Total synthesis and in vitro bioevaluation of clavaminols A, C, H & deacetyl clavaminol H as potential chemotherapeutic and antibiofilm agents

Article abstract of DOI:10.1016/j.ejmech.2016.04.073

A highly concise and expedient total synthesis of bioactive clavaminols (1-4) has been executed using commercially available achiral compound decanol. The synthetic strategy relied on trans-Wittig olefination, Sharpless asymmetric epoxidation, regioselective azidolysis and in situ detosylation followed by reduction as key reactions with good overall yield. Based on biological evaluation studies of all the synthesized compounds, it was observed that the clavaminol A (1) exhibited good cytotoxicity against DU145 and SKOV3 cell lines with IC₅₀ value of 10.8 and 12.5 μM, respectively. Clavaminol A (1) and deacetyl clavaminol H (3) displayed selective promising inhibition towards Gram-positive pathogenic bacterial strains and showed good antifungal activity against the tested Candida strains. In addition, compounds 1 and 3 have demonstrated significant bactericidal activity. Compound 3 was found to be equipotent to the standard drug Miconazole displaying MFC value of 15.6 μg/mL against Candida albicans MTCC 854, C. albicans MTCC 1637, C. albicans MTCC 3958 and Candida glabrata MTCC 3019. Compounds 1 and 3 were also able to inhibit the biofilm formation of Micrococcus luteus MTCC 2470 and Staphylococcus aureus MLS16 MTCC 2940. Clavaminol A (1) increased the levels of reactive oxygen species (ROS) accumulation in M. luteus MTCC 2470.

Full text of DOI:10.1016/j.ejmech.2016.04.073

European Journal of Medicinal Chemistry 120 (2016) 86e96
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Total synthesis and in vitro bioevaluation of clavaminols A, C, H &
deacetyl clavaminol H as potential chemotherapeutic and antibiofilm
agents
T. Vijai Kumar Reddy , A. Jyotsna , B.L.A. Prabhavathi Devi *, R.B.N. Prasad a,
a
a
a,
b
b
Y. Poornachandra , C. Ganesh Kumar
Centre for Lipid Research, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, 500007, India
a
b
Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, 500007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 March 2016
Received in revised form
A highly concise and expedient total synthesis of bioactive clavaminols (1e4) has been executed using
commercially available achiral compound decanol. The synthetic strategy relied on trans-Wittig olefi-
nation, Sharpless asymmetric epoxidation, regioselective azidolysis and in situ detosylation followed by
reduction as key reactions with good overall yield. Based on biological evaluation studies of all the
synthesized compounds, it was observed that the clavaminol A (1) exhibited good cytotoxicity against
26 April 2016
Accepted 27 April 2016
Available online 6 May 2016
DU145 and SKOV3 cell lines with IC50 value of 10.8 and 12.5
mM, respectively. Clavaminol A (1) and
deacetyl clavaminol H (3) displayed selective promising inhibition towards Gram-positive pathogenic
bacterial strains and showed good antifungal activity against the tested Candida strains. In addition,
compounds 1 and 3 have demonstrated significant bactericidal activity. Compound 3 was found to be
Keywords:
Clavaminols
Cytotoxic
Antimicrobial
Antibiofilm
equipotent to the standard drug Miconazole displaying MFC value of 15.6 mg/mL against Candida albicans
MTCC 854, C. albicans MTCC 1637, C. albicans MTCC 3958 and Candida glabrata MTCC 3019. Compounds 1
and 3 were also able to inhibit the biofilm formation of Micrococcus luteus MTCC 2470 and Staphylococcus
aureus MLS16 MTCC 2940. Clavaminol A (1) increased the levels of reactive oxygen species (ROS)
accumulation in M. luteus MTCC 2470.
Reactive oxygen species
©
2016 Elsevier Masson SAS. All rights reserved.
1
. Introduction
been isolated from the Mediterranean ascidian, namely, Clavelina
phlegraea, and they displayed promising cytotoxic activity [8]. Their
structures were well characterized by spectroscopic methods and
chemical correlation with the CD spectra of benzoyl derivatives
with stereoisomers of known standards for the determination of
the absolute configuration of these compounds [9]. These mole-
cules have an anti-(2R,3S)-configuration and which is opposite to
the absolute configuration of most common natural sphingolipids
[10]. Most of the clavaminols possess a C12 alkyl chain sphingoid
backbone with anti-2-amino-3-alkanol motif and have shown
cytotoxic and apoptotic properties [11]. In particular, clavaminol A
Sphingoid-type bases or marine-derived long-chain 2-amino-3-
alkanols are important functional motifs which are found in a
diverse range of bioactive natural products and synthetic molecules
[1,2]. They are structural backbone of sphingolipids (ceramides,
sphingosine, sphingomyelin, cerebrosides and gangliosides) which
are essential components of the cell membrane; play unique and
crucial role in many physiological processes like cell recognition,
signal transduction and apoptosis [3e5]. In addition long-chain
amino alcohols from marine organisms exhibit prominent anti-
fungal, cytotoxic, antiviral, immunostimulatory, neuritogenic,
antitumor, antidiabetic, and protein kinase inhibitor activities [6,7].
Among these sphingoids, a family of fourteen new marine
sphingoid-type bioactive compounds, called clavaminols A-N have
was found to be the most potent cytotoxic compound (IC50 z 5 mg/
mL), promoting cell death against three different cell lines (A549-
lung carcinoma; T47D-breast carcinoma and AGS-gastric carci-
noma) by activation of apoptosis [8a,12]. Whereas, clavaminols C &
H were not active in comparison to other clavaminols. Interestingly,
deacetyl clavaminol H retained significant activity against AGS
(gastric carcinoma) [8b]. There is a close structure-function
*
Corresponding author.
E-mail address: prabhavathi@iict.res.in (B.L.A. Prabhavathi Devi).
http://dx.doi.org/10.1016/j.ejmech.2016.04.073
223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
0

T. Vijai Kumar Reddy et al. / European Journal of Medicinal Chemistry 120 (2016) 86e96
87
relationship between clavaminols and cytotoxic properties, which
2. Results and discussion
make these type of compounds an attractive scaffold for anticancer
drug discovery. In view of their biological significance, as well as
the unusual stereochemical configuration of these bioactive com-
pounds, and unique structure with an array of functionalities;
clavaminols family became the interesting synthetic goals and in
this regard considerable efforts have been devoted towards their
synthesis. The varied biological property of these compounds has
ensured that very few reports are available in the literature towards
the synthesis of clavaminols A, C, H and deacetyl clavaminol H
2.1. Chemistry
We envisioned the retrosynthetic analysis of bioactive clav-
aminols (1e4) as depicted in Scheme 1. From a retrosynthetic
perspective, we proposed that the target compounds (1e4) were
anticipated to be synthesized from the (2R,3S)-2-azidododecan-
1,3-diol (9), which serves as a common intermediate. This key in-
termediate 9 would afford from enantiomerically pure epoxy
alcohol (8) by C-2 selective endo mode epoxy opening with high
regioselectivity. The substrate 8 can be easily accessed from a
commercially available decanol (5) by adopting oxidation, Wittig
olefination, reduction, and Sharpless asymmetric epoxidation
sequence.
Moving forward with our proposed strategy, synthesis of clav-
aminols (1e4) was commenced from commercially available 1-
decanol (5) (Scheme 2). Initially the free hydroxyl group of 5 was
subjected to oxidation with pyridinium chlorochromate (PCC) in
dichloromethane (DCM) [15] gave the corresponding aldehyde,
which subsequently underwent to a two carbon homologation by a
[12,13].
In 2009, Seguin and coworkers reported the synthesis of
deacetyl clavaminol H in six steps which involved the stereo-
selective addition of racemic 3-alkoxy allenylzinc to a enantio-
pure N-tert-butylsulfinyl imines [13a]. Ait-Youcef et al. reported
the synthesis of clavaminol H in nine steps from fatty acids by
involving Ru-catalyzed hydrogenation and followed by organo-
catalytic electrophilic amination as the key steps [13b]. In 2011,
Sutherland and Zaed reported the title compounds from (R)-
glycidol in 14 steps with an overall yield of 28%, which involved
palladium(II)-catalyzed directed Overman rearrangement and
tin-mediated reduction reactions as the key steps [13c]. Chen and
coworkers have recently reported an improved four step
approach for the stereoselective synthesis of clavaminol A and
Wittig
triphenylphosphorane stable ylide in dry tetrahydrofuran (THF) at
room temperature to afford the desired (E)- -unsatuareted ester
reaction
with
ethoxycarbonyl-methylene-
a,b
deacetyl clavaminol H from
D
-alanine and
D
-serine, respectively
(6) as the only product with 89% yield over two steps [14a,16]. The
H NMR spectrum of 6 revealed a characteristic coupling constant
1
[
12]. In 2012, Prchalova and coworkers described the synthesis of
fluorinated clavaminol
congener by involving microwave induced cross-metathesis of
perfluoroalkylpropenes with a chiral terminal allylic alcohol
H
derivative and its deacetylated
(J ¼ 15.8 Hz) for the trans-olefinic protons. The (E)-ester (6) was
selectively reduced to (E)-allylic alcohol (7) employing diisobuty-
ꢁ
laluminium hydride (DIBAL-H) in DCM at ꢀ78 C in excellent yield
[
13d]. Very recently, Pandey and coworkers reported stereo-
selective approach for the preparation of clavaminols A, C and H
by proline-catalyzed eamination of aldehydes and then fol-
(93%) [16,17]. Incorporation of the required chirality was achieved
by Sharpless enantioselective epoxidation [18]. Accordingly, the
allylic alcohol (7) was treated with L-(þ)-diisopropyltartrate as the
a
ꢁ
lowed by indium mediated allylation as the key steps [13e].
Another recent report for synthesis of clavaminol H and deacetyl
clavaminol H was achieved from the chiral sulfonium salts
chiral source and interrupting the reaction after 24 h at ꢀ25 C in
i
presence of t-BuOOH and Ti(O Pr)
4
, furnished (2S,3S)-epoxy alcohol
2
8
(8) [a
]
D
ꢀ34.5 (c 1.0, CHCl
3
) in 91% yield.
derived from
D
-methionine [13f]. The main drawbacks of all
The next crucial step in the strategy is C-2 regioselective
these above methods are low levels of stereoselection and the
purification of stereoisomers. Most of these routes involved a
large number of steps and they did not show the same level of
stereocontrol. Moreover, an incomplete stereoselection is
observed, and in many cases, the syn isomers are preferentially
formed, while the opposite anti relative stereochemistry is
required to build up the backbone of these compounds. There-
fore, a simple practical, concise and straight forward new syn-
thetic approach of these target molecules from inexpensive and
achiral starting material with high level of regioselectivity is
always still in demand.
