T- and B-cell immunosuppressive activity of novel α-santonin analogs with humoral and cellular immune response in Balb/c mice

Article abstract of DOI:10.1039/c6md00527f

In continuation of our endeavours to synthesize immunosuppressive agents from α-santonin, we report herein the design and synthesis of a new series of α-santonin derived O-aryl/aliphatic ether, ester and amide analogs and the evaluation of their immunosuppressive activities. The in vitro studies led to several analogs with significant immunosuppressive effects by inhibiting ConA and LPS stimulated T- and B-cell proliferation in a dose dependent manner. The more significant compounds 4d, 4e, 4f, 4h, 6a and 6b displayed potent inhibitory activity on the mitogen-induced T- and B-cell proliferation in comparison to α-santonin 1. Compound 4e displayed stupendous in vitro immunosuppressive effects with ～80% suppression of B and ～75% suppression of T lymphocyte proliferation, respectively. The in vivo investigation on BALB/c mice revealed that non-cytotoxic compound 4e suppresses both humoral and cellular immunity.

Full text of DOI:10.1039/c6md00527f

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Page 1 of 25
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DOI: 10.1039/C6MD00527F
Tꢀ and Bꢀcell immunosuppressive activity of novel αꢀsantonin analogs with humoral and
cellular immune response in Balb/c mice†
Nisar A. Dangrooa, Jasvinder Singhb,c, Nidhi Guptaa, Shashank Singhb,c, Anapurna Kaulb,
Mohmmed A. Khurood, and Payare L. Sangwana,c,*
b
aBioorganic Chemistry Division, Cancer Pharmacology Division, CSIR-Indian Institute of
Integrative Medicine, Canal Road Jammu-180001, India
cAcademy of Scientific and Innovative Research (AcSIR), CSIR-IIIM Campus, Jammu, India
dUniversity of Kashmir, Hazaratbal Road, Srinagar, India
#IIIM publication no. IIIM/1970/2016
*Corresponding authors. Tel.: +91 191 2585006ꢀ13 Extn. 371; fax +91 191 2586333.
E-mails; plsangwan@iiim.ac.in (P.L. Sangwan)
†
The authors declare no competing interests.

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Abstract
In continuation of our endeavours to synthesize immunosuppressive agents from αꢀsantonin,
herein, a new series of αꢀsantonin derived Oꢀaryl/aliphatic ether, ester and amide analogs
were designed and synthesized to evaluate their immunosuppressive activity. The in-vitro
studies revealed in finding of several analogs with significant immunosuppressive effects by
inhibiting ConA and LPS stimulated Tꢀ and Bꢀcell proliferation in a dose dependent manner.
More significant compounds 4d, 4e, 4f, 4h, 6a and 6b displayed potent inhibitory activity on
the mitogenꢀinduced Tꢀ and Bꢀcell proliferation in comparison to αꢀsantonin 1. Compound 4e
displayed the stupendous in vitro immunosuppressive effects with ~80% suppression of B
and ~75% suppression of T lymphocyte proliferation, respectively. The in vivo investigation
on BALB/c mice revealed that nonꢀcytotoxic compound 4e suppresses both humoral and
cellular immunity.
Key words: αꢀsantonin, immunosuppressive agents, Bꢀ and Tꢀcell, humoral, cellular immune
response
1. Introduction
Natural products (NPs) have been proved the promising source of leads for drug discovery,
and in fact oneꢀthird of the topꢀselling drugs in the world are either NPs or NPꢀderived.1, 2 In
the field of immunology, the discovery of cyclosporin A from fungus Cylindrocarpon
lucidum revolutionised organ transplantation.3 Since then, NPs have played a dominant role
in the development of most efficient immunosuppressive drugs including cortisol, tacrolimus,
rapamycin, rituximab, everolimus as well as a number of NPs and their analogs which are
under clinical trials.4 The T lymphocytes undoubtedly play a key role for the initiation of an
immune response in transplant rejection and other autoimmune diseases. Tꢀcell proliferation
results from activation by antigenꢀpresenting cells (APCs) in combination with the major
histocompatibility complex class II and B7 complex. This mechanism results in activation of

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calcineurin, which leads to production of interleukinꢀ2 (IL2). Autocrine stimulus by
interleukinꢀ2 results in Tꢀcell proliferation.5 Immunosuppressive drugs for instance
cyclosporin A inhibits the abnormal activation and proliferation of T lymphocytes and the
immune system associated with organ transplantation and other cellꢀmediated autoimmune
diseases.6 Side effects associated with these drugs along with their high cost have enforced
the need for investigation of alternative drugs, especially those belonging to the traditional
system of medicine with better safety profile.
The plant Artemisia laciniata (family asteraceae) usually found at altitudes from 2700 to
6300 m in Himalayan regions and widely distributed in Jammu and Kashmir, Leh, Ladakh,
Uttar Pradesh area of India.7 The plant has been traditionally used to treat jaundice, gall
bladder, high fever and blood purification.8 αꢀSantonin, a major chemical constituent of the
plant possess several biological activities including anticancer, antifungal and antiꢀ
inflammatory.9ꢀ11 We have explored the immunosuppressive activity of αꢀsantonin derived
triazoles in our previous communications.12,13 These findings indicate that αꢀsantonin
symbolized an ideal scaffold for synthesis of diverse analogues for exploration of their
structure activity relationship (SAR).
Acetyl αꢀdesmotroposantonin 2 exhibited potent antiꢀ
proliferation effects against lymphocyte than parent molecule12 and this indicated that
compound 2 could be a useful scaffold for synthesis of new chemical entities (NCEs) to
understand SAR for the immunosuppressive activity. Therefore, in continuation to our
research interest for the design and synthesis of potent NCEs through the structural
modification of NPs,12-15 compound 2 was used as a starting material and a series of new αꢀ
santonin derived Oꢀaryl/aliphatic ether, ester and amide analogs were synthesized for
evaluation of carbon chain length effect on immunosuppressive activity in the present study.
Several analogs displayed potent immunosuppressive activity and compound 4e showing the
stupendous in vitro activity was further investigated for in vivo activity in BALB/c mice

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models for cellular immune response and sheep red blood cell (SRBC) induced Humoral
antibody production. Both in vitro and in vivo results demonstrate immunosuppressive
activity of the compound 4e.
2. Result and discussion
2.1. Chemistry
αꢀSantonin 1 was isolated from dichloromethaneꢀmethanol extract of the aerial parts of
Artemisia laciniata. Compounds 2 and 3 were synthesised as reported in our previous
communication12. Briefly, NP santonin 1 was subjected to dienone phenone type reaction
using Ac2O/H2SO4 to get acetyl αꢀdesmotroposantonin 2, which on deacetylation in NH3:
MeOH furnished αꢀdesmotroposantonin 3. The ester analogs 4aꢀe were prepared by treating 3
with appropriate anhydride in dry dichloromethane (DCM) in presence of pyridine at room
while analogs 4fꢀh were synthesized by treatment of 3 with appropriate acid chloride in dry
dichloromethane in good to excellent yields (scheme 1).
Scheme 1. Synthetic route for compounds 4aꢀh
O
R
O
O
O
4aꢀe
2
CH3CH2CH2CH2ꢀ
a
b
c
R =
CH3CH2CH2CH2CH2ꢀ
O
Ac2O, H2SO4
0 °C
NH3:MeOH (1:1)
CH3CH2CH2CH2CH2CH2CH2ꢀ
(CH3)2CHꢀ
O
O
HO
d
e
O
O
O
(CH3)3Cꢀ
O
1
O
O
2
3
O
R
O
O
O
4fꢀh
CH3CH2ꢀ
C6H5ꢀ
R = f
g
CH3CH2CH2ꢀ
h
Compound 5 was prepared by reacting 3 with succinic anhydride in dry DCM by employing
different bases like NaHCO3, K2CO3, pyridine, Et3N, dimethyl amino pyridine (DMAP) and
1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene (DBU). However, best results were obtained in DMAP.
Compound 5 on reaction with SOCl2 in dry DCM at refluxing resulted in situ generation of

