SAR-based optimization of a 4-quinoline carboxylic acid analogue with potent antiviral activity

Article abstract of DOI:10.1021/ml300464h

It is established that drugs targeting viral proteins are at risk of generating resistant strains. However, drugs targeting host factors can potentially avoid this problem. Herein, we report structure-activity relationship studies leading to the discovery

Full text of DOI:10.1021/ml300464h

Letter
pubs.acs.org/acsmedchemlett
SAR-Based Optimization of a 4‑Quinoline Carboxylic Acid Analogue
with Potent Antiviral Activity
Priyabrata Das,† Xiaoyi Deng,‡ Liang Zhang,§ Michael G. Roth,† Beatriz M. A. Fontoura,§
Margaret A. Phillips,‡ and Jef K. De Brabander*,†
‡
§
†Department of Biochemistry, Department of Pharmacology, and Department of Cell Biology, University of Texas Southwestern
Medical Center, Dallas, Texas 75390, United States
S
* Supporting Information
ABSTRACT: It is established that drugs targeting viral proteins
are at risk of generating resistant strains. However, drugs
targeting host factors can potentially avoid this problem.
Herein, we report structure−activity relationship studies leading
to the discovery of a very potent lead compound 6-fluoro-2-(5-
isopropyl-2-methyl-4-phenoxyphenyl)quinoline-4-carboxylic
acid (C44) that inhibits human dihydroorotate dehydrogenase
(DHODH) with an IC50 of 1 nM and viral replication of VSV
and WSN-Influenza with an EC50 of 2 nM and 41 nM. We also
solved the X-ray structure of human DHODH bound to C44, providing structural insight into the potent inhibition of biaryl
ether analogues of brequinar.
KEYWORDS: High-throughput screening, structure−activity relationship, antiviral, human DHODH inhibitor
n the last 50 years, several antiviral drugs with high
enzymes. Nonetheless, complete dependence of the viruses on
Itherapeutic value for the treatment of several life threatening the cellular functions of the host cell, availability of only a
handful of broad spectrum antiviral drugs,9−11 and the presence
of several unexplored host cell targets interested us in this
diseases such as AIDS, Influenza, Herpes, Hepatitis C, and/or
Hepatitis B infections have emerged.1 Most of these drugs
approach.
inhibit specific virus-encoded enzymes essential for viral
replication. However, this type of antiviral drug therapy has
several drawbacks. First, because these drugs are designed to
target and/or inhibit specific viral enzymes, their efficacy can be
quite narrow, sometimes targeting only a subtype of a specific
virus. Although this strategy reduces the risks associated with
the toxicity caused by targeting viral enzymes nonspecifically, it
restricts the usage of these agents.2 Furthermore, most viruses
mutate rapidly, leading to drug resistance and thereby limiting
the usage of viral specific inhibitors. Resistance to viral
inhibitors is a major problem especially for important small
RNA viruses such as HIV, HCV, and influenza virus. For
viruses with genomes encoding only 10−12 genes, of which
only a few function as enzymes, it is difficult to find drugable
targets for small molecules despite substantial medicinal
chemistry efforts.3
Virus physiology and often their pathogenicity are highly
dependent upon host cell factors. Viral proteins and genomes
often use host cell machinery to generate highly infectious viral
progeny and, more often than not, evade and antagonize host
antiviral immune response.4 This raises the possibility that host
genes, proteins, and/or pathways that are needed for viral
replication and pathogenesis, can be used as targets for antiviral
drug discovery. Some examples of potential host cell targets
include protein kinases, cell surface receptors, the proteasome,
and nuclear receptors.5−8 One inherent problem might be the
toxicity associated with inhibiting host cell pathways and/or
Among potential viral drug targets, dihydroorotate dehydro-
genase (DHODH) is unique. It is the fourth enzyme of
pyrimidine de novo synthesis that catalyzes the conversion of
dihydroorotate to orotate.12 Pyrimidines are essential for the
biosynthesis of DNA, RNA, and phospholipids.13 Inhibition of
DHODH causes reduced levels of pyrimidine nucleotides that
in turn trigger various activities such as anticancer,14
immunosuppressive,15 antimalarial,16 and antifungal.17 Addi-
tionally, inhibition of DHODH can be beneficial for spinal cord
injury and rheumatoid arthritis.18 Various DHODH inhibitors
have evolved over the years out of which brequinar15 and
leflunomide18 have gained a lot of attention due to their high
potency. Recently, we described the discovery of 4-quinoline
carboxylic acids with antiviral (IC50 = 110 nM for VSV) activity
via a high-throughput screen and brief SAR study19 and
disclosed the target enzyme for the antiviral activity to be
human DHODH.19 Other inhibitors of pyrimidine biosynthesis
were also reported to have antiviral activity.20,21 Herein, we
report an extensive structure−activity relationship study leading
to the identification of compound C44 with excellent inhibitory
activity for viruses (EC50 = 1.9 nM for VSV and 41 nM for
WSN) and human DHODH (IC50 = 1.0 nM).
