The design and synthesis of N-1-alkylated-5-aminoaryalkylsubstituted-6-methyluracils as potential non-nucleoside HIV-1 RT inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.07.058

Novel compounds 1a-u, which can be considered as hybrid analogues of MKC-442 and pyridinon, have been synthesized and evaluated as inhibitors of HIV-1 reverse transcriptase (HIV-1 RT). Starting from 6-methyuracil 2, 1-alkylated-5-bromomethyl-6-methyluracils 8 was prepared in four steps by hydroxylmethylation, etherification, N-1 alkylation, and bromination. Finally, compounds 1a-u were achieved in the displacement of 5-bromomethyl group by nucleophiles with amino compounds. Some of compounds 1a-u showed potent inhibitory activity against HIV-1 RT. The most active compounds showed activity in the low micromolecular range with IC₅₀ values (IC₅₀ 0.82-5.09 μM) comparable to that of nevirapine (IC₅₀ 10.60 μM). The biological testing results are in accordance with the docking.

Full text of DOI:10.1016/j.bmc.2007.07.058

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7399–7407
The design and synthesis of
N-1-alkylated-5-aminoaryalkylsubstituted-6-methyluracils
as potential non-nucleoside HIV-1 RT inhibitors
Xiao Lu,a Yanli Chen,a Ying Guo,a Zhenming Liu,b Yawei Shi,b Yang Xu,a
Xiaowei Wang,a Zhili Zhanga and Junyi Liua,b,
*
aDepartment of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China
bState key Laboratory of Natural and Biomimetic Drug, Peking University, Beijing 100083, China
Received 18 April 2007; revised 16 July 2007; accepted 18 July 2007
Available online 28 August 2007
Abstract—Novel compounds 1a–u, which can be considered as hybrid analogues of MKC-442 and pyridinon, have been synthesized
and evaluated as inhibitors of HIV-1 reverse transcriptase (HIV-1 RT). Starting from 6-methyuracil 2, 1-alkylated-5-bromomethyl-
6-methyluracils 8 was prepared in four steps by hydroxylmethylation, etherification, N-1 alkylation, and bromination. Finally,
compounds 1a–u were achieved in the displacement of 5-bromomethyl group by nucleophiles with amino compounds. Some of
compounds 1a–u showed potent inhibitory activity against HIV-1 RT. The most active compounds showed activity in the low
micromolecular range with IC50 values (IC50 0.82–5.09 lM) comparable to that of nevirapine (IC50 10.60 lM). The biological testing
results are in accordance with the docking.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Non-nucleoside reverse transcriptase inhibitors (NNR-
TIs) of HIV are a very broad class of structurally di-
verse molecules5,6 but they exhibit similarity in 3D
structures and show remarkable potential and selective
Following the discovery of human immunodeficiency
virus (HIV) as causative acquired immunodeficiency
syndrome (AIDS),1 enormous efforts have been made
to search the potential targets for antiviral chemother-
apy. HIV-1 reverse transcriptase (HIV-1 RT) has been
successful to date. Using as inhibitors of this enzyme,
the administration of Nevirapine, AZT, DDI, and
DDC are useful drugs against HIV.2 The inhibition
of RT is considered as one of the most valuable and
practicable approaches to suppress virus spreading.3,4
However, their long-term use leads to significant toxic-
ity and viral resistance. Therefore, the development of
very specific and highly active inhibitors of HIV-RT
was envisioned to be the potential solution for HIV-1
infection.
inhibition
profiles
such
as
1-[(2-hydroxy-eth-
oxy)methyl]-6-phenylthio thymine (HEPT) (i)7,8 and
the 2-pyridinon derivatives (L-697, 661) (ii)9 (Fig. 1).
Structure–activity studies in the HEPT series have
led to the synthesis of serial new promising analogues,
of which 6-benzyl-1-(ethoxymethyl)-5-isopropyluracil
(MKC-442) (iii),10,11 and TNK-651 (iv)12 (Fig. 1) have
been chosen as candidates for clinical trials with AIDS
patients. The reason for the increased activity is that
the 5-substituent in the uracil ring of MKC-442 in-
duces a Tyr181 switch in the HIV-RT.12 A major
problem with NNRTIs therapy is the rapid develop-
ment of resistance mediated by mutations of residues
that line the NNRTI binding pocket of the viral RT
and reduce drug binding, even though they are more
active against HIV-RT with general low toxicity and
favorable pharmacokinetic properties. Therefore, in
NNRTIS areas, interests are focused on finding new
analogues with higher binding affinity and the capabil-
ity of inhibiting clinically resistant mutants.13–16
Keywords: HIV-1 reverse transcriptase; Non-nucleoside reverse ran-
scriptase inhibitors (NNRTIs); HEPT analogues.
*
Corresponding author. Tel.: +86 010 8280 1706; fax: +86 010 6201
5584; e-mail addresses: Jyliu@bjmu.edu.cn; ard@bjmu.edu.cn
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.058

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X. Lu et al. / Bioorg. Med. Chem. 15 (2007) 7399–7407
O
O
Cl
O
O
HN
H
N
HN
HN
O
S
N
O
O
O
Cl
HO
i (HEPT)
ii (L-697,661)
O
iii (MKC-442)
O
O
NH Cl
HN
N
HN
HN
O
O
N
O
N
O
O
O
Cl
Cl
1n
1a
iv (TNK651)
Figure 1. Structures of HEPT, L-697,661, MKC-442, TNK-651, and designed target compounds 1a, 1n.
