Regioselective synthesis of folate receptor-targeted agents derived from epothilone analogs and folic acid

Article abstract of DOI:10.1016/j.bmcl.2010.06.016

Efficient regioselective syntheses of conjugates of folic acid and cytotoxic agents derived from natural epothilones are described. These folate receptor (FR) targeting compounds are water soluble and incorporate a hydrophilic peptide-based spacer unit and a reducible self-immolative disulfide-based linker system between the FR-targeting ligand and the parent drug.

Full text of DOI:10.1016/j.bmcl.2010.06.016

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4578–4581
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Regioselective synthesis of folate receptor-targeted agents derived
from epothilone analogs and folic acid
Iontcho R. Vlahov a, , Gregory D. Vite , Paul J. Kleindl , Yu Wang , Hari Krishna R. Santhapuram , Fei You ,
b
a
a
a
a
*
a
b
b
a
Stephen J. Howard , Soong-Hoon Kim , Francis F. Y. Lee , Christopher P. Leamon
Endocyte Inc., 3000 Kent Ave., Suite A1-100, West Lafayette, IN 47906, USA
a
b
Bristol-Myers Squibb Research, PO Box 4000, Princeton, NJ 08543, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Efficient regioselective syntheses of conjugates of folic acid and cytotoxic agents derived from natural
epothilones are described. These folate receptor (FR) targeting compounds are water soluble and incor-
porate a hydrophilic peptide-based spacer unit and a reducible self-immolative disulfide-based linker
system between the FR-targeting ligand and the parent drug.
Received 30 April 2010
Revised 28 May 2010
Accepted 2 June 2010
Available online 8 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Folate
Folate receptor targeting
Folate–epothilone conjugate
Disulfide linkers
The major aims of targeted chemotherapies are twofold: to im-
prove the efficacy of currently available methods and to reduce the
inherent collateral toxicity to normal tissues. Receptor-specific tar-
geting is an innovative approach that can potentially herald a new
era in selective delivery of cytotoxic drugs to pathologic cells.
Folic acid (FA) binds with a high affinity cell-surface glycopro-
tein called the folate receptor. After binding, FA is transported into
sis.5 On account of their higher affinity, the epothilones displace
paclitaxel from microtubules and show activity against cancer cells
resistant to paclitaxel. Several epothilones and epothilone analogs
6
have entered human clinical trials, with ixabepilone being the first
7
drug in this class to receive FDA approval.
In this Letter, we report the design and regioselective synthesis
of FA–Epo conjugates 1a–c (Scheme 1). As indicated in the retro-
synthetic analysis, 1 can be assembled by tethering a FA-spacer
unit 2 to epothilone derivatives (3a–c) via a self-immolative linker
system containing a reducible disulfide bond, which is important
for drug delivery applications. A recent study involving real-time
imaging using a fluorescence resonance energy transfer technique
has demonstrated that reduction-mediated release of the drug car-
go from a disulfide linked FA-conjugate efficiently occurs within
1
the cell via FR-mediated endocytosis. The FR is expressed at rela-
tively high levels in human epithelial cancers, but has limited
expression in normal tissues. Additionally, conjugation of biologi-
cally active compounds to FA does not significantly affect FR bind-
ing and FR-mediated endocytosis in a vast number of cases.
Consequently, FA can be exploited as a molecular ‘Trojan horse’
for the targeted delivery of covalently-attached, biologically active
2
8
molecules. In pursuing this strategy, scientists at Endocyte have
the endosomes of cancer cells.
synthesized a series of FA conjugates of single chemotherapeutic
drugs, including desacetyl-vinblastine hydrazide, mitomycin, and
tubulysin; as well as a dual drug combination of mitomycin and
The peptide-based spacer 2 was designed to be bifunctional
containing both acidic (Asp) and basic (Arg) amino acids to provide
solubility of the final drug conjugate under physiological condi-
tions. This unit was assembled using standard fluorenylmethyloxy-
3
desacetyl-vinblastine hydrazide.
4
3
Epothilones (Epo) are 16-membered macrolides. Their activity,
carbonyl-based solid phase peptide synthesis (Fmoc SPPS).
3
like that of paclitaxel, is based on the inhibition of cell division by
binding to microtubules resulting in their stabilization. Microtu-
bules are responsible for various cellular activities, such as mitosis,
motility and trafficking of vesicles, organelles and proteins. Agents
which disrupt the dynamics of microtubules, can induce apopto-
Activated carbonate 5 (Scheme 2) served as universal heterobi-
functional crosslinker for the preparation of all conjugates de-
scribed below. The synthesis of our first folate conjugate involved
21-amino-epothilone B 4 as the parent drug. As shown in Scheme 1,
the amine 4 was treated with the activated carbonate 5 and
diisopropylethylamine (DIPEA) in dichloromethane to yield 2-pyr-
idyldisulfanylethyl carbamate 3a. After chromatographic purifica-
tion on silica gel, 3a was isolated in 80% yield. Treatment of a
*
Corresponding author. Tel.: +1 765 463 7175.
E-mail address: ivlahov@endocyte.com (I.R. Vlahov).
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.016

I. R. Vlahov et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4578–4581
4579
Scheme 1.
2 3 2 2 2 2 2
Scheme 2. Reagents and conditions: (a) 2-bromoethanol, K CO , acetonitrile, 80 °C; (b) TFA/CH Cl ; (c) 5, DMAP, CH Cl ; (d) 2, H O, THF.

