Biphenyl-4-ylcarbamoyl thiophene carboxylic acids as potent DHODH inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.10.011

A previously discovered DHODH inhibitor series was further improved by replacing the cyclopentene ring by aromatic heterocycles. Different isomers of these compounds were prepared by the directed ortho-metallation procedure. The compounds are more active than the corresponding cyclopentene analogs and show potent effects on PBMC's proliferation.

Full text of DOI:10.1016/j.bmcl.2005.10.011

Bioorganic & Medicinal Chemistry Letters 16 (2006) 267–270
Biphenyl-4-ylcarbamoyl thiophene carboxylic acids as potent
DHODH inhibitors
Johann Leban,* Martin Kralik, Jan Mies, Roland Baumgartner,
Michael Gassen and Stefan Tasler
4SC AG, Am Klopferspitz 19a, 82152 Martinsried, Germany
Received 24 June 2005; revised 14 September 2005; accepted 5 October 2005
Available online 21 October 2005
Abstract—A previously discovered DHODH inhibitor series was further improved by replacing the cyclopentene ring by aromatic
heterocycles. Different isomers of these compounds were prepared by the directed ortho-metallation procedure. The compounds are
more active than the corresponding cyclopentene analogs and show potent effects on PBMCÕs proliferation.
Ó 2005 Elsevier Ltd. All rights reserved.
We have developed a novel series of DHODH inhibitors
based on a lead, which came out of a docking procedure
using 4ScanÒ technology and medicinal chemistry
exploration. The activity of the initial lead was im-
proved by a QSAR method and yielded low nanomolar
inhibitors.1
a second carboxylic group, like in C or E, were either
built up by 2-fold DoM/CO2-quenching or from 3,4-thi-
ophene dicarboxylate upon its conversion into the
anhydride with acetic anhydride and nucleophilic anhy-
dride opening with the respective 4-phenylaniline to give
compounds 13–15. Regiochemistry for the carboxylate
introduction in 3-thiophenamides A was in analogy to
a literature protocol4 and was finally deduced from
NMR analysis to be in the 2-position.
Such compounds promise to have great potential as
treatment for autoimmune diseases such as rheumatoid
arthritis, graft versus host disease, and multiple sclero-
sis.2 To further explore the scope of DHODH inhibitors
of types 1–3 (Table 1), a replacement of the cyclopentene
ring with other small aromatic systems was envisaged.
Our X-ray structure indicated that the cyclopentene
ring lies virtually planar in a pocket close to the FAD
cofactor.3 Flat small aromatic rings might therefore
be of benefit for binding and activity.
Introduction of a third carboxylic group into B yielded a
major and a minor component, which were separated by
HPLC. Major product C possessed a completely identi-
cal NMR spectrum as the major product E from carbox-
ylation of 3-thiophene-2-carboxylate D by the DoM
protocol. Therefore, the structure has to be as depicted
in Figure 1.
Compound B (Fig. 1) was synthesized by the directed
ortho-metallation procedure (DOM)4 at the stage of
the amide using butyl lithium and dry ice as CO2 source
for the introduction of the carboxylic group. Thiophene
amides A were prepared from commercial 3-thiophene-
carboxylic acid by conversion into the acid chloride
and reaction with the corresponding 4-phenylaniline,
which in turn were obtained by the Suzuki cross-
coupling method as described earlier.1 Compounds with
Enzyme inhibition was measured in an in vitro enzyme
assay. For the assay N-terminally truncated recombi-
nant human DHODH was used and the data are pre-
sented in Table 1.5
Direct comparison of the previously described com-
pounds 1–3 with their thiophene analogs 4, 5, and 8
showed a clear trend toward an increased inhibitory
activity of the thiophene analogs. As with the cyclopen-
tene series, the activity increases with fluoro constituents
in the aromatic ring adjacent to the amide bond and
with a methoxy group in the terminal aromatic, but
the trend is more pronounced in the thiophene series.
A X-ray structure investigation of the cyclopentene
Keywords: DHODH inhibitors.
*
Corresponding author. Tel.: +49 89 700763 0; fax: +49 89 700 763
29; e-mail: leban@4sc.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.011

