Synthesis and evaluation of new peptide-linked doxorubicin conjugates as prodrugs activated by prostate-specific antigen

Article abstract of DOI:10.1007/s00044-020-02573-w

The targeted delivery of cytotoxic agents to prostate cancer cells via selective activation of peptide-linked prodrugs by prostate-specific antigen (PSA) has been previously demonstrated. In our continued efforts to design prodrugs with improved prostate

Full text of DOI:10.1007/s00044-020-02573-w

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02573-w
ORIGINAL RESEARCH
Synthesis and evaluation of new peptide-linked doxorubicin
conjugates as prodrugs activated by prostate-specific antigen
1,2
Herve Aloysius1 Longqin Hu
●
Received: 15 April 2020 / Accepted: 25 May 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
The targeted delivery of cytotoxic agents to prostate cancer cells via selective activation of peptide-linked prodrugs by
prostate-specific antigen (PSA) has been previously demonstrated. In our continued efforts to design prodrugs with improved
prostate tumor specificity, we developed GABA ← mGly-Ala-Ser-Chg-Gln and glutaryl-Ser-Ala-Ser-Chg-Gln as
promoieties with enhanced PSA specificity starting from the known substrate sequence glutaryl-Hyp-Ala-Ser-Chg-Gln.
Based on their PSA cleavage rates and resistance to non-PSA-mediated hydrolysis in plasma, GABA ← mGly-Ala-Ser-Chg-
Gln and glutaryl-Ser-Ala-Ser-Chg-Gln were selected as optimal promoieties and coupled to doxorubicin (Dox) as PSA-
targeted prodrugs, using Ser-Leu linkers. Following 72-h incubations with Dox prodrugs, there was insignificant cytotoxicity
in non-PSA-producing DU145 cells. The Dox prodrugs, glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox (I), glutaryl-Ser-Ala-
Ser-Chg-Gln-Ser-Leu-Dox (II) and GABA ← mGly-Ala-Ser-Chg-Gln-Ser-Leu-Dox (III) demonstrated comparable PSA
cleavage rates (t1/2 values <23 min), and were cytotoxic against PSA-producing LNCaP cells with IC50 values of 0.18, 0.27
and 0.082 μM, respectively. To mitigate neprilysin-mediated hydrolysis of the Ser-Leu linker in prodrugs I–III and further
improve PSA specificity, 3-aminooxypropionate was incorporated between the promoiety and Dox (prodrug IV). Despite its
slower PSA cleavage rate (t1/2 value of 67 min), GABA ← mGly-Ala-Ser-Chg-Gln-NH-O-CH2-C(Me)2C(O)-14-O-Dox (IV)
was equipotent (IC50 value of 0.19 μM) to prodrug I at killing PSA-producing LNCaP cells due to its ability to release free
Dox through a cyclization activation mechanism. Further metabolism and PK/PD studies will be conducted to evaluate the
tumor specificity of the novel Dox prodrugs (II–IV) reported herein.
Abbreviations for common amino acids are in accordance with IUPAC
recommendations
Dedicated to Professor Robert P. Hanzlik on the occasion of his
retirement after 49 years of dedicated service on the faculty of the
Department of Medicinal Chemistry, University of Kansas School of
Pharmacy
* Longqin Hu
2
Rutgers Cancer Institute of New Jersey, New Brunswick, NJ
longhu@pharmacy.rutgers.edu
08901, USA
1
Department of Medicinal Chemistry, Ernest Mario School of
Pharmacy, Rutgers, The State University of New Jersey,
Piscataway, NJ 08854, USA

Medicinal Chemistry Research
Graphical Abstract
●
●
●
Keywords Prostate-specific antigen Doxorubicin Prodrug Peptide-linked drug conjugate
Abbreviations
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide
internal standard
AMC
ACN
7-amino-4-methylcoumarin
acetonitrile
IS
Alloc-OSu (N-allylcarbonyloxy)succinimide
NMP
NMR
PSA
N-methyl-2-pyrrolidone
Boc
tert-butyloxycarbonyl
nuclear magnetic resonance
prostate-specific antigen
(benzotriazol-1-yloxy)tripyrrolidinophospho-
nium hexafluorophosphate
Roswell Park Memorial Institute
tissue culture medium
selected reaction monitoring
tetrathydrofuran
trifluoroacetic acid
CDI
carbonyldiimidazole
Chg
L-cyclohexylglycine
PyBOP
DBU
DCM
DEA
DIAD
DIEA
DMF
DMAP
Dox
1,8-diazabicycloundec-7-ene
dichloromethane
diethylamine
diisopropyl azodicarboxylate
N,N-diisopropylethylamine
dimethylformamide
4-dimethylaminopyridine
doxorubicin
RPMI
TCM
MRM
THF
TFA
TLC
thin-layer chromatography.
DMSO
EDC
dimethyl sulfoxide
1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide
Introduction
EDU
FCC
Fm
Fmoc
FUDR
GABA
HBTU
1-Ethyl-3-(3-dimethylaminopropyl)urea
flash column chromatography
fluorenylmethyl
fluorenylmethoxycarbonyl
floxuridine or 5-fluoro-2’-deoxyuridine
γ-aminobutyric acid
O-benzotriazole N,N,N’,N’-tetramethyluronium
hexa-fluorophosphate
N-hydroxysuccinimide
high performance liquid chromatography
high-resolution mass spectrometry
trans-4-hydroxy-L-proline
liquid chromatography-mass spectrometry
Prostate-specific antigen (PSA) is a kallikrein-related pep-
tidase secreted in the epithelial cells of the prostate gland as
a semen liquefaction enzyme. Elevated serum PSA levels
are routinely screened as a serological marker for prostate
cancer although low systemic concentrations of the antigen
are also detectable in healthy males (Balk et al. 2003;
Catalona et al. 1991; Diamandis 1995; Levesque et al.
1995; Ayyıldız and Ayyıldız 2014). Additionally, increased
systemic levels of PSA in males are associated with chronic
inflammation in other prostate diseases (McDonald et al.
2014). In prostate cancer, hyperplasia-induced disruption of
the epithelium causes PSA to leak into the interstitial space
of tumor cells and subsequently diffuse into the systemic
circulation (Herschman et al. 1997; Nadler et al. 1995;
HOSu
HPLC
HRMS
Hyp
LC-MS

