2-Aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridines as broad-spectrum inhibitors of human herpesvirus polymerases

Article abstract of DOI:10.1016/j.bmcl.2007.03.102

A novel series of 2-aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamides have been identified as potent antivirals against human herpesviruses. These compounds demonstrate broad-spectrum inhibition of the herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases.

Full text of DOI:10.1016/j.bmcl.2007.03.102

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3349–3353
2-Aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno-
[2,3-b]pyridines as broad-spectrum inhibitors
of human herpesvirus polymerases
Mark E. Schnute,a,* David J. Anderson,b Roger J. Brideau,c Fred L. Ciske,c
Sarah A. Collier,b Michele M. Cudahy,b MariJean Eggen,b Michael J. Genin,b
Todd A. Hopkins,d Thomas M. Judge,b Euibong J. Kim,b Mary L. Knechtel,b
Sajiv K. Nair,d James A. Nieman,d Nancee L. Oien,b Allen Scott,e Steven P. Tanis,d
Valerie A. Vaillancourt,b Michael W. Wathenb and Janet L. Wieberb
aGlobal Research and Development, Pfizer Inc., 700 Chesterfield Parkway West, St. Louis, MO 63017, USA
bGlobal Research and Development, Pfizer Inc., 7000 Portage Road, Kalamazoo, MI 49007, USA
cGlobal Research and Development, Pfizer Inc., 2800 Plymouth Road, Ann Arbor, MI 48105, USA
dGlobal Research and Development, Pfizer Inc., 10770 Science Center Drive, San Diego, CA 92121, USA
eResearch API, Pfizer Inc., 7000 Portage Road, Kalamazoo, MI 49007, USA
Received 10 February 2007; revised 28 March 2007; accepted 30 March 2007
Available online 5 April 2007
Abstract—A novel series of 2-aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamides have
been identified as potent antivirals against human herpesviruses. These compounds demonstrate broad-spectrum inhibition of
the herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases.
Ó 2007 Elsevier Ltd. All rights reserved.
Infections by viruses of the Herpesvirus family pose a
significant health burden to the general population,
leading to diminished quality of life for millions, and
are a leading contributor to morbidity in immunocom-
promised patients. Human cytomegalovirus (HCMV)
is associated with clinical symptoms such as pneumonia,
retinitis, and graft rejection in the immunocompro-
mised, as well as congenital birth defects in neonates.1
The existing therapies to treat HCMV infection are
hampered by significant drug-associated toxicities which
limit their duration of use. As a consequence and be-
cause of the nature of the patient population, emergence
of drug resistance is a common concern.2 The prevalence
of other herpesviruses such as herpes simplex virus
(HSV), Epstein–Barr virus (EBV), and varicella-zoster
virus (VZV) is widespread among the human popula-
tion.3 The adoption of routine childhood vaccination
against VZV has significantly reduced the incidence of
primary disease, chickenpox. However, the impact on
corresponding epidemiological outcome and extent of
acceptance of vaccination in the elderly toward inci-
dence of reactivation leading to herpes zoster (shingles),
a highly debilitating illness characterized by persistent
neuropathic pain, remains unclear. Antiviral therapy
in the immunocompromised to address complications
to VZV infections will likely still be a necessity in the fu-
ture. Unfortunately, the availability of a well-tolerated,
orally bioavailable, broad-spectrum agent to address
OH
O
O
N
O
N
N
N
H
S
Cl
1
Keywords: Cytomegalovirus; HCMV; Varicella-zoster; VZV; HSV.
*
Figure 1. Optimization of lead 4-oxo-4,7-dihydrothieno[2,3-b]pyridine
carboxamide 1 by replacement of the morpholine substituent.
Corresponding author. Tel.: +1 636 247 3662; fax: +1 636 247
5234; e-mail: mark.e.schnute@pfizer.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.102

3350
M. E. Schnute et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3349–3353
O
N
O
O
N
O
R
HO
a
N
N
H
N
H
S
S
OH
Cl
Cl
O
2
3a-f
O
O
N
O
O
O
O
N
Cl
N
b
c
N
N
N
R
H
H
H
S
S
S
OH
Cl
N
Cl
N
Cl
1
4
5a-s
Scheme 1. Synthesis of 2-hydroxyethylamine-substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridines 3a–f and 5a–r. Reagents and condition: (a) MsCl,
2,4,6-collidine, DMAP, DMF; PhCH(OH)CH2NHR (6a–f); (b) ethyl chloroformate, CHCl3, reflux; (c) i-Pr2EtN, DMF, RCH(OH)CH2N(CH3)H
(7a–s).
herpesvirus infections in the growing immunocompro-
mised population remains an unmet medical need.
order to explore the structure–activity relationships
surrounding the critical morpholine substituent, an
alternative 2-hydroxyethylamine motif was identified.
