Synthesis, structure-activity relationships, and pharmacokinetic properties of dihydroorotate dehydrogenase inhibitors: 2-cyano-3-cyclopropyl- 3-hydroxy. N-[3'-methyl-4'-(trifluoromethyl)phenyl]propenamide and related compounds

Article abstract of DOI:10.1021/jm9604437

The active metabolite (2) of the novel immunosuppressive agent leflunomide (1) has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. A series of analogues of the active metabolite 2 have been synthesized. Their in vivo biological activity determined in rat and mouse delayed type hypersensitivity has been found to correlate well with their in vitro DHODH potency. The most promising compound (3) has shown activity in rat and mouse collagen (II)-induced arthritis models (ED₅₀ = 2 and 31 mg/kg, respectively) and has shown a shorter half-life in man when compared with leflunomide. Clinical studies in rheumatoid arthritis are in progress.

Full text of DOI:10.1021/jm9604437

4608
J . Med. Chem. 1996, 39, 4608-4621
Syn th esis, Str u ctu r e-Activity Rela tion sh ip s, a n d P h a r m a cok in etic P r op er ties
of Dih yd r oor ota te Deh yd r ogen a se In h ibitor s: 2-Cya n o-3-cyclop r op yl-3-h yd r oxy-
N-[3′-m eth yl-4′-(tr iflu or om eth yl)p h en yl]p r op en a m id e a n d Rela ted Com p ou n d s
Elizabeth A. Kuo, Philip T. Hambleton, David P. Kay, Phillip L. Evans, Saroop S. Matharu, Edward Little,
Neil McDowall, C. Beth J ones, Charles J . R. Hedgecock, Christopher M. Yea, A. W. Edith Chan,
Peter W. Hairsine, Ian R. Ager, W. Roger Tully, Richard A. Williamson, and Robert Westwood*
Hoechst Marion Roussel, Kingfisher Drive, Covingham, Swindon, Wiltshire SN3 5BZ, England
Received J une 19, 1996X
The active metabolite (2) of the novel immunosuppressive agent leflunomide (1) has been shown
to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the
fourth step in de novo pyrimidine biosynthesis. A series of analogues of the active metabolite
2 have been synthesized. Their in vivo biological activity determined in rat and mouse delayed
type hypersensitivity has been found to correlate well with their in vitro DHODH potency.
The most promising compound (3) has shown activity in rat and mouse collagen (II)-induced
arthritis models (ED50 ) 2 and 31 mg/kg, respectively) and has shown a shorter half-life in
man when compared with leflunomide. Clinical studies in rheumatoid arthritis are in progress.
In tr od u ction
Rheumatoid arthritis (RA) is a chronic inflammatory
disease characterized by painful and swollen joints with
possible progression to irreversible joint damage. It is
believed to have an underlying autoimmune pathology.
Initial treatment is with nonsteroidal antiinflammatory
drugs (NSAIDs) which provide symptomatic relief but
do not modify the course of the disease. Except for the
mildest cases, they are used in combination with
disease-modifying antirheumatic drugs (DMARDs). Re-
quence of this work we were able to identify analogues
with equivalent activity to leflunomide (1) but which
had differential pharmacokinetic properties and were
therefore candidates for clinical development in areas
where it is not appropriate to develop leflunomide itself.
During these studies we reported the ability of the
active metabolite 2 to inhibit the enzyme dihydroorotate
dehydrogenase (DHODH),10 the enzyme which catalyzes
the fourth step in the synthesis of the pyrimidine bases
necessary for cell proliferation.
cent recommendations have focused on the early and
aggressive use of these agents because of the serious
long-term outcome of RA. This group of compounds
includes gold salts, sulfasalazine, D-penicillamine, hy-
droxychloroquine, azathioprine, and methotrexate, but
their modes of action remain largely unknown.1 RA
synovitis is currently regarded as an immunological
process involving T-cells, antigen-presenting cells, mac-
rophages, synoviocytes, and cytokines.2,3 New and
potentially disease-modifying therapies for RA are
under investigation based on these components and
include anti-cytokine treatments, such as tenidap4 and
neutralizing antibodies,5 and immunomodulatory com-
pounds, such as cyclosporine6 and leflunomide.7
This paper reports the structure-activity relation-
ships between a series of analogues of the leflunomide
metabolite 2 with regard to their inhibition of DHODH
and relates this activity to the observed in vivo phar-
macological activity of this series. One compound from
the series, 3, has progressed into phase II clinical trials
for RA.
Leflunomide (1) was first reported in 1976,8 and its
pharmacological, kinetic, and clinical profile has been
described in the literature.9 The species responsible for
the biological activity of leflunomide (1) has been
reported to be the ring-opened metabolite (2).9 Despite
the abundance of data on leflunomide (1) itself, at the
outset of this work the mode of action was not under-
stood. Consequently, rationalization of the available
structure-activity data which related largely to its in
vivo activity was difficult.
Ch em istr y
Several general synthetic routes have been employed
in the synthesis of this series of compounds (Tables 1
and 2). Two methods (Schemes 1 and 2) have been used
for the preparation of the intermediate cyanoaceta-
mides, which were then acylated to afford the â-hydroxy
enamides using sodium hydride and the required acyl
halide. The preparation of 3-acetylenic derivatives was
carried out using acyl fluorides for the acylation,11 while
We therefore established a program of further work,
with the objective of determining the mode of action of
a series of molecules related to 2 and the rationalization
of their structure-activity relationships. As a conse-
* Address for correspondence: Roussel Uclaf, 102 Route de Noisy,
93235 Romainville Cedex, France.
X Abstract published in Advance ACS Abstracts, October 15, 1996.
S0022-2623(96)00443-8 CCC: $12.00 © 1996 American Chemical Society

Synthesis, SAR, and Properties of DHODH Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23 4609
for the preparation of the 3-olefinic derivative 4, the
acylation using propenoyl chloride afforded only very
poor yields of the required product. The acylation was
best performed by method C12 (Scheme 3) in which the
acid chloride is substituted with a potential leaving
group, phenylseleno, thus allowing the generation of the
carbon-carbon double bond as a last step in the
synthesis.
Ta ble 1. Physical Properties of â-Hydroxy Enamides

4610 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
Ta ble 1 (Continued)
Kuo et al.
a
Acid imidazolide instead of acid chloride (method E, step 1, for preparation of imidazolide). Added at -30 °C to cyanoacetamide.
Sodium hydride (80% dispersion in oil) and catalytic imidazole. Methacryloyl chloride added to reaction mixture at -78 °C. Stirred for
b
30 min at -78 °C and quenched with glacial acetic acid. Stirred for 30 min and poured onto aqueous HCl at 0 °C. Product filtered,
washed with water and ether, and then dried in vacuo. c Propargoyl fluoride11 distilled directly into cold (-70 °C) reaction mixture via an
d
air condenser. Reaction mixture stirred at -78 °C for 1 h. Product purified by trituration with ether. Crotonyl chloride added at -78
°C. Reaction mixture stirred at -78 °C for 2 h. Reaction quenched with iced aqueous HCl. e Acid chloride added to reaction mixture at
g
-50 °C. f Dichloromethane as solvent with dicyclohexylcarbodiimide. Temperature increased to 40 °C during addition of DCC. Calcd:
h
j
C, 56.76; F, 19.24. Found: C, 57.27; F, 18.76. Calcd: C, 59.44. Found: C, 59.93. Prepared from 54 by standard literature procedures.
k
l
Product extracted with ethyl acetate and the organic phase washed with brine, dried MgSO4, and concentrated in vacuo. Product
purified by trituration with ether. Calcd: N, 10.62. Found: N, 10.09.
m
Ta ble 2. Physical Properties of Pyridyl Amides
compd no.
A
B
C
X
method
mp (°C)
empirical formula
C12H10ClN3O2
anal.
79
80
81
82
83
C
C
C
C
N
CH
CH
CH
N
N
N
N
CH
CH
Cl
Br
CF3
Cl
Bra
A
223-5
202-3
CHN
C
12H10BrN3O2
CHBrb
CHNF
CHNCl
CHN
Ba
202-3 dec
196-8
C13H10N3F3O2
C12H10N3ClO2
C12H11N3O2
Ba
CH
Dc,d
>300
a
b
Dichloromethane as solvent with dicyclohexylcarbodiimide. Temperature increased to 40 °C during addition of DCC. Calcd: N,
13.64. Found: N, 13.19. c Pyridine (0.72 g, 1 equiv) was added during the reaction of the isoxazole acid chloride with 4-aminopyridine
d
(1.1 equiv). The product was filtered and used without further purification. Ring opening of isoxazole using triethylamine in methanol
at reflux for 1 h. Reaction mixture treated with concentrated HCl to pH 1 and product crystallized at 4 °C for 16 h.
Sch em e 1
Sch em e 2a
a
DIPCDI, diisopropylcarbodiimide.
Sch em e 3
Occasionally it was necessary to prepare the â-hy-
droxy enamides by ring opening of the corresponding
isoxazoles (method D in Scheme 4). This was particu-
larly useful where the aryl substituents were groups
such as hydroxyl, amino, or pyridyl.
Most anilines required for the synthesis of the cy-
anoacetamides were either commercially available or
prepared by standard methods. However, new synthetic
routes were developed for the synthesis of some of the
anilines, in particular a variety of 3-methyl 4-substi-
tuted anilines (5) (Scheme 5). For example, diazotiza-
tion of 2-methyl-4-nitroaniline followed by treatment
with the appropriate potassium halide13 or potassium
cyanide afforded the halo- or cyano-substituted ni-
trobenzenes in good yield which could then be reduced
to the corresponding anilines. The 4-iodo-3-methylni-

