Stereoselective synthesis and biological evaluation of (R)-rugulactone, (6R)-((4R)-hydroxy-6-phenyl-hex-2-enyl)-5,6-dihydro-pyran-2-one and its 4S epimer

Article abstract of DOI:10.1016/j.ejmech.2010.07.036

A simple and highly efficient synthetic route has been developed for synthesis of (R)-rugulactone (1a), (6R)-((4R)-hydroxy-6-phenyl-hex-2-enyl)-5,6- dihydro-pyran-2-one (1b) and its 4S epimer 1c by employing proline-catalyzed α-aminooxylation, Sharpless epoxidation, Mitsunobu reaction as chirality introuducing steps. The antibacterial and antifungal activity of the compounds 1a, 1b and 1c were evaluated. 1a and 1b showed better antibacterial activity against Pseudomonas aeroginosa (MIC =12.5 μg/ml for 1a, 25 μg/ml for 1b) Klebsiella pneumonia (MIC =25 μg/ml for 1a). Compounds (1a, 1b, 1c) exhibited good to moderate antifungal activity.

Full text of DOI:10.1016/j.ejmech.2010.07.036

European Journal of Medicinal Chemistry 45 (2010) 4657e4663
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Stereoselective synthesis and biological evaluation of (R)-rugulactone,
(6R)-((4R)-hydroxy-6-phenyl-hex-2-enyl)-5,6-dihydro-pyran-2-one and
its 4S epimer
D. Kumar Reddy a, V. Shekhar a, P. Prabhakar a, B. Chinna Babu a, B. Siddhardha b, U.S.N. Murthy b,
,
Y. Venkateswarlu a
*
a Natural Products Laboratory, Organic Division-I, Indian Institute of Chemical Technology, Hyderabad 500 007, India
b Division of Biology, Indian Institute of Chemical Technology, Hyderabad 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple and highly efficient synthetic route has been developed for synthesis of (R)-rugulactone (1a),
(6R)-((4R)-hydroxy-6-phenyl-hex-2-enyl)-5,6-dihydro-pyran-2-one (1b) and its 4S epimer 1c by
employing proline-catalyzed a-aminooxylation, Sharpless epoxidation, Mitsunobu reaction as chirality
introuducing steps. The antibacterial and antifungal activity of the compounds 1a, 1b and 1c
were evaluated. 1a and 1b showed better antibacterial activity against Pseudomonas aeroginosa
Received 14 May 2010
Received in revised form
10 June 2010
Accepted 18 July 2010
Available online 24 July 2010
(MIC ¼12.5
m
g/ml for 1a, 25
m
g/ml for 1b) Klebsiella pneumonia (MIC ¼25
mg/ml for 1a). Compounds (1a,
1b, 1c) exhibited good to moderate antifungal activity.
Keywords:
(R)-rugulactone
Ó 2010 Elsevier Masson SAS. All rights reserved.
(6R)-(4R)-hydroxy-6-phenyl-hex-2-enyl)-5,
6-dihydro-pyran-2-one
Inhibitor of NF-kB activation pathway
Antibacterial activity
Antifungal activity
1. Introduction
25 mm/mL [6]. Inspired by its interesting biological activity, previ-
ously we reported the synthesis of natural compound 1a [7]. We
wish to synthesize 1a, 1b and 1c (Fig. 1) by employing proline-
The development of drug resistance to clinically used agents has
increased the demand for discovery of new chemical scaffolds with
antimicrobial activity. In addition increase in immuno compro-
mised patients and hospital acquired infections has necessitated
the exploration of new biological targets and discovery of effective
antibacterial and antifungal agents. The natural products 5,6-
dihydro-a-pyrone derivatives having substituted alkyl side chain at
the C-6 position have been attracted much attention over the last
decade due to their medicinal potential due to Michael acceptor
nature of the a,b-unsaturated a-pyrones for amino acid residues of
receptors [1]. They inhibit HIV protease [2], induce apoptosis [3],
and have even proven to be atileukemic [4], anticancer agents [5].
Compound like 6-(4-hydroxy-6-phenyl-hex-2-enyl)-5,6-dihydro-
pyran-2-one (1a), (R)-rugulactone was isolated from Cryptocarya
rugulosa by Cardellinah and co-workers [6]. This pyrone was found
catalyzed
a-aminooxylation, Sharpless epoxidation, Mitsunobu
reaction as chirality introducing steps and evaluate antibacterial
activity against Bacillus subtilis (MTCC 441), Staphylococcus aureus
(MTCC 96), Staphylococcus epidermidis (MTCC 437), Pseudomonas
aeruginosa (MTCC 741), Klebsiella pneumonia (MTCC 39) and
Escherichia coli (MTCC 443) and antifungal activity against Rhizopus
oryzae (MTCC 262), Aspergillus (MTCC 1344), Candida albican (MTCC
227), and Saccharomyces cerevisiae (MTCC 171). Here we are
reporting the synthesis and biological evaluation of natural product
1a, as well as unnatural derivatives 1b, and 1c of same series.
2. Chemisty
The synthesis of 1a, 1b, and 1c was initiated starting from
4-benzyloxybutanal 2 [14] and 3-phenylpropanal 10 as depicted in
Schemes 1 and 2 respectively. The 4-benzyloxybutanal 2 was
to inhibit nuclear factor-kB (NF-kB) activation pathway active in
many types of cancers, exhibiting up to 5-fold induction of IkB at
reacted with nitrosobenzene in the presence of D-proline in CH3CN
at ꢀ20 ꢁC followed by treatment with NaBH4 in MeOH gave the
* Corresponding author. Tel.: þ91 40 27193167; fax: þ91 40 27160512.
E-mail address: luchem@iict.res.in (Y. Venkateswarlu).
crude aminooxy alcohol [8]. Subsequent reduction of the crude
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.036

4658
D.K. Reddy et al. / European Journal of Medicinal Chemistry 45 (2010) 4657e4663
O
O
O
O
O
OH
O
OH
O
1a
1b
1c
Fig. 1.
aminoxy product with 30 mol% CuSO4 yielded (R)-diol 3 in 85% [9].
