Identification of compounds with anti-West Nile virus activity

Article abstract of DOI:10.1021/jm051229y

The lack of antiviral compounds targeting flaviviruses represents a significant problem in the development of strategies for treating West Nile Virus (WNV), Dengue, and Yellow Fever infections. Using WNV high-throughput screening techniques developed in o

Full text of DOI:10.1021/jm051229y

J. Med. Chem. 2006, 49, 2127-2137
2127
Identification of Compounds with Anti-West Nile Virus Activity
John R. Goodell,† Francesc Puig-Basagoiti,‡ Brett M. Forshey,‡ Pei-Yong Shi,*,‡ and David M. Ferguson*,†
Department of Medicinal Chemistry and Center for Drug Design, UniVersity of Minnesota, Minneapolis, Minnesota 55455, and
Wadsworth Center, New York State Department of Health, Albany, New York 12208
ReceiVed December 8, 2005
The lack of antiviral compounds targeting flaviviruses represents a significant problem in the development
of strategies for treating West Nile Virus (WNV), Dengue, and Yellow Fever infections. Using WNV high-
throughput screening techniques developed in our laboratories, we report the identification of several small
molecule anti-WNV compounds belonging to four different structural classes including pyrazolines, xanthanes,
acridines, and quinolines. The initial set of “hits” was further refined using cell viability-cytotoxicity assays
to two 1,3,5-triaryl pyrazoline compounds: 1-(4-chlorophenylacetyl)-5-(4-nitrophenyl)-3-(thiophen-2-yl)-
4,5-dihydro-1H-pyrazole and 1-benzoyl-5-(4-chlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole. On
the basis of their activity and favorable therapeutic indexes, these compounds were identified as viable
leads and subjected to additional evaluation using an authentic viral titer reduction assay employing an
epidemic strain of WNV. The compounds were further evaluated in a transient replicon reporting system to
gain insight into the mechanism of action by identifying the step at which inhibition takes place during viral
replication. The results indicate the pyrazolines inhibit RNA synthesis, pointing to viral RNA polymerase,
RNA helicase, or other viral replication enzymes as potential targets. Progress was also made in understanding
the structural requirements for activity by synthesizing a focused chemical library of substi-
tuted pyrazolines. Preliminary SAR data are presented that show the aryl-rings are required for activity against
WNV. More importantly, the results indicate WNV activity is tolerant to aryl-substitutions paving the way
for the design and development of much larger combinatorial libraries with varied physicochemical properties.
Introduction
then processed by viral and host proteases.12 In addition, the
plus-sense RNA is transcribed into minus-sense RNA, which
subsequently acts as a template for the production of more plus-
sense RNA.5
Since the 1999 West Nile Virus (WNV) outbreak, there has
been an increased interest in identifying compounds with anti-
WNV activity.1-4 The WNV is a member of the FlaViVirus
genus, which belongs to the FlaViVidae family. The FlaViVirus
genus includes a number of additional significant human
pathogens such as yellow fever virus (YF), dengue virus (DEN),
Japanese encephalitis virus (JE), Murray Valley encephalitis
virus (MVE), St. Louis encephalitis virus (SLE), and tick-bourne
encephalitis virus (TBE).5-8 Viruses belonging to the FlaViVirus
genus are typically transmitted to vertebrates by mosquitoes or
ticks and are responsible for severe morbidity and mortality in
both humans and animals.5-8
Over the past 6 years, the WNV has spread to nearly every
state in the continental U.S. and parts of Canada and Mexico.9-11
During 2003 and 2004, the CDC reported more than 12,000
human cases in the United States, with at least 362 resulting in
death.10 Discovering compounds that can be used for prevention
and treatment of WNV infections is becoming a general public
health priority.6,11 Currently, there are no viable drugs or vac-
cines available to treat or prevent WNV infection in humans.3,14
Recently, we have developed three separate systems for
conducting high-throughput screening (HTS) assays: a cell line
containing a persistently replicating subgenomic replicon, a full-
length reporting virus, and packaged virus-like particles (VLPs)
containing replicon RNA.12,13 All three assays were validated
using three known inhibitors of the WNV: ribavirin, glycyr-
rhizin, and mycophenolic acid.13 Because each assay encom-
passes multiple yet distinct steps of the viral life cycle, these
assays can be used to help elucidate the mechanism of action
for potential inhibitors.13 These new techniques offer an
attractive alternative to traditional whole cell cytopathic assays,
which are more time-consuming and offer little information
about possible mechanisms of action. Because these assays are
cell-based, it is also possible to investigate cellular uptake and
chemical modification within the cell. Therefore, hits generated
with these methods stand a greater chance of success in animal
studies as compared to hits generated from target-based assays.13
In the current study, an initial library of 108 compounds was
screened in the three HTS WNV assays. The library is a
collection of compounds that have been synthesized in our
laboratory over the last several years. The structural classes
represented have a history of antimicrobial and antiviral activity
and fall into six categories: pyrazolines, xanthones, xanthanes,
acridones, acridines, and quinolines. Primary screening identified
24 “hits” from four of the structural classes. This set was further
refined on the basis of cell cytotoxicity assays, resulting in the
identification of two 1,3,5-triaryl pyrazoline lead compounds.
Here, we describe the library synthesis and biological evaluation
of the 24 potential WNV inhibitors and report preliminary
structure-activity relationship (SAR) data for a second genera-
tion set of pyrazoline compounds. In addition, we discuss the
The WN virion consists of a single plus-sense RNA genome
approximately 11 kb in length. The genomic RNA is composed
of a 5′ and 3′ untranslated region (UTR) and a single open
reading frame (ORF). The ORF encodes a total of 10 viral
proteins: three structural (capsid, membrane, and envelope) and
seven nonstructural, which are essential for viral replication.5,12
Upon viral infection and release of genomic RNA, the plus-
sense RNA can be translated into a single polyprotein that is
* Corresponding authors. D.M.F.: phone, (612) 626-2601; fax, (612)
626-4429; e-mail, ferguson@umn.edu. P.-Y.S.: phone, (518) 473-7487; fax,
(518) 473-1326; e-mail, ship@wadsworth.org.
† University of Minnesota.
‡ New York State Department of Health.
10.1021/jm051229y CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/24/2006

2128 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Scheme 1. Synthesis of Pyrazoline Compoundsa
Goodell et al.
a Reagents and conditions: (a) NaOH, methanol or ethanol, room temperature; (b) N2H4‚H2O, ethanol, room temperature; (c) acid chloride, slight heat.
Scheme 2. Synthesis of Xanthane Compoundsa
a Reagents and conditions: (a) phenol, Cu, CuI, pyridine, DBU, DMF, reflux; (b) H2SO4, 100 °C; (c) SOCl2, reflux; (d) CH2Cl2, N-methylpiperazine; (e)
DIBAL, THF.
validation of the prazoline hit 5a and the initial steps toward
the elucidation of the mechanism by which this compound
inhibits WNV replication.
modification were unsuccessful. Separation of 10 and 11 was
achieved by silica chromatography.
The synthesis of acridines also began with a modified
Ullmann-Goldberg coupling. In this case, however, aniline (12)
was used in place of phenol, yielding the N-phenylanthranilic
acid (13) as shown in Scheme 3.17,18 Cyclization with POCl3
generated the 9-chloroacridine intermediate in addition to
producing an acid chloride of the second carboxylic acid.19 This
intermediate was then reacted with an amine, yielding the
unstable 3-carboxamide-substituted-9-chloroacridine intermedi-
ate. Subsequent reaction with phenol and mild heating results
in the stable 3-amide-9-phenoxyacridine (14). The final step
entailed reacting the intermediate 14 with the desired amine,
producing the substituted 9-aminoacridine (15).20 To synthesize
acridines substituted in the 2- and 4-positions, different reagents
are required in the Ullmann-Goldberg coupling. Hence, the
reaction between 2-chlorobenzoic acid (16) and the appropriate
aminobenzoic acid (17) generated the desired substitutions. It
should be noted that derivatives substituted in the 3-position
cannot be made from 2-chlorobenzoic acid and 3-aminobenzoic
acid because the reaction yields a mixture of 1- and 3-substituted
products, which cannot be separated easily.
Chemistry
The synthesis of the pyrazolines began with a Claisen-
Schmidt condensation between a methyl aryl ketone (1) and an
aromatic aldehyde (2) affording the stable chalcone intermediate
(3) as demonstrated in Scheme 1.15 This reaction was conducted
in the presence of a strong base, such as NaOH, in a minimal
volume of methanol or ethanol. With little purification required,
the chalcone was reacted with hydrazine in ethanol, which
afforded the 3,5-diaryl pyrazoline (4).16 Subsequent reaction with
an acid chloride yielded the 1,3,5-trisubstituted pyrazoline (5).16
The xanthanes were synthesized from xanthones and hence
followed the same synthetic path as the xanthones. As shown
in Scheme 2, the first reaction was a modified Ullmann-
Goldberg coupling between bromoterephthalic acid (6) and
phenol, which was catalyzed by copper.17,18 In this reaction,
CuI and pyridine are cocatalysts responsible for maintaining
the catalytic cycle of Cu0, reducing the reaction time and
increasing yields. The use of a nonnucleophilic base, such as
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), was essential for
coupling with phenol to form the O-phenylsalicylic acid (7).
