Synthesis and investigation of conformationally restricted analogues of lavendustin A as cytotoxic inhibitors of tubulin polymerization

Article abstract of DOI:10.1021/jm0202270

A series of conformationally restricted analogues were synthesized in order to elucidate the possible effects of different amide conformations of lavendustin A derivatives on cytotoxicity in cancer cell cultures and on inhibition of tubulin polymerization. The conformationally restricted analogues were based on the oxazinedione and isoindolone ring systems. In addition, the amide bond was replaced by both cis and trans alkene moieties. Surprisingly, the results indicated very little effect of conformational restriction on biological activity. Because all of the compounds synthesized had similar cytotoxicities and potencies as tubulin polymerization inhibitors, the side chain present on the aniline ring system does not appear to be important in the biological effects of the lavendustins. The hydroquinone ring of lavendustin A may be a more important determinant of the biological activity than the structure surrounding the aniline ring.

Full text of DOI:10.1021/jm0202270

4774
J . Med. Chem. 2002, 45, 4774-4785
Syn th esis a n d In vestiga tion of Con for m a tion a lly Restr icted An a logu es of
La ven d u stin A a s Cytotoxic In h ibitor s of Tu bu lin P olym er iza tion
Fanrong Mu,† Debbie J . Lee,‡ Donald E. Pryor,‡ Ernest Hamel,‡ and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences,
Purdue University, West Lafayette, Indiana 47907, Screening Technologies Branch, Developmental Therapeutics Program,
Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National Institutes of Health,
Frederick, Maryland 21702
Received May 28, 2002
A series of conformationally restricted analogues were synthesized in order to elucidate the
possible effects of different amide conformations of lavendustin A derivatives on cytotoxicity
in cancer cell cultures and on inhibition of tubulin polymerization. The conformationally
restricted analogues were based on the oxazinedione and isoindolone ring systems. In addition,
the amide bond was replaced by both cis and trans alkene moieties. Surprisingly, the results
indicated very little effect of conformational restriction on biological activity. Because all of
the compounds synthesized had similar cytotoxicities and potencies as tubulin polymerization
inhibitors, the side chain present on the aniline ring system does not appear to be important
in the biological effects of the lavendustins. The hydroquinone ring of lavendustin A may be a
more important determinant of the biological activity than the structure surrounding the aniline
ring.
In tr od u ction
variety of esters, alkoxyamides, and amides of general
structure 3 that were found to inhibit PTK activity both
in vitro and in cellular systems.14,15,21,23 Although
compounds of general structure 3 are cytotoxic, it seems
unlikely for the following four reasons that this cyto-
toxicity is due to PTK inhibition: (i) Whereas MCF-10A
cells are EGF-dependent, while MCF-7 cells are not, a
series of amides of general structure 3 were equally
effective as inhibitors of DNA synthesis in both breast
cancer cell lines.21 (ii) Because almost all of the EGFR
tyrosine kinase activity must be inhibited before effects
are seen on cell growth,24 it is unlikely that the potencies
of the amides 3 as EGFR inhibitors could possibly be
responsible for the effects seen on cancer cell growth,
because the IC50 values for EGF PTK inhibition are
close to their IC50 values for cytotoxicity.21 (iii) The fact
that lavendustin A (1) did not inhibit the PTK activity
of the mutant protein pp60F527, but nevertheless did
exhibit antiproliferative activity, suggests that the
antiproliferative effects of 1 could be due to actions on
cellular targets downstream from pp60F527 or on unre-
lated receptors.16 (iv) The potencies of amides 3 as
inhibitors of both the EGF receptor and the nonreceptor
PTK Syk did not correlate particularly well with their
cytotoxicities toward cancer cell lines.21 In view of these
considerations, a COMPARE analysis25 of the cytotox-
icity profiles of amides 3 was performed, and the results
suggested that the cytotoxicities of these compounds
could be due to inhibition of tubulin polymerization.21
When tested as inhibitors of tubulin polymerization,
amides of general structure 3 were in fact found to be
active, and their IC50 values for both inhibition of
tubulin polymerization and for cytotoxicity were rela-
tively close. The most cytotoxic compound in the prior
series was the lavendustin A analogue 4, which dis-
Because the activities of protein-tyrosine kinases
(PTKs) are elevated in diseases that are characterized
by rapid cellular proliferation, the design and synthesis
of PTK inhibitors are a logical strategy for the develop-
ment of new anticancer drugs.1-4 Recent examples of
clinically useful PTK inhibitors include the Bcr-Abl
tyrosine kinase inhibitor Gleevec (imatinib mesylate,
STI571)5,6 and the HER2/neu receptor monoclonal an-
tibody Herceptin (trastuzumab).7,8 Interest in the Strep-
tomyces griseolavendus metabolite lavendustin A (1;
Chart 1) has been stimulated by its ability to function
as a PTK inhibitor.9 Evaluation of the synthetic inter-
mediate 2 as an inhibitor of the epidermal growth factor
(EGF) PTK revealed that it is as potent as the natural
product 1.9 Lineweaver-Burke plots indicated that
inhibition of the EGF PTK by lavendustin A was
competitive with respect to adenosine 5′-triphosphate
(ATP) and noncompetitive with respect to substrate.9
However, a subsequent kinetic analysis of the inhibition
of the EGF PTK by lavendustin A suggested that it is a
hyperbolic mixed type inhibitor with respect to both the
substrate and the ATP; therefore, it binds to the kinase
domain at a location that is distinct from both the ATP
and substrate binding sites.10
These initial reports on lavendustin A (1) and its
biologically active fragment 2 stimulated a great deal
of research on the synthesis of 1 and a variety of
structural analogues.11-21 Although it was soon realized
that lavendustin A (1) was inactive as a PTK inhibitor
in cellular systems, the methyl ester of 1 inhibited PTK
activity and internalization of the EGF receptor in cell
culture.22 This observation led to the synthesis of a
* To whom correspondence should be addressed. Tel: 765-494-1465.
Fax: 765-494-1414. E-mail: cushman@pharmacy.purdue.edu.
† Purdue University.
‡ National Institutes of Health.
10.1021/jm0202270 CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/10/2002

Cytotoxic Inhibitors of Tubulin Polymerization
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4775
Ch a r t 1
Ch a r t 2
through insertion of a carbonyl would lead to the
oxazinedione structure 9. The methyl ether 8 was also
considered in order to investigate the possibility that
differences in activity between the isoindolone 7 and the
oxazinedione 9 could be due to the modification of the
phenolic hydroxyl group. Two additional conformations
of each rotamer 5 and 6 are possible due to rotation
about the amide bond (e.g., E and Z conformations 10
and 11 (Chart 2), respectively). To investigate the
relative contributions of these conformations to the
biological effects, trans alkene analogues 12, as well as
their cis counterparts 13, were also considered for
synthesis as conformationally restricted analogues. The
compounds synthesized in the present study were
examined for cytotoxicity in the National Cancer Insti-
tute (NCI) screen of human cancer cell lines and for
inhibition of tubulin polymerization.
Ch em istr y
played a mean graph midpoint (MGM) of 0.35 µM in
the screen of approximately 55 human cancer cell
lines.21
Because in the prior series of lavendustin A amide
derivatives maximal activity was observed with the 2-(4-
fluorophenyl)ethyl substituent present in compound 4,
it was decided to incorporate that substituent, along
with the corresponding defluorinated substituent, in the
rigid analogues considered for synthesis.21 Because the
n-hexyl derivative (5 and 6, R ) n-hexyl) was particu-
larly potent relative to 2 as an EGF receptor tyrosine
kinase inhibitor, it was also decided to utilize that
substituent in the present series of compounds.18
The preparation of the desired conformationally re-
stricted analogues 20a -c is outlined in Scheme 1. The
synthesis relies on the ring contraction reaction of the
unstable 1,3-benzoxazepine-2,3,5-triones 16a -c to form
the desired 1,3-benzoxazine-2,4-dione intermediates
17a -c.26,27 Conversion of the starting material, 5-nitro-
2-hydroxybenzoic acid 14, to the corresponding amide
derivatives 15a -c, was carried out by reaction with the
required primary amines in the presence of DCC in
The lavendustin A analogue 4 and related compounds
can exist in different conformations resulting from
rotation around the bond connecting the amide carbonyl
to the aromatic ring (see structures 5 and 6). Both of
these conformations could be stabilized through in-
tramolecular hydrogen bonding between the phenolic
hydroxyl group and either the amide nitrogen or the
carbonyl. This raises the question of the relative con-
tributions of these two conformers to the observed
biological activity. One approach to investigating the
general question of conformational effects is to synthe-
size conformationally restricted analogues. In the present
case, the conformation represented by structure 5 could
be stabilized by attaching the amide nitrogen to the
aromatic ring through a methylene linker, suggesting
the isoindolone 7. On the other hand, the connection of
the amide nitrogen to the adjacent phenolic oxygen

4776 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Mu et al.
Sch em e 1
F igu r e 1. ORTEP plot of the X-ray crystal structure of
diacetate 22.
