Identification of Trypanosoma brucei leucyl-tRNA synthetase inhibitors by pharmacophore- and docking-based virtual screening and synthesis

Article abstract of DOI:10.1016/j.bmc.2011.12.035

Human African trypanosomiasis (HAT), caused by the protozoan parasite Trypanosoma brucei, is a neglected fatal disease. Leucyl-tRNA synthetase (LeuRS), which has been successfully applied in the development of antifungal agent, represents a potential antiprotozoal drug target. In this study, a 3D model of T. brucei LeuRS (TbLeuRS) synthetic active site was constructed and subjected to virtual screening using a combination of pharmacophore- and docking-based methods. A new 2-pyrrolinone scaffold was discovered and the structure-activity relationship (SAR) studies aided by the docking model and organic synthesis were carried out. Compounds with various substituents on R¹, R² and R³ were synthesized and their SAR was discussed.

Full text of DOI:10.1016/j.bmc.2011.12.035

Bioorganic & Medicinal Chemistry 20 (2012) 1240–1250
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of Trypanosoma brucei leucyl-tRNA synthetase inhibitors
by pharmacophore- and docking-based virtual screening and synthesis
Yaxue Zhao a, , Qing Wang a, , Qingqing Meng a, Dazhong Ding a, Huaiyu Yang b, Guangwei Gao a,
Dawei Li a, Weiliang Zhu b, Huchen Zhou a,
⇑
a School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China
b Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Human African trypanosomiasis (HAT), caused by the protozoan parasite Trypanosoma brucei, is a
neglected fatal disease. Leucyl-tRNA synthetase (LeuRS), which has been successfully applied in the
development of antifungal agent, represents a potential antiprotozoal drug target. In this study, a 3D
model of T. brucei LeuRS (TbLeuRS) synthetic active site was constructed and subjected to virtual screen-
ing using a combination of pharmacophore- and docking-based methods. A new 2-pyrrolinone scaffold
was discovered and the structure–activity relationship (SAR) studies aided by the docking model and
organic synthesis were carried out. Compounds with various substituents on R1, R2 and R3 were synthe-
sized and their SAR was discussed.
Received 28 September 2011
Revised 16 December 2011
Accepted 17 December 2011
Available online 30 December 2011
Keywords:
Human African trypanosomiasis
Trypanosoma brucei
Leucyl-tRNA synthetase
Virtual screening
Pharmacophore
Ó 2012 Elsevier Ltd. All rights reserved.
Docking
1. Introduction
sets of sequence motifs that reflect distinct active site topologies:
the class I synthetase active site contains a Rossmann-fold domain
The protozoan parasite Trypanosoma brucei causes human
African trypanosomiasis (HAT) which is also known as sleeping
sickness. It is a neglected fatal disease and continues to pose a ma-
jor threat to 60 million people in 36 countries in sub-Saharan Afri-
ca.1,2 Nevertheless, only a limited number of clinically useful drugs
are available for HAT.3 They are pentamidine (discovered in 1941),
suramin (discovered in 1921), melarsoprol (discovered in 1949)
and eflornithine (registered in 1990). All of the four licensed com-
pounds are generally unsatisfactory due to a combination of low
efficacy, severe side effects, and difficulty in administration. Thus,
new therapeutics for the treatment of HAT are urgently needed.
Aminoacyl-tRNA synthetases (aaRSs) play a central role in the
process of protein synthesis. They are responsible for catalyzing
the attachment of the correct amino acid to its cognate tRNA by
a two-step aminoacylation reaction.4 The first step is the formation
of an aminoacyl-adenylate (aa-AMP), as an activated intermediate,
from amino acid and ATP. In the second step, the activated amino
acid is transferred from aa-AMP to the 30-terminal adenosine of
tRNA to form aminoacyl-tRNA (aa-tRNA). The aaRSs are divided
into two classes (class I and class II) based on mutually exclusive
while the class II contains an antiparallel b-fold domain.5 Leucyl-
tRNA synthetase (LeuRS) belongs to class I and contains two con-
served characteristic motifs, with the consensus sequences of
His-Ile-Gly-His (HIGH) and Lys-Met-Ser-Lys-Ser (KMSKS).4 LeuRS
contains a synthetic domain and a connective polypeptide 1
(CP1) editing domain. The synthetic domain is in charge of the
aminoacylation reaction, whereas, the editing domain takes
responsibility for hydrolyzing mischarged tRNA to ensure the high
fidelity required by translation.6,7
LeuRS is a clinically validated drug target that has been success-
fully used for the development of antifungal agent.8,9 We recently
reported a class of inhibitors that target the editing active site of T.
brucei LeuRS (TbLeuRS) as novel antitrypanosomal agents.10 There
are also some LeuRS synthetic active site inhibitors, but none is
for T. brucei.11–15 Despite of the fact that TbLeuRS could serve as
a valuable therapeutic target for the treatment of trypanosomia-
sis,16–18 inhibitors targeting at its synthetic active site have never
been reported to date. In the present study, we will discuss the first
discovery of TbLeuRS synthetic active site inhibitors.
In silico screening has been widely applied in the discovery of lead
compounds in the initial stage of drug development,19,20 and it has
been successfully used in the identification of aaRS inhibitors as dem-
onstrated by the discovery of methionyl-tRNA synthetase (MetRS)21–23
and tryptophanyl-tRNA synthetase (TrpRS)24 inhibitors whereas either
⇑
Corresponding author. Tel.: +86 21 34206721; fax: +86 21 34204457.
E-mail address: hczhou@sjtu.edu.cn (H. Zhou).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.035

Y. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 1240–1250
1241
Table 1
pharmacophore-based or docking-based virtual screening was em-
ployed. In this study, we combined both pharmacophore- and dock-
ing-based approaches to discover TbLeuRS inhibitors. The three
dimensional (3D) structure of TbLeuRS was modeled using the known
Pyrococcus horikoshii LeuRS (PhLeuRS) structure as a template and was
screened against SPECS database which consists of approximately
197,000 commercially available compounds. A novel 2-pyrrolinone
scaffold represented by compounds 1 and 8 (Table 1) were discovered.
Base on the docking model of compound 8 in TbLeuRS, compounds
with various substituents on R1, R2 and R3 were synthesized and the
structure–activity relationship was analyzed. In general, the experi-
mental observations corroborated the docking model, which showed
the R2 phenyl inserted into a new hydrophobic pocket that was not
occupied by the endogenous substrate, and the R3 indolyl was essential
due to its interaction with the leucine-recognition pocket.
Inhibitory effect of hit compound 1 from virtual screening and its related analogs
from SPECS against TbLeuRS (average error within 0.05 lM)
R2
O
HN
N
R1
O
OH
Compd
R1
R2
TbLeuRS IC50 (lM)
F
1
170.3
OCH3
2
3
268.6
64.7
F
OCH3
OCH3
F
2. Results and discussion
2.1. Construction of the 3D structure of T. brucei LeuRS synthetic
active site
4
5
6
7
125.3
86.9
Cl
OCH3
OCH3
The currently known LeuRS X-ray crystal structures containing
the synthetic domain include P. horikoshii and Thermus thermophi-
lus LeuRS (PhLeuRS and TtLeuRS) structures in the Protein Data
Bank (PDB).25,26 Both PhLeuRS and TbLeuRS are the eukaryal/archa-
eal subtype, while the TtLeuRS is the bacterial subtype. Thus, the
PhLeuRS structure (PDB ID: 1WKB, 2.05 Å, the active sites of
PhLeuRS and TbLeuRS share 70% sequence identities)25 was chosen
as the template in the construction of the TbLeuRS structure. Due
to the absence of a ligand in PhLeuRS (1WKB), the coordinates of
Leu-AMS, which is a stable analog of endogenous substrate Leu-
AMP, from TtLeuRS (PDB ID: 1H3N, 2.00 Å, the active sites of
TtLeuRS and TbLeuRS share 36% sequence identities)26 was used
to generate a hypothetical ligand space in PhLeuRS and TbLeuRS.
LeuRS is a large protein consisting of about 1000 amino acid res-
idues. As shown by the sequence alignment in Figure 1, the homol-
ogy is about 30% between the full length TbLeuRS and PhLeuRS.
However, the synthetic active sites within 5 Å of the Leu-AMS li-
gand share about 70% sequence identities (red shaded, Fig. 1),
and the characteristic HIGH and KMSKS motifs of class I aaRS4
are also conserved (black stars, Fig. 1), which made it possible to
construct a reliable TbLeuRS active site for the purpose of virtual
screening. Therefore, although the synthetic domain consists of
four discontinuous fragments (orange boxes, Fig. 1) separated by
several domains including the CP1 editing domain (green residues,
Fig. 1) and presented a challenge for homology modeling, the
highly conserved active site provided an opportunity to simulate
a practical TbLeuRS synthetic active site by mutation method.
The core synthetic active site of TbLeuRS was constructed by
mutating the active site residues within 8 Å of Leu-AMS in PhLeuRS
to their corresponding TbLeuRS residues. The ligand–protein inter-
actions involved in the following pharmacophore and docking
models were found to be well within 5 Å of Leu-AMS, and the layer
between 5 and 8 Å served as a buffer environment for the inner
core sphere.
