Design, (Radio)Synthesis, and in Vitro and in Vivo Evaluation of Highly Selective and Potent Matrix Metalloproteinase 12 (MMP-12) Inhibitors as Radiotracers for Positron Emission Tomography

Article abstract of DOI:10.1021/acs.jmedchem.8b00200

Dysregulated levels of activated matrix metalloproteinases (MMPs) are linked to different pathologies, such as cancer, atherosclerosis, neuroinflammation, and arthritis. Therefore, imaging of MMPs with positron-emission tomography (PET) represents a powerful tool for the diagnosis of MMP-associated diseases. Moreover, to distinguish between the distinct functions and roles of individual MMPs in particular pathophysiological processes, their specific imaging must be realized with radiolabeled tracers, such as fluorine-18-labeled MMP inhibitors (MMPIs). Therefore, fluorinated dibenzofuransulfonamide-based MMPIs showing excellent inhibition of MMP-12 and selectivity for MMP-12 over other MMPs were prepared. MMP-12 is a key enzyme in diseases such as chronic obstructive pulmonary disease (COPD) and atherosclerosis. Because of their promising in vitro properties, three candidates (4, 9, and 19) were selected from this library, and radiofluorinated analogues ([¹⁸F]4, [¹⁸F]9, and [¹⁸F]19) were successfully synthesized. Initial in vitro serum stability and in vivo biodistribution studies of the radiolabeled MMPIs with PET demonstrated their potential benefit for preferable MMP-12 imaging.

Full text of DOI:10.1021/acs.jmedchem.8b00200

Subscriber access provided by UNIV OF DURHAM
Design, (Radio)Synthesis, in Vitro and in Vivo Evaluation of
highly selective and potent Matrix Metalloproteinase (MMP-12)
Inhibitors as Radiotracers for Positron Emission Tomography
Viktoria Butsch, Frederik Börgel, Fabian Galla, Katrin Schwegmann, Sven Hermann,
Michael Schäfers, Burkhard Riemann, Bernhard Wünsch, and Stefan Wagner
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00200 • Publication Date (Web): 16 Apr 2018
Downloaded from http://pubs.acs.org on April 16, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
Design, (Radio)Synthesis, in Vitro and in Vivo Evaluation of highly
6
7
8
selective and potent Matrix Metalloproteinase 12 (MMPꢀ12)
9
Inhibitors as Radiotracers for Positron Emission Tomography
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
Viktoria Butsch,a Frederik Börgel,b Fabian Galla,b Katrin Schwegmann,c Sven Hermann,c
Michael Schäfers, a,c,d Burkhard Riemann, a Bernhard Wünsch,b,d,# and Stefan Wagner a,#,
*
aDepartment of Nuclear Medicine, University Hospital Münster, AlbertꢀSchweitzerꢀCampus 1,
Building A1, 48149 Münster, Germany
bInstitute of Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstr. 48,
48149 Münster, Germany
cEuropean Institute for Molecular Imaging, University of Münster, Waldeyerstraße 15, 48149
Münster, Germany
dCellsꢀinꢀMotion Cluster of Excellence (EXC 1003 – CiM), Westfälische WilhelmsꢀUniversität
Münster, Germany
# Both authors contributed equally to this work and share senior authorship
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 71
1
2
3
4
ABSTRACT
5
6
7
8
Dysregulated levels of activated matrix metalloproteinases (MMPs) are linked to different
pathologies such as cancer, atherosclerosis, neuroinflammation and arthritis. Therefore imaging
of MMPs with positron emission tomography (PET) represents a powerful tool for the diagnosis
of MMP associated diseases. Moreover, to distinguish between the distinct functions and roles of
individual MMPs in particular pathophysiological processes their specific imaging must be
realized with radiolabeled tracers e.g. fluorineꢀ18 labeled MMP inhibitors (MMPIs). Therefore,
fluorinated dibenzofuransulfonamideꢀbased MMPIs showing excellent inhibition of MMPꢀ12
and selectivity over other MMPs were prepared. MMPꢀ12 is a key enzyme in e.g. chronic
obstructive pulmonary disease (COPD) and atherosclerosis. Due to promising in vitro properties,
three candidates (4, 9 and 19) were selected from this library and the radiofluorinated analogs
([18F]4, [18F]9 and [18F]19) were successfully synthesised. Initial in vitro serum stability and in
vivo biodistribution studies of the radiolabeled MMPIs with PET demonstrated their potential
benefit for preferable MMPꢀ12 imaging.
9
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
INTRODUCTION
Matrixins also known as matrix metalloproteinases (MMPs) belong to the metzincin
superfamily of proteases that also include serralysins, adamalysins, pappalysins and astacins. The
MMP protease family consists of at least 23 members in vertebrates and shows proteolytic
activity towards extracellular matrix (ECM) components (collagen, gelatin, elastin, laminin etc.)
as well as nonꢀmatrix proteins (cytokines, chemokines, growth factors etc.).
Structurally MMPs are Ca2+ꢀcontaining endopeptidases with a Zn2+ꢀion in the active site that
is coordinated by three histidineꢀresidues located in the highly conserved HEXGHXXGXXH
ACS Paragon Plus Environment
2

