Absolute configurations of tubulin inhibitors taltobulin (HTI-286) and HTI-042 characterized by X-ray diffraction analysis and NMR studies

Article abstract of DOI:10.1016/j.bmcl.2010.01.047

The stereochemistry of the tubulin inhibitors taltobulin HTI-286 (2) and HTI-042 (3) was determined by utilizing the DPFGSE 1D NOE experiment. Single crystal X-ray diffraction analysis further confirmed the absolute configuration of these two compounds, w

Full text of DOI:10.1016/j.bmcl.2010.01.047

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1535–1538
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Bioorganic & Medicinal Chemistry Letters
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Absolute configurations of tubulin inhibitors taltobulin (HTI-286) and HTI-042
characterized by X-ray diffraction analysis and NMR studies
b
a
a
Chuansheng Niu a, , Douglas M. Ho , Arie Zask , Semiramis Ayral-Kaloustian
*
a Wyeth Research, Pearl River, NY 10965, USA
b Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The stereochemistry of the tubulin inhibitors taltobulin HTI-286 (2) and HTI-042 (3) was determined by
utilizing the DPFGSE 1D NOE experiment. Single crystal X-ray diffraction analysis further confirmed the
absolute configuration of these two compounds, which carry the (S,S,S)-configuration necessary for bio-
logical activity.
Received 21 December 2009
Accepted 15 January 2010
Available online 20 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Tubulin inhibitors
HTI-286
DPFGSE 1D NOE
X-ray diffraction analysis
The natural product hemiasterlin,1 (1, Fig. 1), isolated from
marine sponges,2 is one member of a family of tripeptides consist-
ing of three sterically congested amino acids. Hemiasterlin and its
relatives are potent antimitotic agents that effectively inhibit tubu-
lin polymerization by binding to the Vinca-peptide-binding site.3
The synthetic analog taltobulin (2, HTI-286),4 in which the N-
methylindole ring was replaced with a phenyl group, was found
to be a potent cytotoxin and mitotic blocker like 1, vincristine
and paclitaxel (Taxol™).5 In particular, compound 2 exhibited
more potent activity than 1 against paclitaxel-resistant cell lines
in vitro and in vivo.5,6 Therefore, taltobulin (2) was selected for fur-
ther development for the treatment of advanced malignant solid
tumors.7 The absolute stereochemistry of 2 was proposed as the
(S,S,S)-configuration at its three chiral centers based on the syn-
thetic strategy and the stereochemistry of 1.
Extensive structure–activity relationship (SAR) studies of HTI-
286 analogs were conducted in our laboratories. One superior ana-
log, HTI-042 (3), was discovered.8 Compound 3, which incorporated
a methoxy functional group on the phenyl ring, not only retained the
potency of 2, but also showed a better safety window than 2 and
complete cures in certain xenograft models. HTI-042 was also se-
lected as a development candidate.
O
O
N
N
H
OH
NH
O
N
C-piece D-piece
Hemiasterlin (1)
B-piece
A-piece
O
O
N
N
H
OH
O
NH
O
Taltobulin (HTI-286, 2)
O
N
N
OH
H
NH
O
O
HTI-042 (3)
One of the important aspects of our research was to confirm the
stereochemistry of taltobulin (2) and HTI-042 (3). An efficient and
straightforward synthetic approach for the total synthesis of HTI-
286, HTI-042, and other analogs of 2 for SAR studies is illustrated
in Scheme 1.8,9 Methylation of the pyruvic acid derivative 4 with
Figure 1.
iodomethane in the presence of sodium hydride gave the gem-
dimethyl compound 5. Reductive amination of 5 produced amino
acid 6. Coupling 6 to dipeptide 7 in the presence of PyBOP provided
a mixture of two diastereoisomers 8 and 9, which were easily sep-
arated by HPLC. The desired ester 8, with the right stereochemistry
* Corresponding author. Tel.: +1 845 602 5996; fax: +1 845 602 5561.
E-mail address: niuc@wyeth.com (C. Niu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.047

