Synthesis and biological evaluation of dibenz[b,f][1,5]oxazocine derivatives for agonist activity at κ-opioid receptor

Article abstract of DOI:10.1016/j.ejmech.2011.02.024

A short and high yield synthetic route to dibenz[b,f][1,5]oxazocines has been developed using Pd catalyzed intramolecular cycloamination reaction. Receptor binding assay using [¹²⁵I]-dynorphin demonstrated that one of the derivative, 5b showed

Full text of DOI:10.1016/j.ejmech.2011.02.024

European Journal of Medicinal Chemistry 46 (2011) 1713e1720
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of dibenz[b, f][1, 5]oxazocine derivatives for
agonist activity at -opioid receptor
k
Sudipta Mitra a, Tuhin Suvro Banerjee b, Sandip K. Hota a, Debleena Bhattacharya a,
,
Sumantra Das b, Partha Chattopadhyaya
*
a Chemistry Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India
b Cell biology & Physiology Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A short and high yield synthetic route to dibenz[b, f][1, 5]oxazocines has been developed using Pd
catalyzed intramolecular cycloamination reaction. Receptor binding assay using
demonstrated that one of the derivative, 5b showed selective -opioidergic property.
[
125I]-dynorphin
Received 12 October 2010
Received in revised form
10 February 2011
Accepted 13 February 2011
Available online 22 February 2011
k
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Dibenz[b, f ][1,5]oxazocines
Aryl amination
Palladium catalyst
k-Opioid receptor
Agonist
1. Introduction
or other methods have been reported in the literature [11]. However,
applications of these methodologies to the preparation of diben-
zoxazocine ring are scarce. A few methods for the synthesis of this
useful system have been reported as in References [12,13]. Thus, the
development of a suitable procedure for the synthesis of this ring
system remains a valid preoccupation for synthetic organic chem-
ists. Over the past few years’ intramolecular CeN ring closure has
been shown to offer a better alternative to the synthesis of medium
ring heterocycles. As a part of the ongoing program in our laboratory
on benzo and dibenzo fused medium ring compounds [14], we have
developed various Pd catalyzed CeN/O bond forming cyclization
strategies based on intramolecular Buchwald-Hartwig aryl amina-
tion [15,16] or aryl etherification reactions [17]. To the best of our
knowledge, only one example of preparing dibenzoxazocines byaryl
amination reaction was reported as in Reference [18].
We have been engaged in establishing new synthetic methods
to benzo fused medium ring heterocycles [1,2] for quite sometimes,
we noted that dibenz[b,f][1,5]oxazocines are effective as neuro-
tropic agents, e.g. antidepressants, analgesics, and sedatives [3e5].
These are structurally similar to azocine derivatives, some of which
have been shown to possess CNS activities [6,7]. The benzoxazocine
nefopam is frequently used as a non-opioid analgesic and its
analgesic effect on acetic acid-induced visceral nociception has
been shown to be antagonized by the opioid receptor antagonist
naloxone or naltrindole [8]. Among other azocines, ethyl keto
cyclazocine, bremazocine, pentazocine etc. have been widely used
as k-opioid receptor (KOR) agonists. Although various applications
of dibenzo fused medium ring heterocycles are reported in litera-
ture [9,10], their use as KOR agonist is not explored. We therefore
undertook a project on the synthesis of some dibenzoxazocine
derivatives and testing the compounds for activity at KOR.
In continuation of our efforts [15,16], we studied the applica-
bility of such carbon-nitrogen bond forming reactions during the
final cyclization step.
There have been several approaches to the synthesis of dibenzo
fused seven- and eight-membered ring compounds using ring-
closing metathesis, palladium-induced intramolecular cyclization,
2. Results and discussions
2.1. Chemistry
The starting materials 3aed were prepared [14] in 92e95%
yield by benzylation of commercially available substituted
* Corresponding author. Tel.: þ91 33 2473 0492; fax: þ91 33 2473 5197.
E-mail address: partha@iicb.res.in (P. Chattopadhyay).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.024

1714
S. Mitra et al. / European Journal of Medicinal Chemistry 46 (2011) 1713e1720
Br
Br
R1
R3
R2
R1
Br
OH
O
i
+
R2
CHO
CHO
R3
1
2
3
3a, R1 = R2 = R3 = H, 93 %
1
3
2
NH2
3b,
3c,
R = R = H, R = OMe, 92 %
R = OMe, R = R = H, 95 %
1
2
3
ii-iii
3d, R1 = OMe, R2 + R3 = O-CH2-O, 94 %
R
4a, R = R1 = R2 = R3 = H, 72%
4b, R = R1 = R3 = H, R2 = OMe, 68 %
Br
R3
R2
R1
1
2
3
4c,
4d,
4e,
R = 4-OMe, R = R = R = H, 74 %
O
1
2
3
R = 4-OMe, R = OMe, R = R = H, 75 %
1
2
3
H
N
R = 2-OMe, R = OMe, R = R = H, 74 %
4f, R = H, R1 = OMe, R2 + R3 = O-CH2-O, 72 %
4g, R = 4-OMe, R1 = OMe, R2 + R3 = O-CH2-O, 71 %
4h, R = 4-NO2, R1 = OMe, R2 + R3 = O-CH2-O, 48 %
R
4
Scheme 1. Synthesis of O-bromobenzylated aromatic amines. Reagents and conditions: (i) acetone, K2CO3, reflux, 3 h (ii) EtOH, 25 ꢁC, 12 h (iii) NaBH4, EtOH, 0 ꢁC, 2 h.
salicylaldehydes with substituted 2-bromo benzyl bromides in the
presence of anhydrous potassium carbonate (Scheme 1). Imine
formation with substituted anilines and subsequent NaBH4 reduc-
tion in ethanol afforded the desired amines 4aeg in good yield
(Scheme 1). The spectral data of 4aeg are in excellent agreement
with the assigned structures. The lower yield obtained with 4-nitro
aniline, which could not be significantly improved even by
increasing temperature of the reaction, may be attributed to the
electron withdrawing effect of the nitro group.
only 30% product by using KO-tBu, (Entry 3, Table 1), but the
combined effect of KO-tBu and K2CO3 produced significant
increase in the yield. Cs2CO3 was found to be less effective in this
reaction (Entry 6, Table 1). A study of the solvent effect (THF, EtOH,
toluene, 1,4-dioxane) suggested that toluene was the solvent of
choice. Application of the reagents Pd2(dba)3, ꢀBINAP, and KO-
tBu þ K2CO3 in toluene (Scheme 2) gave the desired product 5b in
77% yield.