The primary hydroxyl group in 3 was protected with tosyl chloride
using triethyl amine and catalytic amount of Bu2SnO to give tosy-
lated compound 4 in 81% yield [10], which upon reaction with
2 equiv K2CO3 in MeOH gave epoxide 5. Selective opening of
epoxide 5 with CuI and vinyl magnesium bromide in THF furnished
homoallylic alcohol 6. The secondary hydroxyl group in 6 was
protected as its TPS ether using TPSCl and imidazole in dry DCM to
yield 7. Now the benzyl group in compound 7 was removed using
lithium naphthanlenide (LN) to yield primary alcohol which was
oxidized using IBX in DMSO to yield aldehyde which, was subjected
to StilleGennari modification of the HornereEmmons olefination
reaction [7] to afford unsaturated ester 8 with a Z/E in ratio of 95:05
in 80% yield. Compound 8 on treatment with 3% HCl in MeOH
Grubbs catalyst (15 mol%) in refluxing dichloromethane to afford 1c
25
in 66% yield {[
a
]
¼ þ4.1 (c 1, CHCl3)}.
D
Finally, compounds 1a and 1b were oxidized (Scheme 4) with
MnO2 [14] to afford 1a. Its physical {[
spectroscopic data were identical to those reported for natural
25
a]
¼ ꢀ57.9 (c 1 CHCl3)} and
D
product (R)-rugulactone [7].
3. Biological activity
The minimum inhibitory concentrations (MIC) of the
compounds 1aec were tested against a panel of bacteria by broth
dilution method recommended by National Committee for Clinical
Laboratory (NCCL) standards [15]. The minimum inhibitory
concentration (MIC) values are presented in Table 1. It has been
observed that the test compounds 1aec exhibited antibacterial
activity, however with a degree of variation. Natural product 1a is
afforded 6-allyl-5,6-dihydro-a-pyrone 9 in 75% yield.
Now, the 3-phenylpropanal 10 was subjected to C2-Wittig
reaction using (ethoxycarbonylmethylene)-triphenylphosphor-
anecarbonyl to afford unsaturated ester 11 as mixture of geomet-
rical isomers (E/Z 95:5), which were separated over silica gel
column to give E isomer in 81% yield. The ester 11 was reduced with
DIBAL-H in CH2Cl2 at 0 ꢁC to allylic alcohol 12 in 85% yield. The
allylic alcohol 12 was subjected to Sharpless epoxidation with
(ꢀ)-DET, Ti (OiPr)4, and cumene hydroperoxide in dry CH2Cl2 for
5 h to afford 13 in 78% yield. The epoxide 13 was opened using
triphenylphosphine in CCl4 to give 14, which on treatment with
sodium metal in ether gave allylic alcohol [11] 15 in 82% (Scheme 2).
The allylic alcohol 15 and lactone 9 were subjected to olefine
cross metathesis (Scheme 2) using second generation Grubbs
highly active against P. aeruginosa (MTCC 741) (12.5
K. pneumonia (MTCC 39) (25 g/ml) and moderately active on S.
aureus (MTCC 96), S. epidermidis (MTCC 437), E. coli (MTCC 443)
(50 g/ml). Compound 1b is exhibiting maximum activity on
Pseudomonas aeroginosa (25 g/ml) and moderately active against
S. aureus and S. epidermidis (50 g/ml). Compound 1c was moder-
mg/ml),
m
m
m
m
ately active against the all bacteria tested. Controls were main-
tained with DMSO and penicillin (for Gram positive bacteria),
streptomycin (for Gram negative bacteria) and nitrofurantoin for all
bacteria. MIC values of penicillin, streptomycin and nitrofurantoin
are provided in Table 1.
Agar cup bioassay was employed for testing antifungal activity
of compounds following the standard procedure [16]. The anti-
fungal activity of compounds was studied against R. oryzae (MTCC
262), A. niger (MTCC 1344), C. albican (MTCC 227), and S. cerevisiae
(MTCC 171). All the compounds 1a, 1b, and 1c showed (Table 2)
good to moderate against the organisms, R. oryzae (MTCC 262) and
A. niger (MTCC 1344) and less active against unicellular fungi C.
catalyst (15 mol%) [12] in dichloromethane under reflux to afford
25
1b in 68% {[
a]
¼ ꢀ44.6 (c 1, CHCl3)}.
D
Further, the stereochemistry at C-4 in allylic alcohol 15 was
inverted following Mitsunobu reaction [13] to afford 15a
25
{[a]
¼ þ6.3 (c 1, CHCl3)}, which was subjected to olefine cross
D
metathesis (Scheme 3) with lactone 9 using second generation
OH
c
OH
a
OH
b
H
BnO
OTS
BnO
2
BnO
O
3
4
OTPS
OH
f
e
d
O
BnO
Bno
BnO
MeO
7
6
5
O
OTPS
g
O
O
8
9
Scheme 1. Reagents and conditions: (a) (i) PhNO, D-proline (25 mol %), CH3CN, ꢀ20 ꢁC, 24 h then MeOH, NaBH4; (ii) CuSO4 (30 mol %), MeOH, 0 ꢁC, 10 h, 85% (over two steps); (b)
TsCl, Bu2SnO, Et3N, 0 ꢁC to rt, 4 h, 81%(c) K2CO3, MeOH, rt, 1 h, 93%; (d) Vinyl magnesium bromide, THF, CuI, ꢀ20 ꢁC, 1 h, 88%; (e) TBDPSCl, Imidazole, dry CH2Cl2, 4 h, 95%; (f) (i) Li in
Naphthalene, ꢀ20 ꢁC, 3 h, 80%; (ii) IBX, dry DMSO, dry CH2Cl2, 5 h, 84%; (iii) MeO2CCH2P(O)(OCH2CF3)2, NaH, dry THF, ꢀ78 ꢁ C, 2 h, 76%; (g) 3% HCl in MeOH, 30 min, 75%.

D.K. Reddy et al. / European Journal of Medicinal Chemistry 45 (2010) 4657e4663
4659
O
O
c
a
H
b
OEt
OH
10
12
11
OH
O
O
e
d
OH
Cl
14
13
15
O
O
O
OH
O
9
f
1b
Scheme 2. Reagents and conditions: (a) Ph3P]CHCOOEt, benzene, reflux, 1 h, 81%; (b) DIBAL-H, CH2Cl2, ꢀ78 ꢁC, 30 min, 85%; (c) (ꢀ)-DET, Ti (OPri)4, TBHP, molecular sieves 4 Å,
DCM, ꢀ20 ꢁC, 15 h, 78%; (d) PPh3, CCl4, 80 ꢁC, 2 h; (e) sodium metal, ether, 6 h, 82% yield over two steps; (f) 2, Grubb’s II (15 mol%), CH2Cl2, 50 ꢁC, 4 h, 68%.
albicans (MTCC 227) and S. cerevisiae (MTCC 171). Compounds 1aec
5.1.1. (S)-4-(Benzyloxy)butane-1,2-diol (3)
are good active compounds with zone of inhibition values in the
To a solution of aldehyde 2 (3 g, 16.85 mmol) and nitrosobenzene
range of 12e16 mm at the concentration of 100
mg/ml against R.
