Design and synthesis of MMP inhibitors with appended fluorescent tags for imaging and visualization of matrix metalloproteinase enzymes

Article abstract of DOI:10.1016/j.bmcl.2013.08.050

We describe the synthesis, MMP-2 and 9 potency, and in vitro evaluation of a series of α-sulfone hydroxmate MMP inhibitors conjugated to a series of dyes with different absorption/emission lamina maxima's that can be used to visualize tumors.

Full text of DOI:10.1016/j.bmcl.2013.08.050

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5566–5570
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of MMP inhibitors with appended fluorescent
tags for imaging and visualization of matrix metalloproteinase
enzymes
a
a,b,
John N. Freskos a, , Bethel Asmelash , Kimberly R. Gaston
, Amol Karwa b, Tim A. Marzan a,
⇑
Maureen A. Nickols b, Thomas E. Rogers a,b,à, Tasha Schoenstein a, Carolyn J. Sympson b, Bich Vu a
a Department of Chemistry, Mallinckrodt Pharmaceuticals, 675 McDonnell Blvd., Hazelwood, MO 63042, United States
b Department of Biology, Mallinckrodt Pharmaceuticals, 675 McDonnell Blvd., Hazelwood, MO 63042, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe the synthesis, MMP-2 and 9 potency, and in vitro evaluation of a series of a-sulfone hydrox-
mate MMP inhibitors conjugated to a series of dyes with different absorption/emission lamina maxima’s
that can be used to visualize tumors.
Received 8 July 2013
Revised 6 August 2013
Accepted 9 August 2013
Available online 16 August 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Imaging agents
MMP inhibitors
Tumor visualazation
Matrix metalloproteinases (MMPs) are a class of zinc-depen-
dent endopeptidases having functionality for degradation and
remodeling of the extracellular matrix and subendothelial base-
ment membrane.1 This family of more than twenty enzymes is fur-
ther classified into several main groups, including gelatinases,
interstitial collagenases, stromelysins, and membrane-type matrix
metalloproteinases. In particular, there is considerable evidence
demonstrating a close association between upregulation of certain
MMPs and the proliferation, invasive behavior and metastatic po-
application for diagnosis, staging, and monitoring of cancer. This
approach may also be useful for evaluation of tissue degradation
associated with pathological conditions such as osteoarthritis, ath-
erosclerosis, and wound repair. In addition, optical agents capable
of selective delivery to cells and to tissue expressing MMPs also
have potential for visually differentiating the tissue of a tumor or
lesion from normal tissue during a surgical procedure, such as a
biopsy or therapeutic tissue removal. This approach was used to
conjugate a derivative of the broad spectrum MMP inhibitor
CGS25966 to a NIR dye and in vitro results indicated that this ap-
proach was viable.6
tential of certain types of tumors.2–4 The discovery of the
a-sulfone
hydroxamates by researchers at Searle/Pharmacia illustrates the
focus on selectivity, potency, and achieving suitable ADME proper-
ties. The optimization of these ADME properties led to inhibitors
with good oral bioavailability and an approriate T1/2 to achieve effi-
cacy in various models of osteoarthritis, cancer, and myocardial
remodeling after an ischemic event.5a,b While MMPs are a well rec-
ognized target for the development of therapeutic and diagnostic
agents, their potential for biomedical imaging and visualization re-
mains much less explored. Optical agents for imaging the expres-
sion of MMPs in certain tumors and lesions has potential
Recently we reported the discovery of a novel set of pyrazine
dyes whose properties as pure GFR agents are in the process of
being evaluated in the clinic for noninvasive, realtime monitoring
of kidney function.7a,b It was of interest to see if these pyrazines,
as well as other dyes, could be tethered to the a-sulfone hydroxa-
mate MMP inhibitor class and still retain MMP activity as well as
maintaining good optical fluorescence properties. Indeed from
the data presented in Table 1 we now demonstrate that this is pos-
sible.8 As shown earlier, through numerous X-ray co-crystal struc-
tures of the
a-sulfone hydroxamate series of MMP inhibitors,
substituents attached to the piperidine nitrogen are solvent ex-
posed and thus ammenable to putting large groups off the nitrogen
while still retaining high potency for MMP’s of interest and sparing
MMP-1.5a–c This is demonstrated quite profoundly by the fact that it
is possible to put a 5000 mer Peg-DSPE (Distearylphosphitylester)
off of the piperidine nitrogen and still maintain low nanomolar
⇑
Corresponding author. Tel.: +1 1 314 654 3148; fax: +1 1 314 654 8343.
E-mail addresses: john.freskos@mallinckrodt.com, freskos@covidien.com
(J.N. Freskos).
Current address: Monsanto Co., 800 N. Lindberg Blvd., St. Louis, MO 63167, United
States.
à
Current address: Center for Emerging Technologies, 4041 Forest Park Ave., St.
Louis, MO 63108, United States.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.050

