Psilostachyin, acetylated pseudoguaianolides and their analogues: Preparation and evaluation of their anti-inflammatory potential

Article abstract of DOI:10.1016/j.bmcl.2011.06.037

Naturally occurring acetylated pseudoguaianolides and psilostachyin including their analogues were synthesized. The structure of semi-synthetic psilostachyin was also confirmed by X-ray crystallography. The anti-inflammatory potential of all the derivatives has been evaluated through in vitro expression of TNF-α, IL-1β and IL-6 in murine neutrophils.

Full text of DOI:10.1016/j.bmcl.2011.06.037

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4847–4851
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Psilostachyin, acetylated pseudoguaianolides and their analogues: Preparation
and evaluation of their anti-inflammatory potential
Renu Chib a, Bhahwal Ali Shah ,a, Naveen Anand a, Anjali Pandey a, Kamini Kapoor b, Sarang Bani a,
⇑
b
b
a
,a
⇑
Vivek K. Gupta , Rajnikant , Vijay K. Sethi , Subhash Chandra Taneja
a Natural Product Microbes Division, Indian Institute of Integrative Medicine, Canal Road, Jammu 180 001, J&K, India
b X-ray Crystallography Laboratory, Post-Graduate Department of Physics, University of Jammu, Jammu, Tawi 180 006, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Naturally occurring acetylated pseudoguaianolides and psilostachyin including their analogues were syn-
thesized. The structure of semi-synthetic psilostachyin was also confirmed by X-ray crystallography. The
anti-inflammatory potential of all the derivatives has been evaluated through in vitro expression of
Received 9 March 2011
Revised 27 May 2011
Accepted 10 June 2011
Available online 22 June 2011
TNF-a, IL-1b and IL-6 in murine neutrophils.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Acetylated pseudoguaianolides
Psilostachyin
Parthenin
Anti-inflammatory
The occurrence of sesquiterpene lactones (SLs) is wide spread in
nature, yet their physiological functions in plants and other
sources have not been fully understood, though many are known
to contribute towards defence mechanisms, may act as poisons
for livestock and also act as insect feeding deterrents.1 The natural
SLs, which are having a diverse range of structures and bioactivities
like anti-inflammatory, antibacterial, ameobicidal, anticancer etc.,
constitute interesting targets for further investigations and exploi-
tations in the area of drug development.2 The SLs, psilostachyin (1)
and acetylated pseudoguaianolides (2, 3) have been isolated from
Ambrosia psilostachya3 and Parthenium hysterophorus,4 respectively,
(Fig. 1). Psilostachyin was also isolated from A. artimisifolia,5 A. ten-
uifolia6 and other species of Ambrosia.7 It is reported to possess
anticancer,8,9 anti-inflammatory,10 leishmanicidal,11 and mollusci-
cidal12 activities. Acetylated pseudoguaianolides have also been
shown to possess significant anticancer activity.4
cially in cancer therapy.14 One important SL, that is, parthenin
(4) isolated from Parthenium hysterophorus is known to possess
anti-tumor,15 anti-inflammatory,16,17 ameobicidal activity18 and
trypanocidal activity.19 It is also believed that this lactone is
responsible for the allergic contact dermatitis in humans.20 Since,
P. hysterophorus is an obnoxious weed with an undemanding and
extensive mode of adaptation behavior, its exploitation in the
development of molecules of therapeutic potential will have enor-
mous social and environmental impacts. Thus, in continuation of
our work on the isolation and structural modifications of natural
products to develop bioactive lead molecules particularly in the
area of inflammation and cancer,21 we envisaged semi-synthesis
of naturally occurring acetylated pseudoguaianolides (2 and 3)
and psilostachyin (1) including their analogues and evaluation of
their anti-inflammatory potential through in vitro expression of
TNF-a, IL-1b and IL-6 in murine neutrophils.
