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Article Cite This: J. Med. Chem. 2018, 61, 9347−9359

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Synthesis and Cytotoxic and Antiviral Profiling of Pyrrolo- and FuroFused 7‑Deazapurine Ribonucleosides Anna Tokarenko,†,‡ Barbora Liškova,́ § Sabina Smolen,́ † Nataĺ ie Tab́ orska,́ § Michal Tichý,† Soňa Gurska,́ § Pavla Perlíkova,́ † Ivo Frydrych,§ Eva Tloušt’ova,́ † Pawel Znojek,§ Helena Mertlíkova-́ Kaiserova,́ † Lenka Poštova ́ Slaveť ínska,́ † Radek Pohl,† Blanka Klepetaŕ ǒ va,́ † Noor-Ul-Ain Khalid,∥ Yiqian Wenren,∥ Rebecca R. Laposa,∥ Petr Džubaḱ ,§,⊥ Mariań Hajdúch,*,§,⊥ and Michal Hocek*,†,‡

J. Med. Chem. 2018.61:9347-9359. Downloaded from pubs.acs.org by 193.9.158.86 on 10/28/18. For personal use only.

†

Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic ‡ Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12843 Prague 2, Czech Republic § Faculty of Medicine and Dentistry, Institute of Molecular and Translational Medicine, Palacky University and University Hospital in Olomouc, Hněvotínská 5, CZ-775 15 Olomouc, Czech Republic ∥ Department of Pharmacology and Toxicology, University of Toronto, 1 King’s College Circle, Room 4213, Toronto, Ontario M5S 1A8, Canada ⊥ Cancer Research Czech Republic, Hněvotínská 5, CZ-775 15 Olomouc, Czech Republic S Supporting Information *

ABSTRACT: Three series of isomeric pyrrolo- and furo-fused 7deazapurine ribonucleosides were synthesized and screened for cytostatic and antiviral activity. The synthesis was based on heterocyclizations of hetaryl-azidopyrimidines to form the tricyclic heterocyclic bases, followed by glycosylation and final derivatizations through cross-coupling reactions or nucleophilic substitutions. The pyrrolo[2′,3′:4,5]pyrrolo[2,3-d]pyrimidine and furo[2′,3′:4,5]pyrrolo[2,3-d]pyrimidine ribonucleosides were found to be potent cytostatics, whereas the isomeric pyrrolo[3′,2′,4,5]pyrrolo[2,3-d]pyrimidine nucleosides were inactive. The most active were the methyl, methoxy, and methylsulfanyl derivatives exerting submicromolar cytostatic effects and good selectivity toward cancer cells. We have shown that the nucleosides are activated by intracellular phosphorylation and the nucleotides get incorporated to both RNA and DNA, where they cause DNA damage. They represent a new type of promising candidates for preclinical development toward antitumor agents.

■

INTRODUCTION Modified nucleoside derivatives and analogues are known to exert a wide range of biological activities, and some of them are used in medicine as clinical antineoplastic1 or antiviral2 agents. Even in the recent years, novel nucleoside-based drugs are being developed for the treatment of drug-resistant tumors3 or as antiviral agents for unmet or new emerging viral infections (i.e., ZIKA, tick-borne encephalitis etc.).4 A particularly interesting class of base-modified nucleosides has been derived from 7-deazapurines [systematic name: pyrrolo[2,3-d]pyrimidines,5 for systematic (used in Experimental Section) and purine (used in the text for comparison with natural purine nucleosides) numbering, see Figure 1]. We have recently discovered 7-(het)aryl-7-deazapurine ribonucleosides bearing either an amino group6 (1) or other small substituents including methyl, methoxy, or methylsulfanyl groups7 (2) at position 6, showing nanomolar cytostatic effects against a © 2018 American Chemical Society

broad panel of leukemia and cancer cell lines in vitro and inhibition of human and/or Mycobacterium tuberculosis adenosine kinase (MtB ADK).8 The study of the metabolism and mechanism of action of clinical candidate compound AB61 (1i, R = 2-thienyl, Figure 1) revealed9 an intracellular phosphorylation of the nucleoside to mono-, di-, and triphosphate selectively in tumor cells, and the modified ribonucleotide was shown to get incorporated partly into RNA and partly to DNA causing inhibition of protein synthesis, formation of double-strand breaks, and apoptosis, respectively.9 Results from other laboratories independently showed that some other tubercidin-derived nucleosides were potent inhibitors of human or pathogenic ADK10 or showed antiviral properties.11 Received: August 8, 2018 Published: October 3, 2018 9347

DOI: 10.1021/acs.jmedchem.8b01258 J. Med. Chem. 2018, 61, 9347−9359

Journal of Medicinal Chemistry

Article

dines 7−10 (Scheme 1). The reaction was catalyzed by Pd(PPh3)4 and proceeded well with 2-iodo-1-methyl-1HScheme 1a

Reagents and conditions: (i) (1) (TMP)2Zn·2MgCl2·2LiCl, 0 °C, 1 h, then rt, 1 h, (2) 2-iodo-1-methyl-1H-pyrrole or 2-iodofuran, Pd(PPh3)4, THF, 65 °C, 16 h; (ii) (1) (TMP)2Zn·2MgCl2·2LiCl, 0 °C, 1 h, then rt, 1 h, (2) 3-iodo-1-methyl-1H-pyrrole, Pd2dba3, P(tBu)3·HBF4, THF, 65 °C, 16 h; (iii) NaN3, LiCl, DMF, rt, 16 h. a

