Structure-activity evaluation of new uracil-based non-nucleoside inhibitors of HIV reverse transcriptase

Article abstract of DOI:10.1039/c3md00225j

A new series of potential nonnucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) based on the carbocyclic 5′-nor-uracil scaffold were designed and synthesized. Three different aspects of the scaffold were investigated: the effects of adding a linker between the carbocyclic and phenyl fragments, introduction of different substituents on the benzoyl residue and replacing the central carbocyclic ring with a benzyl group. Analogues of 2′,3′-dideoxy-2′,3′-didehydro-5′-nor-uridine, bearing 3,5-dichloro- or 3,5-dimethylbenzoyl groups, showed inhibitory activity against HIV-RT wild-type (IC₅₀ 5-10 μM) and mutants L100I (IC ₅₀ 1.2-2.1 μM) and K103N (IC₅₀ 8-17 μM).

Full text of DOI:10.1039/c3md00225j

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CONCISE ARTICLE
Structure-activity evaluation of new uracil-based
non-nucleoside inhibitors of HIV reverse transcriptase†
Cite this: DOI: 10.1039/c3md00225j
a
a
b
Elena S. Matyugina, Vladimir T. Valuev-Elliston, Alexander N. Geisman,
b
c
a
Mikhail S. Novikov, Alexander O. Chizhov, Sergey N. Kochetkov,
d
a
Katherine L. Seley-Radtke* and Anastasia L. Khandazhinskaya
A new series of potential nonnucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) based on the
0
carbocyclic 5 -nor-uracil scaffold were designed and synthesized. Three different aspects of the scaffold
were investigated: the effects of adding a linker between the carbocyclic and phenyl fragments,
Received 5th August 2013
introduction of different substituents on the benzoyl residue and replacing the central carbocyclic ring
Accepted 12th September 2013
0
0
0
0
0
with a benzyl group. Analogues of 2 ,3 -dideoxy-2 ,3 -didehydro-5 -nor-uridine, bearing 3,5-dichloro- or
,5-dimethylbenzoyl groups, showed inhibitory activity against HIV-RT wild-type (IC50 5–10 mM) and
mutants L100I (IC50 1.2–2.1 mM) and K103N (IC50 8–17 mM).
DOI: 10.1039/c3md00225j
3
www.rsc.org/medchemcomm
effect of different substituents on the benzoyl residue that had
Introduction
7,8,10–15
shown promise in previous targets
(B), and (C) the feasi-
HIV reverse transcriptase (HIV-RT) is one of the primary enzy- bility of replacing the central carbocyclic ring with a benzyl
matic targets for successful disruption of HIV replication. In group to increase aromatic character. These modications were
that regard, non-nucleoside inhibitors (NNRTIs) are an integral designed to interrogate structural, as well as potential, hydro-
1
part of current HIV treatment regimens. Unlike nucleoside phobic requirements of the HIV-RT binding site.
inhibitors (NRTIs), NNRTIs are widely diverse from the stand-
point of their chemical structures. One common feature
however, is the overall hydrophobicity of the molecules due to
In addition, in terms of the latter modication, the central
ring likely plays a key role in the high affinity observed for 1 in
HIV-RT by maintaining the proper geometry for the inhibitor as
2–4
the presence of several aromatic rings. Among the many well as to help occupy a hydrophobic pocket adjacent to the
1
potential NNRTI scaffolds, the N -substituted uracil diaryl polymerase active site that had been identied in previous
5,6
2,16
derivatives have exhibited signicant activity. Compounds studies.
As a result, we speculated that replacement of the
1
3
with spatially disconnected aryl components at the N and N
cyclopentene with a bulkier aryl group might lead to increased
affinity since this would not only introduce aromatic character
to the central ring, but also increase p-stacking as well as the
overall hydrophobic interactions.
7
,8
positions of uracil have also demonstrated potent activity.
0
Recently we reported that a 5 -norcarbocyclic analogue of
,3 -dideoxy-2 ,3 -didehydrouridine (1, Fig. 1) acts as an NNRTI
with an IC50 value of 13.4 mM. As a result, this compound was
selected for further investigation to potentially improve its
activity against HIV-RT.
0
0 0 0
2
9
Chemistry
As depicted in Fig. 1 we proposed to investigate three
different aspects of the scaffold: (i) the effects of adding a linker
between the carbocyclic and phenyl fragments to potentially
probe the binding pocket requirements (A), as well as (ii) the
As shown in Scheme 1, phenyl acetic and hydrocinnamic acid
analogues 2 and 3 were synthesized by acylation of 1-(4-hydroxy-
17
2
-cyclopenten-1-yl)uracil with the appropriate acid chlorides.
The requisite acid chlorides were prepared by reuxing with
thionyl chloride in 1,2-dichloroethane. Compounds 4–6 pos-
sessing various substituents on the benzoyl fragment were
prepared in a similar manner from 1-(4-hydroxy-2-cyclopenten-1-
a
Engelhardt Institute of Molecular Biology, Russian Academy of Science, Vavilov Str.,
3
2, Moscow 119991, Russia
b
Department of Pharmaceutical & Toxicological Chemistry, Volgograd State Medical yl)uracil and the corresponding chlorides of benzoic acid. Intro-
University, Pavshikh Bortsov Sq., 1, Volgograd 400131, Russia
duction of the benzyl group onto N-3 of the base by previously
c
Zelinsky Institute of Organic Chemistry, Russian Academy of Science, Leninsky pr., 47,
Moscow 119991, Russia
9
described methods gave compounds 7–13 (Scheme 1).
It should be noted that the reactions with 3,5-dimethyl and
d
Department of Chemistry & Biochemistry, University of Maryland, Baltimore County,
3,5-dichlorobenzoyl chlorides gave not only targets 5 and 6 but
1
†
1
000 Hilltop Circle, Baltimore, MD 21250, USA. E-mail: kseley@umbc.edu
Electronic supplementary information (ESI) available. See DOI:
0.1039/c3md00225j
also bisbenzoylated side products 14 and 15 (Fig. 2), while in all
other cases formation of the bis-side products was not observed.
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Fig. 1 Proposed SAR modifications.
ꢀ
ꢀ
Scheme 1 (a) SOCl
2
, 80 C, 3 h; (b) 1-(4-hydroxy-2-cyclopenten-1-yl)uracil, pyridine, 36 C, 2 h; (c) BnBr for 7–11, 3,5-F
2
-BnBr for 12–13, K
2
CO
3
, DMF, 18 h.
1
3
Similarly, N -[(benzoyloxy)benzyl]uracil derivatives 19–21 chloride in presence of potassium carbonate gave N -benzyl
23
were synthesized by the condensation of (benzoyloxy)benzyl derivatives 22–24 (Scheme 2).
bromides 16–18 and 2,4-bis(trimethylsilyloxy)pyrimidine, obenzoyloxy)benzyl]uracil (25) was obtained by the condensa-
in reuxing anhydrous 1,2-dichloroethane over 35 h.
Next, 1-[3-(3,5-dichlor-
18
tion of the appropriate trimethylsilyl uracil derivative with
Bromides 16–18 were prepared from the isomeric methyl- an equimolar amount of 3-(3,5-dichlorobenzoyloxy)benzyl
1
9,20
phenylbenzoates
by treatment with a solution of bromine in bromide, prepared from corresponding 3-methylphenyl 3,5-
21
21
carbon tetrachloride. Conversely, 2,4-bis(trimethylsilyloxy)- dichlorobenzoate as described above. The structures of the
pyrimidine was obtained by reaction of uracil with excess target compounds and their purities were conrmed by NMR
22
1
13
HMDS. The reaction of compounds 19–21 with excess benzyl ( H and C), TLC and HRMS. The 1-[3-(benzyloxy)benzyl]-
derivatives of 5-bromouracil, thymine and cytosine (structures
not shown) were synthesized in an analogous manner.
