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Bioorganic & Medicinal Chemistry Letters 15 (2005) 545–550

New conformationally locked bicyclic N,O-nucleoside analogues
of antiviral drugs
Antonio Procopio,a,* Stefano Alcaro,a Antonio De Nino,b Loredana Maiuolo,b
Francesco Ortusoa and Giovanni Sindonab
a
Dipartimento di Scienze Farmaco-Biologiche, Università della Magna Graecia, Complesso Ninı` Barbieri,
88021 Roccelletta di Borgia (Cz), Italy
b
Dipartimento di Chimica, Università della Calabria, Ponte Bucci, cubo 15C, 87036 Arcavacata di Rende (Cs), Italy
Received 21 October 2004; revised 17 November 2004; accepted 18 November 2004
Available online 23 December 2004

Abstract—In order to obtain rigidity within the sugar moiety of nucleosides, the bicyclic pyrimidine derivatives of N,O-isoxazoli-
dines were designed and synthesized by using 1,3-dipolar cycloaddition of D1-pyrrolidine-1-oxide and the appropriate vinyl-
nucleobases. 
2004 Elsevier Ltd. All rights reserved.

1. Introduction side analogues9 has prompted the exploitation of
strategies aiming at locking the puckering of the fura-
Modified nucleosides containing hydroxyl handles for nose ring into one of the two rotamers. Many anti-
their enzymatic phosphorylation in vivo, such as AZT, HIV nucleoside analogues are, in fact, active when their
ddC, d4T, etc., are currently employed in the multi-drug sugar ring conformational equilibria are centered
protocols adopted in therapeutic treatment of HIV around the (3E, S-type) C 3 0 -exo conformation. Our ori-
infections.1 ginal strategy for the formation of isoxazolidinyl nucle-
osides is based on the 1,3-dipolar cycloaddition of
A new line of research has been recently opened in this azomethinoxides on vinyl-nucleobases2 achievable by a
field, which considers the replacement of the modified ri- straightforward procedure.2,10
bose with an isoxazolidine nucleus.2–5
The structural feature of the bicyclic isoxazolidine
Preliminary in vivo tests have shown a promising bio- model (1, Fig. 1), obtained from D1-pyrrolidine-1-oxide
logical activity for some of the members of this new fam- and ethene, is represented by the geometric constraints
ily of potential antiviral drugs.6 The antiviral activity preventing cis–trans interconversion by nitrogen inver-
exhibited by nucleoside analogues has been correlated sion.11 Moreover, the presence of the second five-
to some extent to preferred conformations achieved by membered ring fused to the isoxazolidine moiety induces
the drug in the formation of the enzyme–inhibitor com- a restricted conformational mobility. If a nucleobase
plex, which temporarily inhibits the DNA strand prolif-
eration.7 Unmodified nucleosides rings, as described by
the pseudorotation cycle,8 exist in either S-type (2 0 - O O B
endo/3 0 -exo) or N-type (2 0 -exo/3 0 -endo) conformations. N
N
The observation that reverse transcriptase is able to dis-
criminate between two conformationally locked nucleo-

(1) (2a-c)
2a; B= thymine
Keywords: Nucleoside analogues; Vinylpyrimidine nucleobases; N, 2b; B= uracil
O-Isoxazolidines. 2c; B= cytosine
* Corresponding author. Fax: +39 0961391143; e-mail: procopio@
unicz.it Figure 1.

0960-894X/$ - see front matter  2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.048

