Novel 5′-Norcarbocyclic Derivatives of Bicyclic Pyrrolo- and Furano[2,3-d]Pyrimidine Nucleosides

Here we report the synthesis and biological activity of new 5′-norcarbocyclic derivatives of bicyclic pyrrolo- and furano[2,3-d]pyrimidines with different substituents in the heterocyclic ring. Lead compound 3i, containing 6-pentylphenyl substituent, displays inhibitory activity with respect to a number of tumor cells with a moderate selectivity index value. Compound 3i induces cell death by the apoptosis pathway with the dissipation of mitochondrial potential.


Introduction
Nucleic acid components are involved in many vitally important metabolic processes (DNA and RNA synthesis, cell signaling, enzyme regulation and metabolism); this is why their synthetic analogues are convenient tools for studying and influencing these processes. Nucleoside and nucleotide analogues can interact with and inhibit essential enzymes such as human and viral polymerases (DNA-dependent DNA polymerases, RNA-dependent DNA polymerases or RNA-dependent RNA polymerases), kinases, ribonucleotide reductase, DNA methyltransferases, purine and pyrimidine nucleoside phosphorylase and thymidylate synthase [1]. As a result, nucleoside analogues have been in clinical use for almost 50 years and have become cornerstones of treatment for patients with cancer or viral infections [1]. However, the clinical use of these compounds is limited by important side-effects and primary or acquired drug resistance [2]. Thus, the development of new antiviral and anticancer agents is of crucial importance.
Bicyclic furano [2,3-d]pyrimidine nucleosides were first developed by McGuigan et al. as herpes virus family inhibitors [3]. The compounds bearing the 2 -deoxyribose residue are non-toxic and are highly effective inhibitors of the Varicella-Zoster virus [4], and analogues containing the 2 ,3dideoxyribose or acyclic fragments suppress human cytomegalovirus [5]. The corresponding carbocyclic analogue was also synthesized by the same group but turned out to be less active [6]. It was shown that in order to display antiviral activity bicyclic furano [2,3-d]pyrimidine nucleosides have to be phosphorylated by viral deoxythymidine kinase, but the complete mechanism of their inhibitory effect has not yet been elucidated [5]. At the same time, significant anticancer activity was found for several small molecules which include a furo [2,3-d]pyrimidine scaffold due to their inhibitory effect against different protein kinases [7,8]. Recent data have shown that some pyrroloand furano [2,3-d]pyrimidine nucleosides are able not only to suppress the growth of various lines tumor cells, but also to induce apoptosis [9][10][11]. The first 5′-norcarbocyclic derivatives of bicyclic furano [2,3-d]pyrimidines with various alkyl substituents at the 6-position of the heterocyclic base have shown antitumor activity against different cell lines [12]. Here we synthesized new representatives of bicyclic furano [2,3-d]pyrimidine nucleosides and novel bicyclic pyrrolo [2,3-d]pyrimidine nucleosides to obtain structure-activity relationship data for this family of compounds and to get additional information on the mechanisms of action and potential cellular targets for these bicyclic nucleosides.