It is worth noting that the method of thymine alkylation by
treatment with 3-(benzoyloxy)benzylbromide in DMSO in presence
24
of potassium carbonate has been previously reported by Aoyama,
but details of the synthesis and the yield of the alkylation product
were not discussed. The use of the pyrimidine-2,4(1H,3H)-dione
silyl-derivatives in Hilbert–Johnson reaction conditions however,
18,25
is a more selective way of accomplishing the alkylation
thereby
avoiding the formation of disubstituted products as has been
26–28
Fig. 2 Compounds 14 and 15.
observed for alkylation via potassium salts.
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2 3
Scheme 2 (a) 1,2-Dichloroethane, reflux, 35 h; (b) BnCl, K CO , DMF, 15 h.
tried, that being the 3,5-dichloro substitution of 25, however this
also proved to be less active than 1, thus no further substitutions
The three types of new N ,N -disubstituted uracil derivatives were tried. Finally, the 1-[3-(benzyloxy)benzyl]derivatives of
were selected to investigate their various effects on HIV-RT. In 5-bromouracil, thymine and cytosine were synthesized as
the rst group, linkers of different lengths were designed to described above however they also exhibited no activity.
Discussion
1
3
estimate the inuence of distance between the cyclopentene
Notably, the most pronounced inhibitory properties were
residue and the benzoyl group on anti-HIV activity. In that found for the 3,5-dimethylbenzoyl (10) and the 3,5-dichlor-
regard, the benzoyl fragment of 1 was replaced with longer obenzoyl (11 and 25) analogues. As a result, the two compounds
residues to give the phenyl acetic (7 and 12) and hydrocinnamic
(8 and 13) acid analogues. Compounds of the second group
(9–11) have additional substituents on the benzoyl moiety, Table 1 Activity of the studied compounds against HIV-RT
while the last group of compounds (19–25) was designed to
evaluate the inuence of replacing the cyclopentene ring by a
benzyl group.
The compounds were tested as potential HIV NNRTIs
(
Table 1) and the results revealed some interesting structure–
activity relationships. The IC50 values increased as follows: 1
13.4 mM) < 7 (52 mM) < 8 (>100 mM), thus it became immediately
(
clear that increasing the linker length led to an undesirable
decrease in anti-HIV activity, thus no additional linkers were
investigated.
HIV-RT
In terms of the second modication, we have previously
9
3
1
2
reported that the presence of an aryl group on the N uracil Comp.
moiety of similar NNRTIs appeared to be important for activity.
R
n
R
Yield, %
IC50 (mM)
3
2
3
4
5
6
7
8
9
1
H
H
1
2
0
0
0
1
2
0
0
0
1
2
0
0
—
—
—
—
—
—
—
0
H
H
H
H
52
49
71
77
52
35
60
79
63
60
51
98
18
20
73
80
71
65
60
58
74
—
—
Because several N -unsubstituted uracils were serendipitously
formed as intermediates on the way to realize the target
compounds in this study, we decided to isolate and assay those
3-CN
3,5-Me2
3,5-Cl
H
H
3-CN
3,5-Me
3,5-Cl2
H
H
3,5-Me
>100
>100
>100
52
>100
>100
8.2
as well to see if our theory held. Thus, compounds 2–6, and 19–
2
H
3
Bn
Bn
Bn
Bn
Bn
3,5-F
3,5-F
3,5-Me
3,5-Cl
H
H
H
Bn
Bn
Bn
H
2
1, as well as the N -3,5-diuorobenzyls (12 and 13) and the
3
N -benzoyls (14 and 15) were screened. Not surprisingly, none
of the unsubstituted compounds exhibited any appreciable
0
2
3
activity, thereby conrming our hypothesis that the N -substi- 11
tution on the uracil ring is indeed necessary for activity. Only 12
5.1
2
2
Bn
Bn
>100
>100
>100
>100
>100
>100
>100
>100
32.3
>100
42
13
14
15
19
compound 25 exhibited antiviral activity with IC50 of 42 mM,
2
2
Bz
which may be due to its close structural similarity with diaryl
3,5-Cl
2
2
Bz
2,6
5,29
ether and benzophenone types of NNRTIs.
2-BzO
3-BzO
4-BzO
2-BzO
3-BzO
4-BzO
3-(3,5-Cl BzO)
2
H
—
Finally, replacement of the carbocyclic ring led to a threefold 20
decrease in activity in the case of the meta-substituted (23) and 21
an even greater decrease for the ortho- and para-substituted
compounds (22 and 24, respectively). We initially began with only
an unsubstituted benzyl ring system for this replacement because
compound 1 was the most active, however in all cases, the activity
22
23
24
25
1
H
13.4
7.2
decreased as compared to 1. One additional modication was NVP
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Table 2 Inhibitory activity of the compounds against selected mutant HIV-1 RTs
10
11
EFV
IC50 (mut)/
IC50 (WT)
RT form
IC50 (mut)
IC50 (mut)/IC50 (WT)
IC50 (mut)
IC50 (mut)/IC50 (WT)
1
NVP
WT
L100I
8.2
2.1
17.3
>20
>20
—
5.1
1.29
8.8
>10
>10
—
13.4
1.2
7.2
273
0.01
0.08
0.58
0.05
0.03
0.06
0.14
—
8
58
5
3
6
0.26
2.11
—
—
—
0.25
1.69
—
—
—
K103N
V106A
Y181C
G190A
K103N/Y181C
33.6
>50
>50
>50
>50
>2000
>2000
>2000
>2000
>2000
[100
>20
[100
>10
—
—
14
(10 and 11) that showed activity higher than parent 1 were antiviral activity. Thus only the 3,5-dimethylbenzoyl and 3,5-
tested against mutant variants of HIV-RT as shown in Table 2. dichlorobenzoyl derivatives (10 and 11) were able to serve
Both compounds inhibited the L100I mutant approximately effectively as HIV NNRTIs against both wild type and mutant
fourfold better than wild type and the K103N mutant only RTs. Notably, both proved to be more potent than the parent
twofold less than wild type. It should be noted that precipitation compound 1. As a result, further studies to explore additional
was observed at concentrations >20 mM for compound 10, scaffold modications for these two potentially interesting
>10 mM for compound 11 and >50 mM for compound 1, thus NNRTIs will be pursued.
additional testing was not carried out.
A possible explanation for the enhanced activity of carbocy-
clic compounds 1, 10 and 11 against L100I mutant (where NVP Experimental
and EFV lose activity either completely or partially) may be that
there are favourable interactions between the isoleucine residue
General
All reagents were obtained at the highest grade available from
and the cyclopentenyl fragment. This may in turn, help stabilize
the conformation of the inhibitor in the hydrophobic pocket,
however further studies are needed to conrm this hypothesis.
To compare inhibitory properties of derivatives 10, 11 and 1
against mutants V106A, Y181C, G190A and double mutant
K103N/Y181C, RT inhibition was evaluated for all three
compounds at a xed concentration of 10 mM. The compounds
showed weak inhibition of the V106A and Y181C mutants, as
well as the double mutant K103N/Y181C, but did not inhibit the
G190A mutant (Table 3).
Sigma and Acros Organics and used without further puri-
cation unless otherwise noted. Anhydrous DMF and
isopropanol were purchased from Sigma-Aldrich Co. Anhy-
drous 1,2-dichloroethane and ethyl acetate were obtained by
1
13
distillation over P
2
O
5
.
