D-piece modifications of the hemiasterlin analog HTI-286 produce potent tubulin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.05.005

Modifications of the D-piece carboxylic acid group of the hemiasterlin analog HTI-286 gave tubulin inhibitors which were potent cytotoxic agents in taxol resistant cell lines expressing P-glycoprotein. Amides derived from proline had potency comparable to HTI-286. Reduction of the carboxylic acid to ketones and alcohols or its conversion to acidic heterocycles also gave potent analogs. Synthetic modifications of the carboxylic acid could be carried out selectively using a wide range of synthetic reagents. Proline analog 3 was found to be effective in a human xenograft model in athymic mice.

Full text of DOI:10.1016/j.bmcl.2004.05.005

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4353–4358
D-piece modifications of the hemiasterlin analog HTI-286
produce potent tubulin inhibitors
Arie Zask,a,* Gary Birnberg,a Katherine Cheung,a Joshua Kaplan,a Chuan Niu,a
Emily Norton,a Ayako Yamashita,a Carl Beyer,b Girija Krishnamurthy,a
Lee M. Greenberger,b Frank Loganzob and Semiramis Ayral-Kaloustiana
aWyeth Research, Chemical and Screening Sciences, 401 North Middletown Road, Pearl River, NY 10965, USA
bWyeth Research, Oncology Research, 401 North Middletown Road, Pearl River, NY 10965, USA
Received 31 March 2004; accepted 5 May 2004
Available online
Abstract—Modifications of the D-piece carboxylic acid group of the hemiasterlin analog HTI-286 gave tubulin inhibitors which
were potent cytotoxic agents in taxol resistant cell lines expressing P-glycoprotein. Amides derived from proline had potency
comparable to HTI-286. Reduction of the carboxylic acid to ketones and alcohols or its conversion to acidic heterocycles also gave
potent analogs. Synthetic modifications of the carboxylic acid could be carried out selectively using a wide range of synthetic
reagents. Proline analog 3 was found to be effective in a human xenograft model in athymic mice.
Ó 2004 Elsevier Ltd. All rights reserved.
Hemiasterlin1 (1), a tripeptide isolated from marine
sponges, and its synthetic analog HTI-2862 (2), induce
microtubule depolymerization, and mitotic arrest in cells
(Fig. 1). These compounds contain highly unusual and
sterically congested amino acids, which give rise to their
stability and in vivo activity. Their relative struc-
tural simplicity allows for diverse modification via total
synthesis.3 In addition to its potent antitumor effects,
HTI-286 has the advantage of circumventing the
P-glycoprotein-mediated resistance that hampers the
efficacy of other antimicrotubule agents such as paclit-
axel and vincristine in animal models.2 HTI-286 is
presently in clinical trials.4 Previously we described an
in-depth study of the structure–activity relationships of
HTI-286 analogs.5 We now report that the carboxylic
acid region of HTI-286 (D-piece) can be efficiently
converted to a wide range of functional groups resulting
in potent tubulin inhibitors.
The carboxylic acid moiety of HTI-286 could be selec-
tively derivatized via a variety of methods without
affecting the other functional groups in the molecule.
Preliminary activation of the carboxylic acid toward
reaction with nucleophiles by treatment with 1-[3-(di-
methylamino)propyl]-3-ethylcarbodiimide (EDC) and 1-
hydroxybenzotriazole (HOBT) was used in the synthesis
of many analogs. Thus, activation followed by treat-
ment with cyclic amino acid esters (Scheme 1) gave the
corresponding tetrapeptides (3–7) (Table 1). Saponi-
fication of the ester groups in these amides with LiOH
gave the acids 8–12. Acid 8 was converted to amide 13
by coupling with phenethylamine under the coupling
conditions described above. Use of HTI-286 analogs
with modifications of the phenyl group in the A-piece
O
O
N
N
N
H
OH
Hemiasterlin (1)
HTI-286 (2)
N
HN
O
O
O
N
H
OH
HN
O
A-Piece
B-Piece C-Piece D-Piece
Figure 1. Hemiasterlin (1) and HTI-286 (2).
similarly gave
(Table 2).
