Synthesis and structure-activity relationship effects on the tumor avidity of radioiodinated phospholipid ether analogues

Article abstract of DOI:10.1021/jm050252g

Radioiodinated phospholipid ether analogues have shown a remarkable ability to selectively accumulate in a variety of human and animal tumors in xenograft and spontaneous tumor rodent models. It is believed that this tumor avidity arises as a consequence of metabolic differences between tumor and corresponding normal tissues. The results of this study indicate that one factor in the tumor retention of these compounds in tumors is the length of the alkyl chain that determines their hydrophobic properties. Decreasing the chain length from C12 to C7 resulted in little or no tumor accumulation and rapid clearance of the compound in tumor-bearing rats within 24 h of administration. Increasing the chain length had the opposite effect, with the C15 and C18 analogues displaying delayed plasma clearance and enhanced tumor uptake and retention in tumor-bearing rats. Tumor uptake displayed by propanediol analogues NM-412 and NM-413 was accompanied by high levels of liver and abdominal radioactivity 24 h postinjection to tumor-bearing rats. Addition of a 2-O-methyl moiety to the propanediol backbone also retarded tumor uptake significantly. A direct comparison between NM-404 and its predecessor, NM-324, in human PC-3 tumor bearing immune-compromised mice revealed a dramatic enhancement in both tumor uptake and total body elimination of NM-404 relative to NM-324. On the basis of imaging and tissue distribution studies in several rodent tumor models, the C18 analogue, NM-404, was chosen for follow-up evaluation in human lung cancer patients. Preliminary results have been extremely promising in that selective uptake and retention of the agent in tumors is accompanied by rapid clearance of background radioactivity from normal tissues, especially those in the abdomen. These results strongly suggest that extension of the human trials to include other cancers is warranted, especially when NM-404 is radiolabeled with iodine-124, a new commercially available positron-emitting isotope. The relatively long physical half-life of 4 days afforded by this isotope appears well-suited to the pharmacodynamic profile of NM-404.

Full text of DOI:10.1021/jm050252g

J. Med. Chem. 2006, 49, 2155-2165
2155
Articles
Synthesis and Structure-Activity Relationship Effects on the Tumor Avidity of Radioiodinated
Phospholipid Ether Analogues
Anatoly N. Pinchuk,†,‡,# Mark A. Rampy,† Marc A. Longino,†,‡,# R. W. Scott Skinner,§ Milton D. Gross,§
Jamey P. Weichert,*,|,‡,# and Raymond E. Counsell†
Department of Pharmacology, The UniVersity of Michigan Medical School, 1301 MSRB III, 1150 W. Medical Center DriVe, Ann Arbor,
Michigan 48109-0632, the Nuclear Medicine SerVice, VA Medical Center, 2215 Fuller Road, Ann Arbor, Michigan 48105-2303,
Department of Radiology, UniVersity of Michigan Medical School, Ann Arbor, Michigan, 48109-0030, and Department of Radiology,
UniVersity of Wisconsin Medical School, E3 Clinical Science Center, 600 Highland AVenue, Madison, Wisconsin 53792-3252
ReceiVed March 18, 2005
Radioiodinated phospholipid ether analogues have shown a remarkable ability to selectively accumulate in
a variety of human and animal tumors in xenograft and spontaneous tumor rodent models. It is believed
that this tumor avidity arises as a consequence of metabolic differences between tumor and corresponding
normal tissues. The results of this study indicate that one factor in the tumor retention of these compounds
in tumors is the length of the alkyl chain that determines their hydrophobic properties. Decreasing the chain
length from C12 to C7 resulted in little or no tumor accumulation and rapid clearance of the compound in
tumor-bearing rats within 24 h of administration. Increasing the chain length had the opposite effect, with
the C15 and C18 analogues displaying delayed plasma clearance and enhanced tumor uptake and retention
in tumor-bearing rats. Tumor uptake displayed by propanediol analogues NM-412 and NM-413 was
accompanied by high levels of liver and abdominal radioactivity 24 h postinjection to tumor-bearing rats.
Addition of a 2-O-methyl moiety to the propanediol backbone also retarded tumor uptake significantly. A
direct comparison between NM-404 and its predecessor, NM-324, in human PC-3 tumor bearing immune-
compromised mice revealed a dramatic enhancement in both tumor uptake and total body elimination of
NM-404 relative to NM-324. On the basis of imaging and tissue distribution studies in several rodent tumor
models, the C18 analogue, NM-404, was chosen for follow-up evaluation in human lung cancer patients.
Preliminary results have been extremely promising in that selective uptake and retention of the agent in
tumors is accompanied by rapid clearance of background radioactivity from normal tissues, especially those
in the abdomen. These results strongly suggest that extension of the human trials to include other cancers
is warranted, especially when NM-404 is radiolabeled with iodine-124, a new commercially available positron-
emitting isotope. The relatively long physical half-life of 4 days afforded by this isotope appears well-
suited to the pharmacodynamic profile of NM-404.
Introduction
not substrates for this enzyme also have the capacity to
accumulate in tumors.4 A recent hypothesis that a still unidenti-
fied cell surface protein may be involved in the specific
incorporation of antitumor lipids into cancer cells has also been
advanced.5
In the late 1960s, Snyder and co-workers1,2 performed a series
of experiments designed to evaluate the lipid composition of
normal and neoplastic tissues. It was discovered that both animal
and human tumor tissue contained much larger quantities of
naturally occurring ether lipids relative to corresponding normal
tissues. When subsequent studies demonstrated that tumor tissue
had less than normal amounts of the enzyme O-alkyl glycerol
monooxygenase (EC 1.14.16.5, AGMO),3 it was proposed that
differences in the tissue concentration of this ether cleavage
enzyme were responsible for the accumulation of ether lipids
in tumors. More recent evidence, however, has argued against
AGMO deficiency being the sole reason for the elevated ether
lipid levels, since certain phospholipid ether analogues that are
We have previously described the remarkable capacity of
certain radioiodinated phospholipid ether (PLE) analogues
(Figure 1) to be selectively retained by a variety of rodent and
human tumor cells.6-14 Moreover, this property made it possible
to obtain images of these tumors in rabbits, rats, and mice using
γ-camera scintigraphy.
The reason for the retention of PLE and analogues in cancer
cells remains unknown. The prevailing hypothesis, however, is
that radioiodinated phospholipid ether analogues such as NM-
324 (2) become trapped in tumor cell membranes because of
their inability to be metabolized and eliminated. On the other
hand, such lipid molecules are metabolized and cleared by
normal tissues, including the liver. Support for this hypothesis
was found when lipid extraction of tumors following adminis-
tration of radioiodinated PLE to tumor-bearing animals showed
only the presence of the intact agent, whereas similar analysis
of urine and feces revealed only the presence of metabolites.10
* Corresponding author. Tel: (608) 262-6376. Fax: (608) 263-4014.
E-mail: j.weichert@hosp.wisc.edu.
† Department of Pharmacology, University of Michigan Medical School.
‡ University of Wisconsin Medical School.
# Current address: Department of Radiology, E3 Clinical Science Center,
600 Highland Avenue, Madison, WI 53792-3252.
§ VA Medical Center.
| Department of Radiology, University of Michigan Medical School.
10.1021/jm050252g CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/14/2006

2156 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Pinchuk et al.
Figure 3.
enhanced tumor retention. In the interest of decreasing liver
uptake and increasing plasma half-life, a series of second-
generation PLE analogues (4-10) was synthesized with varia-
tions in the alkyl chain length. The alkyl chain modifications
made to the phospholipid ether structure are summarized in
Figure 3. Accordingly, this paper analyzes the effect of altering
the structure of iodinated phospholipid ether analogues in order
to determine the resulting influence on (1) tumor retention and
(2) uptake in nontarget tissues such as liver and kidney.
Figure 1.
Results and Discussion
Chemical Synthesis. In the course of the synthesis of
iodinated phospholipid ether analogues, we sought to develop
a general synthetic scheme that would allow altering the chain
length in the target compounds. Our synthetic approach was
based on the copper-catalyzed cross-coupling reaction of
Grignard reagents with alkyl tosylates or halides15 (Schemes 1
and 2). The choice of the building blocks for alkyl chain
elongation was dictated by the commercial availability of the
starting materials. The synthesis was initiated by the conversion
of p-iodobenzyl alcohol 11 to p-iodobenzyl bromide 12, as
shown in Scheme 1. p-Iodobenzyl bromide was further coupled
with Grignard reagent derived from THP-protected 11-bro-
moundecanol 13 in the presence of 0.5-0.7 mol % of Li2CuCl4.
12-(p-Iodophenyl)dodecanol 17 obtained after deprotection of
the first coupling product 16 was used for the synthesis of C12
iodinated phospholipids as described earlier.12,13 Alcohol 17 also
served as a starting material for the synthesis of ω-iodophenyl
alkanols with longer chains. For example, coupling of the
tosylate 18 with Grignard reagents made from bromides 14 and
15 followed by THP deprotection furnished the C15 (20) and
C18 (22) alcohols, respectively. These alcohols were converted
into corresponding alkylphosphocholines 5 (NM-397) and 6
(NM-404) according to published procedures.12,13 Propanediol
phospholipid ethers 7 (NM-413) and 8 (NM-412) were synthe-
sized from 3-benzyloxypropanol 25, and 2-O-methyl-rac-
glycerol phospholipid ethers 9 (NM-414) and 10 (NM-410) were
obtained from 1-O-benzyl-2-O-methyl-rac-glycerol 2616 using
a sequence of reactions we had previously reported.12,13 Radio-
labeling with iodine-125 was accomplished by an isotope
exchange method routinely employed in our laboratory.17
Biology. The avidity of PLE analogues to localize in tumors
was evaluated in several animal models. The PC-3 model
represents a human tumor cell line that was used to determine
the target (tumor) to nontarget ratio of NM404 and NM412 in
head to head comparison in order to select a candidate for an
initial human pharmacokinetic trial in prostate cancer patients.