H and C NMR spectra were regis-
1
tered on a Bruker Avance 400 spectrometer (400 MHz for H,
00 MHz for C) in CDCl , DMSO-d or CD CN with tetra-
3 6 3
1
3
1
1
9
methylsilane as an internal standard. F NMR spectra were
registered on a Bruker Avance II-300 spectrometer with 282.4
MHz working frequency. Chemical shis are given in ppm,
coupling constants in Hz. TLC was performed on Merck TLC
Silica gel 60 F254 plates eluted with ethyl acetate, chloroform,
chloroform–MeOH (98 : 2), chloroform–MeOH (10 : 1), 1,2-
dichloroethane–ethanol (3 : 1), ethyl acetate–hexane (1 : 1)
and developed with UV-lamp VL-6.LC (France). Acros
Organics (Belgium) silica gel (Kieselgur 60–200 mm, 60A) was
used for column chromatography. Melting points were
determined in glass capillaries on a Mel-Temp 3.0 (Laboratory
In conclusion, of the three structural modications pursued
in this preliminary study, only the 3,5-substituted benzoyl
0
residue of the 4 -OH group of the cyclopentene moiety led to an
increase in inhibitory properties. All other modications
3
including the introduction of substituents to the N -benzyl
fragment or extension of the distance between carbonyl func-
tion and the phenyl group, as well as replacement of the
carbocyclic fragment by a benzyl group, led to a decrease in
1
13
Devices Inc., US). Yields refer to spectroscopically ( H and
C
Table 3 Comparison of selected compounds activity against mutant RTs
NMR) homogeneous materials. High resolution mass spectra
were measured on Bruker micrOTOF II instruments using
electrospray ionization (HRESIMS). The measurements
were done in a positive ion mode (interface capillary voltage
RT Inhibition, %
RT form
10 10 mM
11 10 mM
1 10 mM
NVP 10 mM
ꢁ
4500 V) in a mass range from m/z 50 to m/z 3000 Da; external
or internal calibration was done with ESI Tuning Mixꢀ (Agi-
WT
L100I
52
77
37
14
24
59
89
53
20
17
45
83
26
12
11
85
48
lent Technologies). A syringe injection was used for solutions
in acetonitrile (ow rate 3 mL min ). Nitrogen was applied as
a dry gas; interface temperature was set at 180 C. The acti-
vated DNA (standard template + primer model) was purchased
from GE Healthcare Life Sciences Inc. and used directly.
K103N
V106A
Y181C
G190A
K103N/Y181C
<10
<10
<10
<10
<10
ꢁ
1
ꢀ
<10
23
<10
25
<10
15
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Benzoyloxy)benzyl bromides 16–18 were synthesized as
MedChemComm
0 0
a
H -5 ), 5.74–5.73 (1H, m, H-1 ), 5.78–5.76 (1H, m, H-5), 5.91–5.89
(
21
0
0
previously described, and their physical and chemical prop- (1H, m, H-4 ), 6.07–6.04 (1H, dd, J ¼ 2.27, J ¼ 5.57, H-3 ), 6.41–
30,31
0
erties are in agreement with reported literature data.
6.38 (1H, m, H-2 ), 7.24–7.23 (1H, d, J ¼ 8.08, H-6), 7.61–7.58
0
0
00
00
General method of synthesis for the phenylcarbonic acid (1H, m, H-5 ), 7.87–7.84 (1H, m, H-4 ), 8.23–8.21 (1H, m, H-6 ),
0
0
13
chlorides. Phenyl acetic acid (500 mg, 3.60 mmol), hydro- 8.29–8.28 (1H, m, H-2 ), 9.06 (1H, s, NH). C NMR (CDCl
cinnamic acid (800 mg, 5.30 mmol), 3-cyanobenzoic acid 37.78, 58.56, 78.38, 103.31, 113.33, 117.74, 129.71, 131.11,
500 mg, 3.40 mmol), 3,5-dimethylbenzoic acid (1 g, 6.65 mmol) 133.36, 133.63, 134.63, 136.09, 136.40, 140.27, 150.88, 163.00,
3
):
(
+
or 3,5-dichlorobenzoic acid (1 g, 5.24 mmol) were reuxed with 164.01. HRESIMS m/z: calculated for C17
thionyl chloride (20–40 mL) in 1,2-dichloroethane (30–50 mL) 324.0979, found 324.0984; calculated for C17
H
H
13
N
3
O
4
[M + H]
[M + Na]
+
13
N
2
O
4
for 4 h. Purication was performed by distillation under 346.0798, found 346.0804.
vacuum to afford the desired acid chlorides.
1-[4-(3,5-Dimethylbenzoyloxy)-2-cyclopenten-1-yl]uracil (5)
General numbering for target compounds.
and
1-[4-(3,5-dimethylbenzoyloxy)-2-cyclopenten-1-yl]-3-(3,5-
dimethylbenzoyl)uracil (14). In an analogous manner as was
used to synthesize 2, 1-(4-hydroxy-2-cyclopenten-1-yl)uracil and
3
,5-dimethylbenzoic acid chloride were reacted to give a mixture
of 5 and 14. Purication by column chromatography eluting
with CHCl gave 14 as a colorless oil (21 mg, 0.06 mmol, 18%).
R ¼ 0.43 (CHCl ). H NMR (CDCl ): 1.85–1.80 (1H, dt, J ¼ 15.39,
3
1
f
3
3
0
J ¼ 3.89, H -5 ), 2.28 (6H, s, 2 ꢂ CH ), 2.30 (6H, s, 2 ꢂ CH ),
b
3
3
0
3
.05–3.02 (1H, dt, J ¼ 15.77, J ¼ 8.13, H -5 ), 5.68–5.65 (1H, m,
a
0
0
H-4 ), 5.78–5.76 (1H, d, J ¼ 8.14, H-5), 5.82–5.80 (1H, m, H-1 ),
0
0
6
4
.01–5.99 (1H, m, H-3 ), 6.39–6.38 (1H, m, H-2 ), 7.15 (1H, s, H-
0
0
000
), 7.20 (1H, s, H-4 ), 7.37–7.35 (1H, d, J ¼ 8.14, H-6), 7.46 (2H,
0
0
00
000
000 13
3
s, H-2 , H-6 ), 7.55 (2H, s, H-2 , H-6 ). C NMR (CDCl ): 21.26,
3
1
1
7.69, 58.91, 77.42, 103.01, 127.36, 128.26, 129.63, 131.58,
34.03, 135.16, 137.13, 138.40, 139.05, 140.40, 150.01, 162.11,
66.18, 169.11. HRESIMS m/z: calculated for C H N O [M +
2
7
26
2
5
+
1
-[4-(Phenylacetoxy)-2-cyclopenten-1-yl]uracil (2). Phenyl H] 459.1914, found 459.1902; calculated for C H N O [M +
2
7
26
2
5
+
acetic acid chloride (150 mL, 1.00 mmol) was added to a solution Na] 481.1734, found 481.1734. Further elution provided 5 as an
of 1-(4-hydroxy-2-cyclopenten-1-yl)uracil (100 mg, 0.50 mmol) in off-white powder (65 mg, 0.20 mmol, 77%). R ¼ 0.30 (CHCl ).
