Synthesis and antiproliferative and antiviral activity of carbohydrate- modified pyrrolo[2,3-d]pyridazin-7-one nucleosides

Article abstract of DOI:10.1021/jm960631x

Sugar-modified analogs of 4-amino-1-(β-D-ribofuranosyl)pyrrolo[2,3- d]pyridazin-7-one (1) and 4-amino-3-bromo-1-(β-D-ribofuranosyl)pyrrolo[2,3- d]pyridazin-7-one (3) were prepared in an effort to obtain selective antiviral agents. Treatment of ethyl 3-cya

Full text of DOI:10.1021/jm960631x

794
J . Med. Chem. 1997, 40, 794-801
Syn th esis a n d An tip r olifer a tive a n d An tivir a l Activity of
Ca r boh yd r a te-Mod ified P yr r olo[2,3-d ]p yr id a zin -7-on e Nu cleosid es
Eric A. Meade,† Linda L. Wotring, J ohn C. Drach, and Leroy B. Townsend*
Department of Medicinal Chemistry, College of Pharmacy, Department of Chemistry, College of Literature,
Sciences and Arts, and Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan,
Ann Arbor, Michigan 48109-1065
Received September 5, 1996X
Sugar-modified analogs of 4-amino-1-( -D-ribofuranosyl)pyrrolo[2,3-d]pyridazin-7-one (1) and
4-amino-3-bromo-1-( -D-ribofuranosyl)pyrrolo[2,3-d]pyridazin-7-one (3) were prepared in an
effort to obtain selective antiviral agents. Treatment of ethyl 3-cyano-1-(2,3,5-tri-O-benzyl-1-
-D-arabinofuranosyl)pyrrole-2-carboxylate (6) with hydrazine afforded 4-amino-1-(2,3,5-tri-
O-benzyl-1- -D-arabinofuranosyl)pyrrolo[2,3-d]pyridazin-7-one (7). Treatment of 7 with bromine
afforded 4-amino-3-bromo-1-(2,3,5-tri-O-benzyl- -D-arabinofuranosyl)pyrrolo[2,3-d]pyridazin-
7-one hydrobromide (9). The benzyl ether functions of 7 and 9 were removed with boron
trichloride to afford 4-amino-1-( -D-arabinofuranosyl)pyrrolo[2,3-d]pyridazin-7-one (8) and its
3-bromo analog 10. 4-Amino-1-(2-deoxy- -D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyridazin-7-
one (13) was prepared by the sodium salt condensation of ethyl 3-cyanopyrrole-2-carboxylate
(5) with 2-deoxy-3,5-di-O-p-toluoyl-R-D-erythro-pentofuranosyl chloride (11) followed by ring
annulation with hydrazine. Deprotection of ethyl 3-cyano-1-(2-deoxy-3,5-di-O-p-toluoyl- -D-
erythro-pentofuranosyl)pyrrole-2-carboxylate (12) using sodium ethoxide furnished ethyl 1-(2-
deoxy- -D-erythro-pentofuranosyl)-3-cyanopyrrole-2-carboxylate (14) which served as the start-
ing material for the preparation of 4-amino-1-(2,3-dideoxy- -D-glycero-pentofuranosyl)pyrrolo[2,3-
d]pyridazin-7-one (20). Selective protection of the 5′-hydroxyl group of 14 with tert-
butyldimethylsilyl chloride followed by a Barton type deoxygenation sequence of the 3′-hydroxyl
groups afforded ethyl 3-cyano-1-[2,3-dideoxy-5-O-tert-butyldimethylsilyl)- -D-glycero-pento-
furanosyl]pyrrole-2-carboxylate (18). Deprotection of 18 with tetra-n-butylammonium fluoride
and ring annulation with hydrazine afforded 20. The acyclic analog 4-amino-1-[(1,3-dihydroxy-
2-propoxy)methyl]pyrrolo[2,3-d]pyridazin-7-one (24) was prepared via the sodium salt glyco-
sylation of 5 with (1,3-dihydroxy-2-propoxy)methyl bromide (22) followed by a ring annulation
with hydrazine. N-Bromosuccinimide treatment of 13, 20, and 25 afforded the 3-bromo
derivatives 15, 21, and 25. Evaluation of these compounds in L1210, HFF, and KB cells showed
that the sugar-modified analogs all were less cytotoxic than their corresponding ribonucleoside
analogs. The compounds also were less active against human cytomegalovirus (HCMV) and
herpes simplex virus type 1 (HSV-1). The 3-bromo derivatives were much more active than
the 3-unsubstituted analogs in both the cytotoxicity, and antiviral assays. However, there
was only modest separation between activity against HCMV and cytotoxicity and there was
virtually no selectivity for activity against HSV-1.
In tr od u ction
As reported previously from these laboratories,1-3
a
novel series of 4-amino-3-halo-1-( -D-ribofuranosyl)-
pyrrolo[2,3-d]pyridazin-7-ones 2-4 have significant an-
tiviral and antiproliferative activity. However, the
antiviral potential of these compounds was tempered
by their cytotoxicity to uninfected cells. In an effort to
decrease the cytotoxic effects of the compounds and
obtain more selective antiviral (human cytomegalovirus)
agents, an investigation of 4-aminopyrrolo[2,3-d]py-
ridazin-7-one nucleosides with modified glycosyl moi-
eties was undertaken.
We initiated this investigation on the basis of the
antiviral selectivity obtained previously when sugar
modifications were introduced in a series of pyrrolo[2,3-
d]pyrimidine nucleosides and some structurally related
compounds. The ribonucleosides showed potent cyto-
toxic and antiviral effects but with little selectivity. The
antiviral selectivity was improved by replacing the
7-ribofuranosyl moiety with a xylofuranosyl,4 a 2-deoxy-
ribofuranosyl or an arabinofuranosyl moiety.5 More
recently the replacement of the carbohydrate moiety of
several 4,5-disubstituted pyrrolo[2,3-d]pyrimidine nu-
cleosides with the acyclo side chains hydroxyethoxy-
methyl6 or (1,3-dihydroxy-2-propoxy)methyl7 (DHPM)
also led to compounds with greater antiviral selectivity.
Using the above rationale, we have adopted a similar
strategy for the synthesis of 4-aminopyrrolo[2,3-d]-
* Author to whom correspondence should be addressed.
† Present address: Eisa Research Institute, 4 Corporate Dr, An-
dover, MA 01810.
X Abstract published in Advance ACS Abstracts, December 15, 1996.
S0022-2623(96)00631-0 CCC: $14.00 © 1997 American Chemical Society

Sugar-Modified Pyrrolopyridazin-7-one Nucleosides
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 795
Sch em e 1
Sch em e 2
pyridazin-7-one nucleosides in an effort to obtain more
selective antiviral agents by replacing the 1-ribosyl
moiety with 1-(arabinofuranosyl), 1-(2-deoxyribofura-
nosyl), 1-(2,3-dideoxyribofuranosyl), or 1-[(2,3-dihydroxy-
2-propoxy)methyl]. We report herein the synthesis and
biological evaluation of these compounds.
Resu lts a n d Discu ssion
Ch em istr y. In order to obtain various sugar-modi-
fied analogs of the 4-aminopyrrolo[2,3-d]pyridazin-7-one
nucleosides, we elected to use pyrrole glycosides as
intermediates. The various pyrrole glycosides were
prepared via the sodium salt glycosylation8 of ethyl
3-cyanopyrrole-2-carboxylate9 with the appropriate
1-haloglycoside. The various pyrrole glycosides were
then ring annulated with hydrazine to furnish the
4-amino-1-glycosylpyrrolo[2,3-d]pyridazin-7-one nucleo-
sides.
