Synthesis and biological evaluation of amino analogs of Ludartin: Potent and selective cytotoxic agents

Article abstract of DOI:10.1016/j.bmcl.2013.06.068

Diverse amino analogs of Ludartin, a cytotoxic guaianolide and a position isomer of an anticancer drug, Arglabin were prepared through Michael type addition at its highly active α-methylene-γ-lactone motif. The semisynthetic derivatives were subjected to sulphorhodamine B cytotoxicity assay against a panel of four different human cancer cell lines viz. lung (A-549), leukemia (THP-1), prostate (PC-3) and colon (HCT-116) to look into structure-activity relationship. Few of the analogs displayed potent selective cytotoxicity compared to the parent molecule-Ludartin (1). (11R)-13-(Diethyl amine)-11,13-dihydroludartin (6) and (11R)-13-(piperidine)-11,13-dihydroludartin (10) showed almost same cytotoxicity against leukemia cell lines (THP-1) as that of parent molecule-Ludartin, but were more active against colon (HCT-116) cancer cells. (11R)-13-(Morpholine)-11,13-dihydroludartin (11) displayed selectively better cytotoxicity against Leukemia cancer cells (THP-1) exhibiting IC₅₀ of 2.8 μM. (11R)-13-(6-Nitroindazole)-11,13-dihydroludartin (17) was four times more potent than Ludartin with selective cytotoxic effects against prostate cancer cells (2.2 μM) while as (11R)-13-(6-nitroindazole)- 11,13-dihydroludartin (18) exhibited three-fold selective cytotoxicity for Lung (A-549) cancer cell lines exhibiting IC₅₀ of 2.6 μM.

Full text of DOI:10.1016/j.bmcl.2013.06.068

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4931–4934
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of amino analogs of Ludartin:
Potent and selective cytotoxic agents
a
b
b
Shabir H. Lone a, Khursheed A. Bhat a, , Shakeel-u-Rehman , Rabiya Majeed , Abid Hamid ,
⇑
Mohd A. Khuroo c
a Bio-organic Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Srinagar 190005, India
b Cancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Jammu 180001, India
c Department of Chemistry, University of Kashmir, Srinagar 190006, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Diverse amino analogs of Ludartin, a cytotoxic guaianolide and a position isomer of an anticancer drug,
Received 22 March 2013
Revised 12 June 2013
Accepted 22 June 2013
Available online 4 July 2013
Arglabin were prepared through Michael type addition at its highly active a-methylene-c-lactone motif.
The semisynthetic derivatives were subjected to sulphorhodamine B cytotoxicity assay against a panel of
four different human cancer cell lines viz. lung (A-549), leukemia (THP-1), prostate (PC-3) and colon
(HCT-116) to look into structure–activity relationship. Few of the analogs displayed potent selective cyto-
toxicity compared to the parent molecule-Ludartin (1). (11R)-13-(Diethyl amine)-11,13-dihydroludartin
(6) and (11R)-13-(piperidine)-11,13-dihydroludartin (10) showed almost same cytotoxicity against
leukemia cell lines (THP-1) as that of parent molecule-Ludartin, but were more active against colon
(HCT-116) cancer cells. (11R)-13-(Morpholine)-11,13-dihydroludartin (11) displayed selectively better
Keywords:
Ludartin
Michael-addition
Amino analogs
Cytotoxicity
cytotoxicity against Leukemia cancer cells (THP-1) exhibiting IC50 of 2.8
ole)-11,13-dihydroludartin (17) was four times more potent than Ludartin with selective cytotoxic effects
against prostate cancer cells (2.2 M) while as (11R)-13-(6-nitroindazole)-11,13-dihydroludartin (18)
exhibited three-fold selective cytotoxicity for Lung (A-549) cancer cell lines exhibiting IC50 of 2.6 M.
lM. (11R)-13-(6-Nitroindaz-
l
l
Ó 2013 Elsevier Ltd. All rights reserved.