f
3
ꢀ
1
dry pyridine (10 mL). The reaction mixture was stirred for 18 h Mp 166.5–168.3 C. H NMR (CDCl ): 1.83–1.79 (1H, dt, J ¼
3
0
at room temperature, the solvent evaporated, and the residue 15.33, J ¼ 3.50, H -5 ), 2.35 (6H, s, 2 ꢂ CH ), 3.10–3.06 (1H, dt,
b
3
0
0
puried via column chromatography, eluting with J ¼ 15.72, J ¼ 8.06, H -5 ), 5.75–5.73 (2H, m, H-4 , H-5), 5.87–5.85
a
0
0
CHCl
.26 mmol, 52%). R
CDCl
3
: MeOH (98 : 2) to give 2 as a colorless syrup (80 mg, (1H, m, H-1 ), 6.02–6.00 (1H, dd, J ¼ 2.07, J ¼ 5.54, H-3 ), 6.42–
1
0
00
0
(
(
5
5
6
f
¼ 0.45 (CHCl : MeOH, 98 : 2). H NMR 6.40 (1H, m, H-2 ), 7.20 (1H, s, H-4 ), 7.34–7.32 (1H, d, J ¼ 8.05,
13
3
0
00 00
3
): 1.53–1.49 (1H, dt, J ¼ 15.55, J ¼ 3.12, H
b
-5 ), 2.88–2.81 H-6), 7.59 (2H, s, H-2 , H-6 ), 9.31 (1H, s, NH). C NMR (CDCl ):
3
0
1H, m, H -5 ), 3.56 (2H, s, CH ), 5.52–5.50 (1H, d, J ¼ 8.05, H-5), 21.22, 37.62, 58.55, 77.42, 103.02, 127.33, 129.62, 134.14,
a
2
0
0
.60–5.58 (2H, m, H-1 , H-4 ), 5.87–5.85 (1H, dd, J ¼ 3.17, J ¼ 135.10, 136.82, 138.33, 140.74, 151.00, 163.30, 166.17. HRESIMS
+
0
0
.54, H-3 ), 6.19–6.17 (1H, m, H-2 ), 6.89–6.87 (1H, d, J ¼ 8.05, H- m/z: calculated for C H N O [M + Na] 349.1159, found
1
8
18 2 4
0
0
00
), 7.26–7.17 (5H, m, H-2 -6 ), 9.44 (1H, s, NH).
-[4-(3-Phenylpropanoyloxy)-2-cyclopenten-1-yl]uracil (3). In
an analogous manner as was used to synthesize 2, 1-(4-hydroxy- and
-cyclopenten-1-yl)uracil and hydrocinnamic acid chloride were dichlorobenzoyl)uracil (15). In an analogous manner 1-(4-
reacted to give 3 as a colorless oil (40 mg, 0.12 mmol, 49%). R
hydroxy-2-cyclopenten-1-yl)uracil and 3,5-dichlorobenzoic acid
.63 (CHCl : MeOH, 98 : 2). H NMR (CDCl ): 1.53–1.48 (1H, dt, chloride were reacted to give a mixture of 6 and 15. Following
349.1151.
1-[4-(3,5-Dichlorobenzoyloxy)-2-cyclopenten-1-yl]uracil
1-[4-(3,5-dichlorobenzoyloxy)-2-cyclopenten-1-yl]-3-(3,5-
1
(6)
2
f
¼
1
0
3
3
0
J ¼ 15.37, J ¼ 3.61, H -5 ), 2.65–2.63 (2H, m, CH ), 2.96–2.92 purication by column chromatography eluting with CHCl , 15
b
2a
0
3
0
0
(
(
6
7
9
3H, m, CH , H -5 ), 5.64–5.61 (2H, m, H-1 , H-4 ), 5.71–5.69 was obtained as a white foam (60 mg, 0.11 mmol, 20%). R ¼
2
b
a
f
0
1
1H, d, J ¼ 8.04, H-5), 5.91–5.89 (1H, dd, J ¼ 3.27, J ¼ 5.6, H-3 ), 0.41 (CHCl ). H NMR (CDCl ): 1.84–1.79 (1H, dt, J ¼ 15.30, J ¼
3
3
0
0
0
.21–6.19 (1H, m, H-2 ), 7.08–7.06 (1H, d, J ¼ 8.04, H-6), 7.20– 3.46, H -5 ), 3.15–3.07 (1H, dt, J ¼ 15.53, J ¼ 7.95, H -5 ), 5.66–
b
a
0
0
00
00
00
00
0
0
.18 (3H, m, H-3 , H-4 , H-5 ), 7.29–7.27 (2H, m, H-2 , H-6 ), 5.63 (2H, m, H-4 , H-5), 5.84–5.81 (1H, m, H-1 ), 6.08–6.04 (1H,
0
0
.37 (1H, s, NH).
1
m, H-3 ), 6.39–6.37 (1H, m, H-2 ), 7.32–7.30 (1H, d, J ¼ 8.16, H-
0
0
000
000
-[4-(3-Cyanobenzoyloxy)-2-cyclopenten-1-yl]uracil (4). In an 6), 7.52–7.51 (1H, m, H-4 ), 7.57–7.56 (1H, m, H-4 ), 7.71 (2H,
0
0
00
000
13
analogous manner, 1-(4-hydroxy-2-cyclopenten-1-yl)uracil and m, H-2 , H-6 ), 7.81–7.80 (2H, m, H-2 , H-6 ). C NMR
-cyanobenzoic acid chloride were reacted to give 4 as a white (CDCl ): 37.73, 59.05, 78.26, 103.17, 128.10, 128.60, 132.52,
3
3
powder (114 mg, 3.20 mmol, 71%). R ¼ 0.47 (CHCl : MeOH, 133.36, 134.24, 134.84, 135.68, 136.29, 136.78, 140.50, 149.74,
f
3
ꢀ
0
1
9
8 : 2). Mp 197.5–198.5 C. H NMR (CDCl
3
): 1.81–1.78 (1H, dt, 161.78, 163.60, 167.13. HRESIMS m/z: calculated for
+
J ¼ 15.17, J ¼ 4.19, H
b
-5 ), 3.21–3.16 (1H, dt, J ¼ 15.24, J ¼ 7.93, C H Cl N O [M + H] 540.9703, found 540.9709; calculated
2
3
14
4 2 5
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+
00 13
H
14Cl
4
N
2
O
5
[M + Na] 562.9522, found 562.9531. Further H-2 ) C NMR (CDCl
3
): 37.97, 44.71, 59.45, 78.51, 102.87,
elution afforded 6 as a white powder (100 mg, 0.27 mmol, 52%). 113.40, 117.83, 127.85, 128.58, 129.33, 129.77, 131.24, 133.44,
ꢀ
0
1
R ¼ 0.49 (CHCl : MeOH, 98 : 2). Mp 194.0–195.1 C. H NMR 133.71, 134.92, 135.97, 136.46, 136.90, 138.23, 151.72, 162.59,
f
3
+
(
(
CDCl ): 1.81–1.77 (1H, dt, J ¼ 15.23, J ¼ 3.98, H -5 ), 3.17–3.10 164.09. HRESIMS m/z: calculated for C H N O [M + H]
3
b
24 19 3 4
0
0
1H, dt, J ¼ 15.37, J ¼ 7.87, H
a
-5 ), 5.75–5.72 (1H, m, H-4 ), 5.79– 414.1448, found 414.1460.
0
5
(
.77 (1H, d, J ¼ 8.16, H-5), 5.89–5.86 (1H, m, H-1 ), 6.06–6.04
1-[4-(3,5-Dimethylbenzoyloxy)-2-cyclopenten-1-yl]-3-benzy-
0
0
1H, dd, J ¼ 2.29, J ¼ 5.57, H-3 ), 6.40–6.37 (1H, m, H-2 ), 7.27– luracil (10). In an analogous manner, BnBr and 5 were reacted
0
0
7
.25 (1H, d, J ¼ 8.16, H-6), 7.56 (1H, m, H-4 ), 7.85–7.84 (2H, m, to afford 10 as a white powder (26 mg, 0.06 mmol, 63%). R ¼
f
0
0
00
13
ꢀ
0
1
H-2 , H-6 ), 9.08 (1H, s, NH). C NMR (CDCl
3
): 37.71, 58.54, 0.50 (CHCl
3
: MeOH, 98 : 2). Mp 118.3–120.0 C. H NMR
7
1
8.35, 103.27, 128.08, 132.58, 133.28, 134.67, 135.62, 136.16, (CDCl ): 1.82–1.77 (1H, dt, J ¼ 15.36, J ¼ 3.48, H -5 ), 2.35 (6H, s,
3
b
0
40.38, 150.88, 163.09, 163.62. HRESIMS m/z: calculated for 2 ꢂ CH ) 3.09–3.05 (1H, dt, J ¼ 15.61, J ¼ 7.96, H -5 ), 5.15, 5.12
3
a
+
0
C H Cl N O [M + H] 367.0247, found 367.0241; calculated (CH , CH , 2d, J ¼ 13.71, CH Bn), 5.75–5.74 (1H, m, H-1 ), 5.80–
1
6
12
2
2
4
a
b
2
+
0
for C16
H
12Cl
2
N
2
O
4
[M + Na] 389.0066, found 389.0062.
