Striking a Balance between Carbonate/Carbamate Linkage Bond- and Reduction-Sensitive Disulfide Bond-Bearing Linker for Tailored Controlled Release: In Situ Covalent-Albumin-Binding Gemcitabine Prodrugs Promote Bioavailability and Tumor Accumulation

Article abstract of DOI:10.1021/acs.jmedchem.8b00293

To address the challenges of rapid enzyme inactivation, poor tumor targeting, and acquired drug resistance in gemcitabine (GEM) application, we report two groups of maleimide-functionalized GEM prodrugs conjugating covalently in situ with Cys-34 of blood-circulating albumin and then resulting in macromolecular prodrugs after intravenous administration. Tailored and accurate controlled release was achieved through different combinations of linkage bonds, relatively stable and labile (carbamate and carbonate, respectively), and linkers with or without insertion of a disulfide bond. Interestingly, we found that the overall advantages or disadvantages brought by a disulfide bond varied with the stability of the linkage bond. Finally, the carbonate linkage bond-bearing group, especially the one with a linker lacking a disulfide bond, stood out with remarkably increased bioavailability (21-fold greater than GEM) and efficient tumor free-GEM accumulation (8-fold of GEM), which consequently contributed to excellent in vivo antitumor efficacy.

Full text of DOI:10.1021/acs.jmedchem.8b00293

Subscriber access provided by Kaohsiung Medical University
Article
Striking a Balance between Carbonate/Carbamate Linkage Bond
and Reduction-Sensitive Disulfide Bond-bearing Linker for Tailored
Controlled Release: In Situ Covalently Albumin-Binding Gemcitabine
Prodrugs Promote Bioavailability and Tumor Accumulation
Huicong Zhang, Kuanglei Wang, Kexin Na, Dan Li, Zhenbao Li, Dongyang Zhao, Lu Zhong, Menglin
Wang, Longfa Kou, Cong Luo, Haotian Zhang, Qiming Kan, Huaiwei Ding, Zhonggui He, and Jin Sun
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00293 • Publication Date (Web): 16 May 2018
Downloaded from http://pubs.acs.org on May 16, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Original Research Article
Title:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Striking a Balance between Carbonate/Carbamate Linkage Bond and
Reduction-Sensitive Disulfide Bond-bearing Linker for Tailored
Controlled Release: In Situ Covalently Albumin-Binding Gemcitabine
Prodrugs Promote Bioavailability and Tumor Accumulation
1
2,3
1
1
1
Authors: Huicong Zhang , Kuanglei Wang , Kexin Na , Dan Li , Zhenbao Li ,
1
1
1
1
1
4
Dongyang Zhao , Lu Zhong , Menglin Wang , Longfa Kou , Cong Luo , Haotian Zhang ,
4
5
1,*
1,*
Qiming Kan , Huaiwei Ding , Zhonggui He , Jin Sun
Affiliations:
1Department of Pharmaceutics, Wuya College of Innovation, Shenyang Pharmaceutical
University, Shenyang, Liaoning, 110016, P. R. China
2
Wuyi University, Jiangmen, Guangdong, 529020, P. R. China
3International Healthcare Innovation Institute (Jiangmen), Jiangmen, Guangdong,
5
29080, P. R. China
4School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University,
Shenyang, Liaoning, 110016, P. R. China
5School of Pharmaceutical and Engineering, Shenyang Pharmaceutical University,
Shenyang, Liaoning, 110016, P. R. China
ACS Paragon Plus Environment

Page 3 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
ABSTRACT
To address the challenge of rapid enzyme inactivation, poor tumor targeting, acquired
drug resistance in gemcitabine (GEM) application, we report two groups of maleimide-
functionalised GEM prodrugs conjugating covalently in situ with cys-34 of blood-
circulating albumin, resulting in macromolecular prodrugs after intravenous
administration. Specially, tailored and accurate controlled release was achieved through
different combinations of linkage bonds relatively stable and labile (carbamate and
carbonate, respectively) and linkers with or without insertion of a disulfide bond.
Interestingly, we found that the overall advantages or disadvantages brought by a disulfide
bond varied with the stability of the linkage bond. Finally, the carbonate linkage bond-
bearing group especially the one with a linker absent of a disulfide bond standed out
whereby remarkably increased bioavailability (21-fold greater than GEM) and efficient
tumor free-GEM accumulation (8-fold of GEM), which consequently contributed to
excellent in vivo antitumor efficacy.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Key words: gemcitabine, albumin, prodrugs, carbonate/carbamate linkage bonds,
disulfide bond, accurate controlled release
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
INTRODUCTION
GEM (2′, 2′-difluorodeoxycytidine) is a nucleoside analog widely used in chemotherapy
as a first-line treatment of various malignancies such as pancreatic, breast and lung cancer1-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.
limitations such as rapid enzyme deamination to inactive 2′-deoxy-2′, 2′-difluorouridine
However, the clinical efficacy of GEM is dramatically compromised by its intrinsic
3
-4
(
dFdU) by cytidine deaminase (CDA) in blood circulation , poor tumor targeting,
acquired drug resistance mediated by decreased expression of nucleoside transporters
5
-6
especially the human equilibrative nucleoside transporter (hENT) . Prodrug strategy and
7
8
3
nanotechnology, exemplified by PEG-gemcitabine , LY2334737 , NUC-1031 , and
9
10
11
liposomes , polymeric nanoparticles (NPs) (Albumin and Chitosan), inorganic NPs
respectively, are two main technologies applied in GEM improvements. However, despite
benefits in pharmacokinetics or/and pharmacodynamics in the lab, the clinical outcome of
conventional prodrugs and nanoparticulate delivery systems (NDS) remained
unsatisfactory possibly due to limitations such as poor tumor targeting for prodrugs or
1
2
marginal drug payloads reaching the tumor for NDS . Consequently, there is an urgent
need for the application of more promising strategies for better treatment outcomes of
GEM.
Macromolecular prodrug strategy may serve as a robust alternative. The corresponding
strategy applied in our study is based on the click chemistry whereby covalent addition
between maleimide group and sulfydryl from cys-34 of endogenous albumin13-14. Human
serum albumin (HSA) is an ideal drug carrier for chemotherapeutics because of its
extraordinarily long circulatory time of about 19 days15-17 in the bloodstream and favorable
tumor targeting capacity mediated by enhanced permeability and retention (EPR) effect18-
ACS Paragon Plus Environment

