The synthesis of pteroyl-lys conjugates and its application as Technetium-99m labeled radiotracer for folate receptor-positive tumor targeting

Article abstract of DOI:10.1016/j.bmcl.2011.02.014

Aiming to develop a new ^99mTc-labeled folate derivative for FR-positive tumor imaging, a simpler method has been established to synthesize the folate-drug conjugates with free α-carboxyl group. In this study, the conjugate pteroyl-lys-HYNIC was

Full text of DOI:10.1016/j.bmcl.2011.02.014

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2025–2029
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The synthesis of pteroyl-lys conjugates and its application as Technetium-99m
labeled radiotracer for folate receptor-positive tumor targeting
Hongjuan Guo a, Fang Xie a, Meilin Zhu a, Yan Li a,b, Zhi Yang b, Xuebin Wang a, Jie Lu a,
⇑
a Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, PR China
b Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Breast Center, Peking University School of Oncology,
Beijing Cancer Hospital and Institute, Beijing 100142, PR China
a r t i c l e i n f o
a b s t r a c t
Aiming to develop a new 99mTc-labeled folate derivative for FR-positive tumor imaging, a simpler method
Article history:
Received 28 September 2010
Revised 25 January 2011
Accepted 4 February 2011
Available online 13 February 2011
has been established to synthesize the folate-drug conjugates with free a-carboxyl group. In this study,
the conjugate pteroyl-lys-HYNIC was synthesized and labeled with 99mTc using tricine and TPPTS as co-
ligands. The radiochemical purity of the final complex 99mTc(HYNIC-lys-pteroyl)(tricine/TPPTS), 5 was
high (>98%), and it remained stable in saline and plasma over 6 h after preparation. The biologic evalu-
ation results showed that the 99mTc labeled pteroyl-lys conjugate was able to specifically target the
FR-positive tumor cells and tissues both in vitro and in vivo, highlighting its potential as an effective
folate receptor targeted agent for tumor imaging.
Keywords:
99mTc
Folate receptor
Folate-drug conjugates
Pteroyl-lys-HYNIC
Tumor imaging
Ó 2011 Elsevier Ltd. All rights reserved.
The folate receptor (FR), a glycosylphosphatidylinositol-an-
chored membrane glycoprotein, has been respected as a very
attractive molecular target for tumor selective drug delivery, since
it is over-expressed on a wide variety of tumor cells, but highly
restricted in most normal human tissues.1–3 The FR binds the vita-
min folic acid with high affinity (Kd = 10ꢀ10 M), and mediates the
transport of folic acid into cell interior via an endocytic process.4
Thus, the folic acid has been successfully used as a ‘Trojan horse’
to conjugate with drug molecules, peptides, protein, liposomes
and radionuclides for targeting of FR-positive tumors.5–7
ever, the further biodistribution results of the radiotracers suggest
that the free -carboxyl group of glutamic acid moiety may have
a significant influence on the biologic properties in vivo, since the
-derivative possessed more favorable pharmacokinetic features
a
c
than others, and the pteroate derivative showed the lower uptakes
in FR-positive tissues than the folate derivatives did.14 Therefore,
the
to synthesize the folate conjugates with free
Unfortunately, the traditional coupling reactions with folic acid
result in mixtures of - and -derivatives, since both carboxyl
c-carboxyl group should be the preferred site for derivatization
a- carboxyl group.
