Synthesis and Antiviral Activity of 2-Substituted Analogs of Triciribine

Article abstract of DOI:10.1081/NCN-120026873

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

Full text of DOI:10.1081/NCN-120026873

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Synthesis and Antiviral Activity of 2-Substituted
Analogs of Triciribine
Anthony R. Porcari a d , Roger G. Ptak c , Katherine Z. Borysko c , Julie M. Breitenbach c ,
John C. Drach a c & Leroy B. Townsend a b
a Department of Medicinal Chemistry, College of Pharmacy, Ann Arbor, Michigan, USA
b Department of Chemistry, College of Literature, Sciences and Arts, Ann Arbor, Michigan,
USA
c Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan,
Ann Arbor, Michigan, USA
d Pfizer Global Research & Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann
Arbor, Michigan, 48105
Version of record first published: 19 Dec 2011.
To cite this article: Anthony R. Porcari, Roger G. Ptak , Katherine Z. Borysko, Julie M. Breitenbach, John C. Drach & Leroy B.
Townsend (2003): Synthesis and Antiviral Activity of 2-Substituted Analogs of Triciribine, Nucleosides, Nucleotides and Nucleic
Acids, 22:12, 2171-2193
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 12, pp. 2171–2193, 2003
Synthesis and Antiviral Activity of 2-Substituted
Analogs of Triciribine#
3,
3
Anthony R. Porcari,1, Roger G. Ptak, Katherine Z. Borysko,
z
*
Julie M. Breitenbach,3 John C. Drach,1,3 and Leroy B. Townsend1,2
1Department of Medicinal Chemistry, College of Pharmacy,
2Department of Chemistry, College of Literature, Sciences and Arts, and
3Department of Biologic and Materials Sciences, School of Dentistry,
University of Michigan, Ann Arbor, Michigan, USA
ABSTRACT
Triciribine (TCN) and triciribine monophosphate (TCN-P) have antiviral and
antineoplastic activity at low or submicromolar concentrations. In an effort to
improve and better understand this activity, we have conducted a structure-activ-
ity relationship study to explore the effect of substitutions at the 2-position of
triciribine. 2-Methyl-(2-Me-TCN), 2-ethyl-(2-Et-TCN), 2-phenyl-(2-Ph-TCN),
2-chloro-(2-Cl-TCN), and 2-aminotriciribine(2-NH2-TCN) were designed and
synthesized to determine the effects of substitutions at the 2-position which
change the steric, electronic, and hydrophobic properties of TCN, while maintain-
ing the integrity of the tricyclic ring system. These compounds were evaluated for
activity against human immunodeficiency virus (HIV-1), herpes simplex virus
type 1 (HSV-1), and human cytomegalovirus (HCMV) and were found to be
either less active than TCN and TCN-P or inactive at the highest concentrations
#In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.
z
Current affiliation: Southern Research Institute, Frederick, Maryland, USA.
*Correspondence: Anthony R. Porcari, Pfizer Global Research & Development, Ann Arbor
Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105; Fax: (734) 622-5115;
E-mail: Anthony.Porcari@pfizer.com.
2171
DOI: 10.1081/NCN-120026873
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

2172
Porcari et al.
tested, 100 mM. We conclude that substitutions at the 2-position of triciribine
adversely affect the antiviral activity most likely because these analogs are not
phosphorylated to active metabolites.
Key Words: Triciribine; Triciribine monophosphate; 2-Substituted analogs;
HIV; HSV-1; HCMV; Pyrrolo[2,3-d ]pyrimidine nucleosides.
INTRODUCTION
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 prodrug,
triciribine-50-monophosphate[2] (TCN-P) against murine leukemic L1210 cells
revealed their potential as antineoplastic agents. This discovery led to extensive
in vitro[3–17] and in vivo[18–22] studies of TCN and TCN-P as novel antineoplastic
agents. Phase I clinical trials were completed with TCN-P[23–29] and TCN-P was
½27;29ꢀ32ꢁ
advanced to phase II studies as a potential antineoplastic agent.
Early studies revealed that TCN is converted intracellularly to TCN-P by ade-
nosine kinase.[4,6,7] Phosphorylation is essential for antineoplastic activity as demon-
strated 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 adenosine kinase
because extracellular TCN-P is a charged species and does not cross the cell mem-
brane and therefore must be dephosphorylated to TCN by extracellular phospha-
tases or cellular ecto-50-nucleotidases and rephosphorylated to TCN-P by an
intracellular adenosine kinase.[7,12] Unlike other nucleoside analogs, TCN is metabo-
lized only to the monophosphate and not to the di- or triphosphate forms.[9] Further-
more, no incorporation 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.
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 stu-
dies also found no cross resistance to TCN or TCN-P in AZT- or TIBO-resistant
HIV strains[33] 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 specific[3] and was not observed in human cell lines
used to propagate HIV and human cytomegalovirus (HCMV).[33] Even though TCN
was not very cytotoxic in these cell lines, it had to be phosphorylated to TCN-P to be
active against HIV-1.[34] Studies on the antiviral mechanism of action of TCN are
currently underway.
The earliest analogs of TCN that were synthesized and evaluated were the
7-azaTCN analogs,[35–39] which were found[37] 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 requirements 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 of TCN for bio-
logical 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.[40] Our second

2-Substituted Analogs of Triciribine
2173
study explored the hydroxyl requirements of the ribosyl moiety at the N-8 position
of TCN and established that specific dehydroxylation of the ribosyl moiety also
adversely affected phosphorylation and therefore biological activity of the deoxy
TCN derivative.[41]
This manuscript describes the first study in our series of investigations designed
to explore the tolerance and requirements for maintaining biological activity of TCN
when substitutions occur directly on the tricyclic ring system of TCN. We have
synthesized and evaluated new analogs with substitutions at the 2-position of
TCN (e.g., 2-methyl-(2-Me-TCN), 2-ethyl-(2-Et-TCN), 2-phenyl-(2-Ph-TCN), 2-
chloro-(2-Cl-TCN), and 2-aminotriciribine (2-NH2-TCN)) (Fig. 1). These com-
pounds were selected since they would change the steric, electronic, and hydrophobic
properties of TCN, while maintaining the integrity of the tricyclic ring system. Based
on this rationale, we synthesized these analogs for the following reasons: (i) a methyl
or an ethyl group at the 2-position, as representative aliphatic groups, to increase
steric bulk and hydrophobicity while having only a slight electron donating effect;
(ii) a phenyl group at the 2-position as a representative aromatic group to increase
steric bulk and hydrophobicity while having only a slight electron withdrawing
effect; (iii) a chloro group at the 2-position as a representative electron-withdrawing
Figure 1. 2-Substituted analogs of triciribine (TCN).

