Synthesis and Biological Profiling of Quinolino-Fused 7-Deazapurine Nucleosides

A series of quinolino-fused 7-deazapurine (pyrimido[5′,4′:4,5]pyrrolo[3,2-f]quinoline) ribonucleosides were designed and synthesized. The synthesis of the key 11-chloro-pyrimido[5′,4′:4,5]pyrrolo[3,2-f]quinoline was based on the Negishi cross-coupling of iodoquinoline with zincated 4,6-dichloropyrimidine followed by azidation and thermal or photochemical cyclization. Vorbrüggen glycosylation of the tetracyclic heterocycle followed by cross-coupling or substitution reactions at position 11 gave the desired set of final nucleosides that showed moderate to weak cytostatic activity and fluorescent properties. The corresponding fused adenosine derivative was converted to the triphosphate and successfully incorporated to RNA using in vitro transcription with T7 RNA polymerase.


■ INTRODUCTION
Base-modified nucleosides are an important class of biologically active molecules that display antiviral, 1 anticancer, 2 or antiparasitic 3 activities.Several clinically used drugs for the treatment of leukemia or tumors are based on this type of compounds. 4Despite the recent progress in other types of anticancer treatments, 5 there is still a need for new types of base-modified nucleosides to find new mechanisms of action that may overcome the drug resistance and decrease toxicity. 6articularly interesting are modified 7-deazapurine nucleosides, known for their broad biological activities. 7During our systematic research, we discovered 7-(het)aryl-7-deazapurine ribonucleosides, exemplified by 7-thienyl-7-deazaadenosine AB-61, 8 which are active against a broad spectrum of cancer cell lines and show excellent selectivity against nonmalignant cells.Investigation of its mechanism of action revealed that it is phosphorylated only in cancer cells to ribonucleoside triphosphate, which is then incorporated to DNA, where it causes double-strand breaks leading to apoptosis. 9Later on, we found 10 that even other 6-substituted analogues 1 bearing methoxy, methylsulfanyl, methylamino, dimethylamino, or methyl groups at position 6 retain a similar level of cytotoxic activity.Then we studied diverse deazapurines with fused aromatic or heterocyclic rings and found that the furo- 11 or thieno-fused 12 7-deazapurine nucleosides 2 are also very potent cytostatics, whereas the corresponding benzo-fused analogs (pyrimidoindoles) 3 13 are noncytotoxic but exert moderate antiviral activity.Introducing a single nitrogen atom into a specific position on the fused phenyl ring gave pyridofused derivatives 4, 14 which showed submicromolar cytotoxic activity and a similar mechanism of action involving DNA damage and apoptosis.When we increased the size of the heteroaromatic nucleobase and prepared tetracyclic naphthofused 5 15 or even some bulkier pentacyclic 16 deazapurine nucleosides, they showed only weak cytotoxic activity.To further investigate if the introduction of a nitrogen atom into the fused tetracyclic ring-system can improve the cytotoxic activity and to extend the SAR of this class of compounds, we designed and synthesized a series of novel quinolino-fused 7deazapurine ribonucleosides (6) (Figure 1).d 8 , DMF-d 7 , or DMSO-d 6 , both forms, 9a and 9b, can be observed in various ratios (see Table S1 in the SI) In the third step, the azide 9a can be cyclized by three different cyclization reactions: (1) thermal cyclization, (2) catalytic cyclization with different rhodium catalysts, and (3) photocyclization with UV light.The thermal cyclization of 9 in 1,4-dibromobenzene at 170 °C for 10 min gave the desired tetracyclic nucleobase 10 with 8% yield, and 59% of the starting material 9 was recovered.Prolonging the reaction time to 30 min increased the yield by only 3% and reduced the amount of recovered starting material to only 21%.Similar yields were achieved by heating the azide 9 in a microwave reactor in toluene to 170 °C for 60 min; only 10−14% of product 10 was obtained, and all starting azide 9 was consumed (see Table 1).These results suggest that the azide 9 is also decomposing at this temperature to unidentified side products.
As the thermal cyclization gave only low yields and most of the starting material was just decomposed, we tried the second option: rhodium-catalyzed cyclization.We tested three different catalysts: Rh 2 esp 2 , 19 rhodium octanoate dimer (Rh 2 (O 2 CC 7 H 15 ) 4 ), and rhodium heptafluorobutyrate dimer (Rh 2 (O 2 CC 3 F 7 ) 4 ) 20 in toluene or in toluene/TFA (1:1) with and without molecular sieves.But none of the reaction conditions resulted in the formation of 10 (see Table S2 in the SI) We then tried the photocyclization of 9 under our standard conditions 11,12,15 in TFA with UV light (254 nm, 4 W) for 48 h, but it resulted only in decomposition of the azide 9. We then tried DCM and THF as a solvent and used different photosensitizers (see Table S3 in the SI).The best result  was achieved by using THF with UV light (254 nm, 4 W) and 1.0 equiv of pyrene, a singlet photosensitizer, for 72 h, which resulted in 26% of the desired nucleobase 10.The overall yield of this three-step reaction cascade toward the quinolino-fused 7-deazapurine 10 was 18%.
The quinolino-fused nucleobase 10 was subjected to the Vorbruggen glycosylation, 21 which is known to be the best option for heteroaryl-fused nucleosides. 12,14The nucleobase 10 was first silylated in position 7 with N,O-bis(trimethylsilyl)acetamide (BSA) and then underwent glycosylation with 1-Oacetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf), producing the key nucleoside 11 as a pure β-anomer in 52% yield (Scheme 2).An analytical sample was isolated in pure form and fully characterized.However, the purification in a larger scale was difficult, and hence, we used the crude material (ca.75% pure) directly in the next step.The stereochemistry of 11 was confirmed by H,H-ROESY using the relations between H-6 of the nucleobase and H-2′ and H-3′ as well as between H-1′ and H-4′ of the sugar moiety.