Synthesis, SAR, and Properties of DHODH Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23 4611
Sch em e 4
Sch em e 7
Sch em e 5
Sch em e 8
Sch em e 6
Sch em e 9
trobenzene was further reacted with (trifluoromethyl)-
copper14,15 to afford, after reduction, 3-methyl-4-(triflu-
oromethyl)aniline in good yield. This method provides
a far superior route to that available in the literature.16
The synthesis of 3-methyl-4-(trifluoromethoxy)aniline
was developed as shown (Scheme 6). Conversion of the
phenol 6 to the 1-(bromodifluoromethoxy)-2-methyl-4-
nitrobenzene (7) with dibromodifluoromethane17 pro-
ceeded in 29% yield. Reaction with antimony trifluoride/
antimony pentachloride18 afforded the 2-methyl-4-nitro-
1-(trifluoromethoxy)benzene (8) in 83% yield. This was
readily reduced to the required aniline 9, which was
used without purification due to its volatile nature.
The synthesis of 3-methyl-4-[(trifluoromethyl)thio]-
aniline (13) was carried out as shown (Scheme 7). The
thiol 11 was prepared in 71% yield from the fluoride 10
using sodium sulfide nonahydrate. Trifluoromethyla-
tion was carried out using trifluoromethyl iodide. This
gave a poor yield (17%) of 12, which was readily reduced
using iron/HCl in refluxing benzene to the required
aniline 13 (87% yield).
by bromination/dehydrobromination in 94% yield. Hy-
drogenation of the acetylene 15 using Lindlar’s catalyst
gave the cis olefin 16 in 88% yield (Scheme 9).
Biologica l Resu lts a n d Discu ssion
The olefinic and acetylenic linked biphenyl amines
were prepared by the routes shown (Schemes 8-10).
The trans olefin 14 was prepared in 65% yield by Wittig
reaction (Scheme 8) and converted to the acetylene 15
Dihydroorotate dehydrogenase catalyzes the fourth
step in de novo pyrimidine biosynthesis, the conversion
of dihydroorotate to orotic acid. Its inhibition would
lead to pyrimidine nucleotide depletion and conse-

4612 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
Kuo et al.
Ta ble 3. Investigation of 3-Substituents
Sch em e 10
quently inhibition of DNA and RNA synthesis and cell
proliferation. Pyrimidines are also required for glyco-
sylation of lipids and proteins. Cell proliferation is a
critical component of the immune response, during
which the immune cells have a high nucleotide require-
ment. A DHODH inhibitor would thus be a potential
immunosuppressive agent in, e.g., delayed type hyper-
sensitivity (DTH) reponses and in addition would have
potential therapeutic benefits in disorders involving
aberrant cell proliferation such as arthritis. The bio-
logical activities of this series of compounds were
measured after oral administration in both rat and
mouse DTH. For compounds showing DTH activity in
both species, an approximate half-life in mouse and rat
was also determined. Compounds were later tested in
the inhibition of DHODH, and the correlation between
their in vivo DTH activity and in vitro DHODH potency
was studied.
An investigation of different alkyl groups on the
3-position of the molecule showed that increasing the
size of the substituent reduced DTH activity, e.g., 17
vs 19 (Table 3). The most successful replacement for
methyl in 2 was found to be cyclopropyl (17), which
showed an identical pharmacological profile with 1,
including kinetic properties. In the admitted absence
of a full metabolic study, this result was taken as
indicating that there was no major metabolism occur-
ring through the cyclopropyl group. Olefin-containing
substituents were also investigated, with the hope that
this might introduce a metabolic site into the molecule.
Introduction of the 3-isopropenyl group gave a molecule
(26) showing reduced potency in both rat and mouse
DTH but which showed improved kinetic properties.
Other compounds containing olefin groups such as allyl
(29) showed DTH activity but only slightly modified
kinetic properties (Table 3).
Aromatic substituents were investigated for structure-
activity relationships (Table 4) and also to investigate
the metabolism of various functional groups. The most
potent compounds contain a 4-substituent, with 3-sub-
stituted being less active and 2-substituted the least
active, e.g., 17 vs 32 vs 33 (Table 4). The nature of the
substituent was also important with polar, hydrogen
bond donor groups (48, 50, 53) and larger heterocyclic
4-substituents (55-58) being inactive or very weakly
active compounds in DTH. The compounds showing the
best overall pharmacological profile of combined rat and
mouse DTH activity and modified kinetic properties
when compared with 1 were 38, 40, and 49. In the case
of 38 and 49 no metabolites could be observed in either
rate or mouse plasma by HPLC. However, 40 showed
two metabolites in HPLC which were identified by
chemical synthesis as 41 and 42, with 42 itself display-
a
b
Percent inhibition (mg/kg). IC50 (nM). c Negative value in-
dicates potentiation of response. *P < 0.05; **P < 0.01 (Student’s
t-test).
ing potent DTH activity and long kinetics. Many of the
most potent DTH active molecules showed similar long
mouse kinetics to leflunomide (1) (17, 34, 37, 46). By
extending the arylamine group we hoped to investigate
the binding site for the molecule, which was an un-
known protein at the start of this work. For example,
phenyl ethers (68, 69, 70) showed weak DTH activity
as did cis and trans olefin-linked phenyl groups (73, 74)
and the acetylene-linked compound 75. These com-
pounds also showed long pharmacokinetic properties.
The introduction of chemical functional groups ex-
pected to undergo metabolism was investigated for the
molecules containing potent 4-substituents but display-
ing long kinetics. An alkyl group was considered for
this, and the approach proved to be very successful. The
introduction of a 3-methyl group into 17 to give 3
afforded a molecule with identical DTH activity and
greatly modified kinetic profile in both rat and mouse
(Table 5). From an investigation of the chloro,methyl-
substituted series (61-63) the best substitution pattern

Synthesis, SAR, and Properties of DHODH Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23 4613
Ta ble 4. Investigation of Aromatic Substituents

4614 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
Ta ble 4 (Continued)
Kuo et al.
a
b
Percent inhibition (mg/kg). IC50 (nM). *P < 0.05; **P < 0.01 (Student’s t-test).
Ta ble 5. Investigation of Alkyl Substituents as Potential Metabolic Sites
a
b
Percent Inhibition (mg/kg). IC50 (nM). *P < 0.05; **P < 0.01 (Student’s t-test).
was shown to be with the 4-chloro-3-alkyl analogue, e.g.,
61 (Table 5). All compounds with potent DTH activity
and long kinetics, e.g., 34 and 46 (Table 4), also showed
good DTH and reduced kinetics in both rat and mouse
following the introduction of a 3-methyl group, e.g., 61
and 67 (Table 5). The minimum energy conformation
for 3 contains the aryl ring coplanar with the enolic
amide system.19 The introduction of a 2-methyl group
gave less potent compounds as did the introduction of
other 2-substituents, e.g., 33 vs 17 (Table 4). This may
be due to the steric effects of the 2-alkyl group which
cause the conformation of the aromatic ring to be less
coplanar (∼40° out of plane) (Figure 1, 61 vs 62).
The aryl amide group was investigated for its effects
on both DTH and kinetic properties. Use of pyridyl-
substituted molecules (e.g., 81) (Table 2) gave good DTH
and kinetic properties (Table 6).
More recently the target protein for this series of
drugs has been identified as DHODH.10 The structure-
activity for this series has now been investigated by the
in vitro determination of inhibition of both rat and
mouse DHODH. In general, potent DHODH inhibitors
also show good in vivo DTH activity, and poor in vitro
enzyme inhibitors also show poor in vivo pharmacology.
For example, 40 is one of the most potent compounds
in rat DTH (58% inhibition at 1 mg/kg) and is also one
of the most potent in vitro rat DHODH enzyme inhibi-
tors (IC50 ) 5 nM, 2 IC50 ) 13 nM). The excellent
correlation of in vivo to in vitro potency gives support
to the inhibition of DHODH being largely responsible

