6-N-acyltriciribine analogues: Structure - activity relationship between acyl carbon chain length and activity against HIV-1

Article abstract of DOI:10.1021/jm990236h

Triciribine (TCN) and triciribine-5'-monophosphate (TCN-P) are active against HIV-1 at submicromolar concentrations. In an effort to improve and better understand this activity, we have conducted a structure - activity relationship study to explore the to

Full text of DOI:10.1021/jm990236h

J . Med. Chem. 2000, 43, 2457-2463
2457
6-N-Acyltr icir ibin e An a logu es: Str u ctu r e-Activity Rela tion sh ip betw een Acyl
Ca r bon Ch a in Len gth a n d Activity a ga in st HIV-1
Anthony R. Porcari,† Roger G. Ptak,‡ Katherine Z. Borysko, J ulie M. Breitenbach, J ohn C. Drach, and
Leroy B. Townsend*
Department of Medicinal Chemistry, College of Pharmacy, Department of Chemistry, College of Literature, Sciences and Arts,
Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109-1065
Received May 13, 1999
Triciribine (TCN) and triciribine-5′-monophosphate (TCN-P) are active against HIV-1 at
submicromolar concentrations. In an effort to improve and better understand this activity, we
have conducted a structure-activity relationship study to explore the tolerance of TCN to
structural modifications at the 6-position. A number of 6-N-acyltriciribine analogues were
synthesized and evaluated for antiviral activity and cytotoxicity. The cytotoxicity of these
compounds was minimal in three human cell lines (KB, CEM-SS cells, and human foreskin
fibroblasts (HFF)). The compounds were marginally active or inactive against herpes simplex
virus type 1 (HSV-1) and human cytomegalovirus (HCMV). In contrast, most of the compounds
exhibited moderate to high activity against human immunodeficiency virus type 1 (HIV-1),
IC50’s ) 0.03 to 1 µM. This structure-activity relationship study identified the N-heptanoyl
group as having the optimal carbon chain length. This compound was as active against HIV-1
as TCN and TCN-P. Reverse phase HPLC of extracts from uninfected cells treated with 6-N-
acyltriciribines detected sufficient TCN-P to account for anti-HIV activity thereby suggesting
a prodrug effect. Studies in an adenosine kinase deficient cell line showed that the 6-N-acyl
derivative was not phosphorylated directly but first was metabolized to triciribine which then
was converted to TCN-P.
In tr od u ction
propagate HIV and human cytomegalovirus (HCMV).10
Even though TCN was not very cytotoxic in these cell
lines, it must be phosphorylated to TCN-P to be active
against HIV-1.12 The antiviral mechanism of action of
TCN has yet to be elucidated fully, but it involves viral
accessory proteins.13
The tricyclic nucleoside triciribine (TCN) was syn-
thesized by Schram and Townsend in 1971.1 Early
studies revealed that TCN is converted intracellularly
to triciribine-5′-monophosphate (TCN-P) by adenosine
kinase.2-4 Unlike other nucleoside analogues, TCN is
metabolized only to the monophosphate and not to the
di- or triphosphate forms.5 TCN is also not incorporated
into nucleic acids.6 Although the exact mechanism by
which it inhibits the growth of certain cell lines is
unknown, TCN-P inhibits DNA and protein synthesis
in these cells.6,7 Due to poor water solubility and
difficulty in formulating TCN for clinical use, triciribine-
5′-monophosphate (TCN-P) was synthesized8 as a water
soluble prodrug of TCN.9
More recently, we have found that TCN and TCN-P
are selective and potent inhibitors of HIV-1 and HIV-2
in acutely and persistently infected cells.10 These studies
also found no cross resistance to TCN or TCN-P in AZT-
or TIBO-resistant HIV strains,10 suggesting that TCN
and TCN-P have an entirely different mode of action
than AZT and TIBO. We also found that triciribine has
no activity against the HIV encoded enzymes, reverse
transcriptase, RNase H, integrase, and protease.11
Furthermore, cytotoxicity such as that observed in
murine L1210 cells appears to be highly cell line specific
and was not observed in human cell lines used to
As part of our ongoing research with triciri-
bine,10-12,14-19 we have been conducting systematic
structure-activity relationship studies to determine the
tolerance of triciribine to structural changes and how
they affect phosphorylation and activity against HIV-
1.18,19 The current study was designed to explore the
effect that acylation of the 6-amino group would have
on activity against HIV-1. The results uncovered an
interesting structure-activity relationship indicating
that these compounds act as prodrugs of TCN. Herein,
we describe the synthesis and structure-activity rela-
tionship of these 6-N-acyltriciribine analogues.
Resu lts a n d Discu ssion
All 6-N-acyltriciribine analogues were synthesized
from triciribine, as illustrated in Scheme 1. Selective
acetylation of the hydroxyl groups to give 6-amino-4-
methyl-8-(2,3,5-tri-O-acetyl-â-D-ribofuranosyl)pyrrolo-
[4,3,2-de]pyrimido[4,5-c]pyridazine (1) was accomplished
with 3.3 equiv of acetic anhydride in dry pyridine at
room temperature for 4 h. Once the carbohydrate moiety
of TCN was acetylated, the 6-amino group was acylated
with the desired acyl chloride to give intermediates 2a -
2m . Subsequent deprotection with methanolic ammonia
afforded the 6-N-acyltriciribine analogues (3a -3m ),
where compounds 3a -3j have straight chain alkyl acyl
* To whom correspondence should be addressed. Tel: 734-764-7547.
Fax: 734-763-5633. E-mail: ltownsen@umich.edu.
† Present Address: Parke-Davis Pharmaceutical Research, 2800
Plymouth Rd., Ann Arbor, MI 48105.
‡ Present Address: Southern Research Institute, 431 Aviation Way,
Frederick, MD 21701.
10.1021/jm990236h CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/16/2000

2458 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Sch em e 1. Synthesis of 6-N-Acyltriciribinea
Porcari et al.
a
Reagents: (i) Ac2O, pyridine; (ii) acyl chlorides; (iii) NH3, MeOH.
