The DiPPro approach: synthesis, hydrolysis, and antiviral activity of lipophilic d4T diphosphate prodrugs.

CHEMMEDCHEM FULL PAPERS DOI: 10.1002/cmdc.201300500

The DiPPro Approach: Synthesis, Hydrolysis, and Antiviral Activity of Lipophilic d4T Diphosphate Prodrugs Tilmann Schulz,[a] Jan Balzarini,[b] and Chris Meier*[a] Dedicated to Prof. Dr. Joachim Engels on the occasion of his 70th birthday

Bioreversible protection of the b-phosphate group of nucleoside diphosphates (NDPs) as bis(acyloxybenzyl)phosphate esters is presented. To investigate the structure–activity relationship of these potential NDP prodrugs (DiPPro drugs) a series of DiPPro compounds was synthesized bearing fatty acids of various lengths and d4T as a model nucleoside. For synthesis of the lipophilically modified diphosphate group, preformed phosphoramidites were allowed to react with nucleotides, and the b-PIII moiety was subsequently oxidized. The chemical and enzymatic stability of these prodrugs was studied in different media such as phosphate buffer (pH 7.3) or CEM cell extracts. In all media, the hydrolysis rate was clearly

dependent on the acyl moiety and decreased with increasing alkyl chain length. The compounds showed a markedly lower half-life in cell extracts than in pH 7.3 phosphate buffer due to the presence of enzyme-catalyzed cleavage. In all media, the DiPPro compounds released d4T diphosphate (d4TDP) as the main product beside d4TMP. In antiviral assays, the compounds proved to be at least as potent as d4T against HIV-1 and 2 in wild-type CEM/0 cells. As a proof of concept, compounds with longer acyl residues showed very good anti-HIV activities in thymidine-kinase-deficient cells (CEM/TK), indicating their ability to penetrate cell membranes and the delivery of phosphorylated metabolites.

Introduction Nucleoside analogues are commonly used as antiviral and antitumor agents and are the backbone of current anti-HIV therapies.[1–3] The antiviral efficacy of many nucleoside analogues, such as 3’-deoxy-2’,3’-dehydrothymidine (d4T, 1, Scheme 1) and 3’-deoxy-3’-azidothymidine (AZT), depends on their conversion into the ultimately bioactive nucleoside triphosphates (NTPs) via the mono- and diphosphate forms by cellular kinases. However, cellular kinases often insufficiently catalyze the phosphorylation of nucleoside analogues. In the case of d4T 1, the metabolism-limiting step in human cells is the initial phosphorylation event catalyzed by thymidine kinase (TK).[1, 3] If this is the case, antiviral activity can be enhanced by using prodrugs of the phosphorylated parent nucleosides (masked nucleoside monophosphates; NMPs) that are able to penetrate cell membranes and deliver the nucleotide intracellularly, thus bypassing the limiting step and leading to increased intracellular levels of the active metabolites. In some cases drug resistance could be overcome, and the application spectrum of nucleosides was extended to cover different viruses.[4–7] Several

[a] Dr. T. Schulz, Prof. Dr. C. Meier Organic Chemistry, Department of Chemistry Faculty of Sciences, University of Hamburg Martin-Luther-King-Platz 6, 20146 Hamburg (Germany) E-mail: [email protected] [b] Prof. Dr. J. Balzarini Rega Institute for Medical Research Katholieke Universiteit Leuven (KU Leuven) Minderbroedersstraat 10, 3000 Leuven (Belgium) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201300500.

2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Scheme 1. Reagents and conditions: Method A: 1) N,N-diisopropylamine, Et2O, 20 8C, 2 h. Method B: 1) THF, TEA, 78 8C, 1–2 h; 2) RT, 16–24 h. Method C: 1) CH3CN, DCI, RT; 2) tBuOOH, 25 8C, 15 min; 3) RP-18 chromatography.

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NMP prodrugs[8] have been designed over the past 20 years, tion at the pyrophosphate unit is necessary for delivery. In our for example, cycloSal technology.[9] In contrast, it was shown work we focused on the use of esterases/lipases for intracelluthat the phosphorylation of AZT by cellular TK-1 proceeded eflar activation because of their high intracellular concentration. ficiently, but the second step catalyzed by thymidylate kinase The DiPPro approach is based on two identical acceptor-substi(TMPK) was metabolism-limiting, which led to an intracellular tuted benzyl esters linked to the b-phosphate group of the nuaccumulation of AZT monophosphate (AZTMP).[10, 11] Beside cleoside diphosphate (Figure 1). The a-phosphate remained anemia and neutropenia, the latter is the main cause of the unmasked, because a negatively charged a-phosphate led to severe side effects associated with AZT treatment. Additionally, a considerable stabilization of the pyrophosphate unit. Upon AZTMP interferes with lipid glycosylation.[12–14] Therefore, proenzymatic cleavage of the phenolic acyl ester, an Umpolung took place, which converted the acceptor group (ester) into drug systems that deliver nucleoside diphosphates (NDPs) into a strong donor substituent (hydroxy group). As a consequence, intact cells may overcome metabolic hurdles and potential spontaneous cleavage of the benzyl bond occurred in a 1,6side effects caused by the parent nucleoside or its monophoselimination reaction resulting in the formation of monophate. masked acyloxybenzyl-NDP 3. Repetition of this process reOnly very few attempts to design NDP prodrugs have been leased the NDP.[23–25] published so far. Nucleoside pyrophosphate diesters based on glycerides have been reported by Hostetler and co-workers; However, DiPPro-d4TDP compounds 4 a,b with short or however, these compounds do not serve as diphosphate probranched acyl moieties (R) did not show antiviral activity in TKdrugs, as they released the corresponding NMPs due to cleavdeficient CEM cells (CEM/TK).[22] A possible explanation for [15–18] age of the pyrophosphate group. A second approach was this lack of activity is that the remaining polarity of these compublished by Dinh and co-workers,[19] in which an acyl group pounds due to the negatively charged a-phosphate is still too was attached to the b-phosphate of the pyrophosphate, formhigh and prevents effective uptake into cells. One option to ing a mixed anhydride. The concept was based on a rapid and solve this problem is to increase the lipophilicity of the DiPPro selective cleavage of the mixed anhydride rather than cleavage compounds. To achieve this, longer chains in the acyl moieties of the phosphate anhydride bond.[20] The idea was proven in were investigated. Lipophilic aliphatic esters have been extenhydrolysis studies in aqueous buffer. However, no differences sively used for prodrugs and are known to be taken up by the in antiviral activity between the investigated nucleotide dimononuclear phagocyte system (MPS). Besides CD4 + T-lymphocytes, cells of the MPS play an important role in the pathophosphate–carboxylic acids and the parent nucleosides were genesis of AIDS and are considered as reservoirs for HIV.[26–28] observed.[21] The reason for difficulties in the development of lipophilic Long aliphatic acid esters are cleaved by esterases/lipases and NDP prodrugs is due to chemical instability of the pyrophostherefore may enable an efficient intracellular release of the phate bond, because this bond is energetically rich (as eviNDP. denced by the ubiquitous use of ATP as “fuel” for metabolic Herein we report the synthesis, hydrolysis properties, and processes). However, this bond is kinetically stable due to the structure–activity relationships in the antiviral evaluation of lipnegative charges that prevent cleavage by nucleophiles. Thus, ophilic DiPPro-d4TDPs 4 bearing various fatty acyl moieties (R). if the charged oxygen atoms of the NDPs are masked/esterified The nucleoside analogue d4T was used in this study because and therefore neutralized, a nucleophilic attack at the a- or bphosphate groups is possible, and the chemical hydrolysis of the anhydride bond is significantly increased. We have reported on a new concept of lipophilic masking of nucleoside diphosphates shown in bis(acyloxybenzyl)nucleoside diphosphates 2 (DiPPro compounds; Figure 1) endowed with high chemical stability but rapid and efficient conversion into the corresponding NDP in CEM cell extracts.[22] In this approach the release of NDP is driven chemically or enzymatically by cleavage of the phenyl ester moiety within the masking group that initiates a spontaneous second chemical reaction. Thus, no chemical reac- Figure 1. General structure and proposed hydrolysis pathway of DiPPro compounds 2. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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d4T is well known as a model system for nucleoside monophosphate prodrug systems. For a systematic characterization, this nucleoside analogue is also suitable for the development of nucleoside diphosphate prodrugs.

Results and Discussion Chemistry For the synthesis of DiPPro-d4TDP prodrugs 4 a–j a convergent strategy was chosen to build the pyrophosphate moiety, as reported earlier.[22] Briefly, d4T monophosphate (d4TMP) 6 was coupled with phosphoramidites 7 containing the lipophilic acyl-modified bis(acyloxybenzyl) masking groups. The intermediately formed PIII–PV intermediate was then oxidized to give the pyrophosphate moiety (Scheme 1). D4TMP 6, needed as starting material, was prepared by the method of Sowa and Ouchi.[29] The obtained d4TMP was protonated using a Dowex 50WX8 (H + form), column and was titrated with tetra-n-butylammonium hydroxide solution and freeze-dried. The di(nBu4N) + salt of d4TMP 6 was highly hygroscopic. Consequently, this compound was rigorously dried prior to reaction. A reliable procedure for the removal of traces of water was to store d4TMP 6 in an acetonitrile solution over molecular sieves (3 ) directly after the freeze-drying procedure. For the reactions, aliquots containing the calculated amount of 6 were taken, and the solvent was removed in vacuum. After co-evaporating, the material was kept under an inert gas atmosphere as a paleyellow foam. The preparation of bis(4-acyloxybenzyl)-N,N-diisopropylphosphoramidites 7 a–j was carried out by reaction of PIII reagent 8 with the corresponding benzyl alcohols 9 in the presence of triethylamine. The formed triethylamine hydrochloride was removed by filtration, and the crude materials were purified by preparative thin-layer chromatography on a chromatotron. Phosphoramidites 7 were obtained in high yields (Table 1). Next, dried d4TMP 6 was dissolved in acetonitrile and allowed to react with 1.6–1.7 equivalents of phosphoramidites 7 in the presence of 1.7 equivalents dicyanoimidazole (DCI). In some cases, 0.2–0.4 equivalents of phosphoramidite 7 and DCI were added to the reaction mixture after several hours. tert-Bu-

Table 1. Phosphoramidites 7 and DiPPro-d4TDPs 4.

Compd 7 a[a] 7 b[a] 7c 7d 7 e[a] 7f 7g 7h 7i 7j

Phosphoramidites 7 R Yield [%] Me tBu C4H9 C6H13 C7H15 C9H19 C11H23 C13H27 C15H31 C17H33 (8Z)

75 67 90 49 52 56 58 34 80 16

Compd 4 a[a] 4 b[a] 4c 4d 4e 4f 4g 4h 4i 4j

DiPPro-d4TDPs 4 R Yield [%] Me tBu C4H9 C6H13 C7H15 C9H19 C11H23 C13H27 C15H31 C17H33 (8Z)

[a] Taken from ref. [22].


10 46 32 40 65 62 63 67 49 39

tylhydroperoxide was added after completion of the reaction to yield target compounds 4. The solvent was removed, and purification of the crude product was achieved by RP-18 chromatography using gradients of methanol and water. Aqueous extraction was avoided because of the amphiphilic character of the compounds. DiPPro compounds 4 were obtained in yields of 32–67 % (Scheme 1, Table 1). The amount of product was sometimes considerably decreased during the purification because of difficulties in separating the prodrugs 4 from bis(acyloxybenzyl) phosphate 10 formed as by-product. For this reason, we tried various purification methods—Sephadex LH20 column chromatography, crystallization/precipitation, preparative ion-pair HPLC—all of which led to unsatisfactory results. In the case of 4 f, the use of only 1.1 equivalents of phosphoramidite for the reaction led to a better result during chromatography and thus to an increased yield. Unfortunately, this result could not be confirmed in all cases. Finally, it could not be concluded whether corresponding phosphodiesters 10 were formed through hydrolysis and oxidation of unreacted phosphoramidites 7 or by partial hydrolysis of 4 during chromatography. In addition to DiPPro-d4TDPs 4 a–j, attempts to prepare the mono-masked acyloxybenzyl-d4TDP intermediates 5 were conducted (Figure 2). In oligonucleotide chemistry the b-cyano-

Figure 2. Proposed hydrolysis pathway for 4 under chemical conditions: Route A: release of d4TDP 13 via intermediate acyloxybenzyl (AB)-d4TDP 5; Route B: cleavage of anhydride bond. AB-d4TDP 5 a: R = Me, Me-DiPProd4TDP; 5 b: R = tBu, tBu-DiPPro-d4TDP; 5 c: R = C4H9, C4-DiPPro-d4TDP; 5 d: R = C6H13, C6-DiPPro-d4TDP; 5 e: R = C7H15, C7-DiPPro-d4TDP; 5 f: R = C9H19, C9-DiPPro-d4TDP; 5 g: R = C11H23, C11-DiPPro-d4TDP; 5 h: R = C13H27, C13DiPPro-d4TDP; 5 i: R = C15H31, C15-DiPPro-d4TDP; 5 j: R = C17H33, 8Z-C17DiPPro-d4TDP; t1/2 (1): half-life of the first step (half-life of DiPPro-d4TDP 4); t1/2 (2): half-life of the second step (half-life of acyloxybenzyl-d4TDP 5).

ethoxy group is usually used as a phosphate protecting group. Therefore, this group was also considered here as a suitable substituent which should be easily cleaved with a non-nucleophilic base such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). In a first attempt, b-cyanoethoxy-(4-decanoyloxybenzyl)-N,N-diisopropylaminophosphoramidite 12 was synthesized from commercially available b-cyanoethyl-N,N-diisopropylchlorophosphoramidite and the corresponding benzyl alcohol 9 f. After ChemMedChem 2014, 9, 762 – 775

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Scheme 2. Reagents and conditions: Method A: 1) CH3CN, DCI, RT; 2) tBuOOH, 25 8C, 15 min; 3) RP-18 chromatography. Method B: 1) CH3CN, DBU, RT, 15 min.

