key: cord-0866979-bw2go1on
authors: Gazivoda, Tatjana; Raić-Malić, Silvana; Krištafor, Vedran; Makuc, Damjan; Plavec, Janez; Bratulić, Siniša; Kraljević-Pavelić, Sandra; Pavelić, Krešimir; Naesens, Lieve; Andrei, Graciela; Snoeck, Robert; Balzarini, Jan; Mintas, Mladen
title: Synthesis, cytostatic and anti-HIV evaluations of the new unsaturated acyclic C-5 pyrimidine nucleoside analogues
date: 2008-05-15
journal: Bioorg Med Chem
DOI: 10.1016/j.bmc.2008.03.074
sha: 83ea5f7ec8111775eca183f97073fb98b71a6122
doc_id: 866979
cord_uid: bw2go1on

A series of the novel C-5 alkynyl pyrimidine nucleoside analogues (1–14) in which the sugar moiety was replaced by the conformationally restricted Z- and E-2-butenyl spacer between the phthalimido and pyrimidine ring were synthesized by using Sonogashira cross-coupling reaction. Cytostatic activity evaluation of the novel compounds showed that E-isomers exhibited, in general, better cytostatic activities than the corresponding Z-isomers. E-isomer 14 exhibited the best cytostatic effect against all evaluated malignant cell lines, particularly against hepatocellular carcinoma (Hep G2, IC(50) = 4.3 μM). However, this compound was also cytotoxic to human normal fibroblasts (WI 38). Its Z-isomer 7 showed highly specific antiproliferative activity against Hep G2 (IC(50) = 18 μM) and no cytotoxicity to WI 38. Moreover, compounds 3, 4 and 14 expressed some marginal inhibitory activity against HIV-1 and HIV-2.

Synthesis, cytostatic and anti-HIV evaluations of the new unsaturated acyclic C-5 pyrimidine nucleoside analogues 1 

Many nucleoside analogues with interesting biological properties have arisen by substitution at the 5-position of the uracil base in the 2 0 -deoxyuridine series. 1 Pyrimidine nucleosides containing C-5 alkynyl groups have been shown to possess significant antiviral and/or anticancer properties. 2 The 5-alkynyluracil nucleosides with a longer alkynyl chain at the C-5 position expressed appreciable antiviral activity in contrast to the corresponding alkyl derivatives that showed decreasing antiviral activity with increasing C-5 side chain length. [2] [3] [4] [5] Introduction of very rigid allenic moiety as a linker between the heterocyclic base and hydroxymethyl group led to compounds such as adenallene and cytallene which effectively inhibited the replication of HIV. 6 Fur-thermore, thymine with a 2-butenyl spacer was the first acyclic nucleoside analogue exhibiting potent inhibition of thymidine kinase 2 (TK-2) which catalyzes phosphorylation of certain antiviral drugs. 7 We have reported that some pyrimidines and purines containing c-(Z)-ethylidene-2,3-dibenzylbutenolide exhibited antiproliferative effects against malignant human tumour cell lines. [8] [9] [10] The C-5 alkynyl substituted pyrimidine derivatives of L L-ascorbic acid had some, although slight, inhibitory potency against cytomegalovirus (CMV) and varicella-zoster virus (VZV). 11 The 5propynyl uracil derivative of L L-ascorbic acid showed pronounced cytostatic activity against cervical carcinoma (HeLa). 12 We have also reported that the (Z)-4amino-2-butenyladenine nucleoside analogue showed selective activity against HIV-1. 13 These results prompted us to synthesize a series of the novel (Z)and (E)-2-butenyl-N-phthalimido pyrimidines containing an alkynyl longer side chain at C-5 (1-14) (Fig. 1 ) and evaluate their cytostatic and anti-HIV potency in cell culture.

The key precursors (Z)-(IIa) and (E)-1-[4 0 -(N-phthalimido)-2 0 -butenyl]-5-iodouracil (IIb) for cross-coupling with alkynes were synthesized by condensation of the (Z)-4-chloro-2-butenyl-N-phthalimide (Ia) and (E)-4bromo-2-butenyl-N-phthalimide (Ib) with 5-iodouracil. 13 The (Z)-4-chloro-2-butenyl (Ia) and (E)-4-bromo-2-butenyl (Ib) derivatives of N-phthalimide were synthesized by using a classical Gabriel reaction. 14 Different substituents at C-5 position of the pyrimidine ring in 1-7 and 8-14 were introduced by the reaction of the 5iodouracil derivatives IIa and IIb under optimized Sonogashira Pd(0)-catalyzed reactions. [15] [16] [17] [18] [19] [20] It is important to note that this reaction was performed at room temperature in order to avoid the formation of bicyclic furopyrimidine co-products produced by heating the reaction mixture. Reaction of the (Z)-and (E)-1-[4 0 -(N-phthalimido)-2 0 -butenyl]-5-iodouracil with various terminal alkynes in the presence of palladium(0) catalyst and copper(I) iodide as co-catalyst in DMF at room temperature gave 5-substituted Z-and E-uracil derivatives 1-7 and 8-14 (Scheme 1).