The activity of SLs especially cytotoxicity appears to be
associated with their ability to act as alkylating agents by virtue
of Michael addition of biological nucleophiles like cysteine to
Our efforts initiated with the isolation of parthenin (4) and cor-
onopilin (12) from P. hysterophorus by an earlier reported meth-
od.21c For semi-synthetic modifications, we firstly acetylated the
hydroxyl group at C-1 in 4. Since the hydroxyl at C-1 is tertiary,
therefore, Lewis acid catalyzed acetylation at C-1 was visualized,
which proceeded through acylium ion formation, a reaction similar
to Friedel–Craft acylation. Thus, parthenin in presence of InCl3 as
catalyst was treated with acetic anhydride to facilitate the acetyla-
tion, giving the product (5) in almost quantitative yields (Scheme
1). Subsequently propionate and butyrate derivative, that is, 6
and 7 were also synthesized by using propionic and butyric anhy-
dride, respectively.
a
-methylene part of
tential therapeutic agents was hampered because of the irrevers-
ible addition of biological nucleophiles to -methylene moiety
c
-lactone moiety.13 Their development as po-
a
and any changes in its reactivity resulted in complete loss of the
activity. Interestingly, recent studies indicate that irreversible
binding may be an important factor against drug resistance espe-
⇑
Corresponding authors.
E-mail addresses: bashah@iiim.ac.in (B.A. Shah), sctaneja@iiim.ac.in (S.C. Taneja).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.037

4848
R. Chib et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4847–4851
HO
O
AcO
AcO
O
HO
O
O
O
O
O
O
O
O
O
O
O
1
2
3
4
Figure 1. Naturally occurring SLs.
RO
RO
RO
R2O, InCl3
R2O, InCl3
CH3COOH
+
4
O
O
O
O
O
O
O
O
O
5.
6.
7.
2.
8.
9.
R= -COCH3
R= -COC2H5
R= -COC3H7
R= -COCH3
R= -COC2H5
R= -COC3H7
3.
10.
11.
R= -COCH3
R= -COC2H5
R= -COC3H7
Scheme 1. Synthesis of various acylated pseudoguaianolides
The same reaction facilitated the migration of double bond and
acylation at C-3, when performed in presence of acetic acid, acetic
anhydride and InCl3, thus leading to the direct synthesis of acety-
lated pseudoguaianolides (Schemes 1 and 2). The reaction probably
proceeds via protonation of C-1 hydroxyl by acetic acid, which
facilitates its elimination and generation of carbocation at C-3. At
first glance the formation of 2 and 3 appears as a consequence of
the reactivity umpolung. However, the formation of the carbocat-
ion at C-1 followed by 1, 3-shift of the double bond facilitates
the generation of the carbocation at C-3 and finally leading to
the synthesis of both the isomers of the acetylated pseudoguaiano-
lides (2 and 3) in equal proportions. The presence of the Lewis acid
(InCl3) also facilitates the stabilization of the carbocation and the
generation of acetate ion from anhydride, which concomitantly at-
tacks at C-3, resulting in the formation of the desired isomers 2 and
3. The plausible mechanism is depicted in Scheme 2. In order to
clear the ambiguity that the acetate anion is derived from anhy-
dride and not from the corresponding acid, the reaction was car-
ried out in presence of trifluoroacetic acid instead of acetic acid.
The reaction afforded the same products, that is, 2 and 3, thereby
supporting the observation that the acetate ion is derived from
anhydride. Moreover, the formation of propionate and butyrate
analogues (8, 9, 10 and 11) using propionic and butyric anhydride,
respectively, in presence of acetic acid as shown in Scheme 1 also
supports the plausible mechanism.
An ORTEP view of 1 with atomic labeling is shown in Figure 2.22
The XRD studies showed that in 1, bond lengths and bond angles
are in agreement with those reported for other structure determi-
nations of sesquiterpene lactones.23,24 The torsion angles of the
cycloheptane ring have C2 symmetrical values [DC2 = 8.50] and
the ring is therefore in a twist-chair conformation. The pseudo-
twofold axis bisects the C1–C10 bond and passes through C7. The
spiro lactone ring has a half-chair conformation [DC2 = 1.67] with
a pseudo-twofold axis running through the C4 carbonyl atom.