Page 3 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Zn2+ꢀbinding motif.1 MMPs play an important role in physiological processes such as organ
morphogenesis, angiogenesis, wound healing and embryonic development.2 Alongside activated
MMPs are upregulated in many pathological processes3 such as cancer,4,5 atherosclerosis,6,7
neuroinflammation,8 arthritis9 and pulmonary emphysema.10 From this point of view MMPs
represent highly (pre)clinical relevant and important targets for molecular imaging, e.g. with the
nuclear medicine techniques single photon emission computed tomography (SPECT) and
positron emission tomography (PET). Identification and diagnosis of tissues with upregulated
levels of activated MMPs with SPECT and PET, respectively, would be a powerful instrument
for the research of disorders involving MMPs as well as for the therapy of MMP associated
diseases. Hence MMP inhibitors (MMPIs) labeled with SPECTꢀcompatible nuclides (99mTc,
111In, 123I etc.) and radioisotopes suitable for PET (18F, 11C, 68Ga, 124I etc.), respectively, might be
an appropriate tool for MMP imaging in nuclear medicine.11
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
Therefore our group focused on the development of small molecule radiolabeled MMPIs with
broad spectrum inhibition profile based on the lead structures (R)ꢀNꢀhydroxyꢀ2ꢀ((4ꢀmethoxyꢀNꢀ
(pyridinꢀ3ꢀylmethyl)phenyl)sulfonamido)ꢀ3ꢀmethylbutanamide (CGS 27023A, Figure 1)12 and
(R)ꢀ2ꢀ((Nꢀbenzylꢀ4ꢀmethoxyphenyl)sulfonamido)ꢀNꢀhydroxyꢀ3ꢀmethylbutanamide
(CGS
25966).13 We developed SPECTꢀ and PETꢀ compatible MMPIs of several generations that
coordinate the Zn2+ꢀion of the active site with a hydroxamate moiety. The MMPIs of the first
generation contained iodineꢀ123 (HOꢀ[123I]IꢀCGS 27023A) and facilitated imaging of MMP
activity in the arterial wall in mice in vivo,14,15 while representatives of the second generation
incorporated fluorineꢀ1816,17,18 and the PETꢀtracer [18F]BR351 out of this pool provided a
sensitive, noninvasive diagnostic tool of following lesion formation and resolution in murine
experimental autoimmune encephalomyelitis (EAE) and human multiple sclerosis (MS).19,20 The
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 71
1
2
3
4
5
6
7
8
third generation, also labeled with fluorineꢀ18, was modified with hydrophilic structural
elements (e.g. 1,2,3ꢀtriazol ring(s), ethylene glycol units) resulting in a triazolꢀsubstituted
candidate ([18F]HUG38) that lent itself for further (pre)clinical evaluation.21,22,23
9
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
Figure 1. Hydroxamate based MMPIs CGS 27023A and CGS 25966 and radiolabeled
derivatives.
Moreover barbiturate (pyrimidineꢀ2,4,6ꢀtrione) based MMPIs that showed subgroupꢀ
specificity for the gelatinases A and B (MMPꢀ2 and ꢀ9), neutrophil collagenase (MMPꢀ8) and the
membraneꢀbound MMPs MTꢀ1ꢀMMP (MMPꢀ14) and MTꢀ3ꢀMMP (MMPꢀ16)24 were developed
as radiotracers in our group. These compounds can also be subdivided in several classes (class 1:
123Iꢀ , 124Iꢀ and 125Iꢀlabeled25,26; class 2: 18Fꢀlabeled27; class 3: 18Fꢀlabeled with hydrophilic
structural modifications28). The prototype of 18Fꢀlabeled barbiturates (class 2) was systematically
evaluated in a colorectal cancer model but turned out to be not useful for MMPꢀ9 imaging in this
mouse model.29 Furthermore in cooperation with other groups we developed 18Fꢀlabeled prolin
based30 and 11Cꢀ/18Fꢀ and 68Gaꢀlabeled pyrimidine dicarboxamide based MMPIs31 specific for
collagenaseꢀ3 (MMPꢀ13), a major proteolytic enzyme in ECM breakdown of osteoarthritis (OA)
and rheumatoid arthritis (RA).32
ACS Paragon Plus Environment
4

Page 5 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
In this project we aim at the development of MMPI radiotracers that are potent and selective
for macrophage metalloelastase (MMPꢀ12). MMPꢀ12 represents an upregulated key enzyme in
chronic obstructive pulmonary disease (COPD),33,34,35 neurological diseases,36 cancer37,38 and in
atherosclerosis.39,40,41,42 PET imaging of atherosclerosis is a particular focus of our group. In
general the imaging of single MMPs is challenging because in comparison to an imaging
approach of the whole MMP enzyme family the absolute amount of activated enzyme (and for
this reason Bmax) is decreased. Under the assumption that a Bmax/Ki ratio ≥ 10 is sufficient for
MMP imaging with nuclear medicine techniques a potent MMPI radiotracer (with a low Kiꢀ
value) that is specific for the relevant MMP must be available.43,44,45 Under these premises the
imaging of a single MMP gives the great opportunity to evaluate the role of this specific MMP in
specific biological processes (e.g. pathological situations) resulting in possibilities to manipulate
this processes with targeted interactions with the specific MMP (e.g. potential therapy
approaches associated with single MMPs). For MMPꢀ12 the situation is additionally complicated
because the enzyme can be located intraꢀ and extracellular and the availability of the enzyme for
potential radiotracer binding is not equal in both environments.46,47 Nevertheless latest papers
showed that SPECT/PETꢀimaging of specific MMPs (families) is feasible even in a COPD mice
model that is closely associated with the overexpression of activated MMPꢀ12.48,49
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
For our purposes, the dibenzofuransulfonamideꢀbased MMPIs, selectively inhibiting MMPꢀ12
and discovered by Wu et al. (e.g. MMP118 in Figure 2)50, were chosen as lead compounds for
further (radio)chemical modifications.
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 71
1
2
3
4
5
6
7
8
9
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
Figure 2. a) Lead compound MMP118, b) Xꢀray coꢀcrystal structure of MMP118 and
MMPꢀ12 adapted from Wu et al.50
In contrast to the above mentioned agents CGS 27023A and CGS 25966 the
dibenzofuransulfonamideꢀbased MMPIs possess a carboxylic acid moiety as Znꢀbinding group
while the Oꢀatoms of the sulfonamide group interact with the amino acid backbone of the
enzyme via hydrogen bonding. The dibenzofuran scaffold or core substituted with a fiveꢀ
membered heterocycle occupies the S1´ꢀpocket of MMPꢀ12. The thiazolylꢀsubstituted lead
compound MMP118 demonstrates more than 150ꢀfold selectivity over the structurally related
MMPꢀ8 and ꢀ13.
RESULTS AND DISCUSSION
Chemistry. In the first step of the development of potent and selective MMPꢀ12 radiotracers
for PET imaging labeled with fluorineꢀ18 and based on the described dibenzofuransulfonamide
lead structure the corresponding nonꢀradioactive counterparts substituted with fluorinated fiveꢀ
membered heterocycles were synthesized and tested in in vitro MMP assays.
Thus, the aryl bromide key intermediate 1 was synthezised in 5 steps with an overall yield of
52% from dibenzofuran according to a procedure reported in literature.50,51 Aryl bromide 1 was
converted into the corresponding azide 2 with sodium azide and CuI as catalyst. After copper(I)ꢀ
ACS Paragon Plus Environment
6

