Anti-inflammatory chromone alkaloids and glycoside from Dysoxylum binectariferum

Article abstract of DOI:10.1016/j.tetlet.2017.09.005

Herein we report isolation of a new chromone alkaloid chrotacumine K (12) from fruits and a chromone glycoside schumaniofioside A (13) from leaves of Dysoxylum binectariferum Hook f. Schumaniofioside A is reported for the first time from Meliaceae family. Other known alkaloids isolated include rohitukine (1) and chrotacumine E (6). The structure of new alkaloid 12 was elucidated on the basis of extensive 1D and 2D NMR analysis, synthesis and chemical hydrolysis. Chemically, chrotacumine K (12) is a 3′-O-acetyl rohitukine which on chemical or enzymatic hydrolysis produces rohitukine. The new alkaloid 12 is also present in seeds and stem-barks of this plant. The glycoside schumaniofioside A (13) is present only in leaves, and in abundance (～1% w/w of dried leaves). The isolated compounds and extracts were evaluated for in vitro effect on the proinflammatory cytokines (TNF-α and IL-6) in human monocytic THP-1 cells. The alkaloid 12 displayed potent inhibition (57%) of TNF-α at 0.3 μM, and was non-toxic to THP-1 cells up to 40 μM, indicating its excellent therapeutic window. Furthermore, a nitrobenzoyl ester analog 15e showed better inhibition of IL-6 than parent natural product chrotacumine K.

Full text of DOI:10.1016/j.tetlet.2017.09.005

Accepted Manuscript
Anti-inflammatory chromone alkaloids and glycoside from Dysoxylum binec-
tariferum ‖
Vikas Kumar, Mehak Gupta, Sumit G. Gandhi, Sonali S. Bharate, Ajay Kumar,
Ram A. Vishwakarma, Sandip B. Bharate
PII:
S0040-4039(17)31114-0
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.09.005
TETL 49275
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
31 July 2017
30 August 2017
4 September 2017
Please cite this article as: Kumar, V., Gupta, M., Gandhi, S.G., Bharate, S.S., Kumar, A., Vishwakarma, R.A.,
‖
Bharate, S.B., Anti-inflammatory chromone alkaloids and glycoside from Dysoxylum binectariferum , Tetrahedron
Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.2017.09.005
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Anti-inflammatory chromone alkaloids and
glycoside from Dysoxylum binectariferum
Vikas Kumar, Mehak Gupta, Sumit G. Gandhi, Sonali S. Bharate, Ajay Kumar, Ram A. Vishwakarma
and Sandip B. Bharate
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1
Tetrahedron Letters
journal homepage: www.els evier.com
ǁ
Anti-inflammatory chromone alkaloids and glycoside from Dysoxylum binectariferum
a,b
Vikas Kumar, Mehak Gupta, Sumit G. Gandhi, Sonali S. Bharate, Ajay Kumar, Ram A. Vishwakarma and
c
bd
a
c
b,e
be*
Sandip B. Bharate
a
bPreformulation Laboratory, PK-PD Toxicology & Formulation Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India
cAcademy of Scientific & Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India
dCancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India
Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India
e
Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Herein we report isolation of a new chromone alkaloid chrotacumine K (12) from fruits and
a chromone glycoside schumaniofioside A (13) from leaves of Dysoxylum binectariferum
Hook f. Schumaniofioside A is reported for the first time from Meliaceae family. Other
known alkaloids isolated include rohitukine (1) and chrotacumine E (6). The structure of
new alkaloid 12 was elucidated on the basis of extensive 1D and 2D NMR analysis,
synthesis and chemical hydrolysis. Chemically, chrotacumine K (12) is a 3´-O-acetyl
rohitukine which on chemical or enzymatic hydrolysis produces rohitukine. The new
alkaloid 12 is also present in seeds and stem-barks of this plant. The glycoside
schumaniofioside A (13) is present only in leaves, and in abundance (~1% w/w of dried
leaves). The isolated compounds and extracts were evaluated for in-vitro effect on the
proinflammatory cytokines (TNF-α and IL-6) in human monocytic THP-1 cells. The alkaloid
12 displayed potent inhibition (57%) of TNF-α at 0.3 µM, and was non-toxic to THP-1 cells
up to 40 µM, indicating its excellent therapeutic window. Furthermore, a nitrobenzoyl ester
Received in revised form
Accepted
Available online
Keywords:
chrotacumine K,
rohitukine,
schumaniofioside A,
chromone alkaloid,
anti-inflammatory,
Dysoxylum binectariferum
analog 3e showed better inhibition of IL-6 than parent natural product chrotacumine K.