Synthesis, SAR, and Properties of DHODH Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23 4615
Ta ble 6. Investigation of Amides
a
b
Percent inhibition (mg/kg). IC50 (nM). *P < 0.05; **P < 0.01 (Student’s t-test).
F igu r e 2. Overlay of aryl amides to show space available in
drug binding site. Biological activity (DHODH inhibition)
decreases in the order: 74 > 70 > 78 > 75 > 73.
F igu r e 1. Overlay of 61 and 62, showing the conformational
difference between 2-alkyl-substituted (62) and 3-alkyl-
substituted (61) aryl rings.
ether 70, the biphenyl 78, the acetylene 75, and the
trans olefin 73, all of which show decreasing activity.
Molecules such as the cis olefin 74 and the trans olefin
73, with their obviously different spatial requirements,
help to define the space available in the rat and mouse
enzyme binding site for these compounds (Figure 2).
Brequinar sodium (84) inhibits DHODH noncompeti-
tively with respect to either the substrate (dihydrooro-
tate) or the cofactor ubiquinone.20 Studies to determine
whether 3 and 84 bind to the same site on DHODH are
in progress and will be reported later.21 The structure
activity for 84 on the mouse and human enzyme has
for the in vivo pharmacology for this series of com-
pounds. Some examples which do not correlate well are
the cis olefin 74, which is weaker in mouse DTH than
would be expected from its mouse DHODH in vitro
potency. This may be due to short kinetics or poor oral
bioavailability (not determined). Also the biphenyl
compounds 77 and 78 are more potent in vivo than
would be expected based on DHODH inhibition.
A number of compounds in this series are potent
inhibitors of rat DHODH (IC50 < 10 nM). In general
this series provides more potent inhibitors of the rat
DHODH enzyme than of the mouse enzyme. The best
inhibitors of the rat enzyme (40, 42, 46) all contain the
small cyclopropyl ketone group. In addition, all contain
small substituents on the aromatic ring with the best
being 4-substituted. The best substituents are lipophilic
and, also, weak hydrogen bond acceptor groups, e.g., 46.
The most potent inhibitors of the mouse DHODH
enzyme again predominantly contain 3-cyclopropyl
groups, an indication of restricted space in this binding
region. The best substituents on the aromatic ring for
the mouse enzyme are lipophilic in nature (e.g., 34, 37,
74). For both rat and mouse, polar, hydrogen bond
donor groups afford poor inhibitors in both DTH and
DHODH assays (e.g., 48, 50, 53).
been investigated.22,23 Figure 3 shows an overlay of 84
and the structure of 3.19 The critical regions which have
been defined in the literature22,23 for 84 are (1) the
carboxylic acid group which we have overlapped with
the acidic enolic hydroxyl group, indicating that there
is probably an electrostatic interaction between the
acidic groups of the drugs and their binding site, (2)
bulky hydrophobic substituents (biphenyl) which can
overlay the 4-substituted aryl ring, and (3) the benzo
The larger aryl amides give more information about
the space available in the drug binding site. Thus the
cis olefin-linked phenyl groups in 74, which showed good
DHODH inhibition, can be compared with the phenyl

4616 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
Kuo et al.
Met h od A. 2-Cya n o-3-cyclop r op yl-3-h yd r oxy-N-[3′-
m eth yl-4′-(tr iflu or om eth yl)ph en yl]pr open am ide (3). Step
1.26 Cyanoacetic acid (65.5 g, 0.77 mmol) was added to a
stirred suspension of phosphorus pentachloride (159.5 g, 0.77
mmol) in dichloromethane (2.3 L) at ambient temperature. The
reaction mixture was heated at reflux for 30 min. After cooling
3-methyl-4-(trifluoromethyl)aniline (89.4 g, 0.51 mmol) was
added over 10 min and the reaction mixture heated for 2 h at
reflux. The reaction mixture was cooled in an ice/water bath,
and water (1 L) added. After stirring for 30 min, the reaction
mixture was neutralized by the addition of sodium carbonate
solution. The precipitated solid was filtered, washed with
water, and dried to afford 110 g (89%) of 2-cyano-N-[3′-methyl-
4′-(trifluoromethyl)phenyl]acetamide. This product was used
without further purification: mp 142-3 °C; IR (KBr) 3270,
F igu r e 3. Overlay of brequinar sodium (84) and HR 325 (3).
1
2274, 1677, 1620, 1552; H NMR (DMSO-d6) δ 10.54 (1H, s),
7.56 (3H, m), 4.00 (2H, s), 2.46 (3H, s).
portion of the quinoline ring which overlays the 3 nitrile
group. Our structure-activity data for the mouse
DHODH enzyme fits with the literature data for 84,22,23
which shows a hydrophobic group requirement such as
the biphenyl or 4-tert-butylphenyl group, and our most
potent mouse DHODH inhibitors, which contain hydro-
phobic 4-substituents on the aryl amide group e.g., 34,
37, and 74.
The compound with the best overall pharmacological
profile (3) was further tested in animal models of
arthritis. Good inhibition was achieved in mouse col-
lagen II arthritis24 (ED50 ) 31 mg/kg) and in both rat
developing and established collagen II arthritis25 [ED50
) 2 mg/kg for developing arthritis (leflunomide (1) 102%
(p < 0.01) at 10 mg/kg) and 65% (p < 0.01) inhibition
at 25 mg/kg or 32% (p < 0.05) at 10 mg/kg for
established arthritis (leflunomide (1) 29% (p < 0.05) at
10 mg/kg)].
Step 2. The product from step 1 (107 g, 0.44 mmol) was
suspended in THF (4 L) and cooled to <5 °C. Sodium hydride
(80% sodium hydride in oil) (29.2 g, 0.97 mmol) was added
while the temperature was maintained at <10 °C. The
mixture was allowed to warm to ambient temperature and
stirred for 30 min. Cyclopropanecarbonyl chloride (55.4 g, 0.53
mmol) was added over 10 min, and the reaction mixture stirred
at ambient temperature for 1 h. The reaction was quenched
by careful addition of acetic acid (100 mL) and then poured
onto ice/water (4 L) containing hydrochloric acid (100 mL). The
product was filtered off, washed with water, and dissolved in
dichloromethane (5 L). The solution was washed with water,
dried (MgSO4), filtered, and concentrated in vacuo to a thick
slurry. This was cooled and filtered to afford the title
compound (116.2 g, 85%) as colorless crystals: mp 186-8 °C;
IR 3395 (NH), 2204 (CN), 1630, 1600, 1580; 1H NMR (CDCl3)
δ 15.63 (1H, s), 7.59 (2H, d), 7.43 (2H, d), 2.49 (3H, s), 2.15
(1H, m), 1.35 (2H, m), 1.19 (2H, m). Anal. C, H, N, F.
Met h od B. 2-Cya n o-3-cyclop r op yl-3-h yd r oxy-N-[4′-
(tr iflu or om eth yl)p h en yl]p r op en a m id e (17). Step 1. Cy-
anoacetic acid (8.6 g, 0.10 mmol) and 4-(trifluoromethyl)aniline
(13.5 mL, 0.11 mmol) were dissolved in THF (100 mL) and
stirred at room temperature. Diisopropylcarbodiimide (DIPC-
DI) (16.4 mL, 0.10 mmol) was added over 10 min. The reaction
temperature varied between 20 and 60 °C during the addition.
The reaction was stirred at room temperature for 16 h and
filtered and the solvent evaporated. The residue was washed
with ethanol, dichloromethane, and then hexane and dried
under reduced pressure at 60 °C. This afforded the product
(18.85 g, 83%) which was used without further purification:
mp 195-196 °C; IR (KBr) 3280, 1672, 1608, 1551; 1H NMR
(DMSO-d6) δ 10.72 (1H, s), 7.81 (2H, d, J ) 9 Hz), 7.74 (2H, d,
J ) 9 Hz), 4.03 (2H, s).
Single-dose (20 mg) human kinetics have also been
determined for 3 (t1/2 ) 3.5 h), indicating that the
objective to produce a derivative with reduced human
kinetics over leflunomide has been achieved. Clinical
studies are in progress with this molecule.
Con clu sion
The structure-activity relationship of a series of
â-hydroxy enamides on the dihydroorotate dehydroge-
nase enzyme shows good correlation with the in vivo
DTH activity for this series. The pharmacokinetic
properties of the series have been studied and modified.
The most successful change in pharmacokinetics was
achieved with the introduction of a 3-methyl substituent
onto the aryl amide group. The compound 3 was
selected for development.
Step 2. The title compound was obtained in 70% yield by
the method described for method A, step 2: mp 212-3 °C; IR
(KBr) 3280 (NH), 2202 (CN), 1620, 1602, 1575; 1H NMR
(CDCl3) δ 15.64 (1H, s), 7.77 (1H, s), 7.64 (4H, s), 2.16 (1H,
m), 1.37 (2H, m), 1.18 (2H, m). Anal. C, H, N, F.
Meth od C. 2-Cya n o-3-h yd r oxy-N-[4′-(tr iflu or om eth yl)-
p h en yl]p en ta -2,4-d ien a m id e (4). Step 1. As for method
A or B.
Exp er im en ta l Section
In str u m en ta tion a n d Meth od s. Proton NMR spectra
were recorded on a Bruker WP200SY spectrometer, and the
data are reported with the chemical shifts in parts per million
(ppm, δ) relative to tetramethylsilane as an internal standard.
Mass spectra were obtained on a Fisons VG Autospec E mass
spectrometer. Infrared spectra were recorded using a Pye
Unicam SP3-200S instrument. Melting points were obtained
using a Kofler hot stage apparatus and are uncorrected.
Evaporations were done using a Buchi rotary evaporator.
Elemental microanalyses were performed by CHN Analysis
Ltd. of Leicester. Analytical thin layer chromatography (TLC)
was performed on E Merck silica gel 60 F254 commercial plates
to follow the course of reactions. Column chromatography was
performed with Sorbsil C60 silica gel (mesh 40-60 µm).
Analytical HPLC was performed using Hypersil HSAS (25 cm
× 4.6 mm i.d. column). THF was freshly distilled from
potassium benzophenone ketyl. Reagents were obtained com-
mercially and used without further purification.
Step 2. 2-Cyano-N-[4′-(trifluoromethyl)phenyl]acetamide
(7.0 g, 0.0307 mmol) in dry THF (200 mL) was stirred under
nitrogen during the addition of imidazole (0.02 g; catalyst) and
sodium hydride (80% oil dispersion) (2.3 g, 0.077 mmol), and
the suspension stirred for 2 h at room temperature. The
mixture was cooled to -78 °C and treated dropwise with
3-(phenylseleno)propionyl chloride (9.11 g, 0.037 mmol).12 The
mixture was stirred for 90 min at -78 °C, poured onto dilute
hydrochloric acid/ice, and filtered. The collected solid was
dissolved in dichloromethane, washed with water, and dried
(MgSO4) and the solvent removed in vacuo. Trituration with
diethyl ether gave 2-cyano-3-hydroxy-5-(phenylseleno)-N-[4′-
(trifluoromethyl)phenyl]penta-2,4-dienamide as colorless crys-
tals (13.40 g, 99%). This was used without further purification
for step 3: mp 121-3 °C; IR (KBr) 3293, 2222, 1637, 1609,
1589; 1H NMR (CDCl3) δ 15.53 (1H, s), 7.75 (1H, s), 7.58 (6H,
m), 7.29 (3H, m), 3.16 (2H, m), 3.00 (2H, m).

