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Synthesis, Design, and Structure–Activity Relationship of the Pyrimidone Derivatives as Novel Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase Le Xu 1,† , Wenjie Li 1,† , Yanyan Diao 1,† , Hongxia Sun 1 , Honglin Li 1 , Lili Zhu 1, *, Hongchang Zhou 2, * and Zhenjiang Zhao 1, * 1

2

* †

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China; [email protected] (L.X.); [email protected] (W.L.); [email protected] (Y.D.); [email protected] (H.S.); [email protected] (H.L.) Department of Microbiology, Medical School of Huzhou Teachers College, Huzhou 313000, China Correspondence: [email protected] (L.Z.); [email protected] (H.Z.); [email protected] (Z.Z.); Tel.: +86-21-6425-3379 (L.Z.); +86-21-6425-3962 (Z.Z.); Fax.: +86-21-64250213 (L.Z. & Z.Z.) These authors contributed equally to this work.

Received: 2 May 2018; Accepted: 22 May 2018; Published: 24 May 2018







Abstract: The inhibition of Plasmodium falciparum dihydroorotate dehydrogenase (Pf DHODH) potentially represents a new treatment option for malaria, as P. falciparum relies entirely on a de novo pyrimidine biosynthetic pathway for survival. Herein, we report a series of pyrimidone derivatives as novel inhibitors of Pf DHODH. The most potent compound, 26, showed high inhibition activity against Pf DHODH (IC50 = 23 nM), with >400-fold species selectivity over human dihydroorotate dehydrogenase (hDHODH). The brand-new inhibitor scaffold targeting Pf DHODH reported in this work may lead to the discovery of new antimalarial agents. Keywords: P. falciparum; Pf DHODH; pyrimidone; antimalarial agents

1. Introduction Malaria is a mosquito-transmitted disease caused by protozoan parasites of the Plasmodium species [1,2]. The World Health Organization (WHO) estimated that there were nearly 216 million episodes of malaria and 445,000 deaths from malaria globally in 2016 [3]. Artemisinin combination therapies (ACTs) are the current first-line treatment for the deadliest form of malaria. However, the reduced efficacy of first-line treatments using artemisinin (compound 1) [4] and its derivatives (associated with Kelch 13 propeller protein mutations 7-95430) is now prevalent at the Cambodia–Thailand border and poses a serious threat to malaria control programs globally [5–8]. Chloroquine (compound 2) [9] and pyrimethamine (compound 3) [10], used as the mainstay of antimalarial chemotherapy, have now compromised the development of resistance [11]. To tackle the problem of drug resistance, various strategies have been developed to treat malaria [12,13]. For instance, Gilbert’s group discovered that DDD107498 exhibits a novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite [14]. Dihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme that is required for the fourth step of de novo pyrimidine biosynthesis, converting dihydroorotate (DHO) to orotate (ORO) with the participation of the cofactors flavin mononucleotide (FMN) and ubiquinone (CoQ) [15–17]. Pyrimidine-based biosynthesis represents a basic biological and physiological process that is crucial for RNA and DNA production and cell proliferation. The mammalian cells generate pyrimidines through

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both de novo and salvage pathways for survival, while plasmodium parasites lack the necessary genes Molecules 2018,resulting 23, x FOR PEER REVIEW 2 of 14 [18]. for the former, in de novo pyrimidine synthesis as the vital pathway for the parasite Therefore, Pf DHODH has been considered as the prospective target for the conquest of malaria [19–21]. through both de novo and salvage pathways for survival, while plasmodium parasites lack the DHODHs are divided into two families in the light of different amino acid sequences and coenzyme necessary genes for the former, resulting in de novo pyrimidine synthesis as the vital pathway for dependence. Human dehydrogenase DHODH belong the parasite [18]. dihydroorotate Therefore, PfDHODH has been (hDHODH) considered asand thePfprospective target to forfamily the II, located on theof inner mitochondrial membrane, whichinto utilizes ubiquinone the of terminal conquest malaria [19–21]. DHODHs are divided two families in theaslight differentoxidant amino [22]. Many hDHODH been corroborated to be effective against a number of diseases, acid sequences andinhibitors coenzymehave dependence. Human dihydroorotate dehydrogenase (hDHODH) and PfDHODH belong to family II , located on the inner mitochondrial membrane, which utilizes such as cancer, rheumatoid arthritis (RA), psoriasis, and lupus erythematosus [23–25]. Some successes ubiquinone the terminal oxidanton [22]. in identifying Pfas DHODH inhibitors the basis of the modification of reported hDHODH inhibitors Many hDHODH inhibitors have beenAlthough corroborated to be effective against a number of diseases, (e.g., compound 4) are well known [24]. sharing a highly conserved sequence in their such as cancer, rheumatoid arthritis (RA), psoriasis, and lupus erythematosus [23–25]. Some C-terminal domains, Pf DHODH and hDHODH possess huge variation in terms of the amino acid successes in identifying PfDHODH inhibitors on the basis of the modification of reported hDHODH sequence in the N-terminal domain, which facilitates further discovery of specific inhibitors of inhibitors (e.g., compound 4) are well known [24]. Although sharing a highly conserved sequence in Pf DHODH [22]. In addition, many different kinds of selective and promising Pf DHODH inhibitors their C-terminal domains, PfDHODH and hDHODH possess huge variation in terms of the amino have been developed through target-based screening (e.g., compounds 5–7) (as of shown acid sequence in the N-terminal domain,high-throughput which facilitates further discovery of specific inhibitors in Figure 1) [26–28]. What is more, X-ray structures of Pf DHODH in complex with some representative PfDHODH [22]. In addition, many different kinds of selective and promising PfDHODH inhibitors compounds were used tothrough develop more potent, non-cross-resistant antimalarial agents, have been developed target-based high-throughput screeningselective (e.g., compounds 5–7) (as shown in Figure 1) [26–28]. is more,8 X-ray of PfDHODH in complex with some leading to the identification of What compound (DSMstructures 256), which has progressed in phase II clinical compounds were used to develop potent, development non-cross-resistant selective trialsrepresentative [29]. Furthermore, compound 9 (DSM 421) is amore preclinical candidate for the antimalarial agents, leading to the identification of compound 8 (DSM 256), which has progressed in treatment of malaria, which has improved solubility, lower intrinsic clearance, and increased plasma phase II clinical trials [29]. Furthermore, compound 9 (DSM 421) is a preclinical development exposure after oral dosing compared with DSM 265 [30]. candidate for the treatment of malaria, which has improved solubility, lower intrinsic clearance, and In our previous work, we reported the identification of compound 10 as a Pf DHODH increased plasma exposure after oral dosing compared with DSM 265 [30]. inhibitor by the previous structure-based screening strategyofintegrating Glide docking In our work, wevirtual reported the identification compound molecular 10 as a PfDHODH inhibitor with Prime/MM-GBSA re-scoring on the basis of the complex crystal structure of compound (DSM 1) by the structure-based virtual screening strategy integrating molecular Glide docking 7with with Pf DHODH (PDBre-scoring code 3I65) Compound 10 selectively inhibited DHODH7 (IC Prime/MM-GBSA on[31]. the basis of the complex crystal structure of Pf compound (DSM 50 =1)6 nM, with with >14,000-fold species selectivity over hDHODH) and parasite growth in vitro(IC (IC 15 and PfDHODH (PDB code 3I65) [31]. Compound 10 selectively inhibited PfDHODH 50 50 = 6ofnM, with >14,000-fold species over hDHODH) parasite in vitro (IC50as of a15result and 18of its 18 nM against 3D7 and Dd2selectivity cells, respectively), butand it was lessgrowth effective in vivo against properties 3D7 and Dd2 cells, respectively), but it was less effective in vivo as aon result of its poor poor nM metabolic [31]. Recently, several pyrimidone derivatives based compound 10 as a metabolic properties [31]. Recently, several pyrimidone derivatives based on compound 10 a lead lead compound were obtained in this work and showed high selectivity over hDHODH,assuggesting compound were obtained in this work and showed high selectivity over hDHODH, suggesting potential applications as novel antimalarial agents. potential applications as novel antimalarial agents.

Figure 1. Structures of reportedantimalarial antimalarial agents agents 1–3, inhibitors 4–9,4–9, and and the lead Figure 1. Structures of reported 1–3, PfDHODH Pf DHODH inhibitors the lead compound 10 in this study. compound 10 in this study.

2. Results and Discussion

2. Results and Discussion

2.1. Proposed Binding Pose of Lead Compound 10 in the Ubiquinone Binding Pocket of PfDHODH

2.1. Proposed Binding Pose of Lead Compound 10 in the Ubiquinone Binding Pocket of PfDHODH

To thoroughly explore the binding mode of compound 10 in the ubiquinone binding pocket of

To thoroughly explore the binding mode of compound 10ininMaestro the ubiquinone binding pocket PfDHODH, flexible induced-fit docking [32] was implemented v10.1 on the basis of the of Pf DHODH, flexible induced-fit docking [32] was implemented in Maestro v10.1 on the basis of the

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crystal structure of of PfDHODH Pf DHODHinincomplex complexwith withDSM1 DSM1 (PDB code 3I65). shown in Figure crystal structure (PDB code 3I65). As As shown in Figure 2A, 2A, the the entrance of the ubiquinone binding channel is relatively closed in Pf DHODH; thus compound entrance of the ubiquinone binding channel is relatively closed in PfDHODH; thus compound 10 is 10 is totally buried the protein. The naphthyl group sitsregion in the close region to the entrance totally buried insideinside the protein. The naphthyl group sits in the toclose the entrance and fits and fits the pocket well through hydrophobic interactions with adjacent residues such as Met536, the pocket well through hydrophobic interactions with adjacent residues such as Met536, Leu197, Leu197, Ile237, and Phe227. The group ester group is located almost sameplane plane of of the Leu240, Leu240, Ile237, and Phe227. The ester is located almost in in thethesame the dihydrofuranone moiety, with its carbonyl group pointing in the same direction as that on the dihydrofuranone moiety, with its carbonyl group pointing in the same direction as that on the dihydrofuranone Both the the carbonyl dihydrofuranone ring. ring. Both carbonyl groups groups could could form form favorable favorable hydrogen hydrogen bond bond interactions interactions with thethe binding pocket nearnear Arg265 has a has strongly positivepositive electrostatic surface with Arg265. Arg265.Additionally, Additionally, binding pocket Arg265 a strongly electrostatic potential, which is complementary to the two carbonyl groups with negative electrostatic potential surface potential, which is complementary to the two carbonyl groups with negative electrostatic (the minimum values ofESP the two oxygen atoms areoxygen both −58.53 kcal/mol). additional hydrogen potential (the ESP minimum values of the two atoms are bothAn −58.53 kcal/mol). An bond is presumably the bridging and His185, which is considered to additional hydrogenformed bond isbetween presumably formed nitrogen between atom the bridging nitrogen atom and His185, be essential for the activities and selectivities of Pf DHODH inhibitors. The ethyl group extends to the which is considered to be essential for the activities and selectivities of PfDHODH inhibitors. The cavity near the FMNtobinding site,near forming beneficial hydrophobic effects with hydrophobic residues Ile263 and ethyl group extends the cavity the FMN binding site, forming beneficial effects Ile272 that could helpand to enhance thecould potency 10.potency of compound 10. with residues Ile263 Ile272 that helpoftocompound enhance the 2.2. The Structural 2.2. The Structural Optimization Optimization Strategy Strategy using using the the Lead Lead Compound Compound 10 10 According to rational drug design and the proposed binding mode described above, the desired According to rational drug design and the proposed binding mode described above, the desired Pf DHODH inhibitor should should embrace embrace two two functional functional parts, parts, aa hydrogen PfDHODH inhibitor hydrogen bond bond forming forming “head” “head” linked linked via a nitrogen to a hydrophobic “tail”. In the hydrophilic pocket composed by subsites 2 and 3, via a nitrogen to a hydrophobic “tail”. In the hydrophilic pocket composed by subsites 2 and 3, the the functional groups (e.g., carbonyl groups) could form hydrogen bonds with the guanidine group functional groups (e.g., carbonyl groups) could form hydrogen bonds with the guanidine group of of Arg265, which is indispensable for maintaining the inhibitory activity against Pf DHODH. As for Arg265, which is indispensable for maintaining the inhibitory activity against PfDHODH. As for the the hydrophobic pocket of subsite 1, the naphthyl ring is more suitable compared with the reported hydrophobic pocket of subsite 1, the naphthyl ring is more suitable compared with the reported Pf DHODH inhibitors inhibitors in in terms terms of of the the size size that that the can accommodate Consequently, with PfDHODH the cavity cavity can accommodate [31]. [31]. Consequently, with the attempt to discover more potent Pf DHODH inhibitors for further pharmacological study, as the attempt to discover more potent PfDHODH inhibitors for further pharmacological study, as well well as to verify the structure–activity relationship, an optimization strategy using the lead compound as to verify the structure–activity relationship, an optimization strategy using the lead compound 10 10 was carried out through three modifications: (1) hydrophobic modification on the aromatic ring, was carried out through three modifications: (1) hydrophobic modification on the aromatic ring, (2) (2) hydrophilic group modification on the dihydrofuranone ring, and (3) cyclization forming a new hydrophilic group modification on the dihydrofuranone ring, and (3) cyclization forming a new scaffold scaffold to to enhance enhance the the stability stability (as (as shown shown in in Figure Figure 3). 3).