Figure 1. Structures of previously prepared and newly designed deazapurine nucleosides. Systematic IUPAC numbering shown in black and the custom purine numbering in red. In compounds 4a−h and 5a−h: R = 2-furyl (a), 3-furyl (b), 2-benzofuryl (c), methyl (d), dimethylamino (e), amino (f), methoxy (g), or methylsulfanyl (h).

pyrrole and 2- or 3-iodofuran, however, in the case of 3iodo-1-methyl-1H-pyrrole, the yield was very low (44.44 0.46 0.09 0.04 0.37 0.34 16.53 7.83 >44.44 >44.44 27.34 >44.44 5.98 36.44 0.17 0.78 5.34 0.28 >44.44 0.30 0.38 0.28 1.20

>44.44 >44.44 26.91 >44.44 >44.44 40.86 >44.44 >44.44 >44.44 16.35 >44.44 27.46 >44.44 >44.44 34.62 >44.44 43.98 >44.44 >44.44 >44.44 >44.44 >44.44 >44.44 >44.44 >44.44 >44.44 >44.44

>44.44 3.45 32.5 0.12 >44.44 0.22 0.28 0.13 0.06 0.77 >44.44 15.49 >44.44 >44.44 33.86 >44.44 17.00 >44.44 >44.44 >44.44 >44.44 0.24 >44.44 0.33 0.29 0.24 0.99

>44.44 >44.44 >44.44 >44.44 >44.44 >44.44 >44.44 >44.44 >44.44 2.63 >44.44 29.06 >44.44 >44.44 >44.44 >44.44 36.83 >44.44 >44.44 >44.44 >44.44 >44.44 >44.44 >44.44 >44.44 >44.44 >44.44

a

22h showed also DNA synthesis inhibition. The compounds show similar effect on cell cycle as previously reported cytostatic pyrrolopyrimidine nucleoside 1i (AB61)6 even though the changes were less profound than those caused by 1i. Still, both pyrrolo- and furo-fused nucleosides 22 and 26 are likely to show similar mode of action as 1i does. Intracellular Phosphorylation. In analogy with the parent 7-substituted 7-deazaadenosines 2,9 we assumed that these nucleosides are first activated in cells by phosphorylation to their mono-, di-, and triphosphates. To provide experimental evidence, we studied intracellular phosphorylation of methyl nucleosides 22d and 26d (Table 6). The corresponding ribonucleoside monophosphates 22d-MP and 26d-MP (Scheme 5) were used as analytical standards. Intracellular phosphorylation was tested using prototype of normal (BJ) and malignant (HCT116) cells at 1 or 10 μmol/L concentrations of nucleosides after 1 or 3 h of treatment in vitro. The phosphorylation of both nucleosides 22d and 26d was efficient in all cases, however, unlike in previously published thienopyrrolopyrimidine nucleosides 4 and 5,13 the levels of monophosphates are higher in HCT116 cells than in BJ, what might explain the better selectivity of these nucleosides. Also, the ratio of nucleoside monophosphate versus nucleoside was increased at higher concentration of 22d and 26d (10 μM), indicating rate limited phosphorylation in nonmalignant cells. These data are in accordance with the fact that expression of adenosine kinase (ADK), the enzyme that might be involved in the phosphorylation of the fused deazapurine ribonucleosites, is increased in colorectal tumors compared with the normal tissue.30

Unlike the thienopyrrolopyrimidines 5, all pyrrolopyrrolopyrimidines 24 were completely inactive, the only exceptions are 2-benzofuryl derivative 24c showing some negligible activities against all tested cell lines and 2-furyl nucleoside 24a with single-digit micromolar activities against all cell lines including BJ and MRC fibroblasts and multidrug resistant cells (CEM-DNR and K562-TAX). These results suggest that Nmethyl group in position 7 is already too bulky to bind to the same molecular targets as the other derivatives 22 and 26, and thus pyrrolopyrimidines 24a and 24c might have different mechanism of action. Interestingly, the cytotoxic effect of all compounds was independent of p53 status as demonstrated by nearly identical activities on p53 proficient versus deficient HCT116 cells (Table 4), indicating potential antitumor activity in p53 deficient cancers. Cell-Cycle Studies. The most active nucleosides 22d,g,h and 26d,g,h were submitted for cell-cycle study in CCRF-CEM T-lymphoblastic leukemia cells. Compounds were evaluated at 1 × IC50 and 5 × IC50 concentrations, respectively, and 24 h after the treatment (Table 5). All tested nucleosides caused increase in S-phase fraction of cellular population and induced apoptosis at higher concentrations. Prolonged S-phase transition and/or block could be typically caused by interference with DNA replication and/or RNA synthesis. Indeed, inhibition of RNA synthesis was observed in all tested compounds, and nucleosides 22g and 9352



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Table 4. Cytotoxic Activity of Nucleosides 22a−i, 24a−i, and 26a−h MTS, IC50 (μM) compd

