Targeted Polypharmacology: Discovery of a Highly Potent Non-Hydroxamate Dual Matrix Metalloproteinase (MMP)-10/-13 Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.7b01001

Matrix metalloproteinases (MMPs) play a key role in many diseases like cancer, atherosclerosis or arthritis. Interest in MMP inhibition has been revitalized very recently as the knowledge on the underlying network of biological pathways is steadily growing. On the basis of this new insight into the relevance of MMP-10 and MMP-13 within the MMP network and the ban of hydroxamate inhibitors from clinical development, the discovery of non-hydroxamate multitarget drugs against specific MMPs is of foremost interest. Here, we disclose the discovery of a very potent and selective non-hydroxamate MMP-10/-13 inhibitor. The high potency (IC₅₀ of 31 nM [MMP-10] and 5 nM [MMP-13]) and selectivity over MMP-1, -2, -3, -7, -8, -9, -12, and -14 enable this compound to decipher disease causing MMP networks and to generate new treatment options through targeted polypharmacology.

Full text of DOI:10.1021/acs.jmedchem.7b01001

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Article
Targeted Polypharmacology: Discovery of a Highly Potent Non-
Hydroxamate Dual Matrix Metalloproteinase (MMP)-10/-13 Inhibitor
Nicole Senn, Michael Ott, Jan Lanz, and Rainer Riedl
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01001 • Publication Date (Web): 27 Sep 2017
Downloaded from http://pubs.acs.org on September 27, 2017
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Targeted Polypharmacology: Discovery of a Highly
Potent NonꢀHydroxamate Dual Matrix
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Metalloproteinase (MMP)ꢀ10/ꢀ13 Inhibitor
‡
‡
†
‡
Nicole Senn , Michael Ott , Jan Lanz , Rainer Riedl*
AUTHOR ADDRESS
‡
Institute of Chemistry and Biotechnology
Center for Organic and Medicinal Chemistry
Zurich University of Applied Sciences ZHAW
Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland
Eꢀmail: rainer.riedl@zhaw.ch
ABSTRACT
Matrix metalloproteinases (MMPs) play a key role in many diseases like cancer,
atherosclerosis or arthritis. Interest in MMP inhibition has been revitalized very recently as the
knowledge on the underlying network of biological pathways is steadily growing. Based on this
new insight into the relevance of MMPꢀ10 and MMPꢀ13 within the MMP network and the ban of
hydroxamate inhibitors from clinical development, the discovery of nonꢀhydroxamate multiꢀ
target drugs against specific MMPs is of foremost interest. Here, we disclose the discovery of a
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very potent and selective nonꢀhydroxamate MMPꢀ10/ꢀ13 inhibitor. The high potency (IC of 31
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nM [MMPꢀ10] and 5 nM [MMPꢀ13]) and selectivity over MMPꢀ1, ꢀ2, ꢀ3, ꢀ7, ꢀ8, ꢀ9, ꢀ12 and ꢀ14
enable this compound to decipher disease causing MMP networks and to generate new treatment
options through targeted polypharmacology.
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INTRODUCTION
The relevance of matrix metalloproteinase (MMP) inhibition for the treatment of diseases
has been revitalized recently, mostly validated by knockout experiments showing the importance
1
of specifically interconnected MMPs. The necessity for MMP inhibitors with a specific profile
1
for either one or several MMPs has clearly emerged over time. Unfortunately, potent
1–3
hydroxamate MMP inhibitors delivered negative outcomes in clinical trials.
Unspecific
inhibition of metal containing proteins by the hydroxamic acid led to severe side effects and
2
,3
finally to the exclusion of these inhibitors form clinical development. Recently gained
knowledge about the connected functionality within the MMP family asks for inhibitors with
1
,4
defined inhibition profiles. The benefits of polypharmacological drugs are well accepted as
they show more predictable pharmacokinetics and a lower probability of drug interactions than
5,6
mixtures of inhibitors. This is especially important in complex diseases like neurodegenerative
diseases, chronic inflammation or cancer for which disturbed biological networks are the root
7
cause.
MMPs are major enzymes in the degradation and remodeling of the extracellular matrix. 23
4
,8,9
members of the human MMP family are known.
MMPs are zinc containing proteases that
possess, next to the catalytically active zinc ion, a S1’ selectivity loop, which is hydrophobic in
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nature and of different depth among the different MMPs. In healthy organisms, MMPs are
involved in bone modeling and remodeling, mammary development or blood vessel remodeling,
for example. Dysfunction of MMP expression leads to a broad field of devastating diseases like
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cancer, arthritis or fibrosis.
The targeted proteins MMPꢀ10 and ꢀ13, also known as stromelysinꢀ2 and collagenaseꢀ3,
play important roles in those diseases. Both proteins are involved in several types of cancer (lung
10–14
11,15–17
16,17
16,17
16,17
cancer,
head and neck cancer,
breast cancer,
gastric cancer,
skin cancer,
1
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16,20,21
esophageal carcinoma or thymic lymphoma ), and in arthritis.
In addition, MMPꢀ10 is
2
0,22,23
able to activate the proꢀcollagenases.
MMPꢀ13 is thereby super activated by MMPꢀ10 and
2
0
shows about a tenfold higher activity than activated by trypsin or plasmin.
Based on the documented disease relevance of MMPꢀ10 as well as MMPꢀ13, we set out to
develop a dual MMPꢀ10/ꢀ13 inhibitor for a targeted polypharmacology approach. For MMPꢀ10,
no selective and potent inhibitor is described in literature so far, whereas for MMPꢀ13, there are
2
4–28
several selective inhibitors known with nanomolar affinity or even better.
Therefore, we
2
9
started with a rather poor affinity for MMPꢀ10 based on our previous work (1: 6 nM [MMPꢀ13]
and 8.40 µM [MMPꢀ10], Figure 1) and employed structureꢀbased design to improve the affinity
for MMPꢀ10 and MMPꢀ13 in parallel by reaching into the S1’* pocket of the target proteins to
30
design a dual inhibitor for both proteins. The newly generated carboxamide and sulfonamide
derivatives as well as the starting molecule are depicted in figure 1.
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Figure 1. Starting molecule 1 from our previous work and newly designed/synthesized
structures 2 (oxazole), 3-20 (carboxamides) and 21-28 (sulfonamides).
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RESULTS AND DISCUSSION
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Molecular modeling. Docking studies were performed with the combination of Chimera
32
and AutoDock Vina. The docking parameters as well as the design setup can be found in the
3
3
supporting information. The MMPꢀ10 structure with PDB code 1Q3A and the MMPꢀ13
34
structure with PDB code 456C were used.
The design of the synthesized compounds 2-28 was based on compound 1, from which the
zinc chelating acid functionality on the right hand site was removed and the phenyl ring was
substituted by fluorine in para position to enable more stability against degradation. The main
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changes for the optimization in this polypharmacological approach were introduced at the
carboxylic acid moiety on the left hand site of 1. In a first iteration, this carboxylic acid was
converted into the primary carboxamide 3, which indicated the desired positioning into the S1’*
pocket of MMPꢀ10 (Figure 2 (a)). The carboxamide was chosen because of the lower polarity
compared to the carboxylic acid fragment and the possibility for further Hꢀbond interactions. In
the next step, the carboxamide was docked in methylated forms to investigate the accessible
space for enlargements into the S1’* pocket. The docking pose shown in figure 2 (b) illustrates
the corresponding binding orientation of the dimethylated carboxamide 5. Based on these
docking studies, the extended carboxamides 3-20 as well as the related sulfonamides 21-28 were
designed.
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Figure 2. Initial design iteration towards a dual MMPꢀ10/ꢀ13 inhibitor: (a) primary amide 3 and
(b) dimethylated amide 5 docked into the overlay of the MMPꢀ10 catalytic domain (PDB code:
3
3
34
1Q3A ) and MMPꢀ13 catalytic domain (PDB code: 456C ); green spheres: calcium ions, purple
spheres: zinc ions.
