Second generation analogues of the cancer drug clinical candidate tipifarnib for anti-chagas disease drug discovery

Article abstract of DOI:10.1021/jm9013136

We previously reported that the cancer drug clinical candidate tipifarnib kills the causative agent of Chagas disease, Trypanosoma cruzi, by blocking ergosterol biosynthesis at the level of inhibition of lanosterol 14α-demethylase. Tipifarnib is an inhibitor of human protein farnesyltransferase. We synthesized tipifarnib analogues that no longer bind to protein farnesyltransferase and display increased potency for killing parasites. This was achieved in a structure-guided fashion by changing the substituents attached to the phenyl group at the 4-position of the quinoline ring of tipifarnib and by replacing the amino group by OMe. Several compounds that kill Trypanosoma cruzi at subnanomolar concentrations and are devoid of protein farnesyltransferase inhibition were discovered. The compounds are shown to be advantageous over other lanosterol 14α-demethylase inhibitors in that they show only modest potency for inhibition of human cytochrome P450 (3A4). Since tipifarnib displays high oral bioavailability and acceptable pharmacokinetic properties, the newly discovered tipifarnib analogues are ideal leads for the development of drugs to treat Chagas disease.

Full text of DOI:10.1021/jm9013136

J. Med. Chem. 2010, 53, 3887–3898 3887
DOI: 10.1021/jm9013136
Second Generation Analogues of the Cancer Drug Clinical Candidate Tipifarnib for
Anti-Chagas Disease Drug Discovery
James M. Kraus,† Hari Babu Tatipaka,† Sarah A. McGuffin,† Naveen Kumar Chennamaneni,† Mandana Karimi,‡
Jenifer Arif,‡ Christophe L. M. J. Verlinde,§ Frederick S. Buckner,*,‡ and Michael H. Gelb*,†,§
†Department of Chemistry, ‡Department of Medicine, and §Department of Biochemistry, University of Washington, Seattle,
Washington 98195-7185
Received September 2, 2009
We previously reported that the cancer drug clinical candidate tipifarnib kills the causative agent of
Chagas disease, Trypanosoma cruzi, by blocking ergosterol biosynthesis at the level of inhibition of
lanosterol 14R-demethylase. Tipifarnib is an inhibitor of human protein farnesyltransferase. We
synthesized tipifarnib analogues that no longer bind to protein farnesyltransferase and display increased
potency for killing parasites. This was achieved in a structure-guided fashion by changing the
substituents attached to the phenyl group at the 4-position of the quinoline ring of tipifarnib and by
replacing the amino group by OMe. Several compounds that kill Trypanosoma cruzi at subnanomolar
concentrations and are devoid of protein farnesyltransferase inhibition were discovered. The com-
pounds are shown to be advantageous over other lanosterol 14R-demethylase inhibitors in that they
show only modest potency for inhibition of human cytochrome P450 (3A4). Since tipifarnib displays
high oral bioavailability and acceptable pharmacokinetic properties, the newly discovered tipifarnib
analogues are ideal leads for the development of drugs to treat Chagas disease.
Introduction
One of the guiding philosophies in our collaborative re-
search effortfor antiparasitedrugdiscoveryisthe concept that
we can “piggyback” on the large research efforts underway in
the pharmaceutical industry.3 There is extensive medicinal
chemistry and pharmacology data on inhibitors of protein
farnesyltransferase since they were developed as anticancer
agents.4 Our thinking at the time was that we might find a
human protein farnesyltransferase inhibitor that also had
high affinity for the T. cruzi orthologue. Thus, we previously
investigated known protein farnesyltransferase inhibitors
against a number of parasites including T. cruzi, and one
compound that was highly potent at killing T. cruzi amasti-
gotes (the clinically relevant parasite life cycle stage that grows
in mammalian host cells) in an in vitro assay5 is tipifarnib
(compound 1, Figure 1).6 This compound is a candidate
anticancer drug being developed by Johnson and Johnson
Pharmaceuticals.7 Our subsequent studies showed, surpris-
ingly, that compound 1 kills T. cruzi not by blocking protein
farnesylation but by disrupting sterol biosynthesis by inhibi-
tion of lanosterol 14R-demethylase (Tc-L14DM).6 T. cruzi
amastigotes use ergosterol as a critical component of their
membranes and cannot use host cell derived cholesterol. Since
1 shows a high degree of oral bioavailability, has ideal
pharmacokinetic properties, and is well tolerated in humans,7
we considered 1 as an outstanding lead for the development of
anti-Chagas disease drugs.
Chagas disease is caused by infection with the protozoan
parasite, Trypanosoma cruzi (T. cruzia). When not spread by
poorhygiene, infectionis the result of exposureto the blood or
the feces of triatomine bugs, which live in the mud in
substandard housing in an area ranging from the southern
part of South America to the southern part of the United
States. Once infected, individuals usually become a life-long
host to the parasite because no existing therapy is completely
effective in the chronic stage of the disease. Eight to eleven
million people in Latin America are infected with T cruzi, and
30% of those can be expected to develop complications
ranging from mild to terminal.1 There is no effective treatment
for Chagas disease at this time. The two drugs that have been
used are the nitrofuran, nifurtimox, and the nitroimidazole,
benznidazole. The toxicity of these drugs is linked to their
mode of action, and treatment is unavoidably accompanied
by multiple undesirable side effects. Of the more than 1200
new drugs discovered in the period from 1975 to 1997 only 4
were the result of a directed research and development effort
by the pharmaceutical industry to treat a human tropical
disease, and 1 of those 4 (nifurtimox) is no longer in wide-
spread use.2
*To whom correspondence should be addressed. For F.S.B. (on
biology): phone, (206) 598-9148; fax, (206) 685-6045; e-mail, fbuckner@
u.washington.edu. For M.H.G. (on chemistry): phone, (206) 543-7142; fax,
(206) 685-8665; e-mail, gelb@chem.washington.edu.
a Abbreviations: Tc-14DM, lanosterol 14R-demethylase from T.
cruzi; T. cruzi, Trypanosoma cruzi; IC50, concentration of inhibitor
resulting in 50% enzyme inhibition; EC50, concentration of inhibitor
resulting in 50% parasite growth inhibition; ESI-MS, electrospray
ionization mass spectrometry.
Our goal shifted to designing analogues of 1 that no longer
bind to human protein farnesyltransferase and remain as
highly potentinhibitors of Tc-L14DM and as cytotoxic agents
for T. cruzi amastigotes. To this end we used the X-ray
structure of 1 bound to mammalian protein farnesyltransfer-
ase8 and a homology model of Tc-L14DM with 1 docked into
r
2010 American Chemical Society
Published on Web 04/29/2010
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3888 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Kraus et al.
the active site6 to develop novel tipifarnib analogues with the
desired properties. Our modelis shownin Figure 2 withbound
1 shown as a double-radii van der Waals surface. In this
depiction, if the active site residues are in van der Waals
contact with 1, the stick representations of these residues
approximately sit on the doubled van der Waals surface of
the inhibitor. It can be seen from Figure 1 that 1 fills the active
site of Tc-L4DM well with the imidazole nitrogen of 1
coordinated to the heme iron. Our modeling led to compound
2 (Figure 1),9 which is essentially devoid of mammalian
protein farnesyltransferase inhibition (IC50>5000 nM versus
∼1 nM for 1) and is about 10-fold more potent at killing
T. cruzi amastigotes than 1 (EC50=0.6 nM versus 4 nM for 1).
Modeling suggests that the extra methyl group on the
3-chlorophenyl ring of 2 clashes with the wall of the active site
of protein farnesyltransferase but is tolerated in the active site
of Tc-L14M. Also, the amino group of 1 is involved in a
hydrogen-bonding network in the active site of protein farne-
syltransferase but not in the active site of Tc-L14M. Although
2 is one of the most potent anti-T. cruzi compounds reported
to date, subsequent studies showed that there is hindered
rotation about the bond connecting the 3-chlorophenyl group
and the quinolone ring presumably because of a clash bet-
ween the ortho methyl group and the vinylic proton of the
Figure 1
Figure 2. Stereodiagram of tipifarnib (compound 1) docked into the active site of Tc-L14DM. The 3-chlorophenyl group is shown in orange
with its Cl in magenta. The 4-chlorophenyl group is shown in light pink with its Cl in magenta, and the rest of the inhibitor is shown in red. The
heme is in yellow with the heme iron in cyan. The active site protein residues are shown as sticks. The inhibitor is displayed as its doubled-radii
van der Waals surface.