BJ

MRC-5

A549

CCRFCEM

22a 22b 22c 22d 22e 22f 22g 22h 22i 24a 24b 24c 24d 24e 24f 24g 24h 24i 26a 26b 26c 26d 26e 26f 26g 26h 1i gemcitabine

>50 >50 >50 3.91 >50 >50 4.50 5.16 1.2 5.36 >50 44.74 >50 >50 >50 >50 >50 >50 >50 0.52 >50 7.73 >50 >50 1.09 0.49 >50 >50

>50 >50 >50 50.0 >50 >50 50.0 >50 50.0 5.89 >50 45.19 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 13.42 9.37 >50 >50

>50 >50 >50 2.08 >50 >50 >50 >50 2.99 5.73 >50 35.43 >50 >50 >50 >50 >50 >50 >50 >50 49.29 3.19 >50 >50 1.93 4.86 13.14 0.05

>50 >50 16.48 0.21 >50 50.0 0.44 0.34 0.06 1.36 >50 9.48 >50 >50 >50 >50 25.46 >50 0.45 0.21 14.07 0.24 >50 >50 0.18 0.14 0.02 0.02

CEMDNR >50 >50 29.84 >50 >50 >50 >50 >50 50.0 2.17 >50 30.57 >50 >50 >50 >50 28.24 >50 >50 >50 37.12 >50 >50 >50 3.12 5.57 0.06 0.10

XTT, IC50 (μM)

HCT116

HCT116p 53−/−

>50 8.16 46.20 0.33 >50 >50 0.36 0.30 0.17 3.82 >50 36.45 >50 >50 >50 >50 35.93 >50 >50 12.13 48.80 0.45 >50 48.42 0.28 0.22 0.02 0.03

>50 9.00 47.53 0.33 >50 >50 0.62 0.38 0.15 4.01 >50 36.37 >50 >50 >50 >50 >50 >50 >50 10.72 46.45 0.45 >50 >50 0.30 0.21 0.02 0.41

K562

K562TAX

U2OS

HL60

HepG2

HeLaS3

>50 0.94 >50 0.40 >50 >50 0.51 0.39 0.18 4.03 >50 22.14 >50 >50 >50 >50 18.03 >50 7.03 0.42 39.59 0.41 >50 6.04 0.32 0.27 0.02 0.10

>50 >50 29.33 >50 >50 >50 >50 50.0 50.0 2.40 >50 22.98 >50 >50 >50 >50 29.98 >50 7.01 9.78 24.33 6.93 >50 50.0 0.30 2.70 0.04 0.05

7.37 2.41 43.89 0.61 >50 50.0 1.30 1.41 0.62 3.49 >50 28.88 >50 >50 >50 >50 27.45 >50 4.21 0.64 37.80 0.57 >50 7.09 0.43 0.34 0.51 0.18

>50 >50 >50 1.51 >50 2.29 1.44 0.58 0.06 3.00 >50 >50 >50 >50 >50 >50 >50 >50 2.55 1.92 >50 1.20 >50 0.51 0.70 0.29 0.95 nd

>50 >50 >50 1.13 >50 >50 2.59 1.53 0.16 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 6.71 2.83 2.12 >50 4.51 2.41 2.01 nd nd

>50 >50 >50 1.19 >50 >50 1.53 1.36 0.57 2.97 >50 >50 >50 >50 >50 >50 >50 >50 >50 13.14 >50 2.21 >50 1.71 2.31 2.96 0.02 nd

Table 5. Summary of Cell Cycle Analyses, Proportion of Apoptotic and Mitotic Cells (pH3Ser10 Positive), DNA and RNA Synthesis in CCRF-CEM Treated with 22d, 22g, 22h, 26d, 26g, and 26h % of total cellular populations concentration

sub G1 (apoptosis)

G0/G1

S

G2/M

pH3Ser10 (mitosis)

DNA synthesis

RNA synthesis

1 × IC50 5 × IC50

3.45 29.83 58.53

39.70 20.49 32.31

41.08 62.28 38.71

19.22 17.23 28.98

1.36 2.09 0.94

43.39 3.59 10.76

49.89 0.91 57.09

22d

1 × IC50 5 × IC50

5.10 10.99

31.75 34.66

51.03 52.19

17.22 13.14

1.06 0.90

48.49 59.89

53.62 35.91

22g

1 × IC50 5 × IC50

3.68 13.86

37.73 47.62

45.08 36.98

17.20 15.40

1.48 1.50

31.44 12.29

31.19 28.30

22h

1 × IC50 5 × IC50

5.61 12.67

26.28 40.55

56.36 42.94

17.36 16.52

0.33 1.49

30.41 7.80

21.43 3.56

26d

1 × IC50 5 × IC50

7.62 20.53

44.57 36.83

39.31 59.66

16.12 13.52

1.09 0.99

38.11 38.88

21.63 0.61

26g

1 × IC50 5 × IC50

4.23 6.82

41.52 20.54

41.82 65.39

16.65 14.07

0.56 0.58

42.65 52.74

25.36 4.30

26h

1 × IC50 5 × IC50

6.52 14.06

44.43 33.16

38.26 53.55

17.31 13.30

1.26 0.90

39.68 41.80

0.39 6.55

compd control 1i

Human Adenosine Kinase−Phosphorylation and Inhibition. Once we proved that the title nucleosides are efficiently phosphorylated in cells, we assumed that human