Received: December 13, 2012
Accepted: April 24, 2013
Published: April 24, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Given the structural relationship of HTS hit C1 to brequinar,
we recognized that there are several key pharmacophoric
regions where appropriate substituents are necessary (Figure
1).15,22−24 For example, a free carboxylic acid is crucial, whereas
counterparts. Given the beneficial effect of an additional
aromatic ring (2-F-Ph) connected to the C(2)-phenyl ring in
brequinar, we decided to explore the diaryl ether analogue C11.
Although less active than the trifluoromethoxy-substituted
analogue C5 (0.479 μM), diaryl ether analogue C11
demonstrated improved activity versus the other analogues in
Table 1 (1.293 μM versus 1.770−35.32 μM). This observation
opens the possibility to significantly expand SAR studies via
exploration of a large set of diverse diaryl ethers (vide infra).
Our approach thus focused on the optimization of C11. With
a series of analogues displayed in Table 2, we explored
Table 2. SAR of the Left-Hand Ring
Figure 1. SAR starting point.
a
compd
R1
Cl
R2
R3
EC50 (μM)
a hydrophobic moiety is necessary at C(2). Additionally,
electron withdrawing groups such as fluorine, chlorine, and
trifluoromethyl have a beneficial effect at C(7). Lastly, it was
reported that although the methyl group at C(3) of brequinar
did not influence DHODH activity, it did significantly improve
cellular activity in a mixed lymphocyte reaction (MLR) assay.15
Guided by the structure activity relationship insights of
brequinar, we started our structure optimization study of C1.
Initially, we kept the chloroquinoline part of the molecule
constant and synthesized compounds C2 to C11.25 As shown
in Table 1, replacing the propyl ether of C1 by methyl, ethyl, or
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
F
1.3
F
0.1
Br
3.1
OCF3
OCH3
NO2
CF3
H
95.6
2611
2.0
16.6
1.0
H
1.2
H
H
H
158.8
1.6
F
F
a
Inhibition of VSV replication in MDCK epithelial cells.
Table 1. SAR of the Right-Hand Aryl Ring
functional group tolerance in the 4-quinoline carboxylic acid
ring system. Replacement of the C(7)-chlorine with fluorine
(C12) showed a significant 10-fold boost in activity (0.110
μM), whereas bromine (C13) and nitro (C16) functionality at
this position was only moderately tolerated (EC50 = 3.10 and
1.98 μM, respectively). However, trifluoromethoxy (C14),
trifluoromethyl (C17), and methoxy (C15) functionality is
detrimental for activity (EC50 = 95.64, 16.58, and 2611 μM,
respectively). Interestingly, the unsubstituted analogue C18
exhibited increased activity (EC50 = 0.96 μM), indicating that
the presence of small groups like fluorine and hydrogen are
better tolerated. To investigate how crucial the position of the
fluorine atom is, additional compounds were synthesized and
screened against the in vitro assay. The C(9)-fluoro and the
C(7),C(8)-difluoro analogues C19 and C21 were about 10-fold
less potent than the C(7)-fluoro analogue C 12, whereas
introduction of fluorine at the C(8)-position completely
abolished activity (C20).
