Novel diarylheptanoids as inhibitors of TNF-α production

Article abstract of DOI:10.1016/j.bmcl.2011.04.040

Synthesis and anti-inflammatory activity of novel diarylheptanoids [5-hydroxy-1-phenyl-7-(pyridin-3-yl)-heptan-3-ones and 1-phenyl-7-(pyridin-3-yl) hept-4-en-3-ones] as inhibitors of tumor necrosis factor-α (TNF-α) production is described in the present article. The key reactions involve the formation of a β-hydroxyketone by the reaction of substituted 4-phenyl butan-2-ones with pyridine-3-carboxaldehyde in presence of LDA and the subsequent dehydration of the same to obtain the α,β-unsaturated ketones. Compounds 4i, 5b, 5d, and 5g significantly inhibit lipopolysaccharide (LPS)-induced TNF-α production from human peripheral blood mononuclear cells in a dose-dependent manner. Of note, the in vitro TNF-α inhibition potential of 5b and 5d is comparable to that of curcumin (a naturally occurring diarylheptanoid). Most importantly, oral administration of 4i, 5b, 5d, and 5g (each at 100 mg/kg) but not curcumin (at 100 mg/kg) significantly inhibits LPS-induced TNF-α production in BALB/c mice. Collectively, our findings indicate that these compounds may have potential therapeutic implications for TNF-α-mediated auto-immune/inflammatory disorders.

Full text of DOI:10.1016/j.bmcl.2011.04.040

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3784–3787
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel diarylheptanoids as inhibitors of TNF-a production
Sameer Dhuru a, Dilip Bhedi a, Dnyaneshwar Gophane a, Kiran Hirbhagat a, Vijaya Nadar a, Dattatray More a,
Sapna Parikh b, Roda Dalal b, Lyle C. Fonseca b, Firuza Kharas b, Prashant Y. Vadnal b, Ram A. Vishwakarma a,
H. Sivaramakrishnan a,
⇑
a Department of Medicinal Chemistry, Piramal Life Sciences Limited, 1 Nirlon Complex, Off Western Express Highway, Goregaon (E), Mumbai 400 063, India
b Department of Pharmacology, Piramal Life Sciences Limited, 1 Nirlon Complex, Off Western Express Highway, Goregaon (E), Mumbai 400 063, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and anti-inflammatory activity of novel diarylheptanoids [5-hydroxy-1-phenyl-7-(pyridin-3-
yl)-heptan-3-ones and 1-phenyl-7-(pyridin-3-yl)hept-4-en-3-ones] as inhibitors of tumor necrosis
Received 4 January 2011
Revised 22 March 2011
Accepted 12 April 2011
Available online 22 April 2011
factor-
of a b-hydroxyketone by the reaction of substituted 4-phenyl butan-2-ones with pyridine-3-carboxalde-
hyde in presence of LDA and the subsequent dehydration of the same to obtain the ,b-unsaturated
ketones. Compounds 4i, 5b, 5d, and 5g significantly inhibit lipopolysaccharide (LPS)-induced TNF- pro-
duction from human peripheral blood mononuclear cells in a dose-dependent manner. Of note, the
in vitro TNF- inhibition potential of 5b and 5d is comparable to that of curcumin (a naturally occurring
diarylheptanoid). Most importantly, oral administration of 4i, 5b, 5d, and 5g (each at 100 mg/kg) but not
curcumin (at 100 mg/kg) significantly inhibits LPS-induced TNF- production in BALB/c mice. Collec-
tively, our findings indicate that these compounds may have potential therapeutic implications for
TNF- -mediated auto-immune/inflammatory disorders.
a (TNF-a) production is described in the present article. The key reactions involve the formation
a
a
Keywords:
Diarylheptanoids
Inflammation
a
TNF-a
a
a
Ó 2011 Elsevier Ltd. All rights reserved.
