Synthesis of N-(2-diethylamino-ethyl)-4-(4-fluoro-benzamido)-2-methoxybenzamide (desiodo-MIP-1145) by coupling technique and its radioiodination: a potential melanoma imaging agent

Article abstract of DOI:10.1002/jlcr.3415

Radioiodinated MIP-1145, which specifically targets melanin, is an ideal candidate for targeted therapy of melanoma. An analogue of MIP-1145 lacking the iodo-substituent (desiodo-MIP-1145) was synthesized as a labeling precursor in three simple steps. The radioiodination of desiodo-MIP-1145 by iodine-125 was carried out via an electrophilic substitution reaction. An optimization study for the iodination reaction was carried out. The labeled compound was isolated and purified by means of electrophoresis and HPLC. The maximum radiochemical yield, 76%, was obtained with radiochemical purity greater than 99%. The log P value for [¹²⁵I]MIP-1145 was measured as 4.5.

Full text of DOI:10.1002/jlcr.3415

Research article
Received 27 February 2016,
Revised 30 April 2016,
Accepted 18 May 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3415
Synthesis of N-(2-diethylamino-ethyl)
-4-(4-fluoro-benzamido)-2-methoxybenzamide
(desiodo-MIP-1145) by coupling technique
and its radioiodination: a potential melanoma
imaging agent
a
a
b
a
*
H. Aglan, S. A. Kandil, A. F. EL-Kafrawy and U. Seddik
Radioiodinated MIP-1145, which specifically targets melanin, is an ideal candidate for targeted therapy of melanoma. An
analogue of MIP-1145 lacking the iodo-substituent (desiodo-MIP-1145) was synthesized as a labeling precursor in three
simple steps. The radioiodination of desiodo-MIP-1145 by iodine-125 was carried out via an electrophilic substitution
reaction. An optimization study for the iodination reaction was carried out. The labeled compound was isolated and purified
by means of electrophoresis and HPLC. The maximum radiochemical yield, 76%, was obtained with radiochemical purity
greater than 99%. The log P value for [125I]MIP-1145 was measured as 4.5.
Keywords: desiodo-MIP-1145; coupling agent; radioiodination; melanoma imaging agent; N-(2-diethylamino-ethyl)-4-(4-fluoro-
benzamido)-5-[125I]iodo-2-methoxybenzamide
Mass spectrometry was performed using Hewlett Packard 5988
Introduction
spectrometer. The NMR spectra were recorded on a Varian Mercury VX-
Most melanomas are pigmented by the presence of melanin, an 300 NMR spectrometer at 300 MHz for 1H and at 75.46 MHz for 13C. HPLC
intracellular melanocyte pigment.1,2 This makes a convenient target
system (Sykam) and electrophoresis apparatus (3000 p-series) were used
for purification. Radioactivity was measured by γ-counter (Nucleus Model
2010) connected with well-type NaI (Tl) crystal.
for development of radionuclide therapy of metastatic melanoma.
Several biochemical entities have been investigated as potential
imaging and radiotherapeutic agents, including monoclonal
antibodies,3 iodothiouracils,4 melanocortin-1 receptor-targeting
Chemistry
peptides,5 chloroquine,6 methylene blue dye,7 and iodobenzamides.8
N-(2-Diethylamino-ethyl)-4-nitro-2-methoxybenzamide (1)
The value of radiolabeled benzamides for imaging melanoma
through affinity for melanin was first realized in 1986.9 Recently,
4-Nitro-2-methoxybenzoic acid (200 mg, 1.0 mmol), Chloro-dipyrrol-
several studies10–13 have demonstrated that some N-alkylated idinocarbenium hexafluorophosphate (PyCIU, 366 mg, 1.1 mmol), and
diisopropylethylamine (DIPEA, 348.5 μL, 2.2 mmol) were dissolved in
anhydrous dichloromethane (3 mL). The resulting solution was stirred
for 20 min at room temperature to generate acid chloride. Then 2-
derivatives of benzamides can facilitate detection of melanoma
foci when iodinated with radioactive isotopes (123I and 131I).
Following the evaluation of a library of benzamide compounds,
[
131I]MIP-1145 was selected for entry into clinical trials based
diethylaminoethylamine (154.5 μL, 1.1 mmol) was added with
a
continuous stirring for an additional 30 min, followed by column
chromatography with methanol/dichloromehane/aqueous ammonia
solution (4:1:0.005 v/v/v) to yield a yellow wax-like solid (190 mg,
64.3%). 1H NMR [D6]DMSO: δ 0.96 (t, 6H), 2.6 (q, 4H), 2.8 (t, 2H), 3.4 (t,
on its solubility profile, radiochemical stability, antitumor activity
and significant melanin-specific tumor uptake.14 As far as we
know, there is no published data for the synthesis chemistry of
desiodo-MIP-1145. Our strategy was to synthesize desiodo-
MIP-1145 and optimize its labeling by 125I.
aCyclotron Project, Nuclear Research Centre, Atomic Energy Authority, B.O.
13759, Cairo, Egypt
Experimental
bFaculty of Science, Ain Shams University, Cairo, Egypt
Materials and equipment
*Correspondence to: H. Aglan, Cyclotron Project, Nuclear Research Centre,
Atomic Energy Authority, B.O. 13759, Cairo, Egypt.
E-mail: hany3glan2@yahoo.com.
All chemicals used in this study were of the highest purity grade from
Sigma-Aldrich and Pierce Chemical Company and were used without
further purification. In all cases, double distilled water was used. Na125
I
(185 MBq/5 mL) in diluted NaOH, pH 7–11, was purchased from Institute
Additional supporting information may be found in the online version of this
of Isotopes, Budapest, Hungary.
article at the publisher’s web-site.
J. Label Compd. Radiopharm 2016
Copyright © 2016 John Wiley & Sons, Ltd.

