Radiolabeling of paclitaxel with electrophilic 123I

Article abstract of DOI:10.1016/S0968-0896(99)00255-2

¹²³I-Labeled paclitaxel, [¹²³I]-1 was prepared by electrophilic aromatic radioiodination of 3'-N-(p-trimethylstannylbenzoyl)-3'- debenzoylpaclitaxel 2 with ¹²³I^- in the presence of peracetic acid. (C) 2000 Elsev

Full text of DOI:10.1016/S0968-0896(99)00255-2

Bioorganic & Medicinal Chemistry 8 (2000) 65±68
123
Radiolabeling of Paclitaxel with Electrophilic I
Eun Joo Roh, a,d Young Hoon Park, a Choong Eui Song, a,* Seung-Jun Oh, b
Yearn Seong Choe, b,* Byung-Tae Kim, b Dae Yoon Chi c,* and Deukjoon Kim d
aLife Sciences Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul, 130-650, South Korea
bDepartment of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong,
Kangnam-Ku, Seoul, 135-710, South Korea
cDepartment of Chemistry, Inha University, 253 Yonghyun-Dong, Nam-Ku, In-Chon, 402-751, South Korea
dCollege of Pharmacy, Seoul National University, Shinrim-Dong, Kwanak-ku, Seoul, 151-742, South Korea
Received 19 July 1999; accepted 25 August 1999
AbstractÐ123I-Labeled paclitaxel, [123I]-1 was prepared by electrophilic aromatic radioiodination of 30-N-(p-trimethylstannyl-
benzoyl)-30-debenzoylpaclitaxel 2 with 123
I
in the presence of peracetic acid. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
halo-benzoyl)-30-debenzoylpaclitaxel, the 30-N-(p-[123I]-
iodobenzoyl)-30-debenzoylpaclitaxel [123I]-1 was designed
Paclitaxel (Taxol1), a highly functionalized diterpene
natural product isolated from the bark of Paci®c yew
(Taxus brevifolia) demonstrated anticancer activity in
human malignancies such as ovarian, breast, lung, head
and neck and gastrointestinal tract cancers.1 A unique
mechanism of paclitaxel as an antimitotic agent is based
on its interaction with microtubule polymers which
results in the formation of stable bundles of cellular
microtubules that are resistant to depolymerization
back to tubulin.2±6
as a cancer diagnostic radiopharmaceutical for SPECT
(single photon emission computed tomography).
In this paper, we report the synthesis of 30-N-(p-
[
123I]iodobenzoyl)-30-debenzoylpaclitaxel [123I]-1 from
the trimethyltin precursor 2.
Results and Discussion
Many of the paclitaxel derivatives have been evaluated
in microtubule assembly assays and cytotoxicity assays
against cancer cell lines.7 Among them, analogues bear-
ing a halogen substituent at the para-position of 30-N-
benzoyl group exhibited the ability to induce micro-
tubule formation similar to that of paclitaxel.8 From the
strong microtubule binding anity and antitumor
activity against lung and ovarian cancers of 30-N-(p-
Synthesis of trimethyltin precursor 2
Trimethyltin precursor 2 and authentic iodopaclitaxel 1
were prepared as shown in Scheme 1.
Mixed anhydride 5 was prepared by the reaction of
pivaloyl chloride and p-trimethylstannylbenzoic acid in
the presence of NEt3 in CH2Cl2. p-Trimethyl-
stannylbenzoic acid 4 was prepared by the reaction of
ethyl p-bromobenzoate and hexamethylditin in the pre-
sence of catalytic amounts of tetrakis(triphenylpho-
sphine)palladium(0) in xylene, followed by hydrolysis
with LiOH according to the known procedure.9 The
coupling of mixed anhydride 5 with 30-amino-7-tri-
chloroacetylpaclitaxel derivative 7 which was prepared
Keywords:
30-N-(p-trimethylstannylbenzoyl)-30-debenzoylpaclitaxel;
electrophilic aromatic radioiodination; 30-N-(p-[123I]iodobenzoyl)-30-
debenzoylpaclitaxel; SPECT (single photon emission computed tomo-
graphy).