The key pro-inflammatory cytokine tumor necrosis factor-
a
and a number of naturally occurring phenolic compounds have
been discovered as potent anti-inflammatory agents.3 In many
cases, the pronounced anti-inflammatory activity of natural prod-
ucts is mediated through the inhibition of the synthesis of nitric
oxide, prostaglandins and leukotrienes, and the production
(TNF- ) is mainly produced by the activated macrophages and
a
monocytes, which further induces the production of several inflam-
matory cytokines such as interleukin-1b (IL-1b), interleukin-6
(IL-6) and granulocyte-macrophage colony-stimulating factor
(GM-CSF)1,2 thereby enhancing the inflammation in many auto-
immune/inflammatory diseases such as rheumatioid arthritis
and/or signaling of cytokines such as TNF-a, IL-1b, and IL-6. The
antioxidants such as (À)-epigallocatechin-3-gallate,4 resveratrol5
and the flavonoids (epigenin and kaemferol)6 have been reported
to suppress nitric oxide (NO) production through inhibition of nu-
(RA). In RA patients, TNF-
RA synovial fluid, serum and synovial fibroblasts, and, importantly,
these enhanced levels of TNF- correlate well with disease pathol-
a expression is particularly elevated in
a
clear factor-jB (NF-jB).
ogy and severity.2 The treatment profile of RA has significantly
changed after introduction of antibody based protein therapeutic
agents such as the TNF soluble receptor (Enbrel™) and TNF anti-
body (Remicade™) which have shown excellent efficacy in RA pa-
tients.2 However, these biopharmaceuticals and antibodies are
expensive, difficult to administer orally and have major side effects
(susceptibility to infections such as tuberculosis) on prolonged
clinical use. Therefore, there is a great unmet medical need to dis-
cover small molecule agents to deal with higher levels of produc-
The most widely used spice ginger (Zinziber officinale) and its
major chemical constituents (gingerols, b-carotene, capsaicin,
caffeic acid and curcumin) are reported to be robust inhibitors of
immune cell activation and cytokine secretion.7 Various formula-
tions of ginger have also been reported as dual inhibitors of two
key enzymes cyclooxygenase (COX) and lipooxygenase (LOX),8 as
well as the inhibitors of leukotriene synthesis.9 Their anti-inflam-
matory activity has also been demonstrated in carrageenan-
induced rat-paw edema model.10 Closely related medicinal plants
such as the greater-galangal (Alpinia galanga) and lesser-galangal
(Alpinia officinarum) have been used for rheumatic diseases in
Asian medicine producing linear diarylheptanoid group of com-
pounds (general structure Ar-C7-Ar) isolated,11 responsible for
anti-inflammatory activity. Prior studies have shown that the prin-
cipal constituent (7-(4-hydroxy-3-methoxyphenyl)-1-phenylhept-
4-en-3-one) isolated from Alpinia officinarum suppressed the
tion of TNF-
a. Many low molecular weight compounds have been
reported to have inhibitory activity towards the TNF-
a
production.
Historically, natural products have provided the most consis-
tent source of new scaffolds to design new therapeutic agents,
⇑
Corresponding author.
E-mail address: h.sivaramakrishnan@piramal.com (H. Sivaramakrishnan).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.040

S. Dhuru et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3784–3787
3785
lipopolysaccharide (LPS)-induced TNF-
a
production in human
3-(pyridin-3-yl)propan-1-ol 6 with DMSO and oxalyl chloride) in
presence of LDA in THF at À78 °C to yield substituted 5-hydroxy-
1-phenyl-7-(pyridin-3-yl)heptan-3-ones(4a–n, Table 2). These
were further dehydrated using p-toluenesulfonic acid in toluene
under reflux conditions to give the respective 1-phenyl-7-(pyri-
din-3-yl)hept-4-en-3-one derivatives (5a–m). In case of the
reaction of 4-chloro substituted 4-phenyl-butan-2-one (3l) with
3-(pyridin-3-yl)-propanal (7), a side product 8 (Table 3) was also
formed along with the desired product (4l). The structures of var-
ious synthesized compounds were assigned on the basis of differ-
ent spectral data (experimental details are provided in the
Supplementary data).
peripheral blood mononuclear cells (hPBMC), and attenuated
LPS-induced production of NO, iNOS, and COX-2 in mouse
RAW264.7 macrophages.12 In order to investigate the structure–
activity relationship of diarylheptanoid scaffold for the anti-
inflammatory activity, we initiated the design and synthesis of
diarylheptanoid analogues and evaluation of their potential in sup-
pressing LPS-induced TNF-
The synthetic route designed for synthesis of b-hydroxyketones
and subsequent ,b-unstaturated ketones is depicted in Figure 1.
