A novel and efficient one-pot synthesis of symmetrical diamide (bis-amidate) prodrugs of acyclic nucleoside phosphonates and evaluation of their biological activities

European Journal of Medicinal Chemistry 46 (2011) 3748e3754

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Original article

A novel and efficient one-pot synthesis of symmetrical diamide (bis-amidate) prodrugs of acyclic nucleoside phosphonates and evaluation of their biological activities  ski a, Martin Dra k Zídek b, Gina Bahador c, Petr Jansa a, *, Ondrej Baszczyn cínský a, Ivan Votruba a, Zdene c c c a George Stepan , Tomas Cihlar , Richard Mackman , Antonín Holý , Zlatko Janeba a, * a b c

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic v.v.i., Flemingovo nám. 2, 16610 Prague 6, Czech Republic  ská 1083, 142 20 Prague 4, Czech Republic Institute of Experimental Medicine, Academy of Sciences of the Czech Republic v.v.i., Víden Gilead Sciences, Inc., Foster City, CA 94404, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 April 2011 Received in revised form 12 May 2011 Accepted 14 May 2011 Available online 23 May 2011

A novel and efficient method for the one-pot synthesis of diamide (bis-amidate) prodrugs of acyclic nucleoside phosphonates, starting from free phosphonic acids or phosphonate diesters is reported. The approach from phosphonate diesters via their bis(trimethylsilyl) esters is highly convenient, eliminates isolation and tedious purification of the phosphonic acids, and affords the corresponding bis-amidates in excellent yields (83e98%) and purity. The methodology has been applied to the synthesis of the potent anticancer agent GS-9219, and symmetrical bis-amidates of other biologically active phosphonic acids. Anti-HIV, antiproliferative, and immunomodulatory activities of the compounds are discussed including the bis-amidate prodrugs 14 and 17 that exhibited anti-HIV activity at submicromolar concentrations with minimal cytotoxicity. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Prodrugs Phosphonodiamides Bis-amidates Acyclic nucleoside phosphonates GS-9219

1. Introduction Phosphate and phosphonate diacids are deprotonated at physiological pH and consequently exhibit poor cellular permeability. Masking their charge by lipophilic functionalities enables drug delivery limitations such as poor oral bioavailability to be overcome. A variety of prodrugs of phosphates, phosphonates, and phoshinates have been designed, developed, and thoroughly studied over the past two decades and several comprehensive reviews on this topic have been published [1e4]. Acyclic nucleoside phosphonates (ANPs) represent catabolically stable nucleotide analogs with a variety of antiviral properties [5]. The rational exploration of prodrug designs for ANPs resulted in the discovery and subsequent clinical approval of tenofovir disoproxil fumarate (VireadÒ) and adefovir dipivoxil (HepseraÒ for the treatment of HIV/HBV and HBV infections, respectively.

* Corresponding authors. Tel.: þ420 220183109/220183143; fax: þ420 220183560. E-mail addresses: [email protected] (P. Jansa), [email protected] (Z. Janeba). 0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2011.05.040

McGuigan and coworkers have reported a prodrug approach for nucleoside monophosphates in which an amino acid ester moiety is attached to the phosphorus through the nitrogen to form either a mono-phosphoramidate or bis-amidate (ProTide) [6]. Recently, this approach was also applied to a novel cyclic nucleoside phosphonate HIV reverse transcriptase inhibitor GS-9148, and led to the selection of the phenyloxy ethylalaninyl prodrug GS-9131 as a promising clinical candidate [7]. The amidate prodrug strategy has also been successfully applied to ANPs. Phosphonamidate prodrugs of adefovir (PMEA) and tenofovir (PMPA) were prepared by Ballatore et al. [8], and possessed improved anti-HIV activity compared to the parent ANPs. A number of asymmetrical phenyloxy amidates, as well as symmetrical bis-amidates of tenofovir have been explored by Gilead Sciences, leading to the identification of GS-7340, an isopropylalaninyl monoamidate phenyl monoester prodrug of tenofovir with improved biological properties [9]. Recently, cathepsin A was found to be the primary enzyme that activates the phosphonoamidate prodrugs GS-7340 and GS-9131 in human lymphatic tissues [10]. Bis-amidates are also effective prodrugs for ANPs and allowed for efficient and selective cellular delivery of cPrPMEDAP [11]. After prodrug cleavage the cPrPMEDAP is intracellularly deaminated by

P. Jansa et al. / European Journal of Medicinal Chemistry 46 (2011) 3748e3754

N6-methyl-AMP aminohydrolase to yield the potent antiproliferative and antiviral agent PMEG [12] (1, Fig. 1). The antiproliferative bisamidate agents GS-9219 (2) and GS-9191 (3) are drug candidates for the treatment of non-Hodgkins lymphomas and human papillomavirus-associated proliferative disorders, respectively. Recently, a new class of 5-substituted furan-2-ylphosphonates, mimicking AMP, was invented and studied as a promising class of fructose-1,6-bisphosphatase inhibitors. Several prodrugs were investigated and bis-amidate derivatives exhibit highest oral bioavailability and efficacy in the rat model of diabetes [13,14]. Typically, the bis-amidate prodrugs are prepared from free phosphonic acids using 2,20 -dithiodipyridine (AldrithiolÔ-2) and triphenylphosphine activation [15,16] followed by treatment with the corresponding amino acid ester [7,17]. The yields of the isolated bis-amidates are variable (11e74%) and may depend on the amino acid [7]. Another common approach exploits the conversion of the phosphonic acid to its dichloride using oxalyl or thionyl chloride, followed by condensation with the appropriate amino acid ester, to give the desired bis-amidates in 20e55% yields [13,18]. Herein we report on an efficient and simple method of the synthesis of acyclic nucleoside phosphonate bis-amidates. The success of the reported route enabled a selection of novel ANP prodrugs to be prepared and evaluated for their antiviral, immunomodulatory, and antiproliferative activity. 2. Chemistry The free phosphonic acids are commonly prepared from the alkyl diesters. Deprotection of the phosphonate diester with TMShalides [19,20] forms the bis(trimethylsilyl) ester in situ which is further hydrolyzed to the final phosphonic acid before isolation. The purity of the phosphonic acid is often a key factor in efficient and reliable transformations to the prodrug compounds and therefore tedious purifications are often required. Such procedures usually include separations on various ion-exchange resins, followed by either crystallization or laborious HPLC chromatography. It was rationalized that the reaction of the intermediate bis(trimethylsilyl) ester of ANPs with 2,20 dithiodipyridine, triphenylphosphine in the presence of the amine would also yield the corresponding bis-amidates without the need for isolation of the diacid (Scheme 1). The starting compounds 4e13 were prepared according to the literature, except using the bromomethylphosphonate reagent [21] instead of tosyloxymethylphosphonate derivatives. Initially the phosphonic diacids 4e6 (entries 1e3, Table 1, Scheme 1) were treated with excess (5 eq.) of TMSBr in dry acetonitrile overnight to form the corresponding bis(trimethylsilyl) ester intermediate A. TMSBr acts both as a reagent and as a dehydrating agent since traces of moisture were expected to significantly lower the yields of the desired bis-amidates. The residue, after evaporation of volatiles, was treated with ethyl alanine, Aldrithiol-2, and triphenylphosphine in a mixture of triethylamine and pyridine, to afford bisamidates 14e16 in high yields (50e67%; entries 1, 2 and 3,

