key: cord-0990515-5mtmkpfv
authors: Bosc, Jean-Jacques; Latxague, Laurent; Léger, Jean-Michel; Balzarini, Jan; Forfar, Isabelle; Jarry, Christian; Guillon, Jean
title: Synthesis and in vitro cytostatic activity of new β-d-arabino furan[1′,2′:4,5]oxazolo- and arabino-pyrimidinone derivatives
date: 2009-10-29
journal: Eur J Med Chem
DOI: 10.1016/j.ejmech.2009.10.032
sha: 3456d77417741132134d0b3d2e2242ed3d04f3eb
doc_id: 990515
cord_uid: 5mtmkpfv

A series of nucleoside derivatives was obtained via heteroatom annulation of the amino oxazoline of d-(−)-arabinose. Unequivocal proofs for the stereostructure of some new arabinosyl pyrimidinone derivatives were obtained by X-ray structure analysis. These newly synthesized compounds were then evaluated for their cytostatic activity against murine leukemia (L1210), and human T-lymphocytes (Molt 4/C8 and CEM). Of all the compounds in the series, the protected silylated tricyclic fused pyrimidinone 10 showed the most significant antitumor activity against murine leukemia L1210 (IC(50) = 6 μM), and human T-lymphocytes cells Molt 4/C8 (IC(50) = 7.9 μM) and CEM/0 cell lines (IC(50) = 7.5 μM). None of the compounds exhibited significant antiviral inhibitory activities.

Antimetabolites are widely used cytostatics classified according to their chemical structure and mechanism of action. Amongst them new antimetabolite pyrimidine analogues, i.e. Cytarabine [1] [2] [3] [4] , Enocitabine [4] [5] [6] , are related to a 1-b-D-arabinofuranoside synthon (Fig. 1 ). As part of our research program, we developed the synthesis of a new series of original 1-b-D-arabino furan[1 0 ,2 0 :4,5]oxazolo-and arabino-pyrimidinones via 2-amino-2oxazoline derivatives possessing a 1-b-D-arabinosyl parent. 2-Amino-2-oxazolines are heteroamidine synthons useful for the preparation of various bioactive pyrimidinones through ring annulation, by using the two nucleophilic sites of the amidine moiety. We already developed this approach for obtaining various bicyclic compounds [7] [8] [9] .

In this work we reported the synthesis of a series of new 1-b-Darabino furan[1 0 ,2 0 :4,5]oxazolo-and arabino-pyrimidinone derivatives through a molecular biodiversity strategy, and the preliminary results of their cytostatic activity against murine leukemia (L1210), and human T-lymphocytes (Molt 4/C8 and CEM).

The readily available 2-amino-b-D-arabinofuran[1 0 ,2 0 :4,5]-2oxazolines 1-2, either unprotected (compound 1, Schemes 1 and 2) or silylated (compound 2, Scheme 1) were prepared according to previously described procedures [10] [11] [12] [13] [14] , and used as the starting material for the synthesis of b-D-arabino furan[1 0 ,2 0 :4,5]oxazoloand arabino-pyrimidinone derivatives.

N,N-dimethylformamide dimethylacetal (DMFDMA) reacted with 2-amino-b-D-arabinofuran[1 0 ,2 0 :4,5]-2-oxazoline 1 to give the expected amidine 3 (Scheme 2). The N,N 0 -dimethyl-N 0 -alkylformamide 3 was engaged in an alkylation with diketene acetone adduct, followed by spontaneous elimination of dimethylamine, to give the fused b-D-arabinofuran[1 0 ,2 0 :4,5]oxazolopyrimidin-6-one 4. The oxazoline ring opening was then conducted in dilute aqueous ammonia [11, 12] to afford the surprising arabinofuranopyrimidinedione 5 bearing an enamine function. Structural elucidation of 5 was achieved either by 1 H and 13 C NMR, and by X-ray crystallography (Fig. 2) . Especially, 1 H NMR spectrum of 5 revealed two D 2 O exchangeable singlets at 9.93 and 10.84 ppm characteristics of the enamine group in position 5. Moreover, the 13 C NMR spectrum of 5 showed one signal around 98.4 ppm specific for the enamine alkene bond (]C(Me)NH 2 ). The X-ray crystallography data of 5 unambiguously showed the presence two hydrogen on the N(9), whereas the Csp 2 (8)-N(9) bond was found at 1.292(2) Å, as typically observed for C]C-NH 2 bonds [15] . There is an intramolecular hydrogen bond between N(9)-H(9A) and O (7) where the N(9)-O(7) distance is 2.673(2) &angst. Moreover, N(9) is hydrogen bonded to O (11) of the molecule at (x À 1,Ày À 1, þz) with N(9)-H(9B)-O(11) ¼ 2.814(3) Å; other hydrogen bond are found between O (18) and N(5) of the molecule (x, y, z À 1) where the distance is 2.823(2) Å, and O (20) and O (17) of the molecule (xÀ1, y þ 1, z) with O (20) .O(17) ¼ 2.813(2) &angst. The most common type of pucker of sugar rings in nucleosides and nucleotides is with either atom C (14) or atom C(15) displaced from the plane of the other four atoms on the same side as atom C (19) , when the pucker is called C (14) or C(15) endo, respectively. In the structure of 5, the pucker is C(14) endo differing from that observed in previously described uracil-b-D-arabinofuranoside [16] where the pucker is C(14) endo making impossible an intramolecular hydrogen bond between O (20) and O (17) . Finally, spatial structure of 5 also confirmed that the oxazoline ring opening did not modify the C-1 and C-2 absolute configuration of arabinose. Based on the space group determined from crystallographic data (P1), the absolute configurations at C-1 and C-2 of this pure enantiomer were determined to be R and S, respectively. The formation of 5 was probably due to the action of ammonia on the acetyl substituent leading to an imine function which gave the stable enamine group after alkaline oxazoline ring opening.