a
production.12–16
a
Using this synthetic strategy, a number of substituted 4-phenyl-
butan-2-ones (3a–n) were synthesized starting from various
substituted benzaldehydes (1a–n, Table 1) in two steps. First step
involved an aldol condensation of benzaldehydes with acetone in
presence of 10% aqueous NaOH to yield 4-phenyl-but-3-en-2-ones
(2a–n), which further on hydrogenation in presence of 10%
Pd/C gave desired 4-phenyl-butan-2-ones (3a–n). Substituted
4-phenyl-butan-2-ones were subjected to aldol reaction with 3-
(pyridin-3-yl)-propanal (7, prepared by the Swern oxidation of
The compounds 4a–n, 5a–k, 5m (Table 2) and 8 (Table 3) were
evaluated for their TNF-
a inhibitory activity in whole blood cell
culture system (WBCCS) assay14 and the results are as shown in
the Table 2. It was observed that compounds 4i, 5b, 5d, and 5g
showed a dose dependent inhibition of TNF-a production in the
WBCCS assay. Accordingly, these compounds were further studied
for their ability to inhibit TNF-a production in the human periph-
eral blood mononuclear cells (hPBMC) assay.17
Compounds 5b and 5d significantly inhibited the production of
TNF-
2.5
tion of TNF-
a
l
in
M, respectively) (Fig. 2). The inhibitory effects on the produc-
were not due to non-specific toxicity since the viabil-
a concentration dependent manner (IC50 = 2.0 and
O
O
R4
R3
CHO
R4
R3
R4
R3
a
a
b
R1
ity of the cells determined by MTS was not influenced at any of the
concentrations of the compound tested. The compound 4i
R1
R1
R2
1
R2
2
R2
3
(IC50 = 5.7
higher concentrations. The compound 5g was the least active of
the four with an IC50 of 24.1 M. Of note, the TNF- inhibition
lM) also inhibited the production of TNF-a, albeit at
OHC
R4
c
HO
d
l
a
N
7
potential of 5b and 5d was found to be comparable to that of cur-
cumin (a naturally occurring diarylheptanoid)—which inhibited
N
6
LPS-induced TNF-
(Figure 2). Of note, the IC50 values for TNF-
5d, 4i and 5g) were far lower than the IC50 values for cytotoxicity
(38, 80, >100 and >100 M for 5b, 5d, 4i and 5g, respectively).
To assess whether the diarylheptanoid-mediated marked inhi-
bition of TNF- production observed in vitro could be translated
a
production in hPBMC with an IC50 of 2.71
lM
O
O
OH
R4
R3
a
inhibition (for 5b,
e
R1
N
R3
R1
N
l
R2
5
R2
4
a
Figure 1. Synthetic scheme. Reagents and conditions: (a) Acetone/10% NaOH, 0 °C,
rt; (b) H2, Pd/C; (c) (COCl)2, DMSO, Et3N, DCM, À78 °C; (d) i-Pr2NH, n-BuLi, THF,
À78 °C; (e) pTSA, toluene, reflux.
into a meaningful pharmacological effect in vivo, we used an acute
model of inflammation. In these studies, oral administration of
Table 1
List of various substituted benzaldehydes (1), 4-phenyl-but-3-en-2-ones (2) and 4-phenyl-butan-2-ones (3)
O
O
R4
R3
CHO
R1
R4
R3
R4
R3
R1
R1
R2
R2
R2
1
2
3
1
2
3
R1
R2
R3
R4
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
2c
2d
2e
2f
2g
2h
2i
3a
3b
3c
3d
3e
3f
3g
3h
3i
OMe
OMe
OMe
H
H
H
H
H
H
H
OMe
H
H
H
OMe
H
OMe
OMe
H
H
CN
OMe
H
H
H
OMe
H
OMe
H
H
H
H
H
OMe
OMe
Me
OMe
H
OPMBa
H
1j
2j
3j
1k
3l
2k
2l
3k
3l
H
H
H
H
F
Cl
H
H
1m
1n
2m
2n
3m
3n
H
H
H
H
NMe2
OCH2O
H
H
aPMB = para-methoxybenzyl.

