Novel Selective and Potent Inhibitors of Malaria Parasite

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Novel Selective and Potent Inhibitors of Malaria Parasite Dihydroorotate Dehydrogenase: Discovery and Optimization of Dihydrothiophenone Derivatives Minghao Xu, Junsheng Zhu, Yanyan Diao, Hongchang Zhou, Xiaoli Ren, Deheng Sun, Jin Huang, Dongmei Han, zhenjiang zhao, Lili Zhu, Yufang Xu, and Honglin Li J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 27 Sep 2013 Downloaded from http://pubs.acs.org on September 29, 2013

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Journal of Medicinal Chemistry

Novel Selective and Potent Inhibitors of Malaria Parasite Dihydroorotate Dehydrogenase: Discovery and Optimization of Dihydrothiophenone Derivatives

Minghao Xu1,2,†, Junsheng Zhu1,†, Yanyan Diao1,†, Hongchang Zhou1,3, Xiaoli Ren1, Deheng Sun1, Jin Huang1, Dongmei Han4, Zhenjiang Zhao1, Lili Zhu1,*, Yufang Xu1,2,*, Honglin Li1,*

1

Shanghai Key Laboratory of New Drug Design, State Key Laboratory of Bioreactor

Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China. 2

Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China

University of Science and Technology, Shanghai 200237, China. 3

Department of Microbiology, Medical School of Huzhou Teachers College, Huzhou

313000, China. 4

Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai 200240,

China

†

Authors contributed equally to this work

* To whom correspondence should be addressed. Email: [email protected], [email protected], [email protected]

ACS Paragon Plus Environment


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Please address correspondence and requests for reprints to: Prof. Honglin Li School of Pharmacy, East China University of Science and Technology 130 Mei Long Road, Shanghai 200237 Phone/Fax: +86-21-64250213


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Abstract. Taking the emergence of drug resistance and lack of effective anti-malarial vaccines into consideration, it’s of significant importance to develop novel antimalarial agents for the treatment of malaria. Herein, we elucidated the discovery and structure-activity relationships of a series of dihydrothiophenone derivatives as novel specific inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH). The most promising compound 50 selectively inhibited PfDHODH (IC50 = 6 nM, with >14000-fold species-selectivity over hDHODH) and parasite growth in vitro (IC50s = 15 nM and 18 nM against 3D7 cells and Dd2 cells, respectively). Moreover, an oral bioavailability of 40% for compound 50 was determined from in vivo pharmacokinetic studies. These results further indicate that PfDHODH is an effective target for antimalarial chemotherapy and the novel scaffolds reported in this work might lead to the discovery of new antimalarial agents.

Keywords:

Malaria,

Plasmodium

falciparum

dihydroorotate

(PfDHODH), structure-activity relationship (SAR), virtual screening


dehydrogenase


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Introduction Malaria is a global parasitic infectious disease caused by Plasmodium parasites, among which Plasmodium falciparum is the most dangerous one with the highest rates of complications and mortality.1 It has been estimated that there were 216 million episodes of malaria and 655,000 people died from this disease in 2011,1 mostly African children under the age of five. On one hand, treatment of malaria only relies on chemotherapy owing to the absence of effective vaccines,2, 3 on the other hand, the widespread drug-resistance renders many traditional anti-malarial drugs ineffective.4-6 For instance, chloroquine (compound 1),7 previously one of the most commonly used anti-malarial drugs,8 has been withdrawn in many regions as resistant parasite strains have evolved.9 Moreover, the occurrence of “artemisinin (compound 2) resistant” Plasmodium falciparum malaria in western Cambodia and Thailand is a real threat to artemisinin-based therapies.10-13 Other anti-malarial agent such as pyrimethamine (compound 3),5 a typical malarial dihydrofolate reductase-thymidylate synthase (DHFR-TS) inhibitor,14 has been nagged by the problem of gene mutation and inefficacy for a long time.15 Under the circumstance of a current lack of efficient anti-malarial drugs,16 there is an emergency to identify novel parasites biochemical process to fill the research pipeline and develop new, non-cross-resistant, anti-malarial agents.17, 18, 19

Pyrimidine bases biosynthesis represents a basic biological and physiological process which is crucial for RNA and DNA production and cell proliferation.20, 21 In


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most organisms, dihydroorotate dehydrogenase (DHODH) catalyzes the fourth and rate-limiting step in the de novo pyrimidine biosynthesis,22 and it converts dihydroorotate (DHO) to orotate (ORO).22, 23 DHODHs are divided into two families based on different amino acid sequence and coenzyme dependence. Both human and P. falciparum DHODHs belong to family II that are located on the inner mitochondrial membrane and use ubiquinone as the terminal oxidant.23 Unlike human cells which could acquire pyrimidine bases from salvage pathway,20 Plasmodium falciparum relies entirely on its de novo pyrimidine biosynthetic pathway for survival21. Therefore, P. falciparum dihydroorotate dehydrogenase (PfDHDOH) has been regarded as an attractive target for the treatment of malaria.4, 24, 25

Although PfDHODH and hDHODH share highly conserved sequence in their C-terminal domains, there is huge variation of the amino acid sequence in the putative ubiquinone-binding site formed by two α-helices in the N-terminal domain, which contributes to further identification of species-specific DHODH inhibitors.23 Many hDHODH inhibitors have been developed and studied for their use against a number of anti-proliferative and anti-inflammatory diseases such as rheumatoid arthritis,26, 27 psoriasis, and lupus erythematosus.28 However, in the past decade, some success in identifying PfDHODH inhibitors has been reported based on the modification of known human DHODH inhibitors (e.g. compound 4).29 Most notably, in the past decade, a number of potent and selective PfDHODH inhibitors from different chemical scaffolds have been discovered through enzyme-based high-throughput



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screening (e.g. compounds 5, 6 and 7 (DSM1)).30-32 Co-crystal structures of PfDHODH in complex with representative compounds from these series, 732, 33 and 8 (Genz-667348)34 were subsequently solved by Phillips et al.35 Lead optimization of the triazolopyrimidine series led to the identification of 9 (DSM190),36 which was shown to have good efficacy in a P. berghei mouse model and finally to facilitate the discovery of 10 (DSM265),25 which is currently in clinical development (MMV.org).25 (Figure 1)

Figure 1

In our previous work, we reported the discovery of diverse hDHODH inhibitors by structure-based virtual screening method.37 To find novel PfDHODH inhibitors, the same virtual screening strategy integrating molecular Glide docking with Prime/MM-GBSA rescoring was implemented based on the co-crystal structure of PfDHODH complexed with compound 7 (PDB code 3I65), and then the lead compound 11 (IC50 = 1.11 µM) was identified from SPECS database (ID: AG-690/40639878).

After

subsequent

structural

optimization,

a

series

of

dihydrothiophenone derivatives with more potent inhibitory effects against PfDHODH were obtained in this study, which showed an overall selectivity over hDHODH. Additionally, most of the hits, which had been demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, were highly correlated with enzyme inhibitory activity, suggesting their potential applications as novel


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anti-malarial agents.

Binding Modes Predicted by Molecular Docking and Optimization Strategy of Lead Compound 11：：

In the predicted binding pose of the lead compound 11 (Figure 2), the chlorine-substituted phenyl group binds deeply into the hydrophobic pocket of which is close to the entrance of the channel-like ubiquinone-binding site, making favorable hydrophobic effects and van der Waals (VDW) interactions with residues Leu197, Ile237, Leu240, and Met536. An edge-to-face π-π interaction is probably present between Phe227 and the phenyl ring of 11, which could help to enhance the potency of the lead compound. The dihydrothiophenone moiety occupies the hydrophilic section of the ubiquinone-binding site, and could form hydrogen bonds with residues Arg265 and the water molecule (W15) reserved from crystal structure in the molecular docking process, and the bridging nitrogen atom forms an additional hydrogen bond with His185. Moreover, it is noteworthy that the H-bonding between NH and His 185 was a common feature for the binding and contributed to the selectivity of PfDHODH inhibitors over hDHODH.38 Figure 2

Based on the proposed binding mode described above, our lead compound could be divided into two functional parts, the aromatic section as a “Hydrophobic Group” and



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the dihydrothiophenone section as a hydrogen bond-forming “Hydrophilic Group”. In the hydrophobic pocket of subsite 1,27 there is still some space between the chlorine atom and the residues around to accommodate larger substitution. In the hydrophilic pocket composed by subsites 2 and 3,27 the functional groups (carbonyl groups) which could form hydrogen bonds with polar residues or resident waters should be reserved to maintain the inhibitory activity against PfDHODH. Accordingly, to identify more potent PfDHODH inhibitors for further pharmacological study as well as verify the structure-activity relationship in the absence of crystal structure, the lead optimization was carried out through three modifications: 1) hydrophobic modification on the aromatic ring to improve hydrophobic effects, 2) hydrophilic modification on the dihydrothiophenone ring and 3) scaffold hopping of the dihydrothiophenone core (as shown in Figure 3). Figure 3

Chemistry:

Aromatic amine Mimics

Scheme 1

Firstly, to discover some proper hydrophobic moieties, the target inhibitors 11-28 were obtained via a three-step procedure (Scheme 1). In our design, the preparation of dihydrothiophenone analogues started with commercially available amines and aryl


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isothiocyanates served as the key intermediates. Triphosgene was used instead to avoid poisonous and environmentally hazardous reagents such as thiophosgene and phosgene. After several attempts, electron-withdrawing aryl isothiocyanates which were hard to acquire were finally synthesized and then involved in the condensation with ethyl 4-chloroacetoacetate to afford desired products.39

Formate Mimics

Scheme 2

Next we focused on synthesizing various formates. Extensive efforts had been made to control the hydrolysis of these substrates to corresponding acids (30-31) (Scheme 2). To avoid by-products, temperature must be kept below 60 oC and the concentration of the base must be controlled accurately. Moreover, we synthesized 29 to explore the role of the ethoxycarbonyl group in the scaffold.40 At last, after a purification by chromatography over silica gel, the desired products (32-35, 42-44) were re-crystallized to ensure >95% purity.41

Formamide Mimics

Scheme 3 To further study the structure-activity relationships of dihydrothiophenone derivatives and improve their pharmacological properties, target amides (36-41, 45-49) were designed and prepared. The intermediates (30-31) were made using the same



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method described above and the amides were synthesized in the presence of HOBt, EDC and DIPEA which served as condensating agents and base (Scheme 3).