2174
Porcari et al.
group to increase the electron withdrawing effect as well as steric bulk and hydropho-
bicity; and (iv) an amino group at the 2-position as a representative electron donat-
ing group to increase the electron donating effect as well as decrease hydrophobicity.
The 2-amino group can also invoke hydrogen bonding if a hydrogen acceptor is
available at the binding site of the target molecule. Biological analysis of these ana-
logs has provided some important insights into the tolerance and requirements of
these substitutions on the tricyclic ring system of TCN with regards to phosphoryla-
tion and in vitro activity against selected viruses.
RESULTS AND DISCUSSION
6-Amino-2,4-dimethyl-8-(b-D-ribofuranosyl)pyrrolo[4,3,2-de]pyrimido[4,5-c]py-
ridazine (2-methyltriciribine, 2-Me-TCN) was synthesized, as illustrated in Sch. 1,
from 2-amino-5-bromo-3,4-dicyanopyrrole[42] (1) in 8.5% overall yield. Heating 1
with triethylorthoacetate in acetonitrile at reflux temperature for 1 h and sealing
the isolated product in a steel reaction vessel with ethanolic ammonia at 105ꢂC for
6 h afforded 2 in 78% yield. Silylation of compound 2 with 2 equivalents of N,O-bis
(trimethylsilyl)acetamide (BSA) in dry acetonitrile under argon at room temperature
and then glycosylation with 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose
(TBAR) and 3 equivalents of trimethylsilyl trifluoromethanesulfonate (TMSOTf)
at 80ꢂC for 3 h gave an 88% yield of compound 3. Deprotection of 3 with methanolic
ammonia and reprotection of the intermediate 4 with acetic anhydride in pyridine
afforded the intermediate 5, a less hydrophobic and more manageable intermediate
than compound 3. Debromination of 5 with 10% palladium on charcoal under 50 psi
of hydrogen gas gave the intermediate 6 which was diazotized with sodium nitrite in
aqueous acetic acid at 60ꢂC over 4 h to give the intermediate 7. Compound 7 was
treated with phosphorous oxychloride (POCl3) at reflux temperature for 0.5 h to
obtain the intermediate 8. The reaction of 8 with methylhydrazine in ethanol gave
the intermediate 9 which was ring closed in ethanol, under acidic conditions, at reflux
temperature to afford the intermediate 10. Compound 10 was deprotected with
methanolic ammonia to give 2-methyltriciribine (2-Me-TCN).
6-Amino-2-ethyl-4-methyl-8-(b-D-ribofuranosyl)pyrrolo[4,3,2-de]pyrimido[4,5-c]-
pyridazine (2-ethyltriciribine, 2-Et-TCN) was synthesized, as illustrated in Sch. 2,
from 2-amino-5-bromo-3,4-dicyanopyrrole (1) by a different synthetic route than
the one used for 2-Me-TCN in order to avoid the same deprotection and reprotec-
tion steps needed to convert compound 3 to compound 5. After heating 1 with
triethylorthopropionate in acetonitrile at reflux temperature for 1 h, the isolated pro-
duct was placed in a steel reaction vessel with ethanolic ammonia. The vessel was
sealed and the mixture heated at 105ꢂC for 6 h to afford compound 11 in 94% yield.
Crude 6-bromo-5-cyano-2-ethylpyrrolo[2,3-d ]pyrimidin-4-one (12) was obtained in
90% yield by diazotizing compound 11 with sodium nitrite in aqueous acetic acid
at 100ꢂC for 13 h. Chlorination of compound 12 with phosphorous oxychloride at
reflux temperature for 1 h gave an 81% yield of crude 13. Silylation of compound
13 with 3 equivalents of BSA in dry acetonitrile under argon at reflux temperature
was followed by treatment with 1 equivalent of TBAR and 0.5 equivalents of
TMSOTf in dichloroethane (DCE), under argon, at 70ꢂC for 18 h to afford

2-Substituted Analogs of Triciribine
2175
Scheme 1. Synthesis of 2-methyltriciribine (2-Me-TCN). Reagents: i) (EtO)3CCH3, CH3CN,
(NH3-EtOH); ii) BSA, CH3CN then TBAR, TMSOTf, DCE; iii) NH3, MeOH; iv) Ac2O,
pyridine; v) 10% Pd-C, H2, EtOH, EtOAc, 1N NH4OH; vi) NaNO2, H2O, AcOH; vii) POCl3;
viii) NH2NHCH3, EtOH; ix) HCl, EtOH; x) NH3, MeOH.
compound 14. Treatment of compound 14 with methylhydrazine in ethanol at room
temperature for 1 h afforded 15. Debromination was accomplished by heating a mix-
ture of compound 15, ammonium formate, and 10% palladium on charcoal in etha-
nol at reflux temperature for 4 h to give compound 16. Ring annulation of 16 was
accomplished in ethanol at reflux temperature for 16 h, under acidic conditions, to
give 17. Deprotection of 17 with methanolic ammonia afforded 2-ethyltriciribine
(2-Et-TCN) in a 6% overall yield from compound 1.

2176
Porcari et al.
Scheme 2. Synthesis of 2-ethyl (2-Et-TCN) and 2-phenyltriciribine (2-Ph-TCN). Reagents:
i) (EtO)3CR, CH3CN then NH3, EtOH; ii) NaNO2, AcOH, H2O; iii) POCl3; iv) BSA, CH3CN
then TBAR, TMSOTf, DCE; v) NH2NHCH3, EtOH, CHCl3; vi) HCO2NH4, 10% Pd-C,
EtOH; vii) conc. HCl, EtOH, reflux; viii) NH3, MeOH.
6-Amino-4-methyl-2-phenyl-8-(b-D-ribofuranosyl)pyrrolo[4,3,2-de]pyrimido[4,5-
c]pyridazine (2-phenyltriciribine, 2-Ph-TCN) (Sch. 2) was prepared in 23% overall
yield from 2-amino-5-bromo-3,4-dicyanopyrrole (1) and triethylorthobenzoate by
essentially the same procedure as described above for the preparation of 2-Et-TCN.

2-Substituted Analogs of Triciribine
2177
6-Amino-2-chloro-4-methyl-8-(b-D-ribofuranosyl)pyrrolo[4,3,2-de]pyrimido[4,5-c]-
pyridazine (2-chlorotriciribine, 2-Cl-TCN) and 2,6-diamino-4-methyl-8-(b-D-ribo-
furanosyl)pyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine (2-aminotriciribine, 2-NH2-TCN)
(Sch. 3) were synthesized from 5-cyano-2,4-dichloro-7-(2,3,5-tri-O-acetyl-b-D-ribo-
furanosyl)pyrrolo[2,3-d]pyrimidine[43] (28). Compound 28 was synthesized from
toyocamycin, as previously described,[43] by first forming the 3-N-oxide (25) with
meta-chloroperbenzoic acid in acetic acid and then diazotizing the 4-amino group
with sodium nitrite in N,N-dimethylformamide, acetic acid, and water to give com-
pound 26. Selective protection of the ribosyl hydroxyl groups (27) was accom-
plished with acetic anhydride in pyridine and chlorination of the 2- and 4-
positions was achieved with phosphorous oxychloride at reflux temperature to give
compound 28. Treatment of compound 28 with methylhydrazine in ethanol at
room temperature for 1 h afforded 29. Ring annulation of 29 was accomplished in
ethanol, under acidic conditions, at reflux temperature for 3 h to give compound 30
and subsequent deprotection with methanolic ammonia afforded 2-chlorotriciribine
Scheme 3. Synthesis of 2-chloro (2-Cl-TCN) and 2-aminotriciribine (2-NH2-TCN).
Reagents: i) mCPBA, HOAc; ii) NaNO2, DMF, H2O, AcOH; iii) Ac2O, pyridine; iv) POCl3;
v) NH2NHCH3, EtOH; vi) conc. HCl, EtOH, reflux; vii) NH3, MeOH; viii) NH3(l), 105ꢂC.

