Diketo acids and their amino acid/dipeptidic analogues as promising scaffolds for the development of bacterial methionine aminopeptidase inhibitors

Article abstract of DOI:10.1039/c5ra03354c

Using diketoesters as the template, various derivatives were designed and the selected compounds were synthesized as bacterial methionine aminopeptidase (MetAP) inhibitors. The results of in vitro antibacterial screening revealed fifteen compounds (1a-c,

Full text of DOI:10.1039/c5ra03354c

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PAPER
Diketo acids and their amino acid/dipeptidic
analogues as promising scaffolds for the
development of bacterial methionine
aminopeptidase inhibitors
Cite this: RSC Adv., 2015, 5, 34173
Mir Mohammad Masood,a Vijay K. Pillalamarri,b Mohammad Irfan,a Babita Aneja,a
Mohamad Aman Jairajpuri,c Md. Zafaryab,d M. Moshahid A. Rizvi,d Umesh Yadava,e
b
a
*
Anthony Addlagatta and Mohammad Abid
Using diketoesters as the template, various derivatives were designed and the selected compounds were
synthesized as bacterial methionine aminopeptidase (MetAP) inhibitors. The results of in vitro
antibacterial screening revealed fifteen compounds (1a–c, 1e–h, 1j, 1l, 2a–c, 3d, 5c and 5e) as potent
against different bacterial strains. By using the MTT assay on human cell line (HepG2), the viability of cell
proliferation was evaluated and nine compounds (1c, 1e, 1j, 1l, 2a,b, 3d, 5c and 5e) showed no cytotoxic
effect at the concentration range of 50–450 mg mlꢀ1. In the biochemical evaluation against methionine
aminopeptidase (MetAPs) from Streptococcus pneumonia (SpMetAP), Mycobacterium tuberculosis
(MtMetAP), Enterococcus faecalis (EfMetAP) and human (HsMetAP), compounds displayed differential
behaviour against these four enzymes. Moreover, compound 1g showed 84% inhibition of SpMetAP,
while compound 3d selectively inhibited MtMetAP with 79% inhibition and little effect on HsMetAP at
100 mM concentration. At the same concentration, compound 5e exhibited 87% and 85% inhibition of
EfMetAP and SpMetAP, respectively. Understanding the mode of binding through modeling at the active
site provided the structural basis for the possible mode of inhibition. Together, these data will be useful
for further development of diketo acid based inhibitors with improved potency and selectivity.
Received 23rd February 2015
Accepted 2nd April 2015
DOI: 10.1039/c5ra03354c
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cleavage of N-terminal methionine by MetAP is an important
event during protein synthesis and maturation.5,6 Since, the
1. Introduction
N-terminal methionine excision process is crucial for the
metabolism and cell survival, MetAP is an ideal drug target for
designing new inhibitors against bacterial pathogens.
Bacterial infections are responsible for some of the most deadly
diseases and widespread epidemics in the world.1–3 Due to the
rise in resistance of bacteria to current antibacterial chemo-
therapeutics, it is necessary to develop novel approaches and
new inhibitors against resistant bacterial pathogens.1,3 To
overcome this problem, new enzyme targets must be identied
which can be targeted with small molecules. MetAPs represent
one such important enzyme family, which is essential for
bacteria.4 MetAPs cleave the initiator methionine from about
70% of all ribosome assisted newly synthesized proteins. The
MetAPs are rst-row transition metallo-enzymes with ve
conserved metal ion-binding residues in the active site. There-
fore, we have designed organic scaffolds that can serve as the
metal chelators. Compounds with b-diketo pharmacophoric
motif are believed to function as chelators of dinuclear Mn(II) or
Mg(II) active site of HIV-1 integrase enzyme.7 Very recently, a
diketo acid coupled with L-alanine methyl ester has been
reported as EcMetAP inhibitor.8 Considering the large phar-
macological importance of b-diketo acids and in continuation
to our efforts to explore novel biologically active molecules,9,10
in-house database of 201 compounds virtual library was
screened against Escherichia coli MetAP (EcMetAP). Based on
this preliminary data, diketoesters (1a–m), diketo acids (2a–f,
2h, 2m) were selected and synthesized in good yields. Selected
diketo acids were also coupled with methyl ester of L-Ala, L-Phe
and a dipeptide to get their novel amino acid/dipeptidic
analogues (5a–e). All the synthetic compounds were well char-
acterized using various spectroscopic techniques. Antibacterial
aMedicinal Chemistry Lab, Department of Biosciences, Jamia Millia Islamia (A Central
University), Jamia Nagar, New Delhi 110025, India
bCentre for Chemical Biology, Indian Institute of Chemical Technology, Tarnaka,
Hyderabad 500607, India
cProtein Conformation and Enzymology Lab, Department of Biosciences, Jamia Millia
Islamia (A Central University), Jamia Nagar, New Delhi 110025, India
dGenome Biology Lab, Department of Biosciences, Jamia Millia Islamia (A Central
University), Jamia Nagar, New Delhi 110025, India
eDepartment of Biochemistry 409 Ullman Building, Albert Einstein College of Medicine
of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY, USA. E-mail: mabid@jmi.
ac.in; Fax: +91-11-2698-0229; Tel: +91-87-5029-5095
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activity was carried out against gram positive Staphyloccocus All the acids obtained showed a single spot on TLC, therefore
aureus (S. aureus) and gram negative Escherichia coli (E. coli), used without any further purication. Purity of the acids was
Klebsiella pneumoniae (K. pneumoniae) and Salmonella typhimu- also checked by elemental analyses and the structures were
1
rium (S. typhimurium). MIC and MBC values were calculated. conrmed by FT-IR, H, 13C-NMR and mass analyses. For the
The cytotoxicity by MTT assay on active compounds was also synthesis of dipeptides (3a–e), desired boc-protected amino
performed on
a human cell line (HepG2). Most active acid was coupled with methyl ester of L-Phe in acetonitrile at
compounds against bacteria were screened against SpMetAP, room temperature using EDC$HCl as coupling reagent with
MtMetAP, EfMetAP and HsMetAP.
HOBt and N-methyl morpholine (NMM) as a base (Scheme 2).15
The crude product obtained was puried by column chro-
matography using ethyl acetate (30–40%) in petroleum ether to
give pure dipeptides (3a–e) in quantitative yields. Boc group of a
dipeptide (3e) was cleaved using TFA : DCM mixture (1 : 1). The
reaction mixture was stirred for 2 h at room temperature to give
the desired dipeptide Trp-Phe-OCH3 (4e) in quantitative yield
which was used without further purication.16 As shown in
Scheme 3, the coupling of diketo acids (2a, 2e, 2h, 2m) with
methyl ester of L-Ala/L-Phe was done in anhyd. DMF using
PyBOP and HOBt in presence of triethyl amine to give novel
amino acid conjugated to diketo acids (5a–d) in moderate to
good yields.17 Compound 2a was coupled with Trp-Phe-OCH3
(4e) using the same methodology to give the dipeptidic
analogue of diketo acid (5e) in excellent yield (Scheme 4).
Spectral analysis. The structure of b-diketoesters (1a–m) was
in accordance to their spectroscopic and analytical data. In the
IR spectrum, absorption bands in the region 1520–1605 cmꢀ1
suggested the presence of b-diketoesters in enolic form and the
peaks in the region 1721–1739 cmꢀ1 corresponding to a,
b-unsaturated ester also indicated the formation of b-dike-
toesters. In the 1H-NMR spectrum, the proton adjacent to enolic
hydroxyl group showed a sharp singlet in the range d 6.48–
7.57 ppm which conrmed that the b-diketoester is present in
enolic form. The 13C-NMR spectral data were in good agreement
with the assigned structures. The carbons of b-diketone group
(in enolic form) exhibited chemical shi values in the range d
189.22–200.80 ppm and d 180.38–193.26 ppm. The mass spectra
of all the compounds exhibited molecular ion peaks and
contain fragments which further conrmed the formation of
desired compounds. In case of diketo acids (2a–f, 2h, 2m),
peaks for the enolic form occur at their corresponding positions
as in the case of b-diketoesters. But the disappearance of peaks
for a, b-unsaturated ester group and the appearance of peaks in
the region 3410–3494 cmꢀ1 (broad) and 1684–1709 cmꢀ1 cor-
responding to acid functionality and a, b-unsaturated acid
indicate the conversion of diketoesters into their corresponding
acids. All the compounds displayed either [M + H]+ or [M ꢀ H]ꢀ
corresponding to their molecular formulae. The coupling
between diketo acid with amino acid (L-Ala-OCH3/L-Phe-OCH3)
or dipeptide (Trp-Phe-OCH3) can be monitored based on the
disappearance of peaks corresponding to free amino and
carboxylic groups. Two distinct bands were also observed in the
region 1650–1687 cmꢀ1 and 1510–1570 cmꢀ1 related to –CONH
for C]O stretching and N–H bending vibrations, respectively.
