Synthesis of novel 6-substituted amino-9-(β-D-ribofuranosyl)purine analogs and their bioactivities on human epithelial cancer cells

Article abstract of DOI:10.1016/j.bmcl.2017.12.070

New nucleoside derivatives with nitrogen substitution at the C-6 position were prepared and screened initially for their in vitro anticancer bioactivity against human epithelial cancer cells (liver Huh7, colon HCT116, breast MCF7) by the NCI-sulforhodamine B assay. N⁶-(4-trifluoromethylphenyl)piperazine analog (27) exhibited promising cytotoxic activity. The compound 27 was more cytotoxic (IC₅₀ = 1–4 μM) than 5-FU, fludarabine on Huh7, HCT116 and MCF7 cell lines. The most potent nucleosides (11, 13, 16, 18, 19, 21, 27, 28) were further screened for their cytotoxicity in hepatocellular cancer cell lines. The compound 27 demonstrated the highest cytotoxic activity against Huh7, Mahlavu and FOCUS cells (IC₅₀ = 1, 3 and 1 μM respectively). Physicochemical properties, drug-likeness, and drug score profiles of the molecules showed that they are estimated to be orally bioavailable. The results pointed that the novel derivatives would be potential drug candidates.

Full text of DOI:10.1016/j.bmcl.2017.12.070

Accepted Manuscript
Synthesis of novel 6-substituted amino-9-(β-D-ribofuranosyl)purine analogs
and their bioactivities on human epithelial cancer cells
Meral Tuncbilek, Aslıgul Kucukdumlu, Ebru Bilget Guven, Duygu Altiparmak,
Rengul Cetin-Atalay
PII:
S0960-894X(17)31244-1
https://doi.org/10.1016/j.bmcl.2017.12.070
BMCL 25526
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
13 September 2017
29 December 2017
31 December 2017
Please cite this article as: Tuncbilek, M., Kucukdumlu, A., Guven, E.B., Altiparmak, D., Cetin-Atalay, R., Synthesis
of novel 6-substituted amino-9-(β-D-ribofuranosyl)purine analogs and their bioactivities on human epithelial cancer
cells, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.070
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Synthesis of novel 6-substituted amino-9-(β-D-ribofuranosyl)purine analogs
and their bioactivities on human epithelial cancer cells
Meral Tuncbileka,*, Aslıgul Kucukdumlua, Ebru Bilget Guvenb, Duygu Altiparmaka,
Rengul Cetin-Atalayc,*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100
Ankara, Turkey
b Department of Molecular Biology and Genetics, Bilkent University, 06800 Ankara, Turkey
cCancer Systems Biology Laboratory, Graduate School of Informatics, ODTU, Ankara,
06800, Turkey
*Corresponding authors
Meral Tuncbilek: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara
University, 06100 Ankara, Turkey
Phone: +90 312 203 3071 Fax: +90 312 213 1081
e-mail: tuncbile@pharmacy.ankara.edu.tr
Rengul Cetin-Atalay: Cancer Systems Biology Laboratory, Graduate School of Informatics,
ODTU, Ankara, 06800, Turkey
Phone: +90 312 210 7887 Fax: +90 312 2103745
e-mail: rengul@metu.edu.tr

ABSTRACT
New nucleoside derivatives with nitrogen substitution at the C-6 position were prepared and
screened initially for their in vitro anticancer bioactivity against human epithelial cancer cells
(liver Huh7, colon HCT116, breast MCF7) by the NCI-sulforhodamine B assay. N6-(4-
Trifluoromethylphenyl)piperazine analog (27) exhibited promising cytotoxic activity. The
compound 27 was more cytotoxic (IC50=1-4 μM) than 5-FU, fludarabine on Huh7, HCT116
and MCF7 cell lines. The most potent nucleosides (11, 13, 16, 18, 19, 21, 27, 28) were further
screened for their cytotoxicity in hepatocellular cancer cell lines. The compound 27
demonstrated the highest cytotoxic activity against Huh7, Mahlavu and FOCUS cells (IC50=
1, 3 and 1 μM respectively). Physicochemical properties, drug-likeness, and drug score
profiles of the molecules showed that they are estimated to be orally bioavailable. The results
pointed that the novel derivatives would be potential drug candidates.
Keywords: Nucleoside analogs, microwave-assisted synthesis, cytotoxic activity,
hepatocellular carcinoma

