Novel Potent Proline-Based Metalloproteinase Inhibitors: Design, (Radio)Synthesis, and First in Vivo Evaluation as Radiotracers for Positron Emission Tomography

Article abstract of DOI:10.1021/acs.jmedchem.6b01291

As dysregulation of matrix metalloproteinase (MMP) activity is associated with a wide range of pathophysiological processes like cancer, atherosclerosis, and arthritis, MMPs represent a valuable target for the development of new therapeutics and diagnostic tools. We herein present the chiral pool syntheses, in vitro evaluation, and SAR studies of a series of d- and l-proline- as well as of (4R)-4-hydroxy-l-proline-derived MMP inhibitors possessing general formula 1. Some of the synthesized hydroxamic acids were found to be potent MMP inhibitors with IC₅₀ values in the nanomolar range, also demonstrating no off-target effects toward the other tested Zn²⁺-dependent metalloproteases (ADAMs and meprins). Utilizing the structure of the (2S,4S)-configured 4-hydroxyproline derivative 4, a selective picomolar inhibitor of MMP-13, the radiolabeled counterpart [¹⁸F]4 was successfully synthesized. The radiotracer's biodistribution in mice as well as its serum stability were evaluated for assessing its potential use as a MMP-13 targeting PET imaging agent.

Full text of DOI:10.1021/acs.jmedchem.6b01291

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Article
Novel Potent Proline-based Metalloproteinase Inhibitors:
Design, (Radio)Synthesis and First in Vivo Evaluation
as Radiotracers for Positron Emission Tomography
Dmitrii V. Kalinin, Stefan Wagner, Burkhard Riemann, Sven Hermann, Frederike
Schmidt, Christoph Becker-Pauly, Stefan Rose-John, Michael Schäfers, and Ralph Holl
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01291 • Publication Date (Web): 03 Oct 2016
Downloaded from http://pubs.acs.org on October 4, 2016
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Novel Potent Prolineꢀbased Metalloproteinase
Inhibitors: Design, (Radio)Synthesis and First in
Vivo Evaluation as Radiotracers for Positron
Emission Tomography
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a,b
c
c
b,d
e
Dmitrii V. Kalinin, Stefan Wagner, Burkhard Riemann, Sven Hermann, Frederike Schmidt,
e
e
b,d
a,b,f,g,
Christoph Becker-Pauly, Stefan Rose-John, Michael Schäfers, and Ralph Holl
*
a
Institut für Pharmazeutische und Medizinische Chemie der Westfälischen WilhelmsꢀUniversität
Münster, Corrensstr. 48, 48149 Münster, Germany
b
CellsꢀinꢀMotion Cluster of Excellence (EXC 1003 ꢀ CiM), University of Münster, Germany
c
Department of Nuclear Medicine, University Hospital Münster, AlbertꢀSchweitzerꢀCampus 1,
Building A1, 48149 Münster, Germany
d
European Institute for Molecular Imaging, University of Münster, Waldeyerstraße 15, 48149
Münster, Germany
e
Biochemical Institute, ChristianꢀAlbrechtsꢀUniversity Kiel, Germany
f
German Center for Infection Research (DZIF), partner site HamburgꢀLübeckꢀBorstel
g
Institut für Organische Chemie, Universität Hamburg, MartinꢀLutherꢀKingꢀPlatz 6, 20146
Hamburg, Germany
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ABSTRACT
As dysregulation of matrix metalloproteinase (MMP) activity is associated with a wide range
of pathophysiological processes like cancer, atherosclerosis, and arthritis, MMPs represent a
valuable target for the development of new therapeutics and diagnostic tools. We herein present
the chiral pool syntheses, in vitro evaluation and SAR studies of a series of Dꢀ and Lꢀprolineꢀ as
well as of (4R)ꢀ4ꢀhydroxyꢀLꢀprolineꢀderived MMP inhibitors possessing general formula 1.
Some of the synthesized hydroxamic acids were found to be potent MMP inhibitors with IC ꢀ
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values in the nanomolar range, also demonstrating no offꢀtarget effects towards the other tested
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Zn ꢀdependent metalloproteases (ADAMs and meprins). Utilizing the structure of the (2S,4S)ꢀ
configured 4ꢀhydroxyproline derivative 4, a selective picomolar inhibitor of MMPꢀ13, the
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radiolabeled counterpart [ F]4 was successfully synthesized. The radiotracer’s biodistribution in
mice as well as its serum stability were evaluated for accessing its potential use as a MMPꢀ13
targeting PET imaging agent.
INTRODUCTION
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The metzincin superfamily of proteases comprises most of the Zn ꢀdependent
metalloproteases. The metzincins are mainly involved in the degradation and remodeling of
extracellular matrix (ECM) components, such as collagens, but also cleave nonꢀECM proteins
like von Willebrand factor, aggrecan, cytokines, and substance P. Typical representatives of
metzincins are the MMPs (matrix metalloproteinase), the ADAMs (a disintegrin and
1
metalloproteinase), and the astacins, including the meprins. Despite relatively low amino acid
2
+
sequence identity, these enzymes contain the conserved HExxHxxG/NxxH/D Zn ꢀbinding motif
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in their active site and exhibit an analogous threeꢀdimensional organization of their catalytic
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, 3
domains.
MMPs play an essential role in important remodeling events such as tissue turnover, wound
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healing, and angiogenesis. In humans, MMPs currently comprise more than 23 different
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members. Historically MMPs are classified according to their ability to degrade specific
substrates, thus distinguishing collagenases (MMPꢀ1, ꢀ8, ꢀ13), gelatinases (MMPꢀ2, ꢀ9),
stromelysins (MMPꢀ3, ꢀ10, ꢀ11), matrilysins (MMPꢀ7), metalloelastases (MMPꢀ12), membraneꢀ
type MMPs (MTꢀMMPs: MMPꢀ14ꢀ17, MMPꢀ24ꢀ25), and other MMPs (MMPꢀ19ꢀ23, MMPꢀ26ꢀ
8
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8). MMPs share similar general structural features, typically comprising four distinct domains:
a proꢀdomain, which keeps MMPs inactivated until it is removed; a catalytic domain, where the
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+
substrate cleavage takes place and which contains the Zn ꢀbinding motif HExxHxxGxxH; a
linker region of variable length; and a haemopexinꢀlike domain, the soꢀcalled substrate
9
specificity site, which forms a propeller blade structure.
