Stereoselective synthesis and biological evaluation of syn-1-amino-3-[18F]fluorocyclobutyl-1-carboxylic acid as a potential positron emission tomography brain tumor imaging agent

Article abstract of DOI:10.1016/j.bmc.2009.01.032

Amino acid syn-1-amino-3-fluoro-cyclobutyl-1-carboxylic acid (syn-FACBC) 12, the isomer of anti-FACBC, has been selectively synthesized and [¹⁸F] radiofluorinated in 52% decay-corrected yield using no-carrier-added [¹⁸F]fluoride. The key step in the synthesis of the desired isomer involved stereoselective reduction using lithium alkylborohydride/zinc chloride, which improved the ratio of anti-alcohol to syn-alcohol from 17:83 to 97:3. syn-FACBC 12 entered rat 9L gliosarcoma cells primarily via L-type amino acid transport in vitro with high uptake of 16% injected dose per 5 × 10⁵ cells. Biodistribution studies in rats with 9L gliosarcoma brain tumors demonstrated high tumor to brain ratio of 12:1 at 30 min post injection. In this model, amino acid syn-[¹⁸F]FACBC 12 is a promising metabolically based radiotracer for positron emission tomography brain tumor imaging.

Full text of DOI:10.1016/j.bmc.2009.01.032

Bioorganic & Medicinal Chemistry 17 (2009) 1982–1990
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Stereoselective synthesis and biological evaluation of syn-1-amino-3-[18F]-
fluorocyclobutyl-1-carboxylic acid as a potential positron emission
tomography brain tumor imaging agent
Weiping Yu a, Larry Williams a, Vernon M. Camp a, Eugene Malveaux a,
Jeffrey J. Olson b, Mark M. Goodman a,
*
a Department of Radiology, School of Medicine, Emory University, Atlanta, GA 30322, USA
b Department of Neurosurgery, School of Medicine, Emory University, Atlanta, GA 30322, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Amino acid syn-1-amino-3-fluoro-cyclobutyl-1-carboxylic acid (syn-FACBC) 12, the isomer of anti-FACBC,
has been selectively synthesized and [18F] radiofluorinated in 52% decay-corrected yield using no-carrier-
added [18F]fluoride. The key step in the synthesis of the desired isomer involved stereoselective reduction
using lithium alkylborohydride/zinc chloride, which improved the ratio of anti-alcohol to syn-alcohol
from 17:83 to 97:3. syn-FACBC 12 entered rat 9L gliosarcoma cells primarily via L-type amino acid trans-
port in vitro with high uptake of 16% injected dose per 5 Â 105 cells. Biodistribution studies in rats with
9L gliosarcoma brain tumors demonstrated high tumor to brain ratio of 12:1 at 30 min post injection. In
this model, amino acid syn-[18F]FACBC 12 is a promising metabolically based radiotracer for positron
emission tomography brain tumor imaging.
Received 21 November 2008
Revised 14 January 2009
Accepted 15 January 2009
Available online 21 January 2009
Keywords:
syn-FACBC
Fluorine-18
Amino acid transporter
PET
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
inflammatory cells is low.2 This may complement [18F]FDG for
grading and staging tumors.3
The development of radiotracers that accumulate preferentially
in tumor cells has been one of the active radiopharmaceutical
interests. A number of classes of compounds have been investi-
gated, including metabolically based radiotracers such as amino
acids, carbohydrates, and nucleosides. The application of radiola-
beled amino acids using positron emission tomography (PET) to
detect brain tumors has received considerable attention due to po-
tential advantages over other imaging modalities. Conventional
imaging methods such as computed tomography (CT) and mag-
netic resonance imaging (MRI) do not always reliably distinguish
residual and recurring tumor from tissue injury so they are not
optimal for monitoring the effectiveness of surgical treatment, or
for detecting tumor recurrence. The leading PET agent [18F]FDG, a
glucose derivative, shows high uptake in normal brain cortical tis-
sue as well as in inflammatory tissues associated with radiation
necrosis which can complicate the diagnostic evaluation of brain
tumors.1 Many tumor cells have increased amino acid transport
relative to normal cells as well as to inflammatory tissues associ-
ated largely with the utilization of nutrients in tumors. In contrast
to [18F]FDG, the uptake of amino acids in macrophages and other
Many amino acids have been radiolabeled to study potential
imaging characteristics.3–18 These radiolabeled amino acids differ
in ease of synthesis, in vivo biodistribution and formation of
metabolites. Amino acids enter cells mainly from two pathways,
from outside by carrier mediated transporters or are derived from
intracellular protein recycling. For radiotracers used for tumor
imaging, the former pathway is the major consideration. Naturally
occurring amino acids and their closely related derivatives, repre-
sented by
L
-[11C-methyl]methionine (
L
-[11C]MET),12
L
-1-[11C]-tyro-
sine (
L
-[11C]TYR)5,15 and [18F]fluoro-
L
-phenylalanine,19 enter and
accumulate in tumor cells not only by transporters but also by pro-
tein synthesis processes. These amino acid radiotracers are suscep-
tible to in vivo metabolism through multiple pathways, giving rise
to numerous radiolabeled metabolites. Thus, graphical analysis
with the necessary accuracy for reliable measurement of tumor
metabolic activity is not possible. The shortcomings associated
with naturally occurring amino acids may be overcome with
non-natural amino acids. Non-natural amino acids are expected
to have certain degree of metabolic stability. They may not be
effectively incorporated into proteins. These properties can poten-
tially simplify the analysis of tracers’ in vivo kinetics and tumor
cells uptake mechanisms.
A number of [18F] labeled non-natural cyclobutyl amino acids
have been prepared in our lab for PET tumor imaging, including
* Corresponding author. Tel.: +1 404 727 9366; fax: +1 404 727 3488.
E-mail address: mgoodma@emory.edu (M.M. Goodman).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.032

W. Yu et al. / Bioorg. Med. Chem. 17 (2009) 1982–1990
1983
anti-1-amino-3-[18F]fluorocyclobutane-1-carboxylic acid (anti-
ter involved the separation of the minor isomer from the major
isomer in a 87:13 mixture of intermediates (2), gram scale syn-
thesis was impractical. The work presented here demonstrated
the general synthetic route to produce gram quantities of syn-
[
18F]FACBC)1,20 and syn- and anti-1-amino-3-[18F]fluoromethyl-
cyclobutane-1-carboxylic acid (syn- and anti-[18F]FMACBC).21
These compounds showed high tumor uptake in 9L rat gliosar-
coma tumors. Among these amino acids, the PET tracer anti-
[
18F]FACBC precursor (10d), radiosynthesis and the biological
[
18F]FACBC is undergoing human preclinical trials to validate it
evaluation of syn-[18F]FACBC 12 with 9L rat gliosarcoma tumor
as a valuable imaging agent for the diagnosis and management
model.
of treatment of cancer.22,23 The method for preparation of
[
18F]FACBC reported previously20 affords only gram quantities
2. Results and discussion
2.1. Chemistry
of the precursor, cis (syn)-1-(tert-butoxycarbonylamino)-3-(trif-
luoromethylsulfonyloxy)cyclobutane-1-carboxylic methyl ester,
for producing anti-isomer of [18F]FACBC. Since the preparation
of the precursor of syn-[18F]FACBC, trans (anti)-1-carbamate-3-
(trifluoromethylsulfonyloxy)cyclobutane-1-carboxylic methyl es-
The anti-triflate precursor of syn-FACBC for radiolabeling was
synthesized in a series of synthetic steps starting from epi-
O
O
NH
NH
HN
HN
4 steps
Ref.20
O
O
O
+
Br
O
O
Ph
Ph
1
2
2
anti-hydantoin
syn-hydantoin
minor
major
BocHN CO2CH3
BocHN CO2CH3
5 steps
Ref.2
[
18F]KF/K222
O
18F
O
S O
CF3
4
, syn
3, anti
Scheme 1. Synthesis of syn- and anti-hydantoin 2 and anti-triflate 3.