Cl
260.3
117.3
OCH3
OCH3
OCH3
OCH3
8
9
71.4
OCH3
333.5
10
11
189.8
107.0
OCH3
OCH3
Cl
OCH3
OH
12
13
588.5
Cl
Cl
F
F
>1000
14
15
>1000
>1000
LeuRS proteins from different species bind to the same leucine,
ATP and leucyl-adenylate substrates, and catalyze the same
aminoacylation reaction. A highly conserved hydrophobic pocket
in almost all LeuRSs accommodates the substrate leucine side
chain.26 A conserved Asp residue makes hydrogen bond to the
F
a
-amino group of leucine, and the key conserved Glu/Asp and Gln
the excellent superposition of TtLeuRS and PhLeuRS active sites25
even although they belong to bacterial and eukaryal/archaeal
subtypes, respectively. TbLeuRS and PhLeuRS, both belonging to
the eukaryal/archaeal subtype, share 70% sequence identities for
residues make hydrogen bond interactions with the ribose and ade-
nine of the substrate leucyl-adenylate. The class I characteristic mo-
tifs, HIGH and KMSKS, are also highly conserved (Fig. 1). Thus, the
active sites of all LeuRSs are highly conserved as exemplified by

1242
Y. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 1240–1250
Figure 1. Sequence alignment of TbLeuRS and PhLeuRS. The PhLeuRS residues in salmon font are Rossmann-fold domain, in green font are CP1 editing domain, and in blue
font are SC-fold domain. Residues in orange box are the catalytic domain of LeuRS. The red shaded residues are those within 5 Å of Leu-AMS ligand, yellow shaded are within
5–8 Å. The HIGH and KMSKS motifs were labeled by black stars.
the active sites. Therefore, the TbLeuRS synthetic active site
constructed by mutation method from PhLeuRS should serve as a
reasonable model for the following virtual screening.
A set of 20 molecules comprising substrate analog Leu-AMS, 10
known LeuRS inhibitors,11–15 and nine random compounds from
SPECS database were used to evaluate the pharmacophore models.
Pharmacophores I and II returned 9 and 10 hits, respectively, which
is proved to be superior over III and IV which returned 5 and 4 hits,
respectively. Thus, pharmacophores I and II were selected to screen
the SPECS database using Catalyst and resulted in 302 and 1282 hits,
respectively. Subsequently, the hits were submitted to docking eval-
uation using Glide. Finally, the binding poses of the top 600 mole-
cules were manually inspected using the following criteria: the
binding mode should be consistent with Leu-AMS; the HPS feature
of pharmacophore models should be matched; more than two
hydrogen bonds should be found; diversity of scaffolds should be
considered. The selected 26 compounds were tested against
TbLeuRS, and 2-pyrrolinone 1 was found to be active with an IC50
Although the synthetic active sites of TbLeuRS and PhLeuRS
share high sequence identities and the TbLeuRS active site was
built based on PhLeuRS, they are distinct from each other. As
shown in Fig. 1 and 10 of the 33 synthetic active site residues
(red shaded) in TbLeuRS and PhLeuRS are different. Most of the dif-
ferent residue pairs own distinct properties (e.g., F60 (TbLeuRS)/
R55(PhLeuRS), L62/Y57, H720/F643), which makes it feasible to de-
sign inhibitors specific for the synthetic active site of TbLeuRS.
2.2. Inhibitor identification by virtual screening
A combination of pharmacophore- and docking-based ap-
proaches was employed in the virtual screening to identify inhibi-
tors against TbLeuRS synthetic active site.
of 170.3 lM (Table 1). Compound 1 matched well with pharmaco-
phore I (Fig. 2b). The indolyl group acts as the hydrophobic feature
corresponding to the isobutyl group of Leu-AMS. The NH group of
indolyl and the OH group of pyrrolinone act as the hydrogen bond
donor features in the same manners as the amino and the OH groups
in the leucyl and ribose moieties of Leu-AMS, respectively. The
methoxy oxygen acts as the hydrogen bond acceptor feature in place
of the adenine nitrogen of Leu-AMS.
First, based on the analysis of the characteristic interaction pat-
terns found between endogenous substrate analog Leu-AMS and
TbLeuRS,8,26,27 pharmacophores I-IV (Figs. 2a and c and S1) were gen-
erated. These pharmacophore models all incorporated three com-
mon features that are intrinsic to the substrate–protein
interactions (Figs. 2 and S1): the hydrophobic site (HPS) feature at
the isobutyl group; the hydrogen bond donor (HBD) feature at the
amino group of leucyl moiety; the hydrogen bond donor (HBD) fea-
ture at the OH group of ribose which interacts with a conserved Asp
residue. The four pharmacophore models varied in the hydrogen
bond patterns at the purine group as shown in Figure 2 and S1.
2.3. Initial structure–activity relationship
To investigate the initial structure–activity relationship, more 2-
pyrrolinone compounds with related structures were selected from

Y. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 1240–1250
1243
Figure 2. (a) Pharmacophores I and II: light blue, HPS (hydrophobic site); violet, HBD (hydrogen bond donor); green, HBA (hydrogen bond acceptor). Distances are shown in
Å. (b) Overlap of compound 1 with pharmacophore I. (c) Leu-AMS docked in TbLeuRS active site with pharmacophore features labeled in the same color as (a). The HBA feature
of pharmacophore I is show in green dashed arrow while that of pharmacophore II is shown in green solid arrow.
the SPECS database and tested against TbLeuRS (Table 1). A number
of compounds with IC50 values below 100 M were identified and an
initial structure–activity relationship was established.
the improved potency of compound 11 compared to 10, chloro-
substitution gave improved activity. However, fluoro-substitution
did not have such effect, as shown by the abolished activity of
compounds 13 and 14. Removal of the methoxy group in 1 also
resulted in abolished activity (15).
To facilitate the understanding of the interaction between 2-
pyrrolinones and the synthetic active site of TbLeuRS, 2-pyrrolinone
8 was docked into TbLeuRS using Glide. As shown in Figure 3, com-
pound 8 assumed a similar binding pose to the endogenous sub-
strate analog Leu-AMS. The indolyl group partially inserted into a
hydrophobic pocket composed by Pro46, Tyr47, Ser599, Leu596,
His692, and Leu688, and formed favorable hydrophobic interac-
tions. The indolyl NH group formed a hydrogen bond with the back-
bone carbonyl of Tyr47. The exocyclic tautomeric hydroxyl formed a
l
The effect of substitutions on the R2 phenyl group was first
investigated (Table 1). The para-halogen substitutions gave more
favorable activity than meta- or ortho-substitution as demon-
strated by compounds 1–3 and 4–5. The para-substitution with
halogen (3 and 5) or alkyl (7 and 8) gave improved potency as
compared to unsubstituted compound 6. Ethyl-gave better
potency than methyl (8 and 7). However, polar substituents at
para- or meta-position led to diminished activity as shown by
compound 9 and 12. These observations suggest the existence of
a hydrophobic binding pocket near the para-position of R2 phenyl.
Next, the effect of substitutions on R1 was considered. As shown by
a
b
CP1
editing
domain
L54
L62
P48
F45
c
Y47
H55
S599
H58
V721
Synthetic
domain
P46
L596
H692
L688
H720
D687
Figure 3. Binding mode of compound 8 in TbLeuRS. (a) Illustration of full length LeuRS structure containing CP1 editing domain and synthetic domain. Salmon: Rossmann-
fold; blue: SC-fold; green: CP1 editing domain. (b) Superposition of compound 8 (yellow) and Leu-AMS (cyan) in TbLeuRS synthetic active site. (c) Main interactions between
compound 8 and TbLeuRS synthetic active site. Dashed lines represent hydrogen bonds. This figure was generated in PyMoL.

1244
Y. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 1240–1250
Table 2
a
Inhibitory effect of synthesized compounds with various R3 groups against TbLeuRS
H
(average error within 0.05 lM)
Br
O
19
20
O
O
b
O
R3
N
H
NHBoc
OH
N
22
21
O
OH
O
O
Compd
R3
TbLeuRS IC50 (lM)
c
N
H
NH2
HN
8
71.4
23
Scheme 1. Synthesis of aldehyde and amine intermediates. Reagents and condi-
tions: (a) (i) n-BuLi, THF, ꢀ78 °C; (ii) DMF, THF, ꢀ78 °C to rt (b) tert-butyl 3-
aminopropylcarbamate, EDCI, DMAP, DCM, 0 °C to rt; (c) HCl, DCM.
O
26
27
269.0
101.9
O
O
N
H
O
O
N
H
R2
O
O
O
R3
O
R1
c or d
a or b
N
O
R1
28
29
>1000
173.5
R1
N
H
OH
O
O
O
24a-d
25a-d
26-31, 34-45
26, 27
O
e
32, 33
34: R1 = 4-OCH3-Ph, R2 = 4-CF3-Ph,
3 = 2-(indol-3-yl)ethyl
35: R1 = 4-OCH3-Ph, R2 = 4-i-Pr-Ph,
3 = 2-(indol-3-yl)ethyl
36: R1 = 4-OCH3-Ph, R2 = 4-t-Bu-Ph,
3 = 2-(indol-3-yl)ethyl
HO
30
656.3
24a, 25a: R1 = 4-OCH3-Ph
24b, 25b: R1 = 4-Cl-Ph
24c, 25c: R1 = 2, 4-difluorophenyl
24d, 25d: R1 = 1-napthyl
R
O
N
31
32
33
716.1
>1000
>1000
N
R
H2N
H2N
R
37: R1 = 4-OCH3-Ph, R2 = 4-n-Bu-Ph,
R3 = 2-(indol-3-yl)ethyl
26: R1 = 4-OCH3-Ph, R2 = 4-Et-Ph,
R3 = 3-(N-Boc)NH2-Pr
27: R1 = 4-OCH3-Ph, R2 = 4-Et-Ph,
R3 = 4-(N-Boc-NH2CH2)-Ph
28: R1 = 4-OCH3-Ph, R2 = 4-Et-Ph,
38: R1 = 4-OCH3-Ph, R2 = 4-i-Bu-Ph,
R
3 = 2-(indol-3-yl)ethy
39: R1 = 4-OCH3-Ph, R2 = biphenyl,
3 = 2-(indol-3-yl)ethyl
40: R1 = 4-OCH3-Ph, R2 = napth-2-yl,
3 = 2-(indol-3-yl)ethyl
R
and will aid the design of compounds with improved potency
3 = 3-(N-isovaleryl)aminopropyl
29: R1 = 4-OCH3-Ph, R2 = 4-Et-Ph,
3 = (benzo[1, 3]dioxol-5-yl)methyl
against TbLeuRS.