L-proline containing tetrapeptides (14–20)
* Corresponding author. Fax: +1-845-602-5561; e-mail: zaska@wyeth.
com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.005

4354
A. Zask et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4353–4358
Table 1. Cyclic amino acid analogs
O
a, b
c
N
HTI-286 (2)
O
O
N
CO2Me
N
H
X
3 - 7
N
O
H
Com-
pound
X
Tubulina KB-3-1b KB-8-5b KB-V1b
O
N
O
a, d
N
HTI-286 (2)
88c
82d
0.96
1.5
2.3
7.3
77
CO2H
O
N
O
N
H
Ph
8 - 12
13
3
4
5
6
7
291
OMe
Scheme 1. Reagents and conditions: (a) EDC, HOBT, i-Pr2NEt,
DMF; (b) cyclic amino acid ester; (c) LiOH, MeOH, H2O, THF; (d)
PhCH2CH2NH2.
N
96
77
94
93
16
51
1099
150
O
O
OMe
N
Reduction of the carboxylic acid in HTI-286 to the
alcohol was effected by treatment with EDC in CH3CN
followed by addition of NaBH4 to give analog 21
(Scheme 2). Oxidation of the alcohol in 21 with DMSO
and oxalyl chloride6 gave the corresponding aldehyde
22. Aldehyde 22 could also be synthesized by conversion
of the carboxylic acid in HTI-286 to the Weinreb amide
(23), followed by reduction using LiAlH4.
5.2
3.5
3.5
17
17
OMe
N
272
O
OMe
OH
N
N
N
116
>3000
O
O
OMe
OH
The Weinreb amide moiety could also be used for the
preparation of ketones 24–26 by reaction with organo-
metallic reagents (Scheme 3). For example, treatment of
23 with 3 equiv PhMgBr gave phenyl ketone 25. The
hydroxymethyl ketone analog 26 was prepared by
treatment of 23 with LiCH2OSEM followed by removal
of the SEM group with TFA.7 The methyl ketone 27 was
prepared by treatment of the ethyl ester of HTI-286 (28)
with Tebbe reagent,8 followed by acid hydrolysis. The
aldehyde group in 22 could be used as a handle for
further modification by treatment with organometallic
reagents to give the corresponding alcohols (29–32) as
epimeric mixtures. HPLC separation gave the phenyl
alcohol diastereomers 31 and 32 whose stereochemistry
was determined by NMR analysis. ROE correlations
between protons on the CD portion of the molecule
were consistent with the assigned stereochemistry for
each isomer. Treatment of 22 with Wittig reagent
Ph3PC(CH3)CO2Et, gave diene 33 after saponification
with LiOH.
8
97d
62
205
>3000
9
91
89
515
64
1577
270
>3000
>3000
O
O
OH
N
10
OH
N
11
12
90
96
58
184
>3000
>3000
O
OH
OH
N
N
184
1850
O
O
OH
13
86d
6.4
21
1673
N
H
Ph
Oxidation of the double bond in HTI-286 by ozonolysis
gave intermediate 34, which was used to efficiently effect
D-piece transformations, including synthesis of two
analogs not available through stepwise synthesis
(Scheme 4). Thus, ozonolysis of HTI-286 followed by
treatment with Me2S cleaved the olefin to give aldehyde
34. Treatment of 34 with Wittig reagents gave analogs
35–39. Reaction with the hydantoins9 40 and 41 in the
presence of LiOH gave 35 and 36, respectively, as a
mixture of isomers. Reaction with trifluoroethyl phos-
phonate10 42 gave the Z olefin isomer 37 after saponi-
fication with LiOH. Analog 37 could not be prepared in
a stepwise fashion due to the propensity of cis vinyl-
agous amino esters to cyclize to lactams.11 Reaction of
34 with PPh3CHCO2Et gave 38 after saponification with
LiOH. Analog 38 was not readily prepared in a stepwise
a %Inhibition of tubulin polymerization at 0.5 lM.
b IC50 (nM) in cells.
c Average over 10 runs.
d %Inhibition of tubulin polymerization at 0.3 lM.
synthesis due to reactivity of the disubstituted olefin
during the repetitive coupling and deprotection steps
required. Reaction of 34 with 43 gave 39. Ozonolysis of
HTI-286 followed by treatment with NaOCl cleaved the
olefin to give acid 44.