The crude (75% pure) nucleoside intermediate 11 was used in a series of reactions for derivatization in position 11 and final removal of benzoyl protecting groups from the ribose to give the desired 11-substituted quinolino-fused 7-deazapurine ribonucleosides 14a−g (Scheme 3).The Stille cross-coupling of 11 with 2-(tributylstannyl)furan in the presence of PdCl 2 (PPh 3 ) 2 in DMF at 100 °C for 24 h gave the 2-furyl derivative 13a (26%).The Suzuki−Miyaura cross-coupling of 11 with 2-benzofurylboronic acid gave the 2-benzofuryl derivative 13b in high 82% yield.The cross-coupling of 11 with AlMe 3 and Pd(PPh 3 ) 4 in THF at 65 °C for 3 h gave the methyl derivative 13c with 42% yield.The nucleophilic substitution of 11 with dimethylamine in 2-propanol at 60 °C for 24 h gave the N,N-dimethylamino derivative 13d (64%).Then, the sugar moiety of the protected nucleosides 13a−d was deprotected with NaOMe in methanol at 60 °C for 18 h, resulting in the free nucleosides 14a−d (27−64%).The nucleosides 14e−g were obtained from 11 in a single step as the derivatization in position 11 and the deprotection of the sugar moiety happened simultaneously.Treating 11 with aqueous ammonia/1,4-dioxane (5:2) in a screw-cap pressure vial at 120 °C for 18 h resulted in the formation of the free amino derivative 14e (52%).The reaction of 11 with NaOMe in MeOH at 60 °C for 4 h gave the free methoxy derivative 14f in 22% yield.The reaction with NaSMe in THF at 60 °C for 18 h gave the free methylsulfanyl derivative 14g (29%).
Spectroscopic Properties of Quinolino-Fused 7-Deazapurine Nucleosides.Both the naphtho-and the pyrido-fused 7-deazapurine ribonucleoside derivatives 14,15 show interesting fluorescent properties.Anisolo-fused 7deazapurine 2′-deoxyribonucleosides have been used as nucleic acid probes. 23Therefore, we studied the photophysical properties of the nucleosides 14a−g by measuring their absorption and emission spectra in methanol (Table 2).We then determined their molar extinction coefficient ε as well as their quantum yields Φ f (see S4 in the SI). 24The nucleosides 14a and 14e exhibited fluorescence with moderate Φ f of 4.6−  Biological Profiling.All the titled nucleosides 14a−g were tested for their in vitro cytotoxic activity.The following cancer cell lines were used for the study: A549 (lung cancer), CCRF-CEM (acute T-lymphoblastic leukemia), HCT116 and HCT116p53 − (colon carcinoma, parental and p53 deficient), K562 (chronic myelogenous leukemia), and U2OS (bone osteosarcoma) using a colorimetric MTS assay. 25Additionally, HeLa (cervical cancer), HepG2 (hepatocellular liver carcinoma), and HL60 (acute promyelocytic leukemia) cell lines were tested using the luminescent CellTiter-Glo assay.For comparison, nonmalignant fibroblast cell lines (BJ and MRC-5) were included in the MTS assay, whereas noncancerous human dermal fibroblasts (NHDF) were assessed with the CellTiter-Glo assay. 26Initial screenings were done at 50 μM concentration for the MTS assay and 10 μM for the CellTiter-Glo assay.All the results are summarized in Table 3.
Of all the title nucleosides, the dimethylamino derivative 14d is the only one that did not show any cytotoxicity whatsoever, which is consistent with all other heteroaryl-fused nucleosides. 11,12,14,15Both furyl and benzofuryl derivatives 14a and 14b, respectively, showed only weak activity against the CCRF-CEM leukemia line; 14b also exhibited activity against both HCT116/HCT115p53− colon carcinoma lines and pronounced effect on the HepG2 hepatocellular carcinoma cell line, with an IC 50 value of 2.8 μM indicating a specificity not observed in 14a.The nucleoside 14g bearing SMe group in position 11 displayed some moderate cytotoxic activity against a spectrum of tested cell lines including nonmalignant BJ and MRC-5, thus showing no significant selectivity toward cancerous cells.The most promising nucleosides in this series are methyl 14c, amino 14e, and methoxy 14f derivatives, which all showed comparable activities against several cancer cell lines, with CCRF-CEM and HL60 being the most sensitive one with single-digit micromolar IC 50 values and no cytotoxicity against nonmalignant cell lines BJ, MRC-5, and NHDF.Although the nucleosides 14c, 14e, and 14f are more potent against the CCRF-CEM line compared to their naphtho-fused analogs (6−8 vs 20−23 μM), 15 their activities are still 2 orders of magnitude lower than their tricyclic thieno-, 12 furo-, 11 N-methylpyrrolo-, 11 and pyrido-fused 14 analogs.This is in agreement with our previous findings 15,16,27,28 that nucleosides with tetracyclic nucleobases are already too bulky to be activated by phosphorylation and to interact with their biological target (s).
Biochemistry.The amino derivative 14e was used as an adenosine analogue to study its incorporation by in vitro transcription and its fluorescent properties.First, 14e was triphosphorylated at 5′-OH according to standard procedures, 29 resulting in the triphosphate 15 (A Q TP) with good 59% yield (Scheme 4) A Q TP was then used as a substrate for the T7 RNA polymerase in the in vitro transcription (IVT) experiments. 30e used 35DNA_A7 DNA template encoding for 35-mer RNA containing seven A Q modifications (Table 4).We used the T7 High Yield RNA Synthesis Kit with a high concentration of NTPs (7.5 mM each) but without any further additives.The reaction time at 37 °C was 16 h.The positive control experiment was performed with all four natural NTPs giving nonmodified transcript 35RNA_A7 (Figure 2, lane 2).The negative control contained only the natural CTP, GTP, and UTP in the absence of ATP (Figure 2, lane 3).The real IVT experiment was performed with A Q TP and three natural NTPs (Figure 2, lane 4).The transcription products were visualized by denaturing 20% denaturing PAGE (Figure 2) and characterized by LC−MS (see Figures S1 and S2 in the SI).We observed the formation of a full-length RNA resulting in the modified RNA 35RNA_A Q 7 containing seven A Q modifications and partial formation of an n + 1 product containing an additional guanosine at the 3′-end of the RNA strand as a result of nontemplated addition.Unfortunately, also some truncated products were observed indicating that the incorporation of this bulky modified nucleotide by the T7 RNA polymerase was less efficient compared to standard 7substituted 7-deaza-ATP derivatives.We also studied the absorption and emission spectra of the triphosphate 15 (A Q TP) and the oligonucleotide 35RNA_A Q 7 in water, but the fluorescence was very weak, suggesting that this nucleotide is not the best choice for fluorescent labeling of RNA (see Table S5 in the SI).