2
017 Elsevier Ltd. All rights reserved.
Dysoxylum species is a rich source of chromone alkaloids. The
1
widely investigated natural product rohitukine (1) has been isolated
all 3'-O-acyl rohitukine derivatives, most of them are benzoyl
derivatives and few are with 3-5 carbon chain containing aliphatic
acyl units; however simple 3'-O-acetyl-rohitukine has never been
reported in the literature.
2
from barks of Dysoxylum binectariferum Hook (Meliaceae) and is
reported to possess a wide range of biological activities including
3
cytotoxicity, antidyslipidemic, antiadipogenic, gastroprotective,
4
5
6
7
antifertility and antileishmanial activity. Furthermore, this natural
8
During our efforts to explore fruits and leaves of Dysoxylum
binectariferum as an alternative (renewable) source of rohitukine
isolation; a new alkaloid chrotacumine K (3'-O-acetyl-rohitukine)
along with chrotacumine E, schumaniofioside A and rohitukine
have been isolated and characterized. Herein, we are reporting for
the first time isolation of schumaniofioside A from Meliaceae
family. Previously, it was reported from Pancratium maritimum,
Schumanniophyton magnificum and Staphylea bumalda (DNP
product has inspired the discovery of two anticancer clinical
9-10
11
candidates flavopiridol and P276-00. The structural variations
on rohitukine in nature was primarily observed as change in the
th
location of piperidinyl moiety from 8 to 6 position (dysoline)
th
12
and substitution of piperidine hydroxyl with acyl units, a class of
1
3-16
compounds called chrotacumines A-J (2-11).
OH
(Figure 1).
O
OH
O
5
4
OH
O
5
4
6
7
4
a
3
2
4a
search). The chemical structures of newly isolated chrotacumine K
(12), and schumaniofioside A (13) are shown in Figure 2. The
bioactivity evaluation of isolated compounds for inhibition of
proinflammatory cytokines was also performed.
6
7
3
2
HO
8
8a
O
8a
O
8
H
9
O
2'
9
H
4'
O
OR
O
H
5
'
3'
3'
N
4'
6'
2'
N
N
5'
6'
O COPh
Chrotacumine A (2)
OH
Chrotacumine E (6)
Rohitukine (1): R = H
OH
O
4
HOH
7' H O
6'
OH
O
OH
O
5
5
4
6
7
4a
3
Chrotacumine B (3): R = - COC(Me)=CH-Me
Chrotacumine C (4): R = -COPh(3,4,5-tri-OMe)
Chrotacumine D (5): R = -COPh(3,4-di-OMe)
Chrotacumine F (7): R = -CO-CH=CH-Ph
4a
5
4
5'
6
7
3
2
4a
HO
HO 4'
6
3
2
2'
2
O
H 3'
O
O
7
O 2'
HO
8
8a O
O
9
OH
5
HO
8
8
a
O
8a O
H
4'
3'
H
H
4a
8a
4
9
8
1'' 2''
9
6
H
5'
4'
5'
6'
3
OH
H
3'
3'
5'
2'
7
Chrotacumine G (8): R = -CO-CH=CH-Ph (3,4,5-tri-OMe)
N
4'
O
HO
8
2
6'
2'
N
1
H- H COSY
HMBC
1
9
Chrotacumine H (9): R = -CO-CH=C(Me)CH(CH
3
)
2
N
6'
OH
Chrotacumine I (10): R = -COPh(3,5-di-OMe,4-OH)
Chrotacumine J (11): R = -COPh(3-OMe,4-OH)
1
6
12
13
Figure 1. Structures of rohitukine (1) and chrotacumines A-J (2-11)
Figure 2. Chromone alkaloids 1, 6, 12 and chromone glycoside 13 isolated
from D. binectariferum. The key COSY and HMBC correlations of
chrotacumine K (12) are also shown.
Chrotacumine A and E has modified piperidinyl ring; whereas rest
all chrotacumines are 3'-O-acyl derivatives of rohitukine. Amongst
-
-----
*
Corresponding author. E-mail: sbharate@iiim.ac.in (SBB)
ǁ
IIIM Publication Number. IIIM/1933/2016

2
Tetrahedron Letters
The HPLC chromatogram of methanolic extract of Dysoxylum
assignments of chrotacumine K (12) along with rohitukine and its
17
binectariferum fruits showed three key peaks at t
7.5 min, respectively (Figure 3a). The co-TLC with reference
standard available in our laboratory indicated that the peak at 11.5
min belongs to rohitukine; however the peak at t 12.7 min appeared
R
11.5, 12.7 and
closely related chrotacumine H are shown in Table 1. Further, the
presence of chrotacumine K in chloroform and water extracts of
fruits of D. binectariferum was investigated. Chrotacumine K was
present in both these extracts (HPLC chromatograms are depicted in
Section S36 of supporting information). The presence of this
compound was also investigated in seeds, stem-bark and leaves of D.
binectariferum by TLC and HPLC analysis. The seeds, stem-barks
and leaves of D. binectariferum were extracted with chloroform. The
co-TLC of these chloroform extracts with chrotacumine K indicated
its presence in seeds and stem-barks; however it was found absent in
leaves (TLC images are shown in section S14 of supporting
information). HPLC analysis also supported these observations.