ADK is the most likely enzyme to perform this reaction and, therefore, we assessed the in vitro phosphorylation of the final nucleosides by human ADK (Table 7).31 9353



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but were not cytotoxic (although they showed potent antiHCV activity). This drives important conclusion that the title nucleosides themselves are inactive and that the active species are the corresponding nucleotides. The final nucleosides were also tested for their inhibition of ADK. In general, they are not potent inhibitors of ADK, only six derivatives 22a, 22c, 24a, 24c, 26a, and 26c showed some moderate inhibition (Table 7) but mostly with inverse correlation to cytotoxicity (Table 4). Incorporation of Nucleosides into Nucleic Acids of Treated Cells. Knowing that the title nucleosides are phosphorylated preferentially in cancer cells, we focused our further studies on incorporation of both methyl nucleosides 22d and 26d into DNA and RNA in living cells. CCRF-CEM cells were incubated with nucleosides 22d or 26d for 2.5 h and immediately harvested. RNA and DNA were isolated and digested, and the concentration of nucleosides in nucleic acids was determined by HPLC-MS. The results show that pyrrolofused nucleoside 22d is incorporated both into RNA and DNA as a ribonucleoside (similarly to previously reported 1i9). On the other hand, furo-fused nucleoside 26d is also incorporated to RNA as a ribonucleoside and to DNA as both ribonucleoside 26d-MP and 2′-deoxyribonucleoside 32-MP. The molar ratio of 26d-MP and 32-MP found in DNA was approximately 1:1 (Table 8), thus indirectly suggesting that the corresponding nucleoside diphosphate should be a substrate for the ribonucleotide reductase (unlike in case of 1i and 22d)

Table 6. Intracellular Phosphorylation of Nucleosides 22d and 26d dosing (1 μmol/L; 1 h; pmol/5 × 105 cells) cell line BJ HCT116 cell line BJ HCT116 cell line BJ HCT116

22d

22d-MP

26d

87.10 220.92 155.12 54.04 466.96 27.92 dosing (1 μmol/L; 3 h; pmol/5 × 105 cells) 22d

22d-MP

26d

173.96 332.63 247.21 136.87 1079.19 66.31 dosing (10 μmol/L; 1 h; pmol/5 × 105 cells) 22d

22d-MP

26d

220.71 1531.57 338.91 450.10 8868.83 119.93 dosing (10 μmol/L; 3 h; pmol/5 × 105 cells)

26d-MP 223.51 511.81 26d-MP 241.05 1167.40 26d-MP 4488.64 9531.67

cell line

22d

22d-MP

26d

26d-MP

BJ HCT116

753.01 476.53

5041.67 12049.83

464.66 177.90

4920.41 12533.33

Table 7. Phosphorylation of Nucleosides by ADK and Inhibition of ADKa compd

phosphorylation (%)

inhibition IC50 (μM)

adenosine 22a 22b 22c 22d 22e 22f 22g 22h 22i 24a 24b 24c 24d 24e 24f 24g 24h 24i 26a 26b 26c 26d 26e 26f 26g 26h

73 11 9 0 42 0 68 47 33 77 0 0 0 0 0 0 0 0 0 34 38 58 48 7 75 35 56

7.12 ± 0.68 >10 0.30 ± 0.002 >10 >10 >10 >10 >10 >10 3.58 ± 0.03 >10 0.47 ± 0.02 >10 >10 >10 >10 >10 >10 3.94 ± 0.85 >10 0.336 ± 0.027 >10 >10 >10 >10 >10

Table 8. Incorporation of Selected Cytotoxic Nucleosides into RNA and DNA in Treated CCRF-CEM TLymphoblasts compd

sample

22d 22d 26d 26d

RNA DNA RNA DNA

NMP content (fmol/μg of nucleic acid) 3.74 14.1 3.40 14.3

± ± ± ±

0.28 6.51 0.23 4.25

dNMP content (fmol/μg of nucleic acid) nda 15.4 ± 1.27

Not detected. Incorporation analyzed after 2.5-h treatment at 5 × IC50 and subsequent digestion with nuclease P1 (data from 2−4 independent experiments)

a

Induction of DNA Damage in Cells Treated with Nucleoside Analogues. Some anticancer nucleosides are known inducers of DNA damage, and we have previously reported the induction of double-strand (ds) breaks upon incorporation of 1i9 into RNA and DNA as a ribonucleoside.32 Therefore, we have studied the formation of ds DNA breaks in the U2OS osteosarcoma reporter cell line stably transfected with 53BP1-GFP fusion gene, which is known to translocate to DNA damage sites and form foci in nuclei of treated cells.9 Reporter cells were treated with 22d or 26d, and the results were compared with etoposide33 (a known ds break inducer). Figure 2 shows that the pyrrolo-fused nucleoside 22d (incorporated into nucleic acids as a ribonucleoside only) was a potent inducer of DNA damage, while the furo-fused nucleoside 26d (incorporated both as ribonucleoside and 2′deoxyribonucleoside into DNA) was substantially less potent in term of number of DNA foci per nucleus. However, compared to etoposide, both 22d and 26d induced foci were less frequent but gradually increasing over the time and at the same time much larger, thus indicating unreparable DNA damage. The data suggest that the DNA damage caused by

a Phosphorylation: 50 μM solution of compound, 243 ng of enzyme, 30 min, 37 °C. Inhibition: 50 μM solution of adenosine, 10 μM solution of compound, 97 ng of enzyme, 20 min, 37 °C.

