SPF32629A and SPF32629B: Enantioselective synthesis, determination of absolute configuration, cytotoxicity and antibacterial evaluation

Article abstract of DOI:10.1016/j.ejmech.2011.02.039

We report herein an efficient enantioselective synthesis of SPF32629A and SPF32629B through one-pot enantioselective reduction and protecting-group-free regioselective O-acylation strategy. The absolute configuration of the enantiomerically pure isomers w

Full text of DOI:10.1016/j.ejmech.2011.02.039

European Journal of Medicinal Chemistry 46 (2011) 1803e1812
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
SPF32629A and SPF32629B: Enantioselective synthesis, determination of absolute
configuration, cytotoxicity and antibacterial evaluation
,
,
a
,
b
Srinivasa Rao Vegi a b, Shanthaveerappa K. Boovanahalli a , Balaram Patro , K. Mukkanti
*
*
a Medicinal Chemistry Laboratory, GVK Biosciences Private Limited, Plot No.28, IDA, Nacharam, Hyderabad 500 076, AP, India
b Chemistry Division, Institute of Science and Technology, JNT University, Kukatpally, Hyderabad 500 072, AP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein an efficient enantioselective synthesis of SPF32629A and SPF32629B through one-pot
enantioselective reduction and protecting-group-free regioselective O-acylation strategy. The absolute
configuration of the enantiomerically pure isomers was established by Mosher ester analysis. The
inhibitory potencies of the synthesized compounds were assayed in vitro against a panel of microor-
ganisms and against A549 human lung adenocarcinoma cell line. Compounds 2, 11 and 12 displayed
moderate to potent antibacterial activity against all the tested strains and compounds 7, 8, 2, 11 and 12
exhibited significant cytotoxicity in a dose-dependent manner with an IC50 values ranging from 2.92 to
Received 25 October 2010
Received in revised form
14 February 2011
Accepted 15 February 2011
Available online 23 February 2011
Keywords:
SPF32629A
SPF32629B
4.14 mg/ml and 8e11 mM.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Enantioselective synthesis
Regioselective O-acylation
Antibacterial
Cytotoxicity
Mosher analysis
1. Introduction
the disease [12e14]. In this regard, numerous anticancer agents
have been developed for the treatment of cancer; however, these
drugs suffer from various side effects [15e19]. Thus, there is
a pressing need for potent, safe, and selective anticancer agents
with high potency to overcome primary or secondary resistance
mechanisms evolved in the cancer cells [20].
Many antibacterial agents reported in the literature are semi-
synthetic modifications of various natural compounds and
substances produced from bacterial origin. Compounds 1 and 2
(SPF32629A and B respectively, Fig. 1) having 2-pyridone skeletons,
are potent natural products of bacterial origin, isolated from the
cultured broth of Penicillium sp. Both the compounds exhibited
diverse range of interesting biological properties including signifi-
Despite major breakthroughs in many areas of modern medi-
cine, significant rise in multi-drug resistant microbial infections
and cancer has become an economic as well as a serious health care
problem. In some microbial infections, strains of microbes have
become resistant to all available antimicrobial agents. Resistant
organisms cause infections that are more difficult to treat, requiring
drugs that are often less readily available, more expensive and more
toxic [1e6]. For example, the emergence of multi-drug resistant
strains of Gram-positive bacterial pathogens such as methicillin-
resistant Staphylococcus aureus is often a source for severe and life-
threatening infections [7e11]. Consequently, there is an increasing
need for the development of new chemical entities as potent
antimicrobial agents. Likewise, cancer is the leading cause of
human death, after cardiovascular diseases. Although there has
been increasing sophistication of conventional therapeutic strate-
gies, such as surgery, radiotherapy, and chemotherapy, such
approaches are capable of remediating approximately half of
cancer patients, while over 40% of patients are still likely to die from
cant human chymase (IC50 ¼ 0.25 and 0.42
m
g/mL, respectively) and
human elastase inhibitory activities (IC50 ¼ 4.9 and 4.5
mg/mL,
respectively) and therefore expected to be useful for the treatment
of inflammation-related various diseases and procoagulant [21,22].
Based on these findings, we hypothesized that SPF32629A and B
which were primarily isolated from the cultured broth of Penicil-
lium sp. may exert antimicrobial activity. In view of these above
mentioned facts and with the objective to develop a potent anti-
microbial agent coupled with anticancer activities, we were inter-
ested in exploring antimicrobial and cytotoxicity activities of novel
human chymase inhibitors SPF32629A and B. The synthesis of these
* Corresponding authors. Tel.: þ91 40 6451 1333x1163; fax: þ91 40 2715 2999.
E-mail address: sveerappa.boovanahal@gvkbio.com (S.K. Boovanahalli).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.039

1804
S.R. Vegi et al. / European Journal of Medicinal Chemistry 46 (2011) 1803e1812
without further purification. As expected the resulting crude alcohol
6 underwent coupling with isovaleric acid in a regioselective
manner to produce racemic SPF32629A in good yield with high
purity. Then, we undertook the preparation of the enantiomerically
pure isomers via enantioselective reduction of prochiral keto
compound 5. Table 1 illustrates the enantioselective reduction of
prochiral keto compounds 5 and 9 with various chiral reducing
agents including (ꢁ)-CBS in presence of Catecholborane, (ꢁ)-CBS in
presence of BH3-THFand (ꢁ)-DIP-chloride. Initial experiments using
(þ) or (ꢂ)-CBS in presence of catecholborane did not afford any
reduced alcohol (entry 1e2). On the other hand, reduction of ketone
5 with (þ) or (ꢂ)-CBS in presence of BH3-THF followed by subse-
quent regioselective acylation led to the formation of racemic 1
(entry 3e4). Gratifyingly, enantioselective reduction of 5 was
successfully accomplished using (þ) and (ꢂ)-DIP-Chloride. Thus,
reaction of ketone 5 with 1.5 equivalents of (þ) or (ꢂ)-DIP-Chloride
inTHFat 0 ꢀC toroom temperatureover 12 h followed bysubsequent
reaction with isovaleric acid afforded desired compounds 7 and 8 in
78.32% and 84.79% ee., respectively (entries 5 and 7). High enan-
tiomeric purity (96% ee.) of 7 and 8 was achieved by performing the
reduction at low temperatures ꢂ40 to ꢂ10 ꢀC followed by subse-
quent acylation at 0 ꢀC to rt reaction conditions (entries 6 and 8).
Following similar sequence of reactions under aforementioned
reaction conditions, the synthesis of enantiomerically pure enan-
tiomers of SPF32629B (>98% ee., Table 1, entries 9 and 10, respec-
tively) was accomplished starting from compound 9 (Scheme 2). The
optical purity was determined by chiral HPLC on Chiral PAK AD-H
column. The absolute configuration of pure enantiomers of alcohols
6 and 10 was established by following well documented Mosher
protocol [25e27]. Thus, enantiomerically pure isomers of (þ) or
(ꢂ)ꢂ6 and (þ) or (ꢂ)ꢂ10 were reacted individually with (R)-
Fig. 1. Structures of SPF32629A and SPF32629B.
unique structures constitutes a considerable challenge and in view
of their interesting structures coupled with significant biological
properties, we have recently reported the first total synthesis of
racemic 1 and 2 [23,24]. However, there is no report on the deter-
mination of absolute configuration, antimicrobial and cytotoxicity
evaluation of these bacterial origin natural products. Herein, we
wish to report the first enantioselective synthesis, absolute
configuration elucidation, cytotoxicity and antimicrobial evaluation
of these molecules.
2. Chemistry
The enantioselective synthesis of SPF32629A and B was accom-
plished through a one-pot enantioselective reduction and regiose-
lective acylation approach as depicted in synthetic Schemes 1 and 2.
