Synthesis and structural proof of a potent 5-lipoxygenase inhibitor

Article abstract of DOI:10.1007/s10870-010-9956-7

5-Lipoxygenase inhibitor 3-O-acetyl-9,11-dehydro-β-boswellic acid was detected in the extract of Boswellia serrata gum resulting from unstable 11-hydroxy precursor. It was reported more potent than other Boswellic acids in its inhibition of 5-Lipoxygenase. Here, we report the method of conversion of 3-acetoxy-β-boswellic acid to 3-O-acetyl-9,11-dehydro-β-boswellic acid, and the crystal structure of later. This compound crystallizes in orthorhombic space group P2₁2₁2₁ with cell parameters of a = 12.726(1) A, b = 16.597(1) A, c = 27.332(2) A, α = β = γ = 90°, V = 5772.7(5) A³, D _c = 1.143 Mg/m³, and Z = 8. The X-ray structure investigation indicates that the rings A, B, D and E are exhibit chair and the ring C adopts a distorted half chair conformation. The conformational difference of the two structures in the arrangement is due to crystal packing of 3-O-acetyl-9,11-dehydro-β-boswellic acid. The molecular packing is stabilized by C-H...O and O-H...O types of hydrogen bonding interactions. Graphical Abstract: Synthesis and crystal structure analysis of 5-Lipoxygenase Inhibitor 3-O-acetyl-9,11-dehydro-β-boswellic acid are reported. Rings A, B, D and E of the inhibitor adopt chair conformation and the C-ring exhibit a distorted half chair conformation. The molecular packing is stabilized by C-H...O and O-H...O types of hydrogen bonding interactions.

Full text of DOI:10.1007/s10870-010-9956-7

J Chem Crystallogr (2011) 41:697–702
DOI 10.1007/s10870-010-9956-7
ORIGINAL PAPER
Synthesis and Structural Proof of a Potent 5-Lipoxygenase
Inhibitor
•
Devipriya Balu Kumaradhas Poomani
•
•
Kalyanam Nagabushanam Sridhar Balasubramanium
•
•
Rajendran Ramanujam Majeed Muhammed
Received: 28 April 2010 / Accepted: 30 December 2010 / Published online: 19 January 2011
ꢀ Springer Science+Business Media, LLC 2011
Abstract 5-Lipoxygenase inhibitor 3-O-acetyl-9,11-de-
hydro-b-boswellic acid was detected in the extract of
Boswellia serrata gum resulting from unstable 11-hydroxy
precursor. It was reported more potent than other Boswellic
acids in its inhibition of 5-Lipoxygenase. Here, we report
the method of conversion of 3-acetoxy-b-boswellic acid to
3-O-acetyl-9,11-dehydro-b-boswellic acid, and the crystal
structure of later. This compound crystallizes in ortho-
rhombic space group P212121 with cell parameters of
Introduction
Boswellia serrata gum has been a well recognized natural
product for its anti-inflammatory action [1]. The most
active material in the gum was identified [2], to be 3-acet-
oxy-11-keto-b-boswellic acid (1 AKBBA). The mode of
action of AKBBA has been traced towards its novel non-
redox, allosteric inhibition of 5-Lipoxygenase preventing
the formation of inflammatory mediators leukotrienes
[3]. The crude gum, in addition to AKBBA and related
acids, contains several other terpenoid components
often possessing either inhibitory or potentiating effects
on 5-Lipoxygenase. Hence increasing attention is paid
towards characterizing other constituents of Boswellia
serrata gum.
˚
˚
˚
3
a = 12.726(1) A, b = 16.597(1) A, c = 27.332(2) A, a =
3
˚
b = c = 90ꢁ, V = 5772.7(5) A , Dc = 1.143 Mg/m , and
Z = 8. The X-ray structure investigation indicates that the
rings A, B, D and E are exhibit chair and the ring C adopts
a distorted half chair conformation. The conformational
difference of the two structures in the arrangement is due to
crystal packing of 3-O-acetyl-9,11-dehydro-b-boswellic
acid. The molecular packing is stabilized by C–HꢀꢀꢀO and
O–HꢀꢀꢀO types of hydrogen bonding interactions.