the structural mann population for each north (N) and south (S) properties of bicyclic pyrimidine derivatives 2a–c were conformer and. During tures in the solid state and in solution are virtually these two calculations. Procopio et al. investigated by means of molecular modeling studies. our conformational model.16 With the purpose to validate identical conformations in the solid state and in solu. lower than 0. at 300 K (MD300) and at 500 K (MD500). 7.14 Each Monte already experienced with other nucleoside analogues. compounds widely explored founding both south (C2 0 formationally restricted deoxyriboses. com- ationally locked nucleoside probes whose structural fea. as defined identical. lowing considerations: (1) the use of rigid isoxazolidine allowed us to estimate the conformational space of all pyrrolidine bicyclic heterocycles as surrogates for con. glycoside moieties ex- ally restricted by the fused pyrrolidine ring. respectively. have been monitored. / Bioorg. The large average num- heterocyclic nucleoside analogues was based on the fol. in Figures 2 and 3. glycoside moiety. In order to consider in the following analysis mobility of the new compounds compared with the only the most representative conformations. We performed two runs. con- Monte Carlo conformational search taking into account trolled by the v torsion. computed on the heavy atomic coordinates. 15 (2005) 545–550 was inserted in this molecular framework it could be ex. ers. ated structure has been compared to the others includ- ing both geometric and energetic criteria.546 A.9 Carlo generated conformer has been energy minimized with the force field MMFFs15 by means of 5000 itera- The synthesis of the three pyrimidine nucleosides 2a–c tions of the Polak Ribiere conjugate gradient algorithm was therefore designed to exploit the effect of the fused adopting a convergence criterion equal to 0. which are promising precursors for novel types of tion of the base with respect to the glycoside moiety therapeutic nucleoside derivatives.25 Å The design of isoxazolidine nucleosides 2a–c as novel have been considered identical. We have designed the bicyclic nucleoside analogues 2a– The average value of v has been used to locate the posi- c. The geometric properties of compounds 2a–c have been evaluated considering these two descriptors that showed All molecules have been built using the Maestro12 Linux a very low variability indicating. . v and m0–4 torsions. energy conformers have been submitted to molecular tional analysis for evaluating the reverse transcriptase dynamic simulations. ber of duplicate structures. in all cases higher than 147.2-b]isoxazolidine tion P. (2) the basicity endo–C3 0 exo) and north (C3 0 endo–C2 0 exo) conform- of the hydroxylamino nitrogen atom should confer en. tively. with the isoxazolidine part conformation. as global minimum en- ergy south and high energy north conformations of 2a–c In a comparative structural study performed on hexahy. dropyrrolo[1. in the Monte Carlo case.2-b]isoxazolidine11 it was concluded that a fused pyrrolidine ring can impart significant rigidity to The conformer population has been evaluated by means the isoxazolidine portion on the molecule to the extent of the Boltzmann analysis by using the MOLINE ther- that the resulting nucleoside analogues indeed show modynamic module. each gener- monocyclic analogues. have been widely explored dur- the nucleobase rotation and the interconversion of the ing molecular dynamics simulation of both (see Fig. the 2a–c global minimum tion. in Scheme 1. puted for 5 ns using a time step equal to 1.2. Global minimum energy south conformers of 2a–c structures. respec- (RT) potential binding properties starting with conform. one graphical user interface and submitted to 2000 steps of highly constrained conformation. 4). A structural comparison between them is reported hanced stability to glycosidic linkage in acid media. Chem. For this reason. Lett.8 In Table 1 are summarized the conformational ring system. as sum of the Boltz- aim to identify most stable conformations. Med.01 kcal/ ring on the isoxalidine moiety on the conformational Å mol. properties of 2a–c compounds. as P. we have conducted a conforma. Solvent effects have been considered pected that the modified nucleosides 2a–c show a pecu. With the pressed.5 fs. The sugar moiety in while m0–4 to compute the phase angle of pseudorota- 2a–c consists of a hexahydropyrrolo[1. compounds. for all molecules. Base rotamers.1 kcal/ 2. Conformers with an internal energy difference lower than 0. Figure 2. Results and discussions mol and with a root mean square deviation. in the molecular dynamics cases. using the GB/SA13 water method for all calculations liar structure–function relationship similar to that as implemented in MacroModel ver.