Similar to MMPs, ADAMs, which are transmembrane or secreted metalloproteases, contain a
proꢀdomain and a catalytic domain with corresponding functions. But additionally, they contain a
unique disintegrin domain, a cysteineꢀrich domain, an epidermal growth factorꢀlike domain, and
1
0
a transmembrane domain; they also contain a Cꢀterminal cytoplasmic tail. ADAMs shed (“cut
off”) extracellular portions of transmembrane proteins (e.g. receptors), releasing the soluble
1
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ectodomains from the cell surface, thereby activating them.
Finally, meprins, members of the astacin family, represent complex multiꢀdomain
metalloproteases comprising meprin α and meprin β, which form disulfideꢀlinked homoꢀ or
heterodimers. Meprin α and meprin β degrade a variety of biologically active proteins, but
12
exhibit different substrate specificities. For example, whereas gastrointestinal peptides like
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gastrin and cholecystokinin are readily cleaved by meprin β, substance P and cytokines represent
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substrates for meprin α. Moreover, both meprins cleave proteins of the ECM like collagen type
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IV, lamininꢀ1, nidogenꢀ1, and fibronectin. Importantly, meprins together with other members of
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the astacin family induce collagen deposition by cleaving off the Cꢀ and Nꢀterminal prodomains
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of fibrilar collagens I and III.
Many pathophysiological processes are related to the dysregulation of MMPs, ADAMs, and
meprins. For instance, the overexpression of MMPs and ADAMs is related to cardiovascular risk
factors and promotes arterial remodeling (intimalꢀmedial thickening, fibrosis, calcification),
15ꢀ17
resulting in atherosclerosis and cardiovascular disease progession.
Increased levels of MMPꢀ
9
were found to be associated with neuronal damage and apoptosis in animal models of cerebral
1
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ischemia and in human stroke. Furthermore, metalloproteases promote hallmarks of cancer
such as cell proliferation (MMPꢀ1ꢀ3, ꢀ7, ꢀ9, ꢀ11, ꢀ19, ADAM12 etc.), cell migration (MMPꢀ1ꢀ3, ꢀ
7, ꢀ9, ꢀ13, ꢀ14, ADAM10 etc.), cell invasion (MMPꢀ2, ꢀ9, ꢀ14 etc.), metastasis (MMPꢀ8), and
angiogenesis (MMPꢀ2, ꢀ9, ꢀ14), thereby playing a key role in the development of human breast,
19, 20
colon, thyroid, lung and prostate cancers.
Moreover, MMPs have been found to be involved
in joint cartilage destruction associated with osteoarthritis. MMPꢀ13 is considered as the main
contributor to this pathology due to its ability to degrade type II collagen, the major structural
2
1ꢀ23
protein of articular cartilage.
Notably, MMP activity is also associated with wide a range of
respiratory diseases like idiopathic pulmonary fibrosis, asthma, emphysema and the acute
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respiratory distress syndrome.
Regarding ADAMs, sheddases ADAM10 and ADAM17 (also known as TACE ꢀ tumor
necrosis factorꢀαꢀconverting enzyme) are involved in the generation of amyloid plaques and also
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shed inflammatory mediators promoting thereby cancer, inflammation and Alzheimer’s
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disease.
Finally, meprins have been reported to be crucial for the development of inflammatory
disorders like acute renal failure, urinary tract infections and inflammatory bowel disease, as
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well as fibrosis and neurodegeneration.
Therefore, metalloproteinases represent valuable targets for the development of new
therapeutics and diagnostic tools. Additionally, they can serve as determinants for understanding
the pathophysiology of inflammatory conditions. In this respect, the nonꢀinvasive imaging and
the quantification of MMP activity in vivo using (radio)labeled inhibitors are of great
(pre)clinical interest.
Despite evident and convincing data, indicating that MMP inhibitors have a high potential for
treating different pathologies, their further use in the clinic has been stalled, mainly due to their
limited proof of efficacy and their side effects, which might be related to nonꢀselective inhibition
2
4, 27, 28
of metalloproteases.
Therefore, gaining selectivity between the metalloproteases without
decreasing inhibitory potency is a primary goal in the development of MMP inhibitors, which
would help to improve their efficacy and avoid undesirable side effects.
In order to find new effective inhibitors of MMPs, this work is focused on the development of
compounds, that mimick structural features of collagen, the main component of the ECM, which
is a principal and universal substrate for many metalloproteases. Being the most abundant protein
in mammals, collagen exhibits a triple helix (three supercoiled polyproline IIꢀlike chains) with a
repetitive XaaYaaGly sequence, in which Xaa is often (2S)ꢀproline and Yaa is often (2S,4R)ꢀ4ꢀ
2
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hydroxyproline.
In this work, metalloprotease inhibitors were developed, which possess
proline as well as hydroxyproline scaffolds and display general structure 1 (Figure 1). To ensure
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the desired selectivity of the designed inhibitors, apart from alterations of the main scaffold,
particular emphasis was placed on structural variations of the lipophilic side chain, addressing
the S1' pocket of the enzymes, which is located on their “specificity loop” and which is highly
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variable among different MMPs. Xꢀray crystallographic data of different MMP inhibitors in
complex with various MMPs indicate that some MMPs, especially MMPꢀ13 (but also MMPꢀ3,
MMPꢀ8, and MMPꢀ9), are able to accommodate ligands with long lipophilic residues addressing
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the S1' pocket and its unique S1'' side pocket. In contrast, other MMPs, like for instance MMPꢀ
1, MMPꢀ7 and MMPꢀ11 have only a shallow S1' pocket, so compounds that inhibit these
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enzymes must have should have a shorter lipophilic tail. Therefore, in order to achieve
selectivity for MMPꢀ13, the envisaged compounds in this study possess a long lipophilic side
chain, containing a diphenylacetylene moiety, which was varied in length by attaching
substituents on the distal phenyl ring. The activity of the synthesized compounds towards MMPꢀ
2, ꢀ8, ꢀ9, and MMPꢀ13 was investigated and their selectivity over other metzincins, such as
ADAM10, ADAM17, meprin α, and meprin β, was studied.
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18F
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[18
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X = CH2, C=O
R = H, alkyl, alkoxy, NH2, NR2, F
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R = H, OH, NH2, F
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F]FB-ML5
Figure 1. General Structure (1) of the envisaged proline derivatives and structures of
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radiofluorinated MMP and ADAM inhibitors for PET. The Zn ꢀbinding groups are highlighted
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in red.
Besides inhibition of the enzymes, noninvasive imaging of metalloprotease activity in vivo is
of enormous interest in basic research and in clinical applications. Therefore, attempts to prepare
and evaluate radiolabeled MMPꢀ and ADAMꢀtargeting ligands for positron emission tomography
37
(
PET) were undertaken. Some selected examples are shown in Figure 1 with the structures of
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[ F]FBꢀML5, [ F]2 and [ F]3.