1536
C. Niu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1535–1538
O
O
port studies on the absolute configuration of the synthetic analogs
HTI-286 and HTI-042.
1. MeNH2
2. BH3.Py
MeI
OH
OH
OH
R
R
N
The stereochemistry of compounds 2 and 3 was expected to
be S,S,S based on the synthetic strategy, HPLC retention time,
NaOH
O
O
and bioassay data.8,11 To confirm the stereochemistry at the
a-
4
5
carbon of the N-Me-b-dimethyl phenylalanine residue in these
hemiasterlin derivatives and to assign the stereochemistry of 2,
we utilized the double-pulsed field gradient spin echo (DPFGSE)
1D NOE experiment. All the NOE data discussed in this Letter
were acquired using THF-d8. This solvent provided a very well
dispersed 1D 1H NMR spectrum and appeared to encourage the
occurrence of one significant solution conformation. This obser-
vation was evidenced by the extreme magnitudes of the homo-
nuclear coupling constants (both small and large) for 2. An
example of the 1D NOE data acquired from selective excitation
of the proR-methyl group of the N-Me-b-dimethyl phenylalanine
residue is shown in Figure 2. As can be seen, the strong NOE cor-
relations between the proR-methyl (CH3-20), the N-3 Me, and
the tert-butyl group (12a–c; Fig. 3) require that all of these
atoms be on the same face of the molecule. When this informa-
tion is coupled with the fact that the proS-methyl (CH3-19)
shows correlations only to CH3-20 and the H-14 proton, it be-
comes clear that this methyl must be on the opposite face of
the molecule from CH3-20 and the stereochemistry at C-14 must
be S.8,12
O
O
H2N
O
O
7
R
HN
1. PyBOP, DIPEA
2. HPLC
6
O
O
O
N
N
R,S,S-isomer
+
R
H
HN
O
8
(S,S,S)
9
O
O
N
N
LiOH
OH
R
H
O
HN
2
3
HTI-286, R = H
HTI-042, R = OCH3
Some of the most important NOE correlations for the analysis of
HTI-286 are shown in Figure 3. According to these studies, the ste-
reochemistry of HTI-286 was in fact the predicted S,S,S configura-
tion at the three chiral centers, and this compound appeared to
fold back on itself along its length. These observations were con-
firmed by single-crystal X-ray diffraction.
Scheme 1. Synthesis of HTI-286 and analogs.
Crystals of HTI-286 (2) and the hydrochloride salt of HTI-042 (3)
were grown from acetonitrile/water solutions as colorless prisms
for a pentahydrate acetonitrile solvate, and colorless needles for
a tris(acetonitrile) solvate, respectively. The Flack absolute struc-
ture parameter for 3 refined to À0.03(11) for (S,S,S) and 1.01(11)
for (R,R,R). These values are consistent with the correctness of
the (S,S,S) assignment for 3 based on anomalous dispersion. Using
3 as a heavy-atom derivative, the absolute configuration of 2 is also
(S,S,S).13
Perspective views of 2 and 3 are given in Figure 4. Also included
is a view of the methyl ester of 1 to facilitate comparisons. Note
that it was drawn as the (R,R,R) instead of the (S,S,S) isomer in an
earlier publication.10 The tripeptides 1–3 differ only in their A-
piece amino acids, so it is perhaps not surprising that the most
noticeable conformational differences in their X-ray structures ap-
pear near the A-piece ends of these molecules. These differences
are largely due to torsional degrees of freedom in the O@C–C–N
units in the A-piece amino acid. Referring to Figure 4, from top
to bottom, the O@C–C–N torsion angles are 155.7(4)°, À45.5(5)°,
and À45.9(3)°, respectively. Note that the A-piece geminal di-
methyl group is a key component for potent activity, yet the tor-
sion angles for 2 and 3 forces their dimethyl groups to be
completely out of synch with the dimethyl groups in 1. Presumably
these inhibitors assume the same torsion angle upon binding to
tubulin,14 since they have similar biological activity.
7
6
5
4
3
2
1
ppm
Figure 2. DPFGSE 1D NOE with selective excitation of tert-butyl group of HTI-286.
predicted as (S,S,S) was then hydrolyzed with lithium hydroxide,
providing the target analog 10. SAR studies revealed that changing
the stereochemistry at each chiral center of the tripeptides resulted
in reduction of the activity.8 To facilitate the design and synthesis
of novel analogs with demonstrated cytotoxic and tubulin binding
abilities, it was necessary to confirm the stereochemistry of HTI-
286 and its analogs. To date there have been no reports on deter-
mination of the stereochemistry of synthesized analogs such as 2
and 3 by X-ray crystal analysis. However, the structure of the
methyl ester of natural product 1 was reported.10 We hereby re-
12a
N-2
12c
19
20
14
O
O
1
O
CH3
N
O
H
16
3
9
2
4
N
17
18
15
10
13
N
H
OH
N-1
N
H
OH
5
H
NH
NH
O
8
O
CH3
N-3
7
6
Figure 3. Key NOE enhancements observed for the assignment of C-14 stereochemistry.

C. Niu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1535–1538
1537
Figure 4. The molecular structures of hemiasterlin methyl ester (top), HTI-042 (middle) and HTI-286 (bottom) obtained by X-ray diffraction analysis.
5. 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, M. S.; Fojo, T.; Nieman, J. A.; Ayral-Kaloustian, S.; Zask, A.;
Andersen, R. J.; Greenberger, L. M. Cancer Res. 2003, 63, 1838–1845.
6. Ratain, M. J.; Undevia, S.; Janisch, L.; Roman, S.; Mayer, P.; Buckwalter, M.; Foss,
D.; Hamilton, B. L.; Fischer, J.; Bukowski, R. M. Proc. Am. Soc. Clin. Oncol. 2003,
22, 129.
7. Ayral-Kaloustian, S.; Zask, A. Drugs Future 2005, 30, 254–260.
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. 2004, 47,
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Acknowledgment
We greatly thank Dr. Robert Thomas Williamson for his efforts
on the NMR studies of the stereochemistry of HTI-286.
References and notes
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13. For 2Á5H2OÁCH3CN, monoclinic, C2, a = 25.9873(5) Å, b = 11.3310(2) Å,
c = 14.5689(3) Å, b = 122.180(1)°, V = 3630.96(12) Å3, and Z = 4. For
3ÁHClÁ3CH3CN, orthorhombic, P212121, a = 13.6128(3) Å, b = 14.4216(5) Å,
c = 20.3463(6) Å, V = 3994.4(2) Å3, and Z = 4. These structures have been
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