Synthesis and biological evaluation of a series of thieno-expanded tricyclic purine 2′-deoxy nucleoside analogues

Article abstract of DOI:10.1016/j.bmc.2012.03.004

Introducing structural diversity into the nucleoside scaffold for use as potential chemotherapeutics has long been considered an important approach to drug design. In that regard, we have designed and synthesized a number of innovative 2′-deoxy expanded n

Full text of DOI:10.1016/j.bmc.2012.03.004

Bioorganic & Medicinal Chemistry 20 (2012) 3009–3015
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of a series of thieno-expanded tricyclic
purine 20-deoxy nucleoside analogues
Orrette R. Wauchope a, Cameron Johnson a, Pasupathy Krishnamoorthy a, Graciela Andrei b,
Robert Snoeck b, Jan Balzarini b, Katherine L. Seley-Radtke a,
⇑
a Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA
b Rega Institute for Medical Research, K.U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Introducing structural diversity into the nucleoside scaffold for use as potential chemotherapeutics has
long been considered an important approach to drug design. In that regard, we have designed and syn-
thesized a number of innovative 20-deoxy expanded nucleosides where a heteroaromatic thiophene
spacer ring has been inserted in between the imidazole and pyrimidine ring systems of the natural purine
scaffold. The synthetic efforts towards realizing the expanded 20-deoxy-guanosine and -adenosine tricy-
clic analogues as well as the preliminary biological results are presented herein.
Received 6 January 2012
Revised 23 February 2012
Accepted 1 March 2012
Available online 12 March 2012
Keywords:
Tricyclic
Ó 2012 Elsevier Ltd. All rights reserved.
Nucleosides
Expanded purine
Nucleobase
Guanosine
Adenosine
1. Introduction
This structural diversity provides several advantages over
previously studied systems such as Leonard’s lin-benzoadenosine
Interest in the design and synthesis of modified nucleosides has
increased steadily over the past few decades.1–3 The impetus for
such development is a result of several factors.4 For example,
modified nucleoside analogues have been found to be particularly
important in chemotherapeutic applications for the treatment of
disease. In that regard, they have been used to treat diseases by
inhibiting essential enzymes involved with the replication path-
ways of many viruses, parasites, bacteria and cancers. Brivudine
and Idoxuridine, both possessing heterobase modifications, are
used to treat varicella-zoster virus and herpes simplex virus
respectively.5 Additionally, FDA approved drugs such as Gemcita-
bine, Fludarabine and Cytarabine have been found useful in treat-
ing cancers,6 whereas, Zidovudine (AZT), a reverse transcriptase
inhibitor, has provided the foundation for the treatment of HIV.7
As a consequence, we, as many others in the field, have focused
on the design and synthesis of innovative and structurally unique
types of nucleosides with the aim of synthesizing potential chemo-
therapeutics.8–17 One such series involves modified purine nucleo-
sides that possess heteroaromatic spacer rings, for example a
thiophene, between the imidazole and the pyrimidine ring of the
purine scaffold (Fig. 1).10,18
analogues.4,19–23 Some of the expanded nucleosides reported by
our group (shown in Fig. 1), have exhibited promising chemother-
apeutic activity suggesting the increased aromatic surface area
increases biological activity.24–26 Notably, the triphosphates of
the nucleosides in Figure 1 have been recognized and incorporated
by RNA polymerases from T7 and SP6. Moreover, the triphosphate
⇑
Corresponding author. Tel.: +1 410 455 8684; fax: +1 410 455 2608.
E-mail address: kseley@umbc.edu (K.L. Seley-Radtke).
Figure 1. Thieno-expanded nucleosides.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.004

3010
O. R. Wauchope et al. / Bioorg. Med. Chem. 20 (2012) 3009–3015
Figure 2. Proposed 20-deoxythieno-expanded nucleosides.
of the guanosine nucleoside 1 (Fig. 1) has been shown to be a good
inhibitor of HCV NS5B polymerase.27 In the HCV replicon whole
cell assay, the ribofuranose thieno-expanded guanosine and aden-
osine analogues (1 and 2 respectively in Fig. 1) possessed moderate
Scheme 2. Synthesis of 20-deoxy intermediate 18. Reagents and conditions: (a)
MeCN, NaH, 30 min, diiodoimidazole, 24 h, rt ; (b) MeOH, NaOMe; (c) THF, NaH, 3 h,
TBAI, PMBCl, 18 h, rt; (d) (i) EtMgBr, THF, 15 min; (ii) anhydrous DMF; (e) (i)
NaHCO3, hydroxylamine hydrochloride, H2O; (ii) carbaldehyde, EtOH, rt, 18 h; (f)
CDI, THF, reflux, 18 h; (g) NH2C(O)CH2SH, K2CO3, DMF, 60 °C; (h) NaOEt, EtOH.
inhibitory activity with EC50 values of 74 and 87 lM, respec-
tively.27 Another finding was the observation that the nucleoside
and nucleobase transporters in Trypanosoma brucei recognized
both the ribose analogues in their nucleoside form as well as their
corresponding nucleobases.24 Additionally, the nucleobases of the
modified ribonucleosides exhibited interesting cytostatic activity
against three cancer cell lines.26
Although a minute amount of starting material was recovered,
there were a number of other side products that could not be
isolated or characterized due to very similar Rf values. Finally,
deprotection of the silyl groups followed by removal of the isobu-
tyryl group gave the target compound 5 in 60% yield from 9
(Scheme 1), with an overall yield starting from the ribose nucleo-
side 1 of 3.8%. While on the surface this appeared reasonable, when
considering that the overall yield starting from diiodoimidazole
and commercially available protected ribose was only 0.5%, it
rendered this approach less attractive, so attention turned to the
alternative route—starting from a 20-deoxy sugar.
Given the promising activity of the parent ribose analogues, the
analogous 20-deoxy series (5 and 6, Fig. 2) were therefore of inter-
est for investigating 20-deoxy metabolizing enzymes.