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Scheme 1. General procedure for the synthesis of bis-amidates.

Table 1). Further simplification of the procedure included the direct use of the diisopropyl phosphonate esters rather than the diacids, thereby eliminating the laborious isolation, purification and drying of free phosphonic acids. Thus, treatment of diisopropyl phosphonates 7 and 9 (entries 4 and 6, Table 1, Scheme 1) using the above procedure afforded the desired bis-amidates 17 and 18 in 87% and 83% yields, respectively. The analogous reactions starting from the corresponding free phosphonic acids 8 and 10 (entries 5 and 7, Table 1) afforded the same products 17 and 18 but in lower yields of 70% and 65%, respectively. Although these yields are satisfactory, the more practical one-pot procedure starting from the phosphonate diesters was a superior method for prospective larger scale reaction. To establish general applicability, our novel approach to bisamidate prodrugs was applied to several structurally diverse ANPs. Transformation of the compound 11 with a sterically bulky cyclooctylamino substituent at the C-6 position of the purine moiety gave the amidate 19 in 94% yield (entry 8, Table 1), while amidate 20 was prepared from the “open ring” analog 12 in 90% yield (entry 9, Table 1). The new method was also proven by attempting the synthesis of the antiproliferative agent GS-9219 (2, Fig. 1) which was previously prepared from the corresponding diacid in a moderate yield of 43% [22]. The cPrPMEDAP diisopropyl ester 13 (entry 10, Table 1) was treated with the procedure described and afforded prodrug 2 in 92% yield on a 1 mmol scale. A large scale preparation (10 mmol, entry 11, Table 1) also resulted in 98% yield of GS-9219 clearly demonstrating the superiority of this novel method. Finally, a convenient procedure for isolation and purification of the final bis-amidates has also been developed by means of a sequence of extractions followed by flash chromatography. Overall, the improved method of the bis-amidate preparation and the efficient isolation are favorable developments in the synthesis and scale-up of bis-amidate drug candidates. 3. Biological results 3.1. Antiretroviral activity The newly prepared bis-amidates were evaluated for their antiretroviral activity in the MT-2 T-lymphoblastoid cells infected with HIV-1, strain IIIb (Table 2). Several bis-amidates (14, 15, 17)

Fig. 1. PMEG (1) and its bis-amidate double prodrugs GS-9219 (2) and GS-9191 (3).

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P. Jansa et al. / European Journal of Medicinal Chemistry 46 (2011) 3748e3754

Table 1 Synthesis of bis-amidates of ANPs via bis(trimethylsilyl) intermediates. Entry

1

Starting material

Yielda

Product

4

2

Table 2 Anti-HIV-1 (MT2-IIIBLUC) and cytotoxic properties of ANPs and their bis-amidates.

5

14

15

b

67%

64%b

Acid

EC50a (mM)

CC50b (mM)

Bis-amidate

EC50a (mM)

CC50b (mM)

4 5 6 8 Diacid of 12

7.04 >85.3 13.25 0.94 0.93

>100 >100 >100 >100 97.70

14 15 16 17 20

0.54 5.51 10.73 0.29 1.13

>100 >100 >100 >100 73.30

a Effective concentration required to reduce the HIV-1-dependent cytopathic effect in MT-2 by 50%. b Cytotoxic concentration required to inhibit the MT-2 cell proliferation by 50%.

3.2. Antiproliferative activity 3

6

16

50%b

4

7

17

87%b

5

8

17

70%b

6

9

18

83%b

7

10

18

65%b

8

11

19

94%b

9

12

20

90%b

13

2

92%b 98%c

10

11 a b c

Isolated yields (the compounds are hygroscopic). 1 mmol scale. 10 mmol scale.

displayed improved antiviral properties (3e10-fold) compared to the corresponding free phosphonic acids (4, 5, and 8, respectively). Bis-amidates 14 and 17 exhibited anti-HIV activity with submicromolar EC50 values and no detectable cytotoxicity up to 100 uM, the highest concentration tested. Interestingly, there was no improvement in antiviral activity for bis-amidates 16 and 20, suggesting that these prodrugs may not be efficiently activated by cathepsin A or other hydrolases inside target cells.