2. Results and discussions
2.1. Design of target molecule
Therefore, we decided to select N-1-alkylated, 5-bulky
aryalkyl moiety and 6-methyl group uracils 1a, 1n which
can also be considered as HEPT-pyridinone analogue
hydrid molecules with two possible orientations in RT
as new potential NNRTIs.
Studies of crystal structures of the RT complex with
inhibitors TNK-651, MKC-442 suggest that NNRTIs
share a common mode of action and interact with a
hydrophobic pocket. Upon binding, MKC-442 and
TNK-651 distort the RT active site and alter the confor-
mation of Tyr 181, Tyr 183, and Tyr 188 residues with
respect to that of unliganded RT. Thus the aromatic
group of Tyr 181 is faced into a conformation able to
perform a strong interaction with the C-6 benzyl group
of the inhibitors, stabilizing the structure of the com-
plex. This revealed that the C-5 substituent of the pyrim-
idine ring is very important for anti-HIV-1 RT activity.
To verify our design, these compounds (1a, 1n) were
flexibly docked into the binding site of HIV-1 RT
(PDB:1RT2, complexed with TNK-651) using Auto-
Dock 3.05 program.17 Default parameters were used
as described in the AutoDock manual unless otherwise
specified. Each molecule was docked with 100 genetic
algorithm runs of up to 250,000 energy evaluations for
each run in the docking study of 1a and 1n with a RT
non-nucleoside binding site. It was predicted that the
binding modes for 1a, 1n have approximate conforma-
tions as in the TNK-651-RT complex. (Fig. 2) The
docking results showed that the 1a, 1n bind to the
RT with an conformation forming hydrogen bonds
with the Lys101 and Lys103. Meanwhile the 5-substi-
tuted phenyl ring of 1a, 1n occupies a hydrophobic
pocket constructed by the side chains of Try 181,
Try 188, and Trp 229, making favorable p–p interac-
tions with these residues. As seen in Figure 2, the
bulky substituted aryalkyl moiety with two-atom lin-
ker on the side chain of C-5 is positioned in a favored
location between isopropyl group at C-5 and benzyl
group at C-6 in TNK-651, thus it can exert the func-
tion of both of them, which seems to be crucial for
the inhibition of the RT. Based on results of the dock-
ing study, we hypothesized that an efficient use of C-5
substitute by strategically designed functional groups
should yield more potent anti-HIV agents with higher
affinity for the inhibitor binding pocket.
Although structure–activity relationship on the anti-HIV
of HEPT analogues has been studied, no or little informa-
tion is available about 5-bulky substituents and 6-small
alkyl substituents. In the year 2006, Chen et al. have syn-
thesized the 1-(alkoxymethyl)-5-benzyl-6-methyluracils
whose activity showed to be ca. 2-fold higher than HEPT
but 10-fold lower than nevirapine. After inspection of the
above-mentioned work, we think that the key point to de-
velop such compounds is the optimal linking distance be-
tween the pyrimidine ring and the aromatic ring placed at
C-5. This distance is an important prerequisite for po-
tency. It was pointed out in an earlier study that a major
determinant of increased potency in the analogues of
HEPT is an improved interaction between residue
Tyr181 in the protein and the 6-benzyl ring of the inhibi-
tors which stabilizes the structure of the complex. This
arises through a confirmational switching of the protein
structure triggered by the steric bulk of the 5-substituent
of the inhibitor pyrimidine ring.12 Thus we think the aro-
matic ring at C-5 should possess the conformational sim-
ilarity of C-6 benzyl group of MKC-442 or TNK-651: the
chain between pyrimidine ring and aromatic ring at C-5
should possess conformational flexibility, and property
linking length which should be in 2–3 carbon (nitrogen)
units. From this it also can preserve 5-position group’s
‘trigger’ role.
2.2. Chemistry
The use of the 5-bromomethyl-6-methyl 4 as intermedi-
ate gave easy access to 5-amino substitutes 5 by the use
of 6-methyluracil as starting materials with a modified
method of Carbon,18 and when tested this strategy on
a 6-methyluracil we obtained the corresponding 5-amino
substitutes 5 in 50–60% yield (Scheme 1). However, after

X. Lu et al. / Bioorg. Med. Chem. 15 (2007) 7399–7407
7401
O
O
O
OBn
HN
OBn
CH3
HN
OH
CH3
a
b
HN
O
N
7
CH3
O
N
H
O
N
O
H
R
6
3
O
O
R'
Br
HN
c
HN
d
O
N
CH3
O
N
CH3
O
O
R
R
1 a-u
8
b.
a. BnOH, conc.HCl, reflux
c. (33%)HBr-HOAc, rt
R
O
Cl, BSA , CHCl3 , rt
d. R'NH-, dry 1,4-dioxane , rt
Scheme 2. Reagents and conditions.
Therefore it was decided to introduce the N1-substituent
into the pyrimidine before making 5-bromomethyl. The
inspiration came from Chen19 who made a 5-(benzyl-
oxymethyl)-6-methyluracil starting from 6-methyluracil
in 64% yield. The advantage of this method is easy prep-
aration of 1-alkylated-5-bromomethyl-6-methyluracil 8
starting from the reaction of 5-hydroxymethyl-6-methyl-
uracil 3 (Scheme 2).
5-(Benzyloxymethyl)-6-methyluracil
6
prepared by
benzylation of 3 according to a literature procedure19
was used for the synthesis of the 1-alkylated uracil 7.
Therefore 6 was reacted with N,O-bis(trimethylsi-
ly)acetamide (BSA) in dry CHCl3 and subsequently
alkylated with chloromethyl ethyl ether or chloro-
methyl benzyl ether20 to obtain the desired 1-(alkyl-
oxymethyl)-5-(benzyloxymethyl)-6-methyluracil 7a in
92.5% yield after column chromatography purification.
