Studies on 1-O-acetylbritannilactone and its derivative, (2-O-butyloxime-3-phenyl)-propionyl-1-O-acetylbritannilactone ester

Article abstract of DOI:10.1016/j.bmcl.2003.12.078

The crystal structures of 1-O-acetylbritannilactone 6 isolated from Inula britannica, and its derivative (2-O-butyloxime-3-phenyl) propionyl 1-O-acetylbritannilactone ester 7 are reported. The structure of synthesized derivative 7 was established by spectroscopic methods. Both compounds inhibited the growth of human HL-60, Bel-7402 cell lines and compound 7 had the stronger cytotoxic activity.

Full text of DOI:10.1016/j.bmcl.2003.12.078

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1101–1104
Studies on 1-O-acetylbritannilactone and its derivative,
(2-O-butyloxime-3-phenyl)-propionyl-1-O-acetylbritannilactone ester
Shouxin Liu,a,b He Liu,a Weiying Yan,a Li Zhang,c Naisheng Baic and Chi-Tang Hoc,*
aHebei University of Science and Technology, Shijiazhuan 050018, People’s Republic of China
bXi’an Jiaotong University, Xi’an 710049, People’s Republic of China
cDepartment of Food Science, Rutgers, The State University of New Jersey, NJ 08901, USA
Received 16 November 2003; revised 25 December 2003; accepted 25 December 2003
Abstract—The crystal structures of 1-O-acetylbritannilactone 6 isolated from Inula britannica, and its derivative (2-O-butyloxime-3-
phenyl) propionyl 1-O-acetylbritannilactone ester 7 are reported. The structure of synthesized derivative 7 was established by
spectroscopic methods. Both compounds inhibited the growth of human HL-60, Bel-7402 cell lines and compound 7 had the
stronger cytotoxic activity.
# 2004 Elsevier Ltd. All rights reserved.
1.Introduction
may be essential to the bioactivity, because in this assay
1,6-O,O-diacetylbritannilactone is ten times more active
than 1-O-acetylbritannilactone 6. This condition is
similar to the bioassay results in our previously synthe-
sized 1-O-acetylbritannilactone derivatives. In addition,
four other cytotoxic sesquiterpene lactones were identi-
fied8 from I. britannica. According to other reports,9ꢀ14
acylation of the hydroxy group in the fragment is simi-
lar to that of 1-O-acetylbritannilactone, and both can
ameliorate bioactivity. The present paper describes the
crystal structure of 1-O-acetylbritannilactone 6 and its
derivative, (2-O-butyloxime-3-phenyl) propionyl 1-O-
acetylbritannilactone ester 7. To date, numerous pro-
drug strategies that could improve the aqueous solubility
and pharmacological functions have been tried. Some of
themhave reported the synthesis of prodrug esters with
amino acid moieties.15ꢀ17 We designed and synthesized a
novel chain, (2-O-butyloxime-1-phenyl) propionic acid 4,
and introduced it into the structure of 1-O-acetyl-
britannilactone 6 to transformthe 6-hydroxy group into
its ester 7 and to try to influence its activity. The structure
of synthesized derivative 7 was established by spectro-
scopic methods. Their cytotoxicity was also determined
and compound 7 showed stronger cytotoxic activity.
Inula, fromCompositae, has more than 100 species
and is found mainly in Europe, Africa and Asia. Inula
britannica is a wild plant found in Eastern Asia,
including China, Korea and Japan. In traditional
Chinese medicine, both Inula britannica and Inula japonica
are called ‘Xuanfuhua.’ The flowers fromthese have
been used for the treatment of digestive disorders,
bronchitis, and inflammation. Its extracts have been
reported to have anti-inflammatory, anti-bacterial, anti-
hepatitic and anti-tumor activities.1 Various sesquiterpene
lactones have been isolated fromChinese Inula species
such as I. britannica,2 I. salsoloides, I. hupehensis and
I. helianthus-aquatica,3ꢀ5 and several sesquiterpene
lactones have been shown to be cytotoxic. Several
derivatives of 1-O-acetylbritannilactone 6, the major
component in I. Britannica have been synthesized, and
one of themhas displayed potent cytotoxicity in human
HL-60 cells (ED50=4.6 mg/mL).6 Recently, using a
clonogenic assay, it was demonstrated that 1,6-O,O-
diacetylbritannilactone decreased cell growth in breast,
prostate and colorectal cell lines (IC50 200 nM–2 mM)
and G2-M cell cycle arrest. So it was been concluded
that 1,6-O,O-diacetylbritannilactone is cytotoxic in
multiple tumor cell lines.7 The acetyl group in position 6
2.Chemistry
Keywords: Inula britannica; Sesquiterpene lactone; Cytotoxicity; (2-O-
butyloxime-1-phenyl) propionic acid derivative; X-ray crystal structures.
* Corresponding author. Tel: +1-732-932-9611x235; fax: +1-732-932-
6776; e-mail: ho@aesop.rutgers.edu
By comparing the structure and activity of 618 with
those of diacetylbritannilactone,2 multiradiatin9ꢀ11 and
eupaformosanin,12,13 which all contain a similar struc-
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.078

