Tubulin inhibitors. Synthesis and biological activity of HTI-286 analogs with B-segment heterosubstituents

Article abstract of DOI:10.1016/j.bmcl.2004.05.077

Modifications of the B-segment of HTI-286 (2) produced a class of analogs incorporating heteroatom-substituents. The structure-activity relationship was studied. Analogs bearing methylsulfide and fluoride groups exhibited potency comparable to that of the parent compound HTI-286 and to paclitaxel in cytotoxicity assays against KB-3-1 cell lines. These analogs were more potent than paclitaxel against P-glycoprotein expressing KB-8-5 and KB-V1 cell lines. Several analogs showed strong inhibition of tubulin polymerization.

Full text of DOI:10.1016/j.bmcl.2004.05.077

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4329–4332
Tubulin inhibitors. Synthesis and biological activity of
HTI-286 analogs with B-segment heterosubstituents
Chuan Niu,a,* Daniel Smith,a, Arie Zask,a Frank Loganzo,b Carolyn Discafani,b
Carl Beyer,b Lee Greenbergerb and Semiramis Ayral-Kaloustiana
aChemical and Screening Sciences, Discovery Medicinal Chemistry, Wyeth Research,
401 N. Middletown Road, Pearl River, NY 10965, USA
bOncology, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
Received 10 May 2004; accepted 26 May 2004
Available online
Abstract—Modifications of the B-segment of HTI-286 (2) produced a class of analogs incorporating heteroatom-substituents. The
structure–activity relationship was studied. Analogs bearing methylsulfide and fluoride groups exhibited potency comparable to that
of the parent compound HTI-286 and to paclitaxel in cytotoxicity assays against KB-3-1 cell lines. These analogs were more potent
than paclitaxel against P-glycoprotein expressing KB-8-5 and KB-V1 cell lines. Several analogs showed strong inhibition of tubulin
polymerization.
ꢀ 2004 Elsevier Ltd. All rights reserved.
It is well known that taxanes and Vinca alkaloids are the
two classes of tubulin inhibitors clinically used as anti-
cancer drugs. Despite their successes, a major challenge
to the efficacy of these drugs is inherent and acquired
resistance by tumors during clinical use.1 The recently
discovered natural product hemiasterlin (1, Fig. 1),2
isolated from marine sponges, is an antimitotic agent
that effectively inhibits tubulin polymerization by bind-
ing to the Vinca-peptide site.3 It also exhibits potent
cytotoxicity against human breast, ovarian, colon, and
lung cancer cell lines.4 The synthetic analog HTI-286
(2),5 in which a phenyl group has replaced the
N-methylindole ring of 1, was found to be a potent
cytotoxin and mitotic blocker like 1, vincristine and
paclitaxel (Taxole).6 Compound 2 exhibited espe-
cially potent activity against paclitaxel-resistant cell
lines, in vitro and in vivo. These potent effects of HTI-
286 (2) on tubulin make it very attractive as a drug
candidate. Currently HTI-286 is in clinical trials.7
tion of each of the designated A, B, C, D segments.8 The
synthetic analogs were evaluated for inhibition of
tubulin polymerization and for cellular cytotoxicity
against a panel of epidermoid carcinoma cell lines with
varying levels of P-glycoprotein expression (KB-3-1,
KB-8-5, KB-V1). In vivo studies in tumor xenograft
models identified several potent analogs, including one
B
D
A
C
O
O
s
s
N
s
N
H
OH
Hemiasterlin (1)
HTI-286 (2)
NH
O
N
O
O
N
N
OH
H
NH
O
Broad SAR studies of HTI-286 were conducted in our
laboratories, concentrating on the systematic investiga-
O
O
N
N
H
OH
* Corresponding author. Tel.: +1-845-602-5996; fax: +1-845-602-5561;
e-mail: niuc@wyeth.com
HTI-042 (3)
NH
O
O
Current address: Department of Chemistry, University of Virginia,
Charlottesville, VA 22904, USA.
Figure 1.
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.077

