Structural modifications of 5,6-dihydroxypyrimidines with anti-HIV activity

Article abstract of DOI:10.1016/j.bmcl.2012.09.070

A series of 5,6-dihydroxypyrimidine analogs were synthesized and evaluated for their anti-HIV activity in vitro. Among all of the analogs, several compounds exhibited significant anti-HIV activity, especially 1b and 1e, which showed the most potent anti-HIV activity with EC₅₀ values of 0.14 and 0.15 μM, and TI (therapeutic index) values of >300 and >900, respectively. Further docking studies revealed that the representative compounds 1e and 3a could meet the HIV-1 integrase inhibition minimal requirements of a chelating domain (two metal ions) and an aromatic domain (π-π stacking interactions).

Full text of DOI:10.1016/j.bmcl.2012.09.070

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7114–7118
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural modifications of 5,6-dihydroxypyrimidines with anti-HIV activity
Di-Liang Guo a,b,1, Xing-Jie Zhang c,1, Rui-Rui Wang c, Yu Zhou a, Zeng Li a, Jin-Yi Xu b, Kai-Xian Chen a,b
,
a,b,
Yong-Tang Zheng c, , Hong Liu
⇑
⇑
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zu Chong Zhi
Road, Shanghai, PR China
b Department of Medicinal Chemistry, China Pharmaceutical University, 24 TongjiaXiang, Nanjing, PR China
c Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences,
Kunming 650223, Yunnan, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 July 2012
Revised 3 September 2012
Accepted 21 September 2012
Available online 2 October 2012
A series of 5,6-dihydroxypyrimidine analogs were synthesized and evaluated for their anti-HIV activity
in vitro. Among all of the analogs, several compounds exhibited significant anti-HIV activity, especially
1b and 1e, which showed the most potent anti-HIV activity with EC50 values of 0.14 and 0.15 lM, and
TI (therapeutic index) values of >300 and >900, respectively. Further docking studies revealed that the
representative compounds 1e and 3a could meet the HIV-1 integrase inhibition minimal requirements
of a chelating domain (two metal ions) and an aromatic domain (p–p stacking interactions).
Keywords:
Dihydroxypyrimidine
Synthesis
Ó 2012 Elsevier Ltd. All rights reserved.
HIV
Integrase inhibitor
Docking study
Acquired immunodeficiency syndrome (AIDS) and human
immunodeficiency virus (HIV) infection represent global health
hazards, complex scientific challenges, and obvious targets for
drug discovery and vaccination, as well as having enormous social,
economic and ethical ramifications.1 HIV-1 integrase (IN) is one of
the three virally encoded enzymes required for replication and
therefore a rational target for chemotherapeutic intervention in
the treatment of HIV-1 infection.2 After years of sustained effort,
Merck successfully developed raltegravir (RAL, also known as Isen-
tressÒ or MK-0518, 1 Fig. 1), which was approved by the United
States of America’s Food and Drug Administration (FDA) in late
2007 as the first IN inhibitor.3 The good pharmacokinetics, minimal
side effects and safety of raltegravir expanded its broad use in AIDS
patients, but RAL has limited intestinal absorption4 and resistance
cannot be overcome by increasing the administered dose.
Elvitegravir (EVG, 2 Fig. 1), the next most advanced IN inhibitor ap-
pears to share cross-resistance with RAL.5 Mutations that confer
resistance to these inhibitors have been generated in cell culture
and have emerged in clinical studies,6 suggesting that IN inhibitors
will be confronted with the same resistance issue that have
plagued other HIV/AIDS chemotherapies.7 An intensified search
for structurally novel IN inhibitors to combat resistance is
therefore urgently needed.
Since HIV integrase and HCV polymerase share the common
feature of using two magnesium ions in the active site as a key
component of the catalytic machinery,8 dihydroxypyrimidines
(DHP, 3, Fig. 2) that had been investigated as potential HCV NS5B
polymerase inhibitors were screened in the HIV integrase assays.9
Rencently, introduction and optimization of carboxamide substitu-
ents in the 4-position of the dihydroxypyrimidine core resulted in
analogues effective in suppressing the spread of HIV-1 infection in
cells.10–12 To identify more potent scaffolds against HIV integrase,
we designed a series of new DHP analogs (1a–1l, 2a–2c, 3a–3c)
and assessed them as anti-HIV agents. Overall, the binding of IN
inhibitors conforms to a previously proposed interfacial inhibition
mechanism,1 as they bind all three key elements: the enzyme by
hydrophobic and Van Der Waals interactions; the metals by chela-
tion; and the DNA by
p–p
stacking.13 For example, N-methylpyr-
imidone (4, Fig. 2) which is the critical core skeleton of RAL can
engage metal ion cofactors in the IN active site by interactions with
uniquely positioned oxygen atoms of the pharmacophore.14 RAL
also makes hydrophobic interactions by the isopropyl group and
stacking interactions by the methyl-oxadiazole group. Based on
the above considerations, firstly the carboxamide and benzylureido
substituents were introduced in the 4-position of the dihydroxypy-
rimidine core to get the two analogues of dihydroxypyrimidines (A
and B). And we hypothesize that both of these dihydroxypyrimi-
dines have the same binding behavior of the N-methylpyrimidone
⇑
Corresponding author. Tel.: +86 21 50807042; fax: +86 21 50807042.
E-mail addresses: zhengyt@mail.kiz.ac.cn (Y.-T. Zheng), hliu@mail.shcnc.ac.cn
(H. Liu).
1
These authors contributed equally to this study.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.070

