Preparation and evaluation of a 99mTcN-PNP complex of sanazole analogue for detecting tumor hypoxia

Article abstract of DOI:10.1016/j.bmcl.2012.12.079

A sanazole derivative, having a favorable single electron reduction potential (SERP) value compared to that of misonidazole, was synthesized and radiolabeled with [^99mTcN(PNP)] precursor to evaluate its potential as a hypoxia imaging agent. The complex, which was lipophilic, could be prepared in good yields and challenging studies with cysteine showed stability of the complex against trans-chelation. However, despite being lipophilic as well as possessing favorable SERP value, biodistribution studies of this complex in fibrosarcoma tumor bearing Swiss mice showed low uptake in tumor. This observation is possibly attributed to fast clearance of the complex from blood, whereby the complex spends insufficient time in tumor to get reduced and trapped. Though uptake in tumor was low, slow clearance of activity from tumor suggests reduction and trapping of the complex in hypoxic cells. The present ^99mTc-complex demonstrated acceptable values of tumor to blood (TBR) and tumor to muscle (TMR) ratios. However, low uptake in tumor which may not be indicative of the actual hypoxic status of the tumor, limit the utility of the complex to detect tumor hypoxia.

Full text of DOI:10.1016/j.bmcl.2012.12.079

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1394–1397
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Preparation and evaluation of a 99mTcN–PNP complex of sanazole
analogue for detecting tumor hypoxia
c
b
Anupam Mathur a, Madhava B. Mallia b, Sharmila Banerjee b, , H.D. Sarma , M.R.A. Pillai
⇑
a Radiopharmaceuticals Program, Board of Radiation and Isotope Technology, Mumbai 400705, India
b Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai 400085, India
c Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A sanazole derivative, having a favorable single electron reduction potential (SERP) value compared to
that of misonidazole, was synthesized and radiolabeled with [99mTcN(PNP)] precursor to evaluate its
potential as a hypoxia imaging agent. The complex, which was lipophilic, could be prepared in good
yields and challenging studies with cysteine showed stability of the complex against trans-chelation.
However, despite being lipophilic as well as possessing favorable SERP value, biodistribution studies of
this complex in fibrosarcoma tumor bearing Swiss mice showed low uptake in tumor. This observation
is possibly attributed to fast clearance of the complex from blood, whereby the complex spends insuffi-
cient time in tumor to get reduced and trapped. Though uptake in tumor was low, slow clearance of activ-
ity from tumor suggests reduction and trapping of the complex in hypoxic cells. The present 99mTc-
complex demonstrated acceptable values of tumor to blood (TBR) and tumor to muscle (TMR) ratios.
However, low uptake in tumor which may not be indicative of the actual hypoxic status of the tumor,
limit the utility of the complex to detect tumor hypoxia.
Received 27 August 2012
Revised 18 December 2012
Accepted 22 December 2012
Available online 4 January 2013
Keywords:
[
99mTcN(PNP)]
Hypoxia imaging
Sanazole
Ó 2013 Elsevier Ltd. All rights reserved.
In view of the negative influence of hypoxia on the efficacy of
chemotherapy and radiation therapy in cancer management,1
quantitative assessment of hypoxia in cancerous lesions assumes
paramount significance. Information on hypoxic status of a cancer
aids the physicians not only in patient selection for hypoxia-
directed treatment but also in modulating the treatment strategy
to achieve a better clinical outcome. Though, several invasive
methods are available for the detection and quantification of hy-
poxia in tissue,2–6 their inherent limitations encouraged the use
of non-invasive methods. Currently, imaging using radiotracers
and magnetic resonance methods are the preferred non-invasive
modalities for the detection of hypoxia. However, the former with
sensitivity several orders of magnitude higher than the latter, is
more widely used in the clinical practice. [18F]Fluoromisonidazole
([18F]FMISO), a positron emitting 2-nitroimidazole tracer, is proba-
bly the most widely used agent to detect hypoxia in clinical nuclear
medicine.7 However, short half-life, high production cost and lim-
ited availability of cyclotron produced isotope encouraged devel-
opment of a 99mTc alternative for detecting tissue hypoxia. 99mTc
with its optimal nuclear characteristics, ease of availability and
versatile chemistry makes it an ideal diagnostic radionuclide.