Medicinal Chemistry Research
O’Malley et al. 2014). Due to its inhibition by α1-antic-
hymotrypsin and α2-macroglobulin in the serum, the pro-
teolytic activity of PSA is primarily confined to the tumor
microenvironment (Leinonen et al. 1996; Otto et al. 1998).
This tumor-specific biochemical feature and the narrow
substrate specificity of PSA have been conveniently
exploited for the targeted delivery of potent cancer ther-
apeutics as peptide prodrugs (Atkinson et al. 2008; Choi
et al. 2012; Yang et al. 2011; Aloysius and Hu 2015b).
Significant discovery efforts have been invested in design-
ing such targeted prodrugs over the past two decades, with
several PSA-specific peptide substrates engineered as pro-
moieties (DeFeo-Jones et al. 2000; Denmeade et al. 1997;
Garsky et al. 2001; Doan et al. 2015; Kwiatkowska et al.
2019; Lee et al. 2015; Tarvainen et al. 2020). In our con-
tinued efforts to design prodrugs with enhanced tumor
selectivity, we previously developed the modified peptides
glutaryl-Ser-Ala-Ser-Chg-Gln and GABA ← mGly-Ala-
Ser-Chg-Gln as PSA substrates with improved cleavage
rates and/or resistance to non-PSA-mediated hydrolysis in
plasma compared to the reported sequence glutaryl-Hyp-
Ala-Ser-Chg-Gln (DeFeo-Jones et al. 2000; DiPaola et al.
2002a, 2002b; Garsky et al. 2001). In the present study, we
have coupled the optimized peptide substrates to Dox as
prodrugs to further evaluate their PSA specificity.
Dox is highly effective against a wide spectrum of solid
tumors but causes dose-dependent toxicity. The most
commonly reported adverse effect associated with Dox
administration is cardiomyopathy, possibly through
mechanisms including interference with iron metabolism,
contractile failure and increased production of reactive
oxygen species (Xu et al. 2005; Weiss 1992; Abdullah
Chowdhury et al. 2018; Bartlett et al. 2017; Hortobagyi
et al. 1997; Koleini and Kardami 2017). Thus, peptide
prodrugs with high specificity for PSA are anticipated to
restrict the cytotoxic activity of Dox to tumor cells, thereby
improving its therapeutic window. In order to enhance the
selectivity of Dox for prostate tumors, the PSA-cleavable
peptide conjugate, glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-
Dox (I), was synthesized and evaluated as a prodrug
(DiPaola et al. 2002a, 2002b; Garsky et al. 2001). While the
peptide promoiety of prodrug I was highly specific for PSA,
active metabolites Leu-Dox and Dox were observed in both
mice and humans in vivo (DiPaola et al. 2002a, 2002b;
Wong et al. 2001).
Hu 2020). We demonstrate herein that peptide-Dox prodrug
I and its analogs, glutaryl-Ser-Ala-Ser-Chg-Gln-Ser-Leu-
Dox (II) and GABA ← mGly-Ala-Ser-Chg-Gln-Ser-Leu-
Dox (III) are unstable in biological matrices lacking active
PSA due to significant hydrolysis of their shared Ser-Leu
linker. As an alternative to Ser-Leu, a self-immolative 3-
aminooxypropionate linker may mitigate this non-PSA-
mediated hydrolysis, which we suspect to be catalyzed by
human neprilysin. To promote the release of free Dox
through chemical cyclization, the linker was coupled to the
C-14 α-hydroxyl of Dox as previously demonstrated for
floxuridine (FUDR) (Ge et al. 2009). With this modifica-
tion, we anticipated additional improvements in PSA spe-
cificity and activity for the resulting prodrug IV (Scheme 1)
since the active metabolite Leu-Dox was shown to be 10-
fold less toxic than Dox in PSA-secreting LNCaP tumor cell
lines, and not completely converted to Dox in vivo (Garsky
et al. 2001; Wong et al. 2001).
Results and discussion
The necessity of incorporating a linker between the cyto-
toxic drug and the PSA-cleavable promoiety has previously
been demonstrated for various prodrugs (DeFeo-Jones
2000; Garsky et al. 2001; Wong et al. 2001; Jiang and Hu
2007; Ménez et al. 2008). Initially, peptides were coupled to
the aminoglycoside of Dox via a Ser-Leu linker (Scheme 1)
as previously reported (DeFeo-Jones et al. 2000; DiPaola
et al. 2002a, 2002b; Garsky et al. 2001). To further improve
PSA specificity, mitigation of neprilysin-mediated hydro-
lysis (suggested by metabolism studies) was achieved with
prodrug IV by replacing the Ser-Leu linker of prodrug III
with 3-aminooxypropionate which was coupled to the C-14
α-hydroxyl of Dox. As anticipated, insertion of the self-
immolative 3-aminooxypropionate linker resulted in the
release of free Dox (Scheme 1) into prostate cancer cells
leading to enhanced tumor-specific cytotoxic effects despite
its slower PSA cleavage rate compared to prodrugs I–III.
The enhanced activity is in part due to the fact that the
active metabolite Leu-Dox is 10-fold less toxic than Dox in
LNCaP tumor cell lines (Garsky et al. 2001; Wong et al.
2001).
Synthetic strategy
In the preceding paper, we prepared and evaluated three
peptide-NH-2-F-benzyl phosphoramide mustard conjugates
as prodrugs activated by prostate-specific antigen and found
that the optimal combination of the peptide sequence
GABA ← mGly-Ala-Ser-Chg-Gln with an appropriate lin-
ker would be necessary to achieve an adequate balance
between PSA-mediated cleavage/activation and resistance
to non-PSA-mediated premature activation (Aloysius and
All prodrugs were synthesized by first coupling Gln to the
appropriate linkers for ready attachment to various peptide
sequences (Scheme 2). Preparation of Fm-GABA ← mGly-
Ala-Ser-Chg and Fm-GABA ← mGly-Ala-Ser-Chg-Gln-
Ser-Leu could not be achieved following standard auto-
mated peptide synthesis methods. For example, coupling of
Fm-GABA ← mGly to Ala-Ser-Chg-O-resin failed when

Medicinal Chemistry Research
Scheme 1 Proposed PSA activation mechanisms of doxorubicin prodrugs
EDC, HBTU or PyBOP were used to activate the acid.
Furthermore, premature removal of the Fm-protecting group
was observed under conditions involving greater than 1 eq.
of DMAP. Instead, Fm-GABA ← mGly-OSu was prepared
and coupled to H-Ala-Ser-Chg-Gln-Ser or H-Ala-Ser-Chg
in solution. Alternatively, Fm-GABA ← mGly-Ala-Ser-Chg
could be generated by coupling the Fm-GABA ← mGly-
OSu ester to H-Ala-Ser-Chg-O-resin at room temperature in
48 h. All other peptides were synthesized using standard
solid-phase procedures and coupled to the aminoglycoside
of Dox using PyBOP coupling procedures, or the C-14 α-
hydroxyl of Dox via a 3-aminooxypropionate linker as
shown in Scheme 2. The 3-aminooxypropionate linker of
prodrug IV was obtained using our previously reported
procedure with modifications (Ge et al. 2009). Ready access
to the protected linker 9 was achieved in a single step
starting from benzyl 3-hydroxy-2,2-dimethylpropanoate in
greater than 50% yield. Palladium-catalyzed hydrogenolysis
followed by amine-deprotection with anhydrous hydrazine
afforded the 3-aminooxypropionate linker 9 which was
subsequently coupled to Fmoc-Gln-OH via its OSu ester
(Scheme 3). Trityl-protection of the Fmoc-Gln-OH side-
chain amide (10) was introduced in earlier steps to cir-
cumvent the poor solubility of Fmoc-Gln-OH.
K2CO3. Additionally, the removal of a TFA-protecting
group from the C-4 amino group of Dox with Acylase I was
found to proceed in low yield (< 10% deprotection in
100 mM KHPO4 buffer, pH 7.4, in 24 h, data not shown).
Instead, a Fmoc/Alloc orthogonal protection strategy was
used so that the Alloc group could be removed with tetrakis
(triphenylphosphine)palladium (0) in the presence of the
allyl scavenger dimedone, and Fmoc deprotected with a
non-nucleophilic base such as 1,8-diazabicycloundec-7-ene
(DBU). Much effort was dedicated to optimizing the chal-
lenging synthesis of Dox-14-O-esters. A few specific Dox-
14-O-esters were prepared by chemoenzymatic methods
using C. Antarctica lipase (type B) (Cotterill and Rich
2005; Cotterill et al. 2008); however, the conventional
approach for synthesizing Dox-14-O-esters involves a
nucleophilic displacement-type esterification of 14-
bromodaunorubicin under basic conditions. To access 14-
bromodaunorubicin, C-14 bromination of daunorubicin was
efficiently carried out through ketalization of its carbonyl
group at C-13 (Rho et al. 2001; Rho et al. 2000; Rho et al.
2002). Noteworthy, our previous attempts to esterify Fmoc-
Dox via Mitsunobu or Appel/nucleophilic substitution
reactions failed. Adaptation of a previously reported meth-
odology for N-Fmoc-Dox-14-O-hemiglutarate (van Hens-
bergen et al. 2002) involving coupling of the mixed
anhydride with Fmoc-Dox in the presence of TEA at room
temperature in 48 h did not produce the desired ester either.
We also performed CDI/EDC/DMAP coupling with little
success; increasing the equivalents of CDI or DMAP led to
premature removal of the Fmoc-protecting group. More-
over, ester 6 could not be generated using EDC and cata-
lytic 4-pyrrolopyridine or using 1-(mesitylene-2-sulfonyl)-
Since Dox is unstable under acidic deprotection and
hydrogenation conditions, an orthogonal protection strategy
involving an acid- and base-stable protecting group for
either the α-amino or C-4 amino group of Gln or Dox,
respectively, was necessary. While TFA-protection was
stable to organic base for up of 24 h, this approach was not
pursued because of the anticipated hydrolysis of the Dox-
14-O-ester during subsequent deprotection steps with 10%

Medicinal Chemistry Research
Scheme 2 Prodrug synthetic strategy
3-nitro-1H-1,2,4-triazole (MSNT) as an activating agent,
and Fm-GABA ← mGly-Ala-Ser-Chg-Gln-NH-O-CH2-C
thus possible through coupling of compound 12 with 14-
bromodaunorubicin at room temperature for 48 h, followed
by Alloc-protection (Scheme 3). The conversion yield of the
bromo-substitution step affording the unprotected Dox-14-
O-ester (no Alloc group) was improved to 80% when a
minimum of 5 equivalents of the acid was used; never-
theless, longer reaction times led to significant hydrolysis of
the ester to Dox (greater than 20%) and overall yields
greater than 40% could not be achieved for the Alloc-
protected ester. Additionally, flash column chromatography
(FCC) purification of the Alloc-protected ester was difficult
due to its poor solubility in most solvents. Nonetheless, the
deprotected ester 6 could be purified and isolated by HPLC
in sufficient quantities for the next step.
(Me)2COOH
was
unreactive
towards
14-
bromodaunorubicin under neutral conditions. Never-
theless, preliminary studies with hydroxypivalic acid indi-
cated that its Dox-14-O-ester could be readily synthesized
from sodium hydroxypivalate and 14-bromodaunorubicin at
50 °C in dry acetone (data not shown) as previously
reported for various Dox-14-O-esters generated from
aspartic acid derivatives and fatty acids (Rho et al. 2001;
Rho et al. 2000; Rho et al. 2002). Under those conditions,
significant aglycone formation also occurred due to in situ
generation of HBr. In addition, hydrolysis of 14-
bromodaunorubicin to Dox (~10%) was observed leading
to a further reduction of the reaction yield to less than 50%.
Reaction conditions were optimized to afford nearly quan-
titative conversion to the hydroxypivalate Dox-14-O-ester
by increasing the solvent volume, and adding 4 Å molecular
sieves to the reaction mixture to trap HBr (Hayashi and
Cook 2008). Convenient access to Dox-14-O-esters 6 was
Evaluation of Dox prodrugs in PSA- and non-PSA-
producing Cells
As shown in Fig. 1, the stability of Dox peptide prodrugs in
50 mM Tris buffer, pH 8 was studied over a period of 48 h.

