Synthesis of radiolabeled cytarabine conjugates

Article abstract of DOI:10.1016/j.bmcl.2009.06.056

N4-Modified, novel Ara-C conjugate capable of radiolabeling with gamma ray-emitting (^99mTc) as well as positron emitting (¹⁸F) radionuclides, that is, N4-hydrazine derivative was synthesized. The radiolabeling of N4-(hydrazinonicotin

Full text of DOI:10.1016/j.bmcl.2009.06.056

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4764–4767
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of radiolabeled cytarabine conjugates
*
Pallavi Lagisetty, Prachi Vilekar, Vibhudutta Awasthi
Department of Pharmaceutical Sciences, University of Oklahoma Health Science Center, 1110 N. Stonewall Avenue, Oklahoma City, OK 73117, USA
a r t i c l e i n f o
a b s t r a c t
N4-Modified, novel Ara-C conjugate capable of radiolabeling with gamma ray-emitting (99mTc) as well as
positron emitting (18F) radionuclides, that is, N4-hydrazine derivative was synthesized. The radiolabeling
of N4-(hydrazinonicotinyl)-1-b-arabinofuranosyl cytosine (HAra-C) with 99mTc was performed with over
95% labeling yield. To label HAra-C with 18F, 4-fluoro(18F)-benzaldehyde was synthesized from 4-formyl-
N,N,N-trimethylanilinium triflate in 30% radiochemical yield; it quantitatively formed hydrazone deriva-
tive with HAra-C within 45 min. The radiolabeled conjugates were analyzed by radio-UV-RP-HPLC. The
cold precursors were characterized by 1H, 13C NMR. Additionally, HAra-C was evaluated for cytotoxicity
in lung adenocarcinoma (H441) cells and found to be comparable in cell killing efficiency to that of Ara-C.
Uptake of 99mTc-HAra-C in cultures of H441 cells and sensitive pancreatic cancer cells (MIAPaCa-2) was
inhibited by nucleoside transporter inhibitor nitrobenzylthioinosine. The results suggest that 99mTc-
labeled HAra-C is a substrate for the membrane nucleoside transporters, and that it may be used in
molecular imaging of nucleoside transporter expression for the verification of potential anticancer effi-
cacy of nucleoside drugs, such as Ara-C and gemcitabine.
Article history:
Received 8 April 2009
Revised 8 June 2009
Accepted 15 June 2009
Available online 17 June 2009
Keywords:
Ara-C
Nucleoside transporters
Technetium-99m
Cancer
Ó 2009 Elsevier Ltd. All rights reserved.
Nucleoside analogs are an important family of compounds
which are of great interest in view of their pharmacological and
pharmaceutical applications.1 Cytarabine or cytosine arabinoside
(Ara-C) is a pyrimidine nucleoside analogue used for the treatment
of acute myeloid leukemia, meningial leukemia and lymphoma.2
The clinical utility of Ara-C is limited to blood cancers, because of
its unfavorable pharmacokinetics, inactivation by deamination,
and acquisition of resistance to membrane transport. Besides inac-
tivation by deamination to arabino uracil, it is also rapidly cleared
with a half life of 1–3 h, limiting its efficacy in the treatment of so-
lid tumors.3,4 It has been demonstrated that derivatization of N4
amine of cytosine may increase its stability towards deaminase-
mediated inactivation. As such, protection of Ara-C by encapsulat-
ing the drug inside liposomes, or DepoCyt, or by preparing deriva-
tives of Ara-C, such as N4-acyl polyethylene glycol derivatives have
shown promise.5,6
Ara-C enters the cells by such a carrier-mediated process, mainly
involving human equilibrative nucleoside transporter 1 (hENT1).
Once inside, it is activated by a sequence of phosphorylations to
produce cytosine arabinoside triphosphate (Ara-CTP) which is a po-
tent inhibitor of DNA polymerase, and results in DNA chain termi-
nation. One of the mechanisms of acquired resistance to the
nucleoside drugs is the downregulation of nucleoside transporters.7
A vast majority of work has been conducted in vitro, but a technol-
ogy that can assess expression of nucleoside transporters in vivo re-
mains to be developed. Our overall objective has been to radiolabel
Ara-C with imaging radionuclides, and at the same time, impart
resistance to deamination. Here we describe a modification of
amine group N4 amine of Ara-C that can add the capability of radio-
labeling Ara-C with imaging radionuclides. We also demonstrate
the potential of 99mTc-labeled HAra-C in imaging expression of
nucleoside transporters by testing the labeled conjugate in vitro.
Synthesis of Ara-C-SBHN conjugate: Our goal of chemically mod-
ifying Ara-C was to protect it against N4 deamination, and at the
same time, render the new molecule amenable to radiolabeling
with 99mTc and 18F. The dual purpose was accomplished by conju-
gation of succinimidyl-6-boc-hydrazinonicotinate (SBHN) at the
N4 position of Ara-C. SBHN (4) was synthesized according to
Scheme 1 (Fig. 1) from commercially available 6-chloronicotinic
acid.8,9 SBHN was conjugated to Ara-C (5) to yield N4-(6-Boc-
hydrazinonicotinyl)-1-b-arabinofuranosyl cytosine (6) as shown
in scheme 2 (Fig. 2). Several reaction conditions (Table 1) were
tested in order to optimize the yield. Our initial attempts of
Ara-C (5) (0.04 mmol) reaction with SBHN (4), in the presence of
N,N-dimethyl formamide and diisopropylethyl amine at 50 °C,
afforded 6 in poor yields (29%) and the reaction was not reproduc-
ible. Next, we coupled Ara-C (5) with hydrazinonicotinic acid (2)
via DCC (dicyclohexylcarbodiimide). This reaction was also unsat-
isfactory where the starting material remained unreacted even
after 5 days. The best yields of the conjugate 6 were obtained in
presence of pyridine at 70 °C. The conjugate 6; was characterized
with 1H NMR by the appearance of aromatic protons at d 8.80–
6.50, H-10 at d 6.10, and tertiary butyl protons of Boc at d 1.44.
* Corresponding author. Tel.: +1 405 271 6593; fax: +1 405 271 7505.
E-mail address: vawasthi@ouhsc.edu (V. Awasthi).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.056