Sesquiterpene lactones (SLs) display a wide array of biological
activities like antiviral, antibacterial, antiulcer, cytotoxic, anti-
inflammatory, antifungal and effects on the central nervous system
and cardiovascular system.1 The unique chemical properties of SLs
helenalin11 and ambrosin12 and effective structure–activity
relationships have been developed. It is however assumed that
the amino analogs serve as prodrugs which in presence of thiol
groups of proteins, enzymes and glutathione release the amine
group to generate the parent enone motif that defines their mech-
anism of action at the desired targets.9 Approaches have been
made to devise such synthetic methods that make SLs highly target
specific to cancerous cells while sparing the normal cells. At pres-
ent the SL drugs in clinical trials are artemisinin from Artemisia an-
nua L., thapsigargin from Thapsia and parthenolide from Tanacetum
parthenum and/or many of their synthetic derivatives.2
Ludartin, a bioactive natural product of the widely distributed
class of guaianolides was previously isolated as a mixture along
with 11,13-dihydroderivative from A. caruthii13 and then in pure
form from Stevia yaconensis var. subeglandulosa14 and from A. fila-
tovae.15 The heart of this molecule consists of a cycloheptane ring
with five contiguous stereocenters, to which two five membered
rings are trans-annulated. One of the five membered rings is a
like presence of alkylating center reactivity (a-methylene-c-lac-
tone), lipophilicity along with molecular geometry and electronic
features make them highly bioactive with in the living systems.2
However the clinical translation of these lactones is hampered
due to their nonselective binding at undesired targets via the
highly reactive Michael acceptor, that is, a-methylene-c
-lactone.3
The high lipophilicity allows their facile penetration via the cell
membranes increasing their cytotoxicity in vitro, but higher lipo-
philicity dictates their lower drug bioavailability in vivo.2 These
queries have been addressed by synthetic chemists and one such
approach has been the amino-prodrug approach, where in the
aza-Michael adducts display the enhanced aqueous solubility, im-
proved pharmarmacokinetic potential and even retention or aug-
mentation in bioactivity.3 These amino based Michael adducts
have been prepared for various bioactive sesquiterpene lactones
c
-butyrolactone with an exocyclic double bond. Ludartin shows
such as alantolactone,4 costunolide,5 parthenolide,6–9
a
-santonin,10
gastric cytoprotective effect16 and also inhibits aromatase enzyme
which is involved in hormone-dependent breast cancer.17 So far
there are no reports on the SAR studies of this molecule, and keep-
ing in view the bioactivity of the molecule along with the literature
precedent that reductive amination at exocyclic double bond of a
cytotoxic agent can enhance selectivity for malignant cell lines,18
⇑
Corresponding author. Tel.: +91 1942431253; fax: +91 1942441331.
Institute’s publication No.: IIIM/1581/2013.
E-mail address: kabhat@iiim.ac.in (K.A. Bhat).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.068

4932
S. H. Lone et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4931–4934
Table 1
we designed the synthesis of various Michael adducts at the highly
electrophilic -methylene- -lactone moiety with the hope to de-
velop analogs with enhanced bioavailability, lesser toxicity and
better activity. This however would constitute an important step
towards the rationalization of lead properties of Ludartin (1).
