Design, synthesis and antiviral activity of novel 4,5-disubstituted 7-(β-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazines and the novel 3-amino-5-methyl-1-(β-d-ribofuranosyl)- and 3-amino-5-methyl-1-(2-deoxy- β-d-ribofuranosyl)-1,5-dihydro-1,4,5,6,7,8-hexaa

Article abstract of DOI:10.1021/jm0402014

The synthesis of several heterocyclic analogues of the biologically important nucleoside antibiotic toyocamycin and the tricyclic nucleoside triciribine (TCN) were prepared along with their 2′-deoxy counterparts. Coupling of 2-nitropyrrole-3,4-dicarboxami

Full text of DOI:10.1021/jm0402014

3840
J. Med. Chem. 2005, 48, 3840-3851
Design, Synthesis and Antiviral Activity of Novel 4,5-Disubstituted
7-(â-D-Ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazines and the Novel
3-Amino-5-methyl-1-(â-D-ribofuranosyl)- and
3-Amino-5-methyl-1-(2-deoxy-â-D-ribofuranosyl)-1,5-dihydro-1,4,5,6,7,8-hexa-
azaacenaphthylene as Analogues of Triciribine
Michael T. Migawa,†,| John C. Drach,§ and Leroy B. Townsend†,‡,
*
Department of Chemistry, College of Literature, Sciences and Arts, Department of Medicinal Chemistry, College of Pharmacy,
and Department of Biological and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, Michigan 48109
Received November 23, 2004
The synthesis of several heterocyclic analogues of the biologically important nucleoside antibiotic
toyocamycin and the tricyclic nucleoside triciribine (TCN) were prepared along with their
2′-deoxy counterparts. Coupling of 2-nitropyrrole-3,4-dicarboxamide (15) under a variety of
conditions with R-chloro-2-deoxy-3,4-di-O-toluoyl-D-ribofuranose (16a) gave mixtures of the R
and â anomers. A coupling of 15 with 1-chloro-2,3,5-tri-O-benzoyl-D-ribofuranose (18) gave
exclusively the â anomer. Individually, the two pyrrole nucleosides were treated with Pd/C,
H2 to reduce the nitro groups and cyclized with nitrous acid, and the corresponding 4-position
was functionalized as a triazoyl derivative. Nucleophillic displacement was carried out with
ammonia to give a mixture of 4-amino-1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)pyrrolo[2,3-d]-
[1,2,3]triazine-5-carbonitrile (26) and 2-amino-1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)pyrrole-
3,4-dicarbonitrile (27), the latter being formed via a retro-Diels-Alder reaction. The subsequent
addition of hydrogen sulfide, water, methanol, hydroxylamine, cyanamide, hydrazine and
methylhydrazine to the 5-cyano group was carried out to give the corresponding analogues. In
the case of methyl hydrazine, subsequent treatment with NaOMe in methanol gave the title
hexaazaacenaphthylenes. Biological evaluation of the compounds established that the pyrrole
(17â, 19-21) and most of the pyrrolotriazine (22, 24, 28, 32-34) nucleosides were inactive or
weakly active against human cytomegalovirus (HCMV) and herpes simplex virus type 1
(HSV-1). In contrast 29 and 31 were active against one or both of these viruses but activity
was poorly separated from cytotoxicity. In contrast, the 2-aza analogue of sangivamycin (30)
was active against HCMV and HSV-1 but this apparent activity was most likely due to its
high cytotoxicity. The tricyclic nucleoside 12, was active against its target virus, human
immunodeficiency virus type 1 (HIV-1), but this activity was not well separated from
cytotoxicity.
Introduction
therapeutic agents, potentially with novel biological
moles of action.3,4
For several years we have been involved in the
synthesis of structural analogues of the naturally oc-
curring purine nucleosides, adenosine and guanosine.1
Several of these analogues have been synthesized and
subsequently demonstrated to be useful chemothera-
peutic agents. These discoveries by our group and others
have contributed to the vast number of known purine
ribo- and 2-deoxyribonucleoside analogues.2 While modi-
fications of the sugar moiety, the heterocylic base and
the exocyclic substituents have all been reported, our
major research interests have focused on modifications
of the purine ring per se. The preparation of these base-
modified nucleosides are synthetically challenging and
can result in the discovery of new classes of chemo-
While many different types of heterocyclic modifica-
tions are possible, most modifications have been limited
to 5:6 fused heterocycles containing only carbon and
nitrogen. Of these 5:6 fused nitrogenous heterocycles,
three basic categories have evolved, i.e., (1) the substi-
tution of carbon for nitrogen (the deazapurines), (2) the
substitution of nitrogen for carbon (the azapurines), and
(3) the transposition of carbon and nitrogen (the aza-
deazapurines).
One class of deazapurine nucleosides that has re-
ceived extensive attention are the pyrrolo[2,3-d]pyrim-
idines.2,5 Significant interest in the synthesis of pyrrolo-
[2,3-d]pyrimidines was generated by the isolation and
biological activity of the naturally occurring nucleoside
antibiotics, tubercidin (1), toyocamycin (2) and sangi-
vamycin (3). Subsequently, further interest was prompted
by the total synthesis of these naturally occurring
compounds.
* To whom correspondence should be addressed. E-mail: ltownsen@
umich.edu.
† Department of Chemistry.
‡ Department of Medicinal Chemistry.
§ Department of Biological and Materials Sciences.
| Current address: IBIS Therapeutics, A Division of ISIS Pharma-
ceuticals, Inc., 2292 Faraday Avenue, Carlsbad, CA 92008.
Tubercidin (1) (Figure 1) inhibits the growth of several
strains of bacteria, and both tubercidin and several
10.1021/jm0402014 CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/04/2005

Triciribine Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3841
pared, and it was subsequently shown to exhibit potent
antitumor activity.16 The water-soluble 5′-monophos-
phate derivative, TCN-P (9), was in phase-II clinical
trials as an anticancer agent.17 More recently, TCN and
TCN-P have been shown to inhibit HIV (IC50
)
60 nM)4,18,19 and act by a unique mechanism.20 Interest-
ingly, 2-deoxy-TCN (10)4 and other recently prepared
sugar-modified analogues19,21 were inactive against
HIV-1. Although a structure-activity relationship has
been built for TCN analogues modified22,23 at the
exocyclic positions of the TCN aglycone, only one
analogue of TCN with a modified heterocyclic ring has
previously been reported, 8-amino-6-methyl-2-(â-D-ri-
bofuranosyl)-1,2,3,5,6,7-hexaazaacenaphthalene (7-Aza-
TCN, 11). However, compound 11 was shown to be
inactive against HIV and noncytotoxic15 despite being
structurally related to compounds 6, 7 and 8. In view
of the above structure-activity relationship studies we
proposed to synthesize the target compound 3-amino-
5-methyl-1-(â-D-ribofuranosyl)-1,5-dihydro-1,4,5,6,7,8-
hexaazaacenaphthalene (12) and the 2-deoxy analogue
13, which are structurally related to 2-4 and 8.
Figure 1.
A subsequent literature search revealed that the
1,4,5,6,7,8-hexaazaacenaphthalene ring system has never
been reported. Additionally, it was of considerable
interest that the entire class of aza-deazapurine nucleo-
side analogues, the pyrrolo[2,3-d][1,2,3]triazine nucleo-
sides, had not been reported in the literature either.
These facts prompted us to initiate studies designed to
develop a route into pyrrolo[2,3-d][1,2,3]triazine nucleo-
sides which could be expanded to include the synthesis
of certain 1,4,5,6,7,8-hexaazaacenaphthalene nucleo-
sides as well.
Figure 2.
Only two pyrrolo[2,3-d][1,2,3]triazines heterocycles
have been reported24,25 in the literature. Since the
synthetic route for the preparation of these pyrrolo-
[2,3-d][1,2,3]triazine heterocycles is very limited in
regards to exocyclic functional groups, we developed a
synthetic route that provided 7-benzylpyrrolo[2,3-d]-
[1,2,3]triazines which was amenable toward functional
group modifications at the C-4, C-5 and C-6 positions.26
In this previous work we demonstrated that the in-
tramolecular diazo-coupling of 1-benzyl-2-aminopyrrole-
3,4-dicarboxamide gave 7-benzyl-5-carboxamidopyrrolo-
[2,3-d][1,2,3]triazin-4-one. However, we were unable to
expand this methodology to include the â-D-ribofurano-
syl- and 2-deoxy-â-D-ribofuranosyl- derivatives of the
pyrrolo[2,3-d][1,2,3]triazine ring system since we were
unabletoobtainthe1-glycosyl-2-aminopyrrole-3,4-dicarbox-
amides that would be required for the synthesis of the
7-glycosyl-pyrrolo[2,3-d][1,2,3]triazines. To prepare the
â-D-ribofuranosyl- and 2-deoxy-â-D-ribofuranosyl deriva-
tives of the pyrrolo[2,3-d][1,2,3]triazine ring system we
needed the appropriate N1-unsubstituted-2-aminopyr-
role-3-carboxamide derivative which could be coupled
to an appropriately substituted sugar derivative and
then converted into the desired 7-(â-D-ribofuranosyl)-
and 7-(2-deoxy-â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]-
triazine derivatives. We have developed an efficient
synthesis of 2-aminopyrrole-3,4-dicarboxamide.27 We
now report on the synthetic efforts undertaken toward
the glycosylation of 2-aminopyrrole-3,4-dicarboxamide
and its precursors and their conversion into the title
compounds.
5-substituted derivatives inhibit both DNA and RNA
viruses albeit at concentrations that inhibit DNA, RNA,
and protein synthesis in mice and human cell lines.6,7
Toyocamycin (2) is a known antineoplastic antibiotic
with specific antitumor activity.8,9 Sangivamycin (3) is
active against L1210 leukemia, P338 leukemia and
Lewis lung carcinoma and has been in clinical trials
against colon cancer, gall bladder cancer and acute
myelogenous leukemia in humans.10,11
An example of the second type of purine modifications
includes the 2-azapurines. Interest in this area was
generated when some 2-azapurine ribonucleoside ana-
logues demonstrated antitumor activity, e.g. 2-aza-
adenosine (4) exhibits cytotoxicity five times greater
than its 8-azapurine counterpart against human epi-
dermoid carcinoma cells in vitro.12
The third major class of purine modifications is the
aza-deazapurines, e.g. the pyrazolo[3,4-d]pyrimidines,
which have demonstrated antitumor activity. The
adenosine analogue (4-APP ribofuranoside, 5) was found
to possess modest antiproliferative properties. However,
the introduction of certain substituents at the three
position of 5 (i.e., 6 and 7) led to a dramatic increase in
the cytotoxicity.13
Structurally related to the above bicyclic purine
nucleoside analogues are the tricyclic nucleoside TCN14
(8, triciribine) (Figure 2) and 8-amino-6-methyl-2-
â-D-ribofuranosyl-1,2,3,5,6,7-hexaazaacenaphtha-
lene15 (7-Aza-TCN, 11). TCN was the first to be pre

