Design, synthesis and in vitro evaluation of mono (2, 2, 2-trifluoroethyl) esters, mono l -amino acid ester prodrugs of acyclic nucleoside phosphonates as anti-HBV agents

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Chinese Chemical Letters 22 (2011) 1387–1390 www.elsevier.com/locate/cclet

Design, synthesis and in vitro evaluation of mono (2, 2, 2-trifluoroethyl) esters, mono L-amino acid ester prodrugs of acyclic nucleoside phosphonates as anti-HBV agents Xiao Zhong Fu a, Yu Ou a, Jan Xin b, Yu She Yang c,* a

School of Pharmacy, Guiyang Medical College, Guiyang 550004, China b Shanghai Fudan-Yueda Bio-Tech Co., Ltd., Shanghai 201203, China c State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences, Chinese Academy of Science, Shanghai 201203, China Received 11 May 2011 Available online 10 October 2011

Abstract A series of novel mono (2, 2, 2-trifluoroethyl) esters, mono L-amino acid ester prodrugs of acyclic nucleoside phosphonates was synthesized and their in vitro anti-HBVactivity was evaluated in HepG 2 2.2.15 cells. Compound 1d exhibited more potent antiHBV activity and lower cytotoxicity than those of adefovir dipivoxil and alamifovir (MCC-478) with EC50 and CC50 values of 0.01 mmol/L and >8000 mmol/L respectively. # 2011 Yu She Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Acyclic nucleoside phosphonates; L-Amino acid; Prodrug

Acyclic nucleoside phosphonates (ANPs) have been approved by the FDA for the treatment of virus infection [1]. They circumvent the first phosphorylation step, the bottleneck of the nucleoside analogues transformation and suppress replication of HBV that is resistant to nucleoside analogues such as lamivudine, emtricitabine and famciclovir [2–4]. In the structure of ANPs, nucleotide base and phosphonate moiety play important roles in anti HBV innovation research. As the above two moiety can be easily modified, that makes it reachable to design and synthesis of novel anti HBV agents by using some ANPs as lead compounds [5–7]. In our pervious study, we have obtained adefovir bis(L-amino acid) ester prodrugs with increased anti HBV activity and selective index (SI) [8–10]. As bis (2, 2, 2-trifluoroethyl) phosphonates and 4-methoxy phenylthio structural fragment in alamifovir (MCC478) are crucial for its potent anti HBV activity [11–13]. We designed and synthesized a novel series of mono (2, 2, 2trifluoroethyl) esters, mono L-amino acid ester prodrugs of acyclic nucleoside phosphonates via sub-structure combination method, and to preliminarily evaluate their anti-HBV activity. In this letter, five mono (2, 2, 2trifluoroethyl) esters, mono L-amino acid ester prodrugs of acyclic nucleoside phosphonates 1 (1a–e) were obtained for the first time, and their anti-HBV activity were evaluated in HepG2 2.2.15 cells. It is expected that the results of the

* Corresponding author. E-mail address: [email protected] (Y.S. Yang). 1001-8417/$ – see front matter # 2011 Yu She Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2011.09.005

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X.Z. Fu et al. / Chinese Chemical Letters 22 (2011) 1387–1390 Cl

CF3 a PCl3

CF3CH2OH

+

P(OCH2CF3)3

+

Cl

b

Cl

O

O P O O

F3C

2

N

N

c H2N

N

CF3

N

P O

O

N

N

e H2N

O

N H

N

N

CF3

N

CF3

O O

P O

OH

6a: R1 = -NH-Cyclcopropyl 6b: R1 = -SPh(4-OMe)

Br

NHBoc 7a: R2 = Isopropyl 7b: R2 = 2-Methylpropyl 7c: R2 = Benzyl

R1

R1 N

N H2N

N

N

O P O O

N

N

CF3

O f

H2N

5a: R1 = -NH-Cyclcopropyl 5b: R1 = -SPh(4-OMe)

O O

P

N

N +

R1

O

N

CF3

O

CF3

O

4

R2

N

H2N

O O

N

N

d

O 3

R1

Cl

Cl

g NHBoc O

R2

O 8a-e 8a: R1 = -NH-Cyclcopropyl; R2 = Isopropyl 8b: R1 = -NH-Cyclcopropyl; R2 = 2-Methylpropyl 8c: R1 = -SPh(4-OMe); R 2 = Isopropyl 8d: R1 = -SPh(4-OMe); R 2 =2-Methylpropyl 8e: R1 = -SPh(4-OMe); R 2 =Benzyl

