A novel 3,4-dihydropyrimidin-2(1H)-one: HIV-1 replication inhibitors with improved metabolic stability

Article abstract of DOI:10.1016/j.bmcl.2012.01.133

Following the previous SAR of a novel dihydropyrimidinone scaffold as HIV-1 replication inhibitors a detailed study directed towards optimizing the metabolic stability of the ester functional group in the dihydropyrimidinone (DHPM) scaffold is described. Replacement of the ester moiety by thiazole ring significantly improved the metabolic stability while retaining antiviral activity against HIV-1 replication. These novel and potent DHPMs with bioisosteres could serve as advanced leads for further optimization.

Full text of DOI:10.1016/j.bmcl.2012.01.133

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2522–2526
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel 3,4-dihydropyrimidin-2(1H)-one: HIV-1 replication inhibitors
with improved metabolic stability
Junwon Kim a, Taedong Ok a, Changmin Park a, Wonyoung So a, Mina Jo a, Youngmi Kim a, Minjung Seo a,
Doohyun Lee a, Suyeon Jo a, Yoonae Ko a, Inhee Choi a, Youngsam Park b, Jaewan Yoon b, Moon Kyeong Ju b,
JiYe Ahn b, Junghwan Kim b, Sung-Jun Han b, Tae-Hee Kim c,f, Jonathan Cechetto c, Jiyoun Nam c,
Michel Liuzzi d, Peter Sommer e, Zaesung No a,
⇑
a Medicinal Chemistry Group, Institut Pasteur Korea (IP-K), Sampyeong-dong 696, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea
b Drug Biology Group, Institut Pasteur Korea (IP-K), Sampyeong-dong 696, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea
c Screening Technology Platforms Group, Institut Pasteur Korea (IP-K), Sampyeong-dong 696, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea
d Early Discovery Program, Institut Pasteur Korea (IP-K), Sampyeong-dong 696, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea
e Cell Biology of Retroviruses Group, Institut Pasteur Korea (IP-K), Sampyeong-dong 696, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea
f Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Following the previous SAR of a novel dihydropyrimidinone scaffold as HIV-1 replication inhibitors a
detailed study directed towards optimizing the metabolic stability of the ester functional group in the
dihydropyrimidinone (DHPM) scaffold is described. Replacement of the ester moiety by thiazole ring sig-
nificantly improved the metabolic stability while retaining antiviral activity against HIV-1 replication.
These novel and potent DHPMs with bioisosteres could serve as advanced leads for further optimization.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 26 December 2011
Revised 27 January 2012
Accepted 31 January 2012
Available online 8 February 2012
Keywords:
HIV
Inhibitor
3,4-Dihydropyrimidin-2(1H)-one
Biginelli reaction
Bioisostere
Since the discovery of the human immunodeficiency virus (HIV)
as the causative agent of the acquired immunodeficiency syn-
drome (AIDS) in 1983, the virus has rapidly spread around the
world and HIV/AIDS is currently considered a pandemic.1 Accord-
ing to UNAIDS 2009 report [Data.unaids.org], worldwide some 60
million people have been infected, with some 25 million deaths,
and 14 million orphaned children in southern Africa alone since
the epidemic began. This major global health threat triggered
intensive drug discovery efforts and the first FDA-approved anti-
retroviral drug, AZT, was available in 1987. To date, 25 anti-HIV
drugs belonging to six different inhibitor classes have been
approved by FDA for the treatment of HIV infection, including
nucleoside or non-nucleosides reverse transcriptase inhibitors
(NRTIs/NNRTIs), protease inhibitors (PIs), integrase inhibitors,
entry and fusion inhibitors.2 The introduction of highly active anti-
retroviral therapy (HAART)—a regimen combining 3–4 antiretrovi-
rals from different inhibitor classes—has significantly improved the
life quality of patients by delaying the progression of the disease
and reducing disabilities, transforming HIV/AIDS into a chronic
manageable disease. However, the lack of an effective and safe vac-
cine, the emergence and spread of drug-resistant viral variants3
and the inability of current regimens to eradicate the virus enforce
the continuous development of novel anti-HIV drugs.
