Natural Product Research

Formerly Natural Product Letters

ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20

Semi-synthesis of a novel hybrid isoxazolidino

withaferin via chemoselective and
diastereoselective 1,3-dipolar nitrone
cycloaddition reaction

Ramkrishna Mandal, Meenakshi Singh, Amrutha A.V. Krishnan, Yogita H.

Dahat, Yogesh P. Bharitkar, V. Ravichandiran & Abhijit Hazra

To cite this article: Ramkrishna Mandal, Meenakshi Singh, Amrutha A.V. Krishnan, Yogita H.
Dahat, Yogesh P. Bharitkar, V. Ravichandiran & Abhijit Hazra (2019): Semi-synthesis of a novel
hybrid isoxazolidino withaferin via chemoselective and diastereoselective 1,3-dipolar nitrone
cycloaddition reaction, Natural Product Research, DOI: 10.1080/14786419.2019.1582045

To link to this article: https://doi.org/10.1080/14786419.2019.1582045

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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1582045

Semi-synthesis of a novel hybrid isoxazolidino withaferin

via chemoselective and diastereoselective 1,3-dipolar
nitrone cycloaddition reaction
Ramkrishna Mandal
, Meenakshi Singh
, Amrutha A.V. Krishnan, Yogita H.
Dahat, Yogesh P. Bharitkar, V. Ravichandiran and Abhijit Hazra
National Institute of Pharmaceutical Education and Research (NIPER), Kolkata, India

ABSTRACT ARTICLE HISTORY

A facile, atom-economic synthesis of isoxazilidino withaferin, Received 10 January 2019
a novel hybrid of withaferin A, has been accomplished via two- Accepted 8 February 2019
step reaction of nitrone synthesis followed by nitrone 1,3-dipolar
cycloaddition. The reaction is highly chemoselective (preferential KEYWORDS
reaction only on one of the two double bonds present on witha- Withaferin A; nitrone-
cycloaddition; semi-
ferin A) and diastereoselective affording exclusively the cis-fused synthesis; isoxazolidine; 2D
products. The structure was determined by detailed analysis of NMR (HSQC; HMBC;
1D, 2D NMR and mass spectral data. COSY; NOESY)

1. Introduction
The isoxazolidine ring represents one of the privileged structures in medicinal
chemistry, and there has been an increasing number of studies on isoxazolidine and
isoxazolidine-containing compounds. The saturated five-membered heterocyclic ring,
containing adjacent nitrogen and oxygen atoms, being part of several drug candidates
like Terizidone (MDR-TB) (Galietti et al. 1991), Reglixane (antidiabetic) (Miyachi 2005),
Cycloserine (antibiotic for drug resistant TB) (Mulinos 1955) (Figure 1) as well as
natural products with so many biological activity (Figure 2) (Yotsu-Yamashita et al.
2004; Zhang et al. 1995; Koyama et al. 2010; Hirano et al. 2000; Zhao et al. 2011, 2012;
Takahashi et al. 1998; Wang et al. 2012) has shown increasing interest in the past
decade. Other than mentioned above the potential biological applications of the

CONTACT Abhijit Hazra apuhazra@gmail.com; Yogesh Bharitkar yogeshbharitkar@gmail.com

Equal contribution.
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1582045.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 R. MANDAL ET AL.