5.78 (1H, m, H-5), 5.87–5.85 (1H, m, H-4 ), 6.00–5.98 (1H, dd, J ¼
0
0
00
1
CO
-[4-(Phenylacetoxy)-2-cyclopenten-1-yl]-3-benzyluracil (7). 2.25, J ¼ 5.54, H-3 ), 6.40–6.39 (1H, m, H-2 ), 7.20 (1H, s, H-4 ),
0
00 000
K
2
3
(18 mg, 0.13 mmol) and BnBr (17 mL, 0.13 mmol) were 7.30–7.27 and 7.49–7.47 (6H, m, H-6 and H-2 -6 ), 7.59 (2H, s,
0
0
00 13
3
added to solution of 2 (30 mg, 0.10 mmol) in dry DMF (10 mL). H-2 , H-6 ). C NMR (CDCl ): 21.45, 38.00, 44.71, 59.49, 77.51,
The reaction mixture was stirred for 2 h at room temperature, 102.59, 127.44, 127.82, 128.01, 128.58, 128.82, 129.30, 129.77,
the solvent evaporated, and the residue puried via column 134.40, 135.17, 136.72, 136.86, 136.98, 138.41, 138.50, 138.70,
chromatography eluting with CHCl to give 7 as a colorless oil 138.89, 151.76, 162.73, 166.28. HRESIMS m/z: calculated for
3
1
+
(
1
H
15 mg, 0.04 mmol, 35%). R ¼ 0.41 (CHCl ). H NMR (CDCl ): C H N O [M + H] 417.1809, found 417.1801; calculated for
f
3
0
3
25 24
2
4
+
.52–1.47 (1H, dt, J ¼ 15.47, J ¼ 3.15, H
b
-5 ), 2.87–2.83 (1H, m,
C
H
25 24
N
2
O
4
[M + Na] 439.1628, found 439.1626.
0
a
-5 ), 3.55 (2H, s, CH
2
), 5.05, 5.03 (CHa, CHb, 2d, J ¼ 13.72,
1-[4-(3,5-Dichlorobenzoyloxy)-2-cyclopenten-1-yl]-3-benzylur-
0 0
CH Bn), 5.58–5.56 (3H, m, H-1 , H-4 , H-5), 5.85–5.83 (1H, dd, acil (11). In an analogous manner, BnBr and 6 were reacted to
2
0
0
J ¼ 3.29, J ¼ 5.64, H-3 ), 6.17–6.15 (1H, m, H-2 ), 6.84–6.82 (1H, provide 11 as a white powder (45 mg, 0.10 mmol, 60%). R ¼ 0.53
f
0
0
00
00
000 000
ꢀ
1
m, H-6), 7.25–7.15 (8H, m, H-3 , H-4 , H-5 , H-2 -6 ), 7.41– (CHCl
3
: MeOH, 98 : 2). Mp 94.1–96.0 C. H NMR (CDCl
3
):
0
0
00 13
0
7
5
1
1
4
4
.39 (2H, m, H-2 , H-6 ). C NMR (CDCl ): 37.24, 41.61, 44.59, 1.72–1.68 (1H, dt, J ¼ 15.24, J ¼ 3.96, H -5 ), 3.09–3.01 (1H, dt,
3
b
0
9.17, 76.78, 102.57, 127.44, 127.73, 128.48, 128.82, 129.19, J ¼ 15.37, J ¼ 7.88, H -5 ), 5.08, 5.05 (CH , CH , 2d, J ¼ 13.70,
a
a
b
0
29.23, 133.71, 134.68, 135.96, 136.89, 138.33, 151.62, 162.58, CH Bn), 5.66–5.65 (1H, m, H-1 ), 5.68–5.67 (1H, m, H-5), 5.81–
2
+
+
0
0
70.76. HRESIMS m/z: calculated for C24
03.1652, found 403.1655; calculated for C24
H
22
N
N
2
O
4
[M + H]
[M + Na]
5.75 (1H, m, H-4 ), 5.98–5.96 (1H, dd, J ¼ 2.31, J ¼ 5.58, H-3 ),
0
H
22
2
O
4
6.31–6.28 (1H, m, H-2 ), 7.14–7.12 (1H, d, J ¼ 8.06, H-6), 7.23–
0
00 000
00
25.1472, found 425.1477.
7.19 and 7.42–7.41 (5H, m, H-2 -6 ), 7.49 (1H, s, H-4 ), 7.78–
00
00 13
1
-[4-(3-Phenylpropanoyloxy)-2-cyclopenten-1-yl]-3-benzylur- 7.77 (2H, m, H-2 , H-6 ). C NMR (CDCl ): 37.79, 44.63, 59.33,
3
acil (8). In an analogous manner, BnBr and 3 were reacted to 78.40, 102.76, 127.75, 128.07, 128.15, 128.33, 128.36, 128.42,
give 8 as a colorless oil (25 mg, 0.06 mmol, 60%). R ¼ 0.71 128.49, 129.24, 132.61, 133.24, 134.88, 135.60, 135.90, 136.83,
f
1
(
1
CHCl : MeOH, 98 : 2). H NMR (CDCl ): 1.50–1.46 (1H, dt, J ¼ 138.21, 151.63, 162.52, 163.62. HRESIMS m/z: calculated for
3
3
0
+
5.35, J ¼ 3.62, H -5 ), 2.64–2.60 (2H, m, CH ), 2.96–2.92 (3H, C H Cl N O [M + Na] 479.0536, found 479.0532.
b
2a
23 18
2 2 4
0
m, CH2b, H
a
-5 ), 5.17, 5.07 (CH
a
, CH
b
, 2d, J ¼ 13.71, CH
2
Bn),
1-[4-(2-Phenylacetoxy)-2-cyclopenten-1-yl]-3-(3,5-diuorobenzyl)
0
0
5
5
2
7
.66–5.60 (2H, m, H-1 , H-4 ), 5.75–5.73 (1H, d, J ¼ 8.02, H-5), uracil (12). In an analogous manner, 2 and 3,5-diuorobenzyl
0
.90–5.88 (1H, dd, J ¼ 3.79, J ¼ 5.53, H-3 ), 6.20–6.17 (1H, m, H- bromide were reacted to give 12 as a pale yellow oil (18 mg, 0.04
0
00
00
00
1
), 7.02–7.00 (1H, m, H-6), 7.17–7.16 (3H, m, H-3 , H-4 , H-5 ), mmol, 51%). R
f
¼ 0.49 (CHCl
3
: MeOH, 98 : 2). H NMR (CDCl
3
):
0
00 000
00
00 13
0
.30–7.29 (5H, m, H-2 -6 ), 7.49–7.47 (2H, m, H-2 , H-6 ).