P. Lagisetty et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4764–4767
4765
Figure 1. Scheme 1, Synthesis of succinimidyl-Boc-hydrazinonicotinate (SBHN).
Figure 2. Scheme 2 showing the synthesis of N4-(6-tert-boc-hydrazinonicotinyl)-1-b-arabinofuranosyl Cytosine or HAra-C.
Table 1
Coupling of Ara-C with SBHN
S. No
Precursors
Condition
Temp
Time
Yield of 6 based on TLC
1
2
3
4
5
Ara-C and SBHN
Ara-C and SBHN
Ara-C and SBHN
Ara-C and 6-hydrazino nicotinic acid
Ara-C and SBHN
DIPEA (5 equiv), DMF
DMSO
Pyridine
DMF (DCC)
Pyridine
rt-1 day, 50 °C-1 day
rt
rt
rt-1 day, 70 °C-5 days
70 °C
2 days
7 days
7 days
6 days
3 days
29% of 6 formed, but not reproducible
10%
No reaction
10–15%
50–60%
Generally, Boc-deprotection of amines is carried out with triflu-
oroacetic acid (TFA).10 We used HCl to eliminate the possibility of
the formation of trifluoroacetyl derivatives as reported else-
where.11 When HCl-mediated Boc-deprotection of HAra-C (6)
was performed in acetone, we obtained a hydrazone derivative,
7. Formation of 7 was confirmed by the appearance of molecular
ion peak at 419.07 in mass spectrometry; 1H NMR showed CH3
peaks at d 2.15 integrating for 6 protons. However, deprotection
of 6 with HCl in dioxane at 50 °C (Fig. 2, Scheme 2) provided us
the required product N4-(6-hydrazinonicotinyl)-1-b-arabinofur-
anosyl cytosine (8). The compound 8 was confirmed by the disap-
pearance of tertiary butyl protons at d 1.40 in 1H NMR.
where free pertechnetate had an Rf of 0.8 and the labeled com-
pound had an Rf of 0.1. Upon radio-RP-HPLC, 99mTc-labeled HAra-
C eluted at 5.3 min without any contamination with free pertech-
netate (retention time 11 min).
Flourine-18 labeling of HAra-C: Our next goal was to label HAra-C
with positron-emitting radionuclide 18F for Positron Emission
Tomography or PET. We synthesized 4-fluoro(18F)-benzaldehyde
(11) as a radiolabeled synthon using a krytpofix-mediated nucleo-
philic substitution of 4-formyl-N,N,N-trimethylanilinium triflate
(Fig. 4, Scheme 4, >30% radiochemical yield).15 For labeling HAra-
C, an ethanolic solution of 11 was reacted with aqueous solution
of HAra-C at 70 °C for 45 min. The hydrazone (12) formation was
confirmed by comparing with authentic hydrazone of 4-fluoro-
benzaldehyde and HAra-C which eluted at 3.5 min in RP-HPLC (Re-
verse Phase-High Pressure Liquid Chromatography) and showed a
molecular ion peak at 484.09 in the ESI MS (Electron Spray Ioniza-
tion Mass) spectrum. During radiolabeling, the hydrazone was
monitored by following an increasing UV-radio peak at 3.50 min
and vanishing 4-fluoro(18F)-benzaldehyde peak at 8.53 min.
Cytotoxicity of HAra-C and uptake mechanism: After successfully
labeling HAra-C with 99mTc, we investigated the effects of HAra-C
Techenetium-99m labeling of HAra-C: 99mTc radiolabeling of su-
gar part of thymidine has been previously reported,12 but this is
the first report of 99mTc radiolabeling of the base part of Ara-C by
which it may also be protected from deamination. We performed
99mTc labeling of conjugate 8 by following a standard method using
stannous chloride as a reductant and tricine as a co-ligand.13,14
Quantitative radiolabeling (>95%) was obtained within 15 min of
incubation at room temperature (Fig. 3, Scheme 3). The labeling
was followed by Whatman paper chromatography in acetone