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Figure 3 shows the design principle for 2. Among the different docking poses of 2, no
potential binding to the zinc ion was observed as the carboxylic acid functional groups have been
removed from 1 to avoid nonꢀselective interference with metalloenzymes.
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Figure 3. Design principle for 2 in the S1’ sub site reaching into the aromatic S1’* pocket of the
3
3
MMPꢀ10 catalytic domain (purple chain, PDB code: 1Q3A ) and MMPꢀ13 catalytic domain
34
(
blue chain, PDB code: 456C ). Aromatic interactions with the aromatic amino acid residues
PHE242 and PHE248 of MMPꢀ10 as well as TYR246 and PHE252 of MMPꢀ13 are highlighted;
green spheres: calcium ions, purple spheres: zinc ions.
Both proteins possess two aromatic amino acid residues in the S1’/S1’* pocket (PHE242
and PHE248 in MMPꢀ10, and TYR246 and PHE252 in MMPꢀ13), which can interact with the
3
5
oxazole fragment of 2. The analysis of a related crystal structure of MMPꢀ10 (PDB code 3V96 )
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in complex with a tissue inhibitor of metalloproteinase (TIMP) in comparison to the used
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1
Q3A structure highlighted differences within the specificity loop (S1’ and S1’* pockets).
The flexibility in this part of the protein is limited to the amino acids 239ꢀ247 of the MMPꢀ10
sequence. PHE242 is therefore considered to occupy an analogous position compared to the
TYR246 residue in the MMPꢀ13 structure.
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Chemistry. Schemes 1 to 3 show the synthetic procedures for the preparation of compounds
2-28, which were tested for their potency towards MMPꢀ10 and MMPꢀ13. The synthesis of 2
was realized by a four steps synthesis. Starting with the formation of the oxazole 29, followed by
radical bromination and then substitution of the inserted bromine with 5ꢀiodouracil furnished the
building block 30 for the Sonogashira coupling in the final step to yield compound 2 (Scheme
1).
Scheme 1. Synthesis procedure for the dual MMPꢀ10/ꢀ13 inhibitor 2: (a) TOSMIC, K CO ,
2
3
MeOH, 80 °C, 2 h; (b) NBS, AIBN, CCl , reflux, 16 h; (c) 5ꢀiodouracil, Cs CO , DMF, rt, 2.5 h;
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(d) Pd(PPh ) , CuI, NEt , DMF, rt, 3 h.
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The carboxamide derivatives were synthesized starting with the substitution of the
bromine of 4ꢀ(bromomethyl)benzoic acid by 5ꢀiodouracil leading to compounds 32 and 33,
followed by amide coupling reactions to obtain intermediates 34-46. The test compounds 3ꢀ14
and intermediates 47-48 were afforded by Sonogashira coupling, subsequent ester hydrolysis
yielded the products 15-20 (Scheme 2).
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Scheme 2. Synthesis procedure for the carboxamide derivatives: (a) 5ꢀiodouracil, Cs CO , DMF,
2
3
rt, 2.5 h; (b) RꢀNH , COMU, DIPEA, rt, 16 h; (c) 31, Pd(PPh ) , CuI, NEt , DMF, rt, 3 h; (d)
2
3 4
3
KOH 3N, H O, rt, 2 h.
2
O
R
R
R
H
H
a
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N
O
I
b
O
N
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I
O
O
N
N
1
7 - 31 %
17 - 78 %
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2 R= OH
3 R= OMe
34 R= NH2
Br
35 R= NHMe
3
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3
6 R= NMe2
R= OH/OMe
c
c
7 R= NHCH(CH3)2
8 R= NHcycProp
1
1 - 41 %
39 R= NHC(CH3)3
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0 R= NH(CH ) OH
1 R= Gly-OMe
2
2
42 R= L-Ala-OMe
43 R= D-Ala-OMe
44 R= L-Ser-OMe
R
R
H
H
O
N
O
O
O
N
O
O
O
O
d
F
F
4
4
5 R= D-Ser-OMe
6 R= D-Pro-OMe
N
N
H
H
N
7 - 28 %
N
3
R= NH2
1
1
1
1
5 R= Gly-OH
O
4 R= NHMe
5 R= NMe2
O
6 R= L-Ala-OH
7 R= D-Ala-OH
8 R= L-Ser-OH
6
7
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9
R= OMe
R= NHCH(CH3)2
R= NHcycProp
R= NHC(CH3)3
19 R= D-Ser-OH
0 R= D-Pro-OH
2
1
1
0 R= NH(CH ) OH
1 R= Gly-OMe
2
2
12 R= L-Ala-OMe
13 R= L-Ser-OMe
14 R= D-Pro-OMe
4
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7 R= D-Ala-OMe
8 R= D-Ser-OMe
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For the synthesis of the sulfonamide derivatives, two different entries were chosen. Both
started with sulfonyl chlorides to yield the corresponding amides 49-52 and 53, 54, respectively.
After the introduction of the 5ꢀiodouracil fragment, the intermediates 55-62 were converted by
Sonogashira coupling reactions to the final test compounds 21-28 (Scheme 3).
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Scheme 3. Synthesis procedure for the sulfonamide derivatives: (a) RꢀNH , MeOH or THF, ꢀ78
2
1
°
C to 0 °C, 2 h; (b) 5ꢀiodouracil, Cs CO , DMF, rt, 2.5 h; (c) R ꢀNH , THF, rt, 0.25 h; (d) NBS,
2
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DBPO, CCl , reflux, 16 h; (e) 31, Pd(PPh ) , CuI, NEt , DMF, rt, 3 h. Precursors for compounds
4
3 4
3
57 and 61 were commercially available: 4ꢀ(bromomethyl)ꢀN,Nꢀdimethylbenzenesulfonamide, R
=
NMe for 57 and 4ꢀ[4ꢀ(bromomethyl)benzenesulfonyl]morpholine, R = morpholine for 61.
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The biological evaluation of the synthesized compounds 2-28 was conducted at Reaction
TM
Biology Corporation, Malvern, PA, USA in a fluorescence assay using the (5ꢀFAM/QXL )
®
FRET peptide as substrate (sequence = QXL 520 ꢀ γ ꢀ Abu ꢀ Pro ꢀ Cha ꢀ Abu ꢀ Smc ꢀ His ꢀ Ala ꢀ
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Dab(5 ꢀ FAM) ꢀ Ala ꢀ Lys ꢀ NH (Abu = 2ꢀaminobutyric acid, Cha = βꢀcyclohexylalanine, Dab =
2
®
diaminobutyric acid, Smc = SꢀmethylꢀLꢀcysteine, QXL 520 = quencher, 5ꢀFAM = 5ꢀ
carboxyfluorescein, fluorescence dye). IC50 values and single dose residual activities were
measured at least in duplicates with a starting concentration of 10 respectively 100 µM with 3
fold dilution for IC50 values and at 10 µM for single dose measurements. IC50 values were
calculated using a four parameter least squares fit.
Structure-activity relationship. The resulting structureꢀactivity relationship (SAR) showed
a clear trend within the sulfonamide scaffold, as the affinity for MMPꢀ10 is rising with more
lipophilic amide residues, whereas the affinity for MMPꢀ13 is kept at the same level (Table 1).
Not only for the sulfonamide scaffold but also for the carboxamide scaffold the affinity for
MMPꢀ10 could be improved, while keeping the affinity for MMPꢀ13 on a high level. IC values
5
0
were determined for compounds showing less than 20 % residual enzyme activity and are listed
in table 2. The bioisosteric replacement of the carboxamide function with an oxazole (2)
provided the key entry for piꢀpiꢀinteractions with both MMPꢀ10 and MMPꢀ13 in order to reach a
low nanomolar affinity for both target enzymes.
Table 1. Structureꢀactivity relationship of the synthesized compounds 2-28. Single dose
measurements were carried out at an inhibitor concentration of 10 µM in duplicates. IC values
5
0
were determined for compounds showing less than 20 % residual enzyme activity and are given
in table 2.
