Synthesis and affinity of a possible byproduct of electrophilic radiolabeling of [123I]IBZM

Article abstract of DOI:10.1016/j.bmcl.2003.08.063

The iodobenzamide neuroleptic analogue (S)-N-(1-ethylpyrrolidin-2-ylmethyl) -2-hydroxy-5-iodo-6-methoxybenzamide (5-IBZM) was synthesized stereospecifically and its pharmacological properties were compared with the 3-iodo isomer (IBZM) used for imaging D₂ receptors in vivo. The isomer 5-IBZM had 100-fold lower affinity than IBZM and migrated with similar retention time as the byproduct formed during electrophilic iodination of BZM.

Full text of DOI:10.1016/j.bmcl.2003.08.063

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4015–4017
Synthesis and Affinity of a Possible Byproduct of Electrophilic
Radiolabeling of [123I]IBZM
Ronald M. Baldwin,a Xing Fu,a Nora S. Kula,b Ross J. Baldessarini,b Louis Amici,a
Robert B. Innisa and Gilles D. Tamagnana,*
aDepartment of Psychiatry, Yale University and VA CT/HCS, West Haven, CT 06516, USA
bDepartment of Psychiatry and Neuroscience Program, Harvard Medical School and Laboratories for Psychiatric Research,
McLean Research Center, McLean Division of Massachusetts General Hospital, Belmont, MA 02478-9106, USA
Received 19 February 2003; revised 26 August 2003; accepted 27 August 2003
Abstract—The iodobenzamide neuroleptic analogue (S)-N-(1-ethylpyrrolidin-2-ylmethyl)-2-hydroxy-5-iodo-6-methoxybenzamide
(5-IBZM) was synthesized stereospecifically and its pharmacological properties were compared with the 3-iodo isomer (IBZM) used
for imaging D2 receptors in vivo. The isomer 5-IBZM had 100-fold lower affinity than IBZM and migrated with similar retention
time as the byproduct formed during electrophilic iodination of BZM.
# 2003 Elsevier Ltd. All rights reserved.
The dopamine D2 receptor is implicated in the patho-
genesis of Parkinson’s disease and in psychiatric
disorders including schizophrenia and mania. Measure-
ments of occupancy of this receptor in vivo help to bet-
ter understand the pathophysiology and pharmacology
or such disorders as well as to monitor illness progres-
sion and effects of treatment. One of the first and still
most widely used radioligands for this receptor is (S)-N-
(1-ethylpyrrolidin-2-ylmethyl)-2-hydroxy-3-iodo-6-meth-
oxybenzamide (IBZM, 2) labeled with iodine-123 for in
vivo studies.1,2
to the synthesis of a major derivative (IBZM) plus a
minor slightly more polar compound. BZM (1) has two
active labeling sites, ortho and para to the phenol, so in
addition to IBZM (2), there is the possibility of produ-
cing the byproduct (S)-N-(1-ethylpyrrolidin-2-ylme-
thyl)-2-hydroxy-5-iodo-6-methoxybenzamide (3) during
the radiolabeling reaction. This byproduct might repre-
sent a problem for in vivo imaging studies if it competes
for the D2 receptor or if its pharmacokinetics are not
appropriate.
To identify this possible byproduct of radiosynthesis, we
synthesized 5-iodo-BZM (3) by an unambiguous regiospe-
cific method, tested its affinity, and compared it with the
byproduct formed during electrophilic iodination of BZM.
Usually [123I]IBZM (2) is prepared by electrophilic
iodination of the desiodophenol BZM (1) with [123I]NaI
in the presence of peracetic acid.1 This preparation led
*Corresponding author. Tel.: +1-203-932-5711x3590; fax: +1-203-937-3897; e-mail: gilles.tamagnan@yale.edu
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.063

