New antimitotic agents with activity in multi-drug-resistant cell lines and in vivo efficacy in murine tumor models

Article abstract of DOI:10.1021/jm010231w

During a screen for compounds that could inhibit cell proliferation, a series of new tubulin-binding compounds was identified with the discovery of oxadiazoline 1 (A-105972). This compound showed good cytotoxic activity against non-multi-drug-resistant and multi-drug-resistant cancer cell lines, but its utility in vivo was limited by a short half-life. Medicinal chemistry efforts led to the discovery of indolyloxazoline 22g (A-259745), which maintained all of the in vitro activity seen with oxadiazoline 1, but also demonstrated a better pharmacokinetic profile, and dose-dependent in vivo activity. Over a 28 day study, indolyloxazoline 22g increased the life span of tumor-implanted mice by up to a factor of 3 upon oral dosing. This compound, and others of its structural class, may prove to be useful in the development of new chemotherapeutic agents to treat human cancers.

Full text of DOI:10.1021/jm010231w

4416
J . Med. Chem. 2001, 44, 4416-4430
New An tim itotic Agen ts w ith Activity in Mu lti-Dr u g-Resista n t Cell Lin es a n d in
Vivo Effica cy in Mu r in e Tu m or Mod els
Bruce G. Szczepankiewicz,* Gang Liu,* Hwan-Soo J ae, Andrew S. Tasker,† Indrani W.Gunawardana,
Thomas W. von Geldern, Stephen L. Gwaltney, II, J . Ruth Wu-Wong, Laura Gehrke, William J . Chiou,
R. Bruce Credo, J effery D. Alder, Michael A. Nukkala, Nicolette A. Zielinski, Ken J arvis, Karl W. Mollison,
David J . Frost, J oy L. Bauch, Yu Hua Hui, Akiyo K. Claiborne, Qun Li, and Saul H. Rosenberg
Pharmaceutical Products Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064
Received May 23, 2001
During a screen for compounds that could inhibit cell proliferation, a series of new tubulin-
binding compounds was identified with the discovery of oxadiazoline 1 (A-105972). This
compound showed good cytotoxic activity against non-multi-drug-resistant and multi-drug-
resistant cancer cell lines, but its utility in vivo was limited by a short half-life. Medicinal
chemistry efforts led to the discovery of indolyloxazoline 22g (A-259745), which maintained
all of the in vitro activity seen with oxadiazoline 1, but also demonstrated a better
pharmacokinetic profile, and dose-dependent in vivo activity. Over a 28 day study, indolylox-
azoline 22g increased the life span of tumor-implanted mice by up to a factor of 3 upon oral
dosing. This compound, and others of its structural class, may prove to be useful in the
development of new chemotherapeutic agents to treat human cancers.
Cancer is the second leading cause of death in
industrialized nations. Cancer chemotherapy commonly
involves the use of cytotoxic agents that destroy rapidly
dividing cells. Within the past decade, advances in our
understanding of the cell cycle have presented new
targets that may allow for the development of more
selective chemotherapeutic agentssagents that target
only cancer cells. Despite this progress, cytotoxic agents
will remain a mainstay in cancer chemotherapy for the
near future.
and cell death soon follows. Even when concentrations
of microtubule assembly inhibitors are too low to disrupt
the morphology of the mitotic spindle, these agents can
still freeze mitotic cells in metaphase. This can be
explained by the crucial role microtubules play in
maintaining normal cellular functions other than mi-
tosis.7
There are three major classes of antimitotic agents.
Microtubule-stabilizing agents, which bind to fully
formed microtubules and prevent the depolymerization
of tubulin subunits, are represented by paclitaxel and
the taxanes, the epothilones, and eleutherobin. The
other two classes function by binding to tubulin mono-
mers and inhibiting their polymerization into microtu-
bules. Vinca alkaloids such as vinblastine and vincris-
tine represent one of these classes. Colchicine and
colchincine-site binders define the third class of anti-
mitotic agents. The vinca alkaloids and the colchicine-
site binders occupy distinctly different sites when bound
to tubulin. Both the taxanes and vinca alkaloids are
widely used to treat human cancers, while colchicine-
site binders are being vigorously pursued as potential
new chemotherapeutic agents.1
Antimitotic agents constitute a major class of cytotoxic
drugs and have been the subject of several reviews.1-5
At the molecular level, these agents commonly interfere
with the dynamics of tubulin polymerization and depo-
lymerization. Tubulin is a heterodimer of two closely
related and tightly linked globular polypeptides called
R-tubulin and â-tubulin. Tubulin molecules polymerize
to form long stiff microtubules that extend throughout
the cytoplasm and govern the location of membrane-
bound organelles and other cell components. At the
onset of mitosis, cytoplasmic microtubules disassemble,
and the free tubulin molecules rearrange to form the
mitotic spindle, which bridges between the chromosomes
in the center and the centrosomes at opposite poles of
the cell. The spindle remains in dynamic equilibrium
with the pool of free tubulin, and therefore must
constantly add tubulin subunits to function properly.
During anaphase, controlled subtraction of tubulin
subunits leads to contraction of the mitotic spindle and
concurrent migration of the chromosomes to opposite
poles of the dividing cell. However, in the presence of
agents that interfere with tubulin polymerization, the
depolymerization reaction quickly dominates the dy-
namic equilibrium process, and the mitotic spindle
disappears.6 The dividing cell is arrested in metaphase,
During the course of a screen for agents that inhibit
cell proliferation, we discovered A-105972 (1) (Figure
1). Competition experiments with radiolabeled colchi-
cine confirmed that A-105972 binds to tubulin and at
least partially overlaps the same binding site as colchi-
cine.8 Additionally, A-105972 was found to retain the
same activity in multi-drug-resistant (MDR(+)) tumor
cell lines as it displayed in nonresistant (MDR(-)) cell
lines, which is a desirable quality for a chemotherapeu-
tic agent. Herein, we report our work to improve the
potency and in vivo efficacy shown by this new series
of colchicine-site binders.
* To whom correspondence should be addressed. Phone: (847) 935-
1559. Fax: (847) 938-1674. E-mail: bruce.g.szczepankiewicz@abbott.com.
† Current address: Amgen Inc., Thousand Oaks, CA 91320.
10.1021/jm010231w CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/13/2001

Oxadiazoline- and Oxazoline-Based Antimitotic Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4417
drazide 30. Condensation with 3,4,5-trimethoxybenzal-
dehyde (18) in ethanol solution presumably gave hy-
drazone 31, which rapidly ring-closed and then oxidized
in the presence of air to give thiadiazole 32. When the
condensation was carried out in acetic acid, the oxida-
tion reaction was not as rapid, and addition of acetic
anhydride afforded thiadiazoline 33. Oxazoline 34 was
prepared by acylation of hydrazide 35 with 3,4,5-
trimethoxybenzoyl chloride, followed by dehydration of
dibenzoylhydrazine 36 with POCl3 to effect ring closure
(Scheme 6). Dioxolane 37 was prepared starting from
styrene 389 (Scheme 7). Dihydroxylation of styrene 38
with OsO4 and NMO gave diol 39. Condensation of diol
39 with 4-(N,N-dimethylamino)benzaldehyde to close
the B-ring afforded the dioxolane.
F igu r e 1. Structure of A-105972 (1).
Ch em istr y
The tricyclic system was convergently assembled by
first setting the substitution patterns on the flanking
A- and C-rings and then closing the central B-ring.
Compounds containing an oxadiazoline B-ring were
prepared as follows (Scheme 1). The A-ring was derived
from a benzoyl hydrazide, 2, which could be prepared
in turn by coupling a benzoic acid, 3, with N-Boc-
hydrazine, followed by TFA-mediated removal of the Boc
group. The hydrazide 2 could be condensed with a
benzaldehyde, 4, bearing the substitution pattern de-
sired for the C-ring. The N-benzoyl hydrazone 5 was
then heated in acetic anhydride to effect closure of the
oxadiazoline ring and complete the assembly of tricyclic
system 6. Ring closure could also be accomplished with
acetic-formic anhydride to give an N-formyloxadiazo-
line, 7, with ethyl oxalyl chloride to give an N-(ethylox-
alamido)oxadiazoline, 8, with chloroacetic anhydride to
give an N-(chloroacetyl)oxadiazoline, 9, or with dimethyl
pyrocarbonate to give carbamate 10. Other derivatives
could be prepared from chloroacetate 9 by simple SN2
displacement with a variety of nucleophiles to give
compounds such as acetate 11, azide 12, or dimethy-
lamine 13 (Scheme 2). O-Acetate hydrolysis afforded
hydroxyacetate 14. Amido acetates were prepared by
reduction of the azide to amine 15 and formylation to
give formamide 16 or acylation to give acetamide 17.
The preparation of compounds containing an oxazo-
line B-ring started from 3,4,5-trimethoxybenzaldehyde
(18) (Scheme 3). Henry reaction with nitromethane gave
nitro alcohol 19, which was subsequently hydrogenated
to give the corresponding 2-hydroxy-2-phenethylamine
20. N-Acylation with a substituted benzoic acid gave a
2-hydroxy-2-phenethylamide, 21. Treatment of the hy-
droxy amide 21 with Burgess reagent or with thionyl
chloride then gave the desired oxazoline-based ana-
logues 22a -h .
In Vitr o Assa ys
Detailed protocols for the antiproliferation assays
have been reported elsewhere.8 Briefly, HCT-15 or NCI-
H460 cells in 48- or 96-well plates containing growth
medium were treated with compounds at different
concentrations and then incubated for 48 h. Cells were
trypsinized, and the number of cells in each well was
determined using a Coulter counter. Compounds with
ED50 values of 1 µM or less were retested in both assays.
Resu lts a n d Discu ssion
A-105972 (1) showed ED50 values of 0.029 µM against
the HCT-15 (MDR(+)) cell line and 0.049 µM against
the NCI-H460 (MDR(-)) cell line. In comparison, pa-
clitaxel showed an ED50 of 0.015 µM against the MDR-
(-) cell line, but its ED50 value was 0.54 µM against
the MDR(+) cell line. Although the potency of compound
1 was sufficient to display efficacy in rats (see Figure
2), the lead compound had a short in vivo half-life (see
Table 6). Medicinal chemistry efforts focused on modi-
fications that could potentially improve the in vivo
profile, while preserving or improving the potency of the
series.
Compound 1 can be described as being composed of
three subunits, the A-ring, the B-ring or oxadiazoline
core, and the C-ring (Figure 1). Each region was
examined to determine where changes were tolerated,
and what modifications would lead to potent compounds
with improved pharmacokinetic profiles. In all cases,
compound purity was determined either by combustion
analysis or by reversed-phase HPLC analysis.
Our first A-ring analogues were prepared from the
large pool of commercially available substituted benzoic
acids. After several substituents which lacked the
desired potency were explored, the p-(N,N-dimethy-
lamino)-substituted 6a emerged as the best of the series
(Table 1). This represented a molecule that retained all
of the potency of compound 1, but was much easier to
prepare. A positional survey showed that the optimal
placement for the dimethylamino group was in the para
position. When the dimethylamino group was moved to
the ortho position as in 6b, the result was a 1000-fold
loss of potency. The corresponding meta-substituted
compound 6c was slightly less potent than p-(N,N-
dimethylamino) 6a . More detailed studies on the mech-
anism of action for compound 1 and compound 6a ,
including cell cycle analysis, assays of microtubule
assembly, electron microscopy, and apoptosis studies,
have been reported elsewhere.8,10 These studies clearly
Other B-rings were prepared by a variety of methods.
Activation of 3-methyl-4-nitrobenzoic acid (23) with 1,1′-
carbonyldiimidazole followed by adding the lithium
anion of 3,4,5-trimethoxyacetophenone gave 1,3-dike-
tone 24 (Scheme 4). Condensation with hydrazine
afforded pyrazole 25, which could be N-acylated to afford
a mixture of amides 26 and 27. Hydrogenation of the
nitro group and separation of the regioisomers then gave
desired analogue 28. B-rings containing a sulfur atom
were prepared starting with N-[4-(N,N-dimethylamino)-
benzoyl]-N′-Boc-hydrazine (29) (Scheme 5). Installation
of the sulfur atom with Lawesson’s reagent followed by
Boc deprotection with trifluoroacetic acid gave thiohy-