Sch em e 2
pyridine. Reaction of these compounds 15a -c with
oxalyl chloride in refluxing toluene afforded the required
1,3-benzoxazine-2,4-diones 17a -c. Catalytic reduction
of the nitro group present in 17a -c was performed in
the presence of hydrogen and 10% activated palladium
on carbon to provide the amines 18a -c. A reductive
amination reaction involving 2,5-dihydroxybenzalde-
hyde (19) and the three amines 18a -c afforded the
target compounds 20a -c.
It was anticipated that the conformationally restricted
analogues of general type 7 could be synthesized if the
substituted benzoic acid derivative 26 was available. In
considering possible routes for the synthesis of 26, we
decided to try to exploit the fact that the electron-
attracting nitro group in nitrotoluenes inhibits the
oxidation of the methyl group, and the effect is maximal
in o-nitrotoluenes and decreases in the order o > m >
p.28,29 To take advantage of this fact, 2,3-dimethyl-4-
nitroanisole (21) was chosen as the starting material
(Scheme 2). Subjection of 21 to the Thiele reagent
(CrO3-Ac2O-H2SO4) resulted in oxidative attack on the
methyl group that was more remote from the nitro
group, resulting in the formation of the diacetate 22 as
the major product as well as the monoacetate 23 as the
minor product. To be absolutely sure about the regio-
chemistry of the oxidation reaction, an X-ray structure
was obtained on a crystalline sample of the diacetate
22. The resulting ORTEP drawing, which is displayed
in Figure 1, proves that selective oxidation of the methyl
that is meta to the nitro group had in fact taken place.
After it was separated by column chromatography, the
diacetate 22 was converted to the aldehyde 24 under
acidic conditions.30 Oxidation of the aldehyde with
permanganate in aqueous sodium hydroxide at reflux
afforded the desired intermediate 26. To avoid wasting
any of the material, an attempt was also made to
convert the minor product 23 to 26. Hydrolysis of the
acetate 23 with sodium hydroxide in ethanol afforded
the corresponding primary alcohol 25, which was then
oxidized with permanganate to afford 26.
The conversion of intermediate 26 to the conforma-
tionally restricted target compounds 32a ,b is depicted
in Scheme 3. The acid 26 was converted to the expected
methyl ester 27 by treatment with (trimethylsilyl)-
diazomethane in anhydrous methanol at room temper-
ature. Benzylic bromination of 27 under Wohl-Ziegler
conditions (N-bromosuccinimide (NBS), dibenzoyl per-
oxide) yielded the anticipated intermediate 28, which
was converted to the isoindolones 29a ,b when heated
with â-phenethylamine or p-fluoro-â-phenethylamine,
respectively, in methanol in the presence of triethyl-

Cytotoxic Inhibitors of Tubulin Polymerization
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4777
Sch em e 3
Sch em e 4
Sch em e 5
amine. Cleavage of the methyl ethers present in 29a ,b
with boron tribromide provided the phenols 30a ,b.
Catalytic hydrogenation over palladium on carbon led
to the amines 31a ,b. Reaction of the amines 31a ,b with
the aldehyde 19 furnished the Schiff bases, which were
subjected to catalytic hydrogenation over palladium on
carbon to yield the target compounds 32a ,b.
The synthesis of the compounds 34a ,b, having a
methyl ether present in the isoindolone ring system in
place of the phenol located in 32a ,b, is outlined in
Scheme 4. Catalytic reduction of the nitro compounds
29a ,b afforded the aniline derivatives 33a ,b. Reaction
of 33a ,b with the aldehyde 19 resulted in the expected
Schiff bases, which were reduced to yield the required
rigid analogues 34a ,b.
A conformationally restricted analogue 42, which has
a cis alkene in place of the amide linkage in the side
chain, was designed and prepared as outlined in Scheme
5. The coupling partner 37 for the Sonogashira reaction
was made by methoxymethyl (MOM) protection of
2-bromo-4-nitrophenol (36), which was prepared by
selective monobromination of 4-nitrophenol (35) with
NBS in the presence of FSO3H in acetonitrile.31 The
palladium-catalyzed Sonogashira coupling reaction of
37 with 4-phenyl-1-butyne gave the disubstituted alkyne
38 in quantitative yield.32,33 Reduction of the nitro group
of 38 to the amine 39 was carried out with stannous
chloride (SnCl2) in ethanol.34 Stannous chloride was
chosen as the reducing agent because it is selective in
the catalytic reduction of aromatic nitro compounds
containing groups capable of undergoing catalylic hy-
drogenation (the triple bond in our molecule). In addi-
tion, the MOM protecting group was removed under
acidic condition during the workup process of the SnCl2

4778 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Mu et al.
Sch em e 6
Sch em e 7
reduction to furnish 40 without isolation of intermediate
39. Reduction of the alkyne 40 to the cis-substituted
alkene 41 was performed using the Lindlar catalyst
(Pd-CaCO3, poisoned with lead). The reaction was
closely monitored by thin-layer chromatography (TLC)
in order to avoid the reduction of the cis double bond.
Finally, the reductive amination with 2,5-dihydroxy-
benzaldehyde (19) and NaBH3CN reduction afforded
analogue 42.
To synthesize the counterpart of 42 having a trans
double bond, a Stille coupling reaction of the vinylstan-
nane 44 with the aryl bromide 37 was considered. As
shown in Scheme 6, the formation of the E-vinylstan-
nane 44 was first attempted using palladium-catalyzed
hydrostannylation of 4-phenyl-1-butyne (43).35-37 Al-
1
though the product gave one spot on TLC, the H NMR
Biologica l Resu lts a n d Discu ssion
spectrum indicated the formation of a mixture of both
regio- and stereoisomers 44-46. Separation of this
mixture by column chromatography failed.
The lavendustin A analogues were examined for
antiproliferative activity against the human cancer cell
lines in the NCI cytotoxicity screen, in which the activity
of each compound was evaluated using approximately
55 different cancer cell lines of diverse tumor origins.
The GI50 values, which are the concentrations of the
compounds producing 50% growth inhibition, are listed
in Table 1 for nine representative cell lines. The MGM
values listed in Table 1 are based on a calculation of
the average GI50 values for all of the cancer cell lines
tested (approximately 55) in which GI50 values below
and above the test range (10-4 to 10-8 M) are taken as
the minimum (10-8 M) and maximum (10-4 M) drug
concentrations used in the screening test.41 Also listed
in Table 1 are the IC50 values for inhibition of the
polymerization of purified bovine brain tubulin. In
addition to the new compounds synthesized in the
present study, data for the previously prepared ana-
logues 4, 51, and 52 (Chart 3) are also included in Table
1 for comparison purposes.21
It is apparent from the cytotoxicity data presented
in Table 1 that all of the lavendustin A analogues were
more active vs the leukemia cell line CCRF-CEM than
for the other cell lines investigated. This selectivity of
the lavendustin A analogues extended generally to all
of the other leukemia cell lines that were tested,
including HL-60(TB), K-562, RPMI-8226, MOLT-4, and
SR (data not shown).
The conformationally restricted oxazinediones 20a
(MGM 5.5 µM) and 20c (MGM 3.0 µM) were modestly
more potent than their ring-opened counterparts 51
(MGM 14.8 µM) and 52 (MGM 8.7 µM). On the other
In view of the difficulties encountered in the hy-
drostannylation of the alkyne 43, alternative methods
were sought for the synthesis of the desired E-vinyl-
stannane 44. Zhang et al. previously revealed that
1-bromoalkynes can be converted into (E)-1-vinylstan-
nanes in a regioselective manner using 2 equiv of
tributylstannane in the presence of PdCl2(PPh3)2.38 This
method was successfully applied in our synthesis of
analogue 50 (Scheme 7). The 1-bromoalkyne 47 was
easily prepared from the corresponding 4-phenyl-1-
butyne (43) by reaction with NBS in the presence of a
catalytic amount of silver nitrate.39 When solutions of
the 1-bromoalkyne 47 in tetrahydrofuran (THF) were
treated with 2 equiv of tributylstannane at room tem-
perature in the presence of a catalytic amount of Pd-
(PPh3)4, it was smoothly converted into the correspond-
1
ing (E)-1-vinylstannane (44). The H NMR spectrum of
44 displayed a doublet at δ 5.95 ppm with J ) 18.93
Hz and a doublet of triplets at δ 6.03 ppm with J ) 18.84
and 5.71 Hz, suggesting the formation of the E-isomer.
Because 44 was not separable from the byproduct bis-
(tributyltin), an in situ Stille coupling of the crude
vinylstannane 44 with aryl bromide 37 was carried out,
and the desired product 48 was obtained in 76% yield
in two steps.40 Next, following the procedure described
above, the nitro group in 48 was reduced with stannous
chloride, and the MOM protecting group was removed
to yield 49, which then underwent the reductive ami-
nation with 2,5-dihydroxybenzaldehyde (19) to form the
desired analogue 50.

Cytotoxic Inhibitors of Tubulin Polymerization
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4779
Ch a r t 3
hand, the fluorinated ring-opened compound 4 (MGM
0.35 µM) proved to be more active than its conforma-
tionally restricted oxazinedione analogue 20b (MGM 4.7
µM).
Considering the isoindolones 32a (MGM 12.0 µM) and
32b (MGM 12.4 µM), both of these compounds were
slightly less active than the corresponding oxazinedi-
ones 20a (MGM 5.5 µM) and 20b (MGM 4.7 µM). The
effect of methylation of the phenolic hydroxyl group in
the isoindolone ring of 32b, resulting in 34b (MGM 20.4
µM), was to slightly decrease potency.