4016
R. M. Baldwin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4015–4017
The key step in obtaining 3 was the synthesis of 6-
hydroxy-3-iodo-2-methoxybenzoic acid (9). To achieve
differentiation of the two phenolic groups, we used a
bulky protecting group to avoid iodination of the phenyl
ring at the ortho position. The adamantanyl carbonyl
group, which was initially chosen for this purpose, did
not lead to iodination ortho to the methoxy group. To
achieve the desired differentiation of reactivity of the two
phenols, we changed to tert-butyldiphenyl silyl ether.
Table 1. Potency of iodinated-BZM derivatives compared to raclo-
pride (mean IC50ÆSE, nM) and reported vales for brominated analo-
gues at D1 and D2 receptors in rat caudate-putamen tissue
homogenates. Reported affinity1 of 3-IBZM (2) IC50=1.2 nM
Compound
Dopamine D2
Dopamine D1
3-IBZM (2)
5-IBZM (3)
(À)-(S)-Raclopride
3-BrBZM8
1.71Æ0.35
196Æ18
7.2Æ1.3
21
>10,000
>10,000
>10,000
—
5-BrBZM8
56
—
The synthesis began with 2,6-dihydroxybenzoic acid
methyl ester (4). Two iodination methods were applied
to synthesize 2,6-dihydroxy-3-iodo-benzoic acid methyl
ester (6). Iodine and HgO gave a mixture of 2,6-dihy-
droxy-3,5-diiodo-benzoic acid methyl ester (5) and 2,6-
dihydroxy-3-iodo-benzoic acid methyl ester (6) (ratio
40/60),3 then treatment of the mixture of 5/6 with tert-
butyldiphenylsilyl chloride provided 6-(tert-butyldiphe-
nylsilyloxy)-2-hydroxy-3-iodobenzoic acid methyl ester
(7) in 5% yield (two steps) exclusively.4 Use of iodine
monochloride as iodinating agent led to an increase in
the ratio of 6/5 from 60/40 to 90/10, and the total yield
of the first two steps increased from 5 to 26%.
Although this overall yield is moderate, this route
allowed us to obtain sufficient amount of intermediate.
Compound 7, produced from 6, was treated with
methyl iodide to afford 6-(tert-butyldiphenylsilyloxy)-3-
iodo-2-methoxybenzoic acid methyl ester (8) in 72%
yield.5 Deprotection of 8 with Bu4NF gave 6-hydroxy-
3-iodo-2-methoxy-benzoic acid methyl ester (9).6 The
structure of 9 was confirmed by NOE experiment.
Saponification of ester 9 led to 2-hydroxy-5-iodo-6-
methoxybenzoic acid (10).7 The desired product 3 was
synthesized by coupling 10 and (S)-(1-ethyl-pyrrolidin-
2-yl)-methylamine in the presence of dicyclohex-
ylcarbodiimide and HOBt.
The retention time of 3 on reverse-phase HPLC (C18,
MeOH/H2O/Et3N: 75/25/0.1, 1.0 mL/min) was 5.9 min,
which corresponded to that of the byproduct observed
during electrophilic iodination of BZM. 5-IBZM was
evaluated for potency (indicated by inhibition constant Ki)
in competitive radioreceptor assays with tissue homo-
genates of rat forebrain, compared with common ligands
IBZM and raclopride (Table 1). Preliminary comparison
indicated that 5-IBZM (3) was about 100 times less
potent than IBZM (2) at D2; neither isomer showed
appreciable affinity for D1 receptor sites. The disparity
in D2 binding between the two iodo positional isomers
is surprising since it has been reported that the bromo
analogues (3-Br and 5-Br) present similar D2 affinity.8
In summary, we accomplished regiospecific synthesis of
(S) - N - (1 - ethyl - 2 - methyl - pyrrolidin - 2 - ylmethyl) - 2-
hydroxy-5-iodo-6-methoxybenzamide (3). Results of
HPLC coinjection of 5-IBZM (3) with products of elec-
trophilic substitution of BZM confirm that 5-IBZM (3)
was the byproduct obtained during direct electrophilic
iodination. Since 5-IBZM (3) had a 115-fold lower affi-
nity for D2 than IBZM (2), this compound should not
interfere with quantification of D2 receptor density with