86 and 2c 10. Med.83 version also in extreme conditions (i. was related to the ano- meric carbon C2 0 configuration due to synthetic pathway. Chem. 2a MD300 2b MD300 2c MD300 2a MD500 2b MD500 2c MD500 270 N N O anti O N 180 degree 90 N 0 O N syn O N -90 50 500 950 1400 1850 2300 2750 3200 3650 4100 4550 5000 picoseconds Figure 4. in kJ/mol. which. A.10 95.90 179. Notation of the rotatable bonds used for conformational analysis.e.89 180. Procopio et al. .10 widely reducing the probability of puckering intercon- 2b 5.53 94. Lett. Boltzmann probability in percent at room temperature for N only for not populated high energy conformations. anti.27 178.77. the analysis of P clearly indicated the dependence of this parameter on the C4 0 stereochemis- Scheme 1. v4 v0 onto the pyrimidine ring and that of the isoxazolidine O base moiety.49 dynamics simulation at 500 K).. in our compounds. Relative energies. This observation indi- cates that these molecules have a strongly conserved DNA-like shape and therefore they could be considered The syn conformation was energetically less stable than for further optimization studies with the aim to design the anti one. depicted as white and grey areas. of these structures are: 2a 10. 15 (2005) 545–550 547 Figure 3.44 94. The molecular complication of N S P P the glycoside locked this moiety in a S conformation 2a 5. Energy minimized north conformers of 2a–c compounds. try.47 180. respectively.85. likely due to the electrostatic and Van der new or more potent HIV reverse transcriptase Waals repulsion between the sp2 oxygen in position 2 inhibitors. 2b 14.00 184. and S conformers and the average P values In conclusion. compounds 2a–c showed quite similar Compd Monte Carlo MD300 MD500 structural properties.56 178. v rotation during MD300 and MD500 simulations. molecular 2c 4. The puckering interconversion was observed Table 1.and syn-conformations are. / Bioorg. N χ v3 v1 Taking into account the bicyclic structure of the glyco- v2 side moiety.

C3 0 -exo in the ribo nucleoside analogues). which resemble the 3E (C3 0 -exo) con. 50 200 µΜ sively the 2 0 RS. 5). proton NMR in the presence of indicated concentration of the three compounds. Chem. 400. Antiviral efficacy of 2a–c in VERO cells infected with HSV-1 tween different conformational states. H ecules were synthesized and a possible equilibrium be. 200. The configuration and conformation of the bicyclic nucleosides 2a was evaluated by 1H NMR and NOE The synthesized three modified nucleosides were prelim- experiments. 0 0 Figure 5. inhibition of HSV-1 replication in the concentration (11%/11%) supported the assigned structure and conformation.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy- isoxazolidine ring.3-dipolar process and the homogeneity of the 800 µΜ 1 20 H NMR data permit us to consider the extension of 10 these conclusions to the other two cycloadducts 2b and 2c. (8%/8%) and One of these nucleosides (2b) displayed an interesting H-2 0 . 6). 0 2a 2b 2c For the purpose of detecting a possible equilibrium be. by analysis of proton NMR spectra. Here we describe a convenient first synthesis of 2a–c and Any changes was observed for the relevant coupling also discuss their conformation. The spectrum evidenced the presence of a inarily screened in a cell-based system (VERO cells) for NOE effect between the H-6 of the thymine moiety their ability to inhibit HSV-1 replication (Fig.2-b]isoxazolyl) 5-methyl- tween different conformational states were investigated 1H-pyrimidine-2. Figure 6. In and H-4 0 (8%/8%) indicating an anti conformation of addition. Lett. In addition. The 1. phenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-based assay formation in the b-D -ribo configurated nucleoside ana. one of two hydrogen nuclei in 3 0 . 100 0 90 The absence of any coupling constant between H-4 of 80 Percent of Infection the isoxazolidine ring and H-2 0 also indicates the confor- mation of the isoxazolidine ring to be exclusively S-type. The constant reproducibility 400 µΜ 30 of the 1. cytotoxicity was evaluated in parallel in an the thymidine ring and C4 0 -exo conformation of the MTS [3-(4. 15 (2005) 545–550 O H 3C O B toluene dry N + N+ B 110 ˚C 5 4 O- 3 4a-c 2a-c H 6 3 NH 8% 1 2 O O NH2 N B= CH3 HN O HN N 2' O N O N O N N H H O a b H 8% c 4' 3' H Scheme 2. the other hydrogen nucleus in 3 0 .4 SR)-1-(Hexahydropyrrolo[1. logues.3-cycloaddition constants or chemical shifts between the low and high reaction of D1-pyrrolidine-1-oxide 3 with N1-vinyl temperature spectra indicating that the isoxazole ring pyrimidine derivatives 4a–c gave the bicyclic adducts in these compounds does not appears to be involved in 2a–c in good yields (Scheme 2). / Bioorg. The spectra of thymine derivative 2a was recorded in results are expressed as mean values along with those of three DMSO-d6 over the temperature range of 20–80 C. 70 0 µΜ So 2a was assigned as derived from an exo approach of 60 100 µΜ the dipolarophile to the nitrone furnishing almost exclu.548 A. independent experiments. a conformational N « S equilibrium in solution as is the case for conventional nucleosides. Table 2. Percent growth of a VERO cell line exposed at four increasing concentrations (100. Synthetic route to 2a–c. mutual NOEs between H-4 0 and H-3 0 a. Med. (2 RS. Procopio et al. 7' H 5' H H Based on molecular modeling the compounds 2a–c were 11% 6' predicted to exist in a S-type conformation (correspond- H ing to 3E. (Table 2). In H order to confirm the assumed rigidity the modeled mol. 800 lM) Cell line 2a 2b 2c a HSV-1 in VERO cells 100 200 400 800 100 200 400 800 100 200 400 800 5 10 12 55 41 55 70 83 3 3 6 67 a The negative value indicate the percent of cells killed. . and H-3 0 b. 4 0 SR enantiomer where H-2 0 and H-4 0 40 are anti each other (Fig.4-dione (2a).