Medicinal Chemistry Research
Scheme 3 Synthesis of
Conjugate 6. Reagents and
conditions: a NHPI, Ph3P,
DIAD, THF, 0 °C to rt, 24 h;
b H2/ 10% Pd-C, MeOH, rt, 5 h;
c H2NNH2, Et2O, rt, 2 h; d EDC,
HOSu, MeCN, rt, 2 h; e 95%
TFA/MeCN, rt, 5 min; f rt, 24 h;
g 14-bromodaunorubicin,
NaHCO3 (1 eq.), 4 Å mol.
sieves, acetone, rt, 48 h; h Alloc-
OSu, NaHCO3, MeCN, rt, 1 h;
i 1% DBU, DMF, rt, 10 min
To determine PSA cleavage rates, prodrugs were incu-
bated with human PSA over a period of 3 h. As previously
described, substrate disappearance was monitored by LC-
MS/MS analysis using MRM. The percentage of prodrug
remaining at any given time point could be computed by
taking the ratio of its peak area response and that of the 0-
hour time point. Overall, prodrugs I–III exhibited rapid
cleavage rates with comparable profiles (Fig. 2). Our ori-
ginal substrate optimization studies suggested that glutaryl-
Ser-Ala-Ser-Chg-Gln and GABA ← mGly-Ala-Ser-Chg-
Gln were cleaved by PSA 1.3- and 2-fold faster than glu-
taryl-Hyp-Ala-Ser-Chg-Gln, respectively. Although mod-
ifications were carried out across the entire sequence of
glutaryl-Hyp-Ala-Ser-Chg-Gln, only P5 substitutions led to
notable increases (up to a 2-fold) in PSA cleavage rate
(Aloysius and Hu 2015a). Because the most effective amino
acid substitutions were distant from the PSA scissile bond,
we hypothesized that only substrate Km was improved for
the AMC-conjugates developed. However, as shown in
Table 2, modifications in P1’ and P2’ negatively affected
PSA cleavage rate as suggested by the longer half-life
(67 min) of the Dox prodrug IV compared to prodrugs I–III
(half-life values of 22–23 min). A full characterization of
the Michaelis-Menten kinetic curves for prodrugs I–IV
could help further explain the results summarized in Fig. 2.
Nevertheless, for our purpose, the Dox prodrugs were
cleaved at sufficiently fast rates, and some comparable to
that of known prodrug I.
Fig. 1 Prodrug stability in Tris buffer. Each prodrug (100 μM) was
incubated in 50 mM Tris/HCl buffer, pH 8.0 containing 10 mM CaCl2
and 0.1% TWEEN-20 at 37 °C
Prodrug disappearance was monitored by LC-MS/MS ana-
lysis using multiple reaction monitoring (MRM, Table 1).
Most prodrugs were ~80% stable in Tris buffer, pH 8 over a
period of 24 h (data not shown). LC-MS analysis of the
buffer incubates revealed that one of the major degradants
of Dox prodrugs I, II and III was a putative Tris-adduct
(confirmed by accurate mass, data not shown) probably
originating from the condensation of trisaminomethane with
trace amounts of the terminal aldehyde (tautomerized α-
hydroxyketone) of Dox. Another late-eluting degradant was
detected but could not be identified based on mass spectral
data.
To assess cytotoxicity and tumor selectivity, all prodrugs
were incubated in cultured cell lines secreting (LNCaP) or

Medicinal Chemistry Research
Table 1 MRM transitions used for prodrug quantification by LC-MS/
MS
mentioned earlier, proof-of-concept was first demonstrated
for prodrug I in humans, and both Leu-Dox and Dox are
believed to contribute to its cytotoxic effect elicited in
tumor cells in vitro and in vivo. Currently, there is no clear
understanding of the transport mechanisms involved in
shuttling Leu-Dox and Dox across tumor cell membranes,
and it’s difficult to predict the contribution of each cyto-
toxin to overall prodrug effect. In addition, it is not known
whether Dox (and possibly Leu-Dox) efflux out of tumor
cells leads to any significant circulating drug and metabolite
levels in vivo although non-PSA-mediated metabolism is
suspected to be the main culprit (Wong et al. 2001).
Transition 1a
Q1
Transition 2b
Q1
Prodrug
Q3
Q3
I
1418.6
1392.7
1362.7
1255.5
1022.7
996.7
966.6
451.0
982.6
956.5
926.5
–
228.1
342.1
312.3
–
II
III
IV
aMRM transitions used to quantify prodrug/prodrug-sodium adducts
bBase peaks
Prodrug activation mechanisms
In our carcinoma cell culture studies, prodrug III was the
most cytotoxic prodrug against PSA-secreting LNCaP cells
with excellent selectivity against non-PSA-producing
DU145 cells (Fig. 3, Table 3). Since PSA cleavage rates
among most Dox prodrugs I–III were similar (Fig. 2, Table 2)
alternate unknown mechanisms must have contributed to
differences in prodrug selectivity in LNCaP and DU145
tumor cell lines. While tumor cell lines generally possess
lower metabolic capacity compared to cultured hepatocytes,
prodrug instability cannot be ruled out as a possibility. We
previously demonstrated that GABA ← mGly-Ala-Ser-Chg-
Gln was a more specific PSA substrate compared to glu-
taryl-Ser-Ala-Ser-Chg-Gln and glutaryl-Hyp-Ala-Ser-Chg-
Gln based on the PSA cleavage rate and plasma stability of
their AMC-conjugate (Aloysius and Hu 2015a) and the
results in Fig. 3 appear to suggest similar trends although
the various mechanisms involved in prodrug cytotoxicity
are difficult to tease out. Despite its lower PSA cleavage
rate compared to prodrugs I–III, prodrug IV exhibited a
cytotoxicity profile comparable to that of prodrug I. As
mentioned earlier, the cytotoxin released by prodrug IV is
free Dox rather than Leu-Dox. Because Dox is 10 times
more potent than Leu-Dox at killing tumor cells (Garsky
et al. 2001; Wong et al. 2001), its slower release from
prodrug IV was as effective as the relatively faster release
of Leu-Dox from prodrug I (Fig. 4). Moreover, unlike
prodrug I, prodrug IV does not rely on the hydrolytic action
of additional aminopeptidases to generate free Dox (Leu-
Dox conversion to Dox is slow).
Fig. 2 Disappearance of doxorubicin prodrugs during PSA hydrolysis.
Each prodrug (1 μM) was incubated with PSA (1 μM) in 50 mM Tris/
HCl buffer, pH 8.0 containing 10 mM CaCl2 and 0.1% TWEEN-20 at
37 °C
lacking (DU145) PSA for a period of 72 h. Dox was
included in the study as a positive control, and cell viability
determined at the end of the incubation period using the
MTT assay. We anticipated that peptide prodrugs would be
activated extracellularly in LNCaP, but not DU145 cells, by
the proteolytic action of PSA releasing cytotoxic agents that
would ultimately lead to cell death. Various studies sug-
gested that Dox exerts its apoptotic action in tumor cells via
selective binding to nuclear DNA with subsequent free
radical-mediated DNA damage promoted by its metabolic
activation. However, in non-tumor cells, its primary cyto-
toxic mechanism involves the generation of free-radical
intermediates in subcellular compartments (Keizer et al.
1990). Consequently, after being activated in the micro-
environment of prostate cancer tumor cells, transport of
Leu-Dox or Dox across the cell membrane would be
necessary to induce cell death in LNCaP cells. Drug sen-
sitivity, and more importantly, chemoresistance to Dox have
been associated with ATP-binding cassette (ABC) trans-
porters such as P-glycoprotein 1 (P-gp) and ABCB5 (Frank
et al. 2005; Shen et al. 2008; Singhal et al. 2007). As
Non-PSA-mediated metabolism
Initial studies to evaluate prodrug stability were conducted
in blood and cultured hepatocytes, biological matrices
lacking active PSA. Prodrugs I–III were incubated in
heparinized human or mouse blood at 100 μM over a period
of 24 h and the fraction of prodrug remaining at various
time points determined by LC-MS/MS using MRM (Table 1).

