Antitumor agents. Part 232: Synthesis of cyclosulfite podophyllotoxin analogues as novel prototype antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2003.12.096

An 11,13-O,O′-cyclosulfite GL-331 analogue (7) was synthesized in six steps from podophyllotoxin and evaluated as a potential antitumor agent. Compound 7 was significantly cytotoxic against human tumor cell lines, but showed no inhibition against human DN

Full text of DOI:10.1016/j.bmcl.2003.12.096

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1581–1584
Antitumor agents. Part 232: Synthesis of cyclosulfite
podophyllotoxin analogues as novel prototype antitumor agents§
Zhiyan Xiao, Shiqing Han, Kenneth F. Bastow and Kuo-Hsiung Lee*
Natural Products Laboratory, School of Pharmacy, University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599-7360, USA
Received 22 October 2003; accepted 16 December 2003
Abstract—An 11,13-O,O0-cyclosulfite GL-331 analogue (7) was synthesized in six steps from podophyllotoxin and evaluated as a
potential antitumor agent. Compound 7 was significantly cytotoxic against human tumor cell lines, but showed no inhibition
against human DNA topoisomerase II in vitro. This compound represents a novel prototype of antitumor podophyllotoxin
analogues.
# 2004 Elsevier Ltd. All rights reserved.
Podophyllotoxin (1)2 is a bioactive lignan isolated from
Podophyllum peltatum L., P. emodi W., or P. pleianthum
H. Two semisynthetic derivatives of 1, etoposide (2) and
teniposide (3), are currently used in front-line cancer
chemotherapy against various cancers, including small-
cell lung cancer, testicular carcinoma, lymphoma, and
Kaposi’s sarcoma.3,4 Although 1 is known as an
antimicrotubule agent, 2 and 3 inhibit the catalytic
activity of DNA topoisomerase II (topo II).5,6
The trans-fused g-lactone D ring in 2 and its analogues
are susceptible to metabolic inactivation through
* Corresponding author. Tel.: +1-919-962-0066; fax: +1-919-966-3893; e-mail: khlee@unc.edu
§See ref 1
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.096

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Z. Xiao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1581–1584
C2-epimerization or hydrolysis to give the inactive
cis-fused lactone or open-ring hydroxy acid metabolites,
respectively.7 However, SAR studies indicated that the
trans-lactone ring is essential to maintain topo II inhi-
bition, presumably because it forces the molecules into a
twisted conformation that ensures optimal interaction
with the enzyme. Therefore, previous efforts to circum-
vent the metabolic pathways have primarily focused on
bioisosteric approaches to minimize conformational
alteration.7
evaluation against several forms of cancer, especially
etoposide-resistant malignancies.11 In this modification,
the lactone D ring is replaced with a seven-membered
sulfite ring and the trans-configuration is retained.
As shown in Scheme 1, compound 11 was synthesized in
four steps from podophyllotoxin (1). Compound 1 was
treated initially with tert-butyldimethylsilyl chloride
(TBDMSCl) and imidazole to afford 8, in which the
4-hydroxy group is protected. Reduction of 8 with
lithium aluminum hydride (LiAlH4) in tetrahydrofuran
yielded diol 9.12 Subsequent ring closure with thionyl
chloride and 4-dimethylaminopyridine (DMAP) in
methylene chloride provided the seven-membered cyclic
sulfite ester 10. Compound 10 was a mixture of diaster-
eomers with respect to the orientation of the lone pair
electrons of the sulfur atom.13 Deprotection of 10 with
tetrabutylammonium fluoride (Bu4NF) in tetra-
hydrofuran gave 11, which can be separated by silica gel
column chromatography to afford optical active dia-
stereomers 11a and b.14 The 4b-(p-nitroanilino) deriva-
tive 7 was prepared from 11 (a mixture of two
diastereomers) according to the previously described
procedure.15
Recently, a spin-labeled analogue of 1, GP-11 (4) was
reported as a low immunosuppressive antitumor agent,
which increases the mitotic index and results in G2/M,
and to a lesser extent, S arrest.8 In addition, methyl 9-
deoxy-9-oxo-a-apopicropodophyllate (5) was found to
be a highly selective antitumor agent against HT-29
colon carcinoma.9 Several aldehydes related to this
compound but with different configurations have been
synthesized and evaluated for their cytotoxic activities
in neoplastic cell lines. All of them lacked the lactone
ring but maintained their cytotoxicity at, or under, the
mM level.10
These results reaffirm the importance of D ring
structure in 1-related pharmacological profiles and the
feasibility of producing clinically useful agents with
dramatically modified D rings. Accordingly, we have
Compound 7 was evaluated against multiple human
tumor cell lines (HTCL) and showed significant inhibi-
tion against HTCL replication in vitro (Table 1).16
Although compound 7 was similar to GL-331 in overall
potency, its spectrum of activity and dose–response
profile against certain HTCL differed from those of
designed and synthesized
analogue (7) of GL-331 (6), which is a 4b-arylamino
analogue of currently under Phase II clinical
a
11,13-O,O0-cyclosulfite
1
Scheme 1. Synthesis of 7 from podophyllotoxin.