Page 5 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
20
and receptors such as albondin and SPARC (secreted protein, acidic and rich in
cysteine)21-22. Up till now, one of the most promising modification sites of albumin is the -
SH group from cys-34. In blood plasma, cys-34 of HSA accounts for the major amount
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(80-90%) of the total thiol concentration (400-500 µM) and stays as the most reactive thiol
1
3
group in physiological condition because of the low pKa being approximately 7 .
The macromolecular prodrug strategy of in vivo conjugating with HSA would have
several advantages over an alternative strategy of in vitro synthesized albumin-drug
conjugates: (a) avoiding substantial use of commercial and possibly pathogenic albumin;
(b) simpler quality control than that of their albumin conjugate counterparts, just
comparable to any other low-molecular drug candidates; and (c) inexpensive for
manufacture and convenient to use and preserve23.
Besides the promising macromolecular prodrug strategy, special and intensive efforts
need to be paid on another pivotal element directly affecting the final therapeutic efficacy,
that is, controlled drug release. The linkage bond between the drug and the linker is no
doubt the last barrier prohibiting drug release, and its stability decides the drug release rate
to a great extent. GEM, containing both hydroxy and amino functional groups, confers
itself possibilities to construct different linkage bonds from stable to labile, such as
carbamate, amide, carbonate and ester. Among mentioned linkage bonds, carbamate and
carbonate linkage bonds are often used to obtain controlled release whereby their
respective stable and labile nature, and capacity to fine tune release rates for more accurate
controlled release in the joint efforts with a certain linker, such as a disulfide bond-
containing one24-25. Nowadays, many stimulus-responsive strategies, responding to various
2
6
27
28
internal and external stimuli, such as redox status, pH , enzyme activity , light , and
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
29
temperature , have been designed to obtain controlled release. Disulfide bond strategy is
one of the most commonly used yet very effective technology22, 27, 30, exploiting the internal
stimuli of a high redox potential difference (1-10 mM GSH in the cytoplasm and 1-10 μM
in the plasma) between the reduced intracellular space and the oxidized extracellular
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
space . Notably, it is always being neglected chemically that a linker inserted with a
disulfide bond would bring about unfavorable instability for a prodrug in blood circulation
at the same time, thus negatively affecting pharmacokinetics. Therefore, elaborate selection
of linkage bonds and linkers based on the detailed structure of a certain drug would render
tailored and accurate controlled release.
Herein, we designed and synthesized two groups of maleimide-functionalised GEM
prodrugs, namely, (a) carbonate formation at the 5′-OH group of the sugar moiety and (b)
carbamate masking the 4-NH group of the cytosine base. In each group, GEM was bound
2
through an all-carbon or self-immolative disulfide bond-containing linker to maleimide
which was termed as a warhead here. Notably, the X-ray structure of the defatted albumin
structure (PDB ID: 1AO6) revealed that cysteine-34 was located in a hydrophobic crevice
on the surface of the protein being approximately 10-12 Å deep. Then Kratz reported that
23
the optimal length of the polymethylene spacer consisted of five carbon atoms . So we
combined above conclusions and disulfide bond chemistry when choosing the length of the
linker. We aimed at screening out the best combination of a linkage bond/linker in
balancing stability in circulation and proper GEM release rate at the tumor site, thus
achieving optimal therapeutic effect (Scheme 1 and Figure 1). Next, the prodrugs’
albumin-binding ability, stability against CDA, dependence on hENT, tumor free-GEM
accumulation, and anti-tumor effect both in vitro and in vivo were explored.
ACS Paragon Plus Environment