Initially we attempted to synthesize the desired cyclic product
5a through Pd catalyzed intramolecular cycloamination reaction on
4a pursuing the conditions reported by Buchwald et al. [19]. Entry 1
of Table 1 indicates that under these conditions the desired cyclic
compound was not obtained. More recently, this aryl amination
chemistry has undergone optimization, particularly with the
development of new ligand systems, and the intramolecular
version has been utilized for the synthesis of heterocyclic
compounds [20]. We, therefore, tried to optimize the reaction
conditions with different ligands, catalysts, solvents, and bases
(Table 1).
R1
O
R2
R3
i
4a-g
N
R
5a-g
Table 1
Scheme 2. Synthesis of dibenzoxazocines by intramolecular cycloamination reaction.
Reagents and conditions: (i) 10 mol% Pd2(dba)3, 7 mol % ꢀBINAP, 2.0 equiv K2CO3,
2.0 equiv KO-tBu, dry toluene, reflux, 16e18 h.
Optimization of the cycloamination reaction.
Entry Substrate Catalyst Ligand Base
Solvent Product Yield (%)
1
2
3
4
5
6
7
8
4a
4a
4a
4a
4a
4a
4b
4b
4b
4b
4b
Pd(PPh3)4
e
K2CO3
Toluene 5a
ea
72b
30
20
15
25
77b
ea
ea
35
24
After achieving the optimized results, other substrates 4beh
were similarly treated under optimized reaction conditions to
afford the corresponding cyclized products 5beg (Table 2), though
no cyclized product was formed with 4h. The basicity of the amine
seems to be very important for the Pd catalyzed aryl amine cycli-
zation. In the presence of nitro group, probably it was difficult to
form amine coordination complex in the catalytic cycle [21,22].
The structures of the compounds 5 were determined on the
basis of spectroscopic studies supported by a single crystal X-ray
analysis [23e25] on 5b (Fig. 1).
Pd2(dba)3 ꢀBINAP K2CO3þKO-tBu Toluene 5a
Pd2(dba)3 ꢀBINAP KO-tBu
Pd(OAc)2 ꢀBINAP KO-tBu
Toluene 5a
Toluene 5a
Pd2(dba)3 PPh3
K2CO3þKO-tBu Toluene 5a
Pd2(dba)3 ꢀBINAP Cs2CO3
Toluene 5a
Pd2(dba)3 ꢀBINAP K2CO3þKO-tBu Toluene 5b
Pd2(dba)3 ꢀBINAP K2CO3þKO-tBu EtOH
Pd2(dba)3 ꢀBINAP K2CO3þKO-tBu THF
5b
5b
9
10
11
Pd2(dba)3 ꢀBINAP K2CO3þKO-tBu Dioxane 5b
Pd2(dba)3 DPPF
K2CO3þKO-tBu Toluene 5b
a
No desired product was found.
Optimized reaction condition.
b
2.2. Binding studies
Among the various catalysts used in the synthesis of diben-
zoxazocines, Pd2(dba)3 gave the best result (Table 1, entry 2 and 7).
Use of a proper base was also crucial to obtain good yield; we got
The effect of the dibenz[b,f][1,5]oxazocines derivatives on [125I]-
dynorphin and [125I] DAMGO binding to membranes, prepared

S. Mitra et al. / European Journal of Medicinal Chemistry 46 (2011) 1713e1720
1715
Table 2
Reaction times and yields of the cycloamination reactions.
Entry
Precursor
Product
Time (h)
Yielda (%)
Br
O
O
1
18
72
N
NH
Ph
5a
4a
Ph
Br
O
OMe
O
2
17
77
N
NH
Ph
Ph
5b
OMe
4b
Br
O
O
N
NH
3
16
75
4c
5c
OMe
OMe
OMe
OMe
Br
O
O
N
NH
4
15
70
5d
4d
OMe
OMe
OMe
OMe
Br
O
O
5
16
68
NH
N
MeO
MeO
4e
5e
(continued on next page)

1716
S. Mitra et al. / European Journal of Medicinal Chemistry 46 (2011) 1713e1720
Table 2 (continued ).
Entry
Precursor
Product
Time (h)
Yielda (%)
OMe
OMe
Br
O
O
O
6
16
75
O
O
NH
N
O
4f
5f
OMe
Br
OMe
O
O
O
O
O
NH
N
7
17
65
O
4g
5g
OMe
OMe
a
Isolated yield.
from mouse brain, were determined. While the KOR specific ligand
U50488H, at 5
M, inhibited specific [125I]-dynorphin binding by
pERK by the opioid agonists. The above observations were applied
in cultures of C6 glioma with a view to determining the -opioid
m
k
approximately 49%, only 5b showed significant interaction at KOR
binding site at similar concentrations (Table 3). However, none of
the compounds were found to displace the specific [125I] DAMGO
binding significantly.
Affinity studies for the interaction of 5b at the KOR were done by
calculating the IC50 values (Fig. 2). It was observed that 5b had high
agonistic or antagonistic property of the test compounds. Cells
were treated with the test compounds for 10 min and pERK levels
were estimated by western blotting analysis (Fig. 3). Like the k-
opioid ligand U50488H, 5b also stimulated ERK phosphorylation.
However, in presence of the KOR antagonist norBNI, 5b failed to
evoke ERK phosphorylation, suggesting the mediation of KOR in the
action of 5b. Of the other compounds tested, none stimulated ERK
phosphorylation to a significant extent (see Supplementary data
online).
affinity for KOR with an IC50 of 2.17 mM, which was comparable to
that of the KOR selective ligand U50488H (IC50 of 1.99
reported by us earlier [26].
mM) as
KOR agonists and not antagonists cause transient activation of
pERK [27] in cultured cells within a few minutes of application.
Receptor specific antagonists inhibited such stimulatory effect on
Table 3
Effect of synthetic compounds on the specific bindingsa of
k
and
m opioid receptors.
