Design, synthesis and inhibition activity of a novel cyclic enediyne amino acid conjugates against MPtpA

Article abstract of DOI:10.1016/j.bmc.2011.03.024

In course of studies towards the discovery of selective inhibitors of MPtpA, a novel cyclic endiyne malonamic acid has been designed and synthesized. The synthesis involves a crucial intramolecular Knoevenagel reaction. The compound displayed a reversible non-competitive inhibition against MPtpA with inhibition constant K_i of 22.5 μM. The enediyne acts as a recognition framework in inducing the inhibition and not as a reactive functional moiety. This was confirmed by comparing the inhibiting activity with that of the corresponding saturated cyclic non-enediyne analogue.

Full text of DOI:10.1016/j.bmc.2011.03.024

Bioorganic & Medicinal Chemistry 19 (2011) 3274–3279
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and inhibition activity of a novel cyclic enediyne amino
acid conjugates against MPtpA
Koushik Chandra a, Debajyoti Dutta b, Analava Mitra c, Amit K. Das b, , Amit Basak a,
⇑
⇑
a Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India
b Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, India
c School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721302, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In course of studies towards the discovery of selective inhibitors of MPtpA, a novel cyclic endiyne malo-
namic acid has been designed and synthesized. The synthesis involves a crucial intramolecular Knoeve-
nagel reaction. The compound displayed a reversible non-competitive inhibition against MPtpA with
Received 19 January 2011
Revised 9 March 2011
Accepted 10 March 2011
Available online 30 March 2011
inhibition constant Ki of 22.5 lM. The enediyne acts as a recognition framework in inducing the inhibi-
tion and not as a reactive functional moiety. This was confirmed by comparing the inhibiting activity with
that of the corresponding saturated cyclic non-enediyne analogue.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Inhibition
Enediyne
Amino acid
Conjugate
MPtpA
Knoevenagel
1. Introduction
enediyne with the protein MPtpA,6 a key phosphatase enzyme
present in Mycobacterium tuberculosis. The genome sequence anal-
The enediynes1 comprise an important class of powerful antitu-
mor agent with unprecedented biological profiles. This is due to
their unique molecular architecture and precise mode of interac-
tion with biomolecules. Nearly twenty different enediynes have
been tested as potential clinical agent till date. The effort ulti-
mately resulted in the development of the commercial drug Mylo-
targ2 for treatment of leukemia. The enediynes themselves have no
biological significance until triggered to form cytotoxic diradical (a
process known as Bergman cyclization, BC3) capable of cleaving
DNA backbone at low concentration. Thus the modulation of BC
through activation4 of enediyne moiety becomes an important as-
pect of the research of enediynes in recent years. However, only
few studies5 have been made where the enediyne skeleton has
been utilized as a template for recognition by biomacromolecules.
The enediynes, especially the cyclic ones, because of the existence
of alkyne moieties, offer special advantage over the corresponding
saturated analogues with regard to their interaction with biomol-
ecules like proteins. The conformational constraint coupled with
ysis7 of M. tuberculosis has revealed the presence of two protein
tyrosine phosphatases MPtpA and MPtpB8 both of which are be-
lieved to be linked to the pathogenicity of the organism9. Therefore
the design and application of new molecular framework that acts
as a selective inhibitory agent of MPtpA or MPtpB has become an
important research topic in medicinal chemistry. Recently, we
have reported10 the moderate inhibition of MPtpA by a new class
of cyclic peptides. This prompts us to design and synthesis of a no-
vel cyclic enediyne malonamic acid (CEMA) (1) and to evaluate the
inhibitory activity against MPtpA. To substantiate the role of ened-
iyne framework, we have also synthesized a cyclic non endiyne
malonamic acid (CNEMA) (2) for comparison shown in Figure 1.
