Discovery of 3,4-dihydropyrimidin-2(1H)-ones with inhibitory activity against HIV-1 replication

Article abstract of DOI:10.1016/j.bmcl.2011.12.090

3,4-Dihydropyrimidin-2(1H)-ones (DHPMs) were selected and derivatized through a HIV-1 replication assay based on GFP reporter cells. Compounds 14, 25, 31, and 36 exhibited significant inhibition of HIV-1 replication with a good safety profile. Chiral separation of each enantiomer by fractional crystallization showed that only the S enantiomer retained anti-HIV activity. Compound (S)-40, a novel and potent DHPM analog, could serve as an advanced lead for further development and the determination of the mechanism of action.

Full text of DOI:10.1016/j.bmcl.2011.12.090

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2119–2124
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 3,4-dihydropyrimidin-2(1H)-ones with inhibitory activity against
HIV-1 replication
Junwon Kim a, Changmin Park a, Taedong Ok a, Wonyoung So a, Mina Jo a, Minjung Seo a, Youngmi Kim a,
Jeong-Hun Sohn a, Youngsam Park b, Moon Kyeong Ju b, Junghwan Kim b, Sung-Jun Han b, Tae-Hee Kim c,
Jonathan Cechetto c, Jiyoun Nam c, Peter Sommer d, 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 Cell Biology of Retroviruses Group, Institut Pasteur Korea (IP-K), Sampyeong-dong 696, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
3,4-Dihydropyrimidin-2(1H)-ones (DHPMs) were selected and derivatized through a HIV-1 replication
assay based on GFP reporter cells. Compounds 14, 25, 31, and 36 exhibited significant inhibition of
HIV-1 replication with a good safety profile. Chiral separation of each enantiomer by fractional crystalli-
zation showed that only the S enantiomer retained anti-HIV activity. Compound (S)-40, a novel and
potent DHPM analog, could serve as an advanced lead for further development and the determination
of the mechanism of action.
Received 25 August 2011
Revised 5 December 2011
Accepted 20 December 2011
Available online 12 January 2012
Keywords:
HIV
Ó 2012 Elsevier Ltd. All rights reserved.
3,4-Dihydropyrimidin-2(1H)-one
Biginelli reaction
Fractional crystallization
Enantiomers
In 2009 more than 33 million people were infected with the
human immunodeficiency virus (HIV), the causative agent of the
acquired immunodeficiency syndrome (AIDS), which currently ac-
counts for the highest number of deaths by any single infectious
agent.1 There are currently 25 drugs belonging to 6 different inhib-
itor classes approved for the treatment of HIV infection, including
nucleoside or non-nucleosides reverse transcriptase inhibitors
(NRTIs/NNRTIs), protease inhibitors (PIs), integrase inhibitors, en-
try and fusion inhibitors.2 The introduction of highly active antiret-
roviral therapy (HAART)—a regimen combining 3–4 antiretrovirals
from different inhibitor classes—has considerably improved the life
quality of infected people by delaying the progression of the dis-
ease and reducing disabilities, making HIV/AIDS a chronic disease,
not a death sentence. However, there are serious drawbacks of
HAART due to the tendency of HIV-1 to rapidly mutate. Prolonged
HAART treatment leads to the emergence of drug-resistant strains
of the virus.3 Also, the side effects of combination therapy have
limited their clinical effectiveness.4 Therefore, the continued
development of novel anti-HIV drugs with acceptable toxicity
and resistance profiles is clearly needed.
In a high-throughput screening campaign for the discovery of
novel antiretrovirals employing a HIV full replication assay based
on reporter cells harboring an EGFP expression cassette under the
control of the HIV promoter, we identified a series of compounds
containing a dihydropyrimidinone scaffold (Fig 1) that exhibited
inhibitory activities against HIV replication at low micromolar con-
centrations. Dihydropyrimidinone derivatives have been reported
to exhibit diverse biological activities, such as, anti-bacterial,5
anti-fungal,6 anti-cancer,7 and anti-oxidant activities,8 whereas an
anti-HIV activity has not been previously documented.9 Here, we
report the preliminary SAR of dihydropyrimidinones as novel
inhibitors of HIV-1 replication and a scale-up procedure for the
preparation of enantiomerically pure forms using a fractional crys-
tallization method.
R3
O
Dihydropyrimidinones
(DHPMs)
R5
O
N
OR1
N
R2
R4
⇑
Corresponding author. Tel.: +82 31 8018 8160; fax: +82 31 8018 8015.
