Synthesis of radiolabeled biphenylsulfonamide matrix metalloproteinase inhibitors as new potential PET cancer imaging agents

Article abstract of DOI:10.1016/S0960-894X(03)00382-2

Novel matrix metalloproteinase (MMP) inhibitor radiotracers, (S)-3-methyl-2-(2′,3′,4′-methoxybiphenyl-4-sulfonylamino)- butyric acid [¹¹C]methyl ester (1a-c), (S)-3-methyl-2-(2′,3′,4′-fluorobiphenyl-4-sulfonylamino)- butyric acid [¹¹C]methyl ester (1d-f), and (S)-3-methyl-2-(4′-nitrobiphenyl-4-sulfonylamino)-butyric acid [¹¹C]methyl ester (1g), a series of substituted biphenylsulfonamide derivatives, have been synthesized for evaluation as new potential positron emission tomography (PET) cancer imaging agents.

Full text of DOI:10.1016/S0960-894X(03)00382-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2217–2222
Synthesis of Radiolabeled Biphenylsulfonamide Matrix
Metalloproteinase Inhibitors as New Potential PET
Cancer Imaging Agents
Xiangshu Fei,a Qi-Huang Zheng,a,* Xuan Liu,a Ji-Quan Wang,a Hui Bin Sun,b
Bruce H. Mock,a K. Lee Stone,a Kathy D. Miller,c George W. Sledgec
and Gary D. Hutchinsa
aDepartment of Radiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
bDepartment of Anatomy, Indiana University School of Medicine, Indianapolis, IN 46202, USA
cDepartment of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
Received 27 November 2002; accepted 19 March 2003
Abstract—Novel matrix metalloproteinase (MMP) inhibitor radiotracers, (S)-3-methyl-2-(20,30,40-methoxybiphenyl-4-sulfonyl-
amino)-butyric acid [11C]methyl ester (1a–c), (S)-3-methyl-2-(20,30,40-fluorobiphenyl-4-sulfonylamino)-butyric acid [11C]methyl ester
(1d–f), and (S)-3-methyl-2-(40-nitrobiphenyl-4-sulfonylamino)-butyric acid [11C]methyl ester (1g), a series of substituted biphe-
nylsulfonamide derivatives, have been synthesized for evaluation as new potential positron emission tomography (PET) cancer
imaging agents.
# 2003 Elsevier Science Ltd. All rights reserved.
Matrix metalloproteinases (MMPs) are a family of zinc-
containing enzymes that have been shown to play a sig-
nificant physiological role in the degradation and
remodeling of connective tissues.1 Several MMPs are
expressed in cancers at much higher levels than in nor-
mal tissue and the extent of expression has been impli-
cated in tumor growth, stage, invasiveness, metastasis
and angiogenesis in both human and animal cancers.2À4
The overexpression of MMPs in cancers provides a
potential target for tumor imaging by biomedical ima-
ging technique positron emission tomography (PET).5,6
MMP inhibitors (MMPIs) can significantly reduce the
growth rate of both primary and secondary tumors and
can block the process of metastasis.7 MMPIs currently
undergoing clinical trails as therapeutic agents are
represented by two general chemical classes, namely
succinate-type structures, exemplified by Batimastat,
Marimastat and RO 32-3555, and sulfonamides,
including CGS 27023A, AG-3340 and BAY-12-9566
(Fig. 1).8À16 MMPI radiotracers labeled with carbon-
1117À20 or fluorine-1821,22 may enable non-invasive
monitoring of cancer MMP levels and cancer response
to MMPI therapy using PET imaging techniques.
In our previous work,17À20 CGS 27023A was chosen as
the parent compound for the design and synthesis of
radiolabeled MMPIs, and the carbon-11 labeling was
focused on the labeling both at the aminohydroxyl
position of hydroxamic acid CGS 27023A to prepare
[11C]methylated CGS 27023A analogues, and at the
methoxyphenyl position of hydroxamic acid CGS
25966,23,24 an analogue of CGS 27023A, to prepare
*Corresponding author. Tel.: +1-317-278-4671; fax: +1-317-278-
9711; e-mail: qzheng@iupui.edu
Figure 1. Representative MMP inhibitors.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00382-2

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X. Fei et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2217–2222
[11C]CGS 25966. In this ongoing study, we chose
biphenylsulfonamide MMPIs25 as the target molecules
and labeled these compounds with carbon-11 at the methyl
ester moiety to make their [11C]methyl esters. Here, we
report the synthesis of (S)-3-methyl-2-(X-methoxy-
biphenyl-4-sulfonylamino)-butyric acid [11C]methyl ester
(X=20, 1a; X=30, 1b; X=40, 1c), (S)-3-methyl-2-(X-
fluorobiphenyl-4-sulfonylamino)-butyric acid [11C]methyl
ester (X=20, 1d; X=30, 1e; X=40, 1f), and (S)-3-methyl-
2 - (40 - nitrobiphenyl - 4 - sulfonylamino) - butyric acid
[11C]methyl ester (1g) (Schemes 1 and 2).