The key starting materials 3 and 9 were prepared by following our
previously described protocols [23,24]. In subsequent experiments,
step wise conversion of 3 to 5 was achieved in two steps. Thus, when
a solution of 3 in aqueous acetic acid was heated in a sealed tube at
180e190 ꢀC for a period of 6 h afforded debenzylated product 4,
which upon prolonged heating for further 7 h produced 5. Alterna-
tively, compound 5 can be more conveniently prepared by effecting
debenzylation and dechlorination in a single step. This involved the
reaction of 3 with aqueous acetic acid under similar conditions for
extended reaction time (12 h) to obtain 5 in much higher yields
without the need for isolation of intermediate 4. As reported
previously, reduction of 5 under standard sodium borohydride
mediated reduction conditions furnished the requisite crude alcohol
6 in quantitative yield, which upon extractive isolation was acylated
(þ)-
a-Methoxy-a-(trifluoromethyl)phenylacetic acid [(R)-MTPA] to
obtain pure R-MTPA esters in good yield. As per Mosher’s model
[25e27] the conformationwith the CF3 group aligned synperiplanar
(sp) with the carbonyl group is more stable and the substituents L1
and L2 of the esters prepared from (R)-MTPA are located in such
a way that the 1H NMR signal of the L2 residue is shielded by the
phenyl ring of MTPA in the sp conformer (Fig. 2). In our previous
reports [23,24], based on 2D NMRexperiments we have assigned the
signals of pyridone protons as indicated on the spectra. In the
Scheme 1. Reagents and conditions: (a) AcOH, H2O, Sealed tube, 180e190 ꢀC, 6 h, 83%; (b) AcOH, H2O, sealed tube, 180e190 ꢀC, 7 h, 76%; (c) AcOH, H2O, sealed tube, 180e190 ꢀC,
12 h, 90%; (d) (i) for compound 1, NaBH4, THF:MeOH (8:2), 0e10 ꢀC, 2 h, 100%; (ii) for compound 7, (ꢂ)-DIP-Chloride, THF, ꢂ40 to ꢂ10 ꢀC, 12 h, 100%; (iii) for compound 8, (þ)-DIP-
Chloride, THF, ꢂ40 to ꢂ10 ꢀC, 12 h, 100%; (e) isovaleric acid, EDC.HCl, DMAP (cat), THF:DCM (1:2), 0 ꢀC to rt, 16 h, 84%.

S.R. Vegi et al. / European Journal of Medicinal Chemistry 46 (2011) 1803e1812
1805
(Gram ꢂve), Bacillus subtilis (Gram þve), Staphylococcus epidermidis
(Gram þve), Klebsiella pneumonia (Gram ꢂve), Listeria mono-
cytogenes (Gram þve), Salmonella typhimurium (Gram ꢂve), S. aureus
(Gram þve) and Methicillin-resistant S. aureus Gram þve strains
such as MRSA-I (TB 3G), MRSA-II (TB 4G) and MRSA-IV (TB 5G). The
antibacterial tests were carried-out by disc diffusion and micro-
dilution methods [28,29]. Amikacin [30] a powerful aminoglycoside
antibiotic was used as positive reference standard (100 mg/disc) for
comparison, which displayed significantly high inhibitory potency
in all the tested organisms with the exception of MRSA-IV (TB5G).
The antibacterial activity was evaluated by measuring the diameter
of zone of inhibition against test organisms. The MIC values were
determined by the twofold serial dilution technique in Muel-
lereHinton broth. The lowest inhibitory concentrations of samples
were noted by seeing the growth (in terms of turbidity). The
minimum concentration which results in the lowest growth is said
to be as minimum inhibitory concentration. The diameter of zone of
Scheme 2. Reagents and conditions: (f) (i) for compound 2, NaBH4, THF:MeOH (8:2),
0e10 ꢀC, 4 h, 100%; (ii) for compound 11, (ꢂ)-DIP-Chloride, THF, ꢂ40 to ꢂ10 ꢀC, 12 h,
100%; (iii) for compound 12, (þ)-DIP-Chloride, THF, ꢂ40 to ꢂ10 ꢀC, 12 h, 100%; (g)
isovaleric acid, EDC.HCl, DMAP (cat), THF:DCM (1:2), 0 ꢀC to rt, 12 h, 87%.
inhibition in mm and the minimum inhibition concentration in mg of
the tested compounds are tabulated in Tables 2 and 3, respectively.
From the antibacterial data through zone of inhibition studies
(Table 2), it is evident that all of the tested compounds exhibited
good antibacterial activity, with the exception of compound 1, which
displayed moderate activity (13 mm) against only one organism
S. typhimurium. Compounds 7, 8, 2, 11 and 12 emerged as the most
potent antibacterial agents amongst the tested compounds.
Compound 7 exhibited significant inhibitory activity against E. coli
(26 mm), B. subtilis (23 mm) and K. pneumonia (25 mm). Interest-
ingly, compound 7 also displayed good inhibitory activity (zone of
inhibition ¼ 26 mm) against Methcillin resistant S. aureus II (TB4 G),
a multi-drug resistant strain of Gram-positive bacterial pathogen, at
spectrum of (þ)-6-diastereomer, we could notice that the chemical
shiftsof the pyridoneand methoxy protonsof thetwodiastereomers
0
0
0
(H3eH5 and H3 -H5 ) and (H23 and H23 ) respectively, were observed
at higher field than the (ꢂ)-6-diastereomer, indicating that the
pyridone moiety is shielded by phenyl residue of MTPA (Fig. 3).
Based on the structure of the more stable sp conformer, we
could attribute the (R,S) configuration to the product presenting
the more shielded signals of the pyridone protons in the 1HNMR
spectrum ie., (þ)-6-diastereomer is assigned (R,S) configuration. On
the other hand, the 1H signals of the pyridone protons of the
(ꢂ)-6-diastereomer (major sp conformer) are little affected by the
phenyl residue of MTPA, thus we attribute (R,R) configuration to this
isomer. We could also deduce from the 1H NMR spectrum recorded
on the mixtureof both diastereomers that the chemical shifts are not
depending upon the relative ratio between the two isomers. Simi-
larly, based on the chemical shifts of the pyridone moiety in the
1HNMR spectrum of (þ)- and (ꢂ)-10-(R)-MTPA diastereomers, we
could assign (R,S) and (R,R) configuration for (þ)- and (ꢂ)-10-dia-
stereomers, respectively. Thus, we attribute (S) configuration for
compounds 7 and 11, and (R) configuration for compounds 8 and 12.
The spectral data including IR, MASS, 1H NMR, 13C NMR, 19F NMR,
LCMS and SOR of all the synthesized compounds were in consistent
with their structures. This constitutes the first reported enantiose-
lective synthesis and absolute configuration elucidation of
SPF32629A and B.
the tested concentration of 1000 mg/disc. Compound 8 demon-
strated good inhibitory activities against E. coli (20 mm), B. subtilis
(19 mm), S. epidermis (17 mm), K. pneumonia (24 mm) and
S. typhimurium (20 mm). On the other hand, Compounds 2,11 and 12
displayed moderate to potent antibacterial activity against all the
tested organisms with the zone of inhibition ranging from 10 to
21 mm at the tested concentration of 1000 mg/disc. It is worth noting
that, these compounds also moderately inhibited all the tested
Methcillin resistant S. aureus strains MRSA-I (TB3G), MRSA-II (TB4G)
and MRSA-IV (TB5G), when compared to the reference standard
Amikacin which was inactive against MRSA-IV (TB5G) and active
against rest of the Methcillin resistant S. aureus strains. In view of
their significant antibacterial activity evidenced by zone of inhibi-
tion studies against all the tested organisms, the most active
Compounds 2,11 and 12 were further evaluated for their MIC values
and the results are tabulated in
the three compounds exhibited variable inhibition activities against
the tested strains and the good inhibitory activity (25 g/mL) was
found against organisms B. subtilis, E. coli and S. epidermidis for
Compounds 2, 11 and 12, respectively.
mg/mL values. As shown inTable 3, all
3. Antibacterial activity
m
All the newly synthesized compounds were assayed in vitro for
their antibacterial activity against a panel of selected Gram-positive
and Gram negative mircroorganisms including Escherichia coli
Table 1
Enantioselective reduction of prochiral keto compounds 5 and 9.
Entry
Reagents
Reaction conditions
Compound (ee)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
R-(þ)-CBS (0.1 eq), Catecholborane (1.2 eq), Ketone 5, then Isovaleric acid, EDC.HCl
R-(ꢂ)-CBS (0.1 eq), Catecholborane (1.2 eq), Ketone 5, then Isovaleric acid, EDC.HCl
R-(þ)-CBS (0.1 eq), BH3.THF (1.5 eq), Ketone 5, then Isovaleric acid, EDC.HCl
R-(ꢂ)-CBS (0.1 eq), BH3.THF (1.5 eq), Ketone 5, then Isovaleric acid, EDC.HCl
(ꢂ)-DIP-chloride (1.5 eq), Ketone 5, then Isovaleric acid, EDC.HCl
(ꢂ)-DIP-chloride (1.5 eq), Ketone 5, then Isovaleric acid, EDC.HCl
(þ)-DIP-chloride (1.5 eq), Ketone 5, then Isovaleric acid, EDC.HCl
(þ)-DIP-chloride (1.5 eq), Ketone 5, then Isovaleric acid, EDC.HCl
(ꢂ)-DIP-chloride (1.5 eq), Ketone 9, then Isovaleric acid, EDC.HCl
(þ)-DIP-chloride (1.5 eq), Ketone 9, then Isovaleric acid, EDC.HCl
ꢂ10 to rt, 12 h, then 0 ꢀC to rt, 48 h
ꢂ10 to rt, 12 h, then 0 ꢀC to rt, 48 h
ꢂ10 to rt, 4 h, then 0 ꢀC to rt, 48 h
ꢂ10 to rt, 4 h, then 0 ꢀC to rt, 48 h
No reaction
No reaction
1
1
0
ꢀC to rt, 12 h, then 0 ꢀC to rt, 48 h
7 (78.32%)
7 (96.05%)
8 (84.79%)
8 (96.01%)
11 (98.28%)
12 (98.55%)
ꢂ40 to ꢂ10 ꢀC, 12 h, then 0 ꢀC to rt, 48 h
0
ꢀC to rt, 12 h, then 0 ꢀC to rt, 48 h
ꢂ40 to ꢂ10 ꢀC, 12 h, then 0 ꢀC to rt, 48 h
ꢂ40 to ꢂ10 ꢀC, 12 h, then 0 ꢀC to rt, 12 h
ꢂ40 to ꢂ10 ꢀC, 12 h, then 0 ꢀC to rt, 12 h

1806
S.R. Vegi et al. / European Journal of Medicinal Chemistry 46 (2011) 1803e1812
Fig. 2. Shielding effects of (R)- and (S)-MTPA esters.