Four analogs (45–48) were synthesized using methods
described above on C-piece, D-piece, or CD-Piece seg-
ments of HTI-286 (Scheme 5). The resulting segments

A. Zask et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4353–4358
4355
Table 2. Proline amide derivatives
O
O
H
a, b
N
O
O
N
H
HTI-286 (2)
N
HN
O
N
R1
N
H
34
N
O
H
O
O
OR2
O
P
EtO
EtO
O
c
R2 Tubulina KB-3-1b KB-8-
5b
KB-V1b
NH
O
34
+
Com- R1
pound
NH
N
RN
R
O
14
15
16
17
18
19
20
3-Tolyl
3-Tolyl
3-Cl-Ph
3-Cl-Ph
Me 89
95
Me 88
94
1.6
70
2.5
62
1.9
74
3.7
6.2
599
6.2
180
275
>3000
362
35 R = H
40 R = H
36 R = Me
H
41 R = Me
H
>3000
363
O
P
O
d, e
f, e
CF3CH2O
CF3CH2O
3,4-Xylyl Me 71
3,4-Xylyl
C-hexyl
15
519
16
34
34
+
+
O
H 76
Me 88
>3000
493
CO2H
42
37
a %Inhibition of tubulin polymerization at 0.5 lM.
b IC50 (nM) in cells.
CO2H
O
Ph3P
O
38
OH
a, b
HTI-286 (2)
O
P
g
EtO
21
34
+
EtO
a, d
O
c
39
43
O
O
O
e
OMe
N
H
N
O
a, h
N
H
OH
HTI-286 (2)
23
22
HN
44
Scheme 2. Reagents and conditions: (a) EDC, HOBT, i-Pr2NEt,
CH3CN; (b) NaBH4; (c) DMSO, (COCl)2, Et3N, CH2Cl2; (d)
NHMeOMe; (e) LiAlH4, Et2O, ꢀ30 to 0 °C.
Scheme 4. Reagents and conditions: (a) O3, MeOH, ꢀ78 °C; (b) Me2S;
(c) LiOH, EtOH, H2O; (d) KN(TMS)2, 18-C-6, THF; (e) LiOH,
MeOH, H2O; (f) CH2Cl2, 25 °C; (g) NaH, THF; (h) NaOCl.
O
O
Weinreb amide 49 with 2-Li-3-SEM-imidazole12 to give
50, followed by coupling to the A-piece to give 51 and
removal of the SEM group with TFA. Tetrazole 46 was
synthesized by treatment of 52 with PPh3CHCN to give
nitrile 53, which was coupled, sequentially to the B-piece
and A-piece to give Boc-protected intermediate 54.
Treatment with TMSN3 and n-Bu2SnO13 followed by
deprotection gave tetrazole 46. Coupling of Boc-pro-
a
b, c
OH
23
R
26
24 R = 2-thiazolyl
25 R = Ph
O
O
d, e
OEt
Me
tected N-methyl-L-valine with proline gave 55, which
was coupled sequentially to the B-piece and A-piece to
28
27
give ester 47. Saponification of 47 with LiOH gave acid
48.
OH
O
f
g
22
R
OH
33
*
Transformation of the D-piece carboxylic acid into a
range of functional groups was carried out to evaluate
the structural requirements of this region of HTI-286.
Compounds were evaluated for their ability to inhibit
cell-free tubulin polymerization.2 Cytotoxicity was
determined 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, respec-
tively.2 Functional group changes led to analogs with a
wide range of potency including compounds with
activity comparable to HTI-286.
