Design and synthesis of releasable folate-drug conjugates using a novel heterobifunctional disulfide-containing linker

Article abstract of DOI:10.1016/j.bmcl.2008.04.063

Cellular uptake of vitamin folic acid occurs via folate-receptor mediated endocytosis. Many types of cancer cells express high levels of folate receptors as they need continuous supply of this vitamin for their proliferation. With an objective to use foli

Full text of DOI:10.1016/j.bmcl.2008.04.063

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3196–3199
Design and synthesis of releasable folate–drug conjugates using
a novel heterobifunctional disulfide-containing linker
Apparao Satyam*
Endocyte Inc., 3000 Kent Avenue, Suite A1-100, West Lafayette, IN 47906, USA
Received 31 March 2008; revised 22 April 2008; accepted 24 April 2008
Available online 29 April 2008
Abstract—Cellular uptake of vitamin folic acid occurs via folate-receptor mediated endocytosis. Many types of cancer cells express
high levels of folate receptors as they need continuous supply of this vitamin for their proliferation. With an objective to use folic
acid as a ‘Trojan Horse’ to transport anticancer drugs into cancer cells, a novel heterobifunctional disulfide-containing linker was
synthesized and utilized to covalently link an amino- and hydroxyl-containing anticancer drug, and an appropriately functionalized
folic acid to create novel targetable folate–drug conjugates that are shown to release free drugs under biologically relevant pH via
sulfhydryl-assisted cleavage of the self-immolative disulfide-containing linker.
Ó 2008 Elsevier Ltd. All rights reserved.
Tumor cells use unique defensive pathways for their sur-
vival and proliferation. This very intrinsic property of
cancer cells offers great opportunity for identification
of molecular targets or pathways that can be utilized
to achieve targeted delivery of anticancer drugs selec-
tively into cancer cells. Some of the unique characteris-
tics of cancer cells that have been exploited include:
(1) Highly proliferating cancer cells need continuous
supply of vitamins and tend to express high levels of
vitamin receptors on their cell surfaces. For instance,
vitamin folic acid is transported into cells via receptor-
mediated endocytosis.1,2 Because of that reason, folic
acid has been successfully exploited as a ‘Trojan horse’
to transport anticancer drugs selectively into cancer
cells;3 (2) Cancer cells express high levels of detoxifying
enzymes such as glutathione-S-transferases (GSTs)4 and
glutathione (GSH) to protect themselves from toxic
xenobiotics including therapeutic agents. In our earlier
work, we have successfully exploited this intrinsic prop-
erty of cancer cells to our advantage by designing iso-
zyme selective GST inhibitors5 and also GST-activated
latent alkylating agents6,7 that are currently in clinical
trials; and (3) Higher levels of glutathione present in
cancer cells make them resistant to chemotherapeutic
agents and this particular condition of cancer cells has
been exploited by designing novel molecules that are
activated selectively by glutathione to release lethal
amounts of nitric oxide (NO) intracellularly thereby
selectively killing those cancer cells.8
To exploit some of the above-mentioned inherent prop-
erties of cancer cells to our advantage and also to
achieve targeted delivery of effective amounts of chemo-
therapeutic agents selectively into cancer cells, we have
designed and synthesized novel folate–drug conjugates
of the general structure 1 (Scheme 1).
The folic acid moiety in the conjugates 1 is expected to not
only act as a necessary vitamin for the proliferating cells
path a
O
S
Drug
Folate-(AA)n-Cys-S
RS-
O
Y
path b
Folate-Drug Conjugate (1)
RSH pH 7.4 (biological pH)
O
b
S
a
O
S
CO
+
2
Folate-(AA)n-Cys-S-SR+ Drug
YH
Released Drug
Keywords: Heterobifunctional linker; Disulfide-containing linker;
Folate–drug conjugates; Magic carbonate.
Wherein, Y = O or NR (R = H or a bond);
AA = an amino acid; n = 0-8;
RSH = Dithiothreitol
*
Present address: Piramal Life Sciences Limited, Nirlon Complex,
Goregaon E, Mumbai 400 063, India. Tel.: +91 22 4003 2903; fax:
+91 22 3081 8036; e-mail: apparaosvgk@hotmail.com
Scheme 1. Proposed mechanism of drug release.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.063

