Synthesis and Biological Activity of Reversed Pyrimidine Nucleosides

. An efficient approach to reversed nucleosides which enables their synthesis in gram quantities is described. N -1′-Pyrimidine reversed nucleosides were prepared by treating of the sodium salt of pyrimidine bases with protected 5-tosyl ribose. Additionally, N -1 ′ , N -3 ′ -disubstituted reversed nucleosides were isolated in the condensation reactions with the 5-halogen pyrimidines. Using the Sonogashira coupling of 5′-iodouracil reversed nucleoside with ethynyltrimethyl silane gave 5′-ethynyl derivative which was further transformed into 5′-acetyl reversed nucleoside. Biological activity of deprotected reversed nucleo- sides was validated on the panel of six human carcinoma cell lines (HeLa, MIAPaCa2, Hep2, NCI-H358, CaCo-2, and HT-29). 5′-Iodouracil derivative displayed moderate growth inhibition activity against human colon carcinoma cells.


INTRODUCTION
Modified nucleosides represent a well known class of chemotherapeutic agents for treatment of viral 1−4 and cancer 5,6 diseases. In the quest for new derivatives with a potent biological activity, many structural variations at the base and/or sugar moiety of natural nucleosides have been explored. 7,8 The practical applicability of nucleoside analogues in chemotherapy largely depends on the stability of the drug in organism, because their catabolism usually includes degradation of nucleosidic linkage. Reversed or iso-nucleosides constitute a class of nucleoside analogues in which the nucleobase is linked to the sugar moiety through a carbon atom other than ribofuranose-C1. Hence, this class of compounds appears particularly interesting as drug candidates 9−13 due to the lack of glycosidic linkage which makes them more stable to hydrolytic cleavage. In addition, the reversed nucleosides represent the largest pool of chiral synthons for the synthesis of aliphatic nucleoside analogues. 14 −20 In our previous communication we have reported on the synthesis of several partially and fully deprotected reversed and double headed nucleosides the former incorporating uracil or 5-iodouracil attached by N1′ at the C5 position of ribofuranose. 19,21 In this work we present detailed experimental conditions for the synthesis of such reversed nucleosides and extend the synthesis to the highly interesting reversed nucleoside 13 incorporating 5-fluorouracil, the well-known anticancer drug. We also report on the preparation of the novel type of the nucleoside derivatives 9, 11 and 15 containing the ribose fragments attached at both, the N1′ and N3′ positions of 5′-iodo and 5′-fluorouracil bases. The example of further synthetic modification of the reversed 5′-iodouracil nucleoside 10 into protected 5′ethinyl derivative 16 by the Sonogashira coupling reaction is also presented. Upon deprotection it becomes a versatile synthon for the click chemistry. The described synthetic studies enabling preparation of reversed nucleosides in the gram scale quantities are the prerequisite for biological testing and also open new perspectives for their synthetic transformations into novel optically active aliphatic or double headed nucleoside analogues, or sulfonamido and 1,2,3-triazolyl substituted reversed nucleoside derivatives. 22 The prepared reversed nucleosides were tested for the antiproliferative activity on the panel of six human carcinoma cell lines (HeLa, MIAPaCa2, Hep2, NCI-H358, CaCo-2, and HT-29) and 5′-iodouracil derivative 14 showed promising growth inhibition activity against human colon carcinoma (Ca-Co-2) cells.

