Pharmacophore and structure-activity relationships of integrase inhibition within a dual inhibitor scaffold of HIV reverse transcriptase and integrase

Article abstract of DOI:10.1016/j.bmc.2010.05.004

Rational design of dually active inhibitors against human immunodeficiency virus (HIV) reverse transcriptase (RT) and integrase (IN) has proved viable with 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) type of non-nucleoside RT inhibitors (NNRTIs). To establish the pharmacophore and study the structure-activity relationships (SAR) of integrase inhibition within a previously disclosed RT/IN dual inhibitor scaffold, new analogues featuring substitution at different sites of the HEPT ring were designed and synthesized. These studies have revealed an IN inhibition pharmacophore that is merged with the known RT pharmacophore through a shared C-6 benzyl group. Further SAR also demonstrated that optimal IN inhibition within our dual inhibitor scaffold requires a regiospecific (N-1) diketoacid (DKA)-carrying pendant with a certain length.

Full text of DOI:10.1016/j.bmc.2010.05.004

Bioorganic & Medicinal Chemistry 18 (2010) 4202–4211
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pharmacophore and structure–activity relationships of integrase inhibition
within a dual inhibitor scaffold of HIV reverse transcriptase and integrase
*
Zhengqiang Wang , Jing Tang, Christine E. Salomon, Christine D. Dreis, Robert Vince
Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware St. SE, MMC 204, Minneapolis, MN 55455, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Rational design of dually active inhibitors against human immunodeficiency virus (HIV) reverse trans-
criptase (RT) and integrase (IN) has proved viable with 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thy-
mine (HEPT) type of non-nucleoside RT inhibitors (NNRTIs). To establish the pharmacophore and study
the structure–activity relationships (SAR) of integrase inhibition within a previously disclosed RT/IN dual
inhibitor scaffold, new analogues featuring substitution at different sites of the HEPT ring were designed
and synthesized. These studies have revealed an IN inhibition pharmacophore that is merged with the
known RT pharmacophore through a shared C-6 benzyl group. Further SAR also demonstrated that opti-
mal IN inhibition within our dual inhibitor scaffold requires a regiospecific (N-1) diketoacid (DKA)-car-
rying pendant with a certain length.
Received 12 April 2010
Revised 30 April 2010
Accepted 1 May 2010
Available online 7 May 2010
Keywords:
HIV
Integrase
Pharmacophore
SAR
Ó 2010 Elsevier Ltd. All rights reserved.
Diketoacid
1. Introduction
and represent a subject of tremendous challenge and growing
interest in medicinal chemistry.12–14 We have previously disclosed
The clinical management of acquired immunodeficiency syn-
drome (AIDS) caused by HIV1 is complicated by the lack of vac-
cines2 and the persistence of viral reservoirs.3 Treatment of HIV/
AIDS is essentially limited to highly active antiretroviral therapy
(HAART),4 a multi-target regimen that combines antivirals with
orthogonal mechanisms of action, thus creating a larger barrier to
resistance selection than monotherapy. HAART is centered around
inhibitors of RT and protease (PR), and is enhanced by recently ap-
proved antivirals targeting new molecular mechanisms of HIV rep-
lication, such as CCR5 antagonist maraviroc5 and IN inhibitor
raltegravir.6,7 Nevertheless, the adherence required by multi-target
therapy to effectively curb resistance hinges largely on its tolerance
as well as regimen simplicity, which could be difficult to achieve
with HAART cocktails.8,9 Instead, a maximally simplified multi-tar-
get therapy in which a single chemical scaffold confers inhibitory
activities against multiple viral enzymes offers an appealing alter-
native with less problems of dosing complexity, drug–drug interac-
tions and toxicities.10,11 Such multi-functional single-structure
compounds are referred to as designed multiple ligands (DMLs)
the first rationally designed dually active inhibitors against HIV RT
and IN based on a few NNRTI scaffolds.15–17 Amongst these the best
ones were designed based on TNK-651 (1, Fig. 1), an RT inhibitor of
the HEPT family18 that has an N-1 pendant extending from the
NNRTI binding pocket to the protein/solvent interface. This orienta-
tion allowed the introduction of a relatively hydrophilic chelating
group, namely a DKA functionality, to induce inhibitory activity
against IN.15 Compounds thus designed (e.g., 2, Fig. 1) were found
active against both enzymes, and showed exceptional anti-HIV
activity.15 This design, however, was largely based on a well-
known RT pharmacophore with only a minimal DKA functionality
designed in for IN inhibition. It remains unknown as for what other
structural moieties within this dual inhibitor scaffold may have
contributed to the observed anti-IN activity and whether this activ-
ity is optimized. Given the lack of critical structural information on
HIV IN catalysis, studies aimed at unraveling the pharmacophore
and SAR of IN inhibition within a dual inhibitor scaffold would pro-
vide knowledge and insights necessary for future design of novel
RT/IN dual inhibitors with improved IN inhibition.
Notably major chemotypes of IN inhibitors all feature a DKA
functionality or its heterocyclic bioisostere, and selectively inhibit
strand transfer (ST).19–21 Despite success in clinical development of
ST inhibitors as manifested by the recently approved raltegravir,
structure based design of IN inhibitors is prohibitively hindered
by the insufficient characterization of detailed binding mode,
which most likely involves a DNA–IN interface.19 The DNA bound
IN crystal structure has eluded the field until very recently.22 As
Abbreviations: HIV, human immunodeficiency virus; AIDS, acquired immuno-
deficiency syndrome; RT, reverse transcriptase; IN, integrase; HEPT, 1-[(2-hydroxy-
ethoxy)methyl]-6-(phenylthio)thymine; NNRTI, non-nucleoside reverse transcriptase
inhibitors; SAR, structure–activity relationship; DKA, diketoacid; HAART, highly active
antiretroviral therapy; DML, designed multiple ligand; CPE, cytopathic effect.
* Corresponding author. Tel.: +1 612 626 7025; fax: +1 612 625 8154.
E-mail address: wangx472@umn.edu (Z. Wang).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.004

Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4202–4211
4203
H
N
O
O
N
O
O
OH
H
OH
1, TNK-651
O
N
O
RTIC50: 8.0 nM
INIC50: > 100 µM
O
N
O
2
O
OH
OH
RTIC 50: 57 nM
INIC50: 2.4 µM
O
15
RT IC50: > 100 µM
INIC50: 0.09 µM
Figure 1. Rationally designed RT/IN dual inhibitor 2 based on HEPT NNRTI 1 and DKA IN inhibitor 15.15
a result, docking models23,24 constructed based on partial IN struc-
ture or homologous enzymes generally lack this critical DNA–IN
interface, hence the biological relevance and predictability.25 Nev-
ertheless, a vastly simplified pharmacophore model implicated in
major SAR studies has revealed two minimal structural compo-
nents required for IN binding: a chelating triad which binds with
two Mg2+ ions in a relatively hydrophilic region, anchoring the
inhibitor onto the protein surface; and a hydrophobic benzyl
moiety that fits in a highly hydrophobic cavity near the active site
(Fig. 2).26–29 As observed in compound 15, a stereotypical DKA IN
inhibitor has a benzyl group immediately connected to an aromatic
ring on which the DKA functionality is hanging (Fig. 1). Intrigu-
ingly, our dual inhibitor 2 exhibits potent inhibitory activity
against IN without having such a typical pharmacophore. We
hypothesize that the C-6 benzyl group (circled in blue, Fig. 2) of
the HEPT RT pharmacophore may have also contributed to IN inhi-
bition by binding to the hydrophobic cavity. We intend to verify
this pharmacophore hypothesis and explore its SAR by studying
the spatial relationship between the C-6 benzyl group and the
DKA chelator. Such a relationship has proved central to the SAR
of typical DKA IN inhibitors.
Detailed SAR design is delineated in Figure 3. We first designed
compounds 3–6 to probe if an additional benzyl group is required
for IN binding (Fig. 3). Introducing a benzyl in N-1 side chain would
render the right half of these compounds identical to stereotypical
DKA IN inhibitor 15 (Fig. 1). Compounds 7–8 were designed to
evaluate how the length of the N-1 linker affects IN binding,
whereas compounds 9–10 (substituted at C-2) and compounds
11–14 (substituted at N-3 position) were included to study the im-
pact of the angle between the chelator and benzyl on IN inhibition.
As the focus of this study is primarily on understanding IN inhibi-
tion, potential loss of RT binding affinity incurred from structural
modifications is anticipated. Balancing inhibitory activities against
different enzyme targets will be the concern of future studies and
can be best achieved with well defined pharmacophores, which is
the main goal of this particular study.
