Synthesis of MMP inhibitor radiotracers [11C]methyl-CGS 27023A and its analogs, new potential PET breast cancer imaging agents

Article abstract of DOI:10.1002/jlcr.570

[¹¹C]Methyl-CGS 27023A (1a) and its analogs [¹¹C]methyl-2-picolyl-CGS 27023A (1b), [¹¹C]methyl-benzyl-CGS 27023A (1c), [¹¹C]methyl-2-nitro-CGS 27023A (1d), [¹¹C]methyl-3-nitro-CGS 27023A (1e), and [¹¹C]methyl-4-nitro-CGS 27023A (1f), novel radiolabeled matrix metalloproteinase (MMP) inhibitors, have been synthesized for evaluation as new potential positron emission tomography (PET) breast cancer imaging agents. The appropriate precursors for radiolabeling were obtained in four to five steps from starting material amino acid D-valine with moderate to excellent chemical yields. Precursors were labeled by [¹¹C]methyl triflate through ¹¹C-O-methylation method at the aminohydroxyl position under basic conditions and isolated by solid-phase extraction (SPE) purification to produce pure target compounds in 40-60% radiochemical yields (decay corrected to end of bombardment), in 20-25 min synthesis time. Copyright

Full text of DOI:10.1002/jlcr.570

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 449–470.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.570
Research Article
Synthesis of MMP inhibitor radiotracers
[11C]methyl-CGS 27023A and its analogs,
new potential PET breast cancer imaging
agents
Xiangshu Fei1, Qi-Huang Zheng*,1, Gary D. Hutchins1, Xuan Liu1,
K. Lee Stone1, Kathy A. Carlson1, Bruce H. Mock1,
Wendy L. Winkle1, Barbara E. Glick-Wilson1, Kathy D. Miller2,
Rose S. Fife2, George W. Sledge2, Hui Bin Sun3 and
Raymond E. Carr2
1 Departments of Radiology, Indiana University School of Medicine,
Indianapolis, IN 46202, USA
2 Department of Medicine, Indiana University School of Medicine, Indianapolis,
IN 46202, USA
3 Department Anatomy, Indiana University School of Medicine, Indianapolis,
IN 46202, USA
Summary
[11C]Methyl-CGS 27023A (1a) and its analogs [11C]methyl-2-picolyl-CGS
27023A (1b), [11C]methyl-benzyl-CGS 27023A (1c), [11C]methyl-2-nitro-CGS
27023A (1d), [11C]methyl-3-nitro-CGS 27023A (1e), and [11C]methyl-4-nitro-
CGS 27023A (1f), novel radiolabeled matrix metalloproteinase (MMP)
inhibitors, have been synthesized for evaluation as new potential positron
*Correspondence to: Q-H. Zheng, Department of Radiology, Indiana University School of
Medicine, 975 West Walnut Street, Room 028C, Indianapolis, IN 46202-5121, USA.
E-mail: qzheng@iupui.edu
Contract/grant sponsor: Susan G. Komen Breast Cancer Foundation; contract/grant number:
IMG 2000 837.
Contract/grant sponsor: Indiana University American Cancer Society Institutional Grant
Committee; contract/grant number: IRG-84-002-17
Contract/grant sponsor: National Institute of Health/National Cancer Institute; contract/grant
number: P20CA86350
Contract/grant sponsor: Indiana 21st Century Research & Technology Fund
Copyright # 2002 John Wiley & Sons, Ltd.
Received 26 September 2001
Revised 30 November 2001
Accepted 3 January 2002

450
X. FEI ET AL.
emission tomography (PET) breast cancer imaging agents. The appropriate
precursors for radiolabeling were obtained in four to five steps from
starting material amino acid d-valine with moderate to excellent chemical
yields. Precursors were labeled by [11C]methyl triflate through 11C–O-
methylation method at the aminohydroxyl position under basic conditions
and isolated by solid-phase extraction (SPE) purification to produce pure
target compounds in 40–60% radiochemical yields (decay corrected to end
of bombardment), in 20–25 min synthesis time. Copyright # 2002 John Wiley
& Sons, Ltd.
Key Words: matrix metalloproteinase inhibitor; breast cancer; radiotracer;
carbon-11; positron emission tomography; [11C]methyl-CGS 27023A
1. Introduction
Breast cancer is the most commonly diagnosed cancer of women and the
second leading cause of cancer deaths among women. Experiments in
animal models of breast cancer have shown that matrix metalloprotei-
nase (MMP) inhibitors can significantly reduce the growth rate of both
primary and secondary tumors and can block the process of metastasis.1
MMPs are a family of zinc-containing enzymes responsible for the
breakdown of connective tissue proteins. The ability of these enzymes,
present in high concentrations in many tumor types, to modify the
extracellular matrix (ECM) allows tumor cells to establish metastases
and undergo rapid growth.2 The overexpression of MMPs in breast
tumors provides a target for positron emission tomography (PET)
imaging of breast cancer.3 Radiolabeled MMP inhibitor analogs labeled
with positron emitting radionuclides carbon-11 or fluorine-18 may
enable non-invasive monitoring of breast cancer MMP levels and breast
cancer response to MMP inhibitor therapy using PET imaging
techniques.
There has been a great interest in the design and development of
MMP inhibitors (MMPIs) as therapeutic agents.4 The development of
specific bioavailable inhibitors of the MMPs would aid in delineating
the role of these enzymes in normal and disease states.5,6 Several MMP
inhibitors have been entering human trials such as Batimastat (BB-94,
British Biotech),7 Marimastat (BB2516, British Biotech),8 CGS 27023A
(Novartis),9 CT-1746 (Celltech Therapeutics)10 and AG-3340
(Agouron).11 Among these MMP inhibitors, CGS 27023A possesses
the combination of favorable pharmacokinetics and nanomolar IC50
potencies for several MMP subtypes,6,9 and aminohydroxyl and/or
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

SYNTHESIS OF MMP INHIBITOR RADIOTRACERS
451
Figure 1. Structure of [11C]CGS 27023A and [18F]CGS 27023A analog
O-methyl positions amenable to labeling with carbon-11. These same
properties are often beneficial in a diagnostic radiotracer.
A number of carbon-11 and fluorine-18 labeled CGS 27023A analogs
have been developed in this laboratory.12 [11C]CGS 27023A and
[18F]CGS 27023A analog (Figure 1) are our initial target radiotracers,
however, the low radiochemical yields, long reaction time and
complicated purification methods in their radiochemical syntheses have
prevented the reliable routine production and automation of these
tracers. In order to explore novel possible radiolabeled analogs of CGS
27023A as alternative candidate agents, we have synthesized
[11C]methyl-CGS 27023A (1a) and its analogs [11C]methyl-2-picolyl-
CGS 27023A (1b), [11C]methyl-benzyl-CGS 27023A (1c), [11C]methyl-2-
nitro-CGS 27023A (1d), [11C]methyl-3-nitro-CGS 27023A (1e), and
[11C]methyl-4-nitro-CGS 27023A (1f).
Results and discussion
The requirement for a molecule to be an effective inhibitor of the MMP
class of enzymes is a functional group (e.g. carboxylic acid, hydroxamic
acid, and sulfhydryl, etc.) capable of chelating the active-site zinc (II)
ion (this will be referred to as zinc binding group or ZBG), 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.4 It is now apparent
that this requirement can be satisfied by a variety of different structural
classes of MMP inhibitors which have been discovered by a number of
methods including structure-based design13–29 and combinatorial
chemistry.30,31 The mechanism-based inhibition of MMPs by MMPIs
and the reaction mechanism for proteolysis by MMPs has been
rationalized by the structure-based information of MMPI-MMP
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