give an intermediate 4-nitrobiphenylsulfonic acid with-
out further purification, which was converted to the
corresponding 40-nitrobiphenyl-4-sulfonyl chloride (7)
in refluxing thionyl chloride. The sulfonyl chloride 7
was coupled with amino acid ester 3 in the presence of
triethylamine to give unlabeled standard sample (S)-3-
methyl-2-(40-nitrobiphenyl-4-sulfonylamino)-butyric acid
methyl ester (1g). The ester 1g required trifluoroacetic
acid hydrolysis to the precursor (S)-3-methyl - 2 - (40 -
nitrobiphenyl - 4 - sulfonylamino) - butyric acid (5g) as
previously described.
The synthesis of 1a–f was presented in Scheme 1
(method A). The commercially available starting
material amino acid l-valine (2) was converted into its
ester l-valine methyl ester hydrochloride (3). The ester
3 was coupled with 4-iodophenylsulfonyl chloride in
the presence of triethylamine to give 4-iodobenzene-
sulfonamide intermediate (S)-3-methyl-2-(4-iodobenze-
nesulfonylamino)-butyric acid methyl ester (4). Utilizing
Suzuki reaction,26 iodide 4 was coupled with a variety of
methoxyl- and fluoro-substituted benzeneboronic acid
via palladium catalysis in refluxing benzene to yield
substituted biphenylsulfonamide derivatives, (S)-3-
methyl-2-(X-methoxybiphenyl-4-sulfonylamino)-butyric
acid methyl ester (X=20, 1a; X=30, 1b; X=40, 1c) and
(S)-3-methyl-2-(X-fluorobiphenyl-4-sulfonylamino)-
butyric acid methyl ester (X=20, 1d; X=30, 1e; X=40,
1f), as the unlabeled standard samples. Hydrolysis of
the methyl esters (1a–f) using trifluoroacetic acid in the
presence of concentrated hydrochloric acid gave the cor-
responding carboxylic acid derivatives, (S)-3-methyl-2-
The carboxylates (5a–g) were alkylated under basic
conditions using tetrabutylammonium hydroxide
(TBAH) with [11C]methyl triflate27,28 through 11C-O-
methylation method29À32 and isolated by solid-phase
extraction (SPE) purification29,30 to produce pure target
compounds (1a–g) in 40–60% radiochemical yields
(based on 11CO2, decay corrected to end of bombard-
ment), in 20–25 min synthesis time.33 The large polarity
difference between the acid precursor and the labeled O-
methylated product permitted the use of SPE technique
for purification of radiotracer from radiolabeling reac-
tion mixture. The reaction mixture was diluted with
NaHCO3 and loaded onto C-18 cartridge by gas pres-
sure. The cartridge column was washed with water to
remove unreacted acid precursor, non-reacted
[11C]methyl triflate and reaction solvent acetonitrile,
and then final labeled product was eluted with ethanol.
Chemical purity, radiochemical purity, and specific
radioactivity were determined by analytical HPLC
methods, which employed a Prodigy (Phenomenex)
5 mm C-18 column, 4.6 mm  250 mm; 3:1:1 CH3CN/
MeOH/20 mM, pH 6.7 KHPO-4 mobile phase, 1.5 mL/
min flow rate, and UV (240 nm) and g-ray (NaI) flow
detectors. Retention times in this HPLC system were:
RT5a=2.23 min, RT5b=2.25 min, RT5c=2.14 min,
RT5d=2.26 min, RT5e=2.32 min, RT5f=2.40 min,
RT5g=2.06 min; RT1a=5.18 min, RT1b=4.85 min,
RT1c=4.31 min, RT1d=4.69 min, RT1e=5.14 min,
RT1f=5.12 min, RT1g=3.71 min. The chemical purity
of precursors 5a–g, and standard samples 1a–g was
>95%, radiochemical purity of target radiotracers 1a–g
was >99%, and the chemical purity of target radio-
tracers 1a–g was >95%. The average (n=5–8) specific
activity of target radiotracers 1a–g was 0.6–0.8 Ci/mmol
at end-of-synthesis (EOS).
(X-methoxybiphenyl-4-sulfonylamino)-butyric
acid
(X=20, 5a; X=30, 5b; X=40, 5c) and (S)-3-methyl-2-(X-
fluorobiphenyl-4-sulfonylamino)-butyric acid (X=20, 5d;
X=30, 5e; X=40, 5f), as the precursors for radiolabeling.
