Aza-acyclic Nucleoside Phosphonates Containing a Second

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Aza-acyclic Nucleoside Phosphonates Containing a Second Phosphonate Group As Inhibitors of the Human, Plasmodium falciparum and vivax 6‑Oxopurine Phosphoribosyltransferases and Their Prodrugs As Antimalarial Agents Dianne T. Keough,† Dana Hocková,*,‡ Zlatko Janeba,‡ Tzu-Hsuan Wang,† Lieve Naesens,§ Michael D. Edstein,∥ Marina Chavchich,∥ and Luke W. Guddat*,† †

The School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Brisbane 4072, Queensland Australia Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i. Flemingovo nám. 2, CZ-166 10 Prague 6, Czech Republic § Rega Institute for Medical Research, KU LeuvenUniversity of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium ∥ Department of Drug Evaluation, Australian Army Malaria Institute, Enoggera, Brisbane, Queensland 4051, Australia ‡

ABSTRACT: Hypoxanthine−guanine−[xanthine] phosphoribosyltransferase (HG[X]PRT) is considered an important target for antimalarial chemotherapy as it is the only pathway for the synthesis of the purine nucleoside monophosphates required for DNA/RNA production. Thus, inhibition of this enzyme should result in cessation of replication. The aza-acyclic nucleoside phosphonates (aza-ANPs) are good inhibitors of Plasmodium falciparum HGXPRT (Pf HGXPRT), with Ki values as low as 0.08 and 0.01 μM for Plasmodium vivax HGPRT (PvHGPRT). Prodrugs of these aza-ANPs exhibit antimalarial activity against Pf lines with IC50 values (0.8−6.0 μM) and have low cytotoxicity against human cells. Crystal structures of six of these compounds in complex with human HGPRT have been determined. These suggest that the different affinities of these aza-ANPs could be due to the flexibility of the loops surrounding the active site as well as the flexibility of the inhibitors, allowing them to adapt to fit into three binding pockets of the enzyme(s).

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INTRODUCTION

purine bases preformed in the host cell prior to being transported into the parasite. Hence, protozoan parasites such as Plasmodium possess only one metabolic route to synthesize the essential 6-oxopurine nucleoside monophosphates and this is via salvage.2 Though it has long been speculated that Plasmodium falciparum HGXPRT (Pf HGXPRT) could be a target for drugs aimed at curing malaria,3 it is only in recent times that inhibitors have been developed into compounds that possess antimalarial activity.2b,4 These compounds are structural analogues of the nucleoside monophosphates and are derived from a class of successful antiviral agents, the acyclic nucleoside

Hypoxanthine−guanine−[xanthine] phosphoribosyltransferase (HG[X]PRT) is an ubiquitous enzyme whose function is critical in many organisms. It catalyzes the formation of the 6-oxopurine nucleoside monophosphates from a purine base and 5′-phospho-α-D-ribosyl-1-pyrophosphate (PRib-PP).1 Pyrophosphate is also produced in this reaction (Figure 1). The nucleoside monophosphates are a central part of metabolism as they are required for DNA/RNA synthesis. There is only one other route by which they can be produced, and this is via the de novo pathway in which the purine ring is synthesized from small molecules. However, this is a highly energy demanding pathway requiring five molecules of ATP. Thus, some organisms and, in particular, protozoan parasites have evolved to circumvent the de novo pathway and, instead, utilize the © XXXX American Chemical Society

Received: September 16, 2014

A

dx.doi.org/10.1021/jm501416t | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

lipophilic or hydrophobic groups to the phosphorus atom either by a phosphoramidate or ester bond.11 Once inside the cell, the groups have to be hydrolyzed to produce the active parent compound.12 In the red blood cell, hydrolysis can be carried out by inherent lipases or esterases but the preferred option is that hydrolysis occurs only within the parasite itself. However, this goal is difficult to achieve. Thus, the possible cytotoxicity of these prodrugs needs to be considered because a certain proportion of the prodrug is likely to be hydrolyzed to its active component before entering the parasite cell. Therefore, the degree of inhibition of human HGPRT has to be known to determine if this will be a critical factor in the viability of these compounds as potential antimalarial prodrugs. In this study, new aza-acyclic nucleoside phosphonates (aza-ANPs) have been synthesized that contain a trisubstituted nitrogen atom to which two different phosphonate groups have been attached. The hypothesis is that one of these groups would be located in the 5′-phosphate binding pocket while the second phosphonate (with a longer linker) would fit into the PPi binding pocket. These chemical structures were designed and based on the crystal structure of human HGPRT in complex with ([(2-[(guanin-9H-yl)methyl]-propane-1,3-diyl)bis(oxy)]bis(methylene))diphosphonic acid.13 Prodrugs containing hydrophobic groups attached to the phosphonate moieties, either by a phosphoramidate or ester bond, were prepared and tested for their ability to suppress Pf replication in cultured erythrocytes. The cytotoxicity values in human cells, CC50, were then obtained to determine if there is a correlation between the degree of inhibition of the human enzyme and these values. In addition, the crystal structures of six of these compounds in complex with human HGPRT have been determined. On the basis of these structures, it is proposed that the differences in the affinity of the ANPs for the three enzymes lay in the nature of the flexible loops that move to accommodate the inhibitors.

Figure 1. Reaction catalyzed by HG[X]PRT. R = −H (hypoxanthine), −NH2 (guanine), −OH (xanthine).

phosphonates (ANPs).5 The basic structure of these potential antimalarial ANPs consists of a 6-oxopurine base connected to a phosphonate group by a variety of chemical linkers. Such compounds are good inhibitors of Pf HGXPRT and/or Plasmodium vivax (PvHGPRT) and possess a wide range of Ki values for these enzymes.6 Their advantages as potential antimalarial drugs are that they are cost-effective to synthesize and, because of the carbon−phosphorus bond, cannot be hydrolyzed within the cell to metabolites of reduced activity. A number of ANPs have now been synthesized using different chemical modifications to improve both potency and selectivity for the parasite enzyme(s) compared with their human counterpart.2a,7 One difference between these ANPs is the number of atoms in the linker connecting the N9 atom of the purine base to the phosphonate group. The length and chemical nature of this linker contributes to the orientation of the purine base and the phosphonate group in the active site. A number of studies have suggested that the optimum number of atoms in this linker is five for the parasite enzymes.8 This corresponds to the number of atoms between the N9 atom of the purine base and the phosphorus atom of GMP/IMP. Thus, the purine base is placed under the side chain of an aromatic residue (F186 in human HGPRT and F197 in Pf HGXPRT), and the phosphonate group reaches into the 5′-phosphate binding pocket. In some ANPs, this linker consists solely of carbon atoms while, in others, a single oxygen atom replaces a carbon.6,7 A recent advance has been the replacement of one of the carbons by a nitrogen atom that is positioned three atoms away from the N9 atom of the purine ring. A number of chemical attachments to this nitrogen have been made to try to occupy the third binding site of the enzyme which is that of pyrophosphate (PPi) (Figure 1).9 Schramm and colleagues have designed a second series of ANPs which also contain a nitrogen atom in the linker.2a,8a In these compounds, the nitrogen is two atoms away from the N9 of the purine ring, mimicking the location of the nitrogen in the iminoribitol ring of the transition state analogues or the oxygen in the ribose ring of GMP/IMP. These ANPs also exhibit selectivity between the parasite and the human enzymes. Because the critical amino acids in the active site of these enzymes are conserved, an explanation for this difference in selectivity is yet to be advanced. For successful drug design, the negative charges of the phosphonic oxygen atoms need to be masked to increase cell permeability. There are reports that some phosphonates are able to cross the red blood cell membrane, but this is slow and concentration dependent.10 In the case of the bisphosphonates, transport across the cell membranes is even more difficult, presumably because of the additional size, polarity, and charge(s). Increased cell permeability can be achieved by attaching

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RESULTS The Aza-ANPs Containing a Second Phosphonate Group and Their Prodrugs. The goal in the design of these inhibitors is to construct a single molecule containing three separate moieties able to bind in different areas of the active site. These are those of the purine ring, the 5′-phosphate group, and PPi, thus mimicking, in part, the two products of the reaction, GMP/IMP and PPi. The common feature of these ANPs is the presence of a purine base connected via a five atom linkage from the N9 of the purine ring to a phosphonate group. On the basis of previous studies, it is predicted that this phosphonate should reach into the 5′-phosphate binding pocket.9,13 Pivotal to the linkage is the trisubstituted nitrogen atom which allows an additional and variable substituent group to be added to the basic scaffold (Scheme 1). The design of the linker to the second phosphonate group is such that it should be oriented so that this group can occupy, or partially occupy, the PPi binding site. The aza-ANP prodrugs contain hydrophobic groups attached via phosphoramidate bonds to mask the negative charge on the parent compound so that transport across the cell membranes is facilitated (Scheme 1). This type of prodrug appears sufficiently stable to enable transport not only into the erythrocytes but also across a parasitophorous vacuole membrane and a parasite plasma membrane.14 Chemistry. Synthesis of the bisphosphonate type of aza-ANPs was based on our recently published approach.9 At first, aza-Michael reaction of aminoethanol and diethyl B



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Scheme 1. Synthesis of the Aza-ANPs Containing a Second Phosphonate Group and Their Respective Prodrugs

Table 1. Ki Values of the Aza-ANPs for Pf HGXPRT and PvHGPRT and a Comparison with the in Vitro Antimalarial Activity of Their Prodrugs against Pf Lines aza-ANPs Ki (μM) Pf

linker

a

−CH2-CH2-CH2-CH2− −CH2-CH2-O-CH2− −CH2-CH2-O-CH2-CH2−

7a 7b 7c

−CH2-CH2-O-CH2-CH2−

10c

−CH2-CH2-CH2-CH2− −CH2-CH2-O-CH2− −CH2-CH2-O-CH2-CH2−

6a 6b 6c

aza-ANP prodrugs Ki (μM) Pv

Guanine as Base 2 ± 0.6 0.13 ± 0.04 0.03 ± 0.02 8-Bromoguanine as Base 0.08 ± 0.04 0.01 ± 0.01 Hypoxanthine as Base 10 ± 2 20 ± 4 2.9 ± 0.2 1.6 ± 0.1 0.20 ± 0.09 0.04 ± 0.01

1.5 ± 1 0.50 ± 0.05 0.19 ± 0.09

9a 9b 9c

8a 8b 8c

IC50 (μM)a D6

IC50 (μM)b W2

6.0 ± 0.1 4.1 ± 0.2 6.0 ± 0.1

5.9 ± 0.7 2.7 ± 0.5 2.6 ± 0.7

NPD

NPD

1.5 ± 0.1 1.0 ± 0.1 2.1 ± 0.1

1.3 ± 0.5 1.5 ± 0.2 0.8 ± 0.1

Wild-type Pf D6 strain. bChloroquine and pyrimethamine-resistant Pf W2 strain. NPD = Prodrug not available. Values are means ± SD.

vinylphosphonate afforded the 2-(2-hydroxyethylamino)ethylphosphonate.9 The key branched hydroxy derivatives 1a−1c were prepared by the N-alkylation of this starting compound using K2CO3 as a base. Suitable halogeno derivatives with three different linkers were selected for attachment of the second phosphonate group to the scaffold (Scheme 1). The corresponding branched bisphosphonates 1a−1c were attached to the N9-position of 6-chloropurine or 2-amino-6-chloropurine via Mitsunobu reaction.15 The resulting 6-chloropurine phosphonates 2a−2c were transformed to hypoxanthine derivatives 4a−4c by nucleophilic aromatic substitution under acidic conditions (75% aqueous trifluoroacetic acid). In the case of 2-amino-6-chloropurine phosphonates 3a−3c, the Mitsunobu reaction had to be followed by heating

in water/tetrahydrofuran to decompose the triphenylphosphoranylidene intermediate arising from the presence of the free amino group.16 The chlorine atom was next displaced by a hydroxyl group in a quantitative yield as described above to form the guanine phosphonates 5a−5c. To form the free bisphosphonic acids 6a−6c and 7a−7c (for the determination of Ki values in enzyme assays), both phosphonate diester moieties of 4a−4c and 5a−5c were simultaneously hydrolyzed under standard conditions using Me3SiBr/ acetonitrile (Scheme 1). In the case of deprotection of 5c, 8-bromoguanine derivative 10c was isolated as a side product, probably due to the contamination of Me3SiBr by Br2. The tetraesters of bisphosphonates 4a−4c and 5a−5c were also used for the efficient preparation of the phosphoramidate C



Article

Replacement of the oxygen atom in 7b and 6b by a carbon resulted in an increase in the Ki values for all three enzymes (cf. compounds 7a with 7b and 6a with 6b, Tables 1 and 2). The compounds that only contain carbon in the linker bind more tightly to the human enzyme than to either of the parasite enzymes. It is, therefore, concluded that the design of this linker is an important factor in determining the affinity and selectivity of these aza-ANPs for the enzymes. The nature of the purine base also has an effect on the Ki values. In these aza-ANPs, all three enzymes prefer guanine as the base. Pf HGXPRT has a preference of ∼6-fold (cf. 7b and 6b) although, for 7c and 6c which have the lowest Ki values, there is little difference. For human HGPRT, the ratio between 7b and 6b is the same as for Pf HGXPRT (6-fold). However, for 7c and 6c, it is 12-fold. It is surprising that the aza-ANP, 10c, containing 8-bromoguanine as the base, is an effective inhibitor given the fact that this base is not a substrate for human HGPRT or, at best, a very weak substrate. Whether it is a substrate for the parasite enzymes has not been determined. Nonetheless, several moderate Pf HGXPRT inhibitors containing 8-bromoguanine as the base have previously been described.6 On the basis of the fact that the nature of the purine base makes a strong contribution to binding, it would have been expected that the ANPs containing this 8-bromoguanine would bind more weakly, if at all. However, the reverse appears to be the case. A possible explanation for aza-ANP, 10c, being such a good inhibitor is advanced in the light of the crystal structure in complex with human HGPRT. The IC50 values for the prodrugs, 9b, 9c, and 8c, against the Pf wild-type D6 strains are similar (1−6 μM) but are lower in the chloroquine-resistant Pf W2 (IC50 values 0.8−2.7 μM). 6b has the weakest affinity for Pf HGXPRT (Ki = 2.9 μM), but its prodrug, 8b, still has good antimalarial activity of 1 μM (D6 strain) and 1.5 μM (W2 strain). Thus, this prodrug exhibits no significant discrimination in activity between these two strains of malaria. This suggests that this prodrug design is effective and that the attached hydrophobic groups are hydrolyzed to the parent compound which then inhibits Pf HGXPRT within the cell. No obvious relationship between HG[X]PRT inhibition and cytotoxicity was observed. Prodrugs 9c and 8b have low cytotoxicity in the human cells (CC50 > 129 μM). 9b has increased cytotoxicity (CC50 = 59 μM) although it has the lowest Ki value for the Pf enzyme. However, prodrug 8c has the highest cytotoxicity although its parent 6c has the lowest affinity for human HGPRT (Table 2). Attachment of a Cyano Group Instead of a Phosphonate Group to the Second Linker. To evaluate the effect of the second phosphonate group, two inhibitors previously described were selected for comparison.9 These compounds, 9-[(N-(2-cyanoethyl)-N-(2-phosphonoethyl))-2aminoethyl]-guanine (11a) and 9-[(N-(2-cyanoethyl)-N-(2phosphonoethyl))-2-aminoethyl]-hypoxanthine (11b), do not contain a second phosphonate group but instead a cyano group attached to the nitrogen by two carbon atoms (Scheme 2). This side chain with the cyano group was designed so as to occupy the space used by one of the hydroxyl groups in the ribose ring of either GMP/IMP and to partially occupy the Mg2+·PPi binding site. Two types of prodrugs with hydrophobic groups attached via phosphoramidate or ester bonds were synthesized for testing in cell-based assays (Scheme 2). The Ki values of these aza-ANPs for the two parasite enzymes together with the

prodrugs 8a−8c and 9a−9c (suitable for antimalarial testing in the cell-based assays) by our recently published method.17 The selection of this particular type of phosphonate prodrug was based on our previous experience with increasing the membrane permeability of the ANPs.18 In the first step of the one-pot reaction sequence, the deprotection of the phosphonate esters 4a−4c and 5a−5c with Me3SiBr in dry pyridine forms the tetra(trimethylsilyl) esters in situ. In the second step, the reaction of these intermediates with ethyl (L)-phenylalanine in the presence of 2,2′-dithiodipyridine (Aldrithiol) and triphenylphosphine yields the corresponding tetra-amidates 8a−8c and 9a−9c (Scheme 1) without the need for isolation of the free phosphonic acid intermediates. All target compounds were purified by preparative HPLC. Inhibition of Pf, Pv, and Human 6-Oxopurine PRTases, in Vitro Antimalarial Activity and Cytotoxicity Studies. The potency and selectivity of inhibition of the aza-ANPs toward Pf HGXPRT, PvHGPRT, and human HGPRT was investigated. Table 1 gives the Ki values of the aza-ANPs for the two parasite enzymes together with the ability of their prodrugs to act as antimalarials against the blood stage growth of Pf in cell culture. Table 2 relates the Ki values for human HGPRT to the cytotoxicity of the corresponding prodrug in mammalian cells. Table 2. Comparison of the Ki Values of the Aza-ANPs with Human HGPRT and in Vitro Cytotoxicity of Their Prodrugs in Cell Culture base