The C-1 benzyloxymethyl derivative 7b was synthe-
sized using the same method.
Figure 2. Modeled binding mode of 1a (a, yellow) and 1n (b, green)
overlapped to crystal structure (1RT2 in PDB) of TNK651 (pink). The
two inhibitors are docked into the active site of the HIV-1 RT
structure 1RT2 using AutoDock 3.0517 as rendered in Pymol (Delano
Scientific, South San Francisco, CA). The docking modes are chosen
on the basis of binding affinity rank. All inhibitors and important
interacting residues are shown as stick figures. Hydrogen bonds are
shown as black dashed lines.
For compound 7 we observed exactly the same NMR
chemical shift for the N-1-CH2 as was previously ob-
served for similar compounds.19,20 The conversion of
7a to 1-(alkyloxymethyl)-5-(bromomethyl)-6-methylura-
cil 8a was performed by the reaction with a 33% solution
of HBr in glacial acetic acid in 87.8% yield, which not
afforded the cleavage of N-1 ether bond. Finally com-
pound 8 was treated with various amines in dry 1,4-
dioxane to give compounds 1a–u in 9–87% yields
(Scheme 2 and Table 1).
O
O
O
R'
Br
OH
d
b
HN
HN
HN
O
N
H
CH3
O
N
H
CH3
O
N
H
CH3
5
4
3
c
c
a
O
O
O
N
R'
Br
HN
HN
HN
O
N
CH3
O
N
H
CH3
O
R
CH3
O
O
2
To sum up, a simple and efficient procedure for the
synthesis of a family of 1-alkyl-5-aminoaryalkyl-6-meth-
yluracil 1 has been developed. These new HEPT-pyridi-
none analogue hybrid molecules were obtained in an
easy, rapid and profitable way using solution-phase
synthesis. The whole methodology shows combinatorial
potential in the synthesis of larger libraries of
compounds which is ongoing in our laboratory.
R
b. (33%)HBr-HOAc, rt
d. R'NH-, 1,4-dioxane , rt
a. HCHO, Ba(OH)2, reflux
c. R
O
Cl
, BSA , CHCl3 , rt
Scheme 1. A strategy of synthesis 1-(alkoxymethyl)-5-(aryalkylami-
no)-6-methyluracil.
several attempts, it was not possible to obtain the de-
sired target compounds by reaction 4 or 5 with N,O-
bis-(trimethylsily)acetamide (BSA) in CHCl3 at room
temperature followed by reaction with chloromethyl
ethyl ether (Scheme 1).
2.3. Biological activity
The compounds 1a–u were tested for antiviral activity
against HIV-1 RT, using
a
poly(ra)/oligo(dT)15

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X. Lu et al. / Bioorg. Med. Chem. 15 (2007) 7399–7407
Table 1. Preparation of 1 from 8 and RT inhibitory activity valuesa
b
Compound
R
R0
Time (h)
Yield (%)
59
IC50 (lM)
Cl
H
N
Cl
1a
CH3
2
0.82
NAc
Cl
1b
1c
1d
1e
CH3
CH3
CH3
CH3
4
4
5
5
55
44
86
80
NA
>100
NA
H
N
1f
CH3
CH3
CH3
Over night
9
39
26
NA
NA
26.2
1g
1h
4
1.5
1i
CH3
3
32
NA
Cl
H
N
Cl
1j
CH3
CH3
CH3
CH3
Ph
4
4
1
4
9
40
35
20
38
46
NA
54.7
NA
NA
3.49
Cl
H
N
1k
1l
N
N
H
N
N
NH
1m
1n

X. Lu et al. / Bioorg. Med. Chem. 15 (2007) 7399–7407
7403
Table 1 (continued)
b
Compound
R
R0
Time (h)
Yield (%)
37
IC50 (lM)
1o
1p
1q
1r
1s
1t
Ph
9
13.8
Ph
Ph
Ph
Ph
Ph
Ph
9
29
29
16
45
54
28
NA
NA
NA
>100
5.09
NA
10
10
3
Cl
H
N
Cl
Cl
H
N
9
Cl
H
N
Cl
1u
3
Cl
a Nevirapine was used as a reference compound here; IC50 for nevirapine was 10.60 lM.
b Compound dose (lM) required to inhibit the HIV-1 rRT activity by 50%; Data represent mean values for three separate experiments, variation
among triplicate samples was less than 15%.
c No inhibition of reverse transcriptase activity was observed up to a concentration of 100 lM.
homopolymer template with HIV antigen detection
ELISA21 for quantifying expression of HIV-1 RT in cul-
ture medium, and nevirapine as a reference compound.
Oligo(dT) was immobilized via its 50-terminal phosphate
to Covalink-NH microtiter plates. The biotin-dUTP was
incorporated by reverse transcriptase. The reaction mix-
ture contained 50 mmol/L Tris–HCl (pH 8.3), 3 mmol/L
MgCl2, 75 mmol/L KCl2, 5 mmol/L DTT (DL-dithio-
threitol), 0.13 mg/ml BSA (Albumin Bovine V), 10 lg/ml
poly (A), 0.75 lM biotin-11-dUTP, and 1.5 lM dTTP.
After incubation at 37 °C for 1 h, the plate was washed
three times with a wash buffer containing 50 mmol/L
Tris–HCl (pH7.5), 0.15 mol/L NaCl, 0.05 mmol/L
MgCl2, and 0.02% Tween-20. After 100 ll of 1% BSA
was added to each well and incubated for 30 min at
room temperature, the plate was washed with the
same buffer. Subsequently 50 ll of SA-ALP (Alkaline
Phosphatase Streptavidin) solution (100 ng/ml) was
added per well and then incubated for 1 h at 37 °C.