isoxazolidine ring system include cytotoxic activities via DNA intercalation (Rescifina
et al. 2011, 2012) and transcriptional activators (Minter et al. 2004), antiviral activities
(Loh et al. 2010), antifungal/antimicrobial activities (Chen et al. 2014; Chen et al. 2012),
anti-Inflammatory activities (Setoguchi et al. 2013), advanced glycation end inhibitor
activities (Kaur et al. 2013) etc.
Transforming a parent bioactive natural compound to a more/new bioactive one
via semi-synthetic approach by the incorporation of heteroatoms (N, O & S) has
become a modern way of drug development (Amslinger 2010). Examples include
several compounds like paclitaxel/docetaxel, artemether/artisunate, flavopyridol, ixa-
bepilone etc which have emerged as the drug candidate via semi-synthetic modifica-
tion of the parent bioactive natural products. In this strategy of action for
development of therapeutics, Withaferin A (WA), the steroidal lactone purified from
leaves of the medicinal plant Withania somnifera also known by its Sanskrit name
‘‘Ashwagandha’ is also not an exception but in the frontline of semi-synthetic modifi-
cation. Withaferin A, a group of naturally occurring C28 steroidal lactones assembled
on an ergostane skeleton chemically characterised as 5b,6b-epoxy-4b, 27-dihydroxy-
1-oxowitha-2,24-diexolide, reported to show a number of biological activities, antihy-
pothyroid (Wanjari et al. 2012), anti-inflammatory (Kaileh et al. 2007), anabolic
(against osteoporosis) (Khedgikar et al. 2013), cardioprotective (Gupta et al. 2004),
immuno-modulatory (Agarwal et al. 1999) inhibition of human lung, colon, CNS and
breast cancer cell proliferation, (Antony et al. 2014; Srinivasan et al. 2008; Oh et al.
2007; Mayola et al. 2011; Munagala et al. 2011; Koduru et al. 2010), anticonvulsant,
antistress, (Khan and Ghosh 2010, 2010), COX-2 enzyme inhibitor (Min et al. 2011)
and radiosensitizing activity (Devi and Kamath 2003) etc. Till now several types of
modification like hydroxylation, biotin conjugate, epoxide ring opening, thiirane for-
mation, regio-/stereoselective Michael addition, triazole linking and spiro-pyrrolizi-
dinyl-oxindole formation via [3 þ 2] dipolar cycloaddition, acylation, epoxidation,
silylation etc have been performed for the finding of better therapeutics for mankind
(Figure 3). Strategies adopted to prepare the isoxazolidine ring are mainly 1,3-
Dipolar Cycloaddition, Cyclizations of unsaturated hydroxylamines, from
Isoxazolidinones, Isoxazolines, Isoxazolinium Salts via reduction described in the fol-
lowing Figure 4.
Our objective of this work is to introduce a new molecular entity as Isoxazolidino
Withaferin, a novel hybrid of withaferin A and isoxazolidine. The principle of this con-
cept is to combine partial or whole structures of molecules to create new, possibly
more active, molecular entities (Mehta and Singh 2002; Tietze et al. 2003).

O O
O O
NH O HN
N NH
O N
O O O O NH2
N
HN
Terizidone Cycloserine
Reglixane
O

Figure 1. Some of the drug containing isoxazolidine ring system.

 NATURAL PRODUCT RESEARCH 3

2. Results and discussion

Semi-synthesis of the Isoxazolidino Withaferin hybrid is performed in a two-step reac-
tion. The first step is the synthesis of nitrone and the second step is nitrone 1,3-

O O
H
O N
OH Me H
O N O
O O
HH O Me H H
N O N
O
NHN OH O O OMe

OH MeO O OH O OMe
HN OMe O
OH MeO O
HO3SO OMe O
Zetekitoxin AB, potent voltage- Dactylicapnosinine Dactylicapnosine
dependent sodium channel blocker
H H
HO
O
HN Me
O N
N
O 8 O
COMe 8 n O
N N
R N H Pyrinodemin A (n=3) H
HO CO Me
2 Pyrinodemin C (n=1) Lycojaponicumin A
Alsmaphorazine A (R=OH) potent cytotoxicity in vitro against
Alsmaphorazine B (R=H) murine leukemia L1210 with slight
inhibitor of NO production by antimicrobial or antifungalactivities
lipopolysaccharide-stimulated macrophages
HO
OH O
O
O
H O N
H O O OH
O O N O
O N R1
H O
H O
HO R2
N H
O
(-)-Virosaine A (R1=OH, R2=H) Pyridomacrolidin; tyrosine kinase
(-)-Flueggine A; activity
against three different breast (+)-Virosaine B (R1=H, R2=OH) inhibitor
cancer cell lines

Figure 2. Natural product containing isoxazolidine ring system.

Figure 3. Summary of all possible modification on withaferin A.

4 R. MANDAL ET AL.

Figure 4. Synthetic strategies to prepare the isoxazolidine ring.