Page 7 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Gem release mechanism of four albumin-prodrug conjugates after intravenous
administration of the four prodrugs and then their rapid conjugation of circulating albumin.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Schematic diagram to describe the whole process of two carbonate prodrugs 8a and 8b
covalently targeting albumin in situ, accumulating at the tumor target and then releasing pharmacoactive
parent drug after administrated intravenously.
RESULTS AND DISCUSSIONS
Synthesis of four Prodrugs
Maleimide (4) (Scheme 2) was reacted with methyl chloroformate to furnish compound
5
. 5 was then treated with 5-amino-1-pentanol or compound 3 which compound 2 was
treated with cystamine hydrochloride to yield compound 6a or 6b. 6a or 6b was then
transformed to 7a or 7b containing one chloroformate fragment in the presence of
triphosgene and pyridine.
ACS Paragon Plus Environment

Page 9 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
25
According to reported methods with some modifications , GEM was reacted with
compound 7a or 7b in the presence of DBU to afford target compound 8a or 8b.
The two hydroxyl groups of GEM were masked by TBS groups to furnish compound 9.
Then the amino of 9 was then transformed to isocyanato in the presence of triphosgene and
then reacted with 5-amino-1-pentanol or compound 3 to furnish compound 10a or 10b.
Finally, tetrabutylammonium fluoride (TBAF) unveiled the protective groups of 10a or
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0b to give target compound 11a or 11b.
The identities of all target compounds (8a, 8b, 11a and 11b) were confirmed by H NMR
Supporting Information, SI) and HRMS.
1
(
Scheme 2. Synthesis of GEM Prodrugsa.
aReagents and Reactants: (a) cystamine hydrochloride, TEA, methanol. (b) methyl chloroformate,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
TEA, dichloromethane. (c) appropriate amine substituted alcohol, sat. NaHCO . (d) triphosgene, py,
3
chloroform. (e) 7a or 7b, DBU, DMF. (f) TBSCl, imidazole, dichloromethane. (g) triphosgene, toluene.
(h) appropriate amine substituted alcohol, toluene. (i) TBAF, THF.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Albumin-Binding Test and Potential Immunogenicity
An in vitro binding test was employed to investigate the binding ability of four prodrugs
with bovine serum albumin (BSA). The binding ability was observed by the decrease in
the prodrug peak intensity. Figure 2A displays the chromatographic curves of 8b, BSA-8b
and BSAat incubation time-points of 1 and 30 min at 37°C. The major amount (about 90%)
of 8b conjugated with BSA within the first 1 min and the coupling proceeded almost
completely after 30 min incubation. In contrast, marginal 8b bound with BSA whose cys-
3
4 was blocked by preincubation with excess 6-maleimidocaproic acid (EMC), which
confirmed the reaction of 8b with BSA was specific for the -SH group of cys-34. Similar
trends were observed for the other three prodrugs.
Another binding test was performed by in vitro incubation with fresh rat blood to
simulate the in vivo interaction of albumin and prodrugs after intravenous injection. As
shown in Figure 2B, 8b disappeared almost completely within 1 min incubation,
demonstrating a quick albumin-binding ability in the blood. However, when the blood was
preincubated with excessive EMC and no free cys-34 of albumin was left, a large amount
of residual 8b (95%) could be detected after 1 min incubation.
Noticeably, circular dichroism (CD) spectra of BSA and BSA-prodrug conjugates (CD
spectra of BSA and BSA-8a were shown in Figure 5a as representatives) showed highly
similar trends, which suggested that the conjugation of a small molecular prodrug induced
no secondary structure changes to BSA. What’ more, in the chromatogram shown in
ACS Paragon Plus Environment

