Identification of novel and improved antimitotic agents derived from noscapine

Article abstract of DOI:10.1021/jm050674q

Analogues of the natural product noscapine were synthesized and their potential as antitumor agents evaluated. The discovery of a novel regioselective O-demethylation facilitated the synthesis of the potent aniline 6, which arrests mammalian cells in the

Full text of DOI:10.1021/jm050674q

7096
J. Med. Chem. 2005, 48, 7096-7098
Identification of Novel and Improved
Antimitotic Agents Derived from
Noscapine
James T. Anderson,* Anthony E. Ting,*
Sherry Boozer, Kurt R. Brunden, Chris Crumrine,
Joel Danzig, Tom Dent, Laurel Faga,
John J. Harrington, William F. Hodnick,
Steven M. Murphy, Gary Pawlowski, Robert Perry,
Amy Raber, Stephen E. Rundlett,
Alain Stricker-Krongrad, Jianmin Wang, and
Youssef L. Bennani
Athersys, Inc., 3201 Carnegie Avenue,
Cleveland, Ohio 44115-2634
Received July 15, 2005
Figure 1. FACS cell cycle analysis of HEK293 treated for 16
h with (A) 0.5% DMSO or (B) 1 µM 2.
Abstract: Analogues of the natural product noscapine were
synthesized and their potential as antitumor agents evaluated.
The discovery of a novel regioselective O-demethylation fa-
cilitated the synthesis of the potent aniline 6, which arrests
mammalian cells in the G2/M phase of the cell cycle at 0.1
µM and also affects tubulin polymerization. Aniline 6 is orally
bioavailable and is 250-fold more potent than noscapine in
reducing cell proliferation in rapidly dividing cells.
Antitumor agents that affect microtubule dynamics
are of great medical interest and are now commonly
used in current chemotherapy regimens.1,2 However,
several problems associated with existing cancer thera-
peutics of this type remain, such as (1) myelosuppres-
sion, (2) neuropathy, (3) alopecia,3 (4) increased drug
resistance in tumors,4 (5) poor bioavailability that
results in a need for intravenous delivery, and (6) poor
solubility, necessitating the use of agents (i.e., Cremo-
phor) that can possess undesirable qualities.5 Thus,
there is still a need for effective microtubule-directed
drugs with improved solubility and therapeutic index.
Moreover, it would be desirable to have an orally active
drug of this type that might facilitate patient dosing.
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 favorable toxicity profile.6 Additionally, it has
been known for some time that noscapine can act as a
weak anticancer agent in certain in vivo models.7
Recently, Joshi et al. have performed several studies to
evaluate the mechanism of action of this anticancer
effect and found that noscapine can disrupt tubulin
dynamics.8 Although noscapine appears to be a weak
inhibitor of microtubule polymerization, its low cost and
ready availability allow for further exploratory medici-
nal chemistry of this natural product. Accordingly, our
goal was to identify more potent and orally active
analogues of noscapine as potential anticancer agents.
In an earlier communication, we reported an easily
scalable synthesis of phenol 2 as a single diastereomer.9
We examined 2 in a cell cycle assay that measures DNA
content, and it showed nearly complete G2/M arrest of
HEK293 at 1 µM (Figure 1 and Table 1). In addition, 2
Figure 2. HeLa cells were incubated for 30 min with (A) 0.5%
DMSO or (B) 10 µM 2 and then fixed and stained for the
presence of microtubules.
Table 1. Cell Cycle Results for HEK293 Cells
drug
DMSO
R
R′ concn (µM)a % G1 % S % G2/M
0.5%
10
50
1
48
3
39
14
31
9
13
83
50
88
9
63
92
81
67
90
90
colchicine
1, noscapine OMe
H
H
H
H
H
Br
H
19
3
2
OH
OTf
NHBn
NH2
NH2
NHMe
SH
3
50
5
56
8
35
29
7
5
6
0.1
1
0.5
5
1
7
4
15
25
7
8
8
9
3
10
H
1
4
6
a Concentrations 2- to 10-fold lower did not show a difference
in the FACS profile when compared to DMSO control.
was observed to disrupt the microtubule cytoskeleton
when HeLa cells were stained for microtubules using a
fluorescent-labeled anti-tubulin antibody (Figure 2B).
A direct effect on microtubule dynamics was confirmed
in an in vitro tubulin polymerization assay, where 2
* 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/jm050674q CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/13/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7097
Scheme 2. Triflate Displacement and Reductiona
Table 2. Tubulin Polymerization and A549 Cell Growth
Assays
tubulin
polymerization
assay, EC50 (µM)
colony-forming
MTS assay,
EC50 (µM)
assay,
drug
EC50 (µM))
vinblastine
0.2
>50
0.7
0.3
0.0009
25
0.0005
noscapine
not determined
2
6
0.6
0.5
0.084
0.097
a Reaction conditions: (a) NaSH, NMP, 80 °C (19%); (b)
Pd(OAc)2, 1,3-bis(diphenylphosphino)propane, MeOH, Et3N, DMSO,
80 °C (58%).
Scheme 1. Palladium-Catalyzed Aminationa
butyl carbamate18 were investigated, but only HMDS
gave any desired amination product albeit at very low
