Synthesis, Structure and Biological Activity of 6-R3Si(Ge, Sn)-Substituted 5-Fluorouracils

Direct lithiation of 1-(2-tetrahydrofuryl)-5-fluorouracil (Ftorafur) has been investigated. The treatment of ftorafur with lithium diisopropylamide (LDA)at –70℃ in ether-hexane, followed by the reaction with various electrophiles afforded the corresponding 6-substituted silicon, germanium and tin derivatives of ftorafur. The results of biological investigation indicate the ability of germanium–modified nucleoside analogues to interfere with transcription and replication processes.


Introduction
The chemistry of nucleosides is a rapidly growing area of research. Nowadays the synthetic analogues of nucleosides are used as antimetabolites in biochemistry and medicine. Examples are antiviral agents such as the nucleoside analogues 3'-azidothymidine possessing anti-HIV activity and acycloguanosine with antiherpes activity, the antitumor agents Ftorafur and 5-fluorouridine, and "antisense-RNA" and "antisense-DNA" used as gene blockers.

Experimental
Synthesis of 1-(2-tetrahydrofuryl)-5-fluoro-6-trimethylgermyluracil (111) n-Butyllithium (58.2 ml, 1.25 N in hexane) was added dropwise to a solution of freshly distilled i-Pr2NH (7.34 g, 0.073 mol) cooled to -10C. After the addition of butyllithium the reaction mixture was stirred for 30 min then cooled to-78C and dissolved in 100 ml of THF. After cooling a solution of ftorafur (3.4 g, 0.017 mol) in 70 ml of THF was added dropwise. The mixture was stirred for I h at -78C, and a solution of trimethylchlorogermane (11.25 g, 0.073 mol) in 10 ml of THF was added dropwise at temperature kept below-70C. The mixture was allowed to stand overnight. The solvents were removed in vacuo. The residue was dissolved in the saturated ammonium chloride solution in water and neutralized to pH 7 with the diluted HCI. After 1 h the desired product was filtered off (4.15 g, 77%). Compound III was recrystallized from EtOH, m.p. 132-134C, on TLC it gave one spot (CHCI3 EtOH, 5:1, v/v, Rf 0.70).
The compounds I, II, IV and V were prepared in the similar manner. Yields, melting points and analytical data are presented in Table 1. were unique and found to have > 2(. The structures were solved by a direct method using program SHELXS (7). The non-hydrogen atoms were found in a E-map. Initially, the positional parameters and isotropic temperature factors of all non-hydrogen atoms were refined by full-matrix least-squares procedure. At this stage an empirical absorption correction for compound III (program DIFABS) was performed (s). The positions of hydrogen atoms were geometrically generated assuming the appropriate sp 2or sp3-hybridization for the corresponding atoms. Nonhydrogen atoms were refined in the anisotropic but hydrogens in the isotropic approximations. The final reliability factor R with unit weights was 0.0591 for compound and 0.0440 for compound II1. The highest peak in the final difference map was 0.23 e/A 3 and 0.36 e/A 3 for compounds and III, respectively.  (13) 1.386 (7)  The intramolecular bond distances for both structures are reported in Table 2, the bond angles in Table 3, selected torsion angles in Table 4.
Atoms of the uracil ring in molecules of compounds and III deviates from the average plane and have screw ($2) conformation. Torsional angles (see Table 4) show that the THF rings assume conformations near twist-envelope conformation with atoms C(3') and C(4') out of the )lane of the three others by-0.32 (2) , 0.21 (1) , , for and-0.366 (9) , , , 0.156 (8) for III, respectively. The configuration of the THF ring in both two compounds is C(3')-exo--C(4')-endo. In comparison with closely related compounds (11)(12)(13)(14) the main difference found in reported compounds is the relative orientation of the uracil and five-membered rings. In structures and III the uracil moiety exhibits a sin orientation with respect to the THF ring, while anti conformation was found for compounds described in 0 -4).
Possibly this is a result of the influence of the bulky substituent in 5-fluorouracil ring at C(6). The voluminous SiMe3 and GeMe3 groups make changes in molecular conformations and also in the bond lengths. Inspection of the bond lengths ('rable 2) indicates that the C(6) Si and C(6) Ge are significantly longer than Si-C and Ge--C bonds with methyl groups carbon. Similar to crystal structures described in (12)(13)(14) in the crystal structures of and III there were found hydrogen bonds N(3)-H(3)...0(2) of an intermolecular nature. Distances between N(3) and 0(2) are 2.891 , , and 2.887 , , for compounds and III, respectively.
A 90 min long incubation with both germyl derivatives III and IV reduced the 3H-uridine incorporation rate. Trimethylgermyl derivative (111) caused a two-fold decrease (from 9304.5 +_. 3121.5 dpm//g RNA to 4649 _+ 1068.4 dpm//g RNA), triethylgermyl derivative IV was less effective (to 5433.6 _ _ . 1369.6 dpm//g RNA) (Fig. 2). The compounds manifested a tendency to reduce the 3H-thymidine incorporatiuon, however it was less pronounced and the data were not statistically significant (16). For example, two-hour incubation with compound IV decreased 3H-thymidine incorporation from 420.7 _ _ . 130.2 dpm//g DNA to 307.5 +_. 24.75 dpm//g DNA. These preliminary results indicate the ability of germanium-modified nucleoside analogues to interfere with transcription and, possibly, with replication processes. Several hypothetical mechanisms of action can be proposed. The substances in question could incorporate in the synthesized molecules and block polymerase movement or act as enzyme inhibitors. Further studies of molecular mechanisms of action are necessary for the evaluation of significance of the germaniummodified compounds as biologically active substances.