Synthesis and biological activity of analogues of the antimicrotubule agent N,β,β-trimethyl-L-phenylalanyl-N1-[(1S,2E)-carboxy-1- isopropylbut-2-enyl]-N13-dimethyl-L-valinamide (HTI-286)

Article abstract of DOI:10.1021/jm040056u

Hemiasterlin (1), a tripeptide isolated from marine sponges, induces microtubule depolymerization and mitotic arrest in cells. HTI-286 (2), an analogue from an initial study of the hemiasterlins, is presently in clinical trials. In addition to its potent

Full text of DOI:10.1021/jm040056u

4774
J . Med. Chem. 2004, 47, 4774-4786
Syn th esis a n d Biologica l Activity of An a logu es of th e An tim icr otu bu le Agen t
N,â,â-Tr im eth yl-L-p h en yla la n yl-N1-[(1S,2E)-3-ca r boxy-1-isop r op ylbu t-2-en yl]-
N1,3-d im eth yl-L-va lin a m id e (HTI-286)
Arie Zask,* Gary Birnberg, Katherine Cheung, J oshua Kaplan, Chuan Niu, Emily Norton, Ronald Suayan,
Ayako Yamashita, Derek Cole, Zhilian Tang, Girija Krishnamurthy, Robert Williamson, Gulnaz Khafizova,
Sylvia Musto, Richard Hernandez, Tami Annable, Xiaoran Yang, Carolyn Discafani, Carl Beyer,
Lee M. Greenberger, Frank Loganzo, and Semiramis Ayral-Kaloustian
Chemical and Screening Sciences, and Oncology Research,Wyeth Research, 401 North Middletown Road,
Pearl River, New York 10965
Received March 2, 2004
Hemiasterlin (1), a tripeptide isolated from marine sponges, induces microtubule depolymer-
ization and mitotic arrest in cells. HTI-286 (2), an analogue from an initial study of the
hemiasterlins, is presently in clinical trials. In addition to its potent antitumor effects, 2 has
the advantage of circumventing the P-glycoprotein-mediated resistance that hampers the
efficacy of other antimicrotubule agents such as paclitaxel and vincristine in animal models.
This paper describes an in-depth study of the structure-activity relationships of analogues of
2, their effects on microtubule polymerization, and their in vitro and in vivo anticancer activity.
Regions of the molecule necessary for potent activity are identified. Groups tolerant of
modification, leading to novel analogues, are reported. Potent analogues identified through in
vivo studies in tumor xenograft models include one superior analogue, HTI-042 (48).
Modulation of the dynamics of microtubule formation
represents a major therapeutic approach for the treat-
ment of cancer.1 The taxanes and Vinca alkaloids are
the two classes of tubulin inhibiting natural products
currently in use as anticancer agents. Despite their
successes, inherent and acquired resistance by tumors
to these agents limits their utility.2 Hemiasterlin3 (1)
is a member of a recently discovered class of natural
products whose potent effects on tubulin make them
especially attractive as drug targets (Figure 1). These
tripeptides contain highly unusual and sterically con-
gested amino acids, which give rise to their stability and
in vivo activity. Other peptidic antimicrotubule agents
(e.g. dolastatins, cemadotin and cryptophycin), some of
F igu r e 1.
which competitively inhibit4 binding of hemiasterlin to
tubulin, have been reported and several are under
direct effects on extracellular tubulin polymerization,
clinical investigation.1,5 Clinical trials of compounds
and its cytotoxic effects both in the absence and presence
interacting with the colchicine domain (e.g. combret-
of expression of P-glycoprotein transporters. In vivo
astatins) and the taxane site (e.g. epothilones) are also
studies in tumor xenograft models identified several
underway.1 The relative structural simplicity of the
potent analogues including one superior analogue.
hemiasterlins allows for diverse structural manipulation
of the molecule via total synthesis. HTI-2866 (2), an
Syn th esis
analogue from an initial study of the hemiasterlins
wherein a phenyl group replaces the indole ring,7 is
presently in clinical trials8 (Figure 1). In addition to its
potent antitumor effects, 2 has the advantage of cir-
cumventing the P-glycoprotein-mediated resistance which
hampers the efficacy of other antimicrotubule agents
such as paclitaxel and vincristine in animal models.6
This paper describes an in-depth study of the struc-
ture-activity relationships of analogues of 2 in which
each region of the molecule was systematically inves-
tigated. Each analogue was evaluated in terms of its
Several total syntheses of hemiasterlin have been
reported in the literature.9 The synthetic strategy
comprises synthesizing the individual amino acids,
followed by peptide coupling. This route can also be used
for the synthesis of 2 and other analogues (Scheme 1).7
The most challenging synthesis is that of the A-piece
amino acid 3, necessitating construction of the carbon
scaffold and establishing the chirality by means of a
chiral auxiliary group.9a Alternative A-piece syntheses
utilizing SnCl4-mediated ring opening of epoxides by
indoles9b or asymmetric Strecker synthesis9c have ap-
peared in the literature. The chirally pure B-piece (4)
was commercially available and the CD-piece (5) was
* Corresponding author. Tel: 845-602-2836; Fax: 845-602-5561;
e-mail: zaska@wyeth.com.
10.1021/jm040056u CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/03/2004

New Antimicrotubule Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4775
Sch em e 3. Cyclopropyl A-Piece Synthesis
Sch em e 1. Synthesis of Tripeptides 1 and 2
Sch em e 4. Monomethyl A-Piece Synthesis
Sch em e 5. Preparation of the Cyclohexyl A-Piece 15
anocyclohexene and formic acid to generate intermedi-
ate 10, which upon treatment with 6 N HCl gave the
desired amino acid 11 (Scheme 3). The enantiomerically
pure A-pieces (12), bearing a single methyl substituent
at the beta position, were prepared from the corre-
sponding commercially available amino acids 13, which
were N-methylated with iodomethane and base and
saponified (Scheme 4). Hydrogenation of the aromatic
ring in 14 with platinum oxide catalyst gave the
cyclohexyl A-piece 15 (Scheme 5). A bromine substituent
at the 4-position of the phenyl ring could be introduced
by treatment of phenyl pyruvic acid 7 (R ) Me, R1 )
Ph) with bromine (Scheme 6). After reductive amina-
tion, coupling of the resulting 4-bromophenyl amino acid
to 9 and separation of diastereomers by HPLC, the
bromine was used in a biaryl coupling reaction to give
the biphenyl analogue 16. Preparation of the adaman-
tanyl containing A-piece 17 was effected by treating
alpha-iodo adamantyl acetic acid13 with methylamine
(Scheme 7). Coupling of 17 to 9 followed by hydrolysis
and HPLC gave 18. The A-piece 19 where the methy-
lamine group was incorporated into a thiomorpholine
ring reminiscent of milnamide14 was constructed by
methylation of 2,2-dimethyl-thiomorpholine-3-carboxylic
acid15 using formic acid and formalin (Scheme 8).
Coupling of 19 to 9 followed by hydrolysis gave 20.
Sch em e 2. Pyruvic Acid Route
prepared by reduction of the Weinreb amide of D-leucine
followed by Wittig reaction of the resultant aldehyde
with Ph3PdC(Me)CO2Et.
For A-piece modifications, to efficiently prepare ana-
logues, control of the amino acid stereocenter using
chiral auxiliaries was abandoned and the synthesis
replaced with a shorter route developed in house10
utilizing readily available pyruvic acids 6 (Scheme 2).
Dimethylation alpha to the ketone occurred upon treat-
ment of the pyruvic acids with iodomethane and sodium
hydroxide to give 7. Reductive amination then gave
racemic amino acid 8. Coupling of 8 to the BCD-piece
(9),9a followed by hydrolysis of the ester, gave the desired
analogues as a mixture of two diastereomers (SSS and
RSS) separable by chromatography. The stereochemis-
try at the methylamine position was determined by
NMR. Other substituents could be introduced at the
alpha position of the pyruvic acids, including car-
bocycles, by appropriate choice of alkylating agents. In
the case of the cyclopropyl analogue, cyclization was not
successful by this route. Instead, the multicomponent
Ugi condensation reaction was used,11 combining 1-phen-
yl-cyclopropane carbaldehyde,12 methylamine, 1-isocy-
Investigation of the effect of amine substitution in the
A-piece on activity was expedited by the steric conges-
tion around the nitrogen in 2. For alkyl groups larger
than methyl, the rate of the second N-alkylation reac-
tion was very slow. This lack of reactivity made possible
the preparation of N-monosubstitued derivatives (21)
by treatment of the unsubstituted nitrogen in 22 with
alkyl halides (Scheme 9). A cyclic analogue 23 (i.e.
pyrrolidine) could be synthesized by use of dibromopen-
tane as the alkylating agent. Disubstitution could be
forced to some extent by treatment of 24, the ethyl ester
of 2, with excess bromoethanol at elevated temperature
leading to analogue 25 (Scheme 9). In contrast, treat-
ment of 24 with 1 equiv of iodomethane readily gave
the dialkylated analogue 26 after hydrolysis, as well as
starting 24 and its trimethylammonium salt.