2. Results and discussion
2.1. Chemical synthesis
The chemistry involved in this study primarily concerns the
derivatization at N-1, C-2 and N-3 sites of pyrimidine compounds
to prepare analogues proposed in Figure 3. Particularly the regiose-
lective functionalization at N-1/N-3 is not easily accessible. Exclu-
sive N-1 alkylation can be achieved via bissilylated intermediates
24–27 (Scheme 1), though this reaction works with highly reactive
electrophiles only, such as chloromethyl ether (e.g., 19), benzylic/
allylic halide or methyl iodide. This strategy allowed the prepara-
tion of target compounds 3–6 (Scheme 1). The synthesis starts with
a Suzuki coupling reaction to assemble the benzyl alcohol interme-
diate 18, which was converted into chloromethyl ether 19 using
previously reported method.15 The subsequent alkylation provided
the access to key methyl ketone intermediates 28–31. These
methyl ketones were further elaborated into desired DKA com-
pounds 3–6 via a Claisen type condensation and a saponification
reaction (Scheme 1).
A similar synthetic route was employed for the synthesis of tar-
get compounds 7–8 (Scheme 2), wherein the requisite acetophe-
nones 34–35 were prepared from alcohols 32–33 through a
Friedel–Crafts acylation.30
C-2 derivatization was easily achieved through an S-alkylation
of thiopyrimidine compound 43 (Scheme 3). The alkylating agents,
bromides 41–42, were prepared by brominating alcohols 40 and
18. Conversion of alkylated intermediates, acetophenones 44–45,
into DKA target compounds 9–10 was effected through an afore-
mentioned two-step process consisting of a condensation and a
saponification.
O
OH
H
N
O
O
OH
O
N
O
n
Figure 2. Schematic representation of IN binding mode: the DKA chelator (red) anchors onto the hydrophilic surface of IN and binds to two Mg2+ ions, while a terminal benzyl
group (blue) fits in a hydrophobic cavity nearby. These two structural moieties are connected through a linker (green).

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Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4202–4211
O
O
OH
OH
OH
O
OH
O
N
O
n
N
S
NH
O
N
H
O
11, n = 1
12, n = 2
13, n = 3
14, n = 5
10
O
OH
N-3 linker
N
OH
S
O
NH
9
C-2 linker
O
O
OH
H
O
3 N
O
OH
2
N1
O
O
6
2
Longer N-1 linker
Additional benzyl
in N-1 linker
O
OH
O
OH
H
N
H
OH
O
R
O
O
OH
O
N
O
O
N
N
O
O
n
R'
3
4
5
6
, R = iPr, R' = H
, R = iPr, R' = Me
, R = Et, R' = H
, R = Et, R' = Me
7, n = 2
8, n = 3
R'
Figure 3. Design of SAR: minimal pharmacophores are circled (red for the DKA chelating triad and blue for the terminal benzyl group).
O
O
O
i
(HO)2B
ii
Br
+
Cl
O
HO
HO
19
18
16
17
H
N
O
O
TMSO
R
N
OTMS
iv
NH
iii
N
R
R'
R'
24, R = iPr, R' = H
25, R = iPr, R' = Me
26, R = Et, R' = H
27, R = Et, R' = Me
20, R = iPr, R' = H
21, R = iPr, R' = Me
22, R = Et, R' = H
23, R = Et, R' = Me
R'
R'
O
H
N
O
R
O
O
OH
OH
H
N
O
O
R
N
O
v-vi
R'
N
O
O
28, R = iPr, R' = H
29, R = iPr, R' = Me
30, R = Et, R' = H
31, R = Et, R' = Me
R'
R'
3, R = iPr, R' = H
4, R = iPr, R' = Me
5, R = Et, R' = H
6, R = Et, R' = Me
R'
Scheme 1. Synthesis of compounds 3–6. Reagents and conditions: (i) Pd(OAc)2, PPh3, K3PO4, toluene, rt, 81%; (ii) (HCHO)n, TMSCl, rt; (iii) N,O-bistrimethylsilylacetamide
(BSA, 2.2 equiv), CH2Cl2, rt; (iv) 24/25/26/27, TBAI, rt, 77–88%; (v) Na/EtOH, diethyloxalate, rt; (vi) NaOH, rt, 30 min, 61–72%.

Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4202–4211
4205
O
O
ii
i
HO
O
HO
Cl
O
n
n
n
32,
n = 2
n = 3
34, n = 2
36, n = 2
33,
35, n = 3
37, n = 3
H
TMSO
N
OTMS
N
O
iii
N
NH
iv
24
20
O
OH
O
H
N
H
N
OH
O
O
O
O
v-vi
N
O
n
O
N
O
n
7, n = 2
8, n = 3
38, n = 2
39, n = 3
Scheme 2. Synthesis of compounds 7–8. Reagents and conditions: (i) AcCl/AlCl3, hexanes, ꢀ10 °C to rt, 80–81%; (ii) (HCHO)n, TMSCl, rt; (iii) N,O-bistrimethylsilylacetamide
(BSA, 2.2 equiv), CH2Cl2, rt; (iv) 24, TBAI, rt, 70–99%; (v) Na/EtOH, diethyloxalate, rt; (vi) NaOH, rt, 30 min, 49–60%.
O
O
ii
Br
HO
O
i
41
42
HN
40
18
O
O
S
N
H
43
Br
HO
O
OH
OH
N
N
S
O
S
O
NH
iii
NH
O
9
44
O
O
OH
O
OH
N
O
S
N
NH
S
O
NH
45
10
O
Scheme 3. Synthesis of compounds 9–10. Reagents and conditions: (i) NBS/PPh3, THF, 0 °C, 1 h, 80%; (ii) 43, K2CO3, DMF, rt, 6 h, 50–80%; (iii) (a) Na/EtOH, diethyloxalate, rt;
(b) NaOH, rt, 30 min, 62–67%.
Functionalization at N-3 position was accomplished by direct
alkylation (Scheme 4a) in modest yields. Notably direct alkylation
with uracil or thymine generally occurs at N-1 site.31 The unusual
regioselectivity observed herein may be attributed to steric hin-
drance. Nevertheless, the efficiency of this reaction is considerably
compromised by the undesired N-1 and N-3 dialkylation. An alter-
native route32 involving selective N-1 protection and deprotection
was found to produce N-3 regioisomers in 65% yield over three
steps for compound 55 (Scheme 4b). Comparison on NMR spectra
showed that compounds prepared via both routes are identical,
confirming the N-3 regioselectivity of the direct alkylation.
2.2. Biological evaluation
All compounds were tested biochemically against both RT and
IN using recombinant enzymes. Their ability to inhibit HIV replica-

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Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4202–4211
O
O
O
N
i
n
ii
Br
Br
n
n
20
N
H
O
46, n = 1
47, n = 2
48, n = 3
49, n = 5
50, n = 1
51, n = 2
52, n = 3
53, n = 5
54, n = 1
55, n = 2
56, n = 3
57, n = 5
O
OH
OH
O
O
iii-iv
N
n
N
H
O
11, n = 1
12, n = 2
13, n = 3
14, n = 5
O
OTMS
O
NH
N
NH
vi
v
vii
N
O
N
OTMS
N
H
O
O
20
24
58
O
O
N
O
N
viii
N
O
N
H
O
O
O
55
59
Scheme 4. Synthesis of compounds 11–14. Reagents and conditions: (i) AcCl/AlCl3, CS2, 0 °C, 1 h, 75–83%; (ii) Cs2CO3, DMF, 50 °C, 2 h, 20–40%; (iii) Na/EtOH, diethyloxalate,
rt; (iv) NaOH, rt, 30 min, 46–52%; (v) N,O-bistrimethylsilylacetamide (BSA, 2.2 equiv), CH2Cl2, rt; (vi) Ethoxymethylchloride, TBAI, rt, 83%; (vii) Cs2CO3, DMF, 80 °C, 2 h, 93%;
(viii) 90% TFA (aqueous), reflux, 2 h, 84%.
zyl group can fit into the hydrophobic cavity (Fig. 2), and that this
terminal benzyl group is removed too far from the chelating DKA
by the additional benzyl group in N-1 linker. These results lend
support to our hypothesis that within our dual inhibitor scaffold
the C-6 benzyl group on the HEPT ring is an important part of
the pharmacophore for IN inhibition. This is significant as the same
benzyl group is also essential for RT binding, indicating that the
two pharmacophores responsible for dual inhibitory activities are
merged, which is a highly desirable yet not readily achievable fea-
ture for dual inhibitors (or DMLs).13,14
Table 1
Effects of an additional benzyl group in N-1 side chain on IN binding
TIc
a
a
a
b
Compound
RT IC50
M)
IN IC50
M)
HIV EC50
M)
TC50
M)
(l
(
l
(
l
(
l
3
4
5
6
0.16
0.10
0.71
0.028
0.057
0.016
>100
11
22
16
14
2.4
>100
0.093
0.46
0.17
0.56
0.014
0.033
0.016
0.16
>100
96
>220
560
56
>710
>310
>610
>61
32
>100
>10
>10
>10
2d
1d
15d
To further study the impact of the spatial arrangement between
the DKA and the C-6 benzyl group on IN inhibition, more analogues
with variable distances and angles were designed and synthesized.