2. Results and discussion
2.1. Screening using molecular docking
We modelled 201 diketo esters, acids and their amino acid/
peptidic analogues and did a virtual screening using docking
against EcMetAP. Not all ligands showed binding and a great
variation in the binding affinities was observed using Auto dock
Vina11 and Gilde docking.12 Appreciable binding affinity in kcal
molꢀ1 was observed for 14 compounds (1a (ꢀ6.6), 1b (ꢀ6.1), 1c
(ꢀ6.5), 1e (ꢀ7.0), 1f (ꢀ7.0), 1g (ꢀ6.2), 1h (ꢀ6.4), 1j (ꢀ7.0), 2a
(ꢀ7.1), 2b (ꢀ8.1), 2c (ꢀ7.0), 3d (ꢀ6.4), 5c (ꢀ6.7) and 5e (ꢀ8.6)).
The main active site residues which take part in H-bonding
interactions and ionic interactions are Glu204, His171,
His178, His79, Asp97, and Asp108 while several residues were
involved in hydrophobic interactions. Compounds showing
good binding affinity and interactions were further tested for
antibacterial potential and their ability to inhibit various
MetAPs.
2.2. Chemistry
The synthesis of diketoesters (1a–m) and diketo acids (2a–f, 2h,
2m) was accomplished as outlined in Scheme 1. The oxalylation
of variously substituted aryl, heteroaryl or alicyclic methyl
ketones by diethyl oxalate in the presence of freshly prepared
sodium ethoxide afforded b-diketoesters (1a–m).13 These
compounds were puried by column chromatography (petro-
leum ether : ethyl acetate: 9 : 1) to give desired compounds in
moderate to high yields as conrmed by spectroscopic analysis
(Table 1).
Treatment of the selected b-diketoesters with freshly
prepared lithium hydroxide solution (2 M) in THF–H2O mixture
(1 : 4) for 2 h at room temperature yielded corresponding
b-diketo acids (2a–f, 2h, 2m) in quantitative yields (Table 2).14
1
In the H-NMR spectra, broad singlet for the carboxylic group
disappeared and peaks for the amino group was also absent
while the aromatic protons as well as protons belonging to
alicyclic system appeared at their usual chemical shi and
integral values. The 13C-NMR spectra clearly showed coupling
Scheme 1 Reagents and conditions: (a) Na metal, C2H5OH, 0 ꢁC-r.t.,
3–4 h; (b) LiOH (2 M), THF : H2O, r.t., 2 h.
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Table 1 Structure of various diketoesters (1a–m)
Compound
R
Chemical structure
Mol. formula
C12H11ClO4
Ref.
18
1a
1b
C12H11ClO4
19
1c
1d
1e
C12H11BrO4
18
—
19
C9H12O4
C13H14O4
1f
1g
1h
1i
C12H12O4
19
19
—
19
—
C10H10O4S
C13H14O5
C11H11NO4
1j
C12H10Cl2O4
1k
1l
C10H10O5
19
C10H11NO4
—
1m
C14H14O6
—
between diketoacids and amino acids or dipeptide as peaks
corresponding to amide bond appeared at the expected chem-
ical shi values d 169.23–160.85 ppm. The mass spectra of some
of the coupled compounds showed either [M + H]+ and [M ꢀ H]ꢀ
or [M ꢀ COOCH3]+ and [M ꢀ COOCH3]ꢀ which further provided
evidence for the formation of desired compounds.
2.3. Antibacterial activity
Agar well diffusion assay was carried out to access the suscep-
tibility of one gram positive (S. aureus MTCC737) and three
gram negative (E. coli MTCC739, K. pneumoniae MTCC109, and
S. typhimurium MTCC98) bacterial strains. Twenty two
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Table 2 Structure of various diketo acids (2a–f, 2h, 2m)
Compound
R
Structure
Mol. formula
C10H7ClO4
Ref.
2a
—
2b
C10H7ClO4
—
2c
2d
2e
C10H7BrO4
C7H8O4
—
—
—
C11H10O4
2f
C10H8O4
20
20
2h
C11H10O5
2m
C12H10O6
—
Scheme
2 Reagents and conditions: (a) EDC$HCl, HOBt, NMM,
CH3CN, r.t., 2 h; (b) DCM, TFA, r.t., 2 h.
Scheme 3 Reagents and conditions (a) HOBt, DMF, Et3N, PyBOP, r.t.,
24 h.
compounds that displayed signicant zone of inhibition at
0.5 mg mlꢀ1 concentration were further evaluated to calculate
their MIC and MBC values against respective strains by micro
dilution method. Ciprooxacin was used as the positive control
and the results are summarized in Table 3. Most of the
synthesized compounds showed good antibacterial effect
against E. coli and S. aureus. Among all the tested compounds,
1c showed most potent antibacterial effect against all the
strains with MIC value in the range 9.9–39.6 mg mlꢀ1
.
Compounds 1b, 1l, 2a, and 5c selectively inhibited E. coli with
MIC value of 9.9 mg mlꢀ1. Moreover 1a–c, 1f,g, 1j, and 1l also
showed good activity against S. aureus with MIC value of
19.8 mg mlꢀ1. The 5e was the only compound with MIC value of
19.8 mg mlꢀ1 against S. typhimurium. The MBC values of potent
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inhibited by compound 5e (Ki ¼ 21.3) and MtMetAP was
inhibited by compound 3d (Ki ¼ 56.9).
In vitro cytotoxicity prole. To examine the toxicity of most
active compounds based on MIC and MBC on cell proliferation,
the cytotoxicity was checked by MTT assay on HepG2 cell line
and the results are shown in Table 3. Nine compounds (1c, 1e,
1j, 1l, 2a,b, 3d, 5c and 5e) were found to be non cytotoxic at
concentration range 50–450 mg mlꢀ1. Compound 1g, 1h and 2c
showed cytotoxicity in the range of 195–250 mg mlꢀ1. The effect
of compounds on % viability of cell proliferation versus
concentration shown in Fig. 1, indicates toxicity at a very high
Table 4 Inhibition of different MetAPs (in % age) at 100 mg mlꢀ1
concentration
Comp.
SpMetAP
MtMetAP
HsMetAP
EfMetAP
Scheme 4 Reagents and conditions (a) HOBt, DMF, Et3N, PyBOP, r.t.,
24 h.
1a
1b
1c
1e
1f
1g
1h
1j
2a
2b
2c
3d
5c
5e
52 ꢂ 0.32
63 ꢂ 2.52
29 ꢂ 3.00
25 ꢂ 0.79
37 ꢂ 0.91
84 ꢂ 2.71
68 ꢂ 2.24
53 ꢂ 1.59
35 ꢂ 0.45
30 ꢂ 1.23
49 ꢂ 0.82
19 ꢂ 0.41
41 ꢂ 1.52
85 ꢂ 1.7
33 ꢂ 0.3
ꢀ29 ꢂ 2.21
ꢀ12 ꢂ 3.76
26 ꢂ 3.39
47 ꢂ 2.39
22 ꢂ 0.87
29 ꢂ 2.54
20 ꢂ 2.09
ꢀ3 ꢂ 3.28
ꢀ2 ꢂ 1.06
4 ꢂ 2.22
54 ꢂ 3.58
59 ꢂ 3.71
19 ꢂ 5.54
24 ꢂ 2.29
40 ꢂ 3.20
60 ꢂ 3.01
50 ꢂ 4.15
38 ꢂ 2.76
38 ꢂ 2.83
38 ꢂ 1.22
40 ꢂ 4.41
9 ꢂ 2.85
34 ꢂ 1.23
26 ꢂ 26.22
20 ꢂ 0.75
27 ꢂ 2.97
32 ꢂ 2.93
57 ꢂ 1.60
27 ꢂ 2.80
30 ꢂ 2.22
40 ꢂ 0.40
22 ꢂ 2.3
compounds were also calculated which showed their bacteri-
cidal nature.
Methionine aminopeptidase inhibition studies. Compounds
with good antibacterial properties were screened for inhibition
against four selected methionine aminopeptidases (MetAP's);
HsMetAP, SpMetAP, EfMetAP, and MtMetAP. IC50 and Ki values
were determined for the compounds showing greater than 70%
inhibition of MetAP at 100 mM concentration (Tables 4 and 5).