4776 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Sch em e 6. Biaryl A-Piece Analogue Synthesis
Zask et al.
Sch em e 7. Adamantyl A-Piece Analogue Synthesis
tripeptide. Compounds were evaluated for their ability
to inhibit extracellular tubulin polymerization at a
single concentration (0.3 uM) and for their cytotoxicity
in paclitaxel sensitive KB-3-1 cells and in paclitaxel
resistant KB-8-5 and KB-V1 cells expressing moderate
and high levels of P-glycoprotein, respectively. Certain
regions of the molecule could be modified to give
analogues retaining full potency while other changes led
to less potent analogues. Some sites could not be
modified without full loss of biological activity at the
concentrations tested. Evaluation of in vivo biological
activity identified several analogues with potent anti-
tumor activity including one (48) with superior potency.
Sch em e 8. Thiazole A-Piece Analogue Synthesis
Exploration of the importance of each stereocenter to
activity was a goal of high priority (Table 1). Hemi-
asterlin and 2 contain three chiral centers, one at each
of the amino acid subunits (SSS configuration). Of the
three chiral centers, stereochemistry at the one bearing
the methylamine group on the A-piece was least critical
to activity. The RSS isomer 41 was approximately 28-
fold less potent than 2 in the KB-3-1 cells, and it still
retained its ability to potently inhibit tubulin polymer-
ization. Approximately 100-fold loss of potency was
reported for 41 in MCF-7 cells.7 A comparable loss of
potency was seen in RSS isomers isolated from the
synthesis of other analogues. The most profound effect
on biological activity occurred upon altering the stere-
ochemistry of the carbon bearing the tert-butyl group
on the B-piece. The resulting SRS isomer 42 showed
dramatically decreased activity both in cells and in the
tubulin polymerization assay. The key importance of
this position to activity was also clearly evident when
the substituents attached to it were varied (vide infra).
Isomerization of the isopropyl group attached to the
stereocenter on the C-piece gave the SSR analogue 43
which was 2-3 orders of magnitude less potent than 2
in the KB-3-1 cells.
Replacement of the A-piece methylamine group with
nonbasic substituents could be accomplished, using
phenylmethylbutyric acid modified with Evan’s chiral
auxiliary (27) (Scheme 10). Deprotonation of 27 with
sodium hexamethydisilazide, followed by treatment
with iodomethane and removal of the chiral auxiliary
with lithium hydroperoxide, gave the desired chiral
methyl A-piece (28, R ) CH3). Coupling to the BCD-
piece (9) followed by ester hydrolysis gave a single
diastereomer (29), as expected. Alternatively, racemic
A-pieces could be made through formation of the dianion
of phenylmethylbutyric acid, by treatment with 2 equiv
of LDA followed by addition of alkyl, allyl or benzyl
halide to give 28, or addition of dimethyl disulfide to
give 30. The sulfone 31 could be prepared by oxidation
of 30 with MCPBA. The hydroxy A-piece 32 was
prepared by reduction of pyruvic acid ester 33 with
sodium borohydride to give 34, followed by saponifica-
tion with LiOH. Conversion of the hydroxy ester 34 to
the methoxy A-piece 35 was effected by methylation of
the hydroxy group with silver oxide and methyl iodide,
followed by saponification with LiOH.
In addition to stereochemistry, investigation of the
A-piece included modification of the substituents at-
tached to the quaternary carbon (Tables 2 and 3),
variation of the substituents on the nitrogen (Table 4)
and replacement of the nitrogen with carbon or het-
eroatoms (Table 5). While retaining the necessary
geminal dimethyl group on the quarternary carbon, the
substituent corresponding to the phenyl group in 2 was
varied (Table 2). A large reduction in the size of the
group at this position, as in the tert-butyl analogue 44
(methyl for phenyl replacement), gave a marked de-
crease both in cellular activity (a comparable decrease
was reported in MCF-7 cells7) and inhibition of tubulin
polymerization. Interestingly, a hydroxy group in the
place of phenyl gave analogue 45 that had activity
similar to that of the tert-butyl analogue 44, revealing
a tolerance for polar groups at this position. An increase
in steric bulk and lipophilicity relative to methyl at this
position, as in analogue 46 with an n-pentyl group, led
Reduction of the olefin was accomplished by treat-
ment of 2 with 10% Pd/C in acetic acid under one
atmosphere of hydrogen (Scheme 11). A mixture of two
diastereomers was obtained in an 87:13 ratio (36 and
37, respectively). The diastereomers were separable by
HPLC. The stereochemistry of these isomers was as-
signed by NMR.
Other modifications of the tripeptide were made
according to Scheme 1 by substituting the appropriate
amino acid precursor. Esters and amides of 2 were made
by treatment of 2 with the appropriate amine or alcohol
in the presence of EDC. The N,N-dimethyl analogue,
38, was made by treating 39 with NaH and iodomethane
followed by saponification and N-BOC deprotection
(Scheme 12).
Resu lts a n d Discu ssion
Modification of each of the structural components of
2 revealed its respective role in the bioactivity of the