Next, the chrotacumine K (12) was synthetically prepared by
reacting rohitukine with acetyl chloride in presence of triethylamine
1
R
to be the new one. The methanolic extract was loaded on silica gel
column and was eluted with increasing concentration of methanol in
chloroform. At 7-8% methanol in chloroform, a new alkaloid was
isolated followed by isolation of rohitukine at 12-13% methanol in
chloroform. Isolated compounds were characterized by NMR and
1
8
MS analysis. Rohitukine was characterized by co-TLC with
reference standard and by comparison of their spectral data with
1
literature values. The NMR of new alkaloid was recorded in CDCl
3
(
in order to see phenolic OH signals) as well as CD
3
OD (for
comparison with rohitukine).
(
Figure 4). The synthesized 3´-O-acetyl rohitukine and isolated
1
The alkaloid 12 showed dragendorff positive test, and its H NMR
spectra was similar (except few differences) to rohitukine, which
gave indication that it could be a rohitukine class of compound. In
chrotacumine K were identical in all respects viz. TLC, HPLC, and
NMR.
1
the H NMR spectrum of 12, the signal at δ 4.14 ppm (H-3′) of
rohitukine was downfield-shifted to δ 5.18 ppm. Similarly, in the
From the methanolic extract of seed, another dragendorff positive
compound was isolated, but in very minor amount (5 mg). On TLC
analysis, it was found non-polar to both rohitukine and chrotacumine
K. After comparison of spectral data with literature values, it was
13
C
NMR, the signal at δ 66.70 ppm (C-3′) of rohitukine was shifted to δ
1
1.14 ppm. The C NMR also showed the presence of two extra
3
7
1
4
1
13
carbons, one at δ 170.6 ppm and other at δ 21.01 ppm. These
observations clearly indicated the presence of acetyl group at 3'-OH.
The presence of ester group was also confirmed by IR spectra with
identified as chrotacumine E (6). The comparison of H and C
NMR values of chrotacumine K (12) with rohitukine, chrotacumine
E and chrotacumine H is shown in Table 1.
-
1
appearance of stretching vibration at 1737 cm . Furthermore, the
position of acetyl ester at 3′ position of piperidine ring was
ascertained by HMBC studies, wherein a key correlation between H-
Our observations indicated that the conversion of chrotacumine K
into rohitukine is a slow process; and the extraction process involved
cold maceration for 2 hrs. Therefore, there would be conversion of
very small amount of chrotacumine K to rohitukine during
extraction. But still, the ratio between chrotacumine K and
rohitukine that we observed during extraction (as depicted in Figure
3a) may not be the real ratio of them in fruits.
3′ with C-7′ was observed. The key HMBC correlations of
compound 12 are shown in Figure 2.
It is worth reporting that when compound 12 was submitted for
3
NMR studies in CD OD and after checking TLC of the sample after
a week, the portion of the 12 was converted to rohitukine (1).
Furthermore, the HPLC analysis of the methanolic extract of fruits
recorded immediately on preparation and after a month indicated
that the ratio of rohitukine to chrotacumine K get changed, with
significantly increased percentage of rohitukine. The HPLC
comparison of rohitukine, chrotacumine K and partially hydrolyzed
During phytochemical efforts on leaves of D. binectariferum, it
was interesting to observe that the HPLC chromatogram of
hydroalcoholic extract of leaves showed presence of only two peaks,
of equal intensity. The first among these two peaks was rohitukine,
whereas the second peak was observed to be unknown, as its HPLC
and TLC profile does not matched with any of the reference
1
13
chrotacumine K is shown in Figure 3b-c. The H and C NMR
a)
Rohitukine
(
b)
(
ChrotacumineK
257
Rohitukine
226
296
(
c)
ChrotacumineK
(d)
257
227
ChrotacumineK
297
Rohitukine
Figure 3. HPLC chromatogram of (a) fruits of D. binectariferum MeOH extract; (b) rohitukine; (c) chrotacumine K; and (d) chrotacumine K hydrolysis to
rohitukine in CD OD. Insets in Figure 2b and 2c are UV chromatograms of respective compounds.