E-mail address: noxide@ip-korea.org (Z. No).
Figure 1. Hit compounds from cell-based HIV-1 replication assay.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.090

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J. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2119–2124
O
O
O
O
O
O
(a) or (b)
R2
OR1
R2
O
1
R3
N
R3
O
urea
or
R5
Z
(c)
+
+
OR1
4-37
R2
OR1
thiourea
CHO
N
R2
R4
1
2
3
HO
HO
HO
O
O
O
(e)
(d)
HN
OEt
HN
OH
HN
N
H
O
N
H
O
N
H
O
N
H
5
4
9
Scheme 1. Synthesis of dihydropyrimidinones 4À37. Reagents and conditions: (a) cat. I2, R1OH, toluene, reflux 12 h; (b) NaOH, H2O, 25 °C, 12 h; then EDC, DMAP, R1OH,
CH2Cl2, 25 °C, overnight; (c) cat. Yb(OTf)3, THF, reflux, overnight; (d) NaOH, MeOH/H2O, 25 °C, 48 h; (e) EDC, HOBt, cyclohexylmethylamine, DMF, 25 °C, 4 h.
A simple and direct method for the synthesis of dihydropyri-
midinones (DHPMs), first reported by Biginelli in 1893,10 involves
a reaction in which three reactants come together in a one-pot to
form a new product that contains all the components. The classical
Biginelli reaction is a one-pot condensation reaction of an aryl
aldehyde, b-keto ester, and urea or thiourea with catalytic acid in
a protic solvent, which often suffer from low yields especially in
the case of substituted aromatic and aliphatic aldehydes.11 Since
then numerous improved synthetic methods have been reported.12
To explore the SAR of DHPMs, we prepared a series of dihydropyr-
imidone derivatives using Yb(OTf)3 as a catalyst under refluxing
condition in THF (Scheme 1).13 Various b-keto esters 1 were
HO
HO
O
O
O
O
(a)
O
+
+
N
H
NH2
HN
O
CHO
O
N
3a
1a
10
O
O
O
O
O
(c)
(b)
DMB
NH2
N
H
NH2
+
H2N
NH2
HN
O
• HCl
O
3b
O
N
DMB
11a
HO
O
O
O
O
(d)
(e)
N
O
N
O
N
H
O
N
DMB
11b
11
HO
HO
O
O
(f)
HN
O
HN
O
O
N
O
N
H
8
12
Scheme 2. Synthesis of dihydropyrimidinones 10À12. Reagents and conditions: (a) cat. BF3ÁOEt2, CuCl, AcOH, THF, reflux, 12 h; (b) HCl, H2O, reflux, overnight; (c) 1a, 3-
methoxybenzaldehyde, cat. BF3ÁOEt2, CuCl, AcOH, THF, 65 °C, 12 h; (d), NaHMDS (1.0 M in THF), THF, À78 °C, then (CH3O)2SO2, À78 °C to 25 °C, 2 h; (e) BBr3 (1.0 M in CH2Cl2),
CH2Cl2, À78 °C, 1 h to 25 °C, 10 min; (f) CAN, NaHCO3, acetone/H2O, 0–25 °C, overnight.

J. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2119–2124
2121
R6
NH
O2N
HN
H2N
O
O
O
(a)
(b) or (c)
29
HN
HN
O
O
O
from
O
N
H
O
N
H
O
N
H
26
28
27
29
, 3-NO2
, 4-NO2
, 3-NH2
, 4-NH2
30,
31
R
6 = Me
, R6 = Ac
CO2Bn
O
CO2H
O
(d)
HN
O
HN
O
N
H
O
N
H
O
35a
35
Scheme 3. Synthesis of dihydropyrimidinones 26À35. Reagents and conditions: (a) 10% Pd/C, H2 (1 atm), MeOH, 25 °C, 2 h; (b) MeI, DIPEA, CH2Cl2/DMF (1:1), 25 °C,
overnight; (c) EDCÁHCl, DMAP, Ac2O, THF, 25 °C, 12 h (d) 10% Pd/C, H2 (1 atm), MeOH, 25 °C, 2 h.
