Synthesis of MMP inhibitor radiotracer [11C]CGS 25966, a new potential PET tumor imaging agent

Article abstract of DOI:10.1002/jlcr.675

[¹¹C]CGS 25966, a novel radiolabeled matrix metalloproteinase (MMP) inhibitor, has been synthesized for evaluation as new potential positron emission tomography (PET) tumor imaging agent. The precursor was labeled by [¹¹C]methyl triflate through O-[¹¹C]methylation method at the hydroxyl position of phenol under basic conditions and isolated by HPLC purification to produce pure target compound in 15-25% radiochemical yield, based on ¹¹CO₂, decay corrected to end of bombardment. Copyright

Full text of DOI:10.1002/jlcr.675

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 343–351.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.675
Research Article
Synthesis of MMP inhibitor radiotracer
[11C]CGS 25966, a new potential PET tumor
imaging agent
Xiangshu Fei1, Qi-Huang Zheng1,*, Xuan Liu1, Ji-Quan Wang1,
K. Lee Stone1, Kathy D. Miller2, George W. Sledge2
and Gary D. Hutchins1
1 Department of Radiology, Indiana University School of Medicine, Indianapolis,
IN 46202, USA
2 Department of Medicine, Indiana University School of Medicine, Indianapolis,
IN 46202, USA
Summary
[11C]CGS 25966, a novel radiolabeled matrix metalloproteinase (MMP)
inhibitor, has been synthesized for evaluation as new potential positron emission
tomography (PET) tumor imaging agent. The precursor was labeled by
[11C]methyl triflate through O-[11C]methylation method at the hydroxyl position
of phenol under basic conditions and isolated by HPLC purification to produce
pure target compound in 15–25% radiochemical yield, based on 11CO2, decay
corrected to end of bombardment. Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: matrix metalloproteinase inhibitor; tumor imaging agent; radio-
tracer; carbon-11; positron emission tomography; [11C]CGS 25966
Introduction
Matrix metalloproteinases (MMPs) are a family of zinc-containing
enzymes. MMP family of enzymes plays a critical role in cancer
* 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
Copyright # 2003 John Wiley & Sons, Ltd.
Received 15 July 2002
Revised 2 August 2002
Accepted 2 October 2002

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X. FEI ET AL.
invasion, metastasis, and angiogenesis.1–3 The overexpression of MMPs
is implicated in tumor growth and metastasis, which provides a
potential target for tumor imaging by medical imaging technique
positron emission tomography (PET).4,5 MMP inhibitors (MMPIs) can
significantly reduce the growth rate of both primary and secondary
tumors and can block the process of metastasis.6 MMPIs are being
actively studied as therapeutic agents.7–14 MMPI radiotracers labeled
with carbon-11 or fluorine-18 may enable non-invasive monitoring of
cancer MMP levels and cancer response to MMPI therapy using PET
imaging techniques.
In our previous work,15–17 the carbon-11 labeling was focused on the
labeling at the aminohydroxyl position of hydroxamic acid CGS
27023A12 to prepare methylated CGS 27023A analogs. In this ongoing
study, the carbon-11 labeling was focused on the labeling at the
methoxyphenyl position of hydroxamic acid CGS 25966.18,19 Here we
report the synthesis of [11C]CGS 25966, an analog of [11C]CGS 27023A.
Results and discussion
CGS 25966 is a potent MMP inhibitor for several MMP subtypes such
as MMP-1 (Ki 43 nM), MMP-2 (Ki 11 nM), MMP-8 (Ki 13 nM), MMP-
9 (Ki 27 nM), and MMP-12 (macrophage metalloelastase, MME; IC50,
4.9 nM).18,19 New MMPI radiotracer [11C]CGS 29566 was synthesized
as shown in Scheme 1.
The key intermediate N-benzyl ester (2) was synthesized in our
previous work16 from commercially available starting material d-valine
in three steps. 2 was hydrolyzed under acidic conditions with
hydrochloride and trifluoroacetic acid (TFA) into its carboxylic acid
and followed by coupling with O-(tert-butyl)hydroxylamine hydro-
chloride in the presence of N-[(dimethylamino)propyl]-N-ethylcarbodii-
mide hydrochloride (EDCI), 1-hydroxybenzotrizole (HOBT) and
N-methylmorphorine (NMM) to give t-butylated hydroxamic acid
(3).12,15,16 Treatment of 3 with aluminum chloride and ethanethiol in
dichloromethane (DCM) unexpectedly removed two protecting groups
methyl and t-butyl to give the O-desmethylated precursor of CGS 25966
(4).
