Synthesis and evaluation of novel 3-(3,5-dimethylbenzyl)uracil analogs as potential anti-HIV-1 agents

Article abstract of DOI:10.1016/j.bmc.2013.06.061

A novel series of uracil derivatives with a 3,5-dimethylbenzyl group at the N³-position were synthesized and evaluated as non-nucleoside HIV-1 reverse transcriptase inhibitors. Some of these compounds showed good-to-moderate activity with EC₅₀ values in the submicromolar range. Among them, compound 10c showed significant potency against HIV-1 activity with an EC₅₀ value of 0.03 μM and a high selectivity index of 2863. Preliminary structure-activity relationships and molecular modeling analyses were used to explore the major interactions between HIV-1 reverse transcriptase and the potent inhibitor 10c, which may serve as an important lead for further optimization.

Full text of DOI:10.1016/j.bmc.2013.06.061

Accepted Manuscript
Synthesis and evaluation of novel 3-(3,5-dimethylbenzyl)uracil analogs as po‐
tential anti-HIV-1 agents
Norikazu Sakakibara, Takayuki Hamasaki, Masanori Baba, Yosuke Demizu,
Masaaki Kurihara, Kohji Irie, Masatoshi Iwai, Eriko Asada, Yoshihisa Kato,
Tokumi Maruyama
PII:
S0968-0896(13)00601-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.06.061
BMC 10954
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
16 May 2013
25 June 2013
25 June 2013
Please cite this article as: Sakakibara, N., Hamasaki, T., Baba, M., Demizu, Y., Kurihara, M., Irie, K., Iwai, M.,
Asada, E., Kato, Y., Maruyama, T., Synthesis and evaluation of novel 3-(3,5-dimethylbenzyl)uracil analogs as
potential anti-HIV-1 agents, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.
2013.06.061
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Synthesis and evaluation of novel 3-(3,5-dimethylbenzyl)uracil analogs as
potential anti-HIV-1 agents
Norikazu Sakakibara a*, Takayuki Hamasaki b, Masanori Baba b, Yosuke Demizu c, Masaaki Kurihara c,
Kohji Irie a, Masatoshi Iwai a, Eriko Asada a, Yoshihisa Kato a, Tokumi Maruyama a
a
Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, 1314-1 Shido,
Sanuki City, Kagawa 769-2193, Japan
b Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Graduate School of Medical and
Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan
c Division of Organic Chemistry, National Institute of Health Sciences, 1-18, Kamiyoga 1-chome, Setagaya,
Tokyo 158-8501, Japan
* Corresponding author. Tel.: +81 87 894 5111; fax: +81 87 894 0181.
E-mail: bara@kph.bunri-u.ac.jp (N. Sakakibara).
Abstract
A novel series of uracil derivatives with a 3,5-dimethylbenzyl group at the N3-position were synthesized
and evaluated as non-nucleoside HIV-1 reverse transcriptase inhibitors. Some of these compounds showed
good-to-moderate activity with EC50 values in the submicromolar range. Among them, compound 10c
showed significant potency against HIV-1 activity with an EC50 value of 0.03 µM and a high selectivity
index of 2863. Preliminary structure-activity relationships and molecular modeling analyses were used to
explore the major interactions between HIV-1 reverse transcriptase and the potent inhibitor 10c, which may
serve as an important lead for further optimization.
1. Introduction
O
N
O
3
Non-nucleoside
reverse
HN
O
N
6
transcriptase inhibitors (NNRTIs)
are a structurally diverse group of
compounds that bind to the viral
enzyme reverse transcriptase (RT),
where the NNRTIs 1 interact with a
specific allosteric non-substrate
O
SPh
O
1N
R
X
2a: X = H, R = benzyl
2b: X = H, R = cyanomethy
2c: X = H, R = 4-picolyl
2d: X = N3, R = benzyl
2e: X = NH2, R = benzyl
R
1a: R = OH
1b: R = O(PO3)3
Figure 1. Structures of HEPT derivatives (1a, b) and 1-substituted-3-(3,5-
dimethylbenzyl)uracil derivatives (2a-e).
binding pocket site. It is interesting that some NNRTIs have an aromatic group at the 6-position of uracil
skeleton. For instance, in 1989, Baba et discovered that
al.2a
1-[(2-hydroxyethoxy)methyl]-6-phenylthiothymine (1a, HEPT) had strong anti-HIV-1 activity.
Interestingly, HEPT 1a can be regarded as an acyclonucleoside analog, yet its 5'-triphosphate derivative 1b

