Naphthalene carboxamides as inhibitors of human cytomegalovirus DNA polymerase

Article abstract of DOI:10.1016/S0960-894X(00)00402-9

ortho-Hydroxynaphthalene carboxamides have been identified as inhibitors of HCMV DNA polymerase. SAR investigations have demonstrated that both the amide and hydroxy functionalities are required for activity. Substitution on the naphthalene ring has led to inhibitors with submicromolar IC₅₀s against HCMV polymerase. These compounds have been found to be >100-fold selective for inhibition of HCMV polymerase versus human alpha polymerase and display antiviral activity in a cell-based plaque reduction assay. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00402-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2079±2081
Naphthalene Carboxamides as Inhibitors of Human
Cytomegalovirus DNA Polymerase
Valerie A. Vaillancourt,* Michele M. Cudahy, Sandra A. Staley, Roger J. Brideau,
Steven J. Conrad, Mary L. Knechtel, Nancee L. Oien, Janet L. Wieber,
Yoshihiko Yagi and Michael W. Wathen
Pharmacia, 301 Henrietta Street, Kalamazoo, MI 49007, USA
Received 18 April 2000; accepted 7 July 2000
AbstractÐortho-Hydroxynaphthalene carboxamides have been identi®ed as inhibitors of HCMV DNA polymerase. SAR investi-
gations have demonstrated that both the amide and hydroxy functionalities are required for activity. Substitution on the naphtha-
lene ring has led to inhibitors with submicromolar IC50s against HCMV polymerase. These compounds have been found to be
>100-fold selective for inhibition of HCMV polymerase versus human alpha polymerase and display antiviral activity in a cell-
based plaque reduction assay. # 2000 Elsevier Science Ltd. All rights reserved.
Human cytomegalovirus (HCMV) is a ubiquitous
member of the herpes virus family which infects over
forty percent of the population. Like other herpes viru-
ses, HCMV persists in the host in a latent state after
primary infection. Although generally benign in the
immunocompetent host, reactivation of the latent
infection is associated with signi®cant morbidity and
mortality in immunocompromised individuals. Active
HCMV infection is associated with clinical syndromes
such as pneumonia, retinitis, hepatitis, gastrointestinal
disease and congenital birth defects. Ganciclovir, foscar-
net, cidofovir and formivirsen, the only drugs approved
for treatment of HCMV infections, are less than ideal
agents due to their signi®cant toxicity, modest ecacy
and required intravenous or intraocular routes of
administration.1,2 Clearly, less toxic, orally available
alternatives are needed.
HCMV pol. Structure±activity relationship (SAR) stu-
dies were initiated around this template.
SAR investigations of this template were focused on
three major structural features of the molecule, the
amide functionality, the hydroxyl group and substitu-
tion on the naphthalene ring. The reported naphthalene
carboxamides were prepared by condensation of the
corresponding naphthalene carboxylic acid with the
appropriate amine, utilizing reaction conditions of
either PCl3 in re¯uxing xylenes7 or carbonyldiimidazole
in THF.8 Compounds were tested for their ability to
inhibit HCMV pol using a scintillation proximity
assay.9 Initial SAR investigations of the naphthanilides
showed that the presence of an electron withdrawing
substituent on the aniline ring was essential for activity
(Table 1). Unsubstituted or electron rich anilides were
found to be less active, as were pyridyl amides.
In order to address this unmet medical need, the herpes
virus program team at Pharmacia initiated a program to
develop better anti-HCMV agents. The program team
has chosen HCMV DNA polymerase (HCMV pol) as a
molecular target.3 Currently marketed therapies includ-
ing ganciclovir, foscarnet and cidofovir all inhibit
HCMV via inhibition of this viral enzyme.4 6 Through
broad screening of the compound collection, the naph-
thalene carboxamide 1 was identi®ed as an inhibitor of
To further examine the SAR around this template,
optimal placement of the electron-de®cient aromatic ring
was explored. It was found that a one carbon linkage
between the amide nitrogen and the electron-de®cient
aromatic group gave similar HCMV pol inhibitory
activity (Table 2). Longer linkages resulted in diminished
activity.
