Synthesis of dihydropyrimidine α,μ3-diketobutanoic acid derivatives targeting HIV integrase

Article abstract of DOI:10.1016/j.ejmech.2015.09.015

The synthesis and antiviral evaluation of a series of dihydropyrimidinone and thiopyrimidine derivatives bearing aryl α,γ-diketobutanoic acid moiety are described using the Biginelli multicomponent reaction as key step. The most active among 20 synthesized novel compounds were 4c, 4d and 5b, which possess nanomolar HIV-1 integrase (IN) stand transfer (ST) inhibition activities. In order to understand their mode of interactions within the IN active site, we docked all the compounds into the previously reported X-ray crystal structure of IN. We observed that compounds 4c, 4d and 5b occupied an area close to the two catalytic Mg²⁺ ions surrounded by their chelating triad (E221, D128 and D185), DC16, Y212 and the β-diketo acid moiety of 4c, 4d and 5b chelating Mg²⁺. As those compounds lack anti-HIV activities in cell, their prodrugs were synthetized. The prodrug 4c′ exhibited an anti-HIV activity of 0.19 μM in primary human lymphocytes with some cytotoxicity. All together, these results indicate that the new analogs potentially interact within the catalytic site with highly conserved residues important for IN catalytic activity.

Full text of DOI:10.1016/j.ejmech.2015.09.015

European Journal of Medicinal Chemistry 104 (2015) 127e138
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis of dihydropyrimidine
targeting HIV integrase
a,g-diketobutanoic acid derivatives
a
a
b
b
b
ꢀ
Ozkan Sari , Vincent Roy , Mathieu Metifiot , Christophe Marchand , Yves Pommier ,
Stephane Bourg c, Pascal Bonnet a, Raymond F. Schinazi d, Luigi A. Agrofoglio a
a,
, *
ꢀ
a
ꢀ
ꢀ
Univ. Orleans et CNRS, ICOA, UMR 7311, F-45067, Orleans, France
b Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, 20892, USA
c
ꢀ
ꢀ
CNRS, CBM, UPR 4301, Univ. Orleans, F-45071, Orleans, France
d Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Veterans Affairs
Medical Center, Atlanta, GA, 30322, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and antiviral evaluation of a series of dihydropyrimidinone and thiopyrimidine derivatives
Received 21 June 2015
Received in revised form
9 September 2015
Accepted 10 September 2015
Available online 25 September 2015
bearing aryl a,g-diketobutanoic acid moiety are described using the Biginelli multicomponent reaction as
key step. The most active among 20 synthesized novel compounds were 4c, 4d and 5b, which possess
nanomolar HIV-1 integrase (IN) stand transfer (ST) inhibition activities. In order to understand their
mode of interactions within the IN active site, we docked all the compounds into the previously reported
X-ray crystal structure of IN. We observed that compounds 4c, 4d and 5b occupied an area close to the
two catalytic Mg2þ ions surrounded by their chelating triad (E221, D128 and D185), DC16, Y212 and the
Keywords:
b
-diketo acid moiety of 4c, 4d and 5b chelating Mg2þ. As those compounds lack anti-HIV activities in cell,
Dihydropyrimidine
Synthesis
a,g-diketobutanoic acid
their prodrugs were synthetized. The prodrug 4c0 exhibited an anti-HIV activity of 0.19
mM in primary
human lymphocytes with some cytotoxicity. All together, these results indicate that the new analogs
potentially interact within the catalytic site with highly conserved residues important for IN catalytic
activity.
Docking analysis
Antiviral activity
Integrase-HIV
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
which is required for production of future virions.
Since 2007, three compounds have been approved by the FDA as
The genome of HIV in each viral capsid includes two RNA
strands along with three enzymes required for the replication cy-
cle: reverse transcriptase (RT), protease (PR) and integrase (IN).
While some prior treatments like the highly active antiretroviral
therapy (HAART) used to focus solely on RT and PR, IN became, in
the past decade, a crucial target since it lacks a mammalian coun-
terpart [1e5]. IN acts by inserting the viral DNA into host cell
chromosomes. More specifically, in the cytoplasm, IN catalyzes the
30-processing step (30-P), which consists in the removal of a GT
dinucleotide from the 30-end of both extremities of the viral
genome (U3 and U5 long terminal repeats (LTRs)) [6]. Subsequently,
IN stay bound to the LTRs in the preintegration complex and moves
to the nucleus where the strand transfer step (ST) takes place.
Integration is essential for the generation of the proviral genes,
IN inhibitors: Raltegravir 1 (Isentress®, Merck), Elvitegravir 2
(Vitekta®, Gilead) and Dolutegravir 3 (Tivicay®, ViiV Healthcare),
(Fig. 1). These drugs are unique by their ability to selectively inhibit
ST during the integration process by complexing the two catalytic
Mg2þ ions in the active site.
The design of the
the chelation, was inspired by the pioneering studies made with
the -diketobutanoic acid (DKA). In fact, the discovery of DKAs
b-hydroxy-(amide) functions, responsible for
a,g
was decisive in the validation of IN as a therapeutic target toward
the inhibition of HIV replication [7]. Therefore, as part of our drug
discovery program, we have developed a library of diversely
substituted dihydropyrimidine
a
,
g-diketobutanoic acid derivatives
targeting selectively ST. A metal chelating aryl
a,g
-diketobutanoic
acid moiety was attached to these structures via derivation at N-1
for dihydropyrimidinone (4) or C-2 in the case of thiopyrimidines
(5) (Fig. 2).
* Corresponding author.
E-mail address: luigi.agrofoglio@univ-orleans.fr (L.A. Agrofoglio).
http://dx.doi.org/10.1016/j.ejmech.2015.09.015
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

128
O. Sari et al. / European Journal of Medicinal Chemistry 104 (2015) 127e138
Fig. 1. FDA-approved integrase inhibitors: Raltegravir 1, Elvitegravir 2, Dolutegravir 3.
Scheme 1. Reagents and conditions: a) Ce(NO3)3.6H2O (5 mol%), 80 ꢀC, 15 min
(65e92%) or CeCl3.7H2O (25 mol%), EtOH, 80 ꢀC, 24 h (33e76%); b) HNO3 (65%),
30 min, 0 ꢀC to rt (59e92%); c) 4-(bromomethyl)acetophenone, Cs2CO3, DMF, rt, 24 h
(31e56%); d) LiHMDS, THF, -78 ꢀC, 30 min, then diethyl oxalate; e) LiOH (1 M), THF, rt,
1 h (51e98% over two steps).
In order to study the effect of the proximity of the a,g-diketo-
butanoic acid moiety to the dihydropyrimidine building block, a
compound bearing a diaryl spacer was synthesized. The spacer 16
was prepared in 3 steps from the methyl 4-(bromomethyl)benzoate
13 successively by reduction with DIBAL-H, Suzuki coupling with
the 3-acethylphenylboronic acid and bromination (Scheme 2) [14].
Then, the diaryl spacer was attached to the dihydropyrimidine
derivative 10f to generate the methyl ketone intermediate 17 in 52%
yield. It is noteworthy that the derivative 10f, having an ethyl group
at position 6, was chosen to avoid the side reaction discussed above
Fig. 2. Target dihydropirimidine derivatives bearing a
at N-1 (4) or C-2 (5).
a,g-diketobutanoic acid moiety
2. Results and discussion
2.1. Chemical synthesis
The preparation of the N-1 derived compounds starts with the
synthesis of the 3,4-dihydropyrimidin-2(1H)-ones 4a-m (DHPMs)
using the Biginelli multicomponent reaction as key step [8]. This
reaction allowed us to easily introduce diversity at position C-4 and
for the 6-methyl derivatives. The a,g-diketobutanoic acid moiety
was elaborated following the previously described conditions to
afford the compound 19 in 96% yield over two steps.
The analogs 5a-f, bearing the
a
,g
-diketobutanoic acid moiety at
C-6 of the DHPMs by modulation of the aldehyde 6 and the
b-
C-2, were prepared following
a
slightly modified sequence,
ketoester 8. To begin with, the DHPMs 9a-d substituted by a methyl
group at position 6 were synthesized in high yield under solvent-less
conditions using 5 mol% of Ce(NO3)3.6H2O at 80 ꢀC during 15 min
[9].The synthesis of DHPMs 9e substituted by a benzyl group at C-4
and 9f-m substituted either by an ethyl, an isopropyl or a phenyl
group at C-6 has proven to be difficult using the previously described
respectively. Thus, thiopyrimidines were synthesized using the
previously described CeCl3.7H2O catalyzed procedure to afford 21a-
f in good yields (Scheme 3, 70e94%). C-2 derivation was achieved
by selective S-alkylation with 4-(bromomethyl)acetophenone to
afford 22a-f as a mixture of isomers which were subsequently
treated with MnO2 under microwave conditions to yield the
aromatized derivatives 23a-f (35e49% over two steps) [15]. These
intermediates were then converted as described above to the cor-
conditions. The low reactivity of the aliphatic aldehyde or
b-
ketoesters involved in their preparation led us to apply a different
protocol with a higher catalyst loading and a longer reaction time.
Hence, preparation of these DHPMs was performed using 25 mol% of
CeCl3.7H2O in refluxing ethanol during 24 h to generate the desired
derivatives in moderate to good yields (33e76%) [10]. These DHPMs
were then involved in an oxidative aromatization step to isolate the
N-1 for alkylation. Therefore, a treatment with 65% nitric acid
following the methodology described by Kappe et al. [11] generated
efficiently the desired pseudo-aromatized compounds 10a-m in
good to excellent yields (59e92%). The obtained compounds were
then alkylated with 4-(bromomethyl)acetophenone, which was
prepared by radical bromination of 4-methylacetophenone (not
described) [12]. The alkylated derivatives 11a-m were isolated in
moderate yields (31e56%) after 24 h at room temperature. Finally,
the methyl ketone intermediates were treated with LiHMDS and
responding b-diketoacids 5a-f.
2.2. Biological evaluation
The twenty newly synthesized compounds 4a-m, 19 and 5a-f
were evaluated for their ability to inhibit the enzymatic activity of
HIV IN (30-P and ST) in vitro. The results are summarized in Table 1.
As expected, some of these compounds (4c,d, 5b and 19) displayed
selective inhibition of ST with sub-micromolar activities. The ob-
tained results demonstrated that steric hindrance at C-6 was not
well tolerated, since compounds substituted by either a phenyl or
an isopropyl group exhibited the lowest inhibitions. Nevertheless,
compounds substituted at by a methyl or an ethyl group gave
interesting results. Among them, compound 4c, substituted by a
benzyl group at C-4 position, displayed a promising inhibition with
reacted with diethyl oxalate to produce the
a,g-diketoesters de-
rivatives, which were subsequently submitted to base-promoted
hydrolysis (saponification) and acidification to yield the desired
a,g-diketobutanoic acids 4a-m in good yields, over two steps [13]. It
is noteworthy that 6-methyl substituted derivatives led to lower
yields due to side reactions with LiHMDS, which generated unde-
sired diketoacids derivatives (Scheme 1).
an IC50 of 0.19 mM. Concerning the substitution on the aryl group at
C-4 of both compounds, the presence of halogen such as a fluorine,
of the methoxy or 3,5-dimethyl group did not affect significantly
the ST inhibition (IC50 < 6.5
mM). A phenyl group at C-4 and an ethyl
group at C-6 generated compound 4d with an IC50 of 0.64
mM.

