Covalent analogues of DNA base-pairs and triplets. Part 2: Synthesis and cytostatic activity of bis(purin-6-yl)acetylenes, -diacetylenes and related compounds

Article abstract of DOI:10.1016/S0960-894X(02)00077-X

The title bis(purin-6-yl)acetylenes, -diacetylenes, -ethylenes and -ethanes were prepared as covalent base-pair analogues starting from 6-ethynylpurines and 6-iodopurines by cross-coupling and homo-coupling reactions and hydrogenations. The bis(purin-6-yl

Full text of DOI:10.1016/S0960-894X(02)00077-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1055–1058
Covalent Analogues of DNA Base-Pairs and Triplets. Part 2:y
Synthesis and Cytostatic Activity of Bis(purin-6-yl)acetylenes,
-diacetylenes and Related Compounds
Michal Hocek* and Ivan Votruba
´
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam.2,
CZ-16610 Prague 6, Czech Republic
Received 10 December 2001; revised 24 January 2002; accepted 28 January 2002
Abstract—The title bis(purin-6-yl)acetylenes, -diacetylenes, -ethylenes and -ethanes were prepared as covalent base-pair analo-
gues starting from 6-ethynylpurines and 6-iodopurines by cross-coupling and homo-coupling reactions and hydrogenations.
The bis(purin-6-yl)acetylenes and -diacetylenes exhibited significant cytostatic activity in vitro (IC50=0.4–1.0 mmol/l). # 2002
Elsevier Science Ltd. All rights reserved.
The effect of many clinically used antitumor agents is
based on DNA cross-linking2 or on intercalation3 to
DNA. Numerous models and analogues of Watson–
Crick base pairs consisting of annelated4 or cross-
linked5 purine and pyrimidine heterocycles or even
more simple aromatic rings6,7 have been prepared.
Recently, also the first covalently linked analogues of
Hoogsteen triplets were prepared1 in our laboratory.
Such base-pairs/triplets analogues may interact with
DNA (e.g., by intercalation); if incorporated into single
stranded DNA, they are complementary to abasic site
of a damaged DNA strand; or alternatively, if incorpo-
rated to duplex, they form permanent cross-links.
dimers, trimers and tetramers with the linkers of various
lengths were prepared11 and exhibited diverse types of
biological activity (inhibition of adenosine kinase, ribo-
somal peptidyltransferase, etc.).
Purines bearing carbon substituents in positions 2 or 6
possess a broad spectrum of biological activity. Thus,
6-methylpurine is highly cytotoxic,12 while 2-alkynyl-
adenosines are an important class of adenosine receptors
agonists.13 Recently, a cytokinin activity of 6-(arylalky-
nyl)-, 6-(arylalkenyl)- and 6-(arylalkyl)purines,14 a cyto-
static activity of 6-(trifluoromethyl)purine riboside15 and
of 6-arylpurine ribonucleosides,16 a corticotropin-releas-
ing hormone antagonist activity of some 2,8,9-trisub-
stituted-6-arylpurines17 and an antimycobacterial activity
of 9-benzyl-6-arylpurines18 were also reported.
A number of diverse purine–purine conjugates contain-
ing linkage (9–9, 8–8, 9–8, 9–7, 9–6 and 6–6) of various
lengths, including double- and triple-linked pur-
inophanes, have been prepared8 in order to study the
p–p stacking of purine bases. The purine–purine dimers
with a 6,60-pyridine-2,6-bis(carboxamido) linker have
A combination of the unique structural features of the
above mentioned classes of compounds led us to the
design of a new groupof base-apir analogues
A–E
been recently prepared9 to study its H-bonding proper-
(Chart 1) based on covalent purine–purine conjugates
linked through positions 6 and 60 by carbon linkages.
