Structure based drug design, synthesis and evaluation of 4-(benzyloxy)-1-phenylbut-2-yn-1-ol derivatives as 5-lipoxygenase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.11.003

A group of 4-(benzyloxy)-1-phenylbut-2-yn-1-ol derivatives were designed using Site point connection method, synthesized and evaluated for their 5-Lipoxygenase (5-LOX) inhibitory activity. Hydrophobic site points in 5-LOX were considered for the study and substitutions were planned such that 4k will have strong hydrophobic group in the corresponding site point. Biological results supported the in silico prediction with compound 4k exhibiting good inhibition with IC₅₀ value of 8 μM against 5-LOX. The compounds 4j and 4k showed potent cytotoxic effects against various cancer cell lines (COLO-205, MDA-MB-231 and HepG2) but with no effect on normal cell line (HaCaT). The overall trend showed 4k as the most potent compound. Further studies demonstrated the protective effect of 4k in mouse Acute Lung Injury (ALI) model induced by lipopolysaccharide (LPS).

Full text of DOI:10.1016/j.ejmech.2011.11.003

European Journal of Medicinal Chemistry 47 (2012) 351e359
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structure based drug design, synthesis and evaluation of 4-(benzyloxy)-1-
phenylbut-2-yn-1-ol derivatives as 5-lipoxygenase inhibitors
Nimmanapalli P. Reddy a, T. Chandramohan Reddya, Polamarasetty Aparoya, Chandrani Achari a,
,
P. Ramu Sridhar b, Pallu Reddanna a
*
a School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India
b School of Chemistry, University of Hyderabad, Hyderabad 500 046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2011
Received in revised form
13 October 2011
Accepted 1 November 2011
Available online 10 November 2011
A group of 4-(benzyloxy)-1-phenylbut-2-yn-1-ol derivatives were designed using Site point connection
method, synthesized and evaluated for their 5-Lipoxygenase (5-LOX) inhibitory activity. Hydrophobic
site points in 5-LOX were considered for the study and substitutions were planned such that 4k will have
strong hydrophobic group in the corresponding site point. Biological results supported the in silico
prediction with compound 4k exhibiting good inhibition with IC50 value of 8 mM against 5-LOX. The
compounds 4j and 4k showed potent cytotoxic effects against various cancer cell lines (COLO-205, MDA-
MB-231 and HepG2) but with no effect on normal cell line (HaCaT). The overall trend showed 4k as the
most potent compound. Further studies demonstrated the protective effect of 4k in mouse Acute Lung
Injury (ALI) model induced by lipopolysaccharide (LPS).
Keywords:
4-(Benzyloxy)-1-phenylbut-2-yn-1-ol
derivatives
Ó 2011 Elsevier Masson SAS. All rights reserved.
5-LOX
Site point connection method
In silico prediction
Carcinoma cell line
Acute lung injury
1. Introduction
development of potential and novel 5-LOX inhibitors [5]. In this
study we focused on the development of 5-LOX inhibitors and their
Leukotrienes (LTs) are potent lipid mediators of inflammation
[1]. The first step for LT biosynthesis requires 5-lipoxygenase (5-
LOX), which, in the presence of 5-LOX-activating protein (FLAP),
converts arachidonic acid into LTA4 [2]. LTs have been implicated as
inflammatory mediators in experimental models of acute lung
injury (ALI) [3,4] and are known to contribute to the pathophysi-
ology of osteoarthritis, asthma and other cancers including pros-
tate, lung, breast and colon [5]. They were found to be elevated in
pulmonary edema fluid obtained from patients with human acute
respiratory distress syndrome (ARDS) [6]. MK886, a 5-LOX inhibitor
has been shown to improve lung function by decreasing inflam-
matory parameters including LTB4 induced by mechanical venti-
lation in a piglet model of airway lavage [7]. Recently, it has been
shown that 5-LOX (ALOX5) is a critical regulator for leukemia
cancer stem cells (LSCS) in chronic myeloid leukemia (CML) [8].
With an increase in reports illustrating the role of 5-LOX in
various pathological conditions there is a growing demand for the
role in ALI.
ALI is a significant cause of morbidity and mortality in critically
ill patients. Histologically, ALI in humans is characterized by
a severe acute inflammatory response in the lungs and neutrophilic
alveolitis [9,10]. Inflammatory stimuli from microbial pathogens,
such as endotoxin [lipopolysaccharide (LPS)], are well recognized
for their ability to induce pulmonary inflammation and experi-
mental administration of LPS, both systemically and intratracheally,
has been used to induce pulmonary inflammation in animal models
of ALI [11e13], characterized by activation of alveolar macrophages,
infiltration of neutrophils, lung edema and production of inflam-
matory mediators that resemble the inflammation and ALI seen in
ARDS [14]. However, despite decades of research, few therapeutic
strategies for clinical ARDS have emerged and current specific
options for treatment are limited [15e17]. Studies suggest a causal
role for 5-LOX and LTs in LPS-induced ALI [18,19]. It has been re-
ported that mediators including LTB4, are considered to be the
major causes of ALI because of their strong effect on neutrophil
activation and migration [20].
The increase in studies illustrating the role of 5-LOX in
various diseases has increased interest in the development of
* Corresponding author. Tel.: þ91 40 23134542; fax: þ91 40 23010745.