Preparation of different amino analogs of Ludartin (1)
a
c
S. no
R1R2HNa
Product
Yieldb (%)
NH2
NH2
NH2
NH2
1
2
2
3
80
76
3
4
4
5
64
72
O
5
6
67
NH
O
NH2
NH2
6
7
7
8
78
80
O
O
8
9
9
90
92
NH
NH
O
O
Ludartin (1)
10
Arglabin (19)
O
10
11
11
12
90
73
NH
Our synthetic efforts were mainly focussed towards Michael addition
at exocyclic double bond of
a-methylene-c-lactone motif
F
(Scheme 1). Therefore Ludartin was subjected to synthetic modifi-
cations (Michael addition) using a diversity of amines as Michael
donors. Different analogs were prepared by refluxing a solution of
Ludartin (1) in acetonitrile and an appropriate amine for 8–24 h19
(Scheme 1). The reaction was completely clean furnishing products
with satisfactory yields (70–92%) (Table 1). Unlike primary amines
cyclic secondary amines like pyrollidine, piperidine, morpholine re-
acted quickly without using any base. On the other hand azole type
Michael donors like imidazole, benzotriazole, required DBU as base
for the successful completion of the reaction owing to lesser reac-
tivity of N-containing heterocycles. A library of 17 analogs was
prepared whose formation could easily be confirmed by the
disappearance of two diagnostic proton resonances at d 5.38 ppm
NH2
N
12
13
64
N
H
N
13
14
14
15
60
66
NO2
NH
HN
N
N
HN
N
15
16
16
17
75
78
(d, J = 3.5 Hz) and d 6.21 ppm (d, J = 3.5 Hz) of a-methylene-protons
N
(13-H2) of Ludartin (1). Since the molecule Ludartin in itself bears
five contiguous chiral centers (C-3–7), the Michael addition creates
one more chiral center at C-11 position whose configuration was
determined on the basis of correlations deduced from NOESY exper-
iment. In the NOE experiment of compound 9, the NOESY correla-
tion observed between H-6 and H-11 unambiguously established
the H7–H11 anticonfiguration (Fig. 1). Since the configuration of al-
ready existing stereocenter at C-7 is S, the stereocenter at C-11 was
unambiguously put as R, hence staying in tune with the literature
precedent4,6,18,20 that the Michael addition at exocyclic double bond
of sesquiterpene lactones proceeds with high diastereoselectivity at
position-11 giving preferentially the less hindered R isomer.
N
N
NO2
NO2
H
N
17
18
84
N
H
Here R1R2NH refers to any primary, secondary or heteroaromatic amine.
Refers to the yield after isolation of products.
a
b
R1R2NH, ACN
R1
O
O
Reflux, 8-24 hrs
N
R2
O
O
O
O
Ludartin (1)
17 amino Ludartin analogs
Scheme 1.

S. H. Lone et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4931–4934
4933
14
10
2
9
7
1
3
8
5
4
O
6
N
H5
15
11
O
13
12
NOE
O
Figure 1. NOE correlations of compound 9.
Ludartin along with its 17 amino analogs were then studied in a
calorimetric sulphorhodamine B (SRB) cytotoxicity assay21 against
four human cancer cell lines viz. lung (A-549), leukemia (THP-1),
prostate (PC-3) and colon (HCT-116). Preliminary cytotoxic screen-
ing of the analogs was carried out at 50 lM concentration. The ana-
logs which exhibited greater than 50% growth inhibition at that
concentration were further assayed at different concentrations
(10–100 lM) to generate the IC50 values given in Table 2. From
the cytotoxicity profile it is clear that Ludartin exhibited a broad
spectrum cytotoxic activity against all the tested cancer cell lines
displaying IC50 values of 3.1 (THP-1), 7.4 (A-549), 6.9 (HCT-116)
leukemia cell line (THP-1) exhibiting IC50 value of 2.8
l
M. However
the same Morpholino analog was weakly active against other three
cancer cell lines. Among the heteroaromatic amines used imidaz-
ole (13), 4-nitroimidazole (14) and benzotriazole (16) analogs
exhibited complete loss of cytotoxicity displaying very low growth
inhibition at the preliminary screening concentration. However
1,2,4-triazole analog (15) exhibited moderate cytotoxicity profile
against all the tested cancer cell lines. An interesting difference
was observed between 6-nitroindazole (17) and 5-nitroindazole
(18) analogs against the cancer cell lines. 6-Nitroindazole analog
(17) of Ludartin was cytotoxic against prostate cancer cells (PC-
and 7.5 (PC-3)
l
M. However certain analogs prepared from Ludar-
3) and displayed 3 to 4 fold increase in cytotoxicity (IC50 = 2.2 lM)
tin exhibited a bit selective and improved cytotoxicity towards
particular cancer cell lines with some others showing complete
loss of activity. The secondary amino analogs of Ludartin in general
exhibit better activity as compared to the primary analogs. Both
the diethyl (6) and piperidine (10) amino analogs exhibited two-
fold increase in cytotoxicity against the colon cells (HCT-116)
and retained same cytotoxicity level against leukemia (THP-1)
compared to Ludartin (1). However incorporation of O-atom at
the position 4 of piperidine affording Morpholino analog (11) in-
creased both cytotoxity as well as selectivity of this analog towards
compared to Ludartin while its 5-nitro analog exhibited the three
fold increase in cytotoxicity but human lung cancer cell (A-549)
specificity (IC50 = 2.6 lM).