(0.9 g, 8.49 mmol) in acetonitrile (30 mL) at ꢀ20 ꢁC was added
D-
oryzae (MTCC 262) and A. niger (MTCC 1344). Compounds 1aec are
less active compounds with zone of inhibition values in the range of
proline (0.24 g, 25 mol %) and the reaction mixture was stirred at the
same temperature for 24 h, followed by the addition of MeOH
(10 mL) and NaBH4 (0.97 g, 26.21 mmol) to the reaction mixture,
which was stirred for 10 min. After completion of the reaction,
a solution phosphate buffer was added and resulting mixture was
extracted into EtOAc (3 ꢂ 30 mL) and the combined organic phases
were dried over Na2SO4 and concentrated to give the crude amino-
oxy alcohol, which was directly used for the next step without
purification. To a solution of the crude aminooxy alcohol in MeOH
(50 mL) was added CuSO4$5H2O (0.64 g, 2.55 mmol) at 0 ꢁC. The
reaction mixture was allowed to stir for 10 h at this temperature.
After the addition of the phosphate buffer, the resulting mixture was
extracted into CHCl3 (3 ꢂ 30 mL) and the combined organic phases
were dried over anhydrous Na2SO4 and concentrated to give the
crude diol which was then purified by column chromatography over
8e11 mm at the concentration 100
227) and S. cerevisiae (MTCC 171).
mg/ml against C. albicans (MTCC
4. Conclusion
In conclusion, total synthesis of 6-substituted arylalkyl
unsaturated lactones (1aec) has been achieved employing proline-
catalyzed -aminooxylation, Sharpless epoxidation, mitsunobu
a,b-
a
reaction and olefine cross metathesis as key steps. Synthesized
compounds were screened for their antibacterial activity against
B. subtilis MTCC 441, S. aureus MTCC 96, S. epidermidis MTCC 437,
P. aeruginosa MTCC 741, K. pneumonia MTCC 39 and E. coli MTCC
443. Products 1aec were also tested for their antifungal activity
against R. oryzae MTCC 262, A. niger MTCC 1344, C. albican MTCC
227, and S. cerevisiae MTCC 171. Compounds 1a and 1b showed
silica gel using hexane/EtOAc (6/4 v/v) to give 3 (2.80 g, 85%) as
25
a colorless oil. [
a
]
¼ þ6.8 (c 1, CHCl3). IR (Neat): 3682, 3614, 3463,
D
3021, 2924, 2337, 1608, 1519, 1458, 1424, 1213, 1098, 1057, 924, 844,
771 cmꢀ1. 1H NMR (200 MHz, CDCl3):
7.31e7.20 (m, 5H, Ar-H), 4.48
(s, 2H AreCH2eOe), 3.92e3.78 (m, 1H, H-2), 3.61e3.42 (m, 4H, H-1,
better antibacterial activity against P. aeroginosa (MIC ¼ 12.5
mg/ml
d
for 1a, 25 g/ml for 1b) K. pneumonia (MIC 25 g/ml for 1a).
m
m
Molecules 1aec exhibited good to moderate antifungal activity.
H-4),1.71e1.61 (m, 2H, H-3). 13C NMR (75 MHz, CDCl3):
d 137.8, 128.1,
127.4, 127.3, 72.7, 69.7, 67.1, 66.1, 32.7. EIMS: m/z ¼ 196 [M]þ.
5. Experimental
5.1.2. (S)-Toluene-4-sulfonic acid 3-benzyloxy-2-hydroxy-propyl
ester (4)
5.1. General
To a cooled (0 ꢁC) solution of (R)-diol 3 (2.7 g, 13.77 mmol),
catalytic amount of dibutyl tin oxide (5 mg) and triethyl amine
(3.8 mL, 27.52 mmol) in CH2Cl2 (20 mL) was added drop wise
a solution of tosyl chloride (2.62 g, 13.77 mmol) in dichloromethane
(10 mL) and stirred the reaction for 4 h at room temperature. After
completion of reaction, the mixture was diluted with water and
extracted into CH2Cl2 (3 ꢂ 30 mL). The organic layer was washed
with brine solution and dried over anhydrous Na2SO4 and concen-
trated under reduced pressure to give the crude residue which was
purified on a silica gel column, eluted with hexane/EtOAc (7/3 v/v) to
afford compound 4 as a viscous liquid (3.89 g, 81%). IR (neat): 3407,
3019, 2927, 2400, 1719, 1518, 1454, 1364, 1215, 1176, 1047, 928,
Solvents were dried over standard drying agents and freshly
distilled prior to use. The reagents were purchased from Aldrich
and Acros, and were used without further purification unless
otherwise stated. All moisture-sensitive reactions were carried out
under nitrogen. Organic solutions were dried over anhydrous
Na2SO4 and concentrated below 40 ꢁC in vacuo. All column chro-
matographic separations were performed using silica gel (Acme’s
60e120 mesh). 1H NMR (200 MHz and 300 MHz) and 13C NMR
(50 MHz and 75 MHz) spectra were measured with a Varian Gemini
FT-200 MHz and Bruker Avance 300 MHz with tetramethylsilane as
internal standard for solutions in deuteriochloroform. J values are
given in Hertz. IR spectra were recorded on a PerkineElmer IR-683
spectrophotometer with NaCl optics. Optical rotations were
measured with Horiba high sensitive polarimeter SEPA-300 at
25 ꢁC. Mass spectra were recorded on Agilent Technologies 1100
Series (Agilent ChemstationsSoftware).