Utilizing the unique pharmacological profile of the (2S,4S)ꢀ
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ꢀhydroxyproline based compound 4, a selective picomolar inhibitor of MMPꢀ13 (Table 1),
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which was synthesized in the course of the present study, the radiofluorinated PET tracer [ F]4
was successfully developed (Figure 1) and its first in vivo study was performed.
RESULTS AND DISCUSSION
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Chemistry. The envisaged proline derivatives exhibit a lipophilic side chain comprising a
diphenylacetylene moiety, which should be built up via a Sonogashira coupling reaction. In order
to obtain compounds with various substituents at the terminal phenyl ring, a series of substituted
phenylacetylene derivatives was synthesized. Based on the described synthesis of
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morpholinomethylꢀsubstituted phenylacetylene derivative 8d, pyrrolidine (5a), piperidine (5b),
ꢀhydroxypiperidine (5c) and thiomorpholine (5e) were alkylated with 4ꢀbromobenzyl bromide
to yield tertiary amines 6aꢀc,e (Scheme 1).
4
6-8
R
(a)
(b)
(c)
a
b
c
d
e
f
5a: n = 0, X = CH2
5b: n = 1, X = CH2
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c: n = 1, X = CHOH
d: n = 1, X = O
e: n = 1, X = S
f: n = 1, X = SO2
(d)
Scheme 1. Synthesis of phenylacetylene derivatives 8aꢀf. Reagents and conditions: (a) 4ꢀ
bromobenzyl bromide, ACN, ꢁ, 6a 69%, 6b 85%, 6c 90%, 6d 98%, 6e 92%, 6f 40%; (b)
trimethylsilylacetylene, Pd(PPh ) , CuI, NEt , ꢁ, 7a 88%, 7b 90%, 7c 86%, 7d 71%, 7e 71%, 7f
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82%; (c) TBAF, THF, 0 °C, 8a 69%, 8b 70%, 8c 47%, 8d 99%, 8e 82%, 8f 89%; (d) 1. Boc O,
2
38, 39
NEt , CH Cl , rt, 2. mCPBA, CH Cl , 3. HClꢀsaturated MeOH, ꢁ, 75% (3 steps).
3
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2
Subsequently, a Sonogashira coupling with trimethylsilylacetylene was performed to give
phenylacetylene derivatives 7aꢀc,e. Finally the trimethylsilyl protective group of amines 7aꢀc,e
was cleaved with tetrabutylammonium fluoride to yield terminal alkynes 8aꢀc,e. Additionally,
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sulphone 5f was synthesized from thiomorpholine according to a described procedure and
converted analogously to the corresponding phenylacetylene derivative 8f (Scheme 1).
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R
b
d
e
f
(
a)
(c)
(b)
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g
h
H
NH
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Scheme 2. Synthesis of Lꢀproline derivatives 12. Reagents and conditions: (a) 4ꢀiodobenzoyl
chloride, NEt , DMAP, CH Cl , 0 °C → rt, 86%; (b) Pd(PPh ) , CuI, NEt , ACN, rt, 11b 60%,
3
2
2
3 4
3
1
1d 92%, 11e 99%, 11f 92%, 11g 99%, 11h 60%; (c) H NOH·HCl, NaOMe, MeOH, rt, 12b
2
4
9%, 12d 49%, 12e 45%, 12f 56%, 12g 48%, 12h 66%.
The desired prolineꢀderived hydroxamic acids were synthesized in chiral pool syntheses.
Starting from Lꢀproline methyl ester hydrochloride (9), the 3,4ꢀunsubstituted pyrrolidine
derivatives were obtained (Scheme 2). The acylation of secondary amine 9 with 4ꢀiodobenzoyl
chloride yielded amide 10, which was subsequently subjected to Sonogashira couplings with
several of the previously synthesized terminal alkynes as well as some commercially available
phenylacetylene derivatives. The CꢀC coupling reactions gave diphenylacetylene derivatives
1
1b,dꢀh, which were finally transformed into hydroxamic acids 12b,dꢀh by reacting the esters
with hydroxylamine. The (R)ꢀconfigured hydroxamic acid ent-12d, the enantiomer of
morpholinomethylꢀsubstituted diphenylacetylene derivative 12d, was synthesized in the same
way, starting from Dꢀproline methyl ester hydrochloride.
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(h)
2
1
14
18
(i)
(b)
(f)
15,16,19,
R
2
0,22,23
a
b
c
d
e
22
15
19
(
j)
(c)
(g)
g
h
i
H
NH
2
N(CH
OCH
3 2
)
j
3
k
23
16
20
Scheme 3. Synthesis of 4ꢀsubstituted Lꢀproline derivatives 16, 20, and 23. Reagents and
conditions: (a) 4ꢀiodobenzoyl chloride, NEt , DMAP, CH Cl , 0 °C → rt, 97%; (b) Pd(PPh ) ,
3
2
2
3 4
CuI, NEt , ACN, rt, 15d 99%, 15g 75%, 15h 79%, 15i 83%, 15j 100%, 15k 100%; (c)
3
H NOH·HCl, NaOMe, MeOH, rt, 16d 26%, 16g 66%, 16h 54%, 16i 41%, 16j 38%, 16k 51%;
2
(
d) pꢀnitrobenzoic acid, DIAD, PPh , THF, 0 °C → rt, 80%; (e) K CO , MeOH, rt, 84%; (f)
3 2 3
Pd(PPh ) , CuI, NEt , ACN, rt, 19a 92%, 19b 94%, 19c 98%, 19d 69%, 19e 96%, 19g 99%, 19h
3
4
3
7
8%, 19i 93%, 19j 98%, 19k 100%; (g) H NOH·HCl, NaOMe, MeOH, rt, 20a 22%, 20b 47%,
2
20c 20%, 20d 44%, 20e 44%, 20g 61%, 20h 35%, 20i 52%, 20j 49%, 20k 57%; (h) phthalimide,
DIAD, PPh , THF, 0 °C → rt, 61%; (i) Pd(PPh ) , CuI, NEt , ACN, rt, 22d 85%, 22g 90%; (j)
3
3 4
3
H NOH·HCl, NaOMe, MeOH, rt, 23d 55%, 23g 79%.
2
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The 4ꢀsubstituted pyrrolidine derivatives could be accessed from (2S,4R)ꢀconfigured 4ꢀ
hydroxyproline derivative 13 (Scheme 3). Its reaction with 4ꢀiodobenzoyl chloride gave
benzamide 14, which was coupled with various phenylacetylene derivatives and transformed into
hydroxamic acids 16d,gꢀk as described for the 3,4ꢀunsubstituted pyrrolidine derivatives.