a
compd
R
EC50 (μM)
brequinar
C1
see Figure 1
O(CH2)2CH3
OCH3
0.3
4.7
7.1
5.7
6.3
0.5
1.8
2.2
8.7
5.7
35.3
1.3
C2
C3
OCH2CH3
O(CH2)3CH3
OCF3
C4
C5
C6
F
C7
Br
C8
CH3
C9
CH2CH3
CF3
C10
C11
OPh
At this point, it is useful to make some comparisons to
analogous SAR studies reported for brequinar.15 In a cellular
assay (mixed lymphocyte reaction), which presumably captures
similar issues of cell uptake as in our cellular assay (VSV-
replication), the potency of the C(7)-chloro-analogue was
reduced only 2-fold (compared to C(7)-fluoro) versus >10-fold
in our series. In addition, the cellular activity of the
nonsubstituted (C(7)-H) analogue was reduced >100-fold
(compared to C(7)-F) versus a 9-fold difference reported here.
More significantly, where we observe a significantly decreased
activity (∼150-fold) when substituting a trifluoromethyl for the
C(7)-fluorine (C17 vs C12), an increased 2-fold cellular
a
Inhibition of VSV replication in MDCK epithelial cells.
butyl (C2-C4) ethers did not result in improved activity in the
vesicular stomatitis virus (VSV) viral replication assay.26
However, replacing the propoxy group by fluorine (C6),
bromine (C7), or trifluoromethoxy (C5) resulted in significant
higher activity. In order to elucidate the importance of the ether
linkage, we prepared analogues C8−C10 by replacing the
alkoxy groups of C2, C3, and C5 with their respective alkyl
chain counterparts. In general, the alkyl-substituted analogues
were equipotent or less active than their corresponding alkoxy
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ACS Medicinal Chemistry Letters
Letter
activity was reported for a similar replacement in the brequinar
series of compounds.15 Finally, as reported for the brequinar
series,15 methyl ester formation (C12-methyl ester) virtually
abolished activity in the VSV assay (EC50 = 86.46 μM).
With C12 as our best analogue thus far, we decided to return
to the C(2) diaryl ether moiety of the molecule and synthesized
various analogues of C12 (C22−C36, Table 3).25 Following
of the quinoline and C(2)-aromatic ring proved also
unbeneficial (compound C38 in Chart 1).22
With C12 as our most potent analogue, we made a final push
to improve activity by exploring a set of diaryl ether analogues
(Chart 2).25 The activities of C39−C41 were found to be only
a
Chart 2. VSV Inhibition with Diaryl Ether Analogues
Table 3. SAR of the Right-Hand Phenol
a
compd
R
EC50 (μM)
C12
C22
C23
C24
C25
C26
C27
C28
C29
C30
C31
C32
C33
C34
C35
C36
Ph
0.1
6.4
0.6
19.0
2.1
6.8
1.0
2.0
4.9
0.9
0.3
0.1
1.0
22.9
2.5
14.6
CH3
CH2CH3
(CH2)2CH3
(CH2)3CH3
CH2(C3H5)
CF3
4-Cl−Ph
4-NO2−Ph
3,4-(OCH2O)−Ph
2-F−Ph
3-F−Ph
4-F−Ph
a
Inhibition of VSV replication in MDCK epithelial cells.
slightly less than our most potent compound C12. Interest-
ingly, the t-butyl substituted analogue C42 was more potent,
and sterically hindered diaryl ether C43 showed much
improved potency. On the basis of this result, we envisioned
that restriction of rotation across the diaryl ether linkage
coupled with the increase in hydrophobicity might be crucial
for activity. This hypothesis led to our final analogue C44 that
showed a significant 50-fold increase in potency versus C12 (2
nM) in the VSV in vitro assay.