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acid chloride, followed by the addition of appropriate amine afforded compounds 6aꢀc
(scheme 2).
Scheme 2. Synthetic route for compounds 6aꢀc
O
O
O
, DMAP
O
H
HO
i) SOCl2, reflux
N
HO
O
O
R
O
O
O
O
DCM, rt, 2ꢀ6 h
ii) RNH2, DCM, rt
O
O
O
O
3
5
O
6aꢀc
R
Cl
F
O
b
a
c
Ether analogs were synthesised by the treatment of 3 with different alkyl halides in DCM in
the presence of pyridine at room temperature to afford 7aꢀg (scheme 3). All the analogs were
characterized by 1H NMR, 13C NMR and HRESIMS.
Scheme 3. Synthetic route for compounds 7aꢀg
RBr
R
K2CO3, DCM, rt
HO
O
O
O
3
7aꢀg
O
O
a
b
c
R =
CH3 -
CH3CH2 -
CH3CH2CH2 -
d
e
f
CH3CH2CH2CH2 -
CH3CH2CH2CH2CH2 -
PhCH2-
g
p Cl-PhCH2-
2.2. Biological activity
2.2.1. In Vitro Effect on Lymphocyte Proliferation
All synthesized compounds along with αꢀsantonin 1 were screened for their ability to inhibit
lymphocyte proliferation at three different (1, 10, 100 µM) concentrations (Table 1).
Lipopolysaccharide (LPS) and concanavalin A (Con A) were used to selectively stimulate B
and T lymphocytes, respectively. The proliferation of Bꢀ and Tꢀcells was determined by

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measuring the absorbance using microplate reader. Several analogs displayed improved antiꢀ
proliferation activity than parent molecule 1 in a dose dependent manner.
Table 1. In vitro lymphocyte proliferation activity of compound 1 and its analogs at three different
concentrations (1, 10 and 100 µM).
Compounds
Conc.
(µM)
Cytotoxicity
LPS mean
± SE
LPSꢀinduced
Bꢀcell
proliferation
rate (%)
ꢀ
ConꢀA mean
± SE
ConA ꢀ
induced Tꢀcell
proliferation
rate (%)
Blank
Control
1
ꢀ
ꢀ
0.06 ± 0.00
1.13 ± 0.12
0.49 ± 0.35
0.50 ± 0.02
1.23 ± 0.76
0.38 ± 0.04
0.73 ± 0.25
0.50 ± 0.04
0.50 ± 0.02
0.91 ± 0.02
0.89 ± 0.02
0.90 ± 0.12
0.49 ± 0.04
0.94 ± 0.18
1.03 ± 0.10
0.71 ± 0.01
0.74± 0.33
1.51 ± 0.38
1.47 ± 0.36
0.60 ± 0.27
1.46 ± 0.06
1.16 ± 0.34
0.64 ± 0.30
0.22 ± 0.00
0.38 ± 0.13
0.58 ± 0.33
0.34 ± 0.15
0.64 ± 0.08
0.90 ± 0.18
0.79 ± 0.07
0.54 ± 0.02
0.78 ± 0.01
0.04± 0.00
0.60 ± 0.01
0.21 ±0.02
1.10 ±0.45
0.80 ±0.03
0.64 ± 0.19
0.30 ± 0.09
0.16 ± 0.01
0.21±0.00
0.25± 0.00
0.32± 0.08
0.91± 0.16
0.50 ± 0.31
0.15 ± 0.00
1.25 ± 0.07
1.70 ± 0.29
0.19 ± 0.01
0.74±0.14
1.49±0.53
0.21±0.00
1.23 ± 0.09
0.16 ± 0.00
0.14± 0.01
0.15 ± 0.01
0.25 ± 0.01
0.39 ± 0.15
0.35 ± 0.16
0.40 ± 0.18
0.53 ± 0.19
0.88 ± 0.16
0.43 ± 0.20
0.20± 0.01
ꢀ
ꢀ
ꢀ
100
10
1
ꢀ
ꢀ
33.25↓
27.12↓
19.00↓
67.99↓
44.27↓
51.83↓
38.25↑
24.87↓
23.28↓
6.25↑
8.16↓
3.55↑
ꢀ
35.36↓
63.00↓
45.24↓
39.72↓
58.13↑
27.35↓
22.05↓
28.16↑
29.57↓
43.23↓
78.87↓
46.05↓
39.43↓
4.22↓
100
10
1
+
ꢀ
2
ꢀ
100
10
1
+
+
+
+
ꢀ
3
100
10
1
4a
4b
4c
4d
4e
4f
4g
48.95↓
2.08↓
ꢀ
100
10
1
+
+
ꢀ
7.29↑
26.04↓
22.91↓
57.29↓
53.12↑
37.5↓
100
10
1
+
ꢀ
43.12↓
50.42↓
73.23↓
61.46↓
53.28↓
75.00↓
58.11↓
35.11↓
41.00↓
33.00↓
11.66↓
23.94↑
39.43↓
71.83↓
ꢀ
100
10
1
ꢀ
52.08↓
33.33↓
20.83↑
80.53↓
66.00↓
48.67↓
69.00↓
43.00↓
20.00↓
17.70↓
43.75↓
18.75↓
ꢀ
ꢀ
100
10
1
ꢀ
ꢀ
ꢀ
100
10
1
+
+
+
ꢀ
100
10
1
ꢀ
ꢀ

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100
10
1
ꢀ
ꢀ
0.29 ± 0.14
0.62 ± 015
0.84 ± 0.04
1.01 ± 0.11
0.64 ± 0.06
0.55 ± 0.02
0.25 ± 0.01
0.63 ± 0.36
0.89 ± 0.24
0.36 ± 0.00
0.41 ± 0.13
0.63 ± 0.27
0.26 ± 0.00
0.54 ± 0.17
0.93 ± 0.17
0.89 ± 0.13
0.91 ± 0.02
0.65 ± 0.21
0.61 ± 0.03
1.15 ± 0.31
0.33± 0.11
0.64 ± 0.08
0.34 ± 0.15
0.37 ± 0.18
1.11 ± 0.05
1.25 ± 0.10
1.38 ± 0.09
1.70 ± 0.05
1.23 ±0.05
1.32 ± 0.09
1.46 ± 0.27
0.29± 0.05
0.52± 0.28
1.45 ± 0.14
0.71 ± 0.47
0.71 ± 0.47
0.15 ± 0.00
0.37 ± 0.16
0.57 ± 0.28
1.29 ± 0.08
0.21± 0.00
0.21± 0.01
0.20 ± 0.12
0.30 ± 0.10
0.50 ± 0.12
0.21 ± 0.11
0.29 ± 0.32
0.49 ± 0.21
0.25 ± 0.01
0.39 ± 0.07
0.59 ± 0.20
1.30± 0.29
1.34± 0.15
0.90± 0.32
0.70± 0.16
0.57± 0.16
0.77 ± 0.05
0.82 ± 0.19
0.35 ± 0.16
0.40 ± 0.18
1.28 ± 0.16
0.85 ± 0.07
0.77 ± 0.14
0.96 ± 0.13
0.91± 0.09
1.25 ± 0.18
0.24± 0.01
1.30± 0.57
0.25± 0.00
0.93 ± 0.04
0.40± 0.11
0.51± 0.28
4h
5
69.79↓
35.41↓
12.50↓
5.20↓
71.87↓
38.00↓
28.50↓
61.69↓
53.42↓
29.42↓
66.66↓
41.66↓
16.66↓
56.00↓
45.00↓
13.00↓
58.00↓
35.00↓
1.00↓
ꢀ
100
10
1
+
+
ꢀ
42.70↓
33.33↓
77.87↓
44.00↓
21.00↓
69.00↓
51.00↓
27.69↓
68.00↓
52.00↓
17.69↓
21.00↓
19.10↓
42.00↓
46.00↓
1.00↓
100
10
1
ꢀ
6a
6b
6c
7a
7b
7c
7d
7e
7g
7f
ꢀ
ꢀ
100
10
1
ꢀ
ꢀ
ꢀ
100
10
1
+
ꢀ
ꢀ
100
10
1
+
+
+
+
+
ꢀ
16.12↓
23.25↓
50.00↓
16.00↑
5.00↓
100
10
1
35.00↓
43.00↓
49.00↓
47.00↓
39.01↓
33.31↓
25.17↓
9.59↓
28.00↓
36.00↑
41.00↓
33.00↓
33.82↓
51.00↓
32.05↓
29.41↓
33.08↓
11.08↓
16.00↑
60.00↓
58.00↓
45.00↓
33.00↓
15.00↓
100
10
1
+
+
ꢀ
100
10
1
ꢀ
ꢀ
ꢀ
100
10
1
+
+
+
+
ꢀ
32.41↓
29.47↓
29.00↑
74.00↓
53.00↓
28.00↓
37.00↓
37.00↓
100
10
1
ꢀ
100
10
1
+
ꢀ
ꢀ
Sign ↑ indicate immune stimulant agents and ↓ indicate immunosuppressive agents. Sign + indicate cytotoxic
and – indicate nonꢀcytotoxic. Results are shown as mean of three readings and standard error by student‘t’ test
(±S.E.) of three separate experiments, conducted in triplicate at three different 1, 10 and 100 µM concentrations.
The numbers with bold values represent those compounds which have displayed potent immunosuppressive
activity.