R
R
41: R1 = 4-OCH3-Ph, R2 = 2-Br-4-F-Ph,
R3 = 2-(indol-3-yl)ethyl
2.4. Studies of structure–activity relationship via new
compound design and synthesis
R
30: R1 = 4-OCH3-Ph, R2 = 4-Et-Ph,
R3 = 4-COOH-Bn
31: R1 = 4-OCH3-Ph, R2 = 4-Et-Ph,
42: R1 = 4-OCH3-Ph, R2 = pentyl,
R3 = 2-(indol-3-yl)ethyl
2.4.1. Chemistry
43: R1 = 4-Cl-Ph, R2 = 4-i-Bu-Ph,
The aldehydes and amines used in the synthesis of 2-pyrroli-
nones were either purchased or prepared as exemplified by the
synthesis of aldehyde 20 and amine 23 (Scheme 1). Treatment of
1-bromo-4-butylbenzene 19 with n-BuLi at ꢀ78 °C followed by
reaction with DMF gave aldehyde 20.28 Coupling of 3-methylbutyr-
ic acid 21 with tert-butyl 3-aminopropylcarbamate in the presence
of EDCI and DMAP provided N-Boc protected intermediate 22
which was converted to amine 23 after deprotection by HCl. The
synthesis of 2-pyrrolinones was summarized in Scheme 2. First,
diketo esters 25a–d were obtained via Claisen condensation of ke-
tones 24a–d with diethyl oxalate in the presence of LiHMDS or
NaH.29–31 The subsequent three-component condensation reaction
by refluxing diketo esters with appropriate aldehydes and amines
gave 2-pyrrolinones 26–31 and 34–45, while the presence of DIPEA
as a catalyst was needed in the preparation of 26, 28, 38 and 45
(Scheme 2).32–35 Removal of the terminal N-Boc protection of 26
and 27 provided 32 and 33, respectively.
3 = 3-(imidazol-1-yl)propyl
R
3 = 2-(indol-3-yl)ethyl
R
44: R1 = 2,4-difluorophenyl, R2 = 4-i-Bu-Ph,
R3 = 2-(indol-3-yl)ethyl
32: R1 = 4-OCH3-Ph, R2 = 4-Et-Ph,
R3 = 3-NH2-Pr
33: R1 = 4-OCH3-Ph, R2 = 4-Et-Ph,
R3 = 4-(NH2CH2)-Ph
45: R1 = napth-1-yl, R2 = 4-Et-Ph,
R3 = 2-(indol-3-yl)ethyl
Scheme 2. Synthesis of 2-pyrrolinone derivatives 26–45. Reagents and conditions:
(a) diethyl oxalate, LiHMDS, Et2O, ꢀ78 °C to rt, then 1 M HCl (for compd 25a); (b)
diethyl oxalate, NaH, toluene, ꢀ78 °C to rt(for compds 25b–d); (c) R2CHO, R3NH2,
toluene or ethanol, DIPEA, reflux (for compds 26, 28, 38 and 45); (d) R2CHO, R3NH2,
toluene or EtOH, reflux (for compds 27, 29–31, 34–37, 39–44); (e) HCl, DCM.
hydrogen bond with the highly conserved Asp687. The methoxy-
phenyl group was sandwiched by His55, His58, His720, and formed
favorable p–p interactions. The methoxy oxygen formed a hydrogen
bond with the backbone NH of Val721. It is worth mentioning that
the docking model indeed showed the R2 phenyl inserted into a
new hydrophobic pocket consisting of Phe45, Pro48, Leu54 and
Leu62, which was not occupied by Leu-AMS. These observations cor-
roborate the initial structure–activity relationship discussed above
2.4.2. Structure–activity relationship
First, the effect of R3 group on TbLeuRS inhibitory activity was
explored (Table 2). Replacement of the indolyl group in compound

Y. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 1240–1250
1245
Table 3
(42) both resulted in decreased activity. Considering the fact that
the chlorine substitution for methoxy on R1 phenyl group gave im-
proved activity (compound 11 vs 10), the chloride analog of com-
pound 38 (43) was synthesized, but it showed a comparable IC50
of 41.0 lM. The fluoride analog of compound 38 (44) showed de-
creased activity, while the naphthyl analog of compound 8 (45)
Inhibitory effect of synthesized compounds with various R1 and R2 groups against
TbLeuRS (average error within 0.05
l
M)
R2
O
HN
N
R1
O
OH
showed comparable activity.
Compd
R1
R2
TbLeuRS IC50 (lM)
3. Conclusion
F3C
34
68.1
Human African trypanosomiasis, a fatal disease epidemic in
sub-Saharan Africa, is in urgent need of new therapeutics, espe-
cially those with new mechanism of action. Here we reported the
first discovery of inhibitors targeting the synthetic domain of T.
brucei LeuRS. Virtual screening using a combination of pharmaco-
phore- and docking-based approaches against a model of TbLeuRS
synthetic active site was carried out and a number of 2-pyrrolinon-
es were discovered as TbLeuRS inhibitors. Guided by the docking
model of compound 8 in TbLeuRS, 2-pyrrolinones with various sub-
stituents at R1, R2, and R3 were designed and synthesized. The
structure–activity relationship generally corroborated the docking
model, which showed the R2 phenyl explored a new hydrophobic
pocket, and the R3 indolyl is essential for the favorable interaction
with the leucine-recognition pocket. This work provided a new ap-
proach and lead compounds for the exploration of T. brucei LeuRS
as a novel therapeutic target for the discovery of new anti-parasitic
agents.
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
35
36
37
38
39
40
90.5
94.5
43.9
31.9
43.1
85.5
F
41
109.6
4. Experimental section
Br
4.1. Construction of the 3D structure of TbLeuRS synthetic
active site
42
43
110.8
41.0
OCH3
Cl
The synthetic active site of T. brucei LeuRS was constructed
based on the crystal structure of P. horikoshii LeuRS (1WKB).25
The coordinates of ligand Leu-AMS from T. thermophilus LeuRS
(1H3N)26 was used to generate a hypothetical ligand space in
PhLeuRS and TbLeuRS. The active site residues in PhLeuRS within
8 Å of the Leu-AMS ligand were mutated to the corresponding res-
idues of TbLeuRS. Hydrogen atoms were added, bond orders were
designed, and water molecules were deleted in the Protein Prepa-
ration Wizard work flow in the Maestro interface.36 The added
hydrogen atoms were optimized by H-bond Assignment with
exhaustive sampling method. Finally, the structure was subjected
to minimization in MacroModel37 using OPLS_2005 force field.38
The residues within 5 Å of ligand were set to be free, those between
5 and 8 Å were constrained by 200 kcal/(mol ꢁ A2) force, and those
between 8 and 10 Å were frozen.
F
44
45
175.4
80.0
F
8 with either chain structures or different heterocycles with non-
polar or polar moieties all resulted in diminished activity ( 26
and 28–33) except for compound 27 which showed a moderately
decreased activity. It suggests that the interaction of the indolyl
group with the hydrophobic pocket proposed by the docking mod-
el (Fig. 3) may be significant in the binding of 2-pyrrolinones to
TbLeuRS.
4.2. In silico screening
Next, the effect of R2 group on the TbLeuRS inhibitory activity
was explored (Table 3). Since the docking model showed the R2
group inserted into a new hydrophobic pocket (Fig. 3), various
hydrophobic substituents including p-alkylphenyl (34–38), biphe-
nyl (39), naphthyl (40), halogenated phenyl (41), and aliphatic
chain (42) were synthesized. Compound 34 with p-CF3-phenyl
group showed comparable inhibitory activity to compound 8 with
p-ethyl-phenyl. When the p-alkyl group became butyl or iso-butyl
group, the inhibitory activity showed a significant improvement as
The crucial interaction patterns between TbLeuRS and Leu-AMS
were analyzed using LigandScout.39 Four pharmacophore models,
I-IV (Figs. 2 and S1), were generated and subsequently evaluated
by a set of 20 molecules including substrate analog Leu-AMS, 10
known LeuRS inhibitors11–15 (Table S1) and nine random com-
pounds from SPECS database (ꢂ197,000 compounds, http://
www.specs.net). The pharmacophores I and II were selected to
screen against SPECS database using Catalyst with Best Flexible
Search mode. Molecules that fit all the features of pharmacophore
model I or II were retained as hits, and 302 and 1282 hits were re-
turned, respectively.
demonstrated by compounds 37 and 38 (IC50 = 43.9 and 31.9 lM).