Due to availability of multiple binding modes in the binding
sites, the enediyne malonamic acid (CEMA) may be a good choice
which is capable to deliver a number of aspects such as solubility,
p
-electron cloud surrounding the six atoms of enediynes may facil-
HN
itate binding through stacking interactions with side chains of aro-
matic amino acids present in the active site of the enzyme. With
this in mind, we have undertaken a study of interaction of a cyclic
HN
O
O
2
COOH
1
COOH
⇑
Corresponding authors. Tel.: +91 3222283300; fax: +91 3222282252 (A.B.).
E-mail address: absk@chem.iitkgp.ernet.in (A. Basak).
Figure 1. Synthesized cyclic enediyne and non enediyne amino acid conjugates.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.024

K. Chandra et al. / Bioorg. Med. Chem. 19 (2011) 3274–3279
3275
pH, conformational rigidity, recognizable complementary func-
tionality, etc. The probable interaction comprising of multiple
binding modes for structure based inhibitor (CEMA) is proposed
in Figure 2.
Their onset temperature for Bergman Cyclization (BC) is quite high
depending upon the ring size. However, in order to know whether
endocyclic double bond and amide resonance play any role in low-
ering the BC temperature, we performed solid phase thermal reac-
tion kinetics by Differential Scanning Calorimetry (DSC).15 The
onset temperature of BC of CEMA was found to be 248 °C (Fig. 3),
similar to enediynes of same ring size thus ruling out the involve-
ment of any diradical formation at biological temperature and sub-
sequent inhibition during its interaction with the enzyme. The
enediyne thus can provide only a hydrophobic framework around
the cyclic amide for better interaction with the enzyme.
2. Results and discussion
2.1. Synthesis
The synthesis began with Pd-mediated sequential Sonogashira
coupling11 of dibromobenzene with THP-protected propargyl alco-
hol and homopropargyl alcohol that afforded the alcohol 5. The lat-
ter was converted to amine 8 using standard protocol (Scheme 1).
The amine was then acylated with ethylmalonyl chloride, followed
by the deprotection of the THP group (PPTS, EtOH)12 and subse-
quently oxidized to the primary alcohol by Dess–Martin period-
inane.13 Finally, the crucial intramolecular cyclization was
performed in various solvents by varying the reaction condition
shown in Table 1. No such intermolecular Knovenegal condensa-
tion product was formed; rather the elimination product 12 was
obtained as the sole product, confirmed by NMR and Mass spectro-
metric analysis. Table 1 clearly shows that the cyclization becomes
more facile in terms of yield and reaction time when a buffer med-
ium was introduced in THF solvent at high dilution condition. Sole
use of base could not complete the elimination; rather a mixture of
b-hydroxy ester and the elimination product were obtained. Addi-
tion of catalytic amount of acid could complete the elimination
process. The structure of elimination product was further con-
firmed by carrying out Micheal addition reaction with thiophenol
and diethyl malonate (Scheme 2). Ester hydrolysis using controlled
amount of LiOH in aqueous THF resulted in the formation of a no-
vel 11-membered cyclic enedinyl malonamic acid (CEMA) 1. The
corresponding cyclic non-enedinyl analogue (CNEMA) 2 was de-
rived from compound 1 by catalytic hydrogenation using 10% Pd-
charcoal. CEMA was isolated as the only product while CNEMA
was a racemic mixture. All the compounds were characterized by
NMR, Mass and HPLC technique.
2.3. Inhibition study
Recombinant MPtpA was purified as described previously.16 En-
zyme purity was assessed by SDS–PAGE and concentration of pro-
tein was determined from the absorbance at 280 nm. Buffer
containing with 0.1 M Tris–HCl, pH 7.5 with ionic strength
0.15 M of NaCl and 1 mM EDTA was prepared for the kinetic study.
All the inhibition studies were performed using p-nitrophenyl
phosphate (pNPP) as a substrate with a fixed concentration of pro-
tein (25 nM). The release of p-nitrophenol at 405 nm was quanti-
fied both in case of control and in presence of the inhibitors. The
effective inhibitor and substrate concentration were varied from
3–7 lm and 10–30 lm for CEMA and 10–15 lm and 10–30 lm
for CNEMA, respectively. Each assay was repeated in triplicate.