O
R1
R1
N
NH
H2N CO2H
R2
N
CO2H
Ph
HN
R2
CO2CH3
O
a
b
c
O
O
O
O
Ph
Ph
Ph
7
5
6
2
a, R1=Boc, R2=H
b, R1=TFA, R2=H
a, R1=Boc, R2=H
b, R1=TFA, R2=H
c, R1=Bz, R2=H
c,
R1=Bz, R2=H
R1, R2=Phth
d,
d,
R1, R2=Phth
R1
N
R1
N
R1
N
R2
CO2CH3
R2
CO2CH3
R2
CO2CH3
e
f
d
OH
8, anti : syn 17 : 83
O
OH
8, anti as major
9
a,
b,
a,
R1=Boc, R2=H
R1=Boc, R2=H
R1=TFA, R2=H
a, R1=Boc, R2=H
b, R1=TFA, R2=H
c, R1=Bz, R2=H
b, R1=TFA, R2=H
c, R1=Bz, R2=H
d, R1, R2=Phth
c, R1=Bz, R2=H
d, R1, R2=Phth
d,
R1, R2=Phth
Scheme 2. Synthesis of stereoselective anti-alcohol 8. Reagents and conditions: (a) 3 N NaOH, 120 °C, overnight; (b) di-tert-butyl dicarbonate (6a); trifluoroacetic anhydride
(6b); benzoyl chloride (6c); phthalic anhydride (6d); (c) (CH3)3SiCHN2, rt, 30 min; (d) H2, 10% Pd–C, overnight; (e) NMO, TPAP, rt, 5 h; (f) reductant A–E (see Table 1). Notes:
Boc = tert-butoxycarbonyl; TFA = trifluoroacetyl; Bz = benzoyl; Phth = phthaloyl.

1984
W. Yu et al. / Bioorg. Med. Chem. 17 (2009) 1982–1990
bromohydrin (1) and benzyl bromide. The key intermediates, a
83:17 mixture of syn/anti-5-(3-benzyloxycyclobutane)hydantoins
(2) was prepared in four steps as described previously.20 The sep-
aration of the desired anti-hydantoin by chromatography using re-
ported procedure was time consuming and impractical. Even
though we managed to synthesize anti-triflate (3) as the precursor
for syn-[18F]FACBC, the radiofluorination was unsuccessful, due
part to the bulky Boc group which hindered 18FÀ to access reaction
center at C-3 (Scheme 1).
Our approach to solve the problems involved: (1) using less
bulky amino protecting groups or cyclic imide such as phthalimide
instead of Boc so that the C-3 is more exposed for nucleophilic
attacking by 18F anion and (2) selectively synthesizing anti-precur-
sor at the later synthetic stage by using stereoselective reagent to
avoid tedious chromatographic separation procedure. The detailed
syntheses are depicted in Scheme 2. The N-Boc derivatives were in-
cluded for comparison with the other compounds.
The 83:17 mixture of syn/anti-hydantoins (2) was hydrolyzed at
120 °C with 3 N sodium hydroxide (aq) to give syn- and anti-1-ami-
no-3-benzyloxycyclobutane-1-carboxylic acids (5). Subsequent
amino protection with (a) di-tert-butyl dicarbonate; (b) trifluoro-
acetic anhydride; (c) benzoyl chloride; and (d) phthalic anhydride,
respectively, provided corresponding syn/anti-carbamates 6a–d.
syn/anti-1-Carbamate-3-benzyloxy-cyclobutane-1-carboxylic acid
methyl esters 7a–d were prepared by treatment of 6a–d with (tri-
methylsilyl)diazomethane,24 respectively. Debenzylation using
hydrogen and palladium (0) catalyst on carbon gave 83:17 mixture
of syn/anti-1-carbamate-3-hydroxy-cyclobutane-1-carboxylic acid
methyl esters 8a–d. Since the precursor for syn-FACBC was the
anti-isomer, which was the minor component from above synthe-
ses, it was difficult to isolate from the major syn-compound in large
scale by the chromatographic method reported earlier.20 To opti-
mize the synthetic method, the mixture of syn/anti-alcohols was
firstly oxidized to the corresponding ketone 9a–d with N-methyl-
morpholine-N-oxide (NMO) and tetra-n-propylammonium per-
ruthenate (TPAP) in methylene chloride.25–27 Secondly several
reductants have been tested for the selectivity of converting the
ketone 9a–d back to alcohol 8a–d with the anti-alcohols as the ma-
jor products. The ratios of the anti- and syn-isomers were deter-
mined by 1H NMR and the results are summarized in Table 1.
Among the reductants chosen, BH3-THF/(S)- or (R)-CBS (oxaz-
aborolidine) catalyst28 gave little stereoselectivity to all reductive
products 8a–d, although these conditions led to excellent yields.
The reductant LS-SelectrideÒ (lithium trisiamylborohydride) failed
to provide any products in all attempts, except trace of 8b was col-
lected. Sodium tetrahydridoborate, with or without zinc chloride,
showed same and certain degree of stereoselectivity, which gave
3–4:1 ratios of anti- to syn-alcohols 8a–d. However further separa-
tion procedures are inevitable. The best stereoselectivie reductant
Table 1
R1
N
R1
N
R2
CO2CH3
R2
CO2CH3
O
OH
8a, R1=Boc, R2=H
9a, R1=Boc, R2=H
9b, R1=TFA, R2=H
9c, R1=Bz, R2=H
8b,
R1=TFA, R2=H
8c,
R1=Bz, R2=H
8d, R1, R2=Phth
9d,
R1, R2=Phth
Ketones
Reductants
Time
Yield (%)
Ratio of alcohol 8
anti
syn
9a
A
24 h
24 h
24 h
98
100
—
70
70
30
30
A/ZnCl3
B
B/ZnCl3
C
C/ZnCl3
D
D/ZnCl3
E
E/ZnCl3
24 h
24 h
24 h
30 min
30 min
30 min
30 min
—
92
90
97
96
98
96
78
80
55
60
55
55
22
20
45
40
45
45
in this study was -SelectrideÒ (lithium tri-sec-butylborohydride)
L
with zinc chloride as a chelating Lewis acid. Hydride attack came
generally from both sides of the cyclobutanone but the chelates
promoted more effective approach to anti-configurational alcohols
(Scheme 3).
