3'-β-ethynyl and 2'-deoxy-3'-β-ethynyl adenosines: First 3'-β- branched-adenosines substrates of adenosine deaminase

Article abstract of DOI:10.1016/S0960-894X(99)00639-3

The 3'-C-branched-adenosine and 2'-deoxyadenosine analogues 1-7 were tested as substrate of adenosine deaminase. The 9-(3'-C-ethynyl-β-D-ribo- pentofuranosyl)adenine 1 and its 2'-deoxy analogue 7 were deaminated by the enzyme while the vinyl and ethyl derivatives 2 and 3 were not. The 9-(3'-C- branched-β-D-xylo-pentofuranosyl)adenines 4-6 were deaminated by the deaminase.

Full text of DOI:10.1016/S0960-894X(99)00639-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 139±141
0
0
0
3
First 3 -ꢀ-Branched-Adenosines Substrates of Adenosine
-ꢀ-Ethynyl and 2 -Deoxy-3 -ꢀ-Ethynyl Adenosines:
0
Deaminase
Denis Tritsch,* Pierre M. J. Jung, Alain Burger and Jean-FrancË ois Biellmann
Laboratoire de Chimie Organique Biologique UMR 7509 du CNRS, Universite Louis Pasteur, Faculte de Chimie, 1 rue Blaise Pascal,
7008 Strasbourg Cedex, France
6
Received 1 July 1999; accepted 15 November 1999
0
0
AbstractÐThe 3 -C-branched-adenosine and 2 -deoxyadenosine analogues 1±7 were tested as substrate of adenosine deaminase.
0
0
The 9-(3 -C-ethynyl-b-d-ribo-pentofuranosyl)adenine 1 and its 2 -deoxy analogue 7 were deaminated by the enzyme while the vinyl
0
and ethyl derivatives 2 and 3 were not. The 9-(3 -C-branched-b-d-xylo-pentofuranosyl)adenines 4±6 were deaminated by the de-
aminase. # 2000 Elsevier Science Ltd. All rights reserved.
0
Adenosine deaminase (EC 3.5.4.4) plays an important
regulatory role in purine metabolism. It catalyses the
ribo-con®guration, only the C-3 -ethynyl derivative 1
was a substrate of adenosine deaminase. The Km was
only about 2- to 3-fold higher than that for adenosine.
On the other hand the Vmax was 50-fold lower than the
Vmax of adenosine hydrolysis. Compounds 2 and 3 were
0
0
irreversible hydrolysis of (2 -deoxy)adenosine to (2 -
deoxy)inosine and ammonia. It is well established that
some adenosine analogues of chemotherapeutic interest
are rapidly deaminated to inactive or less active inosine
0
resistant to deamination as previously observed with 3 -
8
1
derivatives. Therefore the factors governing the enzyme
b-methyl-adenosine, and both were weak inhibitors. In
speci®city are of considerable importance for the design
of new compounds with preserved chemotherapeutic
ecacy. We present here our study into the action of
contrast all the xylo compounds 4±6 were deaminated
by the enzyme. The Km of the three xylo compounds
were similar, about 3- to 4-fold higher than the Km of
adenosine. The rate of hydrolysis was 3- to 5-fold lower.
These compounds would be transformed in vivo to their
0
adenosine deaminase on the 3 -branched adenosine and
0
2
-deoxyadenosine analogues 1±7.