452
X. FEI ET AL.
complex.26 Therefore, we have assumed that the structural modification
of CGS 27023A to methylated CGS 27023A (1a) and its analogs (1b–f),
is not likely to cause a major change in their inhibitory properties,
because the ZBG of O-methylated hydroxamic acid has the same
function as the ZBG of hydroxamic acid to coordinate with zinc at the
active site. The evidence to support this assumption is provided by the
biological assay of compounds 1a–f in comparison with CGS 27023A
(6a).
CGS 27023A is a potent MMP inhibitor for several MMP subtypes
such as MMP-3 (stromelysin) and MMP-1 (collagenase). In order to
examine effects of modified compounds methyl-CGS 27023A and its
analogs (1a–f) on MMP activity, we performed a fibril degradation
assay32 using fluorogenic substrates specific to MMP-1. Two hundred
nanomolar of individual modified compound was incubated at room
temperature for 2 h with p-aminophenylmercuric acetate (APMA)
activated 100 ng of human MMP-1 and 12.5 mM MMP-1 specific
fluorogenic substrates in a total 100 ml reaction mixture consisting of
500 mM Tris–HCl (pH 7.6), 1.5 M NaCl, 50 mM CaCl2 and 2 mM
sodium azide. The substrate was intensively labeled with fluorophores
and digestion of substrate by MMP-1 would liberate fluorophores from
a quenching effect of nearby fluorophores. Fluorescent intensity was
measured by FluoroMax-2 spectrofluorometer (Instruments S.A., Inc.).
The absorption and emission wavelengths were set to 382 and 441 nm,
respectively. To examine the statistical significance of the inhibition
effects, a student’s t-test was conducted and p-values 50.05 were
considered statistically significant. Distilled water without any addition
of testing compound was used as a negative control. The results show
that all these modified compounds 1a–f exhibit strong inhibitory
effectiveness on MMP-1 ( p50:01) at a level similar to CGS 27023A (6a)
(Figure 2), although their inhibition effect levels are slightly varied.
New MMP inhibitor radiotracers 1a–f were synthesized as shown in
Schemes 1 and 2.
The commercially available starting material amino acid d-valine (2)
was converted into its ester (3). The ester (3) was coupled with various
sulfonyl chlorides to give the corresponding sulfonylamino esters (4)
and (7). The esters (4) and (7) were reacted with different picolyl
chlorides or benzyl bromide to provide the key intermediates (5) and (8).
Hydroxamic acids were synthesized using two methods (A and B).9,13,33
In Method A, esters (5) were reacted directly with hydroxylamine to
produce hydroxamic acids CGS 27023A (6a), 2-picolyl-CGS 27023A
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

SYNTHESIS OF MMP INHIBITOR RADIOTRACERS
453
Figure 2. Relative inhibition effects of modified compounds on MMP-1 activity
determined by a fibril degradation assay. The inhibition levels are normalized with
a negative control, which the distilled water was used to replace tested inhibiting
substance CGS 27023A or any modified compound. Inhibition effect=1-relative
activity; relative activity = tested inhibiting substance/negative control; we
assumed that relative activity for negative control = 1, then inhibition effect of
negative control=0. The bar on each column indicates standard deviation in three
experiments. The asterisk indicates statistically significant difference (p50:01)
from the negative control
(6b), and benzyl-CGS 27023A (6c). In Method B, we adapted a
synthetic method under acidic condition, because the nitro compounds
(8) are unstable under the condition of strong base sodium methoxide
(NaOMe); in this method, the conversion of esters (8) to hydroxamic
acid includes the hydrolysis of esters (8) into acids followed by coupling
with O-tert-butylhydroxylamine hydrochloride in the presence of
N-[(dimethylamino)propyl]-N-ethylcarbodiimide hydrochloride (EDC),
1-hydroxybenzotrizole (HOBT) and N-methylmorphorine (NMM) to
give t-butylated hydroxamic acids (9), and the deprotection of 9 by
hydrogen chloride gas in dichloroethane (DCE) to give hydroxamic
acids 2-nitro-CGS 27023A (10d), 3-nitro-CGS 27023A (10e), and
4-nitro-CGS 27023A (10f). The overall chemical yields of hydroxamic
acids from d-valine were moderate to excellent.
The hydroxamic acids (6a–c, 10d–f) were stirred with methyl triflate
at room temperature under basic condition to give the standard samples
unlabeled 1a–f. The final products were characterized by analytical data
1
mp, HNMR and MS. The alkylation of the hydroxylamine on the
nitrogen position is a potential competing reaction, however, the
1
evidence provided by the HNMR data (there are d 3.63–3.72 for
CH3ONH, and there are not d ꢀ2–3 for HONCH3) of methylated CGS
27023A analogs shows that the compounds are methylated on the
oxygen position of the hydroxylamine. These results are consistent with
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

454
X. FEI ET AL.
OMe
O
O
O
p-MeOPhSO2Cl
iPr2NEt, CH3CN
NH
NH2
2 HCl
NH
SOCl2, MeOH
MeO
HO
MeO
S
O
O
4
2
3
Picolyl chloride or Benzyl chloride
K2CO3, DMF
OMe
OMe
O
R
O
R
11CH3OTf, TBAH
CH3CN
NH2OH/HCl
NaOMe/MeOH
HO
N
N
MeO
NH
S
S
O
O
O
O
5a, R=3-Picolyl
5b, R=2-Picolyl
5c, R=Benzyl
6a, R=3-Picolyl, CGS 27023A
6b, R=2-Picolyl, 2-Picolyl-CGS 27023A
6c, R=Benzyl, Benzyl-CGS 27023A
CH3OTf/CH3CN
TBAH, R.T.
OMe
O
R
OMe
11CH3O
O
R
N
N
NH
CH3O
S
NH
S
O
O
O
O
1a, R=3-Picolyl, [11C]Methyl-CGS 27023A
1b, R=2-Picolyl, [11C]Methyl-2-Picolyl-CGS 27023A
1c, R=Benzyl, [11C]Methyl-Benzyl-CGS 27023A
1a, R=3-Picolyl, Methyl-CGS 27023A
1b, R=2-Picolyl, Methyl-2-Picolyl-CGS 27023A
1c, R=Benzyl, Methyl-Benzyl-CGS 27023A
Scheme 1. Synthesis of [11/12C]Methyl-CGS 27023A, [11/12C]Methyl-2-Picolyl-
CGS 27023A and [11/12C]Methyl-Benzyl-CGS 27023A
the theoretical explanation that the deprotonization at the hydroxyl
position of the hydroxylamine is easier than at the amine position since
the acidity of HO– of the hydroxylamine is greater than the acidity of
HN– of the hydroxylamine, and the methylation of the hydroxylamine
will prefer to occur at the oxygen position rather than at the nitrogen
position. The existence of strong base in methylation reaction of the
hydroxylamine may epimerise amides, fortunately the epimerization did
not occur during the reaction, which was monitored and confirmed by
analytical HPLC method,34 although tetrabutylammonium hydroxide
in acetonitrile is quite forcing.
The stereochemistry of the analogues prepared is important. The
starting stereochemistry is defined by the starting material d-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 analog. In the synthetic approaches shown in Schemes 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 conserved throughout the synthetic sequences
and no racemization occurred during the various steps.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

SYNTHESIS OF MMP INHIBITOR RADIOTRACERS
455
O
O
3-Picolyl Chloride/HCl
K2CO3, DMF
NH2
HCl
NO2
NH
MeO
S
O2NPhSO2Cl, iPr2NEt, CH3CN MeO
O
O
Nitro
7d, 2-
7e, 3-Nitro
7f, 4-
3
Nitro
1. HCl, Reflux
2. t-BuONH2/HCl
EDC, HOBT
O
O
N
N
NO2
NO2
N
N
HCl, DCE
S
MeO
S
tBuON
H
NMM, DCM
O
O
O
O
9d, 2-Nitro
9e, 3-Nitro
9f, 4-Nitro
8d, 2-Nitro
8e, 3-Nitro
8f, 4-Nitro
O
CH3OTf/CH3CN
TBAH, R.T.
N
O
N
NO2
NO2
N
N
S
HON
H
S
CH3ON
H
O
O
O
O
10d, 2-Nitro-CGS 27023A
10e, 3-Nitro-CGS 27023A
10f, 4-Nitro-CGS 27023A
1d, 2-Nitro, Methyl-2-Nitro-CGS 27023A
1e, 3-Nitro, Methyl-3-Nitro-CGS 27023A
1f, 4-Nitro, Methyl-4-Nitro-CGS 27023A
11CH3OTf, TBAH
CH3CN
1d, 2-Nitro, [11C]Methyl-2-Nitro-CGS 27023A
1e, 3-Nitro, [11C]Methyl-3-Nitro-CGS 27023A
1f, 4-Nitro, [11C]Methyl-4-Nitro-CGS 27023A
O
N
NO2
N
11
S
CH3ON
O
O
H
Scheme 2. Synthesis of [11/12C]Methyl-2-Nitro-CGS 27023A, [11/12C]Methyl-3-
Nitro-CGS 27023A and [11/12C]Methyl-4-Nitro-CGS 27023A
The hydroxamic acid precursors CGS 27023A (6a) and its analogs
(6b–c, 10d–f) were labeled by [11C]methyl triflate35 through 11C-O-
methylation method36–38 at aminohydroxyl position under basic
conditions and isolated by solid-phase extraction (SPE) purification36
to produce pure target compounds in 40–60% radiochemical yields
(based on 11CO2, decay corrected to end of bombardment), in
20–25 min synthesis time. The large polarity difference between the
hydroxamic acid precursor and the labeled methylated product
permitted the use of SPE technique for purification of radioligands
from radiolabeling reaction mixture. The reaction mixture was diluted
with NaHCO3 and loaded onto C-18 cartridge by gas pressure. The
cartridge column was washed with water to remove unreacted acid
precursor and reaction solvent, then final labeled product was eluted
with ethanol. Chemical purity, radiochemical purity, and specific
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

