Imidazo[1,2-b]pyridazines, novel nucleus with potent and broad spectrum activity against human picornaviruses: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1021/jm020583i

A novel structural class of picornavirus inhibitors comprising an imidazo[1,2-b]pyridazine nucleus was discovered. 2-Aminoimidazo[1,2-b]pyridazines (6d, (E/Z)-7b, (E)-7d, (Z)-7d, (E/ Z)-8b, (E)-10b, (E)-13a, (Z)-13a, (E)-13b, (Z)-13b, (E)-13c, and (Z)-13c) were designed and synthesized in an effort to identify potent broad spectrum antirhinoviral agents. A practical synthetic route to this chemical scaffold has been developed. The target compounds were evaluated in a plaque reduction assay and in a cytopathic effect assay. Our preliminary SAR studies highlight the minimum structural features required for antirhinovirus activity. Our data suggest that the nature of the linker between the phenyl and the imidazopyridazine moieties has a significant influence on the activity of these compounds. Oximes are slightly better than vinyl carboxamides at this position. The oximes are the most potent analogues against human rhinovirus 14 (HRV-14), and at the concentrations evaluated, no apparent cellular toxicity is noted. Furthermore, the E geometry appears to be a key element for activity; the Z isomer leads to a considerable loss in potency. Of particular interest, analogue 7b exhibits potent broad-spectrum antirhinoviral and antienteroviral activity when evaluated against a panel of seven additional rhino- and enteroviruses. The chemistry and the biological evaluations are discussed.

Full text of DOI:10.1021/jm020583i

J . Med. Chem. 2003, 46, 4333-4341
4333
Im id a zo[1,2-b]p yr id a zin es, Novel Nu cleu s w ith P oten t a n d Br oa d Sp ectr u m
Activity a ga in st Hu m a n P icor n a vir u ses: Design , Syn th esis, a n d
Biologica l Eva lu a tion
Chafiq Hamdouchi,*,† Concha Sanchez-Martinez,‡ J oseph Gruber,† Miriam del Prado,‡ J avier Lopez,‡
Almudena Rubio,† and Beverly A. Heinz†
Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, DC: 0540,
Indianapolis, Indiana 46285, and Centro de Investigacio´n Lilly, Avenida de la Industria,
30.28108 Alcobendas, Madrid, Spain
Received December 27, 2002
A novel structural class of picornavirus inhibitors comprising an imidazo[1,2-b]pyridazine
nucleus was discovered. 2-Aminoimidazo[1,2-b]pyridazines (6d , (E/Z)-7b, (E)-7d , (Z)-7d , (E/
Z)-8b, (E)-10b, (E)-13a , (Z)-13a , (E)-13b, (Z)-13b, (E)-13c, and (Z)-13c) were designed and
synthesized in an effort to identify potent broad spectrum antirhinoviral agents. A practical
synthetic route to this chemical scaffold has been developed. The target compounds were
evaluated in a plaque reduction assay and in a cytopathic effect assay. Our preliminary SAR
studies highlight the minimum structural features required for antirhinovirus activity. Our
data suggest that the nature of the linker between the phenyl and the imidazopyridazine
moieties has a significant influence on the activity of these compounds. Oximes are slightly
better than vinyl carboxamides at this position. The oximes are the most potent analogues
against human rhinovirus 14 (HRV-14), and at the concentrations evaluated, no apparent
cellular toxicity is noted. Furthermore, the E geometry appears to be a key element for activity;
the Z isomer leads to a considerable loss in potency. Of particular interest, analogue 7b exhibits
potent broad-spectrum antirhinoviral and antienteroviral activity when evaluated against a
panel of seven additional rhino- and enteroviruses. The chemistry and the biological evaluations
are discussed.
In tr od u ction
Picornaviruses cause numerous human diseases, in-
cluding poliomyelitis, acute hepatitis, myocarditis, and
the common cold. Family members include several well-
known human pathogens such as polioviruses, hepatitis
A virus, coxsackieviruses, and over 110 serotypes of
human rhinoviruses (HRV). Despite the fact that some
picornaviral infections are mild and self-limiting in
healthy adults, serious sequelae can occur in children
or patients with pre-existing medical problems. There-
fore, the need for new prevention and treatment options
for picornaviral infections has focused on the discovery
of effective vaccines and potent antiviral agents.
The human rhinoviruses are recognized as the most
F igu r e 1.
important etiologic agents of the common cold in adults
and children.1 In addition to causing the common cold,
these viruses also typically precipitate or exacerbate
several chronic conditions such as bronchitis, otitis
media, sinusitis, emphysema, and asthma. Because of
their significant association with disease, the search for
an effective treatment for rhinovirus infections has
attracted considerable attention in the scientific com-
munity. Since the large number of serotypes makes the
development of a broad-spectrum vaccine unlikely,
efforts have focused on the development of effective
antivirals. Several classes of antiviral drugs have been
studied for the treatment of rhinovirus colds, including
those that bind directly to the virus and inhibit virus
uncoating, compounds that block the attachment of
rhinovirus to ICAM-1, and compounds that inhibit
activity of the viral protease. However, despite good in
vitro activity, the majority of these compounds have
been ineffective when dosed by either oral or intranasal
administration, the routes preferred for the treatment
of common colds. Antiviral compounds have been re-
jected as clinical candidates because of problems with
toxicity, unfavorable pharmacology, or insufficient po-
tency. Thus, the treatment of the common cold remains
an elusive goal.
Enviroxime (Figure 1), a benzimidazole derivative
with strong in vitro antirhinoviral activity, is one of the
more extensively studied synthetic agents.2 Enviroxime
and related benzimidazoles3 showed potent broad-
* To whom correspondence should be addressed. E-mail:
Hamdouchi_Chafiq@Lilly.com. Phone: 317-276-9582. Fax: 317-277-
3652.
† Lilly Research Laboratories.
‡ Centro de Investigacio´n Lilly.
10.1021/jm020583i CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/21/2003

4334 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Hamdouchi et al.
Sch em e 1
spectrum antiviral activity against a wide range of both
rhinoviruses and enteroviruses. Noncytotoxic concentra-
tions of enviroxime are associated with complete inhibi-
tion of replication of 81 rhinovirus serotypes, with 50%
inhibitory concentrations (IC50) ranging from 0.05 to
0.12 µg/mL. Although the mechanism of action of
enviroxime is not completely understood, it is believed
that the drug inhibits the formation of the viral RNA
polymerase replication complex.4
The major shortcomings of enviroxime are its poor
oral bioavailability and undesirable side effects. Studies
of oral enviroxime indicated low drug levels in blood and
nasal secretions and an unacceptably high frequency of
nausea and vomiting.5 Thus, despite its potent in vitro
antirhinovirus activity, significant therapeutic benefit
was not found with either oral or intranasal adminis-
tration, and enviroxime clinical studies were termi-
nated. Enviradene, a related benzimidazole, showed
improved pharmacokinetics in dogs and caused no
emesis.6 However, the peak plasma levels did not
surpass the antiviral IC50 value, and the studies on
enviradene were discontinued.
In our laboratory, a series of 3-substituted imidazo-
[1,2-a]pyridines were recently found to have strong
activity against human rhinovirus.7 Structure-activity
relationship (SAR) studies of imidazo[1,2-a]pyridines
demonstrated that substitution at the 3-position is
essential for antiviral activity and that the isopropyl
sulfonyl group present in enviroxime is not critical for
activity.
As part of our continuous efforts toward the identi-
fication of potent broad-spectrum antirhinoviral agents,
structurally related imidazo[1,2-b]pyridazine analogues
were examined (Figure 1). This series has shown
significant improvement in potency relative to the
imidazo[1,2-a]pyridines series as well as broad-spectrum
activity against a variety of rhinovirus serotypes.
[1,2-a]pyridine7 SARs would be transferable to the
imidazo[1,2-b]pyridazine series. On the basis of this
analysis, we undertook the synthesis of 2-aminoimidazo-
[1,2-b]pyridazines (7-13) that would offer the following
potential advantages: (a) potent antiviral activity (struc-
turally related to benzimidazoles), (b) broad spectrum
of activity, (c) enhanced oral bioavailability, and (d)
fewer side effects.
The synthesis started with the condensation of a
series of para-substituted phenylacetonitriles with 3,6-
dichloropyridazine under basic conditions (Scheme 1).
The reaction accommodated a variety of commercially
available para-substituted phenylacetonitriles. Subse-
quent oxidation with hydrogen peroxide afforded the
corresponding 6-chloro-3-benzoylpyridazines, which were
treated with ammonia in ethanol to yield the desired
6-amino-3-benzoylpyridazines 1a -c. Tosylation of the
amino group with p-tolylsulfonyl chloride in pyridine
and subsequent reaction with the 2-bromo-2-arylaceta-
mides 3a ,b in the presence of Hu¨nig’s base in DMF
provided the corresponding pyridazinyl acetamides
4a -d in good yield. The 2-bromo-2-arylacetamides 3a ,b
were prepared from their corresponding 2-arylacetic
acid by bromination in the presence of N-bromosuccin-
imide and subsequent amidation. Conversion of 4a -d
to the desired 2-(N-trifluoracetylamino)imidazopyridazine
ketones 5a -d was accomplished by treatment with
trifluoroacetic anhydride.
We next turned our efforts to the derivatization of the
ketone moiety. The trifluoroacetamide group in com-
pounds 5b,d was hydrolyzed with 3 N NaOH to give
the corresponding aminoimidazopyridazines 6b,d in
quantitative yield. Compounds 6b,d were then treated
with NH2OH‚HCl to yield the desired oximes 7b,d as
mixtures of E and Z isomers. The Z isomers were
typically isolated after recrystallization from a mixture
of ethyl acetate/hexane while the E isomers were
separated from the remaining mother liquors by chro-
matography. The assignment of the geometry of the
double bond was based on the following nuclear Over-
hauser enhancement (NOE) results. Irradiation of HO
at δ 11.87 in (E)-7 gave an enhancement of the aryl
protons. No enhancement was observed for the py-
Ch em istr y
We initiated an SAR exploration of imidazo[1,2-b]-
pyridazine derivatives as potential antirhinoviral agents.