As a part of our HIV program aimed at the discovery of novel
antiretrovirals via HTS using a cell-based assay, we successfully
identified a novel class of 3,4-dihydropyrimidin-2(1H)-one (DHPM)
HIV-1 replication inhibitors (Fig. 1).4 Initial structure–activity rela-
tionship (SAR) studies focused on altering the substitution patterns
of R groups attached to the DHPM core. These studies revealed that
only the S enantiomer retained anti-HIV activity and R2 with an
ethyl group is optimal. Compound 1 was developed as an early lead
with potent anti-HIV activity. These results have encouraged fur-
ther structural optimization studies to search for more potent anti-
viral agents with the DHPM scaffold. Although compound 1 has a
good antiviral activity (EC50 = 38 nM), it lacked metabolic stability
due to the ester moiety in the DHPM scaffold. Analogs (1, 2) with
ester group were metabolically unstable in liver microsomal stabil-
ity test of human and rat and corresponding free acids were inactive
against HIV-1. As the next step in this project, the main focus was
⇑
Corresponding author. Tel.: +82 31 8018 8160; fax: +82 31 8018 8015.
E-mail address: noxide@ip-korea.org (Z. No).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.133

J. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2522–2526
OH
2523
R3
N
HO
Cl
O
O
O
SAR
R5
O
OR1
HN
O
HN
O
N
R2
O
N
H
O
N
H
R4
Dihydropyrimidinones
(DHPMs)
2
EC50 = 19 nM
HLM t1/2 = 36 min
RLM t1/2 = 7 min
1
EC50 = 38 nM
HLM t1/2 = 11 min
RLM t1/2 = 1.5 min
R
R
R
O
N
O
Y
HN
X
X
HN
HN
O
O
N
H
O
N
H
O
N
H
R'
lactone analogs
bioisostere analogs
ketone analogs
Figure 1. Structural modifications of ester moiety in DHPMs to improve metabolic stability.
Table 2
Table 1
Metabolic stability of DHPMs
Cell-based antiviral activity of DHPM analogs against HIV-1
a
a
b
a
b
Compound
EC50
(lM)
Metabolic stability (t1/2, min)b
Compound EC50
M)
CC50
M)
Compound EC50
M)
CC50
M)
(
l
(
l
(
l
(
l
Human
Rat
1
2
6
0.038
0.019
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
16f
16g
16h
16i
0.983
0.480
0.087
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1
2
0.038
0.019
0.403
0.983
0.087
0.116
0.078
11
36
22
18
24
132
682
2
7
5
7
16
54
30
16b
16f
16h
26a
26b
12
>10
16a
16b
16c
16d
16e
0.420
0.403
0.329
>10
16j
1.40
26a
26b
26c
NVPc
0.116
0.078
>10
a
EC50 is the concentration of compound that inhibits HIV-1 replication by 50%.
3.28
0.150
For compounds 1–26b, n = 2 and the values are the geometric mean of two deter-
a
EC50 is the concentration of compound that inhibits HIV-1 replication by 50%.
minations; all individual values are within 25% of the mean.
b
For compounds 1–26c, n = 2 and the values are the geometric mean of two deter-
Liver microsomal stability.
minations; all individual values are within 25% of the mean.
b
CC50 is the cytotoxic concentration of compound that reduces viability of
uninfected cells by 50%.
