Isolation of isoxanthanol and synthesis of novel derivatives as potential cytotoxic agents

Article abstract of DOI:10.1007/s00044-017-1949-z

Abstract: Novel synthetic derivatives of sesquiterpene lactone isoxanthanol (1) have been prepared and bioevaluated against four human cancer cell lines viz. T98G (glioblastoma), A431 (epidermoid carcinoma), NCI-H322 (bronchioloalveolar carcinoma), and A549 (lung adeno carcinoma) for their cytotoxic potential using paclitaxel as the standard. This has resulted in the identification of potent molecules displaying IC₅₀ 1.9 and 5.0 μM, respectively against the A549 cancer cell line. The study has resulted in the identification of potential cytotoxic activity of the analog (compound 10) bearing electron donating aryl alkenoic substituent. Furthermore, the induction of cell death has been assessed for the most active compound (10) using flow cytometric method and sub-G1 cell population determination by propidium iodide staining. The concentration dependent inhibitory effect of 10 on the A549 cells ability did not reproduce and form colonies at 20 μM concentration. Graphical Abstract: Synthesis of isoxanthanol derivatives and their cytotoxic study resulted in identification of potential cytotoxic agents. Compound 10, one of its aryl alkenoic substituent showed potency against NCI-H322 (bronchioloalveolar carcinoma), and A549 (lung adeno carcinoma) cell lines. [InlineMediaObject not available: see fulltext.].

Full text of DOI:10.1007/s00044-017-1949-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1949-z
ORIGINAL RESEARCH
Isolation of isoxanthanol and synthesis of novel derivatives as
potential cytotoxic agents
Praveen K. Chinthakindi1,4 Santosh K. Rath1,2 Jasvinder Singh2,3
●
●
●
Shashank Singh2,3 Surrinder Koul1 Payare L. Sangwan1,2
●
●
Received: 21 December 2016 / Accepted: 3 June 2017
© Springer Science+Business Media, LLC 2017
Abstract Novel synthetic derivatives of sesquiterpene lac-
tone isoxanthanol (1) have been prepared and bioevaluated
against four human cancer cell lines viz. T98G (glio-
blastoma), A431 (epidermoid carcinoma), NCI-H322
(bronchioloalveolar carcinoma), and A549 (lung adeno
carcinoma) for their cytotoxic potential using paclitaxel as
the standard. This has resulted in the identification of potent
molecules displaying IC50 1.9 and 5.0 µM, respectively
against the A549 cancer cell line. The study has resulted in
the identification of potential cytotoxic activity of the ana-
log (compound 10) bearing electron donating aryl alkenoic
substituent. Furthermore, the induction of cell death has
been assessed for the most active compound (10) using flow
cytometric method and sub-G1 cell population determina-
tion by propidium iodide staining. The concentration
dependent inhibitory effect of 10 on the A549 cells
ability did not reproduce and form colonies at 20 µM
concentration.
Graphical Abstract Synthesis of isoxanthanol derivatives
and their cytotoxic study resulted in identification of
potential cytotoxic agents. Compound 10, one of its aryl
alkenoic substituent showed potency against NCI-H322
(bronchioloalveolar carcinoma), and A549 (lung adeno
carcinoma) cell lines.
●
●
●
Keywords Isoxanthanol Sesquiterpene Lactone Human
leukemia
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1949-z) contains supplementary material,
which is available to authorized users.
* Surrinder Koul
Abbreviations
skoul@iiim.ac.in
NP’s
SLs
Natural products
* Payare L. Sangwan
Sesquiterpene lactones
New chemical entities
Food and drugs administration
Coupling constant
plsangwan@iiim.ac.in
NCEs
FDA
J
1
Bioorganic Chemistry Division, CSIR-Indian Institute of
Integrative Medicine, Canal Road, Jammu 180001, India
2
HRMS
PBS
SRB
TCA
DMAP
CC
High resolution mass spectra
Phosphate buffer saline
Sulforhodamine B
Trichloroacetic acid
N,N-dimethyl amino pyridine
Column chromatography
Academy of Scientific and Innovative Research (AcSIR), CSIR-
IIIM Campus, Jammu, India
3
Cancer Pharmacology Division, CSIR-Indian Institute of
Integrative Medicine, Canal Road, Jammu 180001, India
4
Present address: Catalysis and Peptide Research Unit, University
of Kwazulu-Natal, Durban, South Africa

Med Chem Res
Introduction
Natural products (NP’s) are compounds that are commonly
found in nature, having a wide range of pharmacophores,
high degree of stereochemistry and possess various biolo-
gical applications that serve as an excellent source of lead
scaffolds in drug discovery program (Harvey et al. 2015).
There are only a few instances, where NPs have been used
as drugs as such e.g., vinblastine, vincristine, paclitaxel,
camptothecin, colchicine, and silvestrol etc. In the majority
of the cases, the drugs have been developed in the form of
structural analogs and/or derivatives of NPs as a base ske-
leton e.g., flavopiridol, etoposide, teniposide, com-
bretastatin A-4 phosphate, CDDO, topotecan, irinotecan,
vinorelbine, vindesine, minnelide etc. This is well corro-
borated by the fact that, in recent years Food and drugs
administration approved new chemical entities, have only
8% of NPs compared to the NP derivatives (Cragg et al.
2014; Newman and Cragg 2012), which are three times
higher. Realistically, the structural modification of NPs has
been undoubtedly playing pivotal role in the development
of efficient modern medicine. Further docking studies
support in identification of potent molecules (Taha et al.
2016, 2017).
Fig. 1 Active sites (shown in red) of isoxanthanol (1) and xanthanol
(1a) (color figure online)
Fig. 2 The ORTEP diagram indicates numbering scheme and the
molecular conformation of isoxanthanol in crystals. The displacement
ellipsoids are drawn at the 40% probability level. H atoms are shown
as small spheres of arbitrary radii
Dar et al. 2016; Gupta et al. 2016; Majeed et al. 2012, 2013)
in the area of NP’s-based medicinal chemistry.
Natural sesquiterpene lactones (SLs) and their deriva-
tives are of great interest due to their vast pharmacological
properties and this asset has attracted many medicinal
chemists for further tailoring and screening them as novel
therapeutic agents. Many of these SLs are in cancer clinical
trials and also selectively targeting tumor and cancer stem
cells while leaving normal cells untouched (Ghantous et al.
2010, 2013; Jordan 2006; Gach and Dalugosz 2014; Zhang
et al. 2012; Christensen et al. 2009). However, poor cell
permeability and solubility is a major concern for SLs due
to their extensive plasma protein interactions and hydro-
phobic nature. In addition to that, some of them having
allergenic properties and off-target effect (Ghantous et al.
2010; Efferth 2006; Zhou and Zhang 2008; Schmidt 1997).
These issues might be overcome by chemical modification
and pro drug approach.
So far there is no detailed anticancer report on iso-
xanthanol (Nour et al. 2009; Favier et al. 2005; Yoon et al.
2008; Vasas and Hohmann 2011). Isolation of isoxanthanol
has been problematic from Xanthium species (Abdei-Mogib
et al. 1991; Omar et al. 1984; Bohlmann et al. 1982) by
column chromatography (CC) due to its isomerization to
xanthanol (1a) (Fig. 1) and presuming that, this may be one
of the reasons that no literature reports are present with
respect to its chemical modification. Therefore natural
product isoxanthanol (1) was explored for chemical mod-
ification to investigate new hit or lead molecule for cancer
as a part of our ongoing research activities (Khan et al.
2016; Chinthakindi et al. 2013, 2017; Dangroo et al. 2017;
In the present study, the NP was isolated by repeated CC
and re-crystallization process (see experimental data) in
moderate yields (0.12% from 1 Kg) from the areal part of
Xanthium spinosum plant. The absolute stereochemistry of
the isoxanthanol established through X-ray crystallography
see Fig. 2. The chemical modification of 1 was carried out
for the identification of best possible potent antiproliferative
analog.
Materials and methods
Reagents and instruments
All reagents for chemical synthesis were obtained from
Sigma Aldrich and the solvents used in reactions were
distilled and dried prior to use. All chemical reactions were
monitored by thin layer chromatography (TLC) on 0.25 mm
silica gel 60 F254 plates (E. Merck) using 2% ceric
ammonium sulfate solution for detection of the spots. Pur-
ification of compounds was carried out by CC using silica
gel 60–120 mesh stationary phase. All nuclear magnetic
resonance (NMR) (1H NMR and 13C NMR) spectra
were recorded on Bruker DPX 200, 400, and DPX 500
instruments using CDCl3 as the solvents with tetra-
methylsilane as internal standard. Chemical shifts are
expressed in δ and coupling constants (J) in Hertz. High
resolution mass spectra (HRMS) were recorded on Agilent