3786
S. Dhuru et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3784–3787
Table 2
Inhibition of TNF-
5m
IC50 = 2.0 µM
A
600
a in the whole blood cell culture system assay by 4a–n, 5a–k and
4
5
400
200
0
OH
O
O
R4
R4
R3
R3
R2
R1
R2
N
R1
N
R2
Compd R1
R3
R4
% Inhibition of TNF-a at 10 lM
0.03 0.1 0.3
1
3
10 30 100
S
US
R
5b (µM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
OMe OMe
H
OMe
H
H
H
OMe
H
10
22
0
OMe
OMe
H
H
H
H
H
IC50 = 2.5 µM
B
600
400
200
0
OMe
OMe
Me
OMe
H
OMe
OMe
H
18
OMe 12
H
H
H
H
H
H
H
H
11
2
35
44
3
34
16
0
H
H
OPMBa
CN
H
OPMB OMe
H
H
H
H
H
H
H
H
H
H
F
Cl
NMe2
O–CH2–
O
0.03 0.1 0.3
1
3
10 30 100
S
US
R
H
41
5d (µM)
5a
5b
5c
5d
5e
5f
5g
5h
5i
OMe OMe
H
OMe
H
OMe
OMe
H
OPMB
CN
H
H
33
56
0
400
200
IC50= 5.7 µM
C
D
OMe
OMe
H
H
H
H
H
OMe
H
OMe 23
H
H
H
H
H
H
H
OMe
OMe
Me
OMe
H
56
25
40
9
19
0
H
H
H
OPMB OMe
5j
5k
5m
H
H
H
H
H
H
H
F
NMe2
0
35
32
0.03 0.1 0.3
1
3
10 30 100
S
US
R
4i (µM)
Human whole blood cells were treated with various compounds for 30 min. Fol-
lowing 30 min. incubation, cells were stimulated with LPS. After 5 h, supernatants
IC50= 24.1 µM
400
were collected and TNF-
a levels determined using ELISA.
a PMB = para-methoxybenzyl.
200
0
Table 3
Inhibition of TNF-
a
in whole blood cell culture system assay by (8)
O
OH
0.03 0.1 0.3
1
3
10 30 100
S
US
R
5g (µM)
Cl
H
N
E
IC50 = 2.71 µM (1.6 µg/ml)
800
600
400
200
0
N
8
% Inhibition of TNF-
32
a
at 10 lM
compounds 4i, 5b, 5d, and 5g significantly inhibited LPS-induced
production of TNF- (from BALB/c mice) corroborating the
in vitro findings (71%, 79%, 50% and 75% inhibition of TNF- pro-
a
0.01 0.03 0.1 0.3
1
3
10 30
S
US
R
a
Curcumin (µg/ml)
duction with4i, 5b, 5d and 5g, respectively; Figure 3). Interestingly,
curcumin—dosed at similar levels—had no effect on in vivo LPS-
induced TNF-a production (Fig. 3).
Our results clearly demonstrate that the synthesized novel
diarylheptanoids significantly inhibit in vitro and in vivo produc-
Figure 2. Effect of 5b, 5d, 4i, 5g and curcumin on LPS-induced production of TNF-
a
from hPBMC. S = LPS-stimulated cells treated with vehicle (DMSO), US = Unstim-
ulated control, R = Rolipram (standard)-treated LPS-stimulated cells. Pre-treatment:
30 min. LPS-stimulation: 5 h. Values are average S.E.M. of three experiments.
tion of TNF-a. This functional property of diarylheptanoids could
translate in them showing efficacy in various animal models of
arthritis (e.g., collagen induced arthritis model). Future studies
are warranted to investigate this. In this context, it is important
severity of inflammatory collagen-induced arthritis.19 Given that
diaryl heptanoids inhibit in vitro and in vivo LPS-induced TNF-
a
production, combined with the fact that gram negative bacteria-
mediated signal transduction pathway plays a crucial role in ulcer-
ative colitis, it would be of interest to assess the effect of these
compounds in the experimental models of colitis. In this regards,
to note that earlier studies have demonstrated that TNF-
cient mice have reduced susceptibility for developing experimental
arthritis,18 and neutralization of TNF-
decreases the incidence and
a-defi-
a

S. Dhuru et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3784–3787
3787
8000
7000
6000
5000
4000
3000
2000
1000
0
5000
A
B
4000
3000
*
*
2000
*
*
1000
*
0
LPS
-
-
+
+
+
+
+
+
LPS
-
-
+
+
+
Treatment
CMC
R
Curcumin
Treatment
CMC
R
4i
5b
5d
5g
Figure 3. Effect of (A) 4i, 5b, 5d, and 5g and (B) curcumin in in vivo LPS-induced TNF-
a production assay. Compounds 4i, 5b, 5d, 5g and curcumin (each at 100 mg/kg),
rolipram (R) or 0.5% carboxymethylcellulose (CMC) were administered orally to BALB/c mice. After 1 h, mice were treated with LPS. Following 1.5 h. LPS treatment, blood was
collected and, subsequently, plasma levels of TNF-
LPS-treated group.