Chemistry
All the 5′-norcarbocyclic analogs of bicyclic furano-and pyrrolo [2,3d]pyrimidine nucleosides were synthesized starting from the general precursor racemic 1-(4′-hydroxy-2′-cyclopentene-1′-yl)-5-ioduracil 1 (Figure 1) which was obtained as described earlier [12,13]. 1-(2′,3′,4′-Trihydroxycyclopent-1′-yl)-5-iodouracil 2 was synthesized by oxidation of compound 1 using osmium tetroxide in the presence of N-methylmorpholine-N-oxide (NMMO) [14]. This procedure allows the cis-2′,3′-diol to be obtained selectively [15,16]. To prepare furano [2,3d]pyrimidine nucleosides we used Cu/Pd-catalyzed cyclisation of 1 (for 3a-i) or its oxidized derivative 2 (for 5) with corresponding alkynes in refluxing CH3CN. This afforded target furano [2,3d]pyrimidine nucleosides in good yields (36-82%). Such a deviation in yields was due both to the difference in alkyne structures and to the fact that isolation and purification of some products turned to be laborious. Subsequent treatment of compounds 3a-i with 32% ammonia in methanol resulted in corresponding pyrrolo [2,3-d]pyrimidine analogs 4a-i (Scheme 1). The reactions at 40 °C were rather slow, but such mild conditions gave us an opportunity to obtain products 4a-i with good yields (57-89%) without using a bomb. It is worth remarking that preparative liquid chromatography on silica gel plates turned to be more effective for the isolation of pyrrolo [2,3-d]pyrimidine analogues 4a-i than the column chromatography on silica gel, which was our choice in the case of furano [2,3-d]pyrimidine derivatives 3a-i and 5. All the compounds were synthesized as racemic mixtures.  [2,3-d]pyrimidine-2-one 5 were obtained. The last one was synthesized as a first representative of trihydroxycyclopentyl derivatives of furano [2,3-d]pyrimidine-2-one in order to estimate the potential of this modification for antitumor activity and to gain a better structure activity relationship (SAR) understanding. As a result, a set of novel bicyclic furano [2,3-d]pyrimidine nucleosides 3a-i, early unknown bicyclic pyrrolo [2,3-d]pyrimidine nucleosides 4a-i and a new 1-(2 ,3 ,4 -trihydroxycyclopent1yl)6-decyl-3H-furano [2,3-d]pyrimidine-2-one 5 were obtained. The last one was synthesized as a first representative of trihydroxycyclopentyl derivatives of furano [2,3-d]pyrimidine-2-one in order to estimate the potential of this modification for antitumor activity and to gain a better structure activity relationship (SAR) understanding. Molecules 2018, 23, x FOR PEER REVIEW 3 of 12 Figure 1. Quantification of apoptosis with annexin V binding to KB-3-1 cells. Cells were incubated in the presence of 3i (5, 10 or 20 µM), or in the presence of DMSO (0.1% v/v) for 48 h and then Annexin V/PI staining was analyzed by flow cytometry. Etoposide was used as a standard apoptosis inducer to confirm the correct work of the system (data not shown). The results of one of three independent experiments are represented.

Cell Viability Assay
The target compounds were tested on different lines of tumor cells, HuTu-80 (human duodenal cancer), B16 (mouse melanoma), A549 (human lung adenocarcinoma), KB-3-1 (human squamous cell carcinoma), HeLa (human squamous cell carcinoma of the cervix), as well as on human noncancer cells hFF3.

Cell Viability Assay
The target compounds were tested on different lines of tumor cells, HuTu-80 (human duodenal cancer), B16 (mouse melanoma), A549 (human lung adenocarcinoma), KB-3-1 (human squamous cell carcinoma), HeLa (human squamous cell carcinoma of the cervix), as well as on human noncancer cells hFF3.
Compounds 3b and 4a had no toxic effect on either normal untransfected hFF3 cells or on tumor cells in concentrations up to 100 µM. Compounds 3d-f,h and 4f,i were almost equally toxic for cancer and noncancer cells ( Table 1). The pyrrole-containing compounds 4e and 4h were less toxic than the corresponding furan analogues 3e and 3h, but also reduced the viability of all tested cell lines with IC 50 in the range from 11 µM (HeLa) to 63 µM (hFF3) for 4e and from 15 µM (KB-3-1) to 70 µM (hFF3) for 4h. First 2 ,3 -dihydroxy derivative 5, proved to be less toxic than the corresponding 2 ,3 -didehydro-2 ,3 -dideoxy analogue 3e. Compounds 3a, 3c, 3g, 3i, 4b-d, 4g and 5 inhibited the growth of some tested tumor cells, mainly KB-3-1 and HeLa (Table 1), while they were not toxic for normal cells in concentrations up to 100 µM. Melanoma cells B16 were the most resistant towards action of these new nucleoside analogues. Only compounds 3f, 3i, 4e, 4h and 4i had the selective toxic effect on this line with IC 50 4.5, 21, 25, 35 and 13.4 µM, respectively.
The nucleoside analogue 3i was among most active compounds (Table 1) and had the most selective antiproliferative antitumor effect, especially against the HuTu80, KB-3-1 and HeLa cell lines (Table 2). Therefore, we used it as a lead compound to study the mechanism of induced cells death.  The nucleoside analogue 3i was among most active compounds (Table 1) and had the most selective antiproliferative antitumor effect, especially against the HuTu80, KB-3-1 and HeLa cell lines (Table 2). Therefore, we used it as a lead compound to study the mechanism of induced cells death.  The selectivity index (SI) was the ratio of IC hFF3 (cytotoxicity on normal hFF3 cells) to IC 50 of cancer cells.