Turning to the cis and trans alkenes 42 (MGM 13.1
µM) and 50 (MGM 19.5 µM), respectively, it is apparent
that the cis alkene is modestly more active than the
trans isomer, but the effect is very small. Both of the
alkenes were about as active as their amide counterpart
51 (MGM 14.8 µM). This reinforces the conclusion that
the conformation of the amide in lavendustin A amide
analogues is unimportant as far as their biological
activities are concerned, and it demonstrates that both
E and Z alkenes are biologically effective replacements
of the amides.
All of the compounds synthesized in this investigation
were relatively good inhibitors of tubulin polymerization
(IC50 < 10 µM). The striking aspect of their potencies is
how similar they actually are. The total range of IC50
values for inhibition of tubulin polymerization is 3.6-
9.5 µM. Although in general, the range of cytotoxicities
observed in the cancer cell cultures is also relatively
narrow, there is more variability in the MGM values
listed in Table 1 than there is in the IC50 values for
inhibition of tubulin polymerization. For example, at the
extreme, compound 51 is 1.1 times more potent than 4
as an inhibitor of tubulin polymerization, but it is 42
times less cytotoxic as judged by the corresponding
MGM values. This reflects the fact that the values for
inhibition of tubulin polymerization are determined in
a cell-free system, whereas the cytotoxicity values are
established in cellular systems, in which additional
factors such as cellular uptake, metabolism, and ad-
ditional targets may exert significant effects.

4780 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Mu et al.
mmol) and oxalyl chloride (0.15 mL, 1.8 mmol), a similar
procedure as that described for 17a gave 17b as a white
crystalline solid (0.42 g, 77%); mp 170-172 °C. 1H NMR
(CDCl3): δ 8.96 (d, J ) 2.50 Hz, 1 H), 8.58 (dd, J ) 9.07, 2.76
Hz, 1 H), 7.48 (d, J ) 9.16 Hz, 1 H), 7.23 (q, J ) 5.42 Hz, 2 H),
7.00 (t, J ) 8.56 Hz, 2 H), 4.26 (t, J ) 8.10 Hz, 2 H), 3.01 (t,
J ) 7.75 Hz, 2 H). CIMS m/z 331 (MH+). Anal. (C14H11FN2O5)
C, H, F, N.
3-Hexyl-6-n itr o-ben zo[e][1,3]oxa zin e-2,4-d ion e (17c).
From compounds 15c (0.50 g, 1.90 mmol) and oxalyl chloride
(0.19 mL, 2.2 mmol), a similar procedure as that described for
17a gave 17c as a white crystalline solid (0.50 g, 90%); mp
94-96 °C. 1H NMR (CDCl3): δ 8.98 (d, J ) 2.76 Hz, 1 H), 8.56
(dd, J ) 8.98, 2.64 Hz, 1 H), 7.47 (d, J ) 9.01 Hz, 1 H), 4.05
(t, J ) 7.52 Hz, 2 H), 1.72 (quint, J ) 7.42 Hz, 2 H), 1.34 (m,
6 H), 0.25 (t, J ) 6.82 Hz, 3 H). CIMS m/z 293 (MH+). Anal.
(C14H16N2O5) C, H, N.
Overall, the results indicate that the conformation of
the amide side chain in the lead compounds 4, 51, 52,
and related lavendustin A analogues does not exert a
significant effect on their biological activities.
Exp er im en ta l Section
Melting points were determined with a Mel-Temp capillary
melting point apparatus and are uncorrected. Nuclear mag-
netic resonance spectra for proton (1H NMR) were recorded
on a Bruker VXR-300S or DRX-500 spectrometer. The chemi-
cal shift values are expressed in ppm (parts per million)
relative to tetramethylsilane as internal standard; s ) singlet,
d ) doublet, t ) triplet, m ) multiplet, bs ) broad singlet.
Microanalyses were performed at the Purdue Microanalysis
Laboratory, and all values were within (0.4% of the calculated
compositions. Column chromatography was carried out using
Merck silica gel (230-400 mesh). Analytical TLC was per-
formed on silica gel GF (Analtech) glass-coated plates (2.5 cm
× 10 cm with 250 µM layer and prescored), and spots were
visualized with UV light at 254. Most chemicals and solvents
were analytical grade and used without further purification.
Commercial reagents were purchased from Aldrich Chemical
Co. (Milwaukee, WI).
5-Nitr o-N-(2-p h en eth yl)sa licyla m id e (15a ). DCC (1.24
g, 6.0 mmol) and 2-phenethylamine (0.63 mL, 5.0 mmol) were
added to a solution of 2-hydroxy-5-nitrobenzoic acid (14) (0.91
g, 5.0 mmol) in dry pyridine (50 mL). The mixture was heated
at 90-100 °C for 2 h. After it was cooled, the precipitate was
removed by filtration. The pyridine was removed on a rotary
evaporator. The residue was treated with 10% HCl (50 mL)
and stirred for 1 h. The yellow precipitate was filtered, washed
with water (2 × 50 mL), and dried in vacuo. Purification was
achieved by recrystallization from ethanol to yield pure 15a
as a yellow crystalline solid (1.31 g, 83%); mp 196-198 °C. 1H
NMR (CDCl3): δ 13.50 (s, 1 H, OH), 8.86 (bs, 1 H, NH), 8.70
(d, J ) 2.66 Hz, 1 H), 8.30 (dd, J ) 9.18, 2.67 Hz, 1 H), 7.32-
7.18 (m, 5 H), 7.10 (d, J ) 9.18 Hz, 1 H), 3.71 (q, J ) 7.81 Hz,
2 H), 2.98 (t, J ) 7.69 Hz, 2 H). CIMS m/z 287 (MH+). Anal.
(C15H14N2O4) C, H, N.
6-Am in o-3-p h en et h yl-b en zo[e][1,3]oxa zin e-2,4-d ion e
(18a ). Compound 17a (0.38 g, 1.2 mmol) was hydrogenated
over 10% palladium on activated carbon (wet, contains 50%
water, 0.5 g, 0.23 mmol) in ethanol (20 mL) at atmospheric
pressure for 10 h. The mixture was then filtered through Celite
and washed with acetone (50 mL). The combined filtrate was
evaporated on a rotary evaporator to give compound 18a as a
1
white solid (0.30 g, 89%); mp 193-195 °C. H NMR (DMSO-
d6): δ 7.35-7.17 (m, 5 H), 7.12 (d, J ) 8.85 Hz, 1 H), 7.06 (d,
J ) 2.65 Hz, 1 H), 6.98 (dd, J ) 8.85, 2.65 Hz, 1 H), 5.47 (s, 2
H, NH2), 4.05 (t, J ) 7.96 Hz, 2 H), 2.89 (t, J ) 7.96 Hz, 2 H).
CIMS m/z 283 (MH+). Anal. (C16H14N2O3) C, H, N.
6-Am in o-3-[2-(4-flu or o-p h e n yl)e t h yl]b e n zo[e][1,3]-
oxa zin e-2,4-d ion e (18b). Compound 17b (0.38 g, 1.15 mmol)
was hydrogenated over 10% palladium on activated carbon
(wet, contains 50% water, 0.05 g) in ethyl acetate (20 mL) at
atmospheric pressure for 4 h. The mixture was then filtered
through Celite and washed with ethyl acetate (10 mL). The
combined filtrate was evaporated on a rotary evaporator to
give compound 18b as a white solid (0.28 g, 81%); mp 212-
213 °C. 1H NMR (DMSO-d6): δ 7.26 (m, 2 H), 7.14-7.08 (m, 3
H), 7.06 (d, J ) 2.71 Hz, 1 H), 6.99 (dd, J ) 8.77, 2.76 Hz, 1
H), 5.47 (s, 2 H, NH2), 4.04 (t, J ) 7.65 Hz, 2 H), 2.89 (t, J )
7.43 Hz, 2 H). Anal. (C16H13FN2O3) C, H, F, N.
5-Nitr o-N-[2-(4-flu or op h en yl)eth yl]sa licyla m id e (15b).
From compounds 14 (0.91 g, 5.0 mmol), DCC (1.24 g, 6.0
mmol), and 4-fluoro-2-phenethylamine (0.65 mL, 5.0 mmol),
a similar procedure to that described for 15a yielded pure 15b
as a yellow crystalline solid (1.35 g, 89%); mp 206-208 °C. 1H
NMR (CDCl3): δ 13.49 (s, 1 H, OH), 8.32 (d, J ) 2.50 Hz, 1
H), 8.27 (dd, J ) 9.04, 2.57 Hz, 1 H), 7.22 (m, 2 H), 7.07 (t, J
) 8.74, 2 H), 7.01 (d, J ) 8.68 Hz, 1 H), 6.66 (bs, 1 H, NH),
3.75 (q, J ) 6.82 Hz, 2 H), 2.96 (t, J ) 7.19 Hz, 2 H). CIMS
m/z 305 (MH+). Anal. (C15H13FN2O4) C, H, F, N.
6-Am in o-3-h exyl-ben zo[e][1,3]oxa zin e-2,4-d ion e (18c).