Compound
Specific binding (fmol/mg protein)
k
m
e
20.80 ꢀ 0.48 (100)
10.40 ꢀ 1.30* (50)
e
11.04 ꢀ 0.39 (100)
e
U50488H
Naloxone
6.66 ꢀ 0.38* (60)
8.89 ꢀ 0.48 (80)
10.94 ꢀ 0.74 (99)
9.54 ꢀ 0.51 (86)
10.40 ꢀ 0.32 (90)
9.9 ꢀ 0.52 (89)
8.69 ꢀ 1.02 (78)
15.29 ꢀ 0.78* (138)
5a
5b
5c
5d
5e
5f
18.10 ꢀ 1.10 (87)
6.14 ꢀ 0.41* (29)
16.94 ꢀ 0.50 (81)
17.01 ꢀ 1.55 (81)
24.80 ꢀ 0.37 (119)
21.20 ꢀ 0.54 (101)
21.20 ꢀ 1.98 (101)
5g
*p < 0.05 vs untreated control (Total binding) using one way ANOVA. Values within
parenthesis represent percentage binding compared to total binding in absence of
any test compound, which is taken as 100.
a
Bindings at
k and m
opioid receptors were investigated using [125I]-dynorphin
and [125I] DAMGO respectively, in the absence and presence of test compounds. The
test compounds were used at the concentration of 5 M for studying the displace-
ment of both [125I]-dynorphin and [125I] DAMGO. Binding at
and opioid receptors
was determined from the difference in binding in absence and presence of 5 M of
both U50488H ( ) and naloxone ( ) respectively. The binding was carried out in
mouse brain membrane. Data represent Mean ꢀ SEM of at least three experiments.
m
k
m
m
k
m
Fig. 1. ORTEP diagram of compound 5b.

S. Mitra et al. / European Journal of Medicinal Chemistry 46 (2011) 1713e1720
1717
bremazocine, pentazocine etc.), arylacetamides (U50488H,
U69593, R84760 etc.), epoxymorphinan nalfurafine, ibogaine
congeners, salvinorin A etc.
3. Conclusions
In conclusion, we have established a straightforward three-step
synthetic route to dibenzo fused eight-membered oxazacycles
using palladium catalyzed intramolecular cycloamination reaction
as the key step. The present study identifies a novel dibenzox-
azocine derivative having
k-opioid agonist activity. Development of
safe -agonists has become relevant in recent times as a replace-
k
ment of morphine and dibenzoxazocines afford an interesting
structure for further investigation in this area. We also plan to use
the test compound for attenuating physical dependence in animals
exposed chronically to drugs of abuse.
Fig. 2. Competitive inhibition specific [125I]-dynorphin binding to brain membrane by
5b. Each point represents mean of three individual determinations. IC50 concentrations
were determined.
4. Experimental section
4.1. Chemistry
4.1.1. General
It is common that opioids exert their actions through three types
Some reagents were obtained from commercial sources and
used without purification. The solvents used were of technical
grade, and freshly distilled prior to use. All melting points were
obtained on a laboratory devices melting point bath and are
uncorrected. 1H (300 MHz, 600 MHz) and 13C (75 MHz, 150 MHz)
NMR spectra were recorded using CDCl3 and DMSO-d6 as solvent
and tetramethyl silane (TMS) as internal standard on Bruker DPX
300 MHz and Bruker DRX 600 MHz NMR instruments at ambient
temperature. Chemical shifts are stated in parts per million in
of receptors,
receptors and physical and psychic dependences vary with the
receptor subtype [28]. Although opioid receptor (MOR) mediates
most of the opioid actions including analgesia, tolerance and
reward [29,30], increasing evidence indicates that the activation of
m, k and d, but the relationships between opioid
m
the
[31] like analgesia [32], tolerance [33] and withdrawal [34].
Therefore, the use of -opioid agonists [35,36] is an important
k-receptor opposes a variety of m-receptor mediated actions
k
approach towards exploring potential treatments of stimulant
abuse. However, KOR agonists produce side effects including
sedation and vomiting [35], which has led to the search for other
KOR agonists of varied structures like the azocines (cyclazocine,
d
scales. Infrared spectra were recorded on a JASCO-FTIR Model-
410, using KBr pellets. Mass spectra were measured in ESIMS (þ)
and HRMS mode. ESIMS were done on a Waters Micromass Q-TOF
microÔ Mass Spectrometer. X-Ray crystallographic data of single
crystals were collected on Bruker Kappa Apex II with Mo-K
ation (
¼ 0.71073 Å). TLC was performed on pre-coated plates
(0.25 nm, silica gel 60 F254).
a radi-
l
4.1.2. See Reference [3] for the procedure of the compounds 3aed
and for spectral data of the compounds 3aec
4.1.2.1. 2-(6-Bromo-benzo[1,3]dioxol-5-ylmethoxy)-3-methoxy-
benzaldehyde (3d). White crystalline solid, mp 153e156 ꢁC, Yield
94%. IR (KBr, nmax) 3050, 2920, 2889, 1687,1587, 1482, 1380, 1315,
1250, 1224, 1176, 1107, 1037, 1008, 930, 871, 768, 673 cmꢂ1; 1H NMR
(300 MHz, CDCl3):
J ¼ 12.0 Hz, 2H), 7.15e7.17 (m, 2H), 7.39e7.41 (m, 1H), 10.31 (s, 1H).
13C NMR (75 MHz, CDCl3):
56.1 (CH3), 75.2 (CH2),101.9 (CH2),110.3
d 3.95 (s, 3H), 5.18 (s, 2H), 5.99 (s, 2H), 7.01 (d,
d
(CH), 112.8 (CH), 114.5 (C), 118.0 (CH), 119.0 (CH), 124.4 (CH), 129.1
(C), 130.2 (C), 147.5 (C), 148.4 (C), 150.8 (C), 152.9 (C), 190.3 (CH). MS
(ESIþ): m/z ¼ 385, 387 (M þ Naþ for 79Br, 81Br). Anal. calcd. for
C16H13BrO5: C 56.22, H 4.16; Found C 56.40, H 4.04.
4.1.3. General procedure for the synthesis of O-bromobenzylated
aromatic amines 4aeh
To an ethanolic solution of 3, substituted anilines were added
and the reaction mixture was stirred at room temperature for 12 h.