Scaffold Hopping

Scheme 4 The general synthetic scheme for preparation of dihydrofuranone derivatives (50-56) is shown in Scheme 4.42 In these cases, the treatment of diethyl malonate with 2-chloroacetyl

chloride

provided

the

intermediate

ethyl

2-ethoxy-4-oxo-4,5-dihydrofuran-3-carboxylate which could further react with aromatic amines to generate target molecules. All structures of the final products were determined by 1H NMR, 13C NMR and HRMS (ESI).

Results and Discussion：：

Table 1

Hydrophobic Modification on Aromatic Moiety

In the first stage of optimization, we focused on the modification of the aromatic hydrophobic moiety with different substituents on the phenyl ring (Table 1). As exemplified by compound 12, the removal of chlorine from the phenyl ring resulted in a complete loss of activity, which implied that the larger the group occupying the hydrophobic cavity, the better its potency is, i.e. the inhibition potency against


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PfDHODH could be achieved by substitution with larger hydrophobic groups to improve hydrophobic effects. A similar improvement in potency was observed for those with large alkyl group at para-position of the phenyl ring (compounds 13-15), compound 15 was the most active one against PfDHODH with an IC50 value of 0.23 µM. For these compounds containing para-substituted groups on the phenyl ring, the improved activity correlated well with increased hydrophobicity (-C(CH3)3 > -CF3 > -Cl), verifying our previous optimization strategy.

Compared with compound 11, 16 with the introduction of trifluoromethyl group, exhibited slightly improved activity. Compound 17, which was similar to 16, retained comparative

potency

against

PfDHODH.

Compound

18

containing

meta-trifluomethyl substituted phenyl moiety showed huge loss of activity (13 vs 18), which may result from the absent effects of hydrophobicity in the para-position. For compound 19, which had two chlorine atoms in the meta-positions of the phenyl ring, strong PfDHODH inhibition was observed with an IC50 value of 1.19 µM. Substitution with halogen or methyl at the meta- and para-positions yielded more potent compounds 20 and 21, which displayed 4- and 2-fold better potency against PfDHODH compared to lead compound 11, respectively. Some small substituents, such as methyl, fluorine or trifluoromethyl, at the meta-position of the phenyl ring might have some influence on potency improvement. However, no activity against PfDHODH was observed for compound 22, in which methyl was alternatively introduced into the ortho-position. The results summarized above indicated that the



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size and position of substituents on the phenyl ring significantly affected the in vitro inhibitory potency against PfDHODH: a) a hydrophobic substituent at para-position is preferential and a larger alkyl group is well tolerated, b) more attention should be paid to the size of meta-substituent since halogens or small alkyl groups are suitable, c) substituents at ortho-position should be avoided.

Since the PfDHODH inhibition potency was particularly sensitive to substituent pattern of the phenyl ring, an alternative optimization strategy was carried out by the introduction of cyclic systems with strong hydrophobicity and conformational flexibility as the “hydrophobic group”. Thus, we next developed compounds 23-28. Consistent with the hydrophobic nature of the interactions in this area,36 the 2-naphthyl containing compound 25 showed greater fold improvement in potency against PfDHODH with the IC50 value of 0.02 µM, which turned out to be the most potent inhibitor of this series with up to 56-fold improvement in potency relative to the lead compound 11. A demonstration that hydrophilic group/polar functionality was relatively less acceptable at the entrance of the binding site was evidenced by the significantly reduced activity of 24 compared to 25 (IC50 = 0.35 vs 0.02 µM). Notably, all these compounds still retained the inhibitory activity against PfDHODH and were inactive against hDHODH except for 23. It was also noteworthy that the replacement of 5,6,7,8-tetrahydro-2-naphthyl (compound 23) with similar but smaller 5-indanyl (compound 26) would reduce the activity by nearly 10-fold. This significant variety might be caused by the slight difference of the size and shape of the substituent.25


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Larger substituents such as anthracene (27, IC50 = 0.20 µM) and carbazole (28, IC50 = 0.39 µM) were also well tolerated.

Hydrophilic Group Modification on the Dihydrothiophenone Scaffold with Acids, Esters and Amides

On the basis of previously established proper hydrophobic groups (R1 = 3,4-di-CH3-Ph and 2-naphthyl), compounds 29-49 were achieved to explore SAR in the hydrophilic region. As illustrated in the predicted binding mode of 11, the ester group (-COOC2H5) was pointed toward a small deep hydrophobic cavity formed by residues Ile263 and Ile272, and featured hydrogen bonds formed with Tyr528 at the same time. Therefore, the ethoxycarbonyl moiety could interact with PfDHODH via hydrogen bond and hydrophobic contacts simultaneously. In this section, emphasis was laid on the influence of structural variation of the ethoxycarbonyl group on the inhibitory activity against PfDHODH.

The inhibitor without ethoxycarbonyl group on the dihydrothiophenone scaffold (29) displayed poor enzyme inhibitory activity in comparison with 20, which could be concluded that the derivatives without any substituent at 3-position of the scaffold would be strongly disfavored. The addition of carboxyl group to the 3-position on the dihydrothiophenone ring did not make much improvement, as no activity against PfDHODH was observed for compound 30. Although compound 31 showed an IC50



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value of 1.24 µM, the inhibitory activity against PfDHODH was 6-fold lower compared to that of ethoxycarbonyl-substituted derivative 25. The greatly reduced activity was possibly due to the absence of hydrophobic interactions with the small pocket formed by residues Ile263 and Ile272. Next some derivatives with different alkoxycarbonyl groups (32-34 and 42-44) were synthesized to identify the optimal alkyl chain which could fit the small cavity. Notably, this series of derivatives did not display improved potency compared to the corresponding ethoxycarbonyl analogues 20 and 25. On one hand, the decrease of PfDHODH IC50 values observed for 32 and 42 (IC50 = 1.49 µM and 0.033 µM, respectively) should arise from the reduced hydrophobic interaction caused by the shorter length of methoxycarbonyl group. On the other hand, the reduced binding affinity of 33, 34, 43 and 44 (IC50 = 4.03 µM, 2.35 µM, 0.95 µM and 35.57 µM, respectively) was probably resulted from the size constraint of the cavity when increasing chain length. Likewise, the bulkier such as 35 (IC50 = 4.16 µM), could not be well accommodated by the hydrophobic cavity neither.

Formamide derivatives 36-41 and 45-49 were also evaluated. Interestingly, this series of compounds were collectively much less potent in the PfDHODH enzyme activity assay than corresponding ester derivatives. Two representative compounds, 37 and 45, replaced the ester group (20 and 25) with amide group, displayed up to 160and 600-fold decrease in potency against PfDHODH respectively. The pronounced difference in activity against PfDHODH probably resulted from the intramolecular hydrogen-bond in these amide derivatives, the carbonyl group engaging in


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intramolecular NH-C=O interactions with adjacent amide group, which formed additional hydrogen bond with Arg265 of PfDHODH. Meanwhile, the change of substituents led to some selectivity changes against hDHODH (38-41).35, 38 Overall, these amide derivatives seemed not to be appropriate PfDHODH inhibitors.

Scaffold

Hopping

and

Replacement

of

Sulfur with

Oxygen

on

the

Dihydrothiophenone Skeleton

After discussing the SAR through hydrophobic group modification and hydrophilic group modification, the role of sulfur on the skeleton still remained ambiguous even by the aid of molecular docking simulation.43 Replacement of sulfur with oxygen on the dihydrothiophenone skeleton gave compound 50, which had an IC50 value as low as 6 nM, 3-fold more active than matched sulfur derivative 25 and 185-fold than the lead compound 11. To further verify the significance of oxygen supplement, more dihydrofuranone

derivatives

possessing

different

hydrophobic

groups

were

synthesized with R2 fixed to ethoxycarbonyl group. As displayed in Table 1, an increased trend in activities against PfDHODH was found for compounds 51-56 relative

to

corresponding

dihydrothiophenone

analogues.

Notably,

the

dihydrofuranone scaffold made compound 54 approximately 13-fold more potent than its matched dihydrothiophenone derivative 26. Meanwhile, this series of derivatives remained the high species-selectivity over hDHODH, which indicated that our scaffold hopping was quite successful and the novel dihydrofuranone scaffold, could



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be further investigated to discovery more PfDHODH inhibitors.

In Vitro Evaluation of Compound Activity against Pf3D7 and PfDd2 cells

To further evaluate their antiplasmodial activities, these dihydrothiophenone derivatives were tested against CQ-sensitive Pf3D7 and CQ-resistant PfDd2 strains in vitro assay. It was found that the inhibition potencies of these compounds against PfDHODH were maintained in Pf3D7 and PfDd2 cells (Table 1). For hydrophobic group modification, previous conclusion that para-substituted large hydrophobic group on the phenyl ring is preferential could also be evidenced by the comparison of 13 and 18 in the cell line. “Naphthyl-like” derivatives such as 23 and 25 showed good potency against Pf3D7 strains and PfDd2 strains as well (23, Pf3D7 IC50 = 0.17 µM, PfDd2 IC50 = 0.31 µM; 25, Pf3D7 IC50 = 0.077 µM, PfDd2 IC50 = 0.057 µM). Moreover, compounds with hydrophilic group modification were still less effective in Pf3D7 cells and PfDd2 cells and these results also proved that the ethoxycarbonyl group is essential for the inhibitory activity against PfDHODH. The trend of cell inhibitory activity of 42, 43 and 44 was consistent with the enzyme inhibition activity (Figure 4). Amide derivatives such as 37-41, 45-49 also showed IC50 values in micromolar range. To our satisfaction, scaffold hopping was successful which could be evidenced by the great cell potency demonstrated by 50 and 53 in two kinds of Pf cells. Other dihydrofuranone derivatives such as 52, 54, 55, 56 with attractive activity could also be optimized to find more effective PfDHODH inhibitors.