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Compound 2 with aromatized ring A displayed better inhibition against both the mitogens
than the parent compound 1. Compound 4c displayed 57% inhibition against LPS induced Bꢀ
cell proliferation at 100 µM in the ester series (4aꢀh) while 4b and 4h was found more active
against Con A and displayed 39ꢀ78% and 28ꢀ78% suppression of Tꢀcells in the tested dose
range, respectively. Nonꢀcytotoxic compound 4d showed potent and uniform supersession of
Tꢀcell proliferation with 53% inhibition at 1 µM and finally attaining ~73% inhibition at 100
µM. Compound 4e which was found nonꢀtoxic and proved most potent among the ester
analogs with ~80% suppression of Bꢀcells and 75% inhibition of T lymphocyte proliferation,
respectively. Compound 4f displayed a uniform suppression and reaching up to 69% against
LPS induced Bꢀcell and 41% against Con A induced Tꢀcell proliferation at 100 µM but it was
cytotoxic. Compound 4g was non cytotoxic butcould not proved potency while cytotoxic
compound 5 displayed ~61% inhibition of Tꢀcells at 100 µM.
Amide analogs (6aꢀc) were found non cytotoxic and displayed uniform inhibition against
both the mitogens and among them, 6a showed potent inhibition reaching upto 77% at 100
µM concentration against LPS induced B lymphocytes and 66% against ConꢀA induced Tꢀ
cell proliferation. Compound 6b showed 76% suppression of B lymphocytes and 58%
suppression of T lymphocytes at 100 µM concentration. Among ethers, compound 7g
displayed 74% inhibition of B and 66% inhibition of T lymphocytes without cytotoxicity at
10 µM, but the compound was found to be stimulant as well as cytotoxicity at higher doses.
Rest of the ether analogs displayed less than 50% inhibition along with cytotoxicity and were
found less active compare to ester and amide derivatives.
It was evident from the cell proliferation results that ester and amide analogs display better
inhibition activity compare to ether analogs which indicate that carbonyl group present in
these derivatives play an important role. Among the ester analogs as the carbon chain length
increases the activity increases up to compound containing chain of four carbons (4h) and

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then decreases rapidly as the carbon chain increases. Further, isobutyrate (4d) and trimethyl
acetate (4e) analogs proved more potent compare to straight chain analogs. The compound 6a
bearing ortho –F group in the amide moiety exhibited potent activity among the amides
analogs.
Humoral Immune Response
Cell Mediated Immune Response
150
100
50
150
100
50
***
***
0
0
Conc.mg/ml
Conc.mg/ml
Figure 1. Effect of compound 4e on humoral immunity and DTH response. Data is expressed
as means ± SE of five observations.
2.2.2. In Vivo Effect of Compound 4e on DTH Reaction and Humoral Immunity
The compound 4e with potent immunosuppressive properties and as a representative of these
derivatives was further evaluated for in vivo experiments i.e. humoral and cell mediated
immune response16 on immune suppressed Balb/c mice at three different doses. Balb/c mice
were immunized with antigen SRBC. Levamisole, as immunostimulant, cyclophosphamide
and cyclosporine as immunosuppressant were used as standard controls for Bꢀ and Tꢀcells,
respectively for the study17. The DTH reaction is a cell mediated pathologic response
involved with Tꢀcell activation and the production of many cytokines18. Treatment with 4e
suppressed the DTH reaction, as reflected by the decreased footpad thickness compared to the
control group and down regulated the cell mediated immune response by 30% at 12.5 mg/kg
and 35% at a conc. of 25 mg/kg. In humoral immunity, the antibody response to SRBC was

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observed by the hemagglutination titre. Compound 4e showed 28% suppression at a dose of
6.25 mg/kg and 41% at 25 mg/kg/p.o. which was increased about 1.5 fold (Fig. 1). The
findings suggested that 4e is able to suppress the humoral response.
3. Conclusion
In summary, a series of new αꢀsantonin derived Oꢀaryl/aliphatic ether, ester and amide
derivatives were synthesised. All the compounds were investigated for their in vitro
immunosuppressive effect on Tꢀ and Bꢀlymphocyte of BALB/c mice. Many analogs showed
strong inhibition against ConꢀA and LPS stimulated Tꢀcell and Bꢀcell proliferation in a dose
dependent manner. The screening data revealed that the presence of carbonyl group and
appropriate aliphatic side chain play important factors toward contribution of high
immunosuppressive potency in these analogs. More interestingly, compounds 4c, 4d, 4e and
4h exhibited potent in vitro activity and the compound 4e revealed the most
immunosuppressive effects. Further, compound 4e was investigated for in vivo delayedꢀtype
hypersensitivity (DTH) reaction and antibody production in BALB/c mice models. The in
vitro and in vivo immunosuppressive activity suggested that compound 4e suppressed both
humoral and cell mediated immunity.
4. Experimental
4.1. Chemistry
All the reagents and solvents for executing the present work were obtained from Sigma
Aldrich. Dry DCM was prepared by refluxing freshly distilled DCM over CaH2 (5% w/v) for
few hours and reꢀdistilled it. Dry DCM was stored over 4A° molecular sieves for future use.
Pyridine was dried over KOH for one week, then distilled and stored over 4A° molecular
sieves. All the chemical reactions were monitored by TLC on 0.25 mm silica gel 60 F254
plates (E. Merck) using 2% ceric ammonium sulphate solution as a spraying reagent for
detection of the spots. Purification of compounds was carried out either by crystallization or

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1
column chromatography using silica gel 60ꢀ120 mesh stationary phase. H NMR and 13C
NMR spectra were recorded on Bruker DPX 400 and DPX 500 instruments using CDCl3 or
CD3OD as the solvents with TMS as internal standard. The chemical shifts were expressed in
δ ppm and coupling constants in Hertz. High resolution mass spectra (HRESIMS) were
recorded on Agilent Technologies 6540 instrument.
4.1.1. Isolation of αꢀsantonin (1)
αꢀSantonin (1) for present work was isolated in preparative scale from
dichloromethaneꢀmethanol (1:1) extract of the aerial parts of Artemisia laciniata by
column chromatography over silica gel 60ꢀ120 mesh and further purified by
crystallization.12
4.1.2. Preparation of αꢀdesmotroposantonin acetate (2)
The compound
2
was prepared as described in our previous report.12 Briefly, the
mixture of αꢀsantonin (5.0 g, 20 mmol), acetic anhydride (25 mL) at 0°C, H2SO4 (3
mL, 55.8 mmol) was added drop wise and allowed to react and upon purification
furnished white crystalline product (2) with 92% yield, mp 139 °C. HRESIMS m/z
calcd for C17H20O4 [M+H]+ 289.1434, found 289.1451.
4.1.3. Preparation of αꢀdesmotroposantonin (3)
The compound was prepared by treating compound 2 with 1:1 mixture of NH3: MeOH to
furnish αꢀdesmotroposantonin 3 with 98% yield, mp: 197 °C, HRESIMS m/z calcd for
C15H18O3 [M + H]+ 247.13287, found 247.13195.
4.1.4. General procedure for the Synthesis of ester analogs (4aꢀe)
The ester analogs 4aꢀe were prepared by treating
3 (0.40 mmol) with appropriate
anhydride (0.44 mmol) in dry dichloromethane (DCM) in presence of dry pyridine at
room temperature. Upon completion, the reaction mixture was extracted with ethyl acetate