Med Chem Res
Technologie 6540 instrument and infrared (IR) was recor-
ded on an fourier transform infrared Bruker (270–30)
spectrophotometer.
(3H, s, CH3CO–), 1.78–1.88 (1H, m, CH–CH2–), 1.63–1.77
(2H, m, CH–CH2–), 1.56–1.62 (1H, m, CH2–CH2–), 1.28
(3H, d, J = 6.4 Hz, CH3–CH–), 1.19 (3H, d, J = 6.8 Hz,
CH3–CH–); 13C NMR (CDCl3, 125 MHz): δ = 170.67
(C=O), 170.18 (C=O), 147.67 (CH=C), 138.58 (CH2=C),
122.44 (CH2=C), 122.16 (CH=C), 78.96 (CH–O), 73.61
(CH–OAc), 69.32 (CH–OH), 42.10 (O–CH–CH2–CH–O),
41.36 (CH2–CH–CH–O), 36.96 (CH–CH2–CH–O), 32.83
(=CH–CH2–CH), 26.00 (=C–CH–CH3), 22.09 (CO–CH3),
21.42 (CH3–CH–O), 20.38 (CH3–CH); IR (neat) γmax 3467,
2955, 2934, 2872, 1761, 1736, 1658, 1459, 1409, 1373,
1328, 1247, 1209, 1124, 1089, 1049, 1015, 979, 856, 834,
778, 749, 657, 585, 541, 512 cm−1; HRESIMS m/z (pos):
331.1522 C17H23NaO5 (calcd. 331.1522).
RPMI-1640 medium, MEM medium, penicillin, strep-
tomycin, sodium bicarbonate, phosphate buffer saline
(PBS), sulforhodamine B (SRB), trypsin, rhodamine-123,
paclitaxel and dimethyl sulfoxide (DMSO) were purchased
from Sigma Aldrich. Propidium iodide, Rnase, tri-
chloroacetic acid (TCA) were procured from Merck. Glacial
acetic acid was purchased from Fisher Scientific. Ethyle-
nediaminetetraacetic acid was purchased from Himedia.
Fetal bovine serum was from Gibco and erlotinib was from
Selleck Chemicals.
Cell culture, growth conditions and treatment conditions
General procedure for preparation of acyl derivatives of
Human glioblastoma cancer cell line (T98G), lung carci-
noma (A549, NCI-H322) and epidermal carcinoma (A431)
were procured from European collection of cell culture.
Cells were grown in (RPMI-1640/ MEM) medium supple-
mented with 10% fetal bovine serum, 2 mg/ml sodium
bicarbonate, 100 µg/ml streptomycin and 100 units/ml
penicillin in carbon dioxide incubator (Thermo Scientific,
USA) at 37 °C, 5% CO2 and 98% RH. Cells were treated
with different compounds dissolved in DMSO with
untreated control receiving only vehicle (DMSO).
isoxanthanol (2–6)
Compounds 2–6 were synthesized by treating isoxanthanol
(1) with appropriate alkanoic anhydride in presence of
catalytic amount of N, N-dimethyl amino pyridine (DMAP)
and the progress of reaction monitored by TLC. On com-
pletion of the reaction, the contents poured in ice water and
the oily material separated and dissolved in dichlor-
omethane. The organic layer washed with water, dried over
anhydrous Na2SO4 and concentrated under reduced pres-
sure. The acyl derivatives thus obtained were purified by
CC over silica gel 60–120 mesh, using hexane: ethyl acetate
mixture (49:1) as an eluent to give compounds 2–6 in >90%
over all yield. The spectral data of all the derivatives are
given below.
Isolation of isoxanthanol
Isoxanthanol (1) was isolated from the aerial part of Xanthium
spinosum (duly authenticated by the Taxonomist of our
institute) by repeated CC over silica gel 60–120 mesh, and
purified by recrystallization from chilled ether. The NP was
chemically characterized by spectral data analysis and found
in agreement with the literature data (Abdei-Mogib et al.
1991; Omar et al. 1984; Bohlmann et al. 1982; Marco et al.
1993) and finally its absolute configuration confirmed by X-
ray crystallography (see Fig. 2). This is the first report of the
establishment of absolute stereochemistry by X-ray analysis.
(1S,3S)-1-((3aR,7S,8aS)-7-methyl-3-methylene-2-oxo-
3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-6-yl)
butane-1,3-diyl diacetate (2)
Yellowish gummy mass (This compound was prepared by
reacting 100 mg isoxanthanol (1) with 33 µL acetic anhy-
dride and purified by the procedure as described above to
1
obtained 106 mg with 97% yield); H NMR (CDCl3, 400
(2S,4S)-4-hydroxy-4-((3aR,7S,8aS)-7-methyl-3-methylene-
2-oxo-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-6-
yl)butan-2-yl acetate (Isoxanthanol) (1)
MHz): δ = 6.26 (1H, d, J = 3.2 Hz, CH2=C–), 5.81 (1H,
dd, J = 5.6, 9.2 Hz,=CH–CH2–), 5.52 (1H, d, J = 2.8 Hz,
–CH2=C–), 5.08 (1H, dd, J = 5.6, 8.4 Hz, CHOAc), 4.88
(1H, dt, J = 6.0, 12.4 Hz, –CHOCO–), 4.62 (1H, ddd, J =
2.8, 8.4 Hz, –CHOAc), 3.28–2.42 (1H, m, –CH2CH–),
2.55–2.69 (1H, m, CH2–CH–), 2.39–2.2.52 (1H, m,
CH–CH2–), 2.23–2.37 (2H, m, CH & CH2), 2.04 (2 × 3H
each, s, CH3–CO–), 1.84 (1H, m, CH2–CH2–), 1.75 (2H, m,
CH2–CH2–), 1.28 (3H, d, J = 6.4 Hz, CH3–CH–), 1.19 (3H,
d, J = 6.8 Hz, CH3–CH–); 13C NMR (CDCl3, 125 MHz): δ
= 170.38 (C=O), 170.25 (C=O), 170.00 (C=O), 144.20
(C=CH), 138.63 (CH2=C), 124.99 (CH2=C), 122.16
(CH=C), 78.78 (CH–O), 74.98 (CH–OAc), 67.93
The compound was obtained as white crystalline; mp
1
101.5 °C; [α]25D −27.2 (c 0.1, CHCl3); H NMR (CDCl3,
400 MHz): δ = 6.27 (1H, d, J = 3.2 Hz, CH2=C–), 5.73
(1H, dd, J = 5.6, 9.2 Hz,=CH–CH2–), 5.52 (1H, d, J = 2.8
Hz, –CH2=C–), 4.96 (1H, dt, J = 6.0, 12.4 Hz,
–CHOCO–), 4.61 (1H, ddd, J = 2.8, 8.4 Hz, –CHOAc),
4.10 (1H, dd, J = 5.6, 8.4 Hz, CHOH), 3.3–4.2 (1H, m,
–CHCH–), 2.56–2.66 (1H, m, –CH2CH–), 2.43–2.50
(1H, m, CH2–CH–), 2.25–2.33 (1H, m, CH–CH2–), 2.04