Synthesis, SAR, and Properties of DHODH Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23 4617
Step 3. 2-Cyano-3-hydroxy-5-(phenylseleno)-N-[4′-(trifluo-
romethyl)phenyl]penta-2,4-dienamide (8.0 g, 0.018 mmol) in
dichloromethane (200 mL) was cooled to 0 °C and treated with
30% hydrogen peroxide (4.0 mL), and the mixture stirred
vigorously for 30 min giving a colorless suspension of the
intermediate selenoxide. The mixture was diluted with metha-
nol (40 mL) and dichloromethane (200 mL), stirred at room
temperature for 1 h, and passed through a column of silica
gel. The eluent was concentrated under reduced pressure and
diluted with diethyl ether. Colorless crystals of the title
compound were collected (2.8 g, 54%): mp 206-8 °C; IR (KBr)
3300 (NH), 2215 (CN), 1632, 1604; 1H NMR (DMSO-d6) δ 12.17
(1H, s), 7.81 (2H, d), 6.88 (1H, dd), 6.20 (1H, dd), 5.69 (1H,
dd). Anal. C, H, N, F.
8.10 (1H, s), 7.81 (2H, d, J ) 8.6 Hz), 7.67 (2H, d, J ) 8.6 Hz),
4.04 (1H, m), 3.32 (2H, m), 2.12 (3H, m), 1.44 (1H, m). Anal.
C, H, N, F.
Meth od F . 2-Cya n o-3-h yd r oxy-3-(N-m eth ylp yr r olid in -
2-yl)-N-[4′-(tr iflu or om eth yl)ph en yl]pr open am ide (23). The
trifluoroacetic acid salt of 22 (7.5 g, 0.0243 mmol) in THF (300
mL) was treated with sodium hydride (80% dispersion in oil)
(1.5 g, 2 equiv) and stirred at room temperature for 40 min.
Methyl iodide (4.92 mL, 0.078 mmol) was added, and the
reaction mixture stirred for 24 h. The reaction mixture was
acidified (dilute HCl), extracted with ethyl acetate, dried
(MgSO4), and concentrated. Trituration with dichloromethane/
methanol followed by chromatography of the residues (0-1%
methanol in dichloromethane) afforded the required product
1
(4.16 g, 50%): mp 168-70 °C; IR (KBr) 3260, 1650, 1590; H
Meth od D. Step 1. 5-[4′-(Tr iflu or om eth yl)p h en yl]-N-
[4′-(tr iflu or om eth yl)ph en yl]isoxazole-4-car boxam ide (24).
5-[4′-(Trifluoromethyl)phenyl]isoxazole-4-carboxylic acid27 (2.6
g, 10 mmol) was warmed with thionyl chloride (10 mL) at 50
°C for 1 h.27 The excess thionyl chloride was removed in vacuo
by azeotroping with toluene. The residue was taken up with
dichloromethane and 4-aminobenzotrifluoride (2 g, 1.24 equiv,
1.6 mL) added. The reaction mixture was stirred at room
temperature for 30 min, washed with dilute HCl, water,
aqueous sodium carbonate, and water, dried (MgSO4), and
evaporated to dryness. The product was purified by flash
chromatography (dichloromethane) and recrystallized from
diethyl ether by addition of petroleum ether to afford 2.9 g,
72%, of the title compound. This was used without further
purification: mp 211-3 °C; IR (KBr) 3300, 1655, 1605, 1525,
1475, 1410; 1H NMR (CDCl3/MeOH-d4) δ 8.85 (1H, s), 8.2 (2H,
d), 7.8 (4H, m), 7.60 (2H, d).
NMR (CDCl3) δ 10.44 (1H, s), 8.82 (1H, brs), 7.71 (2H, d, J )
8.6 Hz), 7.53 (2H, d, J ) 8.6 Hz), 4.08 (1H, m), 3.64 (1H, m),
3.23 (1H, m), 3.16 (3H, d, J ) 5.4 Hz) (singlet when decoupled
with OH at 8.8 ppm), 2.22 (3H, m), 1.64 (1H, m). Anal. C, H,
N, F.
Met h od G. P r ep a r a t ion of N-[4′-[(Tr iflu or om et h yl)-
su lfin yl]p h en yl]cya n oa cet a m id e a n d N-[4′-[(Tr iflu or o-
m eth yl)su lfon yl]p h en yl]cya n oa ceta m id e. Step 1. N-[4′-
[(Trifluoromethyl)thio]phenyl]cyanoacetamide (4.35 g, 0.0167
mol) in acetic acid (15 mL) was stirred at room temperature
overnight with peracetic acid (8.0 mL of 32%, w/v, 0.035 mol).
The mixture was poured onto ice/water and extracted into
ethyl acetate. The extracts were washed with aqueous sodium
bicarbonate solution, dried (MgSO4), and evaporated to give a
colorless solid (4.6 g). Chromatography on silica gel (1-10%
acetone in dichloromethane) gave N-[4′-[(trifluoromethyl)-
sulfinyl]phenyl]cyanoacetamide (2.7 g, 59%) as colorless crys-
tals: mp 164-5 °C; IR (KBr) 3280, 1678, 1615, 1592; 1H NMR
(DMSO-d6) δ 10.80 (1H, brs), 7.91 (4H, s), 4.03 (2H, s). This
product was used without further purification.
Step 2.27 2-Cya n o-2-[4′-(tr iflu or om eth yl)ben zoyl]-N-
[4′-(tr iflu or om eth yl)p h en yl]eth a n a m id e. The isoxazole
from step 1 (2.5 g, 6.3 mmol) was added to a solution of Na (1
g, 43 mmol) in ethanol (50 mL) and stirred until all the solids
had dissolved (15 min). The reaction mixture was poured onto
aqueous HCl (400 mL, 2 N) and stirred for 20 min. The
product was filtered and washed with dilute hydrochloric acid
and then water. Recrystallization from hot DMF by the
dropwise addition of water afforded the title compound (2.42
g, 97%): mp 218-20 °C; IR (KBr) 3300, 2200, 1635, 1625, 1605,