E-mail addresses: prsl@uohyd.ernet.in, preddanna@yahoo.com (P. Reddanna).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.003

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N.P. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 351e359
Fig. 1. (a) Overlay of best conformations BP-1, 4a and 4k (b) Interactions of BP-1 in 5-LOX active site; the hydrophobic amino acids in the vicinity are shown in ball and stick (c)
Optimization of inactive molecule (IC50 e 760 M) into active molecule (IC50 e 8 M).
m
m
potential and novel 5-LOX inhibitors. There have been numerous
reports recently on the synthesis and development of 5-LOX
inhibitors [21e25]. In our earlier studies, we have reported
a new class of inhibitors, prenylated chalcones [23] designed
with the aid of a theoretical 3D model of potato 5-LOX elabo-
rated by homology modeling [26]. Further, in another study,
chemical feature based pharmacophore modeling of inhibitors of
5-LOX have been carried out. The fact that 5-LOX inhibitors can
be successfully identified by employing pharmacophore based
virtual screening explains its usefulness in predicting activities
of large datasets of molecules. Thus, our earlier studies provided
homology and pharmacophore models which help in designing
the novel 5-LOX inhibitors. Further, in another study, we have
performed 3D-QSAR, using the comparative molecular field
analysis (CoMFA) and comparative molecular similarity indices
analysis (CoMSIA) techniques to design molecules with better 5-
LOX inhibitory activity [24]. As the crystal structure of human 5-
LOX has been recently solved [27], it is now possible to employ
the reliable structure for Structure Based Lead Optimization
studies. It is well known that de novo design will rarely yield
novel lead structures with nanomolar activity in the first
instance. Rather, the designed structures will probably represent
examples of a prospective new lead series with micromolar
activities that require further optimization [28].

N.P. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 351e359
353
OH
O
A
Br
+
MeS
MeS
1
2
3
R4
R1
R3
R2
H
B
O
O
4a
R4
OH
R3
R2
O
O
MeS
4b
OH R1
MeS
4
OH
R1
R2
R3
R4
4c
H
H
OMe
H
H
4d OMe
OMe
OMe
OMe
H
H
H
4e
OMe
H
OMe
OMe
aReagents and conditions: (A) DMSO, KOH,
0 oC to room temperature.
4f
4g
4h
H
H
H
OMe
H
OMe
CN
H
OMe
H
(B) THF, -78 oC n-BuLi, 3.5 h
NO2
4i
4j
H
H
H
H
H
F
H
H
iPr
4k
Scheme 1. aRoute for the synthesis of 4-(benzyloxy)-1-phenylbut-2-yn-1-ol derivatives.
As part of our ongoing search for potent 5-LOX inhibitors,
we describe herein the design, synthesis and structure activity rela-
tionship (SAR) studies for newclass of 4-(benzyloxy)-1-phenylbut-2-
yn-1-ol derivatives as 5-LOX inhibitors. A study on single round lead
optimization on 1-(benzyloxy) hept-2-yn-4-ol [(BP-1) (IC50 of
replaced with phenyl group in compound 4a. Further, methoxy,
cyano, fluoro, isopropyl and nitro derivatives were designed and
investigated (Fig. 1).
In the case of compound 4a, most of the functional groups
interacted favorably with the 5-LOX active site. Strong hydrogen
bond interactions were observed between hydroxyl of compound 4a
760
mM)] [29] was performed. Active site analysis method was
employed to identify various important amino acids in the active site
and Site Point Connection method was employed for lead optimi-
zation of an existing moderately active 5-LOX inhibitor. The designed
molecules were synthesized and tested for 5-LOX inhibition in vitro
and in the prevention of LPS-induced ALI in mouse model.
and His367 (N
d1/HO, 2.9 Å) and also with Gln363 (Oε1/HO, 2.8 Å).
The Compound 4a was expected to be more active than BP-1 as the
substitution with the phenyl group favored stronger hydrophobic
interactions with Leu368 and also much stronger PeP with Phe421.
In comparison, the conformation of active molecules 4j and 4k
deviated little from 4a because of the methylthio substitution.
Unlike the hydrogen bond interactions seen in 5-LOX-4a, the thio
derivatives formed strong interactions with Tyr181 and Gln363
(Fig. 1b). The phenyl group favored stronger hydrophobic interac-
2. Results and discussion
2.1. Computational studies
tions with Leu368 and also much stronger
PeP with Phe421. The
substitutions on the phenyl ring (thio, fluoro, cyano) occupied the
site points adjacent to hydrophobic amino acids in the active site.
Hence, the analogs were expected to be more active than 4a. The
substitutions in 4i, 4j and 4k were planned such that the hydro-
phobicity of the functional group increases from 4i to 4k. Cyano,
fluoro, isopropyl substitutions were made for 4i, 4j and 4k
respectively. This set of substitutions was used to again evaluate the
site points identified. As all the substitutions occupy the same site
point, the activity towards 5-LOX was expected to increase with
increase in hydrophobicity of the functional group.
Prior to the design of a 5-LOX inhibitor, the active site of 5-LOX
was investigated. Because the 3D-structure of 5-LOX has been
recently solved [27], it was used in this study. All of the possible
ligand binding cavities on the 5-LOX were searched by the Binding
Site Analysis program in Insight II. The largest cavity of 5-LOX ob-
tained in Binding Site Analysis correlated with the inhibitor/
substrate binding cavity of crystal structure of 15-LOX. This corre-
lated well with the previous reports explaining the similarity in the
structural fold and substrate/inhibitor binding site of LOXs. 5-LOX-
benzyl propargyl ether complex was used as initial molecule for the
lead optimization studies. The key regions in the active site that
were essential for inhibitor binding were explored. The interaction
sites in the vicinity of the inhibitor and unoccupied were identified.
New leads were designed so that hydrophobic sites in the vicinity
are exploited as shown in Fig. 1. The propyl group in BP-1 was
2.2. Synthesis
The synthetic strategies in this study to prepare the methyl ((4-
(prop-2-ynyloxy) methyl) phenyl) sulfane is illustrated in Scheme 1.

354
N.P. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 351e359
Table 1
Table 2
IC50 values of the compounds against 5-LOX
enzyme.