Med Chem Res
(CH–OH), 41.33 (O–CH–CH2–CH–O), 39.78 (CH2–
CH–CH–O), 36.94 (CH–CH2–CH–O), 33.50 (=CH–
CH2–CH), 26.18 (=C–CH–CH3), 21.64 and 21.31 (2 ×
CO–CH3), 21.30 (CH3–CH–O), 19.94 (CH3–CH); IR (neat)
γmax 3452, 2930, 2873, 1763, 1736, 1660, 1460, 1372,
1328, 1241, 1123, 1088, 1050, 1018, 958, 855, 815, 607,
545, 518, 506 cm−1; HRESIMS m/z (pos): 373.1621
C19H26NaO6 (calcd. 373.1623).
Hz, CH–OAc), 3.34–3.32 (1H, m, –CH–CH–), 2.65 (1H, m,
CH2–CH–), 2.35 (1H, t, J = 6.4 Hz, CH2–CH–), 2.28–2.33
(2H, m, CH–CH2–), 2.05 (3H, s, CH3–CO–), 1.7–1.8 (2H,
m, CH2–CH2–), 1.65 (2H, m, CH2–CH2–), 1.58–1.66 (2H,
m, CH2–CH2–), 1.52–1.48 (1H, m, –CH2–CH2–), 1.3–1.41
(2H, m, –CH2–CH2–), 1.24 (3H, d, J = 6.4 Hz, CH3–CH),
1.13 (3H, d, J = 7.10 Hz, CH3–CH–), 0.91 (3H, q, J = 6.3
Hz, CH3–CH2); 13C NMR (CDCl3, 100 MHz,): δ = 173.01
(C=O), 170.33 (C=O), 169.91 (C=O), 144.23 (C=CH),
138.44 (CH2=C), 124.62 (CH2=C), 122.02 (CH=C), 78.68
(CH–O), 74.45 (CH–OAc), 67.81 (CH–OH), 41.15
(O–CH–CH2–CH–O), 39.61 (CH2–CH–CH–O), 36.69
(CH–CH2–CH–O), 34.06 (CH2–CH2–C=O), 33.63 (=CH–
CH2–CH), 25.97 (=C–CH–CH3), 25.50 (–CH2–CH2–C =
O), 21.98 (CO–CH3), 21.38 (–CH2–CH3), 21.07
(CH3–CH–O), 19.64 (CH3–CH–C=), 13.46 (–CH2–CH3);
IR (neat) γmax 3442, 2959, 2933, 2873, 1764, 1733, 1659,
1460, 1417, 1372, 1328, 1244, 1175, 1109, 1088, 1051,
1017, 981, 956, 855, 814, 775, 609 cm−1; HRESIMS m/z
(pos): 415.20879 C22H32NaO6 (calcd. 415.20911).
(1S,3S)-3-acetoxy-1-((3aR,7S,8aS)-7-methyl-3-methylene-
2-oxo-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-6-
yl)butyl butyrate (3)
Yellowish gummy mass (This compound was prepared by
reacting 100 mg isoxanthanol (1) with 55 µL butyric anhy-
dride and purified by the procedure as described above to
1
obtained 114 mg with 94% yield); H NMR (CDCl3, 400
MHz): δ = 6.24 (1H, d, J = 3.2 Hz, CH2=C–), 5.79 (1H,
dd, J = 5.6, 9.2 Hz, =CH–CH2–), 5.50 (1H, d, J = 2.8 Hz,
CH2=C–), 5.07 (1H, dd, J = 5.6, 8.4 Hz, –CH–OCOCH2–),
4.86 (1H, dt, J = 6.0, 12.4 Hz, CH–OCO), 4.61 (1H, ddd, J
= 2.8, 8.8 Hz, CH–OAc), 3.21–3.33 (1H, m, CH–CH–),
2.57–2.67(1H, m, CH2–CH–), 2.45–2.38 (2H, m,
CH2–CH–), 2.21–2.35 (3H, m, CH2–CH2–, CH), 2.03 (3H,
s, CH3–CO–), 1.82 (1H, m, CH2–CH2–), 1.73 (2H, m,
CH2–CH2–), 1.63 (2H, m, CH2–CH2–), 1.22 (3H, d, J =
6.4 Hz, CH3–CH–), 1.12 (3H, d, J = 6.8 Hz, CH3–CH–),
0.94 (3H, t, J = 6.4 Hz, CH3–CH2–); 13C NMR (CDCl3,
100 MHz): δ = 172.93 (C=O), 170.37 (C=O), 169.98
(C=O), 144.47 (C=CH), 138.63 (CH2=C), 124.75
(CH2=C), 122.11 (CH=C), 78.79 (CH–O), 74.58
(CH–OAc), 67.94 (CH–OH), 41.34 (O–CH–CH2–CH–O),
39.83 (CH2–CH–CH–O), 36.89 (CH–CH2–CH–O), 36.44
(CH2–CH2–C = O), 33.70 (=CH–CH2–CH), 26.14 (=C–
CH–CH3), 21.56 (CO–CH3), 21.25 (CH3–CH), 19.83
(CH3–CH–O), 18.37 (CH2–CH3), 13.63 (CH2–CH3); IR
(neat) γmax 3435, 2963, 2933, 2875, 1764, 1733, 1658,
1459, 1372, 1328, 1244, 1179, 1087, 1050, 1018, 958,
855, 815, 609 cm−1; HRESIMS m/z (pos): 401.1939
C21H30NaO6 (calcd. 401.1934).
(1S,3S)-3-acetoxy-1-((3aR,7S,8aS)-7-methyl-3-methylene-
2-oxo-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-6-
yl)butyl octanoate (5)
Yellowish gummy mass (This compound was prepared by
reacting 100 mg isoxanthanol (1) with 90 µL octanoic
anhydride and purified by the procedure as described above
to obtained 137 mg with 98% yield); 1H NMR (CDCl3, 400
MHz): δ = 6.27 (1H, d, J = 3.2 Hz, CH2=C–), 5.81 (1H,
dd, J = 5.6, 9.2 Hz,=CH–CH2–), 5.52 (1H, d, J = 2.8 Hz,
CH2=C–), 5.08 (1H, dd, J = 5.6, 8.0 Hz, CH–OCOCH2),
4.88 (1H, dt, J = 6.4, 12.4 Hz, CH–OAc), 4.63 (1H, ddd, J
= 2.8, 8.8 Hz, CH–OCO), 3.27–3.43 (1H, m, CH–CH–),
2.58–2.65 (1H, m, CH2–CH–), 2.44 (1H, m, CH2–CH–),
2.27–2.93 (2H, m, CH–CH2–), 2.06 (3H, s, CH3–CO–),
2.01 (2H, m, –CH2–CO–), 1.8 (2H, m, CH2–CH2–), 1.6
(2H,
m,
CH2–CH2–),
1.28–1.59
(9H,
m,
–CH2–CH2–CH2–CH2–CH–), 1.26 (3H, d, J = 6.4 Hz,
CH3–), 1.11 (3H, d, J = 7.5 Hz, CH3–CH–), 0.87 (3H, q, J
= 6.8 Hz, CH3–CH2). 13C NMR (CDCl3, 125 MHz): δ =
173.17 (C=O), 170.41 (C=O), 170.06 (C=O), 144.43
(C=CH), 138.59 (CH2=C), 124.76 (CH2=C), 122.23
(CH=C), 78.83 (CH–O), 74.60 (CH–OAc), 67.93
(CH–OH), 41.34 (O–CH–CH2–CH–O), 39.78 (CH2–
CH–CH–O), 36.89 (CH–CH2–CH–O), 34.54 (CH2–
CH2–C=O), 33.70 (=CH–CH2–CH), 31.69 (–CH2–CH2–),
29.10 (–CH2–CH2–), 28.92 (–CH2–CH2–), 26.15 (=C–
CH–CH3), 24.90 (–CH2–CH2–C=O), 22.59 (CO–CH3),
21.61 (CH3–CH–O), 21.32 (–CH2–CH3), 19.86
(CH3–CH–C=), 14.07 (–CH2–CH3); IR (neat) γmax 3437,
2955, 2928, 2856, 1765, 1735, 1656, 1459, 1373, 1327,
1243, 1159, 1115, 1051, 1018, 957, 855, 813, 773, 609,
(1S,3S)-3-acetoxy-1-((3aR,7S,8aS)-7-methyl-3-methylene-
2-oxo-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-6-
yl)butyl pentanoate (4)
Yellow liquid (This compound was prepared by reacting
100 mg isoxanthanol (1) with 65 µL valeric anhydride and
purified by the procedure as described above to obtained
121 mg with 96% yield); 1H NMR (CDCl3, 400 MHz): δ =
6.26 (1H, d, J = 3.2 Hz, CH2=C–), 5.81 (1H, dd, J = 5.6,
9.2 Hz, =CH–CH2–), 5.52 (1H, d, J = 2.8 Hz, CH2=C–),
5.08 (1H, dd, 5.6, 8.4 Hz, CH–OCOCH2–), 4.88 (1H, dt, J
= 6.0, 12.4 Hz, CH–OCO–), 4.62 (1H, ddd, J = 2.8 and 8.8

