Synthesis and evaluation of novel thymidine analogs as antitumor and antiviral agents

Article abstract of DOI:10.1021/jm9601260

Two series of thymidine analogs with a hydroxyalkylammonium(amine) moiety have been synthesized and evaluated for antitumor and antiviral activities. The hydroxyalkylammonium-(amine) group was introduced at the 5' position of the 2'-deoxyribose residue of thymidine or at a corresponding position in acyclic thymidine analogs. In order to increase the lipophilicity of these compounds and potentially enable them to cross the cell membrane, the free hydroxy group also was esterified with a long hydrocarbon chain. The hexadecanoyl analogs (compounds 1c, 1d, 7c, and 7d) showed moderate antitumor cytotoxicity against SV-28 and KB cell lines (IC₅₀ ~ 20 μM). Compound 1d showed moderate anti-HIV activity (EC₅₀ = 6.8 μM), while compound 5 showed weak anti-HIV activity (EC₅₀ = 55 μM). None of the compounds showed antiherpes simplex virus activity.

Full text of DOI:10.1021/jm9601260

3412
J . Med. Chem. 1996, 39, 3412-3417
Syn th esis a n d Eva lu a tion of Novel Th ym id in e An a logs a s An titu m or a n d
An tivir a l Agen ts
Xiannong Chen,† Kenneth Bastow,‡ Barry Goz,§ Louis S. Kucera,| Susan L. Morris-Natschke,‡ and
Khalid S. Ishaq*,‡
Department of Chemistry, University of Georgia, Athens, Georgia 30602, Division of Medicinal Chemistry and
Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599,
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, and
Department of Microbiology and Immunology, Bowman Gray School of Medicine of Wake Forest University,
Winston-Salem, North Carolina 27157
Received February 8, 1996X
Two series of thymidine analogs with a hydroxyalkylammonium(amine) moiety have been
synthesized and evaluated for antitumor and antiviral activities. The hydroxyalkylammonium-
(amine) group was introduced at the 5′ position of the 2′-deoxyribose residue of thymidine or
at a corresponding position in acyclic thymidine analogs. In order to increase the lipophilicity
of these compounds and potentially enable them to cross the cell membrane, the free hydroxy
group also was esterified with a long hydrocarbon chain. The hexadecanoyl analogs (compounds
1c, 1d , 7c, and 7d ) showed moderate antitumor cytotoxicity against SV-28 and KB cell lines
(IC50 ∼ 20 µM). Compound 1d showed moderate anti-HIV activity (EC50 ) 6.8 µM), while
compound 5 showed weak anti-HIV activity (EC50 ) 55 µM). None of the compounds showed
antiherpes simplex virus activity.
In tr od u ction
herpes simplex virus drug acyclovir. The hydroxyalkyl-
ammonium moiety in either series of analogs will
contain both a positive charge and a free hydroxy group.
The positive charge may result in a favorable interaction
with a potential target and provide a better inhibitor,
while the free hydroxy group may offer a site for
activation to the monophosphate form. This latter
reaction is an obligatory step for further phosphoryla-
tion to the mono-, di-, and tri-phosphate forms.
Interest in nucleoside research has intensified since
the AIDS epidemic, and many nucleoside derivatives
have been synthesized. Therefore, new nucleoside
analogs should be distinctly dissimilar in structure to
the current families of analogs. Accordingly, we selected
the â-hydroxyalkyltrimethylammonium moiety as a new
template for a novel series of nucleoside derivatives.
Previously, incorporation of this functionality into alkyl
ether lipids has resulted in potent inhibitors of in vitro
neoplastic cell growth,1,2 protein kinase C activity,3 and
HIV-1 infectivity,4 although the mechanism(s) of action
is (are) not clearly understood. The effect of this moiety
on nucleosides, however, has not been investigated.
Therefore, its incorporation into thymidine and other
nucleosides is of interest. Since thymine is a nucleic
acid base unique to DNA, it often serves as a model in
the design of novel nucleoside analogs targeting DNA.
Previous research on thymidine analogs has produced
several successful antiviral drugs, such as 3′-azido-2′,3′-
dideoxythymidine (AZT) and 2′,3′-didehydro-2′,3′-dideoxy-
thymidine (d4T), which, after phosphorylation, can
inhibit HIV-1 reverse transcriptase. Both compounds
are used currently in the treatment of AIDS. Further,
other nucleic acid bases also have been used to combat
AIDS, for example, inosine (in dideoxyinosine, ddI) and
cytosine (in dideoxycytidine, ddC, and 2′-deoxy-3′-thia-
cytidine, 3TC).
The nucleoside sugar ring has been modified exten-
sively.5 One such modification is exemplified by acy-
clovir, an open-chain analog of guanosine, which is
highly effective in the treatment of herpes simplex virus
infection. Although acyclovir mimics the conformation
of guanosine quite well, it is recognized preferentially
by the viral thymidine kinase.6 Since the introduction
of acyclovir as an antiviral drug, many acyclic nucleoside
analogs have been synthesized.7,8 Therefore, the open-
chain thymidine analogs (type II) were based on this
model. A conformational search with Sybyl has shown
that the flexible side chain of compound 1a can adopt a
conformation in which the distance between the termi-
nal hydroxy group and the thymine base is similar to
that in thymidine.
