Synthesis of a 3′-C-ethynyl-β-D-ribofuranose purine nucleoside library: Discovery of C7-deazapurine analogs as potent antiproliferative nucleosides

Article abstract of DOI:10.1016/j.ejmech.2018.07.062

A focused nucleoside library was constructed around a 3′-C-ethynyl-D-ribofuranose sugar scaffold, which was coupled to variously modified purine nucleobases. The resulting nucleosides were probed for their ability to inhibit tumor cell proliferation, as well as for their activity against a panel of relevant human viruses. While C6-aryl substituted purine nucleosides were found to be weakly active, several C7-substituted 7-deazapurine nucleosides elicited potent antiproliferative activity. Their activity spectrum was evaluated in the NCI-60 tumor cell line panel indicating activity against several solid tumor derived cell lines. Analog 32, equipped with a 7-deaza 7-chloro-6-amino-purin-9-yl base was evaluated in a metastatic breast tumor (MDA-MB-231-LM2) xenograft model. It inhibited both tumor growth and reduced the formation of lung metastases as revealed by BLI analysis. The dideazanucleoside analog 66 showed interesting activity against hCMV. These results highlight the potential advantages of recombining known sugar and nucleobase motifs as a library design strategy to discover novel antiviral or antitumor agents.

Full text of DOI:10.1016/j.ejmech.2018.07.062

Accepted Manuscript
Synthesis of a 3′-C-ethynyl-β-D-ribofuranose purine nucleoside library: Discovery of
C7-deazapurine analogs as potent antiproliferative nucleosides
Fabian Hulpia, Sam Noppen, Dominique Schols, Graciela Andrei, Robert Snoeck,
Sandra Liekens, Peter Vervaeke, Serge Van Calenbergh, L.P. Jordheim, D.
Durantel, F. Zoulim, C. Dumontet, L.P. Jordheim, C. Dumontet, J. Shelton, X. Lu, J.A.
Hollenbaugh, J.H. Cho, F. Amblard, R.F. Schinazi, D. Rodriguez, S. Chakraborty, E.
Warnick, S. Crane, Z.G. Gao, R. O'Connor, K.A. Jacobson, J. Carlsson, L. Zhou, H.
Zhang, S. Tao, M. Ehteshami, J.H. Cho, T.R. McBrayer, P. Tharnish, T. Whitaker,
F. Amblard, S.J. Coats, R.F. Schinazi, Z. Yin, Y.-L. Chen, W. Schul, Q.-Y. Wang,
F. Gu, J. Duraiswamy, R.R. Kondreddi, P. Niyomrattanakit, S.B. Lakshminarayana,
A. Goh, H.Y. Xu, W. Liu, B. Liu, J.Y.H. Lim, C.Y. Ng, M. Qing, C.C. Lim, A. Yip, G.
Wang, W.L. Chan, H.P. Tan, K. Lin, B. Zhang, G. Zou, K.A. Bernard, C. Garrett, K.
Beltz, M. Dong, M. Weaver, H. He, A. Pichota, V. Dartois, T.H. Keller, P.-Y. Shi, Q.
Li, E. Lescrinier, E. Groaz, L. Persoons, D. Daelemans, P. Herdewijn, S. De Jonghe,
E. Plebanek, E. Lescrinier, G. Andrei, R. Snoeck, P. Herdewijn, S. De Jonghe,
C. McGuigan, M. Serpi, M. Slusarczyk, V. Ferrari, F. Pertusati, S. Meneghesso,
M. Derudas, L. Farleigh, P. Zanetta, J. Bugert, C. Lin, C. Sun, X. Liu, Y. Zhou, M.
Hussain, J. Wan, M. Li, X. Li, R. Jin, Z. Tu, J. Zhang, M.K. Yates, M.R. Raje, P.
Chatterjee, C.F. Spiropoulou, S. Bavari, M. Flint, V. Soloveva, K.L. Seley-Radtke, N.
Bhuma, S.S. Burade, A.V. Bagade, N.M. Kumbhar, K.M. Kodam, D.D. Dhavale, D.B.
Smith, J.A. Martin, K. Klumpp, S.J. Baker, P.A. Blomgren, R. Devos, C. Granycome,
J. Hang, C.J. Hobbs, W.-R. Jiang, C. Laxton, S.L. Pogam, V. Leveque, H. Ma, G.
Maile, J.H. Merrett, A. Pichota, K. Sarma, M. Smith, S. Swallow, J. Symons, D.
Vesey, I. Najera, N. Cammack, P. Perlíková, L. Eberlin, P. Ménová, V. Raindlová, L.
Slavětínská, E. Tloušťová, G. Bahador, Y.-J. Lee, M. Hocek, H. Hattori, M. Tanaka,
M. Fukushima, T. Sasaki, A. Matsuda, P.J. Hrdlicka, N.K. Andersen, J.S. Jepsen,
F.G. Hansen, K.F. Haselmann, C. Nielsen, J. Wengel, P.J. Hrdlicka, J.S. Jepsen,
C. Nielsen, J. Wengel, S.D. Naik, G. Chandra, P.K. Sahu, H.-R. Kim, S. Qu, J.-s.
Yoon, L.S. Jeong, P.J. Hrdlicka, J.S. Jepsen, J. Wengel, S. Schott, M. Wallwiener,
B. Kootz, H. Seeger, T. Fehm, H. Neubauer, F. Hulpia, J. Balzarini, D. Schols, G.
Andrei, R. Snoeck, S. Van Calenbergh, M. Hocek, A. Holý, I. Votruba, H. Dvořáková,
M. Hocek, P. Nauš, R. Pohl, I. Votruba, P.A. Furman, P.M. Tharnish, M.J. Otto, P.
Perlikova, M. Hocek, A. Bourderioux, P. Naus, P. Perlikova, R. Pohl, I. Pichova, I.
Votruba, P. Dzubak, P. Konecny, M. Hajduch, K.M. Stray, T. Wang, A.S. Ray, J.Y.
Feng, G. Birkus, T. Cihlar, M. Hocek, P. Nauš, O. Caletková, P. Konečný, P. Džubák,
K. Bogdanová, M. Kolář, J. Vrbková, L. Slavětínská, E. Tloušt’ová, P. Perlíková,
M. Hajdúch, M. Hocek, F. Seela, X. Ming, X. Peng, F. Seela, W. Yu, E.J. Chory,
A.K. Wernimont, W. Tempel, A. Scopton, A. Federation, J.J. Marineau, J. Qi, D.
Barsyte-Lovejoy, J. Yi, R. Marcellus, R.E. Iacob, J.R. Engen, C. Griffin, A. Aman, E.
Wienholds, F. Li, J. Pineda, G. Estiu, T. Shatseva, T. Hajian, R. Al-awar, J.E. Dick,
M. Vedadi, P.J. Brown, C.H. Arrowsmith, J.E. Bradner, M. Schapira, L.-C. Campeau,
P.D. O'Shea, T. Brückl, I. Thoma, A.J. Wagner, P. Knochel, T. Carell, I.T. Suydam,
S.A. Strobel, X. Wang, P.P. Seth, R. Ranken, E.E. Swayze, M.T. Migawa, Y. Ju,