Though several 99mTc-nitroimidazole agents have been evalu-
ated,8–10 the search is continuing for a 99mTc agent with optimal
pharmacokinetics to target hypoxia.
The basic unit for hypoxia targeting in majority of radiopharma-
ceuticals including [18F]FMISO is the 2-nitroimidazole moiety. This
is due to its favorable SERP compared to the other possible deriv-
atives viz. 4- and 5-nitroimidazole for the same purpose.11 For neu-
tral complexes, SERP and lipophilicity are the important
parameters which decide the efficacy of the agent to detect hypox-
ia. The complex should be lipophilic to cross the cell membrane
and enter the cell. However, high lipophilicity may result in uptake
of the agent in other vital organs and slow clearance from blood.
This in turn may result in poor target-to-non-target ratio. Inside
the hypoxic cells, the efficiency of reduction of the agent and its
subsequent trapping is decided by SERP. The SERP of bare 2-nitro-
imidazole is ꢀ418 mV with respect to standard hydrogen electrode
(SHE)12 and this value may vary depending on the nature of the
substituent in the nitroimidazole ring. Thus SERP of misonidazole,
a 2-nitroimidazole, is ꢀ389 mV13 with respect to SHE. Sanazole, a
3-nitrotriazole, chosen for the present study has a SERP value of
ꢀ330 mV14 with respect to SHE. Based on the observation of
Adams et al., that groups that are separated by two or more bonds
from the nitroaromatic ring had minimal effect on its SERP,15 a san-
azole derivative was prepared by incorporating a cysteine moiety
three bond distances away from the nitrotriazole ring. Such a cys-
teine-conjugate of sanazole could be expected to have SERP not
significantly different from that of sanazole itself. The pendant
⇑
Corresponding author. Tel.: +91 22 2559 3910; fax: +91 22 2550 5345.
E-mail address: sharmila@barc.gov.in (S. Banerjee).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.079

A. Mathur et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1394–1397
1395
cysteine moiety in the sanazole derivative has the –SH and –COOH
groups of cysteine in a suitable stereochemical orientation to form
complex with [99mTcN(PNP)]2+ core. The complex thus obtained,
was evaluated in fibrosarcoma tumor bearing animal model and
the results are discussed.
trans-chelation of the complex with free cysteine was observed
which indicated the stability of the complex. Also, incubation of
the complex in serum up to 3 h did not show any signs of degrada-
tion. Lipophilicity (LogPo/w) of the complex was determined fol-
lowing a reported protocol using octanol/water system21 and the
LogPo/w value of the complex was found to be 1.3.
The nitrotriazole, 2-(3-nitro-1H-1,2,4-triazol-1-yl)acetic acid,
was obtained as a gift from Dr. V. T. Kagia, Health Research Foun-
dation, Kyoto, Japan. The scheme followed for the synthesis of cys-
teine conjugate of nitrotriazole is shown in Figure 1. Briefly, S-trityl
cysteine was esterified (1)16 and coupled with nitrotriazole in pres-
ence of N-ethyl, N0-(3-dimethylamino) carbodiimide hydrochloride
(EDCI) to obtain compound 2.17 The ethyl ester in compound 2 was
hydrolyzed under alkaline conditions to obtain compound 3.18
Subsequent S-trityl deprotection in compound 3 using TFA/Et3SiH
gave the target sanazole derivative 4.19 All the intermediate com-
pounds were characterized using 1H NMR (300 MHz Varian spec-
trophotometer, USA) and ESI-MS (Varian Prostar ESI mass
spectrometer, USA in negative mode). The target ligand 4 was pre-
pared from compound 3 just prior to radiolabeling with 99mTc and
used as such without further purification.