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Residual activity [%]
X
R
MMPꢀ10
2.8
MMPꢀ13
0.4
2
3
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9
C=O
C=O
C=O
C=O
C=O
C=O
C=O
NH
2
13.9
15.4
7.1
4.4
7.5
NHMe
NMe
1.4
2
OMe
18.0
56.6
30.8
55.7
4.6
NHCH(CH
)
2
14.2
5.9
3
NHcycProp
NHC(CH
)
13.3
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9
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
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
Residual activity [%]
X
R
MMPꢀ10
14.2
MMPꢀ13
2.6
10
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
NH(CH ) OH
2 2
11
12
13
GlyꢀOMe
52.8
12.2
2.5
LꢀAlaꢀOMe
LꢀSerꢀOMe
DꢀProꢀOMe
GlyꢀOH
15.5
21.9
3.9
14
17.5
1.8
1
5
6
68.6
5.12
0.8
1
LꢀAlaꢀOH
DꢀAlaꢀOH
LꢀSerꢀOH
28.5
17
26.6
0.4
18
10.4
0.8
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Residual activity [%]
X
R
MMPꢀ10
77.0
68.9
19.1
12.8
47.9
10.2
19.6
6.9
MMPꢀ13
5.6
19
C=O
DꢀSerꢀOH
DꢀProꢀOH
20
21
22
C=O
1.9
S(=O)
S(=O)
S(=O)
S(=O)
S(=O)
S(=O)
S(=O)
NH
3.9
2
2
2
2
2
2
2
2
NHMe
1.8
23
NMe
2
5.8
2
4
5
NHCH(CH
3
)
2
2.2
2
NHcycProp
2.2
26
NHC(CH
3
)
3
1.7
27
morpholine
15.7
1.2
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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
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
Residual activity [%]
X
R
MMPꢀ10
14.3
MMPꢀ13
2.1
28
S(=O)
2
GlyꢀOMe
Table 2. Structureꢀactivity relationship of compounds showing less than 20 % residual enzyme
activity at an inhibitor concentration of 10 µM. IC values are given with their corresponding 95
5
0
%
confidence interval of pIC .
50
X
R
IC (MMPꢀ10) [nM]
IC (MMPꢀ13) [nM]
50
5
0
2
3
31
[pIC50 = 7.61ꢀ7.53]
5
[pIC50 = 8.44ꢀ8.26]
[pIC50 = 7.11ꢀ6.79]
C=O
NH
1210
[pIC50 = 5.32ꢀ4.74]
111
2
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19
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21
22
23
24
25
26
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31
32
33
34
35
36
37
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
X
R
IC (MMPꢀ10) [nM]
IC (MMPꢀ13) [nM]
50
50
4
5
6
1
1
1
1
2
2
C=O
NHMe
635
243
506
347
751
2050
705
7380
531
147
[pIC50 = 6.32ꢀ6.08]
86
89
[pIC50 = 7.19ꢀ6.95]
[pIC50 = 7.15ꢀ6.96]
[pIC50 = 7.00ꢀ6.80]
[pIC50 = 7.35ꢀ7.14]
[pIC50 = 6.99ꢀ6.71]
[pIC50 = 6.88ꢀ6.66]
[pIC50 = 8.11ꢀ7.82]
[pIC50 = 6.69ꢀ6.49]
[pIC50 = 7.59ꢀ7.39]
[pIC50 = 7.82ꢀ7.46]
C=O
C=O
C=O
C=O
C=O
C=O
S(=O)
S(=O)
S(=O)
NMe
OMe
[pIC50 = 6.73ꢀ6.23]
[pIC50 = 6.34ꢀ6.25]
[pIC50 = 6.56ꢀ6.40]
[pIC50 = 6.31ꢀ5.97]
[pIC50 = 5.90ꢀ5.31]
[pIC50 = 6.30ꢀ6.08]
[pIC50 = 5.32ꢀ4.74]
[pIC50 = 6.39ꢀ6.17]
[pIC50 = 7.17ꢀ6.85]
2
128
58
0
2
4
8
1
2
NH(CH ) OH
2 2
LꢀAlaꢀOMe
DꢀProꢀOMe
LꢀSerꢀOH
149
175
14
NH
258
35
2
2
2
2
NHMe
24
NHCH(CH
3
)
2
25
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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
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
X
R
IC (MMPꢀ10) [nM]
IC (MMPꢀ13) [nM]
50
50
2
5
6
S(=O)
2
2
2
2
NHcycProp
110
131
[pIC50 = 7.17ꢀ6.95]
14
26
66
32
[pIC50 = 7.99ꢀ7.93]
[pIC50 = 7.92ꢀ7.68]
[pIC50 = 7.79ꢀ7.44]
[pIC50 = 7.82ꢀ7.46]
2
S(=O)
S(=O)
S(=O)
NHC(CH
3
)
3
[pIC50 = 7.29ꢀ6.85]
[pIC50 = 6.12ꢀ5.77]
[pIC50 = 6.61ꢀ6.36]
27
morpholine
GlyꢀOMe
1930
485
2
8
The most potent nonꢀhydroxamate MMPꢀ10 inhibitor found in literature was originally
2
4
developed as a very potent MMPꢀ13 inhibitor. This compound, T-26C (structure given in the
supporting information), was disclosed with an IC value of 160 nM for MMPꢀ10 and 6.9 pM
50
for MMPꢀ13. In our assay system, values of 358 nM for MMPꢀ10 [pIC50 = 6.51ꢀ6.39] and 0.19
nM for MMPꢀ13 [pIC50 = 9.86ꢀ9.56] were measured. The dual MMPꢀ10/ꢀ13 inhibitor 2 displays
a ten times lower IC50 value making it, to the best of our knowledge, the most potent nonꢀ
hydroxamate MMPꢀ10 inhibitor currently known. Within the MMP family, the selectivity profile
of 2 was determined (MMPꢀ1, ꢀ2, ꢀ3, ꢀ7, ꢀ8, ꢀ9, ꢀ12 and ꢀ14). Except for MMPꢀ3, the remaining
enzyme activity was >85 % at an inhibitor concentration of 10 µM for all of the tested enzymes,
which demonstrates the high selectivity for MMPꢀ10/ꢀ13 (Table 3). The determination of the
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IC50 value of 2 on MMPꢀ3 (2.91 µM [pIC = 6.01ꢀ4.78]) demonstrates high selectivity for
50
MMPꢀ10/ꢀ13, even between the two structurally closely related stromelysins MMPꢀ3 and MMPꢀ
3
6
10.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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31
32
33
34
35
36
37
38
39
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51
52
53
54
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Table 3. Inhibition profile of 2 at an inhibitor concentration of 10 µM. Residual enzyme activity
is given at an inhibitor concentration of 10 µM (measured in duplicate).
Remaining activity [%]
MMP
ꢀ1
ꢀ2
ꢀ3
ꢀ7
ꢀ8
ꢀ9
ꢀ12
ꢀ14
3
7.3
2.1
87.1
96.7
97.8
88.5
97.6
2
100
100
±
± 1.2
± 2.9
± 0.4
± 0.6
± 0.7
Taking into account that both MMPs are validated drug targets, this dual inhibitor represents
3
7
a promising base for polypharmacological approaches in complex systems. Literature evidence
2
0,22,23
demonstrates the mutual relevance of the two targeted proteases.
By using 2, this
interconnection can be examined in complex diseases related to these proteins. MMPꢀ10 was
2
0,22
shown to activate the proꢀcollagenases and thereby to promote collagenolytic activity.
The
here presented dual inhibitor 2 is able to inhibit MMPꢀ10 and thereby can reduce the
collagenolytic activity. In addition, the inhibition of MMPꢀ13 can block one of the collagenases
selectively for the time of administration.