3

3
1
Table 1. The H and C NMR assignments of chrotacumine K (12), chrotacumine E (6), rohitukine (1) and schumaniofioside A (13) and chrotacumine H (9)
13
a
b
c
Positi
on
12 in CDCl
3
12 in CD
δ(H)
3
OD
6 in CDCl
3
1 in CD
δ(H)
3
OD
13 in CD
3
OD/(CD
3
)
2
CO
9 in CD
3
OD
δ(C)
δ(H)
δ(C)
δ(C)
δ(H)
δ(C)
δ(C)
δ(H)
δ(C)
67.17 /
δ(H)
1
2
3
4
-
166.35
108.13
182.76
104.43
-
168.87
108.71
184.31
106.44
-
165.70
-
167.70
107.40
183.00
104.00
-
-
168.3
108.2
184.0
104.2
1
65.69
6
.03 /
.87
111.65 /
111.77
6.01
5.99
6.03
108.61
182.20
104.45
5.97
5.97
5
1
1
1
80.34 /
78.31
-
-
-
-
-
-
-
-
-
-
-
1
09.19 /
09.78
4
a
-
-
1
60.16 /
60.19
-
5
-
160.79
-
161.93
-
-
160.36
-
-
-
160.70
-
-
161.6
-
1
5
-OH
6
12.86
6.27
-
12.63
6.31
-/-
-
6
6
2
.79 /
.82
104.59 /
105.94
99.65
6.13
99.87
98.48
6.15
99.40
6.18
100.7
6
1
1
64.68 /
63.33
7
8
-
162.71
105.23
156.81
20.42
-
164.37
105.17
158.30
20.38
-
-
162.46
99.36
155.08
20.15
86.51
-
163.00
106.40
156.50
18.90
-
-
167.3
107.6
158.7
20.5
.54 /
.50
-
-
-
-
-
-
99.13 / 99.41
61.11 /
-
6
1
8
a
-
-
-
1
60.29
.33 /
.20
9
2.38
2.34
2.36
4.96
2.32
19.88 / 19.78
2.44
2
3
.49,
4.82 /
4.58
105.04 /
106.62
3.03,
2.45
5.18
2
3
4
´
´
´
3.15-3.20
5.18
58.60
71.14
35.71
3.07- 3.17
5.06
57.59
71.90
37.55
60.40
66.70
35.70
60.6
71.3
37.9
3
.38
4.14
3.62-
3.59
3.64-3.65 29.22
3.61-3.63
74.55 / 74.86
1
2
.94-2.00
.07-2.15
3.53-3.56
3.42-3.46
27.29
3.46-3.48
77.30 / 77.19
3.52
2
.24-2.30,
.81
3.17-3.07,
1.71
3.15,
1.75
3.24,
1.71
5
´
24.81
25.72
4.19-4.23 69.77
21.90
3.43-3.46
71.30 / 71.37
26.4
1
3.35,
3.22-
3
.15-3.20,
2
3.07-3.17,
2.53
2.07-2.15
51.97
3.08,
2.20
6
7
´
´
56.19
-
59.88
-
55.40
-
3.48-3.51
78.68 / 78.42
62.53 / 62.92
58.0
-
.49
2.81-2.85
3
.20
-
3
3
.76-3.79,
.95-3.98
-
-
-
-
-
-
1
2
3
4
5
6
7
´´
´´
´´
´´
´´
´´
´´
170.60
-
172.12
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
167.9
114.4
167.0
39.1
1.88
21.01
1.80
20.93
5.63
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2.29
1.02
1.01
1.86
21.2
21.3
16.6
N-
CH
2
.38
45.93
2.35
46.01
2.55
41.52
2.82
42.90
-
-
2.32
46.5
3
a
1
the H and C NMR of chrotacumine K was recorded in CDCl
13
3
as well as CD
3
OD; however NMR of rohitukine was recorded only in CD
3
OD (as rohitukine
b
). the H and C NMR of schumaniofioside A (13) was recorded in CD
1
13
was insoluble in CDCl
group at carbon no C5. data taken Morita et al., 2014.
3
3
3 2
OD as well as in (CD ) CO in order to see the presence of OH
c
13
OH
O
OH
O
standards present with us. It does not showed Dragendorff positive
1 13
test, indicating that it is not a alkaloid. The H, C NMR, COSY,
O
DMF, TEA (2 equiv.), 0-5 °C
+
HO
O
O
HO
O
HMBC and NOESY correlations were recorded, which revealed that
R
Cl
H
H
R
OH
19
it to be a schumaniofioside A, a chromone-5-O-glycoside. The key
COSY, HMBC and NOESY correlation are shown in Section S37 of
supporting information. The chemical hydrolysis was performed, in
order to know the identity of aglycone and glycone as noreugenin
and β-D-glucose, respectively. Schumaniofioside A was found to be
present in 3-5% w/w in the extract of leaves (i.e. ~1% w/w in dried
leaves). Young aged leaves were found to have higher
O
N
N
Rohitukine (1)
2
a-f (1 equiv.)
