Synthesis and Cytostatic and Antiviral Profiling of Thieno-Fused 7

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Synthesis and Cytostatic and Antiviral Profiling of Thieno-fused 7-Deazapurine Ribonucleosides Michal Tichý, Sabina Smole#, Eva Tlouš#ová, Radek Pohl, Tomáš Oždian, Klára Hejtmánková, Barbora Lišková, So#a Gurská, Petr Džubák, Marián Hajdúch, and Michal Hocek J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01766 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 21, 2017

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Journal of Medicinal Chemistry

Synthesis and Cytostatic and Antiviral Profiling of Thieno-fused 7-Deazapurine Ribonucleosides Michal Tichý,a Sabina Smoleń,a Eva Tloušťová,a Radek Pohl,a Tomáš Oždian,b Klára Hejtmánková,b Barbora Lišková,b Soňa Gurská,b Petr Džubák,b Marián Hajdúch,b Michal Hocek*a,c a

Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Gilead

Sciences & IOCB Research Center, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic b

Institute 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 c

Department of Organic Chemistry, Faculty of Science, Charles University in Prague,

Hlavova 8, CZ-12843 Prague 2, Czech Republic. Abstract: Two isomeric series of new thieno-fused 7-deazapurine ribonucleosides (derived from 4-substituted thieno[2',3':4,5]pyrrolo[2,3-d]pyrimidines and thieno[3',2':4,5]pyrrolo[2,3d]pyrimidines) were synthesized by a sequence involving Negishi coupling of 4,6dichloropyrimidine

with

iodothiophenes,

nucleophilic

azidation,

cyclization

of

tetrazolopyrimidines, followed by glycosylation and cross-couplings or nucleophilic substitutions at position 4. Most nucleosides (from both isomeric series) exerted low micromolar or submicromolar in vitro cytostatic activities against a broad panel of cancer and leukemia cell lines and some antiviral activity against HCV. The most active were the 6methoxy, 6-methylsulfanyl and 6-methyl derivatives, which were highly active to cancer cells and less toxic or non-toxic to fibroblasts.

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Introduction Many modified purine nucleosides and their analogs display diverse biological activities and some are even used as clinical antineoplastic1 or antiviral2 drugs. However, despite extensive systematic and comprehensive study of this class of compounds over the last decades, there is still potential for development of new nucleoside anticancer drugs for the treatment of resistant tumors,3 or antiviral agents against new emerging viruses.4 Our systematic study of nucleosides derived from 7-deazapurines (systematic name: pyrrolo[2,3d]pyrimidines,5 for systematic and purine numbering, see Figure 1) resulted in the discovery of several new classes of nucleoside cytostatics and antivirals. The first type was based on 6hetaryl-7-deazapurine ribonucleosides 1 (bearing H or F at position 7), which showed nanomolar cytostatic effects6 against a broad panel of leukemia and cancer cell lines in vitro and inhibition of human and Mycobacterium tuberculosis adenosine kinase (MtB ADK).7 The second class comprised 7-hetaryl substituted 7-deazaadenosines 2. Derivatives bearing small five-membered heterocycles displayed nanomolar cytostatic effects,8 whereas compounds bearing bulkier aryl groups were found to be inhibitors of MtB ADK.9 Later on, the synthesis and profiling of an extended series of 6-substituted 7-hetaryl-7-deazapurine nucleosides 3 revealed10 that also 6-MeO-, 6-MeS and 6-methyl derivatives exhibited cytostatic activity at nanomolar concentrations. Detailed study of the mechanism of action of the most active derivative from that class, compound AB-61 (2i, R = 2-thienyl, Figure 1), showed11 that the nucleoside is selectively phosphorylated to monophosphate (and then to triphosphate) only in proliferating cancer cells but not in non-malignant cells, and the modified ribonucleotide is then incorporated into RNA (where it causes inhibition of proteosynthesis) and to DNA (where it causes double-strand breaks).11 Moreover, some other related derivatives and analogues of tubercidin displayed antiviral activities12 or inhibition of ADK.13


