The synthesis of novel iodinated iminodiacetic acid analogues as hepatobiliary imaging agents

Article abstract of DOI:10.1007/s00706-004-0174-x

The synthesis and characterization of N-[2-[[4-iodo-2,6-bis(1-methylethyl) phenyl]amino]-2-oxoethyl]-N-(carboxymethyl)glycine and N-[2-[(4-iodo-2,6- diethylphenyl)amino]-2-oxoethyl]-N-(carboxymethyl)glycine is presented, as well as a modified and improved

Full text of DOI:10.1007/s00706-004-0174-x

Monatshefte f€ur Chemie 135, 1009–1014 (2004)
DOI 10.1007/s00706-004-0174-x
The Synthesis of Novel Iodinated
Iminodiacetic Acid Analogues
as Hepatobiliary Imaging Agents
1;ꢀ
1
2
´
´
Jasmina S. Brboric , Sote Vladimirov , Mirjana S. Jovanovic ,
3
´
and Nikola Dogovic
1
Institute of Pharmaceutical Chemistry and Drug Analysis, Faculty of Pharmacy,
Belgrade, Serbia and Montenegro
2
ꢀ
Vinca Institute of Nuclear Sciences, Laboratory for Radioisotopes, Belgrade,
Serbia and Montenegro
3
Military Technical Institute, Belgrade, Serbia and Montenegro
Received December 2, 2003; accepted (revised) January 9, 2004
Published online May 21, 2004 # Springer-Verlag 2004
Summary. The synthesis and characterization of N-[2-[[4-iodo-2,6-bis(1-methylethyl)phenyl]amino]-
2-oxoethyl]-N-(carboxymethyl)glycine and N-[2-[(4-iodo-2,6-diethylphenyl)amino]-2-oxoethyl]-N-
(carboxymethyl)glycine is presented, as well as a modified and improved synthesis of N-[2-[(2,
4-diiodo-6-methylphenyl)amino]-2-oxoethyl]-N-(carboxymethyl)glycine. These compounds are new
agents for hepatobiliary imaging.
Keywords. IDA analogues; Halogenation; NMR spectroscopy; IR spectroscopy; Mass spectroscopy.
Introduction
The halogenated analogues of N-(carboxymethyl)glycine (iminodiacetic acid;
IDA) labeled with technetium-99m have been used recently as diagnostic radio-
pharmaceutical for hepatobiliary imaging. Numerous derivatives of IDA have been
synthesized since the discovery of N-[2-[(2,6-dimethylphenyl)amino]-2-oxoethyl]-
N-(carboxymethyl)glycine HIDA [1, 2] in order to obtain compounds of better
hepatobiliary properties. Biokinetics of these compounds depends strongly on
molecular mass, lipophilicity, protein binding, and the substitution of the aromatic
system [3–5]. Introduction of halogen in the aromatic system of N-substituted IDA
derivatives increases the relative molecular mass and lipophilicity leading to
increased biliary excretion and reduction of urinary excretion and competition with
bilirubin. The hepatobiliary agents 3-iodo-2,6-diethyl-IDA (IODIDA) and 3-bromo-
2,4,6-trimethyl-IDA (mebrofenin) are widely used in hepatobiliary imaging [6].
ꢀ
Corresponding author. E-mail: jbrboric@pharmacy.bg.ac.yu

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J. S. Brboric et al.
1010
Unlike chlorine and bromine, iodine is unreactive towards most aromatic sub-
strates; thus, a more powerful iodinating species like iodonium equivalents (Iþ) or
reagent combinations that generate electrophilic iodine species [7, 8] are required
for direct aromatic iodination. The moderate reactivity of iodine with aromatic
substrates mandates for utilisation of an oxidizing agent (H2O2, HIO3, HgO,
CH3CO3H, HgCl2, KI) and a base (NH4OH, NaHCO3, KOH). It has been also
reported that aromatic iodination with molecular iodine was catalyzed by stoichio-
metric amounts of metal halides, such as AlCl3–CuCl2 [9] or SbCl5 [10]. The direct
iodination has been carried out by using NH4I and catalytic amounts of NOBF4 in
CF3COOH=CH2Cl2 or CF3COOH=CH3COOH with molecular oxygen [11]. Direct
iodination methods have been intensively developed in recent years by using iodo-
nium donating systems, such as iodine-nitrogen dioxyde [12], N-iodosuccinimide
[13], ICl (alone or in the presence of ZnCl2, pyridine, or benzyltrimethylammo-
nium chloride), or benzyltriethylammonium dichloroiodate=sodium bicarbonate as
a mild iodinating reagent for benzenamine [14]. Kajigaeshi et al. [15] reported the
use of benzyltrimethylammonium dichloroiodate (BTMA ICl2) as an iodinating
agent for aromatic amines.