R. M. Baldwin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4015–4017
4017
preparations of IBZM (3) if present in small amounts
after purification of electrophilic iodination of BZM.
90 mg (0.31 mmol) of 6-hydroxy-3-iodo-2-methoxy-
benzoic acid followed by 75 mg (0.34 mmol) of DCC
and 45 mg (0.33 mmol) of 1-hydroxy-benzotriazole
hydrate. The mixture was stirred at room temperature
for 18 h. Solids were removed by filtration, the solvent
was removed on a rotary evaporator, and the residue
was purified by flash chromatography on silica gel 50:50
hexane–Et2O–5% Et3N afforded 53 mg (42% yield) of
3. 1H NMR (CDCl3) d 7.69 (d, 1H, J=8.8 Hz), 6.61 (d,
1H, J=8.8 Hz), 3.82 (s, 3H), 3.76 (qd, 1H), 3.34 (dq,
1H), 3,24 (m, 1H), 2.88 (m, 1H), 2.65 (m, 1H), 2.26 (m,
2H), 1.95 (m, 1H), 1.77–1.60 (m, 3H), 1.13 (t, 3H,
J=7.2).
Experimental
2,6-Dihydroxy-3-iodo-benzoic acid methyl ester (6). To a
solution of 200 mg (1.19 mmol) of 2,6-dihydroxybenzoic
acid methyl ester in 3 mL of CCl4 was added 213 mg
(1.31 mmol) of ICl. The mixture was stirred at rt for 48
h, then purified by flash chromatography on silica gel
with 80:20 hexane–Et2O to give 120 mg (34% yield,
1
90% purity) of 6. H NMR (CD2Cl2) d 7.69 (d, 1H,
J=8.8 Hz), 6.31 (d, 1H, J=8.8 Hz), 4.01 (s, 3H).
Binding assays
6-(tert-Butyldiphenylsilyloxy)-2-hydroxy-3-iodobenzoic
acid methyl ester (7). To a solution of 115 mg of 2,6-
dihydroxy-3,5-diiodo-benzoic acid methyl ester and 2,6-
dihydroxy-3-iodo-benzoic acid methyl ester (10:90) in 5
mL of CH2Cl2 was added 107 mg (0.391 mmol) of
TBDPSiCl followed by 26 mg (0.391 mmol) imidazole.
The mixture was stirred at rt for 5 h, then purified by
flash chromatography on silica gel with 70:30 hexane–
CH2Cl2 to give 160 mg (76% yield) of 7, mp 134–136 ꢀC.
1H NMR (CD2Cl2) d 7.79 (m, 4H), 7.23 (m, 6H), 7.04
(d, 1H, J=8.8 Hz), 5.92 (d, 1H, J=8.8 Hz), 3.48 (s,
3H), 1.15 (s, 9H).
Radioreceptor competitive binding was measured as
follows: for D1, radioligand [3H]SCH-23390 (0.3 nM) in
presence of Na+ (150 mM), using cis-flupenthixol (300
nM) to define blank; for D2, radioligand [3H]nemona-
pride (0.075 nM), using haloperidol (10 mM) to define
blank.9
Acknowledgements
We thank Dr. H. F. Kung for helpful discussions and Dr.
Tomas de Paulis for providing (S)-(1-ethylpyrrolidin-2-
yl)methylamine. This research was supported by grants
from the Department of Veterans Affairs (Schizophrenia
Research Center and Mental Illness Research Education
and Clinical Center), as well as a grant from the Bruce J.
Anderson Foundation and the McLean Private Donors
Neuropharmacology Research Fund (to R.J.B.).
6-(tert-Butyldiphenylsilyloxy)-3-iodo-2-methoxybenzoic
acid methyl ester (8). To 350 mg (0.660 mmol) of 6-(tert-
butyldiphenylsilyloxy) - 2 - hydroxy - 3 - iodobenzoic acid
methyl ester in 10 mL of acetone was added 187 mg
(0.660 mmol) of MeI followed by 100 mg (0.726 mmol)
KHCO3. The mixture was heated to reflux for 24 h, then
purified by flash chromatography with 60:40 hexane–
CH2Cl2 to give 260 mg (72% yield) of 8. 1H NMR
(CDCl3) d 7.84 (m, 4H), 7.24 (m, 6H), 7.18 (d, 1H,
J=8.8 Hz), 6.06 (d, 1H, J=8.8 Hz), 3.81 (s, 3H), 3.72
(s, 3H), 1.17 (s, 9H).
References and Notes
1. Kung, H. F.; Kasliwal, R.; Pan, S.; Kung, M.-P.; Mach,
R. H.; Guo, Y.-Z. J. Med. Chem. 1988, 31, 1039.
2. Kung, M.-P.; Kung, H. F. J. Label. Compd. Radiopharm.
1989, 27, 691.
3. Eckelman, W. C.; Adams, H. R.; Paik, C. H. Int. J. Nucl.
Med. Biol. 1984, 11, 163.
4. Kim, J. C.; Park, W. W. Org. Prep. Proceed. Int. 1994, 26,
479.
5. Mackenzie, A. R.; Moody, C. J.; Rees, C. W. Tetrahedron
1986, 42, 3259.
6. Smith, A. B.; Barbosa, J.; Wong, W.; Wood, J. L. J. Am.
Chem. Soc. 1996, 118, 8316.
7. Hecht, S. M.; Kozarich, J. W. Tetrahedron Lett. 1973, 14, 1397.
8. DePaulis, T.; Kumar, Y.; Johannson, L.; Ramsby, S.;
Florvall, L.; Hall, H.; Angeby-Moller, K.; Ogren, S. J. Med.
Chem. 1985, 28, 1263.
6-Hydroxy-3-iodo-2-methoxybenzoic acid methyl ester
(9). To a solution of 260 mg (0.475 mmol) of 6-(tert-
butyldiphenylsilyloxy)-3-iodo-2-methoxybenzoic
acid
methyl ester in 5 mL of THF was added 0.48 mL (0.48
mmol) of 1.0 M TBAF solution in THF. The mixture
was stirred at rt for 2 h, and then purified by flash
chromatography on silica gel. Elution with 90:10 hex-
ane–Et2O afforded 140 mg (95% yield) of 9 as an oil. 1H
NMR (CDCl3) d 7.83 (d, 1H, J=8.8 Hz), 6.64 (d, 1H,
J=8.8 Hz), 4.03 (s, 3H), 3.81 (s, 3H).
(S)-N-(1-ethylpyrrolidin-2-ylmethyl)-2-hydroxy-5-iodo-
6-methoxybenzamide (3). To a solution of 44 mg (0.34
mmol) of (S)-(1-ethyl-pyrrolidin-2-yl)methylamine in 5
mL of CH2Cl2 under a nitrogen atmosphere was added
9. Kula, N. S.; Baldessarini, R. J.; Kebabian, J. W.; Baktha-
vachalam, V.; Xu, L. X. Eur. J. Pharmacol. 1997, 331, 333.