Ta ble 1. Antiviral Activity and Cytotoxicity of 6-N-Acyltriciribine Analogues
50% inhibitory concentration (µM)
antiviral activitya
cytotoxicityb
HIV-1
RT
HSV-1
ELISA
HCMV
plaque
KB
growth
HFF
visual
CEM-SS
visual
compound
R
TCNc
TCN-Pc
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
0.04
0.04
6.8d
8.4d
0.50d
0.80
0.35
0.035
0.1
23
20
2.5
0.8
>100
>100
10
>100
>100
100
30
100
19
>100
>1.0
>100d
>100d
>100d
32
CH3
CH2CH3
>100
>100
>100
>100
>100
>100
>100
>100
>100
45
>100
100
100
>100
100
32
40
10
0.45
8.8
11
12
12
12
12
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)6CH3
(CH2)7CH3
(CH2)8CH3
(CH2)10CH3
CH(CH3)2
C(CH3)3
40
30
40
65
40
75
45
32
32
32
32
32
21
21
21
21
0.8
0.25
1
8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
phenyl
a
Antiviral activity was determined using the amount of reverse transcriptase (RT) activity in culture supernatants in triplicate for
b
HIV-1, an ELISA assay in quadruplicate for HSV-1, and a plaque assay in duplicate for HCMV as described in the text. Inhibition of
KB cell growth was measured as described in the text in quadruplicate assays. Visual cytotoxicity was scored on uninfected HFF and
CEM-SS cells not affected by virus in HCMV plaque and HIV RT assays. c Averages of replicate experiments as published previously in
d
ref 10. Average IC50 from two experiments.
groups, from 2 to 12 carbons, compounds 3k and 3l have
branched chain alkyl acyl groups, and compound 3m
has an aromatic acyl group. Each compound was evalu-
ated for activity against three viruses (HIV-1, HSV-1,
and HCMV) and for cytotoxicity in three human cell
lines (KB, HFF, and CEM-SS cells) (Table 1).
optimum carbon chain length for activity against HIV-
1. Interestingly, the IC50 of compound 3f was the same
as that of TCN and TCN-P, suggesting that compound
3f is either inherently as active as TCN and TCN-P
against HIV-1 or that compound 3f is a prodrug of TCN
or TCN-P.
TCN, TCN-P, and compounds 3a -3m were tested for
activity against HIV-1 in an assay that employed
reverse transcriptase (RT) in culture supernatants as
a marker for the virus. Table 1 presents the results and
shows that all compounds except 3l and 3m were active
against HIV and that there was a relationship between
the 6-N-acyl chain length and activity against HIV. A
comparison of the activity of the analogues to the
activity of TCN clearly revealed that increasing the
carbon chain length of the acyl group increased activty
until the chain length reached seven carbons (3f)
(Figure 1). This established that compound 3f, with a
heptanoyl group (seven-carbon straight chain alkyl acyl
group) on the 6-amino group of triciribine, had the
The 6-N-acyltriciribine analogues also were evaluated
against HSV-1 in an ELISA and HCMV in a plaque
reduction assay. For each compound, no inhibition of
HSV-1 was observed or it was found that inhibition of
HSV-1 occurred at concentrations that were cytotoxic
(Table 1). Compounds 3a , 3b, 3k , 3l, and 3m also were
weakly active or inactive against HCMV, even at
concentrations as high as 100 µM (Table 1). In contrast,
compounds 3c-3j had activity against HCMV (IC50
)
0.45-12 µM) similar to that of TCN and TCN-P (IC50
) 2.5 and 0.8 µM, respectively) (Table 1).
Cytotoxicity of all the 6-N-acyltriciribine analogues
was determined in three human cell lines (KB, HFF,
and CEM-SS cells). Compounds 3a , 3b, 3c, 3k , 3l, and

6-N-Acyltriciribine Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2459
F igu r e 1. Relationship between number of carbon atoms in N-acyl group of TCN analogues and % of TCN activity against HIV.
Activity data are plotted as percentage of IC50 of analogue compared to IC50 of TCN as positive control in the same experiment.
Ta ble 2. Intracellular Phosphorylation of TCN and
6-N-Acyltriciribine Analogues in Uninfected Cells
either TCN or 3f were incubated with CEM-SS cells.
In fact, higher concentrations of TCN-P were detected
extracellular
concentration
(µM)
intracellular
monophosphate
concentration (µM)a
in cultures incubated with 3f (Table 2), suggesting that
3f entered cells more readily than TCN. In contrast,
when the two compounds were incubated with AA-2
cells in which TCN is inactive against HIV-1,12 the
amount of TCN-P found in cell extracts was only about
3 to 6% of that found in CEM-SS cells, indicating that
phosphorylation of each compound was required for
activity. No phosphorylated product of 3f, per se, was
detected by either ultraviolet absorption or mass spec-
trometry of the HPLC effluent in any experiment,
strongly indicating that 3f was deacylated to TCN which
then was phosphorylated to TCN-P.
incubation
time (h)
compound
TCN
CEM-SS Cellsb
10
100
100
100
10
5
5
5
24
24
5
286
323c
318d
710
3d
3f
3f
3h
225
100
10
850d
243
24
AA-2 Cellsb
TCN
3f
100
100
5
5
9d
48d
On this basis, it appears that these compounds act
as prodrugs of TCN. This observation is important for
devising a new strategy for the delivery of TCN. We
envision that these compounds could be formulated in
an oil-based vehicle and delivered orally, as a hydro-
phobic prodrug, instead of intravenously, as the hydro-
philic prodrug TCN-P. Further studies are needed to
confirm this hypothesis. Regardless, this structure-
activity relationship has identified the heptanoyl group
as having the optimal carbon chain length needed to
obtain activity against HIV-1 equal to TCN and TCN-
P.
a
Nucleotide extractions and quantitation by HPLC was per-
b
formed as described in the text. Lymphoblastoid cells used for
propagation of HIV-1. AA-2 cells have approximately 1-3% of
adenosine kinase and deoxycytidine kinase of CEM-SS cells.
c Control data for TCN phosphorylation previously published in
ref 12. Average from two experiments including experiments in
which HPLC peaks were analyzed by mass spectrometry.
d
3m were not cytotoxic at concentrations as high as 100
µM (Table 1), and compounds 3d -3j showed marginal
to minimal toxicity to each cell line (IC50 ) 21-100 µM)
(Table 1), thereby establishing that the activity against
HIV was not a manifestation of cytotoxicity.
To determine whether these compounds were inher-
ently active or a prodrug of TCN, compounds 3d , 3f, and
3h were incubated with CEM-SS cells for 24 h. Intra-
cellular nucleotides were extracted, separated, and
quantitated by reverse phase HPLC. A peak in the
HPLC chromatograph coinciding with TCN-P was ob-
served in each case, strongly indicating that compounds
3d , 3f, and 3h were metabolized to TCN-P (Table 2).