To shed light on synthesis of clavaminols and their associated
bioactive properties and as a continuation of our longstanding in-
terest towards synthesis of N-containing bioactive molecules [14],
we were engaged in the development of a new synthetic and effi-
cient methodology to synthesize anti-2-amino-3-alcohols in order
to obtain the target compounds, namely clavaminol A, C, H and
deacetyl clavaminol H and evaluated their in vitro biological ac-
tivities. We herein disclose a simple, convenient, and efficient
synthetic approach for the synthesis of title compounds, clavaminol
A (1), clavaminol C (2), deacetyl clavaminol H (3) and clavaminol H
opening of the epoxy alcohol (8) by azide nucleophile which was
accomplished by using NaN -(CH O) B system as reported by
3 3 3
Miyashita and his co-workers [19]. Accordingly, the reaction pro-
ceeded through a novel endo-mode opening of the epoxy alcohol 8
via an intramolecular boron chelation with extremely high C-2
regioselectivity rather than C-3 position [14a,17a,20]. Furthermore,
4
we have treated the resulting crude product with NaIO impreg-
nated over silica gel to remove the C-3 opened compound in the
form of aldehyde by the column chromatography [21]. The desired
key intermediate 9 was isolated in 78% yield in two steps, and as a
single diastereomer after purification. The primary hydroxyl group
in compound 9 was selectively tosylated with 1.1 equiv p-TsCl in
presence of Et
3
N
and
a
catalytic amount of 4-
dimethylaminopyridine (DMAP) in DCM to furnish monotosylate
10 with 79% yield [22]. To achieve the complete synthesis of clav-
aminol A (1), we performed one-pot reduction of azido tosylate 10
with lithium aluminium hydride (LAH) in refluxing THF involving
simultaneous reduction of eN to eNH and removal of a tosyloxy
3 2
group into a methyl group [14,17a]. The overall yield of clavaminol A
(1) was found to be 34% after 8 steps. Finally, the selective acety-
lation of amine group of compound 1 with acetic anhydride (Ac O)
2
(
4) using an inexpensive and commercially available decanol,
in dry THF at reflux temperature [14b,23] afforded clavaminol C (2)
in 83% yield and the overall yield of compound 2 was found to be
28.2% after 9 steps. The physical and spectroscopic data of synthetic
clavaminol A (1) and clavaminol C (2) [For clavaminol A (1)
involving the steps with high regiocontrol in which the anti
configuration could easily be accessible. In addition, all the syn-
thesized compounds were screened for different in vitro biological
activities such as cytotoxic, antimicrobial, antifungal, antibiofilm, as
well as for their ability to induce elevation of ROS levels in Micro-
coccus luteus MTCC 2470.
2
8
28
[
a
]
D
ꢀ4.6 (c 1.0, MeOH) and for clavaminol C (2) [
a
]
D
þ11.0 (c
1.0, MeOH)] were in good agreement with those reported for the
natural product [8a] [For clavaminol A (1) [a]
D
2
5
ꢀ4.25 (c 0.0094,
2
5
MeOH) and for clavaminol C (2) [
a
]
D
þ11.4 (c 0.0022, MeOH)].

88
T. Vijai Kumar Reddy et al. / European Journal of Medicinal Chemistry 120 (2016) 86e96
OH
OH
( )
O
R1
OH
( )
OH
( )
OH
8
8
NHR2
8
N3
1
-4
9
8
5
1
2
1
2
R = H, R = H, clavaminol A (1); R = H, R = COCH
3
, clavaminol C (2);
1
2
1
2
R = OH, R = H, deacetyl clavaminol H (3); R = OH, R = COCH
3
, clavaminol H (4).
Scheme 1. Retrosynthetic analysis of the clavaminols (1e4).
O
a
b
c
O
(
)
OH
(
)
8
OEt
( )
8
OH
( )
g
OH
8
8
5
6
7
8
OH
OH
OH
OH
d
e
f
(
)
OH
( )
8
OTs
( )
8
( )
8
8
N3
N3
NH2
NHAc
9
clavaminol A
clavaminol C
1
0
1
2
OH
OH
h
i
(
)
OH
( )
OH
NHAc
8
8
NH2
deacetyl clavaminol H
clavaminol H
3
4
Reagents and conditions: (a) (i) PCC, CH
2
Cl
2
, 0 °C to rt, 1 h, (ii) Ph
3
P=CHCO
, -78 °C to rt, 1 h, 93%; (c) 4Å
, -25 °C, 24 h, 91%; (d) (i) NaN
impregnated over silica gel, CH Cl , 0 °C to rt,
N, DMAP, dry CH Cl , 0 °C to rt, 4h, 79%; (f)
, dry THF, 0 °C to reflux, 6 h, 73%; (g) Ac O, dry THF, rt to reflux, 6 h, 83%; (h) H
0% Pd-C, CH OH, rt, 5 h, 91%; (i) Ac O, dry THF, rt to reflux, 6 h, 85%.
2
Et, dry THF,
rt, 12 h, 89% (over two steps); (b) DIBAL-H, dry CH
2
Cl
2
i
molecular sieves, L-(+)-DIPT, Ti(O Pr)
4
, TBHP, dry CH
2
Cl
2
3
,
(CH
3
O)
3
B, dry DMF, 50 °C, 3 h, (ii) NaIO
4
2
2
1
h, 78.2% (over two steps); (e) p-TsCl, Et
3
2
2
LiAlH
4
2
2
,
1
3
2
Scheme 2. Synthesis of clavaminols A (1), C (2), deacetyl clavaminol H (3) and H (4).
Our next task was the synthesis of deacetyl clavaminol H (3)
0.1, MeOH) and for clavaminol H (4) [
[13f], thus confirming the (2R,3S)-stereochemical assignment of
these natural products.
a
]25
D
þ3.4 (c 1.0, MeOH)]
and clavaminol H (4). The synthesis of these target compounds
was performed as shown in Scheme 2. Initially, compound 3 was
prepared directly from the key intermediate 9 with high yield
(
91%) by the reduction of azide group of 9 with 10% Pd/C under
2.2. Biological evaluations
hydrogenation conditions in methanol [17a]. To access the N-
acetyl group which is required for clavaminol H (4), it was ach-
ieved by the selective acetylation of amine group of compound 3
2.2.1. In vitro cytotoxic activity
All synthesized clavaminol compounds 1e4 were screened for
in vitro cytotoxic activity against a panel of human tumor cell lines.
Cytotoxic data was assessed on the basis of measurement of in vitro
growth of tumor cell lines in 96-well plates by the standard MTT
assay [24] using Doxorubicin as a standard anticancer drug. Four
human tumor cell lines were used: SKOV3 derived from human
ovarian adenocarcinoma cells (ATCC No. HTB-77), DU145 derived
from human prostate cancer cells (ATCC No. HTB-81), HeLa derived
from human cervical cancer cells (ATCC No. CCL-2), MCF7 derived
from human breast cancer cells (ATCC No. HTB-22) and IC50 values
(50% inhibitory concentration) were determined as a preliminary
screen from the plotted absorbance data for the dose response
in dry THF under reflux conditions with Ac
2
O with 85% yield
[
14b,23]. Finally, we successfully achieved the total synthesis of
deacetyl clavaminol H (3) with 53.6% overall yield involving seven
steps and for clavaminol H (4) with 45.6% overall yield involving
eight steps. The spectroscopic data for synthetic deacetyl clav-
aminol H (3) and clavaminol H (4) was consistent with that re-
ported for the natural product [8b]. The optical rotation of
synthetic compounds 3 and 4 [For deacetyl clavaminol H (3)
2
8
28
[
1
a
]
D
þ6.5 (c 1.0, MeOH) and for clavaminol H (4) [
a
]
D
þ3.5 (c
.0, MeOH)] were also in good agreement with previously re-
ported data [12,13] [For deacetyl clavaminol H (3) [
2
5
a
]
D
þ6.3 (c

T. Vijai Kumar Reddy et al. / European Journal of Medicinal Chemistry 120 (2016) 86e96
89
curves. IC50 value (in
mM) for each cell line was determined as the
Table 2
In vitro antibacterial activity of the synthesized clavaminol compounds 1e4.
average of two independent experiments and the results to this
regard are tabulated in Table 1.
Test compounds
Minimum inhibitory concentration (MIC,
mg/mL)
Based on these results, it is evident that clavaminol A (1) and
clavaminol C (2) showed significant reduction in cell viability
against all tested cell lines. Particularly, clavaminol A (1) displayed
good activity against DU145 and SKOV3 cell lines with IC50 value of
Gram-positive Gram-negative
S.aa
B.sb
S. ac
M.ld
K.pe
E.cf
P.ag
Clavaminol A (1)
Clavaminol C (2)
Deacetyl clavaminol H (3)
Clavaminol H (4)
Ciprofloxacin (Control)
7.8
e
7.8
e
3.9
e
1.9
e
15.6
e
7.8
e
7.8
e
h
1
0.8 and 12.5
observed against HeLa and MCF7 cell lines at micromolar concen-
tration with IC50 value of <18.2 M. In comparison, doxorubicin
exhibited IC50 values ranging between 0.6 and 0.8 M against all
mM, respectively, while moderate activity was
7.8
e
3.9
e
3.9
e
3.9
e
15.6
e
7.8
e
7.8
e
m
0.9
0.9
0.9
0.9
0.9
0.9
0.9
m
The highlighted values in bold for the particular compounds signify the better ac-
tivities against the particular cell lines or strains when compared to other synthe-
sized compounds.
tested cell lines. However, compound 2 exhibited moderate activity
against all tested cell lines with IC50 value ranging from 14.1 to
a
Staphylococcus aureus MTCC 96.
18.5
mM. Whereas, the deacetyl clavaminol H (3) showed moderate
b
Bacillus subtilis MTCC 121.
activity against only two cell lines with IC50 value of <34.1
mM. In
c
Staphylococcus aureus MLS16 MTCC 2940.
Micrococcus luteus MTCC 2470.
Klebsiella planticola MTCC 530.
contrast, clavaminol H (4) did not exhibit any cytotoxic activity
against all the tested cell lines. Based on activity results shown in
Table 1, it was observed that compounds having free 2-amino-3-
alkanol moiety played an important role in imparting cytotoxicity
to the compound. For example, compound 1 with free 2-amino-3-
ol motif showed good to moderate cytotoxicity values against all
the tested cell lines. Whereas, compound 3 displayed less activity
due to the presence of an extra alcohol functionality when
compared to compound 1. Compound 2 having N-acyl moiety also
showed lower cytotoxic activity, while compound 4 was completely
inactive against all the tested cell lines.
d
e
f
Escherichia coli MTCC 739.
g
Pseudomonas aeruginosa MTCC 2453.
h
No activity.
against some bacterial strains. In contrast, clavaminol compounds
containing N-acyl motif (2 and 4) did not exhibit any activity
against all the tested pathogenic bacterial strains. The clavaminol A
(
1) with free amino alcohol functionality displayed superior activity
against M. luteus MTCC 2470 with MIC value of 1.9 g/mL and good
inhibitory action against S. aureus MLS16 MTCC 2940 with a MIC
value of 3.9 g/mL, while moderate inhibition was observed against
m
2.2.2. In vitro antimicrobial activity
m
In vitro antimicrobial screening of all the synthesized com-
remaining tested bacterial strains. Whereas, deacetyl clavaminol H
(3) with 2-amino-1,3-diol moiety showed good activity against
Gram-positive pathogenic strains such as B. subtilis MTCC 121,
S. aureus MLS16 MTCC 2940 and M. luteus MTCC 2470 with MIC
pounds (1e4) were examined against total 21 microbial strains,
four Gram-positive bacteria viz; Staphylococcus aureus MTCC 96,
Bacillus subtilis MTCC 121, S. aureus MLS16 MTCC 2940 and M. luteus
MTCC 2470, three Gram-negative bacteria viz; Klebsiella planticola
MTCC 530, Escherichia coli MTCC 1652 and Pseudomonas aeruginosa
MTCC 741, and fourteen fungal strains viz; Candida albicans MTCC
value of 3.9
against rest of the pathogenic strains with MIC values ranging be-
tween 7.8 and 15.6 g/mL in comparison to standard drug, Cipro-
mg/mL, and moderate inhibitory activity was observed
m
1
83, C. albicans MTCC 227, C. albicans MTCC 854, C. albicans MTCC
floxacin. From a structure-activity relationship (SAR) perspective,
the clavaminol compounds (1 and 3) with free amino alcohol motif
showed enhanced antibacterial activity against both Gram-positive
and Gram-negative pathogenic strains, while the clavaminol com-
pounds (2 and 4) having N-acyl moiety did not exhibit antibacterial
activity against all the tested bacterial strains.
