Synthesis and antiviral activity of prodrugs of the nucleoside 1-[2',3'-dideoxy-3'-C-(hydroxymethyl)-β-D-erythropentofuranosyl] cytosine

Article abstract of DOI:10.1016/S0968-0896(98)00020-0

The synthesis and antiviral evaluation of 21 prodrugs of 1-[2',3'-dideoxy-3'-C-(hydroxymethyl)-β-d-erythropentofuranosyl] cytosine 1 is reported. Cytosine N⁴-imine analogues were prepared by condensation of 1 with selected formamide dimethyl acetals. Amino acid substituted prodrugs were prepared from 1 or imine prodrug 2 by coupling with either N-tert-butoxycarbonyl (t-Boc)-l-valine or N-t-Boc-l-phenylalanine in the presence of dicyclohexycarbodiimide (DCC) and 4-dimethylaminopyridine (4-DMAP). Deprotection of the t-Boc protecting group was achieved with trifluoroacetic acid (TFAA) in methylene chloride. Cytosine N⁴-amide analogues were prepared by reaction of 1 with appropriate anhydrides in aqueous dioxane. Triacylated analogue 22 was prepared by reaction of 1 with four equivalents of benzoyl chloride in pyridine. Prodrugs were evaluated for activity against duck hepatitis B virus, herpes simplex virus types 1 and 2, human cytomegalovirus, and human immunodeficiency virus. A number of analogues were found comparable in activity to 1 with the cytosine N⁴-imine series more active than the amino acid substituted and cytosine N⁴-amide prodrugs. Slight to moderate cellular toxicity was observed with some analogues. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00020-0

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 577±585
Synthesis and Antiviral Activity of Prodrugs of the Nucleoside
1-[20,30-Dideoxy-30-C-(hydroxymethyl)-b-D-
Erythropentofuranosyl] Cytosine
Scott C. Mauldin,a,* C. J. Paget, Jr,a,y C. David Jones,a Joseph M. Colacino,a
Angela J. Baxter,a Kirk A. Staschke,a Nils-Gunnar Johanssonb
and Lotta Vrangb
aLilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA
bMedivir, AB, Huddinge, Sweden
Received 14 July 1997; accepted 17 November 1997
AbstractÐThe synthesis and antiviral evaluation of 21 prodrugs of 1-[20,30-dideoxy-30-C-(hydroxymethyl)-b-D-ery-
thropentofuranosyl] cytosine 1 is reported. Cytosine N4-imine analogues were prepared by condensation of 1 with
selected formamide dimethyl acetals. Amino acid substituted prodrugs were prepared from 1 or imine prodrug 2 by
coupling with either N-tert-butoxycarbonyl (t-Boc)-L-valine or N-t-Boc-L- phenylalanine in the presence of dicyclo-
hexycarbodiimide (DCC) and 4-dimethylaminopyridine (4-DMAP). Deprotection of the t-Boc protecting group was
achieved with tri¯uoroacetic acid (TFAA) in methylene chloride. Cytosine N4-amide analogues were prepared by
reaction of 1 with appropriate anhydrides in aqueous dioxane. Triacylated analogue 22 was prepared by reaction of 1
with four equivalents of benzoyl chloride in pyridine. Prodrugs were evaluated for activity against duck hepatitis B
virus, herpes simplex virus types 1 and 2, human cytomegalovirus, and human immunode®ciency virus. A number of
analogues were found comparable in activity to 1 with the cytosine N4-imine series more active than the amino acid
substituted and cytosine N4-amide prodrugs. Slight to moderate cellular toxicity was observed with some analogues.
# 1998 Elsevier Science Ltd. All rights reserved.
Introduction
crossing of the blood±brain barrier.6 In this regard,
novel and safe nucleosides with enhanced pharmacolo-
gical properties are urgently needed.
The need for e€ective therapeutics for the treatment of
viral infections such as those caused by human immuno-
de®ciency virus (HIV), hepatitis B virus, and herpes
viruses, has prompted the synthesis and development of
numerous biologically active nucleoside analogues.1,2
Zidovudine (30-azido-30-deoxythymidine, AZT) was the
®rst nucleoside analogue approved for the treatment of
AIDS, but has undesirable side e€ects that include bone
marrow suppression, leucopenia, and anemia.3 It has
been demonstrated that AZT therapy can result in the
emergence of viral resistance,4 requires frequent dosing
due to its short plasma half-life,5 and does not involve
The mechanism of action of most nucleoside analogues
is dependent, at least in part, upon their phosphoryla-
tion by viral/cellular nucleoside kinases. This results in
the formation of the triphosphate form of the nucleoside
analogue, which is then responsible for inhibition of the
viral nucleic acid polymerase leading to the disruption
of viral nucleic acid replication. AZT is phosphorylated
by a host cell nucleoside kinase to the triphosphate form
which inhibits the reverse transcriptase of HIV. Fur-
thermore, AZT has a 30-azido substituent instead of a
30-hydroxyl group. Because of the lack of a 30-hydroxyl
group, when AZT is incorporated into DNA, 30 to 50
phosphodiester linkages cannot be made leading to the
termination of the nascent DNA chain.
*Corresponding author. Infectious Diseases Research, Lilly
Corporate Center, Indianapolis, IN 46285, USA.
yRetired, Eli Lilly and Company.
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00020-0

578
S. C. Mauldin et al./Bioorg. Med. Chem. 6 (1998) 577±585
Although much work has targeted the treatment of
infections caused by HIV, nucleoside analogues display
activity against other viruses as well. Other nucleoside
analogues, such as (E)-5-(2-bromovinyl)-20-deoxyur-
idine (BVDU) which is selectively potent against herpes
simplex type 1 (HSV-1),7 are active against herpes viru-
ses, which encode a thymidine kinase that selectively
phosphorylates some nucleoside analogues. Another
nucleoside analogue, 5-iodo-20-deoxyuridine (IDU), is
e€ective for the treatment of herpes simplex keratitis
although with lesser selectivity.8 In the area of hepatitis,
nucleoside analogues such as 3-TC9 have shown pro-
mise as inhibitors of viral replication.
the blood±brain barrier.16 Based upon the relationship
of lipophilicity to membrane permeability18,19 many
examples have been reported regarding modi®cation of
nucleosides in an e€ort to enhance CNS penetration.