Cyclization of 7 with concentrated H2SO4 at 100 °C yielded
the substituted xanthone. Refluxing with SOCl2 produced the
acid chloride intermediate of compound 8. Subsequent reaction
of the acid chloride with N-methylpiperazine afforded the
3-carboxamide substituted xanthone (9). Reduction of the
xanthone with a mild reducing agent, such as DIBAL in THF,
produced a mixture of partially and fully reduced xanthanes
(10 and 11, respectively). In this reaction, the fully reduced
product was the predominant product. Attempts to produce the
partially reduced hydroxyl product in greater portions by reaction
The synthesis of 4-aminoquinoline began with a POCl3-
mediated cyclodehydration reaction between anthranilic acid
(18) and substituted acetophenone (19). This afforded the
2-phenly-3-chloroquinoline intermediate (20), as seen in Scheme
4.21 Subsequent reaction of 20 with N-methylpiperazine under
heating conditions yielded the 2-phenyl-4-aminoquinoline ana-
logue (21).22
As shown in Scheme 5, the first step in the synthesis of
2-aminoquinoline involved the reaction of isatoic anhydride (22)
with N,O-dimethylhydroxylamine (23), generating the weinreb
amide (24).23 Upon purification, amide 24 was reacted with the

Compounds with Anti-West Nile Virus ActiVity
Scheme 3. Synthesis of Acridine Compoundsa
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2129
a Reagents and conditions: (a) Cu, CuI, pyridine, K2CO3, H2O, reflux; (b) POCl3, reflux; (c) amine, CH2Cl2, 0 °C; (d) phenol, 60 °C; (e) amine, 120 °C.
Scheme 4. Synthesis of 4-Aminoqiunoline (21)a
Biology
Three cell-based high-throughput screening (HTS) assays
were used to identify and characterize inhibitors of WNV.13 The
first uses a reporting replicon-containing cell line. As compared
to the wild-type genomic RNA, the reporting replicon contains
a deletion of viral structural genes. In addition, a Renilla
luciferase gene (Rluc) and a Neomycin resistance gene (Neo)
were engineered into the replicon, resulting in a reporting
replicon (Rluc-Neo-Rep). The cell lines containing the replicat-
ing Rluc-Neo-Rep RNAs can be used to screen for inhibitors
of viral replication (including viral translation and RNA
synthesis), but not viral assembly and entry. The second applies
a virus-like particle (VLP) infection assay. The VLPs were
prepared by expressing WNV structural proteins in cells
containing the Rluc-Neo-Rep. The structural proteins could
package the Rluc-Neo-Rep RNAs to form infectious VLPs.
Infection of naive cells with such VLPs allows screening
inhibitors of viral entry and replication, but not viral assembly
(because the replicon contains a deletion of viral structural
proteins). The third is a full-length reporting WNV assay. The
full-length reporting WNV contains a Rluc reporter (Rluc-FL-
WNV) and can be used to screen inhibitors against all steps of
a viral life cycle, including viral entry, replication, and virion
assembly. Because luciferase has been engineered into each of
the assays described above, inhibition is measured through
suppression of luciferase signals. A more detailed description
of the three HTS assays has been recently reported.13
a Reagents and conditions: (a) POCl3, reflux; (b) N-methylpiperazine,
120 °C.
Scheme 5. Synthesis of 2-Aminoquinolinea
All compounds were initially subjected to two primary
screens: the reporting-replicon (Rluc-Neo-Rep) BHK cell line
assay and the full-length reporting (Rluc-FL-WNV) infection
assay in Vero cells. This combination can be applied to delineate
inhibition of viral translation/RNA synthesis from entry and
assembly processes. Each compound was preliminarily screened
at 30 µM at a 1% DMSO final concentration. Potential WNV
inhibitors showing greater than 50% inhibition of Rluc activity
were then analyzed for cytotoxicity using an MTT assay.27
Cytotoxicity of the compounds was determined in both Vero
and BHK cells. Hits from the primary screen were then screened
in the replicon-containing VLP infection assay.
a Reagents and conditions: (a) NEt3, ethanol/H2O, reflux; (b) n-BuLi,
THF, -78 °C; (c) acetyl chloride, CH2Cl2, 0 °C; (d) NaOMe, ethanol, reflux;
(e) POCl3, reflux; (f) N-methylpiperazine, heating.
substituted bromobenzene (25) in the presence of n-butyllithium,
generating the benzophenone intermediate (26).23 At this point,
the amino group was actylated with acetyl chloride, generating
27.24 Lactamization of 27 was then achieved by treating with
sodium methoxide in ethanol under reflux conditions.25 Treat-
ment of 28 with POCl3 produced the 2-chloroquinoline (29),
which was then reacted with N-methylpiperazine, yielding the
2-amino-4-phenylquinoline analogue (30).

2130 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Table 1. WNV Screening Results for Pyrazoline-Based Compounds
Goodell et al.
a BHK-21 cells. b Vero cells, ND ) not determined because initial screen was .30 mM.
Table 2. WNV Screening Results for Xanthane-Based Compounds
Rluc-Neo-Rep in BHK cells
Rluc-FL-WNV in Vero cells
VLPa
compound
EC50 µM
CC50 µM
TIa
EC50 µM
CC50 µM
TIb
EC50 µM
10
11
13.5
>30
325
200
24
<6.7
10
>30
50
90
5
<3
4
28
a BHK-21 cells. b Vero cells.
Results
again suggesting inhibition of the replication process. The final
observation to note is the difference in toxicity between BHK
and Vero cells. It appears that compound 10 is approximately
6.5 times more toxic to Vero cells versus BHK cells. Because
compound 10 is racemic and the toxicity may simply be due to
one of the enantiomers, it is possible that chiral separation could
result in a compound with greater activity and reduced toxicity.
Pyrazolines. The initial screen contained 15 compounds
belonging to the pyrazoline structural class. From this small
set, two derivatives showed significant activity in all three HTS
assays, as shown in Table 1. Activity across all three assays
strongly suggests these compounds are most likely disrupting
viral replication (see Biology section). More importantly, the
pyrazolines 5a and 5b show low toxicity in both BHK and Vero
cell lines. In comparison to the known inhibitor ribavirin, it was
observed that both of these pyrazolines showed greater activity.
Given this finding, and the favorable therapeutic index of the
pyrazolines, 5a and 5b have been identified as WNV lead
compounds. These compounds are racemic mixtures, however,
indicating that gains in activity may be afforded through chiral
separation. In addition, the finding that several other 1,3,5-triaryl
pyrazolines (compounds 5c and 5d in Table 1) do not exhibit
significant activity suggests that SAR studies might be valuable
in refining lead compounds 5a and 5b. Although comprehensive
SAR is beyond the scope of this report, preliminary SAR data
are reported below.
Xanthanes and Xanthones. The library screened contained
eight xanthanes and 10 of the parent xanthones. From these
two classes, there were only two compounds that exhibited anti-
WNV activity. Coincidentally, they are both products from the
same reaction: the fully reduced xanthane and the partially
reduced 9-hydroxyxanthane. Further, the 9-hydroxy compound
(10) is racemic and was the only 9-hydroxy compound submitted
to the screen. The results reported in Table 2 show compound
10 as being more active than the fully reduced analogue (11)
and ribavirin. As was the case with the pyrazoline derivatives,
compound 10 exhibited activity over all three HTS assays, once
Acridines and Acridones. The majority of the compounds
submitted to the screen belong to the acridine class or the related
acridone class consisting of 29 and 40 analogues, respectively.
From these two classes, 18 hits were discovered, all belonging
to the acridine class. Structurally speaking, the primary differ-
ences between these two classes are the substitution at the 9
position (R2) of the acridine and the aromaticity of the middle
ring. In the case of the acridone compounds, the middle ring is
not aromatized and bears no substitution, except a ketone at
the 9 position. From the results shown in Table 3, it is clear
that little to no SAR can be drawn, despite having a compre-
hensive set of compounds. The lack of substantial effect by
various substitutions may indicate that the mechanism whereby
these compounds display anti-WNV activity is not specific and
accommodates various functionalities. It is possible that aro-
maticity of the acridine core structure plays a major role, which
would partly explain the lack of activity with similar acridones.
Once again, because the activity is seen in all three assays, it
strongly suggests the acridines target the replication process as
well. Although these analogues demonstrate reasonable activities
as compared to ribavirin, they also appear to have greater
toxicities, particularly in the Vero cells.
Quinolines. The quinoline class of compounds, six in all,
represents the smallest set of compounds in our screening.

