Regioselective synthesis of phenanthrenes and evaluation of their anti-oxidant based anti-inflammatory potential

Article abstract of DOI:10.1016/j.ejmech.2013.06.016

Regioselective synthesis of 9,10-Dihydro-2,5-Dimethoxyphenanthrene-1,7-diol (1) and 9,10-Dihydro-2,7-Dimethoxyphenanthrene-1,5-diol (2) was achieved using regioselective methylation, Wittig reaction, intramolecular cyclization and hydrogenation as key ste

Full text of DOI:10.1016/j.ejmech.2013.06.016

European Journal of Medicinal Chemistry 67 (2013) 454e463
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Regioselective synthesis of phenanthrenes and evaluation of their
anti-oxidant based anti-inflammatory potential
*
Yogesh Kanekar, Khalander Basha, Sharad Duche, Rajan Gupte, Arnab Kapat
Reliance Life Sciences Pvt. Ltd., Dhirubhai Ambani Life Sciences Center, Thane Belapur Road, Rabale, Navi Mumbai 400 701, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Regioselective synthesis of 9,10-Dihydro-2,5-Dimethoxyphenanthrene-1,7-diol (1) and 9,10-Dihydro-2,7-
Dimethoxyphenanthrene-1,5-diol (2) was achieved using regioselective methylation, Wittig reaction,
intramolecular cyclization and hydrogenation as key steps. The synthesis was successfully completed in
total of 15 steps with 3.3% overall yield in case of 1 and in total of 13 steps with 9.0% overall yield in case
of 2. All compounds (1e4) showed good antioxidant and anti-inflammatory activity in in vitro assays and
these activities were found to be due the presence of phenolic hydroxyl groups.
Received 20 March 2013
Received in revised form
3 June 2013
Accepted 7 June 2013
Available online 18 June 2013
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Regioselective synthesis
Anti-oxidant
Anti-inflammatory
TNF-alpha
1. Introduction
[4]. Several Indian traditional medicinal plants are considered
useful for treating inflammation [5]. Eulophia ochreata, an orchid, is
Tumor Necrosis Factor-alpha (TNF-
mediator of inflammation. Various anti-inflammatory drugs,
particularly biologics such as etanercept, infliximab and adalimu-
mab, work on the principle of inhibiting TNF-a. Though these TNF-a
inhibitors are highly specific, their long term use is associated with
serious side effects including tuberculosis and cancer [1]. However,
TNF-a still remains an attractive target for developing new anti-
inflammatory drugs that could be administered orally, would
have fewer side effects and would lead to cost effective therapy [2].
a
) is a cytokine and a primary
a traditional medicinal plant used in India for rejuvenating and
aphrodisiac properties [6e8]. It is also shown to possess anti-
bacterial [9,10], anti-inflammatory [11], anti-oxidant and anti-
diabetic [12] potential. Based on 1,1-Diphenyl-2-picryl-hydrazyl
(DPPH) radical scavenging property, we had recently isolated 9,10-
Dihydro-2,5-Dimethoxyphenanthrene-1,7-diol (1) and 3,7-
dihydroxy-2,4-dimethoxyphenanthrene (4) from E. ochreata tuber
extract [13]. The compound 1 had further been shown to inhibit
inflammatory signaling mediated by toll-like receptor [14]. This
highlights the radical scavenging and anti-inflammatory potential
of compound 1. The compound 1 had also been shown to possess
anti-proliferative potential [15]. Compared to its regio-isomer 9,10-
Dihydro-2,7-Dimethoxyphenanthrene-1,5-diol (eulophiol) (2) [16],
very limited study has been done on 9,10-Dihydro-2,5-
Dimethoxyphenanthrene-1,7-diol (1), possibly due to scarcity of
the sample. Flavanthridin (3) is a 9,10-dihydro-derivative of 3,7-
dihydroxy-2,4-dimethoxyphenanthrene (4). Although, the inhibi-
tory effect of 3 on LPS-induced NO-production in RAW 264.7 cells
has been demonstrated [17], evaluation of anti-oxidant potential of
3 has not been reported so far. In addition, the inhibitory effect of all
of these compounds (1e4) (Fig. 1) on TNF-mediated inflammation
has not yet been reported. Since, isolation of these compounds from
E. ochreata in large scale is not feasible owing to its endangered
status [18,19]; we first synthesized 1, 2 and 3 chemically for
structureeactivity-relationship study and then evaluated these
compounds for their anti-oxidant and anti-inflammatory potential.
TNF-
specific receptor on cell surface. Nuclear-Factor kappa B (NF-
one of the prominent ones. NF- B activation is facilitated by con-
a
activates many signaling cascades upon binding with its
k
B) is
k
ditions associated with increased intracellular oxidative stress.
Reactive oxygen species (ROS) generated during inflammation not
only helps to kill pathogens but also acts as important signaling
molecules by changing the intracellular redox balance. ROS are
important mediators that initiate and propagate inflammatory re-
sponses by stimulating release of pro-inflammatory cytokine such
as TNF-a. However, uncontrolled ROS generation leads to oxidative
stress and become primary cause of a variety of inflammatory
diseases [3]. Therefore attenuating ROS by an antioxidant is bene-
ficial in the management of ROS mediated inflammatory diseases
* Corresponding author. Tel.: þ91 22 6767 8000/8300; fax: þ91 22 6767 8099.
E-mail address: Arnab.Kapat@ril.com (A. Kapat).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.016

Y. Kanekar et al. / European Journal of Medicinal Chemistry 67 (2013) 454e463
455
were unsuccessful which instead resulted in the formation of 15.