■ CONCLUSIONS
We developed the synthesis of the quinolino-fused 7deazapurine 10 with 18% yield over three steps.Nucleosides bearing methyl 14c, amino 14e, and methoxy 14f groups in position 11 on the nucleobase showed moderate cytotoxic activity against several cancer cell lines (especially CCRF-CEM with IC 50 values of 6−8 μM) and no cytotoxicity against nonmalignant fibroblasts.This makes them more potent than their naphtho-fused analogs, suggesting a certain positive effect of a nitrogen atom in the fused ring; however, they are still not potent enough for any further development.Moreover, this series of quinolino-fused nucleosides provides another evidence that the tetracyclic nucleobases are already too bulky for interaction with their biological target, most likely for efficient intracellular phosphorylation and then incorporation into DNA and/or RNA.
The amino derivative 14e was triphosphorylated to 15 (A Q TP) and used as an ATP analog in the in vitro transcription of using the T7 RNA polymerase.Unlike in case of the corresponding naphtho-fused analog, 15 A Q TP was a moderately efficient substrate for the polymerase, and we observed the formation of the corresponding full-length RNA containing seven modifications accompanied by some truncated and extended products.The moderate substrate activity and weak fluorescence do not qualify this nucleotide for a useful RNA modification and fluorescent label.