The amount of intracellular TCN-P detected was suf-
ficient to account for activity against HIV.
To explore the possibility that the 6-N-acyl derivatives
were acting as prodrugs, TCN and 3f were incubated
with uninfected CEM-SS cells and AA-2 cells-a cell line
with 1-3% wild-type adenosine kinase (AK) and deoxy-
cytidine kinase (dCK) activity. Analysis of cell extracts
by HPLC/mass spectrometry revealed the presence of
high concentrations of TCN-P in CEM-SS cells when
Exp er im en ta l Section
Gen er a l P r oced u r es. Reaction mixtures were evaporated
at temperatures less than 60 °C under reduced pressure (water
aspirator) using a Buchi R-151 rotary evaporator. Melting
points (uncorrected) were obtained on a Laboratory Devices
Mel-Temp melting point apparatus. Thin-layer chromatogra-
phy used Analtech GHLF SiO2 prescored plates. TLC plates
were developed using a chloroform:methanol (9:1) mixture and
visualized under ultraviolet light (254 nm). E. Merck silica gel
(230-400 mesh) was used for gravity or flash column chro-
matography. Proton nuclear magnetic resonance (1H NMR)
spectra were obtained with a Brucker Avance DPX 300 or DRX
500 spectrometer (solutions in CDCl3 or DMSO-d6) with the
chemical shifts reported in parts per million (ppm) downfield
from tetramethylsilane as the internal standard. UV spectra
were obtained with
a Kontron UVIKON 860 ultraviolet
spectrometer. Elemental analysis were performed by the
Analytical Laboratory, Department of Chemistry, University
of Michigan, Ann Arbor, MI.

2460 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Porcari et al.
6-Am in o-4-m eth yl-8-(2,3,5-tr i-O-a cetyl-â-D-r ibofu r a n o-
syl)p yr r olo[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (1). Acetic
anhydride (0.34 g, 0.31 mL, 3.3 mmol) was added to a solution
of triciribine (320 mg, 1 mmol) and dry pyridine (10 mL), and
the mixture was stirred for 4 h at room temperature. The
solvent was removed under vacuum at 60 °C, and the oily
residue was coevaporated with toluene (2 × 20 mL) and
ethanol (2 × 20 mL). The resulting brown oil was eluted from
a silica gel column (2.5 cm × 12 cm) using a solution of
methanol:chloroform (1:99). Fractions of 10 mL were collected,
and the UV containing fractions (TLC, 1:9 methanol:chloro-
form) with Rf values of 0.62 were combined and evaporated to
give a red oil. (It was observed that the fractions containing
the product eluted from the column as a clear and colorless
solution, but became light red in color upon exposure to air).
Yield ) 0.39 g (87%); Rf ) 0.62 (TLC, 1:9 methanol:chloroform);
1H NMR (CDCl3) δ 8.24 (1H, s, H-2), 6.70 (1H, s, H-7), 6.21
(1H, d, H-1′), 5.77 (1H, t), 5.56 (1H, dd), 4.45 (2H, bs, NH2),
4.40 (3H, m), 3.51 (3H, s), 2.12 (3H, s), 2.10 (3H, s), 2.04 (3H,
s).
Gen er a l P r oced u r es for th e Syn th esis of 6-N-Acyl-
tr icir ibin e An a logu es. Meth od 1. The acyl chloride (5 mmol)
was added to a solution of compound 1 (0.22 g, 0.5 mmol) in
dry pyridine (40 mL) and stirred for 2 h at room temperature.
The reaction mixture was diluted with ethyl acetate (150 mL),
extracted with 3 N HCl (3 × 125 mL), a saturated solution of
aqueous sodium bicarbonate (2 × 125 mL), and brine (125 mL),
and dried over magnesium sulfate. The magnesium sulfate was
removed by filtration, and the solvent was removed under
vacuum. The residue, presumed to be intermediate 2, was
dissolved in methanol (20 mL) and transferred to a glass
pressure bottle. Methanolic ammonia (100 mL), saturated at
0 °C, was added, and the sealed reaction vessel was stirred
for 18 h at room temperature. The reaction vessel was cooled
to 0 °C and then opened, and the solvent was evaporated to
dryness. The residue was adsorbed onto silica gel and eluted
from a silica gel column (2.5 cm (d) × 12 cm (h)) using a solvent
system of 1:19 methanol:chloroform. Fractions of 10 mL were
collected, and the UV containing fractions (TLC, 1:9 methanol:
chloroform) with the same Rf values were combined and
evaporated. The yellow residue was recrystallized from a hot
mixture of ethyl acetate:hexane. The solid was collected and
dried under vacuum (water aspirator) for 18 h at 80 °C.
Meth od 2. The acyl chloride (3.2 mmol) was added to a
solution of compound 1 (0.73 g, 1.6 mmol) in dry pyridine (10
mL) and stirred for 18 h at room temperature. The solvent
was removed under vacuum, and the residue was coevaporated
with toluene (2 × 20 mL). The residue, presumed to be
intermediate 2, was dissolved in methanol (20 mL) and
transferred to a glass pressure bottle. Methanolysis and
workup as described for method 1 above was followed except
for a different column size. The resulting yellow residue was
either heated at reflux temperature in dichloromethane for 1
h or recrystallized or both. The solid was collected and dried
under vacuum (water aspirator) for 18 h at 80 °C.
6-Acet a m id o-4-m et h yl-8-(â-D-r ib ofu r a n osyl)p yr r olo-
[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3a ). Compound 3a
was synthesized by method 2 using acetyl chloride (0.25 g, 0.23
mL, 3.2 mmol). The column-purified product was recrystallized
from acetone. Yield ) 0.47 g (81%); Rf ) 0.21; mp 200-202
°C; UV [λmax (ꢀ)] (pH 1) 286 (11643), 278 (10906), 238 (17989),
(pH 7) 287 (12577), (pH 11) 285 (15354); 1H NMR (DMSO-d6)
δ 10.47 (1H, bs, NH), 8.10 (1H, s, H-2), 7.20 (1H, s, H-7), 5.84
(1H, d, H-1′), 5.40 (2H, m, 2 × OH), 5.20 (1H, d, OH), 4.48
(1H, q, H-4′), 4.10 (1H, m), 3.95 (1H, m), 3.56 (2H, m, H-5′),
3.35 (3H, s, NCH3), 2.07 (3H, s, CH3). Anal. (C15H18N6O5) C,
H, N.
m), 3.55 (2H, m, H-5′), 3.43 (3H, s, NCH3), 2.35 (2H, q, CH2),
1.06 (3H, t, CH3). Anal. (C16H20N6O5) C, H, N.