4418 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Szczepankiewicz et al.
Sch em e 1a
a
Reagents and conditions: (a) H2NNH2Boc, EDCI, DMF; (b) TFA, CH2Cl2; (c) acetic acid, EtOH; (d) Ac2O (6), Ac2OHCO2H (7), ethyl
oxalyl chloride (8), chloroacetic anhydride (9), or dimethyl pyrocarbonate (10).
Sch em e 2a
a
Reagents and conditions: (a) by hydrolysis of acetate 11 with K2CO3 in aqueous MeOH; (b) by reduction of azide 12 with PPh3 in
THF; (c) by formylation of amine 15 with formic acid and EDAC in CH3CN; (d) by acylation of amine 15 with acetic anhydride.
Sch em e 3a
a
Reagents and conditions: (a) (i) CH3NO2, 10% aqueous NaOH, EtOH; (ii) AcOH (70%); (b) 60 psi of H2/Pd-C, AcOH (94%); (c) RACOCl,
(i-Pr)2EtN, CH2Cl2 or RACOOH, EDAC, i-PrEt2N, CH2Cl2; (d) Burgess reagent, CHCl3 or SOCl2, CHCl3.
demonstrated that both compounds inhibit microtubule
assembly and freeze the cell cycle in metaphase before
inducing apoptosis.
Structure-activity relationship (SAR) studies on the
C-ring demonstrated that the 3,4,5-trimethoxy substitu-
tion pattern was apparently optimal (Table 2). For
example, 2,4,5-trimethoxyphenyl 6d , 2,4,6-trimethox-
yphenyl 6e, and 2,3,4-trimethoxyphenyl 6f were at least
100-fold less potent than 3,4,5-trimethoxyphenyl 1.
Little change was tolerated when we replaced or re-
moved any of the methoxy groups. p-Amide 6g, 3,4,5-
trimethyl 6h , acetoxy compound 6i, and 3,4-dimethox-
yphenyl 6j all showed very weak activity, but 4-carboxylic
ester 6k retained some potency. Even methylenedioxy
substitution for two of the methoxy groups in 6l
produced an unacceptable loss of potency. This SAR
about the C-ring was reminiscent of that observed with
colchicine. The trimethoxyphenyl ring is crucial for
retaining potency in this and in other series of molecules
which occupy the colchicine binding site.11

Oxadiazoline- and Oxazoline-Based Antimitotic Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4419
Sch em e 4a
a
Reagents and conditions: (a) (i) CDI, THF; (ii) LDA, 3,4,5-trimethoxyacetophenone, THF; (b) H2NNH2·H2O, CH2Cl2/EtOH; (c) Ac2O;
(d) H2/Pd-C, EtOAc (100%); (e) separate regioisomers by flash chromatography.
Sch em e 5a
a
Reagents and conditions: (a) (i) Lawesson’s reagent; (ii) TFA, CH2Cl2; (b) 18, EtOH; (c) (i) 18, AcOH, under argon; (ii) Ac2O, 95 °C.
Sch em e 6a
a
Reagents and conditions: (a) 3,4,5-trimethoxybenzoyl chloride; (b) POCl3.
Sch em e 7a
a
Reagents and conditions: (a) OsO4, NMO; (b) 4-(N,N-dimethylamino)benzaldehyde, TsOH, toluene, 4 Å molecular sieves.
Replacement of the N-acetyl group present on the
B-ring in compound 6a gave several compounds that
retained activity (Table 3). N-Formyl 7, N-(hydroxy-
acetyl) 14, N-(formamidoacetyl) 16, and N-(acetoxy-
acetyl) 11 all showed approximately the same potency
as compound 1. N-(azidoacetyl) 12, N-(acetamidoacetyl)
17, and N-(aminoacetyl) 15 were about 5-10-fold less
active than compound 6a , but N-[(N,N-dimethylamino)-
acetyl] 13, N-(methoxycarbonyl) 10, and N-ethyloxala-
mide 8 lost all activity. On this portion of the molecule,

4420 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Szczepankiewicz et al.
F igu r e 2. In vivo efficacy of antimitotic agents in various murine models. Each bar represents a treatment group of 10 mice. In
each model, a lifespan increase of 25% or greater was considered biologically significant. For the P388 model, the tumor was
intraperitoneal, and compounds were dosed ip, qd for 5 days (22a , vincristine, paclitaxel), 7 days (compound 1), or 9 days (compound
6a ). For the B16 model, compound 1 was dosed against an intraperitoneal tumor, while other compounds were dosed against a
subcutaneous tumor. Also in the B16 model, compounds were dosed ip, qd for 5 days. For the M5076 model, paclitaxel and vincristine
were dosed against an intraperitoneal tumor ip, qd. Compounds 1, 22a , and 22g were dosed against a subcutaneous tumor according
to the following regimens: compound 1 was dosed ip, qd; compounds 22a and 22g were dosed po, qd.

Oxadiazoline- and Oxazoline-Based Antimitotic Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4421
Ta ble 1. SAR of A-Ring Substitutions
were active compounds, but they proved to be more
prone to decomposition than the 4-amino analogues. We
tried to make additional gains by taking advantage of
alkyl substitutions on the 4-amino group. It was easy
to prepare a large number of compounds via palladium-
mediated aminations on the corresponding 4-bromide,12
but all of these analogues lost a great deal of activity in
the antimitotic assay. Reasoning that the coplanar
orientation of the N,N-dimethyl groups with the ben-
zene ring might be critical, we investigated fusing
nitrogen-containing heterocycles to the A-ring. Benzox-
azolone 22d , tetrahydroquinoline 22e, and indoline 22f
were all less active than 22a , but indoles 22g and 22h
were each as potent as compound 22a . Clearly, both
N-alkyl substituents were not necessary for activity, but
only the rigid, electron-rich indole maintained activity
out of all the A-ring heterocycles we examined.
In Vivo Resu lts. On the basis of the trends we saw
in vitro, we chose to examine the in vivo profiles of
compounds 1, 6a , 22a , 16, 14, and 22g. We began with
the determination of the pharmacokinetic parameters
for the new compounds. Unfortunately, the physical
properties of substituted acetates 16 and 14 made it
difficult to dose these agents safely. Both of these
compounds were insoluble either in an ethanol/propy-
lene glycol/D5W medium or a DMSO/PEG400/D5W
medium, and attempted dosing of a suspension led to
mortality in rats. Compounds 1, 6a , 22a , and 22g all
displayed pharmacokinetic profiles that allowed us to
proceed with evaluation of their efficacies in murine
tumor models (Table 6).The new antimitotic agents,
paclitaxel, and vincristine were evaluated in P388, B16,
or M5076 murine cancer models. Approximately 8 week
old mice were inoculated with tumor cells on day 0, and
then therapy was initiated on day 1 of the study. Mice
were monitored daily for mortality, and were euthanized
when moribund. The day of death was recorded for each
individual, and was used to calculate the percent
increase in life span (ILS). A 25% or greater increase
in life span was considered biologically significant.
small, uncharged groups were acceptable additions to
the N-acetate group, but such changes did not improve
potency relative to the lead compound.
The potential for the oxadiazoline ring to undergo
hydrolysis in vivo was seen as a major liability with the
lead molecule, so the search for a replacement for the
B-ring was a major goal of our SAR study. Some of the
analogues we prepared bore the A-ring found in com-
pound 1, while others contained the 4-(N,N-dimethy-
lamino)phenyl A-ring found in 6a . Either of these
A-rings was active when attached to the oxadiazoline
B-ring, but analogues with a 4-(N,N-dimethylamino)-
phenyl A-ring were generally easier to prepare. Table
4 gives several examples of heterocyclic replacements
for the oxadiazoline ring. Aromatic heterocycles such as
oxadiazole 34, thiadiazole 32, and pyrazole 28 were
inactive. Thiadiazoline 33 showed modest activity, but
1,3-oxazoline 22a retained most of the activity seen with
the original oxadiazoline B-ring. In contrast, dioxolane
37 was much less potent than oxazoline 22a . These
results demonstrate that both the 1-oxygen and 3-ni-
trogen atoms are required for activity, but the 4-nitro-
gen atom and the pendant acetate are not required.
Additionally, the B-ring must have an sp3 carbon at the
5-position, rendering aromatic analogues inactive.
Following the discovery of oxazoline 22a , we contin-
ued to explore the SAR of the A-ring coupled to the
oxazoline B-ring (Table 5). We explored placing an
unsubstituted amino group in the A-ring 3-position.
Both 3-amino-4-methoxy 22b and 3-amino-4-methyl 22c
Early studies were conducted using the P388 and B16
tumor models (Figure 2). Both compound 1 and com-
pound 22a showed dose-dependent in vivo activity in
the P388 model, but compound 6a showed no significant
activity. Paclitaxel and vincristine were both active in
the P388 model. In the B16 model, again compound 1
showed dose-dependent activity, but compound 22a
failed to show any activity. Paclitaxel retained its
anticancer activity in the B16 model, but vincristine was
not active. M5076 is a faster growing tumor model that
presents a higher standard of efficacy for in vivo agents
than either P388 or B16 tumor models. In the M5076
model, compound 1, compound 22a , paclitaxel, and
vincristine were all inactive. However, compound 22g
showed efficacy at 50 (mg/kg)/day (Figure 2). Signifi-
cantly, this activity was apparent following oral dosing
of compound 22g. Additionally, the compound was
efficacious in a solid tumor model. Inspired by this
result, we evaluated compound 22g in an extended
M5076 study. After a 28 day dosing regimen was
complete, the dose-dependent efficacy of compound 22g
became clear, with an increase in mean survival time
of 120% at 25 (mg/kg)/day and 227% at 50 (mg/kg)/day.

4422 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Ta ble 2. SAR of C-Ring Substitutions
Szczepankiewicz et al.
Con clu sion s
modifications gave us compounds with slightly better
in vitro activity against both MDR(-) and MDR(+) cell
lines, but in vivo efficacy was improved greatly over
those of the lead compound, paclitaxel, and vincristine.
In M5076 cancer models, indolyloxazoline 22g (A-
259745) rose above the other compounds we evaluated
A new series of tubulin polymerization inhibitors with
activity in multi-drug-resistant tumor cell lines has been
discovered. Medicinal chemistry efforts starting from
lead compound 1 led to the development of several
compounds worthy of in vivo evaluation. Structural