This motif imparted to compounds of the DHTP series
differentially improved polymerase inhibition compared
to the same analogs in the earlier 4-oxo-1,4-dihydro-
quinoline carboxamide template.5 Herein we report
optimization of the amine substituent in the thie-
no[2,3-b]pyridine carboxamide series leading to overall
increased antiviral potency.
Previously, we have described a novel series of 4-oxo-
4,7-dihydrothieno[2,3-b]pyridine (DHTP) carboxamides
exemplified by 1 which were broad-spectrum inhibitors
of herpesvirus polymerases including HCMV, HSV-1,
and VZV polymerases, Figure 1.4 During the course of
screening commercially available amine monomers in
Synthesis of 2-hydroxyethylamine-substituted thie-
no[2,3-b]pyridine carboxamides was accomplished by
alkylation of the corresponding aminoethanol with
2-chloromethyl thieno[2,3-b]pyridine 4 either discreetly
or generated in situ, Scheme 1. Alkylchloride 4 could
Table 1. HCMV polymerase and antiviral inhibition for compounds
3a–f
Compound R
HCMV Polymerase HCMV Antiviral
a
IC50 (nM)
a,b
IC50 (nM)
3a
3b
3c
3d
3e
3f
H
CH3
8400
100
>5000
180 ( 64)
450 ( 150)
400 ( 200)
1600
CH2CH3
CH2CH2OH 1580
240 ( 50)
Table 3. Influence of ethylamine C2-substituent on HCMV polymer-
ase and antiviral inhibition for compounds 5a–s
CH(CH3)2
C3H5
2040
8900
>5000
Compound
R
HCMV
polymerase
a
IC50 (nM)
HCMV
antiviral
a,b
IC50 (nM)
a Values are means of at least two experiments where standard devia-
tion is given in parentheses, see Table 5 for reference compounds.
b Determined by plaque reduction assay (Davis strain).
3b
5a
5b
5c
5d
5e
5f
Phenyl
100
100
180 ( 64)
57 ( 11)
200 ( 100)
250 ( 50)
90 ( 10)
60 ( 40)
85 ( 15)
30 ( 0)
400 ( 0)
150 ( 50)
40
4-Hydroxyphenyl
4-Fluorophenyl
3-Chlorophenyl
Pyridin-2-yl
Pyridin-3-yl
Pyridin-4-yl
2-Furan
220 ( 40)
203 ( 31)
86 ( 3)
160 ( 20)
200
OH
O
a
NHR
5g
5h
5i
77 ( 15)
250 ( 10)
12
6c-f
3-Furan
Scheme 2. Synthesis of 2-hydroxyethylamines 6c–f. Reagents and
condition: (a) RNH2, methanol, reflux (R = c, ethyl; d, 2-hydroxyethyl;
e, isopropyl; f, cyclopropyl).
Benzofuran-2-yl
Quinolin-2-yl
2-Thiophene
3-Cyanophenyl
4-Cyanophenyl
3-Methoxyphenyl
1,3-Thiazol-2-yl
Pyrimidin-2-yl
Pyrazin-2-yl
CH3
5j
65 ( 10)
175 ( 25)
66 ( 35)
315 ( 15)
87 ( 14)
235 ( 35)
100 ( 30)
85 ( 25)
3530
5k
5l
250 ( 50)
100 ( 0)
300 ( 100)
20 ( 10)
200
5m
5n
5o
5p
5q
5r
5s
Table 2. Configurational preference of stereogenic center in compound
3b toward herpes polymerase inhibition
a
Polymerase IC50 (nM)
100 ( 0)
65 ( 35)
1500
Compound
HCMV
HSV-1
VZV
(S)-3b
(R)-3b
100
2550
400
10,500
200
5000
CH(CH3)2
17,500
>5000
a Values are means of at least two experiments where standard devia-
tion is given in parentheses, see Table 5 for reference compounds.
b Determined by plaque reduction assay (Davis strain).
a Values represent single determinations, see Table 5 for reference
compounds.