Medicinal Chemistry Research
Table 2 Hydrolysis of prodrugs
by human PSA
Prodrug
% of prodrug at 60 min t1/2 (min)
Glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox (I)
Glutaryl-Ser-Ala-Ser-Chg-Gln-Ser-Leu-Dox (II)
GABA ← mGly-Ala-Ser-Chg-Gln-Ser-Leu-Dox (III)
13
12
9.8
53
23
22
22
67
GABA ← mGly-Ala-Ser-Chg-Gln-NH-O-CH2-C(Me)2C(O)-14-O-
Dox (IV)
Conditions: Each peptide prodrug (1 μM) was incubated with human PSA (1 μM) in 50 mM Tris/HCl buffer,
pH 8, for 360 min, and the % of prodrug determined using LC-MS/MS (MRM)
significant differences in half-life and stability were
observed in human hepatocytes (Table 5). Finally, all pro-
drugs were stable in cultured mouse hepatocytes with half-
life values exceeding 48 h, comparable to mouse blood
stability results.
To elucidate soft spots of non-PSA-mediated prodrug
metabolism, we identified key metabolites formed in blood
and hepatocyte incubations at early time points
(30–60 min). In order to characterize the early steps in non-
PSA-mediated metabolism, prodrugs were incubated in
blood and cultured hepatocytes, and prodrug-related frag-
ments were identified and characterized by LC-MS with
accurate mass measurements. In addition, we leveraged the
unique UV-VIS spectrum of Dox to identify peptide frag-
Fig. 3 Cytotoxicity of doxorubicin prodrugs in LNCaP Cells
ments. Dox and its peptide prodrugs exhibited a maximal
absorbance at 484 nm and could therefore be readily
detected in a complex matrix such as blood. Following a 1-h
incubation of Dox prodrugs in human blood and hepato-
cytes, a key metabolite, M1 with measured accurate mass of
657.2657 was detected in all blood and cultured hepatocytes
samples (Fig. 9, Table 6). M1 was unambiguously identi-
fied to be Leu-Dox suggesting that the first step in prodrug
hydrolysis was cleavage of the Ser-Leu linker. Prolonged
prodrug exposure to blood (8 h) and hepatocyte cultures
(48 h) led to increased levels of M1, which was slowly
converted to Dox (accurate mass: 544.1815). While addi-
tional minor metabolites could be detected by accurate mass
spectral analysis (Table 6), blood and hepatocyte metabolic
profiles clearly indicated that M1 or Leu-Dox was the
earliest and major metabolite originating from the hydro-
lysis of prodrugs I–III. Similar metabolic profiles were
obtained in mouse blood and hepatocytes; however, as
suggested by preliminary stability studies, lower levels of
M1 were produced in mouse blood and hepatocytes (Fig. 10).
Since there were differences in sample processing proce-
dures between the stability and metabolic profiling samples,
parent prodrug and metabolite recovery may not be com-
parable. Nevertheless, the general trends in our stability and
metabolic profiling results indicated that human and mouse
blood and hepatocytes produced structurally similar
metabolites.
Blood samples were treated with sodium heparin since
EDTA is known to inhibit the activity of various metallo-
proteases through chelation of divalent ions such as Zn2+
.
As shown in Table 4 and Fig. 5, most prodrugs were
hydrolyzed in human blood to ~20 μM in less than 10 h
except for prodrug III. Similar experiments in heparinized
mouse blood indicated that prodrugs I–III were more stable
in mouse blood overall, with roughly 50% of prodrug
remaining after 8 h (Fig. 6). Based on half-life measure-
ments, the most stable prodrug in human and mouse blood
was prodrug III (Table 4).
To further evaluate their specificity for PSA, the stability
of prodrugs I–III in cultured hepatocytes was measured.
Our preliminary attempts to determine prodrug stability in
select biological matrices indicated that prodrug I was
stable in hepatic microsomes, cytosolic, and S9 suspensions
(data not shown). Since the metabolic capacity of micro-
somes, cytosolic, and S9 preparations are typically sub-
optimal after 2 h, we set out to further investigate prodrug
stability in matrices with prolonged metabolic activity
lasting beyond 24 h, and cultured hepatocytes were a rea-
sonable choice. Depending on experimental conditions,
cultured hepatocytes can remain metabolically active for at
least 48 h. Similar to blood stability results, all prodrugs
demonstrated improved stability in cultured mouse hepa-
tocytes than human (Figs. 7 and 8). Compared to blood,
half-life values were extended to at least 10 h although
Based on its known cleavage map (Keil 1992), we
hypothesized that a thermolysin-like mammalian protease

Medicinal Chemistry Research
Table 3 Prodrug cytotoxicity
in PSA-producing LNCaP
cell lines
Prodrug
LNCaP IC50 (μM) Selectivityaover DU145
Dox
<0.07
0.18
~1
Glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox (I)
Glutaryl-Ser-Ala-Ser-Chg-Gln-Ser-Leu-Dox (II)
GABA ← mGly-Ala-Ser-Chg-Gln-Ser-Leu-Dox (III)
560
370
1220
560
0.27
0.082
0.19
GABA ← mGly-Ala-Ser-Chg-Gln-NH-O-CH2-C(Me)2C(O)-
14-O-Dox (IV)
Conditions: PSA-producing LNCaP and non-PSA-producing DU145 cell lines were treated with Dox or
prodrugs I–IV at concentrations ranging from 0.07 to 100 μM for 72 h and cell viability determined using the
MTT assay
aSelectivity-fold expressed as the ratio of the LNCaP and DU145 (data not shown) IC50 values
60000
UV Chromatogram
50000
40000
30000
20000
10000
0
H-Ser-Leu-Dox
Prodrug I
13.01
14.01
20.83 21.39
19.62
23.19
23.92
3.47
12.23
14.75 17.28 18.44
3.99
6.25 6.69
9.75 11.30
7.21 9.13
1.03 1.93 3.02
60000
50000
40000
30000
20000
10000
0
Dox
Prodrug IV
12.23
13.59
13.90
20.95 21.55
23.10
3.44
11.57
15.40
18.54 19.60
24.28
24
3.94 6.09 6.55 7.03
9.77
10
0.98 2.02 3.00
2
9.33
4
6
8
12
14
16
18
20
22
Time (min)
744.2961
100
Mass Spectrum
80
60
40
20
H-Ser-Leu-Dox
766.2788
782.2429
546.3990
544.1809
502.3729
590.4251
458.3466
634.4511
678.4773
722.5036
846.4953
0
200000
Dox
150000
100000
50000
0
502.3734
590.4258
458.3471
450
645.2284
678.4781
566.1628
722.5043
796.3436
800
758.4444
750
485.3469
609.3393
528.4106
846.4971
850
500
550
600
650
m/z
700
Fig. 4 PSA-mediated hydrolysis of prodrugs I and IV. Conditions: Each peptide prodrug (1 μM) was incubated with human PSA (1 μM) in 50 mM
Tris/HCl buffer, pH 8, for 4 h. Samples were precipitated with 1 volume of ACN, centrifuged and analyzed by LC-MS-UV
would cleave before Leu (a hydrophobic residue next to
Ser) within the Ser-Leu linker of prodrugs I–III. Using
thermolysin as a starting point, incubations of prodrug I
(100 μM) with purified enzyme (300 μg/ml) generated sig-
nificant levels of metabolite M1 within 20 min (Fig. 11)
suggesting that thermolysin hydrolyzed the Ser-Leu of
prodrug I similar to blood and hepatocyte incubations.
Prolonged exposure to thermolysin led to complete con-
version of prodrug I to metabolite M1 within 60 min. Of
relevance to humans, the thermolysin-like mammalian
protease, neprilysin (50 μg/ml), was also found to convert
prodrugs I–III to significant levels of Leu-Dox (M1) and
trace amounts of M2 (Chg-Gln-Ser-Leu-Dox) in 30 min.
Prolonged incubation led to increased levels of M1 and

