Synthesis of radioiodinated probes targeted toward matrix metalloproteinase-12

Article abstract of DOI:10.1016/j.bmcl.2017.11.027

Matrix metalloproteinase-12 (MMP-12, macrophage elastase) is a member of the MMP family that is responsible for the degradation of extracellular matrix, and is associated with the inflammatory process of chronic obstructive pulmonary disease (COPD). COPD, characterized by progressive and irreversible airflow obstruction, is recently a major cause of mortality and morbidity worldwide. Herein, to develop radioiodinated probes for the early diagnosis of COPD, we designed and synthesized novel MMP-12-targeted dibenzofuran compounds (1–3) with a variety of linker structures (carbamate, amide, and sulfonamide). In competitive enzyme activity assays, it was revealed that the linker structures significantly affected the inhibitory activity against and selectivity for MMP-12. Compound 1, with carbamate linker, demonstrated potent MMP-12 inhibitory activity (IC₅₀ = 8.5 nM) compared to compound 2, with amide linker, and compound 3, with sulfonamide linker. Using bromo-substituted carbamate 13 as a radioiodination precursor, [¹²⁵I]1 was successfully prepared to high radiochemical purity (over 98%) and good specific radioactivity (4.1 GBq/μmol). These results suggest that radioiodinated compound 1 is potent as a novel MMP-12-targeted probe.

Full text of DOI:10.1016/j.bmcl.2017.11.027

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of radioiodinated probes targeted toward matrix
metalloproteinase-12
Masayori Hagimori a,b, Takashi Temma c,d, Shinji Kudo a, Kohei Sano a, Naoya Kondo c,d, Takahiro Mukai a,
⇑
a Kobe Pharmaceutical University, 4-19-1 Motoyamakita Machi, Higashinada-ku, Kobe 658-8558, Japan
b Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
c Department of Investigative Radiology, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan
d Department of Biofunctional Analysis, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Matrix metalloproteinase-12 (MMP-12, macrophage elastase) is a member of the MMP family that is
responsible for the degradation of extracellular matrix, and is associated with the inflammatory process
of chronic obstructive pulmonary disease (COPD). COPD, characterized by progressive and irreversible
airflow obstruction, is recently a major cause of mortality and morbidity worldwide. Herein, to develop
radioiodinated probes for the early diagnosis of COPD, we designed and synthesized novel MMP-12-tar-
geted dibenzofuran compounds (1–3) with a variety of linker structures (carbamate, amide, and sulfon-
amide). In competitive enzyme activity assays, it was revealed that the linker structures significantly
affected the inhibitory activity against and selectivity for MMP-12. Compound 1, with carbamate linker,
demonstrated potent MMP-12 inhibitory activity (IC50 = 8.5 nM) compared to compound 2, with amide
linker, and compound 3, with sulfonamide linker. Using bromo-substituted carbamate 13 as a radioiod-
ination precursor, [125I]1 was successfully prepared to high radiochemical purity (over 98%) and good
Received 21 September 2017
Revised 10 November 2017
Accepted 14 November 2017
Available online xxxx
Keywords:
MMP-12
COPD
Radioiodine
Dibenzofuran
Carbamate
specific radioactivity (4.1 GBq/
lmol). These results suggest that radioiodinated compound 1 is potent
as a novel MMP-12-targeted probe.
Ó 2017 Elsevier Ltd. All rights reserved.
Matrix metalloproteinases (MMPs) are enzyme families of zinc-
dependent endopeptidases, and 24 members of MMPs have been
discovered in humans.1–4 MMPs catalyze the remodeling of several
extracellular matrix molecules, including collagen, proteoglycan,
laminin, and elastin.5,6 In addition, MMPs participate in cleaving
cell surface proteins and mediate the processing of bioactive
molecules.1 Among these, MMP-12, known as a macrophage elas-
tase, is secreted predominantly from alveolar macrophages.7–9
MMP-12 is also associated with pulmonary inflammatory diseases,
including chronic obstructive pulmonary disease (COPD).10,11The
number of COPD patients is recently increasing, and could become
the third leading global cause of death.12,13 Since COPD is consid-
ered to be an irreversible disease,14,15 the early detection of
MMP-12 related to pulmonary inflammation diseases is important
in order to determine appropriate therapeutic strategies to delay
the disease progression,16 thereby leading to improvement in prog-
nosis and quality of life of patients.
