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. 2022 Oct 17;144(42):19437–19446. doi: 10.1021/jacs.2c07517

Synthesis of Polycyclic Hetero-Fused 7-Deazapurine Heterocycles and Nucleosides through C–H Dibenzothiophenation and Negishi Coupling

Chao Yang †,‡, Lenka Poštová Slavětínská ‡, Marianne Fleuti †,‡, Blanka Klepetářová ‡, Michal Tichý ‡, Soňa Gurská §, Petr Pavliš §, Petr Džubák §, Marián Hajdúch §, Michal Hocek †,‡,*
PMCID: PMC9619403  PMID: 36245092

Abstract

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A new approach for synthesizing polycyclic heterofused 7-deazapurine heterocycles and the corresponding nucleosides was developed based on C–H functionalization of diverse (hetero)aromatics with dibenzothiophene-S-oxide followed by the Negishi cross-cooupling with bis(4,6-dichloropyrimidin-5-yl)zinc. This cross-coupling afforded a series of (het)aryl-pyrimidines that were converted to fused deazapurine heterocycles through azidation and thermal cyclization. The fused heterocycles were glycosylated to the corresponding 2′-deoxy- and ribonucleosides, and a series of derivatives were prepared by nucleophilic substitutions at position 4. Four series of new polycyclic thieno-fused 7-deazapurine nucleosides were synthesized using this strategy. Most of the deoxyribonucleosides showed good cytotoxic activity, especially for the CCRF-CEM cell line. Phenyl- and thienyl-substituted thieno-fused 7-deazapurine nucleosides were fluorescent, and the former one was converted to 2′-deoxyribonucleoside triphosphate for enzymatic synthesis of labeled oligonucleotides.

Introduction

Modified nucleoside and nucleotide analogues are important cytostatic1 and antiviral drugs.2 Recent outbreaks of RNA-virus diseases, including the current pandemics caused by SARS-CoV-2 virus, started a renaissance3 of this class of compounds because several modified nucleosides and their prodrugs (i.e., Remdesivir4 or Molnupiravir5) are precursors of metabolites (usually nucleoside triphosphates) that inhibit RNA-dependent RNA polymerases6 or cause mutations in viral replication.7 Clearly there is a great need for novel types of nucleoside and nucleotide derivatives in the development of antiviral drugs against emerging viruses or anticancer agents against drug resistant tumors or leukemias.

Previously, we have discovered several classes of substituted 7-deazapurine ribonucleosides 1 with potent and selective cytotoxic effect8 against cancer cell lines that act through incorporation to DNA causing DNA damage and apoptosis.9 Other related derivatives exerted antiviral10 or antiparasitic11 effects. Recently, we designed and synthesized novel types of tri- and tetracyclic fused 7-deazapurine ribonucleosides and found some benzo-fused derivatives were potent antivirals against RNA viruses.12 More importantly, the thieno-,13 furo-,14 pyrrolo-,14 pyrazolo-,15 and some pyrido-fused16 deazapurine nucleosides 2a–e showed strong cytostatic effects in submicromolar concentrations and their mechanism of action involved DNA damage and apoptosis. The corresponding tetracyclic naphtho-17 and benzothieno-fused18 nucleosides 3a,b (Figure 1) were less or noncytotoxic but still showed antiviral effects suggesting that the increased bulkiness of the fused heteroaromatic system might lead to selectivity in antiviral versus cytotoxic activities. These tri- and tetracyclic fused nucleobases can be synthesized either by multistep heterocyclization approach12,15,16 or through cross-coupling of in situ generated 4,6-dichloropyrimidine-5-zinc reagent with hetaryl halides,18,13,14 but for some heterocycles, the corresponding halides are inaccessible, expensive, or unreactive. Therefore, there is a need for an alternative general approach that would enable synthesis of a wide range of novel fused deazapurine bases for further applications.

Figure 1.

Figure 1

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Structures of previously developed biologically active substituted and fused 7-deazapurine nucleosides and the design of target compounds (in dashed square).