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2
2
2
2
2
2
2
2
2
2
3
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3
3
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3
3
3
3
4
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4
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4
4
4
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5
5
5
6
CONCLUSION
In conclusion, we succeeded in the parallel optimization of a dual MMPꢀ10/ꢀ13 inhibitor to
discover the nonꢀhydroxamate inhibitor 2 with outstanding affinity for MMPꢀ10 and similar
affinity for MMPꢀ13. Selectivity for these two proteins among the MMP family was additionally
shown. The benefit of a dual MMP inhibitor with a clean inhibition profile is based on the
network like organization of MMPs, in which specific proteases activate each other and take
related functions in this complex system. Inhibitor 2 represents a viable tool compound to gain
further insight into this complex system, and provides an excellent starting point for the
development of therapies based on targeted polypharmacology.
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
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9
0
1
2
3
4
5
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8
9
0
EXPERIMENTAL SECTION
Chemistry General Procedures. All reagents and solvents were purchased from Sigma Aldrich,
TCI or Fluorochem and used as received. All NMR spectra were recorded on a Bruker AVANCE
III HD 500 One Bay spectrometer with a magnetic field of 11.75 T and a 5mm SmartProbe
BB(F)ꢀHꢀD. For 1H NMR spectra, a frequency of 500 MHz resulted. Chemical shifts are
reported in ppm from tetramethylsilane as internal standard. Data are reported as follows:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint. = quintet, br =
1
3
broad, m = multiplet), coupling constants (Hz), integration. For C NMR spectra, a frequency of
125 MHz resulted. Chemical shifts are reported in ppm from tetramethylsilane as internal
standard. Highꢀresolution mass spectrometry was performed on an Agilent Technologies 6530
QꢀTOF. Purity was assayed by HPLC (Interchim Strategy Cꢀ18 column, 4.6 mm × 250 mm) with
a gradient of 5 – 100 % methanol in 0.2 % aqueous acetic acid with UV detection at λ = 254 nm.
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All final compounds were obtained with ≥95 % purity. Microwave synthesis was performed in a
Biotage initiator.
General procedure for the nucleophilic substitution by 5-iodouracil. The 4ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
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50
51
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53
54
55
56
57
58
59
60
(
bromomethyl)benzoic acid derivative (4.7 mmol, 1.0 eq) was dissolved in DMF (7 mL) and
added dropwise to a suspension of 5ꢀiodouracil (8.4 mmol, 1.8 eq) and caesium carbonate (5.7
mmol, 1.2 eq) in DMF (18 mL), which has been stirred at room temperature for 15 minutes
before under nitrogen atmosphere. After the complete addition, the mixture was stirred for 2.5 h.
The mixture was then separated between ethyl acetate and 2 M hydrochloric acid. After
concentration, the resulting liquid was purified by flash chromatography (reversed phase C18,
gradient: 10 – 100% methanol in water).
General procedure for the amide formation. Procedure A. Carboxylic acid (0.17 mmol),
amine (0.45 mmol), COMU (0.17 mmol) and diisopropylethylamine (DIPEA, 0.1 mL) were
dissolved in DMF (1.5 mL), and the yellow solution was stirred over night at room temperature
under nitrogen atmosphere. The reaction mixture was then separated between ethyl acetate and 2
M hydrochloric acid, concentrated and then purified by flash chromatography (reversed phase
C18, gradient: 10 – 100% methanol in water). Procedure B. Sulfonyl chloride (2.78 mmol, 1.5
eq) was dissolved in THF or methanol (20 mL) and cooled. At this temperature, the amine (1.86
mmol, 1.0 eq) was added dropwise and the mixture was stirred for 30 min. Then, 6 M
hydrochloric acid was added to the cold solution, which was then allowed to warm up to room
temperature. The mixture was then concentrated in vacuo and purified by flash chromatography
(SiO , gradient: dichloromethane – methanol).
2
General procedure for the Sonogashira coupling. Iodouracil derivative (0.10 mmol, 1.0 eq),
alkyne (0.17 mmol, 1.7 eq), tetrakistriphenylphosphine palladium (0.01 mmol, 0.1 eq), copper
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
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4
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4
4
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5
5
5
5
5
5
5
6
iodide (0.05 mmol, 0.5 eq) and triethylamine (0.2 mL) were dissolved under argon atmosphere in
DMF (1.5 mL), and stirred at room temperature for 3 h. The reaction mixture was separated
between ethyl acetate and 2 M hydrochloric acid, concentrated and purified by flash
chromatography (reversed phase C18, gradient: 10 % – 100% methanol in water).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
0
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5
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7
8
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1
2
3
4
5
6
7
8
9
0
General procedure for the ester hydrolysis. The ester was suspended in potassium hydroxide
solution (3M, 3 mL) and stirred at room temperature for 2 h. The reaction mixture was then
acidified to pH 1 with 6 M hydrochloric acid and extracted with ethyl acetate. The combined
organic layers were concentrated under reduced pressure und purified by flash chromatography.
N-[(4-Fluoro-3-methoxyphenyl)methyl]-3-(1-{[4-(1,3-oxazol-5-yl)phenyl]methyl}-2,4-dioxo-
1,2,3,4-tetrahydropyrimidin-5-yl)prop-2-ynamide (2) was synthesized according to the
general procedure of the Sonogashira coupling from intermediates 30 and 31. Before flash
chromatography, the product was washed with 10 ml of a 1/1 mixture of methanol/water. 2 was
1
obtained with 13 % yield as a white solid. H NMR (500 MHz, DMSOꢀd , 25 °C, TMS): δ 11.80
6
(
br s, 1H), 9.15 (t, J = 6.0 Hz, 1H), 8.44 (s, 1H), 8.43 (s, 1H), 7.74ꢀ7.70 (m, 2H), 7.69 (s, 1H),
.46ꢀ7.41 (m, 2H), 7.14 (dd, J = 11.9 Hz, 8.5 Hz, 1H), 7.07 (dd, J = 8.5 Hz, 2.0 Hz, 1H), 6.80
(ddd, J = 8.2 Hz, 4.2 Hz, 2.0 Hz, 1H), 4.94 (s, 2H), 4.27 (d, J = 6.0 Hz, 2H), 3.81 (s, 3H) ppm.
7
1
3
C NMR (125 MHz, DMSOꢀd , 25 °C, TMS): δ 161.9, 152.2, 151.9, 151.5, 150.6 (d, JCF
=
6
242.0 Hz), 150.2, 150.1, 146.8 (d, JCF = 10.9 Hz), 136.7, 135.5 (d, JCF = 3.4 Hz), 128.5 (2C),
126.9, 124.4 (2C), 122.2, 119.5 (d, JCF = 7.7 Hz), 115.5 (d, JCF = 18.0 Hz), 113.1 (d, JCF = 1.6
+
Hz), 95.5, 85.8, 77.8, 55.9, 50.8, 42.0 ppm. HRMSꢀTOF m/z [M+H] calculated for
C H FN O : 474.134, found: 474.142.
25 19
4
5
4-{[5-(2-{[(4-Fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-1-yl]methyl}benzamide (3) was synthesized according to the general
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procedure of the Sonogashira coupling from intermediates 34 and 31. Before flash
chromatography, the product was washed with 10 ml of a 1/1 mixture of methanol/water. 3 was
1
obtained with 20 % yield as a white solid. H NMR (500 MHz, DMSOꢀd , 25 °C, TMS): δ 11.83
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
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42
43
44
45
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47
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(
s, 1H), 9.14 (t, J = 6.2 Hz, 1H), 8.38 (s, 1H), 7.96 (s, 1H), 7.87ꢀ7.81 (m, 2H), 7.42ꢀ7.36 (m, 2H),
7.36 (s, 1H), 7.15 (dd, J = 11.5 Hz, 8.4 Hz, 1H), 7.08 (dd, J = 8.4 Hz, 1.9 Hz, 1H), 6.81 (ddd, J =
1
3
8
.3 Hz, 4.6 Hz, 2.2 Hz, 1H), 4.95 (s, 2H), 4.28 (d, J = 6.2 Hz, 2H), 3.82 (s, 3H) ppm. C NMR
(125 MHz, DMSOꢀd , 25 °C, TMS): δ 167.9 (2C), 152.8, 151.9, 151.8, 151.0 (d, JCF = 241.0
6
Hz), 147.3 (d, JCF = 10.6 Hz), 140.0, 136.0 (d, JCF = 4.0 Hz), 134.2, 128.3 (2C), 127.8 (2C),
1
4
4
20.0 (d, J = 6.7 Hz), 116.0 (d, J = 17.9 Hz), 113.6 (d, J = 1.6 Hz), 96.0, 87.2, 56.4, 51.3,
CF CF CF
+
2.5 ppm. HRMSꢀTOF m/z [M+H] calculated for C H FN O : 450.134, found: 450.141.