Safayhi et al. identified one such artifact in their study
[4], wherein 3-O-acetyl-9,11-dehydro-b-boswellic acid
(2) (Scheme 1) as also a 5-Lipoxygenase inhibitor,
actually more potent than AKBBA. The formation of (2)
in the extract was traced towards an unstable 11-hydroxy
precursor molecule in the gum leading to the formation
of (2). Since increasing attention is paid towards the
standardization of activity of crude plant drugs, it is
important to secure the structure of (2). In this com-
munication we describe the conversion of 3-acetoxy-b-
boswellic acid (3), an independently well characterized
molecule by single crystal X-ray analysis [5], to 3-O-
acetyl-9,11-dehydro-b-boswellic acid (2) chemically thus
firmly securing the structure of (2). In addition a single
crystal X-ray structure of (2) independently establishes
structure of (2) now available through this study for
biological studies.
Keywords Boswellia serrata gum ꢀ 5-Lipoxygenase
inhibitor ꢀ Medicinal activity ꢀ Hydrogen bonding
D. Balu ꢀ K. Poomani (&)
Department of Physics, Periyar University, Salem 636 011,
Tamil Nadu, India
e-mail: kumaradhas@yahoo.com
K. Nagabushanam ꢀ R. Ramanujam ꢀ M. Muhammed
Research & Development, Sabinsa Corporation,
20 Lake Drive, East Windsor, NJ 08520, USA
Thus 3-acetoxy-b-boswellic acid (3) on reaction with
N-bromosuccinimide [6], directly gave (2) in good yields.
Hydrolysis of the acetate (2) furnished (4). We also found
S. Balasubramanium
Laboratory of X-Ray Crystallography, Indian Institute
of Chemical Technology, Hyderabad 500 007, India
123

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J Chem Crystallogr (2011) 41:697–702
Synthesis of 3-O-Acetyl-9,11-dehydro-b-boswellic
Acid (2)
CH3
E
H3C
CH3
3-Acetyl-b-boswellic acid (2.0 g) was taken in carbontet-
rachloride (40 mL). N-Bromosuccinimide (1.2 g) was
added and the reaction mixture was refluxed for 4–5 h.
TLC indicated completion of the reaction. The reaction
mixture was washed twice with water. The organic layer
was dried over sodium sulfate and the solvent was removed
to yield a crude product weighing 1.6 g; this crude product
was dissolved in methanol (60 mL) and ethyl acetate
(10 mL) and charcolized. The solvents were removed to
yield a light brown powder weighing 1.2 g which was
further purified by column chromatography to yield a white
material (0.7 g). Mp: 290–292 ꢁC (reported [4] 230 ꢁC).
Elem. Analysis (calc. values for C32H48O4 in parenthesis)
%C: 77.48 (77.38), %H: 9.86 (9.74). 1H NMR (CDCl3
solvent): 5.66 (1H, d, J = 5.87 Hz), 5.47 (1H, d,
J = 5.87 Hz), 5.32 (1H, distorted t, J = 3.04 Hz), 2.18
(1H, br t, J = 13.8 Hz), 2.10–2.00 (4H, including a singlet
at 2.07 for 3H, CH3–CO and a, d of t centered at 2.03,
J = 13.3 & 4.4 Hz for 1H), 1.98–1.86 (2H, d of q,
J =*12.5 & *4.5 Hz), 1.86–1.67 (4H, m), 1.62–1.25
(13H, m, including a singlet for 3H at 1.28 for a CH3), 1.20
(3H, s, CH3), 1.12 (3H, s, CH3), 1.07 (1H, br d,
J = 11.6 Hz), 1.00–0.91 (m, 8H, including a singlet at 0.95
for 3H for a –CH3 and another broad singlet at 0.94 for 3H
for a CH–CH3), 0.87 (3H, s, CH3), 0.82 (3H, d,
J = 6.2 Hz, CH–CH3)
D
C
CH3
H3C
A
B
CH3
AcO
HOOC
CH3
Scheme 1 Chemical structure of 3-O-acetyl-9,11-dehydro-b-boswel-
lic acid
CH3
H3C
O
CH3
CH3
CH3
CH3
AcO
HOOC
CH3
1 (AKBBA)
CH3
CH3
H3C
H3C