synthesized by the methods known in the literatures.14 Hydrolysis
of the ester 5 in sodium hydroxide gave the corresponding carbox-
ylic acid 4, which was converted into amide analog 9 using EDC
coupling reaction. In order to synthesize N-methyl substituted
analogs (10, 11), substituted ureas (3a, 3b) were used in BF3ÁOEt2
catalyzed Biginelli reaction (Scheme 2). For compound 11,
Table 1
Cell-based antiviral activity of 4À37 against HIV-1
R1
R2
R3
R4
R5
EC50
(lM)
CC50 (lM)
a
b
compound
Z
4
5
6
7
8
o
o
o
0
o
o
o
o
0
s
o
o
0
o
o
o
o
s
o
o
o
0
o
o
o
0
o
o
o
o
0
o
o
o
H
Et
Bn
c-Hex
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
2-OH
3-OMe
4-OH
4-OH
3-N02
3-NH2
4-NO2
4-NH2
4-NHMe
4-NHAc
4-NMe2
4-CN
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>10
>10
>10
>10
0.529
>10
>10
>10
>10
>10
0.087
0.286
>10
>10
0.359
2.20
>10
>10
>10
1.27
0.431
0.031
0.335
0.159
0.141
0.151
0.108
0.063
0.177
0.061
0.122
>10
>10
NDd
>10
>10
>10
ND
>10
>10
>10
>10
>10
>10
ND
CH2-c-Hex
NHCH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
CH2-c-Hex
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
NVPc
Pr
i-Pr
Cyclopropyl
–CH2Cl
–C„CH
Ph
>10
>3
>1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
Et
Me
Me
Me
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
4-C02Me
4-C02H
4-F
0.024
0.088
0.150
4-CI
a
EC50 is the concentration of compound that inhibits HIV-1 replication by 50%. For compound 4–37, n = 2 and the values are the geometric mean of two determinations; 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%.
Nevirapine (NVP) was used as a positive control.
Not determined.
c
d

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J. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2119–2124
cellular activities. Also the distance of alicyclic group R1 from the
carbonyl group of carboxyl ester is a critical factor and analog 7
without methylene group showed reduced potency. Replacement
of ester moiety with amide 9 did not show any inhibitory activity.
Methyl substitutions on nitrogen (10, 11) resulted in complete loss
of activity. Oxygen atom at position 2 in dihydropyrimidinones was
a mandatory requirement for activity as was shown in compounds
(8, 14) compared with sulfur analogs (13, 21), respectively. The spa-
tial arrangement of phenyl group at C-4 of DHPMs plays a pivotal
Table 2
Cell-based antiviral activity of separated enantiomers against HIV-1
a
b
Compound
R2
Stereochemistry
EC50
(lM)
CC50 (lM)
8
Me
Racemic
(S)-
(R)-
Racemic
(S)-
(R)-
0.529
0.233
>10
0.087
0.038
>10
>10
>10
>10
>10
>10
>10
(S)-38
(R)-39
14
(S)-40
(R)-41
Et
a
EC50 is the concentration of compound that inhibits HIV-1 replication by 50%.
role and an oxidized, achiral analog 12 was inactive up to 10 lM.
For compound, n = 2 and the values are the geometric mean of two determinations;
We next investigated the effect of R2 moiety in DHPM analogs
14À20 and their results displayed clear SAR. Increasing the size of
R2 from methyl, ethyl, and propyl showed cellular activities from
529, 87, and 286 nM, respectively. Other analogs similar to two car-
bon unit (iso-Pr 16, cyclopropyl 17, chloromethyl 18 and ethynyl
19) and phenyl analog 20 exhibited reduced or complete loss of
activities. From these results, it is clear that the R2 position is sen-
sitive to steric and electronic nature of substituents. The optimal
substitution at this position is an ethyl group (14, EC50 = 87 nM).
The same effect was also shown again by 13-fold activity difference
between 24 and 25. After examining the R1 and R2 regions, we eval-
uated the substituent effect on phenyl ring with compounds 22À37.
Both para-OH analog 8 and meta-OH analog 24 showed comparable
activities of 529 and 431 nM, respectively. However, ortho-OH ana-
log 22 suffered the loss of antiviral activity. Methoxy analog 23 was
twofold less active than hydroxyl analog 8. Generally, amino ana-
logs (27, 29) displayed slightly reduced potency compared to corre-
sponding hydroxyl analogs (14, 25). Mono-substituted amine
analogs (NH-Me 30, NH-acetyl 31) exhibited slightly improved
activity than corresponding di-substituted amine analog 32. Ana-
logs with electron-withdrawing group at para-position on phenyl
ring (4-NO2 28, 4-CN 33, 4-CO2Me 34, 4-F 36, 4-Cl 37) maintained
significant inhibitory activities except carboxylic acid analog 35,
which was presumably due to its low cell membrane permeability
in our cell-based assay system.
all individual values are within 25% of the mean.