O
STEP- 1 H
N H
H HO RT N
+ O
R Overnight, Dark
STEP-2
α -(4-substituted phenyl)
R N-phenyl nitrone
R
OH OH
O O
O O
O H butanol:Xylene (1:1) O
H N H H
+ O 120°C, 40 h
H H N H H
O
O H O 3A; R= -NO2,
OH R OH 3B; R= -Br,
1 2A-2C 3C; R= -Cl
Isoxazolidino Withaferin; 3A-3C

Scheme 1. Generalized scheme for synthesis of isoxazolidino withaferin A hybrid.

dipolar cycloaddition reaction on ring A unsaturation of withaferin A (1). Due to

unavailability of the n-phenyl hydroxylamine in commercial market, prior to the semi-
synthesis n-phenyl hydroxylamine was synthesized from nitrobenzene as per the litera-
ture procedure of reduction using Zn, NH4Cl (Kamm 1925). Synthesis of nitrones (2A-
2C) was performed as per the literature report with N-phenyl hydroxylamine and vari-
ous 4-substituted benzaldehyde in ethanol under room temperature in dark condition
for overnight (Bru €ning et al. 1966). The cycloaddition reactions were accomplished
using nitrone analogs (2A-2C) and withaferin-A (1) in 1:1 mixture of refluxing butanol
and xylene at 120  C (Scheme 1). Around 38–40 hours were required for the reaction
to produce products 3A–3C in moderate (60–65%) yields. With increasing the tem-
perature and time of reflux, the yield is not increased. The identity of all the products
of the cycloaddition reaction was confirmed by detailed mass, NMR spectrometric data
analysis. Assignment of the site of 1,3-dipolar cycloaddition on withaferin A was con-
firmed on the basis of disappearance of characteristic double bond signals of ring A of
withaferin A in the NMR spectrum of 3A. The careful analysis of the NMR spectral data
of the products revealed that the signals relating to B, C and D ring system and d
 NATURAL PRODUCT RESEARCH 5

Figure 5. Important correlation of Isoxazolidino withaferin (3A) [HMBC, COSY, NOSEY].

lactone ring remained unaltered. However, the chemical shifts for the nuclei belonging
to the a,b-unsaturated ketone containing A ring were distinctly shifted. The shift was
observed with C-2 and C-3, suffering a profound alteration in the resonance position
from d 131.3 and 141.9 to d 58.5 and 78.1 respectively. It must be pointed out that
withaferin A numbering has been maintained for the basic skeleton for ease in
correlation.
The crucial evidence in support of this addition came from the observed HMBC cor-
relation (Figure 5) in the spectrum of 3A between signals of C-1 (d 206.91) and H-2 (d
3.99), C-4 (d 72.60) and H-3 (d 4.76), C-100 (i.e., the isoxazolidine carbon, d 72.07) and
H-2 (d 3.99) as also H-3 (d 4.76), and C-5 (d 62.7) and H-3 (d 4.76). Further, the COSY
relationship (Figure 5) between H-100 of isoxazolidine (d 5.34) and H-2 (d 3.99) strongly
support the mode of addition. Strong NOESY relationship of H-2 (d 3.99) with both H-
3 (d 4.76) and b-oriented C-19 methyl proton (d 1.18) suggests that both H-2 and H-3
are b oriented which was also deduced from the coupling constant value of the ring
juncture protons (J H2, H3 = 9.6 Hz).
Following the success of the semi-synthesis of isoxazolidino withaferin A hybrids
(3A-3C) with the above mentioned 4-substituted benzaldehydes, a dinitrone (4) spe-
cies was prepared using glyoxal followed by the synthesis of a dinitrone adduct (5) via
double nitrone cycloaddition in one pot condition at around 60% yield (Scheme 2). A
critical observation of the HMBC spectrum of withaferin-dinitrone adduct (5) revealed
the crucial evidence in support of the cycloaddition as the correlation between signals
of C-1 (d 208.4) and H-2 (d 4.03), H-3 (d 5.03), H-100 (d 4.61); between C-200 (i.e., the iso-
oxazolidine carbon attached to nitrogen, d 68.4) and H-2 (d 4.03). Further the COSY
relationship between H-100 of isoxazolidine (d 4.61) and H-2 (d 4.03), and between H-2
(d 4.03) and H-3 (d 5.03) strongly support the mode of addition. Strong NOESY rela-
tionship of H-2 (d 4.03) with both H-3 (d 5.03) and b-oriented C-19 methyl proton (d
1.31) suggests that both H-2 and H-3 are b oriented (Figure 6). From a mechanistic
point of view, it is expected that the phenyl group of C-substituted N-phenyl nitrone
does prefer to be a-oriented to avoid dipole-dipole repulsion with C-1 carbonyl group
of withaferin A. The reaction proceeds in a highly stereoselective manner as it fur-
nished b-orienting cis fused ring junction hydrogens due to the presence of the C-4
b-orienting hydroxyl group, which guides the addition mode from a-face. Moreover,
the reaction also proceeds through a regioselective as well as a chemoselective path-
way; only unsaturation of ring A in withaferin A took part in the reaction, but
6 R. MANDAL ET AL.