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
NBS, CCl4
AcO
HOOC
AcO
HOOC
CH3
CH3
2
3
KOH/CH3OH
KOH/CH3OH
CH3
CH3
3-Hydroxy-9,11-dehydro-b-boswellic Acid (4)
H3C
H3C
NBS, CCl4
3-O-Acetyl-9,11-dehydro-b-boswellic acid (2) (3.5 g) was
refluxed in methanol (70 mL) in the presence of aq. KOH
(5 g in 5 mL water) till the completion of reaction as
indicated by TLC. The reaction time was about 6 h. Water
(70 mL) was added and the pH was adjusted to 2–3 range
using dil. HCl. The reaction mixture was stirred well and
filtered. The solid was washed with water till neutral pH
and dried at 80 ꢁC to yield 3.2 g of the product. One gram
of this product was further purified by column chroma-
tography to obtain 0.8 of analytically pure product. Mp:
240–245 ꢁC (reported [4] 218 ꢁC). Analysis (calc. values
for C30H46O3 in parenthesis) %C: 79.28 (79.25), %H:
10.40 (10.20). 1H NMR (CDCl3 solvent): 5.66 (1H, d,
J = 5.87 Hz), 5.46 (1H, d, J = 5.87 Hz), 4.09 (1H, dis-
torted br t), 2.25 (1H, br t, J = 14.5 Hz), 2.02(1H, d of t,
J = 13.2 & 4.4 Hz), 1.97–1.86 (2H, m), 1.84–1.55 (7H,
m), 1.54–1.45 (2H, m), 1.43–1.35 (5H, m including a sin-
glet at 1.38 for 3H for a –CH3), 1.24–1.35 (4H, m), 1.19
(3H, s, CH3), 1.11 (3H, s, CH3), 1.06 (1H, broad d,
J = 11.7 Hz), 0.98–0.90 (8H, m, including a broad singlet
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
HO
HOOC
HO
HOOC
CH3
CH3
5
4
Fig. 1 Scheme of compounds 1, 2, 3, 4 & 5
that b-boswellic acid (5) itself could be converted to
3-hydroxy-9,11-dehydro-b-boswellic acid (4), without any
protection of the 3-OH function (Fig. 1).
Experimental
Melting points reported are uncorrected. Elemental analy-
sis was done at Robertson Microlit Laboratories, Madison,
NJ. Proton NMR spectrum were taken at 500 MHz in a
CDCl3 solvent with TMS as internal standard.
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J Chem Crystallogr (2011) 41:697–702
699
at 0.93 for 6H for two –CH3), 0.86 (3H, s, CH3), 0.80 ((3H,
d, J = 6.3 Hz, CH–CH3)
results are given in Table 1. Selected bond lengths, bond
angles and torsion angles of (2) are listed in Table 2. The
fractional atomic coordinates of both molecules of the
asymmetric unit and their detailed geometry have been
deposited to Cambridge Crystallographic Data Centre
(CCDC-657235) as a supplementary publication.
3 ? 5 and 2 ? 4 conversions utilized similar proce-
dures described above.
Single Crystal X-Ray Diffraction
The 3-O-Acetyl-9,11-dehydro-b-boswellic acid (2) com-
pound has been crystallized from ethanol by slow evapo-
ration technique at room temperature yielded a white
rectangular block shaped single crystals. X-ray intensity
data collection was performed at room temperature. All the
diffraction intensity measurements were made on Bruker’s
APEX-II CCD Area Detector [7], with graphite mono-
˚
Table 2 Comparison of geometrical parameters (A,ꢁ) of C-ring of
structure (2) with similar ring of the analogous structure (3)
Molecule-I
Molecule-II
Bond lengths
C(9)–C(10)
1.535(3)
1.542
1.537(3)
1.556
˚
chromated Mo Ka radiation (k = 0.71073 A). The data has
been corrected for Lorentz and Polarization effects but not
for absorption and extinction. The structure was solved by
direct methods and refined by full-matrix least-squares
using SHELXS97 [8], and SHELXL97 [9] programs.