O. Sari et al. / European Journal of Medicinal Chemistry 104 (2015) 127e138
129
compounds with antiviral activity are also showing some
cytotoxicity.
2.3. Molecular modeling
In order to investigate the binding mechanism of our com-
pounds, we performed molecular docking experiments. Our studies
relied on the crystal structure of the Prototype Foamy Virus (PFV)
intasome complexed with raltegravir [17] which is an established
model for the development of HIV-1 IN strand transfer inhibitors as
reported by Billamboz et al. [18] and shown by Hare et al. [19,20]
First, we validated our approach by extracting and redocking ral-
tegravir in the binding site, and analyzed the result by super-
imposing the docked poses with the crystallographic conformation.
The enolic tautomeric form was retained for the ligand, as it is the
well-established form in solution for diketo acids [20]. A RMSD
value of 0.3 Å for the best pose considering the heavy atoms was
found. The pharmaphoric features of the crystallographic binding
mode are well reproduced such as the strong chelation of the two
Mg2þ cations by (Glu221), (Asp128) and (Asp185) residues, and two
Scheme 2. Reagents and conditions: a) DIBAL-H, CH2Cl2, 1.5 h, ꢁ78 ꢀC, quant.; b) 3-
acethylphenylboronic acid, Pd(OAc)2, PPh3, K3PO4, toluene, rt, 48 h, 77%; c) NBS,
PPh3, THF, 0 ꢀC, 1 h, 98%; d) 10f, Cs2CO3, DMF, rt, 24 h, 52%; e) LiHMDS, THF, ꢁ78 ꢀC, 1 h,
then diethyl oxalate; e) LiOH (1 M), THF, rt, 1 h, 96% over two steps.
p
e p stacking interactions, one between (Tyr212) and oxadiazol
ring and the other between DC16 and p-fluorobenzyl ring. Next we
docked compounds 4c, 4d and 5b following the same protocol. The
best poses are superimposed and compared to raltegravir on Fig. 3.
Compounds 4c, 4d and 5b chelate the two Mg2þ cations by their
Attaching a diaryl spacer such as in compound 19, or moving the
a
,
g
-diketobutanoic acid moiety at C-2 position as in compounds
5a,f did not affect remarkably the activity providing a slightly less
potent compound with an IC50 of 0.85 M and 2.23 M for 19 and
aryl
a,g-diketobutanoic acid moiety, as does raltegravir with
m
m
(Glu221), (Asp128) and (Asp185) residues. The ester moiety of the
three compounds 4c, 4d and 5b are parallel to the DNA strand and
pointing towards the solvent area. They form hydrogen bond in-
teractions between the carbonyl of the ester and DNA donor
nucleotide, 4c and 4d with DC16 and 5b DA15. In contrast to ral-
tegravir, the central aromatic six-membered ring of the compounds
5a, respectively when compared to the parent structure 4d.
In a first attempt, antiviral evaluation of the DKAs against HIV-1
in CEM cells (data not shown) did not exhibit any anti-HIV activity
(EC50 > 65 mM), which is in total disconnection with enzymatic
potencies. This lack of activity has been imputed to the low cell
penetration of the compounds caused by the presence of a highly
polar carboxylic acid group. Therefore, the best ST inhibitors 4c,d,l,
is not able to interact via
p
e
p
stacking interactions with DC16.
Additionally, no
(Tyr212).
p e p stacking interaction is observed with
5b and 19 were converted back to their
overcome this issue. Recently, Nair et al. have reported the iso-
propyl ester as an efficient prodrug of DKAs [16]. They also showed
a,g-diketoester form to
The Mg2þ chelation and the hydrogen bonds could explain the
activity of the compounds against IN, but those hydrogen bond
interactions are probably not sufficient to counterbalance the loss
that the
a,g-diketoester form was quickly hydrolyzed in human
liver microsomes to produce the active acid form. Thus, the iso-
propyl ester prodrug version of our compounds were prepared via
one-step acid-catalyzed esterification of DKA with 2-propanol
(Scheme 4) and these prodrugs were screened to evaluate their
anti-HIV-1 activity.
The cellular anti-HIV activity of a,g-diketoester prodrugs 4′c,d,l,
19′ and 5'b is reported on Table 2. As expected, a slight improve-
ment in cellular activities were observed especially for the best ST
of the
p e p stacking interactions with DC16 observed with
raltegravir.
3. Conclusions
Twenty newly
cessfully synthesized through the Biginelli reaction to achieve py-
rimidine building blocks. As expected, compounds 4a-m, 19 and
5a-f which were evaluated for their ability to inhibiti HIV-1 IN
a,g-diketobutanoic acid derivatives were suc-
inhibitor 4′c with an EC50 of 17.2
mM, which most likely implies that
the prodrug forms are able to penetrate into cells. However, the
Scheme 3. Reagents and conditions: a) CeCl3.7H2O (25 mol%), EtOH, reflux, 24 h (77e94%); b) 4-(bromomethyl)acetophenone, K2CO3, DMF, 60 ꢀC, 1 h (35e49%); c) MnO2
(10 equiv.), CH2Cl2, MW, 100 ꢀC, 30 min (89e98%); d) LiHMDS, THF, ꢁ78 ꢀC, 30 min, then diethyl oxalate; e) LiOH (1 M), THF, rt, 1 h 89e98% (over two steps).

130
O. Sari et al. / European Journal of Medicinal Chemistry 104 (2015) 127e138
Table 1
Inhibition of integrase activities (30-Processing and Strand transfer).
Compounds
-R2
-R1
IC50 (m
M) S.D.a
30-Processing
Stand transfer
2.4 0.5
4a
4b
4c
4d
4e
4f
-CH3
>111
-CH3
17.4 2.1
16.9 3.4
19.1 4.0
1.3 0.1
0.19 0.12
0.64 0.16
1.1 0.4
5.6 4.6
2.3 0.7
3.6 0.8
3.3 1.4
2.0 0.3
2.4 0.6
1.9 0.8
1.3 0.5
-CH3
-CH2-CH3
-CH2-CH3
-CH2-CH3
-CH(CH3)2
-CH(CH3)2
-CH(CH3)2
35
8
72 4.5
4g
4h
4i
37.5 5.1
40 12
Scheme 4. Synthesis of ester prodrug forms. a) iPrOH, H2SO4 (cat), 85 ꢀC,
(24e38%).
3 h
19.2 5.1
18.4 1.7
16.6 2.4
17.2 3.2
16.3 3.5
4j
Table 2
Evaluation of the antiviral activity against human immunodeficiency virus (HIV) and
4k
4l
cytotoxicity against PBM, CEM, and VERO cells in vitro, expressed in
m
M, of syn-
thesized of
a
,
g-dicetoester analogs.
a
b
Prodrug
EC50
(m
M)
CC50 (mM)
4m
PBM
CEM
VERO
4′c
4′d
4′l
19′
5′b
AZT
17.2
>100
80.6
36.8
25.6
77.0
7.3
21.4
25.3
8.9
5.2
56.1
32.9
>100
>100
47.6
43.2
>100
>100
>100
>100
39.5
19
5a
e
22.5 2.0
21.1 1.2
0.85 0.12
2.23 0.27
-CH2-CH3
5b
5c
5d
5e
5f
-CH2-CH3
-CH2-CH3
29.3 3.5
30.7 2.7
0.92 0.14
2.27 0.28
6.1 1.4
0.0074
39.5
a
Concentration required to induce 50% of inhibition.
Cytostatic Concentration required to reduce cell growth by 50%.
b
30
22
23
e
4
4
4
Silica Gel 60 M (0.040e0.063 mm, E. Merck). The 1H and 13C NMR
spectra were recorded on a Varian InovaUnity 400 spectrometer
(400 MHz) in (d4) methanol, CDCl3, shift values in parts per million
relative to SiMe4 as internal reference. High Resolution Mass
spectra were performed on a Bruker maXis mass spectrometer.
2.8 0.9
1.2 0.3
Raltegravir
e
0.013 0.001
a
Concentration required to induce 50% of inhibition.
4.1.1. General procedure for the Biginelli reactions (9a-e)
Procedure A: A mixture of
b-ketoester (1 mmol), aldehyde
(1 mmol) and urea (1.5 mmol) was heated at 80 ꢀC during 15 min in
the presence of Ce(NO3)3.6H2O (5 mol%). The obtained solid was
filtered and washed with ice-cold water and recrystallized from hot
ethanol to afford the desired 1,3-dihydropyrimidinone derivative.
in vitro and showed IC50 values in the low nanomolar range with
some of them in nanomolar concentration (4c). Assuming that
compounds suffered from low cell penetration, ester prodrug forms
of selected compounds were prepared. Micromolar antiviral ac-
tivities were observed for these prodrugs but unfortunately they
were also associated with some cytotoxicity. The results of molec-
ular docking indicate that the new analogs potentially interact with
the highly conserved residues important for IN catalytic activities.
The data reported in this work should be considered as a starting
point for developing new HIV-IN inhibitors.
Procedure B: A solution of
b-ketoester (1 mmol), aldehyde
(1 mmol) and urea (1.5 mmol) in EtOH (20 mL) was heated at reflux
(80 ꢀC) during 24 h in the presence of CeCl3.7H2O (25 mol%). The
mixture was cooled down to room temperature, poured into
crushed ice and stirred/triturated until precipitation was observed.
The solid was filtered and washed with ice-cold water and
recrystallized from hot ethanol to afford the desired 1,3-
dihydropyrimidinone derivative.
4. Experimental section
4.1.2. General procedure for aromatization reactions (10a-m)
To a solution of 65% nitric acid (5 mL) in an open vessel was
added portion-wise 1,3-dihydropyrimidinone derivative (1 mmol)
at 0 ꢀC. After stirring 5 min at 0 ꢀC, the solution was left to reach
room temperature and stirred for an additional 30 min. The
mixture was poured into crushed ice, neutralized (pH 7e8) with
K2CO3 and extracted with CH2Cl2 (3 ꢂ 20 mL). Combined organic
4.1. Chemistry
Commercially available chemicals were of reagent grade and
used as received. The reactions were monitored by thin layer
chromatography (TLC) analysis using silica gel plates (Kieselgel
60F254, E. Merck). Column chromatography was performed on

O. Sari et al. / European Journal of Medicinal Chemistry 104 (2015) 127e138
131
Fig. 3. Docking results of compounds 4c, 4d and 5b regarding to raltegravir. Integrase and DNA are shown as gray ribbons, Mg2þ metal ions as magenta spheres and chelating triad
(Glu221, Asp128 and Asp185), (DC16) and (Tyr212) as element colored sticks. Ligands are C atom colored: raltegravir in purple, 4c in orange, 4d in cyan and 5b in green. 4c (left), 4d
(center) and 5b (right) are superimposed to raltegravir. Hydrogen bonds are represented by red dashed line (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.).
layers were washed with water (20 mL), brine (20 mL), dried over
MgSO4 and concentrated in vacuo. The resulting solid was recrys-
tallized from hot EtOH to afford the desired aromatized 1,3-
dihydropyrimidinone derivative.
4.1.8. Ethyl 6-ethyl-2-oxo-4-phenyl-3,4-dihydro-1H-pyrimidine-5-
carboxylate (9d)
Yield: 76%, white solid. CAS: 205999-91-3.
4.1.9. Ethyl 6-ethyl-4-(4-fluorophenyl)-2-oxo-3,4-dihydro-1H-
pyrimidine-5-carboxylate (9e)
4.1.3. General procedure for alkylation reactions (11a-m)
To a solution of aromatized dihydropyrimidinone (1 mmol) in
DMF (2 mL) was added 4-(bromomethyl)acetophenone (1 mmol)
and Cs2CO3 (1.5 mmol). The mixture was stirred at room temper-
ature during 24e36 h and evaporated under reduced pressure. The
residue was purified over silica gel column chromatography with
Petroleum ether/EtOAc to afford the desired alkylated derivative as
a colorless oil.
Yield: 75%, white solid. 1H NMR (400 MHz, CDCl3)
d 8.31 (s, 1H),
7.38e7.17 (m, 2H), 6.99 (t, J ¼ 8.6 Hz, 2H), 6.16 (s, 1H), 5.39 (d,
J ¼ 2.2 Hz, 1H), 4.20e3.96 (m, 2H), 2.73 (ddd, J ¼ 30.7, 13.2, 7.2 Hz,
2H),1.22 (t, J ¼ 7.5 Hz, 3H),1.17 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz,
CDCl3)
d
165.2, 162.3 (d, J ¼ 246.5 Hz), 153.9, 151.8, 139.7 (d,
J ¼ 3.2 Hz), 128.3 (d, J ¼ 8.2 Hz), 115.5 (d, J ¼ 21.5 Hz), 100.5, 60.1,
55.0, 25.3, 14.1, 12.5. HRMS (ESI): m/z [MþH]þ calcd for
C15H18FN2O3: 293.1296, found: 293.1298.
4.1.4. General procedure to b-diketoacids (4a-m and 5a-f)
4.1.10. Ethyl 6-ethyl-4-(4-methoxyphenyl)-2-oxo-3,4-dihydro-1H-
pyrimidine-5-carboxylate (9f)
A solution of methyl ketone derivative (1 equiv.) in dry THF
(0.1 M) under positive pressure of nitrogen was treated with
LiHDMS (1.1 equiv.) at ꢁ78 ꢀC. After stirring 30 min at ꢁ78 ꢀC,
diethyloxalate (1.1 equiv.) was added dropwise and the mixture was
left to reach room temperature. Stirring was continued for 2 h and
the solution was concentrated under reduced pressure. The residue
was taken with 1 M HCl and extracted with CH2Cl2. Combined
organic layers were washed with water, brine, dried over MgSO4
and evaporated in vacuo. The remainder was dissolved in THF
(0.1 M) and 1 M LiOH was added. The mixture was stirred 1 h at
room temperature, acidified with 1 M HCl and extracted with DCM.
Combined organic layers were washed with water, brine, dried over
MgSO4 and evaporated in vacuo. The residue was purified over
short reverse phase silica gel column with H2O/ACN to afford the
Yield: 66%, white solid. CAS: 205999-93-5.
4.1.11. Ethyl 6-isopropyl-2-oxo-4-phenyl-3,4-dihydro-1H-
pyrimidine-5-carboxylate (9g)
Yield: 50%, white solid. CAS: 868755-03-7.
4.1.12. Ethyl 4-(4-fluorophenyl)-6-isopropyl-2-oxo-3,4-dihydro-
1H-pyrimidine-5-carboxylate (9h)
Yield: 38%, white solid. 1H NMR (400 MHz, CDCl3)
d 7.38 (s, 1H),
7.34e7.20 (m, 2H), 6.99 (t, J ¼ 8.7 Hz, 2H), 6.12 (s, 1H), 5.37 (d,
J ¼ 2.9 Hz, 1H), 4.28e4.13 (m, 1H), 4.06 (q, J ¼ 7.1 Hz, 2H), 1.21 (d,
J ¼ 7.0 Hz, 2H),1.18 (d, J ¼ 7.0 Hz, 2H),1.15 (t, J ¼ 7.2 Hz, 3H). 13C NMR
desired b-diketoacid derivative.
(101 MHz, CDCl3)
d 165.3, 163.5, 161.1, 154.4, 153.1, 139.7, 139.7,
128.3, 128.2, 115.7, 115.4, 60.1, 55.0, 27.5, 19.7, 19.5, 14.0. HRMS (ESI):
m/z [MþH]þ calcd for C16H20N2O3: 307.1452, found: 307.1452.
4.1.5. Ethyl 6-methyl-2-oxo-4-phenyl-3,4-dihydro-1H-pyrimidine-
5-carboxylate (9a)
4.1.13. Ethyl 6-isopropyl-4-(4-methoxyphenyl)-2-oxo-3,4-dihydro-
Yield: 98%, white solid. CAS: 5395-36-8.
1H-pyrimidine-5-carboxylate (9i)
Yield: 33%, white solid. 1H NMR (400 MHz, DMSO)
d 8.79 (s, 1H),
4.1.6. Ethyl 4-(4-fluorophenyl)-6-methyl-2-oxo-3,4-dihydro-1H-
pyrimidine-5-carboxylate (9b)
7.63 (s, 1H), 7.13 (d, J ¼ 8.6 Hz, 2H), 6.87 (d, J ¼ 8.6 Hz, 2H), 5.08 (d,
J ¼ 3.2 Hz, 1H), 4.11 (dt, J ¼ 14.0, 7.0 Hz, 1H), 4.03e3.89 (m, 2H), 3.71
(s, 3H), 1.13 (d, J ¼ 7.1 Hz, 2H), 1.11 (d, J ¼ 7.1 Hz, 2H), 1.07 (t,
Yield: 92%, white solid. CAS: 5937-24-6.
J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO)
d 165.8, 158.9, 156.5,
4.1.7. Ethyl 4-benzyl-6-methyl-2-oxo-3,4-dihydro-1H-pyrimidine-
5-carboxylate (9c)
153.0, 137.3, 127.8, 114.2, 98.9, 59.7, 55.5, 53.7, 27.4, 19.6, 19.4, 14.4.
HRMS (ESI): m/z [MþH]þ calcd for C17H23N2O4: 349.1547, found:
349.1548.
Yield: 40%, white solid. CAS: 378186-60-8.