456
X. FEI ET AL.
radioactivity were determined by analytical HPLC methods. The
chemical purity of precursors 6a–c, 10d–f, and standard samples 1a–f
was >95%. The radiochemical purity of target radiotracers 1a–f was
>99%. The chemical purities of target radiotracers 1a–f were ꢀ95%
(1a), ꢀ98% (1b), ꢀ98% (1c), ꢀ95% (1d), ꢀ92% (1e), and ꢀ93% (1f).
The average (n ¼ 5210) specific activity of target radiotracers 1a–f was
0.6–0.8 Ci/mmol at end-of-synthesis (EOS).
Conclusion
We have developed synthetic procedures that provide MMP inhibitor
radiotracers 1a and its analogs 1b–f. Preliminary findings of biological
assay indicate the analogs synthesized have strong inhibitory effective-
ness on MMP-1 in comparison with CGS 27023A. The results warrant
further evaluation of these radiotracers as new potential PET breast
cancer imaging agents in vivo.
Experimental
All commercial reagents and solvents were used without further
purification unless otherwise specified. The [11C]methyl triflate was
made according to a literature procedure16 by the metathetical reaction
of [11C]methyl bromide over a hot column of silver triflate supported on
porous graphite beads. One silver triflate column was used for 20–40
[11C]methyl triflate runs before replacement. Melting points were
determined on a MEL-TEMP II capillary tube apparatus and were
uncorrected. 1H NMR spectra were recorded on a Bruker QE 300 NMR
spectrometer using tetramethylsilane (TMS) as an internal standard.
Chemical shift data for the proton resonances were reported in parts per
million (d) relative to internal standard TMS (d 0.0). The low resolution
mass spectra were obtained using a Hewlett–Packard Engine mass
spectrometer, and the high resolution mass measurements were
obtained using a ThermoFinnigan MAT95 mass spectrometer, at
Purdue University Campus-wide Mass Spectrometry Center. Chroma-
tographic solvent proportions are expressed on a volume: volume basis.
Thin layer chromatography was run using Analtech silica gel GF
uniplates (5 ꢁ 10 cm). Plates were visualized by UV light. Normal phase
flash chromatography was carried out on EM Science silica gel 60
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

SYNTHESIS OF MMP INHIBITOR RADIOTRACERS
457
(230–400 mesh) with a forced flow of the indicated solvent system in the
proportions described below. All moisture-sensitive reactions were
performed under a positive pressure of nitrogen maintained by a direct
line from a nitrogen source.
Analytical HPLC was performed using a Prodigy (Phenomenex) 5 mm
C-18 column, 4.6 250 mm; 3:1:3 CH3CN: MeOH: 20 mM, pH 6.7
KHPO-4 mobile phase, flow rate 1.5 mL/min, and UV (185 nm) and
g-ray (NaI) flow detectors. Semi-prep C-18 silica guard cartridge
column 1 ꢁ 1 cm was obtained from E. S. Industries, Berlin, NJ, Part
number 300121-C18-BD 10 mM. Sterile vented Millex-GS 0.22 mM
vented filter unit was obtained from Millipore Corporation, Bedford,
MA.
d-valine methyl ester hydrochloride (3)
To a solution of d-valine (2) (3.58 g, 30.56 mmol) in MeOH (75 ml) was
added thionyl chloride (10 g, 84 mmol) in an ice bath. After the solution
was stirred at room temperature (RT) overnight, the solvent was
removed under vacuum to give a white solid 3 (4.94 g, 96%). The crude
product was used for the next step reaction without further purification.
N-[(4-methoxyphenyl)sulfonyl]-d-valine methyl ester (4)
To a solution of compound 3 (3.26 g, 19.44 mmol) and p-MeOPhSO2Cl
(4.61 g, 22.36 mmol) in CH3CN (100 ml) was added iPr2NEt (5.0 ml)
dropwise in an ice bath. After the resulting solution was stirred at RT
for 2 days, the solvent was evaporated. The residue was diluted with
EtOAc, washed with brine and dried over Na2SO4. The evaporation of
the solution gave the crude product, which was purified by flash
chromatography (1/3 EtOAc/Hexane) to give a solid 4 (5.10 g, 83%).
1
TLC Rf 0.45 (1/3 EtOAc/Hexane), mp 91–928C. H NMR (300 MHz,
CDCl3): 0.86–0.88 (d, J ¼ 6:6 Hz, 3H, CH3CH), 0.93–0.96 (d,
J ¼ 6:6 Hz, 3H, CH3CH), 1.98–2.03 (m, 1H, (CH3)2CH), 3.47 (s, 3H,
CO2CH3), 3.69–3.74 (dd, J ¼ 5:1, 10.3 Hz, 1H, CHN), 3.86 (s, 3H,
PhOCH3), 5.12–5.15 (d, J ¼ 10:3 Hz, 1H, NH), 6.94–6.97 (d,
J ¼ 8:8 Hz, 2H, H–Ph), 7.74–7.77 (d, J ¼ 8:8 Hz, 2H, H–Ph).
Methyl 2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)amino]-
3-methylbutanoate (5a)
A mixture of compound 4 (2.04 g, 6.48 mmol), 3-picolyl chloride/HCl
salt (1.27 g, 7.77 mmol) and K2CO3 (5.36 g, 38.88 mmol) in dry DMF
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

458
X. FEI ET AL.
(50 ml) was stirred at RT under nitrogen overnight. The reaction
mixture was diluted with H2O (75 ml) and extracted with Et2O
(75 ml ꢁ 3). The combined organic phase was dried over Na2SO4 and
evaporated to give the crude product, which was purified by flash
chromatography (1/3 EtOAc/Hexane) to give the product 5a (2.37 g,
93%). TLC Rf 0.50 (EtOAc), mp 63–658C. 1H NMR (300 MHz,
CDCl3): 0.76–0.78 (d, J ¼ 6:6 Hz, 3H, CH3CH), 0.81–0.84 (d,
J ¼ 6:6 Hz, 3H, CH3CH), 1.89–1.93 (m, 1H, (CH3)2CH), 3.44 (s, 3H,
CO2CH3), 3.86 (s, 3H, PhOCH3), 4.14–4.18 (d, J ¼ 11 Hz, 1H, CHN),
4.63–4.64 (d, J ¼ 2:2 Hz, 2H, NCH2Py), 6.90–6.93 (d, J ¼ 8:8 Hz, 2H,
H–Ph), 7.23–7.27 (m, 1H, H–Py), 7.67–7.70 (d, J ¼ 8:8 Hz, 2H, H–Ph),
7.87–7.89 (d, J ¼ 8:1 Hz, 1H, H–Py), 8.49–8.54 (m, 2H, H–Py).
Methyl 2(R)-[[(4-methoxyphenyl)sulfonyl](2-picolyl)amino]-
3-methyl butanoate (5b)
A mixture of compound 4 (0.58 g, 1.77 mmol), 2-picolyl chloride/HCl
salt (0.58 g, 3.54 mmol) and K2CO3 (1.46 g, 10.74 mmol) in dry DMF
(25 ml) was stirred at RT under nitrogen overnight. The reaction
mixture was diluted with H2O (75 ml) and extracted with Et2O
(75 ml ꢁ 3). The combined organic phase was dried over Na2SO4 and
evaporated to give the crude product, which was purified by flash
chromatography (1/3 EtOAc/Hexane) to give a thick oil 5b (0.75 g,
93%), it was converted to be a solid in the freezer. TLC Rf 0.35 (1/2
1
EtOAc/Hexane), mp 61–628C. H NMR (300 MHz, CDCl3): 0.77–0.80
(d, J ¼ 6:6 Hz, 3H, CH3CH), 0.84–0.86 (d, J ¼ 6:6 Hz, 3H, CH3CH),
2.04–2.11 (m, 1H, (CH3)2CH), 3.44 (s, 3H, CO2CH3), 3.86 (s, 3H,
PhOCH3), 4.11–4.14 (d, J ¼ 10:3 Hz, 1H, CHN), 4.69–4.75
(d, J ¼ 16:9 Hz, 1H, NCHaPy), 4.83–4.88 (d, J ¼ 17:2 Hz, 1H,
NCHbPy), 6.91–6.94 (d, J ¼ 8:8 Hz, 2H, H–Ph), 7.10–7.16 (m, 1H,
H–Py), 7.65–7.67 (m, 2H, H–Py), 7.73–7.76 (d, J ¼ 8:8 Hz, 2H, H–Ph),
8.45–8.46 (d, J ¼ 4:4 Hz, 1H, H–Py).
Methyl 2(R)-[[(4-methoxyphenyl)sulfonyl]benzylamino]-
3-methylbutanoate (5c)
A mixture of compound 4 (1.08 g, 3.42 mmol), benzyl bromide (0.70 g,
4.11 mmol) and K2CO3 (2.85 g, 20.8 mmol) in dry DMF (25 ml) was
stirred at RT under nitrogen overnight. The reaction mixture was
diluted with H2O (75 ml) and extracted with Et2O (100 ml ꢁ 3). The
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