The chain length between the free hydroxyl and the
ammonium nitrogen may affect the biological activity;
thus, compounds with both two (1a , 1b, 1c) and three
(1d , 2, 3) carbon chains were made. Compound 3, which
has two free hydroxyls, was synthesized to increase the
possibility of phosphorylation. Hydroxyalkylamine ana-
logs (4 and 5) also were synthesized to explore the
effects of the quaternary ammonium moiety versus a
tertiary amine group. At physiological pH, the tertiary
amine will be protonated and bear a positive charge that
is electrostatically similar to the ammonium analogs.
At the active site of the enzyme, however, the proto-
nated tertiary amine may be neutral since there may
be no water molecules around it. Therefore, analogs 4
and 5 may indicate whether or not the positive charge
We have designed two types of compounds containing
the hydroxyalkylammonium moiety. In type I, it is
introduced at the 5′-position of the 2′-deoxyribose resi-
due of thymidine, and in type II, it is incorporated into
an acyclic chain, which is similar to that of the anti-
* Author to whom correspondence should be addressed.
† University of Georgia.
‡ Division of Medicinal Chemistry and Natural Products, University
of North Carolina.
§ Department of Pharmacology, University of North Carolina.
| Bowman Gray School of Medicine of Wake Forest University.
X Abstract published in Advance ACS Abstracts, August 1, 1996.
S0022-2623(96)00126-4 CCC: $12.00 © 1996 American Chemical Society

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3413
Sch em e 1
were prepared by first treating octanoyl chloride or
hexadecanoyl chloride with 2-bromoethanol or 3-bromo-
1-propanol in benzene in the presence of pyridine.2
Reaction of the corresponding products, 2-bromoethyl
octanoate, 2-bromoethyl hexadecanoate, and 3-bromopro-
pyl hexadecanoate, with dimethylamine in acetonitrile
gave 2-(dimethylamino)ethyl octanoate, 2-(dimethylami-
no)ethyl hexadecanoate, and 3-(dimethylamino)propyl
hexadecanoate. These intermediates then were treated
with 1-[(2-iodoethoxy)methyl]thymine to give the cor-
responding products 1b, 1c, and 1d .
Gen er a l Meth od for th e Syn th esis of Th ym id in e
An a logs a n d Th eir P r od r u gs (Sch em e 1). The
synthesis of N,N-dimethyl-N-(2-hydroxyethyl)-N-[5′-(5′-
deoxythymidinyl)]ammonium iodide (7a ) illustrates this
series. 5′-Iodothymidine, prepared from thymidine and
methyltriphenoxyphosphonium iodide (MTPI),11 was
reacted with the tertiary amine 2-(dimethylamino)-
ethanol in acetonitrile to give the corresponding product
7a . Compounds 5 and 6 were synthesized in the same
manner using 2-(methylamino)ethanol and 3-(dimethyl-
amino)-1-propanol. The prodrugs of 7a were prepared
as outlined for 1b, 1c, and 1d . Thus, treating 5′-
iodothymidine with 2-(dimethylamino)ethyl octanoate,
2-(dimethylamino)ethyl hexadecanoate, or 3-(dimethyl-
amino)propyl hexadecanoate afforded 7b, 7c, and 7d .
at the quaternary ammonium nitrogen is important for
biological activity. These compounds are polar and may
not be able to cross the cell membrane unless they can
be transported by an active nucleoside transport mech-
anism. Therefore, the free hydroxy group was esterified
by two different hydrocarbon chains (octanoyl and
hexadecanoyl) to produce prodrugs (1c, 1d ; 7c, 7d ) with
increased ability to enter the cell through passive
diffusion. The choice of either the octanoyl or the
hexadecanoyl derivatives to serve as model prodrugs
was based on a lipophilicity consideration as well as rate
of ester cleavage.9
Biologica l Resu lts a n d Discu ssion
The antitumor activity of this series of compounds
was evaluated by tissue culture assay against the SV-
28 cell line, an SV40-transformed line of baby hamster
kidney cells. Compounds 1c, 1d , 7c, and 7d were
equipotent with IC50 values of 18 ( 3, 21, 23 ( 3, and
25 µM, respectively. All other compounds were inactive
(no inhibition at >50 µM). Compounds 1c and 7c were
further evaluated against the human nasopharyngeal
KB cell line; the IC50 values were 20 ( 2 and 25 ( 5
µM, respectively.
Since all four active compounds have a hexadecanoyl
ester moiety, increased lipophilicity may offer one of two
possible explanations for the pharmacological activity.
Compounds 1a , 2, and 3 were synthesized first, and
their lack of antitumor activity led to the synthesis of
the ester prodrugs based on the following rationale.
