Synthesis and activity of novel analogs of hemiasterlin as inhibitors of tubulin polymerization: Modification of the a segment

Article abstract of DOI:10.1016/j.bmcl.2004.08.024

Analogs of HTI-286 (1), containing various aromatic rings in the A segment, were synthesized as potential inhibitors of tubulin polymerization, and the structure-activity relationships related to stereo- and regio-chemical effects of substituents on the a

Full text of DOI:10.1016/j.bmcl.2004.08.024

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5317–5322
Synthesis and activity of novel analogs of
hemiasterlin as inhibitors of tubulin polymerization:
modification of the A segment
Ayako Yamashita,* Emily B. Norton, Joshua A. Kaplan, Chuan Niu,
Frank Loganzo, RichardHernandez, Carl F. Beyer, Tami Annable,
Sylvia Musto, Carolyn Discafani, Arie Zask andSemiramis Ayral-Kaloustian
Chemical and Screening Sciences and Oncology Research, Wyeth Research, Pearl River, NY 10965, USA
Received19 May 2004; accepted10 August 2004
Available online 11 September 2004
Abstract—Analogs of hemiasterlin (1) andHTI-286 ( 2), which contain various aromatic rings in the A segment, were synthesizedas
potential inhibitors of tubulin polymerization. The structure–activity relationships relatedto stereo- andregio-chemical effects of
substituents on the aromatic ring in the A segment were studied. Analogs, which carry a meta-substitutedphenyl ring in the A seg-
ment show comparable activity for inhibition of tubulin polymerization to 2, as well as in the cell proliferation assay using KB cells
containing P-glycoprotein, comparedto those of 1 and 2.
ꢀ 2004 Elsevier Ltd. All rights reserved.
2
Hemiasterlin1 (1, Fig. 1) isolatedfrom marine sponges,
is one member of a family of tripeptides consisting of
three sterically congestedamino acisd. Hemiasterlin
andits relatives have been foundto be potent antimi-
totic agents that effectively inhibit tubulin polymeriza-
tion by binding to the Vinca alkaloid site.3 The total
synthesis of 1 was achievedwith a focus on the enantio-
selective synthesis of N-methyl-a,a-dimethyltryptophan
(the A segment).4 The phenyl analog (2, HTI-286),
in which the N-methylindole in the A segment was
replacedwith a phenyl ring, was also synthesized, and
its antimitotic properties were comparedwith those
of 1.5 Although the biological profile of 2 as an inhibitor
of tubulin polymerization is comparable to that of 1, 2
showedbetter activity than 16 against resistant cell lines.
In addition, the synthesis of the A segment of 2 required
fewer andrelatively easier steps than that of 1. Initial
SAR (structure–activity relationship) involving stereo-
chemical modifications of 2 was studied.7 The general
trends observed from the SAR study are: (1) changing
the stereochemistry of the amino acids of the A–B–C
segment from a*,b*,c* = S,S,S resultedin reduction of
the activity. (2) Both the gem-dimethyl and N-methyl
groups in the A segment are necessary. (3) A sterically
hindered alkyl substituent in the B segment is requisite
for activity, as the tert-butyl group showedthe best
activity. (4) Removal of the N-methyl group in the C
segment of 2 decreases the activity. (5) The stereochem-
istry of the olefin in the D segment prefers the E form.
A-segment
B-segment C-segment D-segment
O
O
γ∗
α∗
N
N
H
OH
Hemiasterlin (1)
β∗
NH
O
N
(α∗,β∗,γ∗) = (S,S,S)
O
O
γ∗
α∗
NH
N
N
H
OH
β∗
HTI-286 (2)
O
(α∗,β∗,γ∗) = (S,S,S)
Figure 1.