d4TDPs 5 (Figure 2). However, as discussed blow, d4TMP was also consistently formed in the hydrolysis studies. This means that the half-lives given are the sum of two hydrolysis processes: formation of d4TDP and d4TMP. In all cases for prodrugs 4 bearing linear alkyl residues, the amount of d4TDP was higher than the amount of d4TMP. As expected, the chemical stability of prodrugs increased with the length of the acyl moieties, except for 8Z-C17-DiPPro-d4TDP 4 j (t1/2 = 73 h). For compounds 4 b–f the half-lives of the second hydrolysis step (t1/2), reflecting the stability of the intermediate acyloxybenzyld4TDPs 5 b–f, are also listed in Table 2. These were determined after full conversion of the DiPPro-d4TDPs 4 b–f to intermediates 5 b–f. The ratio of d4TDP and d4TMP formation was determined, and values are given in Table 2. Generally, in all cases the second hydrolysis step proceeded much more slowly than the first one, which became evident by the appearance of intermediates 5 b–f. As an example, the hydrolysis of C9-DiPPro compound 4 f is shown in Figure 3. This is due to the formation of a second negative charge at the pyrophosphate unit in intermediates 5 generating repulsive interactions with the nucleophile. It was shown that tBu-, C4-, C6-, C7- and C9-DiPPro-d4TDP (4 b–f, respectively) were hydrolyzed via acyloxybenzyl intermediates 5 c–f, which were eight- to ninefold more stable than the corresponding parent compounds 4. The branched intermediate 5 b proved to be 25-fold more stable than the parent prodrug 4 b (Table 3). In contrast, only very small amounts of C11-, C13-, C15- and 8Z-C17-acyloxybenzyl intermediates 5 g–j were detected (Supporting Information Figure S4). Half-lives of these intermediates could not be determined. The high lipophilicity of prodrugs 4 g–j in the incubation media (PBS/DMSO) may result in a slow attack by nucleophiles, while the nucleophile accessed the second acyl moiety of the more polar intermediates 5 a–f more easily. One might speculate that prodrugs 4 form micelles in the incubation

RP-18 chromatography, b-cyanoethoxy-(4-decanoyloxybenzyl)d4TDP 13 was obtained as a mixture of two diastereomers in 40 % yield (Scheme 2). Unfortunately, the synthesis of (4-decanoyloxybenzyl)-d4TDP 5 f by elimination of the cyanoethyl group with DBU failed. If traces of water are present during synthesis or workup, the acyl moiety is cleaved rapidly due to the highly basic DBU; decanoylic acid, 4-hydroxybenzyl alcohol, and d4TDP 11 were then detected in the crude mixture based on 1H and 31P NMR spectroscopy. However, selective hydrolysis of C9-DiPPro-d4TDP 4 f in a 1:1 mixture of [D6]DMSO and 50 mm PBS (pH 7.3) led to (4-decanoyloxybenzyl)-d4TDP 5 f. The reaction was followed by 31P NMR spectroscopy and stopped by freeze-drying after a considerable amount of 5 f was Table 2. Hydrolysis data of DiPPro compounds 4, intermediate 5 f, and the mixed bformed. Compound 5 f was isolated by RP-18 chrocyanoethyl-C9-DiPPro compound 13. matography and used as reference compound for the HPLC experiments described below. CharacterizaPBS (pH 7.3) CE[a] [b] [c] [d] [e] Compd R t [min] 4: t [h] 5: t [h] d4TDP/d4TMP t R 1/2 (1) 1/2 (2) 1/2 (1) [h] tion of DiPPro-d4TDP prodrugs 4 a–j and CE-decanoyl1 13 [f] [g] [g] [g] oxybenzyl-d4TDP 13 was performed by H, C, and 4a C1 9.41 10 ND ND ND 0.05 31 4b tBu 11.44 55 1390 0.6 1 6.1 P NMR spectroscopy as well as high-resolution mass 4c C4 11.57 40 330 3 1 0.6 spectrometry. Chemical stability Stability studies of DiPPro-d4TDP prodrugs 4 and CEdecanoyloxybenzyl-d4TDP 13 were conducted in aqueous 25 mm phosphate buffer (PBS, pH 7.3). The hydrolysis products were detected by analytical RP18 HPLC. The chemical hydrolysis half-lives t1/2 (1) of compounds 4 a–j listed in Table 2 mainly reflect the initial hydrolysis step of DiPPro-d4TDPs 4 to acyloxybenzyl 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

4d 4e 4f 4g 4h 4i 4j 5f 13

C6 C7 C9 C11B C13 C15 8Z-C17 C9 CE

13.2 14.05 15.88 17.76 19.23 20.54 19.86 11.0 12.09

36 43 63 100 179 258 73 247 11

330 390 280 NA[h] NA[h] NA[h] NA[h] NA[h] 280

4 2 1.5 1.5 2 1.5 ND[g] NA[h] 3

1 1 1 1 1 1 ND[g] NA[h] 1

3.6 1.6 7.6 4 13 21 9.5 ND[g] 0.46

[a] 33 % CEM cell extract in PBS, pH 6.8. [b] HPLC retention time, HPLC analysis method 1. [c] Half-life of first step (4!5). [d] Half-life of second step (5!d4TDP 11). [e] Ratio of d4TDP/d4TMP after full conversion of DiPPro-d4TDPs and intermediates. [f] See ref. [22]. [g] Not determined. [h] Not applicable.

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Figure 4. Hydrolysis of C4-DiPPro-d4TDP 4 c (^, exponential fit), formation and hydrolysis of intermediate acyloxybenzyl-d4TDP 5 c (&, manual), formation of d4TMP (*, manual) and d4TDP (~, manual) in PBS, pH 7.3.

Figure 3. HPLC profile for C9-DiPPro-d4TDP 4 f after incubation in PBS, pH 7.3 (0–48 days). Peaks were attributed by co-injection and/or tR of reference compounds (Supporting Information Figures S1, S2, S3); HPLC analysis method A.

Table 3. Hydrolysis half-lives of C6- and C15-DiPPro-d4TDP 4 d,i and CEC9-decanoyloxybenzyl-d4TDP 13 in various biological media.

Compd

R

CE[b]

HS[c]

4d 4i 13

C6 C15 CE-C9

3.6 21 0.46

5.0 25 2.6

t1/2 (1) [h][a] RPMI/FCS[d] FCS[e] 5.5 28 1.5

5.5 70 ND

PBS (pH 6.8) 59 407 5.5

[a] Hydrolysis half-life. [b] 33 % CEM cell extract in PBS, pH 6.8; [c] 20 % human serum in PBS, pH 6.8. [d] RPMI/10 % heat-inactivated FCS. [e] 10 % FCS in PBS, pH 6.8.

media, whereas intermediates 5 are unable to form such aggregates. A further example of the chemical hydrolysis is shown below in Figure 5. While C4-DiPPro-d4TDP 4 c is hydrolyzed to C4-acyloxybenzyl-d4TDP 5 c, some concomitant cleavage to d4TMP occurred as well. C4-DiPPro-d4TDP 4 c was completely hydrolyzed at pH 7.3 within 250 h (t1/2 ~ 40 h). The main product after 151 h was intermediate 5 c and not d4TMP, indicating that formation of intermediate 5 c proceeds at a higher rate than cleavage of the anhydride bond. Additionally, the peak amount of d4TMP was observed at this point. Moreover, the formation of d4TMP was clearly dependent on the hydrolysis half-life of the parent d4TDP prodrug 4; the higher the half-life of the initial cleavage, the higher the amount of d4TMP formed. However, intermediates 5 c–f released exclusively d4TDP with half-lives of ~ 280–390 h at pH 7.3 (Table 2). This was proven by incubation of intermediate 5 f (t1/2 ~ 250 h; Figures 3, 4 and Supporting Information Figure S5). Additionally, C6-DiPPro-d4TDP 4 d was hydrolyzed in a 1:1 mixture of [D6]DMSO and 50 mm PBS (pH 7.3) to follow the reaction by 31P NMR (Supporting Information Figure S6). The majority of C6-DiPPro-d4TDP 4 d hydrolyzed to the intermediate 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

heptanoyloxybenzyl-d4TDP 5 d, yet small amounts of d4TMP 6 and phosphate diester 10 were also observed after 12 days. Formation of d4TMP and phosphate diester 10 occurred as a result of cleavage of the anhydride bond (Figure 2; pathway B), but it was clearly proven that d4TDP 11 was formed with marked selectivity via intermediate 5 d (42–68 days of hydrolysis). Hydrolysis of the mixed acyloxybenzyl-b-cyanoethyl prodrug 13 was also studied at different pH values (PBS, pH 7.3 or 8.7). This compound released d4TDP via intermediate C9-acyloxybenzyl-d4TDP 5 f, as proven by co-injection (Supporting Information Figures S7 and S8). Cleavage of the b-cyanoethyl group proceeded with a surprisingly short half-life (t1/2 (1) = 11 h; pH 7.3), followed by hydrolysis of the decanoyloxybenzyl group with t1/2 (2) ~ 270 h. As expected, stabilities decreased at pH 8.7 to half-lives of t1/2 (1) = 6.5 and t1/2 (2) = 133 h, respectively. The d4TDP/d4TMP ratio was found to be 4:1 at pH 7.3 and 8.7. Hydrolysis in biological media The cleavage of compounds 4 and 13 was further investigated in T-lymphocyte CEM cell extracts (Table 2). Moreover, C6DiPPro-d4TDP 4 d and C15-DiPPro-d4TDP 4 i were hydrolyzed in human serum (HS), RPMI-1640 culture medium containing 10 % heat-inactivated fetal calf serum (RPMI/FCS) as well as in FCS (Table 3). The rapid cleavage of 5 a in CEM cell extracts was described previously.[22] Cell extracts were diluted with phosphate buffer to decrease enzyme-mediated degradation of d4TDP formed (33 % CE in PBS, pH 6.8). Half-lives of prodrugs 5 b–j were in the range of 1–21 h. The cleavage was 10to 67-fold faster in CE than chemical hydrolysis, indicating a significant contribution by enzymatic cleavage (Table 2). The formation and further hydrolysis of intermediates 6 b–f were also observed. Again, a predominant formation of d4TDP was observed from DiPPro-d4TDPs, as proven by HPLC experiments (4 f as an example, Figure 5). Unfortunately, it was not possible to quantify the released amount of d4TDP because it was still prone to some enzymatic degradation. In line with data from the chemical hydrolysis studies, rates of hydrolysis decreased with increasing length of the aliphatic chains. ChemMedChem 2014, 9, 762 – 775

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www.chemmedchem.org drolysis may be advantageous for a possible intracellular “lockin” of the intermediate 5.[30, 31]

Antiviral evaluation

Figure 5. HPLC profile for C9-DiPPro-d4TDP 4 f after incubation in cell extracts for the time periods shown and reference traces of d4TDP and d4TMP; degradation products of the extracts are highlighted in the box. HPLC analysis method A.

All compounds were evaluated for their anti-HIV activity in wild-type CEM/0 and mutant thymidine-kinase-deficient cells (CEM/TK ; Table 4). Unfortunately, cells deficient in thymidine monophosphate kinase (TMPK) are not available because TMPK is crucial for supplying thymidine building blocks for DNA synthesis. Deletion of this enzyme would result in nonviable cells. The parent nucleoside d4T 1 was used as reference compound. Antiviral activity in TK-deficient cells is an indication that the intact d4TDP prodrug is able to enter the cell by diffusion, because d4T is only very poorly monophosphorylated in this cell line (Table 4, last entry). The majority of tested compounds proved to be at least equipotent to or slightly less potent (4 h,i) than d4T against HIV-1 and HIV-2 in wild-type CEM/0 cells. All compounds with acyl residues R C6H13 (4 d–j)

Additionally, C6-DiPPro-d4TDP 4 d and C15-DiPProTable 4. Antiviral activity and cytotoxicity of compounds 4 a–j and 13. d4TDP 4 i were incubated in 20 % human serum in PBS (pH 6.8) and the determined half-lives were CC50 [mm][b] DiPPro EC50 [mm][a] found to be slightly higher than in CE (Table 3). The CEM/0 CEM/TK intermediate C6-acyloxybenzyl-d4TDP 5 d accumulatHIV-1 HIV-2 HIV-2 ed, but its stability could not be determined due to [c,d] 4a 1.05 0.92 1.5 0.7 21.0 5.7 > 125 the relatively short incubation time used (9–10 h; 0.78 0.80 0.90 0.16 14 5 66 1.4 4 b[c,d] 4c 2.0 1.8 2.9 2.8 15 7.1 80 0.71 longer incubation was not relevant because of the 4 d 0.47 0.28 1.1 0.14 1.2 0.28 85 2.1 occurring protein denaturation). 0.90 0.44 1.3 0.39 2.6 1.9 81.1 1.4 4 e[d] Additionally, the half-lives of 4 d,i in the incubation 0.13 0.087 0.20 0.006 0.15 0.042 61 11 4 f[d] medium used for the anti-HIV tests (RPMI-1640 with 4f 0.080 0.042 0.32 0.12 0.11 0.042 62 6.4 4g 0.16 0.042 0.35 0.078 0.23 0.064 39 0.71 10 % heat-inactivated FCS) were determined. Relative 4 h 1.8 1.6 2.0 1.2 1.2 0.030 134 21 to their chemical stabilities, lower stability in this 4i 2.0 1.8 1.7 0.42 0.99 0.014 72 9.9 medium was clear (Table 3). A possible explanation 4j 1.1 0.29 1.8 1.5 0.65 0.35 100 3.1 may be residual esterase activity in FCS, as previously 13 0.99 0.16 5.0 2.5 2.8 2.1 86 4.9 d4T 0.86 0.45 2.3 2.4 173 70 > 250 observed by our group.[30] Moreover, compounds 4 d,i were incubated in 10 % FCS in PBS, pH 6.8. Inter[a] Antiviral activity in CD4 + T-lymphocytes: 50 % effective concentration (values are the mean SD of two to three independent experiments). [b] Cytotoxicity: 50 % cytoestingly, an 11-fold shorter half-life for the first hystatic concentration or compound concentration required to inhibit CEM cell proliferadrolysis step was observed for 4 d, and a sixfold retion by 50 % (values are the mean SD of two to three independent experiments). duced half-life was found for 4 i relative to chemical [c] Previously reported results.[22] [d] Tested as ammonium salt. hydrolysis in PBS, pH 6.8. Aminolysis or other side reactions may also occur in the culture medium due to the presence of amino acids, proteins, and other nushowed very pronounced anti-HIV activities in CEM/TK cells, cleophiles such as inorganic salts. As before, a longer alkyl chain led to increased stability. indicating their ability to diffuse across the cell membrane and The hydrolysis of CE-C9-DiPPro-d4TDP 13 was studied in biorelease phosphorylated d4T metabolites. None of the comlogical media (CE, HS, RPMI/FCS, Table 3) as well. Again, in pounds displayed significant cytotoxicity. comparison with the chemical stabilities in PBS (pH 6.8), In particular, C9-DiPPro-d4TDP 4 f demonstrated the good a marked decrease in half-lives was observed in these media. potential of the DiPPro concept, with 1570-fold higher antiviral In all media, the b-cyanoethyl group was cleaved first, leading activity in TK-deficient CEM cells and antiviral activity one to decanoyloxybenzyl-d4TDP 5 f, which was identified by order of magnitude higher in infected wild-type CEM/0 cells HPLC. Interestingly, hydrolysis of the b-cyanoethyl group of relative to the parent nucleoside d4T. We tested both the amDiPPro-d4TDP 13 (t1/2 (1) = 0.46 h) was 16-fold faster than cleavmonium and the tetra-n-butylammonium salts of C9-DiPProd4TDP 4 f to evaluate the possible influence of the counterion. age of one decanoyloxybenzyl moiety of C9-DiPPro-d4TDP (t1/ No effects on antiviral activity or cytotoxicity were observed. 2 (1) = 7.6 h) in CEM cell extract. This marked acceleration in hyThese results demonstrate that the DiPPro approach has been 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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CHEMMEDCHEM FULL PAPERS successfully optimized by increasing the lipophilicity of the prodrugs relative to earlier prepared compounds.[22]

Conclusions In summary, DiPPro-d4TDP prodrugs 4 c–j were synthesized by using a convergent approach and were obtained with high purity and in yields of up to 67 %. Acyl moieties of varying chain length were introduced to study their influence on the hydrolysis mechanism, stability, and antiviral activity of the DiPPro-d4TDP prodrugs. It was proven that tBu-, C4-, C6-, C7and C9-DiPPro-d4TDP 4 b–f hydrolyzed predominantly via acyloxybenzyl intermediates 5 b–f to give d4TDP. The ratio of d4TDP and d4TMP formation was determined and correlated with the stability of the compounds; the higher the half-life of the cleavage of the first masking acyloxybenzyl groups, the higher the amount of d4TMP formed. Interestingly, intermediates 5 did not release d4TMP, but exclusively d4TDP. In the mixed b-cyanoethyl-C9-DiPPro-d4TDP 13, the b-cyanoethyl group was cleaved markedly faster by b-elimination than the decanoyloxybenzyl group, leading to a high amount of d4TDP. In in vitro anti-HIV tests, compounds with acyl residues R C6H13 were found to be highly active in the CEM/TK cell assay, proving that DiPPro-NDP prodrugs are the most effective bioreversibly protected nucleoside diphosphates reported thus far. Using these insights, NDP prodrugs of other nucleoside analogues, such as AZT and ddU, will be synthesized and investigated.

