5-(3-Phosphonated 1H -1,2,3-triazol-4-yl)isoxazolidines: synthesis, DFT studies and biological properties

5-Triazolyl-2-methylisoxazolidin-3-yl 3-phosphonates have been synthesized by 1,3-dipolar cycloaddition of N -methyl- C -diethoxyphosphorylnitrone and vinyl triazoles. The process showed a complete regioselectivity and a nearly exclusive cis stereoselectivity. M062X/6-31G(d,p) calculations rationalize the regio-and the stereochemical results. The formation of a hydrogen bond along a particular reaction channel significantly stabilizes both transition states and products related to cis -adducts. Biological tests indicate that the obtained compounds do not show relevant antiviral and


Introduction
2][3] In this context, structural modifications on the sugar moiety of natural nucleosides and/or modifications of the heterocyclic base have been performed.The first option involved changes in the (2-deoxy)-D-ribofuranose moiety as the inversion of hydroxyl group configuration, their elimination leading to dideoxy-or dideoxy-dydehydro-nucleosides, their substitution/functionalization by various groups or cleavage of the sugar ring leading to acyclic nucleosides. 4][9][10][11][12][13] PCOANS 1 have shown to be potent inhibitors of reverse trascriptase (RT) of different retroviruses; [14][15][16][17] truncated phosphonated azanucleosides 2 are able to inhibit HIV and HTLV-1 viruses at nM concentration; 8 truncated phosphonated N,O-psiconucleosides 3 inhibit HIV infection with low or absent cytotoxicity (Figure 1).Structural modifications concerning the purine or pyrimidine nucleobases have also been investigated [19][20] and shown to led to biologically interesting compounds.In particular, the use of unnatural heterocycles as nucleobases in the design of novel nucleoside analogues not only enhances the in vivo stability of the obtained compounds, but also confers novel mechanisms of action.In this context, the triazole heterocycle has been exploited as mimetic of the natural nucleobase.] Recently, we have synthesized new series of 1,2,3-triazolyl-N,O-nucleosides: 3hydroxymethyl-5-(1H)-1,2,3-triazol)isoxazolidines 4 inhibit the proliferation of follicular and anaplastic human thyroid cancer cell lines, with IC 50 values ranging from 3.87 to 8.76 µM; 23 1,2,3-triazole-appended N,O-nucleosides 5 show a good anticancer activity especially on the U87MG human primary glioblastoma cell line; 24 C-5'-triazolyl N,O-nucleosides 6 inhibit cell proliferation of Vero, HEp-2, MDCK, HFF, BS-C-1 cell 50% (CC 50 ) at concentration between 5.0-40.0µM; 25 C-nucleosides 7, containing a 1,2,3-triazole ring linked to an isoxazolidine system show a significant antiproliferative effect in HepG2, HT-29 and SH-SY5Y cell lines (Figure 2). 26n continuation of these efforts, we have designed a novel series of phosphonated Cnucleosides 8, featured by the presence of a 1,2,3-triazole ring linked to the isoxazolidine moiety by a C-C bond.The rationale of this choice lies on the major stability of these compounds, with respect to N-nucleosides, toward the enzymatic cleavage of the nucleosidic bond, due to the replacement of the C-N bond by the non hydrolyzable C-C bond.
The synthetic route is based on a 1,3-dipolar cycloaddition process: [27][28][29] the stereo-and regiochemistry of the reaction has been assessed in terms of theoretical calculations.The obtained compounds have been tested for their antitumoral and their antiviral activities.

Results and Discussion
The synthesis of 3-phosphonated 5-(1H-1,2,3-triazol-4-yl)isoxazolidines 11a-g and 12a-g was performed by 1,3-dipolar cycloaddition (DC) involving the phosphonated nitrone 10 18 and the corresponding 4-vinyl triazoles 9a-g 26   The reaction was carried out under conventional heating or microwave irradiation.In particular, the reaction in refluxing toluene, ethanol, and acetonitrile for 24-48 h proceeded slowly and in moderate yields (10-20%), leading to a mixture of 5-substituted cis and trans adducts 11 and 12 respectively.When the reaction was performed in CH 2 Cl 2 on microwave irradiation at 100 W for 2 h at 90 °C, an acceleration of the reaction time together with an increased yield was obtained (93-96%.).In all experiments, a 99:1 mixture of cis and trans cycloadducts was observed.
The regiochemistry of the adducts, as 3,5-substituted regioisomers, was readily deduced from 1 H NMR data.In each case, there was one proton signal at 5.30-5.15ppm, resonating as doublet of doublets, which corresponds to the H 5 proton; the alternative 3,4-substituted regioisomers are not reported to show a resonance at this chemical shift value.
The relative stereochemistry of the main compounds 11a-g was determined by 2D NOE experiments.In particular, the 1 H NMR of the adduct 11b, chosen as model compound, shows a positive NOE effect observed for H 4b (the downfield resonance of protons at C-5, 2.97 ppm) upon irradiation of H 5 (δ 5.27 ppm); analogously, irradiation of H 3 (δ 3.23 ppm) in the same compound gives rise to the enhancement of the signal corresponding to H 4b .A NOE effect was also observed when H 4b was irradiated giving rise to an enhancement for the signals corresponding to H 3 and H 5 .These results are clearly indicative of a cis relationship of the PO(OEt) 2 and the triazole units.
The observed stereochemistry is in deep contrast with the precedent results reported for the cycloaddition reactions of this nitrone with various electron-rich, electron-poor and conjugative dipolarophiles, in which the trans adducts are the major stereoisomers. 8,30Then, to rationalize the observed high regio-and stereoselectivity, a computational study at DFT level [31][32][33][34] was performed using the M062X/6-31G(d,p) calculations. 35