3842 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Migawa et al.
Table 1
affecting the reaction rate. Changing the solvent to a
more polar mixture of THF/DMF (entry 3) did not effect
a substantial change in the anomeric ratio; however, in
this case both the pyrrole 15 and its sodium salt were
soluble. Either an increased rate of anomerization of the
chlorosugar 16r or a slow reaction rate could account
for these results.
We then tried a rather novel procedure where the
pyrrole 15 was first silylated with BSA to form an
unidentified silylated intermediate and then treated
sequentially with sodium hydride and the chlorosugar
16r (entry 4). After heating the mixture in aqueous
methanol to effect a desilylation, a 98% yield of
2-nitro-1-[3,5-di-O-(p-toluoyl)-2-deoxy-R-D-ribofuranosyl]-
pyrrole-3,4-dicarboxamide (17r) and 2-nitro-1-[3,5-di-
O-(p-toluoyl)-2-deoxy-â-D-ribofuranosyl]pyrrole-3,4-di-
carboxamide (17â) was obtained in a 1:4 R: â ratio,
respectively. We then found (entry 5) that by cooling
the silylated sodium salt of pyrrole 15 before adding the
chlorosugar (16r) and then allowing the reaction to
warm to ambient temperature gave, after workup, a
98% yield of nucleosides 17r and 17â in a 1:6 R: â ratio,
respectively. Flash chromatography of the R,â-mixtures
was only partially successful in the separation of ano-
mers. However, fractional crystallization, in carbon
tetrachloride and methanol, of the fractions enriched in
the â-anomer gave pure 17â. A small sample of com-
pound 17r was obtained by flash chromatography.
As an additional explanation for our increase in
stereoselectivity when silylated pyrrole 15 is used, it
cannot be ruled out that the unsilylated 2-nitropyrrole
initially reacts with the R-anomer to give an ester of
the expected â-configuration. An intramolecular rear-
rangement would then give the R-glycoside. When the
pyrrole is silylated, the nitro functionality would be
unavailable to participate in the glycosylation and the
esterification-rearrangement mechanism would be un-
available.
entry base
sovent
NaH CH3CN
KOH CH3CN
temp (°C) time (h) R:â yield (%)
1
2
3
4
5
25
25
20
20
20
1
5:3
5:4
5:3
1:4
1:6
74
60
77
98
98
NaH 1:4 DMF/THF 25
NaH CH3CN/BSA
NaH CH3CN/BSA
25
0 then 25
8
Results and Discussion
Chemical. Initial glycosylation attempts were carried
out on 2-nitro-3,4-dicarboxamidepyrrole (15) with
3,5-di-O-toluoyl-2-deoxy-R-D-ribofuranosyl chloride (16r,
Table 1). Compound 16r is readily available by litera-
ture methods28 and is a stable crystalline solid.29 In
contrast, most ribosyl chlorides are difficult to obtain
as a stable solid, and must usually be prepared fresh,
just prior to use.30,31 Therefore, it was convenient to
develop the sodium-salt methodology with this 2-deoxy-
ribofuranosyl chloride rather than its ribofuranosyl
congener.
The standard sodium salt procedure32 for the prepa-
ration of pyrrole nucleosides is carried out by deproto-
nation of the heterocycle with sodium hydride in aceto-
nitrile followed by treatment with the chlorosugar 16r.
This reaction proceeds very rapidly with a complete
Walden inversion and results in the exclusive formation
of a â-nucleoside. 1H NMR studies33 of 16r has demon-
strated that anomerization of this sugar is a function
of both time and solvent polarity; the â-anomer, 16â, is
formed more quickly in more polar solvents than in less
polar solvents. (In CH2Cl2, typically 80% of 16r remains
after 40 min at 25 °C, and 70% remains after 120 min)
Moreover, 16â has been demonstrated to react much
faster than its R-congener. Consequently, relatively slow
reactions, such as the standard Hilbert-Johnson nucleo-
side synthesis,33 react with 16r to give predominately
the R-product. Conversely, relatively rapid reactions,
such as sodium salt reactions, react with 16r to give
predominately the â-product.34
The anomeric configuration of each anomer was
determined by 1H NMR. It has been observed that
the 1H NMR resonance of the anomeric proton of a
R-2-deoxyribonucleoside appears as a quartet with a
peak-width-at-half-height (W1/2) of 10.4 ( 0.4 Hz. Ad-
ditionally the peak for the R-anomeric proton resonance
appears downfield relative to the â-anomeric proton.
1
The H NMR peak for the resonance of the anomeric
proton of compound 17r was observed as a doublet
(JH1,H2a ) O) centered at 6.8 ppm. The W1/2 was
calculated to be 10.5 Hz. Conversely, the 1H NMR
resonance of the anomeric proton of a â-2′-deoxyribo-
nucleoside is usually observed as a ‘pseudo-triplet′ with
a W1/2 of 13.7 ( 0.5 Hz. Compound 17â was in agree-
ment with these observations and a ‘pseudo-triplet’
centered at 0.1 ppm upfield from the 1H NMR resonance
of the anomeric proton of 17r was observed. The W1/2
was calculated to be 13.5 Hz.
The preparation of â-D-ribofuranosyl derivatives by
the sodium salt method generally uses 2,3,5-tri-O-acyl-
D-ribofuranosyl halides.30 Neighboring group participa-
tion of the 2′-acyl group to form an acyloxonium cation
results in the exclusive formation of a â-nucleoside. In
several cases, an attack at the acyloxonium ion by the
heterocycle has been reported to give products substi-
tuted at the 2′-carbonyl carbon.34
It was predicted that 16r would react with the pyrrole
15 under the sodium salt method to give exclusively the
â-anomer. However, as shown in Table 1, the reaction
of 15 under the reported conditions35 (entry 1) gave a
5:3, R/â product ratio. Since both 15 and its correspond-
ing salt are insoluble in acetonitrile, the reaction rate
is reduced and the R-anomer predominates.
Moreover, phase transfer conditions36 (entry 2) gave
similar results, probably also due to the solubility