Cancer is one of the most important causes of death in the world, with almost 14 million new
patients and 8.2 million deaths from cancer in 2012.1 Therefore, development of new potent
and selective anticancer agents is of high interest to medicinal chemistry. Nucleobase and
nucleoside analogues are often exploited as chemotherapeutic agents in both hematologic
malignancies and solid cancers. Nucleobases and nucleosides are the nucleotide precursors;
therefore, they are considered as antimetabolites. Nucleotide compounds of similar structure,
are involved in many cell processes such as cell growth and division, hence nucleobase and
nucleosides have often been exploited as antineoplastic agents.2,3 The mechanism of action of
nucleobase analogs is through induction of apoptosis.4 5-Fluorouracil which is a nucleobase
derivative with fluorine atom, is a frequently preferred anticancer agent for a variety of
malignancies in clinics.5 Similarly, other pyrimidine nucleosides like cytarabine and
gemcitabine have been described as antimetabolite anticancer drugs.6 For the last six decades,
6-mercaptopurine and 6-thioguanine have been used as a nucleic acid metabolism inhibitor
for the treatment of paediatric acute lymphoblastic leukaemia.7 Furthermore, purine
nucleosides such as fludarabine, cladribine, and pentostatine, have become established to be
effective against haematological malignancies.8 These analogs achieve an unbalance in dNTP
pool via inhibition of the ribonucleotide reductase enzyme that induces degradation in DNA
synthesis.9 Therefore, nucleosides with anticancer bioactivities induce apoptotic cell death in
general.6
Fludarabine
Cladribine
Pentostatin

Figure 1. Structures of fludarabine, cladribine and pentostatine.
Primary liver cancer, hepatocellular carcinoma (HCC) is second deadly cancer worldwide
(GLOBOCAN 2012). It is the fifth most common cancer in men and seventh in women,
accounting for 7% of all cancer cases, worldwide with around 700,000 new cases each year.10-
12
Ethological factors for primary liver cancer are mainly HBV or HCV infection, chronic
alcohol consumption, obesity and environmental toxins (aflatoxin B).10,13 Prognosis of HCC
patients is usually very poor due to the resistance against conventional chemotherapeutic
agents. Sorafenib and regorafinib are FDA approved multikinase inhibitors, which extent
patient survival only 3 months with liver cancer.14-17 Therefore, it is essential to identify new
candidate therapeutic agents for hepatocellular carcinoma.18,19
We have previously exploited purine and purine nucleoside derivatives, which have displayed
promising cytotoxic activities in liver cancer cells. The molecules from those studies had
significant
bioactivities
on
liver
cancer
cells.
The
compound
N6-(4-
trifluoromethylphenyl)piperazine nucleoside (IC50=5.2-9.2μM) induced senescence and
purine analogs (IC50=0.1-0.8 μM) lead to apoptotic cell death in HCC cell lines.20,21
Therefore, we designed novel compounds with amine and chlorine electronegative
substituents at the C-2 position of the purine ring. These molecules were then synthesized as a
new series of 6-substituted amino-9-(β-D-ribofuranosyl)purine derivatives (9-22, 27, 28) and
their cytotoxic activities were screened in human epithelial cancer cells (liver Huh7, colon
HCT116, breast MCF7). The bioactivities of the most potent nucleoside derivatives (11, 13,
16, 18, 19, 21, 27, 28) were further analysed in hepatocellular cancer cell lines.
The piperazine-containing nucleoside analogs (9-16) were synthesized as shown in Scheme 1.
In the first transformation, inosine and guanosine are converted to the 6-chloro nucleoside (5,
6) with the trifluoroacetic acid anhydride, thionyl chloride method developed by Robins for