1637, C. albicans MTCC 3017, C. albicans MTCC 3018, C. albicans
MTCC 3958, C. albicans MTCC 4748, C. albicans MTCC 7315, Candida
parapsilosis MTCC 1744, Candida aaseri MTCC 1962, Candida glab-
rata MTCC 3019, Candida krusei MTCC 3020 and Issatchenkia han-
oiensis MTCC 4755. All synthesized target compounds (1e4) were
screened for the determination of preliminary antibacterial and
antifungal activities by the well diffusion method [25]. Ciproflox-
acin and Miconazole were employed as standard drugs for evalu-
ation of antibacterial and antifungal activities, respectively.
2.2.2.2. In vitro antifungal activity. Fungi which include the genus
Candida are most commonly associated with various disease epi-
sodes and among them the most notable one is C. albicans, which is
a predominant commensal and opportunistic fungal pathogen
causing both superficial and systematic infections in humans [26].
Further, different Candida species are pathogenic in nature and
from a clinical perspective they cause severe nosocomial infections
(candidiasis) among immunologically compromised patients un-
dergoing cancer chemotherapy, immunosuppressive treatments,
2.2.2.1. In vitro antibacterial activity. Minimum inhibitory concen-
tration (MIC) of all synthesized compounds (1e4) was measured
against Gram-positive and Gram-negative pathogenic bacteria and
the results are illustrated in Table 2. The clavaminol compounds (1
and 3) having free amino alcohol moiety exhibited potent activity
Table 1
In vitro cytotoxic activity of the target clavaminol compounds 1e4 against human cancer cell lines.
Test compounds
IC50 values (mM)
SKOV3
DU145
HeLa
MCF7
Clavaminol A (1)
Clavaminol C (2)
Deacetyl clavaminol H (3)
Clavaminol H (4)
12.5 ± 0.32
14.1 ± 0.22
33.6 ± 0.44
e
10.8 ± 0.24
18.3 ± 0.42
34.1 ± 0.52
e
15.4 ± 0.18
18.5 ± 0.28
18.2 ± 0.32
16.3 ± 0.20
e
a
e
e
e
Doxorubicin (Control)
0.7 ± 0.09
0.7 ± 0.11
0.6 ± 0.06
0.8 ± 0.12
The highlighted values in bold for the particular compounds signify the better activities against the particular cell lines or strains when compared to other synthesized
compounds.
a
No activity, SKOV3-Ovarian cancer (HTB-77), DU145-Prostate cancer (HTB-81), HeLa-Cervical cancer (CCL-2), MCF7-Breast cancer (HTB-22).

90
T. Vijai Kumar Reddy et al. / European Journal of Medicinal Chemistry 120 (2016) 86e96
Table 3
In vitro antifungal activity of the synthesized clavaminol compounds 1 and 3.
Test strains
Minimum inhibitory concentration (MIC,
m
g/mL)a
Deacetyl clavaminol H (3)
Clavaminol A (1)
Candida albicans MTCC 183
Candida albicans MTCC 227
Candida albicans MTCC 854
Candida albicans MTCC 1637
Candida albicans MTCC 3017
Candida albicans MTCC 3018
Candida albicans MTCC 3958
Candida albicans MTCC 4748
Candida albicans MTCC 7315
Candida parapsilosis MTCC 1744
Candida aaseri MTCC 1962
Candida glabrata MTCC 3019
Candida krusei MTCC 3020
Issatchenika hanoiensis MTCC 4755
31.2
31.2
15.6
31.2
15.6
15.6
31.2
31.2
15.6
31.2
62.5
15.6
7.8
15.6
15.6
15.6
7.8
15.6
15.6
7.8
15.6
31.2
31.2
15.6
15.6
7.8
31.2
15.6
The highlighted values in bold for the particular compounds signify the better activities against the particular cell lines or strains when
compared to other synthesized compounds.
a
MIC value of Miconazole (positive control) for all the tested fungal strains is 7.8 mg/mL.
broad spectrum antibiotics and/or among HIV-infected individuals
27]. Based on these facts, the target clavaminol compounds 1e4
were assayed for in vitro antifungal activity against clinically
important pathogenic fungi and the results to this regard are listed
in Table 3.
The MIC values were compared with Miconazole as a clinically
used standard drug and defined as the lowest concentration of the
antifungal agent exhibiting no fungal growth. Among all the syn-
thesized clavaminol compounds, only two clavaminol compounds
Gram-positive and Gram-negative bacterial strains in comparison
to Ciprofloxacin as standard and the results to this regard are
shown in Table 4. The clavaminol A (1) displayed minimum
[
bactericidal concentration of 7.8
mg/mL against S. aureus MLS16
MTCC 2940 and M. luteus MTCC 2470, and moderate bactericidal
effects against the rest of tested strains. Interestingly, the deacetyl
clavaminol H (3) once again proved to exhibit remarkable bacteri-
cidal effect selectivity against Gram-positive bacterial strains such
as S. aureus MTCC 96, B. subtilis MTCC 121, S. aureus MLS16 MTCC
(
1 and 3) showed potent to moderate antifungal activity against the
tested fourteen pathogenic fungal strains with MIC values ranging
between 7.8 and 62.5 g/mL. Based on the results shown in Table 3,
the clavaminol A (1) showed remarkable activity against C. krusei
MTCC 3020 with an MIC value of 7.8 g/mL which is equipotent to
Miconazole (MIC value of 7.8 g/mL), while moderate inhibitory
activity was observed against the remaining tested fungal strains
with MIC values ranging between 15.6 and 62.5 g/mL. Interest-
2940 and M. luteus MTCC 2470 with MBC value of 7.8 mg/mL, and
showed moderate MBC values against Gram-negative bacterial
strains.
m
m
m
2.2.4. Minimum fungicidal concentration
Based on antifungal activity results of the clavaminol com-
pounds 1 and 3, they were further assayed for minimum fungicidal
concentration (MFC) [29] against several Candida strains in com-
parison to Miconazole as standard drug. The results illustrated in
Table 5 revealed that the clavaminol A (1) and deacetyl clavaminol
H (3) exhibited different levels of fungicidal effects with MFC values
m
ingly, the deacetyl clavaminol H (3) with an extra alcohol moiety
displayed significant inhibition against C. albicans MTCC 1637, C.
albicans MTCC 3958 and C. krusei MTCC 3020 with MIC value of
7
.8 mg/mL comparable to that of Miconazole (MIC value of 7.8 mg/
mL), while moderate inhibitory action was observed towards rest of
the tested fungal strains with MIC values ranging between 15.6 and
3
ranging between 7.8 and 62.5
compound displayed promising fungicidal effect against
C. albicans MTCC 854, C. albicans MTCC 1637, C. albicans MTCC 3958,
and C. glabrata MTCC 3019 with MFC value of 15.6 g/mL which is
equipotent to that of Miconazole (MIC value of 15.6 g/mL), while
mg/mL. Particularly, clavaminol
3
1.2 mg/mL.
m
m
2
.2.3. Minimum bactericidal concentration
Based on promising antibacterial activity results for clavaminol
moderate MFC values were observed against rest of the tested
fungal strains. Whereas, clavaminol A (1) showed moderate
fungicidal effect against all the tested pathogenic fungal strains
A (1) and deacetyl clavaminol H (3), they were further screened for
the minimum bactericidal concentration (MBC) [28] against all the
with MFC values ranging between 15.6 and 62.5 mg/mL.
Table 4
Minimum bactericidal concentration (MBC) of the clavaminol compounds 1 and 3.
Test strains
Minimum bactericidal concentration (MBC, mg/mL)
Clavaminol A (1)
Deacetyl clavaminol H (3)
Ciprofloxacin (control)
Staphylococcus aureus MTCC 96
Bacillus subtilis MTCC 121
Staphylococcus aureus MLS16 MTCC 2940
Micrococcus luteus MTCC 2470
Klebsiella planticola MTCC 530
Escherichia coli MTCC 739
15.6
15.6
7.8
7.8
7.8
7.8
0.9
1.9
0.9
1.9
0.9
0.9
1.9
7.8
7.8
15.6
15.6
15.6
31.2
15.6
15.6
Pseudomonas aeruginosa MTCC 2453
The highlighted values in bold for the particular compounds signify the better activities against the particular cell lines or strains when compared to other synthesized
compounds.

T. Vijai Kumar Reddy et al. / European Journal of Medicinal Chemistry 120 (2016) 86e96
91
Table 5
Minimum fungicidal concentration (MFC) of the clavaminol compounds 1 and 3.
Test strains
Minimum fungicidal concentration (MFC, mg/mL)
Clavaminol A (1)
Deacetyl clavaminol H (3)
Miconazole (control)
Candida albicans MTCC 183
Candida albicans MTCC 227
Candida albicans MTCC 854
Candida albicans MTCC 1637
Candida albicans MTCC 3017
Candida albicans MTCC 3018
Candida albicans MTCC 3958
Candida albicans MTCC 4748
Candida albicans MTCC 7315
Candida parapsilosis MTCC 1744
Candida aaseri MTCC 1962
Candida glabrata MTCC 3019
Candida krusei MTCC 3020
Issatchenkia hanoiensis MTCC 4755
62.5
31.2
31.2
62.5
31.2
31.2
62.5
62.5
31.2
62.5
62.5
31.2
15.6
62.5
15.6
31.2
15.6
15.6
31.2
15.6
15.6
31.2
62.5
62.5
31.2
15.6
15.6
31.2
7.8
15.6
15.6
15.6
7.8
7.8
15.6
7.8
7.8
7.8
7.8
15.6
7.8
7.8
The highlighted values in bold for the particular compounds signify the better activities against the particular cell lines or strains when compared to other synthesized
compounds.
2
.2.5. Antibiofilm activity
Biofilms are cell-cell or solid surface-attached groups of mi-
MTCC 2470 and B. subtilis MTCC 121 (BIC values ranging between
2.1 and 2.6 g/mL). It is noteworthy to mention that the antibiofilm
m
crobes that produce a hydrated, polymeric matrix which acts as a
protective shield enabling the survival of microbes against various
environmental stresses and their recalcitrance towards antibiotics
and disinfectant chemicals [30]. The current estimates suggests
that over 65% of nosocomial infections and over 80% of chronic
bacterial infections are caused due to biofilms [31]. Studies have
shown that during post-surgical nosocomial infections, microor-
ganisms attach to the indwelling medical devices and implants
such as urinary catheters, heart valves, prosthesis, etc. which result
in bacterial adhesion and biofilm formation [32]. To address these
issues, a systematic study was undertaken to identify the target
compounds that can function as biofilm inhibitors. The compounds
activity for all the tested compounds to a maximum extent is in
accordance with antimicrobial activity against the respective strain.