On the other hand, treatment of 4 with sodium ethanolate afforded the expected pyrimidinedione 6 (Scheme 2). Diethyl ethoxymethylene malonate (DEEM) is a well-known bis-electrophile reagent used for heterocyclic ring annelation [9, 17] . When reacted at room temperature with 1, only the fused pyrimidinone 7 bearing a b-keto ester moiety was obtained, probably due to the regioselectivity in term of nitrogen reactivity as previously observed in the 2-amino-2-oxazoline series [17] . Oxazoline ring opening was then conducted with ammonium hydroxyde to give the sugar derivative 8 in 63% yield (Scheme 2).

As depicted in Scheme 3, 2-amino-3 0 ,5 0 -bis(O-tert-butyldimethylsilyl)-b-D-arabinofuran[1 0 ,2 0 :4,5]-2-oxazoline 2 was subsequently reacted with DMFDMA to give 9 as described above. Then, 9 was reacted with diketene acetone adduct in acetone to give 10 as previously described for the synthesis of 4 from the unprotected amino oxazoline 1. The 3D spatial determination of 10, established by X-ray crystallography, confirmed the structure in the solid state (Fig. 3 Alternatively, reaction of 2 with ethyl-2-oxocyclohexanecarboxylate in refluxing styrene led to a mixture of isomeric angularly and linearly annelated nitrogen bridgehead compounds 12 and 13, through a one-step ring annulation. This synthesis illustrates the nucleophilic reactivity of both nitrogen atoms of 2-amino-2-oxazolines [9] . Thus, the endocyclic nitrogen atom attacks first the electrophilic carbonyl carbon of ethyl-2-oxocyclohexane-carboxylate, followed by the exocyclic nitrogen attack on the carboxyl carbon (afterwards) to achieve cyclisation.

The resulting unstable cyclic b-hydroxyketone is then dehydrated to give 12. On the other hand, compound 13 was obtained after dehydration when the two nitrogen atom attacks order was reversed. After separation these two regioisomers were obtained in 41% and 30% yields, respectively. Each structure has been probed by NMR analysis. Thus, a NOE effect between the H-3 0 arabinose proton and one methylene proton of the fused cyclohexyl moiety was observed for compound 13, while no correlation was observed between the two corresponding protons in 12 [9] . For the unequivocal assignment of the two different possible structures, X-ray single crystal analyses were performed with 12 ( Fig. 4) and 13

( Fig. 5) . The 3D structures of 12 and 13 confirmed the isomerization, as anticipated on the basis of NOESY data. Both regioisomers 12 and 13 are characterized by a quasi-planar conformation of the oxazolo[3,2-a]pyrimidine moiety with dihedral angle between the mean planes of the pyrimidine base and the sugar ring ¼ 72.6(7) and 71.4(10) , respectively. In compound 12, the maximum deviation from planarity for the tricyclic cyclohexa-oxazolo-pyrimidinone moiety is found lying 0.368 Å, while in 13, C(5) deviates by 0.189 Å from the plane in the cyclohexenyl cycle. The C(8)-N(7) and C(1)-C(6) bonds in 12 were 1.277(7) and 1.388(9) Å, respectively, as typically observed for double bonds. In compound 13, the distances in the pyrimidine ring were 1.285(10) Å for C(8)-N(9), and 1.346(11) Å for C(1)-C(2). Both regioisomers were then desilylated with tetrabutyl ammonium fluoride (TBAF) in THF to give 14 and 16, respectively. Following oxazoline ring opening with EtONa, the arabinose derivatives 15 and 17 were eventually obtained in relatively poor yields (13 and 25%, respectively).

