2-(3,4-Dihydro-4-Oxothieno[2,3-d]pyrimidin-2-ylthio) Acetamides as a New Class of Falcipain-2 Inhibitors. 3. Design, Synthesis and Biological Evaluation

The cysteine protease falcipain-2 (FP-2) of Plasmodium falciparum is a principal cysteine protease and an essential hemoglobinase of erythrocytic P. falciparum trophozoites, making it become an attractive target enzyme for developing anti-malarial drugs. In this study, a series of novel small molecule FP-2 inhibitors have been designed and synthesized based on compound 1, which was identified by using structure-based virtual screening in conjunction with an enzyme inhibition assay. All compounds showed high inhibitory effect against FP-2 with IC50s of 1.46-11.38 μM, and the inhibitory activity of compound 2a was ~2 times greater than that of prototype compound 1. The preliminary SARs are summarized and should be helpful for future inhibitor design, and the novel scaffold presented here, with its potent inhibitory activity against FP-2, also has potential application in discovery of new anti-malarial drugs.


Introduction
Malaria remains one of the most important infectious disease problems in the world, accounting for 300-500 million clinical cases and up to 2.7 million deaths each year. About 90% of these casualties occur in tropical Africa, and the great majority are children under the age of 5 [1]. Plasmodium falciparum, one of the four species of Plasmodium, is the most lethal protozoan parasite of the genus, which is responsible for malaria. At present no effective vaccines are available due to the high mutability of the genome of Plasmodium falciparum [2], meanwhile, resistance of malaria parasites to available conventional drug therapy is an increasingly serious problem [3][4][5]. Accordingly, the discovery of new effective drugs to counter the spread of malaria parasites that are resistant to existing agents, especially acting on new targets, is an urgent need.
Recently, crystal structures for falcipain-2 have been reported [23][24], and the reservoir of structural and functional information of FP-2 has offered a solid starting point for the rational structure-based design of novel antimalarial drugs targeting FP-2. By using a docking-based virtual screening approach in conjunction with an enzyme inhibition assay, a novel small molecule inhibitor of FP-2 featuring the 2-(3,4-dihydro-4-oxothieno [2,3-d]pyrimidin-2-ylthio) acetamide framework (compound 1) has been discovered [25]. After the identification of compound 1 as a possible prototype for the design of selective inhibitors of FP-2, fifteen new compounds, including 1 and its fourteen analogs (2a-e, 3a-c, and 4a-f), have been synthesized and tested against FP-2. All of the compounds show high inhibitory effect against FP-2 with IC 50 s of 1.46-11.38 μM, and inhibitory activity of compound 2a increases ~2 times than that of prototype compound 1. The preliminary SARs are summarized and should be helpful for future design of inhibitors.

Identification of Prototype (Hit) 1 by Virtual Screening
Targeting the crystal structure of falcipain-2 (PDB entry 2GHU) [23], a total of 80,000 compounds were subsequently docked and ranked according to the software Glide and GAsDock [25]. Finally, 81 compounds were purchased and submitted to biological evaluations against falcipain-2. Among the 81 compounds, the inhibitory activity of compound 1 was concentration-dependent. The collected data indicated that compound 1 can inhibit FP-2 in vitro, with the inhibitory effect against FP-2 is in the micromolar range (IC 50 = 2.81 μM). The substrate (L-3-trans-carboxyoxiran-2-carbonyl)-L-leucylagmatin (E-64), whose inhibitory effect against FP-2 is in the subnanomolar range (IC 50 = 18.1 nM), was used for the positive control. Compound 1 could be designated as a hit of FP-2. The inhibitory rates (%) off compounds 1 and E-64 plotted against the common logarithm of the compound concentrations are shown in Figures 1a and 1b, respectively. On the basis of the framework of compound 1, fourteen compounds (2a-e, 3a-c, and 4a-f) were designed and synthesized. Their chemical structures are shown in Table 1. Retaining the common 2-(3,4-dihydro-4-oxothieno[2,3-d]pyrimidin-2-ylthio) acetamide framework of compound 1, we first changed the allyl group on the pyrimidine ring into cyclohexyl, aryl, and benzyl groups, and obtained analogs 2a-e (Table 1). We then replaced the phenyl group on the thiophene ring with cyclohexyl or substituted phenyl groups to prepare compounds 3a-c (Table 1). Finally, compounds 4a-f (Table 1) were achieved by replacing the amide N-substituents with other aryl groups.
Compounds 1-4 were synthesized through the route outlined in Scheme 1. Using our previous method [26], using basic aluminium oxide as solid support and morpholine as base catalyst, ketones, cyanoacetates, and sulphur were mixed and subjected to microwave irradiation for several minutes, which afforded 2-aminothiophenes 5a-d. Compounds 5a-d were converted to the corresponding thioureas 6a-i by reaction with isothiocyanates, then 6a-i were cyclized under alkaline conditions, giving the key intermediates 7a-i (Scheme 1). Various aromatic amines were chloracetylated with chloroacetic chloride to give compounds 8a-g, which were reacted with compounds 7a-i to produce the target compounds 1-4 smoothly. The details of the synthetic procedures and product characterizations are described in the Experimental section.