Experimental Section Chemistry General: Diethyl ether was dried over sodium/benzophenone and distilled under nitrogen. Tetrahydrofuran (THF) was dried over potassium/benzophenone and distilled under nitrogen. Pyridine, dichloromethane, and CH3CN were distilled from calcium hydride under nitrogen. N,N-Diisopropylethylamine and TEA were distilled from sodium prior to use. Phosphoryl chloride was distilled under nitrogen prior to use. All commercially available reagents were used without further purification. Thin-layer chromatography (TLC): Merck pre-coated 60 F254 plates (0.2 mm layer of silica gel), or precoated plates with a 0.2 mm layer of silica gel by Macherey & Nagel Xtra Sil UV254 were used; sugar-containing compounds were visualized with sugar spray reagent (0.5 mL 4-methoxybenzaldehyde, 9 mL EtOH, 0.5 mL conc. sulfuric acid and 0.1 mL glacial acetic acid). Preparative chromatography: All preparative TLC was performed on a chromatotron (Harrison Research, Model 7924T) using glass plates coated with 1, 2, or 4 mm layers of Merck 60 PF254 silica gel containing a fluorescent indicator. Flash column chromatography: Merck silica gel 60, 230–400 mesh or Merck RP18 silica gel was used. Glass columns were slurry packed using the appropriate eluent. Fractions containing the product were identified by TLC and pooled, and the solvent was removed in vacuo. Water as eluent was removed by freeze drying. High-performance liquid chromatography (HPLC): analytical HPLC was performed on a VWR-Hitachi LaChromElite HPLC system (L-2130, L-2200, L-2455) equipped with a EcoCART 125–3 column containing reversed-phase silica gel Lichrospher 100 RP-18 (5 mm; VWR-Merck, Darmstadt, Germany). The solvents for HPLC were ob 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org tained from Sigma–Aldrich or VWR (CH3CN, HPLC grade). The software used was EzChromElite. Method A: 0–18 min: TBAH ion buffer/CH3CN gradient (5–95 %); 18–26 min: buffer/CH3CN (95 %); 26–30 min: buffer/CH3CN gradient (95–5 %); 30–35 min: buffer/ CH3CN (5 %); flow: 1 mL min1. Method B: 0–24 min: TBAH ion buffer/CH3CN gradient (5–90 %); 24–28 min: buffer/CH3CN (90 %); 28–30 min: buffer/CH3CN gradient (90–10 %); 30–35 min: buffer/ CH3CN (5 %); flow: 1 mL min1. TBAH ion buffer: 0.55 mm tetra-nbutylammonium hydroxide in H2O. Method C: 0–20 min: TBAH ion buffer/CH3CN gradient (5–90 %); 20–24 min: buffer/CH3CN (90 %); 24–29 min: buffer/CH3CN gradient (90–5 %); 29–36 min: buffer/ CH3CN (5 %); flow: 1 mL min1. Method D: 0–15 min: TBAH ion buffer/CH3CN gradient (5–70 %); 15–18 min: buffer/CH3CN (70 %); 18–21 min: buffer/CH3CN gradient (70–5 %); 21–27 min: buffer/ CH3CN (5 %). NMR spectra were recorded with Bruker AMX 400, AV I 400, AV II 400, or Bruker DRX 500 Fourier transform spectrometers. All 1H and 13 C NMR chemical shifts (d) are quoted in parts per million (ppm) downfield from tetramethylsilane and calibrated on solvent signals. The 31P NMR chemical shifts (proton decoupled) are quoted in ppm using H3PO4 as the external reference. The spectra were recorded at room temperature in automation mode. MS: Mass spectra were obtained with a VG Analytical VG/70–250 F [FAB, (double focusing), matrix: m-nitrobenzyl alcohol], ESI mass spectra were recorded with a VG Analytical Finnigan ThermoQuest MAT 95 XL spectrometer or with an Agilent 6224 LC–MS-TOF or with a Bruker maXis spectrometer. IR and UV spectroscopy: IR spectra were recorded on a Bruker Alpha P FT-IR spectrometer at RT in a range of 400–4000 cm1. UV spectra of final compounds 4 were determined using the diode array detector during analytical HPLC. Purity of Final Compounds: The 95 % purity of the final compounds 4, 5 f and 13 was confirmed using HPLC analysis. Melting points are uncorrected. 3’-Deoxy-2’,3’-didehydrothymidinemonophosphate (d4TMP 6) as ammonia salt: The reaction was carried out under nitrogen atmosphere with dried solvents. To a solution of 2.85 mL (30.5 mmol, 4.4 equiv) of distilled phosphoryl chloride in 14 mL CH3CN at 0 8C 2.4 mL (2.41 g, 30.5 mmol, 4.4 equiv) of dry pyridine and 0.27 mL (0.27 g, 15.3 mmol, 2.2 equiv) of H2O were added. After 10 min 1.55 g (6.94 mmol, 1.0 equiv) of d4T were added. After 4 h at RT the reaction was quenched by addition of ice/H2O and stirred at 4 8C for 1 h. By careful addition of solid ammonium bicarbonate the solution was adjusted to pH 8. Solvents were removed by freeze-drying. The residue was dissolved in H2O and purified by RP-18 gradient chromatography (H2O/CH3CN 0–10 %). The pure product was obtained after freeze-drying. Yield: 2.21 g (6.53 mmol, 94 %) of a colorless fluffy powder. 1H NMR (400 MHz, D2O) d = 7.58 (d, 1 H, 4JHH = 1.1 Hz, H-6), 6.93 (ddd, 1 H, 3JHH = 3.4 Hz, 3/4JHH = 2.0 Hz, 4JHH = 1.5 Hz, H-1’), 6.45 (ddd, 1 H, 3JHH = 6.1 Hz, 4JHH = 1.7 Hz, 4 JHH = 1.7 Hz, H-3’), 5.88 (ddd, 1 H, 3JHH = 6.1 Hz, 3/4JHH = 2.3 Hz, 4JHH = 1.4 Hz, H-2’), 5.07–5.02 (m, 1 H, H-4’), 4.00–3.91 (m, H-5’), 1.85 ppm (d, 1 H, 4JHH = 1.1 Hz, Me); 13C NMR (101 MHz, D2O) d = 166.7 (C-4), 152.3 (C-2), 138.2 (C-6), 134.2 (C-3’), 125.2 (C-2’), 111.4 (C-5), 89.9 (C1’), 85.9 (d, 3JCP = 8.6 Hz, C-4’), 65.5 (d, 2JCP = 4.8 Hz, C-5’), 11.41 ppm (Me); 31P NMR (162 MHz, 1H-decoupled, D2O) d = 0.42 ppm; IR: n˜ = 2819, 1661, 1468, 1223, 1164, 1038, 908, 642, 489 cm1; UV (HPLC): lmax [nm] = 265, HPLC: tR [min] = 6.31, method 1; Rf : 0.38 (iPrOH/ H2O/NH3 (25 % in H2O) 14:7:1 v/v); MS (ESI + ): calcd 302.03 [M] + , found 302.99; Mr [Da]: 338.25; C10H19N4O7P. Bis(tetra-n-butyl)-ammonia-d4TMP 6: 133 mg (0.39 mmol) ammonium salt of d4TMP was dissolved in H2O and eluted over a protonChemMedChem 2014, 9, 762 – 775

