key: cord-0704096-iwqqq8xh
authors: Hamon, Nadège; Slusarczyk, Magdalena; Serpi, Michaela; Balzarini, Jan; McGuigan, Christopher
title: Synthesis and biological evaluation of phosphoramidate prodrugs of two analogues of 2-deoxy-d-ribose-1-phosphate directed to the discovery of two carbasugars as new potential anti-HIV leads
date: 2015-02-15
journal: Bioorg Med Chem
DOI: 10.1016/j.bmc.2014.12.039
sha: d170f3ce0214d6b9b4c1b3f79c52213d8cde65d6
doc_id: 704096
cord_uid: iwqqq8xh

2-Deoxy-α-d-ribose-1-phosphate is of great interest as it is involved in the biosynthesis and/or catabolic degradation of several nucleoside analogues of biological and therapeutic relevance. However due to the lack of a stabilising group at its 2-position, it is difficult to synthesize stable prodrugs of this compound. In order to overcome this lack of stability, the synthesis of carbasugar analogues of 2-deoxyribose-1-phosphate was envisioned. Herein the preparation of a series of prodrugs of two carbocyclic analogues of 2-deoxyribose-1-phosphate using the phosphoramidate ProTide technology, along with their biological evaluation against HIV and cancer cell proliferation, is reported.

Glycosyl-1-phosphates are essential constituents of larger biomolecules and play a diverse and important role in many physiological processes. 1 In particular they are key intermediates in the metabolism of carbohydrates, critical in their transformation into nucleosides. 2,3 Among them, 2-deoxy-a-D-ribose-1-phosphate 1 (Fig. 1) is a catabolic product of thymidine phosphorylase (TP, EC 2.4.2.4), a nucleoside phosphorylase (NP) enzyme involved in the pyrimidine nucleoside salvage pathway. 3 However, TP was also shown to be responsible for promoting angiogenesis. 4 Increased TP expression levels, found in many solid tumours, are often correlated with neovascularisation, onset of metastasis and poor prognosis. 5 During previous studies, aimed at preparing novel inhibitors of NPs as anticancer agents, we have identified 3,5-di-p-chlorobenzoyl-2-deoxy-D-ribose-1-phosphate 2 ( Fig. 1) , which was found to inhibit a variety of pyrimidine and purine NPs with preference for uridine-and inosine-hydrolyzing enzymes. 6 This compound efficiently prevented the enzymatic breakdown of therapeutic analogues such as 5-fluoro-2 0 -deoxyuridine (FdUrd). In these studies we also demonstrated the difficulty in synthesizing phosphorami-date prodrugs at the anomeric position of 2-deoxyribose as they were found unstable. To overcome this instability we have shown that introduction of fluorine atoms on the 2-position of 2-deoxyribose enabled the synthesis of phosphoramidate prodrugs of 2fluoro and 2,2-difluoro-2-deoxyribose-1-phosphate 7 (compounds 3a and 3b, respectively, Fig. 1 ). Within this context we have more recently reported on the synthesis of a series of phosphonamidate prodrugs of another stable analogue of 2-deoxy-D-ribose-1-phosphate, in which the anomeric oxygen has been replaced by a methylene group. 8 As a continuation of our interest in preparing prodrugs of stable analogues of 2-deoxy-D-ribose-1-phosphate we report here the application of the ProTide approach to two carbasugar analogues of 2-deoxyribose. The term 'carbasugar' refers to a family of compounds in which the oxygen atom of the furanose sugar ring has been replaced by a methylene group. [9] [10] [11] Carbasugars are chemically more stable towards degradation than their sugar analogues 12 but at the same time, due to their resemblance to natural sugars, they may be still recognized by the same enzymes. 12 It is well known that the therapeutic potential of drugs bearing a phosphate moiety is decreased because of the negative charges of the phosphate group at physiological pH. 13 To overcome this problem, the ProTide technology has been successfully applied in the past to various nucleoside 14 and sugar analogues. 15 With this in mind we herein report the synthesis of a series of prodrugs http://dx.doi.org/10.1016/j.bmc.2014.12.039 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved. as reported in a recent paper by Schinazi et al., 21 it can be removed in neutral conditions, perhaps without affecting the phosphoramidate moiety. Hydroboration-oxidation reaction of 8 was investigated using different borane reagents such as 9-BBN, 16,20,22 dicyclohexylborane 23,24 or BH 3 ÁTHF. 23 The best yield was obtained with BH 3 ÁTHF. As expected in addition to the carbocycle 10, formation of its regio-isomer 12 was also observed. Only the a-epimer of compound 12 was isolated whereas both alpha and beta epimers of compound 10 were observed in a 1:1 ratio. NMR spectra of compounds 10 and 12 were in agreement with the literature. 20, 23 We then adapted these optimized hydroboration conditions to compound 9 obtaining carbocycle 11 (a-epimer) and its regio-isomer 13 (mixture of epimers in a 1:0.5 ratio) in 26% and 28% yield, respectively.