GI50 values of the compounds in various cell lines.
GI50 M)
(m
IC50 (mM)
Comp #
COLO-205
HepG2
MDA-MB-231
Comp #
5-LOX
4g
4h
4i
4j
4k
w100
71
88
22
12
w100
54
91
31
22
w100
62
81
20
15
4a
4b
4c
4d
4e
>100
>100
>100
69
72
4f
80
4g
80
4h
4i
4j
4k
AA-861
41
62
12
8
(hepatoma) and MDA-MB-231 (breast). The experiments were also
carried out under the same experimental conditions in normal
HaCaT (human keratinocyte) cell line. Cytotoxicity assays were
carried out by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl
tetrazolium bromide (MTT) assay. The GI50 values calculated from
the dose-survival curves obtained after 48 h of compound treat-
ment from MTT assay are shown in Table 2. These results showed
significant cytotoxic activity with compounds 4j and 4k against all
the cell lines. In particular, compounds 4j and 4k were the most
active agents against COLO-205 cell line with GI50 values of 22 and
1.7
Compound 3 was prepared by reacting 4-methylthiobenzyl alcohol
with propargyl bromide and KOH in DMSO. The 4-(benzyloxy)-1-
phenylbut-2-yn-1-ol derivatives 4bek were synthesized by
generating the corresponding lithium acetalide of 3 using n-BuLi
at ꢀ78 ꢁC in THF followed by substituted aldehydes. The crude
products were purified by column chromatography over silica gel.
The structures of all the compounds were established on the basis
of IR, 1H, 13C NMR and LCMS spectral data.
12
mM respectively. Furthermore, compounds 4h and 4i showed
moderate activity (<100
mM), the remaining compounds were less
active towards COLO-205 cell line. Compounds 4j and 4k were also
active against HepG2 and MDA-MB-231 cell lines as shown in the
Table 2. No effect was observed on HaCaT cell line treated with 4j
and 4k up to 100 mM (data not shown). These data suggest that 4j
and 4k are compounds with potent cytotoxic effects on cancer cells
2.3. In vitro 5-LOX assay
but without any effect on normal cells.
The potential 5-LOX inhibitory molecules synthesized were
tested in vitro for their inhibitory properties against 5-LOX enzyme
using the assay described by Reddanna et al. (1990) [30]. Out of the
ten molecules tested, compounds 4j and 4k showed inhibition at
2.5. In vivo anti-inflammatory effect of 4k on LPS-induced ALI
a concentration less than 15
around 50 M. All the other compounds showed IC50 greater than
100 M. The most active molecule 4k, showed potent inhibition of
5-LOX with an IC50 value of 8 M (Table 1). The number of hydrogen
mM. Compound 4h and 4i showed IC50
2.5.1. 4k Decreased LPS-induced inflammatory cells count and total
protein concentration in the bronchoalveolar lavage fluid of ALI mice
Alveolar macrophages are one of the main sources of pro-
inflammatory and anti-inflammatory cytokines, and their activa-
tion is critical in the development of ALI [32]. Total cell counts,
differential cell counts and protein concentration in the bron-
choalveolar lavage fluid (BALF) were evaluated at 12 h after LPS
administration. In this study, as expected mice exposed to LPS
exhibited massive recruitment of inflammatory cells, including
neutrophils and macrophages to the airways. In contrast, pre-
administration with 4k at a single dose of 50 mg/kg, significantly
inhibited the LPS-induced increase in the number of total cells,
neutrophils and macrophages in the BALF [Fig. 2]. Similar results
were obtained with celecoxib (CE) pre-treatment at a dose 50 mg/
kg. Furthermore, LPS administration resulted in significant
increase in the protein concentration in the BALF of mice. Pre-
treatment with 4k and CE at a single dose of 50 mg/kg each
effectively reduced the protein concentration in the BALF. Thus,
4k could attenuate LPS-induced ALI in mice by preventing infil-
tration of inflammatory cells and thus decreasing total protein
concentration.
m
m
m
bonds and hydrophobic interactions of the molecules with 5-LOX
showed inverse correlation with the experimental IC50 values.
To elucidate the mechanism of action of compounds 4j and 4k
on 5-LOX inhibition, assays were done as described by McGovern
et al. (2002) [31]. IC50 values of the compounds 4j and 4k did not
deviate upon incubation 5-LOX. Further, our results show that 5-
LOX enzyme remained fully inhibited upon dilution of the
mixture which suggested the binding is irreversible. Inhibition of 5-
LOX by compounds 4j and 4k has showed negligible decrease (w1-
fold) in the presence of 0.1 mg/mL BSA. These results suggest that
inhibition of 5-LOX by compounds 4j and 4k is by a specific
mechanism. To test whether the IC50 value of the compounds 4j and
4k altered upon increase in the concentration of 5-LOX, assays were
carried by incubation of compounds with 5-fold and 10-fold excess
of 5-LOX enzyme. These results showed a slight increase in the IC50
value with 5-fold increase in concentration of the enzyme, 5-LOX
but with no change in IC50 value upon 10-fold increase in the
concentration of 5-LOX (data not shown). The foregoing studies
clearly demonstrate the specific and irreversible inhibition of 5-
LOX by compounds 4j and 4k. A well-studied selective and
competitive inhibitor of 5-LOX, AA-861 was not affected by incu-
bation with the enzyme, 5-LOX.