SYNTHESIS OF MMP INHIBITOR RADIOTRACERS
459
combined organic phase was dried over Na2SO4 and evaporated to give
the crude product, which was purified by flash chromatography (1/4
EtOAc/Hexane) to give a solid 5c (1.35 g, 97%). TLC Rf 0.65 (1/4
1
EtOAc/Hexane), mp 65–668C. H NMR (300 MHz, CDCl3): 0.76–0.78
(d, J ¼ 6:6 Hz, 3H, CH3CH), 0.79–0.81 (d, J ¼ 6:6 Hz, 3H, CH3CH),
1.86–1.98 (m, 1H, (CH3)2CH), 3.40 (s, 3H, CO2CH3), 3.86 (s, 3H,
PhOCH3), 4.13–4.17 (d, 1H, CHN), 4.49–4.54 (d, J ¼ 15:4 Hz, 1H,
NCHaPh), 4.64–4.70 (d, J ¼ 15:4 Hz, 1H, NCHbPh), 6.89–6.92 (d,
J ¼ 8:8 Hz, 2H, H–PhOMe), 7.23–7.30 (m, 3H, H–Ph), 7.36–7.41 (m,
2H, H–Ph), 7.68–7.71 (d, J ¼ 8:8 Hz, 2H, H–PhOMe).
N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)amino]-
3-methylbutanamide (CGS 27023A, 6a)
Ester 5a (0.73 g, 1.94 mmol) was dissolved in anhydrous MeOH (6.0 ml).
To this solution was added hydroxylamine hydrochloride (0.65 g,
9.35 mmol), followed by the addition of sodium methoxide, freshly
prepared from sodium (0.34 g, 14.78 mmol) dissolved in MeOH (5 ml).
After the resulting reaction mixture was stirred at RT for 2 days, the
solvent was removed under vacuum. The residue was diluted with
EtOAc (75 ml), washed with brine, and dried over Na2SO4. The solution
was evaporated to give the crude product, which was purified by flash
chromatography (50/1 EtOAc/MeOH) to give a white solid 6a (0.34 g,
1
46%). TLC Rf 0.35 (1/10 MeOH/EtOAc), mp 107–1088C. H NMR
(300 MHz, CDCl3): 0.62–0.64 (d, J ¼ 6:6 Hz, 3H, CH3CH), 0.82–0.84
(d, J ¼ 6:6 Hz, 3H, CH3CH), 2.10–2.20 (m, 1H, (CH3)2CH), 3.82 (s, 3H,
PhOCH3), 3.83–3.86 (d, J ¼ 10:4 Hz, 1H, CHN), 4.55–4.61
(d, J ¼ 15:1 Hz, 1H, NCHaPy), 4.66–4.71 (d, J ¼ 15:4 Hz, 1H,
NCHbPy), 6.84–6.87 (d, J ¼ 7:8 Hz, 2H, H–Ph), 7.19–7.25 (m, 1H,
H–Py), 7.55–7.58 (d, J ¼ 8:8 Hz, 2H, H–Ph), 7.76–7.78 (d, J ¼ 7:4 Hz,
1H, H–Py), 8.38–8.39 (d, J ¼ 3:7 Hz, 1H, H–Py), 8.49 (s, 1H, H–Py).
MS (DCI, CH4) m/z 394 (M+H)+. CGS 27023A hydrochloride, mp
168–1708C.
N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](2-picolyl)amino]-
3-methylbutanamide (2-picolyl-CGS 27023A, 6b)
Ester 5b (0.49 g, 1.25 mmol) was dissolved in anhydrous MeOH (5.0 ml).
To this solution was added hydroxylamine hydrochloride (0.52 g,
7.50 mmol), followed by the addition of sodium methoxide, freshly
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

460
X. FEI ET AL.
prepared from sodium (0.20 g, 8.79 mmol) dissolved in MeOH (5 ml).
After the resulting reaction mixture was stirred at RT for 2 days, the
solvent was removed under vacuum. The residue was diluted with
EtOAc (75 ml), washed with brine, and dried over Na2SO4. The solution
was evaporated to give the crude product, which was purified by flash
chromatography (100/1 EtOAc/MeOH) to give a white solid 6b (0.15 g,
1
30%). TLC Rf 0.44 (1/20 MeOH/EtOAc), mp 103–1048C. H NMR
(300 MHz, CDCl3): 0.68–0.70 (d, J ¼ 6:6 Hz, 3H, CH3CH), 0.78–0.81
(d, J ¼ 6:6 Hz, 3H, CH3CH), 1.96–2.02 (m, 1H, (CH3)2CH), 3.84 (s, 3H,
PhOCH3), 3.85–3.89 (d, J ¼ 10:1 Hz, 1H, CHN), 4.68–4.74
(d, J ¼ 16:9 Hz, 1H, NCHaPy), 4.82–4.87 (d, J ¼ 16:9 Hz, 1H,
NCHbPy), 6.88–6.91 (d, J ¼ 8:8 Hz, 2H, H–Ph), 7.10–7.12 (m, 1H,
H–Py), 7.65–7.67 (m, 2H, H–Py), 7.73–7.76 (d, J ¼ 8:8 Hz, 2H, H–Ph),
8.45–8.47 (d, J ¼ 4:4 Hz, 1H, H–Py).
N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl]benzylamino]-
3-methylbutanamide (benzyl-CGS 27023A, 6c)
Ester 5c (0.53 g, 1.41 mmol) was dissolved in anhydrous MeOH (5.0 ml).
To this solution was added hydroxylamine hydrochloride (0.63 g,
9.13 mmol), followed by the addition of sodium methoxide, freshly
prepared from sodium (0.24 g, 10.46 mmol) dissolved in MeOH (5 ml).
After the resulting reaction mixture was stirred at RT for 2 days, the
solvent was removed under vacuum. The residue was diluted with
EtOAc (75 ml), washed with brine, and dried over Na2SO4. The solution
was evaporated to give the crude product, which was purified by flash
chromatography (EtOAc) to give a white solid 6c (0.13 g, 24%). TLC Rf
0.45 (1/50 MeOH/EtOAc), mp 110–1128C. 1H NMR (300 MHz,
CDCl3): 0.47–0.49 (d, J ¼ 6:6 Hz, 3H, CH3CH), 0.80–0.82
(d, J ¼ 6:6 Hz, 3H, CH3CH), 2.12–2.20 (m, 1H, (CH3)2CH), 3.69–3.72
(d, J ¼ 10:3 Hz, 1H. CHN), 3.85 (s, 3H, PhOCH3), 4.45–4.50
(d, J ¼ 15:4 Hz, 1H, NCHaPh), 4.63–4.68 (d, J ¼ 15:5 Hz, 1H,
NCHbPh), 6.88–6.90 (d, J ¼ 8:2 Hz, 2H, H–PhOMe), 7.26–7.32
(m, 3H, H–Ph), 7.35–7.38 (m, 2H, H–Ph), 7.59–7.62 (d, J ¼ 8:8 Hz,
2H, H–PhOMe).
N-[(2-nitrophenyl)sulfonyl]-d-valine methyl ester (7d)
To a solution of compound 3 (1.29 g, 7.71 mmol) and 2-nitrophenyl
sulfonyl chloride (2.20 g, 9.93 mmol) in CH3CN (75 ml) was added
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