This observation corroborates the occupation of the new hydro-
phobic pocket by R2 in the docking model. The biphenyl compound
Subsequently, all hits were prepared by LigPrep using OPLS_2005
force field38 and the ionization states at pH 7.0 2.0 were generated
using Epik before they were docked to TbLeuRS using Glide in SP
39 also gave good inhibitory activity (IC50 = 43.1
lM). Halogen sub-
stitutions (41) and replacement of the phenyl with a pentyl chain

1246
Y. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 1240–1250
precision. The TbLeuRS grid was generated in Receptor Grid Genera-
tion and the docking box was defined as a 10 Å ꢁ 10 Å ꢁ 10 Å bound-
ing box with Leu-AMS at the center. The docking box well covered
the complete active site.
mixture was stirred at ꢀ78 °C for 45 min before diethyl oxalate
(5.4 mL, 40 mmol) was added dropwise. After warmed to room
temperature and stirred for another 3 h, yellow precipitation ap-
peared. The mixture was allowed to settle overnight. The yellow
solid was filtered and washed with 60 mL hot ether to afford a pale
yellow powder which was suspended in water and acidified to pH
2–3 with 1 M hydrochloric acid in ice bath. Subsequent filtration
afforded 25a (1.32 g, 70% yield) as a yellow solid. 1H NMR
(400 MHz, CDCl3): d 15.47 (br s, 1H), 7.98 (d, 2H, J = 8.0 Hz), 7.02
(s, 1H), 6.97 (d, 2H, J = 8.0 Hz), 4.39 (q, 2H), 3.89 (s, 3H) and 1.41
(t, 3H) ppm. Mp: 246–248 °C.
4.3. Chemistry
NMR spectra were recorded on Bruker Avance III 400 MHz or
Varian Mercury 300 Hz. Chemical shifts are expressed in parts
per million (d) relative to residual solvents as internal reference
(CDCl3: 7.26; MeOD: 3.31; DMSO-d6: 2.50). High resolution mass
spectra were obtained on an Agilent 6530 Accurate Mass Q-TOF
LC-MS. Column chromatography was performed using Huanghai
4.3.4. General procedure for the preparation of 25b–d
To the solution of acetophenones 24b–d (1.0 g) in 10 mL dry
toluene in ice bath was added NaH (1.5 equiv) under nitrogen
atmosphere. The mixture was allowed to warm to room tempera-
ture and stir for another 1.5 h. After cooled in ice-water bath,
diethyl oxalate (2.0 equiv) dissolved in 7 mL dry toluene was
added dropwise. The mixture was heated at 50 °C for 15 min,
poured into 100 mL ice water, and acidified to pH 5 with 1 M
HCl. The aqueous layer was separated and extracted with ethyl
acetate (30 mL ꢃ 2). The organic layers were combined, washed
with water and brine, dried over anhydrous sodium sulfate, and
evaporated. Further appropriate purification afforded the desired
compounds 25b–d.
silica gel (45–75 lm). Melting points were measured on a SGWX-
4 melting point apparatus. The preparative HPLC was conducted
on an Unimicro EasySep-1010 liquid chromatograph using a C18
column (30 mm ꢃ 250 mm), 1 mL sample loop, a flow rate of
16 mL/min, and a gradient of 10% v/v MeOH in H2O (containing
0.1% v/v TFA) (t = 0.0 min) to 100% MeOH (t = 30.0 min). All re-
agents were commercially available if not indicated otherwise.
Reactions were not optimized for maximum yields.
4.3.1. 4-n-Butylbenzaldehyde (20)
To a solution of 4-bromo-4-butylbenzene 19 (4.0 g, 18.8 mmol)
was added n-butyllithum (35 mL, 1.6 M in hexane, 56.3 mmol)
over 30 min at ꢀ78 °C. After stirring at ꢀ78 °C for 2 h, dimethyl-
formamide (3.7 mL, 56.3 mmol) in 10 mL dry tetrahydrofuran
was added over 15 min. The mixture was warmed to room temper-
ature gradually, poured into ice-water, and extracted by petroleum
ether (100 mL ꢃ 4). The organic layers were combined, washed
with 100 mL brine, dried over anhydrous sodium sulfate, and evap-
orated in vacuo. The residue was purified by column chromatogra-
phy on silica gel using dichloromethane/petroleum ether (1/10, v/
v) as eluent to give 20 (2.62 g, 86.1% yield) as a yellowish oil. 1H
NMR (400 MHz, CDCl3): d 9.97 (s, 1H), 7.79 (d, 2H, J = 7.6 Hz),
7.33 (d, 2H, J = 7.6 Hz), 2.69 (t, 2H, J = 7.6 Hz), 1.62 (m, 2H), 1.36
(m, 2H) and 0.93 (t, 3H, J = 7.2 Hz) ppm.
4.3.4.1. (Z)-Ethyl 4-(4-chlorophenyl)-2-hydroxy-4-oxo-2-bute-
noate (25b).
Compound 25b was prepared from 4-chloroace-
tophenone 24b (1.0 g, 6.47 mmol) and diethyl oxalate (1.890 g,
12.94 mmol) in the presence of NaH (388 mg, 9.71 mmol, 60% in
mineral oil). The residue was washed with dichloromethane to give
compound 25b (0.728 g, 44.0% yield) as a yellowish solid. .1H NMR
(400 MHz, DMSO-d6): d 7.88 (d, 2H, J = 7.2 Hz), 7.49 (d, 2H,
J = 7.2 Hz), 6.51 (br s, 1H), 4.16 (q, 2H) and 1.25 (t, 3H) ppm. Mp:
214–215 °C.
4.3.4.2. (Z)-Ethyl 4-(2,4-difluorophenyl)-2-hydroxy-4-oxo-2-
butenoate (25c).
Compound 25c was prepared from 2,4-
4.3.2. N-(3-Aminopropyl)-3-methylbutyramide hydrochloride
(23)
difluoroacetophenone 24c (1.0 g, 6.41 mmol) and diethyl oxalate
(1.873 g, 12.82 mmol) in the presence of NaH (384 mg, 9.61 mmol,
60% in mineral oil). The residue was washed by hexane to give
compound 25c (287 mg, 11.2% yield) as a yellow solid. 1H NMR
(400 MHz, CDCl3): d 15.05 (br s, 1H), 8.01 (m, 1H), 7.08 (d, 1H),
7.02 (m, 1H), 6.92 (m, 1H), 4.39 (q, 2H) and 1.40 (t, 3H) ppm.
EDCI (220 mg, 2.3 mmol) and DMAP (6.6 mg, 0.054 mmol) in
5 mL dichloromethane was added dropwise to a mixture of
3-methylbutyric acid 21 (140 mg, 1.38 mmol) and tert-butyl
3-aminopropylcarbamate (200 mg, 1.15 mmol) in 4 mL dichloro
methane at 0 °C under nitrogen atmosphere. The mixture was al-
lowed to warm to room temperatureand stirred overnight. The solu-
tion was washed by 1 M NaOH (2 mL), 1 M hydrochloric acid (2 mL),
and saturated sodium bicarbonate solution (2 mL), dried over anhy-
drous sodium sulfate, and concentrated in vacuo. The residue was
purified by column chromatography on silica gel using petroleum
ether/ethyl acetate (1/1, v/v) as eluent to give tert-butyl 3-(3-meth-
ylbutyrylamino)propylcarbamate 22 (190 mg, 64.1% yield) as a
white solid. 1H NMR: (300 MHz, CDCl3): d 6.18 (br s, 1H), 4.94 (br
s, 1H), 3.28 (m, 2H), 3.16 (m, 2H), 2.04–2.26 (m, 3H), 1.56–1.65 (m,
2H), 1.44 (s, 9H) and 0.938 (d, 6H, J = 6.0 Hz) ppm. Mp: 88–90 °C
After treatment of 22 (95 mg, 0.37 mmol) with dry HCl in
dichloromethane, the solvent was evaporated to give compound
23 (68 mg, 94.7% yield) as a white solid. 1H NMR: (400 MHz,
D2O): d 3.25(t, 2H, J = 6.8 Hz), 2.96 (t, 2H, J = 7.6 Hz), 2.07 (d, 2H,
J = 7.2 Hz), 1.94 (m, 1H), 1.83 (m, 2H) and 0.87 (d, 6H, J = 6.8 Hz).
4.3.4.3. (Z)-Ethyl 2-hydroxy-4-(naphthalen-1-yl)-4-oxo-2-bute-
noate (25d).
Compound 25d was prepared from 1-(naphtha-
len-1-yl) ethanone 24d (1.0 g, 5.88 mmol) and diethyl oxalate
(1.718 g, 11.76 mmol) in the presence of NaH (353 mg, 8.82 mmol,
60% in mineral oil). The residue was purified by column chroma-
tography on silica gel using petroleum ether/ethyl acetate (5/1,
v/v) as eluent to give compound 25d (1.497 g, 92.4 % yield) as a yel-
low solid. 1H NMR (400 MHz, CDCl3): d 15.15 (br s, 1H), 7.59 (d, 1H,
J = 8.0 Hz), 8.04 (d, 1H, J = 8.0 Hz), 7.91 (m, 2H), 7.52–7.65 (m, 3H),
7.00 (s, 1H), 4.40 (q, 2H, J = 7.2 Hz) and 1.41 (t, 3H, J = 7.2 Hz) ppm.
Mp: 59–60 °C.
4.3.5. General procedure for the synthesis of compounds 26–31
and 34–45
The appropriate aldehyde (1.0–5.0 equiv) and amine (1.0 equiv)
in ethanol or toluene was stirred at room temperature for 1 h be-
fore the appropriate diketo ester (0.7–2.0 equiv) was added. The
mixture was heated to reflux for 24 h to provide 2-pyrrolinones
26–31 and 34–45. For the synthesis of compounds 26, 28, 38 and
45, DIPEA (0.1–0.2 equiv) was added as a catalyst. Purification:
4.3.3. (Z)-Ethyl 2-hydroxy-4-(4-methoxyphenyl)-4-oxo-2-butenoate
(25a)
To a solution of LiHMDS (41.5 mL, 1.06 M) in 60 mL dry ether at
ꢀ78 °C under nitrogen atmosphere was added dropwise 4-meth-
oxy acetophenone 24a (3.0 g, 20 mmol) in 10 mL dry ether. The

Y. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 1240–1250
1247
Method A, the reaction mixture was filtered, washed, and recrys-
tallized from appropriate solvent to provide the product; Method
B, the solvent was removed by rotary evaporation and the product
was purified by column chromatography on silica gel; Method C,
the solvent was removed by rotary evaporation and the residue
was purified by preparative HPLC.