a
c
The folic acid, a combination of the pteroic acid (Pte) and gluta-
groups of the glutamate moiety have similar reactivities. Separa-
tion of these mixtures is often difficult and some biologic results
have been obtained on mixtures.15 Many efforts have been done
mate (Glu), offers two positions for derivatization, the
boxylic acid (Fig. 1). The role of the Glu residue of folate and the
necessity of a free -carboxyl group to retain binding affinity to
a- and c-car-
a
to synthesize the defined a- and c
-derivatives.10,11,13
FR are still debated in the literature.8–10 The first findings indicated
that the glutamate moiety of folic acid is essential for FR binding,
because pteroic acid (Fig. 1), a fragment of folic acid lacking the dis-
tal glutamic acid residue shows a poor binding affinity to the FR. But
the results of subsequent studies showed that the pteroic-conju-
gates could still selectively bind to the FR-positive tumor cells,
which indicate that the Glu residue of folate is not critical for the fo-
late receptor recognition.10–13 The findings reported by Leamon and
It has been shown that folate-based radiopharmaceutical can be
a useful tool for the detection and characterization of FR-positive tu-
mors by means of noninvasive molecular imaging.15–19 In this study,
in order to develop a new 99mTc-labeled pteroate derivative as a po-
tential radiopharmaceutical for FR-positive tumor imaging, we de-
signed and synthesized pteroyl-lys conjugates ( Fig. 1), in which
the glutamate residue of folate is replaced by a lysine. It is a new
strategy to synthesize pteroyl-lys conjugate with free
a-carboxylic
Müller et al. suggest that the free
a-carboxyl group is not necessary
group, since the -amion group of the lysine moiety can be easily
e
for FR binding either, since both
c
-glutamyl- and
a
-glutamyl-linked
modified and selectively couple with a functional agent.
conjugates were able to bind to FR-positive cells in vitro.11,13 How-
Scheme 1 shows the synthesis of pteroyl-lys-HYNIC conjugate
2. The reaction of pteroyl azide 320 with N(
e)-BOC-Lysine in the
presence of tetramethylguanidine afforded the pteroyl-lys-BOC
conjugate 4. Treatment the BOC protected compound 4 with TFA
⇑
Corresponding author.
E-mail address: ljie74@bnu.edu.cn (J. Lu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.014

2026
H. Guo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2025–2029
Figure 1. The structures of folic acid, pteroic acid, pteroyl-lys 1 and pteroyl-lys-HYNIC 2.
Scheme 1. Reagents and conditions: (a) trifluoracetic anhydride, THF, 0 °C–rt, 10 h, yield 40%; (b) ice, THF, rt, 5 h, yield 98%; (c) hydrazine, DMSO, rt, 12 h, yield 72%; (d)
potassium thiocyanate, trifluoroacetic, 0 °C to ꢀ10 °C; tert-butylnitrite, rt, 5 h, yield 67%; (e) N( )-BOC-Lysine, tetramethylguanidine, DMSO, rt, 7 h, yield 90%; (f)
trifluoroacetic, DMF, pyridine, 0 °C, yield 94%; (g) succinimidyl 6-[2-(4-dimethylamino)benzaldehydehydrazono]nicotinate, DMSO, pyridine, rt, 16 h, yield 65%.
e
yielded the pteroyl-lys 1.21 Compound 1 was then coupled with
The biodistribution and pharmacokinetics of the radiotracer
were evaluated in athymic mice bearing KB tumors to further
investigate the FR binding properties of 99mTc-labeled folate conju-
gate 5 in vivo.27 The radiolabeled complex was purified by HPLC for
the biodistribution studies to remove the excess of cold ligand. A
volume of 0.1 ml of the purified radiotracer solution (ꢁ150 kBq,
ꢁ90 pmol/l) was injected into the mice via the tail vein. Then the
mice (n = 3) were sacrificed by cervical dislocation at 60, 120 and
240 min post-injection. Blocking experiments were performed by
co-injected with excess folic acid (100 lg/mouse), and the mice
were sacrificed at 120 min post-injection. The selected tissues
and organs were removed, weighed and measured in a well-type
NaI(Tl) c-counter. The results were expressed as the percent up-
succinimidyl
6-[2-(4-dimethylamino)benzaldehydehydrazon-
o]nicotinate22 to give the pteroyl-lys-HYNIC conjugate 2.23
The conjugate 2 was labeled with 99mTc using tricine and TPPTS
as co-ligands in three steps (Scheme 2). The resulting solutions
were analyzed by radio-HPLC.24 The retention times of 99mTcO4
,
ꢀ
99mTc-tricine, 99mTc(HYNIC-lys-pteroyl)(tricine) and 99mTc(HY-
NIC-lys-pteroyl)(tricine/TPPTS) 5 were 10.0, 12.8, 14.9 and 21.6
min, respectively. The complex 5 was purified by HPLC and its final
radiochemical purity was over 98% (Fig. 2). In this study, HYNIC has
been chosen as the bifunctional coupling agent due to its high
labeling efficiency at low HYNIC-conjugate concentrations. The
complex 5 was hydrophilic (log P = ꢀ2.89 0.06, n = 5) and re-
mained stable in saline and plasma over 6 h after preparation.
