Xylo-C-nucleosides with a pyrrolo[2,1-f][1,2,4]triazin-4-amine heterocyclic base: Synthesis and antiproliferative properties

Article abstract of DOI:10.1016/j.bmcl.2019.04.023

The synthesis of a xylo-C-nucleoside containing pyrrolo[2,1-f][1,2,4]triazin-4-amine as nucleobase along with that of its 1′-cyano analogue is described. Among different experimental conditions explored in order to optimize a key debenzylation step in the

Full text of DOI:10.1016/j.bmcl.2019.04.023

Accepted Manuscript
Xylo-C-nucleosides with a pyrrolo[2,1-f][1,2,4]triazin-4-amine heterocyclic
base: Synthesis and antiproliferative properties
Peng Nie, Elisabetta Groaz, Dirk Daelemans, Piet Herdewijn
PII:
S0960-894X(19)30243-4
https://doi.org/10.1016/j.bmcl.2019.04.023
BMCL 26390
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 April 2019
11 April 2019
13 April 2019
Please cite this article as: Nie, P., Groaz, E., Daelemans, D., Herdewijn, P., Xylo-C-nucleosides with a pyrrolo[2,1-
f][1,2,4]triazin-4-amine heterocyclic base: Synthesis and antiproliferative properties, Bioorganic & Medicinal
Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.04.023
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Xylo-C-nucleosides with a pyrrolo[2,1-
f][1,2,4]triazin-4-amine heterocyclic base:
Synthesis and antiproliferative properties
Leave this area blank for abstract info.
Peng Nie, Elisabetta Groaz, Dirk Daelemans, and Piet Herdewijn

Bioorganic & Medicinal Chemistry Letters
Xylo-C-nucleosides with a pyrrolo[2,1-f][1,2,4]triazin-4-amine heterocyclic base:
Synthesis and antiproliferative properties
a
a
b
a,
Peng Nie , Elisabetta Groaz , Dirk Daelemans , and Piet Herdewijn
a
Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Herestraat 49-box 1041, Leuven 3000, Belgium
Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, Herestraat 49-box 1043, Leuven 3000, Belgium
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The synthesis of a xylo-C-nucleoside containing pyrrolo[2,1-f][1,2,4]triazin-4-amine as
nucleobase along with that of its 1’-cyano analogue is described. Among different experimental
conditions explored in order to optimize a key debenzylation step in the presented synthetic
route, it was found that palladium catalyzed hydrogen transfer allowed for obtaining the target
compounds in good yields. The resulting mixture of epimers was separated and each was
characterized by NOESY NMR experiments. In vitro antiproliferative assays showed that the 1’-
unsubstituted analogue was active against a panel of tumor cell lines such as the human
leukemia HL-60 (IC50 = 1.9 µM) and lung cancer NCI-H460 (IC50 = 2.0 µM) cells.
Keywords:
Nucleoside analogues
C-nucleosides
Xylo-nucleosides
Antitumor agents
2
009 Elsevier Ltd. All rights reserved.
C-Nucleosides are a unique class of nucleoside analogues in
which a C-C bond is present between the sugar moiety and the
nucleobase in place of the typical C-N linkage. Owing to this
structural feature, C-nucleosides are more stable against chemical
and enzymatical hydrolysis. Some C-nucleosides containing
nucleobases that are structurally different from the canonical
heteroaromatic DNA and RNA bases occur in nature. Examples
include showdomycin and formycin A and B (Figure 1), which
GS-5734, is currently being developed as a promising antiviral
prodrug for the treatment of Ebola virus infections.
6
1,2
act as nucleoside antibiotics. Pyrazomycin (Figure 1), another
3
natural C-nucleoside isolated from Streptomyces candidus,
Figure 2. Structures of synthetic C-nucleosides.
exhibits activity against several tumor cell lines, including
4
Walker carcinosarcoma 256 and Gardner lymphosarcoma.
On the other hand, nucleosides with a xylose-configured sugar
ring, i.e., 3’-epimers of ribonucleosides, are an underexplored
class of nucleoside analogues, although some of them have been
reported to possess antiviral or antitumor properties (Figure 3).
Figure 1. Structures of naturally occurring C-nucleosides.
Over the years, many C-nucleoside analogues have also been
prepared by chemical synthesis. In some cases, interesting
5-8
biologically properties have been found, particularly when the
9
heterocycle pyrrolo[2,1-f][1,2,4]triazin-4-amine (or 4-aza-7,9-
dideazaadenine) was introduced as nucleobase. For example, 4-
aza-7,9-dideazaadenosine (compound 1, Figure 2) has been
found to be active against several cancer cell lines such as
5,10
leukemic and pancreatic adenocarcinoma.
Another analogue,
Figure 3. Examples of bioactive xylo-nucleoside analogues.
—
——