The residual fractions (1.7 g) from the above chromatogra-
phy were combined, dissolved in acetic acid (6 mL), and treated
with peracetic acid (6 mL). The mixture was heated to 50 °C
overnight, poured onto water, and extracted into ethyl acetate.
The extracts were washed with aqueous sodium bicarbonate,
dried (MgSO4), and evaporated. Chromatography on silica gel
(100 g) (2-3% acetone in dichloromethane) gave N-[4′-[(tri-
fluoromethyl)sulfonyl]phenyl]cyanoacetamide as colorless crys-
tals (1.06 g, 22%): mp 135-7 °C; IR (KBr) 3343, 3316, 1724,
1
1595; H NMR (DMSO-d6) δ 12.35 (1H, brs), 7.85-7.55 (8H,
m). Anal. C, H, N, F.
1
Meth od E. 3-[N-(ter t-Bu tyloxyca r bon yl)p yr r olid in -2-
yl]-2-cyan o-3-h ydr oxy-N-[4′-(tr iflu or om eth yl)ph en yl]pr o-
p en a m id e. Step 1. Carbonyldiimidazole (4.21 g, 1.3 equiv,
25.96 mmol) was added at -30 °C to a solution of N-(tert-
butyloxycarbonyl)proline (5.16 g, 1.2 equiv, 23.97 mmol) in
THF (20 mL), and the reaction mixture was warmed to room
temperature for 2 h. The resulting mixture was then used to
acylate the required cyanoacetamide at room temperature
(method A, step 2). Final product was obtained by addition
of water and extraction of the aqueous phase with ethyl
acetate. The organic phase was washed with saturated sodium
chloride with the addition of dilute hydrochloric acid until pH
= 5. The extracts were then washed with saturated NaHCO3,
dried MgSO4, and evaporated. The product was purified by
trituration with diethyl ether to give (6.3 g, 62%) of a colorless
solid: mp 205-10 °C; IR (KBr) 3480, 3387, 2188, 1650, 1601,
1608, 1592; H NMR (DMSO-d6) δ 11.09 (1H, s), 8.15 (2H, d,
J ) 9 Hz), 8.00 (2H, d, J ) 9 Hz), 4.08 (2H, s). This product
was used without further purification.
Step 2. P r ep a r a tion of 2-Cya n o-3-cyclop r op yl-3-h y-
dr oxy-N-[4′-[(tr iflu or om eth yl)su lfin yl]ph en yl]pr op-2-en a-
m id e (41). N-[4′-[(Trifluoromethyl)sulfinyl]phenyl]cyanoacet-
amide (3.1 g, 0.0112 mol) in dry THF (100 mL) was treated
with imidazole (10 mg; catalytic) and sodium hydride (80% oil
dispersion) (0.75 g, 0.0246 mol) and stirred at room temper-
ature for 90 min. The solution was treated dropwise with
cyclopropylcarbonyl chloride (1.32 mL, 0.014 mmol), stirred
at room temperature for 10 min, poured onto ice/1 M HCl, and
filtered. The precipitated product was collected, dissolved in
dichloromethane, and passed through a short silica gel column
with dichloromethane to give 2-cyano-3-cyclopropyl-3-hydroxy-
N-[4′-[(trifluoromethyl)sulfinyl]phenyl]prop-2-enamide (2.54 g,
66%): mp 155-7 °C; IR (KBr) 3280, 2219, 1603, 1575; 1H NMR
(CDCl3) δ 15.49 (1H, s), 7.89 (1H, s), 7.82 (4H, s), 2.18 (1H,
m), 1.37 (2H, m), 1.22 (2H, m). Anal. C, H, N, F, S.
P r ep a r a tion of 2-Cya n o-3-cyclop r op yl-3-h yd r oxy-N-
[4′-[(tr iflu or om eth yl)su lfon yl]ph en yl]pr op-2-en am ide (42).
Treatment of N-[4′-[(trifluoromethyl)sulfonyl]phenyl]cyano-
acetamide by the above method gave 2-cyano-3-cyclopropyl-
3-hydroxy-N-[4′-[(trifluoromethyl)sulfonyl]phenyl]prop-2-ena-
mide (65%): mp 168-70 °C; IR (KBr) 3300, 2218, 1638, 1579;
1H NMR (CDCl3) δ 15.30 (1H, s), 8.03 (2H, d, J ) 8.8 Hz),
7.92 (1H, s), 7.85 (2H, d, J ) 8.8 Hz), 2.18 (1H, m), 1.41 (2H,
m), 1.25 (2H, m). Anal. C, H, N, F, S.
1
1556; H NMR (DMSO-d6) δ 12.40, 12.25 (1H, 2 × s, coales-
cence at 373 K), 7.76 (2H, d, J ) 8.6 Hz), 7.59 (2H, d, J ) 8.6
Hz), 4.64 (1H, m), 3.50 (2H, m), 2.17 (1H, m), 1.83 (3H, m),
1.43, 1.34 (9H, 2 × s, coalescence at 373 K).
Step 2. 2-Cya n o-3-h yd r oxy-3-(p yr r olid in -2-yl)-N-[4′-
(t r iflu or om et h yl)p h en yl]p r op en a m id e (22). The pro-
tected amine (425 mg, 1 mmol) in anisole (2 mL) and
dichloromethane (5 mL) was cooled to 0 °C and trifluoroacetic
acid (5 mL) added. The reaction mixture was stirred at room
temperature for 4 h and evaporated to dryness. The residue
was triturated with diethyl ether and filtered to afford the
trifluoroacetic acid salt of the title compound (330 mg, 75%).
The salt was dissolved in methanol/dichloromethane and
basified with aqueous sodium bicarbonate. The precipitate
was filtered to give the free base: mp 266-8 °C; IR (KBr) 3380,
Met h od H . 2-Cya n o-3-cyclop r op yl-3-h yd r oxy-N-[4′-
(m eth ylsu lfon yl)p h en yl]p r op en a m id e (45). 2-Cyano-3-cy-
clopropyl-3-hydroxy-N-[4′-(methylthio)phenyl]propenamide (43)
(8.0 g, 29.16 mmol) was dissolved in acetic acid (500 mL), and
1
1660, 1640, 1600, 1579; H NMR (DMSO-d6) δ 10.64 (1H, s),