H2N

N

CF3

N O

O P O O 1a-e

NH2 O

R2 O

1a: R1 = -NH-Cyclcopropyl; R2 = Isopropyl 1b: R1 = -NH-Cyclcopropyl; R2 = 2-Methylpropyl 1c: R1 = -SPh(4-OMe); R 2 = Isopropyl 1d: R1 = -SPh(4-OMe); R 2 =2-Methylpropyl 1e: R1 = -SPh(4-OMe); R 2 =Benzyl

Scheme 1. The synthetic method of compounds 1a–1e. Reagents and conditions: (a) 110 8C, 4 h, yield 65%; (b) 160 8C, 7 h, yield 95%; (c) Cs2CO3, anhydrous DMF, 80 8C, 5 h, yield 58%; (d) substituted arylthiol or cyclopropylamine, Et3N, anhydrous DMF, 80–100 8C, yield 45–80%; (e) 1 N NH3
H2O, rt; 3 h, yield 78–97%; (f) N, N-dicyclohexyl-4-morpholine carboxamidine (DCMC), anhydrous DMF, 60 8C, 24 h, yield 19–22%; (g) 4 N hydrochloride-1,4-dioxane, rt, 5 h, yield 62–81%.

reported study might be able to afford some valuable information on new acyclic nucleoside phosphonates prodrug design. As shown in Scheme 1, phosphorus trichloride was treated with trifluoroethanol to afford tris (2, 2, 2trifluoroethyl)phosphine 2. Compound 2 was quantitatively converted into bis (2, 2, 2-trifluoroethyl) (2chloroethoxy)-methylphosphonate 3 by treatment with 2-chloroethyl chloromethyl ether. Subsequently, 2-amino6-chloropurine was treated first with Cs2CO3 and then with compound 3 in anhydrous DMF, 2-amino-6-chloro-9-[2(phosphonomethoxy) ethyl] purine bis (2, 2, 2-trifluoroethyl) ester 4 was obtained as major product, compound 4 coupled with substituted arylthiol or cyclopropylamine to afford compound 5, one 2, 2, 2-trifluoroethyl phosphonate of compound 5 was removed by using 1 N NH3.H2O to afford compound 6 [13,14]. Treatment of compounds 6 with 2bromoethyl ester of N-Boc-L-amino acids 7 in the presence of N, N-dicyclohexyl-4-morpholine carboxamidine (DCMC) in anhydrous DMF afforded compounds 8. Removal of the N-protecting group in 4 mol/L hydrochloride-1, 4-dioxane solution gave target compounds 1 as white foam, yields were in the range of 62–81%. The synthesized compounds were evaluated for their inhibitory effect on the replication of HBV in HepG2 2.2.15 cells according to Ref. [15]. The HepG2 2.2.15 cells were plated at a density of 2  104 cells/well on 24-well plates and incubated for 2 days. Then, the medium was replaced with medium containing test compounds at final concentrations of 0.001–100 mmol/L. The cells were cultured for 9 more days, and the medium was replaced with fresh Dulbecco’s Modified Eagle’s Medium (DEME) medium every 3 days. On day 9 the culture media were collected, viral DNA recovered from the secreted particles in cultured medium was obtained by using polyethylene glycol precipitation method and analyzed by real-time PCR using Icycler (Bio-Rad). Amplification primers were HBVFP: 5TGT CCT GGT TAT CGC TGG-3 and HBVRP: 5-CAA ACG GGC AAC ATA CCT T-3. The TaqMan probe was FAM-5-TGT GTC TGC GGC GTT TTA TCAT-3-TAMRA. The 50% cytotoxic concentration of each compound was determined in HepG2 2.2.15 cell line. After compounds treatments for the evaluation of anti-HBV activity, each well of the 24-well plate was treated with 10 mL of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazdium bromide (MTT)

X.Z. Fu et al. / Chinese Chemical Letters 22 (2011) 1387–1390

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Table 1 Anti-HBV evaluation of mono (2, 2, 2-trifluoroethyl) esters, mono L-amino acid ester prodrugs of acyclic nucleoside phosphonates. Compounds a

EC50b (mmol/L)

CC50c (mmol/L)

SI d

1a 1b 1c 1d 1e MCC-478 Adefovir dipivoxil

11.40 4.95 3.02 0.01 0.69 3.13 0.43

1030 >8000 2940 >8000 >8000 3170 610

90.35 >1616 973 >8  10 5 >11,594 1021 1400

a b c d

Obtained as hydrochloride salts. Concentrations of compounds achieving 50% inhibition of cytoplasmic HBV-DNA synthesis. Concentrations of compounds required for 50% extinction of HepG2 2.2.15 cells. Selective index CC50/EC50.

