Discovery of S-phase arresting agents derived from noscapine

Article abstract of DOI:10.1021/jm0494220

Analogues of the natural product noscapine were synthesized, and their potential as antitumor agents were examined. The discovery of a novel regio- and stereoselective O-demethylation led to the synthesis of several O-alkylated analogues that induced an u

Full text of DOI:10.1021/jm0494220

2756
J. Med. Chem. 2005, 48, 2756-2758
Letters
Discovery of S-Phase Arresting Agents
Derived from Noscapine
James T. Anderson,* Anthony E. Ting,*
Sherry Boozer, Kurt R. Brunden, Joel Danzig,
Tom Dent, John J. Harrington, Steven M. Murphy,
Rob Perry, Amy Raber, Stephen E. Rundlett,
Jianmin Wang, Nancy Wang, and Youssef L. Bennani
Athersys, Inc., 3201 Carnegie Avenue,
Cleveland, Ohio 44115-2634
Received July 20, 2004
Abstract: Analogues of the natural product noscapine were
synthesized, and their potential as antitumor agents were
examined. The discovery of a novel regio- and stereoselective
O-demethylation led to the synthesis of several O-alkylated
analogues that induced an unexpected S-phase arrest of
mammalian cells. Compound 4a was the most potent analogue
inhibiting cell proliferation at an EC50 of 1.9 µM.
The eukaryotic cell cycle is a series of tightly regu-
lated events that result in the transmission of genetic
material from one generation to the next. The fidelity
of these processes is monitored by cell cycle checkpoints
that induce cell cycle arrest when damage is detected.
If the damage cannot be repaired, these checkpoints
may also cause cells to undergo apoptosis. Many che-
motherapeutic agents take advantage of these check-
point controls to preferentially kill rapidly dividing
cancer cells. However, there are several problems com-
mon to existing cancer chemotherapies that mainly
result from a narrow therapeutic index leading to
undesired side effects. Increased drug resistance in
tumors is another problem.1 Thus, there is still a need
for effective drugs with an improved safety profile.
Noscapine 1 is a naturally occurring phthalideiso-
quinoline alkaloid obtained from opium. It has been
used orally in humans as an antitussive agent and
displays a low toxicity profile.2 Additionally, it has been
known for over 60 years that noscapine acts as a weak
anticancer agent.3 Recently, Joshi et al. have reported
that noscapine causes cells to arrest in the G2/M phase
of the cell cycle and can cause tumor regression in
animal models when administered at 120 mg/kg (ip)
daily for 3 weeks.4 Here, we report on efforts to discover
potent noscapine derivatives that may be suitable for
development as anticancer agents.
Figure 1. FACS analysis of HEK293 cells treated for 16 h
with (A) 0.5% DMSO, (B) 50 µM noscapine, and (C) 50 µM 2.
Scheme 1. Benzyloxide Addition
capine was treated with sodium benzyloxide in benzyl
alcohol and DMF and heated to 120 °C for 16 h to give
a 1:1 mixture of diastereomers 2. This substitution
typically proceeded in low yield with epimerization of
the phthalide stereocenter and was limited to electron-
rich alkoxides. The mixture of diastereomers 2 showed
an unexpected S-phase arrest on HEK293 cells that
were analyzed for DNA content by flow cytometry
(Figure 1 and Table 1). It was later revealed after HPLC
purification that each of the diastereomers showed
effects and potency similar to those of the mixture 2 in
the cell cycle assay at 50 µM. Moreover, the mixture 2
was found to inhibit the growth of HEK293 cells with
an EC50 of 15 µM. To determine whether the S-phase
arrest observed in HEK293 cells treated with mixture
2 was due to the induction of apoptosis, the cells were
examined for the presence of apoptotic nuclei following
treatment with mixture 2 or buffer control. In contrast
to HEK293 cells treated with the apoptotic agent
etoposide (p < 0.05), no apoptosis was observed in cells
incubated with mixture 2 (p > 0.5). This was consistent
Because of limited literature outlining procedures for
the chemical modification of noscapine, we initially
chose to probe the structure-activity relationship (SAR)
at the 7-position of noscapine using the chemistry of
Schmidhammer and Klotzer (Scheme 1).5 Thus, nos-
* To whom correspondence should be addressed. For J.T.A.: phone,
216-431-9900; fax, 216-361-9596; e-mail, janderson@athersys.com. For
A.E.T.: phone, 216-431-9900; fax, 216-361-9596; e-mail, ating@
athersys.com.