0.52 mmol) and 25a (125 mg, 0.50 mmol) in 5 mL ethanol. Purifica-
tion with Method A using dichloromethane/hexane (5/4, v/v) as
recrystallization solvent gave compound 29 (55 mg, 21.0% yield)
as a white solid. 1H NMR (400 MHz, CDCl3): d 7.65 (d, 2H,
J = 8.0 Hz), 7.08 (d, 2H, J = 7.6 Hz), 7.01 (d, 2H, J = 8.0 Hz), 6.81 (d,
2H, J = 8.0 Hz), 6.76 (d, 1H, J = 8.0 Hz), 6.68 (s, 1H), 6.61 (d, 1H,
J = 8.0 Hz), 5.98 (s, 2H), 5.33 (s, 1H), 5.06 (d, 1H, J = 14.8 Hz), 3.81
(s, 3H), 3.50 (d, 1H, J = 14.8 Hz), 2.58 (q, 2H, J = 8.0 Hz) and 1.18
(t, 3H, J = 7.6 Hz) ppm. 13C NMR (100 MHz, DMSO-d6): d 187.16,
165.63, 162.77, 147.40, 146.49, 143.64, 131.23, 130.51, 127.92,
127.65, 121.12, 113.33, 108.16, 108.11, 100.95, 60.37, 55.40,
43.58, 27.70, 15.15 ppm. Mp: 263–265 °C. HRMS (ESI): [M+H]+
C28H25NO6 calcd 472.1755, found 472.1783.
4.3.5.1.
methoxybenzoyl)-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)propylcar-
bamate (26). Compound 26 was prepared from 4-ethylbenzal-
tert-Butyl 3-(2-(4-ethylphenyl)-4-hydroxy-3-(4-
dehyde (71 mg, 0.53 mmol), tert-butyl 3-aminopropylcarbamate
(20 mg, 0.11 mmol) and 25a (27 mg, 0.11 mmol) in the presence of
DIPEA (1.9 mg, 0.014 mmol) in 5 mL ethanol. Purification with
Method A using ethanol as recrystallization solvent gave compound
26 (28 mg, 49.2% yield) as a white solid. 1H NMR: (300 MHz, DMSO-
d6): 7.71 (d, 2H, J = 8.7 Hz), 7.20 (d, 2H, J = 8.1 Hz), 7.14 (d, 2H,
J = 7.8 Hz), 6.96 (d, 2H, J = 9.0 Hz), 6.71 (t, 1H, J = 4.2 Hz), 5.44 (s,
1H), 3.81 (s, 3H), 3.53 (m, 1H), 2.85 (m, 2H), 2.63 (m, 1H), 2.55 (q,
2H, J = 7.5 Hz), 1.56 (m, 2H), 1.36 (s, 9H) and 1.12 (t, 3H, J = 7.5 Hz)
ppm. 13C NMR (100 MHz, DMSO-d6): d 187.50, 165.18, 162.91,
155.46, 149.64, 143.67, 133.20, 131.26, 131.16, 130.48, 127.97,
127.53, 120.15, 113.47, 113.41, 77.50, 60.67, 55.49, 38.08, 37.39,
28.19, 27.95, 27.69,15.13 ppm. Mp: 224–226 °C. HRMS (ESI):
[M+H]+ C28H34N2O6 calcd 495.2495, found 495.2508.
4.3.5.5. 4-(2-(4-Ethylphenyl)-4-hydroxy-3-(4-methoxybenzoyl)-
5-oxo-2,5-dihydro-1H-pyrrol-1-yl)methyl benzoic acid (30)
.
Compound 30 was prepared from 4-ethylbenzaldehyde
(134 mg, 1.0 mmol), 4-aminomethylbenzoic acid (151 mg,
1.0 mmol) and 25a (250 mg, 1.0 mmol) in 14 mL toluene. The result-
ing precipitate was filtered and washed with ethyl acetate (2 mL)
and H2O (30 mL) to give 30 (170 mg, 34% yield) as a white solid.
1H NMR (400 MHz, DMSO-d6): d 12.90 (br s, 1H), 11.75 (br s, 1H),
7.86 (d, 2H, J = 8.0 Hz), 7.74 (d, 2H, J = 8.4 Hz), 7.19 (d, 2H,
J = 7.6 Hz), 7.12 (d, 2H, J = 8.0 Hz), 7.09 (d, 2H, J = 8.0 Hz), 6.96 (d,
2H, J = 8.4 Hz), 5.24 (s, 1H), 4.86 (d, 1H, J = 15.6 Hz), 3.87 (d, 1H,
J = 15.6 Hz), 3.81 (s, 3H), 2.53 (q, 2H, J = 7.6 Hz), 1.11 (t, 3H,
J = 7.6 Hz) ppm. 13C NMR (100 MHz, DMSO-d6): d 187.06, 166.97,
162.72, 157.15, 150.71, 143.69, 141.72, 133.20, 131.20, 130.68,
129.73, 129.52, 129.44, 127.90, 127.69, 127.57, 119.50, 113.31,
60.89, 55.39, 43.76, 27.70, 15.18 ppm. Mp: 260–263 °C. HRMS
(ESI): [M+H]+ C28H25NO6 calcd 472.1755, found 472.1783.
4.3.5.2.
methoxybenzoyl)-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)benzyl car-
bamate (27). Compound 27 was prepared from 4-ethyl benz-
tert-Butyl 4-(2-(4-ethylphenyl)-4-hydroxy-3-(4-
aldhyde (67 mg, 0.50 mmol), tert-butyl 4-aminobenzylcarbamate
23b (111 mg, 0.50 mmol) and 25a (125 mg, 0.50 mmol) in 5 mL
ethanol. Purification with Method A using ethyl acetate/hexane
(1/2, v/v) as recrystallization solvent gave compound 27
(13.0 mg, 4.8% yield) as a white solid. 1H NMR (400 MHz, MeOD):
d 7.78 (d, 2H, J = 8.4 Hz), 7.52 (d, 2H, J = 8.4 Hz), 7.22 (d, 2H,
J = 8.0 Hz), 7.20 (d, 2H, J = 7.6 Hz), 7.02 (d, 2H, J = 7.6 Hz), 6.96 (d,
2H, J = 8.4 Hz), 6.19 (s, 1H), 4.15 (s, 2H), 3.86 (s, 3H), 2.49 (q, 2H,
J = 8.0 Hz), 1.44 (s, 9H) and 1.10 (t, 3H, J = 7.6 Hz) ppm. 13C NMR
(100 MHz, DMSO-d6): d 188.16, 165.04, 163.59, 156.23, 148.78,
143.64, 137.59, 135.42, 133.94, 131.84, 130.87, 128.19, 128.00,
127.66, 122.91, 121.32, 114.03, 78.26, 61.60, 55.98, 43.34, 28.70,
28.04, 15.34 ppm. Mp: 216–218 °C. HRMS (ESI): [M+H]+
C32H34N2O6 calcd 543.2495, found 543.2508.
4.3.5.6.
pyl)-4-(4-methoxybenzoyl)-1,5-dihydro-2H-pyrrol-2-one
(31). Compound 31 was prepared from 4-ethylbenzaldehyde
5-(4-Ethylphenyl)-3-hydroxy-1-(3-imidazol-1-yl-pro-
(67 mg, 0.50 mmol), 3-imidazol-1-yl propylamine (62 mg,
0.50 mmol) and 25a (125 mg, 1.0 mmol) in 5 mL ethanol. Purifica-
tion with Method C gave 31 (9 mg, 3.46% yield) as a brown solid. 1H
NMR: (300 MHz, MeOD): d 8.98 (br s, 1H), 7.80 (d, 2H, J = 8.4 Hz),
7.66 (s, 1H), 7.56 (s, 1H), 7.22 (d, 2H, J = 7.5 Hz), 7.16 (d, 2H,
J = 7.2 Hz), 6.94 (d, 2H, J = 7.5 Hz), 5.57 (s, 1H), 4.21 (s, 2H), 3.84
(s, 3H), 3.63 (m, 1H), 2.95 (m, 1H), 2.58 (q, 2H, J = 7.5 Hz), 2.04
(m, 2H) and 1.15 (t, 3H, J = 7.5 Hz) ppm. 13C NMR (100 MHz,
DMSO-d6): d 187.50, 165.48, 162.93, 154.48, 149.47, 143.80,
135.41, 133.03, 131.20, 130.41, 128.00, 127.58, 121.73, 120.30,
120.04, 113.42, 60.42, 55.42, 45.95, 36.83, 27.94, 27.65,
15.11 ppm. Mp: 115–117 °C. HRMS (ESI): [M+H]+ C26H28N2O4 calcd
446.2074, found 446.2092.