After cooling to 0 ꢁC, sodium borohydride was added portionwise
and the reaction mixture was stirred for 2 h (TLC). The solvent was
removed via rotary evaporation. The reaction mixture was extrac-
ted with CH2Cl2 (2 ꢃ 10 ml), and then washed with saturated
sodium bicarbonate solution and water (2 ꢃ 8 ml). The organic
layers were combined, dried over Na2SO4, filtered and concen-
trated. The crude residue was purified via flash column
Fig. 3. Effect of test compound 5b on the induction of pERK activity in C6 glioma cells.
Five-day old cultures were serum starved for 24 h. The effects of k-opioid receptor
agonist U50488H (1
mM) and the test compound 5b (40 mM) were evaluated by
treating cells for 10 min with the drugs and quantitating pERK1/2 by western blot
analysis. Stimulation of pERK by 5b was antagonized by co-treatment with norBNI
(1
mM). The relative intensities of the pERK bands, indicated in the figure, were
obtained by densitometric scanning by ImageJ. Results are mean ꢀ SEM of at least
three blots. *p < 0.01 versus untreated control.

1718
S. Mitra et al. / European Journal of Medicinal Chemistry 46 (2011) 1713e1720
chromatography over silica gel (230e400 mesh) eluting with 2e4%
ethyl acetate in pet. ether to get the compounds 4aeh.
1H), 6.62 (t-like, 1H), 6.76 (d, J ¼ 7.8 Hz, 1H), 6.81 (d, J ¼ 6.0 Hz, 1H),
6.98e7.03 (m, 2H), 7.30 (t-like,1H), 7.42 (t, J ¼ 7.3 Hz, 1H), 7.58 (d,
J ¼ 7.2 Hz, 1H), 7.66 (d, J ¼ 7.8 Hz, 1H). 13C NMR (75 MHz, CDCl3):
4.1.3.1. [2-(2-Bromo-benzyloxy)-benzyl]-phenyl-amine (4a). White
solid, mp 64e66 ꢁC, Yield 72%; IR (KBr, nmax): 3399, 2924, 1602,
d 42.8 (CH2), 55.4 (CH3), 55.9 (CH3), 73.7 (CH2), 109.3 (CH), 110.1
(CH), 111.4 (CH), 116.3 (CH), 120.7 (CH), 121.2 (CH), 122.6 (C), 124.3
(CH), 127.4 (CH), 129.1 (CH), 129.5 (CH), 132.4 (CH), 133.6 (C), 137.4
(C), 138.1 (C), 145.6 (C), 146.8 (C), 152.7 (C). MS (ESIþ): m/z ¼ 450,
452 (M þ Naþ for 79Br, 81Br). Anal. calcd. for C22H22BrNO3: C 61.69,
H 5.18, N 3.27; Found C 61.44, H 4.92, N 3.48.
1500, 1450, 1362, 1245, 1092, 1044, 1026, 750, 688 cmꢂ1 1H NMR
;
(300 MHz, DMSO-d6):
d
4.27 (d, J ¼ 5.4 Hz, 2H), 5.13 (s, 2H), 6.06 (br.
s, 1H), 6.52 (d, J ¼ 7.2 Hz, 2H), 6.91 (t, J ¼ 7.2 Hz, 1H), 6.99e7.09 (m,
3H), 7.19e7.34 (m, 4H), 7.41 (d, J ¼ 6.3 Hz, 1H), 7.67 (dd, J ¼ 7.5,
18.3 Hz, 2H). 13C NMR (75 MHz, CDCl3):
d 43.7 (CH2), 69.4 (CH2),
111.6 (CH), 113.1 (2 ꢃ CH), 117.4 (CH), 121.1 (CH), 122.3 (C), 127.6
(CH), 128.3 (CH), 128.8 (CH), 129.1 (CH), 129.2 (2 ꢃ CH), 129.3 (CH),
132.6 (CH), 136.2 (C), 148.2 (C), 156.1 (C). MS (ESIþ): m/z ¼ 390, 392
(M þ Naþ for 79Br, 81Br). Anal. calcd. for C20H18BrNO: C 65.23, H
4.93, N 3.80; Found C 65.50, H 5.12, N 4.02.
4.1.3.6. [2-(6-Bromo-benzo[1,3]dioxol-5-ylmethoxy)-3-methoxy-
benzyl]-phenyl-amine (4f). White solid, mp 95e98 ꢁC, Yield.72%; IR
(KBr, nmax): 3398,1604,1502,1479,1272,1251,1204,1038,1010, 826,
751, 692 cmꢂ1 1H NMR (300 MHz, DMSO-d6):
d 3.85 (s, 3H), 4.12 (d-
like, J ¼ 5.7 Hz, 2H), 5.02 (s, 2H), 6.08 (s, 2H), 6.41e6.50 (m, 3H),
6.83e6.88 (m, 1H), 6.98e7.02 (m, 4H), 7.08 (s, 1H), 7.25 (s, 1H). MS
(ESIþ): m/z ¼ 464, 466 (M þ Naþ for 79Br, 81Br). Anal. calcd. for
C22H20BrNO4: C 59.74, H 4.56, N 3.17; Found C 60.02, H 4.81, N 2.93.
4.1.3.2. [2-(2-Bromo-5-methoxy-benzyloxy)-benzyl]-phenyl-amine
(4b). White solid, mp 104e107 ꢁC, Yield 68%; IR (KBr, nmax): 3413,
2999, 2934, 2841, 1600, 1481, 1456, 1365, 1277, 1243, 1157, 1055,
1014, 867, 798, 749 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
d
3.71 (s,
4.1.3.7. [2-(6-Bromo-benzo[1,3]dioxol-5-ylmethoxy)-3-methoxy-
benzyl]-(4-methoxy-phenyl)-amine (4g). White solid, mp 120e123 ꢁC,
Yield.71%; IR (KBr, nmax): 3400, 1610, 1506, 1490, 1270, 1265, 1190,
3H), 4.28 (d, J ¼ 5.4 Hz, 2H), 5.15 (s, 2H), 6.03 (br. s, 1H), 6.48e6.55
(m, 3H), 6.90 (d, J ¼ 7.5 Hz, 2H), 6.99e7.09 (m, 3H), 7.22e7.29 (m,
3H), 7.58 (d, J ¼ 8.7 Hz, 1H). 13C NMR (75 MHz, CDCl3):
d
43.7 (CH2),
1030, 1012, 835, 760 cmꢂ1 1H NMR (300 MHz, DMSO-d6):
d 3.60
55.2 (CH3), 69.2 (CH2), 111.6 (CH), 112.1 (C), 112.9 (2 ꢃ CH), 113.8
(CH), 115.1 (CH), 117.3 (CH), 121.1 (CH), 127.5 (C), 128.4 (CH), 129.1
(2 ꢃ CH), 129.2 (CH), 133.1 (CH), 137.1 (C), 148.3 (C), 156.0 (C), 159.1
(C). MS (ESIþ): m/z ¼ 420, 422 (M þ Naþ for 79Br, 81Br). Anal. calcd.
for C21H20BrNO2: C 63.33, H 5.06, N 3.52; Found C 63.05, H 4.87, N
3.77.
