Synthesis and RT inhibitory activity evaluation of new pyrimidine-based Seco-nucleosides

Article abstract of DOI:10.1080/15257770500446931

Eleven new 3 ′,4 ′- seco acyclic nucleosides (4-14) were prepared by nucleophilic substitution of protected pyrimidine bases on ethyl 3,3-diethoxypropanoate (3). Structures were characterized spectroscopically and a brief analysis of their conformation in solution was performed by the vicinal coupling constants 3 J H 2′ aH 3′ and 3 J H 2′ bH 3′. In solid state, compound 6 forms a homodimer linked by hydrogen bonding. In preliminary tests all compounds show low toxicity and gentle activity against HIV-1 RT in vitro. Copyright Taylor & Francis Group, LLC.

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Synthesis and RT Inhibitory Activity
Evaluation of New Pyrimidine-Based
Seco-Nucleosides
a
a
a
Genaro Vargas , Iker S. Escalona , Magali Salas , Barbara
a
b
Gordillo & Adolfo Sierra
a
Departamento de Química , Centro de Investigación y de Estudios
Avanzados del Instituto Politécnico Nacional, México , México, D.F.
b
Sección de Toxicología, Centro de Investigación y de Estudios
Avanzados del Instituto Politécnico Nacional, México , México, D.F.
Published online: 23 Aug 2006.
To cite this article: Genaro Vargas , Iker S. Escalona , Magali Salas , Barbara Gordillo & Adolfo Sierra
(2006) Synthesis and RT Inhibitory Activity Evaluation of New Pyrimidine-Based Seco-Nucleosides,
Nucleosides, Nucleotides and Nucleic Acids, 25:3, 243-257, DOI: 10.1080/15257770500446931
To link to this article: http://dx.doi.org/10.1080/15257770500446931
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Nucleosides, Nucleotides, and Nucleic Acids, 25:243–257, 2006
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770500446931
SYNTHESIS AND RT INHIBITORY ACTIVITY EVALUATION OF NEW
PYRIMIDINE-BASED SECO-NUCLEOSIDES
Genaro Vargas, Iker S. Escalona, Magali Salas, and Barbara Gordillo
2
Departamento de Qu ´ı mica, Centro de Investigaci o´ n y de Estudios Avanzados del Instituto
Polit e´ cnico Nacional, M e´ xico, D.F., M e´ xico
Adolfo Sierra
2
Secci o´ n de Toxicolog ´ı a, Centro de Investigaci o´ n y de Estudios
Avanzados del Instituto Polit e´ cnico Nacional, M e´ xico, D.F., M e´ xico
ꢁ
ꢁ
2
Eleven new 3 ,4 -seco acyclic nucleosides (4–14) were prepared by nucleophilic substitution
of protected pyrimidine bases on ethyl 3,3-diethoxypropanoate (3). Structures were characterized
spectroscopically and a brief analysis of their conformation in solution was performed by the vicinal
3
3
coupling constants JH2ꢁaH3ꢁ and JH2ꢁbH3ꢁ . In solid state, compound 6 forms a homodimer linked by
hydrogen bonding. In preliminary tests all compounds show low toxicity and gentle activity against
HIV-1 RT in vitro.
Keywords Non-nucleosides; Acyclonucleosides; Seco-nucleosides; RT inhibitors
INTRODUCTION
One of the most devasting diseases in the last quarter of a century is
acquired immune deficiency syndrome (AIDS). This often fatal immunode-
ficiency disorder is caused by the retrovirus HIV-1 that targets CD4 receptors
in cell-mediated immune system. During the process of viral infection, the
single-stranded RNA genome of HIV is converted to double-stranded DNA
by the viral encoded reverse transcriptase (RT) enzyme. Thus, a strategy to
control the disease is to target the RT enzyme with drugs that bind to it,
blocking their active site, thus leading to inactivation of the replicative cycle
of the virus.[
1]
Received 27 June 2005; accepted 31 October 2005.
This work was supported by a grant from CONACyT M e´ xico (32221-E). G. V. is grateful to CONACYT
M e´ xico for a Ph.D. fellowship. We thank Dr. Sa u´ l Villa from the Department of Biolog ´ı a Celular at
CINVESTAV for his valuable assistance in the treatment of the biological data. The help given by Georgina
Guzm a´ n and Gabina Dionisio in the purification of some compounds is acknowledged. Elemental analysis
of the compounds were obtained by Geiser Cuellar and Ma. Teresa Cortez; their assistance to obtain these
data is greatly appreciated.
Address correspondence to Barbara Gordillo, Depto. de Qu ´ı mica. CINVESTA-IPN. Av. IPN 2508
0
7000 M e´ xico D.F. E-mail: ggordill@cinvestav.mx
2
43