2.4. Inhibition kinetics
Both the values of the kinetic parameters and inhibitor con-
stants were obtained from the Lineweaver Burk plot (Fig. 4). For
different concentration of same inhibitor, double reciprocal plots
were taken into account to conclude the mode of inhibition. Inhi-
ꢀ
ꢁ
Km
bition constant (Ki) was determined from the plot of
versus
[I] where [I] is the inhibitor concentration, Km is theVMmaxichaelis–
Menten constant and Vmax is the maximum reaction velocity. Both
CEMA and CNEMA were found to behave as reversible non-com-
petitive inhibitors (Fig. 4) with Ki values in micromolar range
(Table 2). In a previous report10 we have shown that the increase
in hydrophobicity in the cyclic peptides reduces the effective inter-
action with MPtpA. Here also, CNEMA, being more hydrophobic as
compared to CEMA, exhibits weak interaction with MPtpA as re-
flected in the Ki values and IC50 values (Table 2).
2.2. Study of thermal reactivity by DSC
It has been demonstrated earlier that enediynes with ring size
greater than ten does not undergo BC under ambient conditions.14
2.5. Docking study
Docking study was performed using Autodock 4.217 to throw
light on the probable interactions of CEMA with MPtpA. Coordi-
nates of CEMA and CNEMA were generated using PRODRG server
(http://davapc1.bioch.dundee.ac.uk/prodrg/). The surface area of
active site groove occupied by CEMA was calculated using PISA ser-
ver.18 The docking clearly reflects that the inhibitor binds effec-
tively in the vicinity of the PTP loop by virtue of multiple
hydrophobic interactions. CEMA consumes 318.10 Å2 of the active
site groove where mostly hydrophobic residues play an important
role in determining the probable mode of binding. The PTP loop in
MPtpA acts as the key mediator for its flexible accessibility of the
substrate to the active site and directs the catalytic mechanism.
However, the aromatic ring of CEMA disposes sufficiently close en-
ough to the PTP loop, thereby restricting the flexible movement of
the loop. Gly13 and Ile15 are two PTP loop residues involved in
strong hydrophobic interactions with aromatic ring of CEMA.
Tyr128 is also located within the hydrophobic interacting distance
with CEMA whereas Tyr129 interacts with carbonyl group of the
enediyne ring via hydrogen bonding interaction. Although Trp48
is involved in H-bonding interaction with CEMA it may indulge
Polar
Trp-48
interaction
H
S
N
H
H
O
O
H
NH
N
H2N
Hydrophobic
interaction
O
H
O
N
O
H
Figure 2. Possible interaction of 1 with the active site residue of MPtpA.

3276
K. Chandra et al. / Bioorg. Med. Chem. 19 (2011) 3274–3279
OMs
OH
Br
Br
OTHP
(i)
(iii)
(iv)
(ii)
OTHP
OTHP
Br
3
4
5
6
H
N
NH2
N3
O
(v)
(vi)
(vii)
OTHP
COOEt
OTHP
OTHP
8
7
9
H
N
H
N
O
O
(ix)
(x)
(viii)
HN
O
COOEt
OH
COOEt
H
COOEt
12
11
10
O
(xi)
HN
HN
O
O
2
1
COOH
COOH
Scheme 1. Synthesis of cyclic enediyne and non-endiyne amino acid conjugates. Reagents and conditions: (i) propargyl-OTHP, Pd(PPh3)4, n-BuNH2, 85 °C, reflux, 3 h, 66%; (ii)
3-butyne-1-ol,Pd(PPh3)4, n-BuNH2, 85 °C, reflux, 10 h, 58%; (iii) MsCl, Et3N, 0 °C, 10 min, 95%; (iv) NaN3, dry DMF, rt, 3 h, 89%; (v) PPh3, moist THF, rt, overnight, 78%; (vi)
ethylmalonyl chloride, dry DCM, Et3N, 0 °C, 15 min, 83%; (vii) PPTS, EtOH, 50 °C, 3 h, 68%; (viii) Dess–Martin periodinane, dry DCM, rt, 2 h, 98%; (ix) see Table 1, 65–91%; (x)
LiOH, moist THF, rt, 1 h, 81%; (xi) H2, 10% Pd-charcoal, dry EtOH, rt, 93%.