The cis-fused lactone ring at C6–C7 has envelope conformation
with C7 at the flap [DCs = 4.78]. Crystallographic data for the struc-
ture has been deposited with the Cambridge Crystallographic Data
Center as supplementary publication No. CCDC-795880.
Parthenin was further subjected to a series of reactions (Scheme
4) to generate a library of analogues based on psilostachyin and re-
lated d-valerolactones. Thus, Bayer–Villiger oxidation21b of parthe-
nin in presence of m-CPBA, gave d-valerolactone (13) (Scheme 4). In
the next step it was treated with TFA under reflux to form a rear-
ranged product, that is, spirolactone 14 in almost quantitative
yield. Besides, 13 was acylated at C-1 using acyl anhydride in pres-
ence of InCl3 as catalyst to afford corresponding acylates 16-18 (
Scheme 4). Modifications were also envisaged in the
a-methy-
lene- -lactone part of the lactones via 1,3-dipolar cycloaddition
c
In the literature, the synthesis of psilostachyin (1) is reported
from coronopilin (12) using peracetic acid.3 In the present work,
we synthesized 1 by subjecting 12 to Bayer–Villiger oxidation21b
using m-CPBA. The intermediate d-lactone (A) formed equilibrated
into the more stable spirolactone 1 in presence m-chlorobenzoic
acid formed in situ (Scheme 3). The higher stability of the spiro
H2O
O
HO
InCl3
-H2O
H+
1
2
3
4
O
O
Cl3In
Cl3In
O
4
O
O
R
c-lactone scaffold prompted us to study the structural features
including the conformations of the three rings in 1. Therefore, it
was envisaged to carry out its X-ray crystallographic analysis.
The energy minimization calculations using impact minimization
tool of Schrodinger software with OPLS_2005 force field disclosed
that the spiro derivative (1) is at a lower energy state (33.3 kcal)
compared to d-lactone (A, 40.5 kcal) which is apparently more
strained.
O
O
R
H2O
O
O
O
O
R
R
O
O
O
Cl3In
2
3
Scheme 2. Plausible mechanism of synthesis of acylated pseudoguaianolides

R. Chib et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4847–4851
4849
O
HO
HO
O
H+
m-CPBA, DCM
HO
O
O
O
O
O
O
O
O
A
1
12
O
Scheme 3. Synthesis of psilostachyin
cals, synthesis of cytokines, chemokines and lipid mediators
causing inflammation. Studies have demonstrated that the recruit-
ment of leukocytes into an inflamed area is mediated by various
peptides and other factors which in turn amplify the inflammatory
response by their effects on macrophages and lymphocytes.25 Ini-
tially, TNF-
ity screening. The analogues displayed a range of TNF-
inhibition from low to high. It was quite interesting to observe that
some of the compounds displayed better TNF- inhibition than
parthenin and were found to be efficacious in suppressing intracel-
lular TNF- level at the dose of 1 g/ml when compared to the LPS
control. Parthenin itself displayed significant inhibition of the
expression of TNF- in the range of 39.45%. From the results given
in Table 1, it is clear that at 1 g/ml concentrations, the analogues
15, 19 and 20 displayed maximum inhibitory effects, that is,
49.21%, 59.76% and 53.12%, respectively, on TNF- cytokine secre-
a
was chosen as the target for anti-inflammatory activ-
a
expression
a
a
l
a
l
a
tion in murine isolated neutrophils in response to LPS stimulant.
Figure 2. ORTEP view of the molecule, showing the atom-labeling scheme. Note:
Displacement ellipsoids are drawn at the 50% probability level and H atoms are
shown as small spheres of arbitrary radii.