showed no inhibitory effect on HIV-1 RT,2b suggesting that HEPT could be undoubtedly considered an
NNRTI despite having a uracil skeleton. Since the first finding of HEPT 1a, related uracil derivatives have
been synthesized.2 We have consistently searched for an anti-HIV-1 agent using the structure-activity
relationships (SAR) of the 1,3-disubstituted and 1,3,6-trisubstituted uracils.3 As a result, we have
demonstrated that the 3,5-dimethylbenzyl group at the 3-position of the uracil skeleton plays an important
role in an enhancement of the anti-HIV-1 activity; notably, 1-substituted (e.g., benzyl, cyanomethyl, or
4-picolyl group) 3-(3,5-dimethylbenzyl)uracil 2a–c showed good antiviral activity.3a Moreover, the
introduction of an azido or amino group at the 6-position of 1-benzyl-3-(3,5-dimethylbenzyl)uracil 2d and
2e showed excellent potency with an EC50 of 0.067 ±0.011 µM and 0.069 ± 0.006 µM, respectively.3c
However, the CC50 and corresponding SI values of compounds 2d and 2e were not satisfactory (CC50 45.9 ±
0.7 µM and 45.6 ±0.9 µM; SI 685 and 661, respectively).3c Taken together, these results prompted us to
evaluate the following newly synthesized 1,3,6-trisubstituted uracils with an aim to develop higher
anti-HIV-1
activity
and
lower
cytotoxicity:
i)
6-alkylamino-introduced
analogs
of
1-benzyl-3-(3,5-dimethylbenzyl)uracil (5 series) to examine the effect of N-alkylation of the C6-amino
group on 2e, and ii) 1-substituted derivatives of 6-azido (or amino)-3-(3,5-dimethylbenzyl)uracil (9 and 10
series) to investigate substituent effect when the N1-benzyl group of 2d or 2e is replaced by an appropriate
alkyl group such as cyanomethyl, 2-picolyl and 4-picolyl groups. We also report on the docking studies of
the most promising inhibitor 10c with an RT nevirapine binding site.
2. Results and discussion
2.1. Chemistry
To obtain a series of 6-amino-substituted analogs of 1-benzyl-3-(3,5-dimethylbenzyl)uracil (5 series),
1-benzyl-6-chloro-3-(3,5-dimethylbenzyl)uracil 4 was prepared in two steps from the commercially
available 6-chlorouracil 3 using our previously reported method shown in scheme 1.3c Then, compound 4
was subjected to nucleophilic substitution with a variety of acyclic or cyclic amines (e.g., dimethylamine,
pyrrolidine, morpholine, or benzylamine) in the presence of Na2CO3 in EtOH at 50–100 °C for 0.5–65 h to
give their 6-alkylamino-substituted analogs in 65–100% yields (Scheme 1, 5b–j). In the case of the
6-acetamide analog 5a, 6-amino derivative 2b prepared from 33c was N-monoacetylated with Ac2O and
pyridine to obtain the corresponding derivative 5a.
The 6-azido (and amino)-1-substituted analogs of 3-(3,5-dimethylbenzyl)uracil synthesis (9 and 10 series)
also began with 6-chlorouracil 4 as a starting material and led to 1-methoxymethyl-6-chlorouracil 6
(Scheme 2). The resulting 6 was condensed with 3,5-dimethylbenzyl alcohol using the Mitsunobu
reaction3c to afford 3-(3,5-dimethybenzyl) congener 7. The product was carried out by deprotection of the
N1-MOM group using B-bromocatecholborane4 in CH2Cl2 to give the corresponding N1-deprotected
inseparable mixture of 8a and 6-bromo-substituted product 8b in 72% combined yield (ratio 8a/8b: 80/20).
Next, the resulting mixture of 8a and 8b was treated with bromoacetonitrile, 2-picolyl chloride or 4-picolyl
chloride to give the N1-alkylated congeners, and subsequently, the mixture of 6-chloro and 6-bromo

5a: R = NHAc
5b: R = NHMe
5c: R = NMe2
5d: R = pyrrolidinyl
5e: R = piperidinyl
5f: R = piperazinyl
5g: R = morpholyl
5h: R = cyclopropylamino
5i: R = cyclohexylamino
5j: R = benzylamino
O
N
O
N
O
N
N
3
HN
i
ii
R
6
O
Cl
O
O
N
Cl
1 H
3
4
Scheme 1. Synthesis of 6-alkylamino-substituted-1-benzyl-3-(3,5-dimethylbenzyl)uracil (5 series). Reagents and conditions:
i, see ref. 3c; ii, K2CO3, EtOH, 50 100 ºC, 0.5 65 h, 65 100%
O
O
O
O
i
ii
iii
HN
3
HN
6
N
N
N
O
N
H
Cl
O
N
Cl
O
N
Cl
O
N
H
Cl
Br
MOM
MOM
8a
7
+
O
O
O
N
iv, v
vi or vii
N
O
N
H
O
N
N3
O
N
NH2
8b
R1
R1
9a: R1 = cyanomethyl
9b: R1 = 2-picolyl
9c: R1 = 4-picolyl
10a: R1 = cyanomethyl
10b: R1 = 2-picolyl
10c: R1 = 4-picolyl
Scheme 2. Synthesis of 6-azido-1-substitueted-3-(3,5-dimethylbenzyl)uracil (9 series) and 6-amino-1-substitueted-3-(3,5-
dimethylbenzyl)uracil (10 series). Reagents and conditions: i, MOMCl, DBU, CH2Cl2, 0 C, 20 min, 90%; ii, 2,6-
dimethylbenzylalcohol, TMAD, PPh3, 50 C, 2 h, 93%; iii, B-bromocatecholborane, CH2Cl2, rt, 1 h, 72%; iv, R-X (R = cyanomethyl, X
= Br; R= 2-picolyl, X = Cl; R = 4-picolyl, X = Cl), K2CO3, DMF, 40 93%; v, NaN3, DMF, rt, 30 min, 81 90%; vi, PPh3, H2O, THF,
rt, 10 h, 90%; vii, LiAlH4, THF, 0 C, 5 min, 69 74%
derivatives was subjected to the nucleophlic substitution at the C6-position in the uracil moiety with
sodium azide to afford the single product of 6-azido substituted analogs 9a–c. The reduction of compounds
9b and 9c was carried out with lithium aluminum hydride to provide 6-amino compound 10b and 10c,
5
whereas the conversion of 9a was performed using PPh3-H2O condition to obtain 10a, avoiding the
excessive reduction of cyanomethyl group of 9a.
2.2. Biological activity
The antiviral activity of the 6-alkylamino-substituted 1-benzyl-3-(3,5-dimethylbenzyl)uracils 5a–j,
6-azido congeners 9a–c, and 6-amino analogs 10a–c of 1-substituted-3-(3,5-dimethylbenzyl)uracils was
determined by examining the inhibitory effects of these compounds on the HIV-1-induced
cytopathogenicity and on cell viability in MT-4 cells. Zidovudine (AZT) was also tested as a positive
control. In the series of 6-alkylamino-substituted analogs 5a–j shown in Table 1, almost every compound
had considerably reduced anti-HIV-1 activity when compared with 6-azido or amino-introduced derivatives
2d and 2e, which displayed EC50 values of 0.067 ±0.011 µM and 0.069 ±0.006 µM, respectively,3c suggesting
that alkylation of the amino group at the 6-position on the uracil moiety diminished anti-HIV-1 activity.
Even N-monomethylation of the amino group at the 6-position, such as for compound 5b, resulted