Compounds with Anti-West Nile Virus ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2131
Table 3. WNV Screening Results for Acridine-Based Compounds
Rluc-Neo-Rep in BHK cells
Rluc-FL-WNV in Vero cells
VLPa
ring
compound position
R1
R2
EC50 µM CC50 µM
TIa
7.5
<5.1
20
44
15
14
16
EC50 µM CC50 µM
TIb
2.5
2
3.6
3.4
<3
2.8
2.5
EC50 µM
14a
14c
15a
15b
15c
15d
15e
15f
14d
15g
15h
15i
15j
14f
15k
15l
15m
15n
4
4
4
4
4
4
4
4
3
3
3
3
3
2
2
2
2
2
N-methy1-piperazinyl phenoxy
morpholino
N-methy1-piperazinyl 4-amino-N-benzylpiperdinyl
diethylamino
morpholino
N-methy1-piperazinyl tryptamino
diethylamino
morpholino
N-methy1-piperazinyl phenoxy
N-methy1-piperazinyl 4-amino-N-benzylpiperdinyl
diethylamino
N-methy1-piperazinyl tryptamino
diethylamino tryptamino
N-methy1-piperazinyl phenoxy
N-methy1-piperazinyl 4-amino-N-benzylpiperdinyl
diethylamino
N-methy1-piperazinyl tryptamino
diethylamino tryptamino
20
>30
15
150
155
300
350
300
150
150
250
100
150
150
150
125
100
100
120
170
110
20
22
50
45
80
75
90
50
50
60
40
80
55
45
45
80
80
50
65
75
20
>30
20
20
20
17
9
23
18
8
6
10
7
20
25
22
27
20
phenoxy
22
22
>30
18
4-amino-N-benzylpiperdinyl
4-amino-N-benzylpiperdinyl
8
20
10.5
9.2
20
tryptamino
tryptamino
20
>30
28
13
<2
28
3.6
9.7
12
11
18
4.7
3.6
5.4
6.1
4.4
1.4
2.7
2.7
<1.5
1.8
4
15.5
12.5
13.7
7
30
20
>30
25
4-amino-N-benzylpiperdinyl
21
28
22
28
20
25
20
25
3.2
2.5
2.6
5
4-amino-N-benzylpiperdinyl
25
15
a BHK-21 cells. b Vero cells.
Table 4. WNV Screening Results for Quinoline-Based Compounds
Rluc-Neo-Rep in BHK cells
Rluc-FL-WNV in Vero cells
VLPa
compound
EC50 µM
CC50 µM
TIa
EC50 µM
CC50 µM
TIb
EC50 µM
21
30
14.5
17
50
110
3.4
6.5
15
30
20
25
1.3
0.8
8
20
a BHK-21 cells. b Vero cells.
Figure 1. Inhibition of an epidemic strain of WNV by compound 5a.
Percent inhibition of viral titer at each concentration of compound is
reported on the top of the columns.
Figure 2. A WNV reporting replicon containing a luciferase reporter
(fused in-frame with the ORF; Rluc-Rep) was used to measure the
effects of 5a on viral translation and RNA synthesis at indicated time
points post-transfection (p.t.). Values above each data point indicate
the percentage of luciferase signals from the compound-treated trans-
fection as compared to that from the mock-treated transfection. Average
results of two to three experiments are shown.
Similar to the acridine derivatives, the quinoline compounds
demonstrated activity in all three HTS assays, as well as
significant toxicities as shown in Table 4. This is not surprising,
considering some of the similarities between the two classes.
In comparing compounds 21 and 30, it should be pointed out
that the only difference is the exchange of the substitutions at
the 2- and 4-positions. This modification does not appear to
have much effect on activity, which may suggest that there is
little structural specificity for these compounds. As with the
acridines, the usefulness of these compounds is limited by their
low TI ratios.
manner with an EC50 value estimated to be 15 µM, which is in
good agreement with the three HTS assay results reported in
Table 1.
Mechanism of Action of 5a. The results taken from the
primary screens (Table 1) indicate that 5a is active in both the
Rluc-VLP and the Rluc-Neo assays, suggesting the compound
functions by either inhibiting viral translation or RNA synthesis.
To further evaluate the mechanism of action, 5a was assayed
using a transient WNV replicon system as previously reported.36
This assay is able to distinguish between compound-mediated
inhibition of viral translation and inhibition of RNA synthesis.37
BHK cells transfected with WN RlucRep RNA were incubated
with compound (at 33 and 100 µM) and assayed for luciferase
Verification of Anti-WNV Activity of 5a. To validate the
anti-WNV potency of 5a, the compound was additionally tested
in an authentic viral titer reduction assay.36 In this assay, Vero
cells were first infected with an epidemic strain of WN virus
followed immediately by treatment with compound. Viral yields
were then assayed 42 h post infection. The results shown in
Figure 1 indicate that 5a inhibited viral titer in a dose response

2132 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Goodell et al.
Table 5. WNV Screening Results for Trisubstituted Pyrazolines Analogues of 5a and 5b
activities at 2, 4, and 72 h post-transfection. The 2 and 4 h time
points represent inhibition of viral translation, whereas the 72
h time point represents inhibition of RNA synthesis. As shown
in Figure 2, there was no significant suppression of luciferase
activity at the 2 and 4 h time points, therefore compound 5a
does not inhibit viral translation. In contrast, the luciferase
activity at the 72 h time point was suppressed by approximately
85%, indicating that 5a inhibits WNV through suppression of
viral RNA synthesis. These results are also supported by more
traditional time-of-addition assays, which are reported elsewhere
for compound 5b.36
Preliminary SAR. To provide insight to potential SAR and
to further evaluate the trisubstituted pyrazolines for development
as WNV inhibitors, a preliminary set of analogues based on 5a
and 5b has been synthesized. The results from this exploratory
set of compounds are reported in Table 5. Even with this small
focused set of compounds, SAR trends are beginning to emerge.
A decrease of activity was observed when R1 and R3 of the
triaryl pyrazoline were substituted with alkyls (34, 42, and 43),
suggesting 1,3,5-triaryl substitutions are key to antiviral activity.
The antiviral activity was also reduced for the R3-4-nitrobenzoyl
substitution (31). While the data presented here are limited (and
should be applied with some caution), a general trend is evident
for the R3-4-chlorophenylacetyl substituted compounds (33, 35,
37, 41, and also 5a from Table 1), which all tend to show similar
or improved activity as compared to their R3-benzoyl analogues.
The activity of the bromo-substituted derivative (39) is particu-
larly interesting and suggests that bulky as well as polar
substitutions to the thiophene ring can be tolerated.
Discussion
The lack of a practical therapeutic agent for the treatment of
WNV is a growing concern. Current inhibitors of WNV are
not viable candidates for various reasons. In particular, the broad
spectrum anti-viral nucleoside, ribavirin, is limited by the dose
required and toxicities associated with size of the dose.3
Therefore, the discovery of new compounds with anti-WNV
activity is of utmost importance. Because of their reasonable
activity and favorable therapeutic index, the pyrazolines, 5a and
5b, exhibit the greatest promise as anti-WNV agents. The status
of the pyrazoline 5a as a genuine anti-WNV lead was
determined by conducting a viral titer reduction assay. The close

Compounds with Anti-West Nile Virus ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2133
Conclusion
agreement between the EC50’s for the HTS assays and the WNV
titer reduction assay is conclusive, indicating that 5a is a valid
WNV antiviral.
In summary, the primary screening of 108 compounds has
led to the discovery of two 1,3,5-triaryl pyrazoline compounds
with promising therapeutic indices and activity against WNV.
The leads were also applied to the design and development of
a focused chemical library to further evaluate the initial “hits”
and gain insight into the requirements for activity. Although
the second generation library reported here is somewhat limited,
the results indicate that additional combinatorial chemistry/SAR
studies may be useful in refining the activity of these compounds
against WNV and for identifying compounds with improved
physicochemical properties (i.e., ADME). In this regard, the
pyrazolines offer significant advantages, due in part by the ease
of synthesis and amenability of chalcone chemistry to combi-
natorial library design.38 Steps were also taken to determine the
molecular target for these compounds using a transient reporting
replicon assay. While the results point to a mechanism that
involves inhibition of RNA synthesis, the details of this
mechanism are not yet known. It is unlikely, however, that the
pyrazolines function through nonspecific binding, such as
intercalation, since the structures do not adopt planar conforma-
tions.
Finally, it is important to point out that this work has shown
that several heterocycles related to well-known drug compounds
(such as amsacrine and chloroquine) are potential inhibitors of
WNV. We did, in fact, screen a short list of known acridine-
and quinoline-based drugs and found that amsacrine and
acriflavin were both active below 3.3 µM. While compounds
of this type suffer from high toxicity, the results are quite
interesting and will undoubtedly spark interest in evaluating
heterocycles of this type in WNV and other flavivirus assays.
In this regard, the xanthanes may be of particular interest.