Scheme 1. Synthesis of Tipifarnib Analogues from 5-Bromoisatoic Anhydridea
a (a) CH3ONHCH3 HCl, pyridine, CH2Cl2; (b) R1PhBr (2 equiv), n-BuLi (2 equiv), THF; (c) Ac2O, toluene, reflux; (d) t-BuOK, 1,2-dimethoxyethane;
(e) (i) BF4OMe3, CH2Cl2; (ii) NaOH; (f) (i) n-BuLi, THF, -78 °C; (ii) N(Me)imidazole-CO-PhenylR2; (g) 6 N HCl, THF, reflux; (h) CH3I, NaOH,
BTEAC, THF; (i) SOCl2, neat, 12 h; (j) NH3 (or CH3NH2), THF, room temp; (k) tosic acid, 1equiv þ cat., MeOH, reflux.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3889
Scheme 2. Synthesis of Tipifarnib Analogues with R1 = Ortho Halogensa
a (a) SOCl2, (neat), reflux, 6 h, then 4-bromoaniline, DIEA (1.5 equiv), CH2Cl2, 0 °C; (b) H2SO4 (conc), 105 °C; (c) DDQ, dioxane, reflux.
Scheme 3. Synthesis of Substituted 5-Benzoyl-N1-alkylimidazolesa
a Route 1: (a) SOCl2, neat; (b) CH3ONHCH3, pyridine, CH2Cl2; (c) N-alkylimidazole, (i) n-BuLi, THF -78°C; (ii) Et3SiCl, -78 °C; (iii) n-BuLi, THF,
-78 °C. Route 2: (a) TrCl, Et3N, CH3CN; (b) (i) Mg, I2, ether, RC6H4Br, room temp to reflux; (ii) MnO2, dioxane, reflux; (c) MeOTf, CH2Cl2.
quinolone ring. Thus, 2 is a mixtureof rotamers with the ortho
methyl group either above or below the plane of the quinolone
ring closure using base gives quinolone 5. Conversion of 5 to
the set of compounds 10 (Scheme 1) was carried out as described
in our earlier study.9
1
ring. This is evident by the H NMR spectrum of 2, which
shows a doubling of resonance peaks (see Supporting In-
formation). Furthermore, the doubling does not collapse even
when the temperature is raised to 65 °C (not shown), indicat-
ing that the rotamers are very stable at physiological tempera-
ture. Compound 2 also contains a chiral center, so it is a
mixture of 4 stable isomers, and this greatly diminishes its
potential as a drug candidate. This is because the different
isomers can be expected to have different affinities for Tc-
L14DM, and it is possible that only 1 of the 4 isomers would
be biologically active. Also, the pharmacokinetic and toxicity
profiles of all 4 compounds would have to be separately
investigated to go forward with drug candidate selection.
We thus went on to design new analogues that either lack
a 2-substitutent on the 3-chlorophenyl ring or have a C2-
symmetric ring in order to avoid rotamer formation. In this
study we reported the result of these two approaches. We also
carried out additional structure-activity studies of these
tipifarnib analogues.
We were also interested in analogues in which R1 is a 2-
halide or a 2,6-dihalide. The use of 2-chlorophenyllithium is
problematic because of benzyne formation. We formed 2,6-
difluorophenyllithium but found that it reacted slugglishly
with Weinreb amide 3 and also formed benzyne in the process.
Thus, we developed Scheme 2 for the preparation of the
additional analogues. Commercially available halogenated
cinnamic acid 11 was converted to the acid chloride and then
reacted with 4-bromoaniline to give amide 12. Intramolecular
Friedel-Crafts alkylation proceeded smoothly with concen-
trated H2SO4 to give lactam 13. Conversion to the quinolone
was accomplished by oxidation of 13 with 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone to give 14. The latter was con-
verted to the desired tipifarnib analogues by using steps from
Scheme 1.
Scheme3 shows two routes to make the methanones needed
for step f in Scheme 1.
Results and Discussion
Chemistry
We synthesized a variety of analogues of 1 and tested them
for their ability to kill T. cruzi amastigotes growing in
mammalian host cells (3T3 cells). As noted above, adding a
Me group at the 2-position of the 3-chlorophenyl ring of 1 and
replacing the NH2 with OMe led to 2, which is devoid of
protein farnesyltransferase inhibition and is ∼10-fold more
potent at killing parasites compared to 1. In an attempt to
make analogues that do not exist as a pair of stable rotamers
(see above), we synthesized analogues of 2 that have changes
to the 3-chloro-2-methylphenyl ring. Values of EC50 for killing
Scheme 1 shows the route used to synthesize some of the
compounds reported in this study. Commercially available
5-bromoisatoic anhydride is converted to Weinreb amide 3,
which reacts with a variety of phenyllithiums (formed in situ
from the phenyl halide using 2 equiv of n-BuLi, 1 equiv needed
to deprotonate the NH) to give ketone 4. The order of
addition of reagents is important to avoid lithium-bromide
exchange between n-BuLi and the aryl-Br bond present in 3.
Acetylation of the amino group followed by intramolecular

3890 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Table 1. Tipifarnib Analogues with Ring 1 and Ring 2 Modifications
Kraus et al.
T. cruzi amastigotes are listed in Table 1. Compound 3 lacks
the ortho methyl group but retains the NH2 to OMe substitu-
tion. The compound is 100-fold less potent on protein farne-
syltransferase compared to 1 and retains low nanomolar
potency on T. cruzi. This result shows that the ortho methyl
group and the OMe group of 2 act together to reduce the
binding to protein farnesyltransferase. We also made comp-
ound 4, which like 2 has the ortho methyl group and the NH2-
to-OMe change but lacks the 3-chloro group. The properties
are very similar to those of 2, showing that the 3-chloro group

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3891
Table 2. Tipifarnib Analogues with X Group Modifications
is not required for potent inhibition of T. cruzi growth and
that the ortho methyl group is important for reducing binding
to protein farnesyltransferase. However, compound 4 still
exists as a pair of stable rotamers, so it does not constitute
the solution that we require. The same picture emerges with
compound 5, which has an ortho CF3 instead of Me.
Wemadecompounds thatlack the orthomethylgroup(and
thus the rotamer problem) but contain replacements for 3-
chloro group hoping to reduce binding to protein farnesyl-
transferase while retaining potency on T. cruzi. Replacing Cl
with F, Me, or CF3 reduced affinity to protein farnesyltrans-
ferase by ∼100- to 2000-fold while retaining good potency on
T. cruzi (see 6-8, Table 1). This result shows that the Cl
substituent gives an optimal fit in the active site of protein
farnesyltransferase. This is apparent from the X-ray structure
of 1 bound to protein farnesyltransferase.
substantial loss of binding to protein farnesyltransferase, with
11 showing the largest loss.
We also reasoned that addition of a non-hydrogen group to
the ortho position is acceptable if both ortho positions are
substituted, since the two rotamers are chemically identical in
this case. Compound 14 contains a 2,6-dimethylphenyl ring,
and like the 2-methylphenyl-containing compound 2, it is
devoid of protein farnesyltransferase inhibition activity and dis-
plays good potency for killing parasites. Compounds 15 and
16 display similar properties as 14. The 2,6-difluorophenyl
compound 16 is the most potent in the series against T. cruzi
with an EC50 of 0.3 nM. This compound is as potent as
posaconazole against T. cruzi in our in vitro amastigote killing
assay. These are the most potent anti-T. cruzi compounds that
we are aware of.
Compound 17 contains a 3,5-dimethyphenyl group. It is
highly potent against parasites and displays a ∼600-fold
reduction in affinity for protein farnesyltransferase compared
to 1. In our previous study we showed that the tipifarnib
analogue with a β-naphthyl replacing the 4-chlorophenyl
group of 1 displayed ∼20-fold less affinity to protein farnesyl-
transferase compared to 1 and displayed an EC50 against
T. cruzi of 45 nM. Given the lowering of EC50 and the loss of
protein farnesyltransferase affinity when the amino group of 1
is replaced with OMe, we prepared compound 18. Indeed this
compound now is a very weak inhibitor of protein farnesyl-
transferase, and the EC50 for killing T. cruzi drops to 2 nM.
Table 2 shows the effect of a wider variation in substitution
of the amino group of 1. Replacing NH2 with OH produces
only a slight reduction in protein farnesyltransferase affinity.