Z. Xiao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1581–1584
Table 1. Inhibition of HTCL replication by GL-331 and 7
1583
Compd
ED50 (mM)a
KB
KB-7d
KB-VIN
MCF-7
HCT-8
1A9
SK-MEL-2
PC-3
HOS
U87-MG
A549
6
7
3.5
5.1
1.5
2.9
4.1
7.1
24.5
2.5b
1.6
10.0
<1.6
4.2
3.0
7.9
8.9
>20b
<1.6
6.0
5.5
8.0
<0.1
8.0
a ED50 is the concentration of drug that afforded 50% reduction in cell number after a 3-day incubation. Cell lines: KB, nasopharyngeal; KB-7d,
etoposide resistant; KB-VIN, vincristine resistant; MCF-7, breast; HCT-8, ileocecal; 1A9, ovarian; SK-MEL-2, melanoma; PC-3, prostate; HOS,
bone; U-87-MG, glioblastoma; A549, lung.
b Plateau dose–response was observed (refer to Fig. 1).
Figure 1. Different dose–response effects of 7 and GL-331.
GL-331. Plateau dose–response was observed in the
replication of drug-treated MCF-7 and PC-3 cells
(Table 1 and Fig. 1).
antitumor podophyllotoxin analogues with a mechanism
different from GL-331. Further synthetic and biological
investigation of this series of compounds is underway.
In spite of the significant activity against HTCL
replication, compound 7 was inactive in topoisomerase
II inhibition in vitro assays (Fig. 2).17 The unique dose–
response profile, activity spectrum, and inability to
induce topoisomerase II-mediated DNA breaks sug-
gested that compound 7 might be a novel prototype of
Acknowledgements
This investigation was supported by a NIH grant CA
17625 and a grant from the Elsa U. Pardee Foundation
(UNC No. 49849) awarded to K. H. Lee.
References and notes
1. Antitumor Agents 232. For part 231, see: Lee, K. H.
J. Nat. Prod., submitted.
2. (a) Schrecker, A. W.; Hartwell, J. L. J. Org. Chem. 1956,
21, 381. (b) Imbert, T. F. Biochimie 1998, 80, 207.
3. Jardine, I. Anticancer Agents Based on Natural Products
Models; Academic Press: New York, 1980; p 319.
4. Issell, B. F. Cancer Chemother. Pharmacol. 1982, 7, 73.
5. Osheroff, N.; Zechiedrich, E. L.; Gale, K. C. BioEssays
1991, 13, 269.
6. Alton, P. A.; Harris, A. L. Br. J. Haematol 1993, 85, 241.
7. Zhang, Y. L.; Lee, K. H. Chin. Pharm. J. 1994, 46, 319.
8. Wang, J.; Tian, X.; Tsumura, H.; Shimura, K.; Ito, H.
Anti-Cancer Drug Des. 1993, 8, 193.
9. Gordaliza, M.; Del Corral, J. M. M.; Castro, M. A.; Lopez-
Vazquez, M. L.; Garcia, P. A.; Feliciano, A. S.; Garcia-
Gravalos, M. D. Bioorg. Med. Chem. Lett. 1995, 5, 2465.
10. Gordaliza, M.; Castro, M. A.; Del Corral, J. M. M.;
Lopez-Vazquez, M. L.; Garcia, P. A.; Garcia-Gravalos,
M. D.; Feliciano, A. S. Eur. J. Med. Chem. 2000, 35, 691.
Figure 2. In vitro topoisomerase II inhibition by compounds GL-331,
7, and etoposide. Lane 1: pBR322 DNA (predominantly supercoiled),
Lane 2: M. Wt. markers, Lane 3: linear BR322 DNA, Lane 4: Enzyme
control, Lane 5: GL-331, Lane 6: compound 7 and Lane 7: etoposide.
o: Origin, n: Nicked, l: Linear, s: Supercoiled, and r: Relaxed.