2178
Porcari et al.
(2-Cl-TCN) in 50% yield from compound 28. Treatment of 2-Cl-TCN with liquid
ammonia sealed in a steel reaction vessel at 105ꢂC for 7 days afforded 2-aminotri-
ciribine (2-NH2-TCN) in 77% yield.
These TCN analogs (2-Me-TCN, 2-Et-TCN, 2-Ph-TCN and 2-Cl-TCN) were
evaluated in assays that use growth inhibition of human cells as indicators of cyto-
toxicity. 2-Me-TCN and 2-Et-TCN were not cytotoxic to KB cells, human foreskin
fibroblasts (HFF) or CEM-SS cells (Table 1). Only 2-Cl- and 2-NH2-TCN showed
marginal cytotoxicity (IC50 ¼ 33 and 60 mM, respectively) toward KB cells and only
2-Ph-TCN showed marginal cytotoxicity in both HFF cells (IC50 ¼ 21 mM) and
CEM-SS cells (IC50 ¼ 15 mM).
The six target compounds also were evaluated for activity against three viruses.
Activity against HIV-1 was measured in an assay that employs reverse transcriptase
(RT) in culture supernatants as a marker for HIV-1. For 2-Me-TCN and 2-Et-TCN,
it was found that after six days of incubation with CEM-SS cells acutely infected
with HIV, the amount of RT activity in the culture supernatant was equal to that
of control (Table 1) thereby establishing that these two analogs have no activity
against HIV. Under the same conditions, 2-Ph-TCN and 2-Cl-TCN were active at
an IC50 of 3 mM and 25 mM, respectively, suggesting that these two analogs have
activity against HIV, however, at a fraction of the activity of TCN (IC50 ¼ 0.04 mM)
and TCN-P (IC50 ¼ 0.04 mM). 2-NH2-TCN has yet to be tested.
The 2-substituted TCN analogs were also evaluated against HSV-1 in an ELISA
and HCMV in a plaque assay. The replication of HSV-1 was equal to that of control
for all the analogs tested, even at concentrations as high as 100 mM (Table 1). Only
Table 1. Cytotoxicity and antiviral activity of 2-substituted TCN analogs.
50% Inhibitory concentration (mM)
Cytotoxicity in human cellsa
Antiviral activityb
KB
growth
HFF
visual
CEM-SS
visual
HIV-1
RT
HSV-1
ELISA
HCMV
plaque
Compound
TCN
TCN-P
> 100
10
> 100
> 100
> 100
33
100
19
> 100
> 100
21
> 100
> 1.0
> 100
> 100
15
0.04
0.04
> 100
> 100
3
23
20
2.5
0.8
> 100
> 100
32
100
2-Me-TCN
2-Et-TCN
2-Ph-TCN
2-Cl-TCN
2-NH2-TCN
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
NTc
25
NTc
60
> 100
aInhibition of KB cell growth was measured in triplicate as described in the text. Visual
cytotoxicity was scored on uninfected HFF and CEM-SS cells used in HCMV plaque and
HIV RT assays.
bAntiviral activity was determined using an ELISA assay in quadruplicate for HSV-1, a
plaque assay in duplicate for HCMV and the amount of reverse transcriptase (RT) activity
in culture supernatants in triplicate for HIV-1 as described in the text.
cNT ¼ Not tested.

2-Substituted Analogs of Triciribine
2179
2-Ph-TCN was found to have marginal activity (IC50 ¼ 32 mM) against HCMV,
which is less active than TCN (IC50 ¼ 2.5 mM) and TCN-P (IC50 ¼ 0.8 mM).
The phosphorylation of two analogs was determined and compared to that of
TCN. After a 24-h incubation of uninfected CEM-SS cells with 100 mM 2-Me-
TCN, a 5 mM intracellular concentration of a phosphorylated product was detected.
After a similar incubation with 10 mM 2-Ph-TCN, no intracellular product was
found. In contrast, incubation with 10 mM TCN for 5 or 12 h gave intracellular con-
centrations of 175–285 mM TCN-P.
We conclude that a substitution at the 2-position of TCN leads to a loss in anti-
viral activity. Limited data with two compounds suggest that the lack of activity is
due to low phosphorylation of the analogs to an active metabolite.
EXPERIMENTAL
General Procedures. Reaction mixtures were evaporated at temperatures less
than 60ꢂC under reduced pressure (water aspirator) using a Buchi R-151 rotary eva-
porator. Melting points (uncorrected) were obtained on a Laboratory Devises Mel-
Temp melting point apparatus. Thin layer chromatography 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 tet-
ramethylsilane 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.
4-Amino-6-bromo-5-cyano-2-methylpyrrolo[2,3-d ]pyrimidine (2). 2-Amino-5-
bromo-3,4-dicyanopyrrole[42] (1) (8.44 g, 40 mmol) was suspended in dry acetonitrile
(150 mL). Triethylorthoacetate (11 mL, 9.73 g, 60 mmol) was added and the mixture
was heated at reflux temperature for 1 h. The dark brown solution was cooled to
room temperature and filtered through filter paper. The acetonitrile was removed
on a rotoevaporator to afford a brown solid. The brown solid was transferred to a
steel reaction vessel, which was cooled to 0ꢂC in ice water. Ethanolic ammonia
(200 mL), saturated at 0ꢂC, was added to the reaction vessel. The vessel was then
sealed and heated at 105ꢂC in an oil bath for 6 h. The vessel was cooled to 0ꢂC, in
ice water and the dark solution poured into a 500 mL erlenmeyer flask. The solution
was treated with decolorizing carbon (5.0 g) and vacuum filtered through a pad of
Celite. The Celite pad was washed with ethanol (100 mL) and the ethanol was
removed from the solution using a rotoevaporator. The resulting solid was sus-
pended in methanol (150 mL) and then dissolved with 28–30% ammonium hydroxide
(40 mL). The solution was again treated with decolorizing carbon (5.0 g) and vacuum
filtered through a pad of Celite. The filtrate was slowly treated with acetic acid
(80 mL) to precipitate a white solid. This suspension was allowed to stand at
ꢀ5ꢂC for 8 h and the solid collected by filtration. The white solid was suspended