The MatLyLu (Dunning R3327 rat) model, a rat prostate tumor
line, was used specifically to screen nine specific analogues prior
Figure 2. Scintigraphic image obtained in lung cancer patient with
131I-NM-324 6 days after administration (T ) lung tumor; L ) liver).
Without knowing the precise mechanism for the tumor
retention of PLE, the strategy for designing agents with
properties superior to NM-324 has been largely empirical. Early
work was conducted with meta-iodinated compounds, because
it was thought that the iodine would be more metabolically stable
in this position. However, synthesis and biological evaluation
of para-iodinated PLE analogues demonstrated an equivalent
or greater tumor avidity than the corresponding meta-isomers
along with minimal in vivo metabolic deiodination.11,12 One of
these compounds, NM-346 (4), the para-isomer of NM-324, also
displayed significant tumor avidity in the Walker tumor-bearing
rat.
As a result of promising rodent studies, a preliminary
pharmacokinetic analysis of NM-324 (2) was undertaken in
cancer patients in order to extend the findings from our animal
models into humans.6 These patient studies demonstrated that
NM-324 was taken up and retained by tumors in concentrations
sufficient for diagnostic imaging (Figure 2). However, the study
results also showed that significant uptake by the liver and
clearance by the kidneys caused unacceptable levels of radio-
activity to localize in these organs. While these studies served
to indicate the relevance of our animal models, they also
demonstrated the need to develop agents with superior tumor/
liver and tumor/kidney ratios. High tumor uptake observed with
NM-324 initiated efforts to make structural modifications in
order to determine the structure-activity relationship for

Radioiodinated Phospholipid Ether Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2157
Scheme 1a
a Reagents and conditions: (a) Me3SiBr, CH2Cl2; (b) BrMg(CH2)11OTHP, Li2CuCl4 (cat.), THF, -78 to 20 °C; (c) PPTS, EtOH, 40 °C; (d) TsCl, DMAP,
CH2Cl2; (e) BrMg(CH2)3OTHP, Li2CuCl4 (cat.), THF, -78 to 20 °C; (g) BrMg(CH2)6OTHP, Li2CuCl4 (cat.), THF, -78 to 20 °C.
Scheme 2
to entering them into control animals for dosimetry and tumor-
bearing animals for determining tumor/background ratios.
Finally, the Walker-256 carcinosarcoma model was used for
quantitative tissue distribution purposes.
tribution analysis. Tissue distribution of radioactivity in rats
bearing Walker-256 carcinosarcoma was assessed at various
time intervals following intravenous administration of the
radioiodinated chain length homologues. The first group of
compounds that was tested included three alkylphospho-
cholines: a shorter chain analogue with seven carbons, 3 (NM-
396), and two analogues with a longer chain length, 5 (NM-
397, C15 alkyl chain length) and 6 (NM-404, C18 alkyl chain
length).
Initial biodistribution experiments performed with 3 (NM-
396, C7 analogue, Table 1) indicated rapid tissue clearance
accompanied by significant in vivo deiodination. By 24 h, the
amount of radioactivity in the thyroid was 213% injected dose/
gram (ID/g), whereas levels of radioactivity in all of the organs
surveyed was <0.10% ID/g. As follows from these studies,
reducing the number of methylene groups to seven apparently
afforded a much more hydrophilic molecule that was rapidly
To expedite the screening process and minimize the number
of tumor-bearing animals utilized in multiple time point tissue
distribution studies, new radioiodinated homologues were im-
aged by γ-camera scintigraphy in the rat Dunning R3337 (MAT
LyLu strain) prostate cancer model. Thus, male Copenhagen
rats received subcutaneous injection of MAT LyLu cells (1 ×
106 cells) in the thigh 10-14 days prior to injection of the
radioiodinated PLE analogues (30-40 µCi) in 2% Tween-20
solution. γ-Camera images were obtained at multiple time points
including 24 and 48 h postinjection. Homologues (NM-410,
NM-413, and NM-414) displaying high hepatic uptake, signifi-
cant abdominal accumulation, and retention or poor tumor
uptake and retention were not submitted to subsequent biodis-

2158 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Pinchuk et al.
Table 1. Tissue Distribution over Time of Phospholipid Ether Analogue
3 (NM-396) in Walker 256-Tumor-Bearing Rats after Iv Injectiona
tissues. The accumulation of radioactivity in the thyroid
increased throughout the course of the study, suggesting the
presence of a low level of in vivo deiodination. Levels of
radioactivity in the duodenum were similar to those of tumor,
with maximum levels being observed at 48 h after administration
(1.38 ( 0.24% ID/g).
Although limited results were obtained with the C12 analogue
4 (NM-346) in this model, results suggest that the tissue
distribution profile of the C15 analogue 5 (NM-397) was similar
to that observed with the C12 analogue 4 (Table 4) with the
exception of a 2-fold increase in tumor uptake at 24 h. The
remaining uptake and clearance in other organs and tissues was
similar between the two compounds.
tissue
6 h (n ) 3)
24 h (n ) 3)
48 h (n ) 3)
adrenal
blood
duodenum
heart
kidney
liver
lung
muscle
ovary
plasma
spleen
thyroid
tumor
0.16 ( 0.01
0.29 ( 0.02
0.20 ( 0.02
0.11 ( 0.00
0.57 ( 0.03
0.40 ( 0.01
0.19 ( 0.01
0.04 ( 0.01
0.17 ( 0.01
0.45 ( 0.03
0.11 ( 0.01
65.33 ( 18.98
0.15 ( 0.01
0.04 ( 0.00
0.03 ( 0.00
0.02 ( 0.00
0.02 ( 0.00
0.09 ( 0.01
0.06 ( 0.00
0.04 ( 0.00
0.02 ( 0.00
0.06 ( 0.00
0.05 ( 0.01
0.03 ( 0.00
212.96 ( 38.40
0.04 ( 0.00
0.02 ( 0.00
0.01 ( 0.00
0.01 ( 0.00
0.01 ( 0.00
0.04 ( 0.00
0.03 ( 0.00
0.02 ( 0.00
0.01 ( 0.00
0.03 ( 0.00
0.02 ( 0.00
0.02 ( 0.00
71.84 ( 4.59
0.03 ( 0.01
The effect of further extending the aliphatic chain to the C18
analogue 6 (NM-404) is displayed in Table 3. The dynamic
profile of this compound was similar to that of the C15 analogue
5 (NM-397), as levels of radioactivity peaked in the tumor 48
h after administration (1.14 (0.01% ID/g), albeit at slightly
lower levels. Quantities of radioactivity detected in liver, kidney,
and duodenum were significantly lower following administration
of the C18 compound 6 as compared to the same organs in the
C15 analogue 5 studies. In addition, the C18 analogue 6 was
retained in the circulation to a much greater extent than the
other chain length homologues surveyed. For example, at 120
h, blood levels for 6 were 0.6 ( 0.1% ID/g as compared to
levels of 0.07 (0.00% ID/g for the C15 (5) analogue. Total
radioactivity levels in the thyroid were relatively low in both 5
and 6 when the extremely small mass of the gland is considered.
Table 4 contains a comparative summary of the data obtained
for the biodistribution of the alkyl chain length homologues.
To examine the transport properties of PLE analogues, plasma
was isolated from Walker 256-tumor-bearing rats 7 days after
administration of iodine-125-labeled 6. The distribution of
radioactivity in the plasma compartment of a rat receiving 6
(NM-404) is shown in Table 5. PAGE analysis revealed that
most of the circulating radioactivity (88%) was associated with
the albumin fraction following administration of the C18
analogue 6. This finding is similar to results reported by Eibl,18
who studied binding of phospholipid ether prototype ET-18-
OCH3 with serum proteins and found that the majority of the
ether lipid (71%) was bound to albumin and about 6% to HDL.
Comparative Imaging Studies. γ-Camera scintigraphy im-
ages shown in Figure 4 directly compare the tumor uptake and
body clearance of second-generation analogue 125I-NM-404 (6)
versus its shorter chain, first-generation predecessor 125I-NM-
324 (2) at 1, 2, and 4 days postadministration in immune-
compromised SCID mice bearing human PC-3 prostate tumor
xenografts. Qualitative scintigraphic comparison of these two
PLE analogues demonstrated a striking difference in tumor
a Results are expressed as mean % administered dose per gram (SEM.
Table 2. Tissue Distribution over Time of NM-397 (5) in Walker
256-Tumor-Bearing Rats after Iv Injectiona
tissue
6 h (n ) 3) 24 h (n ) 4) 48 h (n ) 3) 120 h (n ) 3)
adrenal
blood
duodenum 0.97 ( 0.15
heart
kidney
liver
lung
muscle
ovary
plasma
spleen
thyroid
tumor
0.99 ( 0.07
0.27 ( 0.02
0.73 ( 0.08
0.16 ( 0.01
1.13 ( 0.13
0.31 ( 0.02
1.14 ( 0.11
1.29 ( 0.10
0.97 ( 0.03
0.10 ( 0.02
0.72 ( 0.10
0.16 ( 0.01
0.86 ( 0.05
0.49 ( 0.05
0.14 ( 0.01
1.38 ( 0.24
0.28 ( 0.02
0.91 ( 0.04
0.83 ( 0.04
0.81 ( 0.04
0.08 ( 0.00
0.41 ( 0.04
0.15 ( 0.01
0.70 ( 0.04
0.27 ( 0.01
0.07 ( 0.00
0.55 ( 0.11
0.12 ( 0.01
0.36 ( 0.05
0.51 ( 0.01
0.40 ( 0.02
0.04 ( 0.00
0.23 ( 0.01
0.07 ( 0.01
0.32 ( 0.02
0.37 ( 0.00
1.40 ( 0.08
3.08 ( 0.61
1.07 ( 0.30
0.26 ( 0.15
0.93 ( 0.07
1.06 ( 0.04
1.08 ( 0.03
2.27 ( 0.26 11.60 ( 5.40 13.61 ( 3.19 18.13 ( 3.44
0.72 ( 0.06 1.47 ( 0.10 1.65 ( 0.23 0.87 ( 0.09
a Results are expressed as mean % administered dose per gram (SEM.
excreted by the kidneys. In contrast to compound 4 (NM-346,
the C12 analogue), the C7 analogue 3 cleared rapidly from the
rat and did not localize in tumor tissue at any of the time points
examined.