Page 11 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 2a, the BSA and BSA-8b kept the same retention time, implying marginal
conformational differences between the two kind of biomacromolecules. Similar results
were obtained from the other BSA-prodrug conjugates. Then it was unlikely that BSA-
prodrugs would expose new antigenic determinant when compared with free albumin and
would consequently induce immunogenicity when inside the body.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. The binding abilities of prodrugs with serum albumin. (A) Chromatograms of incubation
study of 8b with BSA or BSA blocked by excessive EMC at 37°C. (B) Incubation study of 8b with fresh
rat blood or blood preincubated with excessive EMC at 37°C (n=3).
Reduction-Sensitive Properties of Prodrugs bound to Cys-34 of BSA
After entering into the body, four prodrugs conjugated with albumin within minutes and
then existed mainly in the form of macromolecular prodrugs which determined all most
the last whole drug delivery process. So we prepared corresponding macromolecular
prodrugs (BSA-8a, BSA-8b, BSA-11a and BSA-11b) in advance by incubating four
prodrugs with BSA to simulate the main existing state of our prodrugs in vivo. The
structures of four BSA-prodrug conjugates were confirmed by HRMS shown in SI.
Dithiothreitol (DTT), a prevailing GSH simulatant, was utilized to investigate the
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
reductive responsiveness of BSA-8b and BSA-11b. As shown in Figure 3, about 33% and
7
% of GEM was released from BSA-8b and BSA-11b within 8 h in PBS (pH 7.4) with 1
μM DTT at 37°C respectively, but nearly 100% and 80% of GEM was released within the
first 2 h in the presence of 1 mM DTT respectively. By contrast, there was marginal GEM
released from BSA-8a and BSA-11a after 8 h incubation in PBS (pH 7.4) either with 1 μM
or 1 mM DTT.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. In vitro reduction-responsive drug release. Release of GEM from BSA-8a, BSA-8b, BSA-
11a and BSA-11b in PBS with 1 mM or 1 μM DTT (n=3).
Obviously, incorporation of a disulfide bond led to significantly accelerated drug release
upon reduction stimuli inside the cell and the corresponding prodrugs would excert
stronger cytotoxicity than their non-reduction-sensitive counterparts. The possible drug
release mechanism of 8b inside the cell was showed in Figure 1, and 11b was in a similar
way. Under the action of high concentration of GSH inside the cell, the disulfide bond in
the linker of BSA-8b was vulnerable enough to cleave. The resulting thiol underwent an
intramolecular nucleophilic substitution at the carbonate moiety, forming a five-membered
ring thiolactone and releasing GEM molecules in a pharmaceutically active form.
Meanwhile, a disulfide bond introduction would bring about more unfavorable
ACS Paragon Plus Environment

Page 13 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
extracellular instability to prodrugs and that was the reason why BSA-8b and BSA-11b
released 2 and 6 times more free GEM than their non-reduction-sensitive counterparts
respectively after incubation for 8 h in 1 μM DTT. When circulating in the bloodstream,
addition of a disulfide bond might be disastrous for a prodrug already with a relatively
labile linkage bond, in that excessive premature drug release would fail in fully exploiting
the carrier and lead to more off-target effect. Overall advantages or disadvantages upon
disulfide bond incorporation needed further experiments.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Stability of BSA-Prodrug Conjugates against Metabolism by CDA in Rat Plasma
To test the stability of BSA-prodrug conjugates against metabolism by CDA, the
-1
conjugates and GEM solution at an equivalent GEM dose of 16 μg·mL were incubated
with fresh rat plasma at 37°C for 12 h. Free GEM and dFdU were detected at the same
time. GEM in the conjugated form could be kept away from CDA by virtue of BSA’s steric
hindrance, that is, the less free GEM was released from the conjugates, the more GEM
would circumvent enzyme deamination into inactive dFdU. As shown in Figure 4A and
Figure 4B, among the four BSA-prodrug conjugates, BSA-8b released the most free GEM
4
-1
-1
(
AUC: 6.7 × 10 ng·mL ·h), correspondingly, it generated the most (AUC: 695 ng·mL ·h)
-1
yet 3 times less dFdU than that of the GEM solution group (AUC: 3038 ng·mL ·h). For
the other three BSA-prodrug conjugates with AUC of dFdU being no more than 380
-1
ng·mL ·h, stronger capacity against CDA inactivation was manifested due to more stable
linkage bond/linker. Above results suggested a successful strategy for GEM to resist
degradation by CDA, implying that longer blood circulation time could be achieved.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Metabolism stability of GEM and four BSA-prodrug conjugates against CDA in fresh rat
blood. (A) Concentration of free GEM at different time points. (B) Concentration of dFdU at different
time points, ***P < 0.001 (n=3).
Cellular Uptake and Endocytosis Pathways of BSA-Prodrug Conjugates
To investigate the cellular uptake, we synthesized Cy5-labelled BSA and BSA-prodrug
conjugates. Cy5-BSA and four Cy5-BSA-prodrug conjugates exhibited similar CD spectra
to that of BSA, indicating that the synthesis process had no significant influence on the
secondary structure of BSA (representatives of BSA, BSA-8a, Cy5-BSA, and Cy5-BSA-
8
a were shown in Figure 5A). 4T1 tumor cells were incubated with Cy5-labelled BSA and
four BSA-prodrug conjugates for different time and temperature. After incubation for 0.5
h at 37°C, 2 h at 37°C or 2 h at 4°C, the intracellular fluorescence intensity was quantified.
As shown in Figure 5B, cellular uptake of BSA and four BSA-prodrug conjugates
depended on both time and temperature and demonstrated no significant difference with
each other. With as low as 1/130 of BSA molecular weight, prodrugs seemed to have
negligible affection on their corresponding albumin-binding macromolecular prodrugs
compared to prodrug-free albumin upon cellular uptake performance.
We next investigated the endocytosis pathways of BSA-prodrug conjugates. BSA-8a
ACS Paragon Plus Environment