Results and Discussions
The synthesis of the compounds 1, 2, and 3 in three steps is outlined in (Scheme 1).
The first step consists of a iodination by means of BTMA ICl2 according to Ref.
[15]. The reaction of the benzenamines with one or two equivalents of BTMA ICl2
in a dichloromethane-methanol mixture in presence of calcium carbonate gave the
4-iodo- or 2,4-diiodobenzenamines. This method gives good yields, no secondary
products are obtained, and the reaction is carried out under neutral conditions at
room temperature.
To obtain the target compounds, the iodinated benzenamines 1a, 2a, and 3a were
reacted with chloroacetylchloride in acetone. The products 1b, 2b, and 3b were
subsequently condensed with IDA in refluxing aqueous ethanol to yield 1, 2, and 3.
N-[2-[(2,4-Diiodo-6-methylphenyl)amino]-2-oxoethyl]-N-(carboxymethyl)gly-
cine (3) has been synthesized recently and labeled with technetium-99m [16]. The
iodination reaction was performed with I2 in aqueous NaHCO3 solution, and the
yield of the precursor 3a was 51%. The synthesis of 3a, using BTMA ICl2 as
iodinating agent as described above resulted in an improvement to 76% yield.
The structures of 1, 2, and 3 could be confirmed by their elemental analyses and
1
spectroscopic data (UV, IR, H NMR, 13C NMR, and GC-mass spectrometry; see
Scheme 1

Iodinated Iminodiacetic Acid Analogues
1011
Experimental). The purity of the compounds was confirmed by melting point, GC,
and TLC. The obtained products 1, 2, and 3 are promising for biological evaluation
studies.
Experimental
Melting points were determined on a Boetius PHMK 05 apparatus. UV spectra were recorded on a
CINTRA 20. IR spectra (KBr): FTS 3000 MX (BIO-RAD, Excalibur). 1H NMR and 13C NMR
spectra were recorded on a GEMINI-200 at 200 and 50MHz using DMSO-d6 as solvent and TMS as
internal standard, chemical shifts in ꢀ (ppm). Mass spectra: Hewlett Packard GCD series II (70 eV)
HP-5MS (cross-linked 5% PH-ME-Siloxane), 30 mꢁ 0.25 mmꢁ 0.25 mm, carrier gas: hydrogen,
0.9 cm3=min, T injector 250ꢂC, T detector 280ꢂC, T column from 150ꢂC to 280ꢂC (10ꢂC=min);
or HP 5090 and 5080 (70 eV), HPL 12m ꢁ 0.2 mm, carrier gas: He, 0.6 cm3=min, T detector 280ꢂC.
Elemental analyses were conducted using the Elemental Analyser Vario EL III CHNS=O, their
results were found to be in good agreement (ꢃ0.16%) with the calculated values. Thin layer
chromatography (TLC): TLC plates (Merck, silica gel 60 F254, 0.25mm, 200 ꢁ 200 mm; the com-
pounds were detected in UV light at 254 nm). All reagents were obtained from commercial sources
and used as received.