The position isomer of Ludartin (1), that is, Arglabin (19), was
approved for its use in several countries to treat lung, liver and
ovarian cancers.23 Derivatives of Arglabin prepared from the reac-
tion of exocyclic double bond show similar or slightly reduced but
cell line dependent cytotoxicity.23 Among all the analogs of Argla-
bin (19), piperizine analog is the selective but potent cytotoxic
agent displaying IC50 = 3.75
lM
towards ovarian cancer cells
Table 2
Cytotoxicity profile of Ludartin and its amino analogs against four different cancer cell lines
Compound
Lung (A-549)
% Inhibitiona
Leukemia (THP-1)
% Inhibitiona
Prostate (PC-3)
Colon (HCT-116)
IC50
IC50
% Inhibitiona
IC50
% Inhibitiona
IC50
1
2
3
4
5
6
7
8
97
10
20
39
00
73
0
36
00
87
42
16
00
00
69
25
84
99
7.4
nd
nd
nd
nd
22
nd
nd
nd
17
nd
nd
nd
nd
32
nd
35
2.6
98
25
68
23
28
98
39
40
67
93
97
26
36
32
83
15
63
72
3.1
nd
29
nd
nd
3.5
nd
nd
26
3.9
2.8
nd
nd
nd
15
nd
29
31
97
12
43
17
14
56
16
33
11
85
59
64
53
15
66
40
99
52
7.5
nd
nd
nd
nd
32
nd
nd
nd
23
nd
nd
nd
nd
35
nd
2.2
34
97
16
36
02
04
95
28
04
15
90
58
00
42
30
70
13
86
57
6.9
nd
nd
nd
nd
3.7
nd
nd
nd
4.0
nd
nd
nd
nd
20
nd
17
40
9
10
11
12
13
14
15
16
17
18
IC50 values are expressed in lM concentration.
nd = Not determined in the given concentration range.
a
% Inhibition refers to growth inhibition measured at 50
lM concentration.

4934
S. H. Lone et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4931–4934
10. Klochkov, S. G.; Afanaseva, S. V.; Pushin, A. N.; Gerasimovr, G. K.; Vlasenkova,
N. K.; Bulychew, Y. N. Chem. Nat. Compd. 2009, 46, 817.
11. Lee, M.; Furukawa, H.; Huang, E. S. J. Med. Chem. 1972, 15, 609.
12. Hejchman, E.; Haugwitz, R. D.; Cushman, M. J. Med. Chem. 1995, 38, 3407.
13. Geissman, T. A.; Griffin, T. S. Phytochemistry 1972, 11, 833.
14. Sosa, V. E.; Oberti, J. C.; Gil, R. R.; Ruveda, E. A.; Goedken, V. L.; Gutierrez, A. B.;
Herz, W. Phytochemistry 1989, 28, 1925.