2. Results and discussion
2.1. Chemistry
Synthesis began with the coupling of 4,5-diiodoimidazole to
Hoffer’s well-known toluoyl-protected chlorosugar30,31 to provide
intermediate 11 in 84% yield (Scheme 2). Sekine et al. were suc-
cessful in synthesizing and isolating 11 not only in good yield,
but also as the beta isomer.30 Due to the harsh conditions present
in subsequent steps however, the toluoyl groups needed to be re-
placed with a more robust protecting group. Although several at-
tempts were made to replace the toluoyl group before coupling,
they all resulted in poor yields, so the decision was made to replace
them after coupling. In that regard, the para-methoxybenzyl (PMB)
group appeared to be a viable option as there exists a plethora of
deprotection procedures in the literature.32–37 More importantly,
a number of those procedures utilized neutral conditions thus
would be less likely to result in glycosidic bond cleavage, some-
thing that had been a problem in the past using other deblocking
protocols. Consequently, the nucleoside was protected with PMB
using standard procedures to provide 13 in 64% yield.
Two potential synthetic routes could be envisioned in order to
realize the 20-deoxynucleosides; one, a linear route stemming di-
rectly from a 20-deoxy sugar, or two, treatment of the ribofuranose
guanosine tricyclic nucleoside9,10,18,26 with the well-known Barton
deoxygenation protocol28,29 to generate the 20-deoxy target. The
latter was initially chosen since a small amount of the ribose
nucleosides were already in hand and could be utilized immedi-
ately. In preparation for the Barton deoxygenation, the exocyclic
amine group on the guanosine nucleoside 110 was protected with
isobutyryl chloride followed by selective bis-protection of the 30-
and 50-hydroxyl groups with 1,3-dichloro-1,1,3,3-tetraisopropyl-
1,3-disiloxane to give 7 in 52% yield for the two steps (Scheme 1).
Using Barton deoxygenation procedures,28,29 the free 20-hydro-
xyl group was then treated with phenyl chlorothionoformate to
give the thiocarbonate 8. Treatment with AIBN and tributyltin hy-
dride then afforded intermediate 9, albeit in only 30% yield.
Addition of the aldehyde was carried out as before10,12 with the
exception that the crude product was taken on directly to the next
reaction without purification due to the observed instability of the
aldehyde on silica gel columns. The protocol for the synthesis of
the aldoxime 15 was also modified due to the decomposition of
the 20-deoxynucleoside under the previously used conditions.10,18
Sodium bicarbonate was used instead of pyridine as the base,
and the order of addition for the reagents was changed, as well
as the reaction conditions, all which served to result in greater
yields. Dehydration was accomplished with 1,10-carbonyldiimidaz-
ole (CDI)38 instead of acetic anhydride10,18 or trichloroacetoni-
trile12 to give 16. CDI appeared superior to the previously used
reagents in terms of the yield of the isolated product as well as
its purification. Ring closure to bicyclic intermediate 18 was then
carried out in an analogous manner as was used for the ribose
series.
Scheme 1. Synthesis of 20-deoxyguanosine analogue 5. Reagents and conditions:
(a) TMSCl, pyridine then isobutyryl chloride, 24 h; (b) 1,3-dichloro-1,1,3,3-tetra-
isopropyldisiloxane, pyridine, rt, 24 h; (c) phenyl chlorothionoformate, DMAP, rt,
24 h; (d) AIBN, Bu3SnH, toluene, reflux, 6 h; (e) (i) 1 M TBAF, THF, rt, 4 h; (ii)
methanolic ammonia, rt.
Formation of the tricyclic intermediate 19 (Scheme 3) was sub-
sequently achieved in a 53% yield for three steps. The previous ring
closure conditions using CS2 and NaOH in MeOH were observed to
be incompatible with the 20-deoxy nucleoside, as an inseparable

O. R. Wauchope et al. / Bioorg. Med. Chem. 20 (2012) 3009–3015
3011
involving the Barton deoxygenation was accompanied by low
yields and difficulties with various purifications, thus the route
using the 20-deoxy sugar was attempted. Starting with the bicyclic
intermediate 18, ring closure to the tricyclic base was accom-
plished with triethyl orthoformate and molecular sieves to give
the inosine intermediate 20 (Scheme 4).
Initially, use of our previously published route of converting the
carbonyl group of 20 to the thiocarbonyl followed by methylation
and subsequent removal with ammonia18,26 to obtain the adeno-
sine analogue was tried. However, reaction of the inosine interme-
diate with phosphorus pentasulfide did not afford any of the
desired product, but instead, resulted in a complex mixture of
unidentifiable compounds. Surmising that the phosphorus penta-
sulfide was reacting with the ether moiety of the PMB protecting
group, akin to the results reportedly observed39 in the synthesis
of Lawesson’s reagent from anisole, a new route was sought. Efforts
to convert 20 to the chloro derivative using phosphorus oxychlo-
ride also proved futile as the starting material decomposed under
these harsh conditions regardless of reaction time and/or temper-
ature. Moreover, converting the carbonyl of 20 to the relatively
labile triisopropylbenzenesulfonyl (TPS) group followed by re-
moval with ammonium hydroxide,40 was disappointing. The yield
of 21 was less than 20% although fortunately a significant amount
of the inosine starting material 20 could be recovered. Speculation
was that the water in the ammonium hydroxide was facile in
replacing the labile TPS group, thus the use of ammonia instead
of ammonium hydroxide provided 21 in 65% yield. Finally, use of
the Lewis acid deprotection of 21 as before provided 6 in 30% yield.
Comparing the two approaches, just as was seen with the gua-
nosine analogue, the overall yield for the route to the tricyclic
adenosine starting with ribose and diiodoimidazole and using the
Barton deoxygenation proved to be only 0.1%, while the route
starting with the 20-deoxy sugar was 2.1%. Thus, while it could
be argued that using the ribose as a starting material would essen-
tially produce both the 20-deoxy and the ribose nucleosides, it was
clear it was not a particularly efficient way to approach their
synthesis.
Scheme 3. Formation of tricyclic 20-deoxyguanosine 5. Reagents and conditions: (a)
(i) KSC(S)OEt, DMF, reflux, 4 h; (ii) H2O2, MeOH, 0 °C, 2 h; (iii) NH3, MeOH, 125 °C,
18 h; (b) BF3ꢁOEt2, EtSH, CH2Cl2.
2.2. Biological results
Scheme 4. Synthesis of tricyclic 20-deoxyadenosine 6. Reagents and conditions: (a)
CH(OEt)3, 4 Å molecular sieves; (b) (i) TPSCl, DMAP, TEA, MeCN, 3 h; (ii) NH3, 18 h,
rt; (c) BF3ꢁOEt2, EtSH, CH2Cl2.