C
1.59–1.55 (1H, dt, J ¼ 15.59, J ¼ 3.08, H
b
-5 ), 2.97–2.89 (1H, dt,
0
NMR (CDCl ): 31.07, 35.94, 37.53, 44.59, 59.25, 76.78, 102.53, J ¼ 15.58, J ¼ 7.84, H -5 ), 3.62 (2H, s, CH ), 5.05, 5.04 (CH , CH ,
3
a
2
a
b
0
0
1
1
26.50, 127.73, 128.35, 128.48, 128.60, 129.23, 134.12, 136.28, 2d, J ¼ 14.03, CH Bn), 5.65–5.63 (3H, m, H-1 , H-4 , H-5), 5.93–
2
0
0
36.88, 138.42, 140.18, 151.60, 162.60, 172.22. HRESIMS m/z: 5.91 (1H, dd, J ¼ 3.92, J ¼ 5.45, H-3 ), 6.25–6.24 (1H, m, H-2 ),
+
000
000
000
calculated for C25
H
H
24
N
2
O
O
4
[M + H] 417.1809, found 417.1819; 6.71–6.67 (1H, m, H-4 ), 6.94–6.92 (3H, m, H-6, H-2 , H-6 ),
+
00
00
00
00
00
13
calculated for C25
-[4-(3-Cyanobenzoyloxy)-2-cyclopenten-1-yl]-3-benzyluracil (CDCl
9). In an analogous manner, BnBr and 4 were reacted to give 9 25.31), 111.82 ( J
as a white solid (43 mg, 0.10 mmol, 79%). R
24
N
2
4
[M + Na] 439.1628, found 439.1650. 7.32–7.24 (5H, m, H-2 , H-3 , H-4 , H-5 , H-6 ). C NMR
C–F
1
3
): 37.21, 41.60, 43.86, 59.30, 77.30, 102.45, 103.23 ( J
¼
C–F
(
¼ 18.54), 127.44, 128.82, 129.18, 133.69,
C–F
f
¼ 0.69 134.49, 136.18, 138.70, 140.41, 151.48, 162.31, 163.01 (J
¼
ꢀ
1
C–F
19
(
1
CHCl : MeOH, 98 : 2). Mp 135.2–137.0 C. H NMR (CDCl ): 248.18, J
¼ 12.60), 170.72. F NMR (CDCl ): ꢁ110.60. HRE-
3
3
3
0
+
.79–1.74 (1H, dt, J ¼ 15.18, J ¼ 4.16, H -5 ), 3.19–3.14 (1H, dt, SIMS m/z: calculated for C H F N O [M + Na] 461.1283,
b
24 20 2 2 4
0
J ¼ 15.32, J ¼ 8.01, H -5 ), 5.17, 5.07 (CH , CH , 2d, J ¼ 13.70, found 461.1285.
a
a
b
0
CH
2
Bn), 5.74–5.73 (1H, m, H-1 ), 5.82–5.80 (1H, m, H-5), 5.90–
1-[4-(2-Phenylpropanoyloxy)-2-cyclopenten-1-yl]-3-(3,5-diuor-
0
0
5
6
7
7
.88 (1H, m, H-4 ), 6.05–6.03 (1H, dd, J ¼ 2.27, J ¼ 5.58, H-3 ), obenzyl)uracil (13). In an analogous manner, 3 and 3,5-
0
.38–6.37 (1H, m, H-2 ), 7.19–7.17 (1H, d, J ¼ 8.03, H-6), 7.31– diuorobenzyl bromide were reacted to afford 13 as a colorless
0
00 000
00
.28 and 7.49–7.47 (5H, m, H-2 -6 ), 7.60–7.57 (1H, m, H-5 ), oil (35 mg, 0.08 mmol, 98%). R
f
¼ 0.57 (CHCl
3
: MeOH, 98 : 2).
0
0
00
1
0
.86–7.83 (1H, m, H-6 ), 8.21–8.20 (1H, m, H-4 ), 8.28 (1H, s,
H NMR (CDCl
3
): 1.45–1.42 (1H, dt, J ¼ 15.35, J ¼ 3.62, H
b
-5 ),
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0
00
00
2
4
.58–2.53 (2H, m, CH2a), 2.90–2.85 (3H, m, CH2b, H
a
-5 ), 5.04, (1H, d, J ¼ 7.80, H-6), 8.13–8.11 (2H, d, J ¼ 8.10, H-2 , H-6 ), 11.37
0
13
.96 (CH , CH , 2d, J ¼ 14.03, CH Bn), 5.58–5.55 (2H, m, H-1 , (1H, s, NH). C NMR (DMSO-d ): 53.20, 104.83, 125.52, 132.14,
a
b
2
6
0
H-4 ), 5.70–5.68 (1H, d, J ¼ 8.05, H-5), 5.85–5.83 (1H, dd, J ¼ 132.22, 132.36, 133.15, 137.45, 138.00, 148.96, 153.45, 154.42,
0
0
+
+
3
.42, J ¼ 5.56, H-3 ), 6.15–6.14 (1H, m, H-2 ), 6.65–6.61 (1H, m, 167.06, 167.96. HRESIMS m/z: calculated for C H N O [M + H]
1
8
14
2
4
0
00
000
000
H-4 ), 6.92–6.89 (2H, m, H-2 , H-6 ), 7.00–6.98 (1H, d, J ¼ 323.1026, found 323.1025; calculated for C18
H
14
N
2
O
4
[M + Na]
00 00 00
8
.05, H-6), 7.11–7.09 (3H, m, H-3 , H-4 , H-5 ), 7.19–7.17 (2H, 345.0846, found m/z 345.0840.
0
0
00 13
m, H-2 , H-6 ). C NMR (CDCl
3
): 31.07, 35.94, 37.53, 43.87,
1-[2-(Benzoyloxy)benzyl]-3-benzyluracil (22). K
9.40, 76.82, 102.44, 103.24 ( J ¼ 25.29), 111.84 ( J ¼ 18.52), 5.60 mmol) was added to a solution of 1-[2-(benzoyloxy)benzyl]-
26.51, 128.36, 128.61, 133.92, 136.53, 138.80, 140.18, 140.42, uracil (19) (1.45 g, 4.50 mmol) in dry DMF (20 mL). The reaction
2 3
CO (0.78 g,
C–F
C–F
5
1
1
C–F
C–F
19
ꢀ
51.49, 162.34, 163.03 ( J
¼ 248.69, J
¼ 12.79), 172.21.
F
mixture was stirred for 1 h at 80 C, then BnCl (0.58 mL, 5.10
NMR (CDCl3): ꢁ110.57. HRESIMS m/z: calculated for mmol) was added and the reaction mixture stirred for 15 h at
+
ꢀ
C H F N O [M + H] 453.1620, found 453.1632; calculated for 80 C. The reaction mixture was ltered, the precipitate washed
2
5
22
2
2
4
+
C
25
H
22
F
2
N
2
O
4
[M + Na] 475.1440, found 475.1435.
with DMF and the ltrate evaporated. The resulting oily residue
ꢀ
1
-[2-(Benzoyloxy)benzyl]uracil (19). A solution of 2-benzoy- was triturated with hexane (30 mL) and kept at 4 C overnight.