Table 1
Reaction parameter of intramolecular Knoevenegal reaction
Solvents
Additives
Concn (M)
Reaction time(h)
% of Yield
THF
THF
0.1(M)Et3N
0.1(M)Et3N then .04(M)AcOH
0.08(M)Et3N
0.1(M)Et3N then .04(M)AcOH
0.1(M)Et3N
0.1(M)Et3N then .04(M)AcOH
0.1(M)Et3N then .04(M)HCl
0.1(M)HCl
4.1 Â 10À3
3.8 Â 10À3
4.1 Â 10À3
3.8 Â 10À3
4.1 Â 10À3
3.8 Â 10À3
3.8 Â 10À3
4.1 Â 10À3
18
2
22
4
25
5
3
65
91
55
84
59
85
72
DCM
DCM
CH3CN
CH3CN
THF
THF
No reaction
PhSH, K CO
2
3
HN
O
DCM, rt., 2hr.
( 80% )
HN
O
13
COOEt
SPh
COOEt
COOEt
12
HN
COOEt
K2CO3
O
DCM, rt., 2hr.
( 85% )
COOEt
EtOOC
COOEt
14
Scheme 2. Michael addition into thiophenol and diethyl malonate.
the molecule to bind via hydrophobic interaction. His49 is also
found to exist within the hydrogen bonding distance from CEMA
(Fig. 5). No direct interactions between the active site residues
(Cys11, Arg17 and Asp126) and CEMA is observed indicating the
additional support in favor of non-competitive mode of interaction.
It seems that CEMA probably inhibits the reaction by creating a
partition between catalytic residues Cys11 and Asp126. The above
observations categorically explain the results of inhibition kinetics.
Observed interactions between CEMA and MPtpA are shown in
Table 3.

K. Chandra et al. / Bioorg. Med. Chem. 19 (2011) 3274–3279
3277
Table 2
Inhibition kinetics parameter of CEMA and CNEMA
Compound
Mode of inhibition
IC50
(
lM)
Ki (lM)
1
2
Non-competitive
Non-competitive
110.2 10.5
235.6 12.4
22.5 1.2
101.4 10.8
Figure 3. DSC plot of CEMA.
Comparative docking analysis also reveals that unlike CNEMA,
CEMA has greater impact on MPtpA mainly because of its confor-
mational preferences controlled by enediyne framework. Actually,
the loop undergoes facile conformational change upon CEMA bind-
ing that allows associated amino acid residues to form an extensive
hydrophobic interaction. Thus, the size, shape and selectivity of the
CEMA towards MPtpA have given a new dimension to utilize the
enediyne framework for the discovery of potent drug molecules.
3. Conclusion
A novel enediyne malonamic acid conjugates has been identi-
fied to be a potential candidate for MPtpA inhibitor and possible
protein modulator. The enediyne behaves as a non-competitive
inhibitor. Since, the non-competitive type inhibition belongs to
reversible inhibition, so the possibility of diradical induced irre-
versible inhibition has been excluded which was also supported
by the very high onset temperature for BC. Thus, a new role of
enediyne as an enzyme inhibitor has been comprehensively estab-
lished after comparison with saturated non enediyne based ana-
logue. Further studies are on the way to bring down the
inhibition to nanomolar range as well as deactivation the MPtpA’s
function by suitable modification in the structure of enediyne
framework.