Since our focus is primarily on IN binding, dose response studies
were carried out against IN only. Inhibition against HIV and RT
was evaluated at single and double concentrations, respectively
(Table 2). Interestingly, a 3–4-fold drop in anti-IN activity was ob-
served with compounds 7–8 compared to compound 2, confirming
that elongating the N-1 linker adversely affects IN binding affinity.
The most drastic loss of IN inhibition was observed with com-
a
b
c
Concentration of 50% inhibition.
Concentration of 50% cytotoxicity.
Therapeutic index, defined as the ratio of TC50/EC50
.
d
Data taken from previous publication.15
tion was evaluated using a cytoprotection (reduction of CPE) assay
with CEM-SS cells infected with the IIIB strain of HIV-1. Table 1
summarizes assay results for compounds 3–6. Significantly, com-
pound 3 which has an additional benzyl group confers an anti-
HIV activity 14-fold lower than compound 2, along with a reduced
anti-RT activity (3-fold) and anti-IN activity (5-fold). The latter is
particularly intriguing as the right half of molecule 3 is identical
to compound 15 which is a potent IN inhibitor (Fig. 1). Apparently
the additional benzyl group in the N-1 side chain severely hinders
IN binding, which strongly suggests that only the terminal C-6 ben-
pounds 9 and 10 (inactive at 100 lM), in which the side chain car-
rying the DKA chelator is placed on the C-2 of the pyrimidine ring.
Such an orientation dramatically alters the angle between the ter-
minal benzyl group and the DKA, preventing the former from bind-
ing to the hydrophobic cavity. By contrast, the placement of the
DKA-bearing pendant at the N-3 position does not seem to prohibit

Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4202–4211
4207
Table 2
4.1.1.1. 1-(4-(2-Hydroxyethyl)phenyl)ethanone (34). AlCl3 (19.56 g,
0.147 mol) was added to 30 mL of pre-cooled (ꢀ10 °C) hexanes fol-
lowed by the slow addition of alcohol 32 (6.0 g, 0.049 mol). To the
resulting slurry was added acetyl chloride dropwise. Upon completion
of addition, the mixture was allowed to stir at ꢀ10 °C for an additional
2 h. The reaction was quenched by pouring the mixture into 50 mL of
ice water. After separation, the aqueous phase was extracted with
CH2Cl2 (40 mL ꢁ 3). The combined organics were concentrated and
the residue was re-dissolved into 50 mL of THF. To the solution was
then added 50 mL of 2 N NaOH and the resulting mixture was stirred
at room temperature for 2 h. After separation the aqueous was ex-
tracted with CH2Cl2 (50 mL ꢁ 3). The combined organic extracts were
washed with saturated aqueous NH4Cl, water and brine, dried over
Na2SO4 and concentrated under reduced pressure. The resultant resi-
due was subjected to flash chromatography (silica gel, hexanes/EtOAc,
4:1) to afford compound 34 (6.5 g, 81%) as a pale yellow oil: 1H NMR
(600 MHz, CDCl3) d 7.76 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 3.75
(q, J = 6.0 Hz, 2H), 3.41 (t, J = 5.4 Hz, 1H), 2.80 (t, J = 6.6 Hz, 2H), 2.44 (s,
3H); 13C NMR (150 MHz, CDCl3) d 198.6, 145.3, 135.3, 129.4, 128.7,
63.1, 39.3, 26.7.
Effects of distance and angle between DKA and C-6 benzyl on IN binding
Compound
% Inhibition against RT
IN IC50
(
lM)
% HIV CPE reduction
at 10 lM
10
lM
100 lM
7
8
9
10
11
12
13
14
100
100
0
100
100
0
7.4
8.2
>100
>100
8.0
12
37
>100
100
91
NAa
NAa
28
0
0
0
0
0
0
0.6
0
9.0
0
0
0
0
a
Compounds are cytotoxic.
IN binding, though a considerable decrease in inhibition was ob-
served (3–15-fold for compounds 11–13), with a 2–3 atom linker
being most active. When a linker as long as six carbons was used
(compound 14), the compound was found devoid of inhibition
against IN. Not surprisingly, the derivatization at N-3 of the pyrim-
idine ring (compounds 11–14) causes a complete loss of activity
against RT (Table 2). The proton at N-3 has proved crucial for RT
binding as it forms a key hydrogen bond with K101 in the NNRTI
binding pocket.33 However, the loss of anti-RT activity observed
with C-2 derivatives (compounds 9, 10) is somewhat unexpected
as similar scaffolds have been found in dihydro(alkylthio)ben-
zyloxopyrimidines (s-DABO) compounds which are generally
active against RT.34,35
4.1.1.2. 1-(4-(3-Hydroxypropyl)phenyl)ethanone (35). This com-
pound was prepared from alcohol 33 following the procedure de-
scribed for the synthesis of 34. 1H NMR (600 MHz, CDCl3) d 7.83
(d, J = 7.8 Hz, 2H), 7.24 (d, J = 7.8 Hz, 2H), 3.63 (q, J = 5.4 Hz, 2H),
2.73 (t, J = 6.0 Hz, 2H), 2.53 (s, 3H), 2.27 (s, 1H), 1.86 (m, 2H); 13C
NMR (150 MHz, CDCl3) d 198.3, 148.2, 135.2, 128.9, 128.8, 62.0,
34.0, 32.3, 26.7.
3. Conclusions
4.1.1.3. 1-(3-(4-(Hydroxymethyl)benzyl)phenyl)ethanone (18).
A
We have designed and synthesized various derivatives of a
previously reported HIV RT/IN dual inhibitor (2) to establish the
pharmacophore of IN inhibition and explore the impact on IN bind-
ing of spatial arrangements between the DKA chelating triad and
the C-6 terminal benzyl group. Through these studies we have
demonstrated that for RT/IN dual inhibitors based on HEPT scaf-
fold, the C-6 benzyl group which constitutes part of the RT phar-
macophore also fulfils the hydrophobic binding requirement for
IN, and that to achieve optimal inhibition against IN the DKA-car-
rying pendant should be at N-1 or N-3 site of the HEPT ring and the
linker between the two minimal pharmacophores should be 2–3
atoms long (compounds 2, 11, 12). It was also observed that the
N-3 substitution causes a complete loss of anti-RT activity, leaving
N-1 as the sole site for the incorporation of the DKA chelator. Due
to the dearth of structural information on IN binding, these find-
ings provide an important source of knowledge and inspiration
for our ongoing efforts in the design and synthesis of improved
HIV RT/IN dual inhibitors.
mixture of alcohol 16 (2.01 g, 10 mmol), boronic acid 17 (2.46 g,
15 mmol), K3PO4 (20 mmol), PPh3 (104 mg, 0.4 mmol) and
Pd(OAc)2 (45 mg, 0.2 mmol) was vacuumed and purged with ar-
gon. To this was then added 30 mL of toluene. The resulting yellow
suspension was stirred at room temperature for 2 days. The reac-
tion mixture was then taken up in 100 mL of ether and washed
with 1 N NaOH and brine. The organic was dried over Na2SO4
and concentrated under reduced pressure. The resultant residue
was subjected to flash chromatography (silica gel, hexanes/EtOAc,
2:1) to afford compound 18 (1.9 g, 81%) as a light yellow solid: 1H
NMR (600 MHz, CDCl3) d 7.80–7.77 (m, 2H), 7.37–7.36 (m, 2H),
7.28 (d, J = 7.8 Hz, 2H), 7.16 (d, J = 7.8 Hz, 2H), 4.63 (d, J = 5.4 Hz,
2H), 4.01 (s, 2H), 2.55 (s, 3H), 2.07 (br s, 1H); 13C NMR (150 MHz,
CDCl3) d 198.6, 141.9, 140.0, 139.3, 137.6, 133.9, 129.3, 129.0,
128.8, 127.6, 126.6, 65.2, 41.7, 26.9.
4.1.1.4. 1-(4-(Bromomethyl)phenyl)ethanone (41). To a solution
of alcohol 40 (0.45 g, 0.003 mol) and PPh3 (0.786 g, 0.003 mol) in
THF was added NBS (0.534 g, 0.003 mol) in one portion at 0 °C.