Med Chem Res
515 cm−1; HRESIMS m/z (pos): 457.2556 for C25H38NaO6
crude product obtained was purified by CC on silica gel
(calcd 457.2560).
column using hexane-ethyl acetate as the eluent.
(1S,3S)-3-acetoxy-1-((3aR,7S,8aS)-7-methyl-3-methylene-
2-oxo-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-6-
yl)butyl 4-methyl-3,5-dinitrobenzoate (7)
(1S,3S)-3-acetoxy-1-((3aR,7S,8aS)-7-methyl-3-methylene-
2-oxo-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-6-
yl)butyl decanoate (6)
Yellowish powder (This compound was prepared by
reacting 100 mg isoxanthanol (1) with 4-methyl-2,5-dinitro
benzoic acid (73 mg) in presence of DCC (74 mg), DMAP
(4 mg) by the procedure as described above to obtained 145
Yellowish gummy mass (This compound was prepared by
reacting 100 mg isoxanthanol (1) with 90 µL decanoic
anhydride and purified by the procedure as described above
to obtained 147 mg with 98% yield); 1H NMR (CDCl3, 400
MHz): δ = 6.26 (1H, d, J = 3.21 Hz, CH2=C–), 5.81 (1H,
dd, J = 5.6, 9.2 Hz,=CH–CH2–), 5.52 (1H, d, J = 2.8 Hz,
CH2=C–), 5.08 (1H, dd, J = 5.6, 8.4 Hz, –CH–OCOCH2),
4.87 (1H, dt, J = 6.0, 12.4 Hz, CH–OAc), 4.62 (1H, m,
CH–OCO–), 3.25–3.38 (1H, m, CH–CH–), 2.51–2.65 (1H,
m, CH2–CH–), 2.40–2.50 (1H, m, CH2–CH–), 2.20–2.30
(2H, m, –CH2–CH–), 2.03 (3H, s, CH3–CO–), 1.81–1.89
(1H, m, CH2–CH2–), 1.72–1.80 (2H, m, –CH–CH2–),
1.52–1.62 (2H, m, CH2–CH2–), 1.26 (17H, m, –(CH2)7−
and CH3–CH–), 1.13 (3H, d, J = 6.90 Hz, CH3–CH–), 0.87
(3H, q, J = 7.0 Hz, CH3–CH2); 13C NMR (CDCl3, 100
MHz): δ = 173.11 (C=O), 170.34 (C=O), 169.96 (C=O),
144.49 (C=CH), 138.65 (CH2=C), 124.74 (CH2=C),
122.10 (CH=C), 78.78 (CH–O), 74.58 (CH–OAc), 67.93
(CH–OH), 41.37 (O–CH–CH2–CH–O), 39.83 (CH2–
CH–CH–O), 36.91 (CH–CH2–CH–O), 34.55 (CH2–
CH2–C=O), 33.70 (=CH–CH2–CH), 31.83 (–CH2–CH2–),
29.43 (–CH2–CH2–), 29.24 (–CH2–CH2–), 29.14 (2 × –
CH2–CH2), 26.18 (=C–CH–CH3), 24.90 (–CH2–CH2
–C=O), 22.63 (CO–CH3), 21.58 (CH3–CH–O), 21.27 (–
CH2–CH3), 19.85 (CH3–CH–C=), 14.06 (–CH2–CH3); IR
(neat) γmax 3436, 2926, 2854, 1766, 1735, 1657, 1459,
1372, 1326, 1243, 1157, 1115, 1050, 1017, 957, 855, 813,
773, 609, 521 cm−1; HRESIMS m/z (pos): 485.2864 for
C27H42NaO6 (calcd 485.28736).
1
mg with 91% yield); mp 240–241 °C; H NMR (CDCl3,
500 MHz): δ = δ 8.57 (2H, s, Ar–H), 6.27 (1H, d, J = 3.21
Hz, CH2=C–), 5.95 (1H, dd, J = 5. 54 and 9.2 Hz,
=CH–CH2–), 5.53 (1H, d, J = 2.77 Hz, CH2=C–), 5.37
(1H, dd, J = 5.81, 8.32 Hz, CH–OCO–), 4.93 (1H, dt, J =
6.0, 12.4 Hz, CH–OAc), 4.64 (1H, ddd, J = 2.8, 8.8 Hz,
CH–OCO–), 3.30–3.40 (1H, m, –CH2–CH–), 2.70–2.75
(1H, m, CH2–CH–), 2.64 (3H, s, Ar–CH3), 2.25–2.29 (2H,
m, CH–CH2–), 2.06–2.13 (2H, m, CH2–CH–), 2.02
(3H, s, CH3–CO–), 1.89–2.0 (2H, m, CH2–CH2–), 1.29
(3H, d, J = 6.28 Hz, CH3–CH–), 1.17 (3H, d, J = 6.9 Hz,
CH3–CH–); 13C NMR (CDCl3, 100 MHz): δ = 170.78
(C=O), 170.32 (C=O), 162.50 (Ar–C=O), 151.80 (2 ×
Ar–C–NO2), 143.36 (C=CH), 138.46 (CH2=C), 131.77
(Ar–C–CH3), 130.39 (Ar–C–), 127.78 (2 × Ar–CH),
126.65 (CH2=C), 122.40 (CH=C), 78.59 (CH–O), 77.87
(CH–OAc), 67.77 (CH–OH), 41.29 (O–CH–CH2–CH–O),
39.93 (CH2–CH–CH–O), 36.84 (CH–CH2–CH–O), 33.70
(=CH–CH2–CH), 26.36 (=C–CH–CH3), 21.74 (CO–CH3),
21.25 (CH3–CH–O), 21.14 (CH3–CH–C=), 15.24 (Ar–
CH3); IR (neat) γmax 3436, 3091, 2927, 2853, 1762, 1729,
1624, 1542, 1458, 1347, 1276, 1243, 1204, 1158, 1123,
1088, 1050, 1018, 956, 904, 857, 814, 744, 722, 609 cm−1
;
HRESIMS m/z (pos): 539.16198 C25H28N2NaO10 (calcd.
539.16361).
(1S,3S)-3-acetoxy-1-((3aR,7S,8aS)-7-methyl-3-methylene-
2-oxo-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-6-
yl)butyl 2-(naphthalen-1-yl)acetate (8)
General procedures for preparation of aryl esters (7–10)
In a two-necked round bottom flask equipped with a cal-
cium chloride drying tube was charged aryl carboxylic acid
(1 equiv.), isoxanthanol (1) (1 equiv.), and DMAP (10 mol
%) in dry dichloromethane. The resulting reaction mixture
cooled in an ice bath to 0 °C and stirred for a while followed
by addition of DCC (1.1 equiv.) at 0 °C over a period of 5
min. The ice bath was removed, and the dark-brown reac-
tion mixture stirred for 3 h at room temperature. After
completion of the reaction, the precipitate of dicyclohex-
ylurea was removed by filtration through a fritted Buchner
funnel, and the filtrate washed with water and extracted with
dichloromethane. The organic layer was dried over anhy-
drous sodium sulfate and concentrated on a rotavapor. The
Colorless liquid (This compound was prepared by reacting
100 mg isoxanthanol (1) with naphthyl acetic acid (60 mg)
in presence of DCC (74 mg), DMAP (4 mg) by the proce-
dure as described above to obtained 149 mg with 97%
1
yield); H NMR (CDCl3, 400 MHz): δ = 8.04 (1H, d, J =
8.31 Hz, Ar–H), 7.85 (1H, d, J = 7.82 Hz, Ar–H), 7.75 (1H,
d, J = 7.84 Hz, Ar–H), 7.52 (1H, d, J = 1.22 Hz, Ar–H)
7.47–7.50 (3H, m, Ar–H) 6.18 (1H, d, J = 3.2 Hz,
CH2=C–), 5.42 (1H, dd, J = 5.6, 9.2 Hz, =CH–CH2–),
5.37(1H, d, J = 3.63 Hz, CH2=C–), 4.98 (1H, dd, J = 5.6,
8.4 Hz, –CH–OCOCH2), 4.88 (1H, dt, J = 6.0, 12.4 Hz,
CH–OAc), 4.17 (1H, ddd, J = 2.8, 8.8 Hz, CH–OCO–),

Med Chem Res
4.04 (2H, s, Ar–CH2–CO–), 2.51–2.65 (1H, m, CH2–CH–),
2.34–2.39 (1H, m, CH2–CH–), 2.11–2.23 (1H, m,
CH–CH2–), 2.0–2.02 (1H, m, CH2–) 2.01(3H, s,
CH3–CO–), 1.70–1.80 (2H, m, CH2–CH2–), 1.60–1.70 (2H,
m, CH2–CH2–), 1.19 (3H, d, J = 6.27 Hz, CH3–CH–), 0.92
(3H, d, J = 6.9 Hz, CH3–CH–); 13C NMR (CDCl3, 125
MHz): δ = 170.80 (Ar–CH2C=O), 170.37 (C=O), 170.00
(C=O), 144.50 (C=CH), 138.68 (CH2=C), 133.81 (Ar–
C–), 131.94 (Ar–C–), 130.32 (Ar–CH), 128.74 (Ar–CH),
128.32 (Ar–CH), 128.09 (Ar–CH), 126.31 (CH2=C),
125.89 (Ar–CH), 125.57 (Ar–C–), 124.11 (Ar–CH), 123.90
(Ar–CH), 121.93 (CH=C), 78.66 (CH–O), 74.63
(CH–OAc), 68.00 (CH–OH), 40.92 (O–CH–CH2–CH–O),
39.89 (CH2–CH–CH–O), 39.75 (Ar–CH2–), 36.56 (CH–
CH2–CH–O), 34.46 (=CH–CH2–CH), 25.99 (=C–
CH–CH3), 21.30 (CO–CH3), 21.15 (CH3–CH–O), 19.63
(CH3–CH–C=); IR (neat) γmax 3442, 2956, 2926, 2871,
1762, 1732, 1658, 1597, 1511, 1459, 1372, 1327, 1245,
1128, 1088, 1050, 1018, 956, 856, 783, 609, 539, 510
cm−1; HRESIMS m/z (pos): 499.2091 C29H32NaO6 (calcd.
499.20911).
HRESIMS m/z (pos): 470.14148 C22H25NNaO9 (calcd.
470.14152).
(E)-(1S,3S)-3-acetoxy-1-((3aR,7S,8aS)-7-methyl-3-
methylene-2-oxo-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta
[b]furan-6-yl)butyl 3-(4-methoxyphenyl)acrylate (10)
Yellowish liquid (This compound was prepared by reacting
100 mg isoxanthanol (1) with 4-methoxycinnamic acid (57
mg) in presence of DCC (74 mg), DMAP (4 mg) by the
procedure as described above to obtained 150 mg with 99%
1
yield); H NMR (CDCl3, 400 MHz): δ = 7.98 (1H, d, J =
16.14 Hz, –CH=CH–), 7.50 (1H, m, Ar–H), 7.35 (1H, m,
Ar–H), 6.94 (2H, m, Ar–H), 6.49 (1H, d, J = 16.14 Hz,
–CH=CH–), 6.26 (1H, d, J = 3.2 Hz, CH2=C–), 5.87 (1H,
dd, J = 5.54, 9.2 Hz,=CH–CH2–), 5.52 (1H, d, J = 2.8 Hz,
CH2=C–), 5.20 (1H, dd, J = 5.6, 8.4 Hz, CH–OCO–CH=),
4.96 (1H, dt, J = 6.0 and 12.4 Hz, CH–OAc), 4.66 (1H,
ddd, J = 2.8, 8.8 Hz, CH–OCO–), 3.89 (3H, s, Ar–OCH3),
3.31 (1H, m, –CH–CH2–), 2.52–2.67 (1H, m, CH2–CH–),
2.32–2.51 (1H, m, CH2–CH–), 2.22–2.31 (1H, m,
CH–CH2–), 2.1–2.20 (1H, m, CH–CH2–), 2.01 (3H, s,
CH3–CO–), 1.86–2.05 (1H, m, CH2–CH–) 1.79–1.83 (2H,
m, CH2–CH2–), 1.25 (3H, d, J = 6.3 Hz, CH3–CH–), 1.20
(3H, d, J = 6.9 Hz, CH3–CH–); 13C NMR (CDCl3, 100
MHz): δ = 170.41 (C=O), 170.05 (C=O), 166.75
(CH=CHC=O), 158.43 (Ar–C–O–), 144.62 (C=CH),
140.78 (Ar–CH=CH–), 138.72 (CH2=C), 131.68 (Ar–CH),
129.03 (Ar–C–), 124.59 (CH2=C), 123.23 (Ar–CH),
122.06 (Ar–CH), 120.73 (Ar–CH), 118.34 (Ar–CH=CH–),
111.20 (Ar–CH), 78.87 (CH–O), 74.73 (CH–OAc), 68.15
(CH–OH), 55.51 (CH3–O–), 41.37 (O–CH–CH2–CH–O),
39.96 (CH2–CH–CH–O), 36.95 (CH–CH2–CH–O), 33.70
(=CH–CH2–CH), 26.21 (=C–CH–CH3), 21.63 (CO–CH3),
21.30 (CH3–CH–O), 19.91 (CH3–CH–C=); IR (neat) γmax
3400, 2929, 2873, 2849, 1762, 1734, 1707, 1628, 1597,
1576, 1488, 1464, 1437, 1372, 1322, 1246, 1161, 1122,
1087, 1049, 956, 869, 814, 757, 609 cm−1; HRESIMS m/z
(pos): 491.20417 C27H32NaO7 (calcd. 491.20402).
(1S,3S)-3-acetoxy-1-((3aR,7S,8aS)-7-methyl-3-methylene-
2-oxo-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-6-
yl)butyl 5-nitrofuran-2-carboxylate (9)
Yellowish liquid (This compound was prepared by reacting
100 mg isoxanthanol (1) with nitro furanoic acid (51 mg) in
presence of DCC (74 mg), DMAP (4 mg) by the procedure
as described above to obtained 140 mg with 97% yield); 1H
NMR (CDCl3, 500 MHz): δ = 7.35 (1H, d, J = 3.7 Hz,
Ar–H), 7.30 (1H, d, J = 3.80 Hz), 6.27 (1H, d, J = 3.3 Hz,
CH2=C–), 5.94 (1H, dd, J = 5.54 and 9.2 Hz,=CH–CH2–),
5.53 (1H, d, J = 2.8 Hz, CH2=C–), 5.34 (1H, dd, J = 5.6,
8.4 Hz, CH–OCO–Ar), 4.96 (1H, dt, J = 6.0, 12.4 Hz,
CH–OAc), 4.65 (1H, ddd, J = 2.8, 8.8 Hz, CH–OCO–),
3.27–3.32 (1H, m, –CH2–CH–), 2.60–2.70 (1H, m,
CH2–CH–), 2.40–2.51 (1H, m, CH2–CH–), 2.15–2.33 (1H,
m, CH–CH2–), 2.0–2.13 (2H, m, CH2–CH–), 2.03 (3H, s,
CH3–CO–), 1.83–1.91 (2H, m, CH2–CH2–), 1.19 (3H, d, J
= 6.89 Hz, CH3–CH–), 0.86 (3H, d, J = 6.9 Hz,
CH3–CH–); 13C NMR (CDCl3, 100 MHz): δ = 170.39
(C=O), 169.91 (C=O), 168.71 (C=O), 163.12 (Ar–
C–NO2), 144.71 (Ar–C–), 143.17 (C=CH), 138.48
(CH2=C), 126.76 (CH2=C), 122.36 (CH=C), 119.05 (Ar–
CH), 111.45 (Ar–CH), 78.62 (CH–O), 77.81 (CH–OAc),
67.72 (CH–OH), 41.24 (O–CH–CH2–CH–O), 39.67 (CH2–
CH–CH–O), 36.88 (CH–CH2–CH–O), 33.5 2(=CH–
CH2–CH), 26.30 (=C–CH–CH3), 21.68 (CO–CH3), 21.25
(CH3–CH–O), 20.12 (CH3–CH–C= IR (neat) γmax 3400,
2923, 2851, 1761, 1734, 1625, 1582, 1539, 1351, 1275,
General procedure for preparation of amine adducts
(11–12) of isoxanthanol
The amine adducts were prepared using 1 equiv. of iso-
xanthanol (1) with 2 equiv. of respective amine in methanol
and the resulting reaction mixture stirred at room tempera-
ture for 6 h. The reaction monitored by TLC, and after
completion of the reaction, the contents poured in water,
extracted with ethyl acetate and concentrated on thin film
evaporator under reduced pressure to give the crude product
which on CC in mobile phase 20% EtoAc in hexane
afforded the amine adducts 11 and 12.
1244, 1190, 1127, 1019, 959, 812, 765, 610 cm−1
;