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O. Sari et al. / European Journal of Medicinal Chemistry 104 (2015) 127e138
4.1.14. Ethyl 2-oxo-4,6-diphenyl-3,4-dihydro-1H-pyrimidine-5-
carboxylate (9j)
4.1.24. Ethyl 6-isopropyl-2-oxo-4-phenyl-1H-pyrimidine-5-
carboxylate (10g)
Yield: 48%, white solid. CAS: 34906-28-0.
Yield: 88%, white solid. CAS: 868755-10-6.
4.1.15. Ethyl 4-(4-fluorophenyl)-2-oxo-6-phenyl-3,4-dihydro-1H-
pyrimidine-5-carboxylate (9k)
4.1.25. Ethyl 4-(4-fluorophenyl)-6-isopropyl-2-oxo-1H-pyrimidine-
5-carboxylate (10h)
Yield: 76%, white solid. CAS: 397882-37-0.
Yield: 74%, white solid. CAS: 1313372-06-3.
4.1.16. Ethyl 4-(4-methoxyphenyl)-2-oxo-6-phenyl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (9l)
4.1.26. Ethyl 6-isopropyl-4-(4-methoxyphenyl)-2-oxo-1H-
pyrimidine-5-carboxylate (10i)
Yield: 82%, white solid. 1H NMR (400 MHz, CDCl3)
d 12.79 (s, 1H),
Yield: 71%, white solid. CAS: 380655-10-7.
7.60 (d, J ¼ 8.6 Hz, 2H), 6.96 (d, J ¼ 8.6 Hz, 2H), 4.12 (q, J ¼ 7.1 Hz,
4.1.17. Ethyl 4-(3,5-dimethylphenyl)-2-oxo-6-phenyl-3,4-dihydro-
2H), 3.86 (s, 3H), 3.32e3.12 (m, 1H), 1.42 (s, 3H), 1.40 (s, J ¼ 6.6 Hz,
3H), 1.05 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 167.0, 161.9,
1H-pyrimidine-5-carboxylate (9m)
Yield: 65%, white solid. 1H NMR (400 MHz, DMSO)
d 9.21 (s, 1H),
158.3, 129.9, 114.0, 61.8, 55.4, 20.7, 13.7. HRMS (ESI): m/z [MþH]þ
7.75 (s, 1H), 7.40e7.30 (m, 5H), 6.97e6.91 (m, 3H), 5.16 (s, 1H), 3.70
calcd for C17H21N2O4: 317.1496, found: 317.1501.
(q, J ¼ 6.6 Hz, 2H), 0.72 (t, J ¼ 6.3 Hz, 3H). 13C NMR (101 MHz, DMSO)
d
165.5, 152.5, 149.1, 144.9, 137.8, 135.6, 129.3, 128.8, 128.2, 124.5,
4.1.27. Ethyl 2-oxo-4,6-diphenyl-1H-pyrimidine-5-carboxylate
(10j)
100.9, 59.5, 54.6, 21.5, 13.8. HRMS (ESI): m/z [MþH]þ calcd for
C
21H23N2O3: 351.1703, found: 351.1703.
Yield: 59%, white solid. CAS: 34906-29-1.
4.1.18. Ethyl 6-methyl-2-oxo-4-phenyl-1H-pyrimidine-5-
carboxylate (10a)
4.1.28. Ethyl 4-(4-fluorophenyl)-2-oxo-6-phenyl-1H-pyrimidine-5-
carboxylate (10k)
Yield: 84%, white solid. CAS: 69207-36-9.
Yield: 71%, white solid. 1H NMR (400 MHz, CDCl3)
d 13.17 (s, 1H),
7.71e7.58 (m, 4H), 7.57e7.43 (m, 3H), 7.15 (t, J ¼ 8.6 Hz, 2H), 3.93 (q,
4.1.19. Ethyl 4-(4-fluorophenyl)-6-methyl-2-oxo-1H-pyrimidine-5-
carboxylate (10b)
J ¼ 7.1 Hz, 2H), 0.87 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
166.2, 164.5 (d, J ¼ 252.3 Hz), 157.8, 131.3, 130.5 (d, J ¼ 8.9 Hz),
Yield: 59%, white solid. CAS: 1091909-58-8.
128.8, 128.0, 115.9 (d, J ¼ 22.0 Hz), 111.9, 61.9, 13.4. HRMS (ESI): m/z
[MþH]þ calcd for C19H16FN2O3: 339.1139, found: 339.1142.
4.1.20. Ethyl 4-benzyl-6-methyl-2-oxo-1H-pyrimidine-5-carboxylate
(10c)
4.1.29. Ethyl 4-(4-methoxyphenyl)-2-oxo-6-phenyl-1H-pyrimidine-
5-carboxylate (10l)
Yield: 92%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 13.54 (s, 1H),
7.31e7.15 (m, 5H), 4.22 (s, 2H), 4.20 (q, J ¼ 7.1 Hz, 2H), 2.49 (s, 3H),
Yield: 74%, white solid. CAS: 913696-98-7.
1.25 (t, J ¼ 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 165.3, 158.2,
129.1, 128.6, 127.0, 111.7, 61.7, 14.0. HRMS (ESI): m/z [MþH]þ calcd
4.1.30. Ethyl 4-(3,5-dimethylphenyl)-2-oxo-6-phenyl-1H-
for C15H17N2O3: 273.1234, found: 273.1235.
pyrimidine-5-carboxylate (10m)
Yield: 85%, white solid. 1H NMR (400 MHz, CDCl3)
d 12.91 (s, 1H),
4.1.21. Ethyl 6-ethyl-2-oxo-4-phenyl-1H-pyrimidine-5-carboxylate
7.66e7.58 (m, 2H), 7.56e7.42 (m, 3H), 7.22 (s, 2H), 7.13 (s, 1H), 3.94
(10d)
(q, J ¼ 7.1 Hz, 2H), 2.35 (s, 6H), 0.90 (t, J ¼ 7.1 Hz, 3H). 13C NMR
Yield: 86%, yellow solid. 1H NMR (400 MHz, CDCl3)
d 13.68 (s,1H,
(101 MHz, CDCl3)
d 166.4, 157.7, 138.3, 132.8, 131.0, 128.6, 128.0,
NH), 7.62e7.56 (m, 2H), 7.51e7.38 (m, 3H), 4.04 (q, J ¼ 7.1 Hz, 2H),
125.7, 111.9, 61.7, 21.3, 13.4. HRMS (ESI): m/z [MþH]þ calcd for
2.88 (q, J ¼ 7.6 Hz, 2H), 1.38 (t, J ¼ 7.6 Hz, 3H), 0.93 (t, J ¼ 7.1 Hz, 3H).
13C NMR (101 MHz, CDCl3)
d 166.2, 158.6, 130.8, 128.39, 128.0, 111.0,
C21H21N2O3: 349.1547, found: 349.1548.
61.7, 13.4. HRMS (ESI): m/z [MþH]þ calcd for C15H17N2O3: 273.1234,
4.1.31. Ethyl 1-[(4-acetylphenyl)methyl]-6-methyl-2-oxo-4-phenyl-
found: 273.1237.
pyrimidine-5-carboxylate (11a)
Yield: 84%, colorless oil. 1H NMR (400 MHz, CDCl3)
d 7.97 (d,
4.1.22. Ethyl 6-ethyl-4-(4-fluorophenyl)-2-oxo-1H-pyrimidine-5-
carboxylate (10e)
J ¼ 8.2 Hz, 2H), 7.65e7.53 (m, 4H), 7.52e7.40 (m, 3H), 5.58 (s, 2H),
Yield: 62%, yellow solid. 1H NMR (400 MHz, CDCl3)
d 13.72 (s,
4.16 (q, J ¼ 7.1 Hz, 2H), 2.61 (s, 3H), 2.59 (s, 3H), 1.04 (t, J ¼ 7.1 Hz,
3H). 13C NMR (101 MHz, CDCl3)
d 197.8, 168.9, 168.1, 166.5, 163.6,
1H), 7.85e7.49 (m, 2H), 7.13 (t, J ¼ 8.3 Hz, 2H), 4.10 (q, J ¼ 7.0 Hz,
141.8, 137.6, 136.7, 130.8, 128.5, 128.4, 128.2, 127.9, 120.4, 68.5, 61.7,
26.6, 22.8, 13.6. HRMS (ESI): m/z [MþH]þ calcd for C23H23N2O4:
391.1652, found: 391.1653.
2H), 2.89 (q, J ¼ 7.3 Hz, 2H), 1.40 (t, J ¼ 7.4 Hz, 3H), 1.03 (t, J ¼ 7.1 Hz,
3H). 13C NMR (101 MHz, CDCl3)
130.3 (d, J ¼ 8.8 Hz), 115.6 (d, J ¼ 22.2 Hz), 110.9, 61.8, 13.6. HRMS
(ESI): m/z [MþH]þ calcd for C15H16FN2O3: 291.1139, found:
291.1144.
d
166.1, 164.4 (d, J ¼ 251.9 Hz),158.5,
4.1.32. Ethyl 1-[(4-acetylphenyl)methyl]-4-(4-fluorophenyl)-6-
methyl-2-oxo-pyrimidine-5-carboxylate (11b)
4.1.23. Ethyl 6-ethyl-4-(4-methoxyphenyl)-2-oxo-1H-pyrimidine-
Yield: 35%, yellow oil. 1H NMR (250 MHz, CDCl3)
d 7.98 (d,
5-carboxylate (10f)
J ¼ 8.4 Hz, 2H), 7.67e7.53 (m, 4H), 7.12 (t, J ¼ 8.7 Hz, 2H), 5.56 (s,
Yield: 81%, yellow solid. 1H NMR (400 MHz, CDCl3)
d 13.58 (s,
2H), 4.17 (q, J ¼ 7.1 Hz, 2H), 2.60 (s, 3H), 2.57 (s, 3H), 1.09 (t,
1H), 7.62 (d, J ¼ 8.3 Hz, 2H), 6.95 (d, J ¼ 8.5 Hz, 2H), 4.13 (q,
J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 197.7,169.0,168.0,165.2,
J ¼ 7.1 Hz, 2H), 3.86 (s, 3H), 2.86 (q, J ¼ 7.5 Hz, 2H), 1.38 (t, J ¼ 7.5 Hz,
164.02 (d, J ¼ 250.9 Hz), 163.6, 141.7, 136.7, 133.7, 130.4 (d,
J ¼ 8.6 Hz), 128.5, 127.8, 120.2, 115.6 (d, J ¼ 21.8 Hz), 68.5, 61.8, 26.6,
22.8, 13.7. HRMS (ESI): m/z [MþH]þ calcd for C23H22FN2O4:
409.1558, found: 409.1560.
3H), 1.06 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 166.7, 162.0,
158.6,130.1, 113.8, 61.7, 55.4,13.7. HRMS (ESI): m/z [MþH]þ calcd for
C
16H19N2O4: 303.1339, found: 303.1341.

