Synthesis and evaluation of new 4-peptidamido-2-fluorobenzyl phosphoramide mustard conjugates as prodrugs activated by prostate-specific antigen

Article abstract of DOI:10.1007/s00044-020-02572-x

In our continued efforts to develop targeted prodrugs activated by prostate-specific antigen (PSA), we designed and synthesized novel phosphoramide mustard peptide conjugates using previously optimized PSA substrates. Initial Nu/Nu mouse PK studies indicated that prodrug I (glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) exhibits high clearance with significant extrahepatic metabolism in vivo. Substrate optimization studies were thus carried out to further improve PSA specificity and enable the design of prodrugs with reduced in vivo clearance and enhanced tumor selectivity. To assess the utility of the newly optimized sequences as promoieties, they were coupled to phosphoramide mustard using a 4-amino-2-fluorobenzyl alcohol linker akin to prodrug I. In the presence of human PSA, prodrug I was rapidly cleaved with a half-life (t_1/2) of 35 min. Prodrugs II (glutaryl-Ser-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) and III (GABA ← mGly-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) were hydrolyzed at slower rates with t_1/2 values of 80 and 107 min, respectively. These results we observed here are different from our previously reported data but may be explained by the fact that PSA-activated release of phosphoramide mustard and reactive quinonimine methides resulted in mechanism-based inhibition of PSA, thereby preventing further hydrolysis of prodrugs I–III. Prodrug I was cytotoxic to PSA-producing LNCaP cells with an IC₅₀ value of 7.3 μM and demonstrated 14-fold selectivity over the non-PSA-producing DU145. Despite its poor in vitro antiproliferative activity (IC₅₀ = 30 μM), prodrug III was found to be more stable against non-PSA-mediated hydrolysis compared with prodrug I as revealed by metabolite profiling studies, which was in agreement with its improved stability in human hepatocyte cultures. These results suggested that a combination of the peptide sequence GABA ← mGly-Ala-Ser-Chg-Gln with optimal linkers and/or other cytotoxic agents can help achieve an adequate balance between PSA cleavage rate and enhanced resistance to non-PSA-mediated hydrolysis. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-020-02572-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2020) 29:1264–1279
https://doi.org/10.1007/s00044-020-02572-x
ORIGINAL RESEARCH
Synthesis and evaluation of new 4-peptidamido-2-fluorobenzyl
phosphoramide mustard conjugates as prodrugs activated by
prostate-specific antigen
Herve Aloysius1 Longqin Hu
1,2
●
Received: 15 April 2020 / Accepted: 25 May 2020 / Published online: 3 June 2020
©
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
In our continued efforts to develop targeted prodrugs activated by prostate-specific antigen (PSA), we designed and
synthesized novel phosphoramide mustard peptide conjugates using previously optimized PSA substrates. Initial Nu/Nu
mouse PK studies indicated that prodrug I (glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) exhibits
high clearance with significant extrahepatic metabolism in vivo. Substrate optimization studies were thus carried out to
further improve PSA specificity and enable the design of prodrugs with reduced in vivo clearance and enhanced tumor
selectivity. To assess the utility of the newly optimized sequences as promoieties, they were coupled to phosphoramide
mustard using a 4-amino-2-fluorobenzyl alcohol linker akin to prodrug I. In the presence of human PSA, prodrug I was
rapidly cleaved with a half-life (t ) of 35 min. Prodrugs II (glutaryl-Ser-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide
1/2
mustard) and III (GABA ← mGly-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard) were hydrolyzed at slower rates
with t1/2 values of 80 and 107 min, respectively. These results we observed here are different from our previously reported
data but may be explained by the fact that PSA-activated release of phosphoramide mustard and reactive quinonimine
methides resulted in mechanism-based inhibition of PSA, thereby preventing further hydrolysis of prodrugs I–III. Prodrug I
was cytotoxic to PSA-producing LNCaP cells with an IC50 value of 7.3 μM and demonstrated 14-fold selectivity over the
non-PSA-producing DU145. Despite its poor in vitro antiproliferative activity (IC = 30 µM), prodrug III was found to be
5
0
more stable against non-PSA-mediated hydrolysis compared with prodrug I as revealed by metabolite profiling studies,
which was in agreement with its improved stability in human hepatocyte cultures. These results suggested that a combination
of the peptide sequence GABA ← mGly-Ala-Ser-Chg-Gln with optimal linkers and/or other cytotoxic agents can help
achieve an adequate balance between PSA cleavage rate and enhanced resistance to non-PSA-mediated hydrolysis.
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
longhu@pharmacy.rutgers.edu
2
Rutgers Cancer Institute of New Jersey, New Brunswick, NJ
8901, USA
0
1
Department of Medicinal Chemistry, Ernest Mario School of
Pharmacy, Rutgers, The State University of New Jersey,
Piscataway, NJ 08854, USA