This observation directed our future efforts to assessing the
effect of increasing the length of the aliphatic chain upon tumor
uptake and retention. The tissue distribution of the C15
homologue 5 (NM-397) was assessed utilizing the same Walker
256 rat tumor model (Table 2). Radioactivity in the tumor
increased with time and peaked at 48 h after administration (1.65
( 0.23% ID/g) as opposed to most normal tissues, which
exhibited their highest levels of radioactivity 6 h after admin-
istration. With the exception of thyroid, the tumor had higher
radioactivity concentrations at 24, 48, and 120 h than any of
the other tissues surveyed. A more rapid washout of radioactivity
occurred in the normal tissues as compared to the tumor,
presumably due to metabolism and elimination by normal
Table 3. Tissue Distribution over Time of NM-404 (6) in Walker 256-Tumor-Bearing Rats after Iv Injectiona
tissue
6 h (n ) 3)
24 h (n ) 3)
48 h (n ) 3)
120 h (n ) 2)
168 h (n ) 1)
adrenal
blood
duodenum
heart
kidney
liver
lung
muscle
ovary
plasma
spleen
thyroid
tumor
0.87 ( 0.08
1.28 ( 0.02
0.85 ( 0.08
0.53 ( 0.02
0.87 ( 0.06
1.05 ( 0.11
1.14 ( 0.10
0.18 ( 0.01
0.98 ( 0.09
1.96 ( 0.05
0.84 ( 0.07
10.73 ( 0.67
0.55 ( 0.04
0.85 ( 0.04
0.96 ( 0.00
0.69 ( 0.05
0.38 ( 0.02
0.65 ( 0.00
0.55 ( 0.04
0.88 ( 0.06
0.13 ( 0.01
0.90 ( 0.03
1.47 ( 0.03
0.59 ( 0.03
13.40 ( 0.46
0.98 ( 0.07
0.92 ( 0.06
0.64 ( 0.07
0.68 ( 0.04
0.32 ( 0.02
0.59 ( 0.04
0.59 ( 0.05
0.76 ( 0.07
0.10 ( 0.01
0.68 ( 0.08
0.95 ( 0.11
0.65 ( 0.06
11.46 ( 0.86
1.14 ( 0.01
0.60 ( 0.01
0.55 ( 0.10
0.42 ( 0.02
0.25 ( 0.02
0.42 ( 0.02
0.34 ( 0.04
0.56 ( 0.00
0.07 ( 0.01
0.47 ( 0.05
0.79 ( 0.10
0.35 ( 0.03
6.08 ( 0.44
0.89 ( 0.08
0.48
0.61
0.45
0.28
0.41
0.32
0.62
0.16
0.46
0.83
0.31
4.60
0.81
a Results are expressed as mean % administered dose per gram (SEM.

Radioiodinated Phospholipid Ether Analogues
Table 4. Biodistribution over Time of Phospholipid Ether Analogues in Walker 256-Tumor-Bearing Rats 24 and 48 h after Iv Injectiona
C7 analogue (NM-396) C12 analogue (NM-346) C15 analogue (NM-397) C18 analogue (NM-404)
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2159
tissue
24 h (n ) 3)
48 h (n ) 3)
24 h (n ) 3)
48 h (n ) 3)
24 h (n ) 3)
48 h (n ) 3)
24 h (n ) 4)
48 h (n ) 3)
adrenal
blood
duodenum
heart
kidney
liver
lung
muscle
ovary
plasma
spleen
thyroid
tumor
0.04 ( 0.00
0.03 ( 0.00
0.02 ( 0.00
0.02 ( 0.01
0.09 ( 0.01
0.06 ( 0.00
0.04 ( 0.00
0.02 ( 0.00
0.06 ( 0.00
0.05 ( 0.01
0.03 ( 0.00
212.96 ( 38.40
0.04 ( 0.00
0.02 ( 0.00
0.01 ( 0.00
0.01 ( 0.00
0.01 ( 0.00
0.04 ( 0.00
0.03 ( 0.00
0.02 ( 0.00
0.01 ( 0.00
0.03 ( 0.00
0.02 ( 0.00
0.02 ( 0.00
71.84 ( 4.58
0.03 ( 0.01
0.33 ( 0.01
0.29 ( 0.02
0.44 ( 0.16
0.21 ( 0.02
0.41 ( 0.04
1.74 ( 0.17
0.63 ( 0.03
0.06 ( 0.01
0.37 ( 0.03
0.39 ( 0.05
0.42 ( 0.01
90.8 ( 14.8
2.05 ( 0.32
0.29 ( 0.01
0.27 ( 0.03
0.77 ( 0.06
0.19 ( 0.01
0.34 ( 0.08
1.37 ( 0.01
0.53 ( 0.01
0.06 ( 0.01
0.33 ( 0.01
0.39 ( 0.05
0.32 ( 0.01
85.2 ( 7.4
0.73 ( 0.08
0.16 ( 0.01
1.13 ( 0.13
0.31 ( 0.02
1.14 ( 0.11
1.29 ( 0.10
0.97 ( 0.03
0.10 ( 0.02
0.72 ( 0.10
0.16 ( 0.01
0.86 ( 0.05
11.60 ( 5.4
1.47 ( 0.10
0.49 ( 0.05
0.14 ( 0.01
1.38 ( 0.24
0.28 ( 0.02
0.91 ( 0.04
0.83 ( 0.04
0.81 ( 0.04
0.08 ( 0.00
0.41 ( 0.04
0.15 ( 0.01
0.70 ( 0.04
13.61 ( 3.19
1.65 ( 0.23
0.85 ( 0.04
0.96 ( 0.00
0.69 ( 0.05
0.38 ( 0.02
0.65 ( 0.00
0.55 ( 0.04
0.88 ( 0.06
0.13 ( 0.01
0.90 ( 0.03
1.47 ( 0.03
0.59 ( 0.03
13.40 ( 0.46
0.98 ( 0.07
0.92 ( 0.06
0.64 ( 0.07
0.68 ( 0.04
0.32 ( 0.02
0.59 ( 0.04
0.59 ( 0.05
0.76 ( 0.07
0.10 ( 0.01
0.68 ( 0.08
0.95 ( 0.11
0.65 ( 0.06
11.46 ( 0.86
1.14 ( 0.01
1.84 ( 0.09
a Results are expressed as mean % administered dose per gram (SEM.
Table 5. Distribution of Radioactivity in the Plasma Samples from
Walker-256-Tumor-Bearing Rats (n ) 3, (SEM) Receiving 6 (NM-404)
Collected 168 Hours after Iv Administration, As Determined by
Polyacrylamide Gel Electrophesis (PAGE)
background tissue ratios steadily increased over the course of
the experiment, due to prolonged retention in tumors coupled
with a steady elimination from normal tissues. Tumor to
background tissue ratios exceeded 4, 6.8, 23, and 9 in blood,
liver, muscle, and prostate, respectively, 3 days after injection
and continued to improve at 5 and 8 days. Again, although
thyroid levels ranged from 26 to 54% injected dose per gram
of tissue, these levels are actually quite low and represent an
extremely small percentage of the injected dose when the
exceedingly small mass of the organ is considered, and the data
are presented on a percent administered dose per organ basis.
stacking gel
LDL
1.17 ( 0.08
2.84 ( 1.37
7.24 ( 1.03
87.81 ( 0.52
0.94 ( 0.01
HDL
albumin
below albumin
Ko¨tting et al have investigated the effects of alkyl chain length
on the biodistribution of three alkyl phosphocholine (APC)
analogues.19 The Ko¨tting study differed from our experiments
in that (1) compounds were orally administered at concentrations
thought to be cytotoxic to tumor and (2) the C22 compound
contained a double bond in the alkyl chain. Therefore, direct
comparisons with the work described herein cannot be made
due to large dose differences and the unknown bioavailability
of the oral agents. In the Ko¨tting study, C16, C18, and C22
analogues were administered orally to rats bearing a methyl-
nitrosourea-induced mammary carcinoma in daily doses of 50-
120 µmol/kg. As alkyl chain length increased, the observed
levels of compound in kidney, liver, and lung decreased. In
contrast to our tracer results obtained with the radioiodinated
PLE analogues, Ko¨tting and co-workers found that levels of
APC in blood decreased with increasing chain length, while
tumor levels increased with increasing chain length.19 It would
be expected that oral administration would result in a substantial
amount of degradation of the ether lipids in the GI tract prior
to absorption.
Figure 4. Scintigraphic comparison of NM-324 (top panel) and NM-
404 (bottom panel) at 1, 2, and 4 days postinjection in a SCID mouse
with human prostate PC-3 tumor (arrow) implanted in the flank. Liver
and abdominal background radioactivities are much lower with NM-
404 relative to NM-324.
Tumors were readily visualized with the C12 (4), C15 (5),
and C18 (6) alkyl phosphocholine homologues via γ-camera
scintigraphy at both 24 and 48 h after injection. Rat imaging
results obtained with C15 (7, NM-413) and C18 (8, NM-412)
propanediol analogues, on the other hand, displayed tumor
uptake accompanied by high liver and abdominal radioactivity
levels (Figure 5). Imaging results obtained with C15 (9, NM-
414) and C18 (10, NM-410) 2-O-Me glycerol analogues in the
MAT LyLu prostate model indicated high radioactivity levels
in the liver and abdomen with little to no uptake of the agent
into tumors (Figure 5). The differences in the clearance and
quantity of radioactivity from nontarget tissues including the
liver and intestinal tract will have significant impact upon the
application of radioiodinated phospholipid ether analogues as
imaging agents in humans. Nontarget tissue uptake can decrease
the efficacy of radiodiagnostic imaging by creating high
background activity or by causing excessive exposure of
uptake and overall body clearance. The longer chain agent, NM-
404, displays rapid tumor uptake and prolonged retention
accompanied by rapid whole body elimination of radioactivity,
whereas tumor uptake and body clearance are substantially
delayed with NM-324, even at 4 days following administration.