Alternative approach via 16 and 17 gave the same result. Though
the presence of methoxyl group is known to cause bromination to
occur at para position [24], formation of 15 was unexpected as high
temperature and non-polar solvent were used in order to facilitate
the side chain bromination via radical mechanism [25] (Scheme 2).
Having faced the problem of ring bromination on TBDMS pro-
tected phenol 13, phenol 12 was acetylated (18), and then bromi-
nated (19). Since in Wittig reaction phosphonium salt interacts
with a strong base (in this case n-butyl lithium), the presence of
labile acetate group was undesirable. Therefore, we decided to
replace it with TBDMS group, which is known to withstand Wittig
conditions [26]. However deacetylation of 19 by various methods
like methanolic HCl, CCl4eHCl or refluxing methanolic HCl, HCl in
acetone, NaOMe in MeOH, either failed or led solely to decompo-
sition. As per Das et al. (2003) [27] phenolic acetates could be
selectively removed using AmberlystÒ-15 in MeOH at room tem-
perature. However, it proved to be slow and generated an un-
identified impurity in our case. Further transformation (22)
followed by Wittig reaction with 7 generated a complex desilylated
mixture (23b instead of 23a) which on catalytic hydrogenation
resulted in the formation of dihydrostilbene 24. Owing to the
perceived problem of removing acetate protecting group at benzyl
bromide stage (19), alternative route b (Scheme 1) was trialed. The
required component 27 was generated from 18 via tribromide 25
and aldehyde 26. The other Wittig component 30 was derived from
7 via 28 and 29. However the outcome of newly tried Wittig re-
action did not change (Scheme 3).
OMe
OMe
OH
OMe
HO
HO
HO
MeO
MeO
MeO
OH
OH
OMe
OH
OH
OMe
3
2
4
1
Fig. 1. Structure of 9,10-Dihydro-2,5-Dimethoxyphenanthrene-1,7-diol (1), 9,10-Dihy-
dro-2,7-Dimethoxyphenanthrene-1,5-diol (eulophiol) (2), Flavanthridin (3) and 3,7-
dihydroxy-2,4-dimethoxyphenanthrene (4).
2. Results and discussion
2.1. Synthesis
2.1.1. 9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol (1)
Since synthesis of 1 has not been cited in the literature, we
selected a reaction sequence comprising regioselective methyl-
ation, Wittig reaction, intramolecular radical cyclization and hy-
drogenation as the key steps due to commercial abundance of
starting materials, o-Vanillin (2-hydroxy-3-methoxybenzaldehyde)
(6) & orcinol (5-methylbenzene-1,3-diol) (8) (Scheme 1). As shown
in Scheme 1, the intramolecular radical cyclization of stilbene
would either give protected phenanthrene derivatives 1c or 1d
which on further transformation would result in 1 or 2 respectively.
Hence we employed bromo orcinol derivative (5a or 5c) rather than
bromo o-Vanillin derivative to achieve the regioselectivity in
cyclization.
The presence of electron-withdrawing groups, Br in particular,
at para position is known to expedite the deprotection of phenolic
TBDMS ethers [28]. Therefore, the observed facile desilylation was
suspected to be due to the presence of para-Br substituent (22, 27).
Also, it is known that the phenolic TBDMS ethers get easily
deprotected by catalytic lithium hydroxide (LiOH) in DMF under
milder conditions. According to Davies et al. (1992) [29], basic
Synthesis of 1 commenced with silylation of o-Vanillin (6) to 7.
Its Wittig counterpart (13) was prepared from orcinol (8) via 9
[20,21] to 12 [22] and then silylation [23] with an overall yield of
39.2% from 8. However, repeated attempts of converting 13 to 14
OP
CHO
+
OP
MeO
CH2PPh3Br
OP
OP
OH
OMe
Br
a
b
MeO
MeO
MeO
5a
5b
Br
or
OP
OP
OH
OP
OMe
CH2PPh3Br
OP
OMe
OMe
+
1b
1
1a
MeO
CHO
OMe
5d
Br
OR1
OP
*
5c
R2O
MeO
P=Protecting group
*
*
* = possible sites for cyclization to
form 6-membered ring
OP
OP
OMe
OMe
1c: R1= P, R2= Me
1d: R1= Me, R2= P
Stilbene
Scheme 1. Retrosynthetic analysis of 1.

456
Y. Kanekar et al. / European Journal of Medicinal Chemistry 67 (2013) 454e463
a
9: R1= OH, R2= OH
10: R1= OH, R2= OTs
CHO
OH
CHO
c
OTBDMS
d
OMe
OMe
11: R1= OMe, R2= OTs
12: R1= OMe, R2= OH
e
6
7
f
13: R1= OMe, R2= OTBDMS
OH
R2
b1 or b2
HO
Me
R1
Me
Br
9-13
8
OTBDMS
R1
f, d
8
MeO
MeO
CH2Br
R2
16-17
Me
Br
14
g
16: R1= OTBDMS, R2= OH
13
OTBDMS
Br
17: R1= OTBDMS, R2= OMe
g
Me
15
Br
15
Scheme 2. Synthesis of intermediates (7, 9e17). Reagents and conditions: (a) TBDMSCl, DIPEA, THF, RT (87.9%); (b1) TBABr3, DCM:MeOH (3:2), 15e20 ꢀC (70.1%); (b2) Dioxane
dibromide, diethyl ether, 15e20 ꢀC (71.4%); (c) TsCl, K2CO3, acetone, reflux; (d) MeI, K2CO3, acetone, RT; (e) KOH, EtOHeH2O, reflux (83.5%); (f) TBDMSCl, Immidazole, DCM; (g) NBS,
CCl4, benzoyl peroxide (cat.), reflux.