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C. Niu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4329–4332
superior analog, HTI-042 (3), which has been selected as
a development candidate. This paper describes the syn-
thesis and biological results of novel analogs of HTI-
286, focusing on the heteroatom containing B segment
analogs as potential inhibitors of tubulin polymeriza-
tion.
groups, was treated with di-tert-butyl dicarbonate to
give the N-Boc-protected amino acids 7.9 A few of the
latter compounds 7 were also commercially available.
Intermediate 7 was then coupled to optically pure CD-
segment 5, producing the N-Boc-BCD segment 8. Re-
moval of the Boc protecting group in 8 with 4 N HCl in
dioxane gave compound 9. Coupling of 9 to the A-
segment 4, in the presence of PyBOP and N,N-diiso-
propylethylamine,10 produced the protected tripeptide
10. Hydrolysis of ester 10 with 1 N lithium hydroxide
solution gave the corresponding acid 11. Final depro-
tection of the Boc protecting group in 11, under acidic
conditions, provided the optically pure tripeptides
12a–k.
The total synthesis of hemiasterlin has been reported in
the literature.4 This synthetic approach was also used
for synthesis of HTI-286 and other analogs.8 The gen-
eral synthesis for the purposes of this paper, involved
preparing amino acids as B-segments, followed by cou-
pling to the CD-segment (5) and the A-segment (4).8
Deprotection of the amino and carboxyl groups pro-
vided the desired analogs (12, Scheme 1).
The synthesis of B-piece analogs incorporating a fluo-
rine atom is outlined in Scheme 2. Tripeptide 14, as a
mixture of two diastereoisomers, was synthesized start-
Commercially available (S)-amino acid 6, containing
either O or S heteroatoms as well as other functional
O
O
HCl HN
OH
O
NH
R3
4 A-segment
5
CD-segment
R1
R2
N
R1
R2
O
R1
R2
O
O
a
b
c
OH
OH
BocHN
O
H2N
BocHN
O
6
7
8
R1
R2
O
R1
R2
O
O
O
O
O
d
N
N
N
H
Boc
O
H2N
HCl
N
R3
9
10
R1
R2
O
O
O
e
f
N
N
H
Boc
OH
N
R3
11
R1
R2
O
O
O
N
N
H
OH
NH
R3
12
12a8 R1 = CH3, R2 = SCH3, R3 = H
12b R1 = CH3, R2 = SOCH3, R3 = H
12c
12d
R
R
1 = CH3, R2 = SO2CH3, R3 = H
1 = CH3, R2 = SCH2C6H4OCH3-p, R3 = H
12e8 R1 = CH3, R2 = C6H4OCH3-p, R3 = H
12f
R1 = H, R2 = OH (R), R3 = H
12g R1 = H, R2 = OH (S), R3 = H
12h
12i
R
1 = H, R2 = OCH3 (R), R3 = H
R1 = CH3, R2 = OH, R3 = H
12j R1 = CH3, R2 = SCH2C6H4OCH3-p, R3 = OCH3
12k R1 = CH3, R2 = SCH3, R3 = OCH3
Scheme 1. Reagents and conditions: (a) Boc2O, NaHCO3, H2O; (b) 5, PyBOP, DIEA, CH2Cl2; (c) 4 N HCl/dioxane; (d) 4, PyBOP, DIEA, CH2Cl2;
(e) 1 N LiOH, H2O, CH3OH; (f) 4 N HCl/dioxane or TFA, CH2Cl2.