The sensitivity of Hep G2, HL60, HeLa S3, and CCRF-CEM cell lines toward the bis-amidates of PMEG (compound 18), and cPrPMEDAP (compound 2; GS-9219) was determined by an XTT-based cell viability test. The data show (Table 3) that the compound 2 exhibits 3- to 4-fold improved antiproliferative activity in HL60 and CCRFCEM cells relative to compound 18. On the other hand, the antiproliferative activity of both compounds is similar in Hep G2 and HeLa S3 cell lines. This could potentially be explained by differences in the intracellular hydrolysis of the two bis-amidate prodrugs or, slower rate of conversion of cPrPMEDAP to PMEG in Hep G2 and HeLa S3 compared to HL60 and CCRF-CEM cells. This process is catalyzed by N6-methylAMP aminohydrolase [23,24] and sufficient intracellular activity of this enzyme is crucial for potent antiproliferative activity of cPrPMEDAP and its prodrug 2. We can also speculate that the reduced growth inhibition of the prodrug 2 in Hep G2 and HeLa S3 cells could be due to the dealkylation of cPrPMEDAP to PMEDAP. This metabolic conversion was detected in the dog liver [25]. These results support the notion that high intracellular concentrations of PMEG after administration of liphophilic prodrug 18 may not be as therapeutically promising as prolonged and controlled releasing of PMEG by its double prodrug 2.

3.3. Immunomodulatory activity It has been previously shown that several ANPs, and especially N6 substituted analogs, possess immunostimulatory activity [26,27]. There was virtually no constitutive production of NO by resident mouse peritoneal cells. In contrast, a positive control of IFN-g alone or in combination with LPS significantly enhanced the NO biosynthesis. The prepared bis-amidate prodrugs were tested at concentrations up to 25 mM and neither inhibited nor significantly enhanced the immune-stimulated NO production. The production of NO has been shown to correlate with the activation of cytokine secretion [28]. The lack of effects of compound 2 (GS-9219) on the NO production suggests that the intact prodrug itself does not possess immunostimulatory activity. Similarly, none of the other prepared prodrugs stimulate the production of NO. It should be noted however that in previous studies [26] the free phosphonic acid of compound 19 showed high potential to stimulate NO production. This could be explained by

Table 3 Antiproliferative activity of compounds 2 and 18 in Hep G2, HL60, HeLa S3 and CCRFCEM cell lines. Bis-amidate

18 2

Cell growth inhibition e IC50, mmol l1 Hep G2

HL60

HeLa S3

CCRF-CEM

2.32  0.17 1.74  0.16

3.02  0.08 0.78  0.01

3.94  0.37 2.15  0.16

1.22  0.15 0.48  0.03

P. Jansa et al. / European Journal of Medicinal Chemistry 46 (2011) 3748e3754

a different or limited metabolic activation of bis-amidate prodrugs in murine macrophages compared to human cell lines used for the antiviral and antiproliferative screening, or by the presence of LPS (or other constituents of bacterial cell wall as e.g. lipoteichoic acid) in the free phosphonic acid samples tested previously [26]. The further study on the effect of the LPS presence in the ANPs samples on the immunostimulatory activity is currently in progress. 4. Conclusion The preparation of bis-amidate prodrugs of acyclic nucleoside phosphonate analogs was significantly improved by the development of a novel and efficient one-pot synthesis that directly transforms the phosphonic acid diesters to their respective prodrugs. The new procedure can also be effectively applied to the phosphonic acids. In this new approach, the reaction of phosphonic acid (or diester) with TMSBr yields the bis(trimethylsilyl) ester intermediate that in turn can be reacted with amino acid esters, in the presence of 2,20 -dithiodipyridine (Aldrithiol-2), and triphenylphosphine, to generate symmetrical bis-amidates. One previously synthesized bis-amidate (GS-9219) and seven novel, structurally diverse bis-amidates were prepared by this efficient methodology and their biological activities were evaluated. Bis-amidates of compounds 4 and 8 exhibited anti-HIV activity at submicromolar concentrations with no observed cytotoxicity at the highest tested concentration. In addition, results of the antiproliferative activity assays in human cancer cell lines of different origin demonstrated slightly more potent antiproliferative activity for compound 2 (GS9219) compared to compound 18. Thus, a double prodrug of PMEG that combines a guanine base modification with masking of the phosphonic acid by bis-amidate moiety (2) provides a benefit in terms of potency compared to the single prodrug of PMEG (18) with the same bis-amidate prodrug part. Finally, none of the prepared compounds displayed any immunomodulatory activities when tested in the NO screening platform. 5. Experimental section 5.1. Materials and methods Solvents were dried by standard procedures. Bromotrimethylsilane, Aldrithiol-2, and triphenylphosphine are commercially available from SigmaeAldrich. L-Alanine ethyl ester hydrochloride was dried in vacuo at 30  C and 0.1 mbar for 1 day. TLC was performed on plates of Kieselgel 60 F254 (Merck). NMR spectra were recorded Bruker Avance 500 (1H at 500.0 MHz, 13C at 126 MHz). Chemical shifts (in ppm, d scale) were referenced to the solvent signal (DMSO-d6 for 1H NMR d ¼ 2.5 ppm and for 13C NMR d ¼ 39.7). Mass spectra were measured on an LCQ classic spectrometer using electrospray ionization (ESI). Optical rotations were measured on an AUTOPOL IV polarimeter (Rudolph research analytical) at 20  C; [a]D values are given [101 deg cm2 g1]. All new compounds were fully characterized by mass spectrometry, NMR spectroscopy, and elemental analysis. 5.2. General procedure for preparation of phosphoramidate prodrugs A mixture of phosphonic acid diester (typically diisopropyl or diethyl ester, 1 mmol), dry acetonitrile (10 mL), and bromotrimethylsilane (5 mmol) was stirred overnight at room temperature under inert atmosphere. After evaporation (without any contact with air) in vacuo (40  C, 2 mbar) and codistillation with dry toluene (without any contact with air), the flask was purged with argon and amino acid ester hydrochloride (4 mmol, dried in

