Deoxy sugar analogues of triciribine: Correlation of antiviral and antiproliferative activity with intracellular phosphorylation

Article abstract of DOI:10.1021/jm990205m

Triciribine (TCN) and triciribine monophosphate (TCN-P) have antiviral and antineoplastic activity at low micromolar or submicromolar concentrations. In an effort to improve and better understand this activity, we have conducted a structure - activity rel

Full text of DOI:10.1021/jm990205m

2438
J . Med. Chem. 2000, 43, 2438-2448
Deoxy Su ga r An a logu es of Tr icir ibin e: Cor r ela tion of An tivir a l a n d
An tip r olifer a tive Activity w ith In tr a cellu la r P h osp h or yla tion
Anthony R. Porcari, Roger G. Ptak, Katherine Z. Borysko, J ulie M. Breitenbach, Sauro Vittori,†
Linda L. Wotring,‡ J ohn C. Drach, and Leroy B. Townsend*
Department of Medicinal Chemistry, College of Pharmacy, Department of Chemistry, College of Literature,
Sciences and Arts, and Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan,
Ann Arbor, Michigan 48109-1065
Received April 23, 1999
Triciribine (TCN) and triciribine monophosphate (TCN-P) have antiviral and antineoplastic
activity at low micromolar or submicromolar concentrations. In an effort to improve and better
understand this activity, we have conducted a structure-activity relationship study to explore
requirements for the number of hydroxyl groups on the ribosyl moiety for biological activity.
2′-Deoxytriciribine (2′-dTCN), 3′-deoxytriciribine (3′-dTCN), 2′,3′-epoxytriciribine (2′,3′-ep-
oxyTCN), 2′,3′-dideoxy-2′,3′-didehydrotriciribine (2′,3′-d4TCN), and 2′,3′-dideoxytriciribine (2′,3′-
ddTCN) were synthesized and evaluated for activity against human immunodeficiency virus
(HIV-1), herpes simplex virus type 1 (HSV-1), and human cytomegalovirus (HCMV). Antipro-
liferative activity of the compounds also was tested in murine L1210 cells and three human
tumor cell lines. All compounds were either less active than TCN and TCN-P or inactive at
the highest concentration tested (100 µM) in both antiviral and antiproliferative assays. Reverse-
phase HPLC of extracts from uninfected cells treated with the deoxytriciribine analogues only
detected the conversion of 3′-dTCN and 2′,3′-ddTCN to their respective monophosphates.
Therefore, either the deoxytriciribine analogues were not transported across the cell membrane
or, more likely, they were not substrates for a nucleoside kinase or phosphotransferase. We
have concluded that the hydroxyl groups on the ribosyl ring system of TCN and TCN-P must
be intact in order to obtain significant antiviral and antineoplastic activity.
In tr od u ction
TCN is metabolized only to the monophosphate and not
to di- or triphosphate forms.9 Furthermore, no incorpo-
ration of TCN into nucleic acids has been observed.16
TCN-P inhibits both DNA and protein synthesis,15,16 but
the exact mechanism is unknown.
Triciribine (TCN) is a tricyclic nucleoside that was
first synthesized by Schram and Townsend in 1971.1
Initial testing of triciribine and the water-soluble pro-
drug, triciribine-5′-monophosphate2 (TCN-P), against
murine leukemic L1210 cells revealed their potential
as antineoplastic agents. This discovery led to extensive
in vitro3-17 and in vivo18-22 studies of TCN and TCN-P
as novel antineoplastic agents. Phase I clinical trials
were completed with TCN-P,23-29 and it was advanced
to phase II studies as a potential antineoplastic
agent.27,29-32
Early studies revealed that TCN is converted intra-
cellularly to TCN-P by adenosine kinase.4,6,7 Phospho-
rylation is essential for antineoplastic activity as dem-
onstrated by the absence of growth inhibition when
adenosine kinase-deficient cells were treated with
TCN.3,11,12 The activity of TCN-P also requires adenos-
ine kinase because extracellular TCN-P is a charged
species and does not cross the cell membrane, and
therefore it must be dephosphorylated to TCN by
extracellular phosphatases or cellular ecto-5′-nucleoti-
dase and rephosphorylated to TCN-P by intracellular
adenosine kinase.7,12 Unlike other nucleoside analogues,
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.33 These studies
also found no cross-resistance to TCN or TCN-P in HIV
strains resistant to the reverse transcriptase (RT)
inhibitors AZT and TIBO33 suggesting that TCN and
TCN-P have a different mode of action than AZT and
TIBO. Furthermore, cytotoxicity such as that observed
in murine L1210 cells appears to be highly cell line-
specific3 and was not observed in human cell lines used
to propagate HIV and human cytomegalovirus (HC-
MV).33 Even though TCN was not very cytotoxic in these
cell lines, it must be phosphorylated to TCN-P to be
active against HIV-1.34 Studies on the antiviral mech-
anism of action of TCN are currently underway and
suggest inhibition of the function of an HIV accessory
protein.35
Since 1971, the only analogues of TCN to have been
synthesized and evaluated have been 7-azaTCN ana-
logues,36-40 which were found39 to be virtually inactive
due to their inability to be phosphorylated. This prompted
us to initiate specific structure-activity relationship
studies to further investigate the structural require-
ments and mode of action of TCN and TCN-P. In a series
of studies designed to specifically explore the structural
requirements for the sugar moiety at the N-8 position
* To whom correspondence should be addressed. Tel: 734-764-7547.
Fax: 734-763-5633. E-mail: ltownsen@umich.edu.
† Dipartimento Scienze Chimiche, Via S. Agostino 1, Universita di
Caerino, 62032 Camerino (MC), Italy.
‡ Michigan Department of Community Health, Bureau of Epidemi-
ology, HIV/AIDS Surveillance Section, Herman Kiefer Health Complex,
Rm 210B, 1151 Taylor, Detroit, MI 48202.
10.1021/jm990205m CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/17/2000

Deoxy Sugar Analogues of Triciribine
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2439
Ch a r t 1. Deoxy Analogues of Triciribine (TCN)
Sch em e 1. Synthesis of 2′-Deoxytriciribine (2′-dTCN)a
a
Reagents: (i) NaH, CH3CN; (ii) NH2NHCH3, EtOH, CHCl3; (iii) 10% Pd-C, 50 psi H2, EtOH, EtOAc, 1 N NH4OH; (iv) concd HCl,
EtOH, reflux; (v) NH3, MeOH.
of TCN for biological activity, our first study explored
the requirements for rigidity of the ribosyl moiety. That
study established that a disruption of the rigidity of the
ribosyl moiety adversely affected phosphorylation and
therefore biological activity.41
triciribine (Chart 1). We also elected to synthesize 5′-
deoxytriciribine (5′-dTCN) to unequivocally confirm the
fact that TCN requires phosphorylation to TCN-P in
order to exhibit biological activity. Biological analysis
of these analogues has provided an important insight
into the hydroxyl requirements of TCN with regards to
phosphorylation and in vitro activity against selected
viruses and cancer cell lines.
This article describes the second study in this series
and was designed to explore the hydroxyl requirements
of the ribosyl moiety at the N-8 position of TCN. A
systematic removal of the hydroxyl groups while main-
taining the rigidity of the furan ring system should
determine which individual and which combination of
hydroxyl groups are necessary for TCN to be phospho-
rylated and active against selected viruses and cancer
cell lines. We designed most of these analogues to
maintain the relative availability and position of the 5′-
hydroxyl group due to the obvious importance of the 5′-
hydroxyl group to phosphorylation and activity. On the
basis of this rationale, 2′-deoxytriciribine (2′-dTCN), 3′-
deoxytriciribine (3′-dTCN), and 2′,3′-epoxytriciribine
(2′,3′-epoxyTCN) were synthesized as monodeoxy ana-
logues of triciribine and 2′,3′-dideoxy-2′,3′-didehydro-
triciribine (2′,3′-d4TCN) and 2′,3′-dideoxytriciribine (2′,3′-
ddTCN) were synthesized as dideoxy analogues of
Resu lts a n d Discu ssion
6-Amino-8-(2-deoxy-â-D-ribofuranosyl)-4-methylpyrrolo-
[4,3,2-de]pyrimido[4,5-c]pyridazine (2′-deoxytriciribine,
2′-dTCN) was synthesized from 6-bromo-4-chloro-5-
cyanopyrrolo[2,3-d]pyrimidine (1), as illustrated in
Scheme 1. The sodium salt of 6-bromo-4-chloro-5-cyan-
opyrrolo[2,3-d]pyrimidine42 (1), formed by treating com-
pound 1 with sodium hydride, was glycosylated with
1-chloro-2-deoxy-3,5-di-O-p-toluoyl-R-D-ribofuranose43 (2)
to afford 6-bromo-4-chloro-5-cyano-7-(2-deoxy-3,5-di-O-
p-toluoyl-R-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine44 (3).
