thiazolidine-2,4-dione. Pale-yellow oil (88% yield). 1H NMR (300 MHz, CDCl3): δ = 2.32 (s, 3H), 3.92 (s, 2H), 4.73 (s, 2H), 7.13 (m, 2H), 7.30 (m, 2H) ppm. HRMS (ESI): m/z [M + H]+ calcd for C11H12NO2S, 222.0583; found, 222.0583. Procedure E: Synthesis of 3-(4-Methylphenyl)thiazolidine-2,4dione. The corresponding 3-(4-methylphenyl)rhodanine (2.5 mmol) was dissolved in ethyl acetate (25 ml), followed by the addition of sodium tungstate (0.25 mmol) and hydrogen peroxide (25 mL, 30% in water). After stirring at room temperature for 15 h, the organic layer was washed two times with brine and a solution of sodium sulfite. After removal of the solvent, the crude product was purified by flash chromatography (cyclohexane/ethyl acetate) to obtain a colorless solid (14% yield). 1H NMR (300 MHz, CDCl3): δ = 2.40 (s, 3H), 4.12 (s, 2H), 7.13 (m, 2H), 7.31 (m, 2H) ppm. HRMS (ESI): m/z [M + Na]+ calcd for C10H9NNaO2S, 230.0246; found, 230.0289. Procedure F: Synthesis of 3-Benzyl-2-thiohydantoin and 3-Phenyl2-thiohydantoin. A mixture of the corresponding benzyl or phenyl isothiocyanate (5 mmol), methyl glycinate hydrochloride (5 mmol), and triethylamine (5 mmol) in diethyl ether (20 mL) was stirred at room temperature for 15 h. The resulting precipitate was filtered, washed with diethyl ether, and used as crude product without further purification. 3-Phenyl-2-thioxoimidazolidin-4-one. Pale-pink solid (85% yield). 1 H NMR (300 MHz, CDCl3): δ = 4.29 (s, 2H), 7.30−7.33 (m, 2H), 7.42−7.54 (m, 3H) ppm. HRMS (ESI): m/z [M − H]− calcd for C9H7N2OS, 191.0285; found, 191.0267. 8400