structure-activity relationship (SAR) studies.17 Although the shape
and size of the specificity loops were similar between MMP-12 and
MMP-13, the dibenzofuran derivatives with a carbamate linker
revealed higher affinity for MMP-12 than for MMP-13 due to a
change in the dihedral angle and a restriction in the rotation of
the core structure associated with the S10 pocket.17 Of these deriva-
tives, MMP-408 revealed potent inhibitory activity against and
selectivity to MMP-12, and prevented lung inflammation induced
by recombinant human MMP-12 in C57BL/6 mice. In accordance
with these reports, we planned to develop a novel radioiodinated
MMP-408 derivative 1 (Fig. 1) as an MMP-12 in vivo imaging probe.
In addition, in order to investigate the linker effective for the inter-
action with the S10 pocket of MMP-12,17 we also designed com-
pound
2 with an amide linker and compound 3 with a
sulfonamide linker. Herein, we synthesized a series of MMP-12-
targeted iodinated compounds (1–3) to diagnose early-stage COPD
and evaluated their in vitro inhibitory activities against MMP-12
and MMP-13. Compound 1, with potent inhibitory activity toward
MMP-12, was labeled with radioiodine.
For the treatment of COPD, Li et al. previously reported selective
inhibitors of MMP-12 based on dibenzofuran scaffolds through
For MMP-12 selective inhibitors based on dibenzofuran scaf-
folds, SAR studies revealed that carboxylic acid was essential
for zinc chelation, and the dibenzofuran structure was strongly
⇑
Corresponding author.
E-mail address: tmukai@kobepharma-u.ac.jp (T. Mukai).
https://doi.org/10.1016/j.bmcl.2017.11.027
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Hagimori M., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.11.027

2
M. Hagimori et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
a yield of 78%. After the coupling reaction of 7 with L-valine tert-
butyl ester, Pd/C catalyzed the hydrogenation generated interme-
diate 9 with a yield of 56%. Carbamate derivatives 10–12 were
obtained through the reaction of intermediate 9 with different
chloroformates. Target compound 1 was obtained through the
deprotection of tert-butyl ester with trifluoroacetic acid, generated
with a yield of 77%. Similarly, fluorinated compound 4 and bromi-
nated compound 13 were synthesized from 11 or 12 with yields of
69 and 89%, respectively. The synthesis of compound 2 with an
amide linker and compound 3 with a sulfonamide linker is pre-
sented in Scheme 2. The condensation reaction of intermediate 9
with 4-iodobenzoyl chloride yielded derivative 14, and the depro-
tection of tert-butyl ester of 14 with trifluoroacetic acid generated
the desired compound 2 with a yield of 79%. The compound 3 was
prepared by the reaction of intermediate 9 with 4-iodophenylsul-
fonyl chloride followed by treatment with trifluoroacetic acid.
To elucidate the in vitro affinity for MMP-12 and MMP-13, we
performed competitive enzyme activity assays by reacting the syn-
thesized fluorogenic peptide substrates with recombinant human
MMP-12 and MMP-13. The IC50 values of compounds 1–3 were
evaluated using compound 4 as a positive control (Table 1). Iodi-
nated compound 1 revealed a higher potent inhibitory activity
against MMP-12 (IC50 = 8.5 nM) than for MMP-13. The IC50 value
of 1 was almost similar to that of fluorinated compound 4, indicat-
ing that replacing the fluorine atom with an iodine atom at the
para position of the phenyl carbamate moiety did not affect the
inhibitory activities against MMP-12 and MMP-13. Replacing the
carbamate linker with an amide or sulfonamide linker (2 and 3)
resulted in the reduction of the inhibitory activities against
MMP-12 and MMP-13. Compound 2, with the amide linker,
demonstrated selectivity toward MMP-12; however, the inhibitory
activity of 2 for MMP-12 was about 60 times lower compared to
that of 1. Compound 3, with the sulfonamide linker, revealed less
selectivity and lower inhibitory activity against both MMP-12
and MMP-13. These results suggested that compound 1 could be
a useful probe for MMP-12. Radiosynthesis was then performed
using compound 1.
