Synthesis of a technetium-99m-labeled thymidine analog: A potential HSV1-TK substrate for non-invasive reporter gene expression imaging

Article abstract of DOI:10.1016/j.tetlet.2004.09.145

{2-[5-(2′-Fluoro-2′-deoxyuridin-5-yl)pent-4(E)-enyl] [2-(2-mercaptoethyl)aminoethyl]aminoethanethiolato(3)-N,N′,S,S′} oxo-[^99mTc]technetium(V), a potential viral thymidine kinase substrate, was synthesized by coupling 2′-fluoro-2′-deoxyuridine

Full text of DOI:10.1016/j.tetlet.2004.09.145

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 8673–8676
Synthesis of a technetium-99m-labeled thymidine analog:
a potential HSV1-TK substrate for non-invasive reporter
gene expression imaging
Yi Zhang,a Xiaoman Dai,a David F. Kallmesb and Dongfeng Pana,*
aThe Department of Radiology, University of Virginia, Charlottesville, VA 22908, USA
bDepartment of Radiology, Mayo Clinic, Rochester, MN 55902, USA
Received 25 August 2004; revised 17 September 2004; accepted 21 September 2004
Available online 8 October 2004
Abstract—{2-[5-(20-Fluoro-20-deoxyuridin-5-yl)pent-4(E)-enyl] [2-(2-mercaptoethyl)aminoethyl]aminoethanethiolato(3)-N,N0,S,S0}-
oxo-[99mTc]technetium(V), a potential viral thymidine kinase substrate, was synthesized by coupling 20-fluoro-20-deoxyuridine
analog with a N2S2 radiometal chelator, followed by [Tc-99m]technetium conjugation. The chemical structure of the radioactive
probe was characterized by 1H NMR and high resolution MS using Re-188 conjugated mimics. The radiochemical purity and yield
were identified as 98% and 42%, respectively.
ꢀ 2004 Published by Elsevier Ltd.
Non-invasive nuclear imaging of Herpes simplex virus
type-1 thymidine kinase (HSV1-TK) expression has
gained broad interests because of its potential in clinical
application.1–3 The biological principal of the imaging
modality involves phosphorylation and, therefore, intra-
cellularly retention of the radiolabeled nucleosides by
the HSV1-TK. The current HSV1-TK substrate of nu-
clear imaging includes radioactive fluorine and iodine
isotope-labeled nucleoside derivatives, such as 9-(4-[F-
pent-4(E)-enyl] [2-(2-mercaptoethyl)aminoethyl]amino-
ethanethiolato(3)-N,N0,S,S0}oxo-[99mTc]technetium(V)
(FTcAU). The fact that among the thymidine substrates
of HSV1-TK the substitution on 5 position of thymine
ring shows the most variability and flexibility7,9–11 leads
us to design our first Tc-99m-labeled probe using 5-sub-
stituted 20-fluoro-20-deoxyuridine as a template. Because
of the requirement of cellular membrane penetration,
the radiometal is labeled through a lipophilic, N2S2
metal chelator, N,N0-bis(2-mercaptoethyl)ethylenediam-
ine. To characterize the chemical structure of the Tc-
99m-labeled probe, a nonradioactive mimic compound,
{2-[5-(20-fluoro-20-deoxyuridin-5-yl)pent-4(E)-enyl] [2-
(2-mercaptoethyl)aminoethyl]aminoethanethiolato(3)-
N,N0,S,S0}oxo-[188Re] rhenium(V) (FReAU) is also
synthesized for NMR and MS analysis and HPLC
identification.
18]fluoro-3-hydroxymethylbutyl)guanine
(FHBG)4,5
and 5-[I-131/124]iodo-1-(2-fluoro-2-deoxy-b-D-arabino-
furanosyl)uracil (FIAU).6–8 However, use of radioactive
fluorine- and iodine-labeled probes in routine proce-
dures is hampered by either the limited supply of radio-
isotopes or by suboptimal imaging characteristics, such
as iodine-131. A [Tc-99m]technetium-labeled TK sub-
strate for nuclear gene imaging would address the con-
cerns because of its ease of production and optimal
imaging characteristics (T1/2 = 6h, 140keV).
A convergent synthetic strategy is employed to obtain 5-
{[2-(4-methoxybenzylsulfanyl)ethyl]{2-[2-(4-methoxybenz-
ylsulfanyl)ethylamino]ethyl}amino} pent-4(E)-enyl-1-(3,
5-diacetyl-2-fluoro-2-deoxy-1-b-D-ribofuranosyl)uracil
9, the precursor of Tc-99m-labeled 12, from two
synthons, 5-(5-bromopent-1(E)-enyl)-1-(3,5-diacetyl-2-
fluoro-2-deoxy-1-b-D-ribofuranosyl)uracil 5 and N,N0-
bis-[2-(4-methoxybenzylsulfanyl)ethyl]ethylenediamine
8.
In this communication we report the synthesis and
characterization of {2-[5-(20-fluoro-20-deoxyuridin-5-yl)-
Keywords: FTcAU; TK substrate; Nuclear imaging; Reporter probe.
*
Corresponding author. Tel.: +1 434 243 2893; fax: +1 434 924
9435; e-mail: dp3r@virginia.edu
0040-4039/$ - see front matter ꢀ 2004 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2004.09.145