Page 7 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
catalyzed Huisgen azideꢀalkyne 1,3ꢀdipolar cycloaddition (“clickꢀreaction”) with 5ꢀfluoropentꢀ1ꢀ
yne22 yielding triazole 3, the tertꢀbutyl ester was cleaved with KSF clay in acetonitrile under
reflux. The resulting target compound 4 was obtained with 37% yield over 3 steps. Moreover
precursor 5 for potential labeling with 5ꢀ[18F]fluoropentꢀ1ꢀyne was also prepared by ester
cleavage of 2 with KSF clay in acetonitrile (Scheme 1).
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
Scheme 1. Synthesis of Nꢀlinked triazole derivative 4. Reagents and conditions: (a) NaN3, Na
ascorbate, CuI, DMEDA, EtOH/H2O (7:3), 100 °C, 30 min, 94%; (b) Na ascorbate, CuI, DMF,
rt, 4 h, 49%; (c) KSF clay, CH3CN, 100 °C, 6 h, 81%; (d) KSF clay, CH3CN, 100 °C, 5 h, 79%.
The synthesis of the Cꢀlinked triazole derivative 9 also started with the aryl bromide 1 (Scheme
2). After Sonogashira coupling of 1 with trimethylsilylacetylene, the silyl group was cleaved
from 6 under basic conditions similar to a procedure described in the literature52 to yield alkyne
7. Subsequent 1,3ꢀdipolar cycloaddition of alkyne 7 with 2ꢀfluoroethyl azide provided the Cꢀ
linked triazole 8. Finally the tertꢀbutyl ester 8 was hydrolyzed with KSF clay and the Cꢀlinked
triazole 9 was obtained in 42% over the the described four reaction steps. Furthermore the
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 71
1
2
3
4
5
potential precursor 10 for radiolabeling with 2ꢀ[18F]fluoroethyl azide22,53 was prepared
analogously from 7 by cleavage of the ester group with KSF clay and 71% yield.
6
7
8
9
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
Scheme 2. Synthesis of Cꢀlinked triazole derivative 9. (a) (Me)3SiC≡CH, PdCl2(PPh3)2, CuI,
Et3N, DMF, 65 °C, 16 h, 68%; (b) K2CO3, MeOH, rt, overnight, 94%; (c) Na ascorbate, CuI,
DMF, rt, 4 h, 83%; (d) KSF clay, CH3CN, 100 °C, 6 h, 80%; (e) KSF clay, CH3CN, 100 °C, 5 h,
71%.
To elucidate the influence of different Znꢀbinding groups, the carboxylic acids 4 and 9 were
converted into the corresponding hydroxamates 12aꢀb by coupling with OꢀTHP hydroxylamine
and subsequent cleavage of the THP protective group with HCl in dioxane (Scheme 3). The
overall yield of the Nꢀand Cꢀlinked triazoles 12a and 12b containing the hydoxamate Znꢀbinding
moiety was 51% and 65%, respectively.
ACS Paragon Plus Environment
8

Page 9 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
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
Scheme 3. Conversion of the carboxylic acids 4 and 9 into hydroxamic acids 12a and 12b. (a)
THPONH2, HOBT, NMM, EDC, DMF, rt, overnight, 11a 96%, 11b 87%; (b) 4 M HCl in
dioxane, rt, 2 h, 12a 53%, 12b 75%.
For the preparation of dibenzofuransulfonamides substituted with fluorinated pyrazoles the
brominated pyrazol derivates 13 and 14 were prepared from 2ꢀfluoroethyl tosylate and the
corresponding 3ꢀbromoꢀ and 4ꢀbromopyrazole via nucleophilic substitution under basic reaction
conditions (Scheme 4).
Scheme 4. Synthesis of brominated (2ꢀfluoroethyl)pyrazoles 13 and 14. (a) Cs2CO3, DMF, 100
°C, 3 h, 51%; (b) Cs2CO3, DMF, 100 °C, 6 h, 56%.
The (S)ꢀenantiomer of the arylboronic acid pinacol ester 15 prepared according to a literature
protocol51 was reacted with bromopyrazoles 13 and 14 in a SuzukiꢀMiyaura reaction to afford the
coupling products 16 and 18. Cleavage of the tertꢀbutyl ester of 16 and 18 with KSF clay in
acetonitrile led to the carboxylic acids 17 and 19 with overall yields of 37 and 38%, respectively
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 71
1
2
3
4
5
6
7
8
(Scheme 5). In order to study the significance of the configuration of the center of chirality in
this class of MMP inhibitors entꢀ19, the (R)ꢀconfigured enantiomer of 19, was prepared in the
same manner starting from the pinacol ester entꢀ15 in two steps with an overall yield of 7%.
9
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
Scheme 5. Synthesis of fluorinated pyrazole substituted derivatives 17, 19 and entꢀ19. (a)
K2CO3, PdCl2(PPh3)2, DME/H2O (7:1), 85 °C, 3ꢀ5 h, 16 51%, 18 47%, entꢀ18 21%; (c) KSF
clay, CH3CN, 100 °C, 5 h, 17 72%, 19 80%, entꢀ19 34%.
Moreover, arylboronate 15 was used for the preparation of the tosylate precursor 22 for the
synthesis of the fluorineꢀ18 labeled MMPꢀ12 inhibitor [18F]19. For this purpose, 4ꢀ
bromopyrazole was alkylated with ethylene sulfate followed by tosylation of the hydroxy group
of alcohol 20. The final SuzukiꢀMiyaura coupling of the arylboronic acid pinacol ester 15 with
tosylate 21 provided the desired precursor 22 with 9% yield over three steps (Scheme 6).
ACS Paragon Plus Environment
10