We made the initial assumption that structural infor-
mation gained from the benzimidazole3 and imidazo-

Imidazo[1,2-b]pyridazines
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4335
Sch em e 2
Sch em e 3
The stereospecificity of the reaction depends on the
substrate, the solvent, and the reaction time. In THF
(35 h), compound 5b gave a mixture of E and Z isomers
(about 4:1). In DMF, the reaction was much faster (5 h)
but the E/Z ratio decreased to 1:5. Interestingly, equi-
librium was produced in favor of the E isomer (about
3:1) when the reaction was performed in DMF and
quenched after 48 h. The isomers were separated either
by crystallization or by chromatography. In the case of
compound 12b, simple crystallization from ethyl acetate
afforded (E)-12b in its pure form. The assignment of
the geometry of the double bond was based on the
following NOE results (Scheme 5). Irradiation of Ha at
δ 7.0 in (E)-12b gave an enhancement of the vinyl
proton (Hd). No enhancement was observed for the
proton (He). Irradiation of Hd at δ 6.7 gave enhancement
of the pyridazine proton (Ha) and the aromatic proton
of the 4-fluorphenyl. Conversion of 12a -c to their
corresponding final targets 13a -c was performed by
cleavage of the trifluoroacetamide group with DIPEA
in refluxing methanol.
ridazine protons. A stability study on the final oximes
7b,d showed an equilibrium between the Z and E
isomers forms in polar solvents such as DMSO and TFA
at room temperature.
Treatment of the ketone 6b with PhNHNH2‚HCl in
refluxing ethanol led to the corresponding phenylhy-
drazone 8b that was shown to exist as a rapidly
equilibrating mixture of E and Z geometric isomers
(Scheme 2).
Efforts to overcome the configurational instability
suggested the need to evaluate additional unsaturated
linkers. Treatment of the ketone 5b with the Horner-
Wadsworth-Emmons reagent, potassium salt of triethyl
phosphonoacetate, afforded the corresponding R,â-
unsaturated ester 9b as a mixture (about 13:1) of E and
Z isomers. Hydrolysis of the trifluoroacetamide group
with DIPEA in refluxing ethanol furnished the final
aminoimidazo[1,2-b]pyridazine (E)-10b (Scheme 3).
The incorporation of the vinyl carboxamide group was
achieved using the protocol outlined in Scheme 4. When
ketones 5a -c were subjected to the Horner-Wad-
sworth-Emmons reaction with the potassium salt of
diethyl (N-methylcarbamoylmethyl)phosphonate 11,7
the desired vinyl carboxamides 12a -c were isolated as
mixtures of E and Z isomers.
Str u ctu r e-Activity Rela tion sh ip An a lysis
2-Aminoimidazo[1,2-b]pyridazines 6d , (E/Z)-7b, (E)-
7d , (Z)-7d , (E/Z)-8b, (E)-10b, (E)-13a , (Z)-13a , (E)-13b,
(Z)-13b, (E)-13c, and (Z)-13c were subjected to biological
evaluation following the procedures described in the
Experimental Section. The activity of new analogues
was compared to that of enviroxime used as a reference
standard. Human rhinovirus 14 (HRV-14) was selected
as the prototype virus for testing because of the amount
of information known about this serotype. Plaque
reduction assays were used to determine the antiviral
activity reported as the IC50 (Table 1). This family of
compounds exhibited potent antirhinoviral activity. Our
Sch em e 4

4336 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Hamdouchi et al.
Ta ble 2. Broad-Spectrum Antiviral Activity Evaluation of 7d a
Sch em e 5
assay
IC50 (µg/mL)
PR-HRV14
PR-HRV1A
PR-HRV2
PR-HRV16
PR-PV1
PR-CA21C
PR-CA21M
PR-CB3
0.04
0.05
0.03
0.06
0.04
0.04
0.02
0.05
0.04
average
a
PRA assay, IC50 (µg/mL), using four human rhinoviruses
(HRV-14, HRV-1A, HRV-2, and HRV-16), a prototypical enterovi-
rus (poliovirus 1 (PV-1)), coxsackievirus A21 (CA21), coxsackievi-
rus A21 mouse muscle adapted (CA21M), and coxsackievirus B3
(CB3).
Ta ble 1. Antiviral Activity Evaluation of Imidazopyridazines
IC50 (µg/mL) TC50 (µg/mL)
compd
X
Y
B-A
PRAa
CPE/XTTb
enviroxime
6d
(E/Z)-7b
(E)-7d
(E/Z)-8b
(E)-10b
(E)-13a
(Z)-13a
(E)-13b
(Z)-13b
(E)-13c
(Z)-13c
0.05
7.19
0.05
0.04
>10
>10
0.6
H
H
H
H
H
H O
F N-OH
H N-OH
F N-NHPh
F CH-CO2Et
that were chosen as representative of the large number
of human rhinoviruses and human enteroviruses. In-
cluded in this panel were two viruses from the minor
rhinovirus receptor subgroup, human rhinovirus 1A
(HRV-1A) and human rhinovirus 2 (HRV-2); one ad-
ditional virus from the major rhinovirus receptor sub-
group, human rhinovirus 16 (HRV-16); a prototypical
enterovirus, poliovirus 1 (PV-1); and three nonpolio
enteroviruses, coxsackievirus A21 (CA21), coxsackievi-
rus A21 mouse muscle adapted (CA21M), and coxsack-
ievirus B3 (CB3). We were pleased to find that com-
pound 7d exhibited potency and broad-spectrum activity
against all these viruses (Table 2).
>10
>10
>10
MeO F CH-CONHMe
MeO F CH-CONHMe
H
H
Me
Me
>10
>10
4.06
4.2
F CH-CONHMe
F CH-CONHMe
F CH-CONHMe
F CH-CONHMe
0.11
>10
0.23
>10
>10
>10
a
b
PRA assay using rhinovirus 14. CPE/XTT assay using rhi-
novirus 14. The concentration of test compound required to cause
50% cytotoxic effect (TC50) relative to a control with no drug and
no virus present and that inhibits the development of virus
cytopathic effect (CPE) by 50% (IC50) was determined from the
linear portion of each dose-response curve.
Con clu sion
Enviroxime and related benzimidazoles with potent
broad-spectrum antirhinoviral and antienteroviral ac-
tivity represent a unique opportunity for the treatment
of the common cold. These compounds are believed to
inhibit the formation of the viral RNA polymerase
replication complex. However, despite good in vitro
activity, they have been ineffective when given in a
manner that would be acceptable for the treatment of
colds (namely, by either oral or intranasal administra-
tion). Therefore, the identification of a novel nucleus
that maintains the potency and broad-spectrum activity
of enviroxime and offers new physiochemical properties
is highly desirable. The compounds described here
represent a novel lead series for the development of
antipicornavirus agents. Members of this series of
inhibitors have shown potency that is comparable to
that of enviroxime. 2-Aminoimidazo[1,2-b]pyridazines
showed a high potency when evaluated against HRV-
14 with no apparent associations between cellular
toxicity and antiviral activity. Furthermore, compound
7d has shown broad-spectrum activity against a panel
of seven additional viruses that were chosen as repre-
sentative of the large number of human pathogens.
Finally, a practical synthetic route to this chemical
scaffold has been developed.
data suggest that the nature of the linker between the
phenyl and the imidazopyridazine moieties has a sig-
nificant influence on the activity of these compounds.
The oximes (E)-7d and (E/Z)-7b showed potency (IC50
) 0.04 and 0.05 µg/mL) comparable to that of en-
viroxime. The vinylcarboxamides that would be expected
to participate in hydrogen-bond formation with their
target in an analogous way to the oximes8 were also
found to be potent ((E)-13b, IC50 ) 0.11 µg/mL). A
complete loss of activity was observed with the vinyl
esters and with larger groups such as phenylhydra-
zones. Furthermore, the E geometry appears to be a key
element for activity; the Z isomer led to a considerable
loss in potency.
To establish a general cellular toxicity of this chemical
platform and to determine whether it correlates to the
antiviral activity, imidazo[1,2-b]pyridazines were also
tested in a cytopathic effect (CPE) assay. The CPE assay
was performed as described previously9 with a modifica-
tion to the method of the quantification of the cytopathic
effect of virus on the cells. Instead of using crystal violet
staining, XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-
2H-tetrazolium-5-carboxanilide) was used as a substrate
to quantify the surviving host cells. We were pleased to
find that, in general, the concentration of test compound
that resulted in a 50% cytotoxic effect (TC50) relative to
a control with no drug and no virus present was
considerably high. Our data indicate that there are no
apparent associations between cellular toxicity and
antiviral activity.
Exp er im en ta l Section
Gen er a l Meth od s. All reagents were purchased from
Aldrich and used without further purification unless stated
otherwise. Column chromatography was carried out on flash
silica gel (Merck 230-400 mesh). TLC analysis was conducted
on Whatman silica gel plates. Melting points were determined
with a Thomas-Hoover melting point apparatus and are
uncorrected. 1H NMR spectra were recorded in the solvent in-
dicated on a Bruker spectrometer at 200 MHz, a Varian Mer-
In addition to screening imidazopyridazines against
HRV-14, we examined the broad-spectrum nature of this
chemical platform. Compound 7d was selected for
screening against a panel of seven additional viruses

Imidazo[1,2-b]pyridazines
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4337
cury spectrometer at 400 MHz, or a GE QE-300 spectrometer
at 300.15 MHz. 13C NMR spectra were recorded in the solvents
indicated on the previously mentioned spectrometers at 50 Mz,
100 MHz, and 75 MHz, respectively. IR spectra were recorded
on a Nicolet 510P FT-IR spectrometer; UV spectra were
recorded on a Shimadzu UV-2101 PC spectrometer; and field
desorption (FD) mass spectra were recorded on either a VG
ZAB-3F or VG 70-SE instrument. High-resolution mass spec-
tra were recorded on a Micromass QTOF mass spectrometer.
IR spectra, UV spectra, FDMS spectra, elemental analyses,
and some 1H NMR spectra were provided by the Physical
Chemistry Department at Lilly Research Laboratories.