Medicinal Chemistry Research
Table 4 Non-PSA-mediated prodrug hydrolysis in mouse and
human blood
Prodrug
t1/2 (h)
Human Mouse
Glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox (I)
Glutaryl-Ser-Ala-Ser-Chg-Gln-Ser-Leu-Dox (II)
1.4
9.1
11
12
0.91
7.0
GABA ← mGly-Ala-Ser-Chg-Gln-Ser-Leu-Dox
(III)
Conditions: Each peptide prodrug (100 μM) was incubated in human
or mouse blood for 24 h, and the % of prodrug remaining as
determined using LC-MS/MS (MRM)
Fig. 7 Stability of prodrugs I–III in cultured human hepatocytes. Each
prodrug (100 μM) was incubated in plated human hepatocytes at 37 °C
over a period of 48 h
Fig. 5 Stability of prodrugs I–III in human blood. Each prodrug
(100 μM) was incubated in human blood at 37 °C over a period of 24 h
Fig. 8 Stability of prodrugs I–III in cultured mouse hepatocytes. Each
prodrug (100 μM) was incubated in plated Mouse hepatocytes at 37 °C
over a period of 48 h
Table 5 Non-PSA-mediated prodrug hydrolysis in mouse and human
hepatocytes
Prodrug
t1/2 (h)
Human Mouse
Glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox (I)
Glutaryl-Ser-Ala-Ser-Chg-Gln-Ser-Leu-Dox (II)
11
9.6
11
>48
>48
>48
GABA ← mGly-Ala-Ser-Chg-Gln-Ser-Leu-Dox
(III)
Fig. 6 Stability of prodrugs I–III in mouse blood. Each prodrug
(100 μM) was incubated in mouse blood at 37 °C over a period of 24 h
Conditions: Each peptide prodrug (100 μM) was incubated in cultured
human or mouse hepatocytes for 48 h, and the % of prodrug remaining
as determined using LC-MS/MS
modest formation of M2, suggesting that the major hydro-
lytic step was cleavage of the Ser-Leu linker. While we did
not evaluate the possible conversion of M2 to M1, this
scenario was unlikely since the intervention of additional
proteases would be required. Moreover, M1 or M2 was not
detected in 4- and 48-h buffer incubations (data not shown)
further confirming the formation of M1 and M2 was likely
catalyzed by human neprilysin. These results were in
agreement with our findings from blood and cultured
hepatocytes which indicated that the key metabolic step in
the degradation of prodrugs I–III was the hydrolysis of
their shared Ser-Leu linker.

Medicinal Chemistry Research
Prodrug I
Blood, 1 h
Blood, 8 h
M1
M1
Prodrug I
M3
M2
M1
Prodrug II
Prodrug II
M2
M1
M3
Prodrug III
Prodrug III
M1
M3
M1
M2
Hepatocytes, 1 h
Hepatocytes, 48 h
M2
Prodrug I
(Exact mass: 1011.4570)
M1
M3
M1
(Exact mass: 657.2657)
Prodrug I
Prodrug II
M1
M3
Prodrug II
M2
M1
M1
Prodrug III
Prodrug III
M3
M2
M1
Fig. 9 Metabolic profiles of prodrugs I–III in human blood and cultured hepatocytes
Using various substrates and inhibitors, several studies
have shown that thermolysin and neprilysin, despite their
weak homology, share similar C-terminal fragments and
substrate specificity (Benchetrit et al. 1988; Murphy et al.
1994; Rawlings and Barrett 1995; Roques et al. 1993;
Schulz et al. 2009). Neprilysin also known as CD10 or
common acute lymphoblastic leukemia antigen (CALLA) is
used a clinical marker for several hematologic disorders
including angioimmunoblastic T cell lymphoma, hairy cell
leukemia and Burkitt lymphoma. As part of its physiolo-
gical function in modulating hypertensive effects, it con-
verts big endothelin-1 (big ET-1) by proteolysis (Murphy
et al. 1994; Rawlings and Barrett 1995). Neprilysin also
hydrolyzes various small endogenous peptides such as the
β-amyloid peptide implicated in the initiation and progres-
sion of Alzheimer’s disease. The involvement of neprilysin
in the regulation of signaling peptides as enkephalins,
endothelin, substance P and atrial natriuretic factor has also
been demonstrated by designing specific inhibitors that
inactivate its function (Oefner et al. 2004; Sahli et al. 2005).
The highest concentrations of neprilysin are found on the
apical (luminal) membrane of kidney and lung cells, but the
enzyme may also be induced in certain types of tumors
(Velazquez et al. 2007; Thong et al. 2014). The lack of
robust hydrolysis in mouse samples may be explained by
substrate specificity differences between mouse and human

Medicinal Chemistry Research
Table 6 Metabolites of prodrugs
I–III
Prodrugs (Base peak, retention time)
Metabolite (theoretical mass)a
M1 (657.2659)
M2 (1011.4563)
M3 (544.1819)
I (1396.6071, 14.1)
II (1370.5920, 14.2)
III (1340.5817, 14.2)
657.2657, 12.5
657.2657, 12.5
657.2657, 12.5
1011.4570, 13.5
544.1815, 12.2
544.1815, 12.2
544.1815, 12.2
1011.4570, 13.5
1011.4570, 13.5
aMeasured as mass of base peak at retention time (amu, min)
Blood, 1 h
Blood, 8 h
Prodrug I
M1
M3
Prodrug I
M2
Prodrug II
M1
Prodrug II
M3
M2
Prodrug III
Prodrug III
M1
M3
M2
Hepatocytes, 1 h
Hepatocytes, 48 h
Prodrug I
Prodrug I
M1
Prodrug II
Prodrug II
M1
Prodrug III
Prodrug III
M1
Fig. 10 Metabolic profiles of prodrugs I–III in mouse blood and cultured hepatocytes
neprilysin given that the topography of neprilysin in non-
lymphoid organs such as liver and lungs were found to be
strikingly similar between mouse and humans (Kalled et al.
1995). The instability of prodrugs I–III could not be
effectively predicted with their corresponding AMC-
conjugates (Aloysius and Hu 2015a) which were more
effectively hydrolyzed in mouse rather than human plasma.
Nevertheless, such preliminary experiments guided our
choice of optimal PSA substrates to be further developed as
promoieties. The exclusive localization of neprilysin on the

Medicinal Chemistry Research
Neprilysin, 4 h
Neprilysin, 1 h
Prodrug I
M1
Prodrug I
M1
M2
M2
Neprilysin, 1 h
Neprilysin, 4 h
Prodrug II
M1
Prodrug II
M1
M2
M2
Neprilysin, 1 h
Neprilysin, 4 h
Prodrug III
M1
Prodrug III
M1
Thermolysin, 20 min
Prodrug I
M1
Thermolysin, 60 min
Fig. 11 Metabolism of prodrugs I–III by thermolysin and neprilysin
luminal membrane (Thong et al. 2014) of hepatocytes may
help further explain the slower formation of M1 in cultured
hepatocytes compared to blood since the prodrug must first
access the luminal side before being cleaved by neprilysin.
This implies potential cytotoxicity of M1 (released by
prodrugs I–III) to hepatocytes; however, as discussed ear-
lier, little is known about mechanisms involved with the
active transport of Dox (M3) and M1.
PSA-producing tissues, we modified its peptide sequence,
glutaryl-Hyp-Ala-Ser-Chg-Gln, towards the design and
synthesis of various Dox prodrugs. Systematic substitution
of the N-terminal segment of glutaryl-Hyp-Ala-Ser-Chg-
Gln with D-retro-inverso-peptides afforded GABA ←
mGly-Ala-Ser-Chg-Gln and glutaryl-Ser-Ala-Ser-Chg-Gln
as optimal promoieties (Aloysius and Hu 2015a) which
were subsequently coupled to Dox using a Ser-Leu linker.
L-377,202 or glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox
(I), glutaryl-Ser-Ala-Ser-Chg-Gln-Ser-Leu-Dox (II) and
GABA ← mGly-Ala-Ser-Chg-Gln-Ser-Leu-Dox (III) were
rapidly cleaved by PSA with comparable half-life values (<
23 min). The in vitro antiproliferative activity profiles of the
Summary
In order to improve the PSA specificity and the stability of
the prostate cancer prodrug L-377,202 (I) in normal non-