O H
H N
OH RT N
H + 2 O
O Dark, overnight N
O
Glyoxal N-phenyl hydroxylamine Dinitrone; 4

OH
OH
O O O
O
O
H HO
O
O N O H
2 H H
H
H
O Butanol:Xylene (1:1)
OH O N
1 120oC, 40 h O O
OH
+
O O
N
N O
O HO Withaferin - Dinitrone Adduct; 5
4

Scheme 2. Synthesis of withaferin–dinitrone adduct.

Figure 6. Important correlations of withaferin–dinitrone adduct (5) [COSY, NOESY and HMBC].

unsaturation present in lactone ring of WA remain unreactive due to steric

hindrance of 24th position methyl group and 25th position hydroxymethyl group on
withaferin A.

3. Experimental
3.1. General experimental procedure
Withaferin A used in reaction was isolated from the methanol extract of leaves of the
medicinal plant Withania somnifera by column chromatography followed by crystalliza-
tion. All chemicals employed for reactions were purchased from Alfa-Aesar/Sigma-
Aldrich Company and used as received without further purification. All other solvents
and chromatographic adsorbents were purchased from E. Merck (Germany) and SRL
(India) Ltd. unless otherwise indicated. TLC analysis was performed on Merck 60 F254
 NATURAL PRODUCT RESEARCH 7

silica gel TLC plates using varying percentages of methanol in chloroform as solvent
system and spots were identified using UV indicator (254 nm) followed by staining in
iodine vapour. Flash chromatography was performed using YAMAZEN AKROS smart
flash chromatography system. NMR spectra were recorded using a Bruker spectropho-
tometer operating at 600 MHz for 1H and 150 MHz for 13C respectively in CDCl3. HPLC
analysis was done in Shimadzu HPLC with SPD-M20A PDA detector using a C18 col-
umn. HRMS data of the compounds were obtained from Agilent 6545 Q-TOF LC/
MS instrument.

3.2. Synthesis of a-(4- substituted phenyl) N-phenyl nitrone

Solution was prepared of freshly prepared 1g. of pure N-phenyl hydroxylamine in
15 ml. of ethanol in a 100 ml. erlenmeyer flask and react with equimolar amount of 4-
substituted (R ¼ NO2, Br, Cl) benzaldehyde (exothermic reaction) in 40–60  C. The flask
was stoppered and kept overnight at room temperature in the dark. The colorless nee-
dles of a-(4-substituted phenyl) N-phenyl nitrone are collected on a Buchner funnel
and washed once with 20 ml. of ethanol. The yield of the product obtained is
1.90g (85–87%).

3.3. Synthesis of withaferin - nitrone hybrids

In a 100 ml. round bottom flask provided with a reflux condenser 500 mg. previously
prepared pure nitrone and an equimolar amount of withaferin A were taken in 25 ml
of butanol and xylene 1:1 mixture. The flask was heated at 120  C for 40 hours. After
compilation of reaction as evident from TLC, the solvent was removed in rotary evap-
orator and the crude product was subjected to flash chromatography using increasing
concentration of methanol in chloroform as eluant. The product was crystallized from
chloroform–methanol mixture.