To explore the SAR of DHPMs with ketone moiety, isosteric
ketone derivatives 15a–15j were prepared in a similar procedure
using Biginelli reaction with various 1,3-diketones 14a–14j, which
were synthesized by two-step protocol via aldol reaction10 and sub-
sequent oxidation.11 The racemic ketone analogs (15a–15j) were
separated into its enantiomer (16a–16j) via chiral separation.12
The next series of ester modifications to be explored was bioiso-
stere replacement. The concept of bioisosterism has been widely
utilized as one strategy for lead optimization in the drug discov-
ery.13 Three different bioisosteric replacements were designed
and synthesized as shown in Scheme 3. To access an oxazole surro-
c
Nevirapine (NVP) was used as a positive control.
shifted to identify metabolically stable equivalents of the ester
group that retained anti-HIV potency as well as continue to explore
SAR. Three different structural modifications were designed to re-
solve this key issue; (i) lactone analogs, (ii) ketone analogs and
(iii) bioisostere analogs (Fig. 1). We describe here our efforts for
the identification of novel DHPM HIV-1 replication inhibitors with
greatly improved metabolic stability.
The target compounds (Tables 1 and 2) were synthesized as out-
lined in Schemes 1–3. The first series of ester modification in
DHPMs focused on lactone analogs (6, 12). For the synthesis of lac-
tone analog 6, Biginelli adduct 5 was obtained in a one-pot conden-
sation reaction of an aldehyde 3, b-keto ester 4, and urea in the
presence of 10 mol % VCl3.5 Subsequent thermolysis of ester 5 at
high temperature gave the five-membered lactone 6 in good yield.6
To synthesize the six-membered lactone analog 12, the required
b-keto ester 9 was prepared in three-steps from 1,3-propanediol.
After the monoprotection of diol with TIPSCl, the resulting alcohol
7 was oxidized to aldehyde 8 and then subjected to SnCl2-pro-
moted Roskamp homologation7 to give b-keto ester 9. Condensa-
tion adduct 10 was synthesized via Yb(OTf)3 catalyzed Biginelli
reaction.8 After deprotection of TIPS group, the resulting alcohol
11 underwent tin-mediated lactonization to afford lactone 12.9
gate,
a-bromo ketone 17 which was prepared from cyclohexyl
methyl ketone by bromination underwent a cyclization with acet-
amide to give 2-methyl-1,3-oxazole 18.14 After the formation of
2-(lithiomethyl)oxazole via regioselective deprotonation with
lithium diethylamide, the resulting anion was reacted with ethyl
propionate to afford acylated product 20 in good yield.15 Similarly,
the synthesis of corresponding thiazole derivative 21 was achieved
using thioacetamide. In order to obtain the substituted imidazole
24, commercially available acetamidine hydrochloride salt was
neutralized with sodium hydroxide and then subjected to cycliza-
tion with
a-bromo ketone 17 to give 2-methyl-5-cyclohexyl
imidazole 22. After the extensive trials with various protecting
groups,16 we found trityl group is suitable for the following regio-
selective lithiation at 2-methyl position. Deprotonation/acylation

2524
J. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2522–2526
HO
HO
O
HO
O
O
(b)
OEt
(a)
+
+
urea
HN
HN
OEt
O
O
O
CHO
O
N
H
6
O
N
H
Cl
4
Cl
3
5
O
O
(c)
(f)
(d)
(e)
HO
OH
TIPSO
HO
OH
H
TIPSO
HO
TIPSO
OEt
7
9
8
HO
O
O
O
(g)
(h)
3
+ 9 + urea
HN
O
OEt
OTIPS
HN
O
OEt
OH
HN
O
N
H
N
H
N
H
12
O
10
11
Scheme 1. Synthesis of lactone analogs 6 and 12. Reagents and conditions: (a) 10 mol % VCl3, CH3CN, reflux, 8 h, 28%; (b) neat, 210 °C, 6 min, 91%; (c) NaH, THF, TIPSCl, 25 °C,
7 h, 92%; (d) (COCl)2, DMSO, Et3N, CH2Cl2, ꢀ78 to 25 °C, 30 min, 93%; (e) 10 mol % SnCl2, ethyl diazoacetate, CH2Cl2, 25 °C, 16 h, 86%; (f) 10 mol % Yb(OTf)3, CH3CN, 90 °C, 24 h,
56%; (g) TBAF, THF, 25 °C, overnight, 80%; (h) (n-Bu3Sn)2O, MeOH, 70 °C, 24 h, 95%.