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vacuo at 30  C and 0.1 mbar for 1 day), dry triethylamine (2 mL), and dry pyridine (8 mL) were added and this mixture was heated to 50  C to obtain a homogenous solution and a solution of Aldrithiol-2 (6 mmol) and triphenylphosphine (6 mmol) in 10 mL of dry pyridine under argon was added immediately. The resulting mixture was heated at 50  C for 3 h to reach the full conversion. After cooling, the bright yellow solution was evaporated on the rotary evaporator in vacuo (40  C, 2 mbar) and then a mixture of methanol (10 mL), water (10 mL), toluene (10 mL), and hexane (10 mL) was added to the dry residue and this mixture was transferred into a separatory funnel. The upper phase containing toluene and hexane was removed and the phase containing methanol and water was extracted with 20 mL of the mixture of toluene and hexane 3 more times. The phase containing methanol and water was then extracted 3 times with 20 mL of the appropriate organic solvent (dichloromethane or ethyl acetate). The combined organic phases were dried over MgSO4, filtered, and evaporated. A solid phase extraction of the dry residue with a mixture of diethyl ether and hexane (1:1) removed from the product the other impurities. The solid residue was then dissolved in a minimal amount of ethanol and diethyl ether was added to induce the precipitation of pyridine-2-thiol. After cooling to 0  C, pyridine-2-thiol was filtered off. The solid residue was washed with an ice cold mixture of ethanol and diethyl ether (1:10). The combined organics were evaporated and the product was isolated by the flash chromatography using 100% ethyl acetate followed by a mixture of ethyl acetate/acetone/ethanol/water (usually 30/3/4/ 3) to elute the desired product. Final products were dried one week at 30  C and 0.1 mbar or were lyophilized. Isolated yields are summarized in Table 1. 5.2.1. (2S,20 S)-Diethyl 2,20 -{[({2-[2-amino-6-(cyclopropylamino)-9Hpurin-9-yl]ethoxy}methyl)phosphoryl]bis(azanediyl)}dipropanoate (2) Prepared from diester 13 [29]. 1H NMR (DMSO-d6, 500 MHz) d 7.69 (s, 1H, H-8), 7.26 (bs, 1H, 6-NH), 5.82 (bs, 2H, NH2), 4.56 (dd, 1H, JHeNeP ¼ 12.2, JNHeCH ¼ 10.4, NHP), 4.48 (t, 1H, JHeNeP ¼ JNHeCH ¼ 10.5, NHP), 4.12e4.00 (m, 6H, COOeCH2 and H10 ), 3.86e3.76 (m, 4H, H-20 and CHCOO), 3.61 (d, 2H, JHeCeP ¼ 7.8, OeCH2eP), 3.08e2.96 (m, 1H, CHCH2), 1.24 (d, 6 H, JCH3eCH ¼ 7.1, NHeCHeCH3), 1.17 (t, 3H, JCH3eCH2 ¼ 7.1, COOeCH2eCH3), 1.16 (t, 3 H, JCH3eCH2 ¼ 7.1, COOeCH2eCH3), 0.66e0.63 (m, 2H, CHCH2), 0.57e0.55 (m, 2H, CHCH2). 13C NMR (DMSO-d6, 126 MHz) d 174.1e173.9 (m, COO), 160.1 (C-2), 155.8 (C-4), 138.6 (C-8), 136.5 (C-6), 113.2 (C-5), 70.4 (d, J20 -P ¼ 11, C-20 ), 66.7 (d, JCeP ¼ 131, OeCH2eP), 60.59 and 60.56 (COOeCH2), 48.70 and 47.40 (NHeCH), 43.3 (C-10 ), 21.8 (d, JCeCeNeP ¼ 4.5, NH-CH-CH3), 20.0 (d, JCeCeNeP ¼ 5.3, NHeCHeCH3), 14.59 and 13.32 (COOeCH2eCH3), 6.42 (CHCH2). MS (ESIþ), m/z (%): 527 (100) [M þ Hþ], 549 (74) [M þ Naþ]. HR-MS (ESIþ) for C21H36O6N8P calculated: 527.2490, found: 527.2491. Anal. for C21H35O6N8P Calcd (%): C 47.90, H 6.70, N 21.28; Found (%): C 47.83, H 6.92, N 21.15. [a20] ¼ 37.6 (c 0.4 g/ 100 mL, water). 5.2.2. (2S,20 S)-Diethyl 2,20 -{[({[(S)-1-(6-amino-9H-purin-9-yl)-3fluoropropan-2-yl]oxy}methyl)phosphoryl]bis(azanediyl)} dipropanoate (14) Prepared from acid 4 [30]. 1H NMR (DMSO-d6, 500 MHz) d 8.14 (s, 2H, H-2, H-8), 7.21 (bs, 2H, 6-NH2), 4.65e4.46 (m, 3H, H-30 a), 4.44e4.28 (m, 3H, H-10 and H-30 b), 4.14e3.99 (m, 5 H, COOeCH2 and H-20 ), 3.87e3.77 (m, 2H, NH-CH), 3.73 (dd, 1H, Jgem ¼ 13.3, JHeCeP ¼ 7.6, OeCH2eP), 3.66 (dd, 1H, Jgem ¼ 13.3, JHeCeP ¼ 8.5, OeCH2eP), 1.24e1.21 (m, 6H, NHeCHeCH3), 1.18 (t, 3H, JCH3eCH2 ¼ 7.1, COOeCH2eCH3), 1.15 (t, 3H, JCH3eCH2 ¼ 7.1, COOeCH2eCH3). 13C NMR (DMSO-d6, 126 MHz) d 174.21e174.13 (m, COO), 156.15 (C-6), 152.63 (C-2), 149.90 (C-4), 141.72 (C-8), 118.62