AzaANP

guanine guanine guanine 8-Br-guanine hypoxanthine hypoxanthine hypoxanthine

7a 7b 7c 10c 6a 6b 6c

Ki (μM)

prodrug

± ± ± ± ± ± ±

9a 9b 9c

0.3 0.03 0.08 0.04 4 0.19 1.00

0.1 0.004 0.01 0.01 1 0.03 0.04

8a 8b 8c

CC50 (μM) (A549 cells)a,b

SIc

242 ± 44 59 ± 7 210 ± 76 NPD 44 ± 6 129 ± 12 39 ± 2

40 15 35 NPD 29 129 19

a

Values for the prodrug. bA549 = human lung carcinoma cells. Selectivity index (SI) = ratio of CC50 (A549)/IC50(D6) values. NPD = prodrug not available.

c

The aza-ANPs are good inhibitors of all three enzymes (Tables 1 and 2). The addition of an extra carbon in the linker containing oxygen increases the affinity of these compounds for the parasite enzymes (cf. 7b with 7c and 6b with 6c). For Pf HGXPRT, this decrease in the Ki value is approximately 2.5fold when guanine is the base (7b and 7c) but around 15-fold with hypoxanthine as the base (6b and 6c). In comparison, lengthening the linker has the reverse effect for the human enzyme, with the Ki increasing around 3-fold with guanine as the base (7b and 7c) and 5-fold with hypoxanthine as the base (6b and 6c). This means that 7b and 6b where the linker contains four atoms have a greater affinity for the human enzyme compared to the parasite enzymes (17- and 15-fold, respectively, for Pf HGXPRT and 4- and 8-fold for PvHGPRT). However, with the lengthening of the linker by one atom (7c and 6c), this ratio drops and/or the preference changes. The ratios of Ki (Pf HGXPRT)/Ki (human HGPRT) are 2.4 and 0.2 for 7c and 6c, respectively, and the ratios of Ki (PvHGXPRT)/Ki (human HGPRT) are 0.4 and 0.04 for 7c and 6c. Thus, this linker is clearly the optimum for the parasite enzymes. D



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Scheme 2. Synthesis of the Two Types of Prodrugs Derived from Aza-ANP Cyanoethyl Derivatives 11a and 11b

Table 3. Ki Values of Aza-ANPs Containing a Cyano Group for Pf HGXPRT and Pv HGPRT and the Antimalarial Activity of These Aza-ANPs in Comparison with the in Vitro Antimalarial Activity of Their Prodrugs Aza-ANP 11a 12a 13a 11b 12b a

guanine as the basea phosphoramidate of 11ab ester prodrug of 11ab hypoxanthine as the basea phosphoramidate of 11bb

Ki (μM) Pf

Ki (μM) Pv

0.2 ± 0.03

0.3 ± 0.03

0.2 ± 0.5

0.7 ± 0.06

IC50 (μM) D6

IC50 (μM) W2

± ± ± ± ±

≥300 10.4 ± 2.0 45 ± 3.1 ≥300 19.5 ± 7.5

145 6.4 24 75 12.5

12 0.9 2.0 10 1.6

The Ki values for the parent compound were previously determined.9 bValues for the prodrug. Values are means ± SD.

differ only within experimental error (Table 2). However, when the IC50 values for the wild-type and chloroquine resistant strains are compared among the cyano containing compounds, they are slightly lower for the wild-type compared to the chloroquine resistant strain (∼2-fold). This suggests a slight preference for the uptake of the cyano group into the wildtype parasite over the chloroquine resistant strain. The IC50 values for the parent cyano compounds are higher than those of their corresponding prodrugs (Table 4). 12b has a favorable selectivity index [CC50 (A549)/IC50(D6)] of >25 (Table 4), although this value is not as high as for the azaANP phosphoramidate prodrug, 8b, whose selectivity index is 129. Crystal Structures of 7b, 7c, 6b, 6c, 10c, and 11a in Complex with Human HGPRT. The crystal structures of 7b, 7c, 6b, 6c, 10c, and 11a in complex with human HGPRT have been determined. The refinement statistics are presented in Table 5. Five of these structures contain a tetramer in the asymmetric unit. The exception is the complex with 7c, which contains two tetramers. The overall fold for each of the subunits and the tetramer are similar in all cases. The electron density for 6c in the active site of human HGPRT is shown in Figure 2. The differences between these complexes lie in four areas: (i) the conformations of the seven mobile loops (residues 65−74, 102−115, 133−134, 137−141, 142−153, 165−171, 186−196), (ii) the orientation of critical amino acid residues in these

antimalarial activity of parent compounds compared with their prodrugs are given in Table 3. For these aza-ANPs, guanine is the preferred base for human HGPRT (Table 4), whereas, for the parasite enzymes, there is little discrimination (Table 3). It is clear that, although the parent compounds themselves possess measurable antimalarial activity, masking of the negative charges significantly improves these values (Table 4). The parent compounds are not cytotoxic (Table 3) in the human cell line A549 and neither is the prodrug of 11b (12b). The prodrug of 11a (12a), which has the lowest Ki value for human HGPRT, has the highest cytotoxicity. For the aza-ANPs containing a second phosphonate the IC50 values of the wild-type (D6) and chloroquine resistant (W2) Pf Table 4. Ki Values of the Aza-ANPs Containing a Cyano Group for Human HGPRT and in Vitro Cytotoxicity of the Parent Compounds and Their Prodrugs in Cell Culture azaANP 11a 12a 11b 12b

Ki (μM) guanine as the base 0.07 ± 0.02 phosphoramidate of 11ab hypoxanthine as the basea 2.4 ± 0.2 phosphoramidate of 11bb a

CC50 (μM) (A549 cells) >300 16 ± 2 >300 >300

SIc 2.6 >25

a

The Ki values for the parent compound were previously determined.9 Prodrug of the parent compound. cSI = ratio of CC50 (A549)/IC50 (D6) values. b

E


F

aza-ANPs water molecules Mg2+ ions

8

4 226

Unit Cell Parameters 52.83 a (Å) 114.80 b (Å) 67.66 c (Å) 90.0, 98.3, 90.0 α, β, γ (deg) space group P 21 Diffraction Dataa resolution 66.95−2.05 (2.16−2.05) range (Å) observations 173628 (14073) unique 48031 (5524) reflections completeness 95.9 (76.2) (%) Rmergeb 0.076 (0.554) Rpimc 0.056 (0.511) 11.4 (1.5) ⟨I⟩/⟨σ(I)⟩ Refinement resolution 41.44−2.05 (2.10−2.05) limits (Å) Rwork % 20.38 (30.86) Rfree % 24.20 (32.43) rmsd bond 0.006 lengths (Å) rmsd angles 0.975 (deg) Clashscored 11.0 Components of the Asymmetric Unit protein chain A 3−102, 113−217 B 3−103, 111−217 C 6−102, 112−217 D 3−102, 115−217

7b

41.18−1.87 (1.91−1.87) 19.86 (24.15) 22.97 (27.32) 0.007 1.22 3.9 A 1−102, 121−216 B 4−102, 115−120 123−170 173−217 C 4−102, 114−217 D 4−104, 112−217

35.56−2.00 (2.05−2.00) 21.52 (31.72) 26.59 (36.51) 0.003 0.862 11.4 A 5−101, 115−170, 174−217 B 4−103, 111−217 C 4−102, 113−215 D 4−101, 114−217 E 3−104, 111−168, 172−217 F 4−103, 113−117 123−169 173−217 G 5−102, 113−169 173−217 H 3−104, 112−217 8 598 9

A 4−103, 116−217 B 4−102, 114−170, 173−217 C 5−102, 113−217 D 4−102, 114−217 A 3−102, 115−119, 122−217 B 3−100, 116−217 C 4−101, 114−217 D 4−101, 115−217 A 3−103, 120−217 B 3−102, 113-217 C 5−101, 116−217 D 3−101, 114−217

0.054 (0.662) 0.023 (0.287) 16.2 (2.1)

0.064 (0.422) 0.043 (0.385) 7.7 (1.7)

7

4 194

9.24

3.8

8.9

99.8 (98.0)

98.7 (99.5)

9

4 114

1.159

17.75 (29.66) 26.91 (33.82) 0.008

58.90−2.59 (2.66−2.59)

0.123 (0.930) 0.054 (0.517) 13.0 (1.4)

97.5 (83.4)

4

4 24

0.698

19.48 (25.40) 24.57 (32.91) 0.003

20.01−2.55 (2.61−2.55)

0.058 (0.411) 0.026 (0.184) 19.9 (4.3)

99.3 (97.0)

7

4 152

0.89

20.92 (28.64) 23.14 (30.94) 0.008

41.53−2.26 (2.33−2.26)

0.097 (0.547) 0.072 (0.450) 8.0 (2.7)

98.7 (93.9)

158717 (12115) 42845 (3511)

496734 (30807) 68208 (4267)

282705 (25990) 109369 (10968)

200736 (26588) 29382 (4128)

46.14−2.21 (2.28−2.21)

20.01−2.55 (2.69−2.55)

92.61−2.53 (2.66−2.53)

46.43−1.87 (1.91−1.87)

35.56−2.00 (2.07−2.00) 188163 (14443) 27294 (3317)

55.73 129.00 62.94 90.0, 103.0, 90.0 P 21

10c

66.09 95.37 139.74 90.0, 90.0, 90.0 P 21 21 21

11a

76.33 92.61 115.01 90.0, 90.0, 90.0 P 21 21 21

6b

76.60 92.85 114.85 90.0, 90.0, 90.0 P 21 21 21

6c

76.05 114.93 97.54 90.0, 97.5, 90.0 P 21

7c

Table 5. X-ray Data Collection and Refinement Statistics for the Human HGPRT·Aza-ANP Structures

Journal of Medicinal Chemistry Article


Journal of Medicinal Chemistry where Ii(hkl) is the observed intensity and is the average intensity obtained from multiple observations of symmetry related reflections. dClashscore is defined as the number of bad overlaps ≥0.4 Å per thousand atoms.

hkl

⎡ ⎤1/2 1 ⎢ ⎥ ∑ |Ii(hkl) − (I(hkl))|/∑ ∑ Ii(hkl) ⎣ [N (hkl) − 1] ⎦ i hkl i

∑ R pim =

where Ii(hkl) is the observed intensity and is the average intensity obtained from multiple observations of symmetry related reflections.

i hkl i hkl

∑ ∑ |Ii(hkl) − (I(hkl))|/∑ ∑ Ii(hkl) R merge =

Values in parentheses are for the outer resolution shell. All data were collected at 100 K. a

Ramachandran Plot (%) favored 95.6 outliers 0.5

Table 5. continued

7b

96.4 0.1

7c

97.2 0.12

b

6c

94.6 0.0

6b

c

96.6 0.13

11a

97.2 0.1

10c

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Figure 2. Connolly surface for the active site of the human HGPRT− 6c complex (pdb code 4RAO). The 2Fo − Fc electron density for 6c is shown in orange mesh.

loops, (iii) the positioning of magnesium ions, and (iv) the location of each of the inhibitors in the active site. These variations occur not only between each of the six structures but also, in some cases, between the subunits of each structure. The latter provides a picture of how the inhibitor moves into the active site and the changes in the enzyme structure as this occurs. The structure of one of the subunits of human HGPRT in complex with 6c is shown in Figure 3, identifying the location of these seven mobile loops and how this compound binds in the active site. The flexibility of both the enzymes and the ligands can be factors that contribute to the binding affinity of a particular ligand for a particular enzyme. Further, this flexibility may also affect the selectivity ratio for the inhibitors when human HGPRT and the two parasite enzymes are compared. An alignment of the amino acid sequence of the human, Pf and Pv enzymes, highlighting their differences and similarities, is given in Figure 3. The location of each of the subunits of 6b, 6c, 7b, 7c, and 11a in the active site is shown in Figure 4. The Location of Magnesium Ions in Each of the Five Structures. In the structures of the five ligands in complex with human HGPRT, magnesium ions can be found in either one or two locations and, in some cases, both (Figure 4). The location of these ions differs between each of the structures and, in some cases, even between subunits of the same structure. In comparison, the unliganded structure of human HGPRT does not contain any metal ions, suggesting that the binding of such ions occurs in combination with the aza-ANPs. Site 1 for Metal Binding. Residues E133 and D134 in human HGPRT are conserved in the Plasmodial enzymes (Figure 3) and in the amino acid sequences of all of the known 6-oxopurine phosphoribosyltransferases (PRTases). In the absence of ligands, the side chains of these residues in the human structure are oriented away from each other (7.3 Å apart). The carboxylate oxygen of E133 can form hydrogen bonds with the NZ atom of K68 (2.8 Å) and with the carbonyl oxygen of I135 (3 Å). However, when the aza-ANPs bind, these bonds are broken and G



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Figure 3. (A) The active site of human HGPRT in complex with 6c (pdb code 4RAO). (B) The sequence alignment of human HGPRT, Pf HGXPRT, and PvHGPRT. Active site loops that are critical for binding are highlighted in different colors. The pyrophosphate binding site is in light brown, the 5′-phosphate binding site is in magenta, the β-sheet connections and the large mobile group are in green, the second loop that encircles the 5′-phosphate group is dark brown, the aromatic ring (F186) that covers the purine binding site is in dark blue, and the loops that interact with the metals are in light green (E133-D134) and red (191−195).

of this divalent metal ion moves up to 1.6 Å depending on the position of the second phosphonate group. This position can be dictated by the chemical nature of the linker which confers flexibility (Scheme 1 and Figure 4). In 6b (Figure 4a) (all subunits), 6c (Figure 4b) (all subunits), and 7b (Figure 4c) (three of the four subunits), magnesium is coordinated to one of the phosphonic oxygens and to the carboxylate oxygen of D193. Coordination to water molecules then contributes to the octahedral geometry. Thus, one of the functions of this divalent metal ion is to anchor the second phosphonate group in the active site. There is no magnesium ion in subunit D for the 7b (Figure 4c) complex as, in this instance, the phosphonate group is located in two positions. This also occurs in five of the eight subunits for 7c (subunits A, C, D, E, and H) (Figure 4d,e), as the second phosphonate group is also mobile and is located in two different positions. Thus, when this second phosphonate group oscillates between two sites, there is no room for a magnesium ion. This magnesium ion can also assist in anchoring the phosphonate group via coordination to a water molecule that then forms a hydrogen bond with N3 of the purine ring. This is found in 6b, subunit A. Although a water molecule is coordinated to magnesium in the other three subunits, it is too far (3.3, 4.4, and 3.7 Å) from N3 to form a hydrogen bond. These differences are due to the slightly different location of the phosphonate group and the magnesium ion. However, this constellation of bonds does exist in 6c (Figure 4b) (subunits A, B, and C) although in subunit D, the distance between the water and N3 is too long (3.5 Å). The triad of magnesium to N3 through water exists in 7b (Figure 4c) (except subunit D which does not have magnesium because of the two positions of the phosphonate group) and 7c (subunit F) (Figure 4e). The structure of each of the subunits in the complex with 7c differs from the other complexes in that magnesium is found in this second site only in two of the subunits (B and F) (Figure 4d,e). Again, this is because the movement of the phosphonate group means that this site is partially occupied. Thus, the primary role of this second magnesium ion is to anchor the aza-ANP in the active site and its presence does not influence movements in the structure of the enzyme itself.

the side chain of K68 rotates by 180°, setting up new interactions within its own subunit and with amino acid residues in the adjacent subunit (Figure 5). A magnesium ion becomes coordinated to the carboxylate atoms of E133 and D134 and, with waters, is able to form the regular octahedral geometry. This coordination brings these two side chains closer together. It is suggested that when magnesium is coordinated to these two residues, the aza-ANPs can bind optimally. Magnesium ion coordination with the side chains of E133 and D134 occurs in all subunits when 6b (Figure 4a), 7b (Figure 4c), and 11a (Figure 4f) are bound and in three of the subunits for the complex with 6c (Figure 4b). This magnesium ion is then positioned to form interactions with one of the phosphonic oxygens of the inhibitor either by direct coordination or via a water molecule. This network of interactions helps to anchor the aza-ANP in position. In subunit B of 6c (Figure 4b), three positions of the side chain of E133 can be seen as it moves to coordinate to the magnesium ion in this site. In the complex with 7c (Figure 4d,e), a diversity of binding is apparent across the eight subunits. In four of the subunits, the magnesium ion is coordinated to E133 and D134. However, in the other four subunits, the side chain of E133 does not rotate toward D134. In this arrangement, the magnesium ion coordinates only to a carboxylate oxygen atom of E133 and the carbonyl of I135. In the remaining subunit, no magnesium ion is observed in the vicinity of these two amino acids. In the complex with 11a (Figure 4f), the side chain of K68 is also rotated outside the active site. This aza-ANP does not contain a second phosphonate group and, so, the cyano group does not occupy the space vacated by the side chain of K68. The trigger that prompts the movement of K68 during catalysis or when inhibitor binds has not been determined. This result, however, suggests that it maybe the coordination of the magnesium ion to E133 and D134 that initiates the rotation of K68, making room for the phosphonate group to bind, rather than the presence of this group into the active site. Site 2 for Metal Binding. In the complexes of human HGPRT with 6b, 6c, 7b, and 7c (all of which contain a second phosphonate group), a magnesium ion can be found in a second site in most subunits (Figure 4). However, the location H



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Figure 4. Location of the six ANP inhibitors in the active site of human HGPRT and their interactions with main chain and side chain atoms of the surrounding amino acid residues. Each panel is the superimposition of the four subunits on each other, showing the different positions that the ANP can adopt to allow it to bind. The figure also shows the flexibility of the active site amino acids that move to accommodate the inhibitor in the active site. (a) Complex with 6b (pdb code 4RAQ). (b) Complex with 6c (pdb code 4RAO). (c) Complex with 7b (pdb code 4RAC). (d) One tetramer of the complex with 7c (pdb code 4RAD). (e) Second tetramer of the complex with 6b (pdb code 4RAD). (f) Complex with 11a (pdb code 4RAN). In all of the panels, the Mg2+ ions and water molecules are shown as green and red spheres, respectively. For all the complexes, the carbon atoms in chains A, B, C, and D are green, cyan, magenta, and yellow, respectively. For 7b, which has a second tetramer, the carbon atoms of the E, F, G, and H chains are colored green, cyan, magenta, and yellow, respectively.