Q. Xiao, Y. Song, H. Ding, Y. Dou, R. Yang, Q. Sun, Y. Ji, T. Brueckl, R.D. Baxter,
Y. Fujiwara, I.B. Seiple, S. Su, D.G. Blackmond, P.S. Baran, J.C. Fennewald, B.H.
Lipshutz, Z. Gonda, S. Kovács, C. Wéber, T. Gáti, A. Mészáros, A. Kotschy, Z.
Novák, G.E. Schiltz, K.A. Scheidt, S.T. Rosen, N.L. Krett, V. Iaroshenko, Y. Wang,
D. Sevenard, D. Volochnyuk, F. Seela, R. Gumbiowski, I. Antonini, F. Claudi, G.
Cristalli, P. Franchetti, M. Grifantini, S. Martelli, D.C.M. Leysen, O.R. Defert, K.J.O.A.
De, E.P.P.R. Fourmaintraux, P. Arzel, W.G.J.H. De, S.L. Holbeck, J.M. Collins,
J.H. Doroshow, R.H. Shoemaker, M.-J. Pérez-Pérez, E.-M. Priego, O. Bueno, M.S.
Martins, M.-D. Canela, S. Liekens, A.J. Minn, G.P. Gupta, P.M. Siegel, P.D. Bos, W.
Shu, D.D. Giri, A. Viale, A.B. Olshen, W.L. Gerald, J. Massagué, M. Stipković Babić,
D. Makuc, J. Plavec, T. Martinović, S. Kraljević Pavelić, K. Pavelić, R. Snoeck, G.
Andrei, D. Schols, K. Wittine, M. Mintas, Fabian Hulpia, Sam Noppen, Dominique
Schols, Graciela Andrei, Robert Snoeck, Sandra Liekens, Peter Vervaeke, Serge Van
Calenbergh, L.P. Jordheim, D. Durantel, F. Zoulim, C. Dumontet, L.P. Jordheim, C.
Dumontet, J. Shelton, X. Lu, J.A. Hollenbaugh, J.H. Cho, F. Amblard, R.F. Schinazi,
D. Rodriguez, S. Chakraborty, E. Warnick, S. Crane, Z.G. Gao, R. O'Connor, K.A.
Jacobson, J. Carlsson, L. Zhou, H. Zhang, S. Tao, M. Ehteshami, J.H. Cho, T.R.
McBrayer, P. Tharnish, T. Whitaker, F. Amblard, S.J. Coats, R.F. Schinazi, Z. Yin,
Y.-L. Chen, W. Schul, Q.-Y. Wang, F. Gu, J. Duraiswamy, R.R. Kondreddi, P.
Niyomrattanakit, S.B. Lakshminarayana, A. Goh, H.Y. Xu, W. Liu, B. Liu, J.Y.H.
Lim, C.Y. Ng, M. Qing, C.C. Lim, A. Yip, G. Wang, W.L. Chan, H.P. Tan, K. Lin, B.
Zhang, G. Zou, K.A. Bernard, C. Garrett, K. Beltz, M. Dong, M. Weaver, H. He, A.
Pichota, V. Dartois, T.H. Keller, P.-Y. Shi, Q. Li, E. Lescrinier, E. Groaz, L. Persoons,
D. Daelemans, P. Herdewijn, S. De Jonghe, E. Plebanek, E. Lescrinier, G. Andrei,
R. Snoeck, P. Herdewijn, S. De Jonghe, C. McGuigan, M. Serpi, M. Slusarczyk, V.
Ferrari, F. Pertusati, S. Meneghesso, M. Derudas, L. Farleigh, P. Zanetta, J. Bugert,
C. Lin, C. Sun, X. Liu, Y. Zhou, M. Hussain, J. Wan, M. Li, X. Li, R. Jin, Z. Tu, J.
Zhang, M.K. Yates, M.R. Raje, P. Chatterjee, C.F. Spiropoulou, S. Bavari, M. Flint, V.
Soloveva, K.L. Seley-Radtke, N. Bhuma, S.S. Burade, A.V. Bagade, N.M. Kumbhar,
K.M. Kodam, D.D. Dhavale, D.B. Smith, J.A. Martin, K. Klumpp, S.J. Baker, P.A.
Blomgren, R. Devos, C. Granycome, J. Hang, C.J. Hobbs, W.-R. Jiang, C. Laxton,
S.L. Pogam, V. Leveque, H. Ma, G. Maile, J.H. Merrett, A. Pichota, K. Sarma, M.
Smith, S. Swallow, J. Symons, D. Vesey, I. Najera, N. Cammack, P. Perlíková, L.
Eberlin, P. Ménová, V. Raindlová, L. Slavětínská, E. Tloušťová, G. Bahador, Y.-J.
Lee, M. Hocek, H. Hattori, M. Tanaka, M. Fukushima, T. Sasaki, A. Matsuda, P.J.
Hrdlicka, N.K. Andersen, J.S. Jepsen, F.G. Hansen, K.F. Haselmann, C. Nielsen, J.
Wengel, P.J. Hrdlicka, J.S. Jepsen, C. Nielsen, J. Wengel, S.D. Naik, G. Chandra,
P.K. Sahu, H.-R. Kim, S. Qu, J.-s. Yoon, L.S. Jeong, P.J. Hrdlicka, J.S. Jepsen,
J. Wengel, S. Schott, M. Wallwiener, B. Kootz, H. Seeger, T. Fehm, H. Neubauer,
F. Hulpia, J. Balzarini, D. Schols, G. Andrei, R. Snoeck, S. Van Calenbergh, M.
Hocek, A. Holý, I. Votruba, H. Dvořáková, M. Hocek, P. Nauš, R. Pohl, I. Votruba,
P.A. Furman, P.M. Tharnish, M.J. Otto, P. Perlikova, M. Hocek, A. Bourderioux,
P. Naus, P. Perlikova, R. Pohl, I. Pichova, I. Votruba, P. Dzubak, P. Konecny, M.
Hajduch, K.M. Stray, T. Wang, A.S. Ray, J.Y. Feng, G. Birkus, T. Cihlar, M. Hocek,
P. Nauš, O. Caletková, P. Konečný, P. Džubák, K. Bogdanová, M. Kolář, J. Vrbková,
L. Slavětínská, E. Tloušt’ová, P. Perlíková, M. Hajdúch, M. Hocek, F. Seela, X. Ming,
X. Peng, F. Seela, W. Yu, E.J. Chory, A.K. Wernimont, W. Tempel, A. Scopton,
A. Federation, J.J. Marineau, J. Qi, D. Barsyte-Lovejoy, J. Yi, R. Marcellus, R.E.

Iacob, J.R. Engen, C. Griffin, A. Aman, E. Wienholds, F. Li, J. Pineda, G. Estiu, T.
Shatseva, T. Hajian, R. Al-awar, J.E. Dick, M. Vedadi, P.J. Brown, C.H. Arrowsmith,
J.E. Bradner, M. Schapira, L.-C. Campeau, P.D. O'Shea, T. Brückl, I. Thoma, A.J.
Wagner, P. Knochel, T. Carell, I.T. Suydam, S.A. Strobel, X. Wang, P.P. Seth, R.
Ranken, E.E. Swayze, M.T. Migawa, Y. Ju, Q. Xiao, Y. Song, H. Ding, Y. Dou, R.
Yang, Q. Sun, Y. Ji, T. Brueckl, R.D. Baxter, Y. Fujiwara, I.B. Seiple, S. Su, D.G.
Blackmond, P.S. Baran, J.C. Fennewald, B.H. Lipshutz, Z. Gonda, S. Kovács, C.
Wéber, T. Gáti, A. Mészáros, A. Kotschy, Z. Novák, G.E. Schiltz, K.A. Scheidt, S.T.
Rosen, N.L. Krett, V. Iaroshenko, Y. Wang, D. Sevenard, D. Volochnyuk, F. Seela, R.
Gumbiowski, I. Antonini, F. Claudi, G. Cristalli, P. Franchetti, M. Grifantini, S. Martelli,
D.C.M. Leysen, O.R. Defert, K.J.O.A. De, E.P.P.R. Fourmaintraux, P. Arzel, W.G.J.H.
De, S.L. Holbeck, J.M. Collins, J.H. Doroshow, R.H. Shoemaker, M.-J. Pérez-Pérez,
E.-M. Priego, O. Bueno, M.S. Martins, M.-D. Canela, S. Liekens, A.J. Minn, G.P.
Gupta, P.M. Siegel, P.D. Bos, W. Shu, D.D. Giri, A. Viale, A.B. Olshen, W.L. Gerald,
J. Massagué, M. Stipković Babić, D. Makuc, J. Plavec, T. Martinović, S. Kraljević
Pavelić, K. Pavelić, R. Snoeck, G. Andrei, D. Schols, K. Wittine, M. Mintas, Fabian
Hulpia, Sam Noppen, Dominique Schols, Graciela Andrei, Robert Snoeck, Sandra
Liekens, Peter Vervaeke, Serge Van Calenbergh
PII:
S0223-5234(18)30627-5
10.1016/j.ejmech.2018.07.062
EJMECH 10597
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 June 2018
Revised Date: 24 July 2018
Accepted Date: 25 July 2018
Please cite this article as: F. Hulpia, S. Noppen, D. Schols, G. Andrei, R. Snoeck, S. Liekens, P.
Vervaeke, S. Van Calenbergh, Synthesis of a 3′-C-ethynyl-β-D-ribofuranose purine nucleoside library:
Discovery of C7-deazapurine analogs as potent antiproliferative nucleosides, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.062.
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ACCEPTED MANUSCRIPT
Table of contents graphic

ACCEPTED MANUSCRIPT
[
Title page]
Synthesis of a 3’-C-ethynyl-β- -ribofuranose purine
D
nucleoside library: discovery of C7-deazapurine analogs as
potent antiproliferative nucleosides
1
2
2
2
2
Fabian Hulpia, Sam Noppen, Dominique Schols, Graciela Andrei, Robert Snoeck, Sandra
2
2
1,*
Liekens, Peter Vervaeke & Serge Van Calenbergh
1
Laboratory for Medicinal Chemistry (Campus Heymans), Ghent University, Ottergemsesteenweg
60, B-9000, Gent, Belgium.
4
2
Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven,
Herestraat 49, B-3000, Leuven, Belgium.
Declarations of interest
Declarations of interest: none.

ACCEPTED MANUSCRIPT
Abstract
A focused nucleoside library was constructed around a 3’-C-ethynyl-
D
-ribofuranose sugar scaffold,
which was coupled to variously modified purine nucleobases. The resulting nucleosides were probed
for their ability to inhibit tumor cell proliferation, as well as for their activity against a panel of
relevant human viruses. While C6-aryl-substituted purine nucleosides were found to be weakly active,
several C7-substituted 7-deazapurine nucleosides elicited potent antiproliferative activity. Their
activity spectrum was evaluated in the NCI-60 tumor cell line panel indicating activity against several
solid tumor derived cell lines. Analog 32, equipped with a 7-deaza 7-chloro-6-amino-purin-9-yl base
was evaluated in a metastatic breast tumor (MDA-MB-231-LM2) xenograft model. It inhibited both
tumor growth and reduced the formation of lung metastases as revealed by BLI analysis. The
dideazanucleoside analog 66 showed interesting activity against hCMV. These results highlight the
potential advantages of recombining known sugar and nucleobase motifs as a library design strategy to
discover novel antiviral or antitumor agents.
Keywords
Antiviral
Antiproliferative
3
7
1
’-C-ethynylnucleoside
-deazapurine nucleoside
,7-dideazapurine nucleoside
Vorbrüggen glycosylation

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Introduction
Nucleoside analogs are the cornerstones for antiviral therapy with notable successes in the treatment of
1
HIV and Hepatitis C virus infections. Furthermore, nucleoside analogs have also found widespread
1
-3
use in oncology, with most approved derivatives active against various forms of lymphomas.
A focused screening library, comprised of nucleoside analogs surrounding around a single modified
ribofuranose moiety could be a viable strategy to discover attractive hits for the aforementioned
D-
4
-14
disease areas, as evidenced by a number of recent publications.
In this paper, a rather
was used as the sugar scaffold to construct a
small library of nucleoside analogs. Previous reports mainly focused on pyrimidine(-like) nucleobase
1
5-18
underrepresented 3’-C-ethynyl-ribofuranose motif
1
7, 19
moieties,
structurally resembling the cytidine analog, ECyd (1, Figure 1), which emerged as the
1
5
most promising derivative from the initial discovery of 3’-C-ethynyl nucleosides in 1996. ECyd has
3
been evaluated in clinical trials as a new antitumoral agent and recently attracted renewed interest as a
3
combination therapy, e.g. with carboplatin, or as part of a ‘duplex drug’, in which it is linked to 2’-
2
0
deoxy-5-fluorouridine.
The purine counterparts on the other hand, exemplified by the 3’-C-ethynyladenosine analog (EAdo,
), have received little attention, which motivated us to combine this peculiar 3’-C-ethynyl sugar
element with different purine nucleobases to build a focused library. Previously we reported a series of
5
21
C2- and C6-substituted purine analogs (4, Figure 1) of 3’-C-ethynyladenosine (EAdo, 5).
2
2,
Considering the interesting biological properties reported for nucleosides comprising a C6 arylpurine
2
3
(
e.g. 2, Figure 1) or a C7-substituted-7-deazapurine base, which was recently coined a ‘privileged
24
24-26
scaffold’ and is part of the natural nucleoside antibiotic tubercidin (3),
in this contribution we
investigate the effect(s) of combining these base moieties with the 3’-C-ethynylribofuranose moiety.