The plate was washed as above and to which then was
added 50 ll of PNPP (p-nitrophenyl phosphate,
disodium) (1 mg/ml, pH 9.5), after 30 min at 37 °C, the
reaction was stopped by addition of 0.5 M NaOH.
The products were detected and quantified using a
colorimetric streptavidin alkaline phosphatase reporter
system. It is interesting to note that some of the reported
compounds showed high inhibitory activity against
HIV-1 RT. It is noteworthy that compounds 1a (IC50
0.82 lM), 1n (IC50 3.49 lM), and 1t (IC50 5.09 lM) were
more active than nevirapine (IC50 10.60 lM) at concen-
trations 2- to 13-fold compared to nevirapine by
introducing the terminal 2,4,5-trichloro-anilino, benze-
nmethanamine, and cyclohexylamino groups at pyrimi-
dine C-5. However, an increase of the other steric
bulkynesses at C-5 showed lower activity compared to
nevirapine.
2.4. Conclusions
We have developed novel HIV-1 RT non-nucleoside
inhibitors characterized by micromolar potency. Among
our study compounds (1a–u), the higher potency of 1a,
1n, and 1t, compared with nevirapine and other synthe-
sizing target molecules (TMs), might depend on switch-
ing the conformation of Tyr 181, Tyr 183, and Tyr 188
residues with respect to that of unliganded RT after
binding. Additional reasons for the relatively high activ-
ity of these novel inhibitors might be the existing
5-substituted phenyl ring which is located at suitable
distance from the pyrimidine ring. In designing the

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X. Lu et al. / Bioorg. Med. Chem. 15 (2007) 7399–7407
new TMs, it was also hoped that the favorable effects of
the hydrogen bonds attributed to the virtual RT/inhibi-
tor complexes. Encouraged by these results, more
detailed SAR studies on these compounds (5-bulky
substituents and 6-small alkyl substituents) are under-
way, with a focus on exploring the important role of
these unique structure features.
3.3. 1-(Ethoxymethyl)-5-(benzyloxymethyl)-6-methyl-
uracil (7a)
Compound 6 (0.35 g, 1.4 mmol) was suspended in dry
CHCl3 (15 ml), and N,O-bis(trimethylsily)acetamide
(BSA) (0.8 ml, 3.2 mmol) was added to the suspension.
Introduced (0.198 ml, 1.4 mmol) (chloromethyl)-ethyle-
ther after the entire solid completely dissolved. The reac-
tion mixture was allowed to stir for 4 h until no change
in the amount of the starting material could be noticed
on TLC. After evaporation of the solvent in vacuum,
the resulting residue was purified by silica gel column
chromatography using CHCl3-MeOH (9:1) to give 7a
3. Experimental
In general, reagents and solvents were used as purchased
without further purification. Melting points were deter-
mined with an X4-type melting-point apparatus and are
left uncorrected. 1H and 13C NMR spectra were re-
corded on a JNM-AL-300 or a VarianINOVA-500 spec-
trometer. Chemical shifts were expressed in d (ppm)
values with tetramethylsilane as an internal standard
(in NMR description, s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; br, broad peak). Mass spectra
were recorded on a VG-ZAB-HS spectrometer. IR Spec-
tra were recorded with Avatar 360 FT-IR and reported
in cmꢀ1. Silica gel (0.040–0.064 mm) was used for col-
umn chromatography.
1
(0.40 g, 31% yield) as white solid. Mp 104–105 °C. H
NMR(300 MHz, CDCl3): d 8.80 (s, 1H, NH), 7.36–
7.26 (m, 5H, ArH), 5.34 (s, 2H, CH2), 4.56 (s, 2H,
CH2), 4.42 (s, 2H, CH2), 3.62 (q, J = 7.2 Hz, 2H,
CH2), 2.42 (s, 3H, CH3), 1.20 (t, 3H, J = 6.9 Hz, CH3).
m/z (MS) found 305.1067 ([MH]+ C16H21N2O4 requires
305.1501), found 327. 0861 ([MNa]+C16H20N2Na O4 re-
quires 327.1321).
3.4. 1-(Benzyloxymethyl)-5-(benzyloxymethyl)-6-methyl-
uracil (7b)
3.1. 5-Hydroxymethyl-6-methyluracil (3)
Compound 6 (2.17 g, 8.8 mmol) was converted to 7b
(0.613 g, 20.6% yield) according to the similar proce-
dure used in the preparation of compound 7a. Mp
143–145 °C. 1H NMR (300 MHz, CDCl3): d 8.29 (s,
1H, NH), 7.35–7.28 (m, 10H, ArH), 5.42 (s, 2H,
CH2), 4.64 (s, 2H, CH2), 4.54 (s, 2H, CH2), 4.40 (s,
2H, CH2), 2.43 (s, 3H, CH3). m/z (MS) found
367.1658 ([MH]+ C16H21N2O4 requires 367.1607),
found 389. 1477 ([MNa]+C16H20N2NaO4 requires
389.1378).
