New inhibitors of dihydroorotate dehydrogenase (DHODH) based on the 4-hydroxy-1,2,5-oxadiazol-3-yl (hydroxyfurazanyl) scaffold

Article abstract of DOI:10.1016/j.ejmech.2011.12.038

Based on some structural analogies with leflunomide and brequinar, two well-known inhibitors of dihydroorotate dehydrogenase (DHODH), a new series of products was designed, by joining the substituted biphenyl moiety to the 4-hydroxy-1,2,5-oxadiazol-3-yl s

Full text of DOI:10.1016/j.ejmech.2011.12.038

European Journal of Medicinal Chemistry 49 (2012) 102e109
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European Journal of Medicinal Chemistry
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Original article
New inhibitors of dihydroorotate dehydrogenase (DHODH) based on the
4-hydroxy-1,2,5-oxadiazol-3-yl (hydroxyfurazanyl) scaffold
*
Marco L. Lolli, Marta Giorgis, Paolo Tosco, Antonio Foti, Roberta Fruttero , Alberto Gasco
Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, via Pietro Giuria 9, 10125 Torino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2011
Accepted 27 December 2011
Available online 3 January 2012
Based on some structural analogies with leflunomide and brequinar, two well-known inhibitors of
dihydroorotate dehydrogenase (DHODH), a new series of products was designed, by joining the
substituted biphenyl moiety to the 4-hydroxy-1,2,5-oxadiazol-3-yl scaffold through an amide bridge. The
compounds were studied for their DHODH inhibitory activity on rat liver mitochondrial/microsomal
membranes. The activity was found to be closely dependent on the substitution pattern at the biphenyl
system; the most potent products were those bearing two or four fluorine atoms at the phenyl adjacent
to the oxadiazole ring. A molecular modeling study suggested that these structures might have a bre-
quinar-like binding mode. The greater potency of fluorinated analogs may depend partly on enhanced
interactions with the hydrophobic ubiquinone channel, and partly on the role of fluorine in stabilizing
the putative bioactive conformation.
Keywords:
Hydroxyfurazan
Dihydroorotate dehydrogenase inhibitors
Leflunomide
Brequinar
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
used in combination with disease-modifying anti-rheumatic drugs
(DMARDs) for the management of established rheumatoid arthritis.
One step in pyrimidine biosynthesis is conversion of
L
-dihy-
Methotrexate is the principal DMARD, but other drugs in use
include sulphasalazine, penicillamine, hydroxychloroquine and
chloroquine, gold salts, cyclosporine and glucocorticoids [9,12].
Hepatotoxicity, blood dyscrasias, intestinal and lung diseases are
serious adverse effects of these drugs. Recently, biological agents
able to activate pro-resolving biochemical pathways have been
introduced onto the market; these include inhibitors of tumor
droorotate (DHO) to orotate (ORO), under action of the enzyme
dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which
contains a flavine (FMN) as redox cofactor [1]. In this trans-
formation, electrons resulting from DHO oxidation are transferred
to ubiquinone, and finally to cytochrome c oxidase. Previous studies
have shown that this enzyme possesses two distinct binding sites,
respectively for DHO/ORO and ubiquinone. Oxidation of DHO to
ORO is the rate-limiting step of the whole pyrimidine biosynthetic
pathway. Inhibitors of DHODH display antiproliferative and
immunomodulator activities, and have shown potential benefits for
treating rheumatoid arthritis (RA) [2e5]. RA is a chronic inflam-
mation characterized by painful joints and progression to irre-
versible joint damage [6]. It is currently regarded as an
immunological process involving a variety of mediators [7,8].
Treatment relies on the traditional non-steroidal anti-inflamma-
tory drugs (NSAIDs) or selective COX-2 inhibitors (Coxibs) [9].
While these drugs may provide symptomatic relief, they do not
modify the course of the disease. The main shortcoming of NSAIDs
is their marked gastrotoxicity, whereas Coxibs potentially increase
the risk of heart attack and stroke [10,11]. Generally, both drugs are
necrosis factor-
a (TNF-a). These inhibitors are used when arthritis
is uncontrolled and serious toxic effects have arisen with DMARDs
[9]. Inhibitors of DHODH have also been considered for treating RA;
leflunomide (1) and brequinar (BQN, 2) are the two most important
such drugs (Fig. 1) [13,14]. The former is an isoxazol derivative,
which behaves as a prodrug: upon absorption, it is rapidly con-
verted into its active metabolite A771726 (3), which was assigned
the Z-configuration. The latter was developed for cancer therapy,
but failed in clinical trials due to limited therapeutic windows [15].
The binding sites for both A771726 and BQN are located at the
narrow end of the channel that ubiquinone uses to accomplish
a redox reaction with FMNH2. Several charged and polar side chains
(Gln47, His56, Tyr356, Thr360, and Arg136) are present at this
position. In the case of A771726, the deprotonated enolic OH group
interacts via hydrogen bonding with Tyr356, while the amide
carbonyl is hydrogen bonded to Arg136 through a water molecule
[16]. Moving to BQN, the binding mode is quite different. Here, the
carboxylate group forms a salt bridge with Arg136, and is hydrogen
* Corresponding author. Tel.: þ39 0116707850; fax: þ39 0116707286.
E-mail address: roberta.fruttero@unito.it (R. Fruttero).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.038

M.L. Lolli et al. / European Journal of Medicinal Chemistry 49 (2012) 102e109
103
of DHODH, in some cases similar to BQN, in some others to A771726
[22,23]. This paper describes a series of hydroxyfurazans (Scheme
1) which display moderate-to-high DHODH inhibitory activity,
largely dependent on the substitution pattern at the biphenyl ring.