■ EXPERIMENTAL PART
Unless otherwise stated, all reactions were carried out under an argon atmosphere.An oil bath was used for reactions requiring heating.Thin-layer chromatography (TLC) was performed on Merck silica gel 60 F-254 aluminum sheets.Visualization was obtained by UV light (λ max = 254 or 366 nm).Highperformance flash chromatography (HPFC) was conducted with a Combi Flash R f instrument from Teledyne Isco Inc. using SiO 2 (particle size 0.040−0.063mm, 230−400 mesh) from Fluorochem in refillable flash columns or HP C18 Redi Sep R f gold flash columns with the solvent gradient indicated in the corresponding procedures.Preparative high-pressure liquid chromatography (prep.HPLC) was performed on a Waters 2535 Quaternary Gradient System with a fraction collector.Melting points (m.p.) were measured by Bohunka S ̌perlichovaT   Coupling constants (J) are given in Hz, and the multiplets are described as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and b (broad). 13C{ 1 H} NMR experiments were broadband proton-decoupled and were performed using APT pulse sequence.DFQ-COSY, HSQC, and HMBC experiments were used to assign the 1 H and 13 C NMR signals where required.ROESY experiments were used to confirm the relative stereochemistry of nucleosides 11 and 12.To simplify the assignment, the benzoyl group attached to the 2′-hydroxy group of the ribofuranose ring was given the letter A, the one attached to the 3′-hydroxy group is considered B, and the benzoyl group at 5′-OH is called C (Figure 3).All spectra can be found in Supporting Information S6.In the ROESY spectra for nucleosides 11, 12-β, and 12-α, the important relations for the determination of the stereochemistry are highlighted.Infrared spectra (IR) were recorded on a Bruker ALPHA FT-IR spectrometer with a single-reflection Platinum ATR module.The compounds were measured in their initial state of appearance at 20 °C, and their absorption bands were reported in wavenumbers (v) in the range between 4000 and 400 cm −1 .Intensities are described as strong (s), medium (m), and weak (w).UV/vis spectra were measured on a Varian Cary 100 Bio UV−visible spectrophotometer in the range 250−800 nm using transparent 1.5 mL quartz cuvettes.Fluorescence spectra were recorded on a Fluoromax 4 spectrofluorimeter from HORIBA Scientific.The sample concentration was adjusted to have a UV absorbance of 0.05−0.10.The excitation was performed at the absorption maximum with the highest wavelength λ abs with the slit set at 2 nm.The emission spectra were recorded from λ abs + 20 nm to 2 × λ abs − 20 nm with a 2 nm slit opening.High-resolution mass spectrometry (HR-MS) was measured by the MS-Service at IOCB Prague on an LTQ Orbitrap XL instrument from Thermo Fisher Scientific using electrospray ionization (ESI).The purity of the final nucleosides (>95%) was confirmed by UPLC-MS on an Agilent 1260 Infinity II LC system with an Agilent 1260 Photodiode Array Detector using a Kinetex EVO C 18 100 Å column (2.1 × 150 mm) from Phenomenex.Samples were dissolved in DMSO (1 μL injection volume).−16,25−28 Single-stranded DNA oligonucleotides for the preparation of the double-stranded DNA template 35DNA_A7 were purchased from Generi Biotech.The T7 Hight Yield RNA Kit, DNase I, EDTA (50 mM), and Monarch RNA purification kit (50 μg) were purchased from New England Biolabs.RNase/DNase free solutions for biochemical reactions were prepared using Milli-Q water that was treated with DEPC and sterilized by autoclaving.The precision RNA mass marker 10− 100 nt was purchased in Future Synthesis.The denaturing PAGE gel was analyzed by fluorescence (λ ex = 532 nm) using a Typhoon FLA 9500 from GE Healthcare Life Sciences.LC-ESI-MS analysis of oligonucleotides was carried out on an Agilent 1260 Infinity II LC system with an Agilent InfinityLab LS/MSD XT Detector using a BioZen C 18 100 Å column (2.1 × 150 mm) from Phenomenex with the mobile phases A (12.2 mM Et 3 N, 300 mM HFIP in water) and B (12.2 mM Et 3 N, 300 mM HFIP in 100% MeOH) and a gradient from 95:5 to 0:100 within 10 min.Deconvolutions of the LC-ESI-MS spectra were carried out using a UniDec program.
Transcription Experiment with T7 Polymerase.Four in vitro transcription reactions were performed in parallel using the HiScribe T7 High yield RNA synthesis Kit: positive control, negative control, modification, and negative control for spectroscopy.Each reaction mixture (100 μL) contained Tris buffer (40 mM, pH 7.9), the three natural NTPs (7.5 mM each), the dsDNA template 35DNA_A7 (1 μg) and the T7 RNA polymerase (7.5 μL).Additionally, the positive control contained natural ATP (7.5 mM), the negative control contained water instead of ATP or A Q TP, the modification contained A Q TP (7.5 mM), and the negative control for spectroscopy contained the modified A Q TP (7.5 mM) but no T7 polymerase.All four reaction mixtures were incubated at 37 °C for 16 h.Then, the reactions were stopped by treatment with DNase I (0.1 U/μL) at 37 °C for 15 min followed by treatment with EDTA (0.05 M) at 70 °C for 10 min.Afterward, the mixtures were purified by the Monarch RNA Cleanup Kit (50 μg) resulting in solutions of 40 μL each.
Aliquots of the first three reactions (50 ng) were separated by denaturating PAGE (20%) with urea at 23 mV for 1 h and visualized by fluorescence imaging.
Aliquots of the positive control (35RNA_A7) and the modified RNA (35RNA_A Q 7) were analyzed by UPLC-ESI-MS confirming the full transcription without any misincorporation (see Figures S1 and S2 in Supporting Information).