Whereas the acridines are thought to function through intercala-
tion (leading to inherent toxicities), the mechanism of action
of xanthanes has not been established, providing some hope
that toxicity can be factored out of the equation.
Since inhibition was seen in all three HTS assays, the
mechanism of action can be characterized as a replication
inhibitor rather than a viral entry or an assembly inhibitor. The
mechanism of action for 5a was further evaluated using the
transient reporting replicon system, which differentiates between
viral translation and RNA synthesis.11,13,36 The results for 5a
indicated that the pyrazoline compound significantly repressed
WNV RNA synthesis without dramatic suppression of viral
translation. Inhibiting replication in this manner could entail
the disruption of RNA synthesis by inhibiting viral RNA
polymerase, RNA helicase, or other encoded viral replication
enzymes. Target assays using the multifunctional WNV proteins
NS3 and NS5 or mapping of mutant genes in resistant virus
would have to be used to offer a more specific determination
of the mechanism.13
In conducting these HTS assays, cell line selection may show
significant influence on assay sensitivity and hence is a
legitimate concern. For example, cellular functions within the
cell are not entirely the same in Vero and BHK-21 cells.
Ribavirin, in particular, requires phosphorylation inside the cell,
which is known to be less efficient in Vero cells than in MA-
104 cells.1 In validating the three HTS assays used here, it was
similarly found that ribavirin demonstrated significantly reduced
activity in Vero versus BHK cells.13 In contrast, mycophenolic
acid, which does not require modification prior to its inhibition
of viral replication through depleting the intracellular GTP pool,
showed comparable activities in both Vero and BHK cell lines.13
Because cell line selection could possibly affect assay sensitivity,
we used both BHK-21 cells and Vero cells in our two primary
screens. One primary screen was performed in BHK-21 cells
containing the Rluc-Neo-Rep. The other screening was con-
ducted in Vero cells infected with the full-length Rluc-WNV.
To verify that the pyrazolines were not experiencing cell line
dependence, they were also screened in Rluc-Neo-Rep contain-
ing Vero cells. Results here indicate that the pyrazolines, unlike
ribavirin, do not show a cell line dependence (Table 1).
Experimental Section
General. All reagents were purchased from commercial suppliers
such as Sigma-Aldrich Chemical Co., Lancaster Chemical Co,
Acros Chemical Co., etc. ACS reagent grade or better solvents were
used without further purification unless otherwise noted. Water was
purified via a Millipore filtration system. Column chromatography
was conducted using silica gel 60 (40-63 µm), and thin-layer
chromatography was conducted using EMD Chemical silica gel
60 F254 on aluminum sheets. 1H nuclear magnetic resonance spectra
were collected on a Varian Mercury 300 MHz instrument and a
Varian Mercury 600 MHz instrument using CDCl3 and DMSO-d6
as solvents. High-resolution mass spectra (HRMS) were collected
from a TOF-ESI Agilant LC-MS and analyzed using the Analyst
QS software. Reversed-phase high-performance liquid chromatog-
raphy (RP-HPLC) was performed on a Beckman Coulter 125S
System Gold using an Agilent Zorbax Eclipse XDB-C8 3.5 µM
3.0 × 150 mm column monitoring UV at 254λ on a 166 detector.
Method A was a gradient method that ran for 14 min at a flow rate
of 0.5 mL/min. Over the first 8 min, the percent ACN/water was
increased from 60% to 100%. Over the next 4 min, the gradient
was decreased back to 60% ACN/water and then held for 2 min.
Method B was an isocratic method using 70% MeOH/water and a
flow rate of 0.4 mL/min. The run duration was 20 min. Results
were analyzed using the 32 Karat software package.
The class of compounds that produced the most hits in the
three screens was the acridines. However, these compounds also
possess the greatest toxicity, which is believed to be due to the
intercalative nature of acridines into DNA. It is well known
that planar polyaromatic compounds, such as acridines, are good
intercalators and have been widely investigated as antitumor
agents.28,29 An example of this is the acridine, amsacrine, which
is a known intercalator with potent topoisomerase II inhibition
and is approved for use as an anti-leukemia drug.28 Upon
intercalation, these compounds can feasibly affect any cellular
enzyme that processes DNA.30,31 Such enzymes would include
polymerases, helicases, topoisomerases, and gyrases.31,32 It is
also possible for these compounds to intercalate in dsRNA stem
loop regions, thus preventing enzyme recognition or pro-
cessing.33-35 With this knowledge, the presumed mechanism
of action for these compounds in the WNV assays is most likely
viral replication. Considering that the acridine derivatives are
demonstrating WNV inhibition and cytotoxicity via seemingly
the same mechanism, it is unlikely that structural modification
of the acridine derivatives will yield compounds with more
favorable therapeutic indices. Presently, we are under the
assumption that the quinolines, which bear several structural
similarities with the acridines, will not benefit from structural
modification as well.
General Procedure for the Chalcone Intermediate (3). Aro-
matic acetyl (1) (5 mmol) and aromatic aldehyde (2) (5 mmol) were
dispersed in either ethanol or methanol (∼3 mL). Next, two NaOH
pellets (∼0.24 g) were added, and the reaction was stirred while
slightly heating until starting materials were completely dissolved.

2134 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Goodell et al.
Note that heating was not always necessary. Once dissolved, the
reaction was removed from heat and stirred overnight. The product
precipitated out and was filtered. Washing with cold methanol and
water affords the chalcone, which was used without further
purification. The product is a mixture of cis and trans alkenes, which
convolutes the NMR spectra.
heated to 100 °C while being stirred for 3 h. The starting material
dissolved upon heating. The reaction was cooled and poured over
ice (300 mL), producing a gray solid that was then filtered and
washed with water. Yield ) 54.9%; 1H NMR (300 MHz, DMSO-
d6) δ 7.51 (t, 1H), 7.7 (d, 1H), 7.91 (t, 1H), 7.95 (d, 1H), 8.09 (s,
1H), 8.20 (d, 1H), 8.28 (d, 1H).
3-(4-Nitrophenyl)-1-thiophen-2-yl-propenone (3a). Yield )
3-(4-Methylpiperazine-1-carbonyl)-xanthen-9-one (9). Thionyl
chloride (∼25 mL) was added to a flask containing 9-oxo-9H-
xanthene-3-carboxylic acid (8) (1.08 mmol) and was set to reflux
for 1 h. Excess thionyl chloride was distilled off under reduced
pressure. The resulting residue was dissolved in CH2Cl2 (∼15 mL)
and cooled to 0 °C on an ice bath. Next, N-methylpiperazine (∼2
mL) was added dropwise while stirring. The reaction was allowed
to reach room temperature under continued stirring for about 1 h.
An additional 30 mL of CH2Cl2 was added, and the reaction was
extracted with 1 M HCl. The aqueous layer was made basic with
5% NaOH and extracted into EtOAc, dried with MgSO4, and
concentrated. Yield ) 71.5%; 1H NMR (300 MHz, CDCl3) δ 2.33
(s, 3H), 2.37 (s br, 2H), 2.52 (s br, 2H), 3.43 (s br, 2H), 3.83 (s br,
2H), 7.39 (m, 2H), 7.51 (m, 2H), 7.74 (t, 1H), 8.32 (d, 1H), 8.37
(d, 1H).
Synthesis of 3-(4-Methylpiperazin-1-ylmethyl)-9H-xanthen-
9-ol (10) and 1-Methyl-4-(9H-xanthen-3-ylmethyl)-piperazine
(11). 3-(4-Methyl-piperazine-1-carbonyl)-xanthen-9-one (9) (0.775
mmol) was dissolved in dry tetrahydrofuran (THF) (∼25 mL) and
cooled under argon to 0 °C on an ice bath. Next, diisobutylalumi-
num hydride (DIBAL) (4.65 mmol, 4.65 mL of 1.0 M solution in
toluene) was added. Use of fewer equivalents only resulted in more
unreduced starting material. The reaction was allowed to warm to
room temperature and stirred overnight (∼14 h). The reaction was
then quenched with distilled water and extracted with diethyl ether
(4×), dried with MgSO4, and concentrated. TLC showed two spots
when eluted with a 9:1 mix of CH2Cl2:MeOH. The product was
purified by silica chromatography, yielding the partially reduced
3-(4-methyl-piperazin-1-ylmethyl)-9H-xanthen-9-ol (10) and the
fully reduced 1-methyl-4-(9H-xanthen-3-ylmethyl)-piperazine (11).
10 yield ∼11%; 1H NMR (300 MHz, CDCl3) δ 2.26 (s, 3H), 2.45
(s br, 8H), 2.97 (s, 1H), 3.52 (s, 2H), 5.76 (s, 1H), 7.15 (m, 4H),
7.36 (m, 1H), 7.49 (m, 1H), 7.59 (m, 1H); HRMS (C21H19N2O2S)
[M + H]+: found m/z 367.0424, calcd 367.0666. 11 yield ∼53%;
1H NMR (300 MHz, CDCl3) δ 2.29 (s, 3H), 2.48 (s br, 8H), 3.48
(s, 2H), 4.03 (s, 2H), 7.00 (m, 4H), 7.10 (d, 1H), 7.17 (m, 2H);
HRMS (C19H23N2O) [M + H]+: found m/z 295.1795, calcd
295.1805.