D.-L. Guo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7114–7118
7115
O
N
F
O
N
O
OH
F
Cl
N
OH
O
H
N
H
N
N
N
O
O
O
HO
H
1
2
Raltegravir
Elivitegravir
Figure 1.
core of RAL with the two metal ions (Mg2+) (See Fig. 3). Secondly,
the isopropyl and methyl-oxadiazole groups of RAL are involved
in hydrophobic and stacking interactions with the side chains of
Pro 214 and Tyr 212, respectively, further stabilizing this drug in
the active site.12 The target compounds (1a–1l, 2a–2c, 3a–3c) fo-
cused on the replacement of the methyl-oxadiazole group by ben-
zyloxycarbonyl group and retention of the isopropyl group. Finally,
carboxylate intermediate 5 was prepared based on a reported
method15 and our optimized preparation of raltegravir16, including
aminonitrile formation, the addition of a benzyloxycarbonyl group,
the conversion of nitrile to amidoxime, and cyclization to dihy-
droxypyrimidine. Firstly, treatment of the key DHP carboxylate
intermediate 5 with the corresponding amine was performed in
ethanol with microwave-assistance, leading to the formation of
1a–1l, or with hydrazine hydrate in refluxing ethanol to generate
2a. The reaction of 2a with 4-fluorobenzaldehyde in refluxing
THF yielded compound 2b. Subsequently, 2a was reacted with so-
dium nitrite in the presence of hydrochloric acid, and then refluxed
in toluene via Curtius rearrangement yielding compound 2c. 3a–3c
were prepared from 2c reacted with the corresponding amine in
THF. All target compounds were characterized by 1H NMR and
MS (see Supplementary Data).
hydrophobic or
p–p stacking interactions can be achieved by ali-
phatic chain or aromatic groups.
As outlined in Scheme 1, target compounds containing the dihy-
droxypyrimidine core skeleton were successfully synthesized from
commercially available reagents. The key dihydroxypyrimidine
OH
O
Target compounds were evaluated for their inhibitory activity
against HIV-1 replication in acutely infected C8166 cells in vitro
according to the previously described method,17–19 and AZT was
used as a positive control. The assay results of the target com-
pounds are presented in Table 1. Among all of the target com-
pounds, 1b and 1e showed the most potent anti-HIV activity
OH
OH
N
N
N
3
N
4
Dihydroxypyrimidines
N-methylpyrimidones
with EC50 values of 0.14 and 0.15
>900, respectively.
lM, and TI values of >300 and
Figure 2.
OH
OH
N
H
H
O
N
N
N
O
O
O
OH
F
N
O
N
H
N
H
A
N
N
O
N
OH
O
OH
N
H
O
O
N
RAL
N
N
H
O
B
M2+
M2+
OH
M2+
O
N
OH
N
OH
N
M2+
M2+
M2+
OH
OH
N
N
N
O
O
O
N
H
HN
HN
A
B
Figure 3.