These include 50 esteri®cation of the parent nucleoside,20
phosphate modi®cation of nucleotides,21,22 or derivati-
zation of the nucleoside base.23
We chose to modify 1 to provide a variety of prodrugs
with derivitization at the cytidine amino group, deoxy
sugar 50 and 60 alcohols, or a combination of both in the
hopes that such a strategy would provide useful infor-
mation relating to the three potential H-donor sites of 1
(cytidine NH2, 50, 60 OH). Initially, we sought a method
for selective derivatization of the cytosine amine and
were especially intrigued by the formation of N,N-di-
alkylaminomethylene derivatives of 20,30-dideoxycytidine
(ddC) and its 30-¯uoro derivative as reported by Kerr
and Kalman.23 We prepared a series of these derivatives
for compound 1. Additionally, we incorporated amino
acid substitution into the nucleosides since the amino
acid transport system for delivery into the CNS is well
de®ned.20 A number of cytosine amine (N4-substituted)
analogues and a triacylated derivative were also synthe-
sized for comparison.24,25 This paper describes the pre-
paration and antiviral activities of the foregoing various
prodrugs of 1.
In the search for nucleoside analogues with greater
antiviral activity and enhanced selectivity, the incor-
poration of a hydroxymethylene group at the 30 position
of the sugar moiety of the nucleoside analogue has been
considered.10,11 It has been known for some time that
branch-chained sugar nucleosides are biologically
active,12 possibly because of greater recognition of these
substrates by polymerase enzymes as compared to non-
branched nucleoside analogues.13 Oxetanocin, a natu-
rally occurring purine nucleoside, and its analogues are
prototypical of this class which demonstrates ecacy
against infections caused by HIV-1,14 and herpes viru-
ses.15 Another branch-chained sugar containing nucleo-
side analogue, 1-[20,30-dideoxy-30-C-(hydroxymethyl)-b-
D-erythropentofuranosyl] cytosine 1 (Figure 1), was found
to be a potent inhibitor of HIV-1 replication in vitro
(inhibition of HIV replication by 50% at a concentra-
tion of 0.01 mM).11 Due to the branch-chained nature of
its sugar moiety, 1 was investigated further for activity
against a broader spectrum of viruses.
Chemistry
Parent nucleoside 1 was prepared according to the pro-
cedure of Svansson et al.11 The preparation of the cyto-
sine N4-substituted imines was accomplished as shown
in Scheme 1. The requisite formamide acetals were
Although 1 was found to be highly water soluble (S. C.
Mauldin, unpublished observation), it was our concern
that it might not be transported into the CNS eciently.
Considerable evidence indicates that transport of certain
nucleosides into the CNS occurs.16,17 However, there
appears to be a preference for transport of thymidine
and uridine analogues rather than cytidine derivatives.
For example, AZT readily enters the cerebrospinal ¯uid
whereas ddC is more limited in its ability to penetrate
Figure 1. Numbering system of 1.
Scheme 1.

S. C. Mauldin et al./Bioorg. Med. Chem. 6 (1998) 577±585
579
prepared from intermediate dialkylformamides and
dimethyl sulfate at room temperature. Reaction of the
intermediate salts with sodium methoxide provided the
amide-acetals in 20±40% yield after distillation.23,26
These were condensed with 1 to provide imines 2±8. In
this way, a variety of straight-chain and cyclic analogues
were prepared for biological comparison.
For the N4-iminyl- 50, 60 disubstituted amino acids, the
cytosine imine was found to be quite labile. In fact, 13
was initially formed during puri®cation of 9 and was
obtained in a column fraction. Apparently, methanol
and the acidity of silica gel were sucient to cleave the
imine to a small extent. This was not observed with the
corresponding phenylalanine derivative 10. It was sub-
sequently found convenient to cleave the imines with
warm aqueous methanol and subsequently remove the
N-tert-butoxycarbonyl protecting groups with TFAA in
methylene chloride. In this manner, the diamino acid
cytosine derivatives 15 and 16 were obtained as tri-
¯uoroacetate salts. Analogously, the trisubstituted N-
tert-butoxycarbonyl-a-amino acids were deprotected
with TFAA to give trisubstituted analogues 17 and
18. Characteristic of the trisubstituted prodrugs was the
1H NMR resonance of the H-5 proton, found to be
considerably further down®eld relative to the corre-
sponding 50,60 disubstituted cytosine analogues. This
e€ect was attributed to the N4 neighboring carboxamide
deshielding e€ect.27 (For example, H-5 for 15 was d 6.02
ppm; for 17 it was d 7.21 ppm).
The amino acid (a-L-valyl and a-L-phenylalanyl) esters
9±18 containing either a free or substituted N4-cytosine
amino function were prepared by coupling of either 1 or
2 with 3.5 and 2.2 equivalents, respectively, of the
desired N-tert-butoxycarbonyl-a-L-amino acid in
a
manner analogous to that reported by Aggarwal et al.20
(Scheme 2).
The non-amino acid N4-amide prodrugs were prepared
by classical acylation of 1 analogous to the method of
Akiyama, et al.24 (Scheme 3). Alkylation of 1 with 4
equivalents of benzoyl chloride in pyridine gave the
triacylated analogue 22.
Scheme 2.
Scheme 3.

580
S. C. Mauldin et al./Bioorg. Med. Chem. 6 (1998) 577±585
Results and Discussion
labeled primers, were retained by the interaction of the
biotin labeled primers with the avidin coated beads and
were quanti®ed by ¯uorescence. DHBV DNA was then
quanti®ed by comparing the experiment with a standard
curve derived from known amounts of template DHBV
DNA.
Antiviral activity of the prodrugs was determined
against duck hepatitis B virus (DHBV), herpes simplex
virus (HSV), human cytomegalovirus (HCMV), and
human immunode®ciency virus (HIV). Results are
shown in Table 1. Initially, 2 was assayed against
DHBV. Using 1 as a comparator, the assay was con-
ducted in G2/G3 cells, a human hepatoblastoma cell
that constitutively produces DHBV28 (obtained from
W. S. Mason, Fox Chase Cancer Center, Philadelphia,
PA). The cells were incubated in test compounds for 11
days, after which time virion DNA in the cell culture
¯uid was analyzed by polymerase chain reaction (PCR)
using primers designed to amplify the pre S region of the
viral genome.29 PCR ampli®ed products were electro-
phoresed through 1% agarose. Antiviral activity was
correlated to a decrease in the amount of PCR product
which could be visualized by ethidium bromide staining.
