DOI: 10.1002/cmdc.201500183

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Bisamidate Prodrugs of 2-Substituted 9-[2(Phosphonomethoxy)ethyl]adenine (PMEA, adefovir) as Selective Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis Michal Cˇesnek,[a] Petr Jansa,[a] Mark¦ta Sˇm†dkov‚,*[a] Helena Mertl†kov‚-Kaiserov‚,[a] Martin Dracˇ†nsky´,[a] Tarsis F. Brust,[d] Petr P‚vek,[b, c] Frantisˇek Trejtnar,[b] Val J. Watts,[d] and Zlatko Janeba*[a] Novel small-molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health threat worldwide. In this study, a series of 2-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, adefovir), in their isopropyl ester bis(l-phenylalanine) prodrug form, were designed and synthesized as potent inhibitors of adenylate cyclase toxin (ACT) isolated from B. pertussis. The series consists of PMEA analogues bearing either a linear or branched aliphatic chain or a heteroatom at the C2 position of the purine moiety. Compounds with a small C2 substituent showed high potency against ACT without cytotoxic effects as well as good selectivity over human adenylate

cyclase isoforms AC1, AC2, and AC5. The most potent ACT inhibitor was found to be the bisamidate prodrug of the 2-fluoro PMEA derivative (IC50 = 0.145 mm). Although the bisamidate prodrugs reported herein exhibit overall lower activity than the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their toxicity and plasma stability profiles are superior. Furthermore, the bisamidate prodrug was shown to be more stable in plasma than in macrophage homogenate, indicating that the free phosphonate can be effectively distributed to target tissues, such as the lungs. Thus, ACT inhibitors based on acyclic nucleoside phosphonates may represent a new strategy to treat whooping cough.

Introduction Pertussis (whooping cough) is a highly contagious respiratory disease caused by a strictly human pathogen, Bordetella pertussis.[1] Despite high levels of vaccination, pertussis remains a serious worldwide health threat, especially for infants.[2] As B. pertussis is generally sensitive to antibiotics, this approach has been the primary recourse for therapy.[3] However, antibiotics are ineffective against toxemia and antibiotic-resistant B. pertussis strains.[4] Moreover, the protection imparted by pertussis vaccinations has been reported to wane after 4–12 years.[5] As [a] Dr. M. Cˇesnek, Dr. P. Jansa, Dr. M. Sˇm†dkov‚, Dr. H. Mertl†kov‚-Kaiserov‚, Dr. M. Dracˇ†nsky´, Dr. Z. Janeba Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic v.v.i. Flemingovo n‚m. 2, 166 10 Prague 6 (Czech Republic) E-mail: [email protected] [b] Prof. P. P‚vek, Dr. F. Trejtnar Charles University in Prague, Faculty of Pharmacy in Hradec Kr‚lov¦ Heyrovsk¦ho 1203, 500 05 Hradec Kr‚lov¦ (Czech Republic) [c] Prof. P. P‚vek Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry, Palacky University Olomouc Hneˇvot†nsk‚ 5, 775 15 Olomouc (Czech Republic) [d] T. F. Brust, Prof. V. J. Watts Department of Medicinal Chemistry and Molecular Pharmacology College of Pharmacy, Purdue University 575 Stadium Mall Drive, West Lafayette, IN, 47907 (USA) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201500183.

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a result, whooping cough still annually affects some 16 million people worldwide, and about 195 000 children died from this disease in 2008.[6] Thus, the need for new effective therapies to combat pertussis remains. B. pertussis is transmitted from infected to susceptible individuals through aerosols from a cough or sneeze. The colonization of the upper respiratory tract begins by adherence of bacteria to the ciliated epithelial cells in the nasopharynx and trachea.[7] Afterward, a host defense response is neutralized by the secretion of several types of toxins.[7, 8] Among these, the adenylate cyclase toxin (ACT or CyaA) is considered an important virulence factor for B. pertussis infection.[9] A study by Weiss et al.[10] was the first demonstration that an ACT- and hemolysin-deficient double mutant B. pertussis strain was nonvirulent in infant mouse models. This provided direct evidence that ACT may be essential for lethal infection in mice. The entry of ACT into phagocyte cytosol is a two-step process:[11] The toxin first binds the integrin CD11b/CD18 and is inserted into the phagocyte membrane to mediate calcium ion influx into cells.[12] This promotes relocation of the toxin–receptor complex into specific lipid microdomains (rafts) within the cell membrane; the increased cholesterol concentrations in rafts and their particular organization then support translocation of the adenylate cyclase domain from ACT directly into cytoplasm.[11] Increased intracellular calcium concentrations lead to a calmodulin-mediated activation of ACT, which converts cy-

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Full Papers tosolic ATP into the key signaling molecule cAMP.[9] This process completely subverts the cellular signaling pathways by establishing non-physiological intracellular cAMP levels. In vitro, ACT has been shown to impair a number of important metabolic functions of human immune effector cells,[13] facilitating an effective invasion and colonization of bacteria in the host organism. Moreover, such disruption of the immune response can clearly make the host organism susceptible to secondary infections. Acyclic nucleoside phosphonates (ANPs)[14] belong to a recognized class of highly effective nucleotide analogues with a broad spectrum of antiviral activity.[15] ANPs also exert a myriad of other biological processes including cytostatic,[16] antibacterial,[17] antiparasitic,[18] and immunomodulatory activities.[19] Furthermore, adefovir diphosphate (PMEApp, 1, Figure 1), the active cellular metabolite of adefovir dipivoxil (bis(POM)PMEA, 2, Figure 1) approved for the treatment of

Figure 1. Structures of adefovir diphosphate (1), adefovir dipivoxil (2), and PMEA bisamidate prodrugs 3 and 4.

chronic hepatitis B virus infection, was shown to effectively inhibit both B. pertussis adenylate cyclase toxin[20] and Bacillus anthracis edema factor (EF).[21] PMEApp (1) also exhibited lower affinity for mammalian adenylate cyclases (ACs),[21] suggesting

that it has the potential for use at concentrations that effectively neutralize the bacterial toxins without pronounced interference with host ACs. Analogously to other ANPs, PMEA prodrugs provide temporary protection of the polar phosphonate moiety, allowing them to enter cells, where they undergo metabolic transformation into free PMEA and subsequent phosphorylation to active PMEApp (1), thus inhibiting adenylate cyclase activity. The drawbacks of adefovir dipivoxil (2) are its rapid hydrolysis to PMEA by plasma esterases[22] and its dose-dependent cytotoxicity.[23] Therefore, a bisamidate series of PMEA prodrugs was recently studied by our research group as potential inhibitors of B. pertussis ACT. Of those, compounds 3 and 4 (Figure 1) were selected as the most promising prodrugs owing to their attractive in vitro activity, bioavailability, and synthetic accessibility.[24] Based on in vivo pharmacokinetic tests, the isopropyl ester bis(l-phenylalanine) moiety (present in 4, Figure 1) was selected for synthesis of the cell-permeable prodrugs of the new series of ACT inhibitors. Based on the ACT crystal structure in the presence of PMEApp,[20] we proposed possible structural modifications of the PMEA molecule, especially at the C2 or C8 positions of the purine moiety. Because none of the 8-substituted PMEA analogues[25] exhibited promising activity,[26] we focused on the 2substituted derivatives of PMEA. Our in silico predictions suggested that substitution at the C2 position of PMEApp (1) could be well tolerated by the ACT active site, as demonstrated with 2-ethyl- and 2-butyl-substituted PMEApp, for example (Figure 2). The two alkyl substituents are nicely accommodated in the hydrophobic pocket of the ACT active site, with possible attractive interactions between the lipophilic substituents and the uncharged portion of the ACT protein (Figure 2). The aim of this work was the synthesis of a series of 2-substituted PMEA prodrugs 5 (Figure 3) and evaluation of their ability to selectively inhibit B. pertussis ACT. Furthermore, the pharmacological and biochemical properties of the lead structure were investigated in vitro and in vivo and were compared with those of the previous generation of compounds, represented by compound 4 (Figure 1).

Figure 2. Docking of A) 2-ethyl-PMEApp and B) 2-butyl-PMEApp with B. pertussis ACT. Images were generated in Discovery Studio 4.0 using the implemented protocols. The protein surface is colored according to interpolated charge (red: negative, blue: positive, grey: uncharged).

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Figure 3. General structure of the target 2-substituted PMEA derivatives 5 in their bisamidate prodrug form for subsequent cell-based assays.

Results Chemistry

(Scheme 1, Table 1 entries 10 and 11) were also prepared by Method C using the corresponding commercially available alkylzinc bromide reagents in 82 and 69 % yields, respectively. Because the preliminary docking studies (Figure 2) suggested that there may be sufficient space in the ACT binding pocket for even bulkier C2 substituents, we decided to explore the spatial tolerance of ACT and to prepare PMEA analogues bearing tert-butyl and adamantyl substituents at the C2 position. However, the reaction of tertiary zinc bromide reagents (1-adamantylzinc bromide or tert-butylzinc bromide) with compound 8 using Method C (Pd(dppf)2Cl2·CH2Cl2, THF) failed, and 2-tetrahydrofuranyl derivative 9 i was formed as the sole reaction product in both cases. Compound 9 i (Scheme 1, Table 1 entry 9) was isolated in 78 % yield in the case of reaction with 1-adamantylzinc bromide; 9 i was not isolated from the reaction with tert-butylzinc bromide. Product 9 i probably results

Because nonaqueous diazotation–dediazonation reactions on purine moieties have been thoroughly studied and optimized,[27a,b] 2-amino-6-chloropurine derivative 6 (Scheme 1)[28] was selected for subsequent modifications of the purine moiety at the C2 position. Compound 6 was converted by diazotation– dediazonation[27a,b] into 6-chloro2-iodo intermediate 7 in 77 % yield. Key 6-amino-2-iodopurine intermediate 8 was then prepared by ammonolysis of compound 7 in 86 % yield (Scheme 1). The coupling reaction of inter- Scheme 1. Synthesis of 2-substituted PMEA analogues and their prodrugs 5 (see Table 1 for R1 groups). Reagents mediate 8 with trimethylalumi- and conditions: a) I2, CH2I2, CuI, THF, isoamylnitrite, 80 8C, 30 min; b) NH3/EtOH, RT, 24 h; c) see Table 1 for methods num and Pd(PPh3)4 in THF af- and yields; d) TMSBr, pyridine, RT, 12 h, then l-phenylalanine isopropyl ester hydrochloride, Et3N, 2,2’-dipyridyldiforded 6-amino-2-methyl deriva- sulfide, PPh3, pyridine, 70 8C, 24–72 h; e) CuSCN, DMF, 120 8C. tive 9 a as its mono-isopropyl ester in 65 % yield (Scheme 1, Table 1. Synthesis of 2-substituted PMEA derivatives 9 and isopropyl ester bis(l-pheTable 1 entry 1). Derivatives 9 b, 9 d, and 9 h nylalanine) prodrugs 5. (Scheme 1, Table 1 entries 2, 4, and 8) were prepared in high yields (78–93 %) from compound 8 and the Yield [%] Compd Yield [%] Entry Compd R1 corresponding alkylmagnesium bromides by a trans[a] 1 9a Me 65 5a 70 metalation reaction using zinc bromide (Method A). 2 9b Et[b] 93 5b 55 3 9c Pr[c] 70 5c 57 The other 2-alkylpurine derivatives were synthe[b] 4 9 d iPr 86 5 d 69 sized by Negishi coupling of the corresponding alkyl5 9e Bu[c] 97 5e 65 zinc bromides with intermediate 8. Derivatives 9 c 6 9f sBu[d] 77 5f 53 and 9 e (Scheme 1, Table 1 entries 3 and 5) bearing 7 9g iBu[e] 49 5g 54 8 9h pentan-3-yl[b] 78 5h 51 linear alkyl groups were prepared in high yields (70 [f] 9 9 i tetrahydrofuran-2-yl 78 5 i 29 and 97 %, respectively) by the standard cross-cou10 9j cyclopentyl[d] 82 5j 70 [29] pling procedure with Pd(PPh3)4 in DMF (Method B). 11 9k bicyclo[2.2.1]heptan-2-yl[d] 69 5k 66 2-sec-Butyl derivative 9 f (Scheme 1) was obtained 12 9 l[31] 2-(cyclopropylamino)[g] 59 5l 41 13 9 m[28] NH2 NA[h] 5m 46 under analogous reaction conditions (Method B) in [28] [h] 14 9 n F NA 5 n 45 poor yield (31 %), as the reaction also afforded 2-n15 9 o[28] Cl NA[h] 5o 61 butyl derivative 9 e (17 %) and the 2-dehalogenated 16 9 p[28] OH NA[h] 5p 48 product (PMEA diester) as the major product (45 %). 5q 55 17 8 I NA[h] Modification of the reaction conditions (Method C), Reaction conditions: [a] Me3Al, Pd(PPh3)4, THF, 70 8C. [b] Method A (alkMgBr, ZnBr2, that is, replacement of solvent (DMF for THF) and Pd(dppf)2Cl2·CH2Cl2, THF). [c] Method B (alkZnBr, Pd(PPh3)4, DMF). [d] Method C (alkZnBr, Pd(dppf)2Cl2·CH2Cl2, THF). [e] tBuZnBr, BHT, Pd(dppf)Cl2·CH2Cl2, NMP, 80 8C. catalyst (Pd(PPh3)4 for Pd(dppf)2Cl2), yielded 2-sec[f] 1-AdamantylZnBr, Pd(dppf)Cl2·CH2Cl2, ¢40 8C, then RT. [g] Cyclopropylamine, CuI, butyl derivative 9 f in 77 % yield (Scheme 1, Table 1 (iPr)2NEt, NMP. [h] Not applicable. entry 6). Subsequently, compounds 9 j and 9 k ChemMedChem 2015, 10, 1351 – 1364

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Full Papers from the solvent trapping the adamantyl or tert-butyl radical formed during the reaction.[30] Thus, 2,6-di-tert-butyl-4-methylphenol (BHT) was used as a radical scavenger in the subsequent experiments, and an increase in BHT concentration actually caused a decrease in yields of 9 i in both cases. Ultimately, the reactions of intermediate 8 with 1-adamantylzinc bromide and tert-butylzinc bromide were carried out with excess BTH and in N-methylpyrrolidone to yield the corresponding 2dehalogenated product (PMEA diester, not isolated) and isomerized 2-isobutyl derivative 9 g (49 % yield; Scheme 1, Table 1 entry 7), respectively. All of the above-mentioned PMEA derivatives 9 a–9 k contain a lipophilic group attached to the C2 position of the purine moiety by a C¢C bond. To extend the structure–activity relationship (SAR) study of this group of compounds, a series of PMEA derivatives bearing various heteroatoms at the C2 position were prepared according to previously published procedures: namely, 2-(cyclopropylamino) derivative 9 l (59 % yield) by copper-catalyzed nucleophilic aromatic substitution with cyclopropylamine (Scheme 1, Table 1 entry 12);[31] 2-amino, 2fluoro, and 2-chloro derivatives 9 m–9 o (Scheme 1, Table 1 entries 13–15) by alkylation of the corresponding 6-amino-2-substituted purines;[28] and 2-hydroxy derivative 9 p (Scheme 1, Table 1 entry 16) by treatment of 2,6-diaminopurine analogue 9 m with isoamylnitrite in 80 % acetic acid.[28] All prepared 2-substituted PMEA analogues 8 and 9 a–9 p were converted into their corresponding isopropyl ester bis(lphenylalanine) prodrugs 5 a–5 q (Scheme 1, Table 1) by standard methodologies.[32] Furthermore, 2-thiocyano derivative 5 r (Scheme 1) was prepared in 21 % yield directly from 2-iodo PMEA bisamidate prodrug 5 q by the reaction using CuSCN in DMF.

