6-Hydrophobic aromatic substituent pyrimethamine analogues as potential antimalarials for pyrimethamine-resistant Plasmodium falciparum

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6-Hydrophobic aromatic substituent pyrimethamine analogues as potential antimalarials for pyrimethamine-resistant Plasmodium falciparum Siriporn Saepua, Karoon Sadorn1, Jarunee Vanichtanankul, Tosapol Anukunwithaya, Roonglawan Rattanajak, Danoo Vitsupakorn, Sumalee Kamchonwongpaisan, ⁎ Yongyuth Yuthavong, Chawanee Thongpanchang National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani 12120, Thailand

ARTICLE INFO

ABSTRACT

Keywords: Dihydrofolate reductase inhibitors Dihydropyrimidines Malaria Plasmodium falciparum

The series of des-Cl (unsubstituted) and m-Cl phenyl analogues of PYR with various flexible 6-substituents were synthesized and studied for the binding affinities with highly resistant quadruple mutant (QM) DHFR. The derivatives carrying 4 atoms linker with a terminal carboxyl substituted on the aromatic ring exhibited good inhibition to the QM enzyme and also showed effective antimalarial activities against resistant P. falciparum bearing the mutant enzymes with relatively low cytotoxicity to mammalian cells. The X-ray crystallographic analysis of the enzyme-inhibitor complexes suggested that the hydrophobic substituent at 6-position was accommodated well in the hydrophobic pocket and the optimal length of the flexible linker could effectively promote the binding of the terminal carboxyl group to the key amino acid residues, Arg59 and Arg122.

1. Introduction Plasmodium falciparum is the most prevalent and lethal malaria parasite affecting humans. More importantly, multidrug resistant strains P. falciparum have emerged, making most available antimalarial drugs less effective. P. falciparum dihydrofolate reductase (PfDHFR) has long been a proven drug target for malarial chemotherapy. The enzyme is responsible for reducing dihydrofolate to tetrahydrofolate in the folate pathway and exists together with thymidylate synthase (TS) as a bifunctional protein.1,2 Pyrimethamine (PYR, Fig. 1), which targets PfDHFR, has been used clinically in the treatment of malaria infection. Unfortunately, resistance of the parasite to this drug occurred rapidly.3 The various point mutations of DHFR, were shown to be the key events in the mechanism of the resistance. The conserved residues that contain in the active site region of pfDHFR, namely Ile14, Ala16, Trp48, Asp54, Phe58, Ser108, Ile64, and Thr185, are important in the activity of the enzyme. These amino acids interact with dihydrofolate and antifolate inhibitors, or the NADPH cofactor. The residues that have been involved in the mutations resulting in the resistance, including Ala16, Cys50, Asn51, Cys59, Ser108, and Ile64, are all located in the active site region. S108N was recognized as the first mutation and the level of resistance was subsequently increased by additional mutations at other

positions.4–7 Nonetheless, the possibilities for the mutation of the parasite are limited since the minimal activity of the DHFR is needed for the survival of the parasite.8–11 Therefore, the prospect for the development of new effective DHFR inhibitors for the resistant parasites is feasible. One of the key successes in rational antifolate design is P218, a new effective antifolate antimalarial currently in clinical development.12 Our previous study showed that removal of the p-Cl or replacement with m-Cl in the phenyl group of PYR (compounds P20 and P30) led to better binding with the mutant PfDHFRs, and PYR analogues with extended 6-substituents exhibited good binding with the mutant PfDHFRs, with very low affinity to human DHFR (hDHFR).13 In addition, it was also shown that the carboxylate group at the end of P218 side chain made charge-mediated hydrogen bonds with the conserved Arg122 residue of PfDHFR resulting in favorable binding characteristics.12 In this paper, a series of des-Cl (unsubstituted) and m-Cl phenyl analogues of PYR with various flexible 6-substituents were extensively studied for the binding affinities with highly resistant quadruple mutant (QM) DHFR. The derivatives with 6-substituent carrying a terminal carboxyl group for interaction with Arg122 were also explored. Moreover, the Xray crystal structures of some representative compounds with QMPfDHFR-TS were studied in order to understand the binding mode of

Corresponding author. E-mail address: [email protected] (C. Thongpanchang). 1 Present Address: Department of Chemistry, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Chalongkrung Road, Ladkrabang, Bangkok 10520, Thailand. ⁎

https://doi.org/10.1016/j.bmc.2019.115158 Received 24 August 2019; Received in revised form 7 October 2019; Accepted 8 October 2019 0968-0896/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Siriporn Saepua, et al., Bioorganic & Medicinal Chemistry, https://doi.org/10.1016/j.bmc.2019.115158

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Fig. 1. Chemical structures of previously reported DHFR inhibitors.

the compounds. The results provide important cue for rational drug design of this type of compounds.

hydrogenation, followed by alkylation with appropriate bromo alkyl ester gave PYR derivatives linked to the corresponding ester 8. The products with carboxylic side chain were achieved by hydrolysis of esters 8 under NaOH conditions. PYR analogues were used as their corresponding hydrochloride salts (10–33) for the testing of binding affinity with DHFRs and antiplasmodial activity. All tested compounds are summarized in Table 1.

2. Results and discussion 2.1. Chemistry Preparation of PYR analogues was carried out using the method described by Ji et al.14 in combination with the modified method reported by our group13 and the synthetic route is shown in Scheme 1. The coupling of nitriles 2 with esters 1 under KOt-Amyl afforded the corresponding β-ketonitriles 3, which upon treatment with diazomethane, followed by condensation of intermediate enol ethers 4 with guanidine finally furnished PYR derivatives 5. For derivatives bearing carboxylic side chain, the additional further steps are outlined in Scheme 2. The cleavage of the benzyl group on the side chain of 6 by

2.2. Enzymatic inhibitory activity In order to avoid unfavorable steric interaction between p-Cl atom of the rigid phenyl side chain of PYR and the side chain of Asn108 of mutant PfDHFR, which has been clearly shown in our previous report on the X-ray crystal structures of PfDHFRs,15 a series of des-Cl (unsubstituted) and m-Cl phenyl analogues of PYR with different substituents at 6-position of pyrimidine ring were synthesized. The

Scheme 1. General procedure for the synthesis of compound 5. 2

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Scheme 2. General procedure for the synthesis of compound 9.