OR1
OR1
R3
OR
MeO
CH2R2
Br
MeO
R2
Br
OMe
12: R1= H, R2= H
18: R1= Ac, R2= H
h
g
7
18-27
7: R= TBDMS, R3= CHO
18: R1= Ac, R2= Me
19: R1= Ac, R2= Br
20: R1= H, R2= Br
n
o
g
i
f
j
28: R= TBDMS, R3=CH2OH
29: R= TBDMS, R3= CH2Br
30: R= TBDMS, R3= CH2PPh3Br
25: R1= Ac, R2= CHBr2
m
f
26: R1= H, R2= CHO
21: R1= TBDMS, R2= Br
j
27: R1= TBDMS, R2= CHO
22: R1= TBDMS, R2= PPh3Br
OH
OR
22 + 7
k
l
MeO
MeO
or
Br
27 + 30
OH
OR
OMe
OMe
24
23a: R=TBDMS
23b: R=H
Scheme 3. Synthesis of Wittig components and their coupling by Wittig reaction. Reagents and conditions: (h) Ac2O, pyr, RT; (i) Amberlyst@15, MeOH, RT, 24 h; (j) PPh3, toluene,
reflux, 4 h; (k) n-BuLi, ꢁ78 ꢀC, THF; (l) H2, 10%Pd(C), MeOH, RT, 24 h; (m) ammonium formate, EtOH:H2O (1:1), reflux, 24 h; (n) NaBH4, IPA, reflux, 2 h, (48.4%); (o) PBr3, DCM, 0 ꢀC,
45 min.

Y. Kanekar et al. / European Journal of Medicinal Chemistry 67 (2013) 454e463
457
conditions favor cleavage of aryl silyl ether. Hence a small amount
of DMF which was used to get a clear solution of compounds (22,
30) in THF and formation of catalytic LiOH from n-BuLi in DMF
could also be responsible for desilylation. Therefore benzyl pro-
tection was chosen because phenolic benzyl ether is known to be
stable under these conditions [30]. Accordingly, 31 was synthesized
from 26 with an overall 13% yield from 8. For its Wittig counterpart,
o-Vanillin (6) was benzylated (32), reduced (33), brominated (34)
and then converted to phosphonium salt 35 with an overall 80.1%
yield from 6. It has been previously shown that the use of lithium
methoxide (LiOMe) in DMF mainly led to Z-alkenes as the Wittig
product [31e34]. Accordingly, Wittig reaction between 31 and 35
gave 36 which on AIBNeBu3SnH induced cyclization resulted in the
desired phenanthrene 37. The catalytic hydrogenation of 37 resul-
ted in the formation of 1. The outcome of the Wittig reaction is
greatly influenced by number of factors which determine whether
the Wittig reaction is under kinetic control or under thermody-
namic control [35]. Electron donating groups make the ylide un-
stable and such unstable ylides react faster leading to kinetically
controlled Z-alkenes. The presence of electron donating sub-
stituents in both the aldehyde and ylide appreciably enhances Z-
selectivities of Wittig reaction [36e39]. In addition, when lithium
salt in DMF is used, products are almost exclusively Z-alkenes
[31,40]. The exclusivity of Z-36 could hence be rationalized on the
above mentioned grounds.
position of OBn. Further, the formation of tetra-ortho-substituted
symmetrical biaryl 43a would be more difficult than tri-ortho-
substituted cross-coupled biaryl 43b due to steric hindrance. It was
shown that the steric hindrance is not a major factor in aryl halides
with one ortho group, however when ortho disubstituted Suzuki
components (similar to 42a) are used, the reaction gets adversely
affected because of the steric hindrance during the trans-
metallation to the Palladium(II) complex [51]. Cross-coupled
product (43b) on subsequent Wittig olefination gave divinyl 44,
which on ring-closing metathesis (45) followed by hydrogenation
resulted in the formation of eulophiol (2) (Scheme 5). Low overall
yield obtained in the above mentioned synthetic sequence
demanded further development in the synthesis of eulophiol. At
this point, we decided to use Wittig reaction since the required
intermediates 35 (Scheme 4) and 42 (Scheme 5) were ready.
Applying similar sequence we were able to synthesize eulophiol (2)
with 93% purity (Scheme 6).
2.1.3. Semi-synthesis of flavanthridin (3)
Hydrogenation of 4 in EtOAc:EtOH (3:1) using Pd(C) followed by
chromatographic purification resulted in the formation of 3 as
white solid with 95% purity (Scheme 6).
2.2. Evaluation of biological functions
Though we reached to 1, low overall yield and difficulty in
final step purification demanded further development. The con-
cerned impurity was generated due to suspected dehalogenation
[37,41] of the stilbene 36 during radical cyclization. To circum-
vent this, the double bond of 36 was selectively reduced [42].
Further cyclization [43] of 38 was however unsuccessful. AIBNe
Bu3SnH induced cyclization of 38 resulted in the formation of
39a along with the desired dihydrophenanthrene 39. However,
due to low yield of the desired dihydrophenanthrene 39 (6.94%)
further work along these lines was abandoned and a hybrid
methodology was adopted to reach effectively to 1. Accordingly,
37 was first treated with p-TsNHNH2, purified and then subjected
to hydrogenation which yielded compound 1 with 97% purity
(Scheme 4).