The resulting yellow solution was stirred at 0 °C for 1 h. The reaction
was then quenched by adding 20 mL of H2O and was extracted with
CH2Cl2 (20 mL ꢁ 3). The combined organic extracts were dried over
Na2SO4 and concentrated under reduced pressure. The resultant res-
idue was subjected to flash chromatography (silica gel, hexanes/
EtOAc, 2:1) to afford compound 41 (0.48 g, 80%) as a pale yellow
oil: 1H NMR (600 MHz, CDCl3) d 7.88 (d, J = 7.8 Hz, 2H), 7.43 (d,
J = 7.8 Hz, 2H), 4.46 (s, 2H), 2.55 (s, 3H); 13C NMR (150 MHz, CDCl3)
d 197.5, 143.0, 137.1, 129.5, 129.0, 32.4, 26.9.
4. Experimental section
4.1. Chemistry
4.1.1. General procedures
All commercial chemicals were used as supplied unless other-
wise indicated. Dry solvents (THF, Et2O, CH2Cl2 and DMF) were dis-
pensed under argon from an anhydrous solvent system with two
packed columns of neutral alumina or molecular sieves. Anhydrous
ethanol was purchased from Sigma–Aldrich. Flash chromatography
was performed on a Teledyne Combiflash RF-200 with RediSep
columns (silica) and indicated mobile phase. All reactions were
performed under inert atmosphere of ultra-pure argon with
oven-dried glassware. 1H and 13C NMR spectra were recorded on
a Varian 600 MHz spectrometer. High resolution mass data were
acquired on an Agilent TOF II TOS/MS spectrometer capable of
ESI and APCI ion sources.
4.1.1.5. 1-(3-(4-(Bromomethyl)benzyl)phenyl)ethanone (42). This
compound was prepared from alcohol 18 as an orange oil following
the procedure described for the preparation of 41. Yield: 80%, 1H
NMR (600 MHz, CDCl3) d 7.81–7.79 (m, 2H), 7.37 (m, 2H), 7.31 (d,
J = 7.8 Hz, 2H), 7.15 (d, J = 7.8 Hz, 2H), 4.45 (s, 2H), 4.01 (s, 2H), 2.56
(s, 3H); 13C NMR (150 MHz, CDCl3) d 198.3, 141.5, 141.1, 137.7,
136.1, 134.0, 129.6, 129.5, 129.1, 128.9, 126.7, 41.7, 33.7, 27.0.

4208
Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4202–4211
4.1.1.6. 1-(4-(2-Bromoethyl)phenyl)ethanone (50). A mixture of
AlCl3 (8.3 g, 0.063 mol), acetyl chloride (4.7 mL) and CS2 (30 mL)
was stirred at 0 °C while a solution of bromide 46 (12.4 g,
0.067 mol) in 9.5 mL of acetyl chloride was added. The resulting
mixture was stirred at 0 °C for 1 h and then poured into a mixture
of concentrated HCl (20 mL) and ice (200 g). This mixture was ex-
tracted with CH2Cl2 (30 mL ꢁ 4). The combined organic extracts
were washed with 1 N NaOH, water and brine, dried over Na2SO4
and concentrated under reduced pressure. The resultant residue
was subjected to flash chromatography (silica gel, hexanes/EtOAc,
9:1) to afford compound 50 (11.4 g, 75%) as a pale yellow oil: 1H
(s, 3H), 1.27 (d, J = 6.6 Hz, 6H); 13C NMR (150 MHz, CDCl3) d
198.5, 162.8, 152.4, 148.6, 141.7, 140.5, 137.6, 135.7, 135.6,
134.0, 129.4, 129.2, 129.0, 128.9, 128.4, 127.5 (2), 126.6, 120.1,
73.2, 71.8, 41.7, 33.7, 28.5, 26.9, 20.7.
4.1.1.11. 1-((4-(3-Acetylbenzyl)benzyloxy)methyl)-6-(3,5-dimeth-
ylbenzyl)-5-isopropyl pyrimidine-2,4(1H,3H)-dione (29). This
compound was prepared from pyrimidine 21 as a pale yellow oil
following the procedure described for the preparation of 38. Yield:
77%, 1H NMR (600 MHz, CDCl3) d 9.58 (s, 1H), 7.80–7.78 (m, 2H),
7.37 (m, 2H), 7.26 (d, J = 7.2 Hz, 2H), 7.15 (d, J = 7.8 Hz, 2H), 6.88
(s, 1H), 6.67 (s, 2H), 5.22 (br, 2H), 4.63 (s, 2H), 4.10 (s, 2H), 4.02
(s, 2H), 2.85 (m, 1H), 2.57 (s, 3H), 2.26 (s, 6H), 1.28 (d, J = 6.6 Hz,
6H); 13C NMR (150 MHz, CDCl3) d 198.5, 162.7, 152.3, 148.8,
141.7, 140.4, 139.0, 137.6, 135.7, 135.3, 134.0, 129.2, 129.1,
129.0, 128.9, 128.4, 126.6, 125.3, 119.9, 73.2, 71.8, 41.7, 33.5,
28.6, 26.9, 21.5, 20.7.
NMR (600 MHz, CDCl3)
d 7.85 (d, J = 7.8 Hz, 2H), 7.24 (d,
J = 7.8 Hz, 2H), 3.52 (t, J = 7.8 Hz, 2H), 3.15 (t, J = 7.2 Hz, 2H), 2.51
(s, 3H); 13C NMR (150 MHz, CDCl3) d 197.8, 144.5, 136.1, 129.1,
128.9, 39.2, 32.5, .26.8.
4.1.1.7. 1-(4-(3-Bromopropyl)phenyl)ethanone (51). This com-
pound was prepared from bromide 47 as a pale yellow oil follow-
ing the procedure described for the preparation of 50. Yield: 81%,
1H NMR (600 MHz, CDCl3) d 7.85 (d, J = 7.8 Hz, 2H), 7.29 (d,
J = 7.8 Hz, 2H), 3.38 (t, J = 6.6 Hz, 2H), 2.84 (t, J = 7.2 Hz, 2H), 2.58
(s, 3H), 2.18 (quintet, J = 7.2 Hz, 2H); 13C NMR (150 MHz, CDCl3)
d 197.9, 146.5, 135.6, 129.0, 128.9, 34.2, 33.9, 33.0, 26.8.
4.1.1.12. 1-((4-(3-Acetylbenzyl)benzyloxy)methyl)-6-benzyl-5-
ethylpyrimidine-2,4(1H,3H)-dione (30). This compound was
prepared from pyrimidine 22 as a pale yellow oil following the pro-
cedure described for the preparation of 38. Yield: 81%, 1H NMR
(600 MHz, CDCl3) d 9.73 (s, 1H), 7.81–7.78 (m, 2H), 7.37 (m, 2H),
7.30–7.23 (m, 5H), 7.15 (d, J = 7.8 Hz, 2H), 7.06 (d, J = 7.2 Hz, 2H),
5.18 (br, 2H), 4.61 (s, 2H), 4.14 (s, 2H), 4.02 (s, 2H), 2.57 (s, 3H),
2.45 (q, J = 7.2 Hz, 2H), 1.04 (t, J = 7.2 Hz, 3H); 13C NMR
(150 MHz, CDCl3) d 198.5, 171.4, 163.6, 152.2, 149.1, 141.7,
140.5, 137.6, 135.6, 135.4, 133.9, 129.5, 129.2, 129.0, 128.8,
128.4, 127.6, 126.6, 117.2, 73.0, 71.8, 41.7, 33.6, 26.9, 19.4, 14.0.
4.1.1.8. 1-(4-(4-Bromobutyl)phenyl)ethanone (52). This com-
pound was prepared from bromide 48 as a pale yellow oil follow-
ing the procedure described for the preparation of 50. Yield: 83%,
1H NMR (600 MHz, CDCl3) d 7.85 (d, J = 7.2 Hz, 2H), 7.23 (d,
J = 7.8 Hz, 2H), 3.38 (t, J = 6.6 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H), 2.54
(s, 3H), 1.85 (quintet, J = 6.6 Hz, 2H), 1.76 (quintet, J = 7.2 Hz,
2H); 13C NMR (150 MHz, CDCl3) d 197.9, 147.8, 135.4, 128.8 (2),
35.2, 33.6, 32.3, 29.6, 26.8.