Although a nucleoside requires a transport carrier to
cross the cell membrane, compounds 1a , 2, and 3 may
not be actively transported by this carrier, and their
hydrophilic character also may impede diffusion across
the cell membrane. However, the hexadecanoyl analogs
(1c, 1d , 7c, 7d ) may be lipophilic enough to diffuse
through the membrane. (See also the dipyridamole
assay below.) Once the compounds are in the cells, the
hydrocarbon ester chain is expected to be cleaved
enzymatically, and the nucleoside analog released.
Lengthening the alkyl chain between the sterically
hindered quaternary ammonium nitrogen and the ester
group might be expected to affect the rate of enzymatic
hydrolysis and increase levels of the nucleoside analog
inside the cell.14 However, since the IC50 values of 1d
and 7d , which have a three-carbon bridge, equal those
of 1c and 7c, which have a two-carbon bridge, the
cytotoxic effect of these hexadecanoyl esters may be
independent of their rate of cleavage. A second expla-
nation for the activity of these compounds is that 1c,
1d , 7c, and 7d may interact with the cellular membrane
Ch em istr y
Gen er a l Meth od for th e Syn th esis of Acyclic
Th ym id in e An a logs a n d Th eir P r od r u gs (Sch em e
1). The synthesis of N,N-dimethyl-N-(2-hydroxyethyl)-
N-[N1-(5-methyl-2,4-dioxopyrimidinyl)methoxyethyl]-
ammonium iodide (1a ) exemplifies the preparation of
compounds 1a , 2, 3, and 4. 1-[(2-Iodoethoxy)methyl]-
thymine, prepared by literature procedures,10,11 was
treated with 2-(dimethylamino)ethanol to give the de-
sired product 1a . Compounds 2, 3, and 4 were obtained
in a similar manner from the corresponding amines
3-(dimethylamino)-1-propanol, 3-(dimethylamino)-1,2-
propanediol, and 2-(methylamino)ethanol in acetonitrile.
We also were interested in synthesizing long-chain
esters of compound 1a , since the design of a prodrug of
this thymidine analog would lead to increased lipophi-
licity and, thus, might facilitate passive diffusion into
the cell. Direct treatment of 1a with octanoyl chloride
or hexadecanoyl chloride failed to give the desired
product. [Similarly, other laboratories12 could not di-
rectly phosphorylate (2,3-distearoyloxypropyl)dimethyl-
(â-hydroxyethyl)ammonium bromide. Our laboratory
also has been unsuccessful in phosphorylating analogs
of this compound.] Therefore, the ester prodrugs were
prepared by direct reaction of an iodo nucleoside inter-
mediate with dimethylaminoalkyl esters. The latter

3414 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Notes
without metabolic activation. Compounds with similar
structures have been shown to exert cytotoxic and
incubation period of 48 h. All the compounds in this
series were then tested for anti-HSV-1 activity; none
showed activity at concentrations up to 100 µM.
antiviral effects through
a membrane-interactive
mechanism.16-18 The inactivity of the octanoyl analogs
(1b, 7b) may be due to their inability to either cross
the membrane or exert a biological effect through
membrane interaction.
The same compounds were tested for anti-HIV-1
activity and for cytotoxicity against the HIV-1 host cell
(CEM-SS cell). Compounds 1c, 1d , 7c, and 7d showed
some toxicity against CEM-SS cell growth with IC50
values of 67.3 ( 10.0, 62.4 ( 0.4, 53.0 ( 24.2, and 22.3
( 1.9 µM, respectively. All other compounds showed
no inhibition at 100 µM. Compound 1d showed moder-
ate anti-HIV-1 activity at a concentration (EC50 ) 6.8
( 1.6 µM) 10 times less than its cytotoxic IC50 value,
while compounds 5, 6, and 7a showed weak anti-HIV-1
activity by inhibiting viral multiplication by 50, 31, and
44% at 55, 100, and 100 µM, respectively. The latter
three compounds have an intact sugar moiety; their
polarity and probable lack of active transport might be
responsible for their weak activity.
The tertiary amine analogs (4 and 5), which were
prepared to ascertain the role of the quaternary am-
monium head, were inactive as antitumor agents.
Although sterically less hindered,9 their inactivity might
be due to the protonation of the tertiary amine group
at physiological pH; this charge would increase polarity
and hinder these compounds from crossing the cell
membrane if not transported by a nucleoside carrier
protein.
To ascertain whether these thymidine analogs are
actively transported across the cellular membrane, 1c
and 7c were tested for cytotoxicity in the presence of 1
µM dipyridamole, an antithrombotic drug that inhibits
nucleoside transport. If compounds 1c and 7c are
transported by the active nucleoside transport mecha-
nism, the presence of dipyridamole should decrease the
amount of compound entering the cells and, therefore,
increase the IC50 values. In this assay, the IC50 values
with dipyridamole (1c: 22 µM, 7c: 20 µM) were
unchanged from those (1c: 18 ( 3 µM, 7c: 23 ( 3 µM)
without dipyridamole, which is evidence that these
compounds do not enter the cell by a nucleoside trans-
port mechanism.