6-Am in o-3-(4-m eth oxyben zoyl)p yr id a zin e (1a ). Step 1:
6-Ch lor o-3-(4-m eth oxyben zoyl)p yr id a zin e. Potassium tert-
butoxide (37.66 g, 335.62 mmol) was added to a stirred solution
of 4-methoxyphenylacetonitrile (167.81 mmol, 22.76 mL) in 200
mL of N,N-dimethylacetamide (DMA) at 0 °C under nitrogen..
The reaction mixture was stirred for 30 min, and 3,6-dichlo-
ropyridazine (25 g, 167.81 mmol) was added in one portion.
The ice bath was then removed, and the reaction mixture was
stirred at room temperature for 1 h. The reaction mixture was
again cooled to 0 °C, and hydrogen peroxide (30 mL of a 30%
solution, 326 mmol) was added dropwise. The reaction mixture
was stirred for an additional hour, then poured onto 1 N HCl
(1 L), and extracted with ethyl acetate (3 × 300 mL). The
organic layers were combined and washed with 1 N NaOH (3
× 200 mL) and brine (3 × 150 mL). After drying over MgSO4,
the solvents were removed in vacuo and the remaining solid
recrystallized from ethyl acetate/hexane to yield 21.92 g (53%)
of the desired product: 1H NMR (300 MHz, DMSO-d6) δ 8.17
(AB system, 2H, J ) 8.9, Het), 7.05 (AA′BB′ system, 4H, Ar),
3.85 (s, 3H, OMe); FDMS m/z 250/248 (M+). Anal. Calcd for
(d, J ) 9.6, 1H, Het), 7.29 (AA′BB′ system, 2H, Ar), 6.97
(AA′BB′ system, 2H, Ar), 3.90 (s, 3H, OMe) 2.42 (s, 3H, Me);
13C NMR (DMSO-d6) δ 187.5, 163.7, 156.0, 142.7, 139.7, 133.4,
132.5, 129.7, 127.0, 126.4, 124.1, 113.9, 55.8, 21.1; MS (EI+)
m/z 383 M+ (9), 318 (63), 229 (10), 135 (100), 107 (6), 91 (34),
65 (12); HRMS calcd for
383.094 24.
C19H17N3O3S 383.093 98, found
N-(6-Ben zoyl)-2H -p yr id a zin -3-ylid en e-4-m et h ylb en -
zen esu lfon a m id e (2b). Compound 2b was prepared accord-
ing to the procedure for 2a : 14.72 g (83%); mp 136 °C; 1H NMR
(300 MHz, DMSO-d6) δ 8.11 (d, J ) 9.8, 1H, Het), 7.93 (m,
3H, Ar + Het), 7.55 (AA′BB′ system, 2H), 7.64 (m,1H), 7.49
(m, 2H, Ar), 2.32 (s,3H); HRMS calcd for C18H15N3O3S 353.0834,
found 353.0829.
N-[6-(4-Meth ylben zoyl)-2H-pyr idazin -3-yliden e)-4-m eth -
ylben zen esu lfon a m id e (2c). Compound 2c was prepared
according to the procedure for 2a : 11.5 g (76%); mp 155 °C; 1H
NMR (300 MHz, DMSO-d6) δ 8.12 (d, J ) 9.7, 1H, Het), 7.95
(d, J ) 9.7, 1H, Het), 7.86 (AA′BB′ system, 2H, Ar), 7.78
(AA′BB′ system, 2H, Ar), 7.36 (AA′BB′ system, 2H, Ar), 7.32
(AA′BB′ system, 2H, Ar), 2.38 (s, 3H, Me), 2.35(s, 3H, Me);
13C NMR (75 MHz, DMSO-d6) δ 188.2, 155.4, 143.5, 142.1,
139.0, 131.9, 131.6, 129.0, 128.7, 128.3, 125.8, 123.4, 114.2,
20.7, 20.4; MS (EI+) m/z 367 M+ (12), 302 (100), 119 (83), 91
(84), 65 (31); HRMS calcd for C19H17N3O3S 367.099 61, found
367.099 15.
2-Br om o-2-(p-flu or op h en yl)a ceta m id e (3a ). Step 1: 4-
F lu or op h en yla ceta m id e. A solution of oxalyl chloride (200
mmol) in CH2Cl2 (25 mL) was added dropwise over 25 min to
a stirred solution of 4-fluorophenylacetic acid (21 g, 100 mmol)
and DMF (3 drops) in dry CH2Cl2 (170 mL) at 0 °C under
nitrogen. The ice bath was removed, and the reaction mixture
was stirred for 3 h. The solvents were removed under vacuo
and then azeotroped with toluene (3 × 25 mL). The remaining
oil was dissolved in 300 mL of toluene and 300 mL of hexane,
and the mixture was stirred vigorously with a mechanical
stirrer. Ammonia gas was then blown through a gas dispersion
tube over the top of this solution for 1 h. The resulting solid
was filtered and then dissolved in EtOAc/H2O. The organic
layer was washed with 1 N HCl, saturated NaHCO3, and brine
and was dried over sodium sulfate. The solvent was removed
in vacuo and the remaining solid was recrystallized from
EtOAc/hexane to yield 4-fluorophenylacetamide (87%) as a
white solid: 1H NMR (200 MHz, CDCl3) δ 7.29 (dd, J ) 8.9, J
) 5.6, 2H, Ar), 7.12 (dd, J ) 9.0, J ) 8.9, 2H, Ar), 6.00 (bs,
1H, NH2), 5.35 (bs, 1H, NH2) 3.59 (s, 2H, CH2).
Step 2: 2-Br om o-2-(p-flu or op h en yl)a ceta m id e (3a ). To
a suspension of 4-fluorophenylacetamide (6.5 mmol, 1 equiv)
in 25 mL of CCl4 was added NBS (6.5 mmol, 1 equiv). The
mixture was refluxed under UV radiation for 6 h. The reaction
mixture was cooled to room temperature and then washed with
water and brine. The residue was purified by flash chroma-
tography (ethyl acetate/hexane, 1:3): mp 112 °C; 1H NMR (200
MHz, CDCl3) δ 7.5 (dd, J ) 8.8, J ) 5.5, 2H, Ar), 7.12 (dd, J
) 8.8, J ) 8.9, 2H, Ar), 6.6 (bs, 1H, NH2), 6.1 (bs, 1H, NH2),
5.43 (s, 1H, CH); 13C NMR (DMSO-d6) δ 168.9, 164.7 134.0,
131.0, 130.8, 115.7, 115.3, 48.5; MS (EI+) m/z 230 M+ (65), 214
(100), 188 (22),186 (21), 152 (77), 136 (24), 124 (56), 109 (100),
108 (64), 107 (48), 85 (47), 83 (74), 80 (15).
C
12H9N2O2Cl: C, 57.96; H, 3.65; N, 11.26. Found: C, 57.98;
H, 3.64; N, 11.29.
Step 2: 6-Am in o-3-(4-m eth oxyben zoyl)p yr id a zin e (1a ).
To a solution of 6-chloro-3-(4-methoxybenzoyl)pyridazine (21.92
g, 88.15 mmol) in 350 mL of EtOH was added 125 mL of
anhydrous ammonia. The mixture was then placed in a bomb
and heated to 145 °C for 16 h. The solvents were removed,
and the solids were recrystallized from ethyl acetate to yield
15.76 g (78%) of 6-amino-3-(4-methoxybenzoyl)pyridazine 1a :
mp 136 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.05 (AA′ of
AA′BB′ system, 2H, J ) 8.9, Ar), 7.8 (A of AB system, 1H, J
) 12, Het), 7.16 (bs, 2H, exchange D2O, NH2), 7.05 (BB′ of
AA′BB′ system, 2H, J ) 8.9, Ar), 6.85 (B of AB system, 1H, J
) 12, Het), 3.82 (s, 3H, Me); HRMS calcd for C12H11N3O2
229.0851, found 353.0846.
6-Am in o-3-ben zoylp yr id a zin e (1b). Compound 1b was
prepared according to the procedure for 1a : 13.35 g (75%);
mp 122 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.95-7.92 (m,
2H, Ar), 7.34 (AB system, 2H, J ) 9.2, Het), 7.60-7.51 (m,
1H, Ar), 7.48-7.46 (m, 2H, Ar), 7.23 (bs, 2H, exchange D2O,
NH2); FDMS m/z 200 (M + H)+. Anal. Calcd for C11H9N3O: C,
66.32; H, 4.55; N, 21.09. Found: C, 66.06; H, 4.52; N, 21.30.
6-Am in o-3-(4-m et h ylb en zoyl)p yr id a zin e (1c). Com-
pound 1c was prepared according to the procedure for 1a :
12.64 g (58%); UV, EtOH, λ ) 295 (ꢀ ) 17 622); IR (KBr): 3354,
1
3170, 1660, 1616, 1458, 1301, 1163, 927, 783 cm-1; H NMR
(300 MHz, DMSO-d6) δ 7.64 (AA′BB′ system, 4H, Ar), 7.37 (AB
system, 2H, J ) 12, Het), 7.21 (bs, 2H, exchange D2O, NH2),
2.41 (s, 3H, Me); FDMS m/z 214 (M + H)+. Anal. Calcd for
2-Br om o-2-p h en yla ceta m id e (3b). Step 1: 2-Br om o-2-
p h en yla cetic Acid . A mixture of phenylacetic acid (0.13 mol),
benzoyl peroxide (0.54 mmol), and N-bromosuccinimide (0.130
mol) in 500 mL of CCl4 was refluxed under UV radiation for 5
h. The reaction mixture was cooled to room temperature, and
the succinimide was filtered away. The CCl4 was removed in
vacuo and the remaining oil was recrystallized from hexane,
yielding the desired product as a yellow solid (82%).
Step 2: 2-Br om o-2-p h en yla ceta m id e (3b). A solution of
oxalyl chloride (200 mmol) in dry CH2Cl2 (25 mL) was added
dropwise over 25 min to a solution of 2-bromo-2-phenylacetic
acid (21 g, 100 mmol) and DMF (3 drops) in CH2Cl2 (170 mL)
at 0 °C under. The ice bath was removed, and the reaction
mixture was stirred for 3 h. The solvents were removed in
vacuo and then azeotroped with toluene (3 × 25 mL). The
C
12H11N3O: C, 67.59; H, 5.20; N, 19.71. Found: C, 67.03; H,
4.80; N, 19.16.