O
(a)
O
O
O
OH
R1
(b)
R1
14a-14j
OH
OH
Cl
Cl
OH
O
Cl
O
O
R1
(c)
(d)
+
+ urea
HN
R1
HN
R1
O
CHO
O
N
H
O
N
H
13
14a-14j
15a-15j
16a-16j
a, R1
=
f, R1
=
1
g
, R
=
1
b,
c,
d,
R
=
h, R1
=
1
R
=
O
1
i
, R
=
1
R
=
=
1
1
e
j
, R
, R
=
Scheme 2. Synthesis of ketone analogs 16a–16j. Reagents and conditions: (a) LDA, ꢀ78 °C, THF, 1 h, then R1CHO, ꢀ78 °C, 1 h; (b) Dess–Martin periodinane, NaHCO3, CH2Cl2,
0 °C, 1 h; (c) 10 mol % Yb(OTf), THF, reflux, overnight, 44–63%; (d) Chiral HPLC separation.
was achieved successfully with 2 equiv of n-BuLi at low tempera-
ture, followed by trapping the resulting anion with ethyl propio-
nate to generate b-keto imidazole 24 in moderate yield. After all
the requisite b-keto heteroazoles were in hand, substituted oxazole
20, thiazole 21, and imidazole 24 were converted into Biginelli ad-
ducts 25a–25c by heating in acetic acid with aldehyde and urea.17
N-Trityl group in imidazole analog 25c was deprotected simulta-
neously during acidic media condensation. The racemic bioisostere
analogs (25a–25c) were also resolved into its enantiomer (26a–
26c) using chiral separation.
Synthesized target compounds (6, 12, 16a–16j, 26a–26c) were
evaluated for their inhibitory activity against HIV-1 replication in
CEM cells and Nevirapine was used as a positive control.18 The as-
say results of compounds are summarized in Table 1. It was clear
that the anti-HIV activity of the DHPMs is sensitive to structural
modifications. Both lactone analogs ( 6, 12) resulted in inactive
compounds, which indicated that a large lipophilic substituent in
the right hand side is essential for anti-HIV activity. We next inves-
tigated the effect of R1 moiety in the DHPM ketone analogs
(16a–16j). Ketone analog 16b revealed moderate antiviral activity

J. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2522–2526
2525
O
O
X
(a)
(b)
(c)
O
N
N
+
NH2
X = O, S
Br
N
X
X
18, X = O
19, X = S
20
, X = O
21, X = S
17
NH
HCl
(d)
(e)
(f)
N
O
N
N
+
17
NH2
N
Tr
23
N
H
Tr
22
24
R3
R3
R3
N
N
X
(g)
(h)
O
N
urea
+
+
X
HN
HN
X
CHO
O
N
H
O
N
20
21
, X = O
, X = S
H
26a-26c
25a-25c
24, X = NTr
a, X = O, R3 = 3-Cl, 4-OH
b, X = S, R3 = 3-Cl, 4-OH
3
c
, X = NH, R = 3-F, 4-OH
Scheme 3. Synthesis of bioisostere analogs 26a–26c. Reagents and conditions: (a) Br2, MeOH, H2O, 25 °C, overnight, 97%; (b) neat, 130 °C, 2.5 h, 18 26%, 19 89%; (c) LiNEt2,
THF, ethyl propionate, ꢀ78 to 25 °C, 2 h, 20 81%, 21 53%; (d) NaOH, MeOH, 0 °C, 3 h; then 17, K2CO3, CH3CN/H2O, 25 °C, overnight, 52%; (e) TritylCl, Et3N, DMF, 25 °C,
overnight, 100%; (f) n-BuLi (2.0 equiv), THF, ꢀ78 to 25 °C, 1 h; then ethyl propionate, ꢀ78 to 25 °C, overnight, 48%; (g) AcOH, 100 °C, 16 h, 25a 28%, 25b 49%, 25c 20%; (h)
Chiral HPLC separation.