(s, 3H), 3.84 (s, 3H), 4.07 (br. s, 2H), 5.00 (s, 2H), 5.58 (br. s, 1H), 6.07
(s, 2H), 6.38 (d-like, J ¼ 8.1 Hz, 2H), 6.63 (d-like, J ¼ 7.0 Hz, 2H), 6.85
(br. s, 1H), 6.96 (br. s, 1H), 7.07 (s, 1H), 7.25 (s, 1H). 13C NMR (75 MHz,
CDCl3):
d 44.3 (CH2), 55.8 (CH3), 55.9 (CH3), 73.8 (CH2), 101.8 (CH2),
110.0 (CH), 111.5 (CH), 112.6 (CH), 113.8 (C), 114.2 (2 ꢃ CH), 114.8
(2 ꢃ CH), 120.9 (CH), 124.3 (CH), 130.4 (C), 133.7 (C), 142.4 (C), 145.5
(C), 147.4 (C), 148.0 (C), 152.1 (C), 152.6 (C). MS (ESIþ): m/z ¼ 472, 474
(M þ Hþ for 79Br, 81Br) and 494, 496 (M þ Naþ for 79Br, 81Br). Anal.
calcd. for C23H22BrNO5: C 58.49, H 4.69, N 2.97; Found C 58.28, H 4.41,
N 3.18.
4.1.3.3. [2-(2-Bromo-benzyloxy)-benzyl]-(4-methoxy-phenyl)-amine
(4c). White solid, mp 105-107 ꢁC, Yield 74%; IR (KBr, nmax): 3386,
2952, 2899, 2846, 1597, 1513, 1455, 1372, 1293, 1245, 1122, 1032,
818, 756 cmꢂ1 1H NMR (300 MHz, DMSO-d6):
d 3.60 (s, 3H), 4.23 (d,
J ¼ 5.7 Hz, 2H), 5.19 (s, 2H), 5.63 (br. s, 1H), 6.48 (d, J ¼ 8.7 Hz, 2H),
6.66 (d, J ¼ 8.7 Hz, 2H), 6.90 (t, J ¼ 7.2 Hz,1H), 7.07 (d, J ¼ 8.1 Hz,1H),
7.19e7.34 (m, 3H), 7.43 (t, J ¼ 7.0 Hz, 1H), 7.66 (dd-like, J ¼ 7.2,
4.1.4. General procedure for the cycloamination reaction
To a stirred solution of O-bromobenzylated amine (1 mmol) in
dry toluene (15 mL/mmol) were added KO-tBu (135 mg, 2 equiv),
anhydrous K2CO3 (167 mg, 2 equiv), Pd2(dba)3 (10 mol %), and
ꢀBINAP (7 mol %), and the reaction mixture was refluxed for the
stipulated time (Table 2) under nitrogen atmosphere. After
completion of the reaction (monitored by TLC), toluene was evap-
orated under vacuum, and the crude reaction mixture was extracted
with ethyl acetate (3 ꢃ 25 ml). The organic layer was washed with
water (3 ꢃ 25 ml) followed by brine (2 ꢃ 15 ml). The total organic
layer was dried over sodium sulfate, filtered, and the solvent was
evaporated under reduced pressure. The crude mass was purified by
flash column chromatography to get the desired compound.
20.7 Hz, 2H). 13C NMR (75 MHz, CDCl3):
d 44.7 (CH2), 55.8 (CH3),
69.4 (CH2), 111.6 (CH), 114.5 (2 ꢃ CH), 114.8 (2 ꢃ CH), 121.1 (CH),
122.4 (C), 127.7 (CH), 128.0 (C), 128.3 (CH), 128.8 (CH), 129.2 (CH),
129.3 (CH), 132.7 (CH), 136.2 (C), 142.6 (C), 152.1 (C), 156.2 (C). MS
(ESIþ): m/z ¼ 420, 422 (M þ Naþ for 79Br, 81Br). Anal. calcd. for
C21H20BrNO2: C 63.33, H 5.06, N 3.52; Found C 63.07, H 4.81, N 3.27.
4.1.3.4. [2-(2-Bromo-benzyloxy)-3-methoxy-benzyl]-(4-methoxy-
phenyl)-amine (4d). White solid, mp 75e78 ꢁC, Yield 75%; IR (KBr,
nmax): 3422, 2932, 1585, 1516, 1473, 1436, 1376, 1305, 1261, 1224,
1183, 1084, 1058, 982, 952, 820, 788, 759 cmꢂ1 1H NMR (300 MHz,
DMSO-d6):
d
3.60 (s, 3H), 3.84 (s, 3H), 4.09 (d, J ¼ 5.7 Hz, 2H), 5.10 (s,
4.1.4.1. 11- Phenyl-11,12-dihydro-6H-dibenz[b,f][1,5]oxazocine (5a).
Yellow solid; mp 70 ꢁC, Yield: 72%; IR (KBr, nmax): 2924, 1596, 1495,
1448, 1371, 1265, 1213, 1104, 973, 766, 744 cmꢂ1 1H NMR (300 MHz,
2H), 5.58 (br. s, 1H), 6.38 (d, J ¼ 8.7 Hz, 2H), 6.63 (d, J ¼ 8.7 Hz, 2H),
6.86 (d, J ¼ 6.6 Hz, 1H), 6.95e7.04 (m, 2H), 7.30 (t, J ¼ 7.2 Hz, 1H),
7.41 (t, J ¼ 7.2 Hz, 1H), 7.58 (d, J ¼ 7.2 Hz, 1H), 7.66 (d, J ¼ 7.8 Hz, 1H).