1
94.8%; H NMR (300 MHz, CDCl3) δ 7.22 (dd, J ) 3.9 and 4.9
Hz, 1H), 7.52 (d, 1H), 7.75 (dd, J ) 1.0 and 4.9 Hz, 1H), 7.79 (d,
J ) 8.8, 2H), 7.82 (d, 1H), 7.90 (dd, J ) 0.98 and 3.8 Hz, 1H),
8.29 (d, J ) 8.8, 2H).
3-(4-Chlorophenyl)-1-thiophen-2-yl-propenone (3b). Yield )
1
80.1%; H NMR (300 MHz, CDCl3) δ 7.20 (dd, J ) 3.8 and 4.9
Hz, 1H), 7.38 (m, 3H), 7.59 (d, 2H), 7.71 (dd, J ) 1.1 and 4.9 Hz,
1H), 7.81 (d, 1H), 7.88 (dd, J ) 1.1 and 3.8 Hz, 1H).
General Procedure for the 3,5-Diaryl Pyrazoline Intermediate
(4). The chalcone intermediate (3) (5 mmol) was stirred in ethanol
(∼5 mL), and hydrazine monohydrate (12.5 mmol) was added. The
reaction will go into solution and fall back out. Some light heating
may be required for the reaction to go into solution. The resulting
product was then passed through a fritted filter and washed with a
cold 50/50 mix of methanol/water, quickly rinsed with methanol,
and then briefly air-dried. The product sometimes undergoes
decomposition and was used in the next reaction as soon as the
product appeared dry without further purification.
General Procedure for 1,3,5-Trisubstituded Pyrazolines (5a-
5d). The 3,5 diaryl pyrazolines (∼5 mmol) were placed in a
medium-sized vial, and excess amounts of the desired acid chloride
were added. The reaction was then heated as HCl gas evolved. After
approximately 5 min, the reaction was cooled and methanol (∼2
mL) was added. The reaction was heated to dissolve any solid and
cooled. The product that crystallized out was filtered and recrystal-
lized in methanol to yield the 1,3,5-trisubstituted pyrazoline.
1-(4-Chlorophenylacetyl)-5-(4-nitrophenyl)-3-(thiophen-2-yl)-
1
4,5-dihydro-1H-pyrazole (5a). Yield ) 60.8%; H NMR (300
MHz, CDCl3) δ 3.12 (dd, J ) 5.1 and 17.6 Hz, 1H), 3.82 (dd, J )
11.9 and 17.6 Hz, 1H), 4.04 (s, 2H), 5.60 (dd, J ) 5.1 and 11.9
Hz, 1H), 7.09 (dd, J ) 3.7 and 5.0 Hz, 1H), 7.22 (dd, J ) 1.0 and
3.7 Hz, 1H), 7.29 (m, 6H), 7.49 (dd, J ) 1.0 and 5.0 Hz, 1H), 8.15
(d, J ) 8.8, 2H); HRMS (C21H17ClN3O3S) [M + H]+: found m/z
426.0644, calcd 426.0673; RP-HPLC method A, tR ) 5.12 min
(>99%); method B, tR ) 5.62 min (>99%).
1-Benzoyl-5-(4-chlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-
1H-pyrazole (5b). Yield ) 47.3%; 1H NMR (300 MHz, CDCl3) δ
3.16 (dd, J ) 5.0 and 17.5 Hz, 1H), 3.81 (dd, J ) 11.8 and 17.5
Hz, 1H), 5.79 (dd, J ) 5.0 and 11.8 Hz, 1H), 7.07 (dd, J ) 3.7
and 5.1 Hz, 1H), 7.24 (dd, J ) 1.1 and 3.7 Hz, 1H), 7.29 (m, 7H),
8.01 (d, J ) 7.0, 2H); HRMS (C20H16ClN2OS) [M + H]+: found
m/z 367.0643, calcd 367.0666; RP-HPLC method A, tR ) 4.82 min
(>99%); method B, tR ) 5.82 min (>99%).
1-Benzoyl-5-(4-bromophenyl)-3-(thiophen-2-yl)-4,5-dihydro-
1H-pyrazole (5c). HRMS (C2OH16BrN2OS) [M + H]+: found m/z
411.0030, calcd 411.0172.
1-Benzoyl-5-(4-methoxyphenyl)-3-(thiophen-2-yl)-4,5-dihydro-
1H-pyrazole (5d). HRMS (C21H19N2O2S) [M + H]+: found m/z
363.1060, calcd 363.1172.
2-Phenoxyterephthalic Acid (7). Bromotherethphalic acid (8)
(12.4 mmol) was added to 100 mL of dimethylformamide (DMF),
followed by phenol (25.2 mmol), 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU) (36.9 mmol), pyridine (0.2 mL), copper(0) (0.1 g), and
copper(I) iodide (0.1 g). The reaction was heated to reflux and
monitored via TLC. After 2 h, TLC showed that all of the
bromotherethphalic acid was consumed. The reaction was cooled
and diluted with 1 M HCl (∼500 mL) until no more precipitate
had formed. The resulting greenish solid was filtered and washed
with water (∼100 mL) and dried under vacuum. Yield ) 93.5%;
1H NMR (300 MHz, acetone-d6) δ 7.05 (d, J ) 7.77, 2H), 7.17 (t,
1H), 7.42 (t, 2H), 7.57 (d, J ) 1.2 Hz, 1H), 7.88 (dd, J ) 1.4 and
8.0 Hz, 1H), 8.03 (d, J ) 8.0, 1H).
General Procedure for Phenylamino-terephthalic Acid (13a-
c). 2-Phenylamino-terephthalic Acid (13a). Potassium carbonate
(30.6 mmol) was first dissolved in 100 mL of distilled water. Next,
2-bromotherethphalic acid (8) (20.4 mmol) was added followed by
aniline (12) (40.8 mmol), pyridine (0.3 mL), copper(0) (0.1 g), and
copper(I) iodide (0.1 g). The reaction was stirred under reflux for
4 h. Once the reaction was cooled, just enough 5% NaOH (aq)
was added to completely dissolve the product. The mixture was
then passed through a fritted filter containing a thin layer of silica
gel between two layers of compressed Celite and was rinsed with
saturated NaHCO3 to remove copper catalyst. The resulting filtrated
was acidified using 3 M HCl (aq) until the pH ) 1. The resultant
yellow precipitate (2-phenylamino-terephthalic acid) was then
filtered and recrystallized from absolute ethanol/water. Yield )
1
64.7%; H NMR (300 MHz, CDCl3) δ 7.09 (t, 1H), 7.27 (d, 2H),
7.41 (m, 1H), 7.82 (d, 1H), 7.95 (d, 1H), 8.13 (s, 1H), 9.60 (s,
1H).
N-(2-Carboxyphenyl)anthranilic Acid (13b). Yield ∼68%; 1H
NMR (300 MHz, DMSO-d6) δ 6.93 (m, 2H), 7.44 (m, 4H), 7.88
(m, 2H).
N-(4-Carboxyphenyl)anthranilic Acid (13c). Yield ∼43.1%;
1H NMR (300 MHz, DMSO-d6) δ 6.92 (m, 1H), 7.26 (d, 2H), 7.48
(m, 2H), 7.85 (d, 2H), 7.93 (d, 1H), 9.79 (s, 1H).
General Procedure for Intermediates 14a-g. (4-Methylpip-
erazin-1-yl)-(9-phenoxyacridin-4-yl)-methanone (14a). 2-Phe-
nylamino-terephthalic acid (13) (7.77 mmol) was dissolved in POCl3
(∼50 mL) and refluxed for 1 h. Excess POCl3 was then recovered
9-Oxo-9H-xanthene-3-carboxylic Acid (8). 2-Phenoxy-tereph-
thalic acid (7) (10.6 mmol) was added to 100 mL of H2SO4 and

Compounds with Anti-West Nile Virus ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2135
via vacuum distillation, being careful not to heat the reaction flask
over 100 °C. The resulting residue was then dissolved in CH2Cl2
(∼50 mL) and cooled to 0 °C on an ice bath. Then excess amounts
of desired amine (for example, N-methylpiperazine) (4 mL) were
added dropwise while stirring. The reaction was allowed to warm
to room temperature and then slightly heated while stirring for
approximately 1 h. The reaction was then condensed and left on
vacuum to remove residual amounts of amine. After 2 days on the
pump, phenol (∼15 g) was added to the reaction flask and heated
to 80 °C for 30 min with manual stirring using a thermometer.
The reaction was then cooled and diluted with 200 mL of CH2Cl2.