6-Bu tyr ylam ido-4-m eth yl-8-(â-D-r ibofu r an osyl)pyr r olo-
[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3c). Compound 3c
was synthesized by method 1 using butyryl chloride (0.53 g,
0.52 mL, 5 mmol). Yield ) 0.10 g (54%); Rf ) 0.35; mp 145 °C
dec; 1H NMR (DMSO-d6) δ 10.41 (1H, bs, NH), 8.09 (1H, s,
H-2), 7.21 (1H, s, H-7), 5.83 (1H, d, H-1′), 5.40 (2H, m, 2 ×
OH), 5.20 (1H, d, OH), 4.49 (1H, m), 4.08 (1H, m), 3.95 (1H,
m), 3.55 (2H, m, H-5′), 3.43 (3H, s, NCH3), 2.35 (2H, t, CH2),
1.60 (2H, m, CH2), 0.93 (3H, t, CH3). Anal. (C17H22N6O5) C, H,
N.
4-Meth yl-6-p en ta n oyla m id o-8-(â-D-r ibofu r a n osyl)p yr -
r olo[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3d ). Compound
3d was synthesized by method 2 using valeryl chloride (0.39
g, 0.38 mL, 3.2 mmol). The column purified product was
recrystallized from acetone. Yield ) 0.21 g (33%); Rf ) 0.39;
mp 236-238 °C; 1H NMR (DMSO-d6) δ 10.43 (1H, bs, NH),
8.09 (1H, s, H-2), 7.19 (1H, s, H-7), 5.83 (1H, d, H-1′), 5.40
(2H, m, 2 × OH), 5.20 (1H, d, OH), 4.49 (1H, m), 4.08 (1H, m),
3.95 (1H, m), 3.55 (2H, m, H-5′), 3.43 (3H, s, NCH3), 2.35 (2H,
t, CH2), 1.57 (2H, m, CH2), 1.27 (2H, m, CH2), 0.85 (3H, t, CH3).
Anal. (C18H24N6O5) C, H, N.
6-Hexa n oyla m id o-4-m et h yl-8-(â-D-r ibofu r a n osyl)p yr -
r olo[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3e). Compound
3e was synthesized by method 2 using hexanoyl chloride (0.43
g, 0.45 mL, 3.2 mmol). The column purified product was heated
in dichloromethane, at reflux temperature, for 1 h. Yield )
0.24 g (36%); Rf ) 0.40; mp 216-218 °C; 1H NMR (DMSO-d6)
δ 10.43 (1H, bs, NH), 8.09 (1H, s, H-2), 7.19 (1H, s, H-7), 5.83
(1H, d, H-1′), 5.40 (2H, m, 2 × OH), 5.20 (1H, d, OH), 4.49
(1H, m), 4.08 (1H, m), 3.95 (1H, m), 3.55 (2H, m, H-5′), 3.43
(3H, s, NCH3), 2.35 (2H, t, CH2), 1.57 (2H, m, CH2), 1.27 (4H,
m, 2 × CH2), 0.85 (3H, t, CH3). Anal. (C19H26N6O5) C, H, N.
6-Hep ta n oyla m id o-4-m eth yl-8-(â-D-r ibofu r a n osyl)p yr -
r olo[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3f). Compound 3f
was synthesized by method 2 using heptanoyl chloride (0.48
g, 0.5 mL, 3.2 mmol). The column purified product was heated
in dichloromethane, at reflux temperature, for 1 h. Yield )
0.27 g (39%); Rf ) 0.44; mp 164-166 °C; 1H NMR (DMSO-d6)
δ 10.43 (1H, bs, NH), 8.09 (1H, s, H-2), 7.19 (1H, s, H-7), 5.83
(1H, d, H-1′), 5.40 (2H, m, 2 × OH), 5.20 (1H, d, OH), 4.49
(1H, m), 4.08 (1H, m), 3.95 (1H, m), 3.55 (2H, m, H-5′), 3.43
(3H, s, NCH3), 2.35 (2H, t, CH2), 1.57 (2H, m, CH2), 1.27 (6H,
m, 3 × CH2), 0.85 (3H, t, CH3). Anal. (C20H28N6O5) C, H, N.
4-Meth yl-6-octan oylam ido-8-(â-D-r ibofu r an osyl)pyr r olo-
[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3g). Compound 3g
was synthesized by method 2 using octanoyl chloride (0.52 g,
0.55 mL, 3.2 mmol). The column purified product was heated
in dichloromethane, at reflux temperature, for 1 h. Yield )
0.16 g (23%); Rf ) 0.48; mp 170-172 °C; 1H NMR (DMSO-d6)
δ 10.43 (1H, bs, NH), 8.09 (1H, s, H-2), 7.19 (1H, s, H-7), 5.83
(1H, d, H-1′), 5.40 (2H, m, 2 × OH), 5.20 (1H, d, OH), 4.49
(1H, m), 4.08 (1H, m), 3.95 (1H, m), 3.55 (2H, m, H-5′), 3.43
(3H, s, NCH3), 2.35 (2H, t, CH2), 1.57 (2H, m, CH2), 1.27 (8H,
m, 4 × CH2), 0.85 (3H, t, CH3). Anal. (C21H30N6O5) C, H, N.
4-Met h yl-6-n on a n oyla m id o-8-(â-D-r ib ofu r a n osyl)p yr -
r olo[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3h ). Compound
3h was synthesized by method 2 using nonanoyl chloride (0.57
g, 0.58 mL, 3.2 mmol). The column purified product was
recrystallized from hot acetone. Yield ) 0.10 g (14%); Rf ) 0.52;
mp 170-172 °C; 1H NMR (DMSO-d6) δ 10.43 (1H, bs, NH),
8.09 (1H, s, H-2), 7.19 (1H, s, H-7), 5.83 (1H, d, H-1′), 5.40
(2H, m, 2 × OH), 5.20 (1H, d, OH), 4.49 (1H, m), 4.08 (1H, m),
3.95 (1H, m), 3.55 (2H, m, H-5′), 3.43 (3H, s, NCH3), 2.35 (2H,
t, CH2), 1.57 (2H, m, CH2), 1.27 (10H, m, 5 × CH2), 0.85 (3H,
t, CH3). Anal. (C22H32N6O5) C, H, N.
