Juraj
Konč
a,
Michal
Tichý
a,
Radek
Pohl
a,
Jan
Hodek
a,
Petr
Džubák
b,
Marián
Hajdúch
b and
Michal
Hocek
*ac
aInstitute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic. E-mail: hocek@uochb.cas.cz
bInstitute of Molecular and Translational Medicine, Palacky University and University Hospital in Olomouc, Faculty of Medicine and Dentistry, Hněvotínská 5, CZ-775 15 Olomouc, Czech Republic
cDepartment of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12843 Prague 2, Czech Republic
First published on 25th August 2017
Three types of sugar modified pyrimido[4,5-b]indole nucleosides (2′-deoxy-2′-fluororibo-, 2′-deoxy-2′-fluoroarabino- and arabinonucleosides) were synthesized by glycosylation of 4,6-dichloropyrimido[4,5-b]indole followed by modification of sugar moiety and introduction of substituents into position 4 by cross-coupling reactions or nucleophilic substitutions. Some 2′-fluororibo- and 2′-fluoroarabinonucleosides displayed interesting anti-HCV activities (IC50 = 1.6–20 μM) and the latter compounds also some anti-dengue activities (IC50 = 10.8–40 μM).
Our long-term research of biological activities of 7-deazapurine nucleosides resulted in discovery of two main groups of cytostatics (6-hetaryl-7-deazapurines 17 and 7-hetaryl 7-deazapurines 28) with nanomolar activities against broad panel of cancer cell lines. These compounds also showed potent anti-HCV effects, which were unfortunately accompanied by cytotoxicity. These results showed the space for modification in the “major groove” part of the molecule and inspired us to design of fused-7-deazapurine nucleosides with the aim of possible selectivity modulating of antiviral and cytostatic activities. First generation of such fused nucleosides, pyrimidoindole ribonucleosides 3a bearing various hetaryl groups in position 4,9 displayed negligible cytostatic activity, however, several derivatives bearing 2-hetaryl groups exerted interesting micromolar activity against dengue virus.9 Benzo-fused 7-deazaadenine analogues 3b showed10 similarly potent anti-dengue effect and anti-HCV activity with 4-methyl derivative being the most active compound with sub-micromolar anti-HCV activity (replicon 1B) and no cytotoxicity. Second generation of fused nucleosides, thienopyrrolopyrimidine ribonucleosides 4,11 were again cytostatic at nanomolar concentrations with potent anti-HCV activity accompanied by cytotoxicity and no effect against dengue virus. In order to complete the SAR of this class of compounds and to gain selectivity to RNA viruses without cytotoxicity, we designed sugar-modified nucleosides derived from 4-substituted 6-chloropyrimido[4,5-b]indole ribonucleosides (Fig. 1). We focused on 2′-deoxy-2′-fluororibo-, 2′-deoxy-2′-fluoroarabino- and arabinonucleosides, because related sugar modified derivatives of 7-hetaryl-7-deazapurine nucleosides were previously shown to be significantly less cytotoxic than corresponding ribonucleosides.12 Moreover, arabino- or 2′-fluoroarabino sugars occur in clinically used cytostatics Clofarabine13 and Fludarabine14 and also some 2′-fluororibonucleotides have displayed biological effects.15
A nucleobase anion glycosylation of the previously reported 4,6-dichloropyrimido[4,5-b]indole (5)9 with the known α-bromo-2-fluoroarabinose 616 furnished the desired key-intermediate fluoroarabinonucleoside 7 in 51% yield (Scheme 1) from which a series of final 4-substituted 2′-deoxy-2′-fluoroarabinonucleosides 9a–i was then synthesized. The selection of substituents and reaction conditions was based on our previous experience with fused-deazapurine nucleosides.9 First, we attempted to deprotect nucleoside 7 to get free 4-chloro 2′-deoxy-2′-fluoroarabinonucleoside, however, the position 4 on pyrimidoindole base was found so reactive, that nucleophilic substitution was easier than debenzoylation and proceeded simultaneously. With the aim to introduce substituents selectively into the position 4 and keep chlorine in position 6 untouched, we applied previously optimized conditions for Suzuki coupling (catalysis by Pd(PPh3)4 in combination with potassium carbonate as a base in toluene) to synthesize 4-phenyl-, 4-(3-thienyl)- and 4-(3-furyl)-derivatives. Isomeric 2-furyl- and 2-thienyl-derivatives were obtained by Stille coupling with 2-(tributylstannyl)furan or 2-(tributylstannyl)thiophene catalyzed by PdCl2(PPh3)2 in DMF. Methyl group was introduced by Pd-catalyzed methylation with trimethylaluminium. All these reactions were performed starting from the benzoylated nucleoside 7 and intermediates 8d–i were then deprotected to desired final free nucleosides 9d–i using the standard Zemplén method – sodium methoxide in methanol. The amino-, methoxy- and methylsulfanyl-derivatives 9a, 9b, and 9c were obtained by nucleophilic substitution with aqueous ammonia in dioxane at 100 °C, sodium methoxide in MeOH or sodium methanethiolate in EtOH, respectively. Benzoyl groups were simultaneously removed under reaction conditions and the final free nucleosides 9 were isolated in good yields (Scheme 1, Table 1).
