Synthesis of LoganVir, a new carbocyclic nucleoside analogue

Abstract Starting from a natural cyclopentanoid monoterpene belonging to the class of iridoid glucosides called loganin, we performed the synthesis of a new carbocyclic nucleoside, allowing the preparation of a new lead compound, with a potential HIV antiviral activity as an reverse transcriptase competitive inhibitor that we named LoganVir. The stereocontrol of the coupling reaction was completed utilizing the procedure described by Mitsunobu with a purinic base.


Introduction
The carbocyclic nucleoside analogues are an important class of antiviral agents, particularly when used against HIV infection. Carbocyclic nucleosides are included in the class of reverse transcriptase (RT) inhibitors (Mehellou & De Clerq 2010;Matyugina et al. 2012). Nucleosides, as lead compounds, are fundamental building blocks of biological systems, and display a wide range of biological activity. Through kinases, they are phosphorylated into their mono-, di-and triphosphates, and the resulting nucleotides are processed into nucleic acids by polymerases (Mehellou & De Clercq 2009). There has been an intense research into nucleoside analogues, which function as non-toxic, selective inhibitors of kinases and polymerases for the control of viral diseases and cancer (Borthwick et al. 1990;Agrofoglio et al. 1994). Consequently, extensive modifications have been made to both the heterocyclic base and the sugar moiety in order to avoid the drawbacks exhibited by nucleosides or analogues in certain applications, mainly due to enzymatic degradations. The carbocyclic nucleosides ABSTRACT Starting from a natural cyclopentanoid monoterpene belonging to the class of iridoid glucosides called loganin, we performed the synthesis of a new carbocyclic nucleoside, allowing the preparation of a new lead compound, with a potential HIV antiviral activity as an reverse transcriptase competitive inhibitor that we named LoganVir. The stereocontrol of the coupling reaction was completed utilizing the procedure described by Mitsunobu with a purinic base. are different from nucleosides since a methylene group is present instead of the oxygen present in the furan of the nucleosides. The presence of a methylene group in the place of the oxygen in the position adjacent to the carbonium in which the heterocyclic base is linked characterises the carbocyclic nucleosides since the bond is no longer a glycosidic one but an aminic one, therefore resulting in stable division of phosphorylases and hydrolases. The structure similarity of carbocyclic nucleosides and normal nucleosides guarantees useful interactions with other enzymes involved in nucleoside metabolism in order to be used in potential therapeutic interventions (Marquez 1996). The understanding of the virus structure and its replication mechanism influences the design of the aforementioned inhibitors. The interaction mechanism of the inhibitors can be understood by analyzing the action of RT. The single viral stranded RNA is used by the RT enzyme as a template for the synthesis of double helix DNA. Firstly, the RT links the template primers, and, subsequently, it links a new deoxyribonucleoside triphosphate (dNTP). The chain elongation, which is catalysed by DNA polymerase, is a nucleophilic attack of the 3′-OH -that is terminus of the primer on the innermost phosphorous atom of a dNTP. The concurrent release of pyrophosphate forms a phosphodiester bridge De Clercq 2005). Nucleoside carbocyclic analogues have the role to act as competitive inhibitors of RT; at the same time, because of the lack of the 3′-OH function, these analogues act as chain terminators. Numerous natural products of biological interest, e.g. prostaglandins and similar derivatives, have the presence of the cyclopentane template. This template has been introduced in the molecular structure of nucleosides as a substitute for furanose moiety and deoxyribose (Bindu Madhavan & Martin 1986). Loganin, a natural monoterpenoid belonging to the class of natural products called iridoids, was chosen by us as a starting material for the synthetic process of enantiomerically pure cyclopentane moiety. This starting synthon has the right chemical function in the right position for the heterocyclic base to be inserted, giving new carbocyclic nucleoside analogues, which have a highly functionalised cyclopentane ring as a feature. In particular, aucubin and antirrhinoside were previously used as synthons for new carbocyclic nucleoside analogues (Bianco & Mazzei 1997;Bianco et. al. 2001a;Bianco et al. 2001b).