4. Cytotoxicity studies
absolute configuration of the synthesized compounds was estab-
lished by following well documented Mosher ester analysis. Thus,
based on the Mosher ester analysis results by comparing 1HNMR
chemical shifts, we could assign (S) configuration for compounds 7
and 11, and (R) configuration for compounds 8 and 12. The newly
synthesized compounds were evaluated for their ability to display
antibacterial activity against a panel of selected microorganisms and
cytotoxicity against A549 human lung adenocarcinoma cell line,
which resulted in the identification of potent antimicrobial and
anticancer agents. Compounds 2, 11 and 12 displayed potent anti-
bacterialactivityagainstE. coli, B. subtilis, S. epidermidis, K. pneumonia,
L. monocytogenes, S. typhimurium and S. aureus with the zone of
inhibition ranging from 13 to 21 mm at the tested concentration of
The newly synthesized compounds were evaluated for their
ability to display cytotoxicity against A549 human lung adenocar-
cinoma cell line and the results are expressed as IC50 values in
mg and
mm (Table 4). Etoposide or VP-16-213, a well known anticancer drug
was used as the positive control for comparison [31]. The viability of
the cells was assessed by MTT ((3, 4, 5-dimethylthiazol-2-yl)-
2-5diphenyltetrazolium bromide) assay, which is based on the
reduction of MTT by the mitochondrial dehydrogenase of intact cells
to a purple formazan product [32]. As shown in Table 4 and Fig. 4, all
the tested compounds have shown remarkably good cytotoxicity
against A549 lung cancer cell line in a dose-dependent manner. In
particular, Compounds 7, 8, 2,11 and 12 dose-dependently displayed
significant inhibitory activity with an IC50 values ranging from
1000
resistant S. aureus strains. Besides, compounds 2,11 and 12 displayed
good minimum inhibitory concentration (25 g/mL) against organ-
mg/disc and moderately inhibited all the tested methicillin-
2.92e4.14
mg/ml and 8e11
mM, superior to the reference compound
m
Etoposide which exhibited IC50 values of 6.67
m
g/ml and 11 M.
m
isms B. subtilis, E. coli and S. epidermidis, respectively. In addition, all
the testedcompoundsdemonstratedremarkablecytotoxicityagainst
A549 lung cancer cell line. In particular, compounds 7, 8, 2, 11 and 12
displayed significant inhibitory activity superior to the reference
compound Etoposide. In summary, on the basis of preliminary anti-
bacterial and cytotoxicity results we suppose that, either these
compounds display antibacterial and cytotoxicity activities through
non-specific mechanism of action or these compounds exert anti-
bacterial action due to their cytotoxicity activity. Taken together,
compounds reported in this paper could serve as potential leads and
further investigations are warranted to know the mechanism of
action of these molecules.
These findings suggest that all of the tested compounds are likely to
be useful as potential leads for the development of a novel class of
anticancer agents, especially against lung cancer.
5. Conclusions
Insummary, we have developeda novelandefficientapproachfor
the synthesis of pure enantiomers of SPF32629A and SPF32629B by
employing direct enantioselective reduction and regioslective acyl-
ation as the key step, wherein no protecting group was employed for
the amide, acid and phenol functional groups. In addition, the
Fig. 3. 1HNMR Spectra of (R)-(ꢂ)- and (S)-(þ)-6 esters of (R)-MTPA.

S.R. Vegi et al. / European Journal of Medicinal Chemistry 46 (2011) 1803e1812
1807
Table 2
The diameter of inhibition zones in mm of tested Compounds 1, 2, 7, 8, 11, 12 and reference antibiotic discs against tested microorganisms by disc diffusion method.
Microorganism
Diameter of zone inhibition (mm)a
1 ( g/disc) 7 ( g/disc)
1000 1000
m
m
8 (
m
g/disc)
1000
2 (
m
g/disc)
1000
11 (
m
g/disc)
1000
12 (
m
g/disc)
1000
Amikacin (
mg/disc)
500
500
500
500
500
500
100
E. coli
B. subtilis
e
e
e
e
e
7
e
e
e
e
e
e
9
e
e
13
e
e
e
e
16
13
e
26
23
e
13
10
8
14
e
20
19
17
24
e
8
7
8
12
11
9
7
5
15
13
17
21
20
20
15
10
12
12
9
5
18
12
18
20
18
19
16
10
12
10
10
8
12
9
7
9
11
7
6
18
15
20
20
15
20
18
13
10
11
22
21
22
21
20
19
20
17
19
e
S. epidermidis
K. pneumonia
L. monocytogenes
S. typhimurium
S. aureus
MRSA-I (TB3 G)
MRSA-II (TB4 G)
MRSA-IV (TB5 G)
11
13
10
11
7
5
7
6
15
e
25
e
e
e
12
e
20
e
e
e
e
e
e
e
14
-
26
e
e
e
7
6
-
e
5
MRSA, methicillin-resistant Staphylococcus aureus.
a
Values are means of three experiments.
6. Experimental
chromatography was performed using Merck silica gel 60 (230e400
mesh) and eluting solvents are indicated in the procedures.
6.1. Chemistry
6.2. (6-Chloro-4-hydroxypyridin-2-yl)(phenyl)methanone (4)
All non-aqueous reactions were performed under an oxygen-free
atmosphere of argon with rigid exclusion of moisture from reagents
and glassware, unless otherwise noted. The solvents were purified
according to standard procedures prior to use and all commercial
chemicals were used as received. Melting points were determined
on a Buchi B-500 apparatus in open capillary tubes and were
uncorrected. IR spectra were registered on a Perkin Elmer-100
spectrophotometer and nmax is given for the main absorption bands.
1H and 13C NMR spectra were recorded on a Varian Unity-400
instrument at room temperature at 400 and 100 MHz, respectively.
A solution of compound 3 [23] (20 g; 61.77 mmol), acetic acid
(300 mL) and water (10 mL) was heated in a sealed tube at
180e190 ꢀC for 6 h. After completion of the reaction as monitored by
TLC, the reaction mixture was cooled and concentrated in vacuo to
obtain crude solid. Obtained crude solid was purified by flash
column chromatography over silica gel 60 (230e400 mesh) using 2%
Methanol: Dichloromethane as eluent to give compound 4 as off
white solid (12 g; 83%). Rf ¼ 0.5 (5% Methanol:Dichloromethane);
mp 142e146 ꢀC; IR (KBr pellet): nmax 3349, 1651, 1594, 1576, 1430
cmꢂ1; 1H NMR (400 MHz, d6-DMSO):
d 11.73 (1H, s, D2O exchange-
Chemical shifts are reported in d parts per million (ppm) downfield
from tetramethylsilane (TMS) with reference to internal solvent and
coupling constants in Hz. Multiplicities are abbreviated as follows:
singlet (s), doublet (d), triplet (t), quartet (q), quintet (qn), septet
(sp), multiplet (m), and broad (br). Mass spectra were obtained on
a high resolution Micro mass QuattroMicroÔAPI-autospectrometer
usingeither APCI or ESI techniques. Optical rotations weremeasured
on a Rudolph Autopol-V in a 10 dm cell of 10 ml capacity at 25 ꢀC and
able OH), 7.93e7.91 (2H, m), 7.70e7.66 (1H, m), 7.57e7.53 (2H, m),
7.31 (1H, d, J 2 Hz), 7.08 (1H, d, J 2 Hz); 13C NMR (100 MHz, d6-
DMSO):
d 191.84 (C), 166.95 (C), 155.67 (C), 150.23 (C), 135.51 (C),
133.23 (CH),130.45 (2CH),128.28 (2CH),113.50 (CH),111.69 (CH); MS
(APCI) m/z 234.37 (MþH)þ; LCMS (ES) m/z calcd for C12H9ClNO2,
[M þ H]þ: 234.65, found: 234.37; Column used: Develosil ODS MG-3
(4.6 ꢃ 33 mm), Mobile Phase: A: 0.1% Aq. HCOOH, B: 0.1% HCOOH
(Acetonitrile), T/%B: 0/30, 4/90,10/90,10.1/30; Flow Rate: 1 mL/min,
Diluent: Acetonitrile; UV: 257 nm, RT ¼ 2.97, Purity ¼ 98.96%; HPLC-
data are reported as follows: [a]D temp., concentration (c g/100 mL)
and solvent. All analytical assays including high performance liquid
chromatography (HPLC), liquid chromatography-mass spectrom-
etry (LCMS) and semipreparative chromatography were performed
under the conditions given in experimental section. All reactions
were monitored by thin layer chromatography using Merck 60 SI
F254 precoated silica gel polyester plates and visualization of the
developed chromatogram was performed by UV absorbance,
aqueous cerium molybdate, ethanolic phosphomolybdic acid,
aqueous potassium permanganate and iodine vapor. Flash column
Column used: Kromasil 100-5-C-18 (4.6 ꢃ 250 mm), 5
m; Mobile
Phase: A: 0.1% Aq. HCOOH, B: Acetonitrile; T/%B: 0/30, 2/30, 6/90,16/
90, 18/30, 20/30; Flow Rate: 1.0 mL/min, Diluent: Acetonitrile; UV:
210 nm; RT ¼ 8.360; Purity ¼ 98.01%.