synthesized compounds lacking of p-substituents at the 5-phenyl side chain presented improvement on their binding affinities with the mutant enzyme by about 10–100 folds and lower ratios of Ki for mutant enzyme to Ki for wild type enzyme as compared to PYR. Pairs of compounds in the series of des-Cl and m-Cl analogues with the same substituent at C-6 showed comparable Ki values against wild-type PfDHFR with Ki-values in the range of 0.21–1.78 nM (mean 0.7) versus 0.24–3.76 (mean 0.87) for des-Cl and m-Cl compounds respectively. The Ki-ratio of des-Cl versus m-Cl compounds is in the range of 0.47–1.77 (mean 1.02) as compared to 2.88 for Ki-ratio of P20 versus P30. As for the mutant enzyme, m-Cl analogues tend to show higher affinities than their des-Cl pairs with Ki-values in the range of 0.5–33.3 (mean 9.9) nM versus 0.41–15.9 (mean 3.8) and the Ki- ratio of des-Cl versus m-Cl compounds of about 1.13–5.45 (mean 2.2) as compared to 9.67 for Kiratio of P20 versus P30. The influence of the aromatic substituents at 6-position of pyrimidine ring and the length of the linker between the rings (m + n) on the binding affinity was observed for both wild type and mutant DHFRs. Compounds 10–15 and 22–25 with short atom linker, 2 methylene groups (m = 2, n = 0) or 1 methylene group and 1 oxygen atom (m, n = 1), bearing any substituents on the aromatic ring, exhibited good binding affinity to wild-type but considerably poorer affinity to mutant DHFRs than the other compounds with longer atom linker. Compounds 22&23, 26&27, and 28&29 carrying different number of linker, m = 1, 3, and 4, respectively, but with same number of n (n = 1) and same substitution on the aromatic ring (R′ = 3-OCH2COOH) exhibited similar good binding affinity against wild-type enzyme (Kiwt = 0.25–1.02 nM) but different range of Ki-values against mutant enzyme (Ki-QM = 0.41–27.48 nM). Compounds with long linker (m = 3 and 4, 26–29) tend to exhibit better Ki-values against mutant enzyme (Ki-QM = 0.41–1.04 nM) and the best optimal number of linker is the 4-atom linker (m = 3 + n = 1), with compounds 26 and 27 as the most active against wild-type and mutant enzyme. Similarly, among the compounds without a substituent (R' = H) on the aromatic ring (compounds 16–21), compounds 16 and 17 with 4-atom linker were the most active inhibitors against both wild type and mutant enzymes which were about 3–30 fold more effective than 6-ethyl analogues (P20, P30). Extension of the aromatic ring with the alkoxy group carrying a terminal carboxylic acid (compounds 26–33) led to enhanced binding to both wild-type and mutant enzymes. In comparison, the length of the alkoxy chain between the aromatic ring and the carboxyl

Table 1 Structures of the synthetic compounds 10–33.

Compounds

Ori code

R

m

n

R′

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

B09015 B09023 B09013 B09014 B11053 B11054 B10041 B10042 B10045 B10046 B10051 B10052 B12125 B12126 B12127 B12128 B12153 B12154 B12148 B12145 B11077 B12155 B12129 B12146

H Cl H Cl H Cl H Cl H Cl H Cl H Cl H Cl H Cl H Cl H Cl H Cl

2 2 2 2 2 2 3 3 4 4 5 5 1 1 1 1 3 3 4 4 3 3 4 4

0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2-OCH2Ph 2-OCH2Ph 2-O(CH2)3Ph 2-O(CH2)3Ph 2-O(CH2)3COOEt 2-O(CH2)3COOEt H H H H H H 3-OCH2COOH 3-OCH2COOH 3-O(CH2)3COOH 3-O(CH2)3COOH 3-OCH2COOH 3-OCH2COOH 3-OCH2COOH 3-OCH2COOH 3-O(CH2)3COOH 3-O(CH2)3COOH 3-O(CH2)3COOH 3-O(CH2)3COOH

hydrophobic aromatic substituents at 6-position were designed with various lengths of linker for proper interaction with the hydrophobic environment in the active site pocket (Leu46, Ile112, Pro113, Phe116 and Leu119).12,15 Meanwhile, the carboxyl group at the end of the 6substitution was introduced for anchoring with Arg122,12 which should lead to the increase in the binding affinity of inhibitors against the mutant enzymes. All these synthesized compounds were subjected to the test for inhibitory activity with wild type (wt), quadruple mutant (QM, N51IC59RS108NI164L), and hDHFRs. Ki values for the synthesized compounds are shown in Table 2 along with Ki values of PYR, P20 and P30 for comparison. As expected, all the 3

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Table 2 Inhibition constants (Ki values) of PYR analogues and reference compounds (PYR, P20 and P30) against wild type (wt) and quadruple mutant (QM) PfDHFRs and hDHFR. compd PYR P20a P30a 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 a b c

Ori code

Ki wt (nM)b

rel to PYRc

Ki QM (nM)b

Ki QM/Ki wt

rel to PYRc

Ki human (nM)b

Ki human/Ki wt

Ki human/Ki QM

B09015 B09023 B09013 B09014 B11053 B11054 B10041 B10042 B10045 B10046 B10051 B10052 B12125 B12126 B12127 B12128 B12153 B12154 B12148 B12145 B11077 B12155 B12129 B12146

0.60 ± 0.20 2.3 ± 0.3 0.8 ± 0.1 0.96 ± 0.05 0.64 ± 0.11 1.78 ± 0.13 3.76 ± 0.29 1.17 ± 0.21 0.66 ± 0.16 0.21 ± 0.02 0.24 ± 0.02 0.28 ± 0.06 0.38 ± 0.04 0.30 ± 0.08 0.55 ± 0.09 1.02 ± 0.10 0.88 ± 0.10 1.48 ± 0.17 1.55 ± 0.24 0.26 ± 0.04 0.25 ± 0.01 0.50 ± 0.01 0.37 ± 0.07 0.29 ± 0.03 0.44 ± 0.10 0.55 ± 0.03 0.45 ± 0.06

1.00 3.83 1.33 1.60 1.07 2.97 6.27 1.95 1.10 0.35 0.40 0.47 0.63 0.50 0.92 1.70 1.47 2.47 2.58 0.43 0.42 0.83 0.62 0.48 0.73 0.92 0.75

385.00 ± 163.0 31.9 ± 7.8 3.3 ± 0.4 8.71 ± 0.22 3.06 ± 0.33 22.44 ± 1.07 15.89 ± 0.43 33.32 ± 2.88 7.50 ± 0.57 1.37 ± 0.33 1.16 ± 0.05 1.73 ± 0.12 1.49 ± 0.10 3.61 ± 0.76 3.20 ± 0.48 27.48 ± 1.82 5.04 ± 0.15 17.08 ± 0.50 4.96 ± 0.20 0.50 ± 0.06 0.41 ± 0.03 1.04 ± 0.10 0.72 ± 0.05 1.26 ± 0.12 0.98 ± 0.11 1.24 ± 0.17 0.93 ± 0.11

641.67 13.87 4.13 9.07 4.78 12.61 4.23 28.48 11.36 6.52 4.83 6.18 3.92 12.03 5.82 26.94 5.73 11.54 3.20 1.92 1.64 2.08 1.95 4.34 2.23 2.25 2.07

1.000 0.083 0.009 0.023 0.008 0.058 0.041 0.087 0.019 0.004 0.003 0.004 0.004 0.009 0.008 0.071 0.013 0.044 0.013 0.001 0.001 0.003 0.002 0.003 0.003 0.003 0.002

28.30 ± 2.50 10.4 ± 0.8 1.6 ± 0.2 22.07 ± 1.12 14.36 ± 0.96 27.27 ± 0.48 High 32.43 ± 2.36 23.42 ± 3.54 12.50 ± 0.25 8.72 ± 0.59 48.67 ± 3.42 12.77 ± 1.06 32.89 ± 3.23 18.41 ± 3.43 508.88 ± 80.91 52.39 ± 1.26 555.55 ± 14.03 74.16 ± 1.52 18.24 ± 1.39 13.59 ± 0.60 45.97 ± 1.37 13.41 ± 1.02 29.05 ± 0.63 19.55 ± 0.73 34.84 ± 2.45 21.72 ± 0.24

47.17 4.52 2.00 22.99 22.44 15.32 N/A 27.72 35.48 59.52 36.33 173.82 33.61 109.63 33.47 498.90 59.53 375.37 47.85 70.15 54.36 91.94 36.24 100.17 44.43 63.35 48.27

0.07 0.33 0.48 2.53 4.69 1.22 N/A 0.97 3.12 9.12 7.52 28.13 8.57 9.11 5.75 18.52 10.39 32.53 14.95 36.48 33.15 44.20 18.62 23.06 19.95 28.10 23.35

Data from Ref. 16. Standard error of the mean (SEM) was reported based on 3 replicates. Ki synthetic compound/Ki PYR.