Presumably the OH group can still engage in the active site
hydrogen bond network. Replacement of the NH2 group
with OMe, OEt, OPr, and NHMe has a much more dramatic
effect. Modeling shows that the NH2 group does not contact
Tc-L14DM residues. It is quite likely that the improved
Next we made compound 9, which contains an ortho F
instead of the ortho Me, hoping that the smaller F would not
sterically interfere with the vinylic hydrogen. Compound 9 is
similar to 2 in that it binds much more weakly to protein
farnesyltransferase compared to 1 and is superior to 1 in its
ability to kill T. cruzi. However, 9 was found to exist as stable
rotamers that are distinguished in the 1H NMR spectrum (not
shown). The NMR peaks coalesce when the temperature is
raised to 75 °C (not shown); however, the rotamers persist at
physiological temperature.
We made compound 10, which lacks an ortho substituent
(so no rotamers are possible) and also lacks the 3-chloro
group, which is important for binding to protein farnesyl-
transferase. This compound ranks among the most potent of
our compounds against T. cruzi and displays an intermediate
loss in affinity for protein farnesyltransferase (∼500-fold).
Compounds 11-13 lack ortho substituents and contain a
single non-hydrogen substituent at the para position. All 3
compounds show good potency for killing T. cruzi and a

3892 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Kraus et al.
Table 3. Tipifarnib Analogues with Imidazole and X Group Modifications
Table 4. Tipifarnib Analogues with Additional Ring 1 and Ring 2 Modifications
potency for killing T. cruzi when NH2 is replaced with OMe is
the result of better penetration of the more hydrophobic
compound across host cell and parasite membranes. This
was our motivation for preparing OEt, OPr and NHEt ana-
logues. However, OMe remains as the best group in terms of
EC50 values. We attempted to prepare the analogue contain-
ing N(Me)2, but itwas unstablepresumablybybreaking down
in an SN1 type reaction.
is superior to 3 in that it binds ∼1500-fold weaker to protein
farnesyltransferase than 1. Again, compounds with the OMe
group are superior against T. cruzi. Compound 26 also lacks
rotamers (no ortho substituent).
We made additional modifications to 1 to further test the
validity of our homology model (Figure 2), and the results are
summarized in Table 4. Modeling shows that the Cl of the
4-chlorophenyl group of 1 fills the active site well. It can be
seen that replacement of this Cl with Me and CF3 is tolerated,
but the larger groups Et and i-Pr lead to less potent com-
pounds against T. cruzi. Modeling also suggests that the Cl of
Table 3 shows data for compounds in which the imidazole
N-Me is replaced with N-Et. Compound 26 is as potent
against T. cruzi as the N-Me comparator, compound 3, but

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3893
enzymes,10 and we also observed relatively weak inhibi-
tion against CYP3A4 (IC50=4060 nM). The tipifarnib ana-
logues designed in these studies had either approximately
micromolar level IC50 values (10-12) or somewhat more
potent activity as observed with 16 (Table 5). It is encoura-
ging that the CYP3A4 inhibition by some of the tipifarnib
analogues is considerably less than for the azole drugs.
In future studies, it will be necessary to profile the com-
pounds against additional liver microsomal enzymes to
more fully establish their potential to cause drug-drug
interactions.
Conclusions
Figure 3. Difference spectra for the binding of 16 to recombinant
Tc-14DM. Compound 16 was added at 200 nM to 2 μM with a Tc-
14DM concentration of 1.4 μM.
In this continued study of analogues of the cancer drug
clinical candidate tipifarnib, 1, as inhibitors of Tc-L14DM
and as anti-T. cruzi agents, we have developed 20 new
compounds that display values of EC50 for killing T. cruzi
of <10 nM and 7 new compounds that display values of
EC50<1 nM. Many of these compounds either fail to bind
to mammalian protein farnesyltransferase or bind with >100-
fold reduced affinity. In this study we have solved the rotamer
problem that we encountered in our earlier study of tipifarnib
analogues containing an ortho substituted phenyl group. The
availability of a large set of highly potent anti-T. cruzi
compounds sets the stage for the next steps of drug develop-
ment which include pharmacokinetic, efficacy, and toxicology
studies.
Table 5. In Vitro Inhibition of Cytochrome P450 CYP3A4 Isoform
compd
IC50 (nM)
SEM (nM)
tipifarnib
10
4060
2345
923
1628
275
82
338.5
847.5
212
594
57
11
12
16
posaconazole
ketoconazole
13
0.5
11
the 3-chlorophenyl group fills the active site of Tc-L14DM
well (Figure 2). Replacement of this Cl with larger groups, Ph
and Bn, lead to a dramatic reduction in potency against
T. cruzi parasites.
Experimental Section
Biological and Modeling Studies. Assays of rat protein farne-
syltransferase to obtain the IC50 for inhibitors and of T. cruzi
amastigote growth to obtain EC50 values for the inhibitors were
carried out as described.9 Binding of inhibitors to recombinant
Tc-L14DM was carried out by difference optical spectroscopy
as described.11 Molecular modeling studies were carried out as
described.6 EC50 values reported in the tables have standard
errors of less than 50% based on duplicate or triplicate indepen-
dent determinations.
Inhibition of recombinant human CYP3A4 enzyme was
determined using a commercial kit (CYP3A4/BFC high through-
put inhibitor screening kit, GenTest, Inc.) following the manu-
facturer’s instructions. The compounds were tested in serial
dilutions in duplicate with the IC50 values calculated by non-
linear regression analysis using the Prism software (GraphPad
Software, Inc.). The assay was performed twice, and average
IC50 values are shown. Ketoconazole was the positive control
and gave results within the expected range provided by the
manufacturer.
Chemistry. Starting materials were purchased from Aldrich,
Acros, Alfa-Aesar, EMD, Fisher, Lancaster, Mallinckrodt,
TCI-America, or VWR and used without further purification
unless otherwise specified. N-Methylimidazole (M50834) was
purchased from Sigma-Aldrich and distilled at reduced pressure
(10 mmHg, bp: 67-69 °C) after being stirred over sodium at
room temperature overnight. Solvents were purified using a J.C.
Meyer type solvent dispensing system utilizing Al2O3 and/or
copper cartridges, depending on the particular solvent. n-BuLi
was titrated with diphenylacetic acid prior to use. Nitrogen gas
used in reactions requiring an inert atmosphere was house-
supplied and run through Dri-Rite desiccant. Glassware for
distillation and water sensitive reactions was flame-dried under
vacuum or dried in an oven. Silica was EMD silica gel 60, 40-
63 μm (11567-1). TLC plates were aluminum backed EMD silica
gel 60 F254 (5554/7). 1H NMR spectra were recorded on a Bruker
Avance AV300. ESI-MS were recorded on a Bruker Esquire ion
trap mass spectrometer. Purification of all final compounds was
We tested our most potent anti-T. cruzi compound, 16, for
binding to recombinant Tc-L14DM in vitro. As shown in
Figure 3, when 1.4 μM Tc-L14DM is titrated with 0.2-2 μM
16, the visible absorption spectrum of the heme Soret band
undergoes a spectral shift (as seen in the difference spectrum,
Figure 3). The spectral features strongly suggest that the
imidazole nitrogen of 16 coordinates directly to the heme
iron. When the difference spectrum is quantified as a function
of the equivalents of 16 added to the enzyme, it is found that
the change in the spectrum ceases when 1 enzyme equivalent
of inhibitor is added (not shown). This shows that the
equilibrium dissociation constant for the enzyme-inhibitor
complex, Kd, is much less than the enzyme concentration of
1.4 μM. From this result we can say that16 binds tightly to Tc-
L14DM, but we cannot determine the value of Kd precisely. It
is for this reason that we prefer to rank our tipifarnib
analogues based on values of EC50 for killing T. cruzi amas-
tigotes (Tables 1-4). EC50 is the most important parameter
for ourdrug discovery effort, sincethe compoundnot only has
to bind to the target enzyme but also has to cross the host cell
and parasite membranes.
As these tipifarnib analogues contain an imidazole ring,
there is some concern that they may be potent inhibitors of
hepatic cytochrome P450s. We tested some of our best
compounds in the series for inhibition of the major drug
metabolizing enzyme, cytochrome P450 CYP3A4 isoform,
using aninvitroassay. The well established CYP3A4inhibitor
ketoconazole was a highly potent inhibitor with an IC50
measured at 11 nM (Table 5). We also tested posaconazole
because it is being considered for clinical testing as an anti-
Chagas disease drug. This compound was the next most
potent inhibitor (IC50 = 82 nM). Tipifarnib was previously
reported to be a weak inhibitor (>10000 nM) of P450

3894 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Kraus et al.
by reverse phase HPLC utilizing an octadecylsilane stationary
phase and a water-to-methanol gradient with 0.08% v/v trifluoro-
acetic acid, (TFA). HPLC was carried out on a Varian Pro-Star
fitted with a Waters YMC Pack-ODS-A column (2 cm ꢀ 10 cm)
running at 12 mL/min using a detector excitation wavelength of
254 nm. All target compounds possessed a purity of g95% as
verified by HPLC.