4766
P. Lagisetty et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4764–4767
Figure 3. Scheme 3 showing 99mTc labeling of HAra-C.
Figure 4. Scheme 4 showing synthesis of 4-fluoro(18F)benzaldehyde and its hydrazone formation with HAra-C.
and Ara-C on human lung adenocarcinoma cell line H441. The total
number of cells after 72 h of treatment were counted using trypan
blue cell exclusion assay. It was observed that there is a dose
cytotoxicity of 99mTc-labeled HAra-C, because it is meant to be
used in tracer doses for imaging studies.
Among the purported mechanisms of resistance to pyrimidine
analogs is the downregulation of nucleoside transporters in cancer
cells.16,17 A close analog of Ara-C is gemcitabine which is pharma-
cologically similar to Ara-C.7,18 Expression of hENT1 has been
shown to be associated with the chemosensitivity of gemcitabine
in human pancreatic adenocarcinoma and biliary tract carcinoma
cells.19 In order to design a molecular imaging agent for quantita-
tion of hENT1 expression, we investigated the effect the nucleoside
transporter inhibition on the uptake of tracer dose of 99mTc-HAra-C
in H441 cells as well as in two pancreatic cancer cell lines MIA-
PaCa-2 and PANC-1. Nucleoside transport is specifically inhibited
by nitrobenzylmercaptopurine ribonucleoside (NBTI).20 It is clear
from Figure 6 that the specific ENT inhibitor NBTI reduces 99mTc-
HAra-C uptake in H441 and MIAPaCa-2 cells, but not in PANC-1
cells. In separate experiments we have observed that MIAPaCa-2
cells are more sensitive to gemcitabine than PANC-1 cells (data
not shown). Resistance of PANC-1 cells to gemcitabine has also
been reported.21 Recently, researchers have shown that the adeno-
viral-mediated overexpression of hENT1 enhances gemcitabine re-
sponse in human pancreatic adenocarcinoma. Our results confirm
these published reports, and provide a novel method for potential
molecular imaging of nucleoside transporters in vivo.
dependent reduction in cell viability; at 2 lM dose both drugs
showed comparable viability of around 55% (Fig. 5). The results
were confirmed by serum lactate dehydrogenase and cellular glu-
cosaminidase assays (not shown). No attempt was made to test the
Figure 5. Cytotoxicity of Ara-C and HAra-C in lung adenocarcinoma H441 cells in
*
**
indicate
culture. Cell viability was studied by trypan blue exclusion. The and
In summary, we synthesized N4-conjugate of Ara-C (HAra-C)
whose cytotoxicity was comparable to that of Ara-C in H441 cells.
significance in difference between Ara-C and HAra-C analyzed by analysis of
variance Bonferroni post-test.