The rate of phosphorylation correlates well with cytotoxic activities, the better phosphorylation, the higher is cytostatic activity. Moreover, all the inactive nucleosides are not phosphorylated at all, which supports the hypothesis that ADK is the enzyme responsible for their phosphorylation and thus intracellular activation. The only exceptions were amino derivatives 22f and 26f, which were efficiently phosphorylated 9354



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Table 9. Stability and PAMPA for Nucleosides 22d, 22h, 26d, and 26h compd remaining (%)

time compd (min)

chemical stability

plasma stability

microsomal stability

plasma protein binding bound fraction (%)

PAMPA log Pe

22d

0 15 30 60 120

100 98 97 93 91

100 98 83 81 79

100 99 98 97

29

−6.87

22h

0 15 30 60 120

100 100 99 98 97

100 100 99 98 98

100 99 99 97

89

−5.69

26d

0 15 30 60 120

100 99 99 99 96

100 99 99 98 96

100 100 99 98

59

−6.88

26h

0 15 30 60 120

100 99 99 99 96

100 99 99 98 96

100 100 99 98

84

−5.99

59%, respectively. All four nucleosides were shown to cross an artificial cellular membrane in the parallel artificial membrane permeability assay (PAMPA), permeability of both methyl derivatives 22d and 26d was rather low and permeability of methylsulfanyl nucleosides is almost one order of magnitude higher as determined by PAMPA (Table 9). All nucleosides were stable in human microsomal fraction (Table 9). Mitochondrial Toxicity. Because a number of nucleosides can be incorporated by human mitochondrial polymerases,34 and because mitochondrial polymerases may be important drug targets in cancer therapy,35,36 selected compounds 22h, 26d, and 26h were tested for their effect on the growth and viability of the leukemia cell line OCI-AML2 because these cells are highly reliant on mitochondrial oxidative phosphorylation (OXPHOS) for growth and viability.37 A 3-day survival experiments were conducted, and the compounds showed submicromolar IC50 values (μM): 22h, 0.16; 26d, 0.34; 26 h, 0.19, consistently with the cytotoxicity data on a broad panel of cancer cells (Table 4). We then proceeded to assess mitochondrial gene expression at 24, 48, and 72 h of treatment with these three compounds (Table S1 in Supporting Information). Two representative mitochondrial genes, MT-CO2 and MT-ND1 were selected, and cells were treated with 1 × IC50 or 5 × IC50 concentration. For 26d and 26h, mitochondrial gene expression was decreased at all time points, with maximal decreases of about 50%. Given the positive performance of 26d in other biological assays, the effect of 26d on mitochondrial protein levels (both mitochondrial DNA-encoded and nuclear DNA-encoded) was assessed further in OCI-AML2 cells (Table S2 in Supporting

Figure 2. 53BP1 foci in U2OS-53BP1-GFP cells exposed to vehicle (untreated cells), etoposide, 22d, or 26d for 11 h. (a) Representative confocal microscopy images (objective magnification 40×) showing 53-BP1 fusion protein (green) in nuclei of reporter cells. Formation of green spots (foci) in nucleus indicates for DNA damage. (b) Time dependence of 53BP1 foci formation during treatment with etoposide, 22d or 26d, respectively.

incorporation of modified ribonucleotides into DNA, and the subsequent apoptosis indeed might be at least partial mechanism of action of these cytostatic nucleosides (although other additional parallel modes of action cannot be ruled out). In Vitro Pharmacology of Nucleoside Analogues. We also studied metabolism, plasma protein binding, and permeability of nucleosides 22d, 22h, 26d, and 26h. All nucleosides were found to be stable in plasma (>95% of compound was present in plasma after 120 min, only derivative 22d showed slightly lower stability with 79% presence in plasma after 120 min) and in microsomes (Table 9). Binding to proteins is significantly higher for both methylsulfanyl derivatives 22h and 26h, 89 and 84%, respectively, while methyl derivatives 22d and 26d were bound only from 29 and 9355