Page 15 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
was taken as an example. As shown in Figure 5B, the cellular uptake of BSA-8a was
inhibited in the presence of chlorpromazine (clathrin-mediated endocytosis inhibitor),
indomethacin (caveolae-mediated endocytosis inhibitor), quercetin (inhibitor of caveolae-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
/
clathrin-independent endocytosis), and sodium azide (energy inhibitor) (Figure 5C). As a
result, it could be deduced that the internalization of BSA-8a in 4T1 cells might be energy-
dependent endocytosis processes, involving clathrin-mediated endocytosis, caveolae-
mediated endocytosis and caveolae-/clathrin-independent endocytosis.
Figure 5. Cellular uptake and endocytosis pathways of BSA-prodrug conjugates. (A) CD spectra of
BSA, BSA-8a, Cy5-BSA and Cy5-BSA-8a. (B) Cellular uptake of Cy5-BSA, Cy5-BSA-8a, Cy5-
BSA-8b, Cy5-BSA-11a and Cy5-BSA-11b after incubation with cells at 37°C for 0.5 or 2 h, or at 4°C
for 2 h, *P < 0.05,**P < 0.01, ***P < 0.001 (n=3). (C) Cellular uptake of Cy5-BSA-8a after incubation
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with chlorpromazine, indomethacin, quercetin, sodium azide and dipyridamole, ns: no significance vs
control, *P < 0.05 vs control, **P < 0.01 vs control, ***P < 0.001 vs control (n=3).
Intracellular Release of GEM from BSA-Prodrug Conjugates
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To determine GEM release rates intracellularly after cellular uptake of four BSA-
prodrug conjugates, the four conjugates were incubated with 4T1 tumor cells at an
-1
equivalent GEM dose of 1 μg·mL for 6, 12 and 18 h. As to all BSA-prodrug conjugates,
GEM release mainly occurred in the first six hours, then slowed down (Figure 6). Free
GEM accumulated with time and stronger cytotoxicity would be exerted. Moreover, the
combination of the most labile linkage bond/linker from BSA-8b especially in the
reductive cytoplasm led to the fastest GEM release rate which would benefit rapid reach to
therapeutic concentration, while the combination of the most stable linkage bond/linker
from BSA-11a led to both slowest and least GEM release which would directly undermine
the anticancer efficacy. Interestingly, BSA-8a liberated GEM in a little faster way than that
of BSA-11b, probably because the labile nature of carbonate bond as well as one or more
elements in the nasty intracellular environment such as strong acid and various enzymes in
the lysosome promoted the cleavage of carbonate bond.
Figure 6. Cellular GEM release from four BSA-prodrug conjugates inside 4T1 cells, ***P < 0.001
ACS Paragon Plus Environment

Page 17 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
n=3).
In vitro Cytotoxicity induced by released GEM
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To fully investigate the cytotoxicity of our prodrugs, MTT method was applied. Table 1
showed IC50 values of GEM, four free prodrugs and four BSA-prodrug conjugates on 4T1
cells for 48 h incubation. Considering decreased release rates and extent of free GEM, both
free prodrugs and BSA-prodrug conjugates demonstrated less cytotoxicity than that of
GEM, but still exhibit IC50 values in the low micromolar range (0.02-0.4 µM) except those
of 11a and BSA-11a. On the whole, the carbonate prodrugs including both free and
conjugated form inhibited cell proliferation more than their carbamate ones, and reduction-
sensitive ones were more potent than their non-reduction-sensitive counterparts, which
closely related to the cellular GEM release rates and would affect the final efficacy in vivo.
Additionally, IC50 values of GEM and prodrugs on A549 cells were listed in supplementary
information (SI).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. IC50 values of GEM, 8a, 8b, 11a and 11b in the form of free prodrugs, BSA conjugates, or
BSA-conjugates in the presence of hENT inhibitor against 4T1 cells (n=3).
BSA
BSA conjugates & hENT
inhibitor
Free prodrugs
nM)
Parameters
GEM
conjugates
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
(nM)
(nM)
9.3 ± 1.0
23.2 ± 2.1
41.3 ± 1.9
57.1 ± 12.6
>2000
-
266.7 ± 55.4
118.1 ± 44.9
174.8 ± 50.3
563.2 ± 15.7
>2000
8
b
a
88.5 ± 24.4
192.8 ± 47.8
331.2 ± 73.3
>2000
8
11b
11a
Nucleoside Transporter Dependence
To investigate the sensitivity of nucleoside transporter-dependent membrane permeation
of free GEM and four conjugates, dipyridamole (DP) was used as an hENT inhibitor during
cytotoxicity evaluation (Table 1). In 4T1 cells, dipyridamole significantly undermined
cytotoxity of by GEM 28-fold, while the cytotoxicity of four BSA-prodrug conjugates were
decreased within 2-fold. The results suggested that after conjugation with albumin, our
prodrugs entered these cells in a nucleoside transporter independent manner, but not for
unmodified GEM.
In addition, in the cellular uptake experiments, uptake of BSA-8a did not show obvious
differences between 4T1 cells preincubated with or without DP, again indicating the hENT
independence of our macromolecular prodrugs in cell entry. Then drug resistance induced
by decreased expression of nucleoside transporters was expected to be bypassed (Figure
5
C).
Pharmacokinetics
ACS Paragon Plus Environment