244
G. Vargas et al.
FIGURE 1 Anti-HIV ddN drugs.
The HIV-1 reverse transcriptase has a highly flexible hydrophobic bind-
ing domain with the shape of a pocket with two inhibitory binding sites
[
2]
ꢁ
named the catalytic and the allosteric site. The metabolically activated 5 -
ꢁ
ꢁ
triphosphates of 2 ,3 -dideoxynucleoside (ddN) analogues are known to be
potent antiviral drugs that interact at the catalytic site of the RT enzyme.[3,4]
However, ddN analogues are toxic to normal cells because they compete with
natural nucleosides for the in vivo phosphorylation step acting as DNA chain
[
5,6]
terminators in the polymerization process.
in Figure 1.
Some antiviral ddN are shown
On the other hand, non-nucleosides reverse transcriptase inhibitors
[
7]
(
NNRTIs), shown in Figure 2, are less toxic since they do not interact
with DNA polymerases but with the RT enzyme at the allosteric site located
a
1
0 A apart from the catalytic site.
A striking feature of allosteric RT pocket is its considerable conforma-
[
8]
tional flexibility, which is believed to be essential for its catalytic action.
The pocket geometries dictate the binding energies of the NNRTIs into the
cavity; therefore, changes of inhibitor potency are accompanied with those
changes in conformation that optimize their interaction with proteins. For
example, the non-nucleoside analogues MKC-442 and TNK-651 (Figure 3)
are better RT inhibitors than HEPT (i.e., IC50 = 17 µM for HEPT vs. IC
=
50
8
nM for MKC-442) because they maximize the interaction of the 6-benzyl
[9]
ring with Tyr 181 residue.
A common feature in all HEPT analogues is the linear substituted nature
ꢁ
of C(1 ) at the N(1) of the pyrimidine ring; however, it is interesting to
ꢁ
ꢁ
ꢁ
[10]
note that branched C(1 ) analogues (called 3 ,4 -seco acyclic nucleosides),

Synthesis and Evaluation of New Seco-Nucleosides
245
FIGURE 2 Anti-HIV NNRTIs drugs.
which resembles more closely to natural nucleosides (Figure 4), are in fact
less active RT inhibitors,[ as well as some others seco nucleosides.
11]
[12,13]
This
behavior may attend to the fact that nucleoside analogues target the catalytic
site of the enzyme and non-nucleoside analogues the allosteric, as mentioned
above.
Taking into consideration that in the allosteric RT pocket, non-covalent
interactions as hydrogen bonding or hydrophobic interactions, among oth-
ers, are of importance to form the RT-NNRTIs complex, we synthesized
ꢁ
ꢁ
several 3 ,4 -seco acyclic nucleosides combining an ether function as ob-
served in MKC-442 and an ester function, as hydrogen bonding acceptor,
ꢁ
in the C(1 ) branch (compounds 4–14 in Figure 5), to test their activ-
ity in vitro against the enzyme RT of HIV-1. The conformation of the
seco nucleosides in solution is reported as well as the X-ray diffraction of
compound 6.
FIGURE 3 NNRTIs, analogues of HEPT.