For the synthesis of 4-amino-1-( -D-arabinofuranosyl)-
pyrrolo[2,3-d]pyridazin-7-one (8) and the 3-bromo ana-
log 10, ethyl 3-cyano-1-(2,3,5-tri-O-benzyl- -D-arabino-
furanosyl)pyrrole-2-carboxylate3 (6) served as the starting
material. Ring annulation of 6 using hydrazine in
ethanol at reflux temperature proceeded smoothly to
afford 4-amino-1-(2,3,5-tri-O-benzyl- -D-arabinofurano-
syl)pyrrolo[2,3-d]pyridazin-7-one (7) in 73% yield. The
deprotection of 7 was accomplished using boron trichlo-
ride. The bromination of 7 using bromine in dichlo-
romethane followed by deprotection afforded 4-amino-
3-bromo-1-( -D-arabinofuranosyl)pyrrolo[2,3-d]pyridazin-
groups to furnish 4-amino-1-(2-deoxy- -D-erythro-pento-
furanosyl)pyrrolo[2,3-d]pyridazin-7-one (13). For larger
scale preparations of 13, it proved to be more convenient
to use a two-step procedure. Using this modification
the p-toluoyl groups of 12 were removed with a solution
of sodium ethoxide in ethanol to afford ethyl 3-cyano-
1-(2-deoxy- -D-erythro-pentofuranosyl)pyrrole-2-carbox-
ylate (14), which was then ring annulated using hydra-
zine to provide 13. Treatment of 13 with N-bromo-
succinimide in acetic acid furnished a good yield of
4-amino-3-bromo-1-(2-deoxy- -D-erythro-pentofuranosyl)-
1
1
7-one (10). An examination of the H NMR spectrum
pyrrolo[2,3-d]pyridazin-7-one (15). From the H NMR
of 10 and 4-amino-3-bromo-1-( -D-ribofuranosyl)pyrrolo-
[2,3-d]pyridazin-7-one (3) allowed us to assign the
anomeric configuration for 8 and 10. In 10, a doublet
corresponding to the anomeric proton was observed at
7.05 which was downfield from the peaks at 6.84
corresponding to the anomeric proton of the ribofura-
nosyl analog 3. The loss of the shielding effect of the
neighboring 2′-hydroxyl group in 10 was consistent with
this observation.10
spectrum of 15 it was apparent that monobromination
had occurred at the 3-position. In the H NMR spec-
trum of 13 the peaks corresponding to H-2 and H-3
1
appeared as doublets at 7.70 and 6.63, respectively.
1
In the H NMR spectrum of the bromo analog 15, we
observed a disappearance of the upfield peak corre-
sponding to H-3 and a small downfield shift to 7.96 of
the singlet corresponding to H-2. This observation was
in good agreement with similar peak patterns observed
1
The preparation of the 2′-deoxyribofuranosyl analogs
of the pyrrolo[2,3-d]pyridazin-7-ones was initiated from
the sodium salt condensation of 5 with 2-deoxy-3,5-di-
O-p-toluoyl-R-D-erythro-pentofuranosyl chloride (11)11
which furnished ethyl 3-cyano-1-(3,5-di-O-p-toluoyl-2-
deoxy- -D-erythro-pentofuranosyl)pyrrole-2-carboxy-
late (12). The -configuration for 12 was determined
in the H NMR spectra of the ribofuranosyl analogs 1
and 3, whose site of bromination had been established
unequivocally by 13C NMR.3
Compound 14 also served as a useful starting mate-
rial for the preparation of the 2′,3′-dideoxy analogs
4-amino-1-(2,3-dideoxy- -D-glycero-pentofuranosyl)pyr-
rolo[2,3-d]pyridazin-7-one (20) and 4-amino-3-bromo-1-
(2,3-dideoxy- -D-glycero-pentofuranosyl)pyrrolo[2,3-d]-
pyridazin-7-one (21), respectively. In order to selectively
manipulate the 3′-hydroxyl, the 5′-primary hydroxyl
group had to be protected. This manipulation was
accomplished by a selective silylation of 14 using tert-
butyldimethylsilyl chloride and imidazole in dimethyl-
1
from the H NMR spectrum. The peaks corresponding
to the anomeric proton appeared as a pseudotriplet
which was in accord with similar observations for other
-2′-deoxyribonucleosides.12 The direct treatment of 12
with hydrazine in ethanol effected ring annulation with
a concomitant deprotection of the toluoyl blocking

796 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Meade et al.
Sch em e 3
obtained using methods similar to those described
above. Alkylation of the sodium salt of 5 with (1,3-
diacetoxy-2-propoxy)methyl bromide (22)14 afforded eth-
yl 3-cyano-1-[(1,3-diacetoxy-2-propoxy)methyl]pyrrole-
2-carboxylate (23). The ring annulation and deprotection
of 23 was accomplished using hydrazine in ethanol at
reflux temperature to furnish 4-amino-1-[(1,3-dihy-
droxy-2-propoxy)methyl]pyrrolo[2,3-d]pyridazin-7-one
(24). The UV spectrum of 24 with a max at 287 nm was
in good agreement with the pyrrolo[2,3-d]pyridazin-7
one structure (vide supra). 4-Amino-3-bromo-1-[(1,3-
dihydroxy-2-propoxy)methyl]pyrrolo[2,3-d]pyridazin-7-
one (25) was prepared via N-bromosuccinimide treat-
ment of 24. A comparison of the shift patterns for the
1
H-2 and H-3 protons in the H NMR spectra of 24 and
25 was in accord with the 3-position being the site of
bromination (vide supra).
An tivir a l a n d An tip r olifer a tive Stu d ies. The
antiproliferative activity of the compounds was evalu-
ated in L1210 murine leukemic cells. The results of
these evaluations, along with those reported previously3
for the corresponding ribosyl derivatives 1-4, are shown
in Table 1. The 3-halo-substituted nucleosides 2-4, 10,
15, and 21 all had more potent antiproliferative activity
than their 3-unsubstituted counterparts. The carbohy-
drate moiety also had a strong influence on antiprolif-
erative activity in this series of compounds. The 3-Br-
pyrrolo[2,3-d]pyridazin-7-ones with a 1-arabinosyl, 1-(2-
deoxyribosyl), or 1-(2,3-dideoxyribosyl) substituent were
on the order of 100-fold less potent than the correspond-
ing 1-ribosyl derivatives. Furthermore, replacing the
furanosyl moiety with the acyclic (1,3-dihydroxy-2-
propoxy)methyl side chain virtually abolished antipro-
liferative activity.
formamide. That the 5′-hydroxyl group was selectively
silylated was determined by the loss of the D2O ex-
changeable triplet corresponding to the 5′-OH in the 1H
NMR spectrum of ethyl 3-cyano-1-[5-O-(tert-butyldi-
methylsilyl)-2-deoxy- -D-erythro-pentofuranosyl]pyrrole-
2-carboxylate (16). The reductive deoxygenation of 16
was accomplished using a Barton type procedure.13
Ethyl 3-cyano-1-[5-O-tert-butyl-3-O-(phenoxythiocarbo-
nyl)-2-deoxy- -D-erythro-pentofuranosyl]pyrrole-2-car-
boxylate (17) was prepared by the treatment of 16 with
phenyl chlorothionoformate in the presence of 4-(di-
methylamino)pyridine. A reduction of the thiocarbonate
moiety of 17 to yield ethyl 3-cyano-1-[5-O-tert-butyldi-
methylsilyl)-2,3-dideoxy- -D-glycero-pentofuranosyl]pyr-
role-2-carboxylate (18) was then accomplished by heat-
ing 17 and tri-n-butyltin hydride in the presence of
AIBN. Treatment of 18 with tetra-n-butylammonium
fluoride successfully removed the 5′-silyl protecting
group and furnished ethyl 3-cyano-1-(2,3-dideoxy- -D-
glycero-pentofuranosyl)pyrrole-2-carboxylate (19). The
ring annulation of 19 with hydrazine provided 4-amino-
1-(2,3-dideoxy- -D-glycero-pentofuranosyl)pyrrolo[2,3-d]-
The antiviral effects of the compounds were investi-
gated using human cytomegalovirus (HCMV) and her-
pes simplex virus type 1 (HSV-1). To determine their
selectivity, the cytotoxicities of compounds were inves-
tigated in uninfected stationary human foreskin fibro-
blasts (HFF cells) and growing KB cells (Table 2). Only
compounds having a 3-halo substituent had significant
antiviral activity. For these compounds the effect of
replacing the ribofuranosyl moiety by a modified sugar
decreased cytotoxicity to HFF and KB cells. Antiviral
activity, however, was significantly lowered as well. It
was of considerable interest that, in this series of
compounds, replacing the ribofuranosyl moiety with the
DHPM moiety (25) completely abolished the antiviral
activity. In contrast, the arabinofuranosyl analog 10
was the most promising antiviral compound in this
study with approximately a 10-fold difference between
activity against HCMV and cytotoxicity in host HFF
cells. Little or no selectivity for HSV-1 was observed
(Table 2).