CDCl3):
d
¼ 4.76 (s, 2H), 4.94 (s, 2H), 6.63e6.68 (m, 3H), 7.02e7.20
13C NMR (75 MHz, CDCl3):
d
44.3 (CH2), 55.8 (2 ꢃ CH3), 73.8 (CH2),
(m, 5H), 7.25e7.32 (m, 2H), 7.37e7.45 (m, 3H). 13C NMR (75 MHz,
111.5 (CH),114.3 (2 ꢃ CH),114.8 (2 ꢃ CH),120.9 (CH), 122.9 (C), 124.3
(CH), 127.5 (CH), 129.2 (CH), 129.7 (CH), 132.5 (CH), 133.7 (C), 137.2
(C), 142.4 (C), 145.6 (C), 152.0 (C), 152.7 (C). MS (ESIþ): m/z ¼ 450,
452 (M þ Naþ for 79Br, 81Br). Anal. calcd. for C22H22BrNO3: C 61.69,
H 5.18, N 3.27; Found C 61.48, H 4.95, N 3.01.
CDCl3):
d
¼ 53.3, 76.0, 113.6, 117.2, 122.4, 123.5, 126.4, 128.8, 129.2,
130.1, 130.6, 131.0, 131.6, 136.6, 147.2, 147.6, 160.7. MS (ESIþ): m/
z ¼ 310 (M þ Na)þ. HRMS: 310.1198 (M þ Na)þ [expected 310.1208
(M þ Na)þ].
4.1.4.2. 2-Methoxy-11- phenyl-11,12-dihydro-6H-dibenz[b,f][1,5]oxa-
zocine (5b). Pale yellow solid, mp 85 ꢁC, Yield 77%; IR (KBr, nmax):
2922, 1724, 1599, 1498, 1372, 1269, 1039, 982 cmꢂ1 1H NMR (CDCl3,
4.1.3.5. [2-(2-Bromo-benzyloxy)-3-methoxy-benzyl]-(2-methoxy-
phenyl)-amine (4e). White solid, mp 108e111 ꢁC, Yield 74%; IR (KBr,
nmax): 3427, 3016, 2930, 2838, 1599, 1518, 1477, 1459, 1351, 1274,
300 MHz):
d 3.84 (s, 3H), 4.71 (s, 2H), 4.84 (s, 2H), 6.57e6.65 (m,
1216, 1092, 1056, 1022, 898, 777, 735 cmꢂ1
;
1H NMR (300 MHz,
3H), 6.91e6.97 (m, 2H), 7.02e7.12 (m, 4H), 7.15e7.24 (m, 2H), 7.42
DMSO-d6):
d
3.74 (s, 3H), 3.84 (s, 3H), 4.20 (d, J ¼ 6.0 Hz, 2H), 5.10 (s,
(d, J ¼ 7.2 Hz, 1H). 13C NMR (CDCl3, 75 MHz):
d 53.8, 55.5, 76.1, 112.9,
115.2, 115.9, 116.7, 122.6, 123.7, 128.8, 130.6, 131.3, 131.6, 138.3, 140.4,
2H), 5.27 (br. s, 1H), 6.22 (d, J ¼ 7.5 Hz, 1H), 6.49 (t-like, J ¼ 7.2 Hz,

S. Mitra et al. / European Journal of Medicinal Chemistry 46 (2011) 1713e1720
1719
147.6, 158.0, 160.8. EI MS (ESIþ): m/z 340 (M þ Na)þ. HRMS:
4.2.2. Animals
340.1314 (M þ Na)þ [expected 340.1313 (M þ Na)þ].
The experimental protocols using animals have been approved
by the Institutional Animal Ethics Committee and meet the
guidelines of the Government of India. For binding experiments,
adult albino Balb/c mice, 20e30 g, were used. Animals were housed
four per cage at room temperature and allowed to adapt to labo-
ratory conditions for at least 2 days before the initiation of any
experiment. The animals were housed under a standard light dark
cycle with free access to food and water, except during testing.
4.1.4.3. 11-[4-Methoxy-phenyl]-11,12-dihydro-6H-dibenz[b,f][1,5]
oxazocine (5c). Yellow solid, mp 84 ꢁC, Yield: 75%; IR (KBr, nmax):
2920, 2853, 1726, 1600, 1495, 1370, 1271, 1042, 979, 858,
746 cmꢂ1 1H NMR (300 MHz, CDCl3):
d
¼ 3.70 (s, 3H), 4.76 (s,
2H), 5.01 (s, 2H), 6.68 (q, J ¼ 9.0 Hz, 4H), 7.02e7.03 (m, 2H),
7.14e7.20 (m, 2H), 7.24 (d, J ¼ 3.4 Hz, 1H), 7.30e7.40 (m, 3H). 13C
NMR (75 MHz, CDCl3):
d 53.5, 55.6, 75.7, 114.5, 116.2, 121.9, 123.3,
125.1, 128.2, 128.8, 129.8, 130.1, 131.0, 131.4, 135.2, 141.4, 148.3,
152.2, 160.2. MS (ESIþ): m/z ¼ 318 (M þ H)þ. Anal. Calcd for
C21H19NO2: C, 79.47; H, 6.03; N, 4.41. Found: C, 79.29; H, 6.21; N,
4.66.
4.2.3. Cell culture
C6 glioma cells were used in the study. Cells were grown in
DMEM containing 10% fetal calf serum, 50
mg/ml gentamicin, pen-
strep in 5% CO2 at 37 ꢁC.