Su m m a r y
Novel thymidine and acyclic nucleoside analogs con-
taining hydroxyalkylammonium moieties were de-
signed, synthesized, and evaluated for in vitro cytotox-
icity and antiviral activity. Prodrugs of both types
containing hexadecanoyl ester groups were cytotoxic in
SV-28, KB, Vero, and CEM-SS cells, while the parent
hydroxy compounds and the octanoyl esters were not.
The increased lipophilicity of the long alkyl chain may
enhance the membrane permeability of these com-
pounds. None of the compounds were active against
HSV-1, and most compounds were either inactive or
weakly to moderately active against HIV-1. Further
structural modification and biological evaluation of this
novel class of nucleoside analogs are in progress.
In order to investigate the mechanism of action of
these thymidine hexadecanoyl ester prodrugs, 1c and
7c were tested in the SV-28 cell assay with excess
thymidine (10 µM) in the medium. Usually, nucleoside
analogs exert their activity by competing with the
natural substrate; thus, the presence of a large amount
of the natural substrate should affect the activity of the
nucleoside analog. However, 10 µM thymidine in the
cell medium did not change the IC50 values of 1c (24
µM, with, vs 18 µM, without) and 7c (21 µM, with, vs
23 µM, without). This suggests that these compounds
may not act as nucleoside analogs of thymidine to exert
their cytotoxic effects, or they may compete with a
nucleoside other than thymidine.
Exp er im en ta l Section
All melting points were taken on a Thomas Hoover melting
point apparatus and are uncorrected. Infrared spectra (IR)
were recorded on a Perkin-Elmer 1320 spectrophotometer. 1H
NMR spectra were obtained using a Bruker AC-300 NMR.
Elemental analyses were performed by Atlantic Microlab Inc.,
Norcross, GA. Due to the hygroscopic nature of the quaternary
ammonium compounds, some of the structures were confirmed
by high-resolution mass spectroscopy by using a VG70-SQ
spectrometer. Thin-layer chromatography (TLC) was per-
formed on 1 × 3 in. fluorescent precoated Whatman Silica Gel
60 Å TLC plates. Silica gel (70-230 mesh) from Fisher
Scientific was used for column chromatography. Reagents
were purchased from Aldrich. Solvents, including acetonitrile,
N,N-dimethylformamide (DMF), and methylene chloride, were
dried by placement over molecular sieves (4 Å) for 2 weeks
before use.
Compounds 1c and 7c also were tested for their
effects on cell plating efficiency. The SV-28 cell has a
plating efficiency between 50 and 60%. Drugs that
target DNA or RNA usually decrease plating efficiency.
At 25 µM, 1c (47.4%) and 7c (58.5%) did not lower cell
plating efficiency after a 24 h exposure.
All compounds also were tested against the herpes
simplex virus 1 (HSV-1, KOS strain) by using a plaque
reduction assay. Compounds first were tested for cell
toxicity against the HSV-1 virus host, Vero, cell line to
determine the appropriate concentrations to be used in
the antiviral assay. Consistent with the antitumor
cytotoxicity assay, only 1c, 1d , 7c, and 7d inhibited the
growth of Vero cells with IC50 values in the range of
35-40 µM after 2 days of continuous treatment. At 200
µM, 1c, 1d , 7c, and 7d were cytotoxic within a 30 min
exposure based on the change in cell morphology
(shrinkage and rounding) visible upon microscopic
examination. Although these compounds inhibited the
growth of proliferating cells, they did not exert visible
cytotoxic effects on preformed cell monolayers after an
2-(Dim eth yla m in o)eth yl Octa n oa te. 2-Bromoethyl oc-
tanoate (0.2 g, 0.72 mmol), prepared by reaction of 2-bromo-
ethanol and octanoyl chloride in 10:1 benzene/pyridine,2 and
40% aqueous dimethylamine (4.2 mL, 36 mmol) were dissolved
in acetonitrile (10 mL). The reaction mixture was stirred at
room temperature for 48 h. After removal of acetonitrile under
vacuum, CHCl3 (30 mL) was added. The CHCl3 layer was
extracted with K2CO3 solution (20 mL, 0.01 N) and water (2
× 20 mL), and then was dried over anhydrous Na2SO4. The
drying agent was suction-filtered and the CHCl3 removed in
vacuo. The resulting material was applied to a silica gel
column (discontinuous gradient of CHCl3/CH3OH 95:5, 8:2 as
eluent), affording 0.13 g of pure product: yield 77%; liquid;
1H NMR (CDCl3) δ 0.85-0.95 (t, CH3-(CH2)4), 1.20-1.40 (m,
CH3-(CH2)4-), 1.55-1.70 (m, CH2-CH2-CO), 2.25-2.40 (t,
CH2-CH2-CO), 2.45-2.55 (s, (CH3)2-N) 2.75-2.85 (t, O-CH2-
CH2-N), 4.25-4.35 (t, O-CH2-CH2-N).