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Figure 1: Overview of previously reported nucleoside analogs featuring a 3’-C-ethynylribofuranose
(
ECyd and EAdo); known purine-modified nucleoside analogs with interesting biological activity (2
and 3), and their combination to construct a small library.
Results and discussion
Chemistry
The target nucleoside analogs were prepared by Vorbrüggen glycosylation using either acetate (6,
21
15
Scheme 1) or benzoate (18, Scheme 2) protected sugar precursors. Reaction conditions depended
2
1
27
on the type of nucleobase (purine vs. 7-deazapurine analogs ). Synthesis of the 6-substituted purine
analogs 13 - 17 was accomplished via Suzuki reaction of the 6-chloropurine nucleoside 7 with
2
2
appropriate phenyl boronic acids. Final compounds were obtained after deprotection with
NH /MeOH (Scheme 1).
3

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Scheme 1: Reagents and conditions: a) (i) 6-chloropurine, HMDS, cat. (NH ) SO , reflux; (ii)
4
2
4
TMSOTf, 1,2-dichloroethane, reflux, 81%; b) (substituted) phenylboronic acid, K CO , Pd(Ph P) ,
2
3
3
4
toluene, 100 °C, 30–52%; c) 7N NH /MeOH, 43–75%.
3
a
For the synthesis of the C7-modified 7-deazanucleosides (Scheme 2), the use of the aforementioned
sugar precursor 6 only provided the desired glycosylation product in low yield after cumbersome
1
5
purification procedures. Switching to the benzoate protected sugar derivative 18 improved the
2
7
coupling yields, but the isolated glycosylation products were contaminated with residues of glycosyl
1
donor degradation products (tentative assignment based on H-NMR data; not shown). Treatment of
2
7
28
the crude intermediates 19-22 with NH /MeOH (or NH OH ) at elevated (> 100 °C) temperatures
3
4
caused decomposition. Therefore, an alternative protocol to introduce the 6-amino group was
29, 30
employed.
Nucleophilic displacement of the 6-chloride with sodium azide efficiently delivered the
corresponding azide derivatives (23-26), which generally could be obtained in pure form due to the
marked difference in polarity induced by the predominating tetrazolo tautomer. Staudinger reduction
furnished the corresponding 6-amino derivatives. Deprotection afforded the final compounds (31, 32,
3
3, 34). To dehalogenate the 7-iodo intermediate 26, it was subjected to I/Mg exchange using
2
6, 31
Knochel’s iPrMgCl.LiCl,
and the magnesiated intermediate was quenched with aqueous acid to
give 35 in good yield. Further conversion to 37 was realized as described above. Introduction of a
2
5
furan-2-yl moiety was achieved via an aqueous Suzuki reaction on 34. Remarkably, Suzuki reaction
on the 7-iodo-7-deazapurine substrate gave significantly lower yields than with 6-chloropurine starting
material.
a
In the body of the text, purine numbering will be used for nucleoside analogs; however in the experimental section, IUPAC
nomenclature and numbering of the pyrrolo[2,3-d]pyrimidine system will be applied.

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3
2, 33
34
34
Scheme 2: Reagents and conditions: a) 4-chloro-5-halo-7H-pyrrolo[2,3-d]pyrimidine (F
, Cl , Br ,
34
I ), TMSOTf, MeCN, 80 °C; b) NaN , DMF, 65 °C; c) (i) 1.0 M PMe in THF, THF; (ii) aq. HOAc,
3
3
MeCN, 65 °C; d) 7N NH /MeOH, 30-95%; e) (i) iPrMgCl.LiCl, toluene, -65 °C; (ii) sat. aq. NH Cl,
3
4
7
6%; f) furan-2-yl-boronic acid, Na CO , Pd(OAc) , TPPTS, MeCN/water (1/2), 100 °C, 29%.
2
3
2
b
A similar strategy was followed to synthetize the C7 (C5) trifluoromethyl analog 49 from 39 (upper
3
5, 36
line in Scheme 3)
by treating commercial 4-chloro-7H-pyrrolo[2,3-d]pyrimidine with the Langlois
3
5
3
1
13
reagent (sodium trifluoromethanesulfinate). The presence of a J
cross peak in the H- C
H-1’-C-8
gHMBC spectrum of compound 48 (see Supporting Information) allowed to ascertain glycosylation at
N9 (N7). However, a large coupling constant (J = 37.8 Hz) observed for C8 (C6) was inconsistent with
the CF group being attached to C7 (C5). This led us to assign the structure trifluoromethylated
3
compound prepared using Langlois’ reagent as the C8 (C6) regio-isomer. The C7 (C5) substituted
b
In order to allow for a more facile comparison with the assigned NMR data provided in the experimental section;
pyrrolo[2,3-d]pyrimidine numbering is indicated for selected derivates in italic and between brackets.

ACCEPTED MANUSCRIPT
3
4
heterocycle 42 could be obtained from the known C7 (C5) iodide 40 by subsequent N-Boc protection
3
7
and trifluoromethylation using the Ruppert reagent (the N-Boc protecting group was lost during the
1
reaction). Comparison of the H-NMR spectra of both regioisomers 39 and 42 (see Supporting
Information) led to the confirmation that 39 and thus also 48, are the C8 (C6) substituted isomers. Of
3
8
note is a recent patent application that describes both regio-isomers 39 and 42, in which the
regiochemical assignment is opposite to our conclusions. The C7 substituted heterocycle 42 was used
to synthetize the desired product 49, following the same reaction sequence as above.
Cl
F
3
C
N
N
Cl
N
N
N
H
N
42
N
a
H
Cl
N
4
-chloro-
pyrrolo[2,3-d]pyrimidine
3
F C
N
H
39
Cl
I
Cl
N
Cl
N
I
3
F C
N
b
N
c
N
N
N
N
N
H
H
O
40
O
41
42
CF
3
5
CF
3
CF
3
CF
N
3
6
BzO
BzO
BzO
HO
7
O
O
O
d
O
Cl
e
f
NH2
g
NH2
N
4
N
N
N
N
18
N
BzO
N1
N
3
BzO
OBz
N
N
BzO
OBz
N
N
HO
OH
N
N
OBz
C-5 CF
C-6 CF
3
: 43
: 44
C-5 CF
C-6 CF
3
: 45; not isolated
: 46
C-5 CF
C-6 CF
3
: 47
: 48
C-5 CF
C-6 CF
3
3
: 49
: 50
3
3
3
not isolated
Remark: pyrrolo[2.3-d]pyrimidine numbering used
Scheme 3: Reagents and conditions: a) Langlois reagent, t-BuOOH, DCM/water; b) Boc O, DBU,
2
DMAP, 1,4-dioxane, 96%; c) Ruppert reagent (TMSCF ), B(OMe) , KF, CuI, 1,10-phenanthroline,
3
3
DMSO, 60 °C, 23%; d) 39 or 42, BSA, TMSOTf, MeCN, 80 °C; e) NaN , DMF, 65 °C, 43% (2 steps,
3
4
6); f) (i) 1.0 M PMe in THF, THF, 21% (3 steps, 47), 55% (48); (ii) aq. HOAc, MeCN, 65 °C; g) 7N
3
NH /MeOH, 80% (49), 87% (50).
3