loxybenzyl bromide (16) (2.62 g, 9.00 mmol) in 1,2-dichloroethane The resulting solid was ltered, air-dried and recrystallized
25 mL) was added to a solution of 2,4-bis(trimethylsilyloxy)- from a 1 : 1 mixture of EtOAc (15 mL) and hexane (15 mL) to give
pyrimidine (2.31 g, 9.00 mmol) in dry 1,2-dichloroethane (25 mL). 22 as colorless crystals (1.21 g, 2.93 mmol, 65%). R
¼ 0.69
The reaction mixture was reuxed for 35 h, cooled to room (EtOAc : hexane, 1 : 1). Mp 102.6–104.6 C. H NMR (DMSO-d ):
(
f
ꢀ
1
6
1
3
temperature, and the resulting precipitate ltered. The ltrate was 4.83 (2H, s, N –CH ), 4.99 (2H, s, N –CH ), 5.72–5.70 (1H, d, J ¼
2
2
0
0
0
0
000 000
then treated with 2-propanol and ltered again. The resulting 7.90, H-5), 7.47–7.19 (9H, m, H-3 , H-4 , H-5 , H-6 , H-2 -6 ),
0
0
00

ltrate was evaporated and the residue recrystallized from EtOH 7.63–7.59 (2H, t, J ¼ 7.80, H-3 , H-5 ), 7.68–7.66 (1H, d, J ¼ 7.90,
0
0
00
(
0
15 mL) to give 19 as yellow needles (2.12 g, 6.58 mmol, 73%). R
f
¼
H-6), 7.78–7.74 (1H, m, H-4 ), 8.15–8.13 (2H, d, J ¼ 7.30, H-2 ,
ꢀ
1
00 13
.71 (EtOAc). Mp 151.9–153.8 C. H NMR (DMSO-d ): 4.93 (2H, s, H-6 ). C NMR (CD CN): 43.42, 47.26, 100.58, 123.19, 126.43,
6
3
CH
2
0
0
), 5.63–5.62 (1H, dd, J ¼ 7.80 and 2.20, H-5), 7.26–7.23 (3H, m, 127.14, 127.45, 128.31, 128.51, 128.69, 128.96, 129.28, 129.40,
0
0
0
H-3 , H-5 , H-6 ), 7.48–7.45 (1H, m, H-4 ), 7.61–7.58 (2H, t, J ¼ 7.60, 129.95, 134.17, 136.99, 144.21, 148.65, 151.06, 162.29, 164.36.
0
00
00
+
H-3 , H-5 ), 7.75–7.72 (1H, td, J ¼ 7.60 and 0.80, H-4 ), 7.79–7.78 HRESIMS m/z: calculated for C H N O [M + H] 413.1496,
2
5
20 2 4
0
0
+
(
1H, d, J ¼ 7.80, H-6), 8.13–8.12 (2H, dd, J ¼ 7.60 and 0.90, H-2 , found 413.1479; calculated for C H N O [M + Na] 435.1315,
2
5
20 2 4
0
0
13
H-6 ), 11.38 (1H, s, NH). C NMR (DMSO-d ): 53.37, 104.91, found m/z 435.1295.
6
124.24, 124.69, 128.43, 132.20, 132.32, 133.17, 133.24, 137.43,
1-[3-(Benzoyloxy)benzyl]-3-benzyluracil (23). In an analogous
1
42.06, 148.93, 154.19, 154.42, 167.09, 167.91. HRESIMS m/z: manner as was used to synthesize 22, 1-[3-(benzoyloxy)benzyl]-
+
calculated for C18
calcd for C18
-[3-(Benzoyloxy)benzyl]uracil (20). In an analogous manner NMR (DMSO-d
as was used to synthesize 19, 3-(benzoyloxy)benzyl bromide (17) (1H, d, J ¼ 7.80, H-5), 7.28–7.18 (8H, m, H-2 , H-4 , H-6 , H-2 -
14 2 4
H N O [M + H] 323.1026, found 323.1022; uracil (20) afforded 23 as colorless crystals (1.11 g, 2.69 mmol,
+
ꢀ
1
H N O
14 2 4
[M + Na] 345.0846, found 345.0830.
60%). R
f
¼ 0.69 (EtOAc : hexane, 1 : 1). Mp 112.4–113.8 C. H
1 3
1
6
): 5.00 (4H, br.s, N –CH
2
, N –CH
2
), 5.84–5.82
0
0
0
000
00
0
00
0
and 2,4-bis(trimethylsilyloxy)pyrimidine were reacted to give 20 6 ), 7.49–7.45 (1H, m, H-5 ); 7.63–7.59 (2H, t, J ¼ 8.00, H-3 ,
0
0
00
as colorless needles (2.32 g, 7.20 mmol, 80%). R ¼ 0.62 (EtOAc). H-5 ), 7.77–7.73 (1H, m, H-4 ), 7.91–7.89 (1H, d, J ¼ 7.80, H-6),
f
ꢀ
1
00
00 13
Mp 159.8–161.5 C. H NMR (DMSO-d
6
): 4.91 (2H, s, CH
2
), 5.53– 8.15–8.13 (2H, d, J ¼ 7.30, H-2 , H-6 ). C NMR (CD
3
CN): 43.51,
0
0
5
.52 (1H, dd, J ¼ 7.90 and 2.20, H-5), 7.34–7.31 (3H, m, H-2 , H-4 , 51.28, 100.82, 120.82, 121.44, 125.13, 127.13, 127.49, 128.32,
0
0
00
00
H-6 ), 7.45–7.42 (1H, m, H-5 ), 7.63–7.58 (3H, m, H-6, H-3 , H-5 ), 128.82, 129.01, 129.83, 129.91, 134.14, 137.10, 138.54, 144.42,
0
0
00
00
7
1
1
1
.77–7.74 (1H, m, H-4 ), 8.16–8.15 (2H, d, J ¼ 7.60, H-2 , H-6 ), 150.85, 151.31, 162.39, 164.55. HRESIMS m/z: calculated for
1
3
+
1.30 (1H, s, NH). C NMR (DMSO-d
6
): 49.21, 104.68, 126.43,
C
25
H
20
N
2
O
4
[M + H] 413.1496, found 413.1503; calculated for
+
29.76, 132.05, 132.12, 132.31, 132.48, 132.55, 133.29, 137.47, C H N O [M + Na] 435.1315, found 435.1318
48.76, 151.92, 154.23, 167.00, 167.72. HRESIMS m/z: calculated
2
5
20 2 4
1-[4-(Benzoyloxy)benzyl]-3-benzyluracil (24). In an analogous
+
for C H N O [M + H] 323.1026, found 323.1025; calculated for manner as was used to synthesize 22, using 1-[4-(benzoyloxy)-
C
1
8
14 2 4
+
18
H
14
N O
2 4
[M + Na] 345.0846, found 345.0840.
benzyl]uracil (21) gave 24 as a white crystalline powder (1.08 g,
¼ 0.69 (EtOAc : hexane, 1 : 1). Mp 109.5–
1
-[4-(Benzoyloxy)benzyl]uracil (21). A solution of 4-benzoy- 2.62 mmol, 58%). R
f
ꢀ
1
1
loxybenzyl bromide (18) (2.62 g, 9.00 mmol) in 1,2-dichloroethane 111.5 C. H NMR (DMSO-d
6
): 4.99 (2H, s, N –CH
2
), 5.02 (2H, s,
3
(25 mL) was added to a solution of 2,4-bis(trimethylsilyloxy) N –CH
2
), 5.83–5.82 (1H, d, J ¼ 7.90, H-5), 7.31–7.23 (7H, m,
0
0
000 000
0
0
pyrimidine (2.31 g, 9.00 mmol) in dry 1,2-dichloroethane (25 mL). H-3 , H-5 , H-2 -6 ), 7.44–7.43 (2H, d, J ¼ 8.50, H-2 , H-6 ),
0
0
00
The reaction mixture was reuxed for 35 h, cooled to room 7.60–7.58 (2H, t, J ¼ 7.80, H-3 , H-5 ), 7.74–7.72 (1H, t, J ¼ 7.40,
0
0
00
temperature and treated with 2-propanol. The precipitate was H-4 ), 7.91–7.90 (1H, d, J ¼ 7.90, H-6), 8.14–8.12 (2H, m, H-2 ,
00
ltered and recrystallized from DMF (15 mL) to give 21 as a white H-6 ). C NMR (DMSO-d ): 46.91, 54.53, 104.11, 125.52,
6
13

powder (2.06 g, 6.39 mmol, 71%). R
f
¼ 0.73 (ethyl acetate). Mp 130.51, 130.94, 131.68, 132.24, 132.31, 133.16, 137.41, 137.76,
ꢀ
1
6
268.8–270.6 C. H NMR (DMSO-d ): 4.91 (2H, s, CH
2
), 5.63–5.61 140.51, 147.70, 153.56, 154.66, 165.72, 167.94. HRESIMS
0
+
(
1H, dd, J ¼ 7.80 and 2.20, H-5), 7.29–7.28 (2H, d, J ¼ 8.50, H-3 , m/z: calculated for C25
H
20
N
2
O
N
4
[M + H] 413.1496, found
0
0
0
+
H-5 ), 7.41–7.39 (2H, d, J ¼ 8.50, H-2 , H-6 ), 7.62–7.59 (2H, m, H- 413.1488; calculated for C25
H
20
2
O
4
[M + Na] 435.1315, found
0
0
00
00
3
, H-5 ), 7.76–7.73 (1H, td, J ¼ 7.50 and 1.10, H-4 ), 7.81–7.80 435.1307.