Notes
2-(Dimethylamino)ethyl hexadecanoate and 3-(dimethyl-
amino)propyl hexadecanoate were prepared similarly from
dimethylamine and the appropriate haloester.
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3415
1
iu m Iod id e (1c): yield 49%; wt 203 mg; mp 144-146 °C; H
NMR (CDCl3) δ 0.80-1.00 (t, CH3-(CH2)12), 1.20-1.40 (m,
CH3-(CH2)12-), 1.50-1.70 (m, CH2-CH2-CO), 1.85-1.95 (s,
CH3-C5 thymine), 2.30-2.45 (m, CH2-CH2-CO), 3.30-3.60
(s, (CH3)2-N), 3.90-4.10 (m, CH2-N-CH2), 4.15-4.30 (m,
CH2-CH2-O-CH2-N), 4.50-4.65 (m, C(O)O-CH2), 5.20-
5.35 (s, O-CH2-N), 7.35-7.40 (s, H-C6 thymine), 9.95-10.25
(s, HN thymine). Anal. (C28H52O5N3I) C, H, N.
N,N-Dim eth yl-N-(2-h yd r oxyeth yl)-N-[N1-(5-m eth yl-2,4-
d ioxop yr im id in yl)m eth oxyeth yl]a m m on iu m Iod id e (1a ).
1-[(2-Iodoethoxy)methyl]thymine (100 mg, 0.32 mmol) and
2-(dimethylamino)ethanol (34.5 mg, 0.38 mmol) were dissolved
in acetonitrile (10 mL). The reaction mixture was heated to a
gentle reflux and stirred for 5 h. After cooling to room
temperature, 77 mg of pure product was obtained through
crystallization by slow addition of Et2O: yield 59%; mp 158-
161 °C; 1H NMR (CD3CN) δ 1.85-1.88 (s, CH3-C5 thymine),
3.10-3.15 (s, (CH3)2-N), 3.30 (s, HO-CH2), 3.45-3.48 (m,
N-CH2-CH2-O), 3.55-3.58 (m, HO-CH2-CH2-N), 3.90-
4.00 (m, HO-CH2-CH2-N-CH2-CH2-O), 5.10 (s, O-CH2-
N), 7.30 (s, H-C6 thymine). Anal. (C12H22N3O4I) C, H, N.
Compounds 2 and 3 were prepared in an analogous manner
with the appropriate amino alcohol.
N,N-Dim eth yl-N-3-(h exa d eca n oyloxy)p r op yl-N-[N1-(5-
m e t h yl-2,4-d ioxop yr im id in yl)m e t h oxye t h yl]a m m on -
1
iu m Iod id e (1d ): yield 29%; wt 120 mg; mp 133-135 °C; H
NMR (CDCl3) δ 0.80-1.00 (t, CH3-(CH2)12), 1.20-1.40 (m,
CH3-(CH2)12-), 1.50-1.70 (m, CH2-CH2-CO), 1.85-1.95 (s,
CH3-C5 thymine), 2.15-2.28 (m, O-CH2-CH2-CH2-N),
2.30-2.40 (m, CH2-CH2-CO), 3.30-3.50 (s, (CH3)2-N), 3.65-
3.72 (m, CH2-N-CH2-CH2-O), 3.90-4.03 (m, CH2-N-CH2-
CH2-O), 4.15-4.35 (m, CH2-CH2-O-CH2-N; C(O)O-CH2),
5.20-5.35 (s, O-CH2-N), 7.35-7.40 (s, H-C6 thymine), 9.80-
9.85 (s, HN thymine); HRMS calcd for C29H54O5N3 524.4064
(M+), found 524.4043.
N,N-Dim et h yl-N-(3-h yd r oxyp r op yl)-N-[N1-(5-m et h yl-
2,4-d ioxop yr im id in yl)m eth oxyeth yl]a m m on iu m Iod id e
(2): Yield 59%; mp 178-180 °C; 1H NMR (DMSO-d6) δ 1.75-
1.80 (s, CH3-C5 thymine), 1.75-1.90 (m, HO-CH2-CH2-
CH2-N), 3.05-3.15 (s, (CH3)2-N), 3.40-3.50 (m, HO-CH2-
CH2-CH2-N), 3.50-3.60 (m, N-CH2-CH2-O), 3.85-3.95 (m,
HO-CH2-CH2-CH2-N-CH2-CH2-O), 4.75-4.85 (t, HO-
CH2), 5.05-5.10 (s, O-CH2-N), 7.55-7.60 (s, H-C6 thymine),
11.35-11.40 (s, HN thymine). Anal. (C13H24N3O4I) C, H, N.
N,N-Dim eth yl-N-(2,3-d ih yd r oxyp r op yl)-N-[N1-(5-m eth -
yl-2,4-d ioxop yr im id in yl)m et h oxyet h yl]a m m on iu m Io-
Compounds 5 and 6 were prepared by using the same
procedure as given for the synthesis of 7a .