Fig. 1. Chemical structures of MMP-12-targeted compounds.
associated with the S10 pocket;17 however, substitution at the C7 or
C8 position of the dibenzofuran structure was effective in affording
MMP-12 selectivity over MMP-13.17 Since fluorinated compound 4
also revealed MMP-12 selectivity over MMP-13,17 we designed (S)-
((8-(2-(4-iodophenyl)acetamido)dibenzo[b,d]furan-3-yl)sulfonyl)-
L
-valine (1) as the MMP-12-targeted iodinated compound (Fig. 1).
As previously reported,17 the key intermediate 9 was prepared
via a four-step reaction, as presented in Scheme 1. Dibenzofuran-
3-sulfonyl chloride (6) was readily prepared from 5 with sulfur
dioxide gas in the presence of CuCl. Nitration of 6 using trifluo-
roacetic acid and nitric acid produced the nitro derivative 7 with
The radioiodinated compound [125I]1 was prepared as pre-
sented in Scheme 3. Due to its relatively long half-life and low
energy gamma-rays, 125I was selected as a radioiodine in this
study. The [125I]10 was radiosynthesized by copper-catalyzed halo-
gen exchange reaction18 of brominated precursor 12 with sodium
[
125I]iodide followed by direct heating at 130 °C for 30 min. Since
compound 13 (the esterolysis product of 12) also revealed MMP-
12 inhibitory activity (IC50 = 11 nM) similar to compound
1
(Table 1), it was necessary to completely separate [125I]10 and 12
after radioiodination. As presented in Fig. 2, the radio-HPLC analy-
sis of the crude mixture indicated that the radioactive peak derived
from the desired [125I]10 was detected at 15 min, which was
Scheme 1. Synthesis of 1, 4, and 13. Reagents and conditions: (a) glacial acetic acid,
cHCl, NaNO2 in H2O, SO2 (gas), CuCl 2H2O, rt, (b) HNO3, TFA, rt, (c) L-valine t-butyl
ester, N,N-diisopropylethylamine, rt, (d) H2, Pd/C, (e) DMAP, 4-iodophenyl chloro-
formate or 4-bromophenyl chloroformate or 4-fluorophenyl chloroformate, rt, (f)
TFA, CH2Cl2.
Scheme 2. Synthesis of 2 and 3. Reagents and conditions: (a) trimethylamine, 4-
iodobenzoyl chloride, (b) TFA, CH2Cl2, (c) trimethylamine, 4-iodophenylsulfonyl
chloride, (d) TFA, CH2Cl2.
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M. Hagimori et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Table 1
In vitro affinity of MMP-12-targeted compounds for MMP-12 and MMP-13.
Fig. 3. HPLC analysis of [125I]1. HPLC conditions: the column was a COSMOSIL
Cholester 4.6 mm ꢀ 250 mm, flow rate was 1.0 mL/min, UV excitation at 254 nm,
mobile phase systems were CH3CN (0.1% TFA): H2O (0.1% TFA) = 60:40.
Compound
X
Y
IC50
MMP-12
MMP-13
1
2
3
4
CO
CO
SO2
CO
CO
O-(4-I)phenyl
(4-I)phenyl
(4-I)phenyl
O-(4-F)phenyl
O-(4-Br)phenyl
8.5 5.7
502 169
297 70
6.2 4.0
11 6.0
69 11
>2000
403 98
65 2.2
51 9.9
halogen exchange reaction was optimized to improve the radio-
chemical yield of [125I]10. As presented in Table 2, Entry 5, the
radiochemical yield of [125I]10 was improved to 13% when the
reaction was conducted at 140 °C for 60 min. Radioiodinated com-
pound [125I]1 was obtained by the deprotection of the tert-butyl
ester of [125I]10 with trifluoroacetic acid, with a yield of 91%. The
radiochemical purity and specific activity of [125I]1 were over
13
Data represent mean SD (n = 3).