6.3. 6-Benzoyl-4-hydroxypyridin-2(1H)-one (5)
Step-b: compound 4 (10 g; 42.79 mmol) was treated with acetic
acid (150 mL) and water (5 mL) for 7 h according to the procedure
described above for compound 4 to provide the title compound as
pale greenish solid (7 g; 76%). Step-c: compound 3 (20 g; 61.9 mmol)
Table 3
The minimum inhibition concentration in mg of the tested compounds 2, 11 and 12.
Microorganism
MIC (
2
m
g/ml)a
11
12
Amikacin
Table 4
The cytotoxicity data of the tested compounds 1, 2, 7, 8, 11 and 12 against A549 lung
cancer cell line.
E. coli
B. subtilis
50
25
25
50
50
100
06
06
S. epidermidis
K. pneumonia
L. monocytogenes
S. typhimurium
S. aureus
MRSA-I (TB 3G)
MRSA-II (TB 4G)
MRSA-IV (TB 5G)
>100
50
50
>100
>100
>100
>100
>100
100
100
25
>100
100
>100
>100
>100
>100
>100
12
06
06
12
Compound % of cell death at various doses
10
IC50
(m
g) IC50 (mM)
>100
>100
>100
>100
>100
>100
1
m
g
2
m
g
5
m
g
m
g
25 mg
1
7
8
8.22 25.54 37.22 50.21 74.02 8.55
9.96 27.71 38.96 47.62 63.20 3.34
3.13 18.59 48.82 50.39 75.78 3.17
28
11
10
11
9
50
>100
>100
>100
2
11
12
Etoposide
7.07 21.53 44
9.53 25.23 47.69 77.23 82.46 3.36
10.46 30.76 44.92 77.84 78.76 2.92
5.54 9.12 25.91 55.33 69.48 6.67
71.07 81.53 4.14
8
11
MRSA, methicillin-resistant Staphylococcus aureus.
a
Values are means of three experiments.

1808
S.R. Vegi et al. / European Journal of Medicinal Chemistry 46 (2011) 1803e1812
ODS MG-3 (4.6 ꢃ 33 mm), Mobile Phase: A: 0.1% Aq. HCOOH, B: 0.1%
HCOOH (Acetonitrile:Methanol (50:50)), T/%B: 0/10, 4/50, 6/90, 10/
90,10.1/10; Flow Rate: 1 mL/min, Diluent: Acetonitrile; UV: 282 nm,
RT ¼ 3.30, Purity ¼ 99.04%; HPLC-Column used: Eclipse-XDB-C18
(4.6 ꢃ 150 mm), 5.0
m; Mobile Phase: A: 0.1% Aq. HCOOH,; B: Meth-
anol; T/%B: 0/30, 8/90,15/90,15.1/30; Flow Rate: 1.0 mL/min, Diluent:
Acetonitrile; UV: 285 nm; RT ¼ 4.80; Purity ¼ 96.71% Chiral HPLC:
racemic mixture; column used: Chiral PAK-IA (4.6 ꢃ 250 mm), 5
m,
mobile phase: A:B: n-hexane:ethanol (80:20), isocratic; flow rate:
0.7 mL/min, diluent: ethanol, run time: 30 min, UV: max-plot,
RT ¼ 9.41 and 11.35.
6.5. (4-Hydroxy-6-oxo-1,6-dihydropyridin-2-yl)(phenyl)methyl
3-methylbutanoate (ꢁSPF32629A, 1)
To a cooled solution ofcompound 6 (1.8 g, 8.28 mmol) ina mixture
of tetrahydrofuran:dichloromethane (1:2, 30 mL) was sequentially
added isovaleric acid (1.04 g, 10.12 mmol), EDC.HCl (2.64 g,
13.81 mmol) and 4-(dimethylamino)pyridine (25 mg) at 10 ꢀC and
stirred at room temperature for 16 h. The reaction mixture was
quenched with ice cold water, separated organic layer and again
extracted aqueous layer with dichloromethane. Combined organic
layer was washed with brine solution, dried over anhydrous sodium
sulfate, filtered and evaporated under reduced pressure to obtain
crude solid. Obtained crude solid was purified by flash column
chromatography over silica gel 60 (230e400 mesh) using 6% meth-
anol: dichloromethane as eluent to afford 2.1 g (84%) of 1 as colorless
solid. Rf ¼ 0.5 (10% Methanol:Dichloromethane); mp 127e130 ꢀC; IR
Fig. 4. Graph of percentage of cell death at various doses.
was reacted with with acetic acid (300 mL) and water (10 mL) for
12 h according to the procedure described for compound 4 to afford
the compound 5 as pale greenish solid (12 g; 90%). Rf ¼ 0.4 (10%
Methanol:Dichloromethane); mp 258e262 ꢀC; IR (KBr pellet): nmax
3438, 1669, 1639, 1596, 1446 cmꢂ1; 1H NMR (400 MHz, d6-DMSO):
11.0 (1H, brs, D2O exchangeable OH), 10.85 (1H, s, D2O exchange-
able CONH), 7.82 (2H, d, J 7.6 Hz), 7.72e7.69 (1H, m), 7.59e7.55 (2H,
m), 6.28 (1H, s), 5.85 (1H, s); 13C NMR (100 MHz, d6-DMSO):
189.55
d
(KBr pellet): nmax 3492 (br), 1759, 1652, 1611 cmꢂ1
;
1H NMR
(400 MHz, CDCl3): 9.2 (brs, D2O exchangeable CONH and OH), 7.34
d
d
(5H, s), 6.62(1H, s), 5.97 (1H, d, J 1.6 Hz), 5.88 (1H, s, D2O exchangeable
pyridine 3-CH), 2.32e2.30 (2H, m), 2.14e2.08 (1H, m), 0.92e0.91 (6H,
(C), 165.95 (C), 163.76 (C), 143.76 (C), 135.4 (C), 133.42 (CH), 129.70
(2CH), 128.63 (2CH), 105.82 (CH), 101.79 (CH); MS (ESI) m/z 216.42
(MþH)þ; LCMS (ES) m/z calcd for C12H10NO3, [M þ H]þ: 216.20,
found: 216.4; Column used: Develosil ODS MG-3 (4.6 ꢃ 33 mm),
Mobile Phase: A: 0.1% Aq. HCOOH, B: 0.1% HCOOH (Acetonitrile), T/%
B: 0/30, 4/90, 8/90, 8.1/30; Flow Rate: 1 mL/min, Diluent: Methanol;
UV: 215 nm, RT ¼ 3.25, Purity ¼ 99.05%; HPLC-Column used:
dd, J 1 Hz, 6.6 Hz); 13C NMR (100 MHz, CDCl3):
d 171.84 (C),169.98 (C),
165.78 (C), 146.11 (C), 135.93 (C), 129.11 (CH), 128.9 (2CH), 127.18
(2CH), 101.84 (CH), 98.45 (CH), 72.78 (CH), 43.09 (CH2), 25.64 (CH),
22.29 (CH3), 22.27 (CH3); MS (ESI) m/z 300.1 [MeH]ꢂ; Anal. calcd for:
C17H19NO4: C, 67.76; H, 6.36; N, 4.65, Found: C, 67.9; H, 6.173; N, 4.551;
LCMS (ES) m/z calcd for C17H18NO4, [M ꢂ H]ꢂ: 300.33, found: 300.2;
m/z calcd for C17H20NO4, [M þ H]þ: 302.14, found: 302; Column used:
Develosil ODS MG-3 (4.6 ꢃ 33 mm), Mobile Phase: A: 0.1% Aq.