SYNTHESIS OF MMP INHIBITOR RADIOTRACERS
461
iPr2NEt (5.0 ml) dropwise in an ice bath. After the resulting solution
was stirred at RT for 2 days, the solvent was evaporated. The residue
was diluted with EtOAc, washed with brine and dried over Na2SO4. The
evaporation of the solution gave the crude product, which was purified
by flash chromatography (1/1 EtOAc/Hexane) to produce a yellow solid
7d (2.10 g, 86%). TLC Rf 0.48 (1/10, MeOH/Hexane), mp 81–828C. 1H
NMR (300 MHz, CDCl3): 0.86–0.88 (d, J ¼ 6:6 Hz, 3H, CH3CH),
0.93–0.95 (d, J ¼ 6:6 Hz. 3H, CH3CH), 2.00–2.10 (m, 1H, (CH3)2CH),
3.52 (s, 3H, CO2CH3), 3.70–3.75 (dd, J ¼ 5:0, 10.0 Hz, 1H, CHN),
5.13–5.15 (d, J ¼ 10:0 Hz, 1H, NH), 7.40–7.48 (m, 2H), 7.57–7.58 (m,
1H), 8.43 (s, 1H).
N-[(3-nitrophenyl)sulfonyl]-d-valine methyl ester (7e)
To a solution of compound 3 (1.23 g, 7.31 mmol) and 3-nitrophenyl
sulfonyl chloride (2.43 g, 10.97 mmol) in CH3CN (75 ml) was added
iPr2NEt (5.0 ml) dropwise in an ice bath. After the resulting solution
was stirred at RT for 2 days, the solvent was evaporated. The residue
was diluted with EtOAc, washed with brine and dried over Na2SO4. The
evaporation of the solution gave the crude product, which was purified
by flash chromatography (1/1 EtOAc/Hexane) to produce a yellow solid
1
7e (2.17 g, 94%). TLC Rf 0.52 (1/2, EtOAc/Hexane), mp 86–888C. H
NMR (300 MHz, CDCl3): 0.84–0.86 (d, J ¼ 6:6 Hz, 3H, CH3CH),
0.90–0.99 (d, J ¼ 6:6 Hz, 3H, CH3CH), 2.02–2.12 (m, 1H, (CH3)2CH),
3.52 (s, 3H, CO2CH3), 3.81–3.86 (dd, J ¼ 4:8, 10.1 Hz, 1H, CHN), 5.28–
5.32 (d, J ¼ 10:2 Hz, 1H, NH), 7.56–7.62 (t, J ¼ 8:1 Hz, 1H), 7.72–7.74
(d, J ¼ 7:4 Hz, 1H), 8.33–8.37 (dd, J ¼ 1:8; 8:1 Hz, 1H), 8.42 (s, 1H).
N-[(4-Nitrophenyl)sulfonyl]-d-valine methyl ester (7f)
To a solution of compound 3 (1.39 g, 8.27 mmol) and 4-nitrophenyl
sulfonyl chloride (2.75 g, 12.41 mmol) in CH3CN (75 ml) was added
iPr2NEt (5.0 ml) dropwise in an ice bath. After the resulting solution
was stirred at RT for 2 days, the solvent was evaporated. The residue
was diluted with EtOAc, washed with brine and dried over Na2SO4. The
evaporation of the solution gave the crude product, which was purified
by flash chromatography (1/2 EtOAc/Hexane) to give a yellow solid 7f
1
(2.58 g, 98%). TLC Rf 0.45 (1/3 EtOAc/Hexane), mp 95–978C. H
NMR (300 MHz, CDCl3): 0.87–0.89 (d, J ¼ 6:6 Hz, 3H, CH3CH),
0.97–0.99 (d, J ¼ 6:6 Hz, 3H, CH3CH), 2.07–2.14 (m, 1H, (CH3)2CH),
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

462
X. FEI ET AL.
3.52 (s, 3H, CO2CH3), 3.82–3.87 (dd, J ¼ 4:8, 10.1 Hz, 1H, CHN), 5.28–
5.31 (d, J ¼ 10:3 Hz, 1H, NH), 8.02–8.05 (d, J ¼ 8:8 Hz, 2H, H-Ar),
8.34–8.36 (d, J ¼ 8:8 Hz, 2H, H-Ar).
Methyl 2(R)-[[(2-nitrophenyl)sulfonyl](3-picolyl)amino]-
3-methylbutanoate (8d)
A mixture of compound 7d (2.18 g, 6.90 mmol), 3-picolyl chloride/HCl
salt (2.26 g, 13.80 mmol) and K2CO3 (5.70 g, 41.40 mmol) in dry DMF
(30 ml) was stirred at RT under nitrogen overnight. The reaction
mixture was diluted with H2O (75 ml) and extracted with Et2O
(100 ml ꢁ 3). The combined organic phase was dried over Na2SO4 and
evaporated to give the crude product, which was purified by flash
chromatography (2/1 EtOAc/Hexane) to give a yellow solid 8d (2.45 g,
87%). TLC Rf 0.55 (EtOAc), mp 91–938C. 1H NMR (300 MHz,
CDCl3): 0.88–0.90 (d, J ¼ 6:6 Hz, 3H, CH3CH), 0.93–0.95 (d,
J ¼ 6:6 Hz, 3H, CH3CH), 2.11–2.18 (m, 1H, (CH3)2CH), 3.58 (s, 3H,
CO2CH3), 4.29–4.33 (d, J ¼ 10:3 Hz, 1H, CHN), 4.60–4.65 (d,
J ¼ 15:4 Hz, 1H, NCHaPy), 4.91–4.96 (d, J ¼ 15:4 Hz, 1H, NCHbPy),
7.09–7.11 (m, 1H), 7.47–7.50 (m, 1H), 7.60–7.62 (m, 2H), 7.68–7.71 (d,
J ¼ 8:1 Hz, 1H), 7.78–7.81 (d, J ¼ 7:4 Hz, 1H), 8.42–8.43 (d,
J ¼ 4:4 Hz, 1H), 8.56 (s, 1H).
Methyl 2(R)-[[(3-nitrophenyl)sulfonyl](3-picolyl)amino]-
3-methylbutanoate (8e)
A mixture of compound 7e (1.22 g, 3.88 mmol), 3-picolyl chloride/HCl
salt (1.27 g, 7.75 mmol) and K2CO3 (2.97 g, 21.52 mmol) in dry DMF
(25 ml) was stirred at RT under nitrogen overnight. The reaction
mixture was diluted with H2O (75 ml) and extracted with Et2O
(100 ml ꢁ 3). The combined organic phase was dried over Na2SO4 and
evaporated to give the crude product, which was purified by flash
chromatography (2/1 EtOAc/Hexane) to give a solid 8e (1.35 g, 77%).
1
TLC Rf 0.54 (2/1, EtOAc/Hexane), mp 94–958C. H NMR (300 MHz,
CDCl3): 0.91–0.93 (d, J ¼ 6:6 Hz, 6H, (CH3)2CH), 2.05–2.10 (m, 1H,
(CH3)2CH), 3.57 (s, 3H, CO2CH3), 4.34–4.37 (d, J ¼ 10:3 Hz, 1H,
CHN), 4.52–4.57 (d, J ¼ 15:4 Hz, 1H, NCHaPy), 4.65–4.71 (d,
J ¼ 16:2 Hz, 1H, NCHbPy), 7.16–7.20 (m, 1H), 7.56–7.62 (t,
J ¼ 8:1 Hz, 1H), 7.72–7.74 (d, J ¼ 7:4 Hz, 1H), 7.91–7.94 (d,
J ¼ 8:1 Hz, 1H), 8.33–8.36 (dd, J ¼ 1:5, 8.1 Hz, 1H), 8.43–8.46 (m, 3H).
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