Med Chem Res
(2S,4S)-4-((3S,3aR,7S,8aS)-3-((dimethylamino)methyl)-7-
methyl-2-oxo-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]
furan-6-yl)-4-hydroxybutan-2-yl acetate (11)
(2S,4S)-4-hydroxy-4-((3R,3aR,7S,8aS)-7-methyl-2-oxo-
2,3a,4,4′,5′,7,8,8a-octahydrospiro[cyclohepta[b]furan-3,3′-
pyrazol]-6-yl)butan-2-yl acetate (13)
Yellowish liquid (This compound was prepared by reacting
100 mg isoxanthanol (1) with dimethylamine (140 mg)
to obtained 103 mg with 91% yield); 1H NMR (CDCl3, 400
MHz): δ = 5.69 (1H, dd, J = 5.53, 9.1 Hz,=CH–CH2–),
4.97 (1H, dt, J = 6.0, 12.4 Hz, CH–OAc), 4.48 (1H, ddd, J
= 2.8, 8.8 Hz, CH–OCO–), 4.08 (1H, dd, J = 5.6, 8.4 Hz,
CHOH), 2.65 (5H, m, –NCH3, –NCH2), 2.42 (4H, m,
–NCH3, –CH–CH2–), 2.24 (6H, m,–CH2–, –CH2–CH–,
–CH2–CH–), 2.04 (3H, s, CH3CO–), 1.89 (2H, m,
–CH–CH–), 1.67 (3H, d, J = 6.6 Hz, CH3–CH–), 1.23
(3H, d, J = 6.9 Hz, CH3–CH–); 13C NMR (CDCl3, 125
MHz): δ = 177.60 (C=O), 170.65 (C=O), 138.58
(CH=C), 124.01 (CH=C), 78.95 (CH–O), 73.59
(CH–OAc), 69.31 (CH–OH), 65.80 (–N–CH2), 59.24
(CH2–CH–CH–O), 45.60 (–CH–CH–O), 43.43 (O–CH–
CH2–CH–O), 35.85 (CH–CH2–CH–O), 32.85 (=CH–
CH2–CH), 26.01 (=C–CH–CH3), 23.63 (–N–CH3), 22.07
(CO–CH3), 21.40 (CH3–CH–O), 20.36 (CH3–CH–C=); IR
(neat) γmax 3443, 2921, 1619, 1415, 1020, 773, 607, 519,
506 cm-1; HRESIMS m/z (pos): 354.2269 C19H32NO5
(calcd. 354.2275).
Yellowish liquid (This compound was prepared by reacting
100 mg isoxanthanol (1) with diazomethane in ether
at 0 °C to obtained 111 mg with 98% yield); 1H NMR
(CDCl3, 400 MHz): δ = 5.66 (1H, dd, J = 5.5, 8.9 Hz,
=CH–CH2–), 4.95 (1H, dt, J = 6.02, 12.27 Hz, CH–OAc),
4.46 (1H, ddd, J = 3.01, 8.78 Hz, CH–OCO–), 4.07
(1H, dd, J = 5.67, 8.72 Hz, –CHOH), 3.49 (2H, m,
–CH2–N=), 2.30 (5H, m, –CH2–, –CH2– and –CH–),
2.3 (3H, m, –CH2– and –CH–), 2.04 (3H, s, CH3CO–), 1.88
(2H, m, –CH2–), 1.65 (3H, d, J = 6.59 Hz, CH3–CH–),
1.21 (3H, d, J = 6.80 Hz, CH3–CH–). 13C NMR (CDCl3,
125 MHz): δ = 177.47 (C=O), 170.76 (C=O), 141.04
(–CH=C–), 131.98 (–CH=C–), 96.72 (–C–COO),
76.13 (CH–O), 75.00 (=NCH2−), 72.86 (CH–OAc),
70.09 (CH–OH), 51.12 (–CH–CH–O), 44.57 (–CH2–C–),
44.01 (O–CH–CH2–CH–O), 32.60 (=CH–CH2–CH),
30.49 (–CH–CH2–CH–O), 29.97 (CH3–CH–), 21.84
(CO–CH3), 21.32 (CH3–CH–O), 21.19 (CH3–CH–C=);
HRESIMS m/z (pos): 351.19144 C18H27N2O5 (calcd.
351.1842).
(2S,4S)-4-hydroxy-4-((3S,3aR,7S,8aS)-7-methyl-3-((4-
methylpiperazin-1-yl)methyl)-2-oxo-3,3a,4,7,8,8a-
hexahydro-2H-cyclohepta[b]furan-6-yl)butan-2-yl acetate
(12)
(3aR,7S,8aS)-6-((1S,3S)-1,3-dihydroxybutyl)-7-methyl-3-
methylene-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]
furan-2-one (14)
Yellowish liquid (This compound was prepared by enzy-
matic deacetylation of 100 mg isoxanthanol (1) using
Lipase AS Amano (1 mg) in water to obtained 84 mg with
Yellowish liquid (This compound was prepared by reacting
100 mg isoxanthanol (1) with N-methyl piperzine (32 mg)
to obtained 100 mg with 89% yield); 1H NMR (CDCl3, 400
MHz): δ = 5.67 (1H, dd, J = 5.51, 9.0 Hz, =CH–CH2–),
4.94 (1H, dt, J = 6.03, 12.29 Hz, CH–OAc), 4.44 (1H, ddd,
J = 3.0, 8.8 Hz, CH–OCO–), 4.05 (1H, dd, J = 5.7, 8.6 Hz,
CHOH), 3.36 (4H, m, –N(CH2)2), 2.63 (4H, m, –N(CH2)2),
2.47 (5H, m, –NCH2, –CH2– and –CH–), 2.42 (4H, –CH2–
and –CH2–), 2.32 (3H, s, –NCH3), 2.04 (3H, s, CH3CO–),
1.89 (2H, m, –CH2), 1.67 (3H, d, J = 6.59 Hz, CH3–CH–),
1.23 (3H, d, J = 6.80 Hz, CH3–CH–); 13C NMR (CDCl3,
125 MHz): δ = 177.81 (C=O), 170.65 (C=O), 144.27
(CH=C), 124.69 (CH=C), 79.24 (CH–O), 73.42
(CH–OAc), 69.36 (CH–OH), 65.72 (–N–CH3), 58.04 (–N–
CH2), 57.75(–N–CH2), 54.77 (–N–CH2), 45.57 (–CH–
CH–O), 43.29 (O–CH–CH2–CH–O), 35.85 (CH–
CH2–CH–O), 32.53 (=CH–CH2–CH), 26.63 (=C–
CH–CH3), 23.71 (CH2–CH–CH–O), 21.86 (CO–CH3),
21.44 (CH3–CH–O), 20.31 (CH3–CH–C=); IR (neat) γmax
3436, 2921, 2851, 1733, 1619, 1374, 1247, 1020, 771, 607,
1
98% yield); H NMR (CDCl3, 400 MHz): δ = 6.26 (1H, d,
J = 3.22 Hz, CH2=C–), 5.81 (1H, dd, J = 5.64, 9.21 Hz,
=CH–CH2–), 5.54 (1H, d, J = 2.83 Hz, –CH2=C–), 4.63
(1H, dt, J = 6.03, 12.42 Hz, –CHOCO–), 4.08 (1H, dd, J =
5.63, 8.42 Hz, CHOH) 3.34 (1H, m, –CHCH–), 2.63 (1H,
m, –CH2CH–), 2.47 (1H, m, CH2–CH–), 2.32 (1H, m,
CH–CH2–), 1.82 (1H, m, CH–CH2–), 1.73 (2H, m,
CH–CH2–), 1.60 (2H, m, CH2–CH2–), 1.24 (3H, d, J =
6.46 Hz, CH3–CH–), 1.19 (3H, d, J = 6.83 Hz, CH3–CH–);
13C NMR (CDCl3, 125 MHz): δ = 170.54 (C=O), 148.32
(CH=C), 138.65 (CH2=C), 122.30 (CH2=C), 121.57
(CH=C), 79.29 (CH–O), 76.24 (CH3–CH–OH),
68.84 (–CH2CH–OH), 44.05 (O–CH–CH2–CH–O),
41.50 (CH2–CH–CH–O), 36.81 (CH–CH2–CH–O), 33.28
(=CH–CH2–CH),
(CH3–CH–O), 21.86 (CH3–CH); IR (neat) γmax 3446, 2921,
2851, 1732, 1620, 1374, 1246, 1019, 607, 503 cm−1
25.95
(=C–CH–CH3),
24.16
;
HRESIMS m/z (pos): 289.1408 C15H22NaO4 (calcd.
289.159).
522, 503 cm−1
C22H37N2O5 (calcd. 409.2624).
.
HRESIMS m/z (pos): 409.2696