2. Chemistry
The synthetic pathway used to obtain the final products is very
straightforward (Scheme 1). The benzyloxy-substituted 1,2,5-
oxadiazolecarboxylic acid 8, a product we have described else-
where (see Experimental), was transformed into the corresponding
carbonyl chloride 9 by action of thionyl chloride. This intermediate
was treated with the appropriately substituted anilines 10aen in
the presence of pyridine, to afford the related amides 11aen. The
same products were also obtained by direct coupling of the acid 8
with the corresponding anilines using HBTU (2-(1H-benzotriazole-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) in DMF.
The amides 11aen, upon hydrogenation on Pd/C in THF, yielded the
expected final compounds 7aen.
Fig. 1. Structures of leflunomide and brequinar, two well-known inhibitors of DHODH.
bonded to Gln47. In addition, the biphenyl moiety establishes
a number of hydrophobic contacts with several lipophilic residues
of the tunnel. In a development of our quest for drugs to use as anti-
inflammatory agents [17e19] we recently explored the possibility
of using the 1,2,5-oxadiazole ring (furazan) as a replacement for the
isoxazole moiety present in 1 [20]. Similarly to isoxazole in 1, the
furazans we designed (general structure 4) are also easily opened
3. Inhibition of DHODH
The final products 7aen were assessed for their DHODH
inhibitory activity on rat liver mitochondrial/microsomal
membranes.
A procedure adapted from the literature was
employed (see Experimental), in which oxidation of DHO to ORO is
monitored by following the concomitant reduction of the chro-
mophore 2,6-dichlorophenolindophenol (DCIP) [24]; A771726 and
BQN were taken as references. The reduction of DCIP was detected
by the decrease in absorbance at 650 nm. The initial rate of the
enzymatic reaction in the presence (v) and in the absence (V)
of inhibitor was measured, and the IC50 value was calculated from
Eq. (1):
under physiological conditions, to yield the related a-oximinoace-
tonitrile derivatives (cyano-oximes) 5 (Fig. 2). These products
proved to be very poor DHODH inhibitors, probably due to the
unfavorable E-configuration of the oxime moiety generated upon
ring opening, which prevents hydrogen bonding to Tyr356 [20].
Since the attempt to restore this interaction by oxidizing the oximes
to nitroderivatives was only partially successful, we decided to
pursue another strategy, consisting in the replacement of the
unsubstituted furazan in 4 with a hydroxyfurazan ring (7a,
Scheme 1). The latter moiety is stable under physiological condi-
tions, and should provide the correct orientation for the deproto-
nated hydroxyl, in order to mimic the enolate group of A771726 and
interact likewise with Tyr356. The activity of 7a, the first hydrox-
yfurazan analog of A771726 that was tested, was not of great
v ¼ V/(1 þ [I]/IC50
)
(1)
where [I] is the concentration of the inhibitor. The results are
collected in Table 1.
4. Results and discussion
interest (4.3 mM), but nonetheless played a key role in the further
Analysis of Table 1 shows that compound 7b, in which the
development of our project. When 7a was docked in silico at the
DHODH active site, a BQN-like pose was obtained with the depro-
tonated furazan hydroxyl facing Arg136 instead of Tyr356. This
amounted to a 180ꢀ flip of the furazan ring with respect to our
expectations. From this observation, the suggestion arose that the
hydroxyfurazan scaffold might have the potential to bind to
DHODH like brequinar itself, thanks to its known bioisosterism
with the carboxylic group [21]. A number of biphenyl derivatives
linked to pentatomic arene- and cyclopentenecarboxylic acids
through an amide bond (6, Fig. 3) were recently proposed as potent
DHODH inhibitors. These inhibitors were found, by X-ray crystal-
lography, to assume different poses in the ubiquinone binding site
biphenyl scaffold does not bear any substituent, displays its
inhibitory potency in the micromolar range (IC50 ¼ 11
mM). If the
amide bridge is moved from position 1 to 3 of the biphenyl system,
product 7c is yielded, whose potency is about 4 times lower. Adding
a CF3 group (which is present in A771726) to 7b, at either position 40
(compound 7d) or position 30 (compound 7e) of the biphenyl
moiety, again decreases the potency, approximately by one quarter
and by one half, respectively. Conversely, considerable modulation
in the inhibitory activity occurs when fluoro substitution is per-
formed on the biphenyl system. Fluorine is a lipophilic substituent
of small size, endowed with high electronegativity; its presence
may affect the binding affinity of a compound to its target protein in
many ways [25,26]. The introduction of two or four fluorine atoms
on the phenyl ring adjacent to the oxadiazole enhances the inhib-
itory potency of compounds 7f and 7g about 12-fold and 166-fold
that of the parent structure. In both 7f and 7g, the introduction of
a CF3 group at position 4 or 3 of the second phenyl ring (products
7h, 7i, 7l, 7m) decreases the inhibitory potency which, neverthe-
less, remains higher than that of 7b. By contrast, the presence of
a trifluoromethoxy group at position 30 (7n) affords the most active
compound of the series, whose potency is comparable to that of
BQN. In an attempt to rationalize the activity profile across the
Fig. 2. Ring opening reaction of monosubstituted furazans (4) yielding the related
a
eoximinoacetonitrile derivatives (cyano-oximes, 5).
series,
a
molecular modeling study was carried out.