4.1.4.4. 10-Methoxy-11-[4-methoxy-phenyl]-11,12-dihydro-6H-
dibenz[b,f][1,5]oxazocine (5d). White solid, mp 88 ꢁC, Yield: 70%;
IR (KBr, nmax): 2924, 2840, 1722, 1598, 1550, 1460, 1360, 1240,
1170, 1130, 1080, 990, 812, 699 cmꢂ1 1H NMR (300 MHz, CDCl3):
4.2.4. Membrane preparation
For opioid receptor binding studies, membrane was prepared
from mouse brain. The mouse was sacrificed, brain was dissected
out, homogenized in ice-cold 50 mM TriseHCl buffer (pH 7.4), and
centrifuged at 20,000 g for 30 min. The pellet was resuspended in
the same buffer, incubated for 20 min at 37 ꢁC, and centrifuged as
above. The pellet was resuspended in ice-cold buffer and used for
binding assay. Protein concentration was determined by the
method described by Lowry et al. [37] (1951) using bovine serum
albumin (BSA) as standard.
d
¼ 3.70 (s, 3H), 3.84 (s, 3H), 4.73 (s, 2H), 5.0 (s, 2H), 6.66 (dd,
J ¼ 8.4 Hz, 3H), 6.80e6.82 (m, 2H), 6.95e6.97 (m, 2H), 7.17e7.25
(m, 2H), 7.31e7.41 (m, 2H). MS (ESIþ): m/z ¼ 370 (M þ Na)þ.
Anal. Calcd for C22H21NO3: C, 76.06; H, 6.09; N, 4.03. Found:
C, 75.87; H, 5.89; N, 4.25.
4.1.4.5. 10-Methoxy-11-[2-methoxy-phenyl]-11,12-dihydro-6H-
dibenz[b,f][1,5]oxazocine (5e). White solid, mp 89 ꢁC, Yield: 68%; IR
(KBr, nmax): 2931, 2835, 1597, 1576, 1496, 1454, 1361, 1346, 1262,
1212, 1183, 1122, 1078, 998, 745 cmꢂ1 1H NMR (300 MHz, CDCl3):
4.2.5. Preparation of 125I-dynorphin
Dynorphin was labeled with [125I] sodium iodide as described by
Gairin et al. [38]. The reaction was initiated by adding 10
chloramine T (2 g/ l) dissolved in a 0.2 M phosphate buffer (pH
7.4) to 10 l of dynorphin (1 g/ l) dissolved in 0.2 M phosphate
buffer and 1 mCi of Na125I. After 40 s of reaction, the reaction was
terminated by adding 40 l of sodium metabisulfite (2 g/ l). The
ml of
d
¼ 3.77 (s, 3H), 3.84 (s, 3H), 4.90 (s, 2H), 5.36 (s, 2H), 6.68e6.98 (m,
7H), 7.06e7.08 (m, 2H), 7.16e7.25 (m, 1H), 7.36e7.40 (m, 1H). 13C
NMR (75 MHz, CDCl3):
m m
m
m m
d
¼ 53.6, 55.6, 56.1, 75.5, 111.5, 112.4, 121.3,
122.2, 123.0, 123.8, 124.4, 127.0, 128.5, 129.0, 130.0, 130.2, 131.1,
136.9, 148.3, 150.4, 151.1, 154.4. MS (ESIþ): m/z ¼ 370 (M þ Na)þ.
Anal. Calcd for C22H21NO3: C, 76.06; H, 6.09; N, 4.03. Found: C,
75.92; H, 6.20; N, 3.88.
m
m m
reaction mixture was diluted with 0.1% aqueous trifluoroacetic acid
(TFA) and absorbed in SepPak. The mixture of labeled and unla-
beled dynorphin were eluted out in a 1 ml solution containing
99.9% acetonitrile with 0.1% TFA. Finally the labeled dynorphin was
separated from the unlabeled dynorphin by column chromatog-
raphy on a C18 column with elution with a mobile phase of
20:0.2:79.8 acetonitrile/TFA/water. Fractions containing the mono-
iodinated compound were pooled and used for subsequent studies.
4.1.4.6. 2,3-(Methylenedioxy)-10-Methoxy-11-phenyl-11,12-dihydro-
6H-dibenz[b,f][1,5]oxazocine (5f). White solid, mp 98 ꢁC, Yield: 75%;
IR (KBr, nmax): 2936, 2840, 1608, 1585, 1453, 1367, 1260, 1179, 1130,
742 cmꢂ1.1H NMR (300 MHz, CDCl3):
d
¼ 3.84 (s, 3H), 4.69 (s, 2H),
4.80 (s, 2H), 6.00 (s, 2H), 6.60e6.68 (m, 3H), 6.75e6.82 (m, 3H), 6.91
(s, 2H), 7.00e7.02 (m, 1H), 7.11 (m, 1H). MS (ESIþ): m/z ¼ 384
(M þ Na)þ. Anal. Calcd for C22H19NO4: C, 73.12; H, 5.30; N, 3.88.
Found: C, 73.35; H, 5.49; N, 3.64.
4.2.6. Radioligand binding
KOR binding was carried out with mouse membrane using [125I]
dynorphin as performed earlier [26]. Briefly, membranes
(100e200
for 30 min at 37 ꢁC in 50 mM TriseHCl buffer, pH 7.4. Non-specific
binding was determined in the presence of 5 M concentration of
unlabeled U50488H. The different test compounds were used at the
concentration of 5
M for studying the displacement of [125I]-
m
g of protein) were incubated with 2 nM [125I] dynorphin
4.1.4.7. 2,3-(Methylenedioxy)-10-Methoxy-11-[4-methoxy-phenyl]-
11,12-dihydro-6H-dibenz[b,f][1,5]oxazocine (5g). White solid, mp
102 ꢁC, Yield: 65%; IR (KBr, nmax): 2933, 2850,1604,1597, 1470, 1380,
m
1302, 1145, 745 cmꢂ1 1H NMR (300 MHz, CDCl3):
d
¼ 3.70 (s, 3H),
m
3.83 (s, 3H), 4.66 (s, 2H), 4.82 (s, 2H), 5.98 (s, 2H), 6.58 (s, 2H),
6.72e6.97 (m, 7H). MS (ESIþ): m/z ¼ 414 (M þ Na)þ. Anal. Calcd for
C23H21NO5: C, 70.49; H, 5.51; N, 3.58. Found: C, 70.72; H, 5.29; N,
3.79.
dynorphin. The dibenzoxazocine, 5b, which showed significant
affinity for KOR was further evaluated for its IC50 value for dis-
placing [125I]-dynorphin using six different concentrations ranging
from 1 nM to 25 mM. Following incubation, bound radioligand was
collected by filtering under vacuum in a Millipore filtration mani-
fold using glass-fiber filters (GF/B; Whatman, Clifton, NJ), pre-
treated with 0.5% polyethyleneimine. The filters were washed
thrice with ice-cold buffer and the radioactive retained on filters
were counted in a liquid scintillation counter (Wallac, model
1049e411, Perkin Elmer, USA).