2-pyridyl
3-pyridyl
2-thiazolyl
a
Inhibition of VSV replication in MDCK epithelial cells.
the same trend as observed for the C(7)-chloro series (Table
1), the diaryl ether C12 was more potent than the
corresponding aryl-alkyl ethers C22−C27. The 3,4-
(methylenedioxy)phenoxy (C30), 4-chlorophenoxy (C28),
and 4-nitrophenoxy (C29) analogues were approximately 9-
to 45-fold less active than the nonsubstituted analogue C12. Of
the three fluorophenyl analogues, C31 and C33 performed
slightly less, whereas the 3-F-Ph analogue C32 displayed an
activity similar to lead C12. Finally, heteroaromatic substitution
was less fruitful as indicated by the weak activity exhibited by
the pyridyl and thiazolyl analogues C34−C36.
Because SAR studies leading to brequinar indicated that a
C(3)-methyl substituent increased cellular activity 10-fold
(DHOD enzyme activity did not change however),15 we
synthesized C(3)-methyl substituted analogue C37 (Chart 1).
Surprisingly, this compound was significantly less potent (∼70-
fold) compared to the nonsubstituted congener C12.
Restricting conformational freedom with a planarizing fusion
In Table 4, we compare several analogues in assays
measuring inhibition of human DHODH enzyme activity and
Table 4. DHODH Enzyme, VSV, and WSN Viral Growth
Inhibition for Selected Analogues
a
compd
C1
DHODH IC50 (nM) VSV EC50 (nM) WSN EC50 (nM)
260
310
1600
5900
36
4650
479
1770
8650
110
1330
65
7600
b
C5
NA
C6
NA
NA
1380
1884
55
C8
C12
C11
C42
C43
C44
brequinar
95
19
16
23
71
1
2
41
12
304
460
a
b
Assay conditions are described in the Supporting Information. NA:
Chart 1. Conformationally Restricted Analogues
not assayed.
in vitro influenza-WSN viral replication. The data for inhibition
of VSV viral replication are included for comparison. Thus,
starting from compound C1 with modest activity, a
submicromolar analogue C12 was developed and further
optimized to the low nanomolar biaryl ether analogue C44.
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In the VSV assay, compound C44 was 2 orders of magnitude
more potent than brequinar at nontoxic concentrations
(cytotoxicity in human bronchial epithelial and Caco-2 cells:
IC50 ≥ 10 μM; see Figures S2 and S3 in the Supporting
Information).
isopropyl moiety appears to add additional hydrophobic
interactions within the pocket that provide additional binding
energy, thus explaining the potent inhibition of this compound.
In conclusion, we have discovered a new class of diaryl ether-
substituted 4-quinolinecarboxylic acid analogues of brequinar
with potent human DHODH inhibitory and antiviral activity.
Analogue C44 was identified as a promising candidate that
showed broad spectrum antiviral activity (2 orders of
magnitude more potent than brequinar) via the inhibition of
human DHODH enzyme. A cocrystal structure of analogue
C44 bound to human DHODH was solved and provides
structural insight into the potent inhibition of this particular
biaryl ether analogue.
The X-ray structure of C44 in complex with human
DHODH was solved to provide insight into the structural
basis for the potent binding.27 Previous X-ray structures of
human DHODH utilized a truncated soluble enzyme
containing amino acids 30−396, and the highest resolution
was obtained for the complex with brequinar,12 solved to 1.6 Å.
In order to improve crystallization, we generated a new
expression construct (pET28b-hDHODH33−396) containing
amino acids 33−396. Using this construct, we were able to
obtain crystals of human DHODH bound to C44 that
diffracted to 1.22 Å resolutions. The crystals formed in a
space group of P3221 with the cell dimension a = b = 90.87 and
c = 122.19, and the structure was refined by molecular
replacement to an Rfree/Rcrysal of 16.1 and 14%, respectively.