2. (A) ubiquinone binding pocket of Figure 2. (A) Proposed binding binding pose of lead compound 10 in the ubiquinone PfDHODH. Pf DHODH. Key residues around the binding pocket are shown as white lines and ligand is shown as sticks. The labeled as as black black dashes. dashes. (B) Electrostatic Electrostatic complementarity complementarity green sticks. The hydrogen bonds are labeled dihydrofuranone moiety of of compound compound 10 10 and and the the binding binding site site near nearArg265. Arg265. The The between the dihydrofuranone potential surface surface of of Pf PfDHODH shown between between − −20 and +20 +20 kT/e kT/e (blue) was electrostatic potential DHODH shown 20 (red) and calculated using usingAPBS APBStool tool [33–35]. of dihydrofuranone the dihydrofuranone fragment was calculated by calculated [33–35]. TheThe ESPESP of the fragment was calculated by Jaguar with DFT at B3LYP/cc-pVTZ(-f) level. level. Jaguar with DFT at B3LYP/cc-pVTZ(-f)

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Figure 3. Diagram for the structural optimization strategy. Figure 3. 3. Diagram Diagram for for the the structural structural optimization optimization strategy. strategy. Figure Figure 3. Diagram for the structural optimization strategy.

2.3. Chemistry 2.3. Chemistry Chemistry 2.3. 2.3. Chemistry The lead compound 10, with a dihydrofuranone core, was regarded as the well-designed The lead lead compound 10,initial with aoptimization. a dihydrofuranone dihydrofuranone core,replaced was regarded regarded as metabolized the well-designed well-designed structural foundation in10, our First, we the ester The compound 10, core, was as the The lead compound withwith a dihydrofuranone core, was regarded as theeasily well-designed structural structural foundation in our initial optimization. First, we replaced the easily metabolized ester group withfoundation anour amide group. preparation theFirst, amide started compound 11, structural inoptimization. our The initial optimization. wederivatives replaced the easilywith metabolized ester foundation in initial First, weofreplaced the easily metabolized ester group with an group with an amide group. The preparation of the amide derivatives started with compound 11, which served as an important intermediate (Scheme 1). To avoid byproducts during ester group with an amide group. The preparation of the amide derivatives started with compound amide group. The preparation of the amide derivatives started with compound 11, which served as 11, an which served served as an an important important intermediate (Scheme 1). 1). ToWith avoid byproducts duringstudy ester hydrolysis, the temperature must controlled accurately [36]. the byproducts purpose to further which as intermediate (Scheme To avoid during ester important intermediate (Scheme 1). be To avoid byproducts during ester hydrolysis, the temperature must hydrolysis, the temperature must be controlled accurately [36]. With the purpose to further study the structure–activity relationships improve the pharmacological properties, a series of hydrolysis, the temperature controlled [36]. the With the purpose to further study be controlled accurately [36]. must With be theand purpose toaccurately further study structure–activity relationships the structure–activity relationships and improve the pharmacological properties, a series of dihydrofuranone derivatives were designed and synthesized (Scheme 1) [37]. Amides were the improve structure–activity relationships and improve thedihydrofuranone pharmacological properties, a designed series of and the pharmacological properties, a series of derivatives were dihydrofuranone derivatives were designed designed and synthesized (Scheme 1)obtained [37]. Amides Amides were synthesized in the presence of HOBt, EDCI, DIPEA. Compound 20 of was replacing dihydrofuranone derivatives were and synthesized (Scheme 1) [37]. were and synthesized (Scheme 1) [37]. Amides wereand synthesized in the presence HOBt, EDCI,by and DIPEA. synthesized in the presence of HOBt, EDCI, and DIPEA. Compound 20 was obtained by replacing alkylamines with hydrazine hydrate and using N, N'-carbonyldiimidazole (CDI) and in routes synthesized 20 in was the80% presence ofbyHOBt, EDCI, and DIPEA. Compound 20 was hydrate obtained bythe replacing Compound obtained replacing alkylamines with 80% hydrazine using N, alkylamines with 80% hydrazine hydrate and using N, N'-carbonyldiimidazole (CDI) in the routes depicted in Scheme 2. alkylamines with 80% hydrazine hydrate usinginN,Scheme N'-carbonyldiimidazole (CDI) in the routes N'-carbonyldiimidazole (CDI) in the routesand depicted 2. depicted in in Scheme Scheme 2. 2. depicted

Scheme 1. Reagents and conditions: (a) sodium hydride, diethyl malonate, 2-chloroacetyl chloride, Scheme 1. Reagents and conditions: (a) sodium hydride, diethyl malonate, 2-chloroacetyl chloride, and Scheme 1.atReagents and conditions: (a) sodium sodium hydride, diethyl malonate, 2-chloroacetyl chloride, and THF,1. rt for 1 h,and at 40–45 °C for 5h, and under reflux for 18 h;malonate, (b) LiOH–H 2O, MeOH–H 2O (55– ◦ Scheme (a) hydride, 2-chloroacetyl chloride, THF, at rt forReagents 1 h, at 40–45 ◦conditions: C for 5h, and under reflux for 18 diethyl h; (b) LiOH–H 2 O, MeOH–H2 O (55–60 C) and THF, at rt for 1 h, at 40–45 °C for 5h, and under reflux for 18 h; (b) LiOH–H 2 O, MeOH–H 2O (55– 60 °C) for 12 h. and12THF, for h. at rt for 1 h, at 40–45 °C for 5h, and under reflux for 18 h; (b) LiOH–H2O, MeOH–H2O (55– 60 °C) °C) for for 12 12 h. h. 60 O O O N O O Ar H O 1 CONHR Ar N N H 12-19CONHR 1 H CONHR 1 12-19 R1 = Me, Et,12-19 n-Pr, n-Bu, cyclo-Pr R1 = and Me, cyclopropylmethyl Et, n-Pr, n-Bu, cyclo-Pr R1 = Me, Et, n-Pr, n-Bu, cyclo-Pr and cyclopropylmethyl and cyclopropylmethyl O O Ar O N O O Ar H O CONH-NH Ar N 2 N H CONH-NH H 20 CONH-NH 2 2 20 20 Ar

c) O O O N O O Ar H O COOH Ar N N H COOH H COOH

Ar

c) c) d) d) d)

Scheme 2. Reagents and conditions: (c) RNH2 , HOBt, EDC, DIPEA, and CH2 Cl2 , overnight at rt; (d) Scheme 2. Reagents and conditions: (c) RNH2, HOBt, EDC, DIPEA, and CH2Cl2, overnight at rt; (d) 80% hydrazine hydrate and N,N 0 -carbonyldiimidazole (CDI), overnight atCH rt. 2Cl2, overnight at rt; (d) Scheme 2. Reagents Reagents and conditions: (c) RNH RNH22,, HOBt, HOBt, (CDI), EDC, DIPEA, DIPEA, and 80% hydrazine hydrate and N,N′-carbonyldiimidazole overnight atCH rt. 2Cl2, overnight at rt; (d) Scheme 2. and conditions: (c) EDC, and 80% hydrazine hydrate and N,N′-carbonyldiimidazole (CDI), overnight at rt. 80% hydrazine hydrate and N,N′-carbonyldiimidazole (CDI), overnight at rt.

According to the predicted binding pose of the lead compound 10, two carbonyl groups that According todirection the predicted predicted bindingfor pose of the lead lead compound 10,we two carbonyl groups that that in the sameto are necessary the potent activity. Therefore, used paraformaldehyde orientAccording the of potent activity. used paraformaldehyde the binding pose the compound 10, two carbonyl groups orient in the same direction are necessary for the potent activity. Therefore, we used paraformaldehyde forminthe two carbonyl groupwe orientations well in in space. space. to ring that could the two carbonyl group orientations well orient therigid samesix-membered direction are necessary for thefix potent activity. Therefore, used paraformaldehyde to form the rigid six-membered ring that could fix the two carbonyl group orientations well in space. this reason, compounds 13–16,18 and 20 were subsequently transformed to the pyrimidone For subsequently to form the rigid six-membered ring that could fix the two carbonyl group orientations well in space. For this this reason, reason, compounds 13–16,18 and 20 were were subsequently transformed towere the pyrimidone pyrimidone derivatives 21–26 compounds by cyclization (Schemeand 3) [38]. All structures of thetransformed final productsto determined For 13–16,18 20 subsequently the derivatives 21–26 by cyclization (Scheme 3) [38]. All structures of the final products were determined spectroscopic by derivatives 21–26techniques. by cyclization (Scheme 3) [38]. All structures of the final products were determined by spectroscopic techniques. by spectroscopic techniques.

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Scheme 3. Reagents and conditions: (e) paraformaldehyde, NaOH and EtOH, at rt for 1 h and at 70 ◦ C Scheme 3. Reagents and conditions: (e) paraformaldehyde, NaOH and EtOH, at rt for 1 h and at for another 8 h. 70 °C for another 8 h.

2.4. 2.4. Inhibitory Inhibitory Activities Activities against against PfDHODH PfDHODH and and SAR SAR Study Study 2.4.1. Hydrophobic Group Modification 2.4.1. Hydrophobic Group Modification Because the Pf DHODH inhibition potency was particularly sensitive to the substitution pattern at Because the PfDHODH inhibition potency was particularly sensitive to the substitution pattern the phenyl ring [39], an optimization strategy was carried out: modification on the hydrophobic moiety at the phenyl ring [39], an optimization strategy was carried out: modification on the hydrophobic substituted by phenyl (compound 12) and 5-aminoindan (compound 13). Compared with compounds moiety substituted by phenyl (compound 12) and 5-aminoindan (compound 13). Compared with 12 and 13, compound 15 containing a 2-naphthyl moiety was more active against Pf DHODH, with compounds 12 and 13, compound 15 containing a 2-naphthyl moiety was more active against an IC50 value of 4.8 µM. Notably, the phenyl ring as the hydrophobic group reversed the species PfDHODH, with an IC50 value of 4.8 µM. Notably, the phenyl ring as the hydrophobic group selectivity of compound 12, which showed an IC50 value of 2.4 µM against hDHODH and was inactive reversed the species selectivity of compound 12, which showed an IC50 value of 2.4 µM against against Pf DHODH. The above results validate that using 2-naphthyl as the hydrophobic group is hDHODH and was inactive against PfDHODH. The above results validate that using 2-naphthyl as beneficial to keep a high potency against Pf DHODH (as shown in Table 1). the hydrophobic group is beneficial to keep a high potency against PfDHODH (as shown in Table 1). 2.4.2. Hydrophilic Group Modification 2.4.2. Hydrophilic Group Modification On the basis of previous results using 2-naphthyl as the hydrophobic group (R1 = 2-naphthyl), On the basis of previous results using 2-naphthyl as the hydrophobic group (R1 = 2-naphthyl), compounds 11 and 14–20 were obtained. In this section, the emphasis is on evaluating the effect of compounds 11 and 14–20 were obtained. In this section, the emphasis is on evaluating the effect of the structural changes of the hydrophilic group on the inhibitory activity against Pf DHODH. In the the structural changes of the hydrophilic group on the inhibitory activity against PfDHODH. In the absence of an ethoxycarbonyl group on the dihydrofuranone scaffold, compound 11 displayed lower absence of an ethoxycarbonyl group on the dihydrofuranone scaffold, compound 11 displayed inhibitory activity in comparison with 10, which suggests that it would be strongly disfavored if the lower inhibitory activity in comparison with 10, which suggests that it would be strongly disfavored derivatives do not possess any substituent at the 3-position of the scaffold (as shown in Figure 3). Next, if the derivatives do not possess any substituent at the 3-position of the scaffold (as shown in Figure derivatives with different amide groups (compounds 12–20) were synthesized to identify the optimal 3). Next, derivatives with different amide groups (compounds 12–20) were synthesized to identify alkyl chain that could fit the binding cavity. Amides with different chain lengths were introduced to the optimal alkyl chain that could fit the binding cavity. Amides with different chain lengths were investigate their effects on the inhibitory activities. Compared with compound 15 (IC50 = 4.8 µM), introduced to investigate their effects on the inhibitory activities. Compared with compound 15 (IC50 compound 14 (IC50 = 9 µM) showed a decline in the inhibitory activity against Pf DHODH; the reason = 4.8 µM), compound 14 (IC50 = 9 µM) showed a decline in the inhibitory activity against PfDHODH; may be attributed to the shorter alkyl chain, which mismatched the size of the cavity. However, the reason may be attributed to the shorter alkyl chain, which mismatched the size of the cavity. compounds 16 and 17 lost their activities because of the long alkyl chains, which may have resulted in However, compounds 16 and 17 lost their activities because of the long alkyl chains, which may collisions with residues such as Ile263 and Ile272. Compound 18 inhibited Pf DHODH with an IC50 have resulted in collisions with residues such as Ile263 and Ile272. Compound 18 inhibited value of 6 µM, which indicated that the size of the cyclopropyl was suitable for the hydrophobic cavity PfDHODH with an IC50 value of 6 µM, which indicated that the size of the cyclopropyl was suitable to some extent. However, compound 19 with cyclopropanemethylamine lost the inhibitory activity for the hydrophobic cavity to some extent. However, compound 19 with cyclopropanemethylamine against Pf DHODH owing to the increased size of the alkyl side chain. According to our present lost the inhibitory activity against PfDHODH owing to the increased size of the alkyl side chain. study, ethyl and cyclopropyl are well matched with the size and shape of the cavity. Compound 20 According to our present study, ethyl and cyclopropyl are well matched with the size and shape of equipped with the amino displayed an IC50 value of 70 nM, likely a result of forming a hydrogen bond the cavity. Compound 20 equipped with the amino displayed an IC50 value of 70 nM, likely a result interaction with the adjacent residue His185. Therefore, a suitable substituent group in the 3-position of forming a hydrogen bond interaction with the adjacent residue His185. Therefore, a suitable and the amino group forming the hydrogen bonding interaction with His185 in Pf DHODH played a substituent group in the 3-position and the amino group forming the hydrogen bonding interaction vital role for potent activities of Pf DHODH inhibitors (as shown in Table 1). with His185 in PfDHODH played a vital role for potent activities of PfDHODH inhibitors (as shown in Table 1).