2.2.1. Free-radical scavenging activity of the compounds
Reactive oxygen species (ROS) have been implicated in a num-
ber of pro-inflammatory signal transduction cascades activated by
cytokines such as IL-1
context, ROS have been considered second messengers. For
example, ROS are generated in response to IL-1 , TNF- , and lipo-
b, TNF-a, and lipopolysaccharide. In this
b
a
polysaccharide, and clearance of intercellular ROS can inhibit the
ability of these ligands to activate downstream signals, including
NF-kB [52]. Hence compounds with antioxidant activity would be
beneficial in the management of inflammatory diseases. The radical
scavenging potential of the compounds is generally assessed by
using stable and colored radicals like DPPH, ABTS, and Galvinoxyl
radicals. Both DPPH and Galvinoxyl radicals are commercially
available whereas ABTS radical needs to be generated by oxidizing
its neutral counterpart by potassium persulfate.
2.1.2. 9,10-Dihydro-2,7-dimethoxyphenanthrene-1,5-diol
(eulophiol) (2)
DPPH is a stable free radical with an absorption maximum at
515 nm. It loses this absorption when reduced by an antioxidant or
a free radical species. The DPPH method is widely used for the
determination of antiradical/antioxidant activity of purified
phenolic compounds as well as natural plant extracts [53]. Hence
we evaluated free radical scavenging potential of compounds 1e4
in DPPH assay. As shown in Table 1, compounds 3 and 4 exhibited
significant DPPH scavenging activities which were higher than all
the standards, such as Curcumin, Ascorbic acid, Catechin and BHT
used in this assay whereas compounds 1 and 2 showed activities
comparable to BHT. The order of reactivity of the compounds was
3 > 4 > 2 > 1 and that of the standards was curcumin > ascorbic
acid > catechin > BHT.
Galvinoxyl is a stable phenoxyl radical and have characteristic
absorbance maximum (lmax ¼ 428 nm) in methanolic solution.
Unlike DPPH which is an N-centered radical, galvinoxyl is an O-
centered radical and hence more closely related to physiologically
acting oxygen radical [54]. When free radical scavenging potential of
compounds 1e4 was tested in galvinoxyl assay, as shown in Table 1,
the orderof reactivity of the compounds was 3 > 4 >1 >2 and that of
the standards was ascorbic acid > curcumin > catechin > BHT.
The bleaching of blue-green solution of ABTSþ, radical
Synthesis of eulophiol (2) has not been cited in the literature. Its
synthesis was started with the preparation of Suzuki coupling
partners: 41 from o-Vanillin (6) [44,45] and 42 from orcinol (8)
[46,47] as per published protocols. Attention was next directed to
the synthesis of arylboronic ester of either 41 or 42 by Miyaura
protocol [48] because arylboronic acid synthesis using Grignard or
lithium reagents requires the protection of functional groups (like
CHO) which are sensitive to these reagents whereas Miyaura pro-
tocol tolerates various functional groups. Also, 2-Formyl group on
arylboronic acid is known to accelerate the rate of hydrolytic
deboronation [49]. However, our attempt to synthesize the boro-
nate 41a in DMF resulted in the formation of only a symmetrical
biaryl 43c. Alternative protocol [50] also resulted in the formation
of symmetrical biaryls 43a and 43c. Formation of homodimers was
presumably due to the Suzuki coupling of the boronate and its
halide precursor under the reaction conditions [50]. Hence, an
alternative reverse strategy was devised wherein boronate forma-
tion was attempted on 42, followed by one-pot Suzuki-coupling
with 41. This yielded the desired cross coupled product (43b) along
with the symmetrical biaryl (43a) (43a:43b 1:3.2).
Although we could not ascertain the exact cause of this defer-
ential behavior of 41 and 42, this could be due to benzyloxy group
(OBn) as both the aldehydes differ from each other only in the
(
lmax ¼ 734 nm) is a widely accepted method for evaluating the
antiradical property of the compounds. ABTSþ, radical is more
reactive than DPPH radical and in contrast to the reactionwith DPPH

458
Y. Kanekar et al. / European Journal of Medicinal Chemistry 67 (2013) 454e463
R
6: R2= OH, R1= CHO
R1
R2
p
32: R2= OBn, R1= CHO
n
o
j
MeO
CHO
OMe
33: R2= OBn, R1= CH2OH
34: R2= OBn, R1= CH2Br
Br
26, 31
6, 32-35
35: R2= OBn, R1= CH2PPh3Br
26: R= OH
p
31: R= OBn
OBn
OBn
q
r
s
MeO
MeO
31
35
+
1
Br
OBn
OMe
OBn
OMe
36
37
Z:E= 100 : 0
OBn
OBn
OBn
t
r
+
36
MeO
MeO
MeO
Br
OBn
OMe
OBn
OBn
OMe
OMe
39a
39
38
OBn
s
24
39a
t,u
r
MeO
37
+
36
s
1
37
OBn
OMe
40
(not isolated)
Scheme 4. Synthesis of 1 via radical cyclization of stilbene 36. Reagents and conditions: (p) BnBr, K2CO3, DMF, 24 h, RT; (q) LiOMe, DMF, reflux, 1 h; (r) AIBNeBu3SnH, Toluene,
reflux, 2 h; (s) H2 (70 psi), 10% Pd(C), EtOAceEtOH (3:1), 24 h; (t) p-TsNHNH2 (5.0 equiv), NaOAc (2.0 equiv), EtOH, reflux, 2 h; (u) Si-gel column chromatography (Pet ether/EtOAc
gradient) (66.6% 37 from 36).
which is an H-atom transfer, its reaction involves a single electron
transfer process [55]. When free radical scavenging potential of
compounds 1e4 was tested in ABTS assay, as shown in Table 1, the
order of reactivityof the compounds was 3 > 4 >1 >2 and thatof the
standards was catechin > curcumin > ascorbic acid > BHT.