Triciribine Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3843
Scheme 1
We next initiated studies designed to develop a
method for the nucleophillic displacement of the triazole
substituent of 24 with ammonia in an effort to prepare
4-amino derivatives related to toyocamycin (2) and
sangivamycin (3). Nucleophilic displacement reactions
of nucleoside 24 in liquid ammonia were unsuccessful,
probably due to decomposition. Subsequently, ammonia
dissolved in several different solvents was used, and this
strategy proved to be successful. Subjecting nucleoside
24 to commercially available 0.5 M ammonia in
1,4-dioxane (Aldrich) and acetonitrile at 45 °C gave an
approximate 1.4:1 mixture of 4-amino-1-(2,3,5-tri-O-
benzoyl-â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazine-
5-carbonitrile (26) and 2-amino-1-(2,3,5-tri-O-benzoyl-
â-D-ribofuranosyl)pyrrole-3,4-dicarbonitrile (27). A similar
reaction has been previously reported26 from our labora-
tory. Recently we have reported26 that 7-benzyl-4-(1,2,4-
triazol-1-yl)pyrrolo[2,3-d][1,2,3]triazine-5-carbonitrile un-
dergoes a thermal elimination of nitrogen in the presence
of NH3/CH3CN to give a mixture of 4-amino-7-benzyl-
pyrrolo[2,3-d][1,2,3]triazine-5-carbonitrile and 2-amino-
1-benzylpyrrole-3,4-dicarbonitrile. Two 15N labeling stud-
ies have suggested that this reaction may proceed by a
retro-Diels-Alder reaction involving the elimination of
N-2 and N-3 from the imino tautomer of compound 26.38
Deprotection of 26 with sodium carbonate in ethanol,
followed by a work up with acetic acid, gave ethyl
4-amino-1-(â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazine-
5-carboxylate (33). This compound (33) is most likely
formed by the hydrolysis of an intermediate ethyl
imidate. It was found that nucleoside 26 could be
deprotected under acyl transfer conditions. Treatment
of 26 with a catalytic amount of potassium cyanide in
methanol gave methyl 4-amino-1-(â-D-ribofuranosyl)-
pyrrolo[2,3-d][1,2,3]triazine-5-formimidate (28).
Attempts to prepare the 5-thioamide analogue by
treating the methyl imidate 28 with sodium hydrogen-
sulfide produced a complex mixture of products. In
direct contrast, a variety of other nucleophiles produced
a clean reaction with the methyl imidate 28 (Scheme
3). Treatment of 28 with hydrazine monohydrate in H2O
at reflux temperature afforded the carboxamidrazone,
4-amino-1-(â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazine-
5-carboxamidrazone (29), in good yield. The target
compound, 4-amino-1-(â-D-ribofuranosyl)pyrrolo[2,3-d]-
[1,2,3]triazine-5-carboxamide (2-azasangivamycin, 30),
was obtained by the treatment of 28 with an aqueous
solution of sodium hydroxide. The carboxamidoxime,
4-amino-1-(â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazine-
5-carboxamidoxime (31) and the cyanoamidine, 4-amino-
1-(â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazine-5-N-
cyanoamidine (32), were prepared by heating 28 in an
aqueous solution of hydroxylamine or cyanamide, re-
spectively.
Therefore, we elected to use the procedure we had
developed for the preparation of the 2-deoxy derivative
in order to circumvent the possibility of substitution at
the 2′-carbonyl carbon. 2-Nitropyrrole-3,4-dicarbox-
amide (15) was silylated with BSA to form an unidenti-
fied silylated intermediate and then treated sequentially
with sodium hydride and freshly prepared 2,3,5-(tri-O-
benzoyl)-D-ribofuranosyl chloride.37 When gylcosylation
was complete, the product was heated in aqueous
methanol to effect a desilylation and furnished a 70%
yield of 2-nitro-1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)-
pyrrole-3,4-dicarboxamide (19). As expected, the â-ano-
mer was the only anomer formed and no attack at the
2′-acylcarbonyl carbon by the sodium salt of pyrrole 15
was observed.
Finally, a reduction of the 2-nitro groups of 19 and
17â was accomplished by hydrogenation over 10%
palladium on carbon in THF/ethanol to give 2-amino-
1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)pyrrole-3,4-di-
carboxamide (20) and 2-amino-1-[3,5-di-O-(p-toluoyl)-
2-deoxy-â-D-ribofuranosyl]pyrrole-3,4-dicarboxamide (21),
respectively (Scheme 2). This demonstrated the feasibil-
ity of accessing the 2-aminopyrrole 2-deoxyribosides and
ribosides by the coupling of an appropriately substituted
carbohydrate with 2-nitropyrrole-3,4-dicarboxamide fol-
lowed by a catalytic hydrogenation reaction. Studies
were then initiated to develop a synthesis of the 1,2,3-
triazine portion of the 4-amino-1-(â-D-ribofuranosyl)-
pyrrolo[2,3-d][1,2,3]triazines by the diazo-coupling con-
ditions that were used successfully for the preparation
of the N-benzyl derivative.26 These initial attempts were
unsuccessful due to the insolubility of the pyrrole
ribonucleoside 20 in HCl (aq) at -35 °C and resulted
in the starting material being recovered. Moreover,
allowing the reaction to warm to higher temperatures
resulted in decomposition of the starting material.
Subsequently, it was found that the use of acetonitrile
as a cosolvent soubilized 20 at the required low tem-
peratures and 5-carboxamido-1-(2,3,5-tri-O-benzoyl-â-
D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazin-4-one (22) was
obtained in a good yield. Likewise, treatment of the
2′-deoxyribonucleoside 21 with sodium nitrite in HCl
(aq) and acetonitrile at -35 °C afforded 1-(3,5-di-O-p-
tolouyl-2-deoxy-â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]-
triazin-4-one-5-carboxamide (23). These compounds were
the first examples of pyrrolo[2,3-d][1,2,3]triazine nu-
cleosides. The treatment of compound 22 and 23 with a
premixed suspension of POCl3, 1,2,4-triazole and tri-
ethylamine afforded 4-(triazol-1-yl)-1-(2,3,5-tri-O-ben-
zoyl-â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazine-5-
carbonitrile (24) and 4-(triazol-1-yl)-1-[3,5-di-O-(p-toluoyl)-
2-deoxy-â-D-ribofuranosyl]pyrrolo[2,3-d][1,2,3]triazine-
5-carbonitrile (25), respectively. These intermediates
were useful in the preparation of other N-4 and N-5
substituted pyrrolo[2,3-d][1,2,3]triazine ribonucleosides.
Since we were unable to prepare 2-azathiosangiva-
mycin directly from 28, we opted to modify our synthetic
route. The thioamide 34 was prepared by heating a
mixture of compound 26 in a solution of sodium hydro-
gensufide, generated in situ, and methanol in a sealed
tube. Deprotection of this benzoylated thioamide inter-
mediate could then be accomplished using potassium
cyanide in methanol to give 4-amino-1-(â-D-ribofur-
anosyl)pyrrolo[2,3-d][1,2,3]triazine-5-thiocarboxamide

3844 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Migawa et al.
Scheme 2
Scheme 3
Scheme 4
(2-azathiosangivamycin, 34), which was isolated as a
mono hydrate.
The final pyrrolopyrimidine analogue target, 2-azato-
yocamycin, was then prepared from 2-azathiosangiva-
mycin. Treatment of 34 with excess methyl iodide in
ammonium hydroxide gave the desired 4-amino-1-(â-D-
ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazine-5-carboni-
trile (2-azatoyocamycin, 35). This reaction presumably
proceeds through a methylation of the thione group
followed by an elimination.
We next turned our attention to the preparation of
2-aza-TCN and 2-deoxy-2-aza-TCN. A displacement of
the triazole group of nucleoside 24 and 25 with methyl-
hydrazine in methanol gave 4-(1-methylhydrazino)-1-
(â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazine-5-carbo-
nitrile (36) and 4-(1-methylhydrazino)-1-[3,5-di-O-
(p-toluoyl)-2-deoxy-â-D-ribofuranosyl]pyrrolo[2,3-d][1,2,3]-
triazine-5-carbonitrile (37), respectively (Scheme 4). It
is interesting to note that very high regioselectivity and
chemoselectivity was achieved and neither the 2-meth-
ylhydrazino adduct was formed nor did any deacylation
occur. Treatment of compound 36 or 37 with sodium
methoxide in methanol effected a removal of the acyl
groups and a concomitant cyclization to afford 3-amino-
5-methyl-1-(â-D-ribofuranosyl)-1,5-dihydro-1,4,5,6,7,8-
hexaazaacenaphthalene (12) and 3-amino-5-methyl-1-
(2-deoxy-â-D-ribofuranosyl)-1,5-dihydro-1,4,5,6,7,8-
hexaazaacenaphthalene (13), respectively. These are the

Triciribine Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3845
Table 2. Antiviral Activity and Cytotoxicity of Pyrrole, Pyrrolotriazine and Tricyclic Nucleosides
50% inhibitory concentratin (µM)a,b
HCMV
HSV-1
ELISA
HIV-1
HFF
KB
compound
heterocycle
plaque
yieldc
RT/CPE
visual
growth
17â
19
20
21
22
24
28
29
30
31
32
33
34
36
12
8
pyrrole
32
-
-
>100
>100
>10d
>100
>10d
>10d
>100
80
-
32
pyrrole
32
-
32
pyrrole
pyrrole
>10d
12
-
-
>10d
32
>10d
100
>10d
>10d
20
-
-
pyrrolotriazine
pyrrolotriazine
pyrrolotriazine
pyrrolotriazine
pyrrolotriazine
pyrrolotriazine
pyrrolotriazine
pyrrolotriazine
pyrrolotriazine
pyrrolotriazine
tricyclic
>10d
>10d
32
-
-
>10d
>10d
21
-
-
-
-
32
0.3
1.0
0.4
-
-
32
0.3
3
2.5
-
0.08
1.5
<2.0e
>100e
>100
8.5
0.9
-
6.6e
>100e
>100
32
-
>100
>100
40
-
5
-
-
80
-
-
2
>10d
14
-
>10d
>100
23
-
>10d
10
>10d
2.5
-
2.2/0.6
0.04/-f
0.02g
tricyclic
(triciribine, TCN)
2.5f
-
100e
100e
a Antiviral activity was determined using a plaque assay in duplicate for HCMV and an ELISA in quadruplicate for HSV-1. Two
different assays were used for HIV-1: The amount of reverse transcriptase (RT) activity in culture supernatants was measured in triplicate
and viral cytopathogenic effect (CPE) was scored by visual inspection of duplicate cultures, all as described in the text. Ganciclovir was
used as a positive control in all HCMV assays, acyclovir was used in all HSV-1 assays, and zidovudine (AZT) was used in the HIV-1
assays. b Visual cytotoxicity was scored on uninfected HFF cells not affected by virus in HCMV plaque assays. Inhibition of KB cell
growth was measured as described in the text in quadruplicate assays; 2-acetylpyridine thiosemicarbazone was used as a positive control.
c 90% inhibitory concentration. d Limit of solubility. e Average of duplicate experiments. f Data previously presented in ref 23. g Data derived
by syncytial plaque assay, presented previously in ref 4.
first reported examples of nucleosides containing the
1,4,5,6,7,8-hexaazanaphthalene ring system.
growth inhibitory. These effects were comparable to
those on HFF cells but somewhat less than on KB cells
(Table 2). The 2-aza analogue of TCN (12) was the least
growth inhibitory, approximately 100-less so than san-
givamycin. However, even though it was less cytotoxic,
it’s activity against HIV-1 occurred at concentrations
only a few fold lower than its growth inhibitory concen-
trations. This is significantly less favorable than TCN
itself, leading us to conclude that 2-aza-TCN is less
promising as an anti-HIV compound than is TCN.
Biological. The target compound 2-aza-TCN (12) and
several precursor nucleosides were evaluated for activity
against two herpesviruses (human cytomegalovirus
(HCMV) and herpes simplex virus type 1 (HSV-1). The
pyrrole nucleosides (17, 19-21) and most of the pyrro-
lotriazine nucleosides (22, 24, 28, 32-34) were inactive
or only weakly active against HCMV and HSV-1 (Table
2). Compounds such as 21 or 34, that had modest
activity against HCMV, were cytotoxic at concentrations
of only two to three times their antiviral concentrations.
Compounds 29 and 31 were active against one or both
of these viruses but their activity was poorly separated
from cytotoxicity. In contrast, the 2-aza analogue of
sangivamycin (30) appeared to be very active against
HCMV and HSV-1 but this activity was most likely due
to its high cytotoxicity (Table 2).
2-Aza-TCN, 12, had only weak activity against HCMV
(most likely related to its cytotoxicity) and no activity
against HSV-1. In contrast, it was active against human
immunodeficiency virus type 1 (HIV-1) in two different
assays (Table 2). But this activity was observed only at
concentrations near those that produced cytotoxicity in
HFF (human diploid fibroblasts) and KB (human can-
cer) cells. Thus in comparison to triciribine (TCN), this
new TCN analogue (12) was less active against HIV-1
and more cytotoxic (Table 2).
Experimental Section
Melting points were determined on a Thomas-Hoover ap-
paratus and are uncorrected. Silica gel, SilicAR 40-63 µm
230-400 mesh (Mallinckrodt) was used for chromatography.
Flash column chromatography refers to the chromatography
technique described by Still (J. Org. Chem. 1978, 43, 2923-
2925). (X% EtOAc/Hex, Y cm × Z cm) means the solvent
system that is used as the eluent, the diameter of the column
(Y) and the height of silica gel (Z). Solvent systems are
expressed as a percentage of the more polar component with
respect to total volume (v/v%). Thin-layer chromatography
(TLC) was performed on prescored SilicAR 7GF plates (Anal-
tech, Newark, DE). Compounds were visualized by illuminat-
ing with UV light (254 nm) or by spraying with 10% meth-
anolic sulfuric acid followed by charring on a hot plate.
Evaporations were carried out under reduced pressure (water
aspirator) with the bath temperature not exceeding 50 C,
unless specified otherwise. The 1H (300, 360, or 500 MHz) 13
C
(75, 90, or 125 MHz) and 15N (50 MHz) NMR spectra were
recorded on Bruker instruments. The chemical shifts are
expressed in parts per millions relative to the standard
chemical shift of the solvent for DMSO-d6; 2.50 ppm (1H NMR),
39.50 ppm (13C NMR), and relative to tetramethylsilane as an
internal standard for CDCl3 (1H NMR), and relative to the
standard chemical shift of the solvent for 13C NMR (77.0 ppm).
Chemical shifts for 15N NMR spectra are recorded in ppm
relative to 2% 15N-benzamide, 0.2% Cr(Acac)3 in DMSO-d6 as
an external standard (100.0 ppm). 1H NMR assignments
reported were made by homonuclear decoupling experiments,
or COSY experiments, or assigned by analogy. 13C NMR
assignments were made by J-modulated decoupling, DEPT-
To more thoroughly examine the cytotoxicity of key
compounds, the effects of sangivamycin (3), 2-aza-
sangivamycin (30) and 2-aza-TCN (12) on the growth
of CEM-SS cells was examined. This cell line was chosen
because HIV-1 was propagated in it thus a direct
comparison of anti-HIV activity to cytotoxicity is most
valid. The data in Figure 3 show that as reported several
times previously,8 sangivamycin was highly toxic, in-
hibiting cell growth at 0.01 to 0.1 µM. In contrast, its
2-aza analogue (30) was approximately 10-fold less