Once the tricyclic targets were in hand, they were evaluated for
their potential inhibitory activity against a broad variety of viruses,
and for their potential cytostatic activity. Moderate inhibition was
observed for 5 and 6 against the proliferation of three cancer cell
lines as summarized in Table 1. The compounds were somewhat
more cytostatic against human embryonic lung fibroblasts (HEL).
In all cases, 6 was ꢀ2-fold more inhibitory to cell proliferation than
5. Both compounds 5 and 6 were evaluated against a wide variety
of viruses (see Table 2 below), including herpes simplex virus type
1 (HSV-1), HSV-2, HSV TK, vaccina virus, vesicular stomatitis virus,
respiratory syncytial virus, Sindbis virus, Punta Toro virus, Reovi-
rus-1, Coxsackie virus B4, parainfluenza-3 virus, influenza virus
types A and B, and human immunodeficiency virus type 1 (HIV-
1) and type 2 (HIV-2). Moderate activity was observed for both
analogues against VZV (Table 2).
mixture of products was obtained. As a result, the first step of the
ring closure protocol was modified and pre-formed potassium eth-
ylxanthic acid salt was used instead of CS2 and NaOH in MeOH.
Surprisingly, in contrast to our reported synthesis of the ribose
analogues, no xanthosine side-product was observed.10
Deprotection of the PMB groups proved to be quite problematic
despite the availability of numerous deprotection strategies that
could be found in the literature.32–37 Several conditions were at-
tempted, including the standard deprotection procedure utilizing
ceric ammonium nitrate (CAN). Unfortunately CAN resulted in sig-
nificant glycosidic bond cleavage. In most cases, either no product
was observed or the product yields proved to be less than 10%.
Ultimately the original Lewis acid mediated deblocking procedure
was employed, which gave 5, albeit in only a 35% yield (Scheme 4).
The overall yield for the linear route starting with the tolouyl sugar
provided to be 2.2%, a significant increase over the yield starting
from ribose.
Table 1
Anticancer activity of tricyclic targets
a
Compound
IC50
(lM)
L1210
CEM
HeLa
HEL
Analogous to the synthetic route to the guanosine analogue, the
expanded adenosine nucleoside 6 could be approached from the
same two routes, and since the synthesis of both 5 and 6 were
being pursued concurrently, both routes were tried for compari-
son. As was the case with the guanosine analogue, the route
5
6
144 15
166 54
82 39
81 10
57 40
22
12
53
4
a
50% inhibitory concentration or compound concentration required to inhibit
cell proliferation by 50%.

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O. R. Wauchope et al. / Bioorg. Med. Chem. 20 (2012) 3009–3015
1H and 13C NMR spectra were referenced to internal tetramethyl-
Table 2
Antiviral activity of targets 5 and 6 in cell culture
silane (TMS) at 0.0 ppm. The spin multiplicities are indicated by
the symbols s (singlet), d (doublet), dd (doublet of doublets), t
(triplet), q (quartet), m (multiplet), and br (broad). Reactions were
monitored by thin-layer chromatography (TLC). Column chroma-
tography was performed using commercial silica gel and eluted
with the indicated solvent system. Yields refer to chromatograph-
ically and spectroscopically (1H and 13C NMR) homogeneous
materials.
a
Virus
EC50 (lM)
5
6
Ribavirin
HEL cells
HCMV (AD-169 & Davis)
VZV (OKA & 07/1)
HSV-1 (KOS)
HSV-1 TK-
HSV-2 (G)
VV
>100
71.3–100
>100
>100
>100
>4
8.9
—
—
>20
>20
>20
>20
>20
>250
>250
>250
>250
146
>100
>100
4.1.1.1. 30,50-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl) [N7-iso-
VSV
butyryl-7-aminoimidazo[40,50:4,5]thieno[3,2-d]pyrimidin-3-yl-
HeLa cells
VSV
100
>100
>100
>100
>100
>100
29
146
10
7-one]-2,3,5-b-
D
-ribofuranose (7).
To a mixture of the triol 1
Coxsackie virus B4
RSV
(0.735 g, 2.17 mmol) in dry pyridine (40 mL) was added TMSCl
(3.5 mL, 27.8 mmol) at 0 °C. The reaction mixture was stirred at
this temperature for 30 min and then for an additional 2 h at room
temperature (rt). The mixture was then cooled to 0 °C and iBuCl
(1.14 mL, 10.85 mmol) was added dropwise. The mixture was al-
lowed to warm up to rt slowly and then stirred overnight. The
reaction was quenched by the addition of H2O (5 mL) at 0 °C and
the mixture was allowed to stir for 15 min followed by the addition
of NH4OH (20 mL). The mixture was stirred at rt for an additional
2 h. The volatiles were then removed under reduced pressure
and the product purified by column chromatography eluting with
CH2Cl2/MeOH (10:1) to give the intermediate as a white solid
(303 mg). 1H NMR (acetone-d6):d 0.93–0.94 (m, 1 H), 1.22–1.24
(m, 6H), 2.02–2.03 (m, 1H), 2.58–2.66 (m, 1H), 2.89–2.97 (m, 1
H), 4.05–4.09 (m, 1H), 4.28–4.30 (m, 1H), 4.94–4.99 (m, 1 H),
6.21 (d, 1 H), 8.17 (s, 1H), 9.99 (s, 1H), 12.24 (s, 1H); 13C NMR (ace-
tone-d6): d 18.3, 18.4, 29.2, 29.4, 63.0, 53.2, 71.1, 71.2, 74.0, 82.4,
90.2, 90.3, 148.6, 149.5, 177.4, 180.4, 180.5, 180.6.