O. Sari et al. / European Journal of Medicinal Chemistry 104 (2015) 127e138
133
4.1.33. Ethyl 1-[(4-acetylphenyl)methyl]-4-benzyl-6-methyl-2-oxo-
4.1.39. Ethyl 1-[(4-acetylphenyl)methyl]-6-isopropyl-4-(4-
methoxyphenyl)-2-oxo-pyrimidine-5-carboxylate (11i)
Yield: 54%, colorless oil. 1H NMR (400 MHz, CDCl3)
pyrimidine-5-carboxylate (11c)
Yield: 33%, colorless oil. 1H NMR (400 MHz, CDCl3)
d
7.92 (d,
d
7.96 (d,
J ¼ 8.3 Hz, 2H), 7.50 (d, J ¼ 8.2 Hz, 2H), 7.32e7.12 (m, 5H), 5.48 (s,
J ¼ 8.3 Hz, 2H), 7.62 (t, J ¼ 8.3 Hz, 4H), 6.95 (d, J ¼ 8.8 Hz, 2H), 5.58
2H), 4.30 (q, J ¼ 7.1 Hz, 2H), 4.14 (s, 2H), 2.60 (s, 3H), 2.49 (s, 3H),
(s, 2H), 4.21 (q, J ¼ 7.1 Hz, 2H), 3.18 (hept, J ¼ 6.7 Hz, 1H), 2.61 (s,
1.30 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
197.7, 169.4,
3H), 1.30 (s, 3H), 1.29 (d, J ¼ 6.7 Hz, 6H), 1.14 (t, J ¼ 7.1 Hz, 3H). 13
C
168.7,167.3,163.5,141.7,137.3, 136.6,129.2,128.4,128.4,127.9,126.7,
120.8, 68.3, 61.7, 41.8, 26.6, 23.2, 14.0. HRMS (ESI): m/z [MþH]þ
calcd for C24H25N2O4: 405.1809, found: 405.1808.
NMR (101 MHz, CDCl3) d 197.8, 176.1, 168.7, 165.7, 164.0, 161.34,
142.2, 136.6, 130.0, 129.9, 128.4, 128.0, 119.2, 113.8, 68.4, 61.7, 55.4,
33.1, 26.6, 21.6, 13.8. HRMS (ESI): m/z [MþH]þ calcd for C26H29N2O5:
449.2071, found: 449.2075.
4.1.34. Ethyl 1-[(4-acetylphenyl)methyl]-6-ethyl-2-oxo-4-phenyl-
pyrimidine-5-carboxylate (11d)
4.1.40. Ethyl 1-[(4-acetylphenyl)methyl]-2-oxo-4,6-diphenyl-
Yield: 46%, colorless oil. 1H NMR (400 MHz, CDCl3)
d
7.95 (d,
pyrimidine-5-carboxylate (11j)
J ¼ 8.3 Hz, 2H), 7.62e7.56 (m, 4H), 7.49e7.39 (m, 3H), 5.58 (s, 2H),
4.14 (q, J ¼ 7.1 Hz, 2H), 2.85 (q, J ¼ 7.5 Hz, 2H), 2.60 (s, 3H), 1.31 (t,
J ¼ 7.5 Hz, 3H), 1.03 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
Yield: 39%, colorless oil. 1H NMR (250 MHz, CDCl3)
d 7.99 (d,
J ¼ 8.4 Hz, 2H), 7.81e7.55 (m, 6H), 7.53e7.37 (m, 6H), 5.62 (s, 2H),
4.06 (q, J ¼ 7.1 Hz, 2H), 2.61 (s, J ¼ 4.3 Hz, 3H), 0.95 (t, J ¼ 7.1 Hz, 3H).
d
197.7, 173.2, 168.2, 166.5, 163.9, 141.9, 137.7, 136.7, 130.1, 128.5,
13C NMR (101 MHz, CDCl3)
d 197.7, 168.1, 167.4, 163.8, 141.8, 137.3,
128.4, 128.24, 127.9, 120.0, 68.5, 61.7, 29.0, 26.6, 13.6, 12.7. HRMS
(ESI): m/z [MþH]þ calcd for C24H25N2O4: 405.1809, found:
405.1809.
136.7, 130.2, 128.5, 128.5, 128.3, 127.9, 120.3, 68.7, 61.8, 26.7, 13.4.
HRMS (ESI): m/z [MþH]þ calcd for C28H25N2O4: 453.1809, found:
453.1811.
4.1.35. Ethyl 1-[(4-acetylphenyl)methyl]-6-ethyl-4-(4-
4.1.41. Ethyl 1-[(4-acetylphenyl)methyl]-4-(4-fluorophenyl)-2-oxo-
6-phenyl-pyrimidine-5-carboxylate (11k)
fluorophenyl)-2-oxo-pyrimidine-5-carboxylate (11e)
Yield: 43%, colorless oil. 1H NMR (250 MHz, CDCl3)
d
7.97 (d,
Yield: 43%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 7.98 (d,
J ¼ 8.3 Hz, 2H), 7.72e7.55 (m, 4H), 7.14 (t, J ¼ 8.7 Hz, 2H), 5.59 (s,
2H), 4.19 (q, J ¼ 7.1 Hz, 2H), 2.85 (q, J ¼ 7.5 Hz, 2H), 2.61 (s, 3H), 1.33
(t, J ¼ 7.5 Hz, 3H), 1.11 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
J ¼ 8.4 Hz, 2H), 7.74e7.58 (m, 6H), 7.55e7.42 (m, 3H), 7.16 (t,
J ¼ 8.7 Hz, 2H), 5.63 (s, 2H), 4.06 (q, J ¼ 7.1 Hz, 2H), 2.62 (s, 3H), 0.97
(t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 197.7, 168.1, 167.5,
d
197.7, 173.3, 168.1, 164 (d, J ¼ 250.1 Hz), 163.9 (s), 141.8, 136.7, 133.8
166.2, 164.0 (d, J ¼ 251.0 Hz), 163.8, 141.7, 137.2, 136.7, 133.4 (d,
J ¼ 3.3 Hz), 130.5 (d, J ¼ 8.7 Hz), 130.3, 128.5, 128.5, 128.3, 127.9,
120.1, 115.6 (d, J ¼ 21.9 Hz), 68.7, 61.9, 26.7, 13.5. HRMS (ESI): m/z
[MþH]þ calcd for C28H24FN2O4: 471.1715, found: 471.1715.
(d, J ¼ 3.2 Hz), 130.4 (d, J ¼ 8.7 Hz), 128.5, 127.9, 119.9, 115.5 (d,
J ¼ 21.9 Hz), 68.5, 61.8, 29.0, 26.6, 13.7, 12.7. HRMS (ESI): m/z
[MþH]þ calcd for C24H24FN2O4: 423.1715, found: 423.1714.
4.1.36. Ethyl 1-[(4-acetylphenyl)methyl]-6-ethyl-4-(4-methoxy
4.1.42. Ethyl 1-[(4-acetylphenyl)methyl]-4-(4-methoxyphenyl)-2-
phenyl)-2-oxo-pyrimidine-5-carboxylate (11f)
oxo-6-phenyl-pyrimidine-5-carboxylate (11l)
Yield: 49%, colorless oil. 1H NMR (400 MHz, CDCl3)
d
7.97 (d,
Yield: 44%, colorless oil. 1H NMR (400 MHz, CDCl3)
d 7.99e7.94
J ¼ 8.3 Hz, 2H), 7.61 (t, J ¼ 7.8 Hz, 4H), 6.95 (d, J ¼ 8.8 Hz, 2H), 5.58 (s,
2H), 4.21 (q, J ¼ 7.1 Hz, 2H), 3.86 (s, 3H), 2.83 (q, J ¼ 7.5 Hz, 2H), 2.61
(s, 3H), 1.31 (t, J ¼ 7.5 Hz, 3H), 1.14 (t, J ¼ 7.1 Hz, 3H). 13C NMR
(m, 2H), 7.75e7.56 (m, 6H), 7.54e7.38 (m, 3H), 6.99e6.93 (m, 2H),
5.62 (s, 2H), 4.06 (q, J ¼ 7.1 Hz, 2H), 3.86 (s, 3H), 2.61 (s, 3H), 0.98 (t,
J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 197.8,168.5,167.3,166.6,
(101 MHz, CDCl3)
d
197.7, 172.9, 168.6, 165.6, 163.8, 161.4, 142.0,
163.7, 161.5, 141.9,137.5, 136.7,130.1, 130.8,129.6,128.5,128.4,128.2,
127.9, 119.8, 113.9, 68.6, 61.8, 55.4, 26.67, 13.5. HRMS (ESI): m/z
[MþH]þ calcd for C29H27N2O5: 483.1914, found: 483.1916.
136.6, 130.0, 129.9, 128.4, 127.9, 119.5, 113.9, 68.4, 61.7, 55.4, 29.0,
26.6, 13.8, 12.7. HRMS (ESI): m/z [MþH]þ calcd for C25H27N2O5:
435.1914, found: 435.1918.
4.1.43. Ethyl 1-[(4-acetylphenyl)methyl]-4-(3,5-dimethylphenyl)-2-
4.1.37. Ethyl 1-[(4-acetylphenyl)methyl]-6-isopropyl-2-oxo-4-
oxo-6-phenyl-pyrimidine-5-carboxylate (11m)
phenyl-pyrimidine-5-carboxylate (11g)
Yield: 36%, colorless oil. 1H NMR (400 MHz, CDCl3)
d 7.98 (d,
Yield: 53%, colorless oil. 1H NMR (400 MHz, CDCl3)
d
7.97 (d,
J ¼ 8.2 Hz, 2H), 7.70e7.58 (m, 4H), 7.54e7.41 (m, 3H), 7.26 (s,
J ¼ 4.5 Hz, 2H), 7.12 (s, 1H), 5.63 (s, 2H), 4.06 (q, J ¼ 7.1 Hz, 2H), 2.62
(s, 3H), 2.36 (s, 6H), 1.00 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz,
J ¼ 8.2 Hz, 2H), 7.70e7.56 (m, 4H), 7.54e7.38 (m, 3H), 5.59 (s, 2H),
4.15 (q, J ¼ 7.1 Hz, 2H), 3.22 (hept, J ¼ 6.7 Hz, 1H),1.31 (s, 3H), 1.30 (s,
3H), 1.04 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
197.7, 176.4,
CDCl3) d 197.7, 168.2, 167.7, 167.1, 163.8, 141.9, 138.0, 137.4, 137.2,
168.3, 166.5, 164.1, 142.0, 137.7, 136.6, 130.1, 128.4, 128.4, 128.2,
128.0, 119.8, 68.4, 61.7, 33.1, 26.6, 21.6, 13.6. HRMS (ESI): m/z
[MþH]þ calcd for C25H27N2O4: 419.1965, found: 419.1967.
136.7, 131.9, 130.1, 128.5, 128.4, 128.3, 127.9, 126.1, 120.3, 68.6, 61.7,
26.6, 21.3, 13.5. HRMS (ESI): m/z [MþH]þ calcd for C30H29N2O4:
481.2122, found: 481.2123.
4.1.38. Ethyl 1-[(4-acetylphenyl)methyl]-4-(4-fluorophenyl)-6-
isopropyl-2-oxo-pyrimidine-5-carboxylate (11h)
4.1.44. 4-[4-[(5-ethoxycarbonyl-6-methyl-2-oxo-4-phenyl-
pyrimidin-1-yl)methyl]phenyl]-2-hydroxy-4-oxo-but-2-enoic acid
(4a)
Yield: 56%, colorless oil. 1H NMR (400 MHz, CDCl3)
d 7.97 (d,
J ¼ 8.2 Hz, 2H), 7.68e7.56 (m, 4H), 7.13 (t, J ¼ 8.6 Hz, 2H), 5.58 (s,
2H), 4.18 (q, J ¼ 7.1 Hz, 2H), 3.20 (hept, J ¼ 6.7 Hz, 1H), 2.61 (s, 3H),
1.31 (s, 3H), 1.29 (s, 3H), 1.10 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz,
Yield: 51%, white solid. 1H NMR (400 MHz, CDCl3)
d 8.02 (d,
J ¼ 8.4 Hz, 1H), 7.67e7.60 (m, 2H), 7.52e7.39 (m, 1H), 7.15 (s,
J ¼ 9.8 Hz,1H), 5.61 (s,1H), 4.17 (q, J ¼ 7.1 Hz, 1H), 2.62 (d, J ¼ 4.3 Hz,
CDCl3)
d
197.7,176.5,168.2,165.3,164.1,164.0 (d, J ¼ 250.8 Hz),141.9,
1H), 1.05 (t, J ¼ 7.1 Hz, 1H). 13C NMR (101 MHz, CDCl3)
d 186.8, 169.0,
136.7, 133.8 (d, J ¼ 3.3 Hz), 130.4 (d, J ¼ 8.6 Hz), 128.4, 128.0, 119.6,
115.5 (d, J ¼ 21.8 Hz), 68.5, 61.8, 33.2, 26.6, 21.6, 13.7. HRMS (ESI): m/
z [MþH]þ calcd for C25H26FN2O4: 437.1871, found: 437.1874.
168.0, 166.6, 163.5, 161.9, 143.0, 137.5, 132.8, 130.3, 128.5, 128.2,
128.2, 128.0, 120.5, 95.5, 68.4, 61.8, 22.7, 13.6. HRMS (ESI): m/z
[MþH]þ calcd for C25H23N2O7: 463.1499, found: 463.1492.