ACCEPTED MANUSCRIPT
To obtain the C7 ethynyl substituted analog 54 (Scheme 4) a Sonogashira reaction was envisioned. To
avoid selectivity issues with the 3’-C-ethynyl group of 18, this group was protected with a TMS group
2
5
(
51) and glycosylated (52). After introduction of the C7 ethynyl chain, 53 was transformed into 54
employing the same reaction sequence as described above. The synthesis of 3’-ethyl analog 59 started
with catalytic hydrogenation of 18, giving rise to 55, which was subjected to glycosylation conditions.
The glycosylation product 56 was directly used and elaborated as described above.
Scheme 4: Reagents and conditions: a) 1. iPrMgCl.LiCl (1.3 M in THF), toluene, -65 °C; 2. TMSCl,
3
4
6
0%; b) 9H-6-chloro-7-iodo-7-deazapurine (40) , BSA, TMSOTf, MeCN, 80 °C, 30%; c)
ethynyltrimethylsilane, CuI, Pd(Ph P) Cl , Et N, DMF, 39%; d) (i) NaN , DMF, 65 °C; (ii) 1.0 M
3
2
2
3
3
PMe in THF, THF; (iii) aq. HOAc, MeCN, 65 °C; (iv) 7N NH /MeOH, 46%; e) Pd/C, H (balloon),
3
3
2
3
4
ethyl acetate, 92%; f) 4,5-dichloro-7H-pyrrolo[2,3-d]pyrmidine , BSA, TMSOTf, MeCN, 80 °C; g)
NaN , DMF, 65 °C, 55% (2 steps); h) (i) 1.0 M PMe in THF, THF; (ii) aq. HOAc, MeCN, 65 °C,
3
3
8
8%; i) 7N NH /MeOH, 87%.
3
c
For the synthesis of 1,7-dideazapurine (7-azaindole or pyrrolo[2,3-b]pyridine) nucleoside analogs
(
Scheme 5 & 6), commercially available 1H-4-chloro-pyrrolo[2,3-b]pyridine was halogenated with the
c
As for the 7-deazapurine analogs, in the body of the text, purine numbering will be used. In the Experimental section the
corresponding pyrrolo[2,3-b]pyridine nomenclature is employed. In order to allow for a more facile comparison with the

ACCEPTED MANUSCRIPT
34
appropriate halosuccinimide. Glycosylation products were obtained using the same conditions as for
their C7-deazapurine counterparts. Lewis acid-mediated glycosylation with this type of heterocycles
39
40
41
has only been reported once. Generally nucleobase-anion glycosylation or acid-catalyzed fusion
1
are employed to ensure this transformation. Both regio- and stereochemistry were ascertained by H-
1
3
C gHMBC and 2D NOESY experiments (see Supporting Information). Deprotection with
NH /MeOH gave final products 66, 67 and 68. De-iodination by I/Mg exchange of 65 gave 69 in good
3
yield, after which deprotection furnished 70. As expected, introduction of the C6 (C4) azido group on
e.g. 63 was problematic due to the higher electron density of the pyrrolo[2,3-b]pyridine system with
respect to the pyrrolo[2,3-d]pyrimidine system. No desired product could be detected after reaction
with NaN at 65 °C, while gradual increase of the temperature to 100 °C (and higher) only led to
3
degradation.
Scheme 5: Reagents and conditions: a) appropriate N-halosuccinimide, DMF, 93% (X = Cl), 96% (X
=
Br), 92% (X = I); b) 60-62, BSA, TMSOTf, MeCN, 80 °C, 25–39%; c) 7N NH /MeOH, 70–80%; d)
3
(
i) iPrMgCl.LiCl (1.3 M in THF), toluene, -65 °C; (ii) sat. aq. NH Cl, 75%.
4
assigned NMR data (Experimental section), pyrrolo[2,3-b]pyridine numbering is indicated for selected compounds in italic
and between brackets.

ACCEPTED MANUSCRIPT
4
2
These issues led us to introduce the azido group before the glycosylation step (Scheme 6).
Glycosylation, employing the same conditions as for the chloride-substituted heterocycles, was first
attempted with 72, which afforded two products, 74 and 75. The identity of each isomer was assigned
1
13
after Staudinger reduction to 76 and 77 (Scheme 6) to facilitate purification. The H- C gHMBC
spectrum of 76 and 77 showed a markedly different cross-peak pattern between H-1’ and the
heterocyclic moiety (see Supporting Information). The synthesis of iodo-substituted 79 was
accomplished using the same conditions (the tentative N3 (N7) isomer could be observed from TLC
analysis, but was not isolated, nor formally characterized). Both intermediates (76 and 79) afforded,
after deprotection with saturated ammonia/MeOH, the desired nucleosides 80 and 81.
Scheme 6: Reagents and conditions: a) NaN , NH Cl, DMF, 110 °C, 66% (71), 71% (72); b) NIS,
3
4
DMF, 93%; c) 72 or 73, BSA, TMSOTf, MeCN, 80 °C, 25% (78); d) (i) 1.0 M PMe in THF, THF; (ii)
3
aq. HOAc, MeCN, 65 °C, 22% (2 steps, 76), 13% (2 steps, 77), 74% (79); e) 7N NH /MeOH, 73%
3
(
80), 74% (81).

ACCEPTED MANUSCRIPT
Biological evaluation
All final nucleoside analogs were assayed for their ability to inhibit cell proliferation of three different
tumor cell lines (L1210, CEM and HeLa; Table 1) and for their antiviral activity against a
representative panel of human viruses, including herpex simplex virus (HSV) 1 and 2,
cytomegalovirus (CMV), varicella zoster virus (VZV), vaccinia virus (VV), adenovirus-2, influenza-A
virus (H1N1, H3N2), influenza B virus, feline corona virus, feline herpes virus, para-influenza virus,
reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus, vesicular stomatitis virus, respiratory
syncytial virus (RSV) (Tables 4 & 5).
Antiproliferative activity
The results of the inhibition of cell line proliferation are depicted in Table 1.
L1210
CEM
HeLa
L1210
CEM
50
HeLa
50
Cpd.
Cpd.
IC (µM)
IC (µM)
IC (µM)
IC (µM) IC (µM) IC (µM)
5
0
50
50
50
a
5
0.73 ± 0.14
>250
0.61 ± 0.08 0.29 ± 0.11
38
49
50
54
30 ± 1
17 ± 5
14 ± 1
0.36 ±
0.75 ±
0.19
1
1
1
3
4
5
>250
154 ± 8
>250
>250
>250
>250
0.11 ± 0.03
>250
0.26
205 ± 45
>250
>250
>250
159 ± 49
63 ± 0
114 ± 20
1
1
3
6
7
1
225 ± 23
>250
124 ± 49
223 ± 21
170 ± 53
220 ± 30
59
66
67
1.2 ± 0.1
75 ± 19
30 ± 6
2.6 ± 1.2 4.4 ± 1.5
1.3 ± 0.1 6.3 ± 3.8
0.56 ±
0.89 ±
0.11
0.035 ± 0.008 0.16 ± 0.02 0.15 ± 0.01
0.07
0
0
0
0
.012 ±
.001
.030 ±
.007
0.051 ±
0.006
0.093 ±
0.009
0.26 ±
0.03
3
3
2
3
0.014 ± 0.009
0.028 ± 0.013
68
70
20 ± 5
1.1 ± 0.1
197 ± 62
86 ± 4
135 ± 1
3
3
4
7
0.056 ± 0.012 0.12 ± 0.02 0.18 ± 0.04
0.38 ± 0.04 0.71 ± 0.13 0.88 ± 0.09
80
81
0.98 ± 0.03 5.4 ± 0.8 1.7 ± 0.4
0.31 ±
0.18 ±
0.03
0.044 ± 0.14
0.27
Table 1: Effects of different 3’-C-ethynyl purine derivatives on the proliferation of three tumor cell
lines. IC50 values represent the concentration of compound able to inhibit proliferation by 50%
a
21
(
Coulter Counter cell count endpoint). Results are taken from reference .

ACCEPTED MANUSCRIPT
Introduction of (substituted) phenyl rings in the C6 position of the purine nucleobase, known to confer
2
2, 23
cytostatic activity in ribofuranosylpurine nucleosides,
failed to display any inhibitory activity on
the cell line proliferation. Remarkably, 3’-C-ethynyl-7-deaza-adenosine (3’-C-ethynyltubercidin) 37,
as well as its 7-halogenated analogs 31–34 inhibited the proliferation of the different tumor cell lines
with nanomolar IC values. The chloro (32) and Br (33) analogs showed the highest antiproliferative
5
0
activity irrespective of the cell line studied, while unsubstituted derivative 37 showed similar activity
as EAdo (5). Interestingly, the observed structure-antiproliferative activity relationship from this small
2
5
subset significantly differs from that observed for the corresponding ribofuranose derivatives. Furan-
2
-yl substituted analog 38, was found to only weakly inhibit tumor cell proliferation, which contrasts
2
5
to the potent activity observed for the corresponding 7-(furan-2-yl)-7-deazaadenosine. Similarly, the
7
-ethynyl analog 54 only showed weak antiproliferative activity, which also contrasts with the activity
2
5
observed for the corresponding ribofuranose analog. Saturation of the ethynyl substituent as in 59,
resulted in two orders of magnitude lower IC 's than those for 32.
5
0
The 7-trifluoromethyl analog 49 gave sub-micromolar activity, while the corresponding C8 isomer 50
was completely devoid of activity, possibly due to a preferred anti-orientation of the purine ring for
activity.
To investigate the importance of N1, we synthetized the 1,7-dideazapurine or pyrrolo[2,3-b]pyridine
analogs 66–70 and 80 and 81. Interestingly, all the analogs elicited antiproliferative effects, most
notably for the CEM cell line (C6 chloride analogs), except for 70. In the C6 chloride series, the
antiproliferative activity correlated with halogen size (I > Br > Cl > H). While analog 80 was
significantly less active than the related 32 (approximately 100-fold); this was not the case for the
iodo-substituted analog 81, which displayed potent antiproliferative activity, especially on the L1210
cell line.
To further explore the potential of these new nucleoside analogs, the most potent analogs (31–34 and
4
3, 44
3
7) were selected for testing in the NCI-60 cell line panel.
Table 2 and 3 summarize the GI50