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Z. Xiao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1581–1584
11. Lee, K. H. Med. Res. Rev. 1999, 19, 569.
7.01 (s, 1H, 5-H), 6.34 (s, 1H, 8-H), 6.16 (s, 2H, 20, 60-H),
5.91 (s, 2H, OCH2O), 4.54 (dd, 1H, J=3.5, 12.5 Hz, 4-H),
4.44–4.30 (m, 2H, 13-H2), 4.07–3.97 (m, 2H, 11-H2), 3.88
(dd, 1H, J=3.0, 12.0 Hz, 1-H), 3.82 (s, 3H, 40-OCH3),
3.76 (s, 6H, 30,50-OCH3), 2.39 (m, 1H, 2-H), 2.22 (m, 1H,
3-H), 1.80 (m, 1H, OH). Anal. calcd C22H24O9S. 3=4
H2O.
12. The protocol used to synthesize 9 was similar to that used
for its 40-demethylated congener as previously described
in: Zhou, X. M.; Lee, K. J. H.; Cheng, J.; Wu, S. S.; Chen,
H. X.; Guo, X.; Cheng, Y. C.; Lee, K. H. J. Med. Chem.
1994, 37, 287.
13. Compound 10: To a solution of 9 (300 mg, 0.564 mmol) in
10 mL of dry CH2Cl2 was added DMAP (60 mg, 0.48
mmol) and SOCl2 (0.15 mL, 1.99 mmol), and the mixture
was stirred at room temperature for 1.5 h. The mixture was
then diluted with EtOAc, washed with water and brine,
dried over anhydrous Na2SO4, and concentrated under
reduced pressure. The residue was purified by flash col-
umn chromatography using EtOAc–hexane (1:3) as an
eluent to give 255 mg (78%) of 10: white solid, mp 119–
120 ꢀC, [a]D27.4 ꢁ193.05 (c 0.47, CHCl3); IR (film) 2960,
1590, 1504, 1483, 1329 cmꢁ1; MS m/e: 578 [M]+; 1H NMR
(CDCl3) d 6.91 (s, 1=2 H, 5-H), 6.90 (s, 1/2 H, 5-H), 6.344
(s, 1=2 H, 8-H), 6.341 (s, 1=2 H, 8-H), 6.21 (s, 2H, 20, 60-
H), 5.91 (d, 2H, J=1.8 Hz, OCH2O), 4.78–4.70 (m, 1=2
H, 13-H), 4.50–4.35 (m, 2H, 4-H, 13-H), 4.21–3.86 (m,
3H, 1-H, 11-H2), 3.84 (s, 3/2H, 40-OCH3), 3.83 (s, 3/2H,
40-OCH3), 3.78 (s, 3H, 30, 50-OCH3), 3.60–3.51 (m, 1=2 H,
13-H), 2.50 (m, 1=2 H, 2-H), 2.38 (m, 1=2 H, 2-H), 2.33
(m, 1=2 H, 3-H), 2.20 (m, 1=2 H, 3-H), 0.97 (s, 9/2H,
t-BuSi), 0.96 (s, 9/2H, t-BuSi), 0.30 (s, 3H, CH3-Si), 0.19
(s, 3H, CH3-Si). Anal. calcd C28H38O9SiS. 1=2 H2O.
14. Compound 11: 200 mg (0.35 mmol) of 10 in 5 mL of THF
was added 365 mg (1.38 mmol) of Bu4NF. The reaction
mixture was stirred at room temperature for 2 h. After the
reaction was completed as monitored by TLC, the reac-
tion mixture was evaporated to dryness and CH2Cl2 was
added. The solution was then washed with H2O and dried
over anhydrous Na2SO4. After the solvent was removed
in vacuo, the crude products was chromatographed on
silica gel (eluent: EtOAc:hexane=1:1) to separate the two
diastereoisomers 11a and 11b. Compound 11a: white
solid, mp 129–130 ꢀC (dec.), [a]2D9.2 +71.9 (c 0.16,
CH2Cl2); IR (film) 3500 (OH), 2938, 1591, 1507, 1203,
15. The protocol used to prepare 7 was similar to that pre-
viously described in: Kamal, A.; Laxman, N.; Ramesh, G.
