4-Oxo-4,7-dihydrothieno[2,3-b]pyridines as non-nucleoside inhibitors of human cytomegalovirus and related herpesvirus polymerases

Article abstract of DOI:10.1021/jm050162b

A novel series of 4-oxo-4,7-dihydrothieno[2,3-6]pyridine-5-carboxamides have been identified as potential antivirals against human herpesvirus infections resulting from human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), and varicella-zoster virus (VZV). Compounds 10c and 14 demonstrated broad-spectrum inhibition of the herpesvirus polymerases HCMV, HSV-1, and VZV. High specificity for the viral polymerases was observed compared to human α polymerase. The antiviral activity of 10c and 14, as determined by plaque reduction assay, was comparable or superior to that of existing antiherpes drugs, ganciclovir (for HCMV) and acyclovir (for HSV-1 and VZV). Drug resistance to compound 14 correlated to point mutations in conserved domain III of the herpesvirus DNA polymerase, but these mutations do not confer resistance to existing nucleoside therapy. In addition, compound 14 maintained potent antiviral activity against acyclovir-resistant HSV-1 strains. Substitution to the pyridone nitrogen (N7) was found to be critical for enhanced in vitro antiviral activity.

Full text of DOI:10.1021/jm050162b

5794
J. Med. Chem. 2005, 48, 5794-5804
4-Oxo-4,7-dihydrothieno[2,3-b]pyridines as Non-Nucleoside Inhibitors of Human
Cytomegalovirus and Related Herpesvirus Polymerases
Mark E. Schnute,* Michele M. Cudahy, Roger J. Brideau, Fred L. Homa, Todd A. Hopkins, Mary L. Knechtel,
Nancee L. Oien, Thomas W. Pitts, Roger A. Poorman, Michael W. Wathen, and Janet L. Wieber
Medicinal Chemistry and Infectious Diseases Biology, Pharmacia Corporation, 301 Henrietta Street,
Kalamazoo, Michigan 49001
Received February 20, 2005
A novel series of 4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamides have been identified
as potential antivirals against human herpesvirus infections resulting from human cyto-
megalovirus (HCMV), herpes simplex virus type 1 (HSV-1), and varicella-zoster virus (VZV).
Compounds 10c and 14 demonstrated broad-spectrum inhibition of the herpesvirus polymerases
HCMV, HSV-1, and VZV. High specificity for the viral polymerases was observed compared to
human R polymerase. The antiviral activity of 10c and 14, as determined by plaque reduction
assay, was comparable or superior to that of existing antiherpes drugs, ganciclovir (for HCMV)
and acyclovir (for HSV-1 and VZV). Drug resistance to compound 14 correlated to point
mutations in conserved domain III of the herpesvirus DNA polymerase, but these mutations
do not confer resistance to existing nucleoside therapy. In addition, compound 14 maintained
potent antiviral activity against acyclovir-resistant HSV-1 strains. Substitution to the pyridone
nitrogen (N7) was found to be critical for enhanced in vitro antiviral activity.
Introduction
enzymes must recognize the drug as a substrate. The
requirement for two viral-specific enzymes in the path-
way for inhibition by the nucleoside analogues has also
led to increased risk of resistance especially in the
growing immunocompromised population.5 Many exist-
ing therapies are also hampered by significant drug-
associated toxicities that limit their duration of use. As
a consequence, there is an increasing realization of the
need for less toxic, orally bioavailable, broad-spectrum
agents to address herpesvirus infections in the growing
immunocompromised population, particularly in AIDS
patients and organ transplant recipients. The identi-
fication of a broad-spectrum, non-nucleoside anti-
viral that directly inhibits the viral DNA polymerase
therefore would offer clear advantages toward existing
therapies.
The herpesvirus DNA polymerase represents a com-
pelling molecular target to identify broad-spectrum
agents because of the high degree of homology among
the family.6 Seven regions (I-VII) have been identified
as being highly conserved by most members of the
family. Through the characterization of HSV polymerase
mutants it has been suggested that regions II and III
play a critical role in substrate recognition.7 Indeed,
point mutations within these regions have been associ-
ated with resistance to established therapies such as
acyclovir and ganciclovir.5,6,8
Diseases resulting from infections by viruses of the
Herpesvirus family continue to play a significant role
in patient morbidity and diminished individual quality
of life. Especially troublesome among viruses of the
family is human cytomegalovirus (HCMV). Although
generally benign in the immunocompetent host, HCMV
infection is associated with clinical symptoms such as
pneumonia, retinitis, and graft rejection in the immuno-
compromised, as well as congenital birth defects in
neonates.1 The prevalence of other herpesviruses such
as herpes simplex virus (HSV) types 1 and 2 and
varicella-zoster virus (VZV) is widespread among the
human population. Infection by HSV is responsible for
labial and genital herpes, while VZV on primary infec-
tion causes chicken pox.2 Reactivation of latent VZV
infection is associated with herpes zoster (shingles), a
highly debilitating illness characterized by persistent
neuropathic pain.3 Existing therapies for the manage-
ment of herpesvirus infections have been dominated by
nucleoside analogues such as acyclovir, ganciclovir, and
famciclovir. The nucleoside-based antivirals require
phosphorylation by a viral specific thymidine kinase and
subsequently cellular enzymes to afford an active drug
form, the nucleoside triphosphate. The resulting tri-
phosphate acts as a substrate for the viral DNA poly-
merase, competing with the binding of natural 2′-
deoxynucleoside triphosphate, and leads to partial or
complete chain termination after incorporation.4 As a
consequence, these therapies have a limited spectrum
of utility among viruses of the family because both
Recently, we have reported a novel series of 4-oxo-
1,4-dihydroquinoline (DHQ) carboxamides, represented
by PNU-181128, PNU-181465, and PNU-183792, that
demonstrated broad-spectrum inhibition of HCMV,
HSV-1, and VZV polymerases (Figure 1).9 These non-
nucleoside agents were found to be mixed competitive
inhibitors of nucleoside binding, however, not cross-
resistant to ganciclovir-resistant HCMV or acyclovir-
* To whom correspondence should be addressed. Address: Pfizer,
700 Chesterfield Parkway West, BB4M, Chesterfield, MO 63017.
Phone: (636) 247-3662. Fax: (636) 247-5234. E-mail: mark.e.schnute@
pfizer.com.
10.1021/jm050162b CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/09/2005

Thieno[2,3-b]pyridine Antivirals
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5795
ine 1, Scheme 1.11 Reduction of 2-nitrothiophene with
tin(II) chloride12 afforded the unstable amine that was
immediately condensed with diethyl ethoxymethylene-
malonate to provide enamine 1. Subsequent thermal
cyclization in degassed diphenyl ether afforded thieno-
[2,3-b]pyridine carboxylate ester 2. Conversion of the
ester to the desired 4-chlorobenzyl amide 3 was ac-
complished by simply heating a mixture of 2 and the
amine at 190 °C with removal of ethanol. To elaborate
the C2 position of the thienopyridine ring, amide 3 was
regioselectively iodinated at this position with iodine
monochloride to provide 4.
Iodide 4 served as the common intermediate for the
synthesis of analogues with diversity at both the C2 and
the N7 positions. In the first series of compounds, the
3-hydroxypropyl and 3-hydroxy-1-propynyl substituents
were maintained at the C2 position while the N7
substituent was surveyed. Sonogashira coupling be-
tween iodide 4 and propargyl alcohol provided alkyne
6a (Scheme 2). The pyridone nitrogen readily underwent
regioselective alkylation with a variety of electrophiles
(iodoethane, b; 2-bromopropane, c; 2-bromoethanol, d;
2-bromo-N,N-diethylethylamine, e) to afford alkylated
products 6b-e. Subsequent hydrogenation of 6a-e
afforded the corresponding 3-hydroxypropyl analogues
7a-e. In the converse approach, the N7 substituent was
maintained as methyl, and the tether length to the
hydroxy moiety at the C2 position was surveyed in the
alkyl and alkynyl series. The shortest homologue, hy-
droxymethyl, was prepared from 4 by palladium-
catalyzed carbonylation in the presence of methanol to
afford methyl carboxylate ester 8 (Scheme 3). Subse-
quent reduction of the ester with lithium aluminum
hydride followed by alkylation of the pyridone nitrogen
with iodomethane provided hydroxymethyl compound
10a. Preparation of the two-carbon homologue, 2-hy-
droxyethyl, required initial alkylation of the pyridone
nitrogen; therefore, iodide 4 was converted to compound
5 with iodomethane (Scheme 1). Subsequent coupling
of 5 with tributylvinylstannane under Stille conditions
yielded vinyl derivative 11 (Scheme 4). The sequence
ofhydroboration-oxidationemploying11and9-borabicyclo-
[3.3.1]nonane afforded hydroxyethyl compound 10b. The
remaining homologues at the C2 position were prepared
by Sonogashira coupling between iodide 5 and a corre-
sponding hydroxyalkyl acetylene (propargyl alcohol,
3-butyn-1-ol, and 4-pentyn-1-ol) to afford alkynes 12a-c
(Scheme 5). Hydrogenation of the resulting alkynes
provided hydroxyalkyl derivatives 10c-e.
Figure 1. Lead 4-oxo-1,4-dihydroquinoline carboxamides.
resistant HSV-1 mutants. Structure-activity relation-
ship studies around these lead structures as well as
related naphthalene carboxamides suggested that the
ortho orientation of the carboxamide and the hydroxy
(or oxo) group was critical for activity and that the C6
substituent played a significant role toward enhanced
potency.9,10
We were intrigued by the possibility that the central
heterocycle may serve an organizational role for these
three pharmacophoric elements and that other isosteric
structures might play a similar role. Noteworthy among
possible isosteres to the quinoline ring is the thieno-
[2,3-b]pyridine heterocycle (Scheme 1, 2) in which a
thiophene now replaces the fused benzene ring. Al-
though the ring size has been contracted, the increased
atomic radius of sulfur versus carbon still allows for
spatial superpositioning between the C6 position of the
quinoline ring and the C2 position of the thieno[2,3-b]-
pyridine ring. This report examines the viability of such
an isosteric replacement toward maintained herpes
DNA polymerase inhibition. Initial structure-activity
relationships have been characterized for the C2 sub-
stituent (hydroxyalkyl, hydroxyalkynyl, or morpholin-
4-ylmethyl) as well as the significance of pyridone
nitrogen substitution.