3846 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Migawa et al.
25 min at 0 °C, the reaction was allowed to proceed at room
temperature for 8 h. At that time the solvent was evaporated
under reduced pressure until a syrup was obtained. 90%
MeOH (aq) (15 mL) was added, and the reaction was then
heated on a steam bath for 10 min. At that time, the whole
reaction mixture was partitioned between H2O and EtOAc
(20 mL/20 mL), the organic layer was separated and washed
with brine (10 mL), dried (Na2SO4) and filtered and the filtrate
was evaporated under reduced pressure. The resultant syrup
was subjected to flash chromatography (2% MeOH/CHCl3 then
6% MeOH/CHCl3, 2 cm × 25 cm). The product, Rf ) 0.32
(10%MeOH/CHCl3), was collected to give a fraction containing
21 mg of 17 with an ratio of 1:0.6, and a 255 mg fraction
containing 17 with an ratio of 1:8.7, as determined by integra-
tion of the 1H NMR resonances for the anomeric protons.
Recrystallization of 250 mg of this latter fraction in CCl4/
MeOH gave 142 mg (57% recovery) of pure 17â.
17â: mp 198-199 °C; Rf ) 0.32 (10%MeOH/CHCl3); 1H
NMR (DMSO-d6, 300 MHz) δ 8.06 (s, 1H, H-5), 7.92 (bd, 3H,
collapses to d, 2H upon addition of D2O, CONH2, Ph), 7.83
(d 2H, j ) 8.0 Hz, Ph), 7.63 (bs, 1H, D2O exchangeable,
CONH2), 7.53 (bs, 1H, D2O exchangeable, CONH2), 7.37
(d, 2H, J ) 8.0 Hz, Ph), 7.30 (d, 2 H, J ) 8.0 Hz, Ph), 7.3 (bs,
1 H, D2O exchangeable, CONH2), 6.72 (t, 1 H, J ) 6.2 Hz, H-1′),
5.59 (m, 1 H, H-3′), 4.68-4.54 (m, 3 H, H-4′, H-5′), 2.96-2.88
(m, 1 H, H2a′), 2.73-2.66 (m, 1 H, H-2b′), 2.40 (s, 3 H, CH3),
2.37 (s, 3 H, CH3); 13C NMR (DMSO-d6, 75 MHz): 165.5, 165.2,
164.3, 162.6, 144.2, 143.9, 133.2, 129.5, 129.4, 129.3, 126.5,
126.4, 124.7, 124.2, 117.4, 88.2, 82.3, 74.0, 63.9; Anal.
(C27H26N4O9): C, H, N. 17r: A small sample of compound 17r
was obtained as an oil by repeated flash chromatography (2%
MeOH/CHCl3 then 6% MeOH/CHCl3, 2 cm × 25 cm): Rf )
0.34 (10%MeOH/CHCl3); 1H NMR (DMSO-d6, 300 MHz) δ 8.14
(s, 1 H, H-5), 7.94 (d, 2 H, J ) 7.9 Hz, Ph), 7.76 (bs, 1 H, D2O
exchangeable, CONH2), 7.63 (d, 2 H, j ) 7.9 Hz, Ph, Ph), 7.50
(bs, 1 H, D2O exchangeable, CONH2), 7.53 (bs, 1 H, D2O
exchangeable, CONH2), 7.38 (d, 2 H, J ) 7.9 Hz, Ph), 7.23
(d, 2 H, J ) 7.4 Hz, Ph), 6.80 (d, 2 H, J ) 6.1 Hz, H-1′), 5.61
(m, 1 H, H-3′), 5.2 (m, 1 H, H-5′), 4.5 (m, 2 H, H-4′), 3.15-3.09
(m, 1 H, H2a′), 2.53-2.47 (m, 1 H, H-2b′), 2.40 (s, 3 H, CH3),
2.35 (s, 3 H, CH3); HRMS (CI,NH3) Calcd. for (M + H)
C27H27N9, predicted: 551.0778, Found: 551.0795.
2-Nitro-1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)pyrrole-
3,4-dicarboxamide (19). Compound 19 was prepared in a
69% yield from bis(trimethylsilyl)acetamide (10.0 mL,
40.6 mmol), 2-nitropyrrole-3,4-dicarboxamide (15, 2.01 g,
10.0 mmol), NaH (60% dispersion in mineral oil, 487 mg,
13.2 mmol) and 2,3,5-(tri-O-benzoyl)-D-ribofuranosyl chloride30
(18, 6.3 g, 13.2 mmol) in CH3CN (100 mL) as described for
compound 17: mp 125 °C (sinters); Rf ) 0.46 (10%MeOH/
CHCl3); 1H NMR (DMSO-d6, 300 MHz) δ 8.1-7.4 (m, 20H,
collapses to 18H upon adddition of H2O, Ph, H-5, CONH2), 6.81
(d, 2H, J ) 1.8 Hz, H-1′), 5.96 (m, 1H, H-2′), 5.79 (m, 1H, H-3′),
4.96 (m, 1H, H-4′), 4.79 (m, 2H, H-5); Anal. (C32H26N4O11):C,
H, N.
Figure 3. Effect of selected compounds on the growth of CEM-
SS cells. Cells were planted in the presence of the indicated
concentrations of compounds, harvested on the days shown
and enumerated by Coulter counter.
2-Amino-1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)pyr-
role-3,4-dicarboxamide (20). Compound 20 was prepared
in a quantitative yield by hydrogenation of compound 19
(2.04 g, 3.17 mmol) with 10% palladium on carbon (95 mg)
and H2 (40 psi) in EtOH/THF (75 mL/35 mL) as described for
compound 21. An analytical sample was prepared by recys-
tallization of 500 mg of 20 with a mixture of EtOH/EtOAc and
drying under reduced pressure at 78 °C for 24 h to give
263 mg (53% recovery) of pure 20: mp 230-232 °C; Rf ) 0.62
(10%MeOH/CHCl3); 1H NMR (DMSO-d6, 300 MHz) δ 9.75 (bs,
1 H, D2O exchangeable, CONH2), 8.0-7.9 (m, 6 H, Ph),
7.7-7.6 (m, 4 H, collapses to 3 H upon addition of D2O,
CONH2, Ph), 7.5-7.4 (m, 7 H, pH, H-5), 7.21 (bs, 1 H, D2O
exchangeable, CONH2), 6.75 (bs, 1 H, D2O exchangeable,
CONH2), 6.64 (bs, 1 H, D2O exchangeable, CONH2), 6.31 (d, 2
H, J ) 4.4 Hz, H-1′), 5.9-5.8 (m, 2 H, H-2′, H-3′), 4.8-4.7
(m, 1 H, H-4′), 4.7 (m, 2 H, H-5); Anal. (C32H28N4O9): C, H, N.
135, DEPT-90 or selective decoupling of fully coupled spectra,
or assigned by analogy. UV spectra were recorded on a Hewlet-
Packard 8450-A UV/VIS spectrophotometer. IR spectra were
recorded on a Nicolet 5 DXB FT spectrophotometer. Mass
spectroscopy and elemental analyses were performed by the
University of Michigan Chemistry Department or by MHW
Laboratories, Phoenix, AZ.
2-Nitro-1-[3,5-di-O-(p-toluoyl)-2-deoxy-â-D-ribofurano-
syl]pyrrole-3,4-dicarboxamide (17â) and 2-Nitro-1-[3,5-
di-O-(p-toluoyl)-2-deoxy-r-D-ribofuranosyl]pyrrole-3,4-
dicarboxamide (17r). Bis(trimethylsilyl)acetamide (0.5 mL,
2.02 mmol) was added to a stirred mixture of 2-nitropyrrole-
3,4-dicarboxamide27 (15, 100 mg, 0.505 mmol) in CH3CN
(10 mL) and stirred at room temperature under argon. After
20 min, a solution had formed and NaH (60% dispersion in
mineral oil, 30 mg, 0.75 mmol) was added in one portion. After
15 min, the reaction mixture was cooled to 0 °C (ice bath) and
then 3,5-di-O-(p-toluoyl)-2-deoxy-R-D-ribofuranosyl chloride28,33
(16r, 266 mg, 0.94 nmmol) was added in one portion. After
2-Amino-1-[3,5-di-O-(p-toluoyl)-2-deoxy-â-D-ribofura-
nosyl]pyrrole-3,4-dicarboxamide (21). A solution of 17â