P. Lagisetty et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4764–4767
4767
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.06.056.
References and notes
1. Hatse, S.; De Clercq, E.; Balzarini, J. Biochem. Pharmacol. 1999, 58, 539.
2. de Vries, J. F.; Falkenburg, J. H.; Willemze, R.; Barge, R. M. Haematologica 2006,
91, 912.
3. Hamada, A.; Kawaguchi, T.; Nakano, M. Clin. Pharmacokinet. 2002, 41, 705.
4. Ohta, T.; Hori, H.; Ogawa, M.; Miyahara, M.; Kawasaki, H.; Taniguchi, N.;
Komada, Y. Oncol. Rep. 2004, 12, 1115.
5. Benesch, M.; Urban, C. Expert Opin. Pharmacother. 2008, 9, 301.
6. Choe, Y. H.; Conover, C. D.; Wu, D.; Royzen, M.; Greenwald, R. B. J. Controlled
Release 2002, 79, 41.
7. Pennycooke, M.; Chaudary, N.; Shuralyova, I.; Zhang, Y.; Coe, I. R. Biochem.
Biophys. Res. Commun. 2001, 280, 951.
8. Abrams, M. J.; Juweid, M.; TenKate, C. I.; Schwartz, D. A.; Hauser, M. M.; Gaul, F.
E.; Fuccello, A. J.; Rubin, R. H.; Strauss, H. W.; Fischman, A. J. J. Nucl. Med. 1990,
31, 2022.
Figure 6. 99mTc-labeled HAra-C uptake in H441 lung adenocarcinoma cells in
*
presence or absence of nucleoside transport inhibitor (NBTI). The
significance in difference in uptake of HAra-C between cells treated with 0
0.1 M NBTI.
indicates
lM and
l
9. Schwartz, D. A.; Abrams, M. J.; Hauser, M. M.; Gaul, F. E.; Larsen, S. K.; Rauh, D.;
Zubieta, J. A. Bioconjugate Chem. 1991, 2, 333.
10. Srinivasan, N.; Yurek-George, A.; Ganesan, A. Mol. Divers. 2005, 9, 291.
11. Surfraz, M. B.; King, R.; Mather, S. J.; Biagini, S. C.; Blower, P. J. J. Med. Chem.
2007, 50, 1418.
12. Teng, B.; Bai, Y.; Chang, Y.; Chen, S.; Li, Z. Bioorg. Med. Chem. Lett. 2007, 17,
3440.
13. Awasthi, V.; Goins, B.; McManus, L.; Klipper, R.; Phillipsa, W. T. Nucl. Med. Biol.
2003, 30, 159.
14. Awasthi, V. D.; Goins, B.; Klipper, R.; Phillips, W. T. Nucl. Med. Biol. 1998, 25,
155.
15. Chang, Y. S.; Jeong, J. M.; Lee, Y. S.; Kim, H. W.; Rai, G. B.; Lee, S. J.; Lee, D. S.;
Chung, J. K.; Lee, M. C. Bioconjugate Chem. 2005, 16, 1329.
16. Clarke, M. L.; Mackey, J. R.; Baldwin, S. A.; Young, J. D.; Cass, C. E. Cancer Treat.
Res. 2002, 112, 27.
Based on our preliminary in vitro cytotoxicity investigations, it is
anticipated that the modification retains cell killing potential of
Ara-C in addition to the nucleoside transporter-mediated intracel-
lular uptake. The amenability to radiolabel HAra-C can be used to
follow its biological disposition in vivo by both SPECT as well as
PET imaging. The non-invasive imaging of the expression of hENT1
in cancer cells may influence clinical interventions dependent on
pyrimidine nucleosides. For instance, gemcitabine or Ara-C therapy
in cancers with no ENT expression may be futile. We are further
exploring the utility of this concept in vivo in animal models of
lung and pancreatic cancers.
17. Cros, E.; Jordheim, L.; Dumontet, C.; Galmarini, C. M. Leuk. Lymphoma 2004, 45,
1123.
18. Maring, J. G.; Groen, H. J.; Wachters, F. M.; Uges, D. R.; de Vries, E. G.
Pharmacogenomics J. 2005, 5, 226.
19. Mori, R.; Ishikawa, T.; Ichikawa, Y.; Taniguchi, K.; Matsuyama, R.; Ueda, M.;
Fujii, Y.; Endo, I.; Togo, S.; Danenberg, P. V.; Shimada, H. Oncol. Rep. 2007, 17,
1201.
20. Lauzon, G. J.; Paterson, A. R. Mol. Pharmacol. 1977, 13, 883.
21. Hering, J.; Garrean, S.; Dekoj, T. R.; Razzak, A.; Saied, A.; Trevino, J.; Babcock, T.
A.; Espat, N. J. Ann. Surg. Oncol. 2007, 14, 3620.
Acknowledgements
The generous supply of 99mTc-pertechnetate by the OUHSC-Nu-
clear Pharmacy and 18F-water supply from Midwest Medical Iso-
topes, Oklahoma City is acknowledged. Authors also thank Dr.
Shrikant Anant for providing pancreatic cancer cell lines PANC-1
and MIAPaCa-2.