Figure 5. Docking of CEMA on MPtpA is showing the major interactions: PTP loop is
shown in red color.
following usual protocols. Purification by Column chromatographic
separations were performed using 60–120, 100–200 and 230–400
mesh size silica gel. Further purification was carried out with the
help of HPLC. TLC was performed on aluminium-backed plates
coated with Silica Gel 60 with F254 indicator. The 1H NMR spectra
were measured on 400 MHz and 13C NMR spectra were measured
with 100 MHz using CDCl3 as a solvent in Brucker NMR instru-
ments unless otherwise mentioned. IR spectra were recorded using
Thermo Nicolet FT-IR using KBr pellet and the peaks are expressed
in cmÀ1. Finally, Mass spectra were analyzed by Waters LCT mass
spectrometer. Enzyme purity was assessed by SDS–PAGE and pro-
tein concentration was determined from the absorbance at
280 nm. 0.1 M Tris–HCl pH 7.5 having ionic strength 0.15 M of
NaCl and 1 mM EDTA was used for the kinetic study with all the
synthesized compounds.
4. Experimental
4.1. General experimental methods
All reactions were conducted with oven-dried glassware under
an atmosphere of argon (Ar). Commercial grade reagents were
used without further purification. Solvents were dried and distilled
2500
2000
1500
1000
500
0
I=200 M
I=150 M
I=60 M
2000
1500
1000
500
I=45 M
I=30 M
I=100 M
I=0 M
I=0 M
0
-0.02
0
0.02 0.04 0.06 0.08 0.1
1/[S] mM-1
-0.02
0
0.02 0.04 0.06 0.08 0.1
1/[S] mM-1
-500
-500
Figure 4. Line Weaver Burk plot of CEMA (left) and CNEMA (right).

3278
K. Chandra et al. / Bioorg. Med. Chem. 19 (2011) 3274–3279
Table 3
Docking interaction analysis during complexation between MPtpA and compound 1
Interacting protein residues
Interacting counterpart of CEMA
Interaction distance (Å)
Remarks on interaction pattern
Gly13
Ile15
Trp48
Trp48 (OH)
His49 (NH2)
Tyr128
Benzene ring and enediyne moiety
Benzene ring and enediyne moiety
Cyclic enediyne moiety
Acid group
Amide hydrogen
Cyclic enediyne moiety
Carbonyl oxygen
3.3
3.5
4.1
2.9
3.5
3.4
3.6
Hydrophobic
Hydrophobic
Hydrophobic
Hydrogen bonding
Hydrogen bonding
Hydrophobic &
Polar (electrostatic)
p-stacking
Tyr129(OH)
4.2. Selected NMR data
4.2.1. Compound (7)
50 °C. The solution was concentrated and the crude residue was
subjected to flash column chromatography using 2:1 EtOAc/Hex-
ane (Rf = 0.3) as eluent, afforded off-white semi-solid compound
10 (68%); cmax 1182, 1675, 2395, 3454; dH (200 MHz) 1.17 (3H, t,
J = 7.2 Hz, CH2CH3), 2.67 (2H, t, J = 6.2 Hz, NHCH2CH2), 3.31 (2H,
s, COCH2CO), 3.50 (2H, t, J = 6.2 Hz, NHCH2CH2), 4.09 (2H, q,
J = 7.2 Hz, CH2CH3), 4.50 (1H, s, CH2OH), 7.16–7.38 (4H, m, Ar-H),
7.62 (1H, br s, CH2OH); dC (50 MHz) 13.9, 20.3, 38.6, 41.6, 51.2,
61.7, 80.7, 83.8, 91.3, 91.5, 125.2, 125.9, 127.6, 128.0, 131.7,
165.8, 169.3; HRMS calcd for C18H19NO4+H+ 314.1314, found
314.1316.
To dry DMF (10 mL) solution of mesylate 6 (250 mg, 0.7 mmol)
and sodium azide (68 mg, 1.1 mmol) was stirred 3 h at room tem-
perature. The mixture was partitioned between EtOAc and water
(50 mL each). The organic layer was washed thoroughly with brine
and dried over Na2SO4 and concentrated in vacuo. Finally, the
crude residue was purified by column chromatography using 1:7
EtOAc/Hexane (Rf = 0.6) as eluent to afford yellowish viscous com-
pound 7 (89%); cmax 1162, 1738, 2131, 2405; dH (200 MHz) 1.53–
1.86 (6H, m, 3 Â CH2-THP), 2.76 (2H, t, J = 7.0 Hz, NHCH2CH2),
3.48–3.61 (3H, m, NHCH2CH2, 1 Â CH2-THP), 3.82–3.92 (1H, m,
CH2-THP), 4.55 (1H, d, J = 3.0 Hz, CH2O-THP), 4.98 (1H, m, CH-
THP), 7.21–7.45 (4H, m, Ar-H); HRMS calcd for C18H19N3O2+H+
310.1477, found 310.1485.