Each concentration was performed in duplicate and
each compound was compared to its own no-drug con-
trol. Parent 1 was found to be active at a concentration
of 4.1 mM and prodrug 2 was comparably active at a
concentration of 1.7 mM.
Compounds 2±22 were evaluated for their ability to
inhibit herpes simplex virus I (HSV-I) Mayo strain
induced cytopathic e€ect (CPE) in BSC-1 cells using
Acyclovir as the positive inhibitor control. Acyclovir
was found active at a minimum inhibitory concentration
of 1.8 mM. Compounds 5, 6 and 8 were found to be the
more active compounds in the HSV-I assay as minimum
inhibitory concentrations of 5.0 mM was observed. All
test compounds demonstrated similar inhibitory values
against herpes simplex virus II (HSV-II) G strain. The
inhibitory e€ect of the prodrugs on HCMV plaque for-
mation in WI-38 cell monolayers was assayed using
Ganciclovir as a positive inhibitor control with the more
active compounds listed. Analogs were evaluated for
activity against wild-type HIV strain IIIb or HIV which
is resistant to the non-nucleoside reverse transcriptase
inhibitor, TIBO in an XTT assay as previously descri-
bed.30,31 An average of two determinations was used
and the more active derivatives are shown.
More quantitative results against duck hepatitis virus
were obtained using a newly developed PCR/PANDEX
assay (K. A. Staschke, J. A. Burgess, A. J. Baxter and J.
M. Colacino, manuscript in preparation). Brie¯y, the
assay involved a solid-phase capture of a PCR product
generated by a primer set designed to amplify a region
within the pre-S gene of the DHBV genome. One primer
of the set was conjugated to biotin while the other was
conjugated to ¯uoroscein. The resulting PCR product
was captured by mixing with avidin coated beads and
added to the Pandex 96-well assay plates. The free,
unincorporated ¯uoroscein labeled primers were washed
through a cellulose acetate ®lter in the bottom of the
well. The PCR products, which contain ¯uoroscein
In these antiviral assays, the prodrugs tested displayed
only minimal toxicity. Using the XTT assay to evaluate
anti-HIV activity in MT4 cells, no toxicity was observed
up to 300 mM. In plaque assays to evaluate activity
against CMV, slight to moderate toxicity in WI-38 cells
was observed for compounds 3, 5±7,10, and 21 at con-
centrations of 100 mM.
Conclusions
A number of prodrugs of 1 were found to be active in
the antiviral assays. Inhibition of DHBV by the imine
prodrugs was comparable to 1. Activity against HSV-1
and 2 was shown by all derivatives in the imine series as
well. Certain amino acids in which the cytosine amine
was unsubstituted showed some activity, but were in
general less active than the imines. Trisubstituted ana-
logue 22 showed some activity, but displayed moderate
toxicity. Also, slight to moderate toxicity was observed
among the amino acid derivatives.
Table 1. Antiviral activities of various prodrugs of 1 (IC50
mM)
,
Compd
DHBV
0.2
HCMV
HIV-wt
HIV-TIBOr
1
2
11.4
4.5
9.1
4.0
3
13.3
4
1.2
0.3
0.3
5
3.4
2.1
2.0
5.0
7.7
12.8
13.1
3.2
6.8
4.5
Activity against HCMV was noted in the imine series as
well as the amino acid series. Slight to moderate toxicity
was observed with the L-valyl analogues, making them
less desirable than the L-phenylalanyl derivatives in this
regard. Signi®cant anti-HIV activity was observed
against both wild-type and TIBO-resistant strains in the
imine series. The esteri®ed prodrugs were essentially
inactive against both HIV strains.
6
7
10.8
9.4
8
9
0.5
2.0
10
19
21
7.2
18.7
22.7

S. C. Mauldin et al./Bioorg. Med. Chem. 6 (1998) 577±585
581
The data obtained in the whole cell assays supports the
concept that antiviral activity can be preserved in con-
junction with decreasing the polarity of parent 1. The
imine series of prodrugs was more active than the amino
acid series of prodrugs, as substitution on the alcohol
sites resulted in a decrease in activity in virtually all
assays. These results could indicate that intracellular
phosphorylation of the 50 alcohol was necessary to
achieve activity. However, it cannot be overlooked that
di€erences in transport, conversion, or extracellular
activation could be responsible for the di€erences in
activities as well. The imine series appears attractive for
design of prodrugs of 1 since compounds of this type
require no derivitization of the 50 and 60 alcohols. Fur-
ther, it has been demonstrated that imines of this type
are hydrolytically cleaved to parent.23 The activity of a
number of cytosine N4-imine analogues is uniform and
synthesis of these compounds is highly ecient. Further
data on membrane permeability, half-lives, and hydro-
lytic stability are needed at this point to determine if the
prodrugs described here have potential utility as clinical
antiviral agents.
N,N-dimethylformamide dimethyl acetal was obtained
from Aldrich.
N4-Dimethylaminomethylene-1-[20,30-dideoxy-30-C-(hydr-
oxymethyl)-ꢀ-D-erythropentofuranosyl] cytosine (2). A
mixture of 1 (50 mg, 0.21 mmol), N,N-dimethylform-
amide dimethyl acetal (125 mg, 1.05 mmol), and DMF
(2 mL) was reacted to give after crystallization 2 (49 mg,
80%): mp 198±200 ꢀC; IR (KBr) 3237, 2895, 1650,
1
1598 cm ; 1H NMR (DMSO-d6): d 1.90±2.00 (m, 1H),
2.18±2.33 (m, 2H), 3.03 (s, 3H), 3.16 (s, 3H), 3.45 (t,
J=5.1 Hz, 2H), 3.55±3.62 (m, 1H), 3.70±3.84 (m, 2H),
4.74 (t, J=5.1 Hz, 1H), 5.05 (t, J=5.1 Hz, 1H), 5.95 (m,
2H), 8.19 (d, J=7.0 Hz, 1H), 8.60 (s ,1H) FD MS 297.
Anal. (C13H20N4O4) C , H, N.