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Y. Zhang et al. / Tetrahedron Letters 45 (2004) 8673–8676
At first, the thymidine analog 4 is synthesized through a
multiple-step reaction from 20-fluoro-20-deoxyuridine 1
(Scheme 1). The hydroxyl groups of 1 are protected by
treating with acetic anhydride in pyridine to give 2,
followed by iodination to 5-iodo-30,50-diacetyl-20-fluoro-
20-deoxyuridine 3 using iodonium chloride in methyl-
ene chloride with the iodination yield of 87%. Stille
coupling reaction of 3 with 1-(tributylstannyl)-1(E)-pen-
ten-5-ol yields 5-(5-hydroxylpent-1(E)-enyl)-1-(3,5-
with Hg(OAc)2 in TFA to give trifluoroacetate salts of
11. The crude air-sensitive compound 11 is conjugated
with technetium immediately, without purification.
Addition of [99mTc]pertechnetate in PBS into the aque-
ous methanol of the crude 11 in the presence of Sn-
glucoheptonate in 80ꢁC water bath for 30min and
thereafter HPLC18 purification yields the target com-
pound, FTcAU 12 with a radiochemical yield of 42%
(Scheme 3). To characterize the chemical structure of
the FTcAU 12, its analog of rhenium-188 conjugate,
FReAU 13, is synthesized with modifying a similar reac-
diacetyl-2-fluoro-2-deoxy-1-b-D-ribofuranosyl)uracil
4
in 92% yield.12 Treatment of 4 with NBS in the presence
of triphenyl phosphorus gives the bromide 5 in 75%
yield.13
19
tion condition by adding tetrabutylammonium tetra-
chlorooxorhenate(V) into a solution of compound 11 in
methanol and stirring for 12h. The rhenium conjugate
13 is purified by flash chromatography and its chemical
The metal chelating moiety, N,N0-bis-[2-(4-meth-
oxybenzylsulfanyl)ethyl]ethylenediamine 8 is prepared
from 2-(4-methoxybenzylsulfanyl)ethylamine 614 and
dibromoethylene 7 in yield of 41% (Scheme 2). Cesium
hydroxide controls the N-alkylation reaction to obtain
secondary amine as the major product.15
1
structure is characterized with H NMR and high reso-
lution ESI-MS.20 The characterization of FTcAU is car-
ried out using reverse phase HPLC by co-injection with
FReAU.
The present work demonstrates the synthesis of a neu-
tral Tc-99m-labeled thymidine analog 12, a potential
HSV1-TK substrate. In vitro verification of the
compound by cell uptake experiments is underway in
our laboratory and the result will be published
elsewhere.
Coupling of thymidine analog 5 and chelator fragment 8
produces 9.16 The removal of acetyl protecting groups
of 9 with potassium carbonate in aqueous methanol
yields 1017 with a two-step yield of 76%. The thiol pro-
tecting groups, 4-methoxybenzyl, of 10 are removed
O
O
O
N
I
HN
HN
HN
O
O
O
HO
AcO
AcO
N
N
O
O
O
i
ii
OH
OAc
OAc
F
F
F
3
2
1
iii
O
O
N
Br
OH
(CH2)3
(CH2)3
HN
HN
O
O
AcO
AcO
N
O
O
iv
OAc
OAc
F
F
4
5
Scheme 1. Reagents: (i) Ac2O, Py; (ii) ICl, CH2Cl2; (iii) 1-(tributylstannyl)-1(E)-penten-5-ol, (CH3CN)2PdCl2, DMF; (iv) NBS, PPh3, DMF.
NH
NH
S
S
PMB
PMB
i
PMB
NH2
+
Br
Br
S
7
6
PMB = p-methoxylbenzyl
8
˚
Scheme 2. Reagents: (i) CeOH, molecular sieves (4A), DMF.