The Interactions of the Carbonyl, Amide, and NZ Atoms of K68 with the Aza-ANPs and Active Site Amino Acid Residues. K68 is part of the flexible loop between residues 65 and 72. In the presence of all five aza-ANPs, the side chain of K68 rotates, allowing the second phosphonate group to occupy the space vacated by this side chain. Further, the backbone structures in this region are identical and all have a cis-peptide bond between L67 and K68. This is not surprising for the structures that contain a second phosphonate, as this group mimics the pyrophosphate product of the chemical reaction. However, 11a does not possess this group or any group capable of interacting with amino acid residues in this region (Figure 4f). Thus, again, the movement of the K68 side chain and its interactions with residues in the adjacent subunit and, possibly, the close approach of the Mg2+ in the first site are

sufficient to promote the trans to cis peptide conformational change. In all of the structures, there are four hydrogen bonds to K68 that are common. These are between: (i) the carbonyl oxygen of K68 and the amide nitrogen of G70, (ii) the carbonyl of K68 and the amide nitrogen of Y71, (iii) the NZ nitrogen of K68 and the side chain atom of E196, and (iv) the NZ nitrogen of K68 and the carbonyl of V96 in the adjacent subunit. The latter bond helps to stabilize each pair of the dimers within the biologically active tetramer. A second hydrogen bond can also be seen to help stabilize the dimer. This is between the NZ atom of K68 and the OD1 atom of D119 in the adjacent subunit. This is only found in 6c (three subunits: A, B, and D) (Figure 4b), 7b (one subunit: B) (Figure 4c), and 11a (two subunits: A and D) (Figure 4f). I



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Figure 5. Superimposition of unliganded human HGPRT (pdb code 17ZG) and human HGPRT in complex with aza-ANPs. For clarity, only the structures in complex with 7c (pdb code 4RAD) and 11a (the four different conformations) (pdb code 4RAN) are shown. This figure shows the movement of the side chains of E133 and D134 as a magnesium ion enters the active site. The side chain of K68 then rotates by 180°, vacating the site required for the second phosphonate. This movement then helps to stabilize each pair of dimers by forming hydrogen bonds with main chain atoms (carbonyl of V96) in the adjacent subunit. The conformational changes to the pyrophosphate binding loop of the unliganded human HGPRT occur irrespective of whether a phosphonate group (or pyrophosphate mimic) is present (6b, 6c, 7b, and 7c) or absent (11a).

For 6c, this hydrogen bond cannot be seen in subunit C as D119 is not visible in subunit A (subunit A pairs with subunit C) (Figure 4b). D119 is located at the C-terminal of the large flexible loop (residues 102−120). These particular intersubunit interactions are not seen when the transition state analogue, (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-Dribitol 5-phosphate (ImmGP), and PPi are bound in the active site of human HGPRT. A third hydrogen bond between subunits can also be observed in some structures. This is between the NZ atom of K68 and the OD2 atom of D97, 7b (subunit A) (Figure 4c), 7c (subunits D, E, and H) (Figure 4d,e), and 11a (subunits B and C) (Figure 4f). This bond is formed in three of the subunits in the complex of human HGPRT with the transition state analogue. Thus, the positioning of K68 not only stabilizes the location of the inhibitor in the active site (when a second phosphonate group is present) but also stabilizes the tetrameric structure. Positioning of the Purine Ring. The purine ring of the aza-ANPs is held in place by a network of hydrogen bonds to main chain and side chain amino acid residues in the active site. These include the carbonyl oxygen atoms of V187, D193, the amide of V187, and the NZ atom of K165 (Figure 6). A second

feature that is seen in a number of subunits is the hydrogen bond between the N3 atom of the purine ring and a ubiquitous water molecule. This water molecule can be coordinated to a magnesium ion in the second site (Figure 6). It is this magnesium ion that is then coordinated to one of the phosphonic oxygens. In some instances, this water molecule can also form a hydrogen bond to a phosphonic oxygen. Thus, although there are a number of common features when these aza-ANPs bind, there are also differences. These occur not only between the five different structures but also occur between subunits within the same structure. The N1 Atom of the Purine Ring. The hydrogen bond with the carbonyl oxygen of V187 is a common feature in all structures and all subunits (Figure 6). The Exocyclic Amino Group Attached to C2 Atom of the Purine Ring. When the purine base is hypoxanthine, there are no interactions between the proton attached the C2 atom and active site residues. However, when the base is guanine, the exocyclic amino group forms a hydrogen bond with the carbonyl of V187 (∼2.8 Å) in 7b (Figure 4c) and 11a (Figure 4f) and in subunits A, B, D, and E in 7c (Figure 4d,e; Figure 6). There is no hydrogen bond in the other four subunits of 7c as the carbonyl oxygen is too far away (∼3.4 Å). This group is also able to form a hydrogen bond with the carbonyl of D193, occurring in three of the subunits of 7b (subunits A, B, and C) (Figure 4c) and in seven of the subunits with 7c (the exception is subunit A) (Figure 4d,e; Figure 6). There is no hydrogen bond between the exocyclic amino group and the side chain of D193 in any of the subunits for 11a as this distance is between 3.5−3.6 Å (Figure 4f). The N3 of the Purine Ring. In many cases, there is a hydrogen bond between this atom and a single water molecule (∼3 Å away) (Figure 6). This water molecule then acts as a bridge between the second magnesium ion and the phosphonic oxygens. This bonding arrangement is one of the differences between the five structures and between individual subunits. In the structures with 6b, 6c, and 7b, this water is coordinated to the magnesium ion in all subunits (Figure 4a−c). However, in subunits B and D of 6b and subunit C of 7b, there is also a hydrogen bond between this water molecule and a phosphonic oxygen (Figure 4a,c). As only subunits B and H in the structure with 7c contain a magnesium ion in the second site, this network of bonds cannot occur. In subunit H,

Figure 6. Purine base binding site for four of the human HGPRTANPs. (a) 6b (pdb code 4RAQ), (b) 6c (pdb code 4RAO), (c) 7b (pdb code 4RAC), (d)7c (pdb code 4RAD). All four images are subunit A of the structures. Hydrogen bonds are shown as yellow dashed lines. D137, K165, V187, and D193 are highlighted in pink. J



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other subunits. This interaction cannot form in the parasite enzymes as arginine is replaced by leucine. Thus, the side chain of this aspartic acid residue in the Pf and Pv enzymes should be freely rotatable to form an ionic interaction with the NZ atom of K165 helping to anchor it in place and, in turn, the inhibitor. Mobility of the Amino Acid Residues around the 5′-Phosphate Binding Pocket (Residues 137−141). This loop is found at the N-terminus of a flexible α-helix (residues 142−152). Thus, the positive charge of this helix dipole contributes to the binding of the phosphonate or 5′-phosphate group. When GMP is bound, these five residues (137-DTGKT-141) surround the 5′-phosphate group of the nucleoside monophosphate. In comparing the sequences between the human and the two parasite enzymes, this region is completely conserved (Figure 3). When the aza-ANPs bind to the human enzyme, these five residues move to setup a network of hydrogen bonds to position the phosphonate group in this binding pocket. These bonds are between the three phosphonic oxygens and the main chain and side chain atoms of the amino acids in this loop. They include the amide nitrogens of D137, T138, and T141 and the OG1 atoms of T138 and T141. Water molecules can also be seen in some of these structures in this binding pocket. These contribute to defining the structure of the amino acid residues surrounding this group and to the positioning of the phosphonate group. The most common hydrogen bond between bridging water molecules and the phosphonic oxygen is with the amide of M142. This occurs in two of the subunits of the complex with 6b (B,D) (Figure 4a), all four of the subunits with 6c (Figure 4b), and in six of the subunits in 7c (A, B, D, E, F, and H) (Figure 4d,e). Although three water molecules can be seen in subunit A of the 11a complex, they do not form hydrogen bonds with any other atoms and there is no electron density for water molecules in the other three subunits. The position of this loop (137−141) is stabilized by interactions between each other and also with interactions with amino acids surrounding this loop. Thus, there are three hydrogen bonds that the amino acid residues in the 5′-phosphate binding loop form with other amino acids. These are between: the carbonyl of K140 and the main chain amide atom of T144 and the OG1 atom of T144, the carbonyl of G139 and the amide of Q143, and the carbonyl oxygen of T141 and the amide nitrogen of L145. Although other hydrogen bonds are formed, these are not universal. These are between the carbonyl of D137 and the OG1 atom of R169 (subunit B, 6c; subunit A, 7b; and subunits B and F, 7c) (Figure 4b−e). The carbonyl oxygen of T138 can also form with the amide nitrogen of V171 (subunit D, 6b; subunits A, B, and C, 6c; subunit D, 7b; subunits C and D, 7c; and subunits A, B and C, 11a) (Figure 4). The Positioning of the Large Mobile Loop (Residues 102−115) in These Five Structures. Electron density for all the amino acid residues in this mobile loop in the structure of human enzyme in complex with the aza-ANPs cannot be completely assigned (Table 4). In all five structures, the amino acid residues between 105 to 109 are not visible. In some structures, some residues between 101 and 104, 111−123, may also not be visible. When present, the secondary structure between residues 98−101 and residues 111−114 can either be that of a β-sheet or a random coil. However, in all the structures, the Cα atoms follow the same path with the main chain atoms being superimposable. This arrangement to form two β-sheets is identical

however, the same network is seen (Figure 4d,e). In subunit B, the magnesium is only coordinated to the OG1 atom of D193 and not to a phosphonic oxygen. There is a hydrogen bond between N3 and a water molecule in subunits A, B, and D, and this water molecule forms a hydrogen bond with the side chain of D193. A water molecule cannot be seen in the vicinity of N3 in the complex with 11a (Figure 4f). This is probably due to the absence of a magnesium ion in this second site. The Exocyclic Oxo Atom Attached to C6 of the Purine Ring. The 6-oxo group of the purine ring forms hydrogen bonds to the NZ atom of K165 and to the amide of V187 in all subunits of all of the structures (Figure 6). In subunits C, D, F, and G of the structure in complex with 7c, there is an additional hydrogen bond with the carbonyl of K185 (Figure 4d,e). The N7 Atom of the Purine Ring. In all the structures and all the subunits (except for subunits B and C, 6b) (Figure 4a), there is a hydrogen bond between this atom and the NZ atom of K165 (Figure 6). In most subunits, this distance varies between 2 and 9−3.1 Å, although in subunit A of 7b (Figure 4c), the distance is longer (3.3 Å). The fact that the NZ atom of K165 now forms a hydrogen bond with N7 suggests that, in the aza-ANPs, this atom is not protonated else it would repel the side chain of K165. The different number of interactions of the purine ring with amino acid residues, and with waters, is one of the critical factors that affect the affinity of these inhibitors for the enzymes. Flexibility of the Side Chain of D137 and the Mobile Loop between K165 and R171. D137, K165, and R171 are part of two different mobile loops, residues D137−T141 and residues K165−V171. The amino acid sequence between D137 and T141 is identical in all three enzymes (−DTGKT−; Figure 3). However, the amino acid sequence between residues 165−171, although the same in the Pf and Pv enzymes (−KRTPLWN−), differs in the human (−KRTPRSV−). K165 is conserved across all the amino acid sequences of the 6-oxopurine PRTases, and the NZ atom forms a hydrogen bond with the exocyclic 6-oxo group of the purine base. When the aza-ANPs bind, the NZ atom of K165 is within hydrogen bond distance of (i) the 6-oxo atom in the purine ring, (ii) the N7 atom of the purine ring, and (iii) the carbonyl of K185. This arrangement differs from that which occurs when the transition state analogue binds as the NZ atom cannot form a hydrogen bond with the protonated N7 and, instead, forms an ionic interaction with the negatively charged OD1 atom of D137. The side chain of D137 can adopt different positions (Figures 4 and 6). D137 is located at the N-terminal of the loop which surrounds the 5′-phosphate binding pocket. When the aza-ANPs bind, it points away from the purine ring (Figure 4) and not toward N7. In the complex with 6b (Figure 4a), 6c (Figure 4b), and 7b (Figure 4c), a carboxylate atom forms a hydrogen bond with either or both of the NH atoms of R169. There are three exceptions. These are subunit C in the 6b structure (Figure 4a), where the side chain of R169 is not visible, subunit B of 6c (Figure 4b) and subunit A of 7b (Figure 4c), where it is the carbonyl of D137 which forms a hydrogen bond with the NH atoms of R169, and subunit D of the 7b complex, where it is more than 4 Å away (Figure 4c). The structure with 7c, which differs in many respects from the other four structures, also differs in the positioning of the side chain of D137 (Figure 4d,e). Thus, the interaction between D137 and R199 is seen only in subunits A, C, and H (Figure 4d,e). In the structure with 11a (Figure 4f), there is a hydrogen bond between these two residues in subunit A but this is not seen in any of the K