Basedon these observations, we next studiedthe SAR
involving stereo- andregio-chemical effects of substitu-
ents on the aromatic ring in the A segment of 2. Toward
this goal, we preparedvarious A segments, in which the
phenyl ring is substitutedby methyl, halogen, andaryl
*
Corresponding author at present address: Department of Chemistry,
Columbia University, New York, NY 10027, USA. Fax: +1 212 854
9720; e-mail: ay2122@columbia.edu
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.024

5318
A. Yamashita et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5317–5322
O
O
a
b
Ar
O
Ar-CHO
3a-l
Ar
OH
N
O
5a-l
4a-l
Ar
Compds
O
6a
6b
6c
6d
6e
6f
3-Tolyl
4-Tolyl
c
Ar
OH
NH
3,4-Dimethylphenyl
3,5-Dimethylphenyl
3-Chlorophenyl
6a-l
4-Chlorophenyl
6g
6h
6i
3-Bromophenyl
3-Fluorophenyl
3,5-Difluorophenyl
3-Trifluoromethylphenyl
3,5-Di-trifluoromethylphenyl
2-Thienyl
6j
6k
6l
6m
3-Thienyl
Scheme 1. Reagents andconditions: (a) i. N-acetylglycine, NaOAc, Ac 2O, reflux, 5h; (b) i. 1N NaOH, 80ꢁC, ii. 5N HCl, reflux, 5h; (c) i. MeI, 5N
NaOH, THF, rt, ii. CH3NH2, THF, 55ꢁC, iii. BH3-pyridine, 60ꢁC, 4h.
groups at various positions (ortho, meta, para). The A
segments thus preparedwere coupledwith the B–C–D
segment, andthe final analogs were evaluatedfor inhibi-
tion of tubulin polymerization. In this paper, we report
our finding that introduction of a substituent at the
meta-position of the phenyl ring, in general, increases
potency relative to 2.
Me
Me
O
Me
Ph
Me
Ph
CHO
a
-
BF4
+
S+
9
3n
8
Me
Me
Me
CN
b
CHO
10
c
NH
The synthesis of a racemic A segment 6 follows the
7
reportedprocedures (Scheme 1). Aldehyde 3 was con-
11
Me
vertedto the corresponding azlactone
4,8 which was
O
then convertedto the a-keto acid 5 by hydrolysis.9
Dimethylation, followedby reductive amination, gave
the desired racemic amino acid 6.10 This process allowed
us to prepare meta- and para-tolyl-derivatives 6a and 6b.
O
NH2
Boc
NH2
d
e
N
NH
12
13
In the synthesis of an ortho-tolyl A segment, ortho-tolu-
aldehyde (3n) was convertedto the corresponding keto
acid using the procedure described earlier. An attempt
to introduce dimethyl group to the keto acid, however,
resultedin only mono-methylation at the benzylic posi-
tion, presumably because the secondmethylation was
inhibitedby steric hindrance from the ortho-methyl in
the phenyl ring as well as by the methyl at the benzylic
position. An alternative route to the A segment 6n
was, therefore, developed (Scheme 2).10 Aldehyde 3n
was convertedto an epoxide 9 by treatment with the
anion of isopropyldiphenylsulfonium tetrafluoroborate
(8).11 Ring opening/rearrangement of 9 in the presence
of Lewis acid, using the conditions reported by Yama-
Me
O
Me
O
Boc
Boc
g
f
N
OH
Boc
N
N
Boc
6n
14
Me
O
Me
O
Boc
N
O
N
13y
10x
Scheme 2. Reagents andconditions: (a) LDA, DME, CH 2Cl2, ꢀ78ꢁC
then ꢀ40ꢁC, 15h, 65%; (b) i. (C6F5)3B, benzene, 60ꢁC, 89%; (c)
MeNH2, KCN, MeOM, H2O, 1–2days, 89%; (d) LiOH, H2O2, 2–5
days, 25%; (e) Boc2O, MeCN, 4–5days; (f) DMAP, i-Pr2EtN; (g)
NaOH, 88%.
12
moto andco-workers, gave aldehyde 10 as the major
product, along with a small amount of ketone 10x,
which was removedby chromatography. Reaction of
10 with potassium cyanide and methylamine gave amino-
nitrile 11. Hydration of the cyano group in 11 to
amide 12 with lithium hydroxide and hydrogen perox-
ide turned out to be very slow. Only a 25% yield of 12
was obtained, along with recovered 11, after 5days.