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S. Liu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1101–1104
ture of sesquiterpene lactone but with different ester
groups, it may be possible to determine that the acyla-
tion of the hydroxy group can improve bioactivity.
Therefore, we synthesized (2-O-butyloxime-1-phenyl)
propionic acid 4 to realize the acylation of the hydroxy
group in 6. In addition an oximno group is a common
and stable functional group frequently employed in
current drugs, so we introduced an O-butoxyloxime
group into compound 4, (2-O-butyloxime-1-phenyl)
propionic acid. 4 was used for the synthesis of com-
pound 7. The general procedure for the preparation of
target compound 7,19,20 (2-O-butyloxime-3-phenyl)
propionyl 1-O-acetylbritannilactone ester, is shown in
Scheme 1.
Scheme 1. Reagents and conditions: (a) NaOEt, PhCH2Cl; (b) EtONO, H2SO4, 0 ꢁC; (c) Bromobutane, K2CO3/acetone, 30–35 ꢁC; (d) NaOH, H2O/
EtOH; (e) SOCl2, CH2Cl2.

S. Liu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1101–1104
1103
Table 1. The activity of compounds 6 and 7 against different cell
bioassays (ID50 mg/mL)
Cell lines
6
7
HL-60a
Bel-7402b
18.4
15.6
4.3
7.2
a HL-60: Promyelocytic leukemia.
b Bel-7402: Hepatocellular carcinoma.
of C13=C15 and C14=O5, which are both connected to
the five-membered ring, are 1.319 (3) A and 1.207(3) A,
respectively. The bond of C (12)–O (3) is at the opposite
of the planar five-membered ring and the long alkyl
chain connected to C7 atom, and can reduce the steric
obstacle.
Figure 1. The structure of 1-O-acetylbritannilactone 6. Selected bond
distances (A) and bond angles (ꢁ): O(3)–C(12) 1.441(2), O(4)–C(14)
1.334(2), O(5)–C(14) 1.207(3), C(7)–C(8) 1.329(2), C(8)–C(16)
1.503(3), C(13)–C(15) 1.319(3), C(13)–C(14) 1.471(3), C(14)–O(4)–C(10)
112.15(15), C(7)–C(6)–C(5) 112.60(15), C(7)–C(6)–C(17) 111.90(16),
C(7)–C(8)–C(16) 126.03(18), C(16)–C(8)–C(9) 114.82(18), C(8)–C(9)–
C(10) 113.31(16), O(4)–C(10)–C(9) 107.82(16), O(4)–C(10)–C(11)
106.31(15), C(13)–C(11)–C(12) 113.52(15), O(3)–C(12)–C(7) 111.45(14),
O(3)–C(12)–C(11) 108.03(14), C(7)–C(12)–C(11) 113.21(14), C(15)–
C(13)–C(14) 122.1(2), C(15)–C(13)–C(11) 129.4(2), C(14)–C(13)–C(11)
108.51(16), O(5)–C(14)–O(4) 121.6(2), O(5)–C(14)–C(13) 128.9(2) O(4)–
C(14)–C(13) 109.52(17).
The crystals of (2-O-butyloxime-3-phenyl) propionyl 1-
O-acetylbritannilactone ester 721 were obtained froma
CH2Cl2 solution. The structure is depicted in Figure 2.