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linker in the 7c structure (Figure 4d,e) resulting in two positions of the phosphonate group, magnesium is only found in the second site in subunits B and F, and it is only in subunit F that this ion is coordinated to the ubiquitous divalent metal ion occupying the second site that forms a hydrogen bond to the N3 atom of the purine ring. The Interactions of the PO3 Oxygen Atom. This oxygen atom forms a hydrogen bond with the amide nitrogen of K68. There are only five out of 24 instances where this bond is not formed: subunit A (6b) (Figure 4a), subunit D (6c) (Figure 4b), one of the two phosphonate groups in subunit D (7b) (Figure 4c), and subunits B, D, and G of 7c (Figure 4d,e). This data suggests that this interaction is one of the important factors for the aza-ANPs to bind efficiently. In some instances, the PO3 oxygen atom can also form a second hydrogen bond. This occurs in subunit A (6b) (Figure 4a) with the NE atom of R100. In 7b (Figure 4c), this occurs via water with the carbonyl of L101 (subunit A) via water with the side chains of R100 and E196 (subunit B) and via water with the side chains of R100, E196, and R199. There are no additional hydrogen bonds in the structure with 6c or 7c (Figure 4b,d−e). From the electron density, the cyano group in the complex with 11a also occupies different sites in each of the four subunits. However, this group does not appear to form interactions either directly or indirectly with either amino acid side chain or main chain atoms residues or indirectly via water in any of the subunits (Figure 4f). The Effect of the Bromine Atom at Position 8 in the Purine Ring. The addition of a bromine atom at position 8 in the purine ring enhances the binding of 10c compared to 7c (Tables 1 and 2). For human HGPRT, the Ki value is 2-fold lower, for Pf HGXPRT, 4-fold lower and, for PvHGPRT, 3-fold lower. Comparison of the structure of the complex of 10c with 7c and 6c (the hypoxanthine counterpart) shows that the location of the two phosphonate groups is different in these structures (Figure 7). In 10c, the phosphonate group, attached to the trisubstituted nitrogen by five atoms and containing an oxygen atom in this linker, is located in the 5′-phosphate

to that when the transition state analogue binds to human HGPRT. In that structure, the highly conserved SY residues, found at the top of one of these sheets, are clearly visible. The hydroxyl of Y104 forms a hydrogen bond with one of the 5′-phosphate oxygens, and the amide of S103 forms a hydrogen bond with one of pyrophosphate oxygens. In the aza-ANP structures in complex with human HGPRT, the side chains of the serine and tyrosine residues can only be assigned in subunit E in the complex of 7c with human HGPRT (Figure 4e). However, the side chain of Y104 points away from the active site and there are no interactions with the inhibitor, suggesting crystal contacts stabilize the loop in this subunit. Thus, in all of these structures with the aza-ANPs, this mobile loop is not closed over the active site. Flexibility of the Second Linker Attached to the Trisubstituted Nitrogen. The linker connecting the second phosphonate group to the trisubstituted nitrogen is designed to be flexible. This results in the phosphonate group being able to occupy different positions in the four structures and, in some cases, alternative positions with reduced occupancy within the same subunit. This is most evident in the structure in complex with 7c, where five of the eight subunits (A, C, D, E, and H) (Figure 4d,e) contain two orientations of this second phosphonate group. In the structure with 6b (Figure 4a), there is electron density for only one orientation in all subunits but there are two different orientations in each of the four subunits in the complex with 6c (subunit B) (Figure 4b) and with 7b (subunit D) (Figure 4c). Figure 4 shows the different positions that this group can occupy in the active site together with the variations in the positions of the divalent metal ion and water molecules. The Interactions of the PO1 Oxygen Atom. In some structures and some subunits, this phosphonic oxygen atom forms a hydrogen bond to a water molecule, which is then coordinated to the magnesium ion in the first site (6b, 6c except subunit B, where the phosphonate group has two orientations; 7b and 7c, subunits C and H) (Figure 4a−e). This network thus links the second phosphonate group to another section of amino acids in the active site (E133 and D134). It can also form a hydrogen bond with either or both of the amide of K68 and G69, although these interactions are not seen in 7c (Figure 4d,e). The Interactions of the PO2 Oxygen Atom. This oxygen atom forms a hydrogen bond to the OD1 atom of D193 and the NH2 atom of R199 in all subunits in the complex with 6b, 6c, and 7b (except subunit D, which contains two orientations of the phosphonate group and, therefore, there is a either a hydrogen bond to the side chain of D193 or R199) (Figure 4a,c). In the complex with 7c, the situation is different because there are two orientations of the phosphonate group in five of the subunits (A, C, D, E, and H) (Figure 4d,e). Thus, there are eight different positions of the phosphonate group in all these subunits in the six structures. The OP2 atom oxygen atom is also coordinated to the magnesium ion that occupies the second site (6b, 6c, and 7b, except for subunit D, which does not contain magnesium because there are two orientations of the phosphonate group; Figure 4a−c). The divalent metal ion is coordinated to a water molecule that then forms a hydrogen bond to the N3 atom of the purine ring. This constellation of bonds with magnesium also occurs when the transition state analogues bind in the active site. However, in the aza-ANPs, only a single molecule is bound in the active site and not two, i.e., ImmGP and PPi. Because of the flexibility of the

Figure 7. Superimposition of the human HGPRT−10c (pdb code 4RAB) and −7c (pdb code 4RAD) complexes. In the structure with 10c, a molecule of phosphate is observed in the location where pyrophosphate binds. In subunit C (drawn above) of the human HGPRT−7c complex, 7c adopts alternative conformations. The phosphate in the 10c complex is in a similar orientation and position as one of the conformations of the second phosphonate in 7c. L



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resistance would impact on the binding of substrates/products of the reaction. As the activity of this enzyme is essential for the survival of the parasite, such attempts are likely to result in compromising its own existence. Furthermore, the interactions between the ANPs and enzyme at the 5′-phosphate and pyrophosphate site predominantly involve main chain atoms. Thus, changes to the side chains of these residues are unlikely to have any impact on the binding of the ANPs. Interactions with the purine base of the ANPs involve the side chain atoms of K168 and F186. However, K168 is absolutely required for binding to the 6-oxo group of the base and an aromatic amino acid (F186) is required for Π stacking to the ring. Mutation of F186 to an alternative aromatic amino acid is unlikely to have any major effect on the binding of the ANPs, but mutation to a nonaromatic amino acid would have a detrimental effect on binding the purine base. The common feature in these aza-ANPs is the atomic connection between the N9 atom of the purine ring and one of the phosphonate groups. This connection is two carbons between N9 and the trisubstituted nitrogen and, then, two carbons between this nitrogen and the phosphorus atom of the phosphonate group. However, these inhibitors have been specifically designed to have flexibility due to the number of freely rotatable bonds (Scheme 1). This flexibility is elucidated in Figure 4, which demonstrates the different locations that the phosphonate group can adopt. This also results in the variability of the location of the second magnesium ion and the water molecules (Figure 4). When a second phosphonate group is attached, the data shows that both parasite enzymes have a higher affinity for those compounds containing an extra atom in linker when an oxygen atom is present (Tables 1 and 3). In comparison, the reverse is true for the human enzyme (Tables 2 and 4). Thus, the chemical nature of the linker in 6c and 7c appears to be the optimum for the parasite enzymes. ANPs which contain only carbon atoms in the linker bind more weakly to all three enzymes compared with the ANPs when an oxygen is substituted for a carbon atom (cf. 7a with 7b and 6a with 7b). One possible explanation for this data is that compounds 7a and 6a adopt a more rigid structure. Another possibility is that the presence of the oxygen atom provides polarity to the linker region. Thus, they do not occupy precisely the same position in the active site as 7b and 6b. This could result in the decreased affinity. Both parasite enzymes have a lower Km for hypoxanthine compared with guanine. Thus, the fact that aza-ANPs containing guanine as the base bind more tightly (Table 1) must mean that this increased affinity is not due to the base alone. Although the occupancy of the three binding sites affects affinity, sometimes these act in synergy while, for other inhibitors, binding of one or two of the groups has more influence than the third. In all the crystal structures of the aza-ANPs in complex with human HGPRT, a magnesium ion is unequivocally identified coordinated to the carboxylate groups of E133 and D134. In four of the structures, this magnesium ion is also coordinated to a water molecule which, in turn, forms a hydrogen bond with the second phosphonate group (Figure 4). This network is a key factor for the tight binding of these inhibitors. However, a different situation exists for Pf HGXPRT when the acyclic immucillin phosphonate binds.2a In this structure, there are no magnesium ions coordinated to the side chains of the two acidic amino acids. Instead, the hydroxyl group, which mimics

binding pocket while the phosphonate group attached by only two carbon atoms points toward the pyrophosphate binding pocket (Figure 7). This is the opposite orientation found when 7c and 6c bind. This appears to be a consequence of the attachment of the bromine atom in the 8-position. In the crystal structure of 10c, the bromine atom is located ∼3 Å away from the carboxylate of D137. The presence of this atom could be the reason why the purine ring in the complex with 10c has rotated compared to its position in the complexes with 7c and 6c (Figures 4 and 7). This movement does not have any significant effect on the interactions of the other atoms of the purine ring with active site residues, but it does influence how the two phosphonate groups bind. This shift of the purine ring causes the trisubstituted nitrogen to move by nearly 2.0 Å (cf. 10c with 7c, Figure 7). There is now extra distance to the 5′-phosphate binding pocket allowing the phosphonate group attached to the longer linker to enter this site. This allows tighter interactions between the phosphonic oxygens and the amino acid backbone atoms and side chain residues (137− 141), resulting in the decreased Ki for 10c compared to 7c. The oxygens on the phosphonate group that points into the area of the pyrophosphate binding pocket do not make any interactions with either main chain or side chain atoms in the active site in subunits E and F, and they are not coordinated to a magnesium ion. It is only in subunits C and G that one of these oxygens is found coordinated to a magnesium ion which, in turn, is then coordinated to the carboxylate of D193. The number of atoms between the phosphorus atom and the nitrogen is only two, and thus this linker is too short to be able to make a significant contribution to the binding of this aza-ANP. A phosphate group has been fitted into the electron density for the complex with 10c. Although phosphate has not been added to the enzyme and is not present in the crystallization conditions, phosphate is a product of the magnesium catalyzed hydrolysis of PRib-PP,19 which is present in the storage buffer. Thus, it is not unlikely that it could be found in the active site. However, though it is not proposed that the presence of phosphate contributes to the affinity of 10c for these enzymes, the structure does suggest chemical modifications to 10c could result in an increase in affinity for the 6-oxopurine PRTases.

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DISCUSSION Human HGPRT, Pf HGXPRT, and PvHGPRT have a high degree of amino acid sequence homology (Figure 3). The critical amino acid residues in the active site are completely conserved as would be expected for enzymes catalyzing the same reaction. However, there are differences in the kinetic properties between these three enzymes including substrate specificity and kcat values. They also exhibit varying Ki values for different the ANPs.2a One of the factors that contribute to their affinity and selectivity lies in the positioning of the amino acids in the flexible loops that surround the active site (Figure 3). Seven new aza-ANPs have been designed so that a single molecule should be able to occupy three binding sites in the active site of the human, Pf, and Pv 6-oxopurine PRTases. These three sites correspond to those occupied by substrates/ products of the catalytic reaction. They are the purine ring (present in the substrate or nucleoside monophosphate product), 5′-phosphate group (present in PRib-PP or nucleoside monophosphate product), and PPi (present in PRib-PP or the second product of the reaction, PPi itself). Such inhibitors should therefore be specific for the 6-oxopurine PRTase enzymes. Further, attempts by the parasite to mutate in order to confer M



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The exception is 10c where the base is 8-bromoguanine. This is a tight binding inhibitor for all three enzymes with a decreased Ki compared with 7c where the base is guanine (3−4 fold; Tables 1 and 3). In the crystal structure of human HGPRT in complex with this compound, there is a rotation around the nitrogen atom so the phosphonate group which normally occupies the 5′-phosphate binding site (Figure 7) points down instead of into the space where pyrophosphate binds. This is probably a result of the 8-bromo group positioning the base in a slightly different position from that of 7c. Therefore, the phosphonate group attached to the trisubstituted nitrogen by a longer linker protrudes further into the 5′-phosphate binding site forming tighter interactions (Figure 7). Thus, this phosphonate moiety reaches further into the space surrounded by the highly conserved −DTGKT− residues (Figure 3) and the interactions in this pocket may result in the decreased Ki. As the ANPs are tight binding inhibitors, prodrugs were synthesized (Schemes 1 and 2). All these compounds exhibited antimalarial activity against Pf in cell culture and have relatively low cytotoxicity (Tables 2 and 4). Currently, different chemical modifications are under investigation to improve the efficacy of these compounds in vivo.

that attached to C2 of the ribose ring, forms a hydrogen bond to the carboxylate of D145. In two of the four subunits, the carboxylate oxygens are only 2.9 Å apart while, in the other two subunits, they are not close together (4.9 Å apart). In the absence of inhibitor, but in the presence of Hx, Pi, and PPi, there is also no magnesium ion coordinated to the side chains of the ED residues though they have moved closer together (∼2.8 Å).2a These structural differences between the human and parasite enzymes in this flexible loop could be another factor contributing to selectivity. Hypoxanthine and PRib-PP are presumed to bind to Pf HGXPRT in the absence of divalent metal ions (the enzyme is inactive in their absence), suggesting that, for Pf HGXPRT, metal ions may not be required for structure but only for catalysis.13 For tight binding inhibitors, occupancy of the site surrounded by residues 137−141 (human) and 148−152 (Pf) appears to be essential. The occupancy of this site maybe even more important for Pf HGXPRT as this enzyme loses activity in the absence of phosphate.13 The backbone amide nitrogens and the side chains of T138 and T141 form hydrogen bonds to the 5′-phosphate oxygens. The backbone carbonyl oxygens are also important as they help stabilize the structure of this loop by forming hydrogen bonds with the amide nitrogens of V171 (N182 in Pf HGXPRT and PvHGPRT) and T144 (K155 in Pf HGXPRT and PvHGPRT). For Pf HGXPRT, when the acyclic immucillin phosphonate inhibitors bind, there are more hydrogen bonds between the carbonyl oxygens and the amino acids that surround this flexible loop. These are between the carbonyl of T149 and the backbone nitrogen and side chain nitrogen of N182 (V176 in human), between the carbonyl of G150 and the amide nitrogen of L153 (M142 in human) and side chain nitrogen of N182, between the carbonyl of K151 and the amide nitrogens of L153 and K155 (T144 in human), and between the amide nitrogen of T152 and the backbone nitrogen of F156 (L145 in human). Thus, there are seven hydrogen bonds between the 5′-phosphate loop (148−152 in Pf HGXPRT) and other amino acids located nearby, 153−182 in Pf and 142−171 in human. These regions are conserved in the parasite enzymes but, apart from residues 165−169, are different in the human and parasite enzymes (Figure 3). Thus, this second network of hydrogen bonds in the parasite enzymes may help to stabilize the 5′-phosphate binding loop and may contribute to the high affinity of some of these inhibitors for the parasite enzymes compared to the human HGPRT. D137 is within hydrogen bonding distance of the protonated N7 when the transition state analogues bind but, in the presence of the ANPs, it is rotated away from N7 and instead forms a hydrogen bond with the side chain of R169. R169 is part of a flexible loop (residues 165−171; Figure 3). In Pf HGXPRT, this residue is replaced by leucine so this hydrogen bond cannot form. When the second attachment is a cyano group, there is little difference in the Ki values irrespective of whether the base is guanine or hypoxanthine for the parasite enzymes. On the other hand, the human enzyme has a marked preference for the aza-ANPs containing hypoxanthine (34-fold). This cyano group occupies a number of positions in the active site. This is because there are no direct contacts between this group and active site residues. Therefore, the differences in Ki values can only be attributed to the positioning of the purine ring and the first phosphonate group. In five of the six aza-ANPs, the purine base is either guanine or hypoxanthine, which are the naturally occurring substrates.

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CONCLUSIONS A number of new aza-ANPs have been designed and synthesized as inhibitors of the 6-oxopurine PRTases. The most potent of these, 10c, has a Ki value of 10 nM for the Pv enzyme and 80 nM for the Pf enzyme. Human HGPRT has been cocrystallized with six aza-ANP inhibitors. The amino acids that form direct interactions with these compounds are all conserved across the human, Pf, and Pv enzymes, suggesting that the structures of the complexes with the human enzyme are a good representation for how these compounds bind to the Pf and Pv enzymes. The highly polar aza-ANPs themselves do not possess strong antimalarial activity. However, when converted to their prodrugs, these compounds exhibit in vitro antimalarial activity against Pf lines with IC50 values as low as 0.8 μM. Thus, the attachment to the parent compound of two hydrophobic groups by a phosphoramidate bond results in a significant improvement to inhibit parasite cell growth. The azaANPs in this study have low cytotoxicity in a human cell line. Coupled with their antimalarial activity, the data demonstrates that there is a therapeutic window for this class of compound.