N-Met h yl-N-(h yd r oxyet h yl)-N-[5′-(2′,5′-d id eoxy)t h y-
m id in yl]a m in e (5): Yield 55%; wt 94 mg; hygroscopic; 1H
NMR (CD3OD) δ 1.88-1.95 (s, CH3-C5 thymine), 2.20-2.35
(m, HR-C2′), 2.48-2.60 (m, Hâ-C2′), 2.68-2.70 (d, HO-C3′),
2.81-2.90 (s, N-CH3), 3.40-3.54 (m, HO-CH2-CH2-N-
CH2), 3.75-3.80 (m, HO-CH2-CH2-N-CH2), 3.82-3.91 (m,
HO-CH2), 4.22-4.31 (H-C4′), 4.35-4.42 (H-C3′), 6.18-6.26
(t, H-C1′), 7.50-7.55 (s, H-C6 thymine); HRMS calcd for
C13H22O5N3 300.1559 (MH+), found 300.1562.
N,N-Dim eth yl-N-(3-h yd r oxyp r op yl)-N-[5′-(5′-d eoxyth y-
m id in yl)]a m m on iu m Iod id e (6): Yield 54%; wt 0.35 g;
hygroscopic; 1H NMR (CD3OD) δ 1.90-1.95 (s, CH3-C5
thymine), 1.95-2.10 (HO-CH2-CH2-CH2), 2.20-2.35 (m,
HR-C2′), 2.42-2.55 (m, Hâ-C2′), 2.85-2.90 (d, HO-C3′),
3.00-3.03 (t, HO-CH2), 3.20-3.35 (s, N-(CH3)2), 3.52-3.62
(m, HO-CH2-CH2-CH2-N-CH2), 3.85-3.95 (m, HO-CH2-
CH2-CH2-N-CH2), 4.00-4.05 (m, HO-CH2), 4.22-4.31 (H-
C4′), 4.35-4.42 (H-C3′), 6.18-6.26 (t, H-C1′), 7.50-7.55 (s,
H-C6 thymine); HRMS calcd for C15H26O5N3 328.1872 (M+),
found 328.1882.
1
d id e (3): Yield 55%; mp 163-165 °C; H NMR (DMSO-d6) δ
1.75-1.80 (s, CH3-C5 thymine), 3.10-3.15 (s, (CH3)2-N),
3.60-3.70 (m, CH2-N-CH2), 3.80-4.10 (m, HO-CH2-
CH(OH)-CH2-N-CH2-CH2-O), 4.95-5.00 (t, HO-CH2),
5.05-5.10 (s, O-CH2-N), 5.20-5.25 (d, HO-CH2-CH(OH)-
CH2), 7.55-7.60 (s, H-C6 thymine), 11.35-11.40 (s, HN
thymine). Anal. (C13H24N3O5I) C, H, N.
N-Met h yl-N-(2-h yd r oxyet h yl)-N-[N1-(5-m et h yl-2,4-d i-
oxopyr im idin yl)m eth oxyeth yl]am in e (4). 1-[(2-Iodoethoxy)-
methyl]thymine (200 mg, 0.65 mmol) and 2-(methylamino)-
ethanol (242 mg, 3.3 mmol) were dissolved in acetonitrile (8
mL). The reaction mixture was heated to a gentle reflux and
stirred for 5 h. After evaporation of acetonitrile, CHCl3 (50
mL) was added, and the solution was washed with NaOH
solution (20 mL, 1 N) and water (2 × 20 mL). The CHCl3 layer
was dried over anhydrous Na2SO4. The drying agent was
suction-filtered, and the CHCl3 was removed under vacuum.
The resulting residue was purified by column chromatography
by using a discontinuous CHCl3/CH3OH (9:1, 7:3) gradient to
give 95 mg of pure product: yield 60%; mp 58-60 °C; 1H NMR
(CD3OD) δ 1.85-1.92 (s, CH3-C5 thymine), 2.25-2.35 (s,
CH3-N), 2.55-2.60 (t, HO-CH2-CH2-N-CH2), 2.60-2.65 (t,
HO-CH2-CH2-N-CH2), 3.55-3.70 (m, HO-CH2-CH2-N-
CH2-CH2-O), 5.10-5.15 (s, O-CH2-N), 7.45-7.50 (s, H-C6
thymine). Anal. (C11H19N3O4) C, H, N.