3.3.1 Spectroscopic data of compound compound 3A

Colour: Obtained as yellowish white solid; Yield-65%; mp: 172-174  C, Rf value: 0.41
(2.5% Methanol in Chloroform); 1H NMR (CDCl3, 600MHz): d 8.17 (2H, dd, J ¼ 18,
9 Hz), 7.74 (2H, d, J ¼ 8.4 Hz), 7.3 (2H, t, J ¼ 7.8 Hz), 7.04 (1H, t, J ¼ 7.2 Hz), 6.96 (2H, d,
J ¼ 8.4 Hz), 5.34 (1H, d, J ¼ 9.6 Hz), 4.76 (1H, t, J ¼ 6 Hz), 4.4 (1H, t, J ¼ 3.6 Hz), 4.38 (1H,
t, J ¼ 4.2 Hz), 4.34 (1H, d, J ¼ 12 Hz), 3.99 (1H, dd, J ¼ 9.6, 7.2 Hz), 3.78 (1H, d, J ¼ 4.8 Hz),
3.26 (1H, s), 3.13 (1H, s), 2.97 (1H, s), 2.54 (1H, dd, J ¼ 17.4, 13.2 Hz), 2.14 (1H, d,
J ¼ 14.4 Hz), 2.07 (3H, s), 2.05 (1H, t, J ¼ 3.6 Hz), 2.02 (1H, d, J ¼ 3 Hz), 1.92-1.9 (1H, m),
1.75 (1H, s), 1.64 (1H, dd, J ¼ 8.4, 3.6 Hz), 1.61 (1H, dd, J ¼ 11.4, 7.8 Hz), 1.3 (1H, d,
J ¼ 12 Hz), 1.27-1.24 (3H, m), 1.18 (3H, s), 1.07 (1H, dd, J ¼ 12, 4.8 Hz), 1.31 (1H, s), 1.01
(1H, d, J ¼ 3 Hz), 0.91 (3H, d, J ¼ 6.6 Hz), 0.69 (1H, d, J ¼ 3 Hz), 0.54 (3H, s), 0.43 (1H, s);
13
C NMR (CDCl3, 150MHz): d 206.9 (-C ¼ O), 171.3 (-C ¼ O), 167.2 (-C), 153.4 (-C), 150.0
(-C), 147.5 (-C), 144.8 (-C), 129.4 (-CH), 129.3 (-CH), 123.5 (-CH), 123.1 (-CH), 114.6 (-CH),
78.7 (-CH), 78.1 (-CH), 72.6 (-CH), 72.1 (-CH), 62.7 (-C), 59.1 (-CH), 58.5 (-CH), 57.5 (-CH2),
56.2 (-CH), 51.9 (-CH), 49.9 (-C), 42.2 (-C), 39.6 (-CH), 38.7 (-CH), 38.7 (-CH2), 31.9 (-CH),
30.6 (-CH2), 29.7 (-CH2), 29.1 (-CH), 27.2 (-CH2), 24.2 (-CH2), 20 (-CH3), 19.9 (-CH2), 17.4
8 R. MANDAL ET AL.

(-CH3), 13.2 (-CH3), 11.3 (-CH3); HRMS [ESI-MS, positive mode]: MF: C41H48N2O9; found
m/z 735.3257 [M þ Na]þ [calcd. 735.3258].

3.3.2. Spectroscopic data of compound compound 3B

Colour: Obtained as yellow solid; Yield-63%; mp: 185-188  C, Rf value: 0.43 (2.5%
Methanol in Chloroform); 1H NMR (CDCl3, 600MHz): d 7.52 (2H, d, J ¼ 8.4 Hz), 7.36-
7.34 (3H, m), 7.32 (1H, s), 7.07 (1H, t, J ¼ 7.8 Hz), 7.03 (2H, d, J ¼ 7.8 Hz), 5.29 (1H, d,
J ¼ 9 Hz), 4.78 (1H, dd, J ¼ 7.2, 5.4 Hz), 4.47 (1H, t, J ¼ 3 Hz), 4.46-4.43 (1H, m), 4.40 (1H,
dd, J ¼ 12.6, 6Hz), 3.96 (1H, dd, J ¼ 9, 7.2 Hz), 3.83 (1H, t, J ¼ 4.2 Hz), 3.31 (1H, s), 2.92
(1H, t, J ¼ 6.6 Hz), 2.56-2.52 (2H, m), 2.19 (1H, dd, J ¼ 11.4, 2.4 Hz), 2.10 (3H, s), 2.05 (1H,
dd, J ¼ 18, 3 Hz), 2 (1H, m), 1.74-1.65 (3H, m), 1.39-1.36 (2H, m), 1.34 (1H, s), 1.31 (1H,
dd, J ¼ 9.36, 3.6 Hz), 1.24 (3H, s), 1.16-1.10 (2H, m), 1.03 (3H, d, J ¼ 6 Hz), 0.81-0.77 (2H,
m), 0.66 (1H, dd, J ¼ 13.8, 3.6 Hz), 0.62 (3H, s), 0.61-0.59 (1H, m); 13C NMR (CDCl3,
150MHz): d 207.5 (-C ¼ O), 167.6 (-C ¼ O), 153.4 (-C), 150.9 (-C), 136.2 (-C), 134.1 (-C),
130.1 (-CH), 129.8 (-CH), 129 (-CH), 126.2 (-C), 123.3 (-CH), 115.3 (-CH), 79.3 (-CH), 78.4
(-CH), 73.6 (-CH), 72.5 (-CH), 63.3 (-C), 59.8 (-CH), 59.1 (-CH), 58.1 (-CH2), 56.5 (-CH), 52.5
(-CH), 50.5 (-C), 42.9 (-C), 39.9 (-CH), 39.3 (-CH), 39.1 (-CH2), 31.3 (-CH), 30.3 (-CH2), 29.7
(-CH), 27.8 (-CH2), 24.8 (-CH2), 20.6 (-CH3), 20.5 (-CH2), 17.9 (-CH3), 13.8 (-CH3), 11.9
(-CH3); HRMS [ESI-MS, positive mode]: MF: C41H48BrNO7; found m/z 768.2510 [M þ Na]þ
[calcd. 768.2512]; found m/z 770.2496 [M þ 2 þ Na]þ [calcd. 770.2491]