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CHEMMEDCHEM FULL PAPERS ated ion-exchange resin (DOWEX 50WX8). The obtained acidic solution was titrated with 511 mg (2.0 equiv) of tetra-n-butylammonium hydroxide solution (40 % in H2O w/w). The resulting slightly basic solution was lyophilized and the resulting material dissolved in dry CH3CN and stored over molecular sieves 3 . Yield: 260 mg (0.33 mmol, 85 %) hygroscopic solid. 1H NMR (400 MHz, CD3OD) d = 7.77 (d, 1 H, 4JHH = 1.2 Hz, H-6), 6.95 (ddd, 1 H, 3JHH = 3.4 Hz, 4JHH = 1.7 Hz, 4JHH = 1.7 Hz, H-1’), 6.56 (ddd, 1 H, 3JHH = 6.0 Hz, 3/4JHH = 1.8 Hz, 3/4JHH = 1.8 Hz, H-3’), 5.80 (ddd, 1 H, 3JHH = 6.0 Hz, 3/4JHH = 2.3 Hz, 4JHH = 1.3 Hz, H-2’), 4.13–4.05 (m, 1 H, H-4’), 4.01–3.94 (m, 1 H, H-5’), 3.28–3.17 (m, 16 H, H-A), 1.93 (d, 3 H, 4JHH = 1.6 Hz, Me), 1.72–1.60 (m, 16 H, H-B), 1.42 (sext., 3JHH = 7.4 Hz, 16 H, H-C), 1.02 ppm (t, 3JHH = 7.4 Hz, 24 H, H-D); 13C NMR (101 MHz, [D6]DMSO) d = 167.4 (C-4), 153.0 (C-2), 135.1–134.9 (m, C-3’), 134.2 (C-6), 126.3 (C-2’), 109.5 (C-5), 88.7 (C-1’), 85.4 (d, J = 7.6 Hz, C-4’), 64.9 (d, J = 5.0 Hz, C-5’), 57.4 (C-a), 23.0 (C-b), 19.1 (C-c), 13.4 (C-d), 13.0 ppm (Me); 31P NMR (162 MHz, 1H-decoupled, CD3OD) d = 3.57 ppm; UV (HPLC): lmax [nm] = 265, HPLC: tR [min] = 6.31, method A; Mr [Da]: 787.10; C42H83N4O7P. General procedure A: Preparation of 4-acyloxybenzyl alcohols 9: All reactions were carried out under nitrogen atmosphere with dry solvents and reagents. To an ice-cold solution of 4-hydroxybenzyl alcohol (1.0 equiv) and TEA (1.1 equiv) in THF or diethyl ether the acyl chlorides (1.1 equiv) were added over a period of 20 min. The mixtures were stirred 2 h at 0 8C followed by stirring overnight at room temperature in some cases. The precipitate was removed by filtration and the solvent was evaporated. The crude products were diluted with EtOAc, washed twice with saturated sodium carbonate solution and twice with H2O. The organic layers were dried (sodium sulfate) and concentrated. The crude products were purified either by column chromatography or by crystallization to give compounds 9. The syntheses of 4-(hydroxymethyl)phenylacetate 9 a, 4-(hydroxymethyl)phenyl-tert-butanoate 9 b and 4-(hydroxymethyl)phenyloctanoate 9 e were described previously.[22] The numbering scheme is shown in Supporting Information Figure S9. 4-(Hydroxymethyl)phenylpentanoate 9 c: General procedure A with 6.0 g 4-hydroxybenzyl alcohol (48 mmol, 1 equiv), 8.08 mL TEA (5.86 g, 58 mmol, 1 equiv) dissolved in 30 mL THF and dropwise addition of 6.99 mL butyryl chloride (2.96 g, 32 mmol, 1 equiv) at 0 8C. Reaction time: 23 h at RT. Column chromatography (PE 50– 70/EtOAc 3:1, v/v). Yield: 4.98 g (23.0 mmol, 46 %) colorless oil. 1 H NMR (400 MHz, [D6]DMSO) d = 7.44–7.25 (m, 2 H, H-3), 7.12–6.96 (m, 2 H, H-2), 5.21 (t, 1 H, 3JHH = 5.6 Hz, OH), 4.49 (d, 2 H, 3JHH = 7.4 Hz, H-Bn), 2.56 (dd, 2 H, 3JHH = 7.4 Hz, 3JHH = 7.4 Hz, H-b), 1.62 (quint., 2 H, 3JHH = 7.4 Hz, H-c), 1.39 (sext., 2 H, 3JHH = 7.4 Hz, H-d), 0.92 ppm (t, 3 H, 3JHH = 7.4 Hz, H-e); 13C NMR (101 MHz, [D6]DMSO) d = 171.8 (C-a), 149.2 (C-4), 140.0 (C-1), 127.4 (2 C-3), 121.3 (2 C2), 62.4 (C-Bn), 33.2 (C-b), 26.5 (C-c), 21.6 (C-d), 13.5 ppm (C-e); IR: n˜ = 3361, 2959, 2933, 2873, 1754, 1507, 1465, 1417 1366, 1198, 1045, 1014, 811 cm1; Rf : 0.22 (PE 50–70/EtOAc 3:1, v/v); MS (FAB): calcd 191.12 [MOH] + , 208.10 [M], 209.12 [M + H] + , 231.09 [M+Na] + ; found 191.1, 208.1, 231.1; Mr [Da]: 208.25; C12H16O3. 4-(Hydroxymethyl)phenylheptanoate 9 d: General procedure A with 8.0 g 4-hydroxybenzyl alcohol (64 mmol, 1 equiv), 10.7 mL TEA (7.82 g, 77.3 mmol, 1.2 equiv) dissolved in 50 mL THF and dropwise addition of 12.0 mL heptanoyl chloride (11.5 g, 77.3 mmol, 1.2 equiv) at 0 8C. Reaction time: 20 h at RT. Column chromatography (gradient of PE 50–70/EtOAc 8:1 to 4:1, v/v). Yield: 5.50 g (23.3 mmol, 56 %) yellowish oil. 1H NMR (400 MHz, [D6]DMSO) d = 7.36–7.31 (m, 2 H, H-3), 7.06–7.02 (m, 2 H, H-2), 5.22 (t, 1 H, 3JHH = 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org 5.7 Hz, OH), 4.48 (d, 2 H, 3JHH = 5.7 Hz, H-Bn), 2.56 (dd, 2 H, 3JHH = 7.4 Hz, 3JHH = 7.4 Hz, H-b), 1.63 (tt, 2 H, 3JHH = 7.4 Hz, 3JHH = 7.4 Hz, Hc), 1.40–1.24 (m, 6 H, H-d, H-e, H-f), 0.87 ppm (t, 1 H, 3JHH = 6.9 Hz, H-g); 13C NMR (101 MHz, [D6]DMSO) d = 171.7 (C-a), 149.1 (C-1), 140.0 (C-4), 127.4 (2 C-3), 121.2 (2 C-2), 62.3 (C-Bn), 33.4 (C-b), 24.2 (C-c), 30.7, 27.9, 21.7 (C-d, C-e, C-f), 13.7 ppm (C-g); IR: n˜ = 3353, 2955, 2929, 2859, 1755, 1507, 1195, 1163, 1140, 1101, 1014, 503 cm1; Rf : 0.28 (PE 50–70/EtOAc 3:1 v/v); MS (FAB): calcd 219.14 [MOH] + , 236.14 [M], 237.14 [M + H] + ; found 219.2, 237.2; Mr [Da]: 236.30; C14H20O3. 4-(Hydroxymethyl)phenyldecanoate 9 f: General procedure A with 8.0 g 4-hydroxybenzyl alcohol (64 mmol, 1 equiv), 8.92 mL TEA (6.52 g, 64.4 mmol, 1.0 equiv) dissolved in 100 mL THF and dropwise addition of 13.4 mL decanoyl chloride (12.3 g, 64.4 mmol, 1.0 equiv) dissolved in 80 mL THF at 0 8C. Reaction time: 20 h at RT. Column chromatography (gradient of PE 50–70/EtOAc 5:1 to 5:2, v/v). Yield: 10.19 g (36.6 mmol, 57 %) colorless solid. 1H NMR (400 MHz, [D6]DMSO) d = 7.35–7.31 (m, 2 H, H-3), 7.06–7.01 (m, 2 H, H-2), 5.21 (t, 1 H, 3JHH = 5.7 Hz, OH), 4.47 (d, 2 H, 3JHH = 5.7 Hz, H-Bn), 2.55 (t, 2 H, 3JHH = 7.3 Hz, H-b), 1.64 (tt, 2 H, 3JHH = 7.3 Hz, 3JHH = 7.3 Hz, H-c), 1.40–1.18 (m, 12 H, H-d, H-e, H-f, H-g, H-h, H-i), 0.85 ppm (t, 3 H, 3JHH = 6.9 Hz, H-j); 13C NMR (101 MHz, [D6]DMSO) d = 171.8 (C-a), 149.1 (C-1), 139.9 (C-4), 127.4 (C-3), 121.3 (C-2), 62.3 (C-Bn), 33.4 (C-b), 24.3 (C-c), 31.2, 28.8, 28.6, 28.5, 28.3, 22.0 (C-d, Ce, C-f, C-g, C-h, C-i), 13.8 ppm (C-j); IR: n˜ = 3326, 2954, 2916, 2848, 1748, 1604, 1211, 1143, 846, 719, 579, 513 cm1; mp: 45–48 8C, Rf : 0.34 (PE 50–70/EtOAc, 3:1 v/v); MS (FAB): calcd 278.19 [M], 279.19 [M + H] + ; found 279.2 [M + H] + ; Mr [Da]: 278.38; C17H26O3. 4-(Hydroxymethyl)phenyldodecanoate 9 g: General procedure A with 6.01 g 4-hydroxybenzyl alcohol (48.5 mmol, 1 equiv), 8.08 mL TEA (5.87 g, 58.0 mmol, 1.2 equiv) dissolved in 30 mL THF and dropwise addition of 13.4 mL dodecanoyl chloride (12.7 g, 58.0 mmol, 1.2 equiv) dissolved in 20 mL THF at 0 8C. Reaction time: 20 h at RT. Column chromatography (PE 50–70/EtOAc 10:1 followed by 3:1, v/v). Yield: 5.81 g (18.9 mmol, 40 %) colorless solid. 1 H NMR (500 MHz, [D6]DMSO) d = 7.37–7.28 (m, 2 H, H-3), 7.07–6.98 (m, 2 H, H-2), 5.20 (t, 1 H, 3JHH = 5.6 Hz, OH), 4.48 (d, 2 H, 3JHH = 5.6 Hz, H-Bn), 2.55 (t, 2 H, 3JHH = 7.4 Hz, H-b), 1.63 (tt, 2 H, 3JHH = 7.2 Hz, 3JHH = 7.2 Hz, H-c), 1.40–1.16 (m, 18 H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k), 0.85 ppm (t, 3 H, 3JHH = 6.7 Hz, H-l); 13C NMR (126 MHz, [D6]DMSO) d = 171.8 (C-a), 149.2 (C-1), 140.04 (C-4), 127.4 (2 C-3), 121.3 (2 C-2), 62.4 (C-Bn), 33.4 (C-b), 24.3 (C-c), 31.3, 28.9, 28.9, 28.6, 28.4, 28.3, 22.2 (C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k), 13.9 ppm (C-l); IR: n˜ = 3332, 2995, 2915, 2848, 1748, 1605, 1509, 1464, 1411, 1384, 1328, 1297, 1267, 1235, 1035, 1013, 719 cm1; mp: 58 8C; Rf : 0.28 (PE 50–70/EtOAc 3:1, v/v); MS (FAB): calcd 183.17 [C12H23OC] + , 289.22 [MOH] + , 306.22 [M] + , 307.23 [M + H] + ; found 183.2 [C12H23OC] + , 289.3 [MOH] + , 307.3 [M + H] + , 329.3 [M + Na] + ; Mr [Da]: 306.44; C19H30O3. 4-(Hydroxymethyl)phenyltetradecanoate 9 h: General procedure A with 8.0 g 4-hydroxybenzyl alcohol (64 mmol, 1 equiv), 8.92 mL TEA (6.52 g, 64.4 mmol, 1.0 equiv) dissolved in 100 mL THF and dropwise addition of 17.5 mL tetradecanoyl chloride (15.9 g, 64.4 mmol, 1.0 equiv) dissolved in 80 mL THF at 0 8C. Reaction time: 16 h at RT. Purification was achieved by crystallization (PE 50–70/EtOAc). Yield: 21.5 g (64.3 mmol, 70 %) colorless solid. 1 H NMR (400 MHz, [D6]DMSO) d = 7.36–7.30 (m, 2 H, H-3), 7.06–7.01 (m, 2 H, H-2), 5.22 (t, 1 H, 3JHH = 5.4 Hz, OH), 4.47 (d, 2 H, 3JHH = 5.0 Hz, H-Bn), 2.54 (t, 2 H, 3JHH = 7.4 Hz, H-b), 1.61 (tt, 2 H, 3JHH = 7.2 Hz, 3JHH = 7.2 Hz, H-c), 1.38–1.17 (m, 20 H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m), 0.85 ppm (t, 3 H, 3JHH = 6.4 Hz, H-n); 13C NMR (101 MHz, [D6]DMSO) d = 171.8 (C-a), 149.1 (C-1), 139.9 (2 C-4), ChemMedChem 2014, 9, 762 – 775

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CHEMMEDCHEM FULL PAPERS 127.4 (2 C-3), 121.3 (C-2), 62.3 (C-Bn), 33.4 (C-b), 24.3 (C-c), 31.3, 29.0, 28.9, 28.8, 28.7, 28.6, 28.5, 28.3, 22.0 (C-d, C-e, C-f, C-g, C-h, Ci, C-j, C-k, C-l, C-m), 13.8 ppm (C-n); IR: n˜ = 2955, 2914, 2847, 1750, 1508, 1252, 1214, 1165, 1014, 924, 719, 496 cm1; mp: 61 8C; Rf : 0.33 (PE 50–70/EtOAc, 5:1 v/v); MS (FAB): calcd 334.25 [M], 335.26 [M + H], found 335.3; Mr [Da]: 334.49; C21H34O3. 4-(Hydroxymethyl)phenylhexadecanoate 9 i: General procedure A with 8.0 g 4-hydroxybenzyl alcohol (64 mmol, 1 equiv), 9.0 mL TEA (6.5 g, 65.0 mmol, 1.0 equiv) dissolved in 50 mL THF and dropwise addition of 19.6 mL hexadecanoyl chloride (17.7 g, 64.4 mmol, 1.0 equiv) dissolved in 20 mL THF at 0 8C. Reaction time: 18 h at RT. Purification was achieved by crystallization (PE 50–70/EtOAc) followed by column chromatography (PE 50–70/EtOAc, 3:1 v/v). Yield: 8.44 g (23.3 mmol, 36 %) colorless solid. 1H NMR (500 MHz, [D6]DMSO) d = 7.36–7.30 (m, 2 H, H-3), 7.06–7.00 (m, 2 H, H-2), 5.20 (t, 1 H, 3JHH = 5.7 Hz, OH), 4.47 (d, 2 H, 3JHH = 5.7 Hz, H-Bn), 2.55 (t, 2 H, 3JHH = 7.4 Hz, H-b), 1.62 (tt, 2 H, 3JHH = 7.3 Hz, 3JHH = 7.2 Hz, H-c), 1.38–1.17 (m, 24 H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m, Hn, H-o), 0.85 ppm (t, 3 H, 3JHH = 6.8 Hz, H-p); 13C NMR (101 MHz, [D6]DMSO) d = 171.5 (C-a), 149.4 (C-1), 137.4 (2 C-4), 128.5 (2 C3), 120.7 (C-2), 63.9 (C-Bn), 33.5 (C-b), 24.1 (C-c), 31.1, 28.7, 28.6, 28.5, 28.4, 28.4, 28.3, 21.7 (C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, Cm, C-n, C-o), 13.2 ppm (C-p); IR: n˜ = 3396, 3073, 2916, 1758, 1509, 1471, 1220, 1152, 1220, 1388, 1471 cm1; mp: 75 8C; Rf : 0.53 (PE 50–70/EtOAc, 1:1 v/v); MS (FAB): calcd 239.23 [C16H31OC] + , 345.27 [C23H37O2C] + [MOH] + , 363.28 [M + H] + ; found 239.3, 345.3, 363.3; Mr [Da]: 362.27; C23H38O3. 4-(Hydroxymethyl)phenyl-Z-octadec-9-enoate 9 j: General procedure A with 8.0 g 4-hydroxybenzyl alcohol (64 mmol, 1 equiv), 10.8 mL TEA (7.82 g, 77.3 mmol, 1.2 equiv) dissolved in 50 mL THF and dropwise addition of 25.5 mL Z-octadec-9-enoyl chloride (23.3 g, 77.3 mmol, 1.2 equiv) dissolved in 20 mL THF at 0 8C. Reaction time: 3 h at RT. Purification was achieved by crystallization (PE 50–70/EtOAc) followed by column chromatography (PE 50–70/ EtOAc, 5:1 v/v). Yield: 13.6 g (36.5 mmol, 57 %) colorless oil. 1 H NMR (400 MHz, CDCl3) d = 7.41–7.33 (m, 2 H, H-2), 7.10–7.01 (m, 2 H, H-3), 5.43–5.31 (m, 2 H, H-i, H-j), 4.68 (s, 2 H, H-Bn), 2.55 (t, 2 H, 3 JHH = 7.5 Hz, H-b), 2.12–1.93 (m, 4 H, H-h, H-k), 1.75 (tt, 2 H, 3JHH = 7.4 Hz, 3JHH = 7.4 Hz, H-c), 1.46–1.21 (m, 20 H, H-d, H-e, H-f, H-g, H-l, H-m, H-n, H-o, H-p, H-q), 0.87 ppm (t, 3 H, 3JHH = 6.8 Hz, H-r); 13 C NMR (101 MHz, CDCl3) d = 172.1 (C-a), 150.1 (C-4), 138.1 (C-1), 129.5 (C-i, C-j), 127.8 (C-2), 121.5 (C-3), 64.6 (C-Bn), 34.1 (C-b), 29.7, 29.6, 29.5, 29.3, 29.1, 29.1, 24.8, 22.6 (C-c, C-d, C-e, C-f, C-g, C-l, Cm, C-n, C-o, C-p, C-q), 27.2 (C-h, C-k), 13.7 ppm (C-r); IR: n˜ = 2922, 2853, 1739, 1609, 1509, 1463, 1199, 1162, 851, 723 cm1; Rf : 0.38 (PE 50–70/EtOAc, 3:1 v/v); MS (FAB): calcd 343.26 [MCH3OH], 374.28 [M], found 343.3, 371.3; Mr [Da]: 374.55; C24H38O3. General procedure B: Preparation of bis-(4-acyloxybenzyl)-N,Ndiisopropylphosphoramidites 7: All reactions were carried out under nitrogen atmosphere with dry solvents and reagents. A solution of dichloro-N,N-diisopropylphosphoramidite (1.0 equiv) in THF was cooled to 78 8C. Over a period of 1–2 h a solution of TEA (2.3 equiv) and corresponding 4-acyloxybenzyl alcohols 9 (2.2 equiv) in THF was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 16 to 24 h. The precipitate was removed by filtration and the filtrate was concentrated. The crude products were purified by preparative TLC (Chromatotron) to give compounds 7. The syntheses of bis(4-acetyloxybenzyl)-N,N-diisopropylphosphoramidite 7 a, bis(4-tert-butyloxybenzyl)-N,N-diisopropylphosphoramidite 7 b, and bis(4-octanoyloxybenzyl)-N,N-diisopropylphosphora 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org midite 7 e were described previously.[22] The numbering scheme is shown Supporting Information Figure S10. Bis(4-pentanoyloxybenzyl)-N,N-diisopropylphosphoramidite 7 c: General procedure B with 1.997 g 4-(hydroxymethyl)phenylpentanoate (9.60 mmol, 1.9 equiv), 1.4 mL TEA (1.0 g, 10 mmol, 2.3 equiv) dissolved in 25 mL THF and 0.882 g dichloro-N,N-diisopropylphosphoramidite (4.36 mmol, 1.0 equiv) dissolved in 25 mL THF. Purification by Chromatotron (PE 50–70, 5 % TEA). Yield: 2.13 g (3.91 mmol, 90 %) colorless oil. 1H NMR (400 MHz, CDCl3) d = 7.36– 7.32 (m, 4 H, H-2), 7.06–7.00 (m, 4 H, H-3), 4.74 (dd, 2 H, 2JHH = 12.6 Hz, 3JHP = 8.1 Hz, H-Bn), 4.66 (dd, 2 H, 2JHH = 12.6 Hz, 3JHP = 8.1 Hz, H-Bn), 3.75–3.61 (m, 2 H, NC-H), 2.54 (t, 4 H, 3JHH = 7.5 Hz, Hb), 1.74 (tt, 4 H, 3JHH = 7.5 Hz, 3JHH = 7.5 Hz, H-c), 1.44 (sext., 4 H, 3 JHH = 7.5 Hz, H-d), 1.19 (d, 12 H, 3JHH = 6.7 Hz, Me), 0.96 ppm (t, 6 H, 3 JHH = 7.3 Hz, H-e); 13C NMR (101 MHz, CDCl3) d = 172.4 (2 C-a), 150.0 (2 C-4), 137.2 (2 C-1), 128.1 (4 C-2), 121.5 (4 C-3), 64.9 (d, 2 JCP = 18.5 Hz, 2 C-Bn), 43.2 (d, 2JCP = 12.9 Hz, 2 C-N), 34.3, 27.2, 24.7, 24.7, 22.4 (C-b, C-c, C-d, 2 Me), 13.8 ppm (C-e); 31P NMR (162 MHz, 1H-decoupled, CDCl3) d = 147.86 ppm, IR: n˜ = 2963, 2931, 2871, 1757, 1507, 1460, 1417, 1364, 1198, 1100, 1026, 851 cm1; Rf : 0.69 (PE 50–70/EtOAc, 5:1 v/v); MS (FAB): calcd 545.3 [M] + , 546.3 [M + H] + ; found 544.3 [MH] + , 546.4 [M + H] + ; Mr [Da]: 545.64; C30H44NO6P. Bis(4-heptanoyloxybenzyl)-N,N-diisopropylphosphoramidite 7 d: General procedure B with 1.8 g 4-(hydroxymethyl)phenylheptanoate (7.6 mmol, 2.2 equiv), 1.11 mL TEA (0.78 g, 7.96 mmol, 2.3 equiv) dissolved in 10 mL THF and 0.69 g dichloro-N,N-diisopropylphosphoramidite (0.64 mL, 3.46 mmol, 1.0 equiv) dissolved in 10 mL THF. Purification by Chromatotron (PE 50–70 and EtOAc gradient 0–50 %, 5 % TEA). Yield: 984 mg (1.66 mmol, 49 %) colorless oil. 1H NMR (400 MHz, CDCl3) d = 7.37–7.32 (m, 4 H, H-2), 7.05–7.00 (m, 4 H, H-3), 4.74 (dd, 2 H, 2JHH = 12.6 Hz, 3JHP = 8.0 Hz, H-Bn), 4.66 (dd, 2 H, 2JHH = 12.6 Hz, 3JHP = 8.0 Hz, H-Bn), 3.74–3.63 (m, 2 H, NC-H), 2.54 (t, 4 H, 3JHH = 7.4 Hz, H-b), 1.75 (tt, 4 H, 3JHH = 7.4 Hz, 3JHH = 7.4 Hz, H-c), 1.45–1.22 (m, 12 H, H-d, H-e, H-f), 1.19 (d, 12 H, 3JHH = 6.7 Hz, Me), 0.90 ppm (t, 6 H, 3JHH = 7.0 Hz, H-g); 13C NMR (101 MHz, CDCl3) d = 173.7 (2 C-a), 154.4 (C-4), 154.3 (C-4), 130.3 (2 C-1), 129.8 (4 C-2), 120.0 (4 C-3), 119.9 (2 C-3), 65.7 (2 C-Bn), 44.1 (d, 2 JCP = 13.7 Hz, 2 -N), 34.2, 27.1, 24.5, 24.5, 22.3 (C-b, C-c, C-d, C-e, C-f, Me), 13.7 ppm (C-g); 31P NMR (162 MHz, 1H-decoupled, CDCl3) d = 147.92 ppm; IR (film): n˜ = 2962, 2930, 2860, 1759, 1608, 1459, 1417, 1199, 1142, 1027, 975, 853, 754 cm1; Rf : 0.55 (PE 50–70/ EtOAc/TEA, 4:1:0.1 v/v/v); MS (FAB): calcd 601.35 [M], 602.36 [M+H] + found 602.4; Mr [Da]: 601.75; C34H52NO6P. Bis(4-decanoyloxybenzyl)-N,N-diisopropylphosphoramidite 7 f: General procedure B with 3.99 g 4-(hydroxymethyl)phenyldecanoate (14.4 mmol, 2.2 equiv), 2.1 mL TEA (1.5 g, 14.6 mmol, 2.2 equiv) dissolved in 10 mL THF and 1.31 g dichloro-N,N-diisopropylphosphoramidite (6.47 mmol, 1.0 equiv) dissolved in 30 mL THF. Purification by Chromatotron (PE 50–70/EtOAc, 9:1 v/v, 5 % TEA). Yield: 2.48 g (3.62 mmol, 56 %) colorless oil. 1H NMR (400 MHz, CDCl3) d = 7.37–7.32 (m, 4 H, H-2), 7.05–7.00 (m, 4 H, H-3), 4.74 (dd, 2 H, 2JHH = 12.6 Hz, 3JHP = 8.0 Hz, H-Bn), 4.66 (dd, 2 H, 2JHH = 12.6 Hz, 3 JHP = 8.0 Hz, H-Bn), 3.75–3.64 (m, 2 H, NC-H), 2.54 (t, 4 H, 3JHH = 7.5 Hz, H-b), 1.75 (tt, 4 H, 3JHH = 7.4 Hz, 3JHH = 7.4 Hz, H-c), 1.45–1.23 (m, 24 H, H-d, H-e, H-f, H-g, H-h, H-i), 1.21 (d, 12 H, 3JHH = 6.7 Hz, Me), 0.88 ppm (t, 6 H, 3JHH = 6.7 Hz, H-j); 13C NMR (101 MHz, CDCl3) d = 172.4 (2 C-a), 150.0 (2 C-4), 137.1 (d, 3JCP = 7.3 Hz, 2 C-1), 128.1 (4 C-2), 121.5 (4 C-3), 64.9 (d, 2JCP = 18.6 Hz, 2 C-Bn), 43.2 (d, 2JCP = 13.0 Hz, 2 C-N), 34.5, 31.9, 29.5, 29.4, 29.2, 26.3, 25.1, 22.7 (C-b, C-c, C-d, C-e, C-f, C-g, C-h, C-i), 24.8 (Me), 24.6 (Me), 14.2 ppm (C-j); 31P NMR (162 MHz, 1H-decoupled, CDCl3) d = 147.94 ppm; IR: ChemMedChem 2014, 9, 762 – 775