CEM cells
HIV-1(IIIB) HIV-2(ROD)
ꢂ250
>10
>50
Vero cells
Parainfluenza-3 virus
Reovirus-1
Sindbis virus
Coxsackie virus B4
Punta Toro virus
>100
>100
>100
>100
>100
>100
100
100
>100
100
85
>250
>250
>250
126
MDCK cells
Influenza virus (AH3N2)
Influenza virus A (H1N1)
Influenza virus B
Cell line MCCb
(lM)
>0.16
>0.16
>0.16
>4
>4
>4
5.1–9.0
2.3–3.4
4.5–9
HEL
>100
100
100
0.8
ꢂ20
>100
>100
20
>250
>250
>250
20
HeLa
Vero
MDCK
a
50% Effective concentration, or compound concentration required to inhibit
To a solution of the tricyclic intermediate (55 mg, 0.134 mmol)
in pyridine (15 mL) was added TIPDSCl (0.052 mL, 0.134 mmol)
dropwise. The reaction was then stirred at rt for 24 h. The solvent
was evaporated and the residue was dissolved in CH2Cl2 (50 mL),
washed with H2O, then saturated NaHCO3. The organic phases
were dried and evaporated to dryness. The residue was purified
by preparative chromatography (CH2Cl2/MeOH, 10:1) to give 7 as
a yellow oil. Yield: 88 mg, 96% (52 % for two steps). 1H NMR
(CDCl3): d 0.93–0.94 (m, 1 H), 1.22–1.24 (m, 34H), 2.02–2.03 (m,
1H), 2.58–2.66 (m, 1H), 2.89–2.97 (m, 1 H), 4.05–4.09 (m, 1H),
4.28–4.30 (m, 1H), 4.94–4.99 (m, 1H), 6.21 (d, 2.5 Hz, 1H), 8.17
(s, 1H), 9.99 (s, 1H), 12.24 (s, 1H); 13C NMR (CDCl3): d 18.3, 18.4,
29.2, 29.4, 63.0, 53.2, 71.1, 71.2, 74.0, 82.4, 90.2, 90.3, 116.6,
127.0, 148.9, 149.5, 151.4, 177.4, 180.4. HRMS (FAB): calcd for
virus-induced cytopathicity by 50%.
Minimal cytotoxic concentration, or compound concentration required to cause
a microscopically visible alteration of cell morphology.
b
3. Summary
Two different routes to the 20-deoxy ribose tricyclic analogues
were developed. Although the Barton deoxygenation has been used
successfully with other nucleosides, it proved to be less efficient
with our analogues. Speculation that the sulfur in the thiophene
ring might be interfering in the radical reaction was one mechanis-
tic possibility we considered, however it was not pursued further
since the other route was reasonable and much higher yielding.
Not surprisingly, some of the conditions for the 20-deoxy’s had to
be altered from our previously reported routes to realize the ribose
analogues due to the inherent differences in stabilities for their
glycosidic bonds, however these modifications may ultimately
prove advantageous to the ribose route as we continue to pursue
additional analogues. Finally, although only moderate cytostatic
and antiVZV activity was noted for the two 20-deoxyanalogues, ef-
forts are currently underway to synthesize other expanded purine
analogues, including the corresponding triphosphates, the results
of which will be reported as they become available.
C
28H46N5O7SSi2 (M+H)+ 652.2657, found 652.2659.
4.1.1.2. 30,50-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl) [N7-iso-
butyryl-7-aminoimidazo[40,50:4,5]thieno[3,2-d]pyrimidin-3-yl-
7-one]-2,3,5-b-
(8). Compound 7 (91.3 mg, 0.143 mmol) was suspended in
D
-ribofuranose- 20-O-thiocarbonate phenyl ester
anhydrous CH3CN (10 mL) and was treated with DMAP (98 mg,
0.804 mmol) followed by phenylchlorothionoformate (0.036 mL,
0.268 mmol). The yellow solution was stirred for 24 h at rt. The sol-
vent was removed and the residue was purified by column chro-
matography eluting with a gradient of 100% hexanes to 25%
EtOAc in hexanes to give yellow crystals. Yield: 39 mg, 39%; mp
132–135 °C. 1H NMR (acetone-d6) d 0.93–0.94 (m, 1 H), 1.22–1.24
(m, 34H), 2.02–2.03 (m, 1H), 2.58–2.66 (m, 1H), 2.89–2.97 (m, 1
H), 4.05–4.09 (m, 1H), 4.28–4.30 (m, 1H), 4.94–4.99 (m, 1H), 6.03
(d, 3.0 Hz, 1H), 6.72–7.32 (m, 5H), 8.30 (s, 1H), 10.34 (s, 1H); 13C
NMR (acetone-d6) d 18.3, 18.4, 29.2, 29.4, 63.0, 53.2, 71.1, 71.2,
74.0, 82.4, 90.3, 90.2, 148.4, 149.8, 177.5, 180.6, 180.5, 180.6,
4. Experimental section
4.1. Synthetic methods
4.1.1. General
All chemicals were obtained from commercial sources and used
without further purification unless otherwise noted. Anhydrous
DMF, MeOH, DMSO, and toluene were purchased or obtained using
a solvent purification system. Melting points are uncorrected. All
205.4, 205.6 HRMS (FAB): calcd for
788.2639, found 788.2641.
C
35H50N5O8S2Si2 [M+H]+

O. R. Wauchope et al. / Bioorg. Med. Chem. 20 (2012) 3009–3015
3013
4.1.1.3. 30,50-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl) [N7-iso-
4.1.1.7.
ethyl)-1-(4,5-diiodoimidazol-3-yl)-b-
(13). To a suspension of NaH (95%, 0.82 g, 32.4 mmol) in
3’-(p-Methoxybenzlyoxy)-5’-(p-methoxybenzyloxym-
butyryl-7-aminoimidazo[40,50:4,5]thieno[3,2-d]pyrimidin-3-yl-
D
-2’-deoxyribofuranose
7-one]-2,3,5-b-
D
-deoxyribofuranose (9).
Under N2,
8
(300 mg, 0.38 mmol) was suspended in 10 mL anhydrous toluene
and AIBN (28 mg, 0.17 mmol) added, followed by n-Bu3SnH
(1.25 mL, 2.07 mmol). The reaction mixture was refluxed for 6 h,
and the solvent was evaporated under vacuum to give a yellow syr-
up. The crude syrup was purified via chromatography eluting with
hexanes/EtOAc (2:1) to afford 9 as a colorless oil. Yield: 72 mg, 30%.
anhydrous THF (20 mL) at 0 °C was added a solution of 12
(5.65 g, 12.96 mmol) in anhydrous THF (20 mL) dropwise over a
period of 10 min. The resulting mixture was stirred at rt for 3 h,
at which point tetrabutylammonium iodide (957 mg, 2.59 mmol)
was added, followed by dropwise addition of p-methoxybenzyl
chloride (5.2 mL, 38.9 mmol). The mixture was further stirred at
rt for an additional 18 h. The reaction was filtered over celite and
repeatedly washed with CH2Cl2. The organic layers were combined
and reduced under vacuum to give a yellow syrup. Column chro-
matography eluting with hexane/EtOAc (3:2) gave 13 as thick oil.
