S Srividya Bommakanti1, Lakshmi Srinivasa Rao Kundeti1, Venkateshwarlu Saddanapu2, Kommu Nagaiah1. 1. Centre for Natural Products & Traditional Knowledge, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India. 2. Department of Applied Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India.
Abstract
Malabaricol is a unique plant natural product, 3-keto tricarbocyclic triterpenoid, isolated from Ailanthus malabarica. Malabaricol underwent reaction with aromatic aldehydes under alkaline conditions to form 2-arylidene analogs. Indoles and pyrazine ring system fused to the 2,3-position of malabaricol were synthesized. In this ring system of tricarbocyclic triterpenoid, the conformation is such that there is no steric hindrance due to C4 and C10 axial methyl groups and other skeletons. Malabaricol and its synthetic analogues show cytotoxic activity toward lung cancer, which was compared to that of standard doxorubicin.
Malabaricol is a unique plant natural product, 3-keto tricarbocyclictriterpenoid, isolated from Ailanthus malabarica. Malabaricol underwent reaction with aromatic aldehydes under alkaline conditions to form 2-arylidene analogs. Indoles and pyrazine ring system fused to the 2,3-position of malabaricol were synthesized. In this ring system of tricarbocyclictriterpenoid, the conformation is such that there is no steric hindrance due to C4 and C10 axial methyl groups and other skeletons. Malabaricol and its synthetic analogues show cytotoxic activity toward lung cancer, which was compared to that of standard doxorubicin.
Triterpenoids
are prolific in the plant kingdom, while malabaricol,
a triterpenoid isolated from the gum exudates of the trunk of Ailanthusmalabaricol by Dev,[1,2] is the first
tricarbocyclictriterpenoid. It is also unusual with a 3-keto group,
having a side chain containing tetrahydrofuran group. Its structure
was established by many chemical reactions and spectral data.[1,2] Further direct correlation of malabaricol with (+)-ambrenolide by
circular dichroism provided the structural proof and also absolute
stereochemistry at C5, C8, C9, and
C10.[1,2] The confirmation and stereochemistry
of malabaricol structure were disclosed in the work of Van Tamelen,[3] who reported nonenzymatic cyclization of squalene-2,3-epoxide
(I) using stannic chloride. Thus, Van Tamelen obtained
a tricarbocyclic-type triterpenoid skeleton, which suggested that
such a structure is feasible.Sharpless also studied the nonenzymatic
cyclization of squalene-2,3-epoxide
by picric acid[4] leading to the synthesis
of d,l-malabaricanediol (II) (C3–OH instead of C=O in malabaricol), which revealed
the stereochemical positions at C17 and C20,
as shown Figure .
To elaborate further, the Sharpless study confirmed that the chemical
tendency of the cyclization of 18,19-dihydroxy squalene-2,3-epoxide I led to the malabaricol skeleton, d,l-malabaricanediol
(II),[5] while the natural enzyme-catalyzed
process in living organisms provided exclusively a vast number of
tetra or pentacyclictriterpenoids or lanostanes or steroids.
Figure 1
Squalene-2,3-epoxide to d,l-malabaracanediol.[4]
Squalene-2,3-epoxide to d,l-malabaracanediol.[4]Further, Sharpless, based on biogenetic reasoning, proposed
the
configuration at C13 and C14.[4] Finally, the structure and stereochemistry of malabaricol
were confirmed by X-ray analysis on the crystal of malabaricol.[6] Subsequent to the isolation of malabaricol 1, a few related tricarbocyclictriterpenoids were isolated
from Ailanthus and other species.[7−14] Natural tricarbocyclic skeleton isomalabaricanes were also isolated
from sponges.[15−17] The structure of isomalabaricanes (III) differs from that of malabaricol 1 in the stereochemistry
of C9 and C8, as shown in Figure .
Figure 2
Structure of malabaricol and isomalabaricanes.[17]
Structure of malabaricol and isomalabaricanes.[17]In 1957, Barton et al.[18−20] explored sterically and/or electronically
the interaction of various functional groups, in tetracyclictriterpenoids,
pentacyclictriterpenoids, steroids, and lanost-8-enone (VI), the four types of structures. Barton conducted a conformational
study based on the steric effects of the above compounds based on
chemical reactions. Thus, Barton studied the condensation of benzaldehyde
under alkaline conditions with triterpinoids having 3-keto function.