J. N. Freskos et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5566–5570
5567
Table 1
IC50 and UV absorption and emission properties (kmax) of MMP–dye conjugates
Compound
hMMP-2 (nM)
hMMP-9 (nM)
k (abs) nM
k (emis) nM
O
O O
S
HO
N
OCF3
OCF3
H
<1
<1
ND
ND
O
O
N
1
O O
S
O
O
HO
N
H
N
5.1
4.1
450
550
NH
N
N
NH2
O
H
N
H2N
O
2
O
O
O O
HO
S
OCF3
N
H
O
N
N
NH2
N
<1
<1
420
520
N
NH2
O
3
O
N
O O
S
HO
HO
OCF3
N
H
O
N
1.6
<1
400
500
N
CN
N
4
O
O O
S
OCF3
O
N
H
O
OH
O
N
H
<1
<1
488
525
N
O
O
NH2
O
5
HO
O
O
O O
S
HO
N
OCF3
N
H
N
2.8
1.6
>700
>700
N
-O3S
SO3
-
6
potency.10 Interestingly, unlike the approach with CGS25966
where a fairly long (3-mer Peg-amide-5-carbon alkyl) linker was
needed to separate the MMP-inhibitor from the dye,6 we have
found that short linkers are acceptable and that the linker may
even be omitted. Compound 1 was our internal standard and its
synthesis has been described elsewhere.5a,b Like compound 1 all
of our MMP–dye’s will spare MMP-1 and be active against not only
MMP-2 and -9 but also against MMP-3, -8, and -13.5a,b This is due
in the S1 binding site of the MMP-1 enzyme, which is much smal-
ler than MMP’s-2, 3, 9, 13, and 14. The MMP-imaging agents 2 and
5 have very short linkers while 3, 4 and 6 have no linker and all yet
still retain excellent potency for MMP-2 and -9. The synthesis of
compounds 2–6 are shown below. The synthesis of MMP-pyrazine
(Scheme 1) 2 starts with commercially available diacid 8, and the
EDC/HOBt mediated coupling affords the desired product plus
the two symmetrical isomers which were separated by Flash
chromatography. Basic hydrolysis (KOH/EtOH 80 °C) to acid 12
followed by standard (EDC–HOBt) coupling to the key amine
intermediate 7 yielded the amide 13. The preparation of 7 has been
0
0
to the phenyl-O-phenyl-OCF3 moiety that lies in the S1 binding
pocket of the various MMP enzymes, unlike the smaller
phenyl-OMe which is present in CGS25966 and enables it to fit

5568
J. N. Freskos et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5566–5570
O
MeO2C
N
N
NH2
HO2C
H2N
N
NH2
EDC
Symmetrical
adducts
N
H
H2N
+
+
CO2Me
H
+
H2N
N
NH2
N
8
CO2H
9
10
O
11
O
O
O
O
S
O
O
O
O
O
KOH
OCF3
HO2C
N
N
NH2
S
OCF3
O
N
H
+
H
N
O
H2N
O
N
N
EDC
O
H
NH
12
7
N
N
NH2
O
H
O
O
N
O
S
O
H2N
HO
OCF3
N
H
O
13
1) KOH
2) THPONH2 / EDC
3) TFA
O
N
NH
N
N
NH2
O
H
N
H2N
O
2
Scheme 1. Synthesis of imaging agent 2.
O
O
O
O
O
O
O
EDC
S
OCF3
S
OCF3
N
N
NH2
OH
O
O
HO
+
Symmetrical
adducts
+
O
N
O
H2N
N
NH2
N
H
O
O
N
7
8
N
O
NH2
O
O
O
HO
S
OCF3
14
N
H
1) KOH
2) THPONH2
3) TFA
O
N
NH2
N
O
N
N
NH2
O
3
Scheme 2. Synthesis of imaging agent 3.
O
O
S
O
OCF3
O
O
O
O
Br
N
CN
Br
N
CN
N
S
OCF3
O
+
O
N
N
NH2
N
O
N
15
N
16
H
7
N
CN
N
O
O
O
17
HO
S
OCF3
N
H
O
N
N
N
CN
N
4
Scheme 3. Synthesis of imaging agent 4.