The refined structure contained residues 34−396, with the
exception that density was not observed for a loop formed by
residues 71−73 and 222−225. Cofactor FMN, substrate L-
DHO, inhibitor C44, 300 water molecules, and a bound
Zwittergent 3−10 detergent molecule were also present in the
structure.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures and characterization data, assay con-
ditions, and details regarding the crystal structure solution of
human DHODH bound to analogue C44. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(J.K.D.B.) E-mail: jef.debrabander@utsouthwestern.edu.
The structure of the bound C44 shows that the inhibitor
binds in the same site as was observed for brequinar (Figure 2),
Funding
Financial support was provided by the National Institutes of
Health through grants GM07159 (to B.M.A.F.), AI079110 (to
B.M.A.F. and M.G.R.), AI089539 (to B.M.A.F. and M.G.R.),
and AI53680 (to M.A.P.). J.K.D.B and M.A.P thank the Robert
A. Welch Foundation for support (grants I-1422 and I-1257).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Results shown in this report are partially derived from work
performed at Argonne National Laboratory, Structural Biology
Center at the Advanced Photon Source. Argonne is operated by
UChicago Argonne, LLC, for the U.S. Department of Energy,
Office of Biological and Environmental Research under
contract DE-AC02-06CH11357. We thank Dr. Soumya
Krishnamurthy, Department of Biochemistry, for generating
the data presented in Supplemental Figure S3. J.K.D.B. holds
the Julie and Louis Beecherl, Jr., Chair in Medical Science,
M.A.P. the Carolyn R. Bacon Professorship in Medical Science
and Education and the Beatrice and Miguel Elias Distinguished
Chair in Biomedical Science, and M.G.R. the Diane and Hal
Brierely Distinguished Chair in Biomedical Research.
Figure 2. X-ray structure of human DHODH bound to C44 solved to
1.2 Å resolutions. The 4 Å shell around C44 is displayed, and the
structure is aligned to the previously reported human DHODH
structure complexed with brequinar (1D3G). The protein chain for
the C44 structure is shown in orange, and the brequinar structure is
shown in turquoise. The bound C44 and brequinar are shown as ball
and stick. The bound FMN cofactor and the product orotate are also
shown.
ABBREVIATIONS
́
■
overlaying with an rmsd of 0.7 Å when the structures were
superimposed with DaliLite.28 The binding site is predom-
inantly hydrophobic, and the only H-bond interaction is
formed between the inhibitor carboxylate and R136. Several
amino acid residues (L46, F62, L67, and Y38) in the binding
site have rotated to accommodate the isopropyl group on the
diaryl ether moiety. Thus, as has been observed previously,29,30
structural plasticity in the DHODH inhibitor binding site
allows it to accommodate a more diverse range of ligands than
would be expected of a rigid structure. The phenyl ring of the
F62 appears to interact with the terminal phenyl ring of the
inhibitor via a T-shaped π-stacking fashion. Additionally, the
DHODH, dihydroorotate dehydrogenase; FMN, flavin mono-
nucleotide; L-DHO, L-dihydroorotate; MDCK, Madin−Darby
canine kidney; rmsd, root-mean-square deviation; SAR,
structure−activity relationship; VSV, vesicular stomatitis virus;
WSN virus, influenza A/WSN/33 (H0N1) virus
REFERENCES
■
(1) De Clercq, E., Ed. Antiviral Drug Strategies, 1st ed.; Wiley-VCH
Verlag GMBH & Co KGaA: Berlin, Germany, 2011; Chapter 1.
(2) Richman, D. D. The implications of drug resistance for strategies
of combination antiviral chemotherapy. Antiviral Res. 1996, 29, 31−33.
520
dx.doi.org/10.1021/ml300464h | ACS Med. Chem. Lett. 2013, 4, 517−521

ACS Medicinal Chemistry Letters
Letter
(3) Magden, J.; Kaariainen, L.; Ahola, T. Inhibitors of virus
R. L.; Jaffee, B. D.; Gardner, T. L.; Jones, E. A.; Magolda, R. L.
Structure−activity relationships (SAR) of some tetracyclic heterocycles
related to the immunosuppressive agent brequinar sodium. Bioorg.