A

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M.L. Lolli et al. / European Journal of Medicinal Chemistry 49 (2012) 102e109
Scheme 1. Synthetic route to compounds object of this work.
conformational search using an implicit solvent model was
accomplished for each of the newly synthesized compounds; this
was followed by refinement of the geometry of local minima
through a quantum-mechanical (QM) method. Subsequently, flex-
ible docking of the compounds was performed in three different
crystallographic structures recovered from the Protein Data Bank
(see Experimental). The complexes of the rat enzyme with BQN
(PDB ID 1UUO), of the human enzyme with a BQN analog (PDB ID
1D3G), and finally of human DHODH with a biphenylamino-
carbonyl derivative of cyclopentenecarboxylic acid (PDB ID 2BXV),
were chosen. The rat isoform was taken into account to explore
whether small inter-species differences may play a role, while the
two human complexes were selected in order to ascertain whether
differences in the conformation of Arg136 and Gln47 side chains
might affect the binding mode, as observed by Baumgartner and
co-workers [23]. Our docking protocol was able to accurately
reproduce the BQN experimental pose in 1UUO and 1D3G (RMSD
0.52 and 0.40 Å, respectively). Moving to derivatives 7aen, no
major differences were observed among the poses obtained on
1UUO and 1D3G; in both cases the compounds were found to bind
in a BQN-like fashion, namely with the deprotonated hydroxyfur-
azan moiety facing Arg136, thus effectively mimicking the carboxyl
group of BQN and related compounds (Fig. 4a). While on 1D3G and
1UUO the hydroxyfurazan was in no case found to interact with
Tyr356 in a leflunomide-like fashion, a significant fraction of such
poses was found when 2BXV was used as the docking target
Table 1
Inhibition of rat liver DHODH activity by A771726, brequinar and compounds
7aen.
Compd.
DHODH inhibition IC50 ꢁ SE [
m
M]a
A771726
Brequinar
0.020 ꢁ 0.002
0.033 ꢁ 0.004
4.3 ꢁ 0.7
7a
7b
7c
7d
7e
7f
7g
7h
7i
11 ꢁ 1
45 ꢁ 3
49 ꢁ 4
25 ꢁ 2
0.88 ꢁ 0.08
0.066 ꢁ 0.006
0.87 ꢁ 0.11
2.3 ꢁ 0.1
7l
7m
7n
0.49 ꢁ 0.04
0.12 ꢁ 0.02
0.050 ꢁ 0.005
Fig. 3. General structure of recently proposed potent DHODH inhibitors where
a biphenyl scaffold is linked to a pentatomic arenee or cyclopentenecarboxylic acid
through an amide bridge.
a
Enzyme activity was measured by DCIP reduction assay and IC50 values
were calculated with non-linear regression analysis.

M.L. Lolli et al. / European Journal of Medicinal Chemistry 49 (2012) 102e109
105
Fig. 5. Putative bioactive conformations of compounds 7n (a) and 7d (b) (thicker
sticks), compared to their respective global minima in solution (thinner sticks).
not only optimize interactions with the hydrophobic amino acids
lining the active site (in particular, Ala59 and leucines 46, 58, 359),
but also stabilize the putative bioactive pose, thus enhancing
inhibition. For compound 7n, the trifluoromethoxy group
protrudes out of the distal benzene ring plane, establishing addi-
tional contacts with Tyr38 (Fig. 4), which probably further
contribute to making it the most active compound of the series.
5. Conclusions
This study shows that it is possible to obtain a new class of
DHODH inhibitors by joining the 4-hydroxy-1,2,5-oxadiazol-3-yl
scaffold to substituted biphenyl moieties through an amide
bridge. SAR analyses show that the most active compounds bear
two or four fluorine atoms at the phenyl ring adjacent to the amide
group. The introduction of the trifluoromethoxy group at the 30-
position of the distal phenyl of 7m affords 7n, which is an inhibitor
as potent as BQN. A molecular modeling study rationalizes the
beneficial role of the fluorine substituents, showing that the latter
enhance interactions with the hydrophobic ubiquinone channel of
the enzyme, and stabilize the putative bioactive conformation.
While the majority of docking results indicate that these inhibitors
adopt a brequinar-like binding mode, additional evidences are
necessary to say a conclusive word on this point.
Fig. 4. a) Brequinar-like docking pose of compound 7n in the 1D3G structure; the co-
crystallized brequinar analog is reported for comparison (thinner sticks). b)
Leflunomide-like docking pose of compound 7n in the 2BXV structure.
(Fig. 4b). In spite of the good qualitative agreement, the scoring
function implemented in AutoDock (see Experimental) was not
able to reproduce the experimentally observed differences in
inhibitory potency across compounds 7aen. However, when the
binding pose of the furazan derivatives in 1D3G was compared with
the QM-refined global minimum obtained from the implicit solvent
conformational search, we found excellent agreement for the
derivatives bearing fluorine atoms on the biphenyl system, espe-
cially with regard to the dihedral angle between the benzene ring
and the amide group (107.1ꢀ in the docking pose, 113.6ꢀ in the
global minimum in solution for 7n, Fig. 5a). Conversely, for
compounds lacking aromatic fluorine atoms, in the putative
bioactive conformation the two moieties are tilted by about 100ꢀ
(106.1ꢀ for 7d, Fig. 5b), while in solution the benzene ring lies in the
same plane as the amide bond. Since such a conformation would
not fit into the ubiquinone site due to steric hindrance, the non-
fluorinated compounds necessarily suffer a conformational strain
penalty to assume a pose compatible with the constraints imposed
by the geometry of the enzyme cavity. The same considerations also
apply to the leflunomide-like poses found on 2BXV, with similar
dihedral angles between the benzene ring and the amide group, the
latter lying on the opposite side of the benzene ring compared to
brequinar-like poses. This suggests that aromatic fluorine atoms
6. Experimental part
6.1. Chemistry
Melting points were determined on a Büchi 530 apparatus. The
compounds were routinely checked by IR spectroscopy (Shimadzu
FT-IR 8101M) and mass spectrometry (Finnigan-Mat TSQ-700
spectrometer, 70 eV, direct inlet). 1H and proton-decoupled 13C-
NMR spectra were recorded on a Bruker AC-300 spectrometer. The
following abbreviations are used: s, singlet; d, doublet; q, quartet;
m, multiplet; br, broad; Fz, furazan. Flash column chromatography
was performed on silica gel (Merck Kieselgel 60, 230e400 mesh
ASTM) using the indicated eluents. Thin layer chromatography
(TLC) was carried out on 5 ꢂ 20 cm plates with a layer thickness of
0.25 mm; when necessary, they were developed with iodine and
KMnO4. Anhydrous magnesium sulphate was used as drying agent
for the organic extracts. Elemental analyses of the new compounds

106
M.L. Lolli et al. / European Journal of Medicinal Chemistry 49 (2012) 102e109
were within ꢁ0.4% of the theoretical values. In case of compounds
7e and 7f, whose elemental analyses were slightly out of range, the
purity was assessed by RP-HPLC (> 99%). Analyses were performed
on a HP1100 chromatograph system (Agilent Technologies, Palo
Alto, CA, USA) equipped with an injector (Rheodyne, Cotati, CA,
USA), a quaternary pump (model G1311A), a membrane degasser
(model G1379A), and a diode-array detector (DAD, model G1315B)
integrated in the HP1100 system. Data analysis was performed
diethyl ether; yield 88%; white solid (m.p. 185.8e186.1 ꢀC, from
diethyl ether); 1H-NMR (DMSO-d6)
5.48 (s, 1H, CH2O), 7.35e7.82
(m, 14H, aromatic protons), 11.1 (s, 1H, NH); 13C-NMR (DMSO-d6)
74.1, 120.7, 126.4, 126.9, 127.7, 128.4, 128.4, 128.7, 128.7, 134.4,
d
d
136.8, 137.1, 139.8, 142.2, 154.0, 163.3; MS (CI) 372 (Mþ1).