Page 11 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
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
Scheme 6. Preparation of the precursor 22 for radiosynthesis of [18F]19. (a) KOtBu, DMF, rt,
overnight, 57%; (b) TsCl, NEt3, CH2Cl2, rt, 20 h, 50%; (c) K2CO3, PdCl2(PPh3)2, DME/H2O
(7:1), 85 °C, 5 h, 30%.
Analog to Scheme 6 the preparation of the corresponding tosylate substituted ester that can serve
as potential precursor for fluorineꢀ18 labeled [18F]17 could not be realized because a product
mixture was obtained that could not be sufficiently purified.
Additionally three fluorinated thiazoles 25, 28 and 31 and a thiophene derivative 34 were
synthesized starting with the SuzukiꢀMiyaura coupling of pinacol ester 15 with the
corresponding brominated hydroxyethyl thiazole or thiophene derivatives and microwave
heating followed by nucleophilic substitution with DAST to yield fluorinated intermediats 24,
27, 30 and 33. Finally, after cleavage of the tertꢀbutyl ester with KSF clay the carboxylic acids
25, 28, 31 and 34 were obtained in overall yields of 17ꢀ37% (Scheme 7).
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 71
1
2
3
4
5
6
R
R
R
O
H
O
O
H
N
H
N
(b)
N
(a)
(c)
O
S
HO
15
O
S
O
S
O
O
O
O
O
O
O
O
7
8
9
N
N
N
OH
23: R =
24: R =
F
25: R =
28: R =
F
S
S
S
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
N
N
N
OH
OH
F
F
26: R =
27
: R =
S
N
S
N
S
N
F
F
29: R =
32: R =
30: R =
33: R =
31: R =
34: R =
S
S
S
OH
F
S
F
S
S
Scheme 7. Synthesis of fluorinated thiazolyl and thienyl substituted derivatives 25, 28, 31 and
34. (a) brominated hydroxyethyl thiazole or thiophene derivative, K2CO3, PdCl2(PPh3)2,
DME/H2O (7:1), microwave, 150 W, 100 °C, 20ꢀ180 min, 23 49%, 26 47%, 29 66%, 32 40%;
(b) DAST, toluene, rt, overnight, 24 82%, 27 64%, 30 80%, 33 83%; (c) KSF clay, CH3CN, 100
°C, 5 h, 25 92%, 28 56%, 31 58%, 34 54%.
In vitro MMP inhibition. The MMP inhibition of synthesized fluorinated fiveꢀmembered
heteroaryl substituted dibenzofuransulfonamides 4, 9, 12aꢀb, 17, 19, entꢀ19, 25, 28, 31 and 34
and the non heteroaryl substituted azide and alkyne derivatives 5 and 10 was recorded. The
inhibition of activated MMPꢀ2, ꢀ8, ꢀ9, ꢀ12 and ꢀ13 was investigated in a fluorometric in vitro
inhibition assay according to a literature procedure.54 Furthermore selected candidates (4, 9)
were tested in MMPꢀ1, ꢀ3 and ꢀ7 assays. The obtained IC50ꢀvalues are listed in Table 1. In order
to analyze changes in the lipophilicity/hydrophilicity caused by structural modifications the
logD7.4ꢀvalues of the nonꢀradioactive test compounds were determined by a microꢀshakeꢀflask
method coupled with LCꢀMS quantification.55 The logD7.4 values of the radiolabeled analogs
were determined by the shakeꢀflask method with γꢀcounting based analysis.
ACS Paragon Plus Environment
12

Page 13 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
Table 1. MMP inhibitory activity of fluorinated fiveꢀmembered hetaryl substituted dibenzofuran
sulfonamides 4, 9, 12aꢀb, 17, 19, entꢀ19, 25, 28, 31 and 34 and the non heterocycle substituted
azide and alkyne derivative 5 and 10.
9
10
11
12
13
14
15
16
17
18
19
IC50 ± SD [nM]
Cpd.
R1
R2
Config.
20
MMPꢀ2
MMPꢀ8
MMPꢀ9
MMPꢀ12
MMPꢀ13
logD7.4
21
22
23
0.59± 0.07a
0.48± 0.01b
4c
OH
(S)
81 ± 3
19 ± 3
2230 ± 1200
0.19 ± 0.02
288 ± 10
24
25
26
27
1170 ±341 2.55± 0.03a
28
NHOH
(S)
2670 ± 449 421 ± 123 8760 ± 2390
62 ± 30
12a
29
30
31
0.09± 0.07a
16 ± 5
9d
0.001 ± 3·10ꢀ5
3 ± 2
32
OH
(S)
(S)
35 ± 6
2.0 ± 0.6
27 ± 11
>10000
>10000
0.00± 0.01b
33
34
35
NHOH
174 ± 21
62 ± 15
2.33± 0.08a
0.73± 0.02a
12b
17
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
OH
OH
(S)
(S)
6.5 ± 3
8.5 ± 0.7
413 ± 317
151 ± 5
0.0008 ± 2·10ꢀ4
2.4 ± 0.4
0.68± 0.02a
0.63± 0.02b
0.84 ± 0.03 0.18 ± 0.02
0.0004 ± 1·10ꢀ4 3.2 ± 0.08
19
entꢀ
19
OH
(R)
2.0 ± 0.4
3.0 ± 0.3
n.d.
2.0 ± 0.1
19 ± 5
0.68± 0.02a
OH
OH
(S)
(S)
26 ± 3
0.22 ± 0.03
2.0 ± 0.6
n.d.
0.12 ± 0.04
332 ± 11
60 ± 4
1.26± 0.01a
1.25± 0.02a
25
28
55 ± 10
195±33
0.081 ± 0.002
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 71
1
2
3
4
5
6
7
8
9
OH
OH
(S)
(S)
0.49 ± 0.01 0.51 ± 0.3
200 ± 49
0.94 ± 0.13
12.0 ± 0.5 1.71± 0.02a
31
34
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
20 ± 12
2.0 ± 0.4
58 ± 4
0.001 ± 2·10ꢀ4
20 ± 3
1.70± 0.02a
N3
OH
OH
(S)
(S)
3 ± 0.5
2 ± 0.1
22 ± 6
250 ± 2
>1000
1.0 ± 0.07
21 ± 6
90 ± 4
ꢀ
ꢀ
5
C≡CH
14 ± 0.3
406 ± 24
10
n.d. not possible to determine due to inhomogenous data
a logD7.4ꢀvalues were determined by MS quantification (n = 9)
b logD7.4ꢀvalues were determined using the radiolabeled analogs (n = 6)
cIC50(MMPꢀ1) > 10 µM, IC50(MMPꢀ3) = 4.2 ± 0.7 µM, IC50(MMPꢀ7) > 10 µM
dIC50(MMPꢀ1) > 10 µM, IC50(MMPꢀ3) = 4.2 ± 2.8 µM, IC50(MMPꢀ7) > 10 µM
In general, a suitable tool to modify MMP selectivity is the variation of the inhibitor P1´ꢀ
substituent that occupies the S1´ꢀcavity of MMPs. The S1´ꢀpocket can be differentiated into
smallꢀ (MMPꢀ1, ꢀ7, ꢀ11, ꢀ20), mediumꢀ (MMPꢀ2, ꢀ8, ꢀ9, ꢀ12, ꢀ14, ꢀ16) and largeꢀsized (MMPꢀ3, ꢀ
10, ꢀ13)56 and previous work of Li et al.57,58 showed for the dibenzofuran based MMPIs that a
C3/C8 substitution pattern is favored over a C2/C7 and a C3/C7 constellation regarding MMPꢀ12
affinity and selectivity.
As shown in Table 1 all fluorinated heteroaryl substituted dibenzofurans with (S)ꢀconfiguration
and carboxylate moiety are excellent MMPꢀ12 inhibitors with IC50ꢀvalues in subnanomolar to
subpicomolar range (0.0004ꢀ0.94 nM for 4, 9, 17, 19, 25, 28, 31 and 34). To the best of our
knowledge, these compounds represent the most potent MMPꢀ12 inhibitors reported so far in
literature. The mentioned dibenzofurans also show MMPꢀ2, ꢀ8 and MMPꢀ13 inhibitory potency
ACS Paragon Plus Environment
14