The reaction of 3 with methylhydrazine gave 6-bromo-
5-cyano-7-(2-deoxy-3,5-di-O-p-toluoyl-â-D-ribofuranosyl)-
4-(1-methylhydrazino)pyrrolo[2,3-d]pyrimidine (4). De-

2440 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Porcari et al.
Sch em e 2. Synthesis of 2′,3′-Epoxytriciribine (2′,3′-epoxyTCN), 2′,3′-Dideoxy-2′,3′-didehydrotriciribine (2′,3′-d4TCN),
and 2′,3′-Dideoxytriciribine (2′,3′-ddTCN)a
a
Reagents: (i) 2-acetoxyisobutyryl bromide, CH3CN; (ii) NH3, MeOH; (iii) Zn-Cu couple, DMF, AcOH; (iv) 40 psi H2, 10% Pd-C,
EtOH, EtOAc; (v) Bu3SnH, AIBN, THF.
bromination of intermediate 4 with 10% palladium on
charcoal under 50 psi of hydrogen gas afforded 5-cyano-
7-(2-deoxy-3,5-di-O-p-toluoyl-â-D-ribofuranosyl)-4-(1-meth-
ylhydrazino)pyrrolo[2,3-d]pyrimidine (5). Ring annula-
tion of intermediate 5 to form 6-amino-8-(2-deoxy-3,5-
di-O-p-toluoyl-â-D-ribofuranosyl)-4-methylpyrrolo[4,3,2-
de]pyrimido[4,5-c]pyridazine (6) was accomplished in
ethanol, under acidic conditions, at reflux temperature.
Deprotection of intermediate 6 with methanolic am-
monia afforded 6-amino-8-(2-deoxy-â-D-ribofuranosyl)-
4-methylpyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine (2′-
deoxytriciribine, 2′-dTCN) in 25% overall yield from
compound 1.
A 47% yield of 2′,3′-epoxytriciribine (2′,3′-epoxyTCN)
was synthesized, using similar procedures as described
with adenosine,45 by sealing compound 7 in a pressure
bottle with methanolic ammonia, saturated at 0 °C, for
18 h at room temperature (Scheme 2).
2′,3′-Dideoxy-2′,3′-didehydrotriciribine (2′,3′-d4TCN)
was synthesized from compound 7, by first introducing
a double bond between the 2′- and 3′-carbons with a zinc
copper couple to give compound 8, using a similar
procedure as described with toyocamycin.46 Subsequent
deprotection of compound 8 with methanolic ammonia
gave 2′,3′-dideoxy-2′,3′-didehydrotriciribine (2′,3′-d4TCN)
in a 57% yield from compound 7 (Scheme 2).
2′,3′-Dideoxytriciribine (2′,3′-ddTCN) was synthesized
by first reducing the double bond in compound 8 with
10% palladium on charcoal in a mixture of ethyl acetate
and ethanol, under 40 psi of hydrogen gas, to give the
intermediate 9. Subsequent deprotection of 9 with
methanolic ammonia afforded 2′,3′-dideoxytriciribine
(2′,3′-ddTCN) in a 73% yield from compound 8 (Scheme
2). Several unsuccessful attempts were made to syn-
thesize 6-amino-8-(3-deoxy-â-D-ribofuranosyl)-4-meth-
ylpyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine (3′-deoxy-
triciribine, 3′-dTCN) from compound 7 using tributyltin
hydride and 2,2′-azobis[isobutyronitrile] (AIBN) (Scheme
2).
The reaction of triciribine with an excess of 2-acetoxy-
isobutyryl bromide in acetonitrile at room temperature
furnished a good yield of 8-[2-O-acetyl-3-bromo-3-deoxy-
5-O-(2,5,5-trimethyl-1,3-dioxolan-4-one-2-yl)-â-D-xylo-
furanosyl]-6-amino-4-methylpyrrolo[4,3,2-de]pyrimido-
[4,5-c]pyridazine (7) (Scheme 2). 6-Amino-8-(2,3-epoxy-
â-D-ribofuranosyl)-4-methylpyrrolo[4,3,2-de]pyrimido[4,5-
c]pyridazine (2′,3′-epoxytriciribine, 2′,3′-epoxyTCN),
6-amino-8-(2,3-dideoxy-2,3-didehydro-â-D-ribofuranosyl)-
4-methylpyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine (2′,3′-
dideoxy-2′,3′-didehydrotriciribine, 2′,3′-d4TCN), and
6-amino-8-(2,3-dideoxy-â-D-ribofuranosyl)-4-methylpyr-
rolo[4,3,2-de]pyrimido[4,5-c]pyridazine (2′,3′-dideoxy-
triciribine, 2′,3′-ddTCN) were all synthesized from this
common intermediate (7), as illustrated in Scheme 2.
Spectral analysis (1H NMR) of what should have been
compound 10 revealed that the methyl protons of the

Deoxy Sugar Analogues of Triciribine
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2441
Sch em e 3. Synthesis of 3′-Deoxytriciribine (3′-dTCN)a
a
Reagents: (i) ref 46; (ii) Bu3SnH, AIBN, THF; (iii) NaNO2, H2O, AcOH; (iv) POCl3; (v) NH2NHCH3, EtOH; (vi) concd HCl, EtOH,
reflux; (vii) NH3, MeOH.
2′-O-acetyl group and the characteristic multiplets for
the 3′-protons, between δ 2.0 and 3.0, were missing.
Subsequent deprotection of this intermediate with
methanolic ammonia, saturated at 0 °C, afforded 2′,3′-
epoxytriciribine instead of 3′-deoxytriciribine. Therefore,
we assumed that excessive steric bulk on the R-face of
the furan ring system prevented a radical reduction of
the 3′-bromo group by the bulky reducing agent, tribu-
tyltin hydride. Unfortunately, we were unable to remove
the 2,5,5-trimethyl-1,3-dioxolan-4-one protecting group
as described in the literature.46 Therefore, we synthe-
sized 3′-deoxytriciribine from toyocamycin using the
route illustrated in Scheme 3. Sequentially treating
toyocamycin with 2-acetoxyisobutyryl bromide, metha-
nol, and acetic anhydride afforded 4-amino-5-cyano-7-
(3-bromo-3-deoxy-2,5-di-O-acetyl-â-D-xylofuranosyl)pyr-
rolo[2,3-d]pyrimidine46 (11). A removal of the 3′-bromo
group on compound 11 with tributyltin hydride and
′AIBN in tetrahydrofuran (THF) at reflux temperature
for 18 h was successful and gave 4-amino-5-cyano-7-(3-
deoxy-2,5-di-O-acetyl-â-D-ribofuranosyl)pyrrolo[2,3-d]-
pyrimidine (12). Diazotization of 12 with sodium nitrite
in aqueous acetic acid at 65 °C over 2.0 h gave 5-cyano-
7-(3-deoxy-2,5-di-O-acetyl-â-D-ribofuranosyl)pyrrolo[2,3-
d]pyrimidin-4-one (13). Chlorination of compound 13
was accomplished with phosphorus oxychloride (POCl3)
at reflux temperature to give 4-chloro-5-cyano-7-(3-
deoxy-2,5-di-O-acetyl-â-D-ribofuranosyl)pyrrolo[2,3-d]-
pyrimidine (14). Displacement of the 4-chloro group
from 14 with methylhydrazine in a mixture of ethanol
and chloroform gave 5-cyano-7-(3-deoxy-2,5-di-O-acetyl-
â-D-ribofuranosyl)-4-(1-methylhydrazino)pyrrolo[2,3-d]-
pyrimidine (15) after 2 h. Ring annulation of 15 was
accomplished in ethanol at reflux temperature for 3 h,
under acidic conditions using concentrated HCl to give
6-amino-8-(3-deoxy-2,5-di-O-acetyl-â-D-ribofuranosyl)-4-
methylpyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine (16).
Deprotection of 16 with methanolic ammonia afforded
3′-deoxytriciribine (3′-dTCN).
6-Amino-8-(5-deoxy-â-D-ribofuranosyl)-4-methylpyrrolo-
[4,3,2-de]pyrimido[4,5-c]pyridazine (5′-deoxytriciribine,
5′-dTCN) was synthesized by treating toyocamycin with
thionyl chloride in hexamethylphosphoramide (HMPA)
to afford 4-amino-5-cyano-7-(5-chloro-5-deoxy-â-D-ribo-
furanosyl)pyrrolo[2,3-d]pyrimidine47 (17) (Scheme 4).