A. Satyam / Bioorg. Med. Chem. Lett. 18 (2008) 3196–3199
3197
but also serve as a targeting ligand to transport the ap-
pended drug selectively in to cancer cells via receptor-
mediated endocytosis. The spacer portion consists of a
bond or a peptide made of 0–8 suitable amino acid resi-
dues and a free sulfhydryl-containing terminal residue
such as cysteine and the spacer is expected to impart desir-
able physicochemical properties such as affinity and
water-solubility, to the resulting conjugate 1. The fo-
late–drug conjugates 1 also contain a novel self-immola-
tive and drug releasable disulfide-containing linker9,10 as
an integral part of their structure. The drug portion of
the conjugate 1 clearly illustrates the broad scope of this
linker technology where any (primary or secondary) ami-
no- or (primary, secondary, tertiary, or phenolic) hydro-
xyl-containing drug can be covalently coupled to the
linker to generate the corresponding releasable folate–
drug conjugates of the general structure 1. Thus, these fo-
late–drug conjugates 1 are expected to be selectively inter-
nalized by the cancer cells having high levels of folate
receptors and release lethal amounts of free drug intracel-
lularly by the action of sulfhydryl-containing species such
as GSH on the disulfide-containing folate–drug conjugate
via a probable mechanism as proposed in Scheme 1.
Thus, we have successfully utilized the novel heterobi-
functional linker 3 to covalently link an (both primary
and secondary) amino-, a (primary, secondary, or phe-
nolic) hydroxyl-containing drug to one side of the linker
to generate drug-linker intermediate 4 (Scheme 3).
Because of its facile reaction even with secondary
alcohols and phenols,9 we often called this heterobi-
functional linker 3 as ‘Magic Carbonate’! Thus, as
shown in Scheme 3 and Figure 1, the drug-linker
intermediates 4 were made by reacting the heterobi-
functional linker 3 with an amino-containing or hy-
droxyl-containing drugs. Further reaction of the
intermediates 4 with an appropriately functionalized
Folate-Cys-OH (5) containing a terminal sulfhydryl
group (most often a cysteine residue) gave the fo-
late–drug conjugates 1.
This novel linker technology was demonstrated by mak-
ing a few representative examples. Thus, the reaction of
an amino-containing drug such as daunomycin gave the
corresponding drug-linker intermediate 4a (Fig. 1),
which upon reaction with Folate-Cys-OH (5) yielded
the desired folate–daunomycin conjugate 1a (Fig. 2).
It was a challenging objective for us to develop linkers
that would release free drugs intracellularly once they
get internalized by the cancer cells. After considerable
experimentation, we have successfully met this objective
by rationally designing, synthesizing, and utilizing a no-
vel heterobifunctional linker 3 and those preliminary re-
sults are reported here. As shown in the Scheme 2, we
have synthesized the linker 3 by reacting 2-(2-hydrox-
yethyldithio)-pyridine (2)11 with 1,1-bis [6-(trifluoro-
methyl)benzotriazolyl]-carbonate (BTBC).12 Obviously,
utilization of BTBC in the synthesis of 3 eliminates the
use of highly toxic phosgene.
As shown in Scheme 4, synthesis of the paclitaxel-linker
intermediate 4b was performed via transient protection
and deprotection of 20-hydroxyl group of paclitaxel.
Thus, the more reactive 20-hydroxyl group of paclitaxel
was selectively protected first with the allyloxycarbonyl
(Alloc) group to give (2-O-Alloc)paclitaxel 6, which
was then treated with the linker 3 to yield the fully pro-
tected paclitaxel-linker derivative 7. Finally, palladium-
catalyzed removal of Alloc protecting group from the
intermediate 7 yielded the desired paclitaxel-linker inter-
mediate 4b.
a
b
S
. HCl
OH
S Cl
SH
N
S
N
N
2 (HCl salt)
A1
A2
c
DMAP. HCl
N
N
N
O
d
S
S
S
OH
N
N
S
O
O
CF3
(free base)
2
3
Scheme 2. Reagents and conditions: (a) SO2Cl2, DCM, 0–5 °C to rt, quant.; (b) 2-mercaptoethanol (A3), DCM, 0–5 °C to rt, 79.4%; (c)
dimethylaminopyridine (DMAP), DCM, chromatography on silica gel, 90–95%; (d) 1,1-bis [6-(trifluoromethyl)benzotriazolyl]-carbonate (BTBC),
acetonitrile, rt, 81%.
N
O
N
N
Drug
S
+
N
HY
S
O
O
3
CF3
a
O
O
S
Drug
+
b
S
Drug
Folate-Cys-S
O
N
Y
S
N
O
X
S
H
Y = O or NR (R = H or a bond)
4
1
Scheme 3. Reagents and conditions: (a) triethylamine/DMAP, acetonitrile, 0 °C to rt, 51–81%; (b) Folate-Cys-OH (5), acetonitrile, 0.5–1.0 N sodium
bicarbonate, water, rt, 30–50%.