The newly synthesized compounds 3-17 were evaluated for their in vitro inhibitory effects on the proliferation of murine leukemia cells (L1210) and human T-lymphocyte cells (Molt 4/C8 and CEM/0) ( Table 1) . For all compounds, the theoretical calculations of lipophilicity as Clog P were performed using the Marvin-Sketch program [18] .

As it appears in Table 1 only compounds 4, 5 and 9-13 showed a cytotoxic activity with IC 50 < 500 mM, with 9-13 belonging to the silylated series of nucleoside derivatives. Compounds 10 and 11 were found to possess interesting activity against leukemia and

human T-lymphocyte cells with IC 50 ranging from 6 to 7.9 mM, and 17 to 30 mM, respectively. The fused tricyclic pyrimidinone 10 was found the most potent compound, whereas ring opening of 10 leading to compound 11, provided a lower activity. It must be noted that during this ring opening process, the heterocyclic ring of 11 is structurally rather different from its precursor in compound 10, since an enamine group now replaces the acetyl group. Both groups could be considered as bioisosteres, and may be involved in the biological binding site of compounds 10 and 11. On the other hand, 12 and 13, lacking such reactive functions on the pyrimidine moiety, were found to be less active. These preliminary results could be related to the lipophilic behaviour of tested compounds, highlighted by the calculated log P parameters ( Table 1) . As expected the silylated compounds are the most lipophilic ones. This lipophilic character of 9-13 could explain, in a first part, a better permeability, leading to a better cell penetration in vitro. On the other hand, comparable Clog P differences are found between corresponding values calculated for 10 and 4, and 5 and 11, respectively. These desilylated pyrimidinones 4 and 5

showed lower activities with IC 50 ranging from 211 to 380 mM on the three cell lines. The tetracylic fused isomeric pyrimidinones 12 and 13 also exhibited moderate antitumor activities, with slightly better IC 50 s for 12 (IC 50 ¼ 201 mM on L1210, IC 50 ¼ 129 mM on Molt 4/C8, and IC 50 ¼ 126 mM on CEM/0). Finally, the moderate results obtained for compound 9 (IC 50 ¼ 215-254 mM) are rather difficult to discuss since this compound appeared in the early stages of the synthesis, and therefore exhibited a quite different chemical structure compared to the other interesting compounds. Nevertheless, it can be compared to its desilylated analog 3 which showed no activity at all, stressing again the important role of the tert-butyldimethylsilyl groups. (H1N1 and H3N2 subtypes) and B in MDCK cell cultures. No specific antiviral effects (i.e. minimal antivirally effective concentration > fivefold lower than the minimal cytotoxic concentration) were noted for any of the compounds against any of the viruses evaluated (data not shown). Nevertheless, only the fused pyrimidinone 12 showed an appreciable antiviral activity against vaccinia virus with an effective concentration (EC 50 ) of 45 mM.

In summary, we reported here the synthesis of new arabinosyl pyrimidinone derivatives together with their cytostatic activity on L1210, Molt 4/C8 and CEM tumor cell lines. The stereostructure of 5, 10, 12 and 13 was unambiguously confirmed by their X-ray structural analysis. In the series, the lipophilic silylated derivatives 10 and 11 were found to be the most biologically active. They can be used now as lead compounds in the search for more active and potent nucleoside analogues to be applied in cancer chemotherapy.

Commercially reagents were used as received without additional purification. Melting points were determined with an SM-LUX-POL Leitz hot-stage microscope and are uncorrected. IR spectra were recorded on a BRUKER IFS-25 spectrophotometer. NMR spectra were obtained on a Bruker Avance 300 spectrometer (300 MHz for 1 H and 75 MHz for 13 C). Deuterated dimethylsulfoxide (DMSO-d 6 ) or chloroform (CDCl 3 ) were used as solvents, with DMSO (d H 2.5) or DMSO-d 6 (d C 39.5), CHCl 3 (d H 7.27) or CDCl 3 (d C 77.0) respectively, being employed as internal standards. Silica gel 60 (70-230 mesh) was used for column chromatography. The homogeneity of all the newly synthesized compounds was routinely checked by TLC on silica gel plates (Polygram Sil G/UV 254 ) and visualized by irradiation with a UV lamp. Elemental analyses were conducted by CNRS, Vernaison, France.

The structure of compounds 5, 10, 12 and 13 has been established by X-ray crystallography (Figs. 1-4) . The data were corrected for Lorentz and polarization effects and for empirical absorption correction [19] . The structure was solved by direct methods Shelx 86 [20] and refined using Shelx 97 [21] suite of programs. White single crystal (0.25 Â 0.10 Â 0. 

To a solution of 4 (1.0 g, 3.7 mmol) in methanol (20 mL) at r.t. was rapidily added ammonium hydroxyde (32% soln., 4 mL).