The structures of these starting 3-ketotriterpinoids have a partial
structure (IV), which, on base-catalyzed condensation
of benzaldehyde with reactive 2-methylene group, led to the formation
of benzylidene structure (V). To determine the rate of
condensation, the reaction of benzaldehyde with lanost-8-enone (VI) is taken as standard in this case study. Barton investigated
the rate of base-catalyzed condensation of the above 3-keto compounds
to 2-benzylidene products. Barton also observed that all of the 3-keto
compounds formed 2-arylidene compounds, but the rates of formation
are different.Based on kinetic studies, the differences in
the rate of formation
may be due to the partial steric hindrance at C3-keto due
to C4 and C10 axial methyl groups, although
these compounds have rigid extended conformations. Thus, the steric
effect, although possible, as shown in the partial structure VII due to the C4 and C10 axial methyl
groups, does not have a direct role. Another possibility to influence
the rate of reaction is by electrostatic effect, space or through
bond induction, which is collectively called the inductive effect.
This possibility also does not seem to have a role in the rate of
formation of 2-benzylidene derivatives. Therefore, the difference
in the rate of formation of 3-benzylidene derivatives is due to “conformational
transmission”. Barton attributed the differences in the rates
of formation of 2-benzylidene formation to the 1:3 interaction between
axial methyl groups at C4 and C10 (VII) (Figure ). Finally,
Barton described that methyl groups at C4, C10, and C8 must interact with each other in a 1:3 manner
to explain the kinetics of the rate of formation. Therefore, this
study provided a method to determine the effects of functional groups
found elsewhere in these structures of tetra or pentacyclictriterpenoids,
etc. on the reactions of functional groups of ring A. As a result
of this study, it is found that the kinetics of this reaction is remarkably
influenced by what Barton termed as conformational transmission. This
effect is primarily due to distortion in a distant part of the molecule
and appears to be transmitted to the reaction site (i.e., 3-CO, 2-CH2– of ring A of triterpenoids or steroids, etc.) through
long-range effects like slight angle and bond distortion.
Figure 3
Condensation
of benzaldehyde with triterpenoide-3-ketone.[18]
Condensation
of benzaldehyde with triterpenoide-3-ketone.[18]
Results and Discussion
Since malabaricol 1 has a tricarbocyclic ring system,
the present study aims at the investigation of the reactivity of the
3-keto group ring and 2-methylene group with various aromatic aldehydes
as well as with aromatic hydrazines and 1,2-diamino aliphatic amine.
The formation of these compounds depends on the steric disposition
of C4 and C10 axial methyl groups and other
factors such as the tricarbocyclic nature of malabaricol. The isolation
of malabaricol 1, which has 3-keto and reactive C2 methylene group, in our laboratories[21] provided us an opportunity to study if 2-arylidene-3-keto compounds
will be formed. Such a study is useful to understand the conformation
of tricarbocyclictriterpenoids. Thus, malabaricol 1 was
condensed with various aryl aldehydes (electron-donating or electron-withdrawing
substituents) under alkaline conditions. Malabaricol 1 was also condensed by bulky naphthalene-1-carboxaldehyde and heterocyclic
aldehydes. The yields of 2-arylidene malabaricol are between 49 and
71% (Scheme ). In
the 1H NMR spectra of malabaricol C2, two multiplets
at δ 2.63–2.47 and δ 2.42–2.29 are observed
due to the mutual coupling of two protons at C2 as well
as the interaction with two vicinal protons of C1. Further,
δ 3.66 (t, J = 6.6 Hz) is due to C17-H and δ 5.08 (m) is due to C24-H. In 2-benzylidene-3-ones,
C1-Ha appeared as a doublet at δ 2.31
(d, J1Ha, 1Hb = 15.8 Hz) and C1-Hb appeared as a doublet at δ 2.94 (d, J1Hb, 1Ha = 15.8 Hz). The triplet at δ 3.70
(J = 7.0 Hz) is due to C17-H, the triplet
δ 5.12 (J = 7.1 Hz) is due to C24-H, and the singlet at δ 7.54 is due to benzylidene CH. Further,
in the IR data of malabaricol, 3-C=O appeared around 1700 cm–1, whereas in benzylidene analogues, 3-C=O appeared
at a reduced frequency, around 1680 cm–1 due to
extended conjugation. This shows that the arylidene ring is coplanar
with ring A, in particular with 3-C=O in all of the arylidene
analogues (Scheme ).