Medicinal Chemistry Research (2020) 29:1264–1279
1265
Graphical Abstract
●
●
●
Keywords Prostate-specific antigen Phosphoramide mustard Prodrug Peptide-linked drug conjugate
Abbreviations
AMC
ACN
Boc
Bn
CDI
7-amino-4-methylcoumarin
acetonitrile
tert-butyloxycarbonyl
benzyl
carbonyldiimidazole
L-cyclohexylglycine
dichloromethane
diethylamine
N,N-diisopropylethylamine
dimethylformamide
Introduction
The feasibility of targeted prodrugs activated by prostate-
specific antigen (PSA) has previously been demonstrated
with various cytotoxic agents (Aloysius and Hu 2015b;
Yang et al. 2011; Giang et al. 2014; Choi et al. 2012).
Prostate disease and cancer progression is characterized by
high systemic PSA levels (McDonald et al. 2014; Hersch-
man et al. 1997; Nadler et al. 1995; O’Malley et al. 2014)
although low serum concentrations of the marker are also
detectable in healthy males (Balk et al. 2003; Catalona et al.
Chg
DCM
DEA
DIEA
DMF
DMSO
EDC
dimethyl sulfoxide
1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide
1991; Diamandis 1995; Levesque et al. 1995; Ayyıldız and
Ayyıldız 2014). PSA is a kallikrein serine protease with
high substrate specificity and a unique ability to cleave the
peptide bond after Gln. In addition, the proteolytic activity
of PSA is confined to the microenvironment of prostate
tumors due to α1-antichymotrypsin and α2-macroglobulin
inhibition in the blood (Leinonen et al. 1996; Otto et al.
FCC
Fm
Fmoc
GABA
HBTU
Flash column chromatography
fluorenylmethyl
fluorenylmethoxycarbonyl
γ-aminobutyric acid
O-benzotriazole N,N,N’,N’-tetramethyluronium
hexa-fluorophosphate
N-hydroxybenzotriazole
N-hydroxysuccinimide
high performance liquid chromatography
high-resolution mass spectrometry
isopropyl chloroformate
trans-4-hydroxy-L-proline
liquid chromatography-mass spectrometry
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide
1998; De Angelis et al. 2007; Nadler et al. 1995) making it
HOBt
HOSu
HPLC
HRMS
IPCF
Hyp
LC-MS
MTT
an attractive target for tumor-specific activation of peptide
prodrugs. Based on the cleavage maps for its natural sub-
strates, semenogelins I and II, several short peptides were
engineered as promoieties for prodrugs activated by PSA
(Aloysius and Hu 2015a; DeFeo-Jones et al. 2000; Den-
meade et al. 1997; Doan et al. 2015; Garsky et al. 2001;
Kwiatkowska et al. 2019; Lee et al. 2015; Tarvainen et al.
2
2
020; Wong et al. 2001; Wu and Hu 2016; DiPaola et al.
002; Jiang and Hu 2007; Jiang et al. 2009).
IS
internal standard
In this study, phosphoramide mustard was selected as a
candidate for peptide conjugation due to its cytotoxic
activity against both cycling and non-cycling cells (Kres
NMP
NMR
NTR
PSA
RPMI
TCM
MRM
THF
TFA
TLC
N-methyl-2-pyrrolidone
nuclear magnetic resonance
nitroreductase
prostate-specific antigen
Roswell Park Memorial Institute
tissue culture medium
multiple reaction monitoring
tetrathydrofuran
1995; Ganesan and Keating 2015), its small molecular
weight and size as well as the non-peptidic nature of the
self-immolative linker (anticipated to resist proteolysis)
required for its attachment to various promoieties (Hu and
Wu 2007; Wu and Hu 2016). We previously reported that
the prodrug 4-nitrobenzyl phosphoramide mustard demon-
strates improved efficacy in SKOV3 ovarian cells expres-
sing E. coli nitroreductase (NTR) and a toxicity profile
trifluoroacetic acid
thin-layer chromatography.

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Medicinal Chemistry Research (2020) 29:1264–1279
Scheme 1 Proposed PSA activation mechanisms of phosphoramide mustard prodrugs
comparable to that of CB1954 (Wu et al. 2011). The
cytotoxic agent released upon NTR activation of 4-
nitrobenzyl phosphoramide mustard is phosphoramide
mustard, an active metabolite of the DNA-alkylating agent
cyclophosphamide (Korkmaz et al. 2007). In combination
with other anticancer agents, cyclophosphamide is used for
the treatment of lymphomas and certain types of leukemia
(Hu et al. 2011; Wu and Hu 2016). Herein we report the
synthesis and evaluation of a series of peptide-linked
phosphoramide mustard conjugates incorporating the opti-
mized PSA substrates glutaryl-Ser-Ala-Ser-Chg-Gln and
GABA ← mGly-Ala-Ser-Chg-Gln (Aloysius and Hu 2015a)
as prodrugs activated by PSA to release phosphoramide
mustard in and around prostate cancer cells and tissues.
(
Shanafelt et al. 2007; Tabchi et al. 2019). The adminis-
tration of cyclophosphamide is associated with moderate
vomiting, diarrhea, and bone marrow suppression) to
(
Results and discussion
severe adverse effects (bloody urine, gonadal toxicity,
mouth sores, and joint pain) (Brock 1996; Emadi et al.
Prodrug design principle
2
009). Thus, phosphoramide mustard prodrugs activated by
PSA (Scheme 1) are anticipated to demonstrate prostate
tumor selectivity and improved safety profiles. The acti-
vated prodrug effectively moves the site of activation to
prostate cancer cells by PSA from the liver where cyclo-
phosphamide is activated by cytochrome P450 enzymes
To minimize steric hindrance near the scissile bond,
attachment of the PSA peptide substrate is typically carried
out by inserting a linker between the cytotoxic drug and the
PSA-cleavable promoiety (DeFeo-Jones et al. 2000; Garsky
et al. 2001; Wong et al. 2001; Jiang and Hu 2007; Ménez
et al. 2008). In the present study, peptides were coupled to
phosphoramide mustard using a 4-amino-2-fluorobenzyl
(
Jiang et al. 2006). The feasibility of coupling the cyclo-
phosphamide metabolite, 4-aminocyclophosphamide, to
various peptides as model prodrugs has been previously
explored and demonstrated (Jiang and Hu 2007; Hu and Wu
alcohol linker (NH -2-F-Bn-OH) as previously reported (Hu
2
and Wu 2007; Hu et al. 2011; Jiang and Hu 2007).
2
007; Wu and Hu 2016). Using a similar strategy, phos-
As proof-of-concept, prodrug I was synthesized using 4-
amino-2-fluorobenzyl alcohol as the linker and the known
PSA substrate, glutaryl-Hyp-Ala-Ser-Chg-Gln (Wu and Hu
2016). Following a single intravenous (IV) administration
to Nu/Nu mice at 2 mg/kg, prodrug I was rapidly cleared
from blood in vivo (Table 1). The high mouse blood
clearance (165 mL/min/kg) of prodrug I suggested that
there was significant extrahepatic metabolism given that
murine hepatic blood flow is ~90 mL/min/kg (Sohlenius-
Sternbeck 2006). These observations led to additional
efforts to further improve the PSA specificity of glutaryl-
Hyp-Ala-Ser-Chg-Gln in terms of cleavage rate and resis-
tance to non-PSA-mediated hydrolysis in matrices that lack
active PSA. Similar to prodrug I, the peptide sequences of
glutaryl-Ser-Ala-Ser-Chg-Gln and GABA ← mGly-Ala-
Ser-Chg-Gln, which we previously optimized (Aloysius and
Hu 2015a), were coupled to phosphoramide mustard using a
4-amino-2-fluorobenzyl alcohol linker to obtain phosphor-
amide mustard prodrugs II and III, respectively (Scheme 2)
phoramide mustard can be readily attached to our optimized
PSA substrates (Aloysius and Hu 2015a). Evaluation of
various D-retro-inverso-7-AMC-peptide surrogates revealed
that the optimal sequences for rapid PSA cleavage were
glutaryl-Ser-Ala-Ser-Chg-Gln-AMC and GABA ← mGly-
Ala-Ser-Chg-Gln-AMC, whereas GABA ← D-Ser-Ψ[NH-
CO-NH]-Ala-Ser-Chg-Gln-AMC and GABA ← mGly-Ala-
Ser-Chg-Gln-AMC were more resistant to non-PSA-
mediated hydrolysis in mouse and human plasma when
compared with the known substrate glutaryl-Hyp-Ala-Ser-
Chg-Gln (Aloysius and Hu 2015a).
The importance of incorporating a linker between the
promoiety and cytotoxic agent has been demonstrated for
several prodrugs (Garsky et al. 2001; Hu et al. 2011; Ménez
et al. 2008; Poreba 2020; Jiang and Hu 2013). We pre-
viously reported a series of self-immolative linkers to
enable ready attachment of highly specific PSA substrates to
small cytotoxic agents such as phosphoramide mustard