2.5.2. Effect of 4k on LPS-induced pulmonary histopathological
changes and W/D ratio
The effect of 4k on the lungs of mice was determined 12 h after
LPS administration by histochemical staining with H & E. As shown
in Fig. 3a, LPS treated lungs showed pro-inflammatory alterations,
characterized by alveolar wall thickening, massive infiltration of
inflammatory cells into the lung interstitium and alveolar space as
well as signs of tissue injury. In contrast, pre-treatment with
a single dose of 4k (50 mg/kg), 1 h before LPS challenge, markedly
abated LPS-induced inflammatory cell infiltration and improved
2.4. In vitro anti-proliferative effects on cancer cell lines
The newly synthesized 4-(benzyloxy)-1-phenylbut-2-yn-1-ol
derivatives were examined for their cytotoxic properties in three
different human cancer cell lines; COLO-205 (colonic), HepG2

N.P. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 351e359
355
Fig. 2. Effects of 4k on LPS-induced inflammatory cell accumulation and total protein in BALF. BALF was prepared from mice 12 h after LPS instillation and the total cell numbers (i)
were counted using a standard hemocytometer. Each cell population including neutrophils (ii) and macrophages (iii) was examined by counting at least 200 cells on a smear
prepared by WrighteGiemsa staining. The results are expressed as the cell numbers for each population in 1 L of BALF. (iv) Total protein level in the BALF was determined 12 h after
the LPS challenge. *P < 0.01 vs. control group; $P < 0.05 vs. LPS group.
alveolar wall thickening which was comparable with effects of
50 mg/kg dose of CE. The lung W/D ratio was evaluated as
described in Materials and Methods. As shown in Fig. 3b,
Compound 4k significantly reduced the W/D ratio of lung 12 h
after LPS instillation. These results corroborated our findings in
BALF which confirmed that 4k may attenuate LPS-induced
pulmonary edema.
4. Experimental section
4.1. Computational studies
Earlier we have reported the application of homology model of
5-LOX for the development of novel inhibitors [26]. The crystal
structure of 5-LOX has been recently reported [22] and has been
used in this Structure based lead optimization study of analogs of
benzyl propargyl ether, a known class of 5-LOX inhibitors. BP-1,
3. Conclusions
having IC50 of 760 mM [24] was used as the initial molecule for
the design studies. The interactions of BP-1 with 5-LOX active site
residues were thoroughly studied to understand the 5-LOX struc-
ture better.
A new class of 4-(benzyloxy)-1-phenylbut-2-yn-1-ol derivatives
have been designed, synthesized and evaluated for their 5-LOX
inhibitory, anti-proliferative effects and in vivo anti-inflammatory
effects. Prior to this work, structure based methods have not
played a major role in the discovery of 5-LOX inhibitors, because
experimental structures of 5-LOX do not exist. This study is
a successful example reported for the 5-LOX inhibitor design using
the de novo design strategy employing the recently solved crystal
The structure of the initial molecule was sketched and mini-
mized using cerius2 and the final structure was obtained in
conformational analysis. GOLD (Genetic Optimization of Ligand
Docking), a docking program based on genetic algorithm [33] was
used to dock the inhibitors. During docking, the default algorithm
speed was selected. The number of poses for the inhibitor was set to
100, and early termination was allowed if the top three bound
conformations of a ligand were within 1.5 Å RMSD.
structure. A molecule of IC50 of 760
mM was optimized to a more
potential molecule with IC50 of 8 M. Site point connection method
m
showed good promise and it can be further used in developing
a very potential 5-LOX inhibitor. This methodology can be further
used by exploiting other site points to yield novel lead structures
with higher activity against 5-LOX enzyme.
After docking, the 5-LOX-BP-1 complex formed was studied. The
interactions were visualized and important amino acids in the
vicinity of the docked inhibitor were elucidated. Site point
connection method is one of the most widely used ligand design

356
N.P. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 351e359
Fig. 3. (a) Histological assessment of the effect of 4k on LPS-induced ALI. 12 h after LPS instillation, the lung tissues were inflated and fixed with 10% buffered formaldehyde;
samples were embedded in paraffin, and then stained with H&E (400ꢂ). i. Control group; ii. LPS group; iii. 4k group; and iv. CE group (b) Effects of 4k on the lung W/D ratio of LPS-
induced ALI in mice. To induce ALI, LPS was instilled intratracheally, 1 h after intra peritoneal administration of 50 mg/kg of 4k. The lung W/D ratio (i) was determined 12 h after the
LPS challenge. *P < 0.01 vs. control group; $P < 0.01, $$P < 0.05 vs. LPS group.
method. A site point can be defined as a space in the active site at
which a suitable ligand atom can make favorable interactions with
one or more enzyme atoms. Site points with appropriate ligand
atoms nearby are said to be satisfied. Site point connection
methods attempt to place small fragments in the active site so that
one or more site points are satisfied and fragments are thereby
placed in favorable regions.
The 5-LOX-benzyl propargyl ether complex discussed earlier
was taken. With the knowledge of interaction sites in the 5-LOX
obtained from active site analysis method, the site points that
were in the vicinity of the docked inhibitor and were unoccupied by
any of the ligand atoms were closely visualized. In this study, only
the site points corresponding to hydrophobic interactions were
considered. Four site points with potential of forming strong
hydrophobic interactions were identified as Leu368, Ile415, Phe421
and Thr364 (Fig. 1b).
docked and their interactions with 5-LOX were further investi-
gated. Ten derivatives were designed using the design method
taking the unsatisfied Site points into consideration. In the first step
the aliphatic chain (propyl) was modified into aromatic ring
(phenyl). The Compound 4a, was designed and docked and inves-
tigated. The phenyl substitution leads to more hydrophobic inter-
actions hence an increased affinity towards 5-LOX. To further
exploit the surrounding amino acids, different substitutions were
planned and studied.