4618 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
Kuo et al.
peracetic acid (25 mL of 32% solution in acetic acid, 105 mmol)
was added. The reaction mixture was stirred overnight at
room temperature, and water (250 mL) was added. The
precipitate was filtered, washed with water, and dried in
vacuo. Crystallization from ethyl acetate/hexane afforded (4.6
g, 51%) of a white solid: mp 199-201 °C; IR (KBr) 3250, 2930,
2220, 1630, 1570; 1H NMR (DMSO-d6) δ 11.42 (1H, s), 7.84
(4H, m), 3.21 (3H, s), 2.23 (1H, m), 1.02 (4H, m). Anal. C, H,
N, S.
Meth od I. N-(4′-Am in op h en yl)-2-cya n o-3-cyclop r op yl-
3-h yd r oxyp r op en a m id e Hyd r och lor id e (50). 2-Cyano-3-
cyclopropyl-3-hydroxy-N-(4′-nitrophenyl)propenamide (49) (4.3
g, 15.7 mmol) was dissolved in ethanol/0.5 N sodium hydroxide
(1:1, 150 mL) and treated with hydrogen (1 atm) for 6 h in the
presence of 10% palladium/carbon (150 mg). The reaction
mixture was filtered through Celite, and the solution was
acidified to pH 1 with concentrated hydrochloric acid. The
product was filtered, washed with water, and dried in vacuo
to afford the title compound (3.7 g, 84%): mp >300 °C; IR
(KBr) 3300, 2840, 2590, 2200 (CN), 1630, 1570. 1H NMR
(DMSO-d6) δ 11.16 (1H, s), 7.66 (2H, m), 7.35 (2H, m), 2.21
(1H, m), 1.04 (4H, m). Anal. C, H, N.
Syn th esis of An ilin es. P r ep a r a tion of 1-Iod o-2-m eth -
yl-4-n itr oben zen e. 2-Methyl-4-nitroaniline (230 g, 1.51 mmol)
was suspended in dilute sulfuric acid (concentrated H2SO4 (189
mL)/H2O (1.38 L)) and cooled to below 5° using an ice/salt bath.
A solution of sodium nitrite (109.6 g, 1.59 mmol in 345 mL of
H2O) was added at a rate to maintain a temperature of 0-5
°C. This diazonium salt solution was added to a solution of
potassium iodide (422.1 g, 2.54 mmol in 1.27 L of H2O)
maintaining a temperature of 0-5 °C during the addition. The
mixture was stirred at ambient temperature for 2 h and then
heated to 50-60 °C for 1 h. The mixture was cooled and
filtered and the solid washed with water. Solid was dissolved
in dichloromethane (4 L) and washed with sodium met-
abisulfite solution (2 × 2 L). The solution was passed down a
column of silica gel to remove the majority of the dark brown
coloration. The solvent was concentrated in vacuo and the
orange crystalline solid triturated with ether, filtered, and
dried to afford the title compound (327 g, 82%): 1H NMR
(CDCl3) δ 8.07 (2H, m), 7.72 (1H, dd, J ) 8.7, 2.8 Hz), 2.55
(3H, s).
(CDCl3) δ 150.05, 145.54, 133.71 (q, J ) 30.5 Hz, C-1), 127.32
(q, J ) 5.5 Hz, C-6), 125.05, 123.48 (q, J ) 273.0 Hz, CF3),
120.72, 25.58, 15.08. Anal. C, H, N. F.
P r ep a r a tion of 1-(Br om od iflu or om eth oxy)-2-m eth yl-
4-n itr oben zen e. 2-Methyl-4-nitrophenol (6 g, 43.13 mmol)
was added to sodium (0.92 g, 40 mmol) dissolved in ethanol
(30 mL). The solvent was evaporated in vacuo, and benzene
(20 mL) was added and reevaporated. The sodium salt was
added to DMF (24 mL), dibromodifluoromethane (30 mL), and
ethanethiol (3 drops; catalyst). The reaction mixture was
heated at 70 °C for 10 h, poured onto ice/water, and extracted
with ethyl acetate. The organic phase was washed with
aqueous sodium hydroxide (0.5 M) and water, dried (MgSO4),
and concentrated in vacuo. The product was purified by flash
chromatography (4% ethyl acetate in hexane) to afford 1-(bro-
modifluoromethoxy)-2-methyl-4-nitrobenzene (3.35 g, 29%)
which was used without further purification: IR (film) 1591,
1
1529, 1490, 1351, 1310, 1237; H NMR (CDCl3) δ 8.15 (2 H,
m), 7.46 (1H, d, J ) 8.9 Hz), 2.43 (3H, s).
P r ep a r a tion of 2-Meth yl-4-n itr o-1-(tr iflu or om eth oxy)-
ben zen e. 1-(Bromodifluoromethoxy)-2-methyl-4-nitrobenzene
(0.3 g, 1.06 mmol), antimony trifluoride (0.12 g, 0.67 mmol),
and antimony pentachloride (0.02 g; catalyst) were heated in
a sealed flask at 175 °C for 4 h. The reaction mixture was
cooled, diluted with ether, washed with water, dried (MgSO4),
and concentrated in vacuo. This afforded the title compound
(0.13 g, 83%) which was used without further purification: IR
(film) 3413, 2928, 1593, 1529, 1494, 1350, 1339; 1H NMR
(CDCl3) δ 8.15 (2H, m), 7.44 (1H, d, J ) 8.9 Hz), 2.43 (3H, s);
13C NMR (CDCl3) δ 120.34 (CF3, J CF ) 260 Hz).
P r ep a r a tion of 4-(Tr iflu or om eth yl)-3-tolu id in e. 2-(Tri-
fluoromethyl)-5-nitrotoluene (145 g, 0.759 mmol) in methanol
(1.7 L) was hydrogenated at 1250 mbar using 5% palladium
on charcoal (14.5 g) as catalyst. When hydrogen uptake had
ceased, the catalyst was filtered off and the solvent removed
under reduced pressure to leave the product as a pale yellow
oil (123 g, 93%): IR (film) 3500, 3400, 3240, 1630, 1590, 1520,
1
1465, 1445, 1320, 1280, 1195, 1163, 1115, 1045, 865, 825; H
NMR (CDCl3) δ 7.36 (1H, d, J ) 8.2 Hz), 6.50 (2H, m), 3.86
(2H, brs), 2.37 (3H, m). Anal. C, H, N, F.
1-Am in o-3-m eth yl-4-(p en ta flu or oeth yl)ben zen e: pre-
pared by the same method, 94% yield; IR (KBr) 3391, 3331,
1
2-E t h yl-1-iod o-4-n it r ob en zen e: prepared by the above
method starting from 4-amino-3-ethyl-1-nitrobenzene,28 60%
yield; mp 32-3 °C; 1H NMR (CDCl3) δ 8.05 (1H, d, J ) 2.7
Hz, H-3), 8.01 (1H, d, J ) 8.6 Hz, H-6), 7.73 (1H, dd, J ) 2.7,
8.6 Hz, H-5), 2.83 (2H, q, J ) 7.5 Hz, -CH2), 1.28 (3H, t, J )
7.5 Hz, -CH3); MS (70 eV) m/e 277 (M+, 100), 262, 204, 133,
104, 103, 78, 77.
3227, 1676, 1650, 1599, 1554, 1342; H NMR (CDCl3) δ 7.30
(1H, s), 6.53 (2H, m), 3.80 (2H, brs), 2.37 (3H, t, J ) 3 Hz).
4-Am in o-2-eth yl-1-(tr iflu or om eth yl)ben zen e: prepared
by the same method, 93% yield, colorless liquid; 1H NMR
(CDCl3) δ 7.37 (1H, d, J ) 8.4 Hz), 6.58 (1H, d, J ) 2.0 Hz),
6.50 (1H, dd, J ) 8.4, 2.0 Hz), 3.88 (2H, brs), 2.72 (2H, q, J )
7.5 Hz), 1.22 (3H, t, J ) 7.5 Hz).
P r ep a r a tion of 2-(Tr iflu or om eth yl)-5-n itr otolu en e. A
450 mL Parr pressure reactor was charged with 2-iodo-5-
nitrotoluene (81.0 g, 0.308 mmol), active copper powder14,15
(62.3 g, 0.98 mol), and dimethylformamide (200 mL). The
reactor was purged with argon and cooled to ca. -25 °C, and
trifluoromethyl iodide (93 g, 0.475 mmol) was introduced via
the dip-tube inlet valve. The reactor was then heated gradu-
ally to 150 °C, with stirring, and maintained at 150 °C for 5
h. The cooled reaction mixture was poured onto ice/water (1.0
L), washing out the reactor with ethyl acetate. The copper
salt was filtered off and washed with ethyl acetate. The
aqueous layer was separated and extracted with ethyl acetate.
All extracts were combined, washed with water, and dried over
magnesium sulfate. The solvent was concentrated in vacuo
and the crude product purified by distillation at ca. 10 mbar,
bp 89-92 °C. The title product was obtained (54.5 g, 95%):
IR (film) 1536, 1355, 1314, 1273, 1178, 1127, 1047, 925, 899,
843, 807; 1H NMR (CDCl3) δ 8.13 (2H, m), 7.81 (1H, d, J ) 8.3
Hz), 2.61 (3 H, m). Anal. C, H, N.
P r epar ation of 4-Am in o-2-m eth yl-1-(tr iflu or om eth oxy)-
ben zen e. 5-Nitro-2-(trifluoromethoxy)toluene (2.5 g, 0.0113
mol) in toluene (100 mL) was treated with iron powder (13 g)
and concentrated HCl (3 drops) and stirred vigorously under
reflux. Water (2.0 mL) was added over 2 h; the mixture was
filtered and concentrated to a low volume. The product was
used in the next step without further purification.
P r ep a r a tion of 4-Am in o-2-m eth yl-1-[(tr iflu or om eth yl)-
th io]ben zen e. 2-Fluoro-5-nitrotoluene (15.5 g, 0.1 mmol) and
sodium sulfide nonahydrate (30 g, 0.125 mmol) in DMSO (50
mL) were heated at 50 °C under a nitrogen atmosphere for 2
h. The reaction mixture was poured onto ice/water and
extracted with dichloromethane. The organic phase was
washed with water, dried (MgSO4), and concentrated in vacuo
to afford 4-mercapto-3-methyl-1-nitrobenzene (12 g, 71%)
which was used without further purification. The product (9
g, 0.053 mmol) was added to sodium (1.22 g, 0.053 mmol) in
ethanol (120 mL) and evaporated to dryness. The residue was
azeotroped with benzene and evaporated to dryness. The
residue was then dissolved in DMF (30 mL) in a Fischer Porter
apparatus, flushed with argon, and cooled to -78 °C and the
flask charged with trifluoromethyl iodide (21 g). The reaction
mixture was stirred for 48 h at room temperature followed by
heating at 75 °C for 24 h. The reaction mixture was cooled,
poured onto ice/water/ether, and filtered. The aqueous phase
was separated and reextracted with ether. The combined
organic phases were dried (MgSO4), evaporated, and purified
3-Met h yl-1-n it r o-4-(p en t a flu or oet h yl)b en zen e: pre-
pared by the same method using pentafluoroethyl iodide, 68%
yield; IR (film) 1536, 1350, 1334, 1304; 1H NMR (CDCl3) δ 8.14
(2H, m), 7.75 (1H, d, J ) 8.4 Hz), 2.65 (3H, t, J ) 3.1 Hz).
3-Eth yl-1-n itr o-4-(tr iflu or om eth yl)ben zen e: prepared
by the same method, 71% yield; IR (film) 2977, 2942, 1535,
1
1352, 1310; H NMR (CDCl3) δ 8.22 (1H, d, J ) 1.5 Hz), 8.14
(1H, dd, J ) 8.6, 1.5 Hz), 7.81 (1H, d, J ) 8.6 Hz); 13C NMR