To overcome the above-mentioned synthetic problems, we turned our attention to recently developed C–H functionalization of (hetero)aromatics with sulfur heterocycles and further cross-coupling of the resulting sulfonium intermediates.19,20 The Ritter group has reported seminal works21 on metal-free C–H thianthrenylation followed by Negishi coupling with aliphatic alkylorganozinc compounds or aromatic nucleophilic substitutions with diverse nucleophiles. Dibenzothiophene (DBT) and dibenzothiophene-S-oxide (DBTO) were also successfully used22 for C–H functionalization and further derivatizations. Diverse substituted benzenes showed good reactivity and selectivity, whereas only several examples of five-membered heterocycles (mostly substituted thiophenes) were reported23 in this reaction sequence suggesting that their reactivity is problematic. Other groups have reported generation and Negishi coupling of alkenyl-24 or alkylsulfonium25 salts with arylzinc reagents, but no example of (het)aryl-(het)aryl cross-coupling and neither applications of this chemistry for the synthesis of biologically active compounds was reported so far. We report here a general approach to a portfolio of novel tri-, tetra-, and even pentacyclic fused 7-deazapurine bases through the C–H functionalization of heterocycles followed by the Negishi cross-coupling of the resulting dibenzothiophenium derivatives, their glycosylations to nucleosides, and the photophysical properties and biochemical and biological profiling of the corresponding nucleosides and nucleotides.

Results and Discussion

Chemistry

As mentioned above, (het)aryl-fused 7-deazapurine heterocycles can be prepared through the following approaches: (1) buildup of the heterocycles from chloronitrobenzene through heterocyclization of 2-amino-1H-indole-3-carboxylate,12,16,26 (2) transition metal-catalyzed intramolecular C–H arylation of arylamino-iodopyrimidine,27 and (3) thermal or photochemical cyclization of 5-aryl-6-azidopyrimidines through nitrene formation.13,14,18,28 However, the starting materials for each approach are not always easily obtained due to the regioselectivity of halogenation or nitrozation of the heterocycles. Therefore, we envisaged an advantageous use of the recently reported C–H functionalization of diverse heterocycles with DBTO followed by the Negishi coupling of sulfonium salts with zincated dichloropyrimidine.

First, we tested the C–H functionalization of a wide range of substituted benzenes and substituted and fused thiophenes, as well as imidazo[1,2-b]pyridazine and a set of other five-membered heterocycles with dibenzothiophene-S-oxide (that was reported23 to perform better on thiophenes compared to thianthrene). The reactions were performed in the presence of trifluoroacetic or triflic anhydride according to literature protocols (Scheme 1).22,23 In the case of all thiophenes, benzenes, imidazopyridazine, and N-methylpyrazole, we obtained the desired (het)arylsulfonium salts in good yields. Only in the case of benzothiophene, the yield was lower and in the case of biphenyl the dibenzothiophenium salt was inseparable and was used directly for the Negishi coupling. On the other hand, the other five-membered heterocycles 4o–4y including furan and diverse azoles did not give any detectable sulfonium products. In all these cases, we also tried C–H thianthrenylation19,21 that also did not provide the desired products. It seems that this C–H functionalization has a severe limitation for most five-membered heterocycles beyond thiophene.

Scheme 1. Preparation of (Het)aryl Sulfonium Salts.

Scheme 1

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With the portfolio of (het)arylsulfonium salts in hand, we then tested the feasibility of the Negishi cross-coupling reaction. Previously, only two examples of Negishi coupling of a substituted phenyl-tetrafluorothianthrenium salt with alkylzinc halides was reported.21 Initially, we tested the model reaction of thienothiophene-derived dibenzothiophenium salt 5a with in situ generated zincated dichloropyrimidine 6(13) under different conditions. Reaction with a 0.5 equiv of 6 in the presence of Pd(PPh3)2Cl2 did not proceed, whereas the same reaction in the presence of Pd(PPh3)4 overnight led to a complex inseparable mixture containing the desired product 7a (TLC-MS). However, simple prolonging of the reaction time to 40 h afforded the desired product 7a with 42% isolated yields. The yield was improved to 60% when using 1 equiv of organozinc 6 (that offers 2 equiv of the arylorganometallic moiety for transmetalation29). Moreover, using the THF/MeCN mixture as the solvent further slightly improved the yield to 64%, since the thienothiophene-derived dibenzothiophenium salt 5a has better solubility in MeCN. Therefore, the conditions outlined in Table 1, entry 5 were chosen as the optimized conditions for subsequent investigations.