3.3.3. Spectroscopic data of compound 3C

Colour: Obtained as brown solid; Yield-68%; mp: 160-164  C, Rf value: 0.40 (2.5%
Methanol in Chloroform); 1H NMR (CDCl3, 400MHz): d 7.36 (2H, d, J ¼ 8.4 Hz), 7.3 (2H,
d, J ¼ 4 Hz), 7.28 (2H, t, J ¼ 2 Hz), 7.18 (1H, t, J ¼ 7.6 Hz), 6.92 (2H, d, J ¼ 6.8 Hz), 4.95
(1H, d, J ¼ 4.8 Hz), 4.89 (1H, s), 4.39 (1H, d, J ¼ 4 Hz), 4.36 (1H, s), 3.77 (2H, t, J ¼ 4 Hz),
3.48 (1H, s), 3.28 (1H, s), 2.85 (1H, s), 2.7 (1H, s), 2.51 (2H, d, J ¼ 6.8 Hz), 2.17 (3H, s),
2.05-1.93 (4H, m), 1.62 (3H, s), 1.42-1.19 (7H, m), 1.1 (2H, t, J ¼ 12.8 Hz), 0.98 (3H, d,
J ¼ 6.4 Hz), 0.87 (1H, s), 0.65 (3H, s), 0.57 (1H, s); 13C NMR (CDCl3, 400MHz): d 207.6
(-C ¼ O), 167.1 (-C ¼ O), 152.9 (-C), 150.5 (-C), 148.7 (-C), 138.6 (-C), 133.8 (-C), 129.4
(-CH), 128.7 (-CH), 128.4 (-CH), 122.4 (-CH), 116 (-CH), 78.8 (-CH), 78.7 (-CH), 70.5 (-CH),
68.9 (-CH), 63.2 (-C), 62.9 (-CH), 57.5 (-CH), 57.3 (-CH2), 56.1 (-CH), 52 (-CH), 49.9 (-C),
42.2 (-C), 41.9 (-CH), 39.2 (-CH2), 38.8 (-CH), 30.69 (-CH2), 29.9 (-CH2), 29.8 (-CH), 28.9
(-CH), 27.3 (-CH2), 24.3 (-CH2), 21.2 (-CH2), 20.1 (-CH3), 16.7 (-CH3), 13.4 (-CH3), 11.7
(-CH3); HRMS [ESI-MS, positive mode]: MF: C41H48ClNO7; found m/z 724.3018 [M þ Na]þ
[calcd. 724.3017].