2’-deoxyinosine.22 Trifluoroacetyl groups were used for transient hydroxyl protection instead
of stability of the glycosidic bond. These groups were readily removed by methanolysis after
the chlorination reaction. The inosine and guanosine derivatives (9-16) were prepared via
nucleophilic aromatic substitution of compounds 7, 8 with 4-substituted piperazines.
Nucleosides substituted with 4-substituted anilines / 2-substituted ethyl amines at the position
C-6 (17-22), were obtained with nucleophilic aromatic substitution reaction of 6-chloro-9-(β-
D-ribofuranosyl)purine (7) with the suitable anilines and amines under basic conditions
(Scheme 1).
The 2-chloro-6-(4-substituted piperazine) / 6-(2-substituted ethyl amino) purine analogs (27,
28) were prepared as shown in Scheme 1. 2,6-Dichloropurine (23) was condensed with the
acetylated ribofuranose under microwave irradiation for 30 minutes to get 2,6-dichloro-
nucleoside derivative (24) in good yield of 79%. The yield obtained as a result of this reaction
was significantly higher than the yield in the previously reported method.23,24 Displacement of
the 6-chloro group was made by nucleophilic aromatic substitution by the substituted
piperazine or ethyl amine. Removal of the acetyl groups as the protecting group was made by
NaOMe to obtain purine nucleoside analogs 27, 28. The structures of the all compounds were
confirmed by 1H, 13C NMR mass spectral data and elemental analysis.

Scheme 1. Reagents: (i) TFAA, CH2Cl2; (ii) SOCl2, CH2Cl2, DMF; (iii) MeOH, 7 50%, 8 56% (yields
over three steps); (iv) 4-substituted piperazines, Et3N, EtOH, 9 35%, 10 38%, 11 60%, 12 23%, 13
11%, 14 14%, 15 16%, 16 35%; (v) 4-substituted anilines, Et3N, abs. EtOH, 17 22%, 18 20%, 19 54%,
20 24%; (vi) 2-substituted ethyl amines, Et3N, EtOH, 21 10%, 22 10%; (vii) 1,2,3,5-tetra-O-acetyl-β-
D-ribofuranose, silica gel 60, EtOAc, microwave irradiation, 120 W, 24 79%; (viii) 1-(α,α,α-trifluoro-
p-tolyl)piperazine, TEA, EtOH, 25 75%; (ix) 2-(1-cyclohexenyl) ethylamine, TEA, EtOH, 2662%; (x)
NaOMe, MeOH, 27 87%, 28 40%.
The in vitro cytotoxicity of the compounds 9-22, 27, 28 were initially analyzed on Huh7
(liver), HCT116 (colon) and MCF7 (breast) cancer cells, using a sulforhodamine B (SRB)
assay.25 The IC50 values for each compound also were calculated in comparison with the
known nucleobase analog 5-fluorouracil (5-FU), nucleoside analogs fludarabine and
cladribine and the results were shown in Table 1. Among the synthesized compounds, analogs
accommodating substituted piperazine moiety at their C-6 position (9-16, 27), the one with
promising IC50 values against Huh7 (2 μM), HCT116 (1 μM) and MCF7 (4 μM) is
trifluoromethylphenyl substituted piperazine analogs (27). Nucleoside 27 displayed
significant cytotoxic activity for all the cell lines screened. When, IC50 values compared with
5-FU, fludarabine, the compound 27 had displayed lower values, which are in micromolar
concentrations. Compound 27 established a better cytotoxic activity on Huh7 cell (2 vs. 30
and 30 for 5-FU, fludarabine respectively), HCT116 (1 vs. 4 and 8 for 5-FU and fludarabine)
and MCF7 cells (4 vs. 3 and 15 for 5-FU and fludarabine). Also compound 16, bearing a
diphenylmethyl substituent at piperazine moiety of the nucleoside, had higher cytotoxic
activities when compared to 5-FU and known nucleoside drug fludarabine, on Huh7 cells. The
substitution of (2-cyclohexenylethyl)amino at C-6 position improved the cytotoxic activity of
compound 28 and the IC50 values for 72 hours of treatment were comparable to those of 5-FU
and fludarabine on Huh7 cell line.