Piet Herdewijn. Tel.: +32 16 32 26 57; e-mail: piet.herdewijn@kuleuven.be

In particular, 9-β-D-xylofuranosyladenine (2) is an antitumor
11
agent active against the proliferation of ascites tumor cells,
while its carbocyclic congener 3 exhibits activity against
12
13
leukemia cells.
After its synthesis in 1971,
9-β-D-
xylofuranosyl-6- thioguanine 4 was found to be able to
1
4
significantly inhibit murine tumor cell growth. The antitumor
effects of 9-β-D-xylofuranosyl-6-mercaptopurine (5) on three
murine tumors including sarcoma 180, Ehrlich, and TA3 ascites
15
tumors
fluorocytosine derivatives 6 with different leaving groups at the
’-position showed good inhibitory activities against mouse
were
also
reported.
1-β-D-Xylofuranosyl-5-
3
16
leukemia cells. Nonetheless, the synthesis and biological
properties of dually modified C-nucleosides comprising a xylosyl
sugar moiety have not been investigated to date. Based on the
above considerations, we identified xylo-C-nucleoside
derivatives 7 and 8 featuring a pyrrolo[2,1-f][1,2,4]triazin-4-
amine nucleobase as targets for this study (Figure 4).
Scheme 1. Synthesis of target xylo-C-nucleoside 7. Reagents and conditions:
a) TfOH, dioxane, 0 °C, 3 h, 90%; (b) 11, LDA, THF, -78 °C, 3 h, 60%; (c)
Et SiH, BF ·OEt , DCM, 0 °C, 1 h, 85%; (d) NH /MeOH, 100 °C, 12 h, 85%.
e) H , Pd(OH) /C, AcOH, rt, 48 h.
(
3
3
2
3
(
2
2
Subsequent removal of the benzyl protecting groups by
Pd(OH) -catalyzed hydrogenolysis in acetic acid led to a mixture
of xylo-C-nucleosides 7 and 7α in poor yield (20%), along with
0% of the ring-opening product 15. In an effort to increase the
yield of the debenzylation reaction, several conditions were
investigated. However, treatment with boron trichloride, a
reagent commonly used for the removal of benzyl groups, only
resulted in a complex mixture of products. Under oxidative
2
Figure 4. Xylo-C-nucleosides developed in this study.
At first, we focused on the development of a suitable synthetic
route for the preparation of both 7 and 8. To this end,
commercially available D-xylono-1,4-lactone 9 was selected as
starting material, as shown in Scheme 1. Initial protection of all
free hydroxy groups of 9 as benzyl ethers was achieved by using
benzyl trichloroacetimidate in the presence of triflic acid (TfOH)
at 0 °C. Lactone 10 was thus obtained in 90% yield. Its coupling
partner thiomethyl substituted nucleobase 11 was prepared
2
debenzylation conditions in the presence of a combination of
19,20
NaBrO
3
and Na
2 2
S O
4
,
no product was formed. Further
attempts to convert the benzyl groups into benzoates by oxidation
1
7
according to a literature procedure. In the presence of lithium
diisopropylamide (LDA), compound 11 was deprotonated to
generate a carbanion, which attacked the carbonyl group of 10 to
afford coupling product 12 as a mixture of two epimers in 60%
yield. Subsequent reduction of the 1’-hydroxy group was
accomplished by treating hemiacetal 12 with triethylsilane and
21,22
with ruthenium tetroxide also met with failure.
BF
3
·OEt , which led to the formation of 13 in good yield (85%).
2
Although a good stereoselectivity was observed in the case of
ribosyl-nucleoside analogues under the same reaction conditions
Scheme 2. Optimization of the synthesis of target xylo-C-nucleoside 7. (f)
cyclohexene, Pd(OH) /C, EtOH, reflux, overnight.
2
1
8
(Et
3
SiH, BF
3
·OEt ), compound 13 was identified as a mixture
2
of two inseparable anomers (α:β = 2:3). The thiomethyl group
was then replaced by an amino group using methanolic ammonia
under heating, thus providing benzyl protected xylo-C-nucleoside
14.
Figure 5. NOESY spectra of compounds 7 and 7α.