Oxadiazoline- and Oxazoline-Based Antimitotic Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4423
Ta ble 3. SAR of N-Acetate Substitutions
then extracted with diethyl ether. The ether layers were
concentrated to 1.09 g (90%) of a tan crystalline solid.
To a solution of 1.09 g (5.31 mmol) of methyl 4-azido-3-
methylbenzoate in 6 mL of ethanol was added 2 mL (19.8
mmol) of 25% aqueous NaOH. The reaction was stirred at
ambient temperature for 1 h, and then the solvents were
removed in vacuo. The residue was taken up in water and
washed with diethyl ether, and then the aqueous layer was
acidified to pH 1 by addition of 1 N H2SO4. The azido acid was
extracted into diethyl ether, and the second set of ether layers
was dried over Na2SO4, filtered, and concentrated to 965 mg
(95%) of 4-azido-3-methylbenzoic acid as a tan crystalline solid.
3-Meth yl-4-a zid oben zoyl Hyd r a zid e (Gen er a l P r oce-
d u r e for th e P r ep a r a tion of Hyd r a zid es 2). To a solution
of 0.965 g (5.45 mmol) of 4-azido-3-methylbenzoic acid in 5 mL
of DMF and 5 mL of THF was added 0.755 g (5.72 mmol) of
tert-butylcarbazate, 1.15 g (5.99 mmol) of 1-[3-(dimethylami-
no)propyl]-3-ethylcarbodiimide hydrochloride (EDCI), and 10
mg (0.08 mmol) of 4-(N,N-dimethylamino)pyridine. The reac-
tion was stirred at ambient temperature for 17 h and then
poured into a mixture of 50 mL of ice cold water and 50 mL of
diethyl ether. The layers were separated, and then the ether
layer was washed with 1 M aqueous NaHSO4, water, saturated
aqueous NaHCO3 solution, and brine. The ether layer was
dried over Na2SO4, filtered, and concentrated to a white solid.
The white solid was suspended in 20 mL of CH2Cl2, and
cooled with an ice bath. Next, 15 mL of trifluoroacetic acid
was added, and the mixture was stirred for 2 h at 0 °C. The
solvents were removed in vacuo, and then the residue was
partitioned between ethyl acetate and saturated aqueous
NaHCO3 solution. The organic phase was washed with water
and then brine, dried over Na2SO4, filtered, and concentrated
to give 1.02 g (98%) of 4-azido-3-methylbenzoyl hydrazide as
a tan crystalline solid.
4-(Azidomethyl)-3-methylbenzoic Acid (3,4,5-Trimethoxy-
b en zylid en e)h yd r a zid e (Gen er a l P r oced u r e for t h e
P r ep a r a tion of Hyd r a zon es 5). To a solution of 1.02 g (5.34
mmol) of 3-methyl-4-azidobenzoyl hydrazide in 50 mL of
ethanol and 2 mL of acetic acid was added 1.05 g (5.34 mmol)
of 3,4,5-trimethoxybenzaldehyde. The solids dissolved, and
then a white precipitate quickly formed. After being stirred
for 20 min, the mixture was cooled to 10 °C. The precipitate
was collected, washed with cold ethanol, and dried in vacuo
to give 1.35 g (67%) of the hydrazone: 1H NMR (CDCl3, 300
MHz) δ 2.25 (s, 3H), 3.90 (s, 9H), 6.97 (br s, 2H), 7.18 (d, 1H,
J ) 7 Hz), 7.73 (br s, 3H), 9.16 (br s, 1H); MS (DCI/NH3) m/z
369 [M + H]+.
2-(4-Azido-3-m eth ylph en yl)-4-acetyl-5-(3,4,5-tr im eth oxy-
p h en yl)-∆2,3-oxa d ia zolin e (Gen er a l P r oced u r e for th e
P r ep a r a tion of Oxa d ia zolin es 6). To 850 mg (2.30 mmol)
of 4-(azidomethyl)-3-methylbenzoic acid (3,4,5-trimethoxyben-
zylidene)hydrazide was added 15 mL of acetic anhydride. The
reaction was heated to 125 °C, and the starting hydrazide
dissolved. After the solution was stirred at 125 °C for 2 h, the
solvent was removed in vacuo. The residue was partitioned
between ethyl acetate and saturated aqueous NaHCO3, and
then the layers were separated. The organic layer was dried
over Na2SO4, filtered, and concentrated. The residue was
purified via silica gel chromatography, eluting with 45% ethyl
acetate/hexanes, to give 605 mg (64%) of 2-(4-amino-3-meth-
ylphenyl)-4-acetyl-5-(3,4,5-trimethoxyphenyl)-∆2,3-oxadiazo-
line.
ED50 (µM) ( SD
compd
R
HCT-15
NCI-H460
7
8
H
0.096 ( 0.06
0.12 ( 0.09
COOC2H5
OCH3
CH2OAc
CH2N3
CH2N(CH3)2
CH2OH
CH2NH2
CH2NHCHO
CH2NHAc
45
40
0.019 ( 0.004
0.15 ( 0.02
80
33
106
0.072 ( 0.005
0.53 ( 0.03
74
10
11
12
13
14
15
16
17
0.026 ( 0.016
0.043 ( 0.028
0.66 ( 0.068
0.040 ( 0.03
0.48 ( 0.01
0.65 ( 0.31
0.035 ( 0.028
0.18 ( 0.04
and increased the life span of mice up to a factor of 3
during a 28 day study. Compound 22g represents a new
tubulin polymerization inhibitor with oral in vivo activ-
ity against solid tumors and a straightforward chemical
synthesis.13 This agent, and others of its structural
class, may prove useful in the development of new
chemotherapeutic drugs.
Exp er im en ta l Section
Analytical HPLC traces were obtained by elution through
a C18 reversed-phase column with a gradient of 5 mM aqueous
ammonium acetate/acetonitrile or 0.1% aqueous TFA/aceto-
nitrile as eluant. The compounds were purified to homogeneity
unless otherwise indicated. 1H NMR and MS data were
recorded at Abbott Laboratories. Combustion analysis data
were obtained from Robertson Microlit Laboratories, Inc.,
Madison, NJ .
2-(4-Amino-3-methylphenyl)-4-acetyl-5-(3,4,5-trimethoxy-
p h en yl)-∆2,3-oxa d ia zolin e (1).14 A solution of 411 mg (1
mmol) of 2-(4-azido-3-methylphenyl)-4-acetyl-5-(3,4,5-trimethox-
yphenyl)-∆2,3-oxadiazoline (see the general experimental
procedures for hydrazides 2, hydrazones 5, and oxadiazolines
6) in 5 mL of acetonitrile and 5 mL of THF was prepared. This
was added to a stirred suspension of 283 mg (1.5 mmol) of
SnCl2, 616 µL (6 mmol) of thiophenol, and 627 µL (4.5 mmol)
of triethylamine in 5 mL of acetonitrile. After being stirred
for 1 h, the reaction was quenched by addition of 10 mL of 1
M NaOH. The solution was poured into additional 1 M NaOH
and then extracted with EtOAc. The organic layers were
washed with water and brine, dried over Na2SO4, filtered, and
concentrated. The product was purified by silica gel chroma-
tography, eluting with a 55-70% gradient of ethyl acetate/
1
hexanes, to give 310 mg (80%) of compound 1. H NMR and
MS data were identical to those of a commercially obtained
sample of compound 1: 1H NMR (300 MHz, d6-DMSO) δ 2.07
(s, 3H), 2.24 (s, 3H), 3.66 (s, 3H), 3.75 (s, 6H), 5.64 (s, 2H),
6.64 (d, 1H, J ) 7.8 Hz), 6.72 (s, 2H), 6.98 (s, 1H), 7.36-7.39
(m, 2H); MS (ESI) m/z 386 [M + H]+.
4-Azid o-3-m eth ylben zoic Acid . To a suspension of 1 g (6.6
mmol) of 4-amino-3-methylbenzoic acid in 1 mL of diethyl ether
was added ethereal diazomethane until effervescence ceased.
The solvent was removed in vacuo, giving 1.05 g (96%) of
methyl 4-amino-3-methylbenzoate as a crystalline solid.
To an ice-cooled suspension of 1.05 g (6.4 mmol) of methyl
4-amino-3-methylbenzoate in 15 mL of 6 N sulfuric acid was
added a solution of 485 mg (7.0 mmol) of NaNO2 in 1.4 mL of
water, keeping the temperature below 5 °C. The solution was
stirred for 45 min, and then a solution of 529 mg (8.1 mmol)
of NaN3 in 1.7 mL of water was added, keeping the temper-
ature below 5 °C, and keeping the vigorous effervescence under
control. The product suspension was stirred for 10 min and
2-[4-(N,N-Dim eth yla m in o)p h en yl]-4-a cetyl-5-(3,4,5-tr i-
m eth oxyp h en yl)-∆2,3-oxa d ia zolin e (6a ). 6a was prepared
according to the general procedures for compounds 2, 5, and
6, starting with 4-(N,N-dimethylamino)benzoic acid: 1H NMR
(CDCl3, 300 MHz) δ 2.37 (s, 3H), 3.03 (s, 6H), 3.82 (s, 3H),
3.85 (s, 6H), 6.68 (d, 2H, J ) 7 Hz), 6.72 (s, 2H), 6.96 (s, 1H),
7.65 (d, 2H, J ) 7 Hz); MS (CDI/NH3) m/z 400 [M + H]+. Anal.
Calcd for C21H25N3O5: C, 63.13; H, 6.31; N, 10.52. Found: C,
63.05; H, 5.95; N, 10.31.
2-[2-(N,N-Dim eth yla m in o)p h en yl]-4-a cetyl-5-(3,4,5-tr i-
m eth oxyp h en yl)-∆2,3-oxa d ia zolin e (6b). 6b was prepared
according to the general procedures for compounds 2, 5, and

4424 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Ta ble 4. SAR of B-Ring Substitutions
Szczepankiewicz et al.
6, starting with 2-(N,N-dimethylamino)benzoic acid: 1H NMR
(CDCl3, 300 MHz) δ 2.40 (s, 3H), 2.84 (s, 6H), 3.82 (s,3H), 3.84
(s, 6H), 6.72 (s, 2H), 6.86 (s, 1H), 7.06 (d, 1H, J ) 9 Hz), 7.42
(t, 1H, J ) 9 Hz), 7.74 (d, 1H, J ) 9 Hz). MS (DCI/NH3) m/z
400 [M + H]+.
2-[3-(N,N-Dim eth yla m in o)p h en yl]-4-a cetyl-5-(3,4,5-tr i-
m eth oxyp h en yl)-∆2,3-oxa d ia zolin e (6c). 6c was prepared
according to the general procedures for compounds 2, 5, and
6, starting with 3-(N,N-dimethylamino)benzoic acid: 1H NMR
(CDCl3, 300 MHz) δ 2.39 (s, 3H), 3.01 (s, 6H), 3.83 (s, 3H),
3.87 (s, 6H), 6.72 (s, 2H), 6.85 (d, 1H, J ) 7 Hz), 6.99 (s,1H),
7.19 (br s, 1H), 7.26-7.33 (m, 2H). MS (CDI/NH3) m/z 400 [M
+ H]+.
2-(3-Meth yl-4-am in oph en yl)-4-acetyl-5-(2,4,5-tr im eth oxy-
p h en yl)-∆2,3-oxa d ia zolin e (6d ). 6d was prepared according
to the general procedures for compounds 2, 5, and 6, starting
with 3-methyl-4-azidobenzoyl hydrazide and 2,4,5-trimethoxy-
benzaldehyde. The product obtained from the oxadiazoline
synthesis was hydrogenated using 1 atm of H2/Pd-C in ethyl
acetate to give the desired product: 1H NMR (d6-DMSO, 300
MHz) δ 2.03 (s, 3H), 2.22 (s, 3H), 3.63 (s, 3H), 3.78 (s, 3H),
3.83 (s, 3H), 5.60 (s, 2H), 6.62 (d, 1H, J ) 8 Hz), 6.72 (s, 1H),
6.76 (s, 1H), 7.08 (s, 1H), 7.32 (d, 1H, J ) 8 Hz), 7.37 (s, 1H);
MS (DCI/NH3) m/z 386 [M + H]+.
2-[4-(N,N-Dim eth yla m in o)p h en yl]-4-a cetyl-5-(2,4,6-tr i-
m eth oxyp h en yl)-∆2,3-oxa d ia zolin e (6e). 6e was prepared
according to the general procedures for compounds 5 and 6,
starting with 4-(N,N-dimethylamino)benzoyl hydrazide and
2,4,6-trimethoxybenzaldehyde: 1H NMR (CDCl3, 500 MHz) δ
7.72 (m, 2H), 7.52 (s, 1H), 6.67 (m, 2H), 6.11 (s, 2H), 3.79 (s,
3H), 3.76 (s, 6H), 3.02 (s, 6H), 2.25 (s, 3H); MS (DCI/NH3) m/z
400 [M + H]+.
2-[4-(N,N-Dim eth yla m in o)p h en yl]-4-a cetyl-5-(2,3,4-tr i-
m eth oxyp h en yl)-∆2,3-oxa d ia zolin e (6f). 6f was prepared
according to the general procedures for compounds 5 and 6,
starting with 4-(N,N-dimethylamino)benzoyl hydrazide and
2,3,4-trimethoxybenzaldehyde: 1H NMR (CDCl3, 500 MHz) δ
7.72 (m, 2H), 7.16 (s, 1H), 7.02 (d, 1H, J ) 8.4 Hz), 6.67-6.64
(m, 3H), 3.94 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.02 (s, 6H),
2.35 (s, 3H); MS (DCI/NH3) m/z 400 [M + H]+.
3,5-Dim eth oxy-4-[[N-(n -bu tyl)a m in o]ca r bon yl]ben za l-
d eh yd e. To a solution of 2.12 g (10 mmol) of 4-(hydroxym-
ethyl)-2,6-dimethoxybenzoic acid in 22 mL of DMF was added
4.05 g (30 mmol) of benzotriazol-1-ol, 1.01 g (10 mmol) of
N-methylmorpholine, and 1.91 g (10 mmol) of EDCI. The
reaction was stirred at ambient temperature for 18 h, then
diluted with saturated aqueous NaHCO3, and extracted with
ethyl acetate. The combined ethyl acetate layers were back-