Yield: 5.6 g, 64%.1H NMR (acetone-d6, 400 MHz): d 1.94 (m, 1H),
2.50–2.72 (m, 2H), 2.80 (s, 2H), 3.76 (dd, 3.2 Hz, 10.56 Hz, 2H),
3.77 (s, 6H), 4.45 (m, 1H), 4.47 (m, 1H), 5.94 (d, 6.40 Hz, 1H),
6.85 (m, 4H), 7.20 (m, 4H), 8.00 (s, 1H); 13C NMR (acetone-d6,
100 MHz) d 39.7, 55.3, 69.5, 71.4, 73.3, 78.6, 84.4, 90.0, 113.9,
114.0, 129.3, 129.4, 129.5, 129.6, 139.7, 159.4. HRMS (ESI): calcd
for C24H27I2N2O5 [M+H]+ 677.0015, found 677.0016.
1H NMR (CDCl3)
d 1.05–1.22 (m, 34H), 2.65–2.74 (m, 3H),
2.75–2.84 (m, 1 H), 3.92–3.94 (m, 1H), 4.05–4.12 (m, 1H), 4.66–
4.72 (m, 1H), 6.39–6.40 (m, 1H), 8.34 (s, 1H), 12.06 (s, 1H); 13C
NMR (CDCl3) d 12.6, 13.1, 13.2, 13.5, 16.9, 17.0, 17.3, 17.4, 17.5,
17.6, 19.1, 36.7, 41.2, 61.5, 69.1, 85.0, 85.6, 148.3, 157.3, 178.5.
HRMS (ESI): calcd for C28H46N5O6SSi2 [M+H]+ 636.2707, found
636.2705.
4.1.1.4. 1-[(5-Aminoimidazo[40,50:4,5]thieno[3,2-d]pyrimidin-3-
yl-7-one)]-b-
D
-20-deoxyribofuranose (5).
To a solution of 9
(72 mg, 0.113 mmol) in 5 ml of anhydrous THF was added TBAF
(1 M in THF, 0.226 ml, 0.226 mmol). The mixture was stirred at rt
until TLC analysis indicated the disappearance of the starting mate-
rial. The THF was removed under vacuum and a solution of meth-
anolic ammonia was added. This mixture was warmed to 40 °C and
stirred for 18 h. The solvent was removed and the product 5 was
purified by column chromatography eluting with EtOAc/acetone/
EtOH/H2O (4:1:1:1) to give a solid. Yield: 22 mg, 60%; mp 136–
138 °C. 1H NMR (DMSO-d6) d 3.64 (s, 1H), 3.75 (dd, 1H), 3.77 (dd,
1H), 3.87 (dd, 1H), 4.10–4.16 (m, 2H), 4.40 (s, 1H), 6.52 (t, 4.5 Hz,
1H), 7.93 (s, 1H), 9.56 (s, 1H), 10.20 (s, 1H); 13C NMR (DMSO-d6)
d 62.2, 70.7, 77.0, 88.6, 89.7, 108.9, 131.7, 150.6, 151.9, 160.6.
4.1.1.8.
ethyl)-1-[(5-iodo-4-carbaldehyde) imidazol-3-yl]-b-
ribofuranose (14). EtMgBr (1.6 mL, 3.0 M in Et2O,
3’-(p-Methoxybenzlyoxy)-5’-(p-methoxybenzyloxym-
D-2’-deoxy-
3.84 mmol) was added to a stirred solution of the starting material
(2.6 g, 3.84 mmol) in anhydrous THF (20 mL) under N2 at 0 °C. The
mixture was then stirred at this temperature for 15 min. Anhy-
drous DMF (1.6 mL, 4.81 mmol) was added, and the mixture was
further stirred for 20 min at 0 °C. The solvent was removed under
reduced pressure. Saturated NH4Cl solution (100 mL) was added
and the mixture extracted with CH2Cl2 (3 ꢃ 50 mL). The organic
extracts were combined, washed with brine (2 ꢃ 20 mL), and dried
over MgSO4. The solvent was removed under reduced pressure to
give 14 as a pale brown syrup which was used in the next step
without further purification. 1H NMR (CDCl3, 400 MHz): d 2.15
(m, 1H), 2.68 (ddd, 3.20 Hz, 6.4 Hz, 13.72 Hz, 1H), 3.53 (m, 1H),
3.71 (dd, 3.2 Hz, 10.56 Hz, 1H), 3.79 (s, 6H), 4.20 (m, 2H), 4.39 (q,
11.44 Hz, 2H), 4.45 (q, 11.92 Hz, 2H), 6.58 (t, 5.04 Hz, 1H), 6.86
(d, 6.44 Hz, 2H), 6.87 (d, 6.44 Hz, 2H), 7.19 (d, 8.64 Hz, 4H), 8.61
(s, 1H), 9.66 (s, 1H); 13C NMR (CDCl3, 100 MHz) d 40.4, 55.3, 68.7,
71.5, 73.2, 77.1, 84.6, 87.9, 114.0, 114.1, 129.4, 129.5, 129.6,
142.2, 159.4, 180.7. HRMS (ESI): calcd for C25H28IN2O6 [M+H]+
579.0992, found 579.0995.
HR-FAB m/z calcd for
C
12H14N5O4S [M+H]+ 324.0767, found
324.0765.
4.1.1.5. 1-[2’-Deoxy-3’,5’-di-O-(4-toluoyl)-b-
D-ribofuranose-1-
yl]-4,5-diiodoimidazole (11)30
.