Methanone Synthesis (Route 1). 4-Chloro-N-methoxy-N-
methylbenzamide. An amount of 20.0 g (0.128 mols) of 4-
chlorobenzoic acid (156.57 g/mol) was placed in a 500 mL round
bottomed flask. Then 120 mL of thionyl chloride was added,
and the mixture was refluxed overnight. Thionyl chloride was
removed under reduced pressure to produce a red oil. Anhy-
drous toluene was added and then removed under reduced
pressure two times. The crude product was dissolved in 200 mL
of anhydrous CH2Cl2. An amount of 13.73 g (0.140 mol) of N,O-
(1-methyl-1H-imidazol-5-yl)methanone. 1H NMR (300 MHz,
CDCl3, δ): 7.81 (m, 2H), 7.75 (s, 1H), 7.58 (s, 1H), 7.50 (m, 2H),
4.03 (s, 1H). ESI-MS m/z 221.4 (M þ Hþ)þ. MW: 234.68 g/mol.
Methanone Synthesis (Route 2). 1-Triphenylmethyl-4-imida-
zole Carboxaldehyde. To a 1 L three-necked round-bottomed
flask with an addition funnel was added 3H-imidazole-4-car-
baldehyde (12 g, 0.125 mol, Aldirch), trityl chloride (38.3 g,
0.137 mol) and acetonitrile (400 mL). The mixture was stirred
at room temperature to give a slurry. Triethylamine (30 mL,
0.125 mol) was added dropwise over 20 min. After the addition
was complete, the reaction mixture was stirred at room tempe-
rature for 20 h. Hexane (40 mL) and water (400 mL) were added.
The slurry was stirred for 30 min and filtered. The cake was
washed with water (3ꢀ100 mL) and dried in an oven at 50 °C for
20 h to give 2 as a white solid. (39.8 g, 94%). 1H NMR (CDCl3,
δ): 9.81 (s, 1H), 7.54 (s, 1H), 7.46 (s, 1H), 7.20-7.29 (m, 10H),
7.09-7.04 (m, 5H). MS m/z 339.4 (M þ H)þ. MW: 338.4.
p-Tolyl-(1-trityl-1-H-imidazol-4-yl)methanone. An oven-dried
250 mL round-bottom flask fitted with reflux condenser was
sealed with a rubber septum under argon atmosphere and
charged with magnesium turnings (1.12 g, 46.7 mmol) and a
pinch of iodine. Anhydrous ether (50 mL) was added, and the
mixture was stirred for 5 min at room temperature. To this
mixture p-bromotoluene (5 g, 29.2 mmol) was added dropwise
in anhydrous ether (10 mL) slowly, at a rate to allow the ether to
barely reflux. The mixture was then refluxed for 30 min and
cooled to 0 °C. 1-Triphenylmethyl-4-imidazole carboxaldehyde
(7.9 g 23.3 mmol) was dissolved in anhydrous ether (20 mL) and
added dropwise with continuous stirring. The reaction mixture
was stirred for 3 h at room temperature and then refluxed for 1 h.
The reaction mixture was cooled and poured into 50 mL of ice
cold 1 M HCl. The phases were separated, and the aqueous phase
was extracted with ether (2ꢀ50 mL). The combined ether layers
were washed with saturated NaHCO3 solution, and the resulting
ether layer was kept in an ice cold bath for 3 h and then filtered.
The resulting white solid (9 g) was transferred to a 200 mL round
bottomed flask fitted with a reflux condenser. The solid was
suspended in 1,4-dioxane (50 mL) and stirred for 5 min, and then
MnO2 (9 g, 104 mmol) was added. The resulting mixture was
refluxed for 3 h and cooled to room temperature and diluted with
CH2Cl2 (50 mL) and then filtered through a Celite pad. Solvent
was removed under reduced pressure toproduce 8.45 g ofproduct
dimethylhydroxylamine HCl (97.54 g/mol) was added. Then
3
52 mL (0.640 mol) of anhydrous pyridine was added over a
period of 10 min. The mixture was stirred under nitrogen at
room temperature overnight. Volatiles were removed under
reduced pressure. The solid was partitioned between CHCl3
and water. The organic phase was washed with brine, then
collected and dried over anhydrous magnesium sulfate. The
solvents were removed to produce a red oil which was purified
on silica with 5% MeOH/CH2Cl2 as eluent. An amount of 22.9 g
of product was produced, yield 90%. 1H NMR (300 MHz,
CDCl3, δ): 7.66 (m, 2H), 7.38 (m, 2H), 3.54 (s, 3H, -OMe), 3.36
(s, 3H, -NMe). ESI-MS m/z 200.4 (M þ Hþ)þ. MW: 199.63 g/mol.
(4-Chlorophenyl)(1-methyl-1H-imidazol-5-yl)methanone. A
flame-dried 125 mL round-bottom flask was charged with a stir
bar and overpressurized with dry nitrogen gas and 3.0 mL
(37.6 mmol) of freshly distilled N-methylimidazole (82.11 g/
mol, 1.035 g/mL). The flask was sealed with a new rubber
septum, and 30 mL of anhydrous THF was added. The solu-
tion was stirred for about 10 min, and then the temperature was
lowered to -78 °C and stirred for an additional 10 min. Then
16.2 mL (41.3 mmol) of freshly titrated n-BuLi (2.5 M in
hexanes) was added dropwise through the septum over a period
of 10 min under an overpressure of dry nitrogen. A slight color
change to pale yellow was observed. The reaction mixture was
allowed to stir at this temperature for 45 min. Then 6.3 mL
(37.6 mmol) of 99% chlorotriethylsilane (Et3SiCl) was added
dropwise over 5 min. The mixture was allowed to stir for 1 h at
-78 °C, at which point 15.0 mL (37.6 mmol) of freshly titrated
n-BuLi (2.5 M in hexanes) was added dropwise through the
septum over a period of 10 min and allowed to stir at -78 °C for
an additional 45 min. A separate flask was flame-dried and
charged with 5.0 g (25.1 mmol) 4-chloro-N-methoxy-N-
methylbenzamide (199.63 g/mol) and sealed with a rubber
septum. A total of 15 mL of anhydrous THF was added, and
the mixture was stirred at room temperature for 10 min and
then at the appropriate time transferred via cannula to the flask
containing the in situ generated C-2 triethylsilyl protected
N-methylimidazole at a slow rate to maintain low temperature
as indicated by the slow sublimation of CO2. The mixture was
left to stir overnight and became deep red. The reaction was
quenched by the addition of 1 M HCl until the pH of the
aqueous phase was no longer basic as indicated by litmus
paper, then allowed to stir for 1 h. The pH of the aqueous
phase was adjusted to above 8 with 1.5 M NaOH, and the
mixture was partitioned between CHCl3 and water. The or-
ganic phase was washed with brine and dried with anhydrous
MgSO4. Solvents were removed under reduced pressure to
produce a reddish solid. Product was purified by recrystalliza-
tion from CH2Cl2 to produce 4.24 g of fluffy golden crystals,
76.6% yield. 1H NMR (300 MHz, CDCl3, δ): 7.83 (d, J =
8.4 Hz, 2H), 7.68 (s, 1H), 7.59 (s, 1H), 7.50 (d, J=8.4 Hz, 2H),
4.03 (s, 1H). ESI-MS m/z 221.4 (M þ Hþ)þ. MW: 220.65 g/mol.
(4-Chlorophenyl)(1-ethyl-1H-imidazol-5-yl)methanone. The com-
pound was prepared in a manner analogous to (4-chlorophenyl)-
1
as a crispy solid, yield 94.4%. H NMR (300 MHz, CDCl3, δ):
8.12 (d, J=8.1 Hz, 2H), 7.69 (s, 1H), 7.54 (s, 1H), 7.43-7.34 (m,
10H), 7.25 (d, J=8.1 Hz, 2H), 7.20-7.12 (m, 5H), 2.55 (s, 3H).
ESI-MS m/z 429.2 (M þ H)þ. MW: 428.56 g/mol.