The results showed that the enzyme SpMetAP was inhibited by
compound 5e (Ki ¼ 35.5) and 1g (Ki ¼ 33.9), EfMetAP was
31 ꢂ 2.04
32 ꢂ 3.73
55 ꢂ 2.63
87 ꢂ 1.11
79 ꢂ 1.41
49 ꢂ 5.32
40 ꢂ 4.53
39 ꢂ 1.71
43 ꢂ 2.55
Table 3 In vitro antibacterial activity (MIC and MBC) and cytotoxicity of synthesized compounds (in mg mlꢀ1
)
Cytotoxicity
E. coli
S. aureus
K. pneumonia
S. typhimurium
Compound
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
IC50 ꢂ S.D.
1a
1b
1c
1d
1e
1f
1g
1h
1j
>312.5
9.9
19.8
>312.5
>312.5
>312.5
39.6
78.1
>312.5
9.9
>312.5
9.9
39.6
19.8
>312.5
39.6
>312.5
>312.5
>312.5
9.9
312.5
>312.5
<1
n.d.
9.9
19.8
19.8
9.9
>312.5
39.6
19.8
19.8
39.6
19.8
19.8
>312.5
39.6
78.1
78.1
78.1
78.1
19.8
n.d.
78.1
78.1
39.6
>156.2
78.1
19.8
156.2
>312.5
19.8
>156.2
>156.2
>312.5
39.6
>312.5
n.d.
78.1
n.d.
n.d.
n.d.
>312.5
n.d.
>312.5
>312.5
n.d.
312.5
n.d.
n.d.
n.d.
n.d.
n.d.
156.2
n.d.
n.d.
n.d.
312.5
<1
>312.5
>156.2
39.6
>312.5
>156.2
>312.5
78.1
39.6
>312.5
39.6
n.d.
n.d.
156.2
n.d.
>312.5
n.d.
>312.5
>156.2
n.d.
156.2
n.d.
156.2
n.d.
n.d.
312.5
>312.5
n.d.
39.6
n.d.
n.d.
n.d.
19.8
<1
300 ꢂ 0.02
300 ꢂ 0.01
>450 ꢂ 0.0
n.d.
>450 ꢂ 0.0
350 ꢂ 0.03
250 ꢂ 0.03
200 ꢂ 0.01
>450 ꢂ 0.0
>450 ꢂ 0.0
n.d.
>450 ꢂ 0.0
>450 ꢂ 0.0
195 ꢂ 0.02
n.d.
n.d.
n.d.
39.6
n.d.
n.d.
n.d.
156.2
>312.5
n.d.
19.8
n.d.
19.8
312.5
78.1
n.d.
312.5
n.d.
n.d.
n.d.
19.8
n.d.
n.d.
<1
>312.5
78.1
1l
>78.1
312.5
156.2
312.5
312.5
>312.5
>312.5
>312.5
78.1
>312.5
>312.5
>312.5
39.6
1m
2a
2b
2c
2e
3b
3c
3d
5a
5c
5d
5e
Cip
n.d.
39.6
312.5
39.6
>312.5
>312.5
312.5
>312.5
n.d.
n.d.
>312.5
n.d.
>312.5
>312.5
156.2
156.2
>312.5
39.6
>312.5
>312.5
>312.5
19.8
78.1
78.1
>312.5
>312.5
78.1
>312.5
>312.5
>312.5
<1
>450 ꢂ 0.0
n.d.
>450 ꢂ 0.0
n.d.
n.d.
n.d.
<1
>450 ꢂ 0.0
<1
<1
n.d.
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Table 5 IC50 and Ki values of lead compounds against bacterial
MetAPs
inhibition with SpMetAP, however its inhibitory activity against
HsMetAP and EfMetAP were comparable, consequent to this it
also showed similar binding affinities. Compound 5e was found
with best binding affinity with all MetAPs but it showed
signicant increase in % inhibition with EfMetAP as compared
to HsMetAP. It also showed a reduced IC50 value and a very low
Ki. Signicantly 5e binds to three different sites on EfMetAP,
MtMetAP and HsMetAP. Compound 3d interacts with the active
site residues (Ser109 and Val111) of MtMetAP and inhibits it
with low IC50 (Table 6).
Compound
Protein
IC50
Ki
1g
3d
5e
5e
SpMetAP
MtMetAP
SpMetAP
EfMetAP
28.7 ꢂ 3.97
28.5 ꢂ 1.69
27.3 ꢂ 3.83
21.3 ꢂ 3.00
33.9 ꢂ 2.44
56.9 ꢂ 3.2
35.5 ꢂ 4.76
25.9 ꢂ 3.31
However in HsMetAP, its binds away from the active site and
there is signicant reduction the % inhibition. Based on the
binding affinity, MIC values, % inhibition, Ki and IC50 values, 3d
and 5e are signicant lead and can be explored with much more
specic synthetic designs around these two compounds.
3. Experimental protocols
3.1. Screening of ligands by molecular docking studies
AutoDock Vina was used for screening of 201 designed
compounds against E. Coli MetAP enzyme. The three dimen-
sional coordinate (2MAT) of EcMetAP was retrieved from PDB
library. AutoDock Vina 4.2 is advanced version which is much
faster and accurate in binding mode prediction if compared with
its older version AutoDock 4.0.10 Default parameters of AutoDock
Vina were used with slight modication. The affinity grid box was
Fig. 1 Cell viability assay on HepG2 cell line.
concentration range to MIC and MBC values of the lead
compounds in antibacterial assay. These results encouraged us
to further examine enzymatic and in vivo studies to give a better
lead in the antibacterial potential.
˚
centred to the whole protein and the grid spacing xed to 1 A. The
Interactions of lead molecules with MetAPs. Comprehensive
inhibitory studies conducted on MetAPs with identied leads
(1g, 3d and 5e) showed appreciable inhibition on puried
proteins. We did a comprehensive docking of these leads with
MtMetAP, EfMetAP and compared it with HsMetAP. The results
shown in Fig. 2 indicated high binding affinity and tight
binding. Compound 1g showed signicant increase in the %
program automatically calculates the grid map and grid centre
coordinates (X ¼ 44, Y ¼ 46, Z ¼ 44). Aer generating receptor
and all ligands' PDB in PyMOL AutoDock/Vina plugin, Vina was
ran to obtained docking score (binding affinity in kcal molꢀ1
)
and RMSD value for all compounds. PyMOL was used for visu-
alization of the polar contacts in between ligands and binding
site of protein.21 The best docking result can be considered to be
the conformation with the lowest docking score and lowest
RMSD. The screening of ligands was also performed by glide
docking using GLIDE 5.8 soware with standard procedure.22,23
3.2. Materials and methods
All the chemicals purchased from Sigma-Aldrich, Spectrochem
and Hi Media were used without further purication. Precoated
Aluminium sheets (Silica gel 60 F254, Merck Germany) were used
for thin-layer chromatography (TLC) and spots were visualized
under UV light. The IR spectra of compounds were taken on
1
Agilent Cary 630 FT-IR spectrometer. H and 13C NMR spectra
were obtained at ambient temperature using Bruker Spectrospin
DPX-300 MHz, 400 MHz, Agilent 500 MHz FT-NMR in CDCl3
using tetramethylsilane (TMS) as an internal standard. Splitting
patterns are designated as follows: s, singlet; d, doublet; t,
triplet; m, multiplet. Chemical shi values are given in parts per
million (ppm) and coupling constants (J) in Hertz (Hz). Mass
spectra were recorded on a Q Star XL hybrid electron spray
ionization high resolution mass spectrometer (Applied bio-
systems) in a scan range of 100 to 1000 atomic mass units (amu).
Melting points were recorded on a digital Buchi melting point
apparatus (M-560) and were reported uncorrected. Purication
Fig. 2 Interactions of 1g, 3d and 5e with different MetAPs.