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D.-L. Guo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7114–7118
NOH
NH2
H
O
CN
NH2
H
N
c
a
b
O
N
CN
O
O
O
3
2
4
OH
N
OH
N
OH
OH
OCH3
N
N
e
d
H
N
H
H
N
O
N
R
O
O
O
O
O
5
1a-1l
R =phenyl: 1a
R =4-FluoroBenzyl: 1g
R =Benzyl: 1h
R =2-Chlorophenyl: 1b
R =4-Methylphenyl: 1c
f
1i
R =Indol-2-methyl:
R =4-Trifluoromethylphenyl: 1d
R =4-Methoxyphenyl: 1e
1j
R =CH2OCH3:
R =CH2CH2OCH3: 1k
R =Cyclopropyl: 1l
R =3,4,5-Trimethoxyphenyl: 1f
OH
N
OH
OH
O
N
N
g
H
N
H
H
N
O
O
N
O
N
H
NH2
N
O
O
O
2a
2c
h
3a
R' =4-Fluorophenyl:
i
R' =4-Methoxyphenyl: 3b
R' =CH2OCH3: 3c
OH
OH
N
OH
OH
O
N
N
H
N
H
H
O
N
O
N
N
N
N
H
N
H
R'
O
O
O
F
3a-3c
2b
Scheme 1. The synthetic route of target compounds. Reagents and conditions: (a) NaCN, NH4Cl, ammonia (30%) in H2O, rt; (b) Cbz–Cl, THF, NaHCO3; (c) NH2OH, IPA; (d) i:
DMAD, MeOH, rt; ii: xylene, refluxed; (e) RCH2NH2, EtOH, microwave 90 °C; (f) NH2NH2.H2O, EtOH, refluxed; (g) i: NaNO2, 2 mol/L HCl; ii: toluene, refluxed; (h) 4-
Fluorobenzaldehyde, THF, refluxed; (i) R0CH2NH2, THF.
In general, the introduction of a phenyl group at position R (1a–
1f) leads to more potent anti-HIV activities than those of a benzyl
group (1g–1h) or indol-2-methyl group (1i). It was evident that an
aromatic ring separated by one sp3 atom was essential for the
showed more potent activity than 2c, especially the EC50 value of
3a, which was approximately 9 lM.
Table 1
activity since the aromatic ethanamine 1g–1i (EC50 > 5 lM, TI <5)
Anti-HIV activity of dihydroxypyrimidines in vitroa
lost significant activity. Then the substitutions of benzyl groups
were explored, when a non-substituted benzyl group (1a) or mon-
osubstituted benzyl groups (1b–1e) were introduced, the EC50 and
b
c
Compounds
CC50
(l
M)
EC50 (lM)
TId
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
2a
2b
2c
3a
41.44
53.94
0.32
0.14
0.55
0.24
0.15
12.32
8.61
11.58
7.12
25.99
8.30
24.45
164.35
27.66
74.19
9.87
128.16
396.10
383.04
244.16
1092
8.89
4.11
3.32
4.76
13.40
31.16
8.33
>3.37
13.66
>7.83
8.21
TI values of the corresponding compounds were <0.6 lM and >100,
233.82
58.13
164.03
109.50
35.40
38.49
34.15
348.19
258.68
203.72
>500
respectively. However, when the multisubstituted benzyl group
was introduced at position R, the anti-HIV activities were reduced
sharply (1f). No significant changes in the anti-HIV activities were
observed with introduction of aliphatic chains at position R (1j–1l)
of the amide. However among these compounds, the TI value of 1k
was >20. After optimization of the amide moiety, the conversion of
amide to hydrazide (2a) and phenylmethylene hydrazide (2b)
caused a substantial loss of the antiviral activity. On the other
hand, the hydroxyl group on the dihydroxypyrimidine is crucial
for the potency. When the hydroxyl group was cyclized to yield
compound 2c, the anti-HIV activity of compound 2c markedly de-
creased. Furthermore, the benzylureido substituents were intro-
duced from compound 2c, the corresponding analogs (3a–3c)
377.82
>500
81.10