0
corresponding inosine derivatives. Compound 7, the 2 -
0
has been described elsewhere. Compounds 3 and 6
Preparation of C-3 substituted nucleosides 1, 2, 4, 5 and
2
deoxy analogue of compound 1, was also a substrate of
the enzyme but the anity was too low to determine the
kinetic constants (Km and Vmax). The rate of hydrolysis
was proportional to the concentration of compound 7,
,3
7
were prepared via catalytic hydrogenation of the ethyn-
yl compounds 1 and 4, respectively, in almost quantita-
4
,5
tive yield.
showing ®rst order kinetics as observed at substrate
9
concentrations much lower than the K . The ecacy of
m
The e€ect of the C , C and Csp3 substituent at the
sp
the enzymatic reaction shown by the ratio (Vmax/K ) is
m
sp2
0
hydroxyl group in compound 7, on adenosine deami-
C-3 stereocenter (ribo 1±3 versus xylo 4±6) and of the
0
higher for the ethynyl deoxy compound 7 than for the
ethynyl compound 1. Compounds 1 and 7 are the ®rst
2
0
strates of adenosine deaminase.
nase activity was studied. The di€erent analogues were
tested on puri®ed adenosine deaminase to compare their
3 -b-branched adenosines (ribo and erythro) to be sub-
0
deoxyadenosine (Table 1). Among the compounds with
kinetic properties against those of adenosine and of 2 -
Adenosine analogues modi®ed on the furanose ring or
on the purine ring have been reported to be substrates
of adenosine deaminase.1
0±12
At C-3 position, analo-
0
gues, in the ribo- and in the xylo-con®guration, where
the hydroxyl group has been replaced by a hydrogen, an
amino group, an azido group or by a halogen atom
*Corresponding author. Fax: +33-388-411524; e-mail: dtritsch@
chimie.u-strasbg.fr
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00639-3

1
40
D. Tritsch et al. / Bioorg. Med. Chem. Lett. 10 (2000) 139±141
12
have been shown to be deaminated by the enzyme.
The C-methyl- and C-hydroxylmethyl-3 -b-adenosine
derivatives were not substrates. These observations raise
the question of why the enzyme tolerates the ethynyl
compound but not the three other derivatives.
0
derivatives are described to be resistant to deamina-
1
1
tion. On the other hand the C-methyl- and the C-
hydroxymethyl-3 -a-adenosine derivatives are reported
0
to be susbstrates of adenosine deaminase. From these
results it appears that the nature of the C-3 substituents
Nucleosides may take several conformations in rapid
13
1
1
equilibrium. Major factors in¯uencing the conforma-
tional ¯exibility are the following: (i) rotation around
0
0
ribose puckering, (iii) rotation around the C4 ±C5
has a dramatic e€ect on their recognition by the
enzyme.
the glycosidic N9±C1 bond (syn/anti orientation), (ii)
0
0
bond. The conformational requirements for an active
enzyme±substrate complex can be deduced from the
three dimensional structure of the binary complex (ade-
nosine deaminase-inhibitor).14 Adenosine would adopt
In the present work, we have shown that the three xylo
compounds 4±6 were deaminated by adenosine deami-
nase. The Km and the Vmax values were similar to those
reported in the literature for other xylo-derivatives.1
Among the tested nucleosides in the ribo series, the
ethynyl analogue 1 was surprisingly substrate of the
enzyme. The Km was only 3-fold higher than the Km of
adenosine, suggesting that analogue 1 ®tted well in
the active site cavity of the enzyme. The deamination
rate was 50-fold lower than that of adenosine. The C-6
carbon of the base in the ethynyl compound may not be
ideally situated in relation to the catalytic groups of the
enzyme. The corresponding methyl,11 ethyl and vinyl
0±12
ꢀ
the anti-conformation (w values from � 106 to � 110 ),
0
0
with the C4 ±C5 bond in a +sc orientation (g values
ꢀ
0
from 54 to 57 ) and with a 3 -endo sugar pucker
(North).14 Therefore only the compounds with the
proper requirements would bind to the active site. As
the tested analogues contain all the sites required for
binding to the enzyme,14 the absence of recognition
could be due to an inappropiate conformation of the
0
analogues. Substituents at position C-3 were reported
to increase the conformational rigidity of the furanose
ring.1
the nucleoside analogue to the active site. In the case of
5,16
This could hinder the productive binding of
0
0
3 -b-methyl-adenosine, known to be resistant to deami-
Table 1. Kinetic constants for the deamination of adenosine, 2 -
deoxyadenosine and compounds 1±7 by adenosine deaminase.6
nation, it was proposed that steric hindrance of the C-8
proton with the bulky methyl group of the 3-endo con-
former prevents the base from adopting the anti-con-
a
max
Compounds
K
m
(mM)
V
Relative Vmax
Vmax/K
m
1
7
formation required for an enzyme±substrate complex.