pyridazin-7-one (20). The
of 288 nm in the UV
max
spectrum of 20 was identical with the
of its
max
corresponding ribofuranosyl analog 1,3 supporting the
fact that ring closure to the pyrrolo[2,3-d]pyridazin-7-
one heterocycle had occurred. Finally, 4-amino-3-bromo-
1-(2,3-dideoxy- -D-glycero-pentofuranosyl)pyrrolo[2,3-
d]pyridazin-7-one (21) was obtained by the bromination
of 20 with N-bromosuccinimide. A comparison of the
chemical shift patterns for the H-2 and H-3 protons in
The goal of this investigation was to find more
selective antiviral agents bearing the 4-amino-3-bro-
mopyrrolo[2,3-d]pyridazin-7-one heterocycle. The find-
ings in this investigation revealed that the sugar-
modified derivatives were much less cytotoxic than their
corresponding ribonucleoside 3. Unfortunately there
was a corresponding or even greater loss of activity
against HCMV and HSV-1 leading to the conclusion
that modification of the sugar did not lead to more
selective antiviral agents.
1
the H NMR spectra of 20 and 21 with the correspond-
ing patterns in the ribofuranosyl analogs 1 and 3
established the 3-position as the site of bromination
(vide supra).
The acyclo analog 4-amino-1-[(1,3-dihydroxy-2-pro-
poxy)methyl]pyrrolo[2,3-d]pyridazin-7-one (24) was again

Sugar-Modified Pyrrolopyridazin-7-one Nucleosides
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 797
Sch em e 4
Ta ble 1. Cytotoxicity of Pyrrolo[2,3-d]pyridazin-7-one
Nucleosides in L1210 Cell Cultures
resulting oil was dissolved in a minimal amount of a solution
of CHCl3:MeOH:acetic acid, 97:2:1, and applied to a column
(3.2 × 20 cm) packed with silica gel (19.7 g) in the same solvent
system. The column was eluted with the same solvent system,
collecting 10 mL fractions. The fractions containing a product
with an Rf ) 0.42 (CHCl3:MeOH, 20:1) were pooled and
concentrated in vacuo to an oil. The oil was coevaporated with
toluene (2 × 10 mL) and then ether (2 × 10 mL). The gum
was further dried in vacuo (25 °C, oil pump) over P2O5 for 2 h
to afford 558 mg (72.6%) of 7. IR (neat): 3500-2700 valley,
substituent
1660, 1630, 1545, 1440, 1215, 745 cm-1
. UV (MeOH):
IC50 ( M)a
max
compd
R′
R′′
nm (ꢀ) 294 (3220). 1H NMR (CDCl3): 10.46 (s, 1H, H-6, D2O
exchangeable), 7.68 (d, J ) 3.0 Hz, 1H, H-2), 7.42-6.93 (m,
16H, phenyl H’s, H-1′), 6.30 (d, J ) 3.2 Hz, 1H, H-3), 4.75-
3.69 (m, 11H, C6H5CH2, H-2′, H-3′, H-4′, H-5′). Anal. Calcd
(C32H32N4O5) C, H, N.
1b
2b
3b
4b
8
ribosyl
ribosyl
ribosyl
ribosyl
H
74
0.23
0.10
0.02
100
Cl
Br
I
arabinosyl
H
4-Am in o-1-(â-D-a r a b in ofu r a n osyl)p yr r olo[2,3-d ]p y-
r id a zin -7-on e Hyd r och lor id e (8). A solution of 7 (573 mg,
1.1 mmol) in CH2Cl2 (6 mL), chilled by an external 2-propanol-
CO2 bath, was treated with a solution of BCl3 (13 mL of 1 M
solution in CH2Cl2) over the course of 40 min. After the
addition was complete, the reaction mixture was stirred for
an additional 1 h at -78 °C. An CH3CN-C02 bath was then
exchanged for the 2-propanol-CO2 bath, and the solution was
stirred for an additional 2.5 h. A solution of CH2Cl2:MeOH,
1:1 (16 mL, prechilled via an external CH3CN-CO2 bath), was
added in three separate portions to quench the reaction. The
reaction mixture was allowed to warm to room temperature
at which point it became a white suspension. The suspension
was concentrated in vacuo, and the resulting concentrate was
coevaporated with MeOH (4 × 20 mL) to remove the excess
BCl3 as methyl borate. The resulting solid concentrate was
suspended in MeOH (3 mL), allowed to stand for 15 h, collected
by filtration, and then washed with ether to afford 8 as a white
solid, 256 mg (75.4%), mp >216 °C dec. IR (KBr pellet): 3670-
2500, 1679.7, 1651.6, 1560.2, 1433.6, 1215.6, 1060.9 cm-1. UV
10
13
15
20
21
24
25
arabinosyl
Br
H
Br
H
Br
H
Br
18
2-deoxyribosyl
2-deoxyribosyl
2,3-dideoxyribosyl
2,3-dideoxyribosyl
DHPM
>100c
9.2
>100c
38
>100c
>100c
0.043
DHPM
5 (tubercidin)b
a
Concentration required to decrease growth rate to 50% of
control. Data for 1-43 and 57 have been reported previously.c At
100 M, the highest concentration tested, cell proliferation was
b
inhibited 0-50%.
Exp er im en ta l Section
Proton magnetic resonance (1H NMR) spectra were obtained
with a Bruker WP270SY spectrometer (solutions in dimethyl
sulfoxide-d6 or deuteriochloroform with tetramethylsilane as
internal standard), with chemical shift values reported in
,
parts per million, relative to the internal standard. Carbon
magnetic resonance (13C NMR) spectra were obtained at 90
MHz in DMSO-d6 with an IBM WM-360 spectrometer. Ul-
traviolet spectra were recorded on a Hewlett-Packard model
8450A UV/vis spectrophotometer. Infrared spectra were re-
corded on a Nicolet 5DXB FTIR spectrophotometer ( , cm-1).
Melting points were determined with a Thomas-Hoover capil-
lary apparatus and are uncorrected. E. Merck silica gel (230-
400 mesh) was used for column chromatography. Thin layer
chromatography was performed on silica gel GHLF-254 plates
(Merck Reagents). Rf’s were determined using the solvent
system used to elute the column unless otherwise specified.
Solvent systems are reported in vol:vol ratios. Compounds of
interest were detected by either ultraviolet lamp (254 nm),
iodine vapors, or treatment with 10% sulfuric acid in MeOH
followed by heating. Evaporations were performed under
reduced pressure with a bath temperature <35 °C, with a
rotary evaporator using a water aspirator unless otherwise
specified. Elemental analyses were performed by M-H-W
Laboratories, Phoenix, AZ.
4-Am in o-1-(2,3,5-t r i-O-b en zyl-â-D-a r a b in ofu r a n osyl)-
p yr r olo[2,3-d ]p yr id a zin -7-on e (7). A solution of ethyl
3-cyano-1-(2,3,5-tri-O-benzyl- -D-ribofuranosyl)pyrrole-2-car-
boxylate3 (6) (789 mg, 1.4 mmol) in EtOH (15 mL) was treated
with anhydrous hydrazine (1.5 mL) in one portion. The
resulting solution was heated at reflux under an argon
atmosphere for 48 h. The reaction mixture was concentrated
in vacuo to an oil which was coevaporated repeatedly with
EtOH (3 × 10 mL) to remove the excess hydrazine. The
(H2O) (pH 7):
nm (ꢀ) 288 (5600); (pH 1) 281 (5600); (pH
max
11) 288 (6020). 1H NMR (DMSO-d6): 7.84 (d, 1H, H-2), 6.95
(br s, 1H, H-1′), 6.80 (d, 1H, H-3), 5.70-4.50 (br s, 4H, OH’s,
NH2, D2O exchangeable), 4.12 (t, 1H, H-2′), 4.00 (t, lH, H-3′),
3.79-3.60 (m, 3H, H-4′, H-5′). Anal. Calcd for (C11H15N4O5-
Cl‚1/2CH3OH) C, H, N.