134
O. Sari et al. / European Journal of Medicinal Chemistry 104 (2015) 127e138
4.1.45. 4-[4-[[5-ethoxycarbonyl-4-(4-fluorophenyl)-6-methyl-2-
oxo-pyrimidin-1-yl]methyl]phenyl]-2-hydroxy-4-oxo-but-2-enoic
acid (4b)
CDCl3) d 187.1, 176.6, 168.2, 166.6, 164.0, 162.0, 143.3, 137.6, 132.9,
130.2, 128.5, 128.4, 128.2, 128.0, 119.9, 95.7, 68.4, 61.8, 33.2, 21.6,
13.6. HRMS (ESI): m/z [MþH]þ calcd for C27H27N2O7: 491.1813,
found: 491.1814.
Yield: 57%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 8.02 (d,
J ¼ 8.3 Hz, 2H), 7.74e7.56 (m, 4H), 7.15 (t, J ¼ 8.6 Hz,1H), 5.60 (s, 2H),
4.20 (q, J ¼ 7.1 Hz, 2H), 2.60 (s, 2H), 1.11 (t, J ¼ 7.1 Hz, 3H). 13C NMR
4.1.51. 4-[4-[[5-ethoxycarbonyl-4-(4-fluorophenyl)-6-isopropyl-2-
oxo-pyrimidin-1-yl]methyl]phenyl]-2-hydroxy-4-oxo-but-2-enoic
acid (4h)
(101 MHz, CDCl3)
d 186.8, 174.4, 169.1, 168.3, 167.9, 165.3, 163.4,
161.9, 161.6, 143.0, 132.8, 130.41 (d, J ¼ 8.7 Hz), 128.1, 128.05, 120.4,
115.64 (d, J ¼ 21.8 Hz), 95.5, 68.4, 61.9, 22.7, 13.7. HRMS (ESI): m/z
[MþH]þ calcd for C25H22FN2O7: 481.1405, found: 481.1406.
Yield: 92%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 8.00 (d,
J ¼ 8.2 Hz, 2H), 7.71e7.59 (m, 4H), 7.15 (t, J ¼ 8.6 Hz, 2H), 7.10 (s, 1H),
5.60 (s, 2H), 4.20 (q, J ¼ 7.1 Hz, 2H), 3.22 (hept, J ¼ 6.7 Hz, 1H), 1.33
(s, 3H), 1.31 (s, 3H), 1.11 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
4.1.46. 4-[4-[(4-benzyl-5-ethoxycarbonyl-6-methyl-2-oxo-
pyrimidin-1-yl)methyl]phenyl]-2-hydroxy-4-oxo-but-2-enoic acid
(4c)
d
188.1, 176.7, 172.4, 168.1, 165.4, 164.0 (d, J ¼ 251.1 Hz), 163.9, 163.0,
Yield: 66%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 7.96 (d,
142.9, 133.5 (d, J ¼ 3.3 Hz), 133.4, 130.4 (d, J ¼ 8.7 Hz), 128.3, 128.1,
119.8, 115.6 (d, J ¼ 21.8 Hz), 96.6, 68.5, 62.0, 33.3, 21.6, 13.7. HRMS
(ESI): m/z [MþH]þ calcd for C25H26FN2O4: 437.1871, found:
437.1870.
J ¼ 8.3 Hz, 5H), 7.55 (d, J ¼ 8.2 Hz, 5H), 7.31e7.16 (m, 16H), 7.13 (s,
2H), 5.50 (s, 5H), 4.31 (q, J ¼ 7.1 Hz, 5H), 4.16 (s, 5H), 2.52 (s, 7H),1.31
(t, J ¼ 7.1 Hz, 8H). 13C NMR (101 MHz, CDCl3)
d 187.5, 169.6, 168.8,
167.2,163.3,162.5,142.8,137.1,133.1,129.2,128.4,128.2,128.0,126.7,
121.0, 99.9, 96.0, 68.3, 61.8, 41.8, 23.0,14.0. HRMS (ESI): m/z [MþH]þ
calcd for C26H25N2O7: 477.1656, found: 477.1657.
4.1.52. 4-[4-[[5-ethoxycarbonyl-6-isopropyl-4-(4-methoxyphenyl)-
2-oxo-pyrimidin-1-yl]methyl]phenyl]-2-hydroxy-4-oxo-but-2-
enoic acid (4i)
4.1.47. 4-[4-[(5-ethoxycarbonyl-6-ethyl-2-oxo-4-phenyl-
pyrimidin-1-yl)methyl]phenyl]-2-hydroxy-4-oxo-but-2-enoic acid
(4d)
Yield: 96%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 8.00 (d,
J ¼ 8.3 Hz, 2H), 7.72e7.57 (m, 4H), 7.11 (s, 1H), 6.96 (d, J ¼ 8.8 Hz,
2H), 5.59 (s, 2H), 4.22 (q, J ¼ 7.1 Hz, 2H), 3.87 (s, 3H), 3.19 (hept,
Yield: 98%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 8.00 (d,
J ¼ 6.7 Hz, 1H), 1.31 (s, 3H), 1.30 (s, 3H), 1.14 (t, J ¼ 7.1 Hz, 3H). 13
C
J ¼ 8.3 Hz, 2H), 7.71e7.57 (m, 4H), 7.12 (s, 1H), 6.97 (d, J ¼ 8.9 Hz,
2H), 5.60 (s, 2H), 4.22 (q, J ¼ 7.1 Hz, 2H), 3.87 (s, 3H), 2.85 (q,
J ¼ 7.5 Hz, 2H),1.33 (t, J ¼ 7.5 Hz, 3H), 1.14 (t, J ¼ 7.1 Hz, 3H). 13C NMR
NMR (101 MHz, CDCl3) d 187.1, 176.2, 174.0, 168.6, 165.8, 163.9, 162.1,
161.4, 143.3, 132.9, 130.0, 129.8, 128.4, 128.0, 119.4, 113.9, 95.6, 68.3,
61.8, 55.4, 33.2, 21.6, 13.8. HRMS (ESI): m/z [MþH]þ calcd for
(101 MHz, CDCl3)
d 187.4, 173.7, 173.0, 168.4, 165.8, 163.6, 162.3,
C28H29N2O8: 521.1918, found: 521.1916.
161.5, 143.1, 133.1, 130.0, 129.7, 128.2, 128.0, 119.6, 113.9, 95.9, 68.3,
61.8, 55.4, 53.4, 28.9, 13.8, 12.8. HRMS (ESI): m/z [MþH]þ calcd for
C
26H25N2O7: 477.1656, found: 477.1656.
4.1.53. 4-[4-[(5-ethoxycarbonyl-2-oxo-4,6-diphenyl-pyrimidin-1-
yl)methyl]phenyl]-2-hydroxy-4-oxo-but-2-enoic acid (4j)
4.1.48. 4-[4-[[5-ethoxycarbonyl-6-ethyl-4-(4-fluorophenyl)-2-oxo-
pyrimidin-1-yl]methyl]phenyl]-2-hydroxy-4-oxo-but-2-enoic acid
(4e)
Yield: 92%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 8.02 (d,
J ¼ 8.3 Hz, 2H), 7.67 (dd, J ¼ 7.7, 1.1 Hz, 6H), 7.60e7.40 (m, 6H), 7.15
(s,1H), 5.66 (s, 2H), 4.05 (q, J ¼ 7.1 Hz, 2H), 0.95 (t, J ¼ 7.1 Hz, 3H). 13
C
Yield: 91%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 8.02 (d,
NMR (101 MHz, CDCl3) d 186.7, 168.0, 167.4, 163.7, 161.8, 143.1, 137.2,
J ¼ 8.3 Hz, 2H), 7.73e7.57 (m, 4H), 7.23e7.07 (m, 3H), 5.61 (s, 2H),
132.8, 130.3, 128.5, 128.3, 128.1, 120.4, 95.4, 68.5, 61.9, 13.4. HRMS
4.21 (q, J ¼ 7.1 Hz, 2H), 2.88 (q, J ¼ 7.5 Hz, 2H), 1.35 (t, J ¼ 7.5 Hz, 3H),
(ESI): m/z [MþH]þ calcd for C30H25N2O7: 525.1656, found: 525.1657.
1.12 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, Chloroform-d)
d 187.5,
173.4, 168.0, 165.4, 164.1 (d, J ¼ 251.3 Hz), 163.7, 162.4, 142.9, 133.5
(d, J ¼ 3.2 Hz), 133.2,130.4 (d, J ¼ 8.7 Hz),128.2,128.1, 120.1, 115.6 (d,
J ¼ 21.9 Hz), 96.0, 68.5, 61.9, 29.0, 13.7, 12.8. HRMS (ESI): m/z
[MþH]þ calcd for C26H24FN2O7: 495.1562, found: 495.1562.
4.1.54. 4-[4-[[5-ethoxycarbonyl-4-(4-fluorophenyl)-2-oxo-6-
phenyl-pyrimidin-1-yl]methyl]phenyl]-2-hydroxy-4-oxo-but-2-
enoic acid (4k)
Yield: 94%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 8.02 (d,
4.1.49. 4-[4-[[5-ethoxycarbonyl-6-ethyl-4-(4-methoxyphenyl)-2-
oxo-pyrimidin-1-yl]methyl]phenyl]-2-hydroxy-4-oxo-but-2-enoic
acid (4f)
J ¼ 8.2 Hz, 2H), 7.76e7.60 (m, 6H), 7.58e7.40 (m, 3H), 7.16 (t,
J ¼ 8.6 Hz, 3H), 5.65 (s, 2H), 4.06 (q, J ¼ 7.1 Hz, 2H), 0.97 (t, J ¼ 7.1 Hz,
3H). 13C NMR (101 MHz, CDCl3)
d 168.0, 167.6, 165.0 (d,
Yield: 93%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 8.00 (d,
J ¼ 259.0 Hz), 143.0, 137.1, 130.6, 130.5, 130.4, 128.5, 128.3, 128.1,
120.3, 115.6 (d, J ¼ 21.9 Hz), 95.5, 68.6, 62.0, 13.5. HRMS (ESI): m/z
[MþH]þ calcd for C30H24FN2O7: 543.1562, found: 543.1560.
J ¼ 8.3 Hz, 2H), 7.72e7.56 (m, 4H), 7.12 (s, 1H), 6.97 (d, J ¼ 8.9 Hz,
2H), 5.60 (s, 2H), 4.22 (q, J ¼ 7.1 Hz, 2H), 3.87 (s, 3H), 2.85 (q,
J ¼ 7.5 Hz, 2H),1.33 (t, J ¼ 7.5 Hz, 3H), 1.14 (t, J ¼ 7.1 Hz, 3H). 13C NMR
(101 MHz, CDCl3)
d 187.4, 173.7, 173.0, 168.4, 165.8, 163.6, 162.3,
161.5, 143.1, 133.1, 130.0, 129.7, 128.2, 128.0, 119.6, 113.9, 95.9, 68.3,
4.1.55. 4-[4-[[5-ethoxycarbonyl-4-(4-methoxyphenyl)-2-oxo-6-
phenyl-pyrimidin-1-yl]methyl]phenyl]-2-hydroxy-4-oxo-but-2-
enoic acid (4l)
61.8, 55.4, 53.4, 28.9, 13.8, 12.9. HRMS (ESI): m/z [MþH]þ calcd for
C
27H27N2O8: 507.1762, found: 507.1762.
Yield: 95%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 8.01 (d,
4.1.50. 4-[4-[(5-ethoxycarbonyl-6-isopropyl-2-oxo-4-phenyl-
pyrimidin-1-yl)methyl]phenyl]-2-hydroxy-4-oxo-but-2-enoic acid
(4g)
J ¼ 8.4 Hz, 2H), 7.76e7.61 (m, 6H), 7.57e7.37 (m, 3H), 7.14 (s, 1H),
7.06e6.90 (m, 2H), 5.65 (s, 2H), 4.08 (q, J ¼ 7.1 Hz, 2H), 3.88 (s, 3H),
0.99 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 197.3, 187.0,
Yield: 93%, yellow oil. 1H NMR (400 MHz, CDCl3)
d
8.01 (d,
168.4, 167.4, 166.6, 163.6,162.1, 161.5, 143.2,137.3, 132.9,130.2,130.1,
129.4, 128.4, 128.2, 128.1, 119.9, 114.0, 95.6, 68.5, 61.9, 55.4, 13.5.
HRMS (ESI): m/z [MþH]þ calcd for C31H27N2O8: 555.1762, found:
555.1760.
J ¼ 8.4 Hz, 2H), 7.71e7.59 (m, 4H), 7.52e7.41 (m, 3H), 7.13 (s, 1H),
5.60 (s, 2H), 4.16 (q, J ¼ 7.1 Hz, 2H), 3.30e3.17 (m, 1H), 1.32 (s, 1H),
1.30 (s, J ¼ 6.7 Hz, 3H), 1.05 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz,