The oxime ether group is in E-configuration to separate
it fromthe fragment that is similar to 6.
The bond lengths and angles of bicyclic structure in
both compounds are nearly equal, as seen by comparing
the X-ray diffraction data of 6 with those of 7. On this
account it can be concluded that the influence of the
introduced side chain is very slight. The steric structure
in the fragment similar to that of 6 varies little, probably
because the introduced group is at the opposite end, far
away fromthe other big groups.
3.X-ray crystal structures of 1- O-acetylbritannilactone
6 and (2-O-butyloxime-1-phenyl)
propionyl 1-O-acetylbritannilactone ester 7
Single crystals of the 1-O-acetylbritannilactone 621 sui-
table for X-ray analysis were obtained froma CH Cl2
2
solution. The structure of 6 is depicted in Figure 1. As
anticipated, the structure of 6 is in accord with the
results obtained fromother methods (NMR, MS, IR).
It is an a,b-unsaturated sesquiterpene lactone containing
a six-membered ring, which adopted a slightly twisted
boat conformation and was fused by a planar five-
membered ring. The dihedral angle between two rings is
58.9ꢁ. The length of double bond C7=C8 in the six-
membered ring is 1.329(2) A, while the bond distances
4.Cytotoxicity assay
The cytotoxicities22 of compounds 6 and 7 against two
cell lines are compared in Table 1. Fromthe results, we
can see that the cytostatic activity of compound 7 is
much higher than that of 6. The data of crystal structure
shows that the cyclic difference between compound 6
and 7 is very small. The improvement of bioactivities is
Figure 2. The structure of (2-O-butyloxime-1-phenyl) propionyl 1-O-acetylbritannilactone ester 2. Selected bond distances (A) and bond angles (ꢁ):
C(8)–C(9) 1.334(6), C(9)–C(16) 1.495(6), C(11)–O(5) 1.483(6), C(12)–O(6) 1.219(6), C(12)–O(5) 1.350(7), C(12)–C(13) 1.467(7), C(13)–C(17) 1.324(8),
C(15)–O(3) 1.473(5), C(18)–O(4) 1.203(6), C(18)–O(3) 1.360(5), C(19)–N(1) 1.279(6), C(27)–O(7) 1.438(7), N(1)–O(7) 1.411(5), C(9)–C(8)–C(6) 126.0(4),
C(8)–C(9)–C(16) 126.7(4), C(11)–C(10)–C(9) 112.1(3), O(5)–C(11)–C(10) 107.3(4), O(5)–C(11)–C(14) 105.5(4), C(13)–C(14)–C(15) 110.8(3), O(3)–C(15)–
C(8) 107.5(3), O(3)–C(15)–C(14) 109.6(3), C(8)–C(15)–C(14) 13.4(3), O(4)–C(18)–O(3) 123.8(4), O(4)–C(18)–C(19) 122.2(4), O(3)–C(18)–C(19) 114.0(4),
N(1)–C(19)–C(18) 117.1(4), N(1)–C(19)–C(20) 127.9(5), C(18)–C(19)–C(20) 114.9(4), C(19)–N(1)–O(7) 110.8(4), C(18)–O(3)–C(15) 15.6(3).