Table 1. Optimization of Negishi Cross-Coupling with 6a.

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Entry Cat. or Base 6 (equiv) Solvent T/°C Time Yield or Result
1 Pd(PPh3)2Cl2 0.5 THF 65 overnight no product
2 Pd(PPh3)4 0.5 THF 65 overnight mixtureb
3 Pd(PPh3)4 0.5 THF 65 40 h 42%
4 Pd(PPh3)4 1 THF 65 40 h 60%
5 Pd(PPh3)4 1 THF/MeCN 65 40 h 64%
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a

Reaction conditions: Sulfonium salt (0.23–0.45 mmol, 1.0 equiv), catalyst (0.05–0.08 equiv), zincated pyrimidine (0.5–1 equiv) and the solvent (THF or MeCN; c = 0.2 M) were stirred at 65 °C for 12–48 h.

b

Inseparable mixture containing product 7a (TLC).

With the optimized reaction conditions, we then examined the substrate scope of this new Negishi coupling (Scheme 2). All the dibenzothiophenium salts derived from thiophene-based heterocycles (thienothiophene, dithienothiophene, phenylthiophene, bithiophene, benzothiophene, and thiophene) 5a–5e and 5n reacted very well to produce the desired products 7a–e and 7n in moderate to good yields (53%–72%). Similarly, most of the sulfonium salts derived from substituted benzenes 5f–5k were amenable to this Negishi coupling reaction with zincated dichloropyrimidine 6 to form the corresponding products 7f–k in acceptable yields (42%–87%) with excellent site selectivity. On the other hand, no reaction was observed with strongly electron-rich methoxybenzene- and imidazopyridazine-derived sulfonium salts 5l and 5m.

Scheme 2. Investigation of the Substrate Scope of the Negishi Cross-Coupling of (Het)arylsulfonium Salts 5a–5u with Dichloropyrimidine-Zinc 6.

Scheme 2

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Since the site selectivity of iodination of some (hetero)aromatics, such as thienothiophene and benzothiophene, is poor, the traditional Negishi coupling using (het)aryl iodides can be quite problematic. Therefore, the regioselective C–H functionalization followed by the Negishi coupling of sulfonium salts and aryl-zinc reagents can be an excellent complementary strategy for the synthesis of complex heterocyclic biaryls.

Based on the biological activity of benzo-, thieno-, benzothieno-, and naphtho-fused deazapurine nucleosides, we designed novel tetracyclic thienothieno- and pentacyclic thienothienothieno-fused deazapurines 9a and 9b, as well as phenyl- and thienyl-substituted thieno-fused 7-deazapurines 9c and 9d as key intermediates in the synthesis of the corresponding nucleosides and their synthesis started from the new thienyl-pyrimidines 7a–d (Scheme 3).

Scheme 3.

Scheme 3

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Reagents and conditions: (i) NaN3 (1 equiv), LiCl (1 equiv), THF, r.t., 40 h; (ii) 1,4-dibromobenzene, 180 °C, 35 min; (iii) for10a, 10c: 1. KOH (2.6 equiv), TDA-1 (1.5 equiv), Hoffer’s chlorosugar (1.5 equiv), MeCN, r.t., 50 min; for10b: 1. BSA (1 equiv), MeCN, 60 °C, 35 min. 2. 1-chloro-3,5-di-(4-chlorobenzoyl)-2-deoxy-α-d-ribose (2 equiv), TMSOTf (2.5 equiv), MeCN, 60 °C, 30 min. (iv) 1. BSA (1 equiv), MeCN, 60 °C, 30 min. 2. 1-O-acetyl-2,3,5-tri-O-benzoyl-β-d-ribofuranose (2 equiv), TMSOTf (2.5 equiv), MeCN, 60 °C, 25–60 min.