M. E. Schnute et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3349–3353
3351
be prepared either from the corresponding 2-hydroxy-
methyl thieno[2,3-b]pyridine 24 by reaction with metha-
nesulfonyl chloride in the presence of 2,4,6-collidine and
DMAP or in high yield (95%) from 2-morpholinylmeth-
yl thieno[2,3-b]pyridine 14 upon treatment with ethyl
chloroformate.
with 4 generated in situ to afford thieno[2,3-b]pyridines
3a–f, Table 1. Aminoalcohols 6c–f were prepared from
styrene oxide by ring opening with the corresponding
primary amine, Scheme 2. HCMV polymerase inhibition
and antiviral activity in an HCMV plaque reduction as-
say was measured for each compound.6 This survey
established that small alkyl substituents (methyl and
ethyl) were preferred with a significant potency reduc-
tion for the unsubstituted (3a) and branched (3e–f) ana-
logs. Ring opening of (R)- and (S)-styrene oxide with
methylamine afforded the corresponding scalemic amin-
oalcohols7 which subsequently provided (R)-3b and (S)-
3b of established configurations (>98% ee), Table 2. The
configuration of the hydroxy-bearing stereogenic center
was found to play a significant role toward polymerase
inhibition with the (S)-isomer being 25-fold more potent
than the (R)-isomer for HCMV, HSV, and VZV poly-
merase. The relevance of substitution at the hydroxy-
bearing carbon toward polymerase inhibition was next
examined. A series of 2-aryl, 2-heteroaryl, and 2-alkyl-
N-methyl-2-hydroxyethylamines (7a–s) were alkylated
with 4 to provide thieno[2,3-b]pyridines 5a–s, Scheme
1 and Table 3. Aminoalcohols 7b–g were prepared from
the corresponding bromoacetyl by reduction to the bro-
mohydrin followed by displacement with methylamine
except for furan-2-yl 7g where the sequence of steps
had to be reversed in order to afford satisfactory yields,
Scheme 3. Aminoalcohols 7h–n were readily prepared
Initial SAR evaluation focused on the role of the
hydroxyethylamine nitrogen substituent by reacting N-
alkyl-substituted 2-hydroxy-2-phenylethylamines (6a–f)
O
O
OH
a,b
H
Br
Br
N
R
R
7b-f
OH
H
c
N
O
O
7g
Scheme 3. Synthesis of 2-hydroxyethyl-1-methylamines 7b–g. Re-
agents and conditions: (a) NaBH4, methanol (R = b, 4-fluorophenyl;
c, 4-chlorophenyl) or NaBH4, methanol; 48% HBr (R = d, pyridin-2-yl;
e, pyridin-3-yl; f, pyridin-4-yl); (b) methylamine, methanol, 60 °C; (c)
methylamine, methanol, 0 °C; NaBH4.
OH
a
O
H
N
R
R
8h-n
7h-n
Table 4. Influence of N7-substituent on HCMV polymerase and
antiviral inhibition for compounds (R)-5g, 11a–c, 11f, and 11g
Scheme 4. Synthesis of 2-hydroxyethyl-1-methylamines 7h–n. Re-
agents and conditions: (a) methylamine, methanol, rt (i, benzofuran-
2-yl; k, 2-thiophene) or 60–100 °C (h, 3-furan; j, quinolin-2-yl; l, 3-
cyanophenyl; m, 4-cyanophenyl;n, 3-methoxyphenyl).
Compound
R
HCMV
polymerase
HCMV
antiviral
a
IC50 (nM)
a,b
IC50 (nM)
(R)-5g
11a
11b
CH3
CH2CH3
35 ( 6)
7 ( 2)
200 ( 20)
180 ( 20)
350
150 ( 50)
35 ( 5)
250 ( 50)
250 ( 50)
300
OH
OH
a
H
CH2CH2CH3
CH2CH2OCH3
CH2CH2OH
CH2CH2CH2OH
Cl
N
11c
11f
R
R
240 ( 40)
280 ( 81)
7o-q
11g
a Values are means of at least two experiments where standard devia-
tion is given in parentheses, see Table 5 for reference compounds.
b Determined by plaque reduction assay (Davis strain).
Scheme 5. Synthesis of 2-hydroxyethyl-1-methylamines 7o–q. Re-
agents and condition: (a) methylamine, methanol, NaI, 60 °C (o, 1,3-
thiazol-2-yl; p, pyrimidin-2-yl; q, pyrazin-2-yl).