Theoretical results
We selected the cycloaddition of 10 with 9a as the reaction model for the theoretical investigation.Nitrone 10 can exist in the Z and E forms with an energetic preference for the Z isomer (∆E=1.7 and ∆G 298 =1.7 kcal mol -1 ).Nevertheless, a not negligible nitrone amount (∼6%) adopts the E configuration and hence its involvement in the cycloaddition cannot be excluded.Other configurations are due to possible rotating groups around the P-C bond for both E and Z nitrones; however, they lie at higher energy.For 1-phenyl-4-vinyl-1H-1,2,3-triazole 9a, the strans and s-cis conformers are almost isoenergetic (∆E=0.6 and ∆G 298 =-0.2 kcal mol -1 ); therefore they are expected to be equally present in the reaction mixture.Because of the presence of E and Z forms of the nitrone and the s-cis and s-trans triazole conformers, parallel models must be proposed to study the cycloaddition reaction.

Prediction of regioselectivity
] Houk's rule.Considering the HOMO-LUMO relative position, the dominant electronic interaction involves the LUMO of the 1,3-dipole and the HOMO of the dipolarophile, i.e. an inverse-electron demand 1,3-dipolar cycloaddition reaction (Figure 3).According to Houk's rule, the regioselectivity is governed by the atoms that bear the largest HOMO and LUMO coefficient orbitals; 33 thus, in the present case, the theoretical formation of the 3,5 regioisomer is in agreement with the experimental findings.Atomic charges.Another way to foresee the regiochemistry of 1,3-dipolar cycloaddition consists of using the charges obtained from Hirshfeld population analysis.The nitrone shows a negative charge on both C1 (∼-0.08 e.u.) and O3 (∼-0.26 e.u.) atoms (Figure 4), and also the carbons of the vinyl triazole carry negative charges (C5 ∼ -0.05 and C4 ∼ -0.10 e.u.).Hence, the cycloaddition will occur with the formation of the 3,5-regioisomer which involves the oxygen atom (the most negative end of the dipole) attached to C5 (less negative end of the vinyl-triazole, with respect to C4).

DFT reactivity indices.
We have also analyzed the cycloaddition reaction using the global indexes, as defined in the context of DFT, which are useful tools to understand the reactivity of molecules in their ground states.Thus, the electronic chemical potential µ, the chemical hardness η, the chemical softness S, and the global electrophilicity power ω were calculated according to the previous reported formulas (Table 2).
The electronic chemical potential of the nitrone is more negative than that of the vinyltriazole.Consequently, electron density transfer will take place from the vinyl-triazole to nitrone; i.e., a HOMO dipolarophile -LUMO dipole interaction occurs.Table 2 reveals that the nitrone acts towards vinyl-triazole as an electrophile due to the larger value of its ω.]   Being the present 1,3-dipolar cycloaddition an inverse electron demand type process, the nitrone undergoes a nucleophilic attack by the vinyl-triazole; thus, the local properties to be used are reported in Table 3.For the dipolarophile, C4 has the higher local nucleophilicity index, ωwith respect to C5.Therefore, C4 will be a preferred site for the electrophilic attack by the dipole.For the dipole carbon atom, higher f+ is found compared to the oxygen atom.Therefore, the C4 of the dipolarophile will be linked to the carbon atom of the dipole, following the favorable interaction between the highest nucleophilic and electrophilic sites of the reagents.These data, therefore, predict the formation of the 3,5-regioisomeric isoxazolidine in the cycloaddition reaction, in complete agreement with the experimental results.
The regioselectivity criteria of four center reactions is explained by Gazquez Mendez rule: 40 the interaction between two chemical species is more favored when the softness difference of two interacting atoms is minimum.Values of ∆S found for this 1,3-dipolar cycloaddition are: where atoms C4 and C5 of the nucleophile interact with atoms O3 and C1 of the electrophile to give rise to the preferred regioisomer and s - C4 , s + O3 , s - C5 , and s + C1 are the respective local softness of the reactive sites.Calculation of softness matching index ensures the simultaneous fulfillment of local HSAB concept at the two reacting termini.The reaction pathway involving lower value of ∆S will be the favored one.The ∆S (3,5) is smaller than ∆S (4,5).This suggests that 3,5substituted isoxazolidine will be generated from the present cycloaddition reaction, in complete agreement with the experimental findings.

