Synthesis and biological evaluation of novel 2-arylalkylthio-4-amino-6-benzyl pyrimidines as potent HIV-1 non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.04.051

Novel 2-aryalkylthio-4-amino-6-benzylpyrimidines (3a-i), which can be considered as S-DABO and TMC-125 analogue hybrid molecules, have been designed and synthesized as inhibitors of HIV-1 RT. The results clearly indicated that the changes at the N₃/C₄ position of pyrimidine ring could affect the hydrogen bonds strength and number between N₃/C₄ and the Lys101 residue which are indispensable for anti-HIV-1 RT activity. The biological activity results are also in accordance with the docking study.

Full text of DOI:10.1016/j.bmcl.2009.04.051

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3003–3005
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel 2-arylalkylthio-4-amino-6-benzyl
pyrimidines as potent HIV-1 non-nucleoside reverse transcriptase inhibitors
Hua Qin a, Chang Liu a, Jianfang Zhang a, Ying Guo b, Siwei Zhang c, Zhili Zhang b, Xiaowei Wang b,
Liangren Zhang c, Junyi Liu b,c,
*
a College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
b Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR China
c State key Laboratory of National and Biomimetic Drug, Peking University, Beijing 100191, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 2-aryalkylthio-4-amino-6-benzylpyrimidines (3a–i), which can be considered as S-DABO and TMC-
125 analogue hybrid molecules, have been designed and synthesized as inhibitors of HIV-1 RT. The
results clearly indicated that the changes at the N3/C4 position of pyrimidine ring could affect the hydro-
gen bonds strength and number between N3/C4 and the Lys101 residue which are indispensable for anti-
HIV-1 RT activity. The biological activity results are also in accordance with the docking study.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 27 October 2008
Revised 13 March 2009
Accepted 15 April 2009
Available online 18 April 2009
Keywords:
Non-nucleoside reverse transcriptase
inhibitors (NNRTIs)
2-Aryalkylthio-4-amino-6-
benzylpyrimidines
Docking
Since the discovery of human immunodeficiency virus (HIV)
caused the acquired immunodeficiency syndrome (AIDS),1 several
potential targets for antiviral chemotherapy such as reverse trans-
criptase (RT), protease enzymes and the fusion process have been
found out during the last decade. Among them the non-nucleoside
reverse transcriptase inhibitors (NNRTIs) serve as a representative
of most frontline HIV combination therapies which are highly
effective drugs with less side effects, such as nevirapine and efavi-
renz.2–4 But NNRTIs are short for rapidly triggering the emergence
of drug resistant HIV-1 variants and the cross-resistance between
these structurally unrelated drugs.5,6 Therefore, one urgent solu-
tion is to discover and develop new highly active agents with im-
proved resistance profiles simultaneous.
TIs share a common mode of action with the HIV-1 reverse trans-
criptase. Substituting with aryl moiety at C-2 and C-6 alkyl chains
of the pyrimidine ring are structural determinant. The extended
side chain at C-2 position point toward into a pocket mainly de-
fined by His235, Pro236 and Val106, and the chain at C-6 position
is located in a hydrophobic region delimited by Tyr181, Tyr188 and
Trp229. Moreover, the NH–C@O moiety at N3/C4 position of pyrim-
O
N
NH2
Br
H
N
O
N
HN
N
O
HN
N
O
N
N
O
Following the dihydro-alkoxy-benzyl-oxopyrimidines (DABOs)
were reported in 1992 as one of the most representative NNRTIs
in the past decade, a number of DABOs were synthesized and
tested as anti-HIV-1 agents with the aim to obtain more potent
and selective drugs, especially the S-DABOs reported to date
(ID50 = 26 nM toward the isolated wt enzyme) with subnanomolar
activity toward both the wt and the pluriresistant virus (IRLL98)
HIV-1 strain (EC50 < 0.14 nM and EC50 = 0.22 nM, respectively)
(Fig. 1).7–10,16 Studies on crystal structures of S-DABOs and other
NNRTIs such as TNK-651 and TMC-125 suggested that these NNR-
CN
TMC-125
CN
Nevirapine
TNK-651
R1
N
O
NH2
HN
N
S
N
X
R
N
S
R
N
R1=Cl, 2a-i
X=O, DABOs
X=S, S-DABOs
X=S, 1a-i
3e
R1=NH2, 3a-i
* Corresponding author. Tel.: +86 010 82801706; fax: +86 010 62015584.