N,N-Dim et h yl-N-(2-h yd r oxyet h yl)-N-[5′-(5′-d eoxyt h y-
m id in yl)]a m m on iu m Iod id e (7a ). A solution of 5′-iodothy-
midine (0.5 g, 1.4 mmol) and 2-(dimethylamino)ethanol (5 mL,
49 mmol) in DMF (30 mL) was heated to 50 °C for 3 h. After
DMF was removed under high vacuum, CHCl3 (2 × 20 mL)
was added to dissolve the unreacted starting material. After
the CHCl3 was decanted, the residue was purified via column
chromatography (CHCl3/CH3OH discontinuous gradient, 6:4
1
to 3:7): yield 52%; wt 0.32 g; hygroscopic; H NMR (CD3OD)
δ 1.90-2.00 (s, CH3-C5 thymine), 2.20-2.35 (m, HR-C2′),
2.42-2.55 (m, Hâ-C2′), 2.85-2.90 (d, HO-C3′), 3.20-3.35 (s,
N-(CH3)2), 3.55-3.65 (t, HO-CH2-CH2-N-CH2), 3.85-3.95
(d, HO-CH2-CH2-N-CH2), 4.00-4.05 (m, HO-CH2), 4.22-
4.31 (H-C4′), 4.35-4.42 (H-C3′), 6.18-6.26 (t, H-C1′), 7.50-
7.55 (s, H-C6 thymine); HRMS calcd for C14H24O5N3 314.1716
(MH+), found 314.1731.
Compounds 7b-7d were prepared from 5-iodothymidine
and the appropriate amino ester by using the same procedure
as for the preparation of 1b.
N ,N -Dim e t h yl-N -[2-(oct a n oyloxy)e t h yl]-N -[5′-(2′,5′-
d id eoxy)th ym id in yl]a m m on iu m Iod id e (7b): Yield 36%;
wt 0.12 g; mp 103-106 °C; hygroscopic; 1H NMR (CDCl3) δ
0.70-0.78 (t, CH3-(CH2)4), 1.11-1.23 (m, CH3-(CH2)4-),
1.42-1.53 (m, CH2-CH2-CO), 1.70-1.75 (s, CH3-C5 thym-
ine), 2.11-2.23 (m, HR-C2′), 2.18-2.25 (t, CH2-CH2-CO),
2.32-2.45 (m, Hâ-C2′), 3.12-3.18 (s, (CH3)2-N), 3.22 (s, HO),
3.65-3.72 (m, O-CH2-CH2-N-CH2), 3.75-3.82 (m, H-C4′),
3.95-4.12 (m, O-CH2-CH2-N-CH2), 4.29-4.42 (m, H-C3′;
C(O)O-CH2), 6.02-6.10 (t, H-C1′), 7.40-7.43 (s, H-C6
thymine); HRMS calcd for C22H38O6N3 440.2761 (M+), found
440.2759.
N,N-Dim et h yl-N-2-(oct a n oyloxy)et h yl-N-[N1-(5-m et h -
yl-2,4-d ioxop yr im id in yl)m et h oxyet h yl]a m m on iu m Io-
d id e (1b). 1-[(2-Iodoethoxy)methyl]thymine (30 mg, 0.097
mmol) and 2-(dimethylamino)ethyl octanoate (100 mg, 3.9
mmol) were dissolved in acetonitrile (10 mL). The reaction
mixture was heated to a gentle reflux and stirred for 24 h.
After the removal of acetonitrile under vacuum, benzene (5 ×
20 mL) was added to the residue, and the solution was
decanted to remove the unreacted starting material. Pure
product (40 mg) was obtained by column chromatography
(discontinuous gradient of CHCl3/CH3OH 9:1, 7:3): yield 74%;
1
mp 96-98 °C; hygroscopic; H NMR (CDCl3) δ 0.80-1.00 (t,
CH3-(CH2)4), 1.20-1.40 (m, CH3-(CH2)4-), 1.50-1.70 (m,
CH2-CH2-CO), 1.85-1.95 (s, CH3-C5 thymine), 2.30-2.45
(m, CH2-CH2-CO), 3.30-3.60 (s, (CH3)2-N), 3.90-4.10 (m,
CH2-N-CH2), 4.15-4.30 (m, CH2-CH2-O-CH2-N), 4.50-
4.65 (m, C(O)O-CH2), 5.20-5.35 (s, O-CH2-N): HRMS calcd
for C20H36O5N3 398.2654 (M+), found 398.2644.
Compounds 1c and 1d were prepared in a similar manner
by using the appropriate amino ester.
N,N-Dim eth yl-N-[2-(h exadecan oyloxy)eth yl]-N-[5′-(2′,5′-
d id eoxy)th ym id in yl]a m m on iu m Iod id e (7c): Yield 30%;
wt 0.12 g; mp 148-150 °C; hygroscopic; 1H NMR (CDCl3) δ
N,N-Dim et h yl-N-2-(h exa d eca n oyloxy)et h yl-N-[N1-(5-
m e t h yl-2,4-d ioxop yr im id in yl)m e t h oxye t h yl]a m m on -

3416 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Notes
0.70-0.78 (t, CH3-(CH2)12), 1.11-1.23 (m, CH3-(CH2)12-),
1.42-1.53 (m, CH2-CH2-CO), 1.70-1.75 (s, CH3-C5 thym-
ine), 2.11-2.23 (m, HR-C2′), 2.18-2.25 (t, CH2-CH2-CO),
2.32-2.45 (m, Hâ-C2′), 3.12-3.18 (s, (CH3)2-N), 3.22 (s, HO),
3.65-3.72 (m, O-CH2-CH2-N-CH2), 3.75-3.82 (m, H-C4′),
3.95-4.12 (m, O-CH2-CH2-N-CH2), 4.29-4.42 (m, H-C3′;
C(O)O-CH2), 6.02-6.10 (t, H-C1′), 7.40-7.43 (s, H-C6
thymine). Anal. (C30H54O6N3I) C, H, N.