2.2.6. Detection of intracellular ROS in mature M. luteus biofilms
Reactive oxygen species (ROS) is an important inducer of
apoptosis in bacteria which may cause oxidative damage to cellular
compounds and lead to cellular dysfunction or cell death [34]. In
order to elucidate whether oxidative stress is involved in the
apoptotic cell death, the intracellular ROS accumulation within the
sessile cells of M. luteus mature biofilms was measured using 2,7-
1
e4 were screened for antibiofilm activity [33] against a panel of
10000
Control Clavaminol A (1) Ciprofloxacin
Gram-positive bacterial strains such as S. aureus MTCC 96, B. subtilis
MTCC 121, S. aureus MLS16 MTCC 2940 and M. luteus MTCC 2470,
and Gram-negative bacterial strains such as K. planticola MTCC 530,
E. coli MTCC 739, and P. aeruginosa MTCC 2453 which are common
and important nosocomial pathogens capable of producing bio-
films. Based on the results depicted in Table 6, it was observed that
the clavaminol A (1) and the deacetyl clavaminol H (3) exhibited
promising antibiofilm activity (BIC values ranging between 0.8 and
1
000
1
00
1
0
1
9
.2 mg/mL) against all tested bacterial strains. The clavaminol A (1)
having free amino alcohol moiety displayed promising biofilm in-
hibition activity against M. luteus MTCC 2470 and S. aureus MLS16
0
.5
1
2
4
MTCC 2940 with BIC values of 0.8 and 2.2 mg/mL, respectively, while
Concentration (μg/mL)
good activity was observed against other tested bacterial species.
Further, the deacetyl clavaminol H (3) also demonstrated promising
anti-biofilm activity against S. aureus MLS16 MTCC 2940, M. luteus
Fig. 1. ROS accumulation measured in sessile cells of Micrococcus luteus MTCC 2470
when treated with clavaminol A (1) and Ciprofloxacin (control). All the experiments
were performed in triplicates and the results were expressed as mean ± S.D, (n ¼ 3).
Table 6
Antibiofilm activity of the clavaminol compounds 1 and 3 against different bacterial species.
Test strains
Biofilm inhibitory concentration (BIC,
Clavaminol A (1)
mg/mL)
Deacetyl clavaminol H (3)
Ciprofloxacin (control)
Staphylococcus aureus MTCC 96
Bacillus subtilis MTCC 121
Staphylococcus aureus MLS16 MTCC 2940
Micrococcus luteus MTCC 2470
Klebsiella planticola MTCC 530
Escherichia coli MTCC 739
4.2 ± 0.32
4.3 ± 0.35
2.2 ± 0.21
0.8 ± 0.11
8.6 ± 0.41
5.2 ± 0.35
3.6 ± 0.26
4.5 ± 0.36
2.6 ± 0.28
2.1 ± 0.16
2.3 ± 0.25
9.2 ± 0.46
4.6 ± 0.33
3.9 ± 0.24
0.6 ± 0.07
0.7 ± 0.08
0.4 ± 0.11
0.5 ± 0.09
0.6 ± 0.08
0.8 ± 0.12
0.7 ± 0.11
Pseudomonas aeruginosa MTCC 2453
The highlighted values in bold for the particular compounds signify the better activities against the particular cell lines or strains when compared to other synthesized
compounds.

92
T. Vijai Kumar Reddy et al. / European Journal of Medicinal Chemistry 120 (2016) 86e96
dichlorofluorescein diacetate dye, in which the dye conversion
depends on the state of the metabolically active cells in the biofilm
otherwise. IR spectra were recorded on a Perkin-Elmer FT-IR
Spectrum BX. Mass spectra were recorded using electron spray
1
13
[35]. After treatment of M. luteus MTCC 2470 cells with different
ionization-mass spectrometry (ESI-MS). H NMR and C NMR
spectra were recorded on Bruker UXNMR (operating at 300 MHz,
concentrations of the test compound clavaminol A (1), a signifi-
cantly increased levels of intracellular ROS accumulation was
observed in the tested M. luteus strain. At a concentration of 2 and
1
13
500 MHz for H and 75 MHz, 125 MHz for C NMR) spectrometer
using CDCl , unless otherwise specified. Chemical shifts and
3
d
4
mg/mL, the clavaminol A (1) treated biofilms showed increased
coupling constants J are given in ppm (parts per million) and Hz
levels of intracellular ROS accumulation which was closer to the
standard drug Ciprofloxacin. The ROS measurements were also
carried out separately in both sessile cells and in the supernatant
(Hertz), respectively. Chemical shifts are reported relative to TMS
(
d
¼ 0.0) as an internal standard. (Abbreviations used in spectra:
s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet,
br ¼ broad, dd ¼ double of doublets, dt ¼ doublet of triplets,
td ¼ triplet of doublets, qd ¼ quartet of doublets, ddd ¼ doublet of
(
data not shown). The ROS induced increase in fluorescence was
observed only for the sessile cells, suggesting that the ROS accu-
mulation is of intracellular origin (Fig. 1). This accumulated ROS
may also be responsible for the bactericidal activity. Oxygen-
containing free radical molecules and their precursors formed in
þ
doublet of doublets). High resolution mass spectra (HRMS) [ESI]
were recorded on QSTAR XL hybrid MS/MS (Applied Biosystems,
Foster City, USA) mass spectrometer. Optical rotations [
D
a] were
biological systems are collectively termed as ROS, comprising of
measured with a JASCO P-2000 digital polarimeter.
ꢀ
superoxides (O
2
$ ), peroxides (H
2
O
2
and ROOH) and free radicals
(
HO$ and RO$). Some of the bactericidal agents stimulate free
4.2. Chemistry
radical formation via the Fenton reaction [36,37]. Recent studies
suggest that when bacterial cells are subjected to oxidative stress,
these free radicals contribute to arrest in cell growth or result in
damage of the specific essential metabolic enzymes (cellular res-
piratory chain), disrupting cellular membrane, and DNA damage
ultimately causing cell lysis and death [38].
4.2.1. (2E)-Ethyl dodec-2-enoate (6)
2 2
To a stirred solution of alcohol 5 (5 g, 31.64 mmol) in CH Cl
(50 mL) was added Celite (25 g) at room temperature and followed
by pyridinium chlorochromate (10.20 g, 47.44 mmol) was added to
ꢁ
the above suspension at 0 C. The reaction mixture was stirred for
1
h at room temperature and mixture was filtered off through Celite
3
. Conclusion
pad. The filtrate was concentrated under reduced pressure afforded
crude aldehyde as a pale yellow color liquid which was directly
used directly for the next step without further purification. To a
solution of the above aldehyde in dry THF (50 mL) was added
In summary, we have accomplished the total synthesis of clav-
aminol A (1), clavaminol C (2), deacetyl clavaminol H (3), and
clavaminol H (4) starting from commercially available decanol in a
highly efficient and concise manner. All steps involved in the pre-
sent synthesis are high yielding and the applied reagents are
inexpensive and readily available. This proposed synthetic route is
straightforward, flexible, effective and superior to those reported in
the literature as it significantly reduces the number of steps
involved in the synthesis of the target molecules. All the synthe-
sized compounds (1e4) were further screened for in vitro cyto-
toxicity against four human cancer cell lines as well as their
antibacterial, antifungal, antibiofilm and intracellular ROS accu-
mulation studies. Based on these assays, it was found that the
clavaminol A (1) with free amino alcohol moiety displayed good
cytotoxicity against DU145 and SKOV3 cell lines. The clavaminol A
Ph
3
P ¼ CHCO
2
Et (19.14 g, 33.34 mmol) and the reaction mixture
was stirred for 12 h at room temperature. After completion of the
reaction, solvent was removed under reduced pressure and the
residue was dissolved in petroleum ether. The triphenylphosphine
oxide crystallized out was filtered off and the filtrate was concen-
trated to dryness. The crude product was purified by silica gel
column chromatography (1% ethyl acetate in hexane) to afford the
pure (E)-a,b-unsaturated ester 6 (6.35 g, 89% yield over two steps)
as a colorless liquid. Spectroscopic data were consistent with the
literature data [14a,16]. IR (neat): 2926, 2855 (CHaliph.), 1723 (C¼O),
ꢀ
1 1
1655 (C¼C) cm
3
. H NMR (300 MHz, CDCl ): d 7.02e6.91 (dt,
J ¼ 15.8, 6.7 Hz, 1H, H-3), 5.81 (dt, J ¼ 15.1, 1.5 Hz, 1H, H-2),
4.23e4.14 (q, J ¼ 6.7 Hz, 2H, -COCH ), 2.24e2.14 (q, J ¼ 6.7 Hz, 2H,
H-4), 1.51e1.39 (m, 2H, H-11), 1.36e1.21 (m, 15H, H-5, H-6, H-7, H-8,
2
(1) and deacetyl clavaminol H (3) have also exhibited broad spec-
13
trum of antimicrobial and antifungal activities. The biofilm inhibi-
tion assay suggested that the clavaminol compounds 1 and 3
inhibited biofilms formed by S. aureus MLS16 MTCC 2940 and
M. luteus MTCC 2470. Furthermore, the clavaminol A (1) showed
increased levels of intracellular reactive oxygen species (ROS) in
M. luteus cells. Finally, it was observed that the compound with free
H-9, H-10, CH
CDCl ): 166.7 (CO), 149.4 (CH), 121.1 (CH), 60.0 (CH
31.8 (CH ), 29.4 (CH ), 29.3 (CH ), 29.2 (CH ), 29.0 (CH
22.6 (CH ), 14.2 (CH ), 14.0 (CH
3
), 0.88 (t, J ¼ 6.7 Hz, 3H, H-12). C NMR (75 MHz,
3
d
2
), 32.1 (CH
2
),
),
2
2
2
2
2
), 27.9 (CH
2
þ
2
3
3
). ESI-MS (m/z): 249 [MþNa] .
4.2.2. (2E)-Dodec-2-en-1-ol (7)
DIBAL-H (28.4 mL, 1.76 M solution in toluene, 50 mmol) was
2
-amino-3-alkanol moiety played a crucial role in imparting bio-
logical activity to the compound.
added drop wise to a stirred solution of (E)-
5.65 g, 25 mmol) in dry CH Cl
was allowed to warm to room temperature for 1 h. After comple-
a
,
b
-unsaturated ester 6
ꢁ
(
2
2
(50 mL) at ꢀ78 C and the mixture
4
. Experimental
tion of the reaction, mixture was quenched with MeOH (5 mL) at
0 C. Then a saturated solution of Rochelle's salt (20 mL) was added,
and stirred for 1 h at room temperature. The organic layer was
ꢁ
4.1. General
All reagents used in this study were obtained from commercial
separated and the aqueous layer extracted with CH
Combined organic layers were dried over anhydrous Na
2
Cl
2
(2 ꢂ 20 mL).
sources without any further purification. All dry reactions were
conducted under a nitrogen atmosphere in oven dried glass appa-
ratus using dry solvents. Reactions were monitored by Thin Layer
2
SO
4
,
concentrated under reduced pressure. The crude product was pu-
rified by silica gel column chromatography (2% ethyl acetate in
hexane) to afford the desired (E)-allylic alcohol 7 (4.28 g, 93%) as a
colorless liquid. Spectroscopic data were consistent with the liter-
ature data [17b]. IR (neat): 3439 (OH), 2923, 2853 (CHaliph.), 1596
Chromatography (TLC) carried out on silica plates (silica gel 60 F254
Merck) employing UV-light, iodine vapors or enaphthol for
visualization. Column chromatography was performed on silica gel
60e120 mesh) procured from Qualigens (India) using freshly
distilled hexane/ethyl acetate mixture as eluent, unless specified
,
b
ꢀ
1 1
(
(C¼C) cm . H NMR (300 MHz, CDCl
3
): d 5.76e5.57 (m, 2H, H-2, H-
3), 4.08 (d, J ¼ 4.9 Hz, 2H, H-1), 2.08e1.99 (q, J ¼ 6.9 Hz, 2H, H-4),

T. Vijai Kumar Reddy et al. / European Journal of Medicinal Chemistry 120 (2016) 86e96
93
1
.66 (br s, 1H, -OH), 1.43e1.21 (m, 14H, H-5, H-6, H-7, H-8, H-9, H-
NMR (500 MHz, CDCl
3
):
d
3.90 (d, J ¼ 4.4 Hz,1H, H-1); 3.81e3.75 (br
13
1
d
0, H-11), 0.88 (t, J ¼ 6.9 Hz, 3H, H-12). C NMR (125 MHz, CDCl
133.2 (CH); 128.7 (CH), 63.5 (CH ), 32.1 (CH ), 31.8 (CH ), 29.5
), 29.1 (CH ), 29.1 (CH ), 22.6 (CH ), 14.0
3
):
m, 1H, H-3), 3.45e3.41 (q, J ¼ 5.1 Hz, 1H, H-2), 2.27 (br s, 1H, -OH),
2
2
2
1.65 (br s, 1H, -OH), 1.59e1.48 (m, 2H, H-4), 1.38e1.22 (m, 14H, H-5,
1
3
(
(
CH
CH
2
3
), 29.4 (CH
). ESI-MS (m/z): 207 [MþNa] .