Compound 17c (0.38 g, 1.15 mmol) was hydrogenated over 10%
palladium on activated carbon (wet, contains 50% water, 0.3
g) in ethyl acetate (25 mL) at atmospheric pressure for 3 h.
The mixture was then filtered through Celite and washed with
ethyl acetate (10 mL). The combined filtrate was evaporated
on a rotary evaporator to give the crude product, which was
purified by recrystallization from ethyl acetate-hexane. Pure
18c was obtained as a white solid (0.40 g, 89%); mp 113-115
1
5-Nitr o-N-(h exyl)sa licyla m id e (15c). From compound 14
(0.91 g, 5.0 mmol), DCC (1.24 g, 6.0 mmol), and hexylamine
(0.66 mL, 5.0 mmol), a similar procedure to that described for
15a yielded product 15c as a yellow crystalline solid (1.04 g,
°C. H NMR (DMSO-d6): δ 7.11 (d, J ) 9.19, 1 H), 7.07 (d, J
) 2.67 Hz, 1 H), 6.98 (dd, J ) 8.77, 2.78 Hz, 1 H), 5.45 (s, 2
H), 3.82 (t, J ) 7.3 Hz, 2 H), 1.57 (quint, J ) 7.09 Hz, 2 H),
1.27 (m, 6 H), 0.85 (t, J ) 6.50 Hz, 3 H). Anal. (C14H18N2O3)
C, H, N.
1
78%); mp 94-95 °C. H NMR (CDCl3): δ 13.51 (s, 1 H, OH),
8.42 (d, J ) 2.61 Hz, 1 H), 8.28 (dd, J ) 9.16, 2.67 Hz, 1 H),
7.08 (d, J ) 9.13 Hz, 1 H), 6.67 (bs, 1 H, NH), 3.50 (q, J )
7.02 Hz, 2 H), 1.67 (quint, J ) 7.44 Hz, 2 H), 0.49 (m, 6 H),
6-[N-(2,5-Dih ydr oxyph en yl)m eth yl]am in o]-3-ph en eth yl-
ben zo[e][1,3]oxa zin e-2,4-d ion e (20a ). 2,5-Dihydroxyben-
zaldehyde (19, 0.15 g, 1.06 mmol) was added to 18a (0.28 g,
0.99 mmol) in methanol (30 mL), and the mixture was heated
to reflux under argon for 6 h. The reaction mixture was then
cooled to room temperature, and the yellow precipitate was
removed by filtration and dried to obtain the imine (0.39 g),
which was hydrogenated over 10% palladium on activated
carbon (wet, contains 50% water, 0.02 g) in methanol (20 mL)
at atmospheric pressure for 15 min. The mixture was then
filtered through Celite and washed with ethyl acetate (10 mL).
The combined filtrate was evaporated on a rotary evaporator
to furnish the crude product, which was further purified by
flash chromatography (ethyl acetate-hexane 1:2). The product
20a (0.37 g, 92%) was isolated as an off-white solid; mp 201-
203 °C. 1H NMR (DMSO-d6): δ 8.86 (s, 1 H, OH), 8.57 (s, 1 H,
OH), 7.33-7.21 (m, 5 H), 7.16 (d, J ) 8.94 Hz, 1 H), 7.05 (dd,
J ) 8.87, 2.84 Hz, 1 H), 6.91 (d, J ) 2.53 Hz, 1 H), 6.65 (d, J
) 8.50 Hz, 1 H), 6.59 (d, J ) 2.63 Hz, 1 H), 6.54 (t, J ) 5.90
0.90 (t, J ) 6.94 Hz, 3 H). CIMS m/z 348 (MH+). Anal. (C13H18
-
INO2) C, H, I, N.
6-Nitr o-3-ph en eth yl-ben zo[e][1,3]oxazin e-2,4-dion e (17a).
A solution of 15a (0.71 g, 2.5 mmol) in toluene (10 mL) was
stirred at room temperature for 10 min. Oxalyl chloride (0.32
g, 2.5 mmol) in toluene (2.5 mL) was added, and the reaction
mixture was heated at reflux under argon for 6 h. After it was
cooled, the precipitate that appeared was removed by filtration
and 17a was recrystallized from ethanol (0.43 g, 55%); mp
1
180-181 °C. H NMR (CDCl3): δ 8.96 (d, J ) 2.53 Hz, 1 H),
8.55 (dd, J ) 9.02, 2.53 Hz, 1 H), 7.46 (d, J ) 9.03 Hz, 1 H),
7.38-7.22 (m, 5 H), 4.29 (t, J ) 8.31 Hz, 2 H), 3.03 (t, J )
7.95 Hz, 2 H). CIMS m/z 313 (MH+). Anal. (C16H12N2O5) C, H,
N.
3-[2-(4-F lu or o-p h e n yl)e t h yl]-6-n it r o-b e n zo[e][1,3]-
oxa zin e-2,4-d ion e (17b). From compound 15b (0.50 g, 1.64

Cytotoxic Inhibitors of Tubulin Polymerization
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4781
Hz, 1 H, NH), 6.45 (dd, J ) 8.56, 2.65 Hz, 1 H), 4.16 (d, J )
4.78 Hz, 2 H), 4.03 (t, J ) 7.57 Hz, 2 H), 2.87 (t, J ) 7.94 Hz,
2 H). CIMS m/z 405 (MH+). Anal. (C23H20N2O5) C, H, N.
60 mL). The combined ether layers were washed with brine
and dried over MgSO4. Evaporation of the solvent gave a
yellow solid, which was recrystallized from ethyl acetate-
hexane. Compound 25 (2.16 g, 87%) was obtained as a yellow
crystalline solid; mp 94-95 °C. 1H NMR (CDCl3): δ 7.90 (d, J
) 9.09 Hz, 1 H), 6.82 (d, J ) 9.09 Hz, 1 H), 4.80 (d, J ) 6.33
Hz, 2 H), 3.92 (s, 3 H), 2.57 (s, 3 H), 1.83 (t, J ) 6.36 Hz, 1 H,
OH). CIMS m/z 198 (MH+). Anal. (C9H11NO4) C, H, N.
6-[N-(2,5-Dih yd r oxyp h en yl)m eth yl]a m in o]-3-[2-(4-flu -
or o-p h en yl)et h yl]b en zo[e][1,3]oxa zin e-2,4-d ion e (20b).
From compounds 19 (0.07 g, 0.5 mmol), 18b (0.15 g, 0.5 mmol),
H2, and 10% Pd-C (wet, contains 50% water, 0.02 g), a similar
procedure as that described for 20a gave pure 20b (0.18 g,
89%) as a light yellow solid; mp 199-200 °C. 1H NMR (DMSO-
d6): δ 8.85 (s, 1 H, OH), 8.57 (s, 1 H, OH), 7.25 (q, J ) 5.77,
2 H), 7.18 (d, J ) 8.94 Hz, 1 H), 7.11 (t, J ) 8.89 Hz, 2 H),
7.05 (dd, J ) 9.01, 2.81 Hz, 1 H), 6.91 (d, J ) 2.64 Hz, 1 H),
6.64 (d, J ) 8.52 Hz, 1 H), 6.59 (d, J ) 2.74 Hz, 1 H), 6.53 (t,
J ) 5.92 Hz, 1 H, NH), 6.43 (dd, J ) 8.50, 2.85 Hz, 1 H), 4.17
(d, J ) 4.99 Hz, 2 H), 4.02 (t, J ) 7.76 Hz, 2 H), 2.87 (t, J )
7.90 Hz, 2 H). CIMS m/z 423 (MH+). ESIMS m/z 421 (M -
H)-. Anal. (C23H19FN2O5) C, H, F, N.
5-Meth oxy-2-m eth yl-3-n itr oben zoic Acid (26) fr om 24.
Compound 24 (1.32 g, 6.8 mmol) was added to a solution of
potassium permanganate (2.14 g, 13.6 mmol) and NaOH (0.5
g, 12.5 mmol) in water (20 mL). The mixture was heated to
reflux for 2 h. After it was cooled, the mixture was acidified
with 3 N HCl and extracted with ethyl acetate (4 × 50 mL).
The combined ethyl acetate solution was dried over MgSO4,
and removal of the solvent furnished 26 (1.2 g, 84%) as a light
yellow solid; mp 152-154 °C.
6-[N -(2,5-Dih yd r oxyp h e n yl)m e t h yl]a m in o]-3-h e xyl-
ben zo[e][1,3]oxa zin e-2,4-d ion e (20c). From compounds 19
(0.17 g, 1.2 mmol), 18c (0.30 g, 1.14 mmol), and NaBH3CN
(0.28 g, 4.5 mmol), a similar procedure as that described for
20a gave 20c (0.33 g, 75%) as a light yellow solid; mp 152-
153 °C. 1H NMR (DMSO-d6): δ 8.85 (s, 1 H, OH), 8.56 (s, 1 H,
OH), 7.16 (d, J ) 8.94 Hz, 1 H), 7.06 (dd, J ) 8.94, 2.82 Hz, 1
H), 6.91 (d, J ) 2.64 Hz, 1 H), 6.63 (d, J ) 8.56 Hz, 1 H), 6.59
(d, J ) 2.76 Hz, 1 H), 6.52 (t, J ) 5.79 Hz, 1 H, NH), 6.43 (dd,
J ) 8.48, 2.91 Hz, 1 H), 4.17 (d, J ) 4.24 Hz, 2 H), 3.81 (t, J
) 7.13 Hz, 2 H), 1.56 (quint, J ) 7.42 Hz, 2 H), 1.27 (m, 6 H),
0.85 (t, J ) 6.45 Hz, 3 H). CIMS m/z 263 (MH+). Anal.