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These results showed that there is a space for some modification at this „majorgroove“ part of the 7-deazapurine ribonucleoside and led us to design of new benzo-fused 7deazapurine (pyrimidoindole) nucleosides, where the (het)aryl group at position 7 of the 7deazapurine moiety is replaced by an aromaric ring fused to the deazapurine. The first generation of pyrimidoindole ribonucleosides 4 bearing 2-furyl or 2-benzofuryl groups in position 4 at pyrimidine ring displayed14 sub-micromolar antiviral activity against Dengue virus, unfortunately accompanied by some cytotoxicity. The corresponding benzo-fused 7deazaadenosine analogues 5 exerted15 sub-micromolar activities against HCV and Dengue viruses, but low cytotoxicity. In order to verify the effect of the size of the fused ring, we designed and report here the synthesis and biological profiling of two isomeric type of thienofused 7-deazapurine (thienopyrrolopyrimidine) nucleosides.



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Figure 1 Previously reported 7-deazapurine nucleoside cytostatics and antivirals 1-5 and the design of thieno-fused analogues. Custom purine numbering (red) is shown in structure 2, systematic numbering of pyrrolo[2,3-d]pyrimidines (black) is shown in structure 1.

RESULTS AND DISCUSSION Chemistry. There are three conceptual approaches to synthesis of pyrimidoindole heterocycle in the literature: (i) build-up of the pyrimidine part starting from 1-chloro-2nitrobenzene,16 (ii) intramolecular C-H arylation of 4-phenylamino-4-iodopyrimidines17 and (iii) thermal of photochemical cyclization from 5-aryl-6-azidopyrimidines.18 For the synthesis of the target thienopyrrolopyrimidines, we have chosen the latter approach both because of availability of starting compounds and because it was reported to successfully produce the only literature example of this rare tricyclic heterocycle.18 Our synthetic plan towards thienofused deazapurines was based on three-step procedure – attachment of thiophene to pyrimidine ring at position 5, introduction of an azido group into position 4 and finally thermal or photocyclization to form thienopyrrolopyrimidine. We decided to link the pyrimidine and thiophene rings through the Negishi crosscoupling.19 4,6-Dichloropyrimidine (6) was first zincated into position 5 using (TMP)2ZnˑMgCl2ˑLiCl (TMP = 2,2,6,6-tetra-methylpiperidyl) following literature procedure20 and was then subjected to Pd(PPh3)4 catalyzed cross-coupling reaction with either 2-iodo- or 3-iodothiophene21 to furnish desired 4,6-dichloro-5-(2- or 3-thienyl)pyrimidines 7 or 9 (Scheme 1). Yields of these two-step one-pot reactions varied from 40 to 80% and heavily depended on the efficiency of the zincation step, whereas the coupling reaction was nearly quantitative. After optimization, an 8 gram-scale reaction gave excellent 70% yield of product 9. The next step was nucleophilic substitution with sodium azide in THF, which proceeded


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smoothly to give tetrazolopyrimidines 8 and 10, cyclic forms of 5-thienyl-6-azidopyrimidines, in excellent 90% yields (Scheme 1).

Cl i)

N N 6

Cl

Cl

Cl

R

N

ii)

N

R

N N N N 7, R = 2-thienyl, 60-80% 8, R = 2-thienyl, 90% 9, R = 3-thienyl, 70-80% 10, R = 3-thienyl, 90% N

Cl

Scheme 1 Reagents and conditions: i) 1. (TMP)2ZnˑMgCl2ˑLiCl, 0 oC, 60 min, then r.t. for 45 min; 2. 2-iodothiophene or 3-iodophiophene, Pd(PPh3)4 (0.1 equiv.), THF, 65 oC, 12 h; ii) NaN3 (1 equiv.), LiCl (1 equiv.), THF, r.t., 2 days.

The next step was the cyclization of the tetrazoles to thienopyrrolopyrimidines. In the original paper, it was described18 either under photochemical or thermal conditions. Attempted photocyclization of 8 in TFA took several days and the product was accompanied by some decomposition side-products. We found, that even traces of DMF in the reaction mixture can cause formation of side-products. Therefore, we used THF in previous nucleophilic substitution step. Finally, we employed a thermal cyclization of 8 in 1,4-dibromobenzene at 180 °C for 30 min to obtain the desired 4-chloro-8H-thieno[2',3':4,5]pyrrolo[2,3-d]pyrimidine (11) in 50% isolated yield (Scheme 2).