Med Chem Res
Biological assays
Clonogenic assay
SRB assay for growth inhibition
The clonogenic assay estimates the reproductive viability of
cells treated with cytotoxic agents or anti-cancer ther-
apeutics by assessing their colony formation ability. A549
cells were seeded in six-well tissue culture grade flasks at a
seeding density of 1.2 × 105 cells/well. After 24 h cells
were exposed to various concentrations of compound 10
and kept at 37 °C, 5% CO2 and 98% RH. Paclitaxel was
used as positive control. After 24 h treatment cells were
harvested and counted. Equal numbers of cells were re-
plated and kept in CO2 incubator at 37 °C for a time
equivalent to six cell divisions so that the untreated well
contains sufficiently large clones of 50 or more cells.
Medium was removed and colonies were gently rinsed with
PBS. Cells were fixed with 4% paraformaldehyde for 30
min and then stained with 0.5% crystal violet solution for
1 h. Excess crystal violet was washed off with water and
plates were dried. Colonies having 50 or more cells were
counted (Bhushan et al. 2007).
SRB is a bright-pink aminoxanthene dye that binds to basic
amino acid residues present on TCA fixed cells. Under mild
acidic conditions it easily binds to cells and can be extracted
under mild basic conditions. The amount of dye extracted is
directly proportional to the cell mass. Briefly, cell suspen-
sions of optimum density (7000–12,000 cells/100 µL) were
seeded and incubated for 24 h. Test compounds in complete
growth medium were added along with Erlotinib and
Paclitaxel as positive controls. After 48 h incubation, cells
were fixed with ice-cold TCA, washed, dried and stained
with SRB dye (0.4% w/v SRB in 1% acetic acid for 30
min). The unbound SRB was removed by washing with 1%
acetic acid and plates were dried. The bound dye was
solubilized with 10 mM Tris buffer (pH = 10.4) and reading
was taken at 540 nm on microplate reader (BioTek Synergy
HT). For preliminary screening, 10 and 50 µM concentra-
tions of test compounds were used whereas for IC50 cal-
culation a range of concentrations were used. IC50 value
was determined by non-linear regression analysis using
Prism, version 5.04 from Graph Pad Software (La Jolla,
CA) (Singh et al. 2013).
Wound healing assay
Wound healing assay is used to study cell migration in vitro
which mimics cell migration during wound healing in vivo.
A wound is induced in cell monolayer and images captured
at beginning and at closure of wound in untreated cells.
A549 cells were seeded in 6-well tissue culture grade flasks
at a seeding density of 8 × 105 cells/well and kept in CO2
incubator for 24 h after which a confluent monolayer was
obtained. Scrape-wound was induced using a 1000 µL
sterile pipette tip. After removing the cellular debris with
PBS, cells were exposed to various concentrations of
compound 10 and kept in CO2 incubator at 37 °C. Paclitaxel
was used as positive control. At 0 h and 24 h treatment,
wounded areas were photographed with (10× objective)
Olympus 70× and percentage of wound closure was esti-
mated (Franken et al. 2006).
Cell cycle analysis by flow cytometry
A549 cells (1.2 × 105 cells/mL/six-well plate) were
exposed to varying concentration of compound iso-
xanthanol cinnamate (10) for 24 h. Cells were then trypsi-
nized, washed with PBS and fixed in 70% ethanol at 20 °C,
overnight. After fixing, cells were again washed, digested
with DNase free RNase (100 µg/mL) at 37 °C for 45 min
and stained with propidium iodide (PI) (5 µg/mL). Cells
were then analyzed on flow cytometer FACS Aria (Becton
Dickinson, USA). Sub-G1 cell fraction represents apoptotic
cell population (Majeed et al. 2013).
Statistical analysis
Measurement of mitochondrial membrane potential loss
Data expressed as mean SD or representative of one of three
similar experiments unless otherwise indicated. Compar-
isons were made between control and treated groups using
Student’s t-test. P > 0.05 values were considered significant.
Changes in mitochondrial transmembrane potential were
measured after staining with rhodamine-123. A549 cells
(1.2 × 105 cells/mL/six-well plate) were exposed to
varying concentration of compound 10 for 24 h. Rh-123
(200 nM) was added to each well before 1 h termination.
Cells were collected at 3000 rpm for 5 min, washed
once with PBS, resuspended in 500 µL of PBS and mito-
chondrial membrane potential loss was analyzed on flow
cytometer FACS Aria (Becton Dickinson, USA) (Singh
et al. 2013).
Results and discussion
Chemistry
Isoxanthanol (1) isolated from the Xanthium spinosum and
well characterized through spectroscopic techniques and

Med Chem Res
data were found in agreement with literature. Further its
absolute configuration was confirmed by X-ray crystal-
lography (see Fig. 2) and this is the first report of the
establishment of absolute stereochemistry by X-ray analy-
sis. Isoxanthanol displayed anti-proliferative activity against
different human cancer cell lines leading thereby to the
establishment of its anticancer activity. Furthermore, che-
mical modification of the parent molecule was undertaken
in a bid to generate more potent molecule/s than the parent
compound 1. The chemical modifications were carried out
at different active sites of the molecule (Fig. 1) and that
included (i) α-methylene part of γ-butyro lactone double
bond (from the literature survey the exocyclic double bond
seems to be as an important functionality for the chemical
modification for anticancer activity) (Winters et al. 1969;
Woods et al. 2013; Baraldi et al. 2004), (ii) on hydroxyl
group, and (iii) acetyl group of the molecule.
The chemical identity of the products was confirmed by
spectral analysis i.e., appearance of additional signals of
alkyl substituent of amine moiety of 11 and 12 in 1H NMR
and 13C spectra along with disappearance of the signal for
α-methylene (=CH2) protons in 11, 12, and 13, which was
fully supported by HRMS data of the assigned chemical
structures.
In an attempt for de-acetylation of 1 to obtain diol product
(14) and bio-evaluate it vis a vis with the parent molecule for
the activity profiling. Our initial attempts of de-acetylation
through different reagents and conditions such as ammonia/
methanol, K2CO3/methanol proved unsuccessful and com-
plex mixture was obtained presuming involvement of
nucleophilic addition as well as lactone ring opening reac-
tions under the basic reaction conditions with perhaps sol-
vent itself acting as a nucleophile. These complex reaction
products were abandoned and not followed further. In order
to get compound 14, commercially available lipases were
used and the reaction carried out under mild and neutral
reaction conditions. The enzymatic reaction (Scheme 3) was
successful and the desired product 14 was isolated in 98%
yields (See Supporting Information, Table-S3).
Chemical modification at the hydroxyl group of com-
pound 1 was carried out to get desired acyl, aryl, and cin-
namyl ester derivatives 2–10 (Scheme 1). The formation of
acyl derivatives were confirmed by IR, NMR, and mass
spectral data, appearance of bands for carbonyl (–CO)
group of ester moiety in IR spectrum at ~1733 cm−1, and
observance of signals for the protons of acetyl (CH3CO),
aryl (aromatic protons) and olefinic protons of the cinna-
Pharmacological evaluation
1
moyl moiety (–CH=CH–CO) in H NMR spectrum. Fur-
Isoxanthanol (1) and its derivatives (2–14) were screened
(Table 1) at 10 and 50 µM concentration for their cytotoxic
potential using sulforhodhamine B assay against four
human cancer cell lines including lung (NCI-H322 and
A549), glioblastoma (T98G) and epidermal (A431). Pacli-
taxel and erlotinib were taken as positive controls for the
experiment. At the 50 µM concentration isoxanthanol deri-
vatives 2–4, 7, 8, 10, and 11 showed significant activity
ther HRESIMS data corroborated with the assigned
structure of the isoxanthanol derivatives.
The chemical modification at the α-methylene group of
the γ-butyro lactone of 1 was carried out to furnish amino
adducts 11–12 (obtained through amination), and pyrazo-
line derivative 13 (obtained by diazomethane reaction) as
shown in Scheme 2.
Scheme 1 Derivatization of
isoxanthanol (1) at hydroxyl
group
Scheme 2 Derivatization at α-
methylene group of isoxanthanol
(1)