15. Jalmakhanbetova, R. I.; Adekenov, S. M. Chem. Nat. Compd. 2007, 43, 347.
16. Giordano, O. S.; Guerreiro, E.; Pestchanker, M. J.; Guzman, J.; Pastor, D.;
Guardia, T. J. Nat. Prod. 1990, 53, 803.
(A2780) compared to the parent Arglabin (19) with IC50 of
12.55 M against the same cell line. On the other hand the most
selective derivative of Ludartin is Morpholino analog which shows
an IC50 = 2.8 M towards leukemia (THP-1) and the most potent
analog is 6-nitroindazole (17) with IC50 = 2.2 M towards prostate
(PC-3) compared to parent Ludartin with IC50 of 3.1 and 6.9 M to-
wards leukemia (THP-1) and prostate (PC-3), respectively. Thus our
study is in fine tune with those of R. Csuk that modification at exo-
cyclic double bond leads to analogs with either modified selectivity
towards particular cell lines or similar/reduced cytotoxicity.
Dimethyl amino analog of Arglabin is a registered antitumor
substance in the Republic of Kazakstan.24
The amino analogs of Arglabin act by impeding the protein farn-
esylation especially those of RAS proteins. The drug inhibits the
incorporation of [(3)H]farnesylpyrophosphate into human H-RAS
protein by FTase.24 Farnesylation of RAS is required for proteolytic
processing and tight binding of RAS to cellular membranes. In the
absence of farnesylation oncogenic forms of RAS cannot oncogeni-
cally transform cells. Thus inhibitors of farnesyl-protein transfer-
ase are considered to be useful anticancer therapeutics for many
types of cancers.25 Since Ludartin is a position isomer of Arglabin,
it is likely that the amino analogs of Ludartin may also follow the
same mechanism of action. However further studies are needed
to explore the exact pathway and the selectivity in action of the
amino analogs of Ludartin. We have already started the work in
this direction.
l
l
l
l
17. Blanco, J. G.; Gil, R. R.; Alvarez, C. I.; Patrito, L. C.; Genti-Raimondi, S.; Flury, A.
FEBS Lett. 1997, 409, 396.
18. Duvvuri, M.; Konkar, S.; Hong, K. H.; Blagg, B. S. J.; Krise, B. J. P. ACS Chem. Biol.
2006, 1, 309.
19. Plant material and isolation of Ludartin: the plant material of Artemisia
amygdalina Decne was collected from the high altitude areas of Gurez valley in
Kashmir and authenticated by Professor A.R. Naqshi, Department of Botany,
University of Kashmir and by comparision with the A. amygdalina growing
within the institute’s gene bank. The shoot and root parts were separately
processed, shade dried, chopped and ground to a fine powder. The powdered
shoot part was extracted thrice with chloroform. The chloroform extracts so
obtained were evaporated under vaccuo on a rotary evaporator to afford a
crude extract of 65.0 g. About 50.0 g of the extract was subjected to normal
silica-gel column chromatography using successively hexane (100%) and
hexane–EtOAc (95:5, 90:10, 85:15, 80:20, 75:25, 70:30) as eluent
(unpublished work). All the 90:10 (hexane/EtOAc) fractions were pooled up
and concentrated to furnish pure white crystals of Ludartin (2.0 g) whose
structure was elucidated on the basis of spectral data analysis and by
comparision with that of literature data.13,14 Synthesis of Amino analogs of
Ludartin (2–18): A solution of 1 (30 mg, 0.121 mmol) in acetonitrile (2 ml) and
amine (0.121 mmol) was heated under reflux for 8–24 h either in presence of
base (DBU) or more often without the base. After cooling the reaction mixture
was evaporated under vaccuo on a rotary evaporator and the residue obtained
was subjected to normal silica-gel column chromatography using Hexane-
EtOAc as eluent to furnish the pure product. Spectral data of reference
compound (9): Yield 90%; Rf = 0.5 (5% MeOH-DCM); IR (KBr cmÀ1) 3013, 2960,
1758, 1205, 780; 1H NMR (400 MHz, CDCl3); d 3.64 (t, J = 10.8, 10.8 Hz, 1H),
3.39 (s, 1H), 3.04 (d, J = 10.7, 1H), 2.83 (d, J = 5.7 Hz, 2H), 2.71–2.68 (m, 1H),
2.52–2.43 (m, 5H), 2.36–2.31 (m, 1H), 2.18–2.15 (m, 3H), 2.02–1.98 (m, 1H),
1.75 (s, 4H), 1.68 (s, 3H), 1.62 (s, 3H), 1.30–1.18 (m, 1H). 13C NMR (126 MHz,
CDCl3) d 175.60, 133.99, 131.79, 78.84, 66.03, 62.59, 53.96, 53.21, 52.74, 50.53,
44.39, 33.04, 32.28, 26.43, 22.44, 21.22, 17.96. ESI-MS (m/z): 318 (M+H)+.