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J. Leban et al. / Bioorg. Med. Chem. Lett. 16 (2006) 267–270
Table 1. (continued)
Table 1. Inhibition of human DHODH
Compound
Structure
IC50 (nM)
>1000
Compound
Structure
IC50 (nM)
410
F
O
HO
O
11
O
HN
O
S
15
HN
HO2C
CO2H
F
HO
O
O
O
F
OH
21
134
HN
O
16
16
HN
O
O
O
F
F
O
OH
F
F
O
O
F
F
HN
O
31
8
17
18
340
HN
O
O
OH
O
O
OH
Brequinar
8
HN
S
4
5
6
7
8
9
303
44
3
O
series revealed a binding mode with the possibility of the
carboxylic acid either having an ion bridge toward Arg
136 of the enzyme or pointing into the opposite direc-
tion toward Tyr 365 and 1473 upon turning by 180°.
To test the possibility that both binding modes could
be obtained within one molecule, compounds with two
free carboxylic acids (compounds 13–15) were synthe-
sized. These compounds, however, were inactive, which
was explained by the fact that for the alternative binding
mode, a conformational change of some residues in the
active site of the enzyme was required.
F
F
O
O
OH
HN
S
O
O
O
OH
HN
S
O
F
F
O
O
O
O
OH
9
HN
S
O
F
F
There was not much difference between the regioisomers
7 and 11 in their inhibitory activity, indicating the rela-
tively diminished importance of the sulfur position.
F
O
OH
1
HN
S
O
F
F
If one compares the X-ray structures of the cyclopentene
analog 2 versus those of the thiophene derivative 5, it is
of significance that the cyclopentene analog 2 displayed
a single binding mode which we termed Ônon-brequinarÕ
like.3 In contrast, the thiophene analog 5 showed a dual
binding mode which was brequinar-like and non-brequ-
inar-like, which can be deduced from the electron densi-
ty pattern in the crystal structure.
O
OH
12
HN
S
O
Cl
HO
O
O
10
1000
S
HN
We have previously shown that the brequinar-like bind-
ing mode leads to a better inhibitory activity, reflecting a
high affinity binding mode. Thus, the higher activity of
the compounds of the thiophene series as compared to
that of the respective representatives of the cyclopentene
series can easily be explained by taking into account their
possibility for binding in this high affinity mode. Com-
pound 3 represents an exception within the cyclopentene
class, as it likewise binds in both modes, resulting in a
comparable excellent activity (cf. compound 3 vs 8). Fur-
thermore, an increased number of fluoro substituents in
the aromatic ring adjacent to the amide bond correlate
with an increased tendency toward a Ôbrequinar-likeÕ
binding mode, thus with a better inhibitory activity.
HO
O
O
F
F
O
11
12
10
10
S
HN
HO
O
O F
O CF3
S
HN
F
F
O
CO2H
HN
S
13
14
>1000
>1000
O
CO2H
F
O
As can be seen from the furan analogs 16 and 17, a
replacement of sulfur by oxygen in the pentacyclic ring
is well tolerated. From the few furan analogs prepared,
CO2H
HN
S
O
HO2C