Table. 1. Antiviral activity of 3-(3,5-dimethylbenzyl)uracil analogs against HIV-1
O
O
NH
N
N
O
O
N
R2
HO
O
R1
AZT
5a-j, 9a-c, 10a-c
(zidovudine)
N3
Compound
EC50 ( M)a
CC50 ( M)b
SIc
R1
R2
AZT
0.04 ± 0.04
> 100
> 100
> 100
> 2769
Bn
Bn
Bn
Bn
Bn
Bn
NHAc
NHMe
NMe2
5a
5b
5c
5d
5e
5f
7 ± 4
65 ± 3
48 ± 5
31 ± 3
47 ± 3
16 ± 4
9
> 48 ± 5
> 31 ± 3
> 47 ± 3
> 16 ± 4
pyrrolidinyl
piperidinyl
piperazinyl
morpholyl
Bn
Bn
Bn
Bn
5g
5h
5i
> 100
> 100
> 100
74 ± 19
> 100
60 ± 1
> 100
59 ± 6
cyclopropylamino
cyclohexylamino
NHBn
> 100
> 74 ± 19
> 100
5j
N3
9a
9b
9c
cyanomethyl
3.5 ± 1
17
N3
2-picolyl
4-picolyl
cyanomethyl
2-picolyl
4-picolyl
0.22 ± 0.10
0.05 ± 0.02
> 457
1162
N3
NH2
NH2
NH2
10a
10b
10c
> 100
> 100
> 100
> 100
0.23 ± 0.06
0.03 ± 0.03
> 443
> 2863
a EC50, effective concentration; the concentration of compound required to protect the cell against viral cytopathogenicity by 50% in MT-4 cells.
b CC50, cytotoxic concentration; the concentration of compound that reduces the normal uninfected MT-4 cell viability by 50%.
c SI, selectivity idex (CC50/EC50).
in centesimal decreased HIV-1 activity (EC50 = 7 ±4 µM) in comparison to the corresponding 6-free amino
analog 2e.
Next, the introduction of an alkyl group (such as cyanomethyl, 2-picolyl, and 4-picolyl) at the N1-position
of the uracil skeleton in place of N1-benzyl group of 2d and 2e was investigated for the SAR. Among the
compounds 9a–c and 10a–c shown in Table 1, N1-2-picolyluracil congeners 9b and 10b showed good
anti-HIV-1 activity with EC50 values of 0.22 ±0.10 µM and 0.23 ±0.06 µM, respectively. Because CC50 values
for both 9b and 10b exceeded 100 µM, their SI values were > 457 and > 443, respectively, which were
comparable to the SI values of the N1-benzyl uracil derivatives 2d and 2e (658 and 661, respectively),
reported in our previous study.3c However, the introduction of an N1-4-picolyl group in 9c and 10c produced
compounds with the strongest anti-HIV-1 potencies (EC50 0.05 ±0.02 µM and 0.03 ±0.03 µM, respectively).
In particular, the 6-amino-1-(4-picolyl) analog 10c not only exhibited significant activity (at EC50 = 0.03 ±
0.03 µM), but also displayed low cytotoxicity with CC50 > 100 µM, resulting in an SI > 2863, which is high

and comparable to those of the positive controls AZT (SI > 2769) or nevirapine3c (SI > 1639). In contrast,
the 6-azido-1-(4-picolyl) congener 9c showed moderate values for CC50 of 59 ±6 µM and for SI of 1162. As
for the similar potencies (defined by their similar EC50 values) among C6-azido analogs 2d, 9b and 9c, and
C6-amino derivatives 2e, 10b and 10c, it is possible that 6-azidouracil developed antiviral activity after
metabolic conversion to the 6-aminocongener. Finally, C6-azidouracil 9a with a cyanomethyl group at the
N1 position was, unfortunately, 100 times less potent (EC50 = 3.5 ±1 µM) than its N1-4-picolyl counterpart,
10c. Moreover, the 6-amino-1-cyanomethyl analog 10a showed no antiviral activity.
2.3. Molecular modeling analysis
The X-Ray co-crystal structure (PDB: 1VRT) of HIV-1 RT with nevirapine was taken from PDB (1VRT)6
and used for docking studies. A docking model of ligand 10c, which showed the most promising HIV-1
activity, bound to HIV-1 RT was constructed by conformational search using MacroModel (ver. 9.1).
AMBER* was used as a force field, and more than 3,000 conformers for 10c were optimized. The most
stable conformation is shown in Figure 2. The hydrogen of the 6-amino group for 10c is hydrogen bonded
to the amide group of Lys101 residue (NH…O=C), and the 3,5-dimethylbenzyl moiety was oriented around
the hydrophobic area (Tyr181, Tyr188, Trp229, and Leu234 residues) of HIV-1 RT. This result was
significantly different from that of the C6-deaminated counterpart 2c in our previous report.3b The nitrogen
of the 4-picolyl formed a hydrogen bond with the amide group of the Lys101 residue (N…H−N), whereas the
3,5-dimethylbenzyl group at the N3 position existed around another hydrophobic area (Val106, Pro225,
Lys101
Trp229
Leu234
Tyr181
Tyr188

Phe227, Leu234, and Pro236 residues) of HIV-1 RT. Most NNRTIs are known to be engaged in the
hydrogen bond with the backbone of the amino acids Lys101.7 Thus, the results from our docking study
with compound 10c suggest a role for the hydrogen bond in the affinity with RT.
2.4. Conclusion
In the present study,
a series of newly synthesized 1-substituted analogs of 6-azido or
6-amino-3-(3,5-dimethylbenzyl)uracil exhibited good potency against HIV-1 activities. In particular,
compound 10c displayed excellent activity with an EC50 value of 0.03 ±0.03 µM and an SI value of 2863.
Furthermore, the simulated binding model of 10c with HIV-1 RT indicated that the hydrogen of the
6-amino group for 10c is hydrogen bonded to the amide group of the Lys101 residue, and that the
3,5-dimethylbenzyl moiety was oriented around the hydrophobic area (Tyr181, Tyr188, Trp229, and
Leu234 residues) of HIV-1 RT. Overall, this compound may serve as the basis for further modification in
the search for more potent candidates for anti-HIV-1 chemotherapy.
3. Experimental section
3.1. Chemistry
1H NMR and 13C NMR spectra were taken with an UltrashieldTM 400 Plus FT NMR System (BRUKER,
Germany). Chemical shifts and coupling constants (J) were given in δand Hz, respectively. Melting points
were determined on a Yanaco MP-500D. High-resolution mass spectrometry was performed on an APEX
IV mass spectrometer (BRUKER) with electrospray ionization mass spectroscopy (ESI-MS).
3.2. 1-Benzyl-3-(3,5-dimethylbenzyl)-6-acetamidouracil (5a)
A solution of compound 2e (33.5 mg, 0.1 mmol) and Ac2O (189.1 µl, 2.0 mmol) in dry pyridine (0.3 mL)
was stirred for 7 days at room temperature. The mixture was then evaporated in vacuo and the residue was
extracted with AcOEt. The organic extracts were washed with water and saturated sodium chloride solution,
dried with sodium sulfate, and then evaporated. The residue was purified by silica gel column
1
chromatography (50% AcOEt in hexane) to a white crystal 5a (13.2 mg, 0.035 mmol, 35%). H NMR
(400MHz, CDCl3): B 11.60 (1H, brs, AcNH), 7.21-7.40 (5H, m, Bn), 7.06 (2H, s, 3,5-Me2-Bn), 6.90 (1H, s,
3,5-Me2-Bn), 5.40 (1H, s, H-5), 5.21 (2H, s, Bn), 5.13 (2H, s, 3,5-Me2-Bn), 2.64 (6H, s, Ac), 2.30 (6H, s,
3,5-Me2-Bn); 13C NMR (100MHz, CDCl ): 199.4, 161.3, 158.2, 150.4, 138.0, 136.9, 133.7, 129.6, 129.2,
3
+
128.7, 126.1, 126.1, 91.9, 46.1, 44.6, 32.5, 21.3; HRMS (ESI) Calcd for C22H23N3NaO3
[M+Na]+:400.16316. Found 400.16349; mp: 212.3 °C.
3.3. General procedure for the synthesis of 5b-j
A solution of compound 4 (177.4 mg, 0.5 mmol), appropriate amine (1.0 mmol) and Na2CO3 (106.0 mg,