Scheme 1
2-Arylidene Derivatives of Malabaricol
Condensation involving the reaction of C2-methylene
with ethylformate under basic conditions yielded an enol (Scheme ).
Scheme 2
Enol Derivative of
Malabaricol
Scheme depicts
the formation of 3-oxime with hydroxylamine hydrochloride in acetic
medium.
Scheme 3
Oxime Derivative of Malabaricol
Scheme shows that
the condensation of malabaricol 1 with 2,4-dinitrophenylhydrazine
in acetic medium at refluxing temperature yielded malabaricol 3-hydrazones.
Fused 2′,3′-indole formation is not observed even at
elevated temperatures and longer hours of reflux. This is due to the
electron-withdrawing effect of the nitro groups.
Scheme 4
Hydrazone Derivative
of Malabaricol
However, at elevated
temperatures, the condensation of phenylhydrazine
in acetic acid medium at 60 °C yielded 2′,3′-fused
indoles. Two examples are given in Scheme .
Scheme 5
Indole Derivatives of Malabaricol
The condensation of malabaricol with 1,2-ethylenediamine
(Scheme ) in the presence
of sulfur in morpholine at the refluxing temperature led to the formation
2′,3′-fused pyrazine, involving both 2 and 3 positions
of malabaricol and subsequent dehydrogenation.
Scheme 6
Pyrazine Derivatives
of Malabaricol
Thus, the present
study revealed that the conformation of malabaricol
is favorable for the formation of 3-arylidene analogues and other
reactions (Schemes –) enumerated
in this paper. This study revealed that the steric hindrance of C4 and C10 axial methyl groups and the tricarbocyclic
system do not interfere in the reactivity of ring A, as observed in
other triterpinoids.[18−20] Malabaricol has been studied for its antifungal activity
and metabolic disorders including diabetes without much success.[21] These new derivatives of this compound were
screened for the cytotoxicity effects on the human cell line.We have tested all compounds (1–7) for their cytotoxicity against A549 (humanlung cancer cell line)
cell line with doxorubicin as control. The data is presented in Table . Surprisingly, several
derivatives including the parent malabaricol showed good activity.
Structural activity relationship suggests that among the benzylidine
derivatives (2a–l), simple para-substituted compounds did not show any activity except
for 2h and 2d. On the other hand ortho, para-dichloro-substituted compound
(2i) displayed better activity. In contrast to benzylidene
derivatives, the heterocyclic furylidene and pyrolidene analogues
showed good activity. While compounds 3 and 5 did not show any cytotoxicity, 4 was active. All 2,3-fused
derivatives (6a, 6b, and 7)
displayed cytotoxicity. On the basis of activity, further synthetic
work on modification of malabaricol 1 will be studied.
Table 1
Cytotoxicity Activity of Malaboricol
and Its Synthetic Analogues on Human Lung Cancer Cell Line
s. no.
compound number
IC50 at 10 μM
1
1
10.91 ± 0.001
2
2a
11.41 ± 0.006
3
2b
>50
4
2c
>50
5
2d
13.24 ± 0.002
6
2e
>50
7
2f
>50
8
2g
>50
9
2h
12.37 ± 0.004
10
2i
10.03 ± 0.003
11
2j
>50
12
2k
>50
13
2l
>50
14
2m
12.81 ± 0.008
15
2n
12.70 ± 0.006
16
2o
12.98 ± 0.004
17
3
>50
18
4
12.07 ± 0.001
19
5
>50
20
6a
13.99 ± 0.005
21
6b
13.40 ± 0.007
22
7
11.98 ± 0.006
23
doxorubicin
9.10 ± 0.003
Conclusions
Malabaricol 1, a multicyclic
tricarbocyclictriterpenoid
framework, is quite intriguing with multiple stereocenters with druglike
structure. A library of compounds were synthesized taking advantage
of the reactive functional groups, 3-keto and 2-methylene groups,
and screened for cytotoxicity. Malabaricol 1 itself and ortho, para-dichloro-substituted compound
(2i) showed promising cytotoxicity comparable to that
of doxorubicin. These observations provided further stimuli for the
synthesis of a number of novel molecules using malabaricol 1 as a scaffold.