SYNTHESIS OF MMP INHIBITOR RADIOTRACERS
463
Methyl 2(R)-[[(4-nitrophenyl)sulfonyl](3-picolyl)amino]-
3-methylbutanoate (8f)
A mixture of compound 7f (0.56 g, 1.76 mmol), 3-picolyl chloride/HCl
salt (0.43 g, 2.64 mmol) and K2CO3 (1.46 g, 10.56 mmol) in dry DMF
(25 ml) was stirred at RT under nitrogen overnight. The reaction
mixture was diluted with H2O (75 ml) and extracted with Et2O
(100 ml ꢁ 3). The combined organic phase was dried over Na2SO4 and
evaporated to give the crude product, which was purified by flash
chromatography (1/1 EtOAc/Hexane) to give a solid 8f (0.69 g, 82%).
1
TLC Rf 0.55 (1/2, EtOAc/Hexane), mp 97–998C. H NMR (300 MHz,
CDCl3): 0.86–0.88 (d, J ¼ 6:6 Hz, 3H, CH3CH), 0.88–0.90 (d,
J ¼ 6:6 Hz, 3H, CH3CH), 2.00–2.08 (m, 1H, (CH3)2CH), 3.52 (s, 3H,
CO2CH3), 4.25–4.29 (d, J ¼ 11:0 Hz, 1H, CHN), 4.54–4.60 (d,
J ¼ 6:2 Hz, 1H, NCHaPy), 4.67–4.72 (d, J ¼ 6:2 Hz, 1H, NCHbPy),
7.17–7.22 (dd, J ¼ 5:1, 8.2 Hz, 1H, H–Py), 7.73–7.76 (d, J ¼ 8:1 Hz, 1H,
H–Py), 7.81–7.84 (d, J ¼ 8:8 Hz, 2H, H-Ar), 8.23–8.26 (d, J ¼ 8:8 Hz,
2H, H-Ar), 8.50 (bs, 2H, H–Py).
N-(tert-butyloxy)-2(R)-[[(2-nitrophenyl)sulfonyl](3-picolyl)amino]-
3-methylbutanamide (9d)
A solution of ester 8d (0.79 g, 2.03 mmol) in concentrated HCl (5.0 ml)
was refluxed for 8 h. The solvent was removed by vacuum and the
residue was dissolved in CH2Cl2 (10 ml). Into this solution were added
EDC (0.43 g, 2.23 mmol), HOBT (0.36 g, 2.67 mmol) and NMM
(1.0 ml). After this solution was stirred at RT for 1 h, tBuONH2/HCl
(0.38 g, 3.00 mmol) was added. The resulting reaction solution was
stirred at RT for 1 day. The solvent was removed by evaporation. The
residue was diluted with EtOAc, washed with brine and dried over
Na2SO4. Evaporation gave the crude product, which was purified by
flash chromatography (EtOAc) to give a white solid 9d (0.78 g, 83%).
1
TLC Rf 0.45 (EtOAc), mp 70–718C. H NMR (300 MHz, CDCl3):
0.56–0.59 (d, J ¼ 5:9 Hz, 3H, CH3CH), 0.87–0.89 (d, J ¼ 5:9 Hz, 3H,
CH3CH), 1.26 (s, 9H, tBu), 2.10–2.13 (m, 1H, (CH3)2CH), 3.70–3.74
(d, J ¼ 11:0 Hz, 1H, CHN), 6.62 (bs, 2H, NCH2Py), 6.86–6.89
(d, J ¼ 8:8 Hz, 1H), 7.16–7.20 (m, 2H), 7.54–7.57 (d, J ¼ 8:1 Hz, 1H),
7.73–7.75 (d, J ¼ 7:6 Hz, 1H), 8.37 (s, 1H), 8.46–8.47 (d, J ¼ 3:0 Hz,
1H), 8.55 (s, 1H).
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

464
X. FEI ET AL.
N-(tert-butyloxy)-2(R)-[[(3-nitrophenyl)sulfonyl](3-picolyl)amino]-
3-methylbutanamide (9e)
A solution of ester 8e (0.48 g, 1.23 mmol) in concentrated HCl (5.0 ml)
was refluxed for 8 h. The solvent was removed by vacuum and the
residue was dissolved in CH2Cl2 (10 ml). Into this solution were added
EDC (0.26 g, 1.35 mmol), HOBT (0.20 g, 1.48 mmol) and NMM
(1.0 ml). After this solution was stirred at RT for 1 h, tBuNH2/HCl
(0.20 g, 1.60 mmol) was added. The resulting reaction solution was
stirred at RT for 1 day. The solvent was removed by evaporation. The
residue was diluted with EtOAc, washed with brine and dried over
Na2SO4. Evaporation gave the crude product, which was purified by
flash chromatography (EtOAc) to give a white solid 9e (0.48 g, 84%).
1
TLC Rf 0.56 (EtOAc), mp 77–798C. H NMR (300 MHz, CDCl3):
0.72–0.74 (d, J ¼ 6:6 Hz, 3H, CH3CH), 0.96–0.98 (d, J ¼ 6:6 Hz, 3H,
CH3CH), 1.38 (s, 9H, tBu), 2.20–2.31 (m, 1H, (CH3)2CH), 3.85–3.89 (d,
J ¼ 11:1 Hz, 1H, CHN), 4.69 (bs, 2H, NCH2Py), 7.16–7.18 (m, 1H),
7.55–7.68 (t, J ¼ 8:2 Hz, 1H), 7.72–7.77 (t, J ¼ 8:1 Hz, 1H), 8.30–8.32
(m, 2H), 8.37 (s, 1H), 8.44–8.46 (m, 1H), 8.50 (s, 1H).
N-(tert-butyloxy)-2(R)-[[(4-nitrophenyl)sulfonyl](3-picolyl)amino]-
3-methylbutanamide (9f)
A solution of ester 8f (0.58 g, 1.44 mmol) in concentrated HCl (5 ml) was
refluxed for 8 h. The solvent was removed under vacuum and the residue
was dissolved in CH2Cl2 (10 ml). Into this solution were added EDC
(0.30 g, 1.58 mmol), HOBT (0.25 g, 1.87 mmol) and NMM (1.0 ml).
After this solution was stirred at RT for 1 h, tBuONH2/HCl (0.36 g,
2.88 mmol) was added. The resulting reaction solution was stirred at RT
for 1 day. The solvent was removed by evaporation. The residue was
diluted with EtOAc, washed with brine and dried over Na2SO4.
Evaporation gave the crude product, which was purified by flash
chromatography (EtOAc) to give a white solid product 9f (0.65 g, 94%).
1
TLC Rf 0.42 (1/1 EtOAc/Hexane), mp 88–898C. H NMR (300 MHz,
CDCl3): 0.73–0.75 (d, J ¼ 6:6 Hz, 3H, CH3CH), 0.93–0.95 (d,
J ¼ 6:6 Hz, 3H, CH3CH), 1.26 (s, 9H, tBu), 2.27–2.33 (m, 1H,
(CH3)2CH), 3.86–3.90 (d, J ¼ 11:0 Hz, 1H, CHN), 4.73 (s, 2H,
NCH2Py), 7.12–7.19 (m, 1H, H–Py), 7.70–7.72 (d, J ¼ 8:1 Hz, 2H, H-
Ar), 8.15–8.18 (d, J ¼ 8:1 Hz, 2H, H-Ar), 8.47–8.48 (d, J ¼ 3:6 Hz, 1H,
H–Py), 8.58 (s, 1H, H–Py), 8.91 (s, 1H, H–Py).
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

SYNTHESIS OF MMP INHIBITOR RADIOTRACERS
465
N-hydroxy-2(R)-[[(2-nitrophenyl)sulfonyl](3-picolyl)amino]-
3-methylbutanamide (2-nitro-CGS 27023A, 10d)
The protected hydroxamic acid 9d (0.60 g, 1.29 mmol) was dissolved in
1,2-dichloroethane (DCE) (20 ml) and EtOH (0.06 g, 1.29 mmol) in a
flask. HCl gas was bubbled through the flask for 0.5 h. The reaction
flask was sealed and stirred at RT for 3 days. The solvent was reduced to
1/3 volume by evaporation and diluted with ether. The mixture was
filtered, and the solid was collected, and dried under vacuum to provide
a white solid hydrochloride salt of 10d (0.51 g, 89%). TLC Rf 0.35 (1/10,
MeOH/EtOAc), mp 150–1518C. It was converted to a free base 10d by
1
Et3N. H NMR (300 MHz, CDCl3): 0.82–0.84 (d, J ¼ 6:6 Hz, 3H,
CH3CH), 0.92–0.94 (d, J ¼ 6:6 Hz, 3H, CH3CH), 2.20–2.36 (m, 1H,
(CH3)2CH), 3.92–3.96 (d, J ¼ 11:0 Hz, 1H, CHN), 4.73–4.79 (d,
J ¼ 15:0 Hz, 1H, NCHaPy), 4.92–4.99 (d, J ¼ 15:0 Hz, 1H, NCHbPy),
7.02–7.10 (m, 1H), 7.39–7.48 (m, 1H), 7.52–7.62 (m, 3H), 7.65–7.67 (bs,
1H), 8.31–8.32 (bs, 1H), 8.51 (s, 1H). HRMS calcd for C17H20N4O6S
408.1104, found 408.1099.
N-hydroxy-2(R)-[[(3-nitrophenyl)sulfonyl](3-picolyl)amino]-
3-methylbutanamide (3-nitro-CGS 27023A, 10e)
The protected hydroxamic acid 9e (0.39 g, 0.85 mmol) was dissolved in
1,2-dichloroethane (DCE) (20 ml) and EtOH (0.04 g, 0.85 mmol) in a
flask. HCl gas was bubbled through the flask for 0.5 h. The reaction
flask was sealed and stirred at RT for 3 days. The solvent was reduced to
1/3 volume by evaporation and diluted with ether. The mixture was
filtered, and the solid was collected, and dried under vacuum to provide
a white solid hydrochloride salt of 10e (0.35 g, 97%). TLC Rf 0.45 (1/10,
MeOH/EtOAc), mp 154–1568C. It was converted to a free base 10e by
1
Et3N. H NMR (300 MHz, CDCl3): 0.77–0.79 (d, J ¼ 6:6 Hz, 3H,
CH3CH), 0.82–0.84 (d, J ¼ 6:6 Hz, CH3CH), 2.22–2.33 (m, 1H,
(CH3)2CH), 3.85–3.88 (d, J ¼ 11:0 Hz, 1H, CHN), 4.73 (bs, 2H,
NCH2Py), 7.17–7.20 (m, 1H), 7.50–7.55 (m, 1H), 7.77–7.79 (m, 1H),
7.84–7.86 (m, 1H), 8.22–8.23 (m, 1H), 8.34–8.47 (m, 3H). HRMS calcd
for C17H20N4O6S 408.1104, found 408.1102.
N-hydroxy-2(R)-[[(4-nitrophenyl)sulfonyl](3-picolyl)amino]-
3-methylbutanamide (4-Nitro-CGS 27023A, 10f)
The protected hydroxamic acid 9f (0.61 g, 1.30 mmol) was dissolved in
1,2-dichloroethane (DCE) (30 ml) and EtOH (0.06 g, 1.30 mmol) in a
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