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and water. The organic layer was concentrated on rota evaporated to furnish the
corresponding desired products in excellent yields (75ꢀ86%).
4.1.4.1. αꢀDesmotroposantonin pentanoate (4a)
White solid; mp: 128 °C; 1H NMR (400 MHz, CDCl3): δ 6.84 (s, 1H, ArꢀH), 5.58 (d, J =
6.4 Hz, 1H, ArCHOꢀ), 2.71ꢀ2.78 (m, 1H, ꢀCHCHCH3), 2.55ꢀ2.61 (m, 2H, ArCH2CH2ꢀ),
2.51ꢀ2.60 (m, 2H, ꢀCH2CH2CO), 2.36ꢀ2.49 (m, 1H, ꢀCHCHCH2ꢀ), 2.21 and 2.19 (s, 3H each,
2x ArꢀCH3), 1.93ꢀ1.96 and 1.69ꢀ1.75 (m, 1H each, ꢀCH2CH2CHꢀ), 1.93ꢀ1.96 (m, 2H, ꢀ
CH2CH2CO), 1.42ꢀ1.45 (m, 2H, ꢀCH3CH2CH2), 1.40 (d, J = 7.0 Hz, 3H, CH3CHꢀ), 0.98 (t, J
= 7.4 Hz, 3H, CH3CH2ꢀ); 13C NMR (100 MHz, CDCl3): δ 179.37, 172.25, 147.45, 134.78,
133.87, 131.27, 129.05, 123.93, 75.51, 41.68, 40.40, 34.23, 31.35, 24.76, 24.02, 22.34,
19.53, 14.45, 13.91, 12.09. HRESIMS m/z calcd for C20H26O4 [M+H]+ 331.1904, found
331.1904.
4.1.4.2. αꢀDesmotroposantonin hexanoate (4b)
White solid; mp: 132 °C; 1H NMR (400 MHz, CDCl3): δ 6.84 (s, 1H, ArꢀH), 5.58 (d, J =
6.4 Hz, 1H, ArCHOꢀ), 2.71 ꢀ2.78 (m, 1H, ꢀCHCHCH3), 2.51ꢀ2.59 (m, 2H each, ArCH2CH2ꢀ
and ꢀOCOCH2CH2ꢀ), 2.39 ꢀ2.43 (m,1H,ꢀCHCHCH2ꢀ), 2.21 and 2.20 (s, 3H each, 2x ArꢀCH3),
1.91ꢀ1.96 and 1.72 ꢀ 1.75 (m, 1H each, ꢀCH2CH2CHꢀ), 1.71ꢀ1.73 (m, 2H, CH3CH2CH2ꢀ), 1.40
(d, J = 7.0 Hz, 3H, CH3CHꢀ), 0.90 ꢀ 0.95 (t, J = 7.4 Hz, 3H, ꢀCH2CH3); 13C NMR (100 MHz,
CDCl3): δ 179.37, 172.25, 147.45, 134.78, 133.87, 131.27, 129.05, 123.93, 75.51, 41.68,
40.40, 34.23, 31.35, 24.76, 24.02, 23.38, 22.34, 19.53, 14.45, 13.91, 12.09. HRESIMS m/z
calcd for C21H28O4 [M+H]+ 345.2060, found 345.2077.
4.1.4.3. αꢀDesmotroposantonin octanoate (4c)
White solid; mp: 135 °C; 1H NMR (400 MHz, CDCl3): δ 6.85 (s, 1H, ArꢀH), 5.58 (d, J =
6.4 Hz, 1H, ArCHOꢀ), 2.71ꢀ2.77 (m, 1H, ꢀCHCHCH3), 2.50ꢀ2.59 (m, 2H each, ArCH2CH2ꢀ
and ꢀOCOCH2CH2ꢀ), 2.39ꢀ2.44 (m, 1H, ꢀCHCHCH2ꢀ), 2.21 and 2.20 (s, 3H each, 2x Arꢀ

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CH3), 1.91ꢀ1.97 and 1.72ꢀ1.75 (m, 1H each, ꢀCH2CH2CHꢀ), 1.40 (d, J = 7.0 Hz, 3H, CH3CHꢀ
), 1.22ꢀ1.46 (m, 10H, ꢀCH2(CH2)5CH3), 0.90 (t, J = 7.4 Hz, 3H, ꢀCH2CH3); 13C NMR (100
MHz, CDCl3): δ 179.37, 172.37, 147.50, 134.73, 133.87, 131.27, 129.06, 123.93, 75.47,
41.68, 40.40, 34.26, 31.65, 29.14, 28.91, 25.06, 24.01, 23.36, 22.59, 19.53, 14.45, 14.05,
12.09. HRESIMS m/z calcd for C23H32O4 [M+H]+ 373.2373, found 373.2406.
4.1.4.4. αꢀDesmotroposantonin isobutyrate (4d)
White solid; mp: 121 °C; 1H NMR (400 MHz, CDCl3): δ 6.83 (s, 1H, ArꢀH), 5.58 (d, J =
6.4 Hz, 1H, ArCHOꢀ), 2.81ꢀ2.89 (m, 1H, ꢀCOCH(CH3)2), 2.71ꢀ2.78 (m, 1H, ꢀCHCHCH3),
2.55ꢀ2.61 (m, 2H, ArCH2CH2ꢀ), 2.36ꢀ2.49 (m, 1H, ꢀCHCHCH2ꢀ), 2.20 and 2.17 (s, 3H each,
2x ArꢀCH3), 1.93ꢀ1.96 and 1.69ꢀ1.75 (m, 1H each, ꢀCH2CH2CHꢀ), 1.39 (d, J = 7.0 Hz, 3H,
CH3CHꢀ), 1.31 (d, J = 6.4 Hz, 6H, CH3CHCH3); 13C NMR (100 MHz, CDCl3): δ 179.37,
175.25, 147.45, 134.78, 133.87, 131.27, 129.05, 123.24, 75.51, 41.68, 40.40, 34.15, 23.97,
23.40, 19.51, 19.10, 19.03, 14.47, 12.0, HRESIMS m/z calcd for C19H24O4 [M+H]+ 317.1747,
found 317.1759.
4.1.4.5. αꢀDesmotroposantonin trimethyl acetate (4e)
White solid; mp: 127 °C; 1H NMR (400 MHz, CDCl3): δ 6.94 (s, 1H, ArꢀH), 5.59 (d, J =
6.4 Hz, 1H, ArCHOꢀ), 2.70 ꢀ2.78 (m, 1H, ꢀCHCHCH3), 2.55ꢀ2.61 (m, 2H, ArCH2CH2ꢀ),
2.51ꢀ2.60 (m, 2H, ꢀCH2CH2CO), 2.36ꢀ2.49 (m, 1H, ꢀCHCHCH2ꢀ), 2.26 and 2.22 (s, 3H each,
2x ArꢀCH3), 1.93ꢀ1.96 and 1.69ꢀ1.75 (m, 1H each, ꢀCH2CH2CHꢀ), 1.93ꢀ1.96 (m, 2H, ꢀ
CH2CH2CO), 1.42ꢀ1.45 (m, 2H, ꢀCH3CH2CH2), 1.55 (s, 9H, C(CH3)3), 1.40 (d, J = 7.0 Hz,
3H, CH3CHꢀ); 13C NMR (100 MHz, CDCl3): δ 179.37, 167.73, 147.69, 134.78, 134.05,
131.20, 129.20, 123.93, 75.46, 41.68, 40.35, 38.04, 27.68 (3x CH3), 23.76, 23.27, 19.17,
13.91, 13.75. HRESIMS m/z calcd for C20H26O4 [M+H]+ 331.1904, found 331.1887.