9b
9c
9d
A
24 h
24 h
24 h
24 h
24 h
24 h
30 min
30 min
30 min
30 min
74
93
<0.1
—
70
68
100
98
95
99
80
95
43
20
5
57
A/ZnCl3
B
B/ZnCl3
C
C/ZnCl3
D
D/ZnCl3
E
The anti-alcohol 8a–d were subsequently reacted with triflic
anhydride to give anti-1-carbamate-3-[(trifluoromethyl)sulfo-
nyl]oxy-cyclobutanecarboxylic acid methyl esters 10a–d as the
radiolabeling precursors, see Scheme 4.
75
78
55
55
62
55
25
22
45
45
38
45
The 19F reference of syn-FACBC 12 was prepared from the anti-
N-phthaloyl amino acid methyl ester anti-8d by treatment of
diethylaminosulfur trifluoride (DAST)29,30 followed by deprotec-
tion with hydrazine hydrate as shown in Scheme 5.
E/ZnCl3
A
24 h
24 h
24 h
24 h
24 h
24 h
30 min
30 min
30 min
30 min
98
77
—
75
93
25
7
A/ZnCl3
B
B/ZnCl3
C
C/ZnCl3
D
D/ZnCl3
E
2.2. Radiolabeling
—
90
91
95
92
96
93
55
60
55
58
55
55
45
40
45
42
45
45
Radiofluorinated syn-[18F]FACBC 12 was prepared in two steps
using no-carrier-added (NCA) nucleophilic substitution with dried
[
18F]KF, potassium carbonate and Kryptofix in acetonitrile using
the triflate 10d (Scheme 6). The precursors of anti-triflates 10a–c
were attempted and failed to give any 18F-labeled products. The
exchange between [18F]fluoride and the leaving group occurred
in 10 min at 90 °C. Unreacted [18F]fluoride and any radiolabeled
charged by-products were eliminated by passing the reaction mix-
ture through a silica SepPakÒ. Removal of protecting groups was
achieved by using hydrazine hydrate at 75 °C for 10 min. The
syn-[18F]FACBC 12 was purified by ion-retardation resin chroma-
tography.24 The procedure required approximately 80 min from
the end of bombardment (EOB) with decay-corrected yields
(DCY) of 51.5 15.6% (n = 7) in over 95% radiochemical purity as
measured by radiometric TLC.
E/ZnCl3
A
17 h
24 h
24 h
79
79
—
82
97
18
3
A/ZnCl3
B
B/ZnCl3
C
C/ZnCl3
D
D/ZnCl3
E
E/ZnCl3
24 h
24 h
24 h
30 min
30 min
30 min
30 min
—
88
91
99
92
89
95
75
82
55
60
58
60
25
18
45
40
42
40
A = L-SelectrideÒ; B = LS-SelectrideÒ; C = NaBH4; D = BH3–THF/(R)-CBS (cat.);
E = BH3–THF/(S)-CBS (cat.).

W. Yu et al. / Bioorg. Med. Chem. 17 (2009) 1982–1990
1985
O
O
H-
O
O
O
O
OH
H
O
R1
N
H
R1
N
+
OH
R1
N
R2
R2
R2
Cl
Cl
O
Zn
O
O
O
O
OH
H
R1
N
R1
N
R2
R2
H-
Scheme 3.
2.3. Cell uptake assays
2.4. In vivo biodistribution studies in tumor-bearing rats
The in vitro studies were performed in 9L rat gliosarcoma cells
in Hank’s Balanced Salt Solution (HBSS) incubated for 30 min at
37 °C with or without inhibitors to evaluate the compounds tumor
cell uptake profile and transport mechanism. 10 mM 2-amino-
bicyclo[2.2.1]-heptane-2-carboxylic acid (BCH) and 10 mM N-
The in vivo biodistribution studies were performed in Fischer
rats with 9L tumors implanted intracranially. Based on the re-
search results we published earlier,1,3,16,21 the tissue distribution
of radioactivity after tail vein injection of amino acid tracers in
the normal tissues of tumor-bearing animals was similar to that
seen in the normal animals of this model. As the result, we only
used tumor-bearing rats for measuring radioactivity biodistrubu-
tion in this study. The radioactivity in tumors and in normal tissues
of tumor-bearing rats (n = 4 each time point) was calculated at 15,
30, 60 and 120 min post injection (p.i.) and normalized as percent
injected dose per gram tissue (%ID/g). The uptake of radioactivity of
syn-[18F]FACBC 12 in tumor and in other tissues is presented in
Table 2.
The experiments showed that this amino acid had rapid and
prolonged accumulation in tumors, ranged from 1.4–2.1%ID/g
and was significantly higher than in normal brain tissue (p < 0.05
at all time points, one-way ANOVA). The uptake in normal brain
tissue was less than 0.25%ID/g at all time points thus the tumor
to normal brain uptake ratios were in the range of 7.2:1–11.5:1.
Except in pancreas, uptake in other tissues tested was lower than
that in tumor (p < 0.02, two-way ANOVA). The low bone uptake
of radioactivity with this radiotracer indicates that compound 12
is metabolically stable so free fluoride was not generated during
the time course of the study. The analogs anti-FACBC, syn-FMACBC
and anti-FMACBC, showed uptake of radioactivity in tumor at
60 min p.i. of 1.72, 1.59 and 2.50%ID/g in the same animal model,
respectively, which resulted in tumor to brain ratios of 6.6:1,
6.9:1 and 8.9:1, respectively.1,21 Thus, compound 12 is comparable
to anti-FACBC, syn-FMACBC and anti-FMACBC, in the terms that all
these amino acids enter 9L cells via L-type transport system
in vitro and show high levels of radioactivity uptake in 9L tumor
in vivo in this animal model.
methyl-a-aminoisobutyric acid (MeAIB) were used as L- and A-
type amino acid transport inhibitors, respectively.13,31–33 We also
used the combination of 10 mM alanine-cysteine-serine (ACS,
equal molar amount) as quality control for our inhibition assays,
since these amino acids can be used as wide-spectra block agents
for amino acid transporters. In the absence of inhibitors, syn-
[
18F]FACBC 12 showed high levels of intracellular accumulation,
15.8 2.4% of the initial dose per 0.5 million cells (%ID/5 Â 105
cells) in 9L gliosarcoma cells. In the presence of BCH, 38.6% of inhi-
bition was observed compared to controls (p < 0.02, 1-way ANO-
VA). On the contrast, no uptake inhibition was detected with
MeAIB relative to controls. When ACS was used, up to 77.8% uptake
reduction occurred compared to controls (p < 0.001, 1-way ANO-
VA). For comparison, we carried out the same cell assays for com-
pound anti-FACBC under the identical condition. anti-FACBC
showed a very similar cell uptake profile to that of syn-FACBC.
Without inhibitors, its cell uptake was 12.3 0.4%ID/5 Â 105 cells.