In order to obtain (4S)ꢀconfigured 4ꢀhydroxyproline derivatives, a Mitsunobu reaction was
performed. The transformation of (4R)ꢀconfigured 4ꢀhydroxyproline derivative 14 with pꢀ
nitrobenzoic acid in the presence of DIAD and triphenylphosphine yielded the (4S)ꢀconfigured
ester 17, which was subsequently cleaved to yield 4ꢀhydroxyproline derivative 18 (Scheme 3).
Analogous to its (4R)ꢀconfigured diastereomer 13, aryl iodide 18 was subjected to Sonogashira
couplings with miscellaneous phenylacetylene derivatives and subsequently transformed into
hydroxamic acids 20aꢀe,gꢀk.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Additionally, the hydroxy group in position 4 of the pyrrolidine ring should be replaced by an
amino group. Therefore, 4ꢀhydroxyproline derivative 14 was reacted with phthalimide under
Mitsunobu conditions (Scheme 3). Subsequently, the resulting (4S)ꢀconfigured pyrrolidine
derivative 21 was coupled with phenylacetylene and the morpholinomethylꢀsubstituted
phenylacetylene derivative 8d, yielding esters 22g and 22d, respectively. In order to obtain the
desired hydroxamic acids, esters 22g and 22d were reacted with hydroxylamine. Under these
conditions, besides the aminolysis of the ester group, the cleavage of the phthalimide moiety
occurred, giving access to primary amines 23g and 23d.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
a)
(b)
(c)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
: R‘ = H
24: R‘ = H
25: R‘ = OH
26d:
26e:
27d:
R‘ = H,O R =
R‘ = H,O R =
R‘ = OH, R =
28d:
28e:
29d:
R‘ = H,O R =
R‘ = H,O R =
R‘ = OH, R =
13: R‘ = OH
Scheme 4. Synthesis of tertiary amines 28d,e and 29d. Reagents and conditions: (a) 4ꢀ
iodobenzyl bromide, NEt , 24 47%, 25 64%; (b) Pd(PPh ) , CuI, NEt , ACN, rt, 26d 85%, 26e
3
3 4
3
9
9%, 27d 93%; (c) H NOH·HCl, NaOMe, MeOH, rt, 28d 53%, 28e 38%, 29d 40%.
2
Additionally, a series of tertiary amines was envisaged. In order to obtain these compounds,
proline derivative 9 and 4ꢀhydroxyproline derivative 13 were Nꢀalkylated with 4ꢀiodobenzyl
bromide yielding amines 24 and 25, respectively (Scheme 4). Sonogashira couplings of aryl
iodides 24 and 25 with phenylacetylene derivatives 8d and 8e gave diphenylacetylene derivatives
2
6d, 26e, and 27d, which were finally transformed into hydroxamic acids 28d, 28e, and 29d via
an aminolysis with hydroxylamine.
Synthesis of fluorinated compounds. To evaluate potential radiofluorinated MMP tracers
based on the proline and 4ꢀhydroxyproline lead structure in vitro and in vivo, the nonꢀradioactive
counterparts were synthesized according to Scheme 5 and 6 for initial in vitro evaluation.
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9
(b)
(c)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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56
57
58
59
60
3
0: X = C=O
32: X = C=O
33: X = CH2
34: X = C=O
35: X = CH2
31: X = CH2
(
a)
(d)
(e)
14: X = C=O
36
37
25: X = CH2
Scheme 5. Synthesis of the fluorinated proline and 4ꢀhydroxyproline derivatives 34, 35, and
7. Reagents and conditions: (a) DAST, CH Cl , ꢀ78 °C → rt, 30 65%, 31 49%; (b)
3
2
2
phenylacetylene, Pd(PPh ) , CuI, NEt , ACN, 32 98%, 33 80%; (c) H NOH·HCl, NaOMe,
3
4
3
2
MeOH, rt, 34 67%, 35 78%; (d) 4ꢀfluorophenylacetylene, Pd(PPh ) , CuI, NEt , ACN, 95%; (e)
3
4
3
H NOH·HCl, NaOMe, MeOH, rt, 27%.
2
First, utilizing key intermediate 14, via a Sonogashira coupling reaction with 4ꢀ
fluorophenylacetylene, diphenylacetylene derivative 36 was obtained, which was subjected to an
aminolysis with hydroxylamine to yield the desired fluorinated hydroxamic acid 37 (Scheme 5).
Then, the 4ꢀfluorine substituted proline derivatives 34 and 35 were synthesized (Scheme 5). At
40
first, alcohols 14 and 25 were fluorinated by treating the compounds with DAST.
Subsequently, the obtained alkyl fluorides 30 and 31, which exhibit (S)ꢀconfiguration in position
of the pyrrolidine ring, were coupled with phenylacetylene and subjected to an aminolysis with
4
hydroxylamine yielding hydroxamic acids 34 and 35 (Scheme 5).
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
a)
(b)
(c)
3
8
39
40
41
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
d)
(e)
(f)
42
43
4
Scheme 6. Synthesis of fluorinated 4ꢀhydroxyproline derivative 4. Reagents and conditions:
(
a) trimethylsilylacetylene, Pd(PPh ) , CuI, NEt , ꢁ, 85%; (b) TBAF, THF, 0 °C, 90%; (c) TsCl,
3
4
3
NEt , CH Cl /pyridine (4:1), 0 °C → rt, 73%; (d) TBAF, 65 °C, 70%; (e) 18, Pd(PPh ) , CuI,
3
2
2
3 4
NEt , ACN, rt, 90%; (f) H NOH·HCl, NaOMe, MeOH, rt, 54%.
3
2
Finally, 4ꢀhydroxyproline derivative 4, possessing a 5ꢀfluoropentoxy substituent in the paraꢀ
position of the terminal phenyl ring, was synthesized (Scheme 6). To build up the desired
4
1
lipophilic tail, aryl bromide 38 was subjected to a Sonogashira coupling reaction with
trimethylsilylacetylene and a subsequent desylilation to give phenylacetylene derivative 40. The
hydroxy group of 40 was then tosylated and the resulting tosylate 41 was subjected to a SN2
4
2
reaction with tetraꢀnꢀbutylammonium fluoride (TBAF)
to give 5ꢀfluoropentoxy
phenylacetylene derivative 42. Lastly, using a Sonogashira reaction, 42 was coupled with 4ꢀ
hydroxyproline derivative 18 to give ester 43, which was transformed into the desired
hydroxamic acid 4 by performing an aminolysis with hydroxylamine.
