Synthesis and biological evaluation of 2-Indolyloxazolines as a new class of tubulin polymerization inhibitors. Discovery of A-289099 as an orally active antitumor agent

Article abstract of DOI:10.1016/S0960-894X(01)00759-4

A series of indole containing oxazolines has been discovered as a result of structural modifications of the lead compound A-105972. The compounds exert their anticancer activity through inhibition of tubulin polymerization by binding at the colchicine site. A-289099 was identified as an orally active antimitotic agent active against various cancer cell lines including those that express the MDR phenotype. The anticancer activity, pharmacokinetics, and an efficient and enantioselective synthesis of A-289099 are described.

Full text of DOI:10.1016/S0960-894X(01)00759-4

Bioorganic & Medicinal Chemistry Letters 12 (2002) 465–469
Synthesis and Biological Evaluation of 2-Indolyloxazolines as a
New Class of Tubulin Polymerization Inhibitors. Discovery of
A-289099 as an Orally Active Antitumor Agent
Qun Li,* Keith W. Woods, Akiyo Claiborne, Stephen L. Gwaltney, II, Kenneth J. Barr,
GangLiu, Laura Gehrke, R. Bruce Credo, Yu Hua Hui, JangLee, Robert B. Warner,
Peter Kovar, Michael A. Nukkala, Nicolette A. Zielinski, Stephen K. Tahir,
Michael Fitzgerald, Ki H. Kim, Kennan Marsh, David Frost,
Shi-ChungNg, Saul Rosenbergand HingL. Sham
Cancer Research, Abbott Laboratories, Abbott Park, IL 60064-6101, USA
Received 12 October 2001; accepted 16 November 2001
Abstract—A series of indole containingoxazolines has been discovered as a result of structural modifications of the lead compound
A-105972. The compounds exert their anticancer activity through inhibition of tubulin polymerization by binding at the colchicine
site. A-289099 was identified as an orally active antimitotic agent active against various cancer cell lines including those that express
the MDR phenotype. The anticancer activity, pharmacokinetics, and an efficient and enantioselective synthesis of A-289099 are
described. # 2002 Elsevier Science Ltd. All rights reserved.
Tubulin binding agents have generated considerable
interest amongcytotoxic agents, due in part to the suc-
cess of the taxanes in clinical oncology.1,2 However,
emerging resistance to antimitotic agents such as pacli-
taxel has limited their ultimate effectiveness.3 Renewed
interest has been generated by the hope that non-MDR
substrates that interact with tubulin at sites different
from those of vinca alkaloids and taxanes can be dis-
covered.4 Recently, compounds which bind to tubulin at
the colchicine bindingsite, as represented by com-
bretastatin A4, demonstrate promisingactivity against
various cancer cell lines, includingthose that express the
MDR phenotype.1,5 The relatively simple structures of
these compounds may provide the added advantage of
beingorally active.
We recently identified the oxadiazoline A-105972 as a
colchicine site binder with an IC50 of 3.4 mM against
tubulin polymerization.6 As a part of our continuing
efforts to identify more stable and orally active com-
pounds, we report here our investigation of indole-based
oxazolines, which led to the discovery of A-289099 (5b-S)
as a potent and orally active antimitotic agent.7
The racemic oxazolines (5) were synthesized by coupling
of the amino alcohol 17 with either the acids (2) in a
two-step transformation or the nitriles (3) in a one-step
reaction as shown in Scheme 1. The required acids (2)
or nitrile (3n) were prepared from known starting
materials8À10 as indicated in Scheme 2.
Synthesis of the pure enantiomers of 5b proved to be
more challenging. Literature methods for the synthesis
of optically active 5-aryl oxazolines are limited. We
eventually developed the enantioselective oxazoline
synthesis shown in Scheme 3. Thus, styrene 14 was
enantioselectively dihydroxylated accordingto the
procedure of Ramacciotti et al.11 usingAD-mix- a to
give S-diol 15. Monotosylation of 15 followed by
nucleophilic displacement of the tosylate with sodium
azide provided 16. Reduction of azide 16 under hydro-
*Correspondingauthor. Fax: +1-847-935-5165; e-mail: qun.li@
abbott.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00759-4