4.2.5. Compound (11)
To the dry DCM solution (10 mL) of compound 10 (50 mg,
0.2 mmol), Dess–Martin periodinate (100 mg, 0.2 mmol) was
added and stirred for 2 h at room temperature. The reaction was
quenched by adding of aqueous solution of Na2S2O3. It was then
washed with NaHCO3 solution followed by brine and finally ex-
tracted with DCM. The DCM was concentrated in vacuo and the
product was isolated by flash column chromatography using 1:1
EtOAc/Hexane (Rf = 0.5) as eluent to afford off-white semi-solid
compound 10 (98%); cmax 1245, 1632, 1710, 2805; dH (200 MHz):
0.99–1.02 (3H, dd, J1 = J2 = 1.8 Hz, CH2CH3), 2.52 (2H, t, J = 6.2 Hz,
NHCH2CH2), 3.16 (2H, s, COCH2CO), 3.32 (2H, t, J = 6.2 Hz,
NHCH2CH2), 3.86–3.94 (2H, ddd, J1 = J2 = J3 = 1.6 Hz, CH2CH3),
6.97–7.49 (5H, m, Ar-H, NH), 9.24 (1H, s, CHO); dC (50 MHz) 14.0,
20.8, 38.5, 41.9, 61.1, 79.4, 91.1, 93.5, 93.7, 121.4, 127.7, 128.1,
128.4, 128.6, 131.0, 165.9, 168.4, 177.2; HRMS calcd for
4.2.2. Compound (8)
To a THF (10 mL) solution of compound 7 (225 mg, 0.7 mmol),
PPh3 (288 mg, 1.1 mmol) followed by two drops of water were stir-
red for overnight. THF was removed by evaporation and the target
compound was isolated pure as off-white solid from the crude
mixture by column chromatography (100–200 silica gel, yield
78%) using 5% MeOH in DCM (Rf = 0.2); dH (200 MHz) 1.55–1.80
(6H, m, 3 Â CH2-THP), 2.72 (2H, t, J = 6.2 Hz, NHCH2CH2), 3.04
(2H, t, J = 6.2 Hz, NHCH2CH2), 3.47–3.59 (1H, ddd, J1 = 2.4 Hz,
J2 = 2.6 Hz, J3 = 3.4 Hz, CH2-THP), 3.80–3.91 (1H, ddd, J1 = 2.4 Hz,
J2 = 2.6 Hz, J3 = 3.4 Hz, CH2-THP), 4.36 (1H, br s, NH), 4.52 (1H, d,
J = 2.6 Hz, CH2O-THP), 4.95 (1H, m, CH-THP), 7.20–7.42 (4H, m,
Ar-H).
C
18H17NO4+H+ 312.1158, found 312.1161.
4.2.6. Compound (12)
4.2.3. Compound (9)
The compound 11 (40 mg, 0.1 mmol) was dissolved under high
dilution condition in distilled THF (30 mL) in presence of a buffer
In dry DCM solution (40 mL) of amine 8 (200 mg, 0.7 mmol) and
triethyl amine (223
lL, 1.6 mmol), ethylmalonyl chloride (100
lL,
mixture of Et3N (35 lL, 2.5 mmol, 0.08 M) and acetic acid (60 lL,
0.7 mmol) was added dropwise at À10 °C for 15 min. The reaction
mixture was stirred for further 15 min at 0 °C. The reaction was
quenched by adding water and then extracted with DCM after
sequential washing with 1 (N) HCl and brine, respectively. The
DCM solution was concentrated in rotavapor and dried in vacuo.