N-[6-(4-Met h oxyb en zoyl)-2H -p yr id a zin -3-ylid en e)-4-
m eth ylben zen esu lfon a m id e (2a ). To a suspension of 6-ami-
nopyridazine 1a (21.80 mmol, 1.0 equiv) in dry pyridine (35
mL) was added p-toluenesulfonyl chloride (34.88 mmol, 1.6
equiv) portionwise. The reaction mixture was heated at 80-
90 °C for 24 h. The pyridine was removed in vacuo, and the
remaining residue was mixed with 100 mL of ice/water and
stirred for 1 h. The solid was collected and recrystallized from
ethyl acetate to yield 2a (85%) as a white solid: mp 148 °C;
1H NMR (200 MHz,CDCl3) δ 8.16 (AA′BB′ system, 2H, Ar),
8.02 (d, J ) 9.6, 1H, Het), 7.88 (AA′BB′ system, 2H, Ar), 7.50

4338 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Hamdouchi et al.
remaining oil was dissolved in 300 mL of toluene and 300 mL
of hexane, and the mixture was stirred vigorously with a
mechanical stirrer. Ammonia gas was then blown through a
gas dispersion tube over the top of this solution for 1 h. The
resulting solid was filtered and then dissolved in ethyl acetate/
H2O. The organic layer was washed with 1 N HCl, saturated
NaHCO3, and saturated NaCl and was then dried over sodium
sulfate. Solvent was removed in vacuo and the remaining solid
was recrystallized from ethyl acetate/hexane to yield 16.8 g
2,2,2-Tr iflu or o-N-[3-(4-flu or op h en yl)-6-(4-m eth oxyben -
zoyl)im id a zo[1,2-b]p yr id a zin -2-yl]a ceta m id e (5a ). To a
suspension of 2-[(4-fluorophenyl)-2-[3-(4-methoxybenzoyl)-6-
(toluene-4-sulfonylimino)-6H-pyridazin-1-yl]acetamide 4a (3.32
mmol, 1 equiv) in 20 mL of dry CH2Cl2 in a flame-dried two-
neck round-bottomed flask fitted with a reflux condenser, was
added trifluoroacetic anhydride until a clear solution was
obtained. The mixture was stirred for 3 h at room temperature.
The solvents were removed, and the residue was taken up in
ethyl acetate, washed with NaHCO3 (2 × 20 mL) and saturated
NaCl (2 × 20 mL), and dried over sodium sulfate. Ethyl acetate
was removed in vacuo, and the residue was crystallized from
ethyl acetate to yield 2,2,2-trifluoro-N-[3-(4-fluorophenyl)-6-
(4-methoxybenzoyl)imidazo[1,2-b]pyridazin-2-yl]acetamide 5a
1
(68%) of the desired product: mp 138-141°C; H NMR (200
MHz, DMSO-d6) δ 7.80 (bs, CONH), 7.54 (dd, J 1 ) 7.8, J 2
)
2.3, 2H, Ar), 7.41 (bs, 1H, CONH), 7.41-7.30 (m, 3H, Ar), 5.54
(s, 1H, ArCHBrCONH2); 13C NMR (50 MHz, DMSO-d6) δ 169.1
(CONH2), 129.0 (ArCH), 133.8 (C), 128.8, 128.7, 49.9 (PhCH-
BrCONH2); MS (EI+) m/z 91.11 (100) [M+ - 122], 134.13
(89.09) [M+ - 79], 169.05 (15.00) [M+ - 44], 170.06 (10.97)
[(M+ - 44) + 1], 171.05 (15.51) [(M+ - 44) + 2], 172.05 (10.49)
[(M+ - 44) + 3], 213.06 (0.38) [M+].
1
as a white solid (82% yield): mp 230-232 °C; H NMR (200
MHz, DMSO-d6) δ 11.95 (bs, 1H, NH), 8.35 (d, J ) 9.4, 1H,
Het), 8.15 (m, 2H, Ar), 7.80 (m, 3H, Ar), 7.34 (m, 2H, Ar), 7.13
(m, 2H, Ar), 3.88 (s, 3H, OMe); 13C NMR (50 MHz, DMSO-d6)
δ 188.2, 163.7, 161.5, 149.0, 137.5, 136.2, 133.4, 130.6, 130.4,
127.6, 125.7, 123.2, 120.6, 117.9, 115.7, 115.4, 55.6; HRMS
calcd for C22H14F4N4O3 458.1002, found 458.1000.
2-[(4-Flu or oph en yl)-2-[3-(4-m eth oxyben zoyl)-6-(tolu en e-
4-su lfon ylim in o)-6H-p yr id a zin -1-yl]a ceta m id e (4a ). To a
suspension of N-[6-(4-methoxybenzoyl)-2H-pyridazin-3-ylidene)-
4-methylbenzenesulfonamide 2a (12.74 mmol, 1 equiv) in 60
mL of dry DMF was added 3.1 mL of DIPEA (17.19 mmol, 1.3
equiv) under an argon atmosphere. The mixture was stirred
for 30 min, and 2-bromo-2-(p-fluorophenyl)acetamide (15.28
mmol, 1.2 equiv) was added. After being stirred for 24 h, the
solution was poured onto 600 mL of water and stirred for 1 h.
Solids were collected and air-dried to give 4a in 92% yield:
2,2,2-Tr iflu or o-N-[3-(4-flu or oph en yl)-6-ben zoylim idazo-
[1,2-b]p yr id a zin -2-yl]a ceta m id e (5b). To a solution of 2-[3-
benzoyl-6-(toluene-4-sulfonylimino)-6H-pyridazin-1-yl]-2-(4-
fluorophenyl)acetamide (12.66 g, 25.12 mmol) in 150 mL of
CH2Cl2 was added 100 mL of TFAA, and the mixture was
refluxed under N2 for 5 h. Solvents were removed, and the
resulting foam was diluted with ethyl acetate, washed with
saturated NaHCO3 (3 × 100 mL) and brine (3 × 100 mL), and
then dried over sodium sulfate. Ethyl acetate was removed
and solids were recrystallized from ethyl acetate to yield 8.4
g (78%) of 2,2,2-trifluoro-N-[3-(4-fluorophenyl)-6-benzoylimi-
dazo[1,2-b]pyridazin-2-yl]acetamide: mp 277 °C; 1H NMR (200
MHz, DMSO-d6) δ 11.96 (bs, 1H, NHCOCF3), 8.39 (d, J ) 9.4,
1H, Het), 8.12 (dd, J ) 7.0, J ) 1.6, 2H, Ar), 7.90 (d, J ) 9.4,
1H, Het), 7.78 (dd, J ) 8.6, J ) 5.5, 2H, Ar), 7.69 (dd, J ) 7.0,
J ) 1.6, 1H, Ar), 7.58 (t, J ) 7.0, 2H, Ar), 7.31 (t, J ) 8.6, Ar);
FDMS m/z 428 (M+); Anal. Calcd for C21H12F4N4O2: C, 58.75;
H, 3.05; N, 13.05. Found: C, 59.03; H, 3.12; N, 13.03.
1
mp 245 °C; H NMR (200 MHz, DMSO-d6) δ 8.08 (s, 2H, Ar),
7.93-6.80 (m, 12H, Ar), 6.64 (s, 1H, CH), 3.83 (s, 3H, OMe),
2.48 (s, 3H, Me); 13C NMR (50 MHz, DMSO-d6) δ 185.7, 168.2,
163.6, 162.5, 155.0, 144.4, 142.5, 140.1, 133.3, 133.1, 131.4,
129.8, 129.6, 127.0, 126.2, 125.9, 115.6, 113.5, 68.7, 55.7, 21.1;
MS (EI+) m/z 535 M+ (1), 517 (10), 399 (8), 362 (11), 171 (14),
155 (31), 135 (100), 107 (16), 91 (73), 65 (18); HRMS calcd for
C
27H23FN4O5S 534.137 32, found 534.136 66.
2-[3-Ben zoyl-6-(tolu en e-4-su lfon ylim in o)-6H-pyr idazin -
1-yl]-2-(4-flu or op h en yl)a ceta m id e (4b). To a suspension of
N-(6-benzoyl)-2H-pyridazin-3-ylidene-4-methylbenzenesulfona-
mide 2b (10.59 g, 30 mmol) in 50 mL of dry DMF, was added
DIPEA (5.75 mL, 33 mmol) followed by R-bromo-p-fluorophen-
ylacetamide 3a (9.66 g, 41.7 mmol). The reaction mixture was
stirred for 72 h, then poured into 3 L of H2O, and stirred
vigorously for 1 h. The precipitate was collected and recrystal-
lized from ethyl acetate to yield 12.66 g (84%) of 2-[3-benzoyl-
6-(toluene-4-sulfonylimino)-6H-pyridazin-1-yl]- 2-(4-fluorophen-
yl)acetamide: mp 226 °C; 1H NMR (200 MHz, DMSO-d6) δ
8.09 (AB system, 2H, J ) 8.9 Hz, Het), 7.789 (bs, 1H, CONH2),
7.77 (d, 2H, J ) 8.2, Ar), 7.59-7.53 (m, 2H, Ar), 7.36-7.24
(m, 7H, Ar), 7.13-7.07 (m, 2H, Ar), 6.62 (bs, 1H, CONH2), 2.34
2,2,2-Tr iflu or o-N-[3-(4-flu or op h en yl)-6-(4-m et h ylb en -
zoyl)im id a zo[1,2-b]p yr id a zin -2-yl]a ceta m id e (5c). Com-
pound 5c was prepared according to the procedure for 5a .
