Total synthesis and evaluation of 22-(3-azidobenzoyloxy)methyl epothilone C for photoaffinity labeling of β-tubulin

Article abstract of DOI:10.1016/j.bmcl.2009.04.077

The total synthesis of 22-(3-azidobenzoyloxy)methyl epothilone C is described as a potential photoaffinity probe to elucidate the β-tubulin binding site. A sequential Suzuki-aldol-Yamaguchi macrolactonization strategy was utilized employing a novel deriva

Full text of DOI:10.1016/j.bmcl.2009.04.077

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3293–3296
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Total synthesis and evaluation of 22-(3-azidobenzoyloxy)methyl epothilone C
for photoaffinity labeling of b-tubulin
Oliver E. Hutt a,c, Jun Inagaki a, Bollu S. Reddy a, Sajiv K. Nair a, Emily A. Reiff a, John T. Henri a,
Jack F. Greiner a, David G. VanderVelde a, Ting-Lan Chiu c, Elizabeth A. Amin c,
Richard H. Himes b, Gunda I. Georg a,c,
*
a Department of Medicinal Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, KS 66045, USA
b Department of Molecular Bioscience, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045, USA
c Department of Medicinal Chemistry, and Institute for Therapeutics Discovery and Development, University of Minnesota, 717 Delaware Street SE, Minneapolis, MN 55414, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The total synthesis of 22-(3-azidobenzoyloxy)methyl epothilone C is described as a potential photo-
affinity probe to elucidate the b-tubulin binding site. A sequential Suzuki-aldol-Yamaguchi macrolacton-
ization strategy was utilized employing a novel derivatized C1–C6 fragment. The C22-functionalized
analog exhibited good activity in microtubule assembly assays, but cytotoxicity was significantly
reduced. Molecular modeling simulations indicated that excessive steric bulk in the C22 position is
accommodated by the large hydrophobic pocket of the binding site. Photoaffinity labeling studies were
inconclusive suggesting non-specific labeling.
Received 5 March 2009
Revised 14 April 2009
Accepted 17 April 2009
Available online 22 April 2009
Keywords:
Epothilone
Photoaffinity labels
Microtubules
Ó 2009 Elsevier Ltd. All rights reserved.
The epothilone class of natural products, typified by epothilone
A (1a), epothilone B (1b), and epothilone C (2), is an extremely
cytotoxic family of macrocyclic compounds. Their isolation from
the myxobacterium Sorangium cellulosum was first reported in
1992 by Gerth et al.1 It is well established that the binding sites
on b-tubulin for the epothilones and taxanes overlap. However,
early observations that the epothilones maintained toxicity against
paclitaxel resistant cell lines, in which the resistance arose from
tubulin point mutations in the paclitaxel binding site, suggested
that the mode of binding was significantly different.2 As a result,
early attempts to find a shared pharmacophore between paclitaxel
and epothilone B were unsuccessful.3–6 The clinical importance7 of
this class of compounds has led to an explosion of synthetic activ-
ity8–10 and a truly multi-disciplinary effort has culminated in two
b-tubulin-epothilone binding models. The first model is based on
combined molecular modeling, NMR, and electron-crystallography
(EC) data of Zn2+ induced tubulin sheets,11 while the second model
is derived from NMR studies of epothilone bound to unpolymer-
In the EC-model the C7 hydroxyl, the C5 carbonyl, and the C3
hydroxyl of epothilone engage with Arg282 and Thr274 via H-
bonds, while the C1 carbonyl interacts with Arg276. The thiazole
ring then forms a hydrogen bond with His227 in the H7 helix.
Although they are remote from the binding site, the residues
Gln292 and Ala231 are important in maintaining the correct con-
formation of the M-loop and H7 helix, respectively.
N3
S
N
R
O
S
N
O
OH
22
7
O
3
5
O
OH
4
22
O
OH
O
O
R = H: Epothilone A (1a)
O
OH O
R = Me: Epothilone B (1b)
R = H, cis double bond in place
of epoxide: Epothilone C (2)
3
ized a
,b-tubulin dimer in solution.12
The NMR-model supports the interaction between the C7 hydro-
xyl and Arg282 and Thr274 and further, that this interaction locks
Arg276 into position to form a salt bridge with Asp224. The thiazole
* Corresponding author. Address: Professor and Department Head, Robert Vince
Endowed Chair, McKnight Presidential Chair in Medicinal Chemistry, Department of
Medicinal Chemistry Director, Institute for Therapeutics Discovery and Develop-
ment, Room 452, College of Pharmacy, University of Minnesota, 717 Delaware
Street SE, Minneapolis, MN 55414, USA. Tel.: +1 612 626 6320; fax: +1 612 626
6318.
and His227 engage in a
p–p interaction and are not hydrogen
bonded. Finally, and perhaps most intriguingly, is the involvement
of the hydrophobic pocket hosting Phe270, which is vital for paclit-
axel activity and also hosts the methyl group of epothilone B and
thus accounts for its 10-fold increase in activity over epothilone A.