2.02–2. conformation.2-b]isoxazolyl) dried over Na2SO4. H5 0 . 1H NMR spectra 4. Jgem = 21. 5. 55. 1H. 17.29. 31. H. 64. H04 ). General procedure 2a–c Green Monkey) cell lines assayed (CC50 > 1000 in every case).4-dione (2a) sidered valuable structural probes for the evaluation of 1 RT binding affinity as well as useful templates for fur. 7.80. JH5H6 = 7.73 (m.06 (m. H7 0 a).66. its spectral data with those reported in literature. cooled to room temperature and the solution separated 18. JH2 0 H3 0 b = 2. 5-methyl-1H-pyrimidine-2.85 (m. Med.2-b]isoxazolyl) The bicyclic N. 2. 127. H6). N. by molecular modeling and NOE spectroscopy sup. 64.72.70–2. 141. 150. H NMR (300 MHz. 136. 2. 4. D1-Pyrrolidine-1-oxide (3) 1H.74–3. the mixture was warmed C NMR (300 MHz. 84 (100). / Bioorg.97 (m.5 h. no apoptosis was detected. (2 0 RS. JH2 0 H3 0 a = 7. resembling molecules 2a–c.84–3. Anal. 41. 1H. carried out with a Perkin–Elmer 240 analyzer.14. 112 (67) [MUra]+. Chem.4 0 SR)-1-(Hexahydropyrrolo[1. 3. 43. 3.02–1. 31.97. (2 0 RS. H. DMSO-d6): d NH2OHÆHCl (8.O-nucleoside analogues 2a–c can be con. H7 0 b.79. 2H. preliminary biological assays seem to re. JH2 0 H3 0 a = 6. ral drugs starting from a locked nucleoside structures DMSO-d6): d (ppm) 164.1. Moreover. 13C NMR (300 MHz.0 equiv) (prepared from its corresponding 3. Conclusions hydroxylamine by HgO oxidation) and some grains of hydroquinone as polymerization inhibitor. 2H.17 significant toxicity was detected in the treatment in the MOLT-3 (human lymphoblastoid) and VERO (African 4. NH2.82.3.4. H. 3. 6. (ppm) 163.88. 84. filtered and concentrated to give a 5-methyl-1H-4-amino-pyrimidine-2-one (2c) crude mixture. 5H. 4. Calcd for C10H13N3O3: C. The reaction mixture was then Anal. 109. 24. 1H. 83.33 (dd. Solvents and reagents were purified by standard proce- dures and were distilled prior to use. Microanalyses were NH). 17. JH2 0 H3 0 b = 3. Chemical shifts are given in ppm from H NMR (300 MHz.53 (m. 13C NMR (300 MHz. Lett. H7 0 b).82 (d.37. DMSO-d6): d (ppm) 7.5. 4. H3 0 ). H7 0 b). ESI/MS m/z 224 (62) Et3N (70 mL). H2 0 . 2. H3 0 b). Cycloaddition reaction for 2a–c preparation lected organic phases were dried over Na2SO4. H6. ther drug design investigations.20–1.15.87. 1H-pyrimidine-2. 13 was added slowly.08 (d. JH2 0 H3 0 b = 4. H.72 (d. CH3). 150.68. (2 0 RS.31.00–3. 1H.61 (m. DMSO-d6): d (ppm) 11.83 g. 1H.87 Furthermore.88.94.69. 4. 46.4-dibromobutane (10. H5. Found: C. tion of 1.2.13. JH2 0 H3 0 a = 5. JH5H6 = 8. H4 0 ). to room temperature and reacted for 4 h as indicated by TLC [CH2Cl2/CH3OH = 9:1]. H3 0 ).70).58.72–2.64.89 (m.2-b]isoxazolyl) were recorded at 300 MHz in DMSO-d6 using a tetra. The crude mixture was then diluted with CH2Cl2 and filtered on Celite. N. 1H.78 (d.02–1. H2 0 . 