Design and regioselective synthesis of a new generation of targeted chemotherapeutics. Part II: Folic acid conjugates of tubulysins and their hydrazides

Article abstract of DOI:10.1016/j.bmcl.2008.07.041

Efficient syntheses of folate conjugates of tubulysins and their hydrazides 1a-d are described. These water soluble folate receptor (FR) targeted conjugates are derivatives of folic acid and the potent cytotoxic agents: tubulysin A, B, or their respective hydrazides, connected in regioselective manner via a hydrophilic peptide spacer and a reducible disulfide linker.

Full text of DOI:10.1016/j.bmcl.2008.07.041

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4558–4561
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and regioselective synthesis of a new generation of targeted
chemotherapeutics. Part II: Folic acid conjugates of tubulysins and their
hydrazides
*
Iontcho R. Vlahov , Yu Wang, Paul J. Kleindl, Christopher P. Leamon
Endocyte Inc., 3000 Kent Avenue, Suite A1-100, West Lafayette, IN 47906, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Efficient syntheses of folate conjugates of tubulysins and their hydrazides 1a–d are described. These
water soluble folate receptor (FR) targeted conjugates are derivatives of folic acid and the potent cyto-
toxic agents: tubulysin A, B, or their respective hydrazides, connected in regioselective manner via a
hydrophilic peptide spacer and a reducible disulfide linker.
Received 13 June 2008
Revised 8 July 2008
Accepted 10 July 2008
Available online 15 July 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Tubulysin
Targeted therapeutics
Folate
Anti-cancer
Disulfide
The tubulysins are members of a new class of natural products
isolated from a myxobacterial species.1 As cytoskeleton interacting
agents, the tubulysins inhibit tubulin polymerization, leading to
cell cycle arrest and apoptosis.2 Tubulysins are exceptionally
potent cytotoxic molecules, exceeding the cell growth inhibition
observed with epothilones, paclitaxel, and vinblastine by 20- to
1000-fold. Furthermore, they are potent against multidrug
resistant cell lines.3 Nevertheless, in vivo studies suggest that the
natural tubulysins alone are unsuitable for clinical development
because of an extremely tight therapeutic window.4
On the other hand, receptor-targeted chemotherapy has
emerged as one of the major approaches in modern drug discovery
as it can potentially satisfy the selective delivery criteria for toxic
agents to pathologic cells. In a previous publication,5 we reported
the design and synthesis of a folate-targeted desacetylvinblastine
conjugate, EC145, which is currently in phase 2 clinical trials. In
EC145, the anti-cancer drug vinblastine was modified and attached
to folic acid (FA) via a water soluble peptide spacer and a reducible
disulfide linker. Once administered, the conjugate targets folate
receptor (FR) over-expressing cancer cells and releases the base
drug after internalization. This receptor-targeted delivery allows
reduced collateral toxicity and hence improves efficacy.
FA-Spacer unit 2 to tubulysins A and B (3a–b) or their hydrazides
via a self-immolative linker 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 cargo from a disulfide linked FA-conjugate
efficiently occurs within the endosomes of cancer cells.6
The peptide-based spacer 2 was designed to be bifunctional
containing both acidic (Asp) and basic (Arg) amino acids to provide
the best potential for water solubility of the final drug conjugate
under physiological conditions. This unit was assembled using
standard fluorenylmethyloxycarbonyl-based solid phase peptide
synthesis (Fmoc SPPS).5 The structure of 2 was confirmed by 1H
NMR and LC/MS [ESI (M+H)+: 931.2] analysis.
Our initial attempt at the regioselective conjugation of tubuly-
sin involved the use of the phenolic oxygen as a nucleophile and
reacting it with the activated carbonate 5 (Scheme 2). However,
the activated tubulysin carbonate 6 proved to be sensitive to water,
indicating that its folate conjugate counterpart might have limited
stability under physiological conditions, and hence an alternate ap-
proach was pursued. Alternatively, the carboxylic group of the
tubulysin was selected to connect to the folate-spacer unit 2 via
a self-immolative disulfanylethyl ester. A scalable and simple to
In this paper we report the design and regioselective synthesis
of FA–tubulysin conjugates 1a–d (Scheme 1). As indicated in
the retrosynthetic scheme, 1 can be assembled by tethering a
Selected 1H NMR data for 2 (DMSO-d6 with D2O exchange, 300 MHz): d 8.64 (s,
1H), 7.63 (d, J = 8.7 Hz, 2H), 6.65 (d, J = 9.0 Hz, 2H), 4.54–4.48 (m, 4H), 4.25 (m, 2H),
4.03 (t, J = 4.2, 1H), 3.03 (br, 2H), 2.93–2.46 (m, 6H), 2.20 (br, 2H), 1.98–1.80 (m, 3H),
1.61 (m, 1H), 1.50 (m, 2H).
* Corresponding author.
E-mail address: ivlahov@endocyte.com (I.R. Vlahov).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.041