E-mail addresses: yxyx@bjmu.edu.cn, oiqh818@sohu.com (J. Liu).
Figure 1. Structures of nevirapine, TNK-651, TMC-125, DABO and designed target
compounds.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.051

3004
H. Qin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3003–3005
idine ring is a crucial factor for the activity because the hydrogen
bonds are formed by N3–H with the carbonyl group of Lys101.11–18
Although the structure–activity relationships (SAR) of DABOs
have been studied for many years, no or little studies on the effect
of varying the hydrogen bond strength and number have been re-
ported. An inspiration prompts us to investigate the importance of
hydrogen bond strength and number between the HIV-1 RT and
the N3/C4 position of the pyrimidine ring. So we designed a series
of novel 2-aryalkylthio-4-amino-6-benzyl-pyrimidines 3 which
can also be considered as S-DABO and TMC-125 analogue hybrid
molecules with two possible orientations in RT as new potential
NNRTIs. The N3H–C4@O group of pyrimidine ring was changed into
N3@C4–NH2 group while the basic structures of the alkyl group
chains with an aryl or substituted aryl moiety at the C-2 and C-6
position were kept to verify whether the changes of hydrogen bond
strength and number at the C-4 position of pyrimidine ring will ef-
fect the activities.
O
O
O
HN
S
O
O
a
d
b
N
H
6
Br
O
4
5
O
Cl
NH2
N
HN
N
N
c
e
S
R
N
S
R
N
S
R
2a-i
1a-i
3a-i
a: R = C6H5CH2
c: R = C6H5CH2CH2CH2
CH2
b: R = C6H5CH2CH2
d: R = (O-CH3)C6H4CH2
f: R = (m-OCH3)C6H4CH2
h: R = 2-naphthyl methyl
e: R = (m-CH3)C6H4
g: R = (p-t-Bu)C6H4CH2
i: R = C6H5(CH3)CH2
To verify our design, the compound of 3e26 was flexibly docked
into the binding site of HIV-1 RT (PDB entry 1RT2, complexed with
TNK-651) using AUTODOCK 3.05 program.19 Default parameters were
used as described in the AUTODOCK manual unless otherwise speci-
fied. The molecule was docked with 100 genetic algorithm runs of
up to 250,000 energy evaluations for each run in the docking study
of 3e with a RT non-nucleoside binding site (NNBS). It was pre-
dicted that the binding mode of 3e has approximate conformation
as in the TNK-651-RT complex (Fig. 2). The result showed that
two chains at C-2 and C-6 of 3e are located in the HIV-1 RT NNBS
as we discussed above. A hydrogen bond is formed between the
N3–H moiety of the TNK-651 and the carbonyl group of Lys101,
but there are two hydrogen bonds are formed between the N3/C4
positions of 3e and the carbonyl group of Lys101(Fig. 2) as the tau-
tomerization between N3H–C4@NH and N3@C4–NH2. In addition,
the hydrogen bonds between 3e and Lys101 could result in closer
interactions (distance C4–NÁ Á ÁO 2.63 Å) than TNK-651 (distance
N3Á Á ÁO 2.76 Å) complex. Based on these results, we hypothesized
that the introduction of NH2 at C-4 position would increase the
affinity in the ligand/enzyme interaction and endow the com-
pounds with higher activities against the HIV-1-RT in a broader
range. To investigate the role of hydrogen bonds and compare the
structure–activity relationship, a Cl substitution was introduced
at the C-4 position of pyrimidine ring which can not form a hydro-
gen bond with Lys101. So we synthesized three series of com-
pounds 1a–i, 2a–i and 3a–i by an efficient route and tested the
activity against HIV-1 RT.