centroid-to-centroid distances of 5.1, 5.6 and 5.8 Å, respectively. These geometry are following the favorable forces described by Hunter et al.17 Thus, this resulted in difference of inhibitory activities of the two compounds by 12 folds (Ki 0.41 and 4.96 nM, for 27 and 25, respectively). The inhibition constant of compound 27 was also much smaller than that of compound 17 (Ki 1.16 nM), in which its flexible linker was the same as that of 27 except for the absence of terminal carboxylate group on the benzene ring, by 3 folds. In comparing compounds 17 and 27, both benzene rings were found to reach out toward Phe116 (Fig. 3), where the hydrophobic interaction could possibly occurred, suggesting that, in addition to the essential interaction of carboxylate group with Arg59 and Arg122, Phe116 might be one of the key residues which could accommodated hydrophobic linker. Interestingly, compound 31, which has 2 more carbon linker than that of compound 27, bound to different residues on the target protein while retaining good Ki values. The crystal structure of the compound 31 complexed with PfDHFR showed that the terminal carboxylate group bound to the backbone nitrogen of Leu 46. However, this pulled the benzene ring away from the favorable distance from Phe58 and Phe116 (Fig. 4). This result suggested an optional position for terminal carboxylate that can be accommodated without substantially disrupting the overall binding.

group, with one to three carbon atoms (compounds 22&24, 23&25, 26 &30, 27&31, 28&32, and 29&33) had little effect on the binding affinity with the enzymes. 2.3. Co-crystal structures of QM-PfDHFR-TS The difference in biological activity urges us to pursuit of high-resolution crystal structure to understand the binding mode of the compounds in this study. Compounds 17, 25, 27, and 31 were able to cocrystallize with quadruple mutant PfDHFR-TS in the presence of cofactor NADPH and dUMP, thus serving as our model systems to investigate the structure activity relationship. All four co-crystals belong to space group P212121 and contain two protein molecules per asymmetric unit (Table 3). All solved crystal structures showed conserved anchoring of 2,4-diaminopyrimidine at the binding site in the same fashion as P218 bound to QM-PfDHFR-TS (PDB:4DP3), as reported earlier.12 The rmsd measurements between the search model (PDB:4DP3) superimposition with the model complex with compounds 17, 25, 27, and 31 are 0.405, 0.206, 0.261, and 0.263 Å, respectively. Among the compounds in this series, compound 27 showed the highest inhibition on QM-PfDHFR-TS with Ki of 0.41 nM, suggesting that flexible linker connected at 6-position was of the optimal length that could effectively promote the binding of the terminal carboxylate group to the key amino acid residues. The crystal structure showed that the terminal carboxylate interacted directly to Arg59 and indirectly to Arg122 via a water-bridge (Fig. 2). Although compounds 25 and 27 had the same number of atoms (m + n + x = 5) and functional group in the flexible linker, the configurations of benzene ring were different (Fig. 2). The three-dimensional pose between 25 and 27 showed differences in both direction of terminal carboxylate group and orientation of benzene ring. It is likely that the benzene ring in the linker of compound 27 was situated in a more favorable position, where it was surrounded by Phe58, Phe116 and internal aromatic ring, with the

2.4. In vitro antiplasmodial activity The in vitro antiplasmodial activities were determined against wild type (TM4/8.2 clone) and resistant parasite (V1/S clone, carrying quadruple amino acid mutation in the DHFR). The results are summarized in Table 4. Most of the compounds exhibited IC50 values against both wild type and resistant parasites at low micromolar levels. Particularly noteworthy were the IC50 values of compounds 27 and 29, with 4-atom linker with the terminal carboxylic substituted on the 4

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Table 3 Data collection and refinement statistics.

PDB code Data collection Space group Molecules/ASU Cell dimensions a, b, c (Å) Resolution (Å) Measured/unique reflections Completeness (%) Redundancy Rsym (%) I/σ Refinement Rwork/Rfree RMSD Bond length (Å) Bond angle (deg) Ramachandran plot (%) Residues in Favored region Allowed region Outlier region

17 (B09042)

25 (B12128)

27 (B12154)

31 (B12155)

6KP2 BL13B1 P212121 2

6KOT BL13B1 P212121 2

6KP7 BL13B1 P212121 2

6KPR BL13B1 P212121 2

56.995, 155.944, 165.340 50–1.97 (2.04–1.97) 391,373/104,189 99.7 (99.9) 3.8 (3.8) 3.2 (16.1) 34.1 (8.0)

57.176, 156.175, 165.080 50–2.15 (2.23–2.15) 257,996/70,259 86.2 (88.2) 3.7 (3.1) 5.8 (40.3) 19.8 (3.0)

57.885, 156.288 165.288 50–1.97 (2.04–1.97) 627,739/105,672 98.8 (91.0) 5.9 (5.3) 5.5 (43.5) 28.6 (3.4)

57.902, 156.504, 165.484 50–2.10 (2.18–2.10) 549,882/88,221 99.1 (96.5) 6.5 (6.0) 5.6 (44.9) 29.6 (3.7)

0.1730/0.2129

0.1936/0.2490

0.1943/0.2326

0.1950/0.2411

0.0126 1.8070

0.0093 1.7018

0.0117 1.7721

0.0107 1.7149

93.68 4.57 1.75

92.79 4.29 2.92

93.33 4.25 2.42

93.92 4.77 1.91

Values in parentheses are for the highest resolution shell. Rsym is∑∣Ij−〈I〉∣/∑I, where Ij is the intensity of an individual reflection, and 〈I〉 is the mean intensity for multiple recorded reflections.

aromatic ring, which were about 200 and 100 times more effective than PYR and 6-ethyl analogues (P20), respectively, against the quadruple mutant resistant parasite. Toxicities of these compounds was also tested to evaluate the selective inhibition against PfDHFR over human DHFR (Table 2) and malaria parasite over the African green monkey kidney fibroblasts (Vero cells, Table 4). All tested compounds were found to bind more selectively to both wild type and mutant PfDHFRs than the binding against hDHFR. Interestingly, the most active compounds (27, 30–31) were relatively nontoxic to Vero cell compared to their activities against malaria parasites (IC50Vero/IC50TM4/8.2 > 200–1000, IC50Vero/IC50V1/S > 20–100). Many of the remaining were, however, quite toxic to Vero cells with the IC50 < 2 μM and the safety ratio (IC50Vero/IC50V1/S) < 1. It should be noted that the selective inhibition of these compounds against PfDHFR were not well-correlated with the in vitro cytotoxicity, which may be affected by other factors such as solubility and permeability of these compounds to access the target in mammalian cells.

3. Conclusion Our study demonstrated that the binding affinities of the des-Cl (unsubstituted) and m-Cl phenyl analogues of PYR with highly resistant quadruple mutant (QM) DHFR can be improved with the flexible substituents at 6 position of the pyrimidine ring. Compound 27, bearing 4 atoms in the linker with a terminal carboxyl substituted on the aromatic ring, exhibited the best inhibition on QM-PfDHFR-TS with Ki of 0.41 nM. The mode of binding of some representative compounds with QM-PfDHFR-TS was revealed by X-ray crystallographic study. It was shown that the hydrophobic substituent at 6-position is accommodated well in the hydrophobic active site pocket and the appropriate length of the flexible linker could effectively promote the binding of the terminal carboxylate group to the key amino acid residues, Arg59 and Arg122. Some of these compounds showed good antimalarial activities against the quadruple-mutant resistant parasite with relatively low cytotoxic to mammalian cells, suggesting that they are potential candidates for further development as new antimalarials for PYR-resistant P. falciparum.