CC50 is the cytotoxic concentration of compound that reduces viability of
uninfected cells by 50%.
b
2,4-dimethoxybenzyl protected urea was prepared in acidic condi-
tion and subjected into following cyclization reaction. Treatment of
Biginelli adduct 11a with NaHMDS and trapping with dimethyl
sulfate afforded N-methylated compound 11b. Subsequent depro-
tection of methoxy and DMB group with BBr3 gave the desired ana-
log 11. To evaluate the effect of chiral center in DHPM analogs,
compound 12 was synthesized via CAN-mediated oxidation from
compound 8. For the synthesis of amine analogs (27–31), nitro
group was reduced to the corresponding amine, which was sub-
jected to alkylation and acylation to give compound 30 and 31,
respectively. Carboxylic acid analog 35 was synthesized from ester
analog 35a by reductive removal of the benzyl group in the pres-
ence of 10% Pd/C under H2 (Scheme 3).
Synthesized compounds 4À37 were evaluated for their inhibi-
tory activity against HIV-1 replication in CEM cells and Nevirapine
was used as a positive control.15 Assay results of compound 4À37
are summarized in Table 1. It was apparent that the anti-HIV activ-
ities of the DHPMs are sensitive to structural perturbations. Com-
pounds 4À8 have the common DHPM core but with varying R1
groups. Results showed that a hydrophobic alicyclic R1 is preferred
and 8 exhibited EC50 of 0.529
ester (6) or polar carboxylic acid (4) itself resulted in the loss of
lM. Typical alkyl ester (5), benzyl
The resolution of 8 and 14 into their enantiomers was per-
formed to evaluate the impact of stereochemistry at C-4.16 The
HO
HO
O
O
O
O
(a)
(b)
+
+
O
HN
OBn
H2N
NH2
OH
*
CHO
O
N
H
42
TBSO
TBSO
TBSO
O
O
(c)
O
O
(d)
*
HN
HN
OBn
chiral baseH
HN
O
N
O
N
H
43
O
N
H
H
44
45
HO
TBSO
TBSO
O
*
O
O
(g)
(e)
(f)
*
HN
O
HN
OH
HN
O
O
N
O
N
O
N
H
47
H
H
-40
(S)
-41
(R)
46
Scheme 4. Synthetic route of enantiomer (S)-40 using fractional crystallization method. Reagents and conditions: (a) cat. Yb(OTf)3, THF, reflux, 24 h, 77%; (b) TBSCl,
imidazole, DMF, 25 °C, overnight, 100%; (c) Pd/C, H2 (3 bars), MeOH/Et3N, 25 °C, 3 h; then 1 N HCl, H2O, 91%; (d) fractional crystallization with chiral amines; (e) 1 N HCl, H2O;
(f) EDC, DMAP, cyclohexylmethanol, 50 °C, overnight; (g) TBAF, CH2Cl2, 0 °C, 30 min, 86% over 2 steps.

J. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2119–2124
2123
TBSO
cinchonidine (1.0 eq)
cinchonine (1.0 eq)
O
MeOH (0.14 M)
76 °C to -20 °C
overnight
MeOH (0.1 M)
76 °C to -20 °C
overnight
HN
OH
76%
87%
N
H
44
O
TBSO
TBSO
O
O
H
N
OH
H
H
H
(S)
(R)
HN
O
O
N
H
HN
O
OH
H
N
N
H
O
N
H
N
86% over 2 steps
HO
HO
O
O
O
(S)
HN
O
(R)
HN
O
N
H
O
N
H
(S)-40
98% ee
(R)-41
94% ee
Scheme 5. Conditions for fractional crystallization of a racemic mixture (44).
absolute stereochemistry of separated enantiomers was confirmed
by circular dichroism (CD) spectroscopy.17 Interestingly, test re-
sults against HIV-1 revealed that only (S)-enantiomers ( 38, 40)
exhibited antiviral activity, whereas (R)-enantiomers (39, 41) were
Dr. Michel Liuzzi for his helpful comments in preparing this
manuscript.
References and notes
completely inactive up to 10 lM (Table 2).