Treatment of compound 17 with tributyltin hydride and
AIBN in THF at reflux temperature for 18 h gave
4-amino-5-cyano-7-(5-deoxy-â-D-ribofuranosyl)pyrrolo-
[2,3-d]pyrimidine47 (18, 5′-deoxytoyocamycin). Acetyla-
tion of compound 18 with acetic anhydride in pyridine
afforded 4-amino-5-cyano-7-(5-deoxy-2,3-di-O-acetyl-â-
D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (19), which was
diazotized with sodium nitrite in aqueous acetic acid at
85 °C for 1.5 h to give 5-cyano-7-(5-deoxy-2,3-di-O-
acetyl-â-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidin-4-one
(20). Chlorination of 20 was accomplished with POCl3
at reflux temperature to give 4-chloro-5-cyano-7-(5-
deoxy-2,3-di-O-acetyl-â-D-ribofuranosyl)pyrrolo[2,3-d]-
pyrimidine (21). Displacement of the 4-chloro group of
intermediate 21 with methylhydrazine in a mixture of
ethanol and chloroform gave 5-cyano-7-(5-deoxy-2,3-di-
O-acetyl-â-D-ribofuranosyl)-4-(1-methylhydrazino)pyrrolo-

2442 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Sch em e 4. Synthesis of 5′-Deoxytriciribine (5′-dTCN)a
Porcari et al.
a
Reagents: (i) SOCl2, HMPA; (ii) Bu3SnH, AIBN, THF; (iii) Ac2O, pyridine; (iv) NaNO2, H2O, AcOH; (v) POCl3; (vi) NH2NHCH3,
EtOH; (vii) concd HCl, EtOH, reflux; (viii) NH3, MeOH.
Ta ble 1. Antiproliferative and Antiviral Activity of Deoxy Analogues of TCN
50% inhibitory concentration (µM)
antiproliferative activity
in murine L1210 cellsa
cytotoxicity
cytotoxicity in human cellsb
antiviral activityc
compound
KB growth HFF visual CEM-SS visual HIV-1 RT HSV-1 ELISA HCMV plaque
TCNd
0.035
0.025
>100
2.2
>100
10
100
19
>100
100
>100
>100
>100
>100
>100
0.04
0.04
23
20
2.5
0.8
>100
>100
>100
>100
>100
>100
TCN-Pd
>1.0
2′-dTCN
3′-dTCN
5′-dTCN
2′,3′-epoxyTCN
2′,3′-d4TCN
2′,3′-ddTCN
>100
>100e
100e
>100e
>100
16e
>100e
>100e
>100
100e
6.0e
>100
>100e
>100
>100
>100e
>100e
>100
>100e
>100e
>100
NTf
65
>100
25
>100
>100
21e
a
Antiproliferative activity was determined as described in the text and is reported as the concentration required to reduce murine
b
L1210 cell growth rate to 50% of control rate. Inhibition of KB cell growth was measured as described in the text in triplicate. Visual
cytotoxicity was scored on uninfected HFF and CEM-SS cells used in HCMV plaque and HIV-1 RT assays. c Antiviral activity was
determined using an ELISA in quadruplicate for HSV-1, a plaque assay in duplicate for HCMV, and amount of RT activity in culture
supernatants in triplicate for HIV-1 as described in the text. Data published previously in ref 33. e Average from two or three separate
d
experiments. f NT ) not tested.
[2,3-d]pyrimidine (22). Ring annulation of 22 was
accomplished in ethanol at reflux temperature for 3 h,
under acidic conditions using concentrated HCl to give
6-amino-8-(5-deoxy-2,3-di-O-acetyl-â-D-ribofuranosyl)-4-
methylpyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine (23).
Deprotection of 23 with methanolic ammonia afforded
5′-deoxytriciribine (5′-dTCN).
These TCN analogues (2′-dTCN, 3′-dTCN, 5′-dTCN,
2′,3′-d4TCN, and 2′,3′-ddTCN) were evaluated in an
assay that uses growth inhibition of murine L1210 cells
as an indicator of cytotoxicity. After 4 days of incubation
with L1210 cells, inhibition of cell proliferation (cyto-
toxicity) was not observed for 2′-dTCN, 5′-dTCN, and
2′,3′-d4TCN at concentrations as high as 100 µM (Table
1). Only 3′-dTCN and 2′,3′-ddTCN were cytotoxic in
L1210 cells, IC50 ) 2.2 and 25 µM, respectively. In the
three human cell lines, KB cells, human foreskin
fibroblasts (HFF), and CEM-SS cells, only 3′-dTCN was
cytotoxic (IC50 ) 16 µM) in KB cells (Table 1). Thus, in
contrast to TCN and TCN-P, which are potent inhibitors
of L1210 cell growth, none of the deoxy analogues
inhibited the growth of the murine or human cells
tested.
The six target compounds also were evaluated for
activity against three viruses. Activity against HIV-1
was measured in an assay that employed reverse tran-

Deoxy Sugar Analogues of Triciribine
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2443
Ta ble 2. Intracellular Phosphorylation of DeoxyTCN
Analogues in Uninfected CEM-SS Cells
activity exhibited by 5′-dTCN unequivocally confirms
the fact that TCN requires phosphorylation to TCN-P
in order to exhibit biological activity.3,11,12,34
extracellular incubation intracellular mono-
compound
TCNb
concn (µM)
time (h) phosphate concn (µM)a
Exp er im en ta l Section
10
100
330
10
100
330
100
100
100
100
100
100
5
5
5
286c
323
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 Devises
Mel-Temp melting point apparatus. Thin-layer chromatogra-
phy used Analtech GHLF SiO2 prescored plates. Developed
TLC plates were visualized under ultraviolet light (254 nm).
E. Merck silica gel (230-400 mesh) was used for gravity or
flash column chromatography. Proton 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 downfield
from tetramethylsilane as the internal standard. UV spectra
775c
TCNb
12
12
12
24
24
24
24
24
24
176c
307c
390c
2′-dTCN
3′-dTCN
5′-dTCN
2′,3′-epoxyTCN
2′,3′-d4TCN
2′,3′-ddTCN
<3-5d
276c
<3-5d
<3-5d
<3-5d
45c
a
Nucleotide extractions and quantitation by HPLC were per-
b
formed as described in the text. Control data for TCN phospho-
rylation previously published in ref 34. c Average from two to five
were obtained with
a Kontron UVIKON 860 ultraviolet
d
experiments. Not detected; limit of detection was 3-5 µM.
spectrometer. Elemental analyses were performed by the
Analytical Laboratory, Department of Chemistry, University
of Michigan, Ann Arbor, MI.
scriptase (RT) in culture supernatants as a marker for
HIV-1. Neither 2′-dTCN, 3′-dTCN, 5′-dTCN, 2′,3′-ep-
oxyTCN, nor 2′,3′-d4TCN significantly reduced the
amount of RT activity in the culture supernatants of
CEM-SS cells acutely infected with HIV-1 (Table 1)
thereby establishing that these five analogues had no
activity against HIV-1. Under the same conditions, 2′,3′-
ddTCN gave an IC50 ) 21 µM, suggesting that this
analogue was active against HIV, but at a small fraction
of the activity of TCN and TCN-P (IC50 of each ) 0.04
µM).
The deoxy analogues were also evaluated against
HSV-1 in an ELISA and HCMV in a plaque assay. With
the exception of 3′-dTCN, all of the analogues also were
inactive, or nearly so, against HSV-1 (Table 1). Activity
of 3′-dTCN (IC50 ) 6.0 µM) against HSV-1 is puzzling
in light of the weak activity of TCN and TCN-P and the
inactivity of the other analogues. This activity could be
related to or a manifestation of the cytotoxicity seen in
L1210 and KB cells and to the fact that it is metabolized
to its 5′-monophosphate (see below). No activity against
HCMV was observed for any of these deoxyTCN ana-
logues, even though TCN and TCN-P are active (Table
1).
Given the results of the biological assays, the lack of
activity of the deoxy derivatives could be due to the
inability of these molecules to be phosphorylated to an
active species, or if the phosphorylated metabolites are
formed, it might be due to the hydroxyl groups of the
ribosyl ring system being necessary for activity. There-
fore the phosphorylation of the deoxyTCN analogues
was studied. The compounds were incubated at a
concentration of 100 µM with CEM-SS cells for 24 h,
and intracellular nucleotides were extracted, separated,
and quantified by reverse-phase HPLC conditions that
also separate TCN-P from TCN. Data in Table 2 show
that in contrast to TCN, intracellular monophosphate
concentrations of 2′-dTCN, 5′-dTCN, 2′,3′-epoxyTCN,
and 2′,3′-d4TCN were not detected. However, the mono-
phosphate forms of 3′-dTCN and 2′,3′-ddTCN were
detected. This observation correlates with the antipro-
liferative and antiviral activity of these compounds
(Table 1) and supports the idea that these analogues
need to be metabolized to the monophosphate in order
to exhibit antiproliferative or antiviral activity. Fur-
thermore, the lack of antiproliferative and antiviral
6-Am in o-8-(2-deoxy-â-D-r ibofu r an osyl)-4-m eth ylpyr r olo-
[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (2′-Deoxytr icir ibin e,
2′-d TCN). Sodium hydride (60% in oil, 470 mg, 11.7 mmol)
was added to a stirred suspension of 6-bromo-4-chloro-5-
cyanopyrrolo[2,3-d]pyrimidine48 (1) (2.0 g, 7.8 mmol) in dry
acetonitrile (150 mL) at room temperature under argon. After
1 h, 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-R-D-ribofuranose43 (2)
(3.0 g, 7.8 mmol) was added and the reaction mixture was
allowed to stir at room temperature for 3 h, at which time TLC
showed the disappearance of starting material and the ap-
pearance of a new spot at Rf ) 0.65 (1:3 ethyl acetate:hexane).