4-Iodo-2,6-bis(1-methylethyl)benzenamine (1a, C12H18IN)
To the solution of 1.77g of 2,6-bis(1-methylethyl)benzenamine (10 mmol) in CH2Cl2 (50 cm3) –
CH3OH (25 cm3), were added 3.48g of BTMA ICl2 (10 mmol) and 1.1 g of CaCO3 (11 mmol). The
mixture was stirred for 6 h at room temperature. During that period, the colour of the solution gradually
changed from yellow to light brown. On completion (monitored by TLC) the excess of CaCO3 was
filtered off and the filtrate was concentrated. To the obtained residue, 30cm3 of NaHSO3 solution (5%)
were added. The mixture was extracted with 4 ꢁ 30cm3 of diethyl ether, the ether layer was dried
(Na2SO4), filtered, and evaporated in vacuo to give 2.6 g (85%) of a yellowish-brown oily mass. The
crude product was used in next synthetic step. UV (methanol, c ¼ 2 ꢄ 10ꢅ5 mol dmꢅ3): ꢁmax (") ¼ 208
(34000), 249 (12200), 295 (3200)nm (molꢅ1 dm3 cmꢅ1); TLC: Rf ¼ 0.45 (n-hexane:benzene¼ 3:7),
Rf ¼ 0.85 (CHCl3); GC: single peak at Rt ¼ 7.4 min; MS (70 eV): m=z (%) ¼ 303 (83.7) [Mþ], 288
(100.0), 161 (7.4), 146 (25.0), 130 (9.8), 115 (7.1), 91 (6.2).
N-[4-Iodo-2,6-bis(1-methylethyl)phenyl]-2-chloroacetamide (1b, C14H19IClNO)
To the solution of crude 1a in 40cm3 of acetone 2 cm3 of chloroacetylchloride (25 mmol) were added.
The mixture was cooled at 0–4ꢂC with constant stirring for 2 h. To that solution, 30cm3 of HCl
(8%) were added and stirred for 30min. The precipited 1b was filtered off under vacuum, dried,
and recrystallized from ethanol to yield 2.47 g (65%). Mp 213–214ꢂC; TLC: Rf ¼ 0.14 (n-
hexane:benzene¼ 3:7), Rf ¼ 0.62 (CHCl3); UV (methanol, c ¼ 4.4 ꢄ 10ꢅ5 mol dmꢅ3): ꢁmax (") ¼ 209
(34200), 233 (13900) nm (molꢅ1 dm3 cmꢅ1); IR (KBr): ꢂꢂ¼ 3231 (b, s), 2960 (s), 2926 (w), 2868 (m),
1661 (s), 1514 (s), 1258 (m), 864 (m), 785 (m), 737 (w) cmꢅ1; GC: single peak at Rt ¼ 10.1min; MS
(70 eV): m=z (%) ¼ 379 (22.9) [Mþ], 331 (15.2), 330 (100.0), 315 (10.8), 286 (10.9), 252 (11.9), 160
(14.0), 144 (16.2), 130 (12.3), 118 (12.6), 91 (9.6), 77 (13.1).
N-[2-[[4-Iodo-2,6-bis(1-methylethyl)phenyl]amino]-2-oxoethyl]-N-
(carboxymethyl)glycine (1, C18H25IN2O5)
A solution of 2.0 g of 1b (5.3mmol) in 140 cm3 of ethanol was mixed with an equimolar amount of
IDA (1.1g) in 65 cm3 of H2O. The pH value of the reaction mixture was adjusted to about 10 by

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J. S. Brboric et al.
1012
addition of 1.25g of Na2CO3. The reaction mixture was refluxed at 84–86ꢂC during 10h. Then it
was cooled and filtered under vacuum. Ethanol was evaporated to leave an aqueous solution. The pH
value of the aqueous solution was adjusted to 2.25, by adding 5 M HCl to precipitate 1. After
filteration 1 was dried and recrystallized from absolute ethanol to yield 1.14g (45%). Mp 182–
184ꢂC; UV (methanol, c ¼ 4.2 ꢄ 10ꢅ5 mol dmꢅ3): ꢁmax (") ¼ 209 (33000), 233 (13500) nm
(molꢅ1 dm3 cmꢅ1); IR (KBr): ꢂꢂ¼ 3251 (b, s), 3052 (b, w), 2961 (s), 2932 (w), 2870 (w), 1690 (s),
1542 (s), 1360 (s), 1330 (b, s), 892 (m), 865 (m), 847 (w), 706 (b, w), 680 (b, w) cmꢅ1 1H
;
NMR (200 MHz, DMSO-d6): ꢀ ¼ 9.518 (s, NH), 7.461 (s, H-3, H-5), 3.571 (s, 4H, N–CH2–CO),
3.516 (s, NHCO–CH2–N), 2.990 (b, m, 2H, –CH(CH3)2), 1.111 (d, 12H, –CH3) ppm; 13C NMR (50
MHz, DMSO-d6): ꢀ ¼ 173.177 (COOH), 171.028 (CO–NH), 149.099 (C-3, C-5), 132.732, 132.259
(C-1, C-6), 94.372 (C-4), 58.179 (CO–CH2–N), 55.648 (N–CH2–COO), 28.258 (–CH<), 23.369
((CH3)2¼) ppm.