Figure 1 .
Figure 1.Structures and biological activity of previously synthesized substituted and fused deazapurine nucleosides and target structures of this study.

able 4 .
Sequences of Oligonucleotides and RNA Products oligonucleotide sequence role in the study

Figure 2 .
Figure 2. Denaturing PAGE analysis of the in vitro transcription reaction with T7 RNA polymerase and 35DNA_A7 template that provides seven incorporations of the modified nucleotide.Lane 1: RNA ladder, lane 2: positive control (all natural NTPs), lane 3: negative control (CTP, GTP, UTP), and lane 4: modification (A Q TP, CTP, GTP, UTP).

Figure 3 .
Figure 3. Numbering used in the assignment of NMR signals (example: key intermediate 11).

Table 2 .
24 and Fluorescence Maxima of Nucleosides 14a− g in MeOH aUV and fluorescence maxima were measured in MeOH.The used excitation wavelengths for fluorescence are in italics.Fluorescence quantum yields Φ f 's were determined using quinine sulfate in 0.1 M H 2 SO 4 as a standard (Φ f = 0.546 at 25 °C).24 a

AUTHOR INFORMATION Corresponding Author Michal
Hocek − Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Prague 2 CZ-12843, Czech Republic; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6 CZ-16610, Czech Republic; orcid.org/0000-0002-1113-2047;Email: hocek@uochb.cas.czDepartment of Organic Chemistry, Faculty of Science, Charles University in Prague, Prague 2 CZ-12843, Czech Republic; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6 CZ-16610, Czech Republic Tania Sanchez-Quirante − Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Prague 2 CZ-12843, Czech Republic; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6 CZ-16610, Czech Republic Lenka Posťová Slaveťínská − Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6 CZ-16610, Czech Republic Eva Tlousť'ová − Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6 CZ-16610, Czech Republic Michal Tichý− Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6 CZ-16610, Czech Republic Sonǎ Gurská − Institute of Molecular and Translational Medicine, Palacky University and University Hospital in Olomouc, Faculty of Medicine and Dentistry, Olomouc CZ-77515, Czech Republic Petr Dzǔbák − Institute of Molecular and Translational Medicine, Palacky University and University Hospital in Olomouc, Faculty of Medicine and Dentistry, Olomouc CZ-77515, Czech Republic; orcid.org/0000-0002-3098-5969Marián Hajdućh − Institute of Molecular and Translational Medicine, Palacky University and University Hospital in Olomouc, Faculty of Medicine and Dentistry, Olomouc CZ-77515, Czech Republic Complete contact information is available at: https://pubs.acs.org/10.1021/acsomega.4c02031 AuthorsMarianne Fleuti −