H. Aglan et al.
2H), 3.8 (s, 3H), 7.6 (s, 1H), 7.7 (d, 1H), 7.9 (d, 1H).13C NMR [D6]DMSO: δ
12.6, 36.1, 47.2, 50.7, 56, 101.2, 117.4, 121.7, 128.8, 140.5, 155.6, 166. MS
(M + H), 296.21 (found) and 295.33 (calculated).
ethanol. The contents were kept for 40 min at room temperature. The
reaction was terminated by the addition of sodium metabisulfite
(100 μL, 10 mg/mL). The radiolabeled compound was purified on
lichrosorb (RP C18, 250 × 4.6 mm, 5 μm) column. The column was eluted
with a mixture of 0.02 M sodium phosphate pH 3/acetonitrile (1:1 v/v) at
flow rate of 1 mL/min. The wavelength of the detector was adjusted to
308 nm. The product was collected, evaporated under reduced pressure,
dissolved in saline solution, filtered, and counted.
The iodination reaction was performed on larger scale (fivefold) using
Na127I for MS and NMR analysis. The product was isolated by HPLC on
discovery ®Bio Wide Pore column (RP C18, 50 × 10 mm, 10 μm) at
2.5 mL/min with the same mobile phase used for the labeled compound.
1H NMR [D6]DMSO: δ 1.0 (t, 6H), 2.6 (q, 4H), 2.8 (t, 2H), 3.5 (t, 2H), 3.8
(s, 3H), 6.1 (s, 1H), 7.4 (dd, 1H), 7.7 (s, 1H), 7.9 (dd, 1H). 13C NMR [D6]DMSO:
δ 11.5, 36, 47.1, 50.6, 56, 90.5, 101.3, 117.2, 125, 129.6, 131.2, 137.5,
143.5, 155.7, 163.3, 165, 165.8. MS (M + H), 514.3 (found) and 513.5
(calculated).
N-(2-Diethylamino-ethyl)-4-amino-2-methoxybenzamide (2)
A 170 mg of compound 1 was dissolved in ethanol/tetrahydrofuran
(1:1 v/v) solution (5 mL). The solution was added to 60 mg of Pd/C 10%
in a dry flask. Then, 100 mg of ammonium formate was added in one
portion. The mixture was stirred for 1 h under an argon atmosphere.
The contents were filtered using a pad of celite and followed by washing
with a 20 mL of ethanol/tetrahydrofuran (1:1 v/v) solution. The excess
solvent was removed, and the residue was dissolved in saturated sodium
hydrogen carbonate solution (10 mL). Finally, the product was extracted
three times with ethyl acetate (10 mL) and the ethyl acetate layers
combined and evaporated to give the desired product in a yield of
88.3%. 1H NMR [D6]DMSO: δ 0.96 (t, 6H), 2.6 (q, 4H), 2.8 (t, 2H), 3.4
(t, 2H), 3.8 (s, 3H), 6.3 (s, 1H), 6.6 (d, 1H), 7.8 (d, 1H). 13C NMR [D6]DMSO:
δ 11.9, 36.3, 46.5, 50.2, 55.7, 103.2, 114.5, 122.2, 129.2, 153.5, 155.6,
165.6. MS (M + H), 266.2 (found) and 265.35 (calculated).
Results and discussion
N-(2-Diethylamino-ethyl)-4-(4-fluoro-benzamido)-2-
methoxybenzamide (3)
In this study, we described the synthesis chemistry of desiodo-
MIP-1145 and its radioiodination by 125I (Scheme 1). The
synthesis was carried out in a sequence of three steps. There
were two PyCIU/DIPEA amide condensations (steps A and C)
and a transfer hydrogenation (step B) using an ammonium
formate hydrogen donor.15–17 Labeling (step D) of desiodo-
MIP-1145 was optimized by adjusting the different parameters,
namely, substrate concentration, medium pH, oxidizing agent,
and reaction time. The reaction was examined in methanol,
acetonitrile, ethanol, Dimethylformamide and Dimethyl sulfoxide.
Ethanol gave the best yield. The maximum radiochemical
yield, 76%, was obtained using 0.5 μmol substrate in
ethanol (200 μL, 1 mg/mL), 0.44 μmol chloramine-T in water
(100 μL, 1 mg/mL), 0.02 mol/L phosphate buffer pH 3 (100 μL),
and 3.7 MBq Na125I at room temperature within 40 min.
4-Fluoro-benzoic acid (84 mg, 0.6 mmol) and PyCIU (220 mg, 0.66 mmol)
were dissolved in anhydrous dichloromethane (3 mL) and DIPEA
(210 μL, 1.2 mmol) was added. The resulting solution was stirred for
20 min at room temperature. This mixture was poured to a flask
containing the compound 2 (125 mg 0.47 mmol). The reaction was
allowed to proceed for a period of 4 h. The product was a pale yellow
wax-like solid, 39%. 1H NMR [D6]DMSO: δ 1.0 (t, 6H), 2.6 (q, 4H), 2.8
(t, 2H), 3.5 (t, 2H), 3.8 (s, 3H), 6.7 (s, 1H), 7.3 (dd, 1H), 7.5 (d, 1H), 7.8
(dd, 1H), 8.1 (d, 1H). 13C NMR [D6]DMSO: δ 11.5, 36, 47.1, 50.6, 56,
103.3,116, 117.2, 122, 128.5, 129.6, 131.2, 139.5, 155.7, 163.3, 165, 165.8.
MS (M + H), 388.2 (found) and 387.45 (calculated).
Radiolabeling of desiodo-MIP-1145
To 200 μg (0.5 μmol) of desiodo-MIP-1145 dissolved in ethanol (200 μL),
a freshly prepared solution of chloramine-T (100 μL, 1 mg/mL in H2O),
0.02 mol/L phosphate buffer pH 3 (100 μL) and Na125I (3–5 MBq) were
The purification of [125I]MIP-1145 was carried out using HPLC
(Figure 1). The unreacted radioiodide was separated at the
added. The mixture was brought to a total volume of 0.5 mL with retention time of 2.5 min, while the precursor was detected at
Scheme 1. Synthesis and radioiodination of desiodo-MIP-1145.
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J. Label Compd. Radiopharm 2016