246
G. Vargas et al.
ꢁ
ꢁ
FIGURE 4 3 ,4 -Seco acyclic nucleosides.
RESULTS AND DISCUSSION
The synthesis of compounds 4–14 was performed by nucleophilic substi-
tution of protected pyrimidine bases on ethyl 3,3-diethoxypropanoate (3).
Compound (3) was prepared according to the procedure reported by Tietze
[
14,15]
(
Scheme 1).
Ethyl vinyl ether was reacted with trichloroacetyl chloride
◦
at 0–4 C for 5 h and then stirred to room temperature for 12 h to produce the
1
,1,1-trichloro-4-chloro-4-ethoxy-butan-2-one (1). The dehydrohalogenation
of 1 was performed by heating and distilling off the product 1,1,1-trichloro-
-ethoxy-buten-2-one (2), obtained in 94% yield. Compound (2) was treated
with an excess of K CO in ethanol to obtain the propanoate (3) in 98%
4
2
3
[
16]
yield.
SCHEME 1

Synthesis and Evaluation of New Seco-Nucleosides
247
FIGURE 5 Pyrimidine based seco acyclic nucleosides (4–14).
In order to obtain the acyclic nucleosides 4–6, compound 3 was
treated with the corresponding persilylated pyrimidine nucleobases under
Vorbr u¨ ggen conditions (Scheme 2).[
17,18]
We found that the products were
obtained in better yields if the reaction mixture was stirred at room tempera-
ture for long periods of time (10–12 days) rather than be heated (Scheme 2).
Compounds 7–14 were obtained by transesterification of 4 or 5 with heating
◦
at 70 C under acidic conditions (Scheme 2). The reaction took place more
efficiently with methanol than with other alcohols as it was carried out at
SCHEME 2

248
G. Vargas et al.
TABLE 1 Seco-Nucleosides 4–14 Synthesized in This Work
Analysis (%)a
H
Isolated
yield (%)
Melting
point ( C)
MS
(m/z)
◦
Compound
C
N
+
4
5
6
7
8
9
0
1
2
3
4
85
80
78
95
93
83
81
81
80
84
76
106–107
149–151
115–116
108–109
119–120
90–91
85–86
45–47
67–68
87–88
256 (M )
51.74 (51.56)
53.46 (53.33)
48.24 (48.17)
49.83 (49.58)
52.03 (51.56)
53.56 (53.33)
55.00 (54.92)
55.34 (54.92)
56.47 (56.37)
55.00 (54.92)
56.59 (56.37)
6.32 (6.25)
6.74 (6.66)
5.58 (5.47)
5.83 (5.78)
6.42 (6.25)
6.78 (6.66)
7.15 (7.04)
7.56 (7.04)
7.49 (7.38)
7.15 (7.04)
7.48 (7.38)
11.05 (10.93)
+
270 (M )
10.68 (10.37)
+
b
274 (M )
+
242 (M )
11.63 (11.57)
11.06 (10.93)
10.21 (10.37)
9.97 (9.85)
9.27 (9.85)
9.39 (9.39)
9.92 (9.85)
9.77 (9.39)
+
256 (M )
+
270 (M )
+
1
1
1
1
1
284 (M )
+
284 (M )
+
298 (M )
+
284 (M )
+
78–79
298 (M )
aCalculated values are given in parentheses.
b
Undetermined.
reflux temperature. However, for all other alcohols, the transesterification
reaction was performed at lower than reflux temperature as the starting
◦
material (3) decomposes at temperatures greater than 70 C. The physical
properties and chemical analysis of compounds 4–14 are summarized in
Table 1.
◦
The spectra were obtained at 27 C using CDCl as solvent. The complete
3
assignments of proton and carbon-13 signals are shown in Tables 2 and 3,
respectively.
ꢁ
In all acyclic nucleosides, the methylene and methine protons at C2 and
ꢁ
C3 respectively, comprise ABX systems centered at around δ 2.72, 2.80 and
.07 ppm (Table 1). The methylene C2 signal is for almost all the compounds
ꢁ
6
3
3
a doublet of doublets with coupling constants J
ꢁ
ꢁ
and J
HH
ꢁ
ꢁ
, which
H2 aH3
H2 bH3
3
differ in 1.5–2.6 Hz. On the basis of the calculated J (anti) = 11.0 Hz
3
and J (gauche) = 3.28 Hz, it can be proposed that the molecule is in
HH
conformational equilibrium between two staggered conformations (A and B
[
19]
of Scheme 3) where conformation C is not likely.
SCHEME 3