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4.1.5. General procedure for the synthesis of ester analogs (4fꢀh)
The ester analogs (4fꢀh) were prepared by treating
3 (0.40 mmol) with appropriate acid
chloride (0.45 mmol) in presence of pyridine at room temperature to afford the desired
products in excellent yields (70ꢀ79%). Acid chlorides were prepared by refluxing
appropriate acid with SOCl2 in dry dichloromethane (DCM) for 1h and dried under
vacuum.
4.1.5.1. αꢀDesmotroposantonin propanoate (4f)
White solid; mp: 132 °C; 1H NMR (400 MHz, CDCl3): δ 6.84 (s, 1H, ArꢀH), 5.58 (d, J =
6.4 Hz, 1H, ArCHOꢀ), 2.71ꢀ2.78 (m, 1H, ꢀCHCHCH3), 2.51ꢀ2.59 (m, 2H, ArCH2CH2ꢀ), 2.55ꢀ
2.61 (m, 2H, ꢀOCOCH2CH3) , 2.39 ꢀ2.43 (m, 1H, ꢀCHCHCH2ꢀ), 2.22 and 2.20 (s, 3H each,
2x ArꢀCH3), 1.93ꢀ1.96 and 1.72 ꢀ1.75 (m, 1H each, ꢀCH2CH2CHꢀ), 1.40 (d, J = 7.0 Hz, 3H,
CH3CHꢀ), 1.30ꢀ1.35 (t, J = 7.4 Hz, 3H, ꢀCH2CH3); 13C NMR (100 MHz, CDCl3): δ 179.37,
173.25, 147.45, 134.78, 133.87, 131.27, 129.05, 123.93, 75.51, 41.68, 40.40, 27.21, 24.01,
23.38, 19.53, 14.45, 12.09, 9.29. HRESIMS m/z calcd for C18H22O4 [M+H]+ 303.1591, found
303.1596.
4.1.5.2. αꢀDesmotroposantonin benzoate (4g)
White solid; mp: 142°C; 1H NMR (400 MHz, CDCl3): δ 8.21ꢀ8.23 (m, 2H, 2x Ar1ꢀH ),
7.60ꢀ7.67 (m, 1H, Ar1ꢀH ), 7.46ꢀ7.55 (m, 2H, 2x Ar1ꢀH ), 6.99 (s, 1H, ArꢀH), 5.62 (d, J = 6.4
Hz, 1H, ArCHOꢀ), 2.74ꢀ2.81 (m, 1H, ꢀCHCHCH3), 2.53ꢀ2.61 (m, 2H, ArCH2CH2ꢀ), 2.42ꢀ
2.48 (m, 1H, ꢀCHCHCH2ꢀ), 2.27 and 2.25 (s, 3H each, 2x ArꢀCH3), 1.93ꢀ1.99 and 1.74 ꢀ1.81
(m, 1H each, ꢀCH2CH2CHꢀ), 1.40 (d, J = 7.0 Hz, 3H, CH3CHꢀ); 13C NMR (100 MHz,
CDCl3): δ 179.37, 165.25, 147.45, 134.78, 134.14, 133.87, 131.27, 130.17, 129.31(2x CH),
129.05, 128 (2x CH), 124.03, 75.51, 41.68, 40.40, 24.01, 23.38, 19.53, 14.45, 12.09.
HRESIMS m/z calcd for C22H22O4 [M+H]+ 351.1591, found 351.1597.

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4.1.5.3. αꢀDesmotroposantonin butanoate (4h)
White solid; mp: 144 °C; 1H NMR (400 MHz, CDCl3): δ 6.84 (s, 1H, ArꢀH), 5.58 (d, J =
6.4 Hz, 1H, ArCHOꢀ), 2.70ꢀ2.77 (m, 1H, ꢀCHCHCH3), 2.51ꢀ2.59 (m, 2H each, ArCH2CH2ꢀ
and ꢀOCOCH2CH2ꢀ), 2.39ꢀ2.43 (m, 1H, ꢀCHCHCH2ꢀ), 2.21 and 2.20 (s, 3H each, 2x Arꢀ
CH3), 1.91ꢀ1.96 and 1.72ꢀ1.75 (m, 1H each, ꢀCH2CH2CHꢀ), 1.71ꢀ1.73 (m, 2H, CH3CH2CH2ꢀ
), 1.40 (d, J = 7.0 Hz, 3H, CH3CHꢀ), 1.08 (t, J = 7.4 Hz, 3H, ꢀCH2CH3); 13C NMR (100 MHz,
CDCl3): δ 179.37, 172.25, 147.45, 134.78, 133.87, 131.27, 129.05, 123.93, 75.51, 41.68,
40.40, 36.11, 24.01, 23.38, 19.53, 18.60, 14.45, 13.76, 12.09. HRESIMS m/z calcd for
C19H24O4 [M+H]+ 317.1747, found 317.1779.
4.1.6. Preparation of compound 4ꢀαꢀdesmotroposantoninꢀ4ꢀoxobutanoic acid (5)
To a solution of 3 (2.0 mmol) in dry DCM, succinic anhydride (2.4 mmol) and DMAP (2.0
mmol) were added and stirred at room temperature till completion of the reaction. The ice
cold water was added to reaction mixture for precipitation of the crude product and finally
washed with cold DCM to furnish the desired product 5 as white solid; mp: 123 °C; 1H
NMR (400 MHz, CDCl3): δ 6.87 (s, 1H, ArꢀH), 5.62 (d, J = 6.4 Hz, 1H, ArCHOꢀ), 2.71ꢀ2.78
(m, 1H, ꢀCHCHCH3), 2.55ꢀ2.61 (m, 2H, ArCH2CH2ꢀ), 2.51(s, 4H, ꢀCOCH2CH2CO), 2.36ꢀ
2.49 (m, 1H, ꢀCHCHCH2ꢀ), 2.25 and 2.23 (s, 3H each, 2x ArꢀCH3), 1.93ꢀ1.96 and 1.69ꢀ1.75
(m, 1H each, ꢀCH2CH2CHꢀ), 1.40 (d, J = 7.0 Hz, 3H, CH3CHꢀ); 13C NMR (100 MHz,
CDCl3): δ 177.37, 172.25, 168.52, 147.45, 134.78, 133.87, 131.27, 129.05, 121.24, 73.51,
39.68, 37.40, 27.21, 26.51, 21.20, 20.53, 16.87, 11.77, 9.36 HRESIMS m/z calcd for
C19H22O6 [M+H]+ 347.1589, found 347.1524.
4.1.7. General procedure for the synthesis of (6aꢀc)
A mixture of 5 (1.44 mmol) and thionyl chloride (85 mmol) in dry dichloromethane (DCM)
(20 mL) was refluxed for 1 h. Excess thionyl chloride and DCM was removed in vacuum.
The crude product (acyl chloride) dissolved in dry DCM (20 mL) and added drop wise with