The linkers differ from rigid linear acetylene-linkage,
bend E- or Z-vinylene-linkers to conformationally flex-
ible saturated ethylene-spacer. Such carbon linkers con-
nected to carbon atoms of the heterocycles are expected
to be stable towards enzymatic degradation. Appar-
ently, such ‘extended’ analogues are larger than the
parent Watson–Crick base-pairs but on the other hand
they could be capable of intercalation within the DNA
0
ties as potential artificial receptors. Methylene N6,N6 -
linked-adenine-adenine dimers are formed by the reac-
tion of carcinogenic formaldehyde with DNA, which
was proved by independent synthesis10 of the products.
0
A
variety of other N6,N6 -linked-adenine-adenine
*Corresponding author. Tel.: +420-2-2018-324; fax: +420-2-33331-
271; e-mail: hocek@uochb.cas.cz
yFor Part I, See ref 1.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00077-X

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M.Hocek, I.Votruba / Bioorg.Med.Chem.Lett.12 (2002) 1055–1058
replication fork and thus inhibit its synthesis ‘de novo’
and cell division. This is a preliminary communication
reporting their synthesis and cytostatic activity.
For the synthesis of the acetylene derivative 5a, an
alternative method based on recently published proce-
dure22 has been used. The reaction of the 6-iodopurine
2a with the terminal acetylene 1a was made in presence
of tetrabutylammonium fluoride (TBAF) as base, cata-
lytic amount of CuI and PdCl2(PPh3)2 in THF at room
temperature to give23 the desired acetylene 5a in good
yield of 57%. This approach has also been used for the
synthesis of other related symmetrically and asymme-
trically disubstituted acetylenes 5b and 5c differing by
the substituent in the positions 9 and 90 starting from
the appropriate iodopurines 2a and 2c and ethynylpur-
ines 1b and 1c. Catalytic hydrogenation of the acetylene
5a on Lindlar catalyst afforded the complementary Z-
ethylene derivative 6a in a low yield of 11% accom-
panied by the fully saturated compound 4a (19%). Oxi-
dative homo-coupling24 of the terminal acetylenes 1a–1c
in presence of CuCl and TMEDA25 afforded26 the 1,4-
bis(purin-6-yl)diacetylenes 7a–7c in good yields of
50–60%.
Chemistry
The synthesis of the target compounds was based on
standard acetylene chemistry (Scheme 1) using the
9-benzyl-6-ethynylpurine19 (1a) as a key starting com-
pound. Attempted Sonogashira reaction of this com-
pound with 9-benzyl-6-iodopurine (2a) in presence of
CuI, Pd(PPh3)4 and Et3N in DMF did not give the
expected bis(purin-6-yl)acetylene 5a but its partly
reduced E-ethylene derivative 3a in 27% yield.20 The
formation of this product could be explained by a
reductive addition21 of the iodopurine 2a on the acetyl-
ene 1a. Catalytic hydrogenation of the ethylene deriva-
tive 3a on Pd/C gave the fully saturated ethane
derivative 4a in good yield of 80%.
Cytostatic activity evaluation
The target base-pair analogues 3–7 were tested27 on
their in vitro inhibition of the cell growth in the follow-
ing cell cultures: mouse leukemia L1210 cells (ATCC
CCL 219); murine L929 cells (ATCC CCL 1); human
cervix carcinoma HeLa S3 cells (ATCC CCL 2.2); and
human T lymphoblastoid CCRF-CEM cell line (ATCC
CCL 119). The results (Table 1) show that while the
bis(purinyl)acetylenes 5a–5c and -diacetylenes 7a–7c
exhibited a significant cytostatic effect in these assays
(IC50=0.3–15 mM), the partly and fully saturated deri-
vatives bearing vinylene and ethylene linkers, compounds
3a, 4a and 6a, were entirely inactive. The nature of sub-
stituents in the positions 9 of the purine rings has only
little effect on the activity (derivatives a–c in each series
of active compounds). Among the assays tested, the T-
lymphoblastoid CCRF-CEM and leukemia L1210 cells
were the most sensitive to the action of these compounds.
Chart 1.
Scheme 1.