N4-Diisopropylaminomethylene-1-[20,30-dideoxy-30-C-(hydr-
oxymethyl)-ꢀ-D-erythropentofuranosyl] cytosine (3). A
mixture of 1 (75 mg, 0.31 mmol), diisopropylformamide
dimethyl acetal (284 mg, 1.62 mmol), and DMF (3 mL)
was reacted to give after chromatography (8% methanol
in methylene chloride) 3 as a foamy resin, (60 mg, 55%):
1
IR (KBr): 3401 (br, OH), 2978, 1653, 1575 cm
;
1H
NMR (DMSO-d6): d 1.23 (d, J=7.0 Hz, 6H), 1.28 (d,
J=7.0 Hz, 6H),1.89±1.98 (m, 1H), 2.17±2.30 (m, 2H),
3.45 (t, J=5.5 Hz, 2H), 3.55±3.69 (m, 1H), 3.70±3.76 (m,
1H), 3.78±3.89 (m, 2H), 4.63±4.78 (m, 2H), 5.04 (t,
J=5.5 Hz, 1H), 5.93±5.97 (m, 2H), 8.18 (d, J=7.0 Hz,
.
1H), 8.71 (s, 1H); FD MS 353. Anal. (C17H28N4O4 O.25
H2O) C, H, N.
Experimental
Melting points were determined with a Thomas±Hoover
melting point apparatus and are uncorrected. IR spectra
were obtained on a Nicolet 510P FT±IR spectrometer.
1H NMR spectra were obtained on a GE QE-300
spectrometer at 300.15 MHZ in the solvent indicated.
Field desorption (FD) mass spectra were recorded on a
VG Analytical ZAB-3F instrument. Elemental analyses
were performed by the Physical Chemistry Department
at Lilly Research Laboratories and were within
‹0.4% of the theoretical values. Chromatography was
performed using silica gel 60, particle size 0.040±
0.063 mm, 230±400 mesh ATM from EM Science. TLC
analyses were done on silica gel 60 precoated plates, EM
Science, with a layer thickness of 250 mm. Spots were
visualized under 254 nm illumination. Products were
dried in high vacuum (<1 mm Hg) for 16 h at either
60 ꢀC for solids melting above 100 ꢀC or at ambient
temperature for those melting below 100 ꢀC, foams, or
oils.
N4-Di-n-propylaminomethylene-1-[20,30 -dideoxy-30 -C-
(hydroxymethyl)-ꢀ-D-erythropentofuranosyl] cytosine (4).
A mixture of 1 (50 mg, 0.21 mmol), di-n-propylform-
amide dimethylacetal (175 mg, 1.00 mmol), and DMF
(2 mL) was reacted to give following crystallization 4 as
a white solid (59 mg, 80%): mp 145±147 ꢀC; IR (KBr):
1
3296 (br,OH), 2965, 2899, 1609 cm
(DMSO-d6):
;
1H NMR
0.86 (dt, J=7.4 Hz, 6H), 1.60 (q,
d
J=7.4 Hz, 4H), 1.89±1.99 (m, 1H), 2.17±2.30 (m, 2H),
3.36±3.46 (m, 6H), 3.54±3.61 (m, 1H), 3.70±3.76 (m,
1H), 3.79±3.85 (m, 1H), 4.74 (t, J=5.2 Hz, 1H), 5.04 (t,
J=5.2 Hz, 1H), 5.95 (m, 2H), 8.18 (d, J=7.4 Hz, 1H),
8.62 (s, 1H); FD MS 353. Anal. (C17H28N4O4) C, H, N.
N4-Pyrrolidinomethylene-1-[20,30-dideoxy-30-C-(hydroxy-
methyl)-ꢀ-D-erythropentofuranosyl] cytosine (5). A mix-
ture of 1 (50 mg, 0.21 mmol), N-(dimethoxymethyl)
pyrrolidine (160 mg , 1.10 mmol), and DMF (3 mL) was
reacted to give following crystallization 5 (60 mg, 90%):
mp 210-212 ꢀC; IR (KBr): 3228 (br,OH), 2891,
1614 cm 1 ; 1H NMR (DMSO-d6): d 1.89 (m, 5H), 2.17±
2.31 (m, 2H), 3.39±3.51 (m, 4H), 3.55±3.77 (m, 2H), 3.65
(m, 2H), 3.83 (m, 1H), 4.74 (t, J=5.2 Hz, 1H), 5.05 (t,
J=4.8 Hz, 1H), 5.95 (m, 2H), 8.19 (d, J=7.4 Hz, 1H),
8.78 (s, 1H); FD MS 323. Anal. (C15H22N4O4) C, H, N.
General procedure for the preparation of the (dialkyl-
amino) methylene derivatives of 1-[20,30-dideoxy-30-C-
(hydroxymethyl)-ꢀ-D-erythropentofuranosyl] cytosine 1.
To a solution of 1 (0.21±0.31 mmol) in dry N,N-di-
methylformamide (DMF) (2±3 mL) was added 5 equiv of
the required dialkylformamide dimethylacetal26 and the
mixture was stirred at room temperature under N2 for
12 h. The solvent was removed under reduced pressure,
the residue was either crystallized from ethanol/ethyl
ether mixture or chromatographed over silica gel.

582
S. C. Mauldin et al./Bioorg. Med. Chem. 6 (1998) 577±585
N4-Piperidinomethylene-1-[20,30-dideoxy-30-C-(hydroxy-
methyl)-ꢀ-D-erythropentofuranosyl] cytosine (6). A mix-
ture or 1 (50 mg, 0.21 mmol), N-(dimethoxymethyl)
piperidine (175 mg, 1.10 mmol), and DMF (2 mL) was
reacted to give following crystallization 6 (58 mg, 82%):
6H), 4.18 (m, 6H), 4.45 (m, 1H), 5.02 (br, 1H,), 5.10 (d,
J=8.5 Hz, 1H), 6.11 (m, 1H), 6.22 (br, 1H), 7.88 (d,
J=6.6 Hz, 1H), 8.86 (s, 1H); FD MS 696. Anal.