Med Chem Res
Scheme 3 Enzyme catalyzed
deacetylation of isoxanthanol (1)
Table 1 In vitro screening of isoxanthanol (1) and its derivatives (2–
14) against various human cancer cell lines
Table 2 IC50 values (in µM) of selected compounds against human
cancer cell lines
Compounds
Conc. (µM)
T98G
% Growth inhibition
Tissue
Glioblastoma Epidermal Lung
Lung A549
compounds
cell line
T98G
A431
NCI-
H322
A431
NCI-322
A549
1
10
50
10
50
10
50
10
50
10
50
10
50
10
50
10
50
10
50
10
50
10
50
10
50
10
50
10
50
5
0
49
0
6
65
34
87
54
100
22
99
4
11
65
16
60
8
9
20
14
81
43
91
55
95
42
89
17
79
6
2
26.8 2.8
35 3.6
35.7 1.7
>50
10 0.5
0.2
12.2 1.3 19.8 2.2
29 5.7
18 1.5
3
6
22 1.7
7
1.2
2
4
7.2 0.6
17 4.2
82
17
44
20
75
0
7
21
9
1
34 4.5
13 0.5
3
8
32
14
1
1
4
0.4
5
0.7
1.9 0.3
>50 14.3
100
14
100
9
10
11
2.3 0.1
16.4
3
0.2
4
48.4
2
2
5
80% and >90% cytotoxicity against T98G and the rest of
the cell lines respectively. Compound 11 exhibited >90%
cytotoxicity against A431 and A549. Screening of the
molecules at 10 µM concentration showed interesting
results, in particular, for the compound 10 which displayed
the best activity profile against A431, NCI-H322, and A549
cells lines and high percentage of growth inhibition (99, 89,
and 95%) against the respective cell lines. The preliminary
cytotoxic screening data of these compounds at the 10 and
50 µM is shown in Table 1. The compounds that were
showed more potent activity than isoxanthanol were further
selected for the determination of their IC50 values (Table 2)
by non-linear regression analysis using Prism, version 5.04
from Graph Pad Software (La Jolla, CA).
Compounds 3, 4, 8, and 10 displayed promising IC50
values of 7, 7.2, 5, and 1.9 µM respectively for the lung
cancer line A549. Compounds 2, 3, 8, and 10 displayed
IC50 values of 10, 6, 9, and 2.3 µM for epidermal carcinoma
A431. Since compound 10 exhibited maximum cytotoxic
effect with IC50 values ranging from 1.9 to 14 µM against
the four cell lines, it was, therefore, chosen for further
mechanistic studies.
18
4
34
0
56
0
6
22
0
14
0
9
7
13
80
52
100
3
7
100
32
100
0
90
56
93
0
8
0
80
0
9
0
8
29
89
100
0
33
95
100
35
95
23
50
8
10
11
12
13
14
25
100
0
99
100
21
93
4
58
0
37
2
9
58
14
27
23
44
86
–
16
13
21
0
0
1
28
14
38
–
1
4
32
78
–
Erlotinib
47
–
Paclitaxel
1
80
Cell death inducing potential of compound 10 was also
assessed using flow cytometry through determination of
sub-G1 cell population by PI staining. A549 cell line treated
with 10 showed concentration dependent increase in
apoptotic population after 24 h of incubation (Fig. 3) (Lowe
and Lin 2000).
As compared to 3% in negative control, sub-G1 popu-
lation was found 6.5, 11.3, 24.4, 38.8, and 51% at 2, 5, 10,
20, and 30 µM concentration of the compound 10. In further
The bold values indicate growth inhibition more than 70%
against two or more human cancer cell lines (epidermal:
A431, and lung: A549) than the parent molecule 1. Com-
pound 2 showed >80% cytotoxicity against T98G, A431
and A549 whereas 3, 4, and 7 (except NCI-H322 displaying
80% inhibition) showed >90% cytotoxicity against A431,
NCI-H322 and A459 cell lines. Compound 8 displayed

Med Chem Res
Fig. 3 Cell cycle analysis of compound 10 treated A549 cancer cells. A549 cells were treated with different conc. (2, 5, 10, 20, and 30 µM) of
compound 10 to determine the sub-G1 (P1) population indicative of DNA damage
study, the effect of 10 on mitochondrial functioning was
also investigated. Cytochrome-c is released on opening of
mitochondrial permeability transition pore resulting in
mitochondrial membrane potential loss (Heiden et al. 1997).
A concentration dependent loss of mitochondrial membrane
potential (Δψm) was established for compound 10 and same
is shown in Fig. 4. While untreated control A549 cells
showed 3.2% MMP loss, 5.1, 12.2, 46.5 65.3, and 96.7%
MMP loss at 2, 5, 10, 20, and 30 µM concentration for
compound 10.
The clonogenic assay was also performed using A549
cells. Compound 10 produced concentration dependent
inhibitory effect on the cells to reproduce and form colo-
nies. The number of colonies formed at lower concentra-
tions was comparable to untreated control but at higher
concentration less number of colonies was formed. No
colony was formed at 20 µM shown in Fig. 5.
Further, in vitro cell migration assay was performed to
estimate the metastatic potential of A549 cells. Compound
10 treated cells were photographed and cell migration was
assessed by comparing the gap size between the untreated
and treated wells. A concentration dependent inhibitory
effect could be seen on the motility of cells as shown in
Fig. 6. The negative control had almost completely filled the
scratched area, whereas treatment with 10 and 15 µM of
compound 10 significantly (p < 0.001) inhibited motility of
A549, as did treatment with paclitaxel.
With respect to structure–activity relationship, some
trends could be observed. For example, influence of nature/
size of the acyl substituent on the activity was observed, and

Med Chem Res
150
100
50
**
**
*
**
*
0
0
2µM
5µM 10µM 20µM 30µM
Fig. 4 a Compound 10 induced mitochondrial membrane potential
loss in A549 cells. Cells were treated with different conc. (2, 5, 10, 20,
and 30 µM) of 10 for 24 h. b Compound 10 induced mitochondrial
membrane potential loss in A549 cells. Cells were treated with dif-
ferent conc. (2, 5, 10, 20, and 30 µM) of compound 10 for 24 h. Cells
were stained with 200 nM Rh-123 and analyzed by flow cytometer

Med Chem Res
Fig. 5 Compound 10 inhibits the colony formation ability of A549
cells. Indicated concentrations of compound 10 were added to A549
cells. After 24 h treatment, cells were harvested; washed and equal
number of cells from each treatment was re-seeded in six-well plate for
a time equivalent to at least six cell divisions. Colonies were then fixed
and stained with 0.5% crystal violet. Number of colonies was counted.
*P < 0.05, **P < 0.01, ***P < 0.005 Compared with untreated control
Fig. 6 Compound 10 inhibits the motility of A549 cells. A549 cells
(8 × 105 cells/mL/six well plate) were grown to confluence and
scratched with sterile tip: compound 10 was added as indicated.
Scratched areas were photographed (Olympus 70×) at 0 h and then at
24 h to assess the degree of wound healing. Scratched areas were
quantified in each treatment and data were calculated from two inde-
pendent experiments. *P < 0.05, **P < 0.001 compared with untreated
control
among the aliphatic acyl substituents, lower homologs
exerted greater influence than the higher homologs resulting
in the increase of activity. Among the aryl moiety bearing
alkenoic substituents, electron releasing group showed
augmentation in the activity of the compound and opposite
effect was observed for the electron withdrawing groups.
Loss of activity was observed for deacylated product
showing thereby that acetyl group apparently contributes
towards the anticancer activity of 1. Similarly, loss of sp2
character of the substituent in lactone ring also lead to
deterrent effect on the activity.
Conclusion
In summary, the cytotoxic activity of sesquiterpene lactone
isoxanthanol (1) and its thirteen analogs has been estab-
lished against various human cancer cell lines and C-3