Fig. 2. A binding modes of (A) compound 27 and (B) compound 25 in mutant PfDHFR; depicted the differences on terminal carboxylate group and positioning of benzene in the linker. Hydrogen bond and hydrophobic interactions are shown as yellow dashed lines. 5

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4.2. Synthesis Pyrimethamine analogues were synthesized by the method described in previous studies13,14 as shown in Scheme 1. General procedure for the synthesis of derivatives bearing carboxylic side chain is outlined in Scheme 2. 4.2.1. Preparation of compound 7 A mixture of 6 (5 mmol) and Pd(OH)2 on charcoal (1.65 g, 0.33 g/ mmol) in EtOAc/CH2Cl2 (1:1, 40 mL) was stirred at room temperature overnight under H2. After filtered through celite, the reaction mixture was purified by preparative HPLC using reverse phase column (SunFire C18 OBD, 10 μm, 19 × 250 mm, step gradient elution with 25–50% MeOH + 0.01%HCOOH/H2O + 0.01%HCOOH) to afford compound 7 as a white solid. 4.2.2. Preparation of compound 8 To a suspension of K2CO3 (1.04 g, 7.5 mmol) and compound 7 (1.5 mmol) in DMF (7.5 mL) was added a solution of appropriated bromo alkyl ester (3 mmol) in DMF (7.5 mL) and the mixture was left stirring at 80 °C overnight. The reaction mixture was neutralized with diluted aqueous HCl, then extracted with CH2Cl2, dried over MgSO4, and evaporated to dryness under reduced pressure to yield the crude product as yellow oil. After purification by preparative HPLC using reverse phase column (SunFire C18 OBD, 10 μm, 19 × 250 mm, step gradient elution with 25–50% MeOH + 0.01%HCOOH/H2O + 0.01% HCOOH), the desired product 8 was obtained as a white solid.

Fig. 3. Positioning of benzene ring without terminal carboxylate group of 17 (pink) were similar to benzene ring of 27 (orange), suggesting the favorable position for benzene ring in this binding pocket.

4.2.3. Preparation of compound 9 Compound 8 (1.0 mmol) was stirred in 10%NaOH (4.5 mL) at 90 °C for 2 h. The reaction mixture was acidified to pH 2 by diluted aqueous HCl then crystallized by MeOH to furnish compound 9 as a white solid. 4.2.4. Preparation of hydrochloride salt of PYR analogues (10–33) The concentrated HCl (37%, 29.6 μL, 0.3 mmol) was added to the solution of PYR analogues (0.3 mmol) in MeOH/CH2Cl2 (1:1, 4 mL) and the mixture was left stirring at room temperature for 15 min. A white solid of the desired hydrochloride salt was obtained after crystallization by EtOH/Et2O. 4.2.4.1. 6-(2-(Benzyloxy)phenethyl)-5-phenylpyrimidine-2,4-diamine (10). White solid (69%); Mp 227.5–229.0 °C; 1H NMR (400 MHz, DMSO-d6) δ 2.54 (2H, t, J = 7.2 Hz, CH2CH2Ar), 2.90 (2H, t, J = 7.1 Hz, CH2CH2Ar), 4.87 (2H, s, ArOCH2Ar), 6.58 (1H, brs, NH), 6.81–7.34 (14H, m, ArH), 7.64 (1H, brs, NH), 8.11 (1H, brs, NH), 12.92 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 28.07, 29.83, 68.83, 109.31, 111.99, 120.38, 127.30, 127.39, 127.69, 127.83, 128.39, 128.50, 129.02, 129.81, 130.33, 130.83, 137.09, 151.80, 154.90, 156.04, 164.00; HRMS (ESI) calcd for C25H24N4O + H 397.2023, found [M + H]+ 397.2022.

Fig. 4. Alternative binding of terminal carboxylate group has shown by compound 31 complexed with PfDHFR. Hydrogen bond and hydrophobic interactions are shown as yellow dashed lines.

4. Experimental 4.1. Methods and materials For the chemical synthesis, solvents (DMSO, ethanol, and dioxane) were dried according to standard methods. Reagents were purchased from Fluka, Merck, Sigma-Aldrich Ltd and were distilled before use. For enzyme studies, chemicals were obtained from Sigma-Aldrich Ltd., Merch, and BDH and were used without further purification. Melting points were measured using a BUCHI M565 melting point apparatus and are uncorrected. Nuclear Magnetic Resonance (NMR) spectra were recorded on Bruker AV400 and Bruker AV500D spectrometers. The chemical shifts (δ) are reported in parts per million (ppm) using tetramethylsilane (TMS) as an internal standard, and the coupling constants (J) are reported in Hertz (Hz). The HRMS data were obtained using a Bruker micrOTOF mass spectrometer. Column chromatography was performed on a silica gel 60 (70–230 Mesh ASTM, Merck). HPLC were performed using Dionex-Ultimate 3000 series equipped with a binary pump, an autosampler, and a diode array detector.

4.2.4.2. 6-(2-(Benzyloxy)phenethyl)-5-(3-chlorophenyl)pyrimidine-2,4diamine (11). White solid (83%); Mp 236.8–238.5 °C; 1H NMR (400 MHz, DMSO-d6) δ 2.56 (2H, t, J = 6.8 Hz, CH2CH2Ar), 2.90 (2H, t, J = 6.8 Hz, CH2CH2Ar), 4.87 (2H, s, ArOCH2Ar), 6.74–7.41 (13H, m, ArH), 6.79 (1H, brs, NH), 7.67 (1H, brs, NH), 8.11 (1H, brs, NH), 13.00 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 28.16, 29.77, 68.83, 108.20, 111.95, 120.39, 127.17, 127.23, 127.72, 127.93, 128.41, 128.66, 129.24, 129.87, 130.20, 130.77, 133.02, 133.57, 137.01, 152.08, 154.96, 156.03, 163.72; HRMS (ESI) calcd for C25H23ClN4O + H 431.1633, found [M + H]+ 431.1638. 4.2.4.3. 5-Phenyl-6-(2-(3-phenylpropoxy)phenethyl)pyrimidine-2,4diamine (12). White solid (62%); Mp 208.5–209.5 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.74 (2H, quin, J = 7.0 Hz, CH2CH2CH2), 2.50–2.57 (4H, m, 2 × CH2CH2Ar), 2.88 (2H, t, J = 7.1 Hz, 6

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Table 4 Antimalarial activities (IC50) against P. falciparum strains carrying wild type (TM4/8.2) and the mutant DHFRs (V1/S, N51I + C59R + S108N + I164L), and cytotoxicity (IC50) against Vero cell lines of PYR analogues and reference compounds (PYR, P20 and P30). compd PYR P20 P30 a 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 a b c d

Ori code

B09015 B09023 B09013 B09014 B11053 B11054 B10041 B10042 B10045 B10046 B10051 B10052 B12125 B12126 B12127 B12128 B12153 B12154 B12148 B12145 B11077 B12155 B12129 B12146

IC50 TM4/8.2 (μM)b

rel to PYRc

IC50 V1/S (μM)b

rel to PYRc

IC50 Vero (μM)b

IC50 Vero/IC50 TM4/8.2

IC50 Vero/IC50 V1/S

0.08 ± 0.01 0.72 ± 0.24 0.42 ± 0.10 0.65 ± 0.09 1.94 ± 0.42 6.73 ± 1.41 6.25 ± 1.48 1.25 ± 0.35 0.71 ± 0.04 0.13 ± 0.01 0.17 ± 0.01 0.36 ± 0.07 0.34 ± 0.07 0.38 ± 0.07 0.43 ± 0.26 52.60 ± 11.0 6.01 ± 1.42 5.24 ± 1.50 1.80 ± 0.60 0.071 ± 0.01 0.048 ± 0.01 0.058 ± 0.01 0.043 ± 0.01 0.052 ± 0.02 0.049 ± 0.02 0.071 ± 0.04 0.051 ± 0.02