2
), 29.2 (CH
2
2
2
2
H-6, H-7, H-8, H-9, H-10, H-11), 0.88 (t, J ¼ 7.1 Hz, 3H, H-12).
NMR (75 MHz, CDCl ): 72.5 (CH), 66.7 (CH ), 62.4 (CH), 33.6 (CH
), 25.5 (CH ), 22.6 (CH
C
),
þ
3
d
2
2
3
1.8 (CH
2
), 29.4 (CH
2
ꢂ 3), 29.2 (CH
2
2
2
), 14.0
þ
4
.2.3. (2S,3S)-3-Nonyloxiran-2-yl)methanol (8)
In a 250 mL two-necked round-bottom flask, 30 mL of dry
Cl was added to 4 Å powdered molecular sieves and the sus-
(CH
3
). ESI-MS (m/z): 266 [MþNa] .
CH
2
2
ꢁ
i
pension mixture was cooled to ꢀ25 C. Next, Ti(O Pr)
4
Cl
(1.18 mL,
(10 mL)
4.2.5. (2R,3S)-2-azido-3-hydroxydodecyl-4-
methylbenzenesulfonate (10)
Anhydrous Et N (1.67 mL, 12 mmol) was added dropwise to a
3
solution of (2R,3S)-2-azidododecan-1,3-diol 9 (2.43 g, 10 mmol) in
4
mmol) and L-(þ) DIPT (0.83 mL, 4 mmol) in dry CH
2
2
were added subsequently at the same temperature and the
resulting mixture was stirred for 30 min under nitrogen atmo-
ꢁ
sphere. The allylic alcohol 7 (3.68 g, 20 mmol) in dry CH
was then added and stirred for 30 min, after which tBuOOH
9.34 mL, 40 mmol, 4.28 M in toluene) was added over 20 min at the
2
Cl
2
(30 mL)
dry CH
2
Cl
2
(20 mL) at 0 C. The resulting solution was treated with
p-TsCl (2.09 g, 11 mmol) and followed by a catalytic amount of 4-
dimethylaminopyridine at the same temperature. The reaction
mixture was stirred for room temperature for 4 h, after which it was
(
same temperature. Then the reaction mixture was stirred for
ꢁ
1
2 h at ꢀ25 C and then kept in a freezer without stirring for
diluted with H
The combined organic layers were washed with brine (20 mL) and
dried over anhydrous Na SO . The solvent was removed under
2
O (20 mL) and extracted with CH
2
Cl
2
(2 ꢂ 20 mL).
ꢁ
another 12 h. It was then warmed to 0 C, quenched with 23.6 mL of
water, and stirred for 1 h at room temperature. After that, a 30%
aqueous NaOH solution (3.93 mL) was then added and the resulting
mixture was stirred vigorously for another 30 min at room tem-
perature. The reaction mixture was filtered through a pad of Celite
rinsing with CH
aqueous phase extracted with CH
organic phases were washed with brine and dried over anhydrous
Na SO . The solvent was removed under reduced pressure and
2
4
reduced pressure and the residue was purified by silica gel column
chromatography (2% ethyl acetate in hexane) to afford the pure
mono tosyl compound 10 (3.14 g, 79%) as a colorless liquid.
2
8
2
Cl
2
. The organic phase was separated and the
[
a
]
D
ꢀ16.5 (c 1.0, CHCl
3
). IR (neat): 3519 (OH), 3035 (CHarom.),
þ
2
Cl
2
(2 ꢂ 20 mL). The combined
2925, 2854 (CHaliph.), 2100 (N ≡N), 1598 (C ¼ Carom.), 1364
ꢀ1 1
(O¼S¼O) cm . H NMR (300 MHz, CDCl
ArH); 7.36 (d, J ¼ 8.3 Hz, 2H, ArH), 4.31 (dd, J ¼ 10.5, 3.0 Hz, 1H, H-
), 4.17 (dd, J ¼ 10.5, 7.5 Hz, 1H, H-1 ), 3.71e3.61 (br m, 1H, H-3),
3.54 (ddd, J ¼ 9.0, 6.0, 3.7 Hz, 1H, H-2), 2.46 (s, 3H, ArCH ), 1.90 (d,
J ¼ 5.6 Hz, 1H, 3-OH), 1.55e1.39 (m, 2H, H-4), 1.35e1.21 (m, 14H, H-
3
):
d
7.82 (d, J ¼ 8.3 Hz, 2H,
2
4
purified by silica gel column chromatography (5% ethyl acetate in
hexane) to afford (2S,3S)-epoxy alcohol 8 (3.64 g, 91%) as a white
solid. m.p. 56.0e58.0 C. [
1
a
b
3
ꢁ
28
a
]
D
ꢀ34.5 (c 1.0, CHCl
3
); IR (KBr): 3440
ꢀ
1
13
(
OH), 2925, 2854 (CHaliph.), 1215 (CeOeCoxirane), 1086 (CeO) cm
H NMR (300 MHz, CDCl
.
5, H-6, H-7, H-8, H-9, H-10, H-11), 0.88 (t, J ¼ 6.9 Hz, 3H, H-12).
NMR (75 MHz, CDCl ):
(Ar CH ꢂ 2), 70.9 (CH), 68.9 (CH), 64.6 (CH
29.6 (CH ), 29.4 (CH ), 29.3 (CH ), 29.2 (CH
21.6 (CH
C
1
3
):
d
3.91 (ddd, J ¼ 12.8, 6.0, 3.0 Hz, 1H, H-
3
d
145.2 (C), 132.5 (C), 129.9 (Ar CH ꢂ 2), 127.9
1
a
), 3.62 (ddd, J ¼ 12.0, 7.5, 4.5 Hz, 1H, H-1
b
), 2.99e2.90 (m, 2H, H-2,
2
), 33.2 (CH
2
), 31.8 (CH
), 22.6 (CH
2
),
),
H-3), 1.62e1.53 (m, 2H, H-4), 1.50e1.39 (m, 2H, H-11), 1.37e1.22 (m,
2
2
2
2
), 25.4 (CH
2
2
1
3
þ
1
2H, H-5, H-6, H-7, H-8, H-9, H-10), 0.88 (t, J ¼ 6.7 Hz, 3H, H-12).
NMR (125 MHz, CDCl 61.7 (CH ), 58.4 (CH), 56.0 (CH), 31.8 (CH
1.5 (CH ), 29.4 (CH ), 29.4 (CH ), 29.3 (CH
), 14.0 (CH
C
),
),
3
), 14.0 (CH
3
). ESI-MS (m/z): 420 [MþNa] . HRMS (ESI):
þ
3
)
d
2
2
2
Calcd for [MþNa] (C19
31 3 4
H N O NaS) requires m/z 420.1932, found
3
2
2
2
2
), 29.2 (CH
). ESI-MS (m/z): 223 [MþNa] .
2
), 25.9 (CH
420.1944.
þ
2
2.6 (CH
2
3
4
.2.4. (2R,3S)-2-azidododecan-1,3-diol (9)
To a stirred solution of (2S,3S)-epoxy alcohol 8 (3.0 g, 15 mmol)
in dry DMF (20 mL) were added subsequently (CH O) B (3.34 mL,
0 mmol) and followed by NaN (1.95 g, 30 mmol) at room tem-
4.2.6. (2R,3S)-2-Aminododecan-3-ol [clavaminol A] (1)
To a stirred suspension of LiAlH
(20 mL) at 0 C, a solution of azido tosylate compound 10 (2.0 g,
4
(0.76 g, 20.12 mmol) in dry THF
ꢁ
3
3
3
3
5.03 mmol) in dry THF (20 mL) was added dropwise. The reaction
ꢁ
perature. The resulting reaction mixture was allowed to warm to
5
cooled to 0 C and was added a saturated solution of NaHCO
and the resulting mixture was stirred for 30 min. The organic phase
was separated and the aqueous phase extracted with ethyl acetate
mixture was refluxed for 6 h and cooled to 0 C, and the mixture
ꢁ
0
C and stirred for 3 h. After completion of the reaction, it was
was quenched with dropwise addition of saturated aqueous solu-
ꢁ
3
(5 mL)
tion of Na
Celite pad and washed thoroughly with hot ethyl acetate
(3 ꢂ 10 mL). The combined organic layers were washed with H
(15 mL), brine (15 mL) and dried over anhydrous Na SO . The sol-
2 4
SO (10 mL). The solid material was filtered off through a
2
O
(
2 ꢂ 20 mL). The combined organic layers were successively
washed with H O (20 mL), aqueous solution of NaHCO (20 mL),
and brine (20 mL), dried over anhydrous Na SO . The solvent was
2
4
2
3
vent was evaporated under reduced pressure and the crude residue
was purified by silica gel column chromatography (20% methanol in
chloroform) to afford the pure clavaminol A (1) (0.74 g, 73%) as a
white solid. Spectroscopic data were consistent with the literature
2
4
removed under reduced pressure and the residue was purified by
silica gel column chromatography (10% ethyl acetate in hexane)
afforded as 84:16 mixture of regioisomeric azido diols (3.50 g, 96%).
ꢁ
ꢁ
data [8a,13e]. m.p. 107e109 C. lit [13e]. m.p. 108e110 C.
2
9
25
To a stirred solution of the above resulting azido diol in CH
2
Cl
2
[a
]
D
ꢀ4.6 (c 1.0, CH
IR (KBr): 3494 (OH), 3388 (NH
bend), 1216 (CeN), 1041 (CeO) cm
CDCl OD):
3
OH). lit [8a]. [
a
]
D
ꢀ4.25 (c 0.0094, CH
3
OH).
ꢁ
(
30 mL) was added silica gel-supported NaIO
4
(4.6 g) at 0 C and
2
), 2927, 2855 (CHaliph.), 1557 (NH
ꢀ
1
1
stirred for 1 h at room temperature. After completion of the reac-
tion, the mixture was filtered through a Celite pad, and the silica gel
washed with CH
anhydrous Na SO
afford the crude compound, which was purified by silica gel column
chromatography (10% ethyl acetate in hexane) furnished the
.