ACCEPTED MANUSCRIPT
values of representative cell lines. Full assay data as well as mean GI graphs are provided in the
5
0
Supporting Information.
Leukemia
HL-60
Lung
Colon
CCRF-
CEM
SR
50
A549
50
NCI-H460
GI (µM)
HCT116
Cpd.
GI (µM) GI (µM) GI (µM)
GI (µM)
5
0
50
50
GI (µM)
5
0
b
3
1
0.338
0.436
0.185
0.377
0.231
0.153
0
0
.248 ±
.009
0.059 ±
0.025
0.074 ±
0.013
0.188 ±
0.050
[<0.01 –
0.046]
0.020 ±
0.002
3
2
3
a
0.213 ±
0.047 ±
0.025
0.064 ±
0.02
0.019 ±
0.097
0.034 ±
0.016
0.028 ±
0.002
3
3
0
.073
0.272 ±
0.050 ±
0.018
0.28 ±
0.098
0.443 ±
0.146
0.098 ±
0.039
4
0.045 ± 0.01
0
.023
b
3
7
2.3
1.57
0.817
1.31
0.473
0.595
Table 2: Summary of the growth inhibitory potential (expressed as GI ) of selected nucleoside
5
0
4
3, 44
analogs against the NCI-60 tumor cell line panel with Sulforhodamine B (SRB) read-out at 48h.
Full details (GI values for all cell lines) can be found in the Supporting Information. Values represent
5
0
a
mean ± SEM of two independent evaluations. Values in brackets represent the obtained GI values
5
0
b
from both experiments. Compounds 31 and 37 were only tested once.
CNS
Melanoma
Lox IMVI
Prostate
Breast
DU145
GI50
MDA-MB-
231
U251
PC-3
GI (µM)
MCF7
50
Cpd
GI (µM)
GI (µM)
GI (µM)
5
0
50
50
(
µM)
GI (µM)
5
0
b
3
1
0.631
0.279
0.293
0.354
0.103
0.379
[
<0.01 –
0.059 ±
0.023
0.192 ±
0.019
0.134 ±
0.025
0.027 ±
0.005
3
2
3
4
a
0.170 ± 0.02
0.136 ± 0.08
0.02]
0
.022 ±
0.067 ±
0.038
0.107 ±
0.060
0.107 ±
0.044
0.023 ±
0007
3
3
0.009
0
.074 ±
0.060 ±
0.021
0.076 ±
0.010
0.131 ±
0.033
[<0.01 –
a
0.186 ± 0.02
1.53
0.006
0.014]
b
3
7
2.44
0.667
0.808
0.563
0.239
Table 3: Summary of the growth inhibitory potential (expressed as GI ) of selected nucleoside
5
0
4
3, 44
analogs against the NCI-60 tumor cell line panel with Sulforhodamine B (SRB) read-out at 48h.

ACCEPTED MANUSCRIPT
Full details (GI values for all cell lines) can be found in the Supporting Information. Values represent
5
0
a
mean ± SEM of two independent evaluations. Values in brackets represent the obtained GI values
5
0
b
from both experiments. Compounds 31 and 37 were only tested once.
Potent growth inhibitory activity was observed for 32, 33 and 34, while 31 and 37 were less active.
The activity spectrum of the former analogs was found to be broad, and especially pronounced for
leukemia cell lines as observed for e.g. clofarabine. GI values for several solid tumor cell lines (e.g.
5
0
MCF-7, HCT-116, U251, NCI-H460) are below 100 nM.
Additionally, these analogs were evaluated for their potential to inhibit the cell proliferation of three
different endothelial cell types. Agents capable of modifying the tumor vasculature, either by
antiangiogenic or vascular-disrupting action, are of interest for antitumor therapies both as single
agents and in combination with other chemotherapeutic drugs. Disruption of vascular networks in
4
5
solid tumors may induce their collapse by deprivation of oxygen and other nutrients. Nucleoside
analogs 31-34, 37 were found to potently inhibit the proliferation of the three endothelial cell types
studied, with the halogenated derivatives 32 (Cl) and 33 (Br) being most potent. Unfortunately, these
analogs also significantly inhibited the proliferation of Hel-fibroblasts.
HMVEC
HMEC-1
IC (µM)
HUVEC
Hel
Cpd.
IC50
µM)
IC (µM)
IC (µM)
5
0
50
50
(
3
1
2
0.061 ± 0.006 < 0.00128
0.067 ± 0.03
0.076 ± 0.019
3
0.0035 ± 0.0009 < 0.00128 0.0049 ± 0.0022
0.0045 ± 0.0003 0.0019 0.018 ± 0.006
0.018 ± 0.015 < 0.00128 0.0099 ± 0.0074
0.061 ± 0.006 N.D. N.D.
0.0092 ± 0.0066
3
3
0.017 ± 0.006
3
3
4
7
0.032 ± 0.023
N.D.
Table 4: Proliferation inhibition on different endothelial cell types: HMEC-1 (Human Dermal
Microvascular Endothelial Cells), HMVEC (Human Microvascular Endothelial Cells) and HUVEC
(
Human Umbilical Vein Endothelial Cells), as well as Hel (Human embryonic lung fibroblasts). IC50

ACCEPTED MANUSCRIPT
values represent the concentration of compound able to inhibit proliferation by 50% (Coulter Counter
cell count endpoint).
In vivo evaluation of compound 32
Encouraged by the strong in vitro anti-proliferative activity of analogs 31-34, 37, 49 and 81 against
various tumor cell lines (Table 1, 2 and 3), analog 32 was selected for in vivo evaluation of its
antitumor activity in a metastatic breast cancer xenograft mouse model employing MDA-MB-231
46
(
LM2) cells expressing firefly luciferase. Antitumor activity was assessed by measurement of both
the BLI signal (Figure 2, Panel A) and the tumor volume (Panel B). LM2 cells were orthotopically
engrafted in SCID mice and treatment commenced once the tumor was palpable (day 10). Compound
3
2 was injected intratumorally (0.3 mg/kg) 3 times a week for two consecutive weeks. The tumor
growth was significantly retarded starting from day 18, i.e. after four i.t. injections, as measured by
bioluminescent radiance (Panel A). The reduced tumor growth was also obvious when the tumor size
was measured using a digital caliper (Panel B). At day 35, mice were sacrificed and the tumors
removed, after which they were macroscopically examined (Panel B). The average control tumor
weight was 551 ± 74 mg versus 241 ± 61 mg for the treatment group.
To analyze the effect of 32 on lung metastasis, mice were covered with a black paper during imaging
to shield the primary tumor. Interestingly, the total metastasis burden in the lungs was significantly
lower in the treated group at day 34 versus the vehicle control group (Panel C). This indicates that the
nucleoside analog not only exhibits antitumor activity but also reduces breast cancer metastasis to
secondary organs.

ACCEPTED MANUSCRIPT
Figure 2: In vivo evaluation of 32: MDA-MB-231-LM2 cells were orthotopically engrafted in the
mammary fat pad of SCID mice. Compound 32 or vehicle were injected i.t. starting 10 days after
inoculation and dosed 3 times a week, for 2 consecutive weeks. Arrows indicate compound
administration. Panel A: BLI signal at regular time intervals. Data are mean ± STDEV, n = 5.
Representative bioluminescence images of vehicle control and 32-treated mice at day 34 are shown.
Panel B: Tumor volumes calculated from caliper measurements. Data are mean ± STDEV, n = 5. At
day 35 mice were sacrificed and the corresponding pictures of dissected tumors are shown. Panel C:
Lung metastasis at day 34 was quantified after shielding the primary tumor. Data are mean ± STDEV,
n = 5. Statistical significance is indicated (multiple t-test).

ACCEPTED MANUSCRIPT
Antiviral evaluation
Most analogs either showed no antiviral activity up to the highest concentration tested (100 µM) when
assayed against a panel of relevant viruses (see Experimental section), or the activity was accompanied
by significant toxicity for the host cell, making the derivatives non-specific (data not shown). Only a
few analogs combined antiviral activities with acceptable selectivity indices. The results are
summarized in Table 5 & Table 6. EAdo (5) showed activity against vaccinia virus (Table 5), although
it also potently inhibited Hel cell line proliferation (Table 6). Activity against vaccinia virus and HSV-
2
was also found for 59, which, however, generally inhibited cell proliferation (see also Table 1).
-
HSV-1
(KOS)
EC50 (µM)
HSV-1 (TK )
vaccinia
virus EC50
(µM)
b
c
HSV-2 (G)
EC50 (µM)
r
a
MCC
CC50
(µM)
Cpd.
5
a
KOS ACY
a
a
(µM)
EC50 (µM)
0
0
.73 ±
.07
N.D.
.25 ±
.80
.35 ±
.02
.16 ±
.0
>4
>4
>4
0.35 ± 0.05
0.73 ± 0.33
>100
20
5
6
9
6
2.10 ± 0.97 0.63 ± 0.09
1.27 ± 0.52
10.57 ± 4.84
≥20
≥100
5
3
0
0
16.3 ± 3.7
6.67 ± 2.67
>100
>100
>100
>100
>100
6
6
7
7
8
0
5.83 ± 3.17
>100
>100
>100
>100
>100
>100
>100
0
0
>100
>100
N.D.
Acyclovir
Cidofovir
0.2
1.5
0.2
1.2
10
2.0
0.5
>250
22
>250
>250
>100
N.D.
N.D.
N.D.
Ganciclovir
0.03
0.03
>100
Table 5: Antiviral activity against herpes virus-1 (HSV-1), herpes virus-2 (HSV-2), acyclovir-resistant
HSV (Thymidine kinase knock-out) and vaccinia virus cultured in Hel (human embryonic lung) cells.
a
Antiviral activity is expressed as EC values (µM) and represent the concentration of test compound
50
b
necessary to reduce viral-induced cytopathogenicity by 50%. MCC or minimal cytotoxic
concentration represents the concentration of test compound that is able to cause a microscopically
c
detectable alteration of normal cell morphology. CC represents the concentration (µM) of test
5
0
compound that reduces cell (Hel) proliferation by 50% as determined by Coulter Counter.