1.0 mmol), in dry EtOH (3.0 mL) was heated at 50–100 °C. After 0.5–65 h stirring, the mixture was
evaporated in vacuo and the residue was extracted with AcOEt. The organic extracts were washed with
water and saturated sodium chloride solution, dried with sodium sulfate, and then evaporated. The residue
was purified by silica gel column chromatography to afford 5b–j in 65–100% yield.
3.3.1. 1-Benzyl-3-(3,5-dimethylbenzyl)-6-methylaminouracil (5b)
Yield 92%; white crystal; 1H NMR (400MHz, CDCl3): B 7.20-7.39 (5H, m, Bn), 7.08 (2H, s, 3,5-Me2-Bn),
6.88 (1H, s, 3,5-Me2-Bn), 5.14 (2H, s, 3,5-Me2-Bn), 5.10 (2H, s, Bn), 4.88 (1H, s, H-5), 4.17 (1H, m, NH),
13
2.69 (3H, d, J 5.2, methylamino), 2.30 (6H, s, 3,5-Me2-Bn); C NMR (100MHz, CDCl3): 162.7, 154.0,
152.1, 137.8, 137.5, 135.1, 129.3, 128.9, 128.2, 126.1, 126.0, 76.0, 45.7, 44.2, 29.9, 21.3; HRMS (ESI)
Calcd for C21H23N3NaO2+ [M+Na]+: 372.16825. Found 372.16855; mp: 186.7 °C.
3.3.2. 1-Benzyl-3-(3,5-dimethylbenzyl)-6-dimethylaminouracil (5c)
1
Yield 100%; pale yellow oil; H NMR (400MHz, CDCl3): B 7.15-7.31 (5H, m, Bn), 6.99 (2H, s,
3,5-Me2-Bn), 6.85 (1H, s, 3,5-Me2-Bn), 5.29 (1H, s, H-5), 5.07 (2H, s, Bn), 5.00 (2H, s, 3,5-Me2-Bn), 2.68
(6H, s, 6-NMe2), 2.25 (6H, s, 3,5-Me2-Bn); 13C NMR (100MHz, CDCl3): 162.9, 160.6, 152.9, 137.7,
137.1, 136.9, 129.1, 128.6, 127.5, 126.8, 126.3, 88.4, 48.8, 44.1, 42.5, 21.3; HRMS (ESI) Calcd for
C22H26N3O3+ [M+H]+:364.20195. Found 364.20256.
3.3.3. 1-Benzyl-3-(3,5-dimethylbenzyl)-6-pyrrolidinyluracil (5d)
Yield 84%; white crystal;1H NMR (400MHz, CDCl3) : B 7.12-7.30 (5H, m, Bn), 7.04 (2H, s, 3,5-Me2-Bn),
6.86 (1H, s, 3,5-Me2-Bn), 5.21 (1H, s, H-5), 5.10 (2H, s, 3,5-Me2-Bn), 5.04 (2H, s, Bn), 3.13 (4H, m,
pirrolidinyl), 2.26 (6H, s, 3,5-Me2-Bn), 1.86 (4H, m, pirrolidinyl); 13C NMR (100MHz, CDCl3): 162.8,
157.2, 153.3, 137.7, 137.3, 136.9, 129.0, 128.6, 127.3, 126.4, 126.0, 84.0, 51.5, 49.9, 44.1, 25.3, 21.3;
HRMS (ESI) Calcd for C24H27N3NaO2+ [M+Na]+: 412.19955. Found 412.19893; mp: 132.2 °C.
3.3.4. 1-Benzyl-3-(3,5-dimethylbenzyl)-6-piperidinyluracil (5e)
1
Yield 96%; pale yellow oil; H NMR (400MHz, CDCl3) : B 7.19-7.29 (5H, m, Bn), 7.00 (2H, s,
3,5-Me2-Bn), 6.86 (1H, s, 3,5-Me2-Bn), 5.30 (1H, s, H-5), 5.04 (2H, s, 3,5-Me2-Bn), 5.00 (2H, s, Bn), 2.85
13
(4H, m, piperidinyl), 2.24 (6H, s, 3,5-Me2-Bn), 1.56 (6H, m, piperidinyl); C NMR (100MHz, CDCl3):
163.1, 160.6, 152.9, 137.8, 137.8, 137.1, 129.1, 128.6, 127.5, 127.0, 126.4, 89.7, 52.3, 48.1, 44.1, 25.3,
23.9, 21.3; HRMS (ESI) Calcd for C25H29N3NaO2+ [M+Na]+: 426.21520. Found 426.21293.
3.3.5. 1-Benzyl-3-(3,5-dimethylbenzyl)-6-piperazinyluracil (5f)
Yield 92%; pale yellow oil; 1H NMR (400MHz, CDCl3): B 7.19-7.32 (5H, m, Bn), 7.01 (2H, s,
3,5-Me2-Bn), 6.86 (1H, s, 3,5-Me2-Bn), 5.33 (1H, s, H-5), 5.07 (2H, s, Bn), 5.01 (2H, s, 3,5-Me2-Bn), 2.93
(4H, s, piperazinyl), 2.87 (4H, s, piperazinyl), 2.25 (6H, s, 3,5-Me2-Bn); 13C NMR (100MHz, CDCl3):