Introduced formaldehyde (37% aq solution) (4.14 ml,
51.0 mmol) and 6-methyluracil (2.0 g, 15.8 mmol) to a
filtered solution of Ba(OH)2Æ8H2O (1.5 g, 36.0 mmol)
in water (20 ml), heated the mixture to gentle refluxing
until the solid completely dissolved. Left to stand for
1 day, CO2 (gas) was bubbled into the reaction mixture
to precipitate BaCO3. Removed the water on a rotary
evaporator, and the viscous residue was dissolved at re-
flux in EtOH (20 ml). Kept the mixture overnight at
room temperature, collected 3 as a pure white solid by
3.5. 1-(Ethoxymethyl)-5-(bromomethyl)-6-methyluracil (8a)
1
filtration. The yield was 80% (1.97 g), mp >300 °C. H
NMR (300 MHz, DMSO): d 10.94 (s, 1H, NH), 10.74
(s, 1H, NH), 4.52 (s, 1H, OH), 4.14 (s, 2H, CH2), 2.12
(s, 3H, CH3). 13C NMR (300 MHz, DMSO): d 164.2
(C4), 151.4 (C2), 150.9 (C6), 109.2 (C5), 62.8 (CH2),
15.8 (CH3). MS (EI) m/z: 156 (M, 70), 155 (38), 138
(100), 127 (39). IR (KBr) cmꢀ1 3414, 2933, 1704, 1665,
1447.
Dry 1,4-dioxane (0.35 ml) and 33% HBr–AcOH (130 ll,
0.34 mmol) were added to 1,3-dibenzyl-5-benzyloxy-6-
methyluracil 7a (0.1 g, 0.33 mmol), resulting in the for-
mation of a clear solution. After the reaction mixture
was stirred for 4 h at room temperature, the solvent
was removed by vacuum to afford an oily residue that
crystallizes after the addition of a few drops of ether.
It was filtered and washed with ether (10 ml). The result-
ing product (80 mg, 80% yield) was analytically pure:
mp 182 °C. 1H NMR (300 MHz, CDCl3): d 8.30 (s,
1H, NH), 5.36 (s, 2H, CH2), 4.42 (s, 2H, CH2), 3.64
(q, J = 7.2 Hz, 2H, CH2), 2.51 (s, 3H, CH3), 1.22 (t,
J = 10.2 Hz, 3H, CH3). MS (EI) m/z: 277 (M, 1), 197
(95), 59 (100).
3.2. 5-Benzyloxymethyl-6-methyluracil (6)
A mixture of 5-hydroxymethyl-6-methyluracil 3 (2.0 g,
12.8 mmol) and aqueous HCl (1 ml) in benzyl alcohol
(50 ml) was refluxed for 1 h. After being cooled to room
temperature, the reaction mixture was poured into ether
(250 ml). The resulting fine precipitate was filtered and
washed several times with ether to give the pure product
3.6. 1-(Benzyloxymethyl)-5-(bromomethyl)-6-methyl-
uracil (8b)
1
6. The yield was 80% (2.51 g). Mp 300 °C. H NMR
(300 MHz, DMSO-d6): d 11.04 (s, 1H, NH), 10.90 (s,
1H, NH), 7.31 (s, 5H, ArH), 4.49 (s, 2H, CH2), 4.22
(s, 2H, CH2), 2.10 (s, 3H, CH3). 13C NMR (75 MHz,
DMSO-d6): d 164.1 (C4), 153.0 (C2), 150.8 (C6), 138.6,
128.2, 127.6, 127.4 (C10–C60), 105.9 (C5), 71.2 (CH2),
61.8 (CH2), 15.9 (CH3). MS (EI) m/z: 247 (M+H, 5),
155 (100), 140 (100). IR (KBr) cmꢀ1 3433, 1721, 1643,
1447, 1071.
Compound 7b (350 mg, 0.96 mmol) was converted to 8b
(150 mg, 46% yield) according to the similar procedure
used in the preparation of compound 8a. Mp 154–
155 °C. 1H NMR (300 MHz, CDCl3): d 9.21 (s, 1H,
NH), 7.26–7.39 (m, 5H, ArH), 5.47 (s, 2H, CH2), 4.67
(d, J = 6.6 Hz, 2H, CH2), 4.39 (s, 2H, CH2), 2.48 (s,
3H, CH3). MS (ES)m/z: 259 (MꢀBr, 85).

X. Lu et al. / Bioorg. Med. Chem. 15 (2007) 7399–7407
7405
3.7. General procedure for the synthesis of 1a–1m
203 °C. 1H NMR (300 MHz, CDCl3): d 5.33 (s, 2H,
CH2), 3.86 (s, 2H, CH2), 3.61 (q, J = 7.2 Hz, 2H,
CH2), 3.65 (s, 3H, CH3), 3.04 (s, 1H, CH), 2.44–2.05
(m, 10H, CH2), 1.18 (t, J = 7.2 Hz, 3H, CH3). MS
(ES) m/z: 296 (M+H, 100), 197 (16).
Mixed 1-(ethoxymethyl)-5-(bromomethyl)-6-methylura-
cil 8a (80 mg, 0.29 mmol) and dry 1,4-dioxane (2 ml),
followed by different amino compounds (1.93 mmol).
The reaction mixture was allowed to stir for several
hours and then was evaporated in vacuum. The resulting
residue was purified by silica gel column chromatogra-
phy using chloroform and methanol (10:1), as the eluent
to give the target compound (1a–1m’s reaction time is
illustrated in Table 1).