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Table 1. 1. Structure Structure and and activity activity profiles profiles of of compounds compounds 11–26 11–26 against against PfDHODH PfDHODH and and human human DHODH DHODH Table 1. Structure and activity profiles of compounds 11–26 against PfDHODH and human DHODH Table Table 1. 1. Structure Structure and and activity activity profiles profiles of of compounds compounds 11–26 11–26 against against Pf PfDHODH and human human DHODH DHODH Table DHODH and (hDHODH) in enzyme assays. (hDHODH) in enzyme enzyme assays. profiles of compounds 11–26 against PfDHODH and human DHODH Table 1. Structure and activity (hDHODH) in assays. (hDHODH) in enzyme assays. (hDHODH) in enzyme assays. (hDHODH) in enzyme assays.

IC50 50 (μM) (μM) IC 50 IC IC50(μM) (μM) IC 5050(μM) IC (µM) a 1 2 PfDHODH aa hDHODH hDHODH bbb Compound R11 R22 PfDHODH hDHODH Compound R R PfDHODH Compound R R PfDHODH a hDHODH b Compound R1 R2 11 2-Naphthyl COOH 0.106 ± 0.009 0.009 >10 b b a 1 1 2 11 2-Naphthyl COOH 0.106 ± 0.009 >10 PfDHODH hDHODH Compound R R a 2 11 2-Naphthyl COOH 0.106 ± >10 Compound Pf0.106 DHODH R R hDHODH 11 2-Naphthyl COOH ± 0.009 >10 12 Phenyl CONHCH 2 CH 3 >10 2.4 0.1 12 Phenyl CONHCH CH33 >10 2.4>10 ±± 0.1 11 2-Naphthyl COOH 0.106 ±0.009 0.009 1112 2-Naphthyl COOH222CH 0.106 ±>10 >10 Phenyl CONHCH 2.4 12 Phenyl CONHCH CH3 >10 2.4±±0.1 0.1 13 5-Aminoindan CONHCH 2CH CH 3 >10 >10 1213 Phenyl CONHCH >10 2.4 ± 0.1 13 5-Aminoindan CONHCH 2 CH 3 >10 >10 12 Phenyl 2.4 ± 0.1 222CH333 5-Aminoindan CONHCH >10 >10 13 5-Aminoindan CONHCH2CH3 >10 >10 1314 5-Aminoindan CONHCH CH >10 >10 14 2-Naphthyl CONHCH 3 9 ± 1 >10 2 3 2-Naphthyl CONHCH >10 13 5-Aminoindan CONHCH 2CH 14 2-Naphthyl CONHCH 333 3 99>10 ±± 111 >10 14 2-Naphthyl CONHCH >10 1415 2-Naphthyl CONHCH 94.8 ±9±1±0.4 3 3 2-Naphthyl CONHCH 2CH CH >10>10 15 2-Naphthyl CONHCH 2 3 4.8 ± 0.4 >10 14 CONHCH 3 9 1 2-Naphthyl CONHCH 22CH 33 4.8 ±0.4 0.4 >10 15 2-Naphthyl CONHCH CH 4.8 ± 0.4 >10>10 1515 2-Naphthyl CONHCH CH 4.8 ± 2 3 16 2-Naphthyl CONHCH CH CH 3 >10 >10 16 2-Naphthyl CONHCH 22CH CH 22CH CH >10 >10 15 CONHCH 2CH 3 33 4.8 ± 0.4 2-Naphthyl CONHCH 2 2 >10 >10 >10 CONHCH CH CH 1616 2-Naphthyl 16 33 2-Naphthyl CONHCH22CH2 2CH >10 >10>10 17 2-Naphthyl CONHCH CH CH CH >10 >10>10 17 2-Naphthyl CONHCH 22CH CH 22CH CH 22CH CH 33 3 >10 >10 16 1717 2-Naphthyl >10 CONHCH CONHCH 2CH CH 2CH CH 3CH 2-Naphthyl CONHCH 2 2 2 3 >10 >10 2 2 2 17 2-Naphthyl CONHCH2CH2CH2CH3 >10 >10 17 2-Naphthyl CONHCH2CH2CH2CH3 >10 >10 18 2-Naphthyl 6 ± 1 >10 18 2-Naphthyl >10>10 1818 2-Naphthyl 6± 2-Naphthyl 666±±1±111 >10 18 2-Naphthyl >10 18 2-Naphthyl 6±1 >10 19 2-Naphthyl >10 >10>10 19 2-Naphthyl >10 >10 1919 2-Naphthyl >10 2-Naphthyl >10 >10 19 2-Naphthyl >10 >10 20 2-Naphthyl CONH–NH 0.07 ±0.01 0.01 >10>10 19 >10 20 2-Naphthyl CONH–NH 22 0.07 ± 0.01 >10 2020 2-Naphthyl CONH–NH 0.07 ± 2-Naphthyl CONH–NH 2 0.07 ± 0.01 >10 22 20 2-Naphthyl CONH–NH 0.07 ± 0.01 >10 20 2-Naphthyl CONH–NH2 0.07 ± 0.01 >10

21 21

5-Aminoindan CH222CH CH333 >10 >10 5-Aminoindan CH CH >10 >10 5-Aminoindan CH >10 >10 5-Aminoindan CH2 CH 2CH >10 >10>10 5-Aminoindan CH >10 33 2-Naphthyl CH 3 8 ± 1 >10>10 2-Naphthyl CH 3 3 >10 5-Aminoindan CH 2CH 2-Naphthyl CH 888>10 ±±1±111 >10 2-Naphthyl CH 8± 333 2-Naphthyl CH >10 2-Naphthyl CH CH 3 >10>10 2-Naphthyl CH 22CH CH 448 ±±±1 111 >10 2-Naphthyl CHCH CH 4± 3 333 2 2-Naphthyl CH 2 4 >10 2-Naphthyl CH2CH3 4±1 >10 2-Naphthyl CH CH CH 3 12 >10>10 2-Naphthyl CH CH 1212 2222CH 2222CH 2-Naphthyl CH CH CH 12 >10 CH 2CH 3 333 4±±±±±6±16666 2-Naphthyl CH >10 24 2-Naphthyl CH 2CH 2CH CH 3 12 >10 24 2-Naphthyl CH2CH2CH3 12 ±16 >10>10 25 2525 2-Naphthyl 5 ± 1 2-Naphthyl 5 ± >10 2-Naphthyl >10 25 2-Naphthyl 555±±±111 >10 25 2-Naphthyl >10 25 2-Naphthyl 5 ± 1 >10>10 2626 2-Naphthyl NH222 0.023 ±± 0.001 26 2-Naphthyl NH 0.023 ± 0.001 >10 2-Naphthyl NH 0.023 0.001 >10 26 2-Naphthyl NH 2 0.023 ±±0.001 >10 26 2-Naphthyl NH 0.023 0.001 >10— DSM1 — 2 0.042 ± ±0.004 DSM1 —— — 0.042 0.004 — DSM1 — — 0.042 ± 0.004 0.004 — 26 2-Naphthyl NH 2 0.023 0.001 >10 DSM1 — — 0.042 ± — — — 0.042 ± 0.004 — a The DSM1 b IC values of the compounds against Pf DHODH, in vitro assay, µM. The IC values of the compounds a b 50 50 The DSM1 IC50 50 values values of of the the compounds compounds against PfDHODH, PfDHODH, in vitro vitro assay, assay, µM. The IC IC50 50 values values of the the aa The bb The IC 50 of the compounds against PfDHODH, in vitro assay, µM. The IC 50 of the — — 0.042 ± 0.004 — IC values against in µM. values aThe b against hDHODH, in vitro assay, µM. The IC50 values of the compounds against PfDHODH, in vitro assay, µM. The IC50 values of of the 21 2121 22 22 21 2222 22 23 23 2323 22 23 24 2424 24 23

compounds against hDHODH, in vitro vitro assay,PfDHODH, µM. a The IC50 values compounds against hDHODH, in vitro assay, µM. of hDHODH, the compounds against in vitro assay, µM. b The IC50 values of the compounds in assay, compoundsagainst against hDHODH, in vitro assay,µM. µM. against hDHODH, vitro assay, µM. 2.4.3.compounds Cyclization Forming a NewinScaffold