Induction of Apoptosis
To examine whether the tested 5 -norcarbocyclic derivatives induce cell death via apoptosis Annexin V and propidium iodide analysis were used ( Figure 1). KB-3-1 cells were exposed to 3i, the most active among tested compounds, for 48 h and then flow cytometric analysis was undertaken. Annexin V binds phosphatidylserine residues, which are asymmetrically distributed toward the inner plasma membrane, and migrate to the outer plasma membrane during apoptosis [17]. The data shows that 3i induces apoptotic cell death in 26% of KB-3-1 cells at concentrations of 5 µM. The increasing of 3i concentration to 20 µM resulted in 55.3% apoptotic cells after 48 h of incubation of KB-3-1 cells with the analogue. Hence, the 5 -norcarbocyclic derivative 3i induced dose-dependent apoptotic cell death.
We next investigated whether 3i utilizes the mitochondrial 'intrinsic' pathway in the apoptotic death of KB-3-1 cells, since the pivotal role of mitochondria in triggering apoptosis is well established. We evaluated the mitochondrial transmembrane potential (∆Ψ m ) in KB-3-1 cells exposed to 3i using cytofluorometric analysis. Cells were stained with the specific mitochondrial cationic dye JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethyl benzimidazole carbocyanine iodide) that accumulates in the transmembrane space of polarized mitochondria and forms the so-called «J-aggregates», emitting red fluorescence. A decrease in ∆Ψ m results in disappearance of J-aggregates and formation of JC-1 monomers, which emit in a green fluorescence. The cytometric analysis of KB-3-1 cells stained with JC-1 is shown in Figure 2. The selectivity index (SI) was the ratio of IChFF3 (cytotoxicity on normal hFF3 cells) to IC50 of cancer cells.

Induction of Apoptosis
To examine whether the tested 5′-norcarbocyclic derivatives induce cell death via apoptosis Annexin V and propidium iodide analysis were used (Figure 1). KB-3-1 cells were exposed to 3i, the most active among tested compounds, for 48 h and then flow cytometric analysis was undertaken. Annexin V binds phosphatidylserine residues, which are asymmetrically distributed toward the inner plasma membrane, and migrate to the outer plasma membrane during apoptosis [17]. The data shows that 3i induces apoptotic cell death in 26% of KB-3-1 cells at concentrations of 5 µM. The increasing of 3i concentration to 20 µM resulted in 55.3% apoptotic cells after 48 h of incubation of KB-3-1 cells with the analogue. Hence, the 5′-norcarbocyclic derivative 3i induced dose-dependent apoptotic cell death.
We next investigated whether 3i utilizes the mitochondrial 'intrinsic' pathway in the apoptotic death of KB-3-1 cells, since the pivotal role of mitochondria in triggering apoptosis is well established. We evaluated the mitochondrial transmembrane potential (∆Ψm) in KB-3-1 cells exposed to 3i using cytofluorometric analysis. Cells were stained with the specific mitochondrial cationic dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazole carbocyanine iodide) that accumulates in the transmembrane space of polarized mitochondria and forms the so-called «J-aggregates», emitting red fluorescence. A decrease in ∆Ψm results in disappearance of J-aggregates and formation of JC-1 monomers, which emit in a green fluorescence. The cytometric analysis of KB-3-1 cells stained with JC-1 is shown in Figure 2.   In the control cells incubated in the presence of 0.1% DMSO the majority of cells shows a high emission of fluorescence in both channels due to the equilibrium between J-aggregates and monomers ( Figure 2). The exposure of KB-3-1 cells to 20 µM of compound 3i leads to a decrease of the red fluorescence value as compared to the control (0.1% DMSO).
Column chromatography was performed on Silica Gel 60 0.040-0.063 mm (Merck, Germany), and systems for elution are indicated in the text. Thin layer chromatography (TLC) was performed on TLC Silica gel 60 F 254 plates (Merck, Germany) in chloroform-methanol, 9:1 (A), or chloroform-methanol, 4:1 (B) systems. Preparative layer chromatography (PLC) was performed on PLC Silica gel 60 F 254 plates (Merck, Germany), systems for elution are indicated in the text. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were registered on a Bruker Avance 400 spectrometer (Bruker, Newark, Germany) using tetramethylsilane (TMS) in CDCl 3 , CD 3 OD, CDCl 3 /CD 3 OD mixture, or DMSO-d 6 as internal standard. Chemical shifts are given in ppm, and the letter "J" indicates normal 3 J HH couplings and all J values are given in Hz.
High-resolution mass spectra (HRMS) were registered on a Bruker Daltonics micrOTOF-Q II instrument using electrospray ionization (ESI). The measurements were acquired in a negative ion mode with the following parameters: interface capillary voltage-3700 V; mass range from m/z 50 to 3000; external calibration (Electrospray Calibrant Solution, Fluka); nebulizer pressure-0.3 Bar; flow rate-3 µL/min; dry gas nitrogen (4.0 L/min); interface temperature was set at 180 or 190 • C. A syringe injection was used.
The absorbance (MTT assay) was measured on a plate reader Multiscan RC (Thermo LabSystems, Vantaa, Finland) at 570 nm. Mitochondrial transmembrane potential and the amount of apoptotic cells in samples were analyzed by flow cytometer «FC500» (Beckman Coulter, Indianapolis, IN, USA).
All compounds were dissolved in dimethylsulfoxide (DMSO) and stock solutions (10 mmol L −1 ) were stored at −20 • C.
After treatments, both floating and adherent scrapped cells were collected by centrifugation and used for further analysis.