29 R = Me (R/S)
30 R = 2-thiazolyl (R/S)
31 R = Ph (S), 32 R = Ph (R)
Scheme 3. Reagents and conditions: (a) THF, 0 °C 24: 2-Li-thiazole,
25: PhMgBr; (b) n-BuSnCH2OSEM, n-BuLi, THF, ꢀ78 to ꢀ60 °C; (c)
TFA, CH2Cl2: (d) (1) Tebbe reagent, THF, ꢀ40 to 25 °C; (2) aq
NaOH; (e) 1 N aq HCl, THF; (f) THF, 0 °C, 29: MeMgBr, 30: 2-Li-
thiazole, 31/32: PhMgBr; (g) (1) PPh3C(Me)CO2Et; (2) LiOH, MeOH,
H2O.
were coupled to the remaining amino acids in a manner
analogous to that reported in the literature.3;5 Thus, the
imidazole analog 45 was synthesized by treating the
We had previously prepared amide analogs using simple
cyclic and acyclic amines.5 Although these compounds

4356
A. Zask et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4353–4358
Table 3. Ketone derivatives
O
O
SEM
N
a
N
OMe
O
N
BOC
N
BOC
O
H
O
N
N
49
50
N
H
X
N
O
O
SEM
N
N
b
Tubulina KB-3-1b KB-8-5b KB-V1b
N
H
Com-
pound
X
45
N
HN
O
51
22
28
45
24
25
26
H
CH3
71
69
454
403
218
582
725
43
447
664
1594
2358
O
c
91c
1364
565
>3000
1787
1805
BOCN
BOCN
CN
2-Imidazolyl
2-Thiazolyl
Phenyl
33
70c
76c
1326
68
52
53
CH2OH
1134
N
N
O
a %Inhibition of tubulin polymerization at 0.3 lM.
b IC50 (nM) in cells.
c %Inhibition of tubulin polymerization at 0.5 lM.
N
d, e
N
CN
N
H
N
H
HN
O
46
BOC
54
O
O
lastatin-10 yield potent tubulin inhibitors.14
L
-Proline
f, g
N
N
BOC
OH
BOC
N
amides with A-piece modifications where the phenyl ring
was replaced with 3-tolyl, 3-Cl-phenyl, 3,4-xylyl, and
cyclohexyl gave analogs (14–20) with comparable bio-
logical activities (Table 2).
CO2Me
55
O
O
N
N
N
H
Conversion of the carboxylic acid to an aldehyde (22)
or a methyl ketone (28) led to greater than two orders of
magnitude decrease in potency in the KB-3-1 cells
(IC50 ꢁ 400 nM) (Table 3). Efforts to increase potency
by incorporation of other R groups (2-imidazolyl, 2-
thiazolyl, phenyl) gave analogs (45, 24, 25, respectively)
with similar activity. However, use of the a-hydroxy-
methyl ketone group, reported to serve as a carboxylic
acid equivalent in other systems,15 led to an analog (26),
HN
O
h
CO2R
47 R = Me
48 R = H
Scheme 5. Reagents and conditions: (a) 2-Li-3-SEM-imidazole, THF,
0 °C; (b) TFA, CH2Cl2; (c) Ph3CHCN, CH2Cl2; (d) TMSN3, n-Bu2SnO,
toluene, 100 °C; (e) HCl, dioxane; (f) EDC, HOBT, i-Pr2NEt, DMF; (g)
L
-proline methyl ester; (h) LiOH, MeOH, H2O, THF.
with a striking 10-fold increase in potency (IC50
43 nM), relative to 28 in the KB-3-1 cells.
¼
were active, they were approximately one to three orders
of magnitude less potent than the corresponding acids in
the KB-3-1 cells. We now report that incorporation of
cyclic amino acids gives analogs with dramatic increases
Reduction of the carboxylic acid to a primary alco-
hol gave analog 21, with greater than two orders of
magnitude loss of potency in the KB-3-1 cells
(IC50 ¼ 454 nM). However, in contrast to the ketone
analogs, addition of simple R groups led to dramatic
increases in potency. Thus, the methyl alcohol analog 29
had an IC50 ¼ 63 nM in the KB-3-1 cells. A further
potency increase was achieved in the 2-thiazolyl con-
taining analog 30 with an IC50 ¼ 13 nM in the KB-3-1
cells. The configuration of the alcohol was revealed to be
important for potency. Thus, in the pair of alcohol
diastereomers incorporating a phenyl group (31, 32),
analog 31 with the S configuration at the carbon bearing
the alcohol, was approximately eight times more potent
than the epimer (32) in the KB-3-1 cells (IC50 ¼ 16 nM).