98% and 4.1 GBq/lmol, respectively (Fig. 3).
We successfully designed and synthesized a series of MMP-12-
targeted compounds (1–3) based on the dibenzofuran scaffold. In
this study, we identified the linker structures that significantly
affected the inhibitory activity against and selectivity for MMP-
12. Of the new compounds, compound 1 with a carbamate linker
demonstrated potent inhibitory activity against MMP-12. Copper-
catalyzed halogen exchange reaction of brominated precursor 12
with [125I]NaI yielded [125I]1 at high radiochemical purity (over
Scheme 3. Synthesis of [125I]1. Reagents and conditions: (a) [125I]NaI, CuSO4 5H2O,
(NH4)2SO4, (b) TFA, CH2Cl2.
98%) and good specific radioactivity (4.1 GBq/lmol). These data
indicate that radioiodinated compound 1 would be a potent
MMP-12-targeted probe for in vivo imaging.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number
24591813 (T.T) and GSK Japan Research Grant 2015 (T.T).
Fig. 2. HPLC analysis of [125I]10 coinjected with precursor compound 12. HPLC
conditions: the column was a COSMOSIL Cholester 4.6 mm ꢀ 250 mm, flow rate
was 1.0 mL/min, UV excitation at 254 nm, mobile phase systems were CH3CN (0.1%
TFA): H2O (0.1% TFA) = 75:25.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2017.11.027.
different from the bromo-substituted carbamates precursor 12 (13
min); however, the radiochemical yield of [125I]10 was only 2%
(Table 2, Entry 1). Therefore, the reaction condition of Cu catalyzed
References
1. Puente XS, Sánchez LM, Overall CM, López-Otín C. Nat Rev Genet. 2003;4:544.
2. Page-McCaw A, Ewald AJ, Werb Z. Nat Rev Mol Cell Biol. 2007;8:221.
3. Jones CB, Sane DC, Herrington DM. Cardiovasc Res. 2003;59:812.
4. Zitka O, Kukacka J, Krizkova S, et al. Curr Med Chem. 2010;17:3751.
5. Parks WC, Wilson CL, Lopez-Boado YS. Nat Rev Immunol. 2004;4:617.
6. Belvisi MG, Bottomley KM. Inf Res. 2003;52:95.
Table 2
Br/I halogen exchange reaction of compound 12.
7. Lamort AS, Gravier R, Laffitte A, Juliano L, Zani ML, Moreau T. Biol Chem.
2016;397:469.
8. Houghton AM, Grisolano JL, Baumann ML, et al. Cancer Res. 2006;66:6149.
9. Egeblad M, Werb Z. Nat Rev Cancer. 2002;2:161.
10. Nénan S, Planquois JM, Berna P, et al. Int Immunopharmacol. 2005;5:511.
11. Shapiro SD, Kobayashi DK, Ley TJ. J Biol Chem. 1993;268:23824.
12. Devereux G. BMJ. 2006;332:1142.
13. Le Quément C, Guénon I, Gillon JY, et al. Br J Pharmacol. 2008;154:1206.
14. Chee A, Sin DD. Int J Chron Obstruct Pulmon Dis. 2008;3:563.
15. Hansen EF, Phanareth K, Laursen LC, Kok-Jensen A, Dirksen A. Am J Respir Crit
Care Med. 1999;159:1267.
Entry
Na125
(MBq)
I
Temperature
(°C)
Time
(min)
Radiochemical
Yield (%)
1
2
3
4
5
6
7
8
9
2.9
3.0
3.8
3.4
3.5
3.7
2.5
2.4
2.5
130
130
130
140
140
140
150
150
150
30
60
90
30
60
90
30
60
90
2
4
5
6
13
9
3
7
7
16. Demedts IK, Morel-Montero A, Lebecque S, et al. Thorax. 2006;61:196.
17. Li W, Li J, Wu Y, et al. J Med Chem. 2009;52:1799.
18. Kiyono Y, Kanegawa N, Kawashima H, et al. Nucl Med Biol. 2003;30:697.
Please cite this article in press as: Hagimori M., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.11.027