Alternatively, 1g was prepared from appropriately sub-
stituted biphenyl starting material 4-nitrobiphenyl (6) as
shown in Scheme 2 (method B). Commercially available
6 was reacted with chlorosulfonic acid in chloroform to
The stereochemistry of radiolabeled biphenylsulfon-
amide analogues prepared here is different from the
stereochemistry of carbon-11 CGS 27023A analogues
prepared in our previous works17À20 and fluorine-
18MMPIs prepared by Furumoto et al.21,22 Compounds
1a–g was prepared starting from l-valine, which is
Scheme 1. Synthesis of 1a–f (method A).
Scheme 2. Synthesis of 1g (method B).

X. Fei et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2217–2222
2219
stereochemical S-form, and they are stereochemical
S-forms.25
Previously reported radiolabeled
The inhibition levels are normalized with the negative
control, which the distilled water was used to replace
tested inhibiting substance parent compound 5c or any
individual compound 1a–g, 5f or 5g. Inhibition Effect
(IE)=1ÀRelative Activity; Relative Activity=Tested
Inhibiting Substance/Negative Control. It was assumed
that Relative Activity for Negative Control=1, then
Inhibition Effect of Negative Control=0. The results
show that all these modified compounds 1a–g (IE 0.9419–
0.8825) exhibit strong inhibitory effectiveness on MMP-13
(p<0.01) with a slight variation at a level greater than or
equal to or similar to the parent compound 5c (IE 0.9380),
at the concentration of 20nM (Table 1).
MMPIs17À22 were prepared starting from d-valine,
which is stereochemical R-form, and they are stereo-
chemical R-forms. The starting stereochemistry in this
report is defined by the starting material l-valine. Based
on the organic reaction mechanism and stereochemistry
theory, the racemization of a chiral compound is due to
the conversion to its non-chiral analogue. In the syn-
thetic approaches shown in Scheme 1 and 2, there is not
any formation of non-chiral intermediates, and the
chiral center is not destroyed by any synthetic step.
Therefore, we can assume that stereochemistry is con-
served throughout the synthetic sequences and no race-
mization occurred during the various steps.
A molecule to be an effective MMP inhibitor requires a
functional group (e.g., carboxylic acid, hydroxamic
acid, and sulfhydryl, etc.) capable of chelating the
active-site zinc (II) ion; this group is called zinc binding
group or ZBG.16 A MMP inhibitor molecule has at
least one functional group, which provides a hydrogen
bond interaction with the enzyme backbone; and one or
more side chains, which undergo effective van der Waals
interactions with the enzyme subsites. It is now appar-
ent that this requirement can be satisfied by a variety of
different structural classes of MMP inhibitors (MMPIs),
which have been discovered and developed by a number
of methods including structure-based design and synth-
esis and combinatorial chemistry.16 Therefore, it was
assumed that the structural modification of carboxylic
acid derivatives 5a–g to methylated ester derivatives 1a–
g, is not likely to cause a major change in their inhibi-
tory properties, because the ZBG of O-methylated car-
boxylic acid has the same function as the ZBG of
carboxylic acid to coordinate with zinc at the active site.
Moreover, the methylation modification may increase
their biological activities, in which in vivo biodistribution
study of methyl ester prodrug showed a better uptake in
tumor and higher tumor/organ uptake ratios in compar-
ison with its acid drug.22 The in vitro biological assay of
the modified compounds (1a–g) in comparison with the
parent compound 5c also provides the evidence to sup-
port this assumption as indicated in Table 1.
Compounds 4, 5 and 1 have analytical data such as mp,
1H NMR and MS in agreement with the indicated
structures.34
Compound 5c was chosen as the parent compound,
since it is a potent MMP inhibitor for several MMP
subtypes such as MMP-1 (IC50, 1.5 mM), MMP-2 (IC50,
0.003 mM), MMP-3 (IC50, 0.008 mM), MMP-7
(IC50, 7.2 mM), MMP-9 (IC50, 2.2 mM), and MMP-13
(IC50, 0.006 mM), and compounds 5f and 5g have similar
activities for these MMP subtypes.25 In order to exam-
ine relative inhibition effects of the modified compounds
methyl esters (1a–g) in comparison with the parent
compound 5c and reference compounds 5f and 5g on
MMP activity, a fibril degradation assay17,18,35 was
performed using fluorogenic substrates specific to
MMP-13. MMP-13 (collagenase) specific fluorogenic
substrates (Molecular Probe) (12.5 mM) and p-amino-
phenylmercuric acetate (APMA) activated human
MMP-13 (Calbiochem) (100 ng) were incubated at room
temperature for 2 h in the presence of individual mod-
ified compound or parent compound or reference com-
pounds (20 nM in DMSO) in a reaction mixture (total
volume of 100 mL) consisting of Tris–HCl (500 mM, pH
7.6), NaCl (1.5 M), CaCl2 (50 mM) and sodium azide
(2 mM). Sodium azide is a stabilizer for MMP enzyme
and substrate and a potent quencher for singlet oxygen,
which led to the biosynthesis of MMP enzyme and
substrate, and the changes of the structures. Based on
the chemical structures of 1a–g, 5c, 5f and 5g, the
methyl ester analogues 1a–g are stable as the carboxylic
acid analogues 5c, 5f and 5g in the media at room tem-
perature throughout the entire time of the assay. The
esters would not hydrolyze in the media, and the activ-
ity observed is due to the esters not the acids. Digestion
of the intensively fluorophore labeled substrate by
MMP-13 would liberate fluorophores from a quenching
effect of nearby fluorophores. Fluorescent intensity was
measured by FluoroMax-2 spectrofluorometer (Instru-
ments S.A., Inc.). The absorption wavelength and the
emission wavelength were set to 382 and 441 nm,
respectively. A Student’s t-test was conducted to exam-
ine the statistical significance of the inhibition effects,
and p values less than 0.05 were considered statistically
significant, in three experiments. An assay without pre-
sence of any to be tested compound or parent com-
pound but distilled water was set as a negative control.