A. Yamashita et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5317–5322
5319
Table 1. Cellular proliferation assay profiles andinhibition of tubulin
polymerization: comparison of analogs with ortho-, meta-, and para-
tolyl substitutedA segment
Considerable effort was put into achieving the conver-
sion of carboxamide 12 into the Boc-protectedA seg-
ment 6n by a one pot process, via the intermediates 13
and 14. We foundthat it was crucial to protect the basic
nitrogen in 12 to form intermediate 13, before the
amide nitrogen could be diacylated with the Boc groups.
Acylation of the N-methyl group couldbe done in the
absence of DMAP, but further acylation of the amide
was extremely slow, andit took 4–5days to form 13
completely. When the whole triacylation process was
attemptedin the presence of DMAP, the hydantoin
13y was isolated, suggesting that under those conditions,
the amide in 12, rather than the N-methyl group, was
first to be Boc-protected, and that the intermediate then
spontaneously cyclizedto 13y. It was eventually found
CompdIC
(nM)
Inhibition of tubulin
polymerization %d (%)e
50
KB-3-1a KB85b
KBV1c
1f
2f
0.3
1.0
1.0
2.4
76
81
NA
91
16a
17a
18a
19a
18b
19b
18n
19n
21.9
263
0.23
525.3
1698
2410
3000
19
58 (94)
26 (94)
90 (89)
88 (89)
95 (94)
95 (94)
91 (96)
85 (96)
0.65
7.9
1.5
19.9
2.6
446
55
17
27.5
1.8
1072
56
0.9
6.9
13.8
501
that if DMAP andHu nigÕs base were added after the
a IC50 (amount of drug needed to kill 50% of cells after 3days con-
tinuous exposure) in human epidermoid cells, which contain very low
levels of P-glycoprotein.
¨
completion of the Boc-protection of the basic amine
to 13, the desired tri-Boc product 14 was obtained. It
couldbe converte,d without isolation, to the corre-
sponding racemic amino acid 6n by treatment with
aqueous base.
b IC50 in KB85 cells, which have moderate levels of P-glycoprotein.
c IC50 in KBV1 cells, which have very high levels of P-glycoprotein.
d Inhibition of MAP-rich tubulin polymerization at concentration
0.3lM.
e Values of (%) are results for 2 in the same assay.
f Compounds 1 and 2 were usedas references.
Coupling between the meta-tolyl containing A segment
6a andthe optically pure B–C–D segment 1513 under
standard conditions (see Section 1) produced the pro-
tected tripeptide as a mixture of two diastereomers.
They were separatedby flash chromatography, to give
the (S,S,S) diastereomer 16a andthe ( R,S,S) isomer
17a.14 Hydrolysis of 16a with aqueous lithium hydroxide
solution, followedby acidification to pH6, gave the tri-
peptide 18a,15 as a white solid. In a similar manner, 17a
was convertedto 19a.15 The racemic A segment 6a was
thus transformedinto two optically pure tripeptides, 18a
and 19a. In parallel sequences, the para-tolyl A segment
6b was convertedto the optically pure 18b and 19b via
intermediates 16b and 17b, respectively. Coupling be-
tween 6n and 15, followedby chromatographic separa-
tion, gave the tripeptides 16n and 17n. Hydrolysis of
the esters, followedby removal of the Boc group, gave
the optically pure tripeptides 18n and 19n, respectively.15
KBV1, 19) than the para- andthe ortho-analogs, 18b
and 18n. In addition, the potency of 18a in KB cell lines
is four-foldbetter than that of 2.