1. Xu, H.; Lv, M. Curr. Pharm. Des. 2009, 15, 2120.
Encouraged by these results, we developed a new synthetic
route to access enantiomers for further evaluation and optimiza-
tion. The most potent enantiomer (S)-40 could be scaled up using
a fractional crystallization method as outlined in Scheme 4. Com-
pound 42 is readily accessible via Biginelli reaction and the result-
ing hydroxyl group was protected with TBSCl. Subsequent
conversion of 43 to carboxylic acid 44 was achieved by reductive
removal of the benzyl group in the presence of 10% Pd/C under
H2 (3 bars). Racemic carboxylic acid 44 was then co-crystallized
with various chiral amines. Enantiomeric excess was determined
after the conversion of resolved carboxylic acid into esters via
EDC coupling and subsequent deprotection of TBS group. After
examining the various chiral amines, we found that cinchonine/
cinchonidine series could separate each enantiomer selectively
with 98% ee for (S)-40 and 94% ee for (R)-41 (Scheme 5).18 This
combination could be applied to other dihydropyrimidinone
compounds.
In summary, a series of dihydropyrimidinone analogs (4–37)
was synthesized and evaluated as HIV-1 replication inhibitors
in vitro. Among the derivatives, compounds 14, 25, 31, and 36
exhibited significant inhibitory activity against HIV-1 in a cell-
based assay. Chiral separation of the enantiomers showed that
the S configuration on the C-4 in dihydropyrimidinone ring is a
crucial factor for antiviral activity. Each enantiomer could be scaled
up and separated selectively via fractional crystallization. Mode of
action and further optimization of this lead compound will be
reported in due course.
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40, 3465; (b) Lu, J.; Bai, Y. J.; Wang, Z. J.; Yang, B. Q.; Ma, H. R. Tetrahedron Lett.
2000, 41, 9075; (c) Hu, E. H.; Sidler, D. R.; Dolling, U. H. J. Org. Chem. 1998, 63,
3454; (d) Kappe, C. O.; Falsone, S. F. Synlett 1998, 718; (e) Kappe, C. O.; Kumar,
D.; Varma, R. S. Synthesis 1999, 1799; (f) Ranu, B. C.; Hajra, A.; Jana, U. J. Org.
Chem. 2000, 65, 6270.
13.
A mixture of Yb(OTf)3 (0.05 mmol, 0.1 equiv), b-ketoester (0.5 mmol,
1.0 equiv), aryl aldehyde (0.5 mmol, 1.0 equiv) and urea (0.5 mmol, 1.0 equiv)
in THF (2 mL) was refluxed 20–30 h under Ar. After adding H2O (5 mL), the
mixture was extracted with EtOAc (3 Â 5 mL). The combined organic layers
were dried over MgSO4, filtered and concentrated in vacuo. The crude product
was purified by flash column chromatography (SiO2, n-Hexanes/EtOAc/AcOH,
50:50:0.5) to afford the desired dihydropyrimidinone compound.
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. We would like to thank
14. (a) Mori, K.; Kisida, H. Tetrahedron 1986, 42, 5281; (b) Chavan, S. P.; Kale, R. R.;
Shivasankar, K.; Chandake, S. I.; Benjamin, S. B. Synthesis 2003, 2695; (c)
Szemes, F.; Marchalin, T.; Pronayova, N.; Daich, A. Heterocycles 2003, 59, 779.

2124
J. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2119–2124
15. HIV full replication assay. CEMx174-LTR-GFP cells (clone CG8) were seeded
with a microplate dispenser (WellMate; Thermo Scientific Matrix; U.S.A.) at a
density of 4000 cells/well into 384-well glass plates (Evotec. Hamburg,
acid 44 (1.00 g, 2.655 mmol) in MeOH (20 mL) was added cinchonine (782 mg,
2.655 mmol) at 76 °C. The resulting suspension was treated with the slow
addition of MeOH (10 mL) at 76 °C. The resulting clear solution was slowly
cooled to room temperature followed by overnight storage at À20 °C. The next
day, the salt was filtered and rinsed with EtOH to give an acid/cinchonine salt
(614 mg, 69%) as a white solid. The filtrate was concentrated in vacuo and the
residue was re-subjected to fractional crystallization in MeOH (10 mL) to give
an additional acid/cinchonine salt (125 mg, 14%). The salt above (80 mg) was
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.; U.S.A.). And see Sommer, P.; Vartanian, J. P.;
Wachsmuth, M.; Henry, M.; Guetard, D.; Wain-Hobson, S. J. Mol. Biol. 2004, 344,
11.