Results and discussion
We report the synthesis of a new carbocyclic nucleoside analogue using loganin 2 as the starting material, which is a methyl-cyclopentanoid glucoside present as loganic acid in Vinca difformis subsp. Sardoa. We chose this plant because it turned out to be rich in loganin and loganic acid (Bianco et al. 2005). V. difformis subsp. Sardoa samples were collected at the beginning of June 2013 from a natural population which grows in southern Sardinia (Diga Corsi, Iglesias (IT); V. sardoa is an endemic species of the Sardinia Island, a sample was stored in the Botanical Garden of the università di Cagliari. This sample of V. sardoa was first dried in a ventilated thermostat oven, then it was put in extraction and the crude extract was lyophilised in order to obtain the dried extract. The isolation of the iridoidic fraction was performed by means of absorption/desorption chromatography through the use of charcoal. This desorption process allowed the isolation of three eluites, as specified in the experimental section, giving three different solutions, one of which contained loganic acid only and the other two a mixture of loganin/loganic acid. The carbocyclic nucleoside 8 has the hydroxymethyl function and shows further hydroxylated functions. We prepared a highly functionalised carbocyclic nucleoside analogue so that we could verify, in the presence of several functions on the cyclopentane ring, whether the nucleoside analogue could be accepted as substrate by cellular kinase, therefore inhibiting the RT. Compound 8, which we named LoganVir, has the structure of the 2-((3R,4R)-4-(6-amino-9H-purin-9-yl)-2-(hydroxymethyl)-3-methylcyclopentyl)propane-1,3-diol. The synthetic strategy is described in scheme 1. The synthetic procedure is described as follows: the isolation process gave spectroscopically pure loganic acid; therefore, we decided to convert it into its derived methylester called loganin 2 by treating it with diazomethane, a specific reagent for the esterification of the carboxylic function group. Loganin was characterised through means of 1 H NMR, 13 C NMR spectroscopy and eSI mass spectrometry. Product 2 was characterised comparing it with the data reported in literature (Güvenalp et al. 2006); signals of the iridoidic nucleus were revealed, namely methyl function in position C-10 at 1.08 ppm, carbomethoxylic function in C-11 at 3.67 ppm, the H-3 olefinic proton at 7.37 ppm and the H-1 hemiacetalyc proton at 5.73 ppm. The synthetic approach used in this procedure was based on six synthetic steps: the enzymatic hydrolysis of the β glycosidic bond led to the loss of the glycosidic residue without compromising the enantiomeric puricy of the iridoic nucleus; the second step concerned the reduction of both the enol-ether cyclic system (also known as cyclic hemiacetal) and of the ester function. The third synthetic step adopted in this procedure regarded the selective protection of the primary alcohol functions maintaining the secondary alcohol function free for the insertion of the heterocyclic base. This insertion was performed only after replacing the secondary alcohol function in its acetylic function, which represents the fourth synthetic step. The fifth step was the link between the carbocyclic nucleus and the heterocyclic base Scheme 1. synthetic protocol applied. by means of Mitsunobu methodology. The heterocyclic base we used in this procedure was N-6 Benzoyl protected adenine. The last step of this synthetic procedure was a one-step total deprotection of all protecting groups giving the target that we called LoganVir. enzymatic hydrolysis was achieved by means of ß-glucosidase. The proton spectrum highlights the absence of the signals linked to the glucosidic residue. The carbon spectrum highlights the C-3 cyclic enol-ether, the carbomethoxylic system and the carbons of the dihydropyranic bridge. The aglicon 3 was treated with an excess of NaBH 4 by using water as a solvent. This reduction gave compound 4 with a 90% yield; NaBH 4 also led to the reduction of the methylester function; this is caused by the usage of water as a solvent which we suppose could improve the reduction features of the NaBH 4 (Bianco et al. 1988) . Through means of NMR, the intermediate 4 revealed the absence of the dihydropyranic signals and of the ester system in C-11 position; the positive ion mode eSI-MS spectrum revealed the presence of the adduct with ion Na. The obtained intermediate 4 contains three primary alcohol functions which were regioselectively protected with an isobutyryl residue giving the triester. The regioselectivity was obtained through a rigorous control of both temperature and reaction time; the former was kept constant at 0 °C during the reaction which was quenched with cold methanol after one hour and a half. A good regioselectivity was obtained thanks to a strict control of the quenching time as well. After exceeding the 1.5 h limit, the secondary alcohol function reacts and the unwanted protection of it is obtained, therefore losing the regioselectivity. The control of these parameters led to the protection of all primary alcohol functions present in the sample. The adopted method allowed us to have the intermediate 5. The compound 9 formed as a minor by-product was isolated during column chromatography, purification of the intermediate 5 was recycled after transesterification to the intermediate 4. The secondary alcohol function of 5 was then acetylated obtaining the key intermediate 6 in quantitative yield. We decided to employ the Mitsunobu methodology to link the nucleobase and the synthons 6 (Mitsunobu 1981;Hughes et al. 1996;Kumara Swamy et al. 2009). The reaction between 6 and N6-benzoyl adenine has been accomplished by Mitsunobu methodology; after 18 h the reaction was complete and the orange oily residue, obtained after evaporation of solvents, was purified by column chromatography to give pure product 7 as white powder. The one-step hydrolysis of all protective groups in 7 was performed by the treatment with DIBAL and the crude product was purified by column chromatography, giving pure LoganVir 8 as a colourless powder. The one-step hydrolysis of all protective groups in 7 was performed by the treatment with DIBAL (Diisobutylaluminium hydride) and the crude product was purified by column chromatography, giving pure LoganVir 8 as a colourless powder. Biological tests are in progress.