New Antimicrotubule Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4777
Sch em e 9. Synthesis of Amine-Substituted Analogues
Sch em e 10. Synthesis of A-Piece Methylamine
Replacements
space in the tubulin binding pocket. Consistent with this
analysis the analogue containing the smaller spirocy-
clopropyl ring (56), showed excellent inhibition of tu-
bulin polymerization (99% at 0.3 uM). However, the
cytotoxicity of 56 fell between that of the spirocyclopen-
tyl analogue (54) and the diethyl analogue (53). Con-
necting the substituents on the quaternary carbon into
a tricyclic adamantyl ring system led to analogue 18
with an IC50 ) 603 nM in the KB-3-1 cells.
Exploration of substitution on the nitrogen in the
A-piece revealed that a degree of variation was tolerated
(Table 4). The N-ethyl analogue 57 and the N,N-
dimethyl analogue 26 were equipotent with 2 in the
cellular assays. Larger groups, such as in the benzyl
analogue 58 or the cyclic analogue 59, led to a decrease
of activity, indicating a small binding pocket at this
position. Incorporation of polar groups such as an
alcohol, acid or amide led to analogues 25, and 60-62
with decreased activity. Removal of the methyl group
led to analogue 63, with a dramatic decrease of cellular
cytotoxicity at the doses tested. N-Acylation led to
compound 64, with 3 orders of magnitude decrease in
activity. Decreased activity was also seen in many of
the N-BOC intermediates tested, and was reported7 for
N-acylhemiasterlin. Incorporation of the nitrogen into
a thiomorpholine ring, reminiscent of the structurally
related natural product milnamide,14 was tolerated and
led to an active, albeit less potent analogue 20 with an
IC50 ) 427 nM in the KB-3-1 cells.
The basic nitrogen in the A-piece was found to be
critical for potent cellular activity (Table 5). Replace-
ment of the methylamine group by alkyl, allyl or benzyl
groups (compounds 65-68) led to a 2-3 order of
magnitude decrease in KB cell activity. However, these
analogues were still inhibitors of tubulin polymeriza-
tion. Analogues 69-72 where the nitrogen was replaced
by other heteroatoms showed similar effects. A methoxy
subtituent gave analogue 69, with activity comparable
to that of analogue 66 bearing an ethyl group. In
contrast, a hydroxy group led to analogue 70 with no
cellular activity and a considerable loss in its ability to
inhibit tubulin polymerization at the concentrations
tested. A thiomethyl group or methylsulfonyl group gave
analogues 71 and 72, respectively, which were compa-
rable in activity to that of analogues with alkyl group
replacements. Lack of a substituent at this position gave
analogue 73 with no activity at the concentration tested.
These results, in combination with the nitrogen acyla-
tion described above, reveal that a basic nitrogen in the
A-piece is a requirement for potent activity. Several
compounds in Table 5 were tested as mixtures of the
SSS and RSS isomers.
Sch em e 11. CD-Piece Olefin Reduction
to regained potency. Other subtituents, both aromatic
and alkyl (47-50), gave potent analogues showing that
a minimum degree of lipophilicity and steric bulk at this
position is required. Saturation of the aromatic ring
gave the cyclohexyl ring analogue 47, which exhibited
an IC50 value of 3.9 nM in the KB-3-1 cells. Replacement
of the phenyl ring with 4-methoxyphenyl, in turn, gave
a potent analogue 48 with improved in vivo properties
(vide infra). This region also showed a tolerance for large
groups, as seen with the potent biphenyl analogue 49,
indicating interaction with a large binding cavity or a
solvent exposed region within tubulin. The tolerance for
large R groups on the A-piece has been exploited to
prepare a photoaffinity probe with a radiolabeled ben-
zophenone group at this position (Table 2, R ) 4-Ph-
COPh) whose binding to tubulin was competitively
inhibited by 2 and dolastatin-10.16 Compared with
indole-containing hemiasterlin (1), 2 showed a lower
level of resistance in P-glycoprotein expressing KB-8-5
and KB-V1 cells.6
The A-piece geminal dimethyl group is a key compo-
nent necessary for potent activity (Table 3). Removal
of both methyl groups was reported to lead to a loss of
potency.7 Stereospecific removal of either methyl group
led to analogues 51 and 52 with a loss of activity of
approximately 2 orders of magnitude in the cellular
assays. Larger groups at this position, such as diethyl,
spirocyclopentyl or spirocyclohexyl led to analogues 53,
54 and 55 with reduced activity, suggesting limited
The nature of the B-piece substituent was critical to
activity (Table 6). As seen previously (vide supra),
isomerization at this position led to profoundly de-

4778 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Sch em e 12. Synthesis of N,N-Dimethyl Analogue 38
Zask et al.
Ta ble 1. Isomers of 2
Ta ble 3. A-Piece Geminal Dimethyl Group Replacement
a
% inhibition of tubulin polymerization at 0.3 µM; 2 was run
as a standard. Over 10 runs its standard deviation was ( 6%.
b
Average IC50 (nM) ( SEM in cells. Where no SEM is shown, the
value is <10% of the average.
a
% inhibition of tubulin polymerization at 0.3 µM; 2 was run
Ta ble 2. Replacement of the A-Piece Phenyl Group
as a standard. Over 10 runs its standard deviation was (6%.
b
Average IC50 (nM) ( SEM in cells. Where no SEM is shown, the
value is <10% of the average.
Ta ble 4. A-Piece Amine Substitution
a
% inhibition of tubulin polymerization at 0.3 µM; 2 was run
as a standard. Over 10 runs its standard deviation was ( 6%.
b
Average IC50 (nM) ( SEM in cells. Where no SEM is shown, the
value is <10% of the average.
creased activity for the SRS isomer (42). Variation of
this substituent revealed that activity was related to
the degree of branching at the beta carbon of the B-piece
amino acid. Thus, while analogue 74 having a methyl
substituent had an IC50 of 381 nM in the KB-3-1 cells,
analogues 75 and 76 having an ethyl or n-propyl
substituent, respectively, showed a dramatic increase
in potency. Addition of a second alkyl group at the beta
carbon (i.e. isopropyl substituent) gave compound 77,
with an additional increase in potency. An illustration
of the dramatic effect of branching at the beta carbon
on activity was the 50-fold greater potency of the
a
% inhibition of tubulin polymerization at 0.3 µM; 2 was run
as a standard. Over 10 runs its standard deviation was ( 6%.
b
Average IC50 (nM) ( SEM in cells. Where no SEM is shown, the
value is <10% of the average.
dimethyl-4-methoxybenzyl analogue 79 relative to the
unmethylated benzyl analogue 78. The tolerance for a
large aromatic or substituted aromatic group at this
position points to a large binding cavity or solvent

New Antimicrotubule Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4779
Ta ble 7. CD-Piece Olefin Reduction
Ta ble 5. A-Piece Methylamine Replacement
a
% inhibition of tubulin polymerization at 0.3 µM; 2 was run
as a standard. Over 10 runs its standard deviation was (6%.
b
Average IC50 (nM) ( SEM in cells. Where no SEM is shown, the
value is <10% of the average. c 87:13 mixture.
Ta ble 8. Amide Mitrogen and CD-Piece Variations
a
% inhibition of tubulin polymerization at 0.3 µM; 2 was run
as a standard. Over 10 runs its standard deviation was ( 6%.
b
Average IC50 (nM) ( SEM in cells. Where no SEM is shown, the
value is <10% of the average.
Ta ble 6. Variation of B-Piece Substituents
a
% inhibition of tubulin polymerization at 0.3 µM; 2 was run
as a standard. Over 10 runs its standard deviation was (6%.
b
Average IC50 (nM) ( SEM in cells. Where no SEM is shown, the
value is <10% of the average.
cant mechanistic question. Conjugation of the olefin to
the carboxylic acid group makes it an electrophile and
potentially reactive with cysteine or lysine residues in
tubulin. Previous results showed a major loss of potency
upon reduction of the olefin in hemiasterlin, using
catalytic reduction conditions.7 For 2, loss of potency was
also seen (Table 7), however, analysis of the reduction
mixture of 2 revealed that one isomer was predominant
(87:13 mixture). Separation of the isomers by HPLC
revealed that the major isomer 36 was weakly active,
while the minor isomer 37 was very potent. This result
supports the view that the olefin serves not as a reactive
group, but as a center of conformational rigidity, pre-
senting the carboxylic acid group in the proper orienta-
tion for binding to tubulin. Presumably, the potent
reduced isomer 37 has a low energy conformation
similar to that of 2. The stereochemistry of the isomers
was established by NMR.
Variation of the substituent on the olefin showed that
2, bearing a methyl group, was 5-fold more potent than
the ethyl analogue 81 (Table 8). The allylic position was
tolerant of larger groups, with the n-butyl group, 82,
giving a 23-fold decrease in activity. However, the
benzyl group gave analogue 83 with a further decrease
in activity. Exploration of the amide linkages revealed
that changes on the B-C amide nitrogen could be
tolerated with removal of the methyl group, or its
replacement with the larger ethyl group, giving rise to
active, albeit substantially less potent compounds 84
a
% inhibition of tubulin polymerization at 0.3 µM; 2 was run
as a standard. Over 10 runs its standard deviation was (6%.
b
Average IC50 (nM) ( SEM in cells. Where no SEM is shown, the
value is <10% of the average. c Purity ∼ 79%. Contains ∼20% of
a diastereomer.
exposure at this site. The SAR information was used to
design and synthesize an analogue with a radiolabeled
benzophenone photoprobe (Table 6, R ) 4-PhCOPh) at
this position whose binding to tubulin was competitively
inhibited by 2 and dolastatin-10.16 Enlargement of the
tert-butyl group via incorporation of the amino acid
penicillamine as the B-piece gave analogue 80, compa-
rable in potency to 2 both in vitro and in vivo (vide
supra). The correlation of branching with potency at this
position is consistent with the reduction of conforma-
tional space accessible to the central portion of the
peptide by bulkier substituents.17 This rigidity preor-
ganizes the molecule into a conformation necessary for
tubulin binding.
The importance of the olefin in the potent antimicro-
tubule effects of the hemiasterlins and 2 was a signifi-