Our idea to make carbon-11 labeled methyl ester pro-
drug, rather than carbon-11 labeled acid drug, was
Table 1. Inhibition effects of biphenylsulfonamide MMP inhibitors
on MMP-13 activity
Compd (concn 20 nM)
Inhibition effect (p<0.01)
Negative control
0
5c
1a
1b
1c
5f
1d
1e
1f
0.9380Æ0.014a
0.9419Æ0.014a
0.9389Æ0.009a
0.9353Æ0.015a
0.9180Æ0.017a
0.9225Æ0.009a
0.9194Æ0.012a
0.9102Æ0.011a
0.8967Æ0.021a
0.8825Æ0.023a
5g
1g
aIt indicated the standard deviation was in three experiments and the
inhibition effect was significant different from negative control based
on the Student’s t-test (p<0.01).

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X. Fei et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2217–2222
based on that (1) it is easy to make the methyl ester
prodrug, and it is hard to make the acid drug, from the
radiochemistry point; (2) the carboxylate ester is
equivalent to carboxylic acid to bind zinc in structure–
activity relationship (SAR) as stated in the literature;16
and (3) the methyl ester prodrug showed a better uptake
in tumor and higher tumor/organ uptake ratios than its
acid drug in the in vivo biodistribution studies as indi-
cated in the literature,22 which has been recently pub-
lished as a full paper by Furumoto et al.,36 so more
details are known about the study. Radio-thin-layer-
chromatographic (radio-TLC) analysis of radiolabeled
methyl ester prodrug metabolites revealed that admi-
nistered methyl ester prodrug was easily converted to
the parent acid drug in vivo and accumulated in tumor
tissue. Therefore, it was concluded radiolabeled methyl
ester is suitable as the prodrug of radiolabeled acid with
potent efficacy. A reviewer of this work criticized that it
is still important to demonstrate the stability of the
methyl ester compounds in the in vitro assay media,
since the reviewer concerned that though azide is pre-
sent in the assay to stabilize the enzyme and the sub-
strate, it is also a potent nucleophile and could catalyze
ester hydrolysis. We feel in the buffer media consisting
of Tris–HCl (500 mM, pH 7.6) at room temperature, the
conditions are moderate and near neutral, azide should
be present as a stabilizer first, although it is a nucleo-
phile, and methyl ester should be stable in these condi-
tions. Following the reviewer’s suggestion, we used
HPLC methods37 to monitor the behavior of the methyl
esters in the assay media, which two different HPLC
systems (acid 5c in the media and methyl ester 1c in the
media) were employed. The results showed the methyl
esters are not converted to the acids or any other deri-
vative in the assay media in light of their retention times
aforementioned in the HPLC chromatograms. We con-
cluded that the methyl esters are stable and would not
hydrolyze to acids in the in vitro inhibition assay as
indicated by HPLC analysis; the methyl esters have
similar inhibition of enzyme activity in comparison with
the acids; and the methyl ester prodrug was converted
to the acid drug in the in vivo metabolites analysis as
shown by radio-TLC.36
the National Institutes of Health/National Cancer
Institute grant P20CA86350 (to G.D.H.), the Indiana
21st Century Research and Technology Fund (to
G.D.H.), and the Lilly Endowment Inc. [to Indiana
Genomics Initiative (INGEN) of Indiana University].
We thank Barbara Glick-Wilson and Michael Sullivan
for the efforts in producing carbon-11 precursors. The
referees’ criticisms and editor’s comments for the revi-
sion of the manuscript are greatly appreciated.