Oxadiazoline- and Oxazoline-Based Antimitotic Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4425
Ta ble 5. SAR of A-Ring Substitutions with an Oxazoline
B-Ring
To a suspension of 1.70 g (6.4 mmol) of N-butyl-4-(hy-
droxymethyl)-2,6-dimethoxybenzamide in 50 mL of CH2Cl2 and
10 mL of CHCl3 was added 2.30 g (10.7 mmol) of pyridinium
chlorochromate. The mixture was stirred at ambient temper-
ature for 2 h, then diluted with 50 mL of diethyl ether, and
filtered to remove the dark solids. The solids were washed with
ethyl acetate, and then the combined organic layers were
concentrated in vacuo to give 1.5 g (89%) of 3,5-dimethoxy-4-
[[N-(n-butyl)amino]carbonyl]benzaldehyde as a brown solid.
This aldehyde was used without further purification for the
preparation of oxadiazoline 6g.
2-(3-Meth yl-4-a m in op h en yl)-4-a cetyl-5-[3,5-d im eth oxy-
4-[[N-(n -b u t yl)a m in o]ca r b on yl]p h en yl]-∆2,3-oxa d ia zo-
lin e (6g). 6g was prepared according to the general procedures
for compounds 5 and 6, substituting 3,5-dimethoxy-4-[[N-(n-
butyl)amino]carbonyl]benzaldehyde for 3,4,5-trimethoxyben-
zaldehyde. The azido product obtained from the oxadiazoline
synthesis was hydrogenated using 1 atm of H2/Pd-C in ethyl
acetate to give the desired product: 1H NMR (d6-DMSO, 300
MHz) δ 0.75 (t, 3H, J ) 7.5 Hz), 1.08-1.17 (m, 2H), 1.32-
1.42 (m, 2H), 2.12 (s, 3H), 2.28 (s, 3H), 3.84 (s, 6H), 5.58 (s,
2H), 6.63 (d, 1H, J ) 7.5 Hz), 7.03 (s, 2H), 7.55 (m, 2H), 8.38
(s, 1H); MS (ESI) m/z 453 [M - H]-; HRMS calcd for
C
24H31O5N4 + H 455.2294, found 455.2285.
2-[4-(N,N-Dim eth yla m in o)p h en yl]-4-a cetyl-5-(3,4,5-tr i-
m eth ylp h en yl)-∆2,3-oxa d ia zolin e (6h ). 6h was prepared
according to the general procedures for compounds 5 and 6,
starting with 4-(N,N-dimethylamino)benzoyl hydrazide and
3,4,5-trimethylbenzaldehyde: 1H NMR (CDCl3, 500 MHz) δ
7.71 (m, 2H), 7.12 (s, 1H), 7.03 (s, 1H), 6.97 (s, 1H), 6.66 (m,
2H), 3.02 (s, 6H), 2.45 (s, 3H), 2.36 (s, 3H), 2.20 (s, 3H), 2.18
(s, 3H); MS (DCI/NH3) m/z 352 [M + H]+.
2-[4-(N,N-Dim eth ylam in o)ph en yl]-4-acetyl-5-(4-acetoxy-
3,5-d im eth oxyp h en yl)-∆2,3-oxa d ia zolin e (6i). 6i was pre-
pared according to the general procedures for compounds 5
and 6, starting with 4-(N,N-dimethylamino)benzoyl hydrazide
and 3,5-dimethoxy-4-hydroxybenzaldehyde: 1H NMR (CDCl3,
500 MHz) δ 7.24 (m, 2H), 6.99 (s, 1H), 6.75 (s, 2H), 6.69 (m,
2H), 3.80 (s, 6H), 3.04 (s, 6H), 2.35 (s, 3H), 2.32 (s, 3H); MS
(DCI/NH3) m/z 428 [M + H]+.
2-[4-(N,N-Dim et h yla m in o)p h en yl]-4-a cet yl-5-(3,4-d i-
m eth oxyp h en yl)-∆2,3-oxa d ia zolin e (6j). 6j was prepared
according to the general procedures for compounds 5 and 6,
starting with 4-(N,N-dimethylamino)benzoyl hydrazide and
3,4-dimethoxybenzaldehyde: 1H NMR (CDCl3, 500 MHz) δ 7.74
(m, 2H), 7.04 (dd, 1H, J ) 2.2, 8.3 Hz), 7.00 (d, 1H, J ) 2.2
Hz), 6.98 (s, 1H), 6.86 (d, 1H, J ) 8.4 Hz), 6.68 (m, 2H), 3.86
(s, 6H), 3.04 (s, 6H), 2.34 (s, 6H); MS (DCI/NH3) m/z 370 [M +
H]+.
2-(3-Met h yl-4-a m in op h en yl)-4-a cet yl-5-[4-(m et h oxy-
ca r bon yl)-3,5-d im eth oxyp h en yl]-∆2,3-oxa d ia zolin e (6k ).
6k was prepared according to the general procedures for
compounds 5 and 6, substituting 3,5-dimethoxy-4-(methoxy-
carbonyl)benzaldehyde for 2,4,5-trimethoxybenzaldehyde. The
azido product obtained from the oxadiazoline synthesis was
hydrogenated using 1 atm of H2/Pd-C in ethyl acetate to give
the desired product: 1H NMR (d6-DMSO, 300 MHz) δ 2.06 (s,
3H), 2.24 (s, 3H), 3.76 (s, 9H), 5.63 (s, 2H), 6.63 (d, 1H, J ) 8
Hz), 6.76 (s, 2H), 7.03 (s, 1H), 7.38 (d, 1H, J ) 8 Hz), 7.40 (s,
1H); MS (DCI/NH3) m/z 439 [M + H]+.
2-(4-Am in o-3-m e t h ylp h e n yl)-4-a ce t yl-5-(7-m e t h oxy-
ben zo[1,3]d ioxol-5-yl)-∆2,3-oxa d ia zolin e (6l). 6l was pre-
pared according to the general procedures for compounds 5
and 6, substituting 3,4-(methylenedioxy)-5-methoxybenzalde-
hyde for 2,4,5-trimethoxybenzaldehyde. The azido product
obtained from the oxadiazoline synthesis was hydrogenated
using 1 atm of H2/Pd-C in ethyl acetate to give the desired
product: 1H NMR (d6-DMSO, 300 MHz) δ 2.04 (s, 3H), 2.22 (s,
3H), 3.84 (s, 3H), 5.66 (s, 2H), 6.02 (s, 2H), 6.58-6.64 (m, 2H),
6.77 (s, 1H), 6.96 (s, 1H), 7.38 (m, 2H); MS (DCI/NH3) m/z 370
[M + H]+.
Ta ble 6. Pharmacokinetic Parameters for Compounds Tested
in Vivo
intravenous
AUCc
oral
t1/2
Vcb
Cmax
Tmax
AUCf
a
d
e
compd (h) (L/kg) [(ng h)/mL] (ng/mL) (h) [(ng h)/mL] Fg (%)
1
6a
22a
22g
1.1
1.1
0.8
1.9
2.2
3.3
3.3
1.4
2109
3170
3407
5119
59
113
891
799
1.7
0.4
0.9
0.4
147
155
1657
1777
7
4.9
49
35
a
Half-life following intravenous dosing. b Volume of distribution
following intravenous dosing. c Area under the curve following
intravenous dosing, integrated drug concentration with respect
d
to time. Maximum plasma concentration following intravenous
dosing. e Time to maximum plasma concentration following oral
dosing. f Integrated drug concentration with respect to time fol-
g
lowing oral dosing. Percent oral bioavailability.
extracted with water and then brine, dried over Na2SO4,
filtered, and concentrated to give 1.70 g (64%) of N-butyl-4-
(hydroxymethyl)-2,6-dimethoxybenzamide as a white solid.
2-[4-(N,N-Dim eth yla m in o)p h en yl]-4-for m yl-5-(3,4,5-tr i-
m eth oxyp h en yl)-∆2,3-oxa d ia zolin e (7). This compound
was prepared by the same general procedure used to prepare