Experimental Section
Material
Malabaricol
plant resin was collected from
Karnataka, India. Solvents and chemicals were purchased from local
vendors and used as received. Reactions were monitored using thin-layer
chromatography (TLC) with 0.25 mm E. Merck precoated silica gel plates
(60 F254), and visualization was accomplished with UV light, iodine
adsorbed on silica gel, or immersion in an ethanolic solution of p-anisaldehyde stain followed by heating. Column chromatography
was carried out on silica gel (60–120 mesh). All 1H NMR spectra were recorded on a Bruker 400 or 500 MHz spectrometer,
and all C13 NMR spectra were recorded on a Bruker 100 or
125 MHz spectrometer. Chemical shifts (δ) were reported in parts
per million (ppm) and calibrated to the residual proton and carbon
resonance of CDCl3 (δH = 7.26 and δC = 77.0 ppm). The coupling constants (J)
are given in hertz. High-resolution mass spectroscopy (HRMS) was conducted
using electrospray ionization (ESI)-time-of-flight techniques. Fourier
transform infrared (FTIR) spectra were recorded with a Bruker α
spectrophotometer and were reported in cm–1. Cellular
viability in the presence of test compounds was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl
tetrazolium bromide (MTT) microcultured tetrazolium assay. The cells
were seeded in flat-bottom (10 000 cells/100 μL) 96-well
plates cultured in a medium containing 10% serum and allowed to attach
and recover for 24 h in a humid chamber containing 5% CO2. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT)
was dissolved in phosphate-buffered saline (PBS) at 5 mg/mL and filtered
to sterilize, and a small amount of insoluble residue present in MTT
was removed. Different concentrations of compounds were added to the
cells. After 48 h, a stock MTT solution (10 μL) was added to
the culture plate. The cells were again kept in a CO2 incubator
for 2 h. After incubation, 100 μL of dimethyl sulfoxide (DMSO)
was added and mixed. The absorbance was read at 562 nm in a plate
reader. The results were represented as a percentage of cytotoxicity/viability.
All of the experiments were carried out in duplicate. From the percentage
of cytotoxicity, the IC50 value was calculated.
Synthesis
of 2-Arylidene Derivatives
To a stirred solution
of malabaricol 1 (100 mg, 218 μmol) in ethanol
(3 mL) was added KOH (73.3 mg, 1.30 mmol) at room temperature. After
half an hour, benzaldehyde (67 μL, 654 μmol) was added
to the reaction mixture. The resulting mixture was stirred at room
temperature for 12 h. The solvent was removed and extracted with diethyl
ether (3 × 50 mL), washed with brine, and dried over Na2SO4. The obtained residue was purified by column chromatography
(9:91, EtOAc/hexane) to get the desired product 2a (79.8
mg, 67%) as a light green viscous liquid. Yield: IR (Neat) νmax 2962, 2874, 1673, 1449, 1028, 904, 665 cm–1; 1H NMR (500 MHz, CDCl3): δ = 7.54 (s,
1H), 7.46–7.38 (m, 4H), 7.33 (m, 1H), 5.12 (t, J = 7.1 Hz, 1H), 3.70 (t, J = 7.0 Hz, 1H), 2.94 (d, J = 15.8 Hz, 1H), 2.31 (d, J = 15.8 Hz,
1H), 2.17–1.31 (m, 25 H), 1.25 (s, 3H), 1.21 (s, 3H), 1.17
(s, 3H), 1.15 (s, 3H), 0.96 (s, 3H), 0.81 (s, 3H); 13C
NMR (100 MHz, CDCl3): δ = 207.7, 137.6, 135.9, 133.8,
131.5, 130.3, 128.4, 128.3, 124.5, 85.6, 82.1, 72.7, 59.6, 57.1, 53.1,
45.1, 44.9, 43.9, 38.1, 37.6, 36.3, 36.0, 29.6, 29.5, 26.0, 25.6,
25.1, 24.3, 23.8, 22.3, 22.2, 21.5, 21.4, 17.6, 15.6; HRMS (ESI+) calculated for [C37H54O3 – H]+: 545.3995, found: 545.4008.
Figure 1
Figure 2
Figure 3
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Scheme 5
Scheme 6