Similarly other compounds were synthesized and characterized using spectral
data analysis.
In conclusion a diversity of amine analogs were prepared and
studied for any structure–activity relationship. The SAR studies
clearly indicate that modifications at a-methylene-c-lactone moi-
ety result in improved and selective cytotoxicity of the bioactive
lactones. Moreover it is the leukemia cell line (THP-1) that forms
the usual target of most of the analogs of Ludartin with enhanced
cytotoxity compared to the parent molecule Ludartin (1). Above all
this approach provides in one synthetic step, an opportunity to en-
hance pharmacokinetic properties to selectively target specific
cells with either retention or augmentation of cytotoxic effect. Fur-
ther efforts to expand the library of such amino analogs, to come
up with a better lead molecule, are underway.
20. Matsuda, H.; Kageura, T.; Inoue, Y.; Morikawa, T.; Yoshikawa, M. Tetrahedron
2000, 56, 7763.
21. Cytotoxicity assay: RPMI-1640 medium, streptomycin, fetal bovine serum,
sodium bicarbonate, phosphate buffer saline, sulphorhodamine, trypsin, 5-
flurouracil, gentamycin sulphate were purchased from Sigma Chemicals Co.
Glacial acetic acid from Fischer scientific and trichloroactetic acid (TCA) from
Merck specialties private limited. All the human cancer cell lines (A-549, THP-
1, PC-3, HCT-116) were obtained from ATCC Sigma. All the cells used were
Acknowledgment
grown in RPMI-1640 medium containing 10% FBS, 100 unit penicillin/100 lg
streptomycin per ml medium. Cells were allowed to grow in carbon dioxide
incubator (Thermoscientific USA) at 37 °C with 98% humidity and 5% CO2 gas
environment. In the present study the cytotoxic effect of the different analogs
was evaluated using sulpharhodamine B (SRB) assay. The SRB dye binds to the
basic protein of cells that have been fixed to tissue-culture plates by
trichloroacetic acid (TCA). As the binding of SRB is stoichiometric, the
amount of dye extracted from stained cells is directly proportional to the cell
number. In the present case, all cells lines seeded in flat-bottomed 96-well
plates were allowed to adhere overnight, and then media containing different
analogs (varying concentrations) were added. The plates were assayed for 48 h.
One of the authors Shabir is thankful to CSIR India for providing
financial Grant in the form of Junior Research Fellowship.
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The cells were fixed by adding 50
60 min. The plates were washed five times in running tap water and stained
with 100 l per well SRB reagent (0.4% w/v SRB) in 1% acetic acid for 30 min.
The plates were washed five times in 1% acetic acid to remove unbound SRB
and allowed to dry overnight. SRB was solubilised with 100 l per well, 10 mM
ll of ice-cold 50% TCA to each well for
l
l
tris–buffer, shaken for 5 min and the optical density was measured at
570 nm.22
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