4.2. Chemistry
4.2.1. General information
Melting points were determined on a Kofler hot-stage apparatus
and are uncorrected. The reactions were monitored by thin layer
chromatography (TLC) on precoated Merck Silica gel 60F254
aluminum plates. IR spectra were recorded in KBr discs on a JASCO
FT/IR-5300. 1H, 13C NMR spectra were recorded on Bruker Avance
To exploit the hydrophobic amino acids round the active site,
modifications of the ligand were proposed. All the molecules were

N.P. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 351e359
357
400 spectrometer using CDCl3 with TMS as internal standard.
Coupling constant (J) values are calculated in hertz (Hz) and spin
multiples are given as s (singlet), d (double), t (triplet), q (quartet),
m (multiplet) and br (broad). Elemental analyses were recorded on
a Thermo Finnigan Flash EA 1112 analyzer. Column chromatog-
raphy was performed on silica gel (Merk) 100e200 mesh.
J ¼ 8.0 Hz, 2H, methoxyphenyl H-2, H-6), 7.30e7.22 (m, 4H,
methylsulfanylphenyl H-2, H-3, H-5, H-6), 6.90 (d, J ¼ 8.0 Hz, 2H,
methoxyphenyl H-3, H-5), 5.47 (s, 1H, CHOH), 4.65 (s, 2H, eOCH2),
4.24 (d, J ¼ 1.2 Hz, 2H, eOCH2), 3.81 (s, 3H, eOMe), 3.10 (br s, 1H,
CHOH), 2.48 (s, 3H, SMe); 13C NMR (100 MHz, CDCl3):
d 159.73,
138.20, 134.15, 132.76, 128.76, 128.08, 126.60, 114.00, 86.59, 82.39,
71.26, 64.24, 57.37, 55.36, 15.87. Anal. (C19H20O3 S): C, H.
4.2.2. Synthesis of methyl (4-((prop-2-ynyloxy) methyl) phenyl)
sulfane (3)
4.2.3.4. 1-(2, 3-Dimethoxyphenyl)-4-(4-(methylthio)benzyloxy) but-
To a suspension of KOH (16.8 g, 0.30 mol) in 100 mL DMSO was
added 4-methylthiobenzyl alcohol (15.4 g, 0.10 mol). After stirring
for 10 min, propargyl bromide (8.8 mL, 0.10 mol) was added at 0 ꢁC.
Stirring was continued for 3 h at room temperature. The resulting
suspension was diluted with 200 mL water and was extracted with
diethyl ether (3 ꢂ 100 mL). The combined organic layers were
washed with water (4 ꢂ 60 mL), brine, dried over Na2SO4, filtered
and concentrated under reduced pressure. The obtained yellowish
residue was purified by silica gel column chromatography eluting
with diethyl ether/hexane (2:8) to afford the title compound 3 as
a colorless liquid (17.10 g, 89.0% yield).
2-yn-1-ol (4d). Yellowish gummy oil (0.255 g); yield 71.2%; IR KBr
nmax: 3429, 2937, 2839, 2248 cmꢀ1 1H NMR (400 MHz, CDCl3):
.
d
7.26e7.21 (m, 4H, methylsulfanylphenyl H-2, H-3, H-5, H-6), 7.12
(dd, J ¼ 9.2,1.6 Hz, 1H, dimethoxyphenyl H-6), 7.07 (t, J ¼ 8.0 Hz,1H,
dimethoxyphenyl H-5), 6.91 (dd, J ¼ 9.6, 1.6 Hz, 1H, dimethox-
yphenyl H-4), 5.69 (s, 1H, CHOH), 4.54 (s, 2H, eOCH2), 4.23 (d,
J ¼ 1.6 Hz, 2H, eOCH2), 3.95 (s, 3H, eOMe), 3.88 (s, 3H, eOMe), 2.90
(br s, 1H, CHOH), 2.47 (s, 3H, SMe): 13C NMR (100 MHz, CDCl3):
d
152.55, 146.42, 138.01, 134.36, 134.12, 128.62, 126.51, 124.20,
119.40, 112.76, 86.71, 81.69, 71.06, 61.07, 57.29, 55.78, 15.78. Anal.
(C20H22O4 S): C, H.
1H NMR (400 MHz, CDCl3):
d
7.29 (d, J ¼ 8.4 Hz, 2H), 7.25 (d,
J ¼ 8.4 Hz, 2H), 4.57 (s, 2H), 4.16 (d, J ¼ 2.4 Hz, 2H), 2.48 (s, 3H, SMe),
4.2.3.5. 4-(4-(Methylthio)benzyloxy)-1-(2, 3, 4-trimethoxyphenyl)-
2.47 (dd, J ¼ 3.0 Hz,1H); 13C NMR (100 MHz, CDCl3):
d
138.15,134.00,
but-2-yn-1-ol (4e). Pale yellow oil (0.273 g); yield 70.3%; IR KBr
128.84, 128.58, 126.68, 126.41, 79.55, 74.62, 71.01, 56.89, 15.80.
nmax: 3433, 2939, 2841, 2247 cmꢀ1 1H NMR (400 MHz, CDCl3):
.
d
7.24e7.18 (m, 5H, methylsulfanylphenyl H-2, H-3, H-5, H-6, tri-
4.2.3. General procedure for the synthesis of 4-(benzyloxy)-1-
phenylbut-2-yn-1-ol derivatives
metoxyphenyl H-6), 6.65 (d, J ¼ 8.8 Hz, 1H, trimetoxyphenyl H-5),
5.61 (s, 1H, CHOH), 4.54 (s, 2H, eOCH2), 4.23 (d, J ¼ 1.6 Hz, 2H,
eOCH2), 3.99 (s, 3H, eOMe), 3.86 (s, 3H, eOMe), 3.85 (s, 3H, eOMe),
3.10 (br s, 1H, CHOH), 2.47 (s, 3H, SMe); 13C NMR (100 MHz, CDCl3):
n-BuLi (1 mmol, 1.6 M in hexane) was added dropwise to
a solution of propargyl benzyl ether (1.0 mmol) in freshly dried THF
(10 mL) at ꢀ78 ꢁC. The reaction mixture was stirred at ꢀ78 ꢁC for
2.5 h, after that aldehyde (1.0 mmol) in dry THF (1 mL) was added
dropwise. The reaction mixture was further stirred at the same
temperature for 1 h and then allowed to warm to 25 ꢁC over 30 min.