2180
Porcari et al.
in methanol (200 mL), with stirring, at reflux temperature for 1 h, cooled to 5ꢂC, and
filtered. The white solid was dried at 120ꢂC in a vacuum oven at reduced
pressure (water aspirator) for 4 h to yield 7.86 g (78%) of 2. Rf 0.58 (9:1 chloroform:
methanol), mp 340ꢂC dec.; 1H-NMR (DMSO-d6) d 13.73 (1H, bs, NH), 7.34 (2H, bs,
NH2), 2.43 (3H, s, CH3); Anal. Calcd. For C8H6BrN5: C, 38.10; H, 2.38; N, 27.78.
Found: C, 38.23; H, 2.57; N, 27.44.
4-Amino-6-bromo-5-cyano-2-methyl-7-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)pyr-
rolo[2,3-d ]-pyrimidine (3). N,O-Bis(trimethylsilyl)acetamide (BSA, 4.1 g, 2.9 mL,
20 mmol) was added to a stirred suspension of 2 (2.52 g, 10 mmol) in dry acetonitrile
(100 mL) at room temperature under argon. After 15 min, 1-O-acetyl-2,3,5-tri-O-
benzoyl-b-D-ribofuranose (5.0 g, 10 mmol) was added along with trimethylsilyl tri-
fluoromethanesulfonate (TMSOTf, 6.7 g, 5.4 mL, 30 mmol). The suspension was stirred
at room temperature for 10 min, during which time the suspension became a clear
yellow solution. Under argon, the solution was heated at 80ꢂC for 3 h and then
cooled. The solution was diluted with ethyl acetate (100 mL) and then added to a
saturated aqueous solution of sodium bicarbonate (100 mL) at room temperature.
The aqueous layer was separated and discarded and the organic layer was washed
with brine (100 mL) and dried over magnesium sulfate. After filtration, the solvent
was removed and the residue was dissolved in chloroform and eluted from a silica
gel column (7 cm (d) ꢃ 9 cm (h)) using a solvent system of 1:9 ethyl acetate:chloro-
form. Fractions of 100 mL were collected and the UV containing fractions (TLC,
1:4 ethyl acetate:chloroform) with an Rf value 0.31 were combined and evaporated
to dryness. The residue was dissolved in dichloromethane (100 mL) and evaporated.
The residue was dried in a vacuum oven at 65ꢂC for 12 h to obtain 6.1 g (88% yield)
of pure 3. mp 196–198ꢂC; Rf ¼ 0.31 (1:4 ethyl acetate:chloroform); 1H NMR
(CDCl3) d 2.60 (s, 3H), 4.71 (1H, m), 4.84 (2H, m), 5.56 (2H, bs), 6.32 (1H, d),
6.61 (1H, m), 6.66 (1H, m), 7.39 (6H, m), 7.56 (3H, m), 7.97 (6H, m). Anal.
Calcd for: C34H26BrN5O7: C, 58.62; H, 3.74; N, 10.06. Found: C, 58.52; H,
3.91; N, 9.96.
6-Amino-2,4-dimethyl-8-(b-D-ribofuranosyl)pyrrolo[4,3,2-de]pyrimido[4,5-c]pyrida-
zine (2-Methyltriciribine, 2-Me-TCN). Saturated methanolic ammonia (300 mL), at
0ꢂC, was added to a pressure bottle containing 3 (6.1 g, 8.76 mmol). The bottle was
sealed and the reaction mixture was allowed to stir at room temperature for 18 h.
The reaction vessel was cooled to 0ꢂC, opened, and the solvent was evaporated to
dryness. TLC showed the disappearance of starting material and the appearance
of a new spot at Rf ¼ 0.37 (9:1 chloroform:methanol). Without further purification,
the residue, presumed to be compound 4 was dissolved in a mixture of acetic anhy-
dride (4.48 g, 4.35 mL, 43.8 mmol) and pyridine (150 mL) and the mixture was stirred
for 6 h, under argon. TLC showed the disappearance of starting material and the
appearance of a new spot at Rf ¼ 0.87 (9:1 chloroform:methanol). The reaction mix-
ture was evaporated, under vacuum, at 60ꢂC and the residue was coevaporated with
toluene (3 ꢃ 50 mL). The residue, presumed to be compound 5, was dissolved in a
mixture of ethyl acetate (100 mL), ethanol (100 mL), and 1N ammonium hydroxide
(9 mL). Palladium on charcoal (10%, 300 mg) was added and the mixture was placed

2-Substituted Analogs of Triciribine
2181
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 ¼ 0.79 (9:1 chloroform:methanol). The palladium on charcoal
was removed by filtration through Celite and the solvent was removed under
vacuum. Without further purification, water (200 mL) was added to the residue, pre-
sumed to be compound 6, and the mixture was heated to 60ꢂC before glacial acetic
acid (100 mL) was added. Sodium nitrite (9.0 g, 130 mmol) was added, in six por-
tions, over a period of 1 h. After 3 h, TLC showed the disappearance of starting
material and the appearance of a new spot at Rf ¼ 0.60 (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 bicarbonate (90 mL). The aqueous layer
was washed with ethyl acetate (2 ꢃ 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
2.6 g (69% yield from 3) of crude product, presumed to be compound 7. Without
further purification or characterization, 7 (2.6 g, 4.6 mmol) was dissolved in phos-
phorous oxychloride (50 mL) and heated at reflux temperature for 0.5 h. The cooled
solution was poured over ice water and shaken vigorously until all the phosphorous
oxychloride was destroyed, leaving a precipitate which was presumed to be com-
pound 8 suspended in the water. TLC of the precipitate showed the disappearance
of starting material and the appearance of a new spot at Rf ¼ 0.95 (9:1 chloroform:
methanol). The precipitate was collected by filtration and air dried for 2 h. Without
further purification or characterization, methylhydrazine (0.276 g, 0.2 mL, 6 mmol)
was added to a solution of 8 in a mixture of ethanol (80 mL) and chloroform
(40 mL) and stirred at room temperature for 2 h. TLC showed the disappearance
of starting material and the appearance of a new spot at Rf ¼ 0.80 (9:1 chloroform:
methanol). The solvent was removed under vacuum and the yellow residue, pre-
sumed to be compound 9, was dissolved in a mixture of ethanol (60 mL) and 1 drop
of conc. 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 ¼ 0.66 (9:1 chloroform:methanol). The solvent was removed under
vacuum and the residue 10 was dissolved in methanol (20 mL) and transferred to
a glass pressure bottle. Methanolic ammonia (180 mL), saturated at 0ꢂC, was added
and the pressure 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 (3 cm (d) ꢃ 12 cm (h)). The residue was eluted from the column using a
mixture of chloroform:methanol (19:1) as the eluting solvent system. Fractions of
10 mL were collected and the UV containing fractions (TLC, 9:1 chloroform:metha-
nol) with Rf values of 0.18 were combined and evaporated to dryness. The residue
was dried at 60ꢂC in a vacuum oven to obtain 0.36 g (18% yield from 7) of 2-methyl-
triciribine (2-Me-TCN). Rf ¼ 0.18 (9:1 chloroform:methanol); mp 211–212ꢂC dec;
1H NMR (DMSO-d6) d 6.98 (1H, s, H-7), 6.22 (2H, s, NH2), 6.02 (1H, d, OH),
5.76 (1H, d, H-1’), 5.36 (1H, d, OH), 5.18 (1H, t, OH), 4.51 (1H, m), 4.10
(1H, m), 3.98 (1H, m), 3.63 (2H, m), 3.33 (3H, s, NCH3), 2.35 (3H, s, CH3). Anal.
Calcd for: C14H18N6O4ꢄ0.50 CH3OH: C, 49.71; H, 5.71; N, 24.00. Found: C,
49.76; H, 5.69; N, 23.80.