The N-benzyl ester (2) was reacted directly with hydroxylamine
hydrochloride to produce the standard sample unlabeled hydroxamic
acid CGS 25966 (1a), which has been described in our previous work.16
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 343–351

SYNTHESIS OF [11C]CGS 25966
345
Scheme 1. Synthesis of [11/12C]CGS 25966 and [11/12C]CGS 25966 Isomer
The treatment of 2 with aluminum chloride and ethanethiol in
dichloromethane (DCM) gave the compound (5), which was hydrolyzed
into its carboxylic acid followed by coupling with methoxylamine
hydrochloride in the presence of EDCI, HOBT and NMM to provide
the standard sample unlabeled CGS 25966 isomer (1b).
The O-desmethylated precursor of CGS 25966 (4) was labeled by
[11C]methyl triflate20,21 through O-[11C]methylation method22À24 at
hydroxyl position of phenol and/or aminohydroxyl position under basic
conditions and isolated by the reversed high performance liquid
chromatography (HPLC) purification to produce pure desired target
compound [11C]CGS 25966 (1a) in 15–25% radiochemical yields based
on 11CO2, decay corrected to end of bombardment (EOB), and
undesired labeled compound [11C]CGS 25966 isomer (1b) in 20–30%
radiochemical yields. Chemical purity, radiochemical purity, and
specific radioactivity were determined by analytical HPLC method.
The chemical purities of precursor 4 and standard samples 1a and 1b
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X. FEI ET AL.
were >95%, the radiochemical purities of target radiotracer 1a and side
product radiotracer 1b were >99%, and the chemical purities of
radiotracers 1a and 1b were ꢀ95% (1a) and ꢀ97% (1b). The average
(n=5–8) specific radioactivity of radiotracers 1a-b was 0.6–0.8 Ci/mmol
at end-of-synthesis (EOS).
Conclusion
The facile synthetic procedure that provide MMP inhibitor radiotracer
[11C]CGS 25966 (1a) and [11C]CGS 25966 isomer (1b) has been
developed. The published in vitro data indicate CGS 25966 has strong
inhibitory effectiveness for several MMP subtypes. Our results
combined with the literature results warrant further evaluation of
[11C]CGS 25966 as a new potential PET tumor imaging agent 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 procedure20. 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 Bruker Biflex III MALDI-Tof mass
spectrometer, and the high resolution mass measurements were
obtained using a Kratos MS80 mass spectrometer, in the Department
of Chemistry at Indiana University. Chromatographic solvent propor-
tions are expressed on a volume: volume basis. Thin layer chromato-
graphy was run using Analtech silica gel GF uniplates (5 Â 10 cm2).
Plates were visualized by UV light. Normal phase flash chromatography
was carried out on EM Science silica gel 60 (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 Â 250 mm2; 3:1:3 CH3CN: MeOH: 20 mM, pH 6.7
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SYNTHESIS OF [11C]CGS 25966
347
KHPOÀ4 mobile phase, 1.5 ml/min flow rate, and UV (240 nm) and g-ray
(NaI) flow detectors. Preparative HPLC was performed using a Prodigy
(Phenomenex) 5 mm C-18 column, 10 Â 250 mm2; 3:1:3 CH3CN: MeOH:
20 mM, pH 6.7 KHPOÀ4 mobile phase, 5.0 ml/min flow rate, and UV
(240 nm) and g-ray (NaI) flow detectors. Sterile vented Millex-GS
0.22 mM vented filter unit was obtained from Millipore Corporation,
Bedford, MA.
N-(tert-Butyoxy)-2(R)-[[(4-methoxyphenyl)sulfonyl]benzylamino]-3-
methylbutanamide (3)
A mixture of ester 2 (0.73 g, 1.80 mmol) in TFA (3 ml) and concentrated
HCl (4 ml) was refluxed overnight. The solvent was removed under
vacuum. The residue was dissolved in DCM (20 ml). Into this solution
were added EDCI (0.45 g, 2.30 mmol), HOBT (0.36 g, 2.70 mmol) and
NMM (1.0 ml). After the resulting solution was stirred at room
temperature (r.t.) for 1 h, O-(tert-butyl)hydroxylamine hydrochloride
(tBuONH2/HCl) (0.38 g, 3.03 mmol) was added. The reaction mixture
was stirred at r.t. for 1 day. The solvent was removed by evaporation.