M.Hocek, I.Votruba / Bioorg.Med.Chem.Lett.12 (2002) 1055–1058
1057
Table 1. Cytostatic activity data for compounds 3–7
IC50 (mM)a
7. (a) Ogawa, A. K.; Abou-Zied, O. K.; Tsui, V.; Jimenez, R.;
Case, D. A.; Romesberg, F. E. J.Am.Chem.Soc. 2000, 122,
9917. (b) Abou-Zied, O. K.; Jimenez, R.; Romesberg, F. E.
J.Am.Chem.Soc. 2001, 123, 4613.
Compd
L1210
L929
HeLa S3
CCRF-CEM
8. (a) Leonard, N. J.; Ito, K. J.Am.Chem.Soc.
1973, 95,
FUDRb <0.02 (ꢃ0.002)
>25
na
na
>25
na
na
na
0.5 (ꢃ0.04)
4010. (b) Akahori, K.; Hama, F.; Sakata, Y.; Misumi, S. Tet-
rahedron Lett. 1984, 25, 2379. (c) Seyama, F.; Akahori, K.;
Sakata, Y.; Misumi, S.; Aida, M.; Nagata, C. J.Am.Chem.
Soc. 1988, 110, 2192.
9. Crisp, G. T.; Jiang, Y. L. Tetrahedron 1999, 55, 549.
10. Chaw, Y. F. M.; Crane, L. E.; Lange, P.; Shapiro, R.
Biochemistry 1980, 19, 5525.
11. (a) Zemlicka, J.; Owens, J. J.Org.Chem. 1977, 42, 517. (b)
Bhuta, P.; Li, C.; Zemlicka, J. Biochem.Biophys.Res.Com-
mun. 1977, 77, 1237. (c) Zemlicka, J. Biochemistry 1980, 19,
163. (d) Murata, M.; Bhuta, P.; Owens, J.; Zemlicka, J. J.
Med.Chem. 1980, 23, 781. (e) Zemlicka, J. Tetrahedron Lett.
1984, 25, 5619. (f) Agathocleous, D. C.; Page, P. C. B.; Cos-
stick, R.; Galpin, I. J.; McLennan, A. G.; Prescott, M. Tetra-
hedron 1990, 46, 2047.
3a
4a
5a
5b
5c
6a
7a
7b
7c
nac
na
na
na
1.8 (ꢃ0.17) 5.3 (ꢃ0.6)
0.9 (ꢃ0.08)
0.9 (ꢃ0.08) 1.9 (ꢃ0.2)
6.0 (ꢃ0.6) 0.32 (ꢃ0.07)
1.5 (ꢃ0.12) 3.0 (ꢃ0.3) 15.0 (ꢃ1.8) 0.36 (ꢃ0.03)
na
na
na
na
na
0.43 (ꢃ0.03)
0.37 (ꢃ0.03) 6.3 (ꢃ0.5)
0.7 (ꢃ0.05) 1.0 (ꢃ0.12) 13.3 (ꢃ1.3) 0.58 (ꢃ0.04)
0.5 (ꢃ0.07) 1.3 (ꢃ0.11) 15.0 (ꢃ1.1) 0.37 (ꢃ0.03)
aValues are means of four experiments, standard deviation is given in
parentheses.
b1-(b-d-2-deoxy-erythro-pentofuranosyl)-5-fluorouracil.
cna, not active (inhibition of cell growth at 10 mM was lower than
20%).
12. Montgomery, J. A.; Hewson, K. J.Med.Chem. 1968, 11,
48.
In conclusion, the substituted bis(purin-6-yl)acetylenes
and -diacetylenes represent a novel class of anti-
neoplastic compounds. They are characterized by a rigid
linear (acetylene or diacetylene) linker between the two
purine rings. It is not yet clear whether the role of this
spacer is just in being a linear linker of certain steric
parameters or whether the alkyne forms covalent or
non-covalent adducts with the target cell system (DNA
or enzyme). Though we suspect that the intercalation or
other interaction with DNA might be the mode of
action of this class of compounds, these problems
remain the subjects for further investigation.