Entry | R | Conditions | Protected nucleoside | Yield [%] | Final nucleoside | Yield [%] |
---|---|---|---|---|---|---|
1 | NH2 | b | — | — | 9a | 78 |
2 | OMe | c | — | — | 9b | 22 |
3 | SMe | d | — | — | 9c | 32 |
4 | Me | e | 8d | 46 | 9d | 78 |
5 | 2-Furyl | g | 8e | 79 | 9e | 69 |
6 | 3-Furyl | f | 8f | 77 | 9f | 33 |
7 | 2-Thienyl | g | 8g | 51 | 9g | 78 |
8 | 3-Thienyl | f | 8h | 53 | 9h | 65 |
9 | Phenyl | f | 8i | 55 | 9i | 65 |
The synthesis of arabinonucleosides and 2′-deoxy-2′-fluororibonucleosides was envisaged by modification of 2′-position of the corresponding 4,6-dichloropyrimidoindole ribonucleoside intermediate 12. It was prepared by stereoselective glycosylation of the pyrimidoindole nucleobase 5 with the protected 1-chlororibose 1017 followed by sugar deprotection. The desired nucleoside 12 was obtained in overall 29% yield as the pure β-anomer (Scheme 2).
Scheme 2 Reagents and conditions: a) KOH, TDA-1, toluene, r.t., 30 min, then 10 in toluene, r.t., 24 h; b) 90% aq. TFA, r.t., 30 min. |
The key 4,6-dichloropyrimido[4,5-b]indole arabinonucleoside intermediate 16 was then prepared by inversion of configuration at the 2′-carbon of the 3′,5′-protected ribonucleoside 13 using a sequence of redox reactions. Nucleoside 13 was first oxidized by Dess–Martin periodinane to oxo-derivative 14 in excellent 91% yield. Then a well known stereoselective reduction of 14 using NaBH4 in ethanol12a,b furnished the desired silylated arabinonucleoside 15, which was deprotected to the free arabinonucleoside 16 in very good 72% yield over 4 steps (Scheme 3).
A series of 4-substituted arabinonucleosides 17a–i was then prepared in good yields by aromatic nucleophilic substitution, Pd-catalyzed cross-coupling reaction with trimethylaluminium or aqueous-phase Suzuki cross-coupling reaction catalyzed by palladium acetate in combination with TPPTS (Scheme 3, Table 2). The only low yielding reaction was the Suzuki coupling with 2-furylboronic acid probably due to limited stability of the reagent.
Entry | R | Conditions | Product | Yield [%] |
---|---|---|---|---|
1 | NH2 | e | 17a | 85 |
2 | OMe | f | 17b | 77 |
3 | SMe | g | 17c | 71 |
4 | Me | h | 17d | 68 |
5 | 2-Furyl | i | 17e | 33 |
6 | 3-Furyl | i | 17f | 62 |
7 | 2-Thienyl | i | 17g | 70 |
8 | 3-Thienyl | i | 17h | 75 |
9 | Phenyl | i | 17i | 58 |
4,6-Dichloropyrimido[4,5-b]indole 2′-deoxy-2′-fluororibonucleoside 22 was selected as the key intermediate for the synthesis of a series of 2′-deoxy-2′-fluororibo derivatives. It was obtained in good 35% overall yield by a 6-step synthesis concluded by stereoselective SN2 fluorination of the bis-THP-protected arabinoside 21 followed by acidic deprotection (Scheme 4). A series of 4-substituted 2′-deoxy-2′-fluororibonucleosides 23a–i was prepared analogously to arabinonucleosides 17 by nucleophilic substitutions or by Pd-catalyzed cross-coupling reactions (Scheme 4, Table 3). Again, the Suzuki reaction with 2-furylboronic acid gave low yield of desired nucleoside 23e.