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S. Liu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1101–1104
a comprehensive result of many factors, but in this case
perhaps the most important reason is the introduction
of the modifying group (2-O-butyloxime-1-phenyl) pro-
pionyl into the target compound. Apparently, this side
chain has the effect of increasing bioactivity of the
parent compound.
18. Compound 6 (1-O-acetylbritannilactone) was isolated
fromthe flowers of Inula britannica var. Chinensis.2 The
flowers were percolated with 95% EtOH at roomtemp-
erature. The CHCl3 soluble part of the EtOH extract was
subject to silica gel column chromatography using
CHCl3–Me2CO to yield product 6. It was previously
characterized by elemental analysis, IR, MS and NMR.
19. Armarego, W. L. F.; Perrin, D. D. Purification of
Laboratory Chemicals, 4th ed.; Butterworth-Heinemann:
Oxford, UK, 1999.
Acknowledgements
20. (2-O-Butyloxime-3-phenyl) propionyl 1-O-acetylbritannil-
actone ester 7: a bright yellow amorphous powder, mp
53–55 ꢁC. [a]D20: ꢀ59.5 (c 0.002 in CH2Cl2). 1H NMR
(300 MHz, CDCl3) d 7.25 (m, 5H), 6.35 (d, J=2.7 Hz,
1H), 5.91 (d, J=2.1 Hz, 1H), 5.29 (d, J=1.8 Hz, 2H), 4.75
(m, 1H), 4.30 (t, 2H), 3.95 (m, 2H), 3.90 (m, 2H), 3.81 (m,
2H), 3.38 (m, 1H), 2.60 (m, 1H), 2.42 (m, 2H), 2.02 (m.
3H), 1.77 (s, 3H), 1.66 (m, 2H), 1.36 (m, 4H), 0.95 (m,
2H), 0.73 (d, J=3.1 Hz, 3H). 13C NMR (75 MHz, CDCl3)
d 13.35, 17.84, 18.85, 19.95, 20.01, 26.02, 30.63 (2 C),
32.52, 34.04 (2 C), 42.11, 63.62, 69.63, 74.15, 75.21, 75.24,
78.42, 124.29, 126.08, 128.14 (2 C), 131.01, 133.94 (2 C),
135.78, 149.38, 162.60, 168.77, 170.40. Found: C, 68.59;
H, 7.51; N, 2.61%. Calc. for C30H39NO7: C, 68.55; H,
7.48; N, 2.66%. IR (KBr pellet): Vmax/cmꢀ1 3029 (alkyl),
1767 (CO), 1734 (CO), 1663 (C¼N), m/z 290, 272, 215,
202, 189 (100%), 143, 117, 91, 43.
21. X-ray crystal structure analysis: Single crystals of 1-O-
acetylbritannilactone 6 and (2-O-butyloxime-3-phenyl)
propionyl 1-O-acetylbritannilactone ester 7, were
obtained in the formof prisms needle and triclinic needle
crystals, respectively. All diffraction measurements were
performed at temperature of 298ꢁK using a Bruker Smart
1000 diffractometer and graphite mono chromated MoKꢀ
radiation (l=0.71073 A). The structure was elucidated
using direct methods and refined by full matrix least-
squares on F2. Crystal data of 1-O-acetylbritannilactone 6:
We are grateful to researchers in the Beijing Institute of
Materia Medica and The Chinese Academy of Sciences
for cytotoxicity testing. This research was partly sup-
ported by grants fromThe Hebei Province Natural
Foundation, P. R. China.
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a=8.0017 (7), b=12.3653 (10), c=16.8794 (14)
A, V=1670.1 (2) A3, T=298 (2) K, Z=4, DC=1.226 Mg
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m ,
ꢀ3, F (000)=664, absorption coefficient=0.089 mmꢀ1
crystal size=0.20ꢂ0.25ꢂ0.30 mm, 6995 reflection mea-
sured, 2949 unique (Rint=0.0241) which were used in all
calculations. The final wR(F2) was 0.0837 (all data).
Crystal data of (2-O-butyloxime-1-phenyl) propionyl 1-O-
acetylbritannilactone ester 7: C30H39NO7, 525.62, Tri-
clinic, space group P1, a=9.185(4), b=9.470(4),
c=9.840(4) A, V=759.4(5) A3, T=298(2) K, Z=1,
14. Hall, I. H.; Lee, K. H.; Williams, W. L., Jr.; Kimura, T.;
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DC=1.149 Mg mꢀ3
, F(000)=282, absorption coeffi-
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cient=0.081 mitymꢀ1, crystal size=0.20ꢂ0.25ꢂ0.30 mm,
2686 reflection measured, 2686 unique which were used in
all calculations. The final wR(F2) was 0.1584 (all data).
22. The in vitro cytotoxicity was evaluated using a system
based on tetrazoliumsalt (MTT), which was reduced by
living cells to yield a soluble formazan product that could
be assayed colorimetrically
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