4.3.5.3.
zoyl)-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)propyl-3-methylbu-
tyramide (28). Compound 28 was prepared from 4-
N-3-(2-(4-Ethylphenyl)-4-hydroxy-3-(4-methoxyben-
ethylbenzaldehyde (38 mg, 0.28 mmol), N-(3-aminopropyl)-3-
methylbutyramide hydrochloride 23 (50 mg, 0.26 mmol) and 25a
(51.3 mg, 0.20 mmol) in the presence of DIPEA (1.9 mg, 0.014 mmol)
in 10 mL ethanol. Purification with Method A using dichlorometh-
ane/hexane (1/3, v/v) as recrystallization solvent gave 28 (10.9 mg,
11.1% yield) as a white solid. 1H NMR: (400 MHz, MeOD): d 7.77
(d, 2H, J = 8.2 Hz), 7.18 (d, 2H, J = 8.0 Hz), 7.14 (d, 2H, J = 8.0 Hz),
6.93 (d, 2H, J = 8.8 Hz), 5.55 (s, 1H), 3.84 (s, 3H), 3.65 (m, 1H),
3.05–3.20 (m, 2H), 2.86 (m, 1H), 2.57 (q, 2H, J = 7.6 Hz), 2.01 (m,
3H), 1.66 (m, 2H), 1.16 (t, 3H, J = 7.6 Hz) and 0.90 (d, 6H, J = 5.2 Hz)
ppm. 13C NMR (100 MHz, DMSO-d6): d 188.89, 171.31, 165.17,
162.77, 143.64, 132.06, 131.18, 131.03, 127.92, 127.50, 123.97,
114.20, 113.36, 60.69, 55.41, 54.83, 44.79, 38.22, 35.92, 27.80,
27.69, 25.40, 22.25, 15.11 ppm. Mp: 210–212 °C. HRMS (ESI):
[M+H]+ C28H34N2O5 calcd 479.2540, found 479.2554.
4.3.5.7. 1-(3-Aminopropyl)-5-(4-ethylphenyl)-3-hydroxy-4-(4-
methoxy-benzoyl)-1,5-dihydro-2H-pyrrol-2-one (32).
Com-
pound 26 (20 mg, 0.04 mmol) was treated with dry hydrochloride
dichloromethane solution. After the reaction was completed, the
solid was filtered and washed by dichloromethane to give 32
(11 mg, 63.2% yield). 1H NMR: (400 MHz, DMSO-d6): d 7.88 (br s,
3H), 7.72 (d, 2H, J = 8.4 Hz), 7.22 (d, 2H, J = 7.6 Hz), 7.16 (d, 2H,
J = 8.0 Hz), 6.96 (d, 2H, J = 8.8 Hz), 5.47 (s, 1H), 3.81 (s, 3H), 3.61
(m, 1H), 2.70 (m, 3H), 2.52 (q, 2H, J = 7.6 Hz), 1.74 (m, 2H) and
1.12 (t, 3H, J = 7.6 Hz) ppm. 13C NMR (100 MHz, DMSO-d6): d
188.06, 165.98, 162.82, 149.50, 143.65, 136.79, 134.10, 131.18,
127.98, 127.52, 119.93, 115.27, 113.35, 60.53, 55.40, 37.44, 36.56,
27.66, 25.68, 15.07 ppm. Mp: 242–243 °C. HRMS (ESI): [M+H]+
C23H27N2O4 calcd 395.1965, found 395.1980.
4.3.5.4. 1-(Benzo[1,3]dioxol-5-yl-methyl)-5-(4-ethylphenyl)-3-
hydroxy-4-(4-methoxybenzoyl)-1,5-dihydro-2H-pyrrol-2-one
(29).
Compound 29 was prepared from 4-ethylbenzaldehyde
(71 mg, 0.53 mmol), (benzo[1,3]dioxol-5-yl)methylamine (80 mg,

1248
Y. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 1240–1250
4.3.5.8. 1-(4-Aminomethylphenyl)-5-(4-ethylphenyl)-3-hydroxy
-4-(4-methoxybenzoyl)-1,5-dihydro-2H-pyrrol-2-one (33)
Treatment of compound 27 (40 mg, 0.074 mmol) with dry
55.42, 40.98, 34.22, 31.02, 23.60 ppm. Mp: 264–266 °C. HRMS
(ESI): [M+H]+ C32H32N2O4 calcd 509.2435, found 509.2443.
.
hydrochloride dichloromethane solution as described above for
the preparation of 32 provided 33 (30 mg, 84.0% yield). 1H NMR:
(400 MHz, DMSO-d6): d 11.53 (br s, 1H), 8.29 (br s, 3H), 7.73 (d,
2H, J = 8.4 Hz), 7.68 (d, 2H, J = 8.0 Hz), 7.42 (d, 2H, J = 8.0 Hz),
7.28 (d, 2H, J = 8.0 Hz), 7.02 (d, 2H, J = 7.6 Hz), 6.99 (d, 2H,
J = 8.4 Hz), 6.29 (s, 1H), 3.93 (s, 2H), 3.82 (s, 3H), 2.43 (q, 2H,
J = 7.6 Hz) and 1.04 (t, 3H, J = 7.6 Hz) ppm. 13C NMR (100 MHz,
DMSO-d6): d 187.07, 165.50, 162.73, 158.55, 142.90, 136.66,
134.20, 131.25, 130.77, 130.48, 130.22, 129.44, 129.31, 127.53,
122.10, 120.43, 113.34, 60.79, 55.46, 41.64, 27.51, 14.94 ppm.
Mp: 248–250 °C. HRMS (ESI): [M+H]+ C27H27N2O4 calcd 443.1971,
found 443.1985.
4.3.5.12. 5-(4-Butylphenyl)-3-hydroxy-1-(2-(indol-3-yl)ethyl)-
4-(4-methoxybenzoyl)-1,5-dihydro-2H-pyrrol-2-one
(37)
.
Compound 37 was prepared from 4-butylbenzaldehyde
(280 mg, 1.5 mmol), tryptamine (240 mg, 1.5 mmol) and 25a
(375 mg, 1.5 mmol) in 15 mL ethanol. Purification with Method A
and use of ether (20 mLꢃ2) to wash the precipitate twice gave
37 (214 mg, 28.1% yield) as an off white solid. 1H NMR:
(400 MHz, DMSO-d6): d 11.48 (br s, 1H), 10.79 (s, 1H), 7.69 (d,
2H, J = 8.4 Hz), 7.33 (d, 1H, J = 8.0 Hz), 7.27 (d, 1H, J = 6.4 Hz),
7.04–7.12 (m, 6H), 6.90–6.97 (m, 3H), 5.35 (s, 1H), 3.81 (s, 4H),
2.96 (m, 2H), 2.72 (m, 1H), 2.51 (t, 2H, J = 7.6 Hz), 1.50 (m, 2H),
1.25 (m, 2H) and 0.85 (t, 3H, J = 7.2 Hz) ppm. 13C NMR (100 MHz,
DMSO-d6): d 187.35, 165.19, 162.77, 142.33, 136.22, 133.44,
131.19, 130.65, 128.41, 127.65, 126.92, 122.71, 120.96, 119.67,
118.18, 118.05, 113.36, 111.39, 110.88, 60.98, 55.42, 40.91, 34.45,
32.92, 23.70, 21.76, 13.70 ppm. Mp 179–182 °C. HRMS (ESI):
[M+H]+ C32H32N2O4 calcd 509.2435, found 509.2444.
4.3.5.9.
zoyl)-5-(4-trifluoromethylphenyl)-1,5-dihydro-2H-pyrrol-2-one
(34). Compound 34 was prepared from 4-trifluoromethyl-
3-Hydroxy-1-(2-(indol-3-yl)ethyl)-4-(4-methoxyben-
benzaldehyde (50 mg, 0.50 mmol), tryptamine (80 mg, 0.50 mmol)
and 25a (125 mg, 0.50 mmol) in 4 mL ethanol. Purification with
Method C gave 34 (12 mg, 4.6% yield) as a brown solid. 1H NMR
(300 MHz, MeOD): d 7.55 (d, 2H, J = 8.7 Hz), 7.40–7.46 (m, 3H),
7.32 (d, 1H, J = 7.8 Hz), 7.14 (dd, 1H, J1 = J2 = 7.5 Hz), 6.75–7.05
(m, 4H), 6.88 (d, 2H, J = 8.4 Hz), 5.04 (s, 1H), 3.97 (m, 1H), 3.83 (s,
3H) and 2.92–3.11 (m, 3H) ppm. 13C NMR (100 MHz, DMSO-d6):
d 187.14, 165.33, 162.78, 151.84, 147.77, 143.92, 141.47, 136.19,
131.07, 128.80, 128.71, 128.62, 126.84, 125.31, 122.75, 120.94,
118.12, 117.90, 113.32, 111.37, 110.73, 60.59, 55.37, 41.05,
23.60 ppm. Mp: 215–217 °C. HRMS (ESI): [M+H]+ C29H23F3N2O4
calcd 521.1688, found 521.1696.
4.3.5.13. 3-Hydroxy-1-(2-(indol-3-yl)ethyl)-5-(4-isobutylphenyl)
-4-(4-methoxybenzoyl)-1,5-dihydro-2H-pyrrol-2-one
(38)
.
Compound 38 was prepared from 4-isobutylbenzaldehyde
(405 mg, 2.5 mmol), tryptamine (80 mg, 0.5 mmol) and 25a
(125 mg, 0.5 mmol) in the presence of DIPEA (6.5 mg, 0.05 mmol)
in 8 mL toluene. Purification with Method A using ethyl acetate/
hexane (1/3, v/v) as recrystallization solvent gave 38 (33 mg,
12.9% yield) as an off-white solid. 1H NMR (300 MHz, CDCl3) : d
8.09 (br s, 1H), 7.38–7.44 (m, 4H), 7.22 (dd, 1H, J1 = J2 = 7.5 Hz),
7.10 (dd, 1H, J1 = J2 = 7.5 Hz), 7.02 (s, 1H), 6.93 (d, 2H, J = 7.8 Hz),
6.76 (m, 4H), 5.13 (s, 1H), 4.02 (m, 1H), 3.79 (s, 3H), 3.10 (m,
2H), 2.90 (m, 1H), 2.35 (d, 2H, J = 6.9 Hz), 1.73 (m, 1H) 0.80 (d,
3H, J = 6.0 Hz) and 0.78 (d, 3H, J = 6.0 Hz) ppm. 13C NMR
(100 MHz, DMSO-d6): d 187.33, 165.18, 162.72, 141.12, 136.21,
133.56, 131.15, 130.69, 129.07, 127.50, 126.90, 122.71, 120.95,
119.56, 118.17, 118.03, 113.34, 111.38, 110.88, 60.98, 55.41,
44.20, 40.93, 29.46, 23.75, 22.12 ppm. Mp: 191–193 °C. HRMS
(ESI): [M+H]+ C32H32N2O4 calcd 509.2440, found 509.2449.