4426 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Szczepankiewicz et al.
N-acetyloxadiazolines 6, substituting a 1:1 mixture of formic
acid and acetic anhydride for acetic anhydride, and then
heating at reflux. The product was obtained as white crystals
from hot EtOAc: 1H NMR (CDCl3, 300 MHz) δ 3.07 (s, 6H),
3.84 (s, 3H), 3.86 (s, 6H), 6.68 (d, 2H, J ) 12 Hz), 6.72 (s, 1H),
6.94 (s, 1H), 7.73 (d, 2H, J ) 12 Hz), 8.77 (s, 6H); MS (DCI/
NH3) m/z 386 [M + H]+.
C23H27N3O7: C, 60.39; H, 5.95; N, 9.18. Found: C, 60.12; H,
5.87; N, 8.84.
2-[4-(N,N-Dim et h yla m in o)p h en yl]-4-(a zid oa cet yl)-5-
(3,4,5-tr im eth oxyp h en yl)-∆2,3-oxa d ia zolin e (12). To a
suspension of 200 mg of chloroamide 9 in 1 mL of DMF was
added 130 mg (2 mmol) of sodium azide. The mixture was
stirred at ambient temperature for 15 h, then poured into 25
mL of 0.25 M NaHCO3 solution, and extracted with ethyl
acetate (3 × 10 mL). The combined ethyl acetate layers were
back-extracted with water (1 × 10 mL) and brine (1 × 10 mL),
dried over MgSO4, filtered, and concentrated to give 203 mg
(100%) of a solid: 1H NMR (300 MHz, CDCl3) δ 3.05 (s, 6H),
3.84 (s, 3H), 3.85 (s, 6H), 4.26 (d, 1H, J ) 12 Hz), 4.31 (d, 1H,
J ) 12 Hz), 6.70 (m, 2H), 6.71 (s, 2H), 6.95 (s, 1H), 7.73 (m,
2H); MS (DCI) m/z 441 [M + H]+, 458 [M + NH4]+. Anal. Calcd
for C21H24N6O5: C, 57.27; H, 5.49; N, 19.08. Found: C, 57.24;
H, 5.53; N, 18.96.
2-[4-(N,N-Dim eth ylam in o)ph en yl]-4-[(N,N-dim eth ylam i-
n o)a ce t yl]-5-(3,4,5-t r im e t h oxyp h e n yl)-∆2,3-oxa d ia zo-
lin e (13). To 150 mg of chloroamide 9 were added 2 mL of
4.07 M dimethylamine in ethanol and 1 mL of DMF. The
reaction was heated to a gentle reflux for 15 min, then poured
into 25 mL of water, and extracted with ethyl acetate (3 × 10
mL). The combined ethyl acetate layers were back-extracted
with brine (1 × 10 mL), dried over MgSO4, filtered, and
concentrated to an oil. This was purified via silica gel chro-
matography, eluting with 95:5 EtOAc/MeOH, to give 40 mg
(26%) of a colorless solid: 1H NMR (300 MHz, CDCl3) δ 2.45
(s, 6H), 3.05 (s, 6H), 3.55 (d, 1H, J ) 15 Hz), 3.68 (d, 1H, J )
15 Hz), 3.84 (s, 3H), 3.85 (s, 6H), 6.68 (m, 2H), 6.73 (s, 2H),
6.99 (s, 1H), 6.74 (m, 2H); MS (DCI) m/z 443 [M + H]+. Anal.
Calcd for C23H30N4O5: C, 62.57; H, 6.62; N, 12.69. Found: C,
62.28, H, 6.72, N, 12.62.
2-[4-(N,N-Dim eth yla m in o)p h en yl]-4-(h yd r oxya cetyl)-
5-(3,4,5-tr im eth oxyp h en yl)-∆2,3-oxa d ia zolin e (14). To 211
mg (0.461 mmol) of 11 were added 9 mL of methanol, 1 mL of
water, and 300 mg (2.2 mmol) of potassium carbonate. The
mixture was stirred at ambient temperature for 3 h, then
diluted with 30 mL of water, and extracted with CH2Cl2 (3 ×
10 mL). The combined organic layers were back-extracted with
brine (1 × 10 mL), dried over MgSO4, filtered, and concen-
trated in vacuo to 180 mg (94%) of a white solid. The product
could be purified by recrystallization from CH3CN: 1H NMR
(300 MHz, CDCl3) δ 3.05 (s, 6H), 3.84 (s, 3H), 3.85 (s, 6H),
4.53 (t, 2H, J ) 4.6 Hz), 6.68 (m, 2H), 6.70 (s, 2H), 6.94 (s,
1H), 7.73 (m, 2H); MS (DCI) m/z 416 [M + H]+, 433 [M +
NH4]+. Anal. Calcd for C21H25N3O6‚0.2CH2Cl2: C, 58.88; H,
5.92; N, 9.72. Found: C, 58.80; H, 5.81; N, 9.50.
4-(Am in oa cet yl)-2-[4-(N,N-d im et h yla m in o)p h en yl]-5-
(3,4,5-tr im eth oxyp h en yl)-∆2,3-oxa d ia zolin e (15). To a
solution of 110 mg (0.250 mmol) of 12 in 2 mL of THF were
added 66 mg (0.25 mmol) of triphenylphosphine and 3 drops
of water. The mixture was stirred at ambient temperature for
16 h and then concentrated in vacuo to a white solid. The
product amine was purified via silica gel chromatography,
eluting with 8% CH3OH in CH2Cl2, to give 40 mg (38%) of a
colorless solid: 1H NMR (300 MHz, CDCl3) δ 3.05 (s, 6H), 3.84
(s, 3H), 3.84 (s, 2H), 3.85 (s, 6H), 6.68 (m, 2H), 6.71 (s, 2H),
6.92 (s, 1H), 7.74 (m, 2H); MS (DCI) m/z 415 [M + H]+. Anal.
Calcd for C21H26N4O5·0.25CH2Cl2: C, 58.58; H, 6.13; N, 12.86.
Found: C, 58.57; H, 6.23; N, 12.77.
2-[4-(N,N-Dim et h yla m in o)p h en yl]-4-[(et h ylca r b oxy)-
ca r b on yl]-5-(3,4,5-t r im e t h oxyp h e n yl)-∆2,3-oxa d ia zo-
lin e (8). To a suspension of 651 mg of the 4-(N,N-dimethyl-
amino)benzoyl hydrazone derived from 3,4,5-trimethoxyben-
zaldehyde (prepared using the general procedure for hydrazones
5) in 8 mL of DMF was added 500 µL of ethyl oxalyl chloride.
The reaction was stirred until dissolution was complete, and
then stirring was continued for another 10 min. The solution
was diluted with 80 mL of water and then extracted with ethyl
acetate (3 × 20 mL). The combined ethyl acetate layers were
back-extracted with water (1 × 20 mL), saturated NaHCO3
solution (1 × 20 mL), water (1 × 20 mL), and brine (1 × 20
mL), dried over MgSO4, filtered, and concentrated to a yellow
oil. This was purified via silica gel chromatography, eluting
with 50% ethyl acetate/hexanes, to give 433 mg of a crystalline
solid. The product could also be crystallized from methanol:
1H NMR (CDCl3) δ (major rotamer) 1.42 (t, 3H, J ) 7.0 Hz),
3.05 (s, 6H), 3.85 (s, 3H), 3.86 (s, 6H), 4.41 (m, 2H), 6.67 (m,
2H), 6.73 (s, 2H), 6.96 (s, 1H), 7.73 (m, 2H); MS (DCI/NH3)
m/z 458 [M + H]+. Anal. Calcd for C23H27N3O7: C, 60.39; H,
5.95; N, 9.18. Found: C, 60.30; H, 5.97; N, 9.19.
4-(Ch lor oa cetyl)-2-[4-(N,N-d im eth yla m in o)p h en yl]-5-
(3,4,5-tr im eth oxyp h en yl)-∆2,3-oxa d ia zolin e (9). To 1.5 g
(4.2 mmol) of the 4-(N,N-dimethylamino)benzoyl hydrazone
derived from 3,4,5-trimethoxybenzaldehyde (prepared accord-
ing to the general procedure used for hydrazones 5) was added
4.5 g (26.3 mmol) of chloroacetic anhydride. The mixture was
stirred at 95 °C for 20 min and then poured into 50 mL of
saturated aqueous NaHCO3 solution, along with 10 mL of ethyl
acetate. After the resulting mixture was stirred for 1 h, the
precipitate was filtered and washed with ethyl acetate and
then with ethanol to give 1.01 g (55%) of a light yellow solid:
1H NMR (CDCl3, 300 MHz) δ 7.74 (m, 2H), 6.95 (s, 1H), 6.71
(s, 2H), 6.68 (m, 2H), 4.54 (d, 1H, J ) 13.6 Hz), 4.44 (d, 1H, J
) 13.6 Hz), 3.84 (s, 6H), 3.84 (s, 3H), 3.05 (s, 6H); MS (DCI/
NH3) m/z 434 [M + H]+.
2-[4-(N,N-Dim eth ylam in o)ph en yl]-4-(m eth oxycar bon yl)-
5-(3,4,5-tr im eth oxyp h en yl)-∆2,3-oxa d ia zolin e (10). To 100
mg (0.28 mmol) of the 4-(N,N-dimethylamino)benzoyl hydra-
zone derived from 3,4,5-trimethoxybenzaldehyde (prepared
according to the general procedure used for hydrazones 5) was
added 2 mL of dimethyl pyrocarbonate. THF (1 mL) was added
to improve solubility, and the mixture was heated in a sealed
tube at 90 °C for 3 h. The tube was cooled with an ice bath
and then opened. (Caution: watch for gas evolution!) The
solvents were removed in vacuo, and the residue was triturated
with a 1:1 mixture of diethyl ether and hexanes to produce a
solid. The solid was washed further with diethyl ether to
provide 75 mg (64% yield) of a white solid: 1H NMR (300 MHz,
CDCl3) δ 3.08 (s, 6H), 3.84 (s, 3H), 3.86 (s,3H), 3.87 (s, 6H),
6.66 (m, 2H), 6.69 (s, 2H), 6.92 (s, 2H), 7.80 (m, 2H), 7.99 (s,
1H); MS (ESI) m/z 416 [M + H]+. Anal. Calcd for C21H25N3O6:
C, 60.71; H, 6.07; N, 10.11. Found: C, 60.48; H, 5.65; N, 9.88.
2-[4-(N,N-Dim eth yla m in o)p h en yl]-4-(a cetoxya cetyl)-5-
(3,4,5-tr im eth oxyp h en yl)-∆2,3-oxa d ia zolin e (11). To a
solution of 100 mg (0.230 mmol) of chloroamide 9 in 1 mL of
DMSO was added 50 mg (0.509 mmol) of potassium acetate.
After 15 h, the reaction was poured into 20 mL of water and
then extracted with ethyl acetate (3 × 5 mL). The combined
ethyl acetate layers were back-extracted with water (1 × 5
mL) and brine (1 × 5 mL), dried over MgSO4, filtered, and
concentrated in vacuo to give 95 mg (90%) of a white solid:
1H NMR (300 MHz, CDCl3) δ 2.18 (s, 3H), 3.05 (s, 6H), 3.83
(s, 3H), 3.85 (s, 6H), 5.05 (d, 1H, J ) 12 Hz), 5.10 (d, 1H, J )
12 Hz), 6.69 (m, 2H), 6.70 (m, 2H), 6.93 (s, 1H), 7.73 (m, 2H);
MS (DCI) m/z 458 [M + H]+, 475 [M + NH4]+. Anal. Calcd for
2-[4-(N,N-Dim et h yla m in o)p h en yl]-4-[(for m yla m in o)-
acetyl]-5-(3,4,5-tr im eth oxyph en yl)-∆2,3-oxadiazolin e (16).
To a mixture of 200 mg (0.483 mmol) of amine 15, 111 mg
(0.580 mmol) of EDCI, and 95 mg (0.580 mmol) of 3-hydroxy-
1,2,3-benzotriazin-4(3H)-one were added 3 mL of DMF and
then 40 µL (0.75 mmol) of 88% formic acid. The pH of the
mixture was adjusted to 8 using triethylamine (15 drops), and
then the mixture was stirred at ambient temperature for 2 h.
The reaction was poured into 30 mL of 0.6 M NaHCO3 solution
and extracted with CH2Cl2 (3 × 10 mL). The combined organic
layers were back-extracted with water (2 × 10 mL) and brine
(1 × 10 mL), dried over MgSO4, filtered, and concentrated to
a white solid. The product was purified by recrystallization