4.1.1.13. 1-((4-(3-Acetylbenzyl)benzyloxy)methyl)-6-(3,5-dimeth-
ylbenzyl)-5-ethylpyrimidine-2,4(1H,3H)-dione (31). This com-
pound was prepared from pyrimidine 23 as a pale yellow oil follow-
ing the procedure described for the preparation of 38. Yield: 88%, 1H
NMR (600 MHz, CDCl3) d 9.75 (s, 1H), 7.80–7.78 (m, 2H), 7.37 (m,
2H), 7.26 (m, 2H), 7.15 (d, J = 7.8 Hz, 2H), 6.87 (s, 1H), 6.66 (s, 2H),
5.21 (br, 2H), 4.62 (s, 2H), 4.07 (s, 2H), 4.02 (s, 2H), 2.57 (s, 3H),
2.45 (q, J = 7.2 Hz, 2H), 2.26 (s, 6H), 1.05 (t, J = 7.2 Hz, 3H); 13C
NMR (150 MHz, CDCl3) d 198.5, 163.7, 152.3, 149.5, 141.7, 140.5,
139.1, 137.6, 135.7, 135.1, 134.0, 129.2 (2), 129.0, 128.9, 128.4,
126.6, 125.3, 117.1, 73.1, 71.8, 41.7, 33.5, 26.9, 21.5, 19.4, 14.0.
4.1.1.9. 1-((4-Acetylphenethoxy)methyl)-6-benzyl-5-isopropyl-
pyrimidine-2,4(1H,3H)-dione (38). To a suspension of pyrimi-
dine 20 (0.23 g, 1.0 mmol) in CH2Cl2 (2.0 mL) was added BSA
(0.538 mL, 2.2 mmol) at room temperature under argon. The
resulting mixture was stirred until a clear solution was achieved
(ca. 30 min, 24 obtained). A separate flask was charged with alco-
hol 34 (246 mg, 1.5 mmol) and 1.0 mL of TMSCl. To this was added
paraformaldehyde (54 mg, 1.8 mmol). The mixture was stirred un-
til a homogeneous solution was observed (ca. 30 min). The solution
was concentrated and the residue was dissolved in 2.0 mL and
added to the freshly prepared intermediate 24, followed by the
addition of a catalytic amount of TBAI. The reaction mixture was
kept overnight and then quenched by adding a saturated aqueous
solution of NaHCO3. The aqueous phase was extracted with CH2Cl2
(10 mL ꢁ 3). The combined organic extracts were dried over
Na2SO4 and concentrated under reduced pressure. The resultant
residue was subjected to flash chromatography (silica gel, hex-
anes/EtOAc, 2:1) to afford compound 38 (0.414 g, 99%) as a pale
yellow solid: 1H NMR (600 MHz, CDCl3) d 10.24 (s, 1H), 7.83 (d,
J = 7.8 Hz, 2H), 7.28–7.20 (m, 5H), 7.02 (d, J = 7.8 Hz, 2H), 5.09
(br, 2H), 3.98 (s, 2H), 3.81 (t, J = 6.0 Hz, 2H), 2.84 (t, J = 5.4 Hz,
2H), 2.77 (m, 1H), 2.51 (s, 3H), 2.54 (s, 3H), 1.22 (d, J = 6.0 Hz,
6H); 13C NMR (150 MHz, CDCl3) d 197.9, 163.0, 152.6, 148.6,
144.7, 135.6 (2), 129.4, 129.3, 128.7, 127.4 (2), 120.0, 73.2, 69.8,
36.2, 33.5, 26.7, 20.6.
4.1.1.14. 1-((3-(4-Acetylphenyl)propoxy)methyl)-6-benzyl-5-iso-
propylpyrimidine-2,4(1H,3H)-dione (39). This compound was
prepared from bromide 35 as a pale yellow oil following the proce-
dure described for the preparation of 38. Yield: 70%, 1H NMR
(600 MHz, CDCl3) d 8.58 (s, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.34 (t,
J = 7.8 Hz, 2H), 7.27 (t, J = 7.2 Hz, 1H), 7.24 (t, J = 7.8 Hz, 2H), 7.10
(d, J = 7.2 Hz, 2H), 5.11 (br, 2H), 4.17 (s, 2H), 3.56 (t, J = 6.6 Hz,
2H), 2.86 (quintet, J = 7.2 Hz, 2H), 2.70 (t, J = 7.8 Hz, 2H), 2.56 (s,
3H), 1.87 (m, 1H), 1.28 (d, J = 7.2 Hz, 6H); 13C NMR (150 MHz,
CDCl3) d 198.0, 163.0, 152.5, 148.6, 147.6, 135.6, 135.4, 129.4,
128.8 (2), 127.5 (2), 120.1, 73.3, 68.6, 33.7, 32.4, 30.8, 28.6, 26.7,
20.6.
4.1.1.15. 3-(4-Acetylphenethyl)-6-benzyl-5-isopropylpyridine-
2,4(1H,3H)-dione (54). A solution of pyrimidine 20 (0.244 g,
1.0 mmol) in DMF (3 mL) was treated with Cs2CO3 (0.326 g,
1.0 mmol) and stirred for 30 min. To this was added a solution of
bromide 50 (0.241 g, 1.0 mmol) in 2 mL of DMF. The resulting yel-
low mix was heated at 50 °C for 2 h. The reaction was quenched by
adding 10 mL of H2O and was extracted with ethyl acetate
(10 mL ꢁ 3). The combined organic extracts were dried over
Na2SO4 and concentrated under reduced pressure. The resultant
residue was subjected to flash chromatography (silica gel, tolu-
ene/EtOAc, 4:1) to afford compound 54 (0.08 g, 20%) as a pale yel-
4.1.1.10. 1-((4-(3-Acetylbenzyl)benzyloxy)methyl)-6-benzyl-5-
isopropylpyrimidine-2,4(1H, 3H)-dione (28). This compound
was prepared from pyrimidine 20 as a pale yellow oil following
the procedure described for the preparation of 38. Yield: 87%, 1H
NMR (600 MHz, CDCl3) d 9.77 (s, 1H), 7.81–7.78 (m, 2H), 7.37–
7.24 (m, 7H), 7.15 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 7.2 Hz, 2H), 5.21
(br, 2H), 4.63 (s, 2H), 4.17 (s, 2H), 4.01 (s, 2H), 2.85 (m, 1H), 2.56

Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4202–4211
4209
low oil: 1H NMR (600 MHz, CDCl3) d 9.81 (s, 1H), 7.86–7.79 (m, 2H),
7.46–7.24 (m, 6H), 4.11 (m, 2H), 3.83 (s, 2H), 3.03–2.95 (m, 3H),
2.56 (s, 3H), 1.25 (d, J = 7.2 Hz, 6H); 13C NMR (150 MHz, CDCl3) d
198.4, 162.7, 152.5, 145.8, 144.5, 139.4, 137.5, 135.5, 134.0,
129.4, 129.3, 129.0, 128.8, 128.7, 127.8, 126.7, 116.3, 41.5, 36.5,
33.9, 27.7, 26.9, 20.5.
4.1.1.20.
2-(4-Acetylbenzylthio)-6-benzyl-5-ethylpyrimidin-
4(3H)-one (44). To a solution of thiopyrimidine 43 (0.246 g,
1.0 mmol) in DMF (1.0 mL) was added bromide 41 (0.235 g,
1.1 mmol) and K2CO3 (0.138 g, 1.0 mmol). The resulting mixture
was stirred at room temperature for 6 h and was quenched by add-
ing 10 mL of H2O. The precipitate was filtered, washed with H2O
and ether, then dried under vacuum to afford compound 44
(0.19 g, 50%) as a white solid: 1H NMR (600 MHz, CDCl3) d 7.76
(br, 2H), 7.27–7.20 (m, 7H), 4.33 (br, 2H), 3.93 (br, 2H), 2.56 (m,
5H), 1.07 (br, 3H); 13C NMR (150 MHz, CDCl3) d 197.8, 165.4,
161.8, 155.8, 143.1, 138.4, 136.3, 129.4, 129.2, 128.7 (2), 126.7,
123.0, 40.6, 34.1, 26.8, 19.0, 13.4.