After completion of reaction (by TLC) saturated aqueous NH4Cl
(10 mL) was added and the mixture was extracted with EtOAc
(3 ꢂ 20 mL). The combined organic layers were dried over Na2SO4,
filtered, and the solvent was evaporated in vacuo. The obtained
crude product was purified by flash column chromatography over
silica gel, eluting with step gradient of hexane/EtOAc to afford pure
1-Aryl-4-benzyloxybut-2-yn-1-ol derivatives 4bek with 70e80%
yield. The reference compound 4a was synthesized from the
starting compound benzyl alcohol using the same procedure.
d
154.04, 151.27, 142.01, 138.03, 134.10, 128.59, 126.66, 126.48,
122.11, 106.96, 86.68, 81.56, 71.05, 61.29, 60.94, 60.66, 57.29, 55.93,
15.75. Anal. (C21H24O5 S): C, H.
4.2.3.6. 1-(3, 4-Dimethoxyphenyl)-4-(4-(methylthio)benzyloxy) but-
2-yn-1-ol (4f). Yellowish gummy oil (0.254 g); yield 70.9%; IR KBr
nmax: 3352, 2935, 2841, 2222 cmꢀ1 1H NMR (400 MHz, CDCl3):
.
d
7.25e7.19 (m, 4H, methylsulfanylphenyl H-2, H-3, H-5, H-6),
7.06e7.04 (m, 2H, dimethoxyphenyl H-2, H-6), 6.83 (d, J ¼ 8.0 Hz,1H,
dimethoxyphenyl H-5), 5.45 (s,1H, CHOH), 4.54 (s, 2H, eOCH2), 4.23
(d, J ¼ 1.6 Hz, 2H, eOCH2), 3.86 (s, 6H, 2ꢂeOMe), 2.71 (br s, 1H,
CHOH), 2.45 (s, 3H, SMe); 13C NMR (100 MHz, CDCl3):
d 149.08,
138.22, 134.07, 133.19, 128.71, 126.57, 119.02, 110.93, 109.86, 86.66,
82.33, 71.23, 64.37, 57.36, 55.96, 55.89,15.82. Anal. (C20H22O4 S): C, H.
4.2.3.1. 4-(Benzyloxy)-1-phenylbut-2-yn-1-ol (4a). Pale Yellow oil
(0.20 g); yield 80.0%; IR KBr nmax: 3439, 3063, 2862, 2227 cmꢀ1. 1H
4.2.3.7. (4-(methylthio)benzyloxy)-1-(3, 4, 5-trimethoxyphenyl)but-
NMR (400 MHz, CDCl3):
4.62 (s, 2H, eOCH2), 4.27 (d, J ¼ 1.2 Hz, 2H, eOCH2), 3.18 (br s, 1H,
CHOH); 13C NMR (100 MHz, CDCl3):
140.59, 137.29, 128.68, 128.54,
d
7.58e7.32 (m, 10H), 5.52 (s, 1H, CHOH),
2-yn-1-ol (4g). Pale yellowish oil (0.278 g); yield 71.6%; IR KBr nmax
3427, 2937, 2841, 2222 cmꢀ1 1H NMR (400 MHz, CDCl3):
d 7.26e7.22 (m, 4H, metyhlsulfanylphenyl H-2, H-3, H-5, H-6), 6.77
:
.
d
128.42,128.24,128.02,126.70, 86.68, 82.47, 71.75, 64.48, 57.47. Anal.
(C17H16O2): C, H.
(s, 2H, trimethoxyphenyl H-2, H-6), 5.45 (s, 1H, CHOH), 4.55 (s, 2H,
eOCH2), 4.23 (d, J ¼ 1.6 Hz, 2H, eOCH2), 3.85 (s, 3H, eOMe), 3.84 (s,
3H, eOMe), 3.83 (s, 3H, eOMe), 2.91 (br s, 1H, CHOH), 2.47 (s, 3H,
4.2.3.2. 1-(4-(Methylthio)benzyloxy)non-2-yn-4-ol (4b). Yellowish
SMe); 13C NMR (100 MHz, CDCl3):
d 153.25, 138.22, 137.85, 136.04,
oil (0.213 g); yield 72.9%; IR KBr nmax: 3414, 2924, 2860, 1900 cmꢀ1
.
133.94, 128.59, 126.50, 103.56, 86.30, 82.48, 71.21, 64.59, 60.77,
57.28, 56.06, 15.75. Anal. (C21H24O5 S): C, H.
1H NMR (400 MHz, CDCl3):
sulfanylphenyl H-2, H-6), 7.24 (d,
d
7.27 (d, J ¼ 8.4 Hz, 2H, methyl-
J
¼
8.4 Hz, 2H, methyl-
sulfanylphenyl H-3, H-5), 4.54 (s, 2H, eOCH2), 4.42 (t, J ¼ 6.4 Hz,
1H), 4.19 (d, J ¼ 1.6 Hz, 2H, eOCH2), 2.48 (s, 3H, SMe), 2.00 (s, 1H,
CHOH), 1.72e1.68 (m, 2H), 1.48e1.44 (m, 2H), 1.33e1.30 (m, 4H),
4.2.3.8. 4-(1-Hydroxy-4-(4-(methylthio)benzyloxy)but-2-ynyl)ben-
zonitrile (4h). Yellowish oil (0.23 g); yield 71.2%; IR KBr nmax: 3418,
3051, 2920, 2856, 2229, 2220 cmꢀ1 1H NMR (400 MHz, CDCl3):
.