Oxadiazoline- and Oxazoline-Based Antimitotic Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4427
from CH3CN to give 101 mg (47%, one crop) of colorless
crystals: 1H NMR (300 MHz, CDCl3) δ 3.05 (s, 6H), 3.84 (s,
3H), 3.85 (s, 6H), 4.51 (d, 2H, J ) 5 Hz), 6.40 (br t, 1H), 6.70
(m, 2H), 6.69 (s, 2H), 6.91 (s, 1H), 7.75 (m, 2H), 8.28 (d, 1H, J
) 1.4 Hz); MS (DCI) m/z 443 [M + H]+. Anal. Calcd for
in small portions, alternating with the addition of 10% aqueous
sodium carbonate solution, to keep the pH between 8 and 9.
The resulting clear solution was stirred at ambient tempera-
ture overnight. The reaction was worked up by the addition
of 3 N HCl. The precipitate was collected, washed with water
and methylene chloride, and dried under vacuum to give the
Fmoc-amino acid (1.54 g, 66%): 1H NMR (d6-DMSO) δ 8.91
(s, 1H), 8.23 (br s, 1H), 7.93 (d, 2H, J ) 9.0 Hz), 7.7 (m, 3H),
7.45 (t, 2H, J ) 7.0 Hz), 7.35 (t, 2H, J ) 7.0 Hz), 7.14 (d, 1H,
J ) 9.0 Hz), 4.25-4.42 (m, 3H), 3.91 (s, 3H); MS (DCI/NH3)
m/z 407 [M + NH4]+.
To a stirred suspension of the Fmoc-amino acid (765 mg,
1.96 mmol) and ethanolamine (446 mg, 1.96 mmol) in tetrahy-
drofuran (20 mL) and DMF (1.5 mL) were added O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexaflu-
orophosphate (HATU) (747 mg, 1.96 mmol) and then N-meth-
ylmorpholine (0.22 mL). The reaction mixture was stirred at
ambient temperature for 18 h. The solvent was removed on a
rotary evaporator, and the residue was dissolved in methylene
chloride, washed with water, dried over MgSO4, and concen-
trated to give the ethanol amide (981 mg, 84%): 1H NMR
(CDCl3) δ 8.46 (br s, 1H), 7.80 (d, 2H, J ) 9.0 Hz), 7.63 (m,
3H), 7.30-7.48 (m, 4H), 6.95 (d, 1H, J ) 9.0 Hz), 6.63 (s, 3H),
6.60 (m, 1H), 4.90 (m, 1H), 4.51 (d, 2H, J ) 9.0 Hz), 4.30 (t,
1H, J ) 6.0 Hz), 3.84 (s, 9H), 3.82 (s, 3H); MS (ESI) m/z 621
[M + Na]+.
A mixture of the ethanol amide (980 mg, 1.63 mmol) and
[(methoxycarbonyl)sulfamoyl]triethylammonium hydroxide, in-
ner salt (Burgess reagent) (507 mg, 2.13 mmol), in THF (25
mL) was heated at reflux for 2 h. After the THF was removed,
the residue was dissolved in methylene chloride, washed with
saturated sodium bicarbonate (2×) and then brine (1×), dried
over MgSO4, and concentrated to give the Fmoc-protected
oxazoline (920 mg, 97%). To a stirred solution of the crude
Fmoc-protected oxazoline (500 mg, 0.86 mmol) in acetonitrile
(5 mL) was added diethylamine. The reaction was stirred at
ambient temperature for 20 min before being concentrated on
a rotary evaporator. The residue was purified by silica gel
chromatography, eluting with methylene chloride/methanol/
ammonium hydroxide (95:6:0.5), to provide oxazoline 22b (227
mg, 74%): 1H NMR (CDCl3) δ 7.4 (m, 2H), 6.81 (d, 1H, J )
9.0 Hz), 6.58 (s, 2H), 5.55 (m, 1H), 4.42 (m, 1H), 3.95 (m, 1H),
3.90 (s, 3H), 3.83 (s, 9H); MS (ESI) m/z 359 [M + H]+. Anal.
Calcd for C19H22N2O5: C, 63.68; H, 6.19; N, 7.82. Found: C,
63.92; H, 6.28; N, 7.81.
2-(3-Am in o-4-m eth yph en yl)-5-(3,4,5-tr im eth oxyph en yl)-
∆2,3-oxa zolin e (22c). This compound was prepared according
to the same procedure as 22b, substituting 3-amino-4-meth-
ylbenzoic acid (910 mg, 6.0 mmol) for 3-amino-4-methoxyben-
zoic acid. Purification via silica gel chromatography, eluting
with CH2Cl2/methanol/ammonium hydroxide (95:5:0.7), gave
438 mg (45% overall yield) of 22c: 1H NMR (CDCl3) δ 7.35
(m, 2H), 7.12 (d, 1H, J ) 9.0 Hz), 6.55 (s, 2H), 5.53 (dd, 1H, J
) 9.0, 12.0 Hz), 4.42 (dd, 1H, J ) 12.0, 15.0 Hz), 3.95 (dd, 1H,
J ) 9.0, 15.0 Hz), 3.84 (s, 9H), 3.17 (br s, 2H); MS (ESI) m/z
343 [M + H]+. Anal. Calcd for C19H22N2O4: C, 66.65; H, 6.48;
N, 8.18. Found: C, 66.80; H, 6.53; N, 7.93.
C
22H26N4O6‚0.35H2O: C, 58.88; H, 6.00; N, 12.48. Found: C,
58.96; H, 6.16; N, 12.35.
2-[4-(N,N-Dim et h yla m in o)p h en yl]-4-[(a cet yla m in o)-
acetyl]-5-(3,4,5-tr im eth oxyph en yl)-∆2,3-oxadiazolin e (17).
To a suspension of 51 mg (0.123 mmol) of amine 15 in 1 mL of
DMF was added 2 drops of acetic anhydride. The suspension
was stirred at ambient temperature for 1.5 h, during which
time the starting material dissolved. The reaction was poured
into 20 mL of 0.6 M aqueous NaHCO3 solution and then
extracted with CH2Cl2 (3 × 3 mL). The combined CH2Cl2 layers
were back-extracted with water (2 × 3 mL) and brine (1 × 3
mL), dried over MgSO4, filtered, and concentrated in vacuo to
56 mg (100%) of a white solid: 1H NMR (300 MHz, CDCl3) δ
2.04 (s, 3H), 3.05 (s, 6H), 3.84 (s, 3H), 3.85 (s, 6H), 4.46 (t, 2H,
J ) 4.1 Hz), 6.26 (br t, 1H), 6.69 (m, 2H), 6.90 (s, 1H), 7.75
(m, 2H); MS (DCI) m/z 457 [M + H]+, 474 [M + NH4]+. Anal.
Calcd for C23H28N4O6: C, 60.52; H, 6.18; N, 12.27. Found: C,
60.53; H, 6.31; N, 12.14.
1-(1-Hydroxy-2-nitroethyl)-3,4,5-trimethoxybenzene (19).
A solution of 3,4,5-trimethoxybenzaldehyde (10 g, 51 mmol)
and nitromethane (10 mL) in ethanol at 0 °C was treated
with 10% sodium hydroxide (21.4 mL, 53.5 mmol), stirred
for 45 s, treated with 2% acetic acid (162 mL), stirred
for 1 h in the ice bath, and filtered. The solid was washed
with water and dried under vacuum to provide 9.0 g of
the desired product as an off-white solid: 1H NMR (300
MHz, CDCl3) δ 6.63 (s, 2H), 5.42 (m, 1H), 4.61 (dd, 1H,
J ) 10.5, 14.4 Hz), 4.51 (dd, 1H, J ) 3.6, 14.4 Hz), 3.85
(s, 3H), 3.89 (s, 6H), 2.88 (d, 1H, J ) 4.5 Hz); MS (CDI/NH3)
m/z ) 275 [M + NH3]+.
2-Am in o-1-(3,4,5-tr im eth oxyp h en yl)eth a n ol (20). Com-
pound 19 was reduced with 10% Pd/C in acetic acid under 4
atm of H2 for 3 h to provide amine 20 as its acetic acid salt:
mp 103-104 °C; 1H NMR (300 MHz, CDCl3) δ 2.08 (s, 3H),
2.82-2.90 (m, 1H), 3.03-3.08 (m, 1H), 3.83 (s, 3H), 3.88 (s,
6H), 4.62-4.67 (m, 1H), 6.60 (s, 2H); MS (CDI/NH3) m/z 228
[M + H]+.
2-[4-(N,N-Dim eth yla m in o)p h en yl]-5-(3,4,5-tr im eth ox-
yp h en yl)-∆2,3-oxa zolin e (22a ). To an ice-cooled solution of
287 mg (1.00 mmol, acetic acid salt) of amine 20 and diiso-
propylethylamine (2 mL) in dichloromethane (15 mL) was
added slowly a solution of 4-(dimethylamino)benzoyl chloride
(219 mg) in dichloromethane (10 mL). The reaction was stirred
for 18 h, gradually warming to ambient temperature, and then
concentrated to near dryness. The crude product was purified
by silica gel chromatography, eluting with 1:1 hexanes/ethyl
acetate, and loading in CH2Cl2 solution, to provide 340 mg
(91%) of the desired amide as a white solid: mp 155-157 °C;
1H NMR (300 MHz, CDCl3) δ 3.03 (s, 6H), 3.44-3.57 (m, 1H),
3.83 (s, 9H), 4.02 (br s, 1H), 4.88-4.95 (m, 1H), 6.62 (s, 2H),
6.64 (d, 2H, J ) 7 Hz), 7.66 (d, 2H, J ) 7 Hz); MS (CDI/NH3)
m/z 375 [M + H]+.
A solution of the amide (320 mg, 0.86 mmol) in chloroform
(15 mL) at -20 °C was treated sequentially with pyridine (0.2
mL) and then triflic anhydride (0.2 mL), stirred for 30 min,
washed with saturated NaHCO3 and brine, dried (Na2SO4),
filtered, and concentrated. The concentrate was purified by
flash chromotography on silica gel with 1:1 hexanes/ethyl
acetate to provide 220 mg of oxazoline 22a as an off-white
solid:
2-(2-Oxob en zoxa zol-6-yl)-5-(3,4,5-t r im et h oxyp h en yl)-
∆2,3-oxa zolin e (22d ). This compound was prepared according
to the same procedure as compound 22e, substituting 2-ox-
obenzoxazole-6-carboxylic acid15 for 1-methyl-1,2,3,4-tetrahy-
droquinoline-6-carboxylic acid. The product was purified by
preparative TLC, eluting with ethyl acetate, to give 36 mg
(50%) of oxazoline 22d : 1H NMR (d6-DMSO, 300 MHz) δ 3.65
(s, 3H), 3.75 (s, 6H), 3.87 (dd, 1H, J ) 8.1, 15.0 Hz), 4.38 (dd,
1H, J ) 9.9, 15.0 Hz), 5.69 (dd, 1H, J ) 8.1, 9.9 Hz), 6.67 (s,
2H), 7.19 (d, 1H, J ) 8.4 Hz), 7.75 (d, 1H, J ) 1.8 Hz), 7.77
(dd, 1H, J ) 1.8, 8.4 Hz); MS (DCI) m/z 371 [M + H]+. Anal.
Calcd for C19H18N2O6: C, 61.62; H, 4.90; N, 7.56. Found: C,
61.34; H, 4.67; N, 7.56.
1H NMR (300 MHz, CDCl3) δ 3.05 (s, 6H), 3.85 (s, 9H), 3.96
(dd, 1H, J ) 7 Hz), 4.43 (dd, 1H, J ) 9 Hz), 5.57 (t, 1H, J ) 7
Hz), 6.58 (s, 2H), 6.70 (d, 2H, J ) 8 Hz), 7.93 (d, 2H, J ) 8
Hz); MS (DCI/NH3) m/z 357 [M + H]+. Anal. Calcd. For
C
20H24N2O4·0.25H2O: C, 66.56; H, 6.84; N, 7.76. Found: C,
66.69; H, 6.88; N, 7.53.
2-(N-Meth yltetr ah ydr oqu in ol-6-yl)-5-(3,4,5-tr im eth oxy-
p h en yl)-∆2,3-oxa zolin e (22e). 1,2,3,4-Tetrahydroquinoline-
6-carboxylic acid was prepared according to the published
procedure.
2-(3-Am in o-4-m eth oxyph en yl)-5-(3,4,5-tr im eth oxyph en -
yl)-∆2,3-oxa zolin e (22b). To a suspension of 3-amino-4-
methoxybenzoic acid (1.00 g, 6.0 mmol) in acetone (15 mL) was
added Fmoc-succinimide (2.63 g, 7.8 mmol) in acetone (15 mL),