(3-Methyl-3H-imidazol-4-yl)-p-tolylmethanone. A flask was
charged with 5.0 g of the previous compound (11.6 mmol) under
nitrogen atmosphere which was suspended in 40 mL of anhy-
drous CH2Cl2 and stirred rapidly for 10 min. A solution of
methyltrifluoromethane sulfonate (1.95 mL, 17.5 mmol) in
anhydrous CH2Cl2 (10 mL) was added dropwise over 10 min,
and the reaction mixture was stirred at room temperature for
20 h. The volume of the mixture was reduced to about 1/3 of its
original volume, and to this was added 30 mL of hexane. The
slurry was stirred for 30 min and filtered. The cake was washed
with hexane and dissolved in 30 mL of 1:2 water/acetone
mixture and stirred for 3 h. The reaction mixture was filtered
to remove trityl alcohol, and the cake was washed with water
(2 ꢀ 10 mL). The filtrate was concentrated under reduced pres-
sure to remove acetone, and the resulting slurry was filtered
again to remove traces of trityl alcohol and the cake washed with
water (10 mL). The filtrate was washed with saturated NaHCO3
and extracted with CH2Cl2 (2 ꢀ 40 mL). Combined organic
layers were dried with MgSO4. Solvent was removed under
reduced pressure to produce 2.09 g of product as a pulpy white
solid, yield 89.6% 1H NMR (300 MHz, CDCl3, δ): 7.78 (d, J=
8.1 Hz, 2H), 7.64 (s, 1H), 7.59 (s, 1H), 7.31 (d, J=8.1 Hz, 2H),
4.02 (s, 3H), 2.45(s, 3H). ESI-MS m/z 201.1 (M þ H)þ. MW:
200.24 g/mol.

Article
(3-Methyl-3H-imidazol-4-yl)(4-trifluoromethylphenyl)methanone.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3895
volatiles were removed under reduced pressure. Approxi-
mately 50 mL of toluene was added and removed at reduced
pressure two times. The crude product was set aside. A
separate flask was fitted with a stir bar and a septum and
loaded with 24.7 g (220 mmol, 6 equiv) of 95% t-BuOK
powder. This was suspended in 120 mL of 1,2-dimethoxy-
ethane and stirred for about 10 min. The temperature was
lowered to 0 °C. The crude product set aside previously was
dissolved in 45 mL of 1,2-dimethoxyethane and then trans-
ferred dropwise by cannula to the flask containing the t-BuOK
suspension. The color of the solution changed to yellow, and
the mixture was allowed to stir under an inert atmosphere
overnight. At this point the volaties were removed under
reduced pressure, and the resulting paste was suspended in
∼300 mL of water. The solid was collected by filtration and
used in the next step without purification. An amount of 8.4 g
(24.10 mmol) of product was produced as a fluffy, white solid,
yield 66%. 1H NMR (300 MHz, CD3OD, δ): 7.62 (dd, J =
8.7 Hz, 1.8 Hz, 1H), 7.53 (d, J=7.8 Hz, 1H), 7.39 (d, J=8.7 Hz,
1H), 7.30 (m, 1H), 7.21 (d, J=1.8 Hz, 1H), 7.11 (d, J=7.2 Hz,
1H), 6.62 (s, 1H), 2.17 (s, 3H). ESI-MS m/z 348.3 (M þ Hþ)þ.
MW: 348.62 g/mol.
The compound was prepared in a manner analogous to the
above methanone. 1H NMR (300 MHz, CDCl3, δ): 7.95 (d, J=
8.7 Hz, 2H), 7.78 (d, J=8.7 Hz, 2H), 7.69 (s, 1H), 7.58 (s, 1H),
4.03 (s, 3H). ESI-MS m/z 255.4 (M þ H)þ. MW: 254.21 g/mol.
(4-Ethylphenyl)(3-methyl-3H-imidazol-4-yl)methanone. The
compound was prepared in a manner analogous to the above
methanone. 1H NMR (300 MHz, CDCl3, δ): 7.80 (d, J =
9.0 Hz, 2H), 7.64 (s, 1H), 7.59 (s, 1H), 7.32 (d, J = 9.0 Hz,
2H), 4.01 (s, 3H), 2.75 (q, J=3.3 Hz, 1.8 Hz, 2H), 1.28 (t, J=
7.2 Hz, 3H). ESI-MS m/z 215.1 (M þ H)þ. MW: 214.26 g/mol.
(4-Isopropylphenyl)(3-methyl-3H-imidazol-4-yl)methanone. The
compound was prepared in a manner analogous to the above
methanone. 1H NMR (300 MHz, CDCl3, δ): 7.82 (d, J=8.6 Hz,
2H), 7.65 (s, 1H), 7.61 (s, 1H), 7.36 (d, J=8.6 Hz, 2H), 4.02 (s, 3H),
3.0 (m, 1H), 1.33 (d, J=6.9 Hz, 6H). ESI-MS m/z 229.1 (M þ H)þ.
MW: 228.29 g/mol.
2-Amino-5-bromo-N-methoxy-N-methylbenzamide. A 2 L flask
was charged with 25.0 g (103.3 mmol) of 5-bromoisatoic anhydride
(242.03 g/mol) and 15.1 g (155 mmol) of N,O-dimethylhydroxyl-
amine hydrochloride (97.54 g/mol). The solids were suspended in
500 mL of anhydrous CH2Cl2 and stirred rapidly. Then 37.5 mL of
pyridine (465 mmol) was added slowly, and the mixture was
allowed to stir until all solids had dissolved, about 36 h. The crude
mixture was partitioned between CHCl3 and water. The organic
phase was washed with brine and dried with anhydrous MgSO4.
Solvents were removed under reduced pressure to produce a gold
oil which crystallized upon standing. Product was triturated with
hexanes, filtered, then used without further purification. An
amount of 23.9 g of a lightly colored crystalline product was
6-Bromo-4-(3-chloro-2-methylphenyl)-2-methoxyquinoline. A
flame-dried 25 mL flask was fitted with a stir bar and charged
with 500 mg (1.43 mmol) of 6-bromo-4-(3-chloro-2-methylphe-
nyl)quinolin-2(1H)-one and 423 mg (2.86 mmol, 2 equiv) of
BF4OMe3 and then sealed with a septum. The solids were
suspended in 5.5 mL of anhydrous CH2Cl2 and stirred for
20 h, at which time 5.5 mL of 1 M aqueous NaOH was added.
The mixture was stirred for about 3 h, then partitioned between
CHCl3 and water. The organic was washed three times with
brine, then dried with MgSO4. Solvents were removed under
reduced pressure to produce a clear yellow oil which crystallized
upon standing. This was purified on silica with 50:50 CH2Cl2/
hexane to produce 329 mg of product as a flaky yellow-green
1
produced, 89% yield. H NMR (300 MHz, CDCl3, δ): 7.52 (d,
J=2.4 Hz, 1H), 7.28 (dd, J=2.4 Hz, 8.1 Hz 1H), 6.61 (d, J=8.7 Hz,
1H), 4.69 (s (broad), 2H), 3.61 (s, 3H), 3.36 (s, 3H). ESI-MS m/z
283.1 (M þ Naþ)þ. MW: 259.10 g/mol.
(2-Amino-5-bromophenyl)(3-chloro-2-methylphenyl)methanone.
A 250 mL flask was flame-dried and charged with a stir bar and a
rubber septum. 2-Bromo-6-chlorotoluene (6 mL, 45.9 mmol)
was added to the flask and dissolved in 60 mL of anhydrous
THF under an atmosphere of dry nitrogen. The solution was
stirred for about 5 min and then cooled to -78 °C and then
stirred for about 10 min. An amount of 18 mL (45.9 mmol, 1
equiv) of n-BuLi (2.5 M in hexanes) was added dropwise at a rate
such that temperature of the reaction remained close to -78 °C,
as indicated by the slow sublimation of CO2. The solution was
allowed to stir for 20 min. Then 5.95 g (22.95 mmol, 0.5 equiv) of
2-amino-5-bromo-N-methoxy-N-methylbenzamide was added
to a separate flask and dissolved in 60 mL of anhydrous THF.