4780 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Ta ble 9. D-Piece Amides
Zask et al.
Ta ble 11. KD Values
a
% inhibition of tubulin polymerization at 0.3 µM; 2 was run
as a standard. Over 10 runs its standard deviation was (6%.
a
standard error < 10% for 2, 25, 47, 48, 80 and 30-35% for 24
b
Average IC50 (nM) ( SEM in cells. Where no SEM is shown, the
b
and 37. % inhibition of tubulin polymerization at 0.3 µM; 2 was
value is <10% of the average.
run as standard. Over 10 runs its standard deviation was (6%.
c Average IC50 (nM) ( SEM in cells. Weak binding, nonsaturable.
d
Ta ble 10. D-Piece Esters
e No evidence for binding.
Tu bu lin Bin d in g Affin ity (KD). Binding affinities
(KD) to tubulin were obtained for representative com-
pounds bearing structural changes in each portion of
the molecule (Table 11). Low micromolar or submicro-
molar binding constants were seen for compounds with
very potent (<5 nM) cellular cytotoxicity, independent
of the position of the structural modification. Two
compounds, with modifications of the methylamine
group (i.e. NH2, 63, and CH3, 65), which had led to a
profound decrease of cellular cytotoxicity, also showed
no evidence for binding. The ethyl ester of 2 (24) had a
KD value of 4 uM, as did the saturated analogue 37, both
somewhat higher than KD values of analogues with
more potent cellular cytotoxicity. The RSS isomer 41
showed evidence for binding, however the binding was
nonsaturable within the concentration range of 0 to 20
uM of 41, consistent with weak binding.
Activity in Resista n t Cell Lin es. Compound 2 and
its analogues are highly effective at inhibiting the
growth of tumors (both in vitro and in vivo) including
those that are resistant to paclitaxel and vincristine due
to expression of P-glycoprotein. In the cellular screening
assays, across the cell lines KB-3-1, KB-8-5 and KB-
V1, where expression of P-glycoprotein varies from none,
to moderate to very high, respectively,18 the cytotoxicity
of the analogues decreases according to the levels of
cellular P-glycoprotein. Certain analogues, representing
modifications in various representative portions of the
tripeptide were tested in other cell lines (Table 12). The
Lox melanoma and KM20 cells, with no P-glycoprotein
expression, gave IC50 values comparable to the KB-3-1
cell values. The NCI-H1299, HCT-15 and S1 cells, which
express P-glycoprotein, in general gave IC50 values
higher than the non-P-glycoprotein expressing cells. The
4-methoxyphenyl analogue 48 was assayed in a panel
of cells representing different tumor types and levels of
P-glycoprotein expression and showed potency compa-
rable to that of 2.6 Both compounds performed dramati-
cally better than paclitaxel in the resistant cell lines.
In Vivo. Select compounds that met the criteria of
good cellular potency (KB-3-1 cells) and activity in the
a
% inhibition of tubulin polymerization at 0.3 µM; 2 was run
as a standard. Over 10 runs its standard deviation was ( 6%.
b
Average IC50 (nM) ( SEM in cells. Where no SEM is shown, the
value is <10% of the average.
and 85, respectively. Methylation of the A-B amide
nitrogen, on the other hand, led to loss of potency (38),
presumably as a result of its effect on the required
conformation of the molecule.
Conversion of the D-piece carboxylic acid group to
amides 86-92 led to substantial retention of tubulin
binding and cellular activity (Table 9). Acyclic and cyclic
amides had comparable activity, however, in the acyclic
series potency decreased with larger groups on nitrogen
(88 and 89). In the cyclic analogues a benzylic group on
the piperazine nitrogen led to the most potent amide
91, with a 2-fold greater cellular potency than that of
the corresponding N-methyl analogue, 92. The hydrox-
amic acid analogue, 93, showed activity similar to that
of the amides.
The ethyl ester of 2 (24) showed good activity in the
cellular assay (Table 10). Longer alkyl groups such as
n-octyl and CH2Ph-4-CH2OBn led to less potent ana-
logues (94 and 95, respectively). The 2-thienylmethyl
ester analogue 96 gave excellent activity in the KB-3-1
cells but with a disproportionate decrease in activity in
the P-glycoprotein expressing KB-8-5 and KB-V1 cells
relative to other analogues, suggesting that it is a better
substrate for this transporter.

New Antimicrotubule Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4781
Ta ble 12. Cytotoxicity in Other Cell Linesa
a
Average IC50 (nM) ( SEM in cells. Where no SEM is shown, the value is <10% of the average.
Ta ble 13. Effect on Lox Melanoma Xenografts in Athymic
Mice
ester 24 to 2 in vivo by plasma esterases could be a
cause of the potent in vivo activity. Qualitative in vitro
plasma stability studies of 24 in CD-1 mouse plasma
revealed low levels of 2, which increased over time
suggesting a prodrug effect on the observed in vivo
potency. The amides 90 and 92 displayed potent anti-
tumor activity in the Lox xenograft model with MED’s
) 3 mpk. Their in vivo potency was approximately 10-
fold less than that of 2, but in line with their higher
cellular IC50 values. Other structural modifications such
as a cyclohexyl ring in the A-piece (47), a thiomethyl
(80) or isopropyl group (77) in the B-piece, an ethyl
group on the olefin (81) or dimethylation of the basic
amine (26) led to potent compounds with comparable
therapeutic windows. One compound, the 4-methoxy-
phenyl analogue (48) of 2, showed superior in vivo
activity (Figure 2). The MED of 0.1 mpk for 48 in the
Lox xenograft model was the most potent of those tested,
and together with an MTD of 1.6 mpk, 48 had the widest
therapeutic window (16×). At day 25-28, the relative
tumor growth (RTG) at 1 mpk, iv dose of 48 was 0.17 (
0.11 (2 RTG ) 3.2 ( 3.6). Therefore, on average, tumor
regression occurred with 48 but not 2 at this time point.
In addition, 48, when administered at the maximum
tolerated dose, displayed cures (no tumor detected) in
many animals in the Lox melanoma model. Compound
48 was also active in vivo using cell lines expressing
P-glycoprotein (HCT-15, DLD1 and MX1W) that are not
responsive to taxanes given at the MTD.
a
b
mg/kg on days 1, 5, 9, i.v. dosing (p < 0.5). Average IC50 (nM)
( SEM in cells. c Also active in HCT 15, DLD1 and MX1W
xenografts. Also active in HCT15 and MX1W xenografts.
d
resistant cell lines (KB-8-5 and KB-V1) were evaluated
in a Lox melanoma human tumor xenograft model in
athymic mice (Table 13). This model is very responsive
to taxanes.18 Both the minimum effective dose (MED)
and the maximum tolerated dose (MTD) were deter-
mined (compounds administered i.v. on a 1, 5, 9 day
schedule, p < 0.5),6 and taken together they were used
to measure the therapeutic window (MTD/MED) of the
compounds. Several compounds were also tested in
xenograft models using cell lines expressing moderate
to high levels of P-glycoprotein transporters (HCT-15,
DLD1 and MX1W). The ethyl ester of 2 (24) showed
excellent in vivo activity in the Lox xenograft model.
The MED (0.4 mpk) was 2-fold less than 2, despite the
40-fold decrease in cellular activity. The conversion of
In conclusion, investigation of the SAR of analogues
of 2 revealed the importance of several key components
to the activity of the molecule. The critical role of
branching in the B-piece was established and its role
in the conformational rigidity of the molecule deter-
mined. The CD-piece olefin was shown to act not as a
group reactive with tubulin, but as a structural element
of the molecule conferring rigidity. The necessity of a
basic group in the A-piece, as evidenced by decrease of
activity upon replacement with alkyl groups or other