4428 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Szczepankiewicz et al.
16
To a solution of 4.4 g (25 mmol) of 1,2,3,4-tetrahydro-
filtered, and concentrated to give 2.65 g (95%) of 1-methylin-
dole-6-carboxylic acid.
quinoline-6-carboxylic acid in 200 mL of methanol were added
10.3 g (75 mM) of K2CO3 and 7.7 mL (120 mmol) of io-
domethane. The resulting mixture was stirred overnight at
ambient temperature. The methanol was removed by concen-
tration in vacuo. Water (200 mL) was added to the residual
oil, and the mixture was extracted with ether (2 × 100 mL).
The aqueous layer was acidified to pH 6 with 6 N HCl, and
the mixture was extracted with ethyl acetate (2 × 200 mL).
The organic layer was dried (MgSO4), filtered, and concen-
trated in vacuo to give 1.8 g (38%) of 1-methyl-1,2,3,4-
tetrahydroquinoline-6-carboxylic acid as a yellow powder.
The acid (0.95 g, 5.0 mM) was coupled with amino alcohol
20 using EDCI, DMAP, and Et3N in THF/DMF. Silica gel
column chromatography (3:1 CH2Cl2/EtOAc and then 1:1 CH2-
Cl2/EtOAc) gave 100 mg (5%) of the amide as a yellow powder.
To an ice-cooled solution of the amide (100 mg, 0.25 mM) in
CHCl3 (2 mL) was added thionyl chloride (36 µL, 0.50 mM),
and the reaction was stirred for 30 min. Saturated aqueous
NaHCO3 (3 mL) was added, and the reaction was extracted
with CH2Cl2 (3 × 4 mL). The combined organic layers were
dried (MgSO4), filtered, and concentrated in vacuo to give a
crude oil. Silica gel column chromatography (3:2 CH2Cl2/
EtOAc) gave 15 mg (16%) of a light yellow oil: 1H NMR (300
MHz, CDCl3) δ 7.89-7.81 (m, 1 H), 7.72 (br s, 1 H), 6.58-6.54
(m, 3H), 5.66-5.53 (m, 1H), 4.50-4.38 (m, 1H), 4.09-3.91 (m,
1H), 3.85 (s, 9H), 3.35 (t, 2H, J ) 6.0 Hz), 2.98 (s, 3H), 2.78 (t,
2H, J ) 6.0 Hz), 2.01-1.93 (m, 2H); MS (DCI/NH3) m/z 383
[M + H]+.
2-(1-Meth yl-2,3-d ih yd r oin d ol-5-yl)-5-(3,4,5-tr im eth oxy-
p h en yl)-∆2,3-oxa zolin e (22f). A suspension of indole-5-
carboxylic acid, N-[2-hydroxy-2-(3,4,5-trimethoxyphenyl)ethyl]-
amide (207 mg, 0.56 mmol), and paraformaldehyde (168 mg,
5.6 mmol) in acetic acid (5 mL) was treated with sodium
cyanoborohydride (176 mg, 2.79 mmol), stirred for 18 h, and
then concentrated. The concentrate was partitioned between
ethyl acetate and 10% NaHCO3, washed sequentially with 10%
NaHCO3 and brine, dried over MgSO4, filtered, and concen-
trated to provide 218 mg of N-methylindoline-5-carboxylic
acid: 1H NMR (300 MHz, CDCl3) δ 7.51 (m, 2H), 6.63 (s, 2H),
6.38 (d, 1H, J ) 8.1 Hz), 4.90 (m, 1H), 3.85 (m, 10H), 3.5 (m,
1H), 3.45 (t, 2H, J ) 8.5 Hz), 3.14 (s, 1H), 2.99 (t, 2H, J ) 8.5
Hz), 2.82 (s, 3H); MS (ESI) m/z 387 [M + H]+.
Compound 22f was prepared according to the same proce-
dure as compound 22h , substituting N-methylindoline-5-
carboxylic acid for indole-5-carboxylic acid. The concentrate
was purified by flash column chromatography on silica gel with
1:1 ethyl acetate/hexanes to provide 25 mg of the desired
product: 1H NMR (300 MHz, CDCl3) δ 7.92 (dd, 1H, J ) 1.7,
8.5 Hz), 7.73 (d, 1H, J ) 1.4 Hz), 6.58 (s, 2H), 6.38 (d, 2H, J
) 8.5 Hz), 5.70 (app t, 1H, J ) 9.2 Hz), 4.53 (dd, 1H, J ) 10.2,
13.2 Hz), 4.22 (dd, 1H, J ) 8.5, 12.9 Hz), 3.88 (s, 6H), 3.86 (s,
3H), 3.59 (t, 2H, J ) 8.5 Hz), 3.06 (t, 2H, J ) 8.5 Hz), 2.90 (s,
3H); MS (DCI/NH3) m/z 369 [M + H]+.
To a solution of 430 mg (2.46 mmol) of 1-methylindole-6-
carboxylic acid in 20 mL of DMF were added 2 mL of
triethylamine and 500 mg (3.69 mmol) of 1-hydroxybenzot-
riazole. Next, 519 mg (2.71 mmol) of EDCI was added, and
then the mixture was stirred at ambient temperature for 15
min. After this time, 1.06 g (3.69 mmol) of amino alcohol 20
was added, along with 10 mg (catalytic amount) of 4-(N,N-
dimethylamino)pyridine. After the resulting solution is stirred
for 18 h, additional EDCI can be added if any carboxylic acid
remains. The reaction was poured into ethyl acetate, then
extracted once with aqueous 1 M NaHSO4 solution, once with
water, twice with saturated aqueous NaHCO3 solution, and
once with brine, dried over MgSO4, filtered, and concentrated
to give 471 mg (50%) of 1-methyl-1H-indole-5-carboxylic acid
N-[2-hydroxy-2-(3,4,5-trimethoxyphenyl)ethyl]amide.
To a solution of 159 mg (0.41 mmol) of 1-methyl-1H-indole-
5-carboxylic acid N-[2-hydroxy-2-(3,4,5-trimethoxyphenyl)ethy-
l]amide in 4 mL of THF was added 128 mg (0.54 mmol) of
[(methoxycarbonyl)sulfamoyl]triethylammonium hydroxide, in-
ner salt (Burgess reagent). The reaction was heated at reflux
for 1 h, then cooled, and poured into saturated aqueous
NaHCO3 solution. The aqueous suspension was extracted with
CH2Cl2, the organic layers were dried over Na2SO4, and the
product was purified via silica gel chromatography, eluting
with 70% ethyl acetate/hexanes, to give 78 mg (52%) of
oxazoline 22g: 1H NMR (300 MHz, d6-DMSO) δ 3.65 (s, 3H),
3.75 (s, 6H), 3.83 (s, 3H), 3.86 (dd, 1H, J ) 8.4, 15.0 Hz), 4.37
(dd, 1H, J ) 9.9, 15.0 Hz), 5.67 (dd, 1H, J ) 8.4, 9.9 Hz), 6.55
(d, 1H, J ) 3.3 Hz), 6.69 (s, 2H), 7.42 (d, 1H, J ) 3.3 Hz), 7.53
(d, 1H, J ) 8.7 Hz), 7.78 (dd, 1H, J ) 1.5, 9.0 Hz), 8.16 (s,
1H); MS (DCI/NH3) m/z 367 [M + H]+. Anal. Calcd for
C
21H22N2O4: C, 68.84; H, 6.05; N, 7.65. Found: C, 68.57; H,
5.92; N, 7.48.
2-(In d ol-5-yl)-5-(3,4,5-t r im et h oxyp h en yl)-∆2,3-oxa zo-
lin e (22h ). A solution of 5-indolecarboxylic acid (2.0 g, 12.4
mmol) in DMF (25 mL) at ambient temperature was treated
portionwise with 1,1′-carbonyldiimidazole (2.1 g, 13.0 mmol).
In a separate reaction, a suspension of amino alcohol 20 in
DMF (20 mL) was treated with diisopropylethylamine (5.0 mL,
28.7 mmol) and 4-(N,N-dimethylamino)pyridine (30.3 mg, 0.25
mmol). When the suspension of the amino alcohol cleared, the
imidazolide solution was transferred dropwise to the amino
alcohol solution. The pink solution was stirred at ambient
temperature for 3 h, treated with water (100 mL), and adjusted
to pH 5.5 with 3 N HCl to cause precipitation of a white solid.
The solid was filtered, washed with cold water, and dried in a
vacuum oven to provide 3.7 g of the desired amide as a white
solid.
A solution of the above amide (200 mg, 0.54 mmol) in THF
(10 mL) was treated with [(methoxycarbonyl)sulfamoyl]tri-
ethylammonium hydroxide, inner salt (Burgess reagent) (142
mg, 0.60 mmol), stirred at reflux for 1 h, cooled, and parti-
tioned between ethyl acetate and brine. The organic layer was
dried (Na2SO4), filtered, and concentrated. The concentrate
was purified by flash column chromotography on silica gel with
1:1 hexanes/ethyl acetate to provide 120 mg of oxazoline 22h
as a light yellow solid: 1H NMR (300 MHz, CDCl3) δ 3.86 (s,
9H), 4.04 (dd, 1H, J ) 8.7, 15.0 Hz), 4.50 (dd, 1H, J ) 9.9,
15.0 Hz), 5.63 (dd, 1H, J ) 8.7, 9.9 Hz), 6.61 (s, 2H), 6.64 (m,
1H), 7.28 (t, 1H, J ) 3.0 Hz), 7.46 (d, 1H, J ) 9.7 Hz), 7.95
(dd, 1H, J ) 1.8, 9.7 Hz), 8.37 (s, 1H), 8.43 (br s, 1H); MS (DCI/
NH3) m/z 353 [M + H]+. Anal. Calcd for C20H20N2O4: C, 68.17;
H, 5.72; N, 7.95. Found: C, 68.42; H, 5.58; N, 7.77.
2-(1-Meth ylin d ol-5-yl)-5-(3,4,5-tr im eth oxyp h en yl)-∆2,3-
oxa zolin e (22g). To a solution of 1.00 g (6.20 mmol) of indole-
6-carboxylic acid in 50 mL of DMF was added 745 mg of dry
NaH, freshly prepared by washing 60% NaH in mineral oil
with hexanes. The reaction was stirred for 25 min, and then
4 mL (62 mmol) of iodomethane was added. After being stirred
for 18 h, the reaction was poured into 1.1 M NaHSO4 and
extracted with ethyl acetate. The organic layers were back-
extracted sequentially with water, saturated aqueous NaHCO3
solution, and brine, dried over MgSO4, filtered, and concen-
trated to give 1.14 g (97%) of 1-methyl-6-(methoxycarbonyl)-
indole.
To a solution of 3.0 g (15.9 mmol) of 1-methyl-6-(methoxy-
carbonyl)indole in 50 mL of THF was added 50 mL of aqueous
1 M LiOH. The reaction was stirred at 50 °C for 24 h and then
diluted with diethyl ether. The reaction was extracted twice
with saturated aqueous NaHCO3 solution, and then 6 M HCl
was added to the combined aqueous layers to adjust the pH
to 1. The acid layer was then extracted with ethyl acetate, and
the combined ethyl acetate layers were dried over Na2SO4,
5-(3-Meth yl-4-am in oph en yl)-2-acetyl-3-(3,4,5-tr im eth oxy-
p h en yl)-∆1,5-p yr a zolin e (28). A mixture of 3-methyl-4-
nitrobenzoic acid (23) (1.80 g, 0.01mol) and 1.1 equiv of 1,1′-
carbonyldiimidazole was dissolved in THF. In a separate flask,
3,4,5-trimethoxyacetophenone (2.10 g, 0.01 mol) was dissolved
in THF at -78 °C, 1.1 equiv of LiHMDS was added to the
ketone solution, and the reaction was stirred for 1 h. The
nitrobenzoic acid solution was added slowly at -78 °C, and
the resulting mixture was warmed to ambient temperature