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Table 1 Summary of
PK parameters
Units
Mouse 1
Mouse 2
Mouse 3
Mean
SD
pharmacokinetic parameters
of prodrug I in Nu/Nu mice
AUC0−t
µM h
0.223
0.231
145
0.181
0.185
182
0.196
0.199
168
0.200
0.205
165
0.021
0.024
19
AUC0−∞
CL
µM h
mL/min/kg
Vss
L/kg
2.24
0.26
0.23
1.95
0.18
0.21
2.28
0.23
0.18
2.16
0.22
0.21
0.18
0.04
0.02
MRT
t½
h
h
Prodrug I was administered to 3 Nu/Nu mice at 2 mg/kg IV in a 0.4 mg/mL solution prepared in DMSO:
HBSS (10:90). Blood samples were drawn at 5, 15, 30 min, 1, 2, 4, 6, 8 and 24 h and analyzed by LC-MS/
MS (Table 2)
Scheme 2 Prodrug synthesis. Reagents and conditions: (a) Peptide 1, 2, or 3 (1.3 eq.), DIEA (2.6 eq.), DMF, 0 °C; (b) Compound 4, 0 °C, 30 min,
then rt, 30 min; (c) 10% DEA, DMF, rt, 30 min
in order to assess PSA-mediated tumor killing activity and
resistance to non-PSA-mediated metabolism.
with 3, were then coupled to the α-amino group of Gln-
Linker-phosphoramide mustard 4 to synthesize prodrugs
I–III, after Fm deprotection.
Synthetic strategy
Evaluation of peptidamidoarylmethyl
phosphoramide mustard prodrugs in PSA- and non-
PSA-producing cells
Peptide conjugates I–III were synthesized by first coupling
Gln to the 4-amino-2-fluorobenzyl alcohol linker, which
was then conjugated to various peptide sequences (Scheme 2).
The synthesis of Gln-linker-phosphoramide mustard 4 has
previously been reported by Wu et al. (Wu et al. 2011). A
combination of solution and solid-phase chemistry proce-
dures was used to synthesize Fm-GABA ← mGly-Ala-Ser-
Chg (3). Briefly, Fm-GABA ← mGly-OSu was prepared
and coupled to H-Ala-Ser-Chg in solution or by coupling
the Fm-GABA ← mGly-OSu ester to H-Ala-Ser-Chg-O-
resin at room temperature in 48 h. Standard solid phase
procedures were used to prepare Fm-glutaryl-Hyp-Ala-Ser-
Chg (1) and Fm-glutaryl-Ser-Ala-Ser-Chg (2) which, along
The stability of peptide-linked phosphoramide mustard
peptide conjugates in 50 mM Tris buffer, pH 8 was first
evaluated over a period of 48 h (Fig. 1). Prodrug dis-
appearance was monitored by LC-MS/MS analysis using
multiple reaction monitoring (MRM) as shown in Table 2.
There was roughly 80, 70 and 60% of prodrugs I, II, and III
remaining over a period of 24 h, respectively (data not
shown). All prodrugs slowly converted to their corre-
sponding 4-peptidamido-2-fluorobenzyl alcohol conjugate
through chemical hydrolysis (see “Non-PSA-Mediated

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Medicinal Chemistry Research (2020) 29:1264–1279
Metabolism”). Overall, peptide conjugate degradation was
insignificant over 8 h and did not affect our enzyme assay
result interpretation.
rates for prodrugs II and III were slower with t1/2 values of
80 and 107 min, respectively. The rank order of prodrug
cleavage rates appears to be reversed when compared with
our initial substrate optimization studies using 7‐amino‐4‐
methylcoumarin (AMC), which suggested that glutaryl-Ser-
Ala-Ser-Chg-Gln-AMC and GABA ← mGly-Ala-Ser-Chg-
Gln-AMC were cleaved by PSA 1.3- and twofold faster
than glutaryl-Hyp-Ala-Ser-Chg-Gln-AMC, respectively
(Aloysius and Hu 2015a). Thus, prodrugs II and III were
unexpectedly no better than prodrug I as suggested by their
longer half-lives. These results were surprising since we
anticipated no effects of peptide conjugation with phos-
phoramide mustard through the 4-amino-2-fluorobenzyl
linker on PSA cleavage due to the small size of 4-amino-2-
fluorobenzyl phosphoramide mustard. A possible explana-
tion for these results is that conjugation of glutaryl-Ser-Ala-
Ser-Chg-Gln and GABA ← mGly-Ala-Ser-Chg-Gln with 4-
amino-2-fluorobenzyl phosphoramide mustard may have
afforded poorer substrates for PSA. However, because the
half-life appeared to increase for our improved peptide
substrate glutaryl-Ser-Ala-Ser-Chg-Gln and GABA ←
mGly-Ala-Ser-Chg-Gln, a more plausible explanation is
that PSA was irreversibly inhibited by the activated
PSA cleavage rates were determined by incubating
peptide-linked phosphoramide mustard prodrugs with
human PSA over a period of 3 h. Substrate disappearance
was monitored by LC-MS/MS analysis using MRM, and
the fraction of prodrug remaining at any given time point
computed by taking the ratio of its peak area response and
that of the 0 h time point. As shown in Table 3 and Fig. 2,
prodrug I exhibited the fastest cleavage rate with a half-life
(
t ) of 35 min. Compared with prodrug I, the cleavage
1/2
Fig. 1 Stability of phosphoramide mustard prodrugs 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
Table 2 MRM transitions used for prodrug quantification by LC-MS/
MS
Transition 1a
Q1
Transition 2b
Q1
Prodrug
Q3
Q3
I
996.6
970.5
940.5
776.4
750.5
720.4
776.4
750.3
720.3
124.1
124.1
124.1
a
II
III
Fig. 2 Disappearance of phosphoramide mustard 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 CaCl and 0.1%
2
MRM transitions used to quantify prodrug/prodrug-sodium adducts or
base peaks
b
TWEEN-20 at 37 °C
Table 3 Hydrolysis of
Prodrug
% of prodrug at 60 min t1/2 (min)
phosphoramide mustard
prodrugs by human PSA
Glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide
mustard (I)
28
68
70
35
80
Glutaryl-Ser-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide
mustard (II)
GABA ← mGly-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide
mustard (III)
107
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)