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EXPERIMENTAL SECTION

Synthesis and Analytical Chemistry. Unless otherwise stated, solvents were evaporated at 40 °C/2 kPa, and the compounds were dried over P2O5 at 2 kPa. NMR spectra were recorded on Bruker Avance 500 (1H at 500 MHz, 13C at 125.8 MHz) and Bruker Avance 400 (1H at 400 MHz, 13C at 100.6 MHz) spectrometers with TMS as internal standard or referenced to the residual solvent signal. Mass spectra were measured on UPLC-MS (Waters SQD-2). The purity of the tested compounds was determined by HPLC (H2O−CH3CN, linear gradient) combustion analysis (C, H, N) and was higher than 95%. The basic chemicals were obtained from commercial sources or prepared according to the published procedures. Preparative HPLC purifications were performed on columns packed with 7 μm C18 reversed phase resin (Waters Delta 600 chromatograph column), 17 nn × 250 mm, in ca. 200 mg batches of mixtures using gradient MeOH/H2O as eluent. Synthesis of Hydroxyderivatives 1a−1c: General Procedure. To the mixture of diethyl 2-(2-hydroxyethylamino)ethylphosphonate9 (6 g, 26.7 mmol, prepared from diethylvinylphosphonate and 2-aminoethanol), K2CO3 (3.7 g, 26.7 mmol), and KI (0.2 g) in dry N



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8.73 s, 1 H (H-2 and H-8); 4.56 m, 2 H (iPr); 4.32 t, 2 H, J(1′,2′) = 5.8 (H-1′); 3.92 m, 4 H (Et); 3.62 d, 2 H, J = 8.1 (H-7′); 3.42 t, 2 H, J(6′,5′) = 5.4 (H-6′); 2.90 t, 2 H, J(2′,1′) = 5.8 (H-2′); 2.64 m, 4 H (H-3′ and H-5′); 1.70 m, 2 H (H-4′); 1.20 m, 18 H (iPr and Et). 13C NMR (DMSO-d6): δ 151.87 (C-4); 151.18 (C-2); 148.71 (C-6); 147.93 (C-8); 130.59 (C-5); 70.83d, J(P,C) = 11.3 (C-6′); 69.95 and 69.89 (iPr); 64.74 d, J(P,C) = 164.5 (C-7′); 60.74 and 60.68 (Et); 52.17 (C-2′); 51.84 and 47.27 (C-3′ and C-5′); 42.13 (C-1′); 23.60 m, 4 C (iPr); 22.35 d, J(P,C) = 134.6 (C-4′); 16.12 and 16.07 (Et). MS (ESI): m/z = 584 [M + H]+. 9-[(N-(Diethyl)phosphonoethyl-N-(diisopropyl)phosphonoethoxyethyl)-2-aminoethyl]-6-chloropurine (2c). Starting from 1c, yield 60%. 1H NMR (DMSO-d6): δ 8.77, 1, and 8.74 s, 1 H (H-2 and H-8); 4.53 m, 2 H (iPr); 4.32 t, 2 H, J(1′,2′) = 5.8 (H-1′); 3.93 m, 4 H (Et); 3.38 m (H-7′); 3.24 t, 2 H, J(6′,5′) = 5.6 (H-6′); 2.89 t, 2 H, J(2′,1′) = 5.8 (H-2′); 2.68 dd, 2 H, J = 15.9 and 8.6 (H-3′); 2.59 t, 2 H, J(5′,6′) = 5.6 (H-5′); 1.90 m, 2 H (H-8′); 1.73 m, 2 H (H-4′); 1.21 m, 18 H (iPr and Et). 13C NMR (DMSO-d6): δ 151.89 (C-4); 151.12 (C-2); 148.66 (C-6); 148.03 (C-8); 130.58 (C-5); 69.20 and 69.14 (iPr); 68.43 (C-6′); 64.31 (C-7′); 60.73 and 60.67 (Et); 52.10 (C-2′); 51.89 (C-3′); 47.25 (C-5′); 42.06 (C-1′); 27.05 d, J(P,C) = 138.3 (C-8′); 23.61 m, 4 C (iPr); 22.17 d, J(P,C) = 134.2 (C-4′); 16.13 and 16.08 (Et). MS (ESI): m/z = 598 [M + H]+. Synthesis of N9-Substituted 2-Amino-6-chloropurines 3a−3c via Mitsunobu Reaction: General Procedure. Starting from 2-amino-6chloropurine and hydroxyderivatives 1a−1c, the procedure was identical as described above for 6-chloropurine. Then after the stirring of reaction mixture for 4 days, water (10 mL) was added and the mixture was heated at 80 °C for 30 h. Solvent was evaporated, the residue was codistilled with toluene or ethanol, and the crude mixture was purified by chromatography on silica gel (MeOH−CHCl3). The pure product was isolated as yellow foam. 9-[(N-(Diethyl)phosphonoethyl-N-(diethyl)phosphonobutyl)-2aminoethyl]-2-amino-6-chloropurine (3a). Starting from 1a, yield 78%. 1H NMR (DMSO-d6): δ 8.11 s, 1 H (H-8); 6.87 s, 2 H (NH2); 4.07 t, 2 H, J(1′,2′) = 5.7 (H-1′); 3.94 m, 8 H (Et); 2.75 t, 2 H, J(2′,1′) = 5.7 (H-2′); 2.62 dd, 2 H, J = 15.6 and 8.1 (H-3′); 2.38 t, 2 H, J(5′,6′) = 6.4 (H-5′); 1.73 m, 2 H (H-4′); 1.58 m, 2 H (H-8′); 1.29 m, 4 H (H-6′ and H-7′); 1.20 m, 12 H (Et). 13C NMR (DMSO-d6): δ 159.54 (C-2); 153.98 (C-4); 149.06 (C-6); 143.62 (C-8); 123.18 (C-5); 60.69 m, 4 C (Et); 51.93 (C-2′); 51.51 (C-3′); 46.53 (C-5′); 41.43 (C-1′); 27.19, J(P,C) = 14.9 (C-7′); 24.21 d, J(P,C) = 138.1 (C-8′); 22.01 d, J(P,C) = 134.3 (C-4′); 19.57 d, J(P,C) = 5.1 (C-6′); 16.14 m, 4 C (Et). MS (ESI): m/z = 569 [M + H]+. 9-[(N-(Diethyl)phosphonoethyl-N-(diisopropyl)phosphonomethoxyethyl)-2-aminoethyl]-2-amino-6-chloropurine (3b). Starting from 1b, yield 65%. 1H NMR (DMSO-d6): δ 8.14 s, 1 H (H-8); 6.89 s, 2 H (NH2); 4.57 m, 2 H (iPr); 4.06 t, 2 H, J(1′,2′) = 5.8 (H-1′); 3.92 m, 4 H (Et); 3.66 d, 2 H, J = 8.1 (H-7′); 3.46 t, 2 H, J(6′,5′) = 5.7 (H-6′); 2.81 t, 2 H, J(2′,1′) = 5.8 (H-2′); 2.64 m, 4 H (H-3′ and H-5′); 1.71 m, 2 H (H-4′); 1.20 m, 18 H (iPr and Et). 13C NMR (DMSO-d6): δ 159.56 (C-2); 153.96 (C-4); 149.05 (C-6); 143.74 (C-8); 123.15 (C-5); 70.88 d, J(P,C) = 11.1 (C-6′); 69.99 and 69.92 (iPr); 64.80 d, J(P,C) = 164.4 (C-7′); 60.78 and 60.72 (Et); 52.13, 52.01, and 47.41 (C-2′, C-3′ and C-5′); 41.51 (C-1′); 23.65 m, 4 C (iPr); 22.49 d, J(P,C) = 134.24 (C-4′); 16.14 and 16.08 (Et). MS (ESI): m/z = 599 [M + H]+. 9-[(N-(Diethyl)phosphonoethyl-N-(diisopropyl)phosphonoethoxyethyl9)-2-aminoethyl]-2-amino-6-chloropurine (3c). Starting from 1c, yield 65%. 1H NMR (DMSO-d6): δ 8.14 s, 1 H (H-8); 6.87 s, 2 H (NH2); 4.54 m, 2 H (iPr); 4.06 t, 2 H, J(1′,2′) = 5.7 (H-1′); 3.92 m, 4 H (Et); 3.46 m, 2 H (H-7′); 3.30 t, 2 H, J(6′,5′) = 5.8 (H-6′); 2.81 t, 2 H, J(2′,1′) = 5.8 (H-2′); 2.68 dd, 2 H, J = 15.9 and 8.4 (H-3′); 2.61 t, 2 H, J(5′,6′) = 5.8 (H-5′); 1.94 m, 2 H (H-8′); 1.74 m, 2 H (H-4′); 1.22 m, 18 H (iPr and Et). 13C NMR (DMSO-d6): δ 159.53(C-2); 153.96 (C-4); 149.01 (C-6); 143.78 (C-8); 123.13 (C-5); 69.23 and 69.16 (iPr); 68.42 (C-6′); 64.37 (C-7′); 60.80 and 60.75 (Et); 52.12 (C-2′); 52.01 (C-3′); 47.34 (C-5′); 41.38 (C-1′); 27.09 d, J(P,C) = 138.1 (C-8′); 23.61 m, 4 C

acetonitrile (80 mL), a corresponding halogenoderivative (30 mmol) was added. The reaction mixture was stirred at 80 °C for 4 days. The solvent was then removed by evaporation. Water and CHCl3 were added, the organic layer separated, washed with brine, and dried over anhydrous MgSO4. After filtration, solvent was evaporated and the residue was purified by column chromatography on silica gel (CHCl3− MeOH); the product was obtained as yellow oil. [N-(Diethyl)phosphonoethyl-N-(diethyl)phosphonobutyl]-2-aminoethanol (1a). Starting from diethyl 4-chlorobutylphosphonate, heated for 10 days, yield 88%. 1H NMR (DMSO-d6): δ 4.36 t, 2 H, J = 5.4 (OH); 3.96 m, 8 H (Et); 3.41 q, 2 H, J = 6.1 (H-1′); 2.65 m, 2 H (H-3′); 2.44 t, 2 H, J(2′,1′) = 6.3 (H-2′); 2.38 t, 2 H, J(2′,1′) = 6.4 (H-5′); 1.70 m, 2 H (H-4′); 1.86 m, 2 H (H-8′); 1.43, 4 H (H-6′ and H-7′);1.22 m, 12 H (Et). 13C NMR (DMSO-d6): δ 60.66 m, 4 C (Et); 59.11 (C-1′); 55.13 (C-2′); 52.53 (C-3′); 46.88 (C-5′); 27.24 d, J(P,C) = 15.4 (C-7′); 24.27 d, J(P,C) = 138.2 (C-8′); 22.05 d, J(P,C) = 134.7 (C-4′); 19.78 d, J(P,C) = 5.0 (C-6′); 16.4 m, 4 C (Et). MS (ESI): m/z = 418 [M + H]+. [N-(Diethyl)phosphonoethyl-N-(diisopropyl)phosphonomethoxyethyl]-2-aminoethanol (1b). Starting from diisopropyl (2-chloroethoxy)methylphosphonate, yield 41%. 1H NMR (DMSO-d6): δ 4.59 m, 2 H (iPr); 4.36 t, 2 H, J = 5.4 (OH); 3.97 m, 4 H (Et); 3.72 d, 2 H, J = 8.1 (H-7′); 3.55 t, 2 H, J(6′,5′) = 5.9 (H-6′); 3.41 dd, 2 H, J = 11.6 and 5.9 (H-1′); 2.72 dd, 2 H, J = 15.6 and 9.5 (H-3′); 2.62 t, 2 H, J(5′,6′) = 5.9 (H-5′); 2.52 t, 2 H, J(2′,1′) = 6.5 (H-2′); 1.88 m, 2 H (H-4′); 1.23 m, 18 H (iPr and Et). 13 C NMR (DMSO-d6): δ 70.83 d, J(P,C) = 11.2 (C-6′); 69.95 and 69.89 (iPr); 64.82 d, J(P,C) = 164.2 (C-7′); 60.73 and 60.67 (Et); 59.11 (C-1′); 55.59 (C-2′); 52.50 and 47.53 (C-3′ and C-5′); 23.65 m, 4 C (iPr); 22.46 d, J(P,C) = 134.7 (C-4′); 16.18 and 16.12 (Et). MS (ESI): m/z = 448 [M + H]+. [N-(Diethyl)phosphonoethyl-N-(diisopropyl)phosphonoethoxyethyl]-2-aminoethanol (1c). Starting from diisopropyl (2-chloroethoxy)ethylphosphonate, yield 31%. 1H NMR (DMSO-d6): δ 4.56 m, 2 H (iPr); 4.35 t, 2 H, J = 5.5 (OH); 3.97 m, 4 H (Et); 3.53 dt, 2 H, J = 7.6 and 11.0 (H-6′); 3.43 m, 4 H (H-1′ and H-7′); 2.72 dd, 2 H, J = 15.6 and 9.7 (H-3′); 2.60 t, 2 H, J(2′,1′) = 6.0 (H-2′); 2.47 m, 2 H (H-5′); 1.96 m, 2 H (H-8′); 1.88 m, 2 H (H-4′); 1.22 m, 18 H (iPr and Et). 13C NMR (DMSO-d6): δ 69.21 and 69.14 (iPr); 68.48 (C-6′); 64.39 (C-7′); 60.70 and 60.67 (Et); 59.10 (C-1′); 55.69 (C-2′); 52.57 (C-3′); 47.52 (C-5′); 27.20 d, J(P,C) = 138.2 (C-8′); 23.62 m, 4 C (iPr); 22.31 d, J(P,C) = 134.6 (C-4′); 16.17 and 16.11 (Et). MS (ESI): m/z = 462[M + H]+. Synthesis of N9-Substituted 6-Chloropurines 2a−2c via Mitsunobu Reaction: General Procedure. To a solution of triphenylphosphine (1.8 g, 6.87 mmol) in dry THF (50 mL) cooled to −30 °C under argon atmosphere diisopropylazadicarboxylate (DIAD, 1.2 mL, 6.61 mmol) was added slowly. The mixture was stirred for 30 min, and this preformed complex was added to chloropurine (0.75 g, 4.84 mmol), dry THF (50 mL), and the corresponding hydroxyderivative 1a−1c (3.8 mmol) at −30 °C under argon. The resulting mixture was slowly warmed to room temperature and stirred for 3 days. Solvent was evaporated and the crude mixture was purified by chromatography on silica gel (MeOH−CHCl3). The pure product was obtained as yellowish foam. 9-[(N-(Diethyl)phosphonoethyl-N-(diethyl)phosphonobutyl)-2aminoethyl]-6-chloropurine (2a). Starting from 1a, yield 73%. 1H NMR (DMSO-d6): δ 8.77, 1, and 8.71 s, 1 H (H-2 and H-8); 4.33 t, 2 H, J(1′,2′) = 5.9 (H-1′); 3.94 m, 8 H (Et); 2.84 t, 2 H, J(2′,1′) = 5.9 (H-2′); 2.63 dd, 2 H, J = 15.9 and 8.3 (H-3′); 2.37 t, 2 H, J(5′,6′) = 6.5 (H-5′); 1.73, 2, and 1.53 m, 2 H (H-8′ and H-4′); 1.20 m, 16 H (H-6′, H-7′ and Et). 13C NMR (DMSO-d6): δ 151.90 (C-4); 151.17 (C-2); 148.73 (C-6); 147.80 (C-8); 130.59 (C-5); 60.65 m, 4 C (Et); 52.75 (C-2′); 51.57 (C-3′); 46.35 (C-5′); 42.00 (C-1′); 27.11 d, J(P,C) = 15.4 (C-7′); 24.18 d, J(P,C) = 138.4 (C-8′); 21.85 d, J(P,C) = 134.3 (C-4′); 19.47 d, J(P,C) = 5.0 (C-6′); 16.12 m, 4 C (Et). MS (ESI): m/z = 554 [M + H]+. 9-[(N-(Diethyl)phosphonoethyl-N-(diisopropyl)phosphonomethoxyethyl)-2-aminoethyl]- 6-chloropurine (2b). Starting from 1b, yield 73%. 1H NMR (DMSO-d6): δ 8.78, 1, and O