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BIOLOGICAL EVALUATION
In Vitro MMPs Assays and SAR. The synthesized proline hydroxamates were assayed in
vitro against the activated collagenases MMPꢀ8 and MMPꢀ13 as well as against gelatinases
MMPꢀ2 and MMPꢀ9 using synthetic fluorogenic substrates according to a previously reported
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
3
procedure.
ylmethyl)phenyl]sulfonamide}ꢀ3ꢀmethylbutanamide (44, CGS 27023A)
in vivo efficacy, was used as a positive control. The IC ꢀvalues obtained in the performed in
The potent MMP inhibitor (R)ꢀNꢀhydroxyꢀ2ꢀ{[4ꢀmethoxyꢀNꢀ(pyridinꢀ3ꢀ
4
4, 45
possessing proven
5
0
vitro inhibition assays of hydroxamates 12, 16, 20, 23, 28, and 29 are summarized in Table 1.
Table 1. MMP inhibitory activity of the synthesized prolineꢀbased hydroxamates 12, 16, 20, 23,
28, and 29 compared to 44.
IC ± SD [nM]
50
Stereoꢀ
isomer
1
Comp
X
R
R
MMPꢀ2
2000 ± 182
47 ± 5
MMPꢀ8
MMPꢀ9
2000 ± 120
146 ± 9
MMPꢀ13
2000 ± 497
29 ± 1
1
2
1
2
1
2
1
2
6g
0g
6j
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
H
H
OH
OH
OH
OH
OH
OH
OH
OH
(2S,4R)
(2S,4S)
(2S,4R)
(2S,4S)
(2S,4R)
(2S,4S)
(2S,4R)
(2S,4S)
392 ± 68
11 ± 1
OCH
173 ± 5
56 ± 4
n.d.
161 ± 11
52 ± 3
n.d.
3
3
0j
OCH
98 ± 30
103 ± 8
45 ± 14
651 ± 203
585 ± 122
33 ± 2
6h
0h
6i
NH
2
2
500 ± 27
57 ± 4
610 ± 36
61 ± 2
709 ± 32
35 ± 7
NH
N(CH
N(CH
)
538 ± 30
92 ± 4
612 ± 77
108 ± 4
141 ± 44
8 ± 7
3
2
2
0i
3
)
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2
2
2
2
2
2
3
3
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3
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3
3
3
3
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4
4
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5
5
5
5
5
5
5
6
1
6k
0k
C=O CH
2
2
(CH
(CH
N
2
)
)
3
3
CH
3
3
OH
OH
(2S,4R)
(2S,4S)
151 ± 4
3 ± 1
282 ± 101
15 ± 4
845 ± 46
120 ± 10
11 ± 7
2
C=O CH
2
CH
0.70 ± 0.01
1000 ± 363
16d
20d
20b
C=O
C=O
C=O
C=O
C=O
OH
OH
OH
OH
OH
(2S,4R)
(2S,4S)
(2S,4S)
(2S,4S)
(2S,4S)
1000 ± 149
55 ± 3
2000 ± 628 9000 ± 70
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
N
198 ± 10
n.d.
1000 ± 6
57 ± 9
n.d.
334 ± 32
87 ± 12
2000 ± 130
20e
20a
1000 ± 319 1000 ± 432
330 ± 121 1000 ± 106
19 ± 1
139 ± 21
S
N
526 ± 43
1
2g
2h
C=O
C=O
H
NH
N
H
H
(2S)
(2S)
168 ± 22
26 ± 1
250 ± 21
955 ± 32
130 ± 8
1
2
1000 ± 133
636 ± 114
1000 ± 78
12d
C=O
C=O
C=O
H
H
H
(2S)
(2R)
(2S)
570 ± 141
503 ± 390
450 ± 21
2000 ± 409 4000 ± 126
280 ± 27
636 ± 57
171 ± 42
O
O
S
entꢀ
1
N
N
50 ± 15
n.d.
731 ± 293
2d
12e
4000 ± 168
N
O
C=O
S
H
H
(2S)
457 ± 37
n.d.
5000 ± 102
219 ± 38
1
2f
O
N
12b
23g
23d
C=O
C=O
C=O
(2S)
807 ± 149
4630 ± 650
>10000
3000 ± 159 6000 ± 237
573 ± 44
3090 ± 526
>10000
H
NH
NH
(2S,4S)
(2S,4S)
n.d.
4060 ± 142
>10000
2
N
>10000
2
O
O
S
O
N
N
N
2
9d
8e
CH
CH
CH
2
2
2
OH
H
(2S,4R)
(2S)
>10000
>10000
n.d.
>10000
n.d.
>10000
n.d.
2
4000 ± 1000
2
8d
4
H
(2S)
3000 ± 415
4 ± 0.001
5000 ± 474
11 ± 2
>10000
11 ± 2
939 ± 92
16 ± 4
4
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n.d. – not determinable
As shown in Table 1, the newly synthesized proline derivatives are potent MMP inhibitors
with IC ꢀvalues mostly in the nanomolar range. Notably, when comparing the inhibitory
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
0
activities of the different hydroxamic acids toward the investigated MMPs, most of the
compounds were found to preferentially inhibit MMPꢀ13. Among them, the (2S,4S)ꢀconfigured
4ꢀhydroxyproline derivative 20k, bearing a long and flexible lipophilic tail, effectively inhibited
MMPꢀ13 in the picomolar range (IC (MMPꢀ13) = 0.7 nM) with selectivities over the other
5
0
tested MMPs ranging from 4 to 170. It was established, that the structure of the lipophilic tail as
well as the configuration of the pyrrolidine scaffold significantly affected MMP inhibitory
activity and selectivity. Thus, (2S,4R)ꢀ4ꢀhydroxyproline derivative 16g showed weak inhibition
against MMPꢀ2, ꢀ9, and ꢀ13 in the micromolar range with a slightly more pronounced activity
toward MMPꢀ8 (IC (MMPꢀ8) = 392 nM). In contrast, its diastereomer 20g, possessing an
50
unnatural (2S,4S)ꢀ4ꢀhydroxyproline scaffold, was strikingly 14ꢀ to 69ꢀfold more active toward all
four tested MMPs with a comparable selectivity toward MMPꢀ8 (IC (MMPꢀ8) = 11 nM).
5
0
Irrespective of the substituent at the terminal phenyl ring of the lipophilic side chain, a similar
trend was apparent for all pairs of diastereomers that were compared (e.g. 16h and 20h, 16i and
20i as well as 16k and 20k, Table 1).