13. Review: Muller, C. E. Curr.Med.Chem. 2000, 7, 1269.
14. Brathe, A.; Gundersen, L. L.; Rise, F.; Eriksen, A. B.;
Vollsnes, A. V.; Wang, L. N. Tetrahedron 1999, 55, 211.
15. Hockova, D.; Hocek, M.; Dvorakova, H.; Votruba, I.
Tetrahedron 1999, 55, 11109.
16. (a) Hocek, M.; Holy, A.; Votruba, I.; Dvorakova, H. J.
Med.Chem. 2000, 43, 1817. (b) Hocek, M.; Holy, A.; Votruba,
I.; Dvorakova, H. Collect.Czech.Chem.Commun.
2000, 65,
1683. (c) Hocek, M.; Holy, A.; Votruba, I.; Dvorakova, H.
Collect.Czech.Chem.Commun. 2001, 66, 483.
17. Cocuzza, A. J.; Chidester, D. R.; Culp, S.; Fitzgerald, L.;
Gilligan, P. Bioorg.Med.Chem.Lett. 1999, 9, 1063.
18. Bakkestuen, A. K.; Gundersen, L. L.; Langli, G.; Liu, F.;
Nolsøe, J. M. J. Bioorg.Med.Chem.Lett. 2000, 10, 1207.
19. Tanji, K.; Higashino, T. Chem.Pharm.Bull.
1935.
1988, 36,
Acknowledgements
20. Its E-configuration has been assigned based on NOE of its
cycloadduct with cyclopentadiene. The details are beyond the
scope of this communication and will appear in a forthcoming
full-paper.
21. An analogous reductive addition has been recently
observed for the reaction of aryl halides with substituted
acetylenes in presence of NaOMe: Wu, M.-J.; Wei, L.-M.; Lin,
F.-C.; Leou, S.-P.; Wei, L.-L. Tetrahedron 2001, 57, 7839. In
our case, apparently Et3N was the reducing agent.
This work is a part of a research project Z4055905. It
was supported by the Grant Agency of the Czech
Republic (grant No. 203/00/0036). NMR spectra were
measured and interpreted by Dr. Hana Dvorakova
(Prague Institute of Chemical Technology).
References and Notes
22. Mori, A.; Shimada, T.; Kondo, T.; Sekiguchi, A. Synlett
2001, 649.
1. Hocek, M.; Stara, I. G.; Stary, I.; Dvorakova, H. Tetra-
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2. Review: Rajski, S. R.; Williams, R. M. Chem.Rev. 1998,
98, 2723.
23. Typical procedure: A degassed 0.165 M solution of TBAF
trihydrate in THF (24 mL, 4 mmol) was added dropwise to an
argon purged flask containing 1a (407 mg, 1.67 mmol), 2a (560
mg, 1.67 mmol), CuI (60 mg, 0.3 mmol), PdCl2(PPh3)2 (100
mg, 0.14 mmol) at ambient temperature and the mixture was
stirred for 4 h. The solvent was evaporated and the residue
was chromatographed on silica gel (200 g, ethyl acetate–light
petroleum 1:1) to give the 1,2-bis(9-benzylpurin-6-yl)acetylene
(5a) (420 mg, 57%), mp254–257 ꢀC (CH2Cl2/heptane), FAB
MS, m/z (rel.%): 443 (5) [M+H], 91 (100). IR (CHCl3):
n=1583, 1448, 1403, 1330 cmꢁ1. 1H NMR (500 MHz, CDCl3):
5.49 (s, 4H, CH2Ph); 7.30–7.40 (m, 10H, H–arom); 8.15 (s, 2H,
H-8); 9.07 (s, 2H, H-2). 13C NMR (100.6 MHz, CDCl3): 47.52
(CH2Ph); 90.89 (Cꢂ); 127.89, 128.80 and 129.28 (CH–arom.);
134.83 and 135.09 (C-5 and C–i-arom); 140.40 (C-6); 145.83
(CH-8); 152.04 (C-4); 152.84 (C-2). EI HR MS, found:
442.1691; C26H18N8 [M] requires: 442.1654.