Enzyme Inhibition Assay
For the primary assay, the percent inhibitions of the compounds at 10 μM were measured. The results are listed in Table 1. All compounds can remarkably inhibit the activity of FP-2 (Percent inhibition at 10 μM > 50%), indicating that these fifteen compounds (including compound 1) are FP-2 inhibitors. Therefore, we determined their IC 50 values (Table 1). From the data in Table 1, we can see that the percent inhibitory rates at 10 μM of compounds 2a, 2e, 4a-d, and 4f obviously increased (more than 90%, Table 1). Among five anologs varied on R 3 substituents (2a-2e, Table 1), it is remarkable that the inhibitory activity of compound 2a increased ~2 times than that of compound 1 (IC 50 decreases from 2.81 μM down to 1.46 μM, Table 1), the former replace the allyl group on pyrimidine ring with a cyclohexyl group. The percent inhibitory rates at 10 μM of analogs 3a-c, which vary on R 1 and R 2 substituents, were decreased in different extent, especially analogue 3c which showed a marked decrease from 88.7% to 53.0% (Table 1). Changes of R 4 substituents did not have a distinct improvement for the IC 50 s of analogs 4a-f (Table 1).

Scheme 1 a
The synthetic route to compounds 1-4.

Structure and Activity Relationships
After testing the inhibitory activities of compounds 1, 2a-e, 3a-c, and 4a-f against FP-2, from the data in Table 1, we can deduce the preliminary structure-activity relationships (SARs) as follows: (1) Replacing the allyl group of compound 1 with proper substituent, such as a cyclohexyl (2a) group, moderately improves potency, as seen with the IC 50 of compound 2a which is decreased to 1.46 μM (Table 1); (2) Replacing the phenyl group on the thiophene ring of 1 with cyclohexyl (3a) or substituted phenyl (3b-3c) can not be tolerated and leads to the loss of inhibitory activity; (3) Nsubstituents of amide of compound 1 have considerable adaptability and alterability. This encouraging result and primary SARs are helpful for future inhibitors design.

Conclusions
In this study, we have discovered a new kind of scaffold of FP-2 inhibitor by using a structurebased virtual screening approach in conjunction with chemical synthesis and bioassay. The preliminary SARs were obtained, which show changes of substituents on 3, 5, and 6-positions of thieno [2,3d]pyrimidine moiety have very important influence on inhibitory activity, and appropriate structural optimizations on the above regions can substantially improve potency. These primary SARs are helpful for future inhibitors design, and the novel scaffold presented here with potent inhibitory activity against FP-2 also provides potential application in discovery of anti-malarial drugs.

Experimental
Reagents were purchased from Alfa, Acros and Shanghai Chemical Reagent Company, and used without further purification. Analytical thin-layer chromatography (TLC) was performed on HSGF 254 plates (150-200 µm thickness, Yantai Huiyou Company, P.R. China). Yields were not optimized. Melting points were measured in capillary tube on a SGW X-4 melting point apparatus without correction. Nuclear magnetic resonance (NMR) spectra were given on a Bruker AVANCE 500 NMR (TMS as internal standard). Chemical shifts were reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). Low-and high-resolution mass spectra (LRMS and HRMS) were given with electrospray (ESI) produced by LCQ-TOF spectrometer. Microwave experiments were carried out in a domestic microwave oven (Haier MA-2270EGC)

Virtual Screening by Molecular Docking
The crystal structure of falcipain-2 (PDB entry 2GHU) [23] from Plasmodium falciparum was retrieved from the Protein Database Bank [27]. Residues located within 14 Å from the catalytic thiolate of Cys42 were defined as part of the binding site for docking studies. All crystallographic water molecules were removed from the coordinate set. The pipeline of virtual screening based on molecular docking method is presented in detail in the reference 25.

Enzyme Inhibition Assay
The purification and refolding of recombinant protein Falcipain-2 was performed as described by Shenai et al [6]. IC 50 values against recombinant Falcipain-2 were determined as described previously [8,28]. FP-2 (30 nM) was incubated for 30 min at room temperature in 100 mM sodium acetate, pH 5.5, 10 mM DTT, with different concentrations of tested inhibitors. Inhibitor solutions were prepared from stock in DMSO (maximum concentration of DMSO in the assay was 1%). After 30 min incubation, the substrate Z-Leu-Arg-AMC (benzoxycarbonyl-Leu-Arg-7-amino-4-methylcoumarin, Bachem AG) in the same buffer was added to a final concentration of 25 μM. The increase in fluorescence (excitation at 355 nM and emission at 460 nM) was monitored for 30 min at room temperature with an automated microtiter plate spectrofluorimeter (Molecular Devices, Flex station).