10.1021/jm0494220 CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/30/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2757
Scheme 3. Phenol Alkylation
Table 1. Effects on Cell Cycle Distribution
high-boiling properties of benzyl alcohol may be advan-
tageous, although other high-boiling alcohols were not
investigated. The use of benzyl alcohol did not detract
from the synthesis because it could be easily removed
by acid-base extraction.
We were gratified that under these novel O-demeth-
ylation conditions little to no epimerization occurred.8
This allowed for the alkylation of phenol 3 using alkyl
halides to give products as pure stereoisomers (Scheme
3). Although the yields of alkylated products were
generally low (<30%), this new derivatization route
significantly widened the scope of the SAR at the
7-position. Minimal effort was made to improve the
alkylation of 3 because novel derivatives could be
accessed in two steps as single diastereomers. In one
example, the 3,4,5-trimethoxybenzyl derivative 4a
showed S-phase arrest activity at 1 µM by fluorescence-
activated cell sorting (FACS) analysis and inhibition of
HEK293 proliferation with an EC50 of 1.9 µM. Other
benzyl isosteres such as the O-3-thiophene 4b and
O-thiazole derivative 4c (Scheme 3) showed S-phase
activity at higher concentrations (25 and 10 µM, respec-
tively; see Table 1).
It was generally observed that O-benzyl-substituted
derivatives 4 (and isosteres) were S-phase arresters.
Minor changes in the O-aryl group resulted in dramatic
changes in activity, thus complicating the SAR. For
example, replacing the 3,4,5-trimethoxy group in 4a
with a 3,4-dimethoxy (4d), 3,4-methylenedioxy-5-meth-
oxy (4e), or a 3,4,5-triethoxy group (4f) (Scheme 3) did
not show a significant difference in the FACS profile at
50 µM when compared to DMSO control. Other ex-
amples of this sensitive structure-activity behavior
were observed with the O-2-thiophene (4g) and the O-3-
benzothiophene (4h) derivatives (compare both to 4b),
which also were morphologically similar to DMSO
control at 50 µM.
a 1:1 mixture of diastereomers. b Concentrations 2- to 10-fold
lower did not show a difference in the FACS profile when compared
to DMSO control.
Scheme 2. 7-O-Demethylation of Noscapine
with the fact that cells treated with mixture 2 displayed
a similar morphology to DMSO-treated cells. The ob-
servation of an S-phase arrest was an unexpected result
because noscapine has been reported to arrest cells in
the G2/M-phase of the cell cycle. The fact that a change
in size of the 7-alkoxy group gave such a dramatic result
warranted further SAR exploration.
We first examined the optimization of the substitution
reaction to address the problem of epimerization, low
yield, and general limitations of the SAR. A series of
reaction conditions focusing around the metal counter-
ion were investigated. Heating a DMF solution of 1 in
the presence of potassium benzyloxide (BnOH, KHMDS)
gave results similar to those from the original sodium
salt conditions, and the lithium analogue (BnOH, nBuLi)
resulted in no reaction after 3 h at 110 °C. However,
the use of MeMgBr and BnOH in toluene-THF at 110
°C for 3-4-h did not result in benzyloxide substitution
but instead resulted in the regioselective O-demethyla-
tion at the 7-position to give phenol 3 (Scheme 2).5,6 The
regio- and stereoselective nature of the reaction was
proven by X-ray crystallography.7 It is reasonable to
hypothesize that the source of regioselectivity is due to
the conjugative stabilization of the resultant phenoxide
by the carbonyl group. However, it is evident that
magnesium plays an important role in the outcome of
the reaction. Ethanol and 2-propanol were investigated
as replacements for benzyl alcohol in the demethylation;
however, no reaction was observed at refluxing temper-
atures over a 24 h period. Thus, the nucleophilic and
Phenol 3 was converted into its corresponding triflate
5, which did not show a significant difference in the
FACS profile at 50 µM when compared to DMSO
control. Direct displacement of the triflate 5 using
benzyl mercaptan gave the S-benzyl derivative 6 (Scheme
4), which also showed S-phase arrest at 10 µM (Table
1) but little activity at 1 µM (data not shown). Additional
noscapine derivatives were synthesized using triflate 5
via Suzuki couplings that yielded biaryl compounds 7
(Scheme 4). Unfortunately, none of the biaryl com-
pounds showed significant S-phase arrest in HEK293
cells at 50 µM.