conversion (∼5%). An optimal catalyst-ligand system
was found in the conveniently prepared palladacycle 4.19
The amination was highly efficient and proceeded to
completion within 15-30 min at 110 °C. More impor-
tantly, no epimerization was detected using the opti-
mized conditions, and the reaction could be easily
performed on a multigram scale (up to 20 g of starting
3).
Aniline 6 was observed to inhibit tubulin polymeri-
zation in vitro (Table 2). Further examination of 6 in
a Reaction conditions: (a) MeMgBr, BnOH, toluene-THF, 120
°C, 60%; (b) Tf2O, pyridine, CH2Cl2, 0 °C, 93%; (c) BnNH2 (or
aqueous MeNH2), 4 (4 mol %), Ba(OH)2‚8H2O, R,R,R-trifluorotolu-
ene-H2O (2:1), 110 °C, 80% for 5 (51% for 8); (d) 10% Pd/C, H2,
MeOH, 43%; (e) Br2, H2O, 48% HBr, 50%.
the MTS (EC50 ) 97 nM) and colony-forming (EC50
)
84 nM) assays indicated that it was more active than 2
and at least 2 orders of magnitude more potent than
noscapine (Table 2). Like the phenol 2, aniline 6 was
specific for dividing Swiss 3T3 cells (EC50 ≈ 50 nM) with
no effects on nondividing Swiss 3T3 cells at 3 µM.
Interestingly, stereochemistry was much more im-
portant for activity for noscapine-derived G2/M inhibi-
tors than for S-phase inhibitors.9 The phthalide dias-
tereomer of 6 was isolated and showed significantly less
activity in the cell-cycle assay (partial G2/M arrest at
25 µM).
Compound 6 was easily brominated using a known
protocol20 to give analogue 7, which was less potent than
the parent compound (Table 1). The 7-NHMe analogue
of noscapine, 8, was synthesized by the amination of 3
using aqueous MeNH2 with catalyst 4 and Ba(OH)2‚
8H2O.
Compound 8 showed G2/M arrest activity at 0.5 µM.
Interestingly, the facile displacement of triflate 3 with
sodium hydrosulfide gave the thiophenol analogue 9
(with ∼10% epimerization of the phthalide ring), which
also showed G2/M arrest at 5 µM (Scheme 2). The source
of activity was postulated to be from an H-donor group
at the 7-position. However, this hypothesis was refuted
when 10 displayed good G2/M arrest at 1 µM (Scheme
2, Table 1).21
As noted, it would be desirable to have orally active
drugs that affect cancer cell growth through an alter-
ation of microtubule dynamics. The in vivo pharmaco-
kinetics of aniline 6 and phenol 2 were investigated in
mice and compared with noscapine (Table 3). Noscapine
and 6 showed similar exposure, volume of distribution
(Vd), half-life, and clearance. Compound 2 appeared to
have an increased half-life and volume of distribution
relative to the other two compounds. All three com-
pounds showed readily measurable oral bioavailability
ranging from 11% to 22%, although 2 appeared to have
somewhat better oral absorption. It should be noted that
the bioavailability of noscapine in humans has been
had an EC50 of 0.7 µM. These pronounced effects on
mammalian cells suggested that phenol 2 would also
inhibit cell growth, and proliferation of the lung epi-
thelial cell line A549 was assessed using two different
assays. In an MTS assay,10 2 had an EC50 of 0.6 µM,
while it showed an EC50 of 0.5 µM in a colony forming
assay (Table 2). These effects were specific for dividing
cells, since an MTS assay using 2 in dividing Swiss 3T3
cells gave an EC50 of 3 µM while nondividing Swiss 3T3
cells were not affected at 50 µM (data not shown).
The readily accessible phenol 2 provided new op-
portunities for functionalization via conversion to its
triflate 3, which itself was not active at 50 µM in the
cell cycle assay. We first explored palladium-catalyzed
amination chemistry11 on triflate 3 to give nitrogen
analogues of noscapine at the 7-position (Scheme 1).
Initially, amination using benzylamine proceeded with
epimerization of the phthalide stereocenter (∼15-25%),
most likely due to the strong base NaOtBu.12,13 How-
ever, the diastereomers could be separated on small
scale using reversed-phase HPLC. The pure (natural)
stereoisomer N-benzyl derivative 5 showed moderate
G2/M arrest activity (63% at 5 µM, Table 1), differing
from the O-benzyl analogue that showed S-phase ar-
rest.9 The activity was dramatically improved when the
benzyl group was removed to give aniline 6, which
arrested HEK293EBNA cells in the G2/M phase at 0.1
µM (Table 1).
The problem of racemization in the triflate amination
was solved after the discovery of improved reaction
conditions. Replacing NaOtBu with barium hydroxide
and using a two-phase system consisting of either R,R,R-
trifluorotoluene-H2O14 or toluene-H2O proved to be
best when using benzylamine. Other bases were ex-
plored such as LiOtBu, K2CO3, Cs2CO3, and aqueous
KOH (biphasic),15 but aminations using benzylamine
were very sluggish. Other ammonia equivalents such
as benzophenone imine,16 LiHMDS,17 HMDS, and tert-