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CHEMMEDCHEM FULL PAPERS n˜ = 2955, 2916, 2848, 1749, 1508, 1210, 1143, 1120, 719, 579, 514 cm1; Rf : 0.53 (PE 50–70/EtOAc, 5:1 v/v); MS (FAB): calcd 685.44 [M], 686.45 [M + H] + , found 686.5; Mr [Da]: 685.91; C40H64NO6P. Bis(4-dodecanoyloxybenzyl)-N,N-diisopropylphosphoramidite 7 g: General procedure B with 3.01 g 4-(hydroxymethyl)phenyldodecanoate (9.82 mmol, 2.2 equiv), 1.43 mL TEA (1.01 g, 10.3 mmol, 2.3 equiv) dissolved in 25 mL THF and 0.902 g dichloro-N,N-diisopropylphosphoramidite (0.821 mL, 4.45 mmol, 1.0 equiv) dissolved in 25 mL THF. Purification by Chromatotron (PE 50–70 and EtOAc gradient 0–50 %, 5 % TEA). Yield: 1.92 g (2.59 mmol, 58 %) colorless solid. 1H NMR (400 MHz, CDCl3) d = 7.36–7.31 (m, 4 H, H-2), 7.05– 7.00 (m, 4 H, H-3), 4.74 (dd, 2 H, 2JHH = 12.8 Hz, 3JHP = 8.0 Hz, H-Bn), 4.66 (dd, 2 H, 2JHH = 12.8 Hz, 3JHP = 8.0 Hz, H-Bn), 3.74–3.63 (m, 2 H, NC-H), 2.54 (t, 4 H, 3JHH = 7.4 Hz, H-b), 1.75 (tt, 4 H, 3JHH = 7.5 Hz, 3 JHH = 7.5 Hz, H-c), 1.45–1.22 (m, 32 H, H-d, H-e, H-f, H-g, H-h, H-i, Hj, H-k), 1.19 (d, 12 H, 3JHH = 6.7 Hz, Me), 0.87 ppm (t, 6 H, 3JHH = 6.7 Hz, H-l); 13C NMR (101 MHz, CDCl3) d = 172.4 (2 C-a), 150.0 (2 C-4), 137.1 (d, 3JCP = 7.5 Hz, 2 C-1), 128.1 (4x C-2), 121.5 (4x C-3), 64.9 (d, 2JCP = 18.5 Hz, 2 C-Bn), 43.2 (d, 2JCP = 13.0 Hz, 2 C-N), 34.5, 32.0, 29.5, 29.5, 29.5, 29.4, 29.2, 25.1, 22.7 (C-b, C-c, C-d, C-e, C-f, Cg, C-h, C-i, C-j), 24.7, 24.6 (Me), 14.2 ppm (C-k); 31P NMR (162 MHz, 1 H-decoupled, CDCl3) d = 147.88 ppm; IR: n˜ = 2958, 2915, 2849, 1753, 1508, 1463, 1412, 1387, 1362, 1328, 1298, 1267, 1236, 1083, 1017, 728 cm1; mp: 47 8C; Rf : 0.87 (PE 50–70/EtOAc, 5:1 v/v); MS (FAB): calcd 741.50 [M], 780.47 [M + K] + ; found 780.6 [M + K] + ; Mr [Da]: 742.02; C44H72NO6P. Bis(4-tetradecanoyloxybenzyl)-N,N-diisopropylphosphoramidite 7 h: General procedure B with 2.00 g 4-(hydroxymethyl)phenyltetradecanoate (5.98 mmol, 2.0 equiv), 0.84 mL TEA (0.59 g, 5.98 mmol, 2.0 equiv) dissolved in 15 mL diethyl ether and 603 mg dichloro-N,N-diisopropylphosphoramidite ((0.549 mL, 2.99 mmol, 1.0 equiv) dissolved in 30 mL diethyl ether. Purification by Chromatotron (PE 50–70 and EtOAc gradient 0–50 %, 5 % TEA). Yield: 811 mg (1.01 mmol, 34 %) colorless solid. 1H NMR (400 MHz, CDCl3) d = 7.38–7.32 (m, 4 H, H-2), 7.05–7.00 (m, 4 H, H-3), 4.74 (dd, 2 H, 2 JHH = 12.5 Hz, 3JHP = 8.0 Hz, H-Bn), 4.66 (dd, 2 H, 2JHH = 12.5 Hz, 3JHP = 8.0 Hz, H-Bn), 3.74–3.63 (m, 2 H, NC-H), 2.54 (t, 4 H, 3JHH = 7.4 Hz, Hb), 1.74 (tt, 4 H, 3JHH = 7.5 Hz, 3JHH = 7.5 Hz, H-c), 1.45–1.22 (m, 40 H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m), 1.19 (d, 12 H, 3JHH = 6.7 Hz, Me), 0.88 ppm (t, 6 H, 3JHH = 6.7 Hz, H-o); 13C NMR (101 MHz, CDCl3) d = 172.5 (2 C-a), 150.1 (2 C-4), 137.1 (d, 3JCP = 7.6 Hz, 2 C-1), 128.1 (4 C-2), 121.5 (4 C-3), 64.5 (d, 2JCP = 18.1 Hz, 2 C-Bn), 43.3 (d, 2JCP = 13.0 Hz, 2 C-N), 34.5, 34.4, 32.1, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 22.7 (C-b, C-c, C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, Cm), 25.1 (2 Me), 14.3 ppm (C-n); 31P NMR (162 MHz, 1H-decoupled, CDCl3) d = 147.84 ppm; IR: n˜ = 2956, 2914, 2847, 1752, 1508, 1463, 1361, 1218, 1197, 1091, 968, 851, 819, 512 cm1; mp: 47 8C; Rf : 0.82 (PE 50–70/EtOAc, 5:1 v/v); MS (FAB): calcd 797.57 [M], 798.57 [M+H] + , found 798.5; Mr [Da]: 798.12; C48H80NO6P. Bis(4-hexadecanoyloxybenzyl)-N,N-diisopropylphosphoramidite 7 i: General procedure B with 4.018 g 4-(hydroxymethyl)phenylhexadecanoate (11.07 mmol, 2.2 equiv), 1.61 mL TEA (1.17 g, 11.5 mmol, 2.3 equiv) dissolved in 10 mL THF and 0.92 mL dichloroN,N-diisopropylphosphoramidite (1.01 g, 4.99 mmol, 1.0 equiv) dissolved in 10 mL THF. Purification by Chromatotron (PE 50–70 and EtOAc gradient 0–50 %, 5 % TEA). Yield: 3.43 g (4.15 mmol, 80 %) colorless solid. 1H NMR (400 MHz, CDCl3) d = 7.37–7.32 (m, 4 H, H-2), 7.05–7.00 (m, 4 H, H-3), 4.74 (dd, 2 H, 2JHH = 12.8 Hz, 3JHP = 8.1 Hz, HBn), 4.66 (dd, 2 H, 2JHH = 12.8 Hz, 3JHP = 8.1 Hz, H-Bn), 3.75–3.62 (m, 2 H, NC-H), 2.54 (t, 4 H, 3JHH = 7.4 Hz, H-b), 1.74 (tt, 4 H, 3JHH = 7.5 Hz, 3 JHH = 7.5 Hz, H-c), 1.45–1.22 (m, 48 H, H-d, H-e, H-f, H-g, H-h, H-i, Hj, H-k, H-l, H-m, H-n, H-o), 1.19 (d, 12 H, 3JHH = 6.7 Hz, Me), 0.88 ppm 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org (t, 6 H, 3JHH = 6.7 Hz, H-p); 13C NMR (101 MHz, CDCl3) d = 172.5 (2 C-a), 150.1 (2 C-4), 137.1 (d, 3JCP = 7.4 Hz, 2 C-1), 128.1 (4 C-2), 121.5 (4 C-3), 64.9 (d, 2JCP = 18.3 Hz, 2 C-Bn), 43.3 (d, 2JCP = 13.0 Hz, 2 C-N), 34.5, 32.4, 32.1, 29.8, 29.7, 29.7, 29.5, 29.4, 29.3, 29.3, 25.1, 22.4 (C-b, C-c, C-d, C-e, C-f, C-g, C-h, C-i, C-j, C-k, C-l, Cm, C-n, C-o), 24.7 (Me), 24.6 (Me), 14.3 ppm (C-p); 31P NMR (162 MHz, 1H-decoupled, CDCl3) d = 147.94 ppm; IR: n˜ = 2916, 2848, 1754, 1223, 1153, 971, 849, 819, 763 cm1; mp: 53 8C; Rf : 0.70 (PE 50–70/EtOAc, 5:1 v/v); MS (FAB): calcd 853.63 [M], 854.64 [M + H] + , found 854.9; Mr [Da]: 854.24; C52H88O6NP. Bis(4-Z-octadec-9-enoyloxybenzyl)-N,N-diisopropylphosphoramidite 7 j: General procedure B with 2.00 g 4-(hydroxymethyl)phenylZ-octadec-9-enoate (5.33 mmol, 2.1 equiv), 0.69 mL TEA (0.48 g, 4.81 mmol, 1.9 equiv) dissolved in 25 mL diethyl ether and 0.455 mL dichloro-N,N-diisopropylphosphoramidite (0.499 g, 2.47 mmol, 1.0 equiv) dissolved in 15 mL diethyl ether. Purification by Chromatotron (PE 50–70 and EtOAc gradient 0–50 %, 5 % TEA). Yield: 795 mg (0.877 mmol, 16 %) colorless solid. 1H NMR (400 MHz, CDCl3) d = 7.36–7.32 (m, 4 H, H-2), 7.05–7.01 (m, 4 H, H-3), 5.43–5.28 (m, 4 H, H-i, H-j), 4.75 (dd, 2 H, 2JHH = 12.8 Hz, 3JHP = 8.2 Hz, H-Bn), 4.67 (dd, 2 H, 2JHH = 12.8 Hz, 3JHP = 8.2 Hz, H-Bn), 3.74–3.63 (m, 2 H, NC-H), 2.54 (t, 4 H, 3JHH = 7.4 Hz, H-b), 2.09–1.93 (m, 8 H, H-h, H-k), 1.75 (tt, 4 H, 3JHH = 7.5 Hz, 3JHH = 7.5 Hz, H-c), 1.46–1.23 (m, 40 H, H-d, H-e, H-f, H-g, H-l, H-m, H-n, H-o, H-p, H-q), 1.19 (d, 12 H, 3JHH = 6.7 Hz, Me), 0.87 ppm (m, 6 H, H-r); 13C NMR (101 MHz, CDCl3) d = 172.5 (2 C-a), 150.1 (2 C-4), 137.1 (d, 3JCP = 8.0 Hz, 2 C-1), 130.2, 129.8 (2 C-i, 2 C-j), 128.1 (4 C-2), 121.4 (4 C-3), 64.9 (d, 2JCP = 18.5 Hz, 2 C-Bn), 43.3 (d, 2JCP = 12.6 Hz, 2 C-N), 34.5 (C-b), 32.1, 29.8, 29.8, 29.6, 29.5, 29.5, 29.4, 29.3, 29.3, 29.2, 29.1, 27.4, 27.3, 22.7 (C-c, C-d, C-e, C-f, C-g, C-l, C-m, C-n, C-o, C-p, C-q), 25.1 (C-h, C-k), 24.7 (d, 3JCP = 7.5 Hz, 4x Me), 14.8 ppm (C-r); 31P NMR (162 MHz, 1H-decoupled, CDCl3) d = 147.91 ppm; IR: n˜ = 2923, 2853, 1761, 1198, 1164, 1129, 1051, 1026, 1008, 974, 915, 754, 553 cm1; Rf : 0.94 (PE 50–70/EtOAc, 5:1 v/v), MS (FAB): calcd 905.66 [M], 906.67 [M + H] + found 906.4; Mr [Da]: 906.31; C56H92NO6P. 2-Cyanethoxy-(4-decanoyloxybenzyl)-N,N-diisopropylphosphoramidite 12: General procedure B with 113 mg 4-(hydroxymethyl)phenyldecanoate (0.405 mmol, 0.9 equiv), 70.7 mL diisopropylethylamine 52.3 mg, 0.405 mmol, 0.9 equiv) dissolved in 10 mL THF and 100 mg chloro-(2-cyanoethyloxy)-N,N-diisopropylphosphoramidite (0.449 mmol, 1.1 equiv) dissolved in 10 mL THF. The mixture was stirred at RT for 90 min. Yield: 150 mg (0.313 mmol, 78 %) colorless oil. 1H NMR (400 MHz, CDCl3) d = 7.39–7.31 (m, 2 H, Haryl-3), 7.08– 7.02 (m, 2 H, Haryl-2), 4.74 (dd, 2 H, 2JHH = 12.6 Hz, 3JHP = 8.0 Hz, H-Bn), 4.66 (dd, 2 H, 2JHH = 12.6 Hz, 3JHP = 8.0 Hz, H-Bn), 3.89–3.78 (m, 2 H, H-1’), 3.70–3.59 (m, 2 H, NC-H), 2.62 (t, 2 H, 3JHH = 6.5 Hz, H-2’), 2.54 (t, 2 H, 3JHH = 7.5 Hz, H-b), 1.75 (tt, 2 H, 3JHH = 7.4 Hz, 3JHH = 7.4 Hz, Hc), 1.45–1.23 (m, 12 H, H-d, H-e, H-f, H-g, H-h, H-i), 1.19 (d, 12 H, 3 JHH = 5.2 Hz, Me), 0.87 ppm (t, 3 H, 3JHH = 6.8 Hz, H-j); 31P NMR (162 MHz, 1H-decoupled, CDCl3) d = 148.54 ppm; Rf : 0.93, (PE 50– 70/EtOAc, 1:1 v/v); Mr [Da]: 478.60; C26H43N2O4P. General procedure C: Preparation of DiPPro-d4T-diphosphates 4: All reactions were carried out under nitrogen atmosphere with dry solvents and reagents. D4T monophosphate 6 was dried in stock solution over molecular sieves before starting the reactions. D4T monophosphate (1.0 equiv), phosphoramidites 7 (1.1– 2.3 equiv) and DCI (1.1–2.3 equiv) were dissolved in up to 2.5 mL CH3CN. The reaction mixture was stirred for several hours and then cooled to 25 8C. Oxidation was achieved by addition of tert-butylhydroperoxide (TBHP, 5.5 m in n-decane; 1.1–2.3 equiv). The mixture was allowed to warm to room temperature. After 15 min the solvents were removed in vacuo. The crude products were purified ChemMedChem 2014, 9, 762 – 775