Table 2. ACT inhibition and cytotoxic effects of bis(POM)PMEA (2), PMEA bisamidate prodrug 4, and 2-substituted PMEA bisamidate prodrugs 5. Compd

2 4 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q 5r

IC50 [mm][a]

Viability [%][b]

0.006 œ 0.001 0.16 œ 0.01 1.14 œ 0.34 5.43 œ 0.10 > 10 > 10 1.56 œ 0.35 > 10 > 10 > 10 5.46 œ 0.60 > 10 > 10 2.68 œ 0.75 0.97 œ 0.13 0.14 œ 0.04 0.54 œ 0.06 10.93 œ 1.05 1.24 œ 0.42 4.38 œ 0.88

59 93 94 105 ND ND 93 ND ND ND 102 ND ND 87 160 83 142 182 81 101

[a] Data are the mean œ SD of at least three independent experiments. [b] Data are the percent cell viability at a fixed prodrug concentration (10 mm) versus untreated control; ND: not determined.

the murine J774A.1 cell line, but also in human tumor (CCRFCEM, HL-60, HeLa-S3, HepG2) and normal (HUVEC-2, NHDF-Ad) cells. From the compounds tested, only 5 m caused higher cytotoxic effects than 4 in all cell lines. This effect was similar to that of adefovir dipivoxil (2, Table 3). The rest of the compounds of series 5 showed a marked improvement in cytotoxic properties, and, unlike compound 4, no cytotoxicity toward normal human cell lines was observed (Table 3). Compound 5 n showed no cytotoxicity for either cell line (Table 3).

Biological activity Inhibition of ACT

Selectivity for ACT over mAC

Bisamidate prodrugs 5 a–r were tested for their ability to inhibThe ability of 2-substituted PMEA prodrugs 5 to inhibit host it ACT activity in J774A.1 macrophage cells (Table 2). Murine mammalian adenylate cyclases (mACs) was also examined macrophage cells J774A.1 were incubated with various concentrations of bisamidate prodrugs 5 a–r and Table 3. Long-term cytotoxic effects of bis(POM)PMEA (2), PMEA bisamidate prodrugs subsequently exposed to B. pertussis ACT. The cells 4, and 2-substituted PMEA bisamidate prodrugs 5 in tumor and normal cells. were lysed, and the cAMP content was determined. Compd Viability [%][a] Compounds that showed activity in the low microCCRF-CEM HL-60 HepG2 HeLa-S3 J774A.1 HUVEC-2 NHDF-Ad molar and sub-micromolar range (Table 2) were also 2 10 11 44 18 34 38 32 assessed for their effects on the viability of J774A.1 4 19 14 65 40 68 72 65 cells under conditions identical to those of the cAMP 5n 114 84 92 83 93 91 93 assay, in order to exclude cytotoxic effects during the 5a 100 101 100 96 92 98 102 5b 107 111 93 89 75 92 93 experiment. In contrast to bis(POM)PMEA (2), no sig5 e 31 84 106 42 77 89 90 nificant cytotoxic effects were observed if the cells 5i 87 90 92 92 65 95 96 were treated with bisamidate prodrugs 5 at a concen5l 73 25 99 91 93 86 87 tration of 10 mm for 5 h (Table 2). 5m 9 11 41 12 29 33 29 5o 5p 5q

Long-term cytotoxicity The three-day cytotoxic effect of prodrugs 2, 4, and 5 at a concentration of 10 mm was tested not only in ChemMedChem 2015, 10, 1351 – 1364

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74 115 91

92 117 95

100 101 133

74 92 94

96 72 91

107 99 98

95 91 101

[a] Data are the percent cell viability at a fixed prodrug concentration (10 mm) versus untreated control.

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Full Papers Table 4. Mammalian AC1, AC2, and AC5 inhibition by bisamidate prodrugs 5.

Compd 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q W300 SKF83566 loratadine

AC1 3 mm A23187

% mAC inhibition[a] AC2 100 nm PMA

180 œ 21 163 œ 34 178 œ 31 168 œ 12 118 œ 12 122 œ 15 156 œ 23 94 œ 10 189 œ 15 119 œ 12[b] 39 œ 4 141 œ 26 161 œ 21 ND 189 œ 29 125 œ 15 204 œ 28 23 œ 5 95 œ 9 102 œ 25

147 œ 15 161 œ 16 164 œ 23 159 œ 30 172 œ 34 146 œ 10 149 œ 26 149 œ 27 193 œ 48 90 œ 12[b] 31 œ 9 166 œ 24 130 œ 26 151 œ 27 196 œ 27 119 œ 23 253 œ 78 336 œ 83 ¢2 œ 4 84 œ 5

AC5 300 nm FSK 97 œ 8 98 œ 14 110 œ 16 103 œ 10 121 œ 23 114 œ 18 101 œ 5 97 œ 11 118 œ 18 96 œ 10[b] 84 œ 19 106 œ 7 101 œ 5 75 œ 6 110 œ 13 97 œ 6 116 œ 8 192 œ 58 83 œ 10 64 œ 1

Figure 4. Susceptibility of prodrug 5 n (100 mm) to enzymatic hydrolysis in macrophage homogenate. PMEA-F, free PMEA-F retention time: 7.9 min; intermediate products IP1, IP2, and IP3 respective retention times: 8.2, 8.5, and 10.2 min; 5 n: parent prodrug.

spective retention times of 8.2, 8.5, and 10.2 min (Figure 4). The free phosphonate PMEA-F (retention time 7.9 min) was also observed; interestingly, this was further partially metabolized to PMEA (Figure 4). Thus, the bisamidate prodrug 5 n was shown to be more stable to enzymatic degradation than bis(POM)PMEA (2), which was completely degraded to free PMEA within one hour (data not shown).

[a] Test compound concentration: 30 mm; data are the mean œ SD of three independent experiments; ND: not determined. [b] Tested at 18 mm; data listed are the percentage of the mAC response treated with stimulant only.

Pharmacokinetic study in rats

(Table 4). These assays were carried out with HEK293 cells stably expressing mAC1, mAC2, or mAC5. Each of these mACs represents one of the three major mAC families. Each compound was tested on three separate occasions at both 30 and 3 mm, with one exception: compound 5 j was tested at 18 and 3 mm. Specific activation of the mAC overexpressed in the cells was accomplished as previously described.[33] Briefly, the calcium ionophore A23187 was employed to stimulate AC1, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) to stimulate AC2, and forskolin (FSK) to stimulate AC5. None of the compounds significantly inhibited any mAC at 3 mm (data not shown). Most of the compounds did not inhibit the mACs, with the exception of prodrugs 5 k and 5 n. In fact, several compounds potentiated the selectively stimulated cAMP response at AC1 and AC2, but not AC5.

The results of a pilot experiment in a rat model revealed that prodrug 5 n is more stable in plasma (Figure 5 A) than in macrophage homogenate (Figure 4). Compared with macrophage homogenate, only the IP3 intermediate was detected in plasma, not free phosphonate (Figure 5 A). Moreover, prodrug 5 n and/or its metabolites were distributed to the organs (liver, kidney, lung) within 2 h. The IP3 intermediate was detected in these tissues predominantly (Figure 5 B). These findings indicate that significant amounts of free phosphonate could be distributed to the target tissue.

Discussion 2-Substituted PMEA analogues 5, in their isopropyl ester bis(lphenylalanine) prodrug form, were efficiently synthesized, and their ACT inhibitory properties were studied in vitro in J774A.1 macrophages. Results of this SAR study demonstrate that small substituents at the C2 position of PMEA are well tolerated and that this trend is consistent in both 2-alkylpurine and 2-halo-

Hydrolysis of 5 n in macrophage homogenate Hydrolysis of isopropyl ester bis(l-phenylalanine) prodrug 5 n into free phosphonate in macrophage homogenate (i.e., in the target tissue) was next evaluated. During the course of the experiment (18 h), hydrolysis of 5 n led to the appearance of the partially hydrolyzed intermediates IP1, IP2, and IP3 with re-

Figure 5. Susceptibility of prodrug 5 n to enzymatic hydrolysis in A) plasma and B) specific organs. PMEA-F; IP3 intermediate (monoester/monoamidate) retention time: 10.2 min; 5 n: parent prodrug.

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Full Papers purine analogues. 2-Methylpurine derivative 5 a (IC50 = 1.14 mm) is the most active compound in the aliphatic series, as is the 2fluoropurine derivative 5 n (IC50 = 145 nm) in the halogenated series. Among the aliphatic compounds, the inhibitory properties decrease in the following order: Me 5 a > Et 5 b > Pr 5 c ~ iPr 5 d ~ sBu 5 f ~ iBu 5 g, whereas in the 2-halogenated series, the order is: F 5 n > Cl 5 o > I 5 q. Interestingly, the 2-butyl derivative 5 e retained some inhibitory activity (IC50 = 1.56 mm), suggesting that the AC binding pocket accommodates even longer linear lipophilic substituents, providing some space for further investigation in this direction. Moreover, unlike bis(POM)PMEA (2), prodrugs of the series 5 maintain low cytotoxicity. The prepared compounds 5 were also tested in cell-based assays for mammalian ACs, namely mAC1, mAC2, and mAC5. Most of the 2-substituted PMEA analogues (with the exception of 5 k) did not inhibit mACs at the concentrations tested, indicating these compounds are selective inhibitors of the bacterial AC. The observed selectivity for ACT over mAC suggests that 2-substituted PMEA derivatives 5, or related compounds, may be used therapeutically at concentrations that effectively neutralize bacterial toxins without deleterious side effects through interference with host ACs. As the main target of B. pertussis infection are lung phagocytes, the most important property of an ideal prodrug for potential oral administration of an ACT inhibitor seems to be its plasma stability as well as its ability to be cleaved in the target tissue. Recently, monoester monoamidate and bisamidate prodrugs of PMEA have been shown to exhibit good stability, with t1/2 > 1 h in human plasma, but undergo rapid hydrolysis in lymphoid cells and cellular extracts.[16a, 34] This pharmacokinetic profile is in contrast with that of adefovir dipivoxil (bis(POM)PMEA), which displays limited stability in both rat and human plasma (t1/2 < 10 min) and no selectivity for intracellular activation over systemic hydrolysis.[35] Based on our previous study,[24] bisamidate PMEA prodrugs 3 (ethyl ester bis(l-alanine)) and 4 (isopropyl ester bis(l-phenylalanine)) (Figure 1) were chosen for pilot pharmacokinetic studies in rats because they displayed optimal stability profiles in macrophage homogenate. Both 3 and 4 exhibited rapid hydrolysis in macrophage homogenate, but 4 manifested higher stability in rat plasma (Figures S1–S4, Supporting Information). Thus, for the present study, the prodrug moiety of 4 (i.e., isopropyl ester bis(l-phenylalanine)) was chosen for the novel series of 2-substituted PMEA derivatives 5. Because the stability of prodrugs can be affected not only by the prodrug moiety, but also by substitution on the purine moiety of PMEA, the stability of the most potent prodrug of series 5, 2-fluoro PMEA derivative 5 n, was evaluated. The hydrolysis of 5 n in macrophage homogenate is rapid, as only some 10 % of 5 n remains uncleaved after 1 h, and ~ 30 % of free 2-fluoro PMEA is present (Figure 4). Interestingly, free dehalogenated PMEA also appeared in 2 h. Evidently, 2fluoro PMEA is slowly metabolized by cellular enzymes to PMEA. The pilot pharmacokinetic study in rats showed that a large portion of the parent prodrug 5 n remained unchanged in rat plasma over the course of a 2 h experiment (Figure 5). ChemMedChem 2015, 10, 1351 – 1364

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Thus, the stability of 5 n far exceeds that of bis(POM)PMEA (2). Although prodrug 5 n did not exceed the potency of bis(POM)PMEA (2; IC50 = 6 nm) for ACT inhibition, an increased in vivo bioavailability of its active metabolite is expected. Moreover, the in vivo study in rats confirmed that the prodrug 5 n and/or its metabolites are distributed to different tissues, including lungs. Compared with compound 4 (IC50 = 158 nm), 5 n (IC50 = 145 nm) demonstrates similar ACT inhibitory activity. Likewise, there is no significant difference in stability in rat plasma between 4 and 5 n. However, modification at the C2 position of 5 n caused a decrease in the rate of release of the free phosphonate in macrophage homogenate. This may be expected to result in steady tissue concentrations of the active component. The high level of adefovir (PMEA) in kidneys is connected with its nephrotoxicity, which has been shown to be mediated by inhibition of mtDNA replication associated with damage to mitochondrial function.[36] Although the compounds of series 5 did not decrease the viability of J774A.1 cells during the first 5 h, they were evaluated for their possible long-term cytotoxic effects on several cancer and normal cell lines. Relative to the previous PMEA series,[24] the substitution at C2 resulted in a decrease in cytotoxicity toward all cancer lines. Moreover, unlike 4, the compounds of series 5 were not cytotoxic toward normal cell lines (HUVEC-2, NHDF-Ad). The only exception is compound 5 m with a 2,6-diaminopurine moiety. The introduction of other substituents at the C2 position appears to weaken the effect on mtDNA replication. Thus, presence of a fluorine atom at the C2 position of PMEA together with the isopropyl ester bis(l-phenylalanine) prodrug moiety substantially improved the cytotoxic profile. In summary, compound 5 n is a lead structure of series 5, with sufficient plasma stability and capacity to deliver the active component to lung tissue without the side effects connected with PMEA analogues.