compared with corresponding ester analog 2. Variations in the ring
size of R1 displayed moderately improved antiviral activities
depending on the ring sizes from five-membered 16a (EC50
=
420 nM), six-membered 16b (EC50 = 403 nM), and seven-mem-
bered 16c (EC50 = 329 nM), respectively. However, the phenyl
analog 16d completely lost its antiviral activity. This result exem-
plified that a hydrophobic alicyclic R1 group is a critical moiety for
maintaining anti-HIV activity. Reducing the linker size from ethyl-
ene to methylene markedly increased the sensitivity of molecules
to steric nature of substituents. Six-membered analog 16f was
three-time more active than five-membered analog 16e. Variations
in steric bulkiness at the 4-position of the cyclohexyl ring exhibited
clear SAR. Attachment of a methyl group showed twofold increased
potency in analog 16g. Increasing substituents from methyl (16g)
to ethyl group (16h) led to fivefold increase in potency. Further in-
crease in size from ethyl to iso-propyl group (16j) and replacing an
ethyl group with a methoxy group (16i) resulted in the loss of
activity. These results clearly showed the subtleness at this posi-
tion. Based on the SAR, the optimal substituent in the ketone ana-
logs is a methylene linker and a cyclohexyl ring with an ethyl
group at the 4-position (16h, EC50 = 87 nM).
After examining lactone and ketone derivatives, we evaluated
the effect of replacing ester with bioisosteres for compounds
26a–26c. The ester group was replaced with bioisosteric fragments
such as oxazole and thiazole, and those compounds were mini-
mized using CHARMM force field19 implemented in Discovery Stu-
dio (ver. 3.1).20 Minimized conformation of compounds 2 (Fig. 2a),
26a (data not shown) and 26b (Fig. 2b) were well aligned to each
other when aligned according to steric and electrostatic fields.
The carboxylic group and thiazole overlapped well with each other
retaining similar physicochemical properties (Fig. 2c). In respect to
minimized energy, compound 26b (13.21 kcal/mol) was more sta-
ble than compound 2 (14.74 kcal/mol). Indeed, oxazole and thia-
zole analogs (26a, 26b) exhibited similar activities with EC50 of
116 and 78 nM, respectively. On the other hand, imidazole analog
Figure 2. Lowest energy conformers of 2 (a) and 26b (b) are well superimposed (c).
All compounds are shown in atom-colored stick form with different colored
carbons: yellow for compound 2, and pink for compound 26b. Hydrogens were
removed for clarity purpose.
26c did not show any antiviral activity, which is consistent with
the results from previously reported amide analogs.4

2526
J. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2522–2526
G. A.; La Voie, E. J. Chem. Rev. 1996, 96, 3147; (e) Chen, X.; Wang, W. Annu. Rep.
Compounds which exhibited significant potency against HIV-1
Med. Chem. 2003, 38, 333; (f) Meanwell, N. A. J. Med. Chem. 2011, 54, 2529.
14. (a) Epple, R.; Cow, C.; Xie, Y. P.; Azimioara, M.; Russo, R.; Wang, X.; Wityak, J.;
Karanewsky, D. S.; Tuntland, T.; Nguyen-Tran, V. T. B.; Ngo, C. C.; Huang, D.;
Saez, E.; Spalding, T.; Gerken, A.; Iskandar, M.; Seidel, H. M.; Tian, S. S. J. Med.