6.1.2.5. 4-Benzyloxy-N-(biphenyl-2-yl)-1,2,5-oxadiazole-3-
carboxamide (11c). The general procedure from the carboxylic acid
was followed (method A). Yield 70%; white solid (m.p.
using
a HP ChemStation system (Agilent Technologies). The
analytical column was a Zorbax Eclipse XDB-C8 (150 ꢂ 4.6 mm,
122.6e123.0 ꢀC); 1H-NMR (CDCl3)
d
5.29 (s, 1H, CH2O), 7.22e8.48
(m, 14H, aromatic protons), 8.51 (br s, 1H, NH); 13C-NMR (CDCl3)
74.8, 121.4, 125.4, 128.3, 128.5, 128.6, 128.7, 129.1, 129.2, 129.3,
5 mm particle size) (Agilent Technologies). Compounds were dis-
solved in mobile phase and injected through a 20
mL loop. The
d
mobile phase consisted of MeOH/water with 0.1% trifluoroacetic
acid 65/35 v/v. HPLC retention times (tR) were obtained at flow
rates of 1.0 mL minꢃ1, and the column effluent was monitored at
226 and 260 nm referenced against 360 nm. The
moistureesensitive reactions were carried out under dry N2.
Tetrahydrofuran (THF) was distilled immediately before use from
sodium and benzophenone under N2. Compounds 8 [21], A771726
[27], BQN [28] and the biphenyl-substituted anilines [29] were
synthesized as described in the literature.
130.4, 132.7, 133.6, 133.8, 137.4, 141.4, 153.4, 162.7; MS(CI) 372
(Mþ1).
6.1.2.6. 4-Benzyloxy-N-[40-(trifluoromethyl)biphenyl-4-yl]-1,2,5-
oxadiazole-3-carboxyamide (11d). Method A was followed. The
solid was triturated with diisopropyl ether/hexane 1/1 v/v; yield
71%; white solid (m.p. 186.2e186.6 ꢀC, from diisopropyl ether/
hexane 1/1 v/v); 1H-NMR (DMSO-d6-CDCl3)
d 5.52 (s, 2H, CH2O),
7.43e7.76 (m, 11H, aromatic protons), 8.48 (s, 1H, NH); 13C-NMR
(DMSO-d6-CDCl3)
d
74.5, 121.1, 124.5 (q, CF3, 1JCF 271.9 Hz), 128.5 (q,
3
2
6.1.1. 4-Benzyloxy-1,2,5-oxadiazole-3-carbonyl chloride (9)
C(30); JCF 3.9 Hz), 128.5 (q, C(40), JCF 31.2 Hz), 127.2, 127.6, 128.7,
128.7, 129.1, 134.6, 135.4, 138.3, 142.4, 143.9 (br), 154.4, 163.6; MS
(CI) 440 (Mþ1).
A solution of 8 (500 mg, 2.27 mmol) and a drop of DMF in freshly
distilled thionyl chloride (15 mL) were stirred at room temperature
for 24 h. The solution was concentrated under reduced pressure,
then distilled under reduced pressure in a Kugelrohr apparatus
(2,10ꢃ5 bar, external bath 110 ꢀC) to afford to the pure title
compound as a low-melting-point white solid, used in the next step
6.1.2.7. 4-Benzyloxy-N-[30-(trifluoromethyl)biphenyl-4-yl]-1,2,5-
oxadiazole-3-carboxyamide (11e). Method A was followed. Yield
19%; white solid (m.p. 117.0e117.3 ꢀC, from diisopropyl ether); 1H-
without any further purification, or stored under inert atmosphere.
NMR (CDCl3) d 5.50 (s, 2H, CH2O), 7.41e7.81. (m, 13H, aromatic
1
Yield 87%; H-NMR (CDCl3)
aromatic protons).
d
5.44 (s, 2H, CH2O), 7.40e7.49 (m, 5H,
protons), 8.45 (s,1H, NH); 13C-NMR (CDCl3) 75.1,120.6,123.6 (q, 3JCF
d
5.1 Hz),124.0 (q, 3JCF 4.6 Hz) (C(40)/C(20)),124.1 (q, CF3, 1JCF 273.8 Hz),
2
127.9, 128.6, 128.9, 129.1, (q, C(30), JCF 34.3 Hz), 129.3, 129.4, 130.1,
6.1.2. General procedure for amide synthesis
124.0, 136.5, 136.8, 140.9, 141.2, 153.6, 163.3; MS (CI) 440 (Mþ1)þ.