Significant tumor uptake and retention of C18 analogue NM-
404 accompanied by rapid whole body elimination clearly
defined the superior imaging properties of NM-404 in this
model.
Extensive quantitative tissue distribution results obtained at
1, 3, 5, and 8 days following administration of radioiodinated
NM-404 in this model (Table 6) indicated rapid elimination of
radioactivity from all normal tissues over the 8-day evaluation
period. Tumor uptake, however, continued to increase up until
day 5, when it reached 18% ID/g of tumor. Tumor to

2160 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Pinchuk et al.
Table 6. Biodistribution over Time of 125I-NM-404 in Male SCID Mice Bearing PC-3 Prostate Cancer Xenograftsa
day 1
day 3
day 5
day 8
tumor/
bkgb
tumor/
bkg
tumor/
bkg
tumor/
bkg
tissue
% dose/g
% dose/g
% dose/g
% dose/g
adrenal
blood
fat
heart
kidney
liver
7.83 ( 0.78
5.74 ( 0.20
2.39 ( 0.33
2.81 ( 0.10
4.22 ( 0.14
3.69 ( 0.22
5.36 ( 0.33
0.80 ( 0.03
9.93 ( 0.53
2.61 ( 0.15
4.99 ( 0.37
42.68 ( 10.72
9.14 ( 0.69
1.2
1.6
3.8
3.3
2.2
2.5
1.7
11.5
0.9
3.5
1.8
0.2
1.0
4.65 ( 0.40
3.10 ( 0.13
1.26 ( 0.21
1.45 ( 0.04
2.15 ( 0.11
1.93 ( 0.10
2.60 ( 0.20
0.57 ( 0.04
5.27 ( 0.16
1.40 ( 0.27
2.24 ( 0.12
54.53 ( 12.54
13.14 ( 0.40
2.8
4.2
10.5
9.1
6.1
6.8
5.1
23.0
2.5
9.4
5.9
0.2
1.0
4.76 ( 0.51
3.08 ( 0.09
1.11 ( 0.09
1.38 ( 0.06
2.28 ( 0.09
1.63 ( 0.06
2.27 ( 0.09
0.49 ( 0.03
5.41 ( 0.14
1.96 ( 0.25
1.99 ( 0.10
26.48 ( 4.00
18.06 ( 0.81
3.8
5.9
16.3
13.1
7.9
11.1
8.0
37.0
3.3
9.2
9.1
0.7
1.0
3.00 ( 0.51
2.17 ( 0.09
0.87 ( 0.09
0.97 ( 0.06
1.46 ( 0.09
1.02 ( 0.06
1.54 ( 0.09
0.40 ( 0.03
3.98 ( 0.14
1.41 ( 0.25
1.36 ( 0.10
33.90 ( 11.02
14.96 ( 0.63
5.0
6.9
17.2
15.4
10.3
14.7
9.7
37.3
3.8
10.6
11.0
0.4
lung
muscle
plasma
prostate
spleen
thyroid
tumor
1.0
a Results are expressed as mean % administered dose per gram (SEM (n ) 4 for each time point). b Tumor to background tissue ratio based on %
administered dose per gram basis.
Figure 5. Representative γ-camera scans of the propanediol analogues
7 (NM-413, C15), 8 (NM-412, C18), 9 (NM-414, C15), and 10 (NM-
410, C18) in MAT-LyLu tumor-bearing rats 24 h after iv administration.
The presence of the 2-O-methyl moiety in 9 and 10 retards tumor uptake
significantly in this series. Rats were injected with I-125-labeled PLE
(35-50 µCi) and scanned for 20 min (A ) abdominal activity
associated with liver, kidney, and GI tract. T ) tumor).
radiosensitive tissues to the injected radioactivity. A preliminary
clinical trial with 2 (NM-324, the m-iodo isomer of NM-346)
in cancer patients, while affording excellent tumor uptake, was
Figure 6. Posterior whole-body planar nuclear medicine image (A) 4
limited by the radiation dosimetry associated with accumulation
in nontarget tissues including liver, kidneys, and bladder (Figure
2).6
days after iv administration of 131I-NM-404 (0.8 mCi) to a patient with
non-small-cell lung cancer (6 cm dia., arrow). Lung tumor is easily
detected in corresponding axial (B) and coronal (C) computed tomog-
raphy (CT) scans.
Our strategy was to examine the alkyl portion of phospholipid
ether analogue structure and determine its role in tumor
localization and retention. Qualitative rat whole body screening
scans acquired in MAT-LyLu tumor bearing rats with radioio-
dinated PLE analogues with longer chain lengths revealed
sufficient tumor uptake to permit detection. However, follow-
up tissue distribution studies have shown that sequential
increases in the chain length from C12 to C15 to C18, resulted
in a rapid decline in the amount of radioactivity detected in the
nontarget organs. This substantial decrease in nontarget tissue
activity was accompanied by a relatively small reduction in the
levels of radioactivity present in the tumor. In addition, the C18
analogue 6 (NM-404) displayed a propensity to remain in the
circulation much longer than the C12 (4) and C15 (5) analogues.
A longer plasma half-life may be expected to result in additional
opportunities for uptake of the C18 compound 6 by the tumor
as it continually circulated through the vasculature. This
extended plasma half-life may be a result of strong binding of
the probe to albumin (Table 5). Uptake and transport of
radiolabeled PLE by plasma components may also be an
important factor related to the tumor retention of these com-
pounds. Certainly, increase of the chain length from C7 to C18,
results in an increase in the lipophilicity of the PLE analogues.
Greater lipophilicity may increase the affinity of these com-
pounds for the cell membrane and may alter their binding to
plasma components. Uptake and transport in the circulation by
endogenous lipoproteins such as LDL and HDL may also impact
the biological distribution into the tumor.
In preparation for human clinical trials, unlabeled NM404
was subjected to independent (University of Buffalo Toxicology
Research Center) acute toxicity evaluation at 1200 times the
anticipated imaging mass dose in rats and rabbits. The agent
was well-tolerated and no acute toxicities were found at this
dose level. Due to its selective tumor uptake and retention
properties in a variety of rodent tumor models and subsequent
excellent safety profile in rats and rabbits, NM-404 was selected
to undergo initial human pharmacokinetic evaluation in non-
small-cell lung cancer (NSCLC) patients. Patients underwent
planar γ-camera scintigraphy after receiving an injection of 131I-
NM-404 (<1 mCi). Preliminary human results (n ) 3)

Radioiodinated Phospholipid Ether Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2161
Experimental Section
demonstrated tumor uptake and prolonged retention in the
primary lung tumors (Figure 6). Relative to the high liver uptake
values observed with its predecessor, NM-324, however, liver
and abdominal radioactivity levels were much lower with NM-
404, suggesting the feasibility of evaluating this agent in other
abdominal tumors, including those associated with the colon,
prostate, and pancreas.
Chemistry. All chemicals were purchased from Aldrich Chemi-
cal Co. (Milwaukee, WI). 1-O-Benzyl-2-O-methyl-rac-glycerol 26
was synthesized as described previously.16 Thin-layer chromatog-
raphy was performed using DC-Alufolien Kieselgel 60 F254 plates
(E. Merck, Darmstadt, Germany). Visualization was achieved by
UV light and/or charring after spraying with 5% H2SO4 in ethanol.
For flash chromatography, silica gel 32-63 µm from Fisher
Scientific (Hanover Park, IL) was used. Melting points were taken
in glass capillary tubes on a Melt-Temp apparatus and are
uncorrected. NMR data were collected on Bruker AM-360 or Varian
Unity Inova 400 and 500 spectrometers. Chemical shifts are reported
in parts per million (ppm) relative to tetramethylsilane (TMS), and
spin multiplicities are given as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), or br m (broad multiplet). Elemental
analyses were performed by the analytical laboratory at the
University of Michigan Department of Chemistry. The elemental
analyses of the compounds agreed to within (0.4% of the calculated
value. High-resolution mass spectra were obtained on an IonSpec
7 T HiResMALDI FT-mass spectrometer at the Analytical Instru-
mentation Center of UW School of Pharmacy.
Conclusions
In summary, the rationale behind the design of new iodinated
PLE analogues described in this paper was exploited in an effort
to evaluate the effect of chain length and other structural
modifications in the hydrophobic part of the phospholipid
molecule on tumor uptake and retention. Decreasing the chain
length from C12 to C7 resulted in little or no tumor accumula-
tion and rapid clearance of the compound in tumor-bearing rats
by 24 h after administration. Increasing the chain length had
the opposite effect, with the C15 and C18 analogues displaying
delayed plasma clearance and enhanced tumor uptake and
retention in tumor-bearing rats. Tumor uptake displayed by
propanediol analogues 7 and 8 was accompanied by fairly high
levels of liver and abdominal radioactivity 24 h postinjection
to tumor-bearing rats. Addition of a 2-O-methyl moiety in 9
and 10 retarded tumor uptake significantly. A direct comparison
between NM-404 and its predecessor, NM-324, in human PC-3
bearing immune-compromised mice revealed a dramatic en-
hancement in both tumor uptake and total body elimination of
NM-404 relative to NM-324. NM-404 afforded superior imaging
properties to the other analogues examined in several animal
models, thus warranting further evaluation of this second-
generation PLE analogue in human lung cancer patients.