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(iPr); 22.30 d, J(P,C) = 134.4 (C-4′); 16.13 and 16.07 (Et). MS (ESI): m/z = 613 [M + H]+. Synthesis of Hypoxanthine Derivatives 4a−4c and Guanine Derivatives 5a−5c: General Procedure. The corresponding 6-chloropurine derivative 2a−2c (2 mmol) or the corresponding 2-amino-6-chloropurine derivative 3a−3c was dissolved in trifluoroacetic acid (aqueous, 75%, 20 mL) and stirred overnight. The solvent was evaporated and the residue codistilled with water (3×) and ethanol. After chromatography on silica gel (MeOH−CHCl3) the pure products were colorless foams. 9-[{N-(Diethyl)phosphonoethyl-N-(diethyl)phosphonobutyl}-2aminoethyl]hypoxanthine (4a). Starting from 2a, yield 98%. 1H NMR (DMSO-d6): δ 12.25 s, 1 H (NH); 8.07, 1, and 8.02 s, 1 H (H-2 and H-8); 4.16 t, 2 H, J(1′,2′) = 6.0 (H-1′); 3.94 m, 8 H (Et); 2.77 t, 2 H, J(2′,1′) = 6.0 (H-2′); 2.63 dd, 2 H, J = 15.8 and 8.1 (H-3′); 2.39 t, 2 H, J(5′,6′) = 6.4 (H-5′); 1.75 m, 2 H (H-4′); 1.60 m, 2 H (H-8′); 1.31 m, 4 H (H-6′ and H-7′); 1.21 m, 12 H (Et). 13C NMR (DMSO-d6): δ 156.52 (C-6); 148.26 (C-4); 145.15 (C-2); 140.56 (C-8); 123.69 (C-5); 60.69 m, 4 C (Et); 52.13 (C-2′); 51.90 (C-3′); 46.51 (C-5′); 41.61 (C-1′); 27.19, J(P,C) = 15.2 (C-7′); 24.21 d, J(P,C) = 138.2 (C-8′); 21.96 d, J(P,C) = 134.1 (C-4′); 19.58 d, J(P,C) = 4.9 (C-6′); 16.15 m, 4 C (Et). MS (ESI): m/z = 536 [M + H]+. 9-[(N-(Diethyl)phosphonoethyl-N-(diisopropyl)phosphonomethoxyethyl)-2-aminoethyl]hypoxanthine (4b). Starting from 2b, yield 89%. 1H NMR (DMSO-d6): δ 12.27 s, 1 H (NH); 8.10, 1, and 8.03 s, 1 H (H-2 and H-8); 4.57 m, 2 H (iPr); 4.15 t, 2 H, J(1′,2′) = 6.0 (H-1′); 3.93 m, 4 H (Et); 3.67 d, 2 H, J = 8.2 (H-7′); 3.46 t, 2 H, J(6′,5′) = 5.5 (H-6′); 2.84 t, 2 H, J(2′,1′) = 6.0 (H-2′); 2.65 m, 4 H (H-3′, H-5′); 1.72 m, 2 H (H-4′); 1.21 m, 18 H (iPr and Et). 13C NMR (DMSO-d6): δ 156.53 (C-6); 148.21 (C-4); 145.16 (C2); 140.69 (C-8); 123.66 (C-5); 70.90 d, J(P,C) = 11.4 (C-6′); 69.98 and 69.91 (iPr); 64.79 d, J(P,C) = 164.5 (C-7′); 60.77 and 60.71 (Et); 52.68, 51.98, and 47.41 (C-2′, C-3′ and C-5′); 41.72 (C-1′); 23.63 m, 4 C (iPr); 22.48 d, J(P,C) = 134.1 (C-4′); 16.15 and 16.10 (Et). HRMS calcd for C22H42N5O8P2: 566.25031; found: 566.25035. MS (ESI): m/z = 566 [M + H]+. 9-[(N-(Diethyl)phosphonoethyl-N-(diisopropyl)phosphonoethoxyethyl)-2-aminoethyl]hypoxanthine (4c). Starting from 2c, yield 98%. 1H NMR (DMSO-d6): δ 12.25 s, 1 H (NH); 8.10, 1, and 8.03 s, 1 H (H-2 and H-8); 4.54 m, 2 H (iPr); 4.16 t, 2 H, J(1′,2′) = 6.0 (H-1′); 3.94 m, 4 H (Et); 3.48, 2, and 3.34 m, 2 H (H-6′ and H-7′); 2.84 m, 2 H, 2.68, 2, and 2.63 m, 2 H (H-2′, H-3′ and H-5′); 1.96 m, 2 H (H-8′); 1.76 m, 2 H (H-4′); 1.22 m, 18 H (iPr and Et). 13C NMR (DMSO-d6): δ 156.52 (C-6); 148.24 (C-4); 145.18 (C-2); 140.70 (C-8); 123.67 (C-5); 69.28 and 69.22 (iPr); 69.01 (C-6′); 64.39 (C-7′); 60.82 and 60.76 (Et); 52.61, 52.00, and 47.41 (C-2′, C-3′ and C-5′); 27.11 d, J(P,C) = 138.6 (C-8′); 23.64 m, 4 C (iPr); 22.07 d, J(P,C) = 134.0 (C-4′); 16.16 and 16.10 (Et). MS (ESI): m/z = 580 [M + H]+. 9-[(N-(Diethyl)phosphonoethyl-N-(diethyl)phosphonobutyl)-2aminoethyl]guanine (5a). Starting from 3a, yield 98%. 1H NMR (DMSO-d6): δ 10.74 s, 1 H (NH); 7.69 s, 1 H (H-8); 6.59 s, 2 H (NH2); 3.96 m, 10 H (H-1′ and Et); 2.73m, 2 H (H-2′); 2.44 m, 5 H (H-3′ and H-5′); 1.83 m, 2 H (H-4′); 1.69 m, 2 H (H-8′); 1.40 m, 4 H (H-6′ and H-7′); 1.21 m, 12 H (Et). 13C NMR (DMSO-d6): δ 156.70 (C-6); 153.52(C-2); 150.99 (C-4); 137.64 (C-8); 116.30 (C-5); 60.72 m, 4 C (Et); 51.90 (C-2′); 51.88 (C-3′); 46.74 (C-5′); 41.59 (C-1′); 27.56, J(P,C) = 15.2 (C-7′); 24.13 d, J(P,C) = 138.0(C-8′); 22.38 d, J(P,C) = 118.2 (C-4′); 19.55 d, J(P,C) = 4.8 (C-6′); 16.16 m, 4 C (Et). MS (ESI): m/z = 551 [M + H]+. 9-[(N-(Diethyl)phosphonoethyl-N-(diisopropyl)phosphonomethoxyethyl)-2-aminoethyl]guanine (5b). Starting from 3b, yield 97%. 1H NMR (DMSO-d6): δ 10.62 s, 1 H (NH); 7.68 s, 1 H (H-8); 6.51 s, 2 H (NH2); 4.57 m, 2 H (iPr); 3.93 m, 6 H (H-1′ and Et); 3.69 d, 2 H, J = 8.1 (H-7′); 3.49 t, 2 H, J(6′,5′) = 5.5 (H-6′); 2.77 t, 2 H, J(2′,1′) = 5.1 (H-2′); 2.65 m, 4 H (H-3′, H-5′); 1.74 m, 2 H (H-4′); 1.22 m, 18 H (iPr and Et). 13C NMR (DMSOd6): δ 156.72 (C-6); 153.42 (C-2); 150.99 (C-4); 137.90 (C-8); 116.31 (C-5); 70.93d, J(P,C) = 11.4 (C-6′); 70.04 and 69.99 (iPr); 64.83 d, J(P,C) = 164.4 (C-7′); 60.83 and 60.78 (Et); 52.57 (C-2′);

52.10 and 47.51 (C-3′ and C-5′); 41.23 (C-1′); 23.73, 23.70, 23.64, and 23.60 (iPr); 22.49 d, J(P,C) = 134.0 (C-4′); 16.18 and 16.14 (Et). MS (ESI): m/z = 581 [M + H]+. 9-[(N-(Diethyl)phosphonoethyl-N-(diisopropyl)phosphonoethoxyethyl)-2-aminoethyl]guanine (5c). Starting from 3c, yield 98%. 1H NMR (DMSO-d6): δ 10.66 s, 1 H (NH); 7.68 s, 1 H (H-8); 6.55 s, 2 H (NH2); 4.54 m, 2 H (iPr); 3.94 m, 6 H (H-1′ and Et); 3.49, 2, and 3.35 m, 2H (H-6′ and H-7′); 2.76 m, 2 H, 2.67, 2, and 2.61 m, 2 H (H-2′, H-3′ and H-5′); 1.97 m, 2 H (H-8′); 1.76 m, 2 H (H-4′); 1.22 m, 18 H (iPr and Et). 13C NMR (DMSO-d6): δ 156.67 (C-6); 153.43 (C-2); 150.98 (C-4); 137.86 (C-8); 116.27 (C-5); 69.30 and 69.23 (iPr); 68,48 (C-6′); 64.40 (C-7′); 60.81 and 60.75 (Et); 52.56 (C-2′); 52.09 and 47.50 (C-3′ and C-5′); 41.10 (C-1′); 27.14 d, J(P,C) = 138.1 (C-8′); 23.60 m, 4 C (iPr); 22.33 d, J(P,C) = 134.6 (C-4′); 16.16 and 16.13 (Et). MS (ESI): m/z = 595 [M + H]+. Synthesis of the Free Bisphosphonic Acids 6a−6c and 7a−7c: General Procedure. A mixture of the corresponding tetraester 4a−4c or 5a−5c (1 mmol), acetonitrile (20 mL), and BrSiMe3 (1 mL) was stirred for 2 days at room temperature. After evaporation and codistillation with acetonitrile, the residue was treated with aqueous methanol (2:1, 30 mL) for 1 h and evaporated. The residue was purified by preparative HPLC (water−methanol). 9-[(N-Phosphonoethyl-N-phosphonobutyl)-2-aminoethyl]hypoxanthine (6a). Starting from 4a, yield 69%. 1H NMR (D2O): δ 8.30, 1, and 8.24 s, 1 H (H-2 and H-8); 4.75 t, 2 H, J(1′,2′) = 6.4 (H-1′); 3.81 t, 2 H, J(2′,1′) = 6.4 (H-2′); 3.40 m, 2 H (H-2′); 3.50 dd, 2 H, J = 16.6 and 8.4 (H-3′); 3.35 t, 2 H, J(5′,6′) = 7.8 (H-5′); 2.06 m, 2 H (H-4′); 1.82 m, 2 H, 1.74, 2, and 1.61 m, 2 H (H-6′, H-7′ and H-8′). 13C NMR (D2O): δ 156.99 (C-6); 147.90 (C-4); 145.65(C-2); 140.73 (C-8); 121.74 (C-5); 51.67 (C-2′); 50.25 (C-3′); 48.52 (C-5′); 38.29 (C-1′); 25.11 d, J(P,C) = 134.94 (C-4′); 22.96 d, J(P,C) = 16.4 (C-7′); 21.43 d, J(P,C) = 129.8 (C-8′); 18.71 d, J(P,C) = 4.3 (C-6′). HRMS calcd for C13H22N5O7P2, 422.09999; found, 422.10015. MS (ESI): m/z = 422 [M − H]−. 9-[(N-Phosphonoethyl-N-ph osphonomethoxyethyl)-2aminoethyl]hypoxanthine (6b). Starting from 4b, yield 71%. 1H NMR (DMSO-d6): δ 12.40 s, 1 H (NH); 8.15, 1, and 8.08 s, 1 H (H-2 and H-8); 4.52 t, 2 H, J(1′,2′) = 5.3 (H-1′); 3.82 m, 2 H (H-7′); 3.55 m, 4 H (H-2′ and H-6′); 3.16 m, 4 H (H-3′, H-5′); 1.90 m, 2 H (H-4′). 13C NMR (DMSO-d6): δ 156.48 (C-6); 148.28 (C-4); 145.71(C-2); 140.30 (C-8); 123.86 (C-5); 66.84 d, J(P,C) = 8.9 (C-6′); 66.82 d, J(P,C) = 157.5 (C-7′); 52.66, 51.55, and 49.44 (C-2′, C-3′ and C-5′); 38.67 (C-1′); 22.64 d, J(P,C) = 131.0 (C-4′). HRMS calcd for C12H20N5O8P2, 424.07926; found, 424.07932. MS (ESI): m/z = 424 [M − H]−. 9-[(N-Phosphonoethyl-N-phosphonoethoxyethyl)-2-aminoethyl]hypoxanthine (6c). Starting from 4c, yield 38%. 1H NMR (DMSOd6): δ 12.40 s, 1 H (NH); 8.16, 1, and 8.07 s, 1 H (H-2 and H-8); 4.48 m, 2 H (H-1′); 3.64 m, 4 H (H-6′ and H-7′); 3.40 m, 2 H (H-2′); 3.20 m, 4 H (H-3′, H-5′); 1.84 m, 2 H (H-4′ and H-8′). 13C NMR (DMSO-d6): δ 156.47 (C-6); 148.23 (C-4); 145.60 (C-2); 140.38 (C-8); 123.81 (C-5); 65.35 (C-6′); 65.05 (C-7′); 52.19 (C-2′); 51.11 (C-3′); 49.23 (C-5′); 28.36 d, J(P,C) = 136.0 (C-8′); 23.58 d, J(P,C) = 129.9 (C-4′). HRMS calcd for C13H22N5O8P2, 438.09491; found, 438.09486. MS (ESI): m/z = 438 [M − H]−. 9-[(N-Phosphonoethyl-N-phosphonobutyl)-2-aminoethyl]guanine (7a). Starting from 5a, yield 35%. 1H NMR (D2O): δ 8.11 s, 1 H (H-8); 4.58 t, 2 H, J(1′,2′) = 6.1 (H-1′); 3.74 t, 2 H, J(1′,2′) = 6.1 (H-2′); 3.54, 2, and 3.32 m, 2 H (H-3′ and H-5′); 2.08 m, 2 H (H-4′); 1.82 m, 2 H (H-8′); 1.62 m, 4 H (H-6′ and H-7′). 13C NMR (D2O): δ 159.25 (C-6); 155.84 (C-2); 152.88 (C-4); 140.45 (C-8); 115.25 (C-5); 54.49 (C-2′); 52.73 (C-3′); 51.01 (C-5′); 40.54 (C-1′); 28.39 d, J(P,C) = 134.1 (C-4′); 25.60 d, J(P,C) = 16.3 (C-7′); 24.21 d, J(P,C) = 129.4 (C-8′); 21.72 d, J(P,C) = 4.3 (C-6′). HRMS calcd for C13H23N6O7P2, 437.11089; found, 437.11120. MS (ESI): m/z = 437 [M − H]−. 9-[(N-Phosphonoethyl-N-ph osphonomethoxyethyl)-2aminoethyl]guanine (7b). Starting from 5b, yield 48%. 1H NMR (D2O): δ 8.66 s, 1 H (H-8); 0.4.68 t, 2 H, J(1′,2′) = 6.4 (H-1′); 3.92 t, 2 H, J(6′,5′) = 5.0 (H-6′); 3.83 t, 2 H, J(2′,1′) = 6.4 (H-2′); 3.67 d, P