In order to biologically evaluate the parent carbasugars and to eventually compare their activities with those of the corresponding phosphoramidate prodrugs, we also prepared triol 14 and its regio-isomer 15 by hydrogenation reaction of compounds 11 and 13. NMR data of compound 15 showed that it is symmetrical meso system, which confirmed that we have prepared only the a-epimer of 15. 25 With both carbocyclic intermediates 10 and 11 in hand we then studied the reaction of an appropriate phosphorochloridate with compound 11 (Scheme 2 and Table 1) in the presence of different bases (tBuMgCl, 26, 27 NMI 26, 27 pK a = 7, Et 3 N pK a = 10.6, n-BuLi pK a ca. 50) and different solvents (THF, toluene). As shown in Table 1 the best results were obtained when Et 3 N and NMI were used (entry 4). 28 In particular, NMI (2.5% mol) was first added to a solution of carbocycle 11 in toluene. The reaction mixture was then cooled at 0°C, followed by addition of Et 3 N (3 equiv) and the desired phosphorochloridate (3 equiv). The reaction was monitored by 31 P NMR analysis until formation of the desired phosphorylated compounds was completed (signals between 1 and 3 ppm by 31 P NMR). The crude was purified by column chromatography to yield the Cbz-protected prodrug, which was submitted to a hydrogenation reaction to afford the final phosphoramidate prodrug 16.

Following this methodology, six prodrugs (16a-f) of the carbasugar 14 were synthesized with yields ranging from 2% to 13% over two steps (Scheme 3). This strategy was then applied to the regioisomer 13 affording the two prodrugs 17a (2% yield) and 17b (3% yield). 

Prodrugs 16a-f were subjected to biological evaluation as antiviral and antiproliferative agents. None of the prodrugs showed any anti-HIV-1 or anti-HIV-2 activity or any cytostatic effect in CEM cells ( Table 2 ). The parent carbocyclic analogue 14 and its Cbz-protected derivative 11 were not active either. However the regio-isomer 15 and its Cbz-protected derivative 13 showed pronounced anti-HIV activity in CEM cell cultures (2-12 lM range) when compared to the corresponding C1-regio-isomers in CEM cells (Table 2 ). When 15 was evaluated against HIV in MT-4 cell cultures, the antiviral activity against both HIV-1 and HIV-2 was confirmed at low micromolar concentrations without marked cytotoxicity (CC 50 : 90 lM). Unfortunately the corresponding prodrugs 17a and 17b were devoid of any significant antiviral activity ( Table 3 ). When assessed for the antiproliferative activity 17a and 17b did not show pronounced inhibition of the proliferation of murine leukemia cells (L1210) and human T-lymphocyte cells (CEM) or cervix carcinoma (HeLa) cells (Table 4) .