2182
4-Amino-6-bromo-5-cyano-2-ethylpyrrolo[2,3-d ]pyrimidine
Porcari et al.
(11). 2-Amino-5-
bromo-3,4-dicyanopyrrole[42] (1) (8.44 g, 40 mmol) was suspended in dry acetonitrile
(150 mL). Triethylorthopropionate (12 mL, 10.58 g, 60 mmol) was added and the
mixture was heated at reflux temperature for 1 h. The dark brown solution was
cooled to room temperature and filtered through filter paper. The acetonitrile was
removed on a rotoevaporator to afford a brown solid. The brown solid was trans-
ferred to a steel reaction vessel which was cooled to 0ꢂC, in ice water. Ethanolic
ammonia (200 mL), saturated at 0ꢂC, was added to the reaction vessel. The vessel
was then sealed and heated at 105ꢂC in an oil bath for 6 h. The vessel was cooled
to 0ꢂC, in ice water and the dark solution poured into a 500 mL erlenmeyer flask.
The solution was treated with decolorizing carbon (5.0 g) and vacuum filtered
through a pad of Celite. The Celite pad was washed with ethanol (100 mL) and
the ethanol was removed from the solution using a rotoevaporator. The resulting
solid was suspended in methanol (150 mL) and then dissolved with 28–30% ammo-
nium hydroxide (40 mL). The solution was again treated with decolorizing carbon
(5.0 g) and vacuum filtered through a pad of Celite. The filtrate was slowly treated
with acetic acid (80 mL) to precipitate a white solid. This suspension was allowed
to stand at ꢀ5ꢂC for 8 h and the solid collected by filtration. The white solid was
suspended in methanol (200 mL), with stirring, at reflux temperature for 1 h, cooled
to 5ꢂC, and filtered. The white solid was dried at 120ꢂC in a vacuum oven at reduced
pressure (water aspirator) for 4 h to yield 8.62 g (81%) of 11. Rf 0.63 (9:1 chloro-
1
form:methanol), mp 327–329ꢂC dec.; H-NMR (DMSO-d6) d 13.73 (1H, bs, NH),
7.27 (2H, bs, NH2), 2.67 (2H, q, CH2), 1.24 (3H, t, CH3); Anal. Calcd. For
C9H8BrN5: C, 40.60; H, 3.01; N, 26.32. Found: C, 40.83; H, 3.04; N, 26.04.
6-Amino-2-ethyl-4-methyl-8-(b-D-ribofuranosyl)pyrrolo[4,3,2-de]pyrimido[4,5-c]-
pyridazine (2-Ethyltriciribine, 2-Et-TCN). Compound 11 (2.13 g, 8 mmol) was sus-
pended in a mixture of water (100 mL) and glacial acetic acid (50 mL) and heated
to 100ꢂC. Aqueous sodium nitrite (6.9 g, 100 mmol in 100 mL water) was added
dropwise to the suspension over a period of 1 h and then the mixture was allowed
to stir at 100ꢂC for another 12 h. The reaction mixture was cooled to room tempera-
ture and then stored at 5ꢂC for 4 h. The precipitate was collected by vacuum filtra-
tion, resuspended in water, and heated at reflux temperature for 3 h. The reaction
mixture was again cooled to room temperature and then stored at 5ꢂC for 4 h.
TLC of the precipitate showed the disappearance of starting material and the
appearance of a new spot at Rf ¼ 0.44 (9:1 chloroform:methanol). The precipitate,
presumed to be compound 12, was collected by vacuum filtration and dried at
80ꢂC for 12 h in a vacuum oven (crude yield 1.92 g (90%)). Compound 12 (0.80 g,
3 mmol) was suspended in phosphorous oxychloride (50 mL) and heated at reflux
temperature for 1 h. The reaction mixture was cooled to room temperature and then
poured over ice water (300 mL). The mixture was vigorously stirred and the tempera-
ture maintained at 0ꢂC until all of the phosphorous oxychloride was destroyed. The
suspension was extracted with ethyl acetate (3 ꢃ 100 mL). The ethyl acetate extracts
were combined and washed with a saturated solution of sodium bicarbonate
(2 ꢃ 100 mL) and dried over magnesium sulfate. The solvent was removed and the
1
yellow residue, compound 13 [crude yield 0.70 g (81%); H-NMR (DMSO-d6) d

2-Substituted Analogs of Triciribine
2183
2.90 (q, CH2), 1.23 (t, CH3)], was dried at 70ꢂC for 12 h in a vacuum oven and used
without further purification. N,O-Bis(trimethylsilyl)acetamide (BSA, 1.34 g,
0.95 mL, 6.6 mmol) was added to a stirred suspension of 13 (0.63 g, 2.2 mmol) in
dry acetonitrile (50 mL) and then heated at reflux temperature for 20 min, under
argon. The reaction mixture was cooled to room temperature and the solvent was
removed. The residue and 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose (1.12 g,
2.2 mmol) were dissolved in dry dichloroethane (DCE) (50 mL), under argon, and
cooled to 0ꢂC in an ice bath. Trimethylsilyl trifluoromethanesulfonate (TMSOTf,
0.25 g, 0.2 mL, 1.1 mmol) was added to the reaction mixture and the reaction mixture
was stirred at room temperature for 1 h and at 70ꢂC for 18 h. TLC of the reaction
mixture showed the disappearance of starting material and the appearance of a
new spot at Rf ¼ 0.78 (1:1 ethyl acetate:hexane). The reaction mixture was cooled
to room temperature, diluted with dichloromethane (60 mL), and poured over an
ice-cold solution saturated with sodium bicarbonate (100 mL). The aqueous layer
was separated and discarded and the organic layer was washed with water
(50 mL ꢃ 2), brine (50 mL) and dried over magnesium sulfate. After filtration, the
solvent was removed and the residue, presumed to be 14, was suspended in ethanol
(50 mL) and then treated with methylhydrazine (0.14 g, 3 mmol, 0.1 mL). The reac-
tion mixture was allowed to stir for 1 h, at which time a TLC of the reaction mixture
showed the disappearance of starting material and the appearance of a new spot at
Rf ¼ 0.52 (1:1 ethyl acetate:hexane). The solvent was removed under vacuum and the
residue, presumed to be 15, ammonium formate (2.0 g, 30 mmol) and 10% palladium
on activated charcoal (140 mg) were heated in ethanol (100 mL) at reflux temperature
for 4 h. TLC of the reaction mixture showed the disappearance of starting material
and the appearance of a new spot at Rf ¼ 0.49 (1:1 ethyl acetate:hexane). The hot
reaction mixture was filtered through Celite and washed with hot ethanol
(100 mL). The solvent was removed and the residue was dissolved in chloroform,
applied to a silica gel column (5 cm (d) ꢃ 5 cm (h)) and eluted from the column using
a solvent system of 1:1 ethyl acetate:hexane. Fractions of 100 mL were collected and
the UV containing fractions (TLC, 1:1 ethyl acetate:hexane) with Rf values of 0.49
were combined and evaporated to dryness. The residue was dissolved in dichloro-
methane (100 mL) and evaporated. The residue, presumed to be 16, was dissolved
in a mixture of ethanol (100 mL) and 1 drop of conc. HCl. The solution was heated
at reflux temperature for 16 h, at which time TLC showed the disappearance of start-
ing material and the appearance of a new spot at Rf ¼ 0.37 (1:1 ethyl acetate:hexane).
The solvent was removed under vacuum and the residue, presumed to be 17, was dis-
solved in methanol (20 mL) and transferred to a glass pressure bottle. Methanolic
ammonia (180 mL), saturated at 0ꢂC, was added to the pressure bottle and sealed.
The sealed reaction vessel was warmed to room temperature and the mixture was
stirred for 24 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 (3 cm
(d) ꢃ 12 cm (h)). The residue was eluted from the column using a mixture of chlor-
oform:methanol (9:1) as the eluting solvent system. Fractions of 10 mL were col-
lected and the UV containing fractions (TLC, 9:1 chloroform:methanol) with Rf
values of 0.18 were combined and evaporated to dryness. The residue was heated
at reflux temperature in dichloromethane (50 mL) for 2 h, cooled to room tempera-
ture, collected by vacuum filtration, and dried at 70ꢂC in a vacuum oven to