4-Iodo-2,6-diethylbenzenamine (2a, C10H14IN)
Compound 2a was prepared from 2,6-diethylbenzenamine in the same manner as 1a was obtained
from 2,6-bis(1-methylethyl)benzenamine. Yield 2.4 g (87%), yellowish-brown oily mass. The crude
product was used in next synthetic step. TLC: Rf ¼ 0.36 (n-hexane:benzene ¼ 3:7), Rf ¼ 0.83 (CHCl3);
UV (methanol, c ¼ 1 ꢄ 10ꢅ4 mol dmꢅ3): ꢁmax (") ¼ 211 (31100), 248 (17000), 292 (2900) nm
(molꢅ1 dm3 cmꢅ1); IR (KBr): ꢂꢂ¼ 3482 (w), 3399 (m), 2964 (s), 2932 (m), 2873 (m), 1619 (s),
1449 (s), 1376 (w), 1343 (w), 1283 (w), 1207 (w), 866 (m), 843 (w) cmꢅ1
.
N-(4-Iodo-2,6-diethylphenyl)-2-chloroacetamide (2b, C12H15IClNO)
Compound 2b was prepared from 2a in the same manner as 1b was obtained from 1a. A 10 mmol run
yielded 2.3 g (65%) of 2b. Mp 191–192ꢂC; TLC: Rf ¼ 0.10 (n-hexane:benzene¼ 3:7), Rf ¼ 0.58
(CHCl3); UV (methanol, c ¼ 4.8 ꢄ 10ꢅ5 mol dmꢅ3): ꢁmax (") ¼ 210 (34400), 235 (14200) nm
(molꢅ1 dm3 cmꢅ1); IR (KBr): ꢂꢂ¼ 3235 (b, s), 3028 (m), 2962 (s), 2925 (m), 2864 (m), 1665 (s),
1530 (s), 1456 (m), 1404 (m), 1327 (m), 1217 (s), 970 (m), 860 (s), 793 (s), 719 (m), 669 (m) cmꢅ1
.
N-[2-[(4-Iodo-2,6-diethylphenyl)amino]-2-oxoethyl]-N-(carboxymethyl)glycine
(2, C16H21IN2O5)
Compound 2 was prepared from 2b in the same manner as 1 was obtained from 1b. A 4.6 mmol run
yielded 1.18 g (58%) of 2. Mp 221–223ꢂC; UV (methanol, c ¼ 3.7 ꢄ 10ꢅ5 mol dmꢅ3): ꢁmax (") ¼ 209
(37200), 235 (15500) nm (molꢅ1 dm3 cmꢅ1); IR (KBr): ꢂꢂ¼ 3298 (s), 3060 (b, m), 3012 (s), 2961 (s),
1
2876 (m), 1704 (s), 1665 (s), 1532 (s), 1408 (s), 1353 (s), 961 (s), 865 (s), 677 (s) cmꢅ1; H NMR
(200 MHz, DMSO-d6): ꢀ ¼ 9.523 (s, NH), 7.455 (s, H-3, H-5), 3.571 (s, 4H, N–CH2–CO), 3.499 (s,
NHCO–CH2–N), 2.463 (q, J ¼ 7.6 Hz, 4H, –CH2), 1.063 (t, J ¼ 7.6 Hz, 6H, –CH3) ppm; 13C NMR
(50 MHz, DMSO-d6): ꢀ ¼ 173.186 (COOH), 170.564 (CO–NH), 144.466 (C-3, C-5), 135.008,
134.098 (C-1, C-6), 93.407 (C-4), 58.206 (CO–CH2–N), 55.657 (N–CH2–COO), 24.280 (–CH2–),
14.621 (–CH3) ppm.