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Table 6 Docking score and interaction of lead with MtMetAP (1YJ3), HsMetAP (2G6P), and EfMetAP (3TB5)
Ligand
Protein
1YJ3
1g
3d
5e
ꢀ5.4 kcalꢀ1
His212
ꢀ6.7 kcalꢀ1
ꢀ7.5 kcalꢀ1
Thr203
Ala58, Arg164
ꢀ6.9 kcalꢀ1
2 G6P
3TB5
ꢀ5.6 kcalꢀ1
Tyr196
ꢀ8.9 kcalꢀ1
His310
Glu128, Thr311, Ala312, Asn314
ꢀ7.6 kcalꢀ1
ꢀ5.3 kcalꢀ1
His108
ꢀ5.8 kcalꢀ1
Arg28, Ser109, Val111, Tyr231
Glu251
of the compounds was done by column chromatography using H 5.64, found: C 62.40 H 5.66. IR (neat): 2948, 2844, 2386, 2125,
silica gel (230–400 mesh size) with cyclohexane/ethyl acetate as 1898, 1676, 1596, 1512, 1462, 1423, 1361, 1289, 1240, 1175,
1
eluent. The optical rotation of compounds 5a–e was recorded on 1140, 1119, 1020, 851, 830, 778, 696 cmꢀ1. H-NMR (300 MHz,
Anton Paar MCP-200 polarimeter at 20 ꢁC using sodium D light. CDCl3) (d, ppm): 7.99–7.90 (m, 2H, Ar-H), 7.18–7.08 (m, 2H, Ar-
H), 6.91 (s, 1H, CH), 4.42 (q, 2H, J ¼ 6.9 Hz, CH2), 3.89 (s, 3H,
OCH3), 1.42 (t, 3H, J ¼ 7.2 Hz, CH3). 13C-NMR (75 MHz, CDCl3)
3.3. General procedure for the synthesis of diketo esters (1a–m)
(d, ppm): 196.94, 190.34, 168.06, 162.47, 130.61, 127.66, 114.21,
97.73, 62.50, 55.45, 14.09. LC-MS: m/z [M + H]+ 251.2.
In a two neck round bottom ask, sodium metal (21 mmol) was
dissolved in anhydrous ethanol at 0 ꢁC to give sodium ethoxide
(Z)-Ethyl 2-hydroxy-4-oxo-4-(pyridin-2-yl)but-2-enoate (1i).
solution. To this freshly prepared solution, a mixture of diethyl
oxalate (20 mmol) and ketone (21 mmol) was added slowly with
the help of dropping funnel over a period of 20 minutes. Thick
precipitate was formed and reaction mixture was stirred for 3–
4 h at room temperature. Aer completion of the reaction
checked by TLC, the precipitate obtained was dissolved in 2 N
sulphuric acid (72 ml) and the compound was extracted with
diethyl ether, dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The characterization of compounds 1a,
1b, 1c, 1e, 1f, 1g, 1i, and 1k is previously reported.18,19
Yield: 63%.
(Z)-Ethyl 4-(2,4-dichlorophenyl)-2-hydroxy-4-oxobut-2-enoate
(1j). Brown solid, M.pt.: 60 ꢁC, yield: 70%, Rf (cyclo-
hexane : ethyl acetate ¼ 7 : 3): 0.86, anal (C12H11ClO4) calc. C
49.85 H 3.49, found: C 49.86 H 3.46. IR (neat): 3101, 2983, 1737,
1628, 1583, 1475, 1456, 1391, 1385, 1253, 1225, 1106, 1089,
1039, 1020, 883, 819, 778, 687 cmꢀ1. 1H-NMR (400 MHz, CDCl3)
(d, ppm): 14.94 (s, 1H, OH) 7.92 (d, 1H, J ¼ 8.4 Hz, Ar-H), 7.76
(s, 1H, Ar-H), 7.68 (d, 1H, J ¼ 8.4 Hz, Ar-H), 7.57 (s, 1H, CH), 4.71
(q, 2H, J ¼ 7.2 Hz, CH2), 1.72 (t, 3H, J ¼ 7.2 Hz, CH3). 13C-NMR
(75 MHz, CDCl3) (d, ppm): 194.78, 189.22, 161.88, 140.14,
136.76, 135.45, 133.12, 129.09, 128.73, 98.67, 63.57, 13.93.
LC-MS: m/z [M ꢀ H]ꢀ 288.9.
(Z)-Ethyl
4-(4-chlorophenyl)-2-hydroxy-4-oxobut-2-enoate
(1a). Yield: 72%.
(Z)-Ethyl 4-(2-cholorophenyl)-2-hydroxy-4-oxobut-2-enoate
(1b). Yield: 69%.
(Z)-Ethyl 4-(furan-2-yl)-2-hydroxy-4-oxobut-2-enoate (1k).
Yield: 72%.
(Z)-Ethyl
4-(4-bromophenyl)-2-hydroxy-4-oxobut-2-enoate
(1c). Yield: 54%.
(Z)-Ethyl 2-hydroxy-4-oxo-4-(1H-pyrrol-2-yl)but-2-enoate (1l).
Black solid, M.pt.: 62 ꢁC, yield: 68%, Rf (petroleum ether : ethyl
acetate ¼ 7 : 3): 0.60, anal (C10H11NO5) calc. C 57.41 H 5.30 N
6.70 found: C 57.42 H 5.32 N 6.71. IR (neat): 3265, 3116, 1721,
1633, 1547, 1510, 1428, 1398, 1324, 1264, 1130, 1078, 1045,
1022, 974, 929, 843, 767, 750 cmꢀ1. 1H-NMR (400 MHz, CDCl3)
(d, ppm): 7.03 (d, 1H, J ¼ 6 Hz, Ar-H), 6.91 (d, 1H, J ¼ 6.3 Hz, Ar-
H), 6.63 (s, 1H, CH), 6.29 (t, 1H, J ¼ 7.2 Hz, Ar-H), 4.25 (q, 2H, J ¼
7.23 Hz, CH2), 1.54 (t, 3H, J ¼ 7.23 Hz, CH3). 13C-NMR (75 MHz,
CDCl3) (d, ppm): 200.80, 193.26, 161.67, 131.3, 128.9, 125.09,
113.74, 98.67, 61.87, 14.96. LC-MS: m/z [M + 2H]+ 211.3.
(Z)-Ethyl 4-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-hydroxy-4-
oxobut-2-enoate (1m). Light yellow oil, yield: 54%, Rf (cyclo-
hexane : ethyl acetate ¼ 7 : 3): 0.40, anal (C14H14O6) calc. C
60.43 H 5.07, found: C 60.48 H 5.10. IR: 2958, 2920, 2853, 1736,
1605, 1577, 1508, 1460, 1369, 1330, 1289, 1253, 1184, 1127,
1024, 912, 890, 819, 780, 726 cmꢀ1.1H-NMR (300 MHz, CDCl3)
(d, ppm): 7.49 (s, 1H, CH), 7.21 (s, 1H, Ar-H), 6.91 (d, 2H, J ¼ 9
Hz, Ar-H), 4.93 (q, 2H, J ¼ 15.3 Hz, CH2), 4.21 (d, 4H, J ¼ 17.7 Hz,
alicyclic ring), 1.22 (t, 3H, CH3). 13C-NMR (75 MHz, CDCl3)
(Z)-Ethyl 4-cyclopropyl-2-hydroxy-4-oxobut-2-enoate (1d).
Colourless oil, yield: 52%, Rf (cyclohexane : ethyl acetate ¼
7 : 3): 0.47, anal (C9H12O4) calc. C 58.69 H 6.57, found: C 58.72 H
6.54. IR (neat): 2948, 2872, 1719, 1678, 1655, 1579, 1508, 1430,
1350, 1322, 1290, 1207, 1130, 1065, 1013, 901, 879, 843, 767, 707
1
cmꢀ1. H-NMR (400 MHz, CDCl3) (d, ppm): 14.62 (s, 1H, OH)
6.48 (s, 1H, CH), 4.34 (q, 2H, J ¼ 2.4 Hz, CH2), 1.92–1.86 (m, 1H,
cyclopropyl ring), 1.38 (t, 3H, J ¼ 5.2 Hz, CH3), 1.24 (q, 2H, J ¼
4.4 Hz, cyclopropyl ring), 1.07 (q, 2H, J ¼ 4.4 Hz, cyclopropyl
ring). 13C-NMR (75 MHz, CDCl3) (d, ppm): 198.89, 185.24,
161.98, 99.76, 62.84, 14.03, 10.04. LC-MS: m/z [M + H]+ 185.34.
(Z)-Ethyl 2-hydroxy-4-oxo-4-o-tolylbut-2-enoate (1e). Yield:
78%.
(Z)-Ethyl 2-hydroxy-4-oxo-4-phenylbut-2-enoate (1f). Yield:
43%.
(Z)-Ethyl 2-hydroxy-4-oxo-4-(thiophen-2-yl)but-2-enoate (1g).
Yield: 43%.