General Procedures for the Preparation of Reversed Nucleosides 7−11
The sodium salt of base was prepared by stirring a suspension of an equimolar amount of the pyrimidine base 4−6 (1 mmol) and sodium hydride (50 % in oil suspension, 1 mmol) in DMF (3−4 mL/mmol) at room temperature for 1 h and warming at 60−80 °C for 0.5 h. A solution of the methyl 2,3-O-isopropylidene-5-O-ptoluenesulfonyl--D-ribofuranoside (3) (0.8 mmol) in DMF (1.7 mL/mmol of sugar) was added dropwise to this suspension at room temperature. The reaction mixture was stirred and heated at 100 o C for 20 hours. The resulting clear solution was evaporated and the residue was dissolved in hot chloroform. The suspension was filtered through Celite and filtrate was washed with water, dried over Na 2 SO 4 and evaporated. Calcd. mass fractions of elements, w/%, for C 13 H 18 N 2 O 6 (M r = 298.29) are: C 52.34, H 6.08, N 9.39; found: C 52.14, H 6.21, N 9.5. Method B: Compound 10 (143 mg, 0.34 mmol) was dissolved in methanol (50 mL) and 0.1 M aqueous NaOH (3.4 mL) was added. The reaction mixture was cooled to 5 o C and purged with argon. Palladium on carbon catalyst (79 mg) was added and the reaction mixture was treated with hydrogen gas (42 psi) in a Parr hydrogenation apparatus for 4 h. The mixture was filtered through a Celite pad and washed with boiling methanol (20 mL). The combined methanol filtrates were concentrated under reduced pressure, dissolved in dichloromethane, washed with water, dried over Na 2 SO 4 and evaporated. The product was crystallized from methanol to afford 82.6 mg (82 %) of 3. The spectral properties were identical with a sample synthesized by method A. (8) and 5fluoro-1,3-bis(tetrahydro-4-methoxy-2,2-dimethylfuro [3,4-d] [1,3]dioxol-6-yl)methylpyrimidine-2,4(1H,3H)-dione (9) Following the general procedure from 5-fluorouracil 5 (1.  (11) Following the general procedure from 5-iodouracil 6 (1.55 g, 6.5 mmol) and after purification of the crude mixture by flash chromatography (CH 2 Cl 2 /MeOH 60:1), N-1′-regioisomer 10 (1.27 g) was obtained in a yield of 58 % as a white solid and N-1′,N-3′-disubstituted nucleoside 11 (47 mg) was obtained in a yield of 1.5 % as a yellow foam. To a solution of reversed nucleoside (1 mmol) in methanol (11−15 mL/mmol) Amberlite IR-120 (H + ) ion exchange resin (3.3 g/mmol), that has been washed several times with absolute methanol, was added. The mixture was refluxed for 8 h, cooled and filtered through a Celite pad, and the resin was washed with methanol (≈20 mL). The filtrate and washings were combined and evaporated.
For the MTT test, cells were seeded on 96 micro well flat bottom plates (Greiner, Austria) at 2×10 4 cells/mL. After 72 hours of incubation with the tested compounds MTT (Merck, Germany) was added. DMSO (Merck, Germany) was used to dissolve the formed MTT-formazane crystals. Absorbency was measured at 570 nm on Stat fax 2100 plate reader (Awareness Technology Inc. USA). All experiments were performed three times in triplicates. The percentage of treated tumor cells growth inhibition was calculated relative to the growth of untreated (control) cells.