The mixture was then washed with 30% w/w NaOH in water (150
mL × 2) to remove phenol, washed with brine (150 mL), dried
over MgSO4, and concentrated. TLC showed multiple spots, but
the product is the major spot as seen by elution with 15:1 CH2-
Cl2:MeOH. Product was purified by silica chromatography eluting
with 15:1. Fractions containing product were combined and
concentrated. The resulting solid was dissolved in minimal amounts
of EtOAc and precipitated out with petroleum ether or hexanes,
yielding the 9-phenoxy-acridine-3-carboxamide. Yield ) 67.6%;
1H NMR (300 MHz, CDCl3) δ 2.22 (m br, 1H), 2.35 (s, 3H), 2.44
(m br, 1H), 2.60 (m br, 1H), 2.74 (m br, 1H), 3.24 (m br, 2H) 3.96
(m br, 1H), 4.16 (m br, 1H), 6.84 (d, 2H), 7.05 (t, 1H), 7.3 (m,
2H), 7.46 (t, 2H), 7.76 (m, 2H), 8.10 (t, 2H), 8.24 (d, 1H); HRMS
(C25H24N3O2) [M + H]+: found m/z 398.1836, calcd 398.1863.
9-Phenoxy-acridine-4-carboxylic Acid Diethylamide (14b).
br, 2H), 2.06 (m br, 4H), 2.18 (s br, 1H), 2.32 (s, 3H), 2.42 (s br,
1H), 2.58 (s br, 1H), 2.71 (s br, 1H), 2.88 (s br, 2H), 3.17 (s br,
1H), 3.26 (s br, 1H), 3.51 (s, 2H), 3.82 (s br, 1H), 3.89 (s br, 1H),
4.17 (s br, 1H), 4.65 (s br, 1H), 7.26 (m, 1H), 7.32 (m, 4H), 7.42
(m, 2H), 7.67 (m, 2H), 8.05 (d, 1H), 8.10 (m, 2H); HRMS
(C31H36N5O) [M + H]+: found m/z 494.2876, calcd 494.2914.
9-(1-Benzylpiperidin-4-ylamino)-acridine-4-carboxylic Acid
Diethylamide (15b). Yield ) 17.7%; 1H NMR (600 MHz, CDCl3)
δ 1.17 (s br, 3H), 1.30 (s br, 3H), 1.75 (m br, 2H), 2.07 (m br,
4H), 2.88 (m br, 2H), 3.41 (s br, 2H), 3.52 (s, 2H), 3.63 (s br, 2H),
3.87 (s br, 1H), 4.72 (s br, 1H), 7.26 (m, 1H), 7.34 (m, 4H), 7.44
(m, 2H), 7.67 (m, 2H), 8.06 (d, 1H), 8.11 (m, 2H); HRMS
(C30H35N4O) [M + H]+: found m/z 467.2781, calcd 467.2805.
[9-(1-Benzylpiperidin-4-ylamino)-acridin-4-yl]-morpholin-4-
yl-methanone (15c). Yield ) 60.7%; 1H NMR (600 MHz, CDCl3)
δ 1.74 (m br, 2H), 2.07 (m br, 4H), 2.88 (s br, 2H), 3.17 (s br,
1H), 3.23 (s br, 1H), 3.46 (s br, 1H), 3.51 (s br, 2H), 3.82 (m br,
4H), 4.01 (s br, 1H), 4.26 (s br, 1H), 4.68 (s br, 1H), 7.26 (m, 1H),
7.32 (m, 4H), 7.42 (m, 2H), 7.67 (m, 2H), 8.05 (d, 1H), 8.10 (m,
2H); HRMS (C30H33N4O2) [M + H]+: found m/z 481.2576, calcd
481.2598.
{9-[2-(1H-Indol-3-yl)-ethylamino]-acridin-4-yl}-(4-methylpip-
erazin-1-yl)-methanone (15d). Yield ) 71.2%; 1H NMR (600
MHz, CDCl3) δ 2.18 (s br, 1H), 2.32 (s, 3H), 2.43 (s br, 1H), 2.58
(s br, 1H), 2.69 (s br, 1H), 3.25 (s br, 4H), 3.88 (s br, 1H), 4.2 (s
br, 3H), 5.28 (s br, 1H), 7.07 (s br, 1H), 7.15 (t, 1H), 7.25 (m,
3H), 7.42 (d, 1H), 7.61 (m, 2H), 7.64 (d, 1H), 7.97 (m br, 3H),
8.14 (s br, 1H); HRMS (C29H30N5O) [M + H]+: found m/z
464.2414, calcd 464.2445.
9-[2-(1H-Indol-3-yl)-ethylamino]-acridine-4-carboxylic Acid
Diethylamide (15e). Yield ) 52.6%; 1H NMR (600 MHz, CDCl3)
δ 1.02 (s br, 3H), 1.41 (s br, 3H), 3.22 (s br, 2H), 3.75 (m br, 2H),
4.18 (s br, 2H), 5.22 (s br, 2H), 7.03 (s br, 1H), 7.14 (t, 1H), 7.25
(m, 3H), 7.40 (d, 1H), 7.56 (m, 2H), 7.65 (d, 1H), 7.96 (m br, 3H),
8.23 (s br, 1H); HRMS (C28H29N4O) [M + H]+: found m/z
437.2320, calcd 437.2336.
{9-[2-(1H-Indol-3-yl)-ethylamino]-acridin-4-yl}-morpholin-4-
yl-methanone (15f). Yield ) 72.0%; 1H NMR (600 MHz, CDCl3)
δ 3.23 (s br, 4H), 3.46 (s br, 1H), 3.87 (m br, 4H), 4.19 (s br, 3H),
5.29 (s br, 1H), 7.06 (s br, 1H), 7.16 (t, 1H), 7.25 (m, 3H), 7.41 (d,
1H), 7.62 (m, 2H), 7.65 (d, 1H), 7.95 (m br, 3H), 8.18 (s br, 1H);
HRMS (C28H27N4O2) [M + H]+: found m/z 451.2106, calcd
451.2128.
[9-(1-Benzylpiperidin-4-ylamino)-acridin-3-yl]-(4-methylpip-
erazin-1-yl)-methanone (15g). Yield ) 40.1%; 1H NMR (600
MHz, CDCl3) δ 1.76 (m br, 2H), 2.08 (m br, 4H), 2.34 (s, 3H),
2.41 (s br, 2H), 2.54 (s br, 2H), 2.88 (s br, 2H), 3.52 (s, 2H), 3.61
(s br, 2H), 3.88 (s br, 3H), 4.74 (s br, 1H), 7.26 (m, 1H), 7.31 (m,
4H), 7.46 (m, 2H), 7.73 (t, 1H), 8.07 (d, 1H), 8.08 (s, 1H), 8.12 (d,
1H), 8.14 (d, 1H); HRMS (C31H36N5O) [M + H]+: found m/z
494.2894, calcd 494.2914.
9-(1-Benzylpiperidin-4-ylamino)-acridine-3-carboxylic Acid
Diethylamide (15h). Yield ) 10.4%; 1H NMR (600 MHz, CDCl3)
δ 1.17 (s br, 3H), 1.30 (s br, 3H), 1.75 (m br, 2H), 2.07 (m br,
4H), 2.88 (m br, 2H), 3.41 (s br, 2H), 3.52 (s, 2H), 3.63 (s br, 2H),
3.87 (s br, 1H), 4.72 (s br, 1H), 7.26 (m, 1H), 7.31 (m, 4H), 7.43
(m, 2H), 7.72 (t, 1H), 8.07 (m, 2H), 8.12 (m, 2H); HRMS
(C30H35N4O) [M + H]+: found m/z 467.2779, calcd 467.2805.
{9-[2-(1H-Indol-3-yl)-ethylamino]-acridin-3-yl}-(4-methyl-
piperazin-1-yl)-methanone (15i). Yield ) 29.6%; 1H NMR (600
MHz, CDCl3) δ 2.33 (s, 3H), 2.39 (s br, 2H), 2.52 (s br, 2H), 3.25
(t, 2H), 3.56 (s br, 2H), 3.87 (s br, 2H), 4.23 (t, 2H), 7.09 (s, 1H),
7.17 (t, 1H), 7.26 (t, 1H), 7.31 (m, 2H), 7.43 (d, 1H), 7.67 (m,
2H), 7.92 (d, 1H), 7.96 (d, 1H), 8.02 (s, 1H), 8.06 (d, 1H), 8.21 (s
br, 1H); HRMS (C29H30N5O) [M + H]+: found m/z 464.2414, calcd
464.2445.
1
Yield ) 41.5%; H NMR (300 MHz, CDCl3) δ 0.96 (s br, 3H),
1.24 (s br, 3H), 3.21 (s br, 2H), 3.53 (s br, 2H), 6.84 (d, 2H), 7.05
(t, 1H), 7.3 (m, 2H), 7.46 (t, 2H), 7.76 (m, 2H), 8.10 (t, 2H), 8.24
(d, 1H).