2758 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Letters
Supporting Information Available: X-ray structure of
3, experimental procedures, and characterization data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Scheme 4. Thiobenzyl and Biaryl Noscapine
Analoguesa
References
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a Reaction conditions: (a) BnSH, K2CO3, DMF, 110 °C, 10%;
(b) Pd(PPh3)4, ArB(OH)2, 2 M aqueous Na2CO3, LiCl, toluene,
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antitumor agent that arrests metaphase and induces apoptosis
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(6) Compound 3 was shown to arrest cells in the G2/M phase
(unpublished results).
(7) See Supporting Information for the crystal structure of 3.
(8) Some epimerization (∼10-15%) was seen at longer reaction
times and higher temperatures.
We investigated the possibility that the novel nos-
capine derivatives cause S-phase arrest through a
disruption of microtubule structures because noscapine
affects tubulin dynamics.4d While the majority of studies
indicate the essential role of tubulin in mitosis, there
is a report of an S-phase arrester that binds tubulin.9
Additionally, it has been reported that a 3,4,5-tri-
methoxy-substituted aryl ring is important for tubulin
binding.10 However, in contrast to noscapine, 4a had no
effect on chromosome alignment during mitosis (data
not shown). We were unable to elucidate the mechanism
of action for noscapine-derived S-phase inhibitors. None-
theless, anticancer agents that arrest in S-phase are of
potential utility. Other S-phase arresters that show
promising anticancer activity are (1) 7-hydroxystauro-
sporine, which targets the cell cycle checkpoint Chk1,11
(2) resveratrol, which works through a yet undiscovered
mechanism,12 and (3) â-lapachone, which acts on topo-
isomerase I and is currently in phase 1 clinical trials.13
Camptothecin analogues, which target topoisomerase I,
are the most well-known S-phase arresters of cells and
are used clinically.14
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interaction of chalcones with tubulin. Anti-Cancer Drug Des.
2000, 15, 135-141.
In conclusion, we have synthesized novel analogues
of noscapine as pure diastereomers, facilitated by a
selective 7-O-demethylation of the natural product. In
contrast to noscapine, these new analogues showed
S-phase arrest in the cell cycle with the most potent
compound being the 3,4,5-trimethoxybenzyl analogue
4a. Cell cycle potency was highly dependent on the
structure of the 7-O-benzyl group. The mechanism of
how these compounds inhibit cell proliferation is cur-
rently unknown and awaits further biological testing.
(11) Shi, Z.; Azuma, A.; Sampath, D.; Li, Y.-X.; Huang, P.; Plunkett,
W. S-phase arrest by nucleoside analogues and abrogation of
survival without cell cycle progression by 7-hydroxystaurospo-
rine. Cancer Res. 2001, 61, 1065-1072.
(12) Joe, A. K.; Liu, H.; Suzui, M.; Vural, M. E.; Xiao, D.; Weinstein,
I. B. Resveratrol induces growth inhibition, S-phase arrest,
apoptosis, and changes in biomarker expression in several
human cancer cell lines. Clin. Cancer Res. 2002, 8, 893-903.
(13) Li, Y.; Sun, X.; LaMont, J. T.; Pardee, A. B.; Li, C. J. Selective
killing of cancer cells by â-lapachone: Direct checkpoint activa-
tion as a strategy against cancer. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 2674-2678.
(14) Holden, J. A. DNA topoisomerases as anticancer drug targets:
From the laboratory to the clinic. Curr. Med. Chem.: Anti-Cancer
Agents 2001, 1, 1-25.
Acknowledgment. The authors thank Judith Gal-
lucci of The Ohio State University for providing the
X-ray crystal structure of 3.
JM0494220