Synthesis of andrographolide cyclophosphate derivatives and their antitumor activities

Article abstract of DOI:10.1080/00397910500377594

A series of cyclophosphate andrographolide derivatives were synthesized from andrographolide, the cytotoxic constituent of the plant Andrographis paniculata. The derivative (P_R, P′_S)-S and (P _S, P′_S)-6 were synthesized and the proposed mechanism for their formation was discussed. The structures of all compounds were elucidated by IR, NMR, MS (ESI), and HR-MS. The stereochemistry of 6 was confirmed by X-ray analysis. All the derivatives were tested for antitumor activity in vitro, and some of them showed good results. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/00397910500377594

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Synthesis of Andrographolide Cyclophosphate
Derivatives and Their Antitumor Activities
Hai‐Wei Xu a b , Jianye Zhang a b , Hong‐Min Liu a b & Jun‐Feng Wang a b
a Department of Chemistry, Zhengzhou University, Zhengzhou, China
b New Drug Research & Development Center, Zhengzhou University, Zhengzhou, China
Published online: 19 Aug 2006.
To cite this article: Hai‐Wei Xu , Jianye Zhang , Hong‐Min Liu & Jun‐Feng Wang (2006): Synthesis of Andrographolide
Cyclophosphate Derivatives and Their Antitumor Activities, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 36:4, 407-414
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Synthetic Communicationsw, 36: 407–414, 2006
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910500377594
Synthesis of Andrographolide
Cyclophosphate Derivatives and
Their Antitumor Activities
Hai-Wei Xu, Jianye Zhang, Hong-Min Liu, and
Jun-Feng Wang
Department of Chemistry, Zhengzhou University, Zhengzhou, China and
New Drug Research & Development Center, Zhengzhou University,
Zhengzhou, China
Abstract: A series of cyclophosphate andrographolide derivatives were synthesized
from andrographolide, the cytotoxic constituent of the plant Andrographis paniculata.
The derivative (PR, PS0 )-5 and (PS, PS0 )-6 were synthesized and the proposed mechanism
for their formation was discussed. The structures of all compounds were elucidated by
IR, NMR, MS (ESI), and HR-MS. The stereochemistry of 6 was confirmed by X-ray
analysis. All the derivatives were tested for antitumor activity in vitro, and some of
them showed good results.
Keywords: Andrographolide, cyclophosphate, 14-deoxy-11,12-didehydroandrogra-
pholide, synthesis
INTRODUCTION
Andrographolide 1 is one of the major labdane diterpenoidal constituents of
Andrographis panicutate (Burm) Nees, which is widely used in traditional
Chinese and Indian medicine. It has been reported to exhibit a wide
spectrum of biological activities, such as anti-inflammatory,[1–3] antialler-
gic,[4,5] antiplatelet aggregation,[6] hepatoprotective,[7,8] and anti–human
immunodeficiency virus (HIV) activities.[9,10] This compound has been also
Received in Japan June 16, 2005
Address correspondence to Hong-Min Liu, Department of Chemistry, Zhengzhou
University, Zhengzhou 450052, China. Tel./Fax: 86-371-67767200; E-mail: liuhm@
public.zz.ha.cn
407

408
H.-W. Xu et al.
widely used in clinics for the treatment of fever, cold, inflammation, diarrhea,
and infectious diseases, so it has aroused the interest of pharmacologists.[11–14]
Furthermore, organic phosphate plays an important role in vivo and has gained
enormous attention for its ability to serve as potent antiviral, anticancer, and
immunostimulatory drugs.[15–17] To develop new drugs and investigate
their structure–activity relationship, a series of andrographolide cyclopho-
sphate derivatives were synthesized from andrographolide.
RESULTS AND DISCUSSION
Deoxyandrographolide (2) was prepared from andrographolide (1) by
refluxing in pyridine with the presence of Al2O3 at high yield. Reaction of 2
with POCl3 in the mixture of CH2Cl2 and pyridine afforded cyclophosphour-
soxychloride (3) at a 89% yield. Without further purification, the crude
compound 3 was used in the synthesis of 4 and 7 (Scheme 1). Compound 7
was also synthesized from 2 in one pot as shown in Scheme 2.