Page 15 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
in the nanomolar range (IC50ꢀvalues: 0.49ꢀ332 nM), but most of them (4, 9, 17, 19, 34) possess
>100ꢀfold selectivity for MMPꢀ12 compared to the structurally related MMPꢀ8 and ꢀ13 and the
gelatinases MMPꢀ2 and ꢀ9. Replacing the carboxylate Znꢀbinding group by a hydroxamate
moiety led to a dramatic loss of MMPꢀ12 inhibition (IC50(4) = 0.19 nM; IC50(12a) = 62 nM;
IC50(9) = 0.001 nM; IC50(12b) = 3 nM). This is a remarkable fact because a contrary effect was
observed for a selective peptideꢀbased MMPꢀ12 inhibitor.59 Maybe this opposite effect for these
different MMPꢀ12 inhibitor classes (peptideꢀbased and dibenzofuranꢀbased) is caused by varied
sterical interactions of the different S1´substituents in both inhibitor classes that must be shifted
when the carboxylate moiety is displaced by a hydroxamate group. An additional effect is
observed by inversion of the configuration. While (S)ꢀconfigurated pyrazole 19 is a highly potent
MMPꢀ12 inhibitor (IC50 = 0.0004 nM), the (R)ꢀconfigured enantiomer entꢀ19 displays
considerably decreased potency (IC50 = 2.0 nM). A similar observation was also reported by Wu
et al.50 for other derivatives of this compound class. Comparison of the compounds 28 and 31
reveals that the introduction of an additional methyl substituent at the terminal thiazole ring
caused a 10ꢀfold loss in MMPꢀ12 inhibitory potency (IC50(28) = 0.081 nM, IC50 (31) = 0.95 nM),
but increased inhibitory activities against MMPꢀ2, ꢀ8 and ꢀ13. The fundamental importance of
the fiveꢀmembered heterocycle for the MMPꢀ12 selectivity was confirmed by azide 5 and alkyne
10. Both compounds are potent inhibitors of MMPꢀ2, ꢀ8 and ꢀ12 (IC50 = 1.0ꢀ22 nM) without
designated inhibitory activities against MMPꢀ9 and MMPꢀ13. Compounds 4 and 9 were also
tested against MMPꢀ1, ꢀ3 and ꢀ7 with a designated smallꢀsized (MMPꢀ1, ꢀ7) and largeꢀsized
(MMPꢀ3) S1´ꢀpocket to demonstrate broad MMPꢀ12 selectivity exemplarily. Consistent with
previous results50,57,58 both compounds showed little to no affinity for MMPꢀ1 and ꢀ7 (IC50 > 10
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
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 71
1
2
3
4
5
6
µM) while a moderate inhibition of MMPꢀ3 with a largeꢀsized S1´ꢀcavity was observed (IC50(4)
> 4.2 ± 0.7 µM, IC50(9) > 4.2 ± 2.8 µM).
7
8
9
The hetaryl substituted dibenzofurans showed moderate lipophilicity with logD7.4 values ranging
from 0.09 to 2.55. Additionally, the logD7.4 values of radiofluorinated analogues [18F]4, [18F]9
and [18F]19 were determined (see section Radiochemistry). The logD7.4 values recorded with the
LCꢀMS for the nonꢀradioactive analogs 4, 9, and 19 were in good accordance with the
radiochemically determined values and differed by ≤0.11 units.
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
Based on these results the 18Fꢀlabeled counterparts of Nꢀlinked triazole 4, which was the first
potent and selective MMPꢀ12 inhibitor in our hands, Cꢀlinked triazole 9 and the most potent and
extremely selective MMPꢀ12 inhibitor 19 were chosen for radiosynthesis and further in vitro/in
vivo evaluation. Radiosynthesis of [18F]17 whose nonꢀradioactive analog 17 showed comparable
MMPꢀ12 affinty and selectivity to 19 was prevented due to the failed synthesis of a potential
labeling precursor (see above).
Radiochemistry. The radiosyntheses of [18F]4, [18F]9 and [18F]19 were carried out in a
semiautomated two step procedure using a modified PET tracer radiosynthesizer TRACERLab
FXFDG (GE Healthcare).
The first step of the preparation of [18F]4 was the nucleophilic substitution at the precursor pentꢀ
4ꢀynꢀ1ꢀyl 4ꢀmethylbenzenesulfonate with anhydrous [18F]fluoride yielding 5ꢀ[18F]fluoropentꢀ1ꢀ
yne according to previous literature.22 The second step consisting of the Cu(I)ꢀcatalyzed Huisgen
azideꢀalkyne 1,3ꢀdipolar cycloaddition of azide 5 and 5ꢀ[18F]fluoropentꢀ1ꢀyne was conducted
outside the automated system after distillation of 5ꢀ[18F]fluoropentꢀ1ꢀyne in a separate flask with
ACS Paragon Plus Environment
16