Chem. 2010, 53, 77; (b) Hammar, W. J.; Rustad, M. A. J. Heterocycl. Chem. 1981,
18, 885.
15. Evans, D. A.; Cee, V. J.; Smith, T. E.; Santiago, K. J. Org. Lett. 1999, 1, 87.
16. Other protecting groups tried in this reaction were THP, 2,4-dimethoxybenzyl,
4-methoxybenzyl, MOM, Boc groups.
replication in a cell-based assay were further evaluated for their
metabolic stability in vitro.21 Test results were summarized in
Table 2. Ketone analogs (16b, 16f, 16h) displayed poor metabolic
stability similar to the corresponding ester analog 2. Gratifyingly,
however, oxazole analog 26a and thiazole analog 26b exhibited
markedly improved metabolic stability in vitro while retaining
significant antiviral activity against HIV-1 replication.
17. General procedure for Biginelli reaction with b-keto heteroazoles: To a mixture of
aryl aldehyde (1.0 equiv), urea (1.5 equiv), and b-keto oxazole (thiazole, or
imidazole) (1.0 equiv) was added acetic acid (0.2 M) and stirred at 110 °C for
16 h. The reaction was slowly quenched with saturated aqueous K2CO3
(caution: gas evolution!) and extracted with EtOAc (3ꢁ). The combined
organic layers were dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was redissolved with CH2Cl2 and crystallized by the slow addition
of hexanes. The resulting precipitate was filtered and washed with cold CH2Cl2/
n-hexanes (1:1) solution. Alternatively, the concentrated residue was directly
subjected to flash column chromatography (SiO2, CH2Cl2/MeOH = 19:1) to give
Biginelli adducts. Compound 26a 1H NMR (400 MHz, CD3OD) d 7.34 (s, 1H),
7.25 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.4, 2.0 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 5.44
(s, 1H), 2.70 (m, 2H), 2.40 (m, 1H), 1.92 (m, 2H), 1.72 (m, 2H), 1.65 (m, 1H),
1.35–1.24 (m, 5H), 1.94 (t, J = 7.4 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 159.6,
152.4, 146.4, 143.3, 136.1, 131.5, 128.0, 125.8, 120.1, 116.2, 98.0, 54.6, 35.6,
31.9, 31.4, 25.8, 25.7, 23.8, 20.2, 11.7; Enantiomerically pure forms were
obtained by chiral HPLC (OD-H preparative column, 20% i-PrOH in n-hexanes,
In summary, novel dihydropyrimidinone analogs (6, 12, 16a–16j,
26a–26c) were designed and synthesized to optimize metabolic sta-
bility. In particular, replacement of the ester moiety by bioisosteres
dramatically improved the metabolic liability while retaining anti-
viral activity against HIV-1. These novel and potent DHPMs contain-
ing ester bioisosteres could serve as advanced lead compounds for
further development. Mode of action and further optimization of
this lead compound will be reported in due course.
Acknowledgments
This work was supported by the National Research foundation
of Korea (NRF) grant funded by the Korea government (MEST)
(No. 2011-00244), Gyeonggi-do and KISTI.
6.5 mL/min, tR = 20.5 min): 26b mp 186 °C; ½a D26
ꢂ
+8.1 (c 0.001, MeOH); 1H NMR
(400 MHz, CD3OD) d 7.22 (d, J = 2.0 Hz, 1H), 7.06 (dd, J = 8.2, 2.2 Hz, 1H), 6.86 (s,
1H), 6.79 (d, J = 8.4 Hz, 1H), 5.39 (s, 1H), 2.63 (m, 3H), 1.96 (m, 2H), 1.79 (m,
2H), 1.71 (d, J = 12.4 Hz, 1H), 1.46–1.25 (m, 5H), 1.22 (t, J = 7.6 Hz, 3H); 13C
NMR (100 MHz, CD3OD) d 164.7, 163.2, 155.7, 153.9, 142.1, 137.1, 129.8, 127.5,
122.5, 117.6, 111.5, 105.4, 58.6, 41.7, 34.1, 33.7, 27.4, 25.0, 12.7; TLC Rf (CH2Cl2/
MeOH 19:1) = 0.42; HRMS (EI) m/z calcd for C21H24ClN3O2S (M)+ 417.1277,
found 417.1282; Enantiomerically pure forms were obtained by chiral HPLC
(OD-H preparative column, 20% i-PrOH in n-hexanes, 6.5 mL/min,
tR = 18.1 min).