3.7.7. 1-(Ethoxymethyl)-5-[(methyl-phenylamino)-meth-
yl]-6-methyluracil (1g). Yield: 39%. Yellowish brown so-
lid. Mp 161–163 °C. 1H NMR (300 MHz, CDCl3): d
7.29–7.24 (m, 2H, ArH), 6.97 (m, 2H, ArH), 6.82 (m,
1H, ArH), 5.35 (s, 2H, CH2), 4.22 (s, 1H, CH2), 3.62
(q, J = 7.2 Hz, 2H, CH2), 2.80 (s, 3H, CH3), 2.38 (s,
3H, CH3), 1.20 (t, J = 6.9 Hz, 3H, CH3). 13C NMR
(75 MHz, CDCl3): d 163.3 (C4), 153.5 (C2), 151.6
(C10), 150.5 (C1), 129.1, 118.3, 114.8 (C20–C60), 109.7
(C5), 73.0 (CH2), 65.0 (CH2), 46.7 (CH2), 37.4 (CH3),
15.5 (CH3), 15.0 (CH3). MS (ES) m/z: 304 (M+H,
100), 197 (30).
3.7.1. 1-(Ethoxymethyl)-5-[(2,4,5-trichloro-phenylamino)-
methyl]-6-methyluracil (1a). Yield: 59%. White solid. Mp
1
>300 °C. H NMR (300 MHz, CDCl3): d 7.29 (s, 1H,
ArH), 6.83 (s, 1H, ArH), 5.37 (s, 2H, CH2), 4.49 (s,
1H), 4.12 (d, J = 4.5 Hz, 2H, CH2), 3.65 (q,
J = 7.2 Hz, 2H, CH2), 2.25 (s, 3H, CH3), 1.21 (t,
J = 7.2 Hz, 3H, CH3). MS (ES) m/z: 392 (M, 35), 197
(100).
3.7.8. 1-(Ethoxymethyl)-5-[(2,5-dimethyl-phenylamino)-
methyl]-6-methyluracil (1h). Yield: 26%. Deep orange
solid. Mp 67–69 °C. 1H NMR (300 MHz, CDCl3): d
7.18–6.54 (m, 3H, ArH), 5.35 (s, 2H, CH2), 4.14 (s,
2H, CH2), 3.63 (q, J = 7.2 Hz, 2H, CH2), 2.33 (s, 6H,
CH3), 2.25 (s, 3H, CH3), 1.19 (t, J = 3.0 Hz, 3H, CH3).
MS (ES) m/z: 318 (M+H, 100), 197 (22).
3.7.2. 1-(Ethoxymethyl)-5-(p-tolylamino-methyl)-6-meth-
yluracil (1b). Yield: 55%. Purple solid. Mp 44–45 °C. 1H
NMR (300 MHz, CDCl3): d 8.63 (s, 1H, NH), 6.99 (d,
J = 8.1 Hz, 1H, ArH), 6.68 (d, J = 8.4 Hz, 2H, ArH),
5.33 (s, 2H, CH2), 4.10 (s, 2H,CH2), 3.61 (q,
J = 7.2 Hz, 2H, CH2), 2.48 (s, 3H, CH3), 2.25 (s, 3H,
CH3), 1.20 (t, J = 7.2 Hz, 3H, CH3). MS (ES) m/z: 304
(M+H, 100), 197 (20).
3.7.9. 1-(Ethoxymethyl)-5-[(2,6-dimethyl-phenylamino)-
methyl]-6-methyluracil (1i). Yield: 32%. Light brown
1
solid. Mp 168–170 °C. H NMR (300 MHz, CDCl3): d
3.7.3. 1-(Ethoxymethyl)-5-[(4-nitro-phenylamino)-meth-
yl]-6-methyluracil (1c). Yield: 44%. Yellow solid. Mp
6.99 (d, J = 7.5 Hz, 2H, ArH), 6.88 (m, 1H, ArH),
5.32 (s, 2H, CH2), 3.89 (s, 2H, CH2), 3.58 (q,
J = 7.2 Hz, 2H, CH2), 2.33 (s, 6H, CH3), 2.25 (s, 3H,
CH3), 1.18 (t, J = 7.2 Hz, 3H, CH3).13C NMR
(75 MHz, CDCl3): d 163.6 (C4), 151.6 (C2), 144.9
(C6), 131.1, 128.7, 123.0, 111.4 (C10–C60), 73.1 (CH2),
64.8 (CH2), 43.4 (CH2), 18.3 (CH3), 15.0 (CH3), 14.9
(CH3). MS (ES) m/z: 318 (M+H, 100), 197 (45).
1
147–148 °C. H NMR (300 MHz, DMSO-d6): d 11.53
(s, 1H, NH), 8.00 (d, J = 9.0 Hz, 2H, ArH), 6.73 (d,
J = 9.0 Hz, 2H, ArH), 5.28 (s, 2H, CH2), 4.06 (s, 2H,
CH2), 3.52 (q, J = 7.2 Hz, 2H, CH2), 2.37 (s, 3H,
CH3), 1.12 (t, J = 7.2 Hz, 3H, CH3). 13C NMR
(75 MHz, DMSO-d6): d 162.7 (C4), 154.3 (C2), 153.3
(C6), 151.2, 135.8, 126.2, 110.9 (C10–C60), 108.0 (C5),
72.3 (CH2), 63.8 (CH2), 37.39 (CH2), 15.3 (CH3), 14.9
(CH3). MS (ES) m/z: 335 (M+H, 25), 197 (50).
3.7.10. 1-(Ethoxymethyl)-5-[(2,4,6-trichloro-phenylami-
no)-methyl]-6-methyluracil (1j). Yield: 40%. Brown solid.