Page 17 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
cooled DMF (ꢀ10 °C) and addition of Cu(II)ꢀsulfate pentahydrate and sodium ascorbate (Scheme
8).
7
8
9
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
Scheme 8. Radiosynthesis of [18F]4. (a) K(K222)[18F]F, K2CO3, CH3CN, 110 °C, 180 s; (b)
CuSO4 ·5H2O, Na ascorbate, DMF, 40 °C, 30 min.
After stirring for 30 min at 40 °C, purification by semipreparative HPLC, concentration and
formulation, the product [18F]4 was obtained with overall radiochemical yield (rcy) of 37 ± 8%
(decay corrected (d. c.), n = 6) in 126 ± 3 min with radiochemical purity (rcp) >99% and molar
activity (Am) of 3ꢀ59 GBq/µmol. The radiochemically determined logD7.4ꢀvalue of [18F]4 was
0.48 and differed just by 0.11 from the logD7.4ꢀvalue determined by MS quantification of
compound 4.
The radiosythesis of the Cꢀlinked triazole [18F]9 was performed in a similar way as the synthesis
of the Nꢀlinked derivative [18F]4. After preparation of 1ꢀazidoꢀ2ꢀ[18F]fluoroethane from 2ꢀ
azidoethyl 4ꢀmethylbenzenesulfonate following the procedure of Glaser and Årstad with the
improvement of Hugenberg et al.22,53 Cu(I)ꢀcatalyzed 1,3ꢀdipolar cycloaddition of precursor 10
with 1ꢀazidoꢀ2ꢀ[18F]fluoroethane yielded the desired radiofluorinated triazole [18F]9. (Scheme 9)
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 71
1
2
3
4
5
6
7
8
9
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
Scheme 9. Radiosynthesis of [18F]9. (a) K(K222)[18F]F, K2CO3, CH3CN, 110 °C, 180 s; (b)
CuSO4 ·5H2O, Na ascorbate, DMF, 40 °C, 30 min.
After purification and formulation, the radiotracer [18F]9 was obtained in overall radiochemical
yield of 43 ± 7% (decay corrected, n = 6) in 140 ± 6 min with radiochemical purity >99% and
molar activity of 7ꢀ57 GBq/µmol. The logD7.4ꢀvalue of [18F]9 was 0.00 and differed only slightly
from the logD7.4ꢀvalue determined for the nonꢀradioactive analog 9 (0.09).55
The radiolabeling of the pyrazole derivative [18F]19 was also performed in two steps via
nucleophilic substitution of the tosyloxy moiety of precursor 22 with anhydrous [18F]fluoride and
subsequent hydrolysis of the tertꢀ butyl ester with TFA (Scheme 10).
rcy: 22 ± 9% (d. c., n = 9)
rcp: > 99%
Am: 3ꢀ68 GBq/ꢁmol
t: 161 ± 15 min
logD7.4: 0.63 ± 0.02
N
N
18F
N
N
OTs
O
O
(a), (b)
H
N
H
N
O
HO
S
S
O
O
O
O
O
O
22
[
18F]19
ACS Paragon Plus Environment
18

Page 19 of 71
Journal of Medicinal Chemistry
1
2
3
4
Scheme 10. Radiosynthesis of [18F]19. (a) K(K222)[18F]F, K2CO3, CH3CN, 100 °C, 30 min; (b)
5
6
TFA, 40 °C, 5 min.
7
8
9
After purification and formulation, the pyrazole derivative [18F]19 was received in overall
radiochemical yield of 22 ± 9% (decay corrected, n = 9) in 161 ± 15 min with radiochemical
purity >99% and molar activity of 3ꢀ68 GBq/µmol. The logD7.4ꢀvalues of 19 and [18F]19
determined by different methods were almost identical (0.68 vs. 0.63).
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
In vitro stability in human and mouse serum. The in vitro stability was investigated by
incubation of the three radiotracers with human and mouse blood serum. All three tracers [18F]4,
[18F]9 and [18F]19 revealed excellent serum stability upon incubation up to 90 min at 37 °C.
Only the parent compounds without any radiometabolites or decomposition products were
detected by analytical radioꢀHPLC. Figure 3 shows exemplarily the radioꢀHPLC chromatograms
of [18F]4, [18F]9 and [18F]19 after incubation with mouse serum for 90 min.
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 71
1
2
3
4
5
6
7
8
9
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
Figure 3. Serum stability: radioꢀHPLC chromatograms after incubation of [18F]4 (top), [18F]9
(middle) and [18F]19 (bottom) with mouse blood serum at 37°C for 90 min. RadioꢀHPLC:
Method D.
In vitro biotransformation using mouse liver microsomes. To identify metabolically labile
structural elements as early as possible, the carboxylic acids 4, 9 and 19 as well as the
hydroxamic acids 12a and 12b were incubated with mouse liver microsomes and NADPH/H+
with and without UDPGA (UDPꢀactivated glucuronic acid) for 2 h. The formation of metabolites
was monitored by LC coupled with quadrupoleꢀtimeꢀofꢀflightꢀMS (qToF) allowing identification
of metabolite structures by analysis of the exact masses of separated compounds and generated
fragments ions.
ACS Paragon Plus Environment
20