The solvent was removed and the residue was adsorbed onto
silica gel and applied to the top of a silica gel column. The
residue was eluted from the column (7 cm (d) × 9 cm (h)) using
a mixture of ethyl acetate:hexane (1:3) as the eluting solvent
system. Fractions of 25 mL were collected and the fractions
containing spots on TLC with an Rf value of 0.65 (1:3 ethyl
acetate:hexane) were combined and evaporated to dryness to
give 2.90 g of a gummy white solid, presumed to be 6-bromo-
4-chloro-5-cyano-7-(2-deoxy-3,5-di-O-p-toluoyl-â-D-ribofurano-
syl)pyrrolo[2,3-d]pyrimidine44 (3). Without further purification,
this intermediate (3) was dissolved in a mixture of ethanol
(40 mL), chloroform (40 mL), and methylhydrazine (0.49 g, 0.35
mL, 10 mmol) and the reaction mixture was stirred for 18 h
at room temperature. TLC showed the disappearance of
starting material and the appearance of a new spot at Rf )
0.35 (1:3 ethyl acetate:hexane). The solvent was removed and
the residue, presumed to be compound 4, was dissolved in a
mixture of ethyl acetate (100 mL), ethanol (100 mL), and 1 N
ammonium hydroxide (5 mL). Palladium on charcoal (10%, 300
mg) was added and the mixture was placed under 50 psi of
hydrogen gas and shaken on a Parr hydrogenator for 12 h at
room temperature. TLC showed the disappearance of starting
material and the appearance of a new spot at Rf ) 0.39 (1:1
ethyl acetate:hexane). The palladium on charcoal was removed
by filtration through Celite, the solvent was removed under
vacuum, and the residue was adsorbed onto silica gel and
applied to the top of a silica gel column. The residue was eluted
from the column (5 cm (d) × 12 cm (h)) using a mixture of
ethyl acetate:hexane (1:1) as the eluting solvent. Fractions of
25 mL were collected and the fractions containing spots on
TLC with an Rf value of 0.39 (1:1 ethyl acetate:hexane) were
combined and evaporated to dryness to give 1.38 g of a white
solid, presumed to be compound 5. Without further purifica-
tion, this intermediate (5) was suspended in a mixture of
ethanol (100 mL) and 1 drop of concentrated HCl and heated
at reflux temperature for 12 h. TLC showed the disappearance
of starting material and the appearance of a new spot at Rf )
0.19 (1:1 ethyl acetate:hexane). The solvent was removed and
the residue, presumed to be compound 6, was suspended in
methanolic ammonia (100 mL), saturated at 0 °C, and sealed
in a pressure bottle, and the reaction mixture was stirred at

2444 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Porcari et al.
room temperature for 18 h. TLC showed the disappearance of
starting material and the appearance of a new spot at Rf )
0.21 (9:1 chloroform:methanol). The solvent was removed
under vacuum; the residue was adsorbed onto silica gel and
applied to the top of a silica gel column. The residue was eluted
from the column (3 cm (d) × 12 cm (h)) using a mixture of
chloroform:methanol (9:1) as the eluting solvent system.
Fractions of 10 mL were collected and the fractions containing
spots on TLC with an Rf value of 0.21 (9:1 chloroform:
methanol) were combined and evaporated to dryness to give
0.60 g (25% overall yield from compound 1) of 2′-deoxytricir-
ibine (2′-dTCN) as a light brown solid: Rf ) 0.21 (9:1
chloroform:methanol); mp 123-125 °C effervesced, 181-183
°C melted; UV [λmax (ꢀ)] (pH 1) 286 (10239), 278 (10239), (pH
7) 293 (11456), (pH 11) 289 (14370); 1H NMR (DMSO-d6) δ
8.01 (1H, s, CH-2), 7.07 (1H, s, H-7), 6.30 (1H, pseudo t, H-1′),
6.24 (2H, s, NH2), 5.36 (1H, t, OH), 5.31 (1H, d, OH), 4.38 (1H,
m), 3.86 (1H, m), 3.52 (2H, m, H-5′), 3.37 (3H, s, CH3), 2.58
(1H, m, H-2′), 2.25 (1H, m, H-2′). Anal. (C13H16N6O3‚0.25H2O)
C, H, N.
mg (71% yield) of compound 8: Rf ) 0.47 (9:1 chloroform:
methanol); 1H NMR (DMSO-d6) δ 8.06 (1H, s, H-2), 6.97 (1H,
s), 6.91 (1H, s), 6.46 (1H, d), 6.22 (1H, d), 6.20 (2H, bs, NH2),
4.97 (1H, s), 4.30 (1H, m), 4.02 (1H, m), 3.38 (3H, s, CH3), 1.94
(3H, s, CH3), 1.41 (6H, s, 2 × CH3).
Methanolic ammonia (200 mL), saturated at 0 °C, was added
to a pressure bottle containing compound 8 (1.33 g, 2.4 mmol).
The sealed reaction vessel was warmed to room temperature
and the mixture was stirred for 18 h. The solvent was removed
under vacuum and the residue was adsorbed onto silica gel
and applied to the top of a silica gel column. The residue was
eluted from the column (3 cm (d) × 12 cm (h)) using a mixture
of chloroform:methanol (9:1) as the eluting solvent system.
Fractions of 10 mL were collected and the fractions containing
spots on TLC with an Rf value of 0.32 (9:1 chloroform:
methanol) were combined and evaporated to dryness. The
residue was recrystallized in a hot mixture of ethyl acetate
and hexane to give 0.55 g (80% yield) of 2′,3′-d4TCN as a
light brown solid: Rf ) 0.32 (9:1 chloroform:methanol); mp 65
1
°C shrank, 126 °C effervesced, 165 °C dec; H NMR (DMSO-
8-[2-O-Acetyl-3-br om o-3-d eoxy-5-O-(2,5,5-tr im eth yl-1,3-
d ioxola n -4-on e-2-yl)-â-D-xylofu r a n osyl]-6-a m in o-4-m eth -
ylp yr r olo[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (7). 2-Ace-
toxyisobutyryl bromide (7.52 g, 36 mmol, 5.3 mL) was added
to a stirred suspension of triciribine (0.96 g, 3 mmol) in
acetonitrile (40 mL), under argon at room temperature. After
18 h, the reaction mixture was poured into a saturated solution
of sodium bicarbonate (100 mL) and carefully extracted with
ethyl acetate (2 × 75 mL). The aqueous layer was removed
and discarded and the ethyl acetate layer was washed with a
saturated solution of sodium bicarbonate (100 mL) and a brine
solution (100 mL) and then dried over magnesium sulfate. The
magnesium sulfate was removed by filtration and the solvent
was removed under vacuum to yield 1.13 g (71%) of compound
7. The product was used without further purification: Rf )
0.60 (9:1 chloroform:methanol); 1H NMR (DMSO-d6) δ 8.25
(1H, s, H-2), 7.11 (1H, s, H-7), 6.28 (1H, d, H-1′), 5.71 (1H, m),
4.54 (6H, m, NH2, 2 × H-5′, 2H), 3.55 (3H, s, CH3), 2.15 (3H,
s, CH3), 2.08 (3H, s, CH3), 1.60 (3H, s, CH3), 1.58 (3H, s, CH3).
8-[2,3-Ep oxy-5-O-(2,5,5-tr im eth yl-1,3-d ioxola n -4-on e-2-
yl)-â-D-r ibofu r a n osyl]-6-a m in o-4-m eth ylp yr r olo[4,3,2-d e]-
p yr im id o[4,5-c]p yr id a zin e (2′,3′-E p oxyt r icir ib in e, 2′,3′-
ep oxyTCN). Methanolic ammonia (200 mL), saturated at 0
°C, was added to a pressure bottle containing 7 (0.83 g, 1.5
mmol). The sealed reaction vessel was warmed to room
temperature and the mixture was stirred for 18 h. The solvent
was removed under vacuum and the residue was adsorbed onto
silica gel and applied to the top of a silica gel column. The
residue was eluted from the column (3 cm (d) × 12 cm (h))
using a mixture of chloroform:methanol (19:1) as the eluting
solvent. Fractions of 10 mL were collected and the fractions
containing spots on TLC with an Rf value of 0.18 (9:1
chloroform:methanol) were combined and evaporated to dry-
ness to give 0.21 g (47% yield) of 2′,3′-epoxyTCN as a light
brown solid: Rf ) 0.18 (9:1 chloroform:methanol); mp 245 °C
dec; 1H NMR (DMSO-d6) δ 8.07 (1H, s, CH-2), 7.08 (1H, s, H-7),
6.29 (2H, s, NH2), 6.20 (1H, s, H-1′), 5.06 (1H, bs, OH), 4.42
(1H, d), 4.24 (1H, d), 4.13 (1H, t, H-4′), 3.50 (2H, m, H-5′), 3.39
(3H, s, CH3). Anal. (C13H14N6O3‚0.5H2O) C, H, N.
d6) δ 8.04 (1H, s, H-2), 6.96 (2H, s, H-7 and H-1′), 6.49 (1H, m,
H-2′), 6.24 (2H, bs, NH2), 6.12 (1H, m, H-3′), 5.10 (1H, t, OH),
4.84 (1H, m, H-4′), 3.50 (2H, m, H-5′), 3.38 (3H, s, CH3). Anal.