Y. Zhang et al. / Tetrahedron Letters 45 (2004) 8673–8676
8675
O
(CH2)3
N
S
S
PMB
PMB
HN
i
O
5
8
AcO
N
+
NH
O
9
OAc
F
ii
O
N
O
N
(CH2)3
(CH2)3
N
S
S
PM
B
N
SH
HN
HN
O
HO
O
HO
NH
PM
B
NH SH
O
O
11
10
iii
OH
OH
F
F
v
iv
O
N
O
N
(CH2)3
(CH2)3
O
99mTc
O
S
N
S
S
N
N
HN
HN
Re
O
O
HO
HO
N
S
O
O
12
FTcAU
13
FReAU
OH
OH
F
F
Scheme 3. Reagents: (i) DIEA, CH3CN; (ii) K2CO3; (iii) Hg(OAc)2/TFA, H2S; (iv) [99mTc]NaTcO4, Sn-glucoheptonate; (v) (Bu4N)+(ReOCl4)ꢀ.
7. Tjuvajev, J. G.; Stockhammer, G.; Desai, R.; Uehara, H.;
Watanabe, K.; Gansbacher, B.; Blasberg, R. G. Cancer
Res. 1995, 55(24), 6126–6132.
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9. Morin, K. W.; Atrazheva, E. D.; Knaus, E. E.; Wiebe, L.
I. J. Med. Chem. 1997, 40(14), 2184–2190.
Acknowledgements
This work was supported in part by grants from the
Nihon Medi-Physics Co. Ltd (NMP) and the Depart-
ment of Radiology, University of Virginia.
References and notes
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12. Compound 4. 1H NMR (300MHz, CDCl3): d 1.72 (m,
2H), 2.13 (s, 3H), 2.17 (s, 3H), 2.25 (m, 2H), 3.68 (m, 2H),
4.34 (m, 1H), 4.44 (d, 2H), 5.17 (m, 1H), 5.40(dq, 1H),
5.82 (d, 1H), 6.10(d, 1H, J = 15.9Hz), 6.47 (dt, 1H,
J = 15.9Hz), 7.31 (s, 1H). HRMS (ESI) [M + H]+, obsd:
415.1514, calcd: 415.1517.
13. Compound 5. 1H NMR (300MHz, CDCl3): d 1.99 (m,
2H), 2.13 (s, 3H), 2.17 (s, 3H), 2.32 (m, 2H), 3.42 (m, 2H),
4.37 (m, 1H), 4.44 (d, 2H), 5.17 (m, 1H), 5.39 (dq, 1H),
5.82 (d, 1H), 6.10(d, 1H, J = 16.2Hz), 6.48 (dt, 1H,
J = 16.2), 7.32 (s, 1H). HRMS (ESI) [M + H]+, obsd:
477.0664, calcd: 477.0673.
14. Oya, S.; Plossl, K.; Kung, M. P.; Stevenson, D. A.; Kung,
H. F. Nucl. Med. Biol. 1998, 25(2), 135–140.
15. Salvatore, R. N.; Nagle, A. S.; Jung, K. W. J. Org. Chem.
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8676
Y. Zhang et al. / Tetrahedron Letters 45 (2004) 8673–8676
16. Compound 9. 1H NMR (300MHz, CDCl3): d 1.59 (m,
2H), 2.09 (s, 3H), 2.15 (s, 3H), 2.47 (m, 4H), 2.61 (s, 4H),
2.77 (m, 6H), 2.97 (t, 2H), 3.64 (s, 2H), 3.69 (s, 2H), 3.76
(s, 3H), 3.77 (s, 3H), 4.28–4.48 (m, 3H), 5.22 (m, 1H), 5.43
(dq, 1H), 5.81 (dd, 1H), 6.10(d, 1H, J = 15.9Hz), 6.44 (dt,
1H, J = 15.9Hz), 6.82 (d, 4H), 7.22 (d, 4H), 7.33 (s, 1H).
HRMS (ESI) [M + H]+, obsd: 817.3322, calcd: 817.3316.
18. NOTE: HPLC purification: column: Econosil C18 10u,
250 · 10mm; gradient: acetonitrile/PBS (pH7.4) (35/65);
flow rate: 3mL/min.; retention time: 10.5min.
19. Meegalla, S. K.; Plossl, K.; Kung, M. P.; Chumpradit, S.;
Stevenson, D. A.; Kushner, S. A.; McElgin, W. T.;
Mozley, P. D.; Kung, H. F. J. Med. Chem. 1997, 40(1),
9–17.
1
1
17. Compound 10. H NMR (300MHz, THF-d8): d 1.57 (m,
20. Compound 13. H NMR (300MHz, THF-d8): d 1.42 (m,
2H), 2.13 (m, 2H), 2.42–2.73 (m, 12H), 2.88 (t, 2H), 3.63
(s, 2H), 3.67 (s, 2H), 3.75 (s, 3H), 3.76 (s, 3H), 3.94 (d,
2H), 4.35 (m, 1H), 4.91 (dq, 1H), 6.05 (d, 1H), 6.15 (d, 1H,
J = 15.0Hz), 6.44 (dt, 1H, J = 15.0Hz), 6.83 (d, 4H), 7.23
(d, 4H), 8.36 (s, 1H). HRMS (ESI) [M + H]+, obsd:
733.3272, calcd: 733.3105.
2H), 2.17 (m, 2H), 2.62 (m, 2H), 2.77 (m, 2H), 3.04
(m, 2H), 3.29 (m, 6H), 3.43 (m, 2H), 3.95 (m, 1H), 4.03 (m,
2H), 4.30(m, 1H), 5.22 (m, 1H), 4.93 (dq, 1H), 6.00(dd,
1H), 6.15 (d, 1H, J = 15.9Hz), 6.57 (dt, 1H, J = 15.9Hz),
8.21 (s, 1H). HRMS (ESI) [M + H]+, obsd: 693.1214,
calcd: 693.1226.