After being stirred for about 5 min, this solution was added
dropwise by canula to the flask containing the in situ generated
aryllithium. The solution was allowed to stir at this temperature
for 2 h, at which time the cooling bath was removed allowing the
flask to rise to room temperature. An amount of 50 mL of 1 M
aqueous HCl was added, and the biphasic mixture was allowed
to stir for 30 min. The mixture was partitioned between CHCl3
and water. The organic phase was washed three times with
saturated aqueous NaHCO3, then separated and dried with
MgSO4. Solvents were removed under reduced pressure to
produce an orange-brown oil. This was purified on silica with
20% EtOAc/hexanes to produce 6.3 g of yellow crystalline
1
solid, yield 63%. H NMR (300 MHz, CDCl3, δ): 7.79 (d, J=
9.0 Hz, 1H), 7.69 (dd, J=8.7 Hz, 2.3 Hz, 1H), 7.50 (dd, J=7.8
Hz, 1.2 Hz, 1H), 7.40 (d, J=2.1 Hz, 1H), 7.26 (m, 1H), 7.09 (dd,
J=7.5 Hz, 1.2 Hz, 1H), 6.77 (s, 1H), 4.10 (s, 3H), 2.08 (s, 3H).
ESI-MS m/z 362.3 (M þ Hþ)þ. MW: 362.65 g/mol.
4-(3-Chloro-2-methylphenyl)-6-((4-chlorophenyl)(hydroxy)(1-
methyl-1H-imidazol-5-yl)methyl)-2-methoxyquinoline. A flame-
dried flask was charged with a stir bar and 4.2 g (11.58 mmol) of
6-bromo-4-(3-chloro-2-methylphenyl)-2-methoxyquinoline and
sealed with a rubber septum. An amount of 20 mL of anhydrous
THF was added and the solution stirred at room temperature
for 10 min. The temperature was lowered to -78 °C, and the
mixture was stirred for another 10 min. Then 5.0 mL (1.1 eq
12.7 mmol) of 2.5 M n-BuLi was added dropwise accompanied
by a color shift to dark yellow-orange. This was allowed to stir
for 20 min. A separate flask was flame-dried and charged with
2.8 g of (4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methanone
(1.1 equiv, 12.7 mmol). This was dissolved in 55 mL of THF
and added to the flask containing quinoline in three increments
by syringe, rapidly dropwise over 10 min. The mixture was
stirred overnight and allowed to warm slowly to room tempera-
ture, and the color shifted to gold. The reaction was quenched by
addition of 1 volume of a saturated aqueous solution of NH4Cl.
This was partitioned between 1 M NH4OH and CHCl3. The
organic phase was collected and solvents were removed under
reduced pressure to produce a foamy, white semisolid. This was
purified on silica with a mobile phase consisting of MeOH/CH2Cl2,
1:10 v/v, to produce 3.52 g (6.91 mmol) of product, 60% yield. TLC
(CH2Cl2/MeOH, 9:1 v/v): Rf=0.45. 1H NMR (300 MHz, CDCl3,
δ): 7.86 (m, 1H), 7.59 (m, 1H), 7.43 (m, 1H), 7.20 (m, 6H), 7.01
(m, 2H), 6.79 (s, 1H), 6.17 (m, 1H), 4.12 (s, 3H), 3.30 (m, 3H), 1.90
(m, 3H). ESI-MS m/z 504.3 (M þ Hþ)þ. MW: 504.41 g/mol.
4-(3-Chloro-2-methylphenyl)-6-((4-chlorophenyl)(hydroxy)(1-
methyl-1H-imidazol-5-yl)methyl)quinolin-2(1H)-one. An amount
of 3.5 g (6.91 mmol) of the previous compound was dissolved in
1
product, yield 85%. H NMR (300 MHz, CDCl3, δ): 7.47 (d,
J=7.8 Hz, 1H), 7.36 (dd, J=8.7 Hz, 2.1 Hz, 1H), 7.25 (d, J=
2.1 Hz 1H), 7.21 (m, 2H), 7.11 (d, J=7.5 Hz 1H), 6.64 (d, J=
8.7 Hz, 1H), 6.49 (s (broad), 2H), 2.27 (s, 3H). ESI-MS m/z 324.3
(M þ Hþ)þ. MW: 324.60 g/mol.
6-Bromo-4-(3-chloro-2-methylphenyl)quinolin-2(1H)-one. A
250 mL flask was fitted with a stir bar, and 11.9 g of (2-amino-
5-bromophenyl)(3-chloro-2-methylphenyl)methanone was added.
A water condenser was attached, and then 50 mL of anhydrous
toluene and 24.3 mL (257 mmol, 7 equiv) of acetic anhydride
were added. The solution was heated to reflux for 6 h. Then

3896 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Kraus et al.
15 mL of THF. Then 30 mL of 6 N HCl (25 equiv) was added
dropwise. The flaskwas fittedwitha water-cooledcondenser, and
the mixture was stirred at 60 °C for 5 h. Most of the THF was
removed by a stream of nitrogen, and the heterogeneous mixture
that remained was made basic with excess aqueous 1 M NH4OH
and then partitioned between water and CHCl3. The organic
was dried with MgSO4, and solvents were removed at reduced
pressure. Product was purified on silica with a mobile phase con-
sisting of MeOH/CH2Cl2, 1:10 v/v, to produce 2.1 g (4.28 mmol)
of product as a white solid, 62% yield. TLC (CH2Cl2/MeOH, 9:1
v/v): Rf=0.30. 1H NMR (300 MHz, CD3OD, δ): 7.68 (m, 1H),
7.59 (m, 1H), 7.47 (m, 2H), 7.28 (m, 3H), 7.12 (m, 3H), 6.73 (m,
1H), 6.51 (m, 1H), 6.1 (m, 1H), 3.41 (m, 3H), 1.96 (m, 3H). ESI-
MS m/z 490.4 (M þ Hþ)þ. MW: 490.38 g/mol.
546.6 (M þ Hþ)þ. MW: 546.49 g/mol. Mono-TFA salt FW:
660.51 g/mol.
4-(3-Chloro-2-methylphenyl)-6-((4-chlorophenyl)(methylamino)-
(1-methyl-1H-imidazol-5-yl)methyl)-1-methylquinolin-2(1H)-one
(23). An amount of 10.0 mg (0.02 mmol) of 4-(3-chloro-2-
methylphenyl)-6-((4-chlorophenyl)(hydroxy)(1-methyl-1H-imidazol-
5-yl)methyl)-1-methylquinolin-2(1H)-one was dissolved in 5 mL
of SOCl2. The flask was covered with aluminum foil, and the
reaction mixture was stirred overnight. SOCl2 was removed at
reduced pressure to produce a whitish solid. A total of 10 mL of
THF was added. Stirring was done with the flask open, and
anhydrous methylamine was bubbled through the solution for a
period of 30 min. Then the flask was sealed and stirred overnight
under an overpressure of methylamine. Solvents were removed
at reduced pressure, and the crude mixture was dissolved in 1 mL
of MeOH and centrifuged. The supernatant was collected and
injected onto the HPLC column. Product was purified using a
water-methanol gradient with 0.08% v/v trifluoroacetic acid:
0-5 min 20% MeOH, 5-25 min 20-65% MeOH, 25-30 min
65-100% MeOH. Product elutes at 24.4 min, and 1.4 mg
(0.0018 mmol) was produced as a bis-TFA salt. Yield 9%.
TLC (CH2Cl2/MeOH, 9:1 v/v): Rf=0.55. 1H NMR (300 MHz,
CDCl3, δ): 8.86 (m, 1H), 7.82 (m, 2H), 7.54 (m, 1H), 7.41-7.27
(m, 6H), 7.15 (m, 1H), 7.00 (m, 1H), 6.64 (s, 1H), 3.86 (m,
3H), 3.57 (m, 3H), 2.16 (m, 3H), 2.01 (m, 3H). ESI-MS m/z 517.5
(M þ Hþ)þ. MW: 517.45 g/mol. Bis-TFA salt FW: 745.5 g/mol.
4-(3-Chloro-2-methylphenyl)-6-((4-chlorophenyl)(hydroxy)(1-
methyl-1H-imidazol-5-yl)methyl)-1-methylquinolin-2(1H)-one (20).
The title compound was injected onto the HPLC column using a
water-methanol gradient with 0.08% v/v trifluoroacetic acid:
0-5 min 20% MeOH, 5-25 min 20-65% MeOH, 25-30 min
65-100% MeOH. Product elutes at 23.9 min and was produced
as a mono-TFA salt. TLC (CH2Cl2/MeOH, 9:1 v/v): Rf=0.55.
1H NMR (300 MHz, CD3OD, δ): 8.94 (m, 1H), 7.86 (dd, J=9.0,
2.1 Hz, 1H), 7.79 (m, 2H), 7.51 (m, 1H), 7.37 (m, 2H), 7.31-7.18
(m, 3H), 7.09 (m, 1H), 6.85-6.76 (m, 2H), 6.62 (m, 1H), 3.85 (s,
3H), 3.65 (m, 3H), 1.96 (m, 3H). ESI-MS m/z 504.6 (M þ Hþ)þ.