Medicinal Chemistry Research
newly synthesized Dox prodrugs, when compared to that of
prodrug I, showed a slight improvement in cytotoxicity.
Metabolism studies suggested that the Ser-Leu linker of
prodrugs I–III was more efficiently cleaved (compared to
P5 cleavages) in blood and cultured hepatocytes pre-
sumably by neprilysin which may have contributed to the
instability of prodrugs I–III in vivo. To mitigate neprilysin-
mediated hydrolysis of prodrugs I–III, Ser-Leu was
replaced with a self-immolative 3-aminooxypropionate lin-
ker coupled to Dox through its C-14 α-hydroxyl group,
affording GABA ← mGly-Ala-Ser-Chg-Gln-NH-O-CH2-C
(Me)2C(O)-14-O-Dox (IV). While the PSA cleavage rate of
prodrug IV was slower than that of prodrug I, its ability to
release free Dox contributed to a cytotoxic profile com-
parable to that of prodrug I. Additional stability, metabo-
lism and mouse PK studies will be conducted to further
evaluate prodrug II–IV as potential PSA-activated prostate
cancer prodrugs.
pumps, a SIL-20ACXR autosampler and SPD-M20A
diode array (Shimadzu, Columbia, MD). Metabolite
separation was achieved with a 2.1×100 mm, 1.7 µm BEH
C18 column (Waters Corporation, Huntingdon Valley,
PA) using a water (A)/acetonitrile (B) mobile phase sys-
tem containing 0.1% formic acid (v/v) and UV detection
at 484 nm. Prodrug quantification was performed on a
Transcend LX2 system (Thermo Fisher, Waltham, MA)
coupled to an API 4000 (AB Sciex, Framingham, MA)
using MRM transitions for both sodium adduct and base
peak from each prodrug (Table 1). While results were
consistent with either transition, only data generated with
the sodium adduct MRM transitions are shown.
Prodrug synthesis
Synthesis of prodrug II (Glutaryl-Ser-Ala-Ser-Chg-Gln-Ser-
Leu-Dox)
Fm-glutaryl-Ser-Ala-Ser-Chg-Gln-Ser-Leu-OH was pre-
pared using standard automated peptide synthesis proce-
Experimental section
dures.
Fm-glutaryl-Ser-Ala-Ser-Chg-Gln-Ser-Leu-OH
Materials and methods
(88 mg, 0.086 mmol) and Dox (25 mg, 0.043 mmol) were
dissolved in 2 mL anhydrous DMF in the presence of 2 eq.
DIEA. PyBOP (54 mg, 0.10 mmol) was added, and the
mixture stirred at room temperature under nitrogen atmo-
sphere for 2 h. Same-pot removal of the Fm-protecting
group was achieved with the addition of 200 μL of DEA
(10%). Trituration from DMF with 20 mL of ACN followed
by HPLC purification (10 mM ammonium acetate/ACN)
afforded the final peptide prodrug in 34% yield (20 mg).
Glutaryl-Ser-Ala-Ser-Chg-Gln-Ser-Leu-Dox; HRMS (ESI
+): m/z calc’d for C63H88N9O25: [M + H]+ = 1370.5891,
found: 1370.5920.
Purified PSA (99%, 1.37 mg protein/ml) was purchased
from Fitzgerald (North Acton, MA). Cell growth medium
was prepared by adding L-glutamine (2 mM), fetal bovine
serum (10%), penicillin G (100 units/mL) and strepto-
mycin sulfate (100 units/mL) to phenol red-containing
RPMI 1640. Cryopreserved human (lot EBP) and mouse
(lot Mc559) hepatocytes were supplied by Celsis (Chi-
cago, IL). HEPES buffer, Cryopreserved Hepatocyte
Recovery Media (CHRM), Cryopreserved Hepatocyte
Plating Media (CHPM) and all cell medium additives
were purchased from Invitrogen (Life Technologies,
Grand Island, NY). Cell maintenance buffer was prepared
by supplementing 500 mL of William’s E medium with
5 mL Penn-Strep Glutamine (100×), 5 μL Dexamethasone
(10 mM in DMSO), 5 mL Insulin-Transferrin-Selenium
and 7.5 mL 1 M HEPES pH 7.4. Reactions were mon-
itored by TLC and/or LC-MS. Flash column chromato-
graphy (FCC) was conducted on a Teledyne ISCO
CombiFlash Companion Automated Flash Chromato-
graphic System (Teledyne Technologies, Thousand Oaks,
Synthesis of Fm-GABA←mGly-Ala-Ser-Chg-Gln-Ser-
Leu-OH (2)
The Fm-GABA ← mGly-OSu ester was freshly prepared as
in the preceding paper (Aloysius and Hu 2020) and coupled
to the amino end of H-Ala-Ser-Chg-Gln-Ser-Leu-OH. The
peptide intermediate was obtained using standard automated
peptide synthesis procedures. To prepare the OSu ester, Fm-
GABA ← mGly (320 mg, 0.87 mmol) and HOSu (100 mg,
0.87 mmol) were dissolved in 6 mL of ACN. EDC (420 mg,
2.2 mmol) was added and the mixture stirred at room tem-
perature under nitrogen for 1 h. ACN was removed, the
residue dissolved in 40 mL of DCM, washed with 10 mL
water (3×), brine and dried over sodium sulfate. The crude
OSu ester (220 mg, 0.48 mmol) was directly coupled
(< 1 min) to H-Ala-Ser-Chg-Gln-Ser-Leu-OH (310 mg,
0.48 mmol) in the presence of 1 eq. DIEA in 1 mL ACN/
NMP (1:2). Trituration with 30 mL of ice-cold ether
CA) using pre-packed silica gel columns. All H and 13C
1
NMR spectra were recorded on a 400 MHz Bruker spec-
trometer at ambient temperature and calibrated using
residual un-deuterated solvents as the internal reference.
Accurate mass measurements and metabolite identifica-
tion were conducted using an LTQ-Orbitrap mass spec-
trometer (Thermo Fisher, Waltham, MA) equipped with
an electrospray ionization source operated in positive
ionization mode interfaced with Shimadzu LC-20ADXR

Medicinal Chemistry Research
afforded 310 mg of the crude peptide which was used for
the next step without further purification.
dimethylpropanoate (590 mg, 2.8 mmol), triphenylphosphine
(820 mg, 3.1 mmol) and N-hydroxyphthalimide (510 mg,
3.1 mmol) prepared in 20 mL anhydrous THF at 0 °C. After
stirring the mixture at room temperature under a nitrogen
atmosphere for 24 h, THF was removed under reduced pres-
sure and the residue re-dissolved in 40 mL of ethyl acetate.
The ethyl acetate mixture was washed with water (10 mL)
three times, brine and dry-loaded onto a 40-g silica column.
The final product was obtained in 73% yield (730 mg) as a
white solid after FCC purification (hexane/ethyl acetate).
Benzyl 3-((1,3-dioxoisoindolin-2-yl)oxy)-2,2-dimethylpro-
panoate. 1H NMR (CDCl3, 400 MHz): δ 7.84 (m, 2H), 7.76 (s,
Synthesis of prodrug III (GABA←mGly-Ala-Ser-Chg-
Gln-Ser-Leu-Dox)
Fm-GABA ← mGly-Ala-Ser-Chg-Gln-Ser-Leu-OH (96 mg,
0.096 mmol) and Dox (28 mg, 0.048 mmol) were dissolved in
the presence of 2 eq. DIEA in 2 mL anhydrous DMF. PyBOP
(60 mg, 0.12 mmol) was added and the mixture stirred at room
temperature under nitrogen atmosphere for 2 h. Same-pot
removal of the Fm-protecting group was achieved with the
addition of 200 μL of DEA (10%). Trituration from DMF with
20 mL of ACN and HPLC purification (10 mM ammonium
acetate/ACN) afforded the final peptide prodrug in 28% yield
2H), 7.39 (m, 5H), 5.19 (s, 2H), 4.32 (s, 2H), 1.42 (s, 6H); 13
C
NMR (CDCl3, 100 MHz): δ 175.0, 163.1, 136.0, 134.4,
129.0, 128.5, 128.1, 123.5, 83.6, 66.7, 43.2, 22.2, MS (ESI+):
m/z (intensity), 354.4 ([M + H]+, 100%).
Hydrogenolysis of benzyl 3-((1,3-dioxoisoindolin-2-yl)
oxy)-2,2-dimethylpropanoate (570 mg, 1.6 mmol) was
accomplished in the presence of 10% Pd-C in 20 mL de-
aerated methanol at room temperature for 5 h. The mixture
was filtered through a layer of celite and concentrated to a
crude oil (345 mg, 82%), which was used for the next step
without further purification.
To a solution of 3-((1,3-dioxoisoindolin-2-yl)oxy)-2,2-
dimethylpropanoic acid (150 mg, 0.57 mmol) prepared in
10 mL of anhydrous diethyl ether was added 36 μL (1.1 mmol)
of 98% anhydrous hydrazine, and the mixture stirred at room
temperature under a nitrogen atmosphere for 2 h. The mixture
was filtered and the filtrate concentrated to generate 78 mg of
crude NH2-O-CH2-C(Me)2COOH (white solid) which was
used for the next step without further purification.
(18 mg).
GABA ← mGly-Ala-Ser-Chg-Gln-Ser-Leu-Dox;
HRMS (ESI+): m/z calc’d for C62H86N9O24: [M + H]+ =
1340.5786, found: 1340.5817.
Synthesis of 14-bromodaunorubicin HCl
Timethylorthoformate (100 μL) was added to a solution of
daunomycin HCl (100 mg, 0.18 mmol) prepared in 6 mL of
Dioxane/MeOH (2:1) and the mixture stirred at room tem-
perature for 20 min. A Br2/CHCl3 (0.22 mmol in 370 μL)
solution was added dropwise and the mixture stirred at
room temperature for an additional 40 min. Precipitation
with 100 mL of ice-cold ether, followed by an ether wash
(100 mL) and filtration afforded a red solid. The final pro-
duct was obtained in 89% yield (102 mg) by recrystalliza-
tion from 5 mL acetone/ether (1:1).
14-Bromodaunorubicin HCl
Synthesis of Fmoc-Gln-OSu (11)
1H NMR (DMSO-d6, 400 MHz) δ 13.92 (s, 1H), 13.13
(s, 1H), 7.81 (m, 2H), 7.75 (s, 2H), 7.56 (dd, 1H, J1 = 2.1,
J2 = 8.2 Hz), 5.22 (m, 1H), 4.85 (m, 1H), 4.12 (q, 1H, J =
7.7 Hz), 3.90 (s, 5H), 3.52 (s, 1H), 3.31 (m, 1H), 3.06 (d,
1H, J = 19.1), 2.83 (d, 1H, J = 33.1 Hz), 2.43 (q, 2H, J =
2.0 Hz), 2.21 (d, 1H, J = 14.3), 1.89 (dd, 1H, J1 = 3.9, J2 =
12.7 Hz), 1.68 (d, 1H, J = 11.6 Hz)), 1.18 (d, 3H, J =
5.7 Hz); 13C NMR (DMSO-d6, 100 MHz): δ 213.7, 205.6,
186.4, 186.3, 160.7, 156.0, 154.4, 154.3, 136.2, 135.1,
134.6, 133.8, 119.9, 119.7, 119.0, 110.7, 99.1, 96.5, 89.4,
75.4, 69.7, 66.1, 56.6, 54.0, 40.1, 39.7, 34.1, 18.5, 16.7, MS
(ESI+): m/z (intensity), 606.3 ([M + H]+, 100%).
To a stirred solution (20 mL of ACN) of Fmoc-Gln(Trt)-OH
(611 mg, 1.0 mmol) and HOSu (150 mg, 1.3 mmol) was
added EDC (290 mg, 1.5 mmol), and the mixture stirred at
room temperature under nitrogen atmosphere for 2 h. ACN
was removed under reduced pressure and the residue dis-
solved in 40 mL of DCM. The organic layer was washed
with 10 mL water (2×), brine and dried over sodium sulfate.
DCM was removed under reduced pressure, and the residue
treated with 10 mL of 95%TFA at room temperature for
5 min. FCC purification (20% MeOH/DCM) afforded
471 mg of crude Fmoc-Gln-OSu (11) as a white solid.
Synthesis of (S)-5-(3-amino-3-oxopropyl)-1-(9H-
fluoren-9-yl)-10,10-dimethyl-3,6-dioxo-2,8-dioxa-
4,7-diazaundecan-11-oic acid (Fmoc-Gln-NH-O-CH2-C
(Me)2COOH, 12)
Synthesis of 3-(aminooxy)-2,2-dimethylpropanoic
acid (NH2-O-CH2-C(Me)2COOH, 9)
A solution of diisopropylazodicarboxylate (DIAD, 0.52 mL,
2.8 mmol) prepared in 2 mL anhydrous THF) was slowly
added to a stirred mixture of benzyl 3-hydroxy-2,2-
Crude NH2-O-CH2-C(Me)2COOH (9) (450 mg, 3.3 mmol)
and Fmoc-Gln-OSu (11) (466 mg, 1.0 mmol) were