Conclusions A series of 18 PMEA analogues substituted at the C2 position of the purine moiety and in their isopropyl ester bis(l-phenylalanine) prodrug (compounds 5) form were synthesized and studied as potent inhibitors of B. pertussis ACT. The SAR study showed that a small substituent at C2 is well tolerated, for which 2-fluoro derivative 5 n was found to be the most potent inhibitor of the entire series (IC50 : 145 nm). The bisamidate prodrug 5 n was shown to be more stable in rat plasma than in macrophage homogenate, and 5 n, and/or its metabolites, were efficiently distributed to organs, including lungs. The tested compounds demonstrated promising selectivity for ACT over mammalian ACs and no cytotoxicity. As ACT is an significant invasive toxin secreted by B. pertussis, its successful neutralization could prevent toxemia in the host. Thus, potent inhibitors of ACT activity based on acyclic nucleoside phosphonates could be used for the prophylaxis or even treatment of whooping cough (and possibly of anthrax), especially in combination with the appropriate antibacterial agent.

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Full Papers Experimental Section Unless otherwise stated, solvents were evaporated at 40 8C/2 kPa, and the compounds were dried over P2O5 at 2 kPa. Solvents were dried by standard procedures. Tetrahydrofuran (THF) was freshly distilled from sodium/benzophenone under Ar. N,N-Dimethylformamide (DMF) and acetonitrile were distilled from P2O5 and stored over molecular sieves (4 A). TLC was performed on plates of Kieselgel 60 F254 (Merck). Mass spectra were measured on an LCQ classic spectrometer using electrospray ionization (ESI). 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. Reagents were obtained from commercial sources (Sigma–Aldrich). Starting compounds were prepared according to published procedures.[32] Preparative HPLC purifications were performed on columns packed with 10 mm C18 reversed-phase resin (Phenomenex Gemini 10 mm 21 Õ 250 mm) on a Waters Delta 600 chromatograph system, in ~ 200 mg batches of mixtures using a gradient of MeOH/H2O as eluent. Flash chromatography on normal phase and deionization on reversed phase was performed on a Reveleris Flash Chromatography System. Deionization was performed on a Redisep Rf Gold C18 Teledyne ISCO column. All tested ANPs were characterized by 1H NMR, 13C NMR, and mass spectrometry. The purity of the compounds was determined by combustion elemental analysis (C, H, N). Compound 6 was prepared according to a published procedure.[28]

General cross-coupling reaction procedures Method A: A solution of ZnBr2 (1 m in THF; 7 mL, 7 mmol) was added dropwise to the appropriate Grignard reagent (6 mmol) in 10 mL THF at ¢78 8C under Ar. The reaction mixture was stirred at ¢78 8C for 15 min, allowed to warm to room temperature (30 min), and stirred at 50 8C for 30 min. The resulting mixture was cooled to room temperature. The solution of compound 8 (0.48 g, 1 mmol) and Pd(dppf)Cl2·CH2Cl2 (0.04 g) in THF (15 mL) under Ar was added dropwise to the reaction mixture at room temperature. Saturated aqueous NH4Cl (10 mL) was added to the reaction mixture, and it was stirred for 15 min. The reaction was poured into EtOAc (150 mL). The organic layer was washed with a saturated EDTA solution (30 mL), brine (30 mL), and then dried over Na2SO4. Volatiles were removed in vacuo, and the residue was purified by flash chromatography. Method B: Compound 8 (0.48 g, 1 mmol) and Pd(PPh3)4 (0.06 g) were dissolved in DMF (5 mL) under Ar. A solution of alkylZnBr (0.5 m in THF; 10 mL, 5 mmol) was added dropwise to the reaction mixture, and it was stirred at room temperature for 2 h. Saturated NH4Cl solution (10 mL) was added to the reaction mixture, and it was stirred for 15 min and then poured into EtOAc (150 mL). The organic layer was washed with a saturated solution of EDTA (30 mL), brine (30 mL), and then dried over Na2SO4. Volatiles were removed in vacuo, and the residue was purified by column chromatography. Method C: A solution of alkylZnBr (0.5 m in THF; 10 mL, 5 mmol) was added dropwise to a solution of compound 8 (0.48 g, 1 mmol) and Pd(dppf)Cl2·CH2Cl2 (0.04 g) in THF (15 mL) under Ar. Saturated NH4Cl solution (10 mL) was added to the reaction mixture and stirred for 15 min. The reaction was poured into EtOAc (150 mL). The organic layer was washed with a saturated solution of EDTA (30 mL), brine (30 mL), and dried over Na2SO4. Volatiles were reChemMedChem 2015, 10, 1351 – 1364

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moved in vacuo, and the residue was purified by column chromatography. 6-Chloro-2-iodo-9-{2-[(diisopropoxyphosphoryl)methoxy]ethyl}9H-purine (7). CH2I2 (55 mL, 682 mmol) was added to a mixture of 6 (18.3 g, 21.3 mmol), I2 (16.5 g, 65 mmol), and CuI (13.2 g, 69 mmol) in dry THF (330 mL) under Ar at room temperature. Isoamylnitrite (57 mL, 424 mmol) was subsequently added dropwise to the reaction mixture. The resulting slurry was placed on a preheated oil bath (80 8C) and held at reflux for 30 min. The reaction mixture was cooled to room temperature, and the solid was filtered off. The filtrate was evaporated in vacuo, dissolved in EtOAc (300 mL), and filtered once again. The filtrate was washed with saturated aqueous Na2S2O3 (2 Õ 100 mL), brine (100 mL), and dried over Na2SO4. Volatiles were removed in vacuo, and the residue was purified by flash chromatography (MeOH/CHCl3 3:97) to give 18 g (77 %) of 7 as a pale-yellow solid. ESIMS m/z (%): 503 (35) [M + H] + ; 1 H NMR (400 MHz, CDCl3): d = 1.26 (d, J = 6.2 Hz, 6 H, CH3), 1.29 (d, J = 6.2 Hz, 6 H, CH3), 3.72 (d, J = 8.2 Hz, 2 H, P-CH2), 3.91–3.96 (m, 2 H, O-CH2), 4.47–4.44 (m, 2 H, N-CH2), 4.68 (m, 2 H, CH-iPr), 8.21 ppm (s, 1 H, H-8); 13C NMR (100 MHz, CDCl3): d = 23.97 (2C, d, JP,C = 4.6 Hz, CH3), 23.98 (2C, d, JP,C = 3.6 Hz, CH3), 44.08 (C1’), 66.10 (d, JP,C = 168.0 Hz, P-C), 70.52 (d, JP,C = 9.7 Hz, C2’), 71.26 (2C, d, JP,C = 6.7 Hz, POC), 116.31 (C2), 131.42 (C5), 146.25 (C8), 150.29 (C6), 152.53 ppm (C4); HRMS (ESI + ): m/z [M + H] + calcd for C14H21ClN4O4P: 503.0112, found: 503.0110. 6-Amino-2-iodo-9-{2-[(diisopropoxyphosphoryl)methoxy]ethyl}9H-purine (8). A mixture of compound 7 (15 g, 30 mmol) and EtOH/NH3 (250 mL) was stirred at room temperature for 24 h. The solvent was removed, and the residue was purified by flash chromatography (MeOH/CHCl3, 2:98) to give 12 g (83 %) of compound 8. ESIMS m/z (%): 484 (45) [M + H] + ; 1H NMR (400 MHz, CDCl3): d = 1.27 (d, J = 6.2 Hz, 6 H, CH3), 1.30 (d, J = 6.2 Hz, 6 H, CH3), 3.73 (d, J = 8.3 Hz, 2 H, P-CH2), 3.90 (t, 2 H, O-CH2), 4.35 (t, 2 H, N-CH2), 4.63– 4.76 (m, 2 H, CH-iPr), 7.92 ppm (s, 1 H, H-8); 13C NMR (100 MHz, CDCl3): d = 23.98 (2C, d, JP,C = 4.5 Hz, CH3), 24.02 (2C, d, JP,C = 3.8 Hz, CH3), 43.61 (C1’), 66.04 (d, JP,C = 168.0 Hz, P-C), 70.97 (d, JP,C = 10.4 Hz, C2’), 71.24 (2C, d, JP,C = 6.7 Hz, POC), 118.61 (C5), 119.65 (C2), 141.20 (C8), 150.21 (C4), 154.99 ppm (C6); HRMS (ESI + ): m/z [M + H] + calcd for C14H24IN5O4P: 484.0611, found 405.0614. 6-Amino-2-methyl-9-{2-[(isopropoxyphosphoryl)methoxy]ethyl}9H-purine (9 a). Compound 8 (0.48 g, 1 mmol) and Pd(PPh3)4 (0.1 g) were purged with Ar and dissolved in THF (20 mL). A solution of Al(CH3)3 in toluene (2 m, 1 mL, 2 mmol) was added dropwise to the above solution. The reaction mixture was heated at 70 8C for 3 h, cooled and poured into saturated aqueous NaH2PO4 (20 mL). The precipitated solid was removed by filtration, the filtrate was evaporated in vacuo, and the residue was purified by flash chromatography (CHCl3/MeOH/H2O, 5:4:1) to give 9 a (0.21 g, 65 %). ESIMS m/z (%): 330 (100) [M + H] + ; 1H NMR (400 MHz, [D6]DMSO): d = 0.96 (d, J = 4.5 Hz, 6 H, CH3), 2.37 (s, 3 H, H-1’’), 3.41 (d, J = 8.7 Hz, 2 H, PCH2), 3.77 (t, J = 5.0 Hz 2 H, O-CH2), 4.12–4.21 (m, 1 H, CH-iPr), 4.27 (t, J = 5.0 Hz, 2 H, N-CH2), 8.09 ppm (s, 1 H, H-8); 13C NMR (100 MHz, [D6]DMSO): d = 24.18 (2C, d, JP,C = 3.7 Hz, CH3), 25.12 (C1’’), 42.47 (C1’), 67.41 (d, JP,C = 147.4 Hz, P-C), 70.14 (d, JP,C = 11.2 Hz, C2’), 66.57 (C, d, JP,C = 5.8 Hz, POC), 116.55 (C5), 140.83 (C8), 150.06 (C4), 155.18 (C6), 160.44 ppm (C2); HRMS (ESI + ): m/z [M + H] + calcd for C12H21N5O4P: 330.1326, found: 330.1326. 6-Amino-2-ethyl-9-{2-[(diisopropoxyphosphoryl)methoxy]ethyl}9H-purine (9 b). Prepared from compound 8 by Method A (reaction time 4 h), yield 0.70 g (93 %). ESIMS m/z (%): 386 (100) [M + H] + ; 1H NMR (400 MHz, CDCl3): d = 1.24 (d, J = 6.2 Hz, 6 H, CH3), 1.27