The alcohols 30 and 31 were also poorer substrates for
P-glycoprotein than other analogs as indicated by their
enhanced activity in the resistant KB-8-5 and KB-V1
cell lines relative to the non-P-glycoprotein expressing
KB-3-1 cells (Table 4).
in potency (Table 1). Thus, in the case of the L-proline
ester analog 3, the IC50 in KB-3-1 cells was 1.5 nM,
comparable to the activity of HTI-286 (IC50 ¼ 0:96 nM).
It was also active in P-glycoprotein expressing cell lines
(KB-8-5 and KB-V1) resistant to paclitaxel. The binding
affinity (KD) of 3 to tubulin was 0.9 lM, approximately
threefold higher than that of HTI-286 (KD ¼ 0:26 nM).2
The analog 4 made from
(KB-3-1 IC50 ¼ 16 nM). Keeping the
D
-proline ester was less potent
-amino acid
L
configuration constant and expanding the ring size to
cyclohexane (5), introducing unsaturation in the ring
(6), or substituting the ring with a hydroxy group (7)
gave potent analogs with IC50 values below 10 nM in the
KB-3-1 cells. Relative to HTI-286 and analogs 3–6, the
hydroxyproline ester 7 was less effective in the resistant
cell lines (KB-8-5, KB-V1) indicating that it was a better
substrate for P-glycoprotein. The corresponding car-
boxylic acid of each of these analogs (8–12) was 10- to
50-fold less potent than the ester. The
L
-proline phen-
Incorporation of the carboxylic acid into an acidic het-
erocycle gave the potent hydantoin analog 36 (KB-3-1
cell IC50 ¼ 5:1 nM). A decrease in potency was seen
upon the demethylation of the hydantoin to give 35.
Weak activity was seen with the tetrazole carboxylic
ethyl amide moiety gave a potent analog 13 with an
IC50 ¼ 6:4 nM in the KB-3-1 cells. These proline analogs
are structurally related to dolastatin-10 as hybrids of
HTI-286 and the proline derived amine portion of do-

A. Zask et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4353–4358
Table 5. Carboxylic acid transformations and translations
4357
Table 4. Alcohol analogs
O
O
H
OH
N
X
N
N
H
N
H
* X
N
O
N
O
H
Com-
pound
X
Tubu-
lina
KB-3-
1b
KB-8-
5b
KB-V1b
>3000
230
Com-
pound
X
*
Tubulin KB-3-1a KB-8-5a KB-V1a
21
29
H
CH3
–– 71b
R/ 75c
S
454
63
447
172
1594
1455
O
NH
O
35
36
59
1072
1738
N
H
30
2-Thiaz- R/ 97c
13
19
364
olyl
Ph
S
S
R
O
95c
5.1
9.9
31
32
63b
83c
16
124
22
186
190
632
NH
O
Ph
N
a IC50 (nM) in cells.
b %Inhibition of tubulin polymerization at 0.3 lM.
c %Inhibition of tubulin polymerization at 0.5 lM.
NH
N
46
38
39
31
53
13
1148
1819
479
>3000
>3000
>3000
N
N
CO2H
191
acid bioisostere 46, although the lack of a methyl group
on the olefin probably contributed to the low potency as
in desmethyl analog 38. Conversion of the carboxylic
acid to a lipophilic phenyl ring (39), in contrast to the
acidic tetrazole, led to a loss of potency at 3000 nM.