During the refinement, all non-hydrogen atoms were trea-
ted anisotropically and the H-atoms were fixed geometri-
cally and refined as a riding model gave final residual index
R(F) = 0.037 and wR(F2) = 0.101 for 5628 observed
reflections [I [ 2r(I)]. The crystal data and the refinement
C(9)–C(11)
C(10)–C(14)
C(11)–C(12)
C(12)–C(13)
C(13)–C(14)
1.581(3)
1.598
1.582(3)
1.601
1.334(3)
1.528
1.334(3)
1.530
1.533(3)
1.533
1.539(3)
1.529
1.330(3)
1.331
1.325(3)
1.325
1.451(4)
1.488
1.452(3)
1.502
Bond angles
C(10)–C(9)–C(11)
109.0(2)
108.4
109.5(2)
106.3
Table 1 Crystal data and the structure refinement of 3-O-acetyl-9,11-
dehydro-b-boswellic acid
C(14)–C(10)–C(9)
C(12)–C(11)–C(9)
C(13)–C(12)–C(11)
C(12)–C(13)–C(14)
C(10)–C(14)–C(13)
115.4(2)
110.5
116.5(2)
109.6
Empirical formula
CCDC No.
C32H48O4
657235
496.7
109.3(2)
109.5
110.0(2)
109.2
Formula weight
Temperature
273(2) K
116.8(2)
119.8
116.5(2)
119.6
˚
Radiation type and wavelength
Crystal system, space group
Unit cell dimensions
MoKa; 0.71073 A
Orthorhombic, P212121
˚
a = 12.726(1) A
122.7(2)
126.8
123.1(2)
126.4
˚
b = 16.597(1) A
122.8(2)
114.1
122.8(2)
113.9
˚
c = 27.332(2) A
3
˚
5772.7(5) A
Volume
Torsion angles
Z, Calculated density
Absorption coefficient
F(000)
8, 1.143 Mg/m3
0.073 mm-1
C(10)–C(9)–C(11)–C(12)
54.8(2)
55.7
52.4(2)
59.4
2176
C(11)–C(9)–C(10)–C(14)
C(9)–C(10)–C(14)–C(13)
C(10)–C(14)–C(13)–C(12)
C(14)–C(13)–C(12)–C(11)
C(13)–C(12)–C(11)–C(9)
-42.2(3)
-61.3
7.4(4)
36.3
-37.6(3)
-64.2
4.1(4)
36.6
Crystal shape and colour
Crystal size
Rectangular block, colourless
0.17 9 0.1 9 0.07 mm
1.77–25.59ꢁ
h = -15 ? 15, k = -19 ? 19,
l = -32 ? 32
Theta range for data collection
Limiting indices
16.9(5)
-5.6
16.4(3)
-3.06
2.2(3)
1.2
Reflections collected/unique
Refinement method
Goodness-of-fit on F2
41973/5628
Full-matrix least-squares on F2
-0.6(4)
-0.8
1.033
R(F) = 0.037, wR(F2) = 0.101
-34.7(3)
-26.4
-36.1(3)
-28.1
Final R indices [I [ 2r(I)]
Largest diff. peak and hole
-3
˚
0.18 and -0.13 eA
123

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J Chem Crystallogr (2011) 41:697–702
˚
Table 3 Hydrogen bonding (A, )
˚
D–HꢀꢀꢀA
D–H
HꢀꢀꢀA
DꢀꢀꢀA
D–HꢀꢀꢀA
C(210)–H(210)…O(1)i
O(20)–H(20)ꢀꢀꢀO(1)ii
O(2)–H(2)ꢀꢀꢀO(30)iii
C(4)–H(4A)ꢀꢀꢀO(10)iv
C(27)–H(27B)ꢀꢀꢀO(1)
C(270)–H(27F)ꢀꢀꢀO(10)
0.98
0.82
0.82
0.97
0.96
0.96
2.97(2)
1.90(2)
1.90(2)
2.77(2)
2.75(2)
2.81(2)
3.831(3)
2.711(2)
2.689(3)
3.602(4)
3.526(3)
3.605(3)
147.0(2)
168.6(1)
161.0(2)
144.4(2)
138.5(2)
140.8(2)
Symmetry codes: (i) x ? 1/2, -y ? 3/2, -z (ii) x, y - 1, z (iii) -x ? 1, y ? 1/2, -z ? 1/2 (iv) x, y ? 1, z
Fig. 2 View of the molecule
(2) showing atom-labeling
scheme with thermal ellipsoid
atoms drawn at 50% probability
level, H atoms are drawn as
small circles of arbitrary radii
˚
Results and Discussion
1.582(3)*] A (* indicates [bond lengths/angles of mole-
cule (2)]), Csp2–Csp3: 1.523(3) to 1.545(3) [1.525(3) to
˚
1.550(3)*] A and Csp2–Csp2: 1.330(3) to 1.451(4) [1.325(3)
˚
to 1.452(4)*] A. The observed major difference in bond
The title compound 3-O-Acetyl-9,11-dehydro-b-boswellic
acid (2) crystallizes two molecules in the asymmetric unit
as its analogous [5]. Figure 2 is the ORTEP [10] plot of
molecular structure, showing, thermal ellipsoid atoms with
atom numbering scheme. Although, the compound crys-
tallizes two molecules in the asymmetric unit, their bond
lengths and angles are approximately same. The different
types of bond distances in the molecules I and II and their
ranges are; Csp3–Csp3: 1.504(3) to 1.581(3) [1.502(4) to
angles are around the atoms C(10), C(13), C(14) and
C(15); this difference is attributed to the conformation
of C-ring. The C(16)–C(17)–C(18) angle is 107.6(2)ꢁ
[108.3(2)ꢁ]* which is small on compared with all other
bond angles of the rings in the molecules. This may be due
to the presence of bulk groups attached at C(6) and C(17).