4.3.5.10. 3-Hydroxy-1-(2-(indol-3-yl)ethyl)-5-(4-isopropylphe-
nyl)-4-(4-methoxybenzoyl)-1,5-dihydro-2H-pyrrol-2-one (35)
.
Compound 35 was prepared from 4-isopropylbenzaldehyde
(74 mg, 0.50 mmol), tryptamine (80 mg, 0.50 mmol) and 25a
(125 mg, 0.50 mmol) in 8 mL toluene. The precipitate was filtered
and washed by toluene (1 mL ꢃ 2) to give 35 (78 mg, 31.6% yield)
as a white solid. 1HNMR (400 MHz, CDCl3) : d 8.06 (br s, 1H), 7.48
(d, 2H, J = 8.8 Hz), 7.40 (d, 2H, J = 8.0 Hz), 7.22 (dd, 1H,
J1 = J2 = 8.0 Hz), 7.10 (dd, 1H, J1 = J2 = 7.2 Hz), 7.04 (s, 1H), 7.03 (d,
2H, J = 7.6 Hz), 6.84 (d, 2H, J = 8.0 Hz), 6.77 (d, 2H, J = 8.8 Hz),
5.16 (s, 1H), 4.02 (m, 1H), 3.81 (s, 3H), 3.11 (m, 2H), 2.90 (m,
1H), 2.79 (m, 1H) and 1.15 (d, 6H, J = 6.0 Hz) ppm. 13C NMR
4.3.5.14. 5-Biphenyl-4-yl-3-hydroxy-1-(2-(indol-3-yl)ethyl)-4-
(4-methoxybenzoyl)-1,5-dihydro-2H-pyrrol-2-one
(39)
.
Compound 39 was prepared from 4-phenylbenzaldehyde
(91 mg, 0.50 mmol), tryptamine (80 mg, 0.50 mmol) and 25a
(125 mg, 0.50 mmol) in 2.5 mL toluene. Purification with Method
A using dichloromethane/MeOH/ether (1/1/8, v/v/v) as recrystalli-
zation solvent gave 39 (32.0 mg, 12.1% yield) as a yellowish solid.
1H NMR (400 MHz, DMSO-d6): d 10.83 (s, 1H), 7.59–7.65 (m, 4H),
7.49 (d, 2H, J = 7.2 Hz), 7.42 (dd, 2H, J1 = J2 = 7.2 Hz), 7.30–7.34
(m, 3H), 7.03–7.14 (m, 4H), 6.92 (1H, dd, J1 = J2 = 7.6 Hz), 6.78 (d,
2H, J = 7.6 Hz), 5.24 (s, 1H), 3.80 (m, 1H), 3.75 (s, 3H), 2.97 (m,
1H), 2.83 (m, 1H) and 2.71 (m, 1H) ppm. 13C NMR (100 MHz,
DMSO-d6): d 186.18, 166.77, 160.27, 142.35, 140.61, 139.98,
138.63, 136.21, 133.92, 130.18, 129.31, 128.80, 128.12, 127.14,
126.59, 126.46, 126.03, 123.66, 123.26, 122.62, 120.90, 118.17,
112.17, 111.35, 111.31, 60.56, 55.05, 40.80, 23.53 ppm. Mp: 258–
260 °C. HRMS (ESI): [M+H]+ C34H28N2O4 calcd 529.2127, found
529.2140.
(100 MHz, DMSO-d6 ): 187.47, 164.90, 162.86, 149.88, 148.31,
d
136.28, 133.28, 131.17, 130.48, 127.68, 126.88, 126.43, 122.67,
120.90, 119.98, 118.12, 117.96, 113.39, 111.33, 110.81, 60.96,
55.39, 40.93, 32.96, 23.63 ppm. Mp: 239–241 °C. HRMS (ESI):
[M+H]+ C31H30N2O4 calcd 495.2284, found 495.2306.
4.3.5.11.
ethyl)-4-(4-methoxybenzoyl)-1,5-dihydro-2H-pyrrol-2-one (36)
Compound 36 was prepared from 4-tert-butylbenzaldehyde
5-(4-tert-Butylphenyl)-3-hydroxy-1-(2-(indol-3-yl)
.
(81 mg, 0.5 mmol), tryptamine (80 mg, 0.5 mmol) and 25a
(125 mg, 0.5 mmol) in 8 mL toluene. Purification with Method A
using ethanol as recrystallization solvent gave 36 (36 mg, 14.2%
yield) as a white solid. 1H NMR (400 MHz, CDCl3): d 8.04 (br s,
1H), 7.48 (d, 2H, J = 8.8 Hz), 7.40 (dd, 2H, J1 = 8.4 Hz, J2 = 2.0 Hz),
7.22 (dd, 1H, J 1 = J2 = 8.0 Hz), 7.19 (d, 2H, J = 8.4 Hz), 7.10 (dd,
4.3.5.15. 3-Hydroxy-1-(2-(indol-3-yl)ethyl)-4-(4-methoxyben-
1H,
J 1 = J2 = 7.6 Hz),, 7.04 (d, 1H, J = 2.0 Hz), 6.85 (d, 2H,
zoyl)-5-naphthalen-2-yl-1,5-dihydro-2H-pyrrol-2-one
(40)
J = 8.4 Hz), 6.77 (d, 2H, J = 8.8 Hz), 5.17 (s, 1H), 4.02 (m, 1H), 3.81
(s, 3H), 3.11 (m, 2H), 2.90 (m, 1H) and 1.22 (s, 9H) ppm. 13C
NMR (100 MHz, DMSO-d6): d 187.45, 164.96, 162.86, 150.61,
136.18, 133.00, 131.22, 130.51, 127.49, 126.88, 125.31, 122.71,
120.93, 119.86, 118.12, 117.98, 113.41, 111.35, 110.80, 60.88,
.
Compound 40 was prepared from 2-naphthaldehyde
(156 mg, 0.5 mmol), tryptamine (40 mg, 0.25 mmol) and 25a
(62.5 mg, 0.25 mmol) in the presence of DIPEA (6.5 mg, 0.05 mmol)
in 8 mL toluene. Purification with Method B using ethyl acetate/
MeOH/AcOH (20/1/0.4, v/v/v) as eluent to gave 40 (15.4 mg,

Y. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 1240–1250
1249
11.9% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6): d
11.65 (br s, 1H), 10.84 (s, 1H), 7.81–7.86 (m, 3H), 7.74 (s, 1H), 7.70
(d, 2H, J = 8.4 Hz), 7.50 (m, 2H), 7.33(d, 1H, J = 8.4 Hz), 7.27 (d, 1H,
J = 8.4 Hz), 7.19 (d, 1H, J = 8.0 Hz), 7.12 (s, 1H), 7.04 (dd, 1H,
J1 = J2 = 7.6 Hz), 6.94 (d, 2H, J = 8.8 Hz), 6.79 (dd, 1H,
J1 = J2 = 7.6 Hz), 5.47 (s, 1H), 3.86 (m, 1H), 3.79 (s, 3H), 2.73–3.05
(m, 2H) and 2.76 (m, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): d
187.45, 172.54, 162.77, 148.52, 142.31, 137.58, 136.20, 132.74,
132.68, 131.12, 129.91, 128.33, 128.14, 127.62, 127.45, 126.84,
126.26, 126.20, 123.98, 122.78, 120.92, 118.14, 118.05, 113.34,
111.37, 110.85, 61.38, 55.38, 40.90, 23.83 ppm. Mp: 233–234 °C.
HRMS (ESI): [M+H]+ C32H26N2O4 calcd 503.1971, found 503.1980.
4.3.5.19.
ethyl)-5-(4-isobutylphenyl)-1,5-dihydro-2H-pyrrol-2-one (44)
Compound 44 was prepared from 4-isobutylbenzaldehyde
4-(2,4-Diflorophenyl)-3-hydroxy-1-(2-(indol-3-yl)
.
(81 mg, 0.50 mmol), tryptamine (80 mg, 0.50 mmol) and 25c
(114 mg, 0.50 mmol) in 15 mL ethanol. Recrystallization gave 44
(57 mg, 22.1 % yield) as an off white solid. 1H NMR: (400 MHz,
CDCl3): 8.15 (dd, 1H, J1 = 8.8 Hz, J2 = 6.0 Hz), 8.06 (br s, 1H), 7.35–
7.39 (m, 3H), 7.13–7.21 (m, 2H), 7.01–7.10 (m, 4H), 6.96 (d, 2H,
J = 8 Hz), 5.17 (s, 1H), 4.19 (m, 1H), 2.95–3.28 (m, 3H), 2.43 (d,
2H, J = 7.2 Hz), 1.83 (m, 1H), 0.88 (d, 6H, J = 6.4 Hz) ppm. 13C
NMR (100 MHz, DMSO-d6): d 172.25, 166.93, 164.42, 161.17,
157.72, 157.58, 155.70, 142.32, 136.73, 131.47, 129.79, 128.57,
128.47, 128.31, 127.83, 127.34, 123.41, 122.56, 121.50, 118.92,
118.73, 118.55, 118.46, 115.27, 115.04, 111.93, 111.16, 106.79,
106.53, 59.88, 44.74, 41.58, 30.00, 24.24, 22.61 ppm. Mp: 172–
174 °C. HRMS (ESI): [M+H]+ C31H28F2N2O3 calcd 515.2146, found
515.2142.