ACCEPTED MANUSCRIPT
The most promising antiviral activity was found for compound 66, which exhibited potent activity
against human cytomegalovirus (hCMV), with a reasonable selectivity index (10-fold). Other halogen-
substituted derivatives 67 (Br) and 68 (I) displayed elevated cytotoxicity and are therefore non-
selective. Apparently, the observed activity is specific for halogen bearing compounds, as the parent
compound without halogen (70) was inactive. Furthermore, changing the 6-chloride for an amino
group, mimicking a natural adenine, was detrimental for the activity (compare 66 and 80).
VZV
TK
+
-
CMV
CMV
VZV TK MCC
c
e
f
b
CC50
(AD-169) (Davis) MCC
CC50
Cpd.
5
(OKA)
(07_01)
d
d
a
a
(µM)
EC50
EC50
(µM)
(µM)
EC50
EC50 (µM) (µM)
(
µM)
(µM)
(
µM)
.82 ±
.24
.71 ±
.63
1
0
0.73 ±
0.07
1.64 ± 0.44
20
0.7 ± 0.1
≥4
≥4
20
4
1
0
3.41 ±
2.25
5
6
6
6
9
6
7
8
3.74 ± 2.69 ≥20
1.79
>4
N.D.
0
0
.53 ±
.09
5.25 ±
3.80
16.55
3.06
20
N.D.
0.51 ± 0.0
0.36 ± 0.0
≥20
2.76 ±
0.35 ±
0.02
0.35 ±
0.02
>10
0
0.35 ±
0.02
2.27 ± 1.74 ≥100
0.09
9.82 ±
2.31 ±
1.70
0.66 ±
0.15
8.26 ± 2.74
20
0.16 ± 0.0
20 0.16 ± 0.0
2.21
7
8
0
0
>100
>100
>100
17.49
>100
>100
N.D.
N.D.
>100
>100
>100
20
20
N.D.
N.D.
100
1
0
.26 ±
.73
36.74 ±
2.95
Acyclovir
Cidofovir
>440
N.D.
N.D.
>440
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
1.59 ±
1.45 ±
0.18
>30
0
N.D.
N.D.
N.D.
N.D.
0.35
Ganciclovi
11.75 ±
0.32
6.52 ±
0.53
>35
0
r
a
Table 6: Activity against varicella-zoster virus (VZV) in HEL (Human Embryonic Lung) culture;
-
TK : thymidylate kinase knock-out strain; EC values (µM) represent the concentration of compound
5
0
b,e
that reduces virus-induced cytopathicity by 50%.
MCC or minimal cytotoxic concentration
represents the concentration of test compound that causes a microscopically detectable alteration of
c,f
normal cell morphology. CC represents the concentration (µM) of test compound that reduces cell
5
0

ACCEPTED MANUSCRIPT
d
(
Hel) proliferation by 50% as determined by Coulter Counter.
Antiviral activity against
cytomegalovirus (CMV) in HEL (Human Embryonic Lung) culture; EC values (µM) represent the
5
0
concentration of compound that reduce virus-induced cytophathicity by 50%.
Conclusion
We have developed a nucleoside library around a 3’-C-ethynyl ribofuranose moiety, which was
combined with several purine nucleobases that bear substituents, which were previously found to
confer biological activity when combined with other sugar motifs. While 6-aryl purine nucleoside
analogs 13-17 were devoid of antiproliferative or antiviral activity, the 7-halogenated 7-deazapurine
analogs (31-34), as well as trifluoromethylated derivative 49 and the C7 unsubstituted analog 37 were
found to significantly inhibit the growth of three tumor cell lines. Their spectrum of activity was
thoroughly investigated (except for 49) by assaying against the NCI-60 panel, which also revealed
nanomolar activity against several solid tumor derived cell lines. Removal of the N7 nitrogen and
introduction of substitutions at C7 (particularly halogens, derivatives 31-34) was shown to lead to
significantly more potent antiproliferative nucleosides than the C2/C6 modified derivatives we have
previously reported. Additionally, analogs 31-34 potently inhibited proliferation of endothelial cell
types, which could be particularly interesting for the treatment of solid tumors. As a proof-of-concept,
analog 32 was investigated in a metastatic breast cancer xenograft mouse model. This derivative
inhibited both tumor growth as well as metastasis as assessed by means of BLI. However, 32 also
potently inhibited in vitro proliferation of Hel-fibroblasts, requiring further optimization to improve on
selectivity. Several 1,7-dideaza-3-C-ethynyl analogs were prepared and some of them found to be
potent inhibitors of human cytomegalovirus (hCMV) in vitro. Particularly, analog 66 requires further
evaluation.
In conclusion, the results presented in this paper showcase the utility of screening a focused nucleoside
library for both antiviral as well as antiproliferative activity as a means of finding novel hits for further
elaboration.

ACCEPTED MANUSCRIPT
Experimental
General experimental
All reagents and solvents were obtained from standard commercial sources and were of analytical
2
1
15
grade. Unless otherwise specified, they were used as received. 6, 18, were prepared according to
3
2,
literature procedures. Halogenated heterocycles 4-chloro-5-halo-pyrrolo[2,3-d]pyrimidine: 5-fluoro,
3
3
34
34
34
5
-chloro, 5-bromo, 5-iodo (40), were prepared from commercially available 4-chloro-7H-
pyrrolo[2,3-d]pyrimidine employing literature conditions.
All moisture sensitive reactions were carried out under an argon atmosphere. Reactions were carried
out at ambient temperature unless otherwise indicated. Analytical TLC was performed on Machery-
Nagel® pre-coated F254 aluminum plates and were visualized by UV followed by staining with basic
aq. KMnO or sulfuric acid-anisaldehyde spray. Column chromatography was performed using
4
Davisil® (40 – 63 µm) or on a Reveleris X2 (Grace/Büchi) automated Flash unit employing pre-
packed silica columns. Exact mass measurements were performed on a Waters LCT Premier XE™
Time of Flight (ToF) mass spectrometer equipped with a standard electrospray (ESI) and modular
Lockspray™ interface. Samples were infused in a MeCN / water (1:1) + 0.1% formic acid mixture at
1
00 µL / min. NMR spectra were recorded on a Varian Mercury 300 MHz spectrometer. Chemical
shifts (δ) are given in ppm and spectra are referenced to the residual solvent peak. Coupling constants
1
9
are given in Hz. In F-NMR, signals were referenced to CDCl or DMSO-d lock resonance frequency
3
6
according to IUPAC referencing with CFCl set to 0 ppm. After glycosylation, both the correct
3
stereochemistry at C1’ (β) and the correct regiochemistry (N9; purine numbering) of the glycosylation
1
13
products was ascertained by means of 2D NMR techniques (2D NOESY, H- C gHMBC,
respectively), either on the protected or on the deprotected derivative (depending on peak resolution).
Melting points were determined on a Büchi-545 apparatus and are uncorrected. Purity was assessed by
means of analytical LC-MS employing either

ACCEPTED MANUSCRIPT
(
1) Waters Alliance 2695 XE separation Module using a Phenomenex Luna® reversed-phase C18
(
2) column (3 ꢀm, 100x2.00 mm) and a gradient system of HCOOH in H2O (0.1%,
v/v)/HCOOH in MeCN (0.1%, v/v) at a flow rate of 0.4 mL / min, 10:90 to 0:100 in 9
minutes. High-resolution MS spectra were recorded on a Waters LCT Premier XE Mass
spectrometer.
(
2) Waters AutoPurification system (equipped with ACQUITY QDa (mass; 100 – 1000 amu)) and
2
998 Photodiode Array (220 – 400 nm)) using a Waters Cortecs® C18 (2.7 µm 100x4.6mm)
column and a gradient system of HCOOH in H O (0.2%, v/v) / MeCN at a flow rate of 1.44
2
mL / min, 95:05 to 00:100 in 6.5 minutes.
All obtained final compounds had purity > 95%, as assayed by analytical HPLC (UV) unless
otherwise indicated.
Chemistry
General procedure A (Suzuki Coupling (6-Cl-purine derivatives)):
In a flame-dried 25 mL round-bottom flask, equipped with a stir bar was added under argon, 6-chloro
purine nucleoside derivative 6 (0.5 mmol, 1 eq.), the corresponding boronic acid (1.5 eq.), anhydrous
K CO (1.5 eq.) and Pd(Ph P) (0.05 eq.). The flask was evacuated and refilled with argon three times.
2
3
3
4
Then, anhydrous degassed toluene (5 mL, 10 mL / mmol SM) was added and the mixture stirred for
approximately 5 min before being heated to 100 °C. After TLC monitoring showed full conversion of
the starting material (~ 2 – 4 H), the mixture was allowed to cool to room temperature, filtered and
evaporated till dryness. The residue was purified by column chromatography (0 → 5% acetone /
DCM).
General procedure B (nucleoside deprotection (ester hydrolysis)):
The ester protected nucleoside (1 eq.) was dissolved in 7N NH in MeOH and stirred at ambient
3
temperature until TLC showed full conversion (generally overnight to 36H). Then, the mixture was