Adenosine
42
110
289
6
100
2
6.9
0.05
1
2
3
4
5
b
b
b
b
NS
NS
NS
NS
NS
NS
NS
NS
Preliminary NMR conformational analyses showed no
signi®cant di€erences between the ribo-derivatives 1±3.
b
b
b
b
140
140
160
31
95
75
55
33
26
0.7
0.5
0.3
11.4
2.6
0
anosyl)-adenine, a coupling constant J
They had, as reported for 9-(3 -C-methyl-b-d-ribo-fur-
18
0
19
0
of about
6
2
19
122
H1 ±H2
0
3,4
-Deoxyadenosine
353
c
8 Hz. Moreover, on irradiation of H-8, comparable
c
c
7
Ð
Ð
Ð
0
0
NOE intensities for H1 and H2 (6.2±6.7% and 3.5±
20
a
4.2%, respectively) were measured from the adiabatic
o€ resonance ROESY.21 So the sugar moiety of the
three ribo derivatives adopted predominantly a South
mmoles/min/mg prot.
Not substrate.
b
c
m
First order kinetics were observed {v=(Vmax/K ) [S]}.

D. Tritsch et al. / Bioorg. Med. Chem. Lett. 10 (2000) 139±141
141
conformation to limit the sterical interactions.22 In this
puckering mode, the C-substituent was located in a
pseudo-equatorial location where its in¯uence on the
syn and anti equilibrium is the weakest. On the other
was synthesized from 9-(3 -C-ethynyl-b-d-xylo-furanosyl)
adenine 4 as described above for compound 3. Mp=206±
0
3
ꢀ
� 1
1
2
08 C; IR (KBr) 3476, 3303, 2975, 1679 cm
;
); 5.96 (s; 1H; H
H NMR
0
(
CD
3
OD) d 8.37 (s; 1H; H
2
); 8.20 (s; 1H; H
0
8
1
);
0
0
4
1
.81 (s; 1H; H ); 4.03 (m; 1H; H ); 3.93 (m; 2H; H ); 1.83 (m;
5
2
4
hand, the J
vatives 4±6 were about 0 to 1 Hz, suggesting that they
0
0
coupling constants for the xylo-deri-
3
H1 ±H2
H; CH ); 1.62 (m; 1H; CH ); 1.02 (t; J=7.2 Hz; 3H; CH );
2
,5
2
3
13
C RMN (DMSO-d
); 118.85 (C
6
) d 156.0 (C
); 90.41 (C
6
); 152.2 (C
0
2
); 148.6 (C
0
4
);
0
exist predominantly, as 9-(3 -C-methyl-b-d-xylo-fur-
0
1
(
(
39.8 (C
0
8
5
1
); 86.47 (C
4
); 80.9 (C
2
); 80.5
anosyl)-adenine, in the North conformation.18 These
results did not show conclusively why compound 1, in
contrast to compounds 2 and 3, was recognized by ade-
nosine deaminase. Additional NMR, molecular model-
ing and crystallographic studies (structure of the
0
C ); 60.2 (C ); 24.5 (CH ); 7.8 (CH ); SM (CI; NH ) 296
3
MH ; 40), 295 (M ; 63), 135 (BH ; 100).