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Full Papers (d, J = 6.2 Hz, 6 H, CH3), 1.31 (t, J = 7.6 Hz, 3 H, H-2’’), 2.78 (q, J = 7.6 Hz, 2 H, H-1’’), 3.71 (d, J = 8.3 Hz, 2 H, P-CH2), 3.92 (t, J = 5.1 Hz, 2 H, O-CH2), 4.36 (t, J = 5.1 Hz, 2 H, N-CH2), 4.61–4.72 (m, 2 H, CHiPr), 6.04 (br s, 2 H, NH2), 7.90 ppm (s, 1 H, H-8); 13C NMR (100 MHz, CDCl3): d = 13.05 (C2’’), 23.89 (2C, d, JP,C = 6.9 Hz, CH3), 23.97 (2C, d, JP,C = 6.9 Hz, CH3), 32.51 (C1’’), 43.13 (C1’), 66.00 (d, JP,C = 167.8 Hz, PC), 71.24 (d, JP,C = 10.5 Hz, C2’), 71.09 (C, d, JP,C = 6.7 Hz, POC), 117.55 (C5), 140.93 (C8), 150.69 (C4), 155.20 (C6), 166.73 ppm (C2); HRMS (ESI + ): m/z [M + H] + calcd for C16H29N5O4P: 386.1952, found: 386.1950. 6-Amino-2-propyl-9-{2-[(disopropoxyphosphoryl)methoxy]ethyl}9H-purine (9 c). Prepared from compound 8 by Method B (reaction time 2 h), yield 0.27 g (70 %). ESIMS m/z (%): 400 (100) [M + H] + ; 1 H NMR (400 MHz, CDCl3): d = 0.98 (t, J = 7.4 Hz, 3 H, H-3’’), 1.26 (d, J = 6.2 Hz, 6 H, CH3), 1.29 (d, J = 6.2 Hz, 6 H, CH3), 1.75–1.87 (m, 2 H, H-2’’), 2.77 (t, J = 7.7 Hz, 2 H, H-1’’), 3.72 (d, J = 8.3 Hz, 2 H, P-CH2), 3.93 (t, J = 4.9 Hz, 2 H, O-CH2), 4.37 (t, J = 4.9 Hz, 2 H, N-CH2), 4.66– 4.72 (m, 2 H, CH-iPr), 5.95 (br s, 2 H, NH2), 7.90 ppm (s, 1 H, H-8); 13 C NMR (100 MHz, CDCl3): d = 13.93 (C3’’), 22.31 (C2’’), 23.94 (2C, d, JP,C = 6.9 Hz, CH3), 23.99 (2C, d, JP,C = 5.7 Hz, CH3), 41.06 (C1’’), 43.26 (C1’), 66.06 (d, JP,C = 167.8 Hz, P-C), 71.20 (d, JP,C = 10.4 Hz, C2’), 71.14 (C, d, JP,C = 6.8 Hz, POC), 117.49 (C5), 141.27 (C8), 150.62 (C4), 154.71 (C6), 165.14 ppm (C2); HRMS (ESI + ): m/z [M + H] + calcd for C17H31N5O4P: 400.2108, found: 400.2107. 6-Amino-2-isopropyl-9-{2-[(disopropoxyphosphoryl)methoxy]ethyl}-9H-purine (9 d). Prepared from compound 8 by Method A (reaction time 2 h), yield 0.34 g (86 %). ESIMS m/z (%): 400 (100) [M + H] + ; 1H NMR (400 MHz, CDCl3): d = 1.25 (d, J = 6.2 Hz, 6 H, H2’’), 1.29 (d, J = 6.2 Hz, 6 H, CH3), 1.30 (t, J = 6.9 Hz, 6 H, CH3), 3.04 (td, J = 13.7, J = 6.8 Hz, 1 H, H-1’’), 3.72 (d, J = 8.3 Hz, 2 H, P-CH2), 3.94 (t, J = 5.0 Hz, 2 H, O-CH2), 4.37 (t, J = 4.9 Hz, 2 H, N-CH2), 4.66– 4.71 (m, 2 H, CH-iPr), 5.90 (br s, 2 H, NH2), 7.88 ppm (s, 1 H, H-8); 13 C NMR (100 MHz, CDCl3): d = 21.93 (C2’’), 23.94 (2C, d, JP,C = 6.9 Hz, CH3), 23.99 (2C, d, JP,C = 6.1 Hz, CH3), 37.23 (C1’’), 43.18 (C1’), 66.03 (d, JP,C = 167.8 Hz, P-C), 71.19 (d, JP,C = 10.5 Hz, C2’), 71.13 (C, d, JP,C = 6.8 Hz, POC), 117.56 (C5), 141.16 (C8), 150.65 (C4), 154.82 (C6), 169.56 ppm (C2); HRMS (ESI + ): m/z [M + H] + calcd for C17H31N5O4P: 400.2108, found: 400.2107. 6-Amino-2-butyl-9-{2-[(disopropoxyphosphoryl)methoxy]ethyl}9H-purine (9 e). Prepared from compound 8 by Method B (reaction time 2 h), yield 0.40 g (97 %). ESIMS m/z (%): 414 (100) [M + H] + ; 1 H NMR (400 MHz, CDCl3): d = 0.92 (t, J = 7.4 Hz, 3 H, 4’-CH3), 1.24 (d, J = 6.2 Hz, 6 H, CH3), 1.28 (d, J = 6.2 Hz, 6 H, CH3), 1.33–1.45 (m, 2 H, H-3’’), 1.69–1.81 (m, 2 H, H-2’’), 2.75 (t, J = 7.9 Hz, 2 H, H-1’’), 3.71 (d, J = 8.3 Hz, 2 H, P-CH2), 3.91 (t, J = 4.9 Hz, 2 H, O-CH2), 4.36 (t, J = 4.9 Hz, 2 H, N-CH2), 4.61–4.74 (m, 2 H, CH-iPr), 6.01 (br s, 2 H, NH2), 7.87 ppm (s, 1 H, H-8); 13C NMR (100 MHz, CDCl3): d = 13.92 (C4’’), 23.96 (2C, d, JP,C = 6.0 Hz, CH3), 23.92 ppm (2C, d, JP,C = 6.7 Hz, CH3): d = 22.58 (C3’’), 31.18 (C2’’), 38.90 (C1’’), 43.21 (C1’), 66.02 (d, JP,C = 167.8 Hz, P-C), 71.19 (d, JP,C = 10.5 Hz, C2’), 71.13 (C, d, JP,C = 6.7 Hz, POC), 117.38 (C5), 141.09 (C8), 150.56 (C4), 154.89 (C6), 165.51 ppm (C2); HRMS (ESI + ): m/z [M + H] + calcd for C18H33N5O4P: 414.2265, found: 414.2263. 6-Amino-2-(sec-butyl)-9-{2-[(disopropoxyphosphoryl)methoxy]ethyl}-9H-purine (9 f). Prepared from compound 8 by Method C (reaction time 1.5 h), yield 0.32 g (77 %). ESIMS m/z (%): 414 (100) [M + H] + ; 1H NMR (400 MHz, CDCl3): d = 0.86 (t, J = 7.4 Hz, 3 H, H4’’), 1.26 (d, J = 6.2 Hz, 6 H, CH3), 1.29 (d, J = 6.2 Hz, 6 H, CH3), 1.28 (d, 3 H, H-1’’), 1.60 and 1.86 2x (m, 1 H, H-3’’), 2.77–2.85 (m, 1 H, H2’’), 3.72 (d, J = 8.3 Hz, 2 H, P-CH2), 3.94 (t, J = 5.0 Hz, 2 H, O-CH2), 4.38 (t, J = 5.0 Hz, 2 H, N-CH2), 4.68–4.72 (m, 2 H, CH-iPr), 5.93 (br s, ChemMedChem 2015, 10, 1351 – 1364

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2 H, NH2), 7.92 ppm (s, 1 H, H-8); 13C NMR (100 MHz, CDCl3): d = 12.09 (C4’’), 19.68 (C1’’), 23.96 (2C, d, JP,C = 6.6 Hz, CH3), 24.00 (2C, d, JP,C = 6.0 Hz, CH3), 29.24 (C3’’), 43.27 (C1’), 44.32 (C2’’), 66.06 (d, JP,C = 167.8 Hz, P-C), 71.15 (d, JP,C = 10.6 Hz, C2’), 71.15 (C, d, JP,C = 6.6 Hz, POC), 117.43 (C5), 141.37 (C8), 150.63 (C4), 154.59 (C6), 168.82 ppm (C2); HRMS (ESI + ): m/z [M + H] + calcd for C18H33N5O4P: 414.2265, found: 414.2264. 6-Amino-2-isobutyl-9-{2-[(disopropoxyphosphoryl)methoxy]ethyl}-9H-purine (9 g). A solution of tert-butylzinc bromide in THF (0.5 m, 10 mL, 5 mmol) was added to the mixture of 8 (0.48 g, 1.0 mmol), 2,6-di-tert-butyl-4-methylphenol (BHT) (0.22 g, 1 mmol), and Pd(dppf)Cl2·CH2Cl2 (0.04 g) in N-methylpyrrolidone (50 mL) under Ar at 80 8C. The reaction mixture was stirred at 80 8C for 4 h and cooled to room temperature. Saturated NH4Cl solution (100 mL) was added to the reaction mixture and stirred for 15 min. The reaction mixture was poured into EtOAc (150 mL). The organic layer was washed with saturated solution of EDTA (30 mL), brine (30 mL), and dried over Na2SO4. Volatiles were removed in vacuo, and the residue was purified by flash chromatography (MeOH/ CHCl3, 3:97) to give 0.21 g (49 %) of 9 g as a pale-yellow solid. ESIMS m/z (%): 414 (100) [M + H] + ; 1H NMR (400 MHz, CDCl3): d = 0.95 (d, J = 6.6 Hz, 6 H, CH3-iBu), 1.25 (d, J = 6.2 Hz, 6 H, CH3), 1.29 (d, J = 6.2 Hz, 6 H, CH3), 2.22–2.29 (m, 1 H, CH-1’’), 2.67 (d, J = 7.3 Hz, 2 H, C1’’), 3.72 (d, J = 8.3 Hz, 2 H, P-CH2), 3.93 (t, J = 4.9 Hz, 2 H, NCH2), 4.37 (t, J = 4.9 Hz, 2 H, O-CH2), 4.64–4.74 (m, 2 H, CH-iPr), 5.97 (s, 2 H, NH2), 7.90 ppm (s, 1 H, H-8); 13C NMR (100 MHz, CDCl3): d = 22.36 (2CCH3), 23.95 (2C, d, JP,C = 6.5 Hz, CH3), 24.00 (2C, d, JP,C = 5.8 Hz, CH3), 28.52 (C2’’), 43.31 (C1’), 47.64 (C1’’), 66.08 (d, JP,C = 167.8 Hz, P-C), 71.15 (2C, d, JP,C = 6.9 Hz, POC), 71.18 (d, JP,C = 10.1 Hz, C2’), 117.43 (C5), 141.38 (C8), 150.54 (C4), 154.50 (C6), 164.33 ppm (C2); HRMS (ESI + ): m/z [M + Na] + calcd for C18H32N5O4PNa: 436.2084, found: 436.2082. 6-Amino-2-(pentan-3-yl)-9-{2-[(disopropoxyphosphoryl)methoxy]ethyl}-9H-purine (9 h). Prepared from 8 by Method A, (reaction time 4.5 h), yield 0.33 g (78 %). ESIMS m/z (%): 428 (100) [M + H] + ; 1 H NMR (400 MHz, CDCl3): d = 0.81 (t, J = 7.4 Hz, 6 H, H-3’’), 1.26 (d, J = 6.2 Hz, 6 H, CH3), 1.29 (d, J = 6.2 Hz, 6 H, CH3), 1.61–1.87 (m, 4 H, H-2’’-CH2), 2.55–1.65 (m, 1 H, H-1’’), 3.72 (d, J = 8.3 Hz, 2 H, P-CH2), 3.94 (t, J = 5.0 Hz, 2 H, O-CH2), 4.38 (t, J = 5.0 Hz, 2 H, N-CH2), 4.65– 4.74 (m, 2 H, CH-iPr), 5.96 (br s, 2 H, NH2), 7.90 ppm (s, 1 H, H-8); 13 C NMR (100 MHz, CDCl3): d = 12.09 (C3’’-CH3), 23.96 (2C, d, JP,C = 6.9 Hz, CH3), 24.00 (2C, d, JP,C = 6.2 Hz, CH3), 27.50 (C2’’-CH2), 43.30 (C1’), 51.79 (C1’’), 66.08 (d, JP,C = 167.9 Hz, P-C), 71.15 (C, d, JP,C = 6.4 Hz, POC), 71.16 (d, JP,C = 10.9 Hz, C2’), 117.52 (C5), 141.34 (C8), 150.59 (C4), 154.48 (C6), 168.83 ppm (C2); HRMS (ESI + ): m/z [M + H] + calcd for C19H35N5O4P: 428.2421, found: 428.2419. 6-Amino-2-(tetrahydrofuran-2-yl)-9-{2-[(disopropoxyphosphoryl)methoxy]ethyl}-9H-purine (9 i). A solution of 1-adamantylzinc bromide in THF (0.5 m, 10 mL, 5 mmol) was added to the mixture of 8 (0.48 g, 1.0 mmol) and Pd(dppf)Cl2·CH2Cl2 (0.04 g) in THF under Ar at ¢40 8C. The reaction mixture was allowed to warm to room temperature and stirred for 2 h at room temperature. Saturated NH4Cl solution (100 mL) was added to the reaction mixture and stirred for 15 min. The reaction was poured into EtOAc (150 mL) and the organic layer was washed with saturated solution of EDTA (30 mL), brine (30 mL), and dried over Na2SO4. Volatiles were removed in vacuo and the residue was purified by flash chromatography (MeOH/CHCl3 6:94) to give 0.33 g (78 %) of 9 i as a white solid. ESIMS m/z (%): 450 (100) [M + Na + ]; 1H NMR (400 MHz, CDCl3): d = 1.28 (d, J = 6.2 Hz, 3 H, CH3), 1.28 (d, J = 6.2 Hz, 3 H, CH3), 1.31 (d, J = 6.2 Hz, 6 H, CH3), 1.98–2.07 (m, 2 H, H-4’’), 2.14–2.35 (m, 2 H, H3’’), 3.74 (d, J = 8.4 Hz, 2 H, P-CH2), 3.94 (t, J = 5.0 Hz, 2 H, O-CH2),