Preliminary clinical results in humans indicated desired tumor
uptake and retention properties similar to those seen previously
in animal models. In contrast to its shorter chain predecessor
NM-324, however, NM-404 displayed significantly lower liver,
kidney, and abdominal background radioactivity levels, which
in addition to providing promise for lung tumor imaging,
suggests further evaluation of this agent in human colorectal,
pancreatic, and prostate cancer patients is warranted. Moreover,
a lack of urinary bladder radioactivity suggests little renal
clearance of the agent or its metabolites over the time points
examined. This represents a significant advantage over 18F-
fluorodeoxyglucose (FDG), a PET agent used routinely for
tumor imaging today, which undergoes significant renal elimi-
nation, thus prohibiting imaging in the area of the prostate.
p-Iodobenzyl Bromide (12). Bromotrimethylsilane (2.5 mL,
19.28 mmol) was slowly added with stirring to a solution of
p-iodobenzyl alcohol 11 (3 g, 12.82 mmol) in anhydrous di-
chloromethane (15 mL). The mixture was stirred at room temper-
ature for 12 h, quenched with water, and extracted with ethyl
acetate. The extract was washed successively with water, Na2S2O3
solution, and water and dried over Na2SO4. Evaporation of solvent
yielded a solid which was crystallized from methanol to give white
1
crystals. Mp: 77-78.5 °C. Yield: 3.207 g (84%). H NMR (400
MHz, CDCl3): 7.67 and 7.13 (2 dt, 2H each, IC6H4), 4.42 (s, 2H,
CH2). 13C NMR (100 MHz, CDCl3): 137.95, 137.41, 130.82, 94.12,
32.45. Anal. (C7H6BrI) C, H.
Tetrahydro-2-(11-bromoundecyloxy)-2H-pyran (13). A solu-
tion of 11-bromoundecanol (6 g, 24 mmol) and dihydropyran (3.3
mL, 36 mmol) in methylene chloride (20 mL) containing pyridinium
p-toluenesulfonate (600 mg, 2.4 mmol) was stirred at room
temperature for 5 h. The solution was diluted with hexane, washed
with water, and dried over Na2SO4. Flash chromatography in
hexanes-ether (150:5) afforded the product (7.93 g, 99%) as a clear
1
oil. H NMR (500 MHz, CDCl3): 4.57 (dd, 1H, anomeric 2-H of
THP), 3.90-3.84 (m, 1H, 6-Heq of THP), 3.73 (dt, 1H, CHAHB-
OTHP), 3.54-3.47 (m, 1H, 6-Hax of THP), 3.40 (t, 2H, CH2Br),
3.38 (dt, 1H, CHAHBOTHP), 1.85 (quintet, 2H, BrCH2CH2), 1.87-
1.79 (m, 1H, 4-Heq of THP), 1.74-1.68 (m, 1H, 3-Heq of THP),
1.62-1.48 (m, 6H, THPOCH2CH2, 3-Hax, 4-Hax, 5-Heq and 5-Hax
of THP), 1.45-1.38 (m, 2H, BrCH2CH2CH2), 1.38-1.26 (m, 12H,
(CH2)6). 13C NMR (100 MHz, CDCl3): 98.81, 67.64, 62.31, 33.97,
32.80, 30.76, 29.72, 29.49, 29.43, 29.42, 29.37, 28.72, 28.14, 26.20,
25.49, 19.68. Anal. (C16H31BrO2) C, H.
Tetrahydro-2-(3-bromopropyloxy)-2H-pyran (14). Following
the procedure described for the preparation of 13, the title compound
was obtained from 3-bromopropanol (1 g, 7.2 mmol) in 98% yield
Because the tumor-targeting strategy of PLE analogues
appears to involve selective tumor retention over time, relatively
short-lived nuclides such as 18F or even 99mTc are not practical
for labeling NM-404 at the current time. Given the preliminary
success of 131I-NM-404 in our current lung cancer imaging trial,
it is now imperative to radiolabel NM-404 with iodine-124, a
relatively new positron-emitting isotope with a 4.2-day half-
life, and to evaluate its tumor detection efficacy by PET. It has
been reported that PET imaging with 124I affords over 40 times
the sensitivity of planar 131I-γ-imaging.20 PET, unlike traditional
γ-camera imaging, also offers significant resolution enhance-
ment and three-dimensional imaging capabilities, as well as
superior quantitation properties21 relative to planar scintigraphic
imaging. The long, 4-day physical half-life of this PET isotope
is well-suited to the tumor uptake and retention kinetics of NM-
404 and we are in the process of extending our imaging studies
with NM-404 to include PET.
1
as a clear oil. H NMR (360 MHz, CDCl3): 4.62-4.59 (m, 1H,
anomeric 2-H of THP), 3.90-3.83 (m, 1H, 6-Heq of THP), 3.88
(dt, 1H, CHAHBOTHP), 3.56-3.48 (m, 1H, 6-Hax of THP), 3.54
(t, 2H, BrCH2), 3.52 (dt, 1H, CHAHBOTHP), 2.14 (quintet, 2H,
BrCH2CH2), 1.84-1.66 (m, 2H, THP), 1.64-1.49 (m, 6H, 4H,
THP). Anal. (C8H15BrO2) C, H.
Tetrahydro-2-(6-bromohexadecyloxy)-2H-pyran (15). This
compound was prepared from 6-bromohexanol (1.38 g, 7.62 mmol)
in 92% yield (clear oil) according to the general procedure described
for 13. 1H NMR (400 MHz, CDCl3): 4.57 (dd, 1H, anomeric 2-H
of THP), 3.90-3.83 (m, 1H, 6-Heq of THP), 3.74 (dt, 1H, CHAHB-
OTHP), 3.53-3.47 (m, 1H, 6-Hax of THP), 3.41 (t, 2H, CH2Br),
3.39 (dt, 1H, CHAHBOTHP), 1.87 (quintet, 2H, BrCHCH2), 1.88-
1.67 (m, 2H, THP), 1.66-1.36 (m, 10H, (CH2)3CH2OTHP and 4H
of THP). 13C NMR (100 MHz, CDCl3): 98.87, 67.37, 62.35, 33.84,
32.72, 30.74, 29.53, 27.97, 25.46, 25.44, 19.67. Anal. (C11H21BrO2)
C, H.

2162 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Pinchuk et al.
Tetrahydro-2-[12-(p-iodophenyl)dodecyloxy]-2H-pyran (16).
Magnesium powder (300 mg, 12.5 mmol) was suspended in THF
(2.5 mL), and dibromoethane (0.05 mL) was added for activation.
After 10 min, the THF solution was withdrawn by syringe and
replaced with 2 mL of fresh THF. A solution of the bromide 13
(3.1 g, 9.25 mmol) in THF (12 mL) was added dropwise over 1 h
at room temperature. Then, a solution of Grignard reagent was
transferred into a round-bottom flask via cannula and cooled to
-78 °C. A solution of Li2CuCl4 in THF (0.4 mL of 0.12 mmol/
mL solution, 0.048 mmol) was added to the Grignard reagent with
stirring followed by a solution of p-iodobenzyl bromide 12 (2.45
g, 8.25 mmol) in THF (15 mL). The reaction mixture was allowed
to warm to room temperature during 5 h followed by continuous
stirring for an additional 12 h. The reaction mixture was quenched
with ammonium chloride solution and extracted with ethyl acetate.
The extract was washed with water and dried over Na2SO4. The
solvent was removed in vacuo and the residue was chromatographed
on silica gel, first eluting with hexane-chloroform (8:2) and then
temperature. When all the halide had been added, stirring was
continued for an additional 15 min, whereupon a small aliquot was
hydrolyzed and analyzed by TLC in hexanes-ether (150:6), which
revealed no presence of starting bromide. The Grignard reagent
was transferred to a round-bottom flask and cooled to -78 °C.
The organometallic reagent was stirred for 10 min at this temper-
ature before a solution of Li2CuCl4 in THF (0.5 mL of 0.077 mmol/
mL solution, 0.0385 mmol) was added followed by 12-(p-
iodophenyl)dodecyl tosylate 18 (2.69 g, 4.96 mmol) dissolved in
THF (10 mL). The reaction mixture was allowed to gradually warm
to room temperature for 3 h and was kept at this temperature for
an additional 12 h before a saturated ammonium chloride solution
was added to quench the reaction. The mixture was extracted with
hexane, washed with water, and dried over Na2SO4. The solvent
was removed in a vacuum, and silica gel chromatography with
hexane-ether (150:3) gave the product (1.77 g, 64%) as a white
wax. Mp: 26-27 °C. 1H NMR (360 MHz, CDCl3): 7.58 and 6.92
(2 dt, 2H each, IC6H4), 4.59-4.57 (m, 1H, anomeric 2-H of THP),
3.91-3.84 (m, 1H, 6-Heq of THP), 3.73 (dt, 1H, CHAHBOTHP),
3.54-3.46 (m, 1H, 6-Hax of THP), 3.38 (dt, 1H, CHAHBOTHP),
2.55 (t, 2H, ArCH2), 1.82-1.67 (m, 2H, THP), 1.64-1.45 (m, 8H,
ArCH2CH2, CH2CH2OTHP and 4H of THP), 1.39-1.22 (m, 28H,
(CH2)14). Anal. (C29H49IO2) C, H.
Tetrahydro-2-[15-(p-iodophenyl)pentadecyloxy]-2H-pyran (19).
This compound was obtained in a manner analogous to that of
compound 21 from tosylate 18 (300 mg, 0.55 mmol) and bromide
14 (136 mg, 0.61 mmol). Silica gel chromatography performed as
described above gave the product 19 (165 mg, 52%). 1H NMR (360
MHz, CDCl3): 7.58 and 6.92 (2 dt, 2H each, IC6H4), 4.59-4.57
(m, 1H, anomeric 2-H of THP), 3.91-3.84 (m, 1H, 6-Heq of THP),
3.73 (dt, 1H, CHAHBOTHP), 3.54-3.46 (m, 1H, 6-Hax of THP),
3.38 (dt, 1H, CHAHBOTHP), 2.55 (t, 2H, ArCH2), 1.82-1.67 (m,
2H, THP), 1.64-1.45 (m, 8H, ArCH2CH2, CH2CH2OTHP and 4H
of THP), 1.39-1.22 (m, 22H, (CH2)11). Anal. (C26H43IO2) C, H.