2184
Porcari et al.
obtain 0.07 g (10% yield from 13) of 2-ethyltriciribine (2-Et-TCN). Rf ¼ 0.18 (9:1
1
chloroform:methanol); mp 130–132ꢂC dec; H NMR (DMSO-d6) d 6.98 (1H, s,
H-7), 6.21 (2H, s, NH2), 5.93 (1H, d, OH), 5.75 (1H, d, H-10), 5.37 (1H, d, OH),
5.19 (1H, t, OH), 4.52 (1H, q), 4.08 (1H, m), 3.97 (1H, m), 3.63 (1H, m), 3.55
(1H, m), 3.40 (3H, s, NCH3), 2.60 (2H, q, CH2), 1.21 (3H, t, CH3). Anal. Calcd
for: C15H20N6O4ꢄ1.0 H2O: C, 49.18; H, 6.01; N, 22.95. Found: C, 49.26; H, 5.77;
N, 22.57.
4-Amino-6-bromo-5-cyano-2-phenylpyrrolo[2,3-d ]pyrimidine (18). 2-Amino-5-
bromo-3,4-dicyanopyrrole[42] (1) (8.44 g, 40 mmol) was suspended in dry acetonitrile
(150 mL). Triethylorthobenzoate (13.6 mL, 13.46 g, 60 mmol) was added and the
mixture was heated at reflux temperature for 1 h. The dark brown solution was
cooled to room temperature and filtered through filter paper. The acetonitrile was
removed on a rotoevaporator to afford a brown solid. The brown solid was trans-
ferred to a steel reaction vessel which was cooled to 0ꢂC, in ice water. Ethanolic
ammonia (200 mL), saturated at 0ꢂC, was added to the reaction vessel. The vessel
was then sealed and heated at 105ꢂC in an oil bath for 6 h. The vessel was cooled
to 0ꢂC, in ice water and the dark solution poured into a 500 mL erlenmeyer flask.
The solution was treated with decolorizing carbon (5.0 g) and vacuum filtered
through a pad of Celite. The Celite pad was washed with ethanol (100 mL) and
the ethanol was removed from the solution using a rotoevaporator. The resulting
solid was suspended in methanol (150 mL) and then dissolved with 28–30% ammo-
nium hydroxide (40 mL). The solution was again treated with decolorizing carbon
(5.0 g) and vacuum filtered through a pad of Celite. The filtrate was slowly treated
with acetic acid (80 mL) to precipitate a white solid. This suspension was allowed
to stand at ꢀ5ꢂC for 8 h and the solid collected by filtration. The white solid was sus-
pended in water (200 mL), with stirring, at reflux temperature for 1 h, cooled to 5ꢂC,
and filtered. The white solid was dried at 120ꢂC in a vacuum oven at reduced pres-
sure (water aspirator) for 4 h to yield 11.30 g (90%) of 18. Rf 0.79 (9:1 chloroform:
1
methanol), mp 284–285ꢂC dec.; H-NMR (DMSO-d6) d 13.70 (1H, bs, NH), 8.31
(2H, m,), 7.48 (3H, m), 6.92 (2H, bs, NH2); Anal. Calcd. For C13H8BrN5: C,
49.68; H, 2.55; N, 22.29. Found: C, 49.75; H, 2.77; N, 22.29.
6-Amino-4-methyl-2-phenyl-8-(b-D-ribofuranosyl)pyrrolo[4,3,2-de]pyrimido[4,5-c]-
pyridazine (2-Phenyltriciribine, 2-Ph-TCN). Compound 18 (1.88 g, 6 mmol) was sus-
pended in glacial acetic acid (150 mL) and heated at 60ꢂC. Aqueous sodium nitrite
(20.7 g, 300 mmol in 120 mL water) was added in 40 mL portions to the suspension
over a period of 3 h and then the mixture was allowed to stir at 60ꢂC for another
48 h. The reaction mixture was cooled to 0ꢂC and the precipitate was collected by
vacuum filtration and washed with water (3 ꢃ 50 mL). TLC of the precipitate showed
the disappearance of starting material and the appearance of a new spot at Rf ¼ 0.26
(19:1 chloroform:methanol). The precipitate, 6-bromo-5-cyano-2-phenylpyrrolo[2,3-
d ]pyrimidin-4-one (19) [crude yield 1.80 g (96%); 1H-NMR (DMSO-d6) d 13.91 (1H,
s, NH), 12.65 (1H, s, NH), 8.08 (2H, m), 7.56 (3H, m)], was collected by vacuum fil-
tration and dried at 80ꢂC for 18 h in a vacuum oven. Compound 19 (1.80 g, 6 mmol)