Medicinal Chemistry Research (2020) 29:1264–1279
1269
Table 4 Prodrug cytotoxicity
Prodrug
a
LNCaP IC50 (μM) Selectivity over DU145
in PSA-producing LNCaP
cell lines
Glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-2-F-Bn-
phosphoramide mustard (I)
7.3
14
ND
1
Glutaryl-Ser-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide >100
mustard (II)
GABA ← mGly-Ala-Ser-Chg-Gln-NH-2-F-Bn-
phosphoramide mustard (III)
30
Conditions: PSA-producing LNCaP and non-PSA-producing DU145 cell lines were treated with prodrugs
I–III at concentrations ranging from 0.07 to 100 μM for 72 h, and cell viability determined using the
MTT assay
ND not determined
aSelectivity-fold expressed as the ratio of the LNCaP and DU145 (data not shown) IC50 values
Table 5 Non-PSA-mediated prodrug hydrolysis in mouse and
human blood
Prodrug
t1/2 (h)
Human Mouse
Glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-2-F-Bn-
phosphoramide mustard (I)
0.91
0.54
1.1
9.4
12
Glutaryl-Ser-Ala-Ser-Chg-Gln-NH-2-F-Bn-
phosphoramide mustard (II)
GABA ← mGly-Ala-Ser-Chg-Gln-NH-2-F-Bn-
phosphoramide mustard (III)
6.5
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. 3 Cytotoxicity of phosphoramide mustard prodrugs in LNCaP cells
results in Fig. 2, prodrug I showed cytotoxic response in
LNCaP with an IC50 of 7.3 μM and a 14-fold selectivity
over DU145 (Table 4, Fig. 3). Prodrug III was equally
cytotoxic in both cell lines (DU145 data not shown)
whereas prodrug II was inactive. These results can be
partially explained by the lack of robust PSA activation of
prodrugs II and III (Table 3, Fig. 2). The cytotoxicity
observed with prodrug III in both LNCaP and DU145 cell
lines is unlikely mediated by PSA and may be due to slow
prodrug hydrolysis and concomitant premature phosphor-
amide mustard release as suggested by its lower stability in
buffer as compared with prodrugs I (Fig. 1).
phosphoramide mustard or electrophilic quinonimine
methide released upon proteolysis, via mechanism-based
inhibition. Thus, the faster the release of the aforementioned
PSA inactivators, the slower the PSA cleavage rate due to
mechanism-based inhibition of the enzyme. It may, thus, be
beneficial to explore combinations of our improved PSA
peptide substrates with previously synthesized 4-
aminoarylmethyl alcohol linkers (Hu and Wu 2007; Hu
et al. 2011) to reach the optimal balance between PSA
cleavage rate and its mechanism-based inhibition.
In order to assess cytotoxicity and tumor selectivity,
prodrugs I–III were incubated in cultured PSA-producing
(
LNCaP) and non-PSA-producing (DU145) cell lines for a
Non-PSA-mediated metabolism
period of 72 h. Peptide conjugates were designed to be
activated extracellularly by PSA secreted from the LNCaP
cells, but not in DU145 cells. 4-Amino-2-fluorobenzyl
phosphoramide mustard conjugate or phosphoramide mus-
tard itself is expected to be released into the extracellular
space around tumor cells upon PSA activation of prodrugs
I–III, producing selective cytotoxicity. Thus, increased
hydrolysis of intracellular phosphoramide mustard by
phosphoramidases, which are typically induced in tumors,
should increase prodrug effectiveness. Consistent with our
To evaluate non-PSA-mediated metabolism, prodrugs I–III
were incubated in blood and cultured hepatocytes, biolo-
gical matrices lacking active PSA. Prodrugs 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.
Experiments in human blood indicated that prodrugs I and
III were the most stable in human blood with t1/2 values of
~1 h (Table 5 and Fig. 4). In heparinized mouse blood,

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Medicinal Chemistry Research (2020) 29:1264–1279
Table 6 Non-PSA-mediated prodrug hydrolysis in mouse and human
hepatocytes
Prodrug
t1/2 (h)
Human Mouse
Glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-2-F-Bn-
phosphoramide mustard (I)
13
20
>48
>48
>48
Glutaryl-Ser-Ala-Ser-Chg-Gln-NH-2-F-Bn-
phosphoramide mustard (II)
GABA ← mGly-Ala-Ser-Chg-Gln-NH-2-F-Bn-
phosphoramide mustard (III)
>48
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 (MRM)
Fig. 4 Stability of phosphoramide mustard prodrugs in human blood.
Each prodrug (100 μM) was incubated in human blood at 37 °C over a
period of 24 h
Fig. 6 Stability of phosphoramide mustard prodrugs 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 phosphoramide mustard prodrugs in mouse blood.
Each prodrug (100 μM) was incubated in mouse blood at 37 °C over a
period of 24 h
mouse and human hepatocytes (Table 6, Figs 6 and 7).
While the metabolic rates of prodrugs I–III in cultured
mouse hepatocytes (Fig. 7) were unexpectedly lower com-
pared with humans (Fig. 6) for unknown reasons, relative
comparison of prodrug stability was still possible within
each in vitro system. Soft spot metabolite identification
conducted in hepatocyte incubations enabled the char-
acterization of early steps in non-PSA-mediated metabo-
lism. Prodrugs were incubated in blood and cultured
hepatocytes, and prodrug-related fragments were identified
and characterized by LC-MS with accurate mass measure-
ments. In human blood and hepatocyte cultures, the earliest
degradants were M1a-c, also observed in Tris buffer incu-
bations (~30% formed, Scheme 3) after 48 h (data not
shown). Consequently, M1a-c likely resulted from chemical
degradation of phosphoramide mustard prodrugs I–III,
respectively. It is possible that the chemical degradation
observed in Tris buffer was accelerated in cultured human
and mouse hepatocytes thereby generating M1a-c, which
could be further converted to metabolites M2a-c (Scheme 3)
prodrugs I and II were the most stable in mouse blood
overall, with t1/2 values of 9.4 and 12 min, respectively
(
Table 5 and Fig. 5). As suggested by our metabolic pro-
filing studies, these results can be explained by chemical
instability and/or the involvement of different hydrolases in
human and mouse blood. In previous substrate optimization
studies, the sequence GABA ← mGly-Ala-Ser-Chg-Gln
coupled to 7-AMC was the most stable to non-PSA-
mediated metabolism in mouse and human plasma (Aloy-
sius and Hu 2015a).
To assess the relative propensity of prodrugs I–III to be
cleared by the liver, their metabolic stability in cultured
hepatocytes was evaluated. In contrast to blood stability
results, prodrugs II and III showed marked improvements
in stability in cultured human hepatocytes (Table 6, Fig. 6).
As expected from substrate optimization studies (Aloysius
and Hu 2015a), prodrug III elicited the highest resistance to
non-PSA-mediated metabolism with a t1/2 value >48 h in