4.2. Biology
4.2.1. Materials
DMEM, F12 and FBS were obtained from Gibco-BRL, Life Tech-
nologies. Trypsin, soybean trypsin inhibitor, U50488H, Naloxone
hydrochloride, Dynorphin, DAMGO, and anti-mouse IgG-HRP
conjugated 2nd antibody were from Sigma Chemical Co. (USA).
Primary antibody of pERK1/2 was purchased from Santacruz (USA).
Na125I was from Perkin Elmer (Boston, MA). All other reagents were
of analytical grade and obtained locally.
4.2.7. ERK assay
ERK phosphorylation was measured by immunoblotting as
described in Reference [39]. Following starvation for 24 h, cells
were treated as indicated. Antagonists were added to the medium

1720
S. Mitra et al. / European Journal of Medicinal Chemistry 46 (2011) 1713e1720
[13] X. Ouyang, Z. Chen, L. Liu, C. Dominguez, A.S. Kiselyov, Tetrahedron 56 (2000)
2369e2377.
[14] For our recent efforts on the synthesis of benzo- and dibenzo fused nine
membered medium ring heterocycles, see: T.P. Majhi, A. Neogi, S. Ghosh,
A.K. Mukherjee, M. Helliwell, P. Chattopadhyay Synthesis (2008) 94e100 (and
the references cited there in).
30 min before stimulation with agonist and compounds. After the
indicated stimulation period, medium was removed, and wells of
the flasks were washed with ice-cold PBS. Cell lysates were
collected in lysing buffer (20 mM HEPES, 10 mM EGTA, 40 mM
glycerophosphate, 2.5 mM MgCl, 2 mM sodium vanadate, 1%
Nonidet P-40, 1 mM PMSF, 20 g/ml aprotinin, and 20 g/ml leu-
b-
[15] A. Neogi, T.P. Majhi, R. Mukhopadhyay, P. Chattopadhyay, J. Org. Chem. 71
(2006) 3291e3294.
m
m
peptin, pH 7.5). Cell lysates were centrifuged at 14,000 g for 20 min
[16] N. Das Adhikary, P. Chattopadhyay, Eur. J. Org. Chem. (2010) 1754e1762 (and
the references cited there in).
[17] A. Neogi, T.P. Majhi, B. Achari, P. Chattopadhyay, Eur. J. Org. Chem. (2008)
330e336.
at 4 ꢁC, and protein concentration of the supernatants was deter-
mined. Samples (40 mg of protein/lane) were separated by 10% SDS-
PAGE. Proteins were blotted on PVDF membranes (Millipore Corp;
Bedford, MA). Ponceau staining of blots was carried out, prior to
immunoblotting, to confirm loading of equal amount of proteins in
the lanes. Non-specific binding to membrane was blocked with 5%
non-fat dry milk in Tris-buffered saline (20 mM Tris, 0.9% NaCl, pH
7.5) for 1 h at 37 ꢁC. The membranes were probed with monoclonal
antibody against pERK (1:1000 dilution) for 2e3 h, followed by
treating with a peroxidase conjugated secondary antibody.
[18] The example showed the formation of dibenzoxazocine derivatives from
primary amine precursors in lower yield: Fink B.E., Gavai A.V., Vite G.D.,
HanW.-C., Misra R.N., Xia H.-Y., Norris D.J., Tokarski J.S., US Patent US2005/
0250753.
[19] J.P. Wolfe, S. Wagaw, S.L. Buchwald, J. Am. Chem. Soc. 118 (1996) 7215e7216.
[20] D.S. Surry, S.L. Buchwald, Angew. Chem. Int. Ed. Engl. 47 (2008) 6338e6361
(and the references cited therein).
[21] J.P. Wolfe, S. Wagaw, S.L. Buchwald, Acc. Chem. Res. 31 (1998) 805e818.
[22] K.U. Singh, R.E. Strieter, G.D. Blackmond, S.L. Buchwald, J. Am. Chem. Soc. 124
(2002) 14104e14114.
[23] Crystal data of compound 5b: C21H19NO2, FW ¼ 317.37, triclinic, space group
P-1, 0 ¼ 9.781 (2) Å, b ¼ 9.806 (2) Å, c ¼ 10.530 (4) Å,
a
¼ 105.362 (19)ꢁ,
Acknowledgments
b
¼ 106.85 (2)ꢁ,
g
¼ 111.933 (13)ꢁ, V ¼ 813.6 (4) Å3, Z ¼ 2, T ¼ 296(2) K,
dcalcd ¼ 1.296 g cmꢂ3, F (000) ¼ 336. Diffraction data were measured with Mo
K
a
(
l
¼ 0.71073 Å) radiation at 296 K using a Bruker
k Apex 2 CCD system. A
Financial support from the Department of Science and Tech-
nology (DST), New Delhi, Government of India is gratefully
acknowledged. We thank CSIR (India) for financial assistance.
Thanks are also due to Dr. B. Achari, Emeritus Scientist, IICB, for
valuable suggestions, Dr. R. Mukhopadhyay and K.K. Sarkar for
spectral analysis.
total of 1585 unique reflections were measured (qmax ¼ 24.48ꢁ). Data analyses
were carried out with the Fast Fourier Transform program. The structures
were solved by direct methods using the SHELXS-97 program [24]. Refine-
ments were carried out with a full matrix least squares method against F2
using SHELXL-97 [25]. Non-hydrogen atoms were refined with anisotropic
thermal parameters. The final R value was R1 ¼ 0.0686 and wR2 ¼ 0.1754
with I > 2s (I). Crystallographic data have been deposited at the Cambridge
Crystallographic Data Centre with reference number CCDC 746587.
[24] G.M. Sheldrick, SHELXS-97, Program for Solution of Crystal Structures.
University of Gottingen, Germany, 1997, G.M. Sheldrick, Acta. Crystallogr. A,
46 (1990) 467e473.
[25] G.M. Sheldrick, SHELXL-97, Program for Refinement of Crystal Structures.
University of Gottingen, Germany, 1997.
Appendix. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.02.024.
[26] I. Deb, P. Paira, A. Hazra, S. Banerjee, P.K. Dutta, N.B. Mondal, S. Das, Bioorg.
Med. Chem. 17 (2009) 5782e5790.
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