To assess the ability of the complex 5 to target the folate recep-
tor, in vitro experiments were performed with KB cells, a human
oral cancer cell line over expressing the FR.13,14,25,26 As shown in
take of injected dose per gram of tissue (%ID/g).
The results of biodistribution are shown in Table 1. As noted
from Table 1, the tumor accumulation of radioactivity was high
already at 1 h after injection (5.39 0.40%ID/g). The maximal tumor
accumulation was found after 2 h (9.60 1.46%ID/g) and was
almost completely retained over the time of investigation
(7.85 1.37%ID/g, 4 h after injection). Clearance from the blood
was fast and the nonspecific retentions of radioactivity in nontar-
geted tissues (lung, spleen, liver, muscle) were low, leading to
increasing tumor-to-blood and tumor-to-muscle ratios over time
Figure 3, the complex
5 displayed a high cell binding of
28.38 4.15% of the total added radioactivity. The internalized
fraction was 20.18 3.43% of total activity. Pre-incubation of the
cells with excess folic acid resulted in a significant blockade (<2%
of the total activity, p <0.05). These data indicate that the complex
5 can target the FR specifically and be internalized in the KB cells.

H. Guo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2025–2029
2027
Figure 3. In vitro experiment of the 99mTc-radiotracer 5 with KB cells; cell total
binding, internalization and blocked by excess folic acid (p <0.05). The cell binding
were calculated per 0.5 mg protein and expressed as percentage of total added
radioactivity.
(from 5.04 and 3.05 at 1 h p.i. to 25.32 and 8.01 at 4 h p.i., respec-
tively). A significant accumulation and retention of radioactivity
were also found in the kidneys (63.26 3.40%ID/g at 1 h p.i. and
88.61 9.17%ID/g at 4 h p.i.), since FRs are abundant in the renal
proximal tubules.28,29 Co-injection of excess folic acid almost
completely blocked the tumor uptake (0.36 0.10%ID/g at 2 h p.i.
p <0.05), as well as the accumulation of radioactivity in the kidneys
(3.79 0.71%ID /g at 2 h p.i. p <0.05), indicating the FR-specific
uptakes of radiofolate in these FR-positive tissues. Compared to
Scheme 2. Reagents and conditions: (a) tricine 40 mg, SnCl2ꢂ2H2O 20
lg, rt,
15 min; (b) conjugate 2 20 lg, pH 3–5, 100 °C, 40 min; (c) trisodium triphenyl-
phosphine-3,30,300-trisulfonate (TPPTS) 1 mg, pH 4.5, 100 °C, 20 min.
Figure 2. The HPLC profiles of 99mTcO4
(A, Rt = 10.0 min), 99mTc-tricine (B, Rt = 12.8 min), 99mTc-(HYNIC-lys-pteroyl)(tricine) (C, Rt = 14.9 min) and
ꢀ
99mTc(HYNIC-lys-pteroyl)(tricine/TPPTS) 5 (D, Rt = 21.6 min).