Cell Viability Analysis by MTT Assay
Cells growing in the logarithmic phase were seeded in triplicate in 96-well plates at a density of 5 × 10 3 cells per well for HeLa and HuTu-80 cells, 7 × 10 3 cells per well for KB-3-1 and hFF3, 10 × 10 3 for A549 and 20 × 10 3 for B16. The plates were incubated at 37 • C in a humidified 5% CO 2 atmosphere. Cells were allowed to adhere to the surface for 24 h and then tested compounds were added at different concentrations and incubation was continued for 48 h. Then [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) solution (10 µL, 5 mg mL −1 ) was added to each well and the incubation was continued for an additional 3 h. The dark blue formazan crystals formed within the healthy cells were solubilized with DMSO and the absorbance was measured using a Multiscan RC plate reader at 570 nm. The IC 50 was determined as the compound concentration required to decrease the A 570 to 50% compared to the control (no tested compounds, DMSO) and was determined by interpolation from dose-response curves.

Apoptosis Detection by Annexin V Staining
Exponentially growing KB-3-1 cells in 6-well plates (5 × 10 5 cells per well) were treated with 3i (5, 10 and 20 µM) or with 0.1% (v/v) DMSO as a control for 48 h. The cells were stained with Annexin V-FITC and propidium iodide by the Annexin-FITC apoptosis staining/detection kit (Abcam) according to the instruction of the manufacturer. Briefly, cells were collected by scrapping, washed twice with cold PBS, and centrifuged (400 g, 5 min). Cells were resuspended in binding buffer (500 µL) and Annexin V-FITC (5 µL) and PI (5 µL) were added. Cells were incubated for 5 min at 20 • C in the dark. Finally, binding buffer (300 µL) was added to each tube, and the amount of apoptotic cells in samples were analyzed by flow cytometry. For each sample, 10,000 ungated events were acquired. Annexin V + PI − cells represent the early apoptotic populations. Annexin V + PI + cells represent either late apoptotic or secondary necrotic populations.

Mitochondria Depolarization Analysis
Mitochondria involvement in apoptosis was measured by mitochondrial depolarization occurring early during onset of apoptosis. KB-3-1 cells were incubated with 3i (5, 10 and 20 µM) or 0.1% (v/v) DMSO as a control for 48 h. Then, cells were collected and incubated in complete media in the dark with mitochondrial potential sensor JC-1 (5 µg mL −1 ) at 37 • C for 30 min, washed with cold PBS and resuspended in PBS (400 µL). Fluorescences of J-aggregate and J-monomer were recorded in the fluorescence channels 2 (FL2) and 1 (FL1), respectively, with flow cytometer «FC500». Necrotic fragments were electronically gated out, on the basis of morphological characteristics on the forward light scatter versus side light scatter dot plot.

Conclusions
The comparative evaluation of the effects of synthesized nucleoside analogues on the growth and viability of tumor cell cultures from various origins and on normal cells has revealed that cytotoxicity depends on both the type of bicyclic system (pyrrolo-or furano [2,3-d]pyrimidine) and the structure of a substituent in the 6th position of the heterocyclic base. Furano [2,3-d]pyrimidine 3i, bearing pentylphenyl substituent, is the most promising among synthesized 5 -norcarbocyclic derivatives of 6-substituted bicyclic pyrrolo-and furano [2,3-d]pyrimidines. This demonstrated inhibitory activities with respect to tumor cells with the selectivity index value about 15-20 depending on the nature and origin of tumor cells. In an attempt to understand the mechanism of the action, we showed that 3i induces cell death by apoptosis pathway with the dissipation of mitochondrial potential.