The cell uptake was reduced to 2.9 0.6%ID/5 Â 105 cells
(p < 0.0001, 1-way ANOVA) and 0.18 0.02%ID/5 Â 105 cells
(p < 0.0001, 1-way ANOVA) by BCH and by ACS, respectively. The
inhibition by MeAIB (6%) was not significant. These results, which
are depicted in Figure 1, demonstrate that both compounds syn-
FACBC 12 and anti-FACBC are selective substrates for L-type amino
acid transporter in 9L gliosarcoma cells in vitro.
R1
N
R1
N
R2
CO2CH3
R2
CO2CH3
3. Conclusions
a
A new PET tumor imaging ligand, syn-FACBC 12 has been selec-
tively synthesized in gram quantities, [18F] labeled in high radio-
chemical yield (>50% DCY) and high radiochemical purity, and
biologically evaluated in rodent 9L gliosarcoma brain tumor model.
The compound demonstrated high levels of tumor uptake in vitro
and in vivo with good tumor to brain ratios ranging from 7:1 to
12:1. It transported into cells primarily through L-type amino acid
transporter with little if any A-type transport property. These re-
sults are comparable to its analogues of anti-FACBC, syn-FMACBC
and anti-FMACBC in the same animal model, which support the
candidacy of syn-FACBC 12 as promising PET brain tumor imaging
agent.
OH
O
S
O
O
CF3
10
R1=Boc, R2=H (= )
b, R1=TFA, R2=H
c, R1=Bz, R2=H
d, R1, R2=Phth
8
a,
3
a,
R1=Boc, R2=H
b, R1=TFA, R2=H
c, R1=Bz, R2=H
d, R1, R2=Phth
Scheme 4. Syntheses precursors of anti-triflates 10a–d. Reagents and conditions:
(a) Tf2O, Py, DCM, 0 °C, 15 min.

1986
W. Yu et al. / Bioorg. Med. Chem. 17 (2009) 1982–1990
O
O
H2N CO2H
a
b
N
CO2CH3
N
CO2CH3
O
O
F
OH
anti-8d
F
11
12
Scheme 5. Synthesis of F-19 reference of syn-FACBC 12. Reagents and conditions: (a) DAST, DCM, À70 °C to rt, overnight; (b) N2H4–H2O, 75 °C, 30 min.
O
O
N
CO2CH3
H2N CO2H
a
b
O
N
CO2CH3
O
18F
O
S
O
O
18F
CF3
10d
18F]12
[
18F]11
[
Scheme 6. Radiosynthesis of syn-[18F]FACBC 12. Reagents and conditions: (a) [18F]KF, K222, K2CO3, 90 °C, 10 min; (b) N2H4–H2O, 75 °C, 10 min.
Figure 1. 9L cell uptake and inhibition assays with (A) [18F]12 and (B) anti-[18F]FACBC (%ID/5 Â 105 cells).
ried out using Merck Kieselgel silica gel 60 (230–400 mesh).
Melting points were measured in capillary tubes using a Mel-
Temp II apparatus (Laboratory Devices, Inc., Holliston, MA USA)
and are uncorrected. 1H NMR spectra were recorded on Varian
400 MHz or 300 MHz spectrometers at NMR Center at Emory
University, and chemical shifts (d values) were reported as parts
per million (ppm) downfield from tetramethylsilane (TMS). Ele-
mental analyses were performed by Atlantic Microlabs, Inc. (Nor-
cross, GA USA) and were within 0.4% of the theoretical values.
Mass Spectra were done on JEOL JMS-SX102/SX102A/E or VG
70-S double focusing mass spectrometers at Mass Spectroscopy
Center at Emory University using high-resolution electrospray
ionization (ESI).
4. Experimental
4.1. Chemistry
All chemicals, solvents and materials used were obtained from
commercially available sources and used without purification.
Chemicals were purchased from Aldrich Chemicals Co. (Milwaul-
kee, WI USA) and Sigma Chemical Co. (St. Louis, MO USA), or
otherwise indicated, and solvents were purchased from Aldrich
Chemicals and VWR Scientific Products (West Chester, PA USA).
Thin-layer chromatography (TLC) analyses were performed with
250
lm UV254 silica gel backing on aluminum plates (Whatman
Ltd.; Maidstone, Kent England). Flash chromatography was car-

W. Yu et al. / Bioorg. Med. Chem. 17 (2009) 1982–1990
1987
Table 2
hydroxide was added benzoyl chloride (1.2 equiv) at 0 °C over
15 min. The mixture was stirred at 0 °C for 2 h then at room tem-
perature overnight. Ethyl acetate (40 mL) was added to the mix-
ture and the aqueous layer was adjusted to pH 1–2 with
hydrochloric acid (concd). The organic phase was retained; the
aqueous phase was saturated with sodium chloride and extracted
with ethyl acetate (4 Â 40 mL). The combined organic phases were
dried over sodium sulfate (anhyd) and the solvent was removed
under reduced pressure. The crude N-Bz acid 6c, 1.22 g (73%),
was used in next step without further purification. 1H NMR
(400 MHz, CDCl3) d 2.54 (syn-, 83%) and 2.80 (anti, 17%) (2H, m,
CH2), 3.01 (anti, 17%) and 3.22 (syn-, 83%) (2H, m, CH2), 4.28 (1H,
m, CH), 4.49 (2H, s, OCH2Ph), 6.86 (1H, br s, NH), 7.32–8.14 (10H,
m, aromatic).
Biodistribution of radioactivity in tissues of 9L tumor-bearing Fischer rats following
intravenous administration of syn-[18F]12
Tissue
15 min
30 min
60 min
120 min
Blood
Heart
Lung
Liver
Pancreas
Spleen
Kidney
Muscle
Brain
Tumor
Bone
Testis
0.36 0.08
0.43 0.09
0.40 0.13
0.73 0.18
2.37 0.96
0.68 0.18
0.76 0.16
0.35 0.16
0.20 0.14*
1.40 0.49*
0.25 0.12
0.17 0.06
7.2
0.37 0.10
0.47 0.15
0.57 0.22
0.60 0.22
3.36 0.24
0.61 0.12
0.80 0.18
0.43 0.002
0.18 0.02**
2.10 0.96**
0.26 0.07
0.27 0.03
11.5
0.32 0.02
0.38 0.02
0.42 0.04
0.41 0.03
2.83 0.59
0.52 0.03
0.70 0.02
0.42 0.01
0.20 0.01
1.48 0.09
0.28 0.05
0.27 0.02
7.6
0.26 0.04
0.30 0.05
0.48 0.05
0.40 0.10
2.78 0.48
0.40 0.06
0.56 0.08
0.39 0.06
0.25 0.03*
1.93 0.72*
0.20 0.03
0.21 0.03
7.7
Tumor to brain ratio
4.5. syn/anti-1-[N-(Phthaloyl)amino]-3-benzyloxy-
cyclobutanecarboxylic acid (6d)
Values are reported as mean percent injected dose per gram tissue (%ID/g) stan-
dard deviation; n = 4 at each time point; p values were calculated using one-way
ANOVA; *p < 0.02, **p < 0.05, p < 0.002.