Bioorg. Med. Chem. Lett. 2000, 10, 2059. Compound 7: 49
mg (0.1 mmol) of a mixture of 11a and 11b was dissolved
in 2 mL of dry CH2Cl2. Sodium iodide (45 mg, 0.3 mmol)
was added and the mixture was stirred for 5 min.
MeSO3H (29 mg, 0.3 mmol) was added in dropwise at
0
ꢀC and stirred for 5 h at room temperature. Nitrogen
was bubbled through the solution to drive off the excess
HI; the solution was then evaporated in vacuo and used
for the next step without further purification. To the
above crude product, anhydrous BaCO3 (40 mg, 0.2
mmol) and 4-nitroaniline (17 mg, 0.12 mmol) in 2 mL of
dry THF under nitrogen were added and stirred for 8 h at
room temperature. The reaction mixture was diluted with
EtOAc, filtered, washed with water and brine, dried over
anhydrous Na2SO4, and purified by column chromato-
graphy (eluent: EtOAc:hexane=1:1) to afford 11 mg (19%)
of 7: Yellow power. Mp 144–146 ꢀC, [a]D30 ꢁ192.5 (c 0.2,
acatone); IR (film) 2936, 1585, 1366, 1216, 1115 cmꢁ1; MS
m/e: 570 [MꢁH]+; 1H NMR (CDCl3) d 8.15 (d, 1H,
J=9.0 Hz, 300,500-H), 8.13 (d, 1H, J=9.0 Hz, 300,500-H),
6.72 (d, 1H, J=9.0 Hz, 200, 600-H), 6.70 (d, 1H, J=9.0 Hz,
200, 600-H), 6.60 (s, 1/2 H, 5-H), 6.58 (s, 1=2 H, 5-H), 6.38
(s, 1=2 H, 8-H), 6.37 (s, 1=2 H, 8-H), 6.10 (s, 1H, 20, 60-H),
6.09 (s, 1H, 20, 60-H), 5.92, 5.90 (d, 2H, J=1.4 Hz,
OCH2O), 4.98 (m, 1H, NH), 4.41–4.25 (m, 2H, 4-H, 13-
H), 4.24–4.11 (m, 2H, 1-H, 13-H), 4.02–3.96 (m, 2H, 11-
H2), 3.80 (s, 3H, 30,50-OCH3), 3.79 (s, 3H, 30,50-OCH3),
2.80 (m, 1=2 H, 2-H), 2.62 (m, 1H, 3-H), 2.55 (m, 1=2 H,
2-H).
16. Cell growth inhibition was assayed using the sulforhoda-
mine B (SRB) protocol developed by Rubinstein et al.
(Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.;
Simon, R. M.; Tosini, S.; Skehan, P.; Scudiero, D. A.;
Monks, M. R. J. Natl. Cancer Inst. 1990, 82, 1113). Drug
exposure was for 3 days, and the ED50 value was inter-
polated from dose–response data.
17. The plasmid DNA relaxation assay was carried out
according to the procedure described previously (Krish-
nan, P.; Bastow, K. F. Anti-Cancer Drug Des. 2000, 15,
255). Assays were performed with drug concentrations of
50 mM.
1
1125 cmꢁ1; MS m/e: 487 [M+Na]+; H NMR (CDCl3) d
7.03 (s, 1H, 5-H), 6.36 (s, 1H, 8-H), 6.17 (s, 2H, 20, 60-H),
5.93 (s, 2H, OCH2O), 4.80 (dd, 1H, J=10.5, 12.0 Hz, 13-
H), 4.50–4.29 (m, 3H, 4-H, 11-H2), 4.09–3.98 (m, 1H, 1-
H), 3.83 (s, 3H, 40-OCH3), 3.78 (s, 6H, 30,50-OCH3), 3.55
(dd, 1.5H, J=10.3, 12.5 Hz, 13-H), 2.50 (m, 1H, 2-H),
2.40–2.24 (m, 1H, 3-H), 2.17 (d, 1H, J=8.1 Hz, OH).
Anal. calcd C22H24O9S 1=2 H2O. Compound 11b: white
solid, mp 122–123 ꢀC (dec.), [a]2D8 ꢁ215.7 (c 0.055,
CH2Cl2); IR (film) 3500 (OH), 2938, 1591, 1507, 1203,
1
1125 cmꢁ1; MS m/e: 487 [M+Na]+; H NMR (CDCl3) d