3.4. Synthesis of dinitrone

Dinitrone was obtained by treating glyoxal and 2 equivalent amount of freshly pre-
pared pure N-phenyl hydroxylamine in ethanol. The flask was stoppered and kept
overnight at room temperature in the dark. The colorless needles of dinitrone are col-
lected on a Buchner funnel and washed once with 20 ml. of ethanol.
 NATURAL PRODUCT RESEARCH 9

3.5. Synthesis of withaferin–dinitrone adduct

In a 50 ml. round bottom flask provided with a reflux condenser 500 mg. previously
prepared pure dinitrone and 2 equivalent amount of withaferin A were taken in 20 ml
of butanol and xylene 1:1 mixture. The flask was heated at 120  C for 40 hours. After
compilation of reaction as evident from TLC, the solvent was removed in rotary evap-
orator and the crude product was subjected to flash chromatography using increasing
concentration of methanol in chloroform as eluant. The product was crystallized from
chloroform–methanol mixture.

3.5.1. Spectroscopic data of compound 5

Colour: Obtained as white solid; Yield-60%; mp: 195-198  C, Rf value: 0.43 (2.5%
Methanol in Chloroform); 1H NMR (CDCl3, 600MHz): d 7.32 (4H, d, J ¼ 12 Hz), 7.28-
7.23 (4H, m), 6.9-6.88 (2H, m), 5.03 (2H, t, J ¼ 9 Hz), 4.61 (2H, s), 4.47-4.35 (5H, m), 4.03
(2H, d, J ¼ 8.4 Hz), 3.51 (2H, d, J ¼ 7.8 Hz), 3.22 (2H, s), 2.53 (4H, dd, J ¼ 17.4, 13.8 Hz),
2.15-2.13 (2H, m), 2.09 (6H, s), 2.05-2.01 (4H, m), 1.93 (2H, d, J ¼ 12.6 Hz), 1.78-1.63 (6H,
m), 1.48-1.36 (6H, m), 1.31 (6H, s), 1.26-1.18 (6H, m), 1.16-1.09 (6H, m), 1.03 (6H, d,
J ¼ 6.6 Hz), 0.92 (1H, t, J ¼ 6.6 Hz), 0.87 (2H, dd, J ¼ 18.6, 11.4 Hz), 0.68 (6H, s); 13C NMR
(CDCl3, 150MHz): d 208.4 (-C ¼ O), 160.9 (-C ¼ O), 152.8 (-C), 151.3 (-C), 128.6 (-CH),
125.6 (-C), 121 (-CH), 113.3 (-CH), 81.7 (-CH), 78.6 (-CH), 68.4 (-CH), 68.4 (-CH), 62.3 (-C),
57.3 (-CH2), 56.7 (-CH), 56.3 (-CH), 55.7 (-CH), 51.8 (-CH), 49.5 (-C), 42.6 (-C), 40.8 (-CH),
38.9 (-CH2), 38.6 (-CH), 29.8 (-CH2), 28.2 (-CH), 27.1 (-CH2), 24.1 (-CH2), 21.3 (-CH2), 19.9
(-CH3), 16.8 (-CH3), 13.3 (-CH3), 11.6 (-CH3); MS [ESI-MS, positive mode]: MF:
C70H88N2O14; found m/z 1181.6342 [M þ H]þ [calcd. 1181.6314].

4. Conclusion
In conclusion, a facile, atom-economic synthesis of isoxazilidino withaferin, a novel
hybrid of withaferin A has been achieved via two-step reaction, generation of nitrone
and nitrone 1,3-dipolar cycloaddition on withaferin A. The reaction is highly chemose-
lective (preferential reaction only on one of the two double bonds present on witha-
ferin A), regioselective and diastereoselective affording exclusively the cis-fused
products. The structure was determined by thorough analysis of 1D and 2D NMR and
mass spectral data.

Acknowledgements
A.H and B.Y. are recipients of Research Fellowships in the form of Young Scientist Grant (YSS/
2015/001141) and NPDF/2016/000088 from SERB-DST, India. R.M., M.S. and A.K. thank DOP,
Ministry of Chemical and Fertilizers, Govt. of India, for providing fellowship. We also thank R.
Gajbhiye of NIPER for his assistance in acquiring HRMS spectra. Our thanks are due to Drs. N.B.
Mondal and B. Achari (Ex-Emeritus Scientist, CSIR-IICB) for helpful suggestions.

Disclosure statement
The authors declare no competing financial interest.
10 R. MANDAL ET AL.

Funding
Young Scientist Grant (YSS/2015/001141) and NPDF/2016/000088 from Science and Engineering
Research Board, India.

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