756 cmꢀ1. 1H NMR (300 MHz, CDCl3):
d
7.8 (d, 2H, J ¼ 8.6Hz, Ts-H),
7.41e7.25 (m, 7H, TS-H, Ar-H), 4.5 (s, 2H, Ar-CH2-O-), 4.11e3.93
(m, 3H, H-1, H-2), 3.71e3.52 (m, 2H, H-4), 2.45 (s, 3H, Ts-CH3),
1.72e1.69 (m, 2H, H-3). 13C NMR (75 MHz, CDCl3): 145.0, 141.0, 137.6,

4660
D.K. Reddy et al. / European Journal of Medicinal Chemistry 45 (2010) 4657e4663
O
O
OH
O
OH
OH
O
b
9
a
1c
15
15 a
Scheme 3. Reagents and conditions: (a) (i) PPh3, DIAD, p-NO2C6H4COOH, THF, rt, 2 h, 86%; (ii)Na (catalytic), MeOH, rt, 1 h, 83%; (b) 2, Grubb’s II (15 mol%), CH2Cl2, 50 ꢁC, 4 h, 66%.
128.4, 127.9, 127.8, 127.6, 126.0, 73.8, 68.6, 67.5, 32.43, 37.3, 21.6.
wise and stirred for 4 h. After the completion of reaction, the reac-
tion mixture was diluted with water (20 mL) and extracted into
dichloromethane (3 ꢂ 30 mL). The combined organic layer was
washed with brine solution (10 mL) and dried over anh. Na2SO4 and
concentrated under vacuum to furnish the crude residue, which was
purified by column chromatography on silica gel eluting with
ESIMS: m/z ¼ 373 [M þ Na]þ.
5.1.3. (S)-4-(Benzyloxy)-1,2-epoxybutane (5)
To a solution of tosyl compound 4 (3.7 g, 10.58 mmol) in MeOH
(30 mL) was added K2CO3 (2.92 g, 21.17 mmol) and the mixture was
stirred at 25 ꢁC for 1 h. After completion of reaction, the solvent was
evaporated, diluted with water and extracted with ether
(3 ꢂ 50 mL). The combined organic layer was dried over anhydrous
Na2SO4 and concentrated to give the crude product, which was
purified by column chromatography over silica gel using hexane/
hexane/EtOAc (1/19 v/v) to afford the pure compound 7 (3.521, 95%).
25
[a]
¼ ꢀ6.5 (c 1, CHCl3). 1H NMR (300 MHz, CDCl3):
¼ 7.71e7.65
d
D
(m, 5H, Si-Ar-H), 7.42e7.18 (m,10H, Ar-H, Si-Ar-H), 5.78e5.64 (m,1H,
H-2), 4.92 (m, 2H, H-1), 4.33 (br s, 2H, Ar-CH2e), 3.96 (m, 1H, H-4),
3.48 (AB, JAB ¼ 4.3 Hz, 2H, H-6), 2.25 (m, 2H, H-3), 1.82e1.77 (m, 2H,
H-5), 1.05 (s, 9H, t-Bu). ESIMS: m/z ¼ 467 [M þ Na]þ.
EtOAc (8/2 v/v) to give epoxide 5 (1.74 g, 93%) as colorless oil.
25
[
a]
D
¼ ꢀ16.8 (c 1, CHCl3). IR (neat): 3015, 2925, 2864, 2402, 1725,
1496, 1455, 1362, 1217, 1102, 1028, 910, 831, 766 cmꢀ1
(300 MHz, CDCl3)
.
1H NMR
5.1.6. (R)-5-(tert-Butyl-diphenyl-silanyloxy)-octa-2,7-dienoic acid
methyl ester (8) [7]
d
¼ 7.40e7.22 (m, 5H, Ar-H), 4.51 (s, 2H, Ar-
CH2eOe), 3.62 (t, 2H, J ¼ 4.8 Hz, H-4), 3.12e3.01 (m, 1H, H-2), 2.53
(dd, 1H, J ¼ 2.7, 5.06 Hz, H-1), 2.0e1.89 (m, 1H, H-1), 1.85e1.71 (m,
To solution of naphthalene (9.94 g, 77.69 mmol) in dry THF
(20 mL) was added lithium metal (558 mg, 93.23 mmol). After
30 min, a dark green color developed which turned dark after 1.25 h.
This solution was cooled to ꢀ25 ꢁC and to this, a solution of
compound 7 (3.45 g, 7.76 mmol) in dry THF (3 mL) was added using
canula. The resulting mixture was stirred at ꢀ25 ꢁC for 3 h. After
completion of reaction as noticed by TLC, the reactionwas quenched
with saturated aqueous ammonium chloride (15 mL) and water
(15 mL). The resulting solution was extracted into ether (3 ꢂ 30 mL)
and washed with brine, dried over Na2SO4. The solvent was removed
under reduced pressure to yield curde compound which, was puri-
fied over silica gel column chromatography using hexane/EtOAc(1/9
v/v) to afford primary alcohol (2.2 g, 80%). The primary alcohol was
oxidized using the following procedure. To a stirred solution of IBX
(2.54 g, 9.1 mmol) in dry DMSO (10 mL) was added a solution of
alcohol (2.15 g, 6.07 mmol) in DCM (50 ml) at roomtemperature and
stirred for 5 h at room temperature. After completion of the reaction,
the mixture was filtered, diluted with water (25 mL) and extracted
into dichloromethane (2 ꢂ 50mL). The combined organic layer was
washed with brine (20 mL), dried (Na2SO4) and the solvent was
removed under reduced pressure to give crude aldehyde, which was
purified on column chromatography using EtOAc/hexane (1/9 v/v)
2H, H-3). 13C NMR (50 MHz, CDCl3):
d 138.1, 128.8, 128.1, 127.3, 72.7,
66.7, 49.6, 46.6, 32.7. EIMS: m/z ¼ 178 [M]þ.
5.1.4. (R)-1-benzyloxy-hex-5-en-3-ol (6)
The copper iodide (0.174 g, 0.982 mmol) was gently heated under
vacuum and slowly cooled under nitrogen atmosphere, then THF
(20 mL) was added and the resulting suspension was cooled to
ꢀ20 ꢁC and added vinyl magnesium bromide (1 M in THF, 19.04 mL,
19.04 mmol) at same temperature while stiring. A solution of
epoxide 5 (1.74 g, 9.82 mmol) in dry THF (15 mL) was added to the
above reagent and the mixture was stirred at ꢀ20 ꢁC for 1 h. After
completion of the reaction, the reaction was quenched with satu-
rated aqueous NH4Cl, extracted into EtOAc (3 ꢂ 30 mL), the
combined organic layer was washed with brine, dried over Na2SO4
and concentrated to give the crude product which was purified by
column chromatography oversilica gel using hexane/EtOAc(8/2 v/v)
25
toafford6 (1.79 g, 88%) asa colorless liquid. [
a]
D
¼ þ1.82 (c 1, CHCl3).