(Z)-Ethyl-2-hydroxy-4-(4-methoxyphenyl)-4-oxobut-2-enoate
(1h). Orange solid, M.pt.: 92 ꢁC, yield: 55%, Rf (petroleum
ether : ethyl acetate ¼ 7 : 3): 0.60, anal (C13H14O5) calc. C 62.39
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(d, ppm): 190.93, 189.46, 161.24, 153.53, 148.37, 129.88, 121.90,
114.81, 99.84, 64.34, 61.71, 14.52. LC-MS: m/z [M + H]+ 279.3,
[M ꢀ H]ꢀ 277.2.
(Z)-2-Hydroxy-4-oxo-4-o-tolylbut-2-enoic acid (2e). Yellow oil,
yield: 99%, Rf (cyclohexane : ethyl acetate ¼ 5 : 5): 0.0, anal
(C11H10O4) calc. C 64.07 H 4.89, found C 64.09 H 4.88. IR: 2931,
2507, 1709, 1596, 1492, 1456, 1387, 1233, 1169, 1127, 1028, 914,
832, 763, 728, 693 cmꢀ1 1H-NMR (400 MHz, acetone-D6)
.
(d, ppm): 7.66 (d, 1H, J ¼ 7.2 Hz, Ar-H), 7.45 (t, 1H, J ¼ 7.2 Hz,
Ar-H), 7.33–7.26 (m, 2H, Ar-H), 6.93 (s, 1H, CH), 2.53 (s, 3H,
CH3). 13C-NMR (75 MHz, acetone-D6) (d, ppm): 194.02, 187.52,
163.74, 138.60, 134.64, 132.46, 132.09, 129.21, 126.13, 100.75,
21.20. LC-MS: m/z [M ꢀ H]+ 205.1.
3.4. General procedure for the synthesis of diketo acids
(2a–f, 2h, 2m)
To a solution of diketo ester (1.0 mmol) in THF : water mixture
(1 : 4), freshly prepared 2 M LiOH solution (4.5 mmol) was
added with the help of a dropping funnel. The reaction was
continued for 2 h until white precipitate is obtained. Aer
completion of the reaction, pH of the reaction was adjusted to
2–3 by adding 1 N HCl. The compound was extracted with
diethyl ether, dried over sodium sulphate, concentrated to give
diketo acids (2a–f, 2h, 2m) in moderate to excellent yields (42–
99%). The characterization of compounds 2f and 2h is previ-
ously reported.20
(Z)-2-Hydroxy-4-oxo-4-phenylbut-2-enoic acid (2f). Yield:
83%.
(Z)-2-Hydroxy-4-(4-methoxyphenyl)-4-oxobut-2-enoic
(2h). Yield: 71%.
acid
(Z)-4-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-hydroxy-4-oxobut-
2-enoic acid (2m). Lemon yellow oil, yield: 91%, Rf (cyclo-
hexane : ethyl acetate ¼ 5 : 5): 0, anal (C12H10O6) calc. C 57.60 H
4.03, found: C 57.55 H 4.01. IR: 3408, 2967, 2912, 2847, 1732,
1610, 1575, 1504, 1455, 1364, 1321, 1284, 1247, 1181, 1121, 1021,
908, 887, 816, 765, 723 cmꢀ1. 1H-NMR (300 MHz, CDCl3) (d, ppm):
7.55 (s, 1H, CH), 7.19 (s, 1H, Ar-H), 6.89 (d, 2H, J ¼ 8.8 Hz, Ar-H),
4.33 (d, 4H, J ¼ 8.7 Hz, alicyclic ring). 13C-NMR (75 MHz, CDCl3)
(d, ppm): 189.97, 189.56, 163.24, 152.53, 148.74, 130.88, 122.90,
115.81, 114.90, 99.84, 64.54, 62.71, 14.52. LC-MS: m/z [M + H]+
251.97.
(Z)-4-(4-Cholorophenyl)-2-hydroxy-4-oxobut-2-enoic acid (2a).
Light yellow solid, M.pt.: 102 ꢁC yield: 75%, Rf (cyclo-
hexane : ethyl acetate ¼ 7 : 3): 0.0, anal (C10H7ClO4) calc. C
53.00 H 3.11, found C 53.01 H 3.09. IR (neat): 3410, 2974, 2866,
1684, 1585, 1395, 1358, 1256, 1090, 1010, 956, 907, 823, 762,
620, 584, 519 cmꢀ1. 1H-NMR (400 MHz, CDCl3) (d, ppm): 15.07
(s, 1H, OH), 7.90 (d, 2H, J ¼ 8.4 Hz, Ar-H), 7.45 (d, 2H, J ¼ 8.8 Hz,
Ar-H), 7.14 (s, 1H, CH). 13C-NMR (75 MHz, CDCl3) (d, ppm):
197.04, 189.24, 163.12, 138.96, 133.47, 129.27, 128.63, 97.99. LC-
MS: m/z [M + H]+ 227.2, [M ꢀ H]ꢀ 225.0.
3.5. General procedure for the synthesis of boc-dipeptides
(3a–e)
(Z)-4-(2-Chlorophenyl)-2-hydroxy-4-oxobut-2-enoic acid (2b).
Light yellow solid, M.pt.: 154 ꢁC, yield: 86%, Rf (cyclo-
hexane : ethyl acetate ¼ 7 : 3): 0.0, anal (C10H7ClO4) calc. C 53.00 In a 50 ml round bottom ask, L-phenylalanine methyl ester
H 3.11, found C 53.02 H 3.12. IR: 3457, 2925, 1685, 1585, 1358, hydrochloride (1.0 mmol) and desired boc amino acid
1
1261, 1127, 1067, 1001, 823, 746, 475 cmꢀ1. H-NMR (300 MHz, (1.2 mmol), were dissolved in acetonitrile (10 ml) and cooled to
ꢁ
acetone-D6) (d, ppm): 8.05 (d, 1H, J ¼ 8.4 Hz, Ar-H), 7.93 (d, 1H, J ¼ 0 C. To this solution, NMM (1.2 mmol), HOBt (2.0 mmol) and
8.7 Hz, Ar-H), 7.79 (d, 1H, J ¼ 8.4 Hz, Ar-H) 7.71 (d, 1H, J ¼ 8.7 Hz, EDC$HCl (1.2 mmol) were added in small portions. The reaction
Ar-H), 7.15 (s, 1H, CH). 13C-NMR (75 MHz, acetone-D6) (d, ppm): mixture was brought to room temperature and the progress of
196.32, 189.62, 162.38, 134.00, 132.26, 131.75, 130.06, 129.68, the reaction was checked by TLC. Aer completion of the reac-
128.34, 97.54. LC-MS: m/z [M + H]+ 227.45, [M ꢀ H]ꢀ 225.18.
tion, solvent was removed under vacuum and ethyl acetate was
(Z)-4-(4-Bromophenyl)-2-hydroxy-4-oxobut-2-enoic acid (2c). added to the residue, washed with 10% citric acid solution, 5%
Orange oil, yield: 82%, Rf (cyclohexane : ethyl acetate ¼ 7 : 3): sodium bicarbonate solution, water and then with brine and
0.0, anal (C10H7BrO4) calc. C 44.31 H 2.60, found C 44.35 H 2.63. nally dried over anhydrous MgSO4. The organic layer was
IR: 3494, 1721, 1613, 1583, 1485, 1451, 1398, 1233, 1183, 1139, concentrated under vacuum. The dipeptide obtained was puri-
1107, 1069, 1051, 903, 819, 773 cmꢀ1 1H-NMR (300 MHz, ed by column chromatography using cyclohexane : ethyl acetate
.
acetone-D6) (d, ppm): 8.15 (d, 2H, J ¼ 8.7 Hz, Ar-H), 7.31 (d, 2H, (7 : 3) as eluent to obtain pure dipeptides in 83–97% yields.
J ¼ 8.7 Hz, Ar-H), 7.02 (s, 1H, CH). 13C-NMR (75 MHz, acetone-
Boc-Ala-Phe-OCH3 (3a). White powder, M.pt.: 80 ꢁC, yield:
7 : 3): 0.24, anal
(C18H26N2O5) calc. C 61.70 H 7.48 N 7.99, found C 61.72 H 7.46
D6) (d, ppm): 205.03, 186.49, 162.24, 142.06, 136.02, 133.53, 91%, Rf (cyclohexane : ethyl acetate
¼
129.09, 99.04. LC-MS: m/z [M + H]+ 270.87.