3a-f, 12 (50-60% yield)
2: R = -Me
1
a: R =
b: R =
c: R =
f: R =
OMe
O
O
d: R =
OMe
OMe
e: R =
N
schumaniofioside A content compared to older leaves. The HPLC
chromatogram of hydroalcoholic extract of leaves is provided in
section S37 of supporting information.
Figure 4. Synthesis of chrotacumine K (12) and its ester analogs 3a-f
Further, in order to establish preliminary SAR for rohitukine
esters for cytokine inhibition activity, a series of rohitukine-3-O-
esters 3a-f (chrotacumine K analogs) were prepared (Figure 4). All
prepared esters were characterized by NMR, mass and IR
Dysoxylum binectariferum and its reported constituents have been
12,20
reported to possess anti-inflammatory activity.
Therefore, the
effect of isolated compounds and crude extract on inhibition of
21
proinflammatory cytokines TNF-α and IL-6 in THP-1 cells was
investigated; and results are shown in Table 2. THP-1 cells were
stimulated for release of cytokines by LPS. After treatment of LPS-
treated cells with test samples, the cytokine levels were measured.
The methanolic extract of fruits (DBFM ext) and hydroalcoholic
extract of leaves (DBLHA ext) showed promising inhibition (43-
spectroscopy and details are provided in the supporting information.
6
0% inhibition) of both cytokines (TNF-α and IL-6) at 2.5-3.12
µg/ml. Rohitukine at 5 µM, showed 50 and 82% inhibition of TNF-α
and IL-6, respectively. A newly isolated alkaloid 12 showed potent
inhibition of TNF-α; and a moderate inhibition of IL-6, at 10 µM,

4
Tetrahedron Letters
while chrotacumine E (6) and schumaniofioside A (13) showed
moderate inhibition of both cytokines. In case of synthesized esters,
varying degree of cytokine inhibition was observed and results are
included in Table 3. All the synthesized esters except compound 3f
showed good TNF-α inhibition. The compound 3e (p-nitro-phenyl
ester derivative) showed comparatively better inhibition of both the
cytokines compared to all isolated natural compounds. Compound 3e
inhibited cytokine IL-6 level up to 91% at 10 µM. Because of the
promising cytokine inhibition activity of compound 3e and
be 2.08 mg/ml. The stability of chrotacumine K in rat plasma was
also studied, in order to understand whether it gets hydrolyzed to the
rohitukine under enzymatic conditions. It was observed that after 4 h
of incubation of compound in rat plasma, 80% of the chrotacumine
was converted to rohitukine. This is indicative of the fact that in in-
vivo efficacy studies, chrotacumine K will act as a prodrug of
rohitukine (which is a bioactive natural product).
Table 4. Aqueous solubility, log P and log D of rohitukine (1), chrotacumine
a
K (12) and Schumaniofioside A (13)
chrotacumine K (12), the concentration-dependent inhibition of IL-6
by compound 3e while the concentration-dependent inhibition of
TNF-α by MeOH extract and chrotacumine K (12) was studied and
results are shown in section S38 of supporting information. All of
them showed concentration-dependent effect on cytokines.
Parameters
1
12
13
Solubility in water
(
>
10
> 10
2.081±0.176
mg/ml)
Log P (water/ n-
octanol)
−1.102 ±
0.063
0.413± 0.020
0.588 ± 0.016
−0.447±0.0314
−0.846±0.007
Log D (PBS pH 7.4/ n-
octanol)
−0.414 ±
0.098
Since this bioactivity is performed in a cell based assay, the
compound should remain intact in the buffer solution (in fact, the
stability of 12 was studied in PBS pH 7.2 and compound was found
to be stable up to 24 hrs). Therefore, it is speculated that esters
remain intact and play a role in the activity in in-vitro assay.
a
values are shown as average of three independent determinations (± SD)
In summary, we have isolated a new chromone alkaloid
Next, the cytotoxic effect of DBFM extract, compound 12 and 3e
was assessed on the THP-1 cells. For toxicity evaluation, seven
different concentrations were used viz. 0.625, 1.25, 2.5, 5, 10, 20 and
chrotacumine K along with three known compounds chrotacumine
E, schumaniofioside A and rohitukine from the fruits/seeds/leaves of
Dysoxylum binectariferum. Schumaniofioside A was isolated for the
first time from Meliaceae family and reported carbon assignments
was corrected on the bases of 2D-NMR spectroscopy.