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the appropriate aniline (2.16 mmol) in dry pyridine at room temperature. After the addition
was complete, stirring was continued for another half an hour and upon purification afforded
the desired products 6aꢀc.
4.1.7.1. 4ꢀαꢀDesmotroposantoninylꢀ((2ꢀfluorophenyl)ꢀ4ꢀoxobutanmide (6a)
White solid; mp: 123 °C; 1H NMR (400 MHz, CDCl3): δ 8.02 (s, 1H, NH), 7.27 (m, 4H, 4x
Ar1ꢀH), 6.90 (s, 1H, ArꢀH), 5.65 (d, J = 6.4 Hz, 1H, ArCHOꢀ), 3.01ꢀ3.04 (m, 2H, ꢀ
NHCOCH2CH2), 2.93ꢀ2.97 (m, 2H, ꢀCH2CH2COO), 2.73ꢀ2.79 (m, 1H, ꢀCHCHCH3), 2.59ꢀ
2.65 (m, 2H, ArCH2CH2ꢀ), 2.36ꢀ2.49 (m, 1H, ꢀCHCHCH2ꢀ), 2.25 and 2.24 (s, 3H each, 2x
ArꢀCH3), 1.91ꢀ1.95 and 1.63ꢀ1.71 (m, 1H each, ꢀCH2CH2CHꢀ), 1.41(d, J = 6.4 Hz, 3H,
CH3CHꢀ); 13C NMR (100 MHz, CDCl3): δ 180.56, 171.87, 169.52, 147.30, 134.94, 134.22,
130.96, 129.88, 128.92, 124.78, 123.82, 121.24, 117.55, 115.12, 114.96, 75.99, 41.76, 40.61,
31.45, 28.50, 23.89, 23.26, 19.29, 14.19, 11.86. HRESIMS m/z calcd for C25H26FNO5
[M+H]+ 440.1868, found 440.1808
4.1.7.2. 4ꢀαꢀDesmotroposantoninylꢀ(2ꢀchlorophenyl)ꢀ4ꢀoxobutanmide (6b)
White solid; mp: 142 °C; 1H NMR (400 MHz, CDCl3): δ 8.35 (s, 1H, NH), 7.36 (d, J = 8.2
Hz, 1H, Ar1ꢀH), 7.26ꢀ7.29 (m, 2H, 2x Ar1ꢀH), 7.03ꢀ7.06 (m, 1H, Ar1ꢀH), 6.88 (s, 1H, ArꢀH),
5.57 (d, J = 6.4 Hz, 1H, ArCHOꢀ), 3.04ꢀ3.07 (m, 2H, ꢀNHCOCH2CH2), 2.94ꢀ2.97 (m, 2H, ꢀ
CH2CH2COO), 2.71ꢀ2.78 (m, 1H, ꢀCHCHCH3), 2.55ꢀ2.61 (m, 2H, ArCH2CH2ꢀ), 2.36ꢀ2.49
(m, 1H, ꢀCHCHCH2ꢀ), 2.25 and 2.23 (s, 3H each, 2x ArꢀCH3), 1.93ꢀ1.96 and 1.69ꢀ1.75 (m,
1H each, ꢀCH2CH2CHꢀ), 1.40 (d, J = 6.4 Hz, 3H, CH3CHꢀ); 13C NMR (100 MHz, CDCl3): δ
179.45, 171.44, 169.52, 147.45, 134.86, 134.46, 134.16, 131.27, 130.82 129.88, 129.07,
127.72, 124.78, 123.82, 121.24, 75.51, 41.62, 40.44, 32.18, 29.32, 24.02, 23.37, 19.56, 14.49,
12.17. HRESIMS m/z calcd for C25H26ClNO5 [M+H]+ 456.1572, found 456.1589.

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4.1.7.3. 4ꢀαꢀDesmotroposantoninylꢀ((4ꢀmethoxyphenyl)ꢀ4ꢀoxobutanmide (6c)
White solid; mp: 147 °C; 1H NMR (400 MHz, CDCl3): δ 7.05 (s, 1H, NH), 7.37 (d, J = 8.4
Hz, 2x Ar1ꢀH), 6.86 (s, 1H, ArꢀH), 6.83 (d, J = 8.4 Hz, 2x Ar1ꢀH), 5.55 (d, J = 6.0 Hz, 1H,
ArCHOꢀ), 3.78 (s, 3H, OCH3), 3.01ꢀ3.04 (m, 2H, ꢀNHCOCH2CH2), 2.71ꢀ2.75 (m, 2H, ꢀ
CH2CH2COO), 2.65ꢀ2.70 (m, 1H, ꢀCHCHCH3), 2.50ꢀ2.61 (m, 2H, ArCH2CH2ꢀ), 2.39ꢀ2.41
(m, 1H, ꢀCHCHCH2ꢀ), 2.19 and 2.18 (s, 3H each, 2x ArꢀCH3), 1.91ꢀ1.95 and 1.69ꢀ1.74 (m,
13
1H each, ꢀCH2CH2CHꢀ), 1.38 (d, J = 6.4 Hz, 3H, CH3CHꢀ); C NMR (125 MHz, CDCl3) δ
179.56, 171.93, 169.30, 156.34, 147.34, 134.92, 134.18, 131.25, 130.95, 129.04, 123.82,
121.77 (2x CH), 114.07 (2x CH), 75.51, 55.48, 41.61, 40.47, 31.74, 29.46, 24.02, 23.36,
19.56, 14.50, 12.18. HRESIMS m/z calcd for C26H29NO6 [M+H]+ 452.2093, found 452.2068
4.1.8. General procedure for the synthesis of ether analogs (7aꢀg)
The ether analogs 7aꢀg were prepared by treating
3 (0.40 mmol) with appropriate alkyl
halide (0.44 mmol) in DCM in presence of pyridine (0.40 mmol) at room temperature.
Upon completion, the reaction mixture was extracted with ethyl acetate and water. The
organic layer was concentrated under vacuum to afford the desired ethers derivatives in
excellent yields (90ꢀ95%).
4.1.8.1. αꢀDesmotroposantonin methyl ether (7a)
White solid; mp: 141 °C; 1H NMR (400 MHz, CDCl3): δ 6.75 (s, 1H, ArꢀH), 5.63 (d, J =
6.4 Hz, 1H, ArCHOꢀ), 3.81 (s, 3H, OCH3), 2.70ꢀ2.75 (m, 1H, ꢀCHCHCH3), 2.55ꢀ2.61 (m,
2H, ArCH2CH2ꢀ), 2.37ꢀ2.49 (m, 1H, ꢀCHCHCH2ꢀ), 2.25 and 2.23 (s, 3H each, 2x ArꢀCH3),
1.85ꢀ1.93 and 1.69ꢀ1.75 (m, 1H each, ꢀCH2CH2CHꢀ), 1.38 (d, J = 7.0 Hz, 3H, CH3CHꢀ); 13C
NMR (100 MHz, CDCl3): δ 179.55, 155.67, 134.12, 130.74, 127.43, 125.58, 113.35, 75.83,
55.8, 41.86, 40.54, 23.70, 23.65, 20.04, 14.53, 11.52. HRESIMS m/z calcd for C16H20O3
[M+H]+ 261.1485, found 261.1491.