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2 H, J = 8.8 (H-7′); 3.57 m, 4 H (H-3′, H-5′); 2.08 m, 2 H (H-4′). 13C NMR (D2O): δ 155.07 (C-6); 154.06 (C-2); 149.60 (C-4); 137.24 (C-8); 117.04 (C-5); 65.92 d, J(P,C) = 164.3 (C-7′); 64.80 (C-6′); 52.35 (C-2′); 49.80 and 49.14 (C-3′ and C-5′); 38.73 (C-1′); 21.28 d, J(P,C) = 123.0 (C-4′). HRMS calcd for C12H21N6O8P2, 439.09016; found, 439.09026. MS (ESI): m/z = 439 [M − H]−. 9-[(N-Phosphonoethyl-N-phosphonoethoxyethyl)-2-aminoethyl]guanine (7c). Starting from 5c, yield 25%. 1H NMR (D2O): δ 8.73 s, 1 H (H-8); 4.72 t, 2 H, J(1′,2′) = 6.1 (H-1′); 3.85 m, 4 H (H-6′ and H-7′); 3.72 m, 2 H (H-2′); 3.60 m, 4 H (H-3′, H-5′); 2.11 m, 2 H (H-8′); 1.98 m, 2 H (H-4′). 13C NMR (D2O): δ 155.06 (C-6); 154.19 (C-2); 149.69 (C-4); 137.29 (C-8); 116.36 (C-5); 64.97 d, J(P,C) = 3.5 (C-7′); 62.55 (C-6′); 52.23 (C-2′); 50.16 (C-3′); 49.05 (C-5′); 39.01 (C-1′); 26.76 d, J(P,C) = 133.9 (C-8′). 21.32 d, J(P,C) = 130.1 (C-4′). HRMS calcd for C13H23N6O8P2, 453.10581; found, 453.10567. MS (ESI): m/z = 453 [M − H]−. 9-[(N-Phosphonoethyl-N-phosphonoethoxyethyl)-2-aminoethyl]-8-bromoguanine (10c). Starting from 5c, side product, yield 20%. 1 H NMR (D2O): δ 4.47 t, 2 H, J(1′,2′) = 6.4 (H-1′); 3.85 t, 2 H, J(6′,5′) = 4.5 (H-6′); 3.71 m, 6 H (H-2′, H-3′ and H-7′); 3.56 t, 2 H, J(5′,6′) = 4.5 (H-5′); 2.12 m, 2 H (H-4′); 1.99 m, 2 H (H-8′). 13C NMR (D2O): δ 156.35 (C-6); 152.89 (C-2); 151.74 (C-4); 122.54 (C-8); 114.99 (C-5); 64.83 d, J(P,C) = 2.9 (C-7′); 62.41 (C-6′); 51.89, 49.59, and 48.55 (C-2′, C-3′ and C-5′); 37.68 (C-1′); 26.65 d, J(P,C) = 133.6 (C-8′). 21.37 d, J(P,C) = 129.8 (C-4′). HRMS calcd for C13H22N6O8P2, 531.01632; found, 531.01674. MS (ESI): m/z = 531 and 533 [M − H]−. Synthesis of Tetraphosphoramidate Prodrugs of Bisphosphonic Acids 8a−8c and 9a−9c: General Procedure. A mixture of corresponding tetraester 4a−4c or 5a−5c (0.5 mmol), dry pyridine (8 mL), and BrSiMe3 (1 mL) was stirred overnight at room temperature under argon. After evaporation and codistillation with pyridine under argon atmosphere, the residue was dissolved in dry pyridine (8 mL) and ethyl (L)-phenylalamine hydrochloride (1.4 g, 6.1 mmol) and triethylamine (2.5 mL) were added. The mixture was heated to 70 °C under argon atmosphere, and then a solution of Aldrithiol (1.76 g, 8 mmol) and triphenylphosphine (2.1 g, 8 mmol) in dry pyridine (8 mL) was added. The reaction mixture was heated at 70 °C for 3 days, the solvent was evaporated and the residue was purified by column chromatography on silica gel and the crude product further purified by preparative HPLC. The phosphoramidate prodrug was obtained as foam. Tetra-(ethyl L-phenylalanine) Prodrug of 9-[(N-PhosphonoethylN-phosphonobutyl)-2-aminoethyl]hypoxanthine (8a). Starting from 4a, yield 38%. 1H NMR (DMSO-d6): δ 12.28 s, 1 H (NH); 8.03, 1, and 8.02 s, 1 H (H-8 and H-2); 7.24 m, 20 H (Ar); 4.52 m, 1 H (NH); 4.35 m, 1 H (NH); 4.12 m, 2 H (NH); 3.98 m, 12 H (H-1′, NHCH and Et); 3.84 m, 2 H (NHCH); 2.87 m, 8 H (CH2Ph); 2.61 m, 2 H (H-2′); 2.45 m, 2 H (H-3′); 2.13 m, 2 H (H-5′); 1.50, 2, and 1.32 m, 2 H (H-4′ and H-8′); 1.06 m, 16 H (H-6′, H-7′ and Et). 13C NMR (DMSO-d6): δ 173.01 m, 4 C (CO); 156.51 (C-6); 148.17 (C-4); 145.18 (C-2); 140.45 (C-8); 137.09, 2 C, 137.05, 2 C, 129.27, 4 C, 129.21, 4 C, 127.96, 4 C, 127.92, 4 C, 126.32, 2 C and 126.26, 2 C (Ar); 123.69 (C-5); 60.09 m (Et); 53.84 m (NHCH); 52.36 (C-2′); 52.33 (C-5′); 46.99 (C-3′); 41.23 (C-1′); 28.42 d, J(P,C) = 134.6 (C-4′); 26.14 d, J(P,C) = 25.9 (C-7′); 24.43 d, J(P,C) = 137.9 (C-8′); 19.89 d, J(P,C) = 6.1 (C-6′); 13.81 and 13.76, 4 C (Et). HRMS calcd for C57H76N9O11P2, 1124.51340; found, 1124.51316. MS (ESI): m/z = 1125 [M + H]+. Tetra-(ethyl L-phenylalanine) Prodrug of 9-[(N-PhosphonoethylN-phosphonomethoxyethyl)-2-aminoethyl]hypoxanthine (8b). Starting from 4b, yield 54%. 1H NMR (DMSO-d6): δ 12.29 s, 1 H (NH); 8.07, 1, and 8.01 s, 1 H (H-8 and H-2); 7.23 m, 20 H (Ar); 4.45 m, 4 H (NH); 4.00 m, 14 H (H-1′, NHCH and Et); 3.18 m, 4 H (H-6′ and H-7′); 2.87 m, 8 H (CH2Ph); 2.66 m, 2 H (H-2′); 2.44 m, 2 H (H-3′); 1.34 m, 2 H (H-4′); 1.07 m, 12 H (Et). 13C NMR (DMSO-d6): δ 172.69 m (CO); 156.53 (C-6); 148.15 (C-4); 145.15 (C-2); 140.58 (C-8); 137.10, 2 C, 137.06, 2 C, 129.22, 4 C, 129.95, 4 C, 127.98, 4 C, 127.94, 4 C, 126.31, 2 C and 126.25, 2 C (Ar); 123.66 (C-5); 70.78 d, J(P,C) = 11.1 (C-6′); 67.53 d, J(P,C) = 133.9

(C-7′); 60.18 m (Et); 53.80 m (NHCH); 52.76 (C-2′); 51.65 (C-5′); 47.70 (C-3′); 41.20(C-1′); 22.87, J(P,C) = 140.8 (C-4′); 13.79 and 13.76 (Et). HRMS calcd for C56H74N9O12P2, 1126.49267; found, 1126.49258. MS (ESI): m/z = 1126 [M + H]+. Tetra-(ethyl L-phenylalanine) Prodrug of 9-[(N-PhosphonoethylN-phosphonoethoxyethyl)-2-aminoethyl]hypoxanthine (8c). Starting from 4c, yield 50%. 1H NMR (DMSO-d6): δ 12.28 s, 1 H (NH); 8.02 m, 2 H (H-8 and H-2); 7.20 m, 20 H (Ar); 4.48 m, 2 H (NH); 4.17 m, 1 H (NH); 4.01 m, 13 H (NH, H-1′, NHCH and Et); 3.86 m, 2 H (NHCH); 3.24 m, 2 H (H-7′); 3.12 t, 2 H, J(6′,5′) = 6.0 (H-6′); 2.85 m, 6 H (CH2Ph); 2.65 m, 2 H (CH2Ph); 2.64 t, 2 H, J(2′,1′) = 5.9 (H-2′); 2.45 m, 2 H (H-3′); 2.37 t, 2 H, J(5′,6′) = 6.0 (H-5′); 1.53, 2, and 1.32 m, 2 H (H-4′ and H-8′); 1.09 m, 12 H (Et). 13C NMR (DMSO-d6): δ 172.89 m, 4 C (CO); 156.51 (C-6); 148.14 (C-4); 145.15 (C-2); 140.47 (C-8); 137.08, 2 C, 137.06, 2 C, 129.25, 4 C, 129.21, 4 C, 127.94, 4 C, 127.90, 4 C, 126.32, 2 C and 126.26, 2 C (Ar); 123.63 (C-5); 68.15 (C-6′); 64.70 (C-7′); 60.16 m (Et); 53.85 m (NHCH); 52.75 (C-2′); 51.81 (C-5′); 47.71 (C-3′); 41.21 (C-1′); 26.65, J(P,C) = 128.03 (C-8′); 20.24, J(P,C) = 121.9 (C-4′); 13.80 and 13.74, 4 C (Et). HRMS calcd for C57H76N9O12P2, 1140.50832; found, 1140.50853. MS (ESI): m/z = 1141 [M + H]+. Tetra-(ethyl L-phenylalanine) Prodrug of 9-[(N-PhosphonoethylN-phosphonobutyl)-2-aminoethyl]guanine (9a). Starting from 5a, yield 26%. 1H NMR (DMSO-d6): δ 10.56 s, 1 H (NH); 7.62 s, 1 H (H-8); 7.118 m, 20 H (Ar); 6.48 s, 2 H (NH2); 4.39 m, 4 H (NH); 4.00 m, 12 H (H-1′, NHCH, Et); 3.85 m, 2 H (NHCH); 2.88 m, 10 H (H-2′ and CH2Ph); 2.43 m, 2 H (H-3′); 2.12 m, 2 H (H-5′); 1.30 m, 4 H (H-4′ and H-8′); 1.23 m, 16 H (H-6′, H-7′ and Et). 13C NMR (DMSO-d6): δ 172.94 m, 4 C (CO); 156.67 (C-6); 153.39 (C-2); 150.96 (C-4); 137.51 (C-8); 137.15, 2 C, 137.13, 2 C, 129.22 m, 8 C, 127.93, 8, and 126.28 m, 4 C (Ar); 116.32 (C-5); 60.18 m (Et); 54.09 m (NHCH); 52.33 (C-2′); 50.78 (C-5′); 47.44 (C-3′); 39.93 (C-1′); 27.75 d, J(P,C) = 150.1 (C-4′); 25.77 d, J(P,C) = 22.7 (C-7′); 23.62 d, J(P,C) = 142.0 (C-8′); 19.34 d, J(P,C) = 6.7 (C-6′); 13.81 and 13.78, 4 C (Et). HRMS calcd for C57H77N10O11P2, 1139.52430; found, 1139.52412. MS (ESI): m/z = 1140 [M + H]+. Tetra-(ethyl L-phenylalanine) Prodrug of 9-[(N-PhosphonoethylN-phosphonomethoxyethyl)-2-aminoethyl]guanine (9b). Starting from 5b, yield 30%. 1H NMR (DMSO-d6): δ 10.31 s, 1 H (NH); 7.61 s, 1 H (H-8); 7.20 m, 20 H (Ar); 6.20 s, 2 H (NH2); 4.05 m, 18 H (H-1′, NH, NHCH, Et); 3.41 m, 4 H (H-6′ and H-7′); 2.91 m, 8 H (CH2Ph); 2.72 m, 2 H (H-2′); 2.57 m, 4 H (H-3′ and H-5′); 1.61 m, 2 H (H-4′); 1.11 m, 12 H and (Et). 13C NMR (DMSO-d6): δ 172.51 m (CO); 156.52 (C-6); 153.25 (C-2); 150.94 (C-4); 137.24 (C-8); 137.06, 2 C, 136.84, 2 C, 129.09, 4 C, 129.04, 4 C, 127.81, 4 C, 127.76, 4 C, 126.15, 2 C and 126.10, 2 C (Ar); 116.66 (C-5); 70.72 (C-6′); 67.78 d, J(P,C) = 135.2 (C-7′); 60.03 m (Et); 53.75 m (NHCH); 52.70 (C-2′); 52.07 (C-5′); 47.77 (C-3′); 40.69 (C-1′); 40.0 (CH2Ph); 26.40 m (C-4′); 13.59 and 13.54 (Et). HRMS calcd for C56H75N10O12P2, 1141.50357; found, 1141.50285. MS (ESI): m/z = 1142 [M + H]+. Tetra-(ethyl L-phenylalanine) Prodrug of 9-[(N-PhosphonoethylN-phosphonoethoxyethyl)-2-aminoethyl]guanine (9c). Starting from 5c, yield 28%. 1H NMR (DMSO-d6): δ 10.53 s, 1 H (NH); 7.63 s, 1 H (H-8); 7.20 m, 20 H (Ar); 6.45 s, 2 H (NH2); 4.49 m, 2 H (NH); 4.28 m, 2 H (NH); 4.01 m, 10 H (NHCH, Et); 3.85 m, 4 H (NHCH, H-1′); 3.27, 2, and 3.15 m, 2 H (H-6′ and H-7′); 2.87, 6, and 2.74 m, 2 H (CH2Ph); 2.57 m, 2 H (H-2′); 2.44, 2, and 2.37 m, 2 H (H-3′ and H-5′); 1.55, 2, and 1.35 m, 2 H (H-4′ and H-8′); 1.08 m, 12 H and (Et). 13C NMR (DMSO-d6): δ 172.92 m (CO); 156.67 (C-6); 153.33 (C-2); 150.95 (C-4); 137.84 (C-8); 137.01, 2 C, 136.89, 2 C, 129.26, 4 C, 129.15, 4 C, 127.97, 4 C, 127.92, 4 C, 126.34, 2 C and 126.28, 2 C (Ar); 116.25 (C-5); 68.16 (C-6′); 64.68 (C-7′); 60.14 m (Et); 53.79 m (NHCH); 52.88 (C-2′); 51.83 (C-5′); 47.71 (C-3′); 40.57 (C-1′); 24.45, J(P,C) = 160.6 (C-8′); 22.42, J(P,C) = 137.7 (C-4′); 13.80 and 13.74, 4 C (Et). HRMS calcd for C57H77N10O12P2, 1155.51922; found, 1155.51984. MS (ESI): m/z = 1156 [M + H]+. Synthesis of Bisphosphoramidate Prodrugs 12a and 12b of Cyanoethyl Aza-ANPs: General Procedure. A mixture of corresponding diethyl ester9 14a or 14b (1 mmol), dry acetonitrile (15 mL), and Q



Article

J(P,C) = 133.9 (C-4′); 15.48 (C-6′). HRMS calcd for C26H43N6O7PS2, 644.24484; found, 644.24493. MS (ESI): m/z = 644 [M + H]+. Determination of Ki Values. Human HGPRT and PvHGPRT were stored in 0.1 M Tris-HCl, 0.01 M MgCl2, pH 7.4, 200 μM PRib-PP, 1 mM dithiothreitol (DTT), −80 °C. Pf HGXPRT was stored in 0.01 M phosphate, 60 μM hypoxanthine, 200 μM PRib-PP, pH 7.2, 1 mM DTT as previously described.20 This difference is because the Pf enzyme is completely inactive under the conditions used to store the human and Pv enzymes. For enzyme assays, the buffer was 0.1 M Tris-HCl, 0.01 M MgCl2, pH 7.4. For the Pf and Pv enzymes, the assays were performed in this buffer and also in 0.01 M phosphate, 5 mM DTT as described by Hazelton and colleagues.2a This was done for direct comparison of the Ki values and also because, in the phosphate buffer, the rates with the parasite enzymes became completely linear for 60 s. The Ki values were calculated by Hanes’ plots at a fixed concentration of guanine (60 μM) and at variable concentrations of PRib-PP (14−1000 μM) depending on the Km(app) in the presence of the inhibitor. Crystallization and Structure Determination. For crystallization experiments, human HGPRT was concentrated and stored as previously described.13 The hanging drop method was used where 1 μL of enzyme in complex with the inhibitor was added to 1 μL of well solution. The trays were then incubated at 18 °C. The reservoir solutions were: 6b and 6c, 0.2 M calcium acetate, 0.1 M Tris-HCl, pH 7.5, 20% PEG3000; 7b and 7c, 1 M sodium/potassium tartrate, 0.2 M NaCl, 0.1 M imidazole, pH 8.0; 11a, 20% PEG 3000, 0.2 M calcium acetate; 10c, 20% PEG 3350, 0.2 M sodium acetate. The crystals were placed in liquid nitrogen and then transported to the Australian synchrotron. X-ray data were collected remotely by BLU-ICE21 using beamline MX1. Data were scaled and merged using XDS.22 The structure was solved by molecular replacement with the program PHASER23 within PHENIX 1.8−106924 with the protein coordinates of the tetramer of human HGPRT in complex with ([(2-[(guanine9H-yl)methyl]propane-1,3-diyl)bis(oxy)bis9mehtylene))diphosphonic acid (pdb code 4IJQ). Subsequent refinement of the coordinates was with PHENIX1.8−106924 and model building was with COOT 0.7.25 The structural restraints files for the inhibitors were generated by use of the PRODRG2 Dundee server.26 Evaluation of in Vitro Antimalarial Activity of ANPs. Pf D6 (Sierra-Leone) laboratory line, sensitive to most antimalarial drugs and W2 (Indochina) line, resistant to chloroquine and pyrimethamine, were maintained in RPMI-1640-LPLF complete medium, containing 10% human plasma, at 4% hematocrit and 1−8% parasitemia as previously described.27 Cultures were routinely synchronized using 28 D-sorbitol. To evaluate the antimalarial activity of the ANPs, the [3H]-hypoxanthine growth inhibition assay29 was utilized, where the uptake of [3H]-hypoxanthine by malaria parasites is used as a surrogate marker for parasite growth. For these assays, stock solutions of ANPs were made to concentrations of 20−40 mM in DMSO or water and subsequently diluted in hypoxanthine-free complete media prior to assay. The assays (in 96-well plate format) were initiated when the majority of parasites (>90%) were at early trophozoite (ring) stage. Parasite cultures (100 μL per well) at 0.5% initial parasitemia and 2% hematocrit in hypoxanthine-free RPMI1640-LPLF medium were exposed to ten 2-fold serial dilutions of the ANPs and chloroquine (CQ) (reference drug) for 96 h, with [3H]-hypoxanthine (0.2 μCi/well) added ∼48 h after beginning of the experiment. The [3H]-hypoxanthine incorporation data were analyzed and sigmoidal growth inhibition curves were produced by nonlinear regression analysis of the [3H]-hypoxanthine incorporation data versus log-transformed concentrations of the compounds using Graphpad Prism V5.0 software (GraphPad Software Inc. USA). The inhibitory concentration (IC50) that results in 50% inhibition of parasite growth was determined. The IC50 values were based on three independent experiments with mean ± SD calculated. Evaluation of in Vitro Cytotoxicity of ANP Prodrugs. The inhibitory effect of the test compounds on cell proliferation was determined in A549 human lung carcinoma cells.30 To determine the cytostatic effect of the test compounds, the cells were seeded in 96-well plates at 7500 cells per well and, 24 h later, the compounds were added at serial