2-Substituted Analogs of Triciribine
2185
was suspended in phosphorous oxychloride (40 mL) and heated at reflux
temperature for 1.5 h. The reaction mixture was cooled to room temperature and
then poured over ice water (200 mL). The mixture was vigorously stirred and the
temperature was maintained at 0ꢂC until all of the phosphorous oxychloride was
destroyed. The precipitate, 6-bromo-4-chloro-5-cyano-2-phenylpyrrolo[2,3-d ]pyri-
1
midine (20) [crude yield 2.05 g (100%); Rf ¼ 0.23 (19:1 chloroform:methanol); H-
NMR (DMSO-d6) d 13.90 (1H, s, NH), 8.33 (1H, m), 8.07 (1H, m), 7.54 (3H, m)],
was collected by vacuum filtration and dried at 80ꢂC for 48 h in a vacuum oven.
Without further purification, N,O-bis(trimethylsilyl)acetamide (BSA, 1.62 g,
1.15 mL, 8 mmol) was added to a stirred suspension of compound 20 (1.33 g, 4 mmol)
in dry acetonitrile (100 mL) and heated at reflux temperature for 30 min, under
argon. The reaction mixture was cooled to room temperature and the solvent was
removed. The residue and 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose (2.04 g,
4 mmol) were dissolved in dry dichloroethane (DCE) (100 mL), under argon, and
cooled to 0ꢂC in an ice bath. Trimethylsilyl trifluoromethanesulfonate (TMSOTf,
0.63 g, 0.5 mL, 2.75 mmol) was added to the reaction mixture and the reaction mix-
ture was stirred at room temperature for 20 min and at 80ꢂC for 18 h. TLC of the
reaction mixture showed the disappearance of starting material and the appearance
of a new spot at Rf ¼ 0.27 (1:3 ethyl acetate:hexane). The reaction mixture was
cooled to room temperature, diluted with dichloromethane (100 mL), and poured
over an ice-cold solution saturated with sodium bicarbonate (100 mL). The aqueous
layer was separated and discarded and the organic layer was washed with water
(2 ꢃ 50 mL), brine (50 mL) and dried over magnesium sulfate. After filtration, the
solvent was removed and the residue, presumed to be compound 21 [crude
yield ¼ 2.6 g (83%)], was dissolved in a mixture of ethanol (100 mL) and chloroform
(50 mL) and then treated with methylhydrazine (0.31 g, 6.6 mmol, 0.35 mL). The
reaction mixture was allowed to stir for 2 h, at which time TLC of the reaction mix-
ture showed the disappearance of starting material and the appearance of a new spot
at Rf ¼ 0.60 (1:1 ethyl acetate:hexane). The solvent was removed under vacuum and
the residue, presumed to be 22, ammonium formate (2.1 g, 33 mmol) and 10% palla-
dium on activated charcoal (260 mg) were heated in ethanol (100 mL) at reflux tem-
perature for 4 h. TLC of the reaction mixture showed the disappearance of starting
material and the appearance of a new spot at Rf ¼ 0.55 (1:1 ethyl acetate:hexane).
The hot reaction mixture was filtered through Celite and washed with hot ethanol
(100 mL). The solvent was removed and the residue was dissolved in chloroform,
applied to a silica gel column (5 cm (d) ꢃ 5 cm (h)) and eluted from the column using
a solvent system of 1:1 ethyl acetate:hexane. Fractions of 100 mL were collected and
the UV containing fractions (TLC, 1:1 ethyl acetate:hexane) with Rf values of 0.55
were combined and evaporated to dryness. The residue was dissolved in dichloro-
methane (100 mL) and evaporated. The residue, presumed to be 23 [crude
yield ¼ 1.45 g (62% yield from 21)], was dissolved in a mixture of ethanol (100 mL)
and 1 drop of conc. HCl. The solution was heated at reflux temperature for 16 h,
at which time TLC showed the disappearance of starting material and the appear-
ance of a new spot at Rf ¼ 0.41 (1:1 ethyl acetate:hexane). The solvent was removed
under vacuum and the residue, presumed to be 24, was dissolved in methanol
(20 mL) and transferred to a glass pressure bottle. Methanolic ammonia (200 mL),
saturated at 0ꢂC, was added to the pressure bottle and sealed. The sealed reaction

2186
Porcari et al.
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 (3 cm (d) ꢃ 12 cm (h)). The residue
was eluted from the column using a mixture of chloroform:methanol (9:1) as the
eluting solvent system. Fractions of 10 mL were collected and the UV containing
fractions (TLC, 9:1 chloroform:methanol) with Rf values of 0.15 were combined
and evaporated to dryness. The residue was heated at reflux temperature in dichlor-
omethane (50 mL) for 20 min, cooled to room temperature, collected by vacuum fil-
tration, and dried at 70ꢂC in a vacuum oven for 18 h to obtain 0.42 g (52% yield from
23) of 2-phenyltriciribine (2-Ph-TCN). Rf ¼ 0.15 (9:1 chloroform:methanol); mp
1
186ꢂC dec; H NMR (DMSO-d6) d 8.35 (2H, m), 7.44 (3H, m), 7.12 (1H, s, H-70),
6.27 (2H, s, NH2), 5.96 (1H, d, H-10), 5.47 (1H, d, OH), 5.27 (1H, d, OH), 5.09
(1H, t, OH), 4.59 (1H, m), 4.17 (1H, m), 3.97 (1H, m), 3.63 (2H, m), 3.35 (3H, s,
NCH3). Anal. Calcd for: C19H20N6O4: C, 57.58; H, 5.05; N, 21.21. Found: C,
57.23; H, 5.20; N, 20.83.
6-Amino-2-chloro-4-methyl-8-(b-D-ribofuranosyl)pyrrolo[4,3,2-de]pyrimido[4,5-c]-
pyridazine (2-Chlorotriciribine, 2-Cl-TCN). 5-Cyano-2,4-dichloro-7-(2,3,5-tri-O-acetyl-
b-D-ribofuranosyl)pyrrolo[2,3-d ]pyrimidine[43] (28) (1.2 g, 2.55 mmol), synthesized
from toyocamycin as previously described (Sch. 3), was dissolved in ethanol
(50 mL) and then treated with methylhydrazine (0.23 g, 5.1 mmol, 0.27 mL). The
reaction mixture was allowed to stir for 1 h, at which time TLC of the reaction
mixture showed the disappearance of starting material and the appearance of a
new spot at Rf ¼ 0.82 (9:1 chloroform:methanol). The solvent was removed under
vacuum to obtain a yellow solid, presumed to be 29 [crude yield ¼ 1.10 g, (90%)].
Intermediate 29 (0.55 g, 1.1 mmol) was dissolved in ethanol (100 mL) and 1 drop
of conc. HCl. The solution was heated at reflux temperature for 2 h, at which time
TLC showed the disappearance of starting material and the appearance of a new
spot at Rf ¼ 0.67 (9:1 chloroform:methanol). The solvent was removed under
vacuum and the residue, presumed to be 30, was dissolved in methanol (20 mL)
and transferred to a glass pressure bottle. Methanolic ammonia (180 mL), satu-
rated at 0ꢂC, was added to the pressure bottle and sealed. The sealed reaction ves-
sel was warmed to room temperature and the mixture was stirred for 24 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 (3 cm (d) ꢃ 12 cm (h)). The residue
was eluted from the column using a mixture of chloroform:methanol (9:1) as the
eluting solvent system. Fractions of 10 mL were collected and the UV containing
fractions (TLC, 9:1 chloroform:methanol) with Rf values of 0.14 were combined
and evaporated to dryness. The residue was heated at reflux temperature in
dichloromethane (50 mL) for 0.5 h, cooled to room temperature, collected by
vacuum filtration, and dried at 100ꢂC in a vacuum oven to obtain 0.22 g (55% yield
from 29) of 2-chlorotriciribine (2-Cl-TCN). Rf ¼ 0.14 (9:1 chloroform:methanol);
1
mp 205–207ꢂC dec; H NMR (DMSO-d6) d 7.13 (1H, s, H-7), 6.46 (2H, s, NH2),
5.78 (1H, d, H-10), 5.43 (1H, d, OH), 5.22 (1H, d, OH), 5.10 (1H, t, OH), 4.37
(1H, q), 4.05 (1H, m), 3.93 (1H, m), 3.60 (2H, m), 3.53 (3H, s, NCH3). Anal. Calcd
for: C13H15ClN6O4ꢄ0.2 CH2Cl2: C, 42.67; H, 4.14; N, 22.24. Found: C, 42.83; H,
4.36; N, 22.23.