3198
A. Satyam / Bioorg. Med. Chem. Lett. 18 (2008) 3196–3199
OH
OH
Similarly, direct reaction of the linker 3 with paclitaxel
O
O
OH
Me
selectively gave the drug-linker intermediate 4c (Fig. 1)
as the major product. The minor product from the reac-
tion was identified as the disubstituted paclitaxel deriva-
tive where both the 20-hydroxyl and 7-hydroxyl groups
of paclitaxel were derivatized with the linkers.
O
OMe
O
O
Me
HO
HN
O
S
N
S
Further reaction of the intermediate 4b with Folate-Cys-
OH (5) gave the expected folate–paclitaxel conjugates 1b
(Fig. 2). The Folate-Cys-OH (5) was made by using the
standard manual fluorenylmethyloxycarbonyl-based so-
lid-phase peptide synthesis (FMOC-based SPPS) on
Wang resin. We have synthesized and evaluated the fo-
late–Cys(CO2Me)–paclitaxel conjugate 1c by an alterna-
tive method starting from the paclitaxel-linker
intermediate 4c and Folic acid-OSu (9)13,14 as shown in
Scheme 5.
O
4a
O
S
N
S
O
O
O
AcO
Me
Me
Me
Me
O
O
H
O
Me
O
H
N
Ph
OH
O
H
OH
OBz
O
4b
O
As a ‘Proof of the Principle’, all the three folate–drug con-
jugates 1a–c released their respective free drugs upon
treatment with sulfhydryl-containing compounds such
as dithiothreitol (DTT) or dithioerythritol (DTE) or glu-
tathione (GSH) at biologically relevant pH of ꢀ7.4. This
drug release study was monitored by reverse-phase
HPLC.15
AcO
OH
Me
Me
Me
Me
O
O
H
O
H
N
O
OH
Me
O
H
OBz
O
O
O
O
S
N
S
4c
Interestingly, a few promising preclinical16,17 and clini-
cal18,19 candidates have already been identified by the
Figure 1. Structures of intermediates 4a–c.
OH
O
Folate-Cys(CO2H)
OH
O
O
S
S
Me
O
O
AcO
Me
O
O
OH
Me
Me
OMe
O
O
Me
O
O
Me
O
H
O
H
O
N
HO
HN
OH
Me
O
S
H
OH
S
OBz
Cys(CO2H)-Folate
O
O
1b
1a
O
AcO
OH
Me
CO2H
O
H
Me
Me
Me
N
O
N
N
S
CO2H/CO2Me
O
O
H
N
H
HN
H2N
O
N
O
OAc
H
H
N
O
N
OH
H
OBz
Cys(CO2Me)-Folate
Folate-Cys(CO2H/CO2Me)-S-
O
O
S
S
O
1c
Figure 2. Structures of folate–drug conjugates 1a–c.
a
Paclitaxel
Paclitaxel (2'-O-Alloc)
6
b
O
S
N
S
O
O
O
H
AcO
Me
Me
Me
c
Me
4b
O
O
O
O
H
N
H
Me
OH
O
O
O
O
O
O
O
7
Scheme 4. Reagents and conditions: (a) allyloxycarbonyl chloride, diisopropylethyl amine, DCM, 0–5 °C, 98%; (b) 3, DMAP, acetonitrile, reflux,
58%; (c) diethylamine, Pd (PPh3)4, THF, rt, 64%.