IR (Neat): 3444, 3069, 3030, 2921, 2862,1640,1492,1451,1363,1207,
1096, 1025, 914 and 739 cmꢀ1 1H NMR (300 MHz, CDCl3):
7.39e7.25 (m, 5H, Ar-H), 5.93e5.75 (m,1H, H-2), 5.14e5.07 (m, 2H,
.
d
H-1), 4.52 (br s, 2H Ar-CH2eOe), 3.93e3.82 (m,1H, H-4), 3.67 (d, 2H,
J ¼ 5.8 Hz, H-6), 2.23(dt, 2H, J ¼ 7.6 Hz,1.6 Hz, H-3),1.85e1.73 (m, 2H,
Table 1
H-5). 13C NMR (75 MHz, CDCl3):
d 137.9, 134.8, 128.4, 127.7, 127.6,
Antibacterial activity of compounds 1aec.
117.5, 73.3, 70.3, 68.9, 41.9, 35.8. EIMS: m/z ¼ 206 [M]þ.
Microorganism
Minimum inhibitory concentration (
mg/ml)
1a 1b 1c Penicillin Streptomycin Nitrofurantoin
5.1.5. (R)-[1-(2-Benzyloxy-ethyl)-but-3-enyloxy]-tert-butyl-
diphenyl-silane (7)
Gram positive bacteria
B. subtilis (MTCC 441) 100 100 50 1.562
100
To cooled (0 ꢁC) solution of compound 6 (1.72 g, 8.34 mmol) and
imidazole (1.41 g, 20.80 mmol) in dry dichloromethane (30 mL) was
added tertiary-butyl-diphenylsilylchloride (2.75 g, 9.89 mmol) drop
S. aureus (MTCC 96)
S. epidermides
(MTCC 437)
50
50
50 100 1.562
50 100 1.562
50
50
Gram negative bacteria
E. coli (MTCC 443)
P. aeruginosa
50 100 100
12.5 25 50
1.562
1.562
25
100
O
(MTCC 741)
O
O
K. pneumonia
(MTCC 39)
25 100 100
1.562
50
a
or
1b
1c
Zone of inhibition diameter are in mm. Negative control DMSO-No activity.
Note: penicillin for gram positive bacteria.
1a
Streptomycin for gram negative bactiria.
Scheme 4. Reagents and conditions: (a) MnO2, CH2Cl2, rt, 12 h, 82%.
Nitrofurantoin for both microorganisms.

D.K. Reddy et al. / European Journal of Medicinal Chemistry 45 (2010) 4657e4663
4661
Table 2
Antifungal activity compounds 1aec.
Microorganism
Zone of inhibition (mm)
1a
1b
1c
Claotrimazole
30 mg/ml
Fungi
30
ml
m
g/
100
ml
mg/
30
ml
m
g/
100
ml
m
g/
30
ml
m
g/
100
ml
mg/
R. oryzae (MTCC 262)
A. niger (MTCC 1344)
C. albicans (MTCC 227)
S. cerevisiae (MTCC 171)
11
14
7
12
16
8
12
11
8
14
14
10
11
12
9
9
16
12
8
21
18
22
23
7
8
8
8
10
Zone of inhibition diameter are in mm. Negative control DMSO-No activity.
Note: Claotrimazole for all fungi.
to give pure aldehyde (1.79 g, 92%) as colorless liquid. The aldehyde
was directly used for the next reaction. To a cooled (0 ꢁC) suspension
of NaH (233 mg, 10.17 mmol) in dry THF (10 mL) under N2 atmo-
sphere was added bis-(2,2,2-trifluoromethyl)(methoxy carbonyl
methyl) phosphonate (1.61 g, 5.08 mmol) in dry THF (10 mL) and
was allowed to stir for 30 min and the reaction temperature was
brought to ꢀ78 ꢁC, then a solution aldehyde (1.79 g, 5.08 mmol) in
dry THF (10 mL) was added drop wise over a period of 10 min. The
resulting mixture was stirred for 2 h at ꢀ78 ꢁC. After completion of
the reaction, the reaction mixture was quenched with saturated
NH4Cl and extracted into ether (3 ꢂ 20 mL). The combined organic
phase was dried overanh. Na2SO4 and solvent was evaporated under
reduced pressure to obtain crude product, which was purified over
d 166.4, 148.2, 140.8, 128.4, 128.3, 125.5, 121.8, 59.9, 34.3, 33.8, 14.2.
EIMS: m/z ¼ 204 [M]þ.
5.1.9. 5-Phenyl-pent-2-en-1-ol (12)
To a cooled (0 ꢁC) solution of ester 11 (3.5 g, 17.15 mmol) in dry
CH2Cl2 (40 mL) was added slowly DIBAL-H (1M in toluene,
25.58 ml, 25.58 mmol) for 15 min. The reaction mixture was stirred
at room temperature for 2 h, cooled to 0 ꢁC, and quenched with
methanol (1 mL) and sodium potassium tartarate solution (10 mL).
The reaction mixture was passed through a short pad of celite. The
filtrate was concentrated and the residue was purified by column
chromatography over silica gel using Hexane/EtOAc (8/2 v/v) to
afford 12 (2.36, 85%) as colorless liquid. IR (neat): 3443, 3068, 3029,
2923, 2861, 1641, 1491, 1450, 1362 cmꢀ1. 1H NMR (300 MHz, CDCl3):
silica gel column chromatography using EtOAc/hexane (1/19 v/v) to
25
afford (Z)-acrylate 8 (1.57 g, 76%) as yellow oil. [
a]
D
¼ þ12.8 (c 1,
d
7.31e7.23 (m, 2H, Ar-H), 7.21e7.14 (m, 3H, Ar-H), 5.77e5.59 (m,
CHCl3). 1H NMR (300 MHz, CDCl3):
d
¼ 7.76e7.64 (m, 4H, Si-Ar-H),
2H, H-2, H-3), 4.05 (d, 2H, J ¼ 3.1Hz, H-1), 2.70 (t, 2H, J ¼ 7.2Hz, H-
7.47e7.33 (m, 6H, Si-Ar-H), 6.34 (dd, J ¼ 11.5, 7.3 Hz, 1H, H-3), 5.78
(dt, J ¼ 11.5, 1.6 Hz,1H, H-2), 5.77e5.63 (m, 1H, H-7), 4.97 (m, 2H, H-
8), 3.98e3.85 (m,1H, H-4), 3.65 (s, 3H, eCO2CH3), 2.96e2.84 (m,1H,
H-4), 2.82e2.70 (m, 1H, H-4), 2.19 (m, 2H, H-6), 1.08 (br s, 9H, t-Bu).