Chemistry
The synthesis of the thieno[2,3-b]pyridine nucleus was
accomplished by following an adaptation of existing
Gould-Jacob cyclization methodology employing enam-
Scheme 1

5796 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Schnute et al.
Scheme 2
Scheme 5
Scheme 6
Scheme 3
Table 1. Influence of N7 Substituent on HCMV Polymerase
Inhibition and HCMV Antiviral Activity
HCMV polymerase HCMV antiviral
R
compd
IC50 (µM)a
IC50 (µM)a,b
Scheme 4
H
6a
12a
6b
6c
6d
6e
2.7 ( 0.2
1.0
>20.0
CH3
1.4 ( 0.8
1.7 ( 0.4
2.7 ( 0.2
2.3
CH2CH3
CH(CH3)2
CH2CH2OH
CH2CH2NEt2
1.8
4.2
1.1 ( 0.2
1.8 ( 0.2
3.8 ( 0.3
H
7a
10c
7b
7c
7d
7e
2.2 ( 0.1
0.40
>20.0
CH3
0.6 ( 0.0
2.3 ( 0.1
1.3 ( 0.0
1.8
Synthesis of the corresponding 2-(morpholin-4-yl-
methyl) analogue in the thieno[2,3-b]pyridine series
proved to be straightforward utilizing Mannich chem-
istry. Carboxamide 3 was reacted with morpholine in
the presence of acetic acid to provide 13 (Scheme 6).
Subsequent alkylation of the pyridone nitrogen with
iodomethane provided the targeted analogue 14.
CH2CH3
CH(CH3)2
CH2CH2OH
CH2CH2NEt2
0.67
2.4
0.75
0.81
0.9 ( 0.2
a Value represents the mean IC50 ( SEM derived from two
determinations (single determination where SEM is absent).
b Determined by plaque reduction assay (Davis strain).
could be replaced by the thieno[2,3-b]pyridine nucleus
and still maintain inhibitory activity against a viral
polymerase. To answer this question, the direct struc-
tural analogues to PNU-181128 and PNU-181465 were
prepared and tested, 6a and 7a, respectively (Table 1).
As anticipated, both compounds demonstrated inhibi-
tion of HCMV polymerase; however, antiviral activity
in cell culture was poor (IC50 > 20 µM) compared to
quinoline derivatives PNU-181128 (IC50 ) 1.1 µM) and
PNU-181465 (IC50 ) 0.4 µM). Nonetheless, these results
suggested that the thieno[2,3-b]pyridine series war-
ranted further examination. Further structural modi-
Results and Discussion
For primary screening, the ability of compounds to
inhibit HCMV polymerase activity was assessed by
determining the reduction in 3H-labeled nucleotide
(dTTP) incorporation into a primer template (biotin-
ylated oligo(dT)16-poly(dA)) through a scintillation prox-
imity assay. The antiviral activity of select compounds
was then evaluated in cell culture plaque reduction
assays employing HCMV (Davis strain).
The first milestone to be realized was whether the
quinoline ring system found in previous lead compounds

Thieno[2,3-b]pyridine Antivirals
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5797
Table 2. HCMV Polymerase Inhibition for C2-Hydroxyalkyl
butyl) methylenes. Both compounds 10c and 10d ex-
hibited similar potency in whole cells against HCMV
(IC50 ) 0.6 and 0.8 µM, respectively). In the alkynyl
series (12a-c), the shortest homologue examined (12a,
3-hydroxy-1-propynyl) proved to be the most potent
against HCMV polymerase. Mirroring the behavior
observed in the hydroxyalkyl series, activity diminished
as the tether length was increased. Consistent with the
previous experience in the DHQ carboxamide series,
morpholin-4-ylmethyl derivative 14 also demonstrated
inhibition of HCMV polymerase (IC50 ) 1.1 µM).
Compounds 10c and 14 were subsequently evaluated
against an expanded set of herpesvirus polymerases
(HSV-1 and VZV) and human DNA polymerases to
assess the activity spectrum and specificity (Table 3).
Compound 10c demonstrated broad-spectrum inhibition
of the herpesvirus polymerases with activity comparable
to that observed for the 4-hydroxyquinoline PNU-
181465. High specificity for the viral polymerases was
observed compared to human R polymerase. The mor-
pholin-4-ylmethyl derivative 14 demonstrated a similar
profile of viral DNA polymerase specificity (human R,
γ, and δ evaluated). The broad-spectrum profile of these
compounds against the polymerases was similarly ob-
served when 10c and 14 were examined for antiviral
effects against HCMV, HSV-1, and VZV, employing
plaque reduction assays. The activity of 10c and 14 was
comparable to that of ganciclovir against HCMV and
acyclovir against HSV-1, while it was significantly more
potent against VZV compared to acyclovir. A high
therapeutic index was also observed with respect to
cytotoxicity in both human foreskin fibroblast (HFF) and
Vero cells (CC50 > 100 µM).
To more fully characterize the mechanism of antiviral
activity derived from the 4-oxo-4,7-dihydrothieno[2,3-
b]pyridine series, enzyme kinetics and the consequence
of polymerase mutation were assessed for the repre-
sentative compound 14. Enzyme kinetics were examined
with respect to inhibition of dTTP incorporation into the
primer template with HCMV polymerase. In contrast
to previously examined 4-hydroxyquinoline antivirals
that demonstrated mixed competitive behavior,9a 14 was
found to be a competitive inhibitor of substrate binding
to the polymerase (Ki ) 1.0 µM) (Figure 2). Previously,
we have reported the identification of HSV-1 and HSV-2
resistant mutants to the 4-oxo-1,4-dihydroquinoline
antiviral class.14 The resulting mutants carried a single
point mutation to the viral DNA polymerase character-
ized by a V823A (HSV-1) or V826A (HSV-2) amino acid
change. These amino acid residues correspond to con-
served region III of the herpes DNA polymerase wherein
valine is conserved in six (HSV-1, HSV-2, HCMV, VZV,
Epstein-Barr virus, and HHV-8 (human herpes virus
8)) of the eight human herpes viruses. In the case of
HCMV, serial passage experiments were unsuccessful
in isolating a resistant strain. A mutant resistant to
members of the 4-oxo-1,4-dihydroquinoline antiviral
class was identified after transfecting cells with HCMV
polymerase carrying two point mutations (V823A/
V824L) corresponding to the valine residues in the
region analogous to that of HSV-1. The three resulting
mutant strains were subsequently used to further verify
the mode of antiviral activity exhibited by compound
14. Indeed, plaque reduction assays conducted with
and Hydroxyalkynyl Homologues
n
compd
IC50 (µM)a
compd
IC50 (µM)a
1
2
3
4
5
10a
10b
10c
10d
10e
18.5
4.0
0.40
0.43
9.7
12a
12b
12c
1.0
9.5
22.0
a IC50 value derived from a single determination. 15-25%
historical average SEM for controls.
fication focused on the N7 (pyridone nitrogen) substit-
uentwhilemaintainingthe3-hydroxypropyland3-hydroxy-
1-propynyl substituents at C2. As shown in Table 1,
substitution was well tolerated at N7 in both series with
respect to HCMV polymerase inhibition. Simple alkyl
as well as functionalized alkyl (hydroxyethyl and N,N-
diethylaminoethyl) substitution demonstrated good in-
hibition with only alkyl branching adjacent to the ring
(isopropyl, 6c and 7c) appearing to be detrimental. In
contrast to 6a and 7a, HCMV antiviral activity for the
substituted series correlated well with HCMV poly-
merase inhibition showing a greater than 10-fold im-
provement in antiviral activity over the unsubstituted
analogues. The stark difference between the cellular
antiviral activity of 7a and the corresponding alkylated
derivatives such as 10c may be associated with the
intrinsic Lewis acidity of the 4-hydroxythieno[2,3-b]-
pyridine (3, pKa ) 6.8), which may impede cellular
transport. Alkylation at nitrogen therefore eliminates
the acidic proton and affords a neutral molecule.
The observation that the inhibitory activity of the
alkylated derivatives against HCMV polymerase was
comparable or superior to that of 6a or 7a also sheds
light on the physiologically significant tautomeric form
of the molecules at the binding site and a refinement of
the role the C4 oxygen substituent plays as a pharma-
cophoric element. Although the relative position of the
keto/enol tautomer population for 6a and 7a under
physiological or assay conditions is unknown,13 the
potency of the alkylated series, where the enol tautomer
population has been excluded, suggests that the keto
tautomer is the dominant species binding to the poly-
merase in the cases of 6a and 7a. In addition, it suggests
that the oxygen substituent at the 4-position of the ring
serves a common pharmacophoric role as either an
inter- or intramolecular hydrogen bond acceptor in both
series.
A more noticeable structural impact on the HCMV
polymerase inhibition was realized as the length and
degrees of freedom to the tether between the thieno-
pyridine C2 position and the terminal hydroxyl group
was surveyed (Table 2). For this series of analogues, the
7-methyl group was maintained for consistency. A
parabolic relationship was observed in the hydroxyalkyl
series (10a-e) as the distance was increased. Optimal
positioning of the hydroxyl group was in the region of
three (10c, 3-hydroxypropyl) to four (10d, 4-hydroxy-

5798 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Schnute et al.
Table 3. Broad-Spectrum Activity of 10c and 14 Compared to 4-Oxo-1,4-dihydroquinolines and Established Therapies
polymerase IC50 (µM)a
antiviral IC50 (µM)e
CC50 (µM)g
compd
HCMV
HSV-1
VZV
R
γ
δ
HCMV
HSV-1
VZV
HFF
Vero
10c
14
0.4
1.1b
0.4
0.7
0.5
1.2
0.3
0.6
0.46
0.77
0.07
0.37
>40.0
>40.0
>40.0
>40.0
ndh
>40.0
>40.0
>40.0
ndh
>40.0
>40.0
>40.0
0.6
0.8f
3.0
3.7
7.4
3.5
ndh
2.1
0.09
0.77
0.17
0.34
ndh
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
PNU-181465c
PNU-183792c
ganciclovir
acyclovir
1.0
0.9
1.3
>20.0
8.1
foscarnet
2.5
0.4
22.1
ndh
0.5
3.3
ndh
0.60
5.8
>20.0
2.6
13.4
< 0.28
2.6
12.0
>20.0
>40.0
2.3
aphidicolin
AZT-TPd
a IC50 value derived from a single determination unless otherwise noted. 15-25% historical average SEM for controls. b n ) 5, SEM (
0.4 µM. c Reference 9a. d AZT-TP ) zidovudine triphosphate. e Determined by plaque reduction assay (HCMV, Davis strain; HSV-1, KOS
strain; VZV, Webster strain). Value represents the mean IC50 value derived from two determinations unless otherwise noted. Average
SEM ( 25%. f n ) 6, SEM ( 0.3 µM. g CC50, 50% cellular cytotoxicity, derived from single determination. h nd: not determined.