Synthesis and Evaluation of New Seco-Nucleosides
249
TABLE 2 1H NMR Chemical Shifts (in ppm) for Compounds 4–14 in CDCl3
Compound
Chemical shifts (δ)
4
5
6
7
8
9
10
11
12
13
14
ꢁ
H2 a
2.73
2.80
6.07
3.58
1.28
4.18
4.18
1.22
—
—
—
9.17
5.80
7.41
—
2.73
2.80
6.09
3.56
1.27
4.18
4.18
1.21
—
—
—
9.25
—
7.20
1.96
2.72
2.78
6.03
3.59
1.26
4.14
4.14
1.22
—
—
—
9.83
—
7.46
—
2.73
2.81
6.06
3.58
1.21
3.72
3.72
—
—
—
—
9.38
5.82
7.40
—
2.72
2.81
6.08
3.56
1.21
3.72
3.72
—
—
—
—
8.19
—
2.72
2.77
6.05
3.55
1.18
4.05
4.05
1.62
0.91
—
2.72
2.80
6.09
3.55
1.20
4.07
4.07
1.65
0.94
—
—
9.50
—
7.20
1.95
2.72
2.78
6.05
3.55
1.19
4.10
4.10
1.59
1.35
0.91
—
2.72
2.81
6.09
3.57
1.22
4.13
4.13
1.62
1.38
0.94
—
2.75
2.81
6.07
3.59
1.22
3.90
3.93
1.95
0.94
—
0.94
8.94
5.82
7.41
—
2.73
2.81
6.09
3.56
1.21
3.88
3.92
1.93
0.92
—
0.92
8.94
—
7.20
1.96
ꢁ
H2 b
ꢁ
H3
ꢁ
H5
ꢁ
Me (H6 )
ꢁ
H8 a
ꢁ
H8 b
ꢁ
H9
ꢁ
ꢁ
H10
H11
Me (9 )
ꢁ
—
H3
H5
H6
9.89
5.80
7.39
—
9.95
5.82
7.40
—
9.23
—
7.20
1.97
7.19
1.96
Me(5)
A splitting of the H signal (δ∼5.8 ppm) of around 1.4–2.0 Hz that
5
corresponds to a cross-ring N H coupling in the uracils substituted acyclic
13
nucleosides (4, 7, 9, and 13) was observed. In C NMR spectra, the chemical
ꢁ
ꢁ
ꢁ
ꢁ
shifts of C2 , C3 or C5 , C6 are similar for all the compounds (Table 3).
The conformation of compound 6 was analyzed in solid state, the X-ray
structure indicates that the molecule is in a preferred staggered conforma-
◦
ꢁ
ꢁ
tion B (Figure 6), with a gauche geometry (60 ) between H2b and H3
and an anti geometry (179 ) between H2a and H3 (Table 4). The ability
◦
ꢁ
ꢁ
of compound 6 to form hydrogen bonding through the geometry of the
.
. .
homodimer in Figure 7. The arrangement [N H O] is symmetrical with
◦
. . .
bond angle equal to 169.0 and bond lengths for the [H O] bonding of
ꢁ
. . .
ꢁ
1
.96 and for the non-bonded distance [N O] of 2.84 . It is remarkable that
these two molecules are even closer in distance than the same in the het-
. .
.
erodimer n-octylthymine/n-octyladenine with bond lengths for the [H O]
. . .
ꢁ
ꢁ [20]
bonding of 2.09 and for the non-bonded distance [N O] of 2.97 .
Non-
covalent interactions are important to support the activity of NNRTIs in the
[
21,22]
hydrophobic cavity of the allosteric site of the RT enzyme.