*Corresponding author. Tel.: +82-2-958-5143; fax: +82-2-958-5189;
e-mail: s1673@kist.re.kr
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00255-2

66
E. J. Roh et al. / Bioorg. Med. Chem. 8 (2000) 65±68
Conclusion
Electrophilic aromatic radioiodination of 2 a€orded the
123I-labeled paclitaxel [123I]-1 in 63±65% radiochemical
yield with e€ective speci®c activity greater than 37 GBq/
mmol. To the best of our knowledge, this is the ®rst
reported example of the radioiododestannylation on a
paclitaxel analogue. Biological studies with [123I]iodo-
paclitaxel are currently in progress and electrophilic
aromatic radio¯uorinations of 2 with both [18F]CH3
COOF and [18F]F2 are also being currently pursued.
Experimental
Materials and methods
High radionuclidic purity Na123I was obtained from
Korea Cancer Center Hospital (Seoul, South Korea).
Peracetic acid (32 wt% in acetic acid) was
purchased from Aldrich Chemical Company (Milwau-
kee, WI, USA) and HPLC solvents from J. T. Baker,
Inc. (Phillipsburg, NJ, USA). HPLC was carried out on
a Thermo Separation Products System (Fremont, CA,
USA) with a semipreparative column (Alltech Econosil
silica gel, 10 m, 10Â250 mm) and an analytical column
(Alltech Econosil C18, 5 m, 4.6Â250 mm). The eluent
was simultaneously monitored by a UV detector
(254 nm) and a NaI(T1) radioactivity detector. Thin
layer chromatography (TLC) was performed on Merck
F254 silica plates and visualized by UV illumination. For
radiolabeled compounds, TLC was analyzed on a Bios-
can TLC scanner (Washington, DC, USA). Radio-
activity was measured in a dose calibrator, and all
radiochemical yields are not decay-corrected unless
Scheme 1. Reagents and conditions: (i) Me3SnSnMe3, Pd(PPh3)4,
ꢀ
.
xylene, 115 C, 15 h; (ii) LiOH H2O, THF-MeOH; (iii) pivaloyl chloride,
NEt3, CH2Cl2; (iv) c-HCl, then aqueous NaHCO3, EtOAc; (v) 5, pyr-
idine; (vi) NH4OAc, MeOH-THF; (vii) NaI, peracetic acid, EtOH.
by oxazolidine cleavage of the compound 6 with concd
HCl in ethyl acetate succeeded smoothly in pyridine to
give 8 in 58% yield. Trimethylstannylpaclitaxel 2 was
then obtained in 85% yield by deprotection of tri-
chloroacetyl group with excess of ammonium acetate.
The 1H NMR (300 MHz, CDCl3) and HRMS (FAB) of
2 were consistent with the assigned structure. Electro-
philic aromatic iodination of 2 with NaI in the presence
of peracetic acid was then carried out to give the
authentic compound 1, and the reaction conditions for
electrophilic aromatic radioiodination of 7 with 123I
were optimized. Iodopaclitaxel 1 was obtained in good
1
noted. H NMR (300 MHz) spectra were recorded on a
Varian Gemini 300 spectrometer using tetramethylsilane
as an internal standard. IR spectra were recorded on a
MIDAC 101025 FT±IR spectrometer and main
absorption frequencies were given in cm 1. HRMS
(FAB) analysis was carried out by Mass Spectrometry
Analysis Group at Korea Basic Science Institute.
1
yield and its structure was con®rmed by H NMR and
HRMS (FAB) analyses.
123I-Labeling of paclitaxel
Synthesis of trimethyltin precursor 2
Electrophilic aromatic radioiodination on 2 was carried
out using 123I and peracetic acid in ethanol (Scheme
2).10 Progress of the reaction was monitored by radio-
TLC, and the substitution reaction gave essentially
one major radioactive product which corresponded to
123I-labeled paclitaxel, [123I]-1. After completion of the
reaction (ca. 30 min) and work up, the crude product was
puri®ed by high performance liquid chromatography
(HPLC) to give the pure radioactive product [123I]-1 in
63±65% radiochemical yield with e€ective speci®c activ-
ity greater than 37 GBq/mmol (1000 mCi/mmol).