Triciribine Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3847
(439 mg, 0.80 mmol) and 10% palladium on carbon (10%,
143 mg) was hydrogenated at 40 psi in MeOH (60 mL) and
EtOAc (20 mL) for 18 h. At that time, the solution was filtered
through Celite and the filtrate was evaporated under reduced
pressure to dryness. Recrystallization of the solid from EtOAc/
hexanes and drying under reduced pressure at 78 °C for 24 h
gave 292 mg (70%) of 21: mp 119-120 °C (sinters); Rf ) 0.45
(10%MeOH/CHCl3); 1H NMR (DMSO-d6, 300 MHz) δ 9.79 (bs,
1 H, D2O exchangeable, CONH2), 7.96 (d, 2 H, J ) 8.0 Hz,
Ph), 7.87 (d, 2 H, J ) 8.1 Hz, Ph), 7.63 (bs, 3 H, D2O
exchangeable, CONH2, NH2), 7.61 (bs, 1 H, D2O exchangeable,
CONH2), 7.14 (bs, 1 H, D2O exchangeable, CONH2), 6.16 (t, 1
H, J ) 6.7 Hz, H-1′), 5.64 (m, 1 H, H-3′), 4.53-4.47 (m, 3 H,
H-4′, H-5′), 2.65 (m, 2 H, H2′), 2.40 (s, 3 H, CH3), 2.37 (s, 3 H,
CH3); Anal. (C27H28N4O7): C, H, N.
5-Carboxamido-1-(2,3,5-tri-O-benzoyl-â-D-ribofurano-
syl)pyrrolo[2,3-d][1,2,3]triazin-4-one (22). A stirred mix-
ture of 20 (6.12 g, 9.99 mmol) and CH3CN (200 mL) was cooled
to -40 °C (dry ice/CH3CN). 6 N HCl (100 mL, pre-cooled to
0 °C) was added to the cold, stirred mixture. At that time, a
solution of NaNO2 (1.26 g, 18.3 mmol in 10 mL of H2O) was
added dropwise over a period of 10 min, while maintaining
the cold bath between -30 °C and -40 °C. The reaction was
then allowed to stir at room temperature until an internal
temperature of 0 °C was reached. The reaction mixture was
poured onto H2O/EtOAc (1:1, 500 mL), the organic layer was
separated, dried (Na2SO4) and filtered and the filtrate was
evaporated to dryness. The resultant red solid was purified
by flash chromatography (3% MeOH/CHCl3, 5 cm × 20 cm),
and the fractions with Rf ) 0.78 (10% MeOH/CHCl3) were
evaporated to 50 mL. Hexanes (250 mL) was then added to
the solution with vigorous stirring. The precipitate which had
formed was collected by vacuum filtration and dried under
reduced pressure at 78 °C for 48 h to give 4.45 g (71%) of 22
as a white solid:: mp 185-187 C (decomp); Rf ) 0.78 (10%
MeOH/CHCl3); 1H NMR (DMSO-d6, 300 MHz) δ 15.24 (bs, 1H,
D2O exchangeable, NH), 9.16 (bs, 1H, D2O exchangeable,
CONH2), 8.53 (s, 1 H, C-6), 8.0-7.9 (m, 6H, Ph), 7.6 (m, 4H,
collapses to 3H upon addition of D2O, CONH2, Ph), 7.5-7.4
(m, 6 H, Ph), 6.87 (d, 1 H, J ) 4.8 Hz, H-1′), 6.30 (m, 1 H,
H-2′), 6.11 (m, 1 H, H-3′), 4.92 (m, 1 H, H-4′), 4.76 (m, 2 H,
H-5); Anal. (C32H25N5O9): C, H, N.
mixture was allowed to stir for 1.5 h at room temperature and
then filtered through Celite. The Celite was washed with
CH3CN (100 mL), and the combined fitrate and washings were
evaporated under reduced pressure until a thick oil was
obtained. This oil was dissolved in CHCl3 (200 mL), washed
with H2O (100 mL), dried (MgSO4) and filtered, and the filtrate
was evaporated under reduced pressure to give a brown foam.
This was purified by flash chromatography (15% EtOAc/
toluene, 5 cm × 20 cm), and the product, Rf ) 0.31 (20% EtOAc/
toluene, 5 cm × 20 cm), was evaporated to dryness. Sequential
addition of CHCl3 (10 mL) followed by hexanes (100 mL) gave
a precipitate which was collected by filtration and dried under
reduced pressure at 70 °C for 24 h to give 1.23 g (58%) of 24
as a white solid: mp 120 °C; Rf ) 0.0.31 (20% EtOAc/toluene);
1H NMR (DMSO-d6, 300 MHz) δ 9.88 (s, 1 H), 9.48 (s, 1 H),
8.75 (s, 1 H), 8.0-7.9 (m, 6 H, Ph), 7.7-7.6 (m, 3 H, Ph), 7.5-
7.4 (m, 6 H, Ph), 7.08 (d, 1 H, J ) 4.1 Hz, H-1′), 6.40 (m, 1 H,
H-2′), 6.23 (m, 1 H, H-3′), 5.0 (m, 1 H, H-4′), 4.9-4.8 (m, 2 H,
H-5); Anal. (C34H24N8O7): C, H, N.
4-(Triazol-1-yl)-1-[3,5-di-O-(p-toluoyl)-2-deoxy-â-D-ribo-
furanosyl]-pyrrolo[2,3-d][1,2,3]triazine-5-carbonitrile (25).
A stirred suspension of 1,2,4-triazole (1.64 g, 0.66 mmol) in
CH3CN (30 mL), under argon, was treated with phosphorus
oxychloride (0.49 mL, 5.3 mmol), the white suspension was
cooled to 0 C. Triethylamine (3.32 mL, 23.8 mmol) was added
and the mixture was allowed to stir at 0 °C for 1 h, at which
time compound 23 (350 mg, 0.659 mmol) was added in one
portion. The reaction mixture was stirred for 5 h at room
temperature and filtered through Celite, and the filter cake
was washed with CH3CN (20 mL). The filtrate and washing
were evaporated under reduced pressure, and the oily residue
was purified by flash chromatography (30% EtOAc/hexanes
then 50% EtOAc hexanes, 2 cm × 25 cm). The resultant solid,
180 mg (48%) Rf ) 0.44 (50% EtOAc/hexanes), was recrystal-
lized from EtOAc/hexanes and dried under reduced pressure
at 78 C for 24 h to give 87 mg (48% recovery) of 25 as white
crystals: mp 205-206 °C; 1H NMR (DMSO-d6, 300 MHz) δ
9.88 (s, 1H), 9.38 (s, 1H), 8.63 (s, 1H), 7.97 (d, 1 H, J ) 7.9 Hz,
Ph), 7.75 (d, 1 H, J ) 8.0 Hz, Ph), 7.37 (d, 1 H, J ) 7.8 Hz,
Ph), 7.21 (d, 1 H, J ) 7.8 Hz, Ph), 7.02 (t, 1 H, J ) 6.4 Hz,
H-1′), 5.86 (m, 1 H), 4.7-4.6 (m, 3 H), 3.3-3.2 (m, 2 H), 2.40
and 2.30 (s, 3 H, CH3, CH3); Anal. (C27H25N5O7): C, H, N.
4-Amino-1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)pyr-
rolo[2,3-d][1,2,3]triazine-5-carbonitrile (26) and 2-Amino-
1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)pyrrole-3,4-di-
carbonitrile (27). A sealed vessel containing Compound 24
(100 mg, 0.15 mmol), NH3 (Aldrich, 0.5 M in 1,4-dioxane,
4 mL) and CH3CN (6 mL) was sealed and stirred at 45 °C for
7 days. At that time the solvent was evaporated to a yellow
oil which was dissolved in EtOAc (20 mL) and washed
successively with H2O (20 mL) and brine (20 mL). The aqueous
layers were back extracted with EtOAc (20 mL), and the
combined organic layers were dried (Na2SO4), filtered and
evaporated under reduced pressure to give a yellow oil which
was purified by flash chromatography (30% EtOAc/hexanes
then 40% EtOAc/hexanes then 50% EtOAc/hexanes, 2 cm ×
20 cm). The appropriate fractions, Rf ) see below, were
evaporated to dryness and then dried under reduced pressure
at room temperature for 48 h.
5-Carboxamido-1-[3,5-di-O-(p-toluoyl)-2-deoxy-â-D-ri-
bofuranosyl]pyrrolo[2,3-d][1,2,3]triazin-4-one (23). A
stirred mixture of 20 (1.37 g, 2.63 mmol) and CH3CN (32 mL)
was cooled to -40 °C (dry ice/CH3CN). 6 N HCl (16 mL, pre-
cooled to 0 °C) was added to the cold, stirred mixture, and a
solution of NaNO2 (316 mg, 5.26 mmol in 1 mL of H2O) was
added dropwise over a period of 5 min, while maintaining the
bath between -30 °C and -40 °C. The reaction was allowed
to stir at room temperature until an internal temperature of
0 °C was reached. The reaction mixture was poured onto H2O/
EtOAc (1:1, 200 mL). At that time, a precipitate had formed
between the aqueous and organic layers. Collection of this
precipitate by filtration and drying under reduced pressure
at 50 °C gave 945 mg (68%) of 23 as a white solid. An
additional 70 mg (5%) of 23 could be obtained after the organic
layer was separated, dried (Na2SO4) and filtered, and the
filtrate was evaporated to dryness: mp 209-210 °C; Rf ) 0.75
1
(10% MeOH/CHCl3); H NMR (DMSO-d6, 300 MHz) δ 15.36
Pyrrolotriazine (26): Yield 45 mg (49%): mp > 105 °C
1
(bs, 1H, D2O exchangeable, NH), 9.16 (bs, 1H, D2O exchange-
able, CONH2), 8.41 (s, 1 H, C-6), 7.97 (m, 2 H, Ph), 7.85 (m,
2 H, Ph), 7.61 (bs, 1H, D2O exchangeable, CONH2), 7.37 (m,
2 H, Ph), 7.28 (m, 2 H, Ph), 6.86 (t, 1 H, J ) 6.7 Hz, H-1′),
5.74 (m, 1 H, H-3′), 4.62-4.56 (m, 3 H, H-4′, H-5′), 3.20-2.82
(m, 2 H, H-2′); Anal. (C27H25N5O7): C, H, N.
4-(Triazol-1-yl)-1-(2,3,5-tri-O-benzoyl-â-D-ribofuranosyl)-
pyrrolo[2,3-d][1,2,3]triazine-5-carbonitrile (24). A suspen-
sion of 1,2,4-triazole (7.9 g, 115 mmol) in CH3CN (100 mL),
stirred under argon, was treated with phosphorus oxychloride
(2.4 mL, 26 mmol), and the white suspension was cooled to
0 °C. Triethylamine (16 mL, 115 mmol) was then added, and
the mixture was allowed to stir at 0 °C for 1 h. Compound 22
(2.00 g, 3.21 mmol) was then added in one portion. The reaction
(decomp); Rf ) 0.39 (50% EtOAc/toluene); H NMR (DMSO-
d6, 300 MHz) δ 8.85 (s, 1 H, C-6), 8.0-7.9 (m, 6 H, Ph), 7.7-
7.6 (m, 3 H, Ph), 7.58 (bs, 2 H, D2O exchangeable, NH2), 7.5-
7.4 (m, 6 H, Ph), 6.81 (d, 1 H, J ) 4.2 Hz, H-1′), 6.34 (m, 1 H,
H-2′), 6.22 (m, 1 H, H-3′), 4.93 (m, 1 H, H-4′), 4.8-4.7 (m, 2 H,
H-5); IR (KBr) 3459-2856, 2229 (CN), 1731, 1635 cm-1; MS
(CI,NH3) 594 (M + H - N2); HRMS (CI,NH3) Calcd. for (M +
H) C32H25N6O7, predicted: 605.1785, found: 605.1791. Anal.
(C32H24N6O7): C, H, N.
Pyrrole (27): Yield 32 mg (36%): mp 200-201 °C; Rf ) 0.
61 (50% EtOAc/toluene); 1H NMR (DMSO-d6, 300 MHz) δ 8.0-
7.8 (m, 6 H, Ph), 7.7-7.6 (m, 4 H, Ph, C-5), 7.6-7.4 (m, 6 H,
Ph), 6.93 (bs, 2 H, D2O exchangeable, NH2), 6.30 (d, 1 H, J )
3.9 Hz, H-1′), 5.87 (m, 2 H, H-2′, H-3′), 4.8-4.7 (m, 3 H, H-4′,