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Scheme 2 Cyclizations of tetrazolopyrimidines 8 and 10 Isomeric 3-thienyl-substituted tetrazolopyrimidine 10 was first cyclized in 1,4dibromobenzene at 180 °C in the same manner as 8. Although there are two possible products of cyclization, only isomer 12 was isolated in low 15% yield accompanied by products of degradation of starting material. Since changing solvent to dichlorobenzene or naphthalene did not help, we tried the photochemical cyclization. Photocyclization of tetrazole 10 was performed in TFA under irradiation by 4W 254 nm UV lamp. The reaction was rather slow and took 3 days to reach full conversion. However, the reaction was relatively clean and provided both possible isomers 12 and 13 in ratio 2:1 (Scheme 2). Their separation by column chromatography was difficult but feasible. However, there were always some mixed fractions containing both isomers, which affects the total isolated yield. After optimization, the desired 4-chloro-8H-thieno[3',2':4,5]pyrrolo[2,3-d]pyrimidine (12) was prepared in multigram scale in isolated yield of 56% and we were also able to get the other isomer 13 in pure form in 18%. Thienopyrrolopyrimidine bases 11 and 12 were then subjected to classical Vorbrüggen glycosylation according to published procedure previously used for pyrimidoindoles,14,15 but no nucleosides were obtained. So we tried to first heat the tricyclic bases 11 or 12 with (N,Obis(trimethylsilyl)acetamide (BSA) and then add trimethylsilyltriflate and 1-O-acetyl-2,3,4tri-O-benzoyl-β-D-ribofuranose. This procedure proceeded smoothly and afforded the desired protected nucleoside intermediates 14 and 15 in 62 and 40% yields as pure β-anomers (Scheme 3). The anomeric configuration of protected nucleoside 14 and 15 was unambiguously determined by H,H-ROESY experiment. In both cases, a strong NOE contact between H-1' and H-4' was observed confirming the β configuration.


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Cl

Cl

S

i)

N

N N

N

N H

N

S

O BzO

11

BzO OBz 14, 62 % Cl

Cl N

S

i)

N

12

N

N

N H

N

S O

BzO BzO

OBz

15, 40 %

Scheme 3. Reagents and conditions: i) 1. BSA (1 equiv.), MeCN, 60 oC, 30 min; 2. 1-Oacetyl-2,3,4-tri-O-benzoyl-β-D-ribofuranose (2 equiv.), TMSOTf (2 equiv.), 60 °C, 8 h.

The 4-chloro-thienopyrrolopyrimidine nucleosides 14 and 15 were then used as key intermediates in the synthesis of series of derivatives bearing diverse substituents at position 4 of the pyrimidine ring (corresponding to position 6 of purines). The selection of substituents was based on previous experience with 7-deazapurine nucleosides.6-11,14,15 They comprised smaller and bulkier heterocycles, amino, dimethylamino, methoxy, methylsulfanyl and methyl groups. 2-Furyl derivatives 16a and 18a were prepared from 4-chloro-thienopyrrolopyrimidine nucleosides 14 or 15 by the Stille cross-coupling with 2-furylSnBu3 catalyzed by PdCl2(PPh3)2 in DMF (Scheme 4, Table 1). The 3-furyl and 2-benzofuryl substituted nucleosides 16b, 16c, 18b and 18c were synthesized by the Suzuki-Miyaura cross-coupling with the corresponding hetarylboronic acid in toluene using Pd(PPh3)4 as a catalyst in presence of potassium carbonate as a base. The 4-methyl derivative 16d was obtained by palladium-catalyzed alkylation of 14 with trimethylaluminium. The isomeric methyl



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derivative 18d was prepared analogously but was not isolated as a pure compound and was directly deprotected. Dimethylamino derivatives 16e and 18e were synthesized by nucleophilic substitution with dimethylamine. Standard Zemplén deprotection of benzoylated nucleosides 16a-e and 18a-e in presence of NaOMe in MeOH furnished desired unprotected nucleosides 17a-e and 19a-e in good yields. Amino, methoxy and methylsulfanyl groups were introduced into position 4 of chloronucleoside 14 or 15 by nucleophilic substitution with aqueous ammonia in 1,4-dioxane, sodium methoxide or sodium thiomethoxide in methanol, respectively. In all instances, the ribose moiety was simultaneously deprotected under reaction conditions and the desired target unprotected nucleosides 17f-h and 19f-h were obtained in good yields (Scheme 4, Table 1).