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Figure 4 The IC50 values of these compounds in both cell lines (Table 1) and linear regession analysis (Figure 4) showed an excellent correlation between inhibitory activity against PfDHODH and anti-malarial potency in Pf3D7 cells and PfDd2 cells (For Pf3D7 cells, slope = 1.256 and r2 = 0.750; For PfDd2 cells, slope = 1.347 and r2 = 0.746). Novel inhibitors described in Table 1 displayed promising activity in both cells with IC50 values in micromolar range either. Meanwhile, consistent with the previous inhibitory activity against PfDHODH (Table 1), compounds which showed poor activity on PfDHODH also exhibited less inhibitory potency against Pf3D7 cells and PfDd2 cells. On the basis of above data and analysis, it could be demonstrated that cell killing by these inhibitors results from the inhibition of PfDHODH, which confirmed PfDHODH was an effective target for antimalarial chemotherapy.

Table 2

In Vivo Pharmacokinetic Studies of compounds 25 and 50 in Sprague-Dawley Rats

Pharmacokinetic studies with potent and selected PfDHODH inhibitors (25 and 50) were carried out in rats at intravenous dose of 1 mg/kg and oral dose of 10 mg/kg (Table 2). After IV dosing, 25 exhibited high clearance of 129 mL/min/kg and short half-life (0.3 h), whereas 50 showed a little lower clearance of 84 mL/min/kg and



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half-life of 0.26 h. Neither compounds had high volume of distribution (3.35 and 1.91 L/kg, respectively). After oral administration, 25 showed very low exposure (AUC0-last) of 39 ng/mL*hr and maximum plasma concentration (Cmax) of 25 ng/mL, giving rise to an oral bioavailability of only 3%. However, 50 displayed pharmacokinetic properties with systemic exposure (AUC0-last) of 790 ng/mL*hr and maximum plasma concentration (Cmax) of 608 ng/mL, which lead to an oral bioavailability of 40%. The half-lives (T1/2) and the time to reach the maximum concentration (Tmax) for both inhibitors were short. The obvious individual difference in overall exposure and pharmacokinetic parameters of 25 and 50 indicated that these compounds might be instable. In contrast with 25, 50 showed higher bioavailability and better pharmacokinetic properties (AUC, Cmax, Clearance) which strongly suggested that sulfur atom in the core was a soft spot and scaffold hopping on the dihydrothiophenone skeleton could improve the stability of the compound.

Conclusion

In summary, a novel series of dihydrothiophenone derivatives as Plasmodium falciparum dihydroorotate dehydrogenase inhibitors were identified through virtual screening, and subsequent structural optimization and SAR analysis led to dozens of novel PfDHODH-specific inhibitors that achieved excellent inhibitory potency. Compounds in this series with three preferential structural fragments were regarded as


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potent PfDHODH inhibitors: 1) para-substituted larger alkyl group on the phenyl ring or bicyclic systems such as “naphthyl-like” substituents as the hydrophobic group, 2) ethoxycarbonyl

group

at

3-position

on

the

dihydrothiophenone

ring

or

dihydrofuranone ring as the hydrophilic group, 3) dihydrofuranone ring was more potent skeleton than dihydrothiophenone ring. The potency of compounds described in this study was particularly encouraging for the development of anti-malarial agents to conquer the widespread drug-resistance. The most potent inhibitors 25, 50 and 53 showed excellent inhibitory activity against PfDHODH (with IC50 values 20 nM, 6 nM, 18 nM) and high species-selectivity over hDHODH. Moreover, the high inhibition potencies of these PfDHODH inhibitors were maintained in the low double-digit nanomolar range against Pf3D7 and PfDd2 cells strains in vitro assay. Furthermore, excellent correlation between inhibitory activity against PfDHODH and anti-malarial potency in Pf3D7 cells and PfDd2 cells were observed for the dihydrothiophenone derivatives and dihydrofuranone derivatives. These results further suggested that PfDHODH is an effective target for antimalarial chemotherapy and novel scaffolds found in this work might lead to the discovery of new antimalarial agents. Study on the crystal structure of the PfDHODH inhibitors complex and efforts to improve pharmacokinetic properties are underway to further understand SAR and make them “druggable”.



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Experimental Section

Materials:

A771726 and chloroquine (CQ) were obtained from J&K and Sigma-Aldrich. Albumax II, SYBR Green I and 1640 incomplete medium were acquired from Invitrogen. DSM1 was synthesized in-house and at least 97% pure. Materials for enzyme and cells assay which were not listed out were purchased from Sigma.


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DHODH inhibition assays. Plasmid construction, protein expression and purification of PfDHODH (Phe158-Ser569) and hDHODH (Met30-Arg396) were performed following the protocols of Liu Shengpin et al44 and Xiaoyi Deng et al35 respectively. The enzyme was diluted into a final concentration of 10 nM with assay buffer containing 50 mM HEPES (pH 8.0), 150 mM KCl, 0.1% Triton X-100 supplemented with 100 µM UQ0, 120 µM DCIP. The mixture was transferred into a 96-well plate and incubated for 5 min at room temperature, and then the dihydroorotate was added to a final concentration of 500 µM to initiate the reaction. The reaction was monitored by measuring the decrease of DCIP in the absorption at 600 nm for each 30 s over a period of 6 min. Inhibition studies were performed in this assay with additional various amounts of inhibitors. A771726 and DSM1 were measured as the positive control for hDHODH and PfDHODH respectively. Percent inhibition relative to no inhibitor control was calculated from (1-Vi/V0) ×100. For the determination of the IC50 value of the inhibitor, 8-9 different concentrations were applied. Each inhibitor concentration point was tested in triplicate and the IC50 values were calculated using the sigmoidal fitting option of the program Origin 8.0.



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In Vitro Plasmodium falciparum whole cell assays. CQ-sensitive strain 3D7 and CQ-resistant strain Dd2 of Plasmodium falciparum were used in vitro blood stage culture to test the antimalarial efficacy. The cultures were maintained according to the method described by Trager and Jensen45 in a medium containing 0.5% Albumax II instead of human serum. Stock solution of CQ was prepared in water (milli-Q grade) with the concentration of 50 µg/mL and stored at -20 oC. The test compounds stock solution was prepared in DMSO with the concentration of 10 µmol/mL and stored at room temperature. All of the stock solution was diluted with 1640 incomplete medium to achieve the required concentration during the experiment. (In all cases except CQ, the highest final concentration of DMSO was 0.2%, which was found to be non-toxic to the parasite). The antiplasmodial activity of the compounds was determined by a fluorometric method using SYBR Green I. In brief, asynchronous parasites were plated in triplicate at 2% hematocrit and 1% parasitemia in 100 µL with the absence or presence of a series of concentration ranging from 0.15625 to 20 µM of each compound by serial dilution. A series of concentration of CQ were used as the positive control and 0.2% DMSO was used as the negative control. Plates were incubated at 37 o

C for 72 h. After that, the supernate was removed and 100 µL of SYBR Green I in

lysis buffer (8.26 g/L NH4Cl, 1 g/L KHCO3, 0.037 g/L EDTA and 5×SYBR Green I) was added to each well. Plates were covered and incubated in the dark at room temperature for 1 h. Plates were read at 485/520 nm on a Synergy MX, Biotek fluorescent plate reader. This experiment was repeated twice. Data analysis was processed as described by Michael J. B et al46 with a little modification. Briefly,


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fluorescent counts from untreated parasites represent the maximum amount of DNA in viable parasites while those from uninfected erythrocytes represent background fluorescence. The mean fluorescent counts in test or control wells were adjusted by deducting the background. The growth inhibition rate at each drug concentration was obtained as described below. % Growth Inhibition = 1-

Mean fluorescence counts in test wells ×100% Mean fluorescence counts in control wells

IC50 values were determined using a Growth/Sigmoidal option of Origin 8.0.

In Vivo Pharmacokinetic Assay. Single dose pharmacokinetic studies on the most active compounds were performed in male Sprague-Dawley Rats in compliance with the Guide for the Care and Use of Laboratory Animals. Blood samples were collected with heparin sodium as anti-coagulate at specified intervals (5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 12 h). PK parameters were calculated by non-compartment model using WinNonlin5.2 based on the LC-MS/MS quantitation data.



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Chemistry: Reagents and Methods. All chemical reagents and solvents were obtained from commercial sources and used without further purification. Thin-layer chromatography (TLC) was carried out to monitor the process of reactions. Purification of compounds was achieved by column chromatography with silica gel (HaiYang, Qingdao) 200-300 mesh. 1H NMR and 13C NMR spectra were recorded on a Bruker AM-400 spectrometer with chemical shifts expressed as ppm (in CDCl3 or DMSO-d6, Me4Si as internal standard). Melting points were recorded on a WRS-1B-digital melting point apparatus. The mass spectra were measured at The Institute of Fine Chemistry of ECUST. Analytical HPLC was performed on a Hewlett-Packard 1100 chromatography system equipped with photodiode array detector and a Zorbax XDB-C18 or Bonus-RP (compounds 30-35, 42-44) column (250 mm×4.6 mm) was used to determine the purity of the products. The mobile phase A was 10 mM NH4OAc in water (pH 6.0) and mobile phase B was acetonitrile. A gradient of 10-100% B over 20 minutes was run at a flow rate of 1.0 mL/min. Compounds synthesized in our laboratory were generally varied from 95% to 99% in purity, and the biological experiments were employed on compounds whose purity is at least 95%.