O. Sari et al. / European Journal of Medicinal Chemistry 104 (2015) 127e138
135
4.1.56. 4-[4-[[4-(3,5-dimethylphenyl)-5-ethoxycarbonyl-2-oxo-6-
4.1.62. Ethyl 6-ethyl-4-(4-fluorophenyl)-2-thioxo-3,4-dihydro-1H-
phenyl-pyrimidin-1-yl]methyl]phenyl]-2-hydroxy-4-oxo-but-2-
pyrimidine-5-carboxylate (21b)
enoic acid (4m)
Yield: 94%, white solid. 1H NMR (400 MHz, CDCl3)
d
8.35 (s, 1H),
Yield: 89%, yellow oil. 1H NMR (400 MHz, CDCl3)
d
8.02 (d,
7.87 (s, 1H), 7.27 (dd, J ¼ 8.5, 5.0 Hz, 2H), 7.01 (t, J ¼ 8.7 Hz, 2H), 5.39
(d, J ¼ 3.0 Hz, 1H), 4.18e4.05 (m, 2H), 2.77 (q, J ¼ 7.5 Hz, 2H), 1.24 (t,
J ¼ 7.5 Hz, 3H), 1.18 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
J ¼ 8.1 Hz, 2H), 7.77e7.57 (m, 4H), 7.56e7.39 (m, 3H), 7.26 (s, 2H),
7.14 (d, J ¼ 10.5 Hz, 2H), 5.65 (s, 2H), 4.06 (q, J ¼ 7.1 Hz, 2H), 2.37 (s,
6H), 1.00 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
168.1, 167.8,
d
174.5, 164.8, 162.5 (d, J ¼ 247.4 Hz), 148.3, 138.4 (d, J ¼ 3.2 Hz),
167.2, 163.6, 143.2, 138.10, 137.3, 137.0, 132.0, 130.2, 128.5, 128.3,
128.1, 126.1, 120.4, 95.5, 77.2, 68.5, 61.79, 21.3, 13.5. HRMS (ESI): m/z
[MþH]þ calcd for C32H29N2O7: 553.1969, found: 553.1969.
128.6 (d, J ¼ 8.3 Hz), 115.8 (d, J ¼ 21.6 Hz), 102.0, 60.5, 55.3, 24.9,
14.0, 12.5. HRMS (ESI): m/z [MþH]þ calcd for C15H18FN2O2S:
309.1067, found: 309.1071.
4.1.63. Ethyl 6-ethyl-4-(4-methoxyphenyl)-2-thioxo-3,4-dihydro-
4.1.57. 4-Bromomethylbenzyl alcohol (14)
1H-pyrimidine-5-carboxylate (21c)
To a solution of methyl 4-bromomethylbenzoate 13 (3.09 g,
13 mmol) in dry CH2Cl2 (80 mL) cooled to ꢁ78 ꢀC with stirring
under nitrogen was added dropwise a solution of DIBAL-H (47 mL,
1.0 M solution in THF). Stirring was continued for 1.5 h at ꢁ78 ꢀC,
and the reaction mixture was then allowed to warm to 0 ꢀC and
quenched with H2O. The organic layer was separated and the
aqueous was extracted with CH2Cl2. The combined organic extracts
were dried over MgSO4 and evaporated to yield quantitatively the
desired alcohol as a white solid. CAS: 71831-21-5.
Yield: 77%, white solid. 1H NMR (400 MHz, DMSO-d6)
d 10.29 (s,
1H), 9.58 (s, 1H), 7.13 (d, J ¼ 8.6 Hz, 2H), 6.91 (d, J ¼ 8.6 Hz, 2H), 5.11
(d, J ¼ 3.6 Hz, 1H), 4.02 (q, J ¼ 7.1 Hz, 2H), 3.73 (s, 3H), 2.82e2.61 (m,
2H), 1.11 (t, J ¼ 7.1 Hz, 6H). 13C NMR (101 MHz, DMSO-d6)
d 174.8,
165.2, 159.2, 150.7, 136.14, 128.0, 114.4, 100.6, 60.0, 55.5, 53.8, 23.9,
14.4, 13.5. HRMS (ESI): m/z [MþH]þ calcd for C16H21N2O3S:
321.1267, found: 321.1268.
4.1.64. Ethyl 4,6-diphenyl-2-thioxo-3,4-dihydro-1H-pyrimidine-5-
carboxylate (21d)
4.1.58. 1-(3-(4-(Hydroxymethyl)benzyl)phenyl)ethanone (15)
Yield: 90%, white solid. CAS: 154866-93-0.
To a solution of alcohol 14 (2.01 g, 10 mmol) in toluene (30 mL)
were added 3-acethylphenylboronic acid (2.46 g, 15 mmol), K3PO4
(20 mmol), PPh3 (104 mg, 0.4 mmol) and Pd(OAc)2 (45 mg,
0.2 mmol). The resulting yellow suspension was stirred at room
temperature for 2 days under Ar. The reaction mixture was then
poured into 100 mL of ether and washed with 1 N NaOH and brine.
The organic was dried over MgSO4 and concentrated under reduced
pressure. The resultant residue was subjected to flash chromatog-
raphy (silica gel, petroleum ether/EtOAc, 7:3) to afford the title
compound (1.8 g, 77%) as a light yellow solid. CAS: 1237521-77-5.
4.1.65. Ethyl 4-(4-fluorophenyl)-6-phenyl-2-thioxo-3,4-dihydro-
1H-pyrimidine-5-carboxylate (21e)
Yield: 89%, white solid. 1H NMR (400 MHz, CDCl3)
d 7.75 (s, 1H),
7.53e7.31 (m, 8H), 7.09 (t, J ¼ 8.6 Hz, 2H), 5.55 (d, J ¼ 3.1 Hz, 1H),
3.97e3.77 (m, 2H), 0.84 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz,
CDCl3)
d
174.8, 164.7, 162.7 (d, J ¼ 247.7 Hz), 143.7, 138.1 (d,
J ¼ 3.2 Hz), 133.9, 130.0, 128.6 (d, J ¼ 8.4 Hz), 128.5, 128.0, 116.0 (d,
J ¼ 21.7 Hz), 103.7, 60.5, 55.9, 13.4. HRMS (ESI): m/z [MþH]þ calcd
for C19H18FN2O2S: 357.1067, found: 357.1068.
4.1.66. Ethyl 4-(4-methoxyphenyl)-6-phenyl-2-thioxo-3,4-dihydro-
1H-pyrimidine-5-carboxylate (21f)
4.1.59. 1-(3-(4-(Bromomethyl)benzyl)phenyl)ethanone (16)
To a solution of alcohol 15 (450 mg, 1.87 mmol) and PPh3
(786 mg, 1.87 mmol) in THF (12 mL) was added NBS (0.534 g,
1.87 mmol) 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 ꢂ 3). The
combined organic extracts were dried over MgSO4 and concen-
trated under reduced pressure. The resultant residue was subjected
to flash chromatography (Petroleum ether/EtOAc, 95:5 to 90:10) to
afford the title compound (559 mg, 98%) as a pale yellow oil. CAS:
1237521-89-9.
Yield: 77%, white solid. CAS: 134074-40-1.
4.1.67. General procedure for alkylation and aromatization of
thiopyrimidines (23a-f)
A 2e5 mL microwave vial was charged with a DMF solution
(0.5 M) of thiopyrimidine (1 mmol), 4-(bromomethyl)acetophe-
none (1.2 mmol) and K2CO3 (2 mmol). The mixture was irradiated
at 60 ꢀC during 1 h. Volatiles were evaporated and the residue was
purified by silica gel column chromatography (EtOAc/Petroleum
ether, 2:8) to afford the alkylated derivative as a mixture of isomers.
Subsequently, in a 10e20 mL microwave vial, the crude alkylated
product was dissolved in CH2Cl2 (0.05 M) and MnO2 (10 equiv.) was
added. The mixture was irradiated at 100 ꢀC during 30 min, filtrated
through a pad of Celite© and concentrated under reduced pressure.
The remainder was purified by a short silica gel column (EtOAc/
petroleum ether, 1:9) to afford the desired aromatized derivative.
4.1.60. (Z)-4-[3-[[4-[(5-ethoxycarbonyl-6-ethyl-2-oxo-4-phenyl-
pyrimidin-1-yl)methyl]phenyl]methyl] phenyl] -2-hydroxy-4-oxo-
but-2-enoic acid (19)
Yield: 96%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 7.85 (d,
J ¼ 7.1 Hz, 1H), 7.70e7.61 (m, 3H), 7.53e7.40 (m, 7H), 7.23 (d,
J ¼ 7.9 Hz, 2H), 7.02 (s, 1H), 5.50 (s, 2H), 4.18 (q, J ¼ 7.1 Hz, 2H), 4.09
(s, 2H), 2.92 (q, J ¼ 7.5 Hz, 2H), 1.35 (t, J ¼ 7.5 Hz, 3H), 1.07 (t,
4.1.68. Ethyl 2-[(4-acetylphenyl)methylsulfanyl]-4-ethyl-6-phenyl-
pyrimidine-5-carboxylate (23a)
J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 189.2,173.3,171.5,168.2,
166.7, 163.9, 162.7, 142.2, 139.9, 137.6, 134.5, 134.3, 130.2, 129.3,
129.3, 129.1, 128.5, 128.3, 125.7, 119.9, 97.0, 69.5, 61.8, 41.1, 28.9, 13.6,
13.1. HRMS (ESI): m/z [MþH]þ calcd for C33H31N2O7: 567.2126,
found: 567.2126.
Yield: 48% in two steps, yellow oil. 1H NMR (400 MHz, CDCl3)
d
7.91 (d, J ¼ 8.3 Hz, 2H), 7.64e7.53 (m, 4H), 7.52e7.40 (m, 3H), 4.51
(s, 2H), 4.16 (q, J ¼ 7.1 Hz, 2H), 2.85 (q, J ¼ 7.5 Hz, 2H), 2.60 (s, 3H),
1.33 (t, J ¼ 7.5 Hz, 3H), 1.05 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz,
CDCl3)
d 197.7, 171.1, 170.0, 167.9, 163.9, 143.7, 137.6, 136.0, 130.1,
4.1.61. Ethyl 6-ethyl-4-phenyl-2-thioxo-3,4-dihydro-1H-
pyrimidine-5-carboxylate (21a)
129.2, 128.49, 128.46, 128.3, 121.2, 61.8, 34.9, 29.0, 26.6, 13.6, 12.8.
HRMS (ESI): m/z [MþH]þ calcd for C24H25N2O3S: 421.1580, found:
421.1582.
Yield: 77%, white solid. CAS:134074-29-6.

