Design, synthesis and enzymatic activity of highly selective human mitochondrial thymidine kinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(01)00207-4

Highly selective arabinofuranosyl nucleosides, which inhibit the mitochondrial thymidine kinase (TK-2) without affecting the closely related herpes simplex virus type 1 thymidine kinase (HSV-1 TK), varicella-zoster virus thymidine kinase (VZV-TK), cytosolic thymidine kinase (TK-1) or the multifunctional Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK), have been obtained. SAR studies indicate a close relation between the length of the substituent at the 2′ position of the arabinofuranosyl moiety and the inhibitory activity.

Full text of DOI:10.1016/S0960-894X(01)00207-4

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1329–1332
Design, Synthesis and Enzymatic Activity of Highly Selective
Human Mitochondrial Thymidine Kinase Inhibitors
Stefano Manfredini,a,* Pier G. Baraldi,a Elisa Durini,a Luca Porcu,a Angela Angusti,a
Silvia Vertuani,a Nicola Solaroli,a,b Erik De Clercq,c Anna Karlssonb and Jan Balzarinic
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, via Fossato di Mortara 17–19, I-44100 Ferrara, Italy
bDivision of Clinical Virology, Huddinge University Hospital, S-141 86 Huddinge/Stockholm, Sweden
cRega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received 19 December 2000; revised 19 March 2001; accepted 20 March 2001
Abstract—Highly selective arabinofuranosyl nucleosides, which inhibit the mitochondrial thymidine kinase (TK-2) without affect-
ing the closely related herpes simplex virus type 1 thymidine kinase (HSV-1 TK), varicella-zoster virus thymidine kinase (VZV-TK),
cytosolic thymidine kinase (TK-1) or the multifunctional Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK), have
been obtained. SAR studies indicate a close relation between the length of the substituent at the 20 position of the arabinofuranosyl
moiety and the inhibitory activity. # 2001 Elsevier Science Ltd. All rights reserved.
The majority of nucleoside analogues in clinical use is
phophorylated by human deoxyribonucleoside kinases
(dNKs) and biochemical studies showed that there are
four distinct dNKs in the human salvage pathway,1 with
different subcellular location: the thymidine kinase 1
(TK-1) cytosolic enzyme, the cytosolic/nuclear con-
stitutively expressed deoxycytidine kinase (dCK), and
deoxyguanosine kinase (dGK) and thymidine kinase 2
(TK-2) both being constitutive mitochondrial enzymes.
Among the deoxyribonucleoside kinases, TK-2 is the
less characterized and up to now no selective nucleoside
analogues recognizing TK-2, have been synthesized
except for AraT. TK-2 is an interesting deoxy-
ribonucleoside kinase for several reasons:
During a study aimed at discovering new potential
inhibitors for dNKs, we have identified a new class of
highly selective arabinofuranosyl nucleosides that inter-
act with mitochondrial TK-2 without affecting the clo-
sely related HSV-1 TK, VZV TK, cytosolic TK-1 or
Dm-dNK.4 SAR studies indicate a close relation
between the length of the substituent at the arabinofur-
anosyl moiety and their inhibitory activity. This is the first
time that such highly effective compounds are described
thus opening new perspectives in the design of selective
mitochondrial TK-2 inhibitors. A new synthetic route, the
biological activity, and the results of a molecular modeling
study on the obtained 20-O-acyl and -alkyl derivatives of
the arabinofuranosyl nucleosides will be briefly reported.
i. It phosphorylates pyrimidine deoxyribonucleo-
sides such as dThd, dUrd, dCyd2 and also the anti-
viral nucleoside analogues AZT and FIAU but at
much lower rates than the natural substrates2,3
ii. The mitochondrial localization of TK-2 may be
important for the mitochondrial toxicity observed
for several pyrimidine nucleoside anti-viral analo-
gues and may also represent a target to induce
DNA depletion.