C. Niu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4329–4332
4331
F
F
O
O
a, b, c, d, e
f
N
OH
N
H
Boc
OH
H2N
N
O
O
13
14
F
N
O
F
O
O
O
O
N
+
N
H
OH
N
H
OH
NH
NH
O
15
16
Scheme 2. Reagents and conditions: a, b, c, d, e refer to Scheme 1; (f) (i) TFA, CH2Cl2; (ii) HPLC.
ing from racemic fluorovaline (13), following the pro-
cedure described in Scheme 1. Removal of the Boc
protecting group, using trifluoroacetic acid, followed by
preparative HPLC separation, gave the (S,S,S) diaste-
reoisomer 15 and the (S,R,S) isomer 16.
showed potent activity against the KB-3-1 cell line, as
well as the paclitaxel resistant cell lines KB-8-5 and KB-
V1. The sulfoxide, 12b, showed no activity against the
KB cell lines below 3 lM concentration. However, the
corresponding sulfone, 12c, retained moderate potency
against KB-3-1 and KB-8-5 cell lines. Analog 15, having
The analogs thus prepared were evaluated for their
antimitotic activities by measuring their inhibition of
tubulin polymerization, as well as their cytotoxicity
against KB cell lines (KB-3-1, KB-8-5, and KB-V1,
Table 1). Most of the analogs showed moderate to
strong inhibition of MAP-rich tubulin polymerization in
comparison with the inhibition by the lead compound
HTI-286 (average 88%). The sulfur containing analogs,
12a,8 12c, 12d, and 12k, oxygen containing analogs 12g,
12h, and 12i, as well as the fluoro-substituted analog 15,
strongly inhibited tubulin polymerization, comparable
to HTI-286. In the cellular assays, the KB-3-1 cell line
does not express P-glycoprotein (Pgp), while the pacli-
taxel-resistant cell lines KB-8-5 and KB-V1 express
moderate to very high levels of Pgp, respectively. HTI-
286 and paclitaxel were used as reference compounds.
Both analogs 12a and 12k, incorporating methylsulfide,
one methyl group of the
tuted with fluorine, retained good potency against KB
cell lines. However, when one methyl group of the
tert-leucine moiety was replaced with a hydroxyl group,
the resulting analog 12i showed moderate potency
against KB-3-1 and KB-8-5 cell lines, and was inactive
versus the KB-V1 cell line at or below 3 lM concen-
L-tert-leucine moiety substi-
L
-
tration. Replacing the
L-tert-leucine moiety with L-
threonine gave analog 12f, which exhibited very poor
inhibition of tubulin polymerization and lost activity
under the given assay conditions. Interestingly, replac-
ing the L-tert-leucine moiety with L-allo-threonine pro-
vided analog 12g, which showed excellent inhibition of
tubulin polymerization and moderate potency in KB-3-1
and KB-8-5 cells. With O-methyl L-threonine in the
place of the L-tert-leucine moiety, the analog 12h
strongly inhibited tubulin polymerization and showed
moderate activity versus KB-3-1 cell line. Analogs 12d
and 12j, containing the bulky functional group
SCH2C6H4OCH3-p, maintained good activity against
the KB cell lines.
Table 1. In vitro potency and inhibition of tubulin polymerization of
hemiasterlin analogs versus paclitaxel
Compound Inhibition of
tubulin poly-
IC50 (nM)
KB-8-5
The pharmaceutical profiling of oxygen containing
analogs 12c, 12g, and 12h revealed that these polar
analogs exhibited very poor permeability11 at pH 7.4.
Presumably, these compounds cannot penetrate into the
cell sufficiently to achieve good potency even though
they are good inhibitors of cell-free tubulin polymeri-
zation.
KB-3-1
KB-V1
merization (%)a
12a
12b
12c
95
43
1.2
2.2
144
>3000
177.8
58.8
>3000
588.8
93.7
54.9
>3000
691.8
1737
95.0
43.7
1.8
>3000
>3000
2291
87
94
12d
12e
56
25.5
1794
12f
9
91
>3000
275.4
870.9
57.9
>3000
>3000
>3000
>3000
2089
12g
12h
12i
SAR studies in our laboratories had confirmed that the
S-configuration and branching on the b-carbon of the B-
segment are necessary for potent activity.8 The SSS
isomer, 2, exhibited high potency against KB cell lines,
but the SRS isomer was much less active. The same
trend was seen in the fluorinated analogs 15 and 16.
Variation of the tert-butyl group revealed that potency
was related to the branching, with the order in potency
as (CH3)3C > (CH3)2CH > CH3CH2 > CH3. Enlarge-
ment of the tert-butyl group, via incorporation of a p-
methoxyphenyl functional group, gave analog 12e,8
95
84
12j
77
88
15.8
12k
15
0.642
1.6
2–6
62.4
117.3
>3000
91
3.5
30–80
Paclitaxel
Promotes
tubulin poly-
merization
HTI-286 (2) 88b
0.6–2
1.2–5
40–140
a % Inhibition of MAP-rich tubulin polymerization at 0.3 lM.
b Average over 10 runs.