3848 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Migawa et al.
H-5′); IR (KBr) 3406-2944, 2230 (CN), 1718 cm-1; Anal.
pressure at 60 °C for 2 days. This solid was recrystallized from
H2O and then dried under reduced pressure at 78 °C for 24 h
to give 125 mg (92%) of pure 31: mp 258-260 °C (eff.); 1H
NMR (DMSO-d6, 300 MHz) δ 9.82 (bs, 1 H, D2O exchangeable),
9.23 (bs, 1 H, D2O exchangeable), 8.18 (s, 1 H, C-6), 7.84
(bs, 1 H, D2O exchangeable), 6.24 (d, 1 H, J ) 5.7, H-1′), 6.16
(bs, 1 H, D2O exchangeable), 5.51 (d, 1 H, D2O exchangeable),
5.26 (d, 1 H, D2O exchangeable), 5.06 (d, 1 H, D2O exchange-
able), 4.39 (m, 1 H, H-2′), 4.13 (m, 1 H, H-3′), 3.95 (m, 1 H,
H-4′), 3.7-3.5 (m, 2 H, H-5′); Anal. (C11H15N7O5): C, H, N.
(C32H24N4O7): C, H, N.
Methyl 4-Amino-1-(â-D-ribofuranosyl)pyrrolo[2,3-d]-
[1,2,3]triazine-5-formimidate (28). Compound 26 (810 mg),
KCN (64 mg, 0.99 mmol) and MeOH (30 mL) were stirred at
room temperature for 5 h. At that time, the reaction was cooled
to -10 °C, and the resultant precipitate was collected by
filtration and washed with diethyl ether (2 × 30 mL). Drying
the collected solid under reduced pressure at 60 °C for 48 h
gave 295 mg (68%) of 28, which was pure as determined by
1H NMR. An analytical sample was prepared by suspending
100 mg of 28 in CH2Cl2 (15 mL), heating at reflux temperature
for 10 min and then collecting the solid by filtration. Drying
as before gave 96 mg (96% recovery) of pure 28 as a white
4-Amino-1-(â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]-
triazine-5-N-cyanoamidine (32). Compound 28 (146 mg,
0.45 mmol), cyanamide (95 mg, 2.25 mmol) and H2O (5 mL)
were heated on a steam bath for 1 h. The reaction was then
allowed to stand at room temperature so that crystallization
could commence. After 20 h at room temperature, the precipi-
tate was collected by filtration, washed with cold diethyl ether
(20 mL) and dried under reduced pressure at 60 °C for 2 days.
This solid was recrystallized from H2O and then dried under
reduced pressure at 78 °C for 24 h to give 77 mg (51%) of pure
1
solid: mp 187-189 °C (eff.); H NMR (DMSO-d6, 300 MHz) δ
9.87 (d, 1 H, J ) 3.4 Hz, D2O exchangeable, NH2), 9.87 (s,
1 H, D2O exchangeable, NH), 8.23 (s, 1 H, C-6), 7.83 (d, 1 H,
J ) 3.3 Hz, D2O exchangeable, NH2), 6.25 (d, 1 H, J ) 6.0 Hz,
H-1′), 5.48 (d, 1 H, D2O exchangeable, OH), 5.2 (m, 3 H, D2O
exchangeable, OH × 2), 4.4 (m, 1 H, H-2′), 4.1 (m, 1 H, H-3′),
4.0 (m, 1 H, H-4′), 3.7-3.6 (m, 2 H, H-5′); UV [λmax (ꢀ)] (PH1)
313 (5100), 268 (7600), 238 (11 600); (EtOH) 321 (5500), 274
(8300); (pH11) 320 (5900), 273 (7900), 238 (7100);. Anal.
(C12H16N6O5): C, H, N.
1
32: mp > 220 °C (decomp); H NMR (DMSO-d6, 300 MHz) δ
9.22 (bs, 1 H, D2O exchangeable), 8.67 (bs, 3 H, collapses to
[s, 1 H] upon addition of D2O), 8.23 (bs, 1 H, D2O exchange-
able), 6.29 (d, 1 H, J ) 5.4, H-1′), 5.60 (d, 1 H, D2O
exchangeable), 5.29 (d, 1 H, D2O exchangeable), 5.02 (t, 1 H,
D2O exchangeable), 4.4 (m, 1 H, H-2′), 4.1 (m, 1 H, H-3′), 4.0
(m, 1 H, H-4′), 3.7-3.6 (m, 2 H, H-5′); Anal. (C12H14N8O4‚0.5
H2O): C, H, N.
4-Amino-1-(â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]-
triazine-5-carboxamidrazone (29). Compound 28 (150 mg,
0.46 mmol), hydrazine monohydrate (0.44 mL, 9.3 mmol) and
methanol (3 mL) were heated on a steam bath for 25 min, and
then H2O was slowly added until a clear solution had formed.
Heating was continued for 10 min, and then the reaction was
allowed to stand at room temperature so that crystallization
could commence. After 20 h at room temperature, the precipi-
tate was collected by filtration, washed with cold diethyl ether
(20 mL) and dried under reduced pressure at 60 °C for 2 days
to give 134 mg (89%) of 29. This solid was recrystallized from
H2O and then dried under reduced pressure over P2O5 at
78 °C for 24 h to give 111 mg (74% yield, 83% recovery) of
pure 29: mp > 240 °C (decomp); 1H NMR (DMSO-d6, 300
MHz) δ 9.80 (bs, 1 H, D2O exchangeable, NH2), 8.05 (s, 1 H,
C-6), 7.66 (bs, 1 H, D2O exchangeable, NH2), 6.22 (d, 1 H, J )
6.0, H-1′), 5.87 (bs, 1 H, D2O exchangeable), 5.48 (d, 1 H, D2O
exchangeable), 5.23 (d, 1 H, D2O exchangeable), 5.1-5.0
(m, 3 H, D2O exchangeable), 4.40 (m, 1 H, H-2′), 4.12 (m, 1 H,
H-3′), 3.94 (m, 1 H, H-4′), 3.7-3.6 (m, 2 H, H-5′); IR (KBr)
3397-3198, 1638, 1465 cm-1; UV [λmax (ꢀ)] (PH1) 312 (5800),
268 (8500); (EtOH) 323 (1600), 282 (2900); (pH11) 321 (6500),
276 (11 700), 228 (7100);. HRMS (CI, methane) Calcd. for
(M + H) C11H18N8O4, predicted: 325.1373, Found: 325.1377.
Ethyl 4-Amino-1-(â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]-
triazine-5-carboxylate (33). Compound 26 (500 mg,
0.83 mmol), Na2CO3 (701 mg, 6.61 mmol) and EtOH/H2O
(10:1, 33 mL) were stirred at room temperature for 52 h. At
that time, AcOH (glacial, 3 mL) was added and the solution
was evaporated under reduced pressure until a volume of
10 mL was obtained. H2O (50 mL) was then added, the clear
mixture was extracted with hexanes (3 × 50 mL) and the
aqueous layer was evaporated under reduced pressure at
60 °C to 1/2 of its original volume. The resultant aqueous
suspension was heated on a steam bath until a clear solution
was observed. Cooling to room temperature produced white
crystals which were collected by filtration and then dried under
reduced pressure at 78 °C for 48 h to give 175 mg (62%) of 33:
1
mp 130-131 °C (sinters); Rf ) 0. 16 (10% MeOH/CHCl3); H
NMR (DMSO-d6, 300 MHz) δ 8.63 (s, 1 H, C-6), 8.1 (bs, 1 H,
D2O exchangeable, CONH2), 7.7 (bs, 1 H, D2O exchangeable,
CONH2), 6.28 (d, 1 H, J ) 5.3 Hz, H-1′), 5.57 (bs, 1 H, D2O
exchangeable, OH), 5.3-5.2 (m, 3 H, D2O exchangeable, OH
× 2), 4.43 (m, 2 H, H-2′), 4.34 (q, 2 H, CH2), 4.15 (m, 1 H,
H-3′), 4.00 (m, 1 H, H-4′), 3.8-3.6 (m, 2 H, H-5′), 1.33 (t, 3 H,
CH3); IR (KBr) 3416-3152, 1690, 1662 cm-1; UV [λmax (ꢀ)]
(PH1) 310 (6500), 265 (1 0000), 229 (13 000); (EtOH) 311
(6500), 276 (10 000); (pH11) 313 (8000), 273 (10 000);. Anal.
(C13H17N5O6): C, H, N.
4-Amino-1-(â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]-
triazine-5-carboxamide (2-AzaSangivamycin, 30).
A
1.25 N solution of NaOH (8 mL) and compound 28 (200 mg,
0.62 mmol) were stirred at room temperature for 1.5 h. At that
time, the reaction was allowed to stand for 15 min at room
temperature. The precipitate was collected by filtration and
washed with diethyl ether (20 mL). Drying this solid under
reduced pressure at 60 °C for 24 h gave 128 mg (67%) of 30 as
a white solid. Recrystallization from H2O and drying under
reduced pressure at 78 °C for 48 h gave 126 mg (64%) of pure
30 as a hemihydrate: mp > 270 °C (decomp); 1H NMR (DMSO-
d6, 300 MHz) δ 8.5-7.5 (bs, 2 H, D2O exchangeable, NH2), 8.43
(s, 1 H, C-6), 8.13 (bs, 1 H, D2O exchangeable, CONH2), 7.64
(bs, 1 H, D2O exchangeable, CONH2), 6.24 (d, 1 H, J ) 5.6,
H-1′), 5.58 (d, 1 H, D2O exchangeable, OH), 5.30 (d, 1 H, D2O
exchangeable, OH), 5.06 (t, 1 H, D2O exchangeable, 5′-OH),
4.40 (m, 1 H, H-2′), 4.13 (m, 1 H, H-3′), 3.98 (m, 1 H, H-4′),
3.7-3.6 (m, 2 H, H-5′); Anal. (C11H14N6O5‚H2O): C, H, N.
4-Amino-1-(â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]-
triazine-5-thiocarboxamide (2-Azathiosangivamycin, 34).
H2S (g) was bubbled into a solution of MeOH (70 mL) and
NaOMe (358 mg, 6.62 mmol), held at 0 °C in an ice bath. After
30 min, the solution was added to a pressure tube containing
Compound 26 (400 mg, 0.66 mmol). The tube was sealed and
heated at 90 °C for 1 h. At that time, the reaction was cooled
to room temperature, the pH was adjusted to 7 with 1 N HCl
and the solvent was evaporated under reduced pressure until
a yellow oil was obtained. H2O was added to the oil, and the
precipitate was collected by filtration, washed with cold H2O
(20 mL) and dried under reduced pressure at 70 °C for 48 h to
give 336 mg (79%) of crude 4-amino-1-(2,3,5-tri-O-benzoyl-â-
D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]triazine-5-thiocarboxam-
ide, Rf ) 0.60 (10% MeOH/CHCl3). The crude benzoylated
compound (259 mg, 0.41 mmol), KCN (78 mg, 1.2 mmol) and
(MeOH 15 mL) were stirred at room temperature for 4 h. At
that time, the reaction mixture was evaporated to dryness and
purified by preparative TLC (Silica, 4 × 500 µm plates, eluting
with 20% MeOH/EtOAc, 30% MeOH/EtOAc, then 50% MeOH/
EtOAc). The most prevalent band was eluted off of the plates
4-Amino-1-(â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]-
triazine-5-carboxamidoxime (31). Compound 28, 135 mg,
0.42 mmol), hydroxylamine (50% aqueous solution, 500 mg)
and H2O (4 mL) were heated on a steam bath for 1 h. The
reaction was then allowed to stand at room temperature so
that crystallization could commence. After 20 h at room
temperature, the precipitate was collected by filtration, washed
with cold diethyl ether (20 mL) and dried under reduced