Chemistry
The 20-acyl derivatives 5a–c and 6c–e of BVaraU (1) and
AraT (2), were obtained by reaction of the correspond-
ing acyl halide on the 30 and 50 protected nucleosides (3
and 4). The deprotection at position 30 and 50 of the
acylated derivatives, gave the final 7a–c and 8c–e in 30–
90% yield (Scheme 1).
iii. TK-2 can be useful for gene therapy studies: the
dNKs are investigated as suicide gene in anti-can-
cer chemotherapy.
The 20-octyl- and -benzyl-ethers were synthesized by a
different synthetic strategy. The alternative synthetic
pathway required the preparation of the parent 1-b-d-
ribofuranosyl-thymine (9)5 and the protection of 30,50-
hydroxyl functions (13) with 1,2-dihydropyrane (DHP),
*Corresponding author. Tel.:+39-532-29-1292; fax: +39-532-29.1296;
e-mail: mv9@dns.unfie.it
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00207-4

1330
S. Manfredini et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1329–1332
which was carried out on 2,20-anhydro-1-(b-d-arabino-
furanosyl)thymine (11), obtained from 9 as described by
Schinazi et al.6 (Scheme 2).
inhibitory activity of AraT than HSV-1 and VZV TK
(Table 1).
The introduction of bulky acyl groups (i.e., decanoyl
and dodecanoyl esters 8c and 8e) at the 20-OH position
of AraT increased the inhibitory effect of the derivatives
on mitochondrial TK-2 by 10-fold, whereas the inhibi-
tion of HSV-1 and VZV TK by these compounds, was
decremented by >50-fold. TK-1 was insensitive to
AraT and the 20-O-acyl AraT derivatives. Thus, intro-
duction of a bulky acyl group at the 20 position of AraT
increased its selectivity by more than 500-fold for TK-2
versus the related viral thymidine kinases. Shortening
the 20-O-acyl chain to a pentanoyl (8d) resulted in a
complete loss of its inhibitory activity against TK-2.
The inhibitory activity of the 20-O-alkyl-AraT deriva-
tives (21 and 23) was lower than that found for the
20-O-acyl ester derivatives (Table 1). 20-O-Octanoyl
and -decanoyl BVaraU derivatives (7a and 7c) mark-
edly increased the inhibitory effect of BVaraU on
TK-2 7-fold (Table 1). As observed for the AraT deri-
vatives, the 20-O-acyl BVaraU derivatives lost their affi-
nity for HSV-1 and VZV TK. The less bulky 20-O-acyl
derivative of BVaraU lost 10- to 20-fold activity against
TK-2, HSV-1 TK and VZV TK. Again, TK-1 remained
insensitive to all substituted BVaraU derivatives (Table
1). The 20-O-alkyl-AraC( 24) derivative did not show any
increase in selectivity in comparison to AraCor the other
20-O-acyl/alkyl derivatives. Despite the structural
Finally, the 30,50 THP protected derivative 13 gave,
by treatment with 0.1 M KOH in 95% ETOH, com-
pound 15 (Scheme 2).7 The 20-octyl derivative of
30,50-protected AraT (17) was obtained using octyl
iodide in the presence of NaH (Scheme 3). A milder
procedure with benzylbromide at room temperature
was adopted to obtain compound 19 because of the
formation of side products. The next deprotection step
was carried out with p-toluenesulfonic acid (TsOH) in
MeOH to give, in 40% yield, the compounds 21 and 23.
The 20-alkyl modification of the sugar moiety has been
extended also to AraC. The preparation of compound
24 required a modified synthetic approach. Therefore,
the synthetic steps leading to the 20-alkyl-derivative of
AraCwere carried out starting on uridine ( 10) (Scheme
2). Compound 18 was then converted to the AraCderi-
vative 24 by the procedure of Divakar and Reese8
(Scheme 3).
Biology
AraT is recognized as a good substrate by the nucleo-
side kinases encoded by HSV-1 and VZV and the mito-
chondrial TK-2 showed a lower sensitivity to the
Scheme 1. (i) TPDSCl2, pyr, rt; (ii) RCl, pyr, rfx; (iii) NH4F, MeOH, Dowex H+ form 50Â2–100, rt.