(C19H24N2O5) C, H, N.
5-Meth oxy-2-m eth yl-3-n itr oben zoic Acid (26) fr om 25.
Compound 25 (2.15 g, 10.9 mmol) was added to a solution of
potassium permanganate (3.4 g, 21.5 mmol) and NaOH (1.0
g, 25 mmol) in water (50 mL). The mixture was heated to
reflux for 2 h. After it was cooled, the mixture was acidified
with 3 N HCl and extracted with ethyl acetate (4 × 80 mL).
The combined ethyl acetate solution was dried over MgSO4,
and removal of the solvent furnished 26 (2.20 g, 96%) as a
light yellow solid; mp 152-154 °C. 1H NMR (DMSO-d6): δ 13.5
(bs, 1 H, OH), 8.11 (d, J ) 9.14 Hz, 1 H), 7.17 (d, J ) 9.14 Hz,
1 H), 3.90 (s, 3 H), 2.40 (s, 3 H). CIMS m/z 212 (MH+). Anal.
(C9H9NO5) C, H, N.
Meth yl 6-Meth oxy-2-m eth yl-3-n itr oben zoa te (27). A 2
M solution of (trimethylsilyl)diazomethane in hexane (10 mL)
was added dropwise to a solution of 26 (2.45 g, 11.6 mmol) in
methanol (40 mL) until the solution stayed yellow. After it was
stirred for 30 min at room temperature, the reaction mixture
was poured into ice-water (50 g) and extracted with ethyl
acetate (3 × 50 mL). The combined organic layers were washed
with brine (20 mL) and dried over MgSO4, and the solvent was
removed to furnish 27 (2.49 g, 95%) as a white crystalline solid;
6-Meth oxy-2-m eth yl-3-n itr oben zylid en e Dia ceta te (22)
a n d 6-Meth oxy-2-m eth yl-3-n itr oben zyl Aceta te (23). Con-
centrated H2SO4 (8.5 mL) was added to a cooled solution of
2,3-dimethyl-4-nitroanisole (21) (7.0 g, 38.6 mmol) in acetic
anhydride (45 mL) and glacial acetic acid (55 mL). The mixture
was cooled to 0 °C, and a solution of chromium trioxide (10 g,
65.8 mmol) in acetic anhydride (20 mL) was added to the
stirred solution at a rate such that the temperature was
maintained at 5-10 °C. Stirring was continued for a further
2 h at below 10 °C, and the mixture was then poured into ice
(500-700 g) and set aside overnight. The precipitate was
removed by filtration, washed with water until the washings
were no longer colored, and then dried in a vacuum at room
temperature to give a pale yellow solid, which showed two
spots on a TLC plate (SiO2, EtOAc-hexane 1:3, Rf (22) ) 0.41,
Rf (23) ) 0.55). Separation and purification were accomplished
by silica gel column chromatography (ethyl acetate-hexane
1:4). Compound 23 was eluted first, followed by compound 22.
Evaporation of the solvent gave 23 (1.95 g, 21%) as a white
1
mp 45-46 °C. H NMR (DMSO-d6): δ 8.06 (d, J ) 9.17 Hz, 1
H), 6.84 (d, J ) 9.16 Hz, 1 H), 3.91 (s, 3 H), 3.88 (s, 3 H), 2.46
(s, 3 H). Anal. (C10H11NO5) C, H, N.
Meth yl 2-(Br om om eth yl)-6-m eth oxy-3-n itr oben zoa te
(28). A mixture of 27 (1.5 g, 6.6 mmol) and NBS (1.32 g, 3.7
mmol) in CCl4 (30 mL) was preheated to 50 °C, and dibenzoyl
peroxide (0.2 g, 0.8 mmol) was then added. The mixture was
stirred and heated at reflux for 3 h, and then, another portion
of dibenzoyl peroxide (0.05 g, 0.2 mmol) was added. The
mixture was stirred and heated at reflux for another 10 h.
After it was cooled to room temperature, the precipitate was
removed by filtration, and the filtrate was washed with cold
diethyl ether (2 × 30 mL). The combined filtrate and washings
were concentrated on a rotary evaporator without the applica-
tion of heat to yield a brown oily liquid. TLC indicated the
presence of both product and starting material. Column
chromatography (silica gel, ethyl acetate-hexane 1:3) yielded
starting material 27 (0.38 g) and pure 28 (0.81 g, 53%) as a
1
crystalline solid; mp 98-100 °C. H NMR (CDCl3): δ 7.97 (d,
J ) 9.13 Hz, 1 H), 6.84 (d, J ) 9.14 Hz, 1 H), 5.26 (s, 2 H),
3.91 (s, 3 H), 2.52 (s, 3 H), 2.07 (s, 3 H). CIMS m/z 240 (MH+).
Anal. (C11H13NO5) C, H, N. Compound 22 (5.1 g, 44%) was
isolated as a light yellow crystalline solid; mp 132-134 °C.
1H NMR (CDCl3): δ 8.35 (s, 1 H), 7.91 (d, J ) 9.12 Hz, 1 H),
6.84 (d, J ) 9.14 Hz, 1 H), 3.94 (s, 3 H), 2.68 (s, 3 H), 2.09 (s,
6 H). CIMS m/z 298 (MH+). Anal. (C13H15NO7) C, H, N.
1
light yellowish oil. H NMR (CDCl3): δ 8.12 (d, J ) 9.20 Hz,
6-Meth oxy-2-m eth yl-3-n itr oben za ld eh yd e (24). Com-
pound 22 (3.0 g, 10.0 mmol) was added to a solution of 50%
ethanol (80 mL) and concentrated H2SO4 (1.5 mL). The
mixture was heated to reflux for 2 h. After it was cooled, the
ethanol was removed on a rotary evaporator, and the precipi-
tate was removed by filtration, washed with water until the
washings were no longer acidic, and dried in vacuo at room
temperature. The product 24 (1.87 g, 96%) was obtained as a
1 H), 6.92 (d, J ) 9.22 Hz, 1 H), 4.70 (s, 2 H), 3.92 (s, 3 H),
3.87 (s, 3 H). ESIMS m/z 304 (MH+). Anal. (C10H10BrNO5) C,
H, N, Br.
7-Meth oxy-4-n itr o-2-p h en eth yl-2,3-d ih yd r oisoin d ol-1-
on e (29a ). â-Phenethylamine (0.085 mL, 0.66 mmol) and Et3N
(0.2 mL, 1.2 mmol) were added to a solution of 28 (0.2 g, 0.66
mmol) in methanol (10 mL). The mixture was heated at 80 °C
under argon for 24 h. After it was cooled to room temperature,
the solvent was removed. The solid was dissolved in methylene
chloride (50 mL) and washed with 0.1 N HCl (2 × 50 mL).
The organic phase was isolated, washed with brine (30 mL),
and dried over MgSO4. Removal of the solvent gave an oily
residue, which was recrystallized from ethyl acetate-hexane
(1:1). Compound 29a (0.19 g, 93%) was obtained as a yellowish
1
light yellow solid; mp 120-121 °C. H NMR (CDCl3): δ 10.57
(s, 1 H), 8.08 (d, J ) 9.15 Hz, 1 H), 6.94 (d, J ) 9.16 Hz, 1 H),
3.99 (s, 3 H), 2.66 (s, 3 H). CIMS m/z 196 (MH+). Anal. (C9H9-
NO4) C, H, N.
6-Meth oxy-2-m eth yl-3-n itr oben zyl Alcoh ol (25). Com-
pound 23 (3.0 g, 12.5 mmol) was added to a solution of 50%
ethanol (50 mL) containing NaOH (0.5 g, 12.5 mmol). The
mixture was heated to reflux for 2 h. After it was cooled to
room temperature, it was extracted with diethyl ether (2 ×
1
solid; mp 173-175 °C. H NMR (CDCl3): δ 8.35 (d, J ) 9.13
Hz, 1 H), 7.28-7.19 (m, 5 H), 7.00 (d, J ) 9.12 Hz, 1 H), 4.59

4782 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Mu et al.
(s, 2 H), 4.06 (s, 3 H), 3.84 (t, J ) 7.47 Hz, 2 H), 2.99 (t, J )
to give a solid, which was purified by column chromatography
(ethyl acetate as elute). Compound 32a (0.27 g, 63%) was
obtained as a yellowish solid; mp 201-203 °C. 1H NMR
(DMSO-d6): δ 8.77 (s, 1 H, OH), 8.53 (s, 1 H, OH), 8.42 (bs, 1
H, OH), 7.32-7.20 (m, 5 H), 6.60 (m, 3 H), 6.49 (d, J ) 8.37
Hz, 1 H), 6.44 (dd, J ) 8.38, 2.43 Hz, 1 H), 5.44 (bs, 1 H, NH),
4.27 (s, 2 H), 4.15 (s, 2 H), 3.73 (t, J ) 7.10 Hz, 2 H), 2.93 (t,
J ) 7.20 Hz, 2 H). ESIMS m/z 391 (MH+), 389 (M - H-). Anal.