6.1.2.1. Method A (from 8). To a stirred solution of 8 (1 mmol), kept
under inert atmosphere, and the appropriate substituted aniline
(1 mmol) in dry DMF (25 mL), HBTU (2 mmol) was added por-
tionwise. The resulting suspension was stirred for 6 h, then poured
into chilled 2 M HCl (20 mL). The mixture was extracted exhaus-
tively with Et2O, and the collected organic layers were dried and
concentrated under reduced pressure. The residue was purified by
flash chromatography, obtaining the title compound. For details
about the purification of the crude product, see the specific method.
6.1.2.8. 4-Benzyloxy-N-[(3,5-difluoro)biphenyl-4-yl]-1,2,5-
oxadiazole-3-carboxyamide (11f). Method B was followed. Yield
62%; white solid (m.p. 144.8e145.0 ꢀC, from diisopropyl ether/EtOH
9/1 v/v); 1H-NMR (CDCl3)
d
5.50 (s, 2H, CH2O), 7.20e7.55 (m, 12H,
75.0,
aromatic protons), 8.01 (s, 1H, NH); 13C-NMR (CDCl3)
d
110.3e110.6 (m, C(3)), 110.8 (t, C(2), 2JCF 17.1 Hz), 126.9, 128.5, 128.7,
128.8,129.1,129.1,133.9,138.2 (t, C(10), 4JCF 2.8 Hz),142.6 (t, C(4), 3JCF
9.3 Hz), 140.5, 153.7, 157.8 (dd, C(1), 1JCF 251.8 Hz, 3JCF 5.3 Hz), 163.4;
MS (CI) 408 (Mþ1)þ.
6.1.2.2. Method B (from 9). A solution of the appropriate aniline
(0.67 mmol) and dry pyridine (2 eq) in dry CH2Cl2 (4 mL) was added
to a solution of 9 (1.2 eq) in dry CH2Cl2 (4 mL). The mixture was
stirred overnight under inert atmosphere, then diluted with CH2Cl2
(15 mL), washed with 1 M HCl (10 mL), 1 M NaOH (10 mL), brine
(10 mL), dried and concentrated under reduced pressure. For details
about the purification of the crude product, see the specific method.
6.1.2.9. 4-Benzyloxy-N-[(2,3,5,6-tetrafluoro)biphenyl-4-yl]-1,2,5-
oxadiazole-3-carboxyamide (11g). Method B was followed. Yield
55%; white solid (m.p. 180.6e181.0 ꢀC, from diisopropyl ether/EtOH
9/1 v/v); 1H-NMR (CDCl3)
d
5.50 (s, 2H, CH2O), 7.47e7.72 (m, 10H,
aromatic protons), 8.44 (s, 1H, NH); 13C-NMR (CDCl3)
d
75.2,
109.6e151.2 (aromatic carbons), 140.2, 153.6, 163.4; MS (CI) 444
(Mþ1).
6.1.2.3. 4-Benzyloxy-N-[(1-trifluoromethyl)phenyl-4-yl]-1,2,5-
oxadiazole-3-carboxamide (11a). Method A was followed. Flash
chromatography eluent: petroleum ether 40e60/ethyl acetate 6/4
v/v. The solid was triturated with hexane/diisopropyl ether 2/8 v/v;
yield 25%; white solid (m.p. 142.4e142.6 ꢀC, from hexane/diiso-
6.1.2.10. 4-Benzyloxy-N-[(3,5-difluoro-30-trifluoromethyl)biphenyl-
4-yl]-1,2,5-oxadiazole-3-carboxyamide (11h). Method B was fol-
lowed. Yield 60%; the crude solid was triturated with diisopropyl
ether/hexane 1/1 v/v; white solid (m.p. 120.5e120.9 ꢀC, from dii-
propyl ether 2/8 v/v); 1H-NMR (CDCl3)
d
5.57 (s, 2H, CH2O),
sopropyl ether); 1H-NMR (CDCl3)
d
5.49 (s, 2H, CH2O), 7.23e7.77 (m,
7.42e7.53 (m, 9H, aromatic protons), 11.36 (s, 1H, NH); 13C-NMR
11H, aromatic protons), 8.04 (s, 2H, NH); 13C-NMR (CDCl3)
d
75.0,
1
(CDCl3)
d
75.6, 120.2, 124.3 (q, CF3, 1JCF 271.7 Hz), 127.0 (q, C(3), 3JCF
110.6e141.8/156.1e159.5 (aromatic carbons), 123.9 (q, CF3, JCF
272.5 Hz), 140.4, 153.7, 163.4; MS (CI) 476 (Mþ1)þ.
2
3.8 Hz), 127.7 (q, C(4), JCF 32.9 Hz), 129.1, 129.3, 129.8, 134.3,
139.0e140.0 (m), 141.3, 154.2, 163.8; MS (CI) 364 (Mþ1).
6.1.2.11. 4-Benzyloxy-N-[(3,5-difluoro-40-trifluoromethyl)biphenyl-4-
yl]-1,2,5-oxadiazole-3-carboxyamide (11i). Method B was followed.