The crude residue was finally purified by flash column chromatog-
raphy using 1:1 EtOAc/Hexane (Rf = 0.3) as eluent to afford yellow-
ish viscous compound 9 (83%); cmax 1251, 1744, 2229, 2455; dH
(200 MHz) 1.23 (3H, m, CH2CH3), 1.54–1.78 (6H, m, 3 Â CH2-
THP), 2.68 (2H, t, J = 6.4 Hz, NHCH2CH2), 3.31 (2H, s, COCH2CO),
3.53 (2H, t, J = 6.4 Hz, NHCH2CH2), 3.79–4.21 (4H, m, CH2CH3,
CH2-THP), 4.51 (2H, ABq, J1 = J2 = 2.6 Hz, CH2O-THP), 4.94 (1H, m,
CH-THP), 7.18–7.42 (4H, m, Ar-H); dC (50 MHz) 14.0, 19.0, 20.4,
25.3, 30.2, 38.5, 41.5, 54.7, 61.5, 61.9, 84.7, 88.6, 91.2, 96.6,
125.1, 126.0, 127.5, 128.1, 131.8, 132.0, 165.3, 169.0; HRMS calcd
for C23H27NO5+H+ 398.1889, found 398.1893.
1 mmol, 0.03 M AcOH). The mixture was stirred at room tempera-
ture as indicated. The reaction was quenched by adding water. It
was then washed with 1 N HCl followed by brine and finally ex-
tracted with EtOAc before drying over anhydrous Na2S2O3 solution.
The organic layer was concentrated in vacuo and crude mixture
was then purified in flash chromatography (230–400 silica gel)
using EtOAc/Hexane mixture (1:2) solvent (Rf = 0.4) yielding 12
as a off-white solid compound (65–91%); mp 143 °C; cmax 1196,
1665, 1764, 2205; dH (200 MHz) 1.36 (3H, t, J = 7.2 Hz, CH2CH3),
2.86 (2H, t, J = 6 Hz, NHCH2CH2), 3.62 (2H, t, J = 6 Hz, NHCH2CH2),
4.34 (2H, q, J1 = J2 = 7 Hz, CH2CH3), 6.71 (1H, br s, NH), 7.16 (1H,
s, CCCH), 7.30–7.59 (4H, m, Ar-H); dC (50 MHz) 14.0, 31.8, 38.5,
61.7, 81.7, 88.1, 93.2, 103.5, 121.4, 125.2, 127.3, 127.7, 129.8,
130.2, 132.1, 137.7, 163.7, 164.2; HRMS calcd for C18H15NO3+H+
294.1052, found 294.1055.
4.2.7. Compound (1)
4.2.4. Compound (10)
To a THF (10 mL) solution of compound 12 (50 mg, 0.2 mmol),
LiOH (6 mg, 0.2 mmol) followed by two drops of water were stirred
for 1 h. Then THF was removed in rotavapour and the crude
mixture was transferred to filtration column chromatography
To the ethanolic solution (15 mL) of compound 9 (100 mg,
0.2 mmol) and pyridinium p-toluene sulphonate (PPTS) (64 mg,
0.2 mmol) were added and the solution was stirred for 2 h at

K. Chandra et al. / Bioorg. Med. Chem. 19 (2011) 3274–3279
3279
(230–400 silica gel) and compound 1 was isolated as pure off-white
Acknowledgments
solid (81%) using 4:1 EtOAc/Hexane (Rf = 0.2) as eluent; mp 128 °C;
cmax 1210, 1678, 2205, 3285; dH 2.81 (2H, t, J = 6 Hz, NHCH2CH2),
3.74 (2H, q, J1 = J2 = 6 Hz, NHCH2CH2), 7.29–7.67 (4H, m, Ar-H),
7.93 (1H, s, CCCH), 8.65 (1H, br s, COOH); dC 31.9, 37.6, 83.6,
89.2, 93.0, 110.7, 121.4, 124.2, 127.2, 127.5, 128.5, 130.5, 131.2,
133.3, 165.1, 166.1; HRMS calcd for C16H11NO3+H+ 266.0739,
found 266.0741.