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O. Sari et al. / European Journal of Medicinal Chemistry 104 (2015) 127e138
4.1.69. Ethyl 2-[(4-acetylphenyl)methylsulfanyl]-4-ethyl-6-(4-
fluorophenyl)pyrimidine-5-carboxylate (23b)
4.1.75. (Z)-4-[4-[[5-ethoxycarbonyl-4-ethyl-6-(4-fluorophenyl)
pyrimidin-2-yl]sulfanylmethyl]phenyl]-2-hydroxy-4-oxo-but-2-
enoic acid (5b)
Yield: 43% in two steps, yellow oil. 1H NMR (400 MHz, CDCl3)
d
7.91 (d, J ¼ 8.3 Hz, 2H), 7.63e7.59 (m, 2H), 7.56 (d, J ¼ 8.2 Hz, 2H),
Yield: 94%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 7.95 (d,
7.20e7.08 (m, 2H), 4.50 (s, 2H), 4.19 (q, J ¼ 7.1 Hz, 2H), 2.84 (q,
J ¼ 8.1 Hz, 2H), 7.66e7.56 (m, 4H), 7.17e7.12 (m, 3H), 4.51 (s,
J ¼ 14.7 Hz, 2H), 4.20 (q, J ¼ 7.1 Hz, 2H), 2.85 (q, J ¼ 7.5 Hz, 2H), 1.32
(t, J ¼ 7.5 Hz, 3H), 1.11 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
J ¼ 7.5 Hz, 2H), 1.32 (t, J ¼ 7.5 Hz, 3H), 1.11 (t, J ¼ 7.1 Hz, 3H). 13C NMR
(101 MHz, CDCl3)
d
197.6, 171.2, 170.1, 167.9, 164.0 (d, J ¼ 251.0 Hz),
162.6, 143.6, 136.0, 133.6 (d, J ¼ 3.3 Hz), 130.4 (d, J ¼ 8.6 Hz), 129.2,
128.5, 121.0, 115.6 (d, J ¼ 21.8 Hz), 61.9, 34.9, 29.0, 26.6, 13.7, 12.8.
HRMS (ESI): m/z [MþH]þ calcd for C24H24FN2O3S: 439.1486, found:
439.1486.
d
187.8, 171.0, 170.2, 167.8, 164.1 (d, J ¼ 229.8 Hz), 162.8, 162.7, 145.0,
133.5 (d, J ¼ 3.2 Hz), 132.5, 130.4 (d, J ¼ 8.7 Hz), 129.6, 128.1, 121.1,
115.6 (d, J ¼ 21.9 Hz), 96.2, 62.0, 35.0, 29.0,13.7,12.8. HRMS (ESI): m/
z [MþH]þ calcd for C26H24FN2O6S: 511.1334, found: 511.1335.
4.1.70. Ethyl 2-[(4-acetylphenyl)methylsulfanyl]-4-ethyl-6-(4-
methoxyphenyl)pyrimidine-5-carboxylate (23c)
4.1.76. (Z)-4-[4-[[5-ethoxycarbonyl-4-ethyl-6-(4-methoxyphenyl)
pyrimidin-2-yl]sulfanylmethyl]phenyl]-2-hydroxy-4-oxo-but-2-
enoic acid (5c)
Yield: 49% in two steps, yellow oil. 1H NMR (400 MHz, CDCl3)
Yield: 89%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 7.91 (d,
d
7.91 (d, J ¼ 8.3 Hz, 2H), 7.61 (d, J ¼ 8.7 Hz, 2H), 7.57 (d, J ¼ 8.3 Hz,
2H), 6.96 (d, J ¼ 8.8 Hz, 2H), 4.51 (s, 2H), 4.22 (q, J ¼ 7.1 Hz, 2H), 3.88
(s, 3H), 2.82 (q, J ¼ 7.5 Hz, 2H), 2.60 (s, 3H), 1.31 (t, J ¼ 7.5 Hz, 3H),
J ¼ 5.2 Hz, 2H), 7.60e758 (m, 4H), 7.13 (s, 1H), 6.95 (d, J ¼ 8.5 Hz,
2H), 4.50 (s, 2H), 4.22 (q, J ¼ 7.0 Hz, 2H), 3.86 (s, 3H), 2.81 (q,
J ¼ 7.4 Hz, 2H), 1.30 (t, J ¼ 7.5 Hz, 3H), 1.14 (t, J ¼ 7.1 Hz, 3H). 13C NMR
1.15 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 197.7, 170.8,
169.8, 168.4, 163.0, 161.4, 143.8, 136.0, 130.0, 129.8, 129.2, 128.5,
120.7, 113.9, 61.8, 55.4, 34.9, 29.0, 26.6, 13.8, 12.9. HRMS (ESI): m/z
[MþH]þ calcd for C25H27N2O4S: 451.1686, found: 451.1687.
(101 MHz, CDCl3)
d 170.6, 169.8, 168.3, 163.1, 161.42, 130.0, 129.7,
129.6, 128.0, 120.7, 113.9, 61.8, 55.4, 34.9, 28.9, 13.8, 12.9. HRMS
(ESI): m/z [MþH]þ calcd for C27H27N2O7S: 523.1533, found:
523.1533.
4.1.71. Ethyl 2-[(4-acetylphenyl)methylsulfanyl]-4,6-diphenyl-
pyrimidine-5-carboxylate (23d)
4.1.77. (Z)-4-[4-[(5-ethoxycarbonyl-4,6-diphenyl-pyrimidin-2-yl)
Yield: 35% in two steps, yellow oil. 1H NMR (400 MHz, CDCl3)
sulfanylmethyl]phenyl]-2-hydroxy-4-oxo-but-2-enoic acid (5d)
d
7.90 (d, J ¼ 8.3 Hz, 2H), 7.72e7.61 (m, 4H), 7.56 (d, J ¼ 8.2 Hz, 2H),
Yield: 98%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 7.94 (d,
7.52e7.39 (m, 6H), 4.52 (s, 2H), 4.06 (q, J ¼ 7.1 Hz, 2H), 2.58 (s, 3H),
J ¼ 8.3 Hz, 2H), 7.75e7.57 (m, 6H), 7.57e7.40 (m, 6H), 7.15 (s, 1H),
0.95 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 197.6, 171.3,
4.54 (s, 2H), 4.05 (q, J ¼ 7.1 Hz, 2H), 0.94 (t, J ¼ 7.1 Hz, 3H). 13C NMR
167.9, 164.6, 143.5, 137.2, 136.0, 130.2, 129.3, 128.5, 128.4, 121.4, 61.9,
35.1, 26.6, 13.5. HRMS (ESI): m/z [MþH]þ calcd for C28H25N2O3S:
469.1580, found: 469.1581.
(101 MHz, CDCl3) d 187.2, 171.0, 167.8, 164.7,162.3,145.1, 137.1, 132.2,
130.3, 129.8, 128.5, 128.3, 128.0, 121.4, 95.6, 61.9, 35.1, 13.4. HRMS
(ESI): m/z [MþH]þ calcd for C30H25N2O6S: 541.1428, found:
541.1425.
4.1.72. Ethyl 2-[(4-acetylphenyl)methylsulfanyl]-4-(4-
fluorophenyl)-6-phenyl-pyrimidine-5-carboxylate (23e)
Yield: 49% in two steps, yellow oil. 1H NMR (400 MHz, CDCl3)
4.1.78. (Z)-4-[4-[[5-ethoxycarbonyl-4-(4-fluorophenyl)-6-phenyl-
pyrimidin-2-yl]sulfanylmethyl]phenyl]-2-hydroxy-4-oxo-but-2-
enoic acid (5e)
d
7.91 (d, J ¼ 8.1 Hz, 2H), 7.70e7.61 (m, 4H), 7.56 (d, J ¼ 8.2 Hz, 2H),
7.53e7.44 (m, 3H), 7.16 (t, J ¼ 8.5 Hz, 2H), 4.52 (s, 2H), 4.06 (q,
Yield: 92%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 7.94 (d,
J ¼ 7.1 Hz, 2H), 2.60 (s, 3H), 0.97 (t, J ¼ 7.1 Hz, 3H). 13C NMR
J ¼ 8.3 Hz, 2H), 7.71e7.57 (m, 6H), 7.55e7.43 (m, 3H), 7.17 (t,
(101 MHz, CDCl3)
d
197.6, 171.3, 167.8, 164.8, 164.03 (d, J ¼ 251.1 Hz),
J ¼ 8.7 Hz, 2H), 7.14 (s, 1H), 4.53 (s, 2H), 4.06 (q, J ¼ 7.1 Hz, 2H), 0.97
163.4, 143.4, 137.1, 136.1, 133.26 (d, J ¼ 3.3 Hz), 130.58 (d, J ¼ 8.7 Hz),
130.3, 129.3, 128.5, 128.5, 128.3, 121.2, 115.65 (d, J ¼ 21.9 Hz), 62.0,
35.1, 26.6, 13.5. HRMS (ESI): m/z [MþH]þ calcd for C28H24FN3O3S:
487.1486, found: 487.1486.
(t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 187.8, 172.8, 171.1,
167.8, 164.84, 164.06 (d, J ¼ 251.2 Hz), 163.5, 163.0, 144.9, 137.0,
133.14 (d, J ¼ 3.3 Hz),132.5,130.6 (d, J ¼ 8.7 Hz), 130.35,129.7, 128.5,
128.3, 128.1, 121.2, 115.7 (d, J ¼ 21.9 Hz), 96.3, 62.1, 35.1, 13.5. HRMS
(ESI): m/z [MþH]þ calcd for C30H24FN2O6S: found: 559.1328.
4.1.73. Ethyl 2-[(4-acetylphenyl)methylsulfanyl]-4-(4-
methoxyphenyl)-6-phenyl-pyrimidine-5-carboxylate (23f)
Yield: 39% in two steps, yellow oil. 1H NMR (400 MHz, CDCl3)
4.1.79. (Z)-4-[4-[[5-ethoxycarbonyl-4-(4-methoxyphenyl)-6-
phenyl-pyrimidin-2-yl]sulfanylmethyl]phenyl]-2-hydroxy-4-oxo-
but-2-enoic acid (5f)
d
7.91 (d, J ¼ 8.3 Hz, 2H), 7.69e7.60 (m, 4H), 7.56 (d, J ¼ 8.2 Hz, 2H),
7.53e7.43 (m, 3H), 6.98 (d, J ¼ 8.9 Hz, 2H), 4.53 (s, 2H), 4.08 (q,
Yield: 90%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 7.92 (d,
J ¼ 7.1 Hz, 2H), 3.89 (s, 3H), 2.61 (s, 3H), 0.99 (t, J ¼ 7.1 Hz, 3H). 13
C
J ¼ 8.2 Hz, 2H), 7.70e7.54 (m, 6H), 7.53e7.42 (m, 3H), 7.12 (s, 1H),
NMR (101 MHz, CDCl3) d 197.7,171.0,168.2,164.6,163.8,161.4,143.6,
6.99 (d, J ¼ 8.9 Hz, 2H), 4.52 (s, 2H), 4.08 (q, J ¼ 7.1 Hz, 2H), 3.87 (s,
137.4, 136.0, 130.1, 130.0, 129.4, 129.3, 128.5, 128.4, 128.3, 120.8,
3H), 0.98 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 188.2,172.4,
114.0, 61.9, 55.4, 35.1, 26.6, 13.5. HRMS (ESI): m/z [MþH]þ calcd for
170.8, 168.2, 164.7, 163.9, 163.3, 161.5, 145.0, 137.2, 132.6, 130.2,
129.7, 129.3, 128.5, 128.3, 128.1, 120.9, 114.0, 96.6, 62.0, 55.4, 35.1,
13.5. HRMS (ESI): m/z [MþH]þ calcd for C31H27N2O7S: 571.1533,
found: 571.1530.
C
29H27N2O4S: 499.1686, found: 499.1687.
4.1.74. (Z)-4-[4-[(5-ethoxycarbonyl-4-ethyl-6-phenyl-pyrimidin-2-
yl)sulfanylmethyl]phenyl]-2-hydroxy-4-oxo-but-2-enoic acid (5a)
Yield: 96%, yellow oil. 1H NMR (400 MHz, CDCl3)
d 8.05e7.88 (m,
2H), 7.67e7.55 (m, 4H), 7.55e7.39 (m, 3H), 7.14 (s, 1H), 4.52 (s, 2H),
4.16 (q, J ¼ 7.1 Hz, 2H), 2.86 (q, J ¼ 7.5 Hz, 2H), 1.32 (t, J ¼ 7.5 Hz, 3H),
4.1.80. General procedure for the preparation of prodrug forms
To a stirred solution of
b-diketocid derivative (50 mg) in 2-
1.05 (t, J ¼ 7.1 Hz, 3H).13C NMR (101 MHz, CDCl3)
d
187.9,172.9,170.9,
propanol (3 mL) was added conc. H2SO4 (1 drop) and the mixture
was heated for 3 h at 85 ꢀC. The reaction was concentrated under
reduced pressure and the remainder (unstable on silica gel) was
purified by preparative reversed phase HPLC.
170.2,167.9,164.01,163.1,145.1,137.5,132.5,130.2,129.7,128.5,128.3,
128.1,121.3, 96.3, 61.9, 35.0, 29.0,13.6,12.9. HRMS (ESI): m/z [MþH]þ
calcd for C26H25N2O6S: 493.1428, found: 493.1428.