HCOOH, B: 0.1% HCOOH (Acetonitrile:MeOH (50:50)), T/%B: 0/30, 4/
90, 8/90, 8.1/30; flow rate: 1 mL/min, diluent: acetonitrile; UV:
283 nm, RT ¼ 3.63, purity ¼ 98.56%; HPLC-Column used: Eclipse-
Eclipse-XDB-C8 (4.6 ꢃ 150 mm), 5.0
m; Mobile Phase: A: 0.01 M Aq.
Ammonium acetate; B: Acetonitrile; T/%B: 0/10, 2/10, 6/90,16/90,18/
10, 20/10; Flow Rate: 1.0 mL/min, Diluent: Acetonitrile; UV: 215 nm;
RT ¼ 5.387; Purity ¼ 98.44%.
6.4. 4-hydroxy-6-(hydroxy(phenyl)methyl)pyridin-2(1H)-one (6)
XDB-C18 (4.6 ꢃ 150 mm), 5
m; mobile phase: A: 0.1% Aq. HCOOH; B:
methanol; T/%B: 0/30, 8/90, 15/90, 15.1/30; flow rate: 1.0 mL/min,
diluent: acetonitrile; UV: 285 nm; RT ¼ 8.741; purity ¼ 99.04%, Chiral
HPLC: racemic mixture; Column used: Chiral PAK-AD-H
To a cooled solution of compound 5 (2 g, 9.29 mmol) in tetrahy-
drofuran (16 mL) and methanol (4 mL) was portion wise added
sodium borohydride (0.36 g, 9.29 mmol) at 0 ꢀC. The resulting reac-
tion mixture wasstirred at 0e10 ꢀC for 2 h and treated drop wisewith
concentrated hydrochloric acid until pH ¼ 4 at 0 ꢀC. The resulting
reactionmixturewasstirredfor15 min, filteredtoremoveboratesalts
and the filtrate was concentrated under vacuo at 50 ꢀC to obtain crude
solid. Obtained crude solid was purified by flash column chroma-
tography over silica gel 60 (230e400 mesh) using 8% methanol: DCM
as eluent to afford the title compound 6 as colorless solid (1.8 g
89.19%).Rf ¼ 0.25(10%Methanol:Dichloromethane);mp119e123 ꢀC;
IR (KBr pellet): nmax 3363,1645,1610,1428 cmꢂ1; 1H NMR (400 MHz,
(4.6 ꢃ 250 mm),
5 m, Mobile Phase: A:B: n-hexane:isopropanol
(90:10), isocratic; flow rate: 0.7 mL/min, diluent: ethanol, run time:
30 min, UV: 206 nm, RT ¼ 9.99 and 12.57.
6.6. (þ)-SPF32629A (7)
To a cooled suspension of compound 5 (1 g, 4.64 mmol) in
tetrahydrofuran (10 mL) was portion wise added (ꢂ)-DIP-Cl (1.8 g,
5.61 mmol) at ꢂ40 ꢀC. The resulting reaction mixture was stirred
at ꢂ40 to ꢂ10 ꢀC for 12 h, quenched with ice cold water and
extracted with ethyl acetate. Combined organic layer was washed
with brine solution, dried over anhydrous sodium sulfate, filtered
and evaporated under reduced pressure to obtain crude compound
6, which was further reacted with isovaleric acid according to the
proceduredescribed for racemic SPF32629Atoobtain 0.92 g(66%) of
d6-DMSO): d 10.75 (1H, brs, D2O exchangeable Pyridone 4-OH),10.41
(1H, s, D2O exchangeable CONH), 7.43e7.41 (2H, m,), 7.35e7.32 (2H,
m), 7.29e7.24 (1H, m), 6.125 (1H, d, J 4 Hz, D2O exchangeable OH),
5.863 (1H, d, J 1.2 Hz) 5.409 (1H, d, J 4 Hz) 5.343 (1H, d, J 2 Hz); 13C
NMR (100 MHz, d6-DMSO): d 167.59 (C),164.26 (C),152.79 (C),142.32
(C), 128.2 (2CH), 127.59 (CH), 126.41 (2CH), 96.63 (CH), 95.76 (CH),
70.82 (CH); MS (ESI) m/z 216.1 (MeH)þ; LCMS (ES) m/z calcd for
C12H10NO3, [MeH]þ: 216.23, found: 216.1; Column used: Develosil
(þ)-SPF32629A as colorless solid. mp 128e132 ꢀC; IR (KBr pellet):
23ꢀC
nmax 3394 (br), 1748, 1645, 1612 cmꢂ1; [
a]
¼ þ24.834 (c 0.5879,
Chloroform); 1H NMR (400 MHz, d6-DMSO):
d 11.23 (1H, brs, D2O

S.R. Vegi et al. / European Journal of Medicinal Chemistry 46 (2011) 1803e1812
1809
exchangeable OH),10.55 (1H, s, D2O exchangeable CONH), 7.44e7.33
(5H, m), 6.43 (1H, s), 5.77 (1H, s), 5.42 (1H, s, D2O exchangeable
pyridine 3-CH), 2.32 (2H, d, J 7.2 Hz), 2.06e1.99 (1H, m), 0.88 (6H, d, J
[MeH]þ: 260.23 found: 260.3; Column used: Acquity-BEH-C18
m, mobile phase: A: 0.1% Aq. HCOOH, B: 0.1%
(50 ꢃ 2.1 mm) 1.7
m
HCOOH in Acetonitrile, T/%B: 0/10, 1.5/100, 2.9/100, 3/10; Flow
Rate: 0.5 mL/min, diluent: acetonitrile; UV: 302 nm, RT ¼ 1.07,
Purity ¼ 97.68%; HPLC-column used: Kromasil C18 (4.6 ꢃ 250 mm),
6.8 Hz); 13C NMR (100 MHz, CDCl3):
d 171.87 (C), 170.01 (C), 165.71
(C), 146.03 (C), 135.88 (C), 129.16 (CH), 128.93 (2CH), 127.19 (2CH),
101.83 (CH), 98.44 (CH), 72.78 (CH), 43.1 (CH2), 25.64 (CH), 22.28
(2CH3); MS (ESI) m/z 300.1 [MꢂH]ꢂ; LCMS (ES) m/z calcd
for C17H18NO4, [MꢂH]ꢂ: 300.33, found: 300.2; Column used:
Develosil ODS MG-3 (4.6 ꢃ 33 mm), Mobile Phase: A: 0.1% Aq.
HCOOH, B: 0.1% HCOOH (Acetonitrile:MeOH (50:50)), T/%B: 0/30,
4/90, 8/90, 8.1/30; flow rate: 1 mL/min, diluent: acetonitrile; UV:
284 nm, RT ¼ 3.62, Purity ¼ 99.59%; HPLC-Column used: Eclipse-
5.0 m; mobile phase: A: 0.05% Aq. TFA; B: Methanol; T/%B: 0/30, 2/30,
8/90,16/90,18/30, 20/30; flow rate: 1.0 mL/min, diluent: acetonitrile;
UV: 210 nm; RT ¼ 9.83; purity ¼ 96.43%. Chiral HPLC: racemic
mixture; Column used: Chiral PAK-AD-H (4.6 ꢃ 250 mm), 5
m, Mobile
Phase: A:B: n-hexane:ethanol (70:30), flow rate: 0.8 mL/min, diluent:
ethanol, run time: 40 min, UV: 302 nm, RT ¼ 8.37 and 10.74.
XDB-C18 (4.6 ꢃ 150 mm), 5
m
; Mobile Phase: A: 0.1% Aq. HCOOH; B:
6.9. 4-Hydroxy-6-((3-methylbutanoyloxy)(phenyl)methyl)-2-oxo-
1,2-dihydropyridine-3-carboxylic acid, (ꢁ)-SPF32629B (2)
methanol; T/%B: 0/30, 8/90, 15/90, 15.1/30; flow rate: 1.0 mL/min,
Diluent: Acetonitrile; UV: 286 nm; RT ¼ 8.73; purity ¼ 99.68%;
Chiral HPLC-Column used: Chiral PAK-AD-H (4.6 ꢃ 250 mm), 5
m,
To a cooled solution of compound 10 (1 g, 3.82 mmol) in
dichloromethane (12 mL) and tetrahydrofuran (8 mL) was
sequentially added isovaleric acid (0.47 g, 4.59 mmol), EDC.HCl
(1.17 g, 6.11 mmol) and 4-(dimethylamino)pyridine (20 mg) at
10 ꢀC and stirred at room temperature for 12 h. The reaction
mixture was quenched with ice cold water, separated organic layer
and again extracted aqueous layer with dichloromethane.