4-Meth yl-6-p r op ion yla m id o-8-(â-D-r ibofu r a n osyl)p yr -
r olo[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3b). Compound
3b was synthesized by method 1 using propionyl chloride (0.46
g, 0.43 mL, 5 mmol). Yield ) 0.10 g (55%); Rf ) 0.30; mp 235
°C dec; 1H NMR (DMSO-d6) δ 10.41 (1H, bs, NH), 8.09 (1H, s,
H-2), 7.21 (1H, s, H-7), 5.83 (1H, d, H-1′), 5.40 (2H, m, 2 ×
OH), 5.20 (1H, d, OH), 4.49 (1H, m), 4.08 (1H, m), 3.95 (1H,
6-Deca n oyla m id o-4-m et h yl-8-(â-D-r ib ofu r a n osyl)p yr -
r olo[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3i). Compound 3i
was synthesized by method 2 using decanoyl chloride (0.61 g,
0.66 mL, 3.2 mmol). The column purified product was recrys-
tallized from hot acetone. Yield ) 0.22 g (29%); Rf ) 0.55; mp
1
171-173 °C; H NMR (DMSO-d6) δ 10.43 (1H, bs, NH), 8.09
(1H, s, H-2), 7.19 (1H, s, H-7), 5.83 (1H, d, H-1′), 5.40 (2H, m,

6-N-Acyltriciribine Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2461
2 × OH), 5.20 (1H, d, OH), 4.49 (1H, m), 4.08 (1H, m), 3.95
(1H, m), 3.55 (2H, m, H-5′), 3.43 (3H, s, NCH3), 2.35 (2H, t,
CH2), 1.57 (2H, m, CH2), 1.27 (12H, m, 6 × CH2), 0.85 (3H, t,
CH3). Anal. (C23H34N6O5) C, H, N.
virus adsorption and then diluted to 5 × 105 cells/mL with
RPMI 1640 containing 10% fetal bovine serum. One-tenth
milliliter was then added to each well of a 96-well cluster dish
which had been pretreated with poly-L-lysine. Fresh medium
(0.1 mL with 10% fetal bovine serum) containing test com-
pounds in twice the desired final concentration was added to
triplicate wells at seven concentrations ranging from 100 µM
to 0.14 µM. After 6 days incubation, supernatant samples were
taken, and the amount of RT activity was measured by the
incorporation of [3H]dTTP into acid insoluble material using
the assay described by White et al.25
HCMV P la qu e Assa y. The Towne strain, plaque-purified
isolate Po, of HCMV was kindly provided by Dr. Mark Stinski,
University of Iowa. HFF cells in 24-well cluster dishes were
infected with approximately 100 plaque forming units (pfu)
of HCMV per well using the procedures detailed earlier.20
Following virus adsorption, compounds dissolved in MEM(E)
containing 0.5% methyl cellulose and 5% fetal calf serum were
added to duplicate wells in four to eight selected concentra-
tions. After incubation at 37 °C for 8-10 days, cell sheets were
fixed and stained with crystal violet, and microscopic plaques
were enumerated. Drug effects were calculated as a percentage
of reduction in number of plaques in the presence of each drug
concentration compared to the number observed in the absence
of drug.
HSV-1 ELISA. An enzyme-linked immunosorbent assay
(ELISA)26 was employed to detect HSV-1. Briefly, 96-well
cluster dishes were planted with 10 000 BSC-1 cells per well
in 200 µL per well of MEM(E) plus 10% calf serum. After
overnight incubation at 37 °C, selected drug concentrations
in triplicate and HSV-1 (KOS strain kindly provided by Dr.
Sandra K. Weller, University of Connecticut) at a concentra-
tion of 100 pfu/well were added. Following a 3 day incubation
at 37 °C, medium was removed, plates were fixed, blocked,
and rinsed, and horseradish peroxidase conjugated rabbit anti-
HSV-1 antibody was added. Following removal of the antibody
containing solution, plates were rinsed and then developed by
adding a solution of tetramethylbenzidine as substrate. The
reaction was stopped with H2SO4, and absorbance was read
at 450 and 570 nm. Drug effects were calculated as a
percentage of the reduction in absorbance in the presence of
each drug concentration compared to absorbance obtained with
virus in the absence of drug.
6-Dod eca n oyla m id o-4-m et h yl-8-(â-D-r ib ofu r a n osyl)-
p yr r olo[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3j). Com-
pound 3j was synthesized by method 2 using lauroyl chloride
(0.70 g, 0.74 mL, 3.2 mmol). The column purified product was
recrystallized from hot acetone. Yield ) 0.21 g (26%); Rf ) 0.60;
mp 176-178 °C; 1H NMR (DMSO-d6) δ 10.43 (1H, bs, NH),
8.09 (1H, s, H-2), 7.19 (1H, s, H-7), 5.83 (1H, d, H-1′), 5.40
(2H, m, 2 × OH), 5.20 (1H, d, OH), 4.49 (1H, m), 4.08 (1H, m),
3.95 (1H, m), 3.55 (2H, m, H-5′), 3.43 (3H, s, NCH3), 2.35 (2H,
t, CH2), 1.57 (2H, m, CH2), 1.27 (16H, m, 8 × CH2), 0.85 (3H,
t, CH3). Anal. (C25H38N6O5) C, H, N.
6-Isobu tyr yla m id o-4-m eth yl-8-(â-D-r ibofu r a n osyl)p yr -
r olo[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3k ). Compound
3k was synthesized by method 1 using isobutyryl chloride (0.53
g, 0.52 mL, 5 mmol). Yield ) 0.12 g (63%); Rf ) 0.38; mp 186-
188 °C; 1H NMR (DMSO-d6) δ 10.41 (1H, bs, NH), 8.09 (1H, s,
H-2), 7.21 (1H, s, H-7), 5.83 (1H, d, H-1′), 5.40 (2H, m, 2 ×
OH), 5.20 (1H, d, OH), 4.49 (1H, m), 4.08 (1H, m), 3.95 (1H,
m), 3.55 (2H, m, H-5′), 3.43 (3H, s, NCH3), 2.72 (1H, m, CH),
1.10 (6H, d, 2 × CH3). Anal. (C17H22N6O5) C, H, N.