23 19
4
5
-{[5-(2-{[(4-Fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-1-yl]methyl}-N-methylbenzamide (4) was synthesized according to the
general procedure of the Sonogashira coupling from intermediates 35 and 31. Before flash
chromatography, the product was washed with 10 ml of a 1/1 mixture of methanol/water. 4 was
1
obtained with 22 % yield as a white solid. H NMR (500 MHz, DMSOꢀd , 25 °C, TMS): δ 11.83
6
(s, 1H), 9.16 (t, J = 6.2 Hz, 1H), 8.43 (s, 1H), 8.42 (q, J = 4.5 Hz, 1H), 7.84ꢀ7.77 (m, 2H), 7.43ꢀ
7
.35 (m, 2H), 7.15 (dd, J = 11.7 Hz, 8.5 Hz, 1H), 7.08 (dd, J = 8.5 Hz, 2.0 Hz, 1H), 6.81 (ddd, J
=
8.0 Hz, 4.5 Hz, 2.0 Hz, 1H), 4.97 (s, 2H), 4.28 (d, J = 6.0 Hz, 2H), 8.32 (s, 3H), 2.78 (d, J =
1
3
4.5 Hz, 3H) ppm. C NMR (125 MHz, DMSOꢀd , 25 °C, TMS): δ 166.7, 162.3, 152.7, 152.0,
6
1
51.1 (d, JCF = 243.6 Hz), 150.5, 147.3 (d, JCF = 9.3 Hz), 139.6, 135.9 (d, JCF = 4.1 Hz), 134.5,
27.8 (4C), 120.0 (d, J = 6.9 Hz), 116.0 (d, J = 17.9 Hz), 113.6 (d, J = 1.5 Hz), 96.0, 87.3,
1
CF
CF
CF
+
7
8.3, 56.4, 51.3, 42.5, 26.7 ppm. HRMSꢀTOF m/z [M+H] calculated for C H FN O : 464.150,
24 21 4 5
found: 464.158.
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-{[5-(2-{[(4-Fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-1-yl]methyl}-N,N-dimethylbenzamide (5) was synthesized according to
the general procedure of the Sonogashira coupling from intermediates 36 and 31. Before flash
0
1
2
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7
8
9
0
1
2
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7
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9
0
chromatography, the product was washed with 10 ml of a 1/1 mixture of methanol/water. 5 was
1
obtained with 28 % yield as a white solid. H NMR (500 MHz, DMSOꢀd , 25 °C, TMS): δ 11.83
6
(s, 1H), 9.14 (t, J = 6.3 Hz, 1H), 8.41 (s, 1H), 7.42ꢀ7.35 (m, 4H), 7.15 (dd, J = 11.6 Hz, 8.6 Hz,
1H), 7.08 (dd, J = 8.6 Hz, 2.0 Hz, 1H), 6.81 (ddd, J = 8.3 Hz, 4.3 Hz, 2.0 Hz, 1H), 4.95 (s, 2H),
1
3
4.28 (d, J = 6.3 Hz, 2H), 3.82 (s, 3H), 2.97 (s, 3H), 2.89 (s, 3H) ppm. C NMR (125 MHz,
DMSOꢀd , 25 °C, TMS): δ 170.2, 163.0, 151.9, 151.0, 151.0 (d, J = 241.4 Hz), 147.3 (d, J =
6
CF
CF
1
0.3 Hz), 138.1, 136.4, 136.0 (d, J = 2.5 Hz), 127.8 (2C), 127.8 (2C), 120.0 (d, J = 6.5 Hz),
CF CF
116.0 (d, JCF = 18.8 Hz), 113.6 (d, JCF = 2.0 Hz), 96.0), 87.7, 78.7, 56.4, 51.2, 42.5, 39.5, 35.2
+
ppm. HRMSꢀTOF m/z [M+H] calculated for C H FN O : 478.165, found: 478.174.
2
5
23
4
5
Methyl 4-{[5-(2-{[(4-fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-dioxo-
,2,3,4-tetrahydropyrimidin-1-yl]methyl}benzoate (6) was synthesized according to the
1
general procedure of the Sonogashira coupling from intermediates 33 and 31. Before flash
chromatography, the product was washed with 10 ml of a 1/1 mixture of methanol/water. 6 was
1
obtained with 11 % yield as a white solid. H NMR (500 MHz, DMSOꢀd , 25 °C, TMS): δ 11.85
6
(br s, 1H), 9.16 (t, J = 6.0 Hz, 1H), 8.45 (s, 1H), 7.97ꢀ7.91 (m, 2H), 7.48ꢀ7.41 (m, 2H), 7.15 (dd,
J = 11.6 Hz, 8.2 Hz, 1H), 7.07 (dd, J = 8.4 Hz, 1.9 Hz, 1H), 6.80 (ddd, J = 8.2 Hz, 4.2 Hz, 1.9
13
Hz, 1H), 5.00 (s, 2H), 4.27 (d, J = 6.1 Hz, 2H), 3.85 (s, 3H), 3.82 (s, 3H) ppm. C NMR (125
MHz, DMSOꢀd , 25 °C, TMS): δ 166.4, 153.1, 152.1, 151.1 (d, J = 245.4 Hz), 147.3 (d, J =
6
CF
CF
9
.2 Hz), 142.1, 135.9 (d, J = 2.6 Hz), 130.0 (2C), 129.5, 129.3, 129.2, 128.1 (2C), 120.0 (d, J
CF
CF
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6.0 Hz), 116.0 (d, JCF = 17.4 Hz), 113.6 (d, JCF = 1.1 Hz), 96.1, 87.4, 78.5, 56.4, 52.7, 51.4,
+
42.5 ppm. HRMSꢀTOF m/z [M+H] calculated for C H FN O : 465.134, found: 465.141.
24 20
3
6
4-{[5-(2-{[(4-Fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-dioxo-1,2,3,4-
10
11
12
13
14
15
16
17
18
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60
tetrahydropyrimidin-1-yl]methyl}-N-(propan-2-yl)benzamide (7) was synthesized according
to the general procedure of the Sonogashira coupling from intermediates 37 and 31. Before flash
chromatography, the product was washed with 10 ml of a 1/1 mixture of methanol/water. 7 was
1
obtained with 12 % yield as a white solid. H NMR (500 MHz, DMFꢀd , 25 °C, TMS): δ 11.79
7
(br s, 1H), 9.23 (t, J = 6.7 Hz, 1H), 8.48 (s, 1H), 8.21 (d, J = 7.4 Hz, 1H), 7.98ꢀ7.92 (m, 2H),
7.55ꢀ7.48 (m, 2H), 7.20 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 7.17 (dd, J = 11.8 Hz, 8.4 Hz, 1H), 6.92
(ddd, J = 7.8 Hz, 4.1 Hz, 1.9 Hz, 1H), 5.11 (s, 2H), 4.42 (d, J = 6.2 Hz, 2H), 4.20 (octett, 7.0 Hz,
1
3
1H), 3.89 (s, 3H), 1.22 (d, J = 6.7 Hz, 6H) ppm. C NMR (125 MHz, DMFꢀd , 25 °C, TMS): δ
7
1
1
1
66.4, 163.7, 153.9, 152.6, 152.3 (d, JCF = 243.8Hz), 151.8, 148.5 (d, JCF = 10.3 Hz), 140.6,
37.0 (d, JCF = 3.6 Hz), 136.0, 128.7 (4C), 120.8 (d, JCF = 7.6 Hz), 116.6 (d, JCF = 18.6 Hz),
14.5 (d, J = 2.0 Hz), 97.3, 88.0, 78.8, 56.8, 52.2, 43.5, 42.5, 23.0 (2C) ppm. HRMSꢀTOF m/z
CF
+
[
M+H] calculated for C H FN O : 492.181, found: 492.188.