The complex was evaluated in Swiss mice (20–25 g body
weight) bearing fibrosarcoma tumor. Solid tumor models were
developed in Swiss mice by implantation of HSDM1C1 murine
fibrosarcoma tumor cells. About 106 cells in 100
lL volume were
injected subcutaneously on the dorsum of every animal. The tu-
mors were allowed to grow till they reached approximately
10 mm in diameter after which the animals were used for in vivo
studies. Complex 5 purified by HPLC (100 lL, 20 lCi/740 KBq)
was administered intravenously through the tail vein of each ani-
mal. Individual sets of animals (n = 3) were sacrificed at different
time points (30, 60 and 180 min) and relevant organs and tissue
were excised to measure the associated radioactivity. The organs
were weighed and the activity associated with each organ was
measured in a flat-bed type NaI (Tl) counter with energy window
set for 99mTc (140 keV 10%). Activity associated with each organ
was expressed as percentage of the total injected activity per gram
of the organ or tissue. All the procedures performed were in accor-
dance with the national laws pertaining to the conduct of animal
experiments.
The distribution of complex 5 in different organs or tissue at dif-
ferent time points is shown in Table 1. The uptake of complex 5 in
tumor at different time points is shown in Figure 4. For comparison
tumor uptake obtained with standard agent [18F]FMISO, carried
out earlier (unpublished result), is also shown in the same figure.
The preparation of 99mTcN(PNP) complex (5) of sanazole deriv-
ative 4 is schematically shown in Figure 2. The procedure involves
prior preparation of [99mTcN]2+ precursor complex following a re-
ported protocol.16 This radiosynthon was subsequently reacted
simultaneously with bis[(diethoxypropylphosphanyl)ethyl]eth-
oxyethylamine (PNP6) and target ligand 4 to yield complex 5.
The radiochemical purity of complex 5 was assessed by HPLC using
a C18 reversed phase column using water/acetonitrile solvent sys-
tem following a gradient elution program.20 The HPLC elution pro-
file obtained with complex 5 is shown in Figure 3. Two closely
spaced peaks, corresponding to a syn–anti isomeric pair, could be
observed with the peak representing the predominant isomer elut-
ing at 22.2 min. The stereochemistry of the individual isomers,
however, was not ascertained in the present study. Ligands similar
to compound 4 has been used to prepare 99mTcN(PNP) complexes
both at the tracer level as well as macroscopic level. Structural
characterization of these complexes at macroscopic level revealed
square pyramidal geometry.16 Hence, complex 5 prepared in the
present study is also presumed to have similar geometry. Peak area
measurements from the chromatogram indicated that the complex
(syn–anti isomers together) could be obtained in more than 88%
radiolabeling yield. The complex was purified using C18 reversed
phase analytical column. The fraction containing the complex
was collected and elution solvent was removed under vacuum.
The complex was then reconstituted in 10% aqueous ethanol and
subsequently used for further studies.
Initial tumor uptake of complex
5 observed at 30 min p.i.
[0.41(0.05)%ID/g] remained steady at 60 min p.i. Even after
60 min p.i. clearance of activity from tumor was slow (only ꢁ22%
activity observed in tumor at 60 min p.i. cleared at 3 h p.i.) which
indicated reduction and consequent trapping of the complex in the
hypoxic cells. It could be noted that, during the same interval,
ꢁ60% of activity observed in muscle at 60 min p.i. was cleared
which ruled out the possibility that activity associated with the tu-
mor is due to non-specific binding of the complex. On the contrary,
[
18F]FMISO showed significantly higher uptake in tumor at all the
time points studied. However, the rate of clearance of activity from
tumor was observed to be higher for [18F]FMISO (ꢁ44% cleared be-
tween 60 and 180 min p.i.) than complex 5.
For hypoxia targeting agents’, adequate lipophilicity is essential
for the radiotracer to enter into the cell by passive diffusion. Tumor
vasculature is often chaotic which makes it difficult to directly
deliver the radiotracer to the entire tumor mass. For distribution
across the entire tumor mass, the radiotracer may have to diffuse
To evaluate in vivo stability, the purified complex 5 was chal-
lenged with excess cysteine following a reported protocol.16 No
Figure 1. Scheme for the synthesis of nitrotriazole–cysteine conjugate.

1396
A. Mathur et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1394–1397
Figure 2. Synthesis of [99mTcN(PNP6)]–nitrotriazole complex.