Synthesis, SAR, and Properties of DHODH Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23 4619
by flash chromatography (5% dichloromethane in hexane) to
afford 2-methyl-4-nitro-1-[(trifluoromethyl)thio]benzene (2.15
g, 17%): 1H NMR (CDCl3) δ 8.18 (1H, d, J ) 2.5 Hz), 8.07
(1H, dd, J ) 8.5, 2.5 Hz), 7.85 (1H, d, J ) 8.5 Hz), 2.66 (3H,
s).
g, 162 mmol, 3 equiv) and (E)-(4-chlorophenyl)(4′-nitrophenyl)-
ethene (14.0 g, 53.9 mmol) in ethanol (100 mL) and water (100
mL). The mixture was refluxed for 4 h and allowed to cool to
room temperature and then basified to ca. pH 11 with 10%
aqueous sodium hydroxide. The suspension was filtered and
the residue washed with hot ethyl acetate (3 × 200 mL). The
solution was evaporated to remove the organic solvents, and
the aqueous phase was extracted with ethyl acetate (3 × 200
mL). The combined organic phases were washed with water
(2 × 200 mL) and brine (100 mL) and then dried (MgSO4),
filtered, and evaporated to give an orange solid. Flash column
chromatography (10-100% ethyl acetate in petroleum ether)
gave the required product (8.82 g, 71%): 1H NMR (CDCl3) δ
7.42-7.25 (6H, m), 7.00 (1H, d, J ) 16.3 Hz), 6.85 (1H, d, J )
16.3 Hz), 6.68 (2H, d), 3.8 (2H, brs).
The product (2.15 g, 9.14 mmol) in benzene (50 mL) was
treated with iron powder (11 g) and stirred with concentrated
hydrochloric acid (3 drops). The reaction mixture was stirred
vigorously and heated at reflux during the addition of water
(1.5 mL) over 2 h. The mixture was heated at reflux for a
further 2 h, cooled, filtered, and evaporated in vacuo to give
the title compound (1.65 g, 87%) which was used without
further purification: IR (film) 3487, 3399, 1626, 1598, 1573,
1
1485, 1321; H NMR (CDCl3) δ 7.40 (1H, d, J ) 8.3 Hz), 6.60
(1H, m), 6.50 (1H, m), 3.87 (2H, brs), 2.43 (3H, s).
(4-Am in op h en yl)(4′-ch lor op h en yl)eth yn e: prepared by
a similar method, 70% yield; 1H NMR (CDCl3) δ 7.44-7.26
(6H, m), 6.64 (2H, d, J ) 8.64 Hz), 3.84 (2H, brs).
P r ep a r a tion of (E)-(4-Ch lor op h en yl)(4′-n itr op h en yl)-
eth en e. Sodium (2.21 g, 96.1 mmol, 1.05 equiv) was added
in lumps over 1.75 h to ethanol (100 mL). The solution was
stirred for 20 min following dissolution of sodium and then
added dropwise over 50 min to a solution of diethyl 4-nitroben-
zyl phosphonate (25 g, 91.5 mmol) and 4-chlorobenzaldehyde
(12.9 g, 91.5 mmol) in ethanol (150 mL). The mixture was
stirred for 16 h and then filtered. The yellow solid was washed
with petroleum ether (2 × 50 mL) and dried in vacuo to afford
the required product (15.35 g, 65%): IR (KBr) 1583, 1498,
1336, 1096, 1084, 962; 1H NMR (CDCl3) δ 8.23 (2H, d, J ) 8.9
Hz), 7.63 (2H, d, J ) 8.9 Hz), 7.49 (2H, d, J ) 8.6 Hz), 7.37
(2H, d, J ) 8.6 Hz), 7.23 (1H, d, J ) 16 Hz), 7.11 (1H, d, J )
16 Hz).
P r ep a r a tion of 1-(4-Ch lor op h en yl)-1,2-d ibr om o-2-(4′-
n itr op h en yl)eth a n e. A solution of bromine (14.17 g, 4.57
mL, 88.9 mmol, 1.5 equiv) in chloroform (50 mL) was added
dropwise over 100 min to a solution of (E)-(4-chlorophenyl)-
(4′-nitrophenyl)ethene (15.4 g, 59.1 mmol) in CHCl3 (700 mL).
The suspension was stirred for 22 h and then evaporated.
Dichloromethane (2 × 100 mL) was evaporated off the solid,
to remove any remaining bromine, to give 23.4 g (95%) of
product as a cream-colored solid: 1H NMR (CDCl3/CD3OD) δ
8.29 (2H, d, J ) 8.8 Hz), 7.69 (2H, d, J ) 8.8 Hz), 7.49-7.37
(4H, m), 5.48 (1H, d, J ) 11.4 Hz), 5.40 (1H, d, J ) 11.4 Hz).
P r epar ation of (4-Ch lor oph en yl)(4′-n itr oph en yl)eth yn e.
Aqueous sodium hydroxide (50%, 150 mL) was added dropwise
over 1.25 h to a suspension of Bu4NHSO4 (68.3 g, 201 mmol,
3 equiv) and 1-(4-chlorophenyl-1,2-dibromo-2-(4′-nitrophenyl)-
ethene (28.1 g, 67.0 mmol) in CH2Cl2 (200 mL) and hexane
(200 mL). After 30 min water (500 mL) and then CH2Cl2 (200
mL) were added. The mixture was filtered, the layers were
separated, and the aqueous fraction was extracted with CH2-
Cl2 (2 × 100 mL). The combined organics were washed with
water (4 × 250 mL) and brine (100 mL) and then dried
(MgSO4), filtered, and evaporated to give 29.9 g of dark green
solid. Flash column chromatography (5-100% ethyl acetate
in 40-60 petroleum ether and then 60% acetone in ethyl
acetate) gave 16.29 g (94%) of product: 1H NMR (CDCl3) δ
8.23 (2H, d), 7.67 (2H, d), 7.50 (2H, d), 7.37 (2H, d).
P r ep a r a tion of (Z)-(4-Ch lor op h en yl)(4′-n itr op h en yl)-
eth en e. A mixture of (4-chlorophenyl)(4′-nitrophenyl)ethyne
(9.0 g, 34.9 mmol), palladium on calcium carbonate poisoned
with lead (Lindlar catalyst; 900 mg), and quinoline (902 mg,
825 µL, 7.00 mmol, 0.2 equiv) was stirred under hydrogen in
ethyl acetate (1 L) until hydrogen uptake ceased. The mixture
was filtered and evaporated. 1H NMR indicated a mixture of
starting material and product. Thus the reaction mixture was
dissolved in EtOAc (175 mL), quinoline (1 mL) and Lindlar
catalyst (1.5 g) were added, and hydrogenation was restarted.
After hydrogen uptake had finished the mixture was filtered,
evaporated, and purified by flash column chromatography (5-
20% ethyl acetate in 40-60 petroleum eluent) to give 7.98 g
(88%) of product: 1H NMR (CDCl3) δ 8.09 (2H, d, J ) 8.9 Hz),
7.36 (2H, d, J ) 8.9 Hz), 7.23 (2H, d, J ) 8.5 Hz), 7.12 (2H, d,
J ) 8.5 Hz), 6.75 (1H, d, J ) 12.2 Hz), 6.63 (1H, d, J ) 12.2
Hz).
(Z)-(4-Am in op h en yl)(4′-ch lor op h en yl)eth en e: prepared
1
by a similar method, 53% yield; H NMR (CDCl3) δ 7.21 (2H,
d), 7.16 (2H, d), 7.03 (2H, d, J ) 8.4 Hz), 6.50 (2H, d, J ) 8.4
Hz), 6.48 (1H, d, J ) 12.2 Hz), 6.33 (1H, d, J ) 12.2 Hz), 3.61
(2H, brs).
Dela yed Typ e Hyp er sen sitivity P a w Ed em a in th e
Mou se (DTH-M).29 On day 0 male CD-1 mice (25-30 g, n )
8-16) were sensitized by subcutaneous injection of 1 mg of
methylated bovine serum albumin (MBSA) in saline/Freund’s
complete adjuvant (FCA) emulsion. Negative control mice
received saline/FCA. On day 7 mice were challenged by
injection into the right hind footpad with MBSA (0.1 mg in
0.05 mL of saline). The left paws received saline. DTH paw
edema formation was measured after 24 h. Control vehicle
or test compounds were administered orally once daily on days
4-6 and twice on day 7, 1 h before and 6 h after challenge.
Dela yed Typ e Hyp er sen sitivity P a w Ed em a in th e Ra t
(DTH-R).30 On day 0 male CFHB rats (160-180 g, n ) 7-12)
were sensitized by subcutaneous injection in the tail base of
0.1 mL of FCA (0.4 mg of Mycobacterium tuberculosis sus-
pended in liquid paraffin). Negative control rats received
Freund’s incomplete adjuvant (liquid paraffin). On day 7 rats
were challenged by injection into the right hind footpad with
soluble M. tuberculosis extract (0.4 mg in 0.2 mL of saline).
The left paws received saline. DTH paw edema formation was
measured after 24 h. Control vehicle or test compounds were
administered orally once daily on days 4-6 and twice on day
7, 1 h before and 6 h after challenge.
Colla gen II Ar th r itis in th e Ra t (CA-R).25 On day 0
female DA rats (130-160 g, n ) 10-17) were sensitized by
subcutaneous injection in the tail base with 0.4 mg of chick
type II collagen in acetic acid/Freund’s incomplete adjuvant
(FIA) emulsion. Negative control rats received acetic acid/FIA.
Arthritic hind paw edema formtion was measured on day 18.
Control vehicle or test compound was administered orally once
daily either throughout the experiment on days 0-4, 7-11,
and 14-17, or to rats with established arthritis on days 14-
17.
Colla gen II Ar th r itis in th e Mou se (CA-M).24
On days
0 and 21 male DBA/1 mice (15-20 g, n ) 12) were sensitized
by intradermal injection in the tail base with 0.1 mg of chick
type II collagen in acetic acid/FCA emulsion. Negative control
mice received acetic acid/FCA. Arthritis severity was mea-
sured on day 32 based on a scoring system of 0-3/paw for
edema and redness (maximum ) 12/mouse). Control vehicle
or test compound was administered orally once daily on days
21-25 and 28-31.
Sta tistica l An a lysis. Paw edema data were compared
using the Student’s t-test. Arthritis severity scores were
compared using the Mann-Whitney U-test. The levels of
statistical significance were as follows: *P < 0.05 and **P <
0.01.
Ra t Kin etic P r otocol. Male Wistar CFHB rats (190-220
g, n ) 3/time point) were used. The drug was administered
orally by gavage at a dose of 10 mg/kg in a dose volume of 10
mL/kg. Blood samples were obtained by cardiac puncture
under ether anesthesia at 0, 1, 3, 6, 24, 48, and 72 h postdose.
The samples were collected individually into heparinized tubes,
P r ep a r a tion of (E)-(4-Am in op h en yl)(4′-ch lor op h en yl)-
eth en e. A solution of concentrated hydrochloric acid (5.4 mL,
ca. 54 mmol) in ethanol (50 mL) and water (50 mL) was added
dropwise over 1 h to a refluxing suspension of iron filings (9.03