Scheme 1. Reagents and conditions for the chemical synthesis: (a) benzyl cyanide,
Zn/THF, reflux, 95%; (b) thiourea, EtONa, EtOH, reflux, 86%; (c) substituted alkyl
halide, Na/MeOH, rt, 65–88%; (d) POCl3, rt, 79–98%; (e) NH3ÁH2O, dixone, 80–90 °C,
12–24 h, 58–95%.
the presence of sodium ethoxide in boiling ethanol gave the
6-benzyl thiouracil 6,21 which was subjected to S-alkylation with
appropriate substituted alkyl halide in MeONa to afford the
S-DABO analogues 1a–i.22 By chlorination of 1a–i with an excess of
POCl3 obtained the products 2a–i.23 The replacement of chlorine
with amine in dioxane by heated at about 80–90 °C in a sealed tube,
gave the desired compounds 3a–i.24 The yields of all compounds are
showed in Table 1. Both analytical and spectral data of all the target
compounds are accordant with the proposed structures.
The compounds 1a–3i were tested for antiviral activity against
HIV-1 RT, using a poly(ra)/oligo(dT)15 homopolymer template with
HIV antigen detection ELISA for quantifying expression of HIV-1 RT
Table 1
The yields of all target compounds (1a–3i)
R1
O
HN
S
N
R
R
N
1a-i
S
N
2a-3i
Compds
R
R1
Yields (%)
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
2c
2d
2e
2f
2g
2h
2i
3a
3b
3c
3d
3e
3f
3g
3h
3i
C6H5CH2
C6H5CH2 CH2
—
—
—
—
—
—
—
—
—
Cl
Cl
Cl
Cl
Cl
Cl
Cl
80
77
88
88
59
78
79
65
65
98
95
79
93
94
95
91
86
95
85
83
79
94
95
95
68
70
58
The synthesis of the newly designed compounds is described in
Scheme 1. The b-ketoesters 5 was prepared by reaction between
benzyl cyanide with zinc and ethyl 2-bromoacetate 4 using the
method of Hannick and Kishi.20 Condensation of 5 and thiourea in
C6H5CH2 CH2 CH2
(O-CH3)C6H4CH2
(m-CH3)C6H4CH2
(m-OCH3)C6H4CH2
(p-t-Bu)C6H4CH2
2-Naphthyl methyl
C6H5(CH3)CH
C6H5CH2
C6H5CH2 CH2
C6H5CH2 CH2 CH2
(O-CH3)C6H4CH2
(m-CH3)C6H4CH2
(m-OCH3)C6H4CH2
(p-t-Bu)C6H4CH2
2-naphthyl methyl
C6H5(CH3)CH
Cl
Cl
C6H5CH2
C6H5CH2 CH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
C6H5CH2 CH2 CH2
(O-CH3)C6H4CH2
(m-CH3)C6H4CH2
(m-OCH3)C6H4CH2
(p-t-Bu)C6H4CH2
2-Naphthyl methyl
C6H5(CH3)CH
Figure 2. Binding mode of 3e (green) and TNK-651 (white) with HIV-RT. Hydrogen
bonds are shown as red (3e) and black (TNK-651) dashed lines.

H. Qin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3003–3005
3005
in culture medium, and nevirapine as a reference compound (Table
2).25 It is interesting to note that three series of compounds reflect
obvious structure–activity relations.
role of substituted groups on the aromatic ring at the arylalkylthio
substituent of C-2 and C-6 position.