2.4.3. Cyclization Cyclization Forming Forming aaa New New Scaffold Scaffold 2.4.3. Cyclization New 2.4.3. 2.4.3. CyclizationForming Forming a NewScaffold Scaffold After cyclization forming a new scaffold, a six-membered ring could stabilize two carbonyl groups 2.4.3.After Cyclization Forming a New Scaffold cyclization forming a new scaffold, aaa six-membered six-membered ring ring could could stabilize stabilize two two carbonyl carbonyl After cyclization cyclization forming forming aa new new scaffold, scaffold, six-membered ring could stabilize two carbonyl After After in cyclization forming awhich new formed scaffold,hydrogen a six-membered ring couldofstabilize two It carbonyl orientated the same direction, bonds with Arg265 Pf DHODH. is clear groups orientated in the the same direction, direction, which formed formed hydrogen bonds bonds with stabilize Arg265 of of PfDHODH. groups orientated in the same direction, which formed hydrogen bonds with Arg265 of PfDHODH. After cyclization forming a new scaffold, a six-membered ring could two carbonyl groups orientated in same which hydrogen with Arg265 PfDHODH. groups orientated in the same direction, which formed hydrogen bonds with Arg265 of PfDHODH. that compound 22–26 showed inhibitory activities compared with the corresponding uncyclized It is clear clear that compound compound 22–26higher showed higher inhibitory activities bonds compared with the corresponding corresponding It is that compound 22–26 showed higher inhibitory activities compared with the corresponding groups orientated in the same direction, whichinhibitory formed hydrogen withwith Arg265 of PfDHODH. It that showed activities the Itisisclear clear that compound 22–26 showed higher inhibitory activities compared with the corresponding amide mimics (Table 1). 22–26 The reason forhigher all these compounds withcompared the new scaffold presenting high uncyclized amide mimics22–26 (Table 1). The The reason for all all activities these compounds compounds with the thecorresponding new scaffold scaffold uncyclized amide mimics (Table 1). The reason for all these compounds with the new scaffold Ituncyclized is clear that compound showed higher inhibitory compared with uncyclized amide mimics (Table 1). reason for these new amide mimics (Table 1). The reason for allfrom these compounds with the the new scaffold inhibitory activities against Pf DHODH can be elucidated the predicted binding pose in our work. presenting high inhibitory activities against PfDHODH can compounds be elucidated elucidated from the predicted presenting high inhibitory activities against PfDHODH can be elucidated from the predicted uncyclized amide mimics (Table 1). The reason for all these with thethe newpredicted scaffold presenting high inhibitory activities against PfDHODH can be from presenting high inhibitory activities against PfDHODH canbridge be elucidated from the predicted The predicted binding poseThe of compound 10 revealed that the nitrogen atom not only linked binding pose in our work. predicted binding pose of compound 10 revealed that the bridge binding pose in our our work. The The predicted binding pose of of can compound 10 revealed revealed the bridge presenting high inhibitory activities against PfDHODH be elucidated fromthat thethe predicted binding pose in work. predicted binding 10 bridge binding pose in group our work. The predicted binding posealso of compound compound 10 revealed that bridge the hydrophilic to the hydrophobic group, pose but interacted with His185 that via a the hydrogen nitrogen atomin notour only linked the hydrophilic group to the the hydrophobic group, but that also the interacted nitrogen atom not only linked the hydrophilic group to the hydrophobic group, but also interacted binding pose work. The predicted binding pose of hydrophobic compound 10group, revealed bridge nitrogen atom only linked the group to but nitrogen atomisnot not only linked the hydrophilic group to the hydrophobic group, butalso alsointeracted interacted bond, which very important forhydrophilic the inhibitory activity. We designed new Pf DHODH inhibitors with His185 via a hydrogen bond, which is very important for the inhibitory activity. We designed with His185 via a hydrogen bond, which is very important for the inhibitory activity. We designed nitrogen atom not only linked the hydrophilic group to the for hydrophobic group, but also interacted with His185 via bond, which isisvery important activity. We designed withcontained His185 via ahydrogen hydrogen bond, which very important forthe theinhibitory inhibitory activity. Wecompound designed that aainhibitors new hydrogen bond donor interacting withbond His185. In our present study, new PfDHODH that contained new hydrogen hydrogen donor interacting with His185. In new PfDHODH inhibitors that contained aa new new hydrogen bond donor interacting with His185. In with His185 via a hydrogen bond, which is very important for the inhibitory activity. We designed new PfDHODH inhibitors that contained a bond donor interacting with His185. In new PfDHODH inhibitors that contained a new hydrogen bond donor interacting with His185. In 26 was the most potent candidate against Pf DHODH, which had an IC value equal to 23 nM and 50 our present study, compound 26 was the the most most potent candidate against PfDHODH,with which had an an our present study, compound 26 was the most potent candidate against PfDHODH, which had an new PfDHODH inhibitors that26 contained amost newpotent hydrogen bond against donor interacting His185. In our present study, compound was candidate PfDHODH, which had our present study, compound 26 was the potent candidate against PfDHODH, which had an was 3-foldequal moretoactive than 20. The predicted binding pose compound is shown in IC 50 value value 23 nM andcompound was 3-fold more active than compound 20.of The predicted26 binding pose IC 50 equal to 23 23 nM and was 3-fold more active than compound 20. The predicted binding pose our present study, compound 26 was the most potent candidate against PfDHODH, which had an IC equal to nM and 3-fold more active than compound 20. The predicted binding pose IC5050value value equal to 23 nM andwas was 3-fold more active than compound 20. The predicted binding pose Figure 4. Two carbonyl groups formed hydrogen bonds with residues Arg265, and the nitrogen atom of compound 26to is shown shown in Figure Figure 4. Two Two carbonyl groups formed hydrogen hydrogen bonds with with residues of compound 26 is shown in Figure 4. Two carbonyl groups formed hydrogen bonds with residues IC value equal nM and was 3-fold more active than compound 20. The predicted binding pose of compound 26 in 4. carbonyl groups formed bonds residues of50 compound 26isis23 shown inanFigure 4. Two carbonyl groups formed hydrogen bonds with residues in the amino group formed additional hydrogen bond with His185. Briefly, cyclization enhanced Arg265, and the nitrogen atom in the amino group formed an additional hydrogen bond with Arg265, and the nitrogen atom in the amino group formed an additional hydrogen bond with of compound 26 is shown in Figure 4. Two carbonyl groups formed hydrogen bonds with residues Arg265, and nitrogen in group an hydrogen bond with Arg265, and the the nitrogen atom in the the amino amino group formed formed an additional additional hydrogen bond with the structural stability andatom maintained the hydrogen bond interactions between the two carbonyl His185. Briefly, cyclization enhanced the structural stability and maintained the hydrogen bond His185. Briefly, cyclization enhanced the structural stability and maintainedhydrogen the hydrogen hydrogen bond Arg265, and thecyclization nitrogen atom in thethe amino groupstability formedand an additional bond bond with His185. Briefly, enhanced structural maintained His185.and Briefly, cyclization enhanced the structural stability and maintained the the hydrogen bond groups the key residue Arg265. interactions between the two two carbonyl carbonyl groups groups and the the key key residue residue Arg265. interactions between the two carbonyl groups and the key residue Arg265. His185. Briefly, cyclization structural andArg265. maintained the hydrogen bond interactions between the and interactions between the twoenhanced carbonyl the groups and thestability key residue Arg265. interactions between the two carbonyl groups and the key residue Arg265.

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4. Proposed binding pose of compound compound 26 in in the the ubiquinone ubiquinone binding binding pocket of Pf PfDHODH. Figure 4. DHODH. Key residues residuesaround aroundthe thebinding binding pocket shown as white and ligand is shown as sticks. green Key pocket areare shown as white lines,lines, and ligand is shown as green sticks. The hydrogen bonds are labeled black dashes. The hydrogen bonds are labeled as blackasdashes.

3. Materials Materials and and Methods Methods 3. All chemical chemical reagents reagents and and solvents solvents were were obtained obtained from from commercial commercial sources sources and and were were used used All without further furtherpurification. purification.Melting Melting points (Mp’s) were recorded a WRS-1B-digital melting without points (Mp’s) were recorded on a on WRS-1B-digital melting point point apparatus and are uncorrected. High-resolution electron mass spectra (ESI-TOF) were apparatus and are uncorrected. High-resolution electron mass spectra (ESI-TOF) were produced 1 13 1 13 produced using a Micromass LC-TOF spectrometer (Waters, H- and C-NMR using a Micromass LC-TOF spectrometer (Waters, Milford, MA,Milford, US). H-MA, and US). C-NMR spectra were 13C at 100 MHz) spectrometer (Bruker, 13 400 spectra were on a Bruker (1H at recorded on arecorded Bruker AM-400 (1 H AM-400 at 400 MHz; C atMHz; 100 MHz) spectrometer (Bruker, Fallanden, Fallanden, Switzerland)) with DMSO-d6 or CDCl3 as the solvent and TMS as the internal standard. Switzerland)) with DMSO-d 6 or CDCl3 as the solvent and TMS as the internal standard. Chemical Chemical shifts are in million). δ (parts Analytical per million). Analytical thin-layer chromatography (TLC) shifts are reported in reported δ (parts per thin-layer chromatography (TLC) was performed was performed on precoated plates (silica gel 60 F254), and spots were visualized with ultraviolet on precoated plates (silica gel 60 F254), and spots were visualized with ultraviolet (UV) light. The (UV) light.are Theused following are used explain the multiplicities: d =t doublet, q= following to explain the to multiplicities: s = singlet, ds == singlet, doublet, = triplet,t q= triplet, = quartet, quartet, m = multiplet, and integration. m = multiplet, couplingcoupling constantconstant (Hz), and(Hz), integration. 3.1. In Vitro Enzyme Assay

Protein expression, expression,purification, purification, enzyme activity determination of PfDHODH (Phe158– Protein andand enzyme activity determination of Pf DHODH (Phe158–Ser569) Ser569) and hDHODH (Met30–Arg396) were performed to our previous [31]. The and hDHODH (Met30–Arg396) were performed accordingaccording to our previous work [31].work The enzymes enzymes were to a final concentration of the 10 nM using the assay buffer containing 50 mM were diluted to adiluted final concentration of 10 nM using assay buffer containing 50 mM HEPES (pH 8.0), HEPES mM KCl,supplemented 0.1% Triton X-100 supplemented µM UQ 0, and 120 was µM 150 mM (pH KCl, 8.0), 0.1%150 Triton X-100 with 100 µM UQ0 , andwith 120 100 µM DCIP. The mixture DCIP. The mixture was transferred into a 96-well plate and incubated with or without various transferred into a 96-well plate and incubated with or without various amounts of inhibitors for 5 min amounts of inhibitors and for 5then minthe at room temperature, thentothe dihydroorotate was at room temperature, dihydroorotate wasand added a final concentration of added 500 µMtotoa final concentration 500 µM to was initiate the reaction. The reaction was monitored the initiate the reaction. of The reaction monitored by measuring the decrease in DCIPby in measuring the absorption decrease in sthe absorption nm Brequinar at each 30and s over a period of 6 min. as Brequinar and at 600 nminatDCIP each 30 over a period at of 600 6 min. DSM1 were measured the positive DSM1 were measuredand as the positive controls for hDHODH and PfDHODH, controls for hDHODH Pf DHODH, respectively. Percent inhibition relative torespectively. no inhibitor Percent control inhibition relative inhibitor control was calculated by Vi50 /Vvalue 0) × 100. Forinhibitor, the determination of was calculated by (1to−no Vi /V For the determination of (1 the−IC of the eight or nine 0 ) × 100. the IC50 value of the inhibitor, eightEach or nine different concentrations were applied. Each inhibitor different concentrations were applied. inhibitor concentration point was tested in triplicate, and the concentration point was tested in triplicate, and the IC 50 values were calculated using the sigmoidal IC values were calculated using the sigmoidal fitting option of the program Origin 8.0 (Originlab, 50 fitting option of theUS). program Origin 8.0 (Originlab, Northampton, MA, US). Northampton, MA, 3.2. Chemistry Experiment 3.2.1. General Procedure for the Synthesis of Intermediates Synthesis of the ethyl 2-(substituted arylamino)-4-oxo-4,5-dihydrofuran-3 carboxylate Diethyl malonate (63 mmol) dissolved in dry THF (15 mL) was added to a stirred solution of sodium hydride (71 mmol, 60%) in dry THF (10 mL), and the mixture was stirred for 20 min at 0–10

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3.2. Chemistry Experiment 3.2.1. General Procedure for the Synthesis of Intermediates Synthesis of the ethyl 2-(substituted arylamino)-4-oxo-4,5-dihydrofuran-3 carboxylate Diethyl malonate (63 mmol) dissolved in dry THF (15 mL) was added to a stirred solution of sodium hydride (71 mmol, 60%) in dry THF (10 mL), and the mixture was stirred for 20 min at 0–10 ◦ C. Molecules 2018, 23, x FOR PEER REVIEW 8 of 14 Then the reaction mixture was stirred at room temperature for 10 min, and 2-chloroacetyl chloride (32 in dry THF (15 mL) was added. The was kept at room for 1 h and at °C. mmol) Then the reaction mixture was stirred atsolution room temperature for 10temperature min, and 2-chloroacetyl ◦ C for another 5 h, and substituted aniline (11 mmol) in dry THF (100 mL) was added dropwise 40–45 chloride (32 mmol) in dry THF (15 mL) was added. The solution was kept at room temperature for 1 over The mixture wassubstituted stirred at room temperature and then heated under h and20atmin. 40–45 °Creaction for another 5 h, and aniline (11 mmol)overnight in dry THF (100 mL) was added reflux for 18 h. After excess was evaporated the resulting was extracted with EtOAc dropwise over 20 min. TheTHF reaction mixture wasoff, stirred at roomresidue temperature overnight and then (3 × 10 mL) and washed with brine (3 × 10 mL). The organic layer was dried (Na SO ), concentrated 2 4 residue was heated under reflux for 18 h. After excess THF was evaporated off, the resulting under reduced pressure, column chromatography (PE: The 1:1 v/v EtOAc) with 30–35% extracted with EtOAc (3 and × 10 purified mL) andby washed with brine (3 × 10 mL). organic layer was dried yield as a white solid. (Na2SO4), concentrated under reduced pressure, and purified by column chromatography (PE: 1:1