We then analyzed the cytotoxic activities of the most potent nucleoside derivatives (11, 13,
16, 18, 19, 21, 27, 28) in a panel of HCC cells: Huh7, HepG2, Mahlavu, and FOCUS (Table
2). N6-Trifluoromethyl nucleoside analog 27 demonstrated the best cytotoxic activity, with
IC50 values of 1-3 μM against Huh7, Mahlavu and FOCUS cells (Table 2). The 2-
Cyclohexenylethyl amino derivative 21 was also found to be significantly bioactive (IC50 1
μM) on HepG2 cell line. Compounds 27 and 21 had a better cytotoxic activity than the known
cytotoxic drugs 5-FU and fludarabine on HepG2 cells. When there was a bigger
diphenylmethyl group at the piperazine (16), we observed that compound 16 had displayed
lower values in micromolar concentrations. Furthermore, nucleoside 13, which had no
substitution at the phenyl ring, were cytotoxic to FOCUS cell line with an IC50 values of 9
μM.
Nucleoside 21 being one of the most active compound, was showed noteworthy IC50 values
(IC50=6 μM) on HCT116 which were comparable to that of 5-FU (IC50=4 μM) and to that of
nucleoside analog fludarabine (IC50=8 μM) (Table 1). Similarly, the cytotoxic activity on
MCF7 cancer cells was significantly low with nucleoside 21 (IC50=3 μM), which was five
times more than the known cytotoxic drug fludarabine.

Table 1. In vitro cytotoxicity of the compounds 9-22, 27, 28 on different human cancer cell
lines (Huh7, HCT116, MCF7)
Cancer cell lines, IC50 (μM)a
Compound
Huh7
NI
HCT116
>100
>100
7010
NI
MCF7
NI
9
NI
10
11
12
13
14
15
16
17
18
19
20
21
22
27
28
402
>100
NI
NI
NI
NI
5020
NI
300.2
NI
NI
>100
101
NI
NI
303
200.5
>100
4010
102
NI
200.9
NI
8080
6040
NI
308
5020
8060
31
>100
NI
61
>100
40.1
308
3010
10.2
90100
20.5
202
5-FU
Fludarabine
Cladribine
302
3020
0.90.7
40.3
83
<0.1
30.7
150.1
22
aIC50 values were calculated from the cell growth inhibition curves obtained from the
treatments done with increasing concentrations of each molecule (40, 20, 10, 5, and 2.5 μM)
for 72h. Experiments are done in duplicate. NI: No inhibition

Table 2. IC50 values of 11, 13, 16, 18, 19, 21, 27, 28 against hepatocellular carcinoma (HCC)
cell lines: Huh7, HepG2, MAHLAVU, FOCUS.
HCC cell line, IC50(μM)a
Compound
Huh7
NI
HepG2
NI
MAHLAVU
>100
FOCUS
409
90.5
203
206
203
NI
11
13
16
18
19
21
27
28
>100
204
3010
408
10.2
30.4
4010
NI
5020
101
8080
6040
>100
10.06
202
71
302
405
>100
30.2
308
10.01
5020
5-FU
302
50.8
102
40.5
Fludarabine
Cladribine
3020
0.40.01
206
0.040.003
105
0.10.01
101
0.40.01
aIC50 values were calculated from the cell growth inhibition curves obtained from the
treatments done with increasing concentrations (40, 20, 10, 5, and 2.5 μM) for the molecules
with IC50 values above 2.5 µM and (4, 2, 1, 0.5, 0.25, 0.125, 0.0625 and 0.03125 µM) for IC50
values below 2.5 µM for 72h. Experiments are done in triplicate. NI: No inhibition
In silico ADME parameters of the new nucleoside analogs 9-22, 27-28, were used to calculate
Lipinski’s rules, solubility, percentage of absorption (%ABS) and topological polar surface
area (TPSA) (Table 3) (see supplementary documentation). All compounds have molecular
weights smaller than 500 (377.44 > MW < 447.47), with the exception of the compounds 16
and 27. The % ABS values were between the range of 49.72% and 68.73%, predicting that the
synthesized nucleosides might penetrate through cell membrane.26 Majority of the synthesized
compounds possess the values of TPSA theoretically compatible with acceptable passive oral
absorption. The results pointed that the novel derivatives would be potential drug candidates.
To further support ourin silico predictions, we calculated drug-likeness and drug-scores of