Medicinal Chemistry Research (2020) 29:1264–1279
1271
t1/2 value of 0.23 h (Table 1). This marked difference in t1/2
values points to a disconnect between in vitro and in vivo
elimination rates if hydrolysis in blood were a major
clearance route for prodrug I in vivo. Thus, it is possible
that the in vivo clearance of prodrug I is driven by meta-
bolism by organs other than the liver (e.g., kidney).
Although all prodrugs were slowly metabolized in cultured
mouse hepatocytes, enhanced resistance to non-PSA-
mediated cleavage was clearly observed with the new pro-
drugs in human cultures.
In summary, we evaluated novel peptide-linked phos-
phoramide mustard conjugates designed as PSA-targeted
prodrugs for their PSA-mediated tumor killing activity and
propensity to undergo non-PSA-mediated metabolism. Nu/
Nu mouse PK studies indicated that prodrug I (glutaryl-
Fig. 7 Stability of phosphoramide mustard prodrugs in cultured mouse
hepatocytes. Each prodrug (100 μM) was incubated in plated mouse
hepatocytes at 37 °C over a period of 48 h
Hyp-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphoramide
mus-
tard), despite its excellent in vitro antiproliferative activity
in PSA-producing LNCaP cells (IC50 of 7.3 μM) and ade-
quate selectivity over non-PSA-producing DU145, exhib-
ited high clearance with significant extrahepatic metabolism
in vivo. In our continued efforts to design prodrugs with
reduced non-PSA-mediated metabolism, improved tumor
selectivity and toxicity profiles, the newly optimized PSA
substrates GABA ← mGly-Ala-Ser-Chg-Gln and glutaryl-
Ser-Ala-Ser-Chg-Gln (Aloysius and Hu 2015a) were cou-
pled to phosphoramide mustard and evaluated. In human
PSA experiments, prodrug I was cleaved by PSA with a t1/2
value of 35 min, followed by glutaryl-Ser-Ala-Ser-Chg-
Gln-NH-2-F-Bn-phosphoramide mustard (II, 80 min) and
GABA ← mGly-Ala-Ser-Chg-Gln-NH-2-F-Bn-phosphor-
amide mustard (III, 107 min). Time-dependent and
mechanism-based inactivation of PSA by the released
phosphoramide mustard and/or electrophilic quinonimine
methide may be responsible for the unexpectedly slower
cleavage rates of prodrugs II and III. Consistent with their
slower cleavage rates compared with prodrug I, prodrugs II
and III showed insignificant tumor killing activity. To
assess non-PSA-mediated hydrolysis of prodrugs I–III, we
conducted stability and metabolism studies in blood and
cultured hepatocytes, which contain no active PSA. Hepa-
tocyte stability and metabolism results confirmed that
P5 substitutions within the peptide promoiety of prodrug I
improved prodrug resistance to non-PSA-mediated meta-
bolism. The improvements were more apparent in human
hepatocytes as indicated by significant differences in t1/2
values between prodrug I and the newly synthesized pro-
drug III. Future studies will explore the profiles of prodrugs
derived from the combination of the sequence GABA ←
mGly-Ala-Ser-Chg-Gln with previously reported self-
immolative linkers (Hu et al. 2011) to achieve an ade-
quate balance between PSA cleavage rate and resistance to
non-PSA-mediated hydrolysis.
by enzymatic intervention as indicated by the results in
Table 7, Figs 8 and 9. Metabolic profiling of blood samples
from initial mouse PK studies with prodrug I also indicated
that M1a and M2a were major circulating metabolites in
vivo (data not shown). Thus, the instability of phosphor-
amide mustard prodrugs appeared to result from chemical
degradation and/or phosphoramidase-mediated hydrolysis to
the corresponding 2-fluoro-4-peptidylaminobenzyl alcohol
conjugates which were subsequently hydrolyzed by ami-
dases. Despite the anticipated resistance of their amino-
benzyl alcohol linker to endopeptidase action, peptide-linked
phosphoramide mustard prodrugs I–III may be susceptible
to hydrolysis by phosphoramidases which are typically
elevated in tumors. Prodrugs become vulnerable to degra-
dation by aminopeptidases and carboxypeptidases, once
hydrolyzed by endopeptidases.
Overall, in human cell cultures, phosphoramide mustard
prodrugs II and III demonstrated improved stability com-
pared with prodrug I, suggesting that non-PSA-mediated
metabolism was mitigated. Further improvement of che-
mical stability and resistance to phosphoramidases can be
achieved by exploring additional linkers reported in
structure-activity relationship studies (Hu et al. 2011) and/or
coupling the peptide sequences to cytotoxic agents other
than phosphoramide mustard (Aloysius and Hu 2020).
Results from exploratory PK studies with prodrug I in Nu/
Nu mice suggest that additional improvements in chemical
stability can be beneficial towards reducing in vivo clear-
ance given that significant resistance to non-PSA-mediated
metabolism in human hepatocytes was achieved with pro-
drugs II and III. Noteworthy, the in vitro blood t1/2 value of
prodrug I generated in fresh heparinized blood was 9.4 h
(
Table 5) which is roughly 40-fold longer than its in vivo