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CHEMMEDCHEM FULL PAPERS by RP-18 flash chromatography and purified by RP-18 flash chromatography a second and third time if necessary (with different H2O/MeOH gradients). Product containing fractions were pooled and MeOH was evaporated. The remaining aqueous solutions were freeze-dried and the products were obtained as brown syrups. The syntheses of ammonium bis-(4-acetyloxybenzyl)-d4TDP 4 a, bis-(4-pivaloyloxybenzyl)-d4TDP 4 b, and ammonium bis-(4-octanoyloxybenzyl)-d4TDP 4 e were described previously.[22] The numbering scheme is shown in Supporting Information Figure S11. (N[nBu]4)-Bis-(4-pentanoyloxybenzyl)-d4TDP 4 c: General procedure C with 115 mg bis-(tetra-n-butylammonium)-d4TMP (0.146 mmol, 1 equiv), 129 mg bis-(4-pentanoyloxybenzyl)-N,N-diisopropylphosphoramidite (0.235 mmol, 1.6 equiv) and 27.7 mg DCI (0.235 mmol, 1.6 equiv) in 4 mL CH3CN at 0 8C. After 2 h stirring at RT 32 mg amidite (0.058 mmol, 0.4 equiv) and 6.9 mg DCI (0.058 mmol, 0.4 equiv) were added. The reaction mixture was stirred for 23 h and cooled to 25 8C followed by oxidation with 53 mL TBHP (0.28 mmol, 1.8 equiv). The crude product was purified by RP-18 flash chromatography (MeOH/H2O, 1:1, than 5:1 v/v, finally MeOH). Yield: 47 mg (0.0467 mmol, 32 %) colorless syrup. 1 H NMR (400 MHz, CD3OD) d = 7.65 (d, 1 H, 4JHH = 1.3 Hz, Hhet-6), 7.43–7.35 (m, 4 H, H-2), 7.09–7.02 (m, 4 H, H-3), 6.94 (ddd, 1 H, 3 JHH = 3.5 Hz, 3/4JHH = 1.7 Hz, 3/4JHH = 1.7 Hz, H-1’), 6.37 (ddd, 1 H, 3 JHH = 6.1 Hz, 4JHH = 1.7 Hz, 4JHH = 1.7 Hz, H-3’), 5.83 (ddd, 1 H, 3JHH = 6.0 Hz, 3/4JHH = 2.2 Hz, 4JHH = 1.5 Hz, H-2’), 5.01 (dd, 4 H, 2JHH = 8.2 Hz, 3 JHP = 6.3 Hz, H-Bn), 4.97–4.92 (m, 1 H, H-4’), 4.25–4.12 (m, 2 H, H-5’), 3.26–3.19 (m, 8 H, H-A), 2.53 (t, 4 H, 3JHH = 7.2 Hz, H-b), 1.89 (d, 3 H, 4 JHH = 1.3 Hz, Mehet), 1.76–1.60 (m, 12 H, H-B, H-c), 1.51–1.36 (m, 12 H, H-C, H-d), 1.05–0.94 ppm (m, 16 H, H-D, H-e); 13C NMR (126 MHz, CD3OD) d = 173.6 (2 C-a), 166.5 (Chet-4), 152.7 (Chet-2), 152.4 (2 C-4), 138.6 (Chet-6), 135.3 (C-3’), 134.8 (d, 3JCP = 7.0 Hz, C1), 134.8 (d, 3JCP = 7.0 Hz, C-1), 130.4 (d, 4JCP = 3.5 Hz, 4 C-2), 127.5 (C-2’), 122.8 (4 C-3), 112.1 (Chet-5), 90.7 (C-1’), 86.9 (d, 3JCP = 8.1 Hz, C-4’), 70.2 (d, 2JCP = 5.5 Hz, C-Bn), 70.2 (d, 2JCP = 5.5 Hz, C-Bn), 68.0 (d, 2JCP = 6.0 Hz, C-5’), 59.5 (pt, 4 C-A), 34.6 (2 C-b), 28.1 (2 C-c), 24.7 (4 C-B), 23.2 (2 C-d), 20.6 (4 C-C), 14.1 (2 C-e), 13.8 (4 CD), 12.5 ppm (Mehet); 31P NMR (162 MHz, 1H-decoupled, CD3OD) d = 12.08 (d, 1P, 2JPP = 20.6 Hz, P-a), 12.96 ppm (d, 1P, 2JPP = 21.3 Hz, P-b); UV (HPLC): lmax = 265 nm, HPLC: tR = 11.57 min, method A; Rf : 0.35 (EtOAc/MeOH, 7:3 v/v); HRMS (ESI): calcd 820.2737 [M] , 819.2665 [MH] , found 819.2665; Mr [Da]: 1006.11; C50H77N3O14P2. (N[nBu]4)-Bis-(4-heptanoyloxybenzyl)-d4TDP 4 d: General procedure C with 190 mg bis-(tetra-n-butylammonium)-d4TMP (0.348 mmol, 1 equiv), 251 mg bis-(4-heptanoyloxy-benzyl)N,N-diisopropylphosphoramidite (0.417 mmol, 1.1 equiv) and 49.0 mg DCI (0.418 mmol, 1.2 equiv) in 3 mL CH3CN. The reaction mixture was stirred for 1 h and cooled to 25 8C followed by oxidation with 76 mL TBHP (0.42 mmol, 1.1 equiv). The crude product was purified by RP-18 flash chromatography (MeOH/H2O, 1:1, than 5:1 v/v, finally MeOH). This first chromatography yielded 53 mg (0.049 mmol) pure product. After a second chromatography (MeOH/H2O, 5:1 v/v) another 96 mg (0.090 mmol) product were isolated. Yield: 149 mg (0.140 mmol, 40 %) colorless syrup. 1H NMR (400 MHz, CD3OD) d = 7.65 (d, 1 H, 4JHH = 1.2 Hz, Hhet-6), 7.42–7.35 (m, 4 H, H-2), 7.07–7.02 (m, 4 H, H-3), 6.95 (ddd, 1 H, 3JHH = 3.5 Hz, 3/4JHH = 1.9 Hz, 3/4JHH = 1.9 Hz, H-1’), 6.37 (ddd, 1 H, 3JHH = 6.5 Hz, 4JHH = 1.9 Hz, 4JHH = 1.9 Hz, H-3’), 5.83 (ddd, 1 H, 3JHH = 6.2 Hz, 4JHH = 1.3 Hz, 3/4JHH = 2.3 Hz, H-2’), 5.01 (dd, 4 H, 2JHH = 8.5 Hz, 3JHP = 6.2 Hz, H-Bn), 4.97–4.92 (m, 1 H, H4’), 4.25–4.13 (m, 2 H, H-5’), 3.25–3.17 (m, 8 H, H-A), 2.57 (t, 4 H, 3 JHH = 7.4 Hz, H-b), 1.88 (d, 3 H, 4JHH = 1.3 Hz, Mehet), 1.76–1.59 (m, 12 H, H-B, H-c), 1.48–1.32 (m, 20 H, H-C, H-d, H-e, H-f), 1.02 (t, 12 H, 3 JHH = 7.3 Hz, H-D), 0.92 ppm (t, 6 H, 3JHH = 6.9 Hz, H-g); 13C NMR 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org (101 MHz, CD3OD) d = 173.6 (2 C-a), 166.5 (Chet-4), 152.7 (Chet-2), 152.4 (C-4), 152.4 (C-4), 138.6 (Chet-6), 135.3 (C-3’), 134.8 (d, 3JCP = 7.3 Hz, C-1), 134.8 (d, 3JCP = 7.3 Hz, C-1), 130.4 (d, 4JCP = 2.7 Hz, 4 C2), 127.5 (C-2’), 122.8 (4 C-3), 112.1 (Chet-5), 90.7 (C-1’), 86.9 (d, 3 JCP = 8.1 Hz, C-4’), 70.2 (d, 2JCP = 5.8 Hz, C-Bn), 70.2 (d, 2JCP = 5.8 Hz, C-Bn), 68.0 (d, 2JCP = 6.2 Hz, C-5’), 59.5 (pt, 4 C-A), 35.0 (2 C-b), 32.5, 29.7, 25.8, 24.7, 23.5, 20.6, 19.3 (2 C-c, 2 C-d, 2 C-e, 2 C-f, 4 C-B), 14.4 (2 C-g), 13.8 (4 C-D), 12.5 ppm (Mehet); 31P NMR (162 MHz, 1H-decoupled, CD3OD) d = 12.11 (d, 1P, 2JPP = 20.3 Hz, Pa), 12.92 ppm (d, 1P, 2JPP = 21.4 Hz, P-b); IR: n˜ = 2959, 2931, 2873, 1755, 1688, 1508, 1465, 1262, 1007, 910, 836, 804, 502 cm1; UV (HPLC): lmax = 265 nm, HPLC: tR = 13.2 min, method A; Rf : 0.80 (EtOAc/MeOH, 7:3 v/v); HRMS (ESI): calcd 820.2737 [M] , 819.2665 [MH] , found 819.2665; Mr [Da]: 1062.21; C54H85N3O14P2. (N[nBu]4)-Bis-(4-decanoyloxybenzyl)-d4TDP 4 f: General procedure C with 148 mg bis-(tetra-n-butylammonium)-d4TMP (0.271 mmol, 1 equiv), 208 mg bis-(4-decanoyloxybenzyl)N,N-diisopropylphosphoramidite (0.303 mmol, 1.1 equiv) and 37.0 mg DCI (0.313 mmol, 1.2 equiv) in 4 mL CH3CN. The reaction mixture was stirred for 3 h and cooled to 25 8C followed by oxidation with 53 mL TBHP (0.28 mmol, 1.0 equiv). The crude product was purified by RP-18 flash chromatography (MeOH/H2O, 1:1, than 5:1 v/v, finally MeOH). Yield: 121 mg (0.105 mmol, 39 %) colorless syrup. 1 H NMR (400 MHz, CD3OD) d = 7.65 (d, 1 H, 4JHH = 1.2 Hz, Hhet-6), 7.42–7.34 (m, 4 H, H-2), 7.07–7.02 (m, 4 H, H-3), 6.95 (ddd, 1 H, 3 JHH = 3.5 Hz, 3/4JHH = 1.7 Hz, 3/4JHH = 1.7 Hz, H-1’), 6.38 (ddd, 1 H, 3 JHH = 6.0 Hz, 4JHH = 1.6 Hz, 4JHH = 1.6 Hz, H-3’), 5.84 (ddd, 1 H, 3JHH = 6.0 Hz, 4JHH = 1.6 Hz, 3/4JHH = 2.0 Hz, H-2’), 5.10 (dd, 4 H, 2JHH = 8.5 Hz, 3 JHP = 6.2 Hz, H-Bn), 4.97–4.92 (m, 1 H, H-4’), 4.25–4.15 (m, 2 H, H-5’), 3.25–3.17 (m, 8 H, H-A), 2.57 (t, 4 H, 3JHH = 7.4 Hz, H-b), 1.89 (d, 3 H, 4 JHH = 1.0 Hz, Mehet), 1.77 (tt, 4 H, 3JHH = 7.5 Hz, 3JHH = 7.5 Hz, H-c), 1.68–1.58 (m, 8 H, H-B), 1.46–1.25 (m, 32 H, H-d, H-e, H-f, H-g, H-h, H-i, H-C), 1.01 (t, 12 H, 3JHH = 7.3 Hz, H-D), 0.90 ppm (t, 6 H, 3JHH = 6.9 Hz, H-j); 13C NMR (101 MHz, CD3OD) d = 173.6 (2 C-a), 166.5 (Chet-4), 152.7 (Chet-2), 152.4 (C-4), 152.4 (C-4), 138.5 (Chet-6), 135.3 (C-3’), 134.8 (d, 3JCP = 7.4 Hz, C-1), 134.8 (d, 3JCP = 7.4 Hz, C-1), 130.4 (d, 4JCP = 2.7 Hz, 4 C-2), 127.5 (C-2’), 122.5 (4 C-3), 112.1 (Chet-5), 90.7 (C-1’), 86.9 (d, 3JCP = 9.0 Hz, C-4’), 70.2 (d, JCP = 5.8 Hz, C-Bn), 70.2 (d, JCP = 5.8 Hz, C-Bn), 68.0 (d, 2JCP = 6.3 Hz, C-5’), 59.5 (pt, 4 CA), 35.0 (2 C-b), 33.0, 30.5, 30.4, 30.3, 30.1, 25.9, 24.7, 23.6, 20.6, 19.3 (2 C-c, 2 C-d, 2 C-e, 2 C-f, 2 C-g, 2 C-h, 2 C-i, 4 C-B, 4 C-C), 14.4 (2 C-j), 13.8 (4 C-D), 12.5 ppm (Mehet); 31P NMR (162 MHz, 1H-decoupled, CD3OD) d = 12.15 (d, 1P, 2JPP = 20.6 Hz, P-a), 12.93 ppm (d, 1P, 2JPP = 20.5 Hz, P-b); IR: n˜ = 2926, 2855, 1758, 1691, 1509, 1465, 1379, 1200, 1167, 1140, 1008, 981, 838, 505 cm1; UV (HPLC): lmax = 265 nm, HPLC: tR = 15.88 min, Method A; Rf : 0.44 (EtOAc/MeOH, 7:3 v/v); HRMS (ESI): calcd 903.3604 [MH] , found 903.36035; Mr [Da]: 1146.37; C60H97N3O14P2. (N[nBu]4)-Bis-(4-dodecanoyloxybenzyl)-d4TDP 4 g: General procedure C with 153 mg bis-(tetra-n-butylammonium)-d4TMP (0.280 mmol, 1 equiv), 228 mg bis-(4-dodecanoyloxybenzyl)N,N-diisopropylphosphoramidite (0.319 mmol, 1.1 equiv) and 40.6 mg DCI (0.344 mmol, 1.2 equiv) in 3 mL CH3CN. The reaction mixture was stirred for 2 h and cooled to 25 8C followed by oxidation with 58 mL TBHP (0.32 mmol, 1.0 equiv). The crude product was purified by RP-18 flash chromatography (MeOH/H2O, 1:1, than 5:1 v/v). Yield: 211 mg (0.175 mmol, 63 %) colorless oil. 1H NMR (400 MHz, CD3OD) d = 7.65 (d, 1 H, 4JHH = 1.2 Hz, Hhet-6), 7.39–7.35 (m, 4 H, H2), 7.06–7.02 (m, 4 H, H-3), 6.95 (ddd, 1 H, 3JHH = 3.4 Hz, 3/4JHH = 1.7 Hz, 3/4JHH = 1.7 Hz, H-1’), 6.38 (ddd, 1 H, 3JHH = 6.0 Hz, 4JHH = 1.7 Hz, 4JHH = 1.7 Hz, H-3’), 5.84 (ddd, 1 H, 3JHH = 6.0 Hz, 3/4JHH = 2.3 Hz, 4JHH = 1.4 Hz, H-2’), 5.09 (dd, 4 H, 2JHH = 8.4 Hz, 3JHP = 6.4 Hz, ChemMedChem 2014, 9, 762 – 775