Table 4. Conferred Resistance to 14 after Induced Mutation to Conserved Region III of Herpes Polymerases and Activity against
Drug-Resistant HSV-1 Isolates
antiviral plaque reduction IC50 (µM)c
HCMV Ad169
HSV-1 KOS
AraAr13
HSV-1 Patton
WT
BWr
2.5
> 10.0
HSV-2
V826A
compd
WTa
V823Ab
WT
V823A
PAAr5
PFAr2
WT
14
1.5
6.2
5.6
3.5
> 50.0
2.1
> 10.0
0.8
> 10.0
2.5
> 10.0
5.4
5.4
0.8
2.4
> 50.0
acyclovir
4.4
2.8
ganciclovir
0.8
0.8
a WT: wild-type parental virus. b Strain contains two residue mutations (V823A/V824L). c Value represents the mean IC50 value derived
from two determinations, average SEM ( 25%.
Conclusion
A novel series of 4-oxo-4,7-dihydrothieno[2,3-b]pyri-
dine-5-carboxamides have been identified as non-
nucleoside inhibitors of herpesvirus DNA polymerases
including HCMV, HSV-1, and VZV. Alkylation of the
pyridone nitrogen (N7) affords compounds that demon-
strate in vitro antiviral activity against these three
viruses. These results indicate that indeed the thieno-
[2,3-b]pyridine ring system is a competent isosteric
replacement for the quinoline ring found in previously
reported 4-oxo-1,4-dihydroquinoline antivirals such as
PNU-183792. Compounds 10c and 14 exhibited broad-
spectrum antiviral activity against HCMV, HSV-1, and
VZV with activity comparable to that of ganciclovir
against HCMV and that of acyclovir against HSV-1.
Both compounds were found to be significantly more
potent against VZV when compared to acyclovir. In
addition, compound 14 was shown not to be cross-
Figure 2. Enzyme kinetics analysis for compound 14.
these mutant strains resulted in a 4- to 10-fold increase
in IC50 for compound 14 with respect to wild type (Table
4). The resulting mutant strains, however, did not
demonstrate a reduced sensitivity to the nucleoside
analogues ganciclovir (HCMV) and acyclovir (HSV-1 and
HSV-2). The results further support that compound 14
elicits an antiviral response through binding to and
subsequent inhibition of the viral DNA polymerase. It
also suggests that 14 makes critical contacts within
conserved domain III of the polymerase (implicated in
nucleotide binding); however, the binding mode is
distinct from that of nucleoside analogues such as
ganciclovir and acyclovir. The marked difference in
antiviral profile from the nucleoside analogues is also
evident when 14 was evaluated against acyclovir-
resistant HSV-1 KOS strains (AraAr13, PAAr5, and
PFAr2) and an HSV-1 Patton strain (BWr).15 The
acyclovir-resistant strains were found to be equally
sensitive (or in some cases hypersensitive) to compound
14 compared to the corresponding wild-type strains.
resistant with existing nucleoside analogues. As a class,
4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxam-
ides offer an exciting platform to identify antivirals that
may address the unmet medical needs still present
resulting from herpesvirus infection.
Experimental Section
All reagents were obtained from commercial suppliers and
used without further purification. N,N-Dimethylformamide
(DMF) was of anhydrous grade. Column chromatography was
performed using either 40S or 40M Biotage KP-Sil silica
prepacked cartridges. Solvents for extraction and chromatog-
raphy were of HPLC grade. Brine refers to a saturated aqueous
1
solution of sodium chloride. Chemical shifts for H NMR and
13C NMR are given in ppm (δ); multiplicities are indicated by
s (singlet), d (doublet), t (triplet), q (quartet), qt (quintet), m
(multiplet), or bs (broadened singlet). Data for mass spectra
analysis are reported in the form m/z (intensity relative to base
) 100). Melting points were determined on a Thomas-Hoover
capillary melting point apparatus and are uncorrected.
Diethyl 2-((2-Thienylamino)methylene)malonate (1).
2-Nitrothiophene (85%, 100 g, 0.77 mol) was suspended in

Thieno[2,3-b]pyridine Antivirals
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5799
concentrated hydrochloric acid (550 mL) in a 2 L, three-necked,
round-bottom flask, and the mixture was heated to 40-45 °C
with vigorous stirring. Stannous chloride dihydrate (350 g, 1.55
mol, 2.0 equiv) was added portionwise to the mixture, main-
taining the reaction temperature between 45-50 °C by im-
mersion in an ice bath. After the addition was complete, the
cooling bath was removed, and the mixture was allowed to
cool to 30-35 °C over 1 h. The mixture was further cooled to
below 5 °C, and the precipitate was filtered and washed with
concentrated hydrochloric acid (20 mL). The brown solid was
dried in a stream of air for 20 min, washed with diethyl ether
(50 mL), and dried for an additional 30 min. The above
procedures were repeated, and the two lots were combined to
afford 169.6 g (0.52 mol) of the hexachlorostannate salt as a
brown solid. The resulting salt was dissolved in water (3 L)
and filtered through a Celite plug. In 500 mL portions, the
solution was neutralized with ammonia hydroxide (50 mL)
followed by 2 N sodium hydroxide (50 mL) and was extracted
with CH2Cl2 (3 × 100 mL). The combined organic layers were
immediately mixed with diethyl ethoxymethylenemalonate
(106 mL, 0.52 mol) and dried (Na2SO4). The solution was
filtered and concentrated in vacuo (40 °C). The crude product
was purified by column chromatography (heptane/2-propanol;
30/1, 20/1, 10/1) and mixed fractions were further purified
(heptane/2-propanol; 50/1, 30/1). The product-containing frac-
tions were concentrated and the enamine was crystallized from
hexane/ethyl acetate (300 mL, 9/1) to afford 72.2 g as an off-
white solid. Mixed fractions from the chromatography contain-
ing diethyl ethoxymethylenemalonate were concentrated and
crystallized from hexane/ethyl acetate (100 mL, 9/1) to afford
21.2 g as a yellow solid. The filtrate from these crystallizations
was concentrated and purified by column chromatography
employing the same conditions as above. The product-contain-
ing fractions were concentrated and crystallized from hexane/
ethyl acetate (100 mL, 9/1) to afford an additional 11.0 g as a
white solid. The above procedure afforded a combined yield of
104.4 g (29%). Mp 39-40 °C; 1H NMR (300 MHz, DMSO-d6) δ
10.90 (br, 1 H), 8.06 (s, 1 H), 7.15 (dd, J ) 5.4, 1.5 Hz, 1 H),
6.97 (dd, J ) 3.7, 1.5 Hz, 1 H), 6.91 (dd, J ) 5.3, 3.7 Hz, 1 H),
4.15 (br, 4 H), 1.24 (t, J ) 7.1 Hz, 6 H); 13C NMR (75 MHz,
DMSO-d6) δ 166.7, 164.7, 152.6, 144.2, 126.5, 118.7, 114.9,
93.4, 59.6, 59.5, 14.2. Anal. (C12H15NO4S) C, H, N, S.
Ethyl 4-Hydroxythieno[2,3-b]pyridine-5-carboxylate
(2). Enamine 1 (25.0 g, 92.8 mmol) and diphenyl ether (250
mL) were combined and degassed (freeze-pump-thaw). The
reaction mixture was heated to reflux for 10 min. The mixture
was allowed to cool to room temperature and was then purified
by column chromatography (CH2Cl2; CH2Cl2/methanol, 99/1)
to afford 15.56 g (75%) of 2 as a pale-yellow solid. 1H NMR
(300 MHz, DMSO-d6) δ 8.72 (s, 1 H), 7.70 (s, 1 H), 7.50-7.48
(m, 1 H), 4.38 (q, J ) 7.0 Hz, 2 H), 1.35 (t, J ) 7.1 Hz, 3 H).
N-(4-Chlorobenzyl)-4-hydroxythieno[2,3-b]pyridine-5-
carboxamide (3). A mixture of ester 2 (74.8 g, 0.335 mol)
and 4-chlorobenzylamine (237.2 g, 1.675 mol) was heated to
190 °C for 1 h with removal of ethanol via a Dean-Stark trap
and then allowed to cool to 120 °C. A solution of toluene (800
mL) and ethanol (15 mL) was added, and the mixture was
allowed to cool to room temperature. The resulting white
precipitate was filtered, washed with toluene (200 mL), and
recrystallized from acetic acid (800 mL)/water (800 mL) to yield
95.69 g (90%) of 3 as a white solid. Mp 238-240 °C; 1H NMR
(300 MHz, DMSO-d6) δ 13.4 (s, 1 H), 10.6 (br, 1 H), 8.72 (s, 1
H), 7.45-7.34 (m, 6 H), 4.55 (d, J ) 5.9 Hz, 2 H); 13C NMR (75
MHz, CF3CO2D) δ 169.0, 167.1, 153.6, 139.5, 134.7, 133.2,
128.8, 128.8, 127.8, 127.6, 107.9, 43.8; MS (ESI-) m/z 317 (100,
(M - H)-). Anal. (C15H11ClN2O2S) C, H, N, Cl, S.
sodium bisulfite (400 mL). After being stirred vigorously for
30 min, the aqueous mixture was partially concentrated in
vacuo to remove chloroform. The resulting aqueous suspension
was filtered, washed with water (20 mL), and dried. The crude
product was recrystallized by dissolving in methanol (1 L), and
the resulting solution was filtered, concentrated in vacuo to
∼400 mL, and cooled to 0 °C. Filtration afforded 6.33 g (62%)
of 4 as white solid. Mp 230 °C (dec); 1H NMR (300 MHz,
DMSO-d6) δ 13.40 (bs, 1 H), 10.40 (t, J ) 5.9 Hz, 1 H), 8.68 (s,
1 H), 7.63 (s, 1 H), 7.41-7.33 (m, 4 H), 4.52 (d, J ) 5.9 Hz, 2
H). Anal. (C15H10ClIN2O2S) C, H, N, Cl, S.