(C23H22N2O4) C, H, N.
4-[N-[(2,5-Dih yd r oxyp h en yl)m eth yl]a m in o]-2-[2-(4-flu -
or op h en yl)et h yl]-7-h yd r oxy-2,3-d ih yd r oisoin d ol-1-on e
(32b). From compounds 19 (0.18 g, 1.30 mmol), 31b (0.30 g,
1.04 mmol), H2, and 10% Pd-C (wet, contains 50% water, 0.1
g), a similar procedure as that described for 32a gave com-
pound 32b (0.32 g, 76%) as a white crystalline solid; mp 178-
180 °C. 1H NMR (DMSO-d6): δ 8.77 (s, 1 H, OH), 8.53 (s, 1 H,
OH), 8.42 (bs, 1 H, OH), 7.28 (m, 2 H), 7.12 (t, J ) 8.87 Hz, 2
H), 6.60 (m, 3 H), 6.50 (d, J ) 8.17 Hz, 1 H), 6.44 (dd, J )
8.34, 2.33 Hz, 1 H), 5.40 (bs, 1 H, NH), 4.27 (s, 2 H), 4.23 (s,
2 H), 3.72 (t, J ) 7.09 Hz, 2 H), 2.92 (t, J ) 7.04 Hz, 2 H).
ESIMS m/z 409 (MH+). Anal. (C23H21FN2O4) C, H, F, N.
4-Am in o-7-m eth oxy-2-(2-p h en eth yl)-2,3-d ih yd r oisoin -
d ol-1-on e (33a ). Compound 29a (0.15 g, 0.48 mmol) was
hydrogenated over 10% palladium on activated carbon (wet,
contains 50% water, 0.05 g) in ethanol (20 mL) at atmospheric
pressure for 3 h. The mixture was then filtered through Celite
and washed with ethanol (10 mL). The combined filtrate was
evaporated on a rotary evaporator to give 33a (0.12 g, 91%)
as an off-white solid; mp 188-190 °C. 1H NMR (DMSO-d6): δ
7.30-7.19 (m, 5 H), 6.76 (d, J ) 8.44 Hz, 1 H), 6.71 (d, J )
8.49 Hz, 1 H), 4.82 (bs, 2 H, NH2), 4.10 (s, 2 H), 3.68 (m, 5 H),
2.88 (t, J ) 7.16 Hz, 2 H). Anal. (C17H18N2O2) C, H, N.
7.53 Hz, 2 H). CIMS m/z 313 (MH+). Anal. (C17H16N2O4) C, H,
N.
2-[2-(4-F lu or op h en yl)et h yl]-7-m et h oxy-4-n it r o-2,3-d i-
h yd r oisoin d ol-1-on e (29b). From 4-fluoro-2-phenethylamine
(0.25 mL, 1.9 mmol), Et3N (0.44 mL, 3.3 mmol), and 28 (0.5 g,
1.64 mmol), a similar procedure as that described for 29a gave
29b (0.43 g, 84%) as a yellowish solid; mp 209-211 °C. 1H
NMR (DMSO-d6): δ 8.41 (d, J ) 9.16 Hz, 1 H), 7.31 (m, 3 H),
7.11 (t, J ) 8.88 Hz, 2 H), 4.77 (s, 2 H), 4.00 (s, 3 H), 3.73 (t,
J ) 7.21 Hz, 2 H), 2.92 (t, J ) 7.19 Hz, 2 H). Anal. (C17H15
FN2O4) C, H, F, N.
-
7-Hyd r oxy-4-n itr o-2-p h en eth yl-2,3-d ih yd r oisoin d ol-1-
on e (30a ). Compound 29a (0.45 g, 1.45 mmol) was dissolved
in anhydrous CH2Cl2 (30 mL) and cooled to -78 °C. BBr3 (2.0
mL, 1 M solution in CH2Cl2, 2.0 mmol) was added to the
reaction mixture under argon. The solution was warmed to
0-10 °C and stirred for 2 h. After it was recooled to -78 °C,
H2O (20 mL) was added, followed by ethyl acetate (30 mL),
and the solution was warmed to room temperature. The
resulting solution was extracted with ethyl acetate (3 × 50
mL), and the combined organic layer was washed with brine
(10 mL) and dried over MgSO4. The solvent was removed, and
the crude oily residue was further purified by flash chroma-
tography (ethyl acetate-hexane 1:1). The product 30a (0.38
g, 88%) was isolated as an off-white crystalline solid; mp 135-
1
137 °C. H NMR (DMSO-d6): δ 9.50 (bs, 1 H, OH), 8.27 (d, J
) 9.05 Hz, 1 H), 7.30-7.21 (m, 5 H), 7.05 (d, J ) 9.03 Hz, 1
H), 4.76 (s, 2 H), 3.75 (t, J ) 7.34 Hz, 2 H), 2.94 (t, J ) 7.47
Hz, 2 H). Anal. (C16H14N2O4) C, H, N.
2-[2-(4-F lu or op h en yl)et h yl]-7-h yd r oxy-4-n it r o-2,3-d i-
h yd r oisoin d ol-1-on e (30b). From compounds 29b (0.50 g,
1.60 mmol) and BBr3 (3.0 mL, 1 M solution in CH2Cl2, 3.0
mmol), a similar procedure as that described for 30a gave
product 30b (0.41 g, 86%) as a yellowish crystalline solid; mp
188-190 °C. 1H NMR (DMSO-d6): δ 11.31 (bs, 1 H, OH), 8.27
(d, J ) 9.04 Hz, 1 H), 7.39 (m, 2 H), 7.11 (t, J ) 8.80 Hz, 2 H),
7.04 (d, J ) 9.03 Hz, 1 H), 4.77 (s, 2 H), 3.73 (t, J ) 7.27 Hz,
2 H), 2.93 (t, J ) 7.28 Hz, 2 H). Anal. (C16H13FN2O4) C, H, F,
N.
4-Am in o-2-[2-(4-flu or op h en yl)eth yl]-7-m eth oxy-2,3-d i-
h yd r oisoin d ol-1-on e (33b). From compound 29b (0.20 g, 0.6
mmol), H2, and 10% Pd-C (wet, contains 50% water, 0.05 g),
a similar procedure as that described for 33a gave pure 33b
1
(0.16 g, 87%) as a yellowish solid; mp 198-200 °C. H NMR
(DMSO-d6): δ 7.25 (m, 2 H), 7.08 (t, J ) 8.76 Hz, 2 H), 6.76
(d, J ) 8.53 Hz, 1 H), 6.73 (d, J ) 8.51 Hz, 1 H), 5.05 (bs, 2 H,
NH2), 4.08 (s, 2 H), 3.69 (s, 3 H), 3.66 (t, J ) 7.17 Hz, 2 H),
2.86 (t, J ) 7.06 Hz, 2 H). Anal. (C17H17FN2O2) C, H, F, N.
4-Am in o-7-h yd r oxy-2-p h en eth yl-2,3-d ih yd r oisoin d ol-
1-on e (31a ). Compound 30a (0.35 g, 1.2 mmol) was hydroge-
nated over 10% palladium on activated carbon (wet, contains
50% water, 0.05 g) in methanol (50 mL) at room temperature
and atmospheric pressure for 30 min. The mixture was then
filtered through Celite and washed with methanol (10 mL).
The combined filtrate was evaporated on a rotary evaporator
to give 31a (0.29 g, 90%) as an oily residue, which was used
4-[N-[(2,5-Dih ydr oxyph en yl)m eth yl]am in o]-7-m eth oxy-
2-(2-p h en yleth yl)-2,3-d ih yd r oisoin d ol-1-on e (34a ). From
compounds 19 (0.23 g, 1.70 mmol), 33a (0.48 g, 1.70 mmol),
H2, and 10% Pd-C (wet, contains 50% water, 0.1 g), a similar
procedure as that described for 32a yielded compound 34a
1
(0.22 g, 87%) as an off-white solid; mp 172-174 °C. H NMR
(DMSO-d6): δ 8.76 (s, 1 H, OH), 8.51 (s, 1 H, OH), 7.30-7.18
(m, 5 H), 6.76 (d, J ) 8.65 Hz, 1 H), 6.60 (d, J ) 8.54 Hz, 1 H),
6.57 (d, J ) 2.05 Hz, 1 H), 6.49 (d, J ) 8.67 Hz, 1 H), 6.40 (dd,
J ) 8.36, 2.42 Hz, 1 H), 5.53 (t, J ) 5.80 Hz, 1 H, NH), 4.19 (s,
2 H), 4.16 (d, J ) 5.73 Hz, 2 H), 3.71 (q, J ) 7.80 Hz, 2 H),
3.69 (s, 3 H), 2.89 (t, J ) 7.30 Hz, 2 H). CIMS 404 (MH+), 283.
Anal. (C23H24N2O3‚2.2 H2O) C, H, N.
1
without further purification. H NMR (DMSO-d6): δ 8.37 (s,
1 H, OH), 7.30-7.21 (m, 5 H), 6.64 (d, J ) 8.30 Hz, 1 H), 6.56
(d, J ) 8.39 Hz, 1 H), 4.65 (bs, 2 H, NH2), 4.15 (s, 2 H), 3.71
(t, J ) 7.12 Hz, 2 H), 2.93 (t, J ) 7.09 Hz, 2 H).