ACCEPTED MANUSCRIPT
evaporated to dryness and the residue purified by column chromatography (typically 2 → 10% MeOH
/
DCM).
General procedure C (Vorbrüggen glycosylation of pyrrolo[2,3-d]pyrimidine and pyrrolo[2,3-
b]pyridine derivatives):
In a flame dried 2-neck round bottom flask, equipped with a stir bar was added the appropriate
heterocycle (1.1 eq.) under argon. Then, anhydrous MeCN (7.5 mL / mmol SM) was added, followed
by BSA (1.2 eq.). The resulting suspension was stirred at ambient temperature for approximately 10
15
min; after which a clear solution was obtained. Then, glycosyl donor, 18 (1 eq.) was added in one
portion, immediately followed by TMSOTf (1.25 eq.). The resulting solution was stirred at ambient
temperature for another 15 min, and then transferred to a pre-heated oil bath at 80 °C. Heating was
continued until TLC analysis showed full consumption of the starting material (~ 2 – 3 H), after which
the mixture was cooled to ambient temperature. EA was added, and the mixture poured into a sat. aq.
NaHCO solution. The layers were separated and the water layer extracted twice more with EA. The
3
organic layers were combined, dried over Na SO , filtered and evaporated till dryness. The residue
2
4
was purified by column chromatography 15% EA / Hexanes.
General procedure D (nucleophilic displacement with sodium azide):
The appropriate chloro-nucleoside (1 eq.) was dissolved in DMF (10 mL / mmol). Then, NaN (2 eq.)
3
was added and the mixture stirred at 65 °C for 30 min. Then, the mixture was cooled to ambient
temperature after which is was poured into half-saturated aq. NaHCO solution and EA. The layers
3
were separated and the water layer extracted two more times with EA. The organic layers were
combined, dried over Na SO , filtered and evaporated till dryness. The residue was purified by column
2
4
chromatography (EA / Hexanes) to yield the protected azido-nucleoside.
General procedure E (Staudinger reduction & iminophosphorane hydrolysis):
The appropriate azido-nucleoside (1 eq.) was dissolved in THF (10 mL / mmol). Then, PMe solution
3
(
1M in THF; 2 eq.) was added and the mixture stirred at ambient temperature until TLC analysis
showed full conversion of starting material (generally 30 min to 1H). Next, the solution was

ACCEPTED MANUSCRIPT
evaporated till dryness, and subsequently re-dissolved in MeCN (10 mL / mmol). To this solution was
added a 1M aq. HOAc solution (3.33 eq.), and the mixture heated in a pre-heated oil bath at 65 °C for
1
H. Next, the mixture was cooled to ambient temperature and poured into sat. aq. NaHCO solution.
3
DCM was added, layers were separated, and the water layer extracted two more times with DCM. The
organic layers were combined, dried over Na SO , filtered and evaporated till dryness. Purification by
2
4
column chromatography (EA / Hexanes) gave rise to the protected nucleoside aminopurine.
2
2
22
N9-β-
D-ribofuranosyl-6-phenylpurine (2) . 2 was prepared according to a literature procedure.
2
2 1
Spectral data are in accordance with literature values. H NMR (300 MHz, DMSO-d ) δ: 3.56 – 3.64
6
(
m, 1H, H-5’’), 3.69 – 3.76 (m, 1H, H-5’), 4.00 (dd, J = 7.8, 3.8 Hz, 1H, H-4’), 4.20 – 4.24 (m, 1H, H-
’), 4.66 (dd, J = 10.8, 5.7 Hz, 1H, H-2’), 5.13 (t, J = 5.7 Hz, 1H, OH-5’), 5.25 (d, J = 5.1 Hz, 1H,
OH-3’), 5.56 (d, J = 6.0 Hz, 1H, OH-2’), 6.10 (d, J = 5.4 Hz, 1H, H-1’), 7.58 – 7.65 (m, 3H, HPhe),
3
8
.82 – 8.85 (m, 2H, H ), 8.93 (s, 1H, H-8), 9.02 (s, 1H, H-2).
Phe
6
-chloro-N9-(2’,3’,5’-tri-O-acetyl-3’-C-ethynyl-β-D-ribofuranosyl)-purine (7). 6-chloropurine
(
0.46 g, 2.94 mmol, 1.5 eq.) was suspended in HMDS (16 mL, 8 mL / mmol SM) and a catalytic
amount of (NH ) SO was added. The mixture was then refluxed overnight. After cooling to ambient
4
2
4
temperature, the resulting solution was carefully evaporated till dryness, and the resulting oil further
dried at high vacuum (~ 1H). Then, anhydrous 1,2-dichloroethane (16 mL, 8 mL / mmol SM) was
21
added to dissolve the silylated heterocycle, after which 6 (0.67 g, 1.96 mmol, 1 eq.) was added via
syringe, immediately followed by TMSOTf (0.71 mL, 3.91 mmol, 2 eq.). The resulting solution was
subsequently refluxed for approximately 30 minutes and cooled to ambient temperature. Then, the
mixture was poured into sat. aq. NaHCO , and DCM was added. The layers were separated, and the
3
water layer extracted twice more with DCM. The organic layers were combined, dried over Na SO ,
2
4
filtered and evaporated till dryness. The residue was purified by column chromatography 5 → 7.5%
1
acetone / DCM to give 7 (0.693 g, 1.59 mmol) as a white foam in 81% yield. H NMR (300 MHz,
CDCl ) δ: 2.11 (s, 3H, acetyl-CH ), 2.14 (s, 3H, acetyl-CH ), 2.20 (s, 3H, acetyl-CH ), 2.86 (s, 1H,
3
3
3
3
ethynyl-H), 4.54 (dd, J = 14.7, 5.7 Hz, 1H, H-5’’), 4.61–4.68 (m, 2H, H-5’, H-4’), 6.05 (d, J = 4.5 Hz,

ACCEPTED MANUSCRIPT
1
3
1
H, H-2’), 6.30 (d, J = 4.8 Hz, 1H, H-1’), 8.52 (s, 1H, H-8), 8.77 (s, 1H, H-2). C NMR (75 MHz,
CDCl ) δ: 20.5 (acetyl-CH ), 20.9 (acetyl-CH ), 21.0 (acetyl-CH ), 63.3 (C-5’), 75.3, 76.0, 77.0, 79.9
3
3
3
3
(
C-2’), 81.6 (C-4’), 86.4 (C-1’), 131.9 (C-5), 143.1 (C-8), 151.7, 152.6 (2C), 168.4 (C=O), 168.7
+
(
C=O), 170.4 (C=O). HRMS (ESI): calculated for C H Cl N O ([M+H] ): 452.0968, found:
18
19
1
5
7
4
52.0970. [Remark: correct stereo- and regiochemistry was ascertained by transforming a small
1
5
amount into 3’-C-ethynyladenosine (5) and comparing NMR data to literature reference; which
confirmed the correct structure]
6
-phenyl-N9-(2’,3’,5’-tri-O-acetyl-3’-C-ethynyl-β-D-ribofuranosyl)-purine (8). 8 was prepared
according to General procedure A. 7 (0.22 g, 0.5 mmol) gave rise to 8 (0.101 g, 0.211 mmol) as a
white foam in 42% yield. Purification: 0 → 5% acetone / DCM; second column 0 → 35% EA /
1
Hexanes. H NMR (300 MHz, CDCl ) δ: 2.12 (s, 3H, acetyl-CH ), 2.16 (s, 3H, acetyl-CH ), 2.22 (s,
3
3
3
3
H, acetyl-CH ), 2.86 (s, 1H, ethynyl-H), 4.52–4.58 (m, 1H, H-5’’), 4.62 – 4.69 (m, 2H, H-4’, H-5’),
3
6
.12 (d, J = 4.8 Hz, 1H, H-2’), 6.40 (d, J = 4.8 Hz, 1H, H-1’), 7.54 – 7.61 (m, 3H, H ), 8.55 (s, 1H,
Phe
+
H-8), 8.75–8.79 (m, 2H, H ), 9.04 (s, 1H, H-2). HRMS (ESI): calculated for C H N O ([M+H] ):
Phe
24 23
4
7
4
79.1561, found: 479.1562.
-(4-methylphenyl)-N9-(2’,3’,5’-tri-O-acetyl-3’-C-ethynyl-β-D-ribofuranosyl)-purine (9). 9 was
6
prepared according to General procedure A. 7 (0.22 g, 0.5 mmol) gave rise to 9 (0.107 g, 0.217 mmol)
as a white foam in 43% yield. Purification: 0 → 5% acetone / DCM; second column 0 → 35 EA /
1
Hexanes. H NMR (300 MHz, CDCl ) δ: 2.12 (s, 3H, acetyl-CH ), 2.16 (s, 3H, acetyl-CH ), 2.22 (s,
3
3
3
3
H, acetyl-CH ), 2.46 (s, 3H, CH ), 2.86 (s, 1H, ethynyl-H), 4.51 – 4.58 (m, 1H, H-5’’), 4.61 – 4.70
3 3
(
m, 2H, H-4’, H-5’), 6.12 (d, J = 4.8 Hz, 1H, H-2’), 6.39 (d, J = 5.1 Hz, 1H, H-1’), 7.36 – 7.39 (m, 2H,
H-3 , H-5 ), 8.53 (s, 1H, H-8), 8.67–8.70 (m, 2H, H-2 , H-6 ), 9.01 (s, 1H, H-2). HRMS (ESI):
Phe
Phe
Phe
Phe
+
calculated for C H N O ([M+H] ): 493.1718, found: 493.1732.
2
5
25
4
7
6
-(4-methoxyphenyl)-N9-(2’,3’,5’-tri-O-acetyl-3’-C-ethynyl-β-D-ribofuranosyl)-purine (10). 10
was prepared according to General procedure A. 7 (0.22 g, 0.5 mmol) gave rise to 10 (0.131 g, 0.258
mmol) as a white foam in 52% yield. Purification: 0 → 5% acetone / DCM; second column 0 → 50
1
EA / Hexanes. H NMR (300 MHz, CDCl ) δ: 2.11 (s, 3H, acetyl-CH ), 2.15 (s, 3H, acetyl-CH ), 2.22
3
3
3