Starting from compound 4c, the imidazopyridazine 5c was
obtained in 85% yield: mp 220 °C; 1H NMR (200 MHz, DMSO-
d6) δ 11.96 (bs, 1H, NH), 8.36 (d, J ) 9.4, 1H, Het), 8.04
(AA′BB′ system, 2H, Ar), 7.80 (m, 3H, Ar), 7.34 (m, 4H, Ar),
2.42 (s, 3H, Me); 13C NMR (50 MHz, DMSO-d6) δ 189.5, 159.4,
146.1, 137.7, 136.2, 132.5, 131.0, 130.5, 128.8, 125.7, 123.1,
122.9, 121.5, 118.6, 117.7, 115.3, 21.2; MS (EI+) m/z 442 M+
(52) 119 (100), 91 (54), 84 (10), 65 (18); HRMS calcd for
(s, 3H, Me); FDMS m/z 504 (M+). Anal. Calcd for C26H21
-
C
22H14F4N4O2 442.1053, found 442.1060.
FN4O4S: C, 61.90; H, 4.20; N, 11.10. Found: C, 62.11; H, 4.34;
N, 10.97.
2-N-[6-Ben zoyl-3-p h en ylim id a zo[1,2-b]p yr id a zin -2-yl]-
2,2,2-tr iflu or oa ceta m id e (5d ). Compound 5d was prepared
according to the procedure for 5a . Starting from compound 4d ,
the imidazopyridazine 5d was obtained in 92% yield: mp 248
°C; 1H NMR (200 MHz, DMSO-d6) δ 11.83 (bs, 1H, NHCOCF3),
8.31 (d, J ) 9.2, 1H, Het), 7.90 (d, J ) 9.2, 1H, Het), 7.80-
7.65 (m, 2H, Ar), 7.60-7.51 (m, 1H, Ar), 7.48-7.46 (m, 2H,
Ar), 7.78 (m, 2H, Ar), 7.69 (m, 1H, Ar), 7.58 (m, 2H, Ar), HRMS
calcd for C21H13F3N4O2 410.0991, found 410.0981.
2-[3-(4-Meth ylben zoyl)-6-(tolu en e-4-su lfon ylim in o)-6H-
p yr id a zin -1-yl]-2-(4-flu or op h en yla ceta m id e (4c). Com-
pound 4c was prepared according to the procedure for 4a .
Starting from the tosylate 2c and 2-bromo-2-(4-fluorphenyl)-
acetamide 3a , compound 4c was obtained in 74% yield: mp
1
237 °C; H NMR (200 MHz, DMSO-d6) δ 8.09-7.12 (m, 14H,
Ar), 6.64 (s, 1H, CH), 2.37 (s, 3H, Me), 2.35 (s, 3H, CH3); 13C
NMR (50 MHz, DMSO-d6) δ 186.4, 167.7, 161.8, 154.3, 143.4,
143.2, 141.8, 139.3, 132.3, 131.2, 130.5, 130.1, 128.9, 128.1,
125.5, 125.2, 114.7, 67.9, 20.7, 20.4; MS (EI+) m/z 519 M+(2),
501 (15), 399 (13), 320 (37), 155 (38), 119 (87), 91 (100); HRMS
calcd for C27H23FN4O4S 518.142 404, found 518.142 820.
2-Am in o-3-(4-flu or op h en yl)-6-b en zoylim id a zo[1,2-b]-
p yr id a zin e (6b). A solution of 2,2,2-trifluoro-N-[3-(4-fluo-
rophenyl)-6-benzoylimidazo[1,2-b]pyridazin-2-yl]acetamide 5b
(204 mg, 0.476 mmol) in 3 N NaOH (3.2 mL) was heated at
50 °C for 72 h. The reaction mixture was poured into ethyl
acetate (150 mL) and washed with saturated NaCl (2 × 100
mL). The organic layer was dried (sodium sulfate) and
evaporated. The residue was triturated with Et2O (7 mL) for
2 h to give 144 mg (93%) of the desired amine (6b): mp 218
°C; 1H NMR (300 MHz, DMSO-d6) δ 8.06 (AA′ of AA′BB′
system, 2H, Ar), 7.9 (m, 3H), 7.7 (m, 2H), 7.58 (m, 2H), 7.22
(m, 2H), 6.05(b, 2H); HRMS calcd for C21H13F3N4O2 410.0991,
found 4102.0980.
2-[3-Ben zoyl-6-(tolu en e-4-su lfon ylim in o)-6H-pyr idazin -
1-yl]-2-p h en yla ceta m id e (4d ). Compound 4d was prepared
according to the procedure for 4a . Starting from the tosylate
2d and 2-bromophenylacetamide 3b, compound 4d was ob-
1
tained in 89% yield: mp 211 °C; H NMR (200 MHz, DMSO-
d6) δ 8.09 (AB system, 2H, Het), 7.90 (bs, 1H, CONH2), 7.77
(d, 2H, J ) 8.1, Ar), 7.57-7.54 (m, 4H, Ar), 7.34-7.25 (m, 8H,
Ar), 6.68 (bs, 1H, CONH2), 2.35 (s, 3H, Me); HRMS calcd for
C
26H22N4O4S 486.1362, found 486.1365.

Imidazo[1,2-b]pyridazines
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4339
2-Am in o-3-p h e n yl-6-b e n zoylim id a zo[1,2-b]p yr id a -
zin e (6d ). Compound 6d was prepared according to the pro-
cedure for 6b. Starting from compound 5d , the aminoimida-
zopyridazine 6d was obtained in 87% yield: mp 199-201 °C;
1H NMR (300 MHz, DMSO-d6) δ 8.05 (d, 2H, Ar), 7.9 (A of an
AB system, 1H, Het), 7.85 (d, 2H, Ar),7.68 (m, 2H), 7.58 (m,
2H), 7.4 (m, 2H), 7.22 (m, 1H), 6.1(b, 2H, NH2); HRMS calcd
for C19H14N4O 314.1168, found 314.1177.
A solution of the ketone 5b (400 mg, 0.934 mmol) was added
dropwise, the reaction mixture was allowed to warm to room
temperature, and the mixture was stirred for 5 h. The solvents
were evaporated, and the residue was dissolved in ethyl
acetate (150 mL), washed with 10% NH4Cl (4 × 100 mL) and
saturated NaCl (6 × 100 mL), and dried over anhydrous
sodium sulfate. Upon evaporation of the solvent, the crude
material was triturated with Et2O, yielding 250 mg (54%) of
the product as an isomeric mixture (E/Z, 13:1). The mixture
of isomers was used without further purification for the next
step: 1H NMR (300 MHz, DMSO-d6) δ 1.01 (t, J ) 7.09, 3H,
OEt), 3.99 (q, J ) 7.09, 2H, OEt), 6.95 (s, 1H, R1R2CdCHR3),
7.21 (t, J ) 8.9, 2H, Ar), 7.29-7.26 (m, 2H, Ar), 7.44-7.40 (m,
2-Am in o-3-(4-flu or op h en yl)-6-b en zoylim id a zo[1,2-b]-
p yr id a zin e Oxim e [(E)-7b] a n d [(Z)-7b]. A suspension of
6b (129 mg, 0.399 mmol), NH2OH‚HCl (339 mg, 4.878 mmol),
and NaOAc (408 mg, 4.973 mmol) in 80% EtOH (15 mL) was
refluxed under argon atmosphere for 24 h. The reaction
mixture was monitored by TLC (neutral aluminum oxide, CH2-
Cl2/CH3CN). The ethanol was evaporated, and the crude
residual solid was dissolved in ethyl acetate (150 mL). The
organic layer was washed with 5% NaHCO3 (2 × 100 mL) and
saturated NaCl (4 × 100 mL) and dried over anhydrous sodium
3H, Ar), 7.51 (d, J ) 9.6, 1H, Het), 7.64 (dd, J 1 ) 5.56, J 2
)
8.77, 2H, Ar), 8.21 (d, J ) 9.6, 1H, Het), 11.8 (s, 1H,
NHCOCF3); 13C NMR (75 MHz, DMSO-d6) δ 115.47 (d, J )
21.6, Ar(F)CH), 118.15, 122.91 (CH), 123.25 (d, J ) 3.2, Ar-
(F)C), 125.55, 128.05, 128.36, 129.26 (CH), 130.15 (d, J ) 8.3,
Ar(F)CH), 135.73, 136.51, 149.01, 152.28 (C), 156.08 (d, J )
37.3, NHCOCF3), 161.8 (d, J ) 246, Ar(F)C), 165.23 (COOEt);
FAB-MS (rel abundance) m/z 499.4 (100) [M+ + 1], 997.6 (2.25)
[2M+ + 1]; HRMS calcd for C25H18F4N4O3 498.131 50, found
499.139 33 [M+ + 1].
1
sulfate, and the solvent was evaporated. H NMR analysis of
the crude material showed compound 7b to be a mixture of E
and Z isomers in a 1:1 ratio. The residue was recrystallized
from hot ethyl acetate/hexane, affording 48 mg (36%) of the Z
isomer in its pure form. The mother liquor was concentrated
and purified by flash column chromatography (CH2Cl2/CH3-
CN, 2:1, neutral aluminum oxide) to yield 63 mg (47%) of a
solid that was shown by 1H NMR analysis to be predominantly
(90:10) the E isomer.
(E)-7b: 1H NMR (200 MHz, DMSO-d6) δ 11.87 (s, 1H, OH),
7.77 (d, J ) 9.3, 1H), 7.68 (dd, J ) 8.9, J ) 5.5, Ar), 7.64 (d,
J ) 9.3, 1H), 7.53-7.36 (m, 5H, Ar), 7.00 (t, J ) 8.9, 2H), 5.77
(b, 2H); 13C NMR (50 MHz, DMSO-d6) δ 166.0, 159.9, 153.3,
150.7, 146.5, 135.9, 132.1, 129.4, 128.2, 128.1, 127.7, 126.9,
125.4, 120.5, 114.8, 112.9, 106.9; MS (FAB+) m/z 348.3 (M +
H)+.
(Z)-7b: 1H NMR (200 MHz, DMSO-d6) δ 11.79(s, 1H), 7.89
(d, J ) 8.6, H), 7.79 (dd, J ) 9, 2H), 7.53-7.36 (m, 5H), 722 (t,
J 1 ) 5.5, J 2) 9.0, 2H), 7.15 (d, J ) 9.0, 1H), 5.8 (b, 2H); 13C
NMR (50 MHz, DMSO-d6) δ 166.1, 160.4, 151.9, 151.0, 144.1,
135.7, 135.1, 129.4, 128.7, 128.6, 127.7, 126.6, 120.4, 116.6,
115.3, 107.2.