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4.1.8.2. αꢀDesmotroposantonin ethyl ether (7b)
White solid; mp: 113 °C; 1H NMR (400 MHz, CDCl3): δ 6.71 (s, 1H, ArꢀH), 5.60 (d, J =
6.4 Hz, 1H, ArCHOꢀ), 3.99 (q, 2H, J = 5.6 Hz, ꢀOCH2CH3), 2.66ꢀ2.71 (m, 1H, ꢀCHCHCH3),
2.47ꢀ2.54 (m, 2H, ArCH2CH2ꢀ), 2.37ꢀ2.39 (m, 1H, ꢀCHCHCH2ꢀ), 2.27 and 2.21 (s, 3H each,
2x ArꢀCH3), 1.87ꢀ1.89 and 1.68ꢀ1.71 (m, 1H each, ꢀCH2CH2CHꢀ), 1.36ꢀ1.41 (m, 3H each,
CH3CHꢀ OCH2CH3); 13C NMR (100 MHz, CDCl3): δ 179.74, 155.20, 134.00, 130.71,
127.79, 125.97, 114.76, 75.90, 64.23, 41.87, 40.59, 23.73, 23.70, 19.99, 15.08, 14.53, 11.57.
HRESIMS m/z calcd for C17H22O3 [M+H]+ 275.1642, found 275.1638.
4.1.8.3. αꢀDesmotroposantonin propyl ether (7c)
White solid; mp: 128 °C; 1H NMR (400 MHz, CDCl3): δ 6.72 (s, 1H, ArꢀH), 5.60 (d, J =
6.4 Hz, 1H, ArCHOꢀ), 3.89 (t, 2H, J = 6.4 Hz, OCH2CH2ꢀ), 2.66ꢀ2.72(m, 1H, ꢀCHCHCH3),
2.45ꢀ2.55 (m, 2H, ArCH2CH2ꢀ), 2.36ꢀ2.42 (m, 1H, ꢀCHCHCH2ꢀ), 2.27 and 2.21 (s, 3H each,
2x ArꢀCH3), 1.87ꢀ1.92 and 1.68ꢀ1.75 (m, 1H each, ꢀCH2CH2CHꢀ), 1.80 (q, 2H, J = 6.0 Hz, ꢀ
CH2CH3), 1.38 (d, J = 7.6 Hz, 3H, CH3CHꢀ), 1.04 (t, J = 7.2 Hz, 3H, CH3CH2ꢀ); 13C NMR
(100 MHz, CDCl3): δ 179.77, 155.30, 134.02, 130.69, 127.67, 125.86, 114.57, 75.91, 70.13,
41.85, 40.59, 23.73, 23.69, 22.82, 19.98, 14.52, 11.57, 10.73. HRESIMS m/z calcd for
C18H24O3 [M+H]+ 289.1789, found 289.1812.
4.1.8.4. αꢀDesmotroposantonin butyl ether (7d)
White solid; mp: 118°C; 1H NMR (400 MHz, CDCl3) δ 6.72 (s, 1H, ArꢀH), 5.61 (d, J = 6.4
Hz, 1H, ArCHOꢀ), 3.93 (t, J = 7.8 Hz, 2H, ꢀCH2CH2O), 2.67ꢀ2.72 (m, 1H, ꢀCHCHCH3),
2.46ꢀ2.57 (m, 2H, ArCH2CH2ꢀ), 2.36ꢀ2.42 (m,1H,ꢀCHCHCH2ꢀ), 2.27 and 2.22 (s, 3H each,
2x ArꢀCH3), 1.86ꢀ1.94 and 1.67ꢀ1.75 (m, 1H each, ꢀCH2CH2CHꢀ), 1.76ꢀ1.82 (m, 2H, ꢀ
CH2CH2CH2O), 1.42ꢀ1.45 (m, 2H, CH3CH2CH2ꢀ), 1.38 (d, J = 7.0 Hz, 3H, CH3CHꢀ), 0.97 (t,
J = 7.8 Hz, 3H, CH3CH2ꢀ); 13C NMR (125 MHz, CDCl3): δ 179.70, 155.49, 133.96, 130.69,

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127.77, 125.75, 114.65, 75.90, 68.26, 41.86, 40.49, 31.36, 23.55, 23.52, 19.93, 19.31, 14.43,
13.71, 11.45. HRESIMS m/z calcd for C19H26O3 [M+H]+ 303.1955, found 303.1981.
4.1.8.5. αꢀDesmotroposantonin pentyl ether (7e)
White solid; mp: 131 °C; 1H NMR (400 MHz, CDCl3) δ 6.72 (s, 1H, ArꢀH), 5.62 (d, J = 6.4
Hz, 1H, ArCHOꢀ), 3.81 (t, J = 7.8 Hz, 2H, ꢀCH2CH2O), 2.67ꢀ2.75 (m, 1H, ꢀCHCHCH3),
2.52ꢀ2.57 (m, 2H, ArCH2CH2ꢀ), 2.37ꢀ2.42 (m, 1H, ꢀCHCHCH2ꢀ), 2.28 and 2.22 (s, 3H each,
2x ArꢀCH3), 1.85ꢀ1.90 and 1.67ꢀ1.75 (m, 1H each, ꢀCH2CH2CHꢀ), 1.76ꢀ1.82 (m, 2H, ꢀ
CH2CH2CH2O), 1.42ꢀ1.45 (m, 2H, ꢀCH2CH2CH2O), 1.38 (d, J = 7.0 Hz, 3H, CH3CHꢀ), 0.93
(t, J = 7.8 Hz, 3H, CH3CH2CH2ꢀ); 13C NMR (100 MHz, CDCl3): δ 179.74, 155.35, 134.00,
130.69, 127.63, 125.90, 114.54, 75.79, 68.60, 41.87, 40.55, 29.17, 28.36, 23.73, 232.69,
22.47, 20.00, 14.53, 14.10, 11.56. HRESIMS m/z calcd for C20H28O3 [M+H]+ 317.2011,
found 317.2087
4.1.8.6. αꢀDesmotroposantonin benzyl ether (7f)
White solid; mp: 124 °C; 1H NMR (400 MHz, CDCl3): δ 7.30ꢀ7.45 (m, 5H, 5x Ar1ꢀH), 6.80
(s, 1H, ArꢀH), 5.62 (d, J = 6.4 Hz, 1H, ArCHOꢀ), 5.05 (s, 2H, Ar1CH2O), 2.70ꢀ2.75 (m, 1H, ꢀ
CHCHCH3), 2.55ꢀ2.61 (m, 2H, ArCH2CH2ꢀ), 2.37ꢀ2.49 (m, 1H, ꢀCHCHCH2ꢀ), 2.25 and 2.21
(s, 3H each, 2x ArꢀCH3), 1.93ꢀ1.96 and 1.69ꢀ1.75 (m, 1H each, ꢀCH2CH2CHꢀ), 1.40 (d, J =
7.0 Hz, 3H, CH3CHꢀ); 13C NMR (100 MHz, CDCl3): δ 179.93, 137.57, 134.09, 130.90,
130.74, 128.68 (2x CH), 128.26, 127.84, 127.14 (2x CH), 126.90, 114.83, 75.74, 70.05,
41.85, 40.64, 23.77, 23.73, 20.07, 14.57, 11.83. HRESIMS m/z calcd for C22H24O3 [M+H]+
337.1798, found 337.1807
4.1.8.7. αꢀDesmotroposantonin pꢀchlorobenzyl ether (7g)
White solid; mp: 139 °C; 1H NMR (400 MHz, CDCl3): δ 7.33ꢀ7.38 (m, 4H, 4x Ar1ꢀH), 6.76
(s, 1H, ArꢀH), 5.63 (d, J = 6.4 Hz, 1H, ArCHOꢀ), 5.01 (s, 2H, Ar1CH2O), 2.70ꢀ2.75 (m, 1H, ꢀ
CHCHCH3), 2.55ꢀ2.61 (m, 2H, ArCH2CH2ꢀ), 2.37ꢀ2.49 (m, 1H, ꢀCHCHCH2ꢀ), 2.25 and 2.21

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(s, 3H each, 2x ArꢀCH3), 1.93ꢀ1.96 and 1.69ꢀ1.75 (m, 1H each, ꢀCH2CH2CHꢀ), 1.40 (d, J =
7.0 Hz, 3H, CH3CHꢀ); 13C NMR (100 MHz, CDCl3): δ 179.37, 154.73, 136.02, 134.20,
133.51, 130.99, 129.20, 128.72 (2x CH), 128.48 (2x CH), 126.08, 114.86, 75.80, 69.70,
41.83, 40.36, 23.73, 23.68, 20.04, 14.54, 11.79 HRESIMS m/z calcd for C22H23ClO3 [M+H]+
371.1408, found 371.1412.
4.2. Biology
4.2.1. Animals, Mitogens and reagents
Animals: Female Balb/c mice 10ꢀ12 week old and weighing 20ꢀ24 g were obtained from
animal house of Indian Institute of Integrative Medicine (IIIM), Jammu which were taken up
in groups of six and employed for study. These animals were maintained in standard sized
polycarbonated cages with controlled condition of temperature of 26 ±2°C, humidity 50ꢀ60%
and 12 h light/dark cycle. The animals were fed on pellet food (Ashirwad Industries India
Ltd.) and Ad libitum autoclaved water. The study was carried out in normal mice as per
OECD guidelines no. 42318. The experimental protocols and animals used in the experiments
were approved by the Institutional Animal Ethics Committee. The approval no. is CPCSEA:
99/65/758/8/15.
Mitogens and reagents: ConcanavalinꢀA (ConꢀA), Lipopolysaccharide (LPS) and 3ꢀ[4,5ꢀ
dimethylthiazolꢀ2ꢀyl]ꢀ2,5ꢀdiphenylꢀtetrazolium bromide (MTT) were procured from Sigma
Aldrich. RPMI (Roswell Park Memorial Institute) 1640 medium and fetal bovine serum
(FBS) were purchased from Gibco BRL, Life Technologies (USA).
4.2.2. Determination of in vitro cytotoxic effect of different samples by MTT assay
MTT [3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide] assay, reported in by
Mosmann 198319, is a colorimetric assay, which was used to determine the ability of viable
cells to convert soluble MTT [3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide]
dye into insoluble blue coloured formazan precipitate, which is dissolved by the addition of a