p-Trimethylstannylbenzoic acid 4 was prepared by the
reaction of ethyl p-bromobenzoate and hexamethylditin
in the presence of catalytic amounts of tetrakis(tri-
phenylphosphine)palladium(0) in xylene, followed by
hydrolysis with LiOH according to the known proce-
dure.9 The oxazolidine-protected 7-trichloroacetyl-
paclitaxel 6 was prepared according to the reported
procedure.11
Pivaloyl p-trimethylstannylbenzoate 5. To a stirred mixture
of p-trimethylstannylbenzoic acid (0.1 g, 0.35 mmol),
triethylamine (0.07 mL, 0.53 mmol) in dichloromethane
(10 mL) was added dropwise pivaloyl chloride (0.05 mL,
0.38 mmol) at 0 ꢀC and stirring was continued for 3 h at
room temperature. The mixed anhydride 5 generated in
situ as above was used without further puri®cation for
the coupling reaction with 7-trichloroacetyl-30-deben-
zoylpaclitaxel 7.
Scheme 2. Reagents: Na123I, CH3CO3H, EtOH.

E. J. Roh et al. / Bioorg. Med. Chem. 8 (2000) 65±68
67
7-Trichloroacetyl-30-debenzoylpaclitaxel 7. To a solution
of oxazolidine-protected 7-trichloroacetylpaclitaxel 6
(0.2 g, 0.19 mmol) in ethyl acetate (5 mL) was added
concd HCl (0.05 mL) at 0 ꢀC. After stirring for 30 min,
the reaction mixture was neutralized by addition of
aqueous NaHCO3 and the organic phase was washed
with brine. The organic layer was dried over anhydrous
MgSO4 and concentrated in vacuo to a€ord the crude
product of 7 which was used without further puri®ca-
tion for the coupling reaction with pivaloyl p-trimethyl-
stannyl benzoate 5.
1710, 1621, 1561, 1222 cm
;
1H NMR (300 MHz,
1
CDCl3) d 8.06 (d, J=7.4 Hz, 2H), 7.67 (d, J=8.3 Hz,
2H), 7.60±7.25 (m, 10H), 6.96 (d, J=8.9 Hz, 1H), 6.19
(s, 1H), 6.14 (t, J=8.8 Hz, 1H), 5.69 (dd, J=8.8, 2.1 Hz,
1H), 5.59 (d, J=7.0 Hz, 1H), 4.87 (d, J=8.2 Hz, 1H),
4.71 (d, J=2.4 Hz, 1H), 4.32 (dd, J=6.7, 10.7 Hz, 1H),
4.23 (d of ABq, J=8.4 Hz, 1H), 4.11 (d of ABq,
J=8.4 Hz, 1H), 3.71 (d, J=7.0 Hz, 1H), 2.5 (sym.m,
1H), 2.31 (s, 3H), 2.17 (s, 3H), 1.71 (s, 3H), 1.61 (s, 3H),
1.16 (s, 3H), 1.07 (s, 3H); HRMS (FAB) m/z calcd for
C47H51INO14: 980.2354. Found: 980.2407.
7-Trichloroacetyl-30-(p-trimethylstannylbenzoyl)-30-de-
benzoylpaclitaxel 8. To a stirred solution of the crude
product of 7 (50 mg, 0.05 mmol) in pyridine (5 mL) was
added dropwise p-trimethylstannylbenzoyl pivaloyl
anhydride (0.17 mL) at 0 ꢀC. After stirring for 2 h at the
same temperature, the reaction mixture was diluted with
ethyl acetate and washed with brine. The organic layer
was dried over anhydrous MgSO4 and concentrated in
vacuo. The crude residue was puri®ed by ¯ash column
chromatography on silica gel (EtOAc:n-hexane=1:2) to
Radiolabeling
30-(p-[123I]Iodobenzoyl)-30-debenzoylpaclitaxel
[
123I]-1.