ESIMS: m/z ¼ 431 [M þ Na]þ.
5), 2.41e2.31 (m, 2H, H-4). 13C NMR (75 MHz, CDCl3):
d 141.6, 132.0,
129.4, 128.4, 128.29, 125.8, 63.5, 35.5, and 33.9. EIMS: m/z 162 [M]þ.
5.2. (2R,3R)-5-phenyl-2,3-epoxypentanol (13)
To a cooled (ꢀ25 ꢁC) suspension of powdered activated 4 Å
molecularsievesindryCH2Cl2 (50 mL)wasaddedTi(OiPr)4 (1.205 mL,
4.073 mmol) and (ꢀ)-DET (0.9 ml, 4.07 mmol) in dry CH2Cl2 (10 mL)
and the resulting mixture was stirred for 30 min at ꢀ25 ꢁC. To this
suspension compound 12 (2.2 g, 13.58 mmol) in dry CH2Cl2 (20 mL)
wasadded and theresulting mixturewasstirred foranother30 minat
ꢀ25 ꢁC followed by addition of cumene hydroperoxide (2.38 mL,
16.29 mmol) and the resulting mixture was stirred at the same
temperature for 25 h. It was then warmed to 0 ꢁC, quenched with
1 mL of water and stirred for 1 h at rt and diluted with 30% aqueous
NaOH (10 mL) and the reaction mixture was stirred vigorously for
another 30 min at rt. The resulting mixture was then filtered through
celite rinsing with CH2Cl2. The organic phase was separated and the
aqueous phase was extracted with CH2Cl2 (3 ꢂ 10 mL). Combined
organic layer was washed with brine and dried over anhydrous
Na2SO4. The solvent was removed under reduced pressure and the
residue was purified by column chromatography over silica gel using
5.1.7. 6-Allyl-5, 6-dihydro-pyran-2-one (9)
The ester 8 (1.52 g, 3.72 mmol) was taken in 3% HCl in methanol
solution (10 mL) and stirred for 30 min. After completion of the
reaction as monitored by TLC, reactionwas quenched with saturated
NaHCO3 and extracted in to in ethyl acetate (3 ꢂ 25mL). The
combined organic extract was washed with brine solution, dried
over anh. Na2SO4 and evaporated under reduced pressure to give
crude lactone, which was purified over silica gel column chroma-
tography using EtOAc/hexane (2/8 v/v) to afford pure compound 9 as
25
clearliquid (358 mg, 75%). [
a
]
¼ ꢀ117.4 (c 1, CHCl3). IR (Neat): 3075,
2921, 2853,1721,1644 cmꢀ1.1DH NMR (300 MHz, CDCl3):
d 6.92e6.84
(m, 1H, H-3), 6.03 (d, 1H, J ¼ 9.1 Hz, H-2) 5.88e5.76 (m, 1H, H-7),
5.24e5.12 (m, 2H, H-8), 4.56e4.43 (m,1H, H-5), 2.63e2.47 (m, 2H, H-
4), 2.45e2.28 (m, 2H, H-6). 13C NMR (75 MHz, CDCl3):
d 166.7, 144.9,
133.9, 28.7, 121.3, 118.8, 77.1, 39.0. EIMS: m/z ¼ 138 [M]þ.
5.1.8. 5-Phenyl-pent-2-enoic acid ethyl ester (11)
Hexane/EtOAc (8/2 v/v) to afford 13 (1.87 g, 78%) as colorless liquid.
25
To a solution of the aldehyde 10 (3 g, 22.38 mmol) in dry
benzene (30 mL) was added Ph3P]CH2COOEt (8.54 g, 24.62 mmol)
and stirred at reflux for 1 h. After completion of the reaction, the
solvent was removed under reduced pressure and the residue
purified by column chromatography was eluted with hexane/EtOAc
(9/1 v/v) to afford 11 as colorless syrup as a mixture of geometrical
isomers (E/Z 95:5). The mixture was separated by silica gel column
chromatography to give pure E isomer (3.69 g, 81%). IR (neat): 1721,
[a]
¼ þ46.8 (c 1, CHCl3). IR (Neat): 3441, 3039, 2881, 1752, 1612,
D
1505,1460,1107,1039, 883, 748, 698 cmꢀ1.1H NMR (300 MHz, CDCl3):
d
7.28e7.13 (m, 5H, Ar-H), 3.86 (m, lH, H-2), 3.54 (m, lH, H-3), 3.01 (dt,
1H, J ¼ 6, 2 Hz, H-1), 2.91e2.7 (m, 2H, H-5), 2.1e1.81 (m, 2H, H-4). 13C
NMR (75 MHz, CDCl3): d 145.1,128.7,128.5,126.7, 61.3, 58.5, 55.4, 33.1,
31.8, and EIMS: m/z ¼ 178 [M]þ.
5.2.1. (3R)-5-Phenyl-pent-1-en-3-ol (15)
1639, 1254 cmꢀ1. 1H NMR (300 MHz, CDCl3):
d
7.28e7.11 (m, 5H, Ar-
To a solution of compound 13 (1.75 g, 9.83 mmol) in dry CCl4
(30 mL) was added triphenylphosphine (3.93 g, 14.74 mmol) and
NaHCO3 (0.82 g, 9.83 mmol). The resulting mixture was vigorously
refluxed for 2 h. Solids were filtered and washed with ether. The
H), 6.99e6.91(m, 1H, H-3), 5.80 (d, 1H, J ¼ 17.3Hz, H-2), 4.16 (q, 2H,
J ¼ 7.4, eCOOCH2CH3), 2.76 (t, 2H, J ¼ 7.6 Hz, H-5), 1.28 (t, J ¼ 7.4,
eCOOCH2CH3), 2.52 (m, 2H, H-4). 13C NMR (75 MHz, CDCl3):

4662
D.K. Reddy et al. / European Journal of Medicinal Chemistry 45 (2010) 4657e4663
solvent was removed under reduced pressure and the residue was
then purified by flash column chromatography over silica gel using
hexane/EtOAc (9:1) to afford chloro compound 14 as a colorless
viscous liquid, which was used for next step without purification.