To a suspension of syn/anti-1-amino-3-benzyloxy-cyclobutane-
carboxylic acid (5)20 (669 mg, 3.03 mmol) in 15 mL of toluene was
added phthalic anhydride (2 equiv) and triethylamine (2 equiv).
The mixture was heated to reflux at 120 °C for 5 h. After cooled
to room temperature, 10 mL of water was added and 3 N HCl
was used to adjust the pH of the aqueous solution to 2. The aque-
ous phase was separated, saturated with sodium chloride and ex-
tracted with ethyl acetate (2 Â 15 mL). The combined organic
phases were dried over sodium sulfate (anhyd), filtered and con-
centrated to dryness. The crude N-Phth acid 6d was used in next
step without further purification. 1H NMR (400 MHz, CDCl3) d
2.81 (syn-, 83%) and 2.93 (anti, 17%) (2H, m, CH2), 3.32 (syn-,
83%) and 3.56 (anti, 17%) (2H, m, CH2) 4.45 (1H, m, CH), 4.48
(2H, s, OCH2Ph), 7.32–7.85 (9H, m, aromatic).
4.2. syn/anti-1-[N-(tert-Butoxycarbonyl)amino]-3-benzyloxy-
cyclobutanecarboxylic acid (6a)
syn/anti-1-Amino-3-benzyloxy-cyclobutanecarboxylic acid (5)
was prepared according to the method described earlier.20 To a
suspension of 5 (2.0 g, 9 mmol) in 50 mL of 9:1 methanol/triethyl-
amine (v/v) was added a 1.5 equiv portion of di-tert-butyl dicar-
bonate (14 g), and the solution was stirred at room temperature
overnight. The solvent was removed under reduced pressure and
the resulting residue was re-dissolved in 50 mL of water. The pH
of the solution was adjusted to 2 with 3 N HCl and the aqueous
phase was extracted with ethyl acetate (2 Â 50 mL). The combined
organic layers were washed with water (1 Â 50 mL), dried over
magnesium sulfate (anhyd). The N-Boc acid 6a (2.1 g, 69%) was ob-
tained as a white solid suitable for use in the next step without fur-
ther purification. 1H NMR (400 MHz, CDCl3) d 1.43 (9H, s, C(CH3)3),
2.31 (syn-, 83%) and 2.50 (anti-, 17%) (2H, m, CH2), 2.64 (anti-, 17%)
and 2.93 (syn-, 83%) (2H, m, CH2), 4.20 (1H, m, CH), 4.45 (2H, s,
OCH2Ph), 5.14 (1H, br s, NH), 7.27–7.37 (5H, m, aromatic).
4.6. syn/anti-1-Carbamate-3-benzyloxy-cyclobutanecarboxylic
acid methyl ester (7a–d)
Compound 6a–d was dissolved in 15 mL of 4:1 benzene/meth-
anol (v/v), respectively, and (trimethylsilyl) diazomethane (2.0 M
in hexanes, 1.2 equiv) was added.24 After stirred at room tempera-
ture for 30 min, the solvent was removed under reduced pressure.
The crude 7a–d was purified using silica gel chromatography (20%
ethyl acetate in hexanes).
4.3. syn/anti-1-[N-(Trifluoroacetyl)amino]-3-benzyloxy-
cyclobutanecarboxylic acid (6b)
To a suspension of syn/anti-1-amino-3-benzyloxy-cyclobutane-
carboxylic acid (5)20 (1 g, 4.5 mmol) in 30 mL of dichloromethane
was added triethylamine (5 equiv). The mixture was cooled to
À15 °C and trifluoroacetic anhydride (3 equiv) was added drop-
wise under an argon atmosphere. The mixture was warmed up
to room temperature and stirred overnight. Ammonium chloride
(1 M, 10 mL) was added to the mixture and two phases separated
after stirred for 15 min. The organic layer was retained, and the
aqueous phase was extracted with ethyl acetate (2 Â 30 mL). The
combined organic layers were washed with water (2 Â 30 mL),
dried over magnesium sulfate (anhyd). The crude N-TFA acid 6b
was purified using silica gel chromatography (30% ethyl acetate
in hexanes) to provide 6b as a clear oil, 1.11 g (82%). 1H NMR
(400 MHz, CDCl3) d 2.47–2.52 (syn-, 83%) and 2.71–2.76 (anti,
17%) (2H, m, CH2), 2.85–2.90 (anti, 17%) and 3.00–3.05 (syn-,
83%) (2H, m, CH2), 4.27 (syn-, 83%) and 4.36 (anti, 17%) (1H, m,
CH), 4.47 (syn-, 83%) and 4.50 (anti, 17%) (2H, s, OCH2Ph), 5.34
(1H, br s, NH), 7.30–7.37 (5H, m, aromatic).
4.6.1. syn/anti-1-[N-(tert-Butoxycarbonyl)amino]-3-benzyloxy-
cyclobutanecarboxylic acid methyl ester (7a)
White crystalline solid, 2.0 g from 2.0 g (9.1 mmol) of 5 (66%, 2
steps). 1H NMR (400 MHz, CDCl3) d 1.43 (9H, s, C(CH3)3), 2.31 (syn-,
83%) and 2.50 (anti-, 13%) (2H, m, CH2), 2.64 (anti-, 13%) and 2.93
(syn-, 83%) (2H, m, CH2), 3.76 (3H, s, OCH3), 4.12–4.27 (1H, m,
CH), 4.45 (2H, s, OCH2Ph), 5.14 (1H, br s, NH), 7.29–7.38 (5H, m,
aromatic).
4.6.2. syn/anti-1-[N-(Trifluoroacetyl)amino]-3-benzyloxy-
cyclobutanecarboxylic acid methyl ester (7b)
Oil, 1.44 g from 1.17 g (3.7 mmol) of 6b (92%). 1H NMR
(400 MHz, CDCl3) d 2.48–2.53 (syn-, 83%) and 2.65–2.70 (anti-,
17%) (2H, br m, CH2), 2.75–2.80 (anti-, 17%) and 2.95–3.00 (syn-,
83%) (2H, m, CH2), 3.77 (syn-, 83%) and 3.78 (anti-, 17%) (3H, s,
OCH3), 4.23–4.29 (1H, m, CH), 4.45 (anti-, 17%) and 2.46 (syn-,
83%) (2H, s, OCH2Ph), 7.06 (1H, br s, NH), 7.30–7.37 (5H, m,
aromatic).
4.4. syn/anti-1-[N-(Benzoyl)amino]-3-benzyloxy-
cyclobutanecarboxylic acid (6c)
4.6.3. syn/anti-1-[N-(Benzoyl)amino]-3-benzyloxy-
cyclobutanecarboxylic acid methyl ester (7c)
To a suspension of syn/anti-1-amino-3-benzyloxy-cyclobutane-
carboxylic acid (5)20 (1.14 g, 5.2 mmol) in 50 mL of 2 N sodium
Clear oil, 1.0 g from 1.14 g (5.2 mmol) of 5 (58%, 2 steps). 1H
NMR (400 MHz, CDCl3) d 2.54 (syn-, 83%) and 2.80 (anti, 17%)

1988
W. Yu et al. / Bioorg. Med. Chem. 17 (2009) 1982–1990
(2H, m, CH2), 3.01 (anti, 17%) and 3.22 (syn-, 83%) (2H, m, CH2), 3.83
(anti-, 17%) and 3.85 (syn-, 83%) (3H, s, OCH3), 4.30 (1H, m, CH),
4.51 (2H, s, OCH2Ph), 6.66 (1H, br s, NH), 7.30–8.11 (10H, m,
aromatic).