J. N. Freskos et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5566–5570
5569
O
Cbz
O
S
O
HN
OCF3
O
O
O
S
O
OCF3
+
EDC
O
O
N
H
N
Boc
O
O
Cbz
N
H
CO2H
N
H
HN
O
7
18
Boc
19
O
O
HO
S
OCF3
O
O
O
O
O
N
H
1) KOH
2) THPONH2
3) H2 / Pd-C
1) EDC
2) TFA
O
O
S
OCF3
N
H
O
OH
O
N
H
N
O
N
O
O
NH2
NH2
O
HN
Boc
5
HO
20
Scheme 4. Synthesis of imaging agent 5.
O
O
O
S
O
O
O
S
HO
N
OCF3
Cl
HO
OCF3
N
N
H
N
H
N
+
O
O
N
-
-O3S
N
SO3
H
21
7
N
-
-O3S
SO3
6
Scheme 5. Synthesis of imaging agent 6.
Figure 1. Compound 5 uptake in MCF7 cells at 37 °C.
described elsewhere.5a The conversion from 13 to the desired
hydroxamate 2 was accomplished by the three-step sequence
shown below involving hydrolysis, EDC-mediated coupling with
THP-protected hydroxylamine, and removal of the THP protecting
group with trifluroacetic acid.
The synthesis of compound 3 is shown in Scheme 2 and involves
the direct coupling of 7 and 8 in the presence of 1 equivalents of
pyyrolidine to afford, after chromatography, the unsymmetrical
amide 14, which was converted to 3 as described above.
The synthesis of imaging agent 4 is shown in Scheme 3. The
commercially available pyrazine 15 was alkylated with methyl
iodide and Cs2CO3 in DMF at room temperature to afford 16. Palla-
dium catalyzed coupling afforded, after RPHPLC purification, 60% of
the desired adduct 17. The conversion to hydroxamate 4 was
accomplished as described above.
The synthesis of imaging agent 5 is shown in Scheme 4. The
amine 7 was coupled with Boc-Lys(Z)-OH under standard condi-
tions to afford amide 19. This was then hydrolyzed and coupled
to afford the protected hydroxamate that was then hydrogenated
with Pd–C to afford the amine 20. EDC mediated coupling with
5-FAM followed by de-protection afforded imaginng agent 5.
The synthesis of the Near Infared Red (NIR) dye-MMP inhibitor 6
is shown in Scheme 5. Reaction of the amine 7 with the cy5.5 deriv-
ative 21 afforded the desired imaging agent in 10–20% yield and the
main product obtained was from displacement of chloride with
hydroxide. No attempts were made to optimize these conditions.
In Figure 1 we show the specificity of compound 5 when
incubated with parental MCF7 and MMP-14 (MT-1 MMP)
overexpressing MCF7 cells as visualized by confocal microscopy.
This compound showed preferential accumulation in the MMP-14