4-Am in o-2-[2-(4-flu or op h en yl)eth yl]-7-h yd r oxy-2,3-d i-
h yd r oisoin d ol-1-on e (31b). From compound 30b (0.40 g, 1.3
mmol), H2, and Pd-C (wet, contains 50% water, 0.05 g), a
similar procedure as that described for 31a afforded 31b (0.34
g, 89%) as a dark yellow solid; mp 78-80 °C. 1H NMR (DMSO-
d6): δ 8.37 (s, 1 H, OH), 7.28 (m, 2 H), 7.11 (t, J ) 8.84 Hz, 2
H), 6.66 (d, J ) 8.33 Hz, 1 H), 6.57 (d, J ) 8.41 Hz, 1 H), 4.72
(bs, 2 H, NH2), 4.15 (s, 2 H), 3.70 (t, J ) 7.12 Hz, 2 H), 2.90 (t,
J ) 7.13 Hz, 2 H). Anal. (C16H15FN2O2) C, H, F, N.
4-[N-[(2,5-Dih ydr oxyph en yl)m eth yl]am in o]-7-h ydr oxy-
2-p h en et h yl-2,3-d ih yd r oisoin d ol-1-on e (32a ). 2,5-Dihy-
droxybenzaldehyde 19 (0.25 g, 1.80 mmol) was added to 31a
(0.29 g, 1.1 mmol) in anhydrous methanol (40 mL), and the
mixture was heated to reflux under argon for 6 h. After it was
cooled to 0 °C, the precipitate was removed by filtration,
washed with cold methanol (5 mL), and dried. The solid was
dissolved in a mixture of ethyl acetate (100 mL) and methanol
(100 mL) and hydrogenated over 10% palladium on activated
carbon (wet, contains 50% water, 0.1 g) at room temperature
and atmospheric pressure for 10 min. The mixture was then
filtered through Celite and washed with methanol (40 mL).
The combined filtrate was evaporated on a rotary evaporator
4-{[(2,5-Dih yd r oxyp h en yl)m eth yl]a m in o}-2-[2-(4-flu o-
rophenyl)ethyl]-7-methoxy-2,3-dihydroisoindol-1-one (34b).
From compounds 19 (0.16 g, 1.16 mmol), 33b (0.35 g, 1.16
mmol), H2, and 10% Pd-C (wet, contains 50% water, 0.05 g),
a similar procedure as that described for 32a afforded com-
pound 34b (0.33 g, 69%) as an off-white solid; mp 205-207
°C. 1H NMR (DMSO-d6): δ 8.75 (s, 1 H, OH), 8.50 (s, 1 H,
OH), 7.26 (m, 2 H), 7.10 (t, J ) 8.85 Hz, 2 H), 6.76 (d, J )
8.65 Hz, 1 H), 6.60 (d, J ) 8.52 Hz, 1 H), 6.57 (d, J ) 2.55 Hz,
1 H), 6.49 (d, J ) 8.69 Hz, 1 H), 6.41 (dd, J ) 8.45, 2.46 Hz,
1 H), 5.53 (t, J ) 5.75 Hz, 1 H, NH), 4.19 (s, 2 H), 4.16 (d, J )
5.73 Hz, 2 H), 3.71 (q, J ) 7.80 Hz, 2 H), 3.69 (s, 3 H), 2.88 (t,
J ) 7.03 Hz, 2 H). ESIMS m/z 423 (MH+). Anal. (C24H23FN2O4)
C, H, F, N.
2-Br om o-4-n itr op h en ol (36). Fluorosulfonic acid (3.16 g,
31.6 mmol) and NBS (6.16 g, 34.4 mmol) were slowly added
to a cold (-30 °C) solution of 4-nitrophenol (35) (4 g, 28.8
mmol) in CH3CN (40 mL) under argon at a rate that kept the
temperature below -20 °C. After it was added, the reaction
mixture was allowed to warm to room temperature and stirred

Cytotoxic Inhibitors of Tubulin Polymerization
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4783
for 48 h. NaHSO3 (38%, 20 mL) was added, and the reaction
mixture was extracted with diethyl ether (2 × 100 mL). The
combined ether layer was washed with H2O (3 × 50 mL) and
brine (25 mL), dried over MgSO4, and concentrated under
reduced pressure. The crude product was subjected to flash
chromatography (silica gel 60, ethyl acetate-hexane 1:4). The
product 36 (5.97 g, 95%) was isolated as a yellow solid; mp
1 H), 6.44 (dd, J ) 8.43, 2.70 Hz, 1 H), 6.27 (d, J ) 11.29 Hz,
1 H), 6.18 (d, J ) 2.58 Hz, 1 H), 5.78 (dt, J ) 11.29, 7.31 Hz,
1 H), 3.96 (bs, 2 H, NH2), 2.65 (t, J ) 7.39 Hz, 2 H), 2.40 (q, J
) 7.36 Hz, 2 H).
cis-1-{5-[N-[(2,5-Dih yd r oxyp h en yl)m et h yl]a m in o]-2-
h yd r oxy}p h en yl-4-p h en ylbu ten e (42). 2,5-Dihydroxyben-
zaldehyde 19 (0.11 g, 0.82 mmol) was added to 41 in ethanol
(20 mL), and the mixture was stirred at room temperature
under argon for 6 h. While it was stirred, NaBH3CN (0.13 g,
2.06 mmol) was added, and the reaction mixture was stirred
for another 1 h. The reaction mixture was concentrated, and
H2O (100 mL) was added to it. The mixture was extracted with
ethyl acetate (3 × 60 mL). The combined organic layers were
washed with brine (20 mL), dried over MgSO4, and concen-
trated to furnish the crude product, which was further purified
by flash chromatography (silica gel 60 g, ethyl acetate-hexane
1:2). The product 42 (0.11 g, 58% in two steps) was isolated as
1
114-115 °C (lit.31 mp 112-114 °C). H NMR (CDCl3): δ 8.44
(d, J ) 2.31 Hz, 1 H), 8.17 (dd, J ) 9.01, 2.37 Hz, 1 H), 7.13
(d, J ) 9.01 Hz, 1 H), 6.24 (bs, 1 H, OH).
2-Br om o-1-(m eth oxym eth oxy)-4-n itr oben zen e (37). A
suspension of 36 (2.0 g, 9.17 mmol) and NaH (0.44 g, 18.3
mmol) in dry THF (50 mL) was stirred for 1 h. Chloromethyl
methyl ether (1.38 mL, 18.3 mmol) was added to the mixture,
and the reaction mixture was stirred for 24 h at room
temperature. Water (50 mL) and saturated NH4Cl were added
to adjust the pH to 7.0, and the solvent was evaporated. The
residue was extracted with diethyl ether (3 × 50 mL), and the
organic phase was washed with water (50 mL) and brine (20
mL). The ether layer was dried over MgSO4 and evaporated
to afford the MOM ether 37 (2.16 g, 90%) as a yellow
crystalline solid; mp 86-88 °C. 1H NMR (CDCl3): δ 8.47 (d, J
) 2.67 Hz, 1 H), 8.17 (dd, J ) 9.14, 2.68 Hz, 1 H), 7.25 (d, J
) 9.15 Hz, 1 H), 5.35 (s, 2 H), 3.53 (s, 3 H). CIMS m/z 262,
264 (MH+). Anal. (C8H8BrNO4) C, H, Br, N.
1
a brownish liquid. H NMR (DMSO-d6): δ 8.72 (s, 1 H, OH),
8.51 (s, 1 H, OH), 8.38 (s, 1 H, OH), 7.20-7.04 (m, 5 H), 6.67
(d, J ) 8.44 Hz, 1 H), 6.66 (d, J ) 8.42 Hz, 1 H), 6.59 (m, 2 H),
6.52 (d, J ) 2.25 Hz, 1 H), 6.30 (d, J ) 2.20 Hz, 1 H), 6.25 (d,
J ) 11.24 Hz, 1 H), 5.80 (dt, J ) 11.59, 7.33 Hz, 1 H), 4.50
(bs, 1 H, NH), 4.17 (s, 2 H), 2.62 (t, J ) 7.21 Hz, 2 H), 2.31 (q,
J ) 7.17 Hz, 2 H). ESIMS m/z 362 (MH+). Anal. (C23H23
NO3‚CH3COOCH2CH3) C, H, N.
-
1-(2-Me t h oxym e t h oxy-5-n it r o)p h e n yl-4-p h e n ylb u -
tyn e (38). 2-Bromo-1-(methoxymethoxy)-4-nitrobenzene (37)
(1 g, 3.8 mmol), bis(triphenylphosphine) palladium (II) chloride
(0.08 g, 0.11 mmol), triphenylphosphine (0.06 g, 0.23 mmol),
and copper(I) iodide (0.015 g, 0.076 mmol) were added to a
dry round-bottomed flask, which was then sparged with argon
and charged with diethylamine (30 mL). 4-Phenyl-1-butyne
(1.07 mL, 7.6 mmol) was added via syringe. The stirred
reaction mixture was heated at 50-60 °C for 6 h. During the
reaction, precipitation of [H2N(i-Pr)2]Br was observed. After
it was cooled to room temperature, the reaction mixture was
diluted with EtOAc (30 mL), filtered through a small pad of
silica gel with EtOAc rinsings, concentrated in vacuo, and
purified by flash chromatography (silica gel 180 g, ethyl
acetate-hexane 1:6) to give 38 (1.20 g, 100%) as a dark brown
liquid. 1H NMR (CDCl3): δ 8.13 (d, J ) 2.78 Hz, 1 H), 7.99
(dd, J ) 9.18, 2.80 Hz, 1 H), 7.25-7.15 (m, 5 H), 7.09 (d, J )
9.19 Hz, 1 H), 5.20 (s, 2 H), 3.41 (s, 3 H), 2.87 (t, J ) 7.37, 2
H), 2.69 (t, J ) 7.34 Hz, 2 H). Anal. (C18H17NO4) C, H, N.