To probe this result, incubation of compound 16d with carboxypeptidase Y in d 6 -acetone and Trizma buffer was performed and the assay was followed by 31 P NMR (Fig. 2) . Compound 16d was slowly metabolized and after 13 h the starting material was still present. This slow processing may explain the lack of activity of this kind of prodrugs.

The anti-HIV activity of 13 and 15 at low micromolar concentrations is surprising and worth further exploring with respect to its molecular mechanism of antiviral action. Indeed, it should be noticed that compound 15 did not show any measurable activity at 100 lM against a wide variety of other viruses including herpes simplex virus type 1 (HSV-1), HSV-2 and vaccinia virus in HEL cell cultures, vesicular stomatitis virus (VSV), Coxsackie virus B4 and respiratory syncytial virus (RSV) in HeLa cell cultures, parainfluenza virus-3, reovirus-1, Sindbis virus and Punta Toro virus in Vero cell cultures, influenza virus A (H1N1, H3N2) and influenza virus B in MDCK cell cultures, and feline corona virus and feline herpesvirus in CrFK cell cultures. These findings make the compounds 13 and 15 highly selective for HIV. Reverse transcriptase has been excluded as a direct target since the compounds were found inactive against this enzyme (data not shown).

In summary, we have prepared several prodrugs of two carbasugar analogues of 2-deoxyribose-1-phosphate. Biological evaluation of these prodrugs showed that they had no inhibitory activity against HIV and cancer cell proliferation. However we note that parent carbocycle 15 and its synthetic intermediate 13 showed micromolar activity against HIV. Further investigations to elucidate the underlying mechanism by which 13 and 15 exert their antiviral activity against HIV will be performed. In particular, the synthetic route to (1R,2R,3S)-3-hydroxy-2-(hydroxymethyl)cyclopentyl-1-phosphate 5 is under investigation in order to confirm whether this compound can indeed act as an inhibitor against NPs. Application of other prodrug approaches is also under consideration. 

2. A suspension of (1S,2S)-2-((isopropoxydimethylsilyl)methyl) cyclopent-3-enol (5.28 g, 24.6 mmol), potassium fluoride (7.25 g, 124.9 mmol, 5.07 equiv), KHCO 3 (7.42 g, 74.1 mmol, 3.01 equiv) and hydrogen peroxide 30% (27 mL) in methanol/THF 1:1 (154 mL) was heated at reflux for 15 h. The reaction mixture was diluted with AcOEt (800 mL) and sodium thiosulfate (1.87 g) and magnesium sulfate (6.34 g) were added. The reaction mixture was stirred at room temperature for 30 min before filtration though a pad of Celite. The filtrate was then evaporated to dryness. The crude was purified by column chromatography (Eluent Hexane/AcOEt 5:95 to 0:100) in order to give compound 7 (2.689 g, 23.6 mmol, 96%) as a colorless oil. 1 To a solution of compound 7 (150 mg, 1.31 mmol) in THF (4.7 mL) at 0°C was added NaH (60% slurry, 152 mg, 1.87 mmol, 2.8 equiv). The reaction mixture was stirred at room temperature for 1 h before addition of BnBr (560 lL, 4.72 mmol, 3.6 equiv).

The reaction mixture was heated at reflux for 3 h before addition of crushed ice. The reaction mixture was stirred for 30 min and was then diluted with AcOEt (20 mL). The organic phase was washed with H 2 0 (2 * 20 mL) and brine (2 * 20 mL) and dried over 

To a solution of compound 7 (1.983 g, 17.4 mmol) and DMAP (12.735 g, 104.2 mmol, 6 equiv) in CH 2 Cl 2 (100 mL) at 0°C was added dropwise CbzCl (11.02 mL, 78.2 mmol, 4.5 equiv). The reaction mixture was stirred at room temperature for 4 h and was then diluted with CH 2 Cl 2 (100 mL) and washed with HCl 1 N (30 mL) and H 2 O (50 mL). The organic phase was dried over MgSO 4 and solvents were evaporated to dryness. Purification of the crude by column chromatography (eluent: PE/AcOEt 98:2 to 90:10) gave compound 9 (6.54 g, 17.1 mmol, 98%) as a colorless oil. 1 