4-Meth yl-8-(â-D-r ibofu r a n osyl)-6-tr im eth yla cetyla m i-
d op yr r olo[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3l). Com-
pound 3l was synthesized by method 1 using trimethylacetyl
chloride (0.60 g, 0.62 mL, 5 mmol). Yield ) 0.11 g (55%); Rf )
0.51; mp 209-211 °C; 1H NMR (DMSO-d6) δ 9.91 (1H, bs, NH),
8.11 (1H, s, H-2), 7.08 (1H, s, H-7), 5.85 (1H, d, H-1′), 5.42
(2H, m, 2 × OH), 5.18 (1H, d, OH), 4.49 (1H, m), 4.10 (1H, m),
3.95 (1H, m), 3.50 (2H, m, H-5′), 3.48 (3H, s, NCH3), 1.23 (9H,
s, 3 × CH3). Anal. (C18H24N6O5‚0.25EtOAc) C, H, N.
6-Ben zoylam ido-4-m eth yl-8-(â-D-r ibofu r an osyl)pyr r olo-
[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3m ). Compound 3m
was synthesized by method 1 using benzoyl chloride (0.70 g,
0.58 mL, 5 mmol). The column purified product was recrystal-
lized from a hot mixture of ethyl acetate:hexane. Yield ) 0.10
g (47%); Rf ) 0.52; mp 265 °C dec; 1H NMR (DMSO-d6) δ 10.90
(1H, bs, NH), 8.14 (1H, s, H-2), 8.00 (2H, d), 7.60 (3H, m), 7.16
(1H, s, H-7), 5.85 (1H, d, H-1′), 5.36 (2H, m, 2 × OH), 5.18
(1H, d, OH), 4.49 (1H, m), 4.10 (1H, m), 3.94 (1H, m), 3.55
(2H, m, H-5′), 3.48 (3H, s, NCH3). Anal. (C20H20N6O5) C, H, N.
Cytotoxicity Assa ys. Several different assays were used
to explore the cytotoxicity of selected compounds using meth-
ods we have detailed previously. (i) Cytotoxicity produced in
stationary HFF cells and in CEM-SS cells was determined by
microscopic inspection of cells not affected by the virus used
in the respective assays.20 (ii) The effect of compounds during
two population doublings of KB cells was determined by crystal
violet staining and spectrophotometric quantitation of dye
eluted from stained cells as described earlier.27 Briefly, 96-
well cluster dishes were planted with KB cells at 5000 cells
per well. After overnight incubation at 37 °C, test compound
was added in triplicate at eight concentrations. Plates were
incubated at 37 °C for 48 h in a CO2 incubator, fixed with 95%
ethanol, and stained with 0.1% crystal violet. Acidified ethanol
was added, and plates read at 570 nm in a spectrophotometer
designed to read 96-well ELISA assay plates.
Da ta An a lysis. Dose-response relationships were con-
structed by linearly regressing the percent inhibition of
parameters derived in the preceding sections against log drug
concentrations. Fifty percent inhibitory (IC50) concentrations
were calculated from the regression lines. Samples containing
positive controls [acyclovir, ganciclovir, and zidovudine (AZT),
respectively] for HSV-1, HCMV, and HIV were used in all
assays.
In Vitr o An tivir a l Stu d ies. Cell Cu ltu r e P r oced u r es.
The routine growth and passage of KB, HFF, and BSC-1 cells
was performed in monolayer cultures using minimal essential
medium (MEM) with either Hanks salts [MEM(H)] or Earle
salts [MEM(E)] supplemented with 10% calf or fetal calf serum
as detailed previously.20 The sodium bicarbonate concentration
was varied to meet the buffering capacity required. Cells were
passaged at 1:2 to 1:10 dilutions according to conventional
procedures by using 0.05% trypsin plus 0.02% EDTA in a
HEPES buffered salt solution. CEM-SS and AA-2 cells were
obtained through the AIDS Research and Reference Reagent
Program, Division of AIDS, NIAID, NIH. CEM-SS cells were
from Dr. P. Nara;21,22 AA-2 cells were from Dr. M. Hershfield.
AA-2 cells are a subclone of AA cells derived from the WIL-2
human splenic EBV+ B lymphoblastoid line. The AA-2 sub-
clone was selected for resistance to 6-methylmercaptopurine
ribonucleoside and arabinosylcytosine to give cells with <1-
3% wild-type AK dCK activity.23 Cells were passaged twice
weekly at 1:10 dilutions using RPMI 1640 with 10% fetal calf
serum supplemented with nonessential amino acids and 1 mM
pyruvate.
HIV-1 Assa y. The HIV strain IIIB producer cell line H9IIIB
was obtained through the courtesy of Dr. R. C. Gallo. HIV
strain IIIB was propagated in CEM-SS cells as described
previously by Kucera et al.10,24 To evaluate the activities of
compounds in cells acutely infected with HIV, reverse tran-
scriptase (RT) was employed as a marker for HIV-1. CEM-SS
cells were infected at a multiplicity of infection of approxi-
mately 0.001 plaque forming units (pfu) per cell with strain
IIIB of HIV-1 in a minimal volume of stock virus in growth
medium. Cultures were incubated at 37 °C for 2 h to permit
Extr a ction P r oced u r es for Ch r om a togr a p h ic An a ly-
sis. CEM-SS and AA-2 cells were grown in suspension culture
with concentrations of compounds given in Table 2 for 5 to 24
h. Cells were harvested by centrifugation (250g, 5 min) at 25
°C, the resulting cell pellet was resuspended in 10 mL of cold
Puck’s saline with glucose (Gibco), and the cell suspension was
again centrifuged (250g, 5 min). The extraction procedures

2462 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Porcari et al.
used to obtain the aqueous phase of supernatant were previ-
ously described.28 Briefly, nucleotides were extracted by vor-
texing the resulting cell pellet in 0.4 mL of 0.6 N TCA (4 °C)
which was kept on ice for 15 min. This suspension was
transferred to a microfuge tube and microfuged for 30 s at
12000g (4 °C). The supernatant was carefully removed to an
Eppendorf tube, volume was measured, and an equal amount
of ice cold freon containing 0.5 M trioctylamine was added to
the tube. The mixture was vortexed for 5 or more seconds and
then microfuged for 30 s at 12000g (4 °C). The lower phase
was removed by aspiration and discarded. The aqueous phase
was frozen at -76 °C for high performance liquid chromatog-
raphy (HPLC) analysis.