162.9, 160.0, 152.8, 137.8, 136.9, 129.1, 128.7, 127.6, 126.8, 126.3, 90.1, 52.2, 48.1, 45.3, 44.2, 21.3;
HRMS (ESI) Calcd for C24H29N4NaO2+ [M+Na]+:405.22850. Found 405.22688.
3.3.6. 1-Benzyl-3-(3,5-dimethylbenzyl)-6-morpholyluracil (5g)
Yield 100%; white crystal; 1H NMR (400MHz, CDCl3): B 7.24-7.33 (5H, m, Bn), 7.02 (2H, s, 3,5-Me2-Bn),
6.87 (1H, s, 3,5-Me2-Bn), 5.35 (1H, s, H-5), 5.09 (2H, s, Bn), 5.02 (2H, s, 3,5-Me2-Bn), 3.74 (4H, s,
morpholyl), 2.88 (4H, s, morpholyl), 2.25 (6H, s, 3,5-Me2-Bn); 13C NMR (100MHz, CDCl ): 162.8, 159.5,
3
152.7, 137.8, 136.9, 136.8, 129.2, 128.8, 127.7, 126.7, 126.4, 90.3, 66.1, 51.4, 48.1, 44.2, 21.3; HRMS
(ESI) Calcd for C24H27N3NaO3+ [M+Na]+:428.19446. Found 428.19306; mp: 133.2 °C.
3.3.7. 1-Benzyl-6-cyclopropylamino-3-(3,5-dimethylbenzyl)uracil (5h)
Yield 94%; white crystal; 1H NMR (400MHz, CDCl3) : B 7.26-7.38 (5H, m, Bn), 7.10 (2H, s, 3,5-Me2-Bn),
6.89 (1H, s, 3,5-Me2-Bn), 5.31 (1H, s, H-5), 5.10 (2H, s, Bn), 5.08 (2H, s, 3,5-Me2-Bn), 4.46 (1H, brs, NH),
2.29 (7H, m, 3,5-Me2-Bn and cyclopropylamino), 0.42-0.75 (4H, m, cyclopropylamino); 13C NMR
(100MHz, CDCl3): 161.2, 154.1, 151.5, 137.9, 137.1, 136.5, 128.4, 128.2, 127.1, 126.1, 124.9, 75.5, 44.3,
+
42.9, 24.2, 20.9, 6.6; HRMS (ESI) Calcd for C23H25N3NaO2 [M+Na]+ : 398.18390. Found 398.18193; mp:
214.4 °C.
3.3.8. 1-Benzyl-6-cyclohexylamino-3-(3,5-dimethylbenzyl)uracil (5i)
Yield 89%; white crystal; 1H NMR (400MHz, CDCl3) : B 7.22-7.34 (5H, m, Bn), 7.10 (2H, s, 3,5-Me2-Bn),
6.88 (1H, s, 3,5-Me2-Bn), 5.16 (2H, s, 3,5-Me2-Bn), 5.10 (2H, s, Bn), 4.88 (1H, s, H-5), 4.13 (1H, m,
cyclohexylamino), 2.30 (6H, s, 3,5-Me2-Bn), 2.05 (1H, brs, NH), 1.53-1.76 (4H, m, cyclohexylamino),
1.11-1.47 (6H, m, cyclohexylamino); 13C NMR (100MHz, CDCl3): 162.8, 152.4, 151.9, 137.8, 137.6,
135.3, 129.4, 129.0, 128.4, 126.3, 126.2, 76.2, 51.3, 46.0, 44.2, 31.9, 25.3, 24.0, 21.3; HRMS (ESI) Calcd
for C26H31N3NaO2+ [M+Na]+ : 440.23085. Found 440.22996; mp: 71.8 °C.
3.3.9. 1-Benzyl-6-benzylamino-3-(3,5-dimethylbenzyl)uracil (5j)
1
Yield 64%; white crystal; H NMR (400MHz, CDCl3) : B 7.26-7.36 (5H, m, Bn), 7.18-7.19 (2H, s,
3,5-Me2-Bn), 6.89-6.95 (5H, m, benzylamino), 5.18 (2H, s, Bn), 5.10 (2H, s, 3,5-Me2-Bn), 4.92 (1H, s, H-5),
4.46 (1H, m, NH), 4.11 (2H, d, J 5.2, benzylamino), 2.29 (6H, s, 3,5-Me2-Bn); 13C NMR (100MHz, CDCl ):
3
162.7, 152.7, 152.2, 137.8, 137.4, 135.9, 135.2, 129.3, 129.0, 128.9, 128.3, 128.0, 127.1, 126.3, 126.2,
+
76.8, 47.1, 45.8, 44.2, 21.3; HRMS (ESI) Calcd for C27H27N3NaO2 [M+Na]+ : 448.19955. Found
448.19661; mp: 189.1 °C.
3.4. 6-Chloro-1-methoxymethyluracil (6)
6-Chlorouracil (3) (1.46 g, 10.0 mmol) was dissolved in dry CH2Cl2 (30 ml), and DBU (1646.3 µl, 11.0
mmol) was added to the solution at room temperature, and after stirring an additional 10 min, the mixture