Na123I in 0.01 N NaOH (220 mL) was treated with 0.05
N H3PO4 (40 mL). To this solution were added succes-
sively the solution of 2 (2 mg, 0.002 mmol) in EtOH
(300 mL) and peracetic acid (10 mL of 10 mL 0.32 wt%/
2.4 mL H2O, 0.2 mmol) at room temperature. After
30 min, the reaction was quenched by addition of aqu-
eous NaHSO3 solution (10 mL of 1 mg/mL H2O,
0.096 mmol) and the reaction mixture was diluted with
CH2Cl2. After washing with water, the organic layer
was passed through a short plug ®lled with 7 cm of
anhydrous Na2SO4. The solvents were evaporated
under a gentle stream of N2, and the residue was redis-
solved in 1 mL of CH2Cl2 and injected onto a semi-
preparative HPLC column, which was eluted with a
35:65 mixture of CH2Cl2 (5% i-PrOH) and n-hexane at a
¯ow rate of 4 mL/min. The desired product [123I]-1 eluted
at 26±28 min was collected (Fig. 1) and the solvents were
removed. The residue was resolubilized in ethanol and
passed through a sterile membrane ®lter (0.22 mm), and
then diluted with sterile saline to give a ®nal solution of
10% ethanol in saline. Radiochemical purity of [123I]-1
was determined to be greater than 99% by a radio-TLC
developed in a 1:1 mixture of ethyl acetate and n-hexane
(Fig. 2). E€ective speci®c activity of [123I]-1 was deter-
mined by HPLC method (Fig. 3). An aliquot of the ®nal
1
give 8 (38 mg, 58% yield) as a white solid: H NMR
(300 MHz, CDCl3) d 8.05 (d, J=7.1 Hz, 2H), 7.30±7.72
(m, 11H), 6.95 (d, J=8.3 Hz, 1H), 6.23 (m, 2H), 5.74
(dd, J=8.9, 2.2 Hz, 1H), 5.62 (d, J=7.2 Hz, 1H), 4.82
(d, J=2.8 Hz, 1H), 4.76 (m, 1H), 4.23 (d, J=8.4 Hz,
1H), 4.12 (d, J=8.5 Hz, 1H), 3.82 (d, J=6.9 Hz, 1H),
2.35 (s, 3H), 2.22 (s, 3H), 1.73 (s, 3H), 1.63 (s, 3H), 1.22
(s, 3H), 0.21 (s, 9H).
30-(p-Trimethylstannylbenzoyl)-3-debenzoylpaclitaxel 2.
To a stirred solution of 8 (38 mg, 0.03 mmol) in MeOH
(5 mL) and THF (5 mL) was added ammonium acetate
(13 mg, 0.16 mmol) at room temperature and the reac-
tion was monitored by TLC. After completion of the
reaction (ca. 2 h), the reaction mixture was diluted with
ethyl acetate and washed with brine. After concentra-
tion in vacuo, the crude product was puri®ed by ¯ash
column chromatography on silica gel (EtOAc:n-hexane
=1:1) to give 2 (28 mg, 85% yield) as a white solid: IR
(KBr) 3400±3500, 2900, 1700, 1650, 1516, 1431, 1381,
1
1227 cm
;
1H NMR (300 MHz, CDCl3) d 8.07 (d,
J=7.0 Hz, 2H), 7.25±7.70 (m, 11H), 6.92 (d, J=8.3 Hz,
1H), 6.23 (m, 2H), 5.72 (dd, J=2.8, 2.9 Hz, 1H), 5.61 (d,
J=7.2 Hz, 1H), 4.93 (d, J=2.8 Hz, 1H), 4.72 (m, 1H),
4.35 (m, 1H), 4.23 (d, J=8.4 Hz, 1H), 4.12 (d,
J=8.5 Hz, 1H), 3.78 (d, J=6.9 Hz, 1H), 2.53 (m, 1H),
2.35 (s, 3H), 2.23 (s, 3H), 1.72 (s, 3H), 1.61 (s, 3H), 1.21
(s, 3H), 0.22 (s, 9H); HRMS (FAB) m/z calcd for
C50H60NO14Sn: 1018.3048. Found: 1018.3036.
30-(p-Iodobenzoyl)-30-debenzoylpaclitaxel 1. To a solution
of 2 (2 mg, 0.002 mmol) in EtOH were added NaI
(0.32 mg, 0.002 mmol) and peracetic acid (32% in acetic
acid, 0.62 mL, 0.003 mmol) at 0 ꢀC and the reaction was
monitored by TLC. After completion of the reaction
(ca. 1 h), the reaction mixture was diluted with ethyl
acetate, washed with brine and dried over anhydrous
MgSO4. After evaporation of organic volatiles, the crude
product was puri®ed by ¯ash column chromatography
on silica gel (EtOAc:n-hexane=1:1) to give 1 (1.5 mg,
80% yield) as a white solid: IR (KBr) 3400±3500, 1740,
Figure 1. HPLC trace of puri®cation of [123I]iodopaclitaxel, [123I]-1.