H. Aglan et al.
Figure 1. HPLC radiochromatogram and ultraviolet (UV) absorbance profile of reaction mixture of desiodo-MIP-1145 with 125I+ separated on lichrosorb (RP C18,
250 × 4.6 mm, 5 μm) column. Mobile phase: 0.02 M sodium phosphate pH 3/acetonitrile (1:1 v/v). The flow rate is 1 mL/min.
6.5 min, and the labeled [125I]MIP-1145 was detected away at
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uptake characteristics is 1.4,19 while the measured log P value
for [125I]MIP-1145 was found to be 4.5, indicating that
[
125I]MIP-1145 may be useful as a melanoma imaging agent.
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Conclusion
A simple synthesis scheme has been developed leading to the
procurement of desiodo-MIP-1145 as a precursor for the
production of a potential melanoma imaging agent, [125I]MIP-
1145. The maximum labeling yield for [125I]MIP-1145 was 76%
with purity greater than 99%. The measured log P value for
[
125I]MIP-1145, 4.5, is higher than the minimally suggested log
P value of 1.4 that is necessary for the enhanced tumor uptake
characteristics.
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Conflicts of interest
The authors declare that they have no conflict of interest.
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Supporting information
Additional supporting information may be found in the online
version of this article at the publisher’s web-site.
J. Label Compd. Radiopharm 2016
Copyright © 2016 John Wiley & Sons, Ltd.
www.jlcr.org