1.00 9.00 5.25 8.13 24.25 84.13 78.13 15.63 8.88 1.63 2.13 4.50 4.25 4.75 5.38 657.50 75.13 53.50 22.50 0.89 0.60 0.73 0.54 0.65 0.61 0.89 0.64

> 100 > 50 9.11 ± 2.76 4.37 ± 0.89 4.81 ± 0.52 6.15 ± 1.82 5.50 ± 1.69 > 10 5.24 ± 0.59 15.10 ± 5.87 3.42 ± 0.95 11.20 ± 3.85 6.94 ± 1.35 7.89 ± 2.96 9.17 ± 2.41 > 100 11.40 ± 2.60 31.10 ± 9.15 6.20 ± 1.10 2.71 ± 0.94 0.43 ± 0.01 >1 1.42 ± 0.40 0.54 ± 0.11 0.44 ± 0.15 3.91 ± 1.28 3.00 ± 0.51

1.00 > 0.5 0.09 0.04 0.05 0.06 0.06 0.10 0.05 0.15 0.03 0.11 0.07 0.08 0.09 1.00 0.11 0.31 0.06 0.03 0.004 0.01 0.01 0.005 0.004 0.04 0.03

32.0 NTd 7.0 1.87 ± 0.80 0.40 ± 0.16 4.96 ± 0.40 0.65 ± 0.10 > 10 8.13 ± 2.61 1.39 ± 0.29 0.47 ± 0.21 15.38 ± 6.39 5.15 ± 2.32 7.91 ± 0.92 7.21 ± 2.25 > 100 > 10 > 50 > 100 > 10 > 50 >1 > 50 > 25 > 10 > 100 > 100

400.00 N/A 16.67 2.88 0.21 0.74 0.10 > 8.00 11.45 10.69 2.76 42.72 15.15 20.82 16.77 > 1.90 > 1.66 > 9.54 > 55.56 > 140.85 > 1041.67 > 17.24 > 1162.79 > 480.77 > 204.08 > 1408.45 > 1960.78

> 0.32 N/A 0.77 0.43 0.08 0.81 0.12 1.00 1.55 0.09 0.14 1.37 0.74 1.00 0.79 1.00 > 0.88 > 1.61 > 16.13 > 3.69 > 116.28 1.00 > 35.21 > 46.30 > 22.73 > 25.58 > 33.33

Data from Ref. 16. Standard error of the mean (SEM) was reported based on 3 replicates. IC50 synthetic compound/IC50 PYR. Not tested.

CH2CH2Ar), 3.71 (2H, t, J = 6.4 Hz, CH2CH2OAr), 6.54 (1H, brs, NH), 6.76–7.32 (14H, m, ArH), 7.68 (1H, brs, NH), 8.09 (1H, brs, NH), 13.04 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 28.10, 29.78, 30.30, 31.52, 66.36, 109.26, 111.25, 120.05, 125.82, 127.19, 127.87, 128.34, 128.41, 128.99, 129.83, 130.30, 130.88, 141.48, 151.87, 155.00, 156.39, 163.97; HRMS (ESI) calcd for C27H28N4O + H 425.2336, found [M + H]+ 425.2339.

4.2.4.6. Ethyl 4-(2-(2-(2,6-diamino-5-(3-chlorophenyl)pyrimidin-4-yl) ethyl)phenoxy)butanoate (15). White solid (49%); Mp 199.5–200.5 °C; 1 H NMR (400 MHz, DMSO-d6) δ 1.16 (3H, t, J = 7.1 Hz, OCH2CH3), 1.72 (2H, quin, J = 6.8 Hz, CH2CH2CH2), 2.26 (2H, t, J = 7.3 Hz, CH2CH2C]O), 2.48–2.52 (2H, m, ArCH2CH2), 2.83 (2H, t, J = 6.5 Hz, ArCH2CH2), 3.70–3.74 (2H, m, CH2CH2OAr), 4.04 (2H, q, J = 7.1 Hz, CH3CH2OC]O), 6.66–7.43 (8H, m, ArH), 7.64 (2H, brs, 2 × NH), 8.07 (1H, brs, NH), 12.92 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 14.12, 24.04, 28.09, 29.78, 30.19, 59.89, 66.24, 108.17, 111.35, 120.19, 126.96, 127.97, 128.58, 129.21, 129.82, 130.17, 130.75, 133.02, 133.53, 152.17, 154.97, 156.21, 163.70, 172.47; HRMS (ESI) calcd for C24H27ClN4O3 + H 455.1844, found [M + H]+ 455.1842.

4.2.4.4. 5-(3-Chlorophenyl)-6-(2-(3-phenylpropoxy)phenethyl)pyrimidine2,4-diamine (13). White solid (65%); Mp 211.5–213.5 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.76 (2H, quin, J = 7.0 Hz, CH2CH2CH2), 2.55 (4H, t, J = 7.6 Hz, 2 × CH2CH2Ar), 2.87 (2H, t, J = 6.5 Hz, CH2CH2Ar), 3.70–3.72 (2H, m, CH2CH2OAr), 6.64–7.37 (13H, m, ArH), 6.77 (1H, brs, NH), 7.68 (1H, brs, NH), 8.10 (1H, brs, NH), 12.93 (1H, brs, NH); 13 C NMR (100 MHz, DMSO-d6) δ 33.00, 34.48, 35.08, 36.30, 71.13, 112.96, 116.01, 124.82, 130.59, 131.66, 132.74, 133.07, 133.10, 133.33, 133.94, 134.60, 134.91, 135.47, 137.74, 138.28, 146.18, 156.87, 159.69, 161.11, 168.47; HRMS (ESI) calcd for C27H27ClN4O + H 459.1946, found [M + H]+ 459.1949.

4.2.4.7. 6-(3-Phenoxypropyl)-5-phenylpyrimidine-2,4-diamine (16). White solid (70%); Mp 219.5–221.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.98 (2H, brs, CH2CH2CH2), 2.38 (2H, t, J = 7.0 Hz, ArCH2CH2), 3.81 (2H, t, J = 5.6 Hz, CH2CH2OAr), 6.75–7.43 (10H, m, ArH), 7.37 (1H, brs, NH), 12.90 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 27.02, 27.51, 66.18, 108.66, 114.28, 120.50, 128.42, 129.18, 129.43, 130.50, 131.83, 154.00, 156.01, 158.20, 163.74; HRMS (ESI) calcd for C19H20N4O + H 321.1710, found [M + H]+ 321.1708.

4.2.4.5. Ethyl 4-(2-(2-(2,6-diamino-5-phenylpyrimidin-4-yl)ethyl)phenoxy) butanoate (14). White solid (34%); Mp 191.5–193.2 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.17 (3H, t, J = 7.1 Hz, OCH2CH3), 1.70 (2H, quin, J = 6.8 Hz, CH2CH2CH2), 2.26 (2H, t, J = 7.4 Hz, CH2CH2C]O), 2.48–2.51 (2H, m, ArCH2CH2), 2.84 (2H, t, J = 7.2 Hz, ArCH2CH2), 3.72 (2H, t, J = 6.3 Hz, ArOCH2CH2), 4.04 (2H, q, J = 7.1 Hz, CH3CH2OC]O), 6.54 (1H, brs, NH), 6.77–7.37 (9H, m, ArH), 7.66 (1H, brs, NH), 8.08 (1H, brs, NH), 13.06 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 14.13, 24.02, 27.98, 29.79, 30.18, 59.87, 66.19, 109.21, 111.32, 120.15, 127.21, 127.86, 128.42, 129.01, 129.81, 130.32, 130.90, 151.82, 155.00, 156.23, 163.95, 172.53; HRMS (ESI) calcd for C24H28N4O3 + H 421.2234, found [M + H]+ 421.2230.