The residue was diluted by EtOAc (200 ml), washed by brine (20 ml  2)
and dried over Na2SO4. The evaporation of the solution gave the crude
product, which was purified by flash chromatography (1:5 EtOAc:Hex-
ane) to give a white solid 3 (0.65 g, 78%), TLC Rf 0.50 (1:5
EtOAc:Hexane), m.p. 134–1358C. 1H NMR (300 MHz, CDCl3):
0.73–0.75 (d, J=6.6 Hz, 3 H, CH3CH), 0.82–0.84 (d, J=6.6 Hz, 3 H,
CH3CH), 1.22 (s, 9 H, tBu), 1.96–2.10 (m, 1 H, (CH3)2CH), 3.80 (s, 3 H,
PhOCH3), 3.84–3.87 (d, J=11.1 Hz, 1 H, CHN), 4.45–4.50 (d,
J=15.4 Hz, 1 H, NCHaPh), 4.60–4.65 (d, J=15.1 Hz, 1 H, NCHbPh),
6.89–6.92 (d, J=8.8 Hz, 2 H, H-PhOMe), 7.20-7.23 (m, 3 H, H-Ph),
7.36–7.40 (m, 2 H, H-Ph), 7.66-7.70 (d, J=8.8 Hz, 2 H, H-PhOMe).
N-Hydroxy-2(R)-[[(4-hydroxyphenyl)sulfonyl]benzylamino]-3-methyl-
butanamide (4)
A mixture of ethanethiol (1 ml) and DCM (2 ml) was cooled to 0oC.
Aluminum trichloride (0.14 g, 1.05 mmol) was added, and the mixture
was warmed to r.t. Compound 3 (0.10 g, 0.22 mmol) was added into the
above solution. The reaction mixture was stirred at r.t. for 3 h, and then
quenched with cool water (1 ml). The mixture was diluted with EtOAc
(150 ml), and washed with brine and dried over Na2SO4. The
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X. FEI ET AL.
evaporation gave the crude product, which was purified by flash
chromatography (1:10 MeOH:EtOAc) to give a white solid 4 (66 mg,
1
78%), TLC Rf 0.30 (1:10 MeOH:EtOAc), m.p. 157–1588C. H NMR
(300 MHz, DMSO-d): 0.67–0.69 (d, J=6.6 Hz, 3 H, CH3CH), 0.75–0.77
(d, J=6.6 Hz, 3 H, CH3CH), 1.86–1.98 (m, 1 H, (CH3)2CH), 3.79–3.83
(d, J=11.1 Hz, 1 H, CHN), 4.55–4.60 (d, J=16.1 Hz, 1 H, NCHaPh),
4.67–4.72 (d, J=16.1 Hz, 1 H, NCHbPh), 6.73–6.76 (d, J=8.8 Hz, 2 H,
H-PhOH), 7.19–7.20 (m, 3 H, H-Ph), 7.36–7.39 (m, 2 H, H-Ph),
7.45–7.47 (d, J=8.8 Hz, 2 H, H-PhOH). MS: m/z 378 (M+).
Methyl-2(R)-[[(4-hydroxyphenyl)sulfonyl]benzylamino]-3-methyl-
butanoate (5)
A mixture of ethanethiol (3 ml) and DCM (4 ml) was cooled to 0oC.
Aluminum trichloride (0.73 g, 5.47 mmol) was added, and the mixture
was warmed to r.t. Compound 2 (0.74 g, 1.82 mmol) was added into the
above solution. The reaction mixture was stirred at r.t. for 1 h, and then
quenched with cool water (1 ml). The mixture was diluted with EtOAc
(200 ml), and washed with brine and dried over Na2SO4. The
evaporation gave the crude product, which was purified by flash
chromatography (1:2 EtOAc:Hexane) to give a white solid 5 (0.48 g,
1
67%), TLC Rf 0.40 (1:2 EtOAc:Hexane), m.p. 144–1468C. H NMR
(300 MHz, CD3OD): 0.76–0.78 (d, J=6.6 Hz, 3 H, CH3CH), 0.80–0.82
(d, J=6.6 Hz, 3 H, CH3CH), 1.90–1.98 (m, 1 H, (CH3)2CH), 3.41 (s,
3 H, CO2CH3), 4.11–4.15 (d, J=10.2 Hz, 1 H, CHN), 4.52–4.57 (d,
J=15.5 Hz, 1 H. NCHaPh), 4.64–4.69 (d, J=15.5 Hz, 1 H, NCHbPh),
6.84–6.87 (d, J=8.8 Hz, 2 H, H-PhOH), 7.23–7.29 (m, 3 H, H-Ph),
7.36–7.40 (m, 2 H, H-Ph), 7.61–7.64 (d, J=8.8 Hz, 2 H, H-PhOH).