(C33H54N6O10) C, H, N. Further elution provided 13 as
a white foam ®ltered with the aid of ethyl ether (35 mg,
12%); IR (KBr): 3386, 2968, 1747, 1717 cm 1; 1H NMR
(CDCl3): d 0.90 (d, J=4.0 Hz, 3H), 0.92 (d, J=4.0 Hz,
3H), 0.98 (d, J=2.6 Hz, 3H), 1.00 (d, J=2.6 Hz, 3H),
1.44 (s, 18H), 2.12 (m, 2H), 2.29±2.52 (m, 3H), 4.19 (m,
6H), 4.37±4.50 (m, 1H), 5.30 (d, J=8.8 Hz, 2H), 5.94 (d,
J=7.4 Hz, 1H), 6.04 (m, 1H), 7.83 (d, J=6.6 Hz, 1H);
mp 170±174 ꢀC ; IR (KBr): 3295, 2922, 1651 cm 1; H
1
NMR (DMSO-d6): d 1.49±1.70 (m, 6H) , 1.93 (m, IH),
2.24 (m, 2H), 3.44 (t, J=5.2 Hz, 2H), 3.53 (m, 2H), 3.61
(m, 1H), 3.65±3.76 (m, 3H), 3.82 (m, 1H), 4.76 (t,
J=5.5 Hz, 1H), 5.04 (t, J=5.5 Hz, 1H), 5.94 (m, 2H),
8.18 (d, J=7.4 Hz, 1H), 8.62 (s, 1H); FD MS 337. Anal.
(C16H24N4O4) C, H, N.
.
FD MS 640. Anal. (C30H49N5O10 0.5C4H10O) C, H, N.
N4-Hexamethyleneiminylmethylene-1-[20,30-dideoxy-30-C-
(hydroxymethyl)-ꢀ-D-erythropentofuranosyl] cytosine
(7). A mixture of 1 (50 mg, 0.21 mmol), N-(dimethoxy-
methyl)hexamethyleneimine (190 mg, 1.10 mmol), and
DMF (2 mL) was reacted to give following crystal-
N4-(Dimethylaminomethylene)-1-[20,30 -dideoxy-50,60 -O-
(N-ꢁ-tert-butoxycarbonyl-L-phenylalanyl)-ꢀ-D-erythro-
pentofuranosyl] cytosine (10). A mixture of dicyclohexyl-
carbodiimide (227 mg, 1.10 mmol), compound
2
(135 mg, 0.46 mmol), 4-dimethylaminopyridine (167 mg,
1.37 mmol), and N-tert-butoxycarbonyl-L-phenylalanine
(266 mg, 1.00 mmol) in anhydrous ethyl acetate (20 mL)
was stirred for 36 h at room temperature. The solids
were ®ltered and the ®ltrate was concentrated and
chromatographed (2.5% methanol in methylene chlor-
ide) to give a white amorphous solid. Crystallization
from ethyl ether/hexanes gave 10 as a white solid
lization 7 (56 mg, 76%): mp 194±196 ꢀC; IR (KBr): 3249,
1
2936, 1647 cm
;
1H NMR (DMSO-d6): d 1.52 (m,
4H), 1.72 (m, 4H), 1.93 (m, 1H), 2.24 (m, 2H), 3.45 (t,
J=5.2 Hz, 2H), 3.60 (m, 5H), 3.69±3.76 (m, 1H), 3.82
(m, 1H), 4.74 (t, J=5.2 Hz, 1H), 5.04 (t, J=5.2 Hz, 1H),
5.95 (m, 2H), 8.19 (d, J=7.0 Hz, 1H), 8.65 (s, 1H); FD
MS 351. Anal. (C17H26N4O4) C, H, N.
(310 mg, 86%): mp 120±125 ꢀC; IR (KBr): 3349, 2927,
N4-Morpholinomethylene-1-[20,30-dideoxy-30-C-(hydroxy-
methyl)-ꢀ-D-erythropentofuranosyl] cytosine (8). A mix-
ture of 1 (50 mg, 0.21 mmol), N-(dimethoxymethyl)
morpholine (177 mg, 1.37 mmol), and DMF (3 mL) was
reacted to give after crystallization 8 (47 mg, 66%): mp
225±228 ꢀC; IR (KBr): 3279, 2908, 1652 cm 1; 1H NMR
(DMSO-d6): d 1.94 (m, 1H), 2.25 (m, 2H), 3.45 (t,
J=5.2 Hz, 2H), 3.55±3.77 (m, 10H), 3.83 (m, 1H), 4.75
(t, J=5.2 Hz, 1H), 5.05 (t, J=5.2 Hz, 1H), 5.94 (m, 2H),
8.22 (d, J=7.4 Hz, 1H), 8.67 (s, 1H) ; FD MS 339. Anal.
(C15H22N4O4) C, H, N.
1737, 1691 cm
;
1H NMR (CDCl3): d 1.42 (s, 18H),
1
1.85 (brm, 1H), 2.02±2.29 (m, 2H) , 3.05 (m, 4H), 3.17
(s, 6H), 3.78 (m, 1H), 3.95 (d, J=5.9 Hz, 2H), 4.22 (m,
2H), 4.54 (m, 2H), 5.05 (m, 2H), 5.99 (dd, J=3.3,
6.6 Hz, 1H), 6.17 (d, J=7.0 Hz, 1H), 7.11±7.34 (m,
10H), 7.74 (d, J=7.4 Hz , 1H), 8.86 (s, 1H); MS FD
792. Anal (C41H54N6O10) C , H , N.
N4-(N0 -ꢁ-tert-butoxycarbonyl-L-valyl)-1-[20,30-dideoxy-
50,60-O-(N00-ꢁ-tert-butoxycarbonyl-L-valyl)-ꢀ-D-erythro-
pentofuranosyl] cytosine (11). To
a solution of 1
(120 mg, 0.50 mmol), 4-dimethylaminopyridine (275 mg,
2.25 mmol), N-tert-butoxycarbonyl-L-valine (380 mg,
1.75 mmol), and anhydrous ethyl acetate (25 mL) under
N2 was added diclohexylcarbodiimide (365 mg,
1.77 mmol). The solution was stirred 2 days at room
temperature, ®ltered, and concentrated. The residue was
chromatographed (2% methanol in methylene chloride)
N4-Dimethylaminomethylene-1-[20,30-dideoxy-50,60-O-(N-
ꢁ-tert-butoxycarbonyl-L-valyl)-ꢀ-D-erythropentofuranosyl]
cytosine (9) and 1-[20,30-dideoxy-50,60-O-(N-ꢁ-tert-but-
oxycarbonyl - L - valyl) - ꢀ- D - erythropentofuranosyl]
cytosine (13). To
a
stirring solution of
2
(135 mg, 0.46 mmol), 4-dimethylaminopyridine (167 mg,
1.37 mmol), and N-tert-butoxycarbonyl-L-valine (218 mg,
1.00 mmol) in dry ethyl acetate (20 mL) under N2 was
added dicyclohexylcarbodimide (227 mg, 1.10 mmol).