First, the azidation of dichloropyrimidines 7a–d with sodium azide in THF was performed to get tetrazolopyrimidines 8a–d, which have poor solubility in organic solvents and were used directly for the next step without chromatographic purification. The NMR spectra in DMSO showed that crude compounds 8a–d exist mainly as the form of tetrazolopyrimidines. Based on our previous experience with the synthesis of heteroaryl-fused 7-deazapurine nucleobases, the thermal condition was applied for the heterocyclization of tetrazolopyrimidines to nucleobases. For compounds 8a, 8c, and 8d, the robust thermal cyclization at 180 °C successively gave us the desired fused deazapurine products 9a, 9c, and 9d in 75%, 63%, and 67% yield over 2 steps, respectively. In the case of nucleobase 9b, the poor solubility did not allow chromatographic purification; hence, the crude compound 9b was used directly for the next glycosylation step.

The glycosylation of nucleobases 9a,c,d with the 1-chloro-3,5-bis-O-(4-chlorobenzoyl)-2-deoxy-α-d-ribofuranose (Hoffer’s chlorosugar) under basic conditions gave the desired protected deoxynucleosides 10 in moderate to good yields (10a 49%, 10c 72%, 10d 62%) and with exclusive stereoselectivity to form β-anomers. The less soluble crude heterocycle 9b was first treated with BSA at 60 °C and then TMSOTf and chlorosugar, which resulted in the formation of the desired β-anomeric nucleoside 10b in a low yield of 5% over 3 steps and its α-anomer 10bα in 14% yield over 3 steps. Next we performed the glycosylation of nucleobases 9 with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-d-ribofuranose to prepare the corresponding ribonucleosides. We used a modified procedure of Vorbrüggen glycosylation as in our previous works.13,14,18 A MeCN solution of nucleobase 9a–d and BSA was heated to 60 °C for 30 min. After addition of TMSOTf and 1-O-acetyl-2,3,5-tri-O-benzoyl-β-d-ribofuranose, the mixture was stirred at the same temperature for 25–60 min. The protected ribonucleosides 11a, 11c, 11d were obtained in good yields of 51%–65%, and also the ribonucleoside 11b was synthesized from crude 9b in an acceptable yield of 35% (over 3 steps) as the pure β-anomer. All nucleosides 11a–11d were obtained as pure β-anomers.

The protected chloro derivatives of deoxyribonucleosides 10a–d and 10bα were converted to the corresponding analogues of 2′-deoxyadenosine (Scheme 4). Their nucleophilic substitution reaction with aqueous ammonia at 120 °C in dioxane substituted the chlorine at position 4 with the amino group with concomitant deprotection of the sugar part to form the desired fused 2′-deoxy-7-deazaadenosine derivatives 12a–d and 12bα in good yields (58%–90%). Triphosphorylation of 12c by the standard procedure30 furnished the desired phenylthieno-fused 7-deazapurine 2′-deoxynucleoside triphosphate 12cTP (dAPTTP) in 41% yield after HPLC purification (Scheme 4).

Scheme 4.

Scheme 4

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Reagents and conditions: (i) NH3 (aq.), dioxane, 120 °C, 24 h; (ii) 1. POCl3 (1.5 equiv), PO(OMe)3, 0 °C, 3 h; 2. (NHBu3)2H2P2O7 (5 equiv), Bu3N, DMF, 0 °C, 2 h; 3. TEAB.

In the ribonucleoside series, we designed 4-amino-, 4-methoxy-, and 4-methyl derivatives in each type of fused deazapurine nucleoside, as these were the most active in the related tricyclic fused nucleosides in our previous works. Starting from the protected 4-chloro nucleosides 11a–d, the amino group was introduced to position 4 using the same procedure and conditions as those for the 2′-deoxynucleosides with aqueous ammonia in dioxane to give the desired products 13a–d (fused analogues of adenosine) in 41–74% yields. Reactions of intermediates 11a–d with sodium methoxide displaced the chloro group with methoxy at position 4 and simultaneously cleaved the benzoyl protecting groups to give a series of 4-methoxy nucleosides 14a–d in moderate to good yields. Due to the poor solubility of the intermediates 11, the mixture of 1,4-dioxane and methanol and refluxing at 65 °C were used to accelerate the reaction and increase the yield. The introduction of a methyl group to position 4 was achieved through cross-coupling reactions of 11a–d with trimethylaluminum and Pd(PPh3)4 in THF to obtain a set of protected 4-methyl nucleosides 15a–d (62–80% yields) that were deprotected by MeONa in MeOH/dixane to obtain free nucleosides 16a–d in 65–86% yield. All the target free nucleosides 13a–d, 14a–d, and 16a–d were obtained in sufficient amounts and purity for further biological profiling (Scheme 5).