To a solution of compound 8 (141 mg, 0.48 mmol) in THF (2.4 mL) at 0°C was added dropwise a 1 M solution of BH 3 ÁTHF (960 lL, 0.96 mmol, 2 equiv). The reaction mixture was stirred at 0-5°C for 24 h and then at room temperature for 19 h before addition at 0°C of 120 lL of NaOH 3 N and 120 lL of H 2 O 2 30%. The reaction mixture was stirred at room temperature for 24 h. The reaction mixture was diluted in H 2 O (10 mL) and AcOEt (10 mL). The aqueous phase was extracted with AcOEt (3 * 10 mL) and the combined organic phases were dried over MgSO 4 . Solvents were evaporated to dryness. Purification of the crude by column chromatography (eluent: PE/AcOEt 9:1 to 7:3) gave compound 10 (46 mg, 0.15 mmol, 31%) and compound 12 (18 mg, 0.6 mmol, 12%) as colorless oils. 16.2 mmol, 2 equiv). The reaction mixture was stirred at 0-5°C for 18 h before addition at 0°C of 5.05 mL of NaOH 3 N and 5.05 mL of H 2 O 2 30%. The reaction mixture was stirred at room temperature for 24 h. The reaction mixture was diluted in H 2 O (100 mL) and AcOEt (100 mL). The aqueous phase was extracted with AcOEt (3 * 100 mL) and the combined organic phases were dried over MgSO 4 . Solvents were evaporated to dryness. Purification of the crude by column chromatography (eluent: PE/AcOEt 9:1 to 75:25) gave compound 11 (822 mg, 2.05 mmol, 26%) and compound 13 (904 mg, 2.25 mmol, 28%) as colorless oils. To a solution of 11 in EtOH (C SM = 0.014 M) was added Pd/C 10% (1 mg/mmol of 11) and the reaction mixture was stirred at room temperature under H 2 atmosphere until all 11 was consumed (1-2 h). Pd was filtrated through a pad of Celite and solvents were evaporated to dryness. To afford the desired final compounds 14 in quantitative yield. 1 To a solution of 13 in EtOH (C SM = 0.014 M) was added Pd/C 10% (1 mg/mmol of 13) and the reaction mixture was stirred at room temperature under H 2 atmosphere until all 13 was consumed (1-2 h). Pd was filtrated through a pad of Celite and solvents were evaporated to dryness. To afford the desired final compounds 15 in quantitative yield. This compound has been synthesized according to the method described above and was obtained with 13% yield as a a/b/P S /P R mixture (4 diastereoisomers). 1 This compound has been synthesized according to the method described above and was obtained with 8% yield as an a-epimers mixture according the NOESY experiments (no signal between H1 and H4 on NOESY experiments). The ratio P S /P R is 1:0.5 but we do not know which one is P S and which one is P R . 1 . This compound has been synthesized according to the method described above and was obtained with 2% yield as a mixture of two diastereoisomers. 1 

CH 3 Ala of one diastereoisomer), 1.34 (d, J = 7.5 Hz, CH 3 Ala of one diastereoisomer), 0.93, 0.90 (two s, 9H, C(CH 3 ) 3 of both diastereoisomers). 13 C NMR (126 MHz, MeOD): d 175.1 (d, J = 4.6 Hz, C@O of one diastereoisomer), 175.0 (d, J = 4.6 Hz, C@O of one diastereoisomer), 148.2 (d, J = 2.7 Hz, Cq of one diastereoisomer), 148.1 (d, J = 2.8 Hz, Cq of one diastereoisomer)