ACCEPTED MANUSCRIPT
(
s, 3H, acetyl-CH ), 2.86 (s, 1H, ethynyl-H), 3.91 (s, 3H, OCH ), 4.54 (dd, J = 14.4 Hz, 5.4 Hz, 1H, H-
3
3
5
7
’’), 4.62 – 4.66 (m, 2H, H-4’, H-5’), 6.11 (d, J = 4.8 Hz, 1H, H-2’), 6.39 (d, J = 5.1 Hz, 1H, H-1’),
.06 – 7.09 (m, 2H, H-3 , H-5 ), 8.51 (s, 1H, H-8), 8.80–8.83 (m, 2H, H-2 , H-6 ), 8.97 (s, 1H,
Phe
Phe
Phe
Phe
+
H-2). HRMS (ESI): calculated for C H N O ([M+H] ): 509.1667, found: 509.1654.
2
5
25
4
8
6
-(4-chlorophenyl)-N9-(2’,3’,5’-tri-O-acetyl-3’-C-ethynyl-β-
D-ribofuranosyl)-purine (11). 11 was
prepared according to General procedure A. 7 (0.22 g, 0.5 mmol) gave rise to 11 (0.09 g, 0.175 mmol)
1
as a slightly yellow foam in 35% yield. Purification: 0 → 37% EA / Hexanes. H NMR (300 MHz,
CDCl ) δ: 2.12 (s, 3H, acetyl-CH ), 2.16 (s, 3H, acetyl-CH ), 2.22 (s, 3H, acetyl-CH ), 2.87 (s, 1H,
3
3
3
3
ethynyl-H), 4.55 (dd, J = 14.7 Hz, 5.7 Hz, 1H, H-5’’), 4.62–4.69 (m, 2H, H-4’, H-5’), 6.10 (d, J = 4.8
Hz, 1H, H-2’), 6.39 (d, J = 4.8 Hz, 1H, H-1’), 7.52–7.56 (m, 2H, H-3 , H-5 ), 8.55 (s, 1H, H-8),
Phe
Phe
8
.76–8.81 (m, 2H, H-2 , H-6 ), 9.02 (s, 1H, H-2). HRMS (ESI): calculated for C H ClN O
Phe Phe 24 22 4 7
+
(
[M+H] ): 513.1172, found: 513.1170
6
-(4-fluorophenyl)-N9-(2’,3’,5’-tri-O-acetyl-3’-C-ethynyl-β-D-ribofuranosyl)-purine (12). 12 was
prepared according to General procedure A. 7 (0.22 g, 0.5 mmol) gave rise to 12 (0.075 g, 0.152 mmol)
1
as a slightly yellow foam in 30% yield. Purification: 0 → 37% EA / Hexanes. H NMR (300 MHz,
CDCl ) δ: 2.11 (s, 3H, acetyl-CH ), 2.16 (s, 3H, acetyl-CH ), 2.22 (s, 3H, acetyl-CH ), 2.86 (s, 1H,
3
3
3
3
ethynyl-H), 4.55 (dd, J = 15, 5.7 Hz, 1H, H-5’’), 4.62–4.69 (m, 2H, H-4’, H-5’), 6.10 (d, J = 4.8 Hz,
1
8
1
H, H-2’), 6.38 (d, J = 4.8 Hz, 1H, H-1’), 7.21 – 7.27 (m, 2H, H-3 , H-5 ), 8.54 (s, 1H, H-8), 8.82 –
Phe Phe
1
9
.87 (m, 2H, H-2 , H-6 ), 9.01 (s, 1H, H-2). F-NMR (282 MHz, CDCl ) δ: -108.6 – -108.5 (m,
Phe
Phe
3
+
F). HRMS (ESI): calculated for C H FN O ([M+H] ): 497.1467, found: 497.1469.
2
4
22
4
7
6
-Phenyl-N9-(3’-C-ethynyl-β-D-ribofuranosyl)-purine (13). 13 was prepared according to General
Procedure B. 8 (0.1 g, 0.209 mmol) gave rise to 13 (0.032 g, 0.091 mmol) as a white solid in 43%
1
yield. Purification: 3% MeOH / DCM. Melting point: 221 °C. H NMR (300 MHz, DMSO-d ) δ: 3.61
6
(
s, 1H, ethynyl-H), 3.73 – 3.84 (m, 2H, H-5’, H-5’’), 4.06 (dd, J = 4.5, 3.0 Hz, 1H, H-4’), 4.91 (t, J =
.2 Hz, 1H, H-2’), 5.18 (t, J = 5.1 Hz, 1H, OH-5’), 6.00 (d, J = 6.9 Hz, 1H, OH-2’), 6.09 (d, J = 7.5
Hz, 1H, H-1’), 6.14 (s, 1H, OH-3’), 7.59 – 7.65 (m, 3H, H-3 , H-4 , H-5 ), 8.81 – 8.87 (m, 2H, H-
7
Phe
Phe
Phe
1
3
2Phe, H-6 ), 8.95 (s, 1H, H-8), 9.03 (s, 1H, H-2). C NMR (75 MHz, DMSO-d ) δ: 61.9 (C-5’), 72.8,
Phe
6

ACCEPTED MANUSCRIPT
7
7.3, 78.0 (C-2’), 82.6, 86.2 (C-1’), 87.8 (C-4’), 128.7 (2C, C-3 , C-5 ), 129.4 (2C, C-2 , C-6 ),
Phe
Phe
Phe
Phe
1
30.8 (C-5), 131.2 (C-4 ), 135.2 (C-1 ), 145.2 (C-8), 152.0 (C-2), 152.5 (C-4), 153.1 (C-6). HRMS
Phe
Phe
+
(
ESI): calculated for C H N O ([M+H] ): 353.1244, found: 353.1259. [Remark: The final product
18 17 4 4
contained ~1.5 eq. of acetamide; which was added to the MW of the product.]
-(4-Methylphenyl)-N9-(3’-C-ethynyl-β- -ribofuranosyl)-purine (14). 14 was prepared according
6
D
to General Procedure B. 9 (0.1 g, 0.203 mmol) gave rise to 14 (0.040 g, 0.109 mmol) as a white solid
1
in 54% yield. Purification: 3% MeOH / DCM. Melting point: 221 °C. H NMR (300 MHz, DMSO-d )
6
δ: 2.42 (s, 3H, CH ), 3.61 (s, 1H, ethynyl-H), 3.71 – 3.87 (m, 2H, H-5’, H-5’’), 4.06 (dd, J = 4.5, 3.0
3
Hz, 1H, H-4’), 4.90 (t, J = 7.2 Hz, 1H, H-2’), 5.19 (dd, J = 5.7, 4.8 Hz, 1H, OH-5’), 5.99 (d, J = 6.9
Hz, 1H, OH-2’), 6.08 (d, J = 7.5 Hz, 1H, H-1’), 6.13 (s, 1H, OH-3’), 7.41 – 7.44 (m, 2H, H-3 , H-
Phe
1
3
5Phe), 8.74 – 8.77 (m, 2H, H-2 , H-6 ), 8.92 (s, 1H, H-8), 8.99 (s, 1H, H-2). C NMR (75 MHz,
Phe
Phe
DMSO-d ) δ: 21.1 (CH ), 61.9 (C-5’), 72.8, 77.3, 78.0 (C-2’), 82.6, 86.2 (C-1’), 87.8 (C-4’), 129.4
6
3
(
4C, C ), 130.6 (C-5), 132.5 (C-1 ), 141.3 (C-4 ), 144.9 (C-8), 151.9 (C-2), 152.4 (C-4), 153.2 (C-
Phe Phe Phe
+
6
). HRMS (ESI): calculated for C H N O ([M+H] ): 367.1401, found: 367.1387. [Remark: The
19 19 4 4
final product contained ~2.5 eq. of acetamide; which was added to the MW of the product.]
-(4-Methoxyphenyl)-N9-(3’-C-ethynyl-β- -ribofuranosyl)-purine (15). 15 was prepared according
6
D
to General Procedure B. 10 (0.131 g, 0.258 mmol) gave rise to 15 (0.060 g, 0.157 mmol) as a white
1
solid in 61% yield. Purification: 0 → 5% MeOH / DCM. Melting point: 219 °C. H NMR (300 MHz,
DMSO-d ) δ: 3.60 (s, 1H, ethynyl-H), 3.71 – 3.85 (m, 2H, H-5’, H-5’’), 3.87 (s, 3H, OCH ), 4.05 (dd,
6
3
J = 4.5, 3.3 Hz, 1H, H-4’), 4.90 (dd, J = 7.5, 6.9 Hz, 1H, H-2’), 5.20 (dd, J = 5.7, 4.8 Hz, 1H, OH-5’),
5
2
.96 (d, J = 6.9 Hz, 1H, OH-2’), 6.07 (d, J = 7.5 Hz, 1H, H-1’), 6.11 (s, 1H, OH-3’), 7.15 – 7.18 (m,
1
3
H, H-3 , H-5 ), 8.84 – 8.87 (m, 2H, H-2 , H-6 ), 8.89 (s, 1H, H-8), 8.95 (s, 1H, H-2). C NMR
Phe
Phe
Phe
Phe
(
75 MHz, DMSO-d ) δ: 55.3 (OCH ), 61.9 (C-5’), 72.8, 77.3, 77.9 (C-2’), 82.6, 86.2 (C-1’), 87.8 (C-
6 3
4
1
3
’), 114.2 (2C, C-3 , C-5 ), 127.6 (C-1 ), 130.2 (C-5), 131.2 (2C, C-2 , C-6 ), 144.7 (C-8),
Phe Phe Phe Phe Phe
+
51.9, 152.2, 152.9 (C-4), 161.8 (C-4 ). HRMS (ESI): calculated for C H N O ([M+H] ):
Phe
19 19
4
5
83.1350, found: 383.1359. [Remark: The final product contained ~1.5 eq. of acetamide; which was
added to the MW of the product.]