Figure 3. HPLC profile of (a) [99mTcN(PNP6)] intermediate complex and (b) [99mTcN(PNP6)]–nitrotriazole complex.
through the cells along the concentration gradient between the
blood vessels and the intracellular environment. This requires the
concentration gradient to hold for sufficient period of time to facil-
itate the distribution of the radiotracer to cells which are away
from the blood vessels. It could be observed from Figure 5 that
complex 5 cleared very fast from blood, compared to [18F]FMISO,
through hepatobiliary route. This is evident from the presence of
significant level of activity in intestine as early as 30 min p.i.
(Table 1).
We have observed that fast clearance of the complex from blood
significantly limits the distribution of the complex in the tumor
(unpublished results). In addition, fast clearance of the complex
from blood may have possibly reversed the concentration gradient,
whereby the complex taken up in the tumor may be cleared before
Table 1
Bio-distribution studies of
bearing fibrosarcoma tumor
[
99mTcN(PNP6)]–nitrotriazole complex in Swiss mice
Organs
30 min
60 min
180 min
Blood
Liver
Int + GB
Heart
Lung
Spleen
Muscle
Kidney
Tumor/blood
Tumor/muscle
0.33 (0.02)
3.67 (1.37)
39.36 (11.80)
0.62 (0.15)
0.80 (0.30)
0.32 (0.13)
0.54 (0.04)
0.56 (0.12)
1.22 (0.11)
0.76 (0.05)
0.30 (0.07)
8.91 (3.53)
54.4 (5.55)
0.36 (0.07)
0.36 (0.08)
0.31 (0.05)
0.38 (0.07)
0.43 (0.30)
1.37 (0.25)
1.07 (0.25)
0.05 (0.01)
1.58 (0.13)
60.15 (7.58)
0.23 (0.06)
0.39 (0.10)
0.50 (0.16)
0.15 (0.01)
0.54 (0.17)
6.96 (1.40)
2.13 (0.31)
Figure 4. Uptake and retention behavior of [99mTcN(PNP6)]–nitrotriazole complex
Figure 5. Clearance of activity of
18F]FMISO from blood with time.
[
99mTcN(PNP6)]–nitrotriazole complex and
and [18F]FMISO in fibrosarcoma tumor.
[

A. Mathur et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1394–1397
1397
7. Lee, S. T.; Scott, A. M. Semin. Nucl. Med. 2007, 37, 451.
8. Ballinger, J. R.; Judy, W. M. K.; Rauth, A. M. J. Nucl. Med. 1996, 37, 1023.
9. Melo, T.; Duncan, J.; Ballinger, J. R.; Rauth, A. M. J. Nucl. Med. 2000, 41, 169.
10. Mallia, M. B.; Banerjee, S.; Venkatesh, M. Technetium-99m Radiopharmaceuticals;
Status and Trends, IAEA, 2009. 295.
11. Mallia, M. B.; Subramanian, S.; Mathur, A.; Sarma, H. D.; Venkatesh, M.;
Banerjee, S. J. Labelled Compd. Radiopharm. 2010, 53, 535.
12. Wardman, P. J. Phys. Chem. Ref. Data 1989, 18, 1637.
13. Krohn, K. A.; Link, J. M.; Mason, R. P. J. Nucl. Med. 2008, 49, 129S.
14. Kapoor, S.; Mathew, R.; Huilgol, N. G.; Kagiya, T. V.; Nair, C. K. J. Radiat. Res.
2000, 41, 355.
it could get reduced and trapped in hypoxic cells despite having a
favorable SERP. Though it cannot be conclusively stated, low tumor
uptake observed with the present complex could be due to these
reasons in which case, the present complex is unable to depict
the actual hypoxic status of the tumor.
The distribution of activity in other organs as well as the TBR
and TMR obtained with complex 5 are shown in Table 1. It could
be observed that both TBR and TMR improved significantly with
time attaining a maximum value of 6.96 (1.40) and 2.13 (0.31),
respectively.