Basedon these biological data, various A segments 6c–k,
which carry a substituent at the meta position, were pre-
pared( Scheme 1). Similarly, the A segments carrying two
regioisomeric thienyl groups 6l and 6m were also pre-
pared. They were converted to the optically pure pep-
tides 18c–m and 19c–m by the procedures described
above (Scheme 3). The meta bromophenyl derivative
(16g + 17g) was further convertedto optically pure ana-
logs, such as the biphenyl (18o), the acetylphenyl (18p),
the vinylphenyl (18q), andthe ethylphenyl ( 18r) by the
Pd(0) mediated coupling reactions and deprotection
sequences (Scheme 4).15
The antimitotic activities of 18a, 19a, 18b, 19b, 18n, and
19n, were measuredby the inhibition of tubulin polym-
erization, as well as by proliferation assays with KB cell
lines (KB-3-1, KB85, KBV1), using 1 and 2 as reference
compounds (Table 1). Comparison of activities between
18a, 19a, 18b, 19b, 18n, and 19n shouldshow the effect
of a methyl group at the ortho-, the meta-, or the para-
position, the effect of the configuration of the tripeptides
(SSS vs RSS), as well as the effect of protecting groups
(the protectedvs the unprotectedtripeptides). Excellent
inhibition of tubulin polymerization was observedwith
all analogs mentionedabove, with the exception of
17a. For the proliferation assays with KB cell lines,
the SSS diastereomers 18a, 18b, 18n, as expected,7 were
foundto be 10-foldmore active (IC 50 nM: KB-3-1 0.23–
1.5) than the RSS isomers 19a, 19b, 19n (IC50 nM: KB-
3-1 6.9–17). The unprotectedpeptides 18a, 19a are more
active than their esters 16a, 17a, although 16a showed
unusually high activity on the KB cell assay (IC50 nM:
KB-3-1 22), comparedwith 17a.16 Among the SSS ana-
logs, 18a, 18b, and 18n, the meta-tolyl analog 18a shows
better KB activities (IC50 nM: KB-3-1, 0.23; KB85, 0.65;
The analogs thus preparedwere evaluatedfor their anti-
mitotic activities (Table 2). The analogs carrying the
meta-substitutedA segment showedconsistently good
activity in KB cell lines. The electronic effect of the phen-
yl ring of the A segment appears to have little influence
on their activity, as seen in IC50 nM: KB 1.9 for 18p and
IC50 nM: KB-3-1 0.86 for 18q. Among the analogs car-
rying a halogen substitutedphenyl A segment (Cl, Br,
F), 18e (meta-Cl) and 18f (para-Cl) showedbetter activi-
ties (IC50 nM: KB-3-1 0.58–1.7) than those of 18g (Br)
and 18h (F) (IC50 nM: KB-3-1 1.8 and0.8–1.2, respec-
tively). The analogs carrying a sterically bulky substitu-
ent on the A segment, such as 18o, 18p, showedsome
reduction of activity (IC50 nM: KB 1.9), while substitu-
tion by a vinyl or ethyl group maintainedactivity (IC
50
nM: KB-3-1 0.7–0.9). In order to increase the potential
metabolic stability of these compounds, the analog 18j
carrying the A segment substitutedwith a trifluorometh-
ylphenyl group was prepared. The potency of 18j was,
however, slightly less (IC50 nM: KB-3-1 0.65) than that
of 18a. Activities of the analogs carrying the A segment

5320
A. Yamashita et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5317–5322
O
S
N
a
OR1
+
H2N
HCl
6a,b
S
O
15 : R1 = Et
O
O
O
O
S
R
N
S
S
OR1
N
Ar
N
H
Ar
N
OR1
S
S
H
NH
O
+
NH
O
: R1 = Et
: R1 = H
17a,b
19a,b
: R1 = Et
: R1 = H
16a,b
18a,b
b
b
a
6n
15
+
O
O
O
O
N
N
OR1
OR1
N
H
N
H
O
N
O
N
R2
R2
: R1 = Et
: R1= H
: R2 = Boc
: R2 = H
: R1 = Et : R2 = Boc
17n
19n
16n
18n
c
c
: R2 = H
: R1= H
Scheme 3. Reagents andconditions: (a) i. HOBT, EDC, NMM, DMF, rt, ii. chromatographic separation; (b) i. LiOH, MeOH, H 2O, rt, 15h, ii.
pH = 6, citric acid; (c) i. LiOH, MeOH, H2O, rt, 15h, ii. 4N HCl, p-dioxane, 30min, rt.