6. Adenosine deaminase from calf spleen was purchased from
Sigma. The activity was measured in a 50 mM sodium phos-
phate bu€er pH 7.5 at 30 C by following the decrease of the
absorbance at 262 nm. To determine the kinetic constants (K
and Vmax), the concentration of adenosine and its analogues
was varied between 0.05 and 0.1 mM according to the proce-
dure of Murphy et al. All the double reciprocal substrate±
initial velocity plots were linear. Enzyme (0.14 mg for adeno-
5
2
3
3
+
+
+
ꢀ
0
complex of adenosine deaminase with 3 -b-C-ethynyl-
adenosine or 3 -b-C-ethynyl-6-hydroxyl-1,6-dihydro-
m
0
purine ribonucleoside) should help to explain why the
enzyme deaminated compound 1 and not compounds 2
and 3.
7
0
sine and 2 -deoxy-adenosine; 1.4 mg for compounds 4±7; 5.6 mg
for 1 and until 22.4 mg for 2 and 3) was added to initiate the
enzymatic reaction.
0
The very high activity of the 3 -C-ethynyl-nucleosides as
The ade-
anticancer agents is now well established.2
nosine analogue was less ecient than the uridine and
3,24
7. Murphy, J.; Baker, D. C.; Behling, C.; Turner, R. A. Anal.
Biochem. 1982, 122, 328±337.
8. Inhibition of 16 and 23% were observed when adenosine
3
cytidine derivatives. This could be due to the in vivo
0
(
39 mM) was incubated with 3 -C-vinyl-(ribo)-adenosine (60
deamination of compounds 1 and 7 by adenosine dea-
minase. Addition of adenosine deaminase inhibitors
could be essential to potentiate the e€ectiveness of ade-
nosine analogues 1 and 4±7 as anticancer reagents.
0
mM) and 3 -C-ethyl-(ribo)-adenosine (60 mM) respectively.
9
. Segel, I. H. In Enzyme Kinetics; John Wiley and Sons/
Wiley-Interscience: New York, 1975; pp 41±43.
0. Zielke, C. L.; Suelter, C. H. In The Enzymes, 3rd ed.;
1
Boyer, P. D., Ed.; Academic Press: New York, 1971; Vol. 4,
pp 47±78.
Acknowledgements
11. Tronchet, J. M. J.; Tronchet, J. F. Helv. Chim. Acta 1979,
2, 689±695.
6
We wish to thank Dr. Roland Gra€, Michelle Mar-
tigneaux and Jean-Daniel Sauer for the NMR analyses
and the ANRS for ®nancial support.
12. Mikhailopulo, I. A.; Wiedner, H.; Cramer, F. Biochem.
Pharmacol. 1981, 30, 1001±1004.
13. Saenger, W. In Principles of Nucleic Acid Structure; Can-
tor, C. R. Ed.; Springer-Verlag: New York, 1984.
14. Wang, Z.; Quiocho, F. A. Biochemistry 1998, 37, 8314±
8324, and references cited therein.
15. Kalinichenko, E. N.; Beigelman, L. N.; Mikhailov, S. N.;
References and Notes
Mikhailopulo, I. A. Bioorg. Khim. 1988, 14, 1157±1161.
16. Iimori, T.; Murai, Y.; Ohuchi, S.; Kodama, Y.; Ohtsuka,
Y.; Oishi, T. Tetrahedron Lett. 1991, 32, 7273±7276.
17. Mikhailov, S. N. Nucleosides Nucleotides 1998, 17, 1915±
1918.
1
. Montgomery, J. A. Med. Res. Rev. 1982, 2, 271±308.
2. Jung, P. M. J.; Burger, A.; Biellmann, J.-F. Tetrahedron
Lett. 1995, 36, 1031±1034.
3
1
4
. Jung, P. M. J.; Burger, A.; Biellmann, J.-F. J. Org. Chem.
997, 62, 8309±8314.