Table 1. Antiproliferative activity of xylo-C-nucleoside 7.
IC50 (µM)a
Capan-1
Hap-1
HCT-116
NCI-H460
DND-41
HL-60
K-562
Z-138
Compound 7
Docetaxel
2.7±1.0
9.3±4.2
8.4±0.8
2.0±0.1
2.8±0.4
1.9±0.1
4.3±2.3
4.0±3.7
0.019±0.014
0.048±0.002
0.018±0.009
0.039±0.017
0.004±0.0004
0.037±0.016
0.007±0.004
0.024±0.002
0.014±0.009
0.023±0.014
0.047±0.005
0.019±0.009
0.016±0.0004
0.083±0.013
0.009±0.0006
0.018±0.010
Staurosporine
aIC50 indicates the half maximal inhibitory concentration.
It has been reported that the Birch reduction can also be used for
pancreatic adenocarcinoma (Capan-1), Hap-1 (chronic myeloid
leukemia), colorectal carcinoma (HCT-116), lung carcinoma
(NCI-H460), DND-41 (acute lymphoblastic leukemia), HL-60
(acute myeloid leukemia), K-562 (chronic myeloid leukemia),
and Z-138 (non-Hodgkin lymphoma). The inhibition of cell
proliferation induced by the tested compounds was calculated as
IC50, which is the half maximal inhibitory concentration. The IC50
was calculated by interpolation based on the semi-log dose
response. Compound 7 displayed antiproliferative activity against
23,24
the cleavage of benzyl groups.
sodium in liquid NH led only to the decomposition of the
starting material. Nonetheless, deprotected C-nucleosides 7 and
α could be obtained in a pleasing 80% overall yield by using
Pd(OH) on carbon (Pearlman’s catalyst) and cyclohexene as
hydrogen donor under reflux (Scheme 2).
However, treatment of 14 with
3
7
2
2
5,26
The two epimers
were separated by silica chromatography and target xylo-C-
nucleoside 7 was eventually isolated in 62% yield.
all above cell lines (Table 1), in particular exhibiting an IC50
=
The structures of compound 7 and its epimer 7α were
determined by NOESY NMR experiments. The main difference
between the two epimers is the direction of the nucleobase,
which is pointed upward and downward in 7 and 7α,
respectively, therefore resulting in different H,H-correlations. As
shown in Figure 5 (spectrum on the left), correlations between 8-
H and 2’-H as well as 1’-H and 4’-H were observed, which
provided the evidences needed for the characterization of
compound 7 as the β anomer. The existence of a NOESY
correlation between 1’-H and 5’-H (Figure 5, spectrum on the
right) together with the absence of any correlation between 8-H
and other protons further confirmed the configuration of 7α.
1.9 and 2.0 µM against HL-60 and NCI-H460 cells, respectively.
In contrast, no significant cytotoxicity was observed for 1’-
cyano-xylo-C-nucleoside 8.
In summary, a synthetic route was developed for the
preparation of novel xylo-C-nucleosides 7 and 8 featuring a
pyrrolo[2,1-f][1,2,4]triazin-4-amine base. Suitable reaction
conditions were established to accomplish a key debenzylation
step in the synthetic route. Furthermore, 2D NMR experiments
were performed to analyze the structures of the different C-
nucleoside epimers formed upon removal of the benzyl
functionalities. Both xylo-C-nucleosides 7 and 8 were evaluated
against different tumor cell lines. The results of this screening
Next, a cyano group was introduced at the 1’-position of
compound 12 upon reaction with TMSCN in the presence of
revealed that compound
7
possessed
a
micromolar
antiproliferative activity against a variety of tumor cells such as
HL-60 and NCI-H460, while compound 8 displayed no
significant cytotoxicity.
27
TMSOTf as promotor to provide nitrile compound 16 in 80%
yield (Scheme 3). Replacement of the thiomethyl group with an
amino functionality at the 4-position of 16 led to compound 17,
which then underwent removal of the benzyl protecting groups
Acknowledgments
by using BCl in DCM to afford a mixture of 8 and 8α (α:β = 2:1)
3
in an overall 45% yield. Benzyl deprotection by palladium
mediated hydrogenolysis was disappointing in this case, leading
to compounds 8 and 8α in poor yield. C-Nucleoside 8 was
isolated by silica chromatography in 15% and then further
purified by reverse phase HPLC chromatography.
P.N. would like to thank the China Scholarship Council (CSC)
for funding (grant No. 201506200062). We are grateful to Prof.
Jef Rozenski for recording HRMS spectra, Luc Baudemprez for
running the 2D NMR experiments, and Leentje Persoons for
excellent technical assistance.
References and notes
(
(
(
(
1) Darnall, K.; Townsend, L. B.; Robins, R. K. Proc. Natl. Acad. Sci. U. S.
A. 1967, 57, 548.
2) Robins, R. K.; Townsend, L. B.; Cassidy, F.; Gerster, J. F.; Lewis, A. F.;
Miller, R. L. J. Heterocycl. Chem. 1966, 3, 110.
3) Gutowski, G. E.; Chaney, M. O.; Jones, N. D.; Hamill, R. L.; Davis, F.
A.; Miller, R. D. Biochem. Biophys. Res. Commun. 1973, 51, 312.
4) Sweeney, M. J.; Davis, F. A.; Gutowski, G. E.; Hamill, R. L.; Hoffman,
D. H.; Poore, G. A. Cancer Res. 1973, 33, 2619.
(
(
5) Patil, S. A.; Otter, B. A.; Klein, R. S. Tetrahedron Lett. 1994, 35, 5339.
6) Warren, T. K.; Jordan, R.; Lo, M. K.; Ray, A. S.; Mackman, R. L.;
Soloveva, V.; Siegel, D.; Perron, M.; Bannister, R.; Hui, H. C. Nature
Scheme 3. Synthesis of target xylo-C-nucleoside 8. Reagents and conditions:
2
016, 531, 381.
(g) TMSOTf, TMSCN, DCM, 0 °C, 2.5 h, 80%; (h) NH
3
/MeOH, 100 °C,
(
(
7) Lim, M. I.; Klein, R. S. Tetrahedron Lett. 1981, 22, 25.
8) Warren, T. K.; Wells, J.; Panchal, R. G.; Stuthman, K. S.; Garza, N. L.;
Van Tongeren, S. A.; Dong, L.; Retterer, C. J.; Eaton, B. P.; Pegoraro, G.
Nature 2014, 508, 402.
9) Patil, S. A.; Otter, B. A.; Klein, R. S. J. Heterocycl. Chem. 1994, 31, 781.
10) Li, Q.; Lescrinier, E.; Groaz, E.; Persoons, L.; Daelemans, D.;
Herdewijn, P.; De Jonghe, S. ChemMedChem 2018, 13, 97.
overnight, 86%; (i) BCl , DCM, 0 °C to rt, 2.5 h.
3
In order to evaluate the biological properties of the
synthesized xylo-C-nucleosides 7 and 8, both compounds were
screened against eight different tumor cell lines including
(
(