7098 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Table 3. In Vivo Pharmacokinetics in Micea
Letters
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AUC
Vd
T1/2
â
CL
F
drug
route (mg‚h/L) (L/kg) (min) ((mL/min)/kg) (%)
noscapine iv
noscapine po
0.345
0.524
0.331
0.740
0.350
0.391
15.53
28.12 167.7
8.47 62.6
50.0
96.8
100.6
95.3
14.1
22.0
11.4
2
2
6
6
iv
po
iv
po
a Dosing: 2 mg/kg (iv); 20 mg/kg (po).
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J.; Dent, T.; Harrington, J. J.; Murphy, S. M.; Perry, R.; Raber,
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measured to be ∼30%.6 Thus, it is possible that 2 and 6
may show greater oral absorption in humans than in
mice.
In conclusion, we have synthesized several potent
derivatives of noscapine. In particular, phenol 2 and
aniline 6 showed significant improvement in microtu-
bule inhibition and cytotoxicity relative to noscapine.
Like noscapine, both 2 and 6 appear to be orally
absorbed in mice. Importantly, the more potent 6 can
be synthesized in only four steps from noscapine, which
is abundant and inexpensive. The potent activity of
these natural product derivatives and their ready
synthesis lend hope that one or more novel anticancer
agents may emerge from these efforts. Toward this goal,
select noscapine derivatives described here will be
examined in established cancer models.
Acknowledgment. This manuscript is dedicated to
Dr. Stephen Hanessian on the occasion of his 70th
birthday.
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Supporting Information Available: Experimental pro-
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via the Internet at http://pubs.acs.org.
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JM050674Q