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CHEMMEDCHEM FULL PAPERS H-Bn), 4.97–4.93 (m, 1 H, H-4’), 4.25–4.15 (m, 2 H, H-5’), 3.25–3.19 (m, 8 H, H-A), 2.57 (t, 4 H, 3JHH = 7.4 Hz, H-b), 1.89 (d, 3 H, 4JHH = 1.2 Hz, Mehet), 1.77–1.61 (m, 12 H, H-c, H-B), 1.46–1.26 (m, 40 H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-C), 1.01 (t, 12 H, 3JHH = 7.4 Hz, HD), 0.89 ppm (t, 6 H, 3JHH = 6.9 Hz, H-l); 13C NMR (101 MHz, CD3OD) d = 173.6 (2 C-a), 166.5 (Chet-4), 152.7 (Chet-2), 152.4 (2 C-4), 138.5 (Chet-6), 135.3 (C-3’), 134.8 (d, 3JCP = 5.4 Hz, 2 C-1), 130.4 (d, 4JCP = 2.7 Hz, 4 C-2), 127.5 (C-2’), 122.8 (4 C-3), 112.1 (Chet-5), 90.7 (C-1’), 86.9 (d, 3JCP = 9.3 Hz, C-4’), 70.2 (d, JCP = 5.2 Hz, C-Bn), 70.2 (d, JCP = 5.2 Hz, C-Bn), 68.1 (d, 2JCP = 6.3 Hz, C-5’), 59.5 (pt, 4 C-A), 35.0 (2 C-b), 33.1, 30.6, 30.5, 30.4, 30.4, 30.2, 25.9, 24.7, 23.4, 20.6 (2 C-c, 2 C-d, 2 C-e, 2 C-f, 2 C-g, 2 C-h, 2 C-i, 2 C-j, 2 C-k, 4 C-B, 4 C-C), 14.4 (2 C-l), 13.8 (4 C-D), 12.5 ppm (Mehet); 31P NMR (162 MHz, 1H-decoupled, CD3OD) d = 12.10 (d, 1P, 2JPP = 20.4 Hz, P-a), 12.94 ppm (d, 1P, 2JPP = 20.9 Hz, P-b); IR: n˜ = 2926, 2855, 1758, 1691, 1509, 1379, 1200, 1140, 1008, 981, 914, 838, 505 cm1; UV (HPLC): lmax = 265 nm, HPLC: tR = 17.76 min, Method A; Rf : 0.46 (EtOAc/MeOH, 7:3 v/v); HRMS (ESI): calcd 959.4224 [MH] , found 959.42295; Mr [Da]: 1202.48; C64H105N3O14P2. (N[nBu]4)-Bis-(4-tetradecanoyloxybenzyl)-d4TDP 4 h: General procedure C with 149 mg bis-(tetra-n-butylammonium)-d4TMP (0.189 mmol, 1 equiv), 242 mg bis-(4-tetradecanoyloxybenzyl)N,Ndiisopropylphosphoramidite (0.304 mmol, 1.5 equiv) and 35.8 mg DCI (0.344 mmol, 1.2 equiv) in 3 mL CH3CN giving a suspension due to the more or less insoluble amidite. The reaction mixture was stirred for 91.5 h and cooled to 35 8C followed by oxidation with 56 mL TBHP (0.31 mmol, 1.5 equiv). The crude product was purified by RP-18 flash chromatography (MeOH/H2O, 1:1, than 5:1, than 6:1, finally 8:1 v/v). Yield: 159.9 mg (154.6 mmol, 67 %) colorless fluffy solid. 1H NMR (400 MHz, CD3OD) d [ppm] = 7.66 (d, 1 H, 4 JHH = 1.2 Hz, Hhet-6), 7.40–7.33 (m, 4 H, H-2), 7.06–7.02 (m, 4 H, H-3), 6.95 (ddd, 1 H, 3JHH = 3.5 Hz, 3/4JHH = 1.8 Hz, 3/4JHH = 1.8 Hz, H-1’), 6.38 (ddd, 1 H, 3JHH = 6.0 Hz, 4JHH = 1.6 Hz, 4JHH = 1.6 Hz, H-3’), 5.84 (ddd, 1 H, 3JHH = 5.9 Hz, 3/4JHH = 2.2 Hz, 4JHH = 1.3 Hz, H-2’), 5.08 (dd, 4 H, 2 JHH = 8.5 Hz, 3JHP = 6.5 Hz, H-Bn), 4.97–4.93 (m, 1 H, H-4’), 4.25–4.13 (m, 2 H, H-5’), 3.26–3.19 (m, 8 H, H-A), 2.57 (t, 4 H, 3JHH = 7.4 Hz, H-b), 1.89 (d, 3 H, 4JHH = 1.2 Hz, Mehet), 1.77–1.61 (m, 12 H, H-c, H-B), 1.46– 1.25 (m, 48 H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m, H-C), 1.02 (t, 12 H, 3JHH = 7.3 Hz, H-D), 0.89 (t, 6 H, 3JHH = 6.7 Hz, H-n); 13 C NMR (126 MHz, CD3OD, CH2Cl2) d = 173.7 (2 C-a), 166.5 (Chet-4), 152.7 (Chet-2), 152.4 (2 C-4), 138.5 (Chet-6), 135.3 (C-3’), 134.8 (2 C1), 130.4 (d, 4JCP = 4.3 Hz, 4 C-2), 127.5 (C-2’), 122.8 (4 C-3), 112.1 (Chet-5), 90.7 (C-1’), 86.9 (d, 3JCP = 9.3 Hz, C-4’), 70.2 (d, 2JCP = 5.2 Hz, C-Bn), 70.2 (d, 2JCP = 5.2 Hz, C-Bn), 68.1 (d, 2JCP = 6.3 Hz, C-5’), 59.5 (pt, 4 C-A), 54.7 (CH2Cl2), 35.0 (2 C-b), 25.9 (2 C-c), 24.7 (4 C-B), 33.1, 30.7, 30.7, 30.6, 30.5, 30.5, 30.4, 30.2, 23.6, 20.6, 19.3 (2 C-d, 2 C-e, 2 C-f, 2 C-g, 2 C-h, 2 C-i, 2 C-j, 2 C-k, 2 C-l, 2 C-m, 4 C-C), 14.4 (2 C-n), 13.8 (4 C-D), 12.5 ppm (Mehet); 31P NMR (162 MHz, 1H-decoupled, CD3OD) d = 12.09 (d, 1P, 2JPP = 20.9 Hz, P-a), 12.91 ppm (d, 1P, 2JPP = 19.9 Hz, P-b); IR: n˜ = 2916, 2849, 1756, 1687, 1508, 1466, 1252, 1142, 1111, 1003, 974, 914, 836, 503 cm1; UV (HPLC): lmax = 265 nm, HPLC: tR = 19.23 min, Method A; Rf : 0.73 (EtOAc/MeOH, 7:3 v/v); MS (FAB): calcd 1016.48 [M], 1039.47 [M + Na] + , 1061.45 [MH + + 2 Na + ] found 1039.5, 1061.5; Mr [Da]: 1258.58; C68H113N3O14P2. (N[nBu]4)-Bis-(4-hexadecanoyloxybenzyl)-d4TDP 4 i: General procedure C with 160 mg bis-(tetra-n-butylammonium)-d4TMP (0.203 mmol, 1 equiv), 280 mg bis-(4-hexadecanoyloxy-benzyl)N,Ndiisopropylphosphoramidite (0.328 mmol, 1.6 equiv) and 50.4 mg DCI (0.427 mmol, 2.1 equiv) in 3 mL CH3CN and 2 mL dichloromethane. The reaction mixture was stirred for 116 h and cooled to 30 8C followed by oxidation with 60 mL TBHP (0.31 mmol, 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org 1.5 equiv). The crude product was purified by RP-18 flash chromatography (MeOH/H2O, 1:1, than 5:1, than 6:1, finally 15:1 v/v). Yield: 132 mg (0.100 mmol, 49 %) colorless fluffy solid. 1H NMR (400 MHz, CD3OD) d = 7.66 (d, 1 H, 4JHH = 1.2 Hz, Hhet-6), 7.39–7.34 (m, 4 H, H-2), 7.06–7.02 (m, 4 H, H-3), 6.95 (ddd, 1 H, 3JHH = 3.4 Hz, 3/4 JHH = 1.7 Hz, 3/4JHH = 1.7 Hz, H-1’), 6.38 (ddd, 1 H, 3JHH = 6.0 Hz, 4 JHH = 1.7 Hz, 4JHH = 1.7 Hz, H-3’), 5.84 (ddd, 1 H, 3JHH = 5.9 Hz, 4JHH = 1.3 Hz, 3/4JHH = 2.2 Hz, H-2’), 5.08 (dd, 4 H, 2JHH = 8.5 Hz, 3JHP = 6.4 Hz, H-Bn), 4.98–4.92 (m, 1 H, H-4’), 4.27–4.12 (m, 2 H, H-5’), 3.27–3.18 (m, 8 H, H-A), 2.56 (t, 4 H, 3JHH = 7.4 Hz, H-b), 1.89 (d, 3 H, 4JHH = 1.1 Hz, Mehet), 1.77–1.61 (m, 12 H, H-c, H-B), 1.46–1.25 (m, 56 H, H-d, H-e, H-f, H-g, H-h, H-i, H-j, H-k, H-l, H-m, H-n, H-o, H-C), 1.02 (t, 12 H, 3JHH = 7.5 Hz, H-D), 0.89 ppm (t, 6 H, 3JHH = 6.7 Hz, H-p); 13 C NMR (101 MHz, CD3OD) d = 173.6 (2 C-a), 166.5 (Chet-4), 152.7 (Chet-2), 152.4 (2 C-4), 138.6 (Chet-6), 135.3 (C-3’), 134.8 (2 C-1), 130.4 (d, 4JCP = 3.3 Hz, 4 C-2), 127.5 (C-2’), 122.8 (4 C-3), 112.1 (Chet-5), 90.7 (C-1’), 86.9 (d, 3JCP = 9.4 Hz, C-4’), 70.2 (d, JCP = 5.6 Hz, C-Bn), 70.2 (d, JCP = 5.6 Hz, C-Bn), 68.0 (d, 2JCP = 6.1 Hz, C-5’), 59.5 (pt, 4 C-A), 35.0 (2 C-b), 25.9 (2 C-c), 24.7 (4 C-B), 33.0, 30.7, 30.6, 30.6, 30.5, 30.4, 30.4, 30.2, 23.6, 20.6, 19.3 (2 C-d, 2 C-e, 2 C-f, 2 C-g, 2 C-h, 2 C-i, 2 C-j, 2 C-k, 2 C-l, 2 C-m, 2 C-n, 2 C-o, 4 C-C), 14.4 (2 C-p), 13.8 (4 C-D), 12.4 ppm (Mehet); 31P NMR (162 MHz, 1H-decoupled, CD3OD) d = 12.08 (d, 1P, 2JPP = 20.07 Hz, P-a), 12.92 ppm (d, 1P, 2JPP = 20.21 Hz, P-b); IR: n˜ = 2915, 2848, 1757, 1687, 1466, 1143, 979, 915, 878, 780, 764, 721, 574, 503 cm1; UV (HPLC): lmax = 265 nm, HPLC: tR = 20.54 min, Method A; Rf : 0.61 (EtOAc/MeOH, 7:3 v/v); HRMS (ESI): calcd 1072.555 [M] , 1071.5478 [MH] , found 1071.5470 [MH] ; Mr [Da]: 1314.69; C72H121N3O14P2. (N[nBu]4)-Bis-(4-(Z)-octadec-9-enoyloxybenzyl)-d4TDP 4 j: General procedure C with 144 mg bis-(tetra-n-butylammonium)-d4TMP (0.264 mmol, 1 equiv), 263 mg bis-(4-(Z)-octadec-9-enoyloxybenzyl)-N,N-diisopropylphosphoramidite (0.290 mmol, 1.1 equiv) and 37.1 mg DCI (0.314 mmol, 1.2 equiv) in 2 mL CH3CN and 2 mL dichloromethane. The reaction mixture was stirred for 16 h and cooled to 30 8C followed by oxidation with 58 mL TBHP (0.26 mmol, 1.0 equiv). The crude product was purified by RP-18 flash chromatography (MeOH/H2O, 1:1, than 10:1 v/v). Yield: 105 mg (76.0 mmol, 29 %) colorless oil. 1H NMR (400 MHz, CD3OD) d = 7.65 (d, 1 H, 4JHH = 1.1 Hz, Hhet-6), 7.40–7.33 (m, 4 H, H-2), 7.06– 3 JHH = 3.4 Hz, 7.00 (m, 4 H, H-3), 6.95 (ddd, 1 H, 3/4 3/4 JHH = 1.7 Hz, JHH = 1.7 Hz, H-1’), 6.38 (ddd, 1 H, 3JHH = 7.8 Hz, 4 JHH = 1.8 Hz, 4JHH = 1.8 Hz, H-3’), 5.84 (ddd, 1 H, 3JHH = 6.0 Hz, 3/4JHH = 2.2 Hz, 4JHH = 1.4 Hz, H-2’), 5.39–5.34 (m, 4 H, H-i, H-j), 5.08 (dd, 4 H, 2 JHH = 8.5 Hz, 3JHP = 6.1 Hz, H-Bn), 4.97–4.93 (m, 1 H, H-4’), 4.25–4.13 (m, 2 H, H-5’), 3.25–3.18 (m, 8 H, H-A), 2.56 (t, 4 H, 3JHH = 7.4 Hz, H-b), 2.13–1.98 (m, 8 H, H-h, H-k), 1.89 (s, 3 H, Mehet), 1.77–1.61 (m, 12 H, H-c, H-B), 1.47–1.25 (m, 48 H, H-d, H-e, H-f, H-g, H-l, H-m, H-n, H-o, H-p, H-q, H-C), 1.02 (t, 12 H, 3JHH = 7.3 Hz, H-D), 0.88 ppm (t, 6 H, 3 JHH = 6.5 Hz, H-r); 13C NMR (101 MHz, CD3OD) d = 173.6 (2 C-a), 166.5 (Chet-4), 152.4 (Chet-2), 150.5 (2 C-4), 138.6 (Chet-6), 135.3 (C3’), 130.9, 130.7 (2 C-i, 2 C-j), 130.4 (d, 4JCP = 3.1 Hz, 4 C-2), 129.6, 127.5 (C-2’), 122.8 (4 C-3), 112.0 (Chet-5, hmbc), 90.7 (C-1’), 86.9 (d, 3JCP = 9.4 Hz, C-4’), 70.2 (m, 2 C-Bn), 68.7 (d, 2JCP = 5.5 Hz, C-5’), 59.5 (pt, 4 C-A), 35.0 34.9 (2 C-b), 33.0, 30.7, 30.7, 30.6, 30.5, 30.4, 30.4, 30.3, 30.3, 30.2, 28.1, 28.1, 25.9, 24.7, 23.4, 20.6 (2 C-c, 2 C-d, 2 C-e, 2 C-f, 2 C-g, 2 C-h, 2 C-k, 2 C-l, 2 C-m, 2 C-n, 2 C-o, 2 C-p, 2 C-q, 4 C-C, 4 C-B), 14.4 (2 C-r), 13.8 (4 C-D), 12.4 ppm (Mehet); 31P NMR (162 MHz, 1H-decoupled, CD3OD) d = 12.06 (d, 1P, 2JPP = 20.98 Hz, P-a), 12.91 ppm (d, 1P, 2 JPP = 20.24 Hz, P-b); 31P NMR (162 MHz, 1H-decoupled, CDCl3) d = 11.73 ppm (s, 2P, P-a, P-b); IR: n˜ = 2959, 2925, 2874, 2854, 1755, 1687, 1464, 1244, 1044, 503 cm1; UV (HPLC): lmax = 265 nm; HPLC: ChemMedChem 2014, 9, 762 – 775