The removal of the hydroxy group of the (2S,4S)ꢀconfigured 4ꢀhydroxyproline derivatives
leads to a loss of inhibitory activity, resulting in (2S)ꢀproline hydroxamates with inhibitory
activities generally ranging between those of the respective diastereomeric 4ꢀhydroxyproline
derivatives. Thus, for instance, poorly active (2S,4R)ꢀconfigured compound 16g (IC ꢀvalues
5
0
ranging from 0.39 to 2 ꢂM against the four tested MMPs) can be transformed into the more
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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5
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5
5
5
5
5
6
potent hydroxamic acid 12g, with IC ꢀvalues from 26 to 250 nM, by removing its hydroxyl
5
0
group (Table 1).
These data confirm that the synthesized (2S,4S)ꢀ4ꢀhydroxyproline derivatives are more potent
inhibitors of the investigated MMPs than compounds with a (2S,4R)ꢀ4ꢀhydroxyproline or a (2S)ꢀ
proline scaffold. In order to rationalize these observations, the compounds were docked into the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
6
available crystal structure of MMPꢀ13 (PDB: 1KRY). As the hydroxy groups of both
diastereomeric 4ꢀhydroxyproline scaffolds point out from the active site and are highly exposed
to the solvent (Figure 2A) without directly interacting with the enzyme, no clear explanation for
that phenomenon can be given based on the obtained docking scores and poses. Additionally, no
obvious clashes were observed between the hydroxy groups of the (2S,4R)ꢀ4ꢀhydroxyproline
derivatives and the lipophilic residue Leu184 of the MMPꢀ13 enzyme. However, the close
proximity of these two groups (ca. 3.2 Å for the (4R)-configured diastereomer compared to ca.
4.5 Å for the (4S)ꢀconfigured stereoisomer, Figure 2) in the active site is unfavorable and might
result in the decreased potency of the (2S,4R)ꢀ4ꢀhydroxyprolineꢀbased compounds. This could
also explain the partial recovery of inhibitory activity in case of the (2S)ꢀproline derivatives,
which lack the 4ꢀhydroxy group.
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
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58
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60
Figure 2. A: Compound 20g (in yellow), 16g (in cyan), and 12g (in magenta) docked in the
4
6
2+
active site of MMPꢀ13 (PDB: 3KRY). The Zn ꢀion is shown as a gray sphere; amino acid
residues in proximity to the surface are depicted as white stick models (Leu184 is depicted as
yellow stick model). Oxygen and nitrogen atoms are colored in red and blue, respectively. The
surface is colored as follows: lipophilic regions are in green, hydrophilic in magenta and neutral
in white B: LigPlotꢀgenerated twoꢀdimensional schematic diagram of MMPꢀ13 – hydroxamic
acid 20g interactions. Hydrogen bonds are dashed orange lines, hydrophobic contacts are
2
+
represented by an arc with spokes radiating towards the ligand atoms. The Zn ꢀion is shown as a
green sphere.
The exchange of the hydroxy group in position 4 of the pyrrolidine ring with an amino group
leading to (2S,4S)ꢀconfigured primary amines 23g and 23d was found to be detrimental for MMP
inhibitory activity.
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4
4
4
4
5
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5
5
5
5
5
5
5
5
6
In case of the Lꢀproline derivative 12d, possessing a morpholinomethylꢀsubstituted
diphenylacetylene moiety as lipophilic fragment, the inversion of configuration in position 2 of
the pyrrolidine ring from (S) to (R), leading to compound entꢀ12d, has ambiguous effects.
Whereas no alteration of MMPꢀ2 inhibitory potency was observed, MMPꢀ8 and MMPꢀ9 were
significantly more susceptible to inhibition by (R)ꢀproline derivative entꢀ12d (Table 1). By
contrast, in the case of MMPꢀ13, compound 12d, possessing a Lꢀproline scaffold, showed a
lower IC ꢀvalue (280 nM) compared to its (R)ꢀconfigured enantiomer entꢀ12d (IC (MMPꢀ13) =
0
1
2
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5
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7
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9
0
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0
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9
0
1
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3
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5
6
7
8
9
0
5
0
50
636 nM).
Whereas the synthesized amides are generally able to strongly inhibit the proteolytic activity of
the examined MMPs, the tertiary amines 28d, 29d, and 28e exhibit little to no inhibitory activity.
This finding can be attributed to the absence of the carbonyl oxygen atom in these compounds,
which in the case of the amides undergoes strong interactions with Leu185 or Ala186. Similar
interactions with Leu185 or Ala186 can be observed in Xꢀray crystal structures of MMPꢀ13 in
complex with sulfonamideꢀbased inhibitors like Nꢀhydroxyꢀ1ꢀ(2ꢀmethoxyethyl)ꢀ4ꢀ({4ꢀ[4ꢀ
46
(trifluoromethoxy)phenoxy]phenyl}sulfonyl)piperidineꢀ4ꢀcarboxamide (SCꢀ78080).
The substituent in the paraꢀposition of the distal phenyl ring of the synthesized
diphenylacetylene derivatives was found to influence the inhibitory potency of the compounds.
For Lꢀproline derived hydroxamic acids 12g, 12h, 12d, 12e, 12f, and 12b, the introduction of a
substituent at the terminal phenyl ring generally leads to a decrease in inhibitory potency against
all tested MMPs. Whereas for the primary amine 12h the observed loss in potency is most
pronounced against MMPꢀ2 and MMPꢀ13, the piperidine derivatives 12d, 12e, 12f, and 12b are
particularly less active against MMPꢀ8 and MMPꢀ9 compared to the unsubstituted
diphenylacetylene derivative 12g.
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For the 4ꢀhydroxyproline derivatives contrary observations were made. For the (2S,4R)ꢀ
configured diastereomer, a substituent in the paraꢀposition of the distal phenyl ring generally led
to an increase in inhibitory activity against all tested MMPs compared to the unsubstituted
compound 16g. Whereas for hydroxamic acid 16h the introduction of the amino group led to a
three to four fold enhancement in inhibitory potency against all tested MMPs, compound 16i,
bearing a more lipophilic N,Nꢀdimethylamino group showed a distinct increase in inhibitory
activity against MMPꢀ13. The trend is even more pronounced for the nꢀpentylꢀsubstituted
compound 16k, resulting in a 13ꢀfold increase in inhibitory potency against MMPꢀ2, a 2.4ꢀfold
increased inhibitory activity against MMPꢀ9 and a 181ꢀfold increased affinity toward MMPꢀ13,
when compared to unsubstituted compound 16g. In this series of compounds, only the
morpholinomethyl substituted hydroxamic acid 16d was equally or even less active than
compound 16g.