Entry | R | Conditions | Product | Yield [%] |
---|---|---|---|---|
1 | NH2 | g | 23a | 82 |
2 | OMe | h | 23b | 80 |
3 | SMe | i | 23c | 87 |
4 | Me | j | 23d | 65 |
5 | 2-Furyl | k | 23e | 12 |
6 | 3-Furyl | k | 23f | 50 |
7 | 2-Thienyl | k | 23g | 70 |
8 | 3-Thienyl | k | 23h | 40 |
9 | Phenyl | k | 23i | 41 |
The anti-RSV activity was tested based on methods published previously.19 All the title arabinonucleosides and fluororibonucleosides were completely inactive against RSV. Few fluoroarabino derivatives (9c, 9e, 9f, 9i) showed moderate micromolar activity (13.2, 34.6, 25.2 and 11.3 μM, respectively) against RSV. 2-Thienyl derivative 9g was the most active compound with EC50 = 5.2 μM.
The anti-dengue activity was measured by determining the extent to which the test compounds inhibited replication in Vero cells as previously described.10 Fluoroarabinonucleosides 9a, 9e, 9g, 9h and arabino derivative 16 inhibited dengue virus with EC50 = 10–33 μM, however, their selectivity index was rather low (Table 4).
Compd | HCV (1B) | HCV (2A) | Dengue type 2 | ||||||
---|---|---|---|---|---|---|---|---|---|
EC50 (μM) | CC50 (μM) | SI | EC50 (μM) | CC50 (μM) | SI | EC50 (μM) | CC50 (μM) | SI | |
9a | 6.7 | >44.4 | >6.6 | >44.4 | 33.1 | 0.75 | 10.8 | 12.7 | 1.2 |
9b | 3.1 | >44.4 | >14.3 | 10.8 | >44.4 | >4.1 | >50 | >50 | — |
9c | 1.6 | 22.9 | 14.3 | 6.9 | 20.2 | 2.9 | >50 | >50 | — |
9d | 6.3 | >44.4 | >7.0 | 23.2 | >44.4 | >1.9 | >50 | >50 | — |
9e | 4.6 | >44.4 | >9.7 | 14.7 | 34.6 | 2.4 | 10.5 | 39.0 | 3.7 |
9f | 23.0 | >44.4 | >1.9 | >44.4 | >44.4 | — | >50 | >50 | — |
9g | 2.5 | >44.4 | >17.8 | 13.9 | 34.3 | 2.5 | 27.9 | 39.1 | 1.4 |
9h | 4.1 | 26.5 | 6.5 | 16.2 | 30.3 | 1.9 | 33.3 | >50 | 1.5 |
9i | 5.2 | >44.4 | >8.5 | 15.7 | 34.4 | 2.2 | >50 | >50 | — |
16 | 13.7 | 32.5 | 2.4 | 17.4 | 21.4 | 1.2 | 17.4 | 40.9 | 2.4 |
17a | 22.3 | >44.4 | >2.0 | >44.4 | >44.4 | — | >50 | >50 | — |
17c | 17.4 | >44.4 | >2.6 | 38.5 | >44.4 | >1.2 | >50 | >50 | — |
17d | 18.5 | 40.2 | 2.2 | >44.4 | >44.4 | — | >50 | >50 | — |
17e | 3.0 | >44.4 | >14.8 | 24.0 | >44.4 | >1.9 | >50 | >50 | — |
22 | 4.7 | 9.1 | 1.9 | 9.5 | 10.3 | 1.1 | >50 | >50 | — |
23a | 4.7 | >44.4 | >9.4 | >44.4 | >44.4 | — | >50 | >50 | — |
23b | 8.7 | >44.4 | >5.1 | >44.4 | >44.4 | — | >50 | >50 | — |
23c | 6.6 | 29.0 | 4.5 | 17.0 | >44.4 | >2.6 | >50 | >50 | — |
23d | 5.2 | >44.4 | >8.5 | 16.1 | >44.4 | >2.8 | >50 | >50 | — |
23e | 2.3 | >44.4 | >19.3 | 15.7 | >44.4 | >2.8 | >50 | >50 | — |
23f | 5.2 | >44.4 | >8.5 | 21.8 | >44.4 | >2.0 | >50 | >50 | — |