1
with hexane-ether (150:3) to give a clear oil (2.16 g, 55%). H
NMR (500 MHz, CDCl3): 7.58 and 6.92 (2 dt, 2H each, IC6H4),
4.57 (dd, 1H, anomeric 2-H of THP), 3.90-3.84 (m, 1H, 6-Heq of
THP), 3.73 (dt, 1H, CHAHBOTHP), 3.52-3.47 (m, 1H, 6-Hax of
THP), 3.38 (dt, 1H, CHAHBOTHP), 2.53 (t, 2H, ArCH2), 1.88-
1.79 (m, 1H, 4-Heq of THP), 1.75-1.68 (m, 1H, 3-Heq of THP),
1.62-1.48 (m, 8H, ArCH2CH2, THPOCH2CH2, 3-Hax, 4-Hax, 5-Heq,
5-Hax of THP), 1.38-1.22 (m, 16H, (CH2)8). 13C NMR (100 MHz,
CDCl3): 142.52, 137.20, 130.54, 98.84, 90.46, 67.68, 62.34, 35.42,
31.27, 30.79, 29.75, 29.59, 29.56 (2C), 29.52, 29.47, 29.43, 29.17,
26.23, 25.50, 19.70. HRMS: calculated for C23H37IO2Na (M+
Na) 495.1736, found 495.1744. Anal. (C23H37IO2) C, H.
+
12-(p-Iodophenyl)dodecanol (17). A solution of THP ether 16
(4.068 g, 8.62 mmol) and pyridinium p-toluenesulfonate (216 mg,
0.86 mmol) in ethanol (20 mL) was stirred at 50 °C for 3 h until
TLC showed no starting material. The reaction mixture was diluted
with water, extracted with ethyl acetate, washed with water, and
dried over Na2SO4, and the solvent was evaporated. Silica gel
chromatography of the residue in hexane-ethyl acetate (85:15) gave
the product (3.01 g, 90%) as a white solid. In cases when
contamination by the aliphatic alcohol derived from Grignard
reagent was revealed by NMR, the product was crystallized from
acetonitrile at 15 °C (for alcohols 20 and 22 at room temperature).
Mp: 61.5-63 °C. 1H NMR (400 MHz, CDCl3): 7.58 and 6.92 (2
dt, 2H each, IC6H4), 3.64 (q, 2H, CH2OH), 2.54 (t, 2H, ArCH2),
1.62-1.50 (m, 4H, ArCH2CH2 and CH2CH2OH), 1.40-1.22 (m,
16H, (CH2)8), 1.21 (t, 1H, OH). 13C NMR (100 MHz, CDCl3):
142.55, 137.24, 130.56, 90.49, 63.11, 35.45, 32.82, 31.29, 29.61,
29.59, 29.56, 29.54, 29.45, 29.43, 29.18, 25.74. Anal. (C18H29IO)
C, H.
15-(p-Iodophenyl)pentadecanol (20). Compound 19 (150 mg,
0.26 mmol) was converted to the desired product by the procedure
1
described for 17. Alcohol 20 was obtained in a yield of 91%. H
NMR (360 MHz, CDCl3): 7.57 and 6.92 (2 dt, 2H each, IC6H4),
3.64 (t, 2H, CH2OH), 2.54 (t, 2H, ArCH2), 1.60-1.55 (m, 4H,
ArCH2CH2 and CH2CH2OH), 1.35-1.22 (m, 22H, (CH2)11). Anal.
(C21H35IO) C, H.
18-(p-Iodophenyl)octadecanol (22). By the procedure described
for 17, THP ether 21 (1.4 g, 2.5 mmol) was converted to the desired
1
product 22 in 85% yield. Mp: 72-73 °C (from acetonitrile). H
NMR (CDCl3) 7.58 and 6.92 (2 dt, 2H each, IC6H4), 3.64 (t, 2H,
CH2OH), 2.54 (t, 2H, ArCH2), 1.60-1.55 (m, 4H, ArCH2CH2 and
CH2CH2OH), 1.35-1.20 (m 28H, (CH2)14). Anal. (C24H41IO) C,
H.
12-(p-Iodophenyl)dodecyl Tosylate (18). A solution of 12-(p-
iodophenyl)dodecanol 17 (2.01 g, 5.18 mmol), tosyl chloride (1.09
g, 5.7 mmol), and N,N-(dimethylamino)pyridine (0.72 g, 5.9 mmol)
in dichloromethane (15 mL) was stirred for 6 h. The clear reaction
mixture was diluted with 10 mL of hexane and carefully poured
directly onto the top of a silica gel column. The column was eluted
with hexane-chloroform (1:1) and then with chloroform to give
the tosylate 18 (2.69 g, 96%) as a white solid. Mp: 42-43 °C. 1H
NMR (400 MHz, CDCl3): 7.58 and 6.92 (2 dt, 2H each, IC6H4),
7.79 and 7.34 (2 dt, 2H each, CH3C6H4SO3), 4.02 (t, 2H, CH2-
OTs), 2.53 (t, 2H, ArCH2), 2.45 (s, 3H, CH3C6H4SO3), 1.66-1.52
(m, 4H, ArCH2CH2 and CH2CH2OTs), 1.32-1.18 (m, 16H, (CH2)8).
13C NMR (100 MHz, CDCl3): 144.57, 142.50, 137.21, 133.28,
130.54, 129.77, 127.86, 90.47, 70.68, 35.42, 31.25, 29.53, 29.49,
29.44, 29.41, 29.34, 29.15, 28.90, 28.81, 25.31, 21.62. Anal.
(C25H35IO3S) C, H.
18-(p-Iodophenyl)octadecyl Methanesulfonate (24). To a solu-
tion of 18-(p-iodophenyl)octadecanol 22 (150 mg, 0.317 mmol)
and triethylamine (0.07 mL, 0.48 mmol) in methylene chloride (2
mL) was added methanesulfonyl chloride (0.03 mL, 0.38 mmol)
at 0 °C. Stirring was continued for 40 min, whereupon the reaction
mixture was quenched by addition of water. The reaction mixture
was diluted with chloroform and washed several times with
NaHCO3 solution and water. The chloroform layer was dried over
Na2SO4 and the solvent was removed in vacuo. The residue was
chromatographed with hexane-ethyl acetate (9:1) to afford pure
24 (142 mg, 82%). Mp: 61-62 °C (from ethanol). 1H NMR (360
MHz, CDCl3): 7.58 and 6.92 (2 dt, 2H each, IC6H4), 4.22 (t, 2H,
CH2OMs), 3.00 (s, 3H, CH3S), 2.53 (t, 2H, ArCH2), 1.75 (quintet,
2H, CH2CH2OMs), 1.60-1.5 (m, 2H, ArCH2CH2), 1.40-1.20 (m,
28H, (CH2)14). Anal. (C25H43IO3S) C, H.
Tetrahydro-2-[18-(p-iodopheny)octadecyloxy]-2H-pyran (21).
Magnesium powder (370 mg, 15.4 mmol) in THF (2.5 mL) was
activated by addition of 1,2-dibromoethane (0.03 mL) and stirring
for 10 min. After the reaction with dibromoethane had ceased, the
solution was removed via syringe and replaced with fresh THF (5
mL). This procedure was followed by the addition of bromide 15
(1.373 g, 5.18 mmol) in THF (10 mL) over 0.5 h at room
15-(p-Iodophenyl)pentadecyl Methanesulfonate (23). By the
above procedure, alcohol 20 (150 mg, 0.35 mmol) was converted
to the desired product 23 (163 mg, 92%). H NMR (360 MHz,
CDCl3): 7.58 and 6.92 (2 dt, 2H each, IC6H4), 4.22 (t, 2H, CH2-
OMs), 3.00 (s, 3H, CH3S), 2.53 (t, 2H, ArCH2), 1.75 (quintet, 2H,
CH2CH2OMs), 1.60-1.50 (m, 2H, ArCH2CH2), 1.40-1.20 (m,
22H, (CH2)11). Anal. (C22H37IO3S) C, H.
1

Radioiodinated Phospholipid Ether Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7 2163
1-O-[18-(p-Iodophenyl)octadecyl]-3-O-benzyl-1,3-propane-
diol (29). Sodium hydride (8 mg of 60% suspension in oil, 0.2
mmol) was added at room temperature to a solution of 3-benzyl-
oxypropanol 25 (0.03 mL, 0.18 mmol) and 18-(p-iodophenyl)-
octadecyl methanesulfonate 24 (66 mg, 0.12 mmol) in dimethyl-
formamide (3 mL). The reaction mixture was stirred for 12 h,
quenched with water, and extracted with ethyl acetate. The extract
was washed with brine and dried over Na2SO4, and the solvent
was removed in a vacuum. Column chromatography with hexane-
ethyl acetate (gradient from 95:5 to 85:15) afforded 29 (60 mg;
81%). 1H NMR (360 MHz, CDCl3): 7.58 and 6.92 (2 dt, 2H each,
IC6H4), 7.36-7.30 (m, 5H, C6H5), 4.50 (s, 2H, PhCH2), 3.57 (t,
2H, alkyl-OCH2(CH2)2O), 3.52 (t, 2H, CH2OBn), 3.39 (t, 2H,
CH2O(CH2)3)OBn), 2.53 (t, 2H, ArCH2), 1.90 (quintet, 2H,
OCH2CH2CH2O), 1.65-1.55 (m, 4H, ArCH2CH2 and CH2CH2O-
(CH2)3O), 1.35-1.20 (m, 28H, (CH2)14). Anal. (C34H53IO2) C, H.