Plant material and isolation
V. difformis subsp. sardoa samples were collected at the beginning of June 2013 from a natural population which grows in southern Sardinia (Diga Corsi, Iglesias (IT)); V. sardoa is an endemic species of the Sardinia Island. The voucher specimen was identified by Dr. Cinzia Sanna (Consorzio Interuniversitario di Ricerca 'Co.S.Me.Se' , Dipartimento della Vita e dell'Ambiente, università di Cagliari) which was then stored (Herbarium CAG 736) in the General Herbarium of the botanical garden of the university of Cagliari. About 500.0 g of the aerial parts of V. sardoa were dried about 48 h in a ventilated thermostat oven at 40 °C. The dried plant materials were extracted with 96% ethanol (3 × 500 mL each). The obtained extract was concentrated under reduced pressure in order to eliminate the ethanol. The resulting water suspension was first frozen at −20 °C and the residue was lyophilised to recover 35 g of the crude extract. The total crude extract was suspended in a water solution with charcoal (about 100 g) until the negative Vanilline test, and the resulting suspension was filtered through a gooch funnel. elution with water and water-10% etOH removed all the salts and sugars, whereas 30, 60 and 90% etOH eluted the iridoid-contanining fraction. The fraction eluted with 60% ethanol was purified by chromatography with CHCl 3 -CH 3 OH (7:3) system, obtaining 220 mg of pure loganic acid, which was identified through the 1 H NMR-13 C NMR and eSI-MS, in line with the data present in literature.

Loganin (2)
468 mg of compound 1 and 10 mL of CH 3 OH, were added to 20 mL of a CH 2 N 2 /et 2 O solution giving a yellow cloudy mixture, and the mixture was left at room temperature for 20 min until slow complete decolouration. The reaction mixture was bubbled with CO 2 for 10 min until complete removal of excess diazomethane . The crude product was purified by column chromatography on silica gel (BuOH sat ) to give 428 mg of an amorphous brown powder (87.9% yield). 1 H NMR-13 C NMR and eSI-MS data and spectra are shown in S1, S2 and S3.

2-((1S,2S,3R,4S)-4-hydroxy-2-(hydroxymethyl)-3-methylcyclopentyl)propane-1,3diol.
Compound 3 (47 mg) was dissolved in water (5 mL) and a 10 fold excess of NaBH 4 was added port wise to the stirred solution. After 20 min, the reaction was complete, as shown by TLC using chloroform/methanol (7:3). The solution was neutralised with CO 2 gas. Charcoal was then added to the solution to absorb organic materials and the level of absorption was checked by H 2 SO 4 test. The resulting mixture was filtered through a gooch funnel and first washed to eliminate the salts, for desorbtion was then washed with boiling methanol. The methanolic solution was concentrated in vacuo and the product 4 was purified by chromatography, eluting it with chloroform/methanol (8:2). The product was obtained as yellow powder (36 mg, 85.5% yield). 1 (S7, S8, S9).

Loganinol recycled from tetrabutyroyl-derivative (9)
Compound 9 (50 mg) was dissolved in MeOH (10 ml), and treated with Na; at 0 °C for 5 min. The reaction mixture was left at room temperature. After 15 min the reaction was complete, as shown by TLC using chloroform/methanol (8:2). The solution was neutralised with CO 2 gas. Charcoal was the added to the solution to absorb organic materials. The adsorption of organic material was obtained by adding charcoal to the solution, and the adsorption degree was checked by TLC with H 2 SO 4 test. The resulting suspension was filtered on a gooch funnel and then washed first to eliminate the salts, and then with boiling methanol. The methanolic solution was concentrated in vacuo and the product 4 was purified by chromatography, eluting with chloroform/methanol (8:2). The product was obtained as yellow powder (23 mg, 100% yield).

Conclusion
In this work, we synthesised a new carbocyclic nucleoside analogue which we named LoganVir, starting from a substrate of natural product that is called loganin, a monoterpenoid belonging to the class of iridoids. The synthetic procedure is a linear synthesis, which consists of few synthetic steps, namely six steps, and each step has some high yields; this makes the process particularly interesting. every reaction had the dual purpose of maintaining the enantiomerically pure moiety unchanged and we performed aimed modifications to the dihydropiranic system with the goal of obtaining a carbocylic system; this carbocyclic system has shown to be variously functionalised with a good synthetic flexibility. The carbocyclic alcohol intermediate four presents synthetic flexibility since its free alcohol functions can be replaced with the possibility of giving new analogues which can show different functions. Once we obtained the carbocyclic intermediate, we carried out the insertion of the heterocyclic base in it. Finally, by performing the removal of the isobutyryl and benzoyl systems in one step, we reached our goal, namely the new carbocyclic nucleoside analogue, LoganVir. It is currently being tested for antiviral activity as a competitive inhibitor of RT.

Disclosure statement
No potential conflict of interest was reported by the authors.