18. Koole, L. H.; Buck, H. M.; Vial, J. M.; Chattopadhyaya,
J. Acta Chem. Scand. 1989, 43, 665±669.
0
0
. 9-(3 -C-ethyl-b-d-ribo-furanosyl) adenine 3. 9-(3 -C-ethynyl-
3
b-d-ribo-furanosyl) adenine 1 (0.37 g; 1.25 mmol) was
hydrogenated overnight in methanol (73 mL) at 25 C and 1
bar, in the presence of palladium on charcoal (10%, 0.15 g).
The catalyst was removed by ®ltration on Celite (2Â6 cm,
methanol). The solution was concentrated to 2 mL then ace-
tonitrile (10 mL) was added. Evaporation of the solvents gave
19. Rosemeyer, H.; Toth, G.; Golankiewicz, B.; Kazimierczuk,
Z.; Bourgeois, W.; Kretschmer, U.; Muth, H.-P.; Seela, F. J.
Org. Chem. 1990, 55, 5784±5790.
ꢀ
1
20. H NMR and Roesy analyses were performed in DMSO-
d
6
at a 0.1 M concentration.19
21. Desvaux, H.; Berthault, P.; Birlirakis, N.; Goldman, M.;
Piotto, M. J. Magn. Reson. 1995, 113, 47±52.
ꢀ
compound 3 quantitatively. Mp=193±194 C; UV(MeOH):
�
1
� 1
l
M
max=214 nm; e=14000 M cm ; lmax=262 nm; e=13600
22. For a discussion relative to the conformational energy
di€erence between axial and equatorial monosubstituted
cyclohexanes by an ethynyl, a vinyl, an ethyl or a methyl
group, see Eliel, E. L.; Wilen, H. S.; Mander, L. N. in Stereo-
chemistry of Organic Compounds; John Wiley and Sons/Wiley-
Interscience: New York, 1994, pp 690±700.
23. Hattori, H.; Nozawa, E.; Iino, T.; Yoshimura, Y.; Shuto,
S.; Shimamoto, Y.; Nomura, M.; Fukushima, M.; Tanaka,
M.; Sasaki, T.; Matsuda, A. J. Med. Chem. 1998, 41, 2892±
2902 and references cited therein.
�
1
� 1
� 1
24
d
cm ; IR (KBr) 3422, 3339, 2993, 1674 cm ; ‰aŠ � 63
1
(c=1; DMSO); H NMR (DMSO-d ) d 8.35 (s; 1H; H ); 8.12
6
2
(
J
s; 1H; H ); 7.39 (s; 1H; NH ); 5.96 (s; 1H; OH); 5.88 (d;
0
8
2
0
0
=7.9 Hz; 1H; H
0
); 5.40 (s; 1H; OH); 4.60 (s; 1H; OH);
0
H
1H
2
1
0
4
2
CH
.50 (d; J =7.9 Hz; 1H; H
0
H; H ); 1.73 (q; J=7.5 Hz; 2H; CH ); 0.97 (t; J=7.5 Hz; 3H;
H
1H
0
2
2
); 3.91 (m; 1H; H
4
); 3.61 (m;
5
1
2
3
3
); C NMR (DMSO-d
); 140.6 (C
0
6
) d 156.2 (C
6
); 152.0 (C
0
2
); 149.0
0
0
(
C
4
8
); 119.5 (C ); 87.8 (C
5
4
); 87.7 (C
1
); 78.2 (C );
). Anal. calcd for
3
0
7
6.5 (C
C H N O : C 48.81, H 5.76, N 23.73. Found: C 48.98, H
2
); 61.3 (C
5
); 25.5 (CH
2
); 7.7 (CH
3
24. Weltin, D.; Jung, P. M. J.; Holl, V.; Dauvergne, J.; Burger,
A.; Dufour, P.; Aubertin, A. M.; Bischo€, P.; Biellmann, J.-F.
unpublished results.
1
2
17
5
4
+
5
5
.93, N 23.55.; SM (CI) 296 (MH ; 100).
0
. 9-(3 -C-ethyl-b-d-xylo-furanosyl) adenine 6. Compound 6