Mp 145–147 °C. 1H NMR (300 MHz, CDCl3): d 7.26 (s,
2H, ArH), 5.30 (s, 2H, CH2), 4.45 (s, 1H), 4.25 (s, 2H,
CH2), 3.58 (q, J = 7.2 Hz, 2H, CH2), 2.28 (s, 3H,
CH3), 1.17 (t, J = 7.2 Hz, 3H, CH3). 13C NMR
(75 MHz, CDCl3): d 163.4 (C4), 151.5 (C2), 151.4
(C6), 140.9, 128.8, 128.5, 127.3 (C10–C60), 110.8 (C5),
73.0 (CH2), 64.8 (CH2), 42.4 (CH2), 15.1 (CH3), 15.0
(CH3). MS (ES) m/z: 394 (M+H, 20), 197 (100).
3.7.4. 1-(Ethoxymethyl)-5-[(2-nitro-phenylamino)-meth-
yl]-6-methyluracil (1d). Yield: 86%. Orange solid. Mp
1
138–140 °C. H NMR (300 MHz, CDCl3): d 8.17 (m,
1H, ArH), 7.48 (m, 1H, ArH), 7.03 (d, J = 8.7 Hz, 1H,
ArH), 6.70 (t, J = 7.8 Hz, 1H, ArH), 5.37 (s, 2H,
CH2), 4.33 (d, J = 5.1 Hz, 2H, CH2), 3.65 (q,
J = 7.2 Hz, 2H, CH2), 2.48 (s, 3H, CH3), 1.21 (t,
J = 7.2 Hz, 3H, CH3). MS (ES) m/z: 335 (M+H, 30),
197 (60).
3.7.11. 1-(Ethoxymethyl)-5-(pyridin-2-ylaminomethyl)-6-
methyluracil (1k). Yield: 35%. White solid. Mp 161–
1
3.7.5. 1-(Ethoxymethyl)-5-[(3-nitro-phenylamino)-methyl
]-6-methyluracil (1e). Yield: 80%. Yellow solid. Mp 169–
163 °C. H NMR (300 MHz, CDCl3): d 8.06 (m, 1H,
60H), 7.42 (m, 1H, 40H), 6.65 (m, 1H, 30H), 6.53 (m,
1H, 50H), 5.34 (s, 2H, CH2), 4.39 (d, J = 6.0 Hz, 2H,
CH2), 3.62 (q, J = 7.2 Hz, 2H, CH2), 2.62 (s, 3H,
CH3), 1.19 (t, J = 7.2 Hz, 3H, CH3). MS (ES) m/z: 314
(M+Na, 50), 292 (M+H, 100), 197 (35).
1
171 °C. H NMR (300 MHz, CDCl3): d 7.55 (m, 1H,
ArH), 7.44 (m, 1H, ArH), 7.27 (m, 1H, ArH), 6.92 (m,
1H, ArH), 5.36 (s, 2H, CH2), 4.45 (s, 1H), 4.17 (d,
J = 5.1 Hz, 2H, CH2), 3.58 (q, J = 7.2 Hz, 2H, CH2),
2.54 (s, 3H, CH3), 1.21 (t, J = 7.2 Hz, 3H, CH3). MS
(ES) m/z: 335 (M+H, 70), 197 (90).
3.7.12. 1-(Ethoxymethyl)-5-(pyrimidin-2-ylaminomethyl)-
6-methyluracil (1l). Yield: 20%. Light yellow solid. Mp
1
3.7.6. 1-(Ethoxymethyl)-5-(cyclohexylaminomethyl)-
6-methyluracil (1f). Yield: 9%. White solid. Mp 201–
>300 °C. H NMR (300 MHz, CDCl3): d 11.42 (s, 1H,
NH), 8.31 (m, 2H, 40 and 60H), 6.51 (m, 1H, 50H),

7406
X. Lu et al. / Bioorg. Med. Chem. 15 (2007) 7399–7407
5.34 (s, 2H, CH2), 4.39 (d, J = 6.3 Hz, 2H, CH2), 3.36 (q,
J = 7.2 Hz, 2H, CH2), 2.62 (s, 3H, CH3), 1.19 (t,
J = 7.2 Hz, 3H, CH3). MS (ES) m/z: 291 (M+H, 100),
197 (30).
7.48 (m, 3H, ArH), 7.32–7.27 (m, 5H, ArH), 7.03 (m,
1H, ArH), 5.46 (s, 2H, CH2), 4.66 (s, 2H, CH2), 4.26
(d, J = 5.1 Hz, 2H, CH2), 2.52 (s, 3H, CH3). MS (ES)
m/z: 419 (M+Na, 10), 274 (28).
3.7.13. 1-(Ethoxymethyl)-5-(naphthalen-1-ylaminometh-
yl)-6-methyluracil (1m). Yield: 38%. Purple solid. Mp
3.8.6. 1-(Benzyloxymethyl)-5-[(2,4,5-trichloro-phenylami-
no)-methyl]-6-methyluracil (1s). Yield: 45%. White solid.
Mp 180–182 °C. 1H NMR (300 MHz, CDCl3): d 8.73 (s,
1H, NH), 7.30–7.18 (m, 6H, ArH), 6.74 (s, 1H, ArH),
5.38 (s, 2H, CH2), 4.60 (s, 2H, CH2), 4.00 (s, 2H,
CH2), 2.40 (s, 3H, CH3). MS (ES) m/z: 478 (M+Na,
70), 454 (M, 30), 259 (95), 274 (82).
1
69–71 °C. H NMR (300 MHz, CDCl3): d 9.24 (s, 1H,
NH), 7.83–7.29 (m, 6H, 30, 40, 50, 60, 70, and 80H), 6.55
(m, 1H, 20H), 5.33 (s, 2H, CH2), 4.24 (s, 2H, CH2),
3.62 (q, J = 7.2 Hz, 2H, CH2), 2.47 (s, 3H, CH3), 1.20
(t, J = 7.2 Hz, 3H, CH3). MS (ES) m/z: 362 (M+Na,
40), 340 (M+H, 90), 197 (30).