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Medicinal Chemistry Research (2020) 29:1264–1279
Scheme 3 Metabolism of prodrugs I–III and the metabolites M1a-c and M2a-c identified
Table 7 Metabolites of
Prodrug (Base peak, RT)
M1a-c
M2a-c
phosphoramide mustard
Founda
prodrugs I–III
Theoretical mass
Founda
Theoretical mass
I (996.3577, 14.1)
II (970.3417, 14.1)
III (940.3311, 14.2)
873.3559
847.3403
817.3297
873.3553, 12.1
847.3407, 12.2
817.3303, 11.5
794.3736
768.3580
738.3474
794.3732, 12.9
768.3569, 12.9
738.3478, 12.5
aMeasured as mass of base peak at retention time (amu, min)
Experimental section
Teledyne ISCO CombiFlash Companion Automated Flash
Chromatographic System (Teledyne Technologies, Thou-
1
Materials and methods
sand Oaks, CA) using pre-packed silica gel columns. All H
1
3
and C NMR spectra were recorded on a 400 MHz Bruker
spectrometer 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
pumps, a SIL-20ACXR autosampler, and SPD-M20A
diode array detector (Shimadzu, Columbia, MD). Meta-
bolite separation was achieved with a 2.1 × 100 mm,
1.7 µm BEH C18 column (Waters Corporation, Hunting-
don Valley, PA) using a water (A)/acetonitrile (B) mobile
phase system containing 0.1% formic acid (v/v) and UV
detection at 484 nm. Prodrug quantification was per-
formed on a Transcend LX2 system (Thermo Fisher,
Waltham, MA) coupled to an API 4000 (AB Sciex, Fra-
mingham, MA) using MRM transitions for both sodium
adduct and base peak from each prodrug (Table 2). While
results were consistent with either transition, only data
generated with the sodium adduct MRM transitions
are shown.
Male Nu/Nu mice were ordered from Charles River
Laboratories (Wilmington, MA). Prodrug I was previously
synthesized in our laboratory (Hu and Wu 2007). Purified
PSA (99%, 1.37 mg protein/ml) was purchased from Fitz-
gerald (North Acton, MA). Cell growth medium was pre-
pared by adding L-glutamine (2 mM), fetal bovine serum
(
10%), penicillin G (100 units/mL), and streptomycin sul-
fate (100 units/mL) to phenol red-containing RPMI 1640.
Cryopreserved human (lot EBP) and mouse (lot Mc559)
hepatocytes were supplied by Celsis (Chicago, IL). HEPES
buffer, Hank’s Balanced Salt Solution (HBSS), Cryopre-
served Hepatocyte Recovery Media (CHRM), Cryopre-
served 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 monitored by TLC and/or LC-MS. Flash
column chromatography (FCC) was conducted on a

Medicinal Chemistry Research (2020) 29:1264–1279
1273
Prodrug II
Prodrug I
Exact mass: 996.3577)
(
M1a
M1b
(
Exact mass: 873.3533)
M2a
M2b
(
Exact mass: 794.3722)
Prodrug III
M1c
M2c
Fig. 8 Metabolic profiles of prodrugs I–III in cultured human hepatocytes at 1 h
Synthesis of Fm-GABA←mGly-Ala-Ser-Chg-OH (3)
-((tert-Butoxycarbonyl)amino)butanoic acid (Boc-GABA)
brine, and dried over sodium sulfate. Ethyl acetate was
removed under reduced pressure to afford the intermediate
Boc-GABA collected as a white solid in 84% yield (1.7 g).
4
GABA (1.0 g, 9.8 mmol) was dissolved in 24 mL of 6%
4-((tert-Butoxycarbonyl)amino)butanoic acid (Boc-GABA)
NaHCO solution at 0 °C. A pre-cooled solution of Boc-
3
1
anhydride (15 mmol in 10 mL dioxane) was added slowly
and the mixture stirred at 0 °C for 1 h, then overnight at room
temperature. After removing dioxane under reduced pressure,
the aqueous mixture was acidified to pH 1 with 1 N sodium
bisulfate and extracted with 50 mL ethyl acetate three times.
The combined organic layer was washed with 10 mL water,
H NMR (CDCl , 400 MHz): δ 3.21 (m, 2H), 2.42 (t, 2H,
3
J = 7.2 Hz), 1.84 (q, 2H, J = 7.0 Hz, J = 6.4 Hz), 1.46 (s,
1
2
1
3
9H); C NMR (CDCl , 100 MHz): δ 166.7, 141.3, 125.0,
3
120.1, 67.8, 46.6, 40.8, MS (ESI+): m/z (intensity), 203.7
([M + H]+, 45%), 406.8 ([2M + H]+, 74%), 306.8 ([2M +
H-Boc]+, 100%).

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Medicinal Chemistry Research (2020) 29:1264–1279
Prodrug I
Prodrug II
M1a
M1b
M2a
M2b
Prodrug III
M1c
M2c
Fig. 9 Metabolic profiles of prodrugs I–III in cultured mouse hepatocytes
(
9H-Fluoren-9-yl)methyl 4-((tert-butoxycarbonyl)amino)
group was carried out with 50% TFA/DCM at room tem-
perature in 30 min. All solvents were removed under a
gentle nitrogen stream to afford the intermediate Boc-
GABA-Fm, which was washed repeatedly with hexanes
(5 × 20 mL) and used for the next step.
butanoate (Boc-GABA-Fm)
Boc-GABA (1.0 g, 9.8 mmol) was pre-activated with CDI
(
1.6 g, 9.8 mmol) in 2 mL of anhydrous DCM at room
temperature under nitrogen atmosphere for 1 h. Fluor-
enylmethanol (1.3 g, 6.5 mmol) was added and the reaction
carried out at room temperature under nitrogen atmosphere
for 4 h. The mixture was diluted to 20 mL with DCM,
(9H-Fluoren-9-yl)methyl 4-((tert-butoxycarbonyl)amino)
butanoate (Boc-GABA-Fm)
1
washed sequentially with 20 mL 5% NaHCO , brine and
H NMR (CDCl , 400 MHz): δ 7.80 (d, 2H, J = 8.0 Hz),
3
3
dried over sodium sulfate. After removing DCM under
reduced pressure, the product was collected as an off-white
solid in 95% yield (1.2 g). Removal of the Boc-protecting
7.61 (d, 2H, J = 7.4 Hz), 7.44 (t, 2H, J = 7.3 Hz), 7.35 (t,
2H, J = 7.9 Hz), 4.56 (s, 1H), 4.45 (d, 2H, J = 6.7 Hz), 4.24
(t, 1H, J = 7.3 Hz), 3.13 (m, 2H), 2.43 (t, 2H, J = 7.3 Hz),