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CHEMMEDCHEM FULL PAPERS tR = 19.86 min, Method A; Rf : 0.68 (EtOAc/MeOH, 7:3 v/v); HRMS (ESI): calcd 1123.5795 [MH] , found 1123.5795 [MH] ; Mr [Da]: 1366.76; C76H125N3O14P2.

Bioassays Chemical hydrolysis studies of DiPPro-d4TDP compounds 4 b–j and 13: 50 mm DMSO stock solutions of the DiPPro-d4TDP compounds were prepared; 300 mL of a 1.9 mm hydrolysis solution of the appropriate compound were prepared by dilution of 11 mL of stock solution in 100 mL Millipore water and 189 mL [D6]DMSO. The reaction was started by addition of 300 mL of 50 mm phosphate buffered saline (PBS, pH 6.8, 7.3, 8.7) at room temperature in order to reach the final concentrations (0.94 mm of BAB-d4TDP 5, 24.8 mm buffer salts). This kinetic solution was incubated at 37 8C. To monitor the reaction, aliquots of 30 mL were taken from the kinetic solution and frozen (liquid nitrogen); 20 mL of these samples, including a starting sample directly taken after addition of PBS, were analyzed by analytical HPLC at 263–265 nm. Calculation of exponential decay curves with commercially available software (Microsoft excel) yielded the half-life (t1/2) of the prodrug compounds in hours. For each compound, two to three determinations of t1/2 were performed and the resulting values were averaged. Hydrolysis studies of DiPPro-d4TDP compounds 4 b–j and 13 in CEM/ 0, FCS, human serum, fetal calf serum and RPMI culture medium: Hydrolysis in CEM/0 cell extract: 30 mL of the appropriate 50 mm DMSO stock solution was diluted to 6.0 mm hydrolysis solution by addition of 220 mL DMSO. Human CEM/0 cell extract (100 mL, 33 % cell extract in PBS, pH 6.8) were mixed with 20 mL of a 70 mm aqueous magnesium chloride solution. The reaction was started by addition of 20 mL of 6.0 mm hydrolysis solution of the appropriate compound 4 to the cell extract solution; 6–8 different samples of the above mentioned mixture were incubated at 37 8C for different periods of hydrolysis (depending on rate of hydrolysis). The reaction was stopped by addition of 300 mL MeOH and the solutions were kept on ice for 5 min followed by ultrasonification for 10– 15 min. After centrifugation the supernatant was filtered (Schleicher & Schuell Spartan 13/30, 0.2 mm) and stored in liquid nitrogen. Samples were defrosted and 90 mL were subject of HPLC analysis. The calculation of t(1/2) was performed analogously to that for the chemical hydrolysis studies using the absolute integral values. Identification of the hydrolysis products was based on the retention times of the reference compounds under identical analytical conditions or by co-injection. Hydrolysis studies in FCS, human serum (obtained from pooled blood samples from the University Medical Center (UKE) in Hamburg), and RPMI culture medium were done as described for the hydrolysis of compounds 4 b--j and 13 in CEM/0. FCS (10 %) and human serum (20 %) were diluted in PBS (pH 6.8). Preparation of CEM cell extracts: Human CD4 + T-lymphocyte CEM cells were grown in RPMI-1640-based cell culture medium to a final density of ~ 3 106 cells mL1. Then, cells were centrifuged for 10 min at 1250 rpm, 4 8C, washed twice with cold phosphatebuffered saline (PBS) and the pellet was resuspended at 108 cells mL1 and sonicated (3 10 sec) to destroy the cell integrity. The resulting cell suspension was then centrifuged at 10 000 rpm to remove cell debris and the supernatant divided in aliquots before being frozen at 80 8C and used. Antiviral assays: Inhibition of HIV-1(IIIB)- and HIV-2(ROD)-induced cytopathicity in wild-type CEM/0 or thymidine kinase-deficient CEM/ TK cell cultures was measured in microtiter 96-well plates containing ~ 3 105 CEM cells/mL infected with 100 CCID50 of HIV per milli 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org liter and containing appropriate dilutions of the test compounds. After 4–5 days of incubation at 37 8C in a CO2-controlled humidified atmosphere, CEM giant (syncytium) cell formation was examined microscopically. The EC50 (50 % effective concentration) was defined as the compound concentration required to inhibit HIV-induced giant cell formation by 50 %.

Supporting Information HPLC hydrolysis studies of compounds 4 f, 4 i, 5 f, and 13 as well as an NMR study following the hydrolysis of DiPPro compound 4 d.

Acknowledgements C.M. and T.S. thank Sandra Mhmel and Thiago Dinis de Oliveira (University of Hamburg, Germany) for practical assistance, and the Deutsche Forschungsgemeinschaft (DFG) and the University of Hamburg for financial support of this work. J.B. is grateful to Ria Van Berwaer (Rega Institute for Medical Research, Belgium) for technical assistance. Financial support to J.B. was provided by the KU Leuven (GOA 10/014). Keywords: antivirals · bioreversible protection · nucleoside diphosphates · prodrugs · pronucleotides [1] J. Balzarini, P. Herdewijn, E. De Clercq, J. Biol. Chem. 1989, 264, 6127 – 6133. [2] J. Balzarini, Pharm. World Sci. 1994, 16, 113 – 126. [3] T. Cihlar, A. S. Ray, Antiviral Res. 2010, 85, 39 – 58. [4] C. Meier, T. Knispel, E. De Clercq, J. Balzarini, J. Med. Chem. 1999, 42, 1604 – 1614. [5] C. Meier, A. Lomp, A. Meerbach, P. Wutzler, J. Med. Chem. 2002, 45, 5157 – 5172. [6] Y. Mehellou, J. Balzarini, C. McGuigan, ChemMedChem 2009, 4, 1779 – 1791. [7] M. Derudas, D. Carta, A. Brancale, C. Vanpouille, A. Lisco, L. Margolis, J. Balzarini, C. McGuigan, J. Med. Chem. 2009, 52, 5520 – 5530. [8] S. J. Hecker, M. D. Erion, J. Med. Chem. 2008, 51, 2328 – 2345 and citations therein. [9] a) C. Meier, Eur. J. Org. Chem. 2006, 1081 – 1102; b) C. Meier, M. Lorey, E. De Clercq, J. Balzarini, J. Med. Chem. 1998, 41, 1417 – 1427; c) H. J. Jessen, J. Balzarini, C. Meier, J. Med. Chem. 2008, 51, 6592 – 6598; d) N. Gisch, J. Balzarini, C. Meier, J. Med. Chem. 2009, 52, 3464 – 3473; e) E. H. R. Morales, J. Balzarini, C. Meier, Chem. Eur. J. 2011, 17, 1649 – 1659; f) E. H. R. Morales, C. A. Romn, J. O. Thomann, C. Meier, Eur. J. Org. Chem. 2011, 4397 – 4408. [10] A. Lavie, I. Schlichting, I. R. Vetter, M. Konrad, J. Reinstein, R. S. Goody, Nat. Med. 1997, 3, 922 – 924. [11] A. Lavie, I. R. Vetter, M. Konrad, R. S. Goody, J. Reinstein, I. Schlichting, Nat. Struct. Biol. 1997, 4, 601 – 604. [12] J. P. Sommadossi, R. Carlisle, Z. Zhou, Mol. Pharmacol. 1989, 36, 9 – 14. [13] J. A. Harrington, J. E. Reardon, T. Spector, Antimicrob. Agents Chemother. 1993, 37, 918 – 920. [14] J.-P. Yan, D. D. Ilsley, C. Frohlick, R. Steet, E. T. Hall, R. D. Kuchta, P. Melancon, J. Biol. Chem. 1995, 270, 22836 – 22841. [15] K. Y. Hostetler, L. M. Stuhmiller, H. B. M. Lenting, H. Van den Bosch, D. D. Richman, J. Biol. Chem. 1990, 265, 6112 – 6117. [16] G. M. T. van Wijk, K. Y. Hostetler, H. van den Bosch, Biochim. Biophys. Acta Lipids Lipid Metab. 1991, 1084, 307 – 310. [17] K. Y. Hostetler, D. D. Richman, D. A. Carson, L. M. Stuhmiller, G. M. T. van Wijk, H. van den Bosch, Antimicrob. Agents Chemother. 1992, 36, 2025 – 2029. [18] K. Y. Hostetler, S. Parker, C. N. Sridhar, M. J. Martin, J. L. Li, L. M. Stuhmiller, G. M. T. van Wijk, H. van den Bosch, M. F. Gardner, Proc. Natl. Acad. Sci. USA 1993, 90, 11835 – 11839.

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Received: December 2, 2013 Published online on March 11, 2014

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