The carbonyl bonds C(23)–O(2) and C(25)–O(2) are
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J Chem Crystallogr (2011) 41:697–702
701
Fig. 3 Hydrogen bonding
interactions
significantly shorter than the similar type of bonds
˚
C(5)–O(4): 1.451(3) [1.461(3)*] A, C(23)–O(2):1.324(3)
Figure 3 depicts the hydrogen bonding interactions of
the molecules in the crystal. The molecular packing is
predominantly stabilized by C–HꢀꢀꢀO and O–HꢀꢀꢀO type of
hydrogen bonding interactions. Among the C–HꢀꢀꢀO type of
interactions, the C(210)–H(210)ꢀꢀꢀO(1)i interaction is con-
˚
˚
[1.324(3)*] A and C(25)–O(4): 1.319(4) [1.323(4)*] A. As
expected, the double bond distances C(25)–O(3): 1.179(5)
˚
˚
[1.192(5)*] A and C(23)–O(1): 1.203(3) [1.198(3)*] A in
the molecule are almost equal. However, both the mole-
cules exhibit almost similar geometry, but they are con-
formationally slightly different. This is due to the effect of
the mode of crystallization. Similar trend appears in the
reported structure (3) [5]. The geometry and conformation
of both (2) and (3) are differing by C-ring. These confor-
mational differences can be determined from their torsion
angles. We have analyzed individual rings of (2) and (3),
on the basis of formalism reported by DUAX et al. [11].
The average torsion angles of ring A of I and II in the
asymmetric unit are 54.2 and 53.6ꢁ, respectively, indicates,
a chair conformation and further the rings B, D and E also
exhibit the same; but unlikely the ring C adopt distorted
half chair conformation [12]. Table 2, shows the difference
in torsion angles of C-ring of (2) and (3). Precisely, the
C-ring altered the molecular conformation significantly;
perhaps, the enhanced medicinal activity of the structure
(2) may be attributed to the C-ring geometry.
sidered as a strong interaction and the H-bond parameters
i
0
0
˚
˚
are; C(21)–H(21 ): 0.980 A, H(21 )ꢀꢀꢀO(1) : 2.970(2) A,
C(21)ꢀꢀꢀO(1)i: 3.831(3) A and the angle is C(210)–
˚
H(210)ꢀꢀꢀO(1)i: 147.0(2)ꢁ. The O(20)–H(20)ꢀꢀꢀO(1)ii hydro-
gen0 bonding parameters are; O(20)–H(20): 0.820 A,
˚
ii
ii
0
˚
˚
H(2 )ꢀꢀꢀO(1) : 1.900(2) A, O(2 )ꢀꢀꢀO(1) : 2.711(2) A and
the angle is O(20)–H(20)ꢀꢀꢀO(1)ii : 168.6(1)ꢁ. [Symmetry
codes: (i) x ? 1/2, -y ? 3/2, -z (ii) x, y - 1, z]. Some of
the weak inter and intra molecular interactions are listed in
Table 3.
Supporting Information
CCDC-657235 contains the supplementary crystallo-
graphic data of this paper. These data can be obtained from
the Cambridge crystallographic data centre (CCDC), 12,
Union Road, Cambridge CB2 1EZ, UK.
123

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J Chem Crystallogr (2011) 41:697–702
7. Bruker (2000) APEX-II and SAINT. Bruker AXS Inc, Madison,
WI, USA
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