v/v EtOAc) with 30–35% yield as a white solid. Synthesis of the 2-(substituted arylamino)-4-oxo-4,5 dihydrofuranone-3-carboxylic acid Synthesis of the arylamino)-4-oxo-4,5 acid arylamino)LiOH-H (10 mmol) was slowly added to dihydrofuranone-3-carboxylic a solution of ethyl 2-(substituted 2 O 2-(substituted 4-oxo-4,5-dihydrofuran-3-carboxylate (2 mmol) in MeOH–H2 O (18 mL, 5:1 v/v MeOH/H2 O) at 0 ◦ C LiOH-H2O (10 mmol) was slowly added to a solution of ethyl 2-(substituted arylamino)-4-oxoover 30 min. The reaction mixture was allowed to warm to 55–60 ◦ C for 12 h with stirring. After MeOH 4,5-dihydrofuran-3-carboxylate (2 mmol) in MeOH–H2O (18 mL, 5:1 v/v MeOH/H2O) at 0 °C over 30 was evaporated off, the aqueous residual was acidified to pH 1–2 with 1 N HCl and precipitated solid min. The reaction mixture was allowed to warm to 55–60 °C for 12 h with stirring. After MeOH was was filtered, washed with water, and dried under vacuum with 70–80% yield as a yellow solid. evaporated off, the aqueous residual was acidified to pH 1–2 with 1 N HCl and precipitated solid was filtered, washed with Synthesis of compound 11 water, and dried under vacuum with 70–80% yield as a yellow solid. LiOH–H (10 11 mmol) was slowly added to a solution of ethyl Synthesis of compound 2O 2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxylate (2 mmol) in MeOH–H2 O (18 mL, 5:1 LiOH–H2O (10 mmol) was slowly added to a solution of ethyl 2-(naphthalen-2-ylamino)-4-oxov/v MeOH/H2 O) at 0 ◦ C over 15 min. The reaction mixture was allowed to warm to 55–60 ◦ C for 12 h 4,5-dihydrofuran-3-carboxylate (2 mmol) in MeOH–H2O (18 mL, 5:1 v/v MeOH/H2O) at 0 °C over 15 with stirring. After MeOH was evaporated off, the aqueous residual was acidified to pH 1–2 with 1 N min. The reaction mixture was allowed to warm to 55–60 °C for 12 h with stirring. After MeOH was HCl and precipitated solid was filtered, washed with water, and dried under vacuum with 70–80% evaporated off, the aqueous residual was acidified to pH 1–2 with 1 N HCl and precipitated solid yield as a yellow solid. was filtered, washed with water, and dried under vacuum with 70–80% yield as a yellow solid.

2-(Naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxylic acid (11); Mp: 164.4–165.0 °C. 1H-NMR (400 MHz,

2-(Naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxylic acid (11); Mp: 164.4–165.0 ◦ C. 1 H-NMR 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, (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). 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 13 C-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, (ESI): [M + H]+ calcd for C15H11NO4, 270.0688; found, 270.0688. 123.7, 123.4, 98.6, 38.7. HRMS (ESI): [M + H]+ calcd for C15 H11 NO4 , 270.0688; found, 270.0688.

3.2.2. General Procedure for Target Compounds 12–19 3.2.2. General Procedure for Target Compounds 12–19 HOBt (1.1 mmol), EDC (1.1 mmol), and DIPEA (1 mmol) were added to a solution of amine (1 HOBt (1.1 mmol), EDC (1.1 mmol), and DIPEA (1 mmol) were added to a solution of amine mmol) amino)-4-oxo-4,5dihydrofuranone-3-carboxylic dihydrofuranone-3-carboxylic acid (1 mmol) dry (1 mmol)and and2-(substituted 2-(substituted amino)-4-oxo-4,5 acid (1 mmol) in dryinDCM DCM (5 mL) at 0 °C. The reaction mixture was stirred overnight at room temperature and then (5 mL) at 0 ◦ C. The reaction mixture was stirred overnight at room temperature and then washed with washed with 5% aqueous HCl (2 × 15 mL), 5% aqueous NaHCO3 (2 × 15 mL), and brine (2 × 15 mL) 5% aqueous HCl (2 × 15 mL), 5% aqueous NaHCO3 (2 × 15 mL), and brine (2 × 15 mL) and was dried and was dried (Na2SO4) and concentrated under reduced pressure with purification by column (Na2 SO4 ) and concentrated under reduced pressure with purification by column chromatography (PE: chromatography (PE: 6:1, v/v EtOAc) with 20–25% yield as a white solid. 6:1, v/v EtOAc) with 20–25% yield as a white solid.

N-Ethyl-4-oxo-2-(phenylamino)-4,5-dihydrofuran-3-carboxamide (12); Mp: 146.9–147.4 °C. 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). 13C-NMR (100 MHz, DMSO-d6): δ 191.0, 183.3, 165.5, 137.8, 130.0, 128.3, 125.6, 97.4, +

HOBt (1.1 mmol), EDC (1.1 mmol), and DIPEA (1 mmol) were added to a solution of amine (1 HOBt mmol), EDCamino)-4-oxo-4,5 (1.1 mmol), and DIPEA (1 mmol) were added toacid a solution of amine (1 mmol) and(1.1 2-(substituted dihydrofuranone-3-carboxylic (1 mmol) in dry mmol) amino)-4-oxo-4,5 dihydrofuranone-3-carboxylic (1 mmol) in then dry DCM (5and mL)2-(substituted at 0 °C. The reaction mixture was stirred overnight at room acid temperature and DCM (5 mL) at 0 °C. The reaction mixture was stirred overnight at room temperature and then washed with 5% aqueous HCl (2 × 15 mL), 5% aqueous NaHCO3 (2 × 15 mL), and brine (2 × 15 mL) washed with aqueous HCl (2 × 15 mL), 5% aqueous NaHCO3 (2 × 15 mL), and brine (2 × 159 mL) Molecules 2018, 23,5% 1254 of 14 and was dried (Na 2SO4) and concentrated under reduced pressure with purification by column and was dried (Na 2SO4) and concentrated under reduced pressure with purification by column chromatography (PE: 6:1, v/v EtOAc) with 20–25% yield as a white solid. chromatography (PE: 6:1, v/v EtOAc) with 20–25% yield as a white solid.

◦ C. 1 H-NMR (400 N-Ethyl-4-oxo-2-(phenylamino)-4,5-dihydrofuran-3-carboxamide (12); 146.9–147.4 Mp: 146.9–147.4 N-Ethyl-4-oxo-2-(phenylamino)-4,5-dihydrofuran-3-carboxamide (12); Mp: °C. 1H-NMR (400 MHz, 1H-NMR (400 MHz, N-Ethyl-4-oxo-2-(phenylamino)-4,5-dihydrofuran-3-carboxamide (12); Mp: 146.9–147.4 °C. MHz, ): 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, J3.67 = 7.2 CDCl3):CDCl 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), (s, 3 13 C-NMR CDCl 3): 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, 13 Hz, 2H), 3.67 (s, 2H), 1.42 (t, J = 7.2 Hz, 3H). (100 MHz, DMSO-d ): δ 191.0, 183.3, 165.5, 2H), 1.42 (t, J = 7.2 Hz, 3H). C-NMR (100 MHz, DMSO-d6): δ 191.0, 183.3, 165.5, 137.8, 6 130.0, 128.3, 125.6, 97.4, 13C-NMR (100 MHz, DMSO-d6): δ 191.0, 183.3, 2H), (t, J 128.3, = HRMS 7.2 Hz, 3H).97.4, 137.8, 130.0, 128.3, 125.6, 97.4, 137.8, 130.0, 125.6, 38.4,for 14.9. HRMS [M + H]+ 165.5, calcd for C13 H14 N 59.7, 1.42 38.4, 14.9. (ESI): [M +59.7, H]+ calcd C13H 14N2O3,(ESI): 247.1004; found, 247.1009. 2 O3 , 247.1004; + calcd for C13H14N2O3, 247.1004; found, 247.1009. 59.7, 38.4, 14.9. HRMS (ESI): [M + H] found, 247.1009.

Molecules 2018, 23, x FOR PEER REVIEW Molecules 2018, 23, x FOR PEER REVIEW Molecules 2018, 23, x FOR PEER REVIEW

9 of 14 9 of 14 9 of 14

2-[(2,3-Dihydro-1H-inden-5-yl)amino]-N-ethyl-4-oxo-4,5-dihydrofuran-3-carboxamide (13); (13); Mp: 127.7–128.2 °C. 2-[(2,3-Dihydro-1H-inden-5-yl)amino]-N-ethyl-4-oxo-4,5-dihydrofuran-3-carboxamide 127.7–128.2 2-[(2,3-Dihydro-1H-inden-5-yl)amino]-N-ethyl-4-oxo-4,5-dihydrofuran-3-carboxamide (13); Mp: Mp: 127.7–128.2 °C. ◦12-[(2,3-Dihydro-1H-inden-5-yl)amino]-N-ethyl-4-oxo-4,5-dihydrofuran-3-carboxamide 1 H-NMR (400 MHz, DMSO-d 6): 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, (13); Mp: 127.7–128.2 °C. C. H-NMR (400 MHz, DMSO-d ): δ 11.08 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.27 (s, 1H), 7.16 (d, J = 8.0 1H-NMR 6 (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 =138.0 Hz, 1H), 4.22 (q, 1H-NMR JHz, = 7.2 Hz, 2H), 3.65 (s, 2H), 2.89 (t, J = 7.6 Hz, 4H), 2.09-2.02 (m, 2H), 1.26 (t, J = 7.2 Hz, 3H). C-NMR (100 MHz, DMSO-d 62H), ): δ(t,11.08 (s,(s,Hz, 1H), 7.32 (d,(t, J =J 8.0 Hz,Hz, 1H),4H), 7.27(t, (s,J 1H), J =138.0 Hz, 4.22 (q, 1H), 4.22 (q, J =(s, 7.2 Hz, 2H), 2.89 = (m, 7.6 2.09-2.02 (m,(d, 2H), 1.26 (t,1H), J(100 = 7.2 Hz, J = 7.2 Hz,(400 2H),MHz, 3.65 2H), 2.89 J3.65 = 7.6 4H), 2.09-2.02 2H), 1.26 = 7.27.16 Hz, 3H). C-NMR MHz, 13C-NMR DMSO-d 6):2H), 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 JDMSO-d = 7.213Hz, 3.65 (s, 2H), 2.89 (t, J = 7.6 Hz, 4H), 2.09-2.02 (m, 2H), 1.26 (t, J = 7.2 Hz, 3H). (100 MHz, 3H). C-NMR (100 MHz, DMSO-d ): δ 190.9, 183.5, 165.6, 145.7, 144.2, 135.8, 125.4, 123.6, 121.6, 97.1, 6 6): 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 + calcd for C16H18N2O3, 287.1317; found, 287.1320. (ESI): [M6+): H] DMSO-d δ 190.9, 183.5, 145.7, 144.2, 135.8, 59.7, 38.4, 32.8, 32.4, 25.7, 14.9. HRMS + calcd 59.7, 38.4, 32.4, 25.7, 14.9. HRMS (ESI): [M125.4, + 287.1320. H]+123.6, calcd121.6, for C97.1, 16 H18 N2 O3 , 287.1317; found, 287.1320. (ESI): [M + 32.8, H] for C165.6, 16H 18N2O 3, 287.1317; found, (ESI): [M + H]+ calcd for C16H18N2O3, 287.1317; found, 287.1320.

◦ C. N-Methyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide Mp: °C. 126.3–126.9 1H-NMR (400 N-Methyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide (14); Mp: (14); 126.3–126.9

(14); Mp: 126.3–126.9 °C. 1(s, H-NMR (400 1N-Methyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide H-NMR MHz,(s,CDCl 1H), 7.67–7.79 (m, 4H), 7.42–7.51 (m, 3H),13C-NMR 4.62 2H),MHz, 3.32 3 ): δ 11.65 MHz, CDCl(400 3): δ 11.65 1H), 7.67–7.79 (m,(s, 4H), 7.42–7.51 (m, 3H), 4.62 (14); (s, 2H), 3.32 (s, 3H). (100 N-Methyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide Mp: 126.3–126.9 °C. 1H-NMR (400 MHz, CDCl 3): δ 11.65 (s, 1H), 7.67–7.79 (m, 4H), 7.42–7.51 (m, 3H), 4.62 (s, 2H), 3.32 (s, 3H). 13C-NMR (100 MHz, 13 (s, 3H). (100 MHz, CDCl δ 4H), 192.4, 181.3,129.8, 165.9, 133.6, 134.9, 133.4, 131.8, 129.8, 127.8, 13 CDCl 3): δ C-NMR 192.4, 181.3, 165.9, 133.6, 134.9, 133.4,7.42–7.51 131.8, 127.8, 127.2, 126.5, 121.8, 120.6, 99.9, 78.3,127.2, 35.7. 3 ): MHz, CDCl 3): δ 11.65 (s, 1H), 7.67–7.79 (m, (m, 3H), 4.62 (s, 2H), 3.32 (s, 3H). C-NMR (100 MHz, CDCl3): δ 192.4, 181.3, 165.9, 133.6, 134.9, 133.4, 131.8, 129.8, 127.8, 127.2, 126.5, 121.8, 120.6, 99.9, 78.3, 35.7. + calcd 126.5, 120.6, 78.3, (ESI): [M + 283.1011. H]127.8, for126.5, C18 H121.8, found, HRMS (ESI): [M +181.3, H]++99.9, calcd for133.6, C1835.7. H14134.9, N2HRMS O3,133.4, 283.1004; found, 14 N2 O120.6, 3 , 283.1004; CDCl3):121.8, δ 192.4, 165.9, 131.8, 129.8, 127.2, 99.9, 78.3, 35.7. HRMS (ESI): [M + H] calcd for C18H14N2O3, 283.1004; found, 283.1011.