References and notes
1. Xu, H.; Lv, M. Curr. Pharm. Des. 2009, 15, 2120.
2. Mehellou, Y.; De Clercq, E. J. Med. Chem. 2010, 53, 521. and references cited
therein.
3. (a) Brenner, B.; Wainberg, M. A.; Salomon, H.; Rouleau, D.; Dascal, A.; Spira, B.;
Sekaly, R. P.; Conway, B.; Routy, J. P. Int. J. Antimicrob. Agents 2000, 16, 429; (b)
Si-Mohamed, A.; Kazatchkine, M.; Heard, I.; Goujon, C.; Prazuck, T.; Aymard, G.;
Cessot, G.; Kuo, Y. H.; Bernard, M. C.; Diquet, B.; Malkin, J. E.; Gutmann, L.;
Belec, L. J. Infect. Dis. 2000, 182, 112; (c) Kiertiburanakul, S.; Sungkanuparph, S.
Curr. HIV Res. 2009, 7, 273; (d) Wensing, A. M. J.; van de Vijver, D. A.; Angarano,
G. J. Infect. Dis. 2005, 192, 1501.
4. Kim, J.; Park, C.; Ok, T.; So, W.; Jo, M.; Seo, M.; Kim, Y.; Sohn, J.-H.; Park, Y.; Ju,
M. K.; Kim, J.; Han, S.-J.; Kim, T.-H.; Cechetto, J.; Nam, J.; Sommer, P.; No, Z.
Bioorg. Med. Chem. Lett. 2012, 22, 2119.
5. Sabitha, G.; Reddy, G. S. K. K.; Reddy, K. B.; Yadav, J. S. Tetrahedron Lett. 2003, 44,
6497.
6. Kappe, C. O.; Perez, R.; Beryozkina, T.; Zbruyev, O. I.; Haas, W. J. Comb. Chem.
2002, 4, 501.
7. (a) Holmquist, C. R.; Roskamp, E. J. J. Org. Chem. 1989, 54, 3258; (b) Kopecky, D.
J.; Rychnovsky, S. D. J. Am. Chem. Soc. 2001, 123, 8420.
8. General procedure for Biginelli reaction with b-keto esters: The b-keto ester 9
(200 mg, 0.632 mmol), 3-hydroxybenzaldehyde (77 mg, 0.632 mmol), urea
(57 mg, 0.948 mmol), and Yb(OTf)3 (39 mg, 0.063 mmol) were dissolved in
acetonitrile (1.6 mL) and stirred under Argon for 24 h at 90 °C. After cooling to
room temperature, the reaction was quenched by the addition of saturated
aqueous NaHCO3 (10 mL) and extracted with CH2Cl2 (4 ꢁ 20 mL). The
combined organic layers were dried over Na2SO4. After filtration and
concentration in vacuo, the residue was purified by flash column
chromatography (SiO2, n-hexanes/EtOAc = 5:1?1:1 then CH2Cl2/MeOH =
20:1) to give Biginelli adduct 10 (164 mg, 56%) as a yellow solid: 1H NMR
(400 MHz, CD3OD) d 7.88 (s, 1H), 7.01 (t, J = 7.6 Hz, 1H), 6.76–6.73 (m, 2H), 6.64
(d, J = 8.0 Hz, 1H), 6.39 (s, 1H), 5.22 (s, 1H), 4.04–3.94 (m, 4H), 3.07–2.97 (m,
2H), 1.12–1.02 (m, 24H); 13C NMR (100 MHz, CD3OD) d 165.7, 156.5, 153.1,
148.8, 144.9, 129.7, 117.8, 114.9, 113.7, 101.5, 62.0, 60.1, 55.1, 33.6, 17.9, 14.0;
TLC Rf (CH2Cl2:MeOH 10:1) = 0.42.