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In summary, the synthetic procedures that provide new
biphenylsulfonamide MMP inhibitor radiotracers 1a–g
have been developed. Preliminary findings from in vitro
biological assay indicate the synthesized analogues have
strong inhibitory effectiveness on MMP-13 in compar-
ison with the parent compound 5c. These results war-
rant further evaluation of these radiotracers as new
potential PET cancer imaging agents in vivo.
Acknowledgements
This work was partially supported by the Susan G.
Komen Breast Cancer Foundation grant IMG 2000 837
(to Q.H.Z.), the Indiana University American Cancer
Society (ACS) Institutional Grant Committee grant
IRG-84-002-17 (to Q.H.Z.), the Indiana University
Cores Centers of Excellence in Molecular Hematology
(CCEMH) pilot and feasibility (P/F) grant (to Q.H.Z.),
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3H, CH3CH), 0.82–0.84 (d, J=6.6 Hz, 3H, CH3CH), 1.88–
1.99 (m, 1H, ðCH3Þ2CH), 3.52–3.58 (dd, J=5.1, 10.3 Hz, 1H,
CHN), 3.83 (s, 3H, CH3OPh), 5.21–5.25 (d, J=10.3 Hz, 1H,
NH), 6.98–7.01 (d, J=8.1 Hz, 1H, H-PhOMe), 7.25 (s, 1H, H-
PhOMe), 7.27–7.30 (d, J=8.1 Hz, 1H, H-PhOMe), 7.38–7.43
(t, J=8.1 Hz, 1H, H-PhOMe), 7.81–7.84 (d, J=8.8 Hz, 2H, H-
Ph), 8.07–8.10 (d, J=8.8 Hz, 2H, H-Ph). LRMS (CI, CH4, m/
z): 363.1 (M+, 25%), 183.1 (100%). HRMS (CI, CH4, m/z):
calcd for C18H21NO5S 363.1140, found 363.1141. 5c, a gray
solid, 99% yield, mp 169–170 ꢀC. 1H NMR (300 MHz,
DMSO-d6): d 0.78–0.80 (d, J=6.6 Hz, 3H, CH3CH), 0.82–0.84
(d, J=6.6 Hz, 3H, CH3CH), 1.88–2.00 (m, 1H, ðCH3Þ2CH),
3.51–3.55 (d, J=5.1, 9.7 Hz, 1H, CHN), 3.80 (s, 3H,
CH3OPh), 5.14–5.18 (d, J=10.3 Hz, 1H, NH), 7.03–7.06 (d,
J=8.1 Hz, 2H, H-Ph), 7.67–7.70 (d, J=8.1 Hz, 2H, H-Ph),
7.76–7.78 (d, J=9.5 Hz, 1H, H-Ph), 7.79 (s, 2H, H-Ph), 8.01–
8.05 (d, J=9.5 Hz, 1H, H-Ph). 5d, a gray solid, 93% yield, mp
141–143 ꢀC. H NMR (300 MHz, DMSO-d6): d 0.98–1.00 (d,
1
31. Zheng, Q.-H.; Mulholland, G. K. J. Labelled Cpd. Radio-
pharm. 1997, 40 (Suppl. 1), 551.
J=6.6 Hz, 3H, CH3CH), 1.02–1.03 (d, J=6.6 Hz, 3H,
ðCH3Þ2CH), 2.10–2.20 (m, 1H, ðCH3Þ2CH), 3.74–3.79 (dd,
J=6.0, 9.5 Hz, 1H, CHN), 5.07–5.11 (d, J=10.3 Hz, 1H, NH),
7.51–7.58 (m, 2H, H-FPh), 7.66–7.68 (m, 1H, H-FPh), 7.74–
7.79 (t, J=8.1 Hz, 1H, H-FPh), 7.91–7.93 (d, J=8.1 Hz, 2H,
H-Ph), 8.04–8.07 (d, J=8.1 Hz, H-Ph). LRMS (CI, CH4, m/z):
351.1 (M+, 2.2%), 171.0 (100%). HRMS (CI, CH4, m/z):
calcd for C17H18FNO4S 351.0941, found 351.0934. 5e, a gray
solid, 97% yield, mp 147–148 ꢀC. 1H NMR (300 MHz,
DMSO-d6): d 0.98–1.00 (d, J=6.6 Hz, 3H, CH3CH), 1.01–1.03
(d, J=6.6 Hz, 3H, CH3CH), 2.02–2.20 (m, 1H, ðCH3Þ2CH),
3.72–3.77 (dd, J=5.1, 10.3 Hz, 1H, CHN), 5.10–5.13 (d,
J=10.3 Hz, 1H, NH), 7.43–7.48 (t, J=7.4 Hz, 1H, H-FPh),