To a solution of 4,5-diiodo-
imidazole (35.5 g, 111.2 mmol) in anhydrous CH3CN (200 mL) at
0 °C was added NaH (95%, 2.8 g, 111.2 mmol) portion wise. After
the mixture was stirred for 30 min at rt, 10 (32 g, 101.1 mmol)
was added portion wise at rt over a period of 1 h. After stirring
at the same temperature for 18 h the reaction mixture was filtered
over celite and washed with EtOAc (3 ꢃ 150 mL). The organic layer
was concentrated under reduced pressure. The product was iso-
lated by flash chromatography on silica gel using hexane/EtOAc
(3:1) to give 11 as a pale yellow syrup. Yield: 57 g, 84%. 1H NMR
(CDCl3, 400 MHz): d 2.39 (s, 3H), 2.42 (s, 3H), 2.54 (ddd, 6.40 Hz,
7.80 Hz, 14.20 Hz, 1H), 2.80 (ddd, 2.32 Hz, 5.96 Hz, 14.24 Hz, 1H),
4.60 (m, 1H), 4.64 (d, 3.64 Hz, 2H), 5.63 (dt, 2.76 Hz, 6.4 Hz, 1H),
6.06 (dd, 5.48 Hz, 7.76 Hz, 1H), 7.22 (d, 8.24 Hz, 2H), 7.27 (d,
8.24 Hz, 2H), 7.84 (d, 8.24 Hz, 2H), 7.85 (s, 1H), 7.93 (d, 8.24 Hz,
2H);13C NMR (CDCl3, 100 MHz) d 21.81, 21.87, 39.8, 63.8, 74.6,
79.8, 83.2, 89.5, 97.2, 126.3, 126.5, 129.4, 129.5, 129.6, 129.9,
138.9, 144.4, 144.7, 165.9, 166.2. HRMS (ESI): calcd for
4.1.1.9.
ethyl)-1-[(5-iodo-4-carbaldehydeoxime) imidazol-3-yl]-b-
deoxyribofuranose (15). To NaHCO3 (2.9 g, 34.6 mmol) in
3’-(p-Methoxybenzlyoxy)-5’-(p-methoxybenzyloxym-
D
-2’-
H2O (15 mL) was carefully added hydroxylamine hydrochloride
(1.97 g, 28.3 mmol), after which a solution of crude aldehyde 14
(2.22 g, 3.84 mmol) in THF/EtOH (1:3, 16 mL) was added dropwise.
To the resulting turbid solution was added THF until the mixture
turned homogeneous. The reaction mixture was then stirred at rt
overnight. The solvent was removed under reduced pressure; the
residue was treated with H2O (10 mL) and the mixture extracted
with CH2Cl2 (3 ꢃ 75 mL). The organic extracts were combined,
washed with brine (10 mL), and dried over Na2SO4. The solvent
was removed under reduced pressure to afford 15 as a pale yellow
syrup which was used directly without further purification. Yield:
1.8 g, 79%.1H NMR (CDCl3, 400 MHz): d 2.15 (dt, 5.92 Hz, 13.72 Hz,
1H), 2.56 (ddd, 4.92 Hz, 5.96 Hz, 13.98 Hz, 1H), 3.50 (dd, 3.20 Hz,
10.52 Hz, 1H), 3.67 (dd, 2.76 Hz, 10.56 Hz, 1H), 3.78 (s, 6H), 4.18
(m, 2H), 4.36 (q, 11.44 Hz, 2H), 4.44 (q, 11.92 Hz, 2H), 5.23 (s,
1H), 6.83 (d, 8.72 Hz, 2H), 6.87 (d, 8.68 Hz, 2H), 7.15 (d, 8.72 Hz,
2H), 7.19 (d, 8.34 Hz, 2H), 8.05 (s, 1H), 8.17 (s, 1H); 13C NMR
(CDCl3, 100 MHz) d 40.3, 55.3, 68.9, 71.5, 73.2, 77.6, 84.4, 88.0,
90.1, 113.9, 114.0, 125.0, 129.4, 129.5, 129.6, 139.4, 141.3, 142.8.
C
24H23I2N2O5 (M+H)+ 672.9696, found 672.9693.
4.1.1.6. 1-(2’-Deoxy-b-
D-ribofuranose-1-yl)-4,5-diiodoimidazole
(12)30
.
To a solution of 11 (20.34 g, 30.3 mmol), in anhydrous
MeOH (200 mL) was added NaOMe (3.38 g, 60.5 mmol) was added
and the resulting solution was stirred for 4 h at rt. Then the solvent
was evaporated and the product 12 was isolated as a white solid by
flash chromatography on silica gel eluting with EtOAc/MeOH (9:1).
Yield: 8.2 g, 87%; mp 58 °C–60 °C. 1H NMR (CD3OD, 400 MHz): d
2.42 (m, 2H), 3.75 (m, 2H), 3.95 (m, 1H), 4.42 (m, 1H), 6.02 (t,
6.40 Hz, 1H), 8.20 (s, 1H).13C NMR (CD3OD, 100 MHz): d 41.3,
61.3, 70.5, 88.0, 89.6, 94.5, 140.0. HRMS (ESI): calcd for
C8H11I2N2O3 [M+H]+ 436.8859, found 436.8856.

3014
O. R. Wauchope et al. / Bioorg. Med. Chem. 20 (2012) 3009–3015
HRMS (ESI): calcd for C25H29IN3O6 [M+H]+ 594.1101, found
594.1104.
4.1.1.13. 1-[(5-Aminoimidazo[40,50:4,5]thieno[3,2-d]pyrimidin-
3-yl-7-one)]-b- -2’-deoxyribofuranose (5). Intermediate 19
D
(360 mg, 0.638 mmol) was dissolved at 0 °C in CH2Cl2, followed
by the addition of BF3ꢁOEt2 (0.27 mL, 1.275 mmol) and EtSH
(0.96 mL, 1.275 mmol). The mixture was left to stir at rt overnight.
The solvent was removed under reduced pressure and H2O (10 mL)
was added. The aqueous layer was then extracted with CH2Cl2
(4 ꢃ 10 mL). After which, the aqueous layers were combined and
evaporated to give 5 as a colorless amorphous solid. Yield:
72 mg, 35%; mp 136–138 °C. 1H NMR (DMSO-d6) d 3.68 (s, 1H),
3.74 (dd, 2.4 Hz, 10.5 Hz. 1H), 3.76 (dd, 1.8 Hz, 10.5 Hz, 1H), 3.82
(dd, 1.9 Hz, 10.7 Hz, 1H), 4.08–4.16 (m, 2H), 4.37 (s, 1H), 6.32 (t,
4.5 Hz, 1H), 7.92 (s, 1H), 9.55 (s, 1H), 10.19 (s, 1H); 13C NMR
(DMSO-d6) d 61.2, 70.2, 76.1, 86.7, 89.5, 108.7, 131.6, 150.4,
151.7, 160.4. HRMS (FAB) m/z calcd for C12H14N5O4S [M+H]
324.0767, found 324.0764.