18. HIV full replication assay. CEMx174-LTR-GFP cells (clone CG8) were seeded
with a microplate dispenser (WellMate; Thermo Scientific Matrix; USA) at a
density of 4000 cells/well into 384-well glass plates (Evotec. Hamburg,
Germany) pre-dispensed with 10 lL of compound diluted in DMSO and
incubated for 1 h at 37 °C, 5% CO2. Then cells were infected with HIV-1LAI at a
multiplicity of infection (MOI) of 3 and incubated for 5 days at 37 °C, 5% CO2.
Fluorescence intensities were the determined using a multilabel plate reader
(Victor3; PerkinElmer, Inc.; USA). And see: Sommer, P.; Vartanian, J. P.;
Wachsmuth, M.; Henry, M.; Guetard, D.; Wain-Hobson, S. J. Mol. Biol. 2004, 344,
11.
19. Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.;
Karplus, M. J. Comput. Chem. 1983, 4, 187–217.
20. Discovery Studio, version 3.1; Accelrys Inc.: San Diego, CA.
21. (a) Materials: Human, Rat Microsomes and NADPH regenerating system were
purchased from BD Gentest (Woburn, MA). Liquid chromatographic analysis
was performed on the instrument of Agilent 1200 series equipped with a
diode-array detector and autosampler (Agilent Technology, Piscataway, NJ). An
analytical column was applied after the trapping cartridges (Phenomenex,
Gemini C18 50 mm ꢁ 2.0 mm, 3
lm, Torrance, CA). A quadrupole LC mass
spectrometer (Agilent Technology, Piscataway, NJ), with electrospray
ionization (ESI) was employed for sample analysis. Instruments were
controlled by ChemStation software (Version 2.0, Agilent Technology,
Piscataway, NJ).; (b) Methods: Compounds (2 lM final concentration) are
incubated with liver microsomes (rat and human) in potassium phosphate
buffer. The microsomal protein concentration in the assay is 0.5 mg/mL and the
final percent DMSO is 0.2%. Reaction is started by the addition of NADPH and
stopped either immediately or at 5, 10, 30, 60 and 120 min for a precise
estimate of clearance. The corresponding loss of parent compound is
determined by LC/MS. The mobile phases were (A) water with 0.1% of formic
acid and (B) acetonitrile with 0.1% of formic acid at a flow rate of 0.3 mL/min.
The LC conditions were 5% B at 0 min, a linear gradient from 5% to 50% B over
1 min, held at 50% for 0.5 min, then ramped from 50% to 95% over 0.5 min,
followed by 95% B for 1.5 min and back to 5% B over 0.5 min, then held at 5% B
for the remaining 3.5 min.; (c) Data analysis: % remaining of compound is
calculated compared to the initial quantity at time zero. Half-life is then
calculated based on first-order reaction kinetics.
9. Steliou, K.; Poupart, M. A. J. Am. Chem. Soc. 1983, 105, 7130.
10. Menche, D.; Arikan, F.; Li, J.; Rudolph, S. Org. Lett. 2007, 9, 267.
11. Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277.
12. Enantiomerically pure forms were obtained by chiral HPLC (Daicel Chiralcel AD
column).
13. (a) Burger, A. Prog. Drug Res. 1991, 37, 287; (b) Thornber, C. W. Chem. Soc. Rev.
1979, 8, 563; (c) Lipinski, C. A. Annu. Rep. Med. Chem. 1986, 21, 283; (d) Patani,