J. Leban et al. / Bioorg. Med. Chem. Lett. 16 (2006) 267–270
269
F
O
1. nBuLi
2. CO2
F
O
CO2H
HN
HN
O
S
S
O
A
B
1. nBuLi
2. CO2
F
O
F
O
CO2H
CO2H
HN
S
HN
S
O
HO2C
O
CO2H
:
2
8
C
F
O
1. nBuLi
2. CO2
HN
O
S
D
OH
O
F
O
F
O
CO2H
HN
HN
O
S
S
HO2C
O
CO2H
CO2H
:
1
9
E
Figure 1.
it can be deduced that this series will have a similar SAR
compared with that of the thiophene compounds. A
somewhat lower activity can be expected from higher
hydrophilicity which results in a hindered diffusion into
the hydrophobic environment of the active site, a phe-
nomenon we have encountered with all analogs.
compounds have activities comparable to those of
brequinar and promise to be useful to treat autoimmune
diseases.
Acknowledgments
Inhibition of DHODH is reflected by an antiprolifera-
tive effect on peripheral blood mononuclear cells
(PBMCs), which can be measured by the inhibitory
effect on their phytohemagglutinin (PHA) stimulated
growth. The test was performed as described in the ref-
erence.6 Data for few selected compounds are presented
in Table 2. Compounds 8 and 12 displayed potent anti-
proliferatory activities on PBMCs and suggest the po-
tential of such compounds to have an effect in animal
models for autoimmune diseases.
We thank Matthias Dormeyer for helpful discussions,
Tanja Wieber for synthetic assistance, and Gerhard
Keilhauer and Daniel Vitt for support.
References and notes
1. Leban, J.; Saeb, W.; Garcia, G.; Baumgartner, R.; Kramer,
B. Bioorg. Med. Chem. Lett. 2004, 14, 55.
2. Allred, A.; Emery, P. Exp. Opin. Pharmacother. 2001, 2,
125.
In conclusion, we have found that pentacyclic aromatic
heterocycles can be a potent substitute for a cyclopen-
tene ring for the design of DHODH inhibitors. Such
3. Baumgartner, R.; Walloschek, M.; Kralik, M.; Gotschlich,
A.; Leban, J. J. Med. Chem., in press.
4. (a) Hammer, K.; Benneche, T.; Hope, H.; Undheim, K.
Acta Chem. Scand. 1997, 51, 392; (b) Muenster, P.; Steiner,
G.; Freund, W.; Wuerzer, B.; Westphalen, K.-O.; Patent
DE 3933573 A1, 1991; (c) Chadwick, D. J.; Plant, A.
Tetrahedron Lett. 1987, 28, 6085.
Table 2. Inhibition of human PBMC proliferation
Compound
IC50 (lM)
5. Davis, J. P.; Cain, G. A.; Pitts, W. J.; Magolda, R. L.;
Copeland, R. A. Biochemistry 1996, 35, 1270, Recombinant
human, rat and murine DHODH were obtained from Prof.
Dr. M. Lo¨ffler, Univ. of Marburg, Karl-von-Frisch-Str.1
35043 Marburg.
5
8
20
2
12
16
2
15

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J. Leban et al. / Bioorg. Med. Chem. Lett. 16 (2006) 267–270
6. Cells (mononuclear cells) were isolated from human periph-
eral blood by AccuspinTM System-HistopaqueÒ-1077 (Sig-
ma,Germany). After washing, the cells were diluted to
approximately 1,00,000–2,00,000 cells/well in a sterile 96-
well flat-bottomed MP (Corning, Netherlands). T-lympho-
cytes were stimulated by addition of 20 lg/ml Phytohemag-
glutinin-L (Roche, Germany). The incubation at 37 °C, 5%
CO2, 90% humidity was made in the presence of different
concentrations of the compounds. All cells were incubated
for 48 h at 37 °C, 5% CO2, 90% relative humidity over a
concentration range of 0.4–50 lM compound solutions with
a final volume per well of 100 ll. After the initial incubation
period, 10 lM BrdU (Roche, Germany) was added for an
additional 4 h incubation. The culture medium employed
was RPMI 1640 which contained 10% heat-inactivated fetal
bovine serum, 2 mM L-glutamine, 100 units/ml penicillinG,
and 100 lg/ml streptamycin sulfate. After 48 h incubation,
the cells were labeled by adding 10 ll BrdU-Solution (Roche,
Germany) and reinsulated for further 4 h. Following incu-
bation, the media plus BrdU and drug were removed, the
cells were fixed, and the DNA was denatured in a single step
using FixDenat (Roche, Germany) The anti-BrdU-POD
(Roche, Germany) binds to the BrdU incorporated in a
newly synthesized, cellular DNA. The immune complexes
were detected by the subsequent substrate reaction. The
reaction product was quantified by measuring the absor-
bance at the respective wavelengths using an ELISA reader.
The EC50 values were determined using a fitting function.