Morpholin-4-yl-(9-phenoxyacridin-4-yl)-methanone (14c). Yield
1
) 45.1%; H NMR (300 MHz, CDCl3) δ 3.22 (m, 1H), 3.28 (m,
1H), 3.51 (m, 1H), 3.77 (m, 1H), 3.85 (m, 1H), 3.90 (m, 1H), 4.04
(m, 1H), 4.26 (m, 1H), 6.84 (d, 2H), 7.06 (t, 1H), 7.28 (t, 2H),
7.47 (m, 2H), 7.77 (m, 2H), 8.09 (d, 1H), 8.13 (d, 1H), 8.25 (d,
1H); HRMS (C24H21N2O3) [M + H]+: found m/z 385.1529, calcd
385.1547.
(4-Methylpiperazin-1-yl)-(9-phenoxy-acridin-3-yl)-meth-
anone (14d). Yield ) 47.5%; 1H NMR (300 MHz, CDCl3) δ 2.34
(s, 3H), 2.41 (s br, 2H), 2.55 (s br, 2H), 3.58 (s br, 2H), 3.89 (s br,
2H), 6.87 (d, 2H), 7.07 (t, 1H), 7.28 (t, 2H), 7.50 (m, 2H), 7.81 (t,
1H), 8.11 (d, 1H), 8.16 (d, 1H), 8.25 (s, 1H), 8.27 (d, 1H); HRMS
(C25H24N3O2) [M + H]+: found m/z 398.1840, calcd 398.1863.
9-Phenoxyacridine-3-carboxylic Acid Diethylamide (14e).
1
Yield ) 55.5%; H NMR (300 MHz, CDCl3) δ 0.97 (s br, 3H),
1.26 (s br, 3H), 3.23 (s br, 2H), 3.54 (s br, 2H), 6.87 (d, 2H), 7.07
(t, 1H), 7.27 (t, 2H), 7.50 (m, 2H), 7.82 (t, 1H), 8.11 (d, 1H), 8.17
(d, 1H), 8.25 (s, 1H), 8.28 (d, 1H).
(4-Methylpiperazin-1-yl)-(9-phenoxy-acridin-2-yl)-meth-
anone (14f). Yield ) 41.3%; 1H NMR (300 MHz, CDCl3) δ 2.18
(s br, 2H), 2.30 (s, 3H), 2.46 (s br, 2H), 3.36 (s br, 2H), 3.79 (s br,
2H), 6.85 (d, 2H), 7.06 (t, 1H), 7.28 (m, 2H), 7.50 (m, 1H), 7.83
(m, 2H), 8.12 (m, 2H), 8.29 (t, 2H); HRMS (C25H24N3O2) [M +
H]+: found m/z 398.1837, calcd 398.1863.
9-Phenoxy-acridine-2-carboxylic Acid Diethylamide (14g).
1
Yield ) 42.7%; H NMR (300 MHz, CDCl3) δ 0.96 (s br, 3H),
1.24 (s br, 3H), 3.21 (s br, 2H), 3.53 (s br, 2H), 6.85 (d, 2H), 7.06
(t, 1H), 7.28 (m, 2H), 7.50 (m, 1H), 7.83 (m, 2H), 8.12 (m, 2H),
8.29 (t, 2H).
General Procedure for Compounds 15a-n. [9-(1-Benzylpi-
peridin-4-ylamino)-acridin-4-yl]-(4-methylpiperazin-1-yl)-meth-
anone (15a). (4-Methyl-piperazin-1-yl)-(9-phenoxy-acridin-4-yl)-
methanone (14a) (10 mmol) was dissolved in 2 mL of 4-amino-
N-benzylpiperidine and heated to 120 °C (reflux) for 15 min. The
reaction was cooled and run on a silica column to purify the product
and remove excess amine. A solvent ratio of 15:1 CH2Cl2:MeOH
was used for the first 200 mL followed by 15:1:0.1 CH2Cl2:MeOH:
NEt3 until the product eluted from the column. The fractions with
product were collect and concentrated. The yellow product was
dissolved in EtOAc and precipitated out with petroleum ether or
9-[2-(1H-Indol-3-yl)-ethylamino]-acridine-3-carboxylic Acid
Diethylamide (15j). Yield ) 45.5%; 1H NMR (600 MHz, CDCl3)
δ 1.14 (s br, 3H), 1.30 (s br, 3H), 3.32 (s br, 4H), 3.60 (s br, 2H),
4.29 (s br, 2H), 7.13 (m, 2H), 7.24 (m, 3H), 7.43 (d, 1H), 7.59 (t,
1H), 7.63 (d, 1H), 7.84 (s br, 1H), 7.94 (d, 1H), 8.06 (d, 1H), 8.10
1
hexanes. Yield ) 74.1%; H NMR (600 MHz, CDCl3) δ 1.74 (s

2136 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Goodell et al.
(s, 1H), 8.48 (s br, 1H); HRMS (C28H29N4O) [M + H]+: found
m/z 437.2308, calcd 437.2336.
with water, washed with brine, dried with MgSO4, and concentrated.
Product was purified on a silica column using 20:1 CH2Cl2:MeOH
as the eluting solvent. Fractions containing product were concen-
[9-(1-Benzylpiperidin-4-ylamino)-acridin-2-yl]-(4-methylpip-
erazin-1-yl)-methanone (15k). Yield ) 73.6%; 1H NMR (600
MHz, CDCl3) δ 1.75 (m br, 2H), 2.08 (m br, 4H), 2.35 (s, 3H),
2.48 (s br, 4H), 2.88 (s br, 2H), 3.52 (s, 2H), 3.61 (s br, 2H), 3.88
(m br, 3H), 4.84 (s br, 1H), 7.26 (m, 1H), 7.31 (m, 4H), 7.45 (t,
1H), 7.69 (d, 1H), 7.73 (t, 1H), 8.07 (d, 1H), 8.13 (m, 3H); HRMS
(C31H36N5O) [M + H]+: found m/z 494.2882, calcd 494.2914.
9-(1-Benzyl-piperidin-4-ylamino)-acridine-2-carboxylic Acid
Diethylamide (15l). Yield ) 50.7%; 1H NMR (600 MHz, CDCl3)
δ 1.23 (s br, 6H), 1.75 (m br, 2H), 2.07 (m br, 4H), 2.87 (m br,
2H), 3.41 (s br, 2H), 3.51 (s, 2H), 3.63 (s br, 2H), 3.88 (s br, 1H),
4.76 (s br, 1H), 7.26 (m, 1H), 7.30 (m, 4H), 7.44 (t, 1H), 7.68 (d,
1H), 7.74 (t, 1H), 8.07 (d, 1H), 8.12 (m, 2H), 8.22 (s, 1H); HRMS
(C30H35N4O) [M + H]+: found m/z 467.2776, calcd 467.2805.
{9-[2-(1H-Indol-3-yl)-ethylamino]-acridin-2-yl}-(4-methylpip-
1
trated, resulting in a yellow solid. Yield ) 53.8%; H NMR (300
MHz, CDCl3) δ 1.36 (s, 9H), 6.01 (s br, 2H), 6.61 (t, 1H), 6.73 (d,
1H), 7.28 (t, 1H), 7.46 (d, 2H), 7.49 (d, 1H), 7.60 (d, 2H).
(4-tert-Butylphenyl)-(2-isopropenylaminophenyl)-meth-
anone (27). Acetyl chloride (4.74 mmol) was slowly added to a
stirring solution of (2-amino-phenyl)-(4-tert-butyl-phenyl)-metha-
none (26) (3.95 mmol) in CH2Cl2 at 0 °C. The reaction was allowed
to warm to room temperature and stirred overnight. The reaction
was quenched with saturated NaCO3 (∼15 mL) and extracted with
CH2Cl2, washed with brine, dried over MgSO4, and concentrated.
Product was purified on a silica column using 40:1 CH2Cl2:MeOH
as the eluting solvent. Fractions containing product were concen-
trated and recrystallized in hexane, resulting in a pale yellow solid.
Yield ) 50.5%; 1H NMR (300 MHz, CDCl3) δ 1.37 (s, 9H), 2.17
(s, 3H), 7.08 (t, 1H), 7.50 (d, 2H), 7.56 (t, 1H), 7.58 (d, 1H), 7.66
(d, 2H), 7.60 (d, 1H), 10.74 (s br, 1H).
1
erazin-1-yl)-methanone (15m). Yield ) 60.3%; H NMR (600
MHz, CDCl3) δ 2.32 (s, 3H), 2.42 (s br, 4H), 3.23 (t, 2H), 3.49 (s
br, 2H), 3.82 (s br, 2H), 4.23 (t, 2H), 7.14 (m, 2H), 7.24 (t, 1H),
7.30 (t, 1H), 7.42 (d, 1H), 7.64 (m, 3H), 7.94 (d, 1H), 8.04 (t, 2H),
8.07 (s, 1H), 8.43 (s br, 1H); HRMS (C29H30N5O) [M + H]+: found
m/z 464.2415, calcd 464.2445.