4.8. 1-Carbamate-3-oxo-cyclobutanecarboxylic acid methyl
ester (9a–d)
syn/anti-1-Carbamate-3-hydroxy-cyclobutanecarboxylic
acid
methyl ester 8a–d in 5 mL of 10% acetonitrile in dichloromethane
containing N-methylmorpholine N-oxide (1.5 equiv) and 4 Å
molecular sieves (500 mg/1 mmol, powder), respectively, was stir-
red for 10 min at 22 °C under argon and then treated with tetra-n-
propylammonium perruthenate (10% molar equiv). The resulting
solution was stirred for 5 h. The solvent was removed and the res-
idue was purified by flash column chromatography (30% ethyl ace-
tate in hexanes) to afford the ketone 9a–d, respectively.
4.6.4. syn/anti-1-[N-(Phthaloyl)amino]-3-benzyloxy-
cyclobutanecarboxylic acid methyl ester (7d)
Clear oil, 723 mg from 669 mg (3.0 mmol) of 5 (65%, 2 steps). 1H
NMR (400 MHz, CDCl3) d 2.78–2.83 (syn-, 83%) and 2.91–2.96 (anti,
17%) (2H, m, CH2), 3.29–3.34 (syn-, 83%) and 3.54–3.59 (anti, 17%)
(2H, m, CH2), 3.73 (syn-, 83%) and 3.74 (anti, 17%) (3H, s, OCH3),
4.43–4.67 (1H, m, CH), 4.48 (2H, s, OCH2Ph), 7.27–7.34 (5H, m, aro-
matic), 7.71–7.73 and 7.82–7.84 (4H, m, aromatic). HRMS, m/z,
Calcd for C21H20NO5 [M+H]+, 366.1342. Found, 366.1363. Anal.
Calcd for (C21H19NO5): C, 69.03; H, 5.24; N, 3.83. Found: C, 68.64;
H, 5.28; N, 3.86.
4.8.1. 1-[N-(tert-Butoxycarbonyl)amino)amino]-3-oxo-
cyclobutanecarboxylic acid methyl ester (9a)
White solid, 162 mg from 183 mg (0.75 mmol) of 8a (89%), mp
117–119 °C (ethyl acetate/hexane). 1H NMR (400 MHz, CDCl3) d
1.47 (9H, s, C(CH3)3), 3.50–3.66 (4H, m, 2CH2), 3.84 (3H, s, OCH3),
5.48 (1H, br s, NH). Anal. Calcd for (C11H17NO5): C, 54.31; H,
7.04; N, 5.76. Found: C, 54.50; H, 6.96; N, 5.61.20
4.7. syn/anti-1-Carbamate-3-hydroxy-cyclobutanecarboxylic
acid methyl ester (8a–d)
To a solution of 7a–d in 5–10 mL of methanol, respectively, was
added 40% (w/w) of 10% palladium on carbon. The reaction mixture
was stirred overnight at room temperature under a hydrogen
atmosphere. The suspension was then filtered over Celite and con-
centrated under reduced pressure. Purification via silica gel col-
umn chromatography (30% ethyl acetate in hexane) provided the
alcohol 8a–d.
4.8.2. 1-[N-(Trifluoroacetyl)amino]-3-oxo-
cyclobutanecarboxylic acid methyl ester (9b)
Semi solid, 90 mg from 114 mg (0.47 mmol) of 8b (80%). 1H
NMR (400 MHz, CDCl3) d 3.65–3.93 (4H, m, 2CH2), 3.92 (3H, s,
OCH3), 7.50 (1H, br s, NH). HRMS, m/z, Calcd for C8H12F3N2O4
[M+NH4]+, 257.07437, found, 257.07437.
4.7.1. syn/anti-1-[N-(tert-Butoxycarbonyl)amino]-3-hydroxy-
cyclobutanecarboxylic acid methyl ester (8a)
4.8.3. 1-[N-(Benzoyl)amino]-3-oxo-cyclobutanecarboxylic acid
methyl ester (9c)
White crystalline solid, 968 mg from 1389 mg (4.1 mmol) of 7a
(95%), mp 128–131 °C (ethyl acetate/hexane). 1H NMR (400 MHz,
CDCl3) d 1.44 (anti-, 17%) and 1.45 (syn-, 83%) (9H, s, C(CH3)3),
2.48 (anti-, 17%) and 2.56 (syn-, 83%) (2H, m, CH2), 3.00 (syn-,
83%) and 3.35 (anti-, 17%) (2H, m, CH2), 3.77 (syn-, 83%) and 3.79
(anti-, 17%) (3H, s, OCH3), 4.30 (syn-, 83%) and 4.46 (anti-, 17%)
(1H, m, CH), 5.63 (1H, br s, NH).
Semi solid, 288 mg from 340 mg (1.4 mmol) of 8c (85%). 1H
NMR (400 MHz, CDCl3) d 3.67–3.86 (4H, m, 2CH2), 3.87 (3H, s,
OCH3), 7.30 (1H, br s, NH), 7.44–8.11 (5H, m, aromatic). HRMS,
m/z, Calcd for C13H14NO4 [M+H]+, 248.09173, found, 248.09176;
m/z, Calcd for C13H17N2O4 [M+NH4]+, 265.11828, found, 265.11831.
4.8.4. 1-[N-(Phthaloyl)amino]-3-oxo-cyclobutanecarboxylic
acid methyl ester (9d)
4.7.2. syn/anti-1-[N-(Trifluoroacetyl)amino]-3-hydroxy-
cyclobutanecarboxylic acid methyl ester (8b)
White crystalline solid, 112 mg from 125 mg (0.45 mmol) of 8d
(91%). mp 175–176 °C (ethyl acetate/hexane). 1H NMR (400 MHz,
CDCl3) d 3.80 (3H, s, OCH3), 3.98–4.01 (4H, m, 2CH2), 7.78–7.81
(2H, m, aromatic), 7.89–7.92 (2H, m, aromatic). HRMS, m/z, Calcd
for C14H12NO5 [M+H]+, 274.07100, found, 274.0114. Anal.
(C14H11NO5) C, H, N.
Clear oil, 1.0 g from 1.39 g (4.2 mmol) of 7b (99%). 1H NMR
(400 MHz, CDCl3) d 2.69–2.74 (anti-, 17%) and 2.75–2.80 (syn-,
83%) (2H, m, CH2), 2.89–2.94 (anti-, 17%) and 3.01–3.11 (syn-,
83%) (2H, m, CH2), 3.86 (anti-, 17%) and 3.88 (syn-, 83%) (3H, s,
OCH3), 4.36–4.42 (syn-, 83%) and 4.62–4.68 (anti-, 17%) (1H, m,
CH), 7.47 (1H, br s, NH).