2
6
25
4
5
N-Cyclopropyl-4-{[5-(2-{[(4-fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-
dioxo-1,2,3,4-tetrahydropyrimidin-1-yl]methyl}benzamide (8) was synthesized according to
the general procedure of the Sonogashira coupling from intermediates 38 and 31. Before flash
chromatography, the product was washed with 10 ml of a 1/1 mixture of methanol/water. 8 was
1
obtained with 17 % yield as a white solid. H NMR (500 MHz, DMFꢀd , 25 °C, TMS): δ 11.65
7
(br s, 1H), 9.41 (t, J = 5.8 Hz, 1H), 8.770 (s, 1H), 8.63 (d, J = 4.0 Hz, 1H), 8.14ꢀ8.08 (m, 2H),
7.74ꢀ7.68 (m, 2H), 7.38 (dd, J = 8.5 Hz, 1.6 Hz, 1H), 7.34 (dd, J = 11.8 Hz, 8.5 Hz, 1H), 7.09
(ddd, J = 8.1 Hz, 4.3 Hz, 2.0 Hz, 1H), 5.29 (s, 2H), 4.60 (d, J = 6.1 Hz, 2H), 4.07 (s, 3H), 1.54ꢀ
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13
1
.43 (m, 1H), 0.97ꢀ0.88 (m, 2H), 0.86ꢀ0.78 (m, 2H) ppm. C NMR (125 MHz, DMFꢀd , 25 °C,
7
TMS): δ 168.4, 163.7, 162.8, 153.8, 152.8, 152.3 (d, J = 243.2 Hz), 151.4, 148.5 (d, J = 10.7
CF
CF
Hz), 140.5, 137.0 (d, J = 3.2 Hz), 135.6, 128.8 (2C), 128.7 (2C), 120.8 (d, J = 6.9 Hz), 116.6
CF
CF
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(
d, J = 18.2 Hz), 114.5 (d, J = 1.8Hz), 97.3, 88.1, 78.4, 56.8, 52.3, 43.5, 24.2, 6.5 (2C) ppm.
CF CF
+
HRMSꢀTOF m/z [M+H] calculated for C H FN O : 490.165, found: 490.173.
2
6
23
4
5
N-tert-Butyl-4-{[5-(2-{[(4-fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-
dioxo-1,2,3,4-tetrahydropyrimidin-1-yl]methyl}benzamide (9) was synthesized according to
the general procedure of the Sonogashira coupling from intermediates 39 and 31. Before flash
chromatography, the product was washed with 10 ml of a 1/1 mixture of methanol/water. 9 was
1
obtained with 40 % yield as a white solid. H NMR (500 MHz, DMSOꢀd , 25 °C, TMS): δ 11.82
6
(br s, 1H), 9.14 (t, J = 5.7 Hz, 1H), 8.39 (s, 1H), 7.77ꢀ7.73 (m, 2H), 7.72 (s, 1H), 7.40ꢀ7.33 (m,
2
H), 7.14 (dd, J = 11.6 Hz, 8.2 Hz, 1H), 7.07 (dd, J = 8.5 Hz, 2.0 Hz, 1H), 6.80 (ddd, J = 8.3 Hz,
1
3
4
.4 Hz, 2.1 Hz, 1H), 4.95 (s, 2H), 4.27 (d, J = 6.1 Hz, 2H), 3.82 (s, 3H), 1.36 (s, 9H) ppm. C
NMR (125 MHz, DMFꢀd , 25 °C, TMS): δ 166.3, 162.7, 152.9, 151.4, 151.3, 151.2 (d, JCF
=
7
2
40.4 Hz), 147.5 (d, JCF = 11.1 Hz), 139.5, 136.0 (d, JCF = 3.9 Hz), 136.0, 127.7 (2C), 127.6
(2C), 119.8 (d, J = 5.7 Hz), 115.6 (d, J = 18.7 Hz), 113.4 (d, J = 1.1 Hz), 96.2, 85.9, 78.3,
CF
CF
CF
+
5
5.8, 51.2, 51.1, 42.5, 28.3 (3C) ppm. HRMSꢀTOF m/z [M+H] calculated for C H FN O :
27 27 4 5
5
06.197, found: 506.201.
4-{[5-(2-{[(4-Fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-1-yl]methyl}-N-(2-hydroxyethyl)benzamide (10) was synthesized
according to the general procedure of the Sonogashira coupling from intermediates 40 and 31.
Before flash chromatography, the product was washed with 10 ml of a 1/1 mixture of
1
methanol/water. 10 was obtained with 13 % yield as a white solid. H NMR (500 MHz, DMFꢀd ,
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5 °C, TMS): δ 11.84 (br s, 1H), 9.24 (t, J = 6.3 Hz, 1H), 8.56ꢀ8.49 (m, 2H), 8.01ꢀ7.96 (m, 2H),
.57ꢀ7.51 (m, 2H), 7.20 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 7.17 (dd, J = 11.6 Hz, 8.4 Hz, 1H), 6.92
7
(ddd, J = 8.3 Hz, 4.3 Hz, 2.1 Hz, 1H), 5.12 (s, 2H), 4.87 (d, J = 5.7 Hz, 1H), 4.42 (d, J = 6.1 Hz,
10
11
12
13
14
15
16
17
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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
2
H), 3.89 (s, 3H), 3.66 (q, J = 5.9 Hz, 2H), 3.47 (q, J = 5.9 Hz, 2H) ppm. C NMR (125 MHz,
DMFꢀd , 25 °C, TMS): δ 167.4, 162.8, 153.8, 152.8, 152.3 (d, JCF = 243.6 Hz), 151.4, 148.5 (d,
7
J = 11.2 Hz), 140.5, 137.0 (d, J = 3.0 Hz), 135.7, 128.8 (2C), 128.8 (2C), 120.8 (d, J = 7.4
CF
CF
CF
Hz), 116.6 (d, JCF = 18.1 Hz), 114.5 (d, JCF = 1.8 Hz), 97.3, 88.1, 78.4, 61.6, 56.8, 52.3, 43.8,
+
43.5 ppm. HRMSꢀTOF m/z [M+H] calculated for C H FN O : 494.160, found: 494.169.
2
5
23
4
5
Methyl
dioxo-1,2,3,4-tetrahydropyrimidin-1-yl]methyl}phenyl)formamido]acetate
synthesized according to the general procedure of the Sonogashira coupling from intermediates
2-[(4-{[5-(2-{[(4-fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-
(11) was
4
1 and 31. Before flash chromatography, the product was washed with 10 ml of a 1/1 mixture of
1
methanol/water. 11 was obtained with 17 % yield as a white solid. H NMR (500 MHz, DMFꢀd ,
7
2
5 °C, TMS): δ 11.93 (br s, 1H), 9.28 (t, J = 6.1 Hz, 1H), 9.04 (t, J = 5.7 Hz, 1H), 8.56 (s, 1H),
.05ꢀ8.00 (m, 2H), 7.63ꢀ7.57 (m, 2H), 7.24 (dd, J = 8.5 Hz, 1.9 Hz, 1H), 7.20 (dd, J = 11.6 Hz,
8
8.3 Hz, 1H), 6.95 (ddd, J = 8.3 Hz, 4.4 Hz, 2.1 Hz, 1H), 5.17 (s, 2H), 4.46 (d, J = 6.0 Hz, 2H),
1
3
4
.18 (d, J = 5.8 Hz, 2H), 3.93 (s, 3H), 3.74 (s, 3H) ppm. C NMR (125 MHz, DMFꢀd , 25 °C,
7
TMS): δ 171.5, 167.6, 163.8, 158.7, 154.7 (d, J = 244.4 Hz), 152.7, 151.3, 148.4 (d, J = 10.6
CF
CF
Hz), 141.0, 136.8 (d, J = 3.6 Hz), 134.7, 128.8 (2C), 128.7 (2C), 120.7 (d, J = 6.0 Hz), 116.5
CF
CF
(
d, JCF = 17.4 Hz), 114.3 (d, JCF = 2.5 Hz), 97.2, 88.0, 78.3, 56.7, 52.4, 52.2, 43.4, 42.3 ppm.