4620 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
Kuo et al.
(2) Firestein, G. S.; Zvaifler, N. J . How Important are T cells in
Chronic Rheumatoid Synovitis? Arthritis Rheum. 1990, 33,
768-773.
after which the plasma was separated and stored at -20 °C
until assayed.
Mou se Kin etic P r otocol. Male CD-1 mice (25-32 g, n )
5/time point) were used. The drug was administered orally
by gavage at a dose of 30 mg/kg in a dose volume of 10 mL/kg.
Blood samples were obtained by exsanguination under ether
anesthesia at 0, 1, 3, 6, 24, 48, and 72 h postdose. Blood
samples from all the animals at each time point were collected
into a single heparinized tube, after which the plasma was
separated and stored at -20 °C until assayed.
HP LC Assa y. Ca libr a tion . Prior to analysis of the
samples, calibration curves were constructed by spiking blank
plasma samples with appropriate levels of drug and then
subjecting them to the assay procedures described below.
Chromatograms were recorded via a Trivector Trio data
system with drug concentrations being calculated from the
peak areas. Where samples were found to contain concentra-
tions well outside the calibration range, the analysis was
repeated using an appropriately modified injection volume.
(3) Panayi, G. S.; Lanchbury, J . S.; Kingsley, G. H. The Importance
of the T cell in initiating and maintaining the chronic synovitis
of rheumatoid arthritis. Arthritis Rheum. 1992, 35, 729-735.
(4) Madhok, R. Tenidap. Lancet 1995, 346, 481-485.
(5) Maini, R. N.; Elliott, M. J .; Brennan, F. M.; Feldmann, M.
Beneficial effects of tumour necrosis factor-alpha (TNF-R)
blockade in rheumatoid arthritis (RA). Clin. Exp. Immunol.
1995, 101, 207-212.
(6) Tugwell, P.; Pincus, T.; Yogum, D.; Stein, M.; Gluck, O.; Kraag,
G.; McKendry, R.; Tesser, J .; Baker, P.; Wells, G. Combination
therapy with cyclosporine and methotrexate in severe rheuma-
toid arthritis. N. Engl. J . Med. 1995, 333, 137-140.
(7) Parnham, M. J . Leflunomide: a potential new disease-modifying
anti-rheumatic drug. Exp. Opin. Invest. Drugs 1995, 4, 777-
779.
(8) Heubach, G.; Hoerlein, G.; Sachse, B. Fungicidal isoxazole
derivatives. DE2525023, 1975.
(9) (a) Bartlett, R. R.; Campion, G.; Musikic, P.; Schleyerbach, R.;
Zielinski, T.; Schorlemmer, H. U. Leflunomide: A Novel Immu-
nomodulating Drug. In Nonsteroidal anti-inflammatory drugs:
mechanisms and clinical uses, 2nd ed.; Lewis, A. J ., Furst, D.
E., Eds.; Marcel Dekker, Inc.: New York, 1994; pp 349-366.
(b) Carlson, R. P. Newer immunosuppressive drugs and other
agents for the treatment of rheumatoid arthritis - An update.
Exp. Opin. Invest. Drugs 1995, 4, 853-859.
(10) (a) Williamson, R. A.; Yea, C. M.; Robson, P. A.; Curnock, A. P.;
Gadher, S.; Hambleton, A. B.; Woodward, K. A.; Bruneau, J .-
M.; Hambleton, P. T. Moss, D.; Thomson, T. A.; Spinella-J aegle,
S.; Morand, P.; Courtin, O.; Saute´s, C.; Westwood, R.; Hercend,
T.; Kuo, E. A.; Ruuth, E. Dihydroorotate Dehydrogenase Is a
High Affinity Binding Protein for A77 1726 and Mediator of a
Range of Biological Effects of the Immunomodulatory Com-
pound. J . Biol. Chem. 1995, 270, 22467-22472. (b) Greene, S.;
Watanabe, K.; Braatz-Trulson, J .; Lou, L. Inhibition of dihydro-
orotate dehydrogenase by the immunosuppressive agent lefluno-
mide. Biochem. Pharmacol. 1995, 50, 861-867. (c) Zielinki, T.;
Zeitter, D.; Mu¨llner, S.; Bartlett, R. R. Leflunomide, a reversible
inhibitor of pyrimidine biosynthesis? Inflammatory Res. 1995,
44 (Suppl. 2), S207-S208. (d) Davis, J . P.; Cain, G. A.; Pitts,
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1273.
HR 325 An a lysis. An aliquot of plasma (0.5 mL) was
spiked with internal standard (20 µL of a solution containing
200 µg/mL of 46 in methanol) and then diluted with acetoni-
trile (100 µL) with mixing by vortexing after each addition. A
Bond-Elut extraction tube (3 mL, ODS) was conditioned with
methanol (1 mL) and water (1 mL), and then the plasma
sample was passed slowly through the tube. The tube was
washed with 1 M saline (1 mL) and water (1 mL), after which
the drug and internal standard were extracted with methanol
(0.5 mL), and a 10 µL aliquot of extract was injected for HPLC
analysis.
HPLC Conditions
column
mobile phase
125 × 4.6 mm 5 µm Nucleosil ODS
0.1 M pH 7 phosphate buffer
acetonitrile
65%
35%
flow rate
1.2 mL/min
detection
UV at 300 nm
retention times
HR 325 (3)
46 (standard 1)
metabolite
3.6 min
2.9 min
1.9 min
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Dih yd r oor ota te Deh yd r ogen a se Assa y. Dihydroorotate
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literature10 on mouse or rat spleen enzymes. For the rat
enzyme assay, 0.13 mg of membrane proteins was used. Drug
concentrations were generally increased in full log intervals
with each concentration tested in duplicate.
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Molecu la r Mod elin g. Geometry optimization and confor-
mational search of 3 and 84 were done using the semiempirical
method AM1 supplied by Spartan.31 The lowest energy
conformation of 3 was used as a template to construct other
structures. Each molecule was first built using Quanta32 and
then subjected to minimization. All the minimizations were
performed using the CHARMM33 force field with the adopted-
basis Newton Raphson method until the rms gradient was
equal to or less than 0.05 kcal/mol Å. Overlaps (molecular
similarity) of all the molecules were also done using Quanta.32
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Ack n ow led gm en t. We would like to thank P. Rob-
son and K. Woodward for the DHODH assays and H.
Hidden, S. J ohns, K. McDonald, and F. Parker for the
DTH, collagen arthritis, and pharmacokinetic data. We
would also like to thank G. Danswan and C. Hidden
for the large scale synthesis of intermediates and P.
Kennewell for his assistance in the preparation of this
paper. We would like to thank S. Hughes for her help
and patience in the preparation and typing of this paper.
(18) Hudlicky, M. Chemistry of Organic Fluorine Compounds, 2nd
ed.; Ellis Horwood: London, 1992; p 100. McClinton, M. A.;
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(19) See method in the Molecular Modeling section of this paper.
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dihydroorotate dehydrogenase by A77 1726, the active metabo-
lite of leflunomide. Submitted for publication.
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