Med Chem Res
Dar AA, Dangroo NA, Raina A, Qayum A, Singh SK, Kumar A,
Sangwan PL (2016) Biologically active xanthones from Codo-
nopsis ovata. Phytochemistry 132:102–108
Efferth T (2006) Molecular pharmacology and pharmacogenomics of
artemisinin and its derivatives in cancer cell. Curr Drug Targets
7:407–421
Favier LF, Maria AOM, Wendel GH, Borkowski EJ, Giordano OS,
Pelzer L, Tonn CE (2005) Anti-ulcerogenic activity of xantha-
nolide sesquiterpenes from Xanthium cavanillesii in rats. J Eth-
nopharmacol 100:260–267
Franken NAP, Rodermond HM, Stap J, Haveman J, Bree CV (2006)
Clonogenic assay of cells in vitro. Nat Protoc 5:2315–2319
Gach K, Dalugosz A (2014) α-Methylene-γ-lactones as a novel class
of anti-leukemic agents. Anticancer Agents Med Chem
14:688–694
cinnamyl analog (10) found most active displaying single
digit IC50 value for lung cancer cell line. Concentration
dependent increase in apoptotic population after 24 h of
incubation with 51% sub-G1population at 30 µM con-
centration against A549 cell line has been established for
compound 10 (using flowcytometry). Colony formation on
lung cancer cell line at 20 µM concentration has been
established through clonogenic assay. In in vitro cell
migration, the metastatic potential of A549 cells, has been
arrested significantly (p < 0.001) at 10 and 15 µM con-
centration of potent molecule 10.
Acknowledgements The CSIR is well acknowledged for financial
support under 12th five-year project BSC0108. The authors thank
Botany division of the Institute for providing the plant material,
Instrumentation division for spectral data, and CSIR-UGC for the
award of Fellowship to one of the author (PKC). IIIM publication no.
IIIM/2011/2017.
Ghantous A, Gali-Muhtasib H, Vuorela H, Saliba NA, Darwichem N
(2010) What made sesquiterpene lactones reach cancer clinical
trials? Drug Discov Today 15:668–678
Ghantous A, Sinjab A, Herceg Z, Darwiche N (2013) Parthenolide:
from plant shoots to cancer roots. Drug Discov Today
18:894–905
Gupta N, Sharma S, Raina A, Dangroo NA, Bhushan S, Sangwan PL
(2016) Synthesis and anti-proliferative evaluation of novel 3,4-
dihydro-2H-1,3-oxazine derivatives of bakuchiol. RSC Adv 6
(108):106150–106159
Harvey AL, Edrada-Ebe R, Quinn RJ (2015) The re-emergence of
natural products for drug discovery in the genomics era. Nat Rev
Drug Discov 14:111–129
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
Heiden MGV, Chandel NS, Williamson EK, Schumacker PT,
Thompson CB (1997) Bcl-xL regulates the membrane potential
and volume homeostasis of mitochondria. Cell 91:627–637
Jordan CT (2006) Searching for leukemia stem cells-not yet the end of
the road? Cancer Cell 10:253–254
Khan I, Guru SK, Rath SK, Chinthakindi PK, Singh B, Koul S,
Bhushan S, Sangwan PL (2016) A novel triazole derivative of
betulinic acid induces extrinsic and intrinsic apoptosis in human
leukemia HL-60 cells. Eur J Med Chem 108:104–116
Lowe SW, Lin AW (2000) Apoptosis in cancer. Carcinogenesis
21:485–495
Marco JA, Sanz-Cervera JF, Corral J, Carda M, Jakupovic J (1993)
Xanthanolides from Xanthium: absolute configuration of xan-
thanol, isoxanthanol and their C-4 epimers. Phytochemistry
34:1569–1576
Majeed R, Reddy MV, Chinthakindi PK, Sangwan PL, Hamid A,
Chashoo G, Saxena AK, Koul S (2012) Bakuchiol derivatives as
novel and potent cytotoxic agents: a report. Eur J Med Chem
49:55–67
References
Abdei-Mogib M, Dawidar AM, Metwally MA, Abou-Elzahab M
(1991) Xanthanolides from Xanthium spinosum. Phytochemistry
30:3461–3462
Baraldi PG, Nunez MC, Tabrizi MA, Clercq ED, Balzarini J, Bermejo
J, Estevez F, Romagnoli R (2004) Design, synthesis, and biolo-
gical evaluation of hybrid molecules containing α-methylene-γ-
butyrolactones and polypyrrole minor groove binders. J Med
Chem 47:2877–2886
Bhushan S, Kumar A, Malik F, Andotra SS, Sethi VK, Kaur IP, Taneja
SC, Qazi GN, Singh J (2007) A triterpenediol from Boswellia
serrata induces apoptosis through both the intrinsic and extrinsic
apoptotic pathways in human leukemia HL-60 cells. Apoptosis
12:1911–1926
Bohlmann F, Singh P, Joshi KC, Singh CL (1982) Xanthanolides from
Xanthium indicum. Phytochemistry 21:1441–1443
Majeed R, Sangwan PL, Chinthakindi PK, Dangroo NA, Thota N,
Hamid A, Sharma PR, Saxena AK, Koul S (2013) Synthesis of 3-
O-propargylated betulinic acid and its 1,2,3-triazoles as potential
apoptotic agents. Eur J Med Chem 63:782–792
Newman DJ, Cragg GM (2012) Natural products as sources of new
drugs over the 30 years from 1981 to 2010. J Nat Prod
75:311–335
Nour AMM, Khalid SA, Kaiser M, Brun R, Abdallah WE, Schmidt TJ
(2009) The antiprotozoal activity of sixteen asteraceae species
native to sudan and bioactivity-guided isolation of xanthanolides
from Xanthium brasilicum. Planta Med 75:1363–1368
Omar AA, Elrashidy EM, Ghazy NA, Metwally AM, Ziesche J,
Bohlmann F (1984) Xanthanolides from Xanthium spinosum.
Phytochemistry 23:915–916
Schmidt TJ (1997) Helenanolide-type sesquiterpene lactones-III Rates
and stereochemistry in the reaction of helenalin and related
helenanolides with sulfhydryl containing biomolecules. Bioorg
Med Chem 5:645–653
Chinthakindi PK, Sangwan PL, Farooq S, Aleti RR, Kaul A, Saxena
AK, Murthy YLN, Vishwakarma RA, Koul S (2013) Diminutive
effect on T and B- cell proliferation of non-cytotoxic α-santonin
derived 1,2,3-triazoles: a report. Eur J Med Chem 60:365–375
Chinthakindi PK, Singh J, Gupta A, Nargotra A, Mahajan P, Kaul A,
Ahmed Z, Koul S, Sangwan PL (2017) Synthesis of α-santonin
derivatives for diminutive effect on T and B-cell proliferation
and their structure–activity relationship. Eur J Med Chem
127:1047–1058
Christensen SB, Skytte DM, Denmeade SR, Dionne C, Moller JV
(2009) A Trojan horse in drug development: targeting of thap-
sigargins towards prostate cancer cells. Anticancer Agents Med
Chem 9:276–294
Cragg GM, Grothaus PG, Newman DJ (2014) New horizons for old
drugs and drug leads. J Nat Prod 77:703–723
Dangroo NA, Singh J, Gupta N, Singh SK, Koul A, Khuroo MA,
Sangwan PL (2017) T- and B-cell immunosuppressive activity of
novel α-santonin analogs with humoral and cellular immune
response in mice. Med Chem Commun 8:211–219

Med Chem Res
Singh B, Guru SK, Kour S, Jain SK, Sharma R, Sharma PR, Singh SK,
Bhushan S, Bharate SB, Vishwakarma RA (2013) Synthesis,
antiproliferative and apoptosis-inducing activity of thiazolo[5,4-
d]pyrimidines. Eur J Med Chem 70:864–874
Taha M, Ismaila NH, Imrana S, Mohamada MH, Wadood A, Rahimd
F, Saade SM, Rehman AU, Khane KM (2016) Synthesis, α-
glucosidase inhibitory, cytotoxicity and docking studies of 2-aryl-
7-methylbenzimidazoles. Bioorg Chem 65:100–109
Taha M, Shah SAA, Afifi M, Zulkeflee M, Sultan S, Wadood A,
Rahim F, Ismail NH (2017) Morpholine hydrazone scaffold:
synthesis, anticancer activity and docking studies. Chin Chem
Lett 28:607–611
Vasas A, Hohmann J (2011) Xanthane sesquiterpenoids: structure,
synthesis and biological activity. Nat Prod Rep 28:824–842
Winters TE, Geissman TA, Safir D (1969) Sesquiterpene lactones of
Xanthium species Xanthanol and isoxanthanol, and correlation of
xanthinin with ivalbin. J Org Chem 34:153–155
Woods JR, Mo H, Bieberich AA, Alavanja T, Colby DA (2013)
Amino-derivatives of the sesquiterpene lactone class of natural
products as prodrug. Med Chem Commun 4:27–33
Yoon JH, Lim HJ, Lee HJ, Kim HD, Jeon R, Ryu J-H (2008) Dis-
covery of betrixaban (PRT054021), N-(5-chloropyridin-2-yl)-2-
(4-(N,N-dimethylcarbamimidoyl)benzamido)-5-methox-
ybenzamide, a highly potent, selective, and orally efficacious
factor Xa inhibitor. Bioorg Med Chem Lett 18:2179–2185
Zhang Q, Lu Y, Ding Y, Zhai J, Ji Q, Ma W, Yang M, Fan H,
Long J, Tong Z, Shi Y, Jia Y, Han B, Zhang W, Qui C,
Ma X, Li Q, Shi Q, Zhang H, Li D, Zhang J, Lin J, Li L,
Gao Y, Chen Y (2012) Guaianolide sesquiterpene lactones, a
source to discover agents that selectively inhibit acute myelo-
genous leukemia stem and progenitor cells. J Med Chem
55:8757–8769
Zhou J, Zhang Y (2008) Cancer stem cells: models, mechanisms and
implications for improved treatment. Cell Cycle 7:1360–1370