4.1.1.16. 3-(3-(4-Acetylphenyl)propyl)-6-benzyl-1-(ethoxymeth-
yl)-5-isopropylpyrimidine-2,4(1H,3H)-dione (59). A solution of
58 (40 mg, 1.0 mmol) in DMF (1.0 mL) was treated with Cs2CO3
(65 mg, 1.5 mmol) and stirred for 30 min. To this was added bro-
mide 50 (38 mg, 1.2 mmol). The resulting yellow mix was heated
at 80 °C for 2 h. The reaction was quenched by adding 10 mL of
H2O and was extracted with ethyl acetate (10 mL ꢁ 3). The com-
bined organic extracts were dried over Na2SO4 and concentrated
under reduced pressure. The resultant residue was subjected to
flash chromatography (silica gel, hexane/EtOAc, 3:1) to afford com-
pound 59 (57 mg, 93%) as a pale yellow oil: 1H NMR (600 MHz,
CDCl3) d 7.88 (d, J = 7.8 Hz, 2H), 7.30–7.20 (m, 3H), 7.25 (m, 2H),
7.11 (d, J = 7.8 Hz, 2H), 5.13 (s, 2H), 4.16 (s, 2H), 4.04 (t, J = 7.8 Hz,
2H), 3.61 (q, J = 7.2 Hz, 2H), 2.88 (septet, J = 7.2 Hz, 1H), 2.78 (t,
J = 7.2 Hz, 2H), 2.57 (s, 3H), 2.04 (quintet, J = 7.8 Hz, 2H), 1.28 (d,
J = 6.6 Hz, 6H), 1.19 (t, J = 7.2 Hz, 2H), 13C NMR (150 MHz, CDCl3)
d 198.1, 161.9, 152.5, 147.6, 146.7, 135.8, 135.4, 129.4, 128.8,
128.7, 127.5, 127.4, 119.1, 73.9, 65.2, 41.5, 33.7, 33.6, 28.8, 28.7,
26.8, 20.6, 15.2.
4.1.1.21. 2-(4-(3-Acetylbenzyl)benzylthio)-6-benzyl-5-ethylpyr-
imidin-4(3H)-one (45). This compound was prepared from bro-
mide 42 as a white solid following the procedure described for
the preparation of 44. Yield: 80%, 1H NMR (600 MHz, CDCl3) d
12.78 (br, 1H), 7.79 (m, 2H), 7.35 (t, J = 7.8 Hz, 2H), 7.22 (m, 4H),
7.18–7.13 (m, 3H), 7.01 (d, J = 8.4 Hz, 2H), 4.28 (s, 2 H), 3.97 (s,
2H), 3.91 (s, 2H), 2.58–2.54 (m, 5H), 1.05 (t, J = 7.2 Hz, 3H); 13C
NMR (150 MHz, CDCl3) d 198.5, 165.5, 161.9, 156.4, 141.8, 139.8,
138.6, 137.6, 135.4, 134.0, 129.6, 129.3, 129.2, 129.0, 128.9,
128.7, 126.7, 122.7, 41.7, 40.7, 34.3, 27.0, 19.0, 13.5.
4.1.1.22. (Z)-4-(4-(2-((6-Benzyl-5-isopropyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)methoxy)ethyl)phenyl)-2-hydroxy-4-
oxobut-2-enoic acid (7). Sodium (92 mg, 4.0 mmol) was added
to 2 mL of anhydrous EtOH at room temperature under argon.
The mixture was stirred until a clear solution was obtained. To this
was added diethyl oxalate (0.219 mL, 1.6 mmol) and a suspension
of compound 38 (347 mg, 0.8 mmol) in 2 mL of anhydrous EtOH.
The solution turned yellow immediately and precipitate formed.
This mixture was stirred for 2 h and then quenched with a satu-
rated aqueous solution of NH4Cl. The resulting white precipitate
was collected by filtration, washed with water and then dried un-
der vacuum to give an ester intermediate as off-white solid. This
intermediate was then dissolved into EtOH/CH2Cl2 (1:1, 2 mL)
and treated with 1 N NaOH (2 mL). The resulting mixture was stir-
red for 30 min and then thoroughly extracted with 20% MeOH in
CH2Cl2. The aqueous phase was then acidified with 1 N HCl to pH
4. The precipitate was collected by filtration, washed with water
and dried under vacuum to give compound 7 (195 mg, 49%) as a
pale yellow solid: 1H NMR (600 MHz, CDCl3) d 10.05 (s, 1H), 7.85
(d, J = 7.8 Hz, 2H), 7.25 (m, 3H), 7.19 (m, 2H), 7.07 (s, 1H), 6.98
(d, J = 7.8 Hz, 2H), 5.05 (s, 2H), 3.97 (s, 2H), 3.83 (t, J = 6.6 Hz,
2H), 2.83 (t, J = 6.6 Hz, 2H), 2.73 (m, 1H), 1.17 (d, J = 7.2 Hz, 6H);
13C NMR (150 MHz, CDCl3) d 189.8, 170.6, 164.8, 163.8, 152.0,
149.6, 145.8, 135.2, 132.9, 129.8, 129.5, 128.4, 127.6, 127.4,
119.8, 97.7, 73.5, 70.3, 36.4, 33.7, 28.5, 20.5; HRMS (ESI-) calcd
for C27H27N2O7 [MꢀH]ꢀ 491.1823, found 491.1828 (E = 0.9 ppm).
4.1.1.17. 3-(3-(4-Acetylphenyl)propyl)-6-benzyl-5-isopropylpyr-
imidine-2,4(1H,3H)-dione (55). Method (a): This compound was
prepared from bromide 51 as a pale yellow oil following the proce-
dure described for the preparation of 54. Yield: 40%.
Method (b): Compound 59 (50 mg) was dissolved in 90% aque-
ous CF3COOH (3 mL). The resulting mixture was refluxed for 2 h
and then allowed to cool to room temperature and evaporated to
dryness, the residue was subjected to flash chromatography (silica
gel, Hexane/EtOAc, 1.5:1) to afford compound 55 (37 mg, 84%): 1H
NMR (600 MHz, CDCl3) d 9.97 (s, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.30–
7.20 (m, 7H), 3.92 (t, J = 7.2 Hz, 2H), 3.77 (s, 2 H), 3.03 (septet,
J = 7.2 Hz, 1H), 2.72 (t, J = 7.8 Hz, 2H), 2.53 (s, 3H), 1.98 (quintet,
J = 7.8 Hz, 2H), 1.28 (d, J = 7.2 Hz, 6H); 13C NMR (150 MHz, CDCl3)
d 198.1, 162.9, 152.7, 147.5, 145.9, 135.6, 135.3, 129.2, 128.8,
128.7, 127.7, 116.1, 40.4, 36.5, 33.6, 28.7, 27.7, 26.7, 20.5.
4.1.1.18. 3-(4-(4-Acetylphenyl)butyl)-6-benzyl-5-isopropylpyr-
imidine-2,4(1H,3H)-dione (56). This compound was prepared
from bromide 52 as a pale yellow oil following the procedure de-
scribed for the preparation of 54. Yield: 33%, 1H NMR (600 MHz,
CDCl3) d 10.07 (s, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.29–7.23 (m, 7H),
3.89 (br, 2H), 3.78 (s, 2 H), 3.02 (septet, J = 7.2 Hz, 1H), 2.70 (br,
2H), 2.56 (s, 3H), 1.66 (br, 4H), 1.28 (d, J = 6.6 Hz, 6H); 13C NMR
(150 MHz, CDCl3) d 198.1, 162.9, 152.8, 148.4, 145.9, 138.1,
135.8, 135.2, 129.3, 129.2, 128.9, 128.8, 128.4, 127.6, 125.5,
116.2, 40.3, 36.5, 35.7, 28.7, 27.8, 27.5, 26.8, 20.5.
4.1.1.23. (Z)-4-(3-(4-(((6-Benzyl-5-isopropyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)yl)methoxy)methyl)benzyl)phenyl)-2-
hydroxy-4-oxobut-2-enoic acid (3). This compound was pre-
pared from intermediate 28 as a pale yellow solid following the
procedure described for the preparation of 7. Yield: 65%, 1H NMR
(600 MHz, CDCl3) d 9.73 (s, 1H), 7.84 (s, 2H), 7.42 (d, J = 3.6 Hz,
2H), 7.31 (t, J = 7.8 Hz, 2H), 7.25 (d, J = 7.2 Hz, 4H), 7.15 (d,
J = 8.4 Hz, 3H), 7.05 (d, J = 7.2 Hz, 2H), 5.21 (br, 2H), 4.63 (s, 2H),
4.18 (s, 2H), 4.03 (s, 2H), 2.85 (m, 1H), 1.25 (d, J = 6.6 Hz, 6H);
13C NMR (150 MHz, CDCl3) d 189.7, 171.5, 164.3, 163.5, 152.1,
149.5, 142.1, 140.3, 135.6, 135.3, 134.9, 134.7, 129.5, 129.4,
129.2, 128.6, 128.4, 127.6, 127.5, 126.2, 119.9, 97.5, 73.3, 71.9,
41.6, 33.8, 28.5, 20.5; HRMS (ESI-) calcd for C33H31N2O7 [MꢀH]ꢀ
567.2131, found 567.2204 (E = 13 ppm).