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J. N. Freskos et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5566–5570
Yao, J. J. Med. Chem. 2010, 53, 6653; (c) Barta, T. E.; Becker, D. P.; Bedell, L. J.;
Easton, A. E.; Hockerman, S. L.; Kiefer, J.; Munie, G. M.; Mathis, K. J.; Li, M. H.;
Rico, J. G.; Villamil, C. I.; Williams, J. M. Bioorg. Med. Chem. Lett. 2011, 21, 2820.
6. Faust, A.; Waschkau, B.; Waldeck, J.; Holtke, C.; Breyholz, H. J.; Wagner, S.;
Kopka, K.; Schober, O.; Heindel, W.; Schafers, M.; Bremer, C. Bioconjugate Chem.
2009, 20, 904.
7. (a) Rajagopalan, R.; Neumann, W. L.; Poreddy, A. R.; Fitch, R. M.; Freskos, J. N.;
Asmelash, B.; Gaston, K. R.; Galen, K. P.; Shieh, J.-J.; Dorshow, R. B. J. Med. Chem.
2011, 54, 5048; (b) Poreddy, A. R.; Neumann, W. L.; Freskos, J. N.; Rajagopalan,
R.; Asmelash, B.; Gaston, K. R.; Fitch, R. M.; Shieh, J. J.; Dorshow, R. B. Bioorg.
Med. Chem 2012, 20, 2490.
8. Freskos, J. N., WO 2013/039851 Optical Agents for Imaging and Visualization of
Matrix Metalloproteinase Enzymes.
9. The MMP-14 overexpressing MCF7 cells where kindly provided by Professor
Alex Strongin from the Burnham Research Institue.
10. Remacle, A. G.; Shiryaev, S. A.; Golubkov, V. S.; Freskos, J. N.; Brown, M.; Karwa,
A.; Naik, A. D.; Howard, C. P.; Sympson, C. J.; Strongin, A. Y. J. Biol. Chem. 2013,
288, 20568.
11. MMP-2 and MMP-14 enzyme activities were measured using recombinant
human proteins with substrate and protocols provided by R&D Systems,
overexpressing compared to parental cells when incubated at
37 °C.9,10 Compound 5 was tested against MMP-14 and found to
have an IC50 of ꢀ100 nM.11
MCF7 parental cells are shown at left and the MMP-14 over-
expressing MCF7 cells at right.
Summary:We have described a novel series of MMP–dye conju-
gates that possess the ability to visualize MMP-bearing cells
in vitro. Compounds having increased potency toward MMP-14
to elaborate this technology will be described in a susquent
disclosure.
Acknowledgements
The authors would like to thank Professor Alex Strongin from
the Burnham Research Institute for kindly providing the MMP-14
overexpressing MCF7 cells that were employed in the confocal
microscopy experiments. The authors would like to thank Ms.
Laura Lorenz and Ms. Michelle Rieken for help in the preparation
of this manuscript.
Minneapolis, MN. For the assays, 25
activated recombinant human protein were combined in 96 black well plates
at ambient temperature, and 50 L of substrate (Mca-Lys-Pro-Leu-Gly-Leu-
lL of 5ꢁ test samples and 50 lL of
l
Dpa-Ala-Arg-NH2, R&D ES010) was then added to start the reaction. (All
components were prepared at concentrations and in appropriate buffers as
listed below.) The plates were then read at 320 nm excitation/405 nm emission
using the kinetic mode of a fluorescence plate reader. A substrate blank was
subtracted from duplicate determinations and IC50 values from six-point dose
response curves were calculated using GraphPad Prism software, La Jolla, CA.
References and notes
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2. Heath, E. I.; Grochow, L. B. Drugs 2000, 59, 1043.
3. Hidalgo, M.; Eckhardt, G. J. Natl. Cancer Inst. 2001, 93, 178.
4. Stamenkovic, I. Emin. Cancer Biol. 2000, 10, 415.
5. (a) Becker, D. P.; Barta, T. E.; Bedell, L.; Boehm, T. L.; DeCrescenzo, G. A.; Freskos,
J. N.; Getman, D. P.; Hockerman, S. L.; Heintz, R. M.; Howard, S. C.; Li, M.;
McDonald, J. J.; Mehta, P.; Munie, G. E.; Swearingen, C. A.; Carron, C. P.;
Funckes-Shippy, C. L. J. Med. Chem. 2005, 48, 6713; (b) Becker, D. P.; Barta, T. E.;
Bedell, L. J.; Boehm, T. L.; Bond, B. R.; Caroll, J.; Carron, C. P.; DeCrescenzo, G. A.;
Easton, A. M.; Freskos, J. N.; Funckes-Shippy, C. L.; Heron, M.; Hockerman, S. L.;
Howard, C. P.; Kiefer, J. R.; Li, M.; Mathis, K. J.; McDonald, J. J.; Mehta, P.; Munie,
G. E.; Sunyer, T.; Swearingen, C.; Vilamil, C. I.; Welsch, D.; Williams, J. M.; Yu, Y.;
For the MMP-14 enzyme assay, 40
activated with 0.86 g/mL rhFurin (R&D 1503-SE) in activation buffer (50 mM
Tris, 1 mM CaCl2, 0.05% (v/v) Brij-35, pH 9) and incubated at 37 °C for 1.5 °h.
The activated protein was then diluted to 1.24 g/mL in assay buffer (50 mM
Tris, 3 mM CaCl2, 1 M ZnCl2, pH 8.5), for use in the assay. The substrate was
used at 20 M in assay buffer. For the MMP-2 enzyme assay, 100 g/mL
lg/mL of rhMMP-14 (R&D 918-MP) was
l
l
l
l
l
of rhMMP2 (R&D 902-MP) was activated with 1 mM APMA (p-
aminophenylmercuric acetate) in assay buffer (50 mM Tris, 10 mM CaCl2,
150 mM NaCl, 0.05% (v/v) Brij-35, pH 7.5) and incubated at 37 °C for 1 h. The
activated protein was then diluted to 248 ng/mL in assay buffer for use in the
assay. The substrate was used at 25 lM in assay buffer.