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Full Papers 3.97–4.24 (m, 1 H, H-5’’), 4.43 (m, 2 H, N-CH2), 4.67–4.76 (m, 2 H, CHiPr), 4.95 (dd, 1 H, J = 7.2 Hz, J = 6.4 Hz, H-3’’), 6.64 (br s, 2 H, NH2), 8.21 ppm (s, 1 H, H-8); 13C NMR (100 MHz, CDCl3): d = 23.95–24.03 (m, CH3iPr), 25.68 (C4’’), 32.16 (C3’’), 43.68 (C1’), 66.04 (d, JP,C = 168.2 Hz, P-C), 69.39 (C5’’), 70.86 (d, JP,C = 10.4 Hz, C2’), 71.35 (2C, d, JP,C = 6.6 Hz, POC), 80.97 (C2’’), 116.73 (C5), 142.66 (C8), 150.27 (C4), 154.35 (C6), 164.30 ppm (C2); HRMS (ESI + ): m/z [M + H] + calcd for C18H31N5O5P: 428.2057, found: 428.2057. 6-Amino-2-cyclopentyl-9-{2-[(disopropoxyphosphoryl)methoxy]ethyl}-9H-purine (9 j). Prepared from 8 by Method C (reaction time 1.5 h), yield 0.35 g (82 %). ESIMS m/z (%): 426 (100) [M + H] + ; 1 H NMR (400 MHz, CDCl3): d = 1.26 (d, J = 6.2 Hz, 6 H, CH3), 1.30 (d, J = 6.2 Hz, 6 H, CH3), 1.60–1.72 (m, 2 H, CH2), 1.80 (m, 2 H, (m, 2 H, CH2), 1.86–1.95 (m, 2 H, CH2), 1.98–2.08 (m, 2 H, CH2), 3.18 (pent, J = 8.3 Hz, 1 H, H-1’), 3.73 (d, J = 8.3 Hz, 2 H, P-CH2), 3.94 (t, J = 5.0 Hz, 2 H, N-CH2), 4.39 (t, J = 5.0 Hz, 2 H, O-CH2), 4.66–4.74 (m, 2 H, CHiPr), 6.26 (s, 2 H, NH2), 8.05 ppm (s, 1 H, H-8); 13C NMR (100 MHz, CDCl3): d = 23.94 (2C, d, JP,C = 5.6 Hz, CH3), 24.01 (2C, d, JP,C = 4.9 Hz, CH3), 32.87 (C2’’, C3’’), 43.45 (C1’), 48.19 (C1’’), 66.07 (d, JP,C = 167.9 Hz, P-C), 70.95 (d, JP,C = 10.4 Hz, C2’), 71.25 (2C, d, JP,C = 6.7 Hz, POC), 116.64 (C5), 141.76 (C8), 150.57 (C4), 154.40 (C6), 168.67 ppm (C2); HRMS (ESI + ): m/z [M + H] + calcd for C19H33N5O4P: 426.2265, found: 426.2263. 6-Amino-2-(bicyclo[2.2.1]heptan-2-yl)-9-{2-[(disopropoxyphosphoryl)methoxy]ethyl}-9H-purine (9 k). Prepared from 8 by Method C (reaction time 1.5 h), yield 0.31 g (69 %). ESIMS m/z (%): 452 (100) [M + H] + ; 1H NMR (400 MHz, CDCl3): d = 1.09 (dm, J = 9.2 Hz, 1 H, H-7b’’-CH2), 1.25 (d, J = 6.2 Hz, 6 H, CH3), 1.29 (d, J = 6.2 Hz, 6 H, CH3), 1.23 (m, 1 H, endo H-5’’), 1.32 (m, 1 H, endo H-6’’), 1.46–1.55 (m, 3 H, endo H-3’’, exo H-5’’, exo H-6’’), 1.72 (dm, J = 9.2 Hz, 1 H, H7a’’-CH2), 2.15–2.18 (m, 1 H, exo H-3’’), 2.28 (m, 1 H, H-4’’), 2.83–2.87 (m, 1 H, H-1’’), 3.72 (d, J = 8.3 Hz, 2 H, P-CH2), 3.94 (t, J = 5.0 Hz, 2 H, O-CH2), 4.34 (t, J = 5.0 Hz, 2 H, N-CH2), 4.60–4.76 (m, CHiPr), 5.84 (br s, 2 H, NH2), 7.87 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 23.91 (2C, d, JP,C = 6.9 Hz, CH3), 23.97 (2C, d, JP,C = 5.7 Hz, CH3), 29.10 (C5’’), 30.16 (C6’’), 35.97 and 36.18 (C3’’, C7’’), 36.58 (C4’’), 42.87 (C1’), 42.95 (C1’’), 50.09 (C2’’), 66.06 (d, JP,C = 167.8 Hz, P-C), 71.13 (d, JP,C = 9.9 Hz, C2’), 71.17 (2C, d, JP,C = 7.2 Hz, POC), 117.34 (C5), 141.16 (C8), 150.49 (C4), 154.52 (C6), 168.15 ppm (C2); HRMS (ESI + ): m/z [M + H] + calcd for C21H35N5O4P: 452.2421, found: 452.2419. 6-Amino-2-cyclopropylamino-9-{2-[(disopropoxyphosphoryl)methoxy]ethyl}-9H-purine (9 l). Prepared from 8 (0.60 g, 1.24 mmol) according to lit.[31] yield 0.30 g (59 %). ESIMS m/z (%): 413 (100) [M + H] + ; 1H NMR (400 MHz, CDCl3): d = 0.57–0.46 (m, 2 H) and 0.82–0.69 (m, 2 H, CH2-cypr), 1.26 (d, J = 6.2 Hz, 6 H, CH3), 1.29 (d, J = 6.2 Hz, 6 H, CH3), 2.79–2.72 (m, 1 H, CH-1’’), 3.73 (d, J = 8.4 Hz, 2 H, P-CH2), 3.92 (t, J = 5.0 Hz, 2 H, N-CH2), 4.24 (t, J = 5.0 Hz, 2 H, OCH2), 4.67–4.71 (m, 2 H, CH-iPr), 5.24 (s, 1 H, NH), 5.73 (s, 2 H, NH2), 7.64 ppm (s, 1 H, H-8); 13C NMR (100 MHz, CDCl3): d = 7.32 (2Ccypr), 23.95 (2C, d, JP,C = 7.9 Hz, CH3), 23.99 (2C, d, JP,C = 7.2 Hz, CH3), 24.19 (C1’’), 42.92 (C1’), 66.03 (d, JP,C = 167.8 Hz, P-C), 71.17 (d, JP,C = 10.7 Hz, C2’), 71.13 (2C, d, JP,C = 6.8 Hz, POC), 113.85 (C5), 138.83 (C8), 152.11 (C4), 155.05 (C6), 159.70 ppm (C2); HRMS (ESI + ): m/z [M + H] + calcd for C17H30N6O4P: 413.2061, found: 413.2062.

General procedure for preparation of the bisamidate prodrugs 5 Method D: Diester 9 (1 mmol) was dissolved in dry pyridine (10 mL), and TMSBr (1 mL) was added. The reaction mixture was ChemMedChem 2015, 10, 1351 – 1364

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stirred at room temperature overnight. After evaporation of the volatiles and co-distillation with dry pyridine (20 mL) in vacuo (without any contact with air), the flask was purged with Ar and amino acid ester hydrochloride (4 mmol, dried in vacuo at 30 8C and 0.1 mbar for 1 day), dry triethylamine (2 mL) and dry pyridine (8 mL) were added, and the mixture was heated at 70 8C to obtain a homogenous solution. Then, a solution of 2,2’-dipyridyldisulfide (6 mmol) and triphenylphosphine (6 mmol) in dry pyridine (10 mL) under Ar was added. The resulting mixture was heated at 70 8C. After cooling, the solvent was removed in vacuo, and the residue was purified by flash chromatography (2 % MeOH in a mixture of hexane/EtOAc, 60:40) to remove impurities, followed by hexane/ EtOAc 60:40 and gradient of MeOH (2–30 %) to obtain the desired product. Subsequently, polar impurities were removed by reversedphase chromatography on C18 silica gel (aqueous MeOH 1–100 %). The products were lyophilized from dioxane. Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2methyl-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 a). Starting compound 9 a (0.11 g, 0.40 mmol), Method D, reaction time 48 h, yield 0.18 g (70 %) of 5 a. ESIMS m/z (%): 688 (100) [M + Na] + ; 1 H NMR (500 MHz, [D6]DMSO): d = 1.01 (d, J = 6.3 Hz, 3 H, CH3), 1.06 (d, J = 6.3 Hz, 3 H, CH3), 1.11 (d, J = 6.3 Hz, 3 H, CH3), 1.16 (d, J = 6.3 Hz, 3 H, CH3), 2.71–2.90 (m, 4 H, CH2-Bn), 3.24 (dd, J = 8.1 Hz, J = 13.3 Hz, 1 H, Pb-CH2), 3.31 (dd, J = 7.8 Hz, J = 13.3 Hz, 1 H, Pa-CH2), 3.68 (t, J = 5.3 Hz, 2 H, O-CH2), 3.84–3.98 (m, 2 H, NH-CH), 4.11 (dd, J = 10.6 Hz, J = 12.1 Hz, 1 H, NH), 4.21 (t, J = 5.3 Hz, 2 H, N-CH2), 4.42 (dd, J = 10.8 Hz, J = 12.0 Hz, 1 H, NH), 4.78–4.82 (sept, J = 6.3 Hz, CHiPr), 7.04 (bs, 2 H, NH2), 7.09–7.26 (m, 10 H, H-arom), 8.01 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 21.45, 21.52, 21.58 and 21.65 (CH3-iPr), 25.65 (C1’’), 40.17 (CH2-Bn), 42.45 (C1’), 54.02 and 54.21 (NH-CH), 67.51 (d, JP,C = 135.3 Hz, P-C), 67.97 and 68.10 (2C, POC), 70.47 (d, JP,C = 11.3 Hz, C2’), 116.90 (C5), 126.57 and 126.63 (C4’’ arom), 128.20 and 128.24 (C3’’ arom), 129.62 (C2’’ arom), 137.18 and 137.26 (C1’’ arom), 140.74 (C8), 150.45 (C4), 155.75 (C6), 161.11 (C2), 172.30–172.44 ppm (m, COO); Anal. calcd for C33H44N7O6P·5/4 H2O: C (57.59), H (6.81), N (14.15), P (4.50), found: C (57.94), H (6.73), N (13.70), P (4.30). Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2ethyl-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 b). Starting compound 9 b (0.50 g, 1.3 mmol), Method D, reaction time 72 h, yield 0.49 g (55 %) of 5 b. ESIMS m/z (%): 680 (100) [M + H] + ; 1 H NMR (500 MHz, CDCl3): 1.27 (d, J = 6.3 Hz, 3 H, CH3), 1.32 (d, J = 6.3 Hz, 6 H, CH3), 1.37 (d, J = 6.3 Hz, 3 H, CH3), 1.46 (t, J = 7.6 Hz, 3 H, H-2’’), 2.86–3.20 (m, 8 H, CH2-Bn, P-CH2, H-1’’), 3.33–3.45 (m, 2 H, OCH2), 3.80–3.83 (m, 2 H, NH-CH), 4.10–4.18 (m, 1 H, NH), 4.27–4.35 (m, 1 H, NH), 4.39 (t, J = 4.9 Hz, 2 H, N-CH2), 5.03–5.15 (m, 2 H, CHiPr), 5.79 (bs, 2 H, NH2), 7.18–7.38 (m, 10 H, H-arom), 7.91 ppm (s, 1 H, H-8); 13C NMR (125 MHz, CDCl3): d = 13.28 (C2’’), 21.62, 2x 21.72 and 21.80 (CH3-iPr), 32.58 (C1’’), 40.67 (CH2-Bn), 43.10 (C1’), 53.65 and 54.02 (NH-CH), 68.04 (d, JP,C = 135.9 Hz, P-C), 69.97 and 69.17 (2C, POC), 71.12 (d, JP,C = 12.1 Hz, C2’), 117.66 (C5), 126.86 and 126.89 (C4’’ arom), 128.33 and 128.40 (C3’’ arom), 129.60 and 129.62 (C2’’ arom), 136.30 and 136.56 (C1’’ arom), 141.02 (C8), 150.75 (C4), 155.12 (C6), 166.80 (C2), 172.37–172.61 ppm (m, COO); Anal. calcd for C34H46N7O6P·1/2 H2O: C (59.29), H (6.88), N (14.24), P (4.50), found: C (59.68), H (7.08), N (14.07), P (4.44). Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2(propyl)-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 c). Starting compound 9 c (0.26 g, 0.67 mmol), Method D, reaction time 24 h, yield 0.26 g (57 %) of 5 c. ESIMS m/z (%): 694 (100) [M + H] + ; 1 H NMR (500 MHz, [D6]DMSO): d = 0.91 (t, J = 7.4 Hz, 3 H, H-3’’-CH3), 1.01 (d, J = 6.2 Hz, 3 H, CH3), 1.07 (d, J = 6.2 Hz, 3 H, CH3), 1.11 (d, J =