However, expected compound 8 was not obtained when andrographolide
reacted with POCl3 in the same condition, and compound 7 was formed as
main product (Scheme 2).
Compound (PR, PS0 )-5 and (PS, PS0 )-6 were also synthesized from 1 and 2
by adjusting the concentration and the reaction time respectively as shown in
Scheme 3. In the reaction of the preparation for compound 7, a little of (PR,
PS0 )-5 and (PS, PS0 )-6 were also separated as by-products. It was interesting
that the (PR, P’R) product was not obtained. The proposed mechanism was
that the asymmetric synthesis of (PR, PS0 )-5 and (PS, PS0 )-6 was carried out
from the product (PS)-3 and resonant 7, both of which existed in the
reaction system at the same time. The product (PS)-3 was probably obtained
by the asymmetric induction of the chiral center of andrographolide.
The conjugated olefinic protons of compound 3 were detected at about
d 6.8 (H-11), 6.1 (H-12), and 7.2 (H-14). The multilets at d 3.7–4.0 (H-19)
and d 4.0–4.3 (H-3) in 1H NMR spectrum of compound 3 showed the
structure of cyclophosphate. The doublets at d 39.4 (C-4), 71.4 (C-19), and
91.7 (C-3) in 13C NMR spectrum of compound 3 further verified the
structure of cyclophosphate. In the HR-MS of compound 3, the pesudo
Scheme 1.

Andrographolide Cyclophosphate Derivatives
409
Scheme 2.
molecular ion [M þ H]þ, m/z 413.1281, confirmed that the molecular formula
of 3 was C20H26ClO5P. The 31P NMR signal of 3 was detected as a sharp
singlet at d 2 5.8, which corresponded to the six-member cyclophosphate.[18]
Accordingly, the structure of 3 could be identified.
There were similar signals to 3 in 1H NMR, 13C NMR, and 31P NMR spectra
of 4 and 7. Their 31P NMR signals were detected at d 2 4.5 and d 2 8.6 respect-
ively, which suggested a structure similar to 3. On the basis of HR-MS, the
molecular formula of 4 and 7 could be identified as C22H31O6P and C20H27O6P.
In the HR-MS of compound 5 and 6, the peaks at m/z 793.2883 and 793.2873
[M þ Na]þ confirmed that 5 and 6 had the same molecular formula C40H52O11P2.
The 31P NMR signal of 5 appeared at d 2 18.6 and d 2 23.6, and 6 at d 2 22.7,
which were different from those of 3, 4, and 7 and suggested the phosphate struc-
tures of 5 and 6 were different from the latter three compounds. The doublets at
d 2 18.9 and 223.6 in the 31P NMR of 5 showed the presence of two kinds of
phosphorus and that they were coupling with each other. Although the 13C
NMR of 5 showed money more carbon signals than those of 3, 4, and 7, its 1H
NMR spectra was similar to the latter three. Accordingly, the structure of 5
was assumed to be (PR, PS0 )-14-deoxy-11,12-didehydroandrographolide-3,19-
dipolycyclophosphate. The 1H NMR and 13C NMR spectrum of 6 were similar
to 3 and 7, and the 31P NMR signal at d 2 22.7 showed only one kind of
phosphorus existed in 6, which suggested it had a structure of phosphate similar
to 5. Therefore, the structure of 6 was assumed to be (PS, PS0 )-14-deoxy-11,
12-didehydroandrographolide-3,19-dipolycyclophos- phate. At last, the stereo-
chemistry of 6 was confirmed by x-ray diffraction analysis (Figure 1).
Scheme 3.

410
H.-W. Xu et al.
Figure 1. Structure of compound 6.