Medicinal Chemistry Research (2020) 29:1264–1279
1275
.81 (q, 2H, J = 6.8 Hz), 1.46 (s, 9H); 3C NMR (CDCl3,
00 MHz): δ 143.8, 141.3, 127.8, 127.1, 125.0, 120.0, 66.2,
6.9, 38.4, 31.5, 28.4, 25.3, MS (ESI+): m/z (intensity),
81.7 ([M + H]+, 10%), 282.0 ([M + H-Boc], 100%).
1
1.7 g of the free acid Fm-GABA ← mGly, which was carried
forward to the next step without further purification.
1
1
4
3
Fm-GABA ← mGly-Ala-Ser-Chg-OH (3)
t-BuO-mGly
The Fm-GABA ← mGly-OSu ester freshly prepared above
was coupled to the amino end of H-Ala-Ser-Chg-OH. The
tripeptide was generated using standard automated peptide
synthesis procedures. The OSu ester (410 mg, 0.87 mmol)
was directly coupled (within 1 min) to H-Ala-Ser-Chg-OH
(230 mg, 0.73 mmol) in 1 mL ACN/NMP (1:2) in the pre-
sence of 1 eq. DIEA. After FCC purification, the final
peptide was obtained in 56% yield (270 mg).
Malonic acid (5.0 g, 48 mmol) and t-BuOH (1.8 mL,
1
9 mmol) were dissolved in 150 mL of ACN at room
temperature under nitrogen. EDC (9.2 g, 48 mmol) was
added and the reaction conducted at room temperature
under nitrogen for 30 min. ACN was removed under
reduced pressure and the residue dissolved in 200 mL of
ether. The product was back-extracted with two 50 mL
portions of saturated NaHCO , and the combined aqueous
Synthesis of 2-fluoro-4-glutaminylaminobenzyl
3
layer acidified to pH 2 with 1 N sodium bisulfate. Finally,
the product was extracted with three 200 mL portions of
DCM which were combined and washed with water, brine,
and dried over sodium sulfate. DCM was removed under
reduced pressure to afford the intermediate t-BuO-mGly as
a white solid in 76% yield (2.3 g).
phosphoramide mustard (4)
All intermediates leading to compound 4 were synthesized
as previously described (Wu et al. 2011). TFA-Gln was
coupled to 4-azido-2-fluorobenzyl phosphoramide mustard
linker using a selenocarboxylate/azide amidation strategy to
afford 58% yield (462 mg) of crude 2-fluoro-4-
glutaminylaminobenzyl phosphoramide mustard.
t-BuO-mGly
1
H NMR (CDCl , 400 MHz): δ 3.28 (s, 2H), 1.42 (s, 9H);
Synthesis of prodrug II (Glutaryl-Ser-Ala-Ser-Chg-
Gln-NH-2-F-Bn-phosphoramide mustard)
3
1
3
C NMR (CDCl , 100 MHz): δ 168.5, 162.1, 83.5, 39.7,
3
2
7.9, 27.8, 25.6.
Fm-glutaryl-Ser-Ala-Ser-Chg-OH was prepared using
standard automated peptide synthesis procedures. Fm-glu-
taryl-Ser-Ala-Ser-Chg-OH (40 mg, 0.058 mmol) and HBTU
(22 mg, 0.058 mmol) were dissolved in the presence of 2.6
eq. DIEA in 250 μL of anhydrous DMF at 0 °C. 2-Fluoro-4-
glutaminylaminobenzyl phosphoramide mustard (21 mg,
0.044 mmol) was added and the mixture stirred at 0 °C for
30 min, and allowed to warm-up to room temperature in
30 min. The mixture was cooled to 0 °C and 4 mL of ice-
cold water added dropwise. After stirring the resulting
suspension at 0 °C for an additional 30 min, the precipitate
was collected by centrifugation, washed with 2 mL ice-cold
water (2×) and dried under reduced pressure. The crude
solid was re-dissolved in 250 μL of anhydrous DMF and de-
protected with 10% DEA. The mixture was triturated with
ice-cold ether and the final peptide prodrug obtained in 26%
yield (11 mg) after HLPC purification. Glutaryl-Ser-Ala-
Ser-Chg-Gln-NH-2-F-Bn-phosphoramide mustard; HRMS
t-BuO-mGly-OSu
To a solution (7 mL, ACN) of t-BuO-mGly (2.3 g,
1
5 mmol) and HOSu (1.7 g, 15 mmol) was added EDC
(
3.1 g, 16 mmol), and the mixture stirred at room tempera-
ture under nitrogen atmosphere for 1 h. ACN was removed
under reduced pressure and the residue dissolved in 200 mL
of DCM. The organic layer was washed with 50 mL water
(
2×), brine and dried over sodium sulfate. DCM was
removed under reduced pressure to afford the desired
intermediate t-BuO-mGly-OSu as a white solid in 84%
yield (3.1 g).
Fm-GABA←mGly
A solution of t-BuO-mGly-OSu (1.8 g, 6.9 mmol) and
GABA-Fm (2.0 g, 6.9 mmol) was prepared in 25 mL of
anhydrous DCM in the presence of DIEA (1.2 mL,
+
(ESI+): m/z calc’d for C H Cl FN O P: [M + H] =
3
8
60
2
9
13
6
.9 mmol) and stirred at room temperature under nitrogen
atmosphere for 1 h. The reaction mixture was diluted to
0 mL with DCM, washed with 10 mL water (2×), brine and
970.3409, found: 970.3417.
4
Synthesis of prodrug III (GABA←mGly-Ala-Ser-Chg-
dried over sodium sulfate. DCM was removed under reduced
pressure and the crude mixture purified by FCC to afford the
final product in 64% yield (1.9 g). Removal of the t-butyl-
protecting group was achieved with 50%TFA/DCM to afford
Gln-NH-2-F-Bn-phosphoramide mustard)
Fm-GABA ← mGly-Ala-Ser-Chg-OH (3, 40 mg, 0.060 mmol)
and HBTU (23 mg, 0.060 mmol) were dissolved in the