N-(4-Chlorobenzyl)-2-iodo-7-methyl-4-oxo-4,7-dihy-
drothieno[2,3-b]pyridine-5-carboxamide (5). Potassium
carbonate (1.76 g, 12.7 mmol, 3.0 equiv) and iodomethane (0.79
mL, 12.7 mmol, 3.0 equiv) were added to a solution of 4 (2.00
g, 4.23 mmol) in DMF (14 mL). The reaction mixture was
heated to 90 °C and stirred for 18 h. The mixture was
concentrated in vacuo. The resulting residue was partitioned
between water (100 mL) and CH2Cl2 (200 mL). The aqueous
layer was extracted with CH2Cl2 (3 × 150 mL). The combined
organic layers were dried (MgSO4), filtered, and concentrated
in vacuo. The resulting solid was recrystallized from ethanol
to yield 1.78 g (92%) of 5 as an off-white solid. Mp 236-237
1
°C; H NMR (300 MHz, DMSO-d6) δ 10.45 (t, J ) 5.9 Hz, 1
H), 8.69 (s, 1 H), 7.71 (s, 1 H), 7.41-7.32 (m, 4 H), 4.54 (d, J
) 5.9 Hz, 2 H), 3.93 (s, 3 H); 13C NMR (75 MHz, DMSO-d6) δ
171.1, 164.5, 154.8, 145.9, 139.0, 133.2, 132.5, 131.9, 129.6,
128.8, 115.0, 73.5, 43.5, 41.9; MS (ESI+) m/z 459 ((M + H)+,
100). Anal. (C16H12ClIN2O2S) C, H, N, Cl, S.
N-((4-Chlorophenyl)methyl)-4-hydroxy-2-(3-hydroxy-
1-propynyl)-thieno[2,3-b]pyridine-5-carboxamide (6a).
Copper(I) iodide (0.128 g, 0.675 mmol, 0.3 equiv) and PdCl2-
(PPh3)2 (0.032 g, 0.045 mmol, 0.02 equiv) were added to a
suspension of 4 (1.00 g, 2.25 mmol) in diethylamine (28 mL).
Propargyl alcohol (0.16 mL, 2.70 mmol, 1.2 equiv) was added,
and the mixture was stirred at room temperature for 3 days.
The reaction mixture was partitioned between water (100 mL)
and ethyl acetate (100 mL), and the aqueous layer was
extracted with ethyl acetate (2 × 100 mL). The combined
organic layers were washed with a saturated aqueous am-
monium chloride solution (100 mL), dried (MgSO4), filtered,
and concentrated in vacuo. The resulting solid was purified
by column chromatography (CH2Cl2/methanol; 98/2, 95/5)
followed by recrystallization from CH2Cl2/methanol to yield
1
0.165 g (20%) of 6a as an off-white solid. Mp 233-236 °C; H
NMR (300 MHz, DMSO-d6) δ 13.32 (bs, 1 H), 10.41 (bs, 1 H),
8.75 (s, 1 H), 7.49 (s, 1 H), 7.41-7.33 (m, 4 H), 5.44 (bs, 1 H),
4.53 (d, J ) 5.9 Hz, 2 H), 4.34 (s, 2 H); 13C NMR (75 MHz,
DMSO-d6) δ 172.8, 164.9, 148.8, 143.0, 131.9, 130.9, 129.6,
128.8, 126.8, 117.1, 114.2, 96.4, 76.6, 50.0, 41.9; MS (ESI-)
m/z 371 (100, (M - H)-). Anal. (C18H13ClN2O3S) C, H, N, Cl,
S.
N-((4-Chlorophenyl)methyl)-4,7-dihydro-7-ethyl-2-(3-
hydroxy-1-propynyl)-4-oxo-thieno[2,3-b]pyridine-5-car-
boxamide (6b). Potassium carbonate (0.149 g, 1.08 mmol, 2.0
equiv) and iodoethane (0.09 mL, 1.08 mmol, 2.0 equiv) were
added to a solution of 6a (0.200 g, 0.54 mmol) in DMF (10 mL).
The mixture was stirred at room temperature for 3 days. The
reaction mixture was poured into water (20 mL). The resulting
solid was filtered and recrystallized from ethanol to yield 0.140
1
g (65%) of 6b as an off-white solid. Mp 220-221 °C (dec); H
NMR (400 MHz, DMSO-d6) δ 10.42 (t, J ) 5.9 Hz, 1 H), 8.79
(s, 1 H), 7.57 (s, 1 H), 7.41-7.33 (m, 4 H), 5.49 (br, 1 H), 4.55
(d, J ) 5.9 Hz, 2 H), 4.36 (s, 2 H), 4.31 (q, J ) 7.2 Hz, 2 H),
1.44 (t, J ) 7.2 Hz, 3 H); 13C NMR (100 MHz, DMSO-d6) δ
171.7, 163.8, 149.1, 144.9, 138.4, 131.3, 130.9, 129.0, 128.3,
127.4, 116.3, 115.0, 96.3, 75.6, 51.8, 49.4, 41.3, 13.6; MS (ESI+)
m/z 401 ((M + H)+, 100). Anal. (C20H17ClN2O3S‚0.1EtOH) C,
H, N, Cl, S.
N-(4-Chlorobenzyl)-4-hydroxy-2-iodothieno[2,3-b]-
pyridine-5-carboxamide (4). Carboxamide 3 (7.30 g, 22.9
mmol) and sodium acetate (5.64 g, 68.7 mmol, 3.0 equiv) were
dissolved in a mixture of chloroform/methanol (10/1, 440 mL).
A solution of iodine monochloride (11.15 g, 68.7 mmol, 3.0
equiv) in methanol (10 mL) was then added to the mixture.
The reaction mixture was stirred at room temperature for 1 h
and then poured into a mixture of ice and saturated aqueous
N-(4-Chlorobenzyl)-2-(3-hydroxy-1-propynyl)-7-iso-
propyl-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carbox-
amide (6c). Potassium carbonate (0.298 g, 2.16 mmol, 2.0
equiv) and 2-bromopropane (0.20 mL, 2.16 mmol, 4.0 equiv)
were added to a solution of 6a (0.200 g, 0.54 mmol) in DMF

5800 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Schnute et al.
(10 mL). The mixture was heated to 60 °C for 18 h and then
was partitioned between water (20 mL) and CH2Cl2 (25 mL).
The aqueous layer was extracted with CH2Cl2 (3 × 25 mL).
The combined organic layers were washed with brine (50 mL),
dried (MgSO4), filtered, and concentrated in vacuo. The
resulting oil was purified by column chromatography (CH2Cl2/
methanol, 99/1) followed by recrystallization from methanol
to yield 0.043 g (19%) of 6c as a yellow solid. Mp 191-197 °C
(dec); 1H NMR (400 MHz, DMSO-d6) δ 10.42 (t, J ) 6.0 Hz, 1
H), 8.71 (s, 1 H), 7.58 (s, 1 H), 7.41-7.34 (m, 4 H), 5.48 (t, J )
6.0 Hz, 1 H), 4.55 (d, J ) 5.8 Hz, 2 H), 4.55-4.48 (m, 1 H),
4.35 (s, 2 H), 1.57 (d, J ) 6.6 Hz, 6 H); 13C NMR (100 MHz,
DMSO-d6) δ 171.6, 163.7, 149.1, 140.7, 138.4, 131.3, 131.1,
129.1, 128.2, 127.4, 116.3, 115.1, 96.3, 75.5, 59.0, 49.4, 41.4,
20.8; MS (ESI+) m/z 415 ((M + H)+, 100). Anal. (C21H19-
ClN2O3S) C, H, N, Cl, S.
N-(4-Chlorobenzyl)-7-ethyl-2-(3-hydroxypropyl)-4-oxo-
4,7-dihydrothieno[2,3-b]pyridine-5-carboxamide (7b).
Compound 6b (0.197 g, 0.491 mmol) in a mixture of CH2Cl2/
methanol (1/1, 50 mL) was hydrogenated (35 psi) in the
presence of 10% Pd/C (59 mg). The reaction mixture was
filtered through a Celite pad, and the filtrate was concentrated
in vacuo. The resulting solid was purified by column chroma-
tography (CH2Cl2; CH2Cl2/methanol; 98/2, 95/5) to yield 0.114
g (57%) of 7b as a white solid. Mp 144-146 °C; 1H NMR (300
MHz, DMSO-d6) δ 10.62 (t, J ) 5.9 Hz, 1 H), 8.72 (s, 1 H),
7.41-7.33 (m, 4 H), 7.20 (s, 1 H), 4.56 (t, J ) 5.2 Hz, 1 H),
4.55 (d, J ) 5.8 Hz, 2 H), 4.31 (q, J ) 7.2 Hz, 2 H), 3.47 (q, J
) 6.0 Hz, 2 H), 2.90 (t, J ) 7.5 Hz, 2 H), 1.80 (qt, J ) 7.5 Hz,
2 H), 1.44 (t, J ) 7.2 Hz, 3 H); 13C NMR (75 MHz, DMSO-d6)
δ 172.3, 164.8, 148.6, 144.2, 140.5, 139.1, 132.1, 131.8, 129.6,
128.8, 120.0, 115.1, 60.0, 52.1, 41.9, 34.3, 26.5, 14.4; MS (ESI-)
m/z 403 ((M - H)-, 100). Anal. (C20H21ClN2O3S) C, H, N, Cl,
S.
N-(4-Chlorobenzyl)-2-(3-hydroxypropyl)-7-isopropyl-
4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamide
(7c). Compound 6c (0.225 g, 0.542 mmol) in ethanol (50 mL)
was hydrogenated (35 psi) in the presence of 10% Pd/C (68
mg). The reaction mixture was filtered through a Celite pad,
and the filtrate was concentrated in vacuo. The resulting solid
was recrystallized from ethyl acetate/heptane to yield 0.104 g
(46%) of 7c as an off-white solid. Mp 84-92 °C; 1H NMR (300
MHz, DMSO-d6) δ 10.61 (t, J ) 5.9 Hz, 1 H), 8.66 (s, 1 H),
7.41-7.33 (m, 4 H), 7.21 (s, 1 H), 4.59-4.50 (m, 4 H), 3.47 (q,
J ) 6.2 Hz, 2 H), 2.91 (t, J ) 7.5 Hz, 2 H), 1.80 (qt, J ) 7.6
Hz, 2 H), 1.57 (d, J ) 6.6 Hz, 6 H); 13C NMR (75 MHz, DMSO-
d6) δ 172.2, 164.8, 148.6, 140.4, 140.0, 139.1, 132.2, 131.9,
129.7, 128.8, 120.0, 115.1, 60.0, 59.0, 41.9, 34.3, 26.4, 21.5; MS
(FAB) m/z 419 (MH+, 99); HRMS (FAB) calcd for C21H23-
ClN2O3S + H (M + H)+ m/z 419.1196, found 419.1172. Anal.