Oxadiazoline- and Oxazoline-Based Antimitotic Agents
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4429
and stirred for 2 h. After aqueous workup, 1,3-diketone 24 was
purified through silica gel column chromatography, eluting
with hexane/EtOAc (3:2): yield 2.80 g (75%).
suspended in ethanol, filtered, washed with water and then
ethanol, and dried in vacuo to give diacylhydrazine 36.
To 100 mg of diacylhydrazine 36 was added 1 mL of POCl3.
The reaction was stirred at ambient temperature for 24 h and
then heated at 85 °C for 20 min. The mixture was poured over
20 mL of ice, then 25 mL of 2 M NaOH was added, and the
mixture was extracted with ethyl acetate (3 × 10 mL). The
combined ethyl acetate layers were back-extracted with brine
(1 × 10 mL), dried over MgSO4, filtered, and concentrated to
an oil. The product was recrystallized from toluene/hexanes
to give 20 mg of a colorless solid: 1H NMR (300 MHz, CDCl3)
δ 3.08 (s, 6H), 3.93 (s, 3H), 3.98 (s, 6H), 6.80 (d, 2H, J ) 9.2
Hz), 7.34 (s, 2H), 7.99 (d, 2H, J ) 8.8 Hz); MS (DCI) m/z 356
[M + H]+. Anal. Calcd for C19H21N3O4·0.05H3PO4: C, 63.34;
H, 5.92; N, 11.66. Found: C, 63.36; H, 5.79; N, 11.49.
2-[4-(N,N-Dim eth yla m in o)p h en yl]-5-(3,4,5-tr im eth oxy-
p h en yl)-1,3-d ioxola n e (37). To 1.07 g (5.51 mmol) of 3,4,5-
trimethoxystyrene9 in 3 mL of acetone and 968 mg (8.27 mg)
of N-morpholine N-oxide in 1 mL of water was added 1 mL of
a 0.02 M solution of OsO4 in t-BuOH. The mixture was stirred
at ambient temperature for 71 h, and then 1.5 g of Na2S2O5
was added. After 1 h, the reaction was filtered, the salts were
washed with acetone, and the solvents were removed in vacuo.
The brown residue was taken up in 10 mL of ethyl acetate
and extracted with 1 M H2SO4 (2 × 5 mL), and then the
combined aqueous layers were back-extracted with ethyl
acetate (2 × 10 mL). The combined ethyl acetate layers were
washed with brine (1 × 5 mL), dried over MgSO4, filtered, and
concentrated to give 1.22 g of a brown oil.
To a solution of 1.22 g (5.5 mmol) of the crude 1,2-diol, 900
mg (6.0 mmol) of 4-(N,N-dimethylamino)benzaldehyde, and 1.4
g (7.4 mmol) of p-toluenesulfonic acid in 5 mL of toluene was
added 1.2 g of crushed 4 Å molecular sieves. The reaction was
stirred at ambient temperature for 3 h, and then 10 mL of
saturated aqueous NaHCO3 solution was added. The mixture
was filtered, and then the layers were separated. The aqueous
layer was extracted with ethyl acetate (2 × 5 mL), and then
the combined organic layers were washed with water (1 × 5
mL) and then brine (1 × 5 mL), dried over MgSO4, filtered,
and concentrated to an oil. This was purified via silica gel
chromatography, eluting with 30% ethyl acetate/hexanes, to
give the product as a mixture of acetal diastereomers: 1H NMR
(300 MHz, CDCl3) δ 3.09 (s, 6H), 3.84 (s, 3H major), 3.84 (s,
6H major), 3.86 (s, 3H minor), 3.88 (s, 6H, minor), 3.96 (dd,
1H major, J ) 5.9, 8.0 Hz), 4.33 (dd, 1H major, J ) 7.1, 7.8
Hz), 4.50 (m, 1H minor), 5.11 (dd, 1H major, J ) 6.6, 7.0 Hz),
5.93 (s, 1H major), 6.12 (s, 1H major), 6.63 (s, 2H minor), 6.65
(s, 2H major), 6.74 (m, 2H major), 7.41 (m, 2H minor), 7.44
(m, 2H major); MS (DCI/NH3) m/z 360 [M + H]+. Anal. Calcd
for C20H25NO5: C, 66.84; H, 7.01; N, 3.90. Found: C, 66.94;
H, 7.14; N, 3.89.
To a solution of 1,3-diketone 24 in CH2Cl2 and EtOH was
added 1.1 equiv of hydrazine monohydrate, and then the
solution was stirred at room temperature overnight. After
aqueous workup, acetic anhydride was added and heated. After
aqueous workup, the nitro group was hydrogenated on Pd/C
(10%) in EtOH. The catalyst was filtered away, and the solvent
was removed in vacuo to give regioisomeric amides 26 and 27.
The isomers were separated and purified by silica column
chromatography. The slower moving compound was pyrazole
28: 1H NMR (CDCl3, 300 MHz) δ 1.54 (s, 2H), 2.23 (s, 3H),
2.79 (s, 3H), 3.89 (s, 3H), 3.96 (s, 6H), 6.59 (s, 1H), 6.75 (d,
1H, J ) 7.5 Hz), 7.10 (s, 2H), 7.18 (d, 1H, J ) 7.5 Hz), 7.20 (s,
1H); MS (DCI/NH3) m/z 382 [M + H]+. Anal. Calcd for
C
21H23N3O4·0.25H2O: C, 65.36; H, 6.14; N, 10.89. Found: C,
65.58; H, 5.83; N, 10.76.
N-Boc-N′-[4-(N,N-d im et h yla m in o)b en zoyl]h yd r a zin e
(29). 29 was prepared according to the general synthesis for
hydrazides 2: 1H NMR (300 MHz, CDCl3) δ 7.71 (m, 2H), 7.60
(m, 1H), 6.66 (m, 3H), 3.04 (s, 6H), 2.49 (s, 9H); MS (DCI/NH3)
m/z 280 [M + H]+.
N-Boc-N′-[4-(N,N-d im eth yla m in o)th ioben zoyl]h yd r a -
zin e (30). To a solution of 1.5 g (5.15 mmol) of 29 in 35 mL of
THF was added 2.08 g (5.15 mmol) of 2,4-bis(4-methoxyphe-
nyl)-1,3-dithia-2,4-diphosphetane 2,4-disulfide (Lawesson’s re-
agent). The mixture was warmed to 45 °C, stirred for 3 h, then
cooled, filtered to remove Lawesson’s reagent, and concen-
trated under reduced pressure. The thiohydrazide was crystal-
lized from hot absolute EtOH to give a yellow solid: yield 825
mg (54%).
To 700 mg (2.28 mmol) of the thio-Boc-hydrazide in 10 mL
of CH2Cl2 was added 10 mL of trifluoroacetic acid. The solution
was stirred for 2 h at ambient temperature, and then the
solvents were evaporated under reduced pressure to give
thiohydrazide 30 as its TFA salt.
2-[4-(N,N-Dim eth yla m in o)p h en yl]-5-(3,4,5-tr im eth oxy-
p h en yl)-∆2,3-th ia d ia zolin e (32). To hydrazine 30 was added
447 mg (2.28 mmol) of 3,4,5-trimethoxybenzaldehyde dissolved
in 20 mL of EtOH. This mixture turned red, and then a yellow
solid precipitated after 30 min. This was collected and washed
with EtOH to give the desired product 32 (677 mg, 80%): 1H
NMR (CDCl3, 300 MHz) δ 3.05 (s, 6H), 3.92 (s, 3H), 3.95 (s,
6H), 6.75 (d, 2H, J ) 12 Hz), 7.26 (s, 2H), 7.87 (d, 2H, J ) 12
Hz); MS (DCI/NH3) m/z 372 [M + H]+. Anal. Calcd for
C
19H21N3O3S·0.25H2O: C, 60.70; H, 5.76; N, 11.18. Found: C,
60.99; H, 5.81; N, 11.03.
2-[4-(N,N-Dim eth yla m in o)p h en yl]-4-a cetyl-5-(3,4,5-tr i-
m eth oxyp h en yl)-∆2,3-th ia d ia zolin e (33). A 300 mg (1.02
mmol) sample of thio-Boc-hydrazine 30 and 1 mmol of 3,4,5-
trimethoxybenzaldehyde were dissolved in 5 mL of CH2Cl2 and
5 mL of trifluoroacetic acid and stirred for 2 h at ambient
temperature, and then the solvents were evaporated under
reduced pressure. Acetic acid (2 mL) was added, and the
mixture was stirred for 20 min under argon. Acetic anhydride
(4 mL) was added, and then the reaction was heated to 95 °C
and stirred for 30 min. The solvents were evaporated, and then
the residue was suspended in saturated aqueous NaHCO3 and
extracted with EtOAc. The product was purified via silica gel
chromatography, eluting with hexane/EtOAc (4:1 to 3:2), and
crystallized from ether to give 212 mg (50%) of compound 33:
1H NMR (CDCl3, 300 MHz) δ 2.43 (s, 3H), 3.04 (s, 6H), 3.82
(s, 3H), 3.84 (s, 6H), 6.57 (s, 2H), 6.70 (d, 2H, J ) 11 Hz), 6.98
(s, 1H), 7.62 (d, 2H, J ) 11 Hz); MS (FAB) m/z 416 [M + H]+.
Anal. Calcd for C21H25N3O4S: C, 60.70; H, 6.06; N, 10.11.
Found: C, 60.34; H, 5.97; N, 9.93.
In Vitr o Assa ys. Cells in 48- or 96-well plates in growth
medium were treated with compounds at different concentra-
tions for 48 h. The cells were trypsinized, and the number of
cells in each well was determined using a Coulter counter.8
In Vivo Stu d ies. Mice were obtained from Charles River,
Wilmington, MA. Female BALB/cAnNCrlBR × male DBA/
2NCrlBR (hereafter referred to as CDF1), female C57BL/
6NcrlBR × male DBA/2NCrlBR (hereafter referred to as
BDF1), and C57BL/6 mice were housed 10 animals/cage on
bedding and given free access to food and water. P388
leukemia cells were obtained from Arthur D. Little, Inc., while
B16 melanoma and M5076 reticulum cell sarcoma were
obtained from the National Cancer Institute (Bethesda, MD).
For iv inoculations, P388, B16, and M5076 tumor cells were
harvested from ascites fluid of previously inoculated mice.
P388 (106 cells/mouse) was injected iv into CDF1 mice, and
M5076 and B16 (106 cells/mouse) were injected iv into BDF1
mice. The drug efficacy of iv tumors was based on the percent
ILS of treated versus untreated mice.
2-[4-(N,N-Dim eth yla m in o)p h en yl]-5-(3,4,5-tr im eth oxy-
p h en yl)oxa d ia zole (34). To a suspension of 896 mg (5.00
mmol) of [4-(N,N-dimethylamino)benzoyl]hydrazine in 10 mL
of ethyl acetate was added 0.8 mL of triethylamine, and then
a solution of 1.15 g (5.00 mmol) of 3,4,5-trimethoxybenzoyl
chloride in 5 mL of ethyl acetate was added. The mixture was
stirred for 3 h at ambient temperature, then concentrated,
For the solid tumor model, M5076 cells were prepared from
homogenization of the solid tumor tissue. A 1:10 (w/v) tumor
homogenate using Hanks buffered salt solution was made, and
0.5 mL was injected sc into C57BL/6 mice. The tumors were

4430 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Szczepankiewicz et al.
(8) Wu-Wong, J . R.; Alder, J . D.; Alder, L.; Burns, D. J .; Han, E.
K.-H.; Credo, R. B.; Tahir, S. K.; Dayton, B. D.; Ewing, P. J .;
Chiou, W. J . Identification and Characterization of A-105972,
an Antineoplastic Agent. Cancer Res. 2001, 61, 1486-1492.
(9) Reddy, K. L.; Sharpless, K. B. From Styrenes to Enantiopure
R-Arylglycines in Two Steps. J . Am. Chem. Soc. 1998, 120,
1207-1217.
(10) Tahir, S. K.; Han, E. K-H.; Credo, B.; J ae, H.-S.; Pietenpol, J .
A.; Scatena, C. D.; Wu-Wong, J . R.; Frost, D.; Sham, H.;
Rosenberg, S. H.; Ng, S.-C. A-204197, A Novel Oxadiazoline
Derivative with Antimitotic Activity in Multidrug-Sensitive and
-Resistant Tumor Cell Lines. Cancer Res. 2001, 61, 5480-5485.
(11) Andreu, J . M.; Perez-Ramirez, B.; Gorbunoff, M. J .; Ayala, D.;
Timasheff, S. N. Role of the Colchicine Ring A and Its Methoxy
Groups in the Binding to Tubulin and Microtubule Inhibition.
Biochemistry 1998, 37, 8356-8368.
(12) Old, D. W.; Wolfe, J . P.; Buchwald, S. L. A Highly Active Catalyst
for Palladium-Catalyzed Cross-Coupling Reactions: Room-Tem-
perature Suzuki Couplings and Amination of Unactivated Aryl
Chlorides. J . Am. Chem. Soc. 1998, 120, 9722-9723.
(13) Detailed studies of the biological mechanism of action for
A-259745 will be the subject of a future publication.
(14) Attempts to prepare compound 1 from (3-methyl-4-nitrobenzoyl)-
hydrazine gave a penultimate nitro compound that could not be
reduced to the p-amine without destruction of the oxadiazoline
ring.
measured with vernier calipers. The drug efficacy was based
on the percent ILS using the mean percent increase in life span
compared to the vehicle in time for a 1 cm3 tumor. For all
models, group sizes consisted of 10 mice for each treatment
and all experiments began on day 1 post tumor inoculation.
Ack n ow led gm en t. We thank the combinatorial
chemistry group (D4CP) and members of D47B for their
support of this effort. This research was sponsored by
a profit-making company, Abbott Laboratories.
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