these compounds which were comparable to those of 5-FU, cladribine and fludarabine (0.06,
0.45 and 0.46 respectively) in general.
Table 3. Lipinski’s rule of 5, %ABS, TPSA, Log S values, druglikeness and drug scores for
the compounds 9-22, 27-28.
Compound
Parameter
Log S at
PH 7.4
% ABS
TPSA
MW
cLog P
nHBA
nHBD
RB
DL
DS
68.73
58.70
67.60
49.72
59.75
58.62
59.75
59.75
66.81
65.69
66.81
62.51
66.81
62.66
68.73
66.81
88.92
41.44
67.84
116.72
145.78
120.00
171.80
142.74
146.02
142.74
142.74
122.27
125.51
122.27
134.74
122.27
134.30
116.72
122.27
58.20
-1.52
-2.99
-4.16
-3.33
-2.62
-4.50
-2.87
-2.98
-3.81
-2.91
-4.50
-3.36
-3.71
-3.05
-4.70
-4.02
-1.16
-2.33
-2.90
420.51
414.42
426.47
429.44
429.48
441.49
447.47
519.60
391.45
388.42
387.44
430.46
377.44
388.42
516.91
411.89
130.07
365.21
285.69
-1.12
-0.66
1.10
-0.81
-1.25
0.95
-1.11
0.36
-0.49
-1.01
0.13
10
11
9
12
11
10
11
11
9
3
3
3
4
4
4
4
4
4
4
4
4
4
5
3
4
2
5
3
3
4
4
4
3
4
3
5
4
4
4
4
5
6
4
5
0
4
2
9
10
11
12
-5.45
0.95
-1.48
3.67
0.39
0.70
0.43
0.77
0.73
0.61
0.60
0.55
0.37
0.14
0.35
0.13
0.39
0.41
0.27
0.35
0.06
0.45
0.46
13
14
15
16
4.13
1.25
1.20
4.05
17
-2.80
-9.11
-5.28
-4.96
-8.29
-5.14
-6.06
-5.48
-4.50
-21.96
0.89
10
9
11
9
10
10
9
2
10
7
18
19
20
21
22
27
28
5-FU
-1.23
-0.95
-1.75
0.91
0.10
-0.66
-1.59
-0.28
195.80
119.30
Fludarabine
Cladribine
%ABS=109−0.345×TPSA; Number hydrogen bond acceptor (NO)=nHBA≤10;Number hydrogen
bond donors (OHNH)=nHBD≤ 5; MW≤500; RB10; Octanol-water partition coefficient=Log P<5;
Solubility=Log S between −1 and −5; TPSA<140 Å; DL: Drug-Likeness score; DS: Drug-Score
To
conclude,
fourteen
novel
nucleoside
derivatives
(9-22)
bearing
substitutedpiperazine/phenyl amino)/ethyl amino at the C-6 position were designed,
synthesized and their bioactivities were assessed in human liver breast and colon epithelial
cancer and a set of liver cancer cells. These compounds were acquired with a multistep
reactions starting from inosine/guanosine. Alternatively, the condensation of 2,6-
dichloropurine with the sugar acetylated β-D-ribofuranose were efficiently used for the
synthesis of 2-chloro nucleoside analogs (27, 28) and their cytotoxicity were also analyzed on