resuspended in water (4 mL) and acidified with 1 N HCl (500 lL, pH <2). The
resulting suspension was sonicated for 10 min and then centrifuged to remove
the upper layer. After repeating the same procedure one more time, the
resulting solid was washed with H2O (3 Â 4 mL) and freeze dried to give a salt-
free acid 46 (43 mg, 97%) as a white solid: 1H NMR (400 MHz, DMSO-d6) d 8.90
(s, 1H), 7.47 (s, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.70 (d, J = 7.6 Hz, 1H), 6.58 (s, 1H),
6.55 (d, J = 8.0 Hz, 1H), 4.90 (d, J = 3.6 Hz, 1H), 2.50–2.47 (m, 2H), 0.94 (t,
J = 7.2 Hz, 3H), 0.77 (s, 9H), 0.00 (s, 6H); 13C NMR (100 MHz, DMSO-d6) d 167.5,
155.8, 153.4, 147.1, 130.1, 120.1, 119.2, 118.1, 54.2, 26.2, 24.6, 18.6, 13.8, À3.8;
TLC Rf (CH2Cl2–MeOH 10:1) = 0.31. To a solution of acid 46 (20 mg, 0.053 mmol)
and alcohol (18 mg, 0.159 mmol) in DMF (5 mL) was added EDC (31 mg,
0.159 mmol) and DMAP (32 mg, 0.266 mmol) at room temperature. The
resulting mixture was heated at 50 °C overnight under Argon. The reaction
was quenched by the addition of saturated aqueous NH4Cl (5 mL) and extracted
with EtOAc (3 Â 10 mL). The combined organic layers were washed with H2O
(2 Â 5 mL) and brine (5 mL) successively, and then dried over Na2SO4. After
filtration and concentration in vacuo, the residue was purified via preparative
TLC (SiO2, 0.5 mm, CH2Cl2–MeOH = 10:1) to give an ester 47 with a quantitative
yield as a colorless oil: 1H NMR (400 MHz, CDCl3) d 7.75 (s, 1H), 7.14 (t,
J = 8.0 Hz, 1H), 6.86 (d, J = 7.6 Hz, 1H), 6.75–6.72 (m, 1H), 6.71 (dd, J = 6.4,
1.6 Hz, 1H), 5.59 (s, 1H), 5.31 (d, J = 2.8 Hz, 1H), 3.85–3.76 (m, 2H), 2.81–2.68
16. Enantiomerically pure forms were obtained by chiral HPLC (Daicel Chiralcel AD
column).
17. (a) Kontrec, D.; Vinkovic, V.; Sunjic, V.; Schuiki, B.; Fabian, W. M.; Kappe, C. O.
Chirality 2003, 15, 550; (b) Uray, G.; Verdino, P.; Belaj, F.; Kappe, C. O.; Fabian,
W. M. J. Org. Chem. 2001, 66, 6685; (c) Krenn, W.; Verdino, P.; Uray, G.; Faber,
K.; Kappe, C. O. Chirality 1999, 11, 659.