Scheme 5.

Scheme 5

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Reagents and conditions: (i) NH3 (aq.), dioxane, 120 °C, 24 h; (ii) MeONa, MeOH/dioxane, 65 °C, overnight; (iii) Me3Al (2.2 equiv), Pd(PPh3)4 (0.15 equiv), THF, 65 °C, overnight; (vi) MeONa (3 equiv), MeOH/dioxane, 60 °C, overnight.

Fluorescence Properties of Polycyclic Fused Deazapurine Nucleosides

Previous studies reported that benzo-31,32 and naphtho-fused17,33 7-deazapurine nucleosides showed useful fluorescence properties and were used for construction of fluorescent DNA probes. Therefore, we have investigated the photophysical properties of the new polycyclic fused 7-deazapurine nucleosides. Table 2 shows the results of measurement of the UV–vis and fluorescence of the fused 2′-deoxyadenosine analogues 12a–12d in three different solvents (Table 2). All of the four amino derivatives exerted fluorescence with emission maxima at 359–424 nm. Among these amino derivatives, the phenyl-thieno-fused 7-deazapurine nucleoside 12c showed the strongest fluorescence with high quantum yields. Interestingly, it exerted the highest fluorescence quantum yield of 48% in water. The thienyl-thieno-fused 7-deazapurine nucleoside 12d also exhibited relatively strong fluorescence with a 16–25% quantum yield. The fluorescence emission maxima and quantum yields of 12c and 12d did not significantly change in different solvents. Surprisingly, the extended tetra- and pentacyclic thienothieno- and thienothienothieno-fused nucleosides 12a and 12b showed only very weak fluorescence.

Table 2. UV Absorption Spectra and Fluorescence Properties of Nucleosides 12a–12da.

    absorption emission
Compd solvent λabs [nm] (ε [103 M–1 cm–1]) λem [nm] Φf
12a MeOH 323 (21.1), 338 (19.8) 359 0.02
Dioxane 325 (21.7), 340 (20.2) 367 0.01
H2O 322 (18.2), 337 (16.4) 367 0.04
12b MeOH 341 (29.6), 358 (28.1) 385 0.03
Dioxane 343(29.9), 360 (28.0) 392 0.02
H2O 341 (21.6), 357 (17.1) 386 0.04
12c MeOH 344 (23.3) 398 0.40
Dioxane 348 (23.0) 405 0.32
H2O 342 (19.8) 406 0.48
12d MeOH 268 (12.4), 357 (22.9) 417 0.16
Dioxane 268 (12.3), 362 (22.3) 424 0.19
H2O 268 (10.3), 356 (21.3) 419 0.25
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a

Fluorescence quantum yields were measured by using anthracene in EtOH (Φf = 0.27) as a reference (excitation wavelength is 320 nm).

Enzymatic Synthesis and Photophysical Properties of Modified Oligonucleotide

Since phenyl-thieno-fused 7-deazapurine nucleoside 12c showed strong fluorescence in water, we decided to study enzymatic incorporation of the modified nucleotide and investigate the photophysical properties of the resulting oligo-2′-deoxyribonucleotides. The enzymatic synthesis was performed using dAPTTP (12cTP) as a substrate in primer extension (PEX) in the presence of KOD XL DNA polymerase with a 19-nt template Temp1A encoding for incorporation of one dAPT-modified nucleotide into the extended primer (for sequences of oligonucleotides, see Supporting Information (SI), Table S1). A FAM-labeled primer (PrimFAM, 15-nt) was used in the PEX to visualize the extension on denaturing polyacrylamide gel electrophoresis (PAGE). Figure 2A confirms that the PEX reaction using dAPTTP was successful giving the full-length product 19ON_1APT which was also characterized by MALDI-TOF mass analysis (found: 6107.4 Da, calculated: 6105.9 Da, Figure S6 in SI). When the PEX reaction was performed with longer template Temp4A encoding for four modifications, the full-length product ON_4A (31ON_4APT) was also obtained, which was proved by both denaturing PAGE (Figure 2B) and MALDI-TOF mass (see SI, section 6), but it was accompanied by shorter products of incomplete primer extension. To gain more insight, we performed kinetic experiments of single nucleotide extension with dAPTTP in comparison with dATP and 7-deaza-dATP (dAHTP) (Figure S4 in SI) that show that indeed the incorporation of the very bulky tricyclic nucleotide was significantly slower, in particular when it was positioned against the 5′-terminal nucleotide in the template. Nevertheless, the fact that the polymerase was able to incorporate even such a bulky nucleotide is remarkable.