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H. Guo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2025–2029
Table 1
Biodistribution of 99mTc(HYNIC-lys-pteroyl)(tricine/TPPTS) in athynic nude mice bearing KB tumor xenograftsa
b
1 h
2 h
2 h blockade
4 h
Tissues
Heart
Liver
2.48 0.65
3.19 0.09
2.30 0.18
63.26 3.40
0.96 0.33
2.17 0.59
1.16 0.27
1.77 0.17
1.60 0.58
1.07 0.03
5.39 0.40
2.71 0.18
2.47 0.06
2.01 0.64
89.60 5.06
1.01 0.16
2.13 0.74
1.64 0.60
1.78 0.48
1.58 0.95
0.73 0.06
9.60 1.46
0.47 0.06
1.83 0.33
0.76 0.11
3.79 0.71c
0.76 0.10
1.62 0.90
0.66 0.10
0.30 0.12
0.63 0.26
0.65 0.10
0.36 0.10c
2.03 0.18
2.08 0.18
1.73 0.13
88.61 9.17
0.60 0.12
1.72 0.72
1.18 0.35
0.98 0.17
0.62 0.17
0.31 0.07
7.85 1.37
Lungs
Kidney
Spleen
Stomach
Bone
Muscle
Intestines
Blood
Tumor
Ratios
Tumor/Muscle
Tumor/Blood
Tumor/Liver
Tumor/Kidney
3.05
5.04
1.69
0.09
5.39
13.15
3.89
0.11
––
––
––
––
8.01
25.32
3.77
0.09
a
All data are the mean percentage (n = 3) of the injected dose of 99mTc(HYNIC-lys-pteroyl)(tricine)(TPPTS) per gram of tissue (%ID/g), the
standard deviation of the mean.
b
Co-injection of excess folic acid.
c
p <0.05, significance comparisons on tumor and kidney uptakes between the radiotracers with or without folate blockade at 2 h post-
injection.
99mTc-HYNIC-folate, a 99mTc-labeled folate-hydrazide-HYNIC con-
jugate reported by Guo et al.,19 complex 5 had a higher tumor up-
take but lower no-target tissues uptakes in liver, spleen and blood
(see Table 2). These results suggest that the Glu moiety of folate
could be replaced by a Lys residue, and the new pteroyl-lys conju-
gate still retained the targeting potential to FR. The Lys residue may
be acting as an efficient spacer between the Pte moiety and 99mTc-
HYNIC chelating moiety, resulting in more favorable biologic char-
acteristics as a FR imaging agent.
In order to visualize the distribution of the radiotracer in a liv-
ing animal, planar imaging studies of complex 5 were performed
in the athymic nude mice bearing KB tumors using a SPECT device.
The mice were injected with approximately 7.4 MBq of complex 5,
and blocking experiment was performed by co-injected with ex-
Table 2
The comparison of the bidistribution data between 99mTc(HYNIC-lys-pteroyl)(tricine/
TPPTS) and 99mTc-HYNIC-folate at 4 h p.i.
Complex
99mTc(HYNIC-lys-pteroyl)
(tricine/TPPTS)
99mTc-HYNIC-folate*
Tumor/%ID/g
Liver/%ID/g
Spleen/%ID/g
Blood/%ID/g
Tumor to liver
Tumor to spleen
Tumor to blood
7.85 1.37
2.08 0.18
0.60 0.12
0.31 0.07
3.77
5.62 0.75
4.03 0.41
2.55 0.39
0.48 0.04
1.39
cess folic acid (100 lg/mouse). The mice were anesthetized with
1.5% isoflurane for imaging. Figure 4 illustrates the planar images
of the KB tumor-bearing mice administered with complex 5 (A)
and complex 5 with excess folic acid (B) at 2 h p.i. The KB tumor
could be clearly visualized and the uptakes of radiotracer in
FR-positive tumor and kidney were significantly blocked by co-
injection of excess folic acid. The images clearly confirmed that
the conjugate 99mTc(HYNIC-lys-pteroyl)(tricine/TPPTS) could
13.08
25.32
2.20
11.71
*
The data for 99mTc-HYNIC-folate are obtained from the studies of Liu et al. in the
same KB tumor-bearing mice model.30
Figure 4. The planar images of the athymic nude mice bearing KB tumors (male, 18–20 g) at 2 h p.i. (A) Control: injection of 7.4 MBq complex 5; (B) blocked: co-injection of
7.4 MBq complex 5 with excess folic acid (100 g).
l

H. Guo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2025–2029
2029
19. Guo, W.; Hinkle, G. H.; Lee, R. J. J. Nucl. Med. 1999, 40, 1563.
20. Luo, J.; Smith, M. D.; Lantrip, D. A.; Wang, S.; Fuchs, P. L. J. Am. Chem. Soc. 1997,
119, 10004.
target the folate receptor efficiently and selectively in vivo, high-
lighting its potential as an effective imaging probe for FR-positive
tumor detection.