[57,58]. The unshared pair of electrons of ortho-OCH3 in the p-
orbital stabilizes the phenoxyl radical through electron delocal-
ization and electron donation. Both 3 and 4 possessed two ortho-
OCH3. This might be the reason for their low IC50 values compared
to that of 1 and 2. This trend in radical scavenging efficiency of
compounds 1e4 was maintained in all three methods utilized in
this study. However, the differences in radical scavenging potential
of two groups (compounds 3e4 versus compounds 1e2) became
more apparent in DPPH assay than in other two assays. Similar slow
reactivity of DPPH with antioxidant is reported in the literature
[53,59]. Both phenanthrene and dihydrophenanthrene backbone
were found to be inactive (IC50 > 100) in all three assays due to
absence of hydroxyl group.
2.2.2. Investigation into the structureeactivity relationship
The compounds 1, 2, 3 and 4 possess two hydroxyl and two
methoxy groups. Hence no significant differences were noted be-
tween IC50 values of 1 and 2 (both possess two OH). In addition to
number of hydroxyl groups, the antiradical activity also depends on
several other factors such as H-donating ability of the hydroxyl
group, stability of phenoxyl radical, etc. [56]. The antiradical po-
tency of the phenolic compounds is primarily governed by the bond
dissociation energy of OeH bond. The presence of electron-
donating group at ortho-position lowers the OeH bond dissocia-
tion enthalpy and increases the rate of H-atom transfer to radicals
2.2.3. Studies on TNF-
a
mediated biological effects
The release of TNF-
a
after stimulation of THP-1 cells with LPS is
a valid model system to test novel compounds for potential

Y. Kanekar et al. / European Journal of Medicinal Chemistry 67 (2013) 454e463
459
Br
OMe
ref.46, 47
BnO
ref.44,45
CHO
OBn
8
6
CHO
OMe
Br
41
42
OMe
OMe
OMe
OBn
CHO
42
+
v
R3
43c
41
R4
R2
CHO
CHO
v
B
O
O
R1
41a
43a
+
42
w
41a
41
43a: R2,R4= OBn, R1,R3= H
43b: R1,R4= OBn, R2,R3= H
43c: R1,R3= OBn, R2,R4= H
w
43c
43a
OMe
41
+
w
42
w
BnO
CHO
43b
B
O
O
42a
OMe
OMe
y
s
BnO
BnO
x
2
43b
OBn
OBn
OMe
OMe
45
44
Scheme 5. Synthesis of 2 via Suzuki-coupling. Reagents and conditions: (v) Bis(pinacolato)diboron, PdOAc, KOAc, DMF, 80 ꢀC; (w) Bis(pinacolato)diboron, PdCl2(dppf), KOAc, DMSO,
80 ꢀC, 2 h; then added other component, K2CO3, 80 ꢀC, 2 h; (x) Ph3PCH3Br, n-BuLi, THF, ꢁ78 ꢀC; (y) Grubbs 2nd generation Ru catalyst, DCM, 40 ꢀC; (s) H2 (70 psi), Pd/C, EtOAC (1.5%
from 42).
antiinflammatory effects [60]. Hence after LPS stimulation of THP-1
cells, TNF- released was quantified by an Enzyme-linked immu-
nosorbent assay (ELISA). Curcumin was treated as a positive control
2.2.3.2. Cytotoxicity of isolated and synthetic compounds in THP-1
cells. The cytotoxicity of isolated and synthetic compounds was
assayed to determine sample concentrations showing non-toxic
effect on THP-1 cells.
As shown in Table 3, all four compounds 1, 2, 3, and 4 along with
their respective phenanthrene and dihydrophenanthrene backbone
werefoundtobe non-toxic toTHP-1 cells, as against curcuminwhich
a
in the present study because it is known to inhibit NF-
different cell types including leukemia cells [61].
k
B in
2.2.3.1. Inhibitory effects of compounds on LPS-induced TNF-
a
pro-
duction in human THP-1 cells. THP-1 cells were exposed to varying
concentrations of the compounds (1e100 M) in a fresh serum-free
medium in both the presence and absence of LPS. TNF- was
showed toxicity at higher concentration (100 mM).
Curcumin is reported to be cytotoxic to THP-1 cells at higher
concentration [62]. Therefore, in the present study, curcumin which
m
a
quantified by sandwich ELISA. As shown in Table 2, compounds 1e4
along with the positive control curcumin inhibited LPS-induced
exhibited the maximum inhibition of LPS-induced TNF-
result of reduced viability of THP-1 cells. As against this, the
inhibitory effects of all four compounds 1e4 on LPS-induced TNF-
a was a
TNF-a
secretion in THP-1 cells in a dose-dependent manner. On
a
the other hand, their respective phenanthrene and dihy-
drophenanthrene backbone were inactive.
production in human THP-1 cells was not a result of reduced cell
viability.

460
Y. Kanekar et al. / European Journal of Medicinal Chemistry 67 (2013) 454e463
(IR) were recorded using KBr pellets on Perkin Elmer spectrum 100
OMe
OMe
series spectrometer. 1H and 13C NMR spectra were recorded with
Varian operating at 400 MHz for 1H and at 100 MHz for 13C with
TMS as the internal reference (Sigma Aldrich). Mass spectra were
obtained using Applied Biosystem MDS SCIEX 3200 QTRAP. Hy-
drogenation was carried out in rocker-shaker hydrogenator by
Amar equipment. Thin-layer chromatography (TLC) was performed
on Kieselgel 60 F254 (0.20 mm layer, Merck) and the plates were
examined under UV light at 254 nm and/355 nm. HPLC was per-
formed on Dionex using analytical columns: column-1 (Waters
42
+
q
(i) r
s
45
2
BnO
(ii) t
Br
35
OBn
46
Symmetry C18 5
(Kromasil C8 5
m
m 4.6 ꢂ 250 mm) with method 1 or column-2
s
mm 4.6 ꢂ 250 mm) with method 2 (Table 4).