Figure 2.

Figure 2

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PEX with 12cTP (dAPTTP) and KOD XL DNA polymerase with templates encoding for incorporation of one (A) or four (B) modified nucleotide(s). A) template Temp1A, (P): FAM-labeled primer; (A+): dATP, dGTP; (A−): dGTP; (AH): dAHTP, dGTP; (APT): dAPTTP, dGTP. B) template Temp4Abio (for original uncut gel, see Figure S2 in SI); (P): FAM-labeled primer; (A+): dATP, dGTP, dCTP, dTTP; (A−): dGTP, dCTP, dTTP; (AH): dAHTP, dGTP, dCTP, dTTP; (APT): dAPTTP, dGTP, dCTP, dTTP.

In order to study the fluorescence properties of dAPT-modified oligonucleotides (ONs), we first measured the fluorescence emission and quantum yields of the 19ON_1APT. Fluorescence properties of the resulting ONs and duplexes were studied with an excitation wavelength of 320 nm. ON_1A in medium salt buffer showed fluorescence with emission maxima at 406 nm and with a quantum yield of 10.2%. Hybridization with matched or mismatched ONs did not show any significant difference either in emission maxima or in quantum yields (see SI for details). Therefore, we believe that this stable fluorescence property makes dAPT promising for DNA labeling and quantification, but it is not an environment-sensitive label for studying changes of secondary structures or hybridization.34

Biological Activity Profiling

Nucleosides 12a–12d, 13a–13d, 14a–14d, and 16a–16d were also tested for in vitro cytotoxic activity on the panel of leukemic cell lines (CCRF-CEM – acute lymphoblastic leukemia, K562 – myelogenous leukemia,)11 and solid tumor cells (A549 – human lung adenocarcinoma, HCT116 and HCT116p53–/– – colon cancer cells with/without p53 gene, U2OS – human osteosarcoma)35 using a 3-day MTS (3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay.36 Nonmalignant fibroblast cell lines MRC-5 and BJ were used in the MTS assay to assess the cancer cell selectivity. Table 3 summarizes the cytotoxic activities of compounds in the MTS assay in comparison with known parent compounds: strongly and nonselectively cytotoxic tubercidin (7-deazaadenosine)37 and 2′-deoxytubercidin (2′-deoxy-7-deazaadenosine).38

Table 3. Cytotoxic Activities of Nucleosides.