Page 21 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Incubation of hydroxamates 12a and 12b with mouse liver microsomes and NADPH/H+ led to
formation of the carboxylic acids 4 and 9 (phase 1 metabolites), respectively. In addition to the
acids 4 and 9, conjugation products 35a and 35b (phase 2 metabolites) were detected after
incubation of 12a and 12b with UDPGA and NADPH/H+. Both types of metabolites originate
from transformation of the hydroxamic acid. (Scheme 11)
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
Scheme 11. Metabolites of hydroxamates 12aꢀb in the mouse liver microsome assay. (a) mouse
liver microsomes, NADPH/H+, rt, 2 h; (b) mouse liver microsomes, NADPH/H+, UDPGA, rt, 2
h.
Incubation of the acids 4, 9 and 19 with mouse liver microsomes and different cofactors did not
lead to metabolites in reasonable amounts. The missing metabolites indicate a very high
metabolic stability that can be explained by the absence of the hydroxamate functional group,
and thus represents a promising starting point for in vivo experiments.
Due to the extraordinarily high potency of the pyrazole 19 (MMPꢀ12: IC50 = 0.4 pM) a sample
was incubated with cofactors NADPH/H+ and UDPGA for 90 min and subsequently analyzed
using higher injection volumes in order to detect metabolites formed only in trace amounts. The
extracted ion chromatogram (EIC, Figure 4) shows in addition to the peak of the parent
compound 19 two small peaks indicating two metabolites in trace amounts. Although peak sizes
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 71
1
2
3
4
can be compared only supposing a similar ability to be ionized in ESI positive mode, the very
5
6
small peaks of the metabolites reveal high metabolic stability of 19 (Figure 4).
7
8
9
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
19
37
36
Figure 4. Extracted ion chromatograms (EICs, ESI positive mode) of parent compound 19 (m/z
460.1319 ± 0.01) and metabolites 36 (m/z 476.1246 ± 0.01) and 37 (m/z 636.1673 ± 0.01),
90 min after incubation with mouse liver microsomes, NADPH/H+ and UDPGA. HPLCꢀMS:
Method F.
The structures of the formed metabolites 36 and 37 were identified by fragmentation experiments
(MRM) (Figure 5). Whereas the first metabolite 36 was generated by oxidation of the isopropyl
moiety (phase 1 metabolite), conjugation with glucuronic acid led to the glucuronide 37 (phase 2
metabolite). The high metabolic activity of the used murine microsomes and incubation
conditions have already been described.60,61
ACS Paragon Plus Environment
22

Page 23 of 71
Journal of Medicinal Chemistry
1
2
3
4
F
F
5
6
7
N
N
N
N
8
9
O
O
HOOC
HO
HO
H
H
O
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
N
N
O
HO
S
O
S
HO
O
OH
O
O
O
O
36
37
[M+H]+ 476.1246 (8.5 ppm)
[MꢀH]ꢀ474.1148 (1.5 ppm)
[M+H]+ 636.1673 (2.4 ppm)
[MꢀH]ꢀ 634.1501 (1.9 ppm)
Figure 5. Postulated metabolites formed upon incubation of 19 with mouse liver microsomes,
NADPH/H+ and UDPGA.
In vitro binding to human serum albumin. In vitro human serum albumin (HSA) binding of
triazoles 4 and 9 as well as pyrazoles 17 and 19 was investigated by high performance affinity
chromatography (HPAC) using an analytical HPLC column coated with HSA. HSA was selected
as it is, together with α1ꢀacid glycoprotein (AGP), mainly responsible for plasma protein binding
(PPB) and represents the main plasma protein.62,63 The retention factor k´ of a compound
correlates with its HSA binding, since it reflects the interactions with the stationary phase. At
first, the retention factors of compounds with known HSA binding were determined to generate a
calibration curve that allowed the calculation of HSA binding (in %) of unknown compounds
after measurement of their k´ꢀvalues.64 According to this procedure more than 90% HSA binding
was recorded for all four compounds (4: 98%; 9: 94%; 17: 97%; 19: >99%) indicating high HSA
binding and thus high PPB of the investigated MMPꢀ12 inhibitors.
In vivo Biodistribution Studies. The in vivo biodistribution studies of all three fluorineꢀ18
labelled compounds [18F]4, [18F]9 and [18F]19 were investigated in adult C57BL/6 mice (n = 2)
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 71
1
2
3
4
5
6
7
8
9
after intravenous injection of 3ꢀ11 MBq as 120 min dynamic PET scans. Representative
horizontal and sagittal slices and maximum intensity projections (MIP) of whole body images at
selected time points after tracer injection are represented in Figure 6.
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
ACS Paragon Plus Environment
24

Page 25 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 6. Horizontal/sagittal slices and maximum intensity projection of the in vivo
biodistribution of tracerꢀassociated radioactivity in an adult C57BL/6 mouse after intravenous
injection of [18F]4 (8.3 MBq, top), [18F]9 (10.8 MBq, middle) and [18F]19 (9.2 MBq, bottom).
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
The biodistributions of the three radiotracers are very similar to each other, no significant
differences were observed. All tracers showed very fast and efficient elimination from the body.
Shortly after injection, a high accumulation of radioactivity was observed in the liver followed
by accumulation in the interstine. On the other side a comparatively low accumulation in the
kidneys and the bladder was found pointing to a predominate hepatobiliary excretion.
Accumulation of radioactivity in the bones, indicating free [18F]fluoride ions, was not observed
in the entire dynamic imaging study. Additionally the PET image data were analyzed
quantitatively for selected regions of interest (Figure 7).
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 71
1
2
3
4
5
6
7
8
9
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
Figure 7. In vivo biodistribution of radioactivity in an adult C57BL/6 mouse after intravenous
injection of [18F]4 (top), [18F]9 (middle) and [18F]19 (bottom). Timeꢀactivity curves illustrate
tracer dynamics in selected regions of interest (ROI). Activity is displayed as percentage of
injected dose per volume during the whole observation period of 120 min.
ACS Paragon Plus Environment
26