4-Am in o-3-br om o-1-(â-D-a r a bin ofu r a n osyl)p yr r olo[2,3-
d ]p yr id a zin -7-on e (10). A solution of 4-amino-1-(2,3,5-tri-
O-benzyl- -D-arabinofuranosyl)pyrrolo[2,3-d]pyridazin-7-one (7;
481 mg, 0.87 mmol) in CH2Cl2 (50 mL) was treated with a
solution of Br2 (1.5 equiv) in CH2Cl2 (25 mL) by an addition
funnel, dropwise, at room temperature, over the course of 3
h. The resulting mixture was allowed to stir at room temper-
ature for 2 days. The yellow solid which precipitated from the
mixture was collected by filtration to furnish 370 mg of the
presumed HBr salt 9.
A suspension of 9 in CH2Cl2 (10 mL) was chilled by an
external 2-propanol-CO2 bath. A solution of 1 M BCl3 in CH2-
Cl2 (7 mL, 14 equiv) was added to the chilled reaction mixture
over the course of 30 min. After the addition was complete,
the external bath was removed and the reaction mixture was
allowed to stir at room temperature over the course of 2 h.
The reaction mixture was then rechilled by the external
2-propanol-CO2 bath and a prechilled solution of MeOH: CH2-
Cl2,1:1 (8 mL, chilled via an external 2-propanol-CO2 bath),
was added to the mixture in one portion. The reaction mixture
was again allowed to warm to room temperature over the
course of 30 min. The mixture was concentrated in vacuo, and

798 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Meade et al.
Ta ble 2. Antiviral Activity and Cytotoxicity of Pyrrolo[2,3-d]pyridazin-7-one Nucleosides
50% or 90% inhibitory concentration ( M)
antiviral activitya
HCMV
cytotoxicityc
substituent
R′
ribosyl
ribosyl
ribosyl
ribosyl
HSV-1b
ELISA
compd
R′′
plaque
yield
HFF cells
KB cells
1d,e
2
3
H
>100f
0.2
90
6.8
>100
2.1
Cl
Br
I
0.7
6.1
<1.2
1.2
0.1
0.4
0.04
0.02
2.0
4.5
2.5
1.0
4
8
arabinosyl
arabinosyl
H
Br
H
Br
H
Br
H
>100
90
20
128
15
>100
10
13
15
20
21
24
25
2.7
6.3
6.3
32
15
>100e
6.7
2′-deoxyribosyl
2′-deoxyribosyl
dideoxyribosylg
dideoxyribosyl
DHPMg
>100
19
>100
10
>100
>100
>100
>100e
10e
12
10e
27
32
>100
>100
7.7h
>100e
>100e
3.5e
>100
>100
>100h
>100e
>100e
>100e
DHPM
Br
ganciclovir (DHPG)
1.8h
a
Plaque and yield reduction assays were performed as described in the text. Results from plaque assays are reported as IC50’s, those
for yield reduction experiments as IC90’s. Assayed by ELISA in quadruplicate wells; given as average IC50.
c Visual cytotoxicity scored
b
on HFF cells at time of HCMV plaque enumeration. Inhibition of KB cell growth was determined as described in the text in quadruplicate
d
wells. Results are presented as IC50’s. Results for compounds 1-4 reported previously as compounds 3, 16, 18, and 19 in ref 3. e Data
from a single experiment; all other data except as indicated for DHPG are averages from two to four separate experiments. f >100 indicates
g
IC50 or IC90 not reached at the noted (highest) concentration tested. Abbreviations: dideoxyribosyl, 2′,3′-dideoxyribosyl; DHPM, (1,3-
h
dihydroxy-2-propoxy)methyl. Average of 88 and 31 experiments, respectively, for plaque and yield experiments.
the concentrate was coevaporated with MeOH (1 × 20 mL).
The resulting concentrate was redissolved in MeOH, and the
solution was neutralized with sufficient NH4OH to effect a pH
) 8. The resulting solution was treated with powdered Na2-
SO4 (4.12 g), and the suspension was concentrated in vacuo
to a powder. The powder was applied to the top of a column
(3.2 × 20 cm) packed with silica gel (12.2 g) in CHCl3:MeOH,4:
1. The column was eluted with the same solvent system,
collecting 5 mL fractions. The fractions containing a product
with an Rf ) 0.28 were pooled and concentrated in vacuo to
an off-white solid. The solid was suspended in a small amount
of MeOH, collected by filtration, washed with ether, and dried
in vacuo (100 °C, oil pump) for 12 h to afford 58 mg of 10.
The fractions containing the product with an Rf ) 0.28 and
column was eluted with the same solvent system, collecting
50-75 mL fractions. The fractions containing a product with
Rf ) 0.34 were pooled and concentrated in vacuo to a white
solid. The solid was suspended in an ether:pentane, 1:1,
mixture, allowed to stand at 5 °C for 30 min, and then collected
by filtration. The solid was dried in vacuo (25 °C, oil pump)
over P2O5 for 15 h to afford 5.21 g (82.7%) of 12 as a white
solid, mp 122-124 °C. IR (KBr pellet): 3149.2, 2980.5, 2917.2,
2228.1, 1714.8, 1609.4 cm-1
.
1H NMR (DMSO-d6):
7.91-
7.81 (m, 4H, Ph H’s), 7.58 (d, J ) 3.0 Hz, 1H, H-5), 7.37-7.29
(m, 4H, Ph H’s), 6.84 (t, 1H, H-1′), 6.75 (d, J ) 3.0 Hz, 1H,
H-4), 5.57 (m, 1H, H-3′), 4.60 (s, 3H, H-4′, H-5′), 4.30 (q, 2H,
OCH2CH3), 2.80-2.50 (m, 2H, H-2′), 2.39-2.36 (2s, 6H, C6H4-
CH3), 1.29 (t, 3H, OCH2CH3). Anal. Calcd (C29H28N2O7) C,
H, N.
an additional product with
a lower Rf were pooled and
concentrated in vacuo to furnish a sticky solid. The solid was
heated on a steam bath with a minimal amount of H2O and
then allowed to cool to room temperature. The resulting white
solid was collected by filtration, washed with MeOH and then
ether, and dried in vacuo (100 °C, oil pump) to afford an
additional 16 mg of pure 10. The combined yield of 10 was
23.6% from 7, mp >244 °C dec. IR (KBr pellet): 3600-2500
br valley, 1635.9, 1622.6, 1563.6, 1064.8 cm-1. UV (H2O) (pH
4-Am in o-1-(2-deoxy-â-D-er yth r o-pen tofu r an osyl)pyr r olo-
[2,3-d ]p yr id a zin -7-on e (13). Meth od A. A suspension of
12 (4.40 g, 8.5 mmol) in EtOH (100 mL) was treated with
anhydrous hydrazine (3 mL). The reaction mixture (which
became a solution when heated) was heated at reflux temper-
ature for 15 h under an argon atmosphere. The solution was
allowed to cool to room temperature, and then an additional
5 mL of hydrazine was added. The solution was heated at
reflux temperature for another 24 h, and then it was concen-
trated in vacuo. The concentrate was coevaporated with EtOH
(5 × 100 mL) to remove the excess hydrazine to afford an off-
white solid. The solid was suspended in acetone (60 mL),
warmed on a steam bath for 5 min, and then stirred at room
temperature for 20 min. The resulting off-white solid was
collected by filtration and recrystallized from boiling MeOH
to afford an off-white solid. The solid was dried in vacuo (56°
C, oil pump) over P2O5 for 18 h to afford 1.23 g (54.4%) of 13
as a white solid, mp 235-237.5 °C.