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Full Papers 6.2 Hz, 3 H, CH3), 1.16 (d, J = 6.2 Hz, 3 H, CH3), 1.73 (m, 2 H, H-2’’), 2.62 (m, 2 H, H-1’’), 2.72–2.90 (m, 4 H, CH2-Bn), 3.24 (dd, J = 8.2 Hz, J = 13.3 Hz, 1 H, Pb-CH2), 3.31 (dd, J = 7.8 Hz, J = 13.3 Hz, 1 H, PbCH2), 3.69 (t, J = 5.3 Hz, 2 H, O-CH2), 3.84–3.98 (m, 2 H, NH-CH), 4.11 (dd, J = 10.6 Hz, J = 11.9 Hz, 1 H, NH), 4.21 (t, J = 5.3 Hz, 2 H, N-CH2), 4.43 (m, 1 H, NH), 4.78–4.82 (sept, J = 6.3 Hz, CH-iPr), 7.01 (br s, 2 H, NH2), 7.09–7.26 (m, 10 H, H-arom), 8.01 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 14.05 (C3’’), 21.45, 21.52, 21.58 and 21.65 (CH3-iPr), 21.92 (C2’’), 40.18 (CH2-Bn), 40.89 (C1’’), 42.47 (C1’), 54.02 and 54.21 (NH-CH), 67.49 (d, JP,C = 134.8 Hz, P-C), 67.97 and 68.10 (2C, POC), 70.39 (d, JP,C = 11.1 Hz, C2’), 117.08 (C5), 126.56 and 126.63 (C4’’ arom), 128.19 and 128.24 (C3’’ arom), 129.63 (C2’’ arom), 137.19 and 137.26 (C1’’ arom), 140.80 (C8), 150.41 (C4), 155.83 (C6), 164.42 (C2), 172.31–172.44 ppm (m, COO); Anal. calcd for C35H48N7O6P·H2O: C (59.06), H (7.06), N (13.77), P (4.35), found: C (58.89), H (7.06), N (13.40), P (4.36). Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2isopropyl-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 d). Starting compound 9 d (0.30 g, 0.75 mmol), Method D, reaction time 48 h, yield 0.36 g (69 %) of 5 d. ESIMS m/z (%): 716 (100) [M + Na] + ; 1H NMR (500 MHz, [D6]DMSO): d = 1.01 (d, J = 6.3 Hz, 3 H, CH3), 1.06 (d, J = 6.3 Hz, 3 H, CH3), 1.11 (d, J = 6.3 Hz, 3 H, CH3), 1.15 (d, J = 6.3 Hz, 3 H, CH3), 1.23 (d, J = 6.9 Hz, 6 H, CH3), 2.72–2.90 (m, 4 H, CH2-Bn), 2.93 (sept, J = 6.9 Hz, 1 H, CH), 3.25 (dd, J = 8.2 Hz, J = 13.3 Hz, 1 H, Pb-CH2), 3.32 (dd, J = 7.8 Hz, J = 13.3 Hz, 1 H, Pa-CH2), 3.70 (t, J = 5.4 Hz, 2 H, O-CH2), 3.84–3.98 (m, 2 H, NH-CH), 4.10 (dd, J = 10.6 Hz, J = 12.0 Hz, 1 H, NH), 4.22 (t, J = 5.3 Hz, 2 H, N-CH2), 4.42 (dd, J = 10.7 Hz, J = 11.9 Hz, 1 H, NH), 4.77–4.82 (sept, J = 6.3 Hz, CHiPr), 6.99 (bs, 2 H, NH2), 7.09–7.26 (m, 10 H, H-arom), 8.01 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 21.46, 21.52, 21.58 and 21.65 (CH3-iPr), 22.22 (2xC2’’), 36.96 (C1’’), 40.18 (CH2-Bn), 42.43 (C1’), 54.03 and 54.21 (NH-CH), 67.48 (d, JP,C = 135.2 Hz, P-C), 67.97 and 68.11 (2C, POC), 70.32 (d, JP,C = 11.1 Hz, C2’), 117.20 (C5), 126.56 and 126.63 (C4’’ arom), 128.19 and 128.24 (C3’’ arom), 129.63 (C2’’ arom), 137.19 and 137.26 (C1’’ arom), 140.85 (C8), 150.36 (C4), 155.91 (C6), 168.73 (C2), 172.31–172.44 ppm (m, COO); Anal. calcd for C35H48N7O6P·1/2 H2O: C (59.82), H (7.03), N (13.85), P (4.41), found: C (59.43), H (7.07), N (13.41), P (4.11). Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2butyl-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 e). Starting compound 9 e (0.36 g, 0.87 mmol), Method D, reaction time 24 h, yield 0.40 g (65 %) of 5 e. ESIMS m/z (%): 730 (100) [M + Na] + ; 1 H NMR (500 MHz, [D6]DMSO): d = 0.89 (t, J = 7.4 Hz, 3 H, H-4’’-CH3), 1.01 (d, J = 6.2 Hz, 3 H, CH3), 1.07 (d, J = 6.2 Hz, 3 H, CH3), 1.11 (d, J = 6.2 Hz, 3 H, CH3), 1.16 (d, J = 6.2 Hz, 3 H, CH3), 1.33 (m, 2 H, H-3’’), 1.69 (m, 2 H, H-2’’), 2.64 (m, 2 H, H-1’’), 2.72–2.89 (m, 4 H, CH2-Bn), 3.23 (dd, J = 8.0 Hz, J = 13.3 Hz, 1 H, Pb-CH2), 3.31 (dd, J = 7.8 Hz, J = 13.3 Hz, 1 H, Pb-CH2), 3.69 (t, J = 5.3 Hz, 2 H, O-CH2), 3.85–3.97 (m, 2 H, NH-CH), 4.10 (dd, J = 10.6 Hz, J = 12.1 Hz, 1 H, NH), 4.21 (t, J = 5.3 Hz, 2 H, N-CH2), 4.42 (dd, J = 10.8 Hz, J = 12.0 Hz, 1 H, NH), 4.77 and 4.82 (sept, J = 6.3 Hz, 2 H, CHiPr), 7.01 (bs, 2 H, NH2), 7.09– 7.26 (m, 10 H, H-arom), 8.01 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 14.06 (C4’’), 21.45, 21.52, 21.57 and 21.64 (CH3-iPr), 22.20 (C3’’), 30.89 (C2’’), 38.62 (C1’’), 40.16 (CH2-Bn), 42.46 (C1’), 54.02 and 54.20 (NH-CH), 67.48 (d, JP,C = 135.1 Hz, P-C), 67.96 and 68.09 (2C, POC), 70.37 (d, JP,C = 11.2 Hz, C2’), 117.05 (C5), 126.55 and 126.62 (C4’’ arom), 128.19 and 128.23 (C3’’ arom), 129.62 (C2’’ arom), 137.18 and 137.26 (C1’’ arom), 140.78 (C8), 150.41 (C4), 155.84 (C6), 164.62 (C2), 172.30–172.43 ppm (m, COO); Anal. calcd for C36H50N7O6P·2/3 H2O: C (60.07), H (7.15), N (13.62), P (4.30), found: C (60.10), H (6.95), N (13.46), P (4.07). ChemMedChem 2015, 10, 1351 – 1364

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Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2(sec-butyl)-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 f). Starting compound 9 f (0.63 g, 1.5 mmol), Method D, reaction time 24 h, yield 0.57 g (53 %) of 5 f. ESIMS m/z (%): 730 (100) [M + Na] + ; 1 H NMR (500 MHz, [D6]DMSO): d = 0.79 (t, J = 7.4 Hz, 3 H, H-4’’), 1.01 (d, J = 6.2 Hz, 3 H, CH3), 1.07 (d, J = 6.2 Hz, 3 H, CH3), 1.11 (d, J = 6.2 Hz, 3 H, CH3), 1.16 (d, J = 6.2 Hz, 3 H, CH3), 1.20 (d, J = 6.9 Hz, 3 H, H-1’’), 1.52–1.79 (m, 1 H, H-3’’), 2.69 (m, 1 H, H-2’’), 2.79 (m, 4 H, CH2Bn), 3.23 (dd, J = 8.1 Hz, J = 13.2 Hz, 1 H, Pb-CH2), 3.31 (m, 1 H, PaCH2), 3.69 (t, J = 5.4 Hz, 2 H, O-CH2), 3.84–3.98 (m, 2 H, NH-CH), 4.11 (dd, J = 10.6 Hz, J = 11.9 Hz, 1 H, NH), 4.21 (t, J = 5.3 Hz, 2 H, N-CH2), 4.43 (dd, J = 10.7 Hz, J = 11.9 Hz, 1 H, NH), 4.78–4.82 (m, CHiPr), 6.98 (br s, 2 H, NH2), 7.08–7.26 (m, 10 H, H-arom), 8.01 ppm (s, 1 H, H-8); 13 C NMR (125 MHz, [D6]DMSO): d = 12.31 (C4’’-CH3), 20.12 (C1’’), 21.49 (2C, d, JP,C = 8.5 Hz, CH3), 21.62 (2C, d, JP,C = 8.5 Hz, CH3), 29.08 (C3’’), 40.16 (CH2-Bn), 42.44 (C1’), 44.13 (C2’’), 54.02 and 54.21 (NHCH), 67.47 (d, JP,C = 135.3 Hz, P-C), 67.97 and 68.10 (2C, POC), 70.30 (d, JP,C = 11.1 Hz, C2’), 117.19 (C5), 126.54 and 126.62 (C4’’ arom), 128.19 and 128.23 (C3’’ arom), 129.62 (C2’’ arom), 137.19 and 137.26 (C1’’ arom), 140.82 (C8), 150.36 (C4), 155.91 (C6), 168.10 (C2), 172.30–172.43 ppm (m, COO); Anal. calcd for C36H50N7O6P·H2O: C (59.57), H (7.22), N (13.51), P (4.27), found: C (59.76), H (7.17), N (13.21), P (4.16). Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2(isobutyl)-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 g). Starting compound 9 g (0.20 g, 0.48 mmol), Method D, reaction time 24 h, yield 0.18 g (54 %) of 5 g. ESIMS m/z (%): 730 (100) [M + Na] + ; 1H NMR (500 MHz, [D6]DMSO): d = 0.89 (t, J = 6.7 Hz, 6 H, H3’’), 1.01 (d, J = 6.3 Hz, 3 H, CH3), 1.07 (d, J = 6.3 Hz, 3 H, CH3), 1.10 (d, J = 6.3 Hz, 3 H, CH3), 1.16 (d, J = 6.3 Hz, 3 H, CH3), 2.19 (m, 1 H, H2’’), 2.52 (d, J = 7.2 Hz, 2 H, H-1’’), 2.72–2.79 (m, 4 H, CH2-Bn), 3.22 (dd, J = 8.2 Hz, J = 13.3 Hz, 1 H, Pb-CH2), 3.30 (dd, J = 7.8 Hz, J = 13.3 Hz, 1 H, Pa-CH2), 3.68 (t, J = 5.3 Hz, 2 H, O-CH2), 3.84–3.98 (m, 2 H, NH-CH), 4.11 (dd, J = 10.6 Hz, J = 12.0 Hz, 1 H, NH), 4.21 (t, J = 5.3 Hz, 2 H, N-CH2), 4.43 (dd, J = 10.8 Hz, J = 12.0 Hz, 1 H, NH), 4.77– 4.82 (sept, J = 6.3 Hz, CHiPr), 7.04 (bs, 2 H, NH2), 7.08–7.26 (m, 10 H, H-arom), 8.02 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 21.45, 21.52, 21.58 and 21.65 (CH3-iPr), 22.64 (C3’’), 28.14 (C2’’), 40.16 (CH2-Bn), 42.50 (C1’), 47.94 (C1’’), 54.02 and 54.21 (NH-CH), 67.49 (d, JP,C = 135.2 Hz, P-C), 67.97 and 68.11 (2C, POC), 70.36 (d, JP,C = 11.0 Hz, C2’), 117.03 (C5), 126.55 and 126.63 (C4’’ arom), 128.19 and 128.24 (C3’’ arom), 129.63 (C2’’ arom), 137.19 and 137.26 (C1’’ arom), 140.88 (C8), 150.34 (C4), 155.67 (C6), 163.78 (C2), 172.30–172.43 ppm (m, COO); Anal. calcd for C36H50N7O6P·H2O: C (59.57), H (7.22), N (13.41), P (4.27), found: C (59.55), H (7.25), N (12.93), P (3.99). Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2(pentan-3-yl)-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 h). Starting compound 9 h (0.31 g, 0.72 mmol), Method D, reaction time 72 h, yield 0.27 g (51 %) of 5 h. ESIMS m/z (%): 722 (100) [M + H] + ; 1H NMR (500 MHz, [D6]DMSO): d = 0.75 (t, J = 7.4 Hz, 6 H, H-3’’), 1.01 (d, J = 6.3 Hz, 3 H, CH3), 1.07 (d, J = 6.3 Hz, 3 H, CH3), 1.11 (d, J = 6.3 Hz, 3 H, CH3), 1.16 (d, J = 6.3 Hz, 3 H, CH3), 1.58 and 1.73 (m, 4 H, H-2’’), 2.47 (m, 1 H, H-1’’), 2.72–2.90 (m, 4 H, CH2-Bn), 3.22 (dd, J = 8.2 Hz, J = 13.2 Hz, 1 H, Pb-CH2), 3.30 (dd, J = 7.7 Hz, J = 13.2 Hz, 1 H, Pa-CH2), 3.68 (t, J = 5.3 Hz, 2 H, O-CH2), 3.84–3.98 (m, 2 H, NH-CH), 4.11 (dd, J = 10.6 Hz, J = 12.0 Hz, 1 H, NH), 4.21 (t, J = 5.3 Hz, 2 H, NCH2), 4.44 (dd, J = 10.7 Hz, J = 12.0 Hz, 1 H, NH), 4.78–4.82 (sept, J = 6.3 Hz, CHiPr), 6.98 (bs, 2 H, NH2), 7.08–7.26 (m, 10 H, H-arom), 8.01 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 12.33 (C3’’), 21.46, 21.53, 21.58 and 21.65 (CH3-iPr), 27.45 (C2’’), 40.15 (CH2-Bn), 42.47 (C1’), 51.66 (C1’’), 54.03 and 54.22 (NH-CH), 67.47