1 H and 13 C NMR data given in Table 1 and Section 5 are in full agreement with the proposed structures 1-14 (Fig. 2) .

Signals of 1 H and 13 C resonances were observed which were characteristic for each of the moieties constituting structures of N1, C5-substituted pyrimidine nucleoside analogues (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) . H1 0 and H4 0 protons exhibit distinct chemical shifts between 4.09 and 4.60 ppm, whereas H2 0 and H3 0 protons along C@C double bond were observed between 5.50 and 5.80 ppm (Table 1) . Chemical shifts of phenyl protons are between 7.1 and 7.53 ppm, while phthalimido protons are between 7.8 and 7.9 ppm. H6 chemical shifts were found in the range from 7.82 to 8.47 ppm and H3 protons in the range from 11.33 to 11.73 ppm. The configuration along C2 0 @C3 0 double bond was evaluated through 3 J H2 0 H3 0 coupling constants ( Table 1) . The values of all coupling constants involved in the six spin system of N1-substituent including 3 J H2 0 H3 0 had to be determined through simulation due to small Dd/J ratios. As an example of our simulations, multiplets of H2 0 and H3 0 in 14 are compared to the experimental spectrum in Figure 3 . Vinyl 4 J H1 0 H3 0 and 4 J H2 0 H4 0 couplings were below signal half width (<1 Hz). Smaller 3 J H2 0 H3 0 coupling constants of 10.6, 10.6 and 11.1 Hz in 2, 3 and 7, respectively, are in accordance with Z-isomers along C2 0 @C3 0 double bonds. On the other hand, larger 3 J H2 0 H3 0 coupling constants of 15.7, 15.5 and 15.7 Hz in 8, 12 and 14, respectively, are in conformity with E-isomers.

E-Isomers exhibit larger chemical shift difference between H2 0 and H3 0 (Dd 0.06-0.07 ppm) than Z-isomers (Dd 0.02-0.04 ppm). The other significant 1 H and 13 C chemical shift differences between E-and Z-isomers were observed, and are presented in Figure 4 . Perusal of Figure 4 shows that there is a clear distinction in several 1 H and 13 C chemical shifts with respect to Z-and Econfigurations along the C2 0 @C3 0 double bond.

In our further analysis, we have focused on conformational properties of novel pyrimidine-2,4-dione nucleoside analogues. 3 J H1 0 H2 0 and 3 J H3 0 H4 0 coupling constants showed unrestricted rotation along the involved single bonds. In full agreement, protons of both methylene groups are isochronous. Evaluation of NOE enhancements between H6 and protons of N1 substituents did not show any interaction that would indicate preference for any of conformational states. In addition, NOE data did not indicate close spatial proximity of moieties attached to N1 and C5 of pyrimidine-2,4-dione.

Compounds 1-14 were evaluated for their cytostatic activities against malignant human tumour cell lines: murine leukemia (L1210), human T-lymphocyte (Molt4/C8 and CEM), cervical carcinoma (HeLa), pancreatic carcinoma (MiaPaCa-2), colorectal adenocarcinoma (SW 620), breast epithelial adenocarcinoma (MCF-7) and hepatocellular carcinoma (Hep G2), as well as normal diploid human fibroblasts (WI 38) ( Table  2 ). Cytostatic activities of compounds 1-14 were compared with those of 5-fluorouracil (5-FU). The novel C-5 alkynyl substituted olefinic pyrimidine compounds (1-14) showed better antiproliferative activities on the malignant tumour cells than their structurally related C-5 unsubstituted uracil and C-5 fluoro-substituted uracil derivatives containing 4 0 -(N-phthalimido)-2 0 -butenyl side chain. 13 In the series of Z-izomers of C-5 alkynyl 1-[4 0 -(N-phthalimido)-2 0 -butenyl] pyrimidine derivatives 1-7, compounds containing a hexynyl (1), octynyl (2) and pbutylphenylethynyl (6) side chain at C-5 exhibited moderate cytostatic effects against all evaluated cell lines, particularly against hepatocellular carcinoma (Hep G2; IC 50 = 16.6-40.2 lM) (Fig. 5) . However, these compounds were also cytotoxic against normal human fibroblasts (WI 38; IC 50 = 16.7-49 lM) (Fig. 6 ).