Triciribine Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3849
with 30% EtOAc/MeOH and the solution was filtered through
Celite. The filtrate was evaporated to dryness, dissolved in a
minimal amount of H2O and then freeze-dried for 48 h to give
76 mg (54%) of 34 as a light yellow solid: mp > 180 °C
(decomp); 1H NMR (DMSO-d6, 300 MHz) δ 9.86 (bs, 1 H, D2O
exchangeable, CSNH2), 9.70 (bs, 1 H, D2O exchangeable,
CSNH2), 8.3 (bs, 3 H, collapses to [s, 1H] upon addition of D2O,
NH2, C-6), 6.30 (d, 1 H, J ) 4.2, H-1′), 5.58 (d, 1 H, D2O
exchangeable), 5.29 (d, 1 H, D2O exchangeable), 5.11 (m, 3 H,
D2O exchangeable), 4.41 (m, 1 H, H-2′), 4.15 (m, 1 H, H-3′),
3.97 (m, 1 H, H-4′), 3.7-3.6 (m, 2 H, H-5′); Anal. (C11H14N6O4S‚
H2O): C, H, N.
temperature. After 2 h, the reaction mixture was evaporated
to dryness and then purified by filtering through a layer of
silica gel (3% MeOH/CHCl3, 2 cm × 2 cm) to obtain 145 mg
(95%) of crude 37. 1H NMR (DMSO-d6, 300 MHz) δ 8.73
(s, 1H), 7.97 (d, 1 H, J ) 8.0 Hz, Ph), 7.7.87 (d, 1 H, J ) 8.2
Hz, Ph), 7.38 (d, 1 H, J ) 8.1 Hz, Ph), 7.30 (d, 1 H, J ) 8.0 Hz,
Ph, 6.87 (t, 1 H, J ) 6.9 Hz, H-1′), 5.78 (bs, 2 H, D2O
exchangeable, NH2), 5.40 (m, 1 H), 5.65-4.56 (m, 3 H), 3.1-
2.8 (m, 2 H), and 2.37 (s, 3 H, CH3, CH3); This crude yellow oil
(37) was dissolved in MeOH (10 mL), NaOMe (65 mg,
1.21 mmol) was added, and the solution was stirred at room
temperature for 1 h. At that time, the yellow solution was
heated at reflux temperature for 16 h, cooled to room temper-
ature and evaporated under reduced pressure to dryness. The
solid was treated with EtOAc (15 mL), filtered and purified
by preparative TLC (SiO2, 2000 µm, 20 cm × 20 cm, eluting
with 30% MeOH/EtOAc). The bright yellow band was collected
to give 45 mg (53%) of 13 as a yellow solid. An analytical
sample was prepared by recrystallization from MeOH and
drying under reduced pressure at 78 °C for 2 days to give
20 mg (44% recovery) of 13: mp > 230 °C (decomp); 1H NMR
(DMSO-d6, 300 MHz) δ 7.39 (s, 1 H), 6.54 (s, 2 H, D2O
exchangeable, NH2), 6.48 (t, 1 H, J ) 6.7 Hz, H-1′), 5.39 (d,
1 H, D2O exchangeable, 3′-OH), 5.08 (t, 1 H, D2O exchangeable,
5′-OH), 4.43 (m, 1 H), 3.91 (m, 1 H), 3.6-3.5 (m, 2 H), 3.52
(s, 3 H, CH3), 2.8-2.3 (m, 2 H); IR (KBr) 3418-3079, 1695,
1636, 1474 cm-1; HRMS (EI, 70 eV) Calcd. for (M+) C12H15N7O3,
predicted: 305.1236, Found: 305.1238.
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.39 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.4
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.4,40 To evaluate the activities of
compounds in cells acutely infected with HIV, two different
assays were used. In one, 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 (pfu). per cell with strain IIIB of HIV-1 in a
minimal volume of stock virus in growth medium. Cultures
were incubated at 37 °C for 2 h to permit virus adsorption
and then diluted to 5 × 105 cells per mL with RPMI 1640
containing 10% fetal bovine serum. One-tenth milliliter was
then added to each well of a 96-well cluster dish which had
been pretreated with poly-L-lysine. Fresh medium (0.1 mL with
10% fetal bovine serum) containing test compounds in twice
the desired final concentration was added to triplicate wells
at seven concentrations ranging from 100 to 0.14 µM. After 6
days incubation, supernatant samples were taken and the
amount of RT activity was measured by the incorporation of
[3H]dTTP into acid insoluble material using the assay de-
scribed by White et al.41 In the other assay, viral cytopathology
was determined microscopically at 30-fold magnification in
cells infected with HIV-1 infected as described above.
4-Amino-1-(â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]-
triazine-5-carbonitrile (2-Azatoyocamycin, 35). A stirred
mixture of 4-amino-1-â-D-ribofuranosyl)pyrrolo[2,3-d][1,2,3]-
triazine-5-thiocarboxamide (2-azathiosangivamycin) (34,
0.203 mmol) in H2O (1.5 mL), CH3CN (1.5 mL) and NH4OH
(0.5 mL) was treated with MeI (0.14, 1.18 mmol) and stirred
for 1 h at room temperature. At that time, the reaction was
evaporated and coevaporated with toluene. The resulting
yellow solid was recrystallized from EtOH to give 20 mg (34%)
1
of 35 as a tan solid mp > 180 °C (decomp); H NMR (DMSO-
d6, 300 MHz) δ 8.79 (s, 1 H), 7.49 (bs, 2 H, D2O exchangeable,
NH2), 6.25 (d, 1 H, J ) 4.9, H-1′), 6.62 (d, 1 H, D2O
exchangeable), 5.27 (d, 1 H, D2O exchangeable), 5.19 (t, 3 H,
D2O exchangeable), 4.42 (m, 1 H, H-2′), 4.13 (m, 1 H, H-3′),
3.99 (m, 1 H, H-4′), 3.7-3.6 (m, 2 H, H-5′); IR (KBr) 3161, 2066
(CN), 1636 cm-1; HRMS (CI, NH3) Calcd. for (M + H)
C11H13N6O4, predicted: 293.0998, Found: 293.1008.
4-(1-Methylhydrazino)-1-(â-D-ribofuranosyl)pyrrolo-
[2,3-d][1,2,3]triazine-5-carbonitrile (36). A solution of 24
(1.0 g, 1.52 mmol), methylhydrazine (2.0 mL, 38 mmol) and
MeOH (45 mL) were stirred at room temperature. After 3 h,
the reaction mixture was cooled to 0 °C, and the precipitate
was collected by filtration. This solid was washed with cold
MeOH (5 mL) and dried under reduced pressure at 50 °C for
24 h to give 789 mg (82%) of 36, which was sufficiently pure
to be used in subsequent reactions. An analytical sample was
prepared by subjecting 195 mg of 36 to flash chromatography
(20% EtOAc/toluene then 40% EtOAc/toluene, 2 cm × 20 cm),
and collecting the major spot, Rf ) 0.09 (20% EtOAc/toluene).
CHCl3 (5 mL) and hexanes (50 mL) were added sequentially
to the solid, and the solid was collected by filtration and dried
as before to give 163 mg (84% recovery) of pure 36: mp
204-206 °C; 1H NMR (DMSO-d6, 300 MHz) δ 8.75 (s, 1 H,
C-6), 8.0-7.9 (m, 6 H, Ph), 7.6 (m, 3 H, Ph), 7.5-7.4 (m, 6 H,
Ph), 6.79 (d, 1 H, J ) 3.8, H-1′), 6.29 (m, 1 H, H-2′), 6.17 (m,
1 H, H-3′), 5.43 (S, 2 H, D2O exchangeable, NH2), 4.92 (m,
1 H, H-4′), 4.8 (m, 2 H, H-5′), 3.51 (S, 3 H, CH3); Anal.
(C33H27N7O7): C, H, N.
3-Amino-5-methyl-1-(â-D-ribofuranosyl)-1,5-dihydro-
1,4,5,6,7,8-hexaazaacenaphthalene (12). NaOMe (228 mg,
4.22 mmol) was added to a stirred solution 36 (594 mg,
0.94 mmol) in MeOH (35 mL) at room temperature. After
stirring for 1 h, the reaction was heated at reflux temperature
for 17 h at which time a yellow precipitate was observed. The
reaction mixture was cooled to room temperature, and the
precipitate was collected by filtration and dried under reduced
pressure at 60 °C for 24 h. This solid was suspended in
CH2Cl2 (40 mL) and held at reflux temperature for 20 min. At
that time, the suspension of yellow powder was cooled to room
temperature, and the yellow powder was collected by filtration
and dried under reduced pressure at 78 °C for 48 h to give
1
260 mg (86%) of 12: mp 268-270 °C (eff.); H NMR (DMSO-
d6, 300 MHz) δ 7.40 (s, 1 H, H-2), 6.56 (s, 1 H, D2O
exchangeable, NH2), 6.01 (d, 1 H, J ) 6.2, H-1′), 5.63 (bs, 1 H,
D2O exchangeable, 5′-OH), 5.35 (bs, 1 H, D2O exchangeable,
3′-OH), 5.20 (bs, 1 H, D2O exchangeable, 2′-OH), 4.56 (m, 1 H,
H-2′), 4.14 (m, 1 H, H-3′), 4.00 (m, 1 H, H-4′), 3.7-3.5 (m, 2 H,
H-5′), 3.49 (s, 3 H, CH3); Anal. (C12H15N7O4): C, H, N.
3-Amino-5-methyl-1-(2-deoxy-â-D-ribofuranosyl)-1,5-di-
hydro-1,4,5,6,7,8-hexaazaacenaphthalene (13). A stirred
solution of 25 (158 mg, 0.280 mmol) in MeOH (8 mL) was
treated with methylhydrazine (0.37 mL, 7.0 mmol) at room
HCMV Assays. The Towne strain, plaque-purified isolate
Po, of HCMV was kindly provided by Dr. Mark Stinski,
University of Iowa. Both plaque and yield reduction assays
were used. In the former, HFF cells in 24-well cluster dishes
were infected with approximately 100 plaque forming units
(pfu) of HCMV per well using the procedures detailed earlier.39
Following virus adsorption, compounds dissolved in MEM(E)
containing 0.5% methylcellulose and 5% fetal calf serum were
added to duplicate wells in four to eight selected concentra-
tions. After incubation at 37 °C for 8-10 days, cell sheets were