250
G. Vargas et al.
TABLE 3 13C NMR Signal Assignments in Compounds 4–14a
Chemical shifts (δ)
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
C-5
Compound C-1 C-2 C-3 C-5 C-6 C-8 C-9 C-10 C-11 C-2
C-4
C-6 Me-C5
4
5
6
7
8
9
0
1
2
3
4
168.4 40.4 82.0 65.6 14.3 61.3 14.8
168.4 40.5 81.6 65.4 14.3 61.3 14.8
168.2 40.2 82.5 65.8 14.3 61.4 14.8
—
—
—
—
—
—
—
—
—
—
—
—
163.0 150.0 103.4 139.0
164.0 151.0 112.0 134.7 12.9
—
149.0 142.8 122.9 139.6
163.6 150.9 103.4 139.0
164.4 151.3 112.2 134.7 13.0
163.8 151 103.3 139.0
164.6 151.4 112.1 134.8 13.1
—
—
168.9 40.2 82.0 65.6 14.8 52.3
169.2 40.4 81.7 65.6 15.0 52.5
—
—
168.5 40.4 82.0 65.5 14.8 66.9 22.5 10.4
168.7 40.6 81.7 65.5 15.5 67.0 22.5 10.8
168.5 40.5 82.1 65.6 14.9 65.3 30.7 19.2 14.9 163.2 150.7 103.3 139.0
168.5 40.5 81.7 65.1 14.6 64.9 30.5 18.9 13.8 163.9 150.7 111.9 134.7 12.6
168.5 40.4 82.1 65.6 14.9 71.4 27.8 19.2
168.8 40.7 81.5 65.6 14.8 71.6 27.8 19.4
—
1
1
1
1
1
—
b
b
—
—
163.0 150.0 103.4 139.0
164.4 151.2 112.2 134.8 12.8
—
a
Chemical shifts (d) in ppm from TMS in CDCl3.
b
c
ꢁ
Me-C9 (δ = 19.2).
ꢁ
Me-C9 (δ = 19.4).
Biological Assays
The ability of seco nucleosides 4–8, 10, 12–14 to inhibit the HIV-1 re-
verse transcriptase was tested in vitro and compared to Nevirapine. This
ꢁ
TABLE 4 Selected Bond Lengths ( ), Bond Angles (θ) in Deg,
and Torsion Angles (ω) in deg for Compound 6
Bond lengths
Bond and torsion angles
N1-C2
1.385 (3)
1.435 (3)
1.431 (3)
1.439 (3)
1.378 (3)
1.439 (3)
1.258 (3)
0.89 (3)
1.271 (3)
1.399 (3)
1.548 (3)
1.479 (3)
1.379 (3)
N1-C2-N3
113.04 (19)
111.67 (19)
121.85 (16)
108.80 (17)
111.89 (17)
2.64 (0.37)
C2-N3
N3-C4
C4-C5
C5-C6
C6-N1
C2-O2
N3-H3
C4-O4
C5-F8
N3-C4-C5
ꢁ
C6-N1-C3
ꢁ
ꢁ
ꢁ
ꢁ
N1-C3 -C2
N1-C3 -O4
C4-N3-C2-N1
C4-C5-C6-N1
1.04 (0.35)
ꢁ
ꢁ
ꢁ
C6-N1-C3 -C2
−56.73 (0.26)
129.09 (0.21)
179.20 (0.18)
179.13 (1.52)
60.14 (1.46)
58.56 (0.24)
ꢁ
C2-N1-C3 -C2
ꢁ
ꢁ
ꢁ
N1-C3 -C2 -C1
ꢁ
ꢁ
ꢁ ꢁ ꢁ ꢁ
N1-C3
H3 -C3 -C2 -H2 a
ꢁ
ꢁ ꢁ ꢁ ꢁ
H3 -C3 -C2 -H2 b
C3 -C2
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
C3 -O4
O4 -C3 -C2 -C1

Synthesis and Evaluation of New Seco-Nucleosides
251
ꢁ
ꢁ
FIGURE 6 ORTEP projection of the molecular structure of ethyl 3 -ethoxy-3 -(5-fluoro-2,4-dioxo-3,4-
dihydro-1(2H)-pyrimidinyl)propanoate (6). (H3 atom was located from a difference Fourier map and
refined. All others H atoms were placed in calculated positions and refined using a riding model.)
compound was used as a reference because it is already in clinical use
for AIDS treatment.[
23,24]
The RT inhibitor activity for each compound
was obtained as indicated in the experimental part. The range of concen-
trations analyzed for Nevirapine was 0.01–1000 µM. The IC activity of
5
0
[25]
[9,26,27]
Nevirapine was 0.62 ± 0.26 µM.
The IC reported in some studies
50
for Nevirapine ranged from 1.35 ± 0.43 µM to 0.088 ± 0.032 µM, depend-
ing upon the method used for the determination. The dose-effect curves for
FIGURE 7 View of the crystal packing in the unit cell of compound 6.