On the contrary, compound 7 with the C-5 p-pentylphenylethynyl side chain showed selective antiproliferative effect on the growth of hepatocellular carcinoma (Hep G2, IC 50 = 18 lM). However, this compound had lesser cytostatic activity against Hep G2 cell line than standard 5-fluorouracil (5-FU). Among E-isomers of C-5 substituted alkynyl 1-[4 0 -(N-phthalimido)-2 0butenyl]pyrimidine derivatives 8-14, compound 14 containing the p-pentylphenylethynyl side chain at C-5 exhibited the best antiproliferative effect on all the evaluated cell lines (IC 50 in the range of 4.3-39 lM) and normal fibroblasts (IC 50 = 25 lM). Besides, compounds with a C-5 hexynyl (8), heptynyl (9) and phenylbutynyl (12) side chain showed cytostatic activity, particularly against hepatocellular carcinoma (Hep G2; IC 50 = 7.3-9 lM) (Fig. 5) . Thus, compounds 7 and 14 emerged as the most interesting lead compounds with cytostatic activities that could be used for further structural optimization and biological studies on Hep G2 cells. 21-23

Compounds 1-14 were evaluated for their inhibitory activities against HIV-1 and HIV-2 in human T-lymphocyte (CEM) cells (Table 3) . Compounds 3 and 4 showed some specific albeit slight activity against HIV-1, while compound 14 exhibited moderate activity against both HIV-1 and HIV-2. None of the other viruses were sensitive to the inhibitory activity of the tested compounds except for compounds 2 (EC 50 = 2.5-4 lM) and 5 (EC 50 = 20-21 lM) against influenza A (H 3 N 2 ), for compound 14 against Coxsackie B4 virus (EC 50 = 12 lM) and vaccinia virus (EC 50 = 12 lM) and for compound 7 for varicella-zoster virus (EC 50 = 9.4-8.7 lM). However, the EC 50 values of the compounds were close to their toxicity threshold, and therefore, it is unclear whether the observed activity is due to a specific antiviral effect, or indirectly, to a cytotoxic/cytostatic activity of the tested compounds.

The novel (Z)-(1-7) and (E)-(8-14) unsaturated acyclic C-5-alkynyl uracil derivatives were prepared by using Sonogashira coupling of (Z)-and (E)-1-

By comparison of the cytostatic activities of the Z-and E-isomers of C-5 alkynyl olefinic pyrimidine derivatives, we can infer that the E-isomers (8) (9) (10) (11) (12) (13) (14) showed better cytostatic activities. Furthermore, introduction of the phenyl ring in longer side chain caused an increasing cytostatic effect. Thus, the C-5 pentylphenylethynyl substituted olefinic pyrimidine derivative 14 showed the most pronounced inhibitory activity against all the tested tumour cell lines (IC 50 = 4.3-39 lM), including human normal fibroblasts. On the contrary, its Z-isomer (7) showed selective inhibitory activity against the Hep G2 cell line (IC 50 = 18 lM). Thus, the results of the cytostatic examinations indicated that compounds 7 and 14 are suitable candidates for further biological studies on lead drug and structural optimization. Figure 5 . Dose-response curves of the tested compounds 1-14 on hepatocellular carcinoma (Hep G2). 24 the panel cell lines were inoculated onto a series of standard 96-well microtiter plates on day 0 at 1 · 10 4 to 3 · 10 4 cells/mL, depending on the doubling times of specific cell line. Test agents were then added in five, 10-fold dilutions (10 À8 to 10 À4 M) and incubated for further 72 h. Working dilu-tions were freshly prepared on the day of testing. The compounds were prepared as 0.04 M solutions in DMSO, and the solvent was also tested for eventual inhibitory activity by adjusting its concentration to be the same as in the working concentrations. After 72 h of incubation the cell growth rate was evaluated by performing the MTT assay (Sigma) which detects dehydrogenase activity in viable cells. The MTT Cell Proliferation Assay is a colorimetric assay system, which measures the reduction of a tetrazolium component (MTT) into an insoluble formazan product by the mitochondria of viable cells. For this purpose, the substancetreated medium was discarded and MTT was added to each well at a concentration of 20 lg/40 lL. After 4 h of incubation the precipitates were dissolved in 160 lL of DMSO. The absorbance (OD, optical density) was measured on a microplate reader at 570 nm. The absorbancy is directly proportional to the cell viability. The percentage of growth (PG) of the cell lines was calculated according to one or the other of the following two expressions:

If (mean OD test À mean OD tzero ) P 0 then PG ¼ 100 Â ðmean OD test À mean OD tzero Þ= ðmean OD ctrl À meanOD tzero Þ:

If (mean OD test -mean OD tzero ) < 0 then PG ¼ 100 Â ðmean OD test À mean OD tzero Þ=OD tzero where mean OD tzero = the average of optical density measurements before exposure of cells to the test compound; mean OD test = the average of optical density measurements after the desired period of time; and mean OD ctrl = the average of optical density measurements after the desired period of time with no exposure of cells to the test compound.

Each test point was performed in quadruplicate in three individual experiments. The results were expressed as IC 50 , a concentration necessary for 50% of inhibition, which were calculated from dose-response curves using linear regression analysis by fitting the test concentrations that give PG values above and below the respective reference values.

Antiviral activity against HIV-1 and HIV-2 was based on the examination of the virus-induced cytopathicity (syncytia formation) in CEM cell cultures, and determined essentially as described previously. 25, 26 The antiviral activity of the test compounds against herpes simplex virus type 1 (HSV-1 

Allenols derived from nucleic acid bases-a new class of anti-HIV agents: chemistry and biological activity

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.bmc.2008.03.074.