Table 2. Cellular proliferation assay profile andinhibition of tubulin
polymerization of analogs
O
O
R3
N
N
H
OH
CompdIC
(nM)
Inhibition of tubulin
polymerization %d (%)e
16g + 17g
50
NH
O
a, b, c
KB-3-1a KB85b KBV1c
18o R3 = Ph
2
1.0
9.1
2.4
24
81
191
39
91
18p R3 = COCH3
18q R3 = CH=CH2
18r R3 = Et
16c
18c
18d
18e
18f
18g
18h
18i
18j
18k
18l
18m
18o
18p
18q
18r
49 (92)
88 (88)
85 (88)
91 (88)
95 (96)
90 (84)
103(93)
90 (93)
98 (96)
60 (93)
92 (88)
88 (96)
86 (84)
93 (87)
88 (90)
88 (90)
0.4
1.13
1.23
3.2
0.55
1.14
0.56
1.8
44
91
d
1.7
4.1
53
Scheme 4. Reagents andconditions: (a) for 18o: phenylboronic acid,
Pd(Ph3P)4, Na2CO3, DME, H2O, reflux, 18h; for 18p: i. n-
Bu3Sn(CH2@CHOEt), Pd(Ph3P)4, toluene 120ꢁC, 4h, ii. 2N HCl,
THF, rt, 15h; for 18q: n-Bu3Ti(CH@CH2), Pd(Ph3P)4, CH3CN, reflux,
20h; (b) chromatographic separation; (c) i. LiOH, MeOH, 50ꢁC, 4–
15h, ii. citric acid, pH6.0; (d) 10% Pd/C, EtOH.
148
77
1.0
6.2
1.8
16.2
1.7
19
457
0.65
42.7
17
457
162
724
149
370
130
62
2.0
2.1
3.9
6.6
5.4
5.8
2.0
1.8
1.9
1.9
substitutedwith dimethylphenyl groups ( 18c, 18d) were
as goodas that of 2 (IC50 nM: KB-3-1 0.6–1.7), but
18k, carrying the di-trifluoromethylphenyl A segment,
somehow lost its potency (IC50 nM: KB-3-1 17). Against
KB85 andKBV1 cells, which contain moderate andvery
high levels of P-glycoprotein, respectively, 18a, 18c, and
18d showedbetter activity than 2 (IC50 nM: KBV1 19
for 18a, 23–55 for 18c, 38–50 for 18d vs 81 for 2).
0.86
0.79
a IC50 (amount of drug needed to kill 50% of cells after 3days con-
tinuous exposure) in human epidermoid cells, which contain very low
levels of P-glycoprotein.
b IC50 in KB85 cells, which have moderate levels of P-glycoprotein.
c IC50 in KBV1 cells, which have very high levels of P-glycoprotein.
d Inhibition of MAP-rich tubulin polymerization at 0.3lM
concentration.
Selectedcompounds ( 16c, 18a, 18c, 18e, 18j, 18l), which
met criteria of goodcellular potency (KB-3-1) andactiv-
ity in resistant cell lines (KB85, KBV1), were further
evaluatedin in vivo assays using Lox melanoma human
xenograft models in athymic mice. Compound 2 was
usedas a reference ( Table 3). Both the minimum effec-
tive dose (MED) and the maximum tolerated dose
e Values of (%) are results for 2 in the same assay.
(MTD) were determined. Several compounds were fur-
ther evaluatedin xenograft moedls, using cell lines
expressing P-glycoprotein transporters (HCT-15,

A. Yamashita et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5317–5322
5321
Table 3. Effect on human tumor xenografts in athymic mice
analysis. Compounds, which were serially diluted into
media as 2 · stocks, were added to cells in duplicate.
After 3days of incubation, cell survival was assessed
by the SRB assay as described.19
a
CompdMED
MTDa
HCT-15
DLD1
MX1W
2
0.2
1.6
>2
1.5
>1
3
Yb
Y
Y
16c
18a
18c
18e
18g
18j
18l
0.5
<0.1
0.2
0.3
1
NA
Y
NA
Y
NA
Y
In vivo model. Human tumor cell lines Lox melanoma
(1.5–7 · 106) are injected(sc) into athymic mice. When
tumors attain a mass of 80–100mg, animals are random-
izedinto groups of 5 or 10 animals. Animals were trea-
ted (iv) with the indicated doses of compound in a saline
formulation, that represent 80–95% of the maximal tol-
erated doses for the indicated schedule. Tumor growth is
assessedby weekly measurement of the length ( L) and
width (W) of subcutaneous tumors [mg = (L · W2)/2].