The mixture was stirred at room temperature for 36 h
and the solids were ®ltered. The ®ltrate was con-
centrated in vacuo and the residue was chromato-
graphed (2.5% methanol in methylene chloride).
Initially obtained was 9 as a foam (70 mg, 22%): IR
to give 11 as a white foam (225 mg, 54%); IR (KBr):
1
3448, 2976, 1719 cm
;
1H NMR (CDCl3): d 1.01 (m,
18H), 1.44 (m, 27H), 2.12±2.50 (m, 6H), 4.18 (m, 6H),
4.30±4.68 (m, 2H), 5.02 (m, 3H), 6.06 (m, 1H), 7.49 (t,
J=9.9 Hz, 1H), 8.20 (d, J=6.6 Hz, 1H), 8.78 (s, 1H) ;
MS FD 840. Anal. (C40H66N6O13) C, H, N.
N4-(N0 -ꢁ-tert-butoxycarbonyl-L-phenylalanyl)-1-[20,30-
dideoxy-50,60 -O-(N00 -ꢁ-tert-butoxycarbonyl-L -phenyl-
alanyl)-ꢀ-D-erythropentofuranosyl] cytosine (12). A mix-
ture of dicyclohexylcarbodiimide (365 mg, 1.77 mmol),
compound 1 (120 mg, 0.50 mmol), 4-dimethylaminopyr-
1
(KBr): 3380, 2970, 1725, 1715 cm 1; H NMR (CDCl3):
d 0.90 (d, J=2.6 Hz, 3H), 0.92 (d, J=2.6 Hz, 3H), 0.98
(d, J=3.7 Hz, 3H), 1.00 (d, J=3.7 Hz, 3H), 1.45 (s,
18H), 2.13 (m, 2H), 2.30 (m, 1H), 2.46 (m, 2H), 3.18 (s,

S. C. Mauldin et al./Bioorg. Med. Chem. 6 (1998) 577±585
583
idine (275 mg, 2.25 mmol), N-tert-butoxycarbonyl-L-
phenylalanine (465 mg, 1.75 mmol), and dry ethyl acet-
ate (25 mL) was stirred at room temperature for 2 days
under N2. The solids were ®ltered, the ®ltrate con-
centrated, and the resin chromatographed to give 12 as
10H), 7.69 (d, J=8.1 Hz, 1H), 8.00±8.60 (brs, 9H); MS
.
FD 536 (free base). Anal. (C28H33N5O6 3CF3COOH) C,
H, N.
N4-(ꢁ-L-valyl)-1-[20,30-dideoxy-50,60-O-(ꢁ-L-valyl)-ꢀ-D-
erythropentofuranosyl] cytosine tri¯uoroacetate (17). To
a solution of tri¯uoroacetic acid (2 mL) and methylene
chloride (2 mL) was added compound 11 (194 mg,
0.23 mmol) in methylene chloride (2 mL). The solution
was stirred at room temperature for 1 h and the solvent
removed in vacuo to give a resin. The resin was crystal-
lized from ethanol/ethyl ether to give 17 as a white solid
(163 mg, 80%): mp 160±165 ꢀC (d); IR (KBr): 3480,
2978, 1753, 1678 cm 1; 1H NMR (DMSO-d6): d 0.98 (m,
18H), 2.17 (m, 4H), 2.51 (m, 2H), 3.95 (m, 3H), 4.25 (m,
3H), 4.52 (m, 2H), 6.03 (m, 1H), 7.21 (m, 1H), 8.23 (d,
J=7.7 Hz, 1H) , 8.25±8.59 (br, 9H), 11.45 (br, IH); MS
an amorphous solid (200 mg, 41%). IR (KBr): 3420,
1
3370, 2979, 1720 cm
;
1H NMR (CDCl3): d 1.39 (m,
27H), 2.24 (m, 3H), 2.99±3.27 (m, 6H), 3.74±3.97 (m,
5H), 4.61 (m, 3H), 5.00 (m, 3H), 5.95 (m, 1H), 7.10±7.35
(m, 15H), 7.47 (m, 1H), 8.03 (d, J=7.4 Hz, 1H), 8.84 (s,
1H); MS FD 983. Anal. (C52H66N6O13) C, H, N.
1-[20,30 -Dideoxy-50,60 -O-(N-ꢁ-tert-butoxycarbonyl-L-
phenylalanyl)-ꢀ-D-erythropentofuranosyl] cytosine (14).
Compound 10 (280 mg, 0.35 mmol) was heated and
stirred at 60 ꢀC in 1:1 methanol:water (20 mL) for 16 h.
The solvent was removed in vacuo and the residue was
chromatographed (2% methanol in methylene chloride)
.
FD (539, free base). Anal. (C25H42N6O7 3CF3COOH)
C, H, N.
to give 14 as a white solid (45 mg, 17%); mp 115±120 ꢀC;
1
IR (KBr): 3430, 3346, 2980, 1749 cm
;
1H NMR
(DMSO-d6): d 1.32 (s, 18H), 1.91 (m, 1H), 2.14 (m, 2H),
2.93 (m, 4H), 3.89 (brs, 1H), 4.02 (m, 2H), 4.15±4.31 (m,
4H), 5.78 (d, J=7.7 Hz, 1H), 5.97 (m, 1H), 7.09±7.40
(m, 14H), 7.64 (d, J=7.4 Hz, 1H); MS FD 737. Anal.
(C38H49N5O10) C, H, N.
N4-(ꢁ-L-phenylalanyl)-1-[20,30 -dideoxy-50,60 -O-(ꢁ-L-
phenylalanyl)-ꢀ-D-erythropentofuranosyl] cytosine (18).
To a solution of tri¯uoroacetic acid (2 mL) and methyl-
ene chloride (2 mL) was added 12 (165 mg , 0.17 mmol)
and the mixture was stirred 1 h at room temperature.