H NMR (300 MHz,
3
þ CD
3
d
3.49e3.40 (m, 1H, H-3); 2.83 (qd, J ¼ 6.4, 3.3 Hz,
2
Cl
2
(3 ꢂ 10 mL). The solvent was dried over
1H, H-2), 1.56e1.46 (m, 2H, H-4), 1.44e1.21 (m, 14H, H-5, H-6, H-7,
H-8, H-9, H-10, H-11), 1.03 (d, J ¼ 6.6 Hz, 3H, H-1), 0.88 (t, J ¼ 6.9 Hz,
2
4
and concentrated under reduced pressure to
1
3
3H, H-12). C NMR (75 MHz, CDCl
3
þ CD
), 30.1 (CH
), 16.7 (CH ), 14.3 (CH
3
3
OD):
d
75.6 (CH), 51.2
), 30.0 (CH ), 29.8
). ESI-MS (m/z):
H28NO) requires m/
(CH), 33.2 (CH
2
), 32.3 (CH
2
), 30.2 (CH
2
2
2
(
2R,3S)-2-azidododecan-1,3-diol 9 (2.85 g, 78.2% over two steps) as
(CH ), 26.6 (CH
2
2
), 23.1 (CH
2
3
2
8
þ
þ
a pale yellow viscous liquid. [
3
a]
D
ꢀ30.9 (c 1.0, CHCl
3
). IR (neat):
202 [MþH] . HRMS (ESI): Calcd for [MþH] (C12
z 202.2165, found 202.2157.
þ
ꢀ1 1
365 (OH), 2925, 2855 (CHaliph.), 2098 (N ≡N), 1072 (CeO) cm . H

94
T. Vijai Kumar Reddy et al. / European Journal of Medicinal Chemistry 120 (2016) 86e96
ꢁ
ꢁ
]28
3
OH). IR (KBr): 3353 (NH/
4
.2.7. N-((2R,3S)-3-hydroxydodecan-2-yl)acetamide [clavaminol C]
[8b,13f]. m.p. 106.5e108.5 C. lit [13f]. m.p. 106e108 C. [
a
D
3.5 (c
2
5
(2)
1.0, CH
3
OH). lit [13e]. [
a]
D
þ3.4 (c 1.0, CH
ꢀ
1
To a solution of clavaminol A 1 (0.5 g, 2.48 mmol) in dry THF
10 mL) was added Ac O (0.28 mL, 2.98 mmol) under nitrogen at-
OH), 2947, 2832 (CHaliph.), 1651 (CO), 1218 (CeN), 1028 (CeO) cm
.
1
(
2
H NMR (300 MHz, CDCl
3
þ CD OD): 3.85e3.75 (m, 2H, H1a, H-2),
3
d
mosphere at room temperature. The resulting mixture was stirred
at the same temperature for 1 h, and stirring was continued for
additional 5 h at 65 C. The reaction mixture was diluted with H
3.74e3.69 (m, 1H, H-3), 3.68e3.60 (m, 1H, H1b), 2.01 (s, 3H,
-COCH ), 1.57e1.43 (m, 2H, H-4), 1.37e1.21 (m, 14H, H-5, H-6, H-7,
H-8, H-9, H-10, H-11), 0.88 (t, J ¼ 6.7 Hz, 3H, H-12) ppm. C NMR
(75 MHz, CDCl OD) 172.7 (CO), 72.6 (CH), 61.8 (CH ), 55.9
þ CD
(CH), 34.5 (CH ), 32.5 (CH ), 30.2 (CH ), 29.9 (CH ),
), 23.2 (CH ), 22.8 (CH ) ppm. ESI-MS (m/z): 260
3
ꢁ
13
2
O
(
10 mL) and ethyl acetate (10 mL). The organic phase was washed
3
3
d
2
with brine solution (10 mL), dried over anhydrous Na SO and
2
4
2
2
2
ꢂ 2), 30.2 (CH
2
2
removed under reduced pressure to afford the crude residue, which
was purified by silica gel column chromatography (2% methanol in
chloroform) furnished the pure clavaminol C (2) (0.5 g, 83%) as a
white solid. Spectroscopic data were consistent with the literature
26.5 (CH
2
2
2
),14.3(CH
3
þ
þ
[MþH] . HRMS (ESI): Calcd for [MþH] (C14
3
H30NO ) requires m/z
260.2220, found 260.2222.
ꢁ
ꢁ
data [8a,13e]. m.p. 104.5e106.5 C. lit [13e]. m.p.104e106 C.
4.3. Biological assays
2
9
25
[
a
]
D
þ10.5 (c 0.5, CHCl
3
), lit [8a]. [
a
]
D
þ11.4 (c 0.0022, CH
3
OH).
IR (KBr): 3345 (NH/OH), 2950, 2845 (CHaliph.), 1650 (CO), 1563 (NH
bend), 1215 (CeN), 1035 (CeO) cm
d
4.3.1. In vitro cytotoxic assay
ꢀ
1 1
.
H NMR (500 MHz, CDCl
3
):
The cytotoxicity of the synthesized clavaminols was determined
on the basis of measurement of in vitro growth inhibition of tumor
cell lines in 96 well plates by cell-mediated reduction of tetrazo-
lium salt to water insoluble formazan crystals using Doxorubicin as
a standard. The cytotoxicity was assessed against a panel of four
different human tumor cell lines: HeLa derived from human cer-
vical cancer cells (ATCC No. CCL-2), DU145 derived from human
prostate cancer cells (ATCC No. HTB-81), MCF7 derived from human
breast cancer cells (ATCC No. HTB-22), and SKOV3 derived from
human ovarian adenocarcinoma cells (ATCC No. HTB-77) using the
MTT assay [24]. The IC50 values (50% inhibitory concentration) were
calculated from the plotted absorbance data for the dose-response
5.89 (br s, 1H, -NH); 4.04e3.97 (m, 1H, H-2), 3.67e3.61 (m, 1H, H-
), 2.40 (br s, 1H, -OH), 1.99 (s, 3H, -COCH ), 1.43e1.37 (m, 2H, H-4),
3
3
1.34e1.22 (m, 14H, H-5, H-6, H-7, H-8, H-9, H-10, H-11), 1.09 (d,
13
J ¼ 6.8 Hz, 3H, H-1), 0.88 (t, J ¼ 7.1 Hz, 3H, H-12). C NMR (75 MHz,
CDCl 170.0 (CO), 73.9 (CH), 49.4 (CH), 33.5 (CH ), 31.8 (CH ), 29.6
CH ), 29.5 (CH ), 22.6 (CH ),
4.0 (CH
3
)
d
2
2
(
1
2
2
ꢂ 2), 29.2 (CH
2
), 25.9 (CH
2
), 23.3 (CH
2
2
þ
3
), 13.7 (CH
3
). ESI-MS (m/z): 244 [MþH] . HRMS (ESI):
þ
Calcd for [MþH] (C14
2
H30NO ) requires m/z 244.2271, found
2
44.2260.
4.2.8. (2R,3S)-2-Aminododecan-1,3-diol [deacetyl clavaminol H]
(3)
curves. IC50 values (in mM) are expressed as the average of three
To a solution of compound 9 (1.5 g, 6.17 mmol) in MeOH (20 mL)
was added a catalytic amount of 10% palladium adsorbed on carbon
0.015 g, 10% w/w) under hydrogen ballon pressure and stirred at
independent experiments.
(
4.3.2. In vitro antimicrobial activity assay
room temperature for 5 h. After completion of the reaction, it was
filtered through a Celite pad and the filtrate was dried over anhy-
drous Na SO and concentrated to dryness under reduced pressure.
2 4
The crude residue was purified by silica gel column chromatog-
raphy (10% methanol in chloroform) afoorded pure deacetyl clav-
aminol H (3) (1.21 g, 91%) as a white solid. Spectroscopic data were
Antimicrobial activity of the synthesized clavaminols were
screened using well diffusion method [25] against a panel of
pathogenic bacterial strains, including B. subtilis MTCC 121,
S. aureus MTCC 96, S. aureus MLS16 MTCC 2940, M. luteus MTCC
2470, E. coli MTCC 739, K. planticola MTCC 530, P. aeruginosa MTCC
2453 and different Candida strains such as C. albicans MTCC 183, C.
albicans MTCC 227, C. albicans MTCC 854, C. albicans MTCC 1637, C.
albicans MTCC 3017, C. albicans MTCC 3018, C. albicans MTCC 3958,
C. albicans MTCC 4748, C. albicans MTCC 7315, C. parapsilosis MTCC
1744, C. aaseri MTCC 1962, C. glabrata MTCC 3019, C. krusei MTCC
3020 and I. hanoiensis MTCC 4755 which were procured from the
Microbial Type Culture Collection (MTCC), CSIR-Institute of Mi-
crobial Technology, Chandigarh, India. The pathogenic reference
strains were seeded on the surface of Muller-Hinton agar Petri
plates with 0.1 mL of previously prepared microbial suspensions
ꢁ
consistent with the literature data [8b,13f]. m.p. 93e95 C.
2
8
25
[
(
a
]
D
þ6.5 (c 1.0, CH
3
OH). lit [13f]. [
a
]
D
þ6.3 (c 0.1, CH
3
OH). IR
KBr): 3498 (OH), 3382 (NH
2
), 2923, 2856 (CHaliph.), 1574 (NH
ꢀ
1
1
bend), 1215 (CeN), 1049 (CeO) cm
CD ): 3.69 (dd, J ¼ 11.1, 3.9 Hz, 1H, H-1
OD þ CDCl
), 3.38e3.34 (m, 1H, H-3), 2.72 (dt, J ¼ 8.4,
.1 Hz, 1H, H-2), 1.56e1.42 (m, 2H, H-4), 1.38e1.21 (m, 14H, H-5, H-
.
H
NMR (300 MHz,
3
3
d
a
), 3.58 (dd,
J ¼ 11.1, 6.7 Hz, 1H, H-1
b
4
6
13
, H-7, H-8, H-9, H-10, H-11), 0.88 (t, J ¼ 6.7 Hz, 3H, H-12). C NMR
73.9 (CH), 63.4 (CH ), 56.2 (CH), 33.8
), 29.8 (CH ), 29.8 (CH
(
(
(
(
75 MHz, CD
3
OD þ CDCl
), 32.1 (CH ), 29.9 (CH
), 22.8 (CH
3
)
d
2
8
CH
CH
2
2
2
2
2
), 29.5 (CH
2
), 26.2
individually containing 1.5 ꢂ 10 CFU/mL (equal to 0.5 McFarland
þ
2
2
), 14.1 (CH
3
). ESI-MS (m/z): 218 [MþH] ; HRMS
standard). Wells of 6.0 mm diameter were prepared in the media
plates using a cork borer and the synthesized clavaminols at a dose
þ
ESI): Calcd for [MþH] (C12
2
H28NO ) requires m/z 218.2114, found
2
18.2118.
range of 250e0.48 mg/well were added in each well under sterile
conditions in a laminar air flow chamber. Standard antibiotic so-
lution of Ciprofloxacin and Miconazole at a dose range of
4.2.9. N-((2R,3S)-1,3-dihydroxydodecan-2-yl)acetamide
[clavaminol H] (4)
250e0.48
mg/well and the well containing methanol served as
To a solution of compound 3 (1.0 g, 4.60 mmol) in dry THF
positive and negative controls, respectively. The plates were incu-
ꢁ
ꢁ
(
15 mL) was added Ac
2
O (0.52 mL, 5.52 mmol) at room temperature
bated for 24 h at 37 C for bacterial and 30 C for different Candida
strains and the well containing the least concentration showing the
inhibition zone was considered as the minimum inhibitory con-
centration. All experiments were carried out in duplicates and
mean values are represented.