(C21H23ClN2O3S) C, H, N, Cl, S.
N-(4-Chlorobenzyl)-7-(2-hydroxyethyl)-2-(3-hydroxy-1-
propynyl)-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-car-
boxamide (6d). Potassium carbonate (0.739 g, 5.35 mmol, 5.0
equiv) and 2-bromoethanol (0.76 mL, 10.7 mmol, 10.0 equiv)
were added to a solution of 6a (0.400 g, 1.07 mmol) in DMF
(20 mL). The reaction mixture was heated to 100 °C and stirred
for 5 h. The mixture was partitioned between water (50 mL)
and CH2Cl2 (50 mL). The aqueous layer was extracted with
CH2Cl2 (3 × 50 mL). The combined organic layers were washed
with brine (50 mL), dried (MgSO4), filtered, and concentrated
in vacuo. The resulting solid was purified by column chroma-
tography (CH2Cl2/methanol; 99/1, 97/3, 96/4) followed by
recrystallization from ethanol to yield 0.131 g (29%) of 6d as
1
an off-white solid. Mp 225-227 °C (dec); H NMR (400 MHz,
DMSO-d6) δ 10.43 (t, J ) 6.0 Hz, 1 H), 8.70 (s, 1 H), 7.56 (s, 1
H), 7.41-7.33 (m, 4 H), 5.48 (t, J ) 5.9 Hz, 1 H), 5.17 (t, J )
5.4 Hz, 1 H), 4.55 (d, J ) 5.9 Hz, 2 H), 4.36 (d, J ) 5.6 Hz, 2
H), 4.31 (t, J ) 4.8 Hz, 2 H), 3.81-3.78 (m, 2 H); 13C NMR
(100 MHz, DMSO-d6) δ 171.8, 163.8, 149.9, 146.1, 138.4, 131.3,
130.8, 129.0, 128.3, 127.2, 116.1, 114.4, 96.2, 75.6, 59.2, 58.2,
49.4, 41.3; MS (ESI+) m/z 417 ((M + H)+, 100). Anal. (C20H17-
ClN2O4S) C, H, N, Cl, S.
N-(4-Chlorobenzyl)-7-(2-hydroxyethyl)-2-(3-hydroxy-
propyl)-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carbox-
amide (7d). Compound 6d (0.200 g, 0.48 mmol) in THF (90
mL) was hydrogenated (35 psi) in the presence of 10% Pd/C
(60 mg). The reaction mixture was filtered through a Celite
pad, and the filtrate was concentrated in vacuo. The resulting
solid was purified by column chromatography (CH2Cl2/
methanol, 97/3) followed by recrystallization from acetonitrile
to yield 0.066 g (33%) of 7d as a pale-yellow solid. Mp 185-
N-(4-Chlorobenzyl)-7-(2-(diethylamino)ethyl)-2-(3-hy-
droxy-1-propynyl)-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-
5-carboxamide (6e). Potassium carbonate (2.22 g, 16.0 mmol,
15.0 equiv) and 2-bromo-N,N-diethylethylamine hydrobromide
(2.79 g, 10.7 mmol, 10.0 equiv) were added to a solution of 6a
(0.400 g, 1.07 mmol) in DMF (20 mL). The reaction mixture
was heated to 100 °C and stirred for 18 h. The mixture was
poured into water (40 mL). The resulting solid was filtered
and purified by column chromatography (CH2Cl2/methanol, 98/
2) followed by recrystallization from ethanol to yield 0.351 g
1
191 °C (dec); H NMR (400 MHz, DMSO-d6) δ 10.62 (t, J )
5.9 Hz, 1 H), 8.63 (s, 1 H), 7.41-7.33 (m, 4 H), 7.19 (s, 1 H),
5.15 (t, J ) 5.4 Hz, 1 H), 4.58 (t, J ) 5.1 Hz, 1 H), 4.55 (d, J
) 6.0 Hz, 2 H), 4.29 (t, J ) 4.8 Hz, 2 H), 3.81-3.77 (m, 2 H),
3.48-3.44 (m, 2 H), 2.90 (t, J ) 7.5 Hz, 2 H), 1.83-1.76 (m, 2
H); 13C NMR (100 MHz, DMSO-d6) δ 171.8, 164.3, 148.6, 144.9,
139.7, 138.5, 131.4, 131.3, 129.0, 128.2, 119.3, 113.8, 59.4, 58.8,
58.1, 41.3, 33.7, 25.8; MS (ESI+) m/z 421 ((M + H)+, 100). Anal.
(C20H21ClN2O4S) C, H, N, Cl, S.
1
(53%) of 6e as a yellow solid. Mp 139-141 °C; H NMR (400
MHz, DMSO-d6) δ 10.40 (t, J ) 5.9 Hz, 1 H), 8.71 (s, 1 H),
7.55 (s, 1 H), 7.41-7.34 (m, 4 H), 5.47 (t, J ) 6.0 Hz, 1 H),
4.54 (d, J ) 5.9 Hz, 2 H), 4.36 (d, J ) 6.0 Hz, 2 H), 4.29 (t, J
) 5.4 Hz, 2 H), 2.77 (t, J ) 5.5 Hz, 2 H), 2.46 (t, J ) 7.0 Hz,
4 H), 0.78 (t, J ) 7.0 Hz, 6 H); 13C NMR (100 MHz, DMSO-d6)
δ 171.7, 163.8, 149.7, 146.4, 138.3, 131.3, 130.7, 129.0, 128.3,
127.3, 116.0, 114.0, 96.2, 75.6, 55.2, 50.2, 49.4, 46.4, 41.4, 11.9;
MS (ESI+) m/z 472 ((M + H)+, 100). Anal. (C24H26ClN3O3S) C,
H, N, Cl, S.
N-(4-Chlorobenzyl)-7-(2-(diethylamino)ethyl)-2-(3-hy-
droxypropyl)-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-
carboxamide (7e). Compound 6e (0.200 g, 0.42 mmol) in THF
(80 mL) was hydrogenated (35 psi) in the presence of 10% Pd/C
(60 mg). The reaction mixture was filtered through a Celite
pad, and the filtrate was concentrated in vacuo. The resulting
oil was purified by column chromatography (CH2Cl2/methanol;
98/2, 96/4) to yield 0.123 g (62%) of 7e as a pale-yellow solid.
N-(4-Chlorobenzyl)-4-hydroxy-2-(3-hydroxypropyl)-
thieno[2,3-b]pyridine-5-carboxamide (7a). Compound 6a
(0.300 g, 0.805 mmol) in CH2Cl2/methanol (1/1, 70 mL) was
hydrogenated (35 psi) in the presence of 10% Pd/C (90 mg).
The reaction mixture was filtered through a Celite pad, and
the filtrate was concentrated in vacuo. The resulting solid was
purified by column chromatography (CH2Cl2/methanol; 98/2,
95/5) followed by recrystallization from ethanol to yield 0.083
g (27%) of 7a as a white, crystalline solid. Mp 185-186 °C; 1H
NMR (300 MHz, DMSO-d6) δ 13.29 (bs, 1 H), 10.61 (br, 1 H),
8.64 (s, 1 H), 7.42-7.33 (m, 4 H), 7.11 (s, 1 H), 4.54 (d, J ) 5.9
Hz, 2 H), 4.53 (br, 1 H), 3.46-3.43 (m, 2 H), 2.87 (t, J ) 7.4
Hz, 2 H), 1.83-1.74 (m, 2 H); 13C NMR (75 MHz, DMSO-d6) δ
173.1, 165.2, 147.3, 141.3, 140.5, 139.1, 131.9, 129.6, 128.8,
118.7, 113.8, 60.0, 41.9, 34.3, 26.5; MS (ESI-) m/z 376 ((M -
H)-, 100). Anal. (C18H17ClN2O3S) C, H, N, Cl, S.
1
Mp 78-82 °C; H NMR (400 MHz, DMSO-d6) δ 10.59 (t, J )
5.9 Hz, 1 H), 8.63 (s, 1 H), 7.41-7.33 (m, 4 H), 7.18 (s, 1 H),
4.57 (t, J ) 5.1 Hz, 1 H), 4.54 (d, J ) 5.9 Hz, 2 H), 4.27 (t, J
) 5.6 Hz, 2 H), 3.47-3.43 (m, 2 H), 2.90 (t, J ) 7.4 Hz, 2 H),
2.77 (t, J ) 5.7 Hz, 2 H), 2.46 (q, J ) 7.0 Hz, 4 H), 1.83-1.76
(m, 2 H), 0.78 (t, J ) 7.0 Hz, 6 H); 13C NMR (100 MHz, DMSO-
d6) δ 171.7, 164.3, 148.4, 145.2, 139.6, 138.5, 131.3, 129.0,
128.2, 119.4, 113.5, 59.3, 54.9, 50.3, 46.4, 43.2, 41.3, 33.7, 25.9,
11.9; MS (ESI+) m/z 476 ((M + H)+, 100). Anal. (C24H30-
ClN3O3S) C, H, N, Cl, S.
Methyl 5-(((4-Chlorobenzyl)amino)carbonyl)-4-hydroxy-
thieno[2,3-b]pyridine-2-carboxylate (8). Triethylamine (15.6
mL, 112 mmol, 2.0 equiv) and methanol (91 mL, 2.25 mol, 40

Thieno[2,3-b]pyridine Antivirals
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5801
equiv) were added to a solution of 4 (25.0 g, 56.2 mmol) in
DMF (400 mL). Nitrogen was bubbled through the solution
for 15 min to degas the reaction mixture. PdCl2(PPh3)2 (7.86
g, 11.2 mmol, 0.20 equiv) was added. Carbon monoxide was
bubbled through the solution. The reaction mixture was stirred
at 70 °C with continuous carbon monoxide addition for 18 h.
The mixture was allowed to cool to room temperature and was
poured into ice/water (2000 mL). The resulting mixture was
filtered through Celite. The filtrate was divided into two equal
portions, and each was extracted with CH2Cl2 (4 × 500 mL).