252
G. Vargas et al.
FIGURE 8 Relation between RT inhibition percentage and Ln of concentration for seco acyclic
nucleosides.
acyclic nucleosides and Nevirapine in the range 0.01–10 µM were practically
indistinguishable from a straight line (Figure 8). In basis of the slope val-
ues, it is observed that Nevirapine is at least three times more active than
the acyclic nucleoside 8. In this incipient series of moderately active RT in-
hibitors, the maximum potency was obtained with derivatives belonging to
the thymine series (8 and 14), whereas derivatives belonging to the uracil
series (7 and 13) or fluorouracil (6) were less potent (m = 3.24 and 2.56 for
8
and 7, respectively; m = 1.56 and 1.46 for 14 and 13, respectively). The
antiviral activity is related inversely with the presence of bulky substituents at
R of the ester group. Thefore, the slope of the lines decreased in the order:
1
methyl > propyl > butyl > isobutyl for the thymine series (compounds 8, 10,
1
2, and 14 correspondingly) and in the order: methyl > ethyl > isobutyl for
the uracil series (compounds 7, 4, and 13). The acyclic nucleoside 8 was the
most active RT inhibitor, followed by compounds 4 and 7 (Figure 8).
The cytotoxicity of acyclic nucleosides 6, 7, and 14 at 60 µM in Vero
cells[ was similar to that of Nevirapine (Table 5). In this study was included
28]
[
24,29−31]
AZT, another drug used in AIDS infected people,
which was more
toxic than compounds 6, 7, 14, and Nevirapine. AZT is more potent anti HIV-
1
0
compound than Nevirapine; however, it is also more toxic [EC (AZT) =
[32]
5
0
.01 µM; EC (Nevirapine) = 0.3 µM].
According to the results summa-
5
0
rized in Table 5, the acyclic nucleosides 6, 7, and 14 are less toxic than AZT
and comparable in toxicity to Nevirapine at 60-µM doses.

Synthesis and Evaluation of New Seco-Nucleosides
253
TABLE 5 Cytotoxicity in vero Cells of
a
Some Acyclic Nucleosides
Compound
% of Survival
AZT
Nevirapine
6
7
35.3 ± 1.7
72.7 ± 2.3
72.3 ± 3.1
77.7 ± 2.5
77.0 ± 5.0
1
4
a
¯
Data represent X ± SD (n = 3).
In summary, the seco nucleosides reported here have RT inhibitor activity
and similar cytotoxicity than Nevirapine at 60-µM doses. The introduction of
bulky substituents at the ester functionality led to a decrease in potency. This
preliminary studies of activity and cytotoxicity are encouraging for further
studies on structure-activity relationships to optimize their inhibition against
HIV-1 multiplication.
EXPERIMENTAL
Chemistry
Melting points are uncorrected. Nuclear magnetic resonance spectra
1
were recorded on Bruker Avance 300 spectrometer in CDCl (δ 7.26, H; δ
3
13
1
13
7
7.0, C), H at 300.1 MHz, and C at 75.5 MHz. The abbreviations used for
1
the H NMR spectra are: s = singlet, d = doublet, dd = double of doublets, t =
triplet, q = quartet, m = multiplet, bs = broad signal. Mass spectra (El) were
measured on a Hewlett Packard 5989A spectrometer using electron impact
(El) at 70 eV. All chemical and solvents used here were of reagent grade; sol-
vent 1,2-dichloroethane was dried and distilled immediately before use from
calcium hydride. Products were purified by succesive recrystallization.[
Yields are given for isolated products. Microanalyses of 4, 5, and 7–14 were
recorded in a Thermo Finnigan Flash 1112 analyzer. Galbraith Laboratories,
Inc., Knoxville TN performed microanalyses of 6.
33]
1
,1,1-Trichloro-4-ethoxy-buten-2-one (2).[14] In a round-bottomed 250-
mL flask fitted with dropping funnel and a stirbar were placed 86.5 g (0.48
◦
mol) of trichloroacetyl chloride. The liquid was stirred at 0 C and 68.5 g
(
0.95 mol) of ethyl vinyl ether were added dropwise through the dropping
◦
funnel. The mixture was kept on stirring at 0 C for 5 h and then at room
temperature for 12 h. The dropping funnel was replaced by distiller appara-
tus and the mixture was heated under vacuum. The product was obtained as
◦
1
a transparent liquid of bp 84–86 C/1 mm Hg in 94% yield (78 g). H NMR