Changing the orientation of the carboxylic acid through
isomerization of the olefin to the Z configuration was
tolerated giving analog 37, with an IC50 value of 28 nM
in the KB-3-1 cells. Moving the carboxylic acid longi-
tudinally through elongation of the molecule, by adding
a second olefin, gave 33 having an IC50 ¼ 27 nM.
However, truncation of the molecule gave carboxylic
acid 44 with an IC50 > 3000 nM. Conversion of the acid
in 44 to the proline ester gave analog 47, having good
potency in the KB-3-1 cells (IC50 ¼ 18 nM). The five-
membered ring may serve as a rigid scaffold placing the
ester group in the proper orientation for tubulin bind-
ing. We had previously shown that the olefin in HTI-286
serves as a structural component as its hydrogenation
led to a potent saturated analog.5 Analog 47 bears a
structural relationship to cemadotin as the hybrid of 44
with the proline containing amine portion of cemadotin
produces a potent tubulin inhibitor.14 The correspond-
ing acid (48) had an IC50 > 3000 nM in the KB-3-1 cells
(Table 5).
>3000
>3000
37
11
28
58
2630
CO2H
CO2H
33
44
90c
0c
27
53
1582
–CO2H
>3000
>3000
>3000
O
N
47
48
19c
27c
18
53
1533
CO2Et
O
N
>3000
>3000
>3000
CO2H
a %Inhibition of tubulin polymerization at 0.3 lM.
b IC50 (nM) in cells.
c %Inhibition of tubulin polymerization at 0.5 lM.
the carboethoxy group in a defined orientation. There
appears to be some flexibility in the location of the polar
group as seen in 33 and 37 but not in 44. Replacement of
the carboxylic acid with a nonpolar phenyl group (39)
leads to loss of potency in contrast to replacement with
the acidic tetrazole ring (46). The tubulin polymerization
assay results were not always consistent with the cellular
cytotoxicity data. Cell permeability and concentration
within cells16 may play a role in cytotoxicity. The
polymerization assay also measures the effects of
inhibitors on microtubule formation but not their
affinity to the tubulin heterodimer,17 although it is un-
likely that tubulin is not the target as photoaffinity
probes specifically label alpha tubulin.18 For analog 3,
the KD value was consistent with the polymerization
data.
The above results indicate the need for a hydrogen
bonding group in the D-piece of HTI-286 and a
requirement for a correct spatial orientation of the
group. In the series of cyclic amino acid amide analogs
the additional polar carboethoxy group leads to in-
creased potency over simple amides. In addition, the
configuration at the carbon bearing the carboethoxy
group is critical for optimum potency. For the ketone
analogs, there is greatly reduced potency compared to
analogous amides or esters,5 perhaps due to the lower
electron density on the carbonyl oxygen. However,
adding a hydrogen bonding hydroxy group to the ke-
tone (26) gives a sharp increase in potency. The alcohols
were quite potent, with the orientation of the hydroxy
group playing an important role in potency as seen in
the enhanced potency of the S diastereomer (31).
Replacing the olefin with a pyrrolidine ring (47) places
In addition to having potent cellular activity, the
-proline analog 3 was active in a Lox melanoma
human tumor xenograft model2 in athymic mice. The
minimum effective dose (MED) was 1 mpk administered
L

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A. Zask et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4353–4358
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i.v. on a 1, 5, 9 day schedule (p < 0:5). HTI-286 (2) had
an MED of 0.2 mpk in the xenograft model.
In summary, conversion of the carboxylic acid group in
HTI-286 to other polar functionality gave potent tubu-
lin inhibitors with activity in taxane resistant cell lines
expressing P-glycoprotein. Potency was dependent on
the nature of the functional group. The ability of the
group to form hydrogen bonds and a correct spatial
orientation were factors critical to activity. In vivo
activity in a xenograft model in athymic mice was
achieved with proline analog 3.
Acknowledgements
The authors gratefully acknowledge the Wyeth Chemi-
cal Development Group and Discovery Synthesis Group
for supplies of HTI-286 and intermediates. The authors
thank Dr. Carolyn Discafani for in vivo testing of 3 in
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