283.1011. HRMS (ESI): [M + H]+ calcd for C18H14N2O3, 283.1004; found, 283.1011.

N-Ethyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide (15); Mp:(15); 121.8–123.0 H-NMR (400 ◦ C. 1H-NMR (400 N-Ethyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide Mp: °C. 121.8–123.0 N-Ethyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide (15); Mp: 121.8–123.0 °C. CDCl3): δ 11.05 (s, 1H), 7.88–7.79 (m, 4H), 7.53–7.44 (m, 3H), 4.78 (s, 2H), 3.40−3.47 (m, °C. 2H),1H-NMR 1.24 (t, J =(400 7.2 1MHz, N-Ethyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide (15); Mp: 121.8–123.0 H-NMR (400 CDCl δ 11.05 (s, 4H), 4.78 7.53–7.44 3H), 4.78 2H), 3.40 MHz, CDCl 3): δMHz, 11.05 (s, 1H),3 ):7.88–7.79 (m,1H), 4H),7.88–7.79 7.53–7.44 (m, (m, 3H), (s, 2H),(m, 3.40−3.47 (m,(s, 2H), 1.24 (t, − J =3.47 7.2 13C-NMR (100 MHz, CDCl3): δ 190.5, 176.5, 164.3, 133.6, 132.7, 131.1, 129.5, 127.7, 127.6, 127.0, 125.9, Hz, 3H). 13 MHz, CDCl 3): δ 11.05 (s, 1H), 7.88–7.79 (m, 4H), 7.53–7.44 (m, 3H), 4.78 (s, 2H), 3.40−3.47 (m, 2H), 1.24 (t, J = 7.2 13 (m, 2H), 1.24 (t, J = 7.2 Hz, 3H). C-NMR (100 MHz, CDCl ): δ 190.5, 176.5, 164.3, 133.6, 132.7, 131.1, Hz, 3H). C-NMR (100 MHz, CDCl3): δ 190.5, 176.5, +164.3, 133.6,3 132.7, 131.1, 129.5, 127.7, 127.6, 127.0, 125.9, 13C-NMR 120.2, 118.1, 89.5, 75.7, 33.4, 15.1. HRMS [M + H]+164.3, calcd 133.6, for C17132.7, H16N2O 3, 297.1239; found,127.6, 297.1241. Hz, 3H). (100 MHz, CDCl 3): δ(ESI): 190.5, 176.5, 131.1, 129.5, 127.7, 125.9, + calcd 129.5, 127.7, 127.6, 127.0, 125.9, 120.2, 118.1, 75.7,for 33.4, (ESI): [M 297.1241. + H]127.0, for 120.2, 118.1, 89.5, 75.7, 33.4, 15.1. HRMS (ESI): [M +89.5, H] calcd C17H15.1. 16N2OHRMS 3, 297.1239; found, + 120.2, 118.1, 89.5, 75.7, 33.4, 15.1. HRMS (ESI): [M + H] calcd for C 17H16N2O3, 297.1239; found, 297.1241. C17 H16 N2 O3 , 297.1239; found, 297.1241. 1

2-(Naphthalen-2-ylamino)-4-oxo-N-propyl-4,5-dihydrofuran-3-carboxamide (16); Mp: 126.2–127.2 °C. 11H-NMR (400 2-(Naphthalen-2-ylamino)-4-oxo-N-propyl-4,5-dihydrofuran-3-carboxamide (16); Mp: 126.2–127.2 °C. H-NMR (400 ◦ C. 1(q, MHz, CDCl3): δ 12.95 (s, 1H), 8.91 (s, 1H), 7.92–7.84 (m, 4H), 7.56–7.29(16); (m, 3H), 3.75 (s,Mp: 2H), 3.37 J = 6.4 (400 Hz, 2-(Naphthalen-2-ylamino)-4-oxo-N-propyl-4,5-dihydrofuran-3-carboxamide 126.2–127.2 2-(Naphthalen-2-ylamino)-4-oxo-N-propyl-4,5-dihydrofuran-3-carboxamide Mp:(16); 126.2–127.2 °C. H-NMR MHz, CDCl 3): 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, 12H), 13C-NMR (100 MHz, CDCl3): δ 193.8, 181.3, 165.9, 135.0, 133.4, 1.69-1.63 2H),(s, 1.02 (t,38.91 Hz,(s, 3H). H-NMR (400 CDCl ):J =δ 7.2 12.95 1H), 8.91 1H), 7.92–7.84 (m, 4H), 7.56–7.29 3.75 MHz, CDCl 3): (m, δ MHz, 12.95 1H), (s, 1H), 7.92–7.84 (m,(s, 4H), (m, 33H), 3.75 (s, 2H), 165.9, 3.37(m, (q,135.0, J3H), = 6.4 Hz, 13C-NMR 2H), 1.69-1.63 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H). (1007.56–7.29 MHz, CDCl ): δ 13 193.8, 181.3, 133.4, + calcd for 13 131.8, 129.8, 127.8, 127.2, 121.8, 120.6, 40.1, 38.5, 11.5. (ESI): [M165.9, +MHz, H]135.0, (s, 2H), 3.37 (q, J =2H), 6.4 Hz,126.5, 2H), 1.69-1.63 (m,99.6, 2H), 1.02(100 (t, J MHz, = 23.0, 7.2 Hz, 3H). C-NMR (100 CDCl 2H), 1.69-1.63 (m, 1.02 (t, J = 7.2 Hz, 3H). C-NMR CDCl 3):HRMS δ 193.8, 181.3, 133.4, 3 ): 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): [M + H]+ calcd for + 23.0, 18 H18N 2181.3, O3, 333.1317; found, 333.1311. δC 193.8, 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, 11.5. 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): [M + H] calcd for C18H18N2O3, 333.1317; found, 333.1311. + calcd C18H18N(ESI): 2O3, 333.1317; found, 333.1311. HRMS [M + H] for C18 H18 N2 O3 , 333.1317; found, 333.1311.

2-(Naphthalen-2-ylamino)-4-oxo-N-propyl-4,5-dihydrofuran-3-carboxamide (17); Mp: 140.1–141.0 °C. 1H-NMR (400

2-(Naphthalen-2-ylamino)-4-oxo-N-propyl-4,5-dihydrofuran-3-carboxamide (16); Mp: 126.2–127.2 °C. 11H-NMR (400 2-(Naphthalen-2-ylamino)-4-oxo-N-propyl-4,5-dihydrofuran-3-carboxamide (16); Mp: 126.2–127.2 °C. H-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, 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, 2H), 1.69-1.63 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H). 13C-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): [M + H]++ calcd for 131.8, 129.8, 127.8, Molecules 2018, 23, 1254 127.2, 126.5, 121.8, 120.6, 99.6, 40.1, 38.5, 23.0, 11.5. HRMS (ESI): [M + H] calcd 10 of for 14 C18H18N2O3, 333.1317; found, 333.1311. C18H18N2O3, 333.1317; found, 333.1311.

1H-NMR (400 ◦ C. 2-(Naphthalen-2-ylamino)-4-oxo-N-propyl-4,5-dihydrofuran-3-carboxamide (17); Mp:(17); 140.1–141.0 2-(Naphthalen-2-ylamino)-4-oxo-N-propyl-4,5-dihydrofuran-3-carboxamide Mp: °C. 140.1–141.0 1H-NMR (400 2-(Naphthalen-2-ylamino)-4-oxo-N-propyl-4,5-dihydrofuran-3-carboxamide (17); Mp: 140.1–141.0 °C. 1MHz, CDCl 3 ): 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, H-NMR (400 MHz, CDCl ): δ 12.94 (s, 1H), 8.89 (s, 1H), 7.93–7.84 (m, 4H), 7.58–7.44 (m, 3H), 3.75 MHz, CDCl 3): δ 12.94 (s, 1H),38.89 (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, 13 C-NMR 2H), 1.66-1.59 (m,J 2H), (m,1.66-1.59 2H), 0.99 (t, J =2H), 7.2 Hz, 3H). 13 C-NMR (100 MHz, δ 193.8, 165.9, (s, 2H), 3.41 (q, = 6.81.50-1.41 Hz, 2H), 1.50-1.41 (m, 2H), 0.99 (t, J CDCl = 7.233):): Hz, 3H).181.3, 2H), 1.66-1.59 (m, 2H), 1.50-1.41 (m, 2H), 0.99(m, (t, J = 7.2 Hz, 3H). 13C-NMR (100 MHz, CDCl 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): [M + Na]++ (100 δ 193.8, 133.4,99.6, 131.9, 129.8, 127.2, 126.5, 121.8, 120.6, 135.0,MHz, 133.4,CDCl 131.9,3 ): 129.8, 127.8,181.3, 127.2,165.9, 126.5, 135.0, 121.8, 120.6, 38.5, 38.1,127.8, 31.8, 20.2, 13.8. HRMS (ESI): [M +99.6, Na] calcd38.1, for C19 H20N20.2, 2O3, 347.1474; found, 347.1470. + calcd for C H N O , 347.1474; found, 347.1470. 38.5, 31.8, 13.8. HRMS (ESI): [M + Na] 19 20 2 3 calcd for C19H20N2O3, 347.1474; found, 347.1470.

O O N N H H

O O CONH CONH

Molecules 2018, 23, x FOR PEER REVIEW Molecules 2018, 23, x FOR PEER REVIEW

10 of 14 10 of 14

N-Cyclopropyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide (18); Mp: °C. H-NMR ◦ C. N-Cyclopropyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide (18);140.8–41.6 Mp: 140.8–41.6 1H-NMR N-Cyclopropyl-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide (18); Mp: 140.8–41.6 °C. MHz,(400 DMSO-d 6): 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), 1(400 H-NMR 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 (400 MHz,(m, DMSO-d 6): δ 12.62 (s, 1H), 8.78 (d, J = 3.6 Hz,131H), 8.08–7.93 (m, 4H), 7.69–7.54 (m, 3H), 3.89 (s, 2H), 2.82–2.77 1H), 0.78–0.73 (m, 2H), 0.56–0.50 (m, 2H). C-NMR (100 MHz, CDCl 3): δ 193.7, 181.2, 167.5, 134.9, 13 C-NMR (m, 3H), 3.89 (s, 2H), 2.82–2.77 (m, 1H), 0.78–0.73 (m, 2H), 0.56–0.50 (m, 2H). (100 MHz, CDCl 3 ): 13 2.82–2.77 (m, 129.9, 1H), 0.78–0.73 (m, 2H), 0.56–0.50 (m, 2H). (10021.6, MHz, 3): δ 193.7, 181.2, 167.5, 134.9, 133.4, 131.8, 127.9, 127.2, 126.5, 121.7, 120.5, 99.4,C-NMR 38.6, 29.7, 6.5.CDCl HRMS (ESI): [M + H]+ calcd for δ133.4, 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, 29.7, 21.6, 6.5. 131.8, 129.9, 127.9, 127.2, 126.5, 121.7, 120.5, 99.4, 38.6, 29.7, 21.6, 6.5. HRMS (ESI): [M + H]+ calcd for C18H16N 2O3, 309.1161; found, 309.1165. + calcd HRMS (ESI): [M + H] for C18 H16 N2 O3 , 309.1161; found, 309.1165. C18H16N2O3, 309.1161; found, 309.1165. 1