Stereochemistry
The reaction of nitrone 10 with alkenes usually gives rise to trans adducts preferentially; however, in the present case, the almost exclusive formation of the isomer occurs unexpectedly.To rationalize this behavior, quantum chemical calculations have been carried out to evaluate the activation and the formation energies for 3,5-regioisomers.
Calculations show that the reaction occurs throught labile intermediates (I) because of the formation of weak bonds between the nitrone and alkene with long distances for the newly forming C1---C4 and O3---C5 bonds (> 3 Å).The formation of intermediates occurs without any energy barrier and their shape depend on the initial structure of geometry optimization (Table 4 and Figures S1 and S2).It is evident that the relative orientation of nitrone and alkene substituents determine, even at this stage, some energetical preference (Table 4) and this issues will be better clarified in the transition state discussion, where steric constraints become more stringent.
The intermediate formation is an endoergonic process, mainly because of the reduction in entropy contents due to the degrees of freedom lost in the nitrone-alkene coupling (-T∆S: 10 -15 kcal mol -1 ).The next stage of reaction concerns the transition state (TS) that lies at high energy and hence it is the rate limiting step.
The E/Z-nitrone and s-cis/s-trans vinyl-triazole isomers can be combined in both exo or endo modes, giving rise to eight possible transition states (Table 4 and Figure 5).Furthermore, the compounds under analysis are prochiral molecules, thus transition states and final products, formed in the transformation, can adopt both S and R configurations, doubling the number of possible structures.Here, transition states and products of S,S and S,R compounds are reported, while the related R,R and R,S enantiomers are energetically the same and are not further considered.The newly forming isoxazolidine ring has three substituents at C1, N2, and C5 atoms and, of course, the relative stability of the transition states depends on the interactions among these groups.The activation energy found for the eight transition states clearly defines a preferred conformation (Z) -exo -s-trans for the transition state, which leads to the formation of the cisadduct.At about 5 and 2 kcal mol -1 higher in electronic and Gibbs energies, respectively, there are two reaction channels in which the nitrone in the (E) conformation leads to the formation of both cis and trans-adducts.Other reaction pathways lie at higher energy and cannot be reached even at 90 °C.The Boltzmann distribution, calculated at 90 °C for the three reaction channels, assuming a ∆∆G of 2 kcal mol -1 , predicts a cis:trans ratio of 94:6, in reasonable agreement with the experimental trend.
The nitrone cycloaddition with monosubstituted conjugative dipolarophiles (such as vinyl triazoles), capable of secondary orbital interactions, proceeds through endo transition states. 41ccording to this rule, one might expect that the preferred path involves an endo attack on the (E)-nitrone, and not an exo attack on the (Z)-nitrone.On the contrary, in our case, the cycloaddition takes place involving the (Z) nitrone with an exo transition state.It is not easy to rationalize the computed activation energies on the basis of simple arguments.For example, if we assume that interactions between the three substituents at the isoxazolidine ring are steric in nature, the (E) -endo -s-cis and (E) -endo -s-trans attacks should be very high in energy, because the three substituents are on the same side of the ring.Actually, the (E)endo -s-trans attack lies high in energy, but the (E) -endo -s-cis attack is energetically accessible.Similarly, it is not obvious that a change in s-cis / s-trans orientation of the (Z)-exo attack implies a ∆∆E and ∆∆G of 6.3 and 3.3 kcal mol -1 .However, for the (Z) -exo -s-trans process, the molecular structure reveals the formation of a weak intramolecular H-bond involving the oxygen atom of the P=O group and the hydrogen atom of the triazole ring (the P=O …….H triazole distance is 2.33 A).It is well known that the phosphoryl group is a good proton acceptor and forms strong hydrogen bonds with various substrates.Furthermore, it has been found that several 1,4-diaryl-1,2,3-triazole compounds can form C-H . . . .][44] The bond distances of the five-membered rings are similar for all the transition states.In particular, the newly forming bonds C1--C4 and O3--C5 spread in a narrow range 2.104-2.191Å, thus indicating that the reaction is essentially concerted.These bonds almost lie in the same plane, with ring puckering of few degrees (the C1C4C5O3 torsion angle deviate few degrees from zero).The nitrogen atom lies out of this plane, thus the newly forming isoxazolidine ring adopts an envelope conformation with the methyl in the axial position.
The eight kinetic evaluated products spread in a narrow energy range (∆G°2 98 =-17.7 / -23.6 kcal mol -1 ) and indicate an high exoergonic process.The (Z) -exo -s-trans product is the most stable and the molecular structure reveals the formation of the intramolecular H-bond involving the P=O as acceptor and the triazole ring as donor (P=O---H-C 2.147 Å), similarly to the Hbond observed in the related transition state.Therefore, the (Z) -exo -s-trans pathway, that leads to the formation of cis-adducts, is kinetically and thermodynamically preferred.
The cytoxicity of the tested compounds towards uninfected host cells, defined as the minimum cytotoxic concentration (MCC) that causes a microscopically detectable alteration of normal cell morphology, was also assayed.Moreover, to determine if the compounds have any effect on cell proliferation, the cytostatic activity was evaluated by measuring the 50% cytostatic inhibitory concentration (CC 50 ), using the MTT test on Vero, HEp2 and HFF-1 cells.All the tested compounds except 11d were not able to inhibit cell proliferation at 200µM concentrations.