O
O
O
O
O
O
HO
Cl
N
c
a,b
N
N
N
H
H
H
S
S
O
S
OH
N
H
Cl
N
R
Cl
N
R
Cl
9
10a-e
11a-e
O
N
O
N
N
d
H
11d-e
O
S
OH
Cl
OH
( )n
11f-g
Scheme 6. Synthesis of 7-substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridines 11a–c, 11f, and 11g. Reagents and conditions: (a) RX (a, iodoethane; b,
iodopropane; c, 2-bromoethyl methyl ether), K2CO3, DMF, rt or RX (d, THPOCH2CH2I; e, THPOCH2CH2CH2I), Cs2CO3, DMF, 60–100 °C; (b)
MsCl, 2,4,6-collidine, DMAP, DMF; (c) i-Pr2EtN, DMF, (R)-7g; (d) 65% perchloric acid, THF (f, n = 1; g, n = 2).

3352
M. E. Schnute et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3349–3353
Table 5. Broad-spectrum activity of (R)-5d and (R)-5q compared to morpholinyl-substituted DHTP 1 and established therapies
a
Polymerase IC50 (nM)
a,b
Antiviral IC50 (nM)
c
Compound
CC50 (lM)
HCMV
HSV-1
VZV
a/c/d
HCMV
HSV-1
VZV
1
(R)-5d
(R)-5q
1100 ( 400)
61 ( 1)
37 ( 3)
1200
76
770
21
>40,000
>40,000
>40,000
800 ( 300)
100
15 ( 5)
5530 ( 160)
3000
1600
770 ( 180)
2
>100
>100
>100
>100
>100
76 ( 9)
19 ( 1)
7
Ganciclovir
Acyclovir
Foscarnet
Aphidicolin
AZT-TPd
1300 ( 300)
>20,000
nd
2100
nd
8100
2500
487 ( 74)
22,100
nd
438 ( 136)
3300
nd
473 ( 90)
5800
<280 (c)
2600 (a)
2300 (d)
a Values are means of at least two experiments where standard deviation is given in parentheses (nd, not determined).
b Determined by plaque reduction assay (HCMV, Davis strain; HSV-1, KOS strain; VZV, Webster strain).
c CC50, 50% cellular cytotoxicity in HFF cells derived from single determination, Ref. 14.
d AZT-TP, zidovudine triphosphate.
through ring opening of the corresponding oxiranes,8
Scheme 4. Lastly, aminoalcohols 7o–q were prepared
from the corresponding chlorohydrins9,10 by reaction
with methylamine, Scheme 5. The resulting phenyl and
heteroaromatic substituted thieno[2,3-b]pyridines all
showed strong HCMV polymerase inhibition and corre-
sponding HCMV antiviral activity in contrast to the
corresponding alkyl derivatives (5r and 5s) suggesting
the aromatic ring plays an important role in the en-
hanced activity of these analogs. Several analogs dem-
onstrated HCMV polymerase and antiviral IC50 values
below 100 nM including substitution by pyridin-2-yl
(5d), 2-furan (5g), and pyrazin-2-yl (5q). Analogs 5d
and 5q demonstrated good aqueous solubility (9 and
72 lM, respectively); however, furan derivative 5g
unfortunately showed poor solubility (0.4 lM). From
previous work in the series,4 polymerase inhibition was
tolerant of substitution in the 7-position of the thie-
no[2,3-b]pyridine ring system, and we felt it may be pos-
sible to modify the compound’s physical properties by
functionalization in this region. Alcohol 94 was alkyl-
ated by a series of alkyl and alkyl ether halides and sub-
sequently converted to the corresponding chloride 10,
Scheme 6. The resulting chlorides were reacted with chi-
ral 2-furyl aminoalcohol (R)-7g (>96% ee)9,11 to afford
thieno[2,3-b]pyridines 11a–e, Table 4. THP-ether deriva-
tives 11d and 11e were deprotected to provide alcohols
11f and 11g, respectively. Resolution by chiral chroma-
tography afforded (R)-5g.12 By incorporating a hydroxyl
group into the N7-substituent, compounds 11f and 11g
showed improved aqueous solubility (22 and 72 lM,
respectively) over the alkyl derivatives for which solubil-
ity remained poor. Unfortunately, for these analogs as
well as alkyl derivatives 11a–c, HCMV polymerase
and antiviral potency was reduced compared to the ste-
rically smaller methyl substituent.