Relative tumor growth (mean tumor mass on day meas-
ured divided by mean tumor mass on day 0) and %T/C
(mean relative tumor growth of treated divided by that
of control multipliedby 100) are calculated. Data are
analyzedby Student Õs t-test.
NA
Nc
NA
NA
Y
NA
Y
4
NA
NA
NA
NA
NA
NA
1
>5
>5
NA
NA
1
NA: not available.
a MED: the minimum effective dose; MTD: the maximum tolerated
dose.
b Y = active.
c N = inactive.
DLD1, MX1W). The 3,4-dimethylphenyl derivative 18c
andthe meta-chlorophenyl analog 18e showedsimilar in
vivo activity where MEDs were 0.2 and0.3mpk (milli-
gram per kilogram), andMTDs were 1 and3, respec-
tively. Among the compounds tested, the meta-tolyl
analog 18a demonstrated the best in vivo activity.
The MED of 18a was <0.1mpk, which was the most
potent among the compounds tested, including 2
(MED = 0.2mpk). In addition, the MTD value of 18a
was 1.5mpk, which is ideal for a potential drug. In addi-
tion, 18a showedalmost zero tumor growth in the xeno-
graft model, and was active in vivo using cell lines
expressing P-glycoprotein (HCT-15, DLD1, MX1W).
1. Experimental
1.1. General procedure for coupling between 6 and 15
To a cooled(0 ꢁC) solution of 6a (1.1mmol), hydroxy-
benzotriazole (1.1mmol), and1-(3-dimethylaminoprop-
yl)-3-ethylcarbodiimide hydrochloride (1.2mmol) in
anhydrous DMF (3–5mL) was added N-methylmorpho-
line (1.4mmol) via syringe, under an inert atmosphere.
After stirring for 15min at 0ꢁC, the cooling bath was re-
moved, and the resulting mixture was stirred for 2–24h
(the reaction time depends on the solubility of 6 in
DMF andthe time for activation of the acid). The solu-
tion was cooledat 0 ꢁC (ice bath), and was added a solu-
tion of 15 (1.0mmol) in anhydrous DMF (3mL). The
cooling bath was removed, and the resulting mixture
was stirredfor 15–36h at room temperature, under an
inert atmosphere. The mixture was diluted with water,
andthe aqueous layer was extractedwith ethyl acetate.
The combinedextracts were washedwith saturated
sodium chloride solution, dried over Na2SO4, filtered,
andconcentratedin vacuo. The residue was chromato-
graphed(silica gel, flash column), to give 16 (SSS) and
17 (RSS). In general, the first eluate was a diastereomer
of the SSS configuration, andthe secondan isomer of
the RSS configuration.14
In summary, we have synthesizedvarious analogs
relatedto 1 and 2, in which the aromatic ring in the A
segment is substitutedwith a variety of functional
groups in various positions. The meta-tolyl substitution
consistently showedincreasedactivity both in a prolifera-
tion assay with KB cell lines, as well as comparable
activity in inhibition of tubulin polymerization to that
of 2. Among the analogs thus synthesized, 18a showed
the best biological profile; in particular, its activity
against resistant KB-3-1 cell lines seems ideal for a
potential drug for the treatment of cancer.
Determination of inhibition of tubulin polymerization.17
Bovine MAP-rich tubulin, PEM buffer [80mM Na-
PIPES (pH6.9), 1mM MgCl2, and1mM EGTA], and
GTP were purchasedfrom Cytoskeleton (Denver,
CO). MAP-rich tubulin (final concentration 1.5mg/mL)
was dissolved in cold PEM buffer containing 1mM
GTP (GPEM) andcentrifugedat 12,000 g for 10min at
4ꢁC. The tubulin solution (100lL/well) was added rap-
idly to wells of a low-volume, 96-well plate already con-
taining duplicate aliquots (10lL) of test compounds in
GPEM. Final compoundconcentrations were 0.3 lM.