18. The b-keto ester (1.0 g, 4.84 mmol), 3-hydroxybenzaldehyde (590 mg,
4.84 mmol), urea (290 mg, 4.84 mmol), and Yb(OTf)3 (30 mg, 0.484 mmol)
were dissolved in THF (9.6 mL) and stirred under Argon for 24 h at 90. After
cooling to room temperature, the reaction was quenched by the addition of
saturated aqueous NaHCO3 (10 mL) and extracted with CH2Cl2 (4 Â 20 mL). The
combined organic layers were dried over Na2SO4. After filtration and
concentration in vacuo, the residue was purified via flash column
chromatography (SiO2, n-Hexanes/EtOAc/AcOH = 5:1:0.5) to give Biginelli
adduct 42 (1.31 g, 77%) as a pale yellow solid. To a solution of Biginelli adduct
42 (1.30 g, 3.69 mmol) in DMF (19 mL) was added TBSCl (834 mg, 5.53 mmol)
and imidazole (377 mg, 5.53 mmol) at 25 °C. After stirring for overnight at
25 °C, the reaction mixture was quenched by the addition of H2O (30 mL),
extracted with EtOAc (3 Â 30 mL). The combined organic layers were washed
with brine (30 mL) and dried over Na2SO4. After filtration and concentration in
vacuo, the residue was purified via flash column chromatography (SiO2, n-
Hexanes/Et2O = 5:1 ? n-Hexanes/EtOAc = 2:1 to 1:1 ? CH2Cl2/MeOH = 20:1) to
give a TBS protected product 43 with a quantitative yield as a white solid: 1H
NMR (400 MHz, CDCl3) d 7.81 (s, 1H), 7.14–7.12 (m, 3H), 7.01–6.98 (m, 3H), 6.70
(d, J = 7.2 Hz, 1H), 6.61 (bs, 1H), 6.60 (d, J = 8.4 Hz, 1H), 5.48 (bs, 1H), 5.21 (s, 1H),
(m, 2H), 1.65–0.78 (m, 11H), 1.22 (t, J = 7.2 Hz, 3H), 0.94 (s, 9H), 0.14 (s, 6H); 13
C
NMR (100 MHz, CDCl3) d 157.9, 148.7, 145.8, 144.5, 137.6, 122.4, 112.3, 112.0,
110.7, 92.9, 62.0, 48.1, 29.8, 22.3, 18.9, 18.5, 18.3, 17.9, 10.8, 5.1; TLC Rf (CH2Cl2–
MeOH 10:1) = 0.47. To a 0 °C solution of the above ester 47 (0.053 mmol) in
CH2Cl2 (2 mL) was added dropwise TBAF (1 M in THF, 64 lL, 0.064 mmol). After
10 min at 0 °C, the reaction was quenched by the addition of saturated aqueous
NaHCO3 (2 mL) and extracted with EtOAc (3 Â 10 mL). The combined organic
layers were washed with brine (10 mL) and dried over Na2SO4. After filtration
and concentration in vacuo, the residue was purified via preparative TLC (SiO2,
0.5 mm, CH2Cl2–MeOH = 10:1) to give a desilylated product (S)-40 (17 mg, 86%
over 2 steps) as a white solid. The enantiomeric excess was determined to be
98% ee by chiral HPLC (Daicel Chiralcel AD column, 0.85 mL/min, n-Hexanes/i-
PrOH = 75:25): 1H NMR (400 MHz, DMSO-d6) d 9.36 (s, 1H), 9.15 (s, 1H), 7.63 (s,
1H), 7.09 (t, J = 8.0 Hz, 1H), 6.66–6.61 (m, 3H), 5.04 (d, J = 3.2 Hz, 1H), 3.83 (dd,
J = 10.8, 6.0 Hz, 1H), 3.72 (dd, J = 10.8, 6.0 Hz, 1H), 2.76–2.68 (m, 1H), 2.66–2.57
(m, 1H), 1.61–1.41 (m, 6H), 1.40–1.00 (m, 6H), 0.87–0.75 (m, 2H); 13C NMR
(100 MHz, DMSO-d6) d 165.7, 158.1, 154.7, 152.9, 146.7, 129.9, 117.6, 114.8,
113.8, 98.8, 68.8, 54.6, 37.4, 29.72, 29.65, 26.4, 24.7, 13.7.
4.91 (s, 2H), 2.67–2.57 (m, 2H), 1.09–1.05 (m, 3H), 0.81 (s, 9H), 0.00 (s, 6H); 13
C
NMR (100 MHz, CDCl3) d 164.9, 156.1, 153.2, 152.5, 144.9, 136.0, 129.8, 128.4,
128.0, 127.9, 119.7, 119., 118.1, 99.9, 65.9, 55.4, 25.7, 25.3, 18.2, 12.5, À4.4; TLC
Rf (CH2Cl2–MeOH 10:1) = 0.51. To a solution of TBS protected product 43
(721 mg, 1.545 mmol) in MeOH (16 mL) was added 10% Pd on carbon (72 mg)
and Et3N (215 lL, 1.545 mmol) at room temperature. The reaction mixture was
hydrogenated with H2 gas (3 bars) for 3 h at 25 °C. The mixture was filtered
through a pad of Celite and concentrated in vacuo. The residue was resuspended
in water (20 mL) and acidified with 1 N HCl (ꢀ3 mL, pH <2). The resulting
suspension was sonicated for 10 min and then filtered and washed with H2O.
After freeze drying in vacuo, the resulting acid 44 (white solid, 529 mg, 91%)
was used in the following step without further purification. To a suspension of