N-Methoxyl-2(R)-[[(4-hydroxyphenyl)sulfonyl]benzylamino]-3-
methylbutanamide (CGS 25966 isomer, 1b)
A mixture of compound 5 (0.28 g, 0.74 mmol) in TFA (2 ml) and
concentrated HCl (3 ml) was refluxed overnight. After the solvent was
removed under vacuum, the residue was dissolved in DCM (10 ml). Into
this solution was added EDCI (0.21 g, 1.11 mmol), HOBT (0.16 g,
1.19 mmol) and NMM (1.0 ml). After the reaction solution was stirred
at r.t. for 1 h, methoxylamine hydrochloride (MeONH2/HCl) (0.10 g,
1.48 mmol) was added. The reaction mixture was stirred at r.t. for 1 day.
The solvent was removed by evaporation. The residue was diluted by
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SYNTHESIS OF [11C]CGS 25966
349
EtOAc (200 ml), washed by brine (20 ml  2) 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 white
solid 1b (0.11 g, 39%), TLC Rf 0.30 (1:3 EtOAc:Hexane), m.p.
1
164–1678C. H NMR (300 MHz, CD3OD): 0.61–0.63 (d, J=6.6 Hz,
3 H. CH3CH), 0.69-0.71 (d, J=6.6 Hz, 3 H, CH3CH), 1.98–2.11 (m, 1 H,
(CH3)2CH), 3.41 (s, 3 H, CH3ONH), 3.62–3.66 (d, J=11.0 Hz, 1 H,
CHN), 4.68 (bs, 2 H, NCH2Ph), 6.67–6.70 (d, J=8.8 Hz, 2 H, H-PhOH),
7.10–7.14 (m, 3 H, H-Ph), 7.29–7.31 (m, 2 H, H-Ph), 7.43–7.45 (d,
J=8.1 Hz, 2 H, H-PhOH). MS: m/z 393 (MH+).
[11C]CGS 25966 (1a) and [11C]CGS 25966 isomer (1b)
Precursor 4 (0.6–1.0 mg) was dissolved in CH3CN (300 ml). To this
solution was added TBAH (2–8 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 -208C, which was generated by a Venturi cooling device powered with
100 psi compressed air, until radioactivity in solution 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) and 1:1 CH3CN:H2O (0.6 ml), and injected onto the
preparative HPLC column. The product fraction was collected, the
solvent was removed by rotatory evaporation, and the final product 1a
was formulated in saline containing 5% ethanol, 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 (1–3 ml) was noted. The overall synthesis and formulation time
was 30–40 min EOB. The undesired labeled compound 1b was also
isolated by the preparative HPLC purification from radiolabeling
reaction mixture. Retention times in the analytical HPLC system were:
RT4 = 4.72 min, RT1a = 7.34 min, RT1b = 6.40 min. Retention times
in the preparative HPLC system were: RT4 = 5.85 min, RT1a =
9.34 min, RT1b = 8.06 min.
Acknowledgements
This work was partially supported by the Susan G. Komen Breast
Cancer Foundation grant IMG 2000 837 (to QHZ), the Indiana
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University American Cancer Society (ACS) Institutional Grant
Committee grant IRG-84-002-17 (to QHZ), the Indiana University
Cores Centers of Excellence in Molecular Hematology (CCEMH) pilot
and feasibility (P/F) grant (to QHZ), the NIH/NCI grant P20CA86350
(to GDH), the Indiana 21st Century Research and Technology Fund (to
GDH), and the Lilly Endowment Inc. (to the Indiana Genomics
Initiative (INGEN) of Indiana University). We thank Dr. Bruce Mock
and Barbara Glick-Wilson for the efforts in producing carbon-11
precursors. The reviewer and editor’s comments for the revision of the
manuscript are greatly appreciated.
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