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NMR signals was performed using a combination of H,H-COSY, H,H-ROESY, H,C-HSQC, and H,C-HMBC experiments. Lowresolution mass spectra were measured on LCQ Fleet using electron (EI) or electrospray ionization (ESI). High resolution mass spectra were measured on LTQ Orbitrap XL. UV spectra were measured on CARY 100 BIO UV−visible spectrophotometer in Microcell 80 μL, 4 mm × 10 mm at room temperature. Fluorescence spectra were measured on a Fluoromax 4 spectrofluorimeter. UV spectra, fluorescence measurements, and quantum yield determination were performed according to a published procedure.38 High performance flash chromatography (HPFC) was performed with ISCO Combiflash Rf system on RediSep Rf Gold Silica Gel disposable columns or reverse-phase (C18) RediSep Rf column. Purity of all final nucleosides (>95%) was determined by analytical HPLC. X-ray diffraction experiments of single crystals were carried out on an X-ray diffractometer using Cu Kα radiation (λ = 1.54180 Å). Compounds 22d,f,g and 24a−c were analyzed for palladium content using energy dispersive X-ray fluorescence (ED-XRF) method. The measurements were carried out on SPECTRO iQ II spectrometer (Spectro, Germany) with a detection limit of 5 ppm. Using this method, no traces of palladium was detected in the analyzed samples. General Procedure A: Negishi Cross-Coupling Reaction. Solution of 4,6-dichloropyrimidine (6) (3.4 g, 23 mmol) in dry THF (35 mL) was added dropwise into an ice-cooled solution of (TMP)2Zn·2MgCl2·2LiCl (0.35 M in THF/toluene 9:1, 33 mL, 11.6 mmol). The mixture was stirred at 0 °C for 1 h, then allowed to warm to rt for 1 h and added to a prestirred mixture of 2- or 3iodoheterocyclic compound (23 mmol) and Pd(PPh3)4 (2.66 g, 2.3 mmol) in dry THF (10 mL). The resulting solution was stirred at 65 °C for 16 h and then concentrated under reduced pressure. General Procedure B: Nucleophilic Substitution with NaN3. Compounds 7, 8, 9, or 10 (2 mmol), NaN3 (130 mg, 2 mmol), and LiCl (85 mg, 2 mmol) were dissolved in dry DMF (5 mL), and the resulting solution was stirred at rt for 16 h. Next, the reaction mixture was diluted with water and extracted with EtOAc. Combined organic layers were dried over anhydrous MgSO4 and concentrated under reduced pressure. General Procedure C: Stille Cross-Coupling Reaction. Protected nucleoside (0.3 mmol), tributyl(2-furyl)stannane (0.11 mL, 0.36 mmol), and PdCl2(PPh3)2 (21 mg, 0.03 mmol) were dissolved in anhydrous DMF (3 mL) and heated to 100 °C for 3 h. After cooling to rt, the mixture was filtered through a plug of silica gel with 10% of KF, washed with EtOAc, and then concentrated under reduced pressure. General Procedure D: Suzuki−Miyaura Cross-Coupling Reaction. Protected nucleoside (0.3 mmol), boronic acid (0.45 mmol), K2CO3 (83 mg, 0.6 mmol), and Pd(PPh3)4 (35 mg, 0.03 mmol) were dissolved in toluene (2 mL) and heated to 100 °C for 1− 10 h. Then the mixture was filtered through a plug of Celite and solvents were removed under reduced pressure. General Procedure E: Cross-Coupling with AlMe3. AlMe3 (2 M in toluene, 0.3 mL, 0.6 mmol) was added to a solution of a protected nucleoside (0.3 mmol) and Pd(PPh3)4 (35 mg, 0.03 mmol) in dry THF (4 mL), and the resulting mixture was stirred at 70 °C for 3−24 h. Next, dry MeOH (0.5 mL) was added, and the reaction mixture was filtered through a plug of Celite and concentrated under reduced pressure. General Procedure F: Zemplén Deprotection. MeONa (4.37 M in MeOH, 46 μL, 0.2 mmol) was added to a suspension of a protected nucleoside (0.2 mmol) in MeOH (4 mL), and the mixture was stirred at rt for 2−16 h and then concentrated under reduced pressure. The residue was coevaporated several times with MeOH. General Procedure G: Synthesis of Nucleoside Monophosphates. Free nucleoside (0.15 mmol) was dried in vacuo at 75 °C for 1 h and then suspended in trimethyl phosphate (0.58 mL). The suspension was cooled to 0 °C, and, subsequently, POCl3 (21 μL, 0.23 mmol) was added. The mixture was stirred at 0 °C for 2−20 h, and an aqueous solution of TEAB (2 M, 2.5 mL, 5 mmol) was added, and the solvents were removed under reduced pressure. The residue was coevaporated several times with water, then subjected to a

Information). Nuclear DNA-encoded mitochondrial proteins ATP5A and UQCRC2 were unaffected by 26d at these time points, while mitochondrial DNA-encoded protein CO2 and nuclear DNA-encoded protein SDHB were decreased at all time points. The mitochondrial protein results are consistent with the interpretation that 26d decreases mitochondrial gene expression. Considering the fact that the synthesis of cellular RNA is significantly depleted in the treated cells and large portion of cells is already in apoptosis or dead after 24 h (Table 5), the observed mitochondrial toxicity may make a minor contribution to cell death induced by these compounds.