Medicinal Chemistry Research
dissolved in 5 mL of anhydrous DMF and the mixture
stirred at room temperature under nitrogen atmosphere
for 24 h. DMF was removed under reduced pressure and
the residue dry-loaded onto a 40 g silica column. FCC
purification (DCM/ACN/(20%MeOH/DCM)) afforded
Fmoc-Gln-NH-O-CH2-C(Me)2COOH as a white solid in
66% yield (321 mg).
2-((2S,4S)-4-(((2R,4S,5S,6S)-4-(((allyloxy)carbonyl)
amino)-5-hydroxy-6-methyltetrahydro-2H-pyran-2-
yl)oxy)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-
1,2,3,4,6,11-hexahydrotetracen-2-yl)-2-oxoethyl 3-
(((S)-2,5-diamino-5-oxopentanamido)oxy)-2,2-
dimethylpropanoate (Fmoc-Gln-NH-O-CH2-C(Me)2C
(O)-14-O-(Alloc-Dox))
(S)-5-(3-amino-3-oxopropyl)-1-(9H-fluoren-9-yl)-10,10-
dimethyl-3,6-dioxo-2,8-dioxa-4,7-diazaundecan-11-oic acid
(12)
1H NMR (DMSO-d6, 400 MHz): δ 11.32 (s, 2H), 7.90 (m,
2H), 7.69 (m, 2H), 7.41 (m, 2H), 7.30 (m, 3H), 5.61 (s, 1H),
5.24 (m, 3H), 4.94 (s, 1H), 4.54 (d, 2H, J = 5.4 Hz), 4.46
(m, 1H), 3.99 (s, 3H), 3.87 (s, 2H), 3.81 (m, 1H), 3.64 (m,
1H), 2.96 (d, 1H, J = 18.5 Hz), 2.10 (m, 8H), 1.27 (s, 6H),
1.11 (d, 3H, J = 2.9 Hz); 13C NMR (DMSO-d6, 100 MHz):
δ 210.0, 202.0, 174.5, 173.4, 172.7, 156.0, 154.5, 143.7,
140.6, 133.6, 127.6, 127.0, 125.3, 120.0, 116.8, 75.2, 66.7,
65.7, 64.1, 56.5, 46.6, 42.4, 40.1, 39.9, 39.7, 38.9, 31.2,
25.2, 22.1, 22.0, MS (ESI+): m/z (intensity), 1093.2 ([M +
H]+, 100%).
1H NMR (CD3OD, 400 MHz): δ 7.81 (d, 2H, J = 7.61 Hz),
7.68 (d, 2H, J = 5.71 Hz), 7.40 (t, 2H, J = 6.66 Hz), 7.33
(t, 2H, J = 7.61 Hz), 4.39 (d, 2H, J = 3.76 Hz), 4.23 (t, 1H,
J = 6.88 Hz), 3.94 (s, 2H), 2.29 (m, 2H), 2.06 (m, 2H), 1.26
(s, 6H); 13C NMR (CD3OD, 100 MHz): δ 145.3, 145.2,
142.6, 134.0, 128.8, 128.2, 126.6, 126.2, 120.9, 83.5, 68.0,
53.8, 49.6, 43.8, 32.4, 28.9, 22.9, MS (ESI+): m/z (inten-
sity), 484.3 ([M + H]+, 100%).
Synthesis of prodrug IV (GABA←mGly-Ala-Ser-Chg-
Gln-NH-O-CH2-C(Me)2C(O)-14-O-Dox)
Synthesis of 2-((2S,4S)-4-(((2R,4S,5S,6S)-4-
(((allyloxy)carbonyl)amino)-5-hydroxy-6-
methyltetrahydro-2H-pyran-2-yl)oxy)-2,5,12-
trihydroxy-7-methoxy-6,11-dioxo-1,2,3,4,6,11-
hexahydrotetracen-2-yl)-2-oxoethyl 3-(((S)-2,5-
diamino-5-oxopentanamido)oxy)-2,2-
dimethylpropanoate (Fmoc-Gln-NH-O-CH2-C(Me)2C
(O)-14-O-(Alloc-Dox))
Fm-GABA ← mGly-Ala-Ser-Chg-OH (3) was synthesized
as reported in the preceding paper (Aloysius and Hu 2020).
Fm-GABA ← mGly-Ala-Ser-Chg-OH
(3,
7.6 mg,
0.011 mmol) and H-Gln-NH-O-CH2-C(Me)2C(O)-14-O-
(Alloc-Dox) (6) (5 mg, 0.0057 mmol) were dissolved in
300 μL of anhydrous DMF in the presence of DIEA (3 eq.)
and the mixture allowed to stir under nitrogen atmosphere at
0 °C for 10 min. A solution of HATU (4.1 mg, 0.011 mmol,
200 μL in anhydrous DMF) was added dropwise, and the
mixture stirred at 0 °C for 1 h to warm-up to room tem-
perature in 1 h. Trituration with 5 mL of ice-cold water,
followed by centrifugation afforded a dark orange solid
which was dissolved in 5 mL of methanol and dried over
sodium sulfate. After removing methanol under reduced
pressure, the residue was dissolved in 500 μL of anhydrous
THF in the presence of dimedone (11 mg, 0.08 mmol), and
catalytic amount of tetrakis(triphenylphosphine)palladium
(0) added. The mixture was stirred at room temperature for
1 h to allow complete deprotection of the Alloc-protecting
group. Addition of 5 mL of ice-cold ether followed by
centrifugation at 10,000 RPM (repeated 3 three times) gave
an orange solid which was re-dissolved in 500 μL of
anhydrous DMF. Fm-deprotection was performed with 2%
DBU at room temperature in 10 min. The mixture was tri-
turated with ice-cold ether as previously described, and the
final peptide prodrug obtained in 18% yield (1.3 mg) after
HLPC purification. GABA ← mGly-Ala-Ser-Chg-Gln-NH-
O-CH2-C(Me)2C(O)-14-O-Dox; HRMS (ESI+): m/z calc’d
Fmoc-Gln-NH-O-CH2-C(Me)2COOH (335 mg, 0.66 mmol)
was converted to its sodium salt as previously described and
added to a 10-mL acetone solution of 14-daunorubicin HCl
(53 mg, 0.083 mmol) in the presence of 4 Å molecular
sieves, and the mixture stirred at room temperature for 48 h.
After adding 10 mL of methanol, the mixture was cen-
trifuged at 3000 rpm for 15 min. The supernatant was col-
lected, dried in vacuo and reconstituted in 4 mL of
anhydrous ACN. Protection of the aminoglycoside was
carried out with the addition of Alloc-OSu (33 mg,
0.17 mmol) in the presence of 2 eq. of NaHCO3 and stirring
the mixture for 1 h at room temperature. Two-step FCC
purification from DCM/ACN; DCM/(20%MeOH/DCM)
afforded the desired compound Fmoc-Gln-NH-O-CH2-C
(Me)2C(O)-14-O-(Alloc-Dox) in 40% yield (40 mg, greater
than 90% pure by LC-UV). Fmoc-deprotection was per-
formed with 1% DBU in 1 mL of anhydrous DMF at room
temperature in 10 min. Trituration with 10 mL of ice-cold
ether followed by centrifugation at 3000 RPM afforded a
dark orange solid, which was purified by HPLC (10 mM
ammonium acetate/ACN) to afford 17 mg of H-Gln-NH-O-
CH2-C(Me)2C(O)-14-O-(Alloc-Dox) (6), which was used to
prepare prodrug IV without further purification.
for
C58H79N8O23:
[M + H]+ = 1255.5258,
found:
1255.5254.