466
Q. Li et al. / Bioorg. Med. Chem. Lett. 12 (2002) 465–469
gen in the presence of catalytic amount of 10% Pd/C
provided the amino alcohol 17 (95%). Cyclization of the
amide of 17 (4) using Burgess reagent resulted in partial
racemization. Reaction of 17 with methyl indole-5-car-
boximidate failed to give the desired product. No cou-
plingproduct could be isolated by condensation of
azide 16 with indole-5-carboxaldehyde. Finally, 17 was
successfully condensed with cyanoindole 18 to yield
the optically pure oxazoline 5b-S in 68% yield and
>99% ee.12 The same reaction sequence was repeated
to give the R-isomer (5b-R)13 by replacingAD-mix- a
with AD-mix-b. The method described here should
be of wide applicability to other 5-substituted chiral
oxazolines.
Although N-methylation of the indole ringoften
decreases the observed potency, N-methyl analogue 5a
is 10-fold more active than desmethyl compound 5b.
However, N-substitution on the 5-indolyl group with
substituents larger than methyl resulted in a significant
loss of activity (Table 2). The N-cyclopropyl analogue
(5n), which is less metabolically labile, shows improved
oral bioavailability (56%).
All attempts to replace the indolyl group of 5b failed
(Table 3), with the closest one beingthe electron rich
indolizine (5r), which is only 3-fold less active than the
similar indole analogue 5p.
The two enantiomers of the overall best compound
5b were evaluated for their antiproliferative activity
and pharmacokinetic properties. The S-isomer (5b-S,
A-289099) was found to be significantly more potent
than the R-isomer (5b-R) (Table 4). The IC50 values of
A-289099 against HCT-15 and NCI-H460 are 8.6 and
6.2 nM, respectively, which are comparable to com-
bretastatin A4 and up to 74-fold more potent than the
R-isomer. A-289099 is also active against a human lung
cancer with mutated b-tubulin (A549-T2415) with an
IC50 of 6.3 nM. As a comparison, paclitaxel exhibits an
IC50 of >10 mM against the same cell line. A-289099
inhibits tubulin polymerization6a with an IC50 of 2.3
mM, versus 1.6 mM for combretastatin A4. The binding
constant6a of A-289099 at the colchicine site is compar-
able to colchicine at 0.63 mM but is 3-fold higher than
that of combretastatin A4.
Compounds were evaluated for their antiproliferative
activity against the human lung carcinoma cell line
NCI-H460 and the MDR positive human colon adeno-
carcinoma cell line HCT-15.6a The most promising
analogues were then screened for their oral bioavail-
ability in rat.14 The 5-indolyl analogue (5b) was the
most potent amongthe six regio isomers (Tables 1 and
2). It also possesses good oral bioavailability of 35%.
The ability of A-289099 to depolymerize microtubules
in cultured cells6a is shown in Figure 1. Microtubule
depolymerization in human HCT-15 colon carcinoma
cells was observed 4 h after treatment with A-289099 at
a concentration of 44 nM.
Scheme 1. (a) EDCI, THF, rt, overnight; (b) Burgess reagent, THF,
heat, 2 h; (c) K2CO3, ethylene glycol/glycerol (2:1), 140 ꢀC, 40 h.
Molecular modeling suggests that the structure of A-
289099 is similar to that of combretastatin A4. The tri-
Scheme 2. (a) Cyclopropylamine, MeOH, 60 ꢀC, 20 h; (b) ethyl
ethynyl ether, catecholborane, hexanes, rt, 2 h, reflux, 2 h; then 7,
Pd(dppf)2Cl2, CsF, THF, reflux, 42 h, H3O+; (c) bromoacetaldehyde
acetal, HCl, water, 90 ꢀC, 1 h, then 8, NaHCO3, 60 ꢀC, 1 h; (d) LiOH,
THF/H2O (3:1), rt, 30 min; (e) POCl3, CH2Cl2, reflux, 3 h; (f) (i)
bromoacetone, acetone, reflux, overnight; (ii) Na2CO3, ethanol, reflux,
3 h.
Scheme 3. (a) AD-mix-a, t-BuOH/H2O (1:1), then 14, 0 ꢀC, 3 h, then
rt, overnight; (b) TsCl, Py, 0 ꢀC, 2 h; (c) NaN3, DMF, 85 ꢀC, 2 h; (d)
H2, 10% Pd/C, rt, overnight; (e) 18, K2CO3, ethylene glycol/glycerol
(2:1), 140 ꢀC, 40 h.