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Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers (d, JP,C = 135.0 Hz, P-C), 67.97 and 68.11 (2C, POC), 70.28 (d, JP,C = 11.2 Hz, C2’), 117.17 (C5), 126.54 and 126.62 (C4’’ arom), 128.18 and 128.23 (C3’’ arom), 129.63 (C2’’ arom), 137.20 and 137.26 (C1’’ arom), 140.80 (C8), 150.34 (C4), 155.86 (C6), 167.15 (C2), 172.31– 172.44 ppm (m, COO); Anal. calcd for C37H52N7O6P·1/2 H2O: C (60.81), H (7.31), N (13.42), P (4.24), found: C (60.68), H (7.18), N (13.20), P (4.08). Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2(tetrahydrofuran-2-yl)-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 i). Starting compound 9 i (0.34 g, 0.78 mmol), Method D, reaction time 48 h, yield 0.16 g (29 %) of 5 i. ESIMS m/z (%): 744 (100) [M + Na] + ; 1H NMR (500 MHz, [D6]DMSO): d = 1.00 (d, J = 6.3 Hz, 3 H, CH3), 1.06 (d, J = 6.3 Hz, 3 H, CH3), 1.10 (d, J = 6.3 Hz, 3 H, CH3), 1.15 (d, J = 6.3 Hz, 3 H, CH3), 1.87 (m, 1 H, H-4b’’), 2.00–2.10 (m, 2 H, H3b’’, H-4a’’), 2.15 (m, 1 H, H-3a’’), 2.71–2.90 (m, 4 H, CH2-Bn), 3.22– 3.32 (m, 2 H, P-CH2), 3.70 (t, J = 5.3 Hz, 2 H, O-CH2), 3.77–4.02 (m, 4 H, NH-CH, C5’’), 4.12 (m, 1 H, NH), 4.23 (t, J = 5.3 Hz, 2 H, N-CH2), 4.43 (m, 1 H, NH), 4.74–4.84 (m, 3 H, C2’’, CH-iPr), 7.09–7.26 (m, 10 H, H-arom), 8.07 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 21.45, 21.52, 21.58 and 21.65 (CH3-iPr), 25.79 and 31.56 (C3’’, C4’’), 40.17 (CH2-Bn), 42.53 (C1’), 54.02 and 54.22 (NHCH), 67.46 (d, JP,C = 135.3 Hz, P-C), 67.97 and 68.10 (2C, POC), 68.22 (C5’’), 70.34 (d, JP,C = 11.2 Hz, C2’), 81.62 (C2’’), 117.75 (C5), 126.56 and 126.63 (C4’’ arom), 128.20 and 128.24 (C3’’ arom), 129.62 (C2’’ arom), 137.19 and 137.26 (C1’’ arom), 141.35 (C8), 150.11 (C4), 156.00 (C6), 164.34 (C2), 172.31–172.44 ppm (m, COO); Anal. calcd for C36H48N7O7P·4/3 H2O: C (57.98), H (6.85), N (13.15), P (4.15), found: C (58.13), H (6.84), N (12.68), P (3.89). Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2cyclopentyl-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 j). Starting compound 9 j (0.35 g, 0.81 mmol), Method D, reaction time 24 h, yield 0.41 g (70 %) of 5 j. ESIMS m/z (%): 721 (100) [M + H] + ; 1H NMR (500 MHz, [D6]DMSO): d = 1.01 (d, J = 6.3 Hz, 3 H, CH3), 1.06 (d, J = 6.3 Hz, 3 H, CH3), 1.11 (d, J = 6.3 Hz, 3 H, CH3), 1.15 (d, J = 6.3 Hz, 3 H, CH3), 1.59 and 1.74 2x (m, 2 H, H-3’’), 1.84–1.95 (m, 4 H, H-2’’), 2.72–2.89 (m, 4 H, CH2-Bn), 3.09 (p, J = 8.3 Hz, 1 H, H-1’’), 3.25 (dd, J = 8.1 Hz, J = 13.3 Hz, 1 H, Pb-CH2), 3.31 (dd, J = 7.7 Hz, J = 13.4 Hz, 1 H, Pa-CH2), 3.70 (t, J = 5.4 Hz, 2 H, O-CH2), 3.85–3.97 (m, 2 H, NH-CH), 4.10 (dd, J = 10.6 Hz, J = 12.1 Hz, 1 H, NH), 4.21 (t, J = 5.4 Hz, 2 H, N-CH2), 4.42 (dd, J = 10.7 Hz, J = 12.0 Hz, 1 H, NH), 4.77 and 4.82 (sept, J = 6.3 Hz, 2 H, CH-iPr), 7.08–7.26 (m, 10 H, H-arom), 8.01 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 21.44, 21.51, 21.57 and 21.64 (CH3-iPr), 25.66 (C3’’), 32.60 (C2’’), 40.16 (CH2-Bn), 42.43 (C1’), 54.02 and 54.19 (NH-CH), 67.47 (d, JP,C = 135.0 Hz, P-C), 67.96 and 68.09 (2C, POC), 70.31 (d, JP,C = 11.0 Hz, C2’), 117.17 (C5), 126.54 and 126.62 (C4’’ arom), 128.18 and 128.23 (C3’’ arom), 129.61 (C2’’ arom), 137.18 and 137.25 (C1’’ arom), 140.82 (C8), 150.32 (C4), 155.79 (C6), 167.53 (C2), 172.30– 172.42 ppm (m, COO); Anal. calcd for C37H50N7O6P·H2O: C (60.23), H (7.10), N (13.29), P (4.20), found: C (60.26), H (7.10), N (12.91), P (4.00). Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2(bicyclo[2.2.1]heptan-2-yl)-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 k). Starting compound 9 k (0.26 g, 0.57 mmol), Method D, reaction time 48 h, yield 0.28 g (66 %) of 5 k. ESIMS m/z (%): 746 (100) [M + H] + ; 1H NMR (500 MHz, [D6]DMSO): d = 1.03 (dm, J = 9.2 Hz, 1 H, H-7b’’-CH2), 1.01 (d, J = 6.2 Hz, 3 H, CH3), 1.06 (d, J = 6.2 Hz, 3 H, CH3), 1.10 (d, J = 6.2 Hz, 3 H, CH3), 1.15 (d, J = 6.2 Hz, 3 H, CH3), 1.23 (m, 1 H, endo H-5’’), 1.32 (m, 1 H, endo H-6’’), 1.46–1.55 (m, 3 H, endo H-3’’, exo H-5’’, exo H-6’’), 1.70 (dm, J = 9.2 Hz, 1 H, H-7a’’-CH2), 2.20 (m, 1 H, exo H-3’’), 2.28 (m, 1 H, H-4’’), 2.44 (m, 1 H, H-1’’), 2.71–2.88 (m, 5 H, H-2’’, CH2-Bn), 3.21–3.32 (m, ChemMedChem 2015, 10, 1351 – 1364

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2 H, P-CH2), 3.70 (t, J = 5.3 Hz, 2 H, O-CH2), 3.84–3.97 (m, 2 H, NHCH), 4.09 (m, 1 H, NH), 4.21 (t, J = 5.3 Hz, 2 H, N-CH2), 4.42 (m, 1 H, NH), 4.77–4.82 (sept, J = 6.3 Hz, CHiPr), 6.98 (br s, 2 H, NH2), 7.08– 7.26 (m, 10 H, H-arom), 8.00 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 21.45, 21.52, 21.58 and 21.64 (CH3-iPr), 29.01 (C5’’), 29.85 (C6’’), 35.56 and 35.64 (C3’’, C7’’), 36.17 (C4’’), 40.16 (CH2-Bn), 42.46 (C1’), 42.95 (C1’’), 49.95 (C2’’), 54.02 and 54.19 (NH-CH), 67.47 (d, JP,C = 135.1 Hz, P-C), 67.96 and 68.10 (2C, POC), 70.29 (d, JP,C = 11.2 Hz, C2’), 116.99 (C5), 126.54 and 126.62 (C4’’ arom), 128.18 and 128.23 (C3’’ arom), 129.62 (C2’’ arom), 137.18 and 137.26 (C1’’ arom), 140.84 (C8), 150.23 (C4), 155.67 (C6), 167.35 (C2), 172.29– 172.42 ppm (m, COO); Anal. calcd for C39H52N7O6P·4/3 H2O: C (60.84), H (7.16), N (12.74), P (4.02), found: C (61.22), H (7.04), N (12.37), P (3.86). Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2(cyclopropylamino)-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 l). Starting compound 9 l (0.30 g, 0.72 mmol), Method D, reaction time 24 h, yield 0.21 g (41 %) of 5 l. ESIMS m/z (%): 707 (100) [M + H] + ; 1H NMR (500 MHz, [D6]DMSO): d = 0.42 (m, 2 H, H-2’’-CH2), 0.58 (m, 2 H, H-2’’-CH2), 1.01 (d, J = 6.3 Hz, 3 H, CH3), 1.07 (d, J = 6.3 Hz, 3 H, CH3), 1.11 (d, J = 6.3 Hz, 3 H, CH3), 1.16 (d, J = 6.3 Hz, 3 H, CH3), 2.70–2.90 (m, 5 H, CH2-Bn, H-1’’), 3.24–3.31 (m, 2 H, P-CH2), 3.67 (t, J = 5.4 Hz, 2 H, O-CH2), 3.85–3.98 (m, 2 H, NH-CH), 4.07 (t, J = 5.4 Hz, 2 H, N-CH2), 4.10 (dd, J = 10.6 Hz, J = 12.1 Hz, 1 H, NH), 4.41 (dd, J = 10.8 Hz, J = 11.9 Hz, 1 H, NH), 4.78–4.82 (sept, J = 6.3 Hz, CHiPr), 6.41 (bd, J = 3.9 Hz, 2-NH), 6.60 (br s, 2 H, NH2), 7.09–7.27 (m, 10 H, H-arom), 7.68 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 6.67 (C2’’), 21.46, 21.53, 21.59 and 21.65 (CH3-iPr), 24.40 (C1’’), 40.17 (CH2-Bn), 42.10 (C1’), 54.04 and 54.18 (NH-CH), 67.54 (d, JP,C = 135.0 Hz, P-C), 67.99 and 68.12 (2C, POC), 70.38 (d, JP,C = 11.0 Hz, C2’), 113.41 (C5), 126.58 and 126.64 (C4’’ arom), 128.22 and 128.25 (C3’’ arom), 129.52 and 129.63 (C2’’ arom), 137.18 and 137.27 (C1’’ arom), 137.90 (C8), 151.76 (C4), 156.02 (C6), 160.47 (C2), 172.31–172.45 ppm (m, COO); Anal. calcd for C35H47N8O6P·H2O: C (58.00), H (6.86), N (15.40), P (4.27), found: C (58.10), H (6.80), N (14.78), P (4.13). Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(2,6-diamino9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 m). Starting compound 9 m[32] (0.48 g, 1.3 mmol), Method D, reaction time 72 h, yield 0.40 g (46 %) of 5 m. ESIMS m/z (%): 689 (100) [M + Na] + ; 1 H NMR (500 MHz, CDCl3): d = 1.26 (d, J = 6.3 Hz, 3 H, CH3), 1.30 (d, J = 6.3 Hz, 3 H, CH3), 1.32 (d, J = 6.3 Hz, 3 H, CH3), 1.36 (d, J = 6.3 Hz, 3 H, CH3), 2.89–3.18 (m, 4 H, CH2-Bn), 3.26–3.33 (m, 1 H, Pb-CH2), 3.46 (dd, J = 13.1 Hz, 8.0 Hz, 1 H, Pa-CH2), 3.52 (m, 2 H, P-CH2), 3.52 (m, 2 H, O-CH2), 3.67–3.79 (m, 2 H, NH-CH), 4.15–4.23 (m, 1 H, NH), 4.20 (t, J = 4.91 Hz, 2 H, N-CH2), 4.26–4.36 (m, 1 H, NH), 5.04–5.14 (m, 2 H, CH-iPr), 5.23 (bs, 2 H, NH2), 5.91 (bs, 2 H, NH2), 7.20–7.38 (m, 10 H, H-arom), 7.64 ppm (s, 1 H, H-8); 13C NMR (125 MHz, CDCl3): d = 21.61, 2 Õ 21.70 and 21.79 (CH3-iPr), 40.64 (CH2-Bn), 42.81 (C1’), 53.82 and 54.05 (NH-CH), 67.97 (d, JP,C = 135.9 Hz, P-C), 69.07 and 69.12 (2C, POC), 71.12 (d, JP,C = 12.3 Hz, C2’), 114.19 (C5), 126.81 and 126.84 (C4’’ arom), 128.31 and 128.36 (C3’’ arom), 129.60 and 129.64 (C2’’ arom), 136.43 and 136.59 (C1’’ arom), 138.58 (C8), 151.99 (C4), 155.98 and 160.03 (C2, C6), 172.72–172.74 ppm (m, COO); Anal. calcd for C32H43N8O6P: C (57.65), H (6.50), N (16.81), P (4.65), found: C (57.31), H (6.80), N (16.58), P (4.54). Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2fluoro-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 n). Starting compound 9 n[28] (0.44 g, 1.2 mmol), Method D, reaction time 72 h, yield 0.35 g (45 %) of 5 n. ESIMS m/z (%): 692 (100) [M + Na] + ; 1 H NMR (500 MHz, [D6]DMSO): d = 1.00 (d, J = 6.3 Hz, 3 H, CH3), 1.05 (d, J = 6.3 Hz, 3 H, CH3), 1.10 (d, J = 6.3 Hz, 3 H, CH3), 1.15 (d, J =

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Full Papers 6.3 Hz, 3 H, CH3), 2.73–2.87 (m, 4 H, CH2-Bn), 3.23 (dd, J = 8.1 Hz, J = 13.3 Hz, 1 H, Pb-CH2), 3.30 (dd, J = 7.8 Hz, J = 13.3 Hz, 1 H, Pa-CH2), 3.68 (t, J = 5.3 Hz, 2 H, O-CH2), 3.86–3.93 (m, 2 H, NH-CH), 4.14 (dd, J = 10.6 Hz, J = 12.2 Hz, 1 H, NH), 4.18 (t, J = 5.3 Hz, 2 H, N-CH2), 4.45 (dd, J = 10.8 Hz, J = 12.0 Hz, 1 H, NH), 4.76–4.81 (sept, J = 6.3 Hz, 2 H, CH-iPr), 7.09–7.27 (m, 10 H, H-arom), 7.75 (bs, 2 H, NH2), 8.08 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 21.48, 21.55, 21.61 and 21.68 (CH3-iPr), 40.10 (CH2-Bn), 42.87 (C1’), 54.06 and 54.24 (NH-CH), 67.48 (d, JP,C = 135.5 Hz, P-C), 68.01 and 68.14 (2C, POC), 70.24 (d, JP,C = 11.0 Hz, C2’), 117.13 (d, JC¢F = 4.0 Hz, C5), 126.62 and 126.68 (C4’’ arom), 128.24 and 128.29 (C3’’ arom), 129.66 (C2’’ arom), 137.22 and 137.30 (C1’’ arom), 141.78 (d, JC¢F = 2.2 Hz, C8), 151.03 (d, JC¢F = 20.3 Hz, C4), 157.75 (d, JC¢F = 21.3 Hz, C6), 158.87 (d, JC¢F = 203.2 Hz, C2), 172.37–172.48 ppm (m, COO); HRMS (ESI + ): m/z [M + Na] + calcd for C32H41FN7O6P: 692.2732, found: 692.2730. Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2chloro-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 o). Starting compound 9 o[28] (0.50 g, 1.3 mmol), Method D, reaction time 72 h, yield 0.53 g (61 %) of 5 o. ESIMS m/z (%): 708 (100) [M + Na] + ; 1 H NMR (500 MHz, CDCl3): d = 1.27 (d, J = 6.3 Hz, 3 H, CH3), 1.32 (d, J = 6.3 Hz, 3 H, CH3), 1.33 (d, J = 6.3 Hz, 3 H, CH3), 1.38 (d, J = 6.3 Hz, 3 H, CH3), 2.90–3.26 (m, 6 H, CH2-Bn, NH-CH), 3.34–3.45 (m, 2 H, PCH2), 3.79 (t, J = 4.9 Hz, 2 H, O-CH2), 4.09–4.17 (m, 1 H, NH), 4.17– 4.27 (m, 1 H, NH), 4.36 (m, 2 H, N-CH2), 5.06–5.22 (m, 2 H, CH-iPr), 6.72 (bs, 2 H, NH2), 7.19–7.41 (m, 10 H, H-arom), 7.94 ppm (s, 1 H, H8); 13C NMR (125 MHz, CDCl3): d = 21.61, 21.70, 21.72 and 21.80 (CH3-iPr), 40.59 (CH2-Bn), 43.42 (C1’), 53.57 and 54.03 (NH-CH), 68.07 (d, JP,C = 136.0 Hz, P-C), 69.23 and 69.32 (2C, POC), 71.00 (d, JP,C = 12.3 Hz, C2’), 118.15 (C5), 126.90 (C4’’ arom), 128.34 and 128.40 (C3’’ arom), 129.59 and 129.61 (C2’’ arom), 136.23 and 136.55 (C1’’ arom), 141.80 (C8), 150.92 (C4), 154.01 and 156.28 (C2, C6), 172.44–172.70 ppm (m, COO); Anal. calcd for C32H41ClN7O6P·1/ 2 H2O: C (55.29), H (6.09), Cl (5.10), N (14.10), P (4.46), found: C (55.59), H (5.88), Cl (4.82), N (13.96), P (4.49). Bis(l-phenylalanine isobutyl ester) prodrug of ((2-(6-amino-2oxo-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 p). Starting compound 9 p[28] (0.30 g, 1.03 mmol), Method D, reaction time 72 h, yield 0.35 g (48 %) of 5 p. ESIMS m/z (%): 696 (100) [M + H] + ; 1 H NMR (500 MHz, [D6]DMSO): [80 8C] d = 0.82–0.85 (m, 12 H, CH3), 1.74–1.86 (m, 2 H, CHiPr), 2.81–2.94 (m, 4 H, CH2-Bn), 3.38 (d, J = 7.8 Hz, 2 H, P-CH2), 3.68 (t, J = 5.4 Hz, 2 H, O-CH2), 3.75–3.79 (m, 4 H, COO-CH2), 3.90–4.20 (m, 6 H, NH, NH-CH, N-CH2), 7.08–7.27 (m, 12 H, H-arom, NH2), 7.65 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): [80 8C] d = 18.54 and 18.58 (CH3-iPr), 26.96 and 26.97 (CH-iPr), 41.91 (C1’), 53.81 and 53.88 (NH-CH), 67.57 (d, JP,C = 135.4 Hz, P-C), 70.08–70.20 (m, C2’, COOCH2), 126.18 (C4’’ arom), 127.86 (C3’’ arom), 129.08 (C2’’ arom), 136.89 and 136.98 (C1’’ arom), 139.25 (C8), 152.07 (C2), 156.00 (C4), 172.38–172.50 ppm (m, COO); HRMS (ESI + ): m/z [M + H] + calcd for C34H47N7O67P: 696.3269, found: 696.3270. Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2iodo-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 q). Starting compound 8 (1.00 g, 2.1 mmol), Method D, reaction time 24 h, yield 0.88 g (55 %) of 5 q. ESIMS m/z (%): 800 (100) [M + Na] + ; 1 H NMR (500 MHz, [D6]DMSO): d = 1.04 (d, J = 6.3 Hz, 6 H, CH3), 1.14 (d, J = 6.3 Hz, 6 H, CH3), 2.71–2.90 (m, 4 H, CH2-Bn), 3.23 (dd, J = 8.1 Hz, J = 13.3 Hz, 1 H, Pb-CH2), 3.30 (dd, J = 7.7 Hz, J = 13.3 Hz, 1 H, Pa-CH2), 3.67 (t, J = 5.3 Hz, 2 H, O-CH2), 3.84–3.97 (m, 2 H, NH-CH), 4.12 (dd, J = 10.6 Hz, J = 12.1 Hz, 1 H, NH), 4.19 (m, 2 H, N-CH2), 4.43 (dd, J = 10.7 Hz, J = 12.0 Hz, 1 H, NH), 4.77–4.82 (sept, J = 6.3 Hz CHiPr), 7.09–7.27 (m, 10 H, H-arom), 7.60 (br s, 2 H, NH2), 8.03 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 21.46 and 21.53 (2C, ChemMedChem 2015, 10, 1351 – 1364