General procedure for aryl isothiocyanate: A mixture of 1,4-diazabicyclo[2.2.2]octane (9 mmol), aniline (3 mmol), carbon disulfide (5 mL) in acetone (3 mL) was stirred overnight at room temperature. The precipitated solid was filtered, washed with hexane and dried under vacuum. To a stirred solution of the solid in chloroform (10 mL), a solution of triphosgene (1 mmol)


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in chloroform (10 mL) was added dropwise over 30 min. The stirring was continued for 10 h at room temperature. After resulting mixture was filtered, the filtrate was concentrated under reduced pressure and purified by column chromatography (100% PE) with 45%-70% yield as white solid or colorless oil. General procedure for compounds 11-28: Ethyl 4-chloroacetoacetate (1 mmol) was added to a solution of sodium hydride (2 mmol, 60%) in THF (5 mL) , and the mixture was stirred for 1 h at room temperature before a solution of aryl isothiocyanate (1 mmol) in THF (5 mL) was added dropwise over 10 min. After the mixture was stirred overnight under argon, 5% HCl solution (20 mL) was added and the resulting oil was extracted with EtOAc (2×15 mL). The organic layer was washed with brine (2×15 mL), dried (Na2SO4), concentrated under reduced pressure and purified by column chromatography (PE : EtOAc = 2 : 1, v/v) with 40-45% yield as white solid. General procedure for compounds 30 and 31: LiOH-H2O (5 mmol) was slowly added to a solution of 20 or 25 (1 mmol) in MeOH-H2O (3 mL, MeOH : H2O = 5:1, v/v) at 0 oC over 15 min. The reaction mixture was allowed to warm to 55-60 oC for 12 h with stirring. After MeOH was evaporated off, the aqueous residual was acidified to pH 1-2 with 1N HCl and precipitated solid was filtered, washed with water and dried under vacuum with 70-80% yield as yellow solid. General procedure for compound 29: After a solution of 30 (1 mmol) in pyridine (2 mL) had been heated for 2 h, the



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reaction mixture was washed by 5% aqueous HCl (2×10 mL) and extracted with EtOAc (2×10 mL). The organic layer was washed with brine (2×15 mL), dried (Na2SO4) and concentrated under reduced pressure with purification by column chromatography (PE : EtOAc = 4 : 1, v/v) with 45% yield as yellow solid. General procedure for compounds 32-35 and 42-44: Diethyl azodicarboxylate (DEAD, 1.1 mmol) was slowly added to a solution of 30 (or 31) (1 mmol), triphenyl phosphine (1.1 mmol), and alcohol (1.2 mmol) in toluene (5 mL) with a pressure equalized dropping funnel under an argon atmosphere. The mixture was stirred overnight at 45 oC, then extracted with EtOAc (2×15 mL), washed with brine (2×15 mL), dried (Na2SO4) and concentrated under reduced pressure with purification by column chromatography (PE : EtOAc = 3 : 1, v/v) with 20-25% yield as white solid. General procedure for compounds 36-41 and 45-49: HOBt (1.1 mmol), EDC (1.1 mmol) and DIPEA (1 mmol) were added to a solution of amine (1 mmol) and 30 (or 31) (1 mmol) in dry CH2Cl2 (5 mL) at 0 oC were added. The reaction mixture was stirred overnight at room temperature, then washed with 5% aqueous HCl (2×15 mL), 5% aqueous NaHCO3 (2×15 mL), brine (2×15 mL), dried (Na2SO4) and concentrated under reduced pressure with purification by column chromatography (PE : EtOAc = 6 : 1, v/v) with 20%-25% yield as white solid. General procedure for compounds 50-56: Diethyl malonate (2 mmol) was added to a solution of sodium hydride (2 mmol, 60%) in dry THF (20 mL) and the mixture was stirred for 30 min at 0-10 oC. Then the


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reaction mixture was stirred at room temperature for 10 min and 2-chloroacetyl chloride (1 mmol) in dry THF (5 mL) was added. The solution was kept at room temperature for 1 h, at 40-45 oC for another 1 h and aniline (1 mmol) in dry THF (100 mL) was added dropwise over 20 min. The reaction mixture was stirred at room temperature overnight, heated under reflux for 4 h. After excess THF was evaporated off, the resulting residue was extracted with EtOAc (2×15 mL) and washed with brine (2×15 mL). The organic layer was dried (Na2SO4), concentrated under reduced pressure and purified by column chromatography (PE : EtOAc = 2.5 : 1, v/v) with 30-35% yield as white solid. Ethyl 2-(4-chlorophenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (11). mp 213.2-213.7 oC. 1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 4.22 (q, J = 7.2 Hz, 2H), 3.69 (s, 2H), 1.25 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 191.2, 183.2, 165.3, 136.9, 132.5, 129.9, 127.8, 97.7, 59.8, 38.5, 14.9. HRMS (ESI) calcd for C13H12ClNO3S [M+H]+ 298.0305, found 298.0307. Purity: 99.59% (tR 13.95 min). Ethyl

2-(phenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate

(12).

mp

146.8-147.5 oC. 1H NMR (400 MHz, CDCl3) δ 11.51 (s, 1H), 7.47 (t, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.35 (s, 1H), 4.39 (q, J = 7.2 Hz, 2H), 3.67 (s, 2H), 1.42 (t, J = 7.2 Hz, 3H).

13

C NMR (100MHz, DMSO-d6) δ 191.0, 183.3, 165.5, 137.8, 130.0,

128.3, 125.6, 97.4, 59.7, 38.4, 14.9. HRMS (ESI) calcd for C13H13NO3S [M+H]+ 264.0694, found 264.0694. Purity: 99.89% (tR 12.21 min). Ethyl

2-(4-(trifluoromethyl)phenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxyl



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ate (13). mp 180.1-181.0 oC. 1H NMR (400 MHz, DMSO-d6) δ 11.34 (s, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 4.23 (q, J = 7.2 Hz, 2H), 3.74 (s, 2H), 1.26 (t, J = 7.2 Hz, 3H).

13

C NMR (100 MHz, DMSO-d6) δ 191.1, 182.7, 165.4, 141.4,

127.1, 127.1, 125.9, 98.4, 59.9, 38.5, 14.8. HRMS (ESI) calcd for C14H12F3NO3S [M+H]+ 332.0568, found 332.0559. Purity: 99.42% (tR 14.83 min). Ethyl

2-(4-pentafluorosulfenylphenyl)-4-oxo-4,5-dihydrothiophene-3-carboxylate

(14). mp 177.3-178.1 oC. 1H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H), 8.03 (d, J = 9.2 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 4.25 (q, J = 6.8 Hz, 2H), 3.76 (s, 2H), 1.26 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 191.1, 182.6, 165.4, 141.1, 127.74, 127.7, 127.7, 125.7, 98.7, 59.9, 38.6, 14.8. HRMS (ESI) calcd for C13H12F5NO3S2 [M+H]+ 390.0257, found 390.0262. Purity: 99.49% (tR 15.69 min). Ethyl 2-(4-tert-butylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (15). mp 167.7-168.2 oC. 1H NMR (400 MHz, CDCl3): δ 11.44 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 9.6 Hz, 2H), 4.40 (q, J = 6.8 Hz, 2H), 3.66 (s, 2H), 1.43 (t, J = 6.8 Hz 3H), 1.36 (s, 9H).

13

C NMR (100 MHz, CDCl3): δ 191.2, 183.2, 166.6, 151.0,

134.30, 126.6, 123.6, 97.7, 60.4, 38.0, 34.7, 31.3, 14.5. HRMS (ESI) calcd for C17H21NO3S [M+H]+ 320.1320, found 320.1318. Purity: 99.49% (tR 17.37 min). Ethyl 2-(4-chloro-3-(trifluoromethyl)phenylamino)-4-oxo-4,5-dihydrothiophene-3carboxylate (16). mp 211.0-211.6 oC. 1H NMR (400 MHz, CDCl3) δ 11.67 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.73 (s, 1H), 7.45 (d, J = 8.4 Hz, 1H), 4.39 (q, J = 6.8 Hz, 2H), 3.71 (s, 2H), 1.42 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 191.1, 183.0, 165.1, 137.8, 136.5, 131.5, 129.8, 129.4, 125.9, 125.9, 124.3, 121.6, 118.0,


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98.5, 59.8, 38.6, 14.8. HRMS (ESI) calcd for C14H11ClF3NO3S [M+H]+ 366.0179, found 366.0177. Purity: 95.77% (tR 15.64 min). Ethyl 2-(4-bromo-3-(trifluoromethyl)phenylamino)-4-oxo-4,5-dihydrothiophene-3carboxylate (17). mp 223.8-224.5 oC. 1H NMR (400 MHz, CDCl3): δ 11.66 (s, 1H), 7.73 (s, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 4.40 (q, J = 7.2 Hz, 2H), 3.70 (s, 2H), 1.42 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 191.2, 183.1, 165.1, 137.5, 133.1, 131.6, 129.9, 127.9, 127.6, 125.9, 125.8, 125.7, 98.4, 59.8, 38.6, 14.9. HRMS (ESI) calcd for C14H11BrF3NO3S [M+H]+ 409.9673, found 409.9675. Purity: 95.72% (tR 15.87 min). Ethyl

2-(3-(trifluoromethyl)phenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxyl

ate (18). mp 190.2-191.2 oC. 1H NMR (400 MHz, DMSO-d6): δ 11.23 (s, 1H), 7.86 (s, 1H), 7.78-7.72 (m, 3H), 4.24 (q, J = 6.8 Hz, 2H), 3.71 (s, 2H), 1.26 (t, J = 6.8 Hz, 3H). 13

C NMR (100 MHz, DMSO-d6): δ 191.1, 183.3, 165.2, 138.7, 131.1, 130.1, 124.9,

124.8, 122.9, 122.8, 98.1, 59.8, 38.5, 14.8. HRMS (ESI) calcd for C14H12F3NO3S [M+H]+ 332.0568, found 332.0572. Purity: 99.04% (tR 14.52 min). Ethyl 2-(3,5-dichlorophenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (19). mp 188.1-188.9 oC. 1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 7.66 (s, 1H), 7.61 (s, 2H), 4.22 (q, J = 7.2 Hz, 2H), 3.71 (s, 2H), 1.25 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 191.2, 183.2, 165.1, 140.2, 134.8, 127.8, 125.0, 98.5, 59.9, 38.7, 14.8. HRMS (ESI) calcd for C13H11Cl2NO3S [M+H]+ 331.9915, found 331.9918. Purity: 99.89% (tR 16.34 min). Ethyl 2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (20).