4.1.1.19. 3-(6-(4-Acetylphenyl)hexyl)-6-benzyl-5-isopropylpyr-
imidine-2,4(1H,3H)-dione (57). This compound was prepared
from bromide 53 as a pale yellow oil following the procedure de-
scribed for the preparation of 54. Yield: 34%, 1H NMR (600 MHz,
CDCl3) d 9.76 (s, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.31–7.23 (m, 7H),
3.84 (t, J = 7.8 Hz, 2H), 3.79 (s, 2 H), 3.02 (septet, J = 7.2 Hz, 1H),
2.63 (t, J = 7.8 Hz, 2H), 2.57 (s, 3H), 1.61 (m, 4H), 1.36 (br, 4H),
1.28 (d, J = 6.6 Hz, 6H); 13C NMR (150 MHz, CDCl3) d 198.1,
162.9, 152.6, 148.8, 145.6, 135.6, 135.2, 129.2, 128.8, 128.7,
127.7, 116.1, 40.7, 36.5, 36.1, 31.2, 29.1, 27.8, 27.7, 27.1, 26.8,
20.6.

4210
Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4202–4211
4.1.1.24. ((Z)-4-(3-(4-(((6-(3,5-Dimethylbenzyl)-5-isopropyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)methoxy)methyl)benzyl)-
phenyl)-2-hydroxy-4-oxobut-2-enoic acid (4). This compound
was prepared from intermediate 29 as a pale yellow solid following
the procedure described for the preparation of 7. Yield: 61%, 1H
NMR (600 MHz, CDCl3) d 9.64 (s, 1H), 7.83 (s, 2H), 7.40 (s, 2H),
7.25 (d, J = 6.6 Hz, 2H), 7.15 (d, J = 6.6 Hz, 3H), 6.87 (s, 1H), 6.65
(s, 2H), 5.22 (br, 2H), 4.63 (s, 2H), 4.10 (s, 2H), 4.02 (s, 2H), 2.84
(m, 1H), 2.26 (s, 6H), 1.26 (d, J = 6.6 Hz, 6H); 13C NMR (150 MHz,
CDCl3) d 189.4, 172.1, 164.3, 163.2, 151.9, 149.4, 141.9, 140.0,
138.9, 135.5, 134.8, 134.6, 134.5, 129.2, 128.9, 128.3, 128.2,
126.0, 125.0, 119.5, 97.3, 73.1, 71.6, 41.4, 33.4, 28.3, 21.3, 20.3;
HRMS (ESI-) calcd for C35H35N2O7 [MꢀH]ꢀ 595.2444, found
595.2451 (E = 1.0 ppm).
J = 7.8 Hz, 2H), 0.87 (t, J = 7.2 Hz, 3H); HRMS (ESI-) calcd for
C
24H21N2O5S [MꢀH]ꢀ 449.1176, found 449.1167 (E = 2.0 ppm).
4.1.1.29. (Z)-4-(3-(4-((4-Benzyl-5-ethyl-6-oxo-1,6-dihydropyr-
imidin-2-ylthio)methyl)benzyl)phenyl)-2-hydroxy-4-oxobut-2-
enoic acid (10). This compound was prepared from intermediate
45 as a pale yellow solid following the procedure described for the
preparation of 7. Yield: 62%, 1H NMR (600 MHz, DMSO-d6) d 7.78
(br, 2H), 7.37 (br, 2H), 7.16–7.02 (m, 9H), 4.19 (s, 2H), 3.91 (s,
2H), 3.81 (s, 2H), 2.37 (q, J = 7.2 Hz, 2H), 0.86 (t, J = 6.6 Hz, 3H);
HRMS (ESI-) calcd for C31H27N2O5S [MꢀH]ꢀ 539.1646, found
539.1689 (E = 8.0 ppm).
4.1.1.30. (Z)-4-(4-(2-(4-Benzyl-5-isopropyl-2,6-dioxo-2,3-dihy-
dropyrimidin-1(6H)yl)ethyl)phenyl)-2-hydroxy-4-oxobut-2-enoic
acid (11). This compound was prepared from intermediate 54 as
a pale yellow solid following the procedure described for the prep-
aration of 7. Yield: 48%, 1H NMR (600 MHz, DMSO-d6) d 12.03 (br,
1H), 7.96 (d, J = 7.8 Hz, 2H), 7.41 (d, J = 7.8 Hz, 2H), 7.27–7.15 (m,
5H), 7.04 (s, 1H), 4.46 (t, J = 6.0 Hz, 2H), 3.80 (s, 2H), 3.03 (m,
3H), 1.10 (d, J = 6.6 Hz, 6H); HRMS (ESI-) calcd for C26H25N2O6
[MꢀH]ꢀ 461.1718, found 461.1722 (E = 0.9 ppm).
4.1.1.25. (Z)-4-(3-(4-(((6-Benzyl-5-ethyl-2,4-dioxo-3,4-dihydro-
pyrimidin-1(2H)-yl)methoxy)methyl)benzyl)phenyl)-2-hydroxy-
4-oxobut-2-enoic acid (5). This compound was prepared from
intermediate 30 as a pale yellow solid following the procedure de-
scribed for the preparation of 7. Yield: 68%, 1H NMR (600 MHz,
CDCl3) d 9.86 (s, 1H), 7.84 (s, 2H), 7.41 (d, J = 3.6 Hz, 2H), 7.30 (t,
J = 7.8 Hz, 2H), 7.25 (d, J = 7.8 Hz, 3H), 7.16–7.14 (m, 3H), 7.05 (d,
J = 7.2 Hz, 2H), 5.19 (br, 2H), 4.62 (s, 2H), 4.15 (s, 2H), 4.03 (s,
2H), 2.44 (q, J = 7.2 Hz, 2H), 1.03 (t, J = 7.2 Hz, 6H); 13C NMR
(150 MHz, CDCl3) d 189.8, 171.4, 164.3, 164.2, 152.1, 150.0,
142.1, 140.3, 135.6, 135.1, 134.9, 134.8, 129.5, 129.4, 129.2,
128.5, 128.4, 127.6, 127.5, 126.2, 117.1, 97.6, 73.1, 71.9, 41.6,
33.7, 19.3, 13.9; HRMS (ESI-) calcd for C32H29N2O7 [MꢀH]ꢀ
553.1974, found 553.2042 (E = 12 ppm).
4.1.1.31. (Z)-4-(4-(3-(4-Benzyl-5-isopropyl-2,6-dioxo-2,3-dihy-
dropyrimidin-1(6H)-yl)propyl)phenyl)-2-hydroxy-4-oxobut-2-
enoic acid (12). This compound was prepared from intermediate
55 as a pale yellow solid following the procedure described for the
preparation of 7. Yield: 52%, 1H NMR (600 MHz, CDCl3) d 9.50 (s,
1H), 7.93 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.34 (t,
J = 7.8 Hz, 2H), 7.28 (d, J = 7.8 Hz, 1H), 7.23 (d, J = 7.2 Hz, 2H),
7.15 (s, 1H), 3.93 (t, J = 7.8 Hz, 2H), 3.80 (s, 3H), 3.03 (septet,
J = 7.2 Hz, 1H), 2.80 (t, J = 7.2 Hz, 2H), 2.07 (quintet, J = 7.2 Hz,
2H), 1.28 (d, J = 6.6 Hz, 6H); HRMS (ESI-) calcd for C27H27N2O6
[MꢀH]ꢀ 475.1869, found 475.1874 (E = 1.0 ppm).
4.1.1.26. (Z)-4-(3-(4-(((6-(3,5-Dimethylbenzyl)-5-ethyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)methoxy)methyl)benzyl)phenyl)-
2-hydroxy-4-oxobut-2-enoic acid (6). This compound was pre-
pared from intermediate 31 as a pale yellow solid following the
procedure described for the preparation of 7. Yield: 72%, 1H NMR
(600 MHz, CDCl3) d 9.87 (s, 1H), 7.83 (s, 2H), 7.40 (s, 2H), 7.25 (d,
J = 6.6 Hz, 2H), 7.15 (d, J = 7.8 Hz, 3H), 6.87 (s, 1H), 6.64 (s, 2H),
5.21 (br, 2H), 4.62 (s, 2H), 4.07 (s, 2H), 4.02 (s, 2H), 2.44 (q,
J = 7.2 Hz, 2H), 2.25 (s, 6H), 1.04 (t, J = 7.2 Hz, 3H); 13C NMR
(150 MHz, CDCl3) d 189.8, 171.6, 164.3, 164.3, 152.2, 150.3,
142.1, 140.3, 139.2, 135.7, 134.9, 134.8, 129.4, 129.3, 129.2,
128.5, 128.4, 126.2, 125.2, 117.0, 97.6, 73.2, 71.9, 41.6, 33.5, 21.5,
19.3, 13.9; HRMS (ESI-) calcd for C34H23N2O7 [MꢀH]ꢀ 581.2247,
found 581.2354 (E = 11 ppm).