and incubated for 2 h at 37 8C. At the end of the incubation period, the supernatant was removed and 150 mL DMSO was added to each well to dissolve the formazan. The OD value was measured at 570 nm. The data was analyzed using Icycler IQ 3.0. EC50, CC50 and SI of these compounds are reported in Table 1. Adefovir dipivoxil and alamifovir (MCC-478) were used as positive controls. As indicated in Table 1, all synthesized compounds [16] exhibited potent anti-HBV activity with EC50 values of 0.01–11.40 mmol/L and lower cytotoxicities with CC50 values of 1030 to >8000 mmol/L compared with those of adefovir dipivoxil and MCC-478. Compound 1d and 1e exhibited more potent anti HBV activity than alamifovir (MCC-478) with EC50 values of 0.01 and 0.69 mmol/L respectively, especially compound 1d exhibited more potent anti-HBV activity than adefovir dipivoxil with EC50 values of 0.01 mmol/L. The preliminary SAR research revealed that the compounds with 4-methoxy phenylthio substitution at 6-position of purine ring 1c, 1d and 1e showed higher anti-HBV activity than that of the corresponding compounds with cyclopropylamine substitution at the same position 1a, 1b. In addition, respective comparisons of 1a with 1b, and 1c with 1d, 1e indicated that compounds with larger bulk of the amino acid alkyl group, 1b, 1d and 1e, showed higher anti-HBV activity and SI than that of the corresponding compounds 1a and 1c with smaller bulk of amino acid alkyl group. All these results suggested that mono (2, 2, 2trifluoroethyl) esters, mono L-amino acid ester prodrugs of acyclic nucleoside phosphonates are worthy of further investigation to discover new anti-HBV agents. Acknowledgments This work was supported by the grants from the National Natural Science Foundation of China (No. 20962004), the Provincial Social Development Foundation of Guizhou, China (No. QKHSYZ [2009] 3081), Provincial Special Assistant Foundation for High-level Talents of Guizhou, China (No. TZJF-2009-36), and Science and Technology Foundation of Guizhou Province, China (No. QKHJZ [2008] 2140). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]

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[14] B.A. Nguyen, WO 0039143 (2000). [15] A.S. Mary, M.L. Cheng, A. George, et al. Proc. Natl. Acad. Sci. U.S.A. 84 (1987) 1005. [16] The spectra data of the synthetic compounds. 1a: 1H NMR (400 MHz, CD3OD): d 7.96 (s, 1H), 4.47–4.39 (m, 2H), 4.33–4.25 (m, 4H), 4.12– 4.08 (m, 2H), 3.98–3.92 (m, 5H), 2.31–2.27 (m, 1H), 1.29–1.26 (m, 1H), 1.19–1.15 (m, 8H), 0.83 (m, 2H); ESI-MS (m/z): [M+H]+: 554.2. 1b: 1 H NMR (400 MHz, CD3OD): d 7.59 (s, 1H), 5.30–5.22 (m, 1H), 4.79 (brs, 2H), 4.32–4.29 (m, 3H), 4.23–4.21 (m, 4H), 4.12 (t, 2H, J = 7.34 Hz), 3.89 (t, 2H, J = 4.77 Hz,), 3.80 (d, 2H, J = 8.25 Hz), 1.86 (m, 1H), 1.43 (s, 9H), 1.31–1.25 (m, 2H), 1.14–1.21 (m, 1H), 0.92–0.87 (m, 6H); ESI-MS (m/z): [M+H]+: 568.2. 1c: 1H NMR (400 MHz, CD3OD): d 8.71 (s, 1H), 7.59 (d, 2H, J = 6.96 Hz), 7.10 (d, 2H, J = 6.97 Hz), 4.51–4.35 (m, 9H), 4.06–3.99 (m, 4H), 3.87 (s, 3H), 2.16 (m, 1H), 1.07–1.05 (m, 6H); ESI-MS (m/z): [M+H]+: 637.2. 1d: 1H NMR (400 MHz, CD3OD): d 8.55 (s, 1H), 7.53 (d, 2H, J = 8.72 Hz), 7.09 (d, 2H, J = 8.89 Hz), 4.55–4.34 (m, 9H), 4.07 (d, 2H, J = 8.06 Hz), 4.01 (t, 2H, J = 4.77 Hz), 2.01 (m, 1H), 1.57–1.51 (m, 1H), 1.41–1.28 (m, 1H), 1.03–0.95 (m, 6H); ESI-MS (m/z): [M+H]+: 651.3. 1e: 1H NMR (400 MHz, CD3OD): d 8.65 (brs, 1H), 7.58 (d, 2H, J = 8.07 Hz), 7.36–7.28 (m, 5H), 7.09 (d, 2H, J = 8.06 Hz), 4.53 (t, 2H, J = 7.70 Hz), 4.42–4.29 (m, 7H), 4.06 (d, 2H, J = 7.33 Hz), 3.98 (m, 2H), 3.86 (s, 3H), 3.18–3.24 (m, 4H); ESI-MS (m/z): [M+H]+: 685.2.