In summary, we have designed three series of novel 2-aryalkyl-
thio-4-amino-6-benzylpyrimidines as inhibitors of HIV-1 RT and
described the convenient and efficient method of synthesis for
the first time. Although all of the IC50 values of the tested com-
pounds were suboptimal comparing to the nevirapine, the SAR
exploration encouraged us to the new rational design and the fur-
ther structure modifications on the N3/C4 position and two chains
of the pyrimidine ring are underway.
If the C-4 position is substituted by OH or NH2 group, hydrogen
bonds between the compounds and the HIV-1 RT could be formed
and obvious activities could be showed. On the contrary, if the C-4
position is substituted by Cl atom, no hydrogen bond and no activ-
ity could be observed. So the ability of forming hydrogen bond at
the N3/C4 position is critical to the potent activity. More com-
pounds in series 3 such as 3d, 3e, 3h and 3i show higher activity
of IC50 values than 1d, 1e, 1h and 1i in series 1, respectively. A pre-
liminary finding could be obtained from the data that the C4–NH2
could increase the hydrogen bonds strength and number with
Lys101 and increased the activity to some extent. We also noted
that some compounds in series 1 such as 1b and 1c have obvious
activities but the relevant 3b and 3c have not any activity. The
explanation could be that the other function groups(such as C-2
chains) of compounds 3b and 3c take place the conformation
change which is different from 1b and 1c when the hydrogen
bonds between N3/C4 and HIV-RT is stronger and closer. Besides,
when we pay attention to the C-2 chain we found that if the size
of substituted groups at the benzene is small (H) or big (t-Bu) are
not beneficial for the activity. More detailed SAR studies on these
compounds are under way with a focus on exploring the important
Acknowledgments
We thank the National Sciences Foundation of China (No.
20672008) and 985 program of Ministry of Education of China
for financial support.
References and notes
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Table 2
Structure and enzymatic activitiesa (IC50) of 1a–3i
8. Gazzard, B. G.; Pozniak, A. L.; Rosenbaum, W.; Yeni, G. P.; Staszewski, S.;
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Ye, H.; Lichtenstein, M. A.; Andries, K.; Pauwels, R.; de Bethune, M. P.; Boyer, P. L.;
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Colla, P. Eur. J. Med. Chem. 1992, 27, 251.
R1
O
HN
N
R
R
S
N
1a-i
S
N
2a-3i
R1
IC50
NAc
(lM)
b
Compds
R
1a
2a
3a
1b
2b
3b
1c
2c
3c
1d
2d
3d
1e
2e
3e
1f
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2 CH2
C6H5CH2 CH2
C6H5CH2 CH2
—
Cl
NH2
—
Cl
NH2
—
Cl
NH2
—
Cl
NH2
—
Cl
NH2
—
Cl
NH2
—
Cl
NH2
—
Cl
11. Mai, A.; Artico, M.; Sbardella, G.; Massa, S.; Loi, A. G.; Tramontano, E.; Scano, P.;
La Colla, P. J. Med. Chem. 1995, 38, 3258.
>100
>100
17.45
NA
12. Das, K.; Lewi, P. J.; Hughes, S. H.; Eddy, A. Prog. Biophys. Mol. Biol. 2005, 88, 209.
13. Mai, A.; Artico, M.; Sbardella, G.; Quartarone, S.; Massa, S.; Loi, A. G.; De Montis,
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14. Mai, A.; Sbardella, G.; Artico, M.; Ragno, R.; Massa, S.; Novellino, E.; Greco, G.;
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Chem. 2001, 44, 2544.
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Marceddu, T.; Cadeddu, A.; La Colla, P. J. Med. Chem. 2004, 47, 928.
16. Manetti, F.; Jose, A. E.; Clotet-Codina, I.; Armand-Ugon, M.; Maga, G.; Crespan,
E.; Cancio, R.; Mugnaini, C.; Bernardini, C.; Togninelli, A.; Carmi, C.; Alongi, M.;
Petricci, E.; Massa, S.; Corelli, F.; Botta, M. J. Med. Chem. 2005, 48, 8000.