The combined organic layers were dried (MgSO4), filtered, and
concentrated in vacuo. The resulting solid was recrystallized
from methanol to yield 14.65 g (69%) of 8 as a white, crystalline
solid. Mp 238-240 °C; 1H NMR (300 MHz, DMSO-d6) δ 13.45
(br, 1 H), 10.30 (t, J ) 5.8 Hz, 1 H), 8.80 (s, 1 H), 7.96 (s, 1 H),
138.5, 136.6, 131.3, 131.1, 129.7, 129.0, 128.2, 121.8, 115.7,
114.6, 42.9, 41.3; MS (ESI+) m/z 359 (100, (M + H)+); HRMS
(FAB) calcd for C18H15ClN2O2S + H m/z 359.0621, found
359.0621. Anal. (C18H15ClN2O2S) C (calcd 60.25, found 59.28),
H, N, Cl, S.
N-(4-Chlorobenzyl)-2-(2-hydroxyethyl)-7-methyl-4-oxo-
4,7-dihydrothieno[2,3-b]pyridine-5-carboxamide (10b).
Carboxamide 11 (1.40 g, 3.90 mmol) was dissolved in THF (280
mL) with heating and then allowed to cool to room tempera-
ture. A solution of 9-BBN (23.4 mL, 0.5 M in THF, 11.7 mmol,
3.0 equiv) was added. The reaction mixture was stirred at room
temperature for 2 h, and an additional 15.6 mL of 9-BBN was
then added. The reaction mixture was stirred at room tem-
perature for 18 h, and 2 N NaOH (140 mL) followed by 30%
hydrogen peroxide (140 mL) were added. The mixture was
stirred at room temperature for 1 h and was then extracted
with CH2Cl2 (3 × 300 mL). The combined organic layers were
washed with brine (150 mL), dried (MgSO4), filtered, and
concentrated in vacuo. The resulting oil was purified by column
chromatography (CH2Cl2/methanol; 99/1, 98/2, 97/3) followed
by sequential recrystallization from acetonitrile and ethanol
to yield 0.155 g (11%) of 10b as a white solid. Mp 213-214 °C;
1H NMR (300 MHz, DMSO-d6) δ 10.63 (t, J ) 5.9 Hz, 1 H),
8.70 (s, 1 H), 7.42-7.33 (m, 4 H), 7.25 (s, 1 H), 4.99 (t, J ) 5.1
Hz, 1 H), 4.55 (d, J ) 5.9 Hz, 2 H), 3.95 (s, 3 H), 3.67 (q, J )
6.0 Hz, 2 H), 3.00 (t, J ) 6.0 Hz, 2 H); 13C NMR (DMSO-d6) δ
171.7, 164.4, 149.8, 144.9, 138.5, 137.4, 131.3, 130.6, 129.0,
128.2, 120.1, 114.0, 60.9, 42.7, 41.2, 33.1; MS (ESI+) m/z 377
(100, (M + H)+); HRMS (FAB) calcd for C18H17ClN2O3S +H
m/z 377.0726, found 377.0723. Anal. (C18H17ClN2O3S‚1.0H2O)
C, H, N, Cl, S.
N-(4-Chlorobenzyl)-2-(3-hydroxy-1-propynyl)-7-meth-
yl-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxam-
ide (12a). Copper(I) iodide (0.210 g, 1.07 mmol, 0.30 equiv), 5
(1.632 g, 3.56 mmol), and Pd(PPh3)2Cl2 (0.125 g, 0.178 mmol,
0.05 equiv) were suspended in diethylamine (40 mL). Propar-
gyl alcohol (0.29 mL, 4.98 mmol, 1.4 equiv) was added, and
the reaction mixture was stirred at room temperature for 18
h. Diethylamine was removed in vacuo and the resulting solid
was purified by column chromatography (CH2Cl2/methanol, 98/
2) followed by recrystallization from ethanol to yield 1.06 g
(77%) of 12a as a yellow solid. Mp 206-208 °C; 1H NMR (300
MHz, DMSO-d6) δ 10.42 (t, J ) 5.8 Hz, 1 H), 8.76 (s, 1 H),
7.57 (s, 1 H), 7.41-7.33 (m, 4 H), 5.47 (t, J ) 6.0 Hz, 1 H),
4.55 (d, J ) 5.9 Hz, 2 H), 4.37 (d, J ) 6.0 Hz, 2 H), 3.95 (s, 3
H); 13C NMR (75 MHz, DMSO-d6) δ 172.2, 164.4, 151.0, 146.8,
138.9, 131.9, 130.9, 129.6, 128.8, 128.0, 116.9, 115.2, 96.8, 76.2,
50.0, 43.6, 41.9; MS (FAB) m/z 387 (MH+, 99); HRMS (FAB)
calcd for C19H15ClN2O3S + H (M + H)+ m/z 387.0570, found
387.0558. Anal. (C19H15ClN2O3S‚0.3H2O) C, H, N, Cl, S.
N-(4-Chlorobenzyl)-2-(4-hydroxy-1-butynyl)-7-methyl-
4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamide
(12b). Copper(I) iodide (0.125 g, 0.654 mmol, 0.30 equiv), 5
(1.00 g, 2.18 mmol), and Pd(PPh3)2Cl2 (0.077 g, 0.109 mmol,
0.05 equiv) were suspended in diethylamine (25 mL). 3-Butyn-
1-ol (0.23 mL, 3.05 mmol, 1.4 equiv) was added, and the
reaction mixture was stirred at room temperature for 18 h.
Diethylamine was removed in vacuo and the resulting brown
solid was purified by column chromatography (CH2Cl2; CH2Cl2/
methanol, 99/1) to provide a yellow solid that was recrystal-
lized from methanol to yield 0.668 g (76%) of 12b as an off-
white solid. Mp 199-206 °C (dec); 1H NMR (400 MHz, DMSO-
d6) δ 10.44 (t, J ) 5.9 Hz, 1 H), 8.75 (s, 1 H), 7.49 (s, 1 H),
7.41-7.33 (m, 4 H), 4.98 (t, J ) 5.0 Hz, 1 H), 4.54 (d, J ) 5.9
Hz, 2 H), 3.94 (s, 3 H), 3.61-3.57 (m, 2 H), 2.63 (t, J ) 6.6 Hz,
2 H); 13C NMR (100 MHz, DMSO-d6) δ 171.6, 163.9, 149.9,
146.0, 138.4, 131.3, 130.3, 129.0, 128.2, 126.5, 117.4, 114.5,
95.7, 73.0, 59.2, 43.0, 41.3, 23.5; MS (ESI+) m/z 401 (100, (M
+ H)+). Anal. (C20H17ClN2O3S‚0.5H2O): C, H, N, Cl, S.
7.41-7.33 (m, 4 H), 4.55 (d, J ) 6.0 Hz, 2 H), 3.87 (s, 3 H); 13
C
NMR (75 MHz, DMSO-d6) δ 173.3, 164.1, 161.7, 151.4, 143.5,
138.5, 131.4, 130.6, 129.1, 128.3, 127.9, 126.4, 114.0, 52.7, 41.4;
MS (ESI-) m/z 375 (100, (M - H)-). Anal. (C17H13ClN2O4S‚
0.9H2O) C, H, N, Cl, S.
N-(4-Chlorobenzyl)-4-hydroxy-2-(hydroxymethyl)-
thieno[2,3-b]pyridine-5-carboxamide (9). Ester 8 (0.506 g,
1.34 mmol) was dissolved in THF (100 mL) with heating, and
then the reaction mixture was cooled in an ice bath. To this
solution was added a solution of lithium aluminum hydride
(1.0 M in THF, 2.4 mL, 2.4 mmol, 1.8 equiv). The reaction
mixture was allowed to warm to room temperature and was
stirred for 2.5 h. The reaction was quenched with water (1 mL),
10% NaOH (1 mL), and water (1 mL). The aluminum salts
were removed by filtration, and the filtrate was concentrated
in vacuo. The resulting oil was purified by column chroma-
tography (CH2Cl2/methanol; 98/2, 95/5) to yield 0.254 g (54%)
of 9 as a pale-yellow solid. Mp 205-210 °C; 1H NMR (300 MHz,
DMSO-d6) δ 13.34 (br, 1 H), 10.57 (br, 1 H), 8.66 (s, 1 H), 7.41-
7.33 (m, 4 H), 7.22 (s, 1 H), 5.69 (br, 1 H), 4.68 (br, 2 H), 4.54
(d, J ) 5.9 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6) δ 172.4,
164.9, 148.5, 141.8, 141.4, 138.6, 131.4, 130.5, 129.2, 128.3,
117.3, 113.2, 58.5, 41.4; MS (ESI-) for m/z 347 (100, (M - H)-).
Anal. (C16H13ClN2O3S) C, H, N, Cl, S.
N-(4-Chlorobenzyl)-2-(hydroxymethyl)-7-methyl-4-oxo-
4,7-dihydrothieno[2,3-b]pyridine-5-carboxamide (10a).
Potassium carbonate (0.567 g, 4.10 mmol, 1.5 equiv) and
iodomethane (0.20 mL, 3.28 mmol, 1.2 equiv) were added to a
solution of 9 (0.955 g, 2.73 mmol) in DMF (20 mL). The
reaction mixture was stirred at room temperature for 1 h. The
mixture was partitioned between water (50 mL) and CH2Cl2
(100 mL). The organic layer was removed in vacuo, and the
resulting precipitate was filtered and recrystallized from
ethanol to yield 0.825 g (83%) of 10a as a white, crystalline
solid. Mp 222-225 °C; 1H NMR (300 MHz, DMSO-d6) δ 10.59
(t, J ) 5.9 Hz, 1 H), 8.70 (s, 1 H), 7.41-7.33 (m, 4 H), 7.30 (s,
1 H), 5.79 (t, J ) 5.7 Hz, 1 H), 4.72 (d, J ) 5.5 Hz, 2 H), 4.55
(d, J ) 5.9 Hz, 2 H), 3.96 (s, 3 H); 13C NMR (75 MHz, DMSO-
d6) δ 172.0, 164.4, 150.1, 145.2, 141.9, 138.6, 131.4, 130.7,
129.1, 128.3, 118.4, 114.3, 58.4, 42.9, 41.4; MS (ESI+) m/z 363
(100, (M + H)+). Anal. (C17H15ClN2O3S) C, H, N, Cl, S.
N-(4-Chlorobenzyl)-7-methyl-4-oxo-2-vinyl-4,7-dihy-
drothieno[2,3-b]pyridine-5-carboxamide (11). PdCl2(PPh3)2
(0.107 g, 0.153 mmol, 0.02 equiv) and tributylvinylstannane
(2.34 mL, 8.01 mmol, 1.05 equiv) were added to a suspension
of 5 (3.50 g, 7.63 mmol) in DMF (110 mL). The reaction
mixture was stirred at 45 °C for 18 h. The mixture was allowed
to cool to room temperature, was poured into water (200 mL),
and was extracted with CH2Cl2 (3 × 200 mL). The combined
organic layers were washed with water (50 mL) and brine (2
× 50 mL), dried (MgSO4), filtered, and concentrated in vacuo.