Page 19 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Pharmacokinetic profile was studied in male Sprague–Dawley rats. GEM, 8a, 8b, 11a
−
1
and 11b were intravenously administrated at an equivalent GEM dose of 1 mg·kg . As
shown in Figure 7A, Figure 7B and Table 2, the GEM from the GEM group was quickly
cleared from the blood circulation with AUC and t1/2 values being 5449.0 ± 929.2 μg·h·L-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
and 4.1 ± 0.6 h respectively. In stark comparison, AUC of total GEM in the 8a and 8b
group was about 21 fold and 7 fold higher than that of the GEM group respectively. Also,
it should be highlighted that GEM in the binding form reached to about 94% and 68% of
the total GEM in 8a and 8b group respectively, which indicated that the vast majority of
the total GEM could be shielded by albumin, and escape renal clearance and deactivation
by CDA efficiently. Additionally, t1/2 for the 8b group did not manifest obvious increase
than that of the GEM group, while that of the 8a group prolonged 3.6 times. Both in AUC
and t1/2, 8b couldn’t be a counterweight against 8a, suggesting that the adverse effects in
pharmacokinetics brought by incorporation of a reduction-sensitive disulfide bond and a
more stable linker would lead to stronger grappling of albumin and better capacity to
employ albumin’s shielding and long circulation character. For technological reasons, we
just determined free GEM for 11a and 11b groups. On the whole, the more stable the
linkage bond/linker was, the less GEM was prematurely dissociated from the albumin-
binding conjugates into the systemic circulation, which not only elevated enzymic stability
and circulation time but also conferred GEM more opportunities to reach the target
following albumin. Finally, we concluded that endogenous albumin could be a promising
drug carrier to improve the in vivo pharmacokinetic behavior of GEM and the extent of
improvement varied in terms of the stability of the linkage bond/linker from a prodrug.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Pharmacokinetic profile of GEM and four prodrugs in SD rats. (A) Plasma concentration–
time curves of total GEM including both free and conjugated form in rats after intravenous injection of
GEM, 8a and 8b. (B) Logarithmic plasma concentration–time curves of free GEM in rats after
intravenous injection of GEM, 8a, 8b, 11a and 11b (n=6).
Table 2. Pharmacokinetic parameters of GEM, 8a and 8b following IV administration at a GEM
equivalent dose of 1 mg/kg, respectivelya.
PK
-1
-1
AUC0-t / (μg·h·L)
t1/2 / h
Cl / (L·h ·kg )
parameters
GEM
5449.0 ± 929.2
119892.2 ± 12093.5
45512.9 ± 12123.6
4.1 ± 0.6
14.8 ± 0.7
5.4 ± 1.7
0.180 ± 0.031
0.008 ± 0.001
0.023 ± 0.006
8
8
a
b
a AUC0-t: area under the plasma concentration-time profiles from time 0 to the last time point. t1/2
elimination half-life. Cl : plasma clearance.
:
Biodistribution of four BSA-Prodrug Conjugates and Tumor Free-GEM
Accumulation in a 4T1 Tumor Xenograft Model
It has been widely reported that albumin is able to accumulate in the tumor and general
elimination processes such as urinary excretion and hepatic uptake are determinants for the
ACS Paragon Plus Environment