the same set of cancer cells. Our results indicated that the compounds 21, 27 were promising
candidates as chemotherapeutic drugs with the IC50 values less than 10 μM in Huh7 liver,
MCF7 breast and HCT116 colon cancer cells .With the aim of investigating their potential
anticancer use in hepatocellular carcinoma (HCC), the bioactivies of the compounds 11, 13,
16, 18, 19, 21, 27, 28 were further tested on a panel of liver cancer cell lines. Compound 21
and 27, which were designed as putative anticancer agents, showed the best biological
activities with IC50 values of 1-3 μM on HepG2 liver cancer cells.
1. Cancer; World Health Organization: http://www.who.int/cancer/en/. Accessed November
4, 2016.
2. Bosch L, Harber, E, Heidelberger C. Cancer Res. 1958; 8: 335–343.
3. Shelton J, Lu X, Hollenbaugh JA, Cho JH, Amblard F, Schinazi RF. Chem Rev. 2016;
116: 14379–14455.
4. Ewald B, Sampath D, Plunkett W. Oncogene. 2008; 27: 6522–6537.
5. Kuhn JG. Ann Pharmacother. 2001; 35: 217–227.
6. Sampath D, Rao VA, Plunkett W. Oncogene. 2003; 22: 9063–9074.
7. Escherich G, Richards S, Stork LJ, Vora AJ. Leukemia. 2011; 25: 953–959.

8. Lech-Maranda E, Korycka A, Robak T. Mini-Rev Med Chem. 2006; 6: 575–581.
9. Wilson PK, Mulligan SP, Christopherson RI. Leukemia Res. 2004; 28: 725–731.
10. Lonardo A, Loria P. J Gastroen Hepatol. 2012; 27: 1654–1664.
11. Corte CL, Aghemo A, Colombo M. World J Gastroenterol. 2013; 19: 1359–1371.
12. Subramaniam A, Shanmugam MK, Perumal E, Li F, Nachiyappan A, Dai X, Swamy SN,
Ahn KS, Kumar AP, Tan BKH, Hui KM, Sethi G. Biochim Biophys Acta. 2013; 1835:
46–60.
13. Irmak MB, Ince G, Ozturk M, Cetin Atalay R. Cancer Res. 2003; 63: 6707–6715.
14. Finn RS. J Hepatol. 2012; 56: 723–725
15. Berasain C. Gut. 2013; 62: 1674–1675.
16. Sugimoto K, Moriyasu F, Saito K, Rognin N, Kamiyama N, Furuichi Y, Imai Y. Liver Int.
2013; 33: 605–615.
17. Gauthier A, Ho M. Hepatol Res. 2013; 43: 147–154.
18. Ersahin T, Ozturk M, Cetin-Atalay R. Molecular Biology of Liver Cancer. Reviews in
Cell Biology and Molecular Medicine, Version of Record online: 27 JUL 2015 | DOI:
10.1002/3527600906.mcb.200400024.pub2
19. Kahraman DC, Hanquet G, Jeanmart L, Lanners S. Med Chem Comm. 2017; 8:81–87.
20. Tuncbilek M, BilgetGuven E, Onder T, Cetin Atalay R. J Med Chem. 2012; 55: 3058–
3065.
21. Demir Z, Bilget Guven E, Ozbey S, Kazak C, Cetin Atalay R, Tuncbilek M. Eur J Med
Chem. 2015; 89: 701–720.
22. Robins MJ, Basom GL. Can J Chem. 1973; 51: 3161–3169.
23. Andrzejewska M, ami ski , Kazimierczuk Z. Nucleos Nucleot Nucl. 2002; 21: 73–78.
24. Korboukh I, Hull-Ryde EA, Rittiner JE, Randhawa AS, Coleman J, Fitzpatrick BJ, Setola
V, Janzen WP, Frye SV, Zylka MJ, Jin J. J Med Chem. 2012; 55: 6467–6477.

25. Shoemaker RH. Nat Rev Cancer. 2006; 6: 813–823.
26. Lipinski CA. J Pharmacol Toxicol Methods. 2000; 44: 235–249.

GRAPHICAL ABSTRACT
Synthesis of novel 6-substituted amino-9-(β-D-ribofuranosyl)purine analogs and their
bioactivities on human epithelial cancer cells
Meral Tuncbilek, Asligul Kucukdumlu, Ebru Bilget Guven, Duygu Altiparmak, Rengul
Cetin-Atalay
IC50 (μM)
27
5-FU
Fludarabine Cladribine
Huh7
10.06
30.2
302
102
40.5
3020
105
101
0.40.01
0.10.01
0.40.01
MAHLAVU
FOCUS
10.01