Conclusions
A novel series of phosphonated C-nucleosides, featured by the presence of a 1,2,3-triazole ring as a mimetic of natural nucleobases, linked to the isoxazolidine moiety by a C-C bond, has been synthesized.The very high regio-and stereo-selectivity of the reaction process, which involves a cycloaddition reaction of a phoshonated nitrone with vinyl 1,2,3-triazoles, has been rationalized on the basis of theoretical calculations.In particular, DFT calculations showed the inverseelectron demand nature of this 1,3-dipolar cycloaddition.The HOMO/LUMO energies, character and reactivity indices, charges on reactive sites, and Fukui functions of reagents support the formation of the 3,5-regioisomer in agreement with experimental results.
Activation and formation energies, calculated for eight different reaction pathways, explain the high stereoselectivity of the cycloaddition process.In particular, the formation of a hydrogen bond between the phosphoryl group and C-H of triazole ring, along the (Z) -exo -s-trans reaction channel, stabilizes both transition states and final products; the formation of cis-adducts is kinetically and thermodynamically favored.Biological tests indicate that the obtained compounds do not show relevant antiviral and anticancer activity.

Experimental Section
General.Solvents and reagents were used as received from commercial sources.Melting points were determined with a Kofler apparatus.Elemental analyses were performed with a Perkin-Elmer elemental analyzer.NMR spectra ( 1 H NMR recorded at 500 MHz, 13 C NMR recorded at 126 MHz) were obtained with Varian instruments, and data are reported in ppm relative to tetramethylsilane.Thin-layer chromatographic separations were carried out on Merck silica gel 60-F254 precoated aluminum plates.Flash chromatography was carried out using Merck silica gel (200-400 mesh).Preparative separations were carried out using an MPLC Büchi C-601 instrument using Merck silica gel 0.040-0.063mm, and the eluting solvents were delivered by a pump at the flow rate of 3.5-7.0mL/min.C-[(tert-Butyldiphenylsilyl)oxy]-N-methyl nitrone 10 was prepared according to described procedures. 8Benzyl/alkyl and aromatic azides were synthesized according to literature procedures. 24neral 1,3-dipolar cycloaddition procedure.A solution of 9a (0.50 g, 2.92 mmol) and nitrone 10 (1.10 g, 3.50 mmol) in CH 2 Cl 2 (5 mL) was put in a sealed tube and irradiated under microwave conditions at 150 W, 80 °C, for 2 h (CEM Discover Microwave reactor).The removal of the solvent in vacuo afforded a crude material which, after flash chromatography purification by using as eluent a mixture of cyclohexane/ethyl acetate 7:3, gave compound 11a, as yellow oil, 93% yield.The 1H NMR spectrum shows the presence of cis and trans isomer respectively in 99:

Figure 3 .
Figure 3. Energies (hartree) and coefficients of HOMO and LUMO, useful for the application of the Houk's rule (energies are not in scale).

Figure 4 .
Figure 4. Hirshfeld charges (e.u.) on reactive atoms of reagent in the various conformations.

Figure 5 .
Figure 5. Molecular structures of the eight transition states for the formation of the cis and transadducts of the 3,5 regioisomer.
a A (N) -P A (N-1) Nucleophilicity of atom A in molecule of N electronsThe related local softness, s ± A , and the local philicity index, ω ± A , are easily obtained multiplying global quantities and condensed Fukui functions: s