a
determined using ELISA. All values are average S.E.M. of at least three mice. ⁄ indicates p <0.05 compared to CMC-fed,
2. Feldmann, M.; Maini, S. R. Immunol. Rev. 2008, 223, 7.
3. Lin, Y. L.; Lin, J. K. Mol. Pharmacol. 1997, 52, 465.
it is important to note that prior studies have demonstrated that (i)
genetic deletion of TNF-a20 protected mice against experimental
4. Tsai, S. H.; Lin-Shiau, S. Y.; Lin, J. K. Br. J. Pharmacol. 1999, 126, 673.
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colitis and (ii) blockade of TNF-
mental colitis.21 Interestingly, a recent study demonstrated a
critical role of TNF- in experimental colitis-associated colon carci-
a production attenuated experi-
a
nogenesis. Thus, it is reasonable to speculate that these
compounds may also have potential implications for inflamma-
tion-associated oncologic disorders. Clearly, future studies probing
the in vivo efficacy of these compounds in various experimental
models of disease are warranted.
7. Mustafa, T.; Srivastava, K. C.; Jensen, K. B. J. Drug Dev. 1993, 6, 25.
8. Kiuchi, F.; Iwakami, S.; Shibuya, M.; Hanaoka, F.; Sankawa, U. Chem. Pharm. Bull.
1992, 40, 387.
9. Mascolo, N.; Jain, R.; Jain, S. C.; Capasso, F. J. Pharmacol. Exp. Ther. 1989, 27, 129.
10. Jana, U.; Chattopadhayay, R. N.; Shaw, B. P. Indian J. Pharmacol. 1999, 31, 232.
11. Kato, N.; Hamada, Y.; Shioiri, T. Chem. Pharm. Bull. 1984, 3223.
12. Yadav, P. N.; Liu, Z.; Rafi, M. M. J. Pharmacol. Exp. Ther. 2003, 305, 925.
13. Rafi, M. M.; Liu, L.; Rosen, R. T.; Rosen, S. L. US Patent 0215635 A1, 2005.
14. Wilson, B. M. G.; Severn, A.; Rapson, N. T.; Chana, J.; Hopkins, P. J. Immunol.
Methods 1991, 139, 233.
15. Jin, W.; Cai, X. F.; Na, M.; Lee, J. J.; Bae, K. Biol. Pharm. Bull. 2007, 30, 810.
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Immunopharmacol. 2010, 10, 520.
17. Jansky, L.; Reymanova, P.; Kopecky, J. Physiol. Res. 2003, 52, 593.
18. Hata, H.; Sakaguchi, N.; Yoshitomi, H.; Iwakura, Y.; Sekikawa, K.; Azuma, Y.;
Kanai, C.; Moriizumi, E.; Nomura, T.; Nakamura, T.; Sakaguchi, S. J. Clin. Invest.
2004, 114, 582.
In summary, we have designed and synthesized a novel series of
diarylheptanoids. Compounds 4i, 5b, 5d, and 5g significantly inhi-
bit in vitro TNF-
oral administration of these compounds significantly inhibits TNF-
production in mice. These compounds may have potential ther-
a production from human cells. More importantly,
a
apeutic implications for TNF-
a-mediated auto-immune/inflamma-
tory diseases.
Supplementary data
19. Williams, R. O.; Feldmann, M.; Maini, R. N. Proc. Natl. Acad. Sci. U.S.A. 1992, 89,
9784.
20. Corazza, N.; Eichenberger, S.; Eugster, H. P.; Mueller, C. J. Exp. Med. 1999, 190,
1479.
21. Zhang, D. K.; Cheng, L. N.; Huang, X. L.; Shi, W.; Xiang, J. Y.; Gan, H. T. Int. J.
Colorectal Dis. 2009, 24, 5.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.040.
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