Med. Chem. Lett. 1998, 8, 307−312.
(23) Batt, D. G.; Petraitis, J. J.; Sherk, S. R.; Copeland, R. A.;
Dowling, R. L.; Taylor, T. L.; Jones, E. A.; Magolda, R. L.; Jaffee, B. D.
Heteroatom- and carbon-linked biphenyl analogs of brequinar as
immunosuppressive agents. Bioorg. Med. Chem. Lett. 1998, 8, 1745−
1750.
(24) Chen, S.-F.; Papp, L. M.; Ardecky, R. J.; Rao, G. V.; Hesson, D.
P.; Forbes, M.; Dexter, D. L. Structure−activity relationship of
quinoline carboxylic acids. A new class of inhibitors of dihydroorotate
dehydrogenase. Biochem. Pharmacol. 1990, 40, 709−714.
(25) See the Supporting Information for the synthesis of all new
compounds.
(26) For assay conditions, see the Supporting Information.
(27) The coordinates have been deposited in the Protein Data Bank,
with ID 4IGH.
(28) Holm, L.; Park, J. DaliLite workbench for protein structure
comparison. Bioinformatics 2000, 16, 566−567.
(29) Deng, X.; Gujjar, R.; El Mazouni, F.; Goldsmith, E. J.; Rathod, P.
K.; Phillips, M. A. Structural plasticity of malaria dihydroorotate
dehydrogenase allows selective binding of diverse chemical scaffolds. J.
Biol. Chem. 2009, 284, 26999−27009.
(30) Walse, B.; Dufe, V. T.; Svensson, B.; Fritzson, I.; Dahlberg, L.;
Khairoullina, A.; Wellmar, U.; Al-Karadaghi, S. The structure of human
dehydroorotate dehydrogenase with and without inhibitor reveal
conformational flexibility in the inhibitor and substrate binding sites.
Biochemistry 2008, 47, 8929−8936.
̈
̈
̈
replication: recent developments and prospects. Appl. Microbiol.
Biotechnol. 2005, 66, 612−621.
(4) Katze, M. G.; He, Y.; Michael, G., Jr. Viruses and interferon: a
fight for supremacy. Nat. Rev. Immunol. 2002, 2, 675−687.
(5) DeFilippis, V.; Raggo, C.; Moses, A.; Fruh, K. Functional
̈
genomics in virology and antiviral drug discovery. Trends Biotechnol.
2003, 21, 452−457.
(6) Fruh, K.; Simmen, K.; Luukkonen, B. G. M.; Bell, Y. C.; Ghazal,
̈
P. Virogenomics: a novel approach to antiviral drug discovery. Drug
Discovery Today 2001, 6, 621−627.
(7) Tan, S. L.; Gangi, G.; Paeper, B.; Proll, S.; Katze, M. G. Systems
biology and the host response to viral infection. Nat. Biotechnol. 2007,
25, 1383−1389.
(8) DeFilippis, V.; Fruh, K. Host cell targets for antiviral therapy: an
̈
update. Future Virol. 2006, 1, 509−518.
(9) Rider, T. H.; Zook, C. E.; Boettcher, T. L.; Wick, S. T.; Pancoast,
J. S.; Zushman, B. D. Broad-spectrum antiviral therapeutics. PLoS One
2011, 6, e22572.
(10) Wolf, M. C.; Freiberg, A. N.; Zhang, T.; Akyol-Ataman, Z.;
Grock, A.; Hong, P. W.; Li, J.; Watson, N. F.; Fang, A. Q.; Aguilar, H.
C.; Porotto, M.; Honko, A. N.; Damoiseaux, R.; Miller, J. P.;
Woodson, S. E.; Chantasirivisal, S.; Fontanes, V.; Negrete, O. A.;
Krogstad, P.; Dasgupta, A.; Moscona, A.; Hensley, L. E.; Whelan, S. P.;
Faull, K. F.; Holbroock, M. R.; Jung, M. E.; Lee, B. A broad-spectrum
antiviral targeting entry of enveloped viruses. Proc. Natl. Acad. Sci.