Since, analogues 15, 19 and 20 displayed maximum inhibitory
effect on TNF-a cytokine secretion; these samples were further
evaluated for their effect on the expression of extracellular IL-b
and IL-6 levels. The results displayed that the three compounds
15, 19 and 20 significantly suppressed extracellular IL-1b expres-
of diazomethane on exocyclic double bond. Thus, lactones 13 and
14 on treatment with diazomethane afforded corresponding aza-
spiro lactones 15 and 19, respectively, (Scheme 4). Furthermore,
sion level in LPS activated neutrophils at dose level of 1 lg/ml.
Rolipram a standard inhibitor showed 64.27% inhibition when
compared with lipopolysaccharide (LPS) stimulated untreated neu-
trophils (Table 2). The molecules 15, 19 and 20 also suppressed
through ene migration the exocyclic double bond in the
a-methy-
lene- -lactone moiety 13 underwent a facile isomerization to but-
c
enolide 20 during catalytic hydrogenation with Pd/C and hydrogen
gas at atmospheric pressure (Scheme 4).
extracellular IL-6 expression level at the dose of 1
inhibition of expression was not significant (Table 3).
lg/ml but the
The flow cytometry (FCM) studies were carried out on all the
analogues to determine TNF-a cytokine expression in LPS activated
human neutrophils. The neutrophils are involved in the phagocyto-
sis, release of proteolytic enzymes, generation of active free radi-
In conclusion, subsequent to the successful semi-synthesis of
three natural SLs viz., acetylated pseudoguaianolides and psilosta-
chyin, a series of analogues were prepared for the evaluation of
their anti-inflammatory potential. Interestingly, the compounds
O
O
HO
O
O
TFA, reflux
CH2N2, ether
m-CPBA, DCM
4
HO
O
O
HO
N
N
O
O
O
15
13
R2O, InCl3
H
,
14
2
O
H
O
P
d
/
O
C
,
M
e
O
RO
HO
HO
O
O
O
O
N
O
O
N
O
O
O
20
19
16. R=-COCH3
17. R=-COC2H5
18. R=-COC3H7
O
O
O
Scheme 4. Synthesis of analogues of parthenin

4850
R. Chib et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4847–4851
Table 1
In vitro tumor necrosis factor-
a (TNF-a) expression
Entry
Compound
Concn (
lg/ml)
Mean SE
%TNF-a expression
Naïve control
LPS control
1
2
3
4
5
6
7
8
9
10
11
12
13
14
–
–
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
100
0.65 0.07
2.56 0.04
1.55 0.04
2.42 0.04
2.50 0.03
2.57 0.02
2.59 0.03
3.00 0.07
2.20 0.07
2.29 0.01
2.73 0.07
2.80 0.05
1.74 0.05
2.74 0.04
1.50 0.07
1.76 0.06
1.30 0.07
2.13 0.04
1.87 0.04
2.24 0.05
1.03 0.07
1.20 0.05
0.73 0.08
–
–
1
2
3
4
5
6
7
8
39.45;
5.46;
2.34;
0.39"
1.17"
17.18"
14.06;
10.54;
6.64"
9
10
11
12
13
14
15
16
17
18
19
20
21
9.37"
32.03;
7.03"
41.41;
31.25;
49.21;
16.79;
26.95;
12.50;
59.76;
53.12;
71.48
15
16
17
18
19
20
Rolipram (standard)
No. of observations-3; LPS: lipopolysaccharide; ; – inhibition of TNF-
a
expression.
Table 2
and its migration during catalytic hydrogenation (20) increased
the activity of the resulting molecules. Thus, is significant to note
Effect of compounds 15, 19 and 20 on expression of extracellular Interleukin-1b (IL-
1b)
that the presence of
a-methylene-c-lactone moiety is not essential
Compd
Concn
g/ml)
Concn of IL-1b (pg/
ml) Mean SE
% IL-1b inhibition
against LPS control
for TNF- activity in the present series of molecules, besides,
retaining of bioactivity even after the modifications of exocyclic
a
(
l
LPS control
15
19
20
–
1
1
1
115.01 3.06
73.55 2.87
67.43 4.01
68.01 2.55
(standard)
–
double bond is also noteworthy. The presence of spiro-diazo group
also did not affect the expression TNF-a in compounds 15 and 19.