Control wells containedthe same final concentration
of DMSO (0.3%). After initiation of the reaction,
absorbance at 340nm was measuredevery minute for
60min at 24ꢁC using a SpectraMax Plus plate reader
(Molecular Devices, Sunnyvale, CA).
1.2. General procedure for preparation of 18 (or 19)
To a cooled(0 ꢁC) solution of 16 (or 17) in MeOH
(24mL/mmol) were added H2O (8mL/mmol) andaque-
ous LiOH solution (8mL/mmol). The cooling bath was
removed, and the resulting mixture was stirred at room
temperature for 15h. The solvent was removedin vacuo
(bath temperature was kept below 30ꢁC), andthe resi-
due was dissolved in a small amount of H2O. The aque-
ous solution was cooledto 0 ꢁC, andacidifiedto pH5.5–
6.0 with aqueous citric acidsolution. The precipitate was
collectedby filtration, andthe solidwas washedwith
cold water, and dried under high vacuum. Alternatively,
the crude acidic residue was chromatographed using
preparative HPLC.
Cellular proliferation assays. KB-3-1 cells18 were plated
in 96-well plates in 100-lL media at densities predeter-
minedto produce 60–90% confluence at the time of

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A. Yamashita et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5317–5322
6. Loganzo, F.; Discafani, C. M.; Annable, T.; Beyer, C.;
Musto, S.; Hari, M.; Tan, X.; Hardy, C.; Hernandez, R.;
Baxter, M.; Singanallore, T.; Khafizova, G.; Poruchysky,
M. S.; Fojo, T.; Nieman, J. A.; Ayral-Kaloustian, S.;
Zask, A.; Andersen, R. J.; Greenberger, L. M. Cancer Res.
2003, 63, 1838.
7. Zask, A.; Birnberg, G.; Cheung, K.; Kaplan, J.; Niu, C.;
Norton, B.; Suayan, R.; Yamashita, A.; Cole, D.; Tang,
Z.; Krishnamurthy, G.; Williamson, R.; Khafizova, G.;
Musto, S.; Hernandez, R.; Annable, T.; Yang, X.;
Discafani, C.; Beyer, C.; Greenberger, L. M.; Loganzo,
F.; Ayral-Kaloustian, S. J. Med. Chem., in press. Other
references citedtherein. Also see Ref. 5.
1.3. Pd(0) mediated coupling reaction: preparation of 18n
To a solution of a mixture of 16g and 17g (0.43g,
0.74mmol) in ethylene glycol dimethyl ether (15mL)
and water (7.5mL) were added tetrakis-(triphenylphos-
phine)palladium(0) (0.086g, 0.074mmol), phenylbo-
ronic acid(0.18g, 1.5mmol) andsoidum carbonate
(0.23g, 2.2mmol), at room temperature under nitrogen
atmosphere. The resulting mixture was heatedat reflux
for 15h, and then cooled. The mixture was diluted with
ether, the organic layer was separated, and the aqueous
layer was extractedwith ether. The combinedextracts
8. (a) Plochl, J. Ber. 1883, 16, 2815; (b) Erlenmeyer, E. Justus
Liebigs Ann. Chem. 1893, 275, 1.
were washedwith saturatedaqueous NaHCO andsat-
3
uratedaqueous NaCl solution, driedover Na 2SO4, fil-
tered, and concentrated in vacuo. The desired ester
16n (SSS configuration) was isolatedby chromatogra-
phy (flash column, silica gel, EtOAc/ether; 0.18g,
42%). Compound 16n (0.18g, 0.31mmol) was hydro-
lyzed with lithium hydroxide using the procedure
described above. The product 18n was purifiedby
reverse-phase HPLC (elution from 5% acetonitrile/
95% water containing trifluoroacetic acidto 100% aceto-
nitrile, 60min) as its trifluoroacetic acidsalt (0.19g,
79%).