under nitrogen atmosphere. The reaction mixture was stirred at
room temperature for 1 h, and stirring was continued for additional
ꢁ
5
h at 65 C. The reaction mixture was diluted with H
2
O (10 mL) and
ethyl acetate (10 mL). The organic phase was washed with brine
solution (10 mL), dried over anhydrous Na SO and concentrated to
2
4
dryness under reduced pressure to afford the crude residue, which
was purified by silica gel column chromatography (3% methanol in
chloroform) afforded pure clavaminol H (4) (1.01 g, 85%) as a white
solid. Spectroscopic data were consistent with the literature data
4.3.3. Minimum bactericidal concentration (MBC) and minimum
fungicidal concentration (MFC) assays
The minimum bactericidal concentration (MBC) and minimum
fungicidal concentration (MFC) assays [28,29] were performed in

T. Vijai Kumar Reddy et al. / European Journal of Medicinal Chemistry 120 (2016) 86e96
95
sterile 2.0 mL microfuge tubes against a panel of pathogenic bac-
terial strains, including B. subtilis MTCC 121, S. aureus MTCC 96,
S. aureus MLS16 MTCC 2940, M. luteus MTCC 2470, E. coli MTCC 739,
K. planticola MTCC 530, P. aeruginosa MTCC 2453 and MFC activity
determined against different Candida strains such as C. albicans
MTCC 183, C. albicans MTCC 227, C. albicans MTCC 854, C. albicans
MTCC 1637, C. albicans MTCC 3017, C. albicans MTCC 3018, C. albi-
cans MTCC 3958, C. albicans MTCC 4748, C. albicans MTCC 7315, C.
parapsilosis MTCC 1744, C. aaseri MTCC 1962, C. glabrata MTCC 3019,
C. krusei MTCC 3020 and I. hanoiensis MTCC 4755 which were
procured from the Microbial Type Culture Collection (MTCC), CSIR-
Institute of Microbial Technology, Chandigarh, India. The patho-
genic Candida strains were cultured overnight in Mueller Hinton
broth. Serial dilutions of prepared compounds were prepared in
Mueller Hinton broth with different concentrations ranging from
treated with clavaminol A (1) and Ciprofloxacin (control) was run in
parallel and incubated for 24 h at 30 C. Later, the biofilm were
ꢁ
incubated with 10
mM DCFHDA dye, simultaneously with the
treated test sample. After 24 h incubation, the fluorescence was
measured on an Infinite M200Pro microtitre plate reader at exci-
tation and emission wavelengths of 485 and 535 nm, respectively.
The level of ROS production was quantified in triplicate. The entire
content in each well was removed and the cells were separated
from the supernatant by centrifugation at 5000 ꢂ g for 10 min. The
fluorescence of the supernatant and that of the resuspended sessile
cells in phosphate buffered saline was measured separately to
determine the generated fluorescence in either intracellular or
extracellular environment. The level of ROS accumulation was
quantified in triplicate and the values are indicated as mean ± S.D.
150 to 0
mg/mL. To the serially diluted clavaminols, 100
mL of
Acknowledgments
overnight cultured bacterial and Candida suspensions were added
8
to reach a final concentration of 1.5 ꢂ 10 CFU/mL (equal to 0.5
The authors T.V.K. Reddy, A. Jyotsna and Y. Poornachandra are
thankful to the Council of Scientific and Industrial research (CSIR,
New Delhi, India for the financial assistance provided in the form of
Senior Research fellow (SRF).
ꢁ
McFarland standard) and incubated at 37 C for 24 h. After 24 h of
incubation, the minimum bactericidal concentration (MBC) or
minimum fungicidal concentration (MFC) values were determined
by sampling 10
m
L of suspension from the tubes onto Mueller
ꢁ
Hinton agar plates and were incubated for 24 h at 37 C to observe
the growth of test organisms. MBC/MFC values are the lowest
concentrations of prepared compounds required to kill a particular
bacterial/Candida strain. All the experiments were carried out in
duplicates.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.04.073.
References
4.3.4. Biofilm inhibition assay
The test compounds were screened in sterile 96 well poly-
[1] S.T. Pruett, A. Bushnev, K. Hagedom, M. Adiga, C.A. Haynes, M.C. Sullards,
D.C. Liotta, A.H.M. Merrill Jr., Thematic review series: sphingolipids. Biodi-
versity of sphingoid bases (“sphingosines”) and related amino alcohols, J. Lipid
Res. 49 (2008) 1621e1639.
styrene microtiter plates using the modified biofilm inhibition
assay [33], against a panel of pathogenic bacterial strains including
S. aureus MTCC 96, S. aureus MLS-16 MTCC 2940, B. subtilis
MTCC121, E. coli MTCC 739, P. aeruginosa MTCC 2453 and
K. planticola MTCC 530, which were cultured overnight in tryptone
soy broth (supplemented with 0.5% glucose). The test compounds
[2] S.C. Bergmeier, The synthesis of vicinal amino alcohols, Tetrahedron 56 (2000)
2561e2576.
[3] Y.A. Hannun, R.M. Bell, Functions of sphingolipids and sphingolipid break-
down products in cellular regulation, Science 243 (1989) 500e507.
[4] Y.A. Hannun, in: Y.A. Hannun (Ed.), Sphingolipid-mediated Signal Trans-
duction, R.G. Landes Company and Chapman & Hall, New York, 1997.
of predetermined concentrations ranging from 250 to 0
mg/mL were
[5] A.H. Merrill, C.C. Sweely, Biochemistry of Lipids, Lipoproteins and Membranes,
mixed with the bacterial suspensions having an initial inoculum
Elsevier, Amsterdam, 1996.
5
concentration of 5 ꢂ 10 CFU/mL. Aliquots of 100
m
L were distrib-
[6] (a) S. Kobayashi, T. Furuta, T. Hayashi, M. Nichijima, K. Hanada, Catalytic
asymmetric syntheses of antifungal sphingofungins and their biological ac-
tivity as potent inhibitors of serine palmitoyltransferase (SPT), J. Am. Chem.
Soc. 120 (1998) 908e919;
ꢁ
uted in each well and then incubated at 37 C for 24 h under static
conditions. The medium was then discarded and washed with
phosphate buffered saline to remove the non-adherent bacteria.
(b) E. Kobayashi, K. Motoki, Y. Yamaguchi, T. Uchida, H. Fukushima,
Each well of the microtiter plate was stained with 100
m
L of 0.1%
Y. Koezuka, Enhancing effects of
killer cell activity, Bioorg. Med. Chem. 4 (1996) 615e619;
c) H. Kamitakahara, T. Suzuki, N. Nishigori, Y. Suzuki, O. Kanie, C.H. Wong,
A lysoganglioside/poly-L-glutamic acid conjugate as a picomolar inhibitor of
influenza hemagglutinin, Angew. Chem. Int. Ed. Engl. 37 (1998) 1524e1528;
a-, b-monoglycosylceramides on natural
crystal violet solution followed by 30 min incubation at room
temperature. Later, the crystal violet solution from the plates was
discarded, washed thoroughly with distilled water for 3e4 times
and air dried at room temperature. The crystal violet stained bio-
(
(d) T. Natori, M. Morita, K. Akimoto, Y. Koezuka, Agelasphins, novel antitumor
and immunostimulatory cerebrosides from the marine sponge Agelas maur-
itianus, Tetrahedron 50 (1994) 2771e2784;
film was solubilized in 95% ethanol (100 mL) and the absorbance
was recorded at 540 nm using TRIAD multimode reader (Dynex
Technologies, Inc, Chantilly, VA, USA). Blank wells were employed
as background check. The inhibition data were interpreted from the
dose-response curves, where IC50 value is defined as the concen-
tration of inhibitor required to inhibit 50% of biofilm formation
under the above assay conditions. All the experiments were carried
out in triplicates and the values are indicated as mean ± S.D.
(e) R. Higuchi, K. Inukai, J.X. Jhou, M. Honda, T. Komori, S. Tsuji, Y. Nagai,
Biologically active glycosides from asteroidea, XXXI. Glycosphingolipids from
the starfish Asterias amurensis versicolor sladen, 2. Structure and biological
activity of ganglioside molecular species, Liebigs Ann. Chem. 1993 (1993)
359e366;
(f) S.A. Summers, D.H. Nelson, A role of sphingolipids in producing the com-
mon features of type 2 diabetes, metabolic syndrome X, and cushings syn-
drome, Diabetes 54 (2005) 591e602;
(g) H.Y. Li, S. Matsunaga, N. Fusetani, 9þHalicylindrosides, antifungal and
cytotoxic cerebrosides from the marine sponge Halichondria cylindrata, Tet-
rahedron 51 (1995) 2273e2280;
4
.3.5. Intracellular ROS accumulation study in mature M. luteus
biofilms
The intracellular reactive oxygen species (ROS) produced in
(h) S. Brodesser, P. Sawatzki, T. Kolter, Bioorganic chemistry of ceramide, Eur.
J. Org. Chem. 2003 (2003) 2021e2034.
[7] M. Menna, Antitumor potential of natural products from Mediterranean as-
cidians, Phytochem. Rev. 8 (2009) 461e472.
8] (a) A. Aiello, E. Fattorusso, A. Giordano, M. Menna, C. Navarrete, E. Munoz,
Clavaminols A-F, novel cytotoxic 2-amino-3-alkanols from the ascidian Clav-
elina phlegraea, Bioorg. Med. Chem. 15 (2007) 2920e2926;
M. luteus MTCC 2470 sessile cells was was detected using 2,7-
dichlorofluorescein diacetate (DCFH-DA) (Sigma), which is con-
verted to highly fluorescent dichlorofluorescein (DCF) in the pres-
ence of intracellular ROS [35]. Briefly, the M. luteus MTCC 2470 cells
was cultured in 24-well microtitre plates. After biofilm formation,
[
(b) A. Aiello, E. Fattorusso, A. Giordano, M. Menna, C. Navarrete, E. Munoz,
Clavaminols GeN, six new marine sphingoids from the Mediterranean
ascidian Clavelina phlegraea, Tetrahedron 65 (2009) 4384e4388.
the mature biofilms were washed with 300
m
L of 0.9% (w/v) NaCl,
[9] M.H. Kossuga, J.B. MacMillan, E.W. Rogers, T.F. Molinski, G.G.F. Nascimento,

96
T. Vijai Kumar Reddy et al. / European Journal of Medicinal Chemistry 120 (2016) 86e96
R.M. Rocha, R.G.S. Berlinck, (2S,3R)-2-Aminododecan-3-ol, a new antifungal
agent from the ascidian Clavelina oblonga, J. Nat. Prod. 67 (2004) 1879e1881.
10] (a) P.M. Koskinen, A.M.P. Koskinen, Sphingosine, an enigmatic lipid: a review
O,O-triacetyl-d-erythro-sphingosine and N, O,O-triacetyl Sphingonine from a
Common Chiral Intermediate Derived from D-mannitiol, ARKIVOC vi, 2012,
pp. 421e436.
[
[
of recent literature syntheses, Synthesis 1998 (1998) 1075e1091;
[21] (a) Y.L. Zhong, T.K.M. Shing, Efficient and facile glycol cleavage oxidation using
improved silica gel supported sodium metaperiodate, J. Org. Chem. 62 (1997)
2622e2624;
(
b) A.R. Howell, R.C. So, S.K. Richardson, Approaches to the preparation of
sphinganines, Tetrahedron 60 (2004) 11327e11347.