3850 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Migawa et al.
fixed and stained with crystal violet, and microscopic plaques
were enumerated. Drug effects were calculated as a percentage
of reduction in number of plaques in the presence of each drug
concentration compared to the number observed in the absence
of drug. Protocols for HCMV yield-reduction experiments have
been previously described.42 Briefly, monolayer cultures of HFF
cells in 96-well culture dishes (Costar, Cambridge, MA) were
infected at a multiplicity of infection (MOI) of 0.5, 0.05, or 0.005
PFU/cell and incubated in the presence of the test compounds
for 6 to 7 days. Following one cycle of freezing at -76 °C and
thawing at 37 °C, the resulting lysates were diluted, and the
amount of infectious virus was quantified on new cultures of
HFF cells.
HSV-1 ELISA. An enzyme-linked immunosorbent assay
(ELISA)43 was employed to detect HSV-1. Briefly, 96-well
cluster dishes were planted with 10 000 BSC-1 cells per well
in 200 L per well of MEM(E) plus 10% calf serum. After
overnight incubation at 37 °C, selected drug concentrations
in triplicate and HSV-1 (KOS strain kindly provided by
Dr. Sandra K. Weller, University of Connecticut) at a concen-
tration of 100 pfu/well were added. Following a 3-day incuba-
tion 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 stopped with H2SO4 and absorbance was read at
450 and 570 nm. Drug effects were calculated as a percentage
of the reduction in absorbance in the presence of each drug
concentration compared to absorbance obtained with virus in
the absence of drug.
Cytotoxicity Assays. 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.39 (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.44 Briefly, 96-
well cluster dishes were planted with KB cells at 5000 cells
per well. After overnight incubation at 37 °C, test compound
was added in triplicate at eight concentrations. Plates were
incubated at 37 °C for 48 h in a CO2 incubator, 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. (iii) The effect of compounds on the growth of CEM-SS
cells was performed in six-well cluster dishes. Cells were
planted at 100 000 cells per well and incubated for 18-24 h.
Growth medium was removed and replaced with medium
containing 0 to 10 µM in duplicate of one of compounds shown
in Figure 1. Daily for 4 days, cells were harvested by standard
techniques and enumerated in a Coulter Counter.
Data Analysis. Dose-response relationships for all the
foregoing assays except HCMV yield assays were constructed
by linearly regressing the percent inhibition of parameters
derived in the preceding sections against log drug concentra-
tions. Fifty-percent inhibitory (IC50) concentrations were in-
terpolated from the regression lines. For HCMV yield assays,
log/log dose-response curves were constructed by linear
regression and 90% inhibitory (IC90) concentrations were
interpolated 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.
Supporting Information Available: Elemental analyses
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Acknowledgment. This study was supported by
research grants U19-AI-31718 and RO1-AI36872 from
the National Institute of Allergy and Infectious Diseases
and by research funds from the University of Michigan.
We thank Kathy Z. Borysko, Julie M. Breitenbach, and
Roger G. Ptak for expert performance of all biological
assays.

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