1-O-[15-(p-Iodophenyl)pentadecyl]-3-O-benzyl-1,3-propane-
diol (27). This compound was synthesized as described above from
mesylate 23 (85 mg, 0.17 mmol) and 3-benzyloxypropanol 25
(0.036 mL, 0.23 mmol). The compound 27 was obtained in a yield
of 79% (76 mg) after chromatographic purification. 1H NMR (360
MHz, CDCl3): 7.58 and 6.92 (2 dt, 2H each, IC6H4), 7.36-7.30
(m, 5H, C6H5), 4.50 (s, 2H, PhCH2), 3.57 (t, 2H, alkyl-OCH2-
(CH2)2O), 3.52 (t, 2H, CH2OBn), 3.39 (t, 2H, CH2O(CH2)3)OBn),
2.53 (t, 2H, ArCH2), 1.89 (quintet, 2H, OCH2CH2CH2O), 1.65-
1.55 (m, 4H, ArCH2CH2 and CH2CH2O(CH2)3O), 1.40-1.20 (m,
22H, (CH2)11). Anal. (C31H47IO2) C, H.
1-O-[15-(p-Iodophenyl)pentadecyl]-2-O-methyl-rac-glycerol (32).
Compound 28 (75 mg, 0.12 mmol) was converted to the alcohol
32 (51 mg, 80%) by the procedure described for 33. 1H NMR (360
MHz, CDCl3): 7.58 and 6.92 (2 dt, 2H each, IC6H4), 3.76 and
3.65 (2 m, 2H, CH2OH), 3.54 (m, 2H, CHCH2OCH2), 3.47 (s, 3H,
OCH3), 3.46-3.41 (m, 3H, CHCH2OCH2), 2.53 (t, 2H, ArCH2),
1.60-1.50 (m, 4H, ArCH2CH2 and OCH2CH2), 1.35-1.20 (m, 22H,
(CH2)11). Anal. (C25H43IO3) C, H.
1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-rac-glycerol (34)
was synthesized as described for 33 from the benzyl ether 30 (58
mg, 0.09 mmol). The alcohol 34 was obtained in a yield of 80%
1
(40 mg). H NMR (360 MHz, CDCl3): 7.58 and 6.92 (2 dt, 2H
each, IC6H4), 3.76 and 3.65 (2 m, 2H, CH2OH), 3.54 (m, 2H,
CHCH2OCH2), 3.47 (s, 3H, OCH3), 3.46-3.41 (m, 3H, CHCH2-
OCH2), 2.53 (t, 2H, ArCH2), 1.60-1.50 (m, 4H, ArCH2CH2 and
OCH2CH2), 1.35-1.20 (m, 28H, (CH2)14). Anal. (C28H49IO3) C,
H.
18-(p-Iodophenyl)octadecylphosphocholine (6). 2-Chloro-2-
oxo-1,3,2-dioxaphospholane (0.025 mL, 0.27 mmol) was added to
the stirred solution of 18-(p-iodophenyl)octadecanol 22 (115 mg;
0.24 mmol) in benzene (3 mL) containing triethylamine (0.042 mL,
0.29 mmol). Stirring was continued overnight. The precipitated
triethylamine hydrochloride was filtered off and the solvent was
removed in vacuo. The residue was transferred into a pressure bottle.
A solution of trimethylamine in acetonitrile (5 mL; 25% w/v) was
added. The bottle was sealed and heated at 75 °C for 24 h. The
acetonitrile was then evaporated and the residue was chromato-
graphed on silica gel with chloroform-methanol (gradient from
10:0 to 5:5) followed by final elution with chloroform-methanol-
water (65:25:4). After evaporation of the solvent, the product was
precipitated by addition of acetone to give a white solid (130 mg,
1-O-[15-(p-Iodophenyl)pentadecyl]-2-O-methyl-3-O-benzyl-
rac-glycerol (28). This compound was synthesized as described
for 29 from mesylate 23 (92 mg, 0.18 mmol) and 1-O-benzyl-2-
O-methyl-rac-glycerol 2616 (43 mg, 0.22 mmol) to give 75 mg
(68%) of the product. 1H NMR (360 MHz, CDCl3): 7.58 and 6.92
(2 dt, 2H each, IC6H4), 7.35-7.25 (m, 5H, C6H5), 4.55 (s, 2H,
PhCH2), 3.60-3.50 (m, 5H, CH2CHCH2), 3.45 (s, 3H, OCH3), 3.42
(t, 2H, CH2OCH2CH), 2.53 (t, 2H, IC6H4CH2), 1.60-1.50 (m, 4H,
ArCH2CH2 and CH2CH2O), 1.35-1.20 (m, 22H, (CH2)11). Anal.
(C32H49IO3), C, H.
1
84%). H NMR (500 MHz, CDCl3-CD3OD-D2O 1:1:0.3): 7.59
and 6.96 (2 dt, 2H each, IC6H4), 4.28 (br m, 2H, POCH2CH2N),
3.89 (q, 2H, CH2OPOCH2CH2N), 3.65 (m, 2H, CH2N), 3.21 (s,
9H, N(CH3)3), 2.57 (2H, t, ArCH2), 1.68-1.56 (m, 4H, ArCH2CH2
and CH2CH2O), 1.40-1.25 (m, 28H, (CH2)14). 13C NMR (100 MHz,
CDCl3-CD3OD-D2O 1:1:0.3): 143.00, 137.63, 131.00, 90.67,
66.73 (d, JC-P ) 5.8 Hz), 66.7 (m), 59.60 (d, JC-P ) 5.0 Hz), 54.42
(t, JC-N ) 3.9 Hz), 35.76, 31.66, 31.09 (d, JC-P ) 7.3 Hz), 30.12
(3C), 30.11, 30.08 (2C), 30.05, 30.01, 29.98, 29.89, 29.86, 29.79,
29.48, 26.12. 31P NMR (161.8 MHz, CDCl3-CD3OD-D2O
1:1:0.3): 0.80. HRMS: calculated for C29H54INO4P (M+ + H)
638.2835, found 638.2828. Anal. (C29H53INO4P), C, H, N, I.
15-(p-Iodophenyl)pentadecylphosphocholine (5). The introduc-
tion of the phosphocholine moiety into 20 (232 mg, 0.54 mmol)
was carried out as described for 6, yielding 5 (231 mg, 72%) as an
1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-3-O-benzyl-rac-
glycerol (30). This compound was synthesized as described for 29
from mesylate 24 (67 mg, 0.12 mmol) and 1-O-benzyl-2-O-methyl-
rac-glycerol 2616 (36 mg, 0.18 mmol) to give 62 mg (78%) of the
1
product. H NMR (360 MHz, CDCl3): 7.58 and 6.92 (2 dt, 2H
each, IC6H4), 7.35-7.25 (m, 5H, C6H5), 4.55 (s, 2H, PhCH2), 3.60-
3.50 (m, 5H, CH2CHCH2), 3.45 (s, 3H, OCH3), 3.42 (t, 2H, CH2-
OCH2CH), 2.53 (t, 2H, IC6H4CH2), 1.60-1.50 (m, 4H, ArCH2CH2
and CH2CH2O), 1.35-1.20 (m, 28H, (CH2)14). Anal. (C35H55IO3)
C, H.
1
amorphous powder. H NMR (360 MHz, CDCl3-CD3OD-D2O
1:1:0.3): 7.57 and 6.94 (2 dt, 2H each, IC6H4), 4.24 (br m, 2H,
POCH2CH2N), 3.85 (q, 2H, CH2OPOCH2CH2N), 3.61 (m, 2H,
CH2N), 3.21 (s, 9H, N(CH3)3), 2.55 (t, 2H, ArCH2), 1.65-1.55
(m, 4H, ArCH2CH2 and CH2CH2O), 1.35-1.25 (m, 22H, (CH2)11).
Anal. (C26H47INO4P) C, H, N.
1-O-[18-(p-Iodophenyl)octadecyl]-1,3-propanediol (33). Pow-
dered aluminum chloride (353 mg, 2.66 mmol) was added at room
temperature to a solution of benzyl ether 29 (413 mg, 0.66 mmol)
and anisole (0.36 mL, 3.33 mmol) in methylene chloride (10 mL).
Stirring was continued for 2 h. The reaction mixture was quenched
by dilution with 1 N HCl, and the aqueous layer was extracted
with ethyl acetate. The organic layer was washed with NaHCO3
solution, dried over Na2SO4, and evaporated. The remaining residue
was chromatographed with hexane-ethyl acetate (gradient from
1-O-[15-(p-Iodophenyl)pentadecyl]-1,3-propanediol-3-phos-
phocholine (7). Alcohol 31 (60 mg, 0.12 mmol) was converted
into the phosphocholine 7 (65 mg, 81%) as described above for 6.
1H NMR (360 MHz, CDCl3-CD3OD-D2O 1:1:0.3): 7.57 and 6.94
(2 dt, 2H each, IC6H4), 4.26 (br m, 2H, POCH2CH2N), 3.93 (q,
2H, CH2OPOCH2CH2N), 3.61 (m, 2H, CH2N), 3.54 (t, 2H, OCH2-
CH2CH2OP), 3.21 (s, 9H, N(CH3)3), 2.55 (t, 2H, ArCH2), 1.65-
1.55 (m, 4H, ArCH2CH2 and CH2CH2O(CH2)3OP), 1.35-1.25 (m,
28H, (CH2)14). Anal. (C29H53INO5P) C, H, N.
1
95:5 to 80:20) to give the product (301 mg, 85%). H NMR (360
MHz, CDCl3): 7.58 and 6.92 (2 dt, 2H each, IC6H4), 3.78 (q, 2H,
CH2OH), 3.62 (t, 2H, OCH2CH2CH2OH), 3.42 (t, 2H, CH2O(CH2)3-
OH), 2.55 (t, 2H, ArCH2, 2H), 1.83 (quintet, 2H, OCH2CH2CH2-
OH), 1.60-1.50 (m, 4H, ArCH2CH2 and CH2CH2O(CH2)3OH),
1.35-1.20 (m, 28H, (CH2)14). Anal. (C27H47IO2) C, H.