Medicinal Chemistry Research
PSA assay
reader. Cell viability was computed as a percentage of
control growth, and IC50 values determined as the con-
centration at which cell growth is inhibited by 50%.
Peptide solution concentrations were determined by mea-
suring the UV absorbance at 484 of 20 μM solutions pre-
pared (based on weight) in water from 10 mM DMSO stock
solutions. Using the extinction coefficients of Dox at
484 nm (10,800 M−1.cm−1), peptide concentrations were
calculated and adjusted accordingly to prepare 100 μM
solutions in 50 mM Tris buffer, pH 8 containing 2 mM
CaCl2 and 0.1% Tween 20. The stability of peptide pro-
drugs in 50 mM Tris buffer, pH 8 was determined from 48-h
incubations in buffer (Fig. 1). Peptide solutions were pre-
pared in Tris buffer pH 8 at a concentration of 1.11 μM from
1 mM DMSO stock solutions, and warmed up to 37 °C.
Reactions were initiated at 37 °C with the addition of 5 μL
aliquots of a 10 μM solution of human PSA to 45 μL ali-
quots of the 1.11 μM peptide solutions in Eppendorf vials
(total volume 50 μL, final PSA concentration 1 μM), and
time points recorded over a period of 3 h. Reactions were
terminated at 0, 10, 20, 30, 60, 120, 180 and 360 min by
quenching 2 μL aliquots of incubates with 20 μL of chilled
50% ACN containing 0.1 μM internal standard (IS) and
0.1% formic acid (FA) pre-added to Eppendorf vials.
Samples were centrifuged at 3000 RPM for 15 min and the
supernatants analyzed by LC-MS/MS in MRM mode as
previously described. The fraction of prodrug remaining at
each time point was calculated by dividing the prodrug/IS
ratio by the 0-h prodrug/IS area ratio and half-life deter-
mined from prodrug disappearance rate.
Blood sample collection
Blood samples were collected in sodium-heparin tubes from
15 male adult C57BL/6 mice; the use of EDTA as antic-
oagulant was avoided due to its anticipated inhibitory effect
on metalloproteases through ion metal chelation. Likewise,
human blood was sampled from three adult male volunteers
in heparinized collection tubes and stored on ice until use.
Hepatocyte cultures
All hepatocyte culture experiments were conducted under
sterile conditions. Cryopreserved mouse or human hepato-
cytes (10–14 million cells) were thawed in 50 mL CHRM
pre-warmed to 37 °C in a water bath. The cell suspensions
were centrifuged at 800 RPM for 10 min and the pellet re-
suspended in 5 mL CHPM by gentle mixing. Cell count and
viability were determined using AO/PI (acridine orange/
propidium iodide) staining on a Nexcelom Cellometer
(Nexcelom Bioscience, Lawrence, MA). Cell suspensions
with viability greater than 80% were diluted with CHPM to
a concentration of 0.4 and 0.7 × 10 ^ 6 cells/mL, for mouse
and human hepatocytes, respectively, and seeded at 2.5 mL
per well on pre-coated collagen plates. Plates were incu-
bated at 37 °C in a 95% humidified incubator at 5% CO2 for
2–4 h to allow adequate cell attachment before replacing
CHPM with the maintenance media spiked with prodrug.
Carcinoma cell culture
Monolayer cultures of PSA-secreting LNCaP and non-PSA-
secreting DU145 human prostate carcinoma cells were
conducted in growth medium in a CO2 incubator under a
humidified atmosphere at 37 °C for 3 weeks prior to initi-
ating cytotoxicity experiments. During cell growth, media
were changed every 3 days and trypsinization carried out at
80% confluence with a 1:4 split for subsequent cultures.
Cells were grown on 96-well plates for 48 h at initial con-
centrations of 5000 cells/well. At the end of the 48-h period,
the growth medium was replaced by serum-free medium
RPMI 1640 medium containing L-glutamine (2 mM), 2%
TCM, penicillin G (100 units/mL) and streptomycin sulfate
(100 units/mL), and prodrugs added to wells at concentra-
tions ranging from 0.07 to 100 μM. The plates were incu-
bated at 37 °C for 72 h under a humidified CO2 atmosphere,
and cell viability determined using the MTT assay. Briefly,
10 μL aliquots of a 12 mM MTT solution were added to
wells and the plates incubated in a CO2 incubator under a
humidified atmosphere at 37 °C for 4 h. Well contents were
then solubilized with 1% SDS (100 μL/well) for 12 h, and
OD570 values measured on a Dynatech MR5000 plate
Metabolite profiling by accurate mass spectrometry
For each prodrug, 200 μL aliquots were drawn at 1, 2, 4, 8,
and 24 h from blood and plated hepatocyte incubations, and
quenched with 400 μL of chilled acetonitrile containing
0.1% formic acid. Samples were centrifuged at 3000 RPM
for 15 min, the supernatant removed and dried under a
gentle stream of nitrogen. Residues were reconstituted in
100 μL of acetonitrile:water (1:1) and analyzed by LC-MS
system with accurate mass determination. Accurate mass
measurements and metabolite identification were performed
using an LTQ-Orbitrap mass spectrometer (Thermo Fisher,
Waltham, MA) equipped with an electrospray source
operated in positive ionization mode interfaced with Shi-
madzu LC-20ADXR pumps, a SIL-20ACXR autosampler
and SPD-M20A diode array (Shimadzu, Columbia, MD).
Metabolite separation was achieved with a 2.1 × 100 mm,
1.7 µm BEH C18 column (Waters Corporation, Huntingdon
Valley, PA) using a water (A)/acetonitrile (B) mobile phase
system containing 0.1% formic acid (v/v) and UV detection
at 254 and 484 nm. The gradient was performed at a total

Medicinal Chemistry Research
flow rate of 200 μL/min as follows, 2% B from 0 to 3 min, 2
to 95% B from 3 to 20 min, 95% B from 20 to 25 min, 95 to
2% B from 25 to 25.1 min, 2% B from 25.1 to 30 min.
Compliance with ethical standards
Conflict of interest The authors have filed patents on some of the
compounds discussed in this paper.
Prodrug quantification by LC-MS/MS in mouse and
human blood
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Prodrug stability to non-PSA-mediated hydrolysis was
evaluated in heparinized mouse or human blood by incu-
bating each prodrug at a concentration of 100 μM for a
period of 24 h at 37 °C. Reactions were initiated by adding
10 μL of a 10 mM solution of each prodrug to 1 mL of
buffer, mouse or human blood and allowing the mixture to
shake at 37 °C in a 95% humidified incubator at 5% CO2.
Reactions were terminated at 0, 0.5, 1, 2, 4, 6 and 24 h by
quenching 50 μL aliquots of incubates with 50 μL of chilled
acetonitrile containing 0.2 μM internal standard (IS) and
0.1% formic acid (FA) pre-added to 96-well plates. Pre-
cipitated samples were centrifuged at 3000 RPM for 15 min
and the supernatants analyzed by LC-MS/MS in MRM
mode (Table 1). Prodrugs were quantified using LC-MS/MS
on a Transcend LX2 system (Thermo Fisher, Waltham,
MA) coupled to an API 4000 (AB Sciex, Framingham,
MA). As shown in Table 1, both MRM transitions for
sodium adduct and base peak from each prodrug were used
for LC-MS/MS quantification. Results were consistent with
either transition although only data generated with the
sodium adduct MRM transitions are shown. The fraction of
prodrug remaining at each time point was calculated by
dividing its prodrug/IS area ratio by the prodrug/IS area
ratio at 0 h.
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