N,N-Dim et h yl-N-[3-(h exa d eca n oyloxy)p r op yl]-N-[5′-
(2′,5′-d id eoxy)th ym id in yl]a m m on iu m Iod id e (7d ): Yield
36%; wt 0.14 g; very hygroscopic; 1H NMR (CDCl3) δ 0.70-
0.78 (t, CH3-(CH2)12), 1.11-1.23 (m, CH3-(CH2)12-), 1.42-
1.53 (m, CH2-CH2-CO), 1.70-1.75 (s, CH3-C5 thymine),
2.18-2.35 (t, CH2-CH2-CO), 2.28-2.68 (m, H2-C2′; O-CH2-
CH2-CH2-N), 3.22-3.42 (s, (CH3)2-N), 3.58-3.72 (m, O-CH2-
CH2-N-CH2; H-C4′), 4.12-4.30 (m, O-CH2-CH2-N-CH2),
4.48-4.56 (m, H-C3′; C(O)O-CH2), 6.02-6.10 (t, H-C1′),
7.50-7.53 (s, H-C6 thymine), 10.20-10.25 (s, HN thymine);
HRMS calcd for C31H56O6N3 566.4169 (M+), found 566.4178.
treated control cultures was the IC50 value. Acyclovir
(50 µM) was used as a positive control. Stock solutions
of drugs were dissolved in DMSO and stored at -20 °C.
HIV-1 Syn cytia l P la qu e Assa y. A 96-well plate
was treated with 100 µL of poly(L-lysine) solution for
30 min at room temperature and washed 3 times with
0.01 M phosphate-buffered 0.85% (w/v) sodium chloride
(PBS) as previously described.4 Aliquots of CEM-SS
cells in log-phase growth were washed 3 times with PBS
and suspended at a density of 50 000 CEM-SS cells per
50 µL of RPMI-1640 medium without serum. The CEM-
SS cells were transferred to each poly(L-lysine) pre-
coated well. After 30 min incubation at 37 °C in a
humidified atmosphere containing 5% CO2, the cell
monolayer was inoculated with 50 µL of HIV-1 diluted
in growth medium. After 1 h for virus attachment, the
cell monolayer was overlaid with 100 µL of growth
medium containing serial concentrations of test com-
pound. The plates were incubated at 37 °C in a
humidified atmosphere containing 5% CO2. After 3
days, each well received a second 100 µL overlay of
growth medium with or without the same concentration
of test compound, and incubation was continued for an
additional 2 days. On day 5, syncytial plaques were
microscopically observed to be light-refractive, large,
multicellular foci (10-25 nuclei per syncytium) that
appeared as either brown and granular or clear. Plaques
were counted with the aid of a 10× gridded ocular lens,
and the concentration of test compound required to
inhibit 50% of the plaque formation (EC50) relative to a
untreated control culture was determined.
Biologica l Eva lu a tion s
Cytotoxicity Assa y w ith th e SV-28 a n d KB Tu -
m or Cell Lin es. Preliminary biological screening for
antitumor activity was done with SV-28 cells. SV-28
cells are baby hamster kidney cells transformed by the
oncogenic simian virus SV40. Cells were grown in
MEM medium with 5% fetal calf serum. Cells were
seeded at a density of 3 × 103 cells/cm2. About 24 h
later, 10, 20, and 50 µM of the test compound was added,
and cells were incubated for another 48 h to allow three
doublings of the control cells. The cells then were
counted with a hemocytometer. Each compound was
tested on at least two separate occasions. IC50 is the
concentration of the test compound which reduced the
cell count to 50% compared with the control culture. For
compounds showing cytotoxicity, the experiments were
repeated 3 times to determine the standard deviation.
Compounds 1c and 7c also were tested against the KB
cell line by the same assay. The doubling time for KB
cells, however, is slightly longer than that for SV-28
cells.
SV-28 Cell P la tin g Efficien cy Assa y. The cell
plating efficiency assay was performed to study the cells’
ability to reproduce after exposure to the cytotoxic
compounds. On day 1, the SV-28 cells were seeded at
a density of 3 × 103 cells/cm2. After 24 h of incubation,
the test compound was added at about the IC50 concen-
tration. On day 3, the cells were counted with a
hemocytometer, diluted 100 times, and seeded into 75
cm2 flasks. On day 9, the cells were fixed and stained
with 0.5% methylene blue in 50% ethanol, and the
number of colonies was counted. This number divided
by the number of cells plated on day 3 gives the plating
efficiency.
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