(Z)-4-Cyclopropyl-2-hydroxy-4-oxobut-2-enoic acid (2d). Light N 7.80. IR (neat): 3326, 2978, 2931, 1737, 1691, 1654, 1520, 1447,
yellow oil, yield: 43%, Rf (cyclohexane : ethyl acetate ¼ 7 : 3): 1391, 1367, 1335, 1251, 1207, 1162, 1127, 1080, 1056, 1035, 968,
1
0.0, anal (C7H8O4) calc. C 53.85 H 5.16, found C 53.88 H 5.20. IR: 907, 871 cmꢀ1. H-NMR (400 MHz, CDCl3) (d, ppm): 7.29–7.22
3341, 2963, 2920, 2851, 1745, 1669, 1520, 1449, 1438, 1393, (m, 3H, Ar-H), 7.10 (d, 2H, J ¼ 6.8 Hz, Ar-H), 6.49 (s, 1H, NH),
1371, 1300, 1274, 1242, 1222, 1168, 1018, 979, 883, 754, 674 4.85 (q, 1H, J ¼ 6 Hz, CH), 4.12 (q, 1H, J ¼ 7.2 Hz, CH), 3.72
1
cmꢀ1. H-NMR (300 MHz, CDCl3) (d, ppm): 14.95 (s, 1H, OH), (s, 3H, OCH3), 3.12 (d, 2H, J ¼ 10.8 Hz, CH2), 1.43 (s, 9H, CH3),
6.50 (s, 1H, CH), 1.85–1.78 (m, 1H, cyclopropyl ring), 1.22 (q, 2H, 1.32–1.22 (d, 3H, J ¼ 4.2 Hz, CH3). 13C-NMR (75 MHz, CDCl3) (d,
J ¼ 4.6 Hz, cyclopropyl ring), 1.08 (q, 2H, J ¼ 4.6 Hz, cyclopropyl ppm): 172.84, 171.83, 154.90, 137.01, 129.09, 128.11, 126.53,
ring). 13C-NMR (75 MHz, CDCl3) (d, ppm): 198.54, 185.96, 78.05, 53.39, 51.81, 49.51, 36.66, 28.16, 18.09. LC-MS: m/z
161.39, 98.89, 43.09, 11.13. LC-MS: m/z [M ꢀ H]ꢀ 155.0.
[M + H]+ 351.3, [M ꢀ H]+ 349.3, [M ꢀ Boc]+ 251.3.
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Boc-Gly-Phe-OCH3 (3b). Colourless oil, yield: 92%, Rf (cyclo-
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dryness of the mixture led to yellowish oil. This residue was
triturated in ether and the precipitate was recovered as a white
powder aer drying in quantitative yield.
hexane : ethyl acetate ¼ 5 : 5): 0.65, anal (C18H26N2O5) calc. C
60.70 H 7.19 N 8.33, found C 60.72 H 7.18 N 8.34. IR (neat): 3339,
2978, 2933, 1655, 1510, 1439, 1369, 1279, 1249, 1216, 1166, 1052,
Scarlet brown solid, M.pt.: 108 ꢁC, yield: 99%, Rf (petroleum
ether : ethyl acetate ¼ 7 : 3):0.42, anal (C21H23N3O3) calc. C
69.02 H 6.34 N 11.50, found C 69.05 H 6.37 N 11.47. IR: 1726,
1687, 1655, 1524, 1453, 1426, 1359, 1343, 1292, 1244, 1166,
1032, 944, 866, 747, 702 cmꢀ1 1H-NMR (400 MHz, CDCl3)
.
(d, ppm): 7.29–7.26 (m, 3H, Ar-H), 7.09 (d, 2H, J ¼ 7.2 Hz, Ar-H),
6.45 (s, 1H, NH), 5.02 (s, 1H, NH), 4.89 (m, 1H, CH), 3.82–3.76 (m,
2H, CH2), 3.72 (s, 3H, OCH3), 3.11 (d, 2H, J ¼ 8.4 Hz, CH2), 1.44
(s, 9H, CH3). 13C-NMR (75 MHz, CDCl3) (d, ppm): 171.69, 171.30,
155.73, 135.70, 129.23, 128.62, 127.16, 79.89, 53.12, 52.28, 37.99,
30.84, 28.30. LC-MS: m/z [M + H]+ 337.4, [M ꢀ H]+ 335.2.
1099, 1045, 1013, 868, 778, 735, 704 cmꢀ1, H-NMR (400 MHz,
1
CDCl3) (d, ppm): 8.35 (s, 1H, NH), 7.45 (d, 1H, J ¼ 7.6 Hz, Ar-H),
7.31 (d, 1H, J ¼ 8 Hz, Ar-H), 7.15 (s, 4H, Ar-H), 7.06 (t, 2H, J ¼ 7.6
Hz, Ar-H), 6.96–6.90 (m, 2H, Ar-H), 6.74 (s, 1H, NH), 5.48 (s, 2H,
NH2), 4.60 (t, 1H, J ¼ 6.8 Hz, CH), 4.28 (t, 2H, J ¼ 7 Hz, CH), 3.63
(s, 3H, OCH3), 3.25 (t, 2H, J ¼ 7.6 Hz, CH2), 2.99 (t, 2H, J ¼ 5.6
Hz, CH2). 13C-NMR (75 MHz, CDCl3) (d, ppm): 172.89, 171.50,
157.10, 139.56, 136.23, 129.89, 128.91, 127.12, 126.31, 122.52,
120.62, 110.98, 56.45, 53.25, 51.37, 37.76, 31.23. LC-MS: m/z [M +
H]+ 366.0, [M ꢀ H]ꢀ 364.0.
Boc-Val-Phe-OCH3 (3c). White solid, M.pt.: 94 ꢁC, yield: 84%,
Rf (cyclohexane : ethyl acetate ¼ 5 : 5): 0.74, anal (C20H30N2O5)
calc. C 63.47 H 7.99 N 7.40, found C 63.48 H 7.98 N 7.41. IR
(neat): 3343, 2963, 2929, 2874, 1745, 1669, 1518, 1449, 1371,
1300, 1274, 1244, 1222, 1168, 1121, 1082, 1018, 981, 938, 883,
1
804, 754, 702, 672 cmꢀ1. H-NMR (400 MHz, CDCl3) (d, ppm):
7.31–7.20 (m, 3H, Ar-H), 7.11 (d, 2H, J ¼ 6.8 Hz, Ar-H), 6.26
(s, 1H, NH), 4.99 (s, 1H, NH), 4.88 (t, 1H, J ¼ 5.6 Hz, CH), 3.88
(d, 1H, J ¼ 8 Hz, CH), 3.71 (s, 3H, OCH3), 3.17–3.07 (m, 2H, CH2),
1.44 (s, 9H, CH3). 0.89 (d, 6H, J ¼ 6.8 Hz, CH3). 13C-NMR (75
MHz, CDCl3) (d, ppm): 191.31, 156.43, 145.30, 137.90, 136.17,
133.07, 130.15, 128.69, 128.58, 122.16, 120.82, 118.03, 115.23.
LC-MS: m/z [M + H]+ 379.4, [M-Boc]+ 279.4.
3.7. General procedure for the synthesis of amino acid/
dipeptide analogues of diketo acids (5a–e)
To a solution of desired diketo acid (1.0 mmol) in anhydrous
DMF under argon were added HOBt (2.0 mmol), PyBOP
(1.2 mmol), L-amino acid methyl ester hydrochloride/dipeptide
(4e) (1.0 mmol) and Et3N (1.6 mmol) and the reaction mixture
was stirred for 24 h at room temperature. Solvent was removed
and the residue was dissolved in 50 ml of ethyl acetate. The
organic phase was washed with a solution of 10% citric acid, 5%
NaHCO3, water and nally with brine solution (each 50 ml). The
organic phase then dried over Na2SO4 and evaporated to
dryness. Purication by column chromatography on silica gel
(cyclohexane : ethyl acetate ¼ 7 : 3) provided desired compound
in 70–99% yields.
Boc-Ile-Phe-OCH3 (3d). White solid, M.pt.: 112 ꢁC, yield:
98%, Rf (cyclohexane : ethyl acetate
¼
7 : 3): 0.71, anal
(C21H32N2O5) calc. C 64.26 H 8.22 N 7.14, found C 64.27 H 8.24
N 7.16. IR (neat): 3343, 3328, 2965, 1743, 1667, 1514, 1454, 1447,
1367, 1277, 1235, 1168, 1102, 1048, 1022, 889, 752, 700 cmꢀ1
.