Puri®cation was carried out on a silica gel column (10 m, 10Â250 mm)
eluted with a 35:65 mixture of CH2Cl2 (5% i-PrOH) and n-hexane at
4 mL/min. The eluent was simultaneously monitored by a UV detector
(254 nm) and a NaI(T1) radioactivity detector. The peak eluted
between 26 and 28 min is [123I]-1 and the major UV peak at 17±18 min
is the trimethyltin precursor.

68
E. J. Roh et al. / Bioorg. Med. Chem. 8 (2000) 65±68
curve obtained from di€erent concentrations of the
unlabeled standard 1 and the corresponding UV peak
area. Radiochemical yield was 63±65% and e€ective
speci®c activity was greater than 37 GBq/mmol. Total
synthesis time including HPLC puri®cation was 90 min.
Authenticity of [123I]-1 was con®rmed by coinjection
with unlabeled standard 1 on HPLC.
Acknowledgements
This research was supported by grant from the Ministry
of Science and Technology (2N16490 to C.E.S.), the
Samsung grant (#SBRI C-97-033 to Y.S.C.) and the
grant of Good Health R&D Project from the Ministry
of Health and Welfare, South Korea (HMP-97-M-2-
0037 to Y.S.C. and D.Y.C.).
Figure 2. Radio-thin layer chromatogram of the puri®ed [123I]iodopa-
clitaxel.
References
1. For review: Nicolaou, K. C.; Dai, W. M.; Guy, R. K.
Angew. Chem., Int. Ed. Engl. 1994, 33, 15.
2. Schi€, P. B.; Fant, J.; Horwitz, S. B. Nature. 1979, 277,
665.
3. Schi€, P. B.; Fant, J.; Horwitz, S. B. Proc. Natl. Acad. Sci.
USA 1980, 77, 1561.
4. Manfredi, J. J.; Parness, J.; Horwitz, S. B. J. Cell. Biol.
1982, 94, 688.
5. Kumar, N. J. Biol. Chem. 1981, 256, 10435.
6. Rowinsky, E. K.; Donehower, R. C.; Jones, R. J.; Tucker,
R. W. Cancer Res. 1988, 48, 4093.
7. For review: Taxane Anticancer Agents: Basic Science and
Current Status; Georg, G. I.; Chen, T. C.; Ojima, I.; Vyas, D.
M., Eds.; ACS Symposium Series No. 583; American Chemi-
cal Society: Washington, DC, 1995.
8. Georg, G. I.; Cheruvallath, Z. S.; Harriman, G. C. B.;
Hepperle, M.; Park, H.; Himes, R. H. Bioorg. Med. Chem.
Lett. 1994, 4, 1825.
9. Gitlitz, M. H.; Engelhardt, J. E.; Mery, C. M. US Pat.
4,138,483, 1979.
Figure 3. HPLC analysis of the puri®ed [123I]iodopaclitaxel in 10%
ethanol-saline. Analysis was performed on an analytical C18 column
(5 m, 4.6Â250 mm) eluted with a 30:70 mixture of 0.1 M NH4HCO3:
methanol at 1 mL/min. The peak at 19±20 min is [123I]-1.
10. Neumeyer, J. L.; Wang, S.; Milius, R. A.; Baldwin, R. M.;
Zea-Ponce, Y.; Ho€er, P. B.; Sybirska, E.; Al-Tikriti, M.;
Charney, D. S.; Malison, R. T.; Laruelle, M.; Innis, R. B. J.
Med. Chem. 1991, 34, 3144.
11. Kim, K. S.; Chai, K. B.; Moon, Y. H.; Lee, K. O.; Kim, N.
D.; Ha, T. H.; Shin, J. A. PCT WO 98/08832, 1998.
solution was analyzed on an analytical C18 column
eluted with a 30:70 mixture of 0.1 M NH4HCO3:
methanol with a ¯ow rate of 1 mL/min. The area of UV
peak coeluted with [123I]-1 was compared to the standard