+
HRMSꢀTOF m/z [M+H] calculated for C H FN O : 522.155, found: 522.161.
26 23
4
7
Methyl (2S)-2-[(4-{[5-(2-{[(4-fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-
dioxo-1,2,3,4-tetrahydropyrimidin-1-yl]methyl}phenyl)formamido]propanoate (12) was
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6
synthesized according to the general procedure of the Sonogashira coupling from intermediates
42 and 31. Before flash chromatography, the product was washed with 10 ml of a 1/1 mixture of
1
methanol/water. 12 was obtained with 23 % yield as a white solid. H NMR (500 MHz, DMSOꢀ
d6, 25 °C, TMS): δ 11.83 (s, 1H), 9.16 (t, J = 6.0 Hz, 1H), 8.79 (d, J = 6.8 Hz, 1H), 8.42 (s, H),
7.91ꢀ7.82 (m, 2H), 7.49ꢀ7.39 (m, 2H), 7.15 (dd, J = 11.6 Hz, 8.0 Hz, 1H), 7.08 (dd, J = 8.8 Hz,
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
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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
2.0 Hz, 1H), 6.81 (ddd, J = 8.0 Hz, 4.4 Hz, 2.0 Hz, 1H), 4.98 (s, 2H), 4.48 (quint., J = 7.2 Hz,
13
1
H), 4.28 (d, J = 6.0 Hz, 2H), 3.82 (s, 3H), 8.64 (s, 3H), 1.40 (d, J = 7.2 Hz, 3H) ppm. C NMR
(125 MHz, DMSOꢀd , 25 °C, TMS): δ 173.6, 166.3, 152.7, 152.0, 151.1 (d, JCF = 242.0 Hz),
6
1
50.5, 147.3 (d, JCF = 10.6 Hz), 140.1, 135.9 (d, JCF = 3.0 Hz), 133.6, 128.3 (2C), 127.9 (2C),
20.0 (d, J = 6.6 Hz), 116.0 (d, J = 18.0 Hz), 113.6 (d, J = 1.4 Hz), 96.0, 87.3, 78.2, 56.4,
1
CF
CF
CF
+
52.4, 51.4, 48.7, 42.5, 17.2 ppm. HRMSꢀTOF m/z [M+H] calculated for C H FN O : 536.171,
2
7
25
4
7
found: 536.177.
Methyl (2S)-2-[(4-{[5-(2-{[(4-fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-
dioxo-1,2,3,4-tetrahydropyrimidin-1-yl]methyl}phenyl)formamido]-3-hydroxypropanoate
(13) was synthesized according to the general procedure of the Sonogashira coupling from
intermediates 44 and 31. Before flash chromatography, the product was washed with 10 ml of a
1
1
/1 mixture of methanol/water. 13 was obtained with 36 % yield as a white solid. H NMR (500
MHz, DMFꢀd , 25 °C, TMS): δ 9.24 (t, J = 6.3 Hz, 1H), 8.83 (d, J = 7.19 Hz, 1H), 8.53 (s, 1H),
7
8.13ꢀ8.08 (m, 2H), 7.58ꢀ7.53 (m, 2H), 7.20 (dd, J = 8.4 Hz, 2.1 Hz, 1H), 7.17 (dd, J = 11.6 Hz,
8.4 Hz, 1H), 6.92 (ddd, J = 8.2 Hz, 4.5 Hz, 2.1 Hz, 1H), 5.13 (s, 2H), 4.69ꢀ4.64 (m, 1H), 4.42 (d,
13
J = 6.1 Hz, 2H), 4.01ꢀ3.93 (m, 2H), 3.89 (s, 3H), 3.70 (s, 3H) ppm. C NMR (125 MHz, DMFꢀ
d7, 25 °C, TMS): δ 172.3, 167.5, 162.8, 153.8, 152.7, 151.4, 149.2 (d, JCF = 345.8 Hz), 141.0,
1
37.0 (d, JCF = 3.6 Hz), 134.8, 129.2 (2C), 129.1, 128.8 (2C), 120.8 (d, J = 6.6 Hz), 116.6 (d,
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JCF = 18.7 Hz), 114.5 (d, JCF = 1.8 Hz), 97.3, 88.1, 78.4, 62.7, 57.5, 56.8, 52.6, 52.3, 43.5 ppm.
+
HRMSꢀTOF m/z [M+H] calculated for C H FN O : 552.166, found: 552.176.
27 25
4
8
Methyl (2R)-1-(4-{[5-(2-{[(4-fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-
dioxo-1,2,3,4-tetrahydropyrimidin-1-yl]methyl}benzoyl)pyrrolidine-2-carboxylate (14) was
synthesized according to the general procedure of the Sonogashira coupling from intermediates
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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43
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60
46 and 31. Before flash chromatography, the product was washed with 10 ml of a 1/1 mixture of
1
methanol/water. 14 was obtained with 41 % yield as a white solid. H NMR (500 MHz, DMSOꢀ
d6, 25 °C, TMS): δ 11.85 (s, 1H), 9.17 (t, J = 6.1 Hz, 1H), 8.47 (s, 1H), 7.58ꢀ7.49 (m, 2H), 7.44ꢀ
7.38 (m, 2H), 7.15 (dd, J = 11.6 Hz, 8.4 Hz, 1H), 7.08 (dd, J = 8.4 Hz, 1.7 Hz, 1H), 6.81 (ddd, J
=
8.1 Hz, 4.4 Hz, 1.7 Hz, 1H), 4.98 (s, 2H), 4.28 (d, J = 6.1 Hz, 2H), 4.10 (q, J = 5.2 Hz, 1H),
1
3
3.82 (s, 3H), 3.67 (s, 3H), 3.55ꢀ3.47 (m, 2H), 2.32ꢀ2.22 (m, 1H), 1.95ꢀ1.80 (m, 3H) ppm. C
NMR (125 MHz, DMSOꢀd , 25 °C, TMS): δ 172.8, 168.3, 162.1, 152.7, 152.1, 151.1 (d, JCF
=
6
2
1
7
42.9 Hz), 150.4, 147.3 (d, JCF = 11.2 Hz), 138.7, 135.9 (d, JCF = 4.3 Hz), 135.9, 128.0 (2C),
27.9 (2C), 120.0 (d, J = 6.1 Hz), 116.0 (d, J = 18.1 Hz), 113.6 (d, J = 1.3 Hz), 96.0, 87.4,
CF
CF
CF
+
8.1, 59.4, 56.4, 52.3, 51.3, 50.0, 42.5, 29.3, 25.5 ppm. HRMSꢀTOF m/z [M+H] calculated for
C H FN O : 562.186, found: 562.195.