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CONCLUSIONS We have synthesized three series of novel isomeric pyrroloand furo-fused 7-deazapurine ribonucleosides and discovered their significant cytostatic effect. The synthesis was based on heterocyclizations of hetaryl-azidopyrimidines to form the tricyclic heterocyclic bases, followed by their glycosylation and final derivatizations through cross-coupling reactions or nucleophilic substitutions at position 6 and deprotection of the sugar part. Biological activity screening revealed that the pyrrolo[2′,3′:4,5]pyrrolo[2,3-d]pyrimidine and furo[2′,3′:4,5]pyrrolo[2,3-d]pyrimidine nucleosides 22 and 26 were potent cytostatics, whereas the isomeric pyrrolo[3′,2’:4,5]pyrrolo[2,3d]pyrimidine nucleosides 24 were inactive, probably due to steric hindrance of the N-methyl group which affects the syn− anti conformational equilibrium of the nucleosides. In 22 and 26, the most active were the methyl (22d, 26d), methoxy (22g, 26g), and methylsulfanyl (22h, 26h) derivatives exerting submicromolar cytostatic effects and good selectivity toward cancer cells. We have shown that the nucleosides are activated by intracellular phosphorylation and the nucleotides get incorporated to DNA and RNA. At least in compound 22d, the incorporation of ribonucleoside to DNA causes significant formation of double-strand breaks and apoptosis. All nucleosides showed good stability in plasma. Therefore, these compounds are a novel class of potent cytostatics and several of them are good candidates for further preclinical development which is currently under way in our laboratories.

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EXPERIMENTAL SECTION

General Remarks. All reactions were carried out under argon atmosphere. Reactions with organometallic reagents as well as all palladium catalyzed reactions were done in flame-dried glassware. All reagents and solvents were purchased from commercial suppliers and used as received. Photochemical cyclizations were carried out using 4W germicidal ultraviolet GTL3 bulb model EUV-13B. Reactions were monitored by thin layer chromatography (TLC) on TLC Silica Gel 60 F254 (Merck) and detected by UV (254 nm) and by solution of 4-anisaldehyde in ethanol with 10% of sulfuric acid. Melting points were determined using Stuart SMP40 automatic melting point apparatus and are uncorrected. Optical rotations were measured in −1 deg· DMSO on Autopol IV polarimeter, [α]20 D values are given in 10 2 −1 cm ·g . IR spectra were recorded on FT-IR spectrometer using attenuated total reflection (ATR), wavenumbers are given in cm−1. NMR spectra were recorded on ae 400 MHz (1H at 400.0 MHz and 13 C at 100.6 MHz), a 500 MHz (1H at 500.0 MHz, 13C at 125.7 MHz and 31P at 202.3 MHz), or a 600 MHz (1H at 600.1 MHz and 13C at 150.9 MHz) spectrometers in DMSO-d6, CDCl3 (referenced to the residual solvent signal), or in D2O (referenced to the signal of 1,4dioxane as an external standard in 1 mm coaxial capillary; δ (1H) = 3.75 ppm, δ (13C) = 69.3 ppm). 31P NMR spectra were referenced externally to the signal of H3PO4. Chemical shifts are given in ppm (δ scale) and coupling constants (J) in Hz. Complete assignment of all 9356



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purification on DEAE-Sephadex column (0 to 1.2 M aq TEAB) and to ion exchange on Dowex 50 (Na+ form). Complete detailed synthetic procedures and characterization of all compounds is given in the Supporting Information. Methods and experiments used for the biological profiling were performed in analogy to previous papers,6−9,34−38 and detailed procedures are given in the Supporting Information.

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Technology Agency of the Czech Republic (TE01020028 to S.S., M.T., and M. Hajduch) by Gilead Sciences, Inc., and by the Czech Ministry of Education, Youth, and Sports (LO1304, LM2015064 to P.D. and M. Hajdúch). The authors thank Drs. Joy Feng and Gina Bahador (Gilead Sciences, Inc.) for antiHCV testing and Dr. Iva Pichová (IOCB) for cloning and expression of ADK.

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ASSOCIATED CONTENT

S Supporting Information *

ABBREVIATIONS USED ADK, adenosine kinase; BSA, N,O-bis(trimethylsilyl)acetamide; HCV, hepatitis C virus; PAMPA, parallel artificial membrane permeability assay; RSV, respiratory syncytial virus; TEAB, triethylammonium bicarbonate; TMP, 2,2,6,6-tetramethylpiperidinyl.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b01258. Additional data on mitochondrial toxicity, experimental procedures for synthesis and characterization of all compounds, HPLC purity of final unprotected nucleosides, azide-tetrazole equilibrium study, ORTEP drawings and crystal data for X-ray crystal structures of 12t and 15, methods and procedures for biological profiling (PDF) Copies of NMR spectra of the products (PDF) Crystal structure of 12t 7-chloro-8-(1-methyl-1H-pyrrol3-yl)tetrazolo[15-c]pyrimidine (CIF) Crystal structure of 15 4-chloro-5-methyl-7,8dihydropyrrolo[2',3':4,5]pyrrolo[2,3-d]pyrimidine (CIF) Molecular formula strings (CSV)