4
3
Where 1H NMR and HPLC were recorded on the diastereoisomeric
mixtures, we had assumed that the more intense resonances
correspond to the major isomer and that the less intense reso-
nances were due to the minor isomer in accordance with the pro-
cedure adopted by Harrowven et al. (2006) [38]. Column
chromatography was run on silica gel (60e120 mesh or 100e200
mesh) from ACME, India. All commercially available chemicals were
used as received.
Scheme 6. Synthesis of 2 (via Wittig) and 3.
3. Conclusion
The synthesis of 9,10-Dihydro-2,5-Dimethoxyphenanthrene-
1,7-diol (1) was achieved in total of 15 steps with 3.3% overall yield
and that of eulophiol (2) in total of 13 steps with 9.0% overall yield
from orcinol (8). The results also corroborate the structure of iso-
lated compound as 9,10-Dihydro-2,5-Dimethoxyphenanthrene-
1,7-diol [13]. The synthesis of 2 reconfirmed the structure of
eulophiol as 9,10-Dihydro-2,7-Dimethoxyphenanthrene-1,5-diol. A
comparative antioxidant study based on three radical scavenging
assays revealed the anti-inflammatory potential of compounds 1e4
and their potential in limiting ROS mediated inflammation. While
their inhibitory potential demonstrated in THP-1 cells, non toxicity
in THP-1 cell lines confirmed their activity profile. Inactivity of their
corresponding phenanthrene and dihydrophenanthrene backbone
in radical scavenging assays and THP-1 assay indicated that
these activities are primarily due to phenolic hydroxyl groups. Our
earlier research [15] toward this has demonstrated that
isolated compound (1) down regulates expression of LPS-
4.2. Cell culture and stimulation
Human acute monocytic leukemia cells (THP-1) were purchased
from ATCC (Manassas, VA, USA) and cultured in Roswell Park Me-
morial Institute (RPMI) 1640 medium containing 10% heat-
inactivated fetal bovine serum (FBS), 1% non-essential amino
acids, 1% glutamine, 100 U/mL penicillin G and 100 mg/mL strep-
tomycin at 37 ꢀC in a humidified 5% CO2 atmosphere. The culture
medium was changed twice a week. THP-1 cells were pre-
incubated with the compounds (1e100
stimulated with the 250 ng/mL of lipopolysaccharide (LPS) (from
Escherichia coli serotype O55:B5, Sigma) for 24 h.
mM) for 1 h, and then
4.3. Procedure for the synthesis of selected compounds
stimulated NF-kB-mediated, inflammatory cytokines via a Toll
like receptor-mediated process. A similar mechanism could thus be
responsible for anti-inflammatory activity shown by all other
compounds 2e4 discussed in the present study. Successful chem-
ical synthesis of these compounds will further reduce the depen-
dence on the tuber extract of the endangered species (E. ochreata).
These results may be helpful in future for the design of inhibitors of
TNF mediated inflammation, and offer potential application in the
discovery of anti-inflammatory drugs.
4.3.1. 9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol (1)
To a solution of Phenanthrene 37 (0.8 g, 1.78 mmol) in EtOAc
(30 mL) was added 10%Pd/C (360 mg), followed by EtOH (10 mL)
and transferred the suspension to a rocker-shaker hydrogenator.
Hydrogenator was pressurized to 70 psi with H2 and shaken for
24 h. After HPLC monitoring, the suspension was passed through a
celite bed and the filtrate was concentrated under reduced pres-
sure. Crude product obtained was purified by silica gel column
chromatography (Pet-ether: EtOAc 75:25) to get 1 (0.3 g, 62.5%) as
white solid: mp 201e203 ꢀC (reported [63] mp 202e203 ꢀC); IR
(KBr) nmax 3445, 2948, 1602, 1498, 1481, 1442, 1276 cmꢁ1; 1H NMR
4. Experimental protocol
4.1. General methods
(400 MHz, CD3COCD3) d 2.61 (m, 2H, CH2), 2.74 (m, 2H, CH2), 3.82 (s,
3H, OMe), 3.85 (s, 3H, OMe), 6.38 (d, 1H, J ¼ 2.4 Hz, Ar-H), 6.45 (d,
1H, J ¼ 2.4 Hz, Ar-H), 6.78 (d, 1H, J ¼ 8.4 Hz, Ar-H), 7.19 (br s, 1H, D2O
exch., OH), 7.72 (d, 1H, J ¼ 8.4 Hz, Ar-H), 8.24 (br s, 1H, D2O exch.,
Melting points were determined using Lab India MReVIS visual
melting point apparatus and were uncorrected. Infrared spectra
OH); 1H NMR (400 MHz, CDCl3)
d 2.69 (m, 2H, CH2), 2.83 (m, 2H,
Table 1
Free-radical scavenging activity (IC50) of the compounds.
CH2), 3.86 (s, 3H, OMe), 3.91 (s, 3H, OMe), 5.67 (br s, 1H, D2O exch.,
OH), 6.36 (m, 1H, Ar-H), 6.42 (m, 1H, Ar-H), 6.77 (d, 1H, J ¼ 8.4 Hz,
Ar-H), 7.78 (d, 1H, J ¼ 8.4 Hz, Ar-H); 1H NMR (400 MHz, CD3SOCD3)
Compound no.