23g | 18.4 | >44.4 | >2.4 | 19.7 | >44.4 | >2.3 | >50 | >50 | — |
23h | 25.1 | >44.4 | >1.8 | >44.4 | >44.4 | — | >50 | >50 | — |
23i | 8.2 | >44.4 | >5.4 | 20.3 | >44.4 | >2.2 | >50 | >50 | — |
Mericitabine | 1.2 | >44.4 | >37 | 0.99 | >44.4 | >44 | nt | nt | — |
Screening of anti-HCV activities was performed as previously described20 and activities compared to standard Mericitabine.21 The series of arabinonuclesides 17, methoxy and hetaryl derivatives was inactive, whilst amino, methylsulfanyl and methyl derivatives showed moderate anti-HCV effect (EC50 = 17–38 μM). On the other hand, fluoroarabino- and fluororibonucleosides 9a–i and 23a–i were all active against both 1B and 2A genotypes of HCV in replicon assay at (mostly) single digit micromolar concentrations (Table 4) and, more importantly, they were not cytotoxic (in contrast to the previously reported corresponding ribonucleosides9,10).
We assume that, similarly to most antiviral nucleosides,1,2 the mechanism of antiviral activity is intracellular phosphorylation of the nucleosides to NTPs and inhibition of the viral RNA polymerase. To elucidate whether the NTPs of our modified nucleosides would even fit into the active site of the polymerase, we performed a very simple docking and modelling of selected nucleotides into the known crystal structure (PDB code 4WTJ)22 of viral RNA-dependent RNA polymerase HCV NS5B genotype 2A in complex with RNA template 5′-AUCC, RNA primer 5′-PGG, Mn2+ and ADP, which binds to polymerase in catalytically relevant conformation but stalls the primer extension. The modelling was performed using program Moloc and the all-atom MAB force field.23 We selected disphosphates of three amino-substituted nucleosides 9a, 17a and 23a and we used the implemented MAB force field to energy minimize them in the active site to mimic ADP in the original crystal structure. For optimization, the protein and RNA coordinates were kept fixed. The modelling clearly showed that there is enough space to accommodate the fused chlorobenzene ring in the nucleobase binding site and it could have even increased cation–π stacking with Arg158 (Fig. 2a). Also the sugar moieties in all three derivatives could adopt similar conformation as in ADP while the orientation of 2′-substituent does not seem to have a significant influence on the binding as it can form hydrogen bonds with the enzyme in ribo-configuration as well as in arabino-configuration (Fig. 2b). The differences in antiviral activities are probably mostly caused by the different efficiency of the intracellular phosphorylation of the nucleosides.