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REFERENCES

(1) (a) Jordheim, L.; Galmarini, C. M.; Dumontet, C. Drug Resistance to Cytotoxic Nucleoside Analogues. Curr. Drug Targets 2003, 4, 443−460. (b) Jordheim, L. P.; Galmarini, C. M.; Dumontet, C. Recent Developments to Improve the Efficacy of Cytotoxic Nucleoside Analogues. Recent Pat. Anti-Cancer Drug Discovery 2006, 1, 163−170. (c) Parker, W. B. Enzymology of Purine and Pyrimidine Antimetabolites Used in the Treatment of Cancer. Chem. Rev. 2009, 109, 2880−2893. (d) Jordheim, L. P.; Durantel, D.; Zoulim, F.; Dumontet, C. Advances in the Development of Nucleoside and Nucleotide Analogues for Cancer and Viral Diseases. Nat. Rev. Drug Discovery 2013, 12, 447−464. (2) (a) De Clercq, E.; Li, G. Approved Antiviral Drugs over the Past 50 Years. Clin. Microbiol. Rev. 2016, 29, 695−747. (b) Seley-Radtke, K. L.; Yates, M. K. The Evolution of Nucleoside Analogue Antivirals: A Review for Chemists and Non-chemists. Part 1: Early Structural Modifications to the Nucleoside Scaffold. Antiviral Res. 2018, 154, 66−86. (3) Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.; Gottesman, M. M. Targeting Multidrug Resistance in Cancer. Nat. Rev. Drug Discovery 2006, 5, 219−234. (4) (a) Bhakat, S.; Karubiu, W.; Jayaprakash, V.; Soliman, M. E. S. A Perspective on Targeting Non-structural Proteins to Combat Neglected Tropical Diseases: Dengue, West Nile and Chikungunya Viruses. Eur. J. Med. Chem. 2014, 87, 677−702. (b) Eyer, L.; Nencka, R.; de Clercq, E.; Seley-Radtke, K.; Ruzek, D. Nucleoside Analogs as a Rich Source of Antiviral Agents Active against Arthropod-Borne Flaviviruses. Antivir. Chem. Chemother. 2018, 26, 1−28. (c) Eyer, L.; Nencka, R.; Huvarová, I.; Palus, M.; Joao Alves, M.; Gould, E. A.; De Clercq, E.; Růzě k, D. Nucleoside Inhibitors of Zika Virus. J. Infect. Dis. 2016, 214, 707−711. (d) Eyer, L.; Š mídková, M.; Nencka, R.; Neča, J.; Kastl, T.; Palus, M.; De Clercq, E.; Růzě k, D. Structure-activity Relationships of Nucleoside Analogues for Inhibition of Tick-borne Encephalitis Virus. Antiviral Res. 2016, 133, 119−129. (5) Reviews: (a) De Coen, L. M.; Heugebaert, T. S.; García, D.; Stevens, C. V. Synthetic Entries to and Biological Activity of Pyrrolopyrimidines. Chem. Rev. 2016, 116, 80−139. (b) Perlíková, P.; Hocek, M. Pyrrolo[2,3-d]pyrimidine (7-Deazapurine) as a Privileged Scaffold in Design of Antitumor and Antiviral nucleosides. Med. Res. Rev. 2017, 37, 1429−1460. (6) Bourderioux, A.; Nauš, P.; Perlíková, P.; Pohl, R.; Pichová, I.; Votruba, I.; Džubák, P.; Konečný, P.; Hajdúch, M.; Stray, K. M.; Wang, T.; Ray, A. S.; Feng, J. Y.; Birkus, G.; Cihlar, T.; Hocek, M. Synthesis and Significant Cytostatic Activity of 7-hetaryl-7-deazaadenosines. J. Med. Chem. 2011, 54, 5498−5507. (7) Nauš, P.; Caletková, O.; Konečný, P.; Džubák, P.; Bogdanová, K.; Kolár,̌ M.; Vrbková, J.; Slavětínská, L.; Tloušt’ová, E.; Perlíková, P.; Hajdúch, M.; Hocek, M. Synthesis, Cytostatic, Antimicrobial, and anti-HCV Activity of 6-Substituted 7-(Het)aryl-7-deazapurine Ribonucleosides. J. Med. Chem. 2014, 57, 1097−1110. (8) Snásě l, J.; Nauš, P.; Dostál, J.; Hnízda, A.; Fanfrlík, J.; Brynda, J.; Bourderioux, A.; Dušek, M.; Dvořaḱ ová, H.; Stolaříková, J.;

AUTHOR INFORMATION

Corresponding Authors

*For Michal Hocek: phone, +420 220183324; E-mail, hocek@ uochb.cas.cz. *For Marián Hajdúch: phone, +420 585632082; E-mail, [email protected]. ORCID

Anna Tokarenko: 0000-0003-0085-026X Michal Tichý: 0000-0002-8460-7680 Ivo Frydrych: 0000-0003-1412-1607 Michal Hocek: 0000-0002-1113-2047 Author Contributions

A.T. and B.L. contributed equally. M. Hocek designed the compounds and supervised the whole project. A.T., S.S., and M.T. contributed to the design of the compounds and performed the synthesis. P.P. performed study of incorporation to nucleic acids. L.P.S. and R.P. performed NMR analysis of all compounds, and B.K. performed X-ray crystallography. B.L., N.T., S.G., I.F., P.Z., E.T., H.M.K., and P.D. performed the biological profiling which was supervised by M. Hajdúch. N.K., Y W,. and R R L. studied the mitochondrial toxicity. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare the following competing financial interest(s): A.T., S.S., P.D., M. Hajduch, and M. Hocek are inventors of a patent application covering the title compounds.

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ACKNOWLEDGMENTS This work was supported by the Academy of Sciences of the Czech Republic (RVO 61388963 and the Praemium Academiae award to M. Hocek), by the Czech Science Foundation (16-001785 to A.T. and M. Hocek), by the 9357



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