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mp 150.0-151.9 oC. 1H NMR (400 MHz, CDCl3) δ 11.36 (s, 1H), 7.21 (d, J = 7.6 Hz, 1H), 7.11 (dd, J1 = 3.2 Hz, J2 = 10.8 Hz, 2H), 4.39 (q, J = 7.2 Hz, 2H), 3.63 (s, 2H), 2.31 (s, 6H), 1.42 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 190.9, 183.3, 165.6, 138.1, 136.7, 135.3, 130.7, 126.4, 122.8, 97.1, 59.7, 38.3, 19.8, 19.4, 14.9. HRMS (ESI) calcd for C15H17NO3S [M+H]+ 292.1007, found 292.1007. Purity: 98.16% (tR 14.95 min). Ethyl

2-(3-fluorine-4-methylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxy

late (21). mp 212.1-212.7 oC. 1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 7.39 (t, J = 8.4 Hz, 1H), 7.30 (d, J = 10.8 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 3.68 (s, 2H), 2.26 (s, 3H), 1.25 (t, J = 7.2 Hz, 3H).

13

C NMR (100 MHz,

DMSO-d6) δ 191.0, 183.4, 165.4, 161.9, 159.5, 136.9, 136.8, 132.6, 132.5, 124.6, 124.4, 121.7, 121.7, 113.0, 112.7, 97.6, 59.8, 38.4, 14.9, 14.4, 14.3. HRMS (ESI) calcd for C14H14FNO3S [M+H]+ 296.0757, found 296.0757. Purity: 98.58% (tR 13.83 min). Ethyl 2-(4-bromo-2-methylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (22). mp 177.7-178.6 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 7.62 (s, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 4.22 (q, J = 6.8 Hz, 2H), 3.32 (s, 2H), 2.22 (s, 3H), 1.25 (t, J = 6.8 Hz, 3H).

13

C NMR (100 MHz, DMSO-d6) δ

191.0, 184.1, 165.3, 138.2, 136.4, 134.0, 130.1, 129.3, 121.8, 97.3, 59.7, 38.4, 17.6, 14.9. HRMS (ESI) calcd for C14H14BrNO3S [M+H]+ 355.9956, found 355.9955. Purity: 99.88% (tR 14.98 min). Ethyl

2-(5,6,7,8-tetrahydronaphthalen-2-ylamino)-4-oxo-4,5-dihydrothiophene-3-


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carboxylate (23). mp 150.5-151.0 oC. 1H NMR (400 MHz, DMSO-d6): δ 11.06 (s, 1H), 7.14 (d, J = 6.8 Hz, 2H), 7.12 (s, 1H), 4.22 (q, J = 7.2 Hz, 2H), 3.66 (s, 2H), 2.74 (br, 4H), 1.75 (br, 4H), 1.26 (t, J = 7.2 Hz, 3H).

13


195.7, 188.0, 170.4, 143.2, 141.7, 139.7, 135.1, 130.4, 127.3, 101.8, 64.5, 43.1, 33.9, 33.6, 27.7, 27.6, 19.6. HRMS (ESI) calcd for C17H19NO3S [M+H]+ 318.1164, found 318.1168. Purity: 95.18% (tR 17.09 min). Ethyl 2-(quinolin-3-ylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (24). mp 216.0-216.6 oC. 1H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 1H), 8.92 (d, J = 2.4 Hz, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.08 (t, J = 8.4 Hz, 2H), 7.87-7.82 (m, 1H), 7.70 (t, J = 7.2 Hz, 1H), 4.27 (q, J = 6.8 Hz, 2H), 3.73 (s, 2H), 1.28 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 191.3, 184.0, 165.1, 149.2, 146.7, 132.2, 131.8, 130.8, 129.3, 128.8, 128.1, 127.5, 98.3, 59.8, 38.7, 14.9. HRMS (ESI) calcd for C16H14N2O3S [M+H]+ 315.0803, found 315.0806. Purity: 99.37% (tR 11.05 min). Ethyl 2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate

(25).

mp 186.7-187.4 oC. 1H NMR(400 MHz, DMSO-d6): δ 11.34 (s, 1H), 8.05-7.96 (m, 4H), 7.60-7.54 (m, 3H), 4.25 (q, J = 7.2 Hz, 2H), 3.70 (s, 2H), 1.27 (t, J = 7.2 Hz, 3H). 13

C NMR (100 MHz, DMSO-d6) δ 191.1, 183.4, 165.5, 135.4, 133.3, 132.3, 129.8,

128.4, 128.2, 127.5, 127.2, 124.2, 123.6, 97.6, 59.8, 38.5, 14.9. HRMS (ESI) calcd for C17H15NO3S [M+H]+ 314.0851, found 314.0849. Purity: 98.55% (tR 15.15 min). Ethyl

2-(2,3-dihydro-1H-inden-5-ylamino)-4-oxo-4,5-dihydrothiophene-3-carboxy

late (26). mp 128.7-129.2 oC. 1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.27 (s, 1H), 7.16 (d, J = 8.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 3.65



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(s, 2H), 2.89 (t, J = 7.6 Hz, 4H), 2.09-2.02 (m, 2H), 1.26 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 190.9, 183.5, 165.6, 145.7, 144.2, 135.8, 125.4, 123.6, 121.6, 97.1, 59.7, 38.4, 32.8, 32.4, 25.7, 14.9. HRMS (ESI) calcd for C16H17NO3S [M+H]+ 304.1007, found 304.1009. Purity: 95.39% (tR 15.67 min). Ethyl 2-(anthracen-2-ylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (27). mp 216.1-217.0 oC. 1H NMR (400 MHz, CDCl3) δ 11.76 (s, 1H), 8.48 (s, 1H), 8.45 (s, 1H), 8.10 (d, J = 9.2 Hz, 1H), 8.04 (d, J = 8.8 Hz, 3H), 7.53 (t, J = 4.8 Hz, 2H), 7.40 (d, J = 9.2 Hz, 1H), 4.44 (q, J = 7.2 Hz, 2H), 3.76 (s, 2H), 1.46 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 191.2, 183.2, 165.5, 134.9, 132.2, 132.0, 131.0, 130.4, 130.1, 128.6, 128.4, 126.9, 126.8, 126.7, 126.5, 124.0, 122.9, 97.8, 59.9, 38.6, 14.9. HRMS (ESI) calcd for C21H17NO3S [M+H]+ 364.1007, found 364.1005. Purity: 95.90% (tR 18.18 min). Ethyl 2-(9-ethyl-9H-carbazol-3-ylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (28). mp 233.9-234.6 oC. 1H NMR (400 MHz, CDCl3) δ 11.42 (s, 1H), 8.10 (d, J = 7.6 Hz, 2H), 7.55 (t, J = 7.2 Hz, 1H), 7.48-7.41 (m, 3H), 7.30 (d, J = 7.2 Hz, 1H), 4.46-4.39 (m, 4H), 3.64 (s, 2H), 1.51-1.48 (m, 6H). 13C NMR (100 MHz, DMSO-d6) δ 191.1, 184.5, 165.5, 140.7, 139.1, 129.3, 129.1, 126.9, 124.2, 122.8, 122.3, 121.3, 119.7, 118.7, 110.1, 110.0, 96.9, 59.6, 38.4, 37.7, 14.9, 14.2. HRMS (ESI) calcd for C21H20N2O3S [M+H]+ 381.1273, found 381.1274. Purity: 96.23% (tR 16.95 min). 5-(3,4-dimethylphenylamino)thiophen-3(2H)-one (29). mp 170.0-170.7 oC. 1H NMR (400 MHz, CDCl3) δ 7.13 (d, J = 6.8 Hz, 1H), 7.04 (s, 1H), 7.00 (d, J = 6.0 Hz, 1H), 5.66 (s, 1H), 3.73 (s, 2H), 2.27 (s, 6H). 13C NMR (100 MHz, CDCl3): δ 196.3, 175.6,


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137.9, 137.6, 133.7, 130.7, 123.3, 119.7, 95.6, 38.6, 19.9, 19.3. HRMS (ESI) calcd for C12H13NOS [M+H]+ 220.0796, found 220.0801. Purity: 95.79% (tR 12.14 min). 2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylic acid (30). mp 169.1-169.9 oC. 1H NMR (400 MHz, DMSO-d6) δ 7.26 (d, J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.17 (d, J = 8.0 Hz, 1H), 4.02 (s, 2H), 2.26 (s, 6H).

13

C NMR (100MHz,

DMSO-d6) δ 197.2, 183.0, 165.4, 138.3, 137.1, 135.3, 130.8, 126.1, 122.4, 98.2, 38.6, 19.8, 19.5. HRMS (ESI) calcd for C13H13NO3S [M+H]+ 264.0694, found 264.0690. Purity: 98.64% (tR 12.59 min). 2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylic acid (31). mp 165.4-166.0 oC. 1H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H), 10.55 (s, 1H), 8.08-7.89 (m, 4H), 7.62-7.40 (m, 3H), 4.07 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 197.3, 183.3, 165.3, 135.1, 133.2, 132.5, 130.0, 128.4, 128.2, 127.6, 127.4, 123.7, 123.4, 98.6, 38.7. HRMS (ESI) calcd for C15H11NO3S [M+H]+ 286.0538, found 286.0541. Purity: 95.71% (tR 12.36 min). Methyl

2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate

(32). mp 182.7-183.4 oC. 1H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.21 (s, 1H), 7.15 (d, J = 8.0 Hz, 1H), 3.72 (s, 3H), 3.67 (s, 2H), 2.26 (s, 6H).