I. R. Vlahov et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4558–4561
4559
Scheme 1.
Scheme 2.
perform one-pot protocol was designed, in which tubulysin 3a or
3b was activated by N-(3-dimethylaminopropyl)-N0-ethylcarbodi-
imide (EDC) with in situ addition of 7,5 followed by treatment with
N,N-diisopropylethylamine (DIPEA) and solution of 2 in DMSO.
Subsequent preparative HPLC purification provided the product
(1 a–b) in ca. 30% overall yield.
Next, another class of folate–tubulysin conjugates 1c–d was
designed. Based on our biological data4,7 and previous success with
a hydrazide-based cytotoxic drug,5 the endosomal release of a
tubulysin hydrazide (9a–b)à was envisioned (Scheme 3). This
approach might provide potentially different interactions between
the base drug and microtubules, and hence lead to a potentially lar-
ger therapeutic window for the hydrazide-based drug in contrast to
an amino acid form of the natural tubulysin drug. The base drugs 9a–
b were prepared directly from the corresponding natural tubulysins
for use in biological control experiments.8 The synthetic yields were
low due to (a) sensitivity of the acyl groups in tubulysin skeleton to
hydrazine and (b) the further reaction of 9 with the activated inter-
mediate 4. To minimize undesired side reactions for the synthesis of
the target conjugates, a heterobifunctional crosslinker 10 was em-
ployed.9a This molecule contains an oxycarbonylhydrazine moiety
with mono-nucleophilic nitrogen atoms and a 2-mercaptopyridyl
leaving group as a specific conjugation site. Compound 10 was pre-
pared in 70–80% yield from 5 or 7 following simple to perform syn-
thetic routes.9 Following our one-pot protocol, the final folate
conjugates 1c–d were synthesized in excellent yields (55–60%) from
tubulysins3a–b.10 Thesynthesisinvolvedactivationofthetubulysin
with isobutyl chloroformate and in situ treatment of the intermedi-
ate at low temperature with 10. The crude reaction mixture was con-
centrated to remove the solvent and re-dissolved in a water miscible
organicsolvent, THF, followedbymixing withan aqueoussolutionof
folate-peptide spacer 2 at neutral pH. HPLC purification gave pure
à
Selected 1H NMR data for 7b (CD2Cl2, 300 MHz): d 8.52 (br, 1H), 7.94 (s, 1H), 7.10
(d, J = 8.7 Hz, 1H), 6.99 (d, J = 8.7 Hz, 4H), 6.77 (d, J = 7.8 Hz, 2H), 5.95 (d, J = 12.0 Hz,
1H), 5.65 (m, 1H), 5.34 (t, J = 12.0 Hz, 1H), 4.51 (t, J = 9.3 Hz, 1H), 4.14 (m, 1H), 3.83
(br, 2H), 3.36 (q, J = 5.7 Hz, 1H), 2.80–2.67 (m, 3H), 2.34–2.04 (m, 13H), 1.91–1.79 (m,
4H), 1.55–1.18 (m, 11H), 1.06 (t, J = 6.9 Hz, 3H), 0.96–0.75 (m, 15H).

4560
I. R. Vlahov et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4558–4561
Scheme 3.
0.100
0.075
0.050
0.025
0.000
0.24
0.18
0.12
0.06
0.00
0.00
0.80
1.60
2.40
3.20
4.00
0.00
0.80
1.60
2.40
3.20
4.00
Minutes
Minutes
Scheme 4. Conjugate 1d in PBS buffer treated with DTT. Left: t = 0 min; right: t = 2 h.
conjugate 1c–d. LC/MS [ESI (M+H)+: 1878 and 1892, respectively]
In brief, the 1H NMR spectrum (300 MHz, D2O) contained ten
aromatic signals in the range from 6.5 to 8.7 ppm (five from the fo-
late moiety and five from the tubulysin moiety). The signals for the
two protons of the intact N,O-acetal appeared at 6.1 ppm and
5.2 ppm.
When 1d was incubated as a 1 mM solution with 10 equivalents
of DTT in PBS buffer at 37 °C, LC/MS studies indicated a full release
of 9b within 2 h (Scheme 4).
and 1H NMR data§ were in agreement with the expected structure.
§
Selected 1H NMR data for 1d (D2O, 300 MHz): d 8.45 (s, 1H), 7.90 (s, 1H), 7.44
(d,J = 8.7 Hz, 2H), 6.74 (d, J = 8.1 Hz, 2H), 6.45 (m, 2H), 5.93 (d, J = 12.0 Hz, 1H), 5.58 (d,
J = 11.4 Hz, 1H), 5.08 (d, J = 12.0 Hz, 1H), 4.53 (m, 2H), 4.39 (m, 2H), 4.29 (br, 2H), 4.14
(m, 5H), 3.89 (br, 1H), 3.64 (d, J = 9.9 Hz, 1H), 3.38 (d, J = 12.0 Hz, 1H), 3.06–2.74 (m,
6H), 2.62–2.22 (m, 14H), 1.99 (s, 3H), 1.95–1.28 (m, 20H), 1.14 (q, J = 7.5 Hz, 2H),
1.01–0.86 (m, 4H), 0.79 (d, J = 6.3 Hz, 3H), 0.71–0.63 (m, 6H), 0.51–0.44 (m, 6H).