Analysis of the stability of the oxime (E)-7b demonstrated
that an equilibrium between the Z and E isomers forms in
polar solvents such as DMSO and TFA at room temperature.
(E )-2-Am in o-3-p h e n yl-6-b e n zoylim id a zo[1,2-b]p yr i-
d a zin e Oxim e (7d ). Compound 7d was prepared according
to the procedure for 7b. Starting from compound 6d , the
aminoimidazopyridazine 7d was obtained in 76% yield as a
mixture of E and Z isomers. The mixture was separated by
flash column chromatography (CH2Cl2/CH3CN, neutral alu-
minum oxide) to yield the desired isomer (E)-7d : 1H NMR (200
MHz, DMSO-d6) δ 11.83 (s, 1H, OH), 7.79 (d, J ) 9.0, 1H),
7.62 (m, 3H, Ar), 7.4(d, 5H), 7.2-7.1(m, 3H, Ar), 5.70 (b, 2H););
HRMS calcd for C19H15N5O 329.1277, found 329.12. Analysis
of the stability of the oxime (E)-7d demonstrated that an
equilibrium between the Z and E isomers forms in polar
solvents such as DMSO and TFA at room temperature.
2-Am in o-3-(4-flu or op h en yl)-6-b en zoylim id a zo[1,2-b]-
p yr id a zin e P h en ylh yd r a zon e (8b). A suspension of 6b (143
mg, 0.422 mmol), PhNHNH2‚HCl (320 mg, 2.213 mmol), and
NaOAc (183 mg, 2.231 mmol) in absolute EtOH (20 mL) was
refluxed for 72 h. The reaction was monitored by 1H NMR.
The crude residual solid was filtered and washed with EtOH
(5 mL) to yield 120 mg (64%) of 8b as a mixture of E and Z
isomers in a 1:1 ratio: 1H NMR (200 MHz, DMSO-d6) δ 5.66
(s, 2H, NH2), 6.98 (t, J ) 8.6, 2H, Ar), 7.15-7.60 (m, 10H, Ar),
7.67 (dd, J 1 ) 5.5, J 2 ) 8.6, 2H, Ar), 7.75 (d, J ) 9.4, 1H, Het),
7.94 (d, J ) 9.4, 1H, Het), 9.42 (s, 1H, NHph); FAB-MS (rel
aboundance) m/z 422.23 (11.74) [M+], 423.24 (14.46) [M+ + 1].
3-(2′-Eth oxyca r bon yl-1′-p h en ylvin yl)-2-(2,2,2-tr iflu or o-
a ceta m id o-3-(4′-flu or op h en yl)im id a zo[1,2-b]p yr id a zin e
(E/Z)-9b. Triethyl phosphonoacetate (414 mg, 1.847 mmol) was
placed in a round-bottom flask under an argon atmosphere. A
solution of KHDMS (5.6 mL, 2.80 mmol, 0.5 M in toluene) was
added. The reaction mixture was stirred for 30 min at -5 °C.
2-Am in o-6-(2′-eth oxyca r bon yl-1′-p h en ylvin yl)-3-(4′-flu -
or op h en yl)im id a zo[12-b]p yr id a zin e (10b). DIPEA (4 mL)
was added to a suspension of 9b (210 mg, 0.421 mmol) (E/Z,
13:1) in 10 mL of EtOH (90%). The reaction mixture was
refluxed for 48 h (TLC; CH2Cl2/CH3CN, 2:1). After evaporation
of the EtOH, the resulting solid was dissolved in ethyl acetate
(100 mL), washed with 10% NH4Cl (4 × 100 mL) and saturated
NaCl (6 × 100 mL), and dried over sodium sulfate. Upon
evaporation of the solvent, the crude residual solid was
triturated in Et2O, yielding 62 mg of the pure E isomer. The
mother liquors were evaporated and the residue was purified
by flash column chromatography (CH2Cl2/CH3CN, 2:1), afford-
1
ing 85 mg of a solid that was shown by H NMR analysis to
be predominantly (>90:10) the Z isomer: total yield 147 mg
(87%); 1H NMR (300 MHz, DMSO-d6) δ 0.99 (t, J ) 7.10, 3H,
OEt), 3.95 (q, J ) 7.10, 2H, OEt), 5.91 (s, 2H, NH2), 7.13 (t,
2H, J ) 8.8, Ar(F)H), 7.14 (d, J ) 9.3, 1H, Het), 7.25-7.19
(m, 5H, Ar), 7.42-7.37 (m, 2H, Ar), 7.70 (d, J ) 9.3 Hz, 1H,
Het), 7.75 (dd, J 1 ) 5.6, J 2 ) 8.8, 2H, Ar(F)H); 13C NMR (300
MHz, DMSO-d6) δ 13.88 (OEt), 59.97 (OEt), 107.17 (C), 114.68
(CH), 115.17 (d, J ) 21.3, Ar(F)CH), 119.97 (CH), 125.37 (d, J
) 3.1, Ar(F)C), 127.93, 127.99 (CH), 128.58 (d, J ) 7.9, Ar-
(F)CH), 136.13, 137.30, 148.18, 150.53, 151.77 (C), 160.31 (d,
J ) 244, Ar(F)C), 165.31 (COOEt); FAB-MS (rel abundance)
m/z 403.0 (100) [M+ + 1], 805.0 (2.12) [2M+ + 1]; HRMS calcd
for C23H19FN4O2 402.149 204,found 402.149 210[M+],403.157 029
[M+ + 1].
Dieth yl (N-Meth ylcar bam oylm eth yl)ph osph on ate (11).
The Horner-Wadsworth-Emmons reagent was prepared ac-
cording to the procedure that we previously described.6
2-(2,2,2-Tr iflu or oacetam ido)-3-(p-flu or oph en yl)-6-[(E,Z)-
2-N-m eth ylcar bam oyl-1-(p-m eth oxyph en yl)vin yl]im idazo-
[1,2-b]p yr id a zin e [(E)-12a a n d (Z)-12a ]. Meth od A. To a
solution of KHMDS (5.27 mmol) in 5 mL of dry DMF at 0 °C
under argon was added a solution of the Horner-Wadsworth-
Emmons reagent 11 (1.86 mmol) in 10 mL of dry DMF. The
reaction mixture was stirred at 0 °C for 45 min. The imida-
zopyridazine 5a (1.55 mmol) in 10 mL of dry DMF was
transferred via cannula. The bath was removed, and the
resulting brown solution was allowed to warm to room tem-
perature. After 44 h, the reaction mixture was quenched with
saturated NH4Cl (30 mL) and extracted with ethyl acetate (3
× 100 mL). The organic layers were combined and washed with
saturated NH4Cl and then with saturated NaCl. After drying
over sodium sulfate, the solvent was removed in vacuo. 1H
NMR analysis of the crude material showed a mixture of E
and Z isomers in a 1:2 ratio (67% yield). The isomers were
separated by column chromatography (silica gel, ACN/CH2-
Cl2, 1:2 then 1:1 then 2:1).
1
(E)-12a : mp 247-249 °C; H NMR (200 MHz, DMSO-d6) δ
8.16 (m, 1H, NHMe), 8.12 (d, J ) 9.5, 1H, Het), 7.77 (dd, J )

4340 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Hamdouchi et al.
5.7, J ) 8.7, 2H, Ar), 7.37-7.18 (m, 4H, Ar), 7.15 (AA′BB′
system, 2H, Ar), 6.95 (d, J ) 9.5, 1H, Het), 6.65 (s, 1H, CHd
C), 3.75 (s, 3H, OMe), 2.56 (d, J ) 4.3 Hz, 3H, NHMe); 13C
NMR (50MHz, DMSO-d6) δ 164.6, 160.2, 153.7, 144.0, 135.5,
131.0, 130.1, 129.1, 128.6, 124.4, 123.4, 122.3, 121.1, 120.3,
115.8, 115.3, 114.3, 113.2, 55.2, 25.4; MS (EI+) m/z 513 M+
(100), 512 (12), 484 (10), 483 (38), 456 (7), 444 (15). 265 (7),
159 (8), 133 (11), 132 (9), 89 (7), 69 (7), 58 (10); HRMS calcd
for C25H19F4N5O3 513.142 40, found 513.142 52.
153.6, 144.2, 135.6, 134.7, 130.1, 130.0, 129.5, 127.0, 124.4,
123.7, 123.4, 121.0, 120.4, 115.8, 115.3, 113.0, 25.4, 20.1; MS
(EI+) m/z 497 M+ (100), 496 (14), 468 (11), 467 (38), 440 (8),
429 (6), 428 (20), 249 (9), 143 (7), 122 (8), 117 (9), 116 (9), 115
(18), 69 (7), 58 (12); HRMS calcd for C25H19F4N5O2 497.1475,
found 497.1477.
1
(Z)-12c: mp 237-239 °C; H NMR (200 MHz, DMSO-d6) δ
8.22 (m, 1H, NHMe), 8.13 (d, J ) 9.5, 1H, Het), 7.80 (dd, J )
5.6, J ) 8.5, 2H, Ar), 7.38-7.06 (m, 7H, Ar + Het), 6.84 (s,
1H, CHdC), 2.53 (d, J ) 4.4, 3H, NHMe), 2.35 (s, 3H, Me).
1
(Z)-12a : mp 228-230 °C; H NMR (200 MHz, DMSO-d6) δ
8.04-6.95 (m, 10H, Ar), 6.82 (s, 1H, CHdC), 3.79 (s, 3H, OMe),
2.48 (s, 3H, NHMe); 13C NMR (50 MHz, DMSO-d6) δ 164.2,
159.3, 159.1, 155.8, 153.6, 142.8, 135.4, 134.5, 130.2, 128.5,
126.6, 125.1, 123.3, 118.7, 118.5, 115.6, 115.2, 112.9, 55.1, 25.4.
2-Am in o-3-(4-flu or op h en yl)-6-[(E)-2-N-m eth ylca r ba m -
oyl-1-(4-m e t h oxyp h e n yl)vin yl]im id a zo[1,2-b]p yr id a -
zin e [(E)-13a ]. To a suspension of (E)-12a (250 mg, 0.517
mmol) in 90% MeOH (12 mL), DIPEA (4.8 mL) was added.