1H-NMR (500 MHz, CDCl3) (d, ppm): 7.29–7.25 (m, 3H, Ar-H),
7.11 (d, 2H, J ¼ 9.84 Hz, Ar-H), 6.35 (s, 1H, NH), 5.0 (s, 1H, NH),
4.88 (q, 1H, J ¼ 5.6 Hz, CH), 3.88 (t, 1H, J ¼ 5.6 Hz, CH), 3.17 (s,
3H, OCH3), 3.17 (d, 2H, J ¼ 5.3 Hz, CH2), 2.87 (m, 1H, CH), 1.44
(m, 11H, CH3, CH2), 0.89–0.85 (m, 6H). 13C-NMR (75 MHz,
CDCl3) (d, ppm): 171.62, 171.24, 155.61, 135.63, 129.21, 128.57,
127.12, 77.31, 59.20, 53.05, 52.28, 37.94, 37.15, 28.26, 24.60,
15.38, 11.36. LC-MS: m/z [M + H]+ 393.6, [M-Boc]+ 293.6.
(S,Z)-Methyl2-(4-(4-chlorophenyl)-2-hydroxy-4-oxobut-2-ena-
mido)-3-phenyl propanoate (5a). Brown solid, M.pt.: 98 ꢁC yield:
70%, Rf (cyclohexane : ethyl acetate ¼ 5 : 5): 0.50, [a]2D0 ¼ ꢀ47.19
(c 0.45, C2H5OH), anal (C20H18ClNO5) calc. C 61.94 H 4.68 N
3.61, found C 61.89 H 4.72 N 3.64. IR (neat): 1737, 1655, 1579,
1572, 1558, 1439, 1400, 1363, 1309, 1246, 1222, 1175, 1113,
Boc-Trp-Phe-OCH3 (3e). Brown solid, M.pt.: 110 ꢁC, yield:
96%, Rf (petroleum ether : ethyl acetate ¼ 7 : 3): 0.35, anal
(C26H31N3O5) calc. C 67.08 H 6.71 N 9.03, found C 67.10 H 6.72 N
9.04. IR: 3404, 3065, 2959, 2127, 1737, 1665, 1544, 1536, 1501,
1091, 1024, 1015, 775, 747, 702 cmꢀ1 1H-NMR (300 MHz,
.
CDCl3) (d, ppm): 10.83 (s, 1H, OH), 7.90 (d, 2H, J ¼ 8.4 Hz, Ar-H),
7.70 (d, 2H, J ¼ 8.1 Hz, Ar-H), 7.44 (d, 2H, J ¼ 8.7 Hz, Ar-H), 7.28
(t, 3H, J ¼ 9.6 Hz, Ar-H), 6.96 (s, 1H, CH), 4.76 (t, 1H, J ¼ 5.4 Hz,
CH), 3.78 (d, 2H, J ¼ 6.3 Hz, CH2), 3.74 (s, 3H, OCH3). 13C-NMR
(75 MHz, CDCl3) (d, ppm): 191.67, 189.95, 170.97, 166.56,
139.67, 135.41, 129.75, 129.25, 128.91, 128.69, 127.42, 53.23,
52.59, 37.20. LC-MS: m/z [M + H]+ 388.95.
(S,Z)-Methyl2-(2-hydroxy-4-(4-methoxyphenyl)-4-oxobut-2-
enamido)-3-phenylpropanoate (5b). Yellow oil, yield: 76%, Rf
(cyclohexane : ethyl acetate ¼ 5 : 5): 0.60, [a]2D0 ¼ ꢀ0.06 (c 0.75,
C2H5OH), anal (C21H21NO6) calc. C 65.79 H 5.52 N 3.65, found C
65.83 H 5.58 N 3.67. IR (neat): 2937, 2842, 1671, 1595, 1508,
1456, 1420, 1357, 1309, 1247, 1169, 1114, 1023, 956, 831, 753,
699, 566, 478 cmꢀ1. 1H-NMR (300 MHz, CDCl3) (d, ppm): 7.93 (d,
1465, 1359, 1141, 1015, 855, 842, 801, 745, 726, 704 cmꢀ1
,
1H-NMR (400 MHz, CDCl3) (d, ppm): 8.08 (s, 1H, NH), 7.66 (d,
1H, J ¼ 7.6 Hz, Ar-H), 7.33 (d, 1H, J ¼ 14.8 Hz, Ar-H), 7.22–7.11
(m, 5H, Ar-H), 7.02 (s, 1H, Ar-H), 6.80 (d, 2H, J ¼ 6.8 Hz, Ar-H),
6.20 (s, 1H, NH), 4.73 (m, 1H, CH), 4.49 (m, 1H, CH), 3.61 (s, 3H,
OCH3), 3.30–3.08 (m, 2H, CH2), 2.94 (d, 2H, J ¼ 5.6 Hz, CH2), 1.42
(s, 9H, CH3). 13C-NMR (75 MHz, CDCl3) (d, ppm): 172.67, 171.98,
156.61, 139.86, 135.63, 129.64, 128.57, 127.32, 126.56, 122.57,
119.62, 111.42, 78.31, 56.20, 53.05, 51.28, 37.82, 31.15, 28.26.
LC-MS: m/z [M + H]+ 466.1, [M ꢀ H]ꢀ 464.1 [M ꢀ Boc]+ 366.0.
3.6. Synthesis of Trp-Phe-OCH3 (4e)
To a solution of 3e (1.0 mmol) in 5 ml of anhydrous dichloro- 2H, J ¼ 8.7 Hz, Ar-H), 7.24–7.15 (m, 5H, Ar-H), 7.04 (d, 2H, J ¼ 8.7
methane was added 5 ml of triuoroacetic acid and stirred the Hz, Ar-H), 6.81 (s, 1H, CH), 4.45 (q, 1H, J ¼ 7.6 Hz, CH), 3.84 (s,
reaction mixture for 2 h at room temperature. Evaporation to 3H, OCH3), 3.76 (s, 3H, OCH3), 3.54 (d, 2H, J ¼ 5.6 Hz, CH2). 13C-
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NMR (75 MHz, CDCl3) (d, ppm): 196.30, 189.43, 172.02, 163.09, 1.0 ꢃ 105 CFU mlꢀ1). Wells of 6 mm diameter were cut into
139.07, 130.46, 129.88, 128.93, 127.53, 126.06, 113.81, 108.41, solidied agar media with the help of sterilized cork borer. An
55.50, 53.79, 51.83, 38.66. LC-MS: m/z [M + H]+ 384.59.
aliquot of 50 ml of each concentration was poured in the
(Z)-Methyl 2-(2-hydroxy-4-oxo-4-o-tolylbut-2-enamido)- respective well and the plates were incubated at 37 ꢁC for
propanoate (5c). Yellow oil, yield: 70%, Rf (cyclohexane : ethyl overnight. DMSO was used as negative control while cipro-
acetate ¼ 5 : 5): 0.66, [a]2D0 ¼ ꢀ37.65 (c 0.80, C2H5OH), anal oxacin (10 mg mlꢀ1) was used as positive control. The experi-
(C31H29N3O6) calc. C 61.85 H 5.88 N 4.81, found C 61.86 H 5.89 ment was performed in triplicate under strict aseptic
N 4.82. IR (neat): 2952, 2861, 2872, 1687, 1678, 1605, 1510, 1451, conditions. The antibacterial activity for each of the compound
1439, 1208, 761, 737 cmꢀ1. 1H-NMR (400 MHz, CDCl3) (d, ppm): evaluated was expressed in terms of the average of the diameter
7.63 (d, 1H, J ¼ 7.6 Hz, Ar-H), 7.42 (t, 1H, J ¼ 7.6 Hz, Ar-H), 7.31– of zone of inhibition (in mm) produced by the respective
7.28 (m, 2H, Ar-H), 6.83 (s, 1H, CH), 4.39 (q, 1H, J ¼ 7.6 Hz, CH), concentration of each compound at the end of incubation
3.57 (s, 3H, OCH3), 2.55 (s, 3H, CH3), 1.41 (t, 3H, J ¼ 7.2 Hz, period.