(C13H14N6O2) C, H, N.
6-Am in o-8-(2,3-dideoxy-â-D-r ibofu r an osyl)-4-m eth ylpyr -
r olo[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (2′,3′-Did eox-
ytr icir ibin e, 2′,3′-d d TCN). Compound 8 (0.80 g, 1.9 mmol)
was dissolved in a mixture of ethyl acetate (50 mL) and ethanol
(50 mL) along with 10% palladium on charcoal (100 mg) and
shaken on a Parr hydrogenator under 40 psi of hydrogen gas
for 1 h at room temperature. TLC showed the disappearance
of starting material and the appearance of a new spot at Rf )
0.48 (9:1 chloroform:methanol). The palladium on charcoal was
removed by filtration through Celite and the solvent was
removed under vacuum. The residue, presumed to be inter-
mediate 9, was transferred to a glass pressure bottle. Metha-
nolic ammonia (200 mL), saturated at 0 °C, was added to the
pressure bottle, the sealed pressure bottle was warmed to room
temperature, and the mixture was stirred for 24 h. The solvent
was removed and the residue was recrystallized in a hot
mixture of ethyl acetate and hexane to give 0.40 g (73% yield
from compound 8) of 2′,3′-ddTCN as a light brown solid:
Rf ) 0.36 (9:1 chloroform:methanol); mp 50 °C shrank, 127-
1
130 °C melted; H NMR (DMSO-d6) δ 8.03 (1H, s, H-2), 7.08
(1H, s, H-7), 6.22 (2H, bs, NH2), 6.17 (1H, t, H-1′), 5.12 (1H, t,
OH), 4.10 (1H, m, H-4′), 3.50 (2H, m, H-5′), 3.38 (3H, s, CH3),
2.37 (2H, m, H-2′or H-3′), 2.05 (2H, m, H-2′or H-3′). Anal.
(C13H16N6O2‚0.75H2O) C, H, N.
6-Am in o-8-(3-deoxy-â-D-r ibofu r an osyl)-4-m eth ylpyr r olo-
[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (3′-Deoxytr icir ibin e,
3′-dTCN). 4-Amino-5-cyano-7-(3-bromo-3-deoxy-2,5-di-O-acetyl-
â-D-xylofuranosyl)pyrrolo[2,3-d]pyrimidine46 (11) (3.20 g, 7.30
mmol) and AIBN (1 g) were sealed in a dry 500-mL round-
bottom flask, purged with argon. Dry tetrahydrofuran (150
mL) was added, via syringe, and the mixture was stirred for
15 min before tributyltin hydride (9.0 g, 9.0 mL, 31 mmol) was
added to the reaction mixture, via syringe, and stirred for 10
min. The flask was fitted with a condenser and the reaction
mixture was heated at reflux temperature, under argon, for
18 h. The solvent was removed under vacuum and the oily
residue was diluted with petroleum ether (300 mL). The
resulting suspension was stirred for 6 h and stored at -5 °C
for 48 h before the residue was collected by filtration. Upon
collection, the white residue, presumed to be compound 12 with
an Rf value of 0.73 (9:1 chloroform:methanol), began to absorb
water from the atmosphere and become sticky so without
further purification, it was added to water (150 mL) and the
mixture was heated to 60 °C before glacial acetic acid (20 mL)
was added. Sodium nitrite (9.0 g, 130 mmol) was added, in
six portions, over a period of 30 min. After 1.5 h, TLC showed
the disappearance of starting material and the appearance of
a new spot at Rf ) 0.63 (9:1 chloroform:methanol). The solvent
was removed and the light yellow solid was extracted with
ethyl acetate (150 mL) and a saturated solution of sodium
6-Am in o-8-(2,3-d id eoxy-2,3-d id eh yd r o-â-D-r ibofu r a n o-
s y l )-4 -m e t h y l p y r r o l o [4 ,3 ,2 -d e ]p y r i m i d o [4 ,5 -c ]p y -
r id a zin e (2′,3′-Did eh yd r o-2′,3′-d id eoxytr icir ibin e, 2′,3′-
d 4TCN). Zinc-copper couple (4 g) was added to a solution of
7 (1.5 g, 2.7 mmol), N,N-dimethylformamide (10 mL), and
glacial acetic acid (1 mL) and stirred for 2 h. The reaction
mixture was filtered through Celite and the Celite was washed
with ethyl acetate (50 mL). The filtrate was diluted with ethyl
acetate (50 mL) and extracted with water (50 mL). The
aqueous layer was extracted with ethyl acetate (3 × 50 mL)
and then discarded. The ethyl acetate layers were combined
and washed with a saturated solution of sodium bicarbonate
(20 mL), water (2 × 20 mL), and brine (20 mL) and then dried
over magnesium sulfate. After removing the magnesium
sulfate by filtration, the solvent was evaporated to give 800

Deoxy Sugar Analogues of Triciribine
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2445
bicarbonate (90 mL). The aqueous layer was washed with ethyl
acetate (2 × 50 mL) and then discarded. The ethyl acetate
layers were combined and dried over magnesium sulfate. The
magnesium sulfate was removed by filtration and the ethyl
acetate was evaporated under vacuum to give 1.40 g (53% yield
from compound 11) of compound 13 as a white solid: Rf ) 0.63
15 min before tributyltin hydride (8 g, 8 mL, 27.5 mmol) was
added to the reaction mixture, via syringe. The flask was fitted
with a condenser and the reaction mixture was heated at reflux
temperature, under argon, for 18 h. The solvent was removed
under vacuum and the oily residue was diluted with petroleum
ether (300 mL). The resulting suspension was stirred for 30
min before the residue was collected by filtration and dried
under vacuum at 60 °C for 18 h to give 1.42 mg (81% yield) of
5′-deoxytoyocamycin (18): Rf ) 0.50 (9:1 chloroform:methanol);
mp 144-147 °C shrank, 186-188 °C melted (lit.47 mp 187-
188); 1H NMR (DMSO-d6) δ 8.39 (1H, s), 8.22 (1H, s), 6.87 (2H,
bs), 6.03 (1H, d), 5.45 (1H, d, OH), 5.16 (1H, d, OH), 4.39 (1H,
q), 3.93 (2H, m), 1.27 (3H, d).
1
(9:1 chloroform:methanol); H NMR (DMSO-d6) δ 12.56 (1H,
bs, NH), 8.34 (1H, s), 8.11 (1H, s), 6.21 (1H, s), 5.45 (1H, m),
4.51 (1H, m), 4.22 (2H, m), 2.21 (1H, m), 2.10 (3H, s), 2.02
(3H, s), 1.98 (1H, m).
Without further purification or characterization, 13 (1.2 g,
3.30 mmol) was dissolved in phosphorus oxychloride (25 mL)
and heated at reflux temperature for 1 h. The cooled solution
was poured over ice water and shaken vigorously until all the
phosphorus oxychloride was destroyed, leaving a precipitate,
presumed to be compound 14, suspended in the water. TLC
showed the disappearance of starting material and the ap-
pearance of a new spot at Rf ) 0.95 (9:1 chloroform:methanol).