4.2.4.8. 5-(3-Chlorophenyl)-6-(3-phenoxypropyl)pyrimidine-2,4-diamine (17). White solid (69%); Mp 246.5–248.2 °C; 1H NMR (400 MHz, DMSO-d6) δ 2.00 (2H, t, J = 6.6 Hz, CH2CH2CH2), 2.41 (2H, t, J = 7.3 Hz, ArCH2CH2), 3.83 (2H, t, J = 5.8 Hz, CH2CH2OAr), 6.74–7.51 (9H, m, ArH), 7.65 (1H, brs, NH), 8.16 (1H, brs, NH), 12.99 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 26.86, 27.00, 66.07, 107.73, 114.24, 120.55, 128.76, 129.46, 130.42, 131.01, 133.31, 133.73, 152.46, 154.97, 158.13, 163.85; HRMS (ESI) calcd 7

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for C19H19ClN4O + H 355.1320, found [M + H]+ 355.1327.

4.2.4.15. 4-(3-((2,6-Diamino-5-phenylpyrimidin-4-yl)methoxy)phenoxy) butanoic acid (24). White solid (96%); Mp 239.0–240.0 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.90 (2H, quin, J = 6.8 Hz, CH2CH2CH2), 2.36 (2H, t, J = 7.3 Hz, CH2CH2C]O), 3.91 (2H, t, J = 6.4 Hz, CH2CH2OAr), 4.64 (2H, s, ArCH2OAr), 6.41–7.52 (9H, m, ArH), 7.60 (1H, brs, NH), 12.20 (1H, brs, NH), 12.40 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 24.62, 30.53, 64.80, 67.07, 102.28, 107.35, 108.31, 109.22, 129.34, 129.76, 130.54, 130.71, 158.86, 160.07, 164.16, 174.53; HRMS (ESI) calcd for C21H22N4O4 + H 395.1714, found [M + H]+ 395.1718.

4.2.4.9. 6-(4-Phenoxybutyl)-5-phenylpyrimidine-2,4-diamine (18). White solid (73%); Mp 241.0–243.0 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.56 (2H, quin, J = 6.7 Hz, CH2CH2CH2), 1.66 (2H, quin, J = 7.6 Hz, CH2CH2CH2), 2.30 (2H, t, J = 7.6 Hz, CH2CH2Ar), 3.76 (2H, t, J = 6.2 Hz, CH2CH2OAr), 6.73 (1H, brs, NH), 6.82–7.47 (10H, m, ArH), 7.61 (1H, brs, NH), 8.17 (1H, brs, NH), 12.84 (1H, brs, NH); 13 C NMR (125 MHz, DMSO-d6) δ 24.51, 28.31, 30.00, 67.03, 109.26, 114.92, 120.93, 129.16, 129.77, 129.87, 130.92, 131.52, 152.95, 155.36, 158.92, 164.55; HRMS (ESI) calcd for C20H22N4O + H 335.1866, found [M + H]+ 335.1868.

4.2.4.16. 4-(3-((2,6-Diamino-5-(3-chlorophenyl)pyrimidin-4-yl)methoxy) phenoxy)butanoic acid (25). White solid (93%); Mp 226.0–228.0 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.90 (2H, quin, J = 6.8 Hz, CH2CH2CH2), 2.36 (2H, J = 7.3 Hz, CH2CH2C]O), 3.92 (2H, t, J = 6.3 Hz, CH2CH2OAr), 4.68 (2H, s, ArCH2OAr), 6.46–7.53 (8H, m, ArH), 7.80 (1H, brs, NH), 8.30 (1H, brs, NH), 12.20 (1H, brs, NH), 12.58 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 24.63, 30.55, 64.31, 67.08, 102.46, 107.51, 108.00, 108.47, 129.60, 130.54, 130.70, 131.58, 134.25, 158.71, 160.07, 164.10, 174.53; HRMS (ESI) calcd for C21H21ClN4O4 + H 429.1324, found [M + H]+ 429.1328.

4.2.4.10. 5-(3-Chlorophenyl)-6-(4-phenoxybutyl)pyrimidine-2,4-diamine (19). White solid (57%); Mp 229.5–231.4 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.58 (2H, quin, J = 6.5 Hz, CH2CH2CH2), 1.66 (2H, quin, J = 7.2 Hz, CH2CH2CH2), 2.30 (2H, t, J = 7.5 Hz, ArCH2CH2), 3.77–3.80 (2H, m, CH2CH2OAr), 6.82–7.50 (9H, m, ArH), 6.94 (1H, brs, NH), 7.64 (1H, brs, NH), 8.17 (1H, brs, NH), 12.85 (1H, brs, NH); 13 C NMR (125 MHz, DMSO-d6) δ 24.49, 28.30, 30.08, 67.07, 108.08, 114.89, 120.93, 129.27, 129.86, 130.88, 131.54, 133.77, 134.25, 153.28, 155.41, 158.91, 164.32; HRMS (ESI) calcd for C20H21ClN4O + H 369.1477, found [M + H]+ 369.1482.

4.2.4.17. 2-(3-(3-(2,6-Diamino-5-phenylpyrimidin-4-yl)propoxy) phenoxy)acetic acid (26). White solid (92%); Mp 230.0–232.0 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.90 (2H, quin, J = 6.6 Hz, CH2CH2CH2), 2.35 (2H, J = 7.5 Hz, ArCH2CH2), 3.79 (2H, t, J = 6.0 Hz, CH2CH2OAr), 4.59 (2H, s, ArOCH2C]O), 6.32–7.47 (9H, m, ArH), 7.35 (2H, brs, 2 × NH); 13C NMR (100 MHz, DMSO-d6) δ 27.79, 28.30, 65.66, 66.99, 101.76, 107.51, 107.79, 109.28, 129.06, 129.87, 130.56, 131.15, 159.71, 159.96, 164.37, 171.30; HRMS (ESI) calcd for C21H22N4O4 + H 395.1714, found [M + H]+ 395.1717.

4.2.4.11. 6-(5-Phenoxypentyl)-5-phenylpyrimidine-2,4-diamine (20). White solid (39%); Mp 228.6–230.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.26 (2H, quin, J = 7.2 Hz, CH2CH2CH2), 1.53 (2H, quin, J = 7.2 Hz, CH2CH2CH2), 1.56 (2H, quin, J = 7.4 Hz, CH2CH2CH2), 2.25 (2H, t, J = 7.6 Hz, ArCH2CH2), 3.84 (2H, t, J = 6.3 Hz, CH2CH2OAr), 6.72 (1H, brs, NH), 6.86–7.49 (10H, m, ArH), 7.64 (1H, brs, NH), 8.16 (1H, brs, NH), 12.93 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 24.81, 26.94, 27.98, 29.80, 66.83, 108.67, 114.41, 120.38, 128.72, 129.30, 129.44, 130.50, 131.13, 152.63, 154.91, 158.59, 164.04; HRMS (ESI) calcd for C21H24N4O + H 349.2023, found [M + H]+ 349.2021.