To a solution of chloro compound 14 (1.6 g, 8.16 mmol) in dry ether
(20 mL) was added drop wise a stirred suspension of freshly
prepared sodium sand (0.37 g, 16.32 mmol) in dry ether (10 mL)
under a nitrogen atmosphere at room temperature for 4 h. After
complete addition, the reaction mixture was allowed to stir for
further 2 h. After completion of the reaction, it was carefully
quenched with MeOH (10 mL) at 0 ꢁC, diluted with brine (10 mL),
and extracted into EtOAc (3 ꢂ 15 mL). The combined organic layer
was dried over Na2SO4 and concentrated under reduced pressure to
afford crude product which was purified over silica gel column
chromatography using EtOAc/hexane (1/9 v/v) to afford pure
5.2.4. (6R)-(4S-Hydroxy-6-phenyl-hex-2-enyl)-5,6-dihydro-pyran-
2-one (1c)
A solution of compounds 15a (0.1 g, 1.85 mmol) and 9 (0.1 g,
0.724 mmol) in CH2Cl2 (30 mL) was bubbled with nitrogen flow and
added Grubbs type II catalyst (78 mg, 0.092 mmol) and the
resulting mixture was heated under nitrogen at 50 ꢁC for 4 h. The
same experimental and work up procedure was followed to afford
25
the compound 1b (0.1106 g, 66%) as light yellow liquid. [
a]
¼ þ4.1
D
(c 1, CHCl3). 1H NMR (300 MHz, CDCl3):
d
7.32e7.11 (m, 5H, Ar-H),
6.90e6.81 (m, 1H, H-3), 6.01(d, 1H, J ¼ 9.7, H-2), 5.73e5.56 (m, 2H,
H-7, H-8), 4.52e4.35 (m, 1H, H-5), 4.16e4.03 (m, 1H, H-9),
2.77e2.61 (m, 2H, H-11), 2.55e2.41 (m, 2H, H-6), 2.40e2.24 (m, 2H,
H-4), 1.9e1.72 (m, 2H, H-10). 13C NMR (75MHz, CDCl3):
d 163.5,
144.3, 141.7, 137.1, 128.3, 125.7, 124.6, 123.1, 121.3, 76.8, 71.6, 38.5,
37.6, 31.7, and 28.7. ESIMS: m/z 295 [M þ Na]þ.
compound 15 (1.08 g, 82% over two steps) as clear liquid.
25
[a]
¼ ꢀ6.2 (c 1, CHCl3). IR (Neat):
y
3349, 1604, 1498, 1455, 1428,
7.33e7.12 (m,
5.2.5. (6R)-(4-Oxo-6-phenyl-hex-2-enyl)-5,6-dihydro-pyran-2-one
(1a)
D
1403, 750 and 700 cmꢀ1. 1H NMR (300 MHz, CDCl3):
d
5H, Ar-H), 5.98e5.81 (m, 1H, H-2), 5.20e5.11 (m, 2H, H-1),
4.14e4.08 (m, 1H, H-3), 2.71 (t, J ¼ 7.2, 2H, H-5), 1.89e1.81 (m, 2H,
To a stirred solution of compound 1a or 1b (50 mg, 0.183 mmol)
in dry dichloromethane (10 mL), was added activated MnO2 (31 mg,
0.356 mmol) and stirred for 12 h at room temperature. After
completion of the reaction, the mixture was filtered through celite
then solvent removed under reduced pressure to give crude residue
product which was purified over silica gel column chromatography
H-4). 13C NMR (75MHz, CDCl3):
d 142.1, 141.2, 128.6, 128.7, 125.7,
114.8, 72.5, 38.6, 31.7. EIMS: m/z ¼ 162 [M]þ.
5.2.2. (3S)-5-Phenyl-pent-1-en-3-ol (15a)
using hexane/EtOAc (8/2 v/v) to afford pure compound lactone 1c.
To a stirred solution of PPh3 (0.98 g, 3.7 mmol), alcohol 15 (0.6g,
3.7 mmol) and p-nitrobenzoic acid (0.618 g, 3.7 mmol) in dry THF
(10 mL) was added DIAD (95%, 0.72 mL, 3.7 mmol) drop wise at
0 ꢁC. The reaction mixture was stirred for 2 h at room temperature.
After the completion of reaction, the mixture was diluted with
water (5 mL) and extracted into ethyl acetate (3 ꢂ 5 mL). The
combined organic layer was washed with NaHCO3 and brine
solution, and dried over anhydrous Na2SO4. Evaporation of the
solvent gave a crude product, which was purified by flash column
chromatography eluted with hexane/EtOAc (9:1), to afford (S)-ester
(0.98 g, 86%) as a light yellow liquid. Thus obtained ester (0.8 g,
2.5 mmol) was taken in methanol (10 mL) and added sodium
(11 mg, 0.514 mmol) and the reaction mixture stirred for 1 h. After
completion of the reaction, solid NH4Cl (30 mg) was added to the
reaction mixture. After removal of methanol under vacuum, the
residue was dissolved in EtOAc (5 mL), washed with brine and dried
over anhydrous Na2SO4. The crude product was purified over silica
gel column chromatography using EtOAc/hexane (1/9 v/v) to afford
pure inverted alcohol 15a (0.345 g, 83%) as colorless liquid. Its
spectroscopic data were identical to those of compound 15, except
that the optical rotation value is opposite.
25
[a]
¼ ꢀ57.9 (c 0.7, CHCl3). 1H NMR (300 MHz, CDCl3):
d 7.32e7.15
D
(m, 5H, Ar-H), 6.96e6.84 (m, 1H, H-3), 6.79 (dt, 1H, J ¼ 16.2 and
7.4 Hz, H-7), 6.20 (d, 1 H, J ¼ 16.7 Hz, H-8), 6.05 (d, 1H, J ¼ 9.5 Hz, H-
2), 4.62e4.51 (m, 1H, H-5), 3.05e2.80 (m, 4H, H-10, H-11),
2.74e2.57 (m, 2H, H-6), 2.43e2.27 (m, 2H, H-4). 13C NMR (75 MHz,
CDCl3):
d 199.0, 163.7, 144.6, 141.0, 140.0, 133.5, 128.5, 128.3, 126.1,
121.5, 76.1, 41.7, 37.5, 29.9, 28.9. ESIMS: m/z ¼ 293 [M þ Na]þ.