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CH3), 21.60 and 21.66 (2C, CH3), 42.86 (C1’), 54.01 and 54.22 (NHCH), 67.47 (d, JP,C = 135.0 Hz, P-C), 67.97 and 68.10 (2C, POC), 70.17 (d, JP,C = 11.0 Hz, C2’), 118.68 (C5), 120.82 (C2), 126.58 and 126.64 (C4’’ arom), 128.21 and 128.25 (C3’’ arom), 129.62 (C2’’ arom), 137.19 and 137.26 (C1’’ arom), 141.31 (C8), 150.13 (C4), 156.02 (C6), 172.31–172.43 ppm (m, COO); Anal. calcd for C32H41IN7O6P·1/2 H2O: C (48.86), H (5.38), N (12.46), P (3.94), found: C (48.99), H (5.42), N (12.02), P (3.86). Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2(thiocyanato)-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 r). A solution of 5 q (0.28 g, 0.36 mmol) and CuSCN (0.13 g, 1.08 mmol) in DMF (10 mL) was heated at 120 8C for 8 h. The solvent was removed in vacuo, the residue was co-distilled with toluene and dissolved in EtOAc (20 mL). The solid was filtered off. The filtrate was washed with a solution that contains saturated EDTA and brine, 1:1 (3 Õ 30 mL). The organic layer was collected, evaporated, and the residue was purified by flash chromatography (MeOH/CHCl3, 3:97) to give 0.07 g (21 %) of 5 r. ESIMS m/z (%): 731 (100) [M + Na] + ; 1H NMR (500 MHz, [D6]DMSO): d = 0.99–1.16 (m, 12 H, CH3), 2.70–2.86 (m, 4 H, CH2-Bn), 3.25 (dd, J = 8.0 Hz, J = 13.2 Hz, 1 H, Pb-CH2), 3.31 (dd, J = 7.9 Hz, J = 13.3 Hz, 1 H, Pa-CH2), 3.71 (m, 2 H, O-CH2), 3.78–3.99 (m, 2 H, NH-CH), 4.12 (dd, J = 10.6 Hz, J = 12.1 Hz, 1 H, NH), 4.21 (m, 2 H, N-CH2), 4.43 (m, 1 H, NH), 4.73–4.84 (m, 2 H, CHiPr), 7.08–7.27 (m, 10 H, H-arom), 7.83 (bs, 2 H, NH2), 8.14 ppm (s, 1 H, H-8); 13C NMR (125 MHz, [D6]DMSO): d = 21.45, 21.52, 21.58 and 21.65 (CH3-iPr), 40.00 (CH2-Bn), 43.01 (C1’), 54.01 and 54.20 (NH-CH), 67.45 (m, P-C), 67.96 and 68.09 (2C, POC), 70.01 (d, JP,C = 11.3 Hz, C2’), 109.19 (CN), 117.86 (C5), 126.58 and 126.63 (C4’’ arom), 128.20 and 128.24 (C3’’ arom), 129.62 (C2’’ arom), 137.17 and 137.27 (C1’’ arom), 141.76 (C8), 150.24 (C4), 154.04 and 156.47 (C2 and C6), 172.30–172.43 ppm (m, COO); HRMS (ESI + ): m/z [M + H] + calcd for C33H42N8O6PS: 709.2680, found: 709.2679.

Biological assays ACT inhibitory activity. J774A.1 cells were seeded in a 96-well plate at 5 Õ 104 cells per well and left to attach overnight. Prior to the experiment, cells were washed with HBSS (135 mm NaCl, 5.9 mm KCl, 1.5 mm CaCl2, 1.2 mm MgCl2, 25 mm glucose, 10 mm HEPES [pH 7.4]) and pre-incubated with compounds at concentrations of 0.001–30 mm for 5 h. Cells were then exposed to ACT (0.4 mg mL¢1) from B. pertussis for 30 min. Finally, the cAMP content was determined by using the CatchPointcAMP immunoassay kit (Molecular Devices, Wokingham, UK). After the addition of lysis buffer (50 mL per well) provided by the manufacturer, the cellular content was extracted by shaking the plate at 250 rpm for 10 min. The plate was then centrifuged to remove cell debris, the supernatant was replaced to the assay plate, and immunoassays were carried out according to the manufacturer’s instructions. Fluorescence signal was acquired using an Infinite M1000 plate reader (Tecan Systems Inc., San Jose, CA, USA). Effect on the viability of J774A.1 cells. J774A.1 cells were plated onto white 96-well assay plates at 5 Õ 104 cells per well and allowed to attach overnight. Cells were then washed with HBSS and treated with compounds at 10 mm for 5 h. Cell viability was then assessed with a Cell Titer-Glo Luminescent Cell Viability assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Measurement of luminescence signal was performed by use of a GENiosmicro plate reader (Tecan Systems). Data were expressed as percent of control, represented by untreated cells.

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Full Papers In vitro long-term viability tests. The sensitivity of the cells to compound 5 n was assessed with XTT cell proliferation kit II (Roche Diagnostics GmbH, Mannheim, Germany) according to manufacturer’s instructions. The following cell lines were used: murine macrophage cell line J774A.1; two normal human cell lines, HUVEC-2 and NHDF-Ad; and four human cancer cell lines, CCRF-CEM, HL-60, HeLa-S3, HepG2. Cells were seeded in a 96-well plate at a density of 13 500 cells per well and left to rest overnight. After 24 h 5 n was added at a concentration of 10 mm to the culture media and incubated for 72 h before addition of the XTT dye. The absorbance at l 495 nm was measured after 1 h incubation with the dye. Preparation of crude macrophage extracts. J774A.1 cells (2 Õ 109) were washed with PBS and suspended in 1.5 mL Tris·HCl buffer (50 mm, pH 7.4) containing 1 mm dithiothreitol, 5 mm MgCl2, and protease inhibitor cocktail. The cells were then homogenized four times for 30 s at speed 4.0 m s¢1 in a FastPrep24 instrument. The homogenate was centrifuged at 30 000 Õ g for 30 min. The supernatant was incubated with streptomycin sulfate for 1 h to remove nucleic acids and centrifuged at 30 000 Õ g for 30 min. The resulting crude cell extract was desalted on PD-10 columns (GE Healthcare, Piscataway, NJ, USA), divided into aliquots, and stored at ¢80 8C. The protein content was determined with a QuantiPro BCA assay kit (Sigma–Aldrich) according to the manufacturer’s instructions. Assays with mACs. HEK293 cells stably expressing AC1, AC2, or AC5 were cultured and frozen as previously described.[33, 37] Cells were thawed and plated in white-bottom 384-well plates (PerkinElmer, Shelton, CT, USA). Inhibitor compounds were added to cells using a Multipette-mounted 384-well pintool and incubated at room temperature for 30 min. Then, the specific mAC stimulator (3 mm A23187 for AC1, 100 nm PMA for AC2, and 300 nm forskolin for AC5) in 500 mm 3-isobutyl-1-methyxanthine was added to the cells. Cells were incubated at room temperature for 1 h, and cAMP accumulation was measured using Cisbio’s dynamic 2 kit (Cisbio Bioassays, Bedford, MA, USA) according to the manufacturer’s instructions. Hydrolysis of bis(POM)PMEA (2) and bisamidate prodrugs 3, 4, and 5 n in macrophage homogenate. A total of 70 mL of cellular extract (5.5 mg mL¢1 of total protein) was mixed with 30 mL reaction buffer (1 mm dithiothreitol, 5 mm MgCl2, 0.4 mm EDTA, 20 mm Tris·HCl [pH 7.5]) and incubated for various time intervals (2–18 h) at 37 8C in the presence of the corresponding prodrug (100 mm). Simultaneously, prodrugs were incubated in reaction buffer only serving as negative (no enzyme) controls. Finally, samples were deproteinized by methanol precipitation and analyzed by ion-pairing reversedphase high-pressure liquid chromatography using a Supelco Discovery C8 HPLC column (150 Õ 4.6 mm [i.d.: 5 mm]) under a gradient of 50!100 mm CH3COONH4 (pH 4.7) and acetonitrile. The flow rate was 0.9 mL min¢1. The absorption spectra of eluted compounds were recorded by a photodiode array detector (PDA) at l 260 nm. The retention times under the conditions used were as follows: 5 n, 18.7 min; bis(POM)PMEA, 14.7 min; MIC-768, 16.6 min; MIC-747, 11.4 min; PMEA-F, 7.9 min; PMEA, 6.7 min. The concentrations of PMEA and intact prodrugs in the samples were calculated from the peak areas by using calibration curves generated from intact prodrug standards. The concentrations of the PMEA-based intermediate metabolites were calculated using the intact prodrug calibration curve. Blank samples were obtained from untreated macrophage homogenate. No interfering peaks were observed, and thus selectivity was ensured.

were housed under a 12 h light/dark cycle at 22 8C. They were fasted for 16 h before experiments, with free access to water. Plasma levels of tested compounds and their metabolites were analyzed after i.v. administration of parent compounds into vena saphena. Animals were anesthetized with pentobarbital (55 mg kg¢1 i.p.). A silastic cannula was inserted into the carotid artery and a blank sample of blood was taken. Tested compounds were then administered as bolus dose into the saphenous vein (5 n and 4, 10 mg kg¢1, or 25 mg kg¢1 for 3 dissolved in DMSO). Blood samples (0.4 mL) were collected from the carotid artery into heparinized tubes at 5, 10, 20, 45, 60, and 120 min post-administration. Between each blood sample, the cannula was filled with heparinized 0.9 % saline. After the experiments, liver, kidney, and lung were sampled and weighed. The blood samples were centrifuged and the plasma separated. All samples were stored at ¢70 8C until analysis. The organs were cut into pieces and homogenized using a Dounce tissue grinder in 50 mm Tris·HCl buffer (pH 7.4) containing 1 mm dithiothreitol, 5 mm MgCl2, and protease inhibitor cocktail. Finally, all blood and organ samples were deproteinized by methanol precipitation and analyzed by HPLC, which was performed as described above for hydrolysis in macrophage homogenate. Research involving animals was performed in accordance with the respective Czech laws concerning animal protection. The experimental protocol was approved by the Ethical Committee of the Pharmaceutical Faculty, Charles University in Prague, Czech Republic as well as by the Ministry of Education, Youth and Sport, Czech Republic.

Acknowledgements The study was supported by the Ministry of Interior of the Czech Republic (VG20102015046) and Gilead Sciences (Foster City, CA, USA). Keywords: adenylate cyclase toxin · bisamidates · Bordetella pertussis · nucleosides · phosphonates · PMEA · prodrugs

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Bisamidate Prodrugs of 2-Substituted 9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA, adefovir) as Selective Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis.

Novel small-molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health thre...
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