The cytotoxicity in vitro of the derivatives against human cancer were
evaluated in two assay systems of Eca109 and CNE cell lines. (In vitro cyto-
toxicity study: CNE and Eca 109 cell lines were purchased from the Institute
of Biochemistry and Cell Biology, SIBS, CAS. Culture medium was RPMI-
1640 supplemented with 10% (v/v) fetal calf serum. Sensitivity test:
Cancer cells, growing in monolayer cultures at 378C under 5% CO2, were
trypsinized, rinsed with PSB (w/o Ca2þ and Mg2þ), and plated into 96
well-plates (6 ꢀ 103 cells/well). The next day tst substances were freshly
dissolved in DMSO, resulting in 10 mg/mL stock solutions. Stock solutions
were diluted in culture medium and added (200 ml/well) at various concen-
trations to the wells, resulting in seven final concentrations between 1 and
100 mg/ml. The test was performed 48 h later.) The results are presented in
Table 1. Antitumor activity of the compounds
No.
2
3
4
5
6
7
CNE/IC-50 (mg/ml)
Eca109/IC-50 (mg/ml)
64.7
31.9
64.5
42.2
.100
.100
.100
a
—
.100
—
75.0
58.6
a— represents a IC50 value of .1000 mg/ml.

Andrographolide Cyclophosphate Derivatives
411
Table 1, which implied that some of them showed good antitumor activities,
and the cytotoxicity was sensitive to the structures of the compounds. Further-
more, compound 7 was tested for anti-inflammatory activity in vivo and
showed good effects. The test of their activities for anti-HIV are in progress.
EXPERIMENTAL
All reagents and solvents were obtained from commercial suppliers. All the
reactions were monitored by TLC. NMR spectra were recorded on a DPX-400
Bruker spectrometer for all compounds. Chemical shift of proton and carbon
nuclear were reported relative to TMS as internal reference. Phosphorus
nuclear was reported relative to 35% phosphoric acid as external reference.
The mass spectra were recorded with Esquire 3000-00142. The HR-MS were
recorded with Waters Micromass Q-TOF Micro. The X-ray diffraction
analysis was held with a Rigaku RAXIS-IV. The melting points were tested in
Beijing Keyi ELEC-OPTI XT5 XMF, and the temperature was not corrected.
Preparation of 14-Deoxy-11,12-didehydroandrographolide (2)
Al2O3 (0.5 g) was added to the solution of andrographolide (3.0 g) in dry
pyridine. The mixture was refluxed for 10 h and then filtrated to remove the
Al2O3. The filter cake was washed with chloroform. The solvent was
removed from the filtrate under vacuum to give a crude deoxyandrographolide.
It was recrystallized with methanol to yield compound 2 as a white powder
1
(2.6 g, 91%). Mp: 203–2058C; H NMR (CDCl3): 1.35 (1H, m, 1-H), 1.68
(1H, m, 1-H), 1.77 (2 H, m, 2-H), 3.48 (1H, m, 3-H), 1.21 (1H, m, 5-H), 1.52
(1H, d, J ¼ 13.6 Hz, 6-H), 1.20 (1H, m, 6-H), 2.51 (1H, d, J ¼ 13.6 Hz,
7-H), 2.09 (1H, m, 7-H), 2.34 (1H, d, J ¼ 14 Hz, 9-H), 6.87 (1H, dd, J ¼ 10,
16 Hz, 11-H), 6.12 (1H, d, J ¼ 16, 12-H), 7.18 (1H, s, 14-H), 4.82 (2H, s,
15-H), 4.78 (1H, s, 17-H), 4.53 (1H, s, 17-H), 1.26 (3H, s, 18–H), 3.35 (1H,
d, J ¼ 10.8 Hz, 19-H), 4.22 (1H, d, J ¼ 10.8, 19 H), 0.82 (3H, s, 20-H); 13C
NMR (CDCl3): d135.0 (C-11), 121.6 (C-12), 146.7 (C-13), 143.2 (C-14). IR
(KBr) cm21: 3479.7, 3434.5, 2934.2, 2868.1, 1738.6, 1635.8, 1443.4,
1351.4, 1111.4, 1050.1, 1030.5, 1001.1. HR-MS m/z: [M þ H]þ 333.2066
(calcd. 333.2060); [M þ Na]þ 355.1891 (calcd. 355.1885).