13

C NMR (100 MHz, DMSO-d6) δ 191.0, 183.4, 165.8, 138.1, 136.8, 135.4,

130.7, 126.6, 122.9, 97.0, 51.2, 38.4, 19.8, 19.4. HRMS (ESI) calcd for C14H15NO3S [M+H]+ 278.0851, found 278.0851. Purity: 96.40% (tR 13.36 min). Propyl

2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate

(33). mp 135.5-136.1 oC. 1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 7.24 (d, J =



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8.0 Hz, 1H), 7.20 (s, 1H), 7.15 (d, J = 8.0 Hz, 1H), 4.13 (t, J = 6.8 Hz, 2H), 3.66 (s, 2H), 2.26 (s, 6H), 1.68-1.61 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 190.9, 183.3, 165.7, 138.1, 136.6, 135.3, 130.8, 130.7, 126.3, 126.1, 122.7, 122.4, 97.1, 65.1, 38.4, 22.2, 19.8, 19.4, 10.8. HRMS (ESI) calcd for C16H19NO3S [M+H]+ 306.1164, found 306.1170. Purity: 96.07% (tR 16.04 min). Butyl 2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (34). mp 108.0-108.9 oC. 1H NMR (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.15 (d, J = 8.0 Hz, 1H), 4.17 (t, J = 6.4 Hz, 2H), 3.65 (s, 2H), 2.25 (s, 6H), 1.66-1.59 (m, 2H), 1.45-1.36 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 190.9, 183.3, 165.7, 138.2, 136.6, 135.3, 130.8, 130.7, 126.3, 126.1, 122.7, 122.4, 97.1, 64.4, 38.4, 30.9, 19.8, 19.4, 19.1, 14.1. HRMS (ESI) calcd for C17H21NO3S [M+Na]+ 342.1140, found 342.1123. Purity: 96.31% (tR 17.22 min). Cyclopropylmethyl

2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrothiophene-3-

carboxylate (35). mp 115.1-116.2 oC. 1H NMR (400 MHz, CDCl3) δ 11.32 (s, 1H), 7.20 (d, J = 8.8 Hz, 1H), 7.13 (s, 1H), 7.10 (d, J = 8.8 Hz, 1H), 4.17 (d, J = 6.8 Hz, 2H), 3.64 (s, 2H), 1.30-1.27 (m, 1H), 0.61 (d, J = 6.8 Hz, 2H), 0.40 (d, J = 4.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 191.3, 183.4, 166.7, 138.3, 136.6, 134.6, 130.6, 125.3, 121.4, 68.9, 62.2, 38.0, 19.9, 19.4, 14.4, 10.1, 3.5. HRMS (ESI) calcd for C17H19NO3S [M+H]+ 318.1164, found 318.1168. Purity: 96.63% (tR 16.08 min). 2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxamide (36). mp 181.1-181.9 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.92 (s, 1H), 8.92 (s, 1H), 7.88 (s, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.13 (s, 1H), 7.06 (dd, J1 = 2.0 Hz, J2 = 8.0 Hz, 1H),


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3.81 (s, 2H), 2.25 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 196.7, 158.0, 139.1, 137.2, 131.3, 130.7, 126.7, 122.9, 93.2, 38.2, 19.9, 19.4. HRMS (ESI) calcd for C13H14N2O2S [M+H]+ 263.0854, found 263.0853. Purity: 99.79% (tR 14.17 min). N-ethyl-2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxamide (37). mp 95.9-96.6 oC. 1H NMR (400 MHz, CDCl3) δ 12.56 (s, 1H), 8.82 (s, 1H), 7.18 (d, J = 7.6 Hz, 1H), 7.12 (s, 1H), 7.10 (dd, J1 = 2.4 Hz, J2 = 6.4 Hz, 1H), 3.70 (s, 2H), 3.45-3.38 (m, 2H), 2.89 (s, 6H), 1.24 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 193.6, 181.4, 165.8, 138.2, 135.9, 135.1, 130.56, 124.4, 120.5, 99.1, 38.4, 33.2, 19.9, 19.4, 15.0. HRMS (ESI) calcd for C15H18N2O2S [M+H]+ 291.1167, found 291.1169. Purity: 99.70% (tR 16.67 min). N-propyl-2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxamide (38). mp 107.7-108.4 oC. 1H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H), 8.82 (t, J = 5.6 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.15 (dd, J1 = 2.0 Hz, J2 = 8.0 Hz, 1H), 3.85 (s, 2H), 3.23 (q, J = 6.8 Hz, 2H), 2.25 (s, 3H), 2.24 (s, 3H), 1.53-1.48 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 199.0, 186.3, 170.3, 143.1, 140.8, 140.1, 135.7, 129.6, 125.8, 103.6, 43.3, 27.8, 24.6, 24.1, 16.6. HRMS (ESI) calcd for C16H20N2O2S [M+Na]+ 327.1143, found 327.1134. Purity: 99.23% (tR 18.23 min). N-butyl-2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxamide (39). mp 90.9-91.6 oC. 1H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H), 8.80 (t, J = 5.6 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.19 (s, 1H), 7.15 (dd, J1 = 2.0 Hz, J2 = 8.0 Hz, 1H), 3.84 (s, 2H), 3.27 (q, J = 6.8 Hz, 2H), 2.25 (s, 3H), 2.24 (s, 3H), 1.50-1.44 (m,



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2H), 1.35-1.30 (m, 2H), 0.91(t, J = 7.2 Hz, 3H).

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13


194.2, 181.5, 165.5, 138.4, 136.0, 135.4, 130.9, 124.8, 121.0, 98.8, 38.9, 37.6, 31.8, 20.1, 19.8, 19.4, 14.1. HRMS (ESI) calcd for C17H22N2O2S [M+Na]+ 341.1300, found 341.1305. Purity: 99.55% (tR 19.71 min). N-cyclopropyl-2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrothiophene-3-carboxa mide (40). mp 139.1-139.7 oC. 1H NMR (400 MHz, DMSO-d6) δ 12.27 (s, 1H), 8.75 (d, J = 7.6 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.20 (d, J = 2.0 Hz, 1H), 7.15 (dd, J1 = 2.0 Hz, J2 = 8.0 Hz, 1H), 3.83 (s, 2H), 2.77-2.73 (m, 1H), 2.25 (s, 3H), 2.24 (s, 3H), 0.75-0.70 (m, 2H), 0.50-0.48 (m, 2H).

13

C NMR (100 MHz, CDCl3) δ 193.6, 181.3,

167.5, 138.2, 136.0, 135.0, 130.6, 124.3, 120.4, 98.9, 38.4, 21.5, 19.9, 19.4, 6.4. (ESI) calcd for C16H18N2O2S [M+H]+ 303.1167, found 303.1158. Purity: 99.28% (tR 16.59min). N-(cyclopropylmethyl)-2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrothiophene-3carboxamide (41). mp 124.6-125.1 oC. 1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H), 8.88 (t, J = 5.6 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.15 (dd, J1 = 2.4 Hz, J2 = 8.0 Hz, 1H), 3.85 (s, 2H), 3.15 (t, J = 6.0 Hz, 2H), 2.25 (s, 3H), 2.24 (s, 3H), 1.02-0.97 (m, 1H), 0.48-0.43 (m, 2H), 0.23-0.19 (m, 2H).

13

C NMR (100 MHz,

CDCl3) δ 193.7, 181.5, 165.8, 138.2, 136.0, 135.0, 130.6, 124.5, 120.5, 99.0, 42.9, 38.4, 19.9, 19.4, 10.9, 3.5. HRMS (ESI) calcd for C17H20N2O2S [M+H]+ 317.1324, found 317.1316. Purity: 98.92% (tR 18.39 min). Methyl 2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (42). mp 203.4-204.2 oC. 1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.06-7.97 (m,


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4H), 7.66-7.54 (m, 3H), 3.76 (s, 3H), 3.71 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 191.1, 183.4, 165.7, 135.3, 133.3, 132.3, 129.8, 128.4, 128.2, 127.5, 127.3, 124.2, 123.7, 97.5, 51.2, 38.5. HRMS (ESI) calcd for C16H13NO3S [M+H]+ 300.0694, found 300.0699. Purity: 95.50% (tR 13.80 min). Propyl 2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate (43). mp 163.9-164.5 oC. 1H NMR (400 MHz, CDCl3) δ 11.67 (s, 1H), 7.95-7.86 (m, 4H), 7.58-7.44 (m, 3H), 4.31 (t, J = 6.8 Hz, 2H), 3.71 (s, 2H), 1.87-1.82 (m, 2H), 1.06 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 191.0, 183.3, 165.7, 135.3, 133.3, 132.3, 129.8, 128.3, 128.2, 127.5, 127.2, 124.0, 123.3, 97.8, 65.2, 38.5, 22.2, 10.8. HRMS (ESI) calcd for C18H17NO3S [M+Na]+ 350.0827, found 350.0833. Purity: 96.82% (tR 16.43 min). Butyl 2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrothiophene-3-carboxylate

(44).

mp 128.5-129.0 oC. 1H NMR (400 MHz, CDCl3) δ 11.66 (s, 1H), 7.95-7.86 (m, 4H), 7.57-7.44 (m, 3H), 4.35 (t, J = 6.8 Hz, 2H), 3.69 (s, 2H), 1.85-1.77 (m, 2H), 1.55-1.45 (m, 2H), 1.00 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 191.2, 183.2, 166.8, 156.8, 134.4, 133.3, 132.2, 129.9, 127.9, 127.9, 127.3, 126.8, 122.3, 122.0, 98.1, 64.4, 38.1, 30.8, 19.2, 13.8. HRMS (ESI) calcd for C19H19NO3S [M+Na]+ 364.0983, found 364.0991. Purity: 97.49% (tR 17.57 min). N-Ethyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrothiophene-3-carboxamide (45). mp 128.0-128.6 oC. 1H NMR (400 MHz, DMSO-d6) δ 12.71 (s, 1H), 8.80 (d, J = 5.6 Hz, 1H), 8.05-7.95 (m, 4H), 7.60-7.53 (m, 3H), 3.90 (s, 2H), 3.31 (q, J = 6.8 Hz, 2H), 1.12 (t, J = 7.2 Hz, 3H).