MW: 504.41 g/mol. Mono-TFA salt FW: 618.43 g/mol.
4-(3-Chloro-2-methylphenyl)-6-((4-chlorophenyl)(hydroxy)(1-
methyl-1H-imidazol-5-yl)methyl)-1-methylquinolin-2(1H)-one.
An amount of 2.1 g (4.28 mmol) of the previous compound was
added to a 100 mL flask and dissolved in 30 mL of THF. Then
487 mg (0.5 eq., 2.14 mmol) of benzyltriethylammonium chlor-
ide was added as a phase transfer catalyst. A total of 25.5 mL of
40% wt NaOH (120 equiv, 17.1 g) was added and the mixture
allowed to stir for approximately 10 min. Then 375 μL (1.4
equiv, 6 mmol) of CH3I was added, and the mixture was allowed
to stir overnight. The THF was removed at reduced pressure,
and the product was partitioned between CHCl3 and 1 M
NH4OH. The product was purified on silica with a mobile phase
consisting of MeOH/CH2Cl2, 1:10 v/v, to produce 2.0 g (3.97
mmol) of product as a colorless semisolid, 59% yield. TLC
(CH2Cl2/MeOH, 9:1 v/v): Rf = 0.45. 1H NMR (300 MHz,
CDCl3, δ): 7.80 (m, 1H), 7.72 (m, 1H), 7.63 (m, 1H), 7.49 (m,
1H), 7.29 (m, 3H), 7.12 (m, 3H), 6.78 (m, 1H), 6.58 (m, 1H), 6.14
(m, 1H), 3.85 (s, 3H), 3.41 (m, 3H), 1.95 (m, 3H). ESI-MS m/z
504.3 (M þ Hþ)þ. MW: 504.41 g/mol.
4-(3-Chloro-2-methylphenyl)-6-((4-chlorophenyl)(ethoxy)(1-
methyl-1H-imidazol-5-yl)methyl)-1-methylquinolin-2(1H)-one (21).
A total of 10.0 mg (0.02 mmol) of the previous compound was
dissolved in 10 mL of EtOH, and approximately 15 mg of tosic
acid was added. The mixture was heated to reflux for 48 h. TLC
analysis indicated only one major product and complete con-
version of starting material. Solvents were removed under
reduced pressure to produce a colorless, oily semisolid. Product
was purified by HPLC using a water-methanol gradient with
0.08% v/v trifluoroacetic acid: 0-5 min 20% MeOH, 5-25 min
20-65% MeOH, 25-30 min 65-100% MeOH. Product elutes
at 27.9 min, and 4.0 mg (0.0062 mmol) was produced as a mono-
TFA salt. Yield 31%. TLC (CH2Cl2/MeOH, 9:1 v/v): Rf=0.55.
1H NMR (300 MHz, CDCl3, δ): 8.98 (s, 1H), 7.85 (m, 1H), 7.75
(m, 1H), 7.57 (m, 1H), 7.52 (m, 1H), 7.35 (m, 5H), 7.17 (m, 1H),
7.12 (m, 1H), 6.63 (m, 1H), 3.82 (m, 3H), 3.52 (m, 3H) 2.05 (s,
3H), 1.13 (m, 3H). ESI-MS m/z 533.6 (M þ Hþ)þ. MW: 532.46
g/mol. Mono-TFA salt FW: 646.45 g/mol.
6-Bromo-N-(E)-2,6-difluorocinnamoylanilide. An amount of
15.0 g (81.5 mmol) of (E)-2,6-difluorocinnamic acid was dis-
solved in 25 mL of SOCl2, and the mixture was heated to reflux
and stirred overnight. Thionyl chloride was removed at reduced
pressure. Then anhydrous toluene was added and removed at
reduced pressure two times to produce a flaky white solid. Crude
product was triturated and transferred to a separate flame-dried
flask, which was placed under vacuum overnight. An amount of
16.1 g (79.5 mmol) was produced and used without further puri-
fication. Yield, 97%. Then 6.50 g (37.8 mmol) of p-bromoaniline
was placed in a separate 500 mL round-bottomed flask and
dissolved in 100 mL of anhydrous CH2Cl2. A total of 13.2 mL
(75.5 mmol, 2 equiv) of diisopropylethylamine was added, and
the solution was allowed to stir for several minutes, at which
time the temperature was lowered to 0 °C. Then 11.5 g (57.0
mmol, 1.5 equiv) of crude cinnamoyl chloride from above was
dissolved in approximately 40 mL of CH2Cl2 and added
rapidly, dropwise. This was allowed to stir overnight. The
color became dark green. The crude mixture was partitioned
between CH2Cl2 and water. The organic phase was collected
and dried with MgSO4. Then solvents were removed under
reduced pressure to produce a solid which was recrystallized
from CHCl3 to produce 9.72 g (28.74 mmol) of long yellowish
4-(3-Chloro-2-methylphenyl)-6-((4-chlorophenyl)(propoxy)(1-
methyl-1H-imidazol-5-yl)methyl)-1-methylquinolin-2(1H)-one (22).
An amount of 10.0 mg (0.02 mmol) of 4-(3-chloro-2-methyl-
phenyl)-6-((4-chlorophenyl)(hydroxy)(1-methyl-1H-imidazol-5-yl)-
methyl)-1-methylquinolin-2(1H)-one was dissolved in 10 mL of
PrOH, and approximately 15 mg of tosic acid was added. The
mixture was heated to reflux for 48 h. TLC analysis indicated
only one major product and complete conversion of starting
material. Solvents were removed under reduced pressure to
produce a colorless, oily semisolid. Product was purified by
HPLC using a water-methanol gradient with 0.08% v/v tri-
fluoroacetic acid: 0-5 min 20% MeOH, 5-25 min 20-65%
MeOH, 25-30 min 65-100% MeOH. Product elutes at 28.6
min, and 3.9 mg (0.0059 mmol) was produced as a mono-TFA
1
needles of product for a yield of 76%. H NMR (300 MHz,
CDCl3, δ): 7.87 (d, J=15.9 Hz, 1H), 7.54 (m, 2H), 7.46 (m, 2H),
7.32 (m, 1H), 6.96 (m, 2H), 6.85 (d, J=15.9 Hz, 1H). MW:
338.15 g/mol.
6-Bromo-4-(2,6-difluorophenyl)-2-oxotetrahydroquinoline. An
amount of 4.35 g (12.9 mmol) of the previous compound was
added to a 250 mL round bottomed flask, which was fitted with a
reflux condenser and a stir bar. Then 45 mL of concentrated
H2SO4 was added, and the mixture was heated to 105 °C for 4 h,
1
salt. Yield 30%. TLC (CH2Cl2/MeOH, 9:1 v/v): Rf=0.55. H
NMR (300 MHz, CDCl3, δ): 8.97 (s, 1H), 7.76 (ddd, J=9.0, 2.4
Hz, 1H), 7.73 (d, J=9.0 Hz, 1H), 7.54 (m, 2H), 7.32 (m, 5H), 7.19
(dd, J=2.4 Hz, 1H), 7.15 (m, 1H), 6.59 (m, 1H), 3.78 (s, 3H), 3.47
(m, 3H), 2.02 (m, 3H), 1.50 (m, 2H), 0.86 (m, 3H). ESI-MS m/z

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3897
at which time the reaction was quenched by pouring the mixture
into ice-water. A white precipitate formed and was collected by
filtration to produce 3.42 g (10.1 mmol) of crude product, yield
79%. 1H NMR (300 MHz, CD3OD, δ): 7.45 (m, 1H), 7.36 (dd,
J=8.4 Hz, 1.2 Hz, 1H), 7.09 (m, 2H), 7.07 (d, J=8.4 Hz, 1H),
6.94 (s, 1H), 3.00 (dd, J=16.5 Hz, 12 Hz, 1H), 2.79 (dd, J=
16.5 Hz, 6.6 Hz, 1H). MW: 338.15 g/mol.
was added and mixture was refluxed for 4 h at which time
reaction was neutralized with a 1 M NH4OH in water. The
mixture was partitioned between 1 M NH4OH and CHCl3. The
aqueous was extracted 3 times with CHCl3, and the organic
fractions were combined and dried with anhydrous MgSO4.