The resulting solid was purified by column chromatography
(CH2Cl2/methanol, 99/1) followed by recrystallization from
acetonitrile to yield 2.372 g (87%) of 11 as a pale-yellow,
crystalline solid. Mp 205-210 °C (dec); 1H NMR (300 MHz,
DMSO-d6) δ 10.52 (t, J ) 5.9 Hz, 1 H), 8.71 (s, 1 H), 7.46 (s, 1
H), 7.41-7.33 (m, 4 H), 7.04-6.95 (m, 1 H), 5.65 (d, J ) 17.3
Hz, 1 H), 5.35 (d, J ) 10.9 Hz, 1 H), 4.56 (d, J ) 5.9 Hz, 2 H),
3.96 (s, 3 H); 13C NMR (DMSO-d6) δ 171.8, 164.1, 149.6, 145.6,
N-(4-Chlorobenzyl)-2-(5-hydroxy-1-pentynyl)-7-meth-
yl-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxam-
ide (12c). Copper(I) iodide (0.114 g, 0.600 mmol, 0.30 equiv),
5 (0.917 g, 2.00 mmol), and Pd(PPh3)2Cl2 (0.070 g, 0.100 mmol,
0.05 equiv) were suspended in diethylamine (33 mL). 4-Pentyn-

5802 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Schnute et al.
1-ol (0.26 mL, 2.80 mmol, 1.4 equiv) was added, and the
reaction mixture was stirred at room temperature for 18 h.
Diethylamine was removed in vacuo, and the resulting solid
was purified by column chromatography (CH2Cl2/methanol, 99/
1) followed by recrystallization from acetonitrile to yield 0.693
g (84%) of 12c as a pale-yellow solid. Mp 213-217 °C; 1H NMR
(400 MHz, DMSO-d6) δ 10.45 (t, J ) 5.9 Hz, 1 H), 8.75 (s, 1
H), 7.48 (s, 1 H), 7.41-7.33 (m, 4 H), 4.58 (t, J ) 5.1 Hz, 1 H),
4.54 (d, J ) 6.0 Hz, 2 H), 3.94 (s, 3 H), 3.52-3.48 (m, 2 H),
2.56-2.51 (m, 2 H), 1.73-1.66 (m, 2 H); 13C NMR (100 MHz,
DMSO-d6) δ 171.6, 163.9, 149.8, 146.0, 138.4, 131.3, 130.4,
129.0, 128.2, 126.4, 117.4, 114.5, 97.5, 72.4, 59.2, 43.0, 41.3,
31.0, 15.6; MS (ESI+) m/z 415 (100, (M + H)+). Anal. (C21H19-
ClN2O3S) C, H, N, Cl, S.
N-(4-Chlorobenzyl)-2-(3-hydroxypropyl)-7-methyl-4-
oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamide (10c).
Compound 12a (0.520 g, 1.34 mmol) in CH2Cl2/ethanol (1/1,
160 mL) was hydrogenated (35 psi) in the presence of 10% Pd/C
(0.156 g). The mixture was filtered through a Celite pad, and
the filtrate was concentrated in vacuo. The resulting solid was
purified by column chromatography (CH2Cl2/methanol, 98/2)
followed by recrystallization from ethanol to yield 0.211 g (40%)
of 10c as an off-white solid. Mp 197-198 °C; 1H NMR (300
MHz, DMSO-d6) δ 10.61 (t, J ) 5.9 Hz, 1 H), 8.69 (s, 1 H),
7.41-7.33 (m, 4 H), 7.21 (s, 1 H), 4.58-4.53 (m, 3 H), 3.95 (s,
3 H), 3.47 (q, J ) 6.1 Hz, 2 H), 2.91 (t, J ) 7.5 Hz, 2 H), 1.80
(qt, J ) 7.4 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6) δ 172.2,
164.9, 149.8, 145.5, 140.6, 139.1, 131.8, 131.5, 129.6, 128.8,
120.0, 114.7, 60.0, 43.4, 41.9, 34.4, 26.5; MS (ESI+) m/z 391
(100, (M + H)+); HRMS (FAB) calcd for C19H19ClN2O3S + H
(M + H)+ m/z 391.0883, found 391.0904. Anal. (C19H19ClN2O3S)
C (calcd 58.38, found 57.42), H, N, Cl, S.
N-(4-Chlorobenzyl)-2-(4-hydroxybutyl)-7-methyl-4-oxo-
4,7-dihydrothieno[2,3-b]pyridine-5-carboxamide (10d).
Compound 12b (0.400 g, 1.00 mmol) in CH2Cl2/ethanol (1/1,
120 mL) was hydrogenated (35 psi) in the presence of 10% Pd/C
(0.120 g). The reaction mixture was filtered through a Celite
pad, and the filtrate was concentrated in vacuo. The resulting
solid was purified by column chromatography (CH2Cl2/
methanol; 99/1, 98/2) followed by recrystallization from metha-
nol to yield 0.279 g (69%) of 10d as a white solid. Mp 186-
188 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.62 (t, J ) 6.0 Hz,
1 H), 8.70 (s, 1 H), 7.41-7.33 (m, 4 H), 7.21 (s, 1 H), 4.55 (d,
J ) 5.9 Hz, 2 H), 4.39 (br, 1 H), 3.95 (s, 3 H), 3.42 (t, J ) 5.8
Hz, 2 H), 2.89 (t, J ) 7.3 Hz, 2 H), 1.72-1.64 (m, 2 H), 1.51-
1.44 (m, 2 H); 13C NMR (100 MHz, DMSO-d6) δ 171.7, 164.3,
149.2, 144.9, 140.3, 138.5, 131.3, 130.9, 129.0, 128.2, 119.4,
114.1, 60.1, 42.8, 41.3, 31.5, 29.0, 27.2; MS (ESI+) m/z 405 (100,
(M + H)+). Anal. (C20H21ClN2O3S‚1.0 H2O) C, H, N, Cl, S.
N-(4-Chlorobenzyl)-2-(5-hydroxypentyl)-7-methyl-4-
oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamide (10e).
Compound 12c (0.400 g, 0.964 mmol) in CH2Cl2/ethanol (1/1,
140 mL) was hydrogenated (35 psi) in the presence of 10% Pd/C
(0.120 g). The reaction mixture was filtered through a Celite
pad, and the filtrate was concentrated in vacuo. The resulting
solid was purified by column chromatography (CH2Cl2/
methanol; 99/1, 98/2) followed by recrystallization from ethanol
to yield 0.251 g (62%) of 10e as a white solid. Mp 160-164 °C;
1H NMR (400 MHz, DMSO-d6) δ 10.62 (t, J ) 5.9 Hz, 1 H),
8.70 (s, 1 H), 7.41-7.31 (m, 4 H), 7.21 (s, 1 H), 4.55 (d, J ) 5.9
Hz, 2 H), 4.37 (t, J ) 5.1 Hz, 1 H), 3.40-3.36 (m, 2 H), 2.87 (t,
J ) 7.3 Hz, 2 H), 1.69-1.62 (m, 2 H), 1.48-1.42 (m, 2 H), 1.39-
1.33 (m, 2 H); 13C NMR (100 MHz, DMSO-d6) δ 171.7, 164.3,
149.2, 144.9, 140.2, 138.5, 131.3, 130.9, 129.0, 128.2, 119.4,
114.1, 60.4, 42.8, 41.3, 32.0, 30.5, 29.3, 24.8; MS (ESI+)
m/z 419 (100, (M + H)+). Anal. (C21H23ClN2O3S) C, H, N,
Cl, S.
was refluxed for 18 h. Additional morpholine (2.7 mL, 31.4
mmol, 10 equiv) and formaldehyde (2.6 mL, 34.5 mmol, 11
equiv) were added, and the reaction mixture was refluxed for
an additional 24 h. The reaction mixture was allowed to cool
to room temperature and was then concentrated in vacuo. The
residue was treated with 25% NaOH (20 mL). The aqueous
layer was extracted with ethyl acetate (50 mL) and then with
CHCl3 (60 mL). Methanol (30 mL) was added to the aqueous
layer, and it was extracted with CHCl3 (2 × 60 mL). This
procedure was repeated three times. The combined organic
layers were dried (MgSO4), filtered, and concentrated in vacuo.
The resulting solid was purified by column chromatography
(CH2Cl2/methanol; 98/2, 96/4) followed by recrystallization
from ethyl acetate/Et2O to yield 0.718 g (55%) of 13 as an off-
white solid. Mp 193-195 °C; 1H NMR (300 MHz, DMSO-d6) δ
13.30 (br, 1 H), 10.59 (t, J ) 5.3 Hz, 1 H), 8.66 (s, 1 H), 7.41-
7.33 (m, 4 H), 7.27 (s, 1 H), 4.54 (d, J ) 5.9 Hz, 2 H), 3.72 (s,
2 H), 3.58 (t, J ) 4.4 Hz, 4 H), 2.44 (t, J ) 3.6 Hz, 4 H); 13C
NMR (75 MHz, DMSO-d6) δ 172.2, 164.8, 148.9, 141.4, 138.5,
137.5, 131.3, 130.3, 129.0, 128.2, 119.5, 113.1, 66.1, 56.7, 52.8,
41.2; MS (FAB) m/z 418 (MH+, 99); HRMS (FAB) calcd for
C20H20ClN3O3S + H m/z 418.0992, found 418.0996. Anal.
(C20H20ClN3O3S) C, H, N, Cl, S.
N-(4-Chlorobenzyl)-7-methyl-2-(4-morpholinylmethyl)-
4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamide
(14). Potassium carbonate (6.50 g, 47.0 mmol, 2.0 equiv) and
iodomethane (1.76 mL, 28.2 mmol, 1.2 equiv) were added to a
solution of 13 (9.80 g, 23.5 mmol) in DMF (150 mL). The
reaction mixture was stirred at room temperature for 18 h.