E-mail address: georg239@umn.edu (G.I. Georg).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.077

3294
O. E. Hutt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3293–3296
end we have synthesized the C22-photoreactive derivative 3. We
LDA, THF,
now report the synthesis of 3 and the resulting microtubule assem-
bly, cytotoxicity studies, and photoaffinity labeling studies.
The synthesis of the C22 analog follows the well precedented al-
dol/macrolactonization approach.22 With the known aldehyde 12
in hand we focused our attention on the synthesis of the modified
C1–C7 fragment 11 (Scheme 1). Starting from the known b-hydro-
xy ester 4 two sequential Frater alkylations where carried out.
Thus, the b-hydroxy ester 4 was treated with 2 equiv of LDA and
the resulting dianion quenched with 1.1 equiv of allyl iodide to
give the anti-allylated compound 5 in 82% yield. The procedure
was subsequently repeated and quenched with MeI to provide
the desired anti-methylated product 6 in 66% yield. The free alco-
hol was protected as the TBS ether (94%) and the alkene function of
6 was ozonolyzed to furnish aldehyde 7 (97%). The aldehyde was
subsequently reduced and the resulting alcohol protected as the
trityl ether.
-78ºC → -20ºC, 2h;
BnO
OMe
OMe
BnO
OMe
OH
4
O
O
OH
O
CH2CHCH2I, HMPA,
-78ºC → -20ºC, 10h
82%
5
LDA, THF
CH3I, HMPA,
66%;
O
1. TBSOTf,
2,6-Lutidine, 94%
BnO
BnO
BnO
BnO
OMe
2. O3, CH2Cl2, -78ºC;
Me2S, 97%
TBSO
OH
6
O
7
1. NaBH3CN, MeOH, 84%
2. TrCl, Et3N, DMAP, 93%
TrO
TrO
1. DIBAL-H, 80%
OMe
2. IBX, EtOAc, 89%
TBSO
O
TBSO
O
9
The C6–C7 ethyl chain was then introduced by first converting
the ester 8 to aldehyde 9 through a two-step reduction (80%)/oxi-
dation (89%) sequence. The aldehyde 9 was then treated with
EtMgBr and the resulting alcohol (71%) was oxidized to provide ke-
tone 10 in 89% yield. Finally, the benzyl group was removed and
the free primary C1 alcohol reprotected as TBS ether 11 in prepara-
tion for the aldol reaction.23
Accordingly, the C1–C7 fragment 11 was treated with LDA and
then the aldehyde fragment 12 was added to give adduct 13 in 38%
yield (Scheme 2). The newly formed alcohol function was pro-
tected as the TBS ether (90%) and the C1 primary TBS ether was re-
moved (72%) subsequently. The resulting alcohol was sequentially
oxidized to the aldehyde (98%) and then to the acid 14 (94%).
The TBS-protecting group on the allylic alcohol 14 was
deprotected (96%) and the macrocycle closed by employing a
8
1. EtMgBr, 71%
2. DMP, 89%
TrO
TrO
1. Pd(OH)2/C, H2,
EtOAc, 86%
BnO
TBSO
2. TBSCl, Et3N, 83%
TBSO
O
TBSO
O
10
11
Scheme 1. Synthesis of C1–C7 fragment.
The underlying consensus of both the EC-model and NMR-mod-
el is a rigidification and alignment of the M-loop and the H7 helix,
which leads to a conformational change in the
a,b-tubulin dimer.