Combined organic layer was washed with brine solution, dried over
anhydrous sodium sulfate, filtered and evaporated under reduced
pressure to obtain crude solid. Obtained crude solid was purified by
flash column chromatography over silica gel 60 (230e400 mesh)
using 5% methanol: dichloromethane as eluent to afford 1.1 g
(83.2%) of (ꢁ)-SPF32629B as colorless solid. Rf ¼ 0.3 (5% Meth-
anol:Dichloromethane); mp 129e132 ꢀC; IR (KBr pellet): nmax 3461,
Mobile Phase: A:B: n-hexanes:isopropanol (90:10), Isocratic; Flow
rate: 0.7 mL/min, diluent: ethanol, run time: 25 min, UV: 286 nm,
RT ¼ 10.27, purity ¼ 99.77%.
6.7. (ꢂ)-SPF32629A (8)
This compound was prepared according to the procedure
described for (þ)-SPF32629A by replacing (ꢂ)-DIP-Cl with (þ)-DIP-
Cl; obtained yield ¼ 70%. mp 128e133 ꢀC; IR (KBr pellet): nmax 3401
23ꢀC
(br), 1748, 1650, 1613 cmꢂ1; [
a
]
¼ ꢂ25.043 (c 0.5870, Chloro-
form); 1H NMR (400 MHz, d6-DMSO):
d 11.2 (2H, brs, D2O
exchangeable OH and CONH), 7.44e7.32 (5H, m), 6.43 (1H, s), 5.77
(1H, s), 5.41 (1H, s, D2O exchangeable pyridine 3-CH), 2.31 (2H, d, J
7.2 Hz), 2.06e1.99 (1H, m), 0.88 (6H, d, J 6.8 Hz); 13C NMR (100 MHz,
1742, 1688, 1639 cmꢂ1 1HNMR (400 MHz CDCl3):
; d 14.24 (1H, brs,
CDCl3):
d
171.86 (C), 169.99 (C), 165.76 (C), 146.09 (C), 135.91 (C),
D2O exchangeable COOH), 13.51 (1H, brs, D2O exchangeable OH),
10.1 (1H, brs, D2O exchangeable CONH), 7.42e7.34 (5H, m), 6.63
(1H, s), 6.22 (1H, s), 2.34 (2H, d, J 7.2 Hz), 2.15e2.09 (1H, m), 0.94
129.14 (CH),128.92 (2CH),127.19 (2CH),101.8 (CH), 98.45 (CH), 72.79
(CH), 43.1 (CH2), 25.64 (CH), 22.29 (CH3), 22.28 (CH3); MS (ESI) m/z
300.2 [MeH]ꢂ;LCMS (ES)m/z calcd. for C17H18NO4, [MeH]ꢂ:300.33,
found: 300.2; Column used: Develosil ODS MG-3 (4.6 ꢃ 33 mm),
Mobile Phase: A: 0.1% Aq. HCOOH, B: 0.1% HCOOH (Acetoni-
trile:MeOH (50:50)), T/%B: 0/30, 4/90, 8/90, 8.1/30; flow rate: 1 mL/
min, Diluent: Acetonitrile; UV: 283 nm, RT ¼ 3.63, Purity ¼ 99.57%;
(6H, d, J 6.8 Hz); 13CNMR (100 MHz, CDCl3):
d 174.94 (C), 171.83 (C),
171.35 (C), 165.93 (C), 151.07 (C), 134.75 (C), 129.82 (CH), 129.34
(2CH), 127.21 (2CH), 101.35 (CH), 96.84 (C), 72.67 (CH); 42.95 (CH2);
25.69 (CH); 22.28 (CH3); 22.26 (CH3); MS (ESI) m/z 344.20 (MeH)ꢂ;
LCMS (ES) m/z calcd for C18H18NO6 [MeH]ꢂ: 344.36, found: 344.2;
Develosil ODS MG-3 (4.6 ꢃ 33 mm), mobile phase: A: 0.1% Aq.
HCOOH, B: 0.1% HCOOH (Acetonitrile:Methanol (50:50)), T/%B:
0/30, 4/90, 10/90, 10.1/30; flow rate: 1 mL/min, diluent: acetoni-
trile; UV: 301 nm, RT ¼ 4.61, purity ¼ 98.72%; HPLC-column used:
HPLC - Column used: Eclipse-XDB-C18 (4.6 ꢃ 150 mm), 5
m; Mobile
Phase: A: 0.1% Aq. HCOOH; B:methanol;T/%B: 0/30, 8/90,15/90,15.1/
30; flow rate: 1.0 mL/min, diluent: acetonitrile; UV: 286 nm;
RT ¼ 8.74; purity ¼ 99.72%, chiral HPLC: column used: chiral PAK-
AD-H (4.6 ꢃ 250 mm),
5
m
,
mobile phase: A:B: n-hex-
eclipse XDB-C18 (4.6 ꢃ 150 mm) 5
m, mobile phase: A: 0.1 M formic
ane:isopropanol (90:10) isocratic, flow rate: 0.7 mL/min, diluent:
ethanol, run time: 25 min, UV: 286 nm, RT ¼ 12.96, purity ¼ 99.02%.
acid; B: methanol; T/%B: 0/50, 8/90, 15/90, 15.1/50; flow rate:
1.0 mL/min, diluent: (A:ACN,1:1); UV: 303 nm; RT ¼ 9.13;
purity ¼ 99.12%. Chiral HPLC-column used: chiral PAK-AD-H
6.8. 4-Hydroxy-6-(hydroxy(phenyl)methyl)-2-oxo-
1,2-dihydropyridine-3-carboxylic acid (10)
(4.6 ꢃ 250 mm), 5
m, mobile phase: A: 0.1%TFA in n-hexanes, B:
ethanol (70:30), isocratic; flow rate: 0.7 mL/min, diluent: ethanol,
run time: 25 min, UV: 304 nm, RT ¼ 8.69 and 12.57.
To a cooled solution of compound 9 [24] (1 g, 3.85 mmol) in
tetrahydrofuran (10.5 mL) and methanol (4.5 mL) was portion wise
added sodium borohydride (0.2 g, 5.28 mmol) at 0 ꢀC. The resulting
reaction mixture was stirred at 0e10 ꢀC for 2 h and treated drop wise
with concentrated hydrochloric acid until pH ¼ 2 and stirred at 10 ꢀC
for 15 min. Separated salts were filtered and the filtrate was
concentrated under reduced pressure to obtain the title compound as
colorless solid (0.95 g, 95%). Rf ¼ 0.15 (10% Methanol:Dichloro-
methane); mp 196e199 ꢀC; IR (KBr pellet): nmax 3343, 1671,1620,
6.10. (þ)-SPF32629B (11)
To a cooled suspension of compound 9 (1 g, 3.85 mmol) in tetra-
hydrofuran (15 mL) was portion wise added (ꢂ)-DIP-Cl (1.48 g,
4.62 mmol) at ꢂ40 ꢀC. The resulting reaction mixture was stirred
at ꢂ40 to ꢂ10 ꢀC for 12 h, quenched with ice cold water and extracted
with ethyl acetate. Combined organic layer was washed with brine
solution, dried over anhydrous sodium sulfate, filtered and evapo-
rated under reduced pressure to obtain crude compound 10, which
was further reacted with isovaleric acid according to the procedure
described for racemic SPF32629B to obtain 1.1 g (83%) of
(þ)-SPF32629B as colorless solid. Rf ¼ 0.3 (5% Methanol:Dichloro-
methane); mp106e108 ꢀC;IR(KBrpellet):nmax 3471, 2961,1744,1684,
1475 cmꢂ1 1H NMR (400 MHz, d6-DMSO):
; d 15.6 (1H, brs, D2O
exchangeable COOH), 13.16 (1H, brs, D2O exchangeable Pyridone-
4-OH), 12.75 (1H, s, D2O exchangeable CONH), 7.48e7.46 (2H, m),
7.38e7.29 (3H, m), 6.43 (1H, s), 5.58 (1H, s); 13C NMR (100 MHz,
d6-DMSO):
d 173.55 (C), 172.11 (C), 165.87 (C), 158.28(C), 141.0 (C),
23ꢀC
24ꢀC
128.45 (2CH), 128.12 (CH), 126.67 (2CH), 98.18 (CH), 95.41(C), 70.92
1637,1484 cmꢂ1; [
0.4, MeOH); 1HNMR (400 MHz CDCl3):
a]
¼ þ50.5 (c 0.4, chloroform); [
a]
¼ þ24 (c
(CH); MS (ESI) m/z 260.3 (MeH)þ; LCMS (ES) m/z calcd for C19H10NO5,
d
14.19 (1H, brs, D2O

1810
S.R. Vegi et al. / European Journal of Medicinal Chemistry 46 (2011) 1803e1812
exchangeable COOH), 13.49 (1H, brs, D2O exchangeable OH), 11.07
(1H, brs, D2O exchangeable CONH), 7.40e7.37 (5H, m), 6.62 (1H, s),
6.29 (1H, s), 2.34 (2H, d, J 6.8 Hz), 2.15e2.09 (1H, m), 0.94 (6H, d, J
(3H, m), 7.37(5H, m), 6.25(1H, d, J1.6 Hz),5.94(1H, s), 5.65(1H,s),3.60
(3H, s); 19F NMR (400 MHz CDCl3):
CDCl3):
d
ꢂ74.237; 13C NMR (100 MHz,
d
165.30 (C),163.61 (C),160.71 (C),151.93 (C),139.47 (C),131.17
6.8 Hz); 13CNMR (100 MHz, CDCl3):
d
174.95 (C),171.80 (C),171.35 (C),
(C), 130.1 (CH), 128.91 (2CH),128.82 (2CH), 128.78 (CH), 127.05 (2CH),
126.74 (2CH),124.36e121.49 (CF3),108.33 (CH),100.37 (CH), 85.00 (C),
72.59 (CH), 55.77 (CH3); MS (ESI) m/z 432.3 (MeH)þ; LCMS (ES) m/z
calcd for C22H17F3NO5, [MeH]þ: 432.37, found: 432.3; Column used:
165.99 (C), 151.02 (C), 134.65 (C), 129.83 (CH), 129.37 (2CH), 127.199
(2CH) 101.46 (CH), 96.86(C), 72.64 (CH); 42.93 (CH2); 25.70 (CH);
22.26 (2CH3); MS (ESI) m/z 346.2(M þ H)ꢂ; LCMS (ES) m/z calcd for
C18H20NO6 [M þ H]ꢂ: 346.34, found: 346.2; column used: acquity-
Acquity e BEH eC 18 (50 ꢃ 2.1 mm) 1.7
m, mobile phase: A: 0.1% Aq.