After stirring for 2 h at r.t., the solution was concentrated under reduced pressure and the residue purified over silica gel using chloroform-methanol (70/30) as eluant to give 5 (0.69 g, 65%); mp 

A solution of compound 4 (2.68 g, 0.01 mol) in ethanol (150 mL), was quickly added to sodium ethanolate (2.04 g, 0.03 mol) at room temperature. The yellow solution was stirred for 4 h at r.t. After concentration under reduced pressure, the residue was poured into water (40 mL) and acidified to pH 1 with aqueous 5% HCl. Water was removed under vacuum, and absolute alcohol (50 mL) was added to the residue. After filtration (NaCl removal) the filtrate was concentrated and the resulting solid recrystallized in methanol to To a solution of 1 (6.96 g, 0.04 mol) in a minimum amount of absolute ethanol, DEEM (8.64 g, 0.04 mol) was added under stirring at room temperature. Stirring was continued for 12 h, the solution was concentrated under reduced pressure, and the resulting residue was applied to a column of silica gel eluted with chloroform-methanol (90/10) to give 7 (5.1 g, 43%); mp 165 C; IR n cm À1 To a stirred solution of 9 (4.12 g, 9.00 mmol) in acetone (150 mL) at 50 C, diketene acetone adduct (6.39 g, 45.00 mmol) was added dropwise After stirring for 4 h, the solution was concentrated under reduced pressure and the resulting visquous residue applied over silica gel with chloroform-methanol (90/10) as eluant. The title compound was isolated as a fluorescent solid which was recrystallized in aqueous ethanol (50/50) to yield pure 10 (1.74 g, 40%); mp 78 C; 1 To a solution of 10 (0.56 g, 1.13 mol) in THF (4 mL) at r.t. was rapidily added ammonium hydroxyde (32% soln., 0.1 mL). After stirring for 24 h at r.t., the solution was concentrated under reduced pressure and the resulting yellow residue purified over silica gel using chloroform-methanol (90/10) as eluant to give 11 (0.32 g, 56%); mp 207 C; 1 To a stirred solution of 14 (0.12 g, 0.43 mmol) in ethanol (20 mL) was added sodium ethylate (0.013 mol) at room temperature. The mixture was then heated at 80 C and then stirred for 6 h at room temperature. After concentration under reduced pressure, the residue was taken up in water (20 mL) and acidified with 5% aqueous HCl until pH 1. After concentration to dryness, absolute ethanol (50 mL) was added, the mixture was filtered and the filtrate evaporated. The crude product was submitted to column chromatography eluted with chloroform-methanol (70/30) to give pure 15 To a stirred solution of 16 (2.80 g, 0.01 mol) in ethanol (30 mL) was added sodium ethylate (0.03 mol) at room temperature. The mixture was then heated at 80 C and then stirred for 6 h at room temperature. After concentration under reduced pressure, the residue was taken up in water (20 mL) and acidified with 5% aqueous HCl until pH 1. After concentration to dryness, absolute ethanol (50 mL) was added, the mixture was filtered and the filtrate evaporated. The crude product was submitted to column chromatography eluted with chloroform-methanol (70/30) to give pure 17 H-2 0 ), 4.68 (t, 1H, J OH,5 0 5.5 Hz, 5 0 -OH D 2 O exchangeable), 5.31 (t, 1H, J OH,3 0 5.7 Hz, 3 0 -OH D 2 O exchangeable), 5.48 (t, 1H, J OH,2 0 5.1 Hz, 3 0 -OH D 2 O exchangeable), 6.12 (d, 1H, J 1 0 ,2 0 7.2 Hz, H-1 0 ); 13 

Murine leukemia L1210, human T-lymphocyte Molt 4/C8, and CEM cells were suspended at 300,000-500,000 cells/mL of culture medium, and an amount of 100 mL of these cell suspensions was added to 200 mL microtiter plate wells containing 100 mL of an appropriate dilution of the test compounds. After 2 days (L1210) or 3 days (Molt 4/C8 and CEM) of incubation at 37 C, the cell number was determined using a Coulter counter. The 50% cytostatic concentration (IC 50 ) was defined as the compound concentration required to inhibit cell proliferation by 50% [22] .

The antiviral activity of the compounds was determined as described [23, 24] . The assays were based on inhibition of virusinduced cytopathicity in HEL [herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus and vesicular stomatitis virus], Vero (parainfluenza-3, reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus), HeLa (vesicular stomatitis virus, Coxsackie virus B4, and respiratory syncytial virus) or MDCK (influenza A (H1N1; H3N2); and influenza B) cell cultures. Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID 50 of virus (CCID 50 being the virus dose to infect 50% of the cell cultures). After a 1 h virus adsorption period, residual virus was removed, and the cell cultures were incubated in the presence of varying concentrations (200, 40, 8 mM) of the test compounds. Viral cytopathicity was recorded as soon as it reached completion in the control virusinfected cell cultures that were not treated with the test compounds.

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