5.2.6. Agar cup diffusion method for testing antifungal activity
The ready made Potato Dextrose Agar (PDA) medium (Himedia,
39 g) was suspended in distilled water (1000 mL) and heated to
boiling until it dissolved completely. The medium and the Petri
dishes were autoclaved at a pressure of 103.4 ꢂ103 Pa for 20 min.
Agar cup bioassay was employed for testing antifungal activity of
compounds following the standard procedure [16]. The medium
was poured into petri dishes under aseptic conditions in a laminar
flow chamber. When the medium in the plates solidified, 0.5 ml of
24 h old culture of test organism was inoculated and uniformly
spread over the agar surface with a sterile L-shaped rod. Solutions
were prepared by dissolving a compound in DMSO and different
concentrations were made (30 and 100 mg/mL). After inoculation,
cups were scooped out with 6 mm sterile cork borer and the lids of
the dishes were replaced. To each cup different concentrations of
test solutions (30, 100 mg) were added. The controls were main-
5.2.3. (6R)-(4R-Hydroxy-6-phenyl-hex-2-enyl)-5,6-dihydro-pyran-
2-one (1b)
A solution of compound 15 (0.3 g, 1.85 mmol) and compound 9
(0.1 g, 0.724 mmol) in CH2Cl2 (30 mL) was bubbled with nitrogen
flow and added Grubbs type II catalyst (78 mg, 0.092 mmol) and
the resulting mixture was heated under nitrogen at 50 ꢁC for 4 h.
After completion of the reaction the solvent was removed and the
crude product was purified over silica gel column chromatography
tained with clotrimazole whose minimum zone of inhibition values
are presented in Table 2. The treated and the controls were kept at
room temperature for 24e96 h and inhibition zones were
measured and diameter was calculated. Three to four replicates
were maintained for each treatment.
using hexane/EtOAc (7/3 v/v) to afford pure lactone 1b (0.114 g,
Acknowledgements
25
68%) as light yellow liquid. [
a]
¼ ꢀ44.6 (c 1, CHCl3). IR (Neat):
D
3431, 2922, 2854, 1710, 1647, 1386, 1250, 1038, 970, 699 cmꢀ1
.
1H
The authors are thankful CSIR, New Delhi, India for the financial
support.
NMR (300 MHz, CDCl3): 7.29e7.13 (m, 5H, Ar-H), 6.91e6.79 (m,
d
1H, H-3), 6.02 (d, 1H, J ¼ 9.7, H-2), 5.76e5.59 (m, 2H, H-7, H-8),
4.51e4.37 (m, 1H, H-5), 4.15e4.05 (m, 1H, H-9), 2.69 (t, 2H,
J ¼ 6.9 Hz, H-11), 2.59e2.43 (m, 2H, H-6), 2.40e2.23 (m, 2H, H-4),
References
1.92e1.76 (m, 2H, H-10). 13C NMR (75 MHz, CDCl3):
d 163.7, 144.1,
[1] S.B. Buck, C. Hardouin, S. Ichikawa, D.R. Soenen, C.M. Gauss, I. Hwang,
M.R. Swingle, K.M. Bonness, R.E. Honkanen, D.L. Boger, J. Am. Chem. Soc.
125 (2003) 15694e15695.
141.5, 137.2, 128.2, 125.7, 124.7, 123.2, 121.4, 76.9, 71.5, 38.6, 37.4,
31.6, and 28.7. ESIMS: m/z ¼ 295 [M þ Na]þ.

D.K. Reddy et al. / European Journal of Medicinal Chemistry 45 (2010) 4657e4663
4663
[2] K.R. Romines, R.A. Chrusciel, Curr. Med. Chem. 2 (1995) 825e838.
[3] S.H. Inayat-Hussain, B.O. Annuar, L.B. Din, N. Taniguchi, Toxicol. Lett. 131
(2002) 153e159.
[4] H. Kikuchi, K. Sasaki, J. Sekiya, Y. Maeda, A. Amagai, Y. Kubohara, Y. Ohsima,
Bioorg. Med. Chem. 12 (2004) 3203e3214.
[10] M.Narasimhulu,T.SrikanthReddy,K.ChinniMahesh,A.SaiKrishna, J. Venkateswara
Rao, Y. Venkateswarlu, Bioorg. Med. Chem. Lett. 19 (2009) 3125e3127.
[11] J. Jon Paul Selvam, K. Rajesh, V. Suresh, D. Chanti Babu, Y. Venkateswarlu,
Tetrahedron: Asymmetry 20 (2009) 1115e1119.
[12] S. BouzBouz, R. Simmons, J. Cossy, Org. Lett. 6 (2004) 3465e3467.
[13] M. Saeed, T. Ilg, M. Schick, M. Abbas, W. Voelter, Tetrahedron Lett. 42 (2001)
7401e7403.
[5] A.D. Fatima, L.K. Kohn, M.A. Antonio, J.E.d. Carvalho, R.A. Pillia, Bioorg. Med.
Chem. 13 (2005) 2927e2933.
[6] T.L. Meragelman, D.A. Scudiero, R.E. Davis, L.M. Staudt, T.G. McCloud,
J.H. Cardellina II, R.H. Shemaker, J. Nat. Prod 7 (2009) 336e339.
[7] D. Kumar Reddy, V. Shekhar, T. Srikanth Reddy, S. Purushotham Reddy,
Y. Venkateswarlu, Tetrahedron: Asymmetry 20 (2009) 2315e2319.
[8] Y. Hayashi, J. Yamaguchi, K. Hibino, M. Shoji, Tetrahedron Lett. 44 (2003)
8293e8296.
[14] P. Prabhakar, S. Rajaram, D. Kumar Reddy, V. Shekar, Y. Venkateswarlu,
Tetrahedron: Asymmetry 21 (2010) 216e221.
[15] National Committee for Clinical Laboratory Standards (NCCLS), Standard
Methods for Dillution Antimicrobial Susceptibility Tests for Bacteria, Which
Grows Aerobically. Nat. Comm Clini. Lab. Stands, Villanova, 1982, p 242.
[16] E. Margery Linday, Practical Introduction to Microbiology. E. & F.N. Spon, UK,
1962, p 177.
[9] N. Momiyama, H. Yamamoto, J. Am. Chem. Soc. 125 (2003) 6038e6039.