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C. Niu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4329–4332
Acknowledgements
We thank the Discovery Technologies of Wyeth Re-
search: Discovery Analytical Chemistry for analytical
support and Discovery Synthetic Chemistry for pro-
viding bulk intermediate 5.
References and notes
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5. (a) Andersen, R. J. WO 99/32509; (b) Nieman, J. A.;
Coleman, J. E.; Wallace, D. J.; Piers, E.; Lim, L. Y.;
Roberge, M.; Andersen, R. J. J. Nat. Prod. 2003, 66, 183.
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.; Poruchynsky,
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Figure 2. Effect of 12a and 12k on growth of human LOX melanoma
xenografts in athymic mice.
which showed good activity against KB cell lines
in vitro.
The two compounds 12a and 12k selected as possible
backup candidates of HTI-286 were further evaluated in
in vivo assays, using standard pharmacological test
procedures, which measure the ability to inhibit the
growth of human tumor xenografts.12 Both the mini-
mum effective dose (MED) and the maximum tolerated
dose (MTD) were determined. The MED of 12a was
0.5 mpk (mg per kilogram) to inhibit the LOX mela-
noma xenograft model and its MTD value was between
3 and 10 mpk. The MED and MTD values of analog
12k were determined to be 0.5 and 4 mpk, respectively.
Zero tumor growth was achieved with analog 12k at
2 mg/kg dose in the LOX melanoma xenograft model
(Fig. 2). Compound 12k was also active in vivo against
the paclitaxel-resistant cell line DLD1.
8. Zask, A.; Birnberg, G.; Cheung, K.; Kaplan, J.; Niu, C.;
Norton, E.; 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.
9. Schmidt, U.; Schumacher, A.; Mittendorf, J.; Riedl, B. J.
Pept. Res. 1998, 52, 143.
10. Frerot, E.; Coste, J.; Pantaloni, A.; Dufour, M.-N.; Jouin,
P. Tetrahedron 1991, 47, 259.
11. Kansy, M.; Senner, F.; Gubernator, K. J. Med. Chem.
1998, 41, 1007–1010.
12. Human Lox Melanoma cells (NCI, Frederick, MD) were
grown in tissue culture in RPMI (Gibco/BRL, Gaithers-
burg, MD) supplemented with 10% FBS (Gemini Bio-
Products Inc., Calabasas, CA). Athymic nu/nu female
mice (Charles River, Wilmington, MA) were injected SC
in the flank area with 1.5 · 106 Lox cells. When tumors
attained a mass of between 100 and 150 mg, the mice were
randomized into treatment groups (day zero), five animals
per group. Animals were treated with iv on days 1, 5 and 9
post staging with vehicle or drug prepared in normal
In summary, we have synthesized a series of analogs of
HTI-286, containing O, S, and F hetero-substituents in
the B-segment. Most of these compounds strongly
inhibited tubulin polymerization. Tolerance to a free
hydroxyl group on the b-carbon of the B-segment was
shown to be strongly dependent on the configuration at
this carbon atom, as evidenced by the difference in
inhibition of tubulin polymerization by diasteroisomers
12f versus 12g. Several compounds showed potent cel-
lular cytotoxicity against KB cell lines in vitro. The
in vivo assays revealed that analogs 12a and 12k also
effectively inhibited the growth of human tumor xeno-
grafts in athymic mice, including tumors resistant to
paclitaxel.
saline. Tumor mass was determined once
a week
[(length · width2)/2] for up to 21 days. Relative tumor
growth (Mean tumor mass on day of measurement divided
by the mean tumor mass on day zero.) was determined for
each treatment group. Statistical analysis (Student-t-test)
of log relative tumor growth was used to compare treated
verses control group in each experiment. A p-value
(p 6 0:05) indicates a statistically significant reduction in
relative tumor growth of treated group compared to the
vehicle control.