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(C-5), 82.46 (d, J30 -F ¼ 169.6, C-30 ), 77.94 (dd, J20 F ¼ 18.7, J20 -P ¼ 10.1, C-20 ), 67.02 (d, JCeP ¼ 133.7, O-CH2-P), 60.58 and 60.55 (COOeCH2), 48.26 and 48.24 (NHeCH), 42.83 (d, J10 F ¼ 7.7, C-10 ), 20.91 (d, JCeCeNeP ¼ 4.2, NHeCHeCH3), 20.66 (d, JCeCeNeP ¼ 5.4, NHeCHeCH3), 14.17 and 14.16 (COOeCH2eCH3). MS (ESIþ), m/z (%): 504 (52) [M þ Hþ], 526 (100) [M þ Naþ]. HR-MS for C19H32O6N7FP calculated: 504.2129, found: 504.2130. Anal. for C19H31FN7O6P.H2O Calcd (%): C 43.76, H 6.38, F 3.64, N 18.80, P 5.94; Found: C 43.49, H 6.55, N 18.63, P 5.82. [a20] ¼ 45.8 (c 0.3 g/ 100 mL, water). 5.2.3. (2S,20 S)-Diethyl 2,20 -{[({[(R)-1-(6-amino-2-fluoro-9H-purin9-yl)propan-2-yl]oxy}methyl)phosphoryl]bis(azanediyl)} dipropanoate (15) Prepared from acid 5. 1H NMR (DMSO-d6, 500 MHz) d 8.10 (s, 1H, H-8), 7.73 (bs, 2H, 6-NH2), 4.49 (dd, 1 H, JHeNeP ¼ 12.1, JNHeCH ¼ 10.3, NH), 4.34 (dd, 1H, JHeNeP ¼ 11.3, JNHeCH ¼ 10.3, NH), 4.20 (dd, 1H, Jgem ¼ 14.4, J10 ae20 ¼ 3.7, H-10 a), 4.12e3.98 (m, 5H, COOeCH2 and H10 b), 3.98 (m, 1 H, H-20 ), 3.86e3.76 (m, 2H, NHeCH), 3.63 (dd, 1H, Jgem ¼ 13.0, JHeCeP ¼ 8.0, OeCH2eP), 3.55 (dd, 1H, Jgem ¼ 13.0, JHeCeP ¼ 9.1, OeCH2eP), 1.24e1.21 (m, 6H, NHeCHeCH3), 1.18 (t, 3H, JCH3eCH2 ¼ 7.1, COOeCH2eCH3), 1.16 (t, 3H, JCH3eCH2 ¼ 7.1, COOeCH2eCH3), 1.02 (d, 3H, J30 e20 ¼ 6.3, H-30 ). 13C NMR (DMSO-d6, 126 MHz) d 174.20e174.16 (m, COO), 158.86 (d, J2-F ¼ 203.2, C-2), 157.71 (d, J6-F ¼ 21.3, C-6), 151.38 (d, J4-F ¼ 20.2, C-4), 142.20 (d, J80 F ¼ 2.7, C-8), 116.78 (d, J5-F ¼ 4.1, C-5), 75.34 (d, J20 P ¼ 11.6, C-2 ), 65.64 (d, JCeP ¼ 135.0, OeCH2eP), 60.55 and 60.50 (COOeCH2), 48.27 and 48.25 (NHeCH), 47.08 (C-10 ), 20.90 (d, JCeCeNeP ¼ 4.7, NHeCHeCH3), 20.69 (d, JCeCeNeP ¼ 5.5, NHeCHeCH3), 16.84 (C-30 ), 14.17 and 14.15 (COOeCH2eCH3). MS (ESIþ), m/z (%): 504 (25) [M þ Hþ], 526 (100) [M þ Naþ]. HR-MS (ESIþ) for C19H32O6N7FP calculated: 504.2129, found: 504.2130. Anal. for C19H31FN7O6P.(0.5H2O) Calcd (%): C 44.53, H 6.29, F 3.71, N 19.13, P 6.04; Found (%): C 44.46, H 6.37, N 19.04, P 6.01. [a20] ¼ 63.9 (c 0.4 g/100 mL, water). 5.2.4. (2S,20 S)-Diethyl 2,20 -[{[({(S)-1-[2-amino-6-(cyclopropylamino)9H-purin-9-yl]-3-fluoropropan-2-yl}oxy)methyl]phosphoryl} bis(azanediyl)]dipropanoate (16) Prepared from acid 6 [30]. 1H NMR (DMSO-d6, 500 MHz) d 7.72 (s, 1H, H-8), 7.32 (bs, 1H, 6-NH), 5.84 (bs, 2H, 2-NH2), 4.58 (ddd, 1H, J30 aeF ¼ 47.7, Jgem ¼ 10.4, J30 ae20 ¼ 3.4, H-30 a), 4.55e4.48 (m, 2H, PeNH), 4.36 (ddd, 1 H, J30 beF ¼ 47.0, Jgem ¼ 10.4, J30 be20 ¼ 4.7, H-30 a), 4.19 (bdd, Jgem ¼ 14.4, J10 ae20 ¼ 4.3, H-10 a), 4.12e3.99 (m, 6H, COOeCH2 and H-20 and H-10 b), 3.88e3.74 (m, 2H, NHeCH), 3.70e3.62 (m, 2H, OeCH2eP), 3.01 (bs, 1H, CH-cycpr.), 1.24e1.15 (m, 12H, CH3), 0.65 (m, 2H, CH2-cycpr.), 0.57 (m, 2H, CH2-cycpr.). 13C NMR (DMSO-d6, 126 MHz) d 174.26 (m, COO), 160.46 (C-2), 156.11 (C-6), 151.71 (C-4), 138.04 (C-8), 113.32 (C-5), 82.66 (d, J30 -F ¼ 169.3, C-30 ), 78.10 (dd, J20 -F ¼ 18.4, J20 P ¼ 10.1, C-20 ), 67.20 (d, JCeP ¼ 134.3, OeCH2eP), 60.68 and 60.64 (COO-CH2), 48.29 and 48.27 (NHeCH), 42.44 (d, J10 -F ¼ 8.0, C-10 ), 20.90 (d, JCeCeNeP ¼ 4.3, NHeCHeCH3), 20.80 (d, JCeCeNeP ¼ 5.2, NHeCHeCH3), 14.23 (COOeCH2eCH3), 6.66 (CH2-cycpr.). MS (ESIþ), m/z (%): 559 (100)[M þ Hþ], 581 (34) [M þ Naþ]. HR-MS (ESIþ) for C22H37O6N8FP calculated: 559.2553, found: 559.2552. Anal. for C22H36FN8O6P.(0.8H2O) Calcd (%): C 46.12, H 6.61, F 3.32, N 19.56, P 5.41; Found: C 46.01, H 6.83, N 19.47, P 5.45. [a20] ¼ þ10.2 (c 0.2 g/100 mL, water). 5.2.5. (2S,20 S)-Diethyl 2,20 -{[({[(R)-1-(2,6-diamino-9H-purin-9-yl) propan-2-yl]oxy}methyl)phosphoryl]bis(azanediyl)}dipropanoate (17) Prepared from diester 7 [31] or from acid 8 [31]. 1H NMR (DMSOd6, 500 MHz) d 7.71 (s, 1H, H-8), 6.65 (bs, 2H, 6-NH2), 5.75 (bs, 2H, 2NH2), 4.51 (dd, 1H, JHeNeP ¼ 12.1, JNHeCH ¼ 10.3, NH), 4.37 (dd, 1H, JHeNeP ¼ 11.5, JNHeCH ¼ 10.4, NH), 4.13e4.01 (m, 5H, COOeCH2 and