4.1.1.10. 3’-(p-Methoxybenzlyoxy)-5’-(p-methoxybenzyloxym-
ethyl)-1-[(5-iodo-4-carbonitrile)imidazol-3-yl]-b-
D-2’-deoxyri-
bofuranose (16). To oxime 15 (2.40 g, 4.05 mmol) dissolved
in dry THF (10 mL) was added 1, 10-carbonyldiimidazole (821 mg,
5.06 mmol) and the reaction refluxed overnight. The solvent was
removed in vacuo and the syrup purified via flash chromatography
on silica gel using hexane: EtOAc (3:2) to give the product 16 as a
thick oil. Yield: 1.40 g, 60%. 1H NMR (CDCl3, 400 MHz): d 2.63 (m,
2H), 3.61 (dd, 3.24 Hz, 10.56 Hz, 2H), 3.62 (s, 6H), 4.31 (m, 2H),
4.46–4.49 (m, 4H), 6.15 (t, 6.60 Hz, 1H), 6.85 (m, 4H), 7.16 (d,
8.72 Hz, 2H), 7.20 (d, 8.88 Hz, 2H), 8.02 (s, 1H); 13C NMR (CDCl3,
100 MHz) d 15.6, 65.9, 68.9, 69.4, 71.4, 72.2, 73.3, 78.5, 80.4,
81.9, 83.6, 92.1, 127.1, 128.1, 129.3, 129.5, 129.6, 163.9. HRMS
(ESI): calcd for C25H27IN3O5 [M+H]+ 576.0995, found 576.0991.
4.1.1.14. 3’-(p-Methoxybenzyloxy)-5’-(p-methoxybenzyloxym-
ethyl)-1-(imidazo[40,5v:4,5]thieno[3,2-d]pyrimidin-3-yl-7-one)-
4.1.1.11. 3’-(p-Methoxybenzlyoxy)-5’-(p-methoxybenzyloxym-
b-D
-2’-deoxyribofuranose (20).
A mixture of 18 (1.0 g,
ethyl)-1-[(5-carboxamide)[2,3-d]imidazol-3-yl]-b-
D-2’-deoxyri-
1.86 mmol), triethylorthoformate (40 mL), and 4 Å molecular
sieves (oven dried before use) was refluxed for 6 h. The reaction
mixture was filtered and the excess solvent was evaporated and
the residue purified by column chromatography eluting with ethyl
acetate/hexane (2:1) to afford 20 as a hygroscopic white foam.
Yield: 900 mg, 88%. 1H NMR (acetone-d6) d 2.64 (dd, 4.1 Hz,
10.5 Hz, 1H), 3.64 (dd, 5.0 Hz, 10.5 Hz, 1H), 3.70–3.71 (m, 6H),
4.30–4.31 (m, 2H), 4.36–4.38 (m, 2H), 4.46 (s, 2H), 4.52 (s, 2H),
6.58 (t, 4.5 Hz, 1H), 6.87–6.89 (m, 4H), 7.19–7.30 (m, 4H), 8.12 (s,
1H), 8.34 (s, 1H); 13C NMR (Acetone-d6) d 54.3, 70.1, 70.4, 72.5,
79.3, 84.3, 86.9, 113.7, 121.7, 129.3, 129.5, 130.5, 142.5, 143.1,
146.4, 159.1, 159.5. HRMS (ESI) m/z calcd for C28H29N4O6S [M+H]
549.1807, found 549.1804.
bofuranose (18). To a solution of 16 (1.12 g, 1.95 mmol)
dissolved in dry DMF (20 mL) was added potassium carbonate
(352 mg, 2.53 mmol) and thioglycolamide (359 mg, 3.9 mmol)
and heated at 60 °C for 19 h. The solvent was removed under re-
duced pressure. The reaction mixture was filtered over celite and
the celite pad washed with MeOH (100 mL). The solvent was re-
moved under reduced pressure to give a thick syrup 17 which
was used directly in the next step without further purification or
characterization.
To the syrup (1.05 g, 1.95 mmol) dissolved in dry EtOH (30 mL)
was added NaOEt (95%, 279 mg, 3.9 mmol) and refluxed for 6 to
9 h. The organic layer was concentrated under reduced pressure.
The product was isolated as thick oil by flash chromatography on
silica gel eluting with EtOAc to give 18 as a syrup. Yield: 650 mg,
62%.1H NMR (CDCl3, 400 MHz): d 2.38 (m, 1H), 3.66 (m, 1H), 3.74
(m, 1H), 3.82 (s, 6H), 4.26 (m, 1H), 4.46–4.47 (m, 1H), 4.50 (s,
1H), 4.45–4.61 (m, 4H), 6.16 (s, 2H), 6.23 (t, 6.44 Hz, 1H), 6.60–
6.72 (s, 2H), 7.31–7.36 (m, 8H), 8.04 (s, 1H); 13C NMR (CDCl3,
100 MHz) d 15.5, 39.9, 55.3, 68.3, 71.7, 73.3, 84.1, 86.2, 98.9,
113.8, 114.0, 114.1, 129.0, 129.4, 129.6, 129.8, 139.7, 145.6,
159.6, 168.5. HRMS (ESI): calcd for C27H30N4O6S [M]+ 538.1886,
found 538.1889.
4.1.1.15. 3’-(p-Methoxybenzyloxy)-5’-(p-methoxybenzyloxym-
ethyl)-1-[(7-aminoimidazo[40,50:4,5]thieno[3,2-d]pyrimidin-3-
yl)]-b-
D
-20-deoxyribofuranose (21).
To a mixture of 20
(900 mg, 1.64 mmol), DMAP (800 mg, 6.56 mmol), TEA (15 ml) in
MeCN (50 ml) was added TPSCl (1.99 g, 6.56 mmol) portion wise
over 10 min. The mixture was then left to stir overnight. After
TLC analysis confirmed the disappearance of the starting material,
the solvent was removed under reduced pressure and THF (55 ml)
added. The mixture was transferred to a bomb and cooled to
ꢄ78 °C and ammonia was bubbled in for 10 min. The bomb was
sealed and then left to stir overnight at rt. After which, the solvent
was removed and the product was purified to give 21 as a yellow
foam. Yield: 584 mg, 65%. 1H NMR (CDCl3) d 2.87 (m, 1H), 3.73
(m, 1H), 3.75–3.76 (m, 6H), 3.96 (m, 1H), 4.29–4.30 (m, 1H),
4.56–4.60 (m, 4H), 4.62–4.89 (m, 2H), 6.00 (s, 2H), 6.34 (t, 4.5 Hz,
1H), 7.10–7.11 (m, 4H), 7.30–7.34 (m, 4H), 8.20 (s, 1H), 8.39 (m,
1H); 13C NMR (CDCl3) d 14.3, 21.4, 29.8, 53.6, 60.5, 72.4, 73.6,
82.1, 88.9, 115.6, 127.9, 128.0, 128.6, 137.2, 137.5, 143.1, 144.4,
151.5, 156.4, 161.5, 171.2. HRMS (ESI) m/z calcd for C28H30N5O5S
[M+H] 548.1967, found 548.1969.