D.-L. Guo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7114–7118
7117
Compounds 1e and 3a were chosen as representative of the tar-
get compounds and further analyzed. These compounds were
docked into homologous models of the HIV integrase core domain
based on original crystal structures of IN from prototype foamy
virus (PFV) (pdb: 3OYA)14,20 using GLIDE 4.0. The docking study
revealed the following information: (1) the anti-HIV activity of
compounds 1e and 3a involved the two-metal chelating mecha-
nism, but the specific binding mode is different from raltegravir.
The C-6 hydroxyl group, 5-ketone and 4-carbonyl group in ralte-
gravir form the chelation (Fig. 4A), but for compound 1e, the C-6,
C-5 hydroxyl and the NH of the 4-carboxamide form the chelation
(Fig. 4B). Moreover, for compound 3a, the C-6, C-5 hydroxyl groups
and the two NHs of 4-ureido form the chelation (Fig. 4C). (2) Sim-
ilar to raltegravir, the substituted benzyl group and pyrimidine
Table 1 (continued)
b
c
Compounds
CC50
(lM)
EC50
(lM)
TId
3b
>500
>500
5110
93.22
52.29
0.019
4.46
9.13
274200
3c
AZTe
a
Values are means of two separate experiments.
CC50 (50% cytotoxic concentration), concentration of drug that causes 50%
b
reduction in total C8166 cell number.
c
EC50 (50% effective concentration), concentration of drug that reduces syncytia
formation by 50%.
d
In vitro therapeutic index (CC50 value/EC50 value).
AZT was used as a positive control.
e
ring of compounds 1e and 3a exhibit
with DC16 and DA17, respectively. (3) No
p
–
p
stacking interactions
stacking interac-
p–p
tions could be detected between the benzyloxycarbonyl group in
the two compounds and Tyr 212, but the benzyloxycarbonyl group
in compound 1e could form a hydrogen bond with Tyr 212, which
could explain why the anti-HIV activity of compound 1e is better
than that of compound 3a. Generally, compounds 1e and 3a could
meet the requirements of the minimal pharmacophore embedded
in major IN inhibitors: specifically, a chelating triad capable of
binding two Mg2+ ions and a hydrophobic benzyl moiety.7
In summary, we designed and synthesized a series of dihy-
droxypyrimidine derivatives (1a–1l, 2a–2c, 3a–3c) that exhibited
obvious anti-HIV activity in vitro. Among all of the analogs, com-
pounds 1a–1e, 1k, and 3a exhibited potent anti-HIV activities.
Especially, 1b and 1e exhibited significant anti-HIV activities with
EC50 values of 0.14 and 0.15 lM, and TI values of >300 and >900,
respectively. Therefore, the dihydroxypyrimidine analogs afforded
advantageous features of improved antiviral potency and de-
creased cytotoxicity with high therapeutic index, giving rise to
the discovery of potent anti-HIV agents.
Acknowledgments
We gratefully acknowledge financial support from National Ba-
sic Research Program of China (Grants 2012CB518005 and
2012CB518005), the National Natural Science Foundation of China
(Grants 20721003 and 81025017), National S&T Major Projects
(2012ZX09103-101-072) and Silver Project (260644).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
09.070.
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