7.72–7.85 (m, 3H, H-FPh), 8.02–8.11 (dd, J=8.1 Hz, 3H, H-
Ph), 9.29–8.32 (d, J=8.8 Hz, 1H, H-Ph). LRMS (CI, CH4, m/
z): 351.1 (M+, 1%), 171.0 (100%). HRMS (CI, CH4, m/z):
calcd for C17H18FNO4S 351.0941, found 351.0938. 5f, a gray
solid, 97% yield, mp 161–163 ꢀC. 1H NMR (300 MHz,
DMSO-d6): d 0.78–0.80 (d, J=6.6 Hz, 3H, CH3CH), 0.82–0.84
(d, J=6.6 Hz, 3H, CH3CH), 1.90–1.98 (m, 1H, ðCH3Þ2CH),
3.52–3.57 (dd, J=5.9, 9.6 Hz, 1H, CHN), 5.13–5.16 (d,
J=10.3 Hz, 1H, NH), 7.30–7.36 (t, J=8.8 Hz, 2H, H-FPh),
7.43–7.48 (t, J=8.8 Hz, 2H, H-FPh), 7.76–7.81 (m, 3H, H-Ph),
8.07–8.10 (d, J=9.6 Hz, 1H, H-Ph). 5g, a yellow solid, 99%
yield, mp 147–148 ꢀC. 1H NMR (300 MHz, DMSO-d6): d
0.79–0.81 (d, J=6.6 Hz, 3H, CH3CH), 0.82–0.84 (d,
J=6.6 Hz, 3H, CH3CH), 1.90–1.98 (m, 1H, ðCH3Þ2CH), 3.54–
3.59 (dd, J=5.3. 10.3 Hz, 1H, CHN), 5.73–5.76 (d, J=9.7 Hz,
1H, NH), 7.88–7.7.91 (d, J=8.8 Hz, 2H, H-Ph), 7.91–7.96 (d,
J=8.8 Hz, 2H, H-Ph), 8.00–8.03 (d, J=8.8 Hz, 2H, H-Ph),
8.32–8.35 (d, J=8.8 Hz, 2H, H-Ph). 1a, a white solid, 88%
32. Zheng, Q.-H.; Winkle, W.; Carlson, K.; Mulholland, G. K.
J. Labelled Cpd. Radiopharm. 1997, 40 (Suppl. 1), 548.
33. Typical experimental procedure for the radiosynthesis of
[11C] 1a–g. Acid precursor (5a–g) (0.6–1.0 mg) was dissolved
in CH3CN (300 mL). To this solution was added tetra-
butylammonium hydroxide (TBAH) (2–3 mL, 1 M solution in
methanol). The mixture was transferred to a small volume,
three-neck reaction tube. [11C]methyl triflate was passed into
the air-cooled reaction tube at À15 to À20 ꢀC, which was
generated by a Venturi cooling device powered with 100 psi
compressed air, until activity reached a maximum (ꢁ3 min),
then the reaction tube was heated at 70–80 ꢀC for 3 min. The
contents of the reaction tube were diluted with 0.1 M NaHCO3
(1 mL). This solution was passed onto a semi-prep C-18 silica
guard cartridge column 1 Â 1 cm, which was obtained from E.
S. Industries, Berlin, NJ, USA, and part number 300121-C18-
BD10 m, by gas pressure. The cartridge was washed with H2O
(2 Â 3 mL), and the aqueous washing was discarded. The
product was eluted from the column with EtOH (2 Â 3 mL),
and then passed onto a rotatory evaporator. The solvent was
removed by evaporation under high vacuum. The labeled
product (1a–g) was formulated with 50 mM NaH2PO4, whose
volume was dependent upon the use of the labeled product
(1a–g) in tissue biodistribution studies (ꢁ6 mL, 3 Â 2 mL) or
in micro-PET imaging studies (1–3 mL) of the breast cancer
athymic mice, sterile-filtered through a sterile vented Millex-
GS 0.22 mm cellulose acetate membrane and collected into a
sterile vial. Total radioactivity was assayed and total volume
was noted. The overall synthesis time was ꢁ20 min. The decay
corrected yield, from 11CO2, was 40–60%.