254
G. Vargas et al.
δ 1.39 (t, J = 7.0 Hz, 3 H), 4.09 (q, J = 7.0 Hz, 2 H), 6.14 (d, J = 12.4 Hz, 1
H), 7.84 (d, J = 12.4 Hz, 1 H).
Ethyl 3,3-Diethoxypropanoate (3).[14] In a round-bottomed 250-mL
flask fitted with a stirbar were placed 50 g (0.23 mol) of 2 and 50 mL (0.86
mol) of ethyl alcohol. The solution was stirred and 3 g of potassium carbon-
ate were added in portions. The mixture was left under stirring for 15 h at
room temperature. The residual potassium carbonate was filtered off and
◦
the product purified by destillation under vacuum 58–60 C/1 mmHg, to
1
give 38.2 g (87%). H NMR δ 1.15 (t, J = 7.0 Hz, 6 H), 1.21 (t, J = 7.0 Hz,
3
2
H), 2.60 (d, J = 6.0 Hz, 2 H), 3.40 (q, J = 7.0 Hz, 2 H), 3.70 (q, J = 7.0 Hz,
H), 4.10 (q, J = 7.0 Hz, 2 H), 4.90 (t, J = 6.0 Hz, 1 H).
ꢁ
ꢁ
General Procedure for the Preparation of Ethyl 3 -Ethoxy-3 -(substituted
Pyrimidinyl)propanoates 4–6.
obases were persilylated by the procedure proposed by Vorbr u¨ ggen:
Thymine, uracil, or 5-fluorouracil nucle-
[
17]
In
a round-bottomed 50-mL flask fitted with reflux condenser and rubber
septa were placed 7.9 mmol of the nucleobase, 23.69 mmol of 1,1,1,3,3,3-
hexamethyldisilazane, and 2.76 mmol of trimethylchlorosilane. The result-
ing suspension was stirred and heated under reflux until the solution was
transparent (between 5 and 12 h). The excess of HMDS was removed under
◦
vacuum (1 mmHg) at 40 C. The procedure continued as follow:
To the flask containing the persylilated nucleobase were added 10 mL
of dry 1,2-dichloroethane. A solution that contained 7.9 mmol of ethyl
3
,3-diethoxypropanoate 3 in 5 mL of dry 1,2-dichloroethane was then
added via syringe, followed by the addition via syringe of 4.0 mmol of
trimehylsilyltrifluoromethane sulphonate in 5 mL of dry 1,2-dichloroethane.
The mixture was stirred at room temperature (warning: heating of the reac-
tion at this point led to several by-products) until no starting material 3 was
observed by thin layer chromatography (10 days for compounds 4 and 5 and
1
2 days for compound 6). The product was washed with 200.0 mL of water,
extracted with methylene chloride (3 × 50 mL), and the organic layer was
dried over sodium sulfate. The solvent was removed with a rotary evaporator
to yield an oily product in all cases. Purification of the crude products was
accomplished by recrystallization in CH Cl /hexane (1:4).
2
2
ꢁ
General Procedure for the Preparation of Alkyl-3 -Ethoxy-3-(substituted
Pyrimidinyl)propanoate 7–14. In a round-bottomed 25-mL flask, fitted
with reflux condenser and rubber septa, were placed 2.0 mmol of the
propanoate 4 or 5 and 10 mL of the corresponding alcohol. The solution
was stirred and four drops of 95–98% sulfuric acid were added via syringe.
◦
The mixture was heated at 70 C and the transesterification reaction was
folowed by thin-layer chromatography of aliquots taken at different times.