9. (a) Meiwes, J.; Schudok, M.; Kretzschmar, G. Tetrahe-
dron: Asymmetry 1997, 8, 527; (b) Audia, J. E.; Evrard, D.
A.; Murdoc, G. R.; Droste, J. J.; Nissen, J. S.; Schenck, K.
W.; Fluzinski, R.; Lucaites, V. L.; Nelson, D. L.; Cohen,
M. L. J. Med. Chem. 1996, 39, 2773, andother references
citedtherein.
10. Optically pure L- and D-amino acids were obtained by
kinetic resolution. Wu, Y.; Megati, S.; Panolil, R.;
Padmanathan, T.; Kendall, J.; Glestos, C.; Wilk, B.,
unpublishedresults.
11. (a) Compound 8 was prepared as described: Corey, E. J.;
Jautelat, M.; Oppolzer, W. Tetrahedron Lett. 1967, 2325;
(b) Matsuyama, H.; Nakamura, T.; Iyoda, M. J. Org.
Chem. 2000, 65, 4796.
12. Ishihara, K.; Hanaki, N.; Yamamoto, H. Synlett 1995,
721.
Acknowledgements
Authors thank analytical support provided by Discovery
Analytical Department, Wyeth Research, Pearl River,
NY. Authors also thank Drs. Lee Greenberger, Jerauld
Skotnicki, andTarek Mansour for their useful discus-
sions andsuggestions during this project.
13. Compound 15 (hydrochloride salt) was prepared in five
steps from commercially available N-tert-butoxycarbonyl-
L-valine-N0,O-dimethylhydroxamide according to the lit-
erature procedures. See Ref. 7.
14. The absolute stereochemistry of the two diastereomeric
isomers was determined by NMR studies and physical
properties of the product as described in the literature: see
Ref. 7.
References and notes
15. All compounds described in this paper gave satisfactory
analytical data (mass, IR, 1H NMR, LC–MS, HPLC
analysis).
1. (a) Talpir, R.; Benayahu, Y.; Kashman, Y.; Pannell, L.;
Schleyer, M. Tetrahedron Lett. 1994, 35, 4453; (b)
Colemann, J. E.; Patrick, B. O.; Andersen, R. J.; Rettig,
S. J. Acta Cryst. 1996, C52, 1525; (c) Coleman, J. E.; de
Silva, E. D.; Kong, F.; Andersen, R. J.; Allen, T. M.
Tetrahedron 1995, 51, 10653.
2. Crews, P.; Faris, J. J.; Emrich, R.; Keifer, P. A. J. Org.
Chem. 1994, 59, 2932.
3. Anderson, H. J.; Coleman, J. E.; Andersen, R. J.;
Roberge, M. Cancer Chemother. Pharmacol. 1997, 39, 223.
4. Andersen, R. J.; Coleman, J. E. Tetrahedron Lett. 1997,
38, 317.
16. Usually, the protectedtripeptides gave activities compa-
rable to those obtainedfor 17a, which showedIC
KB-3-1 263–3000.
nM:
50
17. The abbreviations usedare: MAP, microtubule-assisted
protein; SRB, sulforhodamine B; MDR 1, multidrug
resistance protein-1.
18. Multiple drug-resistant human KB-3-1 cells were provided
by Dr. M. Gottesman, National Cancer Institute. See Ref.
Shen, D. W.; Cardarelli, C.; Hwang, J.; Cornwell, M.;
Richert, N.; Ishii, S.; Pastan, I.; Gottesman, M. M. J. Biol.
Chem. 1986, 261, 7762.
5. (a) Andersen, R. J. WO 99/32509; (b) Nieman, J. A.;
Coleman, J. E.; Wallace, D.; Piers, E.; Lim, L. Y.;
Roberge, M.; Andersen, R. J. J. Nat. Prod. 2003, 66(2),
183.
19. Rabindran, S. K.; He, H.; Singh, M.; Brown, E.; Collins,
K. I.; Annable, T.; Greenberger, L. M. Cancer Res. 1998,
58, 5850, See also Ref. 7, andother references cited
therein.