4782 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Zask et al.
added CH3I (2.5-3.0 mol equivalent) and 5 N aq NaOH (3-
3.5 mol equiv). The resulting mixture was allowed to warm to
room temperature. After 15-48 h, the volatiles were removed
under reduced pressure and the resulting aqueous solution was
extracted with EtOAc. The aqueous phase was cooled to 0 °C,
acidified with 10% aq HCl to pH 1.0, and extracted with EtOAc
(three times). The combined organic extracts were washed with
brine, dried over Na2SO4 or MgSO4, filtered and concentrated
in vacuo. The product thus obtained was recrystallized or used
without further purification.
Gen er a l P r oced u r e 2: P r ep a r a tion of Ra cem ic A-
P ieces (8) fr om r,r-Dim eth ylp yr u vic Acid s (7). To a
solution of R,R-dimethylpyruvic acid 7 in anhydrous THF (0.1
mol/100 mL) was added methylamine (2.0-2.5 mol equiv, 2
M solution in anhydrous THF) at room temperature, under a
nitrogen atmosphere (during this procedure, the formation of
a thick solid may be observed). The resulting mixture was
stirred for 1-2 h, and a solution of borane-pyridine complex
(1.2 mol equiv, 8 M solution) was introduced. The mixture was
then heated at 45-55 °C (bath temperature) for 2.0-2.5 h and
cooled to room temperature. CH3OH (ca 30 mL/0.1 mol) was
added to the mixture with stirring, and the volatiles were
removed in vacuo. The resulting syrupy residue was triturated
with THF. The product precipitated upon cooling with an ice-
water bath. The solid was collected by filtration and dried
under high vacuum.
Gen er a l P r oced u r e 3: Cou p lin g of A-P ieces a n d th e
BCD-P iece (9). To a cooled (0 °C, ice bath) solution of an
A-piece (1.1 mmol) and benzotriazole-1-yl-oxy-tris-pyrrolidi-
nophosphonium hexafluorophosphate (PyBOP) (1.1 mmol) in
DMF (3-5 mL), under a nitrogen atmosphere, was added
diisopropylethylamine (1.0 mmol). To the resulting solution
was added 9 (1.0 mmol) in DMF (3 mL). After stirring at 0 °C
for 5-10 min, the cooling bath was removed, and the resulting
reaction mixture was stirred at room temperature for 15-20
h. The mixture was diluted with water, and the aqueous layer
extracted with EtOAc (three times). The combined extracts
were washed with brine, dried over anhydrous Na2SO4, filtered
and concentrated in vacuo.
F igu r e 2. Compounds 48 (top) and 2 (bottom) inhibit in vivo
growth of Lox melanoma tumors in athymic mice.
heteroatoms, was clearly demonstrated. Replacement of
the aromatic ring with other groups of sufficient steric
bulk and lipophilicity was possible. Many of the ana-
logues described are potent cytotoxic agents, active
against a variety of tumors, both in vitro and in vivo.
Importantly, they also overcame resistance to taxanes
and Vinca alkaloids associated with P-glycoprotein
expression. One analogue (48) demonstrated superior
in vivo activity.
Exp er im en ta l Section
Ch em istr y. 1H NMR spectra were determined with a
Bruker DPX-300 spectrometer at 300 MHz. Chemical shifts
(δ) are reported in parts per million relative to residual
chloroform (7.26 ppm), TMS (0 ppm), or dimethyl sulfoxide
(2.49 ppm) as an internal reference with coupling constants
(J ) reported in Hertz (Hz). The peak shapes are denoted as
follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broad. Electrospray (ES) mass spectra were recorded in
positive or negative mode on a Micromass Platform spectrom-
eter. Electron impact and high-resolution mass spectra were
obtained on a Finnigen MAT-90 spectrometer. Combustion
analyses were obtained using Perkin-Elmer Series II 2400
CHNS/O analyzer. Chromatographic purifications were per-
formed by flash chromatography using EM Science 0.040-
0.063 mm silica gel. Thin-layer chromatography (TLC) was
performed on EM Science silica gel 60 250 mm plates. Unless
otherwise noted preparative reverse phase HPLC was run on
a Phenomenex Prodigy 5 µM ODS column using a gradient
solvent system (Solvent A: 0.01% TFA/water, Solvent B: CH3-
CN). The terms “concentrated” and “evaporated” refer to
removal of solvents using a rotary evaporator at water
aspirator pressure with a bath temperature equal to or less
than 60 °C. Unless otherwise noted, reagents were obtained
from commercial sources and were used without further
purification. Pyruvic acids were either commercially available
or were prepared according to literature methods via the
corresponding azlactones or hydantoins.19
Alternatively, to a cooled (0 °C, ice bath) solution of A-piece
(1.1 mmol), HOBT (1.1 mmol) and 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimine hydrochloride (EDC) (1.2 mmol) in anhy-
drous DMF (3-5 mL) under a nitrogen atmosphere was added
N-methylmorpholine (1.4 mmol) via syringe. After stirring for
15 min at 0 °C, the cooling bath was removed, and the
resulting mixture was stirred for 2-24 h. The solution was
cooled to 0 °C (ice water bath), and to this mixture was added
a solution of 9 (1.0 mmol) in anhydrous DMF (3 mL). The
cooling bath was removed, and the resulting mixture was
stirred for 15-36 h at room temperature under a nitrogen
atmosphere. The mixture was diluted with water, and the
aqueous layer extracted with EtOAc (three times). The com-
bined extracts were washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated in vacuo.
Gen er a l P r oced u r e 4: Ester Hyd r olysis. The ester (1
mmol) was dissolved in CH3OH (24 mL) and cooled to 0 °C
(ice-water bath). To this solution were added water (8 mL)
and 1 M aq LiOH (8 mmol). The cooling bath was removed,
and the resulting mixture was stirred at room temperature
for 15 h. CH3OH was removed in vacuo, and the residual
aqueous mixture was cooled with an ice-water bath, and
acidified to pH 5.5-6.0 with aq 1 N citric acid. The precipitate
was collect by filtration, and the solid was washed with cold
water and dried in vacuo (alternatively, the product could be
purified by reverse phase HPLC).
Gen er a l P r oced u r e 5: D-P iece Am id e F or m a tion . To
a solution of 2 (0.1 mmol) in CH3CN or DMF (5.0 mL) at 25
°C was added HOBT (0.12 mmol) and EDC (1.33 mmol). After
2-18 h, an amine (0.4 mmol) was added. After 1-18 h, the
reaction mixture was concentrated in vacuo and purified by
reverse phase HPLC.
Biology. The tubulin polymerization assay, cellular cyto-
toxicity assays and in vivo xenograft assays were performed
as described previously.6 The cell lines used in the cytotoxicity
assays and the xenograft assays were obtained as described
previously.6 KD values were determined as described in the
literature.20
Gen er a l P r oced u r e 1: P r ep a r a tion of r,r-Dim eth -
ylp yr u vic Acid s (7). To a solution of a pyruvic acid in THF
(ca. 0.1 mol/100 mL) at 0 °C under a nitrogen atmosphere was
Gen er a l P r oced u r e 6: F or m a tion of D-P iece Ester s.
A mixture of 2 (1.0 equiv), EDC (1.2 equiv), an alcohol (1.2
equiv) and DMAP (0.2 equiv) in anhydrous CH2Cl2 (1 mmol/