Thus, SLs with interesting structural profiles and ubiquitous pres-
ence in natural kingdom present an important platform for further
development into the products of therapeutic significance.
36.04;
41.37;
40.86;
100
Rolipram
41.09 3.09
64.27;
No. of observations-3; LPS: lipopolysaccharide; ; – decrease in extracellular IL-1b
expression.
Acknowledgment
The authors thankfully acknowledge Sh. Samar Singh for techni-
cal assistance. One of the authors (Rajnikant) acknowledges the
Department of Science & Technology for single crystal X-ray dif-
fractometer as a National Facility under Project No. SR/S2/CMP-
47/2003.
Table 3
Effect of compounds 15, 19 and 20 on expression of Interleukin-6 (IL-6)
Compd
Concn
g/ml)
Concn of IL-6 (pg/ml) % IL-6 inhibition against
(
l
Mean SE
LPS control
LPS Control
15
19
–
1
1
1
317.39 6.81
281.44 4.51
272.91 3.11
263.23 7.04
202.81 4.71
–
Supplementary data
11.32;
14.01;
17.06;
36.10;
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.037.
20
Rolipram
(standard)
100
References and notes
No. of observations-3; LPS: lipopolysaccharide; ; – decrease in extracellular IL-6
expression.
1. Arnason, J. T.; Isman, M. B.; Philogene, B. J. R.; Wadde, T. G. J. Nat. Prod. 1987, 50,
690.
2. Dictionary of Natural Products; Buckingham, J., Ed.; Chapman and Hall: London,
1994; (b) Picman, A. K. Biochem. Syst. Ecol. 1986, 14, 255.
3. (a) Miller, H. E.; Kagan, H. B.; Renold, W.; Mabry, T. J. Tetrahedron Lett. 1965, 38,
3397; (b) Mabry, T. J.; Miller, H. E.; Kagan, H. B.; Renold, W. Tetrahedron 1966,
22, 1139.
4. Das, B.; Reddy, V. S.; Krishnaiah, M.; Sharma, A. V. S.; Kumar, K. R.; Rao, J. V.;
Sridhar, V. Phytochemistry 2007, 68, 2029.
5. Herz, W.; Hogenauer, G. J. Org. Chem. 1961, 26, 5011.
6. Oberti, J.; Silva, G.; Sosa, V.; Kulanthaivel, P.; Herz, W. Phytochemistry 1986, 25,
1355.
7. Nathan, P. J.; Romo, J. Tetrahedron 1966, 22, 1723.
8. Dirsch, V. M.; Stuppner, E. P.; Ellmerer, M.; Vollmer, A. M. Bioorg. Med. Chem.
2000, 8, 2747.
9. Sturgeon, C. M.; Craig, K.; Brown, C.; Rundlr, N. T.; Andersen, R. J.; Roberg, M.
Planta Med. 2005, 71, 938.
without
anti-inflammatory potential compared to the compounds having
-methylene moiety when evaluated through in vitro expression
of TNF- , IL-1b and IL-6 in murine neutrophils. Moreover, there
was a significant improvement in anti-inflammatory activity com-
pared to the parent molecules, thus, underlining the need for fur-
ther development. As mentioned earlier the development of SLs
a-methylene moiety viz., 15, 19 and 20 displayed better
a
a
with
a-methylene-
c-lactone moiety was hampered because of
the irreversible addition of biological nucleophiles to
a-methylene
group, but in our case it was interesting to observe that the dipolar
cycloaddition on exocyclic double bond (compounds 15 and 19)
10. Cho, J. Y. Curr. Enz. Inhib. 2006, 2, 329.

R. Chib et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4847–4851
4851
11. Sulsen, V. P.; Frank, F. M.; Cazorla, S. I.; Anesini, C. A.; Malchiodi, E. L.; Freixa, B.;
Vila, R.; Muschietti, L. V.; Martino, V. S. Antimicrob. Agents Chemother. 2008, 52,
2415.