became clear. After the mixture was cooled to 0 °C, methyl chloromethyl ether (911.1 Cl, 12.0 mmol,
MOMCl) was added dropwise, and stirring continued for an additional 20 min at 0 °C. The mixture was
evaporated, and the residue was purified by silica gel column chromatography (AcOEt) to white powder 6
(1.71 g, 8.99 mmol, 90%). 1H NMR (400MHz, CDCl3): δ8.58 (1H, s, NH), 5.94 (1H, s, 5-H), 5.43 (2H, s,
MOM), 3.47 (3H, s, MOM).
3.5. 6-Chloro-1-methoxymethyl-3-(3,5-dimethylbenzyl)uracil (7)
A solution of compound 6 (1.44 g, 7.56 mmol), triphenylphosphine (2.58 g, 9.83 mmol),
3,5-dimethylbenzylalcohol (1.17 ml, 7.94 mmol) and TMAD (N,N,N',N'-tetramethylazodicarboxamide, 1.69
g, 9.83 mmol) in THF (28.0 ml) was stirred at 50 °C. After 2 h stirring, the solution was filtered and
concentrated to a small volume. The residual solution was purified by silica gel column chromatography
(30% AcOEt in hexane) to give as a syrup 7 (2.17 g, 7.03 mmol, 93%). 1H NMR (400MHz, CDCl3): δ7.04
(2H, s, 3,5-Me2-Bn), 6.90 (1H, s, 3,5-Me2-Bn), 5.98 (1H, s, 5-H), 5.44 (2H, s, MOM), 5.03 (2H, s,
13
3,5-Me2-Bn), 3.44 (3H, s, MOM), 2.28 (6H, s, 3,5-Me2-Bn); C NMR (100MHz, CDCl3): 160.5, 151.4,
145.2, 138.0, 136.0, 129.5, 126.6, 103.3, 57.5, 44.8, 21.3; HRMS (ESI) Calcd for C15H17ClN2NaO3 [M+Na]+:
331.08199. Found 331.08154.
3.6. 6-Chloro-3-(3,5-dimethylbenzyl)uracil (8a) and 6-bromo-3-(3,5-dimethylbenzyl)uracil (8b) 4
A solution of compound 7 (1.90 g, 6.15 mmol) and B-bromocatecholborane (1.47 g, 7.38 mmol, 0.2 M in
CH2Cl2) in CH2Cl2 (20 mL) was stirred at room temperature. After 2 h stirring, the mixture was evaporated
in vacuo, and the residue was extracted with AcOEt. The organic extracts were washed with saturated
aqueous NaHCO3 and saturated NaCl solution, dried with sodium sulfate, and then evaporated. The residue
was purified by silica gel column chromatography (40% AcOEt in hexane) to give the inseparable mixture
of 8a and 8b as a needle crystal (1.21 g, 4.42 mmol, 72% combined yield), which was employed in the next
1
1
reaction without further purification, in a ratio of 80:20 according to H-NMR spectrum. H NMR
(400MHz, CDCl3): δ7.06 (2H, s, 3,5-Me2-Bn), 6.90 (1H, s, 3,5-Me2-Bn), 6.05 (0.2H, s, 5-H, 8b), 5.89
(0.8H, s, 5-H, 8a), 4.99 (2H, s, 3,5-Me2-Bn), 2.27 (6H, s, 3,5-Me2-Bn); HRMS (ESI) Calcd for
C13H13ClN2NaO2 [M+Na]+: 287.05578. Found 278.05656 (8a), Calcd for C13H13BrN2NaO2 [M+Na]+:
331.00526. Found 331.00300 (8b).
3.7. General procedure for the synthesis of 9a-c
The mixture of 8a and 8b (264.7 mg, 0.97 mmol) was dissolved in dry DMF (5.0 mL) under nitrogen
atmosphere. To this stirred solution, we carefully added the appropriate alkyl halide (bromoacetonitrile,
2-(chloromethyl)pyridine hydrochloride or 4-(chloromethyl)pyridine hydrochloride; 2.0 mmol) and K2CO3
(276.4 mg, 2.0 mmol). Stirring was continued at room temperature for 2–12 h, and the mixture was
extracted with AcOEt. The organic extracts were washed with water and saturated aqueous sodium chloride
solution, dried with sodium sulfate, and then evaporated. The residue was purified by silica gel column

chromatography to give the inseparable mixture of C6-chloro and C6-bromo congeners, ratio 80:20
according to 1H-NMR spectrum.
The resulting mixture (258.1 mg, 0.83 mmol) was dissolved in dry DMF (5.0 mL), and NaN3 (66.3 mg,
1.02 mmol) was added to the solution, which was stirred for 30 min at room temperature. The mixture was
extracted with AcOEt, washed with saturated aqueous sodium chloride solution, dried with sodium sulfate,
and then evaporated. The residue was purified by silica gel column chromatography (70% AcOEt in
hexane) to give C6-azido derivative 9a–c.
3.7.1. 6-Azido-1-cyanomethyl-3-(3,5-dimethylbenzyl)uracil (9a)
1
Combined yield 82%; brown oil; H NMR (400MHz, CDCl3): δ7.04 (2H, s, 3,5-Me2-Bn), 6.91 (1H, s,
3,5-Me2-Bn), 5.64 (1H, s, 5-H), 5.02 (2H, s, 3,5-Me2-Bn), 4.75 (2H, s, cyanomethyl), 2.28 (6H, s,
13
3,5-Me2-Bn); C NMR (100MHz, CDCl3): 160.5, 150.0, 148.9, 138.1, 135.8, 129.6, 126.6, 113.8, 88.9,
44.9, 30.8, 21.3; HRMS (ESI) Calcd for C15H15N6NaO2 [M+Na]+: 333.10704. Found 333.10687.
3.7.2. 6-Azido-3-(3,5-dimethylbenzyl)-1-(2-picolyl)uracil (9b)
Combined yield 32%; brown oil; 1H NMR (400MHz, CDCl3): δ8.54 (1H, d, J 4.0, 2-picolyl), 7.66 (1H, m,
2-picolyl), 7.20 (2H, m, 2-picolyl), 7.02 (2H, s, 3,5-Me2-Bn), 6.88 (1H, s, 3,5-Me2-Bn), 5.61 (1H, s, 5-H),
5.17 (2H, s, 2-picolyl), 5.05 (2H, s, 3,5-Me2-Bn), 2.27 (6H, s, 3,5-Me2-Bn); 13C NMR (100MHz, CDCl ):
3
161.4, 154.9, 151.2, 151.2, 149.6, 137.9, 136.7, 136.5, 129.2, 126.2, 122.6, 121.1, 88.0, 48.0, 44.5, 21.2;
HRMS (ESI) Calcd for C19H18N6NaO2 [M+Na]+: 385.13834. Found 385.13740.
3.7.3. 6-Azido-3-(3,5-dimethylbenzyl)-1-(4-picolyl)uracil (9c)
Combined yield 59%; brown oil; 1H NMR (400MHz, CDCl3): δ8.58 (2H, d, J 6.0, 4-picolyl), 7.16 (2H, d,
J 5.6, 4-picolyl), 7.03 (2H, s, 3,5-Me2-Bn), 6.90 (1H, s, 3,5-Me2-Bn), 5.60 (1H, s, 5-H), 5.06 (2H, s,
4-picolyl), 5.03 (2H, s, 3,5-Me2-Bn), 2.28 (6H, s, 3,5-Me2-Bn); 13C NMR (100MHz, CDCl ): 161.0, 151.1,
3
150.4, 150.3, 144.6, 138.0, 136.3, 129.4, 126.4, 122.1, 88.2, 45.8, 44.7, 21.3; HRMS (ESI) Calcd for
C19H18N6NaO2 [M+Na]+: 385.13834. Found 385.13777.
3.8. 6-Amino-1-cyanomethyl-3-(3,5-dimethylbenzyl)uracil (10a)
A solution of compound 9a (73.0 mg, 0.23 mmol), triphenylphosphine (74.0 mg, 0.28 mmol) and H2O
(6.2 Cl, 0.35 mmol) in THF (1.0 ml) was stirred at room temperature. After stirring for 10 h, the solution
was concentrated to a small volume. The residual solution was purified by silica gel column
chromatography (20% MeOH in CHCl3) to give as a yellowish crystal 10a (60.0 mg, 0.21 mmol, 90%). 1H
NMR (400MHz, CDCl3): δ6.98 (2H, s, 3,5-Me2-Bn), 6.88 (1H, s, 3,5-Me2-Bn), 5.07 (2H, brs, NH2), 5.05
(1H, s, 5-H), 4.99 (2H, s, cyanomethyl), 4.80 (2H, s, 3,5-Me2-Bn), 2.27 (6H, s, 3,5-Me2-Bn); 13C NMR
(100MHz, CDCl3): 163.0, 153.2, 150.8, 138.2, 136.6, 129.3, 125.6, 114.3, 78.4, 44.6, 30.4, 21.2; HRMS
(ESI) Calcd for C15H16N4NaO2 [M+Na]+: 307.11655. Found 307.11537; mp: 350.0 °C.