Scheme 4 Reagents and conditions: i) 2-tributylstannylfuran (1.2 equiv.), PdCl2(PPh3)2 (0.1 equiv.), DMF, 100 oC, 8 h; ii) R-boronic acid (1.5 equiv.), Pd(PPh3)4 (0.05 equiv.), K2CO3 (2


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equiv.), toluene, 100 oC, 8 h; iii) Me3Al (2 equiv.), Pd(PPh3)4 (0.05 equiv.), THF, r.t., 12 h; iv) Me2NH in THF (2 equiv.), propan-2-ol, r.t., 24 h; v) NH3 (aq.), dioxane, 120 oC, 12 h; vi) MeONa (1.3 equiv.), MeOH, r.t., 12 h; vii) MeSNa (1.3 equiv.), MeOH, r.t., 12 h; viii) 1M MeONa in MeOH (0.3 equiv.), MeOH, r.t., 24 h.

Table 1 Synthesis of 4-substituted thienopyrrolopyrimidine nucleosides 16, 17, 18, 19 Entry Reaction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

i) ii) ii) iii) iv) v) vi) vii) i) ii) ii) iii) iv) v) vi) vii)

Protected Yield nucleoside [%] 2-furyl 84 16a 3-furyl 82 16b 2-benzofuryl 87 16c Me 87 16d Me2N 78 16e NH2 MeO MeS 2-furyl 67 18a 3-furyl 82 18b 2-benzofuryl 83 18c Me 18d Me2N 85 18e NH2 MeO MeS R

Final nucleoside 17a 17b 17c 17d 17e 17f 17g 17h 19a 19b 19c 19d 19e 19f 19g 19h

Yield [%] 62 70 68 82 49 75 78 64 68 83 86 70 88 78 65 90

Nucleosides 17h and 19h were converted to their 5'-O-mono-phosphates 17h-MP and 19h-MP following a standard procedure22 using POCl3, followed by treatment with aqueous triethylammonium bicarbonate (TEAB) and ion exchange (Scheme 5).



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SMe

SMe

S i)

N

O NaO P O HO

HO OH

O HO

17h

OH

17h-MP

SMe

SMe

N

S

N

i)

N

N

N

N

O HO

S

N

N

N

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HO OH 19h

N O NaO P O HO

O HO

S N O

HO

OH

19h-MP

Scheme 5 Reagents and conditions: i) 1. P(O)(OMe)3 (100 equiv.), POCl3 (4 equiv.), 0 °C, 3 h; 2. aqueous TEAB (2 M); 3. ion exchange on Dowex 50 (Na+ form).

Fluorescence properties of thienopyrrolopyrimidine nucleosides Related pyrimidoindole nucleosides were previously reported23 to exert strong fluorescence and were used as building block for the synthesis of fluorescent DNA probes. Therefore, we have investigated electronic spectra and photophysical properties of the title novel thienopyrrolopyrimidine nucleosides 17 and 19 (Table 2).

Most of those compounds

exhibited at least some fluorescence (except of 4-amino derivative 17f). The brightest fluorescence was found in nucleosides bearing furyl and benzofuryl groups. Whilst quantum yields of 3-furyl derivatives 17b and 19b are still moderate (~ 0.1), 2-furyl 17a and 19a derivatives exhibited stronger fluorescence with quantum yields 0.19 and 0.30, respectively. The benzofuryl derivative 19c was the most brightly fluorescent compound with an excellent quantum yield 0.79. Most compounds were excitable at >300 nm so they might have the