BrSiMe3 (0.6 mL) was stirred overnight at room temperature under argon. After evaporation and codistillation with acetonitrile under argon atmosphere, the residue was dissolved in dry pyridine (8 mL) and isopropyl (L)-phenylalamine hydrochloride (1 g, 4 mmol) and dry triethylamine (2 mL) were added. The mixture was heated to 60 °C under argon atmosphere, and then a solution of Aldrithiol (1.32 g, 6 mmol) and triphenylphosphine (1.6 g, 6 mmol) in dry pyridine (10 mL) was added. The reaction mixture was heated at 60 °C for 2 days, the solvent was evaporated, and the residue was purified by column chromatography on silica gel (MeOH/CHCl3) and the crude product further purified by preparative HPLC. The phosphoramidate prodrug was obtained as foam. Diisopropyl 2,2′-(((2-((2-(Guanin-9-yl)ethyl)(2-cyanoethyl)amino)ethyl)phosphoryl)bis(azanediyl))(2S,2′S)-bis(3-phenylpropanoate) (12a). Starting from diethyl 9-[(N-(2-cyanoethyl)-N-(2-phosphonoethyl))-2- aminoethyl]guanine (14a),9 yield 56%. 1H NMR (DMSO-d6): δ 10.57 s, 1 H (NH); 7.66 s, 1 H (H-8); 7.21 m, 10 H (Ar); 6.47 s, 2 H (NH2); 4.80 m, 2 H (iPr); 4.49 t, 1 H, J = 11.1 (NH); 4.18 t, 1 H, J = 10.6 (NH); 3.91 t, 2 H, J(1′,2′) = 6.5 (H-1′); 3.88 m, 2 H, (NHCH); 2.86, 3, and 2.73 m, 1 H (CH2Ph); 2.61 m, 2 H, (H-2′); 2.61 t, 2 H, J(5′,6′) = 6.7 (H-5′); 2.50 m, 2 H (H-3′); 2.48 t, 2 H, J(6′,5′) = 6.7 (H-6′); 1.39 m, 2 H (H-4′); 1.17 d, 3 H, 1.12 d, 3 H, 1.06, 3, and 1.01 d, 3 H, J = 6.2 (iPr). 13C NMR (DMSO-d6): δ 172. 63 and 172.47 (CO); 156.71 (C-6); 153.40 (C-2); 150.98 (C-4); 137.50 (C-8); 137.17, 137.09, 129.35, 2 C, 129.29, 2 C, 127.98, 2 C, 127.93, 2 C, 126.36 and 126.27 (Ar); 119.77 (CN); 116.29 (C-5); 67.74 and 67.58 (iPr); 53.99 and 53.83 (NHCH); 51.82 (C-2′); 47.95 (C-5′); 46.49 (C-3′); 40.59 (C-1′); 25.39 d, J(P,C) = 110.7 (C-4′); 21.30 m, 4 C (iPr); 15.27 (C-6′). MS (ESI): m/z = 734 [M + H]+. Diisopropyl 2,2′-(((2-((2-(Hypoxanthin-9-yl)ethyl)(2-cyanoethyl)amino)ethyl)phosphoryl)bis(azanediyl))(2S,2′S)-bis(3-phenylpropanoate) (12b). Starting from diethyl 9-[(N-(2-cyanoethyl)-N-(2-phosphonoethyl))-2- aminoethyl]hypoxanthine (14b),9 yield 37%. 1H NMR (DMSO-d6): δ 12.28 s, 1 H (NH); 8.06, 1, and 8.02 s, 1 H (H-8 and H-2); 7.20 m, 10 H (Ar); 4.81 m, 2 H (iPr); 4.48 t, 1 H, J = 11.6 (NH); 4.13 m, 3 H, (NH and H-1′); 3.94, 1, and 3.85 m, 1 H (CH); 2.84, 3, and 2.73 m, 1 H (CH2Ph); 2.69 t, 2 H, J(2′,1′) = 6.2 (H-2′); 2.61 t, 2 H, J(5′,6′) = 6.7 (H-5′); 2.45 t, 2 H, J(6′,5′) = 6.7 (H-6′); 1.35 m, 2 H (H4′); 1.17 d, 3 H, 1.12 d, 3 H, 1.06, 3, and 1.01 d, 3 H, J = 6.2 (iPr). 13C NMR (DMSO-d6): δ 172. 93 and 172.74 (CO); 156.86 (C-6); 148.50 (C-4); 145.51 (C-2); 140.69 (C-8); 137.46, 137.39, 129.64, 2 C, 129.58, 2 C, 128.27, 2 C, 128.23, 2 C, 126.65 and 126.56 (Ar); 123.98 (C-5); 119.96 (CN); 68.01 and 67.87 (iPr); 54.24 and 54.09 (NHCH); 52.15 (C-2′); 48.13 (C-5′); 46.89 (C-3′); 41.45 (C-1′); 24.85 d, J(P,C) = 135.2 (C-4′); 21.59 m, 4 C (iPr); 15.52 (C-6′). MS (ESI): m/z = 719 [M + H]+. S,S′-(((2-((2-(Guanin-9-yl)ethyl)(2-cyanoethyl)amino)ethyl)phosphoryl)bis(oxy))bis(ethane-2,1-diyl))bis(2,2-dimethylpropanethioate) (13a). A mixture of diethyl ester9 14a (0.41 g, 1 mmol), dry acetonitrile (10 mL), dimethylformamide (5 mL), 2,6-lutidine (0.05 mL), and BrSiMe3 (0.9 mL) was stirred at room temperature for 2 days. After evaporation and codistillation with toluene, the residue was dissolved in water (5 mL) and triethylamine (5 mL) to obtain ammonium salt. After evaporation to dryness and codistillation with ethanol/toluene, the residue was dissolved in dry pyridine (10 mL) and S-(2-hydroxyethyl)2,2-dimethylpropanethioate (0.81 g, 5 equiv) and 2,4,6-triisopropyl benzenesulfonyl chloride (0.91 g) were added. The mixture was stirred at room temperature for 5 days, the solvent was evaporated, and the residue was purified by column chromatography on silica gel (MeOH/CHCl3) and the crude product further purified by preparative HPLC. The pro-drug was obtained as white solid (130 mg), yield 20%. 1H NMR (DMSO-d6): δ 10.53 s, 1 H (NH); 7.70 s, 1 H (H-8); 6.40 s, 2 H (NH2); 3.98 m, 6 H (H-1′ and 2 × CH2O); 3.09 t, 4 H, J = 6.4 (2 × CH2S); 2.74 m, 4 H (H-2′, H-5′); 2.71 m, 2 H (H-3′); 2.54 t, 2 H, J(6′,5′) = 6.8 (H-6′); 1.89 m, 2 H (H-4′); 1.17 s, 18 H (CH3). 13C NMR (DMSO-d6): δ 205.05 (CO); 156.73 (C-6); 153.36 (C-2); 151.01 (C-4); 137.69 (C-8); 119.81 (CN); 116.35 (C-5); 63.32, 2 C (CH2-O); 51.76 (C-2′); 48.15 (C-5′); 46.18 (C-3′); 45.91, 2 C (tBu); 40.91 (C-1′); 28.45, 2 C (CH2−S); 26.81, 6 C (tBu); 22.09 d, R



Article

dilutions. After 4 days incubation at 37 °C, the cells were trypsinized and counted with a Coulter Counter apparatus. The CC50 values, or compound concentrations at which cell proliferation was 50% compared to that in untreated cells, were calculated by extrapolation. Data presented are the mean ± SEM of two or three independent tests. The selectivity indices (SI) were determined as a ratio of CC50 in human A549 cell lines vs the IC50 in the P. falciparum D6 cell line.

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PRib-PP, 5-phospho-α-D-ribosyl-1-pyrophosphate; Pf , Plasmodium falciparum; Pv, Plasmodium vivax; DTT, dithiothreitol; ImmGP, (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-Dribitol 5-phosphate; ImmHP, (1S)-1-(9-deazahypoxanthine-9yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate; SI, selectivity index; aza-ANP, aza-acyclic nucleoside phosphonate; ANP, acyclic nucleoside phosphonate

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ASSOCIATED CONTENT

Accession Codes

The atomic coordinates and structure factors of human HGPRT in complex with the six inhibitors have been deposited in the Protein Data Bank. For the 9-[(N-phosphonoethyl-Nphosphonomethoxyethyl)-2-aminoethyl]guanine (7b) complex, 9-[(N-phosphonoethyl-N-phosphonoethoxyethyl)-2-aminoethyl]guanine (7c) complex, 9-[(N-phosphonoethyl-N-phosphonoethoxyethyl)-2-aminoethyl]hypoxanthine (6c) complex, for the 9-[(N-phosphonoethyl-N-phosphonomethoxyethyl)-2aminoethyl]hypoxanthine (6b) complex, for the 9-[(N-(2cyanoethyl)-N-(2- phosphonoethyl))-2-aminoethyl]-guanine (11a) complex, and 9-[(N-Phosphonoethyl-N-phosphonoethoxyethyl)-2-aminoethyl]-8-bromoguanine (10c) complex the accession codes are 4RAC, 4RAD, 4RAO, 4RAQ, 4RAN, and 4RAB, respectively.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*For D.H.: E-mail, [email protected]. *For L.W.G.: phone, 07 33653549; E-mail, luke.guddat@ uq.edu.au. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The initial crystallographic conditions were determined using the Mosquito and Rockimager facilities at the University of Queensland Remote-Operation Crystallization facility (UQROCX). Preliminary X-ray data were measured at this facility. Final measurements were made at beamline MX1, Australian Synchrotron, Clayton, Victoria, with the assistance of Alan Riboldi-Tunniclife, Christine Gee, and Tom Caradoc-Davies. The views expressed here are those of the authors and not necessarily those of the Australian Synchrotron. We are grateful to Kerryn Rowcliffe and Wim van Dam for their excellent technical assistance with the in vitro antimalarial and cytotoxicity assays. We thank the Australian Red Cross Blood service (Brisbane) for providing human erythrocytes and plasma for the in vitro cultivation of P. falciparum lines. The opinions expressed herein are those of the authors and do not necessarily reflect those of the Australian Defence Force, Joint Health Command, or any extant policy. This work was supported by funds from the National Health and Medical Research Council (grant no. 1030353), by the subvention for development of research organization (Institute of Organic Chemistry and Biochemistry, RVO 61388963), by the Grant Agency of the Czech Republic (grant no. P207/11/0108), and by Gilead Sciences (Foster City, CA, USA).

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ABBREVIATIONS USED HG[X]PRT, hypoxanthine−guanine−[xanthine] phosphoribosyltransferase; PRTase, phosphoribosyltransferase; HGPRT, hypoxanthine−guanine phosphoribosyltransferase; HGXPRT, hypoxanthine−guanine−xanthine phosphoribosyltransferase; S



Article

phosphoribosyltransferases. Bioorg. Med. Chem. 2012, 20 (2), 1076− 1089. (9) Hocková, D.; Keough, D. T.; Janeba, Z.; Wang, T. H.; de Jersey, J.; Guddat, L. W. Synthesis of novel N-branched acyclic nucleoside phosphonates as potent and selective inhibitors of human, Plasmodium falciparum and Plasmodium vivax 6-oxopurine phosphoribosyltransferases. J. Med. Chem. 2012, 55 (13), 6209−6223. (10) Connelly, M. C.; Robbins, B. L.; Fridland, A. Mechanism of uptake of the phosphonate analog (S)-1-(3-hydroxy-2phosphonylmethoxypropyl)cytosine (HPMPC) in Vero cells. Biochem. Pharmacol. 1993, 46 (6), 1053−1057. (11) Pertusati, F.; Serpi, M.; McGuigan, C. Medicinal Chemistry of nucleoside phosphonate prodrugs for antiviral therapy. Antiviral Chem. Chemother. 2012, 22 (5), 181−203. (12) Oliveira, F. M.; Barbosa, L. C. A.; Ismail, F. M. D. The diverse pharmacology and medicinal chemistry of phosphoramidatesa review. RSC Adv. 2014, 4 (36), 18998−19012. (13) Keough, D. T.; Špaček, P.; Hocková, D.; Tichý, T.; Vrbková, S.; Slavětínská, L.; Janeba, Z.; Naesens, L.; Edstein, M. D.; Chavchich, M.; Wang, T. H.; de Jersey, J.; Guddat, L. W. Acyclic nucleoside phosphonates containing a second phosphonate group are potent inhibitors of 6-oxopurine phosphoribosyltransferases and have antimalarial activity. J. Med. Chem. 2013, 56 (6), 2513−2526. (14) Choi, I.; Mikkelsen, R. B. Plasmodium falciparum: ATP/ADP transport across the parasitophorous vacuolar and plasma membranes. Exp. Parasitol. 1990, 71 (4), 452−462. (15) (a) Mitsunobu, O. The use of diethyl azodicarboxylate and triphenylphosphine in synthesis and transformation of natural products. Synthesis 1981, 1−28. (b) Ludek, O. R.; Meier, C. Synthesis of carbocyclic pyrimidine nucleosides, III. Influence of the N3protection group on N1-vs O-2-alkylation in the Mitsunobu reaction. Eur. J. Org. Chem. 2006, 4, 941−946. (16) Zhou, D.; Lagoja, I. M.; Van Aerschot, A.; Herdewijn, P. Synthesis of aminopropyl phosphonate nucleosides with purine and pyrimidine bases. Collect. Czech. Chem. Commun. 2006, 71 (1), 15−34. (17) Jansa, P.; Baszczynski, O.; Dračínský, M.; Votruba, I.; Zídek, Z.; Bahador, G.; Stepan, G.; Cihlar, T.; Mackman, R.; Holý, A.; Janeba, Z. A novel and efficient one-pot synthesis of symmetrical diamide (bisamidate) prodrugs of acyclic nucleoside phosphonates and evaluation of their biological activities. Eur. J. Med. Chem. 2011, 46 (9), 3748− 3754. (18) (a) Mackman, R. L.; Cihlar, T. Prodrug strategies in the design of nucleoside and nucleotide antiviral therapeutics. Annu. Rep. Med. Chem. 2004, 39, 305−321. (b) Hecker, S. J.; Erion, M. D. Prodrugs of phosphates and phosphonates. J. Med. Chem. 2008, 51 (8), 2328− 2345. (19) Meola, M.; Yamen, B.; Weaver, K.; Sandwick, R. K. The catalytic effect of Mg2+ and imidazole on the decomposition of 5phosphoribosyl-alpha-1-pyrophosphate in aqueous solution. J. Inorg. Biochem. 2003, 93 (3−4), 235−242. (20) Keough, D. T.; Ng, A. L.; Winzor, D. J.; Emmerson, B. T.; de Jersey, J. Purification and characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase and comparison with the human enzyme. Mol. Biochem. Parasitol. 1999, 98 (1), 29−41. (21) McPhillips, T. M.; McPhillips, S. E.; Chiu, H. J.; Cohen, A. E.; Deacon, A. M.; Ellis, P. J.; Garman, E.; Gonzalez, A.; Sauter, N. K.; Phizackerley, R. P.; Soltis, S. M.; Kuhn, P. Blu-Ice and the Distributed Control System: software for data acquisition and instrument control at macromolecular crystallography beamlines. J. Synchrotron Radiat. 2002, 9 (Pt 6), 401−406. (22) Kabsch, W. Xds. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 (Pt 2), 125−132. (23) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl. Cryst. 2007, 40, 658−674. (24) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; GrosseKunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R.

J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 (Pt 2), 213−221. (25) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 (Pt 4), 486−501. (26) Schuttelkopf, A. W.; van Aalten, D. M. PRODRG: a tool for high-throughput crystallography of protein−ligand complexes. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60 (Pt8), 1355−1363. (27) Trager, W.; Jensen, J. B. Human malaria parasites in continuous culture. Science 1976, 193 (4254), 673−675. (28) Lambros, C.; Vanderberg, J. P. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J. Parasitol. 1979, 65 (3), 418−420. (29) Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Quantitative assessment of anti-malarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 1979, 16 (6), 710−718. (30) Chen, T. R. Chromosome changes in 6-TG-resistant mutant strains derived from a karyotypically stable human line, C32. Cytogenet. Cell Genet. 1983, 35 (3), 181−189.

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Aza-acyclic Nucleoside Phosphonates Containing a Second

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