U.S.A. 2010, 107, 3157−3162.
(11) Smith, R. A.; Kirkpatrick, W.; Eds. Ribavarin: A Broad Spectrum
Antiviral Agent; Academic Press: New York, 1980; pp 1−21.
(12) Liu, S.; Neidhardt, E. A.; Grossman, T. H.; Ocain, T.; Clardy, J.
Structures of human dihydroorotate dehydrogenase in complex with
antiproliferative agents. Structure 2000, 8, 25−33.
(13) Loeffler, M.; Zameitat, E. Pyrimidine biosynthesis. Encycl. Biol.
̈
Chem. 2004, 3, 600−605.
(14) Shawver, L. K.; Schwartz, D. P.; Mann, E.; Chen, H.; Tsai, J.;
Chu, L.; Taylorson, L.; Longhi, M.; Meredith, S.; Germain, L.; Jacobs,
J. S.; Tang, C.; Ullrich, A.; Berens, M. E.; Hersh, E.; McMahon, G.;
Hirth, K. P.; Powell, T. J. Inhibition of platlet-derived growth factor-
mediated signal transduction and tumor growth by N-[4-(trifluor-
omethyl)-phenyl]-5-methylisooxazole-4-carboxamide. Clin. Cancer Res.
1997, 3, 1167−1177.
(15) Batt, D. G.; Copeland, R. A.; Dowling, R. L.; Gardner, T. L.;
Jones, E. A.; Orwat, M. J.; Pinto, D. J.; Pitts, W. J.; Magolda, R. L.;
Jaffee, B. D. Immunosuppressive structure−activity relationships of
brequinar and related chinchoninic acid derivatives. Bioorg. Med. Chem.
Lett. 1995, 5, 1549−1554.
(16) Phillips, M. A.; Rathod, P. K. Dihydroorotate dehydrogenase is a
promising target for the identification of novel anti-malarial chemo-
therapy. Infect. Disord.: Drug Targets 2010, 10, 226−239.
(17) Gustafson, G.; Davis, G.; Waldron, C.; Smith, A.; Henry, M.
Identification of a new antifungal target site through a dual
biochemical and molecular-genetics approach. Curr. Genet. 1996, 30,
159−165.
(18) Rosse, G. Novel use of leflunomide and malononitrilamides.
ACS Med. Chem. Lett. 2012, 3, 785−785.
(19) Zhang, L.; Das, P.; Schmolke, M.; Manicassamy, B.; Wang, Y.;
Deng, X.; Cai, L.; Tu, B. P.; Forst, V. F.; Roth, M. G.; Levy, D. E.;
Garcia-Sastre, A.; De Brabander, J. K.; Phillips, M. A.; Fontoura, B. M.
A. Inhibition of pyrimidine synthesis reverses viral virulence factor-
mediated block of mRNA export. J. Cell. Biol. 2012, 196, 315−328.
(20) Hoffman, H. H.; Kunz, A.; Simon, V. A.; Palese, P.; Shaw, M. L.
Broadspectrum antiviral that interferes with de novo pyrimidine
biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 5777−5782.
(21) Bernhoff, E.; Tylden, G. D.; Kjerpeseth, L. J.; Gutteberg, T. J.;
Hirsch, H. H.; Rinaldo, C. H. Leflunomide inhibition of BK virus
replication in renal tubular epithelial cells. J. Virol. 2010, 84, 2150−
2156.
(22) Pitts, W. J.; Jetter, J. W.; Pinto, D. J.; Orwat, M. J.; Batt, D. G.;
Sherk, S. R.; Petraitis, J. J.; Jacobson, I. C.; Copeland, R. A.; Dowling,
521
dx.doi.org/10.1021/ml300464h | ACS Med. Chem. Lett. 2013, 4, 517−521