O. Sari et al. / European Journal of Medicinal Chemistry 104 (2015) 127e138
137
4.1.81. Ethyl (Z)-4-benzyl-1-(4-(3-hydroxy-4-isopropoxy-4-oxobut-
4.2. Enzymatic activity evaluation
2-enoyl)benzyl)-6-methyl-2-oxo-1,2-dihydropyrimidine-5-
carboxylate (25c)
HIV-1 IN recombinant protein was expressed in bacteria and
purified as previously described [21]. 30-P and ST activity were
monitored using a gel-based assay in the following condition.
Compounds or an equivalent volume of the drug solvent (100%
Yield: 32%. White solid. 1H NMR (400 MHz, CDCl3)
d 7.97 (d,
J ¼ 8.4 Hz, 2H), 7.55 (d, J ¼ 8.1 Hz, 2H), 7.38e7.15 (m, 5H), 7.06 (s,
1H), 5.51 (s, 2H), 5.26 (p, J ¼ 6.3 Hz, 1H), 4.32 (q, J ¼ 7.1 Hz, 2H), 4.15
(s, 2H), 2.51 (s, 3H), 1.43 (s, 3H), 1.41 (s, 3H), 1.32 (t, J ¼ 7.1 Hz, 3H).
DMSO) was mixed to 20 nM 32P-labeled DNA substrate and 400
mM
13C NMR (101 MHz, CDCl3)
d
190.1, 170.4, 169.5, 168.7, 167.3, 163.5,
IN in a buffer containing 50 mM MOPS pH 7.2, 7.5 mM MgCl2, and
14 mM 2-mercaptoethanol. After 1 h incubation at 37 ꢀC, the re-
action was quenched by addition of an equal volume of loading
buffer (formamide supplemented with 1% sodium dodecyl sulfate,
0.25% bromophenol blue, and xylene cyanol). Products were
separated in 16% polyacrylamide denaturing sequencing gels. Dried
gels were visualized using a Typhoon 8600 (GE Healthcare).
Densitometric analyses were performed using the ImageQuant 5.1
software from GE Healthcare. Data analyses (linear regression, IC50
determination, and standard deviation) were performed using
Prism 5.0c software from GraphPad.
161.7, 142.5, 137.3, 134.4, 129.2, 128.4, 128.2, 128.0, 126.7, 120.9, 97.8,
70.7, 68.3, 61.7, 41.8, 23.2, 21.7, 14.0. HRMS (ESI): m/z [MþH]þ calcd
for C29H31N2O7: 519.2126, found: 519.2130.
4.1.82. Ethyl (Z)-6-ethyl-1-(4-(3-hydroxy-4-isopropoxy-4-oxobut-
2-enoyl)benzyl)-2-oxo-4-phenyl-1,2-dihydropyrimidine-5-
carboxylate (25d)
Yield: 29%. White solid. 1H NMR (400 MHz, CDCl3)
d 8.02 (d,
J ¼ 8.2 Hz, 2H), 7.78e7.57 (m, 4H), 7.53e7.41 (m, 3H), 7.08 (s, 1H),
5.62 (s, 2H), 5.25 (p, J ¼ 6.3 Hz, 1H), 4.17 (q, J ¼ 7.1 Hz, 2H), 2.88 (q,
J ¼ 7.5 Hz, 2H),1.42 (s, 3H),1.41 (s, 3H),1.34 (t, J ¼ 7.5 Hz, 3H),1.05 (t,
4.3. Antiviral activity evaluation
J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 190.1, 173.3, 170.4, 168.1,
166.6, 163.8, 161.7, 142.7, 137.6, 134.5, 130.2, 128.5, 128.2, 128.2,
128.0, 120.1, 97.8, 70.7, 68.4, 61.8, 29.0, 21.7, 13.6, 12.8. HRMS (ESI):
m/z [MþH]þ calcd for C29H31N2O7: 519.2126, found: 519.2134.
The antiviral activity of compounds described in Table 2 was
evaluated against HIV-1 in activated primary human PBM cells [22].
Cytotoxicity was evaluated in normal PBM cells, along with CEM
and Vero cells [23].
4.1.83. Ethyl (Z)-1-(4-(3-hydroxy-4-isopropoxy-4-oxobut-2-enoyl)
benzyl)-4-(4-methoxyphenyl)-2-oxo-6-phenyl-1,2-
dihydropyrimidine-5-carboxylate (25l)
4.4. Molecular modeling
Yield: 24%. White solid. 1H NMR (400 MHz, CDCl3)
d 8.03 (d,
Software: molecular modeling studies were performed with the
Schrodinger Molecular Modeling Suite [24] within Maestro, the
interface piloting different modules. Glide was used to dock ligands.
Analysis and visualization tasks were performed within MOE
software [25].
J ¼ 8.3 Hz, 2H), 7.78e7.61 (m, 6H), 7.55e7.41 (m, 3H), 7.08 (s, 1H),
6.99 (d, J ¼ 8.8 Hz, 2H), 5.66 (s, 2H), 5.26 (p, J ¼ 6.3 Hz, 1H), 4.09 (q,
J ¼ 7.1 Hz, 2H), 3.89 (s, 3H),1.42 (s, 3H),1.41 (s, 3H),1.00 (t, J ¼ 7.1 Hz,
3H). 13C NMR (101 MHz, CDCl3)
d 190.1, 170.4, 168.5, 167.3, 166.6,
163.7,161.7,161.5, 142.8,137.5,134.5,130.1, 129.5,128.4,128.3,128.2,
128.0, 119.9, 113.9, 97.8, 70.7, 68.5, 61.9, 55.4, 29.7, 21.7, 13.5. HRMS
(ESI): m/z [MþH]þ calcd for C34H33N2O8: 597.2231, found: 597.2227.
Structure preparation: crystal structure of IN in complex with
raltegravir (PDB code 3OYA) was retrieved from the Protein Data
Bank. Next this structure was prepared using the Protein Prepara-
tion Wizard workflow of the Schrodinger Molecular Modeling
Suite. Receptor was pre-processed (hydrogen atoms added,
incomplete residues filled), bond orders and connections of ligand
were manually corrected and non structural water removed. An
exhaustive sampling was conducted regarding hydrogen bond
assignment and the complex was finally refined by a minimization
stage with a constraint to converge to a structure with an RMSD of
0.3 Å (OPLS2005 force field), essentially in order to remove steric
clashes. Ligands, other than the one co-crystallized, were built with
Marvin Sketch 5.8.0 [26] and were submitted to Corina [27]
[MM09], a 3D structure generator. Finally, 3D structures were
submitted to the LigPrep module of the Schrodinger Molecular
Modeling Suite in order to take into account tautomerization and
ionization via the Epik module. The resulting structures became the
starting point for docking simulations. Docking parameters: dock-
ing calculations were performed with extra precision. Ligand flex-
ibility was taken into account and the option of sampling of ring
conformation was activated.
4.1.84. Ethyl (Z)-6-ethyl-1-(4-(3-(3-hydroxy-4-isopropoxy-4-
oxobut-2-enoyl)benzyl)benzyl)-2-oxo-4-phenyl-1,2-
dihydropyrimidine-5-carboxylate (26)
Yield: 30%. White solid. 1H NMR (400 MHz, CDCl3)
d 7.91e7.82
(m, 2H), 7.64 (dd, J ¼ 7.9, 1.7 Hz, 2H), 7.51e7.41 (m, 7H), 7.21 (d,
J ¼ 8.0 Hz, 2H), 7.05 (s, 1H), 5.51 (s, 2H), 5.25 (p, J ¼ 6.3 Hz, 1H), 4.16
(q, J ¼ 7.1 Hz, 2H), 4.08 (s, 2H), 2.86 (q, J ¼ 7.5 Hz, 2H), 1.42 (s, 3H),
1.40 (s, 3H), 1.34 (t, J ¼ 7.5 Hz, 3H), 1.05 (t, J ¼ 7.1 Hz, 3H). 13C NMR
(101 MHz, CDCl3)
d 190.7, 173.1, 170.2, 168.3, 166.4, 164.1, 161.8,
142.0, 140.1, 137.8, 135.2, 134.6, 134.4, 130.0, 129.1, 128.9, 128.9,
128.4, 128.3, 128.2, 125.9, 119.7, 97.9, 70.7, 69.1, 61.7, 41.5, 29.7, 29.0,
21.7, 13.6, 12.8. HRMS (ESI): m/z [MþH]þ calcd for C36H37N2O7:
609.2595, found: 609.2590.
4.1.85. Ethyl(Z)-4-ethyl-6-(4-fluorophenyl)-2-((4-(3-hydroxy-4-
isopropoxy-4-oxobut-2-enoyl)benzyl)thio)pyrimidine-5-
carboxylate (27)
Yield: 38%. Pale yellow oil. 1H NMR (400 MHz, CDCl3)
d
7.95 (d,
Acknowledgments
J ¼ 8.4 Hz, 2H), 7.77e7.53 (m, 4H), 7.14 (t, J ¼ 8.7 Hz, 2H), 7.05 (s,1H),
5.25 (p, J ¼ 6.3 Hz, 1H), 4.52 (s, 2H), 4.19 (q, J ¼ 7.1 Hz, 2H), 2.84 (q,
J ¼ 7.5 Hz, 2H), 1.42 (s, 3H), 1.40 (s, 3H), 1.32 (t, J ¼ 7.5 Hz, 3H), 1.11 (t,
This work was supported in part by the NIH Intramural Research
Program, Center for Cancer Research, National Cancer Institute (Z01
J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d
190.1, 171.0, 170.3, 170.2,
BC 007333-09LMP). O.S. is grateful to the French Ministere de
ꢁ
ꢀ
167.8, 164.0 (d, J ¼ 251.0 Hz), 162.6, 161.7, 144.5, 133.8, 133.6 (d,
J ¼ 3.2 Hz), 130.4 (d, J ¼ 8.6 Hz), 129.5, 128.0, 121.1, 115.6 (d,
J ¼ 21.8 Hz), 97.8, 70.7, 61.9, 35.0, 29.0, 21.7, 13.7, 12.8. HRMS (ESI):
m/z [MþH]þ calcd for C29H30FN2O6S: 553.1803, found: 553.1798.
l'Enseignement Superieur MNERST for a Ph.D. scholarship. We
thank the LABEX SynOrg (ANR-11-LABX-0029) for financial sup-
port. RFS is supported in part by NIH Grant 5P30-AI-50409 (CFAR)
and by the Department of Veterans Affairs.

138
O. Sari et al. / European Journal of Medicinal Chemistry 104 (2015) 127e138
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epoxide hydrolase, J. Med. Chem. 54 (2011) 3037e3050;
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dx.doi.org/10.1016/j.ejmech.2015.09.015.
(c) S.Hare, A. M.Vos, R. F.Clayton, J. W.Thuring, M. D.Cummings, Cherepanov P.
Molecular mechanisms of retrieval integrase inhibition and the evolution of
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