4-(4-tert-Butylphenyl)-1H-quinolin-2-one (28). (4-tert-Butyl-
phenyl)-(2-isopropenylamino-phenyl)-methanone (27) (0.812 mmol)
was dissolved in 15 mL of absolute ethanol, and sodium methoxide
(2.44 mmol) was added. The reaction was set to reflux for 1 h.
The reaction was cooled and concentrated. The product was
extracted into CH2Cl2 and washed with water, brine, dried over
MgSO4, and concentrated. TLC showed relatively clean product
eluting with 9:1 CH2Cl2:MeOH, so no purification was performed.
Yield ) 78.8%; 1H NMR (300 MHz, CDCl3) δ 1.41 (s, 9H), 6.67
(s, 1H), 7.17 (t, 1H), 7.50 (m, 7H), 10.97 (s br, 1H).
4-(4-tert-Butylphenyl)-2-chloroquinoline (29). 4-(4-tert-Butyl-
phenyl)-1H-quinolin-2-one (28) (2.41 mmol) was refluxed in 20
mL of POCl3 for 2 h. The reaction started out faint green and turned
clear orange after ∼1 h. The reaction was cooled and poured over
ice. The mixture was then extracted with CH2Cl2 (3 × 50 mL),
dried over MgSO4, and concentrated. A short column was run to
purify product eluting with 9:1 CH2Cl2:MeOH. Yield ) 79.9%;
1H NMR (300 MHz, CDCl3) δ 1.41 (s, 9H), 7.35 (s, 1H), 7.49 (m,
5H), 7.74 (t, 1H), 7.95 (d, 1H), 8.08 (d, 1H).
4-(4-tert-Butylphenyl)-2-(4-methylpiperazin-1-yl)-quinoline (30).
This followed the same procedure as compound 21. Dihydrochloride
salt was made by dissolving the compound in saturated HCl in
ethanol and rotovaping. Yield ) 73.9%; 1H NMR (300 MHz,
CDCl3) δ 1.41 (s, 9H), 2.37 (s, 3H), 2.57 (s br, 4H), 6.91 (s, 1H),
7.16 (t, 1H), 7.42 (m, 2H), 7.53 (m, 3H), 7.68 (d, 1H), 7.76 (d,
1H); HRMS (C24H30N3) [M + H]+: found m/z 360.2429, calcd
360.2434.
9-[2-(1H-Indol-3-yl)-ethylamino]-acridine-2-carboxylic Acid
Diethylamide (15n). Yield ) 82.8%; 1H NMR (600 MHz, CDCl3)
δ 1.11 (s br, 3H), 1.28 (s br, 3H), 3.22 (t, 2H), 3.26 (s br, 2H),
3.58 (s br, 2H), 4.24 (t, 2H), 7.11 (s, 1H), 7.15 (t, 1H), 7.24 (t,
1H), 7.30 (t, 1H), 7.42 (d, 1H), 7.64 (m, 3H), 7.93 (d, 1H), 7.97 (s,
1H), 8.01 (s br, 2H), 8.43 (s br, 1H); HRMS (C28H29N4O) [M +
H]+: found m/z 437.2310, calcd 437.2336.
2-(4-tert-Butylphenyl)-4-chloroquinoline (20). Anthranilic acid
(10.9 mmol) and acetophenone (10.9 mmol) were heated to reflux
in ∼50 mL of POCl3 for 2 h. Excess POCl3 was distilled off under
vacuum, and the remaining was oily solid was poured into a mixture
of 20 mL of NH4OH and 100 mL of ice. When the mixture had
melted, it was extracted with EtOAc. TLC indicated several
products using 15:1 hexane:EtoAc. The same ratio was used for
purification via silica chromatography. 1H NMR (300 MHz, CDCl3)
δ 1.38 (s, 9H), 7.55 (d, 2H), 7.60 (t, 1H), 7.76 (t, 1H), 7.96 (s,
1H), 8.07 (d, 2H), 8.17 (d, 1H), 8.22 (d, 1H).
2-(4-tert-Butylphenyl)-4-(4-methylpiperazin-1-yl)-quinoline (21).
2-(4-tert-Butyl-phenyl)-4-chloro-quinoline (20) was dissolved in
N-methylpiperazine and refluxed overnight. The reaction was cooled
and placed on a vacuum pump to remove excess amine. The
resulting solid was purified using silica gel chromatography eluting
1
with 20:1 CH2Cl2:MeOH. H NMR (300 MHz, CDCl3) δ 1.37 (s,
General Procedure for 1,3,5-Trisubstituted Pyrazolines (31-
43). This followed the same procedure as described for pyrazolines
of general structure 5. As part of a focused library, compounds
were only characterized by APCI-HRMS for product verification.
1-(4-Nitrobenzoyl)-5-(4-chlorophenyl)-3-(thiophen-2-yl)-4,5-
dihydro-1H-pyrazole (31). HRMS (C20H15ClN3O3S) [M + H]+:
found m/z 412.0620, calcd 412.0528.
1-(Phenylacetyl)-5-(4-nitrophenyl)-3-(thiophen-2-yl)-4,5-dihy-
dro-1H-pyrazole (32). HRMS (C21H18ClN2OS) [M + H]+: found
m/z 381.0676, calcd 381.0833.
1-(4-Chlorophenylacetyl)-5-(4-chlorophenyl)-3-(thiophen-2-
yl)-4,5-dihydro-1H-pyrazole (33). HRMS (C21H17Cl2N2OS) [M +
H]+: found m/z 415.0273, calcd 415.0444.
1-(Trimethylacetyl)-5-(4-chlorophenyl)-3-(thiophen-2-yl)-4,5-
dihydro-1H-pyrazole (34). HRMS (C18H20ClN2OS) [M + H]+:
found m/z 347.1005, calcd 347.0990.
9H), 2.44 (s, 3H), 2.74 (s br, 4H), 3.33 (s br, 4H), 7.29 (s, 1H),
7.44 (t, 1H), 7.53 (d, 2H), 7.65 (t, 1H), 8.01 (d, 3H), 8.11 (d, 1H);
HRMS (C24H30N3) [M + H]+: found m/z 360.2536, calcd 360.2434.
2-Amino-N-methoxy-N-methylbenzamide (24). N,O-Dimeth-
ylhydroxylamine‚HCl (23) (6.13 mmol) was dissolved in 10 mL
of 90% ethanol (aq). Triethylamine (9.20 mmol) was then added
and stirred at room temperature for 10 min. Next, isatoic anhydride
(22) (9.50 mmol) was added in portions. The reaction was heated
to reflux and stirred for ∼2 h and then cooled. Ethanol was removed
by rotovap, and the reaction was poured into a 50/50 mix of
saturated NaHCO3/ice (∼40 mL), then extracted with EtOAc, dried
over MgSO4, and condensed. Product was purified on a long column
using a 20:1 CH2Cl2:MeOH elution mixture. Fractions containing
product were concentrated, resulting in a light tan oil. Yield )
1
51.5%; H NMR (300 MHz, CDCl3) δ 3.35 (s, 3H), 3.60 (s, 3H),
4.64 (s br, 2H), 6.70 (d, 2H), 7.18 (t, 1H), 7.36 (t, 1H).
1-(4-Chlorophenylacetyl)-5-(4-bromophenyl)-3-(thiophen-2-
yl)-4,5-dihydro-1H-pyrazole (35). HRMS (C21H17BrClN2OS) [M
+ H]+: found m/z 458.9740, calcd 458.9938.
1-Benzoyl-5-(4-fluorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-
1H-pyrazole (36). HRMS (C20H16FN2OS) [M + H]+: found m/z
351.0815, calcd 351.0972.
1-(4-Chlorophenylacetyl)-5-(4-methoxyphenyl)-3-(thiophen-
2-yl)-4,5-dihydro-1H-pyrazole (37). HRMS (C22H20ClN2O2S) [M
+ H]+: found m/z 411.0771, calcd 411.0939.
(2-Aminophenyl)-(4-tert-butylphenyl)-methanone (26). 2-Amino-
N-methoxy-N-methyl-benzamide (24) (5.55 mmol) and 1-bromo-
4-tert-butylbenzene (25) (5.55 mmol) were dissolved in dry THF
and placed under argon. The reaction mixture was then cooled to
-78 °C and vigorously stirred. Next, 2.87 M n-butyllithium in
cyclohexane (11.1 mmol) was added dropwise, and the reaction
was stirred for ∼30 min at -78 °C. Next, 1 M HCl (aq) (∼15 mL)
was added, and the reaction was allowed to warm to room
temperature. The reaction was then extracted with EtOAc, washed

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hydro-1H-pyrazole (43). HRMS (C18H26ClN2O) [M + H]+: found
m/z 321.1665, calcd 321.1739.
Acknowledgment. This work has been supported in part
by grant AI061193 from the National Institutes of Health.
Note Added after ASAP Publication. This manuscript was
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