Continuing investigations focused on the hydroxyl sub-
stituent, evaluating the necessity and the placement of
this functionality (Table 3). In the aniline series, deriva-
tization of the hydroxyl group as either its methyl ether
*Corresponding author. Tel.: +1-616-833-9552; fax: +1-616-833-2232;
e-mail: valerie.a.vaillancourt@am.pnu.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00402-9

2080
V. A. Vaillancourt et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2079±2081
Table 1. Aryl amides
Table 4. Naphthalene substitution e€ects
Compound
Aryl group
HCMV pol IC50 (mM)
Compound
R
R0
HCMV pol IC50 (mM)
1
2
3
4
5
6
7
8
9
10
3-NO2-Ph
4-NO2-Ph
4-Cl-Ph
4-Br-Ph
4-CN-Ph
Ph
4-OCH3-Ph
3-NH2-Ph
2-pyridyl
3-pyridyl
12
22
69
22
23
24
25
26
27
28
29
NO2
NO2
Cl
NO2
Cl
4,7-di-Br
5-OH
5-OH
7-OH
7-OH
2.9
3.8
3.3
10.6
2.4
0.82
0.76
19
>100
56
>100
>100
>100
NO2
Cl
7-OCH3
7-OCH3
ring system. Toward this end, commercially available
naphthalene carboxylic acids were condensed with 4-
chloro- or 4-nitrobenzylamine to form the desired car-
boxamides. The results of this study are shown in Table
4. In general, substitution at the 5- and 7-positions of
the naphthalene ring had bene®cial e€ects on HCMV
pol inhibitory activity, with the 7-methoxy substituted
analogues being the ®rst compounds in this series to
display submicromolar activity.
Table 2. Linker region
Compound
n
R
HCMV pol IC50 (mM)
11
12
13
14
1
1
2
2
NO2
Cl
NO2
Cl
12
52
>100
>100
Having identi®ed methoxy as a favored substituent at
the 7-position, the identity and necessity of the amide
group was revisited within this series. Heterocyclic
amides were investigated. Again, the desired analogues
could be prepared by PCl3 or carbonyldiimidazole pro-
moted condensations of the naphthalene carboxylic acid
with the appropriate aminoheterocycle. The compounds
prepared and their HCMV pol inhibitory activities are
shown in Table 5. As can be seen in this table, although
electron de®cient heterocyclic amides displayed activity
in the polymerase assay, they appeared to be less potent
than the benzyl amides. To further evaluate the neces-
sity of the amide functionality, the amide of 29 was
reduced with LiAlH4 to provide 35. This led to a
marked decrease in activity.
Table 3. Signi®cance of hydroxy substituent
Compound
R
R0
n
HCMV pol IC50 (mM)
15
16
17
18
19
20
21
22
NO2
NO2
Cl
NO2
Cl
Cl
Cl
NO2
3-OCH3
3-OMOM
3-OCH3
H
0
0
1
0
1
0
1
1
>100
>100
8.0
>100
>100
37
The next step in the biological investigations of these
compounds was to determine the selectivity of the poly-
merase activity. The initial lead 1 was found to only be 2-
to 3-fold selective for inhibition of HCMV pol versus
human alpha polymerase. In contrast, many of the new
analogues showed remarkable selectivity (10- to 100-
fold) for inhibition of HCMV pol versus human alpha
polymerase. Additionally, antiviral activity in a plaque
reduction assay was observed for many of the more
potent analogues, while less potent HCMV pol inhibi-
tors (e.g. 14 and 21) did not exhibit measurable antiviral
activity.10 Data for select analogues is shown in Table 6.
H
1-OH
1-OH
1-OH
13
14.5
(15) or its methoxymethyl ether (16) resulted in reduc-
tion of activity. This was not true for the benzylamide
series, in which the corresponding methyl ether (17) was
actually more active than the free hydroxyl group.
Removal of the hydroxyl functionality resulted in
reduction of activity in both series (18 and 19). Of note
in this study was that the hydroxy group could be
placed at either the 1- or 3-position of the naphthalene
ring ortho to the carboxamide with little di€erence in
activity. Again, both anilides and benzylamides were
active within this new series (20, 21, and 22).
In summary, ortho-hydroxynaphthalenecarboxamides
have been identi®ed as HCMV DNA polymerase
inhibitors. SAR investigations of these compounds
show that both the amide and hydroxy functionalities
are required for activity. Electron-de®cient phenyl, and
benzylic amides provide the highest levels of inhibition.
Substitution at the 5- or 7-postion of the naphthalene
ring with either hydroxy or methoxy groups enhances
The next phase of SAR investigations involved the
study of the e€ects of substitution on the naphthalene

V. A. Vaillancourt et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2079±2081
2081
Table 5. Heterocyclic amides
pol versus human alpha polymerase and shows antiviral
activity in a cell-based antiviral assay.