13

C NMR (100 MHz, CDCl3) δ 194.1, 181.3, 165.9, 134.9,



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133.4, 131.8, 129.8, 127.8, 127.2, 126.5, 121.8, 120.6, 99.9, 38.7, 33.2, 15.0. HRMS (ESI) calcd for C17H16N2O2S [M+H]+ 313.1011, found 313.1011. Purity: 99.69% (tR 16.95 min). N-Propyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrothiophene-3-carboxamide (46). mp 126.2-127.2 oC. 1H NMR (400 MHz, CDCl3) δ 12.95 (s, 1H), 8.91 (s, 1H), 7.92-7.84 (m, 4H), 7.56-7.29 (m, 3H), 3.75 (s, 2H), 3.37 (q, J = 6.4 Hz, 2H), 1.69-1.63 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H).

13

C NMR (100 MHz, CDCl3) δ 193.8,

181.3, 165.9, 135.0, 133.4, 131.8, 129.8, 127.8, 127.2, 126.5, 121.8, 120.6, 99.6, 40.1, 38.5, 23.0, 11.5. HRMS (ESI) calcd for C18H18N2O2S [M+H]+ 327.1167, found 327.1165. Purity: 98.33% (tR 19.86 min). N-Butyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrothiophene-3-carboxamide (47). mp 140.1-141.0 oC. 1H NMR (400 MHz, CDCl3) δ 12.94 (s, 1H), 8.89 (s, 1H), 7.93-7.84 (m, 4H), 7.58-7.44 (m, 3H), 3.75 (s, 2H), 3.41 (q, J = 6.8 Hz, 2H), 1.66-1.59 (m, 2H), 1.50-1.41 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 193.8, 181.3, 165.9, 135.0, 133.4, 131.9, 129.8, 127.8, 127.2, 126.5, 121.8, 120.6, 99.6, 38.5, 38.1, 31.8, 20.2, 13.8. HRMS (ESI) calcd for C19H20N2O2S [M+H]+ 341.1324, found 341.1320. Purity: 97.25% (tR 18.41 min). N-cyclopropyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrothiophene-3-carboxami de (48). mp 140.8-141.6 oC. 1H NMR (400 MHz, DMSO-d6) δ 12.62 (s, 1H), 8.78 (d, J = 3.6 Hz, 1H), 8.08-7.93 (m, 4H), 7.69-7.54 (m, 3H), 3.89 (s, 2H), 2.82-2.77 (m, 1H), 0.78-0.73 (m, 2H), 0.56-0.50 (m, 2H).

13

C NMR (100 MHz, CDCl3) δ 193.7,

181.2, 167.5, 134.9, 133.4, 131.8, 129.9, 127.9, 127.2, 126.5, 121.7, 120.5, 99.4, 38.6,


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29.7, 21.6, 6.5. HRMS (ESI) calcd for C18H16N2O2S [M+Na]+ 347.0830, found 347.0834. Purity: 97.28% (tR 16.84 min). N-(cyclopropylmethyl)-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrothiophene-3-ca rboxamide (49). mp 164.8-165.3 oC. 1H NMR (400 MHz, DMSO-d6) δ 12.68 (s, 1H), 8.91 (t, J = 5.6 Hz, 1H), 8.06-7.97 (m, 4H), 7.62-7.55 (m, 3H), 3.92 (s, 2H), 3.19 (t, J = 6.4 Hz, 2H), 1.06-0.99 (m, 1H), 0.50-0.45 (m, 2H), 0.26-0.22 (m, 2H).

13

C NMR

(100 MHz, CDCl3) δ 193.8, 181.3, 165.8, 134.9, 133.4, 131.8, 129.8, 127.9, 127.2, 126.5, 121.8, 120.6, 99.5, 43.0, 38.5, 10.9, 3.5. HRMS (ESI) calcd for C19H18N2O2S [M+Na]+ 361.0987, found 361.0997. Purity: 99.33% (tR 18.59 min). Ethyl 2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxylate (50). mp 134.7-135.8 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.02-7.90 (m, 4H), 7.63-7.51 (m, 3H), 4.75 (s, 2H), 4.25 (q, J = 7.2 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 189.1, 177.7, 164.5, 133.4, 133.1, 131.4, 129.3, 128.1, 127.4, 126.5, 122.9, 120.8, 87.5, 75.7, 59.7, 14.9. HRMS (ESI) calcd for C17H15NO4 [M+Na]+ 320.0899, found 320.0897. Purity: 97.21% (tR 14.18 min). Ethyl 2-(3,4-dimethylphenylamino)-4-oxo-4,5-dihydrofuran-3-carboxylate (51). mp 120.8-121.4 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 7.23 (s, 1H), 7.17 (d, J = 1.6 Hz, 2H), 4.67 (s, 2H), 4.22 (q, J = 7.2 Hz, 2H), 2.23 (s, 3H), 2.22 (s, 3H), 1.26 (t, J = 7.2 Hz, 3H).

13

C NMR (100 MHz, DMSO-d6) δ 188.9, 177.4, 164.5, 137.5,

134.8, 133.1, 130.4, 124.5, 120.9, 87.0, 75.5, 59.6, 19.9, 19.3, 14.9. HRMS (ESI) calcd for C15H17NO4 [M+Na]+ 298.1055, found 298.1059. Purity: 99.43% (tR 13.93 min).



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Ethyl

2-(quinolin-3-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxylate

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(52).

mp

203.7-204.2 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.62 (s, 1H), 8.99 (d, J = 2.4 Hz, 1H), 8.44 (d, J = 2.4 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.79-7.75 (m, 1H), 7.65 (t, J = 8.0 Hz, 1H), 4.75 (s, 2H), 4.26 (q, J = 7.2 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 189.4, 178.1, 164.2, 147.7, 145.8, 129.9, 129.5, 129.1, 128.9, 128.5, 127.9, 127.7, 87.9, 75.7, 59.7, 14.9. HRMS (ESI) calcd for C16H14N2O4 [M+Na]+ 321.0851, found 321.0843. Purity: 99.57% (tR 10.23 min). Ethyl 2-(5,6,7,8-tetrahydronaphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carbo xylate (53). mp 127.2-127.8 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 7.15 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 1H), 4.68 (s, 2H), 4.22 (q, J = 6.8 Hz, 2H), 2.71 (br, 4H), 1.73 (br, 4H), 1.26 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 188.9, 177.4, 164.5, 137.9, 135.2, 132.8, 129.9, 123.8, 120.9, 87.0, 75.5, 59.6, 29.3, 28.8, 23.1, 22.9, 14.9. HRMS (ESI) calcd for C17H19NO4 [M+Na]+ 324.1212, found 324.1207. Purity: 99.01% (tR 15.89 min). Ethyl

2-(2,3-dihydro-1H-inden-5-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxylate

(54). mp 149.7-151.2 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.19 (s, 1H), 7.31 (s, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.0 Hz, 1H), 4.66 (s, 2H), 4.22 (q, J = 7.2 Hz, 2H), 2.86 (q, J = 7.6 Hz, 4H), 2.04 (t, J = 7.6 Hz, 2H), 1.26 (t, J = 7.2 Hz, 3H). 13

C NMR (100 MHz, DMSO-d6) δ 189.0, 177.4, 164.5, 145.2, 142.3, 133.6, 125.0,

121.8, 119.8, 87.0, 75.5, 59.6, 32.8, 32.3, 25.7, 14.9. HRMS (ESI) calcd for C16H17NO4 [M+Na]+ 310.1055, found 310.1056. Purity: 99.34% (tR 14.56 min).


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Ethyl 2-(4-pentafluorosulfenylphenyl)-4-oxo-4,5-dihydrofuran-3-carboxylate (55). mp 179.6-181.4 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 7.93 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H), 4.76 (s, 2H), 4.24 (q, J = 7.2 Hz, 2H), 1.26 (t, J = 7.2 Hz, 3H).

13

C NMR (100 MHz, DMSO-d6) δ 189.3, 177.9, 164.3, 139.0, 127.3,

127.3, 127.2, 123.6, 88.2, 75.8, 59.8, 14.8. HRMS (ESI) calcd for C13H12F5NO4S [M+H]+ 374.0485, found 374.0490. Purity: 99.77% (tR 14.89 min). Ethyl

2-(4-(trifluoromethyl)phenylamino)-4-oxo-4,5-dihydrofuran-3-carboxylate

(56). mp 167.0-167.7 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 4.75 (s, 2H), 4.24 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 189.3, 177.8, 164.3, 139.3, 126.7, 126.6, 126.6, 123.8, 88.0, 75.8, 59.8, 14.9. HRMS (ESI) calcd for C14H12F3NO4 [M+Na]+ 338.0616, found 338.0607. Purity: 99.07% (tR 14.03 min).



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AUTHOR INFORMATION

Corresponding Author

Phone : +86-21-64250213. Fax: +86-21-64250213. Email: [email protected], [email protected], [email protected]

Author Contributions †

These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We also thank Prof. Weibin Guan and Dr. Lei Shan of the Second Military Medical University for helpful discussions. This work was supported by the Fundamental Research Funds for the Central Universities, the Shanghai Natural Science Fund for Youth Scholars (grant 12ZR144280), the National Natural Science Foundation of China (grants 21372078, 81302697, 21173076, 81102375, 81222046 and 81230076), the Shanghai Committee of Science and Technology (grants 11DZ2260600 and 12401900801), and the 863 Hi-Tech Program of China (grant 2012AA020308). Honglin Li is also sponsored by Program for New Century Excellent Talents in University (grant NCET-10-0378) and Shanghai Rising-Star Tracking Program (grant 13QH1401100).