Yield 59%; the crude solid was triturated with diisopropyl ether/
6.1.2.4. 4-Benzyloxy-N-(biphenyl-4-yl)-1,2,5-oxadiazole-3-carboxamide
(11b). Method A was followed. The solid was triturated with

M.L. Lolli et al. / European Journal of Medicinal Chemistry 49 (2012) 102e109
107
hexane 1/1 v/v; white solid (m.p. 144.6e144.8 ꢀC, from diisopropyl
ether); 1H-NMR (CDCl3)
5.52 (s, 2H, CH2O), 7.25e7.76 (m, 11H,
129.1,129.5,130.6,132.4,133.3,136.7,137.9,157.2,163.7; MS (CI) 282
(Mþ1); anal. (C9H7N3O2) C, H, N.
d
aromatic protons), 8.03 (s, 1H, NH); 13C-NMR (CDCl3)
d 75.0, 123.9
(q, CF3, 1JCF 272.1 Hz), 110.7e141.6/156.1e159.5 (aromatic carbons),
140.4, 153.9, 163.4; MS (CI) 476 (Mþ1)þ.
6.1.3.4. 4-Hydroxy-N-[40-(trifluoromethyl)biphenyl-4-yl]-1,2,5-
oxadiazole-3-carboxyamide (7d). Yield 55%; white solid (m.p.
191.3e191.9 ꢀC dec., from EtOH/water); 1H-NMR (DMSO-d6)
6.1.2.12. 4-Benzyloxy-N-[(2,3,5,6-tetrafluoro-40-trifluoromethyl)
d
7.81e7.92 (m, 8H, aromatic protons), 11.10 (s, 1H, NH); 13C-NMR
(DMSO-d6)
d
124.3 (q, CF3, 1JCF 271.9 Hz), 127.5 (q, C(40), 2JCF 31.9 Hz),
biphenyl-4-yl]-1,2,5-oxadiazole-3-carboxyamide (11l). Method
B
3
125.7 (q, C(30), JCF 3.3 Hz), 127.0, 127.5, 134.5, 138.5, 143.3,
143.3e143.4 (m), 155.0, 162.2; MS (CI) 350 (Mþ1); anal.
(C16H10F3N3O3,1.17H2O) C, H, N.
was followed. Yield 56%; the crude solid was triturated with dii-
sopropyl ether/hexane 1/1 v/v; white solid (m.p. 196.0e196.4 ꢀC,
from diisopropyl ether/isopropanol 10/1 v/v); 1H-NMR (CDCl3)
d
5.47 (s, 2H, CH2O), 7.38e7.99 (m, 5H, aromatic protons), 11.28 (s,
1H, NH); 13C-NMR (CDCl3)
d 74.2, 115.4e145.4 (aromatic carbons),
6.1.3.5. 4-Hydroxy-N-[30-(trifluoromethyl)biphenyl-4-yl]-1,2,5-
oxadiazole-3-carboxyamide (7e). Purification by flash chromatog-
raphy (eluent: CH2Cl2/MeOH 8/2 v/v). Yield 33%; white solid (m.p.
202.5 ꢀC dec; from hexane/diisopropyl ether 1/1 v/v); 1H-NMR
123.9 (q, CF3, 1JCF 272.1 Hz), 140.9, 154.5, 163.5; MS (CI) 512 (Mþ1)þ.
6.1.2.13. 4-Benzyloxy-N-[(2,3,5,6-tetrafluoro-30-trifluoromethyl)
biphenyl-4-yl]-1,2,5-oxadiazole-3-carboxyamide (11m). Method
B
(DMSO-d6)
d 7.66e8.00 (m, 8H, aromatic protons), 12.48 (br s, 2H,
was followed. Yield 56%; the crude solid was triturated with dii-
sopropyl ether/hexane 1/1 v/v; white solid (m.p. 132.6e133.8 ꢀC,
from diisopropyl ether/isopropanol 10/1 v/v); 1H-NMR (CDCl3)
NH), 13C-NMR (DMSO-d6)
d
120.2, 122.6 (q, C(40), 3JCF 3.8 Hz), 123.6
3
1
(q, C(20), JCF 3.6 Hz), 124.2 (q, CF3, JCF 272.5 Hz), 127.5, 129.7 (q,
2
C(30), JCF 31.5 Hz), 130.0, 130.3, 133.9, 138.2, 140.5, 142.9, 157.2,
d
5.43 (s, 2H, CH2O), 7.26e7.76 (m, 11H, aromatic protons), 8.18 (s,
167.8; MS (CI) 338 (loss of CF3 fragment; Mþ1); anal.
(C16H10F3N3O3$1.19H2O); calc.: C, 50.11; H, 3.63; N, 10.96; found: C,
49.66; H, 2.91; N, 10.84. HPLC purity 99% (tR ¼ 8.15 min).
1H, NH); 13C-NMR (CDCl3)
d 75.2, 103.3e145.8 (aromatic carbons),
123.7 (q, CF3, 1JCF 272.6 Hz), 140.1, 140.1, 153.6, 163.4; MS (CI) 512
(Mþ1)þ.
6.1.3.6. 4-Hydroxy-N-[(3,5-difluoro)biphenyl-4-yl]-1,2,5-oxadiazole-
3-carboxyamide (7f). Yield 65%; white solid (m.p. 169.8e170.1 ꢀC,
6.1.2.14. 4-Benzyloxy-N-[(2,3,5,6-tetrafluoro-30-trifluoromethoxy)
biphenyl-4-yl]-1,2,5-oxadiazole-3-carboxyamide (11n). Method
B
from EtOH/water 1/1 v/v); 1H-NMR (DMSO-d6)
d
7.42e7.80 (m,
7H, aromatic protons), 10.78 (s, 1H, eNH); 13C-NMR (DMSO-d6)
110.1e110.4 (m, C(3)), 112.1 (t, C(1), 2JCF 17.5 Hz), 127.1, 128.9, 129.5,
was followed. Yield 60%; the crude solid was triturated with dii-
sopropyl ether/hexane 3/7 v/v; white solid (m.p. 129.1e129.6 ꢀC,
from diisopropyl ether/isopropanol 10/1 v/v); 1H-NMR (CDCl3)
d
4
3
137.4 (t, C(10), JCF 2.2 Hz), 141.4 (t, C(4), JCF 9.7 Hz), 142.4, 155.6,
158.2 (dd, C(2), 1JCF 249.1 Hz, 3JCF 6.0 Hz), 162.5; MS (CI) 318 (Mþ1);
anal. (C15H9F2N3O3$1.3H2O); calc.: C, 52.80; H, 3.44; N, 12.31;
found: C, 52.37; H, 2.79; N, 11.63; HPLC purity 100% (tR ¼ 3.53 min).