Q. Li et al. / Bioorg. Med. Chem. Lett. 12 (2002) 465–469
467
methoxylphenyl group is a common pharmacophore.
The 2,5-disubstituted oxazoline is a good mimic of the
cis-double bond. Figure 2 shows the superimposed
structures of A-289099 and combretastatin A4.
6.5 to 44.0%. In mouse and rat, 5b-R displays better
oral bioavailabilities than A-289099, but its oral AUCs
are lower, especially in mouse, where a 10-fold reduc-
tion is observed. The bioavailability of A-289099 in
monkey is 18.6%.
The pharmacokinetic properties of A-289099 and 5b-R
in four different species are summarized in Table 5.
Overall, the two enantiomers have similar pharmacoki-
netic properties with oral bioavailabilities ranging from
The anticancer activity6b of A-289099 was evaluated in
the syngeneic M5076 murine ovarian sarcoma flank
Table 3. IC50 (nM) of the bicyclic aryloxazolines (5)a
6a
Table 1. IC50 (nM) and oral bioavailability14 of indolyloxazolines
6a
Compd
R
HCT-15b
NCI-H460c
14
(5)a
5b
16
Compd
R
X
HCT-15b
NCI-H460c
F (%)d
5a
5b
H
200
16
100
14
38.1
34.7
5p
5q
5r
5s
5t
170
510
210
810
Me
5c
5d
H
Me
120
280
330
420
10.5
0
520
520
5e
5f
H
Me
540
15,000
470
150,000
0
—
1300
1200
5g
5f
H
Me
61
37
57
53
0
0
6400
7100
5g
5h
H
Me
67
37
82
87
0
2.5
5u
5v
5w
5x
5y
5z
1200
>100,000
16,000
100,000
3200
5i
5j
H
Me
150
660
350
1500
40.0
—
40,000
28,000
2400
aThe compounds were tested as free bases of racemic mixtures.
bAn MDR positive human colon adenocarcinoma cell line.
cHuman non-small cell lungcarcinoma cell line.
dOral bioavailability in rat after a single 10 mg/kg dose.
Table 2. IC50 (nM) and oral bioavailability14 of N-substituted 5-
indolyloxazolines (5)a
6a
Compd
R
HCT-15b
NCI-H460c
F (%)d
160,000
44,000
270,000
37,000
5b
16
14
34.7
5k
5l
70
480
49
11.1
34.3
19.8
55.8
—
aThe compounds were tested as free bases of racemic mixtures.
b,csee Table 1.
680
Table 4. In vitro characterization of the two enantiomers of 5b6a
Compd
Kia
ITPb
HCT-15c
NCI-H460d
IC50 (nM)
5m
5n
3800
2900
16,300
4200
2900
8180
(mM) IC50 (mM) IC50 (nM)
5b-S (A-289099)
5b-R
Combretastatin A4
Colchicine
Vinblastine
0.63
3.69
0.18
0.78
>100
2.3
740
1.6
—
8.6
390
1.7
140
43
6.2
460
3.0
46
1.0
3.4
50
aBindingconstants at colchicine site of bovine brain tubulin.
bInhibition of bovine brain microtubule polymerization.
c,dsee Table 1, footnotes b and c.
a,b,c,dSee Table 1.

468
Q. Li et al. / Bioorg. Med. Chem. Lett. 12 (2002) 465–469
A-289099 exhibits reasonably good physicochemical
properties. Its solubilities in 0.1 M HCl and pH 7.4 phos-
phate buffer are 1.27 mg/mL and 0.7 mg/mL, respec-
tively. A-289099 has a logD of 3.94 and p Ka of 5.0.
In conclusion, a series of indole containingoxazolines
has been discovered as a result of structural modifica-
tions of the lead compound A-105972. The compounds
exert their anticancer activity through inhibition of
tubulin polymerization by bindingat the colchicine site.
A-289099 was identified as an orally active antimitotic
agent active against various cancer cell lines, including
those that express the MDR phenotype.
Figure 1. Effect of A-289099 on microtubule depolymerization in
HCT-15 cells. Left: control cells. Right: cells treated with 44 nM
A-289099 for 4 h. Green: microtubules, Blue: nuclear DNA.
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13. A-289099 (5b-S) has [a]2D5 of +195.7ꢀ (c=1.04, CH2Cl2)
(99% ee) and 5b-R has [a]2D5 of À184.5ꢀ (c=1.04, CH2Cl2)
(98% ee). The ee was determined by HPLC on a Chirapack
AS column elutingwith 15% ethanol in hexane.
Figure 2. Stereoview of the superimposed structures of A-289099
(green) and Combretastatin A4 (yellow). Their geometries were opti-
mized usingDISCOVER force field (CFF98).
Table 5. Pharmacokinetic properties of the two enantiomers of 5b
Species
Mousea
PK
5b-S
(A-289099)
5b-R
t1/2 (h)
AUC (mgh/mL)
F (%)
1.1
5.69
15.1
1.3
0.40
21.0
Ratb
t1/2 (h)
AUC (mgh/mL)
F (%)
2.1
7.18
17.1
1.3
3.04
44.0
Dogb
t1/2 (h)
AUC (mgh/mL)
F (%)
3.3
0.16
6.5
2.9
0.33
7.0
Monkeyb
t1/2 (h)
AUC (mgh/mL)
F (%)
1.3
1.75
18.6
1.4
0.89
16.2
aA single 10 mg/kg dose.
bA single 5 mg/kg dose.
Table 6. In vivo efficacy6b of A-289099 in comparison with E7010
against M5076 murine ovarian sarcoma in mice (PO, QD, d1-28)
Compd
Dose
(mg/kg/day)
Mean tumor delay
to 1 g(day)
ILSa (%)
A-289099
E7010
50
100b
50
150b
19.0
28.4
6.4
139
206
47
27.4
201
aIncrease in life span.
bMaximum tolerated dose.
tumor model. When dosed QD, d1-28, at the maximum
tolerated dose, A-289099 achieved 206% increase in life
span and an impressive 28 day delay to 1 gtumor
volume in mice with M5076 murine ovarian sarcoma.
This efficacy is comparable to that of a previously
reported antimitotic agent, E701016 (Table 6).

Q. Li et al. / Bioorg. Med. Chem. Lett. 12 (2002) 465–469
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