BEH-C18 (50 ꢃ 2.1 mm) 1.7
m
m, mobile phase: A: 0.1% Aq. HCOOH, B:
HCOOH, B: 0.1% HCOOH in acetonitrile, T/%B: 0/3, 0.1/3,1.5/90, 1.8/90,
2.2/95, 3.2/95, 3.8/3; flow rate: 0.4 mL/min, diluent: acetonitrile; UV:
305 nm, RT ¼ 1.72, purity ¼ 99.82%; HPLC-column used: XBridge-C8
0.1% HCOOH in Acetonitrile, T/%B: 0/10, 1.5/100, 2.9/100, 3/10; flow
rate: 0.5 mL/min, diluent: acetonitrile; UV: UV: Max-plot nm,
RT ¼ 1.54, Purity ¼ 96.72%; HPLC-column used: Kromasil C18
(4.6 ꢃ 150 mm), 5.0
m; Mobile Phase: A: 0.01 M Aq. Ammonium
(4.6 ꢃ 250 mm), 5.0
m
; mobile phase: A: 0.05% Aq. TFA; B: methanol;
acetate; B: acetonitrile; T/%B: 0/20, 4/55, 8/90, 15/90, 15.1/20; flow
rate: 0.8 mL/min, diluent: acetonitrile; UV: 305 nm; RT ¼ 8.3;
purity ¼ 98.87%.
T/%B: 0/30, 2/30, 8/90, 16/90, 18/30, 20/30; flow rate: 1.0 mL/min,
diluent: acetonitrile; UV: 210 nm; RT ¼ 9.67; purity ¼ 97.25%. Chiral
HPLC-column used: Chiral PAK-AD-H (4.6 ꢃ 250 mm), 5
m, Mobile
Phase: A: 0.1%TFA in n-hexanes, B: ethanol (70:30), isocratic; flow
rate: 0.7 mL/min, diluent: ethanol, run time: 25 min, UV: 304 nm,
RT ¼ 9.26, purity ¼ 100%.
6.13. (R)-((R)-(4-hydroxy-6-oxo-1,6-dihydropyridin-2-yl)(phenyl)
methyl) 3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (14)
To a cooled solution of enantiopure alcohol (L)-6 (0.25 g,
20ꢀC
6.11. (ꢂ)-SPF32629B (12)
1.15 mmol;
[
a]
¼ ꢂ45.78,
c 0.21, methanol) in tetrahy-
drofuarn:dichloromethane (1:2, 10 mL) was sequentially added
DCC (0.237 g, 1.15 mmol), 4-(dimethylamino)pyridine (5 mg) and
R-MTPA (0.269 g, 1.15 mmol) at 0 ꢀC and stirred at room tempera-
ture for 8 h. The reaction mixture was concentrated under reduced
pressure to obtain crude solid. Obtained crude solid was purified by
flash column chromatography over silica gel 60 (230e400 mesh)
using 3% methanol: dichloromethane as eluent and the resulting
solid was further taken in dry ether, filtered and evaporated to
obtain 0.369 g (74%) of pure desired diastereomer 14 as colorless
solid. Rf ¼ 0.55 (10% Methanol:Dichloromethane); 1H NMR
This compound was prepared according to the procedure
described for (þ)-SPF32629B by replacing (ꢂ)-DIP-Cl with (þ)-DIP-
Cl; obtained yield ¼ 84%. Rf ¼ 0.3 (5% methanol:dichloromethane);
mp 107e110 ꢀC; IR (KBr pellet): nmax 3460, 2961,1743, 1684,
23ꢀC
24ꢀC
1637,1484 cmꢂ1; [
a]
¼ ꢂ47.4 (c 0.4, Chloroform); [
a
]
¼ ꢂ22.6
(c 0.4, methanol); 1HNMR (400 MHz CDCl3):
d
14.17 (1H, brs, D2O
exchangeable COOH), 13.48 (1H, brs, D2O exchangeable OH), 11.49
(1H, brs, D2O exchangeable CONH), 7.39 (5H, s), 6.63 (1H, s), 6.32 (1H,
s), 2.34 (2H, d, J 6.8 Hz), 2.13e2.08 (1H, m), 0.94 (6H, d, J 6.8 Hz);
13CNMR (100 MHz, CDCl3):
d
174.93 (C), 171.76 (C), 171.34 (C), 166.17
(400 MHz, CDCl3): d 7.68 (2H, s), 7.46 (3H, s), 7.35e7.31 (5H, m), 6.54
(C), 151.22 (C), 134.67 (C), 129.77 (CH), 129.32 (2CH), 127.18 (2CH)
101.43 (CH), 96.82 (C), 72.67 (CH); 42.90 (CH2); 25.67 (CH); 22.24
(2CH3); MS (ESI) m/z 346.2(M þ H)ꢂ; LCMS (ES) m/z calcd for
C18H20NO6 [M þ H]ꢂ: 346.34, found: 346.2; Column used:Acquity-
(1H, d, J 6 Hz), 6.41 (1H, s), 5.63 (1H, s), 3.71 (3H, s); 19F NMR
(400 MHz CDCl3):
d
ꢂ74.375; MS (ESI) m/z 434.2 (M þ H)þ; LCMS
(ES) m/z calcd for C22H19F3NO5, [M þ H]þ: 434.37, found: 434.2;
Column used: Acquity e BEH e C 18 (50 ꢃ 2.1 mm)1.7
m, mobile
BEH-C18 (50 ꢃ 2.1 mm) 1.7
m
m, Mobile Phase: A: 0.1% Aq. HCOOH, B:
phase: A: 0.1% Aq. HCOOH, B: 0.1% HCOOH in acetonitrile, T/%B: 0/3,
0.1/3, 1.5/90, 1.8/90, 2.2/95, 3.2/95, 3.8/3; flow rate: 0.4 mL/min,
diluent: acetonitrile; UV: 305 nm, RT ¼ 1.80, purity ¼ 92.62%.
0.1% HCOOH in Acetonitrile, T/%B: 0/10, 1.5/100, 2.9/100, 3/10; flow
rate: 0.5 mL/min, diluent: acetonitrile; UV: max-plot, RT ¼ 1.53,
purity ¼ 99.13%; HPLC-column used: Kromasil C18 (4.6ꢃ250 mm),
5.0
m; mobile phase: A: 0.05% Aq. TFA; B: methanol; T/%B: 0/30, 2/30,
6.14. 4-Hydroxy-2-oxo-6-((S)-phenyl((R)-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoyloxy) methyl)-1,2-dihydropyridine-
3-carboxylic acid (15)
8/90, 16/90, 18/30, 20/30; flow rate: 1.0 mL/min, diluent: acetoni-
trile; UV: 214 nm; RT ¼ 9.62; Purity ¼ 96.25%; Chiral HPLC-column
used: Chiral PAK-AD-H (4.6 ꢃ 250 mm), 5
m, mobile phase: A: 0.1%
TFA in n-hexanes, B: ethanol (70:30), isocratic; flow rate: 0.7 mL/
min, diluent: ethanol, run time: 35 min, UV: 304 nm, RT ¼ 7.65,
purity ¼ 99.85%.
To a cooled solution of enantiopure alcohol (D)-10 (0.1 g,
20ꢀC
0.38 mmol;
[
a]
¼ þ61.57,
c 0.21, methanol) in tetrahy-
drofuarn:dichloromethane (1:2, 5 mL) was sequentially added DCC
(83 mg, 0.4 mmol), 4-(dimethylamino)pyridine (2 mg) and R-MTPA
(94 mg, 0.4 mmol) at 0 ꢀC and stirred at room temperature for 6 h.