Preparation of 14-Deoxy-11,12-didehydro-3,19-cyclophosphours
Oxychorlide Andrographolide (3)
The solution of POCl3 (0.35 ml) in CH2Cl2 (3 ml) was added dropwise to the
solution of deoxyandrographolide 2 (1.0 g) in dry CH2Cl2 (5 ml) and pyridine
(1.0 ml) in an ice bath. The reaction mixture was allowed to stir at 0–58C for

412
H.-W. Xu et al.
1 h and was washed with saturated NaHCO3 aqueous solution and water in turn.
The CH2Cl2 layer was dried with Na2SO4, concentrated, and recrystallized with
1
ethanol to yield white crystals (1.1 g, 89%). Mp: 180.0–181.48C; H NMR
(CDCl3): 1.11 (1H, m, 1-H), 1.64 (1H, m, 1-H), 2.32 (1H, m, 2-H), 2.03 (1H, m,
2-H), 4.17 (1H, m, 3-H), 1.26 (1H, s, 5-H), 1.81 (1H, m, 6-H), 1.34 (1H, m,
6-H), 2.49 (1H, m, 7-H), 2.20 (1H, m, 7-H), 2.34 (1H, m, 9-H), 6.86 (1H, dd,
J ¼ 10.0, 15.6 Hz, 11-H), 6.13 (1H, d, J ¼ 15.6 Hz, 12-H), 7.19 (1H, s, 14-H),
4.82 (3H, s, 15-H, 17-H), 4.58 (1H, d, J ¼ 11.6Hz, 17-H), 1.44 (3H, s, 18–H),
4.91 (1H, m, 19-H), 3.89 (1H, m, 19-H), 0.91 (3H, s, 20-H); 13C NMR (CDCl3):
d 91.8 (d, J ¼ 9.6 Hz, C-3), 71.4 (d, J ¼ 7.2 Hz, C-19) 39.4 (d, J ¼ 7.4 Hz,
C-4). 31P NMR: d 2 5.89; IR (KBr) cm21: 1748.1, 1229.0, 1033.5. MS (ESI)
m/z: [M þ Naþ] 435. HR-MSm/z: [M þ H]þ 413.1281 (calcd. 413.1285).
Preparation of 14-Deoxy-11,12-Didehydro-3,
19-Cyclophosphate Andrographolide (4)
The andrographolide (5.0g) was dissolved in dry pyridine (30.0 ml) and cooled
in an ice-salt bath to 210 to 2158C. POCl3 (3.0ml) was added dropwise to the
solution below 08C and stirred for 1 h. Solid state NaHCO3 (10.0 g) and cooled
acetone (30.0 ml) were added to the reaction mixture with stirring and ice bath.