12. Wang, P. H.; Xu, J.; Wu, M. Y. Zhongguo Zhong Yao Za Zhi 1993, 18, 164.
13. Kupchan, S. M.; Fessler, D. C.; Eakin, M. A.; Giacobbe, T. J. Science 1970, 168,
376.
21. (a) Kaur, R.; Khan, S.; Chib, R.; Kaur, T.; Sharma, P. R.; Singh, J.; Shah, B. A.;
Taneja, S. C. Eur. J. Med. Chem. 2011, 46, 1356; (b) Shah, B. A.; Kaur, R.; Gupta, P.;
Kumar, A.; Sethi, V. K.; Andotra, S. S.; Singh, J.; Saxena, A. K.; Taneja, S. C. Bioorg.
Med. Chem. Lett. 2009, 19, 4394; (c) Shah, B. A.; Chib, R.; Gupta, P.; Sethi, V. K.;
Koul, S.; Andotra, S. S.; Nargotra, A.; Sharma, S.; Pandey, A.; Bani, S.; Purnima,
B.; Taneja, S. C. Org. Biomol. Chem. 2009, 7, 3230; (d) Shah, B. A.; Taneja, S. C.;
Sethi, V. K.; Gupta, P.; Andotra, S. S.; Chimni, S. S.; Qazi, G. N. Tetrahedron Lett.
2007, 48, 955; (e) Shah, B. A.; Kumar, A.; Gupta, P.; Sharma, M.; Sethi, V. K.;
Saxena, A. K.; Singh, J.; Qazi, G. N.; Taneja, S. C. Bioorg. Med. Chem. Lett. 2007, 17,
6411; (f) Shah, B. A.; Qazi, G. N.; Taneja, S. C. Nat. Prod. Rep. 2009, 26, 72.
22. Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
14. Amslinger, S. ChemMedChem 2010, 5, 351.
15. (a) Fraga, B. M. Nat. Prod. Rep. 2006, 23, 943; (b) Modzelewska, A.; Sur, S.;
Kumar, S. K.; Khan, S. R. Curr. Med. Chem.: Anti-Cancer Agents 2005, 5, 477; (c)
Kupchan, S. M. Trans. NY Acad. Sci. 1970, 32, 85; (d) Hartwell, J. L.; Abbott, B. J.
Adv. Pharmacol. Chemother. 1969, 7, 117.
16. Hall, I. H.; Lee, K. H.; Starnes, C. O.; Sumida, Y.; Wu, R. Y.; Waddell, T. G.;
Cocohran, J. W.; Gerhart, K. G. J. Pharm. Sci. 1979, 68, 195.
17. Kupchan, S. M.; Giacobbe, T. J.; Krull, I. S.; Thomas, A. M.; Eakin, M. A.; Fessler,
D. C. J. Org. Chem. 1970, 35, 3539.
23. Kakuda, H.; Helal, A. M.; Nakamura, N.; Hattori, M. Acta Crystallogr. C 1998, 54,
113.
24. Fronczek, F. R.; Vargas, D.; Fischer, N. H.; Chiari, G.; Balza, F.; Towers, G. H. N.
Acta Crystallogr. C 1989, 45, 2006.
18. Sharma, G. L.; Bhutani, K. K. Planta Med. 1988, 54, 120.
19. Talakal, T. S.; Dwivedi, S. K.; Sharma, S. R. Indian J. Exp. Biol. 1995, 33, 894.
20. Lonkar, A.; Mitchell, J. C.; Calnan, C. D. Trans. St. Johns Hosp. Derm. 1974, 60, 43.
25. Oda, T.; Katori, M.; Hatanaka, K.; Yamashina, S. Mediat. Inflamm. 1992, 1, 403.