2-Substituted Analogs of Triciribine
2187
2,6-Diamino-4-methyl-8-(b-D-ribofuranosyl)pyrrolo[4,3,2-de]pyrimido[4,5-c]pyri-
dazine (2-Aminotriciribine, 2-NH2-TCN). 2-Chlorotriciribine (2-Cl-TCN) (0.25 g,
0.7 mmol) was added to a steel reaction vessel and cooled to ꢀ70ꢂC in a bath of iso-
propanol and dry ice. Liquid ammonia (20 mL) was added and the reaction vessel
was sealed and heated at 100ꢂC for 7 days. The reaction vessel was cooled to
ꢀ70ꢂC in a bath of isopropanol and dry ice and then opened. The liquid ammonia
was allowed to evaporate and the residue was adsorbed onto silica gel and applied to
the top of a silica gel column (3 cm (d) ꢃ 9 cm (h)). The residue was eluted from the
column using a mixture of chloroform:methanol (9:1) as the eluting solvent system.
Fractions of 10 mL were collected and the UV containing fractions (TLC, 9:1 chloro-
form:methanol) with Rf values of 0.10 (9:1 chloroform:methanol) were combined and
evaporated to dryness. The residue was heated at reflux temperature in dichloro-
methane (50 mL) for 2 h, cooled to room temperature, collected by vacuum filtra-
tion, and dried at 80ꢂC in a vacuum oven to obtain 0.18 g (77% yield) of
2-aminotriciribine (2-NH2-TCN). Rf ¼ 0.10 (9:1 chloroform:methanol); mp 240ꢂC
1
dec; H NMR (DMSO-d6) d 6.72 (1H, s, H-7), 6.27 (2H, s, NH2), 5.92 (2H, s,
NH2), 5.75 (2H, m, H-10 and OH), 5.36 (1H, d, OH), 5.11 (1H, d, OH), 4.38 (1H,
m), 4.03 (1H, m), 3.95 (1H, m), 3.59 (2H, m), 3.50 (3H, s, NCH3). Anal. Calcd
for: C13H17N7O4ꢄ0.5 H2O: C, 45.35; H, 5.23; N, 28.48. Found: C, 45.61; H, 5.13;
N, 28.20.
In Vitro Antiviral Studies. Cell Culture Procedures. 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.[44]
Sodium bicarbonate concentration was varied to meet the buffering capacity
required. Cells were passaged at 1:2 to 1:10 dilutions according to conventional pro-
cedures 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 Assay. 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.[33,45] To evaluate the activities
of compounds in cells acutely infected with HIV, reverse transcriptase (RT) was
employed as a marker for HIV-1. CEM-SS cells were infected at a multiplicity of
infection of approximately 0.001 plaque forming units (p.f.u.). per cell with strain
IIIB of HIV-1 in a minimal volume of stock virus in growth medium. Cultures were
incubated at 37ꢂC for 2 h to permit virus adsorption and then diluted to 5 ꢃ 105 cells
per mL with RPMI 1640 containing 10% fetal bovine serum. One-tenth mL was then
added to each well of a 96-well cluster dish which had been pre-treated 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 mM to 0.14 mM. After 6 days incubation, super-
natant 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.[46]

2188
Porcari et al.
HCMV Plaque Assay. 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 (p.f.u.) of
HCMV per well using the procedures detailed earlier.[44] Following virus adsorption,
compounds dissolved in growth medium were added to duplicate wells in four to
eight selected concentrations. After incubation at 37ꢂC for 8–10 days, cell sheets were
fixed, stained with crystal violet and microscopic plaques 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)[47] was
employed to detect HSV-1. Briefly, 96-well cluster dishes were planted with 10,000
BSC-1 cells per well in 200 mL per well of MEM(E) plus 10% calf serum. After over-
night incubation at 37ꢂC, 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 p.f.u.=well were added. Following a 3-day incubation at
37ꢂC, medium was removed, plates were blocked, rinsed, and horse radish peroxi-
dase 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 concen-
tration compared to absorbance obtained with virus in the absence of drug.
Cytotoxicity Assays. Several different assays were used to explore the cytotoxi-
city of selected compounds using methods we have detailed previously. (i) Cytotoxi-
city 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.[44] (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.[48] Briefly, 96-well cluster dishes were
planted with KB cells at 5000 cells per well. After overnight incubation at 37ꢂC, test
compound was added in triplicate at eight concentrations. Plates were incubated at
37ꢂC 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 read at 570 nm in a
spectrophotometer designed to read 96-well ELISA assay plates.
Data Analysis. Dose-response relationships were constructed by linearly regres-
sing the percent inhibition of parameters derived in the preceding sections against log
drugconcentrations. Fifty-percentinhibitory(IC50)concentrationswerecalculatedfrom
the regression lines. Samples containing positive controls [acyclovir, ganciclovir and
zidovudine (AZT), respectively] for HSV-1, HCMV, and HIV were used in all assays.
Extraction Procedures for Chromatographic Analysis. The extraction proce-
dures used to obtain the aqueous phase of supernatant were previously described.[40]
Briefly, CEM-SS cells, grown in suspension with 100 mM concentrations of com-
pounds for 24 h, were harvested by centrifugation at room temp. After harvest,

2-Substituted Analogs of Triciribine
2189
the resulting cell pellet was resuspended in cold PBS, again centrifuged, and the
resulting cell pellet resuspended by vortexing in 0.4 mL of 0.6 N TCA (4ꢂC) and kept
on ice for 15 min. This suspension was microfuged for 30 s at 12,000 g (4ꢂC) and the
supernatant 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 or more sec then microfuged for 30 s at
12,0000 g (4ꢂC). The lower phase was removed by aspiration and discarded. The
aqueous phase was frozen at ꢀ76ꢂC until HPLC analysis.
Reverse Phase HPLC Analysis. HPLC analysis used was previously
described.[49] Briefly, HPLC was performed on a Spectra-physics chromatography
system consisting of a model SP8800 ternary pump, a SP8500 dynamic mixer, a
SP8780 autosampler and a SP8490 variable wavelength detector. Peaks were inte-
grated on a model SP4270 integrator System and data management was achieved
using a Compaq model 386 computer with WINner 386 software (Spectra-physics).
Ion pair reverse-phase chromatography was performed on a 3.9 ꢃ 300 mm m Bonda-
pak C18 column (Waters). The solvent was 5 mM tetrabutylammonium hydroxide
(TBA), 5% methanol pH adjusted to 2.5 with formic acid. Separation of nucleoside
monphosphates was carried out at a flow rate of 1 mL=min with dual wavelength
detection set at 254 and 290 nm.
ACKNOWLEDGMENTS
The authors thank Jack M. Hinkley for his large-scale preparation of starting
materials. 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.
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Received August 20, 2003
Accepted September 5, 2003