The precipitate was collected by filtration and air-dried for 18
h. Without further purification or characterization, methyl-
hydrazine (0.11 g, 0.13 µL, 2.4 mmol) was added to a solution
of 14 (0.76 g, 2.0 mmol) in a mixture of ethanol (40 mL) and
chloroform (20 mL) and stirred for 2 h. TLC showed the
disappearance of starting material and the appearance of a
new spot at Rf ) 0.81 (9:1 chloroform:methanol). The solvent
was removed under vacuum and the yellow residue, presumed
to be compound 15, was dissolved in a mixture of ethanol (40
mL) and 1 drop of concentrated HCl. The solution was heated
at reflux temperature for 3 h, at which time TLC showed the
disappearance of starting material and the appearance of a
new spot at Rf ) 0.54 (9:1 chloroform:methanol). The solvent
was removed under vacuum and the residue, presumed to be
compound 16, was dissolved in methanol (20 mL) and trans-
ferred to a glass pressure bottle. Methanolic ammonia (180
mL), saturated at 0 °C, was added to the pressure bottle
containing 16 and the bottle was sealed. The sealed reaction
vessel was warmed to room temperature and the mixture was
stirred for 18 h. The solvent was removed under vacuum and
the residue was adsorbed onto silica gel and applied to the
top of a silica gel column. The residue was eluted from the
column (3 cm (d) × 12 cm (h)) using a mixture of chloroform:
methanol (19:1) as the eluting solvent system. Fractions of 10
mL were collected and the fractions containing spots on TLC
with an Rf value of 0.21 (9:1 chloroform:methanol) were
combined and evaporated to dryness. The residue was recrys-
tallized from a hot mixture of methanol:ethyl acetate:hexane
to give 0.27 g (44% yield from intermediate 14) of 3′-dTCN as
a light brown solid: Rf ) 0.21 (9:1 chloroform:methanol); mp
237 °C dec; 1H NMR (DMSO-d6) δ 8.03 (1H, s, CH-2), 7.05 (1H,
s, H-7), 6.24 (2H, s, NH2), 5.81 (1H, d, H-1′), 5.63 (1H, d, OH),
5.23 (1H, bs, OH), 4.52 (1H, m), 4.35 (1H, m), 3.60 (2H, m),
3.37 (3H, s, NCH3), 2.00 (1H, m, H-3′), 2.15 (1H, m, H-3′). Anal.
(C13H16N6O3‚0.75H2O) C, H, N.
Without further purification, compound 18 (900 mg, 3.27
mmol) was dissolved in a mixture of acetic anhydride (1.34 g,
1.30 mL, 13.08 mmol) and pyridine (50 mL) and the mixture
was stirred for 6 h, under argon. TLC showed the disappear-
ance of starting material and the appearance of a new spot at
Rf ) 0.72 (9:1 chloroform:methanol). The reaction mixture was
evaporated, under vacuum, at 60 °C and the residue was
coevaporated with toluene (2 × 50 mL). The resulting solid,
presumably compound 19, was suspended in water (50 mL)
and the suspension was heated to 60 °C before glacial acetic
acid (5 mL) was added. Sodium nitrite (0.96 g, 14 mmol) was
added, in six portions, over 1 h. The reaction mixture was then
heated to 85 °C for 1.5 h, upon which the suspension became
a solution. TLC showed the disappearance of starting material
and the appearance of a new spot at Rf ) 0.54 (9:1 chloroform:
methanol). The solvent was removed and the light yellow solid
was extracted with ethyl acetate (50 mL) and a saturated
solution of sodium bicarbonate (30 mL). The aqueous layer was
washed with ethyl acetate (2 × 50 mL) and then discarded.
The ethyl acetate layers were combined and dried over
magnesium sulfate. The magnesium sulfate was removed by
filtration and the ethyl acetate was evaporated under vacuum
to give a yellow solid, presumed to be compound 20. Without
further purification or characterization, 20 was dissolved in
phosphorus oxychloride (10 mL) and heated at reflux temper-
ature for 30 min. The cooled solution was poured over ice water
and shaken vigorously until all the phosphorus oxychloride
was destroyed, leaving a light yellow precipitate, presumed
to be compound 21, suspended in the water. TLC showed the
disappearance of starting material and the appearance of a
new spot at Rf ) 0.97 (9:1 chloroform:methanol). The precipi-
tate was collected by filtration and air-dried for 18 h. Without
further purification or characterization, methylhydrazine (60
mg, 70 µL, 1.32 mmol) was added to a solution of 21 (400 mg,
1.1 mmol), ethanol (40 mL), and chloroform (20 mL) and the
mixture was stirred for 2 h. TLC showed the disappearance
of starting material and the appearance of a new spot at Rf )
0.82 (9:1 chloroform:methanol). The solvent was removed
under vacuum and the yellow residue, presumed to be com-
pound 22, was dissolved in a mixture of ethanol (40 mL) and
1 drop of concentrated HCl. The solution was heated at reflux
temperature for 3 h, at which time TLC showed the disap-
pearance of starting material and the appearance of a new
spot at Rf ) 0.51 (9:1 chloroform:methanol). The solvent was
removed under vacuum and the residue, presumed to be
compound 23, was dissolved in methanol (20 mL) and trans-
ferred to a glass pressure bottle. Methanolic ammonia (180
mL), saturated at 0 °C, was added to the pressure bottle
containing 23 and the bottle was sealed. The sealed reaction
vessel was warmed to room temperature and the mixture was
stirred for 18 h. The solvent was removed under vacuum and
the residue was adsorbed onto silica gel and applied to the
top of a silica gel column. The residue was eluted from the
column (3 cm (d) × 12 cm (h)) using a mixture of chloroform:
methanol (19:1) as the eluting solvent system. Fractions of 10
mL were collected and the fractions containing spots on TLC
with an Rf value of 0.30 (9:1 chloroform:methanol) were
combined and evaporated to dryness. The residue was recrys-
tallized from a hot mixture of methanol:ethyl acetate:hexane
to give 0.19 g (57% yield from 21) of 5′-dTCN as a light
brown solid: Rf ) 0.30 (9:1 chloroform:methanol); mp 114-
6-Am in o-8-(5-deoxy-â-D-r ibofu r an osyl)-4-m eth ylpyr r olo-
[4,3,2-d e]p yr im id o[4,5-c]p yr id a zin e (5′-Deoxytr icir ibin e,
5′-d TCN). Toyocamycin49 (4.40 g, 15 mmol) was added to a
solution of thionyl chloride (6 mL) and hexamethylphosphor-
amide (40 mL) previously cooled to 0 °C in an ice bath. The
mixture was allowed to warm to room temperature, under
argon, and then stirred for 18 h. The red solution was poured
over ice water (200 mL) to obtain a precipitate. Concentrated
ammonium hydroxide was added to the reaction mixture to
dissolve the precipitate at pH 7 and reprecipitate the product
at pH 9. The precipitate was collected by vacuum filtration
and the wet solid was washed with water (100 mL) and dried
under vacuum at 60 °C for 3 days to yield 3.6 g (77%) of 5′-
chloro-5′-deoxytoyocamycin (17): Rf ) 0.65 (9:1 chloroform:
methanol); 1H NMR (DMSO-d6) δ 8.42 (1H, s), 8.24 (1H, s),
6.92 (2H, bs), 6.12 (1H, d), 5.61 (1H, d, OH), 5.48 (1H, d, OH),
4.51 (1H, q), 4.10 (2H, m), 3.91 (2H, m).
Without further purification, compound 17 (2.0 g, 6.4 mmol)
and AIBN (0.5 g, 3 mmol) were sealed in a dry 500-mL round-
bottom flask, purged with argon. Dry tetrahydrofuran (100
mL) was added, via syringe, and the mixture was stirred for

2446 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Porcari et al.
116 °C; 1H NMR (DMSO-d6) δ 8.04 (1H, s, CH-2), 7.01 (1H, s,
H-7), 6.20 (2H, s, NH2), 5.81 (1H, d, H-1′), 5.43 (1H, d, OH),
5.16 (1H, bs, OH), 4.39 (1H, q, H-4′), 3.90 (2H, m), 3.37 (3H, s,
NCH3), 1.29 (3H, d, CH3). Anal. (C13H16N6O3‚0.5H2O) C, H,
N.
In Vitr o An tip r olifer a tive Stu d ies. The in vitro cytotox-
icity against murine L1210 leukemic cells was determined in
a cell growth assay described previously.50 L1210 cells were
grown in Fischer’s medium supplemented with 10% heat-
inactivated (56 °C, 30 min) horse serum and subcultured by
serial dilution. Growth rates were calculated from determina-
tions of the number of cells at 0, 24, 48, 72, and 96 h in the
presence of selected concentrations of the test compound. The
50% inhibitory concentration (IC50) was defined as the con-
centration required to decrease the growth rate to 50% of the
untreated control cells. Growth rate was calculated from the
slope of a semilogarithmic plot of cell number against time
for the treated culture as a percent of the control.
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.51 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 cells were passaged twice
weekly at 1:10 dilutions using RPMI 1640 with 10% fetal calf
serum.
HIV-1 a ssa 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.33,52 To evaluate the activities of
compounds in cells acutely infected with HIV, RT was em-
ployed as a marker for HIV-1. CEM-SS cells were infected at
a multiplicity of infection of approximately 0.001 plaque-
forming units (pfu)/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 virus adsorption and then
diluted to 5 × 105 cells/mL with RPMI 1640 containing 10%
fetal bovine serum; 0.1 mL 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 compounds in twice the desired final concen-
tration was added to triplicate wells at seven concentrations
ranging from 100 to 0.14 µM. After 6 days of 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.53
HCMV p la qu e a ssa 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 pfu of HCMV/well using the
procedures detailed earlier.51 Following virus adsorption,
compounds dissolved in growth medium were added to dupli-
cate wells in four to eight selected concentrations. After
incubation at 37 °C for 8-10 days, cell sheets were fixed and
stained with crystal violet and microscopic plaques enumer-
ated. Drug effects were calculated as a percentage of reduction
in number of plaques in the presence of each drug concentra-
tion compared to the number observed in the absence of drug.