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Marturana, F.; Cadeddu, A.; Maga, G.; Colla, P. L. Bioorg. Med. Chem. 2005, 13, 2065.
19. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
NA
C6H5CH2 CH2 CH2
C6H5CH2 CH2 CH2
C6H5CH2 CH2 CH2
(O-CH3)C6H4CH2
(O-CH3)C6H4CH2
(O-CH3)C6H4CH2
(m-CH3)C6H4CH2
(m-CH3)C6H4CH2
(m-CH3)C6H4CH2
(m-OCH3)C6H4CH2
(m-OCH3)C6H4CH2
(m-OCH3)C6H4CH2
(p-t-Bu)C6H4CH2
(p-t-Bu)C6H4CH2
(p-t-Bu)C6H4CH2
2-Naphthyl methyl
2-Naphthyl methyl
2-Naphthyl methyl
C6H5(CH3)CH
8.17
NA
NA
47.72
NA
29.89
91.14
90.77
18.03
90.85
NA
2f
3f
>100
NA
20. Hannick, S. M.; Kishi, Y. J. Org. Chem. 1983, 48, 3833.
21. Danel, K.; Larsen, E.; Pedersen, E. B. Synthesis 1995, 934.
1g
2g
3g
1h
2h
3h
1i
22. Danel, K.; Pedersen, E. B.; Nielsen, C. J. Med. Chem. 1998, 41, 191.
23. Carraro, F.; Naldini, A.; Pucci, A.; Locatelli, G. A.; Maga, G.; Schenone, S.; Bruno,
O.; Ranise, A.; Bondavalli, F.; Brullo, C.; Fossa, P.; Menozzi, G.; Mosti, L.;
Modugno, M.; Tintori, C.; Manetti, F.; Botta, M. J. Med. Chem. 2006, 49,
1549.
24. Nugent, R. A.; Schlachter, S. T.; Murphy, M. J.; Cleek, G. J.; Poel, T. J.; Wishka, D. G.;
Graber, D. R.; Yagi, Y.; Keiser, B. J.; Olmsted, R. A.; Kopta, L. A.; Swaney, S. M.;
Poppe, S. M.; Morris, J.; Tarpley, W. G.; Thomas, R. C. J. Med. Chem. 1998, 41, 3793.
25. Lu, X.; Chen, Y.; Guo, Y.; Liu, Z.; Shi, Y.; Xu, Y.; Wang, X.; Zhang, Z.; Liu, J. Bioorg.
Med. Chem. 2007, 15, 7399.
26. Spectral data for compound (3e) 2-(3-methylbenzylthio)-6-benzylpyrimidin-4-amine:
White solid; mp 126–128 °C; 1H NMR (500 MHz, CDCl3) d (ppm): 2.34 (s, 3H, CH3),
3.92 (s, 2H, C6H5CH2), 4.38 (s, 2H, CH2S), 4.77 (s, 2H, C4–NH2), 5.85 (s, 1H, C5–H),
7.06-7.35 (m, 9H, Ar–H); 13C NMR (125 MHz, CDCl3) d (ppm): 21.37 (CH3), 35.00
(CH2S), 43.78 (C6H5CH2), 99.38 (C-5), 126.13, 126.68, 127.71, 128.24, 128.59,
129.44, 129.80, 137.83, 137.96, 138.21 (C-arom), 162.89 (C-4), 168.58 (C-6), 170.63
(C-2); HRMS (ESI) m/z [M+H]+ calcd for C19H20N3S: 322.1378. Found: 322.1373.
NA
NA
NA
NA
NH2
—
Cl
17.14
>100
NA
2i
3i
C6H5(CH3)CH
C6H5(CH3)CH
NH2
63.90
a
Nevirapine was used as a reference compound here; IC50 for nevirapine was
M.
Compound dose (
4.12
l
b
l
M) required to inhibit the HIV-1 RT 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 concen-
tration of 100 lM.