29
27
4
7
2
-[(4-{[5-(2-{[(4-Fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-dioxo-
,2,3,4-tetrahydropyrimidin-1-yl]methyl}phenyl)formamido]acetic acid (15)
1
was
synthesized according to the general procedure of the ester hydrolysis from intermediate 11. 15
1
was obtained with 17 % yield over two steps as a white solid. H NMR (500 MHz, DMSOꢀd , 25
6
°
C, TMS): δ 11.79 (br s, 1H), 9.17 (t, J = 6.4 Hz, 1H), 8.43 (s, 1H), 8.05 (br s, 1H), 7.84ꢀ7.75 (m,
2
H), 7.45ꢀ7.35 (m, 2H), 7.14 (dd, J = 11.4 Hz, 8.3 Hz, 1H), 7.07 (dd, J = 8.3 Hz, 1.6 Hz, 1H),
.80 (ddd, J = 8.0 Hz, 4.3 Hz, 1.9 Hz, 1H), 4.97 (s, 2H), 4.27 (d, J = 6.1 Hz, 2H), 3.81 (s, 3H),
6
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Journal of Medicinal Chemistry
Page 28 of 58
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9
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5
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13
3
1
1
9
.56 (br s, 2H) ppm. C NMR (125 MHz, DMFꢀd , 25 °C, TMS): δ 166.4, 163.7, 158.8, 152.8,
7
52.3 (d, J = 243.5 Hz), 151.6, 148.5 (d, J = 10.9 Hz), 140.5, 137.0 (d, J = 3.6 Hz), 135.9 ,
CF
CF
CF
28.9 (2C), 128.5 (2C), 120.8 (d, J = 7.6 Hz), 116.6 (d, J = 17.6 Hz), 114.5 (d, J = 2.0 Hz),
CF
CF
CF
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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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
+
7.3, 88.1, 78.6, 56.8, 57.3, 44.6, 43.5 ppm. HRMSꢀTOF m/z [M+H] calculated for
C H FN O : 508.139, found: 508.149.
25
21
4
7
(
2S)-2-[(4-{[5-(2-{[(4-Fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-dioxo-
1
,2,3,4-tetrahydropyrimidin-1-yl]methyl}phenyl)formamido]propanoic acid (16) was
synthesized according to the general procedure of the ester hydrolysis from intermediate 12. 16
1
was obtained with 18 % yield over two steps as a white solid. H NMR (500 MHz, DMSOꢀd , 25
6
°
C, TMS): δ 12.54 (br s, 1H), 11.83 (s, 1H), 9.17 (t, J = 6.0 Hz, 1H), 8.60 (s, 1H), 8.44 (s, 1H),
7.89ꢀ7.82 (m, 2H), 7.47ꢀ7.82 (m, 2H), 7.15 (dd, J = 11.3 Hz, 8.2 Hz, 1H), 7.08 (dd, J = 8.4 Hz,
1
6
1
.6 Hz, 1H), 6.81 (ddd, J = 8.1 Hz, 4.1 Hz, 1.5 Hz, 1H), 4.98 (s, 2H), 4.36 (m, 1H), 4.28 (d, J =
1
3
.0 Hz, 2H), 3.82 (s, 3H), 1.38 (m, 3H) ppm. C NMR (125 MHz, DMSOꢀd , 25 °C, TMS): δ
6
62.1 (2C), 152.7, 152.1, 151.0 (d, J = 242.8 Hz), 150.4, 147.3 (d, J = 9.6 Hz), 139.8, 135.9
CF
CF
(
d, JCF = 3.5 Hz), 134.1, 130.0, 128.2 (2C), 127.1 (2C), 120.0 (d, JCF = 6.9 Hz), 116.0 (d, J =
CF
18.2 Hz), 113.6 (d, J = 1.3 Hz), 96.0, 87.4, 78.1, 56.4, 51.3, 49.1, 42.5, 17.6 ppm. HRMSꢀTOF
CF
+
m/z [M+H] calculated for C H FN O : 522.155, found: 522.161.
2
6
23
4
7
(
2R)-2-[(4-{[5-(2-{[(4-Fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-dioxo-
1,2,3,4-tetrahydropyrimidin-1-yl]methyl}phenyl)formamido]propanoic acid (17) was
synthesized according to the general procedure of the ester hydrolysis from intermediate 47. 17
1
was obtained with 28 % yield over two steps as a white solid. H NMR (500 MHz, MeODꢀd , 25
4
°
C, TMS): δ 8.30 (s, 1H), 7.93ꢀ7.85 (m, 2H), 7.51ꢀ7.44 (m, 2H), 7.09ꢀ7.02 (m, 2H), 6.87 (ddd, J
=
7.8 Hz, 4.0 Hz, 1.8 Hz, 1H), 5.06 (s, 2H), 4.46 (q, J = 7.1 Hz, 1H), 4.40 (s, 2H), 3.88 (s, 3H),
ACS Paragon Plus Environment

Page 29 of 58
Journal of Medicinal Chemistry
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8
9
13
1
.48 (d, J = 7.1 Hz, 3H) ppm. C NMR (125 MHz, MeODꢀd , 25 °C, TMS): δ 167.0, 163.0,
4
1
53.8, 151.7 (d, J = 244.3 Hz), 151.5, 150.1, 147.7 (d, J = 11.6 Hz), 139.0, 134.4, 134.3 (d,
CF
CF
J = 4.0 Hz), 127.6 (2C), 127.5 (2C), 119.7 (d, J = 7.1 Hz), 115.3 (d, J = 19.2 Hz), 113.0 (d,
CF
CF
CF
10
11
12
13
14
15
16
17
18
19
20
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23
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25
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27
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46
47
48
49
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51
52
53
54
55
56
57
58
59
60
+
JCF = 1.9 Hz), 96.1, 86.5, 77.5, 55.3, 51.3, 48.4, 42.6, 17.9 ppm. HRMSꢀTOF m/z [M+H]
calculated for C H FN O : 522.166, found: 522.163.
2
6
23
4
7
(2S)-2-[(4-{[5-(2-{[(4-Fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-dioxo-
1,2,3,4-tetrahydropyrimidin-1-yl]methyl}phenyl)formamido]-3-hydroxypropanoate
(18)
was synthesized according to the general procedure of the ester hydrolysis from intermediate 13.
1
1
8 was obtained with 23 % yield over two steps as a white solid. H NMR (500 MHz, DMFꢀd ,
7
2
5 °C, TMS): δ 9.23 (t, J = 5.7 Hz, 1H), 8.54 (s, 1H), 8.90 (br s, 1H), 7.98ꢀ7.84 (m, 2H), 7.60ꢀ
7.48 (m, 2H), 7.21 (dd, J = 8.6 Hz, 1.7 Hz, 1H), 7.16 (dd, J = 11.6 Hz, 8.3 Hz, 1H), 6.92 (ddd, J
7.7 Hz, 3.8 Hz, 1.5 Hz, 1H), 5.12 (s, 2H), 4.42 (d, J = 5.8 Hz, 2H), 4.26ꢀ4.15 (m, 1H), 3.89 (s,
=
13
3
1
1
H), 3.71ꢀ3.60 (m, 2H) ppm. C NMR (125 MHz, DMFꢀd , 25 °C, TMS): δ 166.6, 163.7, 153.8,
7
52.7, 152.3 (d, J = 243.0 Hz), 151.5, 148.5 (d, J = 10.9 Hz), 140.6, 137.0 (d, J = 3.7 Hz),
CF
CF
CF
35.8, 129.0 (2C), 128.9, 128.5 (2C), 120.8 (d, J = 6.8 Hz), 116.6 (d, J = 18.0 Hz), 114.5 (d,
CF
CF
+
JCF = 2.0 Hz), 97.3, 88.1, 78.5, 64.2, 56.8, 52.2, 49.9, 43.5 ppm. HRMSꢀTOF m/z [M+H]
calculated for C H FN O : 538.150, found: 538.159.
2
6
23
4
8
(
2R)-2-[(4-{[5-(2-{[(4-Fluoro-3-methoxyphenyl)methyl]carbamoyl}eth-1-yn-1-yl)-2,4-dioxo-
1,2,3,4-tetrahydropyrimidin-1-yl]methyl}phenyl)formamido]-3-hydroxypropanoic acid (19)
was synthesized according to the general procedure of the ester hydrolysis from intermediate 48.
1
1
9 was obtained with 7 % yield over two steps as a white solid. H NMR (500 MHz, MeODꢀd ,
4
2
5 °C, TMS): δ 8.29 (s, 1H), 7.96ꢀ7.90 (m, 2H), 7.52ꢀ7.44 (m, 2H), 7.09ꢀ7.02 (m, 2H), 7.09ꢀ7.02
(
m, 2H), 6.87 (ddd, J = 8.3 Hz, 4.2 Hz, 2.1 Hz, 1H), 5.06 (s, 2H), 4.54 (s, 2H), 4.54 (br s, 1H),
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