(
(
(
11) Ellis, D.; LePage, G. Mol. Pharmacol. 1965, 1, 231.
12) Vince, R.; Brownell, J.; Daluge, S. J. Med. Chem. 1984, 27, 1358.
13) Lee, W. W.; Martinez, A. P.; Blackford, R. W.; Bartuska, V. J.; Reist,
E. J.; Goodman, L. J. Med. Chem. 1971, 14, 819.
(21) Schuda, P. F.; Cichowicz, M. B.; Heimann, M. R. Tetrahedron Lett.
1983, 24, 3829.
(22) Ritter, T.; Zarotti, P.; Carreira, E. M. Org. Lett. 2004, 6, 4371.
(23) Yang, Z.; Lai-Ming Wong, E.; Yuen-Ting Shum, T.; Che, C.-M.; Hui,
Y. Org. Lett. 2005, 7, 669.
(24) Yu, X.-J.; Li, G.-X.; Qi, X.-X.; Deng, Y.-Q. Bioorg. Med. Chem. Lett.
2005, 15, 683.
(25) Araki, L.; Morita, K.; Yamaguchi, M.; Zhao, Z.-y.; Wilson, T. J.; Lilley,
D. M.; Harusawa, S. J. Org. Chem. 2009, 74, 2350.
(26) ivković, A.; Engels, J. Nucleosides Nucleotides Nucleic Acids 2003, 22,
1167.
(14) Kaneko, T.; LePage, G. Cancer Res. 1970, 30, 699.
(15) Sato, K.; LePage, G.; Kimball, A. Cancer Res. 1966, 26, 741.
(16) Watanabe, K.; Reichman, U.; Chu, C.; Hollenberg, D.; Fox, J. J. Med.
Chem. 1980, 23, 1088.
(
(
(
(
17) Girijavallabhan, V. M.; Olsen, D. B.; Zhang, Z.; Fu, J.; Tang, B.-Y.
2
015; Vol. WO2015148746.
18) Metobo, S. E.; Xu, J.; Saunders, O. L.; Butler, T.; Aktoudianakis, E.;
Cho, A.; Kim, C. U. Tetrahedron Lett. 2012, 53, 484.
19) Adinolfi, M.; Barone, G.; Guariniello, L.; Iadonisi, A. Tetrahedron Lett.
(27) Cho, A.; Zhang, L.; Xu, J.; Lee, R.; Butler, T.; Metobo, S.;
Aktoudianakis, V.; Lew, W.; Ye, H.; Clarke, M. J. Med. Chem. 2013,
57, 1812.
1
999, 40, 8439.
20) Du, Y.; Zhang, M.; Kong, F. Org. Lett. 2000, 2, 3797.
Supplementary Material
Supplementary material to this article can be found online at

Highlights



The first examples of a new series of antitumor
nucleoside analogues are described
C-nucleosides with a xylose sugar ring were
synthesized and characterized
Compound
7
displayed
a
micromolar
antiproliferative activity