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3-Benzyl-2-thioxoimidazolidin-4-one. Colorless solid (90% yield). H NMR (300 MHz, CDCl3): δ = 4.08 (s, 2H), 5.00 (s, 2H), 7.28−7.34 (m, 3H), 7.47−7.50 (m, 2H) ppm. HRMS (ESI): m/z [M − H]− calcd for C10H9N2OS, 205.0441; found, 205.0393. Procedure G: Synthesis of 5-Arylidenerhodanines, 5-Arylidenethiazolidine-2,4-diones, and 5-Arylidene-2-thiohydantoins with Exemplary Compound Data. A mixture of substituted rhodanine, thiazolidine-2,4-dione, or 2-thiohydantoin (1 mmol), ammonium acetate (2 mmol), and corresponding benzaldehyde (1.1 mmol) in acetic acid (8 mL) was heated in a microwave reactor at 180 °C for 5 min. The mixture was cooled for 30 min at 4 °C, and the resulting precipitate was filtered, washed with water, and dried by air flow. 4-[(3-Allyl-4-oxo-2-thioxothiazolidin-5-ylidene)methyl]benzoic Acid. Yellow solid (98% yield). 1H NMR (300 MHz, acetone-d6): δ = 4.75 (dt, J = 5.5, 1.5 Hz, 2H), 5.19−5.27 (m, 2H), 5.89 (m, 1H), 7.78 (m, 2H), 7.83 (s, 1H), 8.19 (m, 2H) ppm. HRMS (ESI): m/z [M − H]− calcd for C14H10NO3S2, 304.0108; found, 304.0121; Anal. Calcd for C14H11NO3S2: C, 55.06; H, 3.63; N, 4.59. Found: C, 54.78; H, 3.66; N, 4.53. 4-[(3-(4-Fluorobenzyl)-2,4-dioxothiazolidin-5-ylidene)methyl]benzoic Acid. Pale-yellow solid (76% yield). 1H NMR (300 MHz, acetone-d6): δ = 4.92 (s, 2H), 7.13 (m, 2H), 7.48 (m, 2H), 7.78 (m, 2H), 7.98 (s, 1H), 8.18 (m, 2H) ppm. HRMS (ESI): m/z [M − H]− calcd for C18H11FNO4S, 356.0398; found, 356.0125. 4-[(2-Oxo-1-phenyl-5-thioxoimidazolidin-4-ylidene)methyl]benzoic Acid. Yellow solid (38% yield). 1H NMR (300 MHz, acetoned6): δ = 6.76 (s, 1H), 7.44−7.57 (m, 5H), 7.91 (m, 2H), 8.10 (m, 2H) ppm. HRMS (ESI): m/z [M − H]− calcd for C17H11N2O3S, 323.0496; found, 323.0144. Procedure H: Synthesis of [3-(4-Methoxybenzyl)-4-oxo-2-thioxo1,3-thiazolidin-5-yl]acetic Acid. A suspension of 4-methoxybenzylamine (5 mmol), carbon disulfide (10 mmol), and fumaryl chloride (5.25 mmol) in water (15 mL) was stirred for 72 h at room temperature. Aqueous hydrochloric acid (1N, 5 mL) was added, and the precipitate was collected after filtration. After solvation in acetone the crude product was purified by flash chromatography (cyclohexane/ethyl acetate/1% acetic acid) to yield a pale-brown gum (26% yield). 1H NMR (300 MHz, acetone-d6): δ = 3.15 (dd, J = 17.9, 8.5 Hz, 1H), 3.30 (dd, J = 17.9, 3.8 Hz, 1H), 3.76 (s, 3H), 4.75 (dd, J = 8.5, 3.9 Hz, 1H), 5.02 (d, J = 14.4 Hz, 1H), 5.14 (d, J = 14.4 Hz, 1H), 6.83 (m, 2H), 7.33 (m, 2H) ppm. MS (EI, 70 eV): m/z (%), 311.0 (73) [M+]. Procedure I: Synthesis of 2-[3-(4-Methoxybenzyl)-4-oxo-2-thioxothiazolidin-5-ylidene]acetic Acid and Precursor. A suspension of 4methoxybenzylamine (2 mmol), carbon disulfide (10 mmol), and ditert-butyl acetylenedicarboxylate (2 mmol) in water (5 mL) and ethanol (5 mL) was vigorously stirred for 48 h. After separation of the solvent by decantation from the resulting oil, the crude product was dissolved in dichloromethane and purified by flash chromatography (cyclohexane/ ethyl acetate). The obtained precursor tert-butyl 2-[3-(4-methoxybenzyl)-4-oxo-2-thioxothiazolidin-5-ylidene]acetate (0.75 mmol) was stirred for 18 h with trifluoroacetic acid (1 mL) in dichloromethane (10 mL). After addition of acetone (15 mL), all solvents were evaporated to obtain the desired product without further purification. tert-Butyl 2-[3-(4-Methoxybenzyl)-4-oxo-2-thioxothiazolidin-5ylidene]acetate. Yellow oil (96% yield). 1H NMR (300 MHz, CDCl3): δ = 1.51 (s, 9H), 3.77 (s, 3H), 5.20 (s, 2H), 6.74 (s, 1H), 6.82 (m, 2H), 7.37 (m, 2H) ppm. MS (EI, 70 eV): m/z (%), 365.0 (65) [M+]. 2-[3-(4-Methoxybenzyl)-4-oxo-2-thioxothiazolidin-5-ylidene]acetic Acid. Orange solid (quant). 1H NMR (300 MHz, CDCl3): δ = 3.77 (s, 3H), 5.21 (s, 2H), 6.82 (m, 2H), 6.84 (s, 1H), 7.38 (m, 2H) ppm. MS (EI, 70 eV): m/z (%): 309.0 (71) [M+].

terminal capped peptide hybrids, HRMS and HPLC data for all compounds used in biochemical and biological evaluations, and experimental procedures and analytical data for all synthetic intermediates. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (+)49-6221-544875. Fax: (+)49-6221-546430. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Therese Scholz for performing LC-MS analytics, Michael Wacker for technical support, and Christian Jöst for useful hints in molecular modeling and visualization of the results. Christoph Nitsche appreciates financial support by a fellowship of the Studienstiftung des deutschen Volkes. Mira Behnam appreciates financial support from the German Academic Exchange Service. Work of R.B. is supported by a grant of the European Commission for Research and Innovation (FP7 HEALTH-2010 Collaborative Project SILVER; contract 260644).

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ABBREVIATIONS USED DEN, dengue virus; DIPEA, diisopropylethylamine; DMEM, Dulbecco’s Modified Eagle’s Medium; EGTA, ethylene glycolbis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; HBTU, O(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; MW, microwave; PAMPA, parallel artificial membrane permeability assay; RFU, relative fluorescence units; THR, thrombin; WNV, West Nile virus

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The results for the aprotinin competition assay, the inhibitory activity of selected cap molecules against the DEN and WNV proteases and thrombin, and the results and experimental description of the renilla and firefly luciferase interference experiments. A detailed procedure for the synthesis of the N8401



Article

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