H-10 b), 3.96 (dd, 1H, Jgem ¼ 14.3, J10 ae20 ¼ 6.0, H-10 a), 3.87e3.80 (m, 3 H, H-20 and NHeCH), 3.62 (dd, 1H, Jgem ¼ 13.0, JHeCeP ¼ 8.3, OeCH2eP), 3.56 (dd, 1H, Jgem ¼ 13.0, JHeCeP ¼ 9.0, OeCH2eP), 1.25e1.23 (m, 6H, NHeCHeCH3), 1.19 (t, 3H, JCH3eCH2 ¼ 7.1, COOeCH2eCH3), 1.16 (t, 3H, JCH3eCH2 ¼ 7.1, COOeCH2eCH3), 1.01 (d, 3H, J30 e20 ¼ 6.3, H-30 ). 13C NMR (DMSO-d6, 126 MHz) d 174.3e174.2 (m, COO), 160.5 (C-2), 156.3 (C-6), 152.3 (C-4), 138.4 (C-8), 113.0 (C5), 75.6 (d, J20 P ¼ 12, C-20 ), 65.8 (d, JCeP ¼ 135, OeCH2eP), 60.59 and 60.56 (COO-CH2), 48.33 and 48.30 (NHeCH), 46.5 (C-10 ), 21.0 (d, JCeCeNeP ¼ 4.6, NHeCHeCH3), 20.8 (d, JCeCeNeP ¼ 5.4, NHeCHeCH3), 17.0 (C-30 ), 14.20 and 14.19 (COOeCH2eCH3). MS (ESIþ), m/z (%): 501 (100) [M þ Hþ], 523 (64) [M þ Naþ]. HR-MS (ESIþ) for C19H34O6N8P calculated: 501.2333, found: 501.2331. Anal. for C19H33O6N8P Calcd (%): C 45.60, H 6.65, N 22.39; Found (%): C 45.73, H 6.78, N 22.10. [a20] ¼ 55.7 (c 0.3 g/100 mL, water). 5.2.6. (2S,2’S)-Diethyl 2,2’-[({[2-(2-amino-6-oxo-1H-purin-9(6H)yl)ethoxy]methyl}phosphoryl)bis(azanediyl)]dipropanoate (18) Prepared from diester 9 [32] or acid 10 [32]. 1H NMR (DMSO-d6, 500 MHz) d 10.57 (bs, 1H, NH), 7.73 (s, 1 H, H-8), 6.47 (bs, 2 H, NH2), 4.56 (dd, 1 H, JHeNeP ¼ 12.0, JNHeCH ¼ 10.3, NHP), 4.48 (t, 1 H, JHeNeP ¼ JNHeCH ¼ 10.6, NHP), 4.11e4.02 (m, 6 H, COO-CH2 and H10 ), 3.85e3.75 (m, 4H, H-20 and CHCOO), 3.61 (d, 2H, JHeCeP ¼ 7.6, OeCH2eP), 1.22 (d, 6H, JCH3eCH ¼ 6.9, NHeCHeCH3), 1.17 (t, 3H, JCH3eCH2 ¼ 7.1, COOeCH2eCH3), 1.16 (t, 3H, JCH3eCH2 ¼ 7.1, COOeCH2eCH3). 13C NMR (DMSO-d6, 126 MHz) d 173.8e173.7 (m, COO), 156.7 (C-2), 153.4 (C-4), 151.0 (C-6), 137.6 (C-8), 116.3 (C-5), 70.2 (d, J20 -P ¼ 10, C-20 ), 67.4 (d, JCeP ¼ 136, OeCH2eP), 60.2 (COOeCH2), 48.0 (NHeCH), 42.1 (C-10 ), 20.5 (d, JCeCeNeP ¼ 4.3, NHeCHeCH3), 20.5 (d, JCeCeNeP ¼ 5.0, NHeCHeCH3), 13.8 (COOeCH2eCH3). MS (ESIþ), m/z (%): 488 (38) [M þ Hþ], 510 (100) [M þ Naþ]. MS (ESI-), m/z (%): 486 (100) [M-Hþ]. HR-MS (ESIþ) for C18H31O7N7P calculated: 488.2017, found: 488.2016. Anal. for C18H30O7N7P Calcd (%): C 44.35, H 6.20, N 20.11; Found (%): C 44.56, H 6.46, N 20.03. [a20] ¼ 42.7 (c 0.3 g/100 mL, water). 5.2.7. (2S,20 S)-Diethyl-2,20 -{[({2-[2-amino-6-(cyclooctylamino)9H-purin-9-yl]ethoxy}methyl)phosphoryl]bis(azanediyl)} dipropanoate (19) Prepared from diester 11. 1H NMR (DMSO-d6, 500 MHz) d 7.65 (s, 1H, H-8), 6.83 (bs, 1 H, 6-NH), 5.76 (bs, 2H, NH2), 4.56 (t, 1H, JHeNeP ¼ JNHeCH ¼ 10.3, NHP), 4.48 (t, 1H, JHeNeP ¼ JNHeCH ¼ 10.7, NHP), 4.33e4.19 (m, 1 H, CHCH2), 4.12e4.00 (m, 6H, COOeCH2 and H-10 ), 3.87e3.75 (m, 4H, H-20 and CHCOO), 3.61 (d, 2H, JHeCeP ¼ 7.8, OeCH2eP), 1.80e1.40 (m, 14H, 7 CH2), 1.22 (d, 6H, JCH3eCH ¼ 7.3, NHeCHeCH3), 1.18 (t, 3H, JCH3eCH2 ¼ 7.1, COOeCH2eCH3), 1.17 (t, 3H, JCH3eCH2 ¼ 7.1, COOeCH2eCH3). 13C NMR (DMSO-d6, 126 MHz) d 173.9e173.8 (m, COO), 160.0 (C-2), 153.8 (C-4), 137.2 (C-8), 136.5 (C-6), 113.3 (C-5), 70.3 (d, J20 P ¼ 10, C-20 ), 67.6 (d, JCeP ¼ 135, OeCH2eP), 66.2 (CH2), 60.24 and 60.21 (COO-CH2), 48.00 and 47.97 (NH-CH), 41.8 (C-10 ), 26.8, 25.0 and 23.6 (3 CH2), 20.6 (d, JCeCeNeP ¼ 4.8, NHeCHeCH3), 20.5 (d, JCeCeNeP ¼ 5.1, NHeCHeCH3), 14.52 and 13.36 (COOeCH2eCH3), 6.42 (CHCH2). MS (ESIþ), m/z (%): 597 (100) [M þ Hþ], 619 (58) [M þ Naþ]. HR-MS (ESIþ) for C26H46O6N8P calculated: 597.3272, found: 597.3271. Anal. for C26H45O6N8P Calcd (%): C 52.34, H 7.60, N 18.78; Found (%): C 52.54, H 7.82, N 18.63. [a20] ¼ 16.7 (c 0.4 g/100 mL, water). 5.2.8. (2S,20 S)-Diethyl 2,20 -{[({2-[(2,6-diaminopyrimidin-4-yl)oxy] ethoxy}methyl)phosphoryl]bis(azanediyl)}dipropanoate (20) Prepared from diester 12 [33]. 1H NMR (DMSO-d6, 500 MHz) d 6.02 (bs, 2 H, NH2), 5.87 (bs, 2H, NH2), 5.03 (s, 1H, H-5), 4.57 (dd, 1H, JHeNeP ¼ 11.2, JNHeCH ¼ 10.9, NHP), 4.50 (t, 1H, JHeNeP ¼ JNHeCH ¼ 10.3, NHP), 4.20 (t, 2H, H-10 ), 4.10e4.00 (m, 4H, COOeCH2), 3.88e3.82 (m, 2H, CHCOO), 3.71 (t, 2H, H-20 ), 3.62 (d,