This hypothesis is in agreement with a different overall macromo-
lecular structure observed between polymerized and unpolymer-
ized tubulin.13
Mammalian brain tubulin, the most abundant and studied of
the tubulins, is highly heterogeneous due to the existence of seven
Table 1
Biological testing of C22-epothilone photoaffinity analog 3
a
- and eight b-isoforms and several types of post-translational
Compounds
Microtubule
assembly ED50
Cytotoxicity MCF-7 Cytotoxicity NCI-ADR
Ma ED50 Ma,b Ma,b
ED50
modifications. These combinations are responsible for producing
greater than 22 bands when mammalian brain tubulin is separated
on high-resolution isoelectric focusing gels.14 Given this complex-
ity there still remains significant advances to be made in the
understanding of microtubule stabilizing agents. Early information
on the paclitaxel binding site was derived through photoaffinity
labeling.15–20 More recently discodermolide photoaffinity labeling
studies have been reported.21 However, to date the photolabeling
with epothilone derivatives has remained elusive. Toward this
,
l
,
l
, l
Epothilone B 1.0
5.1 (5.1)
0.0015 0.0012
1.2 (632) 1.0 (833)
3
a
ED50 3/ED50 epothilone B is in parenthesis.
ED50 values for growth inhibition of human breast carcinoma cell line MCF-7.
b
ED50 values for growth inhibition of NCI-ADR, a P-glycoprotein over-expressing
multi-drug resistant human ovarian carcinoma cell line. Cell proliferation assays
were performed as previously described.
performed as described in Ref. 26.
25
Microtubule assembly assays were
S
S
N
S
N
1. HF⋅py, 72%
1. 11, LDA,
N
THF, 38%
2. DMP, 98%
TrO
TrO
O
OTBS
OTBS
2. TBSOTf,
3. NaClO2, Na2HPO4
94%
2,6-Lutidine, 90%
TBSO
HO2C
OTBS
TBSO
TBSO
OR1
O
TBSO
O
13 R1 = TBS
12
14
1. TBAF, 96%
N3
N3
2. 2,4,6-trichlorobenzoyl
chloride, DMAP, Ph-Me
77%
S
N
S
N
S
N
1. CSA, MeOH,
Et2O, 50%
O
O
20% TFA
59%
O
O
TrO
OTBS
2. 3-azidobenzoic
acid, (COCl)2, DMF,
DMAP, THF;
82%
OTBS
OH
O
O
O
O
O
OR1
O
O
OR1
O
OH
O
16 R1 = TBS
3
15 R1 = TBS
Scheme 2. Synthesis of arylazide epothilone analog 3.

O. E. Hutt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3293–3296
3295
Figure 1. (a) Docked configuration (Surflex-Dock, Tripos, Inc.) of 22-(3-azidobenzoyloxy)methyl epothilone C (3) shown with MOLCAD (Tripos, Inc.) electron density surfaces
of the tubulin active site (1TVK11), onto which hydrogen bond donor and acceptor regions have been mapped. Red areas represent hydrogen bond donors; blue areas
represent hydrogen bond acceptors; and gray indicates regions in which no hydrogen bonding takes place. Key ligand–receptor interactions are shown. Hydrogen bonding
occurs between the thiazole nitrogen and one of the amino groups of Arg276; between the C1 carbonyl and the other amino group in Arg276; between the C5 carbonyl and
the Thr274 backbone NH; and between the C7 OH and backbone carbonyl of Pro272. (b) The overlay of epothilone B (yellow) and 3 (cyan) in the binding site (PyMOL, 2006
DeLano Scientific LLC). Docking validation was carried out by comparing the docked configuration of EpoA to experiment (RMSD = 1.575 Å). The pre-docked conformation of 3
was prepared by modifying the experimental epothilone A structure and optimizing the resulting geometry using the MMFF94s force field in MOE (Chemical Computing
Group, Inc.).
Yamaguchi macrolactonization to form the core skeleton 15 in 77%
yield. The trityl group was removed with CSA to give the
corresponding primary hydroxyl derivative in 50% yield. Finally,
the meta-azidobenzoic acid was converted to the acid chloride on
treatment with oxalyl chloride, which was subsequently reacted
with the epothilone derivative to form photoaffinity adduct 16 in
82%.24 Removal of the TBS groups with TFA then gave the target
compound 3 (Scheme 2).
The microtubule assembly properties and cytotoxicity of the
meta-azido analog 3 are summarized in Table 1. In the microtubule
assembly assay the photoaffinity analog 3 was found to assemble
microtubules in a concentration-dependent fashion.25 However,
the assembly was not as rapid and did not reach the same extent
as in the presence of epothilone B. Comparison of the ED50 values
shows that 3 is about 20% as effective as epothilone B. Compound
3 is much less potent against NCI-ADR and MCF-7 cancer cell lines.