Refer en ces
(1) Schram, K. H.; Townsend, L. B. The synthesis of 6-amino-4-
methyl-8-(beta-D-ribofuranosyl)(4-H,8-H)pyrrolo[4,3,2-de]pyrimido-
[4,5-c]pyridazine, A new tricyclic nucleoside. Tetrahedron Lett.
1971, 49, 4757-4760.
(2) Roti Roti, L. W.; Roti Roti, J . L.; Townsend, L. B. Studies on the
mechanism of cytotoxicity of tricyclic nucleoside NSC#154020
in L1210 cells. Proc. Am. Assoc. Cancer Res. 1978, 19, 40.
(3) Schweinsberg, P. D.; Smith, R. G.; Loo, T. L. Identification of
the metabolites of an antitumor tricyclic nucleoside (NSC-
154020). Biochem. Pharmacol. 1981, 30, 2521-2526.
(4) Wotring, L. L.; Townsend, L. B.; Crabtree, G. W.; Parks, R. E.,
J r. A possible role for ecto-5′-nucleotidase in cytotoxicity and
intracellular nucleotide formation from the tricyclic nucleoside-
5′-phosphate (TCN-P). Proc. Am. Assoc. Cancer Res. 1981, 22,
257.
(5) Wotring, L. L.; Passiatore, J . E.; Roti Roti, J . L.; Hudson, J . L.;
Townsend, L. B. Effects of the tricyclic nucleoside 6-amino-4-
methyl-8-(beta-D- ribofuranosyl)pyrrolo[4,3,2-de]pyrimido[4,5-
c]pyridazine on the viability and cell cycle distribution of L1210
cells in vitro. Cancer Res. 1985, 45, 6355-6361.
(6) Wotring, L. L.; Townsend, L. B.; J ones, L. M.; Borysko, K. Z.;
Gildersleeve, D. L.; Parker, W. B. Dual mechanisms of inhibition
of DNA synthesis by triciribine. Cancer Res. 1990, 50, 4891-
4899.
(7) Moore, E. C.; Hurlbert, R. B.; Boss, G. R.; Massia, S. P. Inhibition
of two enzymes in de novo purine nucleotide synthesis by
triciribine phosphate (TCN-P). Biochem. Pharmacol. 1989, 38,
4045-4051.
Nu cleotid e An a lysis. TCN-P was separated and quanti-
tated by either anion exchange HPLC as detailed by Wotring
et al.9 or by ion pair reverse phase HPLC as described by
Walseth et al.29 A Spectra-Physics system was employed
consisting of a model SP8800 ternary pump, a SP8500 dynamic
mixer, a SP8780 autosampler, and a SP8490 variable wave-
length detector. Peaks were integrated on a model SP4270
integrator. A Compaq model 386 computer with WINner 386
software (Spectra-Physics) was used for system and data
management.
In an initial experiment with TCN, analysis was performed
using anion exchange HPLC, but superior resolution of mono-
phosphates was achieved by reverse phase HPLC; conse-
quently it was adopted in subsequent experiments with TCN
and TCN-P. Ion pair reverse phase chromatography was
(8) Townsend, L. B.; Lewis, A. F.; Roti Roti, L. W. Synthesis of 6-
amino-4-methyl-8-(beta-D-ribofuranosyl)pyrrolo[4,3,2-de]pyr-
imido[4,5-c]pyridazine-5′-phosphate as a novel compound and
its utility against L-1210 mouse leukemia. United States. Patent
Number: 4,123,524. October 31, 1978.
performed on
a
3.9 × 300 mm µBondapak C18 column
(9) Wotring, L. L.; Crabtree, G. W.; Edwards, N. L.; Parks, R. E.,
J r.; Townsend, L. B. Mechanism of activation of triciribine
phosphate (TCN-P) as a prodrug form of TCN. Cancer Treat.
Rep. 1986, 70, 491-497.
(10) Kucera, L. S.; Iyer, N. P.; Puckett, S. H.; Buckheit, R. W., J r.;
Westbrook, L.; Toyer, B. R.; White, E. L.; Germany-Decker, J .
M.; Shannon, W. M.; Chen, R. C.; Nassiri, M. R.; Shipman, C.;
Townsend, L. B.; Drach, J . C. Activity of triciribine and tri-
ciribine-5′-monophosphate against human immunodeficiency
virus types 1 and 2. AIDS Res. Hum. Retroviruses. 1993, 9, 307-
314.
(Waters). The solvent was 5 mM tetrabutylammonium hy-
droxide in 5% methanol adjusted to pH 2.5 with formic acid.
Separation of nucleotide monophosphates was carried out at
a flow rate of 1 mL/min with dual wavelength detection set at
254 and 290 nm. TCN-P metabolite identification was based
on comparison of retention time of unknown peaks to retention
time of a TCN-P standard and on the ratio of 290/254 nm due
to the absorbance maximum of TCN and TCN-P at 290 nm.9
(11) Ptak, R. G.; J acobson, J . G.; Borysko, K. Z.; Townsend, L. B.;
Drach, J . C. Triciribine inhibits HIV nuclear import and requires
phosphorylation by cellular enzymes for activity. 5th Conf.
Retroviruses & Opportunistic Infections. Chicago, IL. 1998. abst.
354.
(12) Ptak, R. G.; Borysko, K. Z.; Porcari, A. R.; Buthod, J . L.; Holland,
L. E.; Shipman, C.; Townsend, L. B.; Drach, J . C. Phosphoryl-
ation of triciribine is necessary for activity against HIV-1. AIDS
Res. Human Retroviruses. 1998, 14, 1315-1322.
(13) Ptak, R. G.; Drach, J . C.; J acobson, J . G.; Townsend, L. B.; Stup,
T. L.; Russell, J .; Buckheit, R. W. HIV-1 Regulatory Proteins:
Targets of Triciribine? 6th Conf. Retroviruses & Opportunistic
Infections. Chicago, IL. February, 1999. abst. 622.
(14) Drach, J . C.; Shipman, C.; Ptak, R. G.; Borysko, K. Z.; J acobson,
J . G.; Nassiri, M. R.; Wotring, L. L.; Porcari, A. R.; Townsend,
L. B. Activity of triciribine (TCN) against HIV-1 requires cellular
phosphorylation and inhibition of a viral gene product. 36th
Interscience Conf. Antimicrob. Agents Chemother. New Orleans,
LA. 1996. abst. 2099.