The solvent was removed under reduced pressure and
the resulting oil was crystallized from ethanol/ethyl
ether to give 18 (144 mg, 84%); mp 195±200 ꢀC (d); IR
1-[20,30 -Dideoxy-50,60 -O-(ꢁ-L-valyl)-ꢀ-D-erythropento-
furanosyl] cytosine tri¯uoroacetate (15). A solution of 9
(40 mg, 0.06 mmol) in 1:1 methanol:water (10 mL) was
heated at 60 ꢀC for 12 h. The solvent was removed in
vacuo and the residue was chromatographed (5%
methanol in methylene chloride) to give 13, which was
used as such without further puri®cation. The residue
was dissolved in methylene chloride (1 mL) and added
to a stirring solution of tri¯uoroacetic acid (1 mL) and
methylene chloride (1 mL). The mixture was stirred 1 h
at room temperature, the solvent removed in vacuo and
the resulting oil was crystallized from ethanol/ethyl
ether to give 15 (22 mg, 49%, from 9) as an amorphous
1
(KBr): 3428, 3035, 1755, 1677 cm 1; H NMR (DMSO-
d6): d 1.80±2.40 (brm, 3H), 3.14 (m, 6H), 3.65±3.99
(brm, 2H), 4.35 (m, 4H), 5.92 (m, 1H), 7.30 (m, 16H),
8.08 (d, J=8.1 Hz, 1H), 8.20±8.75 (br, 9H), 11.50 (brs,
.
1H); MS FD 683 (free base). Anal. (C37H45N6O7 3CF3-
.
COOH 4H2O) C, H, N.
General procedure for the preparation of N4-amido
derivatives of 1. To a solution of 1 (0.30 mmol) in water
was added the appropriate anhydride (2.2 equiv) and p-
dioxane (1.2 mL). After stirring 12 h at room tempera-
ture, the solvent was removed under reduced pressure,
and the residue chromatographed (7±10% methanol in
methylene chloride) to give, after crystallization from
ethyl ether, the desired products.
1
solid. IR (KBr): 3430, 2980, 1753, 1680 cm 1; H NMR
(DMSO-d6): d 0.97 (m, 12H), 2.17 (m, 2H), 2.26±2.43
(m, 2H), 2.60 (m, 1H), 3.97 (brs, 2H), 4.15 (m, 1H), 4.29
(d, J=5.2 Hz, 2H), 4.49 (m, 2H), 6.02 (m, 2H), 7.93 (d,
J=7.7 Hz, 1H), 8.20±9.20 (brm, 9H). MS FD 440 (parent
N4-Acetyl-1-[20,30 -dideoxy-30 -C-(hydroxymethyl)-ꢀ-D-
.
ion). Anal. (C20H33N5O6 3CF3COOH) C, H, N.
erythropentofuranosyl] cytosine (19). Compound
1
1-[20,30-Dideoxy-50,60-O-(ꢁ-L-phenylalanyl)-ꢀ-D-erythro-
pentofuranosyl] cytosine tri¯uoroacetate (16). A mixture
of 14 (20 mg, 0.027 mmol) and 1:1 tri¯uoroacetic acid:
methylene chloride (2 mL) was stirred 2 h at room tem-
perature and concentrated to dryness. The addition of
ethyl ether gave 16 as an amorphous solid (10.8 mg,
45%). IR (KBr): 3425, 3035, 1757, 1681 cm 1; 1H NMR
(DMSO-d6): d 1.94 (m, 1H), 2.10 (m, 2H), 3.10 (m, 4H),
3.69±3.87 (m, 2H), 4.17±4.28 (m, 2H), 4.28 (m, 1H), 4.36
(m, 2H), 5.90 (m, 1H), 5.98 (d, J=7.4 Hz, 1H), 7.27 (m,
(75 mg, 0.30 mmol) was reacted with acetic anhydride
(0.06 mL, 0.64 mmol) in p-dioxane (1.2 mL) and water
(0.4 mL) to give 19 (35 mg, 41%): mp 150±155 ꢀC (d); IR
1
(KBr): 3392, 2928, 1697, 1652 cm 1; H NMR (DMSO-
d6): d 2.01 (m, 1H), 2.10 (s, 3H), 2.27 (m, 2H), 3.45 (t,
J=4.8 Hz, 2H), 3.61 (m, 1H), 3.77 (m, 1H), 3.87 (m,
1H), 4.74 (t, J=5.2 Hz, 1H), 5.09 (t, J=5.2 Hz, 1H),
5.93 (m, 1H), 7.17 (d, J=7.4 Hz, 1H), 8.51 (d,
J=7.4 Hz, 1H), 10.83 (s, 1H); MS FD 284. Anal.
(C12H17N3O5) C, H, N.

584
S. C. Mauldin et al./Bioorg. Med. Chem. 6 (1998) 577±585
N4-Benzoyl-1-[20,30-dideoxy-30-C-(hydroxymethyl)-ꢀ-D-
erythropentofuranosyl] cytosine (20). Compound
3. (a) Fischl, M. A.; Richman, D. D.; Grieco, M. H.; Gottlieb,
M. S.; Volberding, P. A.; Laskin, O. L.; Leedom, J. M.;
Groopman, J. E.; Mildvan, D.; Schooley, R. T.; Jackson, G.
G.; Durack, D.T.; King, D. New Engl. J. Med. 1987, 317, 185.
(b) Mitsuya, H.; Weinhold, K. J.; Furman, P. A.; St. Clair, M.
H.; Lehr S. N.; Gallo, R. L.; Bolognesi, D. P.; Barry, D. W.;
Broder, S. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 7096.
1
(75 mg, 0.30 mmol) was reacted with benzoic anhydride
(150 mg, 0.66 mmol) in p-dioxane (1.2 mL) and water
(0.04 mL) to give 20 (30 mg, 28%) as an amorphous
1
solid. IR (KBr): 3481, 3372, 2933, 1697, 1650 cm 1; H
NMR (DMSO-d6): d 2.06 (m, 1H), 2.30 (m, 2H), 3.46
(brs, 2H), 3.63 (d, J=9.9 Hz, 1H), 3.79 (d, J=12.5 Hz,
1H), 3.90 (m, 1H), 4.76 (brs, 1H), 5.13 (brs, 1H), 5.97
(m, 1H), 7.33 (d, J=7.4 Hz, 1H), 7.52 (m, 5H), 8.59 (d,
J=7.4 Hz, 1H), 11.20 (brs, 1H). MS FD 346. Anal.