Fig. 2 Modelled overlay of diphosphates derived from nucleosides 9a, 17a, 23a and ADP in the co-crystal structure of the viral RNA-dependent RNA polymerase HCV NS5B genotype 2A in complex with RNA template, primer, Mn2+, and ADP (PDB code 4WTJ, 2.2 Å resolution). a) Detail of the tricyclic base overlayed with ADP; b) detail of ribose binding site, hydrogen bonds of 2′-OH showed as dashed lines and given in Å. Color code: Cenzyme grey, CADP magenta, C9a-DP salmon, C17a-DP yellow, C23a-DP cyan, CRNA green, O red, N blue, F pale cyan. |
Compd. | MTS, IC50 (μM) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
BJ | MRC-5 | A549 | CCRF-CEM | CEM-DNR | HCT116 | HCT116p53−/− | K562 | K562-TAX | U2OS | |
9a | 37.0 | 43.9 | 41.4 | 15.1 | 32.6 | 30.4 | 30.4 | 27.5 | 37.2 | 25.8 |
9b | 47.1 | >50 | >50 | 34.2 | 45.9 | >50 | >50 | >50 | 47.2 | >50 |
9c | 21.3 | 22.6 | 46.8 | 11.6 | 18.7 | >50 | >50 | 17.6 | 27.4 | 29.0 |
9d | 20.9 | >50 | >50 | 5.1 | 7.5 | >50 | 44.9 | 44.2 | 12.4 | 29.2 |
9e | 26.8 | 28.7 | 44.6 | 13.8 | 25.0 | >50 | >50 | 21.7 | 26.3 | 39.9 |
9f | 46.2 | 49.6 | >50 | 7.2 | >50 | 49.9 | 49.9 | >50 | 39.9 | 37.4 |
9g | 32.0 | 27.0 | >50 | 18.4 | 25.8 | >50 | >50 | 28.1 | 28.1 | >50 |
9h | 41.8 | 41.8 | >50 | 21.1 | 27.6 | >50 | >50 | 33.0 | 28.9 | >50 |
9i | 27.0 | 30.9 | >50 | 19.7 | 25.4 | 49.5 | 49.5 | 24.8 | 26.3 | 33.4 |
16 | >50 | 49.0 | 32.6 | 5.1 | 25.1 | 34.0 | 28.5 | 44.6 | 17.7 | 27.4 |
17a | >50 | >50 | >50 | 28.7 | >50 | >50 | 49.1 | >50 | >50 | >50 |
17c | >50 | >50 | >50 | 11.7 | 24.0 | 36.1 | 34.0 | 26.5 | 25.7 | 26.4 |
17d | >50 | >50 | >50 | 16.1 | 37.2 | >50 | >50 | >50 | >50 | >50 |
17e | >50 | >50 | >50 | 27.1 | 36.6 | >50 | >50 | >50 | 42.7 | >50 |
17i | >50 | >50 | >50 | 24.7 | >50 | >50 | >50 | >50 | >50 | >50 |
22 | >50 | >50 | >50 | 3.2 | 36.3 | 44.6 | 46.6 | >50 | >50 | 32.3 |
23b | >50 | >50 | >50 | 34.2 | 37.9 | >50 | >50 | 49.4 | 34.0 | 45.2 |
23c | >50 | >50 | >50 | 22.1 | 24.1 | >50 | >50 | 25.6 | 26.1 | 27.2 |
23d | >50 | >50 | >50 | 10.9 | 10.0 | 44.7 | 48.9 | >50 | 9.5 | >50 |
23e | 39.6 | >50 | >50 | 16.7 | 27.5 | 48.5 | 48.5 | 30.7 | 27.2 | 37.8 |
23f | >50 | >50 | >50 | 45.6 | 39.7 | >50 | >50 | >50 | >50 | >50 |
23g | >50 | >50 | >50 | 16.2 | 31.3 | >50 | 41.6 | 34.7 | 28.1 | 35.2 |
23h | >50 | >50 | >50 | 37.4 | 40.8 | >50 | >50 | >50 | >50 | >50 |
Gemcitabine | >50 | >50 | 0.05 | 0.02 | 0.10 | 0.03 | 0.41 | 0.10 | 0.05 | 0.18 |
Fluoroarabinonucleosides 9 showed only moderate (>10 μM) cytostatic activity and very poor selectivity against fibroblasts. On the other hand, fluororibonucleosides 23 showed similar activity against CEM cell lines and are not toxic to fibroblasts. Arabinonucleosides 17 bearing methoxy and hetaryl groups in position 4 are inactive against most of the cell lines, they displayed only moderate effect against CEM lines. The most cytotoxic compounds were chloro derivatives 16 and 23 with single digit micromolar activity against CCRF-CEM. In general, most of the arabinonucleosides 17, fluoroarabinonucleosides 9 and fluororibonucleosides 23 are much less cytotoxic than corresponding ribonucleosides.
TPPTS | Triphenylphosphine-3,3′,3′′-trisulfonic acid trisodium salt |
TDA-1 | Tris[2-(2-methoxyethoxy)ethyl]amine |
DAST | (Diethylamino)sulfur trifluoride |
Footnote |
† Electronic supplementary information (ESI) available: Experimental part and characterization data for all new compounds, table with HPLC purities of final compounds, details of biological assays and copies of NMR spectra. See DOI: 10.1039/c7md00319f |
This journal is © The Royal Society of Chemistry 2017 |