4.2.4.18. 2-(3-(3-(2,6-Diamino-5-(3-chlorophenyl)pyrimidin-4-yl) propoxy)phenoxy)acetic acid (27). White solid (88%); Mp 220.0–221.5 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.91 (2H, quin, J = 6.8 Hz, CH2CH2CH2), 2.34 (2H, J = 7.4 Hz, ArCH2CH2), 3.80 (2H, t, J = 5.8 Hz, CH2CH2OAr), 4.58 (2H, s, ArOCH2C]O), 6.32–7.49 (8H, m, ArH), 7.38 (1H, brs, 2 × NH); 13C NMR (100 MHz, DMSO-d6) δ 27.72, 28.28, 65.62, 66.95, 101.69, 107.46, 107.73, 107.99, 129.09, 130.11, 130.56, 131.08, 131.60, 134.31, 135.00, 159.72, 159.96, 164.12, 171.39; HRMS (ESI) calcd for C21H21ClN4O4 + H 429.1324, found [M + H]+ 429.1329.

4.2.4.12. 5-(3-Chlorophenyl)-6-(5-phenoxypentyl)pyrimidine-2,4-diamine (21). White solid (25%); Mp 208.2–209.7 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.28 (2H, quin, J = 7.3 Hz, CH2CH2CH2), 1.52 (2H, quin, J = 7.0 Hz, CH2CH2CH2), 1.54 (2H, quin, J = 7.0 Hz, CH2CH2CH2), 2.24 (2H, t, J = 7.8 Hz, ArCH2CH2), 3.85 (2H, t, J = 6.4 Hz, CH2CH2OAr), 6.86–7.50 (9H, m, ArH), 6.95 (1H, brs, NH), 7.52 (1H, brs, NH), 8.15 (1H, brs, NH), 12.34 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 24.77, 26.88, 27.99, 29.79, 66.84, 107.51, 114.41, 120.38, 128.83, 129.44, 130.47, 131.09, 133.37, 133.75, 152.89, 154.93, 158.58, 163.83; HRMS (ESI) calcd for C21H23ClN4O + H 383.1633, found [M + H]+ 383.1637.

4.2.4.19. 2-(3-(4-(2,6-Diamino-5-phenylpyrimidin-4-yl)butoxy)phenoxy) acetic acid (28). White solid (89%); Mp 237.1–239.1 °C; 1H NMR (500 MHz, DMSO-d6) δ 1.52 (2H, quin, J = 6.7 Hz, CH2CH2CH2), 1.59 (2H, quin, J = 7.1 Hz, CH2CH2CH2), 2.25 (2H, t, J = 7.2 Hz, ArCH2CH2), 3.77 (2H, t, J = 5.8 Hz, CH2CH2OAr), 4.61 (2H, s, ArOCH2C]O), 6.37–7.48 (9H, m, ArH), 7.36 (2H, brs, 2xNH); 13C NMR (100 MHz, DMSO-d6) δ 24.27, 27.85, 64.71, 66.77, 101.47, 106.71, 107.25, 108.57, 128.50, 129.22, 129.81, 130.39, 158.95, 159.48, 163.82, 170.39; HRMS (ESI) calcd for C22H24N4O4 + H 409.1870, found [M + H]+ 409.1875.

4.2.4.13. 2-(3-((2,6-Diamino-5-phenylpyrimidin-4-yl)methoxy)phenoxy) acetic acid (22). White solid (85%); Mp 246.3–247.9 °C; 1H NMR (400 MHz, DMSO-d6) δ 4.62 (2H, s, ArCH2OAr), 4.66 (2H, s, ArOCH2C]O), 6.45–7.54 (9H, m, ArH), 7.00 (1H, brs, NH), 7.77 (1H, brs, NH), 8.35 (1H, brs, NH), 12.40 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 64.32, 64.98, 102.62, 107.75, 108.32, 109.34, 129.56, 129.85, 130.59, 130.70, 155.60, 158.71, 159.31, 164.31, 170.45; HRMS (ESI) calcd for C19H18N4O4 + H 367.1401, found [M + H]+ 367.1409.

4.2.4.20. 2-(3-(4-(2,6-Diamino-5-(3-chlorophenyl)pyrimidin-4-yl)butoxy) phenoxy)acetic acid (29). White solid (93%); Mp 238.7–241.0 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.53 (2H, quin, J = 6.1 Hz, CH2CH2CH2), 1.61 (2H, quin, J = 7.5 Hz, CH2CH2CH2), 2.25 (2H, t, J = 7.1 Hz, ArCH2CH2), 3.76 (2H, brs, CH2CH2OAr), 4.58 (2H, s, ArOCH2C]O), 6.37–7.49 (8H, m, ArH), 7.56 (2H, brs, 2xNH); 13C NMR (100 MHz, DMSO-d6) δ 24.67, 28.32, 30.60, 65.36, 67.28, 101.88, 107.20, 107.55, 107.71, 129.03, 129.89, 130.29, 130.88, 131.48, 134.15, 134.50, 156.53, 159.51, 160.01, 164.02, 171.23; HRMS (ESI) calcd for C22H23ClN4O4 + H 443.1481, found [M + H]+ 443.1486.

4.2.4.14. 2-(3-((2,6-Diamino-5-(3-chlorophenyl)pyrimidin-4-yl)methoxy) phenoxy)acetic acid (23). White solid (85%); Mp 232.6–233.8 °C; 1H NMR (400 MHz, DMSO-d6) δ 4.47 (2H, s, ArCH2OAr), 4.57 (2H, s, ArOCH2C]O), 6.06 (2H, brs, 2 × NH), 6.31–7.43 (8H, m, ArH); 13C NMR (100 MHz, DMSO-d6) δ 65.07, 68.87, 101.90, 107.19, 107.29, 107.51, 127.92, 129.65, 130.30, 130.74, 130.95, 133.66, 137.10, 159.30, 159.73, 162.13, 163.03, 170.74; HRMS (ESI) calcd for C19H17ClN4O4 + H 401.1011, found [M + H]+ 401.1017.

4.2.4.21. 4-(3-(3-(2,6-Diamino-5-phenylpyrimidin-4-yl)propoxy) phenoxy)butanoic acid (30). White solid (68%); Mp 192.6–194.3 °C; 1H 8

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NMR (400 MHz, DMSO-d6) δ 1.92 (2H, quin, J = 6.8 Hz, CH2CH2CH2), 1.96 (2H, quin, J = 6.9 Hz, CH2CH2CH2), 2.38 (2H, t, J = 7.0 Hz, ArCH2CH2), 2.39 (2H, t, J = 6.3 Hz, CH2CH2C]O), 3.80 (2H, t, J = 5.8 Hz, CH2CH2OAr), 3.93 (2H, t, J = 6.4 Hz, CH2CH2OAr), 6.28–7.44 (9H, m, ArH), 6.70 (1H, brs, NH), 7.54 (1H, brs, NH), 8.05 (1H, brs, NH), 12.80 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 24.24, 26.92, 27.11, 30.15, 66.22, 66.53, 100.92, 106.58, 106.86, 108.85, 128.57, 129.21, 129.89, 130.49, 131.28, 155.19, 159.40, 159.68, 163.98, 174.14; HRMS (ESI) calcd for C23H26N4O4 + H 423.2027, found [M + H]+ 423.2021.