yield, mp 78–79 ꢀC. H NMR (300 MHz, CDCl3): d 0.90–0.92
1
34. 4, white solid, 97% yield, mp 100–101 ꢀC. 1H NMR
(300 MHz, CDCl3): d 0.86–0.88 (d, J=6.6 Hz, 3H, CH3CH),
0.95–0.97 (d, J=6.6 Hz, 3H, CH3CH), 2.01–2.10 (m, 1H,
ðCH3Þ2CH), 3.48 (s, 3H, CO2CH3), 3.71–3.76 (dd, J=5.1,
9.6 Hz, 1H, CHN), 5.14–5.18 (d, J=10.3 Hz, 1H, NH), 7.53–
7.55 (d, J=8.1 Hz, 2H, H-Ph), 7.84–7.87 (d, J=8.1 Hz, 2H, H-
Ph). 5a, a gray solid, 98% yield, mp 178–179 ꢀC. 1H NMR
(d, J=6.6 Hz, 3H, CH3CH), 0.97–0.99 (d, J=6.6 Hz, 3H,
CH3CH), 1.99–2.11 (m, 1H, ðCH3Þ2CH), 3.43 (s, 3H,
CO2CH3), 3.76–3.79 (dd, J=5.1, 10.3 Hz, 1H, CHN), 3.86 (s,
3H, CH3OPh), 5.10–5.13 (d, J=10.3 Hz, 1H, NH), 6.99–7.02
(d, J=8.8 Hz, 1H, H-Ph), 7.03–7.08 (dd, J=8.1, 8.8 Hz, 1H,
H-Ph), 7.28–7.31 (m, 2H, H-Ph), 7.63–7.66 (d, J=8.8 Hz, 2H,
H-Ph), 7.82–7.85 (d, J=8.8 Hz, 2H, H-Ph). LRMS (CI, CH4,
m/z): 377.1 (M+, 17%), 318.1 (100%). HRMS (CI, CH4, m/z):
calcd for C19H23NO5S 377.1297, found 377.1303. 1b, a white
solid, 89% yield, mp 93–95 ꢀC. 1H NMR (300 MHz, CDCl3): d
0.88–0.90 (d, J=6.6 Hz, 3H, CH3CH), 0.96–0.98 (d,
J=6.6 Hz, 3H, CH3CH), 1.99–2.00 (m, 1H, ðCH3Þ2CH), 3.43
(s, 3H, CO2CH3), 3.76–3.81 (dd, J=5.1, 10.3 Hz, 1H, CHN),
3.88 (s, 3H, CH3OPh), 5.12–5.15 (d, J=10.1 Hz, 1H, NH),
6.95–6.98 (dd, J=1.8, 8.4 Hz, 1H, H-PhOMe), 7.11–7.12 (d,
J=1.6 Hz, 1H, H-PhOMe), 7.37–7.42 (t, J=8.1 Hz, 2H, H-
PhOMe), 7.68–7.71 (d, J=8.8 Hz, 2H, H-Ph), 7.87–7.90 (d,
J=8.8 Hz, 2H, H-Ph). LRMS (CI, CH4, m/z): 377.1 (M+,
15%), 318.0 (100%). HRMS (CI, CH4, m/z): calcd for
(300 MHz, DMSO-d6):
d 0.79–0.81 (d, J=6.6 Hz, 3H,
CH3CH), 0.82–0.84 (d, J=6.6 Hz, 3H, CH3CH), 1.90–2.02 (m,
1H, ðCH3Þ2CH), 3.53–3.58 (dd, J=5.1, 10.3 Hz, 1H, CHN),
3.78 (s, 3H, CH3OPh), 5.11–5.15 (d, J=10.3 Hz, 1H, NH),
7.02–7.07 (t, J=8.1 Hz, 1H, H-PhOMe), 7.12–7.14 (d,
J=8.1 Hz, 1H, H-PhOMe), 7.30–7.33 (d, J=7.4 Hz, 1H, H-
PhOMe), 7.36–7.41 (t, J=7.4 Hz, 1H, H-PhOMe), 7.61–7.65
(d, J=8.1 Hz, 2H, H-Ph), 7.76–7.79 (d, J=8.1 Hz, 2H, H-Ph).
LR/MS (CI, CH4, m/z): 363.1 (M+, 29%), 247.0 (100%).
HRMS (CI, CH4, m/z): calcd for C18H21NO5S 363.1140,
found 363.1157. 5b, a gray solid, 99% yield, mp 156–157 ꢀC.