Page 27 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
According to predominant hepatobiliary excretion of the three compounds the highest activity
concentrations were found in the liver (35ꢀ40%ID/mL) and gallbladder (>40%ID/mL) within the
first 10 minutes after injection. Subsequent decrease of liver activity to less than 10% ID/mL
reflects the continuous clearance of all three compounds until the end of the study. Gallbladder
activity varied between the biodistribution studies, typically due to interꢀindividual different
secretory stimuli under anesthesia. The activity of [18F]19 in the gallbladder persists throughout
the imaging study of 120 minutes. One might speculate that this pattern could be explained by
the interꢀindividual variation or different hydration states at the day of PET imaging. However a
tracer specific characteristic as a cause for the persistance in the gallbladder can not be excluded
After initial activity peaks in blood (18ꢀ31%ID/mL) and lungs (<15%ID/mL) in the first five
minutes, only a low amount of radioactivity (<3%ID/mL) was observed in nonꢀexcretion organs
or tissues such as brain, lung, heart, spleen, and muscles (exemplarily data of muscles are
shown). Similarily, the activity concentration in the kidneys decreased after an initial peak (10ꢀ
15%ID/mL) to <5%ID/mL for the period 10ꢀ120 min while the bladder activity concentration
remained between 10 and 30%ID/mL for the later time points (30ꢀ120 min). The high HSA
binding determined in the section “in vitro binding to human serum albumin” for nonꢀradioactive
counterparts 4, 9, and 19 prevented not the efficient washout of labeled analogs [18F]4, [18F]9
and [18F]19 from the blood shown by limited blood activities of <5%ID/mL already after 20 min.
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
CONCLUSION
This study aimed at the development of PET tracers for imaging of activated MMPꢀ12 that is a
promising target in different pathologies. Therefore, this work presented in the first step the
successful syntheses of dibenzofuransulfonamidecarboxylic acid based MMPꢀ12 inhibitors with
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 71
1
2
3
4
5
6
7
8
9
(S)ꢀconfiguration and terminal triazole (4, 9), pyrazole (17, 19), thiazole (28, 31) and thiophene
(37) rings with fluoroalkyl substituent. The compounds revealed excellent inhibitory potential
(IC50ꢀvalues: 0.0004ꢀ0.19 nM) and high selectivity (≥ 25 fold) for MMPꢀ12. To the best of our
knowledge these compounds represent the most potent MMPꢀ12 inhibitors decribed in literature
so far. Changing the Znꢀbinding group to a hydroxamic acid moiety (12aꢀb), inversion of the
center of chirality to (R)ꢀconfiguration (22), adding an additional substituent at the terminal fiveꢀ
membered heterocycle (34) or substitution of core sytem by an azide (5) or alkyne group (10) led
to dramatic loss of MMPꢀ12 inhibition and selectivity for MMPꢀ12. Moreover, 4, 9 and 19
showed high HSAꢀbinding (≥94%) and high metabolic stability upon incubation with mouse
liver microsomes. Due to this preliminary results compounds 4, 9 and 19 were selected for
radiochemical resyntheses. The fluorineꢀ18 labeled and moderate lipophilic analogs [18F]4,
[18F]9 and [18F]19 (logD7.4 0.00ꢀ0.63) were successfully synthesized radiochemically in two step
procedures with rcy of 22ꢀ43% (d. c.). The triazoles [18F]4 and [18F]9 and the pyrazole [18F]19
showed excellent stability in vitro, when incubated with human and mouse serum without
formation of fragments and/or metabolites. Initial biodistribution studies of all three radiotracers
in C57BL/6 mice using small animal PET exhibited predominantly hepatobiliary elimination of
the radiofluorinated inhibitors without undesired and unspecific accumulation in nonꢀexcretion
organs and bones indicating nonꢀformation of [18F]fluoride. In summary [18F]4, [18F]9 and
[18F]19 featured promising in vitro and in vivo behavior predetermining these MMPꢀ12
inhibitors for further in vivo evaluation with PET in mouse models that are characterized by
elevated levels of activated MMPꢀ12 such as the models of irritant contact determititis (ICD) and
collagen induced arthritis (CIA).
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
ACS Paragon Plus Environment
28

Page 29 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
EXPERIMENTAL SECTION
Chemistry: General Methods
7
8
9
All purchased chemicals, reagents and solvents for synthesis were of analytical grade quality and
applied without further purification. Demineralized water was used if not otherwise mentioned.
For radiosynthesis water of injection was used and DNAꢀgrade CH3CN. Air and moistureꢀ
sensitive reactions were handled under argon atmosphere. The microwave reactions were
performed with Discover SP – Microwave Synthesizer (CEM). Parameters such as solvent,
power, temperature, and time are declared in the synthetic procedures. Melting points (mp) are
uncorrected and were measured with melting point system Stuart SMP 3 (STUART
SCIENTIFIC) and melting point system mp 70 (METTLER TOLEDO) using an open capillary.
GC/MS measurements were performed with GC system GCꢀ2010 couplet with mass
spectrometer GCMSꢀQP2010 (SHIMADZU). Column Rxiꢀ1ms (30.0 m length, 0.25 mm internal
diameter, 0.25 µm thickness, RESTEK) was used and helium as carrier gas. Data acquisition
were realized with GCMS solution version 2.71 software (SHIMADZU). Thin layer
chromatography was performed with TLC silica gel sheets POLYGRAM® SIL G/UV254
(MACHEREYꢀNAGEL). As stationary phase for the flash column chromatography silica gel 60
(40 – 63 µm, Merck) was used. Pressure was applied with compressed air. Eluent and the
retention factor (Rf) are declared in the synthetic procedures. Automatic flash column
chromatography was performed with Reveleris X2 (GRACE, now BÜCHI). As stationary phase
silica flash cartridges were used. Parameters such as cartridge, flow rate and eluent are declared
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
13
in the synthetic procedures. NMR spectra were recorded on an AV300 (1H NMR 300 MHz, C
NMR 75 MHz, 19F NMR 282 MHz) or AV400 (1H NMR 400 MHz, 13C NMR 100 MHz )
spectrometer (Bruker) and a Varian Unity plus 600 (1H NMR 600 MHz, 13C NMR 151 MHz)
ACS Paragon Plus Environment
29