4.1.1.12. 3’-(p-Methoxybenzyloxy)-5’-(p-methoxybenzyloxym-
ethyl)-1-[(5-aminoimidazo[40,50:4,5]thieno-[2,3-d]pyrimidin-3-
yl-7-one)]-b-
D-2’-deoxyribofuranose (19).
Intermediate 18
(650 mg, 1.21 mmol) and ethylxanthic acid potassium salt
(388 mg, 2.42 mmol) were dissolved in DMF (25 mL) and the mix-
ture was refluxed for 4 h. The solvent was removed under reduced
pressure to give a thick syrup. The syrup was then dissolved in a
minimal amount of THF and cooled to 0 °C followed by the drop-
wise addition of H2O2 (8 mL). The mixture was then stirred at
0 °C for 2 h. The resulting mixture was then transferred to a steel
bomb and cooled down to ꢄ78 °C and NH3 was bubbled in for
15 min. The bomb was sealed and heated at 120–130 °C for 18 h.
The solvent was removed under reduced pressure and the product
19 isolated as a colorless oil. Yield: 360 mg, 53 %. 1H NMR (DMSO-
d6, 400 MHz): d 2.38 (m, 2H), 3.50 (m, 1H), 3.60 (m, 1H), 3.70–3.71
(m, 6H), 4.31 (m, 1H), 4.38 (s, 2H), 4.45 (s, 2H), 6.44 (t, 6.44 Hz, 1H),
6.52 (s, 2H), 6.82–6.89 (m, 4H), 7.17–7.18 (m, 2H), 7.26–7.28 (m,
2H), 8.33 (s, 1H), 11.05 (s, 1H); 13C NMR (DMSO-d6, 100 MHz) d
39.9, 55.3, 68.3, 71.9, 73.6, 84.2, 87.0, 99.0, 113.6, 114.2,
114.5, 129.2, 129.6, 129.8, 130.0, 139.8, 146.2, 150.1, 159.4,
159.6. HRMS (ESI): calcd for C28H30N5O6S [M+H] 564.1917, found
564.1915.
4.1.1.16. Synthesis of 1-[(7-aminoimidazo[40,50:4,5]thieno[3,2-
d]pyrimidin-3-yl)]-b-
D
-20-deoxyribofuranose (6).
Interme-
diate 21 (430 mg, 0.785 mmol) was dissolved at 0 °C in CH2Cl2, fol-
lowed by the addition of BF3ꢁOEt2 (1.25 mL, 5.903 mmol) and EtSH
(6.31 mL, 8.38 mmol). The mixture was left to stir at rt for 48 h. The
solvent was removed under reduced pressure and H2O (10 mL) was
added. The aqueous layer was then extracted with CH2Cl2
(4 ꢃ 10 mL). After which, the aqueous layers were combined and
evaporated to give 6 as a colorless amorphous solid. Yield:
72 mg, 30%; mp 148–151.4 °C. 1H NMR (DMSO-d6) d 3.52 (m,
1H), 3.62 (m, 1H), 3.93 (m, 1H), 4.11 (m, 1H), 4.58 (m, 1H), 5.16

O. R. Wauchope et al. / Bioorg. Med. Chem. 20 (2012) 3009–3015
3015
(d, 1H), 5.41–5.43 (m, 2H), 5.85 (t, 4.5 Hz, 1H), 7.32 (s, 2H), 8.09 (s,
Acknowledgment
1H), 8.31 (s, 1H); 13C NMR (DMSO-d6) d 21.3, 61.7, 70.6, 86.2, 112.1,
129.4, 135.7, 145.3, 155.1, 158.8, 162.5. HRMS (ESI) m/z calcd for
This research was supported by the National Institutes of
Health (R01 GM073645, KRS) and the K. U. Leuven (GOA 10/14).
The authors are grateful for this support. The authors would also
like to thank Phil Mortimer at Johns Hopkins University for his gen-
erous assistance with the HRMS, and Leentje Persoons, Frieda De
Meyer, Lies Van den Heurck, Steven Carmans, Anita Camps, Leen
Ingels and Lizette van Berckelaer for excellent technical assistance.
C
12H14N5O3S [M+H] 308.0817, found 308.0815.
4.2. Antiviral assays
The antiviral assays, other than the anti-HIV assays, were based
on inhibition of virus-induced cytopathicity in HEL [herpes simplex
virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus, vesicular sto-
matitis virus, cytomegalovirus (HCMV) and varicella-zoster virus
(VZV)], Vero (parainfluenza-3, reovirus-1, Sindbis and Coxsackie
B4), HeLa (vesicular stomatitis virus, Coxsackie virus B4, and respi-
ratory syncytial virus) or MDCK [influenza A (H1N1; H3N2) and
influenza B] cell cultures. Confluent cell cultures (or nearly conflu-
ent for MDCK cells) in microtiter 96-well plates were inoculated
with 100 CCID50 of virus (1 CCID50 being the virus dose to infect
50% of the cell cultures) in the presence of varying concentrations
(fivefold compound dilutions) of the test compounds. Viral cyto-
pathicity was recorded as soon as it reached completion in the con-
trol virus-infected cell cultures that were not treated with the test
compounds. The minimal cytotoxic concentration (MCC) of the
compounds was defined as the compound concentration that
caused a microscopically visible alteration of cell morphology.
The methodology of the anti-HIV assays was as follows: human
CEM (ꢀ3 ꢃ 105 cells/cm3) cells were infected with 100 CCID50 of
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.03.004.
References and notes
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4.3. Cytostatic assays
Murine leukemia L1210, human T-lymphocyte CEM, human
cervix carcinoma (HeLa) and human embryonic lung fibroblast
(HEL) cells were suspended at 300,000–500,000 cells/mL of culture
medium, and 100 lL of a cell suspension was added to 100 lL of an
appropriate dilution of the test compounds in wells of 96-well
microtiter plates. After incubation at 37 °C for two (L1210) or three
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