RESULTS AND DISCUSSION
The synthetic approach to reversed nucleoside analogues is based on the preparation of the already known, suitably protected methyl ribofuranoside 2 (73 %) and its transformation into 5-tosyl derivative 3 (76 %) by adopting the methods described in the literature (Scheme 1). 23,24 Following our previously described approach to reversed nucleosides, the sodium salts of the uracil derivatives 4−6 were reacted with ribofuranoside 3 giving the corresponding reversed nucleosides 7, 8 and 10 (Scheme 1). 19,21 It was reported that the condensation of thymine sodium salt with the tosyl monosaccharide 3 gave two regioisomers containing the ribofuranoside attached at N1′ or N3′ position of the thymine ring. 30  is apparent from their 1 H NMR spectra. In the spectrum of 7, the signal of C5′ proton appears as a doublet of doublets due to the vicinal H5′-H6′ coupling (J 5',6' = 7.9 Hz) and the additional long-range H5'-NH3' coupling (J 5',NH-3' = 2.1 Hz), the latter excluding the N-3′-regional isomer structure. In addition, in the spectra of 7, 8 and 10 the NH proton signals appear at δ 9.38; 11.7 and 11.87 ppm, respectively, being the characteristic chemical shifts of the uracil NH-3′ protons.
5-Fluorouracil 5 is well known to exhibit a strong antitumor activity but its toxicity largely limits the use of 5 as a practical antitumor agent for humans. 31 We examined the possibility to prepare the reversed nucleoside 8 incorporating 5-fluorouracil fragment. The sodium salt of 5-fluorouracil 5 was condensed with tosyl ribofuranoside 3 giving the N-1′-regioisomer of reversed nucleoside 8 in 23 % yield and also the novel N-1′,N-3′disubstituted nucleoside 9 in 25 % yield. In the 1 H NMR spectra of both, 8 and 9 the signal of H-6′ vinyl proton is split into a doublet (8:  = 8.08 ppm, 3 J H6'-F = 6.9 Hz; 9:  = 8.19 ppm, 3 J H6'-F = 6.5 Hz) due to the H-F coupling.
Since the yields of the reversed nucleosides 7 and 8 prepared from uracil 4 and 5-fluorouracil 5 sodium salts were relatively low, we examined the condensation of tosyl ribofuranoside 3 with the 5-iodouracil 6 which upon N1′-deprotonation should be better nucleophile compared to the corresponding anions of 4 and 5, due to electron donating effect of iodine. The N-1′-regioisomer 10 was obtained in 58 % yield together with the very small amount of the novel N-1',N-3'-disubstituted nucleoside derivative 11 (1.5 %). The hydrogenation of 10 using Pd/C catalyst afforded the reversed nucleoside 7 in 82 % yield (Scheme 1). By the latter two step preparation, 7 could be prepared in higher yield than in the direct condensation of the sodium salt of uracil 4 with 3.
The 1 H NMR spectra of the isopropylidene protected reversed nucleosides 7, 8 and 10 as well as those of the equally protected N-1′,N-3′-disubstituted nucleosides 9 and 11 conclusively show that all posses the configuration. In each spectrum, the anomeric C1 proton appears as the singlet due to small coupling constant with the proton at C2 ribose.
The isopropylidene protecting groups of 7−10 were removed by using of Amberlite IR-120 (H + ) ion exchange resin in refluxing methanol to yield the corresponding methyl ribofuranoside reversed nucleosides (12−15) in 65−89 % yields (Scheme 1). The 1 H NMR spectrum of 14 reveal the presence of duplicate peaks for H-6' proton due to the presence of an anomeric mixture in the ratio / = 1:10 and in the spectrum of 13 signals of protons at C1 position (/ = 3:10) are well separated as shown in the inset of Scheme 1.
The 5'-iodo reversed nucleoside 10 is suitable for further functionalization at the uracil ring. It is well known that the coupling of terminal alkynes with 5-iodouracil nucleosides proceeds in high yields in the presence of palladium catalyst. 32  Using acidic ion exchange resin in methanol or 50 % aqueous TFA for isopropylidene and trimethylsilyl deprotection of 16 gave 5'-acetyl reversed nucleoside 17 in 78 % yield. As it was described in the literature 5ethynyl-2'-deoxyuridine could be hydrated by dilute sulphuric acid to give 5-acetyl derivative in high yield. 33,34 Hence, during deprotection of 16, under acidic conditions besides removal of isopropylidene and trimethylsilyl groups the addition of water on the acetylenic bond occurred giving the 5'-acetyl derivative 17. Treatment of 16 with 0.2 M sodium methoxide in dry methanol effected removal of the trimethylsilyl group giving 5'-ethynyl reversed nucleoside 18 in 88 % yield. The removal of isopropylidene group of 18 by 50 % aqueous TFA gave the 5'-acetyl 17 in almost quantitative yield (Scheme 2).
Among the tested compounds only 5'-iodo reversed nucleoside 14 ( Figure 1) showed a moderate cytostatic activity against CaCo-2 cell line (50 % growth inhibition c =10 −4 M and 30 % growth inhibition c = 10 −6 10 −7 M), which indicates that further synthetic variations of 14 may result in the preparation of derivatives with improved cytostatic potential.

CONCLUSIONS
In this work we describe the synthetic approach to reversed nucleosides which enables their preparation in gram quantities. The reaction of the sodium salt of various pyrimidine nucleobases 4−6 with a suitably protected ribofuranoside 3, enable the efficient preparation of the reversed pyrimidine nucleosides (7, 8, 10). In some cases also N-1,N-3-diribofuranosyl substituted nucleosides 9 and 11 were isolated. The 5'-iodo reversed nucleoside 10 was suitable for further functionalization at the uracil and by using the Sonogashira coupling 5'ethynyl reversed nucleoside 16 was synthesized and transformed to 5'-acetyl derivative 17 under acidic conditions. The reversed nucleosides 12−15 and 17 were tested for the antiproliferative activity on the panel of six cell lines (HeLa, MIAPaCa2, Hep2, NCI-H358, CaCo-2, and HT-29). Modest growth inhibition was