1276
Medicinal Chemistry Research (2020) 29:1264–1279
presence of 2.6 eq. DIEA in 250 μL of anhydrous DMF at
°C. 2-Fluoro-4-glutaminylaminobenzyl phosphoramide
mustard (16 mg, 0.055 mmol) was added and the mixture
stirred at 0 °C for 30 min to warm-up to room temperature in
initiating 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 cul-
tures. Cells were grown on 96-well plates for 48 h at initial
concentrations 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 strep-
tomycin sulfate (100 units/mL), and prodrugs added to
wells at concentrations ranging from 0.07 to 100 μM. The
plates were incubated at 37 °C for 72 h under a humidified
0
3
0 min. The mixture was cooled to 0 °C and 4 mL of ice-cold
water added dropwise. After stirring the resulting suspension at
°C for an additional 30 min, the precipitate was collected by
0
centrifugation, washed with 2 mL of ice-cold water (2×) and
dried under reduced pressure. The crude solid was re-dissolved
in 250 μL of anhydrous DMF and de-protected with 10%
DEA. The mixture was triturated with ice-cold ether and the
final peptide prodrug obtained in 28% yield (10 mg) after
HLPC purification. GABA ← mGly-Ala-Ser-Chg-Gln-NH-2-
F-Bn-phosphoramide mustard; HRMS (ESI+): m/z calc’d for
CO atmosphere, and cell viability determined using the
2
MTT assay. Briefly, 10 μL aliquots of a 12 mM MTT
solution were added to wells and the plates incubated in a
+
C H Cl FN O P: [M + H] = 940.3304, found: 940.3311.
CO incubator under a humidified atmosphere at 37 °C for
37
58
2
9
12
2
4
h. Well contents were then solubilized with 1% SDS
PSA assay
(100 μL/well) for 12 h, and OD570 values measured on a
Dynatech MR5000 plate reader. Cell viability was com-
puted as a percentage of control growth, and IC50 values
determined as the concentration at which cell growth is
inhibited by 50%.
Phosphoramide mustard peptide solution concentrations
were determined by measuring the UV absorbance at
2
54 nm using 50 μM solutions prepared (based on weight)
in water from 10 mM DMSO stock solutions. Using the
extinction coefficients of 2-fluoro-4-glutaminylaminobenzyl
Nu/Nu mouse PK study
−
1
−1
phosphoramide mustard at 254 nm (20,800 M cm ),
peptide concentrations were calculated and adjusted
accordingly to prepare 100 μM solutions in 50 mM Tris
Three Nu/Nu male mice (weight 25–28 g) received prodrug
I at 2 mg/kg by IV injection in a 0.4 mg/mL solution pre-
pared in DMSO: HBSS (10:90). Blood samples (20 μL)
were drawn at 5, 15, 30 min, 1, 2, 4, 6, 8 and 24 h and
collected on a 96-well plate containing 60 μL of 0.5 M
sodium citrate on ice. Prodrug quantification was carried out
as described under “Prodrug Quantification by LC-MS/MS
in Mouse and Human Blood”.
buffer, pH 8 containing 2 mM CaCl and 0.1% Tween 20.
2
The stability of peptide prodrugs in 50 mM Tris buffer, pH
8
was determined from 48 h incubations in buffer (Fig. 1).
Peptide solutions were prepared in Tris buffer pH 8 at a
concentration of 1.11 μM from 1 mM DMSO stock solu-
tions, and warmed up to 37 °C. Reactions were initiated at
3
7 °C with the addition of 5 μL aliquots of a 10 μM solution
of human PSA to 45 μL aliquots 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,
Blood sample collection for in vitro studies
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.
1
80 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
1
5 min and the supernatants analyzed by LC-MS/MS in
MRM mode as previously described. The fraction of pro-
drug remaining at each time point was calculated by dividing
the prodrug/IS ratio by the 0–h prodrug/IS area ratio and
half-life determined from prodrug disappearance rate.
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 CHRM 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
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 CO incubator under a
2
humidified atmosphere at 37 °C for 3 weeks prior to

Medicinal Chemistry Research (2020) 29:1264–1279
1277
with viability greater than 80% were diluted with CHRM 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-
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.
bated at 37 °C in a 95% humidified incubator at 5% CO for
2
2
–4 h to allow adequate cell attachment before replacing
CHRM with the maintenance media spiked with prodrug.
Prodrug quantification by LC-MS/MS in mouse and
Metabolite profiling by accurate mass spectrometry
human plated hepatocytes
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
As previously described for blood incubations, prodrug
incubations in cultured hepatocytes were initiated at a
concentration of 100 μM by diluting 25 μL of 10 mM
DMSO solutions in 2.5 mL of maintenance media added to
1.75 million cells per well. Reactions were carried out at
0
1
.1% FA. Samples were centrifuged at 3000 RPM for
5 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 mea-
surements 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,
37 °C in a 95% humidified incubator at 5% CO . At 0, 0.5,
2
1, 2, 4, 8, 24 and 48 h, 50 μL aliquots of incubates were
sampled and quenched with 50 μL of chilled acetonitrile
containing 0.2 μM IS and 0.1% FA pre-added to 96-well
plates. Precipitated samples were centrifuged at 3000 RPM
for 15 min and the supernatants analyzed by LC-MS/MS in
MRM mode. The fraction of prodrug remaining at each time
point was calculated.
Acknowledgements We gratefully acknowledge the financial support
of grant SNJ‐CCR 700–009 from the State of New Jersey Commission
on Cancer Research, a pilot grant from the Gallo Prostate Cancer
Center of the Cancer Institute of New Jersey, and grant RSG‐
1
.7 µm BEH C18 column (Waters Corporation, Huntingdon
Valley, PA) using a water (A)/acetonitrile (B) mobile phase
system containing 0.1% FA (v/v) and UV detection at
0
3–004–01‐CDD from the American Cancer Society.
2
2
54 nm. The gradient was performed at a total flow rate of
00 μL/min as follows, 2% B from 0 to 3 min, 2–95% B
from 3 to 20 min, 95% B from 20 to 25 min, 95–2% B from
5 to 25.1 min, 2% B from 25.1 to 30 min.
Compliance with ethical standards
2
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
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