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potential for fluorescent labelling of nucleic acids or for fluorescent detection of their metabolites. Table 2 UV and fluorescence spectra of thieno-fused deazapurine nucleosides Absorption Emission ε ε λmax [nm] λmax [nm] λmax [nm] φ λexc.[nm] [L·mol–1·cm–1] [L·mol–1·cm–1] 253 4600 445 0.19 340 17a 322 11500 433 0.13 340 17b 309 13500 271 26700 465 0.28 360 17c 368 17100 250 32300 301 12300 396 0.12 320 17d 314 13800 232 31100 358 0.07 300 17e 327 11600 220 27500 300 10500 17f 229 21300 291 14400 368 0.15 300 17g 241 30800 313 11600 505 0.03 340 17h 319 15600 484 0.30 320 19a 253 27400 469 0.07 300 19b 260 18800 333 16800 437 0.79 320 19c 246 37000 434 0.10 290 19d 220 32800 285 11500 420 0.03 300 19e 242 20900 277 6400 382 0.03 300 19f 237 58300 408 0.14 300 19g 262 25400 232 39300 467 0.02 290 19h 292 14300 -5 UV spectra were measured in methanol at 25 °C. All 5×10 M solutions. Fluorescence quantum yield in MeOH Compd

measured using quinine sulfate in 0.5 M H2SO4 (Φ f = 0.55) as a reference.

Biological activity profiling All the title nucleosides 17a-h and 19a-h were subjected for cytostatic, antiviral and antimicrobial activities screening. Antiviral activity. The antiviral screening was performed against hepatitis C virus (HCV), respiratory syncytial virus (RSV), Dengue, influenza, coxsackie and herpes simplex viruses. Most compounds were inactive in these assays. Only benzofuryl derivatives 17c and 19c, methoxy 17g and 19g, and methylsulfanyl derivatives 17h and 19h displayed low micromolar or sub-micromolar activity against HCV in the replicon assay in the Huh-7 cells. The most active were the methyl derivatives 17d and 19d showing submicromolar anti-HCV effects (Table 3) which are comparable to standard anti-HCV nucleoside Mericitabine.24



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Table 3 Anti-HCV activities of nucleosides Compd 17c 17d 17f 17g 17h 19c 19d 19f 19g 19h Mericitabine

HCV replicon 1B EC50 (µM) CC50 (µM) 2.1 19.59 0.11 >44.44 5.02 17.17 0.95 29.24 0.47 >44.44 2.05 >44.44 0.06 >44.44 17.52 >44.44 0.23 >44.44 0.13 >44.44 1.2 >44.44

HCV replicon 2A EC50 (µM) CC50 (µM) 13.92 26.55 0.06 >44.44 13.48 16.39 15.00 13.91 0.34 >44.44 14.09 >44.44 0.24 >44.44 >44.44 >44.44 0.40 >44.44 0.80 >44.44 0.99 >44.44

Cytotoxic/cytostatic activity. In vitro cytotoxic/cytostatic activity of final nucleosides 17 and 19 was initially evaluated against six cell lines derived from human solid tumors including lung (A549 cells) and colon (HCT116 and HCT116p53−/−) carcinomas, as well as leukemia cell lines (CCRF-CEM, CEM-DNR, K562, and K562-TAX) and, for comparison, nonmalignant BJ and MRC-5 fibroblasts. Concentrations inhibiting the cell growth by 50% (IC50) were determined using a quantitative metabolic staining with 3-(4,5-dimethylthiazol-2yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)25 following a 3-day treatment. In addition, the anti-proliferative effect was tested against a promyelocytic leukemia HL-60, hepatocellular carcinoma HepG2 and cervical carcinoma HeLa S3. Cell viability was determined following a 3-day incubation using 2,3-bis-(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay.26 The data were compared with previously reported 7-deazapurine nucleoside 2i8 and with clinically used cytostatic gemcitabine.27 All four derivatives bearing 2- or 3-furyl group in position 4 17a, 17b, 19a and 19b, as well as dimethylamino derivatives 17e and 19e were inactive. Benzofuryl 17c and 19c and amino