466
X. FEI ET AL.
flask. HCl gas was bubbled through the flask for 0.5 h. The reaction
flask was sealed and stirred at RT for 3 days. The solvent was reduced to
1/3 volume by evaporation and diluted with ether. The mixture was
filtered, and the solid was collected, and dried under vacuum to provide
a white solid hydrochloride salt of 10f (0.57 g, 99%). TLC Rf 0.36 (1/10
MeOH/EtOAc), mp 151–1538C. It was converted to a free base 10f by
1
Et3N. H NMR (300 MHz, CDCl3): 0.56–0.58 (d, J ¼ 6:6 Hz, 3H,
CH3CH), 0.79–0.81 (d, J ¼ 6:6 Hz Hz, 3H, CH3CH), 1.94–2.02 (m, 1H,
(CH3)2CH), 3.75–3.77 (d, J ¼ 10:3 Hz, 1H, CHN), 4.57–4.62 (d,
J ¼ 16:2 Hz, 1H, NCHaPy), 4.72–4.77 (d, J ¼ 16:2 Hz, 1H, NCHbPy),
7.13–7.27 (m, 1H, H–Py), 7.71–7.33 (d, J ¼ 8:1 Hz, 2H, H–Ph), 8.15–
8.18 (d, J ¼ 8:8 Hz, 2H, H–Ph), 8.46–8.47 (d, J ¼ 3:6 Hz, 1H, H–Py),
8.59 (s, 1H, H–Py), 8.90 (s, 1H, H–Py). HRMS calcd for C17H20N4O6S
408.1104, found 408.1098.
Typical experimental procedure for the synthesis of the standard samples
unlabeled methyl-CGS 27023A (1a), methyl-2-picolyl-CGS 27023A
(1b), methyl-benzyl-CGS 27023A (1c), methyl-2-nitro-CGS 27023A
(1d), methyl-3-nitro-CGS 27023A (1e), and methyl-4-nitro-CGS 27023A
(1f)
Hydroxamic acid precursor 6–10e, or 10f (0.1–0.2 mmol) was dissolved
in CH3CN (5–10 ml). To this solution was added tetrabutylammonium
hydroxide (TBAH) (0.3–1.0 ml, 1 M solution in methanol) and methyl
triflate (0.4–0.6 mmol). The mixture was stirred at RT under nitrogen
overnight. The solvent was evaporated. The residue was diluted with
EtOAc, washed with brine and dried over Na2SO4. The evaporation of
the solution gave the crude product, which was purified by flash
chromatography (EtOAc) to give the solid unlabeled target compounds
1a-f. The yields of 1a–f were 60–85%. 1H NMR (300 MHz, CDCl3): 1a,
0.60–0.62 (d, J ¼ 6:6 Hz, 3H, CH3CH), 0.86–0.88 (d, J ¼ 6:6 Hz, 3H,
CH3CH), 2.15–2.22 (m, 1H, (CH3)2CH), 3.65 (s, 3H, CH3ONH),
3.74–3.77 (d, J ¼ 10:3 Hz, 1H, CHN), 3.84 (s, 3H, PhOCH3), 4.57–4.62
(d, J ¼ 16:2 Hz, 1H, NCHaPy), 4.72–4.77 (d, J ¼ 15:5 Hz, 1H,
NCHbPy), 6.84–6.87 (d, 2H, J ¼ 9:8 Hz, H–Ph), 7.17–7.21 (m, 1H,
H–Py), 7.57–7.60 (d, J ¼ 9:6 Hz, 2H, H–Ph), 7.77–7.80 (d, J ¼ 9:1 Hz,
1H, H–Py), 8.46–8.48 (d, J ¼ 5:2 Hz, 1H, H–Py), 8.61 (s, 1H, H–Py).
HRMS calcd for C19H26N3O5S (M+H)+ 408.1593, found 408.1572. 1b.
0.57–0.60 (d, J ¼ 6:6 Hz, 3H, CH3CH), 0.84–0.86 (d, J ¼ 6:6 Hz, 3H,
CH3CH), 2.10–2.20 (m, 1H, (CH3)2CH), 3.63 (s, 3H, CH3ONH),
3.77–3.80 (d, J ¼ 11:0 Hz, 1H, CHN), 3.85 (s, 3H, PhOCH3), 4.55–4.60
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

SYNTHESIS OF MMP INHIBITOR RADIOTRACERS
467
(d, J ¼ 16:2 Hz, 1H, NCHaPy), 4.70–4.75 (d, J ¼ 16:2 Hz, 1H,
NCHbPy), 6.84–6.87 (d, J ¼ 8:8 Hz, 2H, H–Ph), 7.12–7.17 (m, 1H,
H–Py), 7.63–7.67 (m, 2H, H–Py), 7.73–7.76 (d, J ¼ 8:6 Hz, 2H, H–Ph),
8.45–8.47 (d, J ¼ 4:4 Hz, 1H, H–Py). HRMS calcd for C19H26N3O5S
(M+H)+ 408.1593, found 408.1104. 1c, 0.58–0.60 (d, J ¼ 6:6 Hz, 3H,
CH3CH), 0.87–0.89 (d, J ¼ 6:6 Hz, 3H, CH3CH), 2.10–2.20 (m, 1H,
(CH3)2CH), 3.63 (s, 3H, CH3ONH), 3.74–3.77 (d, J ¼ 11:0 Hz, 1H,
CHN), 3.84 (s, 3H, PhOCH3), 4.58–4.63 (d, J ¼ 15:4 Hz, 1H, NCHaPh),
4.64–4.68 (d, J ¼ 15:6 Hz, 1H, NCHbPh), 6.87–6.90 (d, J ¼ 8:8 Hz, 2H,
H–PhOMe), 7.22–7.27 (m, 3H, H–Ph), 7.36–7.41 (m, 2H, H–Ph), 7.68–
7.71 (d, J ¼ 8:7 Hz, 2H, H–PhOMe). HRMS calcd for C20H27N2O5S
(M+H)+ 407.1641, found 407.1621. 1d, 0.87–0.89 (d, J ¼ 6:6 Hz, 3H,
CH3CH), 0.96–0.98 (d, J ¼ 6:6 Hz, 3H, CH3CH), 2.25–2.30 (m, 1H,
(CH3)2CH), 3.69 (s, 3H, CH3ONH), 3.88–3.92 (d, J ¼ 10:3 Hz, 1H,
CHN), 4.76–4.81 (d, J ¼ 15:4 Hz, 1H, NCHaPy), 4.96–5.01
(d, J ¼ 15:4 Hz, 1H. NCHbPy), 7.05–7.09 (m, 1H), 7.40–7.46 (m, 2H),
7.58–7.78 (m, 1H), 7.65–7.68 (d, J ¼ 7:4 Hz, 1H), 8.39–8.41
(d, J ¼ 3:7 Hz, 1H), 8.56 (s, 1H), 8.93 (s, 1H). HRMS calcd for
C18H23N4O6S (M+H)+ 423.1338, found 423.1320. 1e, 0.76–0.78
(d, J ¼ 6:6 Hz, 3H, CH3CH), 0.95–0.97 (d, J ¼ 6:6 Hz, 3H, CH3CH),
2.28–2.32 (m, 1H, (CH3)2CH), 3.72 (s, 3H, CH3ONH), 3.89–3.92
(d, J ¼ 10:1 Hz, 1H, CHN), 4.73 (s, 2H, NCH2Py), 7.16–7.18 (m, 1H),
7.52–7.58 (t, J ¼ 8:1 Hz, 1H), 7.71–7.74 (d, J ¼ 7:3 Hz, 1H), 7.81–7.83
(d, J ¼ 7:3 Hz, 1H), 8.29–8.32 (d, J ¼ 8:8 Hz, 1H), 8.36 (s, 1H), 8.44
(s, 1H), 8.53 (s, 1H). HRMS calcd for C18H23N4O6S (M+H)+
423.1338, found 423.1326. 1f, 0.72–0.74 (d, J ¼ 6:0 Hz, 3H, CH3CH),
0.93–0.95 (d, J ¼ 6:6 Hz, 3H, CH3CH), 1.98–2.10 (m, 1H, (CH3)2CH),
3.72 (s, 3H, CH3ONH), 3.85–3.89 (d, J ¼ 11:1 Hz, 1H, CHN), 4.73–4.75
(d, J ¼ 3:7 Hz, 2H, NCH2Py), 7.14–7.18 (m, 1H, H–Py), 7.71–7.73
(d, J ¼ 8:1 Hz, 2H, H–Ph), 8.15–8.18 (d, J ¼ 8:8 Hz, 2H, H–Ph),
8.46–8.48 (d, J ¼ 3:7 Hz, 1H, H–Py), 8.59 (s, 1H, H–Py), 8.90 (s, 1H, H–
Py). HRMS calcd for C18H22N4O6S 422.1260, found 422.1246.
Typical experimental procedure for the radiosynthesis of [11C]methyl-
CGS 27023A (1a), [11C]methyl-2-picolyl-CGS 27023A (1b), [11C]
methyl-benzyl-CGS 27023A (1c), [11C]methyl-2-nitro-CGS 27023A
(1d), [11C]methyl-3-nitro-CGS 27023A (1e), and [11C]methyl-4-nitro-
CGS 27023A (1f).
Hydroxamic acid precursor 6a–c, 10d, 10e, or 10f (0.6–1.0 mg) was
dissolved in CH3CN (300 ml). To this solution was added tetrabutyl-
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