4R)-3-Hydroxy-4-(hydroxymethyl)cyclopentyl-1-Onaphthyl-(cyclohexoxy-L-alaninyl)-phosphate (16d)

C4 of one diastereoisomer), 49.8 (C4 of one diastereoisomer), 49.7 (C4 of one diastereoisomer), 43.8 (d, J = 5.0 Hz, C2 of one diastereoisomer), 43.6 (d, J = 4.7 Hz, C2 of one diastereoisomer), 43.5 (d, J = 3.8 Hz, C2 of one diastereoisomer

Reverse HPLC eluting with H 2 O/MeOH from 90:10 to 0:100 in 25 min: t R = 22.91 min (95%)

4R)-3-Hydroxy-4-(hydroxymethyl)cyclopentyl-1-Ophenyl-(cyclohexoxy-L-alaninyl)-phosphate (16e)

CH 2 cyclohexyl), 1.50-1.27 (m, 8H, CH 2 cyclohexyl, CH 3 Ala). 13 C NMR (126 MHz, MeOD): d 174.6 (d, J = 5.7 Hz, C@O of one diastereoisomer), 174.5 (d, J = 4.9 Hz, C@O of one diastereoisomer), 174.5 (d, J = 3.5 Hz, C@O of one diastereoisomer), 174.4 (d, J = 3.9 Hz, C@O of one diastereoisomer

4R)-3-Hydroxy-4-(hydroxymethyl)cyclopentyl-1-Ophenyl-(tert-butoxy-L-alaninyl)-phosphate (16f)

1.46, 1.44, 1.44 (three s, 9H, C(CH 3 ) 3 ), 1.31 (dd, J = 0.9, 7.1 Hz, CH 3 Ala), 1.29 (dd, J = 0.9, 7.1 Hz, CH 3 Ala). 13 C NMR (126 MHz, MeOD): d 174.5 (d, J = 5.5 Hz, C@O), 174.4 (d, J = 5.5 Hz, C@O), 174.3 (d, J = 5.5 Hz, C@O)

3S)-3-Hydroxy-2-(hydroxymethyl)cyclopentyl-1-O-naphthyl-(neopentyl-L-alaninyl)-phosphate (17a)

(m, 4H, CH 2 OH, OCH 2 C(CH 3 ) 3 ), 2.11-1.68 (m, 4H, 2Â CH 2 ), 1.38-1.33 (m, 3H, CHCH 3 ), 0.93, 0.92 (2s, 9H, 2Â C(CH 3 ) 3 ). 13 C NMR (125 MHz, CD 3 OD): 175.36 (d, J cp = 4.6 Hz, C@O), 175.36 (d, J cp = 6.25 Hz, C@O), 148.26 (d, J cp = 3.75 Hz, Cq), 148.21 (d, J cp = 3.62 Hz, Cq), 136.33 (Cq), 136.24 (C naph), 128.86, 128.62 (C naph), 128.05 (d, J cp = 6.25 Hz, Cq naph), 128.98 (d, J cp = 6.25 Hz, Cq naph

(m, 2H, CH 2 OH), 2.10-1.88 (m, 4H, H2, 2Â H5, H4), 1.81-1.73 (m, 1H, H4), 1.66-1.61 (m, 2H, CH 2 ester), 1.40-1.34 (m, 7H, 2Â CH 2 ester

To each well were added (5-7.5) Â 10 4 tumour cells and a given amount of the test compound. The cells were allowed to proliferate for 48 h (murine leukaemia L1210 cells) or 72 h (human lymphocyte CEM cells) or 96 h (human cervix carcinoma HeLa cells) at 37°C in a humidified CO 2 -controlled atmosphere

We are grateful to Leentje Persoons, Frieda De Meyer, Leen Ingels and Lizette van Berckelaer for excellent technical assistance. N.H. is grateful to Cardiff University for support. The biological work was supported by the KU Leuven (GOA 10/014).