New Antimicrotubule Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4783
20 mL) was stirred under a nitrogen atmosphere, at room
temperature, for 20 h. The solvent was removed, the residue
was taken up in CH3OH, and the product was purified by
reverse phase HPLC.
reaction mixture was allowed to warm to room temperature.
After 18 h the reaction mixture was cooled to 0 °C and aq
sodium sulfite solution (1.5 M, 1.3 mL) was added. After
stirring for 1 h at room temperature, the reaction mixture was
concentrated under reduced pressure. The residue was ex-
tracted twice with CH2Cl2, acidified to pH 1 with 10% aq HCl,
then extracted 3× with EtOAc. The combined organic extracts
were washed with brine, dried over anhydrous Na2SO4, and
concentrated under reduced pressure to give the title com-
pound as a white crystalline solid (60 mg, 72%). Mp 69-72 °C
MS, m/z: 191.0 (M - H)-.
(4S)-4-Isop r op yl-3-(3-m eth yl-3-p h en ylbu ta n oyl)-1,3-ox-
a zolid in -2-on e (27). To a solution of 3,3-dimethyl-3-phenyl
propionic acid (2.1 g, 12 mmol) in anhydrous THF (24 mL)
under nitrogen was added triethylamine (2.0 mL). The mixture
was cooled to -78 °C and pivaloyl chloride (1.5 mL, 13 mmol)
was added dropwise. The reaction mixture was held for 15 min
at - 78 °C and was then warmed to 0 °C with an ice-water
bath. In a separate flask, a solution of (S)-4-isopropyl-2-
oxazolidinone (1.5 g, 12 mmol) in anhydrous THF (27 mL) was
prepared under inert atmosphere and cooled to - 35 °C. A
small amount of triphenylmethane (<5 mg) was added as an
indicator of deprotonation. n-Butyllithium (1.6 M solution in
hexanes, 7.7 mL, 12 mmol) was added dropwise via syringe.
After 30 min at 0 °C, the flask containing the mixed anhydride
was recooled to -78 °C in a dry ice/acetone bath. The solution
of the lithium anion of the oxazolidinone was added to the
mixed anhydride solution via cannula. The reaction mixture
was stirred at -78 °C for 1 h, and at 0 °C for 1 h, and allowed
to warm to room temperature. After 18 h, water (∼ 15 mL)
was added. After 20 min the aq phase was extracted 3× with
Et2O. The combined extracts were washed with brine, dried
over Na2SO4, and concentrated under reduced pressure to
afford a pale yellow oil (3.6 g). The crude material was purified
by flash chromatography (EtOAc/hexanes) to afford the title
compound as a clear, colorless oil (1.5 g, 44%). MS, m/z: 290.0
(M + H)+.
3-Meth yl-3-(4-br om op h en yl)-2-oxobu ta n oic Acid (7; R
) CH3, R1 ) 4-Br P h ). To a solution of 3-methyl-3-phenyl-2-
oxobutanoic acid21 (1.0 g, 5.2 mmol) in CCl4 (3.5 mL) were
added bromine (0.86 g, 5.4 mmol) and iron powder (0.007 g).
The resulting mixture was heated at reflux overnight. The
deep red reaction mixture was washed with cold 10% aq HCl
and then water. The organic phase was then extracted with
2.5 M aq NaOH (40 mL). The basic extracts were then acidified
to pH 1.0 with 12 N aq HCl. A dark red liquid oiled out, which
was isolated by extraction with EtOAc. The organic extracts
were washed with brine, dried over Na2SO4, decanted, and
concentrated under reduced pressure to afford a straw-colored
semisolid (1.1 g, 79%). MS, m/z: 269.1, 271.1 (M - H)-. Anal.
(C11H11BrO3).
4-Br om o-N,â,â-tr im eth ylp h en yla la n in e (8; R ) CH3, R1
) 4-Br P h , R2 ) CH3). According to General Procedure 2,3-
methyl-3-(4-bromophenyl)-2-oxobutanoic acid (7; R ) CH3, R1
) 4-BrPh) (1.0 g, 3.7 mmol), THF (8 mL), methylamine (4.1
mL, 2 M solution in THF) and borane-pyridine (0.45 mL, 8
M) gave the title compound as a white powder (0.32 g, 49%).
MS, m/z: 284.2, 286.2 (M - H)-. Anal. (C12H16BrNO2).
(Meth yla m in o)(1-p h en ylcyclop r op yl)a cetic Acid (11).
A mixture of 1-phenyl-cyclopropanecarbaldehyde12 (1.46 g, 10.0
mmol), methylamine (6.25 mL of 2 M solution in CH3OH, 12.5
mmol) and CH3OH (7 mL) was stirred for 15 min. Then
1-isocyano-cyclohexene (1.07 g, 10.0 mmol) and formic acid
(600 mg, 96%, 12.5 mmol) were added. After 18 h the reaction
mixture was concentrated in vacuo and triturated with 10:1
hexane/EtOAc, filtered and dried to give N-cyclohex-1-en-
1-yl-2-[formyl(methyl)amino]-2-(1-phenylcyclopropyl)aceta-
mide 10 as a white solid (1.23 g). MS, m/z: 313.2 (M + H)+.
Compound 10 (400 mg, 1.23 mmol) was refluxed with aq 6 N
HCL (3 mL) for 1 h. The mixture was concentrated in vacuo
2-Ben zyl-3-m eth yl-3-p h en ylbu ta n oic Acid (28, R ) Bn ).
In accordance with a modified literature procedure22 a solution
of 3,3-dimethyl-3-phenyl propionic acid (0.22 g, 1.2 mmol) in
THF (2 mL) under nitrogen was cooled to - 40 °C. Lithium
diisopropylamide (2.0 M in THF, 1.4 mL, 2.8 mmol) was added
dropwise via syringe, followed by 1,3-dimethyl-3,4,5,6-tetrahy-
dro-2(1 H)-pyrimidinone (DMPU, 0.16 mL, 1.3 mmol). The
reaction mixture was then allowed to warm to room temper-
ature and to stir for 30 min before being recooled to 0 °C (ice
water bath). Benzyl bromide (0.15 mL, 1.3 mmol) was then
added via syringe, and the reaction mixture was allowed to
warm to room temperature. After 18 h, the reaction was
quenched by the addition of 10% aq HCl and extracted 3× with
EtOAc. The combined extracts were washed twice with 10%
aq HCl and once with saturated NaCl solution, dried over
anhydrous sodium sulfite, and concentrated under reduced
pressure. Flash chromatography (silica gel, ether/hexanes)
gave the title compound as a straw-colored gum (70%). MS,
m/z: 266.9 (M - H)-.
2-Eth yl-3-m eth yl-3-p h en ylbu ta n oic Acid (28, R ) Et).
By the method described for 28 (R ) Bn), 3,3-dimethyl-3-
phenyl propionic acid (0.47 g, 2.6 mmol), lithium diisopropyl-
amide (2.0 M, 2.9 mL, 5.8 mmol), DMPU (0.35 mL, 2.9 mmol)
and ethyl iodide (0.31 mL, 3.9 mmol) gave, after recrystalli-
zation from hexanes, the title compound as a white solid (0.16
g, 30%). MS, m/z: 205.0 (M - H)-.
2-Allyl-3-m eth yl-3-p h en ylbu ta n oic Acid (28, R ) a llyl).
By the method described for 28 (R ) Bn), 3,3-dimethyl-3-
phenyl propionic acid (0.50 g, 2.8 mmol), lithium diisopropyl-
amide (2.0 M, 3.1 mL, 6.2 mmol), DMPU (0.37 mL, 4.2 mmol)
and allyl iodide (0.15 mL, 1.3 mmol) gave, after flash chro-
matography (silica gel, hexane/CH2Cl2/EtOAc), the title com-
pound as a clear blond oil (0.39 g, 64%). MS, m/z: 217.0 (M -
H)-.
and the residue purified by HPLC to give the title compound
+
as a white solid (224 mg). MS, m/z: 220.1 (M + H)
.
Meth yl (âS)-N-(ter t-bu toxyca r bon yl)-N,â-d im eth yl-L-
p h en yla la n in a te (12b). A solution of (âS)-N-(tert-butoxycar-
bonyl)-â-methyl-L-phenylalanine (200 mg, 0.716 mmol) in DMF
at 25 °C was treated with NaH (60% in mineral oil, 60 mg,
1.5 mmol). After 1 h iodomethane (0.13 mL, 2.15 mmol) was
added. After 24 h EtOAc and 1 N aq citric acid was added.
The organic phase was washed with water (5×) and dried over
MgSO4. Concentration in vacuo gave the title compound as a
colorless oil (230 mg). HRMS (ESI) calcd for C17H25NO4:
308.18620 (M + H)+; found 308.18577.
Ad a m a n ta n -1-yl-m eth yla m in o-a cetic a cid (17). Ada-
mantan-1-yl-iodo-acetic acid13 (7.86 g, 24.55 mmol) was treated
with 2 M methylamine (100 mL, 200 mmol) in THF and heated
at reflux for 18 h. Concentration in vacuo followed by addition
of water and acidification to pH 6 with aq HCl gave the title
compound as a white solid (2.16 g). MS, m/z: 224.2 (M + H)+.
(2S)-2,3-Dim eth yl-3-ph en ylbu tan oic Acid (28, R ) CH3).
A solution of 27 (0.23 g, 0.79 mmol) in anhydrous THF (3 mL)
under a nitrogen atmosphere was cooled to -78 °C. Sodium
hexamethylsilazide (1.0 M in THF, 0.95 mL, 0.95 mmol) was
added dropwise via syringe. After 30 min at -78 °C,
iodomethane (0.25 mL, 4.0 mmol) in THF (1 mL) was added
dropwise. The reaction mixture was allowed to warm to room
temperature. After 18 h the reaction mixture was treated with
water (10 mL) and EtOAc (10 mL). The aqueous phase was
extracted 3× with EtOAc. The combined extracts were washed
with saturated aq NaHCO3 and brine, dried over Na2SO4 and
concentrated under reduced pressure to afford an amorphous
solid. The crude material was purified by flash chromatogra-
phy (EtOAc/hexanes) to afford (4S)-3-[(2S)-2,3-dimethyl-3-
phenylbutanoyl]-4-isopropyl-1,3-oxazolidin-2-one as a crystal-
line solid (0.17 g, 71%). MS, m/z: 303.9 (M + H)+. To a solution
of this compound (0.13 g, 0.43 mmol) in THF (6 mL) and water
(2 mL) at 0 °C was added 30% aq hydrogen peroxide (2.4 mL).
After 2 min, LiOH monohydrate (36 mg) was added. The
3-Meth yl-2-(m eth ylsu lfan yl)-3-ph en ylbu tan oic Acid (30).
By the method described for 28 (R ) Bn ) 3,3-dimethyl-3-
phenyl propionic acid (0.50 g, 2.8 mmol), lithium diisopropyl-