d
5.51 (s, 2H, CH2O), 7.33e7.58 (m, 9H, aromatic protons), 8.10 (s, 1H,
NH); 13C-NMR (CDCl3)
d 75.2, 109.6e149.4 (aromatic carbons),
120.4 (q, CF3, 1JCF 257.9 Hz), 140.1, 153.5, 163.4; MS (CI) 528 (Mþ1)þ.
6.1.3. General hydrogenation procedure to obtain the final products
7aen
6.1.3.7. 4-Hydroxy-N-[(2,3,5,6-(tetrafluoro)biphenyl-4-yl)]-1,2,5-
oxadiazole-3-carboxyamide (7g). Yield 80%; white solid (m.p.
247.1e247.8 ꢀC, from EtOH/water 1/1 v/v); 1H-NMR (DMSO-d6)
Pd/C (16 mg) was added to a solution of the appropriate ben-
zyloxyfurazancarboxyamide (11aen, 0.2 mmol) in dry THF (10 mL).
The resulting mixture was vigorously stirred under hydrogen
atmosphere for 15e60 min. The suspension was filtered on a Celite
bed, washing exhaustively with diethyl ether. The collected organic
layers were concentrated under reduced pressure to obtain the title
compound. See the specific method for purification details.
d
7.55e7.58 (m, 5H, aromatic protons), 11.27 (s, 1H, NH); 13C-NMR
(DMSO-d6)
d 114.5e140.7 (aromatic carbons), 141.8, 155.3, 162.4;
MS (CI) 354 (Mþ1); anal. (C15H7F4N3O3) C, H, N.
6.1.3.8. 4-Hydroxy-N-[(3,5-difluoro-30-trifluoromethyl)biphenyl-
4-yl]-1,2,5-oxadiazole-3-carboxyamide (7h). Yield 78%; white solid
(m.p. 110.6e111.1 ꢀC, from EtOH/water 1/1 v/v); 1H-NMR (DMSO-
6.1.3.1. 4-Hydroxy-N-[(1-trifluoromethyl)phenyl-4-yl]-1,2,5-oxadiazole-
3-carboxyamide (7a). Yield 95%; white solid (m.p. 158.7e159.5 ꢀC,
d6)
NMR (DMSO-d6)
d
7.71e8.14 (m, 6H, aromatic protons), 10.84 (s, 1H, NH); 13C-
from toluene); 1H-NMR (DMSO-d6)
d
7.70e8.03 (m, 4H, aromatic
d
110.2e141.5/156.6e160.0 (aromatic carbons),
124.4 (q, CF3, 1JCF 272.6 Hz), 142.5, 155.7, 162.7; MS (CI) 386 (Mþ1);
anal. (C16H8F5N3O3,H2O) C, H, N.
protons), 8.57 (br s, 2H, NH); 13C-NMR (DMSO-d6)
d
120.2, 124.0 (q,
1
2
3
CF3, JCF 271.7 Hz), 124.7 (q, C(4), JCF 31.9 Hz), 126.2 (q, C(3), JCF
3.8 Hz), 141.2e141.3 (m), 142.9, 155.4, 162.2; MS (CI) 274 (Mþ1);
anal. (C10H6F3N3O3) C, H, N.
6.1.3.9. 4-Hydroxy-N-[(3,5-difluoro-40-trifluoromethyl)biphenyl-4-yl]-
1,2,5-oxadiazole-3-carboxyamide (7i). Yield 87%; white solid (m.p.
167.7e168.4 ꢀC, from EtOH/water 1/2 v/v); 1H-NMR (DMSO-d6)
6.1.3.2. 4-Hydroxy-N-(biphenyl-4-yl)-1,2,5-oxadiazole-3-carboxyamide
(7b). Yield 38%; the crude solid was triturated in hexane/
diisopropyl ether 3/7 v/v (m.p. 185.8e186.1 ꢀC, from ethanol); 1H-
d
7.65e8.04 (m, 6H, aromatic protons), 8.86 (s, 2H, NH); 13C-NMR
(DMSO-d6)
d 110.5, 141.2/156.2e159.2 (aromatic carbons), 124.1 (q,
1
CF3, JCF 272.1 Hz), 142.1, 155.3, 162.3; MS (CI) 386 (Mþ1); anal.
NMR (DMSO-d6)
d
7.33e7.83 (m, 9H, aromatic protons), 11.03 (s, 1H,
120.4, 126.3, 127.0, 127.2, 128.8, 136.2,
NH); 13C-NMR (DMSO-d6)
d
(C16H8F5N3O3) C, H, N.
137.1, 139.4, 143.2, 154.8, 162.1; MS (CI) 282 (Mþ1); anal.
6.1.3.10. 4-Hydroxy-N-[(2,3,5,6-tetrafluoro-40-trifluoromethyl)biphenyl-
4-yl]-1,2,5-oxadiazole-3-carboxyamide (7l). Yield 91%; white solid
(m.p. 260.3e263.7 ꢀC, from EtOH/water 1/1 v/v); 1H-NMR
(C15H11N3O3 0.3H2O) C, H, N.