3. Review: Baguley, B. C. Anti-Cancer Drug.Des. 1991, 3, 1.
4. Bhat, B.; Leonard, N. J.; Robinson, H.; Wang, A. H. J.
J.Am.Chem.Soc. 1996, 118, 10744 and references cited therein.
5. (a) Nagatsugi, F.; Uemura, K.; Nakashima, S.; Maeda, M.;
Sasaki, S. Tetrahedron Lett. 1995, 36, 421. (b) Nagatsugi, F.;
Uemura, K.; Nakashima, S.; Maeda, M.; Sasaki, S. Tetra-
hedron 1997, 53, 3035. (c) Nagatsugi, F.; Kawasaki, T.; Usui,
D.; Maeda, M.; Sasaki, S. J.Am.Chem.Soc. 1999, 121, 6753.
(d) Nagatsugi, F.; Usui, D.; Kawasaki, T.; Maeda, M.; Sasaki,
S. Bioorg.Med.Chem.Lett. 2001, 11, 343.
6. (a) Quiao, X.; Kishi, Y. Angew.Chem,. Int.Ed.Engl. 1999,
38, 928. (b) Qiu, Y. L.; Li, H. Y.; Topalov, G.; Kishi, Y. Tet-
rahedron Lett. 2000, 41, 9425. (c) Li, H. Y.; Qiu, Y. L.;
Moyroud, E.; Kishi, Y. Angew.Chem,. Int.Ed.Engl. 2001, 40,
1471. (d) Li, H. Y.; Qiu, Y. L.; Kishi, Y. Chem.Bio.Chem.
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24. Review: Siemsen, P.; Livingston, R. C.; Diederich, F.
Angew.Chem.Int.Ed.Engl. 2000, 39, 2632.
25. Hay, A. S. J.Org.Chem. 1962, 27, 3320.

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M.Hocek, I.Votruba / Bioorg.Med.Chem.Lett.12 (2002) 1055–1058
1491, 1457, 1435 1404, 1330 cmꢁ1 1H NMR (400 MHz,
.
26. Typical procedure: A solution of CuCl (20 mg, 0.2
mmol), TMEDA (37 mL, 0.25 mmol) in DME (2 mL) was
stirred at ambient temperature while a solution of 1a (244
mg, 1 mmol) in DME (8 mL) was added dropwise. The
stirring of the mixture in air atmosphere was continued for
4 h and was allowed to stand overnight. Then the solvent
was evaporated and the residue was chromatographed on
silica gel (100 g, ethyl acetate–light petroleum 1:1) to give
the 1,4-bis(9-benzylpurin-6-yl)butadiyne (7a) (137 mg, 59%);
mp215 ꢀC dec. (CH2Cl2/heptane); FAB MS, m/z (rel.%):
467 (8) [M+H], 91 (100). IR (CHCl3): n=2157, 1575, 1497,
CDCl3): 5.46 (s, 4H, CH2Ph); 7.26–7.38 (m, 10H, H-arom);
8.12 (s, 2H, H-8); 9.01 (s, 1H, H-2). 13C NMR (100 MHz,
CDCl3): 47.55 (CH2Ph); 78.59 and 80.68 (CꢂC); 127.96,
128.83 and 129.27 (CH–arom.); 134.62 and 135.52 (C–arom
and C-5); 139.72 (C-6); 145.80 (CH-8); 151.98 (C-4); 152.81
(CH-2). FAB HR MS, found: 467.1700; C28H19N8 [M+H]
requires: 467.1732. Anal. calcd for C28H18N8 (466.5): C,
72.10; H, 3.89; N, 24.02; found: C, 71.96; H, 3.82; N,
23.81.
27. For experimental details of the assays see ref 16a.