I. R. Vlahov et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4558–4561
4561
9. (a) Kaneko, T.; Willner, D.; Monkovic, I.; Knipe, J. O.; Braslawsky, G. R.;
Conjugates 1a–d are being tested against a variety of FR positive
cell lines as well as in animal models. The results will be reported
in an appropriate scientific journal.7
Greenfield, R. S.; Vyas, D. M. Bioconjug. Chem. 1991, 2, 133; (b)
Procedure: DIPEA (0.60 mL) was added to
a suspension of 5 (HCl salt,
91%, 685 mg) in anhydrous DCM (5.0 mL) at 0 °C under argon, stirred for
2 min, and to which was added anhydrous hydrazine (0.10 mL). The
reaction mixture was stirred at 0 °C under argon for 10 min and at
room temperature for an additional 30 min, filtered, and the filtrate was
purified by flash chromatography (silica gel, 2% MeOH in DCM) to
Acknowledgments
provide 10 as
Alternative procedure: diphosgene (8.9
(HCl salt, 30 mg) and DIPEA (49 L) in anhydrous DCM (1 mL) at 0 °C
under argon, stirred for 15 min, and to which was added anhydrous
hydrazine (8.4 L). The reaction mixture was stirred at 0 °C under argon
a
clear thick oil (321 mg), solidified upon standing.
We thank the analytical group at Endocyte Inc., for LC/MS sup-
port and R&D Biopharmaceuticals GmbH for providing the natural
tubulysins. We are also thankful to Drs. G. Höfle, W. Richter, and
A. Dömling for their insightful discussions.
l
L) was added to solution of 7
a
l
l
for 10 min and at room temperature for an additional 30 min, filtered,
and the filtrate was purified by flash chromatography (silica gel, 2%
References and notes
MeOH in DCM) to provide 10 as
a clear thick oil (23 mg), solidified
upon standing.
1. Sasse, F.; Steinmetz, H.; Höfle, G.; Reichenenbach, H. J. Antibiot. 2000, 53, 879.
2. Steinmetz, H.; Glaser, N.; Herdtweek, E.; Sasse, F.; Reichenenbach, H.; Höfle, G.
Angew. Chem., Int. Ed. 2004, 43, 4888.
3. For a review see: Dömling, A.; Richter, W. Mol. Divers. 2005, 9, 141.
4. Internal communications.
5. Vlahov, I. R.; Santhapuram, H. K.; Kleindl, P. J.; Howard, S. J.; Stanford, K. M.;
Leamon, C. P. Bioorg. Med. Chem. Lett. 2006, 16, 5093.
6. Yang, J.; Chen, H.; Cheng, J.-X. ; Vlahov, I. R.; Low, P. S. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 13872.
10. Typical procedure: DIPEA (7.8
added to solution of 3a (18 mg) in anhydrous EtOAc (0.50 mL) at
ꢀ15 °C. After stirring for 35 min at ꢀ15 °C under argon, to the reaction
mixture was added solution of 10 (5.8 mg) in anhydrous EtOAc
lL) and isobutyl chloroformate (3.1 lL) were
a
a
(0.50 mL). The cooling was stopped and the reaction mixture was stirred
under argon for an additional 45 min, concentrated, vacuumed, and the
residue was dissolved in THF (2.0 mL). Meanwhile, 2 (20 mg) was taken in
deionized water (bubbled with argon for 10 min prior to use) and the pH
of the yellow suspension was adjusted to 6.9 by saturated NaHCO3
(bubbled with argon for 10 min prior to use). Additional deionized water
was added to the solution of 2 to make a total volume of 2.0 mL and to
which was added immediately the THF solution containing the activated
tubulysin. The reaction mixture, which became homogeneous quickly, was
stirred under argon for 50 min and quenched with sodium phosphate
buffer (2 mM, pH 7.0, 15 mL). The resulting cloudy solution was filtered
7. Leamon, C. P.; Reddy, J. A.; Vetzel, M.; Dorton, R.; Westrick, E.; Parker, N.; Wang,
Y.; Vlahov, I.; Cancer Res., 2008, submitted for publication.
8. Typical procedure: DIPEA (6.1
added to a solution of 3b (14 mg) in anhydrous EtOAc (150
ꢀ15 °C under argon. After stirring for 45 min at ꢀ15 °C, to the reaction mixture
was added anhydrous hydrazine (5 L) and the reaction mixture was stirred at
l
L) and isobutyl chloroformate (3.0
lL) were
lL) in tandem at
l
ꢀ15 °C for an additional 30 min, quenched with sodium phosphate buffer
(1 mL, 2 mM, pH 7), and purified by a preparative HPLC to afford 9b (3.1 mg) as
a white powder.
and the filtrate was injected into
Fractions containing 1c were collected and freeze-dried to produce the
product (23 mg) as a pale yellow fluffy solid.
a preparative HPLC for purification.