4.9. anti-1-Carbamate-3-hydroxy-cyclobutanecarboxylic acid
methyl ester (anti-8a–d)
4.7.3. syn/anti-1-[N-(Benzoyl)amino]-3-hydroxy-
cyclobutanecarboxylic acid methyl ester (8c)
To the solution of the ketone 9a–d in 2 mL of THF (anhyd),
respectively, was added ZnCl2 (2 equiv, in THF) at room tempera-
ture under argon. The solution was stirred at room temperature
for 30 min followed by the addition of the L-SelectrideÒ (1.5 equiv)
at À78 °C. The mixture was stirred at À78 °C for 2 h then at room
temperature overnight. Ammonium chloride (1 N aq, 3 equiv)
was added and the mixture was stirred at room temperature for
30 min. The reaction was washed with brine, and aqueous phase
was re-extracted with ethyl acetate (2 Â 3 mL). The combined or-
ganic phases were dried over sodium sulfate (anhyd), filtered and
concentrated to dryness. The product was purified by silica gel
flash column chromatography using 50% ethyl acetate in hexanes
to afford 8a–d (anti-alcohol as major product, see Table 1).
Clear oil, 550 mg from 980 mg (2.9 mmol) of 7c (76%). 1H NMR
(400 MHz, CDCl3) d 2.57 (syn-, 83%) and 2.73 (anti, 17%) (2H, m,
CH2), 2.87 (anti, 17%) and 3.13 (syn-, 83%) (2H, m, CH2), 3.81
(anti-, 17%) and 3.87 (syn-, 83%) (3H, s, OCH3), 4.37 (syn-, 83%)
and 4.59 (anti, 17%) (1H, m, CH), 6.69 (1H, br s, NH), 7.44–7.83
(5H, m, aromatic).
4.7.4. syn/anti-1-[N-(Phthaloyl)amino]-3-hydroxy-
cyclobutanecarboxylic acid methyl ester (8d)
Semi solid, 74 mg from 186 mg (0.51 mmol) of 7d (53%).
1H NMR (400 MHz, CDCl3)
d
2.68–2.76 (syn-, 83%) and
2.82–2.89 (anti-, 17%) (2H, m, CH2), 3.31–3.42 (2H, m,CH2),
3.73 (syn-, 83%) and 3.76 (anti-, 17%) (1H, s, OCH3), 4.38–
4.45 (anti-, 17%) and 4.66–4.76 (syn-, 83%) (1H, m, CH),
7.71–7.76 (2H, m, aromatic), 7.81–7.86 (2H, m, aromatic).
HRMS, m/z, Calcd for C14H14NO5 [M+H]+, 276.0873, found,
276.0849.
4.9.1. anti-1-[N-(tert-Butoxycarbonyl)amino]-3-hydroxy-
cyclobutanecarboxylic acid methyl ester (anti-8a)
White solid, 16 mg from 16 mg (0.066 mmol) of 9a (100%), mp
108–110 °C (ethyl acetate/hexane, 109–110 °C20). 1H NMR

W. Yu et al. / Bioorg. Med. Chem. 17 (2009) 1982–1990
1989
(400 MHz, CDCl3) d 1.44 (9H, s, C(CH3)3), 2.53–2.63 (4H, m, 2CH2),
4.10.4. anti-1-[N-(Phthaloyl)amino]-3-
(trifluoromethylsulfonyloxy) cyclobutanecarboxylic acid
methyl ester (10d)
3.77 (3H, s, OCH3), 4.43–4.50 (1H, m, CH), 5.02 (1H, br s, NH). 13C
NMR (100 MHz, CD3OD) d 28.83, 42.50, 43.07, 53.05, 63.22,
80.71, 158.04, 175.35. Anal. Calcd for (C11H19NO5): C, 53.87; H,
7.81; N, 5.71. Found: C, 53.61; H, 7.79; N, 5.51.20
Clear oil, 15 mg from 11 mg (0.04 mmol) of anti-8d (93%). 1H
NMR (400 MHz, CDCl3) d 3.32–3.37 (2H, m, CH2), 3.83–3.89 (2H,
m, CH2), 3.80 (3H, s, COCH3), 5.40 (1H, m, CH), 7.78–7.80 (2H, m,
aromatic), 7.88–7.90 (2H, m, aromatic). 19F NMR (376MHz, CDCl3)
d À75.64 (s, SCF3).
4.9.2. anti-1-[N-(Trifluoroacetyl)amino]-3-hydroxy-
cyclobutanecarboxylic acid methyl ester (anti-8b)
Semi solid, 181 mg from 194 mg (0.81 mmol mmol) of 9b
(93%). 1H NMR (400 MHz, CDCl3) d 2.69–2.74 (2H, m, CH2),
2.89–2.94 (2H, m, CH2), 3.85 (3H, s, OCH3), 4.62–4.68 (1H, m,
CH), 7.02 (1H, br s, NH). 19F NMR (376 MHz, CDCl3) d À76.43
(s, CF3). HRMS, m/z, Calcd for C8H10F339KNO4 [M+K]+, 280.00370,
found, 280.00372.
4.11. syn-1-[N-(Phthaloyl)amino]-3-fluoro-
cyclobutanecarboxylic acid methyl ester (11)
anti-Alcohol 8d (50 mg, 0.18 mmol) was dissolved in 2 mL of
dichloromethane and diethylaminosulfur trifluoride (DAST,
3 equiv, in 2 mL of dichloromethane) was added at À78 °C under
argon. The mixture was allowed to warm to room temperature
and stirred overnight. The organic layer was washed with water
and brine, dried with sodium sulfate (anhyd), filtered and concen-
trated under reduced pressure. Purification of the crude product by
flash chromatography (silica, 20% ethyl acetate in hexanes) yielded
compound 11 as a clear oil (24 mg, 48%). 1H NMR (CDCl3) d 2.93–
3.04 (2H, m, CH2), 3.36–3.43 (2H, m, CH2), 3.73 (3H, s, CH3), 5.25–
5.32 and 5.39–5.46 (1H, d-p, J = 54.8, 6.8 Hz, CHF), 7.73–7.76 (2H,
m, aromatic), 7.82–7.86 (2H, m, aromatic). 19F NMR (376 MHz,
CDCl3), d À162.775 to À162.454 (m, CHF). HRMS, m/z, Calcd for
C14H13FNO4 [M+H]+, 278.08231, found, 278.08249. Anal. Calcd for
(C14H12FNO4), C, 60.65; H, 4.36; N, 5.05. Found: C, 60.38; H, 4.40;
N, 5.10.
4.9.3. anti-1-[N-(Benzoyl)amino]-3-hydroxy-
cyclobutanecarboxylic acid methyl ester (anti-8c)
Semi solid, 44 mg from 57 mg (0.23 mmol mmol) of 9c (77%). 1H
NMR (400 MHz, CDCl3) d 2.61–2.66 (2H, m, CH2), 2.71–2.77 (2H, m,
CH2), 3.69 (3H, s, OCH3), 4.44–4.51 (1H, m, CH), 6.69 (1H, br s, NH),
7.33–7.77 (5H, m, aromatic).