4.1.1.32. (Z)-4-(4-(4-(4-Benzyl-5-isopropyl-2,6-dioxo-2,3-dihy-
dropyrimidin-1(6H)yl)butyl)phenyl)-2-hydroxy-4-oxobut-2-enoic
acid (13). This compound was prepared from intermediate 56 as
a pale yellow solid following the procedure described for the prep-
aration of 7. Yield: 46%, 1H NMR (600 MHz, DMSO-d6) d 10.95 (s,
1H), 7.92 (d, J = 6.6 Hz, 2H), 7.76 (d, J = 7.8 Hz, 1H), 7.36–7.19 (m,
7H), 3.75 (m, 4H), 2.82 (septet, J = 7.2 Hz, 1H), 2.67 (t, J = 7.2 Hz,
1H), 2.63 (t, J = 7.2 Hz, 1H), 1.52 (m, 4H), 1.03 (d, J = 6.6 Hz, 6H);
HRMS (ESI-) calcd for C28H29N2O6 [MꢀH]ꢀ 489.2026, found
489.2053 (E = 5.5 ppm).
4.1.1.27. (Z)-4-(4-(3-((6-benzyl-5-isopropyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)methox)propyl)phenyl)-2-hydroxy-4-oxo-
but-2-enoic acid (8). This compound was prepared from interme-
diate 39 as a pale yellow solid following the procedure described
for the preparation of 7. Yield: 60%, 1H NMR (600 MHz, CDCl3) d
9.90 (s, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.34 (t, J = 7.8 Hz, 2H), 7.26
(m, 2H), 7.14 (s, 1H), 7.10 (d, J = 7.2 Hz, 2H), 5.13 (br s, 2H), 4.18
(s, 2H), 3.58 (t, J = 6.0 Hz, 2H), 2.87 (m, 1H), 2.72 (t, J = 7.8 Hz,
2H), 1.89 (q, J = 7.2 Hz, 2H), 1.29 (d, J = 6.6 Hz, 6H); 13C NMR
(150 MHz, CDCl3) d 189.6, 171.1, 164.5, 163.6, 152.1, 149.5,
148.7, 135.3, 132.4, 129.5, 129.3, 128.4, 127.6, 127.5, 120.0, 97.4,
73.4, 68.7, 33.8, 32.5, 30.6, 28.6, 20.6; HRMS (ESI-) calcd for
C28H29N2O7 [MꢀH]ꢀ 505.1980, found 505.1995 (E = 2.9 ppm).
4.1.1.33. (Z)-4-(4-(6-(4-Benzyl-5-isopropyl-2,6-dioxo-2,3-dihy-
dropyrimidin-1(6H)-yl)hexyl)phenyl)-2-hydroxy-4-oxobut-2-
enoic acid (14). This compound was prepared from intermediate
57 as a pale yellow solid following the procedure described for the
preparation of 7. Yield: 48%, 1H NMR (600 MHz, DMSO-d6) d 10.94
(s, 1H), 7.94 (d, J = 7.8 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.29 (t, J =
7.2 Hz, 2H), 7.21 (d, J = 7.8 Hz, 3H), 7.01 (br, 1H), 3.75 (s, 2H),
3.69 (t, J = 7.2 Hz, 2H), 2.81 (septet, J = 7.2 Hz, 1H), 2.63 (t, J =
7.2 Hz, 2H), 1.56 (quintet, J = 7.2 Hz, 2H), 1.46 (quintet, J = 7.2 Hz,
2H), 1.28 (m, 4H), 1.03 (d, J = 6.6 Hz, 6H); HRMS (ESI-) calcd for
C30H33N2O6 [MꢀH]ꢀ 517.2339, found 517.2391 (E = 10.0 ppm).
4.2. Biology
4.1.1.28. (Z)-4-(4-((4-Benzyl-5-ethyl-6-oxo-1,6-dihydropyrimi-
din-2-ylthio)methyl)phenyl)-2-hydroxy-4-oxobut-2-enoic acid
(9). This compound was prepared from intermediate 44 as a pale
yellow solid following the procedure described for the preparation
of 7. Yield: 67%, 1H NMR (600 MHz, DMSO-d6) d 7.76 (br, 2H), 7.31
(br, 2H), 7.22–7.15 (m, 5H), 4.30 (s, 2H), 3.83 (s, 2H), 2.39 (q,
4.2.1. RT assay
This assay was performed using the Quan-T-RT Assay System kit
from Amersham Biosciences (TRK 1022). Experiments were done
using 0.65 mL snap cap tubes with a SPA (scintillation proximity
assay) bead, where a short poly (A) tail and a oligo (T) primer
has been coupled via a biotin linkage to a resin bead containing

Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4202–4211
4211
scintillation cocktail. This resin was incubated with a drug com-
pound, the [3H]-TTP tracer and the reaction was initiated by the
addition of the RT (RT) enzyme from Ambion: the RNA Company
(AM2045). The [3H]-TTP was incorporated into the poly (A) tail
by RT enzyme but in the presence of the drug candidate this incor-
poration should be inhibited. This reaction was incubated for 3 h at
37 °C and quenched by the addition of the stop buffer. These tubes
were then transferred to 20 mL scintillation tubes (glass) and
counted for 1 min per sample using a beta counter.
Craigie of NIH for the integrase plasmid and Dr. William Shannon
for consultation.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.05.004.
References and notes
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4.2.2. IN assay
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IN was expressed using Escherichia coli BL21(DE3) and standard
IPTG induction.36 In a typical assay, DNA substrate 50biotin ATG
TGGAAAATCTCTAGCAGT and 30cy5 ACTGCTAGA GATTTTCCACAT
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12.5 L of 600 nM IN in reaction buffer (10 mM HEPES pH 7.5,
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Brij-35) was added to each well in a 96 well plate. 0.5 L of inhib-
itor or DMSO was added to each sample and allowed to equilibrate
with the enzyme for 10 min at room temperature. After the incuba-
a
thermocycler. For each inhibitor concentration
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to each well to initiate the reaction. The reaction was incubated at
37 °C for 2 h, after which 75
lL of 50 nM DNA substrate in reaction buffer was added
l
L of 4ꢁ binding buffer (20 mM Tris
pH 8.0, 1.6 M NaCl, 40 mM EDTA, 0.4 mg/mL salmon sperm DNA)
was added and the entire mixture was transferred to a StreptaWell
High bind plate (Roche). The DNA was allowed to bind to the plate
for 1 h at room temp with mild agitation. Nonspecific DNA was re-
moved from the plate by washing 3 ꢁ 5 min with 200
ing buffer (30 mM NaOH, 0.2 M NaCl, 1 mM EDTA). The plate was
washed briefly with 200 L of TE. To remove the DNA for fluores-
cence detection 100 L of formamide was added to each well and
incubated for 15 min at room temp with mild agitation. 90 L of
lL denatur-
l
l
l
the formamide solution was transferred into a black 384 well plate
and read on a molecular devices analyst using the continuous lamp
excitation 620 nm, emission 665 nm.
4.2.3. HIV-1 assay
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The HIV Cytoprotection assay used CEM-SS cells and the IIIB
strain of HIV-1. Briefly virus and cells were mixed in the presence
of test compound and incubated for 6 days. The virus was pre-ti-
tered such that control wells exhibit 70–95% loss of cell viability
due to virus replication. Therefore, antiviral effect or cytoprotec-
tion was observed when compounds prevent virus replication.
Each assay plate contained cell control wells (cells only), virus con-
trol wells (cells plus virus), compound toxicity control wells (cells
plus compound only), compound colorimetric control wells (com-
pound only) as well as experimental wells (compound plus cells
plus virus). Cytoprotection and compound cytotoxicity were as-
sessed by MTS (CellTiterÒ 96 Reagent, Promega, Madison WI) or
XTT dye reduction and the EC50 (concentration inhibiting virus rep-
lication by 50%), TC50 (concentration resulting in 50% cell death)
and a calculated TI (therapeutic index TC50/EC50) were provided.
Each assay included the HIV RT inhibitor AZT as a positive control.
Acknowledgements
This research was supported by the Center for Drug Design at
the University of Minnesota. We thank Dr. Yuk Sham for helpful
discussion, Daniel Wilson for assistance in IN assay, Roger Ptak at
Southern Research Institute for cell-based HIV assay, Dr. Robert
36. Jenkins, T. M.; Engelman, A.; Ghirlando, R.; Craigie, R. J. Biol. Chem. 1996, 271,
7712.