HSV-1 ELISA: An enzyme-linked immunosorbent assay
(ELISA)54 was employed to detect HSV-1. Briefly, 96-well
cluster dishes were planted with 10 000 BSC-1 cells/well in
200 µL/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 concentration of 100
pfu/well were added. Following a 3-day incubation at 37 °C,
medium was removed, plates were 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 terminated 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.
Cytotoxicity a ssa 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.51 (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.55 Briefly, 96-
well cluster dishes were planted with KB cells at 5000 cells/
well. After overnight incubation at 37 °C, test compound was
added in triplicate at eight concentrations. Plates were incu-
bated at 37 °C for 48 h in a CO2 incubator, rinsed, fixed with
95% ethanol, and stained with 0.1% crystal violet. Acidified
ethanol was added, and plates were 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. 50% 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.
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 cells were grown in suspension culture with
concentrations of compounds given in Table 2 for 5-24 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 used to
obtain the aqueous phase of supernatant were previously
described.56 Briefly, nucleotides were extracted by vortexing
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 TOA (trioctylamine) was added to the tube.
The mixture was vortexed for 5 s or more 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 chromatography (HPLC)
analysis.
Nu cleotid e An a lysis. TCN-P was separated and quanti-
tated by either anion-exchange HPLC as detailed by Wotring
et al.12 or ion pair reverse-phase HPLC as described by
Walseth et al.57 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, analysis was performed using
anion-exchange HPLC but superior resolution of monophos-
phates was achieved by reverse-phase HPLC; consequently it
was adopted in all subsequent experiments. Ion pair reverse-
phase chromatography was performed on a 3.9 × 300 mm
µBondapak C18 column (Waters). The solvent was 5 mM
tetrabutylammonium hydroxide (TBA), 5% methanol adjusted
to pH 2.5 with formic acid. Separation of nucleotide mono-
phosphates 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 unknown
peaks to retention time of TCN-P standard and on the ratio

Deoxy Sugar Analogues of Triciribine
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2447
(16) 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.
(17) Moore, E. C.; Hurlbert, R. B.; Massia, S. P. Inhibition of CCRF-
CEM human leukemic lymphoblasts by triciribine (tricyclic
nucleoside, TCN, NSC-154020). Accumulation of drug in cells
and comparison of effects on viability, protein synthesis and
purine synthesis. Biochem. Pharmacol. 1989, 38, 4037-4044.
(18) Basseches, P. J .; Durski, A.; Powis, G. High-performance liquid
chromatographic assay of the antineoplastic agent tricyclic
nucleoside 5′-phosphate and its disposition in rabbit. J . Chro-
matogr. 1982, 233, 227-234.
of 290/254 nm due to the absorbance maximum of TCN and
TCN-P at 290 nm.12
Intracellular concentrations were calculated based upon the
integrated area under HPLC peaks of TCN-P, conversion of
area to nmol of TCN-P based upon the area under TCN-P
peaks from standards of known concentration, and conversion
of concentration from nmol/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
hemacytometer, measurement of the diameter of 10-20
representative cells, and assumption of spherical geometry.
(19) Basseches, P. J .; Powis, G. Metabolism and disposition of
3-amino-1,5-dihydro-5-methyl-1-beta-D-ribofuranosyl-1,4,5,6,8-
pentaazaacenaphthylene in the rat. Cancer Res. 1984, 44, 3672-
3678.
(20) Liu, C.; Kimler, B. F.; Evans, R. G.; Morantz, R. A. Intracerebral
chemotherapy in the 9L rat brain tumor model. Proc. Ann. Meet.
Am. Assoc. Cancer Res. 1992, 33, 444.
(21) Schilcher, R. B.; Young, J . D.; Baker, L. H. Reversed-phase high-
performance liquid chromatographic determination of tricyclic
nucleoside and tricyclic nucleoside 5′-phosphate in biological
specimens. J . Chromatogr. 1985, 337, 55-62.
(22) Townsend, L. B.; Milne, G. H. Synthesis, chemical reactivity,
and chemotherapeutic activity of certain selenonucleosides and
nucleosides related to the pyrrolo[2,3-d]pyrimidine nucleoside
antibiotics. Ann. N. Y. Acad. Sci. 1975, 255, 91-103.
(23) Mittelman, A.; Casper, E. S.; Godwin, T. A.; Cassidy, C.; Young,
C. W. Phase I study of tricyclic nucleoside phosphate. Cancer
Treat. Rep. 1983, 67, 159-162.
(24) Cobb, W. R.; Bogden, A. E.; Reich, S. D.; Griffin, T. W.; Kelton,
D. E.; LePage, D. J . Activity of two phase I drugs, homohar-
ringtonine and tricyclic nucleotide, against surgical explants of
human tumors in the 6-day subrenal capsule assay. Cancer
Treat. Rep. 1983, 67, 173-178.
(25) Crabtree, G. W.; Spremulli, E. N.; Weiman, M. C.; Smith, D. E.;
Salvatore, J . R.; Cummings, F. J .; Calabresi, P. Phase I clinical
and pharmacokinetic study of tricyclic nucleoside-5′-monophos-
phate (TCN-P; NSC 280594). Proc. Am. Soc. Clin. Oncol. 1983,
2, 33.
Ack n ow led gm en t. The authors thank Viet Lam
and J ames Lee for expert technical performance of
L1210 cell assays, J ack M. Hinkley for his large-scale
preparation of starting materials, and Kimberly J .
Barrett for assistance with manuscript preparation.
This study was supported by Research Grants RO1-
AI33332 and RO1-AI36872 from the National Institute
of Allergy and Infectious Diseases, by Training Grant
T32-GM07767 from the National Institutes of Health,
and research funds from the University of Michigan.
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]py-
rimido[4,5-c]pyridazine, A new tricyclic nucleoside. Tetrahedron
Lett. 1971, 4757-4760.
(2) 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]py-
rimido[4,5-c]pyridazine-5′-phosphate as a novel compound and
its utility against L-1210 mouse leukemia. U.S. Patent No. 4,
123, 524, Oct 31, 1978.
(3) Plagemann, P. G. W. Transport, phosphorylation, and toxicity
of a tricyclic nucleoside in cultured Novikoff rat hepatoma cells
and other cell lines and release of its monophosphate by the cells.
J . Natl. Cancer Inst. 1976, 57, 1283-1295.
(4) Roti Roti, L. W.; Roti Roti, J . L.; Townsend, L. B. Studies on the
mechanism of cytotoxicity of the tricyclic nucleoside NSC#154020
in L1210 cells. Proc. Am. Assoc. Cancer Res. 1978, 19, 40.
(5) Bennett, L. L., J r.; Smithers, D.; Hill, D. L.; Rose, L. M.;
Alexander, J . A. Biochemical properties of the nucleoside of
3-amino-1,5-dihydro-5- methyl-1,4,5,6,8-pentaazaacenaphthyl-
ene (NSC-154020). Biochem. Pharmacol. 1978, 27, 233-241.
(6) 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.
(7) 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.
(8) Bennett, L. L., J r.; Allan, P. W.; Chang, C. H. Phosphorylation
of “tricyclic nucleoside” by adenosine kinases from L1210 cells
and HEp-2 cells. Biochem. Pharmacol. 1983, 32, 2601-2602.
(9) 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.
(10) Moore, E. C.; Hurlbert, R. B.; Ravel, J . M.; Massia, S. Mecha-
nisms of inhibition of protein and purine biosynthesis by tricyclic
nucleoside phosphate (TCN-P, triciribine phosphate, NSC 280594).
Proc. Am. Assoc. Cancer Res. 1986, 27, 303.
(11) Behrens, B. C.; Hamilton, T. C.; Louie, K. G.; Grotzinger, K. R.;
McKoy, W. M.; Tsuruo, T.; Young, R. C.; Ozols, R. F. Activity of
tricyclic nucleoside 5′-phosphate in model systems of human
ovarian cancer. Invest. New Drugs 1986, 4, 295-304.
(12) 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.
(26) Feun, L. G.; Savaraj, N.; Bodey, G. P.; Lu, K.; Yap, B. S.; Ajani,
J . A.; Burgess, M. A.; Benjamin, R. S.; McKelvey, E.; Krakoff, I.
Phase I study of tricyclic nucleoside phosphate using a five-day
continuous infusion schedule. Cancer Res. 1984, 44, 3608-3612.