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-[3-(3,5-Dichlorobenzoyloxy)benzyl]uracil (25). In an analo- Biological evaluation
Concise Article
1
gous manner as was used to synthesize 19, 3-(3,5-dichlor-
obenzoyloxy)benzyl bromide and 2,4-bis(trimethylsilyloxy)-
pyrimidine gave 25 as colorless needles (2.61 g, 6.67 mmol,
Reverse transcriptase plasmids, RT expression and puri-
cation. Plasmid encoding p66 subunits of the wild-type HIV-1
reverse transcriptase (RT) or mutant forms (L100I, K103N,
V106A, Y181C, Y188L, G190A and K103N/Y181C) were described
ꢀ
1
7
4%). R
f
¼ 0.57 (EtOAc). Mp 221.9–223.6 C. H NMR (DMSO-
d ): 4.92 (2H, s, CH ), 5.62–5.60 (1H, dd, J ¼ 7.90 and 1.80, H-5),
5,32
6
2
previously. A similar plasmid encoding wild-type p51-subunit
0
0
0
7
5
.26–7.25 (3H, m, H-2 , H-4 , H-6 ), 7.48–7.45 (1H, t, J ¼ 8.30, H-
33
was a kind gi of Prof. S. LeGrice. RTs were expressed in
0
00
), 7.77–7.76 (1H, d, J ¼ 7.90, H-6), 7.98 (1H, s, H-4 ), 8.03 (2H,
Rosetta (DE3) E. coli strain as individual subunits, RT-harboring
0
0
00
13
6
s, H-2 , H-6 ), 11.35 (1H, s, NH). C NMR (DMSO-d ): 54.16,
cells were mixed together and the p66/p51 heterodimeric RTs
1
1
05.74, 124.75, 125.31, 129.55, 132.44, 134.10, 136.54, 137.46,
38.99, 142.96, 149.78, 154.71, 155.23, 166.50, 167.93. HRESIMS
33
were puried using the standard Ni-NTA agarose procedure.
RT enzyme assay. The RT assays using activated DNA (stan-
dard template + primer) were performed as follows: the stan-
dard reaction mixture (20 mL) contained 0.75 mg of activated
DNA, 0.05 mg p66/p66 RT, 3 mM dATP, 30 mM of dCTP, dGTP and
+
m/z: calculated for C18
91.0236; calculated for C18
found 413.0059.
-[3-(Benzoyloxy)benzyl]-5-bromouracil. In an analogous
manner, 3-(benzoyloxy)benzyl bromide (17) and 2,4-bis-
trimethylsilyloxy)-5-bromopyrimidine gave the target
product as a white powder (2.60 g, 6.48 mmol, 72%). R
¼ 0.75
): 4.94 (2H, s,
), 7.30–7.24 (3H, m, H-2 , H-4 , H-6 ), 7.49–7.46 (1H, t, J ¼
2 2 4
H12Cl N O [M + H] 391.0247, found
+
3
2 2 4
H12Cl N O [M + Na] 413.0066,
1
32
dTTP, 1 mCi [a- P]dATP in a Tris–HCl buffer (50 mM, pH 8.1)
containing also 10 mM MgCl , and 0.2 M KCl. The test
2
(
compounds were dissolved in DMSO and added to both assays
f
to the 10% nal DMSO concentration. The reaction mixtures
ꢀ
1
(
EtOAc). Mp 221.6–223.6 C. H NMR (DMSO-d
6
ꢀ
were incubated for 30 minutes at 37 C, and applied onto
0
0
0
CH
8
2
Whatman 3 MM lters. Aer drying on air the lters were
washed twice with 10% trichloroacetic acid, then twice with 5%
trichloroacetic acid, once with ethanol and dried on air.
Radioactivity was determined in an Intertechnique SL 30 300
liquid scintillation counter. Preliminary investigation of the
mechanism of action of compounds 1, 10 and 11 was performed
0
00
00
.30, H-5 ), 7.62–7.59 (2H, t, J ¼ 7.80, H-3 , H-5 ), 7.77–7.73
0
0
00
00
(
1H, t, J ¼ 7.40, H-4 ), 8.15–8.13 (2H, d, J ¼ 7.30, H-2 , H-6 ),
1
3
8
5
1
.39 (1H, s, H-6), 11.88 (1H, s, NH). C NMR (DMSO-d ):
6
0.49, 95.37, 120.99, 121.46, 125.20, 128.86, 128.99, 129.84,
34.10, 138.35, 145.19, 150.45, 150.81, 159.70, 164.56. HRE-
+
SIMS m/z: calculated for C18
found 401.0124; calculated for C18
H
13BrN
2
O
H
4
[M + H] 401.0131,
5
as described previously and the results indicate they are likely
+
2 4
13BrN O [M + Na]
acting in a non-competitive manner with regards to the dNTP
substrate.
4
22.9951, found 422.9942.
-[3-(Benzoyloxy)benzyl]thymine. In an analogous manner,
-(benzoyloxy)benzyl bromide (17) and 2,4-bis(trimethylsilyloxy)-
1
3
5
-methylpyrimidine gave the target product as colorless crystals Acknowledgements
ꢀ
(1.91 g, 5.68 mmol, 75%). R ¼ 0.84 (EtOAc). Mp 186.0–187.6 C.
f
1
This work was supported by the Russian Foundation For Basic
Research (Grants 12-04-31355 mol-a and 13-04-00742-a), by the
Presidium of the Russian Academy of Sciences (Programme
“Molecular and Cellular Biology”).
H NMR (DMSO-d
6
0
): 1.76 (3H, s, CH
3
), 4.89 (2H, s, CH
2
), 7.25–7.23
0
0
0
(
7
3H, m, H-2 , H-4 , H-6 ), 7.47–7.45 (1H, t, J ¼ 8.00, H-5 ), 7.60–
0
0
00
.58 (2H, t, J ¼ 7.80, H-3 , H-5 ), 7.65 (1H, s, H-6), 7.75–7.72 (1H,
0
0
00
00
t, J ¼ 7.40, H-4 ), 8.13–8.12 (2H, d, J ¼ 7.20, H-2 , H-6 ), 11.35
1
3
(
1
1
1H, s, NH). C NMR (DMSO-d
6
): 15.34, 53.15, 112.55, 124.22,
24.66, 128.44, 132.19, 132.32, 133.17, 133.21, 137.43, 142.26,
44.60, 154.16, 154.41, 167.65, 167.89. HRESIMS m/z: calculated
References and Notes
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for C H N O [M + H] 337.1183, found 337.1179; calculated for
C
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2
0
0
0
.28–7.23 (3H, m, H-2 , H-4 , H-6 ), 7.48–7.44 (1H, t, J ¼ 8.00,
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1
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