K.C. and D.D. thank Council of Scientific and Industrial Research
(CSIR) and Department of Biotechnology (DBT), Government of In-
dia, respectively, for fellowship. Central Research Facility and
Department of Chemistry, IIT Kharagpur are thanked for providing
all the instrumental facilities. A.B. and A.K.D. gratefully acknowl-
edge the support of DBT for providing the necessary research grant.
4.2.8. Compound (2)
Supplementary data
A two-necked RB flask containing a solution of compound 1
(40 mg, 0.2 mmol) in 10 mL dry ethanol was evacuated for
10 min. Then, under stirring condition, hydrogen gas was purged
through the solution for 15 min. A pinch of 10% Pd-charcoal was
poured quickly into it. The reaction was left stirring at room tem-
perature for 2 h under hydrogen atmosphere. The reaction mixture
was filtered through celite bed and the filtrate was concentrated in
vacuo yielding compound 2 as pure white solid (93%); mp 161 °C;
cmax 1640, 1744, 2358, 3443; cmax 1225, 1520, 2992, 3246; dH
1.47–1.71 (6H, m, 2 Â ArCH2CH2, NHCH2CH2), 1.96 (1H, m,
COCHCH2), 2.12 (1H, m, COCHCH2), 2.43–2.71 (4H, m,
2 Â ArCH2CH2), 3.12–3.34 (2H, m, NHCH2CH2), 3.76–3.78 (1H, m,
COCHCH2), 7.03–7.04 (4H, m, Ar-H), 8.54 (1H, br s, COOH); dC
25.9, 28.4, 29.7, 29.8, 32.0, 32.2, 37.1, 54.8, 125.6, 125.7, 129.4,
129.5, 140.2, 140.3, 169.3, 177.2; HRMS calcd for C16H21NO3+H+
276.1521, found 276.1525.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.03.024.
References and notes
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4.2.9. Compound (13)
To dry DCM solution (10 mL) of compound 12 (50 mg,
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(28 mg, 0.2 mmol) were stirred 2 h at room temperature. The mix-
ture was partitioned between DCM and water (50 mL each). The
organic layer was washed thoroughly with brine solution and dried
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pound (13, 80%); cmax 1179, 1761, 2291, 2560; dH (200 MHz): [Ma-
jor isomer] 1.37 (3H, t, J = 7.2 Hz, CH2CH3), 2.71 (2H, dd,
J1 = J2 = 4.4 Hz, NHCH2CH2), 3.46–3.63 (2H, dd, J1 = J2 = 4.4 Hz,
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24H21NO3S+H+ 404.1242, found
404.1248.
4.2.10. Compound (14)
To the stirring solution of compound 12 (50 mg, 0.2 mmol) in
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l
K2CO3 (28 mg, 0.2 mmol) were added at 0 °C. The reaction was
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and evaporated in vacuo. A filtration column chromatography was
performed using 3:1 EtOAc/Hexane (Rf = 0.5) as eluent resulting
yellowish viscous compound 14 (85%); cmax 1640, 1744, 2358,
3443, 1198, 1673, 1515, 2228; dH 1.18–1.33 (9H, m, 3 Â CH2CH3),
2.72–2.83 (2H, m, NHCH2CH2), 3.46–3.63 (2H, m, NHCH2CH2),
3.81 (1H, d, J = 6.4 Hz, COCHCO), 3.92 (1H, d, J = 9.2 Hz, COCHCO),
4.13–4.30 (7H, m, 3 Â CH2CH3, CHCHCH), 7.19–7.33 (4H, m, Ar-
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19.4, 33.9, 37.9, 53.0, 53.9, 54.9, 61.7, 61.9, 62.0, 82.5, 85.0, 88.3,
91.2, 127.8, 128.1, 129.9, 130.1, 130.2, 131.3, 166.0, 167.0, 167.2,
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