3.9. General procedure for the synthesis of 10b and 10c
Compound 9b or 9c (109.4 mg, 0.30 mmol) was dissolved in dry THF (5.4 ml) under nitrogen atmosphere.
To this stirred solution we carefully added LiAlH4 (13.7 mg, 0.36 mmol) at 0 °C. Stirring was continued at
0 °C for 5 min, and the reaction quenched by the addition of AcOEt (5.0 ml) until no effervescence was
observed. Aqueous 1N HCl (2.2 ml) was then added, and the product was extracted with AcOEt. The
combined organic extracts were washed with water and saturated sodium chloride solution, dried with
sodium sulfate, and then evaporated. The residue was purified by silica gel column chromatography (20%
MeOH in CHCl3) to give a white crystal 10b or 10c.
3.9.1. 6-Amino-3-(3,5-dimethylbenzyl)-1-(2-picolyl)uracil (10b)
1
Yield 69%; white crystal; H NMR (400MHz, CDCl3): δ 8.48 (1H, d, J 4.0, 2-picolyl), 7.73 (1H, m,
2-picolyl), 7.61 (1H, d, J 8.0, 2-picolyl), 7.27 (1H, m, 2-picolyl), 7.03 (2H, s, 3,5-Me2-Bn), 6.85 (1H, s,
3,5-Me2-Bn), 6.21 (2H, brs, NH2), 5.13 (2H, s, 2-picolyl), 5.03 (1H, s, 5-H), 5.01 (2H, s, 3,5-Me2-Bn), 2.26
(6H, s, 3,5-Me2-Bn); 13C NMR (100MHz, CDCl ): 162.8, 155.5, 155.3, 152.0, 148.7, 138.0, 137.8, 137.4,
3
128.9, 126.2, 125.0, 123.6, 79.4, 48.7, 44.2, 21.3; HRMS (ESI) Calcd for C19H20N4NaO2 [M+Na]+:
359.14785. Found 359.14714; mp: 189.3 °C.
3.9.2. 6-Amino-3-(3,5-dimethylbenzyl)-1-(4-picolyl)uracil (10c)
1
Yield 74%; white crystal; H NMR (400MHz, CDCl3): δ8.61 (2H, d, J 4.0, 4-picolyl), 7.14 (2H, d, J 8.0,
4-picolyl), 7.04 (2H, s, 3,5-Me2-Bn), 6.88 (1H, s, 3,5-Me2-Bn), 5.14 (2H, s, 4-picolyl), 5.06 (2H, s,
3,5-Me2-Bn), 5.02 (1H, s, 5-H), 4.32 (2H, brs, NH2) 2.28 (6H, s, 3,5-Me2-Bn); 13C NMR (100MHz, CDCl ):
3
165.1, 156.9, 153.2, 150.3, 147.9, 138.9, 138.7, 129.7, 126.5, 122.9, 77.1, 45.8, 45.1, 21.4; HRMS (ESI)
Calcd for C19H20N4NaO2 [M+Na]+: 359.14785. Found 359.14734; mp: 142.0 °C.
3.10.
Anti-HIV-1 Assay
MT-4 cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine
serum (FBS), 100 U/mL of penicillin G, and 100 mg/mL of streptomycin. The IIIB strain of HIV-1 was
used throughout the experiment. The virus was propagated and titrated in MT-4 cells. Virus stocks were
stored at -80 °C until use. The anti-HIV-1 activity of the test compounds was determined by the inhibition
of virus-induced cytopathogenicity in MT-4 cells.8 Briefly, MT-4 cells (1 ×105 cells/mL) were infected with
HIV-1 at a multiplicity of infection (MOI) of 0.1 and were cultured in the presence of various
concentrations of the test compounds. After 4-day incubation at 37°C in 5% CO2, the number of viable cells
was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method.9
The cytotoxicity of the compounds was evaluated in parallel with their antiviral activity, based on the
viability of mock-infected cells, as determined by the MTT method.