468
X. FEI ET AL.
ammonium 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 ꢂ15
to ꢂ208C, 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 70–808C for 3 min.
The contents of the reaction tube were diluted with 0.1 M NaHCO3
(1 ml). This solution was passed onto a C-18 cartridge by gas pressure.
The cartridge was washed with 2 ꢁ 3 ml H2O, and the aqueous washing
was discarded. The product was eluted from the column with 2 ꢁ 3 ml
EtOH, then passed onto a rotary evaporator. The solvent was removed
by evaporation under high vacuum. The labeled product 1a–f was
formulated with 50 mM NaH2PO4, whose volume was dependent upon
the use of the labeled product 1a–f in tissue biodistribution studies
(ꢀ6 ml, 3 ꢁ 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%, and the radiochemical purity was >99% by analytical
HPLC. Retention times in the analytical HPLC system were:
RT6a=2.90 min, RT6b=3.18 min, RT6c=6.13 min, RT 10d=
2.99 min, RT10e=3.21 min, RT10f=3.20 min; RT1a=3.72 min,
RT1b=4.40 min, RT1c=9.56 min, RT1d=3.54 min, RT1e=4.24 min,
RT1f=4.06 min.
Acknowledgements
This work is supported by the Susan G. Komen Breast Cancer
Foundation grant IMG 2000 837 (to QHZ), Indiana University
American Cancer Society Institutional Grant Committee grant IRG-
84-002-17 (to QHZ), National Institutes of Health/National Cancer
Institute grant P20CA86350 (to GDH), and Indiana 21st Century
Research & Technology Fund grant (to GDH). We thank Joanne Dunn
for her technical assistance. The reviewers’ and editor’s comments for
the revision of the manuscript are greatly appreciated.
References
1. Brown PD, Breast Cancer Res Treat 1998; 52: 125–136.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

SYNTHESIS OF MMP INHIBITOR RADIOTRACERS
469
2. Greenwald RA, Zucker S, Golub LM, (ed.). Inhibition of matrix
metalloproteinases, therapeutic applications. Ann NY Acad Sci 1999;
878: 1–761.
3. Hahnel E, Harvey JM, Joyce R, Robbins PD, Sterrett GF, Hahnel R. Int J
Cancer 1993; 55: 771–774.
4. Whittaker M, Floyd CD, Brown P, Gearing AJH. Chem Rev 1999; 99:
2735–2776.
5. Hagmann WK, Lark MW, Becker JW. Annual Report in Medical
Chemistry Bristol JA (ed.), vol 31. Academic Press: New York, 1996;
231–240.
6. Summers JB, Davidsen SK. Annual Report in Medical Chemistry. Bristol
JA. (ed.), vol 33. Academic Press: New York, 1998; 131–140.
7. Rasmussen HS, McCann PP. Pharmacol Ther 1997; 75: 69–75.
8. Steward WP. Cancer Chemother Pharmacol 1999; 43 (Suppl): S56–S60.
9. MacPherson LJ, Bayburt EK, Capparelli MP, et al. J Med Chem 1997; 40:
2525–2532.
10. Afzal S, Lalani el-N, Foulkes WD, et al. Lab Invest 1996; 74(2): 406–421.
11. Price A, Shi Q, Morris D, et al. Clin Cancer Res 1999; 5(4): 845–854.
12. Zheng Q-H., Hutchins GD, Mock BH, Winkle WL J. Label Compd
Radiopharm 2001; 44 (Suppl 1): S104-S106.
13. Natchus MG, Bookland RG, De B, et al. J Med Chem 2000; 43:
4948–4963.
14. Babine RE, Bender SL. Chem Rev 1997; 97: 1359–1472.
15. Tamura Y, Watanabe F, Nakatani T, et al. J Med Chem 1998; 41: 640–649.
16. Pikul S, Dunham KLM, Almstead NG, et al. J Med Chem 1998; 41:
3568–3571.
17. Pikul S, Dunham KLM, Almstead NG, et al. J Med Chem 1999; 42: 87–94.
18. Kiyama R, Tamura Y, Watanabe F, Tsuzuki H, Ohtani M, Yodo M.
J Med Chem 1999; 42: 1273–1278.
19. Almstead NG, Bradley RS, Pikul S, et al. J Med Chem 1999; 42:
4547–4562.
20. Cheng MY, De B., Almstead NG, et al. J Med Chem 1999; 42: 5426–5436.
21. Cheng MY, De B, Pikul S, et al. J Med Chem 2000; 43: 369–380.
22. Robinson RP, Laird ER, Blake JF, et al. J Med Chem 2000; 43: 2293–2296.
23. Scozzafava A, Supuran CT. J Med Chem 2000; 43: 3677–3687.
24. Natchus MG, Bookland RG, De B, et al. J Med Chem 2000; 43:
4948–4963.
25. Brown S, Bernardo MM, Li ZH, et al. J Am Chem Soc 2000; 122:
6799–6780.
26. Natchus MG, Bookland RG, Laufersweiler MJ, et al. J Med Chem 2001;
44: 1060–1071.
27. Pikul S, Ohler NE, Ciszewski G, et al. J Med Chem 2001; 44: 2499–2502.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470

470
X. FEI ET AL.
28. Hanessian S, MacKay DB, Moitessier N. J Med Chem 2001; 44:
3074–3082.
29. Schroder J, Henke A, Wenzel H, et al. J Med Chem 2001; 44: 3231–3243.
30. Shuttleworth S. Advances in Drug Discovery Techniques, Harvey AL (ed.),
Wiley: New York, 1998; p. 115.
31. Whittaker M. Curr Opin Chem Biol 1998; 2: 386–396.
32. Nagase H, Fields CG, Fields GB. J Biol Chem 1994; 269(33): 20952–20957.
33. Jeng AY, Chou M, Parker DT. Bioorg Med Chem Lett 1998; 8: 897–902.
34. Zheng Q-H, Mulholland GK. Syn Commun 2000; 30: 333–340.
35. Mock BH, Mulholland GK, Vavrek MT. Nucl Med Biol 1999; 26(4):
467–471.
36. Zheng Q-H, Mulholland GK. Nucl Med Biol 1996; 23(8): 981–986.
37. ZhengQ-H,MulhollandG.K. JLabelCompdRadiopharm1997;40(Suppl1):
551–553.
38. Zheng Q-H, Winkle W, Carlson K, Mulholland GK. J Label Compd
Radiopharm 1997; 40 (Suppl 1): 548–550.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 449–470