The mixture was poured into water (300 mL). The resulting
precipitate was filtered and recrystallized from acetonitrile to
yield 7.90 g (78%) of 14 as a pale-yellow, crystalline solid. Mp
231-234 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.59 (t, J )
5.9 Hz, 1 H), 8.70 (s, 1 H), 7.41-7.33 (m, 5 H), 4.55 (d, J ) 5.9
Hz, 2 H), 3.96 (s, 3 H), 3.75 (s, 2 H), 3.59 (t, J ) 4.4 Hz, 4 H),
2.45 (br, 4 H); 13C NMR (100 MHz, CDCl3) δ 172.4, 164.4,
150.0, 143.9, 137.5, 136.8, 132.1, 131.0, 128.3, 128.0, 120.7,
115.1, 66.3, 57.1, 52.9, 42.5, 41.9; MS (ESI+) m/z 432 ((M +
H)+, 100). Anal. (C21H22ClN3O3S) C, H, N, Cl, S.
DNA Polymerase Assay. HSV-1, HCMV, VZV, human R,
and human δ DNA polymerases were expressed as C-terminal
histidine-tagged proteins using a baculovirus expression sys-
tem and purified by standard Ni affinity chromatography16 (see
Supporting Information). Plasmid constructs for HCMV
(pwB747), HSV-1 (pDP4), and VZV (puC18) were obtained
from D. Coen (Harvard University). Human R and δ DNA
polymerases were cloned using standard techniques.17 Purified
human γ DNA polymerase was supplied by William Copeland
(National Institutes of Health). Compounds were evaluated
against the purified polymerases using a scintillation proxim-
ity assay (SPA). All compounds were solubilized in dimethyl
sulfoxide (DMSO). Assay conditions for HSV-1, HCMV, VZV,
human R, and human γ DNA polymerases were as follows: 5
µL of compound solution was added to 95 µL of polymerase in
a solution containing 6.4 mM HEPES (pH 7.5), 12 mM KCl,
25 mM NaCl, 5 mM MgCl2, 46 µg/mL of bovine serum albumin
(BSA), 2 mM CHAPS, 5 mM dithiothreitol, 5% glycerol, 1.2
µCi of [methyl,1′,2′-3H]TTP (Amersham Pharmacia Biotech),
and 6 nM biotinylated oligo(dT)16-poly(dA) (Amersham Phar-
macia Biotech). The polymerase reaction was incubated in a
96-well Dynatech Microlite I plate for 12 min at 26 °C and
stopped by the addition of 50 µL of a 0.5 M EDTA (pH 8.0)
solution containing 4 mg/mL SPA beads. The plate was sealed
and counted on a Topcount microplate scintillation counter
(Packard Instruments, Meriden, CT). Percent inhibition was
determined for each drug concentration compared to the value
of the uninhibited control. IC50 values were calculated using
EXCEL software for linear regression. For human δ poly-
merase, 5 µL of compound solution was added to 95 µL of
polymerase in a solution containing 20 mM HEPES (pH 7.5),
40 µg/mL of BSA, 2 mM MnCl2, 1 mM dithiothreitol, 5%
glycerol, 5% DMSO, 0.2 µM [methyl,1′,2′-3H]TTP (Amersham
Pharmacia Biotech), 0.4 µM dATP (Sigma, St. Louis, Mo.), and
10 µg/mL of poly(dA-dT) primer template. Reactions were
N-(4-Chlorobenzyl)-4-hydroxy-2-(4-morpholinylmeth-
yl)thieno[2,3-b]pyridine-5-carboxamide (13). An aqueous
formaldehyde solution (37%, 2.6 mL, 34.5 mmol, 11 equiv) was
added to morpholine (2.7 mL, 31.4 mmol, 10 equiv) at 0 °C.
Ethanol (10 mL) was then added followed by addition of 3 (1.00
g, 3.14 mmol). Acetic acid (2 mL) was added, and the reaction
mixture was allowed to warm to room temperature and then

Thieno[2,3-b]pyridine Antivirals
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5803
performed at 27 °C for 10 min. Incorporated TMP was
precipitated with an equal volume of 10% trichloroacetic acid
and collected on a GF/B Millipore multiscreen filtration plate.
Wells were washed three times with 5% trichloroacetic acid
and dried prior to the addition of 100 µL of Microscint 40
(Packard Instruments) per well. Zidovudine triphosphate
(AZT-TP) (Moravek Biochemicals), phosphonoformic acid (fos-
carnet) (Sigma), and aphidicolin (Biomol) were used as stand-
ards in the DNA polymerase assay.
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ther. 2002, 46, 724-730. (b) Brideau, R. J.; Knechtel, M. L.;
Huang, A.; Vaillancourt, V. A.; Vera, E. E.; Oien, N. L.; Hopkins,
T. A.; Wieber, J. L.; Wilkinson, K. F.; Rush, B. D.; Schwende, F.
J.; Wathen, M. W. Broad-spectrum antiviral activity of PNU-
183792, a 4-oxo-dihydroquinoline, against human and animal
herpesviruses. Antiviral Res. 2002, 54, 19-28. (c) Knechtel, M.
L.; Huang, A.; Vaillancourt, V. A.; Roger, J.; Brideau, R. J.
Inhibition of clinical isolates of human cytomegalovirus and
varicella zoster virus by PNU-183792, a 4-oxo-dihydroquinoline.
J. Med. Virol. 2002, 68, 234-236.
Plaque Reduction Assays. Antiviral activities of com-
pounds against herpesviruses were determined using plaque
reduction assays. HCMV (Davis strain) and VZV (Webster
strain) were grown on human foreskin fibroblast (HFF) cells.
HSV-1 (KOS strain) was grown on African green monkey
kidney cells (Vero). HCMV Ad169 (V823A), HSV-1 KOS
(V823A), and HSV-2 (V826A) were isolated as previously
described.14 Acyclovir-resistant HSV-1 and HSV-2 strains are
denoted as follows with the coresponding location in poly-
merase gene in parentheses: AraAr13 (V813M), PAAr5 (R842S),
PFAr2 (R605V), and BWr (N815S). They were obtained from
D. Coen (Harvard University). The specific point mutations
in the DNA polymerase gene resulting in resistance for these
isolates have been described previously.6,15 Approximately
50 PFU of virus was added to each well of a 24-well culture
dish containing the appropriate cells. After a 1 h incubation
at 37 °C, the virus inoculum was removed and drug-containing
media was added. All compounds were solubilized in dimethyl
sulfoxide (DMSO) as 200-fold stock solutions. Dilutions of
compounds were then added to DMEM containing 10%
FBS and 0.8% carboxymethyl cellulose. Acyclovir (Sigma) or
ganciclovir (obtained from
a wholesale pharmacy) was
used as positive controls. The plates were incubated at 37 °C
until plaques formed. Plaques were counted microscopically
or by staining cells with crystal violet (0.1% in 20% ethanol).
Percent inhibition compared to the value for the uninhibited
control was determined for each drug concentration, and IC50
values were calculated using EXCEL software for linear
regression.
Cell Viability Determinations. The toxicity of com-
pounds on noninfected mammalian cells was determined for
HFF and Vero cells seeded as subconfluent monolayers and
treated with compound for 3 days. Cell viability determinations
were performed using both microscopic evaluation and a
quantitative neutral red dye uptake assay as previously
described.18
Enzyme Kinetics Analysis for Compound 14. The Ki
value of compound 14 for inhibition of [3H]TTP incorporation
into primer template with HCMV polymerase was determined
by assessing various substrate and inhibitor concentrations
in the standard in vitro polymerase assay. Substrate (TTP)
concentrations spanned the calculated Km for HCMV poly-
merase (1.2 µM), ranging from 0.25 to 8.0 µM. Data for dTTP
incorporation was globally analyzed by the competitive inhibi-
tor equation,
(10) Vaillancourt, V. A.; Cudahy, M. M.; Staley, S. A.; Brideau, R.
J.; Conrad, S. J.; Knechtel, M. L.; Oien, N. L.; Wieber, J. L.; Yagi,
Y.; Wathen, M. W. Naphthalene carboxamides as inhibitors of
human cytomegalovirus DNA polymerase. Bioorg. Med. Chem.
Lett. 2000, 10, 2079-2081.
(11) Khan, M. A.; Guarc¸oni, A. E. Thieno[2,3-b]pyridines and thieno-
[3,2-b]pyridines by the method of Gould-Jacobs. J. Heterocycl.
Chem. 1977, 14, 807-812.
(12) Steinkopf, W. 2-Thiophenes. Liebigs Ann. Chem. 1914, 403, 17-
33.
V
maxS
I
Ki
v )
(1)
Km 1 +
+ S
(
)
using nonlinear regression analysis to yield a competitive Ki
of 1.0 ( 0.1 µM.
(13) Khan and Guarc¸oni (ref 11) have suggested that for thieno[2,3-
b]pyridine 5-carboxylate esters the enol tautomer dominates in
solution on the basis of infrared absorption bands; however, the
C5 substituent may play a significant role in determining the
tautomer population. For a discussion of the tautomer equilib-
rium in the related and more studied 4-hydroxyquinoline
heterocycle, see the following. De la Cruz, A.; Elguero, J.; Goya,
P.; Mart´ınez, A. Tautomerism and acidity in 4-quinolone-3-
carboxylic acid derivatives. Tetrahedron 1992, 48, 6135-
6150.
Acknowledgment. We thank the Structural,
Analytical, and Medicinal Chemistry Departments
for analytical data and Craig Barsuhn for pKa
measurements.
Supporting Information Available: Elemental analyses
for compounds 1 and 3-14 and procedures for the expression
and purification of HCMV polymerase. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
(14) Thomsen, D. R.; Oien, N. L.; Hopkins, T. A.; Knechtel, M. L.;
Brideau, R. J.; Wathen, M. W.; Homa, F. L. Amino acid changes
within conserved region III of the herpes simplex virus and

5804 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
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4-oxo-dihydroquinolines, a novel class of herpesvirus antiviral
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(17) (a) Wang, T. S.; Copeland, W. C.; Rogge, L.; Dong, Q. Purification
of mammalian DNA polymerases: DNA polymerase R. Methods
Enzymol. 1995, 262, 77-84. (b) Downey, K. M.; So, A. G.
Purification of mammalian DNA polymerases: DNA polymerase
δ. Methods Enzymol. 1995, 262, 84-92.
(18) Lowik, C. W. G. M.; Alblas, M. J.; van de Ruit, M.; Papapoulos,
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adherent cells cultured in 96 well plates using the supravital
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vitro mutagenesis of the herpes simplex virus type 1 DNA
polymerase gene results in altered drug sensitivity of the
enzyme. J. Virol. 1989, 63, 4913-4918.
(16) Hochuli, E.; Bannwarth, W.; Dobeli, H.; Gentz, R.; Stuber, D.
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