0.90 (t, J ¼ 6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d
138.12, 134.15,
d
7.66 (d, J ¼ 8.0 Hz, 2H, 4-cyanophenyl H-2, H-6), 7.63 (d, J ¼ 8.0 Hz,
128.68, 126.57, 87.81, 80.53, 71.10, 62.46, 57.24, 37.67, 31.40, 24.80,
22.52, 15.83, 13.96. Anal. (C17H24O2 S): C, H.
2H, 4-cyanophenyl H-3, H-5), 7.25e7.20 (m, 4H, methyl-
sulfanylphenyl H-2, H-3, H-5, H-6), 5.55 (s, 1H, CHOH), 4.53 (s, 2H,
eOCH2), 4.22 (d, J ¼ 1.2 Hz, 2H, eOCH2), 2.91 (br s, 1H, CHOH), 2.47
4.2.3.3. 1-(4-Methoxyphenyl)-4-(4-(methylthio)benzyloxy) but-2-yn-
(s, 3H, SMe); 13C NMR (100 MHz, CDCl3):
d 145.39, 138.45, 133.82,
1-ol (4c). Yellow gummy oil (0.244 g); yield 74.3%; IR KBr nmax
:
132.43, 128.72, 127.17, 126.54, 118.61, 112.05, 85.32, 83.46, 71.58,
63.65, 57.28, 15.79. Anal. (C19H17NO2 S): C, H, N.
3398, 2920, 2852, 2227 cmꢀ1. 1H NMR (400 MHz, CDCl3):
d
7.47 (d,

358
N.P. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 351e359
4.2.3.9. 4-(4-(methylthio)benzyloxy)-1-(3-nitrophenyl)but-2-yn-1-
addition of AA (133 mM). In addition, 5-LOX inhibition, in the pres-
ol (4i). Yellowish oil (0.242 g); yield 70.5%; IR KBr nmax: 3400, 3084,
ence of 10-fold excess 5-LOX, 5-LOX and compounds 4j and 4k were
incubated for 5 min and the reaction was initiated by the addition of
2922, 2860, 2235, 1529, 1350 cmꢀ1
.
1H NMR (400 MHz, CDCl3):
d
8.40 (s, 1H, 3-nitrophenyl H-2), 8.17 (d, J ¼ 8.0 Hz, 1H, 3-
AA (133 mM). A well-studied selective and competitive inhibitor of
5-LOX, AA-861 (A3711, Batch No. 036K1441, SigmaeAldrich, St.
Louis, USA) was used as a control in the assays.
nitrophenyl H-4), 7.85 (d, J ¼ 7.2 Hz, 1H, 3-nitrophenyl H-6), 7.54
(t, J ¼ 8.0 Hz, 1H, 3-nitrophenyl H-5), 7.24e7.20 (m, 4H, methyl-
sulfanylphenyl H-2, H-3, H-5, H-6), 5.59 (s, 1H, CHOH), 4.55 (s, 2H,
eOCH2), 4.24 (s, 2H, eOCH2), 3.10 (br s, 1H, CHOH), 2.47 (s, 3H,
4.4. MTT assay
SMe); 13C NMR (100 MHz, CDCl3):
d 148.33, 142.46, 138.42, 133.79,
132.64, 129.59, 128.74, 126.54, 123.25, 121.59, 85.31, 83.59, 71.58,
63.39, 57.25, 15.78. Anal. (C18H17NO4 S): C, H, N.
Cell lines used in this study were maintained in tissue culture
petri dishes. Medium for the cell line was RPMI-1640 supple-
mented with 10% heat inactivated fetal bovine serum (FBS) 100 IU/
4.2.3.10. 1-(4-Fluoropylphenyl)-4-(4-(methylthio)benzyloxy) but-2-
mL penicillin, 100 mg/mL streptomycin and 2 mM L-glutamine. The
yn-1-ol (4j). Yellow oil (0.226 g); yield 71.5%; IR KBr nmax: 3398,
cell line was maintained in a humidified atmosphere with 5% CO2 at
37 ꢁC. The cultured cells were passaged twice a week, seeding at
a density of 5 ꢂ 103 cells per well in 96-well plate before the day of
experiment. Before the treatment with test compound, cells were
washed with PBS and fresh medium was added. Cell proliferation
was assessed using the MTT staining as described by Mosmann
(1983) [34]. The MTT assay is based on the reduction of the tetra-
zolium salt, MTT, by viable cells. The NADH or NADPH generated in
the living cells, convert the yellow form of the MTT salt to insoluble,
purple formazan crystals. In brief, Cells (5 ꢂ 103 cells per well) were
incubated in 96-well plates in the presence or absence of the test
3074, 2920, 2856, 2222 cmꢀ1 1H NMR (400 MHz, CDCl3):
.
d
7.50e7.46 (2H, m, 4-fluorophenyl, H-2, H-6), 7.27e7.19 (4H,
methylsulfanylphenyl, H-2, H-3, H-5, H-6), 7.07e7.03 (m, 2H, 4-
fluorophenyl, H-3, H-5), 5.46 (s, 1H, CHOH), 4.54 (s, 2H, eOCH2),
4.22 (d, J ¼ 1.2 Hz, 2H, eOCH2), 3.10 (br s, 1H, CHOH), 2.47 (s, 3H,
SMe); 13C NMR (100 MHz, CDCl3):
d 161.43, 138.33, 136.36, 133.92,
128.79, 128.53, 128.45, 126.56, 115.57, 115.35, 86.38, 82.61, 71.38,
63.75, 57.31, 15.79. Anal. (C18H17FO2 S): C, H.