6.1.3.3. 4-Hydroxy-N-(biphenyl-2-yl)-1,2,5-oxadiazole-3-carboxyamide
(7c). The general hydrogenation procedure was followed. Yield 68%
(m.p. 138.1e138.5 ꢀC; from trituration with diisopropyl ether);
(DMSO-d6)
d 7.85e7.98 (d, 4H, aromatic protons), 11.40 (s, 1H, NH);
13C-NMR (CDCl3) 109.1e145.0 (aromatic carbons), 123.9 (q, CF3, 1JCF
272.3 Hz), 144.0, 155.3, 162.4; MS (CI) 422 (Mþ1); anal.
(C16H6F7N3O3) C, H, N.
1H-NMR (CDCl3)
d
7.30e8.34 (m, 9H, aromatic proton), 8.56 (s, 1H,
OH), 8.72 (s, 1H, NH); 13C-NMR (CDCl3)
d
121.5, 126.3, 128.7, 128.7,

108
M.L. Lolli et al. / European Journal of Medicinal Chemistry 49 (2012) 102e109
6.1.3.11. 4-Hydroxy-N-[(2,3,5,6-tetrafluoro-30-trifluoromethyl)biphenyl-
4-yl]-1,2,5-oxadiazole-3-carboxyamide (7m). Yield 92%; white
solid (m.p. 171.3e171.6 ꢀC, from EtOH/water 1/1 v/v); 1H-NMR
computations were performed with the Merck Force Field
(MMFF94s) using the GB/SA implicit solvent model. The local
minima obtained from the conformational search were then
refined by ab initio QM optimization (RHF/6e31þG(d) level of
theory, C-PCM polarizable continuum solvent model [31]) using
GAMESS-US [32]. The experimental crystallographic structures of
DHODH complexes were retrieved from the Protein Data Bank [33]
(PDB IDs 1D3G, 1UUO and 2BXV). The coordinates of a small
number of residues were not resolved in these structures; since
they were verified not to belong to the active site, the C- and N-
termini upstream and downstream of the missing loops were
simply capped with N-methyl and acetyl groups, respectively.
Missing hydrogens were added to the three enzymes in standard
positions, then optimized with the SANDER module of the AMBER
10 software package [34], while keeping heavy atoms harmonically
restrained to the initial crystallographic coordinates with a force
(DMSO-d6) d 7.80e8.04 (m, 4H, aromatic protons), 8.04 (s, 1H, NH);
13C-NMR (CDCl3) 115.2e147.5 (aromatic carbons), 123.81 (q, CF3,
1JCF 272.43 Hz), 141.8, 155.3, 162.6; MS (CI) 422 (Mþ1); anal.
(C16H6F7N3O3) C, H, N.
6.1.3.12. 4-Hydroxy-N-[(2,3,5,6-tetrafluoro-30-trifluoromethoxy)
biphenyl-4-yl]-1,2,5-oxadiazole-3-carboxyamide (7n). Yield 85%;
white solid (m.p. 183.9e184.2 ꢀC, from EtOH/water 1/2 v/v); 1H-
NMR (DMSO-d6) d 7.55e7.75 (m, 4H, aromatic protons), 11.32 (s, 1H,
NH); 13C-NMR (DMSO-d6) 109.1e148.3 (aromatic carbons), 120.0 (q,
1
OCF3, JCF 256.8 Hz), 141.8, 155.3, 162.4; MS (CI) 438 (Mþ1); anal.
(C16H6F7N3O4) C, H, N.
6.2. Inhibition of rat liver DHODH activity
constant of 1000 kcal molꢃ1 ꢃ2. AMBER FF99 parameters and
Å
charges were assigned to protein atoms, GAFF parameters coupled
with QM-fitted RESP charges [35] were used for the co-crystallized
inhibitor and ORO, while values for the FMN cofactor were taken
from literature [36]. After removing the co-crystallized inhibitor,
docking of compounds 7aen was carried out using AutoDock 4.2
[37]. A grid with a 0.375 Å step size was centered on the co-
crystallized inhibitor, which was then removed; energy grid maps
were pre-computed with AutoGrid, then flexible docking was
carried out with AutoDock. The target proteins were kept rigid,
while ligands were left free to explore the conformational space
inside the enzyme cavity; 200 separate docking simulations were
run on each protein, using the Lamarckian genetic algorithm with
default parameters.
6.2.1. Preparation of rat liver membranes
Crude mitochondrial/microsomal membranes from livers of
male Wistar rats (200e250 g) were prepared by homogenization
and differential centrifugation. Homogenization buffer was 25 mM
sodium phosphate, 250 mM sucrose and 0.3% v/v Protease Inhibitor
Cocktail for use with mammalian cell and tissue extracts (SIGMA
Catalog Number P8340) at pH 7.4. The homogenate (9 mL
homogenization buffer/g wet tissue) was centrifuged at 470g for
10 min, the supernatant retained, and the pellets re-homogenized
in buffer (4.5 mL/g wet tissue). This homogenate was centrifuged
(470g, 10 min) and the supernatant was combined with that ob-
tained earlier, and centrifuged (50000g, 60 min). The membranes
were washed by re-suspension in homogenization buffer plus
150 mM NaCl, 1 mM EDTA and 1 mM EGTA before final centrifu-
gation (50000g, 60 min) and re-suspension in the homogenization
buffer (2 mL/g wet tissue). All the above steps were performed at
Acknowledgments
Chemical Computing Group is acknowledged for financial
support to computational work.
4
ꢀC and aliquots of the final membrane preparation were stored
at ꢃ80 ꢀC.
References
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was assessed using a DCIP-linked assay. Membranes (0.8 mg
protein mLꢃ1) were incubated at 37 ꢀC in 50 mM TriseHCl, 0.1%
Triton X-100, 1 mM KCN, pH 8.0 with coenzyme Q10 (100 mM) and
the tested compounds at different concentrations (final DMSO
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