1H NMR (300 MHz, DMSO-d6): d 0.79–0.81 (d, J=6.6 Hz,

2222
X. Fei et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2217–2222
C19H23NO5S 377.1297, found 377.1302. 1c, a white solid, 93%
yield, mp 156–157 ꢀC. 1H NMR (300 MHz, CDCl3): d 0.88–
0.90 (d, J=6.6 Hz, 3H, CH3CH), 0.96–0.98 (d, J=6.6 Hz, 3H,
CH3CH), 2.02–2.11 (m, 1H, ðCH3Þ2CH), 3.42 (s, 3H,
CO2CH3), 3.75–3.80 (dd, J=5.1, 10.3 Hz, 1H, CHN), 3.87 (s,
3H, CH3OPh), 5.11–5.15 (d, J=9.7 Hz, 1H, NH), 6.99–7.02
(d, J=8.1 Hz, 2H, H-Ph), 7.54–7.57 (d, J=8.8 Hz, 2H, H-Ph),
7.65–7.67 (d, J=8.1 Hz, 2H, H-Ph), 7.84–7.87 (d, J=8.1 Hz,
2H, H-Ph). LRMS (CI, CH4, m/z): 377.1 (M+, 28%), 183.1
(100%). HRMS (CI, CH4, m/z): calcd for C19H23NO5S
377.1297, found 377.1308. 1d, a white solid, 94% yield, mp
82–83 ꢀC. 1H NMR (300 MHz, CDCl3): d 0.89–0.91 (d,
J=6.6 Hz, 3H, CH3CH), 0.97–0.99 (d, J=6.6 Hz, 3H,
CH3CH), 1.98–2.12 (m, 1H, ðCH3Þ2CH), 3.43 (s, 3H,
CO2CH3), 3.76–3.81 (dd, J=5.1, 10.1 Hz, 1H, CHN),
5.13–5.16 (d, J=10.1 Hz, NH), 7.15–7.36 (m, 2H, H-FPh),
7.38–7.46 (m, 2H, H-FPh), 7.66–7.68 (d, J=8.1 Hz, 2H,
H-Ph), 7.88–7.91 (d, J=8.1 Hz, 2H, H-Ph). LRMS (CI,
CH4, m/z): 366.1 (M++1, 1%), 171.1 (100%). HRMS
(CI, CH4, m/z): calcd for C18H20FNO4S 365.1097, found
365.1173. 1e, a white solid, 87% yield, mp 106–107 ꢀC.
1H NMR (300 MHz, CDCl3): d 0.88–0.90 (d, J=6.6 Hz, 3H,
CH3CH), 0.97–0.99 (d, J=6.6 Hz, 3H, CH3CH), 2.01–2.10 (m,
1H, ðCH3Þ2CH), 3.44 (s, 3H, CO2CH3), 3.77–3.82 (dd, J=5.1,
10.3 Hz, 1H, CHN), 5.18–5.21 (d, J=10.3 Hz, 1H, NH), 7.05–
7.16 (t, J=8.1 Hz, 1H, H-FPh), 7.27–7.32 (dt, J=2.2, 9.6 Hz,
1H, H-FPh), 7.37–7.46 (m, 2H, H-FPh), 7.67–7.70 (d,
J=8.8 Hz, 2H, H-Ph), 7.89–7.92 (d, J=8.8 Hz, 2H, H-Ph).
LRMS (CI, CH4, m/z): 365.1 (M+, 1%), 171.1 (100%).
HRMS (CI, CH4, m/z): calcd for C18H20FNO4S 365.1097,
found 365.1097. 1f, a white solid, 97% yield, mp 127–128 ꢀC.
1H NMR (300 MHz, CDCl3): d 0.88–0.90 (d, J=6.6 Hz, 3H,
CH3CH), 0.97–0.99 (d, J=6.6 Hz, 3H, CH3CH), 2.01–2.11 (m,
1H, ðCH3Þ2CH), 3.44 (s, 3H, CO2CH3), 3.76–3.82 (dd, J=5.1,
10.3 Hz, 1H, CHN), 5.09–5.13 (d, J=10.3 Hz, 1H, NH), 7.57–
7.20 (t, J=8.8 Hz, 2H, H-FPh), 7.55–7.59 (m, 2H, H-FPh),
7.64–7.67 (d, J=8.1 Hz, 2H, H-Ph), 7.87–7.90 (d, J=8.8 Hz,
2H, H-Ph). LRMS (CI, CH4, m/z): 365.1 (M+, 3.3%), 171.1
(100%). HRMS (CI, CH4, m/z): calcd for C18H20FNO4S
365.1097, found 365.1078. 1g, a light yellow solid, 95%
yield, mp 156–157 ꢀC. 1H NMR (300 MHz, CDCl3):
d
0.89–0.91 (d, J=7.3 Hz, 3H, CH3CH), 0.97–1.00 (d,
J=7.3 Hz, 3H, CH3CH), 2.04–2.14 (m, 1H, ðCH3Þ2CH),
3.48 (s, 3H, CO2CH3), 3.80–3.85 (dd, J=5.1, 10.3 Hz, 1H,
CHN), 5.21–5.24 (d, J=10.3 Hz, 1H, NH), 7.74–7.78 (m,
4H, H-Ph), 7.95–7.98 (d, J=8.1 Hz, 2H, H-Ph), 8.33–8.36
(d, J=8.8 Hz, 2H, H-Ph). LRMS (CI, CH4, m/z): 393.0
(M+, 0.3%), 333.1 (100%). HRMS (CI, CH4, m/z): calcd
for C16H17N2O4S (M+ÀCO2CH3) 333.0908, found
333.0920.
35. Nagase, H.; Fields, C. G.; Fields, G. B. J. Biol. Chem.
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