References and Notes
1. deJong, M. D.; Galasso, G. J.; Gazzard, B.; Griths, P. D.;
Jabs, D. A.; Kern, E. R.; Spector, S. A. Antiviral Res. 1998,
39, 141.
Compound
Het
HCMV pol IC50 (mM)
2. Ho€man, V. F.; Skiest, D. J. Exp. Opin. Invest. Drugs 2000,
9, 207.
30
4
3. Yukihiro, N.; Koichiro, M.; Shonen, Y. Virology 1983, 124,
221.
4. Biron, K. K.; Stenbuck, P. J.; Sorrell, J. B. UCLA Syp.
Mol. Cell. Biol., New Ser. 1984, 21, 677.
31
6.4
5. Eriksson, B.; Tao, P. Z.; Wahren, B.; Oeberg, B. In Proc.
13th Int. Congr. Chemother.; Spitzy, K. H.; Karrer, K., Eds.;
Verlag H. Egermann: Vienna, 1983; p 6.
6. Xiong, X.; Smith, J. L.; Chen, M. S. Antimicrob. Agents
Chemother. 1997, 41, 594.
32
33
34
6
59
2.4
7. Singh, H.; Singh, A. K.; Sharma, S.; Iyer, R. N. J. Med.
Chem. 1977, 20, 826 and references cited therein. A typical
experimental procedure for a PCl3 promoted coupling is as
follows: A solution of 3-hydroxy-2-naphthoic acid (2.82 g,
15 mmol) and 4-nitroaniline (2.07 g, 15 mmol) in 80 mL
xylenes was heated to re¯ux. To this was added dropwise PCl3
(0.52 mL, 6 mmol). Re¯uxing was continued until HCl evolu-
tion had ceased (ca. 1 h). The reaction was then cooled and
water was added to destroy excess PCl3. The solvents were
evaporated and the residual solid was crystallized from 3:1
THF:water by the addition of MeOH. The solid was collected
and dried to yield 1.83 g (40%) of 2 as an olive-green solid.
8. Staab, H. A. Angew. Chem., Int. Ed. Engl. 1962, 1, 351. A
typical carbonyldiimidazole promoted coupling is as follows:
To a solution of 1,10-carbonyldiimidazole (1.70 g, 10.5 mmol)
in THF (20 mL) was added 3-hydroxy-2-naphthoic acid
(0.94 g, 5.00 mmol). The resulting solution was stirred at room
temperature for 1 h. The reaction was cooled to 0 ꢀC and a
solution of 4-chlorobenzylamine (1.2 mL, 10.0 mmol) in THF
(10 mL) was added dropwise. The reaction was allowed to
warm slowly to room temperature and was stirred for 18 h.
The reaction mixture was concentrated in vacuo. The resulting
yellow oil was chromatographed eluting with 5% CH3OH/
CH2Cl2. Fractions homogeneous by TLC were combined and
concentrated in vacuo. The resulting yellow solid was recrys-
tallized twice from EtOAc to yield 0.557 g (36%) of 12 as a
yellow crystalline solid.
Table 6. Polymerase selectivity and antiviral activity for select com-
pounds
Compound
HCMV pol
IC50 (mM)
Human a-pol
IC50 (mM)
Plaque reduction
IC50 (mM)
1
14
21
24
25
27
28
31
12.3
>100
13
32
nda
nd
>100
63
18
nd
>2 0
>20
1.5
3.8
3.3
2.4
0.82
6.4
0.4
0.6
1.0
3.7
>100
93
9. The assay was run as previously described: Tucker, J. A.;
Clayton, T. L.; Chidester, C. G.; Schulz, M. W.; Harrington,
L. E.; Conrad, S. J.; Yagi, Y.; Oien, N. L.; Yurek, D.; Kuo,
M.-S. Bioorg. Med. Chem. 2000, 8, 601. Foscarnet was used as
a positive control in this assay. A typical HCMV polymerase
IC50 value for foscarnet is 3.5 mM.
>5
Ð
Aphidicolin
Ganciclovir
3.0
Ð
Ð
0.6
and not determined.
10. The antiviral assay was run in a standard manner (see
Bedard, J.; May, S.; L'Heureux, L.; Stamminger, T.; Copsey,
A.; Drach, J.; Hu€man, J.; Chan, L.; Jin, H.; Rando, R. F.
Antimicrob. Agents Chemother. 2000, 44, 929) using an HFF
cell line and the Davis strain of HCMV.
polymerase inhibition activity, resulting in compounds
(e.g. 28) which display submicromolar IC50s against
HCMV pol. This compound was also found to be
greater than 100-fold selective for inhibition of HCMV