Solvents were removed under vacuum. Crude product was
purified on silica with a mobile phase consisting of MeOH/
CH2Cl2, 1:10 v/v, to produce 71.5 mg (0.1443 mmol) of product,
78% yield. TLC (CH2Cl2/MeOH, 9:1 v/v): Rf=0.35. 1H NMR
(300 MHz, CDCl3, δ): 7.91 (dd, J=8.4 Hz, 1.8 Hz, 1H), 7.64 (dd,
1H), 7.41 (m, 1H), 7.23 (m, 4H), 7.14 (m, 1H), 6.98 (m, 3H), 4.13
(s, 3H), 3.50 (s, 3H), 3.35 (s, 3H). MW: 477.89 g/mol.
4-(2,6-Difluorophenyl)-6-((4-chlorophenyl)(hydroxy)(1-methyl-
1H-imidazol-5-yl)methyl)-1-methylquinolin-2(1H)-one. An amount
of 54 mg (0.11 mmol) of the previous compound was added to a
25 mL flask and dissolved in 5 mL of THF. Then 13 mg (0.5 equiv,
0.057 mmol) of benzyltriethylammonium chloride was added as a
phase transfer catalyst. A total of 0.82 mL of 40% wt NaOH (120
equiv, 540 mg) was added and the mixture allowed to stir for
approximately 10 min. Then 10 μL (1.4 equiv, 0.16 mmol) of CH3I
was added, and the mixture was allowed to stir overnight. The
THF was removed at reduced pressure, and the residue was
partitioned between CHCl3 and 1 M NH4OH. The product was
purified on silica with a mobile phase consisting of MeOH/
CH2Cl2, 1:10 v/v, to produce 27 mg (3.97 mmol) of product as a
colorless semisolid, 48% yield. TLC (CH2Cl2/MeOH, 9:1 v/v):
Rf=0.45. 1H NMR (300 MHz, CDCl3, δ): 7.77 (dd, 1H), 7.73 (d,
1H), 7.64 (s, 1H), 7.55 (m, 1H), 7.31 (m, 2H), 7.18 (m, 2H), 7.11 (m,
2H), 6.97 (d, 1H), 6.73 (s, 1H), 3.85 (s, 3H), 3.42 (m, 3H). MW:
491.92 g/mol.
4-(2,6-Difluorophenyl)-6-((4-chlorophenyl)(methoxy)(1-methyl-
1H-imidazol-5-yl)methyl)-1-methylquinolin-2(1H)-one (16). An
amount of 13 mg (0.027 mmol) of the previous compound was
dissolved in 10 mL of MeOH, and approximately 7 mg of tosic
acid was added. The mixture was heated to reflux for 48 h. TLC
analysis indicated only one major product and complete con-
version of starting material. Solvents were removed under
reduced pressure to produce a colorless, oily semisolid. Product
was purified by HPLC using a water-methanol gradient with
0.08% v/v trifluoroacetic acid: 30-100% over 20 min followed
by 10 min at 100%. Product elutes at 12.2 min, and 6.7 mg
(0.0893 mmol) was produced as a mono-TFA salt. Yield 59%.
TLC (CH2Cl2/MeOH, 9:1 v/v): Rf=0.55. 1H NMR (300 MHz,
CD3OD, δ): 9.02 (s, 1H), 7.85 (dd, J=9.0, 2.1 Hz, 1H), 7.76 (d,
J=9.0 Hz, 1H), 7.63 (m, 1H), 7.58 (d, J=1.5 Hz, 1H), 7.38 (m,
4H), 7.20 (m, 3H), 6.78 (s, 1H), 6.73 (s, 1H), 3.83 (s, 3H), 3.55 (s,
3H), 3.21 (s, 3H). ESI-MS m/z 506.5 (M þ Hþ)þ. MW: 505.94 g/
mol. Mono-TFA salt FW: 619.97 g/mol.
6-Bromo-4-(2,6-difluorophenyl)quinolin-2(1H)-one. A mixture
of the previous compound (1.0 g, 2.95 mmol) and 2,3-dichloro-
5,6-dicyano-1,4-benzoquinone (4.03 g, 17.75 mmol) in dioxane
(50 mL) was stirred under reflux for 48 h and concentrated in
vacuo. The residuewas dissolvedinpotassium carbonatesolution
(2.5%, 50 mL) and extracted with CH2Cl2/MeOH (20:1, 2 ꢀ
50 mL). The combined dried (MgSO4) organic extracts were
evaporated, and the residue was purified with flash chromato-
graphy over silica to produce 678 mg (2.02 mmol) of pure
1
product, 68.4% yield. H NMR (300 MHz, CD3OD, δ): 7.73
(dd, J=9 Hz, 2.1 Hz, 1H), 7.65 (m, 1H), 7.40 (d, J=8.7 Hz, 1H),
7.25 (m, 3H), 6.69 (s, 1H). MW: 336.13 g/mol.
6-Bromo-4-(2,6-difluorophenyl)-2-methoxyquinoline. A 50 mL
round-bottomed flask was charged with 360 mg (1.07 mmol) of
the previous compound. Then 15 mL of CH2Cl2 was added to
the flask followed by 316 mg (2.14 mmol, 2 equiv) of BF4OMe3.
The flask was sealed with a septum and the mixture stirred
overnight with a slight overpressure of N2 vented through a
bubbler. The following day, several milliliters of 1.5 M NaOH
was added and then allowed to stir for about an hour, at which
time the mixture was partitioned between CHCl3 and H2O. The
organic phase was collected, then washed with brine and dried
with anhydrous MgSO4. Solvents were removed under vacuum.
Crude material was adsorbed onto silica, then purified using a
mobile phase of hexanes/CH2Cl2, 1:1 (v/v), to produce 189 mg
(0.54 mmol) of product, 50% yield. TLC (hexanes/CH2Cl2, 1:1
v/v): Rf=0.50. 1H NMR (300 MHz, CDCl3, δ): 7.82 (d, 7.36 (dd,
J=9 Hz, 1H), 1H), 7.73 (dd, J=8.9 Hz, 2.1 Hz, 1H), 7.55 (d, J=
1.8 Hz, 1H), 7.50 (m, 1H), 7.28 (s, 1H), 7.11 (m, 2H), 6.95 (s, 1H),
4.13 (s, 3H). MW: 350.16 g/mol.
4-(2,6-Difluorophenyl)-6-((4-chlorophenyl)(hydroxy)(1-methyl-
1H-imidazol-5-yl)methyl)-2-methoxyquinoline. A 25 mL round-
bottomed flask was flame-dried, fitted with a magnetic stir bar,
and charged with 100 mg of the previous compound. A total of
5 mL of THF was added, and mixture was stirred for 10 min, at
which time the temperature was lowered to -78 °C and the
mixture stirred for an additional 10 min. Then 125 μL (1.05
equiv, 0.3124 mmol) of 2.5 M n-BuLi was added dropwise
accompanied by a color shift to dark yellow-orange. This was
allowed to stir for approximately 5 min after the sublimation of
CO2 subsided as indicated by the evolution of gas from the dry
ice bath. A separate flask was flame-dried and charged with
60 mg of 13 (0.9 equiv, 0.2678 mmol). This was dissolved in
55 mL of THF and added to the flask containing quinoline in
three increments, rapidly dropwise over 10 min. Color shifted
steadily to yellow-gold after stirring overnight and warming
slowly to room temperature. The reaction was quenched by
addition of 1 volume equivalent of a saturated aqueous solution
of NH4Cl. The biphasic mixture was partitioned between 1 M
NH4OH and CHCl3. Organic phase was collected and solvents
were removed under reduced pressure to produce a foamy, white
semisolid. This was purified on silica with a mobile phase
consisting of MeOH/CH2Cl2, 1:10 v/v, to produce 71.5 mg
(0.1443 mmol) of product, 49% yield. TLC (CH2Cl2/MeOH,
9:1 v/v): Rf=0.45. 1H NMR (300 MHz, CDCl3, δ): 7.91 (d, 1H),
7.64 (dd, 1H), 7.41 (m, 1H), 7.23 (m, 4H), 7.14 (m, 1H), 6.98 (m,
3H), 4.13 (s, 3H), 3.50 (s, 3H), 3.35 (s, 3H). ESI-MS m/z 492.5
(M þ Hþ)þ. MW: 491.92 g/mol.
Acknowledgment. This work was supported by a grant
from the National Institutes of Health (Grant AI070218)
and from the Drugs for Neglected Diseases Initiative (DNDi).
Supporting Information Available: Additional synthesis infor-
mation, analytical data, and NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
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