(15) Ickes, D. E.; Shipman, C.; Drach, J . C. Antiviral activity of
triciribine and triciribine monophosphate against human her-
pesvirus type 6. Seventh International Conference on Antiviral
Research. Charleston, SC. 1994.
(16) Musherure, P. R.; Breitenbach, J . M.; Aseltine, K. R.; Shipman,
C.; Drach, J . C. Antiviral activity of triciribine (TCN) in
combination with phosphonoformate (PFA) on human herpes-
virus type 6 (HHV-6). AADR Meeting. San Antonio, TX. 1995.
abst. 391.
Solvent conditions were changed for the analysis of the TCN
analogues (3d , 3f, and 3h ). The ion pairing reagent was
removed because these conditions resulted in a strong reten-
tion of these analogues (3d , 3f, and 3h ) on the column.
Following equilibration with dilute formic acid (pH 2.5), a
linear gradient was used at a flow rate of 1 mL/min with a 20
min elution time beginning with formic acid (pH 2.5) and
ending with 50% methanol in formic acid (pH 2.5) followed by
a 15 min isocratic elution with the final solution. These
conditions also were employed in a separate study in which
electrospray mass spectrometry was performed to identify
peaks using a Finnigan LCQ mass spectrometer interfaced
with a Hitachi HPLC.
Intracellular concentrations were calculated based upon the
integrated area under HPLC peaks of TCN-P, conversion of
area to nanomoles of TCN-P based upon the area under TCN-P
peaks from standards of known concentration, and conversion
of concentration from nanomoles/106 cells to micromolarity
based upon a cell volume of 1.9 pL/cell. Volumes of CEM-SS
cells were determined by microscopic examination of cells in
a hemocytometer, measurement of the diameter of 10-20
representative cells, and assumption of spherical geometry.
(17) Nassiri, M. R.; Achim, C. L.; Martinez, A. J .; Wiley, C. A.; Drach,
J . C.; Townsend, L. B. Antiretroviral activity of TCN-P in HIV-
infected Human Macrophages. Eleventh International Confer-
ence on Antiviral Research. San Diego, CA. 1998.
(18) Porcari, A. R.; Drach, J . C.; Wotring, L. L.; Townsend, L. B. The
design, synthesis and evaluation of a series of acyclic triciribine
analogues for their antiviral and antineoplastic activity. 213th
National Meeting of the American Chemical Society. San
Francisco, CA. 1997. abst. MEDI 269.
(19) Porcari, A. R.; Drach, J . C.; Wotring, L. L.; Townsend, L. B.
Synthesis and structure-activity relationship of a series of deoxy
triciribine analogues against HIV and murine L1210 cells. 214th
National Meeting of the American Chemical Society. Las Vegas,
NV. 1997. abst. MEDI 181.
Ack n ow led gm en t. This study was supported by
Research Grants U19-AI-31718 and RO1-AI36872 from
the National Institute of Allergy and Infectious Dis-
eases, by Training Grant T32-GM07767 from the Na-
tional Institutes of Health, and by research funds from
the University of Michigan. We thank Mr. Donald L.
McKenzie for performing HPLC-mass spectrophoto-
metric analyses and Ms. Kimberly Barrett for assistance
in manuscript preparation.

6-N-Acyltriciribine Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2463
(20) Turk, S. R.; Shipman, C., J r.; Nassiri, M. R.; Genzlinger, G.;
Krawczyk, S. H.; Townsend, L. B.; Drach, J . C. Pyrrolo[2,3-d]-
pyrimidine nucleosides as inhibitors of human cytomegalovirus.
Antimicrob. Agents Chemother. 1987, 31, 544-550.
(21) Nara, P. L.; Hatch, W. C.; Dunlop, N. M.; Robey, W. G.;
Fischinger, P. J . Simple, rapid quantitative, syncytium-forming
microassay for the detection of human immunodeficiency virus
neutralizing antibody. AIDS Res. Hum. Retroviruses. 1987. 3,
283-302,
(25) White, E. L.; Buckheit, R. W.; Ross, L. J .; Germany, J . M.;
Andries, K.; Pauwels, R.; J anssen, P. A. J .; Shannon, W. M.;
Chirigos, M. A. A TIBO derivative, R82913, is a potent inhibitor
of HIV-1 reverse transcriptase with heteropolymer templates.
Antiviral Res. 1991, 16, 257-266.
(26) Prichard, M. N.; Shipman, C. A three-dimensional model to
analyze drug-drug interactions. Antiviral Res. 1990, 14, 181-
206.
(27) Prichard, M. N.; Prichard, L. E.; Baguley, W. A.; Nassiri, M. R.;
Shipman, C. Three-dimensional analysis of the synergistic
cytotoxicity of ganciclovir and zidovudine. Antimicrob. Agents
Chemother. 1991, 35, 1060-1065.
(28) Pogolotti, A. L.; Santi, D. V. High-pressure liquid chromatog-
raphy-Ultraviolet analysis of intracellular nucleotides. Anal.
Biochem. 1982, 126, 335-345.
(29) Walseth, T. F.; Graff, G.; Moos, M. C.; Goldberg, N. D. Separation
of 5′-ribonucleoside monophosphates by ion-pair reverse-phase
high-performance liquid chromatography. Anal. Biochem. 1980,
107, 240-245.
(22) Nara, P. L.; Fischinger, P. J . Quantitative infectivity assay for
HIV-1 and -2. Nature 1988, 332, 469-470.
(23) Chaffee, S.; Leeds, J . M.; Matthews, T. J .; Weinhold, K. J .;
Skinner, M.; Bolognesi, D. P.; Hershfield, M. S. Phenotypic
variation in the response to the human immunodeficiency virus
among derivatives of the CEM T and WIL-2 B cell lines. J . Exp.
Med. 1988, 168, 605-621.
(24) Kucera, L. S.; Iyer, N.; Leake, E.; Raben, A.; Modest, E. J .;
Daniel, L. W.; Piantadosi, C. Novel membrane-interactive ether
lipid analogues that inhibit infectious HIV-1 production and
induce defective virus formation. AIDS Res. Hum. Retroviruses.
1990, 6, 491-501.
J M990236H