7):
nm (ꢀ) 290 (6230); (pH 1) 290 (6780); (pH 11) 296
max
(5970). 1H NMR (DMSO-d6):
11.47 (s, 1H, NH-6), 7.81(s,
1H, H-2), 7.05 (d, J ) 4.9 Hz, 1H, H-1′), 5.45-5.15 (m, 5H,
OH’s, NH2), 4.08 (dd, 1H, H-2′), 3.98 (dd, 1H, H-3′), 3.76-3.62
(m, 3H, H-4′, H-5′). Anal. Calcd (C11H13N4O5Br) C, H, N.
Eth yl 3-Cyan o-1-(2-deoxy-3,5-di-O-p-tolu oyl-â-D-er yth r o-
p en tofu r a n osyl)p yr r ole-2-ca r boxyla te (12). A solution of
ethyl 3-cyanopyrrole-2-carboxylate (5; 2.0 g, 12.2 mmol)9 in
CH3CN (40 mL) was treated with sodium hydride (50%
mineral oil dispersion, 702 mg, 1.2 equiv). The resulting
suspension was allowed to stir under an argon atmosphere
for 30 min before 2-deoxy-3,5-di-O-p-toluoyl- -D-erythro-pento-
furanosyl chloride (11; 4.98 g, 1.2 equiv)11 was added in two
portions. The resulting reaction mixture was further diluted
with CH3CN (80 mL) and then allowed to stir at room
temperature for 4 h. The reaction mixture was filtered
through a thin pad of Celite, and the resulting filtrate was
concentrated in vacuo to an oil. The oil was dissolved in a
minimal amount of EtOAc and applied to a column (5 × 60
cm) packed with silica gel (320 g) in hexane:EtOAc, 4:1. The
An analytical sample of 13 was prepared by recrystallizing
159 mg of 13 from boiling MeOH. The resulting crystals were
dried in vacuo (56 °C, oil pump) over P2O5 for 18 h, mp 238-
239 °C. IR (KBr pellet): 3600-2700 valley, 1625, 1540, 1432,
1275, 1220, 1110, 772 cm-1
.
UV (H2O) (pH 7):
288 (5080); (pH 1) 273 (5720); (pH 11) 288 (4830). 1H NMR
(DMSO-d6): 11.22 (s, 1H, NH-6, D2O exchangeable), 7.70
nm (ꢀ)
max
(d, J ) 3.0 Hz, 1H, H-2), 7.18 (t, J ) 6.8 Hz, 1H, H-1′), 6.63
(d, J ) 3.0 Hz, 1H, H-3), 5.68 (s, 2H, NH2, D2O exchangeable),
5.24 (d, 1H, OH-3′, D2O exchangeable), 4.94 (t, 1H, OH-5′, D2O

Sugar-Modified Pyrrolopyridazin-7-one Nucleosides
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 799
exchangeable), 4.29 (m, 1H, H-3′), 3.80 (m, 1H, H-4′), 3.55 (m,
2H, H-5′), 2.24 (q, 2H, H-2′). Anal. Calcd (C11H14N4O4) C, H,
N.
colorless oil, 3.03 g (67.9%). IR (neat): 3479.7, 2931.2, 2860.9,
2228.1, 1707.8, 1665.6, 1412.5, 1257.8, 1201.6, 1103.1 cm-1
.
1H NMR (CDCl3): 7.56 (d, J ) 3.0 Hz, 1H, H-5), 6.76 (t, J )
5.9 Hz, 1H, H-1′), 6.46 (d, J ) 3.0 Hz, 1H, H-4), 4.47 (m, 1H,
H-3′), 4.34 (q, 2H, OCH2CH3), 4.00 (dd, 1H, H-4′), 3.87 (m, 2H,
H-5′), 2.56 (m, 1H, H-2′), 2.22 (m, 1H, H-2′), 1.89 (d, 1H, OH-
3′, D2O exchangeable), 1.41 (t, 3H, OCH2CH3), 0.88 (s, 9H,
C(CH3)3), 0.09 (s, 3H, SiCH3), 0.08 (s, 3H, SiCH3). Anal. Calcd
(C19H30N2O5Si) C, H, N.
Meth od B. A solution of 14 (1.21 g, 4.3 mmol) in EtOH
(25 mL) was treated with H2NNH2 (anhydrous, 1.2 mL), and
the resulting mixture was heated at reflux under an argon
atmosphere for 22 h. The reaction mixture was allowed to cool
to room temperature, and another portion (1.2 mL) of hydra-
zine was added. The resulting mixture was reheated at reflux
for an additional 6 h, and then it was allowed to stand at room
temperature for 15 h to effect the precipitation of a white solid.
The solid was collected by filtration, washed with EtOH and
then ether, and dried in vacuo (oil pump) to afford 0.87 g (75%)
of 13, mp 229-232 °C. A sample of 13 prepared by this
method comigrated on TLC with a sample of 13 prepared by
method A (CHCl3:MeOH, 4:1; Rf ) 0.18).
Eth yl 3-Cya n o-1-(2-d eoxy-â-D-er yth r o-p en tofu r a n osyl)-
p yr r ole-2-ca r boxyla te (14). Compound 12 (13.72 g, 26.6
mmol) was added to a solution of sodium ethoxide (0.5 equiv)
in EtOH (300 mL). The reaction mixture was allowed to stir
at room temperature for 11 h. The mixture was adjusted to
pH ) 6 with Dowex 50 × 8-100 ion exchange resin. The resin
was removed by filtration, and the resulting filtrate was
concentrated in vacuo (<40 °C, water aspirator) to an oily solid.
The oily solid was suspended in ether (50 mL) and stirred with
a spatula and the solid collected by filtration to afford 5.92 g
(79.5%) of 14 as a light pink solid, mp 118-120 °C.
E t h yl 3-Cya n o-1-[5-O-(ter t-b u t yld im et h ylsilyl)-3-O-
(p h en oxyt h ioca r b on yl)-2-d eoxy-â-D-er yth r o-p en t ofu r a -
n osyl]p yr r ole-2-ca r boxyla te (17). A solution of 16 (3.03 g,
7.68 mmol) and 4-(dimethylamino)pyridine (4.69 g, 5 equiv)
in CH3CN (60 mL) was chilled by an external ice water bath.
Phenyl chlorothionoformate (3.3 mL, 3.1 equiv) was added in
one portion by pipet. The resulting yellow suspension was
stirred in the ice bath for 10 min, and then it was stirred at
room temperature for 2.5 days. The yellow suspension was
concentrated in vacuo to a residue which was suspended in
EtOAc (125 mL) and extracted with 2% aqueous acetic acid (2
× 100 mL). The organic layer was dried over MgSO4 and
concentrated in vacuo to an oil. The oil was dissolved in a
minimal amount of toluene:EtOAc, 30:1, and applied to a
column (9.5 × 9 cm sintered glass funnel) packed with silica
gel (160 g) in the same solvent system. The column was eluted
with this same system, collecting 40 mL fractions. The
fractions that were homogeneous in containing a product with
an Rf ) 0.42 were pooled and concentrated in vacuo to a
colorless oil, which was dried in vacuo (25 °C, oil pump) to
afford 1.94 g of 17.
A small amount (290 mg) of 14 was recrystallized from
EtOH and dried in vacuo (oil pump, 100 °C) for 6 h to afford
190 mg of 14, mp 119-121 °C. IR (KBr pellet): 3600-2700
1
valley, 2228.1, 1707.8, 1581.2, 1426.6, 1053.9, 646.1 cm-1. H
The fractions containing the product with an Rf ) 0.42 and
an additional impurity with a higher Rf were combined and
concentrated in vacuo to a colorless gum (1.1 g). The gum was
dissolved in a minimal amount of a solution of toluene:EtOAc,
30:1, and applied to a column (3.2 × 20 cm) packed with silica
gel (39 g) in the same system. The column was eluted with
the same solvent system collecting fractions (ca. 10 mL sized).