N-(Cyclopropylmethyl)-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide (19); Mp: N-(Cyclopropylmethyl)-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide (19); Mp: 164.8–165.3 °C. ◦ C. 1 H-NMR (400 MHz, DMSO-d ): δ 12.68 (s, 1H), 8.91 (t, J = 5.6 Hz, 1H), 8.06–7.97 164.8–165.3 N-(Cyclopropylmethyl)-2-(naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carboxamide Mp: 164.8–165.3 °C. 1H-NMR (400 MHz, DMSO-d6): δ 12.68 (s, 1H), 8.91 6(t, J = 5.6 Hz, 1H), 8.06–7.97 (m, (19); 4H), 7.62–7.55 (m, 3H), 3.92 1H-NMR (m, 4H),3.19 7.62–7.55 (m, 3H), 3.92 (s, 2H), 3.19 (t,(t, J= 1.06-0.99 (m, 1H), 0.50–0.45 (m, 2H), (400 DMSO-d 6): δ1.06-0.99 12.68 (s, 1H), 8.91 J =6.4 5.6Hz, Hz, 1H), 7.62–7.55 (m,(100 3H),MHz, 3.92 13 (s, 2H), (t,MHz, J = 6.4 Hz, 2H), (m, 1H), 0.50–0.45 (m,2H), 2H),8.06–7.97 0.26–0.22(m, (m,4H), 2H). C-NMR 13 C-NMR 13 (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). C-NMR (100 MHz, 0.26–0.22 (m, 2H). (100 MHz, CDCl ): δ 193.8, 181.3, 165.8, 134.9, 133.4, 131.8, 129.8, 127.9, 3 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. + calcd CDCl 3):126.5, δ 193.8, 181.3, 165.8,for 134.9, 133.4, 131.8, 129.8,3.5. 127.9, 127.2,(ESI): 126.5, [M 121.8, 120.6, 99.5, 43.0, 10.9, 3.5. 127.2, 121.8, 99.5, 43.0, 38.5, 10.9, HRMS + H] for C38.5, N2 O 19 H18 3, HRMS (ESI): [M + H]+120.6, calcd C19H 18N2O 3, 323.1317; found, 323.1315. + calcd for C19H18N2O3, 323.1317; found, 323.1315. HRMS (ESI): [M + H] 323.1317; found, 323.1315. Synthesis of Compound 20 Synthesis Synthesis of of Compound Compound 20 20 CDI (1 mmol) was slowly added to a solution of ethyl 2-(naphthalen-2-ylamino)-4-oxo-4,5CDI (1(1mmol) mmol) slowly added to DCM a ofsolution ethyl waswas slowly added to ain solution ethyl 2-(naphthalen-2-ylamino)-4-oxo-4,5dihydrofuran-3-carboxylic acid (1 mmol) dry (6 mL) atof0 °C for 2 2-(naphthalen-2-ylamino)h with stirring. Then to the 4-oxo-4,5-dihydrofuran-3-carboxylic acid (1inmmol) in dry DCM (6 0mL) 0 2◦ C 2athstirring. with stirring. Then dihydrofuran-3-carboxylic acid (1 mmol) dry DCM (6 mL) at °C at forstirring h for with Then to the reaction mixture was added 80% hydrazine hydrate overnight with room temperature. to the reaction mixture was added 80% hydrazine hydrate overnight with stirring at room temperature. reaction mixture was added 80% hydrazine hydrate overnight with stirring at room temperature. After the reaction, the precipitation was filtered, dried, and purified by column chromatography After the the was dried, After the reaction, reaction, theinprecipitation precipitation wasasfiltered, filtered, dried, and purified purified by by column column chromatography chromatography (EA: 10:1, v/v MeOH) 23–27% in yield a brown solid.and (EA: MeOH) in in 23–27% 23–27% in in yield yield as a brown brown solid. (EA: 10:1, 10:1, v/v v/v MeOH)

2-(Naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carbohydrazide (20); Mp: 167.3–167.9 °C. 1H-NMR (400 MHz,

◦ C. 1 H-NMR (400 1H-NMR 2-(Naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carbohydrazide (20); Mp: 167.3–167.9 2-(Naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carbohydrazide Mp: 167.3–167.9 °C. 13 (400 CDCl3): δ 12.11 (s, 1H), 8.02-7.91 (m, 4H), 7.66–7.51 (m, 3H), (20); 5.58 (s, 2H), 5.19 (s, 2H). C-NMR 13 (100 MHz, MHz, 13 MHz, CDCl ): δ 12.11 (s, 1H), 8.02-7.91 (m, 4H), 7.66–7.51 (m, 3H), 5.58 (s, 2H), 5.19 (s, 2H). C-NMR CDCl 12.11 1H),162.7, 8.02-7.91 4H),131.5, 7.66–7.51 3H),128.2, 5.58 127.5, (s, 2H), 5.19 124.2, (s, 2H). C-NMR (100 MHz, 3 (s, CDCl33): ): δδ 194.7, 185.4, 133.5,(m, 132.6, 132.2,(m, 128.4, 127.2, 120.3, 79.9, 76.2. HRMS (100 CDCl δ 194.7, 185.4, 162.7, 133.5, 132.6, 131.5, 132.2, 128.4, 128.2, 127.5, 127.2, 120.3, CDCl 3): δ 194.7, 185.4, 162.7, 131.5, 132.2, 128.4, 128.2, 127.5, 127.2, 124.2, 120.3, 79.9,124.2, 76.2. HRMS 3 ): for + calcd (ESI):MHz, [M + H] C15H13133.5, N3O3,132.6, 284.0957; found, 284.0954. + + 79.9, HRMS [M calcd forfound, C15 H13284.0954. N3 O3 , 284.0957; found, 284.0954. (ESI):76.2. [M + H] calcd(ESI): for C15 H13+NH] 3O3, 284.0957;

3.2.3. General Procedure for Target Compounds 21–26 3.2.3. General Procedure for Target Compounds 21–26 NaOH (1.5 mmol) was slowly added to a solution of N-substituted-2-(substituted amino)-4-oxo-4,5NaOH (1.5 mmol) was slowly addedin to EtOH a solution of N-substituted-2-(substituted dihydrofuran-3-carboxamide (1 mmol) (6 mL) at 0 °C with stirring. The amino)-4-oxo-4,5reaction mixture dihydrofuran-3-carboxamide mmol) in EtOH (6 mL) 0 °C with stirring. The(POM) reaction was allowed to warm to 45 °C(1for 1 h with stirring, and at then paraformaldehyde (1.2mixture mmol)

2-(Naphthalen-2-ylamino)-4-oxo-4,5-dihydrofuran-3-carbohydrazide (20); Mp: 167.3–167.9 °C. 1H-NMR (400 MHz, CDCl3): δ 12.11 (s, 1H), 8.02-7.91 (m, 4H), 7.66–7.51 (m, 3H), 5.58 (s, 2H), 5.19 (s, 2H). 13C-NMR (100 MHz, Molecules 23, 1254 of 14 CDCl3): δ2018, 194.7, 185.4, 162.7, 133.5, 132.6, 131.5, 132.2, 128.4, 128.2, 127.5, 127.2, 124.2, 120.3, 79.9, 76.2. 11 HRMS (ESI): [M + H]+ calcd for C15H13N3O3, 284.0957; found, 284.0954.

3.2.3. General Procedure for Target Compounds 21–26 3.2.3. General Procedure for Target Compounds 21–26 NaOH (1.5 mmol) was slowly added to a solution of N-substituted-2-(substituted NaOH (1.5 mmol) was slowly added to a solution of N-substituted-2-(substituted amino)-4-oxo-4,5amino)-4-oxo-4,5-dihydrofuran-3-carboxamide (1 mmol) in EtOH (6 mL) at 0 ◦ C with stirring. The dihydrofuran-3-carboxamide (1 mmol) in EtOH (6 mL) at 0 °C with stirring. The reaction mixture reaction mixture was allowed to warm to 45 ◦ C for 1 h with stirring, and then paraformaldehyde was allowed to warm to 45 °C for 1 h with stirring, and then paraformaldehyde (POM) (1.2 mmol) (POM) (1.2 mmol) was added for 8 h at 70 ◦ C. After the resulting mixture was filtered, the filtrate was was added for 8 h at 70 °C. After the resulting mixture was filtered, the filtrate was concentrated concentrated under reduced pressure and purified by column chromatography (PE: 3:1 v/v EA) with under reduced pressure and purified by column chromatography (PE: 3:1 v/v EA) with 13.2–19.7% 13.2–19.7% yield as a white solid. yield as a white solid.

1-(2,3-Dihydro-1H-inden-5-yl)-3-ethyl-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (21); Mp: 128.6–129.4 °C. 1-(2,3-Dihydro-1H-inden-5-yl)-3-ethyl-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (21); Mp: 1H-NMR (400 ◦ C. 1 H-NMR MHz, CDCl3):(400 δ 7.32 (d, J CDCl = 8.0 Hz, 1H), 7.27 (s, 1H), 7.16 (d, J = 8.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 128.6–129.4 MHz, ): δ 7.32 (d, J = 8.0 Hz, 1H), 7.27 (s, 1H), 7.16 (d, J = 8.0 Hz, 3 13C-NMR (100 2H),4.17 (s,(q, 2H), 3.65 (s, 2H), 2.89 (t,(s, J =2H), 7.6 Hz, 4H), 2.09–2.02 1.26 (t, 4H), J = 7.2 Hz, 3H). (m, 1H), 4.22 J = 7.2 Hz, 2H),4.17 3.65 (s, 2H), 2.89(m, (t, J2H), = 7.6 Hz, 2.09–2.02 2H), 1.26 Molecules 2018, 23, x FOR PEER REVIEW 11 Molecules 2018, 23, x FOR PEER REVIEW 11 of of 14 14 (t, J = 7.2 Hz, 3H). 13 C-NMR (100 MHz, CDCl3 ): δ 190.9, 183.5, 165.6, 145.7, 144.2, 135.8, 125.4, 123.6, Molecules 2018, 23, x FOR PEER REVIEW 11 of 14 MHz, ): 145.7, 144.2, 135.8, 125.4, 123.6, 88.3, 38.4, 25.7, 121.6, 97.1, 3388.3, 59.7,183.5, 38.4, 165.6, 32.8, 32.4, HRMS [M + 97.1, H]+ calcd for C O3 , 299.1317; 17 H32.8, 18 N232.4, MHz, CDCl CDCl ): δδ 190.9, 190.9, 183.5, 165.6, 145.7,25.7, 144.2,14.9. 135.8, 125.4, (ESI): 123.6, 121.6, 121.6, 97.1, 88.3, 59.7, 59.7, 38.4, 32.8, 32.4, 25.7, 14.9. 14.9. ++ calcd for C17H18N2O3, 299.1317; found, 299.1325. HRMS (ESI): [M ++ H] found, 299.1325. HRMS (ESI): [M H] calcd for C 17H18N2O3, 299.1317; found, 299.1325. MHz, CDCl3): δ 190.9, 183.5, 165.6, 145.7, 144.2, 135.8, 125.4, 123.6, 121.6, 97.1, 88.3, 59.7, 38.4, 32.8, 32.4, 25.7, 14.9. HRMS (ESI): [M + H]+ calcd for C17H18N2O3, 299.1317; found, 299.1325.

O O

N N O

O O

O N N O N O CH CH33 O N CH3 1 ◦ C. 3-Methyl-1-(naphthalen-2-yl)-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione 127.9–128.5 °C. 3-Methyl-1-(naphthalen-2-yl)-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (22); Mp: 127.9–128.5 3-Methyl-1-(naphthalen-2-yl)-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (22); (22); Mp: Mp: 127.9–128.5 °C. 1H-NMR H-NMR 13 1(400 MHz, CDCl 33): 4H), (m, 3H), (s, 3.27 (s, (100 13C-NMR H-NMR MHz, CDCl3 ): (m, δ 8.11–7.90 (m, 4H), 3H),4.73 5.61(s, (s,2H), 2H), 4.73 2H), 3.271H-NMR (s, 3H). (400 MHz,(400 CDCl ): δδ 8.11–7.90 8.11–7.90 (m, 4H), 7.51–7.33 7.51–7.33 (m,7.51–7.33 3H), 5.61 5.61 (m, (s, 2H), 2H), 4.73 (s, 2H), 3.27 (s,(s,3H). 3H). C-NMR (100 3-Methyl-1-(naphthalen-2-yl)-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (22); Mp: 127.9–128.5 °C. 13 MHz, CDCl 33): δδ MHz, 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, 86.3, 77.8, MHz, CDCl ): 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, 86.3, 77.8, C-NMR (100 CDCl ): 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, 3 (m, 4H), 7.51–7.33 (m, 3H), 5.61 (s, 2H), 4.73 (s, 2H), 3.27 (s, 3H). 13C-NMR (100 (400 MHz, CDCl3): δ 8.11–7.90 ++ calcd for C17H14N2O3, 295.1004; + calcdfound, 38.5. HRMS (ESI): [M ++38.5. H] 295.1011. 38.5. HRMS (ESI): [M H] calcd C17H133.3, 14[M N2O+132.3, 3,H] 295.1004; 295.1011. 123.6, 97.6, 86.3, 77.8, HRMS (ESI): for C17128.2, H14 N127.5, 295.1004; found, 2 O3 , 127.2, MHz, CDCl3): δ 191.1, 183.4, 165.5,for 135.4, 129.8,found, 128.4, 124.2, 123.6, 295.1011. 97.6, 86.3, 77.8, 38.5. HRMS (ESI): [M + H]+ calcd for C17H14N2O3, 295.1004; found, 295.1011.