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2H, JHeCeP ¼ 7.8, OeCH2eP), 1.26 (m, 6H, JCH3eCH ¼ 6.9, NHeCHeCH3), 1.18 (t, 3 H, JCH3eCH2 ¼ 7.0, COOeCH2eCH3), 1.17 (t, 3 H, JCH3eCH2 ¼ 7.0, COOeCH2eCH3). 13C NMR (DMSO-d6, 126 MHz) d 173.9e173.8 (m, COO), 169.7 (C-2), 165.8 (C-4), 162.7 (C-6), 76.1 (C5), 70.7 (d, J20 P ¼ 9, C-20 ), 67.8 (d, JCeP ¼ 138, OeCH2eP), 63.54 (COOeCH2), 60.2 (C-10 ), 48.0 (NHeCH), 20.6 (d, JCeCeNeP ¼ 4.5, NHeCHeCH3), 20.4 (d, JCeCeNeP ¼ 4.8, NHeCHeCH3), 13.9 (COOeCH2eCH3). MS (ESIþ), m/z (%): 463 (100) [M þ Hþ], 485 (57) [M þ Naþ]. HR-MS (ESIþ) for C17H32O7N6P calculated: 463.2065, found: 463.2064. Anal. for C17H31O7N6P Calcd (%): C 44.15, H 6.76, N 18.17; Found (%): C 44.46, H 6.93, N 17.96. [a20] ¼ 20.1 (c 0.3 g/ 100 mL, water). 5.3. HIV antiviral and cytotoxicity assays The MT-2 cells were obtained from the NIH AIDS Research and Reference Reagent Program (Germantown, MD) and maintained in RPMI-1640 medium supplemented with 10 units/mL penicillin, 10 mg/mL streptomycin, and 10% FBS. MT-2 cells were passaged twice a week and maintained at densities below 0.6  106 cells/mL. 3-fold serial dilutions of compounds were prepared in triplicate in 384-well plates starting at a top concentration of 100 mM. MT-2 cells were infected in bulk with HIV-1 IIIB (ABI, Columbia, MD) for 3 h at a multiplicity of infection (MOI) of 0.0055. The preinfected cells were added to 384-well plates at a density of 3  103 cells per well in a final assay volume of 75 mL and a final DMSO concentration of 0.5%. The infected cells were incubated with serially diluted compounds for five days at 37  C after which virus-induced cytopathic effect was determined by adding Cell-titer Glo viability reagents (Promega, Madison, WI) to the treated cells and measuring luminescence on a Victor Luminescence plate reader (PerkineElmer, Waltham, MA). The cytotoxicity of the compounds in MT-2 cells was determined in replicate plates in the same way as in antiviral activity assays, except no virus was added to the cell culture. EC50 and CC50 values were calculated by non-linear regression of multiple data sets using Pipeline Pilot software (Accelrys, San Diego, CA). Antineoplastic activity was followed in Hep G2 hepatocellular carcinoma cells (ATCC HB 8065), CCRF-CEM T lymphoblastoid cells (human acute lymphoblastic leukemia, ATCC CCL 119), human promyelocytic leukemia HL-60 cells (ATCC CCL 240) and human cervix carcinoma HeLa S3 cells (ATCC CCL 2.2). CCRF-CEM cells, HL-60 and HeLa S3 cells were cultivated in RPMI 1640 medium supplemented with 5% calf fetal serum (CFS). Hep G2 cells were propagated in Eagle’s minimum essential medium (MEM) supplemented with 10% CFS. The endpoint of the cell growth was 72 h following the addition of the drug. Subsequently, the cell viability was quantified using XTT standard spectrophotometric assay. 5.4. Nitric oxide assay Female mice of the inbred strain C57BL/6, 8e10 wks old, were purchased from Charles River Deutschland (Sulzfeld, Germany). They were kept in transparent plastic cages in groups of ten, and maintained in an Independent Environmental Air Flow Animal Cabinet (ESI Flufrance, Wissous, France). Lighting was set on 06e18 h, temperature at 22  C. Animals, killed by cervical dislocation, were i.p. injected with 8 mL of sterile saline. Pooled peritoneal cells collected from mice (n ¼ 4) were washed, resuspended in culture medium, and seeded into 96-well round-bottom microplates (Costar) in final 100-mL volumes, 2  106 cells/mL. The viability of cells was checked using trypan blue. Cultures were maintained at 37  C, 5% CO2 in humidified Heraeus incubator. Complete RPMI-1640 culture medium (SigmaeAldrich) contained 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine,