Acknowledgments
This research was partially supported by a Grant-in-Aid for Young Scientists (B), No. 24790123, from the
Japan Society for the Promotion of Science (JSPS).
Reference and notes
1. (a) De Clercq, E. Nature Reviews Drug Discovery 2002, 1, 13; (b) De Clercq, E. J. Med. Chem. 2005,
48, 1297; (c) De Clercq, E. Nature Reviews Drug Discovery 2007, 6, 1001.
2. (a) Baba, M.; Tanaka, H.; De Clercq, E.; Pauwels, R,; Balzarini, J.; Schols, D.; Nakashima, H.; Perno,
C. F.; Walker R. T.; Miyasaka, T. Biochem. Biophys. Res. Commun. 1989, 165, 1375; (b) Miyasaka,
T.; Tanaka, H.; Baba, M.; Hayakawa, H.; Walker, R. T.; Balzarini, J.; De Clercq, E., J. Med.
Chem. 1989, 32, 2507; (c) Tanaka, H.; Baba, M.; Saito, S.; Miyasaka, T.; Takashima, H.; Sekiya, K.;
Ubasawa, M.; Nitta, I.; Walker, R. T. J. Med. Chem. 1991, 34, 1508; (d) Tanaka, H.; Takashima, H.;
Ubasawa, M.; Sekiya, K.; Nitta, I.; Baba, M.; Shigeta, S.; Walker, R. T.; De Clercq, E.; Miyasaka, T. J.
Med. Chem. 1992, 35, 4713; (e) Tanaka, H.; Takashima, H.; Ubasawa, M.; Sekiya, K.; Nitta, I.; Baba,
M.; Shigeta, S.; Walker, R. T.; De Clercq, E.; Miyasaka, T. J. Med. Chem. 1992, 35, 337; (f) Baba, M.;
De Clercq, E.; Tnaka, H.; Ubasawa, M.; Takashima, H.; Sekiya, K.; Nitta, I.; Emuza, K.; Walker, R.
T.; Mori, S.; Ito, M.; Shigeta, S.; Miyasaka, T. Molecular Pharmacol., 1991, 39, 805; (g) Tanaka, H.;
Miyasaka, T.; Sekiya, K.; Takashima, H.; Ubasawa, M.; Nitta, I.; Baba, M.; Walker, R. T.; De Clercq,
E. Nucleosides and Nucleotides, 1992, 11, 447; (h) Buckheit, R. W. J.; Watson, K.; Fliakas-Boltz, V.;
Russell, J.; Loftus, T. L.; Osterling, M. C.; Turpin, J. A.; Pallansch, L. A.; White, E. L.; Lee, J. W.; Lee,
S. H.; Oh, J. W.; Kwon, H. S.; Chung, S. G.; Cho, E. H. Antimicrob. Agents Chemother. 2001, 45, 393;
(i) Debyser, Z.; Pauwels, R.; Baba, M.; Desmyter, J.; De Clercq, E. Mol. Pharmacol. 1992, 41, 963.
3. (a) Maruyama, T.; Kozai, S.; Yamasaki, T.; Witvrouw, M.; Pannecouque, C.; Balzarini, J.; Snoeck, R.;
Andrei, G.; De Clercq, E. Antivir. Chem. Chemother. 2003, 14, 271; (b) Maruyama, T.; Kozai, S.;
Demizu, Y.; Witvrouw, M.; Pannecouque, C.; Balzarini, J.; Snoecks, R.; Andrei, G.; De Clercq, E.
Chem. Pharm. Bull. 2006, 54, 325; (c) Isono, Y.; Sakakibara, N.; Ordonez, P.; Hamasaki, T.; Baba, M.;
Ikejiri, M.; Maruyama, T. Antivir. Chem. Chemother. 2011, 22, 57.
4. Boeckman, R. K. J.; Potenza, J. C. Tetrahedron Lett. 1985, 26, 1411.
5. Srinivasan, N.; Bruce, G. J. Org. Chem. 1987, 52, 5044.
6. Ren, J.; Esnouf, R.; Garman, E.; Somers, D.; Ross, C.; Kirby, I.; Keeling, J.; Darby, G.; Jones, Y.;
Stuart, D. Nature Struct. Biol., 1995, 2, 293.
7. (a) Ribone, S. R.; Quevedo, M. A.; Madrid, M.; Brinon, M. C. J. Chem. Inform. Model. 2011, 51, 130;
(b) Figueiredo, A.; Zelina, S.; Sluis-Cremer, N.; Tachedjian, G. Current HIV Res. 2008, 6, 130; (c) Ren,
J.; Nichols, C. E.; Stamp, A.; Chamberlain, P. P.; Ferris, R.; Weaver, K. L.; Short, S. A.; Stammers, D.
K. FEBS Journal 2006, 273, 3850; (d) David R. M.; Jeffrey A. R.; Richard B. S.; David J. A.; Yanting
H.; Ligaya M. S.; Prakash C. T.; David A. B.; James G. R.; Benoni E. A. O.; Aiying W.; Michael C.; Li
X.; Li T.; Doree F. S.; Mary F. M.; Jack Z. G.; Ashish K.; Qi H.; Song-Ping H.; Rex A. P.; Lawrence G.

H. J. Med. Chem. 2004, 47, 2587; (e) Joseph, H. C.; Jean, S. H.; Robert, N. H. III.; Faye, O.; Jill, R.
C.; Douglas, B. S.; Steven, M. S.; Barbara, E. R.; Webster, A. III.; Richard, J. H.; Marty, S. C.;
Lawrence, R. B.; Rob, G. F.; Katrina, L. Creech.; Grace, B. R.; Steven, A. S.; Kurt, W.; Ronda, J. O.;
Jingshan, Ren.; Andrew, H.; David, I. S.; David, K. S. J. Med. Chem. 2001, 44, 1866; (f) Ren, J.;
Milton, J.; Weaver, K. L.; Short, S. A.; Stuart, D. I.; Stammers, D. K. Structure 2000, 8, 1089.
8. Baba, M.; De Clercq, E.; Tanaka, H.; Ubasawa, M.; Takashima, H.; Sekiya, K.; Nitta, I.; Umezu, K.;
Nakashima, H.; Mori, S.; Shigeta, S.; Walker, R. T.; Miyasaka, T. Proc. Natl. Acad. Sci. 1991; 88,
2356.
9. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.; Desmyter, J.; De Clercq,
E. J. Virol. Methods 1988, 20, 309.