4.2.3.11. 1-(4-Isopropylphenyl)-4-(4-(methylthio)benzyloxy) but-2-
yn-1-ol (4k). Pale yellowish oil (0.245 g); yield 72.0%; IR KBr nmax
:
compounds (0.1e100
end of the treatment, 20
each well and incubated for an additional 4 h at 37 ꢁC. The purple-
blue MTT formazan precipitate was dissolved in 100 L of DMSO.
mM) for 48 h in a final volume of 100
mL. At the
3364, 3043, 2962, 2862, 2227 cmꢀ1 1H NMR (400 MHz, CDCl3):
.
mL of MTT (5 mg/mL in PBS) was added to
d
7.48 (d, J ¼ 8.0 Hz, 2H, 4-isopropylphenyl H-2, H-6), 7.27e7.23 (6H,
methylsulfanylphenyl H-2, H-3, H-5, H-6, 4-isopropylphenyl H-3, H-
5), 5.50 (s, 1H, CHOH), 4.58 (s, 2H, eOCH2), 4.26 (d, J ¼ 1.2 Hz, 2H,
eOCH2), 2.94e2.92 [m, 1H, CH(CH3)2], 2.70 (br s 1H, CHOH), 2.47 (s,
3H, SMe), 1.30e1.26 [m, 6H, CH(CH3)2)]. 13C NMR (100 MHz, CDCl3):
m
The activity of the mitochondria, reflecting cellular growth and
viability, was evaluated by measuring the optical density at 570 nm
on Quant Bio-Tek Instruments, Inc. microtiter plate reader. Each
concentration was tested in three different experiments run in
three replicates.
d
149.34, 138.19, 137.89, 134.17, 128.78, 126.77, 126.70, 126.62, 86.56,
82.42, 71.26, 64.53, 57.37, 33.91, 23.99,15.89. Anal. (C21H24O2 S): C, H.
4.3. In vitro 5-LOX assays
4.5. LPS-induced acute lung injury
5-LOX was purified and assayed by the method described by
Reddanna et al. (1990) [30]. Enzyme activity was measured using
polarigraphic method with a Clark’s oxygen electrode on Strath-
kelvin Instruments, model 782, RC-300. Typical reaction mixture
4.5.1. Animals
Male BALB/c mice, weighing approximately 20e25 g, were
purchased from the National Institute of Nutrition (NIN), Hyder-
abad, India. The mice were housed in micro isolator cages and
received food and water ad libitum. The temperature was main-
tained at 24 ꢃ 1 ꢁC, and relative humidity was 40e80%. Mice were
housed for 2e3 days to adapt them to the environment before
experimentation. All animal experiments were performed in
accordance with the guide for the Care and Use of Laboratory
Animals published by the US National Institute of Health.
contained 50e100 mL of enzyme and 10 mL of substrate (133 mM of
arachidonic acid) in a final volume of 3 mL with 100 mM phosphate
buffer pH 6.3. Rate of decrease in oxygen concentration was taken
as a measure of enzyme activity. Stock solutions of test compounds
prepared immediately before use, were dissolved in DMSO. Various
concentrations of test drug solutions were added and the LOX
reaction was initiated by the addition of substrate. The reaction was
allowed to proceed at 25 ꢁC and the maximum slope generated was
taken for calculating activity. Percent inhibition was calculated by
comparison of LOX activity in the presence and absence of inhibitor.
The concentration of the test compound causing 50% inhibition
(IC50) was calculated from the concentration-inhibition response
curve. Each assay was repeated thrice.
4.5.2. Chemicals
Escherichia coli lipopolysaccharide (LPS) was purchased from
Sigma Chemical Company (St. Louis, USA). Celecoxib was
a generous gift from Dr. Reddy’s Laboratories, Hyderabad, India.
To check whether the inhibition of the compounds 4j and 4k is
irreversible and/or non-specific, assays were performed as
described by McGovern et al. (2002) [31]. Compounds (4j and 4k)
and 5-LOX were incubated for 5 min at 25 ꢁC at high concentrations
(10-fold) and then diluted to below the apparent IC50 of the
4.5.3. Establishment of the animal model and treatment regimen
The mice were divided randomly into a phosphate buffered
saline (PBS) vehicle group (control group), an LPS challenge group
(LPS group), a 4k intervention group (4k group), and a Celecoxib
intervention group (CE group), with 6 mice per group. After fasting
for 8 h, mice were anesthetized with 50 mg/kg of ketamine and
compounds 4j and 4k i.e. 12
mM and 8
mM. Then the reaction was
initiated by the addition of AA (133
m
M). Non-specific binding is
10 mg/kg of xylazine in 100
administered intratracheally in 50
injury. Control mice were given 50
were administrated 4k at 50 mg/kg or CE at 50 mg/kg intra-
peritoneally, 1 h prior to LPS instillation; mice from control and
LPS groups were treated with the same volume of PBS.
m
L of PBS, and 25
L PBS (1 mg/kg), to induce lung
L PBS intratracheally. The mice
mg of LPS was
often detected by decreased inhibition in the presence of BSA
(MB083, Lot No. 0000090581, HiMedia, Mumbai, India). In order to
check the non-specific binding, if any, in the present study, 5-LOX
was incubated with BSA (0.1 mg/mL) and compounds 4j and 4k
before addition of substrate and the reaction was initiated by the
m
m

N.P. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 351e359
359
4.5.4. Bronchoalveolar lavage and Cell counting
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were stored at ꢀ70 ꢁC until required for determination of protein
content. The cell pellets were resuspended in PBS, and the total cell
number was counted using a standard hemocytometer. Differences
in cell numbers were examined by counting at least 200 cells on
a smear prepared by WrighteGiemsa staining.
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Appendix. Supplementary information
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.11.003.