4784 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Zask et al.
amide (2.0 M, 3.1 mL, 6.2 mmol), DMPU (0.37 mL, 3.1 mmol)
and dimethyl disulfide (0.38 mL, 4.2 mmol) gave, after flash
chromatography (silica gel, hexanes/CH2Cl2/CH3OH), the title
compound as a tan solid (0.54 g, 86%). MS, m/z: 222.9 (M -
H)-.
4-P h en yl-N,â,â-tr im eth yl-L-p h en yla la n yl-N1-[(1S,2E)-3-
ca r b oxy-1-isop r op yl-2-b u t en yl]-N1,3-d im et h yl-L-va lin a -
m id e (49). According to General Procedure 3, 4-bromo-N,â,â-
trimethylphenylalanine (8; R ) CH3, R1 ) 4-BrPh, R2 ) CH3)
(0.29 g, 1.5 mmol), PyBOP (0.57 g, 1.1 mmol), diisopropylethyl-
amine (0.35 mL, 2.0 mmol) and 9 (HCl salt) (0.38 g, 1.1 mmol)
in CH2Cl2 (10 mL) gave after purification by preparative
reverse-phase HPLC, the TFA salt of 4-bromo-N,â,â-trimeth-
ylphenylalanyl-N1-[(1S,2E)-4-ethoxy-1-isopropyl-3-methyl-4-
oxo-2-butenyl]-N1,3-dimethyl-L-valinamide. MS, m/z: 580.6,
582.6 (M + H)+. calcd for Anal. (C29H46BrN3O4‚1.6TFA‚CH3-
CN) To a solution of this material (0.25 g, 0.43 mmol) in
ethylene glycol dimethyl ether (9 mL) and water (4 mL) was
added phenylboronic acid (0.10 g, 0.86 mmol), Na2CO3 (0.14
g, 1.3 mmol), and (tetrakis)triphenylphosphine palladium (0.05
mg, 0.04). The reaction mixture was heated at reflux for 18 h
and then allowed to cool to room temperature. Volatiles were
evaporated under reduced pressure, and the residue was
partitioned between ether and water. The aqueous phase was
extracted 3× with ether. The combined organic extracts were
washed with water then brine, dried over anhydrous Na2SO4,
and concentrated under reduced pressure to afford a reddish-
brown semisolid (0.20 g) which was purified by preparative
reverse-phase HPLC to give the TFA salt of 16 (60 mg). MS,
m/z: 578.5 (M + H)+. According to General Procedure 4, 16
(0.06 g, 0.10 mmol) and LiOH (0.30 mmol) in THF/CH3OH/
water (0.5 mL) gave the TFA salt of the title compound (0.020
g, 26%) after purification by preparative reverse-phase HPLC.
MS, m/z: 550.4 (M + H)+. Anal. (C33H47N3O4‚3TFA‚2H2O).
N -E t h yl-â,â-d im e t h yl-L-p h e n yla la n yl-N 1-[(1S ,2E )-3-
ca r b oxy-1-isop r op ylb u t -2-en yl]-N1,3-d im et h yl-L-va lin -
a m id e (57). Treatment of 22 (143 mg, 0.273 mmol) in DMF
(1.5 mL) with iodoethane (0.025 mL, 0.30 mmol) and diiso-
propylethylamine (100 uL, 0.57 mmol) for 6 h, followed by
HPLC gave a solid (32 mg). This material (28 mg) was treated
as described in General Procedure 4 to give, after HPLC, the
TFA salt of 57 as a white solid (20 mg). MS, m/z: 488.3 (M +
H)+.
N-(2-h yd r oxyeth yl)-N,â,â-tr im eth yl-L-p h en yla la n yl-N1-
[(1S,2E)-3-ca r boxy-1-isop r op ylbu t-2-en yl]-N1,3-d im eth yl-
L-va lin a m id e (25). To 24 (300 mg, 0.56 mmol) in DMF (2 mL)
were added 2-bromoethanol (0.04 mL, 0.56 mmol) and diiso-
propylethylamine (0.195 mL, 1.12 mmol). After 18 h, tetrabu-
tylammonium iodide (10 mg) was added. After 4 h 2-bromo-
ethanol (0.1 mL) was added. After 18 h, the reaction mixture
was heated at 70 °C and additional 2-bromoethanol and
diisopropylethylamine were added. The reaction mixture was
purified by reverse phase HPLC. As described in General
Procedure 4, the resulting material (50 mg, 0.0758 mmol) was
treated with LiOH (0.23 mmol) for 18 h. Purification by HPLC
gave the TFA salt of 25 as a white powder (33 mg). MS, m/z:
518.4 (M + H)+. Anal. (C29H47N3O5‚1.5TFA‚0.2H2O).
3-Meth yl-2-(m eth ylsu lfon yl)-3-ph en ylbu tan oic Acid (31).
A solution of 30 (0.28 g, 1.2 mmol) in CH2Cl2 (10 mL) was
cooled to 0 °C and treated with peracetic acid (32% aqueous
solution, 0.76 mL, 3.6 mmol). The reaction mixture was
allowed to warm to room temperature. After 3 h, additional
peracetic acid (0.76 mL) was added. After 18 h, the reaction
mixture was concentrated under reduced pressure and 1.5 M
aq sodium sulfite solution (20 mL) was added. The reaction
mixture was extracted 3× with EtOAc. The combined organic
extracts were washed with 10% aq HCl and brine, dried over
anhydrous Na2SO4, and concentrated under reduced pressure
to give the title compound as a white powder (0.29 g, 94%).
MS, m/z: 255.0 (M - H)-.
2-Hyd r oxy-3-m eth yl-3-p h en ylbu ta n oic Acid (32). To a
solution of 3-phenyl-3-methyl-2-oxobutanoate (9.0 g, 47 mmol)
in Et2O (40 mL) and CH3OH (60 mL) at 0 °C was added
trimethylsilyl diazomethane (2.0 M solution in hexanes, 35 mL,
70 mmol) dropwise. The solvents were removed under reduced
pressure and the residue was taken up in Et2O/hexanes (1:1)
and washed with 2% aq HCl, saturated aq NaHCO3, and brine.
The organic phase was dried over anhydrous Na2SO4 and
concentrated under reduced pressure to give methyl 3-phenyl-
3-methyl-2-oxobutanoate (33) as a straw-colored liquid (8.5 g,
88%). MS, m/z: 206.9 (M + H)+. According to a literature
procedure23 a solution of 33 (2.5 g, 12 mmol) in CH3OH (144
mL) was cooled to 0 °C. Sodium borohydride (0.21 g, 5.5 mmol)
was added in three portions over 3 min. After 10 min, the
reaction mixture was allowed to warm to room temperature.
After 30 min, the reaction mixture was acidified with 10% aq
HCl until pH ) 6 and then extracted 3× with Et2O. The
combined organic extracts were washed with water and brine,
dried over anhydrous sodium sulfite, and concentrated under
reduced pressure. Flash chromatography (silica gel, hexanes/
EtOAc) gave methyl 2-hydroxy-3-methyl-3-phenylbutanoate
(34) (80%) as a clear, colorless oil. MS, m/z: 209.1 (M + H)+.
According to General Procedure 4, 34 (0.27 g, 1.3 mmol), CH3-
OH (6 mL), THF (6 mL), water (3 mL) and LiOH monohydrate
(0.12 g, 2.9 mmol) were heated at 60-65 °C for 16 h to give
the title compound as a solid (0.25 g, 100%). MS, m/z: 193.0
(M - H)-.
2-Meth oxy-3-m eth yl-3-p h en ylbu ta n oic Acid (35). To a
solution of 34 (0.50 g, 2.4 mmol) under nitrogen in diethyl ether
(11 mL) was added silver(I) oxide (4.3 g) and iodomethane (8.7
mL). The reaction mixture was heated at 40 °C. After 26 h,
the reaction mixture was filtered through a plug of Celite. The
filtrate was concentrated in vacuo and purified by chromatog-
raphy (silica gel, hexanes/EtOAc) to give methyl 2-methoxy-
3-methyl-3-phenylbutanoate as a clear, colorless liquid (68%
yield). MS, m/z: 222.9 (M + H)+. According to General
Procedure 4, this compound (0.49 g, 2.2 mmol), CH3OH (10
mL), THF (10 mL), water (5 mL) and LiOH monohydrate (0.18
g, 4.4 mmol) were heated at 60 °C for 16 h to give 35 as a
crystalline solid (0.46 g, 100%). MS, m/z: 207.0 (M - H)-.
N,â,â-Tr im eth yl-L-p h en yla la n yl-N1-[(1R,3S)-3-ca r boxy-
1-isop r op ylbu tyl]-N1,3-d im eth yl-L-va lin a m id e (36) a n d
N,â,â-Tr im eth yl-L-p h en yla la n yl-N1-[(1R,3R)-3-ca r boxy-1-
isop r op ylbu tyl]-N1,3-d im eth yl-L-va lin a m id e (37). A solu-
tion of 2 (200 mg) in acetic acid (10 mL) was treated with 10%
Pd/C under 1 atm of hydrogen for 18 h. Concentration in vacuo,
filtration through diatomaceous earth with 1:1 CH3OH:water
(0.02% TFA) gave an 87:13 mixture of isomers as determined
by analytical HPLC. Separation by preparative reverse phase
HPLC (1:1 CH3OH:water (0.02% TFA) gave the TFA salt of
the major isomer (36) (first off of HPLC column) as a white
powder (56 mg). HRMS (ESI) calcd for C27H45N3O4: 476.34885
(M + H)+; found 476.34796. Anal. (C27H45N3O4. 1.5 TFA. 1.5
H2O) HPLC gave the TFA salt of the minor isomer (37) (7 mg).
MS, m/z: 476.5 (M + H)+. HPLC ) 98.85%.
N ,N ,â,â-Te t r a m e t h yl-L-p h e n yla la n yl-N 1-[(1S ,2E )-3-
ca r b oxy-1-isop r op yl-2-b u t en yl]-N1,3-d im et h yl-L-va lin -
a m id e (26). To 24 (200 mg, 0.372 mmol) in DMF (3 mL) were
added iodomethane (29 uL, 0.44 mmol) and diisopropylethyl-
amine (200 uL, 1.12 mmol). After 30 min, the reaction mixture
was purified by HPLC to give the desired dimethylamine (99
mg). As described in General Procedure 4, this material was
treated with LiOH (0.47 mmol) in CH3OH/THF/water for 18
h. Purification by HPLC gave the TFA salt of 26 as a white
powder (20 mg). HRMS (ESI) calcd for C28H45N3O4: 488.34885
(M + H)+; found 488.3412.
â,â-Dim eth yl-L-p h en yla la n yl-N1-[(1S,2E)-3-ca r boxy-1-
isop r op ylb u t -2-en yl]-N1,3-d im et h yl-L-va lin a m id e (63).
Commercially available (S)-N-BOC-2-amino-3-methyl-3-phen-
yl-butyric acid was converted to the HCl salt of 22 by the use
of General Procedure 3 followed by treatment with HCl in
dioxane to remove the BOC protecting group. Treatment of
the resulting material according to General Procedure 4 gave
the HCl salt of 63. Anal. (C26H41N3O4‚1.5HCl‚0.2CH3OH).
(2E,4S)-2,5-Dim eth yl-4-(m eth yl{3-m eth yl-N-[(2,2,4-tr i-
m eth ylth iom or p h olin -3-yl)ca r bon yl]-L-va lyl}a m in o)h ex-
2-en oic Acid (20). A mixture of 2,2-dimethyl-thiomorpholine-