11] J.H. Choi, M. Yoshida, T. Suzuki, E. Harada, M. Kawade, K. Yazawa,
S. Nishimoto, H. Hirai, H. Kawagishi, A novel sphingosine with osteoclast-
forming suppressing activity, from the edible mushroom Grifola gargal, Tet-
rahedron 69 (2013) 8609e8611.
(b) D.K. Mohapatra, G. Sahoo, D.K. Ramesh, J.S. Rao, G.N. Sastry, Total syn-
theses and absolute stereochemistry of decarestrictines C1 and C2, Tetrahe-
dron Lett. 50 (2009) 5636e5639;
(c) D.K. Mohapatra, N. Gogulothu, J.S. Yadav, The first asymmetric total syn-
theses of 3((1R,2R)- and 3((1S,2R)-2-(12-methyltridecyl)cyclopropyl)prop-
anoic acid, Tetrahedron Lett. 53 (2012) 6718e6720.
[
[
12] B.S. Chen, L.H. Yang, J.L. Ye, T. Huang, Y.P. Ruan, J. Fu, P.Q. Huang, Diaster-
eoselective synthesis and bioactivity of long-chain anti-2-amino-3-alkanols,
Eur. J. Med. Chem. 46 (2011) 5480e5486.
13] (a) C. Seguin, F. Ferreira, C. Botuha, F. Chemla, A.P. Luna, High-yielding syn-
thesis of sphingoid-type bases, J. Org. Chem. 74 (2009) 6986e6992;
[22] (a) J.S. Yadav, T. Swamy, B.V. Subba Reddy, V. Ravinder, Stereoselective syn-
thesis of C19-C27 fragment of bryostatin 11, Tetrahedron Lett. 55 (2014)
4054e4056;
(
b) R. Ait-Youcef, X. Moreau, C. Greck, Asymmetric synthesis of sphinganine
and clavaminol H, J. Org. Chem. 785 (2010) 5312e5315;
c) A.M. Zaed, A. Sutherland, Total synthesis of clavaminol A, C and H, Org.
Biomol. Chem. 9 (2011) 8030e8037;
d) E. Perchalova, I. Votruba, M. Kotora, Sphingosine and clavaminol H de-
rivatives bearing fluorinated chains and their cytotoxic activity, J. Fluor. Chem.
41 (2012) 49e57;
e) M. Pandey, P.S. Chowdhury, A.K. Dutta, P. Kumar, S. Pal, A highly concise
and practical route to clavaminols, sphinganine and (þ)-spisulosine via in-
dium mediated allylation of ehydrazino aldehyde and a theoretical insight
into the stereochemical aspects of the reaction, RSC Adv. (2013)
5442e15448;
f) F. Sarabia, C.V. Garcia, C.G. Ruiz, A.S. Ruiz, M.S.P. Gonzalez, M.G. Castro,
(b) P.R. Krishna, K. Anitha, Total synthesis of fluvirucinine A1, Tetrahedron
Lett. 52 (2011) 4546e4549;
(c) B. Thirupathi, P.P. Reddy, D.K. Mohapatra, A carbohydrate-based total
synthesis of (þ)-pyrenolide D and (-)-4-epi-pyrenolide D, J. Org. Chem. 76
(2011) 9835e9840;
(
(
(d) G. Sabitha, Ch Srinivas, Ch Maruthi, J.S. Yadav, Synthesis of (4R,6S,7R)-7-
hydroxy-4,6-dimethyl-3-nonanone and (3R,5S,6R)-6-hydroxy-3,5-dimethyl-
2-octanone, Tetrahedron Asymmetry 22 (2011) 2071e2079;
(e) P. Srihari, B. Kumaraswamy, D.C. Bhunia, J.S. Yadav, First stereoselective
total synthesis of stagonolide G, Tetrahedron Lett. 51 (2010) 2903e2905.
[23] M.J. Jin, S.H. Kim, S.T. Chung, Lipase-supported preparation of optically active
2-amino alcohols of biological interest, J. Ind. Eng. Chem. 3 (1997) 182e184.
[24] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays, J. Immunol. Methods 65
(1983) 55e63.
1
(
a
3
1
(
S. Chammaa, Exploring the reactivity of chiral glycidic amides for their ap-
plications in synthesis of bioactive compounds, Eur. J. Org. Chem. 2014 (2014)
3
847e3867.
[25] D. Amsterdam, Susceptibility testing of antimicrobials in liquid media, in:
V. Loman (Ed.), Antibiotics in Laboratory Medicine, fourth ed., Williams and
Wilkins, Baltimore, MD, 1996, pp. 52e111.
[
14] (a) T. Vijai Kumar Reddy, B.L.A. Prabhavathi Devi, R.B.N. Prasad, M. Poornima,
C.G. Kumar, Total synthesis and antifungal activity of (2S,3R)-2-amindodecan-
3
-ol, Bioorg. Med. Chem. Lett. 22 (2012) 4678e4680;
[26] G. Molero, R.D. Orejas, F.N. Garcia, L. Monteoliva, L. Pla, C. Gil, M.S. Perez,
C. Nombela, Candida albicans: genetics, dimorphism and pathogenecity, Int.
Microbiol. 1 (1998) 95e106.
(
b) T. Vijai Kumar Reddy, B.L.A. Prabhavathi Devi, R.B.N. Prasad, P. Sujitha,
C.G. Kumar, Total synthesis and biological evaluation of clavaminol-G and its
analogs, Eur. J. Med. Chem. 67 (2013) 384e389;
[27] J.C.O. Sardi, L. Scorzoni, T. Bernardi, A.M.F. Almeida, M.J.S.M. Giannini, Candida
species: current epidemiology, pathogenicity, biofilm formation, natural,
antifungal products and new therapeutic options, J. Med. Microbiol. 62 (2013)
10e24.
(
c) M. Vijay, B.L.A. Prabhavathi Devi, R.B.N. Prasad, S. Ashita, U. Ramesh,
Design, synthesis and anticancer activities of novel unsaturated fatty acid-
based ehydroxy 1,2,3-triazoles, Int. J. Pharm. Sci. Res. 6 (2015) 1635e1649.
b
[
15] (a) G. Satyalakshmi, K. Suneel, D.B. Shinde, B. Das, Stereoselective total syn-
[28] National Committee for Clinical Laboratory Standards, Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically-fifth
Edition: Approved Standard M7eA5, NCCLS, Wayne, PA, 2000.
[29] National Committee for Clinical Laboratory Standards, Reference Method for
Broth Dilution Antifungal Susceptibility Testing for Yeasts; Approved Stan-
dard Second Edition. NCCLS Document M27-A2, NCCLS, Wayne, PA, 2002.
[30] L.H. Stoodley, J.W. Costerton, P. Stoodley, Bacterial biofilms: from the natural
environment to infectious diseases, Nat. Rev. Microbiol. 2 (2004) 95e108.
[31] U. Romling, C. Balsalobre, Biofilm infections, their resilence to therapy and
innovative treatment strategies, J. Int. Med. 6 (2012) 541e561.
[32] J.W. Costerton, L. Montanaro, C.R. Arciola, Biofilm in implant infections: its
production and regulation, Int. J. Artif. Organs 28 (2005) 1062e1068.
[33] A. Kamal, R. Abdul, S. Riyaz, Y. Poornachandra, B. Moku, C.G. Kumar,
S.M. Hussaini, B. Sridhar, P.K. Machiraju, Regioselective synthesis, antimicro-
bial evaluation and theoretical studies of 2-styryl quinolines, Org. Biomol.
Chem. 13 (2015) 1347e1357.
thesis of the piperidine alkaloids, (þ)-coniine, (þ)-pseudoconhydrine, and
(
(
(
(
þ)-sedamine through a common intermediate, Tetrahedron Asymmetry 22
2011) 1000e1005;
b) B. Das, S. Nagenrda, C.R. Reddy, Stereoselective total synthesis of
þ)-cryptofolione and (þ)-goniothalamin, Tetrahedron Asymmetry 22 (2011)
1
249e1254;
(
c) G.C. Reddy, P. Balasubramanyam, N. Salvanna, T.S. Reddy, B. Das, The first
stereoselective total synthesis of (Z)-cryptomoscatone D2, natural G2
a
checkpoint inhibitor, Bioorg. Med. Chem. Lett. 22 (2012) 2415e2417.
16] (a) R.A. Fernades, P. Kumar, Asymmetric dihydroxylation and one-pot epox-
idation routes to (þ)- and (-)-posticlure: a novel trans-epoxide as a sex
pheromone component of Orgyia postica (Walker), Tetrahedron 58 (2002)
[
6685e6690;
(
b) O. Piva, Direct conversion of b,g-unsaturated esters into lactones induced
by TMS-I, Tetrahedron 50 (1994) 13687e13696.
[
17] (a) S.K. Dinda, S.K. Das, G. Panda, Asymmetric total syntheses of spisulosine, its
diastereo- and regio- isomers, Tetrahedron 66 (2010) 9304e9309;
[34] F. Madeo, E. Frohlich, M. Ligr, M. Grey, S.J. Sigrist, D.H. Wolf, K.U. Frohlich,
Oxygen stress; a regulator of apoptosis in yeast, J. Cell Biol. 145 (1999)
757e767.
(
b) Z.B. Zhang, Z.M. Wang, Y.X. Wang, H.Q. Liu, G.X. Lei, M. Shi, A simple
synthetic method for chiral 1,2-epoxides and the total synthesis of a chiral
pheromone epoxide, J. Chem. Soc. Perkin Trans. 1 1 (2000) 53e57;
[35] C.G. Kumar, Y. Poornachandra, Biodirected synthesis of miconazole-
conjugated bacterial silver nanoparticles and their applications as antifungal
agents and drug delivery vehicles, Colloids Surf. B Biointerfaces 125 (2015)
110e119.
(
c) D.K. Mohapatra, S. Kanikarapu, P.R. Naidu, J.S. Yadav, Toward the synthesis
of brevipolide H, Tetrahedron Lett. 56 (2015) 1041e1044.
[
[
18] T. Katsuki, K.B. Sharpless, The first practical method for asymmetric epoxi-
dation, J. Am. Chem. Soc. 102 (1980) 5974e5976.
19] M. Sasaki, K. Tanino, A. Hirai, M. Miyashita, The C2 selective nucleophilic
substitution reactions of 2,3-epoxy alcohols mediated by trialkyl borates: the
first endo-mode epoxide-opening reaction through an intramolecular metal
chelate, Org. Lett. 5 (2003) 1789e1791.
[36] B. Halliwell, O.I. Aruoma, DNA damage by oxygen-derived species. Its mech-
anism and measurement in mammalian systems, FEBS Lett. 281 (1991) 9e19.
[37] D. Kobayashi, K. Kondo, N. Uehara, S. Otokozawa, N. Tsuji, A. Yagihashi,
N. Watanabe, Endogenous reactive oxygen species is an important mediator
of miconazole antifungal effect, Antimicrob. Agents Chemother. 46 (2002)
3113e3117.
[
20] M. Ravinder, T. Narendar Reddy, S.O. Oladoye, V. Ramesh, V. Jayathirtha Rao,
[38] S.J. Dixon, B.R. Stockwell, The role of iron and reactive oxygen species in cell
death, Nat. Chem. Biol. 10 (2013) 9e17.
Stereoselective Synthesis of N, O,O,O-tetraacetyl-d-ribo-phytosphingosine, N,