(C17H19N3O5) C, H, N.
4. Larder, B. A.; Sharon, D. K. Science 1989, 246, 1155.
5. Klecker, R. W.; Collins, J. M.; Yarchoan, R.; Thomas, R.;
Jenkins, J. F.; Broder, S.; Myers, C. E. Clin. Pharmacol. Ther.
1987, 41, 407.
6. Terasaki, T.; Pardrige,W. M. J. Infect. Dis. 1988, 158, 630.
7. DeClercq, E.; Descamps, J.; DeSomer, J.; Barr, P. J.; Jones,
A. S.; Walker, R. T. Proc . Natl. Acad. Sci., U.S.A. 1979, 76,
2947.
N4-Pivaloyl-1-[20,30-dideoxy-30-C-(hydroxymethyl)-ꢀ-D-
erythropentofuranosyl] cytosine (21). Compound
1
8. Kaufman, H. E. Proc. Soc. Exptl. Biol. Med. 1962, 109, 251.
(75 mg, 0.30 mmol) was reacted with pivalic anhydride
(0.14 mL, 0.66 mmol) in p-dioxane (1.2 mL) and water
(0.04 mL) to give 21 as a white foam (60 mg, 60%); IR
9. Doong, S. L.; Tsai, C. H.; Schinazi, R. F.; Liotta, D. C.;
Cheng, Y. C. Proc. Natl. Acad. Sci., U.S.A. 1991, 88, 8495.
1
(KBr): 3416, 3010, 1657 cm 1; H NMR (DMSO-d6): d
10. Bamford, M. J.; Coe, P. L.; Walker, R. T. J. Med. Chem.
1990, 33, 2494.
1.21 (s, 9H), 2.02 (m, 1H), 2.28 (m, 2H), 3.47 (t,
J=5.2 Hz, 2H), 3.61 (m, 1H), 3.76 (m, 1H), 3.88 (m,
1H), 4.76 (t, J=5.2 Hz, 1H), 5.12 (t, J=5.2 Hz, 1H),
5.94 (m, 1H), 7.24 (d, J=7.7 Hz, 1H), 8.51 (d,
J=7.4 Hz, 1H), 10.30 (brs, 1H). MS FD 326. Anal.
(C15H23N3O5) C, H, N.
11. Svansson, L.; Kvarnstrom, I.; Classon, B.; Samuelsson, B.
J. Org.Chem. 1991, 56, 2993.
12. Yoshimura, J. Adv. Carbohydr. Chem. Biochem. 1984, 42,
69.
13. Acton, E. M.; Goerner, R. N.; Uh, H.S.; Ryan, K. J.;
Henry, D. W. J. Med. Chem. 1979, 22, 518.
N4-Benzamido-1-[20,30 -dideoxy-50,60 -O-(benzoyl)-ꢀ-D-
erythropentofuranosyl] cytosine (22). To a solution of 1
(50 mg, 0.21 mmol) in dry pyridine (3 mL) was added
benzoyl chloride (0.10 mL, 0.80 mmol). The mixture was
stirred at room temperature for 2 h, the solvent was
removed in vacuo and the residue was partitioned
between water (50 mL) and ethyl acetate (50 mL). The
organic layer was dried over magnesium sulfate, ®ltered,
and concentrated to a resin. Chromatography (1%
14. Seki, J. I.; Shimada, N.; Takahashi, K.; Takita, T.; Takeu-
chi, T.; Hoshino, H. Antimicrob. Agents Chemother. 1989, 33,
773.
15. Nishiyama, Y.; Yamamoto, N.; Takahashi, K.; Shimada,
N. Antimicrob. Agents Chemother. 1988, 32, 1053.
16. Collins, J. M.; Klecker, R.W.; Kelley, J. A.; Rother, J. S.;
McCully, C. L.; Balis, M.; Poplack, D. G. J. Pharmacol. Exp.
Ther. 1988, 245, 466.
17. Cornford, E. M.; Oldendorf, W. H. Biochim. Biophys.
Acta. 1975, 394, 211.
methanol in methylene chloride) gave 22 as an amor-
1
phous resin (62 mg, 54%). IR (KBr): 3420, 1723 cm
;
18. Hansch, C.; Steward, A. R.; Anderson, S. M.; Bentley, D.
J. Med. Chem. 1968, 11, 1.
1H NMR (DMSO-d6): d 2.51 (m, 1H), 2.72 (m, 2H),
4.40 (m, 2H), 4.55 (dd, J=3.0, 6.0 Hz, 1H), 4.76 (m,
2H), 6.17 (m, 1H), 7.51 (m, 10H); 8.02 (m, 6H), 8.32 (d,
J=7.0 Hz, 1H), 8.75 (brs, 1H). MS FD 554. Anal.
(C31H27N3O7) C, H, N.
19. Oldendorf, W. H. Proc. Soc. Exp. Biol. Med. 1974, 147,
813.
20. Aggarwal, S. K.; Gogu, S. R.; Rangan, S. R. S.; Agrawal,
K. C. J. Med. Chem. 1990, 33, 1505.
21. Henin, Y.; Gouyette, C.; Schwartz, O.; Debouzy, J.; Neu-
mann, J.; Dinh, T. J. Med.Chem. 1991, 34, 1830.
Acknowledgements
22. Curley, D.; McGuigan, C.; Devin, K. G.; O'Conner, T. J.;
Je€ries, D. J.; Kinchington, D. Antiviral Res. 1990, 14, 345.
The authors thank John Munroe for helpful discussions
and support. We also thank the Physical Chemistry
Department at Eli Lilly and Company for spectral and
analytical data.
23. Kerr, S. G.; Kalman, T. I. J. Med. Chem. 1992, 35, 1996.
24. Akiyama, M.; Oh-Ishi, J.; Shirai, T.; Akashi, K.; Yoshida,
K.; Nishikido, J.; Hayashi, H.; Usubuchi, Y.; Nishimura, D.;
Itoh, H.; Shibuya, C.; Ishida, T. Chem. Pharm. Bull. 1978, 26,
981.
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