Scheme 2. (i) (PhO)2CO, NaHCO3, DMF, 150 ꢀC; (ii) DHP, TsOH.H2O, CH3CN, rt; (iii) KOH 0.1 M, EtOH, rt.

S. Manfredini et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1329–1332
1331
similarities between TK-2 and Drosophila melanogaster
dNK,9 no selectivity was achieved on this latter enzyme.
As reported in Table 2, the results of compounds dock-
ing into the active site of HSV-1 TK are in good agree-
ment with the observed IC50s thus validating our
approach aimed at developing a possible general model
for the interaction on structurally related nucleoside
kinases. Ongoing studies will extend these results on
Dm-dNK and TK-2.
Molecular Modeling
Molecular modeling studies have been started on HSV-1
TK, Dm-dNK and TK-2 with the aim to develop a
general model suitable for the description and predic-
tion of the interaction of substrates with the different
kinases. Because only the crystallographic data of HSV-
1 TK are available,10 the structure of Dm-dNK and TK-
2 have been re-built by homology model techniques
using the HSV-1 TK as template. The three enzymes
show several structural analogies, and the amino acids
involved in binding interactions are highly conserved.
Conclusions
In the present study, a synthetic approach devoted to
obtaining 20-O-acyl- and N-alkyl-substituted pyrimidine
arabinosyl nucleosides has been developed with the aim
of exploring substrate specificity requirements for
dNKs. Inhibitory activity was investigated for cytosolic
ꢀ
.
Scheme 3. (i) NaH, I(CH2)7CH3, THF, reflux or NaH, BnBr, THF, rt; (ii) MeOH, TSOH H2O, rt; (iii) POCl3, 1,2,4-triazole, TEA, 0 Cto rt; (iv)
30% NH4OH, rt; (v) Dowex OHꢃ form 1Â2–400.
Table 1. Inhibitory effects of AraT and BVaraU derivatives on 20-deoxynucleoside kinases from different origins
a
Compounds
R
IC50 (mM)
X
Y
TK-1b
TK-2c
HSV-1 TKd
Dm-dNK
VZV TKe
BVaraU
7a
7b
7c
AraT
8c
8d
8e
21
–CH¼CHBr
–CH¼CHBr
–CH¼CHBr
–CH¼CHBr
CH3
–H
–CO
–CO
–CO
–CO
–CO
–CO
–CO
–CO
–CO
–C
>500
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>O1000
>1000
>1000
43ꢁ5.8
6.3ꢁ0.5
402ꢁ234
6.8ꢁ0.7
285ꢁ94
27ꢁ2.3
>1000
4.3ꢁ0.36
ꢂ1000
84ꢁ1.8
>1000
24ꢁ3.1
ꢂ1000
>1000
ꢂ1000
>1000
>1000
>1000
>1000
31.6ꢁ10.5
178
—
3.3ꢁ1.1
718ꢁ59
35ꢁ1.5
845ꢁ30
17ꢁ8.7
>1000
>1000
>1000
>1000
>1000
>1000
>1000
–CO(CH2)6CH3
–COCH2OCH3
–CO(CH2)8CH3
–H
–CO(CH2)8CH3
–CO(CH2)3CH3
–CO(CH2)10CH3
–(CH2)7CH3
–CH2(C5H6)
–NH
163
65ꢁ28
CH3
CH3
CH3
CH3
872
>1000
>1000
>1000
>1000
5319
28ꢁ2
120ꢁ14
801ꢁ71
>1000
23
AraC–H
24
CH3
–H
2
–H
–(CH2)7CH3
–NH2
>1000
—
a50% inhibitory concentration.
bCytosolic thymidine kinase.
cMitochondrial thymidine kinase.
dHerpes simplex virus type 1 thymidine kinase.
eVaricella-zoster virus thymidine kinase.