A. Satyam / Bioorg. Med. Chem. Lett. 18 (2008) 3196–3199
3199
O
AcO
OH
Me
Me
Me
Me
O
O
O
H
O
a
H
c
N
4c
1c
OH
Me
O
H
O
O
OBz
O
O
S
NH2
CO2Me
S
8
Scheme 5. Reagents and conditions: (a) H-Cys-OMe.HCl, 1 N NaHCO3, acetonitrile, water, pH 7.5–8.0, rt, 59%; (b) folic acid-OSu (9),13,14
N-methylmorpholine, DMSO, rt, 14.8%.
2. Rothberg, K. G.; Ying, Y.; Kolhouse, J. F.; Kamen, B. A.;
Anderson, R. G. J. Cell. Biol. 1990, 110, 637.
3. For a recent review see: Leamon, C. P.; Reddy, J. A. Adv.
Drug Delivery Rev. 2004, 56, 1127.
4. Howie, A. F.; Farrester, L. M.; Glancey, M. J.; Schlager,
J. J.; Powis, G. G.; Beckett, G. J.; Hayes, J. D.; Wolf, C.
R. Carcinogenesis 1990, 11, 451.
5. Lyttle, M. H.; Hocker, M. D.; Hui, H. C.; Caldwell, C. G.;
Aaron, D. T.; Goldstein, A. E.; Flatgard, J. E.; Bauer, K.
E. J. Med. Chem. 1994, 37, 189.
6. Satyam, A.; Hocker, M. D.; Kane-Maguire, K. A.;
Morgan, A. S.; Villar, H. O.; Lyttle, M. H. J. Med. Chem.
1996, 39, 1736.
application of this novel disulfide-carbonate linker tech-
nology, which can be fine tuned10 further for improving
stability of the conjugates as well as achieving con-
trolled/sustained release of free drugs from the conju-
gates. We also made a Folate-seo-CBI analog (a
phenolic hydroxyl-containing seo-CBI20 as an example),
a folate–Normustard conjugate, and a folate–NBD con-
jugate (as a model compound), and the data associated
with these folate–drug conjugates9 will be reported in
due course.
In summary, we have designed and synthesized a novel
heterobifunctional disulfide-containing linker 3 which
could be conveniently reacted with representative ami-
no-containing and hydroxyl (including phenolic hydro-
xyl)-containing therapeutic agents under mild
conditions to generate the corresponding drug-linker
intermediates 4, which in turn could be successfully con-
jugated to sulfhydryl-containing folate derivatives 5 to
give the corresponding folate–drug conjugates 1. Thus,
the heterobifunctional disulfide linker 3 has been proved
to be a versatile reagent with a broad scope of applica-
bility to generate targetable as well as releasable fo-
late–drug conjugates that release free drugs at
biologically relevant pH via sulfhydryl-assisted cleavage
of self-immolative disulfide-containing linker.
7. Lyttle, M. H.; Satyam, A.; Hocker, M. D.; Bauer, K. E.;
Caldwell, C. G.; Hui, H. C.; Morgan, A. S.; Mergia, A.;
Kauvar, L. M. J. Med. Chem. 1994, 37, 1501.
8. Saavedra, J. E.; Srinivasan, A.; Buzard, G. S.; Davies,
K. M.; Waterhouse, D. J.; Inami, K.; Wilde, T. C.;
Citro, M. L.; Cuellar, M.; Deschamps, J. R.; Parrish,
D.; Shami, P. J.; Findlay, V. J.; Townsend, D. M.; Tew,
K. D.; Singh, S.; Jia, L.; Ji, X.; Keefer, L. K. J. Med.
Chem. 2006, 49, 1157.
9. Vlahov, I. R.; Leamon, C. P.; Parker, M. A.; Howard, S. J.;
Santhapuram, H. K.; Satyam, A.; Reddy, J. A. US Patent
Application No. 2005/0002942 A1 (10/765,336), 2005.
10. Jones, L. R.; Goun, E. A.; Shinde, R.; Rothbard, J. B.;
Contag, C. H.; Wender, P. A. J. Am. Chem. Soc. 2006, 128,
6526.
11. Kaneko, T.; Willner, D.; Monkovic, I.; Knipe, J. O.;
Braslawsky, G. R.; Greenfield, R. S.; Vyas, D. M.
Bioconjugate Chem. 1991, 2, 133, Also, see supplementary
material for alternative methods of synthesis of this
compound as shown in Scheme 2.
Acknowledgments
12. Takeda, K.; Tsuboyama, K.; Hoshino, M.; Kishino, M.;
Ogura, H. Synthesis 1987, 557.
13. Leamon, C. P.; Low, P. S. S. J. Biol. Chem. 1993, 268,
24847.
14. Santos, M. A.; Enyedy, E. A.; Nuti, E.; Rossello, A.;
Krupenko, N. I.; Krupenko, S. A. Bioorg. Med. Chem.
2007, 15, 1266.
15. Refer to supporting data for details on drug release studies.
16. Henne, W. A.; Doorneweerd, D. D.; Hilgenbrink, A. R.;
Kularatne, S. A.; Low, P. S. Bioorg. Med. Chem. Lett.
2006, 16, 5350.
We are grateful to Endocyte management (Professor
Phil Low, Dr. Chris Leamon and Mr. Ron Ellis) and sci-
entific staff (especially Jennifer and Chakri Abburi for
their technical help) for their support. We also thank
Machhindra Gund, Dattatraya Desai and Ms. Mini
Dhiman for their technical help in the preparation of
this manuscript.
Supplementary data
17. Vlahov, I. R.; Santhapuram, H. K.; Kleindl Wang, Y.;
Kleindl, P. J.; You, F.; Howard, S. J.; Westrick, J. A.;
Reddy, J. A.; Leamon, C. P. J. Org. Chem. 2007, 72, 5968.
18. 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.
Experimental procedures, analytical, and spectral data
can be found, in the online version, at doi:10.1016/
j.bmcl.2008.04.063.
19. Leamon, C. P.; Reddy, J. A.; Vlahov, I. R.; Westrick, E.;
Dawson, A.; Dorton, R.; Vetzel, M.; Santhapuram, H. K.;
Wang, Y. Mol. Pharmaceutics 2007, 4, 659.
References and notes
1. Kamen, B. A.; Capdevila, A. Proc. Natl. Acad. Sci. U.S.A.
1986, 83, 5983.
20. Boger, D. L.; Ishizaki, T.; Kitos, P. A.; Suntornwat, O.
J. Org. Chem. 1990, 55, 5823.