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17f and 19f derivatives showed moderate activity against all cell lines including fibroblasts, suggesting a non-specific cytotoxic effect. On the other hand, nucleosides of both series bearing methyl, methoxy and methylsulfanyl groups in position 4 (17d, 17g, 17h, 19d, 19g and 19h) showed sub-micromolar or even nanomolar activity against acute leukemia and colorectal cancer cell lines with no toxicity to MRC-5 and low toxicity to BJ, showing promising therapeutic index under in vitro conditions (Table 4). Interestingly, these nucleosides are showing low or no activity against multidrug resistant cancer cell lines (CEMDNR and K562-TAX, REF https://www.ncbi.nlm.nih.gov/pubmed/12584592) indicating that an active transport is involved in their mechanism of action. When compared to previously reported 7-thienyl-7-deazaadenosine (2i), the cytostatic activity profiles of the current compounds are different. The amino-derivatives 17f and 19f, which are direct fused analogues of 2i, were much less active than the parent 2i, but the 4-Me, MeO and MeS-derivatives 17d,g,h and 19d,g,h exerted submicromolar activities (in some cases even comparable to 2i8 or gemcitabine27). Interesting is also the comparison of these highly active thieno-fused deazapurine nucleosides 17d,g,h and 19d,g,h with the corresponding more bulky benzo-fused derivatives 4 and 5, which did not show significant cytotoxicities (but were more selective antivirals).14,15 It seems that the five-membered ring fused to the deazapurine moiety might be still tolerated by some enzymes activating these nucleosides, whereas the benzene ring is already too bulky.



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Table 4 Cytotoxic/cytostatic activity of nucleosides 13a-h, 19a-h and 13h-MP, 19h-MP MTS, IC50 (µM) Compd. >50

CCRFCEM >50

CEMDNR >50

>50

>50

3.61

22.97

32.63

21.16

17d

0.15

1.66

17e

>50

17f

XTT, IC50 (µM)

HCT116

HCT116p53-/-

K562

>50

>50

>50

K562TAX >50

>50

>50

>50

>50

6.86

14.38

16.17

12.35

0.21

0.02

>50

0.05

>50

>50

47.62

>50

16.78

30.29

11.90

6.22

17g

0.40

25.17

0.22

17h

1.25

9.01

19a

>50

19b

BJ

MRC-5

A549

U2OS

HL60

HepG2

HeLaS3

17a

>50

>50

>50

>50

>50

>50

17b

32

>50

>50

>50

>50

>50

17c

15.37

17.88

18.50

2.38

4.28

24.3

0.07

0.14

0.72

0.20

0.21

0.44

0.72

>50

>50

>50

>50

>50

>50

>50

>50

15.20

5.39

4.37

6.16

4.28

2.50

0.31

0.56

1.54

0.13

61.75

0.13

0.13

0.20

4.05

0.46

0.11

0.15

0.90

0.66

0.027

1.46

0.05

0.09

0.35

2.2

0.46

0.51

0.36

0.72

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

19c

36.77

37.53

>50

15.90

20.57

47.12

>50

29.68

14.91

33.11

>50

>50

>50

19d

>50

>50

0.72

0.03

33.88

0.40

0.33

0.46

47.58

0.56

0.37

0.47

0.57

19e

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

19f

1.09

>50

30.11

0.30

41.20

46.66

10.88

36.28

0.82

0.51

0.95

1.46

>50

19g

7.49

>50

1.23

0.22

>50

0.61

0.64

1.06

11.14

0.93

0.57

1.33

0.23

19h

6.90

46.00

0.80

0.20

>50

0.20

0.27

0.51

>50

0.35

0.86

0.68

0.30

17h-MP

0.64

>50

>50

0.2

47.62

0.11

0.13

0.39

>50

9.89

0.34

0.54

9.61

19h-MP

0.73

>50

>50

0.19

>50

0.25

0.26

1.32

>50

2.59

0.77

0.77

2.19

2i

>50

>50

13.14

0.02

0.06

0.02

0.02

0.02

0.04

0.51

0.95

nd

0.02

gemcitabine

>50

>50

0.05

0.02

0.10

0.03

0.41

0.10

0.05

0.18

nd

nd

nd


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Cell-Cycle Studies. Selected most active derivatives underwent cell-cycle investigation. Compounds 17d, 17g, 17h induced apoptosis following 24-hour treatment at 1xIC50/5xIC50 concentrations and nucleoside 19g only at 5xIC50 concentration (Table 5). All tested compounds 17d, 17g, 17h, 19d, 19g, 19h exhibit dose-dependent decrease in population of mitotic (pH3Ser10 positive) cells and inhibition of RNA synthesis, whilst no significant impact on DNA synthesis was observed. Table 5 Summary of cell cycle, apoptosis (sub G1), mitosis (pH3Ser10 positive), DNA and RNA synthesis analyses in CCRF-CEM for 17d, 17g, 17h, 19d, 19g, 19h (with IC50

Synthesis and Cytostatic and Antiviral Profiling of Thieno-Fused 7

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