demonstrated with (R)-5d and (R)-5q being substantially
more potent than 1 against HCMV and VZV. Both
compounds also demonstrated good oral bioavailability
in rat (45% and 21%, respectively) and in dog (76% and
66%, respectively).15 Compound (R)-5d was also found
to inhibit Epstein–Barr virus (EBV) polymerase
(IC50 = 70 nM)16 and demonstrate EBV antiviral activ-
ity in a cell culture assay (IC50 = 200 nM).17 As seen
previously with compound 1,4 HSV-1 mutants contain-
ing a V823A mutation in their polymerase gene were sig-
nificantly less sensitive to inhibition by (R)-5d than wild-
type virus.
The replacement of the morpholinyl-substituent in her-
pes antivirals of the thieno[2,3-b]pyridine series such as
1 with a 2-aryl-2-hydroxyethylamine moiety has affor-
ded orally bioavailable compounds with significantly
improved viral polymerase inhibition. Compounds
(R)-5d and (R)-5q exhibit antiviral activity comparable
(HSV-1) or superior (HCMV and VZV) to that of 1
and established therapies. The application of the
2-aryl-2-hydroxyethylamine motif to core ring systems
other than thieno[2,3-b]pyridines offers the hope of fur-
ther improved antiviral potency within this novel class
of broad-spectrum agents.
Acknowledgments
The authors thank Lester Dolak and Eric Seest for ana-
lytical chiral HPLC analysis, Garold Bryant for X-ray
structural determinations, Kevin Stafanski for solubility
measurements, and Paul Herrington, William Perrault,
William McGhee, and Thomas Beauchamp for assis-
tance in the synthesis of chiral aminoalcohols.
The more potent stereoisomer of the pyridin-2-yl and
pyrazin-2-yl derivatives was similarly prepared by reac-
tion of 4 with the corresponding scalemic (R)-aminoal-
cohols9 to provide (R)-5d13 and (R)-5q (>98% ee),
respectively. Both compounds were found to broad-
spectrum inhibitors of herpesvirus polymerases includ-
ing HCMV, HSV, and VZV while not being active
against human DNA polymerases (a, c, and d), Table 5.
Broad-spectrum herpes antiviral activity was also
References and notes
1. (a) Ljungman, P.; Griffiths, P.; Paya, C. Clin. Infect. Dis.
2002, 34, 1094; (b) Griffiths, P. D.; Clark, D. A.; Emery,
V. C. J. Antimicrob. Chemother. 2000, 45, 29, Topic T3;
(c) Ives, D. V. AIDS 1997, 11, 1791; (d) Gaytant, M.
A.; Steegers, E. A. P.; Semmekrot, B. A.; Merkus, H.
M. M. W.; Galama, J. M. D. Obstet. Gynecol. Surv.
2002, 57, 245.

M. E. Schnute et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3349–3353
3353
2. Scott, G. M.; Weinberg, A.; Rawlinson, W. D.; Chou, S.
Antimicrob. Agents Chemother. 2007, 51, 89.
(phenyl vs 2-furan) while the spatial orientation of the
hydroxyl group with respect to the aromatic moiety is the
same in both molecules.
3. (a) Whitley, R. J.; Roizman, B. Lancet 2001, 357, 1513; (b)
Thorley-Lawson, D. A.; Gross, A. N. Eng. J. Med. 2004,
350, 1328; (c) Heininger, U.; Seward, J. F. Lancet 2006,
368, 1365; (d) Gnann, J. W., Jr.; Whitley, R. J. N. Eng.
J. Med. 2002, 347, 340.
4. Schnute, M. E.; Cudahy, M. M.; Brideau, R. J.; Homa, F.
L.; Hopkins, T. A.; Knechtel, M. L.; Oien, N. L.; Pitts, T.
W.; Poorman, R. A.; Wathen, M. W.; Wieber, J. L.
J. Med. Chem. 2005, 48, 5794.
12. Configuration was subsequently confirmed by reacting
(R)-7g with 4 to afford an authentic sample.
13. The configuration for the active enantiomer of 5d was
confirmed by single crystal X-ray determination of the
corresponding 4-bromobenzoate ester. Crystallographic
data have been deposited with the Cambridge Crystallo-
graphic Data Centre as supplementary publication num-
ber CCDC 636553.