4
580
I. R. Vlahov et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4578–4581
Scheme 3. Conjugate 1b in 50 mM NH
4
OAc buffer (pH 5.5) treated with TCEP.
1
suspension of FA-spacer 2 in H
2
O under argon with 0.1 N NaHCO
3
by H NMR: the signal for the proton attached to C7-atom shifted
resulted in a clear yellow solution at pH >6.5. To this mixture was
added at once under extensive stirring a solution of 3a in THF.
According to the HPLC profile, the reaction was completed in less
downfield from 3.76 ppm in 8 to 5.18 ppm in 3c. Treatment of 3c
with folate spacer unit 2 resulted as expected in clean conversion
to conjugate 1c.
A model study that mimics the release of free drug within the
reductive environment in the endosome was performed on conju-
gate 1b. We performed our chemical release study at pH 5.5
9
then 30 min. HPLC purification gave pure conjugate 1a.
1
0
SAR studies performed on semisynthetic derivatives of epo-
thilones showed that some N-substituted derivatives of 12 ,13a-
a
aziridinyl epothilone are more potent than the natural compounds,
so we decided to modify the aziridine moiety by simple N-alkyl-
4
(50 mM NH OAc buffer) to match the acidic environment in
1
7
endosome. A 1 mM solution of 1b was treated with 5 mM of
tris(2-carboxyethyl)phosphine (TCEP) at 37 °C. The HPLC profile
(Scheme 3, UV detection at 280 nm) showed complete cleavage
of the disulfide bond with concomitant release of the parent drug
7 and the FA-spacer 2 within 45 min. These results were confirmed
by LC/MS.
ation. Thus, treatment of suitably protected 12
a
,13
a-aziridinyl
1
0
epothilone A 6 with 2-bromoethanol and K
followed by deprotection of TES in TFA/CH Cl
member of the epothilone family, namely N-hydroxyethyl-
2
CO
3
in acetonitrile,
2
2
, resulted in a novel
11
12a,13a-aziridinyl epothilone A 7. Its cytotoxicity profile shows
that it is among the most potent epothilones. The primary hydroxyl
In summary, we described the expedient and regioselective
synthesis of folate conjugates of a structurally diverse group of
epothilones and demonstrated the release of the parent drug from
one of the intact conjugates in a simple model study. Furthermore,
we presented the new and powerful epothilone derivative 7. We
also were able to react successfully the sterically hindered C7–
OH in the epothilone backbone with our heterobifunctional linker
5, so that the resulting pyridyldisulfanylethyl carbonate can be
used as precursor for folate conjugation. Compound 1b (Epofolate,
group of 7 was reacted with activated carbonate 5 to provide
1
2,13
3
b.
This thiophilic pyridyldisulfanyl-epothilone derivative
was treated with the thiol-containing folate spacer unit 2 as de-
8
scribed above to give the desired product 1b (Epofolate, BMS-
14
7
53493). LC/MS and 1H NMR data were in agreement with the
expected structure.
The next Epo–FA conjugate involved the modification of the ste-
rically hindered C7-hydroxyl group in the epothilone macrolide
skeleton. As shown by electron crystallography studies,15 the C7-
hydroxyl group in the molecular architecture of epothilones is pro-
posed to be part of the hydrogen-bonding network responsible for
the specific binding to b-tubulin. Successful implementation of our
conjugation methodology to the C7–OH would open a wide range
of epothilones to folate conjugation, rather than being limited to
only those epothilones with more accessible nucleophilic moie-
BMS-753493) is in phase
I clinical trials for solid tumors.
Folate–epothilone conjugates are being tested against a variety of
FR positive cell lines as well as in animal models. The results of
the complex biological investigations will be reported in appropri-
ate scientific journals.
Acknowledgment
1
6
ties. 21-Azido-epothilone B 8 was reacted with excess of cross-
linker 5 in the presence of DMAP to give carbonate 3c as a single
product. The regioselectivity of this modification was confirmed
We thank the analytical group at Endocyte, Inc., for LC/MS
support.

I. R. Vlahov et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4578–4581
4581
2
.92 mmol) was added. After 3 h, the reaction mixture was cooled to room
References and notes
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2. General procedure for preparing of drug carbonates: To a solution of epothilone
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(
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M+3H) at 523.86431 (2.5 ppm), (M+4H) at 393.14996 (3.1 ppm).
9
.
2
O (bubbled with argon for 10 min
1
H NMR data (D
2
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1
5
3
2
1
0
3
(
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3
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1.
K
2
CO
to solution of 6 (1.05 g, 1.46 mmol) in acetonitrile (20 mL) and heated to 80 °C.
After 4 h, additional 2-bromoethanol (0.52 mL, 7.3 mmol) and K CO (1.4 g,
0.2 mmol) were added. After 5 h, additional 2-bromoethanol (0.21 mL,
3
(1.4 g, 10.2 mmol) and 2-bromoethanol (0.52 mL, 7.3 mmol) were added
1
2
3
1
291.
1