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For the (2S,4S)ꢀconfigured 4ꢀhydroxyproline derivatives, the introduction of a relatively small
substituent, like an amino or a methoxy group, in the paraꢀposition of the distal phenyl ring has
relatively little effect on inhibitory activity. Like its diastereomer 16i, N,Nꢀdimethylaminoꢀ
substituted compound 20i was found to exhibit selectivity toward MMPꢀ13 over the other tested
MMPs. The potency increase of the N,Nꢀdimethylamino derivatives toward MMPꢀ13 can be
explained by the fact, that, in comparison to the respective primary amines, their two methyl
groups form additional lipophilic interactions with hydrophobic residues Pro255, Phe252 and
Leu218 in the S1’ pocket of MMPꢀ13 (Figure 3, left).
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Figure 3. Compound 20i (cyan, left) and 20d (magenta, right) docked in the active site of
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MMPꢀ13 (PDB: 3KRY). The Zn ꢀion is shown as a gray sphere; amino acid residues in
proximity to the surface are depicted as white stick models (Pro255, Phe252, Leu218, and
Lys249 are depicted as yellow stick models). Oxygen and nitrogen atoms are colored in red and
blue, respectively. The surface is colored as follows: lipophilic regions are in green, hydrophilic
in magenta and neutral in white.
Further variations of the amino group in the paraꢀposition of the diphenylacetylene moiety led
to heterocyclic compounds 20d, 20b, 20e, and 20a. Whereas pyrrolidine derivative 20a and
piperidine derivative 20b are weak MMP inhibitors, their more polar morpholine (20d) and
thiomorpholine (20e) analogs were found to be more active, especially against MMPꢀ2 and
MMPꢀ13. This latter finding is in agreement with docking studies showing that the morpholine
moiety of compound 20d is located in proximity to Lys249 at the bottom of the S1’ pocket of
MMPꢀ13 and that the morpholine oxygen atom is able to form a hydrogen bond with this amino
acid (Figure 3, right).
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Again, the introduction of a flexible lipophilic nꢀpentyl moiety was found to be most beneficial
in increasing inhibitory activity and, more importantly, in improving selectivity toward MMPꢀ13.
(
2S,4S)ꢀ4ꢀHydroxyproline derivative 20k demonstrates an over 15ꢀfold enhanced inhibition of
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MMPꢀ2 (IC (MMPꢀ2) = 3 nM) and an over 40ꢀfold increased inhibition of MMPꢀ13
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0
(IC (MMPꢀ13) = 0.7 nM) compared to the unsubstituted analog 20g. The extraordinarily high
5
0
inhibitory activity of 20k toward MMPꢀ13 can be explained by greasy interactions of the
lipophilic nꢀpentyl chain, protruding deeply into the S1’ pocket, where it interacts with a number
of lipophilic residues like Ile243, Ile218, Pro255, and Ala238.
In vitro MMP inhibitory activity of fluorinated derivatives. To select radiofluorinated
candidates for potentially visualizing in vivo MMPꢀ13 activity, the synthesized series of
fluorinated proline and 4ꢀhydroxyproline derivatives was also assayed for their inhibitory
activity toward MMPꢀ2, MMPꢀ8, MMPꢀ9, and MMPꢀ13.
Table 2. MMP inhibitory activity of the synthesized fluorinated prolineꢀbased hydroxamates 4,
34, 35, and 37 compared to 44.
IC ± SD [nM]
50
1
Stereo
isomer
Comp
X
R
R
MMPꢀ2
5 ± 1
MMPꢀ8
MMPꢀ9
108 ± 11
184 ± 10
MMPꢀ13
0.07 ± 0.02
159 ± 22
4
C=O O(CH
2
)
4
CH
2
F
OH (2S,4S)
(2S,4S)
21 ± 8
34
C=O
H
F
33 ± 14
25 ± 11
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4
CH
2
H
F
F
(2S,4S)
>10000
600 ± 170
4 ± 0.001
>10000
200 ± 25
11 ± 2
>10000
796 ± 89
11 ± 2
>10000
C=O
OH (2S,4R)
394 ± 32
16 ± 4
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As shown in Table 2, all synthesized amides, possessing a fluorine atom at different positions,
inhibit MMPꢀ13 with different selectivities over the other tested MMPs, whereas the fluorinated
tertiary amine 35 exhibits no inhibition toward the studied enzymes. Amongst these amides, the
(
2S,4R)ꢀconfigured 4ꢀhydroxyproline derivative 37, possessing a fluorineꢀsubstituted distal
phenyl ring, shows only moderate inhibitory activity toward the investigated MMPs, whilst
2S,4S)ꢀ4ꢀfluoroproline derivative 34 is a significantly more potent MMPꢀinhibitor.
(
Nevertheless, both compounds demonstrated a lack of selectivity toward MMPꢀ13 and were not
considered as candidates for the development of a MMPꢀ13 targeting PET tracer. By contrast,
the (2S,4S)ꢀconfigured 4ꢀhydroxyproline derivative 4 containing a 5ꢀfluoropentoxy moiety,
mimicking the long lipophilic tail of the potent and selective MMPꢀ13 inhibitor 20k (Table 1),
showed a MMP binding profile suitable for the development of a PET tracer (Table 2). 4
excellently inhibits MMPꢀ13 in the picomolar range (IC (MMPꢀ13) = 0.07 nM) with
5
0
selectivities over the other tested MMPs ranging from 71× to 1543×. The synthesized 4ꢀ
hydroxyprolineꢀbased derivative 4 represents a good example for an inhibitor, which overcomes
to a certain extent the challenging task of obtaining selectivity for MMPꢀ13 over other MMPs
that possess a deep S1’ pocket, without losing inhibitory potency.
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Figure 4. Overall structure of the catalytic domain with compound 4 (in cyan) docked in the
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active site of MMPꢀ13 (PDB: 3KRY). The Zn ꢀion is shown as a gray sphere; amino acid
residues in proximity to the surface are depicted as white stick models. Oxygen and nitrogen
atoms are colored in red and blue, respectively. The surface is colored as follows: lipophilic
regions are in green, hydrophilic in magenta and neutral in white.