CH3). 13C-NMR (125 MHz, CDCl3) (d, ppm): 191.80, 175.82,
Determination of MIC and MBC of the susceptible
172.48, 160.85, 138.10, 134.78, 131.89, 131.82, 129.11, 125.99, compounds. A micro broth dilution technique was employed to
98.28, 52.65, 48.17, 21.12, 18.19. LC-MS: m/z [M ꢀ COOCH3]+ determine the MIC (minimum inhibitory concentration) of
235.2, [M ꢀ COOCH3]ꢀ 233.1.
synthesized compounds along with positive (ciprooxacin) and
(Z)-Methyl
2-(4-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2- negative (10% DMSO) controls.25 The concentration of DMSO
hydroxy-4-oxobut-2-enamido)propanoate (5d). Golden yellow were maintained less than 10% in the nal test volume. Various
oil, yield: 75%, Rf (cyclohexane : ethyl acetate ¼ 5 : 5): 0.47, [a]2D0 concentrations (312.5 . 2.475 mg mlꢀ1) of test compounds and
¼ ꢀ11.75 (c 0.40, C2H5OH), anal (C16H17NO7) calc. C 57.31 H (512.0 . 1.0 mg mlꢀ1) of ciprooxacin were dispensed into
5.11 N 4.18, found C 57.37 H 5.14 N 4.15. IR (neat): 2965, 2868, wells, then inoculated with test organisms with approx. 2.5 ꢃ
6
ꢁ
1745, 1678, 1607, 1581, 1508, 1462, 1430, 1350, 1322, 1292, 10 cells per ml (McFarland standard) and incubated at 37 C
1207, 1132, 1065, 1013, 901, 845, 767, 707 cmꢀ1. H-NMR (400 for 24 h. The effect of 10% DMSO was also checked on all the
1
MHz, CDCl3) (d, ppm): 7.51 (s, 1H, CH), 7.49 (s, 1H, Ar-H), 6.93 strains separately. The MIC values were determined as the
(d, 2H, J ¼ 8.7 Hz, Ar-H), 4.87 (q, 1H, J ¼ 5.4 Hz, CH), 4.33 (d, 4H, lowest concentration resulting in no growth. The MBC of potent
J ¼ 5.1 Hz, alicyclic ring), 3.59 (s, 3H, OCH3), 1.26 (d, 3H, J ¼ 5.4 compounds were also determined by transferring of 10 ml
Hz, CH3). 13C-NMR (125 MHz, CDCl3) (d, ppm): 196.60, 189.47, aliquot on sterile MH agar plate from wells with no growth. The
ꢁ
171.62, 166.56, 147.95, 143.21, 131.05, 122.39, 117.75, 115.88, plates were incubated at 37 C for 24 h. Aer incubation, the
110.02, 64.62, 64.06, 48.54, 46.26, 18.61. LC-MS: m/z [M + H]+ MBC was determined as the lowest concentration of test
336.42.
compound that results no growth. All the experiments were
(Z)-Methyl 2-(2-(2-hydroxy-4-oxo-4-phenylbut-2-enamido)-3- done in triplicate in separate time.
(1H-indol-3-yl) propanamido)-3-phenylpropanoate (5e). Light
Enzymatic inhibition assay. All compounds were dissolved
yellow solid, yield: 99%, M.pt.: 73 ꢁC, Rf (ethyl aceta- in DMSO to a stock concentration of 10 mM. Initially the
te : cyclohexane ¼ 6 : 4): 0.59, [a]2D0 ¼ ꢀ16.47 (c 0.60, C2H5OH), compounds were screened for inhibition with each of the four
anal (C31H29N3O6) calc. C 69.00 H 5.42 N 7.79, found C 69.01 H MetAP's (HsMetAP, MtMetAP, SpMetAP, EfMetAP). Expression,
5.43 N 7.78. IR (neat): 2926, 2853, 1743, 1655, 1601, 1510, 1458, purication and biochemical assays are performed as reported
1439, 1343, 1253, 1214, 1182, 1078, 1028, 819, 775, 745, 702 previously.26,27 The enzyme assays were performed using Met-
cmꢀ1. 1H-NMR (300 MHz, DMSO) (d, ppm): 8.67 (d, 1H, J ¼ 7.5 AMC as substrate. IC50 values were determined by using
Hz, Ar-H), 8.51 (d, 1H, J ¼ 8.4 Hz, Ar-H), 8.01 (d, 2H, J ¼ 7.5 Hz, compound concentration range of 1 mM to 120 mM. The Ki
Ar-H), 7.68–7.54 (m, 4H, Ar-H), 7.33–7.23 (m, 6H, Ar-H, NH), values were determined by Dixon method. The reaction mixture
7.10–6.93 (m, 4H, Ar-H, CH), 4.68 (t, 1H, J ¼ 5.7 Hz, CH) 4.54 (t, contained 50 mM Hepes (pH-7.5 for SpMetAP, EfMetAP,
1H, J ¼ 6.3 Hz, CH), 3.57 (s, 3H, OCH3), 3.15–2.97 (m, 4H, CH2). MtMetAP and pH-8.0 for HsMetAP), 150 mM KCl, CoCl2 (three
13C-NMR (125 MHz, CDCl3) (d, ppm): 176.92, 170.19, 169.23, molar equivalents of the enzyme concentration), 4 mM enzymes
159.28, 135.19, 134.87, 132.28, 132.14, 127.70, 127.50, 126.85, (HsMetAP, MtMetAP, SpMetAP or EfMetAP) and 50, 100, 150 mM
126.03, 125.22, 122.32, 119.76, 117.10, 116.97, 110.02, 107.89, concentrations of L-Met-AMC. All reactions were performed in
92.78, 52.36, 52.19, 50.52, 35.56, 30.22. LC-MS: m/z [M + H]+ triplicate. 2,20 Bipyridine was used as positive control.
540.4, [M ꢀ H]+ 538.3.
In vitro cytotoxicity. HepG2 cells were cultured and main-
tained as a monolayer in Dulbecco's Modied Eagle's Medium
(DMEM, Sigma) supplemented with 10% of fetal calf serum
(Sigma) and antibiotics (100 IU mlꢀ1 of penicillin and
3.8. Antibacterial activity
In vitro bacterial susceptibility assay. In vitro antibacterial 100 mg mlꢀ1 of streptomycin, Sigma). All cells were cultured at
susceptibility of all the compounds against four different 37 C in a 100% humidity atmosphere and 5% CO2. Exponen-
ꢁ
bacterial strains was determined by standard agar well diffusion tially growing viable cells were plated at 1.2 ꢃ 10ꢀ4 cells per well
assay.24 Petri dishes (size 100 mm diameter) containing 18 ml of into 96-well plates and incubated for 48 h before the addition of
cool and molten Mueller Hinton Agar (MHA) (at 40 ꢁC) were the compounds/metronidazole. Stock solutions of compounds
seeded with 50 ml inoculums of bacterial strain (inoculums size were initially dissolved in 20% (v/v) DMSO and further diluted
was adjusted so as to deliver a nal inoculums of approximately with fresh complete medium. The growth-inhibitory effects of
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the compounds were measured using standard tetrazolium
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ꢁ
MTT assay. Aer 48 h of incubation at 37 C, the medium was
removed and 25 ml of MTT (5 mg mlꢀ1) in serum free medium
ꢁ
was added to each well. The plates were incubated at 37 C for
4 h. At the end of the incubation period, the medium was
removed and 100 ml DMSO added to all wells. The metabolized
MTT product dissolved in DMSO was quantied by reading the
absorbance at 570 nm with a reference wavelength of 655 nm in
an ELISA plate reader. All assays were performed in triplicate.
Percent viability was dened as the relative absorbance of
treated versus untreated control cells.
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4. Conclusion
In this study, we successfully designed and synthesized some
diketo acids, their amino acid/dipeptidic analogues as novel
antibacterial agents targeting bacterial MetAPs. The results of in
silico screening and in vitro antibacterial activity support the
above ndings and suggest their candidature to act as small
molecule inhibitors of MetAPs. Compounds 1c, 1e, 1j, 1l, 2a, 2b,
3d, 5c and 5e showed moderate to excellent antibacterial activity
with no cytotoxic effect in the concentration range of 50–450
mg mlꢀ1. In the biochemical evaluation, compounds 1g and 3d
showed 84% and 79% inhibition of SpMetAP and MtMetAP,
respectively at 100 mM concentration. At the same concentra-
tion, compound 5e exhibited 87% and 85% inhibition of
EfMetAP and SpMetAP respectively. Furthermore, efforts in
modifying these and other lead structures with the aim of
improving potency as well as specicity are in progress.
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Conflicts of interest
All authors declare no competing interests.
20 B. Chen, H. F. Yin, Z. S. Wang, J. H. Xu, L. Q. Fan and J. Zhao,
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Acknowledgements
Mohammad Abid gratefully acknowledges funding support in
the form of Young Scientist from Science & Engineering
Research Board (SR/FT/LS-03/2011), New Delhi, India. AA
acknowledges the support from DST (SR/SO/BB-55/2008) and
DBT (BT-BRB-TF-2-2011), India. VKP, MI and BA acknowledge
the fellowship support from UGC, INDIA. The authors are
thankful to Prof. Umar Farooq, Faculty of Biotechnology,
Shoolini University of Biotechnology and Management
Sciences, Solan, H.P. India, for providing laboratory facilities for
antibacterial activities.
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RSC Adv., 2015, 5, 34173–34183 | 34183