The very low cytotoxicity could be due to poor uptake of 3 by the
cells.
able van der Waals interactions with the bulky C22 position. This
relatively minor rearrangement does not significantly disrupt the
hydrogen bonding network between receptor and the C1–C7 por-
tion of the ligand. However, in analog 3, the thiazole hydrogen
binds to Arg276 rather than to His 227 as is found with epothilone
B.
In summary, the photoaffinity analog 3 promotes the assembly
of tubulin into microtubules and the computer modeling studies
revealed key ligand-receptor interactions. Specifically, molecular
modeling simulations indicated that excessive steric bulk in the
C22 position is adequately accommodated by the large hydropho-
bic pocket in the binding site. However, the non-specific covalent
labeling limits the practical utility of this probe for further elucida-
tion of the b-tubulin binding site.
Acknowledgments
This work was supported by the National Institute of Health
grants CA79641 and CA105305. E.A.R. and J.F.G. were supported
by NIH Training Grant GM 07775. E.A.R. was also supported by
DAMD 17-001-0303.
The labeling studies, carried out on assembled microtubules
with 3H-meta-azido analog 3, demonstrated that irradiation led
to incorporation of label into the protein. However, competition
studies showed that although 40
lM epothilone B did not reduce
the labeling by 10 M analog 3, 40
l
l
M paclitaxel was able to dis-
References and notes
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in solution and irradiated, it produces reactive intermediates that
react in a non-specific manner with the protein.
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23. C1–C7 fragment 11. 1H NMR (400 MHz, CDCl3) d 7.38–7.18 (m, 15H), 3.94–3.92
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(m, 2H), 2.12–2.09 (m, 1H), 1.85–1.78 (m, 1H), 1.55–1.37 (m, 2H), 0.94 (s, 3H),
0.88–0.87 (m, 21H), 0.08–0.06 (m, 12H). 13C NMR (100 MHz, CDCl3) d 214.7,
144.3, 128.8, 128.5, 127.9, 127.0, 87.2, 73.9, 60.3, 59.9, 55.5, 37.5, 37.3, 32.4,
26.3, 26.1, 18.6, 18.4, 14.8, 7.6, ꢀ3.5, ꢀ3.6, ꢀ5.1. HRMS m/z calcld for
C41H66NO4Si2 (M+NH4) 692.4530, found 692.4538. ½a D22:5
ꢀ 17:5 (c 2.55, CHCl3).
ꢁ
24. Photoaffinity analog 3: 1H NMR (400 MHz, CDCl3) d 7.75 (dm, J = 7.9 Hz, 1H),
7.64 (t, J = 1.7 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.18 (dm, J = 8 Hz, 1H), 6.98 (s,
1H), 6.58 (s, 1H), 5.38 (m, 2H), 5.34 (d, J = 9.9 Hz, 1H), 4.37 (m, 2H), 4.20 (ddd,
J = 11.2 Hz, J = 9.5 Hz, 5.7 Hz, 1H), 3.73 (d, J = 4.4 Hz, 1H), 3.45 (br s, 1H), 3.18
(qd, J = 6.7 Hz, J = 1.1 Hz, 1H), 3.16 (s, 1H), 2.67 (m + s, 4H), 2.57 (dd, J = 14.9 Hz,
J = 11.5 Hz, 1H), 2.41 (dd, J = 14.9 Hz, 2.4 Hz, 1H), 2.35 (m, 1H), 2.28 (m, 1H),
2.16 (m, 2H), 2.08 (s, 3H), 1.54–1.8 (m, 4H), 1.23 (t, J = 7.2 Hz, 2H), 1.21 (d,
J = 6.9 Hz, 3H), 1.16 (s, 3H), 1.00 (d, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3)
219.7, 170.2, 165.5, 165.1, 151.9, 140.7, 138.6, 133.4, 131.7, 129.9, 126.0, 125.2,
123.6, 119.9, 119.5, 115.9, 78.4, 73.9, 71.9, 61.9, 56.2, 41.6, 38.9, 38.6, 33.5,
32.5, 31.8, 30.7, 19.1, 15.9, 15.2, 14.2, 13.7, 13.1. IR (neat) mmax 3515, 2358,
2115, 1724 cmꢀ1. HRMS m/z calcd for C34H44N4O7S (M+H) 653.3009, found
653.2990. ½a 2D6
ꢀ 51:6 (c 0.480, CHCl3).
ꢁ
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