IC50 (m
g/mL)b
DPPH radical
Galvinoxyl radical
ABTS radical
d
2.61 (m, 2H), 3.29 (m, 2H), 3.76 (s, 3H, OMe), 3.79 (s, 3H, OMe),
1
2
3
4
32.5 ꢃ 2.86
27.8 ꢃ 4.97
10.28 ꢃ 3.44
11.18 ꢃ 3.79
11.41 ꢃ 2.36
15.12 ꢃ 1.71
15.6 ꢃ 1.17
31.51 ꢃ 2.84
17.04 ꢃ 1.31
17.97 ꢃ 2.91
11.66 ꢃ 2.76
11.79 ꢃ 0.74
17.03 ꢃ 4.20
5.23 ꢃ 1.14
18.03 ꢃ 2.01
43.7 ꢃ 6.55
15.62 ꢃ 3.59
16.98 ꢃ 1.92
11.72 ꢃ 4.37
13.39 ꢃ 5.85
28.3 ꢃ 7.16
29.17 ꢃ 0.48
11.46 ꢃ 0.18
36.11 ꢃ 1.94
6.29 (d, 1H, J ¼ 2.4 Hz, Ar-H), 6.36 (d, 1H, J ¼ 2.4 Hz, Ar-H), 6.77 (d,
1H, J ¼ 8.8 Hz, Ar-H), 7.58 (d,1H, J ¼ 8.8 Hz, Ar-H), 8.35 (br s,1H, D2O
exch., OH), 9.40 (br s, 1H, D2O exch., OH); 13C NMR (100 MHz,
Curcumina
Ascorbic acida
Catechina
BHTa
CD3COCD3)
d 22.2 (CH2), 29.7 (CH2), 55.7 (OMe), 56.3 (OMe), 99.2,
108.2, 109.0, 116.3, 120.2, 124.7, 127.6, 141.6, 142.8, 145.0, 157.5,
159.1; EIMS: m/z 273 (M þ H)þ; HPLC purity: 97.5% tR: 22.8 min
(column-1, method-1).
Similarly were synthesized other 9,10-Dihydrophenanthrene
derivatives Viz. eulophiol (2) from (45) and flavanthridin (3) from
a
Positive control.
b
Compounds tested in triplicate, data expressed as mean value ꢃ SD of three
independent experiments.

Y. Kanekar et al. / European Journal of Medicinal Chemistry 67 (2013) 454e463
461
Table 2
Relative % TNF-a
inhibition in THP-1 cells.a,b
Compound concentration
1
2
3
4
Phenanthrene
Dihydrophenanthrene
Curcumin
0
Cells þ LPS
0
0
0
0
0
0
100
m
M
83 ꢃ 5.66
70 ꢃ 1.41
31 ꢃ 1.41
19.5 ꢃ 3.54
94 ꢃ 0.93
79 ꢃ 4.24
30 ꢃ 0
9 ꢃ 7.78
64 ꢃ 2.83
47 ꢃ 7.07
17.5 ꢃ 0.71
8 ꢃ 0
67.5 ꢃ 3.54
8.5 ꢃ 2.12
14.5 ꢃ 6.36
102.5 ꢃ 0.71
95.5 ꢃ 6.36
38 ꢃ 9.90
50
10
m
m
M
M
34 ꢃ 0
6 ꢃ 2.83
5 ꢃ 4.24
15.5 ꢃ 2.12
3.5 ꢃ 0.71
0
0
0
0
1
m
M
1.5 ꢃ 10.61
a
Compounds tested in triplicate, data expressed as mean value ꢃ SD of two independent experiments.
b
TNF-
a
released in Cells þ LPS taken as 100% TNF-
a release (i.e. 0% TNF-a Inhibition).
3,7-dihydroxy-2,4-dimethoxyphenanthrene (4), physical constants
and spectral data of 2 and 3 are summarized below.
517 nm on a microplate reader. The DPPH radical scavenging ac-
tivity was calculated according to the following equation:
Radical scavenging activity ð%Þ ¼ f1 ꢁ ðA1 ꢁ A2Þ=A0g ꢂ 100
4.3.2. 9,10-Dihydro-2,7-dimethoxyphenanthrene-1,5-diol (2)
White solid; Yield ¼ 66.2%; mp 202e203 ꢀC (reported [16] mp
202e203 ꢀC); IR (KBr) nmax 3446, 2964, 1616, 1436, 1465,
where A0 was the absorbance of the control (without test com-
pound), A1 was the absorbance in the presence of the test com-
pound, and A2 was the absorbance of sample blank (without
radical).
1355 cmꢁ1; 1H NMR (400 MHz, CD3COCD3)
d 2.66 (m, 2H, CH2), 2.77
(m, 2H, CH2), 3.75 (s, 3H, OMe), 3.86 (s, 3H, OMe), 6.40e6.43 (m, 2H,
Ar-H), 6.81 (d, 1H, J ¼ 8.8 Hz, Ar-H), 7.27 (s, 1H, exch.D2O., OH), 7.89
(d, 1H, J ¼ 8.8 Hz, Ar-H), 8.45 (s, 1H, exch.D2O, OH); 13C NMR
4.4.2. Galvinoxyl radical scavenging assay
(100 MHz, CD3COCD3)
d 21.6 (CH2), 30.5 (CH2), 54.7 (OMe), 55.6
80
methanolic solution of each compound (10
of compounds 1e100 g/mL) in 96 well plates. The reaction mixture
(200
L) was mixed for 1 min and incubated at 37 ꢀC for 20 min. The
mM galvinoxyl methanolic solution (190
mL) was added to the
(OMe), 100.9, 105.3, 108.4, 115.2, 119.5, 123.9, 127.1, 141.0, 142.3,
145.4, 155.6, 158.9; 1H NMR (400 MHz, CDCl3)
d
2.72 (m, 2H, CH2),
mL) (final concentration
m
2.81 (m, 2H, CH2), 3.81 (s, 3H, OMe), 3.93 (s, 3H, OMe), 5.47 (s, 1H,
exch.D2O, OH), 5.76 (s, 1H, exch.D2O, OH), 6.36e6.46 (m, 2H, Ar-H),
6.81 (d, 1H, J ¼ 8.8 Hz, Ar-H), 7.51 (d, 1H, J ¼ 8.8 Hz, Ar-H); 13C NMR
m
absorbance was measured at 428 nm on a microplate reader. The
radical scavenging activity was calculated as described for DPPH
assay.