1-O-[15-(p-Iodophenyl)pentadecyl]-2-O-methyl-rac-glycero-3-
phosphocholine (9). Using procedure for the synthesis of 6, alcohol
32 (51 mg, 0.1 mmol) was converted into the phosphocholine 9
1-O-[15-(p-Iodophenyl)pentadecyl]-1,3-propanediol (31). By
the above procedure, benzyl ether 27 (76 mg, 0.13 mmol) was
1
(55 mg, 82%). H NMR (360 MHz, CDCl3-CD3OD-D2O 1:1:
1
converted to the alcohol 31 (60 mg, 94%). H NMR (360 MHz,
0.3): 7.57 and 6.95 (2 dt, 2H each, IC6H4), 4.26 (br m, 2H, POCH2-
CH2N), 3.95 and 3.86 (2 m, 2H, CHCH2OP), 3.62 (m, 2H, CH2N),
3.60-3.50 (m, 3H, CHCH2OCH2), 3.47 (s, 3H, OCH3), 3.46 (t,
2H, CHCH2OCH2), 3.21 (s, 9H, N(CH3)3), 2.55 (t, 2H, ArCH2),
1.65-1.55 (m, 4H, ArCH2CH2 and CH2CH2OCH2), 1.35-1.22 (m,
22H, m, (CH2)11). Anal. (C30H55INO6P) C, H, N.
CDCl3): 7.58 and 6.92 (2 dt, 2H each, IC6H4), 3.78 (q, 2H, CH2-
OH), 3.62 (t, 2H, OCH2CH2CH2OH), 3.42 (t, 2H, CH2O(CH2)3-
OH), 2.55 (t, 2H, ArCH2), 1.83 (quintet, 2H, OCH2CH2CH2OH),
1.60-1.45 (m, 4H, ArCH2CH2 and CH2CH2O(CH2)3OH), 1.35-
1.20 (m, 22H, (CH2)11). Anal. (C24H41IO2) C, H.

2164 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 7
Pinchuk et al.
1-O-[18-(p-Iodophenyl)octadecyl]-1,3-propanediol-3-phospho-
choline (8). The phosphocholine 8 was synthesized by an analogous
manner to that of 6 from the alcohol 33 (42 mg; 79 mmol) in 55%
minced with scissors. Tissue samples were weighted in gelatin
capsules and counted with a Searle-1185 well scintillation counter
(88% counting efficiency). The concentration of radioactivity in
each tissue was expressed as a percentage of administered dose
per gram of tissue.
1
yield (45 mg). H NMR (360 MHz, CDCl3-CD3OD-D2O 1:1:
0.3): 7.57 and 6.94 (2 dt, 2H each, IC6H4), 4.26 (br m, 2H, POCH2-
CH2N), 3.93 (q, 2H, CH2OPOCH2CH2N), 3.61 (m, 2H, CH2N),
3.54 (t, 2H, OCH2CH2CH2OP), 3.21 (s, 9H, N(CH3)3), 2.55 (t, 2H,
ArCH2), 1.65-1.55 (m, 4H, ArCH2CH2 and CH2CH2O(CH2)3OP),
1.35-1.25 (m, 28H, (CH2)14). Anal. (C32H59INO5P) C, H, N.
1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-rac-glycero-3-
phosphocholine (10). The phosphocholine 10 was synthesized from
the alcohol 34 (33 mg, 0.06 mmol) by the procedure described
above for 6 in a yield of 75% (32 mg). 1H NMR (360 MHz,
CDCl3-CD3OD-D2O 1:1:0.3): 7.57 and 6.95 (2 dt, 2H each,
IC6H4), 4.26 (br m, 2H, POCH2CH2N), 3.95 and 3.86 (2 m, 2H,
CHCH2OP), 3.62 (m, 2H, CH2N), 3.60-3.50 (m, 3H, CHCH2-
OCH2), 3.47 (s, 3H, OCH3), 3.46 (t, 2H, CHCH2OCH2), 3.21 (s,
9H, N(CH3)3), 2.55 (t, 2H, ArCH2), 1.65-1.55 (m, 4H, ArCH2CH2
and CH2CH2OCH2), 1.35-1.22 (m, 28H, m, (CH2)14). Anal.
(C33H61INO6P) C, H, N.
Due to its production of thyroxine hormones, the thyroid gland
is the major site of accumulation of free iodide ion found in the
plasma. Sequestration of plasma iodide is so efficient that Na131
I
is administered orally for thyroid ablation. Thyroid radioactivity
following injection of radioiodinated radiopharmaceuticals has been
used as an indicator of in vivo deiodination in nuclear medicine
for many years. In fact, in most clinical trials with these agents,
patients receive an oral dose of saturated potassium iodine solution
in order to presaturate the thyroid and thus minimize subsequent
uptake of free radioiodide. In our own clinical trial, presaturation
with saturated potassium iodine solution would not be expected to
effect thyroid activity, if uptake was due to intact PLE analogue
and not to free iodide. Our results showing no subsequent thyroid
radioactivity confirmed the in vivo stability toward deiodination
and direct uptake of the PLE analogue into the thyroid.
Radioiodination of Phospholipid Ether Analogues. For the
biological studies, 7-(p-iodophenyl)heptyl phosphocholine (3, NM-
396) and 12-(p-iodophenyl)dodecyl phosphocholine (4, NM-346),
whose syntheses have been described previously,12 have been
included for comparison purposes. Radioiodination of the PLE
analogues with iodine-125 was accomplished by an isotope
exchange reaction as previously reported.17 Radiochemical purity
was established by radio-thin-layer chromatography with unlabeled
material serving as a standard. Specific activities for the chain length
homologues ranged from 0.08 to 4.0 Ci/mmol. Radiochemical purity
exceeded 95% for all labeled compounds.
Biological Studies. Cell Lines and Culture Conditions. Walker
256 carcinosarcoma cells were maintained in RPMI 1640 medium
containing 10% fetal bovine serum (FBS), 200 units/mL of
penicillin, and 0.2 mg/mL streptomycin. Cells were maintained at
37 °C in a humidified atmosphere of 5% CO2.
Animals. All procedures using animals conformed strictly to the
guidelines set forth by the animal care units of each respective
institution, which reviewed and approved the experimental protocol.
Female Sprague-Dawley rats (200-250 g from Charles River,
Portage, Michigan) were housed in a temperature- and light-
controlled room and had free access to food and water. The rats
were inoculated with Walker 256 carcinosarcoma cells (5.0 × 106
cells) in 0.2 mL of saline in the right hind limb. Tumors were
allowed to develop for 7-14 days prior to injection with PLE
analogues.
γ-Camera Scintigraphy. The radiolabeled compounds were
dissolved in absolute ethanol (50-500 µL), and Tween-20 (0.1 mL/
mg of compound) was added to the solution. Ethanol was removed
by evaporation under a stream of nitrogen. Normal saline was added,
to give a 2-3% Tween-20 solution, which was subsequently mixed
by vortex. The solubilized radiolabeled compound (35-50 µCi,
0.5 mL per rat; 10-15 µCi, 0.2 mL per mouse) was administered
intravenously via tail vein to anesthetized tumor-bearing animals
(n g 3 per time point). Scanning of the animals was performed
using an LEM Mobile Camera, Siemens Corp. (Hoffman Estates,
IL) with a high-sensitivity-low-energy collimator window opti-
mized for the low-energy γ-emissions of iodine-125. Image
acquisition and storage was accomplished with a Siemens
MicroDELTA computer connected to a larger MicroVAX unit.
Animals (n ) 2 per compound) were sedated with a premixed
solution 87 mg/kg ketamine and 13 mg/kg xylazine. Images were
accumulated for 20 min at various times (1, 3, 6, and 24 h and at
24-h intervals thereafter to 120 h) following tail-vein administration
of radiolabeled compound (40-50 µCi, 0.5-1.0 mL). Animals were
euthanized after imaging protocols and were subjected to tissue
distribution analysis.
The human 131I-NM-404 (800 µCi) images were acquired on a
GE Maxxus dual-head SPECT imaging system using a whole body
scanning protocol at 1, 2, and 4 days after intravenous administra-
tion.
Male Copenhagen rats (200-220 g, Harlan) were housed under
the same conditions. The rats were inoculated subcutaneously with
MAT LyLu prostate cancer cells (1.0 × 106 cells) in 0.1 mL of
phosphate-buffered saline in the right hind limb. Tumors were
permitted to grow for 10-14 days prior to initiation of γ-camera
imaging with the radioiodinated PLE homologues (30-50 µCi, iv).
Male SCID mice (19-22 g, Harlan) were housed in a temper-
ature- and light-controlled room and had free access to food and
water. Each was inoculated with human PC-3 prostate tumor cells
(5 × 105 cells) in 0.1 mL of saline in the right hind limb. Tumors
were allowed to develop 10-14 days prior to injection with 125I-
NM-404.
Tissue Distribution. The radiolabeled compounds were dissolved
in absolute ethanol, Tween-20 (0.1 mL/mg of compound) was added
to this solution, and the solvent was evaporated with a stream of
nitrogen. Physiological saline was added, to give a 2-3% Tween-
20 solution. The solubilized radiolabeled compound (5-10 µCi in
0.3 mL or 2-5 µCi in 0.2 mL) was administered intravenously via
tail vein to tumor-bearing rats or mice, respectively. Injected dose
was normalized to the mass of radiolabeled compound and body
weight of the recipient in order to maintain consistency of injected
mass dose between animals in comparative studies. The animals
were euthanized by exsanguination while under anesthesia at the
various time points. Blood was collected and selected tissues were
removed and blotted to remove excess blood. Large organs were
Acknowledgment. The authors wish to thank Dr. James
Varani of the Department of Pathology, University of Michigan
Medical School, for providing the Walker 256 carcinosarcoma
cells and Dr. Ken Pienta, Department of Urology, University
of Michigan Medical School, for providing the prostate tumor-
bearing rats and mice. The authors wish to acknowledge Dr.
Thomas C. Stringfellow and Mr. Gary Girdaukas of the
Analytical Instrumentation Center in the University of Wiscon-
sin, School of Pharmacy, for assistance in obtaining NMR and
mass spectra, respectively. Support for this research was
provided by the National Institute of Health grants CA-08349
(R.E.C.) and CA-92412 (J.P.W.).
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