The reaction mixture was concentrated under reduced pressure to
obtain crude solid. Obtained crude solid was purified by flash
column chromatography over silica gel 60 (230e400 mesh) using
3% methanol: dichloromethane as eluent and the resulting solid
was further taken in dry ether, filtered and evaporated to obtain
0.149 g (82%) of pure desired diastereomer 15 as colorless solid.
Rf ¼ 0.5 (10% methanol:dichloromethane); IR (KBr pellet): nmax
6.12. (R)-((S)-(4-hydroxy-6-oxo-1,6-dihydropyridin-2-yl)(phenyl)
methyl)3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (13)
To a cooled solution of enantiopure alcohol (D)-6 (0.25 g,
20ꢀC
1.15 mmol; [
a]
¼ þ57.48, c 0.21, methanol) in tetrahydrofuarn:di-
chloromethane (1:2, 10 mL) was sequentially added DCC (0.237 g,
1.15 mmol), 4-(dimethylamino)pyridine (5 mg) and R-MTPA (0.269 g,
1.15 mmol) at 0 ꢀC and stirred at room temperature for 8 h. The
reaction mixture was concentrated under reduced pressure to obtain
crude solid. Obtained crude solid was purified by flash column chro-
matography over silica gel 60 (230e400 mesh) using 3% methanol:
dichloromethaneaseluentandtheresultingsolidwasfurthertakenin
dry ether, filtered and evaporated to obtain 0.399 g (80%) of pure
desired diastereomer 13 as colorless solid. Rf ¼ 0.5 (10% Meth-
anol:Dichloromethane); mp 67e70 ꢀC; IR (KBr pellet): nmax 3412,
3402, 1760, 1672, 1643, 1172 cmꢂ1 1H NMR (400 MHz CDCl3):
;
d
14.15 (1H, brs, D2O exchangeable COOH), 13.48 (1H, brs, D2O
exchangeable OH), 10.01 (1H, brs, D2O exchangeable CONH), 7.44
(2H, d, J 4.8 Hz), 7.38e7.30 (8H, m), 6.78 (1H, s), 6.09 (1H, s), 3.47
(3H, s); 19F NMR (400 MHz CDCl3):
d
ꢂ71.038; MS (ESI) m/z 478.2
(M þ H)þ; LCMS (ES) m/z calcd for C23H19F3NO7, [M þ H]þ: 478.38,
found: 478.2; Column used: Acquity e BEH eC 18 (50 ꢃ 2.1 mm)
1776, 1652, 1613, 1180 cmꢂ1; 1H NMR (400 MHz, CDCl3):
brs, D2O exchangeable OH & CONH), 7.53 (2H, d, J 7.6 Hz), 7.44e7.42
d
12.0 (2H,
1.7 m, mobile phase: A: 0.1% Aq. HCOOH, B: 0.1% HCOOH in aceto-
nitrile, T/%B: 0/3, 0.1/3, 1.5/90, 1.8/90, 2.2/95, 3.2/95, 3.8/3; flow

S.R. Vegi et al. / European Journal of Medicinal Chemistry 46 (2011) 1803e1812
1811
rate: 0.4 mL/min, diluent: acetonitrile; UV: 310 nm, RT ¼ 1.90,
purity ¼ 92.54%; HPLC-column used: XBridge-C8 (4.6 ꢃ 150 mm),
3.5 ; mobile phase: A: 0.05% aqueous TFA; B: acetonitrile; T/%B:
6.16.2. Cytotoxicity assay
A549 (human lung adenocarcinoma cell line) was obtained from
National Centre for Cell Science, Pune, India. The cells were grown
m
0/20, 4/55, 8/90, 15/90, 15.1/20; flow rate: 1 mL/min, diluent:
acetonitrile; UV: 310 nm; RT ¼ 9.02; purity ¼ 94.13%.
in DMEM culture medium supplemented with 2 mmol L-I
mine,10% FBS, penicillin (50 IU/mL) and streptomycin (50
L
-gluta-
m
g/mL) at
a temperature of 37 ꢀC in a humidified incubator with a 5% CO2
atmosphere. Etoposide or VP-16-213, a well known anticancer drug
was used as the positive control for comparison [31]. The viability of
the cells was assessed by MTT (3, 4, 5-dimethylthiazol-2-yl)-
2-5diphenyltetrazolium bromide) assay, which is based on the
reduction of MTT by the mitochondrial dehydrogenase of intact
cells to a purple formazan product [32]. Cells (1 ꢃ104) were placed
in a 96-well plate. After 24 h, they were treated with different
6.15. 4-Hydroxy-2-oxo-6-((R)-phenyl((R)-3,3,3-trifluoro-
2-methoxy-2-phenylpropanoyloxy) methyl)-1,2-dihydropyridine-
3-carboxylic acid (16)
To a cooled solution of enantiopure alcohol (L)-10 (0.1 g,
20ꢀC
0.38 mmol; [
a]
¼ ꢂ70.95, c 0.21, methanol) in tetrahydrofuarn:di-
chloromethane (1:2, 5 mL) was sequentially added DCC (83 mg,
0.4 mmol), 4-(dimethylamino)pyridine (2 mg) and R-MTPA (94 mg,
0.4 mmol) at 0 ꢀC and stirred at room temperature for 6 h. The reaction
mixture was concentrated under reduced pressure to obtain crude
solid. Obtained crude solid was purified by flash column chromatog-
raphy over silica gel 60 (230e400 mesh) using 3% methanol:
dichloromethane as eluent and the resulting solid was further taken in
dry ether, filtered and evaporated to obtain 0.142 g (78%) of pure
desired diastereomer 16 as colorless solid. Rf ¼ 0.5 (10% Methanol:Di-
concentration (0e25 mg) of different test compounds diluted
appropriately with culture media for 48 h. Cells grown in media
containing equivalent amount of DMSO served as positive control
and cells in medium without any supplementation were used as
negative control. After the treatment, media containing compound
were carefully removed. Hundred microliters of 0.4 mg mLꢂ1 MTT
in PBS was added to each well and incubated in the dark for 4 h.
Hundred microliters of DMSO was added to each well and kept in
an incubator for 4 h for dissolution of the formed formazan crystals.
Amount of formazan was determined by measuring the absorbance
at 540 nm using an ELISA plate reader. The data were presented as
percent post treatment recovery (% of live cells), whereas the
absorbance from non-treated control cells was defined as 100% live
cells.
chloromethane);IR (KBr pellet): nmax 3427,1761,1673,1634,1171 cmꢂ1
;
1H NMR (400 MHz CDCl3):
d 14.19 (1H, brs, D2O exchangeable COOH),
13.54 (1H, brs, D2O exchangeable OH), 9.73 (1H, brs, D2O exchangeable
CONH), 7.42e7.32 (10H, m), 6.77 (1H, s), 6.22 (1H, s), 3.49 (3H, s); 13C
NMR (100 MHz, CDCl3):
d 174.85 (C), 171.85 (C), 165.9 (C), 164.87 (C),
149.74 (C),133.67 (C),131.01 (C),130.13 (CH),129.27 (CH),129.25 (2CH),
128.58 (2CH), 127.48 (2CH), 127.21 (2CH), 124.48e121.6 (CF3), 101.06
(CH), 96.98 (C), 84.62 (C), 72.42 (CH), 55.55 (CH3); 19F NMR (400 MHz
Acknowledgements
CDCl3):
d
ꢂ 70.955; MS (ESI) m/z 478.2 (M þ H)þ; LCMS (ES) m/z calcd
for C23H19F3NO7, [M þ H]þ: 477.38, found: 478.2; column used:
We are grateful to the Management, GVK Biosciences Private
Limited for the financial support and encouragement. Help from
the analytical department for the analytical data is appreciated. We
are thankful to Prof. Dr. Uma and Mr. Chiranjeevi, JNTU for the
biological data. We are highly indebted to referees for their valuable
comments and suggestions.
acquity e BEH eC 18 (50 ꢃ 2.1 mm) 1.7
m, mobile phase: A: 0.1% Aq.
HCOOH, B: 0.1% HCOOH in acetonitrile, T/%B: 0/3, 0.1/3, 1.5/90, 1.8/90,
2.2/95, 3.2/95, 3.8/3; flow rate: 0.4 mL/min, diluent: acetonitrile; UV:
310 nm, RT ¼ 1.90, purity ¼ 92.10%; HPLC-column used: XBridge-C8
(4.6 ꢃ 150 mm), 3.5
m;mobilephase:A:0.05%TFA(Aq.);B:acetonitrile;
T/%B: 0/20, 4/55, 8/90, 15/90, 15.1/20; flow rate: 1 mL/min, diluent:
acetonitrile; UV: 303 nm; RT ¼ 9.01; purity ¼ 93.50%.
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