Water (30.0 ml) was added to the reaction system (pH ¼ 6 ꢀ 7) that was
warmed slowly to room temperature 30min later and stirred for 24h. The
reaction system was extracted with chloroform/petrotium (v/v ¼ 1/1,
3 ꢀ 25ml). The water layer was then acidified with 6 M HCl aqueous
solution until the products precipitated completely and were certrifugalized to
give the crude compound. Then they were chromatographed on silica gel
with methanol/chloroform (v/v ¼ 1/3) as eluent to afford the compound 4
1
(3.5g, 64%). Mp . 2718C (decomposed); H NMR (DMSO): 1.11 (1H, m,
1-H), 1.49 (1H, d, J ¼ 13.6Hz, 1-H), 2.23 (1H, dd, J ¼ 12.8, 24.4Hz, 2-H),
1.81 (1H, m, 2-H), 3.88 (1H, m, 3-H), 1.24 (1H, m, 5-H), 1.81 (H, m, 6-H),
1.37 (1H, d, J ¼ 12.8Hz, 6-H), 2.05 (1H, m, 7-H), 2.41 (2H, m, 7-H, 9-H),
6.67 (1H, dd, J ¼ 10, 16Hz, 11-H), 6.11 (1H, d, J ¼ 16Hz, 12-H), 7.53 (1H,
s, 14-H), 4.91 (2H, s, 15-H), 4.78 (1H, s, 17-H), 4.50 (1H, s, 17-H), 1.26
(3H, s, 18–H), 4.61 (1H, d, J ¼ 11.2Hz, 19-H), 3.67 (1H, dd, J ¼ 11.2,
26.4Hz, 19-H), 0.91 (3H, d, 20-H); 13C NMR (DMSO): d 91.0 (s), d71.1 (s);
31P NMR(DMSO): d 2 4.5. IR (KBr) cm21: 1756.4, 1233.1, 1049.0. MS
(ESI) m/z: [M þ K]þ 433. HR-MS m/z: [M-H]2 393.1469 (calcd. 393.1467).
The compound 4 could be prepared by the basic hydrolysis of compound of 3.
Preparation of 14-Deoxy-11,12-didehydroandrographolide-3,
19-Dipolycyclophosphate (5) and (6)
The andrographolide (2.0 g) was dissolved in dry pyridine (10.0 ml) and
cooled in an ice-salt bath to 210 to 2158C. POCl3 (1.5 ml) was added

Andrographolide Cyclophosphate Derivatives
413
dropwise to the solution below 08C and allowed to stir for 1 h. NaHCO3 (5.7 g)
and cooled acetone (10.0 ml) to the reaction mixture were added with stirring
and ice bath. After stirring for 20–30 min, water (0.5 ml) was added, and then
the mixture was warmed slowly to room temperature and stirred for 2–3 h.
More water (15.0 ml) was added to the reaction mixture and was extracted
with chloroform (3 ꢀ 10.0 ml). The chloroform layer was washed with
water, dried with Na2SO4, and concentrated under vacuum to give the crude
compound. It was isolated by column chromatography with chloroform/
acetate (v/v ¼ 2/1) as eluent to yield white power 5 (0.79 g 35.9%). Mp:
1
198.3–199.58C. H NMR (CDCl3): d 1.11 (1H, m, 1-H), 1.64 (1H, m, 1-H),
2.32 (1H, m, 2-H), 2.03 (1H, m, 2-H), 4.17 (1H, m, 3-H), 1.26 (1H, s, 5-H),
1.81 (1H, m, 6-H), 1.34 (1H, d, 6-H), 2.49 (1H, d, 7-H), 2.20 (1H, m, 7-H),
2.34 (1H, s, 9-H), 6.86 (1H, m, 11-H), 6.13 (1H, d, J ¼ 15.8 Hz, 12-H),
7.19 (1H, s, 14-H), 4.82 (3H, s, 15-H, 17-H), 4.58 (1H, d, J ¼ 18 Hz, 17-H),
1.44 (3H, d, 18–H), 4.91 (1H, m, 19-H), 3.89 (1H, m, 19-H), 0.91 (3H, d,
20-H); 13C NMR (CDCl3): d90.42 (d, J ¼ 8.7 Hz, C-3), 89.63
(d, J ¼ 6.1 Hz, C-30), 39.24 (d, J ¼ 6.5 Hz, C-4), 39.07 (d, J ¼ 7.2 Hz,
C-40), 71.67 (d, J ¼ 6.9 Hz, C-19), 71.0 (d, J ¼ 4.7 Hz, C-190); 31P NMR
(CDCl3): d 2 18.9 (d), 223.6(d); IR (KBr) cm21: 1740.5, 1299.5,
1082.3,1070.7. MS (ESI) m/z: [M þ Na]þ 793. HR-MS m/z: [M þ Na]þ
793.2883 (calcd. 793.2883). 6 (0.85 g, 38.6%). Mp: 208.0–209.78C.