4.3.5.16. 5-(2-Bromo-4-fluorophenyl)-3-hydroxy-1-(2-(indol-3-
yl)ethyl)-4-(4-methoxybenzoyl)-1,5-dihydro-2H-pyrrol-2-one
(41).
benzaldehyde
Compound 41 was prepared from 2-bromo-4-fluoro-
(102 mg, 0.50 mmol), tryptamine (80 mg,
0.50 mmol), and 20a (125 mg, 0.50 mmol) in 8 mL ethanol. Purifica-
tion with Method A using dichloromethane/MeOH (2/1, v/v) as
recrystallization solvent gave 41 (58 mg, 21.1% yield) as an off white
solid. 1H NMR (400 MHz, DMSO-d6): d 10.79 (s, 1H), 7.84 (d, 2H,
J = 7.6 Hz), 7.46 (dd, 1H, J1 = 8.4 Hz, J2 = 2 Hz), 7.41 (d, 1H,
J = 8.0 Hz), 7.31 (d, 1H, J = 8.0 Hz), 7.02–7.13 (m, 4H), 6.95 (dd, 1H,
J1 = J2 = 7.2 Hz), 6.79 (d, 2H, J = 8.8 Hz), 5.73 (s, 1H), 3.76 (s, 3H),
4.3.5.20. 5-(4-Ethylphenyl)-3-hydroxy-1-(2-(indol-3-yl)ethyl)-4-
(naphthalene-1-carbonyl)-1,5-dihydro-2H-pyrrol-2-one
(45)
.
Compound 45 was prepared from 4-ethylbenzaldehyde
(340 mg, 2.53 mmol), tryptamine (80 mg, 0.50 mmol) and 25d
(135 mg, 0.5 mmol) in the presence of DIPEA (6.5 mg, 0.05 mmol)
in 10 mL toluene. Purification with Method A using dichlorometh-
ane/hexane (1/2, v/v) as recrystallization solvent gave 45 (101 mg,
50.5% yield) as an off white solid. 1H NMR (300 MHz, DMSO-d6): d
10.85 (s, 1H), 7.93–8.00 (m, 2H), 7.83 (d, 1H, J = 7.8 Hz), 7.43–7.49
(m, 4H), 7.35 (d, 1H, J = 8.1 Hz), 7.29 (d, 1H, J = 7.8 Hz), 7.05–7.16
(m, 6H), 6.93 (dd, 1H, J1 = J2 = 7.2 Hz), 5.34 (s, 1H), 3.80 (m, 1H),
2.96 (m, 2H), 2.75 (m, 1H), 2.56 (q, 2H, J = 7.2 Hz) and 1.15 (t, 3H,
J = 7.2 Hz) ppm. 13C NMR (100 MHz, DMSO-d6): d 186.81, 165.46,
162.75, 147.71, 144.08, 137.85, 136.75, 134.28, 133.55, 130.88,
129.96, 128.68, 128.33, 128.14, 127.48, 127.26, 126.93, 126.47,
125.35, 125.11, 123.26, 121.48, 118.72, 118.54, 111.92, 111.44,
61.17, 41.52, 28.27, 24.07, 15.80 ppm. Mp: 203–204 °C. HRMS
(ESI): [M+H]+ C33H28N2O3 calcd 501.2178, found 501.2188.
3.70 (m, 1H), 2.98 (m, 1H), 2.79 (m, 1H) and 2.65 (m, 1H) ppm. 13
C
NMR (100 MHz, DMSO-d6): d 170.08, 168.15, 161.74, 160.33,
159.27, 138.89, 137.56, 136.26, 133.98, 130.24, 129.11, 129.03,
127.10, 125.49, 125.39, 122.61, 120.96, 118.68, 118.44, 118.27,
118.10, 115.32, 115.11, 112.23, 111.44, 111.15, 105.22, 101.19,
58.58, 55.14, 41.36, 23.84 ppm. Mp: 317–319 °C. HRMS (ESI):
[M+H]+ C28H22BrFN2O4 calcd 549.0825, found 549.0844.
4.3.5.17. 3-Hydroxy-1-(2-(indol-3-yl)ethyl)-4-(4-methoxyben-
zoyl)-5-pentyl-1,5-dihydro-2H-pyrrol-2-one
(42).
Com-
pound 42 was prepared from hexanal (50 mg, 0.50 mmol),
tryptamine (80 mg, 0.50 mmol) and 25a (125 mg, 0.50 mmol) in
4 mL toluene. Purification with Method C gave 42 (10 mg, 2.23%
yield) as a brown solid. 1H NMR (400 MHz, CDCl3): d 8.13 (br s,
1H), 7.63 (m, 3H), 7.39 (d, 1H, J = 6.4 Hz), 7.22 (dd, 1H,
J1 = J2 = 7.2 Hz), 7.14 (dd, 1H, J1 = J2 = 7.2 Hz), 7.08 (br s, 1H), 6.91
(d, 2H, J = 8.4 Hz), 4.53 (m, 1H), 4.26 (m, 1H), 3.87 (s, 3H), 3.37
(m, 1H), 3.05–3.21 (m, 2H), 1.64 (m, 2H), 0.98–1.12 (m, 4H), 0.85
(m, 2H) and 0.74 (t, 3H, J = 6.8 Hz) ppm. 13C NMR (100 MHz,
DMSO-d6): d 188.29, 165.03, 162.85, 150.14, 136.22, 131.22,
131.07, 130.57, 127.03, 122.92, 121.11, 118.35, 118.16, 113.61,
111.51, 110.98, 56.52, 55.58, 40.56, 30.96, 27.45, 23.74, 21.79,
21.18, 13.73 ppm. Mp: 80–82 °C. HRMS (ESI): [M+H]+ C27H30N2O4
calcd 447.2284, found 447.2297.
4.4. Aminoacylation assay
Experiments were performed in a final 70 lL reaction mixtures
containing 50 mM HEPES-KOH (pH 7.8), 5 mM MgCl2, 45 mM KCl,
1 mM DTT, 0.2 mg/mL BSA, 0.4 mg/mL brewer’s yeast tRNA
(Roche), 0.5 nM TbLeuRS, 4 mM ATP, and 0.003 mCi/mL [14C]-Leu-
cine. The test compounds were dissolved in 7
and added to the above mixture. The final compound concentra-
tion gradient was 1 mM, 320 M, 100 M, 32 M, 10 M, 3.2 M,
M, 0.32 M, 0.1 M. Reactions were initiated by the addition
of ATP. At specific time intervals, three 20 L aliquots were spotted
lL DMSO (Genview)
l
l
l
l
l
1
l
l
l
l
on 3 mm filter paper (Whatman), washed three times in 5% trichlo-
roacetic acid and three times in ethanol. Filter paper was dried un-
der a heat lamp and the precipitated [14C]-Leucine tRNALeu were
quantified using a Beckman Coulter LS 6500 liquid scintillation
counter. The inhibitory effect for each compound was determined
by incubating TbLeuRS with the compound, tRNA and [14C]-Leucine
for 20 min at 37 °C prior to the addition of ATP to start the amino-
acylation reaction. Triplicates were averaged to generate an IC50
value using GraphPad.
4.3.5.18.
ethyl)-5-(4-isobutylphenyl)-1,5-dihydro-2H-pyrrol-2-one (43)
Compound 43 was prepared from 4-isobutylbenzaldehyde
4-(4-Chlorobenzoyl)-3-hydroxy-1-(2-(indol-3-yl)
.
(97 mg, 0.6 mmol), tryptamine (80 mg, 0.50 mmol) and 25b
(128 mg, 0.50 mmol) in 15 mL ethanol. Purification with Method
B using dichloromethane/MeOH (10/1, v/v) as eluent gave 43
(14 mg, 5.4% yield) as a brown solid. 1HNMR (400 MHz, MeOD): d
7.33–7.43 (m, 6H), 6.98–7.12 (m, 5H), 6.82 (m, 1H), 4.98 (s, 1H),
3.90 (m, 1H), 3.08 (m, 2H), 2.87 (m, 1H), 2.38 (d, 2H, J = 6.0 Hz),
1.76 (m, 1H) and 0.82 (t, 6H, J = 5.2 Hz) ppm. Mp: 215–217 °C.
13C NMR (100 MHz, DMSO-d6): d 175.81, 169.60, 159.69, 140.31,
139.24, 136.24, 134.44, 129.51, 128.53, 127.40, 127.33, 126.91,
123.05, 122.77, 120.97, 118.24, 117.97, 115.59, 111.46, 110.89,
61.47, 44.10, 41.29, 31.48, 29.47, 23.57, 21.99 ppm. HRMS (ESI):
[M+H]+ C31H29N2O3 calcd 513.1945, found 513.1962.
Acknowledgments
We thank National Science Foundation of China (20702031),
Ministry of Science and Technology of China (2009CB918404), E-
Institutes of Shanghai Universities (EISU) Chemical Biology Divi-
sion, and National Comprehensive Technology Platforms for Inno-
vative Drug R&D (2009ZX09301-007) for financial support of this
work.

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Y. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 1240–1250
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.12.035.
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