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50 mg/mL gentamicin, and 5  105 M 2-mercaptoethanol (all Sigma). All protocols were approved by the institutional ethics committee. The 5 mM stock solution was prepared in apyrogenic water and used immediately. It was diluted to required concentrations using complete RPMI-1640 culture medium. The cells were cultured 24 h in presence of tested compound which was applied either alone or concomitantly with NO-priming stimuli, i.e. murine recombinant interferon-g (IFN-g, 5000 pg/ mL; R&D Systems, Minneapolis, MN) and IFN-g plus lipopolysaccharide (LPS from E. coli 0111:B4, 100 pg/mL; Sigma). The concentration of nitrites in supernatants of cells was taken as a measure of NO production [34]. It was detected in individual, cell-free samples (50 mL) incubated 5 min at ambient temperature with an aliquot of a Griess reagent (1% sulphanilamide/0.1% naphtylendiamine/2.5% H3PO4). The absorbance at 540 nm was recorded using a microplate spectrophotometer (Tecan, Austria). A nitrite calibration curve was used to convert absorbance to mM nitrite. It should be noted that the in vitro immunomodulatory assays used in our studies [28] are extremely sensitive, in concentrations less than 20 pM, to the presence of bacterial lipopolysaccharide (LPS). Usually, free phosphonic acids are purified by the sequence of ion-exchange chromatography and by the final crystallization from water. In all these steps, apyrogenic water has to be used to eliminate possible LPS contamination of free phosphonic acid. On the other hand, bis-amidates prepared according to this paper are purified by filtration through the silica gel and eluated with organic solvent, which dramatically reduces the likelihood of sample contamination by residual LPS present in water. Acknowledgments This study was performed as a part of the research project OZ40550506 of the Institute of Organic Chemistry and Biochemistry and was supported by Ministry of the Interior of the Czech Republic (VG20102015046) and by Gilead Sciences & IOCB Research Center. References [1] V.J. Stella, R.T. Borchardt, M.J. Hageman, R. Oliyai, H. Maag, J.W. Tilley, Prodrugs: Challenges and Rewards. Springer, New York, 2007. [2] R.L. Mackman, T. Cihlar, Prodrug strategies in the design of nucleoside and nucleotide antiviral therapeutics, Annu. Rep. Med. Chem. 39 (2004) 305e321. [3] M.E. Ariza, Current prodrug strategies for the delivery of nucleotides into cells, Drug Des. Rev. 2 (2005) 373e387. [4] S.J. Hecker, M.D. Erion, Prodrugs of phosphates and phosphonates, J. Med. Chem. 51 (2008) 2328e2345. [5] E. De Clercq, A. Holý, I. Rosenberg, T. Sakuma, J. Balzarini, P.C. Maudgal, A novel selective broad-spectrum anti-DNA virus agent, Nature 323 (1986) 464e467. [6] D. Cahard, C. McGuigan, J. Balzarini, Aryloxy phosphoramidate triesters as protides, Mini-Rev. Med. Chem. 4 (2004) 371e381. [7] R.L. Mackman, A.S. Ray, H.C. Hui, L. Zhang, G. Birkus, C.G. Boojamra, M.C. Desai, J.L. Douglas, Y. Gao, D. Grant, G. Laflamme, K.-Y. Lin, D.Y. Markevitch, R. Mishra, M. McDermott, R. Pakdaman, O.V. Petrakovsky, J.E. Vela, T. Cihlar, Discovery of GS-9131: design, synthesis and optimization of amidate prodrug of the novel nucleoside phosphonate HIV reverse transcriptase (RT) inhibitor GS-9148, Bioorg. Med. Chem. 18 (2010) 3606e3617. [8] C. Ballatore, C. McGuigan, E. De Clercq, J. Balzarini, Synthesis and evaluation of novel amidate prodrugs of PMEA and PMPA, Bioorg. Med. Chem. Lett. 11 (2001) 1053e1056. [9] W.A. Lee, G.-X. He, E. Eisenberg, T. Cihlar, S. Swaminathan, A. Mulato, K.C. Cundy, Selective intracellular activation of a novel prodrug of the human immunodeficiency virus reverse transcriptase inhibitor tenofovir leads to preferential distribution and accumulation in lymphatic tissue, Antimicrob. Agents Chemother. 49 (2005) 1898e1906. [10] G. Birkus, R. Wang, X. Liu, N. Kutty, H. MacArthur, T. Cihlar, C. Gibbs, S. Swaminathan, W. Lee, M. McDermott, Cathepsin A is the major hydrolase catalyzing the intracellular hydrolysis of the antiretroviral mucleotide phosphonoamidate prodrugs GS-7340 and GS-9131, Antimicrob. Agents Chemother. 51 (2007) 543e550.

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