  MTS, IC50 [μM]
Compd BJ MRC-5 CCRF-CEM K562 A549 HCT116 HCT116 p53–/– U2OS
12a >50 50 6.6 20.9 32.7 18.8 16.2 20.4
12b 18.1 23.8 0.48 1.9 14.2 1.91 1.46 2.8
12bα >50 >50 0.73 3.7 >50 3.2 2.3 4.7
12c 50 50 0.74 2.2 12.6 2.5 2.7 2.1
12d 50 49.9 0.77 3.2 27.2 2.9 2.8 2.5
13a 48.1 43.8 14.2 20.4 27.2 17.2 19.1 16.6
13b 50 >50 1.4 4.2 8.8 2.3 2.5 4.5
13c >50 >50 3.3 13.6 >50 14.8 14.3 10.6
13d >50 >50 3.2 20.7 >50 12.5 9.6 13.5
14a >50 >50 >50 >50 >50 >50 >50 >50
14b >50 >50 >50 >50 >50 >50 >50 >50
14c >50 >50 >50 >50 >50 >50 >50 >50
14d >50 >50 >50 >50 >50 >50 >50 >50
16a >50 >50 15.5 48.5 >50 35.2 46.2 41.7
16b >50 >50 1.8 3.5 >50 5.5 n.d. 44.5
16c >50 >50 2.0 >50 >50 >50 >50 >50
16d >50 >50 2.0 12.3 >50 25.9 14.9 21.5
tubercidin 0.73 0.74 0.017 0.36 0.52 0.08 0.15 0.089
2′-deoxytubercidin >50 >50 >50 >50 >50 >50 >50 >50
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Ribonucleoside 13b showed low micromolar activities against most of cancer cell lines but not nonmalignant fibroblasts in the MTS assay. Ribonucleosides 13a, 13c, 13d, 16b, and 16d showed only moderate effects at micromolar concentrations with little selectivity to cancer cells. The remaining ribonucleosides are inactive against cancer cells. On the other hand, most of the deoxyribonucleosides 12b–12d and the α-anomer 12bα displayed good antitumor activities in micromolar or submicromolar (for CCRF-CEM cell line) concentrations. Compounds 12bα, 12c, and 12d show good selectivity toward cancer cell lines and are nontoxic to fibroblasts as compared to toxic tubercidin. These rather surprising results are interesting because in the previous examples of tubercidin,37 7-substituted8 or fused deazapurine nucleosides,13,14 the ribonucleosides were always more active whereas 2′-deoxytubercidin38 was inactive. This suggests that the mode of action of this class of nucleosides will probably be different from the previously reported tubercidin or tricyclic thieno-, furo-, and pyrrolo-fused 7-deazapurine ribonucleosides that become phosphorylated and incorporated to DNA causing DNA damage and apoptosis. The mechanism of action of these new polycyclic 2′-deoxyribonuclesides will need a separate study in the future.

All the title nucleosides were also screened for their antiviral activity against herpes simplex, influenza, human immunodeficiency virus (HIV), dengue, and SARS-CoV-2 viruses using previously published protocols.10,16 None of the final nucleosides showed any significant activity at 25 μM concentration.

Conclusion

In this paper, we developed a new approach for the synthesis of fused 7-deazapurine heterocycles and nucleosides. The synthesis of the key polycyclic fused heterocycles relied on C–H functionalization of (hetero)aromatics with DBTO followed by the Negishi cross-coupling of the resulting sulfonium salts with bis(4,6-dichloropyrimidin-5-yl)zinc (6) giving a series of 5-(het)aryl-4,6-dichloropyrimidines 7 in good yields and excellent regioselectivity. It is the first example of the Negishi cross-coupling of (het)arylsulfonium salts with hetarylzinc reagent, and this cross-coupling has a very promising potential in synthesis of other complex heterocyclic biaryls. Selected examples of biaryls 7 were then azidated and cyclized to form novel substituted or extended thieno-fused 7-deazapurine heterocycles. These extended nucleobase analogues, which would be hardly accessible by previously known synthetic approaches, were then glycosylated to form 2′-deoxy- or ribonucleosides, and a series of derivatives were prepared by nucleophilic substitutions at position 4. Most of the modified ribonucleosides were inactive or moderately active against a panel of cancer cell lines in the cytotoxic MTS assay, whereas deoxyribonucleosides 12b–12d displayed high anticancer activities, especially for T-lymphoblastic leukemia cells CCRF-CEM. On the other hand, the extended fused nucleosides did not show any significant antiviral activity. Fluorescent properties of the polycyclic deoxyribonucleosides were also studied, and we also prepared a triphosphate of 2′-deoxyribonucleoside 12c (12cTP, as analogue of dATP) and successfully used it for enzymatic synthesis of fluorescently labeled DNA, which showed stable fluorescence in different sequences and was promising for DNA labeling. These polycyclic fused nucleosides and nucleotides also might have potential in the construction of new types of expanded nucleic acids.39

Acknowledgments

The work has been supported by the National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102, funded by the European Union - Next Generation EU). This research was also supported by grants from the Czech Ministry of Education, Youth and Sports (CZ-OPENSCREEN - LM2018130, EATRIS-CZ - LM2018133).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.2c07517.

  • Absorption and emission spectra of modified nucleosides and DNA, sequences of oligonucleotides, full experimental section with synthetic procedures and characterization of all compounds, biochemical methods and procedures, and copies of NMR spectra. (PDF)

The authors declare no competing financial interest.

Supplementary Material

ja2c07517_si_001.pdf (11.9MB, pdf)

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