3-Ethyl-1-(naphthalen-2-yl)-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione °C. ◦ C. 3-Ethyl-1-(naphthalen-2-yl)-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (23); (23); Mp: Mp: 128.3–129.0 °C. 1H-NMR H-NMR 3-Ethyl-1-(naphthalen-2-yl)-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (23);128.3–129.0 Mp: 128.3–129.0 13 MHz, CDCl 3 ): δ 8.11–7.90 (m, 4H), 7.51–7.33 (m, 3H), 5.61 (s, 2H), 4.73 (s, 2H), 3.27 (s, 3H). C-NMR (100 13 1(400 (400 MHz, (400 CDClMHz, 3): δ 8.11–7.90 7.51–7.33 3H), 5.61 (s, 2H), (s,5.61 2H), (s, 3H). C-NMR (100 1H-NMR H-NMR CDCl3 ):(m,δ 4H), 8.11–7.90 (m, (m, 4H), 7.51–7.33 (m, 4.73 3H), (s,3.27 2H), 4.73 (s, 2H), 3.27 3-Ethyl-1-(naphthalen-2-yl)-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (23); Mp: 128.3–129.0 °C. MHz, CDCl 33): δδ 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, 86.3, 77.8, 13 MHz, CDCl ): 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, 86.3, 77.8, 13 (s, 3H). MHz,(m, CDCl δ 191.1, (m, 183.4, 165.5, 135.4, 129.8, (400 MHz,C-NMR CDCl3): δ(100 8.11–7.90 4H), 3H), 5.61 (s, 2H),133.3, 4.73 (s,132.3, 2H), 3.27 (s, 128.4, 3H). 128.2, C-NMR127.5, (100 3 ):7.51–7.33 ++ calcd for C17H14N2O3, 309.1161; found, 309.1158. 38.5. (ESI): [M ++ H] + calcd 38.5. HRMS HRMS [M97.6, H]86.3, calcd for C 17H14N2O3, 309.1161; found, 309.1158. MHz, CDCl ):123.6, δ 191.1, 183.4, 165.5, 135.4, 133.3, 132.3, 129.8, 128.4, 127.5, 123.6, 97.6, 86.3, 77.8, 127.2, 124.2,3(ESI): 77.8, 38.5. HRMS (ESI): [M + H]128.2, for 127.2, C17 H124.2, found, 14 N2 O 3 , 309.1161; 38.5. HRMS (ESI): [M + H]+ calcd for C17H14N2O3, 309.1161; found, 309.1158. 309.1158. 1

1 1-(Naphthalen-2-yl)-3-propyl-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione 1-(Naphthalen-2-yl)-3-propyl-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (24); (24); Mp: Mp: 128.6–129.7 128.6–129.7 °C. °C. 1H-NMR H-NMR (400 MHz, DMSO-d 66): δδ 7.92-7.84 (m, 4H), 7.56–7.29 (m, 3H), 4.51 (s, 2H), 3.57 (s, 2H), 3.05 (q, JJ == 6.4, 2H), 1.69– (400 MHz, DMSO-d ): 7.92-7.84 (m, 4H), 7.56–7.29 (m, 3H), 4.51 (s, 2H), 3.57 (s, 2H), 3.05 (q, 6.4, 2H), 1.69– 1 1-(Naphthalen-2-yl)-3-propyl-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (24); Mp: 128.6–129.7 °C. H-NMR 13 1.63 (m, 2H), 1.33 (t, JJ == 7.2, 3H). (100 MHz, DMSO-d 66): δδ 193.6, 182.4, 162.3, 135.4, 133.3, 132.3, 131.8, 13C-NMR 1.63 (m, 2H), 1.33 (t, 7.2, 3H). C-NMR (100 MHz, DMSO-d ): 193.6, 182.4, 162.3, 135.4, 133.3, 132.3, 131.8, (400 MHz, DMSO-d6): δ 7.92-7.84 (m, 4H), 7.56–7.29 (m, 3H), 4.51 (s, 2H), 3.57 (s, 2H), 3.05 (q, J = 6.4, 2H), 1.69– + 128.4, 128.2, 127.5, 127.2, 124.2, 123.6, 97.6, 38.5, 14.9. HRMS (ESI): 128.4, 128.2, 127.2, 124.2, 123.6, 97.6, 38.6, 38.6, HRMS (ESI): [M [M ++ H] H]+ calcd calcd for for C C17 17H H18 18N N22O O33,, 345.1317; 345.1317; 13C-NMR 1.63 (m, 2H),127.5, 1.33 (t, J = 7.2, 3H). (100 38.5, MHz,14.9. DMSO-d 6): δ 193.6, 182.4, 162.3, 135.4, 133.3, 132.3, 131.8, found, 345.1314. found,128.2, 345.1314. 128.4, 127.5, 127.2, 124.2, 123.6, 97.6, 38.6, 38.5, 14.9. HRMS (ESI): [M + H]+ calcd for C17H18N2O3, 345.1317; found, 345.1314.

Molecules 2018, 23, 1254

12 of 14

◦ C. 1-(Naphthalen-2-yl)-3-propyl-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (24); Mp: 128.6–129.7 1-(Naphthalen-2-yl)-3-propyl-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (24); Mp: 128.6–129.7 °C. 1H-NMR 1 H-NMR (400 MHz, DMSO-d ): δ 7.92-7.84 (m, 4H), 7.56–7.29 (m, 3H), 4.51 (s, 2H), 3.57 (s, 2H), 3.05 (400 MHz, DMSO-d6): δ 7.92-7.846(m, 4H), 7.56–7.29 (m, 3H), 4.51 (s, 2H), 3.57 (s, 2H), 3.05 (q, J = 6.4, 2H), 1.69– 13 C-NMR (100 MHz, DMSO-d ): δ 193.6, 182.4, 162.3, 13C-NMR (q, 6.4, 2H), 1.69–1.63 (m, 2H), 1.33 (t, J(100 = 7.2, 3H).DMSO-d 1.63J = (m, 2H), 1.33 (t, J = 7.2, 3H). MHz, 6): δ 193.6, 182.4, 162.3, 135.4, 133.3, 132.3, 131.8, 6 135.4, 133.3, 127.5, 132.3,127.2, 131.8,124.2, 128.4,123.6, 128.2,97.6, 127.5, 127.2, 123.6, (ESI): 97.6, 38.6, 14.9.for HRMS + H]+ 128.4, 128.2, 38.6, 38.5, 124.2, 14.9. HRMS [M + 38.5, H]+ calcd C17H18(ESI): N2O3,[M 345.1317; calcd C17 H18 N2 O3 , 345.1317; found, 345.1314. found,for 345.1314.

3-Cyclopropyl-1-(naphthalen-2-yl)-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (25); (25); Mp: Mp: 128.8–129.5 °C. 3-Cyclopropyl-1-(naphthalen-2-yl)-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione 128.8–129.5 1H-NMR (400 MHz, DMSO-d6): δ 8.08–7.93 (m, 4H), 7.69–7.54 (m, 3H), 3.89 (s, 2H), 3.64 (s, 2H), 2.82–2.77 (m, ◦ C. 1 H-NMR (400 MHz, DMSO-d ): δ 8.08–7.93 (m, 4H), 7.69–7.54 (m, 3H), 3.89 (s, 2H), 3.64 (s, 2H), 6 1H), 0.78-0.73 (m, 2H), 0.56–0.50 (m, 2H). 13C-NMR (100 MHz,13CDCl3): δ 193.7, 181.2, 167.5, 134.9, 133.4, 131.8, 2.82–2.77 (m, 1H), 0.78-0.73 (m, 2H), 0.56–0.50 (m, 2H). C-NMR (100 MHz, CDCl3 ): δ 193.7, 181.2, 129.9, 127.9, 127.2, 126.5, 121.7, 123.5, 120.5, 99.4, 83.6, 77.4, 29.7, 21.6, 6.5. HRMS (ESI): [M + H]+ calcd for 167.5, 134.9, 133.4, 131.8, 129.9, 127.9, 127.2, 126.5, 121.7, 123.5, 120.5, 99.4, 83.6, 77.4, 29.7, 21.6, 6.5. Molecules 2018, 23, x FORfound, PEER REVIEW 12 of 14 C 19H16N2O 3, 321.1161; 321.1152. HRMS (ESI): [M + H]+ calcd for C19 H16 N2 O3 , 321.1161; found, 321.1152.

◦ C. 3-Amino-1-(naphthalen-2-yl)-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (26);168.4–169.2 Mp: 168.4–169.2 3-Amino-1-(naphthalen-2-yl)-2,3-dihydrofuro[2,3-d]pyrimidine-4,5(1H,6H)-dione (26); Mp: °C. 1H-NMR 1(400 MHz,(400 CDClMHz, 3): δ 10.33 (s, 32H), 7.83–7.67 (m, 4H), 7.65–7.43 3H),7.65–7.43 5.51 (s, 2H), (s, 5.51 2H).13(s, C-NMR (100 H-NMR CDCl ): δ 10.33 (s, 2H), 7.83–7.67 (m,(m,4H), (m,4.88 3H), 2H), 4.88 13 MHz, 3): δ 194.7, 132.6, 185.4, 131.5, 132.2, 120.3, 87.3, 79.9,127.2, 76.2. (s, 2H).CDCl C-NMR (100185.4, MHz,162.7, CDCl133.5, 162.7, 128.4, 133.5,128.2, 132.6,127.5, 131.5,127.2, 132.2,124.2, 128.4, 128.2, 127.5, 3 ): δ 194.7, HRMS120.3, (ESI): 87.3, [M + 79.9, H]+ calcd C16H13(ESI): N3O3, [M 296.0957; 296.0957. 124.2, 76.2.for HRMS + H]+ found, calcd for C16 H13 N3 O3 , 296.0957; found, 296.0957.

4. 4. Conclusions In DHODH-specific-inhibitor pyrimidone In this this work, work, aa series seriesof ofbrand-new brand-newscaffold scaffoldPf PfDHODH-specific-inhibitor pyrimidone derivatives derivatives were were obtained obtained from from docking docking analyses analyses and and structural structural optimizations. optimizations. The most potent inhibitor inhibitor 26 26 showed DHODH (IC = 23 23 nM) and high species showed excellent excellent inhibitory inhibitory activity activity against against Pf PfDHODH (IC50 50 = species selectivity selectivity over studies, three preferential structural fragments were were essential for these over hDHODH. hDHODH.Through ThroughSAR SAR studies, three preferential structural fragments essential for novel potentpotent Pf DHODH inhibitors: (1) bicyclic systems suchsuch as “naphthyl-like” substituents as the these novel PfDHODH inhibitors: (1) bicyclic systems as “naphthyl-like” substituents as hydrophobic group; (2) two groupsgroups in the dihydrofuranone ring oriented in the same direction the hydrophobic group; (2)carbonyl two carbonyl in the dihydrofuranone ring oriented in the same and that formed hydrogen with bonds the polar residue (Arg265); (3) a(Arg265); hydrogen(3) bond donor (NH direction and that formedbonds hydrogen with the polar residue a hydrogen bond 2 in compounds 20 and 26) that was important for the inhibitory activity to interact with the imidazole donor (NH2 in compounds 20 and 26) that was important for the inhibitory activity to interact with group of His185. The of results might valuable for be thevaluable novel scaffold DHODH inhibitors to be the imidazole group His185. Theberesults might for thePfnovel scaffold PfDHODH developed asbe new antimalarial agents. inhibitors to developed as new antimalarial agents. Author L.X., W.L., W.L., H.S., H.S., and and H.Z. H.Z. Author Contributions: Contributions: H.L. H.L. and and Z.Z. Z.Z. conceived conceived and and designed designed the the experiments. experiments. L.X., performed the experiments. L.X., Y.D., and L.Z. analyzed the data. L.X., Y.D. wrote the paper. performed the experiments. L.X., Y.D., and L.Z. analyzed the data. L.X., Y.D. wrote the paper. Funding: This work was supported by the National Key Research and Development Program (Grant Funding: This work was supported the Foundation National Key Research and21372078 Development Programand (Grant 2016YFA0502304), the National Natural by Science of China (Grants and 81302697), the 2016YFA0502304), the National Science Foundation of China (Grants 21372078 and 81302697), and the Fundamental Research Funds forNatural the Central Universities. Fundamental Research Funds for the Central Universities. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 11–26 are available from the authors. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).