3.8.7. 1-(Benzyloxymethyl)-5-(cyclohexylaminomethyl)-
6-methyluracil (1t). Yield: 54%. Yellow solid. Mp 200–
202 °C. 1H NMR (300 MHz, CDCl3): d 7.26–7.19
(m, 5H, ArH), 5.34 (s, 2H, CH2), 4.55 (s, 2H, CH2),
3.65 (m, 2H, CH2), 2.76 (m, 1H, CH), 2.48 (s, 3H,
CH3), 1.94–1.24 (m, 10H, CH2). MS (ES) m/z: 358
(M+H, 55).
3.8. General procedure for the synthesis of 1n–1u
Mixed 1-(benzyloxymethyl)-5-(bromomethyl)-6-methyl-
uracil 8b (45 mg, 0.13 mmol) and dry 1,4-dioxane
(5 ml), followed by different amino compounds
(0.89 mmol). The reaction mixture was allowed to stir
for several hours and then was evaporated in vacuo.
The resulting residue was purified by silica gel column
chromatography using chloroform and methanol
(10:1), as the eluent to give the target compound (1n–
1u’s reaction time is illustrated in Table 1).
3.8.8. 1-(Benzyloxymethyl)-5-[(2,4,6-trichloro-phenylami-
no)-methyl]-6-methyluracil (1u). Yield: 28%. White solid.
Mp 152–154 °C; 1H NMR (300 MHz, CDCl3): d 9.27 (s,
1H, NH), 7.39–7.26 (m, 6H, ArH), 5.42 (s, 2H, CH2),
4.62 (s, 2H, CH2), 4.24 (s, 2H, CH2), 2.30 (s, 3H,
CH3). MS (ES) m/z: 478 (M+Na, 25), 454 (M, 10).
3.8.1. 1-(Benzyloxymethyl)-5-[(methyl-phenylamino)-meth-
yl]-6-methyluracil (1n). Yield: 46%. Brown solid. Mp
1
45–47 °C. H NMR (300 MHz, CDCl3): d 9.11 (s, 1H,
NH), 7.29–7.14 (m, 7H, ArH), 6.85 (d, J = 7.8 Hz, 2H,
ArH), 6.73 (t, J = 7.2 Hz, 1H, ArH), 5.36 (s, 2H,
CH2), 4.58 (s, 2H, CH2), 4.11 (s, 2H, CH2), 2.69 (s,
3H, CH3), 2.27 (s, 3H, CH3). MS (ES) m/z: 366
(M+H, 100), 274 (18), 259 (22).
Acknowledgments
We thank the National Sciences Foundation of China
(No. 20672008) and 985 program of Ministry of Educa-
tion of China for financial support.
3.8.2. 1-(Benzyloxymethyl)-5-(p-tolylamino-methyl)-6-
methyluracil (1o). Yield: 37%. Brown solid. Mp 42–
44 °C. 1H NMR (300 MHz, DMSO-d6): d 7.41–7.29
(m, 5H, ArH), 6.98 (m, 2H, ArH), 6.56 (d, J = 8.7 Hz,
2H, ArH), 5.41 (s, 2H, CH2), 4.62 (s, 2H, CH2), 4.03
(s, 2H, CH2), 2.44 (s, 3H, CH3), 2.22 (s, 3H, CH3).
MS (ES) m/z: 366 (M+H, 75), 274 (20), 259 (20).
References and notes
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3.8.3.
1-(Benzyloxymethyl)-5-[(4-nitro-phenylamino)-
methyl]-6-methyluracil (1p). Yield: 29%. Brown solid.
Mp 147–148 °C. 1H NMR (300 MHz, DMSO-d6): d
11.54 (s, 1H, NH), 7.97 (d, J = 9.3 Hz, 2H, ArH), 7.32–
7.26 (m, 5H, ArH), 6.68 (d, J = 9.3 Hz, 2H, ArH), 5.38
(s, 2H, CH2), 4.57 (s, 2H, CH2), 4.39 (d, J = 4.5 Hz, 2H,
CH2), 2.37 (s, 3H, CH3). MS (ES) m/z: 274 (30).
3.8.4. 1-(Benzyloxymethyl)-5-[(2-nitro-phenylamino)-
methyl]-6-methyluracil (1q). Yield: 29%. Yellow solid.
Mp 216–218 °C. H NMR (300 MHz, CDCl3): d 8.15
(m, 1H, ArH), 7.46 (t, J = 6.9 Hz, 1H, ArH), 7.32–7.24
(m, 5H, ArH), 7.00 (t, J = 8.1 Hz, 1H, ArH), 6.66 (t,
J = 7.2 Hz, 1H, ArH), 5.44 (s, 2H, CH2), 4.66 (s, 2H,
CH2), 4.27 (d, J = 5.1 Hz, 2H, CH2), 2.47 (s, 3H,
CH3). MS (ES) m/z: 397 (M+H, 60), 259 (83).
1
9. Goldman, M. E.; Nunberg, J. H.; O‘Brien, J. A.; Quintero,
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3.8.5. 1-(Benzyloxymethyl)-5-[(3-nitro-phenylamino)-
methyl]-6-methyluracil (1r). Yield: 16%. Orange solid.
Mp 196–198 °C. H NMR (300 MHz, CDCl3): d 7.63–
10. Yuasa, S.; Sadakata, Y.; Takashima, H.; Sekiya, K.;
Inouye, N.; Ubasawa, M.; Bada, M. Mol. Pharmacol.
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