5. (a) Oien, N. L.; Brideau, R. J.; Hopkins, T. A.; Wieber, J.
L.; Knechtel, M. L.; Shelly, J. A.; Anstadt, R. A.; Wells, P.
A.; Poorman, R. A.; Huang, A.; Vaillancourt, V. A.;
Clayton, T. L.; Tucker, J. A.; Wathen, M. W. Antimicrob.
Agents Chemother. 2002, 46, 724; (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.
Antiviral Res. 2002, 54, 19; (c) Knechtel, M. L.; Huang, A.;
Vaillancourt, V. A.; Brideau, R. J. J. Med. Virol. 2002, 68,
234.
14. Toxicity of compounds to non-infected mammalian cells
was determined using replicating HFF 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. Lowik, C. W. G.
M.; Alblas, M. J.; van de Ruit, M.; Papapoulos, S. E.; van
der Pliijm, G. Anal. Biochem. 1993, 213, 426.
15. Compounds were dosed IV at 5 mg/kg and PO at 15 mg/kg
using a vehicle of 80/20 PEG400/0.01 M methanesulfonic
acid.
6. Compounds were assayed for their ability to inhibit the
incorporation of 3H-labeled nucleotide into template-
primer by purified viral DNA polymerase (HCMV,
HSV-1, VZV) and human DNA polymerases (alpha,
gamma, and delta) using a scintillation proximity assay.
Assay conditions and methods to purify the corresponding
DNA polymerases have been previously described in Ref.
4. Antiviral activities against HCMV, VZV, and HSV-1
herpesviruses were determined using plaque reduction
assays utilizing conditions described in Ref. 4. 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).
16. The EBV polymerase gene was isolated from pSVEBV
(obtained from D. Coen, Harvard Univ.) and inserted into
pGEM-3zf (Promega Corp.). Similarly, the sequence for
BMRF-1 accessory protein was isolated from plasmid
p128.5 (obtained from W. Sugden, Univ. Wisconsin) and
inserted into pGEM-3zf at the EcoRI and XbaI sites. The
T7 quick-coupled transcription/translation system (Pro-
mega Corp.) was used to produce active EBV polymerase
and BMRF-1 protein from pGEM + EBVpol and
pGEM + BMRF-1 DNA. The polymerase assay
employed 2.5 lL each of EBV polymerase and BMRF-1
in 45 lL of buffer comprised of 50 mM Tris, pH 8, 70 mM
NH4SO4, 10 mM MgCl2, 300 mM dCTP/dTTP/dGTP,
0.5 lCi 32P-dATP (3000 Ci/mmol), and 100 lg/mL acti-
vated calf thymus DNA. Compounds were added prior to
enzyme addition at a final DMSO concentration of 1 %.
Reaction mixtures were incubated at 37 °C for 30 min and
then the reaction was stopped by adding the reaction
volume to 2 mL of 5% TCA. Precipitated DNA was
harvested onto 2.4 cm GFC glass filters. Filters were
washed twice with 5% TCA and once with 95% ethanol.
Incorporation of 32P-dATP was measured by scintillation
counter.
17. EBV antiviral activity was measured in cell culture
employing EBV lytic replication. GG68 cells (obtained
from W. Sugden, Univ. Wisconsin) were induced with
40 ng/mL TPA and 4 mM n-butyric acid. Compounds
were added at a final DMSO concentration of 0.5%. After
72 h at 37 °C, cells were lysed and DNA was harvested.
Purified DNA extracts were separated by Southern blot
analysis, quantitated using Imagequant software, and IC50
values determined by an Excel program. Acyclovir EBV
antiviral IC50 = 6900 nM.
7. Coote, S. J.; Davies, S. G.; Middlemiss, D.; Naylorb, A.
J. Chem. Soc., Perkin Trans. 1 1989, 2223.
8. (a) Oxiranes 8h–l were prepared from the corresponding
carboxaldehydes, (CH3)3S+IÀ, KOH, CH3CN/H2O; 8m:
(b) Tamura, N.; Takahashi, K.; Shirai, N.; Kawazoe, Y.
Chem. Pharm. Bull. 1982, 30, 1393; 8n: (c) Perrone, R.;
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´
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11. Although (S)-3b and (R)-7g have different configurational
notations, this is due to a change in the Cahn-Ingold-
Prelog priority assignment for the aromatic moiety