In summary, we established a simpler method to synthesize fo-
late-drug conjugates with free a- carboxyl group. In this study, the
conjugate pteroyl-lys-HYNIC, in which the Glu residue of folic acid
was replaced by a lysine as the linker between the pteroic acid and
HYNIC, was synthesized and labeled with 99mTc. As we anticipated,
the 99mTc labeled pteroyl-lys conjugate 5 was able to specifically
target FR-positive tumor cells and tissues both in vitro and in vivo.
The promising biologic evaluation results suggest that this com-
plex could be a potential folate receptor targeted agent for tumor
imaging.
21. Pteroyl-lys,1H NMR (400 MHz, DMSO-d6): d 8.64 (s,1H), 7.66 (d, J = 8.7 Hz, 2H,
Ar), 6.64 (d, J = 8.7 Hz, 2H, Ar), 4.49 (s, 2H), 4.31 (s, 1H), 2.76 (m, 2H), 1.78 (m,
2H), 1.54 (m, 2H), 1.37 (m,2H). 13C NMR (400 MHz, DMSO-d6):174.1, 167.3,
166.3, 153.9, 151.9, 150.7, 149.5, 148.4, 131.0, 127.9, 111.2, 54.0, 53.5, 46.5,
40.1, 39.8, 39.2. ESI-MS: m/z [M+H]+ calcd for C20H25N8O4: 441.2, found 441.3.
22. Harris, T. D.; Sworin, M.; Williams, N.; Rajopadhye, M.; Damphousse, P. R.;
Glowacka, D.; Poirier, M. J.; Yu, K. Bioconjugate Chem. 1999, 10, 808.
23. Pteroyl-lys-HYNIC, 1H NMR (400 MHz, DMSO-d6): d 8.62 (s, 1H), 7.95–8.05 (m,
2H), 7.63 (m, 4H), 7.15 (s, 1H), 6.94 (m, 3H), 6.62 (m, 4H), 4.46 (s, 2H), 4.26 (m,
1H), 3.10 (overlap, 6H), 2.76 (m,2H), 1.35–1.76 (m, 6H). ESI-MS: m/z
[M+H]+,707. HRMS [M+H]+ calcd for C35H39N12O5 707.3166; found 707.3187.
24. HPLC was carried out with
a Venusil XBP C18 reversed-phase column
(250 ꢃ 10 mm, 5 m), Shimadzu SCL-10AVP series, working at a flow rate of
l
1.0 ml/min. 10 mM NH4HCO3(A) and CH3OH(B) mixtures were used as the
mobile phase (0–10 min, B: 5–50%; 10–20 min, B: 50–50%; 20–25 min, B: 50–
5%).
Acknowledgments
25. The human nasopharyngeal KB carcinoma cell line is cultured as monolayers at
37 °C in a humidified atmosphere containing 5% CO2. The cells were cultured in
FFRPMI medium (modified RPMI 1640, without folic acid) supplemented with
This work was supported by the National Natural Science
Foundation of China (20701004).
10% fetal calf serum and antibiotics (penicillin 100 IU/ml, streptomycin 100
l
g/
ml, Fungizone 0.25
lg/ml).Twenty-four hours prior to the experiment, the KB
cells were seeded in 12-well plates (5 ꢃ 105 cells/well) and incubated at 37 °C
to form confluent monolayer. All experiments were performed in triplicate.
After being washed once with FFRPMI medium, the cells were incubated at
37 °C for 1 h with approximately 7.4 KBq of HPLC purified complex 5 in 1 ml of
FFRPMI medium. The blocking studies were performed by addition of free folic
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