Synthesis and Evaluation of New Seco-Nucleosides
255
The reaction was maintained under stirring and heating until completed (8
h for 7 and 8 and 8–12 days for 9–14). After cooling, the suspension was con-
centrated under vacuum and the residue washed with 25.0 mL of an aqueous
solution of 10% sodium bicarbonate. The product was extracted with methy-
lene chloride (3 × 20 mL) and the organic layer was dried over sodium
sulfate. The solvent was removed with a rotary evaporator. Purification of
the crude product was accomplished by recrystallization in CH Cl /hexane
2
2
(1:4).
In Vitro RT Inhibition Assays
The activity of acyclic nucleosides 4–8, 10, 12–14, and Nevirapine to
inhibit the HIV-1 reverse transcriptase was tested in vitro by using the kit
TM
Lenti-RT (Cavidi Tech, Sweden), which contains poly (rA) as the enzyme
template, an oligo (dT) as the primer and BrdUTP as the non-radioactive
triphosphate substrate. Quantification of the BrdUMP incorporated in DNA
by the RT enzyme was performed after reaction with alkaline phosphatase
enzyme-conjugated anti-BrdU monoclonal antibody in the presence of p-
nitrophenylphosphate (pNPP).[
34−36]
The RT inhibitor activity was obtained
through the absorbance measurements at λ = 405 nm for each compound af-
ter 1 h incubation. Seco nucleosides and Nevirapine were dissolved in DMSO
and all experiments were carried out in triplicate.
Cytotoxicity Assays
Compounds 6, 7, and 14, as well as AZT and Nevirapine, were evaluated
for cytotoxicity in Vero[
37,38]
(African green monkey kidney) cells. Cells were
cultured in Dulbecco’s modified Eagle’s basal medium (Sigma Chemical
Co., St. Louis, MO), supplemented with 10% calf serum (Gibco BRL, Grand
◦
Island, NY) in 95% air, 5% CO incubator at 37 C. Cultures in confluence
2
5
were washed twice with PBS and removed using Trypsin-EDTA. Cells (1 × 10
2
cells/cm ) were cultured in 24-well cell culture plates newly. Cultures at sub-
confluence were washed twice with PBS to remove bond-attached cells and
exposed to 60 µM of compounds dissolved in DMSO. At 40 h of exposure cy-
totoxicity assays were carried out in cultures using Trypan blue dye.[
39,40]
Con-
trol cultures contained only DMSO and assays were carried out in triplicate.
X-Ray Crystallography
Crystallographic work was performed in an Enraf-Nonius Kappa CCD
[
41]
diffractometer. Data collection: COLLECTION software.
Cell refinement
and WinGX version 1.7
The structure of compound 6 was solved and refined by SHELXS
[
42]
and data reduction: Denzo/Scalepack software
[
43]
software.
[
44]
[45]
9
7.
Molecular graphics: CAMERON software.
The crystal structure has

256
G. Vargas et al.
been deposited at the Cambridge Crystallographic Data Centre and allocated
the deposition number CCD 292628.
CONCLUSIONS
ꢁ
ꢁ
A simple method for the synthesis of 3 ,4 -seco acyclic nucleosides, ana-
logues of the antiviral compound MKC-442, is reported. The coupling con-
3
3
stants J
ꢁ
ꢁ
and J
ꢁ
ꢁ
suggest that most compounds adopt the same
H2 aH3
H2 bH3
conformation in solution regardless of the substituent (R ) at the ester func-
1
tion. The hydrogen-bonding interactions in the homodimer of the solid
.
. .
structure of compound 6 are of the type [N H O]; they are symmetrical
◦
. . .
with bond angle equal to 169.0 and bond lengths for the [H O] bonding
ꢁ
. . .
ꢁ
of 1.96 and for the non-bonded distance [N O] of 2.84 . All the com-
pounds are, in vitro, less active than Nevirapine against the enzyme RT of
HIV-1. Compounds substituted with thymine nucleobase and with small R1
substituents in the ester group are more active than those substituted with
uracil nucleobase and with bulky R groups. At doses of 60 µM, the seco nu-
1
cleosides 6, 7, and 14 showed similar cytotoxicity than Nevirapine and lower
cytotoxicity than AZT.
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