2. 3.95. 43.30 (s. 5. 53. used in the oxidation step as shown by comparison of H6.20–3.3 mmol) in dry 56.94. 112 (26) [MThy]+. A 0. H04 ). 18. 15 (2005) 545–550 549 range 100 lM (41% of cells killed) to 500 lM (83% of by comparison of its spectral data with those reported cells killed) (Table 2) and for the three compounds no in literature. H3 0 a).71. The product 3 was obtained Uracil (2b) 17 65 90:10 Cytosine (2c) 20 80 98:2 as pure compound with 89% yield and was identified . 1H.43 (s.36. JH6H5 = 7.91–2. 56.78.09. NH). The stirred solution was maintained [M+H]+. N. DMSO-d6): d (ppm) 166. H7 0 a). 2H.34.54 (m. A suspension of N1-vinyl pyrimidine derivatives 4a–c6 in dry toluene (20 mM) was added to D1-pyrrolidine-1- oxide 3 (3. H5 0 . H6 0 .00). 8. (m.36 (m.5. Table 3. 6. filtered Nucleobase Reaction time (h) Yield (%) Epimeric ratio (%) and concentrated. Procopio et al. 1H.80.4 0 SR)-1-(Hexahydropyrrolo[1. 6. 68 (10). 2. H5 0 . The col.16 (dd.4). H5. ESI/MS m/z 260 (44) [M+Na]+.66).70).0 g.95. under reflux for 1. 56 (3). DMSO-d6): d (ppm) 11. The reaction Conformational analysis of nucleoside analogues 2a–c mixture was stirred under reflux for 6–20 h (Table 3).50. JH6H5 = 8.0 g. H2 0 . The collected organic phases were 4. 123 (50). (m. 1H.4-dione (2b) methylsilane (TMS) as internal standard (Bruker ACP 1 300 MHz).82. After 0. 1H. 1H.6 M solution of 1-hydroxypyrrolidine in CH2Cl2 3. 101. 1H.25. The obtained crude products were purified on silica gel by flash-chromatography Thymine (2a) 6 95 98:2 [CH2Cl2/CH3OH = 9:1]. 6. H6 0 . 7.53 (m. then the solution was concentrated under vacuum and ported the assumption of rigid bicyclic structure in the crude mixture purified by flash-chromatography which the isoxazolidine ring was locked in S-type (ethyl acetate/methanol = 9:1). H7 0 a). 5. H6 0 . 5. TMS and coupling constants in Hz. Found: C.05–2.0 mmol) was added to a solu. 238 (100) [M+H]+. 5H.70. 1H. 1H. which afforded the 1-hydroxypyrrolidine 1 in quantitative yield as a yellow oil pure enough to be H NMR (300 MHz.00).4 0 SR)-1-(Hexahydropyrrolo[1. from the insoluble salts was diluted with ethylic ether and filtered on Celite. Experimental 55. 24. N. 2. Calcd for C11H15N3O3: C.99).32.41.73 (m. A.53 veal interesting chances to develop new potential antivi.75). 1H. 8H.95.5 mmol) 2. 1H. 53.64 (s.09 (dd.5 h. 12.52– was cooled at 0 C and yellow HgO (9.02 (d. 1H.17 6.

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