Page 21 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
fate of the normal macromolecular prodrug in vivo. So we took advantage of Cy5-labelled
BSA and BSA-prodrug conjugates to simulate the biodistribution of our prodrugs in vivo.
Cy5-BSA, Cy5-BSA-8a, Cy5-BSA-8b, Cy5-BSA-11a, Cy5-BSA-11b were intravenously
administrated in 4T1 tumor-bearing mice and noninvasive optical imaging system (IVIS)
was applied for visualization and calculation. While free Cy5 cleared quickly and did not
demonstrate obvious accumulation in special organs, Cy5-BSA and four Cy5-BSA-
prodrugs manifested intense fluorescence of tumor sites at 12 and 24 h post-injection for
the in vivo imaging (Figure 8A). Also, the tumor displayed the highest fluorescence
intensity among the dissected organs (Figure 8B), demonstrating the outstanding
performance in tumor-specific delivery of our macromolecular prodrugs. Moreover, the
four BSA-prodrug conjugates could survive the renal clearance and liver phagocytosis to
a great extent, which was confirmed by the ex vivo radiant efficiency of the kidney and the
liver both being less than half of that in the tumor. In addition, above results showed similar
biodistribution in both trend and extent for Cy5-BSA and four Cy5-BSA-prodrug
conjugates, which indicated that trafficking a small molecular prodrug had insignificant
influence on albumin’s tumor targeting ability (Figure 8C and Figure 8D).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Next, we detected the concentration of free GEM in the tumor at different time to explore
the joint action of albumin carrier and different combinations of the linkage bond/linker.
Five groups of 4T1 tumor-bearing mice were intravenously given free GEM, 8a, 8b, 11a
and 11b respectively at a 1 mg/kg GEM equivalent dose. The group order of quantity for
free GEM at the tumor site from large to small was the following, 8a > 8b > 11b > GEM >
11a (Figure 9A, Figure 9B). In accordance with the cytotoxicity and celluar GEM release
-1
results, the AUC of free GEM in the tumor for 11a group (58.7 ng·mg ·h) was inclined to
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
-1
be less than that of the GEM group (69.1 ng·mg ·h) resulting from the greatly hindered
drug release rate at the tumor site albeit its desirable pharmacokinetic profile and tumor
macromolecular-prodrug accumulation capacity by the combination of a stable linkage
bond and a stable linker. The condition was dramatically improved when a self-immolative
disulfide bond was inserted into the stable linker as suggested by two times more GEM
release in the 11b group, implying better antitumor efficacy compared to 11a. Strikingly,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-1
the AUC of free GEM in the tumor for 8a and 8b group (523.9 and 333.5 ng·mg ·h,
respectively) was 7.6 and 4.8 times higher than that of the GEM group respectively,
meaning a overall favorable pharmacokinetic profile, tumor macromolecular-prodrug
accumulation, and GEM release rates in the tumor, which might bring about a sharp
increase in therapeutic effect. It was interesting to find that the tumor free-GEM
accumulation of the carbonate prodrugs was higher than that of the carbamate ones, and
probably the labile nature of carbonate linkage bonds cleaving to set GEM free more easily
at the tumor site constituted the dominant reason. However, compared with 8a, 8b did not
show final benefits from a disulfide bond-inserted linker albeit accelerated GEM release
rate at the tumor site, probably resulting from increased lability in systemic circulation and
decreased bioavailability (less than 2/5 of the bioavailability of 8a). Thereby we concluded
that the combination of a linkage bond/linker would directly relate to the parent-drug
accumulation in tumor through affecting pharmacokinetics, tumor macromolecular-
prodrug accumulation and ensuing parent drug release rate.
ACS Paragon Plus Environment

Page 23 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. The in vivo and ex vivo fluorescence images of 4T1 tumor-bearing mice after intravenously
injection of Cy5, Cy5-labelled BSA and four BSA-prodrug conjugates. (A) Whole body distribution for
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2 and 24 h. (B) Relative dissected major organs harvested at 24 h post-administration. (C) Quantitative
analysis of relative tumor accumulation in a live body. (D) Quantitative analysis of relative dissected
major organs (n=3).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 9. Quantification of tumor free-GEM accumulation for GEM and four prodrugs. (A) Tumor
free-GEM concentration-time curves in mice after intravenous injection of GEM, 8a, 8b, 11a and 11b.
(B) Relative tumor free-GEM AUC, **P < 0.01, ***P < 0.001 (n=3).
Antitumor Efficacy, Histological Staining, and Serum Biochemistry Assay.
To investigate the antitumor efficacy of four prodrugs in vivo, the 4T1 tumor-bearing
mice were randomly sorted into six groups and administrated vehicle, free GEM, 8a, 8b,
11a and 11b at an equivalent GEM dose of 8 mg/kg for a total of 5 injections. In accordance
with the in vivo tumor free-GEM accumulation of the prodrugs, as shown in Figure 10A,
10B, 10C, compared with the vehicle group and GEM group, 11a showed just a slight
inhibition on tumor growth with a tumor inhibition rate of 32.5%. By contrast, 11b, the
reduction-sensitive counterpart of 11a, generated better antitumor efficacy than GEM did
and slowed down tumor growth dramatically. Notably, with over 90% tumor inhibition
rate, the carbonate GEM prodrugs exerted stronger antitumor activity than that of above
ACS Paragon Plus Environment

Page 25 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
carbamate ones. Moreover, 8a, with a carbonate linkage bond and an all-carbon linker
absent of a disulfide bond, was the most potent and the tumor volume reduced by two third.
Also, the outstanding efficacy of 8a was validated by hematoxylin and eosin (H&E)
staining of tumor sections in which not even one live tumor cell could be found (Figure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1).
On the whole, the potent antitumor efficacy of 8b and 8a could be contributed to several
aspects: (a) rapid and selective binding with endogenous albumin through click reaction;
b) prolonged blood circulation time and enhanced bioavailability; (c) substantially
(
diminished metabolism by CDA with the shield of albumin; (d) prominent
macromolecular-prodrug accumulation in the tumor; (e) efficient cellular uptake with less
dependence on hENT; and (f) rapid and differential liberation of active GEM molecules
within tumor cells. These aspects cover almost all stages of drug delivery following
intravenous administration, resulting in potent chemotherapeutic efficacy with diminished
off-target toxicities.
Additionally, no noticeable changes in body weight (Figure 10D) and the hematological
parameters were observed in all the mice tested, Furthermore, no obvious histopathological
abnormalities were observed in H&E-stained tissue sections of major organs. This suggests
that 8a and 8b with potent antitumor activity elicited no significant off-target toxicities to
major organs.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 10. Anticancer efficiency and safety profile of four prodrugs in vivo in a 4T1 tumor xenograft
model. (A) Tumor growth curves after injected with different formulations in 4T1 tumor bearing mice.
(B) Pictures of tumors after last treatment. (C) Excised tumor weight after last treatment. (D) The weight
growth curves of mice after injected with different formulations,*P < 0.05, **P < 0.01, ***P < 0.001
(n=5). (E) Hematological parameters after last treatment (n=3).
ACS Paragon Plus Environment