1332
S. Manfredini et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1329–1332
Table 2. Docking dataa
a
Compounds
Position
el
el, sf
el, sf, en
IC50 (mM)
BVDU
BVaraU
7a
7b
7c
AraT
8c
8d
8e
21
In
In
Out
In
Out
In
In
Out
Out
Out
In
8,783,229
8,529,401
10,877,780
10,314,959
10,943,267
8,599,677
11,780,963
10.323,786
11,706,303
10,762,047
9,378,132
ꢃ4,657,439
ꢃ4,835,129
ꢃ4,768,179
ꢃ5,372,671
ꢃ3,770,147
ꢃ3,594,713
ꢃ6,416,770
ꢃ2,474,657
ꢃ8,093,062
ꢃ3,862,358
ꢃ5,187,049
ꢃ3,749,570
ꢃ4,668,734
ꢃ3,404,947
ꢃ3,613,954
ꢃ2,627,095
ꢃ3,931,787
ꢃ1,637,087
ꢃ2,360,739
ꢃ4,760,073
ꢃ0,707,513
ꢃ5,723,524
ꢃ1,724,202
ꢃ3,886,204
ꢃ2,574,724
ꢃ2,687,980
2.87ꢁ1.54b
4.3ꢁ0.36
>1000
84ꢁ1.8
>1000
24ꢁ3.1
>1000
>1000
>1000
>1000
>1000
>1000
>1000
23
AraCIn
24
7,974,090
Out
11,242,538
aBinding energy was calculated using different terms: el, electrostatic energy; sf, surface term; en, entropic free energy.
bData taken from ref 9.
thymidine kinase (TK-1), mitochondrial thymidine
kinase (TK-2), herpes simplex virus type 1 thymi-
dine kinase (HSV-1 TK), varicella-zoster virus thymi-
dine kinase (VZV TK) and D. melanogaster
deoxynucleoside kinase (Dm-dNK). Very interestingly,
we have observed that substitution of pyrimidine arabi-
nosyl nucleoside derivatives at the 20-OH position of the
sugar moiety by a bulky lipophilic acyl moiety markedly
enhances their affinity for the mitochondrial enzyme
(TK-2). In addition, it dramatically increases the selec-
tivity of these compounds for TK-2 and shifts the capa-
city of these novel compounds from an efficient substrate
(i.e., BVaraU) to an effective (competitive) inhibitor of
the enzymatic reaction (i.e., 20-O-acyl BVaraU).5
design of highly selective mitochondrial TK-2 inhibitors
and, in general, of selective ligands for different kinases.
Acknowledgements
The authors are grateful to Mrs. Lizette van Berckelaer
for excellent technical help. The research was supported
by grants from the University of Ferrara, the European
commission and the ‘Belgische Federatie tegen kanker’.
References and Notes
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However, substitution of the acyl chains for an alkyl
chain proved detrimental for the activity. Moreover,
despite structural similarities between TK-2 and Dm-
dNK, no selectivity was observed toward this latter
enzyme thus precluding any possible application of the
compounds on this interesting target for gene therapy.
Anyway, this result is, in our opinion, of potential use-
fulness in the understanding of the structural require-
ments for the substrate selectivity among the thymidine
kinase classes of enzymes. Moreover, the results of the
docking of the study compound into the active site of
HSV-1 TK are in good agreement with the observed
IC50s thus validating our approach aimed at developing
a possible general model for the interaction on structu-
rally related nucleoside kinases.
9. Johansson, M.; Van Rompary, A. R.; Degreve, B.; Balza-
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rini, J.; Karlsson, A. J. Biol. Chem. 1999, 34, 23814.
10. Bennett, M. S.; Wien, F.; Champness, J. N.; Batuwangala,
T.; Rutherford, T.; Summers, W. C.; Sun, H.; Wright, G.;
Sanderson, M. R. FEBS Lett. 1999, 443, 121.
In summary, this study provides an interesting report on
potent arabinofuranosyl nucleoside inhibitors of mito-
chondrial TK-2 that opens perspectives for the rational