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solvent (i.e. DMSO) results in the liberation of the crystals which are solubilized. The
number of surviving cells is directly proportional to the level of the formazan formed. The
colour can then be quantified using a simple colorimetric assay. The results can be read on a
multiwall scanning spectrophotometer (ELISA reader) in the wavelengths of 540 nm
respectively.
4.2.3. Methodology: Balb/c mice weighing about 18ꢀ22g were sacrificed, their spleens were
removed under aseptic conditions. A single cell suspension was prepared in 5 mL of RPMI.
The cell suspension was centrifuged at 1200 rpm for 10 min and supernatant was discarded.
RBCs were lysed by trisꢀammonium chloride treatment. The cells were centrifuged twice at
1200 rpm for 10 min, supernatant discarded and reꢀsuspended RPMI. The viability of cells
was checked with trypan blue. 1 x 106 cells/mL suspension was prepared and 100 µL of cell
suspension was seeded into 96ꢀwell flatꢀbottomed plate. The cells were stimulated by using
50 µL of standard mitogens (ConꢀA = 10 µg/mL and LPS = 10 µg/mL) to induce Tꢀcells or
Bꢀcells proliferation respectively. Different concentrations of test materials were added
according to the experimental setup. 50 µL of different concentrations (1, 10 and 100 µM) of
different compounds CRPMI were added to each well. The plates were incubated for 48 h at
37°C in a humidified 5% CO2 incubator. After 48 h incubation, plates were taken out from
the CO2 incubator and 10 µL of MTT dye was added to each well and placed on a shaker for
5 min to thoroughly mix the MTT into the media. Plates were then incubated for 4h in CO2
incubator (37°C, 5% CO2 and 90% relative humidity) to allow the MTT to be metabolized.
The plates were centrifuged at 2000 rpm for 10 min. Cellꢀfree supernatants were then
removed and resolved with 100 µL/well DMSO. The optical density was measured at 540 nm
on a microplate reader. The mean of the optical density of plates were calculated and evaluate
the percentage of each value verses control.
4.2.4. Antigen

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Sheep red blood cells (SRBC) were used as a source of T dependent antigen. For this purpose
the blood was withdrawn from a healthy sheep in Alsever's solution, SRBC used for
immunization were prepared in pyrogen free normal saline.20,21
4.2.5. Delayed type hypersensitivity (DTH)
Compound 4e was administered 2 h after SRBC injection and once daily on consecutive
days. Six days later, the thickness of the left hind footpad was measured with a
spheromicrometer (pitch, 0.01 mm) and was considered as a control. The mice were then
challenged by injecting 20 ꢁL of 5×109 SRBC/mL intradermally into the left hind footpad.
The footpad thickness was measured again after 24 and 48 h.21,22
Supporting information available
Spectral data (1H NMR and 13C NMR) and HRESIMS of all compounds are provided in the
supporting information.
Acknowledgements
Council of Scientific and Industrial Research (CSIR), New Delhi is well acknowledged for
funding support under 12th FYP Project BSCꢀ0108. One of the authors (NAD) is greatly
thankful to the CSIRꢀUGC, New Delhi for the award of senior research fellowship. The
authors are also thankful to the staff of instrumentation division of the institute for recording
the spectroscopic (NMR, HRESIMS) data.
References
1.
2.
3.
4.
5.
D. J. Newman and G. M. Cragg, J. Nat. Prod., 2016, 79, 629ꢀ661.
G. M. Cragg, P. G. Grothaus and D. J. Newman, J. Nat. Prod., 2014, 77, 703ꢀ723.
H. Hackstein and A. W. Thomson, Nat. Rev. Immunol, 2004, 4, 24ꢀ35.
J. Mann, Nat. Prod. Rep., 2001, 18, 417ꢀ430.
J. A. Kobashigawa and J. K. Patel, Nature Clinical Practice Cardiovascular Medicine,
2006, 3, 203ꢀ212.

Page 23 of 25
MedChemComm
View Article Online
DOI: 10.1039/C6MD00527F
6.
7.
F. Issa, A. Schiopu and K. J. Wood, Expert. Rev. Clin. Immunol., 2010, 6, 155ꢀ169.
P. Weyerstahl, H. MarschallꢀWeyerstahl, M. Schröder and V. K. Kaul, J. Essent. Oil
Res, 1992, 4, 107ꢀ112.
8.
9.
S. Fahad and A. Bano, Pak. J. Bot., 2012, 44, 165ꢀ170.
J. Khazir, P. P. Singh, D. M. Reddy, I. Hyder, S. Shafi, S. Sawant, G. Chashoo, A.
Mahajan, M. Alam and A. Saxena, Eur. J. Med. Chem., 2013, 63, 279ꢀ289.
10. B. Singh, J. Srivastava, R. Khosa and U. Singh, Folia Microbiol, 2001, 46, 137ꢀ142.
11. G. Blay, L. Cardona, B. Garcia and J. Pedro, Stud. Nat. Prod. Chem., 2000, 24, 53ꢀ129.
12. N. A. Dangroo, J. Singh, A. A. Dar, N. Gupta, P. K. Chinthakindi, A. Kaul, M. A.
Khuroo and P. L. Sangwan, Eur. J. Med. Chem., 2016, 120, 160ꢀ169.
13. P. K. Chinthakindi, P. L. Sangwan, S. Farooq, R. R. Aleti, A. Kaul, A. K. Saxena, Y.
Murthy, R. A. Vishwakarma and S. Koul, Eur. J. Med. Chem., 2013, 60, 365ꢀ375.
14. R. Majeed, P. L. Sangwan, P. K. Chinthakindi, I. Khan, N. A. Dangroo, N. Thota, A.
Hamid, P. R. Sharma, A. K. Saxena and S. Koul, Eur. J. Med. Chem., 2013, 63, 782ꢀ
792.
15. I. Khan, S. K. Guru, S. K. Rath, P. K. Chinthakindi, B. Singh, S. Koul, S. Bhushan and
P. L. Sangwan, Eur. J. Med. Chem., 2016, 108, 104ꢀ116.
16. N. Doherty, Agents and Actions, 1981, 11, 237ꢀ242.
17. D. Nelson and P. Mildenhall, Aust. J. Exp. Biol. Med. Sci., 1967, 45, 113ꢀ130.
18. M. Jonsson, M. Jestoi, A. V. Nathanail, U.ꢀM. Kokkonen, M. Anttila, P. Koivisto, P.
Karhunen and K. Peltonen, Food Chem. Toxicol., 2013, 53, 27ꢀ32.
19. T. Mosmann, J. Immunol. Methods, 1983, 65, 55ꢀ63.
20. K. Kobayashi, K. Kaneda and T. Kasama, Micros. Res. Tech., 2001, 53, 241.
21. F. Malik, J. Singh, A. Khajuria, K. A. Suri, N. K. Satti, S. Singh, M. K. Kaul, A.
Kumar, A. Bhatia and G.N. Qazi, Life Sciences, 2007, 80, 1525.

MedChemComm
Page 24 of 25
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22. A. Gupta, A. Khajuria, J. Singh, K. L. Bedi, N. K. Satti, P. Dutt, K. A. Suri, O. P. Suri
and G. N. Qazi, Int. Immunopharmacol., 2006, 6, 1543.

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T- and B-cell immunosuppressive activity of novel α-santonin analogs with
humoral and cellular immune response in Balb/c mice
N. A. Dangroo, J. Singh, N. Gupta, S. K. Singh A. Koul, M.A. Khuroo, and P. L. Sangwan
O
A
B
O
C
O
O
O
1
O
Artemisia laciniata
O
4e
Synthesis of α-santonin analogs and identification of potent immunosuppressant molecules. In vivo
investigation on BALB/c mice revealed that compound 4e suppresses both humoral and cellular
immunity.