4.9.4. anti-1-[N-(Phthaloyl)amino]-3-hydroxy-
cyclobutanecarboxylic acid methyl ester (anti-8d)
White solid, 65 mg from 82 mg (0.30 mmol) of 9d (79%), mp
70–72 °C (ethyl acetate/hexane). 1H NMR (400 MHz, CDCl3) d
2.82–2.89 (2H, m,CH2), 3.35–3.42 (2H, m,CH2), 3.75 (1H, s, OCH3),
4.38–4.45 (1H, m, CH), 7.73–7.76 (2H, m, aromatic), 7.82–7.86
(2H, m, aromatic). HRMS, m/z, Calcd for C14H14NO5 [M+H]+,
276.08665, found, 276.08698. Anal. (C14H13NO5) C, H, N.
4.12. syn-1-N-Amino-3-fluoro-cyclobutanecarboxylic acid (12)
4.10. anti-1-Carbamate-3-(trifluoromethylsulfonyloxy)
Compound 11 (17 mg, 0.06 mmol) in 300 lL of hydrazine hy-
cyclobutanecarboxylic acid methyl ester (10a–d)
drate was stirred at 75 °C for 30 min. After cooled to room temper-
ature, the mixture was neutralized to pH 7 with 4 N hydrochloric
acid and passed successively through an ion-retardation resin col-
umn (AG 11A8 50–100 mesh, BioRad, Hercules, CA, USA), an alu-
mina N Sep-PakÒ and an HLB Sep-PakÒ (Waters, Inc., Milford, MA
USA) with water. The fractions containing syn-FACBC 12 were col-
lected and dried to give a white solid (4.6 mg, 58%), mp 215 °C
(decomposition). 1H NMR (D2O) d 2.74–2.84 (2H, m, CH2), 3.16–
3.21 (2H, m, CH2), 5.20–5.28 and 5.32–5.40 (1H, d-p, J = 56.4, 6.6
Hz, CHF). Anal. Calcd for (C5H8FNO2): C, 45.11; H, 6.06; N, 10.52.
Found: C, 45.02; H, 6.24; N, 10.68.
anti-Alcohol 8a–d was dissolved in 1 mL of dichloromethane,
respectively, and cooled to 0 °C under argon. Pyridine (15–
20 equiv) and triflic anhydride (5 equiv) were added successively.
The mixture was stirred at 0 °C for 15 min and the solvent was re-
moved under reduced pressure. The mixture was purified by flash
chromatography (silica, 30% ethyl acetate in hexanes) to afford the
anti-triflate 10a–d, respectively.
4.10.1. anti-1-[N-(tert-Butoxycarbonyl)amino]-3-
(trifluoromethylsulfonyloxy) cyclobutanecarboxylic acid
methyl ester (10a, 3)
4.13. Radiosynthesis
Oil, 8 mg from 18 mg (0.072 mmol mmol) of anti-8a (28%). 1H
NMR (400 MHz, CDCl3) d 1.44 (9H, s, C(CH3)3), 3.06 (4H, m,
2CH2), 3.80 (3H, s, COCH3), 5.31 (1H, br s, NH), 5.38 (1H, m, CH).
19F NMR (376 MHz, CDCl3) d À75.64 (s, SCF3).
[
18F]HF was produced at Emory University with a Siemens 11
MeV RDS 112 negative-ion cyclotron (Knoxville, TN, USA) by the
18O (p, n) 18F reaction using [18O]H2O (95%). A fully automated syn-
thesis system developed for the Siemens computer programmable
chemistry process control unit (CPCU) was used for the preparation
of [18F]12. The CPCU is a valve-and-tubing system which is de-
signed to accommodate two glass reaction vessels and a number
of reagent and solvent reservoir containers. Briefly, the automated
4.10.2. anti-1-[N-(Trifluoroacetyl)amino]-3-
(trifluoromethylsulfonyloxy) cyclobutanecarboxylic acid
methyl ester (10b)
Semi solid, 18 mg from 19 mg (0.079 mmol) of anti-8b (61%).
1H NMR (400 MHz, CDCl3) d 3.12–3.25 (4H, m, 2 Â CH2), 3.94
(3H, s, COCH3), 5.76–5.81 (1H, m, CH), 7.22 (1H, br s, NH).
19F NMR (376 MHz, CDCl3) d À76.44 (s, COCF3) and À75.49 (s,
SCF3).
production of [18F]12 was performed by reacting 8 mg (20
triflate precursor 10d in 1 mL of acetonitrile with 600 mCi NCA
18F]fluoride in the presence of Kryptofix 222 (5 mg, 13.3
mol)
and potassium carbonate (1 mg, 7.2 mol) at 90 °C for 10 min.
After passing a silica SepPakÒ (Waters, Inc.), the product was
hydrolyzed with 200 L of hydrazine hydrate at 75 °C for 10 min
lmol) of
[
l
l
4.10.3. anti-1-[N-(Benzoyl)amino]-3-
l
(trifluoromethylsulfonyloxy) cyclobutanecarboxylic acid
methyl ester (10c)
then neutralized with 4 N hydrochloric acid. The radiolabeled ami-
no acid [18F]12 was purified by passing the resulting solution suc-
cessively through an ion-retardation resin (BioRad) column, an
Oil, 8.2 mg from 13.5 mg (0.054 mmol) of anti-8c (40%). 1H
NMR (400 MHz, CDCl3) d 2.60–2.85 (4H, m, 2CH2), 3.84 (3H, s,
OCH3), 5.26 (1H, m, CH), 7.33 (1H, br s, NH), 7.50–8.10 (5H, m, aro-
matic). 19F NMR (376MHz, CDCl3) d À75.52 (s, SCF3).
alumina N Sep-PakÒ, an HLB Sep-PakÒ (Waters), and a 0.2
lm Gel-
man teflon filter (VWR) with sterile saline into a dose vial, which
was ready for use in in vitro and in vivo studies. The isolated radio-

1990
W. Yu et al. / Bioorg. Med. Chem. 17 (2009) 1982–1990
chemical yields were determined using a dose-calibrator (Capintec
CRC-712M). Thin layer chromatograms of the radiolabeled com-
pounds were analyzed with a Raytest System (model: Rita Star.
Germany) using the same type of silica TLC plates from Whatman.
The identity of the radiolabeled product [18F]12 was confirmed by
comparing the Rf value of the radioactive product visualized with
radiometric TLC with the Rf value of the authentic non-radioactive
form of the compound 12 visualized with ninhydrin stain (Rf = 0.4,
4:1:1 CH3CN/H2O/MeOH).
Acknowledgements
We are grateful to Dr. Bing Wang of the NMR Center at Emory
University for his assistance with NMR studies and Zhaobin Zhang
for his assistance with the biological studies. Research supported
by Nihon Medi-Physics Co., Ltd.
References and notes
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lCi
of [18F]-12 in amino acid free HBSS with or without transport
inhibitors (10 mM BCH, 10 mM MeAIB or 10 mM ACS) for 30 min
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