1H NMR (CDCl3): d 1.11 (1H, m, 1-H), 1.64 (1H, m, 1-H), 2.27 (1H, m,
2-H), 1.89 (1H, m, 2-H), 4.17 (1H, m, 3-H), 1.33 (1H, m, 5-H), 1.81 (1H,
m, 6-H), 1.35 (1H, m, 6-H), 2.49 (1H, d, J ¼ 14 Hz, 7-H), 2.05 (1H, m,
7-H), 2.32 (1H, d, J ¼ 10 Hz, 9-H), 6.83 (1H, dd, J ¼ 10 Hz, 15.6 Hz, 11-H),
6.12 (1H, d, J ¼ 15.6 Hz, 12-H), 7.20 (1H, s, 14-H), 4.82 (3H, s, 15-H,
17-H), 4.57 (1H, s, 17-H), 1.42 (3H, s, 18-H), 5.12 (1H, d, 19-H), 3.91 (1H,
m, 19-H), 0.93 (3H, d, 20-H); 13C NMR(CDCl3): d90.6 (s), 71.9 (s);
31P-NMR(CDCl3): d 2 22.7 (s); IR (KBr) cm21: 1751.1, 1312.0, 1054.6,
1078.7. MS (ESI) m/z: [M þ Na]þ 793. HR-MS m/z: [M þ H]þ 771.3060
(calcd. 771.3063); [M þ Na]þ 793.2873 (calcd. 793.2883). Compounds 5 and
6 could be also prepared from compound 2 with the same reaction condition.
Preparation of 14-Deoxy-11,12-didehydroandrographolide-3,
19-Cyclophosphate (7)
Compound 3 (200 mg) and DMAP were dissolved in anhydrous EtOH
(5.0 ml). Pyridine (0.2 ml) was added to the solution and refluxed for 48 h,
which was treated with water (5.0 ml), filtrated to give the crude product,
and recrystallized in ethanol to yield colorless crystals (120 mg, 60%). Mp:
1
195.8–197.0 8C. H NMR (CDCl3): d 1.11 (1H, m, 1-H), 1.62 (1H, m,
1-H), 2.15 (1H, m, 2-H), 1.95 (1H, m, 2-H), 4.02 (1H, m, 3-H), 1.34 (1H,
m, 5-H), 1.84 (1H, m, 6-H), 1.28 (1H, m, 6-H), 2.49 (1H, m, 7-H), 2.08
(1H, m, 7-H), 2.34 (1H, d, J ¼ 10 Hz, 9-H), 6.92 (1H, dd, J ¼ 10, 15.6 Hz,

414
H.-W. Xu et al.
11-H), 6.13 (1H, d, J ¼ 15.6 Hz, 12-H), 7.19 (1H, s, 14-H), 4.82 (1H, s, 15-H),
4.58 (1H, d, J ¼ 13.2 Hz, 17-H), 4.82 (1H, s, 17-H), 1.42 (3H, s, 18–H), 4.68
(1H, d, J ¼ 11.2 Hz, 19-H), 3.80 (1H, m, 19H), 0.91 (3H, s, 20-H), 4.15 (2H,
m, 21-H), 1.34 (3H, m, 22-H); 13C NMR(CDCl3): d88.3 (d, J ¼ 7.9 Hz, C-3),
39.1 (d, J ¼ 6.7 Hz, C-4), 70.1 (d, J ¼ 6.4 Hz, C-19), 63.5 (d, J ¼ 5.5 Hz,
C-21), 16.3 (d, J ¼ 6.3 Hz, C-22); 31P NMR(CDCl3): d 2 8.6; IR (KBr)
cm21: 1748.2, 1288.6, 1027.0. MS (ESI) m/z: [M þ H]þ 423. HR-MS m/z:
[M þ H]þ 423.1924 (calcd. 423.1937).
ACKNOWLEDGEMENTS
The authors are grateful for the support of NSFC (National Natural Science
Foundation of China).
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