The excellent MMPꢀ13 inhibitory activity of 4 can be explained by the fact that it comprises
structural features which were found to be the most beneficial in terms of inhibitory activity
within the synthesized series of prolineꢀderived hydroxamates 12, 16, 20, 23, 28, 29, 34, 35, and
2
+
3
7. Besides a Zn ꢀchelating hydroxamate moiety, these structural elements include a (2S,4S)ꢀ
configured 4ꢀhydroxyproline scaffold and a lipophilic side chain, which is attached to the
scaffold via an amide moiety whose carbonyl group can undergo interactions with e.g. Leu185
and Ala186 near the entrance of the S1’ pocket (Figure 4). Moreover, 4 possesses a
diphenylacetylene moiety, which is directed straight into the S1’ cleft of MMPꢀ13 to form greasy
interactions with e.g. Pro242, Ile243, and Thr245 and which is elongated by the flexible and
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lipophilic 5ꢀfluoropentoxy substituent. The terminal fluorine of this substituent is able to form a
hydrogen bond with Lys249, which is located at the bottom of the S1’ pocket, and represents
another important pharmacophoric element, which additionally contributes to inhibitory activity
and to the desired selectivity (Figure 4).
0
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Selectivity over other metzincins (ADAM10, ADAM17, meprin α, and meprin β). To
investigate the inhibitory potential of the prolineꢀderived hydroxamate inhibitors against other
metzincins, fluorogenic peptide cleavage assays were performed using recombinant ectodomains
of ADAM10, ADAM17, meprin α, and meprin β. For this, 11 compounds (20e, 20j, 20k, 4, 34,
12g, ent-12d, 16j, 23g, 16k, and 35) were tested in two different concentrations (5 and 50 µM,
reconstituted in DMSO). ADAM10, ADAM17 as well as meprin β showed no inhibition upon
incubation with any of these compounds (Supporting Information). Meprin α was inhibited by
some hydroxamates, showing diminished activity at 5 µM inhibitor concentration up to complete
inhibition at 50 µM. Due to this observation, 16 additional compounds (16g, 16i, 16d, 20i, 20h,
20d, 12h, 12d, 12b, 20b, 12e, 20a, 12f, 28d, 29d, and 37; 1ꢀ50 µM) were tested against meprin
α. Again, some inhibitors exhibited inhibitory capacity towards meprin α activity with estimated
IC ꢀvalues in the micromolar range (Supporting Information). Thus, these compounds were
5
0
much weaker in inhibiting meprin α than they were inhibiting MMPs, and they were also less
potent inhibitors of meprin α than the hydroxamate inhibitor actinonin. Overall, the prolineꢀ
derived hydroxamate compounds display little inhibitory potential against meprin α and no
inhibition of the other tested metzincins ADAM10, ADAM17 and meprin β. Therefore, these
inhibitors seem to be specific for MMPs.
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Radiochemistry. Due to its unique biological profile, compound 4 was chosen for the
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18
radiosynthesis of its Fꢀlabeled analog [ F]4. At first, precursor 46 was prepared, which
possesses a tosylate leaving group (Scheme 7). For this purpose, aryl iodide 18 was subjected to
a Sonogashira coupling with phenylacetylene derivative 41 (Scheme 5) to give ester 45, which
was transformed into the desired precursor 46 via an aminolysis with hydroxylamine. Then
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1
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radiolabeled [ F]4 was prepared in one step. Nucleophilic substitution (S 2) of the tosylate
N
18
18
group in the precursor compound 46 with [ F]fluoride provided the desired product [ F]4 in
radiochemical yields (rcy) of 46.3±9.3% (n = 5) (decayꢀcorrected based on cyclotronꢀderived
18
[
F]fluoride ions (d.c.)) in 105 ± 12 min from the end of radionuclide production. The target
compound was isolated with radiochemical purities of >99% and with specific activities of 0.7ꢀ
33.2 GBq/µmol at the end of the synthesis.
(a)
(b)
(c)
18
45
46
[18F]4
1
8
Scheme 7. Synthesis of PET tracer [ F]4. Reagents and conditions: (a) 41, Pd(PPh ) , CuI,
3 4
1
8
NEt , ACN, rt, 52%; (b) H NOH·HCl, NaOMe, MeOH, rt, 29%; (c) K(K )[ F]F, K CO ,
3
2
222
2
3
ACN, 110°C, 20 min, rcy: 46.3 ± 9.3% ( d.c.).
1
8
Partition coefficient (log D (exp.)). [ F]4 represents a lipophilic compound with a log D (exp.)
*
of 2.50 ± 0.01. In comparison to the calculated log D value (clog D) of 1.18 , the real solubility
1
8
*
of [ F]4 in 1ꢀoctanol in the biphasic 1ꢀoctanol/PBS system is increased by factor 21. (
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calculated log D value (clog D) was calculated by ACD/Chemsketch version ACD/Labs 6.00
(log D = log P at physiological pH (7.4))
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Stability in human and mouse serum. An in vitro stability study was carried out using human
and mouse blood serum, respectively. During incubation for up to 90 min at 37°C, compound
18
18
[
F]4 exhibited high serum stability. As shown in Figure 5, only the parent compound [ F]4
was detected by radioꢀHPLC in human serum. Significant radiometabolites or decomposition
products could not be observed. The same results were obtained from the experiments with
mouse serum.
18
Figure 5. RadioꢀHPLC chromatograms of a typical quality control (QC) of a produced [ F]4
1
8
batch (left) and of the in vitro stability of [ F]4 (t = 12.7 min) after incubation in human blood
R
serum at 37°C after 10 min (middle), and 90 min (right) measured at analytical HPLCꢀsystem B,
method B.
In vivo biodistribution study. An in vivo biodistribution study in adult C57/Bl6 mice after
18
intravenous injection of 400 kBq/g bodyweight of [ F]4 was performed as a 90 min dynamic
PET scan. Representative maximum intensity projections (MIP) of whole body images at
selected time points after tracer injection show notable and early accumulation of radioactivity in
the liver followed by transport of radioactivity into the intestines (Figure 6). In contrast, only a
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very low activity concentration was observed in the kidneys and the urinary bladder reflecting
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that renal elimination of [ F]4 is only playing a minor role as compared to hepatobiliary
elimination of the tracer and/or potential metabolites. In the first minutes after injection
accumulation of radioactivity was observed in large vessels and well perfused tissues such as
liver, lung, kidneys, heart and the spleen followed by a continuously decreasing signal until the
end of the study. Moderate activity accumulation was found in harderian glands behind the eyes
and submandibular glands. Visual inspection did not reveal further regions of interest presenting
with an increased uptake of radioactivity. Accumulation of radioactivity in the bones, indicating
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deꢀfluorination of [ F]4, was not observed.
Figure 6. Maximum intensity projection of the in vivo biodistribution of tracerꢀassociated
1
8
radioactivity in an adult C57/Bl6 mouse after intravenous injection of [ F]4.
PET image data was analysed quantitatively for selected regions of interest (Figure 7). Time
curves of regional radioactivity concentrations expressed as the percentage of injected dose per
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