(27) Powis, G.; Basseches, P. J .; Kroschel, D. M.; Richardson, R. L.;
O’Connell, M. J .; Kvols, L. K. Disposition of tricyclic nucleoside-
5′-monophosphate in blood and plasma of patients during phase
I and II clinical trials. Cancer Treat. Rep. 1986, 70, 359-362.
(28) Schilcher, R. B.; Haas, C. D.; Samson, M. K.; Young, J . D.; Baker,
L. H. Phase I evaluation and clinical pharmacology of tricyclic
nucleoside 5′-phosphate using a weekly intravenous regimen.
Cancer Res. 1986, 46, 3147-3151.
(29) Hoffman, K.; Holmes, F. A.; Fraschini, G.; Esparza, L.; Frye,
D.; Raber, M. N.; Newman, R. A.; Hortobagyi, G. N. Phase I-II
study: triciribine (tricyclic nucleoside phosphate) for metastatic
breast cancer. Cancer Chemother. Pharmacol. 1996, 37, 254-
258.
(30) O’Connell, M. J .; Rubin, J .; Hahn, R. G.; Kvols, L. K.; Moertel,
C. G. Phase II clinical trial of tricyclic nucleoside phosphate for
advanced colorectal cancer. Cancer Treat. Rep. 1987, 71, 333-
334.
(31) Lyss, A. P.; Morrell, L. E.; Perry, M. C. Triciribine phosphate
(TCN-P) in advanced non-small cell lung cancer (NSCLC):
Interim analysis of toxicity. Proc. Am. Soc. Clin. Oncol. 1991,
10, 120.
(32) Feun, L. G.; Blessing, J . A.; Barrett, R. J .; Hanjani, P. A phase
II trial of tricyclic nucleoside phosphate in patients with
advanced squamous cell carcinoma of the cervix. A Gynecologic
Oncology Group Study. Am. J . Clin. Oncol. 1993, 16, 506-508.
(33) 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. S.; Nassiri, M. R.; Shipman, C.,
J r.; Townsend, L. B.; Drach, J . C. Activity of triciribine and
triciribine-5′-monophosphate against human immunodeficiency
virus types 1 and 2. AIDS Res. Hum. Retroviruses 1993, 9, 307-
314.
(34) Ptak, R. G.; Borysko, K. Z.; Porcari, A. R.; Buthod, J . L.; Holland,
L. E.; Shipman, C., J r.; Townsend, L. B.; Drach, J . C. Phospho-
rylation of triciribine is necessary for activity against HIV-1.
AIDS Res. Human Retroviruses 1998, 14, 1315-1322.
(35) 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 Conference on Retroviruses & Op-
portunistic Infections, Chicago, IL, February 1999; Abstr. 622.
(13) Wotring, L. L.; Roti Roti, J . L.; Hudson, J . L.; Passiatore, J . E.;
Borysko, K. Z.; Newcomb, R. D.; Townsend, L. B. Triciribine
(TCN), A novel tricyclic adenosine analogue with anticancer
activity. Nucleosides Nucleotides 1987, 6, 95-109.
(14) Wotring, L. L.; Townsend, L. B.; Mandell, K. L.; Borysko, K. Z.
Metabolism of triciribine in L1210 cells. Proc. Am. Assoc. Cancer
Res. 1989, 30, 590.
(15) 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.

2448 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Porcari et al.
(36) Townsend, L. B.; Kawasaki, A. M.; VanSickle, A. P.; Wotring,
L. L. The synthesis and biological activity of certain pen-
taazaacenaphthylenes, hexaazaacenaphthylenes and their cor-
responding nucleosides. Nucleic Acids Symp. Ser. 1986, 17, 41-
44.
(37) Kawasaki, A. M.; Townsend, L. B. An unusual reductive ring-
opening of the 1,2,3,5,6,7-hexaazaacenaphthylene ring system.
Tetrahedron Lett. 1989, 30, 3287-3290.
(38) Van Sickle, A.; Kawasaki, A. M.; Townsend, L. B. The Synthesis
of 8-amino-6-N-methyl-1,2,3,5,6,7-hexaazaacenaphthylene. Het-
erocycles 1990, 30, 963-969.
(39) Kawasaki, A. M.; Wotring, L. L.; Townsend, L. B. Synthesis and
cytotoxicity studies of 8-amino-6-methyl-2-beta-D-ribofuranosyl-
1,2,3,5,6,7-hexaazaacenaphthylene (7-aza-TCN) and the corre-
sponding 2′-deoxy- and arabinonucleoside analogues. J . Med.
Chem. 1990, 33, 3170-3176.
(40) Kawasaki, A. M.; Townsend, L. B. Synthesis of 8-amino-4-
methylthio-6-methyl-2-(beta -D-ribofura nosyl)-2,6-dihydro-
1,2,3,5,6,7-hexaazaacenaphthylene and an unusual reductive
ring-opening of the 1,2,3,5,6,7-hexaazaacenaphthylene ring
system. J . Heterocycl. Chem. 1991, 28, 1-5.
(41) Porcari, A. R.; Borysko, K. Z.; Ptak, R. G.; Breitenbach, J . M.;
Wotring, L. L.; Drach, J . C.; Townsend, L. B. Acyclic sugar
analogues of triciribine: Lack of antiviral and antiproliferative
activity correlates with low intracellular phosphorylation. Nu-
cleosides Nucleotides 1999, accepted for publication.
(42) Porcari, A. R.; Townsend, L. B. An improved synthesis of the
versatile heterocycle, 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]py-
rimidine. Synth. Commun. 1998, 28, 3835-3843.
(43) Bhat, C. C. 2-Deoxy-3,5-di-O-p-toluoyl-D-erythro-pentosyl chlo-
ride. Preparation of a crystaline O-acyl-2-deoxypentofuranosyl
halide. In Synthetic Procedures in Nucleic Acid Chemistry;
Zorbach, W. W., Tipson, R. S., Eds.; Interscience Publishers:
New York, 1968; Vol. 1, pp 521-522.
(44) Gupta, P. K.; Vittori, S.; Townsend, L. B. An unexpected
nucleophilic displacement involving 6-bromo-4-chloro-5-cyan-
opyrrolo[2,3-d]pyrimidine with methanolic ammonia. Nucleo-
sides Nucleotides 1990, 9, 35-41.
(45) Russel, A. F.; Greenberg, S.; Moffatt, J . G. Reactions of 2-acyl-
oxyisobutyryl halides with nucleosides. II. Reactions of adenos-
ine. J . Am. Chem. Soc. 1973, 95, 4025-4030.
(46) Krawczyk, S. H.; Townsend, L. B. 2′,3′-Dideoxyadenosine ana-
logues of the nucleoside antibiotics tubercidin, toyocamycin and
sangivamycin. Nucleosides Nucleotides 1989, 8, 97-115.
(47) Wang, Y.; Hogenkamp, H. P. C.; Long, R. A.; Revankar, G. R.;
Robins, R. K. A convenient synthesis of 5′-deoxyribonucleosides.
Carbohydr. Res. 1977, 59, 449-457.
(48) Tolman, R. L.; Robins, R. K.; Townsend, L. B. Pyrrolopyrimidine
nucleosides. 3. The total synthesis of toyocamycin, sangivamycin,
tubercidin, and related derivatives. J . Am. Chem. Soc. 1969, 91,
2102-2108.
(49) Porcari, A. R.; Townsend, L. B. Total synthesis of the naturally
occurring antibiotic toyocamycin using new and improved syn-
thetic procedures. Nucleosides Nucleotides 1999, 18, 153-159.
(50) Wotring, L. L.; Townsend, L. B. Study of the cytotoxicity and
metabolism of 4-amino-3-carboxamido-1- (beta-D-ribofuranosyl)-
pyrazolo[3,4-d]pyrimidine using inhibitors of adenosine kinase
and adenosine deaminase. Cancer Res. 1979, 39, 3018-3023.
(51) Turk, S. R.; Shipman, C., J r.; Nassiri, 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.
(52) 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.
(53) White, E. L.; Buckheit, R. W., J r.; 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.
(54) Prichard, M. N.; Shipman, C., J r. A three-dimensional model to
analyze drug-drug interactions. Antiviral Res. 1990, 14, 181-
206.
(55) Prichard, M. N.; Prichard, L. E.; Baguley, W. A.; Nassiri, M. R.;
Shipman, C., J r. Three-dimensional analysis of the synergistic
cytotoxicity of ganciclovir and zidovudine. Antimicrob. Agents
Chemother. 1991, 35, 1060-1065.
(56) Pogolotti, A. L., J r.; Santi, D. V. High-pressure liquid chroma-
tography-Ultraviolet analysis of intracellular nucleotides. Anal.
Biochem. 1982, 126, 335-345.
(57) Walseth, T. F.; Graff, G.; Moos, M. C., J r.; Goldberg, N. D.
Separation of 5′-ribonucleoside monophosphates by ion-pair
reverse-phase high-performance liquid chromatography. Anal.
Biochem. 1980, 107, 240-245.
J M990205M