4
0 µM. Similarly, concentrations 0.625, 1.25, 2.5, 5, 10, 20 and 40
µg/ml were used for fruit extract. The results of cell viability assay
indicated that neither extract nor compounds 12 and 3e showed any
toxicity to the THP-1 macrophages at tested concentrations. Results
are shown in Section S39 of supporting information.
Chrotacumine K was found present in other parts of this plant
including seeds and stem barks. The newly isolated compound 12
displayed promising inhibition of pro-inflammatory cytokines,
indicating its promise as an anti-inflammatory agent. Synthetic
analog of chrotacumine K 3e was found to be better inhibitor of
cytokine IL-6 than parent natural product 12. Both chrotacumine K
and compound 3e were found safe (non-toxic) to the THP-1 cells up
to 40 µM, indicating their excellent therapeutic window.
Table 2. Inhibition of proinflammatory cytokines TNF-α and IL-6 by
extracts of D. binectariferum, rohitukine (1), chrotacumine E (6),
chrotacumine K (12) and schumaniofioside A (13) in THP-1 cells
Entry
% inhibition ± SD
TNF-α
IL-6
DBFM ext, 2.5 µg/ml
48.33 ± 2.34
52.18 ± 3.25
50.60 ± 1.25
41.74 ± 2.01
33.17 ± 1.09
81.09 ± 0.98
39.51 ± 1.21
41.40 ± 2.01
43.11 ± 0.78
60.74 ± 0.42
82.59 ± 1.62
86.47± 2.01
16.01 ± 1.06
20.45 ± 2.20
22.21 ± 0.58
23.57 ± 1.18
Supporting information available. Experimental procedures and
spectral data scans. This material is available free of charge via the
internet at http://sciencedirect.com.
DBLHA ext, 3.12 µg/ml
b
Rohitukine (1), 5 µM
Rohitukine (1), 10 µM
b
Chrotacumine E (6), 10 µM
Chrotacumine K (12), 10 µM
Schumaniofioside A (13), 5 µM
Schumaniofioside A (13), 10 µM
ACKNOWLEDGEMENTS
V. K. is thankful to UGC for the award of research fellowship. S.S.B. is a
Women Scientist (DBT-BioCARe) receiving financial support from grant no
th
GAP-2158. The work was supported by CSIR 12 FYP grant # BSC-0205.
Table 3. Inhibition of proinflammatory cytokines TNF-α and IL-6 by
chrotacumine K ester analogs 3a-f in THP-1 cells
References
%
inhibition ± SD
Entry TNF-α
µM
IL-6
1
2
3
4
Harmon, A. D.; Weiss, U.; Silverton, J. V. Tetrahedron Lett. 1979, 8,
21.
7
1
10 µM
1 µM
10 µM
Naik, R. G.; Kattige, S. L.; Bhat, S. V.; Alreja, B.; de Souza, N. J.; Rupp,
R. H. Tetrahedron 1988, 44, 2081.
Safia; Kamil, M.; Jadiya, P.; Sheikh, S.; Haque, E.; Nazir, A.; Lakshmi,
V.; Mir, S. S. PLoS One 2015, 10, e0137991.
3
3
3
3
3
3
a
b
c
d
e
f
35.12 ± 1.57
48.22 ± 1.43
44.45 ± 3.12
35.08 ± 2.89
nd
75.63 ± 2.81
49.36 ± 0.14
62.10 ± 2.31
45.78 ± 1.81
24.11 ± 0.21
28.20 ± 1.73
35.01 ± 0.72
20.78 ± 2.01
70.31 ± 0.56
26.21 ± 1.78
nd
10.31 ± 1.23
23.10 ± 2.13
16.41 ± 1.34
26.31 ± 0.80
91.29 ± 1.87
23.20 ± 1.23
20.45 ± 2.20
a
Mishra, S. K.; Tiwari, S.; Shrivastava, S.; Sonkar, R.; Mishra, V.;
Nigam, S. K.; Saxena, A. K.; Bhatia, G.; Mir, S. S. J. Nat. Med. 2014,
PMID: 24677095.
79.06± 1.78
32.65 ± 1.30
81.09 ± 0.98
32.02 ± 3.02
55.76 ± 1.23
1
a
2
5
Varshney, S.; Shankar, K.; Beg, M.; Balaramnavar, V. M.; Mishra, S. K.;
Jagdale, P.; Srivastava, S.; Chhonker, Y. S.; Lakshmi, V.; Chaudhari, B.