The resulting orange solution was refluxed for 72 h (TLC:
ACN/MeOH, 10:1). The solvents were evaporated in vacuo, and
the crude mixture was subjected to radial chromatography
(CH3CN/CH2Cl2, 7:3) to yield the desired isomer (E)-13a as a
yellow solid in 70% yield: mp 165 °C; 1H NMR (200 MHz,
CDCl3) δ 7.76 (dd, J ) 5.3, J ) 9.1, 1H, Ar), 7.55 (d, J ) 9.1,
1H, Het), 7.22-7.08 (m, 4H, Ar), 6.95 (m, 2H, Ar), 6.84 (d, J
) 9.1 1H, Het), 6.76 (s, 1H, CHdC), 5.36 (m, 1H, NHMe), 4.39
(bs, 2H, NH2), 3.86 (s, 3H, OMe), 2.66 (d, J ) 4.8, 3H, NHMe);
13C NMR (50 MHz, CDCl3) δ 167.0, 160.0, 161.5, 150.2, 149.6,
144.1, 130.7, 128.9, 128.8, 128.2, 125.0, 124.8, 120.9, 116.1,
115.6, 114.2, 109.2, 55.3, 26.2; MS (EI+) m/z 417 M+ (100), 387
(10), 360 (8), 359 (7), 266 (8), 133 (14), 132 (8), 122 (12), 89
(7), 79 (10), 58 (12); HRMS calcd for C23H20FN5O2 417.160 103,
found 417.160 142.
2-(2,2,2-Tr iflu or oa ceta m id o)-3-(4′-flu or om eth yl)-6-(2′-
N-m et h ylca r ba m oyl-1′-p h en ylvin yl)im id a zo[1,2-b]p yr i-
d a zin e [(E)-12b]. Meth od A. A solution of the Horner-
Wadsworth-Emmons reagent 11 (1.86 mmol) in dry DMF (10
mL) was added to a solution of KHMDS (5.27 mmol) in dry
DMF (5 mL) at 0 °C under argon. The reaction mixture was
stirred at 0 °C for 45 min. A solution of the imidazopyridazine
5b (1.55 mmol)) in 10 mL of dry DMF was transferred via
cannula. The bath was removed, and the resulting brown
solution was allowed to warm to room temperature. After 5 h,
the reaction mixture was quenched with saturated NH4Cl (30
mL) and extracted with ethyl acetate (3 × 100 mL). The
organic layers were combined and washed with saturated NH4-
Cl and then with saturated NaCl. After drying over sodium
1
sulfate, the solvent was removed in vacuo. H NMR analysis
of the crude material showed a mixture of E and Z isomers in
1:5 ratio (59%). The reaction was repeated under the same
conditions by increasing the reaction time to 48 h, resulting
in a mixture of E and Z isomers in a 3:1 ratio (65% yield).
2-Am in o-3-(4-flu or op h en yl)-6-[(Z)-2-N-m eth ylca r ba m -
oyl-1-(4-m e t h oxyp h e n yl)vin yl]im id a zo[1,2-b]p yr id a -
zin e [(Z)-13a ]. Compound (Z)-13a was prepared according to
the procedure described for (E)-13a . Starting from (Z)-12a ,
compound (Z)-13a was obtained in 82% yield: mp 140 °C; 1H
NMR (CDCl3) δ 7.72 (dd, J ) 5.4, J ) 8.7, 1H, Ar), 7.56 (d, J
) 9.1, 1H, Het), 7.10 (m, 6H, Ar), 6.77 (d, J ) 9.1, 1H, Het),
6.31 (s, 1H, CHdC), 6.19 (m, 1H, NHMe), 4.00 (bs, 2H, NH2),
3.73 (s, 3H, OMe), 2.53 (d, J ) 4.8, 3H, NHMe); 13C NMR
(CDCl3) δ 166.3, 163.9, 160.5, 149.8, 149.0, 145.0, 135.8, 131.2,
129.1, 129.0, 124.9, 123.0, 121.0, 117.6, 115.8, 114.0, 109.0,
55.3, 26.0; MS (EI+) m/z 417 M+ (100), 387 (10), 360 (8), 359
(7), 266 (8), 133 (14), 132 (8), 122 (12), 89 (7), 79 (10), 58 (12);
HRMS calcd for C23H20FN5O2 417.160 103, found 417.160 142.
Meth od B. A 0.5 M solution of KHMDS (35 mL, 17.5 mmol)
was added to a solution of the Horner-Wadsworth-Emmons
reagent 11 (1.5 g, 7 mmol) in THF (20 mL) at -78 °C under
argon. The mixture was stirred for 2 h at -78 °C followed by
the dropwise addition of imidazpyridazine 5a (1.6 g, 3.5 mmol)
in 20 mL of dry THF. The resulting brown solution was
allowed to warm to room temperature and then stirred at room
temperature for 34 h. The reaction mixture was diluted in 400
mL of ethyl acetate, and an amount of 200 mL of 5% HCl was
added. The layers were separated, and the aqueous layer was
extracted with ethyl acetate (2 × 200 mL). The organic layers
were combined and washed with 5% HCl (2 × 100 mL) and
once with saturated NaCl. After drying over MgSO4, the
2-Am in o-3-(4′-flu or op h en yl)-6-(2′-N-m eth ylca r ba m oyl-
1′-p h en ylvin yl)im id a zo[1,2-b]p yr id a zin e [(E)-13b]. Com-
pound (E)-13b was prepared according to the procedure for
(E)-13a . Starting from (E)-12b, compound (E)-13b was ob-
1
solvent was removed in vacuo to give a brown solid. H NMR
analysis of the crude material showed a mixture of E and Z
isomers in a 4:1 ratio. Crystallization from ethyl acetate
afforded the E isomer 12b in its pure form in 69% yield.
1
tained in 92% yield: mp 161 °C; H NMR (300 MHz, DMSO-
d6) δ 2.53 (d, J ) 4.6, 3H, CONHMe), 5.81 (s, 2H, NH2), 6.85
(s, 1H, R1R2CdCHR3), 6.97 (d, J ) 9.2, 1H, Het), 7.16 (t, J )
8.9, 2H, Ar), 7.40-7.20 (m, 5H, Ar), 7.70 (d, J ) 9.2, 1H, Het),
7.81 (dd, J 1 ) 8.9, J 2 ) 5.5, 2H, Ar), 7.99 (q, J ) 4.6, 1H,
CONHMe); 13C NMR (75 Hz, DMSO-d6) δ 25.56 (MeNHCO),
107.19 (C), 114.62 (CH), 115.22 (d, J ) 21, Ar(F)CH), 120.18,
124.88 (CH), 125.55 (d, J ) 3.1, Ar(F)C), 127.62, 127.78 (CH),
128.57 (d, J ) 7.9, Ar(F)CH), 129.50 (CH), 137.58, 144.63,
149.47, 151.44 (C), 160.28 (d, J ) 244, Ar(F)C), 165.38
(CONHMe); FAB-MS m/z 388.26 [M+ + 1], 775.4 [2M+ + 1].
(E)-12b: mp 243 °C; 1H NMR (200 MHz, DMSO-d6) δ 11.87
(b, 1H, NHCOCF3), 8.20 (d, J ) 9.6, 1H, Het), 8.10 (b, 1H,
NHCOMe), 7.68 (dd, J ) 5.5, J ) 9.0, 2H, Ar), 7.4-7.1 (m,
7H, Ar), 7.3 (d, J ) 9.6, 1H, Het), 6.93 (s, 1H, CHdC), 2.54 (d,
J ) 4.7, 3H, NHMe); FAB-MS (rel abundance) m/z 483.25
(45.23) [M+], 484.25 (100) [M+ + 1], 486.25 (6.71) [M+ + 2],
967.39 (3.8) [2M+ + 1], 968.40 (2.45) [2M+ + 2]; HRMS calcd
for C24H17F4N5O2 483.131 84, found 484.139 66 [M+ + 1].
2-(2,2,2-Tr iflu or oacetam ido)-3-(4-flu or oph en yl)-6-[(E,Z)-
2-N-m eth ylca r ba m oyl-1-(4-m eth ylp h en yl)vin yl]im id a zo-
[1,2-b]p yr id a zin e [(E)-12c) a n d (Z)-12c)]. 2-(2,2,2-Trifluo-
roacetamido)-3-(p-fluorophenyl)-6-(4-methylbenzoyl)imidazo[1,2-
b]pyridazine 5c was converted to its corresponding vinyl
carboxamides 12c in a manner substantially analogous to the
preparation of 12b (method A). Compound 12c was isolated
in 63% yield as a mixture of E and Z isomers that were
separated by a column chromatography (silica gel, ACN/CH2-
Cl2, 1:2 then 1:1 then 2:1) to give both isomers (E)-12c and
(Z)-12c in their pure forms.