2,4-Diiodo-6-methylbenzenamine (3a)
Compound 3a was prepared from 2-methylbenzenamine in the same manner as 1a was obtained from
2,6-bis(1-methylethyl)benzenamine, except that the molar ratio of 2-methylbenzenamine and BTMA
ICl2 was 1:2.1. A 20mmol of 2-methyl benzenamine run yielded 6.25g (87%) of 3a, a light-brown
crystal mass. Crystallization from n-hexane (58–60ꢂC) afforded 5.46g (76%) of 3a as yellowish
needles. Mp 78–79ꢂC (Ref. [15] 80–81ꢂC); TLC: Rf ¼ 0.60 (n-hexane:benzene¼ 3:7), Rf ¼ 0.85

Iodinated Iminodiacetic Acid Analogues
1013
(CHCl3); UV (methanol, c ¼ 4 ꢄ 10ꢅ5 mol dmꢅ3): ꢁmax (") ¼ 220 (33400), 252 (15100), 303 (3200) nm
(molꢅ1 dm3 cmꢅ1); IR (KBr): ꢂꢂ¼ 3397 (b, s), 3303 (b, s), 1619 (s), 1456 (s), 1436 (m), 1053 (w), 852
(m), 716 (w), 651 (m), 541 (w) cmꢅ1; GC: single peak at Rt ¼ 8.01min; MS (70 eV): m=z (%) ¼ 360
(8.32) [Mþ Hþ], 359 (100.0) [Mþ], 232.0 (22.26), 127 (12.41), 105 (28.97), 104 (28.74), 78 (11.59),
77 (10.60).
N-(2,4-Diiodo-6-methylphenyl)-2-chloroacetamide (3b, C9H8I2ClNO)
Compound 3b was prepared from 3a in the same manner as 1b was obtained from 1a. A 14mmol
run yielded 4.45 g (73%) of 3b as white crystals. Mp 223–224ꢂC; TLC: Rf ¼ 0.12 (n-hexane:benzene ¼
3:7), Rf ¼ 0.59 (CHCl3); UV (methanol, c ¼ 2.8 ꢄ 10ꢅ5 mol dmꢅ3): ꢁmax (") ¼ 228 (30100) nm
(molꢅ1 dm3 cmꢅ1); IR (KBr): ꢂꢂ¼ 3237 (b, s), 3195 (b, s), 3016 (m), 1674 (s), 1572 (m), 1547 (w),
1519 (s), 1452 (m), 972 (w), 853 (m), 775 (m), 692 (m) cmꢅ1; GC: single peak at Rt ¼ 20.75 min; MS
(70 eV): m=z (%) ¼ 435 (6), 358 (10), 350 (13), 310 (37), 309 (12), 308 (100), 272 (9), 271 (17), 231 (7),
181 (7), 104 (7).
N-[2-[(2,4-Diiodo-6-methylphenyl)amino]-2-oxoethyl]-N-(carboxymethyl)glycine
(3, C13H14I2N2O5)
Compound 3 was prepared from 3b in the same manner as 1 was obtained from 1b. A 9 mmol
run yielded 2.9 g (61%) of 3 (crystallized from ethanol). Mp 224–225ꢂC; UV (methanol, c ¼
2.7 ꢄ 10ꢅ5 mol dmꢅ3): ꢁmax (") ¼ 228 (30100) nm (molꢅ1 dm3 cmꢅ1); IR (KBr): ꢂꢂ¼ 3299 (b, s),
3014 (s), 2965 (s), 1705 (s), 1674 (s), 1518 (s), 1417 (m), 1355 (m), 963 (m), 860 (m), 675 (m)
1
cmꢅ1; H NMR (200 MHz, DMSO-d6): ꢀ ¼ 9.728 (b, s, NH), 8.040 (s, H-3), 7.670 (s, H-5), 3.618 (s,
4H, N–CH2–CO), 3.511 (s, NHCO–CH2–N), 2.159 (s, –CH3) ppm; 13C NMR (50 MHz, DMSO-d6):
ꢀ ¼ 173.122 (COOH), 169.799 (CO–NH), 143.638, 139.978, 139.077, 138.767 (C-1, C-3, C-5, C-6),
102.611 (C-2), 93.990 (C-4), 58.124 (CO–CH2–N), 55.767 (N–CH2–COO), 18.727 (–CH3) ppm.
Acknowledgments
These results are part of project No 1980, which is financially supported by the Ministry of Science,
Technology and Development of the Republic of Serbia.
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