New Antimicrotubule Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4785
3-carboxylic acid15 (3.51 g, 20 mmol), formic acid (5.12 g of 90%)
and formalin (4.5 mL of 37%) was heated at reflux for 8 h.
The reaction mixture was cooled, treated with 4 N aq HCl (10
mL) and concentrated in vacuo. Purification by HPLC gave
2,2,4-trimethyl-thiomorpholine-3-carboxylic acid. According to
General Procedure 3, this compound (388 mg, 2.05 mmol),
PyBOP (1.07 g, 2.05 mmol), 9 (0.640 g, 2.05 mmol) and
diisopropylethylamine (0.8 mL, 4.4 mmol) gave the coupled
compound (314 mg, 0.65 mmol), which was treated according
to General Procedure 4 with LiOH (3.3 mmol) to provide, after
HPLC, the TFA salt of 20. MS, m/z: 456.3 (M + H)+. Anal.
(C23H41N3O4S‚1.25TFA‚1.25H2O).
analogue 41 and Vincent Sandanayaka for contributions
toward synthesis of analogue 38. The authors thank Dr.
J erauld Skotnicki and Dr. Tarek Mansour for their
support of this work.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectral
data, elemental analyses, analytical HPLC purity determina-
tions, experimental assignment of the stereochemistry of 36,
37 and the RSS and SSS diasteromers by 1H NMR, experi-
mentals for 24, 41-43, 45-48, 50-56, 58-62, 64, 66-85, 88-
92, 95, 96. This material is available free of charge via the
Internet at http://pubs.acs.org.
(2E,4S)-4-[{N-[(2S)-2-(1-a d a m a n t yl)-2-(m et h yla m in o)-
eth a n oyl]-3-m eth yl-L-va lyl}(m eth yl)a m in o]-2,5-d im eth yl-
2-h exen oic Acid (18). According to General Procedures 3 and
4, 17 and 9 gave, after purification by HPLC, the TFA salt of
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18 as
a
white solid. MS, m/z: 490.2 (M + H)+. Anal.
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(E,4S)-4-[((2S)-2-{[(2S)-2,3-Dim eth yl-3-p h en ylbu ta n oyl]-
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N,â,â-Tr im eth yl-L-p h en yla la n yl-N1-[(1S,2E)-3-ca r boxy-
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(E,4S)-4-[((2S)-3,3-d im eth yl-2-{[(2S)-3-m eth yl-2-(m eth -
yla m in o)-3-p h en ylb u t a n oyl]a m in o}b u t a n oyl)(m et h yl)-
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was treated with HOBT (17 mg, 0.127 mmol) and EDC (27
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2H2O).
(E,4S)-4-[((2S)-3,3-Dim eth yl-2-{[(2S)-3-m eth yl-2-(m eth -
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mmol) was added. Purification by preparative reverse phase
HPLC gave the TFA salt of 87 as a white powder (20 mg).
MS, m/z: 487.6 (M + H)+.
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(E,4S)-4-[((2S)-3,3-Dim eth yl-2-{[(2S)-3-m eth yl-2-(m eth -
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