Page 27 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 11. H&E stained tumor sections from different therapy groups.
CONCLUSIONS
Herein we describe a successful process to achieve tailored and accurate controlled
release for in situ covalently albumin-binding GEM prodrugs circumventing obstacles in
GEM delivery. The group with a carbonate linkage bond standed out with over 90% tumor
inhibition rate and similar safety profiles to GEM. Notably, the prodrug 8a, with a
carbonate linkage bond and a linker absent of a disulfide bond, demonstrated remarkably
increased bioavailability (21-fold greater than GEM) and efficient tumor free-GEM
accumulation (8-fold of GEM), which consequently contributed to the most excellent in
vivo antitumor efficacy.
To the best of our knowledge, the present study is the first in depth investigation of how
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 61
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
various combinations of the linkage bond/linker in covalently albumin-binding prodrugs
correlate with their in vivo antitumor activity. The linkage bond plays a crucial role in
controlled release and a stimuli-responsive linker might provide some fine-tuning.
Noticeably, incorporation of a reduction-sensitive disulfide bond is not a technology that
always brings about overall benefits to a desired controlled release, for which both
favorable burst drug release at the target and unfavorable instability in circulation happens
at the same time. In this time, the stability of the linkage bond influences the final results
being advantageous or disadvantageous. Best antitumor efficacy is achieved when the
linkage bond and the linker are well-coordinated, that is, better controlled release leads to
better therapeutic outcomes.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
General Information
Gemcitabine (98.8% purity) was purchased from Nanjing Chemlin Chemical Industry
Co., Ltd (Jiangsu, Nanjing, PR China). Maleimide (98% purity), 5-amino-1-pentanol (50
wt. % aqueous solution), 2, 2'-dithiodiehanol (98% purity), TBSCl (98% purity), TBAF
(1.0 mol/L in THF) and triphosgene (98% purity) were all obtained from Energy Chemical
Co., Ltd (Shanghai, China). Tetrahydrouridine (THU), a cytidine deaminase inhibitor, was
purchased from J&K Scientific (HPLC grade). 2',2'-difluoro-2'-deoxyuridine (dFdU) was
purchased from Pharmcodia Bovine. Serum albumin (BSA, 98%) was purchased from
HarveyBioGene Technology Co., Ltd (Beijing, China) and the protein contained
approximately 52% free thiol groups as assessed with Ellman’s test. Cy5 NHS ester was
purchased from Okeanos Co., Ltd (Beijing, China). All other reagents and solvents were
ACS Paragon Plus Environment

Page 29 of 61
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
of analytical or HPLC grade and were used without further purification. All prodrugs used
in the biological experiments were >95% pure, as measured by HPLC. Melting points (mp.)
were determined on WRX-4 Micro melting point apparatus (Yice). Nuclear magnetic
1
13
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
resonance ( H-NMR and C-NMR) spectra were measured with a Bruker 400/600 MHz
NMR spectrometer. HRMS experiments of 8a, 8b, 11a and 11b and their conjugation with
BSA were performed by FT-ICR MS (ESI) and SYNAPTTM G2-Si HDMS (ESI),
respectively.
1
1
. Synthesis of four Prodrugs
.1 Synthesis of 2-[(2-aminoethyl)disulfanyl]ethan-1-ol (3)
Cystamine hydrochloride (750 mg, 6.6 mmol) was dissolved in methanol. Triethylamine
1.85 mL, 13.2 mmol) and 2, 2'-dithiodiethanol (6.6 mmol, 50% in water) was added
(
dropwise to the solution in an ice bath. The mixture was stirred overnight and allowed to
warm up to room temperature. After reaction completed, the mixture was concentrated in
vacuo and the crude product was purified by column chromatography to yield intermediate
3
58 mg as a yellow oil in the yield of 5.7%.
.2 Synthesis of 5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl carbonochloridate
7a) and 2-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]disulfanyl}ethyl
1
(
carbonochloridate (7b)
Maleimide (2.00 g, 20 mmol) was dissolved in 80 mL ethyl acetate and the solution was
cooled to approximately 0°C. Then a solution of N-methyl morpholine (1.85 g, 21.0 mmol)
in 10 mL ethyl acetate was added dropwise over 10 min. Finally, methyl chloroformate
(2.46 g, 20.0 mmol) in 5 mL ethyl acetate was added dropwise, and the solution was
allowed to warm up to room temperature while stirred for 1 h. After 1 h, the precipitate
ACS Paragon Plus Environment