P.; Bhatta, R. S.; Saxena, A. K.; Gaikwad, A. N. J. Lipid Res. 2014, 55,
compound 3e was tested at 5 µM for TNF-α inhibition; nd: not determined
The physicochemical properties are very crucial for any
1
019.
bioactive compound to move ahead in preclinical studies. Therefore,
6
7
8
Singh, N.; Singh, P.; Shrivastva, S.; Mishra, S. K.; Lakshmi, V.; Sharma,
R.; Palit, G. Naunyn Schmiedebergs Arch. Pharmacol. 2011, 385, 277.
Keshri, G.; Oberoi, R. M.; Lakshmi, V.; Pandey, K.; Singh, M. M.
Contraception 2007, 76, 400.
Lakshmi, V.; Pandey, K.; Kapil, A.; Singh, N.; Samant, M.; Dube, A.
Phytomedicine 2007, 14, 36.
as a preliminary step towards this, we determined the aqueous
2
solubility, lipophilicity (log P) and distribution co-efficient (log
2
23
2
3
D) of chrotacumine K along with rohitukine and schumaniofioside
A. Results are shown in Table 4. Chrotacumine K showed excellent
aqueous solubility. The Log P and Log D values were slightly
towards more polar side (deviating from the acceptable drug-like
properties). Aqueous solubility of schumaniofioside A was found to
9
1
Blachly, J. S.; Byrd, J. C.; Grever, M. Semin. Oncol. 2016, 43, 265.
Wiernik, P. H. Expert Opin. Investig. Drugs 2016, 25, 729.
0

5
1
1
1
1
1
2
3
4
Cassaday, R. D.; Goy, A.; Advani, S.; Chawla, P.; Nachankar, R.;
Gandhi, M.; Gopal, A. K. Clin. Lymphoma Myeloma Leuk. 2015, 15,
(125 MHz, CDCl
108.13, 105.23, 104.43, 99.65, 71.14, 58.60, 56.19, 45.93, 35.71, 24.81,
21.01, 20.42; IR (CHCl ): νmax 3584.10, 3352.59, 2923.01, 2852.49,
3
): δ 182.76, 170.60, 166.35, 162.71, 160,79, 156.81,
3
92.
3
Jain, S. K.; Meenaa, S.; Qazi, A. K.; Hussain, A.; Bhola, S. K.;
Kshirsagar, R.; Pari, K.; Khajuria, A.; Hamid, A.; Shaanker, R. U.;
Bharate, S. B.; Vishwakarma, R. A. Tetrahedron Lett. 2013, 54, 7140.
Morita, H.; Nugroho, A. E.; Nagakura, Y.; Hirasawa, Y.; Yoshida, H.;
Kaneda, T.; Shirota, O.; Ismail, I. S. Bioorg. Med. Chem. Lett. 2014, 24,
2346.36, 1736.94, 1660.76, 1614.91, 1588.33, 1419.56, 1389.74,
1260.17, 1189.03, 1020.38, 845.50, 759.15; ESI-MS: m/z 348.40
+
[M+H]+; HRMS: m/z 348.1453 calcd for C18
Rohitukine (1): yellow powder; TLC: R
chloroform); HPLC purity: > 99% (t = 11.13 min); mp. 215-218 C;
= –17.07 (c 0.12, MeOH); H NMR (400 MHz, CD OD): δ 6.15
H
22NO
6
+H (348.1447).
f
= 0.22 (20% methanol-
o
R
2
[α]
0
1
2
437.
D
3
Lazim, M. I. M.; Ismail, I. S.; Shaari, K.; Latip, J. A.; Al-Mekhlafi, N.
A.; Morita, H. Chem. Biodivers. 2013, 10, 1589.
Khadem, S.; Marles, R. J. Molecules 2012, 17, 191.
(s, 1H), 5.97 (s, 1H), 4.14 (s, 1H), 3.62-3.59 (m, 1H), 3.49 (d, J = 8.4
Hz, 1H), 3.38 (t, J = 2, 4.4 Hz, 1H), 3.35 (d, J = 4 Hz, 1H), 3.22-3.20 (m,
1H), 3.15 (d, J = 12 Hz, 1H), 2.82 (s, 3H), 2.32 (s, 3H), 1.75 (d, J = 8 Hz,
1
1
5
6
1
3
Ismail, I. S.; Nagakura, Y.; Hirasawa, Y.; Hosoya, T.; Lazim, M. I. M.;
Lajis, N. H.; Shiro, M.; Morita, H. J. Nat. Prod. 2009, 72, 1879.