4.5. Parasite culture, antimalarial and cytotoxicity testing in vitro Two P. falciparum strains were used in this study. Parasite clones TM4/8.2 (Wild type DHFR) was generous gifts from S. Thaithong, Department of Biology, Faculty of Science, Chulalongkorn University. Parasite line V1/S (N51I + C59R + S108N + I164L) was from D. Kyle (MRA-157 and MRA-176, respectively, MR4, ATCC Manassas Virginia). These parasites were maintained continuously in human erythrocytes at 37 °C under 3% CO2 in RPMI 1640 culture media supplemented with 25 mM HEPES, pH 7.4, 0.2% NaHCO3, 40 μg/mL gentamicin and 10% human serum. In vitro antimalarial activity was determined by using [3H]-hypoxanthine incorporation method.21 Cytotoxicity tests of some analogues against Vero cell lines were performed according to the protocol described by Skehan et al.22

4.2.4.22. 4-(3-(3-(2,6-Diamino-5-(3-chlorophenyl)pyrimidin-4-yl) propoxy)phenoxy)butanoic acid (31). White solid (95%); Mp 178.2–180.0 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.92 (2H, quin, J = 6.9 Hz, CH2CH2CH2), 1.95 (2H, quin, J = 6.5 Hz, CH2CH2CH2), 2.36–2.39 (4H, m, ArCH2CH2, CH2CH2C]O), 3.81 (2H, t, J = 5.8 Hz, CH2CH2OAr), 3.93 (2H, t, J = 6.4 Hz, CH2CH2OAr), 6.30–7.48 (8H, m, ArH); 13C NMR (100 MHz, DMSO-d6) δ 24.70, 27.43, 28.15, 30.62, 66.77, 66.98, 101.33, 107.01, 107.31, 107.85, 128.88, 129.90, 130.36, 130.88, 131.37, 134.11, 156.80, 159.88, 160.17, 163.92, 174.63; HRMS (ESI) calcd for C23H25ClN4O4 + H 457.1637, found [M + H]+ 457.1643.

4.6. Crystallization, data collection and structure determination Purified QM-PfDHFR-TS (15 mg/ml) was co-crystallized with 2 mM each of inhibitor, NADPH and dUMP by incubation on ice for 1 hr. Crystallization was set up using microbatch technique as described previously.15 Crystals grew in crystallizing solution containing 0.1 M NaOAc, 0.2 M NH2OAc and 15% (w/v) PEG4000. A single crystal was picked up, dipped in cryoprotectant (20% glycerol in crystallizing solution) and flashed frozen in liquid nitrogen. Data were collected at beamline BL13B1 at NSRRC (Taiwan, ROC.) with ADSC Q315r CCD. Data were processed using HKL-2000.23 Structure was determined by the molecular replacement method with MOLREP24 in the CCP4 suite25 using the structure PDB ID 4DP3 as a search model. The model was built using the program COOT26 and refined by REFMAC27 in CCP4. Finally, models were validated using RAMPAGE28 as listed in Table 3. Figures were prepared using PyMOL.29

4.2.4.23. 4-(3-(4-(2,6-Diamino-5-phenylpyrimidin-4-yl)butoxy)phenoxy) butanoic acid (32). White solid (89%); Mp 211.1–213.5 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.54 (2H, quin, J = 6.3 Hz, CH2CH2CH2), 1.62 (2H, quin, J = 7.3 Hz, CH2CH2CH2), 1.91 (2H, quin, J = 6.9 Hz, CH2CH2CH2), 2.28 (2H, t, J = 7.4 Hz, ArCH2CH2), 2.37 (2H, t, J = 7.3 Hz, CH2CH2C]O), 3.75 (2H, t, J = 5.9 Hz, CH2CH2OAr), 3.94 (2H, t, J = 6.4 Hz, CH2CH2OAr), 6.37–7.48 (9H, m, ArH), 6.70 (1H, brs, NH), 8.05 (1H, brs, NH), 12.45 (1H, brs, NH); 13C NMR (100 MHz, DMSO-d6) δ 24.19, 24.23, 27.86, 30.12, 66.52, 66.63, 101.07, 106.64, 106.80, 108.72, 128.62, 129.28, 129.90, 130.47, 159.69, 163.96, 174.15; HRMS (ESI) calcd for C24H28N4O4 + H 437.2183, found [M + H]+ 437.2184.

Declaration of Competing Interest The authors declared that there is no conflict of interest. Acknowledgements

4.2.4.24. 4-(3-(4-(2,6-Diamino-5-(3-chlorophenyl)pyrimidin-4-yl)butoxy) phenoxy)butanoic acid (33). White solid (93%); Mp 199.2–201.9 °C; 1H NMR (400 MHz, DMSO-d6) δ 1.56 (2H, quin, J = 6.2 Hz, CH2CH2CH2), 1.63 (2H, quin, J = 7.5 Hz, CH2CH2CH2), 1.91 (2H, quin J = 6.8 Hz, CH2CH2CH2), 2.26 (2H, t, J = 7.2 Hz, ArCH2CH2), 2.37 (2H, t, J = 7.3 Hz, CH2CH2C]O), 3.76 (2H, brs, CH2CH2OAr), 3.94 (2H, t, J = 6.4 Hz, CH2CH2OAr), 6.38–7.46 (8H, m, ArH), 7.27 (2H, brs, 2xNH); 13C NMR (100 MHz, DMSO-d6) δ 24.63, 24.71, 28.17, 30.62, 66.98, 67.20, 101.50, 107.12, 107.21, 107.66, 128.89, 129.90, 130.34, 130.89, 131.43, 134.12, 160.16, 160.17, 163.86, 174.63; HRMS (ESI) calcd for C24H27ClN4O4 + H 471.1794, found [M + H]+ 471.1790.

We thank the providers of malaria parasites used in this study: S. Thaithong (Chulalongkorn University, Thailand) and MR4 (ATCC Manassas Virginia, contributed by Prof. D. Kyle). We thank the technical services provided by the “Synchrotron Radiation Protein Crystallography Facility of the National Core Facility Program for Biotechnology, Ministry of Science and Technology” and the “National Synchrotron Radiation Research Center”, a national user facility supported by the Ministry of Science and Technology, Taiwan, ROC. This research was supported by grants from BIOTEC and NSTDA’s Cluster and Program Management [Grant number P1450883 and P1850116].

4.3. Enzyme preparation

Appendix A. Supplementary data

The PfDHFR wild type and mutants were expressed in E. coli BL21(DE3)pLysS bearing pET-PfDHFRs expression plasmids.18 Following 0.4 mM IPTG induction at 20 °C for 20 h, the bacterial cells were disrupted by a French Pressure Cell at 18,000 psi and the enzymes (in 0.1 mM EDTA, 10 mM DTT, 50 mM KCl, 20 mM potassium phosphate buffer at pH 7.0, containing 20% v/v glycerol) were purified from the bacterial lysates by affinity chromatography using Methotrexate-Sepharose resin.13,16,18,19

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmc.2019.115158. References 1. Bzik DJ, Li WB, Horii T, Inselburg J. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene. Proc Natl Acad Sci USA. 1987;84:8360–8364. 2. Ivanetich KM, Santi DV. Bifunctional thymidylate synthase-dihydrofolate reductase in protozoa. FASEB J. 1990;4:1591–1597. 3. Corey VC, Lukens AK, Istvan ES, et al. A broad analysis of resistance development in the malaria parasite. Nat Commun. 2016;7:11901. 4. Cowman AF, Morry MJ, Biggs BA, Cross GA, Foote SJ. Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. Proc Natl Acad Sci USA. 1988;85:9109–9113. 5. Hyde JE. Mechanisms of resistance of Plasmodium falciparum to antimalarial drugs. Microb Inf. 2002;4:165–174.

4.4. Enzyme assays and inhibition by PYR derivatives The activity of PfDHFR was determined spectrophotometrically at 25 °C in cuvette format20 and inhibition constant (Ki-values) was in microplate format as described in detail elsewhere.13,16,18 These compounds were assumed as competitive inhibitors.13 9

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