(100 MHz, CDCl3)
d 21.5 (CH2), 30.2 (CH2), 55.3 (OMe), 56.0 (OMe),
100.7, 106.5, 108.2, 114.7, 116.8, 124.4, 126.5, 141.3, 142.5, 145.0,
153.6, 158.9; EIMS m/z 273 (M þ H)þ; HPLC purity: 93.15% tR:
25.5 min (column-1, method-1).
4.4.3. ABTSþ, radical scavenging assay
The stock solution of ABTS (7 mM) was prepared by dissolving
ABTS in water. ABTSþ, was generated by reacting ABTS stock so-
lution with potassium persulfate (2.45 mM, final concentration)
and allowing the mixture to stand in the dark for 16 h at room
temperature. This stock solution was diluted with methanol to an
absorbance of 0.70 (ꢃ0.02) at 734 nm. The diluted ABTSþ, solu-
4.3.3. 3,7-Dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (3)
White solid: mp 74e75 ꢀC (reported [64] mp 75 ꢀC); IR (KBr)
nmax 3207 (OH),1610; 1H NMR (400 MHz, CDCl3)
d 2.71 (m, 4H), 3.70
(s, 3H, OMe), 3.91 (s, 3H, OMe), 5.26 (br s, 1H, D2O exch., OH), 5.65
(br s, 1H, D2O exch., OH), 6.57 (s, 1H, Ar-H), 6.74 (m, 2H, Ar-H), 8.16
(d, 1H, J ¼ 8.8 Hz, Ar-H); EIMS: m/z 295 (M þ Na)þ; HPLC purity:
95.9% tR: 19.8 min (column-1, method-1).
tion (1.0 mL) was added to methanolic solution (20
m
L) of com-
pounds (final concentration of compounds 1e100
mg/mL) and
absorbance was measured (734 nm) exactly after 1 min. The
radical scavenging activity was calculated as described for DPPH
assay.
4.4. Evaluation of the free-radical scavenging activity of the
compounds
Free radical scavenging activities of the compounds were
4.5. Evaluation of inhibitory effects of compounds on LPS-induced
determined as per following published protocols [65e67]:
TNF-a production
4.4.1. DPPH scavenging assay
THP-1 cells were seeded in 96-well plates at a density of 2 ꢂ 105
The solution of each compound (5
DPPH solution (95 L) in 96 well plates. All the compounds were
tested in final concentrations of 1, 5, 12.5, 25, 50 and 100 g/mL
m
L) was added to 316
m
M
cells/well. Cells were then exposed to varying concentrations of the
m
compounds (1e100 mM) in serum-free medium and incubated for
m
1 h. To cells was then added LPS (250 ng/ml) dissolved in media,
respectively. The reaction mixture (100
m
L) was mixed for 1 min
and incubated for 24 h at 37 ꢀC in a humidified 5% CO2 atmosphere.
and incubated at 37 ꢀC for 30 min. The absorbance was measured at
The cell supernatant (150 mL/well) was collected by centrifuging the
Table 3
Relative cell viability in THP-1 cells.a,b
Compound concentration
Untreated cells
1
2
3
4
Phenanthrene
Dihydrophenanthrene
Curcumin
1
1
1
1
1
1
1
100
m
M
M
M
1.0 ꢃ 0.05
1.2 ꢃ 0.21
1.3 ꢃ 0.27
1.3 ꢃ 0.29
1.1 ꢃ 0.27
1.2 ꢃ 0.38
1.4 ꢃ 0.47
1.5 ꢃ 0.50
1.2 ꢃ 0.04
1.3 ꢃ 0.08
1.4 ꢃ 0.16
1.4 ꢃ 0.02
1.0 ꢃ 0.19
1.1 ꢃ 0.18
1.3 ꢃ 0.34
1.4 ꢃ 0.44
1.1 ꢃ 0.16
1.2 ꢃ 0.27
1.3 ꢃ 0.31
1.4 ꢃ 0.27
1.2 ꢃ 0.38
1.3 ꢃ 0.42
1.5 ꢃ 0.44
1.6 ꢃ 0.41
0.5 ꢃ 0.03
0.9 ꢃ 0.00
1.5 ꢃ 0.21
1.8 ꢃ 0.08
50
10
m
m
1
mM
a
Compounds tested in triplicate, data expressed as mean value ꢃ SD of two independent experiments.
Untreated control cells ¼ 1 (100% viable).
b

462
Y. Kanekar et al. / European Journal of Medicinal Chemistry 67 (2013) 454e463
Table 4
Details of HPLC run.
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Acknowledgments
The authors are grateful to Reliance Life Sciences Pvt. Ltd. for
encouragement and financial support of this research. The authors
are thankful to Dr. J. Venkat Raman, Dr. Anil Karnik, Dr. Vikram
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