Plant material was collected between March and May (2013) from Forest
Research Institute, New Forest Division, Deharadun (India). Plant was
identified and authenticated at IIIM and herbarium sheet was prepared
and submitted to Janaki Ammal herbarium, IIIM (Acronym RRLH,
accession no. 22163). Dried samples of fruits were submitted to the
institutional crude drug repository (accession no. 3893).
1H); C NMR (100 MHz, CD
156.50, 107.40, 106.40, 104.00, 99.40, 66.70, 60.40, 55.40, 42.90, 35.70,
21.90, 18.90; IR (CHCl ): νmax 3584, 3312, 2956, 2920, 2870, 1741,
3
OD): δ 183.00, 167.70, 163.00, 160.70,
1
1
7
8
3
-
1
+
1653, 1460, 1378, 1248, 1082, 1020 cm ; ESI-MS: m/z 306.01 [M+H] ;
+
HRMS: m/z 306.1367 calcd for C H NO +H (306.1341).
1
6
20
5
19 Tane, P.; Ayafor, J.F.; Sondengam, B.L.; Connolly, J.D. Phytochemistry.
1990, 29, 1004.
20 Hu, J.; Song, Y.; Li, H.; Yang, B.; Mao, X.; Zhao, Y.; Shi, X. Fitoterapia
2014 99, 86.
Isolation of chrotacumine K (12) and rohitukine (1) from fruits of
Dysoxylum binectariferum: D. binectariferum fruits were air dried, seeds
were removed and remaining parts were grinded to make coarse powder.
The 90 g of fruits powder was extracted with methanol (250 ml x 3) for 2
hrs under continuous stirring. The solvent was evaporated under reduced
pressure to get dried extract (3.26 g). The obtained extract was first
dissolved in methanol (10 ml), followed by addition of chloroform (20
ml), which resulted in precipitation of polar constituents. White
precipitate was filtered and and the filtrate obtained was subjected to
silica gel column chromatography (n-Hexane-EtOAc). The column was
charged with 100-200 mesh size silica gel and was eluted with increasing
concentration of methanol in chloroform. At 7-8% of MeOH in
chloroform, a new compound (non-polar to rohitukine) was eluted. The
obtained compound was further purified by re-crystallization from
chloroform to get cream colored solid (20 mg). At 12% MeOH in
chloroform, a known alkaloid rohitukine (1) was isolated as yellow
21 In-vitro cytokine inhibition. For the in vitro analysis of pro-inflammatory
cytokines (IL-6 and TNF-α), human leukaemia THP-1 cells were seeded
in a 24-well plate at a density of 4 x 10 cells/ml of RPMI medium per
5
well. THP-1 cells were then treated with PMA (20 ng/ml) for
differentiation into macrophages for 18 h followed by a rest period of 48
h. Cells were further co-treated with different doses (50, 25 and 12.5
µg/ml) of hydro-alcohlic extract or compound and 1 µg/ml of
lipopolysaccharide (LPS) in a serum-free medium. Culture supernatant
was harvested after 24 h for analysis of different cytokines by using
OptEIA ELISA Kits from BD Biosciences. Total protein content for all
the samples was calculated by using Bradford reagent from Bio-Rad
Laboratories. All the samples were normalized by dividing cytokine
concentration with total quantity of protein.
22 Bharate, S. S.; Vishwakarma, R. A. Bioorg. Med. Chem. Lett. 2015, 25,
1561.
powder. Chrotacumine K (12): Creamish white solid; TLC: R
f
= 0.67
=12.84 min); m.p.
= –13.46 (c 0.1, MeOH); H NMR (400 MHz,
): δ 12.86 (brs, 1H), 6.27 (s, 1H), 6.01 (s, 1H), 5.18 (brs, 1H),
.57-3.52 (m, 1H), 3.22 – 3.12 (m, 3H), 2.49 (d, J = 12 Hz, 1H), 2.38 (s,
23 Bharate, S. S.; Kumar, V.; Vishwakarma, R. A. Comb. Chem. High
Throughput Screen. 2016, 19, 461.
(
20% methanol-chloroform; HPLC purity: 98.8% (t
R
o
20
1
1
94-196 C; [α]
D
CDCl
3
3
6
13
H), 2.30 – 2.24 (s, 1H), 1.88 (s, 3H), 1.81 (d, J = 12 Mz, 1H); C NMR

6
Tetrahedron Letters
Highlights
•
•
•
•
•
A new chromone alkaloid chrotacumine K
isolated.
Schumaniofioside A reported for the first
time from Meliaceae family.
Biological evaluation of isolated
compounds performed.
Chrotacumine K showed potent inhibition
of TNF-alpha and IL-6.
Physichochemical properties were
determined for bioactive compounds.