2-Am in o-3-(4′-flu or op h en yl)-6-(2′-N-m eth ylca r ba m oyl-
1′-p h en ylvin yl)im id a zo[1,2-b]p yr id a zin e [(Z)-13b]. Com-
pound (Z)-13b was prepared according to the procedure
described for (E)-13a . Starting from (Z)-12b, compound (Z)-
13b was isolated in 86% yield: mp 145 °C; 1H NMR (300 MHz,
DMSO-d6) δ 2.57 (d, J ) 4.7, 3H, CONHMe), 5.64 (s, 2H, NH2),
6.63 (s, 1H, R1R2dCHR3), 6.84 (d, J ) 9.0, 1H, Het-H), 7.21 (t,
J ) 8.9, 2H, Ar), 7.45-7.25 (m, 5H, Ar), 7.69 (d, J ) 9.0, 1H,
Het), 7.81 (dd, J 1 ) 5.6, J 2 ) 8.9, 2H, Ar), 8.13 (q, J ) 4.7, 1H,
CONHMe); 13C NMR (75 MHz, DMSO-d6) δ 25.59 (CONHCH3),
107.14 (C), 115.32 (d, J ) 21.3, Ar(F)CH), 117.43, 120.08,
124.91 (CH), 125.89 (d, J ) 3.0, Ar(F)C), 127.47 (CH), 128.75
(d, J ) 7.9, Ar(F)CH), 128.89, 129.23 (CH), 135,76, 138.92,
145.02, 149.43, 150.57 (C), 160.37 (d, J ) 244, Ar(F)C), 165.38
1
(E)-12c: mp 251 °C; H NMR (200 MHz, DMSO-d6) δ 8.23
(m, 1H, NHMe), 8.12 (d, J ) 9.3, 1H, Het), 7.65 (dd, J ) 5.8,
J ) 8.5, 2H, Ar), 7.36-7.21 (m, 6H, Ar), 7.14 (d, J ) 9.3, 1H,
Het), 6.71 (s, 1H, CHdC), 2.56 (d, J ) 4.4 Hz, 3H, NHMe),
2.29 (s, 3H, Me); 13C NMR (50 MHz, DMSO-d6) δ 164.5, 159.5,
(CONHMe); FAB-MS (rel abundance) m/z 387.2 (100) [M+
+

Imidazo[1,2-b]pyridazines
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4341
1]; HRMS calcd for C22H18FN5O 387.149 539, found 387.149 539
[M+], 388.157 364 [M+ + 1].
incubated at 37 °C (under CO2) for 3-4 h or until a prominent
color change was observed. The absorbance at 450 nm (refer-
ence was 650 nm) was read in a spectrophotometer.11
2-Am in o-3-(4-flu or op h en yl)-6-[(E)-2-N-m eth ylca r ba m -
oyl-1-(4-m eth ylph en yl)vin yl]im idazo[1,2-b]pyr idazin e [(E)-
13c]. Compound (E)-13c was prepared according to the
procedure for (E)-13a . Starting from (E)-12c, compound (E)-
13c was obtained in 78% yield: mp 175 °C; 1H NMR (200 MHz,
CDCl3) δ 7.76 (dd, J ) 5.4, J ) 9.1, 1H, Ar), 7.53(d, J ) 9.1,
1H, Het), 7.26-7.08 (m, 6H, Ar), 6.80 (d, J ) 9.1, 1H, Het),
6.80 (s, 1H, CHdC), 5.37 (m, 1H, NHMe), 4.40 (bs, 2H, NH2),
2.65 (d, J ) 4.8, 3H, NHMe), 2.42 (s, 3H, Me); 13C NMR (50
MHz, CDCl3) δ 166.8, 161.4, 149.9, 144.4, 138.8, 136.1, 133.3,
129.5, 129.2, 128.8, 127.8, 125.6, 124.7, 124.3, 120.8, 115.8,
109.1, 26.2, 21.3; MS (EI+) m/z 401 M+ (100), 400 (7), 371 (9),
344 (8), 343 (7), 250 (7), 122 (13), 117 (9), 116 (9), 115 (16), 79
(9), 58 (12); HRMS calcd for C23H20FN5O 401.1651, found
401.1651.
Ack n ow led gm en t. We are grateful to the Lilly
Infectious Diseases Action Group and Lilly Research
Process and Development for stimulating discussions.
Refer en ces
(1) (a) Couch, R. B. Rhinoviruses. In Virolgy; Fields, B. N., Ed.;
Raven Press: New York, 1990; Chapter 22, pp 607-629. (b)
Rueckert, R. R. Picornaviruses and Their Replication. In Virolgy;
Fields, B. N., Ed.; Raven Press: New York, 1985; Chapter 32,
pp 705-738. (c) Monto, A. S.; Bryan, E. R.; Ohmit, S. Rhinovirus
Infections in Tecumseh, Michigan: Frequency of Illness and
Number of Serotypes. J . Infect. Dis. 1987, 154, 43-49. (d) Blaas,
D.; Hofer, F.; Gruenberger, M.; Kowalski, H.; Machat, H.;
Kuechler, E.; Huettinger, M. Entry of Minor Group Human
Rhinoviruses into the Cell. In Cellular Receptors for Animal
Viruses; Wimmer, E., Ed.; Cold Spring Harbor Laboratory
Press: Plainview, NY, 1994; pp 129-140. (e) Chapman, M. S.;
Rossmann, M. G. Comparison of the Surface Properties of
Picornaviruses. Virology 1993, 195, 745-756.
(2) (a) Wikel, J . H.; Paget, C. J .; DeLong, D. C.; Nelson, J . D.; Wu,
C. Y. E.; Paschal, J . W.; Dinner, A.; Templeton, R. J .; Chaney,
M. O.; J ones, N. D.; Chamberlin, J . W. Synthesis of Syn and
Anti Isomers of 6-[[(Hydroxyimino)phenyl]methyl]-1-[(1-meth-
ylethyl)sulfonyl]-1H-benzimidazol-2-amine. Inhibitors of Rhi-
novirus Multiplication. J . Med. Chem. 1980, 23, 368-372. (b)
DeLong, D. C. Effect of Enviroxime on Rhinovirus Infections in
Humans. In Microbiology; Leive, L., Schlessinger, D., Eds.;
American Society for Microbiology: Washington, DC, 1984; pp
431-434. (c) DeLong, D. C.; Reed, S. E. Inhibition of Rhinovirus
Replication in Organ Culture by a Potential Antiviral Drug. J .
Infect. Dis. 1980, 141, 87-91.
2-Am in o-3-(4-flu or op h en yl)-6-[(Z)-2-N-m eth ylca r ba m -
oyl-1-(4-m eth ylph en yl)vin yl]im idazo[1,2-b]pyr idazin e [(Z)-
13c]. Compound (Z)-13c was prepared according to the
procedure for (E)-13a . Starting from (Z)-12c, compound (Z)-
13c was obtained in 68% yield: mp 137 °C; 1H NMR (200 MHz,
CDCl3) δ 7.80 (dd, J ) 5.4, J ) 9.1, 1H, Ar), 7.63(d, J ) 9.1,
1H, Het), 7.25-7.11 (m, 6H, Ar), 6.83 (d, J ) 9.1, 1H, Het),
6.42 (s, 1H, CHdC), 6.22 (m, 1H, NHMe), 4.31 (bs, 2H, NH2),
2.59 (d, J ) 4.8, 3H, NHMe), 2.36 (s, 3H, Me); 13C NMR (50
MHz, CDCl3) δ 166.2, 161.3, 149.7, 145.1, 139.3, 136.0, 129.4,
129.3, 129.2, 129.0, 127.7, 124.9, 124.1, 120.9, 117.6, 115.7,
109.0, 25.9, 21.1; MS (EI+) m/z 401 M+ (100), 400 (7), 371 (9),
344 (8), 343 (7), 250 (7), 122 (13), 117 (9), 116 (9), 115 (16), 79
(9), 58 (12); HRMS calcd for C23H20FN5O 401.1651, found
401.164 92.
P la qu e Red u ction Assa y. Susceptible H-Hela cells were
grown in 60 mm tissue culture dishes in medium 199 contain-
ing 10% fetal bovine serum (FBS). After overnight incubation
at 37 °C to produce confluent monolayers, the growth medium
was removed and 0.2 mL/well of an appropriate dilution of
virus was added. After adsorption for 1 h at room temperature,
the infected cell sheet was overlaid with equal parts of 1.5%
sterile agarose solution and a 2× concentration of growth
medium containing 5% FBS and supplemented with test
compound dissolved in DMSO (final concentration of DMSO
was 0.01%). Infected cultures were maintained in a CO2
incubator at 37 °C until the DMSO control wells contained
visible viral plaques. A solution containing 10% formalin was
added to each well to inactivate the virus and fix the cell sheet
to the plastic surface. The fixed cell sheets were stained with
0.5% crystal violet in ethanol, and the plaques were counted.
Results from duplicate wells at each concentration were
averaged and compared with DMSO control wells. The inhibi-
tion of plaque formation by 50% or 90% (IC50 or IC90) was
calculated from the linear region of the inhibition concentra-
tion curve using the method of Reed and Muench.10
Cytop a th ic Effect Assa y. Hela cells were dispersed in 96-
well microtiter plates at 10 000 cells per well with medium
199 containing 5% FBS. After overnight incubation at 37 °C,
the cells were infected with virus, and medium containing
serial dilutions of drug or DMSO was added to the wells. The
resultant mixtures were incubated for 2-3 days (until exten-
sive CPE was apparent in DMSO control wells). To assess the
antiviral and cytotoxic effects, a fresh solution (0.4 mg/mL) of
XTT [2,3-bis(methoxy-4-nitro-5-sulfophenyl)-2H-tetraazolium-
5-carboxanilide, inner salt, sodium salt] in warm medium
without FBS was prepared. For each 5 mL of the XTT solution,
25 µL of 5 mM PMS (phenazine methosulfate) in phosphate
buffer saline was added. After removal of the cultured super-
natant, 100 µL of the freshly prepared XTT/PMS mixture was
added to each of the microtiter wells. The wells were then
(3) (a) Victor, F.; Loncharich, R.; Tang, J .; Spitzer, A. W. Synthesis
and Antiviral Activity of C2 Analogs of Enviroxime: An Explo-
ration of the Role of Critical Functionality. J . Med. Chem. 1997,
40, 3478-3483. (b) Victor, F.; Brown, T. J .; Campanale, K.;
Heinz, B. A.; Shipley, L. A.; Su, K. S.; Tang, J .; Vance, L. M.;
Spitzer, W. A. Synthesis, Antiviral Activity, and Biological
Properties of Vinylacetylene Analogs of Enviroxime. J . Med.
Chem. 1997, 40, 1511-1518.
(4) (a) Heinz, B. A.; Vance, L. M. The Antiviral Compound En-
viroxime Targets the 3A Coding Region of Rhinovirus and
Poliovirus. J . Virol. 1995, 69, 4189-4197. (b) Heinz, B. A.; Vance,
L. M. Sequence Determinants of 3A-Mediated Resistance to
Enviroxime in Rhinoviruses and Enteroviruses. J . Virol. 1996,
70, 4854-4857.
(5) (a) Phillpotts, R. J .; Delong, D. C.; Wallace, J .; J ones, R. W.; Reed,
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