To conclude, the present work describes the synthesis of a new
99mTc labeled hypoxia marker using [99mTcN(PNP)]2+ core. The
complex could be prepared in high yield and had high in vivo sta-
bility. 99mTcN(PNP) complex of sanazole derivative was envisaged
considering two distinct advantages of [99mTcN(PNP)] core. It re-
sults in a lipophilic complex, at the same time presence of PNP li-
gand helps in fast clearance of the complex from background
organs, especially liver.22 Though, as expected, a lipophilic complex
with fast liver clearance was obtained, fast clearance of the com-
plex from blood, which was not anticipated, probably resulted in
low uptake in tumor. This feature could limit its potential to indi-
cate the actual hypoxic status of the tumor.
15. Adams, G. E.; Flockhart, I. R.; Smithen, C. E.; Stratford, I. J.; Wardman, P.; Watts,
M. E. Radiat. Res. 1976, 67, 9.
16. Bolzati, C.; Mahmood, A.; Malago, E.; Uccelli, L.; Boschi, A.; Jones, A. G.; Refosco,
F.; Duatti, A.; Tisato, F. Bioconjugate Chem. 2003, 14, 1231.
17. To
a
cooled (0 °C) stirring mixture of triazole (100 mg, 0.58 mmol) and
compound
1
(226 mg, 0.58 mmol) in CH2Cl2 (15 mL), EDCI (123 mg,
0.63 mmol) was added and the resultant mixture kept stirring at 0 °C for 1 h.
Thereafter, it was brought to room temperature and stirred overnight. Upon
completion of the reaction (cf. TLC), the reaction mixture was washed with
water (3 ꢂ 10 mL), dried and concentrated to give the crude product, which
was purified by silica gel chromatography [EtOAc/CHCl3 (1:9 v/v)] to yield pure
2 as colorless oil.
Yield: 291 mg (92%), Rf = 0.3 [EtOAc/CHCl3 (1:9 v/v)]. 1H NMR (CDCl3, d ppm):
8.32 (s, 1H, triazole); 7.22–7.42 (m, 15H, (C6H5)3C–); 6.36 (d, 1H, –CONH–,
J = 7.5 Hz); 4.90 (s, 2H, –NCH2–); 4.48–4.54 (m, 1H, –NHCHCOO–); 4.19 (q, 2H,
CH3CH2O–, J = 7.2 Hz); 2.75–2.81 (m, 1H, –SCHAHB–); 2.60–2.66 (m, 1H, –
SCHAHB–); 1.25 (t, 3H, –CH2CH3, J = 7.2 Hz). MS (ESI, ꢀve mode): Mass (calcd)
C
28H27N5O5S 545.2; m/z (observed) 544.2.
18. To a solution of 2 (100 mg, 0.18 mmol) in MeOH (600
solution (400 L, 0.4 mmol) was added and stirred for 48 h at room
lL), aqueous 1 M KOH
l
Acknowledgment
temperature. Upon completion of the reaction (cf. TLC), MeOH was removed
under vacuum, water (5 mL) was added and the pH of the reaction mixture was
adjusted to 3 using aqueous 2 N HCl to give 3 as white precipitate which was
filtered and dried under vacuum.
The authors are thankful to Professor Adriano Duatti, University
of Ferrara, Italy for providing PNP6 ligand.
Yield: 80% (75 mg). Rf = 0 [EtOAc/CHCl3 (1:9 v/v)]. 1H NMR (CD3OD, d ppm):
8.63 (s, 1H, triazole), 7.22–7.42 (m, 15H, (C6H5)3C–); 5.15 (s, 2H, –NCH2–);
4.26–4.30 (m, 1H, –CHCOO–); 2.61–2.66 (m, 2H, –CH2S–).
Supplementary data
19. Compound 3 (50 mg, 0.096 mmol) was stirred with TFA (2 mL) for 10 min at
room temperature. To the resultant yellow solution, Et3SiH was added
dropwise until the solution turned colorless. Stirring was continued for
another 15 min. The solvent was removed under vacuum to obtain the
desired product 4 which was dried under vacuum and used as such for
radiolabeling without further characterization.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.12.
079.
20. The HPLC of the prepared complex was carried out on a JASCO PU 2080 Plus
dual pump HPLC system, Japan, with a JASCO 2075 Plus tunable absorption
detector and Gina Star radiometric detector system, using a C18 reversed phase
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observing the radioactivity profile.
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