1-(5-Am in o-2-h yd r oxy)p h en yl-4-p h en ylbu tyn e (40). A
mixture of 38 (0.5 g, 1.6 mmol) and SnCl2‚2H2O (1.8 g, 8.0
mmol) in absolute ethanol (10 mL) was heated at 70 °C under
argon for 2 h. The reaction mixture was allowed to cool to room
temperature and then poured into ice. The pH was adjusted
to 7-8 by addition of 5% aqueous NaHCO3, and the mixture
was extracted with ethyl acetate (2 × 30 mL). The combined
organic extracts were washed with brine, dried over MgSO4,
and concentrated. The crude product was purified by flash
chromatography (silica gel, ethyl acetate-hexane 1:2) to give
40 (0.20 g, 52%) as a solid; mp 96-98 °C. 1H NMR (CDCl3): δ
9.98 (s, 1 H, OH), 7.28-7.18 (m, 5 H), 6.63 (d, J ) 8.59 Hz, 1
H), 6.53 (d, J ) 2.62 Hz, 1 H), 6.48 (dd, J ) 8.61, 2.73 Hz, 1
H), 3.60 (bs, 2 H, NH2), 2.85 (t, J ) 7.19 Hz, 2 H), 2.70 (t, J )
7.11 Hz, 2 H). ESIMS m/z 238 (MH+); high-resolution ESI m/z
calcd, 238.1232; found, 238.1237. Anal. (C16H15NO) C, H, N.
1-Br om o-4-p h en yl-1-bu tyn e (47). A solution of 4-phenyl-
1-butyne (43, 0.4 g, 3.0 mmol) in acetone (20 mL) was treated
at room temperature with NBS (0.62 g, 3.5 mmol) and AgNO3
(50 mg). The mixture was stirred at room temperature for 4
h. The precipitate was removed by filtration, and the solvent
was evaporated. The residue was redissolved in hexane (10
mL), and the precipitate was again removed by filtration.
Evaporation of the solvent gave product 47 (0.62 g, 99%) as a
colorless oily liquid, which was used without further purifica-
tion. 1H NMR (CDCl3): δ 7.32-7.20 (m, 5 H), 2.83 (t, J ) 7.47
Hz, 2 H), 2.49 (t, J ) 7.53 Hz, 2 H).
1-Met h oxym et h oxy-2-[(1E)-4-p h en ylb u t -1-en yl]-4-n i-
tr oben zen e (48). Tributyltin hydride (1.89 g, 6.5 mmol) was
added dropwise over 30 min to a stirred solution of the
1-bromoalkyne 47 (0.68 g, 3.25 mmol) and tetrakis(tri-
phenylphosphine)palladium(0) (20 mg, 0.017 mmol) in THF
(10 mL) under argon at room temperature. The mixture was
stirred at room temperature for a further 2 h. The crude
vinylstannane product 44 was then treated with 37 (0.6 g, 2.3
mmol) and additional tetrakis(triphenylphosphine)palladium-
(0) (20 mg, 0.017 mmol). The mixture was heated to reflux for
3 h and then cooled and diluted with an equal volume of
saturated aqueous KF. The two phase mixture was stirred
vigorously overnight. Diethyl ether (30 mL) was added, the
separated organic layer was washed with saturated am-
monium chloride (50 mL) and brine (50 mL), and the solvent
was removed by evaporation. The residue was purified by
column chromatography (silica gel 180 g, ethyl acetate-hexane
1
1:5) to give compound 48 (0.55 g, 76%) as light yellow oil. H
NMR (CDCl3): δ 8.33 (d, J ) 2.73 Hz, 1 H), 8.08 (dd, J ) 9.09,
2.76 Hz, 1 H), 7.35-7.23 (m, 5 H), 7.18 (d, J ) 9.10 Hz, 1 H),
6.72 (d, J ) 15.97 Hz, 1 H), 6.43 (dt, J ) 15.94, 6.86 Hz, 1 H),
5.31 (s, 2 H), 3.52 (s, 3 H), 2.86 (t, J ) 7.35 Hz, 2 H), 2.64 (q,
J ) 7.27 Hz, 2 H). Anal. (C18H19NO4) C, H, N.
2-[(1E)-4-P h en ylbu t-1-en yl]-4-a m in op h en ol (49). A mix-
ture of 48 (0.5 g, 1.6 mmol) and SnCl2‚2H2O (1.8 g, 8.0 mmol)
in absolute ethanol (10 mL) was heated at 70 °C under argon
for 2 h. The reaction mixture was allowed to cool to room
temperature and then poured into ice and stirred for 30 min.
The pH was adjusted to 7-8 by addition of 5% aqueous
NaHCO3, and the reaction mixture was extracted with ethyl
acetate (2 × 30 mL). The combined organic extracts were
washed with brine, dried over MgSO4, and concentrated. The
crude product was subjected to column chromatography (silica
gel 80 g, ethyl acetate-hexane 1:2) to give 49 (0.21 g, 55%) as
an oily residue, which was used without further purification.
1H NMR (CDCl3): δ 9.98 (s, 1 H, OH), 7.31-7.17 (m, 5 H),
6.72 (d, J ) 8.45 Hz, 1 H), 6.55 (dd, J ) 8.41, 2.39 Hz, 1 H),
6.36 (d, J ) 11.21 Hz, 1 H), 6.25 (d, J ) 2.47 Hz, 1 H), 5.90
cis-1-(5-Am in o-2-h yd r oxy)p h en yl-4-p h en ylbu ten e (41).
Compound 40 (0.13 g, 0.55 mmol) was hydrogenated over 5
wt % palladium on calcium carbonate, poisoned with lead (8
mg), in ethanol (15 mL) at room temperature and atmospheric
pressure. The reaction was traced by TLC every 10 min until
the transformation was completed. The mixture was then
filtered through Celite and washed with ethanol (5 mL). The
combined filtrate was used directly in the next step. For the
purpose of identification, a small portion of the above combined
filtrate was evaporated on a rotary evaporator to give the crude
product, which was then purified by flash chromatography
(silica gel 80 g, ethyl acetate: hexane 1:4). The purified product
1
was isolated as a dark brown oily residue. H NMR (CDCl3):
δ 10.50 (s, 1 H, OH), 7.22-7.11 (m, 5 H), 6.59 (d, J ) 8.47 Hz,

4784 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Mu et al.
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(dt, J ) 11.21, 7.34 Hz, 1 H), 3.40 (bs, 2 H, NH2), 2.74 (t, J )
7.39 Hz, 2 H), 2.48 (q, J ) 7.33 Hz, 2 H).
tr a n s-1-{5-[N-[(2,5-Dih yd r oxyp h en yl)m eth yl]a m in o]-2-
h yd r oxy}p h en yl-4-p h en yl-1-bu ten e (50). 2,5-Dihydroxy-
benzaldehyde 19 (0.2 g, 1.5 mmol) was added to 49 (0.36 g,
1.5 mmol) in methanol (30 mL), and the mixture was stirred
at room temperature under argon overnight. While stirring,
NaBH3CN (0.26 g, 4.12 mmol) was added and the reaction
mixture was stirred for another 3 h at room temperature. The
reaction mixture was then concentrated, and H2O (100 mL)
was added to it. The reaction mixture was extracted with ethyl
acetate (3 × 50 mL). The combined organic layers were washed
with brine (30 mL), dried over MgSO4, and concentrated to
furnish the crude product, which was further purified by flash
chromatography (silica gel 150 g, ethyl acetate-hexane 1:2).
The product 49 (0.26 g, 48%) was isolated as a brown liquid.
1H NMR (DMSO-d6): δ 8.99 (s, 1 H, OH), 8.72 (s, 1 H, OH),
8.53 (s, 1 H, OH), 7.26-7.22 (m, 5 H), 6.69 (d, J ) 8.28 Hz, 1
H), 6.65-6.52 (m, 5 H), 6.31 (dd, J ) 8.49, 2.30 Hz, 1 H), 6.06
(dt, J ) 15.92, 6.77 Hz, 1 H), 5.30 (s, 1 H, NH), 4.02 (d, J )
7.03 Hz, 2 H), 2.70 (t, J ) 7.34 Hz, 2 H), 2.44 (q, J ) 7.27 Hz,
2 H). CIMS m/z 362 (MH+). Anal. (C23H23NO3‚3/4H2O) C, H,
N.
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Ack n ow led gm en t. This research was made possible
by grants from the Showalter Trust and Purdue Re-
search Foundation.
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