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Abbreviations: PfDHODH, Plasmodium falciparum dihydroorotate dehydrogenase; DHO, dihydroorotate; ORO, orotate; NAD+, nicotinamide adenine dinucleotide; SAR, structure–activity

relationship;

VDW,

van

der

Waals;

dihydroorotate dehydrogenase; CQ, chloroquine .


hDHODH,

human


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Figure Captions Figure 1. Structures of reported anti-malarial agents 1-3, PfDHODH inhibitors 4-10, and the lead compound 11 in this study. Figure 2. Proposed binding pose of the lead compound 11 in the ubiquinone-binding pocket of PfDHODH. Key residues around the binding pocket are shown as green lines, and the lead compound is presented as sticks. The reserved water (W15) is depicted as a red ball, and the hydrogen bonds are labeled as black dashed lines. Figure 3. The diagram for the structural optimization strategy. Figure 4. Comparison of the series activity on PfDHDODH vs P. falciparum in 3D7 and Dd2 cells assays. The log of the PfDHODH IC50 data are plotted vs the log of the P. falciparum 3D7 and Dd2 cells IC50 data (nanomolar range). The plotted data include compounds described in Table 1 except for 11, 12, 18, 22, 30, 36, 46, 47, 49. Data were fitted by linear regession analysis (For Pf3D7 cells, slope = 1.256 and r2 = 0.750; For Pf Dd2 cells, slope = 1.347 and r2 = 0.746).



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Scheme 1. Reagents and conditions: (a1) CS2, 1,4-diazabicyclo[2.2.2]octane, acetone, overnight at R.T; (a2) Triphosgene, chloroform, overnight at R.T; (b) Sodium hydride, ethyl 4-chloroacetoacetate, THF, overnight at R.T.


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Scheme 2. Reagents and conditions: (c) LiOH-H2O, MeOH-H2O (5:1, v/v), 55-60 oC for 12 h; (d) Alcohols, DEAD, PPh3, toluene, overnight at 45 oC; (e) Pyridine, reflux for 2 h.



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Scheme 3. Reagents and conditions: (c) LiOH-H2O, MeOH-H2O (5:1, v/v), 55-60 oC for 12 h; (f) RNH2, HOBt, EDC, DIPEA, CH2Cl2, overnight at R.T.


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Scheme 4. Reagents and conditions: (g) Sodium hydride, diethyl malonate, 2-chloroacetyl chloride, THF, at R.T. for 1 h, at 40-45 oC for another 1 h, reflux for 4 h.



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Figure 1. Structures of reported anti-malarial agents 1-3, PfDHODH inhibitors 4-10, and the lead compound 11 in this study.


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Figure 2. Proposed binding pose of the lead compound 11 in the ubiquinone-binding pocket of PfDHODH. Key residues around the binding pocket are shown as green lines, and the lead compound is presented as sticks. The reserved water (W15) is depicted as a red ball, and the hydrogen bonds are labeled as black dashed lines.



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Figure 3. The diagram for the structural optimization strategy.


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Page 61 of 66

A 5.0

3D7 2 3D7 r = 0.750

4.5

Log IC50 PfDHODH (nM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Dd2 2 Dd2 r = 0.746

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Log IC50 Pf cells (nM)

Figure 4. Comparison of the series activity on PfDHDODH vs P. falciparum in 3D7 and Dd2 cells assays. The log of the PfDHODH IC50 data are plotted vs the log of the P.falciparum 3D7 and Dd2 cells IC50 data (nanomolar range). The plotted data include compounds described in Table 1 except for 11, 12, 18, 22, 30, 36, 46, 47, 49. Data were fitted by linear regession analysis (For Pf3D7 cells, slope = 1.256 and r2 = 0.750; For Pf Dd2 cells, slope = 1.347 and r2 = 0.746).



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Table 1. Structure and Activity Profiles of Compounds 11-56 against DHODH and P. falciparum in Whole Cell Assays.

IC50 (µM) Compd

R

1

2

R

PfDHODHa

hDHODHb

Pf 3D7c

Pf Dd2d

11

4-Cl-Ph

1.113±0.081

>50

>20

>20

12

Ph

>50

>50

5.903±0.035

6.556±0.889

13

4-CF3-Ph

0.367±0.006

>50

0.946±0.390

1.040±0.146

14

4-SF5-Ph

0.559±0.014

>50

0.307±0.108

0.697±0.101

15

4-t-butyl-Ph

0.227±0.005

>50

1.525±0.059

0.767±0.367

16

3-CF3-4-Cl-Ph

0.751±0.021

>50

2.191±0.738

3.147±1.018

17

3-CF3-4-Br-Ph

1.615±0.176

>50

5.614±0.347

5.539±0.686

18

3-CF3-Ph

>50

>50

14.351±5.742 13.592±1.030

19

3,5-di-Cl-Ph

1.188±0.181

>50

4.031±1.249

5.996±0.636

20

3,4-di-CH3-Ph

0.256±0.004

>50

1.992±0.737

1.317±0.152

21

3-F-4-CH3-Ph

0.536±0.005

>50

5.048±1.291

3.730±0.915

22

2-CH3-4-Br-Ph

>50

>50

>20

>20

23

5,6,7,8-tetrahydro-

0.092±0.001

11.982±0.512

0.169±0.029

0.315±0.040

0.351±0.004

>50 0.253±0.057

0.290±0.030

2-naphthyl 24

3-quinolinyl


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25

2-naphthyl

0.020±0.001

>50

0.077±0.002

0.057±0.004

26

5-indanyl

0.851±0.027

>50

1.401±0.556

1.573±0.192

27

2-anthracenyl

0.197±0.004

>50

0.688±0.232

1.127±0.202

28

9-ethyl-3-carbazolyl

0.392±0.013

>50

1.233±0.063

1.513±0.864

29

3,4-di-CH3-Ph

11.441±0.261

>50

1.907±1.895

2.498±2.730

30

3,4-di-CH3-Ph

>50

>50

>20

>20

31

2-naphthyl

1.238±0.011

>50

1.860±0.871

1.049±0.079

32

3,4-di-CH3-Ph

1.486±0.106

>50

1.018±0.923

1.267±1.270

33

3,4-di-CH3-Ph

4.032±0.164

11.633±0.085

2.107±2.257

1.689±1.665

34

3,4-di-CH3-Ph

2.350±0.081

>50

0.745±0.546

0.889±0.749

35

3,4-di-CH3-Ph

4.159±0.150

>50

3.534±2.236

2.632±0.961

36

3,4-di-CH3-Ph

>50

>50

>20

>20

37

3,4-di-CH3-Ph

39.450±0.717

>50

8.470±1.508

6.625±0.128

38

3,4-di-CH3-Ph

4.633±0.838

6.299±0.111

3.173±0.485

3.927±0.775

39

3,4-di-CH3-Ph

5.733±0.286

4.422±0.277

2.696±0.299

2.778±0.181

40

3,4-di-CH3-Ph

19.114±0.143

4.109±0.077

6.618±4.554

5.532±0.992

41

3,4-di-CH3-Ph

3.662±0.458

1.839±0.040

3.342±2.100

3.500±0.845

42

2-naphthyl

0.033±0.001

>50

0.245±0.001

0.400±0.265

43

2-naphthyl

0.951±0.041

>50

0.890±0.774

1.660±1.863

44

2-naphthyl

35.572±0.683

>50

8.380±5.475

5.228±1.017

45

2-naphthyl

12.061±1.578

>50

11.148±1.525

8.492±2.683

H



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46

2-naphthyl

>50

>50

3.476±2.808

3.547±2.908

47

2-naphthyl

>50

>50

5.831±2.519

4.032±0.025

48

2-naphthyl

13.210±0.104

>50

10.233±1.228

7.944±0.539

49

2-naphthyl

>50

>50

12.402±0.993

>20

50

2-naphthyl

0.006±0.001

>50

0.015±0.002

0.018±0.002

51

3,4-di-CH3-Ph

0.698±0.013

>50

0.372±0.180

0.486±0.012

52

3-quinolinyl

0.079±0.001

>50

0.492±0.060

0.517±0.168

53

5,6,7,8-tetrahydro-

0.018±0.001

>50

0.057±0.012

0.076±0.008

2-naphthyl 54

5-indanyl

0.065±0.002

>50

0.495±0.028

0.373±0.105

55

4-SF5-Ph

0.168±0.002

>50

0.680±0.095

1.063±0.414

56

4-CF3-Ph

0.142±0.001

>50

0.607±0.009

0.867±0.179

DSM 1

--

--

0.031±0.002

--

--

--

A771726

--

--

--

0.137±0.002

--

--

Chloroquine

--

--

--

--

0.015±0.003

0.062±0.002

a

The IC50 values of the compounds against PfDHODH, in vitro assay, µM

b

The IC50 values of the compounds against hDHODH, in vitro assay, µM

c

The IC50 values of the compounds against Pf3D7 strains, in vitro assay, µM

d

The IC50 values of the compounds against PfDd2 strains, in vitro assay, µM


Page 65 of 66

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Table 2. Pharmacokinetic Profile of Selected Potent Inhibitors, Determined in Male Sprague-Dawley Rats after Intravenous (IV) and Oral (PO) Administrationa Parameter

25

50

IV dose (mg/kg)

1

1

Clearance (mL/min/kg)

129.42±13.53

84.67±2.84

T1/2 (h)

0.30±0.00

0.26±0.02

Volume of distribution (L/kg)

3.35±0.34

1.91±0.18

PO dose (mg/kg)

10

10

T1/2 (h)

2.56±0.78

0.75±0.27

AUC0-last (ng/mL*hr)

39.33±19.67

790.65±521.11

Cmax (ng/mL)

25.49±15.84

608.82±415.94

Tmax (h)

0.25±0.00

0.29±0.10

Bioavailability F (%)

3.02±1.51

40.03±26.38

a

Three animals for IV Administration and six animals for PO administration.



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TOC


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Novel Selective and Potent Inhibitors of Malaria Parasite

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