The fractions containing a product with an Rf ) 0.42 were still
contaminated with the impurity. These fractions were pooled
and concentrated in vacuo to a colorless residue (0.59 g). The
residue was dissolved in a minimal amount of toluene and
applied to a column (3.2 × 20 cm) packed with silica gel (23 g)
in toluene (neat). The column was first eluted with toluene
(neat) to remove the nonpolar impurity and then with toluene:
EtOAc/30:1, to elute the desired product. The product frac-
tions containing the product with an Rf ) 0.42 were pooled
and concentrated in vacuo to a colorless oil. The oil was dried
further in vacuo (25 °C, oil pump) to afford an additional 0.41
g of 17. The combined yield of 17 was 61%. IR (neat): 2958.2,
2932.2, 2858.9, 2232.8, 1708.5, 1492.3, 1420.2, 1263.0, 1210.5,
NMR (DMSO-d6): 7.75 (d, J ) 3.0 Hz, 1H, H-5), 6.73 (d, J
) 3.0 Hz, lH, H-4), 6.64 (t, J ) 5.0 Hz, 1H, H-l ′), 5.26 (d, 1H,
OH-3, D2O exchangeable), 5.05 (t, 1H, OH-5′, D2O exchange-
able), 4.34-4.22 (m, 3H, OCH2CH3, H-3′), 3.82 (dd, 1H, H-4′),
3.64-3.54 (m, 2H, H-5′), 2.50-2.08 (m, 2H, H-2′), 1.31 (t, 3H,
OCH2CH3). Anal. Calcd (C13H16N2O5) C, H, N.
4-Am in o-3-br om o-1-(2-d eoxy-â-D-er yth r o-p en tofu r a n o-
syl)p yr r olo[2,3-d ]p yr id a zin -7-on e (15). To a solution of 13
(0.41 g, 1.54 mmol) in glacial acetic acid (25 mL) was added
N-bromosuccinimide (0.41 g, 1.5 equiv) in one portion. The
reaction mixture was stirred at room temperature for 20 h and
then concentrated in vacuo (<40 °C, oil pump). The resulting
concentrate was dissolved in MeOH, and then powdered Na2-
SO4 (11 g) was added. The resulting suspension was concen-
trated in vacuo to afford a yellow solid. The solid was layered
onto the top of a column (3.2 × 20 cm) packed with silica gel
(26 g) in CHCl3:MeOH, 5:1. The column was eluted with the
same solvent system, collecting 10 mL fractions. The fractions
containing a product with Rf ) 0.42 were pooled and concen-
trated in vacuo to a gel. The resulting gel was dissolved in
H2O and lyophilized on a freeze-drying apparatus (0.2 atm)
for 32 h to yield 0.31 g (57%) of 15 as a fluffy, white solid. IR
(KBr pellet): 3650-2500 valley, 1665.9, 1635.9, 1536.3, 1091.4,
1118.8, 837.1, 771.2, 758.4 cm-1 1H NMR (CDCl3): 7.62 (d,
.
J ) 3.2 Hz, H-5), 7.47-7.10 (m, 5H, phenyl H’s), 6.94 (dd, J )
5.7, 6.7 Hz, 1H, H-1′), 6.53 (d, J ) 3.1 Hz, 1H, H-4), 5.77 (d, J
) 6.1 Hz, 1H, H-3′), 4.48-4.37 (m, 3H, OCH2CH3, H-4′), 4.01
(d, J ) 2.0 Hz, 2H, H-5′), 2.96 (m, 1H, H-2′), 2.38 (m, 1H, H-2′),
1.45 (t, 3H, OCH2CH3), 0.90 (s, 9H, C(CH3)3), 0.12 (s, 6H,
SiCH3). Anal. Calcd (C26H34N2O6SSi) C, H, N.
1058.2 cm-1. UV (MeOH):
nm (ꢀ) 300 (5100). 1H NMR
max
(DMSO-d6): 11.53 (s, 1H, NH-6), 7.96 (s, 1H, H-2), 7.19 (t, J
) 6.5 Hz, 1H, H-1′), 5.41 (br s, 2H, NH2), 5.26 (d, 1H, OH-3′),
5.04 (t, 1H, OH-5′), 4.29 (m, 1H, H-3′), 3.82 (m, 1H, H-4′), 3.57
(m, 2H, H-5′), 2.25 (m, 2H, H-2′). Anal. Calcd (C11H13N4O4-
Br‚1/2H2O) C, H, N.
Eth yl 3-Cya n o-1-[5-O-(ter t-bu tyld im eth ylsilyl)-2,3-d i-
d e oxy-â-D-glycer o-p e n t ofu r a n osyl]p yr r ole -2-ca r b oxy-
la te (18). A solution of 17 (1.13 g, 2.3 mmol) in toluene (35
mL) was treated with AIBN (74 mg, 0.2 equiv) and then
tributyltin hydride (1.24 mL, 2 equiv). The solution was
lowered into an oil bath preheated to 83 °C, heated at this
temperature for 2.5 h under an argon atmosphere, and then
concentrated in vacuo (<35 °C, water pump and then oil pump)
to afford a yellow oil. The oil was dissolved in a minimal
amount of CHCl3:EtOAc, 1:1, and applied to a column (2.5 ×
52 cm) packed with silica gel (95 g) in cyclohexane:EtOAc:
CHCl3, 10:1:1. The column was eluted with the same solvent
system, collecting 10 mL-sized fractions. The fractions con-
taining a product with Rf ) 0.28 were pooled and concentrated
in vacuo to an oil. The oil was dried in vacuo (25 °C, oil pump)
over P2O5 for 3 h to afford 0.53 g (61%) of 18 as a colorless oil.
IR (neat): 3142.2, 2953.3, 2931.2, 2860.9, 2228.1, 1707.8,
Eth yl 3-Cyan o-1-[5-O-(ter t-bu tyldim eth ylsilyl)-2-deoxy-
â-D-er yth r o-p en tofu r a n osyl]p yr r ole-2-ca r boxyla te (16). A
solution of 14 (3.17 g, 11.3 mmol), imidazole (1.85 g, 2.4 equiv),
and tert-BuMe2SiCl (1.87 g, 1.1 equiv) in DMF (80 mL) was
stirred under an argon atmosphere for 5 h at room tempera-
ture. The reaction mixture was concentrated in vacuo (<30
°C, oil pump) to an oil. The oil was dissolved in EtOAc (150
mL) and the resulting solution extracted with H2O (2 × 100
mL). The organic layer was dried over MgSO4 and then
concentrated in vacuo to an oil. The oil was dissolved in a
minimal amount of a mixture of CHCl3: MeOH,50:1, and
applied to a column (9 × 7 cm) packed with silica gel (110 g)
in the same solvent system. The column was eluted with the
same solvent system, collecting (10 mL fractions). The frac-
tions containing a product with an Rf ) 0.50 were pooled and
concentrated in vacuo to an oil. The oil was further dried in
vacuo (25 °C, oil pump) over P2O5 for 7 h to afford 16 as a
1475.8, 1412.5, 1257.8, 1208.6, 1131.2, 835.9, 793.8 cm-1 1H
.
NMR (CDC13): 7.79 (d, J ) 2.9 Hz, 1H, H-5), 6.61 (dd, J )
1.6, 6.0 Hz, 1H, H-1′), 6.44 (d, J ) 2 Hz, 1H, H-4), 4.38 (q, 2H,

800 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5
Meade et al.
OCH2CH3), 4.17 (m, 1H, H-4′), 4.08 (dd, 1H, H-5′), 3.75 (dd,
1H, H-5′), 2.52-1.86 (m, 4H, H-2′, H-3′), 1.43 (t, 3H, OCH2-
CH3), 0.93 (s, 9H, C(CH3)3), 0.11 (s, 3H, SiCH3), 0.10 (s, 3H,
SiCH3). Anal. Calcd (C19H30N2O4Si) C, H, N.
5′) 4.07 (m, 1H, H-4′) 3.63 (m, 2H, H-5′) 2.40, 1.95 (2m, 4H,
H-3′, H-2′). Anal. Calcd (C11H13N4O3Br) C, H, N.
E t h yl 3-Cya n o-1-[(1,3-d ia cet oxy-2-p r op oxy)m et h yl]-
p yr r ole-2-ca r boxyla te (23). A solution of 5 (2.48 g,15.1
mmol) in CH3CN (60 mL) was treated with NaH (523 mg of
97% reagent, 1.5 equiv) in one portion. The resulting suspen-
sion was stirred under an argon atmosphere for 25 min, and
then it was treated with (1,3-diacetoxy-2-propoxy)methyl
bromide14 (22; 4.5 mL, 1.5 equiv) by a syringe. The reaction
mixture was allowed to stir at room temperature for 30 min,
and then more 22 (0.33 equiv) was added. The reaction
mixture was allowed to stir for an additional 10 min after the
second addition and then filtered through a thin pad of Celite.
The filtrate was concentrated in vacuo to an oil which was
dissolved in toluene:EtOAc, 8:1, and applied to a column (4.5
× 24 cm) packed with silica gel (135 g) in the same solvent
system. The column was eluted with this same solvent system
and then with toluene:EtOAc, 5:1, collecting fractions. The
fractions containing a product with an Rf ) 0.28 (toluene:
EtOAc, 5:1) were pooled and concentrated in vacuo to a
colorless oil. The oil was dried in vacuo (100 °C, oil pump) for
48 h to afford 4.74 g (89.1%) of 23. IR (neat): 3121.1, 2987.5,
Eth yl 3-Cya n o-1-(2,3-d id eoxy-â-D-glycer o-p en tofu r a n o-
syl)p yr r ole-2-ca r boxyla te (19). A solution of 18 (562 mg,
1.2 mmol) in THF (10 mL) was treated with n-Bu4NF (1.0 M
solution in THF, 1.3 mL, 1.1 equiv). The resulting yellow
solution was allowed to stir at room temperature for 1 h. The
reaction mixture was then concentrated in vacuo to a yellow
oil. The oil was dissolved in CH2Cl2:MeOH, 1:1 (2 mL), and
applied to a column (3.2 × 20 cm) packed with silica gel (32 g)
in CH2Cl2:MeOH, 35:1. The column was eluted with the same
solvent system, collecting 6 mL sized fractions. The fractions
containing a product with an Rf ) 0.22 were pooled and
concentrated in vacuo to afford a white solid. The solid was
suspended in ether, and the resulting suspension was chilled
via an external ice-water bath. The resultant white crystal-
line precipitate was collected by filtration and dried in vacuo
(25 °C, oil pump) to afford 194 mg (61.1%) of 19, mp 98-100
°C. IR (neat): 3142.2, 2952.3, 2931.2, 2860.9, 2228.1, 1707.8,
1475.8, 1412.5, 1257.8, 1208.6, 1131.2, 835.9, 793.8 cm-1 1H
.
2235.2, 1743.0, 1489.8, 1419.5, 786.7 cm-1 1H NMR (CDCl3):
.
NMR (DMSO-d6): 7.86 (d, J ) 3.0 Hz, 1H, H-5), 6.70 (d, J
) 3.0 Hz, 1H, H-4), 6.49 (d, J ) 6.6 Hz, 1H, H-1′), 5.09 (t, 1H,
OH-5′, D2O exchangeable), 4.29 (q, 2H, OCH2CH3), 4.08 (m,
1H, H-4′), 3.73-3.55 (m, 2H, H-5′), 2.46-1.78 (m, 4H, H-2′,
H-3′), 1.30 (t, 3H, OCH2CH3). Anal. Calcd (C13H16N2O4) C,
H, N.
7.07 (d, J ) 2.9 Hz, 1H, H-5), 6.57 (d, J ) 2.9 Hz, 1H, H-4),
5.85 (s, 2H, NCH2O), 4.42 (q, 2H, OCH2CH3), 4.21-3.96 (m,
5H, OCH2O, CH), 2.04 (s, 6H, C(O)CH3), 1.45 (t, 3H, OCH2-
CH3). Anal. Calcd (C16H20N2O7) C, H, N.
4-Am in o-1-[(1,3-d ih yd r oxy-2-p r op oxy)m eth yl]p yr r olo-
[2,3-d ]p yr id a zin -7-on e (24). A mixture of 23 (4.67 g, 13.2
mmol) and anhydrous H2NNH2 (9.6 mL) in EtOH (50 mL) was
heated at reflux for 4 days. The reaction mixture was
concentrated in vacuo (<40 °C) to an oil which was coevapo-
rated with EtOH (3 × 40 mL). The oil was redissolved in
boiling EtOH (50 mL), and the resulting solution was allowed
to stand at room temperature to effect crystallization. The
off-white solid was collected by filtration. This solid was
recrystallized from a boiling solution of EtOH:H2O, 9:1. The
resulting off-white solid was collected by filtration, washed
with a small amount of EtOH, and dried in vacuo (100 °C, oil
pump) to afford 1.82 g (54%) of 24, mp 186-188.5 °C.
An analytical sample of 24 was prepared by recrystallizing
200 mg of 24 in EtOH:H2O, 4:1. The resulting crystals were
collected by filtration and dried in vacuo (100 °C, oil pump) to
afford 80 mg of 24 as white needles, mp 189-191 °C. IR (KBr
pellet): 3389.1, 3276.2, 3209.8, 1635.9, 1543.0, 1443.4, 1410.1,
1290.6, 1224.2, 1104.7, 1051.6, 991.8, 779.3, 746.1 cm-1. UV
4-Am in o-1-(2,3-d id eoxy-â-D-glycer o-p en t ofu r a n osyl)-
p yr r olo[2,3-d ]p yr id a zin -7-on e (20). A solution of 19 (168
mg, 0.64 mmol) in EtOH (8 mL) was treated with anhydrous
hydrazine (0.34 mL). The resulting solution was heated at
reflux under an argon atmosphere for 24 h. The reaction
mixture was allowed to cool to room temperature, and then
another 0.34 mL of hydrazine was added by pipet. The
solution was again heated at reflux for 24 h. The resulting
reaction mixture was concentrated in vacuo to an oil which
was coevaporated with EtOH (5 × 5 mL). The resulting
concentrate was a white solid which was suspended in boiling
EtOH (5 mL). The EtOH suspension was allowed to stand at
room temperature for 6 h, and then the solid was collected by
filtration. The solid was dried in vacuo for 16 h over P2O5 to
afford 99 mg (62%) of 20 as a white solid, mp >200 °C dec. IR
(KBr pellet): 3600-2700 valley, 1640, 1540, 1440, 1280, 1220,
1090, 1072, 1045, 1030, 980, 920, 820, 775 cm-1. UV (H2O)
(pH 7): max nm (ꢀ) 287 (5280); (pH 1) 274 (6590); (pH 11) 286
(5400). 1H NMR (DMSO-d6):
11.18 (s, 1H, NH-6, D2O
(H2O) (pH 7):
nm (ꢀ) 262 (7040), 287 (6450); (pH 1) 272
max
(8750); (pH 11) 262 (6840), 287 (6710). 1H NMR (DMSO-d6):
11.27 (s, 1H, NH-6, D2O exchangeable), 7.50 (d, J ) 3.0 Hz,
1H, H-2), 5.89 (s, 2H, NCH2O), 5.74 (s, 2H, NH2, D2O
exchangeable), 4.59 (s, 2H, OH’s, D2O exchangeable), 3.56-
3.22, (m, 5H, CH, CHCH2OH). Anal. Calcd (C10H14N4O4) C,
H, N.
exchangeable), 7.73 (d, J ) 3.0 Hz, 1H, H-2), 6.99 (dd, J )
2.7, 6.8 Hz, 1H, H-1′), 6.59 (d, J ) 3.0 Hz, 1H, H-3), 5.67 (s,
2H, NH2, D2O exchangeable), 4.95 (t, 1H, OH-5′, D2O ex-
changeable), 4.04 (m, 1H, H-4′), 3.58 (m, 2H, H-5′), 2.42-2.35,
2.02-1.88 (2m, 4H, H-2′, H-3′). Anal. Calcd (C11H14N4O3) C,
H, N.
4-Am in o-3-br om o-1-(2,3-dideoxy-â-D-glycer o-pen tofu r a-
n osyl)p yr r olo[2,3-d ]p yr id a zin -7-on e (21). To a solution of
20 in glacial acetic acid (8 mL) was added N-bromosuccinimide
(232 mg, 1.4 equiv). The resulting reaction mixture became a
solution and was stirred at room temperature for 30 min. The
reaction mixture was concentrated in vacuo to a reddish-
colored residue which was dissolved in MeOH. To this
methanolic solution was added powdered Na2SO4 (6 g), and
the resulting suspension was concentrated in vacuo to a
powder. The powder was layered onto the top of a column (3.2
× 20 cm) packed with silica gel (16 g) in CHCl3:MeOH, 12:1.
The column was eluted with this same solvent system,
collecting 5 mL fractions. The fractions that contained a
product with an Rf ) 0.22 were pooled and concentrated in
vacuo to a gel. The gel was dissolved in a small amount of
hot MeOH, and the solution was allowed to stand at room
temperature for 6 h. The resulting precipitate was collected
by filtration, washed with ether, and dried in vacuo (78 °C,
oil pump) over P2O5 for 24 h to afford 94 mg (30.7%) of 21, mp
213-215 °C. IR (KBr pellet): 3600-2700 valley, 1635, 1535,
4-Am in o-3-br om o-1-[(1,3-dih ydr oxy-2-pr opoxy)m eth yl]-
p yr r olo[2,3-d ]p yr id a zin -7-on e (25). To a solution of 24 (248
mg, 0.97 mmol) in glacial acetic acid (8 mL) was added a
solution of NBS (280 mg, 1.6 equiv) in acetic acid (25 mL)
dropwise, via an addition funnel over the course of 1.5 h. The
resulting reaction mixture was concentrated in vacuo (<30 °C,
oil pump) to an oil. The oil was dissolved in hot MeOH (40
mL), and the resulting solution was treated with powdered
Na2SO4 (7 g). The suspension was concentrated in vacuo to a
powder which was applied to the top of a column (3.2 × 20
cm) packed with silica gel (16.6 g) in CHCl3:MeOH, 6:1. The
column was eluted with this same solvent system and then
with CHCl3:MeOH, 4:1, collecting 5 mL fractions. The frac-
tions containing a product with an Rf ) 0.38 were pooled and
concentrated in vacuo to a solid. The solid was suspended in
a minimal amount of MeOH, collected by filtration, washed
with ether, and dried in vacuo (100 °C, oil pump) over P2O5
for 18 h to afford 119 mg of (36.8%) 25 as a peach-colored solid,
mp 195-196.5 °C. IR (KBr pellet): 3428.9, 3316.0, 3110.1,
3023.8, 2970.7, 2942.2, 1655.9, 1616.0, 1529.7, 1463.3, 1436.7,
1284.0, 1191.0, 1118.0, 1044.9, 1005.1, 845.7, 752.7, 659.8
1455, 1285, 1170, 1110, 1080, 985 cm-1. UV (H2O) (pH 7):
max
nm (ꢀ) 296 (5800); (pH 1) 291 (6690); (pH 11) 296 (5290). 1H
NMR (DMSO-d6): 11.48 (s, 1H, NH-6) 8.05 (s, 1H, H-2) 6.98
(d, J ) 6.2 Hz, 1H, H-1′) 5.40 (br s, 2H, NH2) 5.09 (t, 1H, OH-
cm-1. UV (H2O) (pH 7):
nm (ꢀ) 246 (7090), 294 (3050);
max
(pH 1) 250 (5460), 290 (3500); (pH 11) 246 (6760), 294 (2940).
1H NMR (DMSO-d6): 11.54 (s, 1H, NH-6, D2O exchangeable),

Sugar-Modified Pyrrolopyridazin-7-one Nucleosides
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 5 801
7.80 (s, 1H, H-2), 5.92 (s, 2H, OCH2N), 5.44 (s, 2H, NH2, D2O
exchangeable), 4.59 (t, 2H, OH’s, D2O exchangeable), 3.57-
3.22 (m, 5, CHCH2OH, CHCH2OH). Anal. Calcd (C10H13N4O4-
Br) C, H, N.
Department of Health and Human Services NIAID
Research Grant U19-AI-31718, Contract N01-AI-72641,
and in part by a research grant (DHP-36) from the
American Cancer Society.
In Vitr o An tip r olifer a tive Stu d ies. The in vitro cyto-
toxicity against L1210 was evaluated as described previously.15
L1210 cells were grown in static suspension culture using
Fischer’s medium for leukemic cells of mice with 10% heat-
inactivated (56 °C, 30 min) horse serum. The growth rate was
calculated from determinations of cell number at 0, 48, and
96 h in the presence of various concentrations of the test
compound. Growth rate is defined as the slope of the semi-
logarithmic plot of cell number against time for the treated
culture as a percent of the slope for the control culture. This
parameter was determined experimentally by calculating the
ratio of the population-doubling time (Td) of control cells to
the Td of treated cells. When the growth rate decreased during
the experiment, the rate used was that between 48 and 96 h.
The IC50 is defined as the concentration required to decrease
the growth rate to 50% of the control.
In Vitr o An tivir a l Eva lu a tion . (a ) Cells a n d Vir u ses.
KB cells, an established human cell line derived from an
epidermoid oral carcinoma, were routinely grown in MEM with
Hank’s salts supplemented with 5% fetal bovine serum.
Diploid human foreskin fibroblasts (HFF cells) were grown in
MEM with Earle’s salts supplemented with 10% fetal bovine
serum. Cells were passaged according to conventional proce-
dures as detailed previously.5,7 A plaque-purified isolate, Po,
of the Towne strain of HCMV was used and was a gift of Dr.
M. F. Stinski, University of Iowa. The S-148 strain of HSV-1
was provided by Dr. T. W. Schafer of Schering Corp. Stock
preparations of HCMV and HSV-1 were prepared and titered
as described elsewhere.5,16
(b) Assa ys for An tivir a l Activity. HCMV plaque reduc-
tion experiments were performed using monolayer cultures of
HFF cells by a procedure similar to that referenced above for
titration of HCMV, with the exceptions that the virus inoculum
(0.2 mL) contained approximately 50 PFU of HCMV and the
compounds to be assayed were dissolved in the overlay
medium. Protocols for the HCMV yield reduction assay have
been described previously.16 HSV-1 was assayed using an
enzyme immunoassay described by Prichard and Shipman.17
(c) Cytotoxicity Assa ys. Two basic tests for cellular
cytotoxicity were routinely employed for compounds examined
in antiviral assays. Cytotoxicity produced in HFF cells was
estimated by visual scoring of cells not affected by virus
infection in the plaque reduction assays described above.
Drug-induced cytopathology was estimated at 30-fold magni-
fication and scored on a 0-4+ basis on the day of staining for
plaque enumeration.5 Cytotoxicity in KB cells was determined
by measuring the effects of compounds on the growth of cells.
Growth was measured spectrophotometrically by staining cells
with crystal violet 2 days after drug treatment.18
(d ) Da ta An a lysis. Dose-response relationships were
constructed by linearly regressing the percent inhibition of
parameters derived in the preceding sections against log of
drug concentration. Fifty percent inhibitory (IC50) concentra-
tions were calculated from the regression lines. Samples
containing positive controls (acyclovir, ganciclovir, or 2-acetylpy-
ridine thiosemicarbazone for HSV-1, HCMV, and cytotoxicity,
respectively) were used in all assays. Results from sets of
assays were rejected if inhibition by the positive control
deviated from its mean response by more than 1.5 standard
deviations.
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Ack n ow led gm en t. The authors are indebted to
Allison C. Westerman, Edward D. Kreske